Synthesis of Novel Triazine-Based Chalcones and 8,9-dihydro-7H-pyrimido[4,5-b][1,4]diazepines as Potential Leads in the Search of Anticancer, Antibacterial and Antifungal Agents

This study presents the synthesis of four series of novel hybrid chalcones (20,21)a–g and (23,24)a–g and six series of 1,3,5-triazine-based pyrimido[4,5-b][1,4]diazepines (28–33)a–g and the evaluation of their anticancer, antibacterial, antifungal, and cytotoxic properties. Chalcones 20b,d, 21a,b,d, 23a,d–g, 24a–g and the pyrimido[4,5-b][1,4]diazepines 29e,g, 30g, 31a,b,e–g, 33a,b,e–g exhibited outstanding anticancer activity against a panel of 60 cancer cell lines with GI50 values between 0.01 and 100 μM and LC50 values in the range of 4.09 μM to >100 μM, several of such derivatives showing higher activity than the standard drug 5-fluorouracil (5-FU). On the other hand, among the synthesized compounds, the best antibacterial properties against N. gonorrhoeae, S. aureus (ATCC 43300), and M. tuberculosis were exhibited by the pyrimido[4,5-b][1,4]diazepines (MICs: 0.25–62.5 µg/mL). The antifungal activity studies showed that triazinylamino-chalcone 29e and triazinyloxy-chalcone 31g were the most active compounds against T. rubrum and T. mentagrophytes and A. fumigatus, respectively (MICs = 62.5 μg/mL). Hemolytic activity studies and in silico toxicity analysis demonstrated that most of the compounds are safe.


Introduction
Cancer and infectious diseases caused by the drug resistance of bacteria and fungi are one of the main causes of death worldwide, and this requires highly selective and efficient treatments with low toxicity.Around 10 million people died from cancer in 2020 worldwide [1,2], and according to Pan American Health Organization (PAHO) it is estimated that this value would increase to 57% by 2040 [3].Likewise, infections by resistant bacteria cause around 700,000 deaths annually worldwide, of which 230,000 deaths are due to multi-resistant tuberculosis [4].
efficient treatments with low toxicity.Around 10 million people died from cancer in 2020 worldwide [1,2], and according to Pan American Health Organization (PAHO) it is estimated that this value would increase to 57% by 2040 [3].Likewise, infections by resistant bacteria cause around 700,000 deaths annually worldwide, of which 230,000 deaths are due to multi-resistant tuberculosis [4].
The union of pharmacophoric fragments to generate molecular hybrids has been an attractive and useful strategy in medicinal chemistry to generate lead molecules with potential biological properties [64][65][66][67].Stimulated by valuable bioactive properties of 1,3,5-triazine, chalcone, and diazepine derivatives, and based on molecular hybridization approach, in this study, we are reporting the synthesis of 1,3,5-triazine-based chalcone-and diazepine hybrids through a simple and versatile synthetic pathway.In vitro screening tests were used for determining the anticancer, antibacterial, antifungal, and cytotoxic profiles of the novel compounds synthesized, which showed outstanding results.

Chemistry
Initially, using a three-step synthetic sequence, the trisubstituted triazine precursors 12-15 were synthesized by aromatic nucleophilic substitution reactions (Ar N S) from 2,4,6trichloro-1,3,5-triazine 4 [50,[68][69][70][71][72][73][74][75] (Table 1).Looking for structural diversity, substituents of aliphatic and aromatic nature and functional groups capable of forming hydrogen bonding were incorporated.Various reaction parameters were explored for each compound and Table 1 shows the optimized reaction conditions that allowed these precursors to be obtained in high yields.To assure the monosubstitution of a chlorine atom to prepare the precursors 5-7, it was imperative to perform the reactions at low temperature (−5-0 • C).The second substitutions were carried out at room temperature, except for 11, since the trisubstituted product was favored; at low temperature it was obtained as the only product.Sodium carbonate (20%) was used as a hydrogen chloride acceptor.As the reactions progressed, the medium acidified to a point where they no longer progressed, therefore, the addition of the base was done slowly throughout the reaction and always maintaining a neutral pH.The trisubstituted derivatives were synthesized under heating at reflux (for 13) and at room temperature (for 12, 14, and 15).Initially, for the synthesis of triazines 12 and 13, dioxane and DMF were tested as solvents.In both tests, an excess of ethylenediamine (1.5 equiv.)and stirring at room temperature was used; however, under these reaction conditions complex mixtures of products were obtained.The use of ethylenediamine as a reaction medium allowed the obtaining of compounds 12 and 13 with good yields.and anticancer agents [54,[56][57][58][59][60][61].Their anticancer action mechanism involves the inhibition of Aurora A, Aurora B, and Kinase Insert Domain-containing Receptor (KDR) [60], receptor tyrosine kinases such as Flt3, and c-Kit [62], extracellular-signal-regulated kinase 5 (ERK5), and leucine rich repeat kinase 2 (LRRK2) [63].
The union of pharmacophoric fragments to generate molecular hybrids has been an attractive and useful strategy in medicinal chemistry to generate lead molecules with potential biological properties [64][65][66][67].Stimulated by valuable bioactive properties of 1,3,5triazine, chalcone, and diazepine derivatives, and based on molecular hybridization approach, in this study, we are reporting the synthesis of 1,3,5-triazine-based chalcone-and diazepine hybrids through a simple and versatile synthetic pathway.In vitro screening tests were used for determining the anticancer, antibacterial, antifungal, and cytotoxic profiles of the novel compounds synthesized, which showed outstanding results.

