CDK8/CycC complex has kinase activity towards the carboxyterminal domain of RNA polymerase II, and contributes to the regulation of transcription via association with the mediator complex. Different human malignancies, mainly colorectal and gastric cancers, were produced as a result of overexpression of CDK8/CycC in the mediator complex. Therefore, CDK8/CycC complex represents as a cancer oncogene and it has become a potential target for developing CDK8/CycC modulators.5a-i was synthesized, and biologically evaluated as potential CDK8/CycC complex inhibitors.A series of nine 4-phenylaminoquinoline scaffold-based compounds The scaffold substituent effects on the intrinsic inhibitory activity toward CDK8/CycC complex are addressed trying to present a novel outlook of CDK8/CycC Complex inhibitors with 4-phenylaminoquinoline scaffold in cancer therapy. The secondary benzenesulfonamide analogues proved to be the most potent compounds in suppressing CDK8/CycC enzyme, whereas, their primary benzenesulfonamide analogues showed inferior activity. Moreover, the benzene reversed sulfonamide analogues were totally inactive.5d showed submicromolar potency against CDK8/CycC (IC50 = 0.639 µM) and it can be used for further investigations and to design another larger library of phenylaminoquinoline scaffold-based analogues in order to establish detailed SARs.The titled scaffold showed promising inhibitory activity data and there is a crucial role of un/substituted sulfonamido group for CDK8/CycC complex inhibitory activity. Compound  Cyclin-dependent kinases (CDKs) drive cell cycle through phosphorylation of a variety of vital substrates . AssociaEven though CDK inhibitors have been abundantly described, attempts of discovering selective CDK8 inhibitors have emerged as a promising strategy for cancer therapy as Pan-CDK inhibitor has shown narrow therapeutic window and potential risks . Such se50 = 15 nM) that exhibited anticancer activity in animal models of acute myeloid leukemia (AML)  . Senexinof 24 nM . In 2016ng modes .It is noteworthy that the kinase activity of CDK8 is affected by substrate binding and association with other mediator complex members as well. By utilizing a scaffold hopping strategy on the aforementioned quinoline isosteres, a series of new phenylaminoquinoline derivatives with sulphonamide moiety at position 3 in terminal phenyl ring was designed, synthesized and pharmacologically evaluated as potential small molecule modulators of CDK8/CycC complex.1H and 100.6 MHz 13C NMR). Column chromatography was performed on Merck Silica Gel 60 (230–400 mesh). Thin layer chromatography (TLC) was performed using sheets pre-coated with silica gel 60 F254 supplied from Merck. The purity of compounds was determined by analytical high performance liquid chromatography (HPLC) using a Water ACQUITY UPLC (CORTECSTM) with C18 column  at 40 °C. HPLC data were noted using parameters as follows: 0.1% formic acid in water and 0.1% formic acid in methanol and flow rate of 0.3 mL/min. Waters ACQUITY UPLC BEH C18 1.7 µ–Q-TOF SYNAPT G2-Si High Definition Mass Spectrometry was used to obtain high-resolution spectra. Compounds 3–4 and 5a–c, i were previously reported  and utilized to accomplish this work as received. All reactions were carried out in a dry nitrogen atmosphere. The microwave-assisted synthesis was carried out in a Biotage Initiator apparatus operating in single mode, the microwave cavity producing controlled irradiation at 2.45 GHz . The reactions were run in sealed vessels. These experimentations were carried out by employing magnetic stirring and a fixed hold time applying variable power to reach (during 1 −2 min) and then keep the desired temperature in the vessel for the preset time. On the reactor vial glass, an IR sensor was applied to monitor the temperature. The NMR spectra were obtained on Bruker Avance 400 (400 MHz reported .4a–d.Common procedures for the synthesis of key intermediates 3a–d (1.0 mmol) in POCl3 (six mL) was refluxed for 1 h. Evaporation of the mixture was performed in vacuo and the residue was extracted with methylene chloride, aqueous ammonia, and crushed ice. The methylene chloride layer was dried over anhydrous Na2SO4 and concentrated. Column chromatography  was applied to purify the residue to get compounds 4a-c (A solution of intermediated nds 4a-c .5a-iCommon procedures for the synthesis of the target compounds 4a-c (0.21 mmol), 3-amino-N-methylbenzenesulfonamide , or N-(3-phenyl)methanesulfonamide , 3-aminobenzenesulfonamide , and absolute ethyl alcohol (12 mL). The microwave vial was sealed and heated under microwave conditions at 150 °C for 30 min. The reaction mixture was evaporated in vacuo and the residue was extracted with ethyl acetate and NaHCO3 (aq). The ethyl acetate layer was dried over anhydrous Na2SO4 and concentrated. The residue was purified by column chromatography  to provide quinolines 5a-i Ethyl 6-Bromo-4-(3-sulfamoyl-phenylamino)-quinoline-3-carboxylate  δ ppm: 1.07 , 3.88 , 7.17 , 7.36 , 7.45–7.51 , 7.94 , 8.53 , 8.92 , 9.71 ; 13C NMR  δ ppm: 14.22 (CH3), 61.49 (CH2), 111.61 (phenyl C-2), 116.40 (phenyl C-4), 119.81 (phenyl C-6), 119.95 (quinoline C-6), 121.47 (quinoline C-3), 123.08 (quinoline C-10), 126.60 (quinoline C-5), 130.21 (phenyl C-5), 132.13 (quinoline C-8), 134.76 (quinoline C-7), 144.01 (phenyl C-1), 145.68 (phenyl C-3), 146.37 (quinoline C-2), 148.91(quinoline C-9), 152.06 (quinoline C-4), 166.33 (C =O); HRESI-MS m/z calcd for [M+H]+ C18H16BrN3O4S: 450.0123, found: 450.0127.Yellow solid, yield: 71%, mp: 235.6 −237.2 °C; 5b)Ethyl 6-methoxy-4-((3-sulfamoylphenyl)amino)quinoline-3-carboxylate : 1.13 , 3.73 , 3.99 , 7.14–7.16 , 7.33 , 7.42–7.48 , 7.91–7.93 , 8.84 ; 13C NMR (DMSO-d6): 14.31 (CH2 CH3), 55.92 (OCH3), 61.40 (CH2), 103.51 (quinoline C-5), 111.54 (phenyl C-2), 116.36 (phenyl C-4), 119.51 (phenyl C-6), 121.78 (quinoline C-3), 122.35 (quinoline C-10), 123.78 (quinoline C-7), 130.17 (phenyl C-5), 131.56 (quinoline C-8), 144.36 (quinoline C-9), 145.55 (phenyl C-3), 146.16 (phenyl C-1), 146.59 (quinoline C-2), 148.90 (quinoline C-3), 157.51 (quinoline C-6), 166.90 (C =O); HRESI-MS calcd for [M+H]+ C19H19N3O5S: 402.1124, found: 402.1116.Yellow solid, yield: 65%, 5c)Ethyl 7-chloro-6-fluoro-4-((3-sulfamoylphenyl)amino)quinoline-3-carboxylate : 1.08 , 3.91 , 7.18–7.20 , 7.45–7.49 , 7.36 , 8.18 , 8.25 , 8.91 , 9.68 ; 13C NMR (DMSO-d6): 14.22 (CH2 CH3), 61.58 (CH2), 110.16, 110.40 (phenyl C-2), 111.47, 116.52 (phenyl C-4), 120.18, 121.24 (phenyl C-6), 121.85 (quinoline C-10), 124.41 (quinoline C-8), 130.29 (phenyl C-5), 131.58, 143.76 (phenyl C-3), 145.69 (phenyl C-1), 147.05 (quinoline C-2), 147.52 (quinoline C-5), 152.27 (quinoline C-3), 153.76 (quinoline C-7), 156.21 (quinoline C-6), 166.26 (C =O); HRESI-MS m/z calcd for [M+H]+ C18H15ClFN3O4S: 424.0534, found: 424.0525.Yellowish white solid, yield: 66%, 5d)Ethyl 6-Bromo-4-((3-(N-methylsulfamoyl)phenyl)amino)quinoline-3-carboxylate  δ ppm: 1.08 , 2.98  3.91 , 7.27 , 7.95 , 8.40 , 8.48 , 8.89 , 9.64 ; 13C NMR  δ ppm: 14.20 (CH3), 29.05 (N-CH3), 61.45 (CH2), 111.61 (benzenesulfonamide C-2), 117.40 (benzenesulfonamide C-4), 119.76 (benzenesulfonamide C-6), 121.02 (quinoline C-3), 122.53 (quinoline C-10), 123.04 (quinoline C-6), 126.69 (quinoline C-5), 130.56 (benzenesulfonamide C-5), 132.14 (quinoline C-8), 134.75 (quinoline C-7), 140.80 (benzenesulfonamide C-3), 144.35 (benzenesulfonamide C-1), 146.54 (quinoline C-2), 148.95 (quinoline C-9), 152.03 (quinoline C-4), 166.34 (C =O); HRESI-MS m/z calcd for [M+H]+ C19H19BrN3O4S: 464.0280, found: 464.0273.White solid, yield: 64%, mp: 212.9 −214.3 °C, 5e)Ethyl 6-methoxy-4-((3-(methylsulfonamido)phenyl)amino)quinoline-3-carboxylate : 1.17 , 2.96 , 3.71 , 3.99 , 6.75–6.77 , 6.88–6.90 , 7.22–7.26 , 7.40–7.45 , 7.89 , 8.80 , 9.52 , 9.74 ; 13C NMR (DMSO-d6): 14.35 (CH2 CH3), 55.82 (OCH3) 61.29 (CH2), 103.88 (quinoline C-5), 110.41 (phenyl C-2), 111.21 (phenyl C-4), 114.27 (phenyl C-6),115.58 (quinoline C-3),121.85 (quinoline C-10), 123.45 (quinoline C-7), 130.44 (phenyl C-5), 139.84 (quinoline C-8), 144.51 (quinoline C-9), 146.11 (phenyl C-3), 147.58 (phenyl C-1), 148.88 (quinoline C-2), 157.14 (quinoline C-3), 150.45 (quinoline C-6), 167.41(C = O); HRESI-MS m/z calcd for [M+H]+ C20H21N3O5S: 416.1280, found: 416.1278.Yellow solid, yield: 64%, 5f)Ethyl 7-chloro-6-fluoro-4-((3-(methylsulfonamido)phenyl)amino)quinoline-3-carboxylate : 1.12 , 2.99 , 3.92 , 6.77 , 6.90–6.93 , 7.23–7.27 , 8.12 , 8.21 , 8.87 , 9.63 , 9.79 ; HRESI-MS calcd for [M+H]+ C19H17ClFN3O4S: 438.0691, found: 438.0687.Yellow solid, yield: 55%, 5g)Ethyl 6-Bromo-4-(3-methanesulfonylphenylamino)-quinoline-3-carboxylate  δ ppm: 1.12 , 2.98  3.93 , 6.78 , 6.91–6.94 , 7.24 , 7.90 , 8.40 , 8.88 , 9.67 ; 13C NMR  δ ppm: 14.29 (CH3), 39.35 (SO2CH3), 61.41 (CH2), 110.54, (phenyl C-2), 111.07 (phenyl C-4), 114.67 (phenyl C-6), 115.17 (quinoline C-7), 119.24 (quinoline C-3), 122.54 (quinoline C-10), 127.01 (quinoline C-6), 130.54 (quinoline C-5), 132.07 (phenyl C-5), 134.59 (quinoline C-8), 140.10 (phenyl C-3), 144.10 (phenyl C-1), 147.47 (quinoline C-2), 148.96 (quinoline C-9), 151.91 (quinoline C-4), 166.90 (C =O); HRESI-MS m/z calcd for [M+H]+ C19H19BrN3O4S: 464.0280, found: 464.0276.Yellow solid, yield: 67%, mp: 183.4 −184.5 °C, 5h)Ethyl 6-methoxy-4-((3-(methylsulfonyl)phenyl)amino)quinoline-3-carboxylate : 1.13 , 2.39 , 3.75 , 3.99 , 7.24 , 7.36–7.52 , 7.95 , 8.85 , 9.57 ; 13C NMR (DMSO-d6): 14.28 (CH2 CH3), 28.99 (NHCH3), 55.91 (OCH3) 61.35 (CH2), 103.47 (quinoline C-5), 111.69 (phenyl C-2), 117.23 (phenyl C-4), 120.47 (phenyl C-6), 122.39 (quinoline C-3), 122.52 (quinoline C-10), 123.73 (quinoline C-7), 130.49 (phenyl C-5), 131.60 (quinoline C-8), 140.72 (quinoline C-9), 144.77 (phenyl C-3), 146.17 (phenyl C-1), 146.50 (quinoline C-2), 148.92 (quinoline C-3), 157.54 (quinoline C-6), 166.86 (C =O); HRESI-MS m/z calcd for [M+H]+ C20H21N3O5S: 416.1280, found: 416.1277.Yellow solid, yield: 62%,5i)Ethyl 7-chloro-6-fluoro-4-((3-(methylsulfonyl)phenyl)amino)quinoline-3-carboxylate : 1.07 , 2.40 , 3.90 , 7.27 , 7.41, 7.49–7.53 , 8.19 , 8.26 , 8.91 , 9.70 , 9.79 ; 13C NMR (DMSO-d6): 14.19 (CH2 CH3), 29.00 (NHCH3), 61.52 (CH2), 110.18, 110.42 (phenyl C-2), 111.56,117.44 (phenyl C-4), 121.17, 121.25 (phenyl C-6), 121.33 (quinoline C-10), 122.63 (quinoline C-8), 125.22, 125.43,130.61 (phenyl C-5), 131.58, 140.89, 144.09 (phenyl C-3), 146.97 (phenyl C-1), 147.02 (quinoline C-2), 147.54 (quinoline C-5), 152.26 (quinoline C-3), 153.76 (quinoline