Design, synthesis and evaluation of biological activities of some novel anti-TB agents with bio-reducible functional group

Materials

All solvents and reagents were purchased from Merck (Merck Co., Germany) without any more purification except eugenol oxide that was synthesized from eugenol using the general procedure reviewed by Fieser and Fieser.²⁴ EDC.HCL was prepared from Sigma (Sigma-Aldrich Co., Germany). Dimethyl sulfoxide (DMSO) and 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazoliumbromide (MTT) were bought from Sigma (Sigma-Aldrich Co., Germany). The K562 cell line was obtained from the Pasteur Institute (Tehran, Iran). The cell culture medium (RPMI 1640) and penicillin/streptomycin were purchased from Gibco (Life Technologies, Paisley, Scotland). The culture plates were supplied from SPL (Seoul, South Korea). Mtb (H37Rv strain) ATCC (No.27294) was obtained from the National Reference Center (NRC) in Borstel, Germany. 7H9 medium (cat# 271310) was obtained from Becton Dickinson (BD Difco; Becton Dickinson). Rifampicin was obtained from the National Reference Center (NRC) in Borstel, Germany. Staphylococcus aureus (PTCC 1112), Escherichia coli (PTCC 1047), and Candidakefyr (ATCC 38296) were provided by the laboratory of Microbiology (Department of Biology, Faculty of Natural Science, University of Tabriz, Tabriz, Iran).

Instrumental Measurement

Melting points were recorded using the melting- point meter (Electro thermal 9100, Staffordshire, UK). IR spectra were performed on FT-IR spectrometer (FT IR-8101M, SHIMADZU, Kyoto, Japan). ¹H NMR and ¹³CNMR spectra were recorded with a Bruker SpectrospinAvance 400 spectrometer operating at 400 MHz (Bucker GmbH, Ettlingen, Germany) and chemical shifts were measured in ppm relative tetramethylsilane. Elemental analyses were conducted with Vario EL ΙΙΙ apparatus (Elementar Co, Langenselbold, Germany). The reactions and purities of the compounds were monitored by TLC on silica gel 60 F₂₅₄ aluminum sheets purchased from Merck (Darmstadt, Germany). The TLC plates visualized using UV light (254 and 366nm, CAMAG, Muttenz, Switzerland). Column chromatography was carried out on silica gel 60 F254 glass plates (Merck, Darmstadt, Germany, 60A, 70-230 mesh). Continuously live cell proliferation, morphology and viability with label free assay were performed using xCELLigence Real Time cell analysis instrument (ACEA Bioscience, San Diego, CA, USA). The absorbance was measured for MTT assay by using a Bio-Tek Cytation 3 Cell Imaging Multi-Mode Reader (Elx 800 Microplate Reader, Quant Bio-tek Instruments, Winooski, VT, 195 USA).

Optimized procedure for preparation of 2-pyrazinoicacid esters

DMAP (0.3 mmol) was added to a swirling mixture of 2-pyrazin carboxylic acid (1 mmol) in dichloromethane (2 mL) and stirred for 10 minutes, then alcohol compound (0.7 mmol) was added and resulting solution was cooled to 0^oC, after that EDC.HCl (0.9 mmol) was added gradually to cool the mixture. After 30 minutes, cooling source was removed and the reaction mixture was stirred for further 4 hours at room temperature, and the proceeding of the reaction was followed by thin layer chromatography (TLC) (Ethyl acetate (ETAC)/n-hexane 1:3). Finally, the mixture was rinsed with saturated sodium bicarbonate and 0.05 N HCl solution, respectively, dried over MgSO₄, and the solvent was evaporated to achieve the POE compounds. Some of the obtained solid esters including 3a, 3b, 3c, 3f, and 3i were more purified by recrystallization from appropriate solvents mentioned below,²⁵ and two other compounds (3e, 3g) were purified on silica gel column chromatography. The resultant products are described below(for more details see supplementary section):