Chemistry
Initially, using a three-step synthetic sequence, the trisubstituted triazine precursors 12-15 were synthesized by aromatic nucleophilic substitution reactions (ArNS) from 2,4,6trichloro-1,3,5-triazine 4 [50,[68][69][70][71][72][73][74][75] (Table 1).Looking for structural diversity, substituents of aliphatic and aromatic nature and functional groups capable of forming hydrogen bonding were incorporated.Various reaction parameters were explored for each compound and Table 1 shows the optimized reaction conditions that allowed these precursors to be obtained in high yields.To assure the monosubstitution of a chlorine atom to prepare the precursors 5-7, it was imperative to perform the reactions at low temperature (−5-0 °C).The second substitutions were carried out at room temperature, except for 11, since the trisubstituted product was favored; at low temperature it was obtained as the only product.Sodium carbonate (20%) was used as a hydrogen chloride acceptor.As the reactions progressed, the medium acidified to a point where they no longer progressed, therefore, the addition of the base was done slowly throughout the reaction and always maintaining a neutral pH.The trisubstituted derivatives were synthesized under heating at reflux (for 13) and at room temperature (for 12, 14, and 15).Initially, for the synthesis of triazines 12 and 13, dioxane and DMF were tested as solvents.In both tests, an excess of ethylenediamine (1.5 equiv.)and stirring at room temperature was used; however, under these reaction conditions complex mixtures of products were obtained.The use of ethylenediamine as a reaction medium allowed the obtaining of compounds 12 and 13 with good yields.
The structures of precursors were confirmed by FTIR, 1 H, and 13 C NMR and mass spectra data (Supplementary Material).ethylenediamine (1.5 equiv.)and stirring at room temperature was used; however, under these reaction conditions complex mixtures of products were obtained.The use of ethylenediamine as a reaction medium allowed the obtaining of compounds 12 and 13 with good yields.
The structures of precursors were confirmed by FTIR, 1 H, and 13 C NMR and mass spectra data (Supplementary Material).The structures of precursors were confirmed by FTIR, 1 H, and 13 C NMR and mass spectra data (Supplementary Material).
Subsequently, trisubstituted precursors 12 and 13 were reacted with 4-acetylbenzenesulfonyl chloride 16 under stirring at room temperature in ethanol and using TEA as a base to generate sulfonamides 17 and 18, respectively (Scheme 1).Using carbonyl precursors 14,15 and 17,18 as starting materials, the triazinyloxy-chalcones (20,21)a-g and triazinylamino-chalcones (23,24)a-g were obtained by Claisen-Schmidt condensation reactions with acetophenones 19a-g and benzaldehydes 22a-g, respectively (Scheme 2).These chalcones were obtained in the range of 65% to 93% yield and their structures were elucidated by FTIR, 1 H, and 13 C NMR and mass spectrometry (Supplementary Material).To illustrate the main spectroscopic characteristics of these Using carbonyl precursors 14,15 and 17,18 as starting materials, the triazinyloxychalcones (20,21)a-g and triazinylamino-chalcones (23,24)a-g were obtained by Claisen-Schmidt condensation reactions with acetophenones 19a-g and benzaldehydes 22a-g, respectively (Scheme 2).These chalcones were obtained in the range of 65% to 93% yield and their structures were elucidated by FTIR, 1 H, and 13 C NMR and mass spectrometry (Supplementary Material).To illustrate the main spectroscopic characteristics of these compounds, chalcone 23f was taken as a reference.The mass spectrum confirms the formation of this compound by presenting the molecular ion peak at m/z 555, which corresponds to its expected mass.The 1 H NMR spectrum run at 400 MHz in CDCl 3 shows two doublets at 7.84 and 8.01 ppm (J = 8.4 Hz) corresponding to the protons of the parasubstituted 4-acylbenzenesulfonyl moiety.A triplet and a double doublet are observed at 7.12 (J = 8.6 Hz) and 7.64 ppm (J = 8.6, 5.4 Hz), respectively, corresponding to the protons of the para-F-substituted ring.Finally, at 7.39 and 7.78 ppm, two doublets are observed (J = 15.7 Hz) corresponding to the vinylic protons of the α,β-unsaturated moiety, confirming that the new double bond formed in product 23f possesses an E configuration.The final target products (i.e., triazinyloxy-and triazinylamino-pyrimido[4,5b] [1,4]diazepines) (28-31)a-g (Scheme 3) were obtained with high regioselectivity by reaction of chalcones (20,21)a-g and (23,24)a-g, respectively, with an excess of 2,4,5,6tetraaminopyrimidine dihydrochloride 27 (1,4-dinucleophile) under stirring in refluxing methanol and using BF3•OEt2 as catalyst.In the same way, diazepines (32,33)a-g were obtained starting from the chalcones (25,26)a-g synthesized elsewhere [50].Reaction yields ranged from 50% to 90% and all synthesized diazepines were characterized by FTIR, 1 H, 13 C NMR, and mass spectrometry (Supplementary Material).Particularly, the 1 H NMR spectrum (run in DMSO-d6) of product 32e shows the signals corresponding to the N-H (a singlet at 7.00 ppm) and the diastereotopic protons (AMX system) of the diazepinic ring formed.The signal assigned to the H7A proton appears as a doublet at 2.75 ppm with coupling constant 2 JAM = 14.2 Hz; the signal for the H7M proton appears as a doublet at 3.77 ppm with coupling constants 2 JAM = 14.2 Hz and 3 JMX = 6.0 Hz, while the signal corresponding to the H8X proton appears as an unresolved doublet at 5.05 ppm, confirming the formation of the new diazepine moiety.Additionally, in the 13 C NMR spectrum, the C-7 and C-8 carbon atoms of the diazepine ring were observed at 38.2 ppm and 57.0 ppm, while a molecular ion peak at m/z 602:604 with an isotopic profile [M] + :[M + 2] + 18:6 was observed in the mass spectrum agreeing with the formation of the structure 32e.23,24)a-g, respectively, with an excess of 2,4,5,6-tetraaminopyrimidine dihydrochloride 27 (1,4-dinucleophile) under stirring in refluxing methanol and using BF 3 •OEt 2 as catalyst.In the same way, diazepines (32,33)a-g were obtained starting from the chalcones (25,26)a-g synthesized elsewhere [50].Reaction yields ranged from 50% to 90% and all synthesized diazepines were characterized by FTIR, 1 H, 13 C NMR, and mass spectrometry (Supplementary Material).Particularly, the 1 H NMR spectrum (run in DMSO-d 6 ) of product 32e shows the signals corresponding to the N-H (a singlet at 7.00 ppm) and the diastereotopic protons (AMX system) of the diazepinic ring formed.The signal assigned to the H 7A proton appears as a doublet at 2.75 ppm with coupling constant 2 J AM = 14.2 Hz; the signal for the H 7M proton appears as a doublet at 3.77 ppm with coupling constants 2 J AM = 14.2 Hz and 3 J MX = 6.0 Hz, while the signal corresponding to the H 8X proton appears as an unresolved doublet at 5.05 ppm, confirming the formation of the new diazepine moiety.Additionally, in the 13

