# Fluorine Substituted 1,2,4-Triazinones as Potential Anti-HIV-1 and CDK2 Inhibitors

Authors: Mohammed S. I. Makki; Reda M. Abdel-Rahman; Khalid A. Khan
Journal: Journal of Chemistry
 (2014)

---

## Abstract

Fluorine substituted 1,2,4-triazinones have been synthesized via alkylation, amination, and/or oxidation of 6-(2-amino-5-fluorophenyl)-3-thioxo-3,4-dihydro-1,2,4-triazin-5(2H)-one1 and 4-fluoro-N-(4-fluoro-2-(5-oxo-3-thioxo-2,3,4,5-tetrahydro-1,2,4-triazin-6-yl)phenyl)benzamide 5 as possible anti-HIV-1 and CDK2 inhibitors. Alkylation on positions 2 and 4 in 1,2,4-triazinone gave compounds 6–8. Further modification was performed by selective alkylation and amination on position 3 to form compounds 9–15. However oxidation of 5 yielded compounds 16–18. Structures of the target compounds have been established by spectral analysis data. Five compounds (5, 11, 14, 16, and 17) have shown very good anti-HIV activity in MT-4 cells. Similarly, five compounds (1, 3, and 14–16) have exhibited very significant CDK2 inhibition activity. Compounds 14 and 16 were found to have dual anti-HIV and anticancer activities.


---

## Body

## 1. Introduction

Human immunodeficiency virus (HIV) type-1 is the causative agent of acquired immunodeficiency syndrome (AIDS), which is one of the serious global health problems [1]. The currently approved anti-HIV drugs can be divided into five groups: reverse transcriptase inhibitors (RTIs), protease inhibitors [2] (PIs), fusion inhibitors (FIs), coreceptor inhibitors (CRIs), and integrase inhibitors (INIs). This arsenal of drugs, which is used in combinations, has moved the prognosis of HIV patients from that of high morbidity and mortality to, for many at least, a chronic, manageable but still complex disease [3–5]. However, the use of these drugs has been relatively limited by their toxicity [6], drug resistance development [7], and, more worryingly, the fact that some newly HIV-infected patients carry viruses that are already resistant to the currently approved AIDS treatments [8]. These issues along with drug-related side effects make it apparent that new anti-HIV drugs with novel mechanisms of action are clearly needed.On the other hand, during the past 30 years, a variety of approaches have been taken for cancer chemotherapy and many antitumor drugs have been developed for clinical use. In the treatment of solid tumors, however, the conventional approaches have met with only limited success and cancer still remains as one of the leading causes of human mortality [9].Chemotherapy drugs are sometimes feared because of a patients’ concern about its toxic effects. There are three goals associated with the use of the most commonly used anticancer agents: (a) damage the DNA of the affected cancer cells, (b) inhibit the synthesis of new DNA strands to stop the cells from replicating, and (c) stop the mitosis or the actual splitting of the original cell into two new daughter cell [10].In the past few years fluorine substituted heterocyclic nitrogen systems have been incorporated into drug discovery research [11–23] to improve the physicochemical properties of drugs. Organic fluorine is a prominent tool in the design and improvement of pharmacokinetic properties of drug molecules. Replacing hydrogen and other functional groups with fluorine can have a dramatic effect on the modulation of electronic, lipophilic, and steric parameters, all of which can critically influence both the pharmacodynamic and pharmacokinetic properties of drugs. Substitution of fluorine into a potential drug molecule not only alters the electronic environment, but it also influences the properties of neighboring functional groups. Fluorine can have significant effects on the binding affinity in protein-ligand complexes. The DNA polymerase inhibitors fludarabine (I) (F-ara-A), clofarabine (II), and tezacitabine (III) are used as cancer chemotherapeutic agents [24], while Gleevec (IV) is used as catalytic inhibitor of imatinib mesylate [25]. In addition, BX-1382BS (V) showed a significant activity as protein kinase inhibitor [26] and a cyanopyrimidine scaffold JNJ-17029259 (VI) is an oral inhibitor of VEGF-mediated signal transduction [27] (Figure 1). Recently a great deal of synthetic efforts has been spent on fluorinated uncondensed 1,2,4-triazines by our group searching for new anti-HIV and anticancer agents [28–38] (compoundsA–F; Figure 2).Figure 1(a)
(b)
(c)
(d)
(e)
(f)Fluorine incorporation on key positions plays a significant role to alter the physicochemical and biological characteristics of organic compounds. Frequently, it is found that a fluorine substituent leads to an enhancement of the binding affinity of a molecule with proteins through a noncovalent bond formation. Fluorine increases binding affinity, reduces plasma protein binding leading to a higher free fraction of the drug, and increases cell penetration. The combination of these effects results in a dramatically improved biological activity. Based on these valuable observations and in part of our continuing efforts in drug development, the present work describes an attempt towards the synthesis of fluorine substituted 1,2,4-triazinones. The purpose of the work is to extend the scope of our previous studies [29, 30] and to substitute fluorine on various positions in 1,2,4-triazines in order to obtain effective anti HIV-1 and CDK2 inhibiting agents.


## 2. Experimental

### 2.1. Chemistry

Melting points were determined on an electrothermal Bibby Stuart Scientific melting point apparatus and are uncorrected. The infrared (IR) spectra were recorded on PerkinElmer RXI FT-IR infrared spectrophotometer using the KBr pellet technique. Electronic absorption spectra were recorded in DMF on Shimadzu UV-Visible 3101 PC spectrophotometer.1H and 13C NMR spectra were recorded on a Bruker DPX-400 FT NMR spectrometer using tetramethylsilane as the internal standard DMSO-d
6 as a solvent (chemical shifts in δ, ppm). 19F NMR spectra were determined at 84.25 MHz using hexafluorobenzene as an internal standard. Splitting patterns were designated as follows:s: singlet;m: multiplet. Mass spectra were measured on a GCMS-Q 1000 Ex spectrometer. Elemental analyses were performed on a 2400 PerkinElmer Series 2 analyzer. Follow-up of the reactions and checking the homogeneity of the compounds were made by TLC on silica gel-protected aluminum sheets (Type 60 F254, Merck) and the spots were detected by exposure to UV-lamp at λ 254.The anti-HIV and anticancer activity were evaluated in DTP, DCT, National Cancer Institute, Bethesda, MD 20892, USA.


#### 2.1.1. 6-(2-Amino-5-fluorophenyl)-3-thioxo-3,4-dihydro-1,2,4-triazin-5(2H)-one (1)

A mixture of 5-fluoroisatin (0.001 mol in warm 5% aqueous NaOH) and thiosemicarbazide (0.001 mol, in hot water 10 mL) was refluxed for 2 h. The reaction mixture was then poured onto ice and neutralized with dil. HCl. The solid thus obtained was filtered off and crystallized from ethanol to give1 as pale yellow crystals. Yield 80%, m.p. 285–287°C. UV: λ
max
⁡ (ε): 282.8 (2.06) nm. IR (ν, cm−1): 3528, 3300 (NH, NH2), 1661 (C=O), 1385 (NCSN), 1255 (C–F), 865 (aryl C–H), 658 (C–F). 1H NMR (δ, ppm): 13.47, (s, 1H, NH), 7.52–7.42, 6.93–6.87 (3H, aromatic H), 3.2 (s, 2H, NH2). 13C NMR (δ, ppm): 172.74 (C=S), 156–152 (C=O), 144.53 (C–F), 138.73 (C=N), 119.68, 117.60, 117.45, 116.50, 116.30 (aromatic carbons), 77.27–76.84, 39.91–39.07 (C–C, C–N carbons). MS (relative intensity): 238 (M + H2O, 254, 5%), 206 (207, 36), 182 (10), 108 (1.1), 103 (100), 75 (35). Anal. calcd. C9H7N4SOF (238): C, 45.38; H, 2.93; N, 23.52; S, 13.0; F, 7.98. Found: C, 44.88; H, 2.55; N, 23.41; S, 12.85; F, 7.77. 19F NMR: δ  −127 ppm.


#### 2.1.2. 2-((6-(2-((Carboxymethyl)amino)-5-fluorophenyl)-5-oxo-4,5-dihydro-1,2,4-triazin-3-yl)thio)acetic acid (2)

A mixture of1 (0.001 mol) and monochloroacetic acid (0.002 mol) in ethanolic NaOH (5%, 50 mL) was warmed for 30 min and then cooled and poured onto ice-cold dil. acetic acid. The product is precipitated as pale solid which was then filtered and crystallized from acetic acid to give yellow crystals of compound2. Yield 70%, m.p. 262–264°C. UV: λ
max
⁡ (ε): 283.3 (1.93) nm. IR (ν, cm−1): 3531 (OH), 3300 (NH), 2929, 2861 (2 CH2), 1660 (C=O), 1499, 1439 (deformation CH2), 1385 (NCSN), 1255 (C–F), 865 (aryl C–H), 658 (C–F). 1H NMR (δ, ppm): 13.38, (s, 1H, OH), 11.82 (s, NH), 7.55–7.37, 6.85–6.71 (aromatic H), 3.55-3.54, 3.53–3.51 (each s, 2 CH2). 13C NMR (δ, ppm): 172.67, 152.42 (2 C=O), 145 (C–F), 117.64, 117.50, 116.41, (aromatic carbons), 77.15, 76.94, 76.73 39.91–39.07 (C–S, C–O, and C–N carbons), 39.91, 39.07 (2 CH2). MS (relative intensity): 266 (M-2 (CO2), 100. Anal. calcd. C13H11N4SO5F (354): C, 44.07; H, 3.10; N, 16.0; S, 9.03; F, 5.35. Found: C, 43.78; H, 2.85; N, 15.69; S, 8.81; F, 5.21. 19F NMR: δ  −130 ppm.


#### 2.1.3. 6-(5-Fluoro-2-(methylamino)phenyl)-3-(methylthio)-1,2,4-triazin-5(4H)-one (3)

Procedure A. A mixture of compound2 (0.050 mol) and 10% K2CO3 (20 mL) was refluxed for 10 min and then left to acquire room temperature. The reaction mixture was now poured in an ice-cold dil. HCl. The precipitated solid was filtered and crystallized as faint yellow crystals. Yield 50%, m.p. 163-164°C.Procedure B. A mixture of compound1 (0.001), methyl iodide (0.002 mol), and 1% alcoholic KOH (20 mL) was stirred at room temperature for 4 h. The reaction mixture was then neutralized with dil. HCl. The resulting light brown solid was filtered and crystallized from ethanol to form yellowish crystals of compound3. Yield 65%, m.p. 162-163°C. UV: λ
max
⁡ (ε): 315.4 (1.154) nm. IR (ν, cm−1): 3310 (NH), 2929, 2862 (2 CH2), 1661 (C=O), 1499, 1438 (deformation CH2), 1385 (NCSN), 1255 (C–F), 865 (aryl C–H), 658 (C–F). MS (relative intensity): 266 (M+H2O, 284, 5%), 110 (5.5), 104 (100), 95 (1.18), 88 (100), 47 (65.0). Anal. calcd. C11H11N4SOF (266): C, 49.95; H, 4.13; N, 21.05; S, 12.03; F, 7.14. Found: C, 49.71; H, 4.01; N, 20.99; S, 11.88; F, 6.91. 19F NMR: δ  −126 ppm.


#### 2.1.4. 2,2′-(((3,3′-((Carboxymethylene)bis(sulfanediyl))bis(5-oxo-4,5-dihydro-1,2,4-triazine-6,3-diyl))bis(4-fluoro-2,1-phenylene))bis(azanylylidene))diacetic acid (4)

A mixture of compound1 (0.001 mol) and 1,1-dichloroacetic acid (0.003 mol) in DMF (20 mL) was refluxed for 10 min, cooled, and then poured on ice. The solid thus separated was filtered and crystallized from THF to give deep orange crystals. Yield 66%, m.p. 280–282°C. UV: λ
max
⁡ (ε): 266.5 (1.51) nm. IR (ν, cm−1): 3534, 3480 (OH), 3350 (NH), 2929, 2861 (aliphatic C–H), 1680, 1660 (C=O), 1499, 1439 (deformation C–H), 1386 (NCSN), 1255 (C–F), 865 (aryl C–H), 658 (C–F). MS (relative intensity): 644 (M-208, 3%), 298 (100), 167 (31.01), 149 (100), 85 (5.00). Anal. calcd. C24H14N8S2O8F2 (266): C, 44.72; H, 2.17; N, 17.36; S, 9.9; F, 5.95. Found: C, 44.51; H, 2.11; N, 17.12; S, 9.55; F, 5.71. 19F NMR: δ  −128 ppm.


#### 2.1.5. 4-Fluoro-N-(4-fluoro-2-(5-oxo-3-thioxo-2,3,4,5-tetrahydro-1,2,4-triazin-6-yl)phenyl)benzamide (5)

A mixture of compound1 (0.001 mol) and 4-fluorobenzoyl chloride (0.001 mol) in dry pyridine (10 mL) was warmed for 10 min, cooled, and then poured on ice. The precipitated solid was filtered and crystallized from dioxane to give faint yellow crystals of5. Yield 85%, m.p. 238–240°C. UV: λ
max
⁡ (ε): 288.4 (1.28) nm. IR (ν, cm−1): 3370 (NH), 3320 (NH), 1663, 1610 (C=O, CONH), 1385 (NCSN), 1255 (C–F), 1190 (C–S), 864 (aryl C–H), 658 (C–F). 1H NMR (δ, ppm): 13.75, (NH), 10.07 (NH), 8.60 (NH), 7.98–7.82, 7.53–7.41, 7.10–7.05 (aromatic H). 13C NMR (δ, ppm): 173.15 (C=S), 164.97–163.30, 158.87–158.57 (C=O), 146.91 (C–F), 144.70 (C–F), 138.07 (C–N), 132.21, 130.16, 129.57, 129.51, 125.5, 124.26 (aromatic carbons), 116.63, 115.04, 114.90 (C–S, C–O, C–N) 77.50–77.07, 40.03, 39.08. MS (relative intensity): 360 (M+4, 6%), 265 (78.0), 123 (12.05), 96 (2.0), 89 (100), 76 (75.0). Anal. calcd. C16H10N4SO2F2 (360): C, 53.30; H, 2.78; N, 15.50; S, 9.16; F, 10.55. Found: C, 52.88; H, 2.55; N, 15.02; S, 8.95; F, 10.35. 19F NMR: δ  −126 and −129 ppm.


