Experimental

Chapter 6. Synthesis, in vitro antimicrobial, antioxidant and antidiabetic activities of amino

6.2 Experimental

6.2.1 Materials and methods

All chemicals were supplied by Sigma-Aldrich via Capital Lab, South Africa. All organic solvents were redistilled and dried according to standard procedures. Silica gel 60 F254 plates (Merck) were used for thin layer chromatography. Products were purified by column chromatography using silica gel (60-120 mesh) with ethyl acetate : hexane as the eluent. Melting points were recorded using a Stuart Scientific SMP3 apparatus. UV spectra were obtained on a Varian Cary UV-VIS spectrophotometer using MeOH as a solvent. IR spectra were recorded on a Perkin Elmer 100 FT- IR spectrophotometer with universal attenuated total reflectance sampling accessory. Microwave reactions were performed using a CEM Discover, Explorer-12 Hybrid microwave instrument. ¹H,

13C NMR and all 2D NMR spectra were recorded on a Bruker Avance instrument operating at 400 MHz. Chemical shifts were reported as δ values (ppm) relative to an internal standard of

tetramethylsilane (TMS) or to the solvent line of DMSO-d6 (δH = 2.50, δC = 39.52). High- resolution mass data were obtained using a Bruker micro TOF-Q II ESI instrument operating at ambient temperature.

General procedure for the preparation of amino acid methyl ester hydrochloride (B2) Methanol (20 mL) was added to different amino acids (B1a-c) (0.1 mol) followed by gradual addition of thionyl chloride (0.3 mol) at room temperature. The resulting solution was stirred at 70 °C for 4h and the reaction mixture concentrated on a rotary evaporator producing the respective amino acid ester hydrochlorides in yields of > 90 %.

The general scheme for the reaction sequence is shown in Scheme 6-1 below.

General procedure for the preparation of compounds (B4a-c)

1-Fluoro-2,4-dinitrobenzene B3 (0.50 g, 2.34 mmol), amino acid ester (3.51 mmol) and NaHCO3

(0.005 g, 4.68 mmol) were mixed in THF (10 mL). The reaction mixture was stirred overnight at room temperature. After completion of the reaction (monitored by TLC), the reaction mixture was washed with water (2 × 10 mL), followed by 10% Na2CO3 solution (10 mL). The organic layer was separated from the aqueous layer by adding a small amount of ethyl acetate to distinguish the THF and aqueous layers. The organic layer was then dried over anhydrous Na2SO4 and concentrated under reduced pressure to afford B4a-c, which was subsequently purified by column chromatography (hexane/ethyl acetate) to afford the desired product in good yields (80-90%).

General procedure for the preparation of compounds (B5a-c)

The 2,4-dinitrophenyl amino acid compounds B4a-c (1 mmol) was added to methanol (10 mL), to which 10% Pd/C (50 mg) was added. The reaction mixture was stirred for 4h at room temperature under H2 atmosphere. The reaction mixture was then filtered through Celite 545 to remove the catalyst. The filtrate was evaporated under reduced pressure and the obtained solid was purified by column chromatography (hexane/ethyl acetate) to afford the desired product in good yield (80- 85%).

General procedure for the preparation of amino acid linked thiazolidine-quinoxaline derivatives (B6a-l)

A mixture of 3-substituted-7-amino quinoxalin-2(1H)-one (B5a-c) (1.0 mmol), the appropriate substituted benzaldehyde (1.5 mmol) and thioglycolic acid (5.0 mmol) was placed in a 10 mL reaction vial containing absolute ethanol (3 mL) and a stirring bar. The vial was sealed tightly

with a Teflon septum, placed into the microwave cavity and irradiated at 100 ºC using 100 W as the maximum power for 10-45 min. Upon completion, the reaction mixture was rapidly cooled by gas jet cooling to ambient temperature, the contents dissolved in ethyl acetate and washed with 5%

aqueous citric acid (2 × 5 mL), water and aqueous sodium hydrogen carbonate (2 × 5 mL). The organic layer was then separated and dried over sodium sulphate, evaporated under reduced pressure and the solid obtained purified by column chromatography (hexane/ethyl acetate) to afford the desired product in good yield (80-90%).

