Synthesis and spectra dataSynthesis of 4-bromo-2-hydroxy-N-(5-(1,3,4,5-tetrahydroxycyclohexyl)-1,3,4-thiadiazole-2-yl) benzamide (5a)

Yield 68% (Chloroform, DMSO & Ethanol);m.p (174–175 °C), Rf Value:0.91; IR (KBr) Cm−1: OH (3350), C–Br (655), C = O (1680), Ar (1625–1590), NH (3140). 1H NMR (500 MHz, DMSO) δ ppm: 7.46 (dd, 1H aromatic), 7.16 (dd, 2H aromatic), 5.38(s, 1H aromatic OH), 3.67 (m, 2H aliphatic), 3.32 (s, 1H aliphatic OH), 3.19 (s, 1H aliphatic OH), 2.39 (m, 2H aliphatic), 2.05 (m, 2H aliphatic), 1.80 (m, 2H aliphatic), 1.43 (s, 1H aliphatic OH), 0.99 (s, 1H aliphatic OH).13C NMR (126 MHz, DMSO) δ ppm: 170.31(1C, s), 157.0 (1C, s); m/z: 444.99; Anal. Calcd for C15H16BrN3O6S: C, 40.37; H, 3.61; Br, 17.90; N, 9.42; O, 21.51; S, 7.18 (Table 4).

Table 4 Protox predictionSynthesis of 5-bromo-2-hydroxy-N-[5-(1,3,4,5-tetrahydroxycyclohexyl)-1,3,4-thiadiazole-2-yl] benzamide (5b)

Yield 67% (Chloroform, DMSO & Ethanol); m.p (183–186 °C), Rf Value:0.93.IR (KBr) Cm−1: 3811 (Ar-OH), 3446 (NH), 1643 (C–C), 1521 (C = N), 1274 (C-N) 1109(C-S). 1H NMR (500 MHz, DMSO) δ ppm: 7.46 (dd, 1H aromatic), 7.16 (dd, 2H aromatic), 5.38 (s, 1H aromatic OH), 3.67 (m, 2H aliphatic), 3.32 (s, 1H aliphatic OH), 3.19 (s, 1H aliphatic OH), 2.39 (m, 2H aliphatic), 2.05 (m, 2H aliphatic), 1.80 (m, 2H aliphatic), 1.43 (s, 1H aliphatic OH), 0.99 (S, 1H aliphatic OH). 13C NMR (126 MHz, DMSO) δ ppm: 155.82 (1C, s), 154.34(1C,s). m/z: 444.99. Anal. Calcd for C15H16BrN3O6S: C, 40.37; H, 3.61; Br, 17.90; N, 9.42; O, 21.51; S, 7.18.

Synthesis of 4-amino-N-[5-(1,3,4,5-tetrahydroxycyclohexyl)-1,3,4-thiadiazole-2-yl] benzamide (5c)

Yield 71% (Chloroform, DMSO & Ethanol); m.p (182–185 °C), Rf Value:0.89.IR (KBr) Cm−1: 3240–3367 (NH,NH2), 1643 (C = N), 1290 (C = S). 1H NMR (500 MHz, DMSO) δ ppm: 7.59 (m, 2H aromatic), 6.72 (m, 2H aromatic), 5.65 (s, 1H aliphatic), 3.85 (m, 2H aliphatic), 3.67 (m, 2H aliphatic), 3.30 (s, 1H aliphatic OH), 2.34 (m, 2H aliphatic), 2.01 (m, 2H aliphatic), 1.77 (m, 2H aliphatic), 1.31 (s, 1H aliphatic OH), 0.96 (s, 1H aliphatic OH). 13C NMR (126 MHz, DMSO) δ ppm: 168.50 (1C, s), 156.63(1C, s), 137.77(1C, s), 135.17 (1C, s), 128.98(1C, s), 128.09 (1C, s), m/z: 366.10; Anal. Calcd for: C15H18N4O5S: C, 49.17; H, 4.95; N, 15.29; O, 21.83; S, 8.75.

