Publication: Synthesis and evaluation of biological activity of some novel urea derivatives on VEGFR-2 enzyme
Abstract
Kanser gelişmiş ülkelerde aterosklerozdan sonra en önemli ölüm sebeplerinden birisidirve geleceğe dair yapılan projeksiyonlar kanser görülme sıklığının hızlanarak artacağını öngörmektedir. Kanser tedavisinde kanserli hücrelerin elimine edilmesi için sitotoksik ajanların kullanımını gerektiren bir yöntem olan kemoterapi, en yaygın kullanılan metottur. Bununla birlikte seçimli olmayan kemoterapik ajanların kullanımı sağlıklı hücreleri de etkilediğinden sadece kanser hücrelerine karşı etkili olan yeni, etkili ve düşük yan etkilere sahip ilaçların geliştirilmesi, kanser tedavisinde en önemli çalışma alanı olarak ortaya çıkmaktadır. Anjiyojenez, var olan damarlardan yeni damarların oluşması için organizmada kullanılan bir metabolik yoldur ve tümör hücreleri genellikle düşük oksijenli şartlarda bulunduklarından anjiyojeneze normal hücrelerden çok daha fazla ihtiyaç gösterirler. Bu nedenle vasküler endotelyal büyüme faktörü reseptörü -2 (VEGFR-2) gibi anjiyojenezde görevli tirozin kinazlar seçimli anti-kanser ilaçlarının geliştirilmesinde en önemli hedeflerdir. Diaril üre fonskiyonel grubuna sahip olan sorafenib, ilk keşfedilen VEGFR-2 inhibitörü moleküldür ve günümüzde farklı kanserlerin tedavisinde kullanılmaktadır. Bu bilgiler ışığında bu tez çalışmasında bilinen VEGFR-2 inhibitör yapıları örnek alınarak diaril üre fonksiyonel grubuna sahip, iki yeni pirazol, altı 1,3,4-okzadiazol, dört 1,3,4-tiyadiazol ve on 1,2,4-triazol-3-tiyon türevi olmak üzere toplam 22 yeni bileşik sentezlenerek literatüre kazandırılmıştır. Bileşiklerin karakterizasyonu IR, 1H ve 13C NMR ve MS verileri ile gerçekleştirilmiştir. Sentezlenen bileşiklerin VEGFR-2 aktivitesi üzerine atkileri 10 µM konsantrasyonda taranmış ve 7 bileşiğin yaklaşık %50 inhibisyon aktivitesine sahip olduğu belirlenmiştir. En yüksek aktiviteye sahip üç bileşiğin (10c, 8a ve 11g) VEGFR-2 aktivitesinin yüzde ellisini inhibe ettikleri konsantrasyonlar (IC50 değerleri) deneysel olarak belirlenmiştir. 10c, 8a ve 11g bileşikleri için IC50 değerleri sırasıyla 0,664 1,154 ve 2,531 µM olarak hesaplanmıştır. Bileşiklerin anti-kanser özellikleri göğüs kanseri MCF-7 ve MDA-MB-231 ve kanser olmayan L929 fibroblast hücre hatları üzerinde MTT yöntemi ile tayin edilmiş ve 8a ve 9d bileşiklerinin kanser hücre hatlarına karşı yüksek sağlıklı hücre hatlarına karşı ise göreceli olarak düşük etkiye sahip oldukları belirlenmiştir. Bu bileşiklerin anti-kanser özelliklerini hangi mekanizma ile gösterdiğinin belirlenmesi amacıyla Annexin-V/ PI denemesi gerçekleştirilmis ve 8a bileşiğinin MCF-7 hücrelerinde erken ve geç apoptozu önemli miktarda arttırdıkları, MDA-MB-231 hücrelerinde ise erken apoptozu öenmli miktarda değiştirmedikleri ancak geç apoptoz ve nekrozu arttırdıkları belirlenmiştir. En etkili VEGFR-2 inhibitörü bileşiklerin etkisinin ayrıntılı olarak anlaşılabilmesi amacıyla sorafenibe bağlı VEGFR-2 kristal yapısı kullanılarak doklama çalışmaları gerçekleştirilmiş ve bileşiklerin enzimin aktif bölgesinde hangi amino asit kalıntılarına ne tür bağlanmalar yaptıkları belirlenmiştir. Ayrıca in silico yöntemler kullanılarak bileşiklerin farmakokinetik ve fizikokimyasal özellikleri tahmin edilmiş ve ilaç olarak kullanılabilme potansiyelleri öngörülmüştür. Yapılan çalışmalar sonucunda bir pirazol türevi olan 8a bileşiği (1-{4-[(3,5-Dimetil-1H-pirazol-1-il)karbonil]phenil}-3-fenilüre) hem yüksek VEGFR-2 aktivitesine sahip olması, hem de göğüs kanser hücre hatlarında sağlıklı hücre hattına göre çok daha yüksek anti-kanser etkiye sahip olması nedeniyle belirli kanser türlerinin tedavisinde kullanılabilme potansiyeli bulunan anti-anjiyojenik bir kemoterapik ajan olarak belirlenmiştir.