(B5a) 7-Amino-3-isopropylquinoxalin-2(1H)-one, dark brown solid; mp 238-240 ºC; UV λmax

(MeOH) nm (log ε) 222 (3.40), 282 (2.40), 364 (3.02); IR υmax 3460 (NH2), 3331 (NH), 1650, 1622 (C=O) cm^-1; ¹H NMR (400 MHz, DMSO) δH 11.87 (s, 1H, H-1), 7.33 (d, J = 8.0 Hz, 1H, H-5), 6.51 (dd, J = 8.0, 2.2 Hz, 1H, H-6), 6.32 (d, J = 2.2 Hz, 1H, H-8), 5.80 (s, 2H, H-11), 3.28-3.39 (m, 1H, H-1a), 1.15 (d, J = 6.8 Hz, 6H, H-2a/H-3a); ¹³C NMR (400 MHz, DMSO) δC 157.4 (C, C- 3), 154.9 (C, C-2), 150.3 (C, C-7), 133.6 (C, C-9), 129.1 (CH, C-5), 124.0 (C, C-10), 111.5 (CH, C-6), 95.9 (CH, C-8), 29.3 (CH, C-1a), 20.3 (2CH3, C-2a/3a); HRMS (m/z): calc. for C11H13N3ONa (M + Na)⁺: 226.0956, found: 226.0956.

(B5b) 7-amino-3-(2-(methylthio)ethyl)quinoxalin-2(1H)-one, brown solid; mp 178-180 ºC; UV λmax (MeOH) nm (log ε) 230 (3.14), 283 (2.97), 390 (3.02); IR υmax 3337 (NH), 2809 (C-H), 1671, 1637 (C=O), 1521 cm^-1; ¹H NMR (400 MHz, DMSO) δH 12.01 (s, 1H, H-1), 7.34 (d, J = 8.5 Hz, 1H, H-5), 6.53 (d, J = 8.5 Hz, 1H, H-6), 6.33 (bs, 1H, H-8), 5.89 (s, 2H, H-11), 3.14-3.20 (m, 2H, H-2a), 2.97-3.05 (m, 2H, H-1a), 2.58 (s, 3H, H-4a); ¹³C NMR (400 MHz, DMSO) δC 155.2 (C, C- 3), 150.7 (C, C-2), 150.7 (C, C-7), 133.9 (C, C-10), 129.0 (CH, C-5), 124.0 (C, C-9), 111.7 (CH,

C-6), 95.9 (CH, C-8), 49.9 (CH2, C-2a), 38.0 (CH3, C-4a), 25.2 (CH2, C-1a); HRMS (m/z): calc.

for C11H12N3OS (M+ H)⁺: 234.0701, found: 234.0708.

(B5c) 3-(4-hydroxybenzyl)-7-aminoquinoxalin-2(1H)-one, light yellow solid; mp 164-166 ºC;

UV λmax (MeOH) nm (log ε) 224 (3.51), 249 (3.0), 279 (2.78), 371 (3.15); IR υmax 3461 (NH2), 3332 (NH), 1651, 1622 (C=O) cm^-1; ¹H NMR (400 MHz, DMSO) δH 11.91 (s, 1H, H-1), 9.17 (s, 1H, H-8a), 7.33 (d, J = 8.6 Hz, 1H, H-5), 7.08 (d, J = 8.2 Hz, 2H, H-3a/7a), 6.65 (d, J = 8.2 Hz, 2H, H-4a/6a), 6.51 (dd, J = 8.6, 1.6 Hz, 1H, H-6), 6.31 (d, J = 1.6 Hz, 1H, H-8), 5.84 (s, 2H, H- 11), 3.85 (s, 2H, H-1a); ¹³C NMR (400 MHz, DMSO) δC 155.7 (C, C-3), 155.3 (C, C-5a), 152.6 (C, C-2), 150.6 (C, C-7), 133.9 (C, C-9), 129.8 (2CH, C-3a/7a), 129.1 (CH, C-5), 128.6 (C, C-10), 124.2 (C, C-2a), 115.0 (2CH, C-4a/6a), 111.6 (CH, C-6), 95.9 (CH, C-8), 37.6 (CH2, C-1a); HRMS (m/z): calc. for C15H13N3O2Na (M + Na)⁺: 290.0905, found: 290.0910.

(B6a) 3-isopropyl-7-(4-oxo-2-phenylthiazolidin-3-yl)quinoxalin-2(1H)-one, white solid; mp 280-282 ºC; UV λmax (MeOH) nm (log ε) 231 (3.13), 287 (2.59), 341 (2.81); IR υmax 3151 (NH), 1683 (C=O), 1652, 1616 cm^-1; ¹H NMR (400 MHz, DMSO) δH 12.27 (s, 1H, H-1), 7.60 (d, J = 8.8 Hz, 1H, H-5), 7.38 (dd, J = 7.7, Hz, 2H, H-2c/6c), 7.31 (d, J = 2.2 Hz, 1H, H-8), 7.26 (dd, J = 7.7, 7.7 Hz, 2H, H-3c/5c), 7.22 (dd, J = 7.7, 2.2 Hz, 1H, H-4c), 7.20 (dd, J = 8.8, 2.2 Hz, 1H, H-6), 6.58 (s, 1H, H-5b), 4.06 (d, J = 15.9 Hz, 1H, H-3bi), 3.92 (d, J = 15.9 Hz, 1H, H-3bii), 3.39 (sep, J = 6.9 Hz, 1H, H-1a), 1.16 (d, J = 6.9 Hz, 3H, H-2a), 1.15 (d, J = 6.9 Hz, 3H, H-3a); ¹³C NMR (400 MHz, DMSO) δC 170.8 (C, C-2b), 165.5 (C, C-3), 154.1 (C, C-2), 139.7 (C, C-1c), 138.1 (C, C-7), 131.7 (C, C-9*), 129.4 (C, C-10*), 128.7 (2CH, C-3c/5c), 128.6 (CH, C-4c), 128.3 (CH, C- 5), 126.9 (2CH, C-2c/6c), 119.3 (CH, C-8), 111.3 (CH, C-6), 63.3 (CH, C-5b), 32.8 (CH2, C-3b),

