Benzotiyazol türevi kalkonların sentezi, biyolojik aktivite, teorik hesaplama ve moleküler yerleştirme çalışmaları
Synthesis, biological activity, theoretical calculation, and molecular docking studies of benzothiazole derivative chalcones
Yazar: AHMAD BADREDDIN MUSATAT
Danışman: PROF. DR. MUSTAFA ZENGİN ; DOÇ. DR. ALPARSLAN ATAHAN
Sakarya Üniversitesi / Fen Bilimleri Enstitüsü / Kimya Ana Bilim Dalı / Organik Kimya Bilim Dalı
Derece: Yüksek Lisans, 2023
Konu: Kimya = Chemistry

Özet:
Flavonoid ailesinin üyeleri çok çeşitli biyolojik aktivitelere sahiptir ve doğal bileşiklerin bu grubu meyveler, bitkiler, tohumlar, yapraklar ve birçok çiçekte bulunmaktadır. Doğal veya sentetik olarak elde edilebilen kalkonlar ise, flavonoid ailesinin bir alt grubudur ve anti-oksidan, anti-kanser, anti-inflamatuar özelliklerle ve diyabet, kardiyovasküler hastalıklar ve beyin hasarı gibi durumların tedavisinde etkili olmaları ile tanınırlar. Ayrıca, kükürt ve azot atomlarını içeren benzotiyazol grubu da heteroaromatik bir yapıya sahiptir ve bir çok farmasötik ilacın geliştirilmesinde kullanılan aktif bir çekirdektir. Bir diğer ifade ile, benzotiyazol türevleri kanser, bakteriyel, fungal ve viral enfeksiyonlar, iltihaplanma, AIDS ve enzim ilişkili vb. hastalıkların tedavisinde kullanılan bir çok etken madde içerisinde yer almaktadır. Diğer taraftan, canlı organizmalardaki birçok enzim metabolizmadaki düzenleyici rollerinden dolayı edema, glokoma, obezite, kanser, epilepsi, lösemi ve osteoporoz gibi kritik hastalıklarla ilşkilendirilir ve çeşitli inhibitör/aktivatör/düzenleyici bileşiklerin geliştirilmesi için çok önemlidir. Bu tezde, benzotiyazol yapısı taşıyan kalkon türevlerinin yeni bir serisinin elde edilmesi ve enzimatik aktivitelerinin belirlenmesi amaçlandı. Ayrıca, bu sonuçların bilgisayar destekli teorik kuantum hesaplamaları ve moleküler yerleştirme çalışmaları ile desteklenmesi hedeflendi. Sonuç olarak, kalkon-benzotiyazol hibrit türevlerinin yeni bir serisi yüksek verimlerle başarıyla elde edildi ve bütün bileşikler 1H-NMR, 13C-NMR, FT-IR ve HR-MS spektroskopik teknikleri kullanılarak karakterize edildi. Sentezlenen bileşiklerin enzim aktiviteleri üç farklı enzim (Karbonik anhidraz-I, Karbonik anhidraz-II ve Paraoksonaz-1) üzerinde hidrataz ve esteraz yöntemleri kullanılarak çalışıldı. Hidrataz aktivitesi metodundaki IC50 değerleri, hCA-I ve hCA-II için sırasıyla 4.15-5.47 μM ve 2.56-4.58 μM aralığında gözlendi. Aynı zamanda, esteraz aktivitesi metodunda Ki değerleri hCA-I ve hCA-II için sırasıyla 14.43-59.66 μM ve 26.65-73.34 μM aralığında iken, IC50 değerleri de 24.91-104.00 μM ve 35.25-97.00 μM aralığında bulundu. Buna ilaveten, paraoksonaz enzim inhibisyon çalışmaları 13.28-16.68 μM arasında IC50 değerleri ile inhibisyon etkileri gösterdi. Son olarak, sentezlenen bileşikler için B3LYP, PBE0 teorileri ve SVP, TVZP temel setleri kullanılarak kapsamlı teorik hesaplama ve ardından moleküler yerleştirme çalışmaları gerçekleştirildi. Sentezlenen bileşikler ve enzim inhibisyon sonuçları arasındaki yapı aktivite ilişkisi, uyumlu teorik sonuçlar ile ortaya konuldu.

Abstract:
The members of flavonoid family have a wide range of biological activities, and this group of natural compounds is found in fruits, plants, seeds, leaves, and many flowers. Chalcones are a sub-group of flavonoid family that can be obtained naturally or synthetically, and they are known for their tremendous bioactive properties such as anti-cancer, anti-oxidant, anti-bacterial/anti-fungal, anti-inflammatory, anti-malarial, anti-leishmanial, and enzyme inhibitory. On the other hand chalcones can be synthesized through out different type of reactions and the most famous and commonly used one is Claisen–Schmidt condensation which is involved in this study. 
In addition, the benzothiazole group has a heteroaromatic structure containing sulfur and nitrogen atoms at once, and it is an active core in the development of many pharmaceutical drugs. In other words, the benzothiazole moiety is included in many active pharmaceutical ingredients which are used in the treatment of cancer, bacterial, fungal and viral infections, inflammation, AIDS, and enzyme related diseases etc. Thus, there are various ways to synthesize this core. In general, it can be obtained starting with 2-aminothiophenol or aniline derivatives reacting with different reagents in considerably good yields. Herein, we designed the aldehyde compound starting by mixing 2-aminothiophenol and terephthalaldehyde following by silicagel column purification. 