Anticancer Activity
All the synthesized trisubstituted triazines 14,15 and 17,18, chalcones (20,21)a-g and (23,24)a-g, and diazepines (28-33)a-g were evaluated through in vitro assays at one dose of 10 μM against 60 cancer cell lines of nine cancer types (Leukemia, Lung, Colon, Melanoma, Renal, Prostate, CNS, Ovarian, and Breast cancer) by the National Cancer Institute (NCI) [76].The results were reported as a graph of growth percentages (PC) available for analysis through the COMPARE program, which permits us to know the inhibition of growth (i.e., %IG = 100 − %PC) and lethality (%PC values less than 0).Additionally, the mean of the growth percentages (MGP) of each compound against all the 60 cancer cell lines is also reported, which is used as one of the selection criteria to continue with tests at five concentration doses.An MGP value less than 50% or with negative values indicates that the compound exhibits outstanding anticancer activity.Figure 2 shows the bar charts of the MGP values for all compounds evaluated.These diagrams are separated according to the linker (p-aryloxy, N-(2-aminoethyl)benzenesulfonamide or p-arylamine; orange moiety) between the triazine ring and the α,β-unsaturated carbonyl system or the diazepine ring (green moiety).In red and yellow background, compounds with MGP < 50% are highlighted; in red are the compounds that were evaluated at five concentration doses.The results were reported as a graph of growth percentages (PC) available for analysis through the COMPARE program, which permits us to know the inhibition of growth (i.e., %IG = 100 − %PC) and lethality (%PC values less than 0).Additionally, the mean of the growth percentages (MGP) of each compound against all the 60 cancer cell lines is also reported, which is used as one of the selection criteria to continue with tests at five concentration doses.An MGP value less than 50% or with negative values indicates that the compound exhibits outstanding anticancer activity.Figure 2 shows the bar charts of the MGP values for all compounds evaluated.These diagrams are separated according to the linker (p-aryloxy, N-(2-aminoethyl)benzenesulfonamide or p-arylamine; orange moiety) between the triazine ring and the α,β-unsaturated carbonyl system or the diazepine ring (green moiety).In red and yellow background, compounds with MGP < 50% are highlighted; in red are the compounds that were evaluated at five concentration doses.
None of the trisubstituted triazine precursors showed remarkable anticancer activity; however, several triazinylamino-and triazinyloxy-chalcones from these precursors enhanced their activity, such as 20b,d, 21a-d, 23d-g, and 24a-g, (Figure 2A-C).It should be noted that the triazinylamino-chalcones that contain N-(2-aminoethyl)benzenesulfonamide moiety 24a-g as a linker exhibited outstanding activity with MGP values below 25% and even with negative values.If the latter are compared with chalcones 21a-g, which have the dimethylamino and 4-fluoroanilino substituents on the triazine ring in common, it can be noted that triazinylamino-chalcones 24a-g are more active than those containing the p-aryloxy linker (21a-g), except for derivative 21d.This suggests that the N-(2-aminoethyl)benzenesulfonamide moiety potentiates anticancer activity.In contrast, the MGP of the chalcone series 23a-g evidenced that the presence of the 4-fluoroanilino substituent enhances the activity of the chalcones containing the N-(2-aminoethyl)benzenesulfonamide moiety except for 23d (R 2 = 3,4,5-(OCH 3 ) 3 ).None of the trisubstituted triazine precursors showed remarkable anticancer activity; however, several triazinylamino-and triazinyloxy-chalcones from these precursors enhanced their activity, such as 20b,d, 21a-d, 23d-g, and 24a-g, (Figure 2A-C).It should be noted that the triazinylamino-chalcones that contain N-(2-aminoethyl)benzenesulfonamide moiety 24a-g as a linker exhibited outstanding activity with MGP values below 25% and even with negative values.If the latter are compared with chalcones 21a-g, which have the dimethylamino and 4-fluoroanilino substituents on the triazine ring in common, it can be noted that triazinylamino-chalcones 24a-g are more active than those containing the p-aryloxy linker (21a-g), except for derivative 21d.This suggests that the N-(2-aminoethyl)benzenesulfonamide moiety potentiates anticancer activity.In contrast, the MGP of the chalcone series 23a-g evidenced that the presence of the 4-fluoroanilino substituent enhances the activity of the chalcones containing the N-(2-aminoethyl)benzenesulfonamide moiety except for 23d (R 2 = 3,4,5-(OCH3)3).
Within the family of diazepines containing the N-(2-aminoethyl)benzenesulfonamide linker 31a-g it was observed that compounds 31a,b,e,f,g exhibited potent activity.The most active diazepines 31a-g and 33a-g (moiety in common: R = Cl or F) coincide with the same substituents R 2 = a:-H, b:-CH3, e:-Cl, f:-F, g:-CF3.On the other hand, comparing the MGP values of the diazepines 31a-g with those of their precursors 24a-g, the latter exhibited higher activity.The series of diazepines containing the p-aryloxy moiety 28a-g did not show a MGP < 50%, while their analogous diazepines 29a,e,f,g did show MGP values less than 50%, with 29e,g being selected for five-dose assays.
Chalcones containing the N-(2-aminoethyl)benzenesulfonamide moiety 24a-g were the only ones having activity in the entire series (a-g) and better activity when compared to chalcones 21a-g containing the p-aryloxy linker with 4-F/Cl-anilino substituent.Likewise, the aromatic substituents 4-Cl-anilino and 4-F-anilino on the triazine ring enhanced the activity of chalcones and diazepines in most cases compared to the morpholino substituent (compounds 24a-c,e-g, 29a-g, and 33a-g were more active than 23a-c,e-g, 28ag, and 32a-g, respectively).Regarding the R 2 substituents in chalcones and diazepines, On the other hand, comparing the MGP values of the diazepines 31a-g with those of their precursors 24a-g, the latter exhibited higher activity.The series of diazepines containing the p-aryloxy moiety 28a-g did not show a MGP < 50%, while their analogous diazepines 29a,e,f,g did show MGP values less than 50%, with 29e,g being selected for five-dose assays.
Chalcones containing the N-(2-aminoethyl)benzenesulfonamide moiety 24a-g were the only ones having activity in the entire series (a-g) and better activity when compared to chalcones 21a-g containing the p-aryloxy linker with 4-F/Cl-anilino substituent.Likewise, the aromatic substituents 4-Cl-anilino and 4-F-anilino on the triazine ring enhanced the activity of chalcones and diazepines in most cases compared to the morpholino substituent (compounds 24a-c,e-g, 29a-g, and 33a-g were more active than 23a-c,e-g, 28a-g, and 32a-g, respectively).Regarding the R 2 substituents in chalcones and diazepines, there was not observed a marked chemical pattern that can be related to anticancer activity.
Based on the above results, the most active compounds were evaluated by the NCI at five concentration doses for their cytostatic (GI 50 ) and cytotoxic (LC 50 ) activity against 60 cancer cell lines (see Supplementary Material).Table 2 highlights the four cell lines that were most sensitive to each derivative (ordered from lowest to highest GI 50 ) and contrasts the GI 50 values with those of the antineoplastic standard drug 5-fluorouracil (5-FU, thymidylate synthase inhibitor).Analysis of these data showed that compounds 20b,d,  21a,b,d, 23a,d-g, 24a-g, 29e,g, 30g, 31a,b,e-g, 33a,b,e-g exhibited significant cytostatic activity with GI 50 values between <0.01-100 µM and cytotoxic activity with LC 50 values between 4.09 µM to >100 µM, against all cancer cell lines.Chalcones 20d, 21d, and 24f and diazepine 33g showed the lowest GI 50 range values (highlighted in green), indicating that they were highly active for all cell lines.The latter points out that chalcones 20d, 21a, 21d, 23a, 23d, 24c, 24d and diazepines 29e,g, 30g, 31a-b,e-g, 33a-b,e-g showed higher activity against several cell lines than the standard drug 5-FU (Table 2, highlighted in pink).Remarkably, diazepine 33a exhibited the best anticancer activity, with a GI 50 value < 10 nM against the MOLT-4 cell line of the Leukemia panel.The above results demonstrate that the triazine-based chalcone/diazepine hybrids 20b,d, 21a,b,d, 23a,d-g, 24a-g, 29e,g, 30g,  31a,b,e-g, 33a,b,e-g are important hits and a starting point for further optimization of their anticancer activity.
Additionally, we found that triazinylamino-and triazinyloxy-diazepines 28a-g, 29a,c,d,f, 30d, 31a-f, 32a,b,f,g, 33a,b,c,f showed inhibition against N. gonorrhoeae with MIC values between 0.25 and 62.5 µg/mL (Table 3).This could suggest that the pyrimido[4,5-b] [1,4]diazepine moiety may be associated to the activity against N. gonorrhoeae [80].Moreover, diazepines containing the p-aryloxy linker 28a-g and N-(2-aminoethyl)benzenesulfonamide linker 31a-f showed inhibition with MIC values between 0.25-500 µg/mL and 0.5-8 µg/mL, being, the triazinylamino-diazepine 31f the compound with the highest activity against N. gonorrhoeae with a MIC value 0.25 µg/mL, which is similar to that for penicillin [81].These findings are relevant because N. gonorrhoeae is the second most prevalent sexually transmitted bacterial infection worldwide, and has developed resistance to the first line treatment and has emerged as a thread for public health [82,83].Some of the evaluated compounds could become molecules for future development in this regard.

