T.C. REPUBLIC OF TURKEY HACETTEPE UNIVERSITY GRADUATE SCHOOL OF HEALTH SCIENCES SYNTHESIS AND BIOLOGICAL ACTIVITY STUDIES OF SOME 3-ARYL-2-(4- (SUBSTITUTEDPHENYL)THIAZOL-2-YL)ACRYLONITRILE DERIVATIVES Pharm. Shukurah ANAS, MS Pharmaceutical Chemistry Program MASTER OF SCIENCE THESIS ANKARA 2023 T.C. REPUBLIC OF TURKEY HACETTEPE UNIVERSITY GRADUATE SCHOOL OF HEALTH SCIENCES SYNTHESIS AND BIOLOGICAL ACTIVITY STUDIES OF SOME 3-ARYL-2- (4-(SUBSTITUTEDPHENYL)THIAZOL-2-YL)ACRYLONITRILE DERIVATIVES Pharm. Shukurah ANAS, MS Pharmaceutical Chemistry Program MASTER OF SCIENCE THESIS ADVISOR OF THESIS Assoc. Prof. Keriman ÖZADALI SARI ANKARA 2023 iii SYNTHESIS AND BIOLOGICAL ACTIVITY STUDIES OF SOME 3-ARYL- 2-(4-(SUBSTITUTEDPHENYL)THIAZOL-2-YL)ACRYLONITRILE DERIVATIVES Pharm. Shukurah ANAS, MS Assoc. Prof. Keriman ÖZADALI SARI This thesis study has been approved and accepted as a Master dissertation in “Pharmaceutical Chemistry Program” by the assessment committee, whose members are listed below, on 03.04.2023. Chairman of the Committee: Prof. Nesrin GÖKHAN KELEKÇİ Hacettepe University Advisor of the Dissertation: Assoc. Prof. Keriman ÖZADALI SARI Hacettepe University Member: Prof. Erhan PALASKA Hacettepe University Member: Prof. Oya ÜNSAL TAN Hacettepe University Member: Assoc. Prof. Çiğdem KARAASLAN KIRMIZIOĞLU University of Health Sciences This dissertation has been approved by the above committee in conformity to the related issues of Hacettepe University Graduate Education and Examination Regulation. Prof. Dr. Müge YEMİŞCİ ÖZKAN, MD, PhD Director iv YAYIMLAMA VE FİKRİ MÜLKİYET HAKLARI BEYANI YAYIMLAMA VE FİKRİ MÜLKİYET HAKLARI BEYANI Enstitü tarafından onaylanan lisansüstü tezimin/raporumun tamamını veya herhangi bir kısmını, basılı (kağıt) ve elektronik formatta arşivleme ve aşağıda verilen koşullarla kullanıma açma iznini Hacettepe Üniversitesine verdiğimi bildiririm. Bu izinle Üniversiteye verilen kullanım hakları dışındaki tüm fikri mülkiyet haklarım bende kalacak, tezimin tamamının ya da bir bölümünün gelecekteki çalışmalarda (makale, kitap, lisans ve patent vb.) kullanım hakları bana ait olacaktır. Tezin kendi orijinal çalışmam olduğunu, başkalarının haklarını ihlal etmediğimi ve tezimin tek yetkili sahibi olduğumu beyan ve taahhüt ederim. Tezimde yer alan telif hakkı bulunan ve sahiplerinden yazılı izin alınarak kullanılması zorunlu metinlerin yazılı izin alınarak kullandığımı ve istenildiğinde suretlerini Üniversiteye teslim etmeyi taahhüt ederim. Yükseköğretim Kurulu tarafından yayınlanan “Lisansüstü Tezlerin Elektronik Ortamda Toplanması, Düzenlenmesi ve Erişime Açılmasına İlişkin Yönerge” kapsamında tezim aşağıda belirtilen koşullar haricince YÖK Ulusal Tez Merkezi / H.Ü. Kütüphaneleri Açık Erişim Sisteminde erişime açılır. o Enstitü / Fakülte yönetim kurulu kararı ile tezimin erişime açılması mezuniyet tarihimden itibaren 2 yıl ertelenmiştir. (1) o Enstitü / Fakülte yönetim kurulunun gerekçeli kararı ile tezimin erişime açılması mezuniyet tarihimden itibaren ... ay ertelenmiştir. (2) o Tezimle ilgili gizlilik kararı verilmiştir. (3) …… /………/…… (İmza) Shukurah ANAS i i“Lisansüstü Tezlerin Elektronik Ortamda Toplanması, Düzenlenmesi ve Erişime Açılmasına İlişkin Yönerge” (1) Madde 6. 1. Lisansüstü tezle ilgili patent başvurusu yapılması veya patent alma sürecinin devam etmesi durumunda, tez danışmanının önerisi ve enstitü anabilim dalının uygun görüşü üzerine enstitü veya fakülte yönetim kurulu iki yıl süre ile tezin erişime açılmasının ertelenmesine karar verebilir. (2) Madde 6. 2. Yeni teknik, materyal ve metotların kullanıldığı, henüz makaleye dönüşmemiş veya patent gibi yöntemlerle korunmamış ve internetten paylaşılması durumunda 3. şahıslara veya kurumlara haksız kazanç imkanı oluşturabilecek bilgi ve bulguları içeren tezler hakkında tez danışmanının önerisi ve enstitü anabilim dalının uygun görüşü üzerine enstitü veya fakülte yönetim kurulunun gerekçeli kararı ile altı ayı aşmamak üzere tezin erişime açılması engellenebilir. (3) Madde 7. 1. Ulusal çıkarları veya güvenliği ilgilendiren, emniyet, istihbarat, savunma ve güvenlik, sağlık vb. konulara ilişkin lisansüstü tezlerle ilgili gizlilik kararı, tezin yapıldığı kurum tarafından verilir *. Kurum ve kuruluşlarla yapılan işbirliği protokolü çerçevesinde hazırlanan lisansüstü tezlere ilişkin gizlilik kararı ise, ilgili kurum ve kuruluşun önerisi ile enstitü veya fakültenin uygun görüşü üzerine üniversite yönetim kurulu tarafından verilir. Gizlilik kararı verilen tezler Yükseköğretim Kuruluna bildirilir. Madde 7.2. Gizlilik kararı verilen tezler gizlilik süresince enstitü veya fakülte tarafından gizlilik kuralları çerçevesinde muhafaza edilir, gizlilik kararının kaldırılması halinde Tez Otomasyon Sistemine yüklenir * Tez danışmanının önerisi ve enstitü anabilim dalının uygun görüşü üzerine enstitü veya fakülte yönetim kurulu tarafından karar verilir. v ETHICAL DECLARATION In this thesis study, I declare that all the information and documents have been obtained in the base of the academic rules and all audio-visual and written information and results have been presented according to the rules of scientific ethics. I did not do any distortion in data set. In case of using other works, related studies have been fully cited in accordance with the scientific standards. I also declare that my thesis study is original except cited references. It was produced by myself in consultation with supervisor (Assoc. Prof. Keriman ÖZADALI SARI) and written according to the rules of thesis writing of Hacettepe University Institute of Health Sciences. (Signature) Shukurah ANAS vi ACKNOWLEDGEMENT Firstly, I would like to thank my supervisor, Assoc. Prof. Keriman ÖZADALI SARI, for her invaluable guidance and support throughout the research process. Her expertise, encouragement, and constructive criticism have been instrumental in shaping my research. I would also like to express my appreciation to Prof. Dr. NESRİN GÖKHAN KELEKÇİ, Head of Department, for providing me with the necessary resources and facilities to carry out my research work. I am also thankful to Siva Krishna VAGOLU and Assoc. Prof. Ceren ÖZKUL KOÇAK for their contribution towards the biologic activity aspect of my research. Additionally, I would like to express my appreciation to the faculty staff and research assistants, who provided their valuable support and assistance throughout my research journey. I cannot forget to thank my parents, Alhaji Anas ADAM and Hajia Sadatu ADAMS, and my siblings for their unwavering love, encouragement, and support throughout my academic journey. Finally, I would like to acknowledge my supportive friends, especially Dr. Rashid ALHASSAN, for their encouragement and for always being there to lend a helping hand and my girl Koko, who has been a constant source of comfort and joy. Thank you all once again for your support and encouragement. vii ABSTRACT Anas, S., Synthesis and Biological Activity Studies of Some 3-Aryl-2-(4- (substitutedphenyl)thiazol-2-yl)acrylonitrile Derivatives, Hacettepe University Graduate School of Health Sciences, Faculty of Pharmacy Department of Pharmaceutical Chemistry, Master of Science Thesis, Ankara, 2023. In this study, the synthesis of 16 novel derivatives of 3-aryl-2-(4-(substituted phenyl)thiazol-2- yl)acrylonitrile (1-16), including two compounds (14 and 15) previously reported in the literature, was carried out and their structures were elucidated using 1H/13C-NMR, IR, and HRMS methods. The antimycobacterial activities of the synthesized compounds were investigated against Mycobacterium tuberculosis H37Rv strain, but none of the compounds showed MIC values lower than 50 μM. Additionally, the antibacterial activities of the target compounds against various gram-positive and gram-negative bacteria, and their antifungal activities against Candida species were evaluated using microdilution methods, with ciprofloxacin and fluconazole used as reference drugs. Among the tested compounds, 2-(4-(2,4-dichlorophenyl)thiazol-2-yl)-3-(4- (trifluoromethyl)phenyl)acrylonitrile (10) was found to be the most promising antimicrobial agent, showing an MIC value of 32 µg/ml against C. parapsilosis. Based on these findings, compound 10 was identified as a lead compound for obtaining more active derivatives. Key Words: Antimycobacterial, antimicrobial, antibacterial, antifungal, tuberculosis, acrylonitrile, thiazole. viii ÖZET Anas, S., Bazı 3-Aril-2-(4-(sübstitüefenil)tiyazol-2-il)akrilonitril Türevlerinin Sentezi ve Biyolojik Aktivite Çalışmaları, Hacettepe Üniversitesi Sağlık Bilimleri Enstitüsü, Eczacılık Fakültesi, Farmasötik Kimya Anabilim Dalı, Yüksek Lisans Tezi, Ankara, 2023. Bu çalışmada, 2 tanesi literatürde kayıtlı (14 ve 15) olmak üzere 16 adet yeni 3-aril-2-(4-(sübstitüefenil)tiyazol-2-il)akrilonitril türevinin (1-16) sentezi yapılmış ve yapıları 1H/13C-NMR, IR ve HRMS yöntemleri ile aydınlatılmıştır. Sentezlenen bileşiklerin antimikobakteriyel aktiviteleri Mycobacterium tuberculosis H37Rv suşuna karşı incelenmiş ancak bileşiklerden hiçbiri 50 μM’ dan daha düşük MİK değeri göstermemiştir. Buna ek olarak, hedef bileşiklerin çeşitli gram-pozitif ve gram- negatif bakterilere karşı antibakteriyel ve Candida türlerine karşı antifungal aktiviteleri mikrodilüsyon yöntemleri kullanılarak incelenmiş, referans ilaçlar olarak siprofloksasin ve flukonazol kullanılmıştır. Test edilen bileşikler arasında, 2-(4-(2,4-diklorofenil)tiyazol-2-il)-3-(4- (trifluorometil)fenil)akrilonitril (10), tüm mikroorganizmalar içinde C. parapsilosis'e karşı MİK = 32 µg/ml değeri ile en umut verici antimikrobiyal aktivite gösteren bileşik olmuştur. Bu bulgulara dayanarak, 10 numaralı bileşik daha aktif bileşikler elde etmek için öncü bir bileşik olarak belirlenmiştir. Anahtar Kelimeler: Antimikobakteriyel, antimikrobiyal, antibakteriyel, antifungal, tüberküloz, akrilonitril, tiyazol ix CONTENT APPROVAL PAGE iii YAYIMLAMA VE FİKRİ MÜLKİYET HAKLARI BEYANI iv ETHICAL DECLARATION v ACKNOWLEDGEMENT vi ABSTRACT vii ÖZET viii CONTENT ix ABBREVIATIONS xii FIGURES xiv TABLES xvii 1. INTRODUCTION 1 2. LITERATURE REVIEW 6 2.1 Acrylonitrile Derivatives 6 2.1.1 General Synthesis of Acrylonitrile 6 2.1.2 Chemical Properties of Acrylonitriles 17 2.1.3 Spectral Properties of Acrylonitrile Derivatives 28 2.1.4 Biological Activities of Acrylonitrile Derivatives 31 2.2 Thiazole Derivatives 41 2.2.1 Synthesis of Thiazoles Derivatives 42 2.2.2 Biological activities of Thiazole Derivatives 44 2.3 Tuberculosis, Vaccine and Treatment 48 x 2.4 Infections and Antimicrobial Treatments 62 3. METHOD AND MATERIALS 67 3.1 Chemical Studies 67 3.1.1 Materials 67 3.1.2 General Synthesis Procedure 67 3.1.3 Analytical Methods 69 3.1.4 Spectroscopic Methods 70 3.2 Biological Activity Studies 70 3.2.1 In vitro Antimycobacterial Activity Studies 71 3.2.2 In vitro Antimicrobial Activity Studies 71 4. RESULTS 73 4.1 Chemical Studies 73 4.2 Biological Activity Studies 82 4.2.1 In vitro Antimycobacterial Activity Studies 82 4.2.2 In vitro Antimicrobial Activity Studies 84 5. DISCUSSION 88 5.1 Chemical Studies 88 5.1.1 General Synthesis Procedures 88 5.1.2 Characterization of the Structures of Synthesized Compounds 91 5.2 Biological Activity Studies 95 5.2.1 Antimycobacterial Activity 95 5.2.2 Antimicrobial Activity 96 xi 6. CONCLUSION 89 7. REFERENCES 90 8. APPENDIX 89 APPENDIX-1: Turnitin originality report 89 APPENDIX-2: Digital Turnitin originality report 90 9. CURRICULUM VITAE 91 xii ABBREVIATIONS 