C-7), 156.21 (quinoline C-6), 166.24 (C = O). HRESI-MS calcd for [M+H]+ C19H 17Cl FN3O4S: 438.0691, found: 438.0693.Yellow solid, yield: 58%, http://www.reactionbiology.com) was used for the screening of tested compounds. Kinase Profiling is 10 dose IC50 singlet assay. Activity of kinases were assessed by the HotSpot assay platform, which contained specific kinase/substrate pairs along with required cofactors (Reaction Biology Corp. Kinase HotSpotSM service (ofactors .Molecular Operating Environment (MOE version 2008.10) by Chemical Computing Group (CCG) was used for the docking studies . The pro5a-i are represented in 1a–c were firstly refluxed in ethanol with diethyl ethoxymethylenemalonate to provide substituted phenylaminomethylenemalonates 2a–c. Compounds 2a-c were cyclized thermally in diphenyl ether to the corresponding 4-oxo-1,4-dihydroquinolines 3a–c. Under the anhydrous condition, quinolines 3a–c were chlorinated via heating with excess of POCl3 to provide the key intermediates 4a–c, as reported previously methanesulfonamide with the appropriate key intermediate 4a–c in ethanol  against CDK8/CycC are summarized in The newly prepared phenylaminoquinolines 5d, we obtained a co-crystal structure of 6-isocyano-N-phenethylquinazolin-4-amine (Senexin A ) in complexation with CDK8:Cyclin C with the DMG motif in the “in” conformation at 2.2 Åresolution (PDB: 4F7S).For designing CDK8/CycC type I inhibitor, targeting the hinge residue is essential to inhibit the kinase activity of the complex. To visualize the binding interactions between the promising biologically active compound 1H-NMR, 13C-NMR spectroscopic data and high-resolution mass spectroscopy (HRMS), the structures of all newly synthesized compounds were confirmed. 1HNMR spectra of all finals 5a-i showed new characteristic signals at δ 7.33–7.37 ppm, and 9.68–10.25 ppm corresponding to NH2 and NH groups, respectively, that distinguished the finals 5a-i from chloroquinolines 4a-c. For compound 5d, three characteristic signals at δ 2.42, 8.94, and 9.64 ppm were displayed and assigned to -NHCH3, -SO2NH- and -NH- protons, respectively.Based on According to the inhibition data stated in 5d showed maximal potency among all final compounds with submicromolar activity and IC50value of 0.639 µM, whereas, the corresponding primary benzenesulfonamide analogue 5a exhibited a 6-fold decrease in potency (IC50 = 3.98 µM). On the contrary, the corresponding substituted benzenesulfonamide analogue (5g) exhibited no CDK8/CycC complex inhibitory activity, confirming the crucial role of PKa values of sulfonamide groups for the intrinsic activity of pharmacophore of the phenylaminoquinoline scaffold–based compounds. Moreover, the primary benzenesulfonamide analogues (5a & 5b) exhibited single digit micromolar potency in inhibiting the CDK8/CycC. Whereas, methanesulphonamide analogues (5d & 5e) showed superior potency with IC50 values of 0.639 and 1.42 µM, respectively. Noteworthy, all 7-chloro-6-fluoro substituted quinolines  failed to inhibit the CDK8/CycC enzyme, signifying the remarkable adverse effects of some quinoline substituents on the binding interaction, and hence the intrinsic activity. Therefore, concerning the influence of substitution of the quinoline moiety, the inhibitory activities increased in the order of 7-Cl-6-F <6-OCH3 <6-Br.The methanesulfonamide analogue 5d in the 3D docking pose can anchor in the kinase deep pocket and extended with diverse functional groups toward the hinge region and the front pocket. Two direct hydrogen bonds are formed between the inhibitor 5d and the kinase domain of CDK8/CycC. The quinoline N forms an essential single H-bond with the backbone nitrogen of Ala100CDK8 on the hinge region. The sulfonyl O forms the second direct H-bond to the backbone N of Asp173CDK8 of the DMG motif. Moreover, π–π stacking interaction with the gatekeeper residue (Phe97CDK8) and VDW interactions with several residues at the ATP binding pocket  were shown as depicted in The virtual docking study showed how the compound 5a-i have been synthesized and biologically evaluated as potential CDK8/CycC inhibitors. The methanesulfonamide analogues (5d & 5e) proved to be the most potent compounds in suppressing CDK8/CycC enzyme, whereas, the un/substitutes benzenesulfonamide analogues showed inferior (or no) activity, demonstrating the advantage of highly acidic NH of sulfonamide moiety. Careful selection of quinoline moiety substituents is highly recommended, as 7-chloro-6-fluoro bearing analogues (5c & 5f) showed no inhibition. Moreover, the secondary benzenesulfonamide analogues should be avoided, as they showed no inhibitory activities. We have discovered the most potent analogue 5d with submicromolar potency against CDK8/CycC (IC50 = 0.639 µM) and it can be prepared in four steps with an overall yield of 64% making it suitable for further investigations. Larger library of phenylaminoquinoline scaffold-based analogues are going to be prepared by our team to establish detailed and distinguished SARs.We have demonstrated a new series of phenylaminoquinoline core structure-based compounds. These target compounds In summary, the novel ((3-(N-methylsulfamoyl)phenyl)amino)quinoline derivatives exhibited potent CDK8/CycC inhibitory activity. Developing inhibitors targeting CDK8/CycC offers an exciting approach for treatment of human carcinomas.10.7717/peerj.8649/supp-1Supplemental Information 1Click here for additional data file.


Ki, 16.9 µM), CYP2C9 , and CYP2C19  and the transport activity of OAT3 . It exhibited time-dependent inhibition on CYP3A4 . Other metabolizing enzymes and transporters such as CYP1A2, CYP2A6, CYP2B6, CYP2D6, UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9, UGT2B7, OAT1, OATP1B1, OATP1B3, OCT1, OCT2, P-glycoprotein, and BCRP, exhibited only weak interactions with AB-PINACA. These data suggest that AB-PINACA can cause drug-drug interactions with CYP3A4 substrates but that the significance of drug interactions between AB-PINACA and CYP2C8, CYP2C9, CYP2C19, or OAT3 substrates should be interpreted carefully.Indazole carboxamide synthetic cannabinoid, AB-PINACA, has been placed into Schedule I of the Controlled Substances Act by the US Drug Enforcement Administration since 2015. Despite the possibility of AB-PINACA exposure in drug abusers, the interactions between AB-PINACA and drug-metabolizing enzymes and transporters that play crucial roles in the pharmacokinetics and efficacy of various substrate drugs have not been investigated. This study was performed to investigate the inhibitory effects of AB-PINACA on eight clinically important human major cytochrome P450s (CYPs) and six uridine 5′-diphospho-glucuronosyltransferases (UGT) in human liver microsomes and the activities of six solute carrier transporters and two efflux transporters in transporter-overexpressing cells. AB-PINACA reversibly inhibited the metabolic activities of CYP2C8 ( N-[(2S)-1-amino-3-methyl-1-oxobutan-2-yl]-1-pentyl-1H-indazole-3-carboxamide) was originally developed by Pfizer Inc. as a synthetic cannabinoid receptor agonist -mephenytoin, 4′-hydroxymephenytoin, bufuralol, 1′-hydroxybufuralol, d9-1′-hydroxybufuralol, 1′-hydroxymidazolam, N-desethylamodiaquine, 4′-hydroxydiclofenac, 13C2,15N-acetaminophen, medium 199, fetal bovine serum (FBS), collagen-coated 24-Transwell plates, and poly-d-lysine-coated 96-well plates were purchased from Corning Life Sciences . 3H-Methyl-4-phenylpyridinium (2.9 TBq/mmol), 3H-para-aminohippuric acid (0.13 TBq/mmol), 3H-estrone-3-sulfate (2.12 TBq/mmol), 3H-estradiol-17β-d-glucuronide (2.22 TBq/mmol), 3H-digoxin (1.103 TBq/mmol), and Optiphase scintillation cocktail were purchased from PerkinElmer Inc. . SN-38 was a product of Santa Cruz Biotechnology . SN-38 glucuronide, troglitazone, diclofenac, mycophenolic acid β-d-glucuronide, chenodeoxycholic acid 24-acyl-β-glucuronide, and N-acetylserotonin β-d-glucuronide were purchased from Toronto Research Chemicals . All other chemicals were of the highest quality available. LLC-PK1-BCRP cells (LLC-PK1 cells stably expressing BCRP) were obtained from Dr. A.H. Schinkel . LC-MS grade acetonitrile, methanol, and water were the products of from Fisher Scientific Co. .AB-PINACA and midazolam were purchased from Cayman Chemical Company . Acetaminophen, 2PO4 buffer (pH 7.4), ultrapooled human liver microsomes (0.2 mg/mL), 10 mM MgCl2, 1.0 mM NADPH, and two sets of a cocktail of eight CYP probes ), and AB-PINACA (0.1–100 μM) were incubated for 15 min at 37 °C in a shaking incubator  with or without preincubation with NADPH (30 min). After 15 min, the reaction was quenched by adding 100 μL of ice-cold methanol containing internal standards (IS) , followed by centrifugation at 13,000× g for 8 min at 4 °C. Supernatant from A and B sets (50 μL each) were mixed and 5 μL of the mixed supernatants were analyzed by LC-MS/MS. IC50 values were calculated by mean values of triplicate assays.The effects of AB-PINACA on CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 activities were measured in ultrapooled human liver microsomes as described in our previous report . Human lKi values and inhibition modes for the inhibition of AB-PINACA on CYP2C8-, CYP2C9-, and CYP2C19-catalyzed hydroxylation of amodiaquine, diclofenac, and [S]-mephenytoin were measured as described in our previous report -mephenytoin for CYP2C19  were incubated at 37 °C in a shaking incubator. After 15 min, the reaction was quenched by adding 100 μL of ice-cold methanol containing 13C2,15N-acetaminophen (IS) for CYP2C8 or d9-1′-hydroxybufuralol (IS) for CYP2C9 and CYP2C19, followed by centrifugation at 13,000× g for 8 min at 4 °C.The s report . Human lKi and kinact) for time- and concentration-dependent inhibition of AB-PINACA against CYP3A4 activity in ultrapooled human liver microsomes were evaluated according to our previous report estrone-3-sulfate and AB-PINACA (0–100 μM) on the basal side. Aliquots (100 μL) of transport samples were mixed with 200 μL of Optiphase scintillation cocktail. The radioactivity of the probe substrate in the cocktail mixture was measured using a liquid scintillation counter. The B to A transport of digoxin and ES mediated by MDR1 and BCRP, respectively, was calculated by subtracting the transport of probe substrate in LLC-PK1-mock cells from that in LLC-PK1-MDR1 and LLC-PK1-BCRP cells and the B to A transport rate of probe substrate was calculated from the slope of the B to A transport of probe substrate versus time