#### 2.1.6. N-(2-(2,4-Disubstituted-5-oxo-3-thioxo-2,3,4,5-tetrahydro-1,2,4-triazin-6-yl)-4-fluorophenyl)-4-fluorobenzamide (6–8)

A mixture of5 (0.001 mol) methanol or morpholine or 2,2′-dipyridylamine (0.001 mol) in methanol (20 mL) and formaldehyde (0.001 mol) was refluxed for 2 h. The reaction mixture was cooled to acquire room temperature and then poured onto ice-cold water. The solid product precipitated was filtered and crystallized from methanol to form yellow-orange crystals.6. Yield 72%, m.p. 310–312°C. IR (ν, cm−1): 3527 (OH), 3100 (NH), 2935, 2890 (aliphatic C–H), 1660, 1600 (C=O, CONH), 1498, 1440 (deformation CH2), 1386 (NCSN), 1255 (C–F), 864 (aryl C–H), 658 (C–F). 1H NMR (δ, ppm): 12.54 (s, 1H, NH), 10.15, 8.04 (Two s, 2 OH), 7.96–7.94, 7.87–7.83, 7.64-7.63 (aromatic H), 2.49 and 2.48 (Two s, 2 CH2). 13C NMR (δ, ppm): 164.99 (C=S), 163.32, 157.02 (C=O), 135.99 (C–F), 130.39, 130.20, 129.53, 129.44, 129.36, 123.45, (aromatic carbons), 115.14, 114.99 (C–S, C–O) 77.38–76.95, 39.83, 39.29. Anal. calcd. C18H14N4SO4F2 (420): C, 51.42; H, 3.36; N, 13.30; S, 7.61; F, 9.04. Found: C, 51.23; H, 3.01; N, 13.11; S, 7.51; F, 8.88. 19F NMR: δ  −126 and −129 ppm.7. Yield 70%, m.p. 260–262°C. Anal. calcd. C26H28N6SO4F2 (558): C, 55.90; H, 5.02; N, 15.05; S, 5.70; F, 6.81. Found: C, 55.68; H, 4.98; N, 14.79; S, 5.35; F, 6.66.8. Yield 72%; m.p. 202–204°C. Anal. calcd. C38H28N10SO2F2 (726): C, 62.79; H, 3.88; N, 19.28; S, 4.27; F, 5.23. Found: C, 62.33; H, 3.58; N, 19.11; S, 4.15; F, 4.89.


#### 2.1.7. 4-Fluoro-N-(4-fluoro-2-(3-(substituted-amino)-5-oxo-4,5-dihydro-1,2,4-triazin-6-yl)phenyl)benzamide (9–12)

A mixture of 5 (0.001 mol) and sulfathiazole or sulfaoxazole or 4-fluoroaniline or 2,2′-dipyridylamine (0.001 mol) in absolute ethanol (50 mL) was refluxed for 6 h. On cooling a solid is separated which is filtered and crystallized from ethanol to give yellow crystals of9–12.9. Yield 85%, m.p. 172–174°C. IR (ν, cm−1): 3484−3310 (NH), 1658, 1610 (C=O, CONH), 1386 (NCSN), 1255 (C–F), 864 (aryl C–H), 658 (C–F). 1H NMR (δ, ppm): 13.72, 10.06, 8.00 (each s, 3 NH), 7.94 (s, 1H, NHSO2), 7.89, 7.84 (each s, 2H, thiazole), 7.56, 7.49, 7.39, 7.38, 7.10, 7.09, 7.78, 7.07, 6.79, 6.78, 6.55, 6.53, 6.36 (aromatic H). 13C NMR (δ, ppm): 173.18 (C–SO2), 167.90, 157.28 (C=O), 144.81 (C–F), 132.24, 132.22 (C–N), 130.22, 130.21, 129.62, 129.56, 128.93, 127.50, 125.58, 125.53, 124.40, 123.14, 116.78, 116.73, 116.62, 116.58, 115.03, 114.88, 112.82, 106.46 (aromatic carbons), 77.62–77.19. Anal. calcd. C25H17N7S2O4F2 (581): C, 51.63; H, 2.92; N, 16.86; S, 11.01; F, 6.55. Found: C, 51.34; H, 2.85; N, 16.70; S, 10.88; F, 6.35.10. Yield 82%, m.p. 178–180°C. Anal. calcd. C25H17N7SO5F2 (565): C, 53.09; H, 3.00; N, 17.16; S, 5.66; F, 6.72. Found: C, 52.88; H, 2.89; N, 17.01; S, 5.41; F, 6.55.11. Yield 89%, m.p. 247–249°C. UV: λ
max
⁡ (ε): 288.7 (1.09) nm. IR (ν, cm−1): 3500, 3310, 3290 (NH), 1661, 1620 (C=O), 1385 (NCSN), 1255 (C–F), 865 (aryl C–H), 658 (C–F). 1H NMR (δ, ppm): 13.79, 11.72, 10.92 (each s, 3 NH), 7.94, 7.88, 7.86, 7.70, 7.54, 7.53, 7.47, 7.25, 7.09, 7.07, 7.06 (aromatic H). 13C NMR (δ, ppm): 164.95–163.27, 158.86–157.24 (C=O), 146.34, 144.74, 138.74 (C–F), 132.22 (C–N), 129.56, 129.51, 125.48, 124.46, 116.77, 116.61, 115.02, 114.88 (aromatic carbons), 77.49-77.07. MS (relative intensity): 437 (M-45, 392, 1.0%), 166 (32.0), 148 (100), 122 (1.1), 110 (11), 95 (3.0). Anal. calcd. C22H14N5S2F3 (437): C, 60.41; H, 3.20; N, 16.01; F, 13.04. Found: C, 59.59; H, 3.10; N, 15.66; F, 12.85. 19F NMR: δ  −127 and −129 ppm.12. Yield 80%, m.p. 200–202°C. UV: λ
max
⁡ (ε): 271.8 (1.51) nm. IR (ν, cm−1): 3525–3100 (3 NH), 1659, 1610 (C=O, CONH), 1386 (NCSN), 1255 (C–F), 865 (aryl C–H), 658 (C–F). 1H NMR (δ, ppm): 13.79, 11.72, 10.92 (each s, 3 NH), 9.98–9.74, 8.16-8.15, 7.96-7.95, 7.94 (4H of pyridine), 7.86–7.84, 7.59–7.55, 7.40–7.37, 7.10–7.08, 7.07–7.05, 6.82-6.81, 6.80-6.79 (aromatic H). 13C NMR (δ, ppm): 164.92–163.25, 158.87–157.25 (C=O), 145.44, 144.84 (C–F), 137.71 (C–N), 132.24, 130.23, 129.61, 129.55, 125.58, 125.53, 124.45, 124.39 (aromatic carbons), 116.76, 116.69, 116.54, 115.69, 114.99 (pyridine carbons). Anal. calcd. C26H17N7O2F2 (497): C, 62.75; H, 3.40; N, 19.71; F, 7.62. Found: C, 62.45; H, 3.30; N, 19.55; F, 7.43.


#### 2.1.8. 4-Fluoro-N-(4-fluoro-2-(3-((4-fluorophenyl)amino)-5-oxo-4,5-dihydro-1,2,4-triazin-6-yl)phenyl)benzamide (11)

Procedure A. Equimolar mixture of compound14 and 4-fluoroaniline was refluxed for 6 h then cooled. The solid thus obtained was filtered and crystallized from ethanol to give11 as faint yellow crystals. Yield 78%, m.p. 247–249°C.Procedure B. An equimolar mixture of compound5 and 4-fluoroaniline in ethanol (50 mL) was refluxed for 6 h and then cooled. The resulting solid was filtered and crystallized from ethanol to give faint yellow crystals of11. Yield 75%, m.p. 247–249°C.


#### 2.1.9. 2-((6-(5-Fluoro-2-(4-fluorobenzamido)phenyl)-5-oxo-4,5-dihydro-1,2,4-triazin-3-yl)thio)acetic acid (13)

A mixture of 5 (0.001 mol) and monochloroacetic acid (0.001 mol) in pyridine (20 mL) was refluxed for 15 min, cooled, and then poured onto ice-cold dil. HCl. The resulting solid was filtered and crystallized from ethanol to give13 as yellow crystals. Yield 75%, m.p. 190–192°C. IR (ν, cm−1): 3550–3100 (br, OH and NH), 2919, 2885 (C–H), 1658, 1610 (C=O, CONH), 1489, 1429 (deformation CH2), 1386 (NCSN), 1255 (C–F), 865 (aryl C–H), 658 (C–F). 1H NMR (δ, ppm): 14.45 (NH), 12.58 (NH), 10.25–10.08 (OH), 7.88–7.85, 7.84-7.83, 7.65–7.31, 7.15-7.14, 7.13-7.12, 7.10–7.07 (aromatic H), 3.96 (CH2). 13C NMR (δ, ppm): 168.74 (C=O), 164.99 (C=O), 159.27–157.65 (C=O), 132.87–132.85 (C–S), 130.20, 130.18, 129.61, 129.49, 129.43, 127.19, 126.20, 117.18, 117.01, 116.73, 116.58, 115.17, 115.03, (aromatic carbons), 77.89–77.45, 40.88, 31.84 (CH2). MS (relative intensity): 418 (M-28, 390, 1.0%), 391 (1.5), 149 (100), 132 (2.8), 91 (1.10), 57 (47.00). Anal. calcd. C18H12N4SO4F2 (418): C, 53.11; H, 2.87; N, 13.35; S, 7.65; F, 9.09. Found: C, 52.88; H, 2.58; N, 13.05; S, 7.33; F, 8.79.


#### 2.1.10. 4-Fluoro-N-(4-fluoro-2-(3-(methylthio)-5-oxo-4,5-dihydro-1,2,4-triazin-6-yl)phenyl)benzamide (14)

Procedure A. A mixture of compound13 (0.050 mol) and 10% ethanolic K2CO3 (10 mL) was warmed for 15 min, cooled, and then poured onto ice-cold dil. HCl. The solid product was filtered and crystallized from dioxane to give deep orange crystals of14. Yield 82%, m.p. 228–230°C.Procedure B. A mixture of compound 5 (0.001 mol) and CH3I (0.001 mol) in ethanolic KOH (1%, 20 mL) was stirred at room temperature for 12 h. The resulting reaction mixture was acidified with dil. HCl. A brownish orange precipitate was separated which on crystallization with dioxane gave 14 as orange crystals. Yield 80%, m.p. 228-229°C. Mixed m.p. 228°C. IR (ν, cm-1): 3484–3250 (br, NH, NH), 2930, 2864 (C–H), 1657, 1620 (C=O, CONH), 1489, 1449 (deformation CH2), 1386 (NCSN), 1255 (C–F), 864 (aryl C–H), 658 (C–F). 1H NMR (δ, ppm): 12.65, 12.37–12.35 (each s, NH, NH), 7.89-7.88, 7.87, 7.40–7.25, 7.14-7.13, 7.12, 7.12–7.10, 7.08–7.04 (aromatic H). 13C NMR (δ, ppm): 163.60, 159.38 (C=O), 157.79, 147.45 (C–F), 132.89 (C–S), 129.85, 129.28, 129.26, 129.22, 129.20, 127.43, 126.28, 126.25, 117.19, 117.03, 116.85, 116.82, 116.70, 116.67, 115.14, 115.12, 115.00, 114.97 (aromatic and heteroaromatic carbons), 77.21–76.78, 39.91–39.07 (CH3). Anal. calcd. C17H12N4SO2F2 (374): C, 54.50; H, 3.20; N, 14.89; S, 8.56; F, 10.17. Found: C, 53.89; H, 3.01; N, 14.55; S, 8.19; F, 10.11.


#### 2.1.11. 6-(2-Amino-5-fluorophenyl)-3-((4-fluorophenyl)amino)-1,2,4-triazin-5(4H)-one (15)

A mixture of1 (0.001 mol) and 4-fluoroaniline (0.001 mol) in ethanol (50 mL) was refluxed for 6 h. On cooling a solid was precipitated which was filtered and crystallized from ethanol to form yellowish crystals of15. Yield 80%, m.p. 308–310°C. IR (ν, cm−1): 3534, 3300 (NH2, NH), 1661 (C=O), 1386 (NCSN), 1255 (C–F), 864 (aryl C–H), 658 (C–F). Anal. calcd. C15H11N5OF2 (315): C, 58.00; H, 3.49; N, 22.22; F, 12.06. Found: C, 57.72; H, 3.15; N, 21.88; F, 11.78. 19F NMR: δ  −127 and −129 ppm.


#### 2.1.12. Aroylation of15 to Form 11

An equimolar mixture of15 and 4-fluorobenzoyl chloride in DMF (20 mL) was warmed for 10 min, cooled, and then poured onto ice. The resultant solid was filtered and crystallized from ethanol to give11 as yellowish crystals. Yields 82%, m.p. 248°C. Mixed m.p. 247°C.


#### 2.1.13. N,N′-((3,3′-Disulfanediylbis(5-oxo-4,5-dihydro-1,2,4-triazine-6,3-diyl))bis(4-fluoro-2,1-phenylene))bis(4-fluorobenzamide) (16)

A mixture of compound5 (0.002 mol) and sulfur (0.002 mol) flowers in dry benzene (100 mL) was refluxed for 4 h. On cooling the reaction mixture a solid appeared which was filtered and crystallized from benzene to give16 as orange crystals. Yield 88%, m.p. 246–248°C. UV: λ
max
⁡ (ε): 288.8 (1.14) nm. IR (ν, cm−1): 3527–3320 (br, NH, NH), 1660, 1608 (C=O, CONH), 1386 (NCSN), 1255 (C–F), 1090 (C–S), 864 (aryl C–H), 658 (C–F). MS (relative intensity): 718 (M-359, –H2O, 341, 85%), 309 (55.01), 306 (100), 290 (90.00), 209 (8.11). Anal. calcd. C32H18N8S2O4F4 (718): C, 53.48; H, 2.50; N, 15.60; S, 8.91; F, 10.58. Found: C, 53.15; H, 2.30; N, 15.31; S, 8.56; F, 10.59. 19F NMR: δ  −127 and −130 ppm.


#### 2.1.14. 2,2,2-Tris((6-(5-fluoro-2-(4-fluorobenzamido)phenyl)-5-oxo-4,5-dihydro-1,2,4-triazin-3-yl)thio)acetic acid (17)

A mixture of compounds1 (0.001 mol) and5 (0.001 mol) in DMF (50 mL) was refluxed for 1 h. The reaction mixture was left to cool at room temperature. The solid thus generated was filtered and crystallized from acetic acid to give17 as yellow crystals. Yield 65%, m.p. 230–232°C. UV: λ
max
⁡ (ε): 288.2 (1.23) nm. IR (ν, cm−1): 3544 (OH), 3320 (NH), 2990, 2898 (C–H), 1760–1610 (C=O), 1499, 1449 (deformation CH2), 1386 (NCSN), 1255 (C–F), 1089 (C–S), 864 (aryl C–H), 658 (C–F). 1H NMR (δ, ppm): 13.72, 13.25, 12.61, 12.17 (4s, 4 NH), 10.09, 10.06 (2s, 2 NH), 7.98, 7.97, 7.96, 7.95, 7.87, 7.86, 7.85, 7.84, 7.83, 7.54, 7.40, 7.39, 7.38, 7.35, 7.10, 7.09–7.05 (aromatic protons), 5.49 (s, 1H, OH). 13C NMR (δ, ppm): 164.98, 163.83, 163.32 (3 C=O), 148, 140 (C–F), 132.11 (C–S), 130.23, 130.19, 130.17, 129.62, 129.56, 129.47, 129.42, 125.51, 125.40 and 116.91, 116.81, 116.77, 116.74, 116.64, 116.62, 116.11, 115.12, 115.06, 114.97, 114.91, 77.59–77.16, 39.91–39.21 (aromatic, heteroaromatic and aliphatic carbons). Anal. calcd. C50H28N12S3O8F6 (1134): C, 52.55; H, 2.46; N, 14.81; S, 8.43; F, 10.05. Found: C, 51.98; H, 2.16; N, 14.53; S, 8.19; F, 9.85.