3D-QSAR Three-Dimensional Quantitative Structure-Activity Relationship 5-LOX 5-Lipoxygenase AChE Acetylcholinesterase AIBN Azobisisobutyronitrile AML Acute Myeloid Leukemia AR Antibiotic Resistance BCG Bacillus Calmette-Guérin CD Centers For Disease Control and Prevention CoMSIA Comparative Similarity Indices Analysis COVID-19 Coronavirus Disease 2019 DABCO 1,4-Diazabicyclo[2.2.2]Octane DCM Dichloromethane DHFR Dihydrofolate Reductase DMF Dimethylformamide DMSO Dimethylsulfoxide DR-TB Drug-Resistant Tuberculosis E Ethambutol FQs Fluoroquinolones H Isoniazid HIV/AIDS Human Immunodeficiency Virus/Acquired Immune Deficiency Syndrome IC50 Half-maximal inhibitory concentration IFIs Invasive Fungal Infections IMP Imipenem INH Isoniazid IR Infrared KOtBu Potassium tert-Butoxide LA Lewis Acid xiii LAT Lysine-Aminotransferase LTBI Latent Tuberculosis Infection MA Mycolic Acid MDR-TB Multidrug-Resistant TB MIC Minimum Inhibition Concentration MRP Meropenem MRSA Methicillin-Resistant Staphylococcus Aureus Mtb Mycobacterium tuberculosis NaSCN Sodium thiocyanate NBS N-Bromosuccinimide NCTS N-Cyano-N-Phenyl-p-Methylbenzenesulfonamide NH Nitrile Hydratase NMR Nuclear Magnetic Resonance NSAID Nonsteroidal Anti-Inflammatory Drug NTS Nitrilase PAN Polyacrylonitriles PAS P-Aminosalicylic Acid PBP Penicillin Binding Protein pre-XDR-TB Pre-Extensively Drug-Resistant TB pTsOH p-Toluenesulfonic Acid PZA Pyrazinamide R Rifampicin RR-TB Rifampicin-Resistant SLDs Second-Line Drugs TB Tuberculosis THF Tetrahydrofuran TLC Thin Layer Chromatography WHO The World Health Organization XDR-TB Extensively Drug-Resistant TB Z Pyrazinamide xiv FIGURES Figure Page 1.1. Chemical structures of acrylonitrile and thiazole derivatives possessing antimycobacterial activity 3 2.1. Chemical formula of acrylonitrile 6 2.2. Synthesis Routes of Acrylonitrile Derivatives 7 2.3. Synthesis of acrylonitrile by Knoevenagel condensation of 1-methyl- 1H-imidazole-2-carbaldehyde and 2-(4-nitrophenyl)acetonitrile 8 2.4. One-pot synthesis of acrylonitrile using 2-cyanothioacetamide and benzaldehyde 8 2.5. Synthesis of stereoselective acrylonitrile from aldehydes and ketones 9 2.6. One-pot synthesis of α,β-disubstituted acrylonitrile from substituted and non-substituted acetonitrile 10 2.7. Synthesis of acrylonitrile by addition of dialkylargentates to enynenitriles 11 2.8. Synthesis of acrylonitrile from γ-hydroxy-alkyne-nitriles 11 2.9. Synthesis of acrylonitrile by hydroamination of 3- phenylpropiolonitrile 12 2.10. Synthesis of acrylonitrile from arylvinyl bromides 12 2.11. Synthesis of acrylonitrile from vinyl halide and an alkali cyanide 13 2.12. Synthesis of acrylonitrile by copper-mediated cyanation of vinylsilanes 14 2.13. Synthesis of acrylonitrile by cyanation of alkynes 14 2.14. Synthesis of acrylonitrile from palladium-catalyzed cyanation of alkyne 15 2.15. Synthesis of acrylonitrile Nickel/LewisAcid-Catalyzed Carbocyanation of alkyne 15 2.16. Synthesis of acrylonitrile copper-catalyzed iodocyanation and dicyanation of alkyne 16 2.17. Synthesis of acrylonitrile form Rh(III)-catalyzed cyanation of alkene 16 2.18. Synthesis of acrylonitrile by direct metal-free cyanation of alkene 17 2.19. Reactions of nitrile group of acrylonitrile 17 xv 2.20. Reaction mechanism for the synthesis of acrylic acid from acrylonitrile 17 2.21. Enzymatic hydrolysis of acrylonitrile in Pseudomonas chlororaphis 18 2.22. Ritter reaction of acrylonitrile with cyclohexanol 19 2.23. Ritter reaction of acrylonitrile with olefins 20 2.24. Reaction of acrylonitrile with aldehyde 20 2.25. Diels-Alder reaction 21 2.26. Diels-Alder reaction between (E)-1,3-pentadiene and acrylonitrile 21 2.27. Mechanism of hydrogenation of acrylonitrile 22 2.28. Hydration of acrylonitrile 22 2.29. Halogenation of acrylonitrile 23 2.30. Proposed mechanisms of acrylonitrile chlorination 23 2.31. Reactions of acrylonitrile with various diazonium salts 24 2.32. Reaction of acrylonitrile with carbon monoxide and hydrogen and a saturated alcohol 25 2.33. Reaction of acrylonitrile with alcohol 26 2.34. Cyanoethylation of acrylonitrile 26 2.35. Homoplymerization of acrylonitrile 27 2.36. Copolymerization of acrylonitrile with ATRIF 28 2.37. Some anti-cancer acrylonitrile derivatives in literature 32 2.38. Some antibacterial acrylonitrile derivatives in literature 33 2.39. Some antiviral acrylonitrile derivatives in literature 34 2.40. Antifungal acrylonitrile derivative 35 2.41. Some antiparasitic acrylonitrile derivatives in literature 36 2.42. Acrylonitriles derivatives with AChE inhibiting activity in literature 37 2.43. Molecular structure of TACNBNF 38 2.44. Acrylonitrile derivatives with anti-inflammatory activities 39 2.45. Antimycobacterial acrylonitrile derivatives in literature 41 2.46. Chemical structure or thiazole 42 2.47. Synthesis of thiazole using Hantzsch-Thiazole synthesis 43 2.48. Synthesis of thiazole using Cook-Heilbron synthesis 44 2.49. Synthesis of thiazole using Gabriel synthesis 44 xvi 2.50. Antimicrobial thiazole derivatives in literature 45 2.51. Anticancer thiazole derivatives in literature 47 2.52. Antiinflammatory thiazole derivative 47 2.53. Antimycobacterial thiazole derivatives in literature 48 2.54. INH activation to isonicotinyl radical 53 2.55. Molecular formulars of first-line oral antituberculosis drugs 55 2.56. Molecular structures of rifampicin analogues 56 2.57. Molecular structures of aminoglycosides 57 2.58. Molecular structures of fluoroquinolones 58 2.59. Molecular structures of second line antituberculosis drugs 58 2.60. Molecular structures of beta-lactam antibiotics 59 2.61. Molecular structures of some multi-drug-resistant TB (MDR-TB) drugs 61 2.62. Molecular structures of clarithromycin and thioacetazone 62 2.63. Molecular structures of some thiazole-containing antibiotic 64 2.64. Molecular structures of some examples of azoles 66 3.1. Synthesis strategy of target compounds 1-16. Reagents and conditions: (i) Lawesson's reagent, THF (ii) NBS, pTsOH, DCM, H2O; (iii) L-proline, EtOH 69 5.1. Thionation of 2-cyanoacetamide using Lawesson's reagent 88 5.2. Mechanism of the thionation reaction of 2-cyanoacetamide using Lawesson’s reagent 89 5.3. Plausible mechanism for a-bromination of acetophenones 89 5.4. Mechanism of Hantzsch thiazole synthesis 90 5.5. Mechanism of Knoevenagel condensation 91 5.6. IR spectrum of compound 5 92 5.7. 1H-NMR spectrum of compound 5 93 5.8. 13C-NMR spectrum of compound 5 94 5.9. Mass spectrum of compound 5 95 xvii TABLES Table Page 1.1. The structure of the synthesized compounds (1-16) 4 4.1. Antimycobacterial activity results for synthesized compounds (1-16) 82 4.2. In-vitro antimicrobial activity result for synthesized compounds (1-16) 85 1 1. INTRODUCTION Tuberculosis (TB) is a communicable disease that ranks as a primary contributor to morbidity and mortality on a global scale. Mycobacterium tuberculosis (Mtb), the causative agent of TB, is a bacillus whose genetically identical descendants trace back to the Paleolithic era in East Africa 3.3 million years ago. This bacterial pathogen emerged as an epidemic in Europe and North America during the 18th and 19th centuries (1, 2). Despite a decrease in tuberculosis (TB) prevalence in developed countries over the last century, it remains a persistent challenge in developing nations due to its severe social consequences (3). Prior to the emergence of coronavirus disease 2019 (COVID-19), TB was the leading infectious disease responsible for mortality, surpassing human immunodeficiency virus/acquired immune deficiency syndrome (HIV/AIDS). The latest annual report on TB from the World Health Organization (WHO) reveals that in 2021, an estimated 106 million individuals were afflicted with the disease, compared to 101 million in 2020, and that 16 million individuals died from TB in 2021, an increase from 15 million in 2020 (3). Isoniazid (H) (1952), pyrazinamide (Z) (1954), ethambutol (E) (1963), and rifampicin (R) were the first-line drugs used to treat tuberculosis, but their success was severely impacted by the emergence of drug resistance (4, 5). Drug-resistant tuberculosis (DR-TB) poses a continuing global health threat, leading to increased morbidity and mortality, negative health outcomes, escalated treatment expenses, among other consequences (6). WHO categorizes drug-resistant TB into five main types: isoniazid- resistant TB, rifampicin-resistant (RR)-TB, and multidrug-resistant TB (MDR-TB), pre- extensively drug-resistant TB (pre-XDR-TB), which is MDR-TB with resistance to a fluoroquinolone, and extensively drug-resistant TB (XDR-TB), which is TB resistant to any fluoroquinolone and at least one of the second-line injectables, such as amikacin, capreomycin, or kanamycin (7). More recently, there have been concerns about the emergence of global resistance jeopardizing the effectiveness of novel MDR-TB treatment regimens, such as bedaquiline, a WHO-approved group A drug for treating MDR-TB. 2 This underscores the ongoing need for developing new antituberculosis agents, improving access to diagnosis, and implementing effective treatment regimens for all forms of DR- TB (8). Bacterial and fungal infections can lead to a range of severe illnesses, and antibiotic resistance (AR) is a significant global healthcare challenge, with developing nations often experiencing a greater impact (9). In 2013, the Centers for Disease Control and Prevention (CDC) published a list of emerging antibiotic resistance threats, which was updated in 2019 based on the latest national estimates of death and infection caused by 18 antimicrobial-resistant bacteria and fungi. The list is structured according to the level of urgency, with some microorganisms classified as Serious Threats. These include Drug-resistant Candida Species, extended-spectrum β-lactamase-producing Enterobacterales (ESBL-E), Methicillin-resistant Staphylococcus aureus (MRSA), and Drug-resistant Streptococcus pneumoniae, among others. The nitrile group is a vital functional group that can be found in both pharmaceuticals and natural products. Over the last few decades, the FDA has approved over 30 nitrile-containing pharmaceuticals for the treatment of a wide range of clinical conditions (10). Acrylonitrile, which is composed of a nitrile group attached to a vinylic moiety, is a promising pharmacophore that continues to pique the interest of medicinal chemistry researchers. Acrylonitriles are crucial building blocks for the synthesis of many biologically active compounds because of their wide range of biological activities (11- 16), as well as their chemical importance, which includes improving the solubility of compounds and the potential for the formation of hydrogen bonds or hydrophobic interactions (17). The acrylonitrile moiety is present in a number of drug molecules that have been marketed, including entacapone (18), rilpivirine (19), teriflunomide (20), Luliconazole (21) and others. Among the numerous studies on the biological and pharmacological activities (such as cancer (22-26), antibacterial (11, 14, 27), antiparasitic (16, 28, 29) , analgesic (30, 31), antialzheimer (32, 33) etc.) of acrylonitrile derivatives carrying heterocyclic rings, their antimycobacterial activity has steered extensive studies 3 for their potential as an anti-TB drug candidate (15, 17, 34, 35). Figure 1.1. Chemical structures of acrylonitrile and thiazole derivatives possessing antimycobacterial activity. In a study evaluating