graph -mephenytoin with IC50 values of 32.5 μM, 17.4 μM, and 20.0 μM, respectively, in human liver microsomes, but 30-min preincubation of AB-PINACA with NADPH did not significantly change the inhibitory potential of AB-PINACA toward CYP2C8, CYP2C9, and CYP2C19. However, preincubation of AB-PINACA with NADPH resulted in a 2.7-fold IC50 shift of CYP3A4-catalyzed midazolam 1′-hydroxylation   , Table 1N-deethylation, CYP2C9-catalyzed diclofenac 4′-hydroxylation, and CYP2C19-mediated [S]-mephenytoin 4′-hydroxylation with different substrate concentrations and varying concentrations of AB-PINACA and the results were transformed into Lineweaver–Burk and Dixon plots to determine the inhibition mode of AB-PINACA on the catalytic activities of CYP2C8, CYP2C9, and CYP2C19 along with the Ki values . AB-PINACA significantly inhibited OAT3-mediated ES uptake in a concentration dependent manner with an IC50 value of 11.8 µM. AB-PINACA did not significantly inhibit the transport activities of OCT2, OAT1, OATP1B1, OATP1B3, P-gp, or BCRP in the concentration ranges tested . Before docking, we removed each substrate from the CYP2C8, CYP2C9, CYP2C19, and CYP3A4 co-crystal structures, conducted structure optimization for refinement using the minimization protocol, and performed rigid- and auto-docking with standard protocol. The results yielded docking scores for AB-PINACA of −29.36, −26.99, −33.15, and −32.34 kcal/mol against CYP2C8, CYP2C9, CYP2C19, and CYP3A4, respectively  by Ki (17.6 μM) was 2.3 mL/μmol/min. Similar time-dependent inhibition of CYP3A4 has been reported for EAM-2201 , a halogenated naphthoylindole synthetic cannabinoid [kinact/Ki, 10.3 mL/μmol/min), an indazole carboxamide synthetic cannabinoid [AB-PINACA inhibited CYP3A4-catalyzed midazolam 1′-hydroxylation in a time-dependent manner . The ina1) by Ki .6 μM wasnabinoid . The calnabinoid . These fnabinoid .N-deethylation , and CYP2C9-catalyzed diclofenac 4′-hydroxylation  with a mixed mode of inhibition  of CYP2C8 -1-pentyl-1H-indazole-3-carboxamide) showed time-dependent inhibition on CYP3A4, which was similar to AB-PINACA. AB-FUBINACA (N-(1-amino-3-methyl-1-oxobutan-2-yl)-1-(4-fluorobenzyl)-1H-indazole-3-carboxamide) did not inhibited CYP3A4 but reversibly inhibited CYP2B6, CYP2C8, CYP2C9, CYP2C19, and CYP2D6. AB-PINCACA competitively inhibited CYP2C8, CYP2C9, and CYP2C19 in this study. We also elucidated that the pentyl group at indazole ring and amino-3-methyl-1-oxobutane moiety interact with the active sites of CYP2C8, CYP2C9, CYP2C19, and CYP3A4 through the hydrogen bonding and hydrophobic interaction via molecular docking results (We investigated the in vitro inhibitory effects of AB-PINACA, AB-FUBINACA, and APINACA, structural analogues of indazole carboxamide synthetic cannabinoids on the CYPs, UGTs, and transporter activities in this study and the previous reports ,19. APIN results . ConsideKi values of 145.7 μM and 8.3 μM, respectively (Ki values of AB-PINACA for OCT1 and OAT3 is suspected for AB-PINACA intoxication [AB-PINACA inhibited OCT1-mediated methyl-4-phenylpyridinium uptake with a mixed mode of inhibition and also competitively inhibited OAT3-mediated estrone-3-sulfate uptake with ectively . Althougxication . Thus suKi values.The CYP, UGT, and transporter-mediated drug interaction potentials of AB-PINACA were evaluated based on in vitro inhibitory effects of AB-PINACA on major CYP and UGT enzyme activities and the transport activities of eight drug transporters. Clinically significant drug interaction potentials between AB-PINACA and CYP2C8, CYP2C9, CYP2C19, CYP3A4, or OAT3 substrates should be evaluated carefully, although the plasma concentrations of AB-PINACA reported in drug abusers were much lower than the


The two-step acidic hydrolysis of α-hydroxybenzylphosphonates and a few related derivatives was monitored in order to determine the kinetics and to map the reactivity of the differently substituted phosphonates in hydrolysis. Electron-withdrawing substituents increased the rate, while electron-releasing ones slowed down the reaction. Both hydrolysis steps were characterized by pseudo-first-order rate constants. The fission of the second P-O-C bond was found to be the rate-determining step. Most often, the hydrolyses were performed under acidic conditions ,2,3,4, b3SiBr ,10,11. U3SiBr . Then, t3SiBr . α-Hydro3SiBr . α-Hydro3SiBr , can be 3SiBr , and may3SiBr . The cat3SiBr . Moreove3SiBr . In this1a–j) prepared as described earlier [1) at reflux resulted in complete hydrolysis within 2.5–9.5 h depending on the substituents. The reactions followed a two-step protocol and took place via the corresponding ester–acid intermediate 2, and were monitored by 31P NMR spectroscopy , k1 was 2.03, while k2 was 0.61 (1f) was the slowest, as a complete hydrolysis required 8 h, and the rate constants were found to be 1.64 (k1) and 0.31 (k2)  /Entry 6.1g–j), the hydrolysis of the unsubstituted model (1g) was significantly slower than that of the dimethyl analogue (1a)   required shorter reaction times of 5.5–9.0 h as compared with that (9.5 h) of the unsubstituted instance (1g)  determined the overall reactivity.Regarding the series of substituted diethyl α-hydroxybenzylphosphonates (of 9.5 h /Entry 7 of 9.5 h /Entry 1)4k), diethyl α-phenylethylphosphonate (4l), and diethyl β-phenylethylphosphonate (4m) were also subjected to hydrolysis, under the conditions applied for the α-hydroxybenzylphosphonates (1a–j) above (4k) took longer than that of the α-hydroxy derivative 1g  were obtained in this case (4m) with a reaction time of 20 h and k values of 0.70 h−1 and 0.15 h−1  above . It was his case /Entry 2.0.15 h−1 /Entry 3 N2 mechanism, i.e., by the nucleophilic attack of the water molecule on the P=O function. In the consecutive series, the fission of the second P-O-C bond is the rate-determining step.It is noted that the hydrolyses of the phosphonate function take place via the S1a–j and 4k–m) observed under acidic conditions was summarized in The overall order of reactivity of the phosphonates . High-resolution mass spectrometric measurements were performed using a Thermo Velos Pro Orbitrap Elite hybrid mass spectrometer in positive electrospray mode.The 31P NMR spectra.The composition of the reaction mixture was determined by the integration of the areas under the corresponding peaks of the starting material, intermediate, and product in the k1 and k2. The corresponding differential equations used in the model are the following:k1 and k2 are the pseudo-first-order rate constants of the first and the second step of the hydrolysis.The acidic hydrolysis was modeled assuming pseudo-first-order kinetics. The concentration of water and hydrochloric acid was constant during the reaction, and their initial concentration is incorporated in the pseudo-first-order rate constants k1 ≠ k2):c0 is the initial molarity of the dialkyl α-hydroxyphosphonate.The solution of the differential equations is the following (c0). The calculated time–composition curves are described by the following equations, by leaving the k1 and k2 rate constants as parameters:The relative quantity of the components is their molarity divided by the sum of the three molarities  corresponding to the maximal ratio of the phosphonic ester–acid intermediate in the reaction mixture was found as follows:The reaction time , 1.0 mL (6.0 mmol) of cc. hydrochloric acid, and 2.0 mL of water was stirred at reflux for 2.5–25 h. The concentration of an aliquot part of the reaction mixture, or the whole mixture, afforded an oil that was analyzed by 31P NMR spectroscopy and LC-MS. Identification of the starting materials , intermediates , and products  can be found in 13C and 1H NMR spectral data of the new intermediates  were obtained from the spectra of the corresponding mixtures containing also the phosphonic acids (3A–F).A mixture of 3.8 mmol of phosphonate (13C and 1H NMR characterization of the new ester acids:Methyl hydrogen 1-(4-nitrophenyl)-1-hydroxymethylphosphonate (2b). 13C NMR (DMSO-d6) δ: 52.6 , 69.1 , 122.7 , 128.1 , 146.5 , 147.6 (C1); 1H NMR (DMSO d6) δ: 3.58 , 5.05 , 7.64–7.73 , 8.17–8.23 .Methyl hydrogen 1-(4-chlorophenyl)-1-hydroxymethylphosphonate (2c). 13C NMR (DMSO-d6) δ: 53.0 , 69.4 , 128.1 , 129.5 , 132.1 , 139.0 (C1); 1H NMR (DMSO-d6) δ: 3.55 , 4.85 , 7.32–7.47 .Methyl hydrogen 1-(4-fluorophenyl)-1-hydroxymethylphosphonate (2d). 13C NMR (DMSO-d6) δ: 53.0 , 69.4 , 114.9 , 129.7 , 136.1 , 161.9 ; 1H NMR (DMSO-d6) δ: 3.54 , 4.84 , 7.09–7.19 , 7.38–7.49 .Methyl hydrogen 1-(4-trifluoromethylphenyl)-1-hydroxymethylphosphonate (2e). 13C NMR (DMSO-d6) δ: 53.1 , 69.7 , 124.7–125.0 , 124.9 , 127.3–128.7 , 145.0 (C1); 1H NMR (DMSO-d6) δ: 3.56 , 4.96 , 7.57–7.72 .Methyl hydrogen 1-(4-methylphenyl)-1-hydroxymethylphosphonate (2f). 13C NMR (DMSO-d6) δ: 21.2 (Ar-CH3), 52.8 , 70.0 , 127.7 , 128.7 , 136.5 , 136.9 (C1); 1H NMR (DMSO-d6) δ: 2.28 , 3.52 , 4.76 , 7.06–7.38 .Ethyl hydrogen 1-(4-nitrophenyl)-1-hydroxymethylphosphonate (2h) 13C NMR (DMSO-d6) δ: 16.9 , 62.0 , 69.1 , 122.7 , 128.2 , 146.5 , 147.6 (C1); 1H NMR (DMSO-d6) δ: 1.17 , 3.91 , 5.03 , 7.65–7.72 , 8.16–8.23 .Ethyl hydrogen 1-(4-chlorophenyl)-1-hydroxymethylphosphonate (2i). 13C NMR (DMSO-d6) δ: 16.6 , 61.7 , 69.3 , 127.7 , 129.2 , 131.8 , 138.6 (C1); 1H NMR (DMSO-d6) δ: 1.15 , 3.91 , 4.84 , 7.33–7.47 .Ethyl hydrogen 1-(4-fluorophenyl)-1-hydroxymethylphosphonate (2j). 13C NMR (DMSO-d6) δ: 16.9 , 61.9 , 69.5 , 114.8 , 129.7 , 136.1 , 161.9 ; 1H NMR (DMSO-d6) δ: 1.15 , 3.87–3.95 , 4.82 , 7.11–7.17 , 7.42–7.47 .1-(4-Trifluoromethylphenyl)-1-hydroxymethylphosphonic acid (3e). 13C NMR (DMSO-d6) δ: 70.5 , 124.7–125.0 , 124.9 , 127.2–128.6 , 128.4 , 145.6 (C1); 1H NMR (DMSO-d6) δ: 4.80 , 7.55–7.72 .1 and k2 pseudo-first-order rate constants belonging to the formation of the corresponding monoester monoacids and the phosphonic acids, respectively. Electron-withdrawing substituents increased the rate, while electron-releasing ones slowed down the hydrolyses starting with the nucleophilic attack of the water molecule. It turned out that the fission of the second P-O-C unit is the rate-determining step. The intermediate ester–acid species were identified and characterized.Kinetic study of the two-step acidic hydrolysis of a series of dialkyl α-hydroxybenzylphosphonates and a few related model compounds allowed the mapping of the reactivity of the different substrates. The two-step hydrolyses were characterized by k