Antitubercular Activity
It was determined that the triazinylamino-chalcones 24a-g exhibited anti-TB activity inhibiting the growth of M. tuberculosis H37Rv at concentrations between 25 and 50 µg/mL (Figure 3A).However, as it was observed for N. gonorrhoeae, the most inhibitory compounds were those with a pyrimido [4,5-b] [1,4]diazepine core, being 29a-g and 31a-g series the most active with MIC values between 0.6 and 5 µg/mL (Figure 3B,C).Previous studies have confirmed the anti-TB activity of some pyrimido-diazepine derivatives [84].In addition, it has been shown that some compounds containing this heterocyclic system can inhibit the action of tyrosine [62] and serine-threonine kinases [85].Unlike most bacteria, which use histidine kinases as major components in signal transduction, M. tuberculosis possesses 11 serine-threonine kinases, two of which (PknA and PknB) have been shown to be essential for its growth in vitro [85].Therefore, the activity observed for compounds 29a-g and 31a-g could be due to the inhibition of one of these proteins.In addition to the pyrimido [4,5b] [1,4]diazepine moiety, the 4-fluoroanilino substituent seems to play a crucial role in the interaction with the target, since compounds with morpholino as substituent did not displayed activity.As shown in Figure 3B,C, compounds 29b and 31b exhibited the most outstanding anti-TB activity both containing a methyl group as R 2 substituent, which could favor Van der Waals interactions, giving stability to the molecule when interacting with the target.
Two compounds (29e and 31g) exhibited outstanding antifungal activity with MICs = 62.5 µg/mL.Thus, triazinyloxy-chalcone 29e was active against T. rubrum, while triazinylamino-chalcone 31g was active against T. mentagrophytes and A. fumigatus.These chalcones contain 4-chloro/4-fluorophenylamine and dimethylamine as substituents attached to the triazine ring, and their linker moieties have both H-bond donors and acceptors.This suggests that this structural design can be further tested for the optimization of its antifungal activity.On the other hand, triazine 17, chalcones 23e, 24c,e, 21d, and diazepines 30d,g, 29f,g showed marginal activity (MIC = 125 µg/mL) against various fungal species (Table 4).