#### 2.1.15. 4-Fluoro-N-(4-fluoro-2-(3-((4-fluoro-2-(5-oxo-3-thioxo-2,3,4,5-tetrahydro-1,2,4-triazin-6-yl)phenyl)amino)-5-oxo-4,5-dihydro-1,2,4-triazin-6-yl)phenyl)benzamide (18)

An equimolar mixture of1 and5 (0.001 mol) in isopropyl alcohol (50 mL) was refluxed for 6 h. Some of the solvent of reaction mixture was evaporatedin vacuo. The product was separated as brownish orange solid which was crystallized from ethanol to form orange crystals of compound18. Yield 80%, m.p. 246–248°C. UV: λ
max
⁡ (ε): 287.8 (2.07) nm. IR (ν, cm−1): 3320, 3300 (NH, NH), 1668, 1620 (C=O, CONH), 1385 (NCSN), 1255 (C–F), 1185 (C=S), 864 (aryl C–H), 657 (C–F). MS (relative intensity): 564 (M-196, 368, 7.0%), 247 (0.01), 204 (3.0), 203 (5.0), 196 (1.0), 100 (1.1), 88 (100), 76 (66.0), 42 (0.5). Anal. calcd. C25H15N8SO3F3 (564): C, 53.34; H, 5.67; N, 19.89; S, 5.67; F, 10.10. Found: C, 52.89; H, 5.55; N, 19.51; S, 5.38; F, 9.88. 19F NMR: δ  −126, −128 and −130 ppm.


### 2.2. Biological Evaluation

#### 2.2.1. Anti-HIV-1 Activity

All the new synthesized compounds have been evaluated for theirin vitro anti-HIV activity that was performed on T-4 lymphocytes infected and uninfected with HIV-1 using DMSO as solvent. The assay involves the killing of T-4 lymphocytes by HIV. Uninfected cells with the compound serve as a toxicity control, and infected and uninfected cells without the compound serve as basic controls. Cultures are incubated at 37°C in a 5% carbon dioxide atmosphere for 6 days. The tetrazolium salt, XTT, is added to all wells, and cultures are incubated to allow formazan color development by viable cells. Compounds that degenerate or are rapidly metabolized in the culture conditions may not show activity in this screen. Zidovudine (AZT) at 10 μM was used as a control. The viability of the cells was determined spectrophotometrically to quantitate formazan production and in addition is viewed microscopically for detection of viable cells and confirmation of protective activity. Drug-treated virus-infected cells are compared with drug treated noninfected cells and with other appropriate controls (untreated infected and untreated noninfected cells, drug-containing wells without cells) on the same plate.


#### 2.2.2. CDK2 Inhibition Assay

CDK2-cyclin E kinase was expressed and assayed as previously described [39]. Kinase activity was expressed as a percentage of maximum activity. The concentration of the test compounds required to decrease the CDK activity by 50% was determined from dose-response curves and designated IC50.


## 2.1. Chemistry

Melting points were determined on an electrothermal Bibby Stuart Scientific melting point apparatus and are uncorrected. The infrared (IR) spectra were recorded on PerkinElmer RXI FT-IR infrared spectrophotometer using the KBr pellet technique. Electronic absorption spectra were recorded in DMF on Shimadzu UV-Visible 3101 PC spectrophotometer.1H and 13C NMR spectra were recorded on a Bruker DPX-400 FT NMR spectrometer using tetramethylsilane as the internal standard DMSO-d
6 as a solvent (chemical shifts in δ, ppm). 19F NMR spectra were determined at 84.25 MHz using hexafluorobenzene as an internal standard. Splitting patterns were designated as follows:s: singlet;m: multiplet. Mass spectra were measured on a GCMS-Q 1000 Ex spectrometer. Elemental analyses were performed on a 2400 PerkinElmer Series 2 analyzer. Follow-up of the reactions and checking the homogeneity of the compounds were made by TLC on silica gel-protected aluminum sheets (Type 60 F254, Merck) and the spots were detected by exposure to UV-lamp at λ 254.The anti-HIV and anticancer activity were evaluated in DTP, DCT, National Cancer Institute, Bethesda, MD 20892, USA.


### 2.1.1. 6-(2-Amino-5-fluorophenyl)-3-thioxo-3,4-dihydro-1,2,4-triazin-5(2H)-one (1)

A mixture of 5-fluoroisatin (0.001 mol in warm 5% aqueous NaOH) and thiosemicarbazide (0.001 mol, in hot water 10 mL) was refluxed for 2 h. The reaction mixture was then poured onto ice and neutralized with dil. HCl. The solid thus obtained was filtered off and crystallized from ethanol to give1 as pale yellow crystals. Yield 80%, m.p. 285–287°C. UV: λ
max
⁡ (ε): 282.8 (2.06) nm. IR (ν, cm−1): 3528, 3300 (NH, NH2), 1661 (C=O), 1385 (NCSN), 1255 (C–F), 865 (aryl C–H), 658 (C–F). 1H NMR (δ, ppm): 13.47, (s, 1H, NH), 7.52–7.42, 6.93–6.87 (3H, aromatic H), 3.2 (s, 2H, NH2). 13C NMR (δ, ppm): 172.74 (C=S), 156–152 (C=O), 144.53 (C–F), 138.73 (C=N), 119.68, 117.60, 117.45, 116.50, 116.30 (aromatic carbons), 77.27–76.84, 39.91–39.07 (C–C, C–N carbons). MS (relative intensity): 238 (M + H2O, 254, 5%), 206 (207, 36), 182 (10), 108 (1.1), 103 (100), 75 (35). Anal. calcd. C9H7N4SOF (238): C, 45.38; H, 2.93; N, 23.52; S, 13.0; F, 7.98. Found: C, 44.88; H, 2.55; N, 23.41; S, 12.85; F, 7.77. 19F NMR: δ  −127 ppm.


### 2.1.2. 2-((6-(2-((Carboxymethyl)amino)-5-fluorophenyl)-5-oxo-4,5-dihydro-1,2,4-triazin-3-yl)thio)acetic acid (2)

A mixture of1 (0.001 mol) and monochloroacetic acid (0.002 mol) in ethanolic NaOH (5%, 50 mL) was warmed for 30 min and then cooled and poured onto ice-cold dil. acetic acid. The product is precipitated as pale solid which was then filtered and crystallized from acetic acid to give yellow crystals of compound2. Yield 70%, m.p. 262–264°C. UV: λ
max
⁡ (ε): 283.3 (1.93) nm. IR (ν, cm−1): 3531 (OH), 3300 (NH), 2929, 2861 (2 CH2), 1660 (C=O), 1499, 1439 (deformation CH2), 1385 (NCSN), 1255 (C–F), 865 (aryl C–H), 658 (C–F). 1H NMR (δ, ppm): 13.38, (s, 1H, OH), 11.82 (s, NH), 7.55–7.37, 6.85–6.71 (aromatic H), 3.55-3.54, 3.53–3.51 (each s, 2 CH2). 13C NMR (δ, ppm): 172.67, 152.42 (2 C=O), 145 (C–F), 117.64, 117.50, 116.41, (aromatic carbons), 77.15, 76.94, 76.73 39.91–39.07 (C–S, C–O, and C–N carbons), 39.91, 39.07 (2 CH2). MS (relative intensity): 266 (M-2 (CO2), 100. Anal. calcd. C13H11N4SO5F (354): C, 44.07; H, 3.10; N, 16.0; S, 9.03; F, 5.35. Found: C, 43.78; H, 2.85; N, 15.69; S, 8.81; F, 5.21. 19F NMR: δ  −130 ppm.


### 2.1.3. 6-(5-Fluoro-2-(methylamino)phenyl)-3-(methylthio)-1,2,4-triazin-5(4H)-one (3)

Procedure A. A mixture of compound2 (0.050 mol) and 10% K2CO3 (20 mL) was refluxed for 10 min and then left to acquire room temperature. The reaction mixture was now poured in an ice-cold dil. HCl. The precipitated solid was filtered and crystallized as faint yellow crystals. Yield 50%, m.p. 163-164°C.Procedure B. A mixture of compound1 (0.001), methyl iodide (0.002 mol), and 1% alcoholic KOH (20 mL) was stirred at room temperature for 4 h. The reaction mixture was then neutralized with dil. HCl. The resulting light brown solid was filtered and crystallized from ethanol to form yellowish crystals of compound3. Yield 65%, m.p. 162-163°C. UV: λ
max
⁡ (ε): 315.4 (1.154) nm. IR (ν, cm−1): 3310 (NH), 2929, 2862 (2 CH2), 1661 (C=O), 1499, 1438 (deformation CH2), 1385 (NCSN), 1255 (C–F), 865 (aryl C–H), 658 (C–F). MS (relative intensity): 266 (M+H2O, 284, 5%), 110 (5.5), 104 (100), 95 (1.18), 88 (100), 47 (65.0). Anal. calcd. C11H11N4SOF (266): C, 49.95; H, 4.13; N, 21.05; S, 12.03; F, 7.14. Found: C, 49.71; H, 4.01; N, 20.99; S, 11.88; F, 6.91. 19F NMR: δ  −126 ppm.


### 2.1.4. 2,2′-(((3,3′-((Carboxymethylene)bis(sulfanediyl))bis(5-oxo-4,5-dihydro-1,2,4-triazine-6,3-diyl))bis(4-fluoro-2,1-phenylene))bis(azanylylidene))diacetic acid (4)

A mixture of compound1 (0.001 mol) and 1,1-dichloroacetic acid (0.003 mol) in DMF (20 mL) was refluxed for 10 min, cooled, and then poured on ice. The solid thus separated was filtered and crystallized from THF to give deep orange crystals. Yield 66%, m.p. 280–282°C. UV: λ
max
⁡ (ε): 266.5 (1.51) nm. IR (ν, cm−1): 3534, 3480 (OH), 3350 (NH), 2929, 2861 (aliphatic C–H), 1680, 1660 (C=O), 1499, 1439 (deformation C–H), 1386 (NCSN), 1255 (C–F), 865 (aryl C–H), 658 (C–F). MS (relative intensity): 644 (M-208, 3%), 298 (100), 167 (31.01), 149 (100), 85 (5.00). Anal. calcd. C24H14N8S2O8F2 (266): C, 44.72; H, 2.17; N, 17.36; S, 9.9; F, 5.95. Found: C, 44.51; H, 2.11; N, 17.12; S, 9.55; F, 5.71. 19F NMR: δ  −128 ppm.


### 2.1.5. 4-Fluoro-N-(4-fluoro-2-(5-oxo-3-thioxo-2,3,4,5-tetrahydro-1,2,4-triazin-6-yl)phenyl)benzamide (5)

A mixture of compound1 (0.001 mol) and 4-fluorobenzoyl chloride (0.001 mol) in dry pyridine (10 mL) was warmed for 10 min, cooled, and then poured on ice. The precipitated solid was filtered and crystallized from dioxane to give faint yellow crystals of5. Yield 85%, m.p. 238–240°C. UV: λ
max
⁡ (ε): 288.4 (1.28) nm. IR (ν, cm−1): 3370 (NH), 3320 (NH), 1663, 1610 (C=O, CONH), 1385 (NCSN), 1255 (C–F), 1190 (C–S), 864 (aryl C–H), 658 (C–F). 1H NMR (δ, ppm): 13.75, (NH), 10.07 (NH), 8.60 (NH), 7.98–7.82, 7.53–7.41, 7.10–7.05 (aromatic H). 13C NMR (δ, ppm): 173.15 (C=S), 164.97–163.30, 158.87–158.57 (C=O), 146.91 (C–F), 144.70 (C–F), 138.07 (C–N), 132.21, 130.16, 129.57, 129.51, 125.5, 124.26 (aromatic carbons), 116.63, 115.04, 114.90 (C–S, C–O, C–N) 77.50–77.07, 40.03, 39.08. MS (relative intensity): 360 (M+4, 6%), 265 (78.0), 123 (12.05), 96 (2.0), 89 (100), 76 (75.0). Anal. calcd. C16H10N4SO2F2 (360): C, 53.30; H, 2.78; N, 15.50; S, 9.16; F, 10.55. Found: C, 52.88; H, 2.55; N, 15.02; S, 8.95; F, 10.35. 19F NMR: δ  −126 and −129 ppm.


### 2.1.6. N-(2-(2,4-Disubstituted-5-oxo-3-thioxo-2,3,4,5-tetrahydro-1,2,4-triazin-6-yl)-4-fluorophenyl)-4-fluorobenzamide (6–8)

A mixture of5 (0.001 mol) methanol or morpholine or 2,2′-dipyridylamine (0.001 mol) in methanol (20 mL) and formaldehyde (0.001 mol) was refluxed for 2 h. The reaction mixture was cooled to acquire room temperature and then poured onto ice-cold water. The solid product precipitated was filtered and crystallized from methanol to form yellow-orange crystals.6. Yield 72%, m.p. 310–312°C. IR (ν, cm−1): 3527 (OH), 3100 (NH), 2935, 2890 (aliphatic C–H), 1660, 1600 (C=O, CONH), 1498, 1440 (deformation CH2), 1386 (NCSN), 1255 (C–F), 864 (aryl C–H), 658 (C–F). 1H NMR (δ, ppm): 12.54 (s, 1H, NH), 10.15, 8.04 (Two s, 2 OH), 7.96–7.94, 7.87–7.83, 7.64-7.63 (aromatic H), 2.49 and 2.48 (Two s, 2 CH2). 13C NMR (δ, ppm): 164.99 (C=S), 163.32, 157.02 (C=O), 135.99 (C–F), 130.39, 130.20, 129.53, 129.44, 129.36, 123.45, (aromatic carbons), 115.14, 114.99 (C–S, C–O) 77.38–76.95, 39.83, 39.29. Anal. calcd. C18H14N4SO4F2 (420): C, 51.42; H, 3.36; N, 13.30; S, 7.61; F, 9.04. Found: C, 51.23; H, 3.01; N, 13.11; S, 7.51; F, 8.88. 19F NMR: δ  −126 and −129 ppm.7. Yield 70%, m.p. 260–262°C. Anal. calcd. C26H28N6SO4F2 (558): C, 55.90; H, 5.02; N, 15.05; S, 5.70; F, 6.81. Found: C, 55.68; H, 4.98; N, 14.79; S, 5.35; F, 6.66.8. Yield 72%; m.p. 202–204°C. Anal. calcd. C38H28N10SO2F2 (726): C, 62.79; H, 3.88; N, 19.28; S, 4.27; F, 5.23. Found: C, 62.33; H, 3.58; N, 19.11; S, 4.15; F, 4.89.