Fourier transformer infrared analysis (FTIR), Proton nuclear magnetic resonance (1H NMR) and 13C nuclear magnetic resonance (13C NMR), as well as that of the mass spectral data, were used as the appropriate characterization techniques for the chemical structures of all newly synthesized compounds. The newly prepared compounds were examined as an anti-inflammatory, antibacterial agents (against E. coli (Escherichia coli) and P. aeruginosa (Pseudomonas aeruginosa) as examples for Gram-negative bacteria and S. aureus (Staphylococcus aureus) as examples for Gram-positive bacteria), as well as antifungal ) agents. The newly prepared compound showed high antibacterial, antifungal, and anti-inflammatory activities in comparing with the commercial antibiotics Indomethacin, Nalidixic acid, Imipenem, and Nystatin. Docking of the most active compounds was performed depending on the results of antibacterial screening and the anti-inflammatory assay.Different novel 1,2,4-triazolotetrazines and 1,2,4-triazolotriazines have been obtained from heterocyclization of 3-substituted-4-amino-5-substituted-amino-1,2,4-triazoles ( For example, many of 1,2,4-triazole rings are found into a wide range of pharmaceutical drugs including antimicrobial agents25. Thus, for many years, the biochemistry of these molecules has been investigated26. Also, Heterobicyclic nitrogen systems containing 1,2,4-Triazines derivatives and 1,2,4-Triazines themselves have been found to display a diversity of biological applications such as Lamotrigine as anti-epileptic drug27, Tirapazamine as anti-tumor28, and fused 1,2,4–triazines as antimicrobial30, anti–viral31, antimycobacterial32, anxiolytic33 and antidepressant34 activities. They also have shown anti–HIV and anticancer activities35. Moreover, derivatives of the tetrazine ring have attracted extensive attention from numerous research groups because of their interesting and diverse biological activities as antitumor and antiviral36. In this respect, Abdel-Rahman et al.39 found that 1,2,4-triazines, 1,2,4-triazoles and/or 1,2,4-triazolo-1,2,4,5-tetrazines can be used as a molluscicidal and antimicrobial as well as they have anticancer drugs activity. Due to their novel energetic properties40, organic compounds with high-nitrogen content currently attract the attention of many researchers.1,2,4-triazole rings possess not only diverse pharmacological activities but also to have herbicidal, insecticidal, plant growth regulatory and antifungal activitiesIn light of these remarks, researchers were prompted to design and synthesize new drugs containing heterocyclic compounds, especially, those containing triazoles, triazines and tetrazines rings as a result of the fact that Nitrogen–Nitrogen bond is difficult to be produced in living organisms in addition to its therapeutic activities. Thus, because of these remarks, the target of this work is to synthesize some new 1,2,4-triazole fused with 1,2,4-triazine and/or 1,2,4,5-tetrazine nucleus holding different types of functional groups to enhance their biological activity, in a hope to design a semi-drug. To demonstrate whether COX-II was a potential target for our newly prepared compounds, molecular modeling studies have been conducted on these compounds.1a,b, and 2a,b with 1:1 molar ratio in 100 mL Ethanol-DMF was refluxed for 5 h, then poured into ice and the formed solids were collected by filtration and crystallized to give compounds 3a to 3d, respectively. To prepare the compounds 5a–d, a mixture of (0.01 mol) of 3a–d and (0.01 mol) of chloromethyl-diphenylphosphanoxide in (100 mL) THF with (0.5 mL) TEA was refluxed for 5 h and cooled. The obtained solids were then were collected by filtration and crystallized to give 5a–d, respectively. When (0.01 mol) of 3a–d were mixed with (0.01 mol) of phenacyl bromide in 50 mL from 5% ethanolic KOH and heated under reflux for 3 h, then poured into ice and acidified with few drops of HCl. The yielded solids were filtered and crystallized to give compounds 6a–d respectively. Compounds 7a–d were prepared by mixing a mixture of (0.01 mol) 3a–d and (0.01 mol) sodium pyruvate, in 10 mL H2O with 50 mL 5% aqueous NaOH. After that, the mixture was refluxed for 3 h, then poured into ice and acidified with few drops of HCl. The products filtered and recrystallized to give 7a–d compounds respectively. Equimolar ratio mixture of both compounds 3a–d and diethyl oxalate in 100 mL THF was refluxed for 5 h and cooled. The obtained products were filtered and crystallized from the proper solvent to give 8a–d respectively. A mixture of each of 1a,b (0.01 mol) and hydrazine hydrate (0.04 mol) in ethanol (100 mL) was refluxed for 6 h and cooled. Then a few drops of acetic acid were added. The produced solids were filtered off and recrystallized to give 9a,b respectively42. When the equimolar ratio of each of 9a,b and triethylphosphite in tetrahydrofuran (100 mL) and triethylamine (0.5 mL) was refluxed 6 h and cooled. The obtained solids were filtered off and recrystallized to give compounds 10a,b respectively. A mixture of 9a,b (0.01 mol) and triethylorthoformate (0.012 mol) in tetrahydrofuran (100 mL) was refluxed for 6 h and cooled. The produced solids were filtered off and recrystallized to give 11a,b respectively. The spectral data together with the physical constants of 11a,b are listed below. A mixture of 9a,b (0.01mole) and 2-chloro-6-fluorobenzeldehyde (0.01 mol) in ethanol (100 mL)/piperidine (0.5 mL) was refluxed 12 h and then cooled. The produced solids were filtered off and crystallized to give 12a,b respectively. A mixture of each of 9a,b (0.01 mol), CS2 (0.02 mol) in DMF (50 mL) was refluxed for 3 h and cooled, then poured onto the ice. The resulting solids so formed were filtered off and recrystallized to give 13a,b respectively. A mixture of each of 9a,b (0.01 mol) and ethyl chloroformate (0.012 mol) with tetrahydrofuran (100 mL) and triethylamine  was refluxed for 3 h and then cooled. The solids obtained were filtered off and recrystallized to give 14a,b respectively.A mixture of compounds 1) was prepared by direct hydrazinolysis of 4-pyridyl-CONHNHCS2K according to Reid et al. reported method43.The first compound  spectrometer (a Perkin Lemer Spectrum RXI FT-IR systems No. 55529) was used. FT-IR spectra within the wavenumber ranged from 4000–600 cm1H/ 13C-NMR Spectra were recorded in (DMSO-d6) at 400 MHz with a Bruker NMR Advance DPX 400 MH Spectrometer. TMS was used as an internal reference. Chemical shifts were supposed to be due to the presence of the solvent.The melting points of the synthesized compounds were determined by using SMP (Stuart Scientific melting point) (USA).GCMS-Q 1000 Ex spectrometer was used to measure the mass spectra of the prepared materials. Shimadzu UV-visible 3101 PC Spectrophotometer was used to record the electronic absorption spectra of the synthesized materials in DMF.8 (CFU) colony-forming units/ mL was prepared by aging a single colony on an agar plate for 24 hours. The antibacterial activity of some newly synthesized compounds was investigated on Mueller-Hinton agar using Agar diffusion techniques at concentrations of 100, 50 and 25 μg/mL, respectively. Muller Hinton agar (MHA) plates were inoculated with test inoculum  of standard inoculum (0.5 McFarland). Nalidixic acid and Imipenem were used as a reference drug for bacteria. Briefly, MHA agar plates were inoculated with bacterial strains under sterile conditions, and disc (diameter = 8 mm) was loaded with 50 μL of the tested sample. The plate was incubated at 37 °C for 24 hours. Nalidixic acid and Imipenem were used as a reference drug for bacteria. After the incubation period, the antimicrobial agent usually diffuses into the agar and inhibits the germination and growth of the test microorganism, and then according to the Clinical Laboratory Standards (CLSI), the diameter of the growth-inhibiting zone was measured44. The MICs of the prepared compounds were determined by the agar dilution plate technique following the standard procedure of the Clinical and Laboratory Standards44. The MICs were calculated from the X-axis intercept of the linear graph between log (Inhibitory concentration) and the growth inhibitory zone area.A bacterial suspension having a density of about 1 to 2 ×105 CFU/mL cells. Fungal cultures (0.1 mL) were spread evenly on Sabourad dextrose agar plates. The plates were inoculated with fungal strains under sterile conditions, and disc (diameter = 8 mm) was loaded with 50 μL of the test sample. The plate was then incubated at 30 °C for 3–4 days and according to the Clinical Laboratory Standards (CLSI), the diameter of the growth-inhibiting zone was measured44. Nystatin was used as antifungal standard drug45.The fungal strain was grown in 5 mL Sabourad Glucose Broth  for 3–4 days to reach 1047. Indomethacin drug as a suspension in 24 tweens 80 was used as the reference drug, using Winter et al. method47. The inhibition percentage of inflammation was calculated using the following equation:Carrageenan-induced paw edema method was used to evaluate the anti-inflammatory effect of the selected prepared compoundsMicroanalyses  and anti-inflammatory activity of the prepared compounds were carried out in the pharmaceutical Microbiology Department, National Center for Radiation Research and Technology (NCRRT), Nasr City, Egypt.5d and the positive control, Nalidixic acid, in case of antibacterial screening was performed with the binding site of DNA Gyrase (topoisomerase II) enzyme (PDB ID: 2XCT). Also, docking of compounds 10a,b, 12a,b and the positive control Indomethacin in case of anti-inflammatory effect was performed with the binding site of COX-2 (PDB ID: 1CX2). Docking was done to shed light on their potential binding modes and investigate their similarity to the standard ligand binding modes50.Based on the results of antibacterial screening and the anti-inflammatory assay, docking of the most active compound X-ray crystal structure of DNA Gyrase (topoisomerase II) complexed with a Ciprofloxacin was determined at 3.35 Å resolution and X-ray crystal structure of COX-2 complexed with a selective inhibitor, sc-558 at 3 Å resolution. All molecular modeling calculations and docking studies were carried out using ‘Molecular Operating Environment 2019.0101’ software  running on a Windows 10 PC.2XCT) and cyclo-oxygenase 2 (PDB ID: 1CX2) were downloaded from the protein data bank (http://www.rcsb.org/)51. The enzyme was prepared for docking study by removal of chain B, C and D of its dimmers, water