In Silico Physicochemical Parameter Predictions
The physicochemical features of the most active compounds were determined by in silico analysis using the SwissADME platform [89] (Table 5).All the active compounds showed violations of the Lipinski rule of five and the pharmacokinetics need improve-ment as gastrointestinal absorption and cytochrome interactions was However, the synthetic accessibility indicates the feasibility for analogues synthesis of these compounds.Bioavailability score 0.17 0.17 0.17 0.17 0.17 0.17 >0.1

Toxicity Studies
Toxicity, median lethal dose (LD 50 ), and toxicity classification were predicted for the six most active compounds of each activity studied: 33a (anticancer), 29b, 31b, and 31f (antibacterial) and 29e and 31g (antifungal) using Protox II [91] (Table 7).Most compounds (five/six) showed predicted immunotoxicity, whereas 29b, 31f, and 31g showed predicted carcinogenicity and 31f, 31g, and 33a may have cytotoxic effects; however, these alerts were predicted with low likelihood (cs < 0.7).None of the compounds were predicted as mutagenic or hepatotoxic chemicals.Four out of six compounds were classified as low toxic (class 5).Additionally, compounds 20b,d, 21a,b,d, 23d, 30d,e,g, 33e were tested for toxicity in the Galleria mellonella model.Nine out of ten tested compounds showed LD 50 ≥ 325 mg/Kg and eight of them LD 50 ≥ 650 mg/Kg.Although some compounds (21a, 30d, 30g, 21d) induced mortality at the higher doses, it was only ≤25%.This suggests that most compounds are likely to have a very low toxic nature.

Materials and Methods
All solvent and reagents were obtained from commercial sources and were used without purification.Thin layer chromatography analyses were performed on 0.2 mm pre-coated aluminium plates of silica gel 60 F254 (Merck, Darmstadt, Hesse, Germany).Melting points were taken on a Stuart SMP10 melting point device Ltd., Stone, Staffordshire, UK) and are uncorrected.FTIR spectra were determined on an IRAffinity-1 spectrophotometer (Shimadzu, Columbia, MD, USA). 1 H and 13 C NMR spectra were measured on a Bruker 400 Ultrashield Avance II spectrometer (Bruker, Billerica, MA, USA) operating at 400 and 100 MHz, respectively, using DMSO-6 and CDCl 3 as solvents and TMS as internal standard.Mass spectra were obtained on a Shimadzu-GCMS-QP2010 spectrometer (Shimadzu, Kyoto, Honshu, Japan) equipped with a Rxi-1HT GC Capillary Column (30 m, 0.25 mm ID, 0.25 um df, phase: dimethyl polysiloxane) and operating at 70 eV.Elemental analyses were performed on a Thermo Finnigan Flash EA1112 CHN elemental analyzer (Thermo Fischer Scientific Inc., Madison, WI, USA) and the values are within ±0.4% of the theoretical values.The synthesis of derivatives 5 [68][69][70] and 6 [71,72] was reported in previous studies.Monosubstituted triazine 7 was obtained as follows: A solution of 4-hydroxy-3-methoxybenzaldehyde (13 mmol) in acetone (20 mL) was added slowly to a solution of 2,4,6-trichloro-1,3,5triazine 4 (35.1 mmol) in acetone (35 mL) at −5-0 • C. The mixture was neutralized with 20% Na 2 CO 3 .The content was poured onto crushed ice, filtered, and washed with water.The required equivalents of the reagents and reaction time are reported in Table 1.
3.1.2.General Procedure for the Synthesis of Disubstituted Triazines (8)(9)(10)(11) The synthesis of derivatives 8 [74], 9 [71] and 11 [73] was reported in previous studies.Disubstituted triazine 10 was obtained as follows: 4-hydroxy-3-methoxybenzaldehyde (13 mmol)was added slowly to a solution of monosubstituted triazine 6 (13 mmol) in acetone (35 mL) at room temperature.The mixture was neutralized with 20% Na 2 CO 3 and after 3 h the content was poured onto crushed ice, filtered, and washed with water.The required equivalents of the reagents, the solvent used, the temperature and the reaction time were reported in Table 1.

