N-SULFONYLPURINES AND PEPTIDE PHENANTHRIDINE DERIVATIVES SYNTHESIS, INTERACTIONS WITH POLYNUCLEOTIDES AND BIOLOGICAL TESTING

Abstract

U ovom radu istra.ene su metode priprave i svojstva dvije vrste biolo.ki aktivnih heterocikla iz grupe malih molekula: 1) N-sulfonilpurina (potencijalnih antitumorskih lijekova i inhibitora enzima purin nukleozid fosforilaze) i 2) peptidnih derivata fenantridina (potencijalnih antitumorskih lijekova i fluorescentnih senzora). Pripravljena je serija C6, N9 supstituiranih purina sa sulfonamidnim farmakoforima. Najprije je sintetizirana serija 6-klorpurinskih derivata uvo.enjem sulfonilnog supstituenta u polo.aj N9 purinske baze. Potom su, supstitucijom klora u C6 polo.aju, sintetizirani 6-morfolino- i 6-bis(2- hidroksietil)amino-derivati N-9-sulfonilpurina. Biolo.ka aktivnost 6-morfolino- i 6-bis(2-hidroksietil)amino-derivata purina ispitana je MTT testom na stani.nim linijama leukemije i limfoma (Raji, HuT 78, K-562), karcinoma (CaCo-2, HeLa) i na normalnoj stani.noj liniji (MDCK I). Najja.i citotoksi.ni u.inak pokazao je 6- morfolino-derivat 17 s trans-Ŕ-stirensulfonilnim supstituentom u N9 polo.aju purina na svim ispitanim stani.nim linijama. 6-morfolinski derivati s tosilnim, naftilsulfonilnim i bifenilsulfonilnim supstituentom u N9 polo.aju purina (8, 9 i 10) pokazali su srednje jako antiproliferativno djelovanje na Raji i HuT 78 stani.ne linije, a spojevi 9 i 10 pokazali su i srednje jako djelovanje na stanice K-562. Ispitana je i sposobnost inhibicije PNP enzima izoliranog iz dva soja bakterije Helicobacter pylori te PNP enzima iz bakterije Escherichia coli. Najbolji rezultati dobiveni su s 6-morfolinoderivatima 8 i 18. Peptidni derivati fenantridina pripravljeni su klasi.nim metodama peptidne kemije (sinteza u otopini). Negishijevom reakcijom pripravljen je alaninski derivat fenantridina 22 koji je funkcionaliziran peptidnom okosnicom nadogra.enom preko N-terminalnog kraja. Pripravljeni su di-, tri- i tetrapeptidni derivati s fenantridinom u bo.nom lancu, koji sadr.e gvanidinopirolnu, gvanidinsku (arginin), pirensku ili fluorensku strukturnu jedinicu. Gvanidinopirolni i argininski derivati prevedeni su u hidrokloridne soli. Ispitane su interakcije odabranih spojeva (gvanidinopirolni derivati 28b i 29b) s polinukleotidima pomo.u spektrofotometrijskih (UV/Vis, CD, fluorimetrija) metoda. Spojevi su, ovisno o konformaciji u kojoj se nalaze, pokazali razli.iti tip interakcija. Spoj 28b, .iji vii kromofori nisu u intramolekulskoj interakciji u blago kiselom mediju, najvjerojatnije se ve.e u mali utor DNA i veliki utor RNA. Spoj 29b, .iji kromofori su u intramolekulskoj .-. interakciji, ve.e se uz okosnicu polinukleotida. U neutralnom mediju oba spoja aglomeriraju uz fosfatnu okosnicu.The thesis presents preparation and characteristics of two types of biologically active heterocyclic small molecules: 1) N-sulfonylpurines (potential antitumor agents and inhibitors of the purine nucleoside phosphorylase enzyme, and 2) peptide phenanthridine derivatives (potential antitumor agents and fluorescent probes). Series of C6, N9 substituted purines bearing sulfonamide pharmacophore has been prepared. Series of 6-chloropurine derivatives has been prepared by introduction of sulfonyl substituent at the N9 position of the purine base. Substitution of chlorine at the C6 position yielded 6- morpholino- and 6-bis(2-hydroxyethyl)amino-derivatives of N-9-sulfonylpurine. Biological activities 6-morpholino- and 6-bis(2-hydroxyethyl)aminopurine derivatives have been tested by MTT test on the leukemia and lymphoma (Raji, HuT 78, K-562), carcinoma (CaCo-2, HeLa) and normal (MDCK 1) cell line. Compound 17, bearing trans--styrenesulfonyl substituent at N9 position of the purine base produced the strongest cyctotoxic effect on all of the tested cell lines. 6-morpholino-derivatives with tosyl, naphtylsulfonyl and biphenylsulphonyl substituent at the N9 position of the purine (8, 9 and 10) exhibited moderate antiproliferative effect on the Raji and HuT 78 cell lines, while 9 and 10 exhibted moderate antiproliferative effect on the K-562 cell line too. The inhibition of the PNP enzyme isolated from two strains of Helicobacter pylori and PNP from Escherichia coli was also tested. Best results are obtained with 6-morpholino-derivatives 8 and 18. Peptide phenanthridine derivatives were prepared by classic peptide chemistry methods (synthesis in solution). Alanine derivative of phenanthridine 22 was prepared by Negishi reaction, and subsequently functionalized by peptide backbone constructed starting from the Nterminus of 22. Di-, tri- and tetrapeptide phenanthridine derivatives with guanidinopyrrole, guanidine (arginine), pyrene or fluorene moiety have been prepared as well. Finally, guanidinopyrrole and arginine derivatives have been converted into hydrochloride salts. ix Interactions of chosen compounds (guanidinopyrrole derivatives 28b and 29b) with polynucleotides have been studied via several spectrophotometric methods, namely UV/visible, circular dichroism and fluorescence spectroscopies. The compounds have shown different types of interactions, depending on their conformation. Compound 28b, whose chromophores are not interacting mutually in mildly acidic media, most likely binds to minor groove of DNA and major groove of RNA. Compound 29b, whose chromophores are involved in intramolecular - interaction, binds on the phosphate backbone of the polynucleotide. In neutral media both compounds agglomerate along the phosphate backbone