2-methoxyphenyl pyrazine-2-carboxylate (3a):Recrystalized from (ETAC/n-hexane; 1:3). Transparent crystal (80%), mp 65-68^°C. ¹HNMR(400 MHz, CDCl3): 3.86 (3H, s), 7.02

(2H, m), 7.21 (1H, dd, j= 8, 1.6 Hz), 7.27 (1H, td, j= 8, 1.6 Hz), 8.81 (1H, m), 8.83 (1H, d, j= 2.2 Hz), 9.47 (1H, d, j= 0.65 Hz). ¹³CNMR(400 MHz, CDCl3): 54.76, 111.39, 119.72, 121.51, 126.40, 138.29, 141.82, 143.56, 145.73, 146.93, 149.81, 160.89. FT-IR(KBr, cm^-1): 3068, 2940, 1747, 1611, 1508, 1303, 1264, 1205, 1051, 1025, 777, 760. C₁₂H₁₀N₂O₃ requires; C, 62.60; N, 12.17; H, 4.38. Found: C, 62.72; N, 12.29; H, 4.25.

4-formyl-2-methoxyphenyl pyrazine-2-carboxylate (3b):Recrystalized from (ETAC/n-hexane; 1:3) White solid (75%), mp 111-112^°C. ¹HNMR(400 MHz, CDCl3): 3.91 (3H, S) ,7.42 (1H, d, J=8Hz), 7.54-7.57 (2H, m), 8.83-8.84 (1H, m), 8.87 (1H, d, J=2.4 Hz), 9.48 (1H, d, J=1.2 Hz). ¹³CNMR (400 MHz, CDCl3): 55, 109.9, 122.2, 123.5, 134.6, 141.3, 143.3, 143.6, 145.8, 147.2, 150.7, 160.3, 189.8. FT-IR (KBr, cm^-1): 3047, 1772, 1756, 1705, 1611, 1299, 1277.C_{1 3}H₁₀N₂O₄ requires: C, 60.47; N, 10.85; H, 3.90. Found: C, 60.53; N, 10.63; H, 3.97.

4-formylphenyl pyrazine-2-carboxylate (3c):Recrystalized from (ETAC/n-hexane; 1:3) White solid (80%), mp 134-135^°C. ¹HNMR(400 MHz, CDCl3): 7.48 (2H, d, J=8.4 Hz), 8.01 (2H, m), 8.84 (1H, m), 8.89 (1H, d, J=2.4 Hz), 9.49 (1H, d, J=1.2 Hz). ¹³CNMR (400 MHz, CDCl3): 121.34, 130.39, 133.55, 141.45, 143.72, 145.89, 147.45, 153.96, 160.95, 189,74. FT-IR (KBr, cm^-1): 3068, 2854, 2769, 1760, 1705, 1611, 1316, 1281, 1222, 1166, 1123, 1029, 884, 863, 824, 773. C₁₂H₈N₂O₃ requires: C, 63.16; N, 12.28; H, 3.53. Found: C, 63.26; N, 12.35; H, 338.

5-allyl-2-methoxyphenyl pyrazine-2-carboxylate (3d): White solid (95%), mp 97-99^°C.

¹ HNMR(400 MHz, CDCl3): 3.42 (2H, d, J= 6.8 Hz), 3.81 (3H, s), 5.13 (2H, m), 5.99 (1H, m), 6.84 (2H, m), 7.12 (1H, d, 8 Hz), 8.81 (1H, m), 8.83 (1H, d, J= 2.4 Hz), 8.47 (1H, s). ¹³CNMR(400 MHz, CDCl3): 39.09, 45.80, 111.79, 115.28, 119.21, 121.27, 135.88, 136.63, 138.65, 142, 143.58, 145.81, 146.93, 149.67, 161.08. FT-IR(KBr, cm^-1): 3089, 2940, 1764, 1650, 1615, 1517, 1307, 1286, 1277, 1205, 1132, 1038, 1025,927. C₁₅H₁₄N₂O₃ Requires: C, 66.66; N, 10.36; H, 5.22. Found: 66.49; N, 10.14; H, 5.29.