molecules, and ligands that are not involved in the binding. The enzyme was then prepared using quick preparation protocol in MOE with default options.The X-ray crystallographic structures of DNA Gyrase (topoisomerase II) enzyme , the coordinates of the native ligand in the co-crystallized PDB file were compared with the coordinates of the greatest scoring docking pose of the native ligand. The docking validation results revealed a near-perfect alignment with the original ligand as attained from the resolved X-ray PDB file. In the case of antibacterial screening, docking validation was confirmed from the small root mean square deviation (rmsd = 0.3155) between the docked pose and the co-crystallized ligand (S (energy score) of −11.2658 kcal/mol). Docking validation was also demonstrated by the ability of the docking poses to restore the main interactions that occur between the co-crystallized ligand and the active site’s hot spots, Manganese (Mn) atom and DG 5 of DNA Gyrase enzyme. In case of anti-inflammatory effect, the re-docked ligand showed a small root mean square deviation value (rmsd = 0.0215) between the docked compound and the co-crystallized ligand , they also showed a high ability to repeat the main interactions that occur between the co-crystallized ligand and the active site’s hot spots, Arg513 and His90 of COX-II.Active compounds preparation for docking was done as follows: firstly, Marvin Sketch was used to built up the 2D structures of the docked ligands and copied onto MOE. This step is followed by 3D protonation of the active compounds structure. Then the systemic search was used for the running of the conformational analysis and then the smallest energetic conformer was selected. An identical docking protocol was applied with the ligand.2XCT (PDB ID: 2XCT) and also COX-II enzyme co-crystallized with the native ligand of protein data bank file ID: 1CX2 (PDB ID: 1CX2). Posing compounds 5d and Nalidixic acid, in case of antibacterial screening, and compounds 10a,b, 12a,b and the positive control Indomethacin, in case of anti-inflammatory effect, was scored by London dG scoring function, that was used for docking, and Triangle Matcher placement protocol. The docked compounds of the greatest scoring pose were documented. Interactions between ligand and receptor in the formed complexes were tested in both 3D and 2D styles.Docking studies were performed using DNA Gyrase (topoisomerase II) enzyme co-crystallized with the native ligand of protein data bank file ID: 3-(5-chloropyrimidin-2-yl)-5-phenyl-4H-1,2,4-triazole-3,4-diamine):For compound (3a) : C, 50.10; N, 34.08; H, 3.50; Cl, 12.32. The analyses found for the compound are: C, 50.16; H, 3.57; Cl, 12.30; N, 34.15). The UV spectrum gave [λmax (Log ε)]: 372 nm. IR characteristic peaks appear at (ν cm−1): 3449, 3318 (NH2), 3189 (NH), 1601, 1581(C=N). 1H NMR characteristic peaks appear at δ ppm: 5.87, NH2), 4.0, NH), 8.05-pyrimidine), 7.41–8.28 . 13C NMR characteristic peaks appear at δ ppm: 119.0 , 126.50,129.20 (C2 and C3 of benzene), 131.1 (C4 of benzene), 130.6 (C1 of benzene), 151.1 (C5 of triazole), 157.2 (C2 of triazole), 167.9 (C2 of pyrimidine), 156.8 (C4 and C6 of pyrimidine). MS (Int.%): 287 , 271, 174 , 159 , 128 , 113 .Yield 68% crystal from dioxan; mp 213–215 3-(5-chloropyrimidin-2-yl)-5-(pyridin-4-yl)-4H-1,2,4-triazole-3,4-diamine):For compound (3b) : C, 45.76; H, 3.14; Cl, 12.28; N, 38.81. The analyses found for the compound are: C, 45.70; H, 3.11; Cl, 12.30; N, 38.83). The UV spectrum gave [λmax (Log ε)]: 374 nm. IR characteristic peaks appear at (ν cm−1): 3442, 3325 (NH2), 3200 (NH), 1601,1581(C=N). 1H NMR characteristic peaks appear at δ ppm: 5.77, NH2), 4.0, NH), 8.05-pyrimidine), 8.75, (CH)-pyridine), 7.99, (CH)-pyridine). 13C NMR characteristic peaks appear at δ ppm: 119.0 , 121.3,149.8 (C2 and C3 of pyridine), 134 (C4 of pyridine), 157.2 (C5 of triazole), 151.1 (C2 of triazole), 167.9 (C2 of pyrimidine), 156.8 (C4 and C6 of pyrimidine). MS (Int.%): 288 , 272, 175 , 160 , 128 , 113 .Yield 61% crystal from dioxan; mp 222–225 3-(5-bromopyrimidin-2-yl)-5-phenyl-4H-1,2,4-triazole-3,4-diamine):For compound (3c) : C, 43.39; H, 3.03; Br, 24.06; N, 29.52. The analyses found for the compound are: C, 43.40; H, 3.01; Br, 24.08; N, 29.55). The UV spectrum gave [λmax (Log ε)]: 370(0.80)nm. IR characteristic peaks appear at (ν cm−1): 3440, 3350 (NH2), 3195 (NH), 1600, 1578 (C=N). 1H NMR characteristic peaks appear at δ ppm: 5.71, NH2), 3.7, NH), 8.53-pyrimidine), 7.04–8.25. 13C NMR characteristic peaks appear at δ ppm: 110.7 , 127.5,129.2 (C2 and C3 of benzene), 131.1 (C4 of benzene), 130.6(C1 of benzene), 157.2 (C5 of triazole), 151.1 (C2 of triazole), 168.4 (C2 of pyrimidine), 159.1 (C4 and C6 of pyrimidine). MS (Int.%): 331 , 316 , 177 , 162 , 170 , 155 .Yield 52% crystal from dioxan; mp 288–291 3-(5-bromopyrimidin-2-yl)-5-(pyridin-4-yl)-4H-1,2,4-triazole-3,4-diamine):For compound (3d) : C, 39.66; H, 2.72; Br, 23.98; N, 33.63 C, 39.66; H, 2.72; Br, 23.98; N, 33.63. The analyses found for the compound are: C, 39.62; H, 2.75; Br, 24.00; N, 33.65). The UV spectrum gave [λmax (Log ε)]: 374(0.84) nm. IR characteristic peaks appear at (ν cm−1): 3452, 3367 (NH2), 3188 (NH), 1600,1568 (C=N). 1H NMR characteristic peaks appear at δ ppm: 5.68, NH2), 4.2, NH), 8.56-pyrimidine), 8.82, (CH)-pyridine), 7.95, (CH)-pyridine). 13C NMR characteristic peaks appear at δ ppm: 110.7 , 121.3, 149.8 (C2 and C3 of pyridine), 134.0 (C4 of pyridine), 157.2 (C5 of triazole), 151.1 (C2 of triazole), 168.4 (C2 of pyrimidine), 159.1 (C4 and C6 of pyrimidine). MS (Int.%): 332 , 317, 178 , 163 , 170 .Yield 48% crystal from dioxane; mp 257–259 For compound (5a) (8-(5-chloropyrimidin-2-yl)-3,6,6-triphenyl-5,6,7,8-tetrahydro-triazolo- triazaphosphinin-6-ol):oC. Analysis calculated for C25H21ClN7OP (501): C, 59.83; H, 4.22; Cl, 7.06; N, 19.53; P, 6.17. The analyses found for the compound are: C,59.80; H, 4.23; N,19.56; Cl, 7.08, P, 6.18. IR characteristic peaks appear at (ν cm−1): 3080(aromatic (CH)), 2925), 1650(P-OH), 1522(C=N). 1H NMR characteristic peaks appear at δ ppm: 2.8, 2.0, NH), 3.1, CH2), 7.41–8.05, 8.08-pyrimidine). 13C NMR characteristic peaks appear at δ ppm: 119.0 , 130.6 , 127.5 (C2 of benzene attached with triazole), 129.2 (C3 of benzene attached with triazole), 131.1 (C4 of benzene attached with triazole), 131.2 (C-P of benzene), 84 (N-C-P of triaziphosphine), 157.2 (C5 of triazole), 151.1 (C2 of triazole), 167.9 (C2 of pyrimidine), 156.6 (C4 and C6 of pyrimidine).Yield 56%, crystals from dioxan; mp 277–279 For compound (5b) (8-(5-chloropyrimidin-2-yl)-3-(pyridine-4-yl)-6,6-diphenyl-5,6,7,8-tetrahydrotriazolotriazaphosphinin-6-ol):oC. Analysis calculated for C24H20ClN8OP (502):C, 57.32; H, 4.01; Cl, 7.05; N, 22.28; O, 3.18; P, 6.16. The analyses found for the compound are: C, 57.40; H, 3.98; N,22.30; Cl, 7.07, P, 6.18. IR characteristic peaks appear at (ν cm−1): 3078(aromatic (CH)), 2943), 1647(P-OH), 1518(C=N). 1H NMR characteristic peaks appear at δ ppm: 3.1, 2.3, NH), 3.6, CH2), 7.35–8.09, 8.21-pyrimidine), 8.75, (CH)-pyridine), 7.99, (CH)-pyridine). 13C NMR characteristic peaks appear at δ ppm: 119.0 , 134(C4 of pyridine), 121.3 (C3 and C5 of pyridine), 149.8 (C2 and C6 of pyridine), 131.2 (C-P of benzene), 131.2 (C2 and C6 of benzene), 128.7 , 84.3 (N-C-P of triaziphosphine), 157.2 (C2 of triazole), 151.1 (C5 of triazole), 167.9 (C2 of pyrimidine), 156.6 (C4 and C6 of pyrimidine).Yield 43%, crystals from dioxan; mp 300–302 For compound (5c) (8-(5-Bromopyrimidin-2-yl)-3,6,6-triphenyl-5,6,7,8-tetrahydro triazolo triazaphosphinin-6-ol):oC. Analysis calculated for C25H21BrN7OP (554): C, 54.96; H, 3.87; Br, 14.62; N, 17.95; O, 2.93; P, 5.67. The analyses found for the compound are: C, 54.94; H, 3.86; Br, 14.65; N, 18.00; O, 2.93; P, 5.70. IR characteristic peaks appear at (ν cm−1): 3073(aromatic (CH)), 2952), 1643(P-OH), 1519 (C=N). 1H NMR characteristic peaks appear at δ ppm: 3.3, 2.4, NH), 3.6, CH2), 7.30–8.05, 8.34-pyrimidine). 13C NMR characteristic peaks appear at δ ppm: 110.7 (C-Br of pyrimidine), 130.6 , 127.5 (C2 and C6 of benzene attached with triazole), 129.2 (C3 and C5 of benzene attached with triazole), 131.1 (C4 of benzene attached with triazole), 131.2 (C-P of benzene), 84.3 (N-C-P of triaziphosphine), 157.2 (C5 of triazole), 151.1 (C2 of triazole), 168.4 , 159.1 (C4 and C6 of pyrimidine).Yield 51%, crystals from dioxan; mp 222–225 For compound (5d) (8-(5-Bromopyrimidin-2-yl)-3-(4-pyridin-4-yl)-6,6-triphenyl-5,6,7,8-tetrahydrotriazolotriazaphosphinin-6-ol):oC. Analysis calculated for C24H20BrN8OP (554): C, 54.96; H, 3.87; Br, 14.62; N, 17.95; O, 2.93; P, 5.67. The analyses found for the compound are: C, 54.94; H, 3.86; Br, 14.65; N, 18.00; O, 2.93; P, 5.70. IR characteristic peaks appear at (ν cm−1): 3073(aromatic (CH)), 2952), 1643(P-OH), 1519 (C=N). 1H NMR characteristic peaks appear at δ ppm: 3.3, 2.4, NH), 3.6, CH2), 7.30–8.05, 8.34-pyrimidine). 13C NMR characteristic peaks appear at δ ppm: 110.7 (C-Br of pyrimidine), 130.6 , 127.5 (C2 and C6 of benzene attached with triazole), 129.2 (C3 and C5 of benzene attached with triazole), 131.1 (C4 of benzene attached with triazole), 131.2 (C-P of benzene), 84.3 (N-C-P of triaziphosphine), 157.2 (C5 of triazole), 151.1 (C2 of triazole), 168.4 , 159.1 (C4 and C6 of pyrimidine).Yield 39%, crystals from dioxan; mp 267–269 For compound (6a) (8-(5-chloropyrimidin-2-yl)-6-phenyl-3-(pyridin-2-yl)-7,8-dihydro-triazolo- triazine):oC. Analysis calculated for C19H13ClN8 (388): C, 58.69; H, 3.37; Cl, 9.12; N, 28.82. The analyses found for the compound are: C, 58.7; H, 3.35; N, 28.9; Cl, 9.15. IR characteristic peaks appear at (ν cm−1): 3079.20, 2966 cm−1 ), 1522(C=N). 1H NMR characteristic peaks appear at δ ppm: 3.13, CH2) triazine ring, 8.05-pyrimidine), 7.52–7.94, 8.38 , 8.59, 7.36, 7.85. 13C NMR characteristic peaks appear at δ ppm: 134.7 (C1 of benzene), 128.2 , 128.8 (C3 and C5 of benzene), 131.0 (C4 of benzene), 157.2 (C5 of triazole), 151.1 (C2 of triazole), 164.6 (C6 of Triazine), 62.0 (C5 of Triazine),156.8 (C4 and C6 of pyrimidine), 119 (pyrimidine C-Cl), 155 , 124.2 (C3 pyridine), 137.2 (C4 pyridine), 123.6 (C5 pyridine), 149.2 (C6 pyridine).Yield 51%, crystals from dioxane/diluted by methanol, mp 171–173For compound (6b) (8-(5-chloropyrimidin-2-yl)-6-phenyl-3-(pyrimidin-4-yl)-7,8-dihydrotriazolo- triazine):oC. Analysis calculated for C18H12ClN9 (389): C, 55.46; H, 3.10; Cl, 9.10; N, 32.34. The analyses found for the compound are: C, 55.5; H, 3.08; N, 32.3; Cl, 9.2. IR characteristic peaks appear at (ν cm−1): 3078, 2959 cm−1 ), 1519 (C=N). 