### 2.1.7. 4-Fluoro-N-(4-fluoro-2-(3-(substituted-amino)-5-oxo-4,5-dihydro-1,2,4-triazin-6-yl)phenyl)benzamide (9–12)

A mixture of 5 (0.001 mol) and sulfathiazole or sulfaoxazole or 4-fluoroaniline or 2,2′-dipyridylamine (0.001 mol) in absolute ethanol (50 mL) was refluxed for 6 h. On cooling a solid is separated which is filtered and crystallized from ethanol to give yellow crystals of9–12.9. Yield 85%, m.p. 172–174°C. IR (ν, cm−1): 3484−3310 (NH), 1658, 1610 (C=O, CONH), 1386 (NCSN), 1255 (C–F), 864 (aryl C–H), 658 (C–F). 1H NMR (δ, ppm): 13.72, 10.06, 8.00 (each s, 3 NH), 7.94 (s, 1H, NHSO2), 7.89, 7.84 (each s, 2H, thiazole), 7.56, 7.49, 7.39, 7.38, 7.10, 7.09, 7.78, 7.07, 6.79, 6.78, 6.55, 6.53, 6.36 (aromatic H). 13C NMR (δ, ppm): 173.18 (C–SO2), 167.90, 157.28 (C=O), 144.81 (C–F), 132.24, 132.22 (C–N), 130.22, 130.21, 129.62, 129.56, 128.93, 127.50, 125.58, 125.53, 124.40, 123.14, 116.78, 116.73, 116.62, 116.58, 115.03, 114.88, 112.82, 106.46 (aromatic carbons), 77.62–77.19. Anal. calcd. C25H17N7S2O4F2 (581): C, 51.63; H, 2.92; N, 16.86; S, 11.01; F, 6.55. Found: C, 51.34; H, 2.85; N, 16.70; S, 10.88; F, 6.35.10. Yield 82%, m.p. 178–180°C. Anal. calcd. C25H17N7SO5F2 (565): C, 53.09; H, 3.00; N, 17.16; S, 5.66; F, 6.72. Found: C, 52.88; H, 2.89; N, 17.01; S, 5.41; F, 6.55.11. Yield 89%, m.p. 247–249°C. UV: λ
max
⁡ (ε): 288.7 (1.09) nm. IR (ν, cm−1): 3500, 3310, 3290 (NH), 1661, 1620 (C=O), 1385 (NCSN), 1255 (C–F), 865 (aryl C–H), 658 (C–F). 1H NMR (δ, ppm): 13.79, 11.72, 10.92 (each s, 3 NH), 7.94, 7.88, 7.86, 7.70, 7.54, 7.53, 7.47, 7.25, 7.09, 7.07, 7.06 (aromatic H). 13C NMR (δ, ppm): 164.95–163.27, 158.86–157.24 (C=O), 146.34, 144.74, 138.74 (C–F), 132.22 (C–N), 129.56, 129.51, 125.48, 124.46, 116.77, 116.61, 115.02, 114.88 (aromatic carbons), 77.49-77.07. MS (relative intensity): 437 (M-45, 392, 1.0%), 166 (32.0), 148 (100), 122 (1.1), 110 (11), 95 (3.0). Anal. calcd. C22H14N5S2F3 (437): C, 60.41; H, 3.20; N, 16.01; F, 13.04. Found: C, 59.59; H, 3.10; N, 15.66; F, 12.85. 19F NMR: δ  −127 and −129 ppm.12. Yield 80%, m.p. 200–202°C. UV: λ
max
⁡ (ε): 271.8 (1.51) nm. IR (ν, cm−1): 3525–3100 (3 NH), 1659, 1610 (C=O, CONH), 1386 (NCSN), 1255 (C–F), 865 (aryl C–H), 658 (C–F). 1H NMR (δ, ppm): 13.79, 11.72, 10.92 (each s, 3 NH), 9.98–9.74, 8.16-8.15, 7.96-7.95, 7.94 (4H of pyridine), 7.86–7.84, 7.59–7.55, 7.40–7.37, 7.10–7.08, 7.07–7.05, 6.82-6.81, 6.80-6.79 (aromatic H). 13C NMR (δ, ppm): 164.92–163.25, 158.87–157.25 (C=O), 145.44, 144.84 (C–F), 137.71 (C–N), 132.24, 130.23, 129.61, 129.55, 125.58, 125.53, 124.45, 124.39 (aromatic carbons), 116.76, 116.69, 116.54, 115.69, 114.99 (pyridine carbons). Anal. calcd. C26H17N7O2F2 (497): C, 62.75; H, 3.40; N, 19.71; F, 7.62. Found: C, 62.45; H, 3.30; N, 19.55; F, 7.43.


### 2.1.8. 4-Fluoro-N-(4-fluoro-2-(3-((4-fluorophenyl)amino)-5-oxo-4,5-dihydro-1,2,4-triazin-6-yl)phenyl)benzamide (11)

Procedure A. Equimolar mixture of compound14 and 4-fluoroaniline was refluxed for 6 h then cooled. The solid thus obtained was filtered and crystallized from ethanol to give11 as faint yellow crystals. Yield 78%, m.p. 247–249°C.Procedure B. An equimolar mixture of compound5 and 4-fluoroaniline in ethanol (50 mL) was refluxed for 6 h and then cooled. The resulting solid was filtered and crystallized from ethanol to give faint yellow crystals of11. Yield 75%, m.p. 247–249°C.


### 2.1.9. 2-((6-(5-Fluoro-2-(4-fluorobenzamido)phenyl)-5-oxo-4,5-dihydro-1,2,4-triazin-3-yl)thio)acetic acid (13)

A mixture of 5 (0.001 mol) and monochloroacetic acid (0.001 mol) in pyridine (20 mL) was refluxed for 15 min, cooled, and then poured onto ice-cold dil. HCl. The resulting solid was filtered and crystallized from ethanol to give13 as yellow crystals. Yield 75%, m.p. 190–192°C. IR (ν, cm−1): 3550–3100 (br, OH and NH), 2919, 2885 (C–H), 1658, 1610 (C=O, CONH), 1489, 1429 (deformation CH2), 1386 (NCSN), 1255 (C–F), 865 (aryl C–H), 658 (C–F). 1H NMR (δ, ppm): 14.45 (NH), 12.58 (NH), 10.25–10.08 (OH), 7.88–7.85, 7.84-7.83, 7.65–7.31, 7.15-7.14, 7.13-7.12, 7.10–7.07 (aromatic H), 3.96 (CH2). 13C NMR (δ, ppm): 168.74 (C=O), 164.99 (C=O), 159.27–157.65 (C=O), 132.87–132.85 (C–S), 130.20, 130.18, 129.61, 129.49, 129.43, 127.19, 126.20, 117.18, 117.01, 116.73, 116.58, 115.17, 115.03, (aromatic carbons), 77.89–77.45, 40.88, 31.84 (CH2). MS (relative intensity): 418 (M-28, 390, 1.0%), 391 (1.5), 149 (100), 132 (2.8), 91 (1.10), 57 (47.00). Anal. calcd. C18H12N4SO4F2 (418): C, 53.11; H, 2.87; N, 13.35; S, 7.65; F, 9.09. Found: C, 52.88; H, 2.58; N, 13.05; S, 7.33; F, 8.79.


### 2.1.10. 4-Fluoro-N-(4-fluoro-2-(3-(methylthio)-5-oxo-4,5-dihydro-1,2,4-triazin-6-yl)phenyl)benzamide (14)

Procedure A. A mixture of compound13 (0.050 mol) and 10% ethanolic K2CO3 (10 mL) was warmed for 15 min, cooled, and then poured onto ice-cold dil. HCl. The solid product was filtered and crystallized from dioxane to give deep orange crystals of14. Yield 82%, m.p. 228–230°C.Procedure B. A mixture of compound 5 (0.001 mol) and CH3I (0.001 mol) in ethanolic KOH (1%, 20 mL) was stirred at room temperature for 12 h. The resulting reaction mixture was acidified with dil. HCl. A brownish orange precipitate was separated which on crystallization with dioxane gave 14 as orange crystals. Yield 80%, m.p. 228-229°C. Mixed m.p. 228°C. IR (ν, cm-1): 3484–3250 (br, NH, NH), 2930, 2864 (C–H), 1657, 1620 (C=O, CONH), 1489, 1449 (deformation CH2), 1386 (NCSN), 1255 (C–F), 864 (aryl C–H), 658 (C–F). 1H NMR (δ, ppm): 12.65, 12.37–12.35 (each s, NH, NH), 7.89-7.88, 7.87, 7.40–7.25, 7.14-7.13, 7.12, 7.12–7.10, 7.08–7.04 (aromatic H). 13C NMR (δ, ppm): 163.60, 159.38 (C=O), 157.79, 147.45 (C–F), 132.89 (C–S), 129.85, 129.28, 129.26, 129.22, 129.20, 127.43, 126.28, 126.25, 117.19, 117.03, 116.85, 116.82, 116.70, 116.67, 115.14, 115.12, 115.00, 114.97 (aromatic and heteroaromatic carbons), 77.21–76.78, 39.91–39.07 (CH3). Anal. calcd. C17H12N4SO2F2 (374): C, 54.50; H, 3.20; N, 14.89; S, 8.56; F, 10.17. Found: C, 53.89; H, 3.01; N, 14.55; S, 8.19; F, 10.11.


### 2.1.11. 6-(2-Amino-5-fluorophenyl)-3-((4-fluorophenyl)amino)-1,2,4-triazin-5(4H)-one (15)

A mixture of1 (0.001 mol) and 4-fluoroaniline (0.001 mol) in ethanol (50 mL) was refluxed for 6 h. On cooling a solid was precipitated which was filtered and crystallized from ethanol to form yellowish crystals of15. Yield 80%, m.p. 308–310°C. IR (ν, cm−1): 3534, 3300 (NH2, NH), 1661 (C=O), 1386 (NCSN), 1255 (C–F), 864 (aryl C–H), 658 (C–F). Anal. calcd. C15H11N5OF2 (315): C, 58.00; H, 3.49; N, 22.22; F, 12.06. Found: C, 57.72; H, 3.15; N, 21.88; F, 11.78. 19F NMR: δ  −127 and −129 ppm.


### 2.1.12. Aroylation of15 to Form 11

An equimolar mixture of15 and 4-fluorobenzoyl chloride in DMF (20 mL) was warmed for 10 min, cooled, and then poured onto ice. The resultant solid was filtered and crystallized from ethanol to give11 as yellowish crystals. Yields 82%, m.p. 248°C. Mixed m.p. 247°C.


### 2.1.13. N,N′-((3,3′-Disulfanediylbis(5-oxo-4,5-dihydro-1,2,4-triazine-6,3-diyl))bis(4-fluoro-2,1-phenylene))bis(4-fluorobenzamide) (16)

A mixture of compound5 (0.002 mol) and sulfur (0.002 mol) flowers in dry benzene (100 mL) was refluxed for 4 h. On cooling the reaction mixture a solid appeared which was filtered and crystallized from benzene to give16 as orange crystals. Yield 88%, m.p. 246–248°C. UV: λ
max
⁡ (ε): 288.8 (1.14) nm. IR (ν, cm−1): 3527–3320 (br, NH, NH), 1660, 1608 (C=O, CONH), 1386 (NCSN), 1255 (C–F), 1090 (C–S), 864 (aryl C–H), 658 (C–F). MS (relative intensity): 718 (M-359, –H2O, 341, 85%), 309 (55.01), 306 (100), 290 (90.00), 209 (8.11). Anal. calcd. C32H18N8S2O4F4 (718): C, 53.48; H, 2.50; N, 15.60; S, 8.91; F, 10.58. Found: C, 53.15; H, 2.30; N, 15.31; S, 8.56; F, 10.59. 19F NMR: δ  −127 and −130 ppm.


### 2.1.14. 2,2,2-Tris((6-(5-fluoro-2-(4-fluorobenzamido)phenyl)-5-oxo-4,5-dihydro-1,2,4-triazin-3-yl)thio)acetic acid (17)

A mixture of compounds1 (0.001 mol) and5 (0.001 mol) in DMF (50 mL) was refluxed for 1 h. The reaction mixture was left to cool at room temperature. The solid thus generated was filtered and crystallized from acetic acid to give17 as yellow crystals. Yield 65%, m.p. 230–232°C. UV: λ
max
⁡ (ε): 288.2 (1.23) nm. IR (ν, cm−1): 3544 (OH), 3320 (NH), 2990, 2898 (C–H), 1760–1610 (C=O), 1499, 1449 (deformation CH2), 1386 (NCSN), 1255 (C–F), 1089 (C–S), 864 (aryl C–H), 658 (C–F). 1H NMR (δ, ppm): 13.72, 13.25, 12.61, 12.17 (4s, 4 NH), 10.09, 10.06 (2s, 2 NH), 7.98, 7.97, 7.96, 7.95, 7.87, 7.86, 7.85, 7.84, 7.83, 7.54, 7.40, 7.39, 7.38, 7.35, 7.10, 7.09–7.05 (aromatic protons), 5.49 (s, 1H, OH). 13C NMR (δ, ppm): 164.98, 163.83, 163.32 (3 C=O), 148, 140 (C–F), 132.11 (C–S), 130.23, 130.19, 130.17, 129.62, 129.56, 129.47, 129.42, 125.51, 125.40 and 116.91, 116.81, 116.77, 116.74, 116.64, 116.62, 116.11, 115.12, 115.06, 114.97, 114.91, 77.59–77.16, 39.91–39.21 (aromatic, heteroaromatic and aliphatic carbons). Anal. calcd. C50H28N12S3O8F6 (1134): C, 52.55; H, 2.46; N, 14.81; S, 8.43; F, 10.05. Found: C, 51.98; H, 2.16; N, 14.53; S, 8.19; F, 9.85.


### 2.1.15. 4-Fluoro-N-(4-fluoro-2-(3-((4-fluoro-2-(5-oxo-3-thioxo-2,3,4,5-tetrahydro-1,2,4-triazin-6-yl)phenyl)amino)-5-oxo-4,5-dihydro-1,2,4-triazin-6-yl)phenyl)benzamide (18)

An equimolar mixture of1 and5 (0.001 mol) in isopropyl alcohol (50 mL) was refluxed for 6 h. Some of the solvent of reaction mixture was evaporatedin vacuo. The product was separated as brownish orange solid which was crystallized from ethanol to form orange crystals of compound18. Yield 80%, m.p. 246–248°C. UV: λ
max
⁡ (ε): 287.8 (2.07) nm. IR (ν, cm−1): 3320, 3300 (NH, NH), 1668, 1620 (C=O, CONH), 1385 (NCSN), 1255 (C–F), 1185 (C=S), 864 (aryl C–H), 657 (C–F). MS (relative intensity): 564 (M-196, 368, 7.0%), 247 (0.01), 204 (3.0), 203 (5.0), 196 (1.0), 100 (1.1), 88 (100), 76 (66.0), 42 (0.5). Anal. calcd. C25H15N8SO3F3 (564): C, 53.34; H, 5.67; N, 19.89; S, 5.67; F, 10.10. Found: C, 52.89; H, 5.55; N, 19.51; S, 5.38; F, 9.88. 19F NMR: δ  −126, −128 and −130 ppm.