Procedure for the Synthesis of Trisubstituted Triazine (12)
A mixture of disubstituted triazine 8 (6.2 mmol) and ethylenediamine (99.2 mmol) was stirred at room temperature for 18 h.The mixture was dissolved in chloroform and washed with water.The organic phase was dried with MgSO 4 , filtered, and concentrated under reduced pressure.A mixture of disubstituted triazine 11 (3 mmol) and ethanolamine (4.5 mmol) in dioxane (15 mL) was stirred at room temperature for 24 h.The mixture was poured onto crushed ice, filtered, and washed with water.
3.1.7.General Procedure for the Synthesis of Trisubstituted Triazines (17,18) A mixture of the respective trisubstituted triazine 12/13 (4 mmol), 4-acetylbenzenesulfonyl chloride 16 (4.8mmol) and TEA (0.5 mL) in ethanol (15 mL) was stirred at room tempera-ture for 24 h.The reaction crude was dissolved in chloroform and washed with water.The organic phase was dried with MgSO 4 , filtered, and concentrated reduced pressure.The product was then purified by column chromatography on silica gel employing 10:1 of ethyl acetate:hexane as eluent.
3.1.9.General Procedure for the Synthesis of Chalcones (20a-g) A mixture of the trisubstituted triazine 14 (0.27 mmol), the respective acetophenone 19a-g (0.22 mmol) and 150 µL of a solution of potassium hydroxide (20%) in ethanol (3 mL) were sonicated (US) for 6-8 h.The content of 20a-c,e,g was filtered, and washed with cold ethanol.The content of 20d and 20f were dissolved in chloroform and washed with water.The organic phase was dried with MgSO 4 , filtered, and concentrated under reduced pressure.The product was then purified by column chromatography on silica gel employing 20:1 of dichloromethane:methanol as eluent.
3.1.10.General Procedure for the Synthesis of Chalcones (21a-g) A mixture of the trisubstituted triazine 15 (0.26 mmol), the respective acetophenone 19a-g (0.22 mmol), and 150 µL of a solution of potassium hydroxide (20%) in ethanol (3 mL) was heated under reflux for 2-5 h.The content was dissolved in chloroform and washed with water.The organic phase was dried with MgSO 4 , filtered, and concentrated under reduced pressure.The products were purified by column chromatography on silica gel employing 2:1 hexane:ethyl acetate as eluent.(10 mL) was heated under reflux for 3-8 h.After cooling to room temperature, the content was quenched with NH 4 OH 6% until neutralization.The content was dissolved in chloroform and washed with water.The organic phase was dried with MgSO 4 , filtered, and concentrated under reduced pressure.The product was purified by column chromatography on silica gel employing 10:1 dichloromethane:methanol as eluent.

Anticancer Activity
The human cancer cell lines of the cancer screening panel were grown in an RPMI-1640 medium containing 5% fetal bovine serum and 2 mM L-glutamine.For a typical screening experiment, cells were inoculated into 96-well microtiter plates.After cell inoculation, the microtiter plates were incubated at 37 • C, 5% CO 2 , 95% air, and 100% relative humidity for 24 h prior to the addition of the tested compounds.After 24 h, two plates of each cell line were fixed in situ with TCA to represent a measurement of the cell population for each cell line at the time of sample addition (Tz).The samples were solubilized in dimethyl sulfoxide (DMSO) at 400-fold the desired final maximum test concentration and stored frozen prior to use.At the time of compound addition, an aliquot of frozen concentrate was thawed and diluted to twice the desired final maximum test concentration with complete medium containing 50 µg/mL gentamicin.An additional four 10-fold or 1/2 log serial dilutions were made to provide a total of five drug concentrations plus the control.Aliquots of 100 µL of these different sample dilutions were added to the appropriate microtiter wells already containing 100 µL of medium, resulting in the required final sample concentrations.After the tested compounds were added, the plates were incubated for an additional 48 h at 37 • C, 5% CO 2 , 95% air, and 100% relative humidity.For adherent cells, the assay was terminated by the addition of cold TCA. were fixed in situ by the gentle addition of 50 µL of cold 50% (w/v) TCA (final concentration, 10% TCA) and incubated for 60 min at 4 • C. The supernatant was discarded, and plates were washed five times with tap water and air dried.Sulforhodamine B (SRB) solution (100 µL) at 0.4% (w/v) in 1% acetic acid was added to each well, and plates were incubated for 10 min at room temperature.After staining, unbound dye was removed by washing five times with 1% acetic acid and the plates were air dried.Bound stain was subsequently solubilized with 10 mM trizma base, and the absorbance was read on an automated plate reader at a wavelength of 515 nm.Using the seven absorbance measurements [time zero (Tz), control growth in the absence of drug, and test growth in the presence of drug at the five concentration levels (Ti)], the percentage growth was calculated at each of the drug concentrations levels.Percentage growth inhibition was calculated as: [(Ti − TZ)/(C − TZ)] × 100 for concentrations for which Ti > Tz, and [(Ti − TZ)/TZ] × 100 for concentrations for which Ti < Tz.Two dose−response parameters were calculated for each compound.Growth inhibition of 50% (GI 50 ) was calculated from [(Ti − TZ)/(C − TZ)] × 100 = 50, which is the drug concentration resulting in a 50% lower net protein increase in the treated cells (measured by SRB staining) as compared to the net protein increase seen in the control cells and the LC 50 (concentration of drug resulting in a 50% reduction in the measured protein at the end of the drug treatment as compared to that at the beginning), indicating a net loss of cells; calculated from [(Ti − TZ)/TZ] × 100 = −50).
Values were calculated for each of these two parameters if the level of activity is reached; however, if the effect was not reached or was exceeded, the value for that parameter was expressed as greater or less than the maximum or minimum concentration tested [76-79].