2-methoxy-5-(oxiran-2-ylmethyl)phenylpyrazine-2-carboxylate (3e): Purified with column chromatography (ETAC/n-hexane, 1:3) low melting tan solid (65%).¹HNMR(400 MHz, CDCl3): 2.58 (1H, dd, J= 4.8, 2.8-2.6 Hz), 2.84 (1H, dd, J= 5.8, 4.8 Hz), 2.88-2.90 (2H, m), 3.18-3.20 (1H, m), 3.83 (3H, s), 6.88-6.91 (1H, dd, J= 8, 1.6 Hz), 6.94 (1H, d, J=1.6 Hz), 7.15 (1H, d, J=8 Hz), 8.81 (1H, dd, J= 2.4, 1.6 Hz), 8.84 (1H, d, J= 2.4 Hz), 9.47 (1H, d, J= 1.2 Hz). ¹³CNMR (400 MHz, CDCl3): 37.68, 45.81, 51.26, 54.87, 112.24, 120.13, 121.46, 135.91, 137.11, 141.92, 143.61, 145.83, 146,99, 149.76, 161.04. FT-IR(KBr, cm^-1): 3068, 2940, 1764, 1615, 1521, 1303, 1277, 1205, 1106, 1038, 1025, 978, 957, 940, 871, 841, 798, 779, 777, 760, 734. C₁₅H₁₄N₂O₄ requires: C, 62.93; N, 9.79; H, 4.93. Found: C, 62.88; N, 9.71; H, 4.99.

2-acetylphenyl pyrazine-2-carboxylate (3f):Recrystalized from (ETAC/n-hexane; 1:4) Off-White solid (70%), mp 62-63^°C. ¹HNMR (400 MHz, CDCl3): 2.58 (3H, S), 7.3 (1H, dd, J= 8.4, 1.2 Hz), 7.4 (1H, td, J=8, 1.2 Hz), 7.64 (1H, td, J= 8, 1,6 Hz),7.92 (1H, dd, J=8, 1.6 Hz), 8.82 (1H, dd, J=2.4, 1.6 Hz), 8.86 (1H, d, J=2.4 Hz), 9.48 (1H, d, J=1.2 Hz). ¹³CNMR(400 MHz, CDCl3): 28.13, 122.87, 125,72, 128.85, 129.82, 132.89, 141.87, 143.61, 145.96, 147.12, 147.87, 161.67, 196.15. FT-IR (KBr, cm^-1): 3089, 2940, 2876, 1756, 1699, 1611, 1290, 1264, 1145, 1120, 1055, 1029, 769, 751. C₂₁H₁₈N₂O₅ requires_: C, 66.66; H, 4.79; N, 7.40. Found: C, 66.73; H, 4.84; N, 7.29.

Cinnamyl pyrazine-2-carboxylate (3g):Purified with column chromatography (ETAC/n-hexane; 1:3), yellow solid (75%), mp 49-52^°C. ¹HNMR(400 MHz, CDCl3): 5.10 (2H, dd, j= 6.73, 1.1 Hz), 6.44 (1H, dt, j= 15.85, 6.73 Hz), 6.79 (1H, d, j= 15.85 Hz), 7.26-7.43 (5H, m), 8.78 (1H, d, j= 2.4), 8.74 (1H, m), 9.36 (1H, d, j= 1.4 Hz).¹³CNMR(400 MHz, CDCl3): 65.87, 121.01, 125.72, 127.31, 127.61, 134.79, 134.88, 142.42, 143.42, 145.35, 146.70, 162.74. FT-IR(KBr, cm^-1): 3068, 3047, 2980, 1726, 1307, 1286, 1140, 1055, 1025, 974, 948, 781, 756, 700. C₁₄H₁₂N₂O₂ requires_; C, 69.99; N, 11.66; H, 5.03. Found: C, 69.92; N, 11.50; H, 5.11.