## 2.1.1. 6-(2-Amino-5-fluorophenyl)-3-thioxo-3,4-dihydro-1,2,4-triazin-5(2H)-one (1)

A mixture of 5-fluoroisatin (0.001 mol in warm 5% aqueous NaOH) and thiosemicarbazide (0.001 mol, in hot water 10 mL) was refluxed for 2 h. The reaction mixture was then poured onto ice and neutralized with dil. HCl. The solid thus obtained was filtered off and crystallized from ethanol to give1 as pale yellow crystals. Yield 80%, m.p. 285–287°C. UV: λ
max
⁡ (ε): 282.8 (2.06) nm. IR (ν, cm−1): 3528, 3300 (NH, NH2), 1661 (C=O), 1385 (NCSN), 1255 (C–F), 865 (aryl C–H), 658 (C–F). 1H NMR (δ, ppm): 13.47, (s, 1H, NH), 7.52–7.42, 6.93–6.87 (3H, aromatic H), 3.2 (s, 2H, NH2). 13C NMR (δ, ppm): 172.74 (C=S), 156–152 (C=O), 144.53 (C–F), 138.73 (C=N), 119.68, 117.60, 117.45, 116.50, 116.30 (aromatic carbons), 77.27–76.84, 39.91–39.07 (C–C, C–N carbons). MS (relative intensity): 238 (M + H2O, 254, 5%), 206 (207, 36), 182 (10), 108 (1.1), 103 (100), 75 (35). Anal. calcd. C9H7N4SOF (238): C, 45.38; H, 2.93; N, 23.52; S, 13.0; F, 7.98. Found: C, 44.88; H, 2.55; N, 23.41; S, 12.85; F, 7.77. 19F NMR: δ  −127 ppm.


## 2.1.2. 2-((6-(2-((Carboxymethyl)amino)-5-fluorophenyl)-5-oxo-4,5-dihydro-1,2,4-triazin-3-yl)thio)acetic acid (2)

A mixture of1 (0.001 mol) and monochloroacetic acid (0.002 mol) in ethanolic NaOH (5%, 50 mL) was warmed for 30 min and then cooled and poured onto ice-cold dil. acetic acid. The product is precipitated as pale solid which was then filtered and crystallized from acetic acid to give yellow crystals of compound2. Yield 70%, m.p. 262–264°C. UV: λ
max
⁡ (ε): 283.3 (1.93) nm. IR (ν, cm−1): 3531 (OH), 3300 (NH), 2929, 2861 (2 CH2), 1660 (C=O), 1499, 1439 (deformation CH2), 1385 (NCSN), 1255 (C–F), 865 (aryl C–H), 658 (C–F). 1H NMR (δ, ppm): 13.38, (s, 1H, OH), 11.82 (s, NH), 7.55–7.37, 6.85–6.71 (aromatic H), 3.55-3.54, 3.53–3.51 (each s, 2 CH2). 13C NMR (δ, ppm): 172.67, 152.42 (2 C=O), 145 (C–F), 117.64, 117.50, 116.41, (aromatic carbons), 77.15, 76.94, 76.73 39.91–39.07 (C–S, C–O, and C–N carbons), 39.91, 39.07 (2 CH2). MS (relative intensity): 266 (M-2 (CO2), 100. Anal. calcd. C13H11N4SO5F (354): C, 44.07; H, 3.10; N, 16.0; S, 9.03; F, 5.35. Found: C, 43.78; H, 2.85; N, 15.69; S, 8.81; F, 5.21. 19F NMR: δ  −130 ppm.


## 2.1.3. 6-(5-Fluoro-2-(methylamino)phenyl)-3-(methylthio)-1,2,4-triazin-5(4H)-one (3)

Procedure A. A mixture of compound2 (0.050 mol) and 10% K2CO3 (20 mL) was refluxed for 10 min and then left to acquire room temperature. The reaction mixture was now poured in an ice-cold dil. HCl. The precipitated solid was filtered and crystallized as faint yellow crystals. Yield 50%, m.p. 163-164°C.Procedure B. A mixture of compound1 (0.001), methyl iodide (0.002 mol), and 1% alcoholic KOH (20 mL) was stirred at room temperature for 4 h. The reaction mixture was then neutralized with dil. HCl. The resulting light brown solid was filtered and crystallized from ethanol to form yellowish crystals of compound3. Yield 65%, m.p. 162-163°C. UV: λ
max
⁡ (ε): 315.4 (1.154) nm. IR (ν, cm−1): 3310 (NH), 2929, 2862 (2 CH2), 1661 (C=O), 1499, 1438 (deformation CH2), 1385 (NCSN), 1255 (C–F), 865 (aryl C–H), 658 (C–F). MS (relative intensity): 266 (M+H2O, 284, 5%), 110 (5.5), 104 (100), 95 (1.18), 88 (100), 47 (65.0). Anal. calcd. C11H11N4SOF (266): C, 49.95; H, 4.13; N, 21.05; S, 12.03; F, 7.14. Found: C, 49.71; H, 4.01; N, 20.99; S, 11.88; F, 6.91. 19F NMR: δ  −126 ppm.


## 2.1.4. 2,2′-(((3,3′-((Carboxymethylene)bis(sulfanediyl))bis(5-oxo-4,5-dihydro-1,2,4-triazine-6,3-diyl))bis(4-fluoro-2,1-phenylene))bis(azanylylidene))diacetic acid (4)

A mixture of compound1 (0.001 mol) and 1,1-dichloroacetic acid (0.003 mol) in DMF (20 mL) was refluxed for 10 min, cooled, and then poured on ice. The solid thus separated was filtered and crystallized from THF to give deep orange crystals. Yield 66%, m.p. 280–282°C. UV: λ
max
⁡ (ε): 266.5 (1.51) nm. IR (ν, cm−1): 3534, 3480 (OH), 3350 (NH), 2929, 2861 (aliphatic C–H), 1680, 1660 (C=O), 1499, 1439 (deformation C–H), 1386 (NCSN), 1255 (C–F), 865 (aryl C–H), 658 (C–F). MS (relative intensity): 644 (M-208, 3%), 298 (100), 167 (31.01), 149 (100), 85 (5.00). Anal. calcd. C24H14N8S2O8F2 (266): C, 44.72; H, 2.17; N, 17.36; S, 9.9; F, 5.95. Found: C, 44.51; H, 2.11; N, 17.12; S, 9.55; F, 5.71. 19F NMR: δ  −128 ppm.


## 2.1.5. 4-Fluoro-N-(4-fluoro-2-(5-oxo-3-thioxo-2,3,4,5-tetrahydro-1,2,4-triazin-6-yl)phenyl)benzamide (5)

A mixture of compound1 (0.001 mol) and 4-fluorobenzoyl chloride (0.001 mol) in dry pyridine (10 mL) was warmed for 10 min, cooled, and then poured on ice. The precipitated solid was filtered and crystallized from dioxane to give faint yellow crystals of5. Yield 85%, m.p. 238–240°C. UV: λ
max
⁡ (ε): 288.4 (1.28) nm. IR (ν, cm−1): 3370 (NH), 3320 (NH), 1663, 1610 (C=O, CONH), 1385 (NCSN), 1255 (C–F), 1190 (C–S), 864 (aryl C–H), 658 (C–F). 1H NMR (δ, ppm): 13.75, (NH), 10.07 (NH), 8.60 (NH), 7.98–7.82, 7.53–7.41, 7.10–7.05 (aromatic H). 13C NMR (δ, ppm): 173.15 (C=S), 164.97–163.30, 158.87–158.57 (C=O), 146.91 (C–F), 144.70 (C–F), 138.07 (C–N), 132.21, 130.16, 129.57, 129.51, 125.5, 124.26 (aromatic carbons), 116.63, 115.04, 114.90 (C–S, C–O, C–N) 77.50–77.07, 40.03, 39.08. MS (relative intensity): 360 (M+4, 6%), 265 (78.0), 123 (12.05), 96 (2.0), 89 (100), 76 (75.0). Anal. calcd. C16H10N4SO2F2 (360): C, 53.30; H, 2.78; N, 15.50; S, 9.16; F, 10.55. Found: C, 52.88; H, 2.55; N, 15.02; S, 8.95; F, 10.35. 19F NMR: δ  −126 and −129 ppm.


## 2.1.6. N-(2-(2,4-Disubstituted-5-oxo-3-thioxo-2,3,4,5-tetrahydro-1,2,4-triazin-6-yl)-4-fluorophenyl)-4-fluorobenzamide (6–8)

A mixture of5 (0.001 mol) methanol or morpholine or 2,2′-dipyridylamine (0.001 mol) in methanol (20 mL) and formaldehyde (0.001 mol) was refluxed for 2 h. The reaction mixture was cooled to acquire room temperature and then poured onto ice-cold water. The solid product precipitated was filtered and crystallized from methanol to form yellow-orange crystals.6. Yield 72%, m.p. 310–312°C. IR (ν, cm−1): 3527 (OH), 3100 (NH), 2935, 2890 (aliphatic C–H), 1660, 1600 (C=O, CONH), 1498, 1440 (deformation CH2), 1386 (NCSN), 1255 (C–F), 864 (aryl C–H), 658 (C–F). 1H NMR (δ, ppm): 12.54 (s, 1H, NH), 10.15, 8.04 (Two s, 2 OH), 7.96–7.94, 7.87–7.83, 7.64-7.63 (aromatic H), 2.49 and 2.48 (Two s, 2 CH2). 13C NMR (δ, ppm): 164.99 (C=S), 163.32, 157.02 (C=O), 135.99 (C–F), 130.39, 130.20, 129.53, 129.44, 129.36, 123.45, (aromatic carbons), 115.14, 114.99 (C–S, C–O) 77.38–76.95, 39.83, 39.29. Anal. calcd. C18H14N4SO4F2 (420): C, 51.42; H, 3.36; N, 13.30; S, 7.61; F, 9.04. Found: C, 51.23; H, 3.01; N, 13.11; S, 7.51; F, 8.88. 19F NMR: δ  −126 and −129 ppm.7. Yield 70%, m.p. 260–262°C. Anal. calcd. C26H28N6SO4F2 (558): C, 55.90; H, 5.02; N, 15.05; S, 5.70; F, 6.81. Found: C, 55.68; H, 4.98; N, 14.79; S, 5.35; F, 6.66.8. Yield 72%; m.p. 202–204°C. Anal. calcd. C38H28N10SO2F2 (726): C, 62.79; H, 3.88; N, 19.28; S, 4.27; F, 5.23. Found: C, 62.33; H, 3.58; N, 19.11; S, 4.15; F, 4.89.


## 2.1.7. 4-Fluoro-N-(4-fluoro-2-(3-(substituted-amino)-5-oxo-4,5-dihydro-1,2,4-triazin-6-yl)phenyl)benzamide (9–12)

A mixture of 5 (0.001 mol) and sulfathiazole or sulfaoxazole or 4-fluoroaniline or 2,2′-dipyridylamine (0.001 mol) in absolute ethanol (50 mL) was refluxed for 6 h. On cooling a solid is separated which is filtered and crystallized from ethanol to give yellow crystals of9–12.9. Yield 85%, m.p. 172–174°C. IR (ν, cm−1): 3484−3310 (NH), 1658, 1610 (C=O, CONH), 1386 (NCSN), 1255 (C–F), 864 (aryl C–H), 658 (C–F). 1H NMR (δ, ppm): 13.72, 10.06, 8.00 (each s, 3 NH), 7.94 (s, 1H, NHSO2), 7.89, 7.84 (each s, 2H, thiazole), 7.56, 7.49, 7.39, 7.38, 7.10, 7.09, 7.78, 7.07, 6.79, 6.78, 6.55, 6.53, 6.36 (aromatic H). 13C NMR (δ, ppm): 173.18 (C–SO2), 167.90, 157.28 (C=O), 144.81 (C–F), 132.24, 132.22 (C–N), 130.22, 130.21, 129.62, 129.56, 128.93, 127.50, 125.58, 125.53, 124.40, 123.14, 116.78, 116.73, 116.62, 116.58, 115.03, 114.88, 112.82, 106.46 (aromatic carbons), 77.62–77.19. Anal. calcd. C25H17N7S2O4F2 (581): C, 51.63; H, 2.92; N, 16.86; S, 11.01; F, 6.55. Found: C, 51.34; H, 2.85; N, 16.70; S, 10.88; F, 6.35.10. Yield 82%, m.p. 178–180°C. Anal. calcd. C25H17N7SO5F2 (565): C, 53.09; H, 3.00; N, 17.16; S, 5.66; F, 6.72. Found: C, 52.88; H, 2.89; N, 17.01; S, 5.41; F, 6.55.11. Yield 89%, m.p. 247–249°C. UV: λ
max
⁡ (ε): 288.7 (1.09) nm. IR (ν, cm−1): 3500, 3310, 3290 (NH), 1661, 1620 (C=O), 1385 (NCSN), 1255 (C–F), 865 (aryl C–H), 658 (C–F). 1H NMR (δ, ppm): 13.79, 11.72, 10.92 (each s, 3 NH), 7.94, 7.88, 7.86, 7.70, 7.54, 7.53, 7.47, 7.25, 7.09, 7.07, 7.06 (aromatic H). 13C NMR (δ, ppm): 164.95–163.27, 158.86–157.24 (C=O), 146.34, 144.74, 138.74 (C–F), 132.22 (C–N), 129.56, 129.51, 125.48, 124.46, 116.77, 116.61, 115.02, 114.88 (aromatic carbons), 77.49-77.07. MS (relative intensity): 437 (M-45, 392, 1.0%), 166 (32.0), 148 (100), 122 (1.1), 110 (11), 95 (3.0). Anal. calcd. C22H14N5S2F3 (437): C, 60.41; H, 3.20; N, 16.01; F, 13.04. Found: C, 59.59; H, 3.10; N, 15.66; F, 12.85. 19F NMR: δ  −127 and −129 ppm.12. Yield 80%, m.p. 200–202°C. UV: λ
max
⁡ (ε): 271.8 (1.51) nm. IR (ν, cm−1): 3525–3100 (3 NH), 1659, 1610 (C=O, CONH), 1386 (NCSN), 1255 (C–F), 865 (aryl C–H), 658 (C–F). 1H NMR (δ, ppm): 13.79, 11.72, 10.92 (each s, 3 NH), 9.98–9.74, 8.16-8.15, 7.96-7.95, 7.94 (4H of pyridine), 7.86–7.84, 7.59–7.55, 7.40–7.37, 7.10–7.08, 7.07–7.05, 6.82-6.81, 6.80-6.79 (aromatic H). 13C NMR (δ, ppm): 164.92–163.25, 158.87–157.25 (C=O), 145.44, 144.84 (C–F), 137.71 (C–N), 132.24, 130.23, 129.61, 129.55, 125.58, 125.53, 124.45, 124.39 (aromatic carbons), 116.76, 116.69, 116.54, 115.69, 114.99 (pyridine carbons). Anal. calcd. C26H17N7O2F2 (497): C, 62.75; H, 3.40; N, 19.71; F, 7.62. Found: C, 62.45; H, 3.30; N, 19.55; F, 7.43.


## 2.1.8. 4-Fluoro-N-(4-fluoro-2-(3-((4-fluorophenyl)amino)-5-oxo-4,5-dihydro-1,2,4-triazin-6-yl)phenyl)benzamide (11)

Procedure A. Equimolar mixture of compound14 and 4-fluoroaniline was refluxed for 6 h then cooled. The solid thus obtained was filtered and crystallized from ethanol to give11 as faint yellow crystals. Yield 78%, m.p. 247–249°C.Procedure B. An equimolar mixture of compound5 and 4-fluoroaniline in ethanol (50 mL) was refluxed for 6 h and then cooled. The resulting solid was filtered and crystallized from ethanol to give faint yellow crystals of11. Yield 75%, m.p. 247–249°C.