29.8 (CH, C-1a), 19.99 (CH3, C-2a), 19.97 (CH3, C-3a); HRMS (m/z): calc. for C20H19N3O2SNa (M + Na)⁺: 388.1096, found: 388.1096. *indicates that assignments are interchangeable.

(B6b) 7-(2-(4-fluorophenyl)-4-oxothiazolidin-3-yl)-3-isopropylquinoxalin-2(1H)-one, white solid; mp 252-254 ºC; UV λmax (MeOH) nm (log ε) 230 (3.29), 287 (2.72), 340 (2.95); IR υmax

3178 (NH), 1657 (C=O), 1615, 1510 cm^-1; ¹H NMR (400 MHz, DMSO) δH 12.27 (s, 1H, H-1), 7.61 (d, J = 8.7 Hz, 1H, H-5), 7.45 (dd, J = 8.7, 5.4, Hz, 2H, H-2c/6c), 7.27 (d, J = 2.2 Hz, 1H, H- 8), 7.19 (dd, J = 8.7, 2.2 Hz, 1H, H-6), 7.12 (t, J = 8.8 Hz, 2H, H-3c/5c), 6.59 (s, 1H, H-5b), 4.06 (d, J = 15.8 Hz, 1H, H-3bi), 3.92 (d, J = 15.8 Hz, 1H, H-3bii), 3.42 (septet, J = 6.8 Hz, 1H, H-1a), 1.16 (d, J = 6.8 Hz, 3H, H-2a), 1.15 (d, J = 6.8 Hz, 3H, H-3a); ¹³C NMR (400 MHz, DMSO) δC

170.7 (C, C-2b), 165.6 (C, C-3), 161.9 (d, JC-F = 243.9 Hz, C, C-4c), 154.1 (C, C-2), 137.9 (C, C- 7), 135.9 (d, J = 2.8 Hz, C, C-1c), 131.7 (C, C-9*), 129.5 (C, C-10*), 129.3 (d, J = 8.6 Hz, 2CH, C-2c/6c), 128.4 (CH, C-5), 119.4 (CH, C-6), 115.6 (d, J = 21.7 Hz, 2CH, C-3c/5c), 111.5 (CH, C- 8), 62.6 (CH, C-5b), 32.8 (CH2, C-3b), 29.8 (CH, C-1a), 19.99 (CH3, C-2a*), 19.96 (CH3, C-3a*);

HRMS (m/z): calc. for C20H18N3O2SFNa (M + Na)⁺: 406.1001, found: 406.0999. *indicates that assignments are interchangeable.

(B6c) 3-isopropyl-7-(2-(4-methoxyphenyl)-4-oxothiazolidin-3-yl)quinoxalin-2(1H)-one, brown solid; mp 268-270 ºC; UV λmax (MeOH) nm (log ε) 230 (3.67), 284 (3.03), 341 (3.22); IR υmax 3144 (NH), 1682 (C=O), 1657, 1609 cm^-1; ¹H NMR (400 MHz, DMSO) δH 12.26 (s, 1H, H- 1), 7.59 (d, J = 8.6 Hz, 1H, H-5), 7.31 (d, J = 8.8 Hz, 2H, H-2c/6c), 7.26 (d, J = 2.2 Hz, 1H, H-8), 7.18 (dd, J = 8.6, 2.2 Hz, 1H, H-6), 6.82 (d, J = 8.8 Hz, 2H, H-3c/5c), 6.52 (s, 1H, H-5b), 4.03 (d, J = 15.7 Hz, 1H, H-3bi), 3.91 (d, J = 15.7 Hz, 1H, H-3bii), 3.67 (s, 3H, H-8c), 3.38 (septet, J = 6.8

Hz, 1H, H-1a), 1.16 (d, J = 6.8 Hz, 3H, H-2a), 1.15 (d, J = 6.8 Hz, 3H, H-3a); ¹³C NMR (400 MHz, DMSO) δC 171.1 (C, C-2b), 165.9 (C, C-3), 159.5 (C, C-4c), 154.3 (C, C-2), 138.4 (C, C-7), 131.8 (C, C-1c), 131.2 (C, C-9*), 129.7 (C, C-10*), 129.1 (2CH, C-2c/6c), 128.5 (CH, C-5), 120.2 (CH, C-6), 114.3 (2CH, C-3c/5c), 111.9 (CH, C-8), 63.5 (CH, C-5b), 55.3 (CH3, C-8c), 33.2 (CH2, C- 3b), 30.1 (CH, C-1a), 20.2 (2CH3, C-2a/3a); HRMS (m/z): calc. for C21H21N3O3SNa (M + Na)⁺: 418.1201, found: 418.1213. *indicates that assignments are interchangeable.