Cancer disease is one of the most important causes of mortality after atherosclerosis in developed countries, and projections for the future predict that the incidence of cancer will increase rapidly. Chemotherapy, which is a method that requires the use of cytotoxic agents to eliminate cancerous cells in cancer treatment, is the most widely used method. However, since the use of non-selective chemotherapeutic agents also affects healthy cells, the development of new, effective and low-side effects drugs that are effective only against cancer cells emerges as the most important field of study in cancer treatment. Angiogenesis is a metabolic pathway used in the organism for the creation of new blood veins from existing veins, and tumor cells generally require angiogenesis much more than normal cells because they are in low oxygen conditions. Therefore, tyrosine kinases involved in angiogenesis, such as vascular endothelial growth factor receptor-2 (VEGFR-2), are the most important target biological molecules for the development of selective anti-cancer drugs. Sorafenib, which has a diaryl urea functional group, is the first discovered small molecule with VEGFR-2 inhibiting activity and is presently used in the treatment of different cancers. In light of this information, in this thesis study, following a hybridization plan using the structures of known VEGFR-2 inhibitors, a total of 22 new compounds were synthesized. These novel derivatives includes ten 1,2,4-triazole-3-thiones, two pyrazoles, four 1,3,4-thiadiazoles and six 1,3,4-oxadiazoles with a diaryl urea moeity. The characterization of compounds was performed with IR, 1H and 13C NMR and mass spectral data. Effects of synthesized compounds on VEGFR-2 activity were screened at 10 µM concentration and it was determined that 7 compounds had approximately 50% inhibition activity. The concentrations that inhibited fifty percent of VEGFR-2 activity (IC50 values) were determined experimentally for the three compounds with the highest activity (10c, 8a and 11g). The IC50 values for derivatives 11g, 8a and 10c were calculated and found to be 2.531, 1.154 and 0.664 µM, respectively. The antineoplastic properties of the derivatives were determined against breast cancer MDA-MB231 and MCF-7 and non-cancerous L929 fibroblast cell lines using MTT assay. Derivatives 8a and 9d were discovered to be highly active against cancer cell lines. and relatively less active against healthy cell lines. In order to determine the mechanism by which these compounds show their anti-cancer properties, an Annexin-V/ PI experiment was carried out. Compound 8a was found to dramatically increase both early and late apoptosis in MCF-7 cells, while it had no effect on early apoptosis but increased necrosis and late apoptosis in MDA-MB-231 cells. To comprehend the action of the most effective VEGFR-2 inhibitor compounds in detail, docking studies were carried out using the VEGFR-2 crystal structure bound to sorafenib. It was determined what kind of bonds the compounds made to which amino acid residues in the catalytic site of the VEGFR-2. Furthermore, the compounds' pharmacokinetic and physicochemical properties were estimated using in silico methods and their potential to be used as drugs was predicted. As a result of the studies, the pyrazole derivative, compound 8a(1-{4-[(3,5-Dimethyl-1H-pyrazol-1-yl)carbonyl]phenyl}-3-phenylurea), determined as an antiangiogenic chemotherapy agent with the potential to be used in the treatment of certain cancer types due to its high VEGFR-2 inhibitory activity and substantially stronger cytotoxicity against breast cancer cell lines relative to healthy cell line.
