Saccharin is an aromatic compound containing a benzene ring linked to an imidosulfone ring. It is primarily known as a low- or no-calorie artificial sweetener. However, research in recent decades has demonstrated that saccharin derivatives possess diverse pharmacological properties, including antibacterial, antitumor and inhibitory effects on important enzymes such as carbonic anhydrase (CA IX and XII), which are therapeutic targets in cancer [1]. Saccharin is considered a safe sweetener for human consumption, declared safe by the World Health Organization (WHO) in 1993. The Joint FAO/WHO Expert Committee on Food Additives stated that the acceptable daily intake of saccharin is 5 mg/kg of body weight. In addition to its function as a sweetener, other metabolic effects of saccharin revealed to date include reducing metabolic activity in human cancer cell lines and inhibiting tumor-causing cell growth factors [2]. Saccharin, one of the oldest artificial sweeteners, has attracted significant attention not only for its industrial use but also for its versatile chemical structure that allows further modifications [3]. Derivatives of saccharin are widely studied due to their ability to form stable heterocyclic systems, which enhance their pharmacological potential [4]. The incorporation of heteroaryl and thiadiazole moieties into saccharin scaffolds is considered a promising approach for generating novel bioactive molecules [5]. Saccharin has been used as a sweetener in the form of sodium salt since 1885. It is also a heterocyclic compound of pharmacological importance, being a key structural component of some central nervous system-active drugs. Saccharin compounds have been extensively studied and saccharin-based saccharin inhibitors have been found to have potent inhibitory activity. Some substituted saccharin derivatives have been used as intermediates in the preparation of the 4-hydroxy-1,2-benzothiazine 1,1-dioxide (Oxicam) ring system, an extended-ring saccharin and its derivatives have anti-inflammatory properties [6]. Some toxic saccharin derivatives, such as N-alkyl saccharin, have been used to kill harmful insects. Saccharin is a cheap and readily available substance and is widely used in the food industry (food chemistry) as an artificial sweetener. Saccharin derivatives have been used in many fields, such as pharmaceutical, medical, agricultural, antimicrobial, antitumor, anti-inflammatory, antifungal and antidiabetic, so it can be used as a raw material in laboratory preparations. Saccharin is a Latin word meaning “sugar,” and it is about 300 times sweeter than sugar [7]. The design of saccharin derivatives with thiadiazole rings has also opened new perspectives in antiviral drug discovery [8]. Antioxidant activity is another promising application of saccharin-thiadiazole derivatives, where their electron-donating properties play a role in radical scavenging [9]. Hybrid molecules containing saccharin and thiadiazole have been reported to exhibit promising anti-fungal activities, particularly against resistant strains [10]. Thiadiazole-based saccharin derivatives are being investigated for their potential to inhibit tumor cell proliferation through apoptosis induction [11]. The structural flexibility of 1,3,4-thiadiazole facilitates strong hydrogen bonding interactions, enhancing binding affinity with biological targets [12]. Several saccharin-thiadiazole compounds have shown excellent inhibitory activity against bacterial DNA gyrase and topoisomerase enzymes [13]. Saccharin derivatives containing thiadiazole groups may also function as chelating agents with transition metals, enhancing bioactivity [14]. The antioxidant and enzyme-inhibitory activities of saccharin-thiadiazole hybrids suggest their potential in the management of metabolic disorders [15]. The hybridization of saccharin with thiadiazole rings contributes to enhanced pharmacological profiles compared to either moiety alone [16]. Computational studies support the role of saccharin-thiadiazole hybrids in molecular docking and dynamic simulations, validating their binding efficiency [17]. Recent reports highlight the anti-inflammatory potential of saccharin-thiadiazole hybrids, making them relevant for chronic disease therapies [18]. The thiadiazole ring acts as a bioisostere of amide and ester functionalities, contributing to drug-likeness and metabolic stability [19]. Saccharin-thiadiazole hybrids are also being evaluated as potent inhibitors of enzymes involved in neurological disorders [20]. Novel saccharin derivatives containing thiadiazole scaffolds have demonstrated activity against Mycobacterium tuberculosis, addressing antibiotic resistance [21]. Green chemistry approaches, including microwave-assisted synthesis, have been applied to optimize saccharin-thiadiazole derivative production [22]. The development of saccharin-thiadiazole hybrids aligns with the ongoing search for multifunctional drugs with diverse therapeutic applications [23].