(B6d) 3-isopropyl-7-(2-(4-nitrophenyl)-4-oxothiazolidin-3-yl)quinoxalin-2(1H)-one, light yellow solid; mp 274-276 ºC; UV λmax (MeOH) nm (log ε) 229 (3.48), 341 (3.12); IR υmax 3088 (NH), 1694, 1664 (C=O), 1624 cm^-1; ¹H NMR (400 MHz, DMSO) δH 12.28 (s, 1H, H-1), 8.15 (d, J = 8.7 Hz, 2H, H-3c/5c), 7.70 (d, J = 8.7 Hz, 2H, H-2c/6c), 7.62 (d, J = 8.8 Hz, 1H, H-5), 7.32 (d, J = 2.2 Hz, 1H, H-8), 7.24 (dd, J = 8.8, 2.2 Hz, 1H, H-6), 6.75 (s, 1H, H-5b), 4.13 (d, J = 15.8 Hz, 1H, H-3bi), 3.96 (d, J = 15.8 Hz, 1H, H-3bii), 3.39 (septet, J = 6.8 Hz, 1H, H-1a), 1.16 (d, J = 6.8 Hz, 3H, H-2a), 1.15 (d, J = 6.8 Hz, 3H, H-3a); ¹³C NMR (400 MHz, DMSO) δC 170.7 (C, C-2b), 165.7 (C, C-3), 154.0 (C, C-2), 147.5 (C, C-4c), 147.4 (C, C-1c), 137.8 (C, C-7), 131.8 (C, C-9*), 129.5 (C, C-10*), 128.5 (CH, C-5), 128.1 (2CH, C-2c/6c), 124.1 (2CH, C-3c/5c), 118.9 (CH, C- 6), 110.9 (CH, C-8), 62.1 (CH, C-5b), 32.7 (CH2, C-3b), 29.8 (CH, C-1a), 19.98 (CH3, C-2a), 19.95 (CH3, C-3a); HRMS (m/z): calc. for C20H17N4O4S (M- H)^-: 409.0971, found: 409.0966.

*indicates that assignments are interchangeable.

(B6e) 3-(2-(methylthio)ethyl)-7-(4-oxo-2-phenylthiazolidin-3-yl)quinoxalin-2(1H)-one, brown solid; mp 258-260 ºC; UV λmax (MeOH) nm (log ε) 230 (3.34), 294 (2.74), 344 (2.98); IR υmax 3173 (NH), 1678 (C=O), 1654, 1618 cm^-1; ¹H NMR (400 MHz, DMSO) δH 12.32 (s, 1H, H-

1), 7.61 (d, J = 8.7 Hz, 1H, H-5), 7.38 (d, 1H, J = 7.3 Hz, H-2c/6c), 7.33 (d, 1H, J = 2.2 Hz, H-8), 7.29 (t, J = 7.3 Hz, 2H, H-3c/5c), 7.23 (t, J = 7.3 Hz, 2H, H-4c), 7.21 (dd, J = 8.7, 2.2 Hz, H-6), 6.58 (s, 1H, H-5b), 4.06 (d, J = 15.9 Hz, 1H, H-3bi), 3.92 (d, J = 15.9 Hz, 1H, H-3bii), 3.01 (t, J = 7.7 Hz, 2H, H-2a), 2.82 (t, J = 7.7 Hz, 2H, H-1a), 2.07 (s, 3H, H-4a); ¹³C NMR (400 MHz, DMSO) δC 170.8 (C, C-2b), 160.1 (C, C-3), 154.5 (C, C-2), 139.7 (C, C-1c), 138.3 (C, C-7), 131.9 (C, C- 9*), 129.4 (C, C-10*), 128.7 (2C, C, C-3c/5c), 128.6 (CH, C-5), 128.3 (CH, C-4c), 126.9 (CH, C- 2c/6c), 119.3 (CH, C-6), 111.3 (CH, C-8), 63.3 (CH, C-5b), 32.8 (CH2, C-3b), 32.6 (CH2, C-2a), 29.9 (CH2, C-1a), 14.6 (CH3, C-4a); HRMS (m/z): calc. for C20H18N3O2S2 (M - H)⁺: 396.0840, found: 396.0856. *indicates that assignments are interchangeable.

