Recent Advances in the Biological Activity of s-Triazine Core Compounds

An effective strategy for successful chemotherapy relies on creating compounds with high selectivity against cancer cells compared to normal cells and relatively low cytotoxicity. One such approach is the discovery of critical points in cancer cells, i.e., where specific enzymes that are potential therapeutic targets are generated. Triazine is a six-membered heterocyclic ring compound with three nitrogen replacing carbon-hydrogen units in the benzene ring structure. The subject of this review is the symmetrical 1,3,5-triazine, known as s-triazine. 1,3,5-triazine is one of the oldest heterocyclic compounds available. Because of its low cost and high availability, it has attracted researcher attention for novel synthesis. s-Triazine has a weak base, it has much weaker resonance energy than benzene, therefore, nucleophilic substitution is preferred to electrophilic substitution. Heterocyclic bearing a symmetrical s-triazine core represents an interesting class of compounds possessing a wide spectrum of biological properties such as anti-cancer, antiviral, fungicidal, insecticidal, bactericidal, herbicidal and antimicrobial, antimalarial agents. They also have applications as dyes, lubricants, and analytical reagents. Hence, the group of 1,3,5-triazine derivatives has developed over the years. Triazine is not only the core amongst them, but is also a factor increasing the kinetic potential of the entire derivatives. Modifying the structure and introducing new substituents makes it possible to obtain compounds with broad inhibitory activity on processes such as proliferation. In some cases, s-triazine derivatives induce cell apoptosis. In this review we will present currently investigated 1,3,5-triazine derivatives with anti-cancer activities, with particular emphasis on their inhibition of enzymes involved in the process of tumorigenesis.


Introduction
As far as we know, tumors are the most serious cause of death in the world. Cancers with the highest mortality rates in 2018 were lung cancer (2.1 million new cases and 1.8 million deaths), breast cancer (million new cases and 880 thousand deaths), prostate cancer (1.3 million new cases and 360 thousand deaths), and stomach cancer (1 million new cases and 783 thousand deaths) [1].
The fight against cancer has consumed huge amounts of money to find the cure with little effect. Nevertheless, it cannot be defined as a failure. As Napoleon Hill said, "every adversity, every failure, every heartache carries with it the seed of an equal or greater benefit". Following this thought, we would like to highlight two aspects of the fight against cancer. First, decades of research lead to more and more precise descriptions of the mechanisms taking place in cancer cells, it is possible to determine the most effective aim in targeted therapies. Second and equally important, the development of small molecules. The development of more active, selective and less cytotoxic drugs is due to designing chemical compounds based on a structure-activity relationship (SAR) [2]. In this search, the leading linker is 1,3,5-triazine, a symmetrical heterocyclic aromatic ring enabling the expansion of the structure in a multi-vector manner. Decades of research have revealed a

Topoisomerase Inhibitors
Topoisomerases are a group of enzymes involved in replication, they are responsible for the degree of twist of the double helix. Topoisomerases convert the chemical energy from ATP into the energy of the torsion tension of a molecule with a superhelical structure. In vivo, topoisomerases unravel the DNA double helix, thus providing a template for the replication or transcription of enzymes. Depending on the number of phosphodiester bonds to be broken at one time, there are two types of enzyme. Topoisomerase I hydrolyses one bond, cuts one strand and is responsible for removing superstrands from the DNA molecule (relaxation). Topoisomerase II hydrolyses two bonds, cuts both strands and is responsible for adding supercoils to the DNA molecule [39].
This review presents the current state of knowledge on 1,3,5-triazine derivatives, their structures and anticancer activity, as well as their ability to inhibit different enzymes or their DNA-binding potential. This data could be helpful in the development of new drugs and therapeutic methods. By analysing the presented approach, a series of compounds with high potency and low toxicity can be designed, synthesized, characterized and evaluated for desired pharmacological activity. The collected data are presented in summary Table 1.

