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Saiu-me certo, fui elogiado… Meu coração é um enorme estrado Onde se expõe um pequeno animálculo… A, microscópio de desilusões
Findei, prolixo nas minúcias fúteis… Minhas conclusões práticas, inúteis… Minhas conclusões teóricas, confusões… Que teorias há para quem sente
O cérebro quebrar-se, como um dente Dum pente de mendigo que emigrou? Fecho o caderno dos apontamentos E faço riscos moles e cinzentos Nas costas do envelope do que sou… Álvaro de Campos
Synthesis of novel and rigid 1,4-benzodiazepine-2,5-dione scaffolds derived from spiro bicyclic D- or L-proline analogues, containing a D-fructose or a L-fructose moiety, was accomplished. The D-proline analogue was synthesised from D-fructose in sixteen-steps and 24% overall yield through a three carbon chain elongation at the anomeric position, introduction of the amino function at C-1, cyclization to pyrrolidine and introduction of the carboxylic group. The spiro bicyclic L-proline analogue was obtained either starting from sorbose via a nineteen steps synthesis in 2% overall yield or starting from arabinose through a pathway involving eighteen steps in 3% overall yield. Both pathways make use of the key intermediate 3-C-(3,4,6-tri-O-benzyl- α-L-fructofuranos-2-yl)propene. In this work a new method was developed for its synthesis taking advantage of the stereochemistry of the arabinonolactone, easily obtained by oxidation of the anomeric position. Chain elongation was then accomplished by reaction with 2-lithium-1,3-dithiane, subsequent formation of the aldehyde and final reduction to the primary alcohol gave the target molecule in 13.5% overall yield. This method is more efficient than the procedure reported in the literature for L-fructose synthesis using sorbose. The latter pathway involved various protection and deprotection steps, in addition to oxirane ring formation and opening to build the appropriate sugar stereochemistry and led to the preparation of the key precursor in 8.5% overall yield. Starting from this key precursor, and using the methodology applied for the D-proline analogue, which requires subsequent eleven steps, the desired new spiro bicyclic L-proline analogue was obtained in 24% overall yield.
Conformational analysis, performed by Molecular Modelling calculations and DNMR data, established the absence of conformational equilibrium in the benzodiazepine ring of the fructose-proline-benzodiazepine scaffolds synthesized. These studies showed a unique preferential conformation of the benzodiazepine ring for each enantiomer. The D-proline analogue induced a rigid (P)-helical conformation and the
L-proline analogue induced a rigid (M)-helical conformation on the 1,4-benzodiazepine-2,5-dione ring, where the fused bicyclic moiety is pseudo-
equatorial for both conformers. The conformational rigidity of the pyrrolobenzodiazepine core linked to the fructose by a spiro junction, allowed an accurate evaluation of the effect of conformational changes in the 1,4-benzodiazepine- 2,5-dione ring on the ability of benzodiazepines to bind to the receptor complex, clarifying the stereoselectivity of this binding site.
Several literature reports showed that binding affinities at GABAA R are sensitive to the helical chirality of the 1,4-benzodiazepine ring and that the (M)-helical conformation is required for best binding to GABAA R. However, preliminary biological evaluation of the synthesized enantiomeric benzodiazepine derivatives showed that the conformation of the benzodiazepine ring was not the determining factor for best binding. We observed that both conformers have similar binding affinities. So, it appears that the receptor binding is governed mainly by the pseudo- equatorial preference of the C-11a substituent instead of (M) or (P) conformation.
In addition, we observed that binding is crucially dependent on the type of substituents present in the sugar (benzyl or hydrogen on the fructose moiety), in the nitrogen N10 (hydrogen or methyl) or in the benzene ring (halogen, nitro or amino). We concluded that substituents in position seven are essential for binding affinity, being –Br the less effective one. The D- and L-fructose-based pyrrolobenzodiazepines substituted with amino or nitro groups in the benzene ring showed the highest affinity for GABAA R, in the range of μM. In general, benzodiazepines with an N-methyl in the diazepine and free hydroxyl groups in the sugar showed higher ability to displace tritiated flunitrazepam from the corresponding binding site in the GABAA R.
Moreover, pyrrolobenzodiazepines synthesized without a sugar moiety showed binding activities slightly higher than those linked to a sugar moiety. In particular, the most potent fructose-proline-benzodiazepine derivative showed 30% reduction of the radioligand specific binding, while the most active pyrrolobenzodiazepine derivative without sugar moiety gave a 40% reduction. This difference does not seem to be
considerably relevant, so it can be assumed that the fructose moiety does not cause significant steric hindrance on binding to the receptor.
In addition, synthesis of novel D-fructose-based β-disubstituted γ-butyrolactones and γ-butyrolactam, and a lipophilic GABA analogue was also been accomplished in high overall yield (35%–58% yield range). The preliminary biological evaluation of such compounds as GABA receptor ligands showed that the γ-butyrolactone derivatives have the lowest binding activities when compared to γ-butyrolactam derivatives. However, they were able to displace 25% of tritiated muscimol from the corresponding binding site. N-Boc substituted γ-butyrolactone derivatives were more effective than the open chain GABA analogue. Substituents present on the fructose moiety did not seem to hinder the binding, as both benzylated and debenzylated lactones and lactams had comparable activity. The hydrophobic nature of benzyl groups may facilitate BBB crossing, which is one of the main issues to be addressed for CNS directed drugs.
In summary, this work generated the first library of pyrrolobenzodiazepines, lactones and lactams, spiro linked to a sugar moiety, significantly relevant for the elucidation of determinant aspects on GABAA R binding.
Pair of Shoes, 1888 Vincent van Gogh