(B6f) 7-(2-(4-fluorophenyl)-4-oxothiazolidin-3-yl)-3-(2-(methylthio)ethyl)quinoxalin-2(1H)- one, dark brown solid; mp 264-266 ºC; UV λmax (MeOH) nm (log ε) 231 (3.27), 293 (2.68), 343 (2.92); IR υmax 3173 (NH), 1679 (C=O), 1655, 1618, 1604, 1508 cm^-1; ¹H NMR (400 MHz, DMSO) δH 12.33 (s, 1H, H-1), 7.62 (d, J = 8.7 Hz, 1H, H-5), 7.46 (dd, J = 8.8, 5.4 Hz, 2H, H- 2c/6c), 7.28 (d, J = 2.0 Hz, 1H, H-8), 7.20 (dd, J = 8.7, 2.0 Hz, 1H, H-6), 7.12 (dd, J = 8.8, 8.8 Hz, 2H, H-3c/5c), 6.59 (s, 1H, H-5b), 4.07 (d, J = 15.7 Hz, 1H, H-3bi), 3.93 (d, J = 15.7 Hz, 1H, H-3bii), 3.01 (t, J = 7.0 Hz, 2H, H-2a), 2.83 (t, J = 7.6 Hz, 2H, H-1a), 2.07 (s, 3H, H-4a); ¹³C NMR (400 MHz, DMSO) δC 170.7 (C, C-2b), 161.9 (d, J = 243.7 Hz, C, C-4c), 160.2 (C, C-3), 154.5 (C, C-2), 138.2 (C, C-7), 135.9 (d, J = 2.9 Hz, C, C-1c), 131.9 (C, C-9*), 129.5 (C, C-10*), 129.3 (d, J = 8.4 Hz, 2CH, C-2c/6c), 128.3 (CH, C-5), 119.5 (CH, C-6), 115.7 (d, J = 21.2 Hz, 2CH, C- 3c/5c), 111.5 (CH, C-8), 62.6 (CH, C-5b), 32.8 (CH2, C-3b), 32.6 (CH2, C-2a), 29.9 (CH2, C-1a), 14.6 (CH3, C-4a); HRMS (m/z): calc. for C20H18FN3O2S2Na (M + Na)⁺: 438.0722, found:

438.0722. *indicates that assignments are interchangeable.

(B6g) 7-(2-(4-methoxyphenyl)-4-oxothiazolidin-3-yl)-3-(2-(methylthio)ethyl)quinoxalin- 2(1H)-one, yellow solid; mp 258-260 ºC; UV λmax (MeOH) nm (log ε) 230 (3.09), 279 (2.95), 389 (3.06); IR υmax 3321 (NH), 1685 (C=O), 1616, 1525 cm^-1; ¹H NMR (400 MHz, DMSO) δH 12.36 (s, 1H, H-1), 7.61 (d, J = 8.7 Hz, 1H, H-5), 7.32 (d, J = 8.6 Hz, 2H, H-2c/6c), 7.28 (d, J = 2.1, 1H, H-8), 7.21 (dd, J = 8.7, 2.1 Hz, 1H, H-6), 6.83 (d, J = 8.6 Hz, 2H, H-3c/5c), 6.53 (s, 1H, H-5b), 4.03 (d, J = 15.9 Hz, 1H, H-3bi), 3.91 (d, J = 15.9 Hz, 1H, H-3bii), 3.67 (s, 3H, H-8c), 3.16-3.10 (m, 2H, H-2a), 3.06-2.96 (m, 2H, H-1a), 2.57 (s, 3H, H-4a); ¹³C NMR (400 MHz, DMSO) δC 170.6 (C, C-2b), 159.4 (C, C-4c), 159.2 (C, C-3), 154.4 (C, C-2), 138.4 (C, C-1c), 138.4 (C, C-7), 131.9 (C, C-9*), 131.1 (C, C-10*), 128.7 (2CH, C-2c/6c), 128.2 (CH, C-5), 119.6 (CH, C-6), 113.9 (2CH, C-3c/5c), 111.6 (CH, C-8), 63.1 (CH, C-5b), 55.0 (CH3, C-8c), 49.3 (CH2, C-3b), 37.9 (CH3, C-4a), 32.9 (CH2, C-2a), 25.5 (CH2, C-1a); HRMS (m/z): calc. for C21H22N3O3S2 (M-H)⁺: 428.1103, found: 428.1084. *indicates that assignments are interchangeable.