## 2.1.9. 2-((6-(5-Fluoro-2-(4-fluorobenzamido)phenyl)-5-oxo-4,5-dihydro-1,2,4-triazin-3-yl)thio)acetic acid (13)

A mixture of 5 (0.001 mol) and monochloroacetic acid (0.001 mol) in pyridine (20 mL) was refluxed for 15 min, cooled, and then poured onto ice-cold dil. HCl. The resulting solid was filtered and crystallized from ethanol to give13 as yellow crystals. Yield 75%, m.p. 190–192°C. IR (ν, cm−1): 3550–3100 (br, OH and NH), 2919, 2885 (C–H), 1658, 1610 (C=O, CONH), 1489, 1429 (deformation CH2), 1386 (NCSN), 1255 (C–F), 865 (aryl C–H), 658 (C–F). 1H NMR (δ, ppm): 14.45 (NH), 12.58 (NH), 10.25–10.08 (OH), 7.88–7.85, 7.84-7.83, 7.65–7.31, 7.15-7.14, 7.13-7.12, 7.10–7.07 (aromatic H), 3.96 (CH2). 13C NMR (δ, ppm): 168.74 (C=O), 164.99 (C=O), 159.27–157.65 (C=O), 132.87–132.85 (C–S), 130.20, 130.18, 129.61, 129.49, 129.43, 127.19, 126.20, 117.18, 117.01, 116.73, 116.58, 115.17, 115.03, (aromatic carbons), 77.89–77.45, 40.88, 31.84 (CH2). MS (relative intensity): 418 (M-28, 390, 1.0%), 391 (1.5), 149 (100), 132 (2.8), 91 (1.10), 57 (47.00). Anal. calcd. C18H12N4SO4F2 (418): C, 53.11; H, 2.87; N, 13.35; S, 7.65; F, 9.09. Found: C, 52.88; H, 2.58; N, 13.05; S, 7.33; F, 8.79.


## 2.1.10. 4-Fluoro-N-(4-fluoro-2-(3-(methylthio)-5-oxo-4,5-dihydro-1,2,4-triazin-6-yl)phenyl)benzamide (14)

Procedure A. A mixture of compound13 (0.050 mol) and 10% ethanolic K2CO3 (10 mL) was warmed for 15 min, cooled, and then poured onto ice-cold dil. HCl. The solid product was filtered and crystallized from dioxane to give deep orange crystals of14. Yield 82%, m.p. 228–230°C.Procedure B. A mixture of compound 5 (0.001 mol) and CH3I (0.001 mol) in ethanolic KOH (1%, 20 mL) was stirred at room temperature for 12 h. The resulting reaction mixture was acidified with dil. HCl. A brownish orange precipitate was separated which on crystallization with dioxane gave 14 as orange crystals. Yield 80%, m.p. 228-229°C. Mixed m.p. 228°C. IR (ν, cm-1): 3484–3250 (br, NH, NH), 2930, 2864 (C–H), 1657, 1620 (C=O, CONH), 1489, 1449 (deformation CH2), 1386 (NCSN), 1255 (C–F), 864 (aryl C–H), 658 (C–F). 1H NMR (δ, ppm): 12.65, 12.37–12.35 (each s, NH, NH), 7.89-7.88, 7.87, 7.40–7.25, 7.14-7.13, 7.12, 7.12–7.10, 7.08–7.04 (aromatic H). 13C NMR (δ, ppm): 163.60, 159.38 (C=O), 157.79, 147.45 (C–F), 132.89 (C–S), 129.85, 129.28, 129.26, 129.22, 129.20, 127.43, 126.28, 126.25, 117.19, 117.03, 116.85, 116.82, 116.70, 116.67, 115.14, 115.12, 115.00, 114.97 (aromatic and heteroaromatic carbons), 77.21–76.78, 39.91–39.07 (CH3). Anal. calcd. C17H12N4SO2F2 (374): C, 54.50; H, 3.20; N, 14.89; S, 8.56; F, 10.17. Found: C, 53.89; H, 3.01; N, 14.55; S, 8.19; F, 10.11.


## 2.1.11. 6-(2-Amino-5-fluorophenyl)-3-((4-fluorophenyl)amino)-1,2,4-triazin-5(4H)-one (15)

A mixture of1 (0.001 mol) and 4-fluoroaniline (0.001 mol) in ethanol (50 mL) was refluxed for 6 h. On cooling a solid was precipitated which was filtered and crystallized from ethanol to form yellowish crystals of15. Yield 80%, m.p. 308–310°C. IR (ν, cm−1): 3534, 3300 (NH2, NH), 1661 (C=O), 1386 (NCSN), 1255 (C–F), 864 (aryl C–H), 658 (C–F). Anal. calcd. C15H11N5OF2 (315): C, 58.00; H, 3.49; N, 22.22; F, 12.06. Found: C, 57.72; H, 3.15; N, 21.88; F, 11.78. 19F NMR: δ  −127 and −129 ppm.


## 2.1.12. Aroylation of15 to Form 11

An equimolar mixture of15 and 4-fluorobenzoyl chloride in DMF (20 mL) was warmed for 10 min, cooled, and then poured onto ice. The resultant solid was filtered and crystallized from ethanol to give11 as yellowish crystals. Yields 82%, m.p. 248°C. Mixed m.p. 247°C.


## 2.1.13. N,N′-((3,3′-Disulfanediylbis(5-oxo-4,5-dihydro-1,2,4-triazine-6,3-diyl))bis(4-fluoro-2,1-phenylene))bis(4-fluorobenzamide) (16)

A mixture of compound5 (0.002 mol) and sulfur (0.002 mol) flowers in dry benzene (100 mL) was refluxed for 4 h. On cooling the reaction mixture a solid appeared which was filtered and crystallized from benzene to give16 as orange crystals. Yield 88%, m.p. 246–248°C. UV: λ
max
⁡ (ε): 288.8 (1.14) nm. IR (ν, cm−1): 3527–3320 (br, NH, NH), 1660, 1608 (C=O, CONH), 1386 (NCSN), 1255 (C–F), 1090 (C–S), 864 (aryl C–H), 658 (C–F). MS (relative intensity): 718 (M-359, –H2O, 341, 85%), 309 (55.01), 306 (100), 290 (90.00), 209 (8.11). Anal. calcd. C32H18N8S2O4F4 (718): C, 53.48; H, 2.50; N, 15.60; S, 8.91; F, 10.58. Found: C, 53.15; H, 2.30; N, 15.31; S, 8.56; F, 10.59. 19F NMR: δ  −127 and −130 ppm.


## 2.1.14. 2,2,2-Tris((6-(5-fluoro-2-(4-fluorobenzamido)phenyl)-5-oxo-4,5-dihydro-1,2,4-triazin-3-yl)thio)acetic acid (17)

A mixture of compounds1 (0.001 mol) and5 (0.001 mol) in DMF (50 mL) was refluxed for 1 h. The reaction mixture was left to cool at room temperature. The solid thus generated was filtered and crystallized from acetic acid to give17 as yellow crystals. Yield 65%, m.p. 230–232°C. UV: λ
max
⁡ (ε): 288.2 (1.23) nm. IR (ν, cm−1): 3544 (OH), 3320 (NH), 2990, 2898 (C–H), 1760–1610 (C=O), 1499, 1449 (deformation CH2), 1386 (NCSN), 1255 (C–F), 1089 (C–S), 864 (aryl C–H), 658 (C–F). 1H NMR (δ, ppm): 13.72, 13.25, 12.61, 12.17 (4s, 4 NH), 10.09, 10.06 (2s, 2 NH), 7.98, 7.97, 7.96, 7.95, 7.87, 7.86, 7.85, 7.84, 7.83, 7.54, 7.40, 7.39, 7.38, 7.35, 7.10, 7.09–7.05 (aromatic protons), 5.49 (s, 1H, OH). 13C NMR (δ, ppm): 164.98, 163.83, 163.32 (3 C=O), 148, 140 (C–F), 132.11 (C–S), 130.23, 130.19, 130.17, 129.62, 129.56, 129.47, 129.42, 125.51, 125.40 and 116.91, 116.81, 116.77, 116.74, 116.64, 116.62, 116.11, 115.12, 115.06, 114.97, 114.91, 77.59–77.16, 39.91–39.21 (aromatic, heteroaromatic and aliphatic carbons). Anal. calcd. C50H28N12S3O8F6 (1134): C, 52.55; H, 2.46; N, 14.81; S, 8.43; F, 10.05. Found: C, 51.98; H, 2.16; N, 14.53; S, 8.19; F, 9.85.


## 2.1.15. 4-Fluoro-N-(4-fluoro-2-(3-((4-fluoro-2-(5-oxo-3-thioxo-2,3,4,5-tetrahydro-1,2,4-triazin-6-yl)phenyl)amino)-5-oxo-4,5-dihydro-1,2,4-triazin-6-yl)phenyl)benzamide (18)

An equimolar mixture of1 and5 (0.001 mol) in isopropyl alcohol (50 mL) was refluxed for 6 h. Some of the solvent of reaction mixture was evaporatedin vacuo. The product was separated as brownish orange solid which was crystallized from ethanol to form orange crystals of compound18. Yield 80%, m.p. 246–248°C. UV: λ
max
⁡ (ε): 287.8 (2.07) nm. IR (ν, cm−1): 3320, 3300 (NH, NH), 1668, 1620 (C=O, CONH), 1385 (NCSN), 1255 (C–F), 1185 (C=S), 864 (aryl C–H), 657 (C–F). MS (relative intensity): 564 (M-196, 368, 7.0%), 247 (0.01), 204 (3.0), 203 (5.0), 196 (1.0), 100 (1.1), 88 (100), 76 (66.0), 42 (0.5). Anal. calcd. C25H15N8SO3F3 (564): C, 53.34; H, 5.67; N, 19.89; S, 5.67; F, 10.10. Found: C, 52.89; H, 5.55; N, 19.51; S, 5.38; F, 9.88. 19F NMR: δ  −126, −128 and −130 ppm.


## 2.2. Biological Evaluation

### 2.2.1. Anti-HIV-1 Activity

All the new synthesized compounds have been evaluated for theirin vitro anti-HIV activity that was performed on T-4 lymphocytes infected and uninfected with HIV-1 using DMSO as solvent. The assay involves the killing of T-4 lymphocytes by HIV. Uninfected cells with the compound serve as a toxicity control, and infected and uninfected cells without the compound serve as basic controls. Cultures are incubated at 37°C in a 5% carbon dioxide atmosphere for 6 days. The tetrazolium salt, XTT, is added to all wells, and cultures are incubated to allow formazan color development by viable cells. Compounds that degenerate or are rapidly metabolized in the culture conditions may not show activity in this screen. Zidovudine (AZT) at 10 μM was used as a control. The viability of the cells was determined spectrophotometrically to quantitate formazan production and in addition is viewed microscopically for detection of viable cells and confirmation of protective activity. Drug-treated virus-infected cells are compared with drug treated noninfected cells and with other appropriate controls (untreated infected and untreated noninfected cells, drug-containing wells without cells) on the same plate.


### 2.2.2. CDK2 Inhibition Assay

CDK2-cyclin E kinase was expressed and assayed as previously described [39]. Kinase activity was expressed as a percentage of maximum activity. The concentration of the test compounds required to decrease the CDK activity by 50% was determined from dose-response curves and designated IC50.


## 2.2.1. Anti-HIV-1 Activity

All the new synthesized compounds have been evaluated for theirin vitro anti-HIV activity that was performed on T-4 lymphocytes infected and uninfected with HIV-1 using DMSO as solvent. The assay involves the killing of T-4 lymphocytes by HIV. Uninfected cells with the compound serve as a toxicity control, and infected and uninfected cells without the compound serve as basic controls. Cultures are incubated at 37°C in a 5% carbon dioxide atmosphere for 6 days. The tetrazolium salt, XTT, is added to all wells, and cultures are incubated to allow formazan color development by viable cells. Compounds that degenerate or are rapidly metabolized in the culture conditions may not show activity in this screen. Zidovudine (AZT) at 10 μM was used as a control. The viability of the cells was determined spectrophotometrically to quantitate formazan production and in addition is viewed microscopically for detection of viable cells and confirmation of protective activity. Drug-treated virus-infected cells are compared with drug treated noninfected cells and with other appropriate controls (untreated infected and untreated noninfected cells, drug-containing wells without cells) on the same plate.


## 2.2.2. CDK2 Inhibition Assay

CDK2-cyclin E kinase was expressed and assayed as previously described [39]. Kinase activity was expressed as a percentage of maximum activity. The concentration of the test compounds required to decrease the CDK activity by 50% was determined from dose-response curves and designated IC50.