Antibacterial Activity
Stock solutions (100 mg/mL) of the respective compounds were prepared in dimethylsulfoxide (DMSO) and diluted to a final concentration of 500 µg/mL.An initial screening for bacterial inhibition was performed by the agar diffusion method.Briefly, sterile Mueller Hinton agar was prepared in Petri dishes and inoculated with a bacterial suspension prepared in trypticase soy broth (TSB) and adjusted to 1.5 × 10 8 CFU/mL (0.08-0.1 OD at 600 nm) [92].6 mm diameter wells were drilled into the agar, and 10 µL of each compound (stock solution) was deposited into each well.DMSO and TSB were included as negative controls.Gentamicin and trimethoprim sulfamethoxazole were included as a positive control for growth inhibition.Derivatives showing growth inhibition were tested at least twice before being selected for the microdilution test.For N. gonorrhoeae, the agar diffusion method was also used for screening with some modifications.For this method, 200 µL of a bacterial suspension (1.5 × 10 8 CFU/mL) was inoculated onto Gonococcal Agar (GC) supplemented with 1% isovitalex, and then compounds were added to wells as mentioned above and incubated at 35-36.5 • C in 5% CO 2 for 48 h.Penicillin and tetracycline were used as controls [93].
Antitubercular screening was carried performed using the broth microdilution method [94].Briefly, 100 µL of Middlebrook 7H9 culture medium supplemented with 10% Middlebrook OADC and test compounds at a concentration of 100 µg/mL were placed in clear U-bottom polystyrene 96-well microplates.Wells with culture medium without compounds were prepared as growth control.Bacterial suspensions were prepared from Lowenstein Jensen agar cultures and adjusted to 5 × 10 6 CFU/mL.Once the required density was reached, a 1:20 dilution was realized in Middlebrook 7H9 culture medium supplemented with 10% Middlebrook OADC and 100 µL were inoculated into the wells containing culture medium supplemented with the compounds.Microplates were incubated at 37 • C for 14-21 days.After time, a visual reading of the growth in each well was performed.Those wells in which no growth was observed were taken as positive for anti-TB activity.
Microdilution test: The Minimum Inhibitory Concentration was determined for those compounds with reproducible and visible antibacterial inhibition in the screening test.The bacterial suspensions were adjusted with Mueller Hinton Broth (MHB) to a concentration of 5 × 10 5 to 8 × 10 5 .The stock solution of new compounds was diluted in MHB containing 5% DMSO and 0.1% Tween 80 and added to 90 µL of inoculum.The microplates were incubated for 24 h at 35-37 • C. The determination of the MIC of the new compounds that presented anti-TB activity was developed as described in the previous section, including decreasing concentrations of each compound.The MIC was defined as the lowest concentration with visible inhibition of bacterial growth, and/or detected using Resazurin (125 µg/mL).Isoniazid was included as growth inhibition controls.The experiments were performed in duplicate.
For the analysis of inhibition against N. gonorrhoeae, compounds that showed growth inhibition on evaluative screening were then tested for MIC on agar plates as described by the Center for Disease Control Prevention and the Clinical and Laboratory Standards Institute with modifications [93].Briefly, GC agar supplemented with 1% isovitalex was prepared with increasing concentrations of the new compounds and inoculated with 10 µL of a bacterial suspension (1 × 10 4 CFU).The lowest concentration of the compound that inhibited bacterial growth was determined as the MIC.Bacterial growth was examined and verified using the oxidase test.The experiments were performed in duplicate in at least two independent assays.

Antifungal Activity
Broth microdilution techniques were performed in 96-well microplates according to the Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts, document M27-A3 [86] and of Filamentous Fungi M38-A8 [87].For assay, stock solutions of each compound in DMSO (maximum concentration 1%) were added to test wells and diluted with RPMI-1640, to final concentrations of 250-0.98 µg/mL.An inoculum suspension (100 µL) was added to each well (final well volume = 200 µL).One growth control well (containing medium, inoculum, and the same amount of DMSO as used for each compound) and one sterility control well (sample, medium, and sterile water instead of inoculum) were included for each fungus tested.Microtiter plates were incubated in a dark, humid chamber at 30 • C for the time necessary for each fungus.Amphotericin B, Terbinafine, Fluconazole and Itraconazole were used as a positive control.The tests were performed in triplicate.

Hemolytic Activity
Compounds that showed activity were evaluated for their ability to induce hemolysis following the cytotoxicity method by spectrophotometry.The method was adapted from Conceição et al. [90] with modifications.240 µL of human red blood (huRBC) adjusted at 5% of hematocrit in phosphate-buffered saline were placed in each well of a 96-well plate and subsequently exposed to 200 µg/mL of selected compounds (10 µL of a 5 mg/mL solution of test compound dissolved in 5% DMSO, 0.1% Mueller Hinton broth Tween-80).As a positive control for hemolytic activity, 10 µL of 1% sodium dodecyl sulfate was added.As a negative control, the medium without the compounds to be tested was employing.Free hemoglobin was measured after 24 h of incubation at 37 • C by spectrophotometry (420 nm Cytation 3M, BioteK).Non-specific absorbance was subtracted from a blank.The determinations were made in triplicate in at least two independent experiments.

Toxicity Studies In Vivo
Compounds biologically active were tested for toxicity in the Galleria mellonella model.G mellonella larvae were cultivated in the laboratory and healthy, beige larvae weighing 150-200 mg, were selected for toxicity assays.Groups of ten six-instar larvae were inoculated with 10 µL or 20 µL of each compound at concentration equivalent to 650 mg/Kg and incubated at 37 • C in darkness.The larval survival was monitored every 24 h for 5 days to determine the half lethal doses (LD 50 ).The larvae were initially injected 30 mg/Kg and if most larvae (≥60%) survived after five days, the assay was performed using higher doses up to 650 mg/Kg [95].