The synthesis of the target molecules (1a-7a) is shown in the sequences of reactions depicted in the scheme below. The F.T.IR spectral data were recorded on F.T.IR-8300 Fourier Transform Infrared Spectrophotometer SHIMADZU using potassium bromide disc, Double-beam Uv-Vis spectrophotometer (UV 1700 CP), SHIMADZU, 1H-NMR and 13C-NMR was recorder on Bruker Ultra Shield, 400 MHz, using DMSO as solvent and TMS as internal standard, Melting points (°C) were recorded on hot stage Gallen Kamp melting point apparatus and were uncorrected and Thin-layer chromatography was performed glass plats coated with 0.25 mm layer of silica-gel (Fluka). Chemical names follow the IUPAC nomenclature. Some starting materials were purchased from EMD Millipore Corporation, Germany and were used without purification (Figure 1).       Figure 1: Synthetic Scheme for the Preparation of Novel Benzothiazine-Azo Derivatives (1a-7a)     Preparation of N-allyl Saccharin Sodium saccharin 0.02 mol was dissolved in DMF 50 mL, allyl chloride 0.02 mol was added and then the mixture was refluxed for 4 hours 125°C. After cooling, 50 mL of distilled water was added to the mixture forming a precipitate, the precipitate was filtered and washed with distilled and recrystallized from ethanol. The melting point is (129°C) with 80% yield, Rf (0.25), F.T-IR (KBr): 3314 cm⁻1, 3097 cm⁻1, (2986-2935) cm⁻1, 1735 cm⁻1, 1663 cm⁻1.   Synthesis of 2-(N-allyl Sulfamoyl) Benzoic Acid N-allyl saccharin 0.01 mol were refluxed at 115°C with equimolar potassium hydroxide 0.01 mol solution, the reflux was continued until the solution became clear; a white precipitate was formed after the acidification by hydrochloric acid, the precipitate was filtered and washed with distilled water to give the desired products. The melting point is (210°C) with 80% yield, Rf (0.21), F.T-IR (KBr): (3421-2652) cm⁻1, 3390 cm⁻1, (3323-3227) cm⁻1, 3089 cm⁻1, (2925-2838) cm⁻1, 1735 cm⁻1, 1695 cm⁻1, 1663 cm⁻1.   Synthesis of 2-amino-5-(N-allyl-6-sulfamoyl phenyl)-1,3,4-thiadiazole Equimolar of 2-(N-allyl sulfamoyl) benzoic acid (0.01 mol) and thiosemicarbazide (0.01 mol) were mixed and dissolved in 15 mL of POCl3 using ice bath and stir cautiously. The reaction mixture was then refluxed for three hours 90°C, later distilled water 30 mL was added and the mixture was refluxed again at 80°C for four hours. Finally, the mixture was neutralized using KOH 10% until reaching pH = 6. The solution was then filtered and the resulting precipitate was washed several times with distilled water and allowed to dry to obtain the desired compound [24]. The melting point (225°C) with 82% yield, Rf (0.25), F.T-IR (KBr): (3420-3390) cm⁻1, 3325 cm⁻1, 3096 cm⁻1, (2929-2835) cm⁻1, 1735 cm⁻1, 1660 cm⁻1, 1625 cm⁻1.   Synthesis of N-allyl-2-(5-((Substituted Phenyl) Diazenyl)-1,3,4-thiadiazol-2-yl) Benzene Sulfonamide 2-Amino-5-(N-allyl-6-sulfamoyl phenyl)-1,3,4-thiadiazole 0.01 mol dissolved in 15 mL of 5 M HCl with stirring. The mixture was then cooled to 0°C in an ice bath, then 15 mL of 1 M sodium nitrite solution was added with stirring and the temperature kept below 0°C (solution 1). Substituted phenols (0.01 mol) in 15 mL of 1 M NaOH at 0°C (solution 2). (solution 1) was added slowly with stirring to (solution 2). After 30 min with temperature at 0°C the precipitate was washed with water several times and filtered [25].   (1a) N-allyl-2-(5-((2-chloro-4-hydroxy phenyl) diazenyl)-1,3,4-thiadiazol-2-yl) Benzene Sulfonamide Uv-vis λ: 293 nm as shown in Figure 2, Yield 83%, the melting point 260°C, F.TIR (KBr): 3429 cm⁻1, 3275 cm⁻1, 3073 cm⁻1, (2933-2867) cm⁻1, 1630 cm⁻1, 1601 cm⁻1, 1572 cm⁻1, 1H-NMR, (DMSO) (d6) δ:8.7 (s,1H), 7.2-7.5 (m,8H), 6.5 (t,1H), 6.2 (d,2H), 6.1 (s,1H), 4.1 (d,2H), 13CNMR (DMSO) (d6) δ: 45, 116, 133, 127-133, 149.   Figure 2: Uv-Vis Spectrum of 1a   (2a) N-allyl-2-(5-((3-chloro-4-hydroxy phenyl) diazenyl)-1,3,4-thiadiazol-2-yl) Benzene Sulfonamide Uv-vis λ: 282 nm as shown in Figure 3, Yield 85%, the melting point 264°C, F.TIR (KBr): 3432 cm⁻1, 3276 cm⁻1, 3045 cm⁻1, (2956-2867) cm⁻1, 1630 cm⁻1, 1603 cm⁻1, 1575 cm⁻1, 1H-NMR, (DMSO) (d6) δ: 8.5(s,1H), 7.3-7.6 (m,8H), 6.7 (t,1H), 6.4 (d,2H), 6.2 (s,1H), 3.9 (d,2H), 13CNMR (DMSO) (d6) δ: 47, 112, 122, 127-129, 153.   Figure 3: Uv-Vis Spectrum of 2a   (3a) N-allyl-2-(5-((2-nitro-4-hydroxy phenyl) diazenyl)-1,3,4-thiadiazol-2-yl) Benzene Sulfonamide Uv-vis λ: 291 nm as shown in Figure 4, Yield 86%, the melting point 245°C, F.TIR (KBr): 3423 cm⁻1, 3273 cm⁻1, 3103 cm⁻1, (2937-2864) cm⁻1, 1628 cm⁻1, 1603 cm⁻1, 1589 cm⁻1, 1H-NMR, (DMSO) (d6) δ: 8.1 (s,1H), 7.2-7.8 (m,8H), 6.8 (t,1H), 6.2 (d,2H), 6.1 (s,1H), 3.4 (d,2H), 13CNMR (DMSO) (d6) δ: 45, 112, 128, 122-138, 162.   Figure 4: Uv-Vis Spectrum of 3a   (4a) N-allyl-2-(5-((3-nitro-4-hydroxy phenyl) diazenyl)-1,3,4-thiadiazol-2-yl) Benzene Sulfonamide Uv-vis λ: 288 nm as shown in Figure 5, Yield 81%, the melting point 259°C, F.TIR (KBr): 3419 cm⁻1, 3276 cm⁻1, 3083 cm⁻1, (2932-2887) cm⁻1, 1622 cm⁻1, 1609 cm⁻1, 1596 cm⁻1, 1H-NMR, (DMSO) (d6) δ: 8.3 (s,1H), 7.0-7.2 (m,8H), 6.7 (t,1H), 6.3 (d,2H), 6.1 (s,1H), 3.3 (d,2H), 13CNMR (DMSO) (d6) δ: 48, 113, 127, 129-139, 157.   Figure 5: Uv-Vis Spectrum of 4a   (5a) N-allyl-2-(5-((2-methoxy-4-hydroxy phenyl) diazenyl)-1,3,4-thiadiazol-2-yl) Benzene Sulfonamide Uv-vis λ: 305 nm as shown in Figure 6, Yield 79%, the melting point 264°C, F.TIR (KBr): 3421 cm⁻1, 3292 cm⁻1, 3085 cm⁻1, (2921-2863) cm⁻1, 1629 cm⁻1, 1602 cm⁻1, 1578 cm⁻1, 1H-NMR, (DMSO) (d6) δ: 8.2 (s,1H), 7.2-7.8 (m,8H), 6.6 (t,1H), 6.4 (d,2H), 6.2 (s,1H), 3.5 (d,2H), 13CNMR (DMSO) (d6) δ: 38, 44, 119, 130, 122-129, 152.     Figure 6: Uv-Vis Spectrum of 5a   (6a) N-allyl-2-(5-((3-methoxy-4-hydroxy phenyl) diazenyl)-1,3,4-thiadiazol-2-yl) Benzene Sulfonamide Uv-vis λ: 319 nm as shown in Figure 7, Yield 75%, the melting point 258°C, F.TIR (KBr): 3421 cm⁻1, 3277 cm⁻1, 3090 cm⁻1, (2921-2853) cm⁻1, 1632 cm⁻1, 1600 cm⁻1, 1581 cm⁻1, 1H-NMR, (DMSO) (d6) δ: 8.6 (s,1H), 7.1-7.7 (m,8H), 6.8 (t,1H), 6.4 (d,2H), 6.1 (s,1H), 3.9 (d,2H), 3.2 (s,1H), 13CNMR (DMSO) (d6) δ: 39, 42, 119, 133, 126-132, 149.   