1H NMR characteristic peaks appear at δ ppm: 3.4, CH2, triazine ring), 8.08 -pyrimidine), 7.52–7.94, 8.29 , 9.36, 9.20. 13C NMR characteristic peaks appear at δ ppm: 134.0 (C1 of benzene), 128.2 , 128.8 (C3 and C5 of benzene), 131.0 (C4 of benzene), 157.2 (C5 of triazole), 151.1 (C2 of triazole), 164.6 (C6 of triazine), 62.0 (C5 of triazine),156.8 (C4 and C6 of pyrimidine), 119 (pyrimidine C-Cl), 167.9 , 164 (C4 non-substituted pyrimidine), 115.6 (C5 of non-substituted pyrimidine), 157.1 (C6 of non-substituted pyrimidine).Yield 41%, crystals from dioxan/diluted by methanol, mp 188–191 For compound (6c) (8-(5-bromopyrimidin-2-yl)-6-phenyl-3-(pyridin-2-yl)-7,8-dihydro-triazolo- triazine):oC. Analysis calculated for C19H13BrN8 (432): C, 52.67; H, 3.02; Br, 18.44; N, 25.86. The analyses found for the compound are: C, 52.7; H, 3.01; N, 25.9; Br, 18.5. IR characteristic peaks appear at (ν cm−1): 3089, 2977 cm−1 ), 1520 (C=N). 1H NMR characteristic peaks appear at δ ppm: 3.3 , CH2, triazine ring), 8.5 -pyrimidine), 7.4–7.8 , 8.8 , 8.9 , 7.6 , 7.8 . 13C NMR characteristic peaks appear at δ ppm: 134.0 (C1 of benzene), 128.2 , 128.8 (C3 and C5 of benzene), 131.0 (C4 of benzene), 157.2 (C5 of triazole), 151.1 (C2 of triazole), 164.6 (C6 of triazine), 62.0 (C5 of triazine),159.1 (C4 and C6 of pyrimidine), 110.7 (pyrimidine C-Br), 168.4 , 155 , 124.2 (C3 pyridine), 137.2 (C4 of pyridine), 123.5 (C5 of pyridine), 149.2 (C6 of pyridine).Yield 36%, crystals from dioxan/diluted by methanol, mp 157–160 For compound (6d) (8-(5-bromopyrimidin-2-yl)-6-phenyl-3-(pyrimidin-4-yl)-7,8-dihydro-triazolo-triazine):oC. Analysis calculated for C18H12BrN9 (433): C, 49.79; H, 2.79; Br, 18.40; N, 29.03. The analyses found for the compound are: C, 49.7; H, 2.8; N, 29.1; Br, 18.4. IR characteristic peaks appear at (ν cm−1): 3078, 2959 cm−1 ), 1522 (C=N). 1H NMR characteristic peaks appear at δ ppm: 3.5 , CH2, triazine ring), 8.4 -pyrimidine), 7.5–7.9 , 8.17 , 9.36, 9.20. 13C NMR characteristic peaks appear at δ ppm: 134.0 (C1 of benzene), 128.2 , 128.8 (C3 and C5 of benzene), 131.0 (C4 of benzene), 157.2 (C5 of triazole), 151.1 (C2 of triazole), 164.6 (C6 of triazine), 62.0 (C5 of triazine),159.1 (C4 and C6 of pyrimidine), 110.7 (pyrimidine C-Br), 168.4 , 167.9 , 164 , 115.5 (C5 of non-substituted pyrimidine), 157.1 (C6 of non-substituted pyrimidine).Yield 49%, crystals from dioxan/diluted by methanol, mp 189–191 For compound (7a) (8-(5-chloropyrimidin-2-yl)-6-methyl-3-phenyl-triazolotriazin-7(8H)-one):oC. Analysis calculated for C15H10ClN7O (339): C, 53.03; H, 2.97; Cl, 10.44; N, 28.86. The analyses found for the compound are: C,53.02; H, 3.0; N, 28.9; Cl, 10.4. IR characteristic peaks appear at (ν cm−1): 3089, 2988 cm−1 ), 1725 (C=O), 1590 (C=N). 1H NMR characteristic peaks appear at δ ppm: 2.07, 8.05-pyrimidine), 7.41–8.28. MS : 339 , 341 , 225 , 227 ((M + 2)-5-chloropyrimidine, 4.8%), 114 , 116 .Yield 55% crystal from ethanol; mp 300–303 For compound (7b) (8-(5-chloropyrimidin-2-yl)-6-methyl-3-(pyridin-4-yl)-triazolo-triazin-7(8H)-one):oC. Analysis calculated for C14H9ClN8O (340): C, 49.35; H, 2.66; Cl, 10.41; N, 32.89. The analyses found for the compound are: C,49.4; H, 2.7; N, 32.9; Cl, 10.4. IR characteristic peaks appear at (ν cm−1): 3091, 2979 cm−1 ), 1728 (C=O), 1522 (C=N). 1H NMR characteristic peaks appear at δ ppm: 2.18 , 8.02 -pyrimidine), 7.99, (CH)-pyridine), 8.75, (CH)-pyridine). MS : 340 , 342 , 226 , 228 ((M + 2) - 5-chloropyrimidine, 4.1%), 114 , 116 .Yield 39% crystal from ethanol; mp 289–292 For compound (7c) (8-(5-bromopyrimidin-2-yl)-6-methyl-3-(pyridin-4-yl)-triazolo-triazin-7(8H)-one):oC. Analysis calculated for C15H10BrN7O (383): C, 46.89; H, 2.62; Br, 20.80; N, 25.52. The analyses found for the compound are: C,47.0; H, 2.6; N, 25.5; Br, 20.8. IR characteristic peaks appear at (ν cm−1): 3077, 2987 cm−1 ), 1724 (C=O), 1520 (C=N). 1H NMR characteristic peaks appear at δ ppm: 2.23 , 8.78 -pyrimidine), 7.41–8.22 . MS : 383 , 385 , 225 , 227 ((M + 2) - 5-bromopyrimidine, 3.2%), 158 , 160 .Yield 42% crystal from ethanol; mp 244–246 For compound (7d) (8-(5-chloropyrimidin-2-yl)-3-phenyl-triazolotriazine-6,7-dione):oC. Analysis calculated for C14H9BrN8O (384): C, 43.66; H, 2.36; Br, 20.74; N, 29.09. The analyses found for the compound are: C,43.7; H, 2.4; N, 29.1; Br, 20.7. IR characteristic peaks appear at (ν cm−1): 3079, 2977 cm−1 ), 1730 (C=O), 1518 (C=N). 1H NMR characteristic peaks appear at δ ppm: 2.17, 8.43-pyrimidine), 7.95 , (CH)-pyridine), 8.78 , (CH)-pyridine). MS : 384 , 386 , 226 , 228 ((M + 2) - 5-bromopyrimidine, 3.8%), 159 , 161 .Yield 36% crystal from ethanol; mp 274–276 For compound (8a) (8-(5-chloropyrimidin-2-yl)-3-(pyridin-4-yl)-triazolotriazine-6,7-dione):oC. Analysis calculated for C14H8ClN7O2 (341): C, 49.21; H, 2.36; Cl, 10.38; N, 28.69. The analyses found for the compound are: C, 49.2; H, 2.4; N, 28.7, Cl, 10.4. IR characteristic peaks appear at (ν cm−1): 3233 (NH), 3079 (aromatic (CH)), 1734, 1658 (2C=O), 1522 (C=N). 1H NMR characteristic peaks appear at δ ppm: 7.6 , NH), 8.05 -pyrimidine), 7.03–8.28 .Yield 42% crystalized from ethanol; mp 152–155 For compound (8b) (8-(5-bromopyrimidin-2-yl)-3-phenyl-triazolotriazine-6,7-dione):oC. Analysis calculated for C13H7ClN8O2 (342): C, 45.56; H, 2.06; Cl, 10.35; N, 32.70. The analyses found for the compound are: C, 45.6; H, 2.1; N, 32.7, Cl, 10.3. IR characteristic peaks appear at (ν cm−1): 3197 (NH), 1729, 1666 (C=O), 1520 (C=N). 1H NMR characteristic peaks appear at δ ppm: 7.9 , NH), 8.12 -pyrimidine), 7.96 , (CH)-pyridine), 8.05 , (CH)-pyridine).Yield 38% crystalized from ethanol; mp 215–217 For compound (8c) (8-(5-bromopyrimidin-2-yl)-3-phenyl-triazolotriazine-6,7-dione):oC. Analysis calculated for C14H8BrN7O2 (384): C, 43.54; H, 2.09; Br, 20.69; N, 25.39. The analyses found for the compound are: C, 43.5; H, 2.1; N, 25.4, Br, 20.7. IR characteristic peaks appear at (ν cm−1): 3188 (NH), 3080 (aromatic (CH)), 1731, 1656 (C=O), 1519 (C=N). 1H NMR characteristic peaks appear at δ ppm: 7.81 , NH), 8.42 -pyrimidine), 7.45–8.06 .Yield 52% crystalized from ethanol/acetic acid mixture; mp 197–199 For compound (8d) (8-(5-bromopyrimidin-2-yl)-3-(pyridin-4-yl)-triazolotriazine-6,7-dione):oC. Analysis calculated for C13H7BrN8O2 (385): C, 40.33; H, 1.82; Br, 20.64; N, 28.94. The analyses found for the compound are: C, 40.3; H, 1.8; N, 28.9, Br, 20.6. IR characteristic peaks appear at (ν cm−1): 3180 (NH), 3088 (aromatic (CH)), 1735, 1668 (C=O), 1520 (C=N). 1H NMR characteristic peaks appear at δ ppm: 7.82 , NH), 8.43 -pyrimidine), 7.87 , (CH)-pyridine), 8.15 , (CH)-pyridine).Yield 57% crystalized from diluted ethanol; mp 223–225 For compound (9a) :oC. Analysis calculated for C8H10N6 (190): C, 50.52; H, 5.30; N, 44.18. The analyses found for the compound are: C, 50.5; H, 5.3; N, 44.2. UV : 316 (2.4) nm. IR characteristic peaks appear at (ν cm−1): 3344, (NH2), 3174 (NH), 3076 (aromatic (CH)), 1602 (deformation NH2), 1522 (C=N). 1H NMR characteristic peaks appear at δ ppm: 4.11 , NH2 of hydrazine), 5.77 , NH2 of triazole), 7.41–8.08 , 9.23 .Yield 67% crystal from ethanol; mp 155–157 For compound (9b)(3-hydrazinyl-5-(pyridin-4-yl)-4H-1,2,4-triazol-4-amine):oC. Analysis calculated for C7H9N7 (191): C, 43.97; H, 4.74; N, 51.28. The analyses found for the compound are: C, 44.0; H, 4.7; N, 51.3. UV : 312 (2.1) nm. IR characteristic peaks appear at (ν cm−1): 3356, (NH2), 3181 (NH), 1600 (deformation NH2), 1518 (C=N). 1H NMR characteristic peaks appear at δ ppm: 4.23 , NH2 of hydrazine), 5.53 , NH2 of triazole), 7.83 , (CH)-pyridine), 8.87 , (CH)-pyridine), 9.76 , NH).Yield 58% crystal from ethanol; mp 187–189 For compound (10a) :oC. Analysis calculated for C8H7N6P (218): C, 44.04; H, 3.23; N, 38.52; P, 14.20. The analyses found for the compound are: C, 44.0; H, 3.3; N, 38.50; P, 14.2. IR characteristic peaks appear at (ν cm−1): 3123–2962 , 1650 (P-NH), 1528 (C=N); 1H NMR characteristic peaks appear at δ ppm: 4.25 , 6.32 , 7.41–8.28 . MS : 218 , 194 , 163 , 148 .Yield 58% crystal from THF; mp 267–269 For compound (10b) (7-(pyridin-4-yl)-3,4-dihydro-triazolo[1–5] tetrazaphosphinine):oC. Analysis calculated for C7H6N7P (219): C, 38.37; H, 2.76; N, 44.74; P, 14.13. The analyses found for the compound are: C, 38.4; H, 2.8; N, 44.8; P, 14.1. IR characteristic peaks appear at (ν cm−1): 3120–2965 , 1643 (P-NH), 1522 (C=N). 1H NMR characteristic peaks appear at δ ppm: 4.31 , 6.52 , 7.94 , (CH)-pyridine), 8.79 , (CH)-pyridine). MS : 219 , 195 , 164 , 149 .Yield 44% crystal from THF; mp 226–228 For compound (11a) :oC. Analysis calculated for C9H8N6 (200): C, 53.99; H, 4.03; N, 41.98. The analyses found for the compound are: C, 54.0; H, 4.0; N, 42.0. IR characteristic peaks appear at (ν cm−1): 3184,3132 , 1521 (C=N). 1H NMR characteristic peaks appear at δ ppm: 3.41 , 5.82 , 7.02 , 7.51–7.82 .Yield 37% crystallized from dioxane; mp 252–254 For compound (11b) (3-(pyridin-4-yl)-1,7-dihydro-triazolotetrazine):oC. Analysis calculated for C8H7N7 (201): C, 47.76; H, 3.51; N, 48.73. The analyses found for the compound are: C, 47.7; H, 3.5; N, 48.7. IR characteristic peaks appear at (ν cm−1): 3188, 3129 , 1519 (C=N). 1H NMR characteristic peaks appear at δ ppm: 3.62 , 6.21 , 7.36 , 7.90 , (CH)-pyridine), 8.63 , (CH)-pyridine).Yield 41% crystallized from dioxan; mp 271–273 For compound (12a) (6-(2-chloro-6-fluorophenyl)-3-phenyl-5,6,7,8-tetrahydro-triazolotetrazine):oC. Analysis calculated for C15H12ClFN6 (330): C, 54.47; H, 3.66; Cl, 10.72; F, 5.74; N, 25.41. The analyses found for the compound are: C, 54.5; H, 3.7; N, 25.4; F, 5.7; Cl, 10.7. UV : 265 (1.13) and 263 (1.03) nm. IR characteristic peaks appear at (ν cm−1): 3354,3169 and 3132 cm−1 (3NH), 3078 (aromatic (CH)), 1522 cm−1 (C=N). 1H NMR characteristic peaks appear at δ ppm: 2.31 , 4.32 , 5.04 , 6.32 , 7.07–8.28 . 13C NMR characteristic peaks appear at δ ppm: 157.2 (C3 of triazole), 151.1 (C5 of triazole), 130.6 (C1 of non-substituted benzene), 127.5 , 129.2 , 131.1 (C4 of non-substituted benzene), 73.5 (C3 of tetrazine), 133.8 (C-Cl), 160.8 (C-F), 129.8 (C2 of substituted benzene), 124.2, 129.7, 113.4 (3 C of substituted benzene).Yield 53% crystal from dioxan; mp 234–236 For compound (12b) (6-(2-chloro-6-fluorophenyl)-3-(pyridin-4-yl)-5,6,7,8-tetrahydro-triazolo tetrazine):oC. Analysis calculated for C14H11ClFN7 (331): C, 50.69; H, 3.34; Cl, 10.69; F, 5.73; N, 29.56. The analyses found for the compound are: C, 50.7; H, 3.4; N, 29.5; F, 5.7; Cl, 10.6. UV : 263(1.11) and 261 (1.01) nm. IR characteristic peaks appear at (ν cm−1): 3343, 3154 and 3129 cm−1 (3NH), 3072 (aromatic (CH)), 1518 cm−1 (C=N). 