2-oxo-2H-chromen-6-yl pyrazine-2-carboxylate (3h):White precipitate (87%), mp 192-194^°C.¹HNMR(400 MHz, CDCl3): 6.46 (1H, d, J= 9.56 Hz), 7.23-7.27 (2H, m), 7.59 (1H, d, J= 8.8 Hz), 7.75 (1H, d, J= 9.6 Hz), 8.84 (1H, m), 8.89 (1H, d, J= 2.4 Hz), 9.48 (1H, d, J= 1.2 Hz). ¹³CNMR (400 MHz, CDCl3): 09.54, 115.54, 116.24, 117.23, 127, 141.30, 141.73, 143.73, 145,95, 147.53, 151.73, 153.70, 159.12, 161. FT-IR(KBr, cm^-1): 3068, 3008, 1735, 1709, 1620, 1405, 1307, 1300, 1273, 1234, 1157, 1132, 1098, 1042, 1021, 991, 888, 845, 769, 615. C₁₄H₈N₂O₄ requires_: C, 62.69; N, 10.44; H, 3.01. Found: C, 62.79; N, 10.28; H, 2.91.

Benzhydryl pyrazine-2-carboxylate (3i): Recrystalized from (ETAC/n-hexane; 1: 3) White solid (70%), mp 89-90^°C. ¹HNMR(400 MHz, CDCl3): 7.235 (1H, m), 7.31 (2H, m), 7.37 (4H, m), 7.46 (4H, m), 8.76 (2H, m), 9.38 (1H, s). ¹³CNMR (400 MHz, CDCl3): 77.63, 126.26, 127.25, 127.65, 138.39, 142.65, 143,65, 145,32, 146.64, 161,99. FT-IR(KBr, cm^-1): 3089, 2961, 1735, 1330, 1303, 1273, 1136, 1055, 1025, 969. C₁₈H₁₄N₂O₂ requires: C, 74.47; N, 9.65; H, 4.86. Found: C, 74.40; N, 9.58; H, 4.89.

2-oxo-1,2-diphenylethyl pyrazine-2-carboxylate (3j):White solid (95%), mp 108^°C. ¹HNMR(400 MHz, CDCl3): 7.20 (1H, S), 7.37 (5H, m), 7.52 (1H, m), 7.59 (2H, m), 7.97 (2H, m), 8.75 (2H, m), 9.38 (1H, s). ¹³CNMR(400 MHz, CDCl3): 78, 127.62, 127.77, 127.84, 128.24, 128.6, 131.83, 132.6, 133.15, 141.88, 143.58, 145.44, 146.74, 162.19, 191.52. FT-IR (KBr, cm^-1): 3089, 1752, 1730, 1696, 1602, 1465, 1307, 1294, 1157, 1046, 1025, 961, 858, 764, 709, 704, 602. C₁₉H₁₄N₂O₃ requires_: C, 71.69; N, 8.80; H, 4.43. Found: C, 71.63; N, 8.65; H, 4.49.

(5-(benzyloxy)-4-oxo-4H-pyran-2-yl)methylpyrazine-2-carboxylate (3k):White crystalline powder, Yield (74%), mp: 133-135 ºC. ¹HNMR (400 MHz, CDCl3): 5.07 (2H, s), 5.23 (2H, S), 6.57 (1H, s), 7.35 (5H, m), 7.56 (1H, s), 8.76 (1H, m), 8.829 (1H, d, j= 2.4 Hz), 9.34 (1H, d, j= 1.2 Hz); ¹³CNMR (400 MHz, CDCl3): 61.42, 70.6, 113.89, 126.69, 127.46, 127.71, 134.40, 140.29, 140.40, 143.6, 145.51, 146.42, 147.31, 159.23, 162.35, 173.15.IR (KBr, cm^-1): 3111, 3068, 1735 (C = O_ester), 1658 (C = O_pyrone), 1632 (C = C_pyrone), 1611, 1320, 1273, 1317, 1166, 1140, 1055, 1025, 965, 863. C₁₈H₁₄N₂O₅ requires_: C, 63.90; N, 8.28; H, 4.17. Found: C, 64.05; N, 8.40; H, 4.07.