On the other hand, metabolic processes in living organisms are continued and regulated by some specific enzymes and proteins. For instance, human carbonic anhydrase I and II are two enzymes that play essential roles in regulating the pH of various tissues and fluids in the body. These enzymes are responsible for the conversion of carbon dioxide into bicarbonate ions, which helps to maintain the balance of acid-base levels in the body. By this, they help the removal of carbondioxide which is formed in the metabolic conversion of carbohydrates. 
Primarily, carbonic anhydrases are Zn2+ centered metallo-enzymes and 16 different isoform sub-unit has been found up to now. For example, hCA-I found mainly in the red blood cells, while hCA-II is present in the kidney, lung, and other tissues. These enzymes have been implicated in various diseases, such as glaucoma, epilepsy, and osteoporosis and the regulation of the activity of this enzyme may help in the treatment of mentioned diseases. Secondly, PON-1 is a Ca2+ ion containing enzyme that plays a crucial role in protecting against oxidative stress and inflammation in the body. It is primarily found in the liver and circulates in the blood, where it is responsible for breaking down certain toxins and oxidized lipids. Low levels of PON-1 activity are associated with an increased risk of various diseases, including cardiovascular disease, diabetes, and cancer. The development of specific inhibitor/activator/regulator compounds targeting these enzymes can have therapeutic potential in treating various diseases. For example, carbonic anhydrase inhibitors have been used to treat glaucoma disease to reduce intraocular pressure by decreasing the production of aqueous humor in the eye, while PON-1 activators may be useful in preventing or treating conditions associated with oxidative stress and inflammation. Overall, these enzymes play critical roles in maintaining proper physiological function and understanding their regulation mechanisms can lead to the development of new therapies for disease treatment. In addition, theoretical and molecular docking studies have become essential tools in drug development and the treatment of critical diseases. These studies utilize computational approaches to investigate the interactions between small molecules and their target proteins, which can provide valuable insights into the drug discovery process. Based on the many studies in the literature, theoretical and docking studies have proven to be valuable tools in drug development and critical disease treatment. By providing insights into the molecular interactions between small molecules and their target proteins, these studies have enabled the development of more selective and effective drugs for a range of diseases mentioned above and many more. 
In this thesis, it was aimed to obtain a new series of chalcone derivatives bearing benzothiazole scaffold and examine their enzymatic activities. For this purpose, a new aldehyde compound has been synthesized and purified using column chromatography by acceptible yield. This compound then reacted with different ketone derivatives under basic conditions by Claisen-Schmidt reaction followed by structure characterization using 1H-NMR, 13C-NMR, FT-IR, and HRMS spectroscopic techniques. Purified and structurally characterized compounds have been evaluated for their enzymatic activities towards three different enzymes (carbonic anhydrase-I, carbonic anhydrase-II, and paraoxonase-1). Carbonic anhydrase isoforms have been tested by using two different methods, esterase and hydratase. In addition, we aimed to explain and support the obtained results with computer-aided theoretical quantum calculations and molecular docking studies. In this context, geometric optimization carried out using two different theories B3LYP, PBE0 and two different basis sets def-2 SVP, def-2 TZVP employing gas phase, ethanol and chloroform as C-PCM implicit solvation model. Consequently, a novel series of chalcone-benzothiazole hybrid derivatives were successfully obtained in high yields, and all the compounds were well characterized accordingly. The enzymatic activities of the synthesized derivatives were studied on three different enzymes using two different methods (hydratase and esterase). IC50 values in hydratase activity method were in the range between 4.15-5.47 μM and 2.56-4.58 μM for hCA-I and hCA-II, respectively. At the same time, in esterase activity method, IC50 values were found in the range between 24.91-104.00 μM and 35.25-97.00 μM while Ki values were between 14.43-59.66 μM and 26.65-73.34 μM for hCA-I and hCA-II, respectively. In addition, the paraoxonase-1 enzyme activity study exhibited inhibitory profile with IC50 values between 13.28-16.68 μM. Finally, for the synthesized compounds, a comprehensive theoretical calculations were performed by using B3LYP, PBE0 theories and SVP, TZVP basis sets followed by molecular docking studies of the titled enzymes isoforms. The structure-activity relationships between the synthesized derivatives and the enzyme inhibition results were supported by the harmonical outputs of the theoretical results. Compounds with higher dipole moments have been shown to exhibit increased enzyme activity, possibly due to their ability to form strong critical interactions with important amino acid residues in the active site of the studied enzymes. This suggests that electronic localization plays an important role in the formation of these interactions. Molecular docking studies shows the important formed interactions of the synthesized compounds with the active site of studies enzymes compared to well-known inhibitors by the energy values of binding affiinity and forming Hydrogen bond, direct and indirect interactions with the metal center of the enzyme. In conclusion, in this study, we successfully synthesized a new series that consists of seven compounds containing a chalcone-benzothiazole hybrid scaffold and elucidated their structures. The experimental and theoretical results indicate that these compounds are promising candidates for the treatment of the aforementioned diseases.