Figure 7: Uv-Vis Spectrum of 6a   (7a) N-allyl-2-(5-((3-bromo-4-hydroxy phenyl) diazenyl)-1,3,4-thiadiazol-2-yl) Benzene Sulfonamide Uv-vis λ: 285 nm as shown in Figure 8, Yield 81%, the melting point 273°C, F.TIR (KBr): 3429 cm⁻1, 3293 cm⁻1, 3075 cm⁻1, (2927-2845) cm⁻1, 1620 cm⁻1, 1597 cm⁻1, 1577 cm⁻1, 1H-NMR, (DMSO) (d6) δ: 8.4 (s,1H), 7.3-7.5 (m,8H), 6.8 (t,1H), 6.5 (d,2H), 6.2 (s,1H), 3.7 (d,2H), 13CNMR (DMSO) (d6) δ: 44, 118, 127, 129-138, 156.     Figure 8: Uv-Vis Spectrum of 7a   Biological Activity The test was performed according to the disk diffusion method. The synthesized derivatives (1a-7a) were tested against one strain of Gram-positive bacteria (Staphylococcus aureus) and two Gram negative bacteria (Escherichia coli). Prepared agar and Petridishes were sterilized by autoclaving for (15 min) at 121°C. The agar plates were surface inoculated uniformly from the broth culture of the tested microorganisms. In the solidified medium suitably spaced apart holes were made all (6 mm) in diameter, were filled with 100 μL of the prepared compounds (1 mg of the compound dissolved in 1 mL of DMSO solvent). These plates were incubated at (37°C) for (24 hours). The inhibition zones caused by the various compounds on the bacteria were examined. The results of the preliminary screening test are listed in Table 1.     Table 1: Biological Activity of Derivatives (1a-7a)) Against Selected Bacteria Compound Staphylococcus aureus Escherichia coli 1a ++ ++ 2a ++ ++ 3a ++ +++ 4a ++ +++ 5a + + 6a + + 7a + + Highly active = +++ (inhibition zone 20-25 mm), Moderately active = ++ (inhibition zone 11-20 mm), Slightly active = + (inhibition zone 5-10 mm)   Antioxidant Activity   Ferric ion (Fe+3) Antioxidant Properties (Reducing Activity): Figure 9 showed the Uv-vis spectrum of the complex Fe(II)-ferrozine at 562 nm. The antioxidant properties of the synthesized 1,3,4-thiadiazole derivatives to reduce (Fe+3 to Fe+2) were measured by using ferrozine [26]. The reduction of (Fe+3) by thiadiazole was studied at pH 5.5, due to low solubility of iron at physiological pH, the reaction mixture contained 50 mM sodium acetate buffer (pH 5.5). 1 mM ferrozine, 100 μM of tested compounds and 100 μM of Fe(NO3)3. The reaction was started by the addition of Fe(NO3)3 and the increase of absorbance at 562 nm after 3 minutes was recorded, Fe+2 concentration was determined by using an extinction coefficient for Fe(ferrozine)3+2 complex which is equal to 27900 L.mol⁻1.cm⁻1 at 562 nm [26] (Table 2).     Figure 9: The Uv-Vis Spectrum of the Complex Fe(II)-ferrozine at 562 nm     Table 2: Reducing Activity of Compounds (1a-7a) Against Fe+3 No. Concentration 100 μM Read 1 Read 2 Read 3 Mean Standard deviation RSD % Range Range 95% CI (+) Dixon's Q-Test 1a 0.005 0.004 0.004 0.004 0.000 4.651 0.000 0.001 0.500 2a 0.006 0.006 0.006 0.006 0.000 1.786 0.000 0.000 0.500 3a 0.009 0.009 0.009 0.009 0.000 0.703 0.000 0.000 1.000 4a 0.008 0.008 0.008 0.008 0.000 1.855 0.000 0.000 0.667 5a 0.005 0.005 0.005 0.005 0.000 2.900 0.000 0.000 0.333 6a 0.006 0.007 0.006 0.006 0.001 8.789 0.001 0.001 0.900 7a 0.005 0.005 0.005 0.005 0.000 1.923 0.000 0.000 0.500     Copper ion (Cu+2) Antioxidant Properties (Reducing Activity) Figure 10 showed the Uv-vis spectrum of the complex Cu (I)-neocuproine at 454 nm. The antioxidant properties of the prepared compounds containing 