6-methyl-4-oxo-4H-pyran-3-yl pyrazine-2-carboxylate (3l): White solid (80%), mp 119-120 ⁰C. ¹HNMR (400 MHz, CDCl3): 2.29 (3H, s), 6.39 (1H, d, J= 5.8 Hz), 7.7 (1H, d, J= 5.8Hz), 8.735 (1H, m), 8.78 (1H, d, J= 2.4HZ), 9.37 (1H, d, J= 1.2Hz); ¹³CNMR (400 MHz, CDCl3): 13.99, 115.68, 137.57, 140.85, 143.55, 145.87, 147.26, 153.40, 158.20, 159.59, 170.02.(KBr, cm⁻¹): 3111, 3068, 1752 (C = O_ester), 1662 (C = O_pyrone), 1640 (C = C_pyrone), 1585, 1435, 1303, 1260, 1175, 1102, 1042, 1025, 931, 863, 841, 777. C₁₁H₈N₂O₄ requires_: C, 56.90; N, 12.06; H, 3.47. Found: C, 56.83; N, 12.13; H, 3.40.

Prop-2-ynyl pyrazine-2-carboxylate (3m): White solid (85%), mp 79-80^°C. ¹HNMR(400 MHz, CDCl3): 2.58 (1H, t, J= 2.4 Hz), 5.05 (2H, d, J= 2.4Hz), 8.76 (1H, m), 8.81 (1H, d, J= 2.4Hz), 9.36 (1H, d, J= 1.2 Hz). ¹³CNMR(400 MHz, CDCl3): 52.50, 74.92, 141.76, 143.50, 145.40, 146.97, 162.12. (One of the propargyl carbons eclipsed by that of CDCl₃). FT-IR(KBr, cm^-1): 3065, 2918, 2854, 2106, 1730, 1303, 1136, 1051, 1025, 919, 880, 777, 726, 563, 448. C₈H₆N₂O₂ requires_: C, 59.26; N, 17.28; H, 3.73. Found: C, 59.19; N, 17.12; H, 3.78.

2-methoxyethyl pyrazine-2-carboxylate (3n): Colorless oil (86%). ¹HNMR(400 MHz, CDCl3): 3.49 (3H, s), 3.78 (2H, d, J= 4.8), 4.61 (2H, d, J= 4.8), 8.75 (1H, m), 8.78 (1H, d, J= 2.4), 9.34 (1H, s). ¹³CNMR(400 MHz, CDCl3): 57.94, 63.96, 69.02, 142.24, 143.40, 145.27, 146.60, 162.81. FT-IR(KBr, cm^-1): 3082, 2284, 1735, 1311, 1290, 1153, 1122, 1059, 1025, 781. C₈H₁₀N₂O₃ requires_: C, 52.74; N, 15.38; H, 5.53. Found: C, 52.64; N, 15.15; H, 5.65.

Determination of the anti-TB activity of small-molecules using GFP-expressing M. tuberculosis bacteria

Compounds preparation and dilution : 7H9 (45 mL) (without glycerol and without Tween 80) was supplemented with 5 mL OADC. Starting from a stock solution of 10 mg/mL, 1 μL of each compound was diluted with 800 μL 7H9 test medium in a 1.5 mL Eppendorf Cap. Then, 80 μL of each concentration was added in triplicates to a black 96-well plate with a clear bottom (Corning) and the plates were transferred into the BSL3 facility.