(B6h) 3-(2-(methylthio)ethyl)-7-(2-(4-nitrophenyl)-4-oxothiazolidin-3-yl)quinoxalin-2(1H)- one, reddish brown solid; mp 240-242 ºC; UV λmax (MeOH) nm (log ε) 233 (2.92), 344 (2.60); IR υmax 3175 (NH), 1681 (C=O), 1655, 1619, 1515 cm^-1; ¹H NMR (400 MHz, DMSO) δH 12.34 (s, 1H, H-1), 8.16 (d, J = 8.7 Hz, 2H, H-3c/5c), 7.70 (d, J = 8.7 Hz, 2H, H-2c/6c), 7.63 (d, J = 8.8 Hz, 1H, H-5), 7.34 (d, J = 2.2 Hz, 1H, H-8), 7.25 (dd, J = 8.8, 2.2 Hz, 1H, H-6), 6.76 (s, 1H, H-5b), 4.13 (d, J = 15.9 Hz, 1H, H-3bi), 3.96 (d, J = 15.9 Hz, 1H, H-3bii), 3.01 (t, J = 7.6 Hz, 2H, H-2a), 2.82 (t, J = 7.6 Hz, 2H, H-1a), 2.06 (s, 3H, H-4a); ¹³C NMR (400 MHz, DMSO) δC 170.8 (C, C- 2b), 160.3 (C, C-3), 154.5 (C, C-2), 147.5 (C, C-4c), 147.4 (C, C-1c), 137.9 (C, C-7), 132.0 (C, C- 9*), 129.5 (C, C-10*), 128.5 (CH, C-5), 128.1 (2CH, C-2c/6c), 124.1 (2CH, C-3c/5c), 119.0 (CH,

C-6), 111.0 (CH, C-8), 62.1 (CH, C-5b), 32.7 (CH2, C-3b), 32.6 (CH2, C-2a), 29.9 (CH2, C-1a), 14.6 (CH3, C-4a); HRMS (m/z): calc. for C20H17N4O4S2 (M + H)⁺: 441.0691, found: 441.0696.

*indicates that assignments are interchangeable.

(B6i) 3-(4-hydroxybenzyl)-7-(4-oxo-2-phenylthiazolidin-3-yl)quinoxalin-2(1H)-one, yellow solid; mp 134-136 ºC; UV λmax (MeOH) nm (log ε) 228 (3.39), 346 (3.01); IR υmax 3177 (NH), 1656 (C=O), 1614, 1510 cm^-1; ¹H NMR (400 MHz, DMSO) δH 12.31 (s, 1H, H-1), 9.19 (s, 1H, H- 8a), 7.60 (d, J = 8.5 Hz, 1H, H-5), 7.38 (d, 2H, J = 7.0 Hz, H-2c/6c), 7.32 (s, 1H, H-8), 7.28 (dd, 2H, J = 7.0, 7.0, H-3c/5c), 7.19-7.23 (m, 2H, H-4c, H-6), 7.07 (d, J = 7.6 Hz, 2H, H-3a/7a), 6.64 (d, J = 7.6 Hz, 2H, H-4a/6a), 6.57 (s, 1H, H-5b), 4.06 (d, J = 15.8 Hz, 1H, H-3bi), 3.93 (bs, 2H, H-1a), 3.91 (d, J = 15.8 Hz, 1H, H-3bii); ¹³C NMR (400 MHz, DMSO) δC 170.8 (C, C-2b), 160.6 (C, C-3), 155.9 (C, C-2), 154.4 (C, C-5a), 139.7 (C, C-1c), 138.3 (C, C-7), 132.0 (C, C-9*), 130.0 (2C, CH, C-3a/7a), 129.5 (C, C-10*), 128.8 (2C, CH, C-2c/6c), 128.6 (CH, C-5), 128.3 (CH, C- 4c), 127.2 (C, C-2a), 126.9 (2C, CH, C-3c/5c), 119.3 (CH, C-6), 115.1 (2CH, C-4a/6a), 111.2 (CH, C-8), 63.3 (CH, C-5b), 38.0 (CH2, C-1a), 32.8 (CH2, C-3b); HRMS (m/z): calc. for C24H18N3O3S (M + H)⁺: 428.1069, found: 428.1073. *indicates that assignments are interchangeable.

(B6j) 3-(4-hydroxybenzyl)-7-(2-(4-fluorophenyl)-4-oxothiazolidin-3-yl)quinoxalin-2(1H)- one, yellow solid; mp 268-270 ºC; UV λmax (MeOH) nm (log ε) 230 (3.03), 286 (2.52), 345 (2.75);

IR υmax 3384 (OH), 3125 (NH), 1678 (C=O), 1615, 1509 cm^-1; ¹H NMR (400 MHz, DMSO) δH

12.31 (s, 1H, H-1), 9.18 (s, 1H, H-8a), 7.61 (d, J = 8.7 Hz, 1H, H-5), 7.44 (dd, J = 8.6, 5.4 Hz, 2H, H-2c/6c), 7.27 (d, J = 2.2 Hz, 1H, H-8), 7.19 (dd, J = 8.7, 2.2 Hz, 1H, H-6), 7.11 (dd, J = 8.6, 8.6