the efficacy of acrylonitrile-containing compounds against dormant-phase Mycobacterium at a concentration of 10 µg/ml, some of the compounds exhibited a bacterial log reduction of 2.9-fold. Notably, they displayed greater potency compared to first-line TB drugs isoniazid (1.2 log fold), rifampicin (1.3 log fold), and moxifloxacin (15) (Figure 1.1). In a different investigation, a compound carrying acrylonitrile structure was discovered to have greater effectiveness against Mtb than ethambutol (MIC = 1.56 g/ml) with a MIC of 0.78 g/ml. The bacterial count of dormant forms of mycobacterium was significantly reduced by this compound antibiotic, decreasing it by 2.8 log fold, O OH S N S N MIC=64.43 µg/mL against M. Tuberculosis (15) N N NHN MIC=0.78 µg/mL against M. Tuberculosis (34) MIC=6.25 µg/mL against M. Tuberculosis and MIC=12.5 µg/mL against M. avium (36) MIC=1.03 µg/mL against M. Tuberculosis (51) N H N S NN+ O -O NN O S NCl Cl F F N Br N N N N MIC=2.43 µg/mL against M. Tuberculosis (50) 4 outperforming frequently prescribed first-line medications isoniazid, ciprofloxacin, rifampicin, and moxifloxacin (34) (Figure 1.1). A substituted-2-(1H(2H)-benzotriazol-1(2)-yl)acrylonitrile derivative was discovered to have excellent inhibitory effects against both M. avium and M. tuberculosis. In particular, it showed 93% and 99% inhibitory efficacy against M. avium and M. tuberculosis, respectively, at a MIC = 12.5 g/ml (36) (Figure 1.1). Thiazole has played a variety including its use as a pharmacophore, spacer, or bioisosteric scaffold. Thiazole derivatives demonstrating promising biological activities such as anticancer (37-39), antiviral (40-42), antialzheimer (43, 44), antidiabetic (45, 46), antioxidant (47-49) etc. and in more recent investigations, thiazole has also demonstrated various antitubercular (15, 35, 50, 51) (Figure 1.1) and antimicrobial activities (52-59). On the basis of these findings, we combined the thiazole ring and acrylonitrile moiety to develop a new series of 3-aryl-2-(4-(substitutedphenyl)thiazol-2-yl)acrylonitrile derivatives (1-16) derivatives and tested their antimycobacterial and antimicrobial activities (Table 1.1). Table 1.1. The structure of the synthesized compounds (1-16). Compounds Ar1 Ar2 1 2 3 S NAr1 CN Ar2 ClCl ClCl CH3 ClCl C2H5 5 4 5 6 7 8 9 10 11 12 13 14 15 16 ClCl OH ClCl OCH3 ClCl OC2H5 ClCl F ClCl Cl ClCl Br ClCl CF3 ClCl N(CH3)2 ClCl O ClCl S Cl CF3 Cl S F S 6 2. LITERATURE REVIEW 2.1 Acrylonitrile Derivatives Acrylonitrile, also known as 2-propenenitrile, propenenitrile, acrylic acid nitrile, propylene nitrile, vinyl cyanide, or propenoic acid nitrile, is an organic compound consisting of a vinyl moiety attached to a nitrile. It has a chemical formula of CH2CHCN and a molecular mass of 53.064 g/mol. Figure 2.1. Chemical formula of acrylonitrile 2.1.1 General Synthesis of Acrylonitrile Unlike most organic compounds, acrylonitrile, which is often referred to as α, β- unsaturated in literature, is not found naturally on Earth, necessitating the use of various synthetic procedures and materials (60). Historically, large-scale production of acrylonitrile was done using the ethylene cyanohydrin process, which involved the based- catalyzed addition of HCN to ethylene oxide in the liquid phase at 60˚C (60). Later in 1970, this method was discontinued and was replaced by the newly discovered propylene ammoxidation process, also known as the SOHIO process. Although this method is known to produce a high yield of acrylonitrile at a lower cost, the toxic chemicals and harsh reaction conditions required, combined with the high demand for acrylonitrile, prompted the development of new synthesis strategies (61). The general synthesis of acrylonitrile can be grouped into the main categories listed below (62, 63) (Figure 2.2). H C H C H C N 1 23 7 Figure 2.2. Synthesis Routes of Acrylonitrile Derivatives Condensation of Carbonyl Compounds and Nitriles (I) The availability of carbonyl precursor in aldehydes and ketones makes this approach the most used method in synthesizing acrylonitrile. In this method, a base is used as a nucleophile to eliminate an acidic proton of a nitrile compound, making it less stable and reactive to attack the carbonyl compound to form an enol. This reaction is a type of aldol condensation reaction known as Knoevenagel condensation. The enol intermediate eventually forms an α,β-unsaturated nitrile in a base medium. Nadaf et al employed a multi-step synthetic methodology to prepare 1,2- disubstituted imidazoles in their investigation. The experimental procedure employed piperidine as the base and ethanol as the solvent. The reaction involved the Knoevenagel condensation of 1-methyl-1H-imidazole-2-carbaldehyde and 2-(4-nitrophenyl) acetonitrile, which yielded a condensed product, namely 3-(1-methyl-1H-imidazol-2-yl)- 2-(4-nitrophenyl)acrylonitrile. This synthetic approach has been demonstrated to be uncomplicated, effective, and takes place under gentle conditions (17) (Figure 2.3). R2 R1 CN R3 CNR1 R2 R1 X R2 R1 M R3R1 R2 R1 R3 R1 R2 O V VI I II III IV 8 Figure 2.3. Synthesis of acrylonitrile by Knoevenagel condensation of 1-methyl-1H- imidazole-2-carbaldehyde and 2-(4-nitrophenyl)acetonitrile Kavitha et al further developed a discovery by synthesizing 2-(4-(2-oxo-2H- chromen-3-yl)thiazol-2-yl)-3-phenylacrylonitrile through a one-pot synthesis involving 3- (2-bromoacetyl)-2H-chromen-2-one (2), 2-cyanothioacetamide, and benzaldehyde. They utilized various base mediums such as piperidine, piperazine, N-methylpiperazine, N- ethylpiperazine, morpholine, triethylamine, triphenylphosphine, ammonium acetate, sulfamic acid, and L-proline. Results showed that L-proline was the most effective catalyst, and methanol and ethanol were the optimal solvents for the Knoevenagel condensation. This study revealed that L-proline and polar protic solvents were the best choices for the catalyst and solvent, respectively. Furthermore, the one-pot synthesis method is an environmentally friendly alternative and allows for improved chemical reaction efficiency and reduced reaction time. The results were extended to different heterocyclic and heteryl aldehydes, yielding good product yields, although longer completion times were observed for heteryl aldehydes possibly due to electronic factors (35) (Figure 2.4). Figure 2.4. One-pot synthesis of acrylonitrile using 2-cyanothioacetamide and benzaldehyde A study conducted by A. Lattanzi et al. described the use of a mild base, lithium hydroxide, and activated 4 Å molecular sieves to develop stereoselective α,β-unsaturated N H H N H O CN NO2 N N NC NO2 Piperidine Ethanol Reflux, 2 h O O O Br H2N S Ar O H Ethanol / L-proline Reflux / 2-4 h O O S N Ar N N 9 esters. The researchers attributed the shorter reaction time to the activated 4 Å MS. The predominant E-stereoselectivity observed was attributed to the directing effect of the hydroxyl group present in α-hydroxy ketones. The results showed that LiOH was more effective than commonly used bases such as LDA, LiHMDS, and NaH in this type of reaction (64) (Figure 2.5). Figure 2.5. Synthesis of stereoselective acrylonitrile from aldehydes and ketones Tamioka et al. conducted a study to synthesize stereoselective α,β-disubstituted acrylonitriles using two different approaches. The first approach, known as the linear approach, involved the one-pot olefination of a substituted acetonitrile using bis(diisopropylamino)chloroborane reagent with 2 equiv of a lithiated RCH2CN, followed by the addition of benzaldehyde, resulting in fair to good yield with (Z)-stereoselectivity. However, this approach required the use of expensive and/or not readily available precious nitrile RCH2CN, making it less desirable. To overcome this limitation, the authors proposed an alternative approach, called the divergent approach, which took advantage of the nature of an α-boryl carbanion. This approach involved the reaction of a carbanion with an alkyl halide (RX) to form an alkylated intermediate, which could then be treated with a base and an aldehyde to provide acrylonitriles. This approach, starting from simple acetonitrile (CH3CN), was found to be more flexible and versatile in accessing various acrylonitrile derivatives (65) (Figure 2.6). R R2 O P O EtO EtO CN R R2 R1 CN R1 A= aldehyde; R1= H A= ketone; R1= H, Me A LiOH, THF 10 Figure 2.6. One-pot synthesis of α,β-disubstituted acrylonitrile from substituted and non-substituted acetonitrile Conjugate Additions or Reductions of Alkyne-Nitriles (II) Another popular method for producing acrylonitrile is the conjugate addition or reduction of alkyne-nitriles. Primarily, Grignard reagents were the first organometallics reported to interact conjugately with unsaturated nitriles. In a study by Kleijn et al., it has been demonstrated that dialkylargentates, such as R2AgMgCl, exhibit a high propensity to react with enynyl nitriles when tetrahydrofuran (THF) is present. Upon undergoing protolysis, the intermediate product yields a considerable amount of trans-2,4-alkadienenitriles. However, substitution of the Grignard reagent with RCu reagents results in the production of a Z-isomer (66) (Figure 2.7). RCH2CN R1 CN Ra) n-BuLi, THF b) (i-Pr2N)2BCl c) R1CHO CH3CN 1a) n-BuLi, THF 1b) (i-Pr2N)2BCl 1c) RX (i-Pr2N)2B CN R 2a) n-BuLi, TMEDA,THF 2b) R1CHO Linear approach Divergent approach R1 = aryl; Z-selective R1 = alkyl; E-selective concentration in vacuo R = alkyl R1 = aryl, alkyl 11 Figure 2.7. Synthesis of acrylonitrile by addition of dialkylargentates to enynenitriles Fleming et al. conducted a recent investigation into the stereoselective chelation- controlled conjugate addition of Grignard reagents to γ-hydroxy-alkyne-nitriles. The reaction process begins with the deprotonation of the γ-hydroxy-alkyne-nitriles by t- BuMgCl, followed by the stepwise conjugate addition of a second Grignard that leads to the formation of a cyclic magnesium chelate. It is postulated that the cyclic magnesium chelate generates a more reactive ate complex capable of alkylating both aliphatic and aromatic aldehydes when t-BuLi is added. The chelation-controlled conjugate addition and alkylation are shown to simplify the production of tri- and tetra-substituted acrylonitrile (67) (Figure 2.8). Figure 2.8. Synthesis of acrylonitrile from γ-hydroxy-alkyne-nitriles Michon et al. documented that the intermolecular hydroamination of 3- phenylpropiolonitrile, an internal alkyne, with pyrazole proceeded with high regio- and stereoselectivity in the presence of gold(I) catalyst under solvent-free conditions. The C C CN 2R1 R R1 CN 2 AgMgCl R R1 CN H E R2AgMgCl THF H3O+ 70-90% 1a, 1b RCu THF H3O+ 70-90% R1 R Cu CN R1 R H CN Z R1 = H2C C(Me); R = Me, Et, n-Bu 1a, 1b, R1 = 1-cyclohexenyl HO R1 R1 CN OH R1 R1 R2 CNt-BuMgCl R2MgX 12 outcome was the formation of two (Z)-regioisomers, with a 9/1 ratio and a high degree of isolation (68) (Figure 2.9). Figure 2.9. Synthesis of acrylonitrile by hydroamination of 3-phenylpropiolonitrile Cyanide coupling of vinyl halides (III) The transition-metal-catalyzed cyanation reaction employing KCN, NaCN, or K4[Fe(CN)6 represents a highly attractive and contemporary approach for synthesizing organic cyanides. This method has been utilized in recent developments for coupling reactions of C—CN bonds, including aryl and vinyl halides and pseudohalides, arylboronic acids, as well as alkene, alkyne, and aromatic hydrocarbons (69). The pioneering study