Some Applications Of Combinatorial Chemistry And High-throughput Screening

Partâ… .Design and parallel synthesis of benzo[1,4]oxazin-3-one library in solution-phaseCombinatorial chemistry is a subject that has been developed and grown up quickly in the past twenty years.Its concepts and techniques have been widely accepted and used in various aspects of scientific research and industries,for example drug discovery and optimization,material science and catalysts.Quick development along with high-throughput or high content screening technologies,combinatorial chemistry has caused a big leap in new drug development in particularly and become an indispensable technique in the process of drug discovery and optimization. Combinatorial chemistry originates from solid-phase peptide synthesis.Nowadays, however,combinatorial generation of small heterocyclic molecules has heavily attracted attention for both organic and medicinal chemists due to their high diversities and drug-like properties.Solution-phase combinatorial synthesis,benefited from the development of solidified reagents,scavenging resins and fluorous techniques,has been widely practiced,among which solution-phase parallel single molecule synthesis was especially regarded.1,5-difluoro-2,4-dinitrobenzene(DFDNB,1) is a protein cross-linker,which has four potential reactive sites.Therefore,diversified benzofused heterocyclic skeletons are potentially able to be synthesized through the asymmetric introduction of different substitution groups.This thesis focused on synthesis of 'privileged structure' of benzo[1,4]xazin-3-one scaffolds through four reaction steps in a parallel solution manner including quantitative neucleophilic substitution,reduction of meta-dinitro groups,autocyclization reaction and acylation.In all,one hundred and eleven benzoxazine analogs were synthesized.Sixty five compounds of five new skeletons based on banzoxazine were also generated.A chemical library with 1040 members was prepared.1) Synthesis of benzo[1,4]oxazin-3-one analogs Benzo[1,4]oxazin-3-ones have shown various biological activities,such as being anti-inflammatory,antiulcer,antipyretic,antihypertensive,and antifungal.The autocyclization of 6-amino-3,4-dihydro-benzo[1,4]oxazin-3-ones herein was discussed,following the neucleophilic substitutions of two fluorine atoms of DFDNB (1) with second amines/phenols/thiophenols,hydroxyacetates,and reduction of meta-dinitro groups.The remaining free aromatic amino group of 6-amino-3,4-dihydro-benzo[1,4]oxazin-3-ones was diversified through the acylation by anhydrides,sulfonyl chlorides,isocyanates,isothiocyanates,aldehydes, dicyanoamine.Particularly,Ugi-4CC reaction was successfully carried out at 6-amino group which greatly increased the substitution diversity.Several microwave assisted reactions were well practiced in this thesis to improve the conditions with shorter reaction time and higher purity.The optimized reaction conditions were compatible with chemical library synthesis which allows three or five diversity points.2) Synthesis of tri-heterocycles based on benzo[1,4]oxazin-3-one skeletonThere is always a considerable effort placed on the integration of privileged structures into one molecule for drug design.Quinoxalin-2-ol,benzoimidazole,benzotriazole, benzocarbonylimidazole,quinoxalin-2,3-dione are recognized as the privileged structures.Integrations of these scaffolds with benzo[1,4]oxazin-3-one will not only increased the compounds diversity,but also provide the new possibility to discover novel hits and lead compounds.After the neucleophilic substitution of two fluorine atoms of DFDNB(1) with aminoacetates/amines and hydroxyacetates subsequently, the meta-dinitro groups were reduced either by Pd/C-HCOONH₄ or Na₂S₂O₄/K₂CO₃ to yield 7-hydroxy-3,4,8,9-tetrahydro-2H-[1,4]oxazino[2,3-g]quinoxalin-3-one or 6,7-diamino-7-3,4-dihydro-2H-1,4-benzoxazin-3-one,respectively.The former was further oxidized in air to offer a new tricycle of 6-hydroxy-4H-1-oxa-4,5,8 -triazaanthracen-3-one(â…¦).The latter reacted with aldehydes,sodium nitrite,carbon disulfide,triphosgene,oxalyl chloride to generate additional four new tricycles as: 3,8-dihydro-5-oxa-1,3,8-triazacyclopenta[b]naphthalene-7-one(â…§,â…©),3,8-dihydro -5-oxa-1,2,3,8-tetraazacyclopenta[b]naphthalene-7-one(â…¨),3,8-dihydro-1H-5-oxa -1,3,8-triazacyclopenta[b]naphthalene-2,7-dione(â…ª),and 6-hydroxy-4,8-dihydro-1 -oxa-4,5,8-triazaanthracen-3,7-dione(â…«).The whole reaction routes were involved in four or five reaction steps,and thus three or four diversity points were introduced to the benzo[1,4]oxazin-3-one skeleton.3) Preparation of chemical library ofâ…§Considering the maximum molecular diversity,high quality(purity) and easy operation,sixteen primary amines,one hydroxyacetate and seventy-two aliphatic, aromatic or hetero aldehydes were selected to generate the library in a parallel manner. It was also referenced the reactivities of various reagents through our reaction optimization.The library synthesis was carried out in solution-phase in the aid of scavenging resin for purification.All final compounds were analyzed by LC-MS and LC-ELSD.The results indicated that 80.1%of the library members were over 80%in purity.The whole library synthesis took up two months to generate 1040 compounds individually by one person.This showed obviously the high performance and speediness of combinatorial chemistry.Partâ…¡.Studies on SARS-CoV fusion inhibitors and conjugation of SARS immunogenic peptides with carrier proteins(mcKLH,HBsAg)Severe acute respiratory syndrome(SARS) is a human respiratory infection disease which began to prevail in the end of 2002.It first occurred in Guangdong Province, China,but spread to Hongkong and Canada shortly.SARS had caused an infection of more than thirty countries and regions across five continents.Due to its high infectivity and high mortality,SARS damaged greatly the public sanitation and the ecnomic stability of the whole world.SARS was caused by a new coronavirus,called SARS coronavirus(SARS-CoV), which is a single chain(+) sense RNA virus.After decoding its gene sequence, knowkedge to this virus's structure,receptor,invasion mechanism,origin,evolution and so on were understood step by step.Although some of these findings need more solid proofs,they still provide useful information for the precaution and therapy of SARS.T-20 is a clinical fusion inhibitor for HIV-1.Wondering if this reagent is also effective to SARS-CoV,we synthesized four peptides derived from the HR1 and HR2 domains of SARS-CoV S protein and studied the interaction between these peptides with T-20,respectively.The significant but moderate interaction between T-20 and an SARS-CoV HR1-derived peptide(Pep 1D) was observed,which is not enough to be able to provide therapeutic efficiency.Our group identified five B-cell immunoantigens of SARS-CoV through the synthsis and screening an 'overlapping' peptide library.To explore the possibility of potential peptide vaccine for a clinical purpose,this dissertation primarily investigated (1) the conjugation between three peptides and mcKLH,HBsAg;(2) the immunity of the conjugates.