Topoisomerase Inhibitors
Topoisomerases are a group of enzymes involved in replication, they are responsible for the degree of twist of the double helix. Topoisomerases convert the chemical energy from ATP into the energy of the torsion tension of a molecule with a superhelical structure. In vivo, topoisomerases unravel the DNA double helix, thus providing a template for the replication or transcription of enzymes. Depending on the number of phosphodiester bonds to be broken at one time, there are two types of enzyme. Topoisomerase I hydrolyses one bond, cuts one strand and is responsible for removing superstrands from the DNA molecule (relaxation). Topoisomerase II hydrolyses two bonds, cuts both strands and is responsible for adding supercoils to the DNA molecule [3].

Dual Phosphoinositide 3-Kinase and Mammalian Target of Rapamycin Inhibitors
The phosphoinositide 3-kinase (PI3K) enzymes show a two-way activity including the activity of the lipid kinase and the activity of the protein kinase. They play a crucial role in processes such as proliferation, migration, differentiation, survival, and trafficking. The PI3K family contains eight isoforms divided into three distinct classes (I, II, and III) which may be different in terms of cellular responsibility [5].
The function of the mammalian target of rapamycin (mTor) is to regulate growth, proliferation and cell traffic, and the processes of translation and transcription. The mTOR catalyzes the phosphorylation ribosomal protein S6 kinase β-1 (S6K1), eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1), Akt, protein kinase C (PKC),

Dual Phosphoinositide 3-Kinase and Mammalian Target of Rapamycin Inhibitors
The phosphoinositide 3-kinase (PI3K) enzymes show a two-way activity including the activity of the lipid kinase and the activity of the protein kinase. They play a crucial role in processes such as proliferation, migration, differentiation, survival, and trafficking. The PI3K family contains eight isoforms divided into three distinct classes (I, II, and III) which may be different in terms of cellular responsibility [40].
The function of the mammalian target of rapamycin (mTor) is to regulate growth, proliferation and cell traffic, and the processes of translation and transcription. The mTOR catalyzes the phosphorylation ribosomal protein S6 kinase β-1 (S6K1), eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1), Akt, protein kinase C (PKC), and type-I insulin-like growth factor receptor (IGF-IR), thereby regulating protein synthesis, nutrient metabolism, growth factor signaling, cell growth, and migration [41].
The construction of compounds with dual inhibitory effects contributes to obtaining a more selective effect. Potential anti-cancer drugs that inhibit PI3K and mTor at the same time showed greater efficiency and reduced the likelihood of inducing drug resistance [42].

Dihydrofolate Reductase Inhibitors
Dihydrofolate reductase (DHFR) is an enzyme responsible for reducing dihydrofolic acid to tetrahydrofolic acid by catalyzing the transfer of hydride from NADPH, generating the oxidized form of NADP + [10]. Inhibiting DHRF induces an amount reduction of tetrahydrofolate (THF), consequently decreasing the synthesis of purines, amino acids, and thymidylate, which are crucial in cell growth and proliferation [11].
Singa et al. demonstrated synthesized triazine-benzimidazole analogs 4-7 ( Figure 3) appointed with a hydrogen bond interaction domain, a polar hydrophilic substituent and an intercalating group. The median growth inhibitory (GI50) values for these compounds were measured relative to leukemia, non-small cell lung cancer, colon cancer, central nervous system (CNS) tumor, melanoma, ovarian cancer, renal cancer, prostate cancer, and breast cancer cells with values in the range of 1.91-2.72 µM. The 50% inhibitory concentration value of DHRF activity was lowest for derivative 7 and was 0.002 µM, which was equivalent to methotrexate (MTX) (IC50 = 0.02 µM) [12,13].

Dihydrofolate Reductase Inhibitors
Dihydrofolate reductase (DHFR) is an enzyme responsible for reducing dihydrofolic acid to tetrahydrofolic acid by catalyzing the transfer of hydride from NADPH, generating the oxidized form of NADP + [43]. Inhibiting DHRF induces an amount reduction of tetrahydrofolate (THF), consequently decreasing the synthesis of purines, amino acids, and thymidylate, which are crucial in cell growth and proliferation [44].
Singa et al. demonstrated synthesized triazine-benzimidazole analogs 4-7 ( Figure 3) appointed with a hydrogen bond interaction domain, a polar hydrophilic substituent and an intercalating group. The median growth inhibitory (GI 50 ) values for these compounds were measured relative to leukemia, non-small cell lung cancer, colon cancer, central nervous system (CNS) tumor, melanoma, ovarian cancer, renal cancer, prostate cancer, and breast cancer cells with values in the range of 1.91-2.72 µM. The 50% inhibitory concentration value of DHRF activity was lowest for derivative 7 and was 0.002 µM, which was equivalent to methotrexate (MTX) (IC50 = 0.02 µM) [6,45]. Zhou