Synthesis of N-[5-(1,3,4,5-tetrahydroxycyclohexyl)-1,3,4-thiadiazole-2-yl] benzamide(5d)

Yield 78% (Chloroform, DMSO & Ethanol); m.p (193–169 °C), Rf Value:0.90.IR (KBr) Cm−1: 3210–3352 (NH),1670 (C = O), 1642 (C = N), 1302 (C = S).1H NMR (500 MHz, DMSO) δ ppm: 7.84–7.43 (m, 5H aromatic), 5.30 (s, 1H aliphatic OH), 3.67 (m, 2H aliphatic), 3.33 (s, 1H aliphatic OH), 2.40 (m, 2H aliphatic), 2.07 (m, 2H aliphatic), 1.50 (m, 2H aliphatic), 1.43 (s, 1H aliphatic OH), 1.13 (s, 1H aliphatic OH). 13C NMR (126 MHz, DMSO) δ ppm: 170.30, 155.81, 154.37, 134.50, 130.00, 129.31(2C, s), 128.03 (2C,s), 127.26(s), 127.03(s), 124.45 (s), 119.01(1C, s), 108.40(1C, s), 64.86(1C, s), 56.41(1C, s). m/z: 351.09. Anal. Calcd for C15H17N3O5S: C, 51.27; H, 4.88; N, 11.96; O, 22.77; S, 9.12.

Synthesis of 2-hydroxy-N-[5-(1,3,4,5-tetrahydroxycyclohexyl)-1,3,4-thiadiazole-2-yl] benzamide (5e)

Yield 69% (Chloroform, DMSO & Ethanol); m.p (169–173 °C), Rf Value:0.90.IR (KBr) Cm−1: 3811 (Ar-OH), 3446 (NH), 1643 (C–C), 1521 (C = N), 1274 (C-N) 1109(C-S). 1H NMR (500 MHz, DMSO) δ ppm: 7.64 (dd, 2H aromatic), 7.32 (dd, 2H aromatic), 6.96 (m, 2H aliphatic), 5.59 (s, 1H, aromatic OH), 3.67 (m, 2H aliphatic), 3.51 (s, 1H aliphatic OH), 3.31 (s, 1H aliphatic OH), 2.36 (m, 2H aliphatic), 2.02 (m, 2H aliphatic), 1.32 (s, 1H aliphatic OH), 0.98 (s, 1H aliphatic OH).13C NMR (126 MHz, DMSO) δ ppm: 170.30, 155.81, 154.37, 134.50, 130.00, 129.31, 128.03, 127.26, 127.03 (1C,s), 124.45(1C,s), 119.01(1C,s), 64.86(1C,s), 56.41(1C,s), 18.07(1C,s). m/z: 367.08. Anal. Calcd for C15H17N4O5S: C, 49.04; H, 4.66; N, 11.44; O, 26.13; S, 8.73.

Synthesis of 4-chloro-N-[5-(1,3,4,5-tetrahydroxycyclohexyl)-1,3,4-thiadiazole-2-yl] benzamide (5f)

Yield 68% (Chloroform, DMSO & Ethanol); m.p (172–175 °C), Rf Value:0.92.IR (KBr) Cm−1: C–Cl(540), SC-(700) Ar(1625),C-O(1260), OH(3350), NH(3140), CH(3079), C = O(1680). 1H NMR (500 MHz, DMSO) δ ppm: 7.72 (dd, 2H aromatic), 7.44 (dd, 2H aromatic), 5.62 (s, 1H aliphatic OH), 3.67 (m, 2H aliphatic), 3.30 (s, 1H aliphatic OH), 2.35 (m, 2H aliphatic), 2.04 (m, 2H aliphatic), 1.48 (m, 2H aliphatic), 1.41 (s, 1H aliphatic OH), 1.10 (s, 1H aliphatic OH).13C NMR (126 MHz, DMSO) δ ppm: 168.50(1C,s), 137.77(1C,s), 135.17 (1C,s), 128.98 (1C,s), 128.09 (1C,s), 126.06 (1C,s), 125.64 (1C,s), 124.94 (1C,s), 123.54 (1C,s), 123.24 (1C,s), 121.60 (1C,s), 65.51 (1C,s), 62.44 (1C,s), 19.18. m/z: 385.05. Anal. Calcd for C15H16ClN3O5S: C, 46.70; H, 4.18; Cl, 9.19; N, 10.89; O, 20.73; S, 8.31.