Bacteria : The numbers of aliquots of Mtb bacteria needed were thawed in a heating block at 37°C. The caps were centrifuged for 10 minutes at 4500 rpm (Heraeus, Swingout rotor). The supernatant was discarded and the bacteria were resuspended in 7H9 test medium to reach a concentration of 6 x 10e6/20 μL. Afterward, 20 μL of the bacterial suspension was added to the wells containing 80 μL well in the absence (Ctrl) or presence of the compounds and sealed with an air-permeable membrane (Porvair Sciences) at culture under mild agitation (Heidolph) at 37°C in an incubator. Plates were not stacked. The plate was measured at days 0, 3, and 7. Each plate was prepared with rifampicin as reference compound (dilutions in water) which has known inhibitory activity against M. tuberculosis . Test compounds were diluted in 100% DMSO at a constant concentration of 10 μg/mL. Rifampicin was tested in dose-response for quality control purposes at 1 μg/mL and 0.1 μg/mL. The assay was carried out in 96-well flat bottom microplates in a final volume of 100 μL. Next, 20 μL of the prepared bacterial working solution (s. above) was added to 80 mL of the compound test plate containing 10 μg/mL of the compound to be tested. The plates were incubated at 37°C for 7 days. Finally, bacterial growth was determined by measuring the relative fluorescence intensity using the plate reader Synergy2 (Biotek).

Measurement of in vitro cytotoxic activity on human macrophage cells usingxCELLigenceRTCA

The in vitro cytotoxic effect of the POE compounds was evaluated on the human macrophages using the xCELLigence RTCA system as previously described.²⁷

Antimicrobial activity assay

The MIC (minimum inhibitory concentration) values of the POEs were determined by the two-fold microdilution method. The test POEs (10 mg) were dissolved in DMSO (0.1 mL) to get a stock solution of 100 μg/μL of each compound. Further progressive double dilution with Muller- Hinton broth for bacteria and Sabouraud dextrose broth for fungi were performed to obtain the required concentrations of 1000, 500, 250, 125, 62.5, 32, 16, 8, 4, and 2 μg/mL. To ensure that the solvent had no effect on bacterial growth, a control test was performed with test medium supplemented with DMSO at the same dilutions as used in the experiment. Gentamicin and nystatin were also diluted in the same manner as control for antibacterial and antifungal tests, respectively. Each microwell was inoculated with overnight cultures of test microorganisms to get inoculums with the size of 5 × 10⁵ colony-forming unit(CFU)/mL. After 18-24 hours incubation at 35˚C, the growth was surveyed by turbidity in microwells. The first concentrations of compounds, which inhibited the growth of microorganisms, were considered as MIC.

MTT assay

MTT assay was used to investigate the cytotoxicity of the POE compounds. The K562 cells (2 × 10⁴ cells/well) were cultivated in 96-well plates in the presence of different concentrations of the POEs (25-150 µM) for 24, 48, and 72 hours. The ultimate volume was 200 μL. After that, 20 μL of MTT (5 mg/mL in PBS) was poured into every well and then further incubated for 4 hours at 37°C. The precipitated MTT formazan was dissolved in DMSO (200 μL). Ultimately, the absorbance values were measured at 570 nm on a multi-well plate reader (Quant Bio-tek Instruments, Winooski, VT, 195 USA). Untreated cells and DMSO treated cells (also Doxorubicin) were used as negative and positive controls, respectively.

Chemistry

Cynamon et al synthesized some POEs via acyl intermediate. The necessity for purification of sensitive pyrazinoyl chloride due to side reactions between thionyl chloride and POA was the most drawback of this method.¹⁶ Later synthesis of POEs was reported by using dicyclohexylcarbodiimide (DCC).²⁸ Despite the effectiveness of this coupling reagent, it is hard to remove the by-product dicyclohexylurea (DCU) as well as rearrangement product N -acylurea.²⁹ This obstacle inspired us to use a more effective reagent for the synthesis of new POEs having sensitive functional groups using EDC.HCl (N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride)/DMAP as a nontoxic, water-soluble coupling reagent that prevailed over formerly mentioned disadvantages (Table 1).