1H NMR characteristic peaks appear at δ ppm: 2.42 , 4.51 , 5.32 , 6.50 , 7.01–7.32 , 7.92 , (CH)-pyridine), 8.71 , (CH)-pyridine). 13C NMR characteristic peaks appear at δ ppm: 157.2 (C3 of triazole), 151.1 (C5 of triazole), 134.0 (C4 of pyridine), 121.3 , 149.8 , 73.5 (C3 of tetrazine), 133.8 (C-Cl), 160.8 (C-F), 129.8 (C2 of substituted benzene), 124.2, 129.7, 113.4 (3C of substituted benzene).Yield 46% crystal from dioxan; mp 256–258 For compound (13a) -thione):oC. Analysis calculated for C9H8N6S (232): C, 46.54; H, 3.47; N, 36.18; S, 13.81. The analyses found for the compound are: C, 46.5; H, 3.5; N, 36.2; S, 13.8. UV [λmax (Log ε)]: 314 (2.1) nm. IR characteristic peaks appear at (ν cm−1): 3332, 3178 (NH-NH), 1528 (C=S). 1H NMR characteristic peaks appear at δ ppm: 3.8 , 5.3 , 6.1 , 7.52–8.07 . 13C NMR characteristic peaks appear at δ ppm: 157.2 (C3 of triazole), 151.1 (C5 of triazole), 182.1 (C3 of tetrazine), 130.6 (C1 of benzene), 127.5 , 129 , 131.1 (C4 of benzene).Yield 45% crystal from THF; mp 276–278 For compound (13b) (3-(pyridin-4-yl)-7,8-dihydro-triazolotetrazine-6(5H)-thione):oC. Analysis calculated for C8H7N7S (233): C, 41.19; H, 3.02; N, 42.03; S, 13.75. The analyses found for the compound are: C, 41.2; H, 3.0; N, 42.0; S, 13.8. UV [λmax (Log ε)]: 312 (1.8) nm. IR characteristic peaks appear at (ν cm−1): 3325, 3182 (NH-NH), 1522 (C=S). 1H NMR characteristic peaks appear at δ ppm: 3.2 , 5.1 , 5.9 , 7.99 , 8.77 . 13C NMR characteristic peaks appear at δ ppm: 157.2 (C3 of triazole), 151.1 (C5 of triazole), 182.1 (C3 of tetrazine), 134.0 (C4 of pyridine), 149.8 , 121.3 .Yield 51% crystal from THF; mp 255–258 For compound (14a) -one):oC. Analysis calculated for C9H8N6O (216): C, 50.00; H, 3.73; N, 38.87. The analyses found for the compound are: C, 50.1; H, 3.7; N, 38; 9. UV [λ max (Log ε)]: 324 (1.51) nm. IR characteristic peaks appear at (ν cm−1): 3167 (NH-NH), 1643 (amidic CO), 1563 (C=N); 1H NMR characteristic peaks appear at δ ppm: 2.9 , 4.8 , 5.6 , 7.87–8.08 . 13C NMR characteristic peaks appear at δ ppm: 157.2 (C3 of triazole), 151.1 (C5 of triazole), 152.4 (C3 of tetrazine), 130.6 (C1 of benzene), 127.5 , 129.2 , 131.1 (C4 of benzene). MS : 216 , 188 , 173 .Yield 43% crystal from ethanol; mp 191–193 For compound (14b) (3-(pyridin-4-yl)-7,8-dihydro-triazolotetrazin-6(5H)-one):oC. Analysis calculated for C8H7N7O (217): C, 44.24; H, 3.25; N, 45.14. The analyses found for the compound are: C, 44.2; H, 3.3; N, 45.1. UV [λ max (Log ε)]: 320 (1.21) nm. IR characteristic peaks appear at (ν cm−1): 3172 (NH-NH), 1651 (amidic CO), 1534 (C=N). 1H NMR characteristic peaks appear at δ ppm: 3.1 , 5.1 , 5.9 , 7.98 , 8.67 . 13C NMR characteristic peaks appear at δ ppm: 157.2 (C3 of triazole), 151.1 (C5 of triazole), 152.4 (C3 of tetrazine), 134.0 (C4 of pyridine), 149.8 , 121.3 . MS : 217 , 189 , 174 .Yield 38% crystal from ethanol; mp 170–173 1a) is simply displaced by the amino group (nucleophilic group) in heterocyclic primary amines such as 5-chloropyrimidin-2-amine (2a) in Ethanol-DMF mixture (1:1) under reflux to yield 4-amino-5-substituted amino-1,2,4-triazole derivative (3a). Structure of 3a was elucidated mainly from the disappearance of the mercapto group peak at 2600–2500 cm−1 in the IR spectrum, the new NH proton with NH2 proton of aminotriazole of compound 3a showed through the 1H NMR spectrum at δ 14.22 ppm 5.87 ppm respectively. Also, the UV absorption of 3a shows λ max at 372 nm while the λ max of 1a appears at 317 nm, which proves the structure of compound 3a -phosphanoxide (4) under reflux in tetrahydrofuran (THF) produces 3,8-diaryl-4,5,6,7-tetrahydro-6-hydroxy-1,2,4-triazole-phosphotriazine (5a) , 3.1 , CH2), 2.0 , 7.41–8.05  and 8.08 -pyrimidine).From the above mentioned spectral data and physical constants of the newly synthesized compounds, we concluded that: the mercapto group in the 4-amino-5-phenyl-4H-1,2,4-triazole-3-thiol  have been obtained from the hetero-cyclization of compound 3a with phenacyl bromide dissolved in ethanolic potassium hydroxide, pyruvic acid dissolved sodium hydroxide and diethyloxalate dissolved in DMF/THF under reflux, respectively  and 2966 (cm−1) for aromatic & aliphatic (CH) and band at 1522 for (C=N). While, representing bands of compound 7a were recorded at ν = 2978 (cm−1), 1480 (cm−1) for (stretching and bending vibration of CH3) and a band at 1725(cm−1) for (C=O). In addition to that compound 8a bands were recorded at ν 3233 (cm−1) for NH, 1734(cm−1) and 1658(cm−1) for (2C=O), C=N and substituted pyrimidine and aromatic rings bands appear at 1522(cm−1) and 850–730 (cm−1). Also, 4 amino-5-(4 pyridyl)-4 H-1,2,4-triazole-3-thiol compound (1b) reacted with 5-chloro-pyrimidine-2-amine (2b) in Ethanol/DMF mixture (1:1) under reflux to give 4-amino-5-substituted amino-1,2,4-triazole derivative 3b. Compound 3b in turn, undergo ring closure with each of diphenyl (chloromethyl) - phosphanoxide (4) in THF/TEA, phenacyl bromide in ethanolic KOH, pyruvic acid in NaOH, and/or diethyloxalate in THF/DMF to provide the compounds 5b to 8b, respectively. The chemical structures of compounds 5b-8b were confirmed by considering both spectral measurements and elemental analyses. In a similar way, under the same above-mentioned experimental conditions, the reaction of compound 1a,b with 5-bromopyrimidin-2-amine (2c) takes place to give 3c,d compounds respectively. The triazole-3,4-diamines 3c,d were cyclized with each of diphenyl(choro-methyl)phosphanoxide (4), phenacyl bromide, pyruvic acid, and diethyloxalate to give the corresponding compounds 5c,d–8c,d, respectively, The chemical structures of compounds5c,d–8c,d were elucidated by their spectral measurements and elemental analyses data.Derivatives of 1,2,4-Triazolotriazolo-tetrazine 11a and/or 11b, respectively. Cycloaddition reaction, in boiling ethanol with a few drops of piperidine, of compounds 9a,b with 2-chloro–6-fluorobenzeldehyde furnished the corresponding triazolotetrazine derivatives 12 a,b, respectively : 3123–2962, 3120–2965 of (broad NH-NH), 1650, 1643 of (P-NH), 1528, 1522 of (C=N) for 10a, b, respectively. The IR absorption bands at ν (cm-1): 3184–3132, 3188–3129 of , 1521, 1519 (C=N) for 11a,b, respectively. The IR absorption bands at ν (cm−1): 3354–3132, 3343–3129 (3NH), 3078, 3072 (aromatic (CH)), 1522, 1518 (C=N) for 12a,b, respectively. The mass spectra of each of 10a and 10b gave molecular ion peaks at 218 and 219 which corresponded to the molecular weights of C8H7N6P and C7H6N7P of the assigned structures 10a,b, respectively.Structures of compounds 3a,b and 12a,b explains that the hetero-cyclization would inhibit the electronic transition and this caused the hypsochromic effect “shift to the shorter wavelength”. Thus, λmax of 3a,b was 372 and 374 nm while that of 12a,b was 265 and 263 nm, respectively. 13C NMR (DMSO) of compound 12a showed the presence of thirteen different signals for thirteen different carbon atoms, which, for 12b, showed twelve signals for twelve different carbons. Finally, the compounds 1,2,4-trizolotetrazine-5(4 H)-thiones/ones  were synthesized from refluxing of 9a,b with CS2 in DMF and/or with ethyl chloroformate in THF/TEA, respectively . The 1H NMR spectra of 13a,b recorded signals of 3.8 , 5.3 , 6.1  and 7.52–8.07 . 13C NMR gives us good evidence for the structure of 13a where it showed resonated signals at 157 (C3 -triazole), 151 (C5 -triazole), 182 (C3 -tetrazine) and 130, 131,129, 127 ppm of benzene carbons. Mass spectra of both compounds 14a,14b showed molecular ion peaks at 216 (4.7%) for 14a and at 217 (6.2%) for 14b, which confirmed their structures. In addition, 13C NMR data of 14a and 14b gave convincing evidence to their structures in which compound 14a revealed the presence of seven different carbon signals, while 14b showed six different carbon signals, which were in good agreement with the proposed structures  was presented in Tables The different biological activities of the synthesized compounds have been evaluated by studying their antibacterial activity against E.coli, S. aureus and P. aeruginosa in comparison with Nalidixic acid at concentrations 100, 50 and 25 µg/mL. All the tested Compounds showed higher activity against E. coli bacteria at 100 µg/mL concentrations and higher to moderate activity at 50 µg/mL concentrations. For S. aureus bacteria, all the tested Compounds showed higher activity at 100 µg/mL concentrations. Compounds 5a, 5c, 8a, 8b, 9a, 10a, 11a, 12a, 13a and 14a were not sensitive toward S. aureus while compounds 5d, 10b and 11b were slightly active at 50 µg/mL concentrations. On the other hand, the remaining compounds showed moderate activity at 50 µg/mL concentrations. For P. aeruginosa bacteria, all the tested compounds showed higher activity at 100 µg/mL concentrations. Compounds 5b, 8b, 8d, 9b, 10a and 12b were not sensitive toward P. aeruginosa bacteria while compounds 5a, 5c, 9a, 13a and 14b were slightly active at 50 µg/mL concentrations. On the other hand, the remaining compounds showed moderate activity at 50 µg/mL concentrations. The MICs for all the tested compounds against E.coli, S. aureus, and P. aeruginosa were presented in Table C. albicans in comparison with Nystatin at 100 µg/mL concentration The MIC for all the tested compounds against C. albicans fungi was 100 µg/mL. The higher activity of some of the tested compounds was mainly due to containing chlorine, fluorine, bromine and phosphorus elements within the chemical structure of 1,2,4-triazine and 1,2,4,5-tetrazine55. On the other hand, the obtained results in Table 10a,b, 12a,b and 13b carrying phenyl and pyridine groups having both the chlorine and fluorine elements had good anti-inflammatory activity, in comparison with the standard anti-inflammatory drug used (Indomethacin). Compounds 10a,b, and 12a,b contained mainly triazole and tetrazine rings with the presence of both fluorine and phosphorus elements, incorporated with pyridyl moiety. The activity of the new materials depends on the existence of those new moieties which have a high biological activity55. The higher biological activity of the synthesized compounds was in a good agreement with the previously stated results in the field of fluorine and phosphorus-bearing nitrogen heterocyclic