Synthesis of 4-fluoro-N-[5-(1,3,4,5-tetrahydroxycyclohexyl)-1,3,4-thiadiazole-2-yl] benzamide(5 g)

Yield 68% (Chloroform, DMSO & Ethanol); m.p (179–182 °C), Rf Value:0.87.IR (KBr) Cm−1: c-f(1000), 1372.14 (C–O–C), 1661.54 (C = O), 1450.01(C = N), 3333.43(N = C), 870.28(C–H). 1H NMR (500 MHz, DMSO) δ ppm 7.72 (dd, 2H aromatic), 7.44 (dd, 2H aromatic), 5.62 (s, 1H aliphatic OH), 3.67 (m, 2H aliphatic), 3.30 (s, 1H aliphatic OH), 2.35 (m, 2H aliphatic), 2.04 (m, 2H aliphatic), 1.48 (m, 2H aliphatic), 1.41 (s, 1H aliphatic OH), 1.10 (s, 1H aliphatic OH).13C NMR (126 MHz, DMSO) δ ppm: 168.50(1C,s),, 137.77 (1C,s), 135.17 (1C,s), 128.98(1C,s), 128.09(1C,s), 126.06(1C,s), 125.64(1C,s), 124.94(1C,s), 123.54(1C,s), 123.24(1C,s), 121.60(1C,s), 65.51(1C,s), 62.44(1C,s), 19.18,m/z 369.08: Anal. Calcd for C15H16FN3O5S: C, 48.78; H, 4.37; F, 5.14; N, 11.38; O, 21.66; S, 8.68.

Synthesis of N-[5-(1,3,4,5-tetrahydroxycyclohexyl)-1,3,4-thiadiazole-2-yl] furan-2-carboxamide(5 h)

Yield 76% (Chloroform, DMSO & Ethanol); m.p (166–170 °C), Rf Value:0.91.IR (KBr) Cm−1: 1353.10 (C–O–C), 1652.2 (C = O), 1580.61(C = N), 3452.03 (N = C imine), 8464.39 (C–H).1H NMR (500 MHz, DMSO) δ ppm:): 7.43 (s, 1H aromatic), 6.93 (dd, 2H aromatic), 6.41 (m, 2H aliphatic), 5.55 (s, 1H aliphatic OH), 3.67 (m, 2H aliphatic), 3.32 (s, 1H aliphatic OH), 2.38 (m, 2H aliphatic), 2.07 (m, 2H aliphatic), 1.43 (s, 1H aliphatic OH), 1.14 (s, 1H aliphatic OH). 13C NMR (126 MHz, DMSO) δ ppm: 170.33(1C,s), 155.18 (1C,s), 154.37 (1C,s), 134.50 (1C,s), 130.00 (1C,s), 128.03 (1C,s), 127.36 (1C,s), 126.03 (1C,s), 124.45 (1C,s), 64.36 (1C,s). m/z: 341.07. Anal. Calcd for C13H15N3O6S: C, 45.74; H, 4.43; N, 12.31; O, 28.12; S, 9.39.

Synthesis of N-[5-(1,3,4,5-tetrahydroxycyclohexyl)-1,3,4-thiadiazole-2-yl]-1H-pyrrole-2-carboxamide (5i)