Carbonic Anhydrase Inhibitors
Carbonic anhydrases (CAs), metalloenzymes from the lyase group, are responsible for pH homeostasis and catalyzing the reversible reaction of the formation of the bicarbonate ion HCO from water and carbon dioxide [16].
Among the numerous isoforms we can distinguish the ubiquitous variants CA I and CA II in mammals. In a pathological condition such as hypoxia, increased expression of CA IX and CA XII is observed. These enzyme forms are involved in the regulation of pH homeostasis and intercellular communication and ion transport.

Carbonic Anhydrase Inhibitors
Carbonic anhydrases (CAs), metalloenzymes from the lyase group, are responsible for pH homeostasis and catalyzing the reversible reaction of the formation of the bicarbonate ion HCO − 3 from water and carbon dioxide [46]. Among the numerous isoforms we can distinguish the ubiquitous variants CA I and CA II in mammals. In a pathological condition such as hypoxia, increased expression of CA IX and CA XII is observed. These enzyme forms are involved in the regulation of pH homeostasis and intercellular communication and ion transport. Based on the structure of SCL-0111, new 1,3,5-triazine derivatives 17 and 18 were synthesized ( Figure 4) and their ability to inhibit CA I, II, IX, and XII was investigated. The most promising result was the selective inhibition of CA IX by compound 17 with a KI value = 0.91 nM [11], while compound 18 had a KI value of 14.6 nM [12].

Epidermal Growth Factor Receptor Inhibitors
The role of the epidermal growth factor receptor (EGFR) in the pathogenesis proces is an important topic of scientific research. As a result, it was discovered that mutation leading to overexpression of EGFR genes (e.g., increased regulation or amplification) ar significantly associated with many cancers: lung granuloma (40% of cases), rectal tumors glioblastoma (50%), and epithelial carcinomas of the head and neck (80-100%) [21,22].
Through the "one pot" reaction, 15 novel monastrol-1,3,5-triazine derivatives wer obtained and investigated for anti-cancer properties and cytotoxicity. . This compound was nontoxic to normal epithelial cells MCF 12A while at a concentration of 10 nM the inhibition of EGFR-TK by 19 was equal 96.4% [23].

Epidermal Growth Factor Receptor Inhibitors
The role of the epidermal growth factor receptor (EGFR) in the pathogenesis process is an important topic of scientific research. As a result, it was discovered that mutations leading to overexpression of EGFR genes (e.g., increased regulation or amplification) are significantly associated with many cancers: lung granuloma (40% of cases), rectal tumors, glioblastoma (50%), and epithelial carcinomas of the head and neck (80-100%) [47,48].

Vascular Endothelial Growth Factor
Vascular endothelial growth factor (VEGF) production can be induced in a cell that does not receive enough oxygen [26]. When a cell is deficient in oxygen, it produces hypoxia induced factor (HIF). HIF stimulates the release of VEGF (including the modulation of erythropoiesis). Circulating VEGF then binds to VEGF receptors on endothelial cells, triggering a tyrosine kinase pathway leading to angiogenesis [27]. Expression of angiopoietin-2 in the absence of VEGF leads to endothelial cell death and vascular regression. VEGF acts as the central mediator of tumor angiogenesis, stimulating the growth of new blood vessels from nearby capillaries and allowing tumors to access the oxygen and nutrients they need to grow [28].