## 3. Results and Discussion

### 3.1. Chemistry

The proposed synthetic strategies to obtain the target compounds are outlined in Schemes1–5. The structures of target compounds have been established by physicochemical and spectroscopic techniques.Scheme 1Scheme 2Scheme 3Scheme 4Scheme 5Reaction of compound1 with monochloroacetic acid (1 : 2 by mole) in refluxing ethanolic sodium hydroxide afforded the α-amino acid2. Decarboxylation of2 by warming with aqueous potassium carbonate gave 6-[5′-fluoro-2-methylaminophenyl]-3-methylthio-1,2,4-triazin-5(4H)-one3. Compound3 was also obtained from methylation of1 by stirring it with methyl iodide in aqueous potassium hydroxide for 12 h (Scheme 1).The IR of1 showed strong peaks at 3528 and 3300 cm−1 for NH, NH2 and at 1661 and 1255 cm−1 for C=O and C–F functional groups, respectively. The mass spectrum showed a peak atm/z 254 (M + H2O, 5%) with a base peak at 103. Similarly, compound 3 exhibited in its IR spectrum three peaks at 3310, 2929, and 2862 cm−1 for NH and methyl groups. The mass of3 exhibited a peak atm/z 284 (M + H2O, 5%) with a base peak at 88. The 1H NMR spectra of3 showed two singlets at δ 3.2 and 3.5 ppm for N–CH3 and S–CH3 protons.El-Gendy et al. [37], obtained 1,1-di[4′-amino-6′-substituted-5-oxo-1,2,4-triazin-3-yl)thio]acetic acid as anticancer agent. Similarly refluxing compound1 with 1,1-dichloroacetic acid (2 : 1 by mole) in DMF produced the 1,1-di[heteroaryl]-thioacetic acid4 through nucleophilic displacement of chlorine by thioheterocyclic moieties (Scheme 2). The UV absorption spectra of4 recorded λ
max
⁡ at 266.5 nm while that of1 at 282.8 nm.Reaction of1 with 4-fluorobenzoyl chloride in dry pyridine furnished5 (Scheme 3). IR spectrum of5 showed two carbonyl groups at 1663 and 1610 cm−1. The 13C NMR spectrum showed peaks at δ 173.15, 164.14, and 158.72 ppm attributed to C=S, C=O, and CONH carbons. The mass spectrum exhibited a peak atm/z 360 (M + 4, 6%) with a base peak at 89.Compound5 was used as starting material for building some more N2-substituted and/or C3–NH substituted 1,2,4-triazinones. Thus hydroxymethylation of5 using methanol-formaldehyde yielded 2,4-dihydroxymethyl derivative6 which on its treatment with secondary amine as morpholine and/or 2,2-dipyridylamine under the same reaction conditions Mannich bases of the type7 and/or8 were isolated (Scheme 3). IR spectrum of6 showed absorptions at 3537, 2935, 2890, 1498, and 1440 cm−1 mainly attributed to O–H, C–H stretching, and CH2 bending vibrations. The 1H NMR spectrum of6 showed peaks at δ 12.54, 10.15 and 8.04 ppm for NH and OH protons. The 13C NMR spectrum, however, exhibited three resonance signals at δ 164.99, 163.32, and 157 ppm for C=S, C=O, and CONH groups and at δ 39.0 ppm for CH2 carbon.3-Substituted amino-1,2,4-triazine derivatives have shown chemotherapeutic [40] activities and have been proven as copper corrosion inhibitors [41]. Thus a simple nucleophilic displacement of SH group of 1,2,4-triazines with NH of different amines and sulfa drugs via reaction of compound5 with sulfa drugs (sulfathiazole and sulfoxazoles), 4-fluoroaniline, and 2,2-dipyridylamine gave 3-substituted-amino-6-[5′-fluoro-2′-(4′′-fluorophenylcarbamidophenyl)-1,2,4-triazin-5(4H)ones9–12 (Scheme 3). The IR spectrum of compound11 showed absorptions at 1255 and 658 cm−1 for C–F. The 13C NMR spectrum showed absence of C=S carbon. The mass spectra exhibitedm/z 437 (M-45, 392, 1.0%) with a base peak at 148 for the fragment C7H3N3F.Alkylation of 3-mercapto-1,2,4-triazinone5 using monochloroacetic acid in dry pyridine produced the mercaptoacetic acid derivative13. Decarboxylation of13 by warming it with aqueous K2CO3 afforded 6-[5′-fluoro-2′-(4′′-fluorophenyl-carbamidophenyl)-3-methylthio-1,2,4-triazin-5(4H)-one14. Compound14 was also obtained from treatment of compound5 with methyl iodide in ethanolic KOH (Scheme 4).Structure of13 was deduced from elemental analysis and spectral data. The IR spectrum showed peaks at 3550–3100, 2919, 2882, 1658, and 1610 cm−1 attributed to O–H, N–H, CH2, C=O, and CONH groups. In 1H NMR spectrum signals at δ 14.45, 12.58, and 10.25 ppm for NH, NH, and OH protons. In addition to this, a peak at δ 3.96 appeared for CH2 protons. Further support was given by its 13C NMR where there are three peaks for three carbonyl groups at δ 168.74, 164.99, and 159.27. The mass spectra recorded a peak atm/z 418 (M-28, 390, 1.00%) with a base peak at 149 as C8H4NOF.Full fluorine substituted aryl-1,2,4-triazinones were synthesized by refluxing14 and/or5 with 4-fluoroaniline in ethanol led to the formation of 4-fluoro-N-(4-fluoro-2-(3-((4-fluorophenyl)amino)-5-oxo-4,5-dihydro-1,2,4-triazin-6-yl)phenyl)benzamide one11 (Scheme 4). However, compound 15 was also obtained by treating compound1 with 4-fluoroaniline in boiling ethanol to give 3-(4′-fluorophenyl)-6-(5′-fluoro-2′-aminophenyl)-1,2,4-triazin-5(4H) one15 which upon warming with 4-fluorobenzoyl chloride in DMF yielded11 (Scheme 4). The structure of15 was established by elemental analysis and spectral data. The IR spectra showed absorptions at 3534, 3300, 1661, and 1640 cm−1 for NH2, NH, and C=O functional groups. The spectrum did show two peaks at 1255 and 658 cm−1 for C–F group. The 13C NMR spectrum lacks a signal for C=S carbon.Oxidation of compound5 by refluxing with sulfur flowers in dry benzene yielded the disulfide16 (Scheme 5). Due to higher nucleophilicity of sulfur when compared with oxygen and nitrogen, the removal of H
+ from S–H is easily followed by removal of H
- from other molecules to form a disulfide16 with evolution of hydrogen (Scheme 5). The IR spectrum showed an absorption band at 1090 cm−1 for C–S–S–C group. The mass recordedm/z 341 (M-18, –H2O, 85%) with a base peak atm/z 306 for C16H5N3SOF.Treatment of compound5 with 1,1,1-trichloroacetic acid in warming DMF generates a thioether17 (Scheme 5). The IR spectrum showed absorption bands at 1255 and 1089 cm−1 which are attributed to C–F and C–S of the thioether. The 13C NMR spectrum lacks a signal for C=S carbon. However, resonances at δ 132.11 ppm for C–S carbon in addition to a signal at δ 164.98 ppm for carbonyl carbon.Finally synthesis of fluorine substituted 1,2,4-triazinone bearing other amino-1,2,4-triazinone18 was done by refluxing compounds1 and15 in boiling isopropyl alcohol as amination reaction (Scheme 5). The IR spectrum of18 showed bands at 3320, 3300, 1668, 1620, and 1185 cm−1 for NH, NH, C=O, CONH, and C=S groups. The mass spectrum showed a peak atm/z 368 (M-196, 7%) along with a base peak at 89 for CH2N3S.The presence of fluorine was confirmed by19F NMR. A single fluorine atom attached to phenyl and/or benzoyl ring appeared in the region δ 120–130 ppm using hexafluorobenzene as internal standard.The mass fragmentation patterns of some of the target molecules are mentioned in Schemes6, 7, 8, 9, and 10.Scheme 6
Mass fragmentation pattern of compound1.Scheme 7
Mass fragmentation pattern of compound3.Scheme 8
Mass fragmentation pattern of compound5.Scheme 9
Mass fragmentation pattern of compound11.Scheme 10
Mass fragmentation pattern of compound16.


### 3.2. Biological Evaluation

#### 3.2.1. Anti-HIV-1 Activity

The present work aimed to synthesize fluorine substituted 1,2,4-triazinones as potential anti-HIV-1 compounds as nucleoside, nonnucleoside reverse transcriptase, protease, and fusion inhibitors. The newly synthesized compounds have been evaluated for theirin vitro anti-HIV activities on T4 lymphocytes, uninfected, or infected with HIV using DMSO as solvent. The assay basically involves the killing of T4 lymphocytes by HIV compounds that degenerate or are rapidly metabolized in the culture conditions may not show activity in this screen. The viability of the cells was determined spectrophotometrically using the tetrazolium assay procedure. The results obtained are reported in Table 1.Table 1
Anti-HIV-1 activities of fluorinated 1,2,4-triazinones1–18.
Compounds
EC50 (μM)a
CC50 (μM)b
SIc
log
⁡
P
1
10.8 ± 1.0
25 ± 1.0
2.51
0.64
3
20 ± 1.0
49 ± 1.0
2.50
2.09
5
25 ± 1.0
75 ± 1.0
3.00
2.4
6
30 ± 1.0
60 ± 1.0
2.00
2.39
9
35 ± 1.0
75 ± 1.0
2.20
4.41
10
42 ± 1.0
82 ± 1.0
2.11
3.04
11
6.6 ± 1.0
44 ± 2.0
7.15
4.44
14
5.8 ± 1.0
35 ± 1.0
6.2
3.56
15
14.0 ± 1.0
29 ± 1.0
2.01
2.67
16
6.9 ± 1.0
36 ± 1.1
5.1
7.07
17
25 ± 1.8
76 ± 1.0
3.01
—
18
50 ± 1.1
90 ± 2.1
1.8
3.69
aEC50: inhibiting p24 production in the MT-4 cells.
bCC50: concentration of compound that reduces the viability of mock-infected cells by 50%, as determined by the MTT method.
cSI: selectivity index; CC50/EC50.The results revealed by Table1 suggest that compounds5,11,14,16, and17 displayed very good anti-HIV-1 activity having a favorable selectivity index between 3 and 7. The CC50 and EC50 data are used to calculate the selectivity index (SI) of each compound as an estimate of a therapeutic window and a mechanism to identify candidates for efficacy studies. The other compounds though are active as anti-HIV-1 agents but do not show very good selectivity index ratio. The remaining compounds exhibited an average to poor activity with selective index <3 (Table 1). In particular, a high activity level was observed for compounds11 and14. A close examination of these two compounds reveals that both the structures have4-fluoro-N-(4-fluorophenyl)benzamide part connected at position 6 in triazinone nucleus. Similarly, in compound16 which is an oxidised dimer of the active compound5, the selectivity index is almost doubled. The antiviral activity diminishes if the triazinone ring experience crowding at carbon-3 in triazinone part as in case of15 and18. Sulfonamide moieties, however, could not contribute towards improving the antiviral profile; rather it significantly diminishes the selectivity index ratio as can be seen in compounds9 and10.The influence of fluorine on the acidity, hydrogen bonding, and lipophilicity of these systems can be envisaged not only a biological activity modulator but also influences the bioavailability of the drug. The active fluorine compounds obtained are capable of forming stronger DNA complexes than their nonfluorinated analogs. These results suggest that the electronic nature of the chain of 3-thioxo-1,2,4-triazines tethering an intercalator that not only influences the DNA-binding process but might also be used to tune the new DNA-drug complex. It can be implied that the fluorine substituted-3-thioxo-1,2,4-triazin-5-ones represent suitable prodrug principle leading to higher bioavailability.The biological activity depends not only on the site of fluorination and the geometry of the conjugate carbanion formed but also on the total electronegativity of new heterocyclic nitrogen systems. In view of these characteristics and the results obtained in Table1 we can infer that fluorination invariably increases C–H acidity through a combination of inductive and hyperconjugative resonance stabilization of the carbanion and thus influences the biological activity of this series of compounds (Figure 3).Figure 3
Hyperconjugation between atom and C–H.


#### 3.2.2. Anticancer Activity

The new fluorine substituted 1,2,4-triazinones (1–18) have been evaluated to inhibit activity of CDK2 in a biochemical assay. The inhibitory concentration (IC50) values were obtained according to the reported methods [39, 42]. Olomoucine has been used as a standard. The results are reported in Table 2.Table 2
CDK2 inhibiting activity of fluorinated 1,2,4-triazinones1–18.
Compounds
IC50  ± SD (μM)a
1
3.8 ± 1.7
3
5.0 ± 1.7
5
12.8 ± 1.7
6
12.2 ± 1.7
9
14.0 ± 1.5
10
15.0 ± 2.5
11
15.0 ± 5.0
14
4.5 ± 2.8
15
4.1 ± 2.8
16
4.0 ± 2.8
17
16.2 ± 5.0
18
18.1 ± 4.0
Olomoucine
5.0 ± 1.0
aIC50 in μM/d
m
-
1.
SD: standard deviation.The results from Table2 reveal that compounds1,3, and14–16 show very significant CDK2 inhibitory activity. Compound3 was found to be as active as olomoucine, while compounds1 and14–16 were more potent than the standard compound. Once again the same structural features are present in active compounds as is evident in compounds active as anti-HIV-1.