by Murahashi et al. presented the first instance of palladium- catalyzed vinylnitrile synthesis from vinyl halides employing potassium cyanide-crown ether for cyanidation (70). Li et al. also contributed to this field with a report on a microwave-assisted coupling reaction utilizing palladium-catalyzed arylvinyl bromides and potassium ferrocyanide, resulting in the production of highly stereoselective α,β- unsaturated nitriles. The procedure is straightforward, expeditious, and affords a product that is easily separable (71) (Figure 2.10). Figure 2.10. Synthesis of acrylonitrile from arylvinyl bromides Sakakibara et al. conducted a reaction between 1-bromo-2-ethoxyethene, a vinyl halide, and alkali cyanide KCN in DMF for a duration of 4 hours, utilizing Nickel catalyst to produce 3-ethoxyacrylonitrile. In this study, various solvent systems were investigated Ph CN Ph N CN N NC N Ph N((Au(IPr))2(µ-OH)(BF4) Bu4NOTf Z Z N H N Ar Br K4 Fe(CN)6 Ar CNPdCl2 BMIm BF4 13 to determine the most effective system for the reaction. The results indicated that nearly all of the procedures tested produced high yields and high stereoselectivity at 50 °C when utilizing the KCN-DMF system with intermediate cyanide solubility. The KCN in hexamethyl phosphoric triamide (HMPA) and KCN-MeCN systems with low cyanide solubility accelerated the coupling of the halides to inhibit the cyanation, whereas the NaCN-DMF and NaCN-HMPA systems with high cyanide solubility yielded the lowest yield because the presence of excess cyanide ion inhibited the reaction (72) (Figure 2.11). Figure 2.11.Synthesis of acrylonitrile from vinyl halide and an alkali cyanide Despite its advantages, the transition-metal-catalyzed cyanation reaction suffers from several drawbacks. Cyanation sources commonly utilized in this reaction are hazardous and generate toxic HCN gas. Additionally, the reaction leads to stoichiometric quantities of metal waste. Another issue that needs to be considered is the need for careful control of the concentration of the reaction mixture to minimize the formation of inactive cyano transition metal complexes in situ that can cause catalyst poisoning (73). Cyanation of vinyl anions (IV) In a study conducted by Wang et al., the cyanation of vinylsilanes was achieved using ammonium iodide and DMF as the combined source of nitrogen and carbon atoms for the introduction of the cyano group. The reaction was observed to occur in two distinct steps. The organosilanes were initially converted to their iodo intermediates, followed by the formation of cyanation complexes. The researchers found that the cyanation proceeded smoothly, resulting in the production of the desired acrylonitrile products (74) (Figure 2.12). X MCN CN MX NiBr2(PPh)2-Zn-PPh3 X = Br, Cl M= K, Na 14 Figure 2.12. Synthesis of acrylonitrile by copper-mediated cyanation of vinylsilanes Cyanation of Alkynes (V) The direct cyanation of alkynes is considered a highly efficient and straightforward approach for the synthesis of acrylonitrile. The metal-catalyzed direct addition of X-CN bonds, where X represents C, B, Br, or other similar bonds, into alkynes has been the subject of extensive research over the years due to its conceptual novelty, product utility, and high degree of regio- and stereoselectivity (75-77) (Figure 2.13). Figure 2.13. Synthesis of acrylonitrile by cyanation of alkynes Cheng et al. investigated a Pd-catalyzed three-component arylcyanation of internal alkynes with aryl bromides and K4[Fe(CN)6], which provides a direct and stereoselective method for synthesizing fully substituted, α,β-unsaturated nitriles from simple starting materials. The reaction was compatible with both diphenylacetylene and dialkyl- substituted alkynes, yielding the desired product in satisfactory yields. This method is considered attractive due to the utilization of readily available internal alkynes, aryl bromides, and an environmentally friendly cyanation reagent (75) (Figure 2.14). Si(OEt)3 R NH4I CN R Cu(NO3)2 3H2O KF, DMF O2, 140 ºC, 25 h R1 R2 X CN CN R2R1 X M cat. X = C, Si, B, Sn, Ge, S R1, R2 = alkyl, aryl 15 Figure 2.14. Synthesis of acrylonitrile from palladium-catalyzed cyanation of alkyne Nakao employed the nickel/Lewis acid (LA) cooperative catalysis strategy in carbocyanation reactions that involve the cleavage of C-CN bonds in nitriles, utilizing both the organic and cyano groups. The reaction relied solely on a nickel catalyst and was limited to the use of aryl and allylcyanides as nitrile substrates. However, the introduction of LA cocatalysts significantly accelerated the rate of arylcyanation and extended the range of nitriles employed in the reaction to include alkynyl, alkenyl, and alkylcyanides. The result of these reactions was the creation of a variety of highly stereo- and regioselective acrylonitrile molecules (78) (Figure 2.15). Figure 2.15. Synthesis of acrylonitrile Nickel/LewisAcid-Catalyzed Carbocyanation of alkyne Sakata has reported a regio- and stereoselective iodocyanation and dicyanation reaction of alkynes with cyanogen iodide, catalyzed by copper. This method allowed for the formation of complex acrylonitrile structures with well-controlled regio- and stereoselectivity. The reaction mechanism, including the stepwise processes of diiodide formation, selective monocyanation, and second cyanation, was elucidated. Furthermore, it was noted that the selectivity of the products could be altered by modifying the reaction conditions (79) (Figure 2.16). R = alkyl, aryl Ar Br R R K4 Fe(CN)6 Pd(OAc)2 DMAc, 120 ºC, 5h CN RR Ar R = alkyl, aryl C CN R R Ni/Lewis acid cooperative catalysis CN R C R 16 Figure 2.16. Synthesis of acrylonitrile copper-catalyzed iodocyanation and dicyanation of alkyne Cyanation of alkene (VI) In the direct C(sp2)-H cyanation of alkenes with directing groups, the rhodium(III)- catalyzed C-H reaction is a valuable alternative to other transition metals due to its substrate scope and functional group compatibility (80, 81). A practical method for the synthesis of alkenyl nitriles has been developed using Rh(III)-catalyzed direct vinylic C-H cyanation reaction with N-cyano-N-phenyl-p- methylbenzenesulfonamide as a cyanation reagent for acrylonitrile compound synthesis. This new C-H cyanation process accommodates both acrylamides and ketoximes as substrates (80) (Figure 2.17). Figure 2.17. Synthesis of acrylonitrile form Rh(III)-catalyzed cyanation of alkene Wang et al. conducted a study on transition metal-free cyanation of alkenes using aryl(biscyano)iodine(III) reagent as the activator and trimethylsilyl cyanide as the source of cyanide. The researchers aimed to expand the approach previously mentioned by exploring a new mechanism involving the electrophilic activation of the alkene by cyano iodine(III) species generated in situ from a [bis(trifluoroacetoxy)iodo]arene. The study found that the method could be successfully applied to noncyclic 1,1- and 1,2- R1, R2 = alkyl, aryl R1 R2 I CN R2 CNR1 I R2 CNR1 NC and/or Cu cat. R1, R2 = alkyl, aryl R H NiPr2 O N Ts CN Ph R CN NiPr2 ORhCp*(CH3CN)3(SbF692 Ag2CO3 NaOAc DCE, 120 ºc, Ar, 24 h 17 disubstituted alkenes, achieving high stereoselectivity and proving to be a highly useful approach (82) (Figure 2.18). Figure 2.18. Synthesis of acrylonitrile by direct metal-free cyanation of alkene 2.1.2 Chemical Properties of Acrylonitriles Acrylonitrile is an important chemical intermediate due to its versatile functionality. Its double bond and nitrile group make it amenable to a diverse range of reactions. The nitrile group can undergo hydrolysis, hydrogenation, esterification, or reduction, while the double bond can engage in reactions including polymerization, copolymerization, cyanoethylation, cyclization, and halogenation. Reaction of the Nitrile group Figure 2.19. Reactions of nitrile group of acrylonitrile R1, R2, R3 = alkyl, aryl R2 R3 H R1 TMSCN ıııı-reagent TMSOTf R2 R3 CN R1 Hydrolysis (Ritter reaction) (Bayliss reaction) C C COOH H H H C C H H C H O NH C C H R1 R2 R4 R3 C C CH CNH H HO R or C CH H H C O H N R CH2CHCN Reaction with olefins or alcohols Reaction with aldehydes 18 i) Hydrolysis Reaction of Nitrile Group The nitrile group present in acrylonitrile can be converted to the corresponding carboxylic acid through the action of strong acids and bases. This chemical reaction has diverse applications, such as in the synthesis of acrylamide, biodegradation of acrylonitrile, and treatment of acrylonitrile wastewater (83). Dong et al. conducted a one-pot selective conversion of acrylonitrile to acrylic acid in a hydrothermal system utilizing NaOH as a catalyst. This process involved the hydrolysis of acrylonitrile to acrylamide, which was further hydrolyzed to form acrylic acid. The experimental parameters, including the initial concentration of acrylonitrile, reaction temperature, reaction time, and amount of alkali, significantly influenced the yield of acrylic acid. The optimal conditions for obtaining the highest yield of acrylic acid (55%) were found to be an initial acrylonitrile concentration of 3x103mg/L, a reaction temperature of 300°C, and a reaction time of 90 seconds with a 1.0M NaOH catalyst (83) (Figure 2.20). Figure 2.20. Reaction mechanism for the synthesis of acrylic acid from acrylonitrile. Another study describes a novel method for enzymatic acrylamide synthesis. The process is defined using immobilized Pseudomonas chlororaphis RPZ-18 cells in a system containing concentrated acrylonitrile solutions as the substrate. The hydrolysis product is an acrylamide with has a high chemical purity. However, the hydrolysis reaction may be restricted to solely produce acrylamide by way of the catalytic activity of the nitrile hydratase (NH) component of nitrilase (NTS) (specifically the first component), following destruction of the amidase component in the same strain (84) (Figure 2.21). C C H H H C N C C H H H C NH2 O C C H H H C OH OH2O Acrylonitrile Acrylic acidAcrylamide H2O 19 Figure 2.21. Enzymatic hydrolysis of acrylonitrile in Pseudomonas chlororaphis ii) Reaction of Acrylonitrile with Olefins and Alcohols The chemical transformation of acrylonitrile's nitrile group through reaction with alcohols or olefins, referred to as The Ritter reaction, generates carboxylic amides, including the production of acrylamide (85). This reaction offers an efficient method for the synthesis of amides that feature secondary or tertiary alkyl groups on the nitrogen atom. In a recent study aimed at developing a practical method for preparing a mildewcide, N-cyclohexylisothiazolone, the starting compound was synthesized using the Ritter reaction of acrylonitrile with cyclohexanol in the presence of concentrated sulfuric acid. Subsequently, the intermediate product was subjected to thiolation with thiourea, which yielded acrylamide (85, 86) (Figure 2.22). Figure 2.22. Ritter reaction of acrylonitrile with cyclohexanol Kazantsev et al. conducted a similar experiment in which N-Alkylacrylamides were synthesized from acrylonitrile and commercial olefin fractions in the presence of sulfuric acid using the Ritter reaction. It was also established that, the main and side H2C CH CN 2 H2O H2C CH COOH NH3 NTS Acrylic acidAcrylonitrile H2C CH CONH2 NH Acrylamide H2OH2C CH CN Acrylonitrile H2C CH CN OH H2SO4 H2C CH C O NH H2C CH2 C O NHHS 1) Thiourea 2) NaOH 20 reactions are affected by temperature, reactant ratio, and sulfuric acid concentration. The reaction is widely