Scheme 2 .
Scheme 2. Synthesis of triazinyloxy-chalcones (20,21)a-g and triazinylamino-chalcones (23,24)a-g.The final target products (i.e., triazinyloxy-and triazinylamino-pyrimido[4,5-b][1,4]diazepines) (28-31)a-g (Scheme 3) were obtained with high regioselectivity by reaction of chalcones (20,21)a-g and (23,24)a-g, respectively, with an excess of 2,4,5,6-tetraaminopyrimidine dihydrochloride 27(1,4-dinucleophile)  under stirring in refluxing methanol and using BF 3 •OEt 2 as catalyst.In the same way, diazepines (32,33)a-g were obtained starting from the chalcones (25,26)a-g synthesized elsewhere[50].Reaction yields ranged from 50% to 90% and all synthesized diazepines were characterized by FTIR, 1 H,13 C NMR, and mass spectrometry (Supplementary Material).Particularly, the 1 H NMR spectrum (run in DMSO-d 6 ) of product 32e shows the signals corresponding to the N-H (a singlet at 7.00 ppm) and the diastereotopic protons (AMX system) of the diazepinic ring formed.The signal assigned to the H 7A proton appears as a doublet at 2.75 ppm with coupling constant 2 J AM = 14.2 Hz; the signal for the H 7M proton appears as a doublet at 3.77 ppm with coupling constants 2 J AM = 14.2 Hz and 3 J MX = 6.0 Hz, while the signal corresponding to the H 8X proton appears as an unresolved doublet at 5.05 ppm, confirming the formation of the new diazepine moiety.Additionally, in the13 C NMR spectrum, the C-7 and C-8 carbon atoms of the diazepine ring were observed at 38.2 ppm and 57.0 ppm, while a molecular ion peak at m/z 602:604 with an isotopic profile [M] + :[M + 2] + 18:6 was observed in the mass spectrum agreeing with the formation of the structure 32e.

Figure 2 .
Figure 2. Bar chart of the mean growth percent (MGP) against 60 cancer cell lines for the trisubstituted triazines 14-15 and 17-18, chalcones (20,21)a-g and (23,24)a-g, and diazepines (28-33)a-g.Within the family of diazepines containing the N-(2-aminoethyl)benzenesulfonamide linker 31a-g it was observed that compounds 31a,b,e,f,g exhibited potent activity.The most active diazepines 31a-g and 33a-g (moiety in common: R = Cl or F) coincide with the same substituents R 2 = a:-H, b:-CH 3 , e:-Cl, f:-F, g:-CF 3 .On the other hand, comparing the MGP values of the diazepines 31a-g with those of their precursors 24a-g, the latter exhibited higher activity.The series of diazepines containing the p-aryloxy moiety 28a-g did not show a MGP < 50%, while their analogous diazepines 29a,e,f,g did show MGP values less than 50%, with 29e,g being selected for five-dose assays.Chalcones containing the N-(2-aminoethyl)benzenesulfonamide moiety 24a-g were the only ones having activity in the entire series (a-g) and better activity when compared to chalcones 21a-g containing the p-aryloxy linker with 4-F/Cl-anilino substituent.Likewise, the aromatic substituents 4-Cl-anilino and 4-F-anilino on the triazine ring enhanced the activity of chalcones and diazepines in most cases compared to the morpholino substituent (compounds 24a-c,e-g, 29a-g, and 33a-g were more active than 23a-c,e-g, 28a-g, and 32a-g, respectively).Regarding the R 2 substituents in chalcones and diazepines, there was not observed a marked chemical pattern that can be related to anticancer activity.Based on the above results, the most active compounds were evaluated by the NCI at five concentration doses for their cytostatic (GI 50 ) and cytotoxic (LC 50 ) activity against 60 cancer cell lines (see Supplementary Material).Table2highlights the four cell lines that were most sensitive to each derivative (ordered from lowest to highest GI 50 ) and contrasts the GI 50 values with those of the antineoplastic standard drug 5-fluorouracil (5-FU, thymidylate synthase inhibitor).Analysis of these data showed that compounds20b,d, 21a,b,d, 23a,d-g, 24a-g, 29e,g, 30g, 31a,b,e-g, 33a,b,e-g exhibited significant cytostatic activity with GI 50 values between <0.01-100 µM and cytotoxic activity with LC 50 values between 4.09 µM to >100 µM, against all cancer cell lines.Chalcones 20d, 21d, and 24f and diazepine 33g showed the lowest GI 50 range values (highlighted in green), indicating that they were highly active for all cell lines.The latter points out that chalcones 20d, 21a, 21d, 23a, 23d, 24c, 24d and diazepines 29e,g, 30g, 31a-b,e-g, 33a-b,e-g showed higher

Table 1 .
Reaction conditions toward the synthesis of trisubstituted triazine precursors

Table 1 .
Reaction conditions toward the synthesis of trisubstituted triazine precursors

Table 2 .
Anticancer against the four most sensitive cancer cell lines c and compared to the GI 50 values of the standard drug 5-FU.

Table 2 .
Cont.GI 50 values highlighted in pink (of our compounds) are lower than GI 50 values of 5-FU.b LC 50 is a parameter of cytotoxicity that reflects the molar concentration needed to kill 50% of the cells.c Data obtained from NCI's in vitro disease-oriented human cancer cell lines screen in µM [76-79].d Range of GI 50 values against the 60 cancer cell lines, entries highlighted in green show the lowest GI 50 ranges.e Activity values against human cancer cell lines shown by 5-FU correspond to those reported by: https://dtp.cancer.gov/dtpstandard/cancerscreeningdata/index.jsp, accessed on 12 October 2023.

Table 4 .
[88]mum inhibitory concentrations (MIC) and minimum fungicidal concentrations (MFC) of triazine derivatives with MIC < 250 µg/mL against fungal species a .Aspergillus niger; Afl: Aspergillus flavus.dFirst-linedrugs used for the treatment of diseases caused by fungi[88].The data in bold indicate the most notable MICs values.

Table 5 .
Physicochemical properties, ADME, and medicinal chemistry predictions of most active compounds.

Table 6 .
Results of the hemolytic activity for the synthesized triazine derivatives that showed significant activity.

Table 7 .
In silico toxicity studies of compounds 29e