Sinteza benzotiazepinskih zaviralcev mitohondrijskega Na+/Ca2+ izmenjevalca s protitumornim delovanjem

Zupanič, Eva (author), Tomašič, Tihomir (mentor), Cotman, Andrej Emanuel (comentor)

Membranski proteini so proteini, vgrajeni v biološke membrane, in imajo pomembno vlogo pri različnih celičnih funkcijah. Takšen protein je tudi Na+/Ca2+ izmenjevalec, ki je transportni protein, namenjen prenosu kalcija. Opisani izmenjevalec najdemo tudi v notranji mitohondrijski membrani (mitohondrijski Na+/Ca2+ izmenjevalec). Ker je znotrajcelična homeostaza kalcija izrednega pomena za delovanje celice, lahko zaviralci mitohondrijskega Na+/Ca2+ izmenjevalca preko kopičenja kalcija znotraj mitohondrija povzročijo celično smrt ali apoptozo. Slednji je s tem zanimiva tarča za razvoj protirakavih zdravilnih učinkovin, ki bi z opisanim procesom uničile rakave celice. Namen magistrske naloge je bil sintetizirali in ovrednotiti potencialne zaviralce mitohondrijskega Na+/Ca2+ izmenjevalca. Izhajali smo iz znanega zaviralca CGP37157, na katerega smo prek distančnika pripenjali trifenilfosfonijev kation z namenom dosega selektivnega delovanja na mitohondrijski Na+/Ca2+ izmenjevalec. Vsem končnim spojinam je skupen CGP37157, razlikujejo pa se po verigi n, ki smo jo spreminjali s tvorbo različno dolgih estrov in verigi m, ki smo jo spreminjali s pripenjanjem ustreznega (aminoalkil)trifenilfosfonijevega fragmenta. Potek reakcij smo spremljali s tankoplastno kromatografijo, strukturo končnih spojin smo določali s jedrsko magnetno resonanco in masno spektrometrijo visoke ločljivosti, njihovo čistoto pa smo preverjali s tekočinsko kromatografijo visoke ločljivosti. Končnim spojinam je bilo preverjeno protirakavo delovanje z resazurinskim testom na celični liniji raka mišjega melanoma B16F10 in njihovo zaviralno delovanje smo primerjali z delovanjem spojine UL-EID-2. Za preverjanje selektivnega delovanja končnih spojin na rakave celice pa je bil izveden tudi test na zdravih celičnih linijah mišjih mioblastov C2C12 in mišjih fibroblastov L929. Spojina 9 ima najmočnejše in tudi selektivno zaviralno delovanje in se od spojine UL-EID-2 razlikuje v verigi m, ki je pri spojini 9 daljša za en C atom. Spojina 15 ima najšibkejše zaviralno delovanje in se od UL-EID-2 razlikuje po verigi n, ki je daljša za dva C atoma. Sklepamo lahko, da podaljševanje verige m ugodno vpliva na zaviralni učinek, medtem ko podaljševanje verige n zaviralni učinek zmanjša. Rezultati magistrske naloge potrjujejo zaviralno aktivnost sintetiziranih spojin in kažejo na smiselnost metode za selektivno dostavo v mitohondrije ter smiselnost združitve njihovih strukturnih elementov.

• Language: Slovenian
• Keywords: Rak, membranski proteini, Na+/Ca2+ izmenjevalec, mitohondrij, trifenilfosfonijev kation
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2023
• PID: 20.500.12556/RUL-153172
• Publication date in RUL: 20.12.2023

ZUPANIČ, Eva, 2023, Sinteza benzotiazepinskih zaviralcev mitohondrijskega Na+/Ca2+ izmenjevalca s protitumornim delovanjem [online]. Master’s thesis. [Accessed 5 April 2025]. Retrieved from: https://repozitorij.uni-lj.si/IzpisGradiva.php?lang=eng&id=153172

Secondary language

• Language: English
• Title: Synthesis of the benzothiazepine-based mitochondrial Na+/Ca2+ exchanger inhibitors with anti-tumor activity

Abstract

Membrane proteins are proteins that are embedded in biological membranes and play an important role in various cellular functions. One such protein is the Na+/Ca2+ exchanger, which is an ion transport protein for calcium transport. This exchanger is also found in the inner membrane of the mitochondria (mitochondrial Na+/Ca2+ exchanger). Since intracellular calcium homeostasis is extremely important for cell function, mitochondrial Na+/Ca2+ exchanger inhibitors can cause cell death or apoptosis due to the accumulation of calcium inside the mitochondria. Therefore, the mitochondrial Na+/Ca2+ exchanger is an interesting target for the development of anticancer agents. The purpose of this Masterʹs thesis was to synthesize and evaluate potential inhibitors of the mitochondrial Na+/Ca2+ exchanger. We used an already known inhibitor CGP37157 to which we attached a triphenylphosphnium cation to achieve a selective targeting of mitochondria. CGP37157 is common to all final compounds, while they differ in the chain n, which was modified by the formation of esters of different lengths, and in the chain m, which was modified by attachment of the corresponding (aminoalkyl)triphenylphosphonium fragment. The progress of the reactions was monitored by thin layer chromatography, the structure of the final compounds was determined by nuclear magnetic resonance and high-resolution mass spectrometry and their purity was checked by high performance liquid chromatography. The final compounds were tested for their anticancer activity on the mouse melanoma cell line B16F10 using the resazurin assay and then compared with the compound UL-EID-2. To verify the selective effect of the final compounds on cancer cells, the assay was also performed on healthy cell lines of mouse myoblasts C2C12 and mouse fibroblasts L929. Compound 9 has the strongest and selective inhibitory activity and it differs from UL-EID-2 by the m chain, which is one C-atom longer in compound 9. Compound 15 has the weakest inhibitory activity and it differs from UL-EID-2 by the length of chain n, which is two C-atoms longer in compound 15. We can conclude that lengthening of the chain m has beneficial effect on the inhibitory activity, while lengthening of the chain n reduces it. These results confirm the inhibitory activity of the synthesized compounds and indicate the meaningfulness of the method for selective delivery to mitochondria and the sense of combining their structural elements.

• Keywords: Cancer, membrane proteins, Na+/Ca2+ exchanger, mitochondrion, triphenylphosphonium cation