## 3.1. Chemistry

The proposed synthetic strategies to obtain the target compounds are outlined in Schemes1–5. The structures of target compounds have been established by physicochemical and spectroscopic techniques.Scheme 1Scheme 2Scheme 3Scheme 4Scheme 5Reaction of compound1 with monochloroacetic acid (1 : 2 by mole) in refluxing ethanolic sodium hydroxide afforded the α-amino acid2. Decarboxylation of2 by warming with aqueous potassium carbonate gave 6-[5′-fluoro-2-methylaminophenyl]-3-methylthio-1,2,4-triazin-5(4H)-one3. Compound3 was also obtained from methylation of1 by stirring it with methyl iodide in aqueous potassium hydroxide for 12 h (Scheme 1).The IR of1 showed strong peaks at 3528 and 3300 cm−1 for NH, NH2 and at 1661 and 1255 cm−1 for C=O and C–F functional groups, respectively. The mass spectrum showed a peak atm/z 254 (M + H2O, 5%) with a base peak at 103. Similarly, compound 3 exhibited in its IR spectrum three peaks at 3310, 2929, and 2862 cm−1 for NH and methyl groups. The mass of3 exhibited a peak atm/z 284 (M + H2O, 5%) with a base peak at 88. The 1H NMR spectra of3 showed two singlets at δ 3.2 and 3.5 ppm for N–CH3 and S–CH3 protons.El-Gendy et al. [37], obtained 1,1-di[4′-amino-6′-substituted-5-oxo-1,2,4-triazin-3-yl)thio]acetic acid as anticancer agent. Similarly refluxing compound1 with 1,1-dichloroacetic acid (2 : 1 by mole) in DMF produced the 1,1-di[heteroaryl]-thioacetic acid4 through nucleophilic displacement of chlorine by thioheterocyclic moieties (Scheme 2). The UV absorption spectra of4 recorded λ
max
⁡ at 266.5 nm while that of1 at 282.8 nm.Reaction of1 with 4-fluorobenzoyl chloride in dry pyridine furnished5 (Scheme 3). IR spectrum of5 showed two carbonyl groups at 1663 and 1610 cm−1. The 13C NMR spectrum showed peaks at δ 173.15, 164.14, and 158.72 ppm attributed to C=S, C=O, and CONH carbons. The mass spectrum exhibited a peak atm/z 360 (M + 4, 6%) with a base peak at 89.Compound5 was used as starting material for building some more N2-substituted and/or C3–NH substituted 1,2,4-triazinones. Thus hydroxymethylation of5 using methanol-formaldehyde yielded 2,4-dihydroxymethyl derivative6 which on its treatment with secondary amine as morpholine and/or 2,2-dipyridylamine under the same reaction conditions Mannich bases of the type7 and/or8 were isolated (Scheme 3). IR spectrum of6 showed absorptions at 3537, 2935, 2890, 1498, and 1440 cm−1 mainly attributed to O–H, C–H stretching, and CH2 bending vibrations. The 1H NMR spectrum of6 showed peaks at δ 12.54, 10.15 and 8.04 ppm for NH and OH protons. The 13C NMR spectrum, however, exhibited three resonance signals at δ 164.99, 163.32, and 157 ppm for C=S, C=O, and CONH groups and at δ 39.0 ppm for CH2 carbon.3-Substituted amino-1,2,4-triazine derivatives have shown chemotherapeutic [40] activities and have been proven as copper corrosion inhibitors [41]. Thus a simple nucleophilic displacement of SH group of 1,2,4-triazines with NH of different amines and sulfa drugs via reaction of compound5 with sulfa drugs (sulfathiazole and sulfoxazoles), 4-fluoroaniline, and 2,2-dipyridylamine gave 3-substituted-amino-6-[5′-fluoro-2′-(4′′-fluorophenylcarbamidophenyl)-1,2,4-triazin-5(4H)ones9–12 (Scheme 3). The IR spectrum of compound11 showed absorptions at 1255 and 658 cm−1 for C–F. The 13C NMR spectrum showed absence of C=S carbon. The mass spectra exhibitedm/z 437 (M-45, 392, 1.0%) with a base peak at 148 for the fragment C7H3N3F.Alkylation of 3-mercapto-1,2,4-triazinone5 using monochloroacetic acid in dry pyridine produced the mercaptoacetic acid derivative13. Decarboxylation of13 by warming it with aqueous K2CO3 afforded 6-[5′-fluoro-2′-(4′′-fluorophenyl-carbamidophenyl)-3-methylthio-1,2,4-triazin-5(4H)-one14. Compound14 was also obtained from treatment of compound5 with methyl iodide in ethanolic KOH (Scheme 4).Structure of13 was deduced from elemental analysis and spectral data. The IR spectrum showed peaks at 3550–3100, 2919, 2882, 1658, and 1610 cm−1 attributed to O–H, N–H, CH2, C=O, and CONH groups. In 1H NMR spectrum signals at δ 14.45, 12.58, and 10.25 ppm for NH, NH, and OH protons. In addition to this, a peak at δ 3.96 appeared for CH2 protons. Further support was given by its 13C NMR where there are three peaks for three carbonyl groups at δ 168.74, 164.99, and 159.27. The mass spectra recorded a peak atm/z 418 (M-28, 390, 1.00%) with a base peak at 149 as C8H4NOF.Full fluorine substituted aryl-1,2,4-triazinones were synthesized by refluxing14 and/or5 with 4-fluoroaniline in ethanol led to the formation of 4-fluoro-N-(4-fluoro-2-(3-((4-fluorophenyl)amino)-5-oxo-4,5-dihydro-1,2,4-triazin-6-yl)phenyl)benzamide one11 (Scheme 4). However, compound 15 was also obtained by treating compound1 with 4-fluoroaniline in boiling ethanol to give 3-(4′-fluorophenyl)-6-(5′-fluoro-2′-aminophenyl)-1,2,4-triazin-5(4H) one15 which upon warming with 4-fluorobenzoyl chloride in DMF yielded11 (Scheme 4). The structure of15 was established by elemental analysis and spectral data. The IR spectra showed absorptions at 3534, 3300, 1661, and 1640 cm−1 for NH2, NH, and C=O functional groups. The spectrum did show two peaks at 1255 and 658 cm−1 for C–F group. The 13C NMR spectrum lacks a signal for C=S carbon.Oxidation of compound5 by refluxing with sulfur flowers in dry benzene yielded the disulfide16 (Scheme 5). Due to higher nucleophilicity of sulfur when compared with oxygen and nitrogen, the removal of H
+ from S–H is easily followed by removal of H
- from other molecules to form a disulfide16 with evolution of hydrogen (Scheme 5). The IR spectrum showed an absorption band at 1090 cm−1 for C–S–S–C group. The mass recordedm/z 341 (M-18, –H2O, 85%) with a base peak atm/z 306 for C16H5N3SOF.Treatment of compound5 with 1,1,1-trichloroacetic acid in warming DMF generates a thioether17 (Scheme 5). The IR spectrum showed absorption bands at 1255 and 1089 cm−1 which are attributed to C–F and C–S of the thioether. The 13C NMR spectrum lacks a signal for C=S carbon. However, resonances at δ 132.11 ppm for C–S carbon in addition to a signal at δ 164.98 ppm for carbonyl carbon.Finally synthesis of fluorine substituted 1,2,4-triazinone bearing other amino-1,2,4-triazinone18 was done by refluxing compounds1 and15 in boiling isopropyl alcohol as amination reaction (Scheme 5). The IR spectrum of18 showed bands at 3320, 3300, 1668, 1620, and 1185 cm−1 for NH, NH, C=O, CONH, and C=S groups. The mass spectrum showed a peak atm/z 368 (M-196, 7%) along with a base peak at 89 for CH2N3S.The presence of fluorine was confirmed by19F NMR. A single fluorine atom attached to phenyl and/or benzoyl ring appeared in the region δ 120–130 ppm using hexafluorobenzene as internal standard.The mass fragmentation patterns of some of the target molecules are mentioned in Schemes6, 7, 8, 9, and 10.Scheme 6
Mass fragmentation pattern of compound1.Scheme 7
Mass fragmentation pattern of compound3.Scheme 8
Mass fragmentation pattern of compound5.Scheme 9
Mass fragmentation pattern of compound11.Scheme 10
Mass fragmentation pattern of compound16.


## 3.2. Biological Evaluation

### 3.2.1. Anti-HIV-1 Activity

The present work aimed to synthesize fluorine substituted 1,2,4-triazinones as potential anti-HIV-1 compounds as nucleoside, nonnucleoside reverse transcriptase, protease, and fusion inhibitors. The newly synthesized compounds have been evaluated for theirin vitro anti-HIV activities on T4 lymphocytes, uninfected, or infected with HIV using DMSO as solvent. The assay basically involves the killing of T4 lymphocytes by HIV compounds that degenerate or are rapidly metabolized in the culture conditions may not show activity in this screen. The viability of the cells was determined spectrophotometrically using the tetrazolium assay procedure. The results obtained are reported in Table 1.Table 1
Anti-HIV-1 activities of fluorinated 1,2,4-triazinones1–18.
Compounds
EC50 (μM)a
CC50 (μM)b
SIc
log
⁡
P
1
10.8 ± 1.0
25 ± 1.0
2.51
0.64
3
20 ± 1.0
49 ± 1.0
2.50
2.09
5
25 ± 1.0
75 ± 1.0
3.00
2.4
6
30 ± 1.0
60 ± 1.0
2.00
2.39
9
35 ± 1.0
75 ± 1.0
2.20
4.41
10
42 ± 1.0
82 ± 1.0
2.11
3.04
11
6.6 ± 1.0
44 ± 2.0
7.15
4.44
14
5.8 ± 1.0
35 ± 1.0
6.2
3.56
15
14.0 ± 1.0
29 ± 1.0
2.01
2.67
16
6.9 ± 1.0
36 ± 1.1
5.1
7.07
17
25 ± 1.8
76 ± 1.0
3.01
—
18
50 ± 1.1
90 ± 2.1
1.8
3.69
aEC50: inhibiting p24 production in the MT-4 cells.
bCC50: concentration of compound that reduces the viability of mock-infected cells by 50%, as determined by the MTT method.
cSI: selectivity index; CC50/EC50.The results revealed by Table1 suggest that compounds5,11,14,16, and17 displayed very good anti-HIV-1 activity having a favorable selectivity index between 3 and 7. The CC50 and EC50 data are used to calculate the selectivity index (SI) of each compound as an estimate of a therapeutic window and a mechanism to identify candidates for efficacy studies. The other compounds though are active as anti-HIV-1 agents but do not show very good selectivity index ratio. The remaining compounds exhibited an average to poor activity with selective index <3 (Table 1). In particular, a high activity level was observed for compounds11 and14. A close examination of these two compounds reveals that both the structures have4-fluoro-N-(4-fluorophenyl)benzamide part connected at position 6 in triazinone nucleus. Similarly, in compound16 which is an oxidised dimer of the active compound5, the selectivity index is almost doubled. The antiviral activity diminishes if the triazinone ring experience crowding at carbon-3 in triazinone part as in case of15 and18. Sulfonamide moieties, however, could not contribute towards improving the antiviral profile; rather it significantly diminishes the selectivity index ratio as can be seen in compounds9 and10.The influence of fluorine on the acidity, hydrogen bonding, and lipophilicity of these systems can be envisaged not only a biological activity modulator but also influences the bioavailability of the drug. The active fluorine compounds obtained are capable of forming stronger DNA complexes than their nonfluorinated analogs. These results suggest that the electronic nature of the chain of 3-thioxo-1,2,4-triazines tethering an intercalator that not only influences the DNA-binding process but might also be used to tune the new DNA-drug complex. It can be implied that the fluorine substituted-3-thioxo-1,2,4-triazin-5-ones represent suitable prodrug principle leading to higher bioavailability.The biological activity depends not only on the site of fluorination and the geometry of the conjugate carbanion formed but also on the total electronegativity of new heterocyclic nitrogen systems. In view of these characteristics and the results obtained in Table1 we can infer that fluorination invariably increases C–H acidity through a combination of inductive and hyperconjugative resonance stabilization of the carbanion and thus influences the biological activity of this series of compounds (Figure 3).Figure 3
Hyperconjugation between atom and C–H.


### 3.2.2. Anticancer Activity

The new fluorine substituted 1,2,4-triazinones (1–18) have been evaluated to inhibit activity of CDK2 in a biochemical assay. The inhibitory concentration (IC50) values were obtained according to the reported methods [39, 42]. Olomoucine has been used as a standard. The results are reported in Table 2.Table 2
CDK2 inhibiting activity of fluorinated 1,2,4-triazinones1–18.
Compounds
IC50  ± SD (μM)a
1
3.8 ± 1.7
3
5.0 ± 1.7
5
12.8 ± 1.7
6
12.2 ± 1.7
9
14.0 ± 1.5
10
15.0 ± 2.5
11
15.0 ± 5.0
14
4.5 ± 2.8
15
4.1 ± 2.8
16
4.0 ± 2.8
17
16.2 ± 5.0
18
18.1 ± 4.0
Olomoucine
5.0 ± 1.0
aIC50 in μM/d
m
-
1.
SD: standard deviation.The results from Table2 reveal that compounds1,3, and14–16 show very significant CDK2 inhibitory activity. Compound3 was found to be as active as olomoucine, while compounds1 and14–16 were more potent than the standard compound. Once again the same structural features are present in active compounds as is evident in compounds active as anti-HIV-1.


## 3.2.1. Anti-HIV-1 Activity

The present work aimed to synthesize fluorine substituted 1,2,4-triazinones as potential anti-HIV-1 compounds as nucleoside, nonnucleoside reverse transcriptase, protease, and fusion inhibitors. The newly synthesized compounds have been evaluated for theirin vitro anti-HIV activities on T4 lymphocytes, uninfected, or infected with HIV using DMSO as solvent. The assay basically involves the killing of T4 lymphocytes by HIV compounds that degenerate or are rapidly metabolized in the culture conditions may not show activity in this screen. The viability of the cells was determined spectrophotometrically using the tetrazolium assay procedure. The results obtained are reported in Table 1.Table 1
Anti-HIV-1 activities of fluorinated 1,2,4-triazinones1–18.
Compounds
EC50 (μM)a
CC50 (μM)b
SIc
log
⁡
P
1
10.8 ± 1.0
25 ± 1.0
2.51
0.64
3
20 ± 1.0
49 ± 1.0
2.50
2.09
5
25 ± 1.0
75 ± 1.0
3.00
2.4
6
30 ± 1.0
60 ± 1.0
2.00
2.39
9
35 ± 1.0
75 ± 1.0
2.20
4.41
10
42 ± 1.0
82 ± 1.0
2.11
3.04
11
6.6 ± 1.0
44 ± 2.0
7.15
4.44
14
5.8 ± 1.0
35 ± 1.0
6.2
3.56
15
14.0 ± 1.0
29 ± 1.0
2.01
2.67
16
6.9 ± 1.0
36 ± 1.1
5.1
7.07
17
25 ± 1.8
76 ± 1.0
3.01
—
18
50 ± 1.1
90 ± 2.1
1.8
3.69
aEC50: inhibiting p24 production in the MT-4 cells.
bCC50: concentration of compound that reduces the viability of mock-infected cells by 50%, as determined by the MTT method.
cSI: selectivity index; CC50/EC50.The results revealed by Table1 suggest that compounds5,11,14,16, and17 displayed very good anti-HIV-1 activity having a favorable selectivity index between 3 and 7. The CC50 and EC50 data are used to calculate the selectivity index (SI) of each compound as an estimate of a therapeutic window and a mechanism to identify candidates for efficacy studies. The other compounds though are active as anti-HIV-1 agents but do not show very good selectivity index ratio. The remaining compounds exhibited an average to poor activity with selective index <3 (Table 1). In particular, a high activity level was observed for compounds11 and14. A close examination of these two compounds reveals that both the structures have4-fluoro-N-(4-fluorophenyl)benzamide part connected at position 6 in triazinone nucleus. Similarly, in compound16 which is an oxidised dimer of the active compound5, the selectivity index is almost doubled. The antiviral activity diminishes if the triazinone ring experience crowding at carbon-3 in triazinone part as in case of15 and18. Sulfonamide moieties, however, could not contribute towards improving the antiviral profile; rather it significantly diminishes the selectivity index ratio as can be seen in compounds9 and10.The influence of fluorine on the acidity, hydrogen bonding, and lipophilicity of these systems can be envisaged not only a biological activity modulator but also influences the bioavailability of the drug. The active fluorine compounds obtained are capable of forming stronger DNA complexes than their nonfluorinated analogs. These results suggest that the electronic nature of the chain of 3-thioxo-1,2,4-triazines tethering an intercalator that not only influences the DNA-binding process but might also be used to tune the new DNA-drug complex. It can be implied that the fluorine substituted-3-thioxo-1,2,4-triazin-5-ones represent suitable prodrug principle leading to higher bioavailability.The biological activity depends not only on the site of fluorination and the geometry of the conjugate carbanion formed but also on the total electronegativity of new heterocyclic nitrogen systems. In view of these characteristics and the results obtained in Table1 we can infer that fluorination invariably increases C–H acidity through a combination of inductive and hyperconjugative resonance stabilization of the carbanion and thus influences the biological activity of this series of compounds (Figure 3).Figure 3
Hyperconjugation between atom and C–H.


## 3.2.2. Anticancer Activity

The new fluorine substituted 1,2,4-triazinones (1–18) have been evaluated to inhibit activity of CDK2 in a biochemical assay. The inhibitory concentration (IC50) values were obtained according to the reported methods [39, 42]. Olomoucine has been used as a standard. The results are reported in Table 2.Table 2
CDK2 inhibiting activity of fluorinated 1,2,4-triazinones1–18.
Compounds
IC50  ± SD (μM)a
1
3.8 ± 1.7
3
5.0 ± 1.7
5
12.8 ± 1.7
6
12.2 ± 1.7
9
14.0 ± 1.5
10
15.0 ± 2.5
11
15.0 ± 5.0
14
4.5 ± 2.8
15
4.1 ± 2.8
16
4.0 ± 2.8
17
16.2 ± 5.0
18
18.1 ± 4.0
Olomoucine
5.0 ± 1.0
aIC50 in μM/d
m
-
1.
SD: standard deviation.The results from Table2 reveal that compounds1,3, and14–16 show very significant CDK2 inhibitory activity. Compound3 was found to be as active as olomoucine, while compounds1 and14–16 were more potent than the standard compound. Once again the same structural features are present in active compounds as is evident in compounds active as anti-HIV-1.


## 4. Conclusions

According to the data obtained from the biological assay five compounds5,11,14,16, and17 have shown very good anti-HIV activity in MT-2 cells. Similarly, five compounds (1,3, and14–16) have exhibited remarkable CDK2 inhibition activity. Compounds14 and16 can be considered as a significant matrix for the design and synthesis of novel candidates with dual anti-HIV and anticancer activities. Further investigation into the other aspects of structure activity relationship studies of this series of compounds is required in order to explore the scope and limitation of its biological activities.



---

*Source: 430573-2014-05-21.xml*