Vascular Endothelial Growth Factor
Vascular endothelial growth factor (VEGF) production can be induced in a cell that does not receive enough oxygen [49]. When a cell is deficient in oxygen, it produces hypoxia induced factor (HIF). HIF stimulates the release of VEGF (including the modulation of erythropoiesis). Circulating VEGF then binds to VEGF receptors on endothelial cells, triggering a tyrosine kinase pathway leading to angiogenesis [50]. Expression of angiopoietin-2 in the absence of VEGF leads to endothelial cell death and vascular regression. VEGF acts as the central mediator of tumor angiogenesis, stimulating the growth of new blood vessels from nearby capillaries and allowing tumors to access the oxygen and nutrients they need to grow [51]. Quinazoline

Vascular Endothelial Growth Factor
Vascular endothelial growth factor (VEGF) production can be induced in a cell that does not receive enough oxygen [26]. When a cell is deficient in oxygen, it produces hypoxia induced factor (HIF). HIF stimulates the release of VEGF (including the modulation of erythropoiesis). Circulating VEGF then binds to VEGF receptors on endothelial cells, triggering a tyrosine kinase pathway leading to angiogenesis [27]. Expression of angiopoietin-2 in the absence of VEGF leads to endothelial cell death and vascular regression. VEGF acts as the central mediator of tumor angiogenesis, stimulating the growth of new blood vessels from nearby capillaries and allowing tumors to access the oxygen and nutrients they need to grow [28].
Quinazoline-1,3,5-triazine derivatives 23, 24, and 25 ( Figure 6) demonstrated antitumor activity against HeLa, MCF-7, HL-60, and HepG2 with IC50 values in range of 6-16 µM. In addition, they were non-toxic against the normal cell line of HFF (human foreskin fibroblasts). Molecular docking results demonstrated the high potency of derivatives 23, 24 and 25 to bind the hydrophobic pocket of the N-terminal chain in the ATP binding site of VEGFR [29].

Focal Adhesion Kinase Inhibitors
Focal Adhesion Kinase (FAK) is a 125-kDa cytoplasmic tyrosine kinase. Deregulation of FAK-dependent processes such as cell adhesion, growth, survival, and mobility are a significant component of tumor progression. Overexpression of FAK leads to the inhibition of apoptosis and an increase in the incidence of metastatic tumors [30]. Dao

Focal Adhesion Kinase Inhibitors
Focal Adhesion Kinase (FAK) is a 125-kDa cytoplasmic tyrosine kinase. Deregulation of FAK-dependent processes such as cell adhesion, growth, survival, and mobility are a significant component of tumor progression. Overexpression of FAK leads to the inhibition of apoptosis and an increase in the incidence of metastatic tumors [53]. Dao

Primary Anticancer Studies
Compound 30 (Figure 9) obtained via the click chemistry method showed higher potency than doxorubicin. Derivative 30 exhibited an IC50 against MCF-7 and HepG2 cells of 2.95 µg/mL and 3.70 µg/mL, respectively, and showed no toxic activity against the growth of normal HFB4 cells [33].
Interesting results have emerged from the comparison of the antitumor properties of the two groups of 1,3,5-triazine derivatives. The groups differed only in one substituent, the first group contained chlorine and the second group contained morpholine. In the second case, a noticeable increase in cytotoxic activities was observed. According to cancer cell lines MCF-7, MDAMB-231, HT-29, HGC-27 the derivative 31 ( Figure 9) proved to be most potent with IC50 values of 4.8 µM, 8.3 µM, 9.8 µM, and 15.1 µM [34].