systems56.The results in Table 5d and Nalidixic acid with amino acids of DNA Gyrase enzyme (PDB ID: 2XCT) active site. From these results, we found that the active compound 5d showed extra binding modes to DA13 and Arg 458 in addition to interaction with the essential binding sites Mn metal and DG5. Table Indomethacin, respectively, with active sites of COX-2 (PDB ID: 1CX2).Table 10a showed well interaction with Arg513 residue. Compound 10b showed well interaction with Ser353 and Tyr355 which is going alongside with the screening results. Also, compound 12a showed three binding interactions to Leu352, His90 and Val523 amino acid. Finally, compound 12b binds to Ala527 and Tyr355 residues.From these results, it appears that, generally, the tested compounds and Indomethacin showed a comparable binding pattern. Compound 5a,b–14a,b containing fluorine, chlorine, bromine and phosphorus elements were synthesized utilizing 3-substituted-4-amino-5-substituted amino-1,2,4-triazoles 3a-d and 3-substituted-4-amino-5-hydrazino-1,2,4-triazole 9a,b compounds as building units in the synthesizing process. These newly prepared compounds were fully characterized through the spectral and elemental analyses which were completely fit with the assigned structures. A number of the synthesized compounds were screened against gram-positive bacteria, gram-negative bacteria, and fungi, such as Streptococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Candida albicans. Most of the newly prepared compounds showed a high antibacterial, antifungal, and anti-inflammatory in comparing with the standard commercial antibiotics Imipenem and Nalidixic acid, Nystatin and Indomethacin, respectively. Compounds 10a,b, and 12a,b containing both chlorine and fluorine elements showed high inflammation activity.Some novel heterocyclic compounds


The synthesis of four new 1,3,5-triaryl-1,3,5-triazinane-2,4,6-trithione derivatives (thioisocyanurates) and two new partially thionated analogues from the corresponding 1,3,5-triaryl-1,3,5-triazinane-2,4,6-triones (isocyanurates) is reported, together with their spectroscopic properties. DFT calculations and comparison with the corresponding isocyanurates evidence the impact of the oxygen-for-sulfur replacement on the electronic structure and linear optical properties of these heterocycles. A bathochromic shift of the absorption bands and more efficient quenching of the fluorescence was observed. The5 was lacking and, more generally, that an extensive characterization of the shorter homologue 1,3,5-triaryl-1,3,5-triazinane-2,4,6-trithiones 4-X was needed. Indeed, whereas triphenyl-1,3,5-triazinane-2,4,6-trione (4-H) has been reported several times in the literature . De. De1-Me  0.01 Å) ,27. The 5, a Sonogashira coupling reaction +  δ = 7.68 , 7.23 . 13C{1H}-NMR  δ = 172.1 (C=S), 142.5 (NCAr), 133.1 (CHAr), 130.1 (CHAr), 122.9 (BrArC). IR : ῡ = 1344 , 1255 . Raman : ῡ = 1584 , 1327 , 1220 (C–H), 438 .1,3,5-Tris(4-iodophenyl)-1,3,5-triazinane-2,4,6-trithione (4-I). Following the general procedure with 1,3,5-tris(4-iodophenyl)-1,3,5-triazinane-2,4,6-trione , the title compound was isolated as a yellow solid (150 mg). Yield: 70%. MP: 250–270 °C (Dec). HRMS: (ASAP): m/z = 783.7400 [M + H]+ . 1H-NMR : δ = 7.85 , 7.18 . IR : ῡ = 1338 , 1254 . Raman : ῡ = 1579 , 1332 , 1220 (C–H), 440 .1,3,5-Triphenyl-1,3,5-triazinane-2,4,6-trithione (4-H). Following the general procedure with 1,3,5-triphenyl-1,3,5-triazinane-2,4,6-trione , the title compound was isolated as a yellow solid (116 mg). Yield: 99%. MP: 290–315 °C (Dec). HRMS: (ASAP): m/z = 406.0501 [M + H]+ . 1H-NMR  δ = 7.61–7.41 , 7.41–7.31 . 13C{1H}-NMR  δ = 172.8 (C=S), 143.9 (NCAr), 129.7 (2CHAr), 128.8 (CHAr), 128.4 (2CHAr). IR : ῡ = 1343 , 1247 . Raman : ῡ = 1592 , 1328 , 1213 (C–H), 439 .1,3,5-Tris(4-tolyl)-1,3,5-triazinane-2,4,6-trithione (4-Me). Following the general procedure with 1,3,5-tris(4-tolyl)-1,3,5-triazinane-2,4,6-trione , the title compound was isolated as a yellow solid (125 mg). Yield: 99%. Crystals of the title compound were obtained by slow evaporation of a CHCl3 solution of this compound. MP: 300–325 °C (Dec). HRMS: (ASAP): m/z = 448.0968 [M + H]+ . 1H-NMR  δ = 7.34 , 7.21 , 2.44 . 13C{1H}-NMR  δ = 173.0 (C=S), 141.6 (NCAr), 139.1 (CAr), 130.3 (CHAr), 128.0 (CHAr), 21.0 (CH3). IR : ῡ = 1351 , 1251 . Raman : ῡ = 1602 , 1328 , 1202 , 436 .1-Me; 1.01 mmol; 403 mg), P4S10 , hexamethyldisiloxane , and xylene (5 mL) were added to a Schlenk flask. The mixture was heated at reflux for four days. After cooling to room temperature, the mixture was passed through silica and the solvent was removed under vacuum. The desired mono-, bis-, and tris-thioisocyanurate derivatives were then separated by column chromatography on silica gel (cyclohexane/AcOEt [90:10]).1,3,5-Tris(4-tolyl)-1,3,5-triazinane-2,4,6-trione -1,3,5-triazinane-2,4-dione (6-Me). The title compound was isolated as a yellow solid (82 mg). Yield: 20%. Crystals of the title compound were obtained by slow evaporation of a CHCl3 solution of this compound. HRMS: (ASAP): m/z = 416.1427 [M + H]+ . 1H-NMR  δ = 7.40–7.33 , 7.33–7.20 , 2.46 . 13C{1H}-NMR  δ = 179.0 (C=S), 147.5 (C=O), 139.8 (CAr), 139.4 (CAr), 136.2 (CAr), 131.2 (CAr), 130.2 (CHAr), 130.1 (CHAr), 128.2 (CHAr), 127.9 (CHAr), 21.0 (CH3), 21.0 (CH3). IR : ῡ = 1709 , 1381 , 1270 . Raman : ῡ = 1755 , 1605 , 1315 , 1211 (C–H).4,6-Dithioxo-1,3,5-tris(4-tolyl)-1,3,5-triazinane-2-one (7-Me). The title compound was isolated as a yellow solid (105 mg). Yield: 24%. HRMS: (ASAP): m/z = 432.1201 [M + H]+ . 1H-NMR  δ = 7.41–7.34 , 7.30–7.21 , 2.47 .13C{1H}-NMR  δ = 176.8 (C=S), 145.0 (C=O), 141.4 (CAr), 139.5 (CAr), 139.0 (CAr), 136.2 (CAr), 130.3 (CHAr), 130.2 (CHAr), 128.3 (CHAr), 127.9 (CHAr), 21.1 (CH3), 21.1 (CH3). IR : ῡ = 1729 , 1367 , 1268 . Raman : ῡ = 1729 , 1603 , 1316 .1,3,5-Tris(4-tolyl)-1,3,5-triazinane-2,4,6-trithione (4-Me). This compound was also isolated by chromatography (26 mg). Yield: 6%. See above for the characterization of this compound.H-fluorene  was added to 1,3,5-tris(4-iodophenyl)-1,3,5-triazinane-2,4,6-trithione , CuI  and PdCl2(PPh3)2  in a mixture of DMF/Et3N (2:3). After seven days of stirring at 70 °C, the reaction medium was allowed to cool to room temperature and the reaction mixture was diluted in CH2Cl2 (20 mL) washed with water (3 × 50 mL) and dried over MgSO4. After filtration and evaporation to dryness, the crude product was purified by column chromatography on silica gel using a mixture of petroleum ether/CH2Cl2 (8:2) as the eluent. After recrystallization from pentane, the title compound was isolated as a yellow powder (16 mg). Yield 11%. Rf: 0.42 (petroleum ether/CH2Cl2 [8:2]). HRMS: (ESI): m/z = 1306.6134 [M + H]+ . Anal. Calc. for C90H87N3S3: C, 82.71, H, 6.71, N, 3.22, S, 7.36; found: C, 82.40, H, 6.68, N, 3.13, S, 6.88. 1H-NMR : δ = 7.80–7.66 , 7.53 , 7.42–7.31 , 2.00 , 1.11 , 0.70 , 0.67–0.51 . 13C{1H}-NMR : δ = 172.8, 151.8, 151.5, 143.4, 142.5, 141.0, 133.7, 131.4, 129.2, 128.3, 127.6, 126.7, 125.2, 123.6, 121.7, 120.7, 120.3, 93.0, 89.1, 55.8, 40.8, 26.6, 23.7, 14.5. IR : ῡ = 2201 , 1349 , 1257 . Raman : ῡ = 2198 , 1596 , 1334 , 1224 (C–H), 428 .In an oven-dried Schlenk tube, an excess of 9,9-dibutyl-2-ethynyl-9−6 M, optical density < 0.1) at room temperature (20 °C) using an Edinburgh Instruments (FLS920) spectrometer  in photon-counting mode. Fully corrected excitation and emission spectra were obtained with an optical density at λexc ≤ 0.1. Fluorescence quantum yields were measured according to procedures in the literature [All photophysical measurements were performed with freshly prepared air-equilibrated THF solutions (HPLC grade) at room temperature (298 K). UV–Vis absorption spectra were recorded on a Jasco V-570 spectrophotometer . The samples used to make the solutions were freshly recrystallized or thoroughly washed with cooled ether/pentane prior to the measurements to remove any organic impurity. Steady state fluorescence studies were performed in dilute air-equilibrated solutions in quartz cells of 1 cm path length  PHOTON 100 detector using Mo-K radiation  at T = 150 K. The structures were solved by a dual-space algorithm using the SHELXT program, [2F  = 0.1223 (FR = 0.0485) or 0.1658 (FR = 0.0931) for 8174 or 3498 observed reflections with I > 2σ(I). The data have been deposited at the Cambridge Crystallographic Data Center  [Data collection was undertaken on monocrystals of program,  and then program ). All noctively) .The DFT calculations reported in this work were performed using the Gaussian09  program.N,N’,N”-triarylthioisocyanurates) were synthesized and characterized in this work. 4-I and 4-Br are key synthetic precursors that can be used to access extended isocyanurates such as 5 by cross-coupling reactions. Thioisocyanurates have been previously mentioned in the literature, but to the best of our knowledge, no characterization of any example has been reported so far, apart from the x-ray structure of 4-H [4-X  and 5 should facilitate the straightforward experimental identification of such compounds. For instance, given that the carbonyl stretching motion provides a simple and powerful means to characterize isocyanurates [−1 for all derivatives 4-X and 5. Other intense IR-active ν(C–N) modes are also proposed as diagnostic vibrational markers for these thioisocyanurate derivatives. Finally, it is worth emphasizing that the structural data obtained for 4-Me in the solid state were consistent with the structural data previously reported for 4-H. It confirmed the existence of electron delocalization within the central heterocycle, a feature also supported by DFT calculations which has been previously reported for isocyanurates [Five triaryl-1,3,5-triazinane-2,4,6-trithiones  states, resulting in efficient quenching of the fluorescence in thioisocyanurates. This is seen even with derivatives featuring extended arms terminated by good fluorophores such as 5. Thus, while the reduction of the molecular bandgap seen for thioisocyanurates and the increased polarizability of sulfur over oxygen are important assets in the development of more NLO-active molecules, the non-emissive nature of these compounds precludes the determination of their 2PA cross-sections by two-photon excited fluorescence (TPEF). Z-scan investigations are now required to unravel the nonlinear absorption properties of these promising octupoles.We also report the synthesis and characterization of the mono- and bis-thionated derivatives