1,3,4-thiadiazole ring to reduce (Cu+2 to Cu+1) were measured by using 2,9-dimethyl-1,10-phenanthroline (neocuproine) [37], an indicator molecule that binds specifically to the reduced form of copper (Cu+1 but no the oxidized form Cu+2) [27]. The reaction mixture contained (20 mM) KH2PO4/KOH buffer (pH 7.4), 200 μM Cu(NO3)2, 600 μM 2,9-dimethyl-1,10-phenanthroline, 100 μM of the tested compounds. The mixtures were incubated at room temperature for 120 minutes and then the absorbances were recorded at 455 nm. The copper concentration was determined by using an extinction coefficient for Cu(neocuproine) complex which is 7.9×106 L.mol⁻1.cm⁻1, at 454 nm that was determined by reducing Cu+2 with ascorbate [27] (Table 3).     Figure 10: The Uv-Vis Spectrum of the Complex Cu (I)-neocuproine at 454 nm     Table 3: Reducing Activity of Compounds (1a-7a) Against Cu+2 No. Concentration 100 μM Read 1 Read 2 Read 3 Mean Standard deviation RSD % Range Range 95% CI (+) Dixon's Q-Test 1a 0.460 0.460 0.470 0.463 0.006 1.246 0.010 0.014 1.000 2a 0.380 0.400 0.390 0.390 0.010 2.564 0.020 0.025 0.500 3a 0.760 0.770 0.750 0.760 0.000 0.008 0.000 0.000 0.500 4a 0.730 0.720 0.730 0.727 0.006 0.795 0.010 0.014 0.000 5a 0.550 0.580 0.570 0.567 0.015 2.696 0.030 0.038 0.333 6a 0.510 0.530 0.510 0.517 0.012 2.235 0.020 0.029 1.000 7a 0.490 0.510 0.500 0.500 0.000 0.020 0.020 0.000 0.500

Thiadiazoles synthesized in this work were identified using F.T-IR, 1H-NMR, 13C-NMR, Uv-Vis spectroscopy and tested for their biological activity (anti-bacterial and anti-oxidant) the synthesized thiadiazole were achieved by starting from sodium saccharin the well-known compound that was reacted with allyl chloride in DMF to form N-allyl saccharin, in the other hand N-allyl saccharin was hydrolyzed with KOH then neutralized with HCl to form 2-(N-allyl sulfamoyl) benzoic acid, the product above was reacted with thiosemicarbazide in the presence with POCl3 to form the derivative 2-amino-5-(N-allyl-6-sulfamoyl phenyl)-1,3,4-thiadiazole. The above derivative was diazotized with some aromatic amines in the presence of NaNO2 and HCl to give the desired products (1a-7a). The derivatives (1a-7a) were characterized using F.T-IR and NMR (1H-NMR, 13C-NMR) and in addition to Uv-Vis spectroscopy all the data above indicate the formation of the titled compounds. The spectral data obtained from the F.T-IR and NMR assure the formation of the titled compounds. The anti-bacterial activity for the synthesized compounds shows that the compounds have different anti-bacterial activity and generally the compounds (1a, 2a, 3a and 4a) have moderate activity against Staphylococcus aureus, while the compounds (3a and 4a) exhibited higher activity against Escherichia coli. Thiadiazoles (1a-7a) studied show higher reducing capacity for copper ions than for iron ions, this can be attributed to the standard reduction and oxidation potentials of the metals, the standard reduction potential of the Cu+2/Cu+1 (0.15 V) which is much lower than that for Fe+3/Fe+2 (0.77 V). Derivatives (3a and 4a) showed higher antioxidant activity than the other compounds against both Cu (II) and Fe (III).