Primary Anticancer Studies
Compound 30 (Figure 9) obtained via the click chemistry method showed higher potency than doxorubicin. Derivative 30 exhibited an IC 50 against MCF-7 and HepG2 cells of 2.95 µg/mL and 3.70 µg/mL, respectively, and showed no toxic activity against the growth of normal HFB4 cells [18]. A novel series of triazine-benzimidazole analogs were synthesized and their antiproliferative activity against 60 human cancer cell lines was evaluated. Screening data revealed that triazine substituted with piperidine 35, phenyl 36, 4-fluorophenyl 37, and 4chlorophenyl 38 (Figure 9) presented the highest inhibiting potency [37].  (Figure 10) was designed and synthesized as a potential anticancer agent. An in vitro evaluation of its antiproliferative activity against A549 and MDA-MB231 confirmed the assumption. The test results were not good enough. On the other hand, modifications of the obtained structure may contribute to the improvement of anti-cancer properties [38].
The series of novel hybrid molecules formed from 2,4-diamino-1,3,5-triazine and 2iminocoumarin were tested toward the human pancreatic cancer cell line DAN-G, human A-427, human non-small cell lung cancer cell line LCLC-103H, human cervical cancer cell line SISO, and human urinary bladder cancer cell line RT-4. Compound 40 (Figure 10) presented the following values IC50: 2.14 µM, 1.51 µM, 2.21 µM, 2.60 µM, and 1.66 µM Interesting results have emerged from the comparison of the antitumor properties of the two groups of 1,3,5-triazine derivatives. The groups differed only in one substituent, the first group contained chlorine and the second group contained morpholine. In the second case, a noticeable increase in cytotoxic activities was observed. According to cancer cell lines MCF-7, MDAMB-231, HT-29, HGC-27 the derivative 31 ( Figure 9) proved to be most potent with IC 50 values of 4.8 µM, 8.3 µM, 9.8 µM, and 15.1 µM [19].
A novel series of triazine-benzimidazole analogs were synthesized and their antiproliferative activity against 60 human cancer cell lines was evaluated. Screening data revealed that triazine substituted with piperidine 35, phenyl 36, 4-fluorophenyl 37, and 4-chlorophenyl 38 (Figure 9) presented the highest inhibiting potency [22].
4-Phenethylthio-2-phenylpyrazolo[1,5-a] [1,3,5]triazin-7(6H)-one 39 ( Figure 10) was designed and synthesized as a potential anticancer agent. An in vitro evaluation of its antiproliferative activity against A549 and MDA-MB231 confirmed the assumption. The test results were not good enough. On the other hand, modifications of the obtained structure may contribute to the improvement of anti-cancer properties [23].  Figure 11). Compound 47 significantly reduced tumor tissue in several animal models and decreased PC-3 proliferation with an IC50 value of 20 µM. This analog also arrested PC-3 cells in stage G0/G1 [42].
Via three-components one spot condensation 110 new of 1,3,5-triazine derivatives were obtained. Antiproliferative activity of the most potent compounds 48-51 ( Figure 11) identified in the screening against DU145 prostate-cancer cells had GI 50 values of 3.43 µM, 4.01 µM, 2.38 µM and 0.67 µM, respectively [28]. Subsequent studies generated further derivatives that were tested for three breast tumors. Evaluation led to the determination that the most active structures are 52 and 53 ( Figure 11) and indicated that the group of derivatives were more active against triple negative breast cancer MDA-MB231 [29,30].
Derivatives based on quinazoline combined with a 1,3,5-triazine ring via urea bridge presented antitumor activity against TPC-1 cells (thyroid cancer), MCF-7. Corresponding to the normal cell line (human foreskin fibroblasts), compounds 54-56 ( Figure 11) were non-toxic. In addition, these structures showed the best IC 50 values against carcinoma cells, and demonstrated tyrosine kinase inhibitory potency [31].

Search Strategy and Selection Criteria
The aim of this study was to collect knowledge and data on the synthesized novel 1,3,5-triazine derivatives, their effects on cancer cells, and to identify enzymes as potential targets for these substances. To carry out the study, the following databases were searched: PubMed (NCBI), Web of Science, and Scopus, using the following key words: 1,3,5-triazine, s-triazine, anticancer, antitumor, and enzyme inhibitor. We examined original articles and case studies published between 2015 and 2021. The results of the study include the compounds from papers with the highest activity.

Conclusions
The "hybrid" approach incorporating a triazine framework ensures an improved profile against the target biological pathways pertaining to infectious parasites, microbes, and conditions such as cancer and neurodegeneration. The multi-targeting approach of the hybrid compounds ensures an effective overcoming of the key regulatory pathways contributing to complicacies such as drug resistance. This review presents a comprehensive discussion on the candidature of the 1,3,5-triazine scaffold for a rational development of the hybrid molecules by conjugation with bioactive pharmacophoric moieties. The basis of superior efficacy of 1,3,5-triazine based hybrid molecules by considering their interactions with the cellular targets has also been discussed in a succinct manner. The literature revealed that s-triazine derivatives possess diverse anticancer potential, easy synthetic routes for synthesis, and have attracted researchers for development of new chemotherapeutic agents. Extensive research is required on the 1,3,5-triazine moiety to find novel analogs suitable for clinical applications in cancer treatment.