assumed to follow the scheme illustrated in Figure 2.23 (86). Figure 2.23. Ritter reaction of acrylonitrile with olefins iii) Reaction of Acrylonitrile with Aldehydes Acrylonitriles have also been observed to react with aldehydes and ketones in a base catalyzed reaction known as the Bayliss-Hillman reaction (87, 88), This is shown in a study by Hill and Isaacs. In the study, a good yield of 2-cyanobut-1-en-3-ol (R = Me) is obtained when an acrylonitrile was subjected to acetaldehyde in the presence of a base catalyst, 1,4-diazabicyclo[2.2.2]octane (DABCO) (88) (Figure 2.24). Figure 2.24. Reaction of acrylonitrile with aldehyde Reaction of the Double Bond of Acrylonitrile i) Diels Alder and Related Reactions of Acrylonitrile The Diels-Alder reaction involves the chemical reaction between a conjugated diene and a substituted alkene, called the dienophile, resulting in a substituted cyclohexene derivative. This cycloaddition reaction is well-established and extensively researched in organic chemistry (89) (Figure 2.25). H2C CH CN R1R2C CR3R4 H2O H2SO4 H2C CH C O NH C(R1R2)CHR3R4 R1,R2, R3, R4 = alkyl H2C CH CN RCHO base H2C C CH(OH)R CN R = alkyl, aryl 21 Figure 2.25. Diels-Alder reaction A study by James et al. provides an example of this reaction and examines the concerted and stepwise pathways for the reactions between (E)-1,3-pentadiene and acrylonitrile. The reaction generates a Diels-Alder adduct product, comprising a mixture of the two expected regioisomers (90) (Figure 2.26). Figure 2.26. Diels-Alder reaction between (E)-1,3-pentadiene and acrylonitrile. ii) Hydrogenation and Hydration Reactions of Acrylonitrile The catalytic hydrogenation of acrylonitrile leads to the production of propionitrile (91). This reaction can be catalyzed by several transition metals, including Rh, Ni, and Pd (92-95). The presence of C=C and C≡N bonds in acrylonitrile makes controlling the selectivity of hydrogenation crucial in producing propionitrile. Nickel-based catalysts, such as the nickel boride catalyst (96) and Ni-B/SiO2 amorphous catalyst (97) have shown high efficiency in selectively hydrogenating acrylonitrile. Acrylonitrile, upon undergoing further hydrogenation, leads to the formation of propylamine and other byproducts, such as 3-(propylamino)propanenitrile and 3-(dipropylamino)propanenitrile, which are created via addition and disproportionation reactions as shown in Figure 2.27 (95). CN stepwise CN CN n CN concerted CN trans CN concerted CN CN 22 Figure 2.27. Mechanism of hydrogenation of acrylonitrile. The process of selectively hydrating acrylonitrile to produce acrylamide has been the subject of various studies involving different metal oxide catalysts, with MnO2, CuO, and Co3O4 found to exhibit high levels of activity and selectivity. The catalytic activities are significantly influenced by the preparation method, and the degree of hydration activity displayed by MnO2 is directly related to the quantity of phenol adsorption. The hydration of acrylonitrile results in the production of both acrylamide and ethylenecyano- hydrine by addition to the C=N and C=C bond in acrylonitrile, respectively (98) (Figure2.28). Figure 2.28. Hydration of acrylonitrile iii) Halogenation of Acylonitrile Several studies have reported on the halogenation of acrylonitrile via heat or UV light (99-102). The addition of a halogen molecule produces 2,3-dihalopropionitriles at low temperatures. In the absence of UV light, an increase in temperature results in a second addition of halogen to produce 2,2,3-trihalopropionitrile (102) (Figure 2.29). H2C H C CN H3C H2 C CN H2 H2 H3C H2 C CH NH H2 H3C H2 C CH2NH2 H2C H C C H NH H3C H2 C C H2 H N C H2 CNH2C H C CN H3C H2 C C H2 H N C H2 H2 C CH3 H3C H2 C C H2 N H2C C H2 CN CH2 CH3 H2C H C CN H2 H2C CH CN H2O H2C CH C O NH2 H2C CH2 HO CN acrylamide ethylenecyanohydrine 23 Figure 2.29. Halogenation of acrylonitrile In the presence of ultraviolet light, the main product of the reaction is 2,3- dichloropropionitrile until significant amounts of hydrogen chloride are formed, at which point side reactions dominate, with 3-chloropropionitrile and 2,2,3-trichloropropionitrile as the main products. It was proposed that hydrogen chloride is formed first during the free-radical photochlorination process, but that once formed, it catalyzes a parallel ionic reaction pathway that produces most, if not all of the 3-chloropropionitrile and 2,2,3- trichloropropionitrile by-products(101, 102) (Figure 2.30). Figure 2.30. Proposed mechanisms of acrylonitrile chlorination H2C CH CN X2 XCH2CHXCN XCH2CHXCN H2C CXCN HX H2C CXCN XCH2CX2CN Cl2 2Cl H2C CH CN ClCH2CHCN ClCH2CHCN Cl2 ClCH2CHClCN Cl ClCH2CHClCHNH Cl ClCH2CClCN Cl ClCH2CClCN Cl2 ClCH2CCl2CN Cl ClCH2CCl2CHNH HCl ClCH2CH2CN Cl HCl Free radical mechanism HCl H2C CH CN ClCH2CH2CNH H ClCH2CHC NH ClCH2CH C NH ClCH2CH C NH Cl2 ClCH2CHCl C Cl NH ClCH2CHClCN HCl ClCH2CH2CHNH H-+ Ionic mechanism 24 iv) Reaction of Acrylonitrile with Diazo compounds Figure 2.31. Reactions of acrylonitrile with various diazonium salts Naidan et al. conducted an exploration on the process of acrylonitrile thiocyanatoarylation, wherein they observed that the combination of benzenediazonium sulfates and acrylonitrile in an aqueous acetone medium, in the presence of thiocyanate ions, resulted in a reaction wherein the unsaturated compound underwent addition with thiocyanato- and arene-sulfonyl groups. This reaction occurred over a range of temperatures from -16 to -20 °C (103) (Figure 2.31 I). Moreover, under the influence of copper(I)ethylxanthate and potassium xanthate, benzene- and p-nitrobenzene-diazonium sulfates react with acrylonitrile in an aqueous acetone solution at 5-10 °C. The reaction occurs with the elimination of nitrogen and produces α-(ethoxythiocarbonylthio)-β-phenylpropionitrile (51%) and α- (ethoxythiocarbonylthio)-β-(p-nitrophenyl)propionitrile (40%) (104) (Figure 2.31 II). Acrylonitriles react with diazonium salts in the presence of copper salts in a reaction known as the Meerwein reaction. In a review paper, various reactions of aromatic diazonium salts with diene and monounsaturated compounds in the presence of nucleophiles are described. It was discovered that in an acetone-water reaction medium, arylchloro(bromo)ethanes are formed in 25%-75% yield when arenediazonium H2C CH CN b) NH4SCN b) NaA b) C2H5OC(S)SK ArCH2CH(SCOC2H5)CN ArCH2CH(CN)A ArCH2CH(SCN)CN Ar = C6H5, p-NO2C6H4, p-CH3C6H5 (NH4)2SO4 KHSO4 NaBF4 -N2 -N2 -N2 I II III Ar N N HSO4a) Ar N N HSO4a) Ar N N BF4a) S A = NO2, Cl, Br 25 tetrafluoroborates interact with acrylonitrile in the presence of sodium nitrite, chloride, and bromide and catalytic amounts of copper salts (105) (Figure 2.31 III). v) Reactions with alcohol and carbon monoxide In the presence of a hydrogenation catalyst such as cobalt or ruthenium, acrylonitrile can react with a mixture of carbon monoxide and hydrogen and a saturated alcohol to produce cyanopropionaldehyde acetals. This reaction is most effective when using saturated primary and secondary monohydric and dihydric alcohols such as methanol, ethanol, ethylene glycol, and propylene glycol. At least two alcoholic hydroxyl groups are required for each acrylonitrile ethylenic bond. The reaction is carried out by heating the reactants at temperatures and pressures of 100-200°C and above 600 atmospheres, and the catalyst of choice is typically selected from the VIIIth group of metals in the periodic table (106) (Figure 3.32). Figure 2.32. Reaction of acrylonitrile with carbon monoxide and hydrogen and a saturated alcohol Thiyagarajan et al. describe a low-cost, and easily obtainable potassium tert- butoxide (KOtBu) catalyzed Michael addition reaction with acrylonitrile as a typical Michael acceptor. This catalytic protocol is said to be compatible with a diverse range of heteroatom nucleophiles, including aliphatic and aromatic primary and secondary alcohols, thiols, and amines with acrylonitrile, substituted acrylonitrile, acrylamide, and acrylic esters. The general ionic mechanism for Michael addition reactions begins with the deprotonation of an acidic proton from the Michael donor by a base, which results in the formation of nucleophilic intermediate I. Nucleophile I conjugates with Michael acceptor acrylonitrile to form intermediate II, which provides Michael addition product upon hydrogen abstraction from protonated base (107) (Figure 2.33). H2C CH CN 2ROH CO H2 Co CHCH2CH2CN RO RO H2O 26 Figure 2.33. Reaction of acrylonitrile with alcohol Cyanoethylation of Acrylonitrile The reactive double bond of acrylonitrile readily undergoes cyanoethylation by a range of organic and inorganic compounds possessing labile hydrogen atoms. This reaction results in the introduction of a cyanoethyl (-CH2-CH2-CN) group in place of the original double bond. Cyanoethylation is a well-known reaction and is analogous to a Michael reaction (108) (Figure 2.34). Figure 2.34. Cyanoethylation of acrylonitrile Reactive hydrogen atoms are typically present in the compounds involved in cyanoethylation of acrylonitriles, example ammonia, water, alcohols, phenols, ketones, carboxylic acid, esters, oximes, mercaptans, etc. Although some amines may necessitate an acidic catalyst for cyanoethylation, it is generally required to have an alkaline catalyst, comprising hydroxides, alkoxides, hydrides, etc. The most effective catalysts for cyanoethylation are highly basic quarternary ammonium hydroxides, such as benzyltrimethylammonium hydroxide. Inert solvents, including benzene, dioxane, pyridine, and acetonitrile, are commonly used, and the reactions are often exothermic, making cooling necessary to prevent excessive polymerization (108). Polymerization of Acrylonitrile Acrylonitrile is a monomer with a high degree of reactivity and can undergo polymerization with various vinyl monomers under diverse conditions. Polyacrylonitriles (PAN) have extensive applications in the pharmaceutical industry, including their use as R2 OH R1 CN R2 O R1 CN KOtBu H2C CH CN RH RCH2CH2CN base catalyst inert solvent 27 antioxidants, emulsifying agents, insecticides, solvents, surface coatings, cross-linking agents, and catalyst treatments for medicines (109). Polymerization of acrylonitrile can occur through either homopolymerization or copolymerization. However, due to the difficulty in melting and processing acrylonitrile homopolymer, it is often copolymerized to achieve the desired thermal stability, melt flow, and physical properties (109). Homopolymerization can be achieved rapidly by using initiators such as radiation, free radicals (110), anionic initiators including metal alkyls and alkali metal alkoxides, and azo compounds such as 2,2'-azobis(isobutyronitrile) at moderate temperatures below 100 °C in solvents such as DMSO, DMF, or an aqueous solution of NaSCN (111). According to research conducted by Shi and Jiang, lithium amides have been demonstrated as effective anionic initiators for anionic polymerization of acrylonitrile to produce high molecular weight polyacrylonitrile. Different types of lithium amides derived from diisopropylamine, diethylamine, hexamethyldisilazane, dicyclohexylamine, and 2,2,6,6-tetramethylpiperidine were used as initiators, and polyacrylonitrile with weight-average molecular weights ranging from 1.02x106 g/mol to 1.23x106 g/mol (Mw/Mn = 1.9-2.2) were obtained. The homopolymerization of acrylonitrile was carried out in N,N-dimethylformamide with minimal side