Hz, 2H, H-3c/5c), 7.08 (d, J = 8.5 Hz, 2H, H-3a/7a), 6.64 (d, J = 8.5 Hz, 2H, H-4a/6a), 6.58 (s, 1H, H-5b), 4.07 (d, J = 15.7 Hz, 1H, H-3bi), 3.93 (bs, 2H, H-1a), 3.92 (d, J = 15.7 Hz, 1H, H- 3bii); ¹³C NMR (400 MHz, DMSO) δC 170.7 (C, C-2b), 161.9 (d, J = 238.8 Hz, C, C-4c), 160.6 (C, C-3), 155.9 (C, C-5a), 154.4 (C, C-2), 138.2 (C, C-7), 135.9 (d, J = 2.8 Hz, C, C-1c), 132.0 (C, C-10*), 130.0 (2C, CH, C-3a/7a), 129.5 (C, C-9*), 129.2 (2C, CH, d, J = 8.4 Hz, C-2c/6c), 128.4 (CH, C-5), 127.2 (C, C-2a), 119.4 (CH, C-6), 115.6 (2C, CH, d, J = 21.7 Hz, C-3c/5c), 115.1 (2C, CH, C-4a/6a), 111.4 (CH, C-8), 62.6 (CH, C-5b), 38.0 (CH2, C-1a), 32.8 (CH2, C-3b); HRMS (m/z): calc. for C24H18FN3O3SNa (M + Na)⁺: 470.0951, found: 470.0947. *indicates that assignments are interchangeable.

(B6k) 3-(4-hydroxybenzyl)-7-(2-(4-methoxyphenyl)-4-oxothiazolidin-3-yl)quinoxalin-2(1H)- one, yellow solid; mp 194-196 ºC; UV λmax (MeOH) nm (log ε) 232 (2.91), 348 (2.56); IR υmax

3200 (NH), 1659 (C=O), 1609, 1510 cm^-1; ¹H NMR (400 MHz, DMSO) δH 12.32 (s, 1H, H-1), 9.21 (d, J = 3.5 Hz, 1H, H-8a), 7.60 (d, J = 8.3 Hz, 1H, H-5), 7.30 (d, J = 8.4 Hz, 2H, H-3a/7a), 7.27 (bs, 1H, H-8), 7.17 (d, J = 8.3 Hz, 1H, H-6), 7.06 (d, J = 8.3 Hz, 2H, H-2c/6c), 6.81 (d, J = 8.4 Hz, 2H, H-4a/6a), 6.63 (d, J = 8.3 Hz, 2H, H-3c/5c), 6.52 (s, 1H, H-5b), 4.02 (d, J = 15.8 Hz, 1H, H-3bi), 3.93 (d, J = 8.3 Hz, 2H, H-1a), 3.92 (d, J = 15.8 Hz, 1H, H-3bii), 3.67 (s, 3H, H-8c);

13C NMR (400 MHz, DMSO) δC 170.7 (C, C-2b), 160.6 (C, C-3), 159.3 (C, C-4c), 155.9 (C, C- 5a), 154.4 (C, C-2), 138.4 (C, C-7), 131.9 (C, C-9*), 131.1 (C, C-10*), 130.0 (2C, CH, C-2c/6c), 129.5 (C, C-1c), 128.5 (2C, CH, C-3a/7a), 128.3 (CH, C-5), 127.3 (C, C-2a), 119.7 (CH, C-6), 115.1 (2C, CH, C-3c/5c), 114.1 (2C, CH, C-4a/6a), 111.5 (CH, C-8), 63.1 (CH, C-5b), 55.1 (CH3, C-8c), 38.1 (CH2, C-1a), 32.9 (CH2, C-3b); HRMS (m/z): calc. for C25H20N3O4S (M + H)⁺: 458.1175, found: 458.1187. *indicates that assignments are interchangeable.