Table 1.Reaction of POA with various alcohols and structures of resultant POEs
		3a
		3b
		3c
		3d
		3e
		3f
		3g
		3h
		3j
		3k
		3l
		3m
		3n

Reaction initiates with the nucleophilic attack of pyrazinoate2, on the diimide carbon of EDC.HCl to form O -acylisourea intermediate 4as a highly reactive intermediate. In the next step, reaction proceeds viaO -acylisourea resulting from the activation of POA by EDC through nucleophilic attack of alcohol on this intermediate (path a) that competes with intramolecular acyl rearrangement resulting in the formation of inert N -acylurea 5 as a byproduct (Path b). Furthermore, O -acylisourea may be attacked by POA to produce anhydride derivative of POA (Scheme 1).²⁹

Scheme 1.

Formation pathways of POE andN -acylurea resulted from the reaction of O -acylisourea intermediate.

Anti-mycobacterial activity

Cell-based assays are key tools in the finding and optimization of new chemical entities against Mtb. The availability of a robust in vitro assay for testing the anti-mycobacterial activity of a new chemical entity is an absolute requirement for the success of a program. The microplate broth dilution assay using a M. tuberculosis strain expressing the green-fluorescent protein (GFP) was selected because of delivering highly reproducible results and allowing screening of a large number of compounds.

The compounds were assayed against slowly growing M. tuberculosis (H37Rv strain) at 10 μg/mL concentration. Compound 3ashowed no inhibitory effect on mycobacterial growth, but compound 3b as a vanillin analog reduced mycobacterial growth by 21.7%. Compound3c having side aldehyde group showed higher activity with 45.42% inhibition. In the presence of compound3dand its epoxidized derivative3e,mycobacterial growth was interrupted by 27.9% and 33.5%, respectively. It was notable that replacing double bond with more active epoxide ring in eugenol pyrazinoate3d made it stronger against Mtb. Compound3f as a ketone compound showed no effect on mycobacterial growth. Between compounds 3g and 3h as cynamyl alcohol and coumarin derivatives, the coumarin-derived compound having internal ester moiety showed to be much more active reagent than the cynamyl alcohol-derived one against Mtb as shown by growth inhibition of 9.83% and 22.5%, respectively. Compound 3jcontaining an alpha-hydroxy carbonyl group was able to limit mycobacterial growth by 45.7% which showed a higher activity against mycobacteria than the compound 3i- the analog without possessing carbonyl group, which showed 41% inhibition. Between hydroxypyrone derivatives, due to having α,β-unsaturated carbonyl (enone) moiety, compound 3lshowed higher anti-mycobacterial activity, reducing Mtb growth value by 56% while compound 3k led to an inhibition of 22.4%. Between compounds 3m and 3n, propargyl-derived pyrazinoate 3mshowed greater anti-mycobaterial activity, preventing mycobacterial growth by 51.2% than compound 3n with 31.7% inhibitory effect (Fig. 1, Table 2).

Fig. 1

Mtb growth (H37Rv) subjected to POEs (10 μg/mL) after 7 days using 7H9 (without glycerol/Tween 80) supplemented with 10% OADC.

Note. Untr.: untreated, RIFA: rifampicin.

In vitro cytotoxic activity

The xCELLigence real-time cell analysis (RTCA) was utilized to evaluate the cytotoxicity of the compounds on macrophages. The xCELLigence system is able to monitor the cultured cell viability in a safe way without any invasion, employing unique gold microelectrodes. Electrical impedance output as cell index (CI) value is evaluated to gain notable information about viability, cell number, cell proliferation status, and morphology.²⁷ In Fig. 2, the analysis of putative cytotoxic effects of the compounds 3cand 3l on human macrophages is shown. Prolonged incubation of macrophages with these two compounds for 47 hours, in contrast to staurosporine as a positive control, did not lead to a substantial decrease in normalized cell index values as an indicator of macrophage cell attachment. These data suggest that the tested POEs do not show a remarkable cytotoxic effect on macrophage cell adhesion and survival if present in a concentration range up to 10 μg.