reactions. (112) (Figure 2.35). Figure 2.35. Homoplymerization of acrylonitrile In contrast, copolymerization refers to the incorporation of electron-donating monomer units into the polymer structure, either randomly or using specific strategies such as combining a potent acceptor monomer with a potent donor monomer to obtain alternating equimolar copolymers. Other methods employed in the synthesis of R2N H n-BuLi THF R2N Li H2C CH CN CH2 CH CN H n lithium amide PAN R alkyl 28 acrylonitrile block copolymers include ultrasonic, radiation, and chemical techniques, such as the use of polymer ions, polymer radicals, and organometallic initiators (109). Atlas et al. conducted an investigation aimed at enhancing the dielectric properties of poly(ATRIF), which is a homopolymer made from 2,2,2-trifluoroethyl acrylate (ATRIF). The strategy involved increasing the polarity of poly(ATRIF) by adding monomers with a high dipole moment, such as acrylonitrile. Copolymers that had both C- CN and C-F substituents were produced by copolymerizing acrylonitrile and 2,2,2- trifluoroethyl acrylate (ATRIF) with azobisisobutyronitrile (AIBN) as the initiator in acetonitrile solution at 70°C using free radical polymerization (113) (Figure 2.36). Figure 2.36. Copolymerization of acrylonitrile with ATRIF 2.1.3 Spectral Properties of Acrylonitrile Derivatives Acrylonitrile has several characteristic peaks in its infrared (IR) spectrum and nuclear magnetic resonance (NMR) spectrum. IR Spectra The IR spectrum of acrylonitrile is characterized by the presence of several characteristic peaks. A strong absorption band at around 2150-2259 cm-1, which corresponds to the triple bond between the carbon and nitrogen atoms in the molecule. A strong absorption band at around 1620-1450 cm-1 corresponding to the C=C stretching vibration and then C-H stretch vibration exhibits medium absorption band at around 700- 800 cm-1 in the fingerprint region (17, 34, 36, 114, 115) H2C C CN H H2C C CO2CH2CF3 H AIBN CH2CN H3C CH H CN H2C CH H CO3CH2CF3x y acrylonitrile ATRIF poly (AN-co-ATRIF) coplymer 29 In the FT-IR characterization of the Pyridine-Carbazole Acrylonitrile Derivatives produced by Pérez-Gutiérrez et al., all three synthesized compounds displayed similar characteristics. While the C=C vibrations of the acrylonitrile molecular double bond peaked around 1628–1629 cm-1, the bands at 745–744 cm-1 were attributed to the C–H vibrations of the double bond, and the bands at 2210–2213 cm-1 were caused by the CºN stretching vibrations (115). The target compounds, 2-(1H-benzo[d]imidazol-2-yl)-3-(4-(4- substitutedpiperazin-1-yl)phenyl)acrylonitrile, revealed absorption bands between 2230 and 2250 cm-1 due to CºN stretching in the IR spectra of newly synthesized benzimidazole-acrylonitrile derivatives by Sirim et al (34). In new 2-(4-(2-oxo-2H-chromen-3-yl)thiazol-2-yl)-3-arylacrylonitrile compounds, Kavitha et al. observed a strong, sharp adsorption band extending between 2156 cm-1 and 2336 cm-1 that corresponded to the CºN group (35). 1H-NMR Spectra Due to the presence of the vinyl group (CH2=CH-) and nitrile group, acrylonitrile displays a distinctive NMR spectrum. The nature of the substituents and their positioning with respect to the vinyl proton determine the 1H-NMR spectrum characteristics of the olefinic proton present on the 2,3-substituted acrylonitrile moiety. The 1H-NMR spectra of 2-(4-(2-oxo-2H-chromen-3-yl)thiazol-2-yl)-3- arylacrylonitrile compounds dissolved in DMSO-d6 with tetramethylsilane (TMS) as an internal standard using a 400 MHz spectrometer revealed that the singlet in the downfield regions around 8.17-8.55 ppm corresponding to vinylic proton were the primary evidence for the structures of the prepared compounds (35). Similarly, the olefinic proton found in 3-(naphthalen-1-yl)-2-(4-(naphthalen-1- yl(phenyl)amino)-phenyl)acrylonitrile also exhibited a singlet at 8.10 ppm (115). 30 In another study, the presence of a singlet at 7.88 ppm in the 1H-NMR spectrum of (2'-hydroxy)-4-((naphthalen-1-yl)methyleneamino)phenyl)-3-(1-methyl-1H-imidazol- 2-yl)acrylonitrile corresponded to the olefinic proton (17). Under similar conditions, the target compounds created by Sirim et al., one of them being 2-(1H-Benzo[d]imidazol-2-yl)-3-(4-(4-phenylpiperazin-1-yl)phenyl)acrylonitrile, showed a proton (C3-H) on the acrylonitrile moiety as a singlet at about 8.15 ppm (34). By using NMR techniques, it is challenging to determine the configuration of the double bond in acrylonitrile due to the steric and electrical repulsions between the aromatic and heteroaromatic groups (116). Nevertheless, research has revealed that the Z isomer is less stable for these molecules than the E isomer (117). 13C-NMR Spectra The 13C-NMR spectrum of an alpha, beta-substituted acrylonitrile moiety typically exhibits 3 major signals in the range. The chemical shifts of the carbons are influenced by the nature of the substituents and the electronic environment of the carbon atoms. Benzimidazole-acrylonitrile hybrid derivatives synthesized by Sirim et al exhibited very distinctive peaks around 152 ppm, corresponding to the C3 of the acrylonitrile moiety. Peaks of the C2 of acrylonitrile appeared around 95 ppm. While the carbon atom of the nitrile group was not very distinctive, it is expected to have appeared in the aromatic region between 110-129 ppm (34). In a similar trend, a series of (E)-2-(benzo[d]thiazol-2-yl)-3-arylacrylonitriles synthesized by Trilleras et al. were distinguished by chemical shifts ranging from 143.2 to 146.9 ppm for C3 and 102.2 to 109.5 ppm for C2 as a result of the highly polarized carbon-carbon double bond (C2-C3). The peak of the CN showed up around 115.6 to 117.3 as expected. The solvent of choice was deuterochloroform and a spectrometer with a frequency of 100 MHz was used (118). 31 2.1.4 Biological Activities of Acrylonitrile Derivatives Anticancer Activity Resveratrol, a natural polyphenolic compound, has been studied for its potential anticancer properties (119, 120). In vitro and in vivo studies have shown its chemo- preventive and chemotherapeutic effects, leading to the synthesis and evaluation of its analogs for potential anticancer properties (121-123). A recent study focused on the synthesis of (Z)-benzothiophene acrylonitrile resveratrol derivatives, which exhibited cytotoxic activity in human cancer cell lines with GI50 values between 10-100 nM, possibly through their interaction with tubulin. This makes them a potential treatment target for advanced prostate cancer. Moreover, these compounds were found to reduce the growth of cells resistant to P-glycoprotein (23) (Figure 2.37 A). This study synthesized and evaluated a series of novel compounds related to resveratrol for their anticancer activity against human cancer cell lines, including acute myeloid leukemia (AML) cells. The compounds included diarylacrylonitrile and trans- stilbene analogues with a cyano group and various aromatic substituents to enhance their activity. Molecular docking studies identified two highly effective compounds that shared a binding site on the α,β-tubulin heterodimer, with one having greater binding affinity than the other. Microtubule depolymerization assays confirmed that the more potent compound was better at inhibiting tubulin polymerization in AML cells. Analysis of the binding cavity residues showed minor differences in van der Waals contacts between the compounds and colchicine. The most potent trans-stilbene analogues had better tubulin binding properties than their corresponding cis-stilbene analogues (24) (Figure 2.37 B).Özen et al. synthesized phenylacrylonitrile derivatives and evaluated their potential as anticancer agents against MCF-7 cell lines. The researchers found that compounds with meta-substituted phenyl rings containing methyl, trifluoromethyl, and chloro groups and para-substituted methyl and nitro groups demonstrated the most potent anti-cancer effects at a concentration of 100 µM. Lower doses of the compounds also showed a dose- dependent decrease in cell viability. In addition, the study revealed that meta-substituted 32 compounds were more effective in inhibiting cell viability compared to para-substituted compounds (25) (Figure 2.37 C). Figure 2.37. Some anti-cancer acrylonitrile derivatives in literature Antimicrobial Activity The study conducted by AlNeyadi et al. focused on synthesizing and testing a new series of benzazole acrylonitrile-based compounds for their antimicrobial properties against bacterial strains, including those that are typically resistant to antibiotics. The results showed that one compound, (E)-2-(benzo[d]thiazol-2′-yl)-3-(2″,4″- diaminopyrimidin-5″-yl)acrylonitrile, exhibited strong antibacterial activity against all bacterial strains tested, with a minimum inhibitory concentration (MIC) value of 1.0 g/ml. Docking studies revealed that the compound's mechanism of action involved irreversible inhibition of the penicillin binding protein (PBP) enzyme, which is necessary for bacterial cell wall synthesis. Additionally, the compound was found to enhance the antibacterial activity of amoxicillin, a commonly used penicillin antibiotic, which could potentially help overcome bacterial resistance (11) (Figure 2.38 A). The antibacterial activity of 2,6-disubstituted heteroaryl acrylonitrile was examined on three different bacteria cultures, namely Enterococcus hirae, Staphylococcus aureus, and Staphylococcus epidermidis, in a study looking at the structure-activity relationships of novel heteroaryl-acrylonitriles as cytotoxic and antibacterial agents. S. epidermidis was the most sensitive, while all of the chemicals tested had almost no effect A B N O O OS anti-tumor against the OVCAR8 and NCI/ADR-RES cell lines (23) NC O O O HO O anti-tumor activity against the acute myeloid leukemia (AML) cell line, MV4-11 (24) O O O NC anti-tumor activity against MCF-7 cell line (25) C 33 on E. hirae. The best activity in the two Staphylococcus strains was demonstrated by compound 2-(benzimidazol-2-yl)-3-(5-nitrothiophen-2-yl) acrylonitrile and its imidazo[4,5-b]pyridine analogue (124) (Figure 2.38 B). Perin et al. investigated the antibacterial effects of novel benzazole-derived acrylonitriles with bicyclic heteroaromatic rings on a panel of Gram-positive and Gram- negative bacterial strains. Per collected data, only one acrylonitrile, (E)-2-(1H- benzo[d]imidazol-2-yl)-3-(benzofuran-2-yl) acrylonitrile, showed moderate efficacy against S. aureus ATCC 29213 (MIC 16 µM) and S. pneumoniae ATCC 49619 (MIC 32 µM) bacterial strains (14) (Figure 2.38 C). Figure 2.38. Some antibacterial acrylonitrile derivatives in literature Antiviral Activity The study focused on the benzimidazole acrylonitrile derivatives that target the Vif-A3G interaction and can be used for the treatment of HIV-1. The analysis showed that N,N-alkyl substitution or p-hydroxyl coupled with m-methoxy substitution of ring A, benzimidazole substructures comprising rings B and C, and an alkene linker between rings A and B were necessary for the anti-HIV-1 activity of the compounds. The compound 2- (1H-benzimidazol-2-yl)-3-(4-diethylaminophenyl)acrylonitrile demonstrated the highest potency, with IC50 values of 3.45 nM in the anti-HIV-1 replication assay in H9 cells. This compound also showed acceptable acute toxicity, suggesting that it has potential as a A BN N N H2N NH2 S N MIC= 1.0 μg/ml, against E. coli and P. aeruginosa (11) N S N+ O -O N H N MIC= 