Cancer disease is one of the most important causes of mortality after atherosclerosis in developed countries, and projections for the future predict that the incidence of cancer will increase rapidly. Chemotherapy, which is a method that requires the use of cytotoxic agents to eliminate cancerous cells in cancer treatment, is the most widely used method. However, since the use of non-selective chemotherapeutic agents also affects healthy cells, the development of new, effective and low-side effects drugs that are effective only against cancer cells emerges as the most important field of study in cancer treatment. Angiogenesis is a metabolic pathway used in the organism for the creation of new blood veins from existing veins, and tumor cells generally require angiogenesis much more than normal cells because they are in low oxygen conditions. Therefore, tyrosine kinases involved in angiogenesis, such as vascular endothelial growth factor receptor-2 (VEGFR-2), are the most important target biological molecules for the development of selective anti-cancer drugs. Sorafenib, which has a diaryl urea functional group, is the first discovered small molecule with VEGFR-2 inhibiting activity and is presently used in the treatment of different cancers. In light of this information, in this thesis study, following a hybridization plan using the structures of known VEGFR-2 inhibitors, a total of 22 new compounds were synthesized. These novel derivatives includes ten 1,2,4-triazole-3-thiones, two pyrazoles, four 1,3,4-thiadiazoles and six 1,3,4-oxadiazoles with a diaryl urea moeity. The characterization of compounds was performed with IR, 1H and 13C NMR and mass spectral data. Effects of synthesized compounds on VEGFR-2 activity were screened at 10 µM concentration and it was determined that 7 compounds had approximately 50% inhibition activity. The concentrations that inhibited fifty percent of VEGFR-2 activity (IC50 values) were determined experimentally for the three compounds with the highest activity (10c, 8a and 11g). The IC50 values for derivatives 11g, 8a and 10c were calculated and found to be 2.531, 1.154 and 0.664 µM, respectively. The antineoplastic properties of the derivatives were determined against breast cancer MDA-MB231 and MCF-7 and non-cancerous L929 fibroblast cell lines using MTT assay. Derivatives 8a and 9d were discovered to be highly active against cancer cell lines. and relatively less active against healthy cell lines. In order to determine the mechanism by which these compounds show their anti-cancer properties, an Annexin-V/ PI experiment was carried out. Compound 8a was found to dramatically increase both early and late apoptosis in MCF-7 cells, while it had no effect on early apoptosis but increased necrosis and late apoptosis in MDA-MB-231 cells. To comprehend the action of the most effective VEGFR-2 inhibitor compounds in detail, docking studies were carried out using the VEGFR-2 crystal structure bound to sorafenib. It was determined what kind of bonds the compounds made to which amino acid residues in the catalytic site of the VEGFR-2. Furthermore, the compounds' pharmacokinetic and physicochemical properties were estimated using in silico methods and their potential to be used as drugs was predicted. As a result of the studies, the pyrazole derivative, compound 8a(1-{4-[(3,5-Dimethyl-1H-pyrazol-1-yl)carbonyl]phenyl}-3-phenylurea), determined as an antiangiogenic chemotherapy agent with the potential to be used in the treatment of certain cancer types due to its high VEGFR-2 inhibitory activity and substantially stronger cytotoxicity against breast cancer cell lines relative to healthy cell line.