Fig. 2

(A) Compounds 3c and 3l do not affect human macrophage viability. Over a time period of 47 hours the cellular viability of human macrophage (40.000 cells/well) expressed as cell index was determined by real-time impedance measurement using the xCELLigence system (RTCA, SPACEA). After 3.5 hours of equilibration, the compounds were added at the indicated concentrations (normalization time point), (Staurosporine, 1 μg/mL). (B) Normalized cell index depicted as area under the curve was calculated using RTCA 2.0 software.

Antibacterial assay

PZA possessing a narrow spectrum of activity against Mtb bacteria has no notable bactericidal activity against non-tuberculous as well as gram-positive and gram-negative bacteria. In contrast, the evaluation of antimicrobial activity of the POE compounds showed that these compounds had antimicrobial activity against gram-positive bacteria as well as Candidakefyr . The MIC test results showed that the tested compounds showed an improved activity against gram-positive bacteria and remarkable fungicidal activity on C . kefyr . The POEs 3b,3d, 3e, 3i, and 3m including vanillin, eugenol, eugenol epoxide, diphenylcarbinol, and propargyl alcohol in the alkyl part showed a very good antifungal activity against C.kefyr with MIC values of 250±20, 250±20, 250±20, 62.5±5, and 125±10 μg/mL, respectively. This increased fungicidal activity resulted from either POE`s or alkyl part`s activity. It seems the most efficacy of the compound 3i is more likely related to the presence of benzhydryl moiety in its structure, as this structure is an important part in the antifungal medicine clotrimazole. The compounds3a,guaiacol-derived ester, and 3e, the oxidized-eugenol one,had a remarkable activity against S . aureus with a MIC value of 500±50 μg/mL. The compound 3b exhibited very good bactericidal activity with MIC value of 125±10 μg/mL, respectively. Yeast and gram-positive bacteria are surrounded by a thick layer of polysaccharides but without outer membrane containing lipopolysaccharide found in the gram-negative bacterial cell wall. Therefore, the compounds can be more easily absorbed through S. aureus and C.kefyr cell walls, but cannot penetrate the lipid layer of E. coli envelope. The eugenol epoxide-derived POE (3e) presented a moderate antibacterial activity on E . coli as gram-negative bacteria with MIC value of 2000±200 μg/mL, although the compound 3d, simple analog without epoxide moiety, showed a weak bactericidal activity with MIC value of 4000±400 μg/mL. The rest of the compounds exhibited no great activity against E . coli except for compound 3l that showed intermediate activity with MIC value of 1000±100 μg/mL. This observation is notable because this is the only POE that shows greater bactericidal activity on gram-negative than gram-positive bacteria with a MIC value of 2000 μg/mL. This peculiar activity is probably related to the maltol ability to bind divalent cations located on the E . coli ’s outer membrane that results in the increasing permeability of E . coli to the ester (Table 3).³⁰

Anticancer potency

One of the prevalent ways to assess the in vitro anticancer potency of the synthesized compounds is to evaluate the cytotoxicity in terms of viability in cancer cell by using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The K562 cells were cultured as supplemented with various concentrations (25-150 µM) of the POEs for 3 days to measure the value of each POE`s inhibitory effect on the viability of the cells by MTT assay. The percentages of viability and IC₅₀ (inhibition of concentration 50) values for all compounds at various concentrations are depicted in Table 4. As shown in Table 4, cell viability was reduced in a time and dose dependent manner for all compounds. Among tested POEs,3e and 3j as eugenol oxide and benzoin pyrazinoate, showed favorable cytotoxicity on the K562 cell lines having IC₅₀ values of 25±5.0 and 25±8.0 µM after 72 hours. As understood from Table 4, substitution of double bond located on eugenol ester3d by epoxide motif sharply affected the cytotoxic efficacy and decreased IC₅₀ value by 75 µM. The rest of the compounds had IC50 values about 100 µM or more.