0.95 μg/ml against S. epidermidis (124) N O H N N MIC = 16 μg/ml, against S. aureus (14) C 34 starting point for developing more potent inhibitory compounds for the treatment of HIV- 1 (125) (Figure 2.39). Forero et al. conducted a bio-guided screening of Euphorbiaceae species against influenza A virus (FLUAV) to investigate the usefulness of bioactive compounds present in medicinal herbs in the treatment or prophylactic treatment of influenza. It was discovered that Codiaeum variegatum had significant anti-FLUAV activity. They isolated a cyanoglucoside with the chemical formula 2-(3,4,5)-trihydroxy-6- hydroxymethyltetrahydropyran-2-yloxymethyl)acrylonitrile from C. variegatum, which has been reported to have antiviral activity in previous studies (126) (Figure 2.39). Figure 2.39. Some antiviral acrylonitrile derivatives in literature Antifungal Activity In recent years, the use of the acrylonitrile chain in the development of new nitrile- containing drugs has become increasingly popular due to its attractive properties. The imidazo[1,2-a]pyridine hybrid molecule, which has shown excellent antifungal activity when connected to functional groups or other rings, has also recently been incorporated into the structure of new topical antifungal azoles such as luliconazole and lanoconazole (127, 128). N’Guessan et. al. developed new anti-Candida imidazo[1,2- a]pyridinylarylacrylonitriles, and found that the addition of a chlorine atom at position 3 of imidazo[1,2-a]pyridine improved the antifungal activity against three strains of Candida (C. albicans, C. glabrata, and C. tropicalis). (Z)-3-(3-chloroimidazo[1,2-a] pyridin-2-yl)-2-phenylacrylonitrile was found to have the best antimycotic profile with N N N HN IC50 = 3.45 nM against H9 cells (125) O HO HO O NC OH OH H IC50 = 17 ± 5 μg/mL against FLUAV (126) A B 35 MICs ranging from 1.4 to 357.5 µM. However, the displacement of the nitrile from one position to another along the phenylacrylonitrile chain did not improve antifungal activity. Standards used in this experiment were fluconazole and ketoconazole with MIC values ranging from 0.64-326.5 µM and 23.52-188.17 µM respectively (129) (Figure 2.40). Figure 2.40. Antifungal acrylonitrile derivative Antiparasitic Activity Bethencourt-Estrella et al. conducted a study in which they examined a variety of acrylonitriles for their in vitro activity against Leishmania amazonensis, a parasitic protozoa that causes Leishmaniasis. The results indicated that the acrylonitriles tested were more selective than miltefosine, the standard medication, with a range of activity between 0.57±0.10 and 363.38±20.24 µM against L. amazonensis promastigotes (29) (Figure 2.41 A). Sharma et al. synthesized and evaluated benzimidazole acrylonitrile derivatives as potential antimalarial agents with a dual receptor mechanism to overcome drug resistance. The compounds showed significant antimalarial activity, inhibited the production of hemozoin and falcipain-2 enzymes, and had acceptable cytotoxicity limits. These enzymes are required for the growth of Plasmodium falciparum, the parasite that causes malaria (16) (Figure 2.41 B). Bethencourt-Estrella et al. conducted a study to evaluate the anti-Trypanosoma cruzi activity of newly synthesized acrylonitriles. The Chagas disease is caused by the protozoan parasite Trypanosoma cruzi. The acrylonitrile derivatives investigated N N N Cl MIC= 1.4 μg/ml against C. tropicalis 36 displayed trypanocidal activity against T. cruzi, while exhibiting only moderate cytotoxicity toward murine macrophages. Further investigations revealed that the synthesized acrylonitriles may induce programmed cell death in T. cruzi, as evidenced by changes in mitochondrial membrane potential, ATP levels, reactive oxygen species accumulation, and chromatin condensation in Trypanosoma cruzi epimastigotes 24 hours after treatment (28) (Figure 2.41 C). Figure 2.41. Some antiparasitic acrylonitrile derivatives in literature AChE Inhibitory Activity Acrylonitriles have also been the subject of several studies investigating their potential as inhibitors of acetylcholinesterase (AChE) (32, 130, 131). de la Torre et al. designed and tested a series of compounds called (E)-2- (benzo[d]thiazol-2-yl)-3-heteroarylacrylonitrile derivatives as inhibitors of AChE. They found that the presence of a [5-(4-chlorophenyl)furan-2-yl] substituent at position 3 of the acrylonitrile scaffold led to the most potent inhibitor (E)-2-(Benzo[d]thiazol-2-yl)-3-[5- (4-chlorophenyl)furan-2-yl]acrylonitrile (IC50 = 64 µM). Molecular modeling analysis revealed that hydrogen bond between the nitrogen atom of the acrylonitrile moiety and the backbone nitrogen of Phe288 to at the active site of AChE (130) (Figure 2.42 A). In another study, Parveen et al synthesized a series of (Z)-acrylonitrile analogues to evaluate their AChE inhibitory potential. The study revealed that all of the compounds synthesized exhibited significant AChE inhibitory activity. Compound (Z)-3-(3′,4′,5′- A B C IC50 = 10.61 ± 2.48 µM against L. amazonensis (29) N ON OO IC50 = 0.69 µM against P. falciparum (29) N O H N N N O IC50 = 5.53 ± 1.36 µM against T. cruzi (28) 37 Trimethoxyphenyl)-2-(4′′-nitrophenyl)-acrylonitrile, which contained a 3,4,5-trimethoxy- substituted benzene moiety (ring A), displayed the strongest AChE inhibition with an IC50 value of 0.20 µM. The research findings indicate that the (Z)-isomer is more stable than the (E)-isomer, as confirmed by the crystallization method used to assess their relative stability (32) (Figure 2.42 B) Figure 2.42. Acrylonitriles derivatives with AChE inhibiting activity in literature Although recent literature has identified the heteroaryl-acrylonitrile scaffold as a promising framework for the development of novel AChE inhibitors (AChEIs) (32, 130), here is no established theory on the structure-activity relationship of E/Z-heteroaryl- acrylonitrile derivatives as AChEIs. To address this gap, de-la-Torre et al. used a comparative similarity indices analysis (CoMSIA) to conduct a three-dimensional quantitative structure-activity relationship (3D-QSAR) analysis on both literature- reported and newly synthesized heteroaryl-acrylonitrile derivatives. The results of this analysis revealed that the electrostatic characteristics of the substituents at positions 2 and 3 on the acrylonitrile scaffold, as well as the presence of hydrogen-bond donor groups, were critical determinants of the inhibitory activity of these compounds. Additionally, the CoMSIA results demonstrated that the electrostatic and hydrogen-bond donor fields were most strongly associated with AChE inhibitory activity. These findings provide a more solid theoretical foundation for predicting the affinities of heteroaryl-acrylonitriles, and offer insight into the structural design, creation, and synthesis of novel and highly selective AChEIs in the future(131). N O Cl S N AChE inh IC50 = 64 µM (130) AChE inh IC50 = 0.20 µM (32) N N+ O O- O O O A B 38 Antioxidant Activity Several studies have examined the potential antioxidant activity of compounds containing an acrylonitrile moiety (31, 132-134). In this regard, (E)-3-(benzofuran-2-yl)- 2-(thiophen-2-yl)acrylonitrile (TACNBNF) was found to exhibit noteworthy antioxidant activity (63.47%) in an in vitro antioxidant research. The study evaluated the scavenging capacity of the compound for free radical production using the DPPH assay, with D- ascorbic acid (Vitamin C) as the control antioxidant agent. At a concentration of 500mg/ml, the compound exhibited strong antioxidant activity (63.47%), while D- ascorbic acid (Vitamin C) showed 94.89% (133) (Figure 2.43). Figure 2.43. Molecular structure of TACNBNF Anti-inflammatory Activity In a reported study, newly synthesized acrylonitrile derivatives such as 3-(4- hydroxyphenyl)-2-(6,7,8,9-tetrahydro-5H-[1,2,4]triazolo[4,3-a]azepin-3-yl)acrylonitrile, exhibited significant analgesic and anti-inflammatory activity in mice with carrageenan- induced paw edema and acetic acid-induced writhing’s compared to ketorolac, a nonsteroidal anti-inflammatory drug (NSAID) used to treat pain (30) (Figure 2.44 A). A similar study conducted by Sivaramakarthikeyan et al. investigated the potential anti-inflammatory effects of benzimidazole-tethered pyrazoles using protein denaturation as the evaluation method. The study found that all molecules tested exhibited significant anti-inflammatory activity, with the compound containing para-nitrophenyl moiety on the pyrazole ring (E)-2-(1H-benzo[d]imidazol-2-yl)-3-(3-(4-nitrophenyl)-1-phenyl-1H- pyrazol-4-yl)acrylonitrile, showing the highest activity (95% inhibition) compared to N S O antioxidant DPPH scavenging% = 63.47% 39 other evaluated molecules. Moreover, its activity was superior to that of diclofenac sodium, which is a commonly used anti-inflammatory drug and exhibited a 90% inhibition of protein denaturation (31) (Figure 2.44 B). Figure 2.44. Acrylonitrile derivatives with anti-inflammatory activities Antimycobacterial Activity Acrylonitriles have been extensively studied as potential antituberculosis drugs. Although some promising findings have been reported, the use of acrylonitriles as antituberculosis drugs is still in the early stages of development and requires further investigation. Sanna et al. synthesized and evaluated the antitubercular efficacy of 22 acrylonitrile derivatives, finding that several compounds demonstrated potent activity against Mtb. The researchers observed that the position and nature of substituents on the benzotriazole ring significantly affected the activity of the compounds. Further investigation revealed that one of the most active derivatives, (E)-2-(2H- benzo[d][1,2,3]triazol-2-yl)-3-(4-bromophenyl)acrylonitrile had an MIC of 6.25 g/ml against M. tuberculosis H37Rv and M. avium. These findings suggest that acrylonitrile derivatives could be promising leads for developing new antituberculosis drugs, but A B N N N N OH writhing inhibition percentage = 57.35% (30) N N CN O2N NH N Anti-inflammatory Activity = 93.53 ± 1.37% (31) 40 additional research is necessary to optimize their therapeutic potential (36) (Figure 2.45 A) Reshma et al. have developed a series of twenty-two compounds and tested their effectiveness against both replicative and non-replicating bacterial stages. (E)-4-(5-(2- (Benzo[d]thiazol-2-yl)-2-cyanovinyl)thiophen-2-yl)isophthalicacid was reported to be an exceptionally potent compound that is effective against latent tuberculosis, with a Lysine- aminotransferase (LAT) IC50 of 2.62 µM and a considerable log reduction of 2.9 and 2.3 log fold against nutrient-starved and biofilm-forming mycobacteria, respectively (15) (Figure 2.45 B). Through genetic expression profiling and highlights, Lysine- aminotransferase (LAT) has been identified as a potential therapeutic target for the treatment of latent tuberculosis (15). Sirim et al. synthesized and evaluated several benzimidazole-acrylonitrile hybrid derivatives for their antimycobacterial activity against Mtb H37Rv in vitro. Among the compounds tested, 2-(1H-Benzo[d]imidazol-2-yl)-3-(4-(4-(4-methylphenyl)piperazin-1- yl)phenyl)acrylonitrile exhibited the highest efficacy with a MIC of 0.78 µg/ml against Mtb. Furthermore, this compound was found to be more potent than first-line drugs including isoniazid, ciprofloxacin, rifampicin, and moxifloxacin, demonstrating a 2.8 log fold reduction in bacterial count of dormant forms of Mycobacterium (34) (Figure 2.45 C). Nadaf et al. investigated the antimycobacterial activity of (2Z)-2-((E)-4- (benzylideneamino)phenyl)-3-(1-methyl-1H-imidazol-2-yl)acrylonitrile derivatives against Mtb H37Rv. The compounds containing hydroxy benzaldehyde and salicyaldehyde substituents demonstrated the highest antimycobacterial activity, with a MIC of the three most potent compounds between 0.2 and 0.4 g/ml, while the compound lacking a hydroxyl group showed the lowest activity (17) (Figure 2.45 D). 