(B6l) 3-(4-hydroxybenzyl)-7-(2-(4-nitrophenyl)-4-oxothiazolidin-3-yl)quinoxalin-2(1H)-one, yellow solid; mp 260-262 ºC; UV λmax (MeOH) nm (log ε) 233 (2.58), 344 (2.37); IR υmax 3185 (NH), 1660 (C=O) cm^-1; ¹H NMR (400 MHz, DMSO) δH 12.33 (s, 1H, H-1), 9.18 (s, 1H, H-8a), 8.14 (d, J = 8.1 Hz, 2H, H-3c/5c), 7.68 (d, J = 8.1 Hz, 2H, H-2c/6c), 7.62 (d, J = 8.4 Hz, 1H, H- 5), 7.32 (bs, 1H, H-8), 7.23 (d, J = 8.4 Hz, 1H, H-6), 7.05 (d, J = 7.7 Hz, 2H, H-3a/7a), 6.74 (s, 1H, H-5b), 6.63 (d, J = 7.7 Hz, 2H, H-4a/6a), 4.12 (d, J = 15.8 Hz, 1H, H-3bi), 3.94 (d, J = 15.8 Hz, 1H, H-3bii), 3.92 (bs, 2H, H-1a); ¹³C NMR (400 MHz, DMSO) δC 170.8 (C, C-2b), 160.8 (C, C-3), 155.9 (C, C-2), 154.4 (C, C-5a), 147.5 (C, C-4c), 147.3 (C, C-1c), 137.9 (C, C-7), 132.1 (C, C-9*), 129.9 (2C, CH, C-3a/7a), 129.6 (C, C-10*), 128.6 (CH, C-5), 128.0 (2C, CH, C-2c/6c), 127.2 (C, C-2a), 124.1 (2C, CH, C-3c/5c), 118.9 (CH, C-6), 115.1 (2C, CH, C-4a/6a), 110.9 (CH, C-8), 62.1 (CH, C-5b), 38.0 (CH2, C-1a), 32.6 (CH2, C-3b); HRMS (m/z): calc. for C24H17N4O5S (M + H)⁺: 473.0920, found: 473.0935. *indicates that assignments are interchangeable.

6.2.2 In vitro antimicrobial studies

The microbial cultures were grown overnight at 37 °C in nutrient broth (UKZN Biolab, South Africa), adjusted to 0.5 McFarland standard using distilled water and lawn inoculated onto Mueller-Hinton agar (MHA) plates. A volume of 10 µL of each sample (21.08 - 49.20 µM, 1 mL DMSO) was inoculated onto antibiotic assay discs (6 mm diameter) and placed on the MHA plates which were incubated overnight at 37 °C for 24 hours. After the incubation period, zones of inhibition were measured in mm. Compounds showing an inhibition zone of > 9 mm were selected to determine their MBC values using the broth dilution assay with ampicillin and ciprofloxacin as

the controls following the method in Andrews (2001). Compounds B5a-c, B6a-c, B6f-h, B6j and B6k were chosen for the broth dilution method to determine their MBCs.

For the broth dilution method, the microbial cultures (adjusted to 0.5 McFarland) were prepared as described previously for the disc diffusion method. The test compounds were dissolved in DMSO (10 mg mL^-1) and subject to a 50% serial dilution in 1 mL Eppendorf tubes with Mueller- Hinton Broth (MHB), inoculated with bacterial cultures (20 µL) and then incubated at 37°C for 18 h. The total volume in each Eppendorf was 200 µL. A volume of 10 µL of each dilution was spotted on MHA plates and incubated at 37°C for 18 h to determine the MBC (µM). Ampicillin, ciprofloxacin and tioconazole served as the standard drugs for the antimicrobial and antifungal studies respectively. All experiments were performed in duplicate.

6.2.3 DPPH radical scavenging activity (in vitro antioxidant activity)

The total free radical scavenging activity of the tested compounds was determined and compared to that of ascorbic acid using a slightly modified method described by Tuba and Gulcin (2008). A 0.3 mM solution of DPPH was prepared in methanol and 500 µL added to 50 µL of the compounds (dissolved in DMSO) at different concentrations (50-200 µg mL^-1). These solutions were mixed and incubated in the dark for 30 min at room temperature. Absorbance was then measured at 517 nm against a blank sample lacking scavenger.

6.2.4 Antidiabetic activity

In vitro α-glucosidase inhibitory activity

The α-glucosidase inhibitory activity was determined according to the method described by Ademiluyi et al. (2013) with slight modifications. Briefly, 50 µL of each compound or acarbose dissolved in DMSO at different concentrations (50-200 µg mL^-1), was incubated with 100 µL of 1.0 U mL^–1 α-glucosidase solution in 100 mM phosphate buffer (pH 6.8) at 37 °C for 15 min.

Thereafter, 50 µL of pNPG solution (5 mM) in 100 mM phosphate buffer (pH 6.8) was added and the mixture incubated at 37 °C for 20 min. The absorbance of the released p-nitrophenol was measured at 405 nm and the inhibitory activity expressed as a percentage of the control sample without inhibitors.

In vitro α-amylase inhibitory activity

The α-amylase inhibitory activity was determined according to the method described by Shai et al. (2010) with slight modifications. A volume of 50 µL of each compound dissolved in DMSO or acarbose at different concentrations (50-200 µg mL^-1) was incubated with 100 µL of porcine pancreatic amylase (2 U mL^–1 ) in 100 mM phosphate buffer (pH 6.8) at 37 °C for 20 min. 50 µL of 1 % starch dissolved in 100 mM phosphate buffer (pH 6.8) was then added to the reaction mixture and incubated at 37 °C for 1 h. 100 µL of DNS colour reagent was then added and boiled for 10 min. The absorbance of the resulting mixture was measured at 540 nm and the inhibitory activity expressed as a percentage of the control sample without inhibitors. All assays were carried out in triplicate.