41 Figure 2.45. Antimycobacterial acrylonitrile derivatives in literature 2.2 Thiazole Derivatives Thiazole, a heterocyclic compound containing sulfur and nitrogen heteroatoms, was discovered by Hantzsch and Weber in 1887 (135). It is a vital and versatile component of numerous natural products and biologically active heterocyclic compounds. Various functionally diverse thiazole analogs are considered key azole frameworks found in many natural products (35). The thiazole functional group is frequently incorporated as a fundamental component in the structure of therapeutic agents such as ritonavir, penicillin- G, tiazofurin, abafungin, sulfathiazole, sulfazole, bleomycin, pramipexole, febuxostat, and vitamin-B, among others (136). O OH O HO S N S N LAT IC50 = 2.62 µM against latent M. tuberculosis (15) MIC=6.25 µg/mL against M. Tuberculosis and MIC=12.5 µg/mL against M. avium (36) N Br N N N N N N NH N MIC=0.78 µg/mL against M. Tuberculosis (34) N N N N OH O MIC = 0.2 μg/mL against M. Tuberculosis (17) A B C D 42 Figure 2.46. Chemical structure or thiazole 2.2.1 Synthesis of Thiazoles Derivatives Various pathways have been explored in the development of the thiazole and its derivatives. The synthesis of thiazoles was initiated by Hofmann and Hantzsch employing two principal synthetic routes: addition reaction and cyclization reaction utilizing diverse catalysts and techniques (137). Hantzsch-Thiazole Synthesis The process involves addition of a-halogeno ketones and N-monosubstituted thioureas in a neutral solvent, yielding only 2-(N-substitutedamino)thiazoles. This method provides a simple, quick, and environmentally friendly method for the solvent-free synthesis of 2-aminothiazoles (138, 139) (Figure 2.47). The study by Özkay et al. involved synthesizing hydrazone derivatives of thiazole to assess their anticholinesterase activities. The synthetic approach involved reacting pyrrole-2-carboxaldehydes with thiosemicarbazide in ethanol and then condensing the resulting thiosemicarbazones with α-bromoacetophenone derivatives using the Hantzsch reaction, ultimately yielding 1-substituted pyrrole-2-carboxaldehyde (4-(4-substituted phenyl)-1,3-thiazol-2-yl) hydrazones (140) (Figure 2.47). S N 1 2 3 4 5 43 Figure 2.47. Synthesis of thiazole using Hantzsch-Thiazole synthesis. Cook-Heilbron Thiazole Synthesis Cook-Heilbron synthesis, which was discovered by Cook and Heilbron, can also be used to synthesize the thiazole ring by using -aminonitriles or -aminoamides and carbon disulfide as reactants. Under mild conditions, aminonitrile is reacted with salt and esters of thioacids, carbon disulfide, or isothiocyanates to produce 5-aminothiazoles, with substitution occurring at position 2 via the reaction of aminonitrile with salt and esters of thioacids, carbon disulfide, or isothiocyanates (141, 142). Figure 2.48 A depicts an example of the Cook-Heilbron reaction involving aminonitriles with carbon disulfide to form 5-amino-2-mercapto-thiazoles (141). Castagnolo et al. described a domino alkylation-cyclization reaction for synthesizing 2-aminothiazoles from a variety of substituted propargyl bromides and thiourea derivatives as starting materials. The reaction was carried out in the presence of a stoichiometric amount of potassium carbonate and DMF as a solvent, and was microwave irradiated at a temperature of 130°C for 10 minutes, consisting of two 5-minute cycles (143) (Figure 2.48 B) NH2 S R1 O R3 R2 X reflux N SR1 R2 R3 X = Cl, Br, I, OMs, OTf R1 = NH-alkyl, NH-aryl, CH2CN, NHN=CH-aryl R2 = H, alkyl R3 = alkyl, aryl 44 Figure 2.48. Synthesis of thiazole using Cook-Heilbron synthesis Gabriel synthesis The Gabriel synthesis is an alternative synthetic technique for synthesizing thiazole derivatives that involves the closure of the thiazole ring via a reaction between acylamino-ketone and phosphorus pentasulfide, resulting in 2,5-disubstituted thiazole derivatives (144). In a study by Kotadiya, phosphorus pentasulfide was heated with N-(2- oxopropyl)acetamide to produce the desired compound, 2,5-dimethylthiazole (145) (Figure 2.49). Figure 2.49. Synthesis of thiazole using Gabriel synthesis 2.2.2 Biological activities of Thiazole Derivatives. Antimicrobial Activity Despite significant advances in science and the availability of various antibacterial and antifungal drugs, infectious diseases continue to pose a challenge to healthcare systems worldwide due to the emergence of bacterial resistance to existing drugs. Thiazole NH2 R N S C S N S R SHH2N-H2S Br R1 S H2N N H R2 N S R1 N H R2 A B K2CO3 DMF R1 = H, alkyl, aryl R2 = H, alkyl R1 = H, alkyl, aryl O O NH S P S S P S S N S 45 derivatives have been discovered to have antimicrobial activity against an array of microorganisms (146-148). A series of new thiazole derivatives were recently synthesized and examined for antimicrobial activity against several bacterial strains, including Bacillus subtilis, Staphylococcus aureus, and Escherichia coli, as well as the fungus Candida albicans. These derivatives demonstrated broad-spectrum antimicrobial activity with moderate antifungal activity, making them a promising candidate for further development as antimicrobial agents (146) (Figure 2.50 A). In a study, a group of researchers synthesized and characterized a series of Schiff bases that incorporate both 2,4-disubstituted thiazole and cyclobutane rings, as well as hydrazone moieties within the same molecule. These Schiff bases were evaluated for their antibacterial and antifungal activities against various microorganisms, and the results showed a MIC value of 16 μg/ml against C. tropicalis and Bacillus subtilis, indicating their potential as antimicrobial agents (149) (Figure 2.50 B). Bikobo et al. also conducted an antimicrobial activity screening on novel thiazole derivatives against gram-positive and gram-negative bacteria as well as fungi. The results indicated that these derivatives exhibit antibacterial and antifungal properties and demonstrated superior inhibitory activity against S. aureus when compared to the reference drug spectinomycin (150) (Figure 2.50 C). Figure 2.50. Antimicrobial thiazole derivatives in literature O S N N N N N F Br MIC = 7.8 µg/ml against C. albicans (146) MIC = 16 μg/ml against B. subtilis (149) HO OH N NH S N MIC = 31.25 μg/mL against Gram-positive bacterial strains (150) O NH2 S N S N NH OH A B C 46 Anticancer Activity According to published studies, thiazole-based compounds can suppress the progression and proliferation of cancer cells through multiple mechanisms, thereby targeting multiple therapeutic pathways simultaneously. As a result, it is possible to create a diverse range of molecules based on the thiazole-based scaffold with various functions (151-153). A recent study documents the inhibit Bcl-2 jurkat and A-431 cells by a range of novel thaizole-based compounds. A recent study found that a variety of novel thaizole- based compounds inhibit Bcl-2 jurkat and A-431 cells. At concentrations ranging from 32 to 52 µM, some of the molecules were found to be effective against cell lines while sparing normal cells. Apoptosis in cancer cells may be brought on by the compounds' interaction with the Bcl-2 proteins, according to in silico studies that confirmed this interaction (152) (Figure 2.51 A). Abdel-Maksoud et al. conducted a study to evaluate the effectiveness of imidazo[2,1-b]thiazole derivatives in inhibiting the growth of cancer cells, specifically melanoma and colon cancer cells. The researchers synthesized and tested a new series of these derivatives and found that they exhibited strong inhibitory effects against two melanoma cell lines and two colon cancer cell lines. Additionally, the compounds displayed significant enzyme activity against WTBRAF, V600EBRAF, and CRAF mutation, and showed stronger molecular interactions with the BRAF active site compared to the native ligand dabrafenib (153). V600EBRAF mutation, is the most common mutation in BRAF and is present in several cancer types, including thyroid cancer, melanoma, and colorectal cancer (154) (Figure 2.51 B). 47 Figure 2.51. Anticancer thiazole derivatives in literature Antiinflammatory Activity A new class of enzyme 5-lipoxygenase (5-LOX) inhibitors was designed using the pharmacophore modeling technique and tested for inhibitory potential against the 5-LOX enzyme in vitro. The results showed that these inhibitors had an IC50 value of 0.9±0.1 µM, which was higher than the IC50 value of the commercially available drug Zileuton (IC50 = 1.5±0.3 µM) (155) (Figure 2.52). Figure 2.52. Antiinflammatory thiazole derivative Antimycobacterial Activity Numerous studies on the synthesis and evaluation of thiazole derivatives for their ability to combat Mtb, the causative agent of tuberculosis, have been conducted (41, 156- 158). S N N N H N IC50 = 34.77 μM against Jurkat Bcl-2 IC50 = 34.31 μM against A-431 (152) IC50 = 9.3 nM against V600EBRAF (154) S O O H N H N N N SN N F Br A B O S N O N 5-LOX inhibitor IC50 = 0.9 µM 48 Novel 1,2,3-triazole derivatives with thiazole and pyrazole moieties were tested in vitro for antimycobacterial activity against both dormant and active strains of Mtb H37Ra. The screening results revealed that these derivatives had moderate to good antitubercular activity against Mtb H37Ra dormant and active strains (158) (Figure 2.53 A). In a study by E. Gürsoy et al., a series of novel derivatives of imidazo[2,1- b]thiazole, including acyl-hydrazone and spirothiazolidinone, synthesized and assessed for their antiviral and antimycobacterial activity against Mtb H37Rv strain showed positive results with MIC values equal to or less than 10 µg/ml (41) (Figure 2.53 B). A recent study also detailed the synthesis, characterization, and evaluation of various 5-methyl-4-thiazolidinone derivatives for their potential in vitro antimycobacterial activities against the Mtb H37Rv strain. Among the synthesized compounds, the thiazolidinone compound identified as 2-(4-ethoxyphenyl)-5-methyl-3- (phenylamino)thiazolidin-4-one exhibited notable antimycobacterial activity, with a MIC of 12.5 μg/ml against Mtb (159) (Figure 2.53 C). Figure 2.53. Antimycobacterial thiazole derivatives in literature 2.3 Tuberculosis, Vaccine and Treatment Tuberculosis (TB) remains a significant global public health issue, caused by the infectious agent Mtb. While mycobacteria have supposedly existed for over 150 million years, the first isolation of the bacillus was achieved by the famous German scientist Robert Koch in March 1882 as Tubercle bacillus and was named Mtb a year later (2). S NN N N N N %inhibition = 35.41 against M. tuberculosis H37Ra Dormant %inhibition = 58.35 against M. tuberculosis H37Ra active strains (158) MIC = 0.854 µg/ml against M. tuberculosis H37Rv (41) O HN S N OS NN Br S N OHN MIC = 12.5 µg/ml against M. tuberculosis H37Rv (159) A B C 49 TB has a global presence, yet its incidence is considerably higher in developing countries, primarily due to poverty, maln