Yield 76% (Chloroform, DMSO & Ethanol); m.p (183–185 °C), Rf Value:0.89.IR (KBr) Cm−1: 3388 (NH), 2752 (Ar-CH3), 1525 (C = N), 1259 (C-N), 1118(C-S) 1H NMR (500 MHz, DMSO) δ ppm: 7.52 (s, 1H, NH), 6.89 (dd, 3H aromatic), 6.32 (m, 2H aliphatic), 5.36 (s, 1H aliphatic OH), 3.67 (m, 2H aliphatic), 3.32 (s, 1H aliphatic OH), 2.39 (m, 2H aliphatic), 2.07 (m, 2H aliphatic), 1.43 (s, 1H aliphatic OH), 1.14 (s, 1H aliphatic OH). 13C NMR (126 MHz, DMSO) δ ppm: 168.50(1C,s), 156.63 (1C,s), 148.92 (1C,s), 148.40 (1C,s), 125.65(1C,s), 122.36(1C,s), 121.60(1C,s), 109.59(1C,s), 109.19(1C,s), 107.64(1C,s), 106.93(1C,s), 106.34(1C,s), 103.91(1C,s), 102.56(1C,s), 102.06(1C,s), 64.39(1C,s), 26.57(1C,s). m/z: 340.08. Anal. Calcd for C13H16N4O5S: C, 45.88; H, 4.74; N, 16.46; O, 23.50; S, 9.42.

Synthesis of 2-phenyl-N-[5-(1,3,4,5-tetrahydroxycyclohexyl)-1,3,4-thiadiazole-2-yl] acetamide (5j)

Yield 71% (Chloroform, DMSO & Ethanol); m.p (171–173 °C), Rf Value:0.91.IR (KBr) Cm−1: 3392 (NH), 1629 (C–C), 1525 (C = N), 1284 (C-N), 1103(C-S).1H NMR (500 MHz, DMSO) δ ppm: 7.45–7.34 (m,4H aromatic), 7.31–7.21 (m, 3H aromatic), 6.13 (s, 1H aliphatic OH), 5.28 (s, 1H aliphatic OH), 3.82–3.78 (m, 1H aliphatic OH), 3.69–3.65 (m, 1H aliphatic OH), 2.52–2.30 (m, 2H aliphatic), 2.15–1.94 (m, 2H aliphatic), 1.26 (s, 3H aliphatic), 1.10–1.06 (m, 1H aliphatic NH). 13C NMR (126 MHz, DMSO) δ ppm: 168.21(1C,s), 148.30(1C,s), 130.92(1C,s), 127.26(1C,s), 125.54(1C,s), 123.83(1C,s), 123.01(1C,s), 121.82(1C,s), 83.18(1C,s), 75.20(1C,s). m/z: 365.10. Anal. Calcd for C16H19N3O5S: C, 52.59; H, 5.24; N, 11.50; O, 21.89; S, 8.77.

Cytotoxic evaluation

The half minimum inhibitory concentration or IC50 value is a metric used to assess how effectively a substance inhibits a particular biological or biochemical process. In vitro studies indicate the minimum inhibitory concentration needed to achieve 50% inhibition. Using the SRB assay, the IC50 values for the synthesized compounds were ascertained. Table 5 provides the results of the anticancer assessment together with the IC50 values. Doxorubicin, a well-known anticancer medication, served as a positive control. According to the results, compound 5 g (benzaldehyde replaced) significantly inhibited proliferation against MCF-7 cell lines when compared to the standard [10]. IC50 values of 7.64 µg/ml, 12.01 µg/ml, 12.71 µg/ml, 15.66 µg/ml, and 17.29 µg/ml were reported for compounds 5 g, 5 h, 5b, 5e, and 5i, which were replaced with 4-fluorobenzoic acid, furan-2-carboxylic acid, 5-bromo-2-hydroxybenzoic acid, 2-hydroxybenzoic acid, and 1H-pyrrole-2-carboxylic acid, respectively. The synthetic compound’s cell viability against the seven MCF cell lines is shown in Fig. 6.

Table 5 In vitro evaluation of designed analogs against MCF-7 cell linesFig. 6

Cell viability of the synthesized analogs against MCF–7 cell lines

Acute oral toxicity

Acute toxicity testing was done on mice using the synthesized compounds 5b, 5 g, and 5 h. The doses for acute toxicity testing were 2000 mg/kg & and 300 mg/kg, by OECD standards 423. Table 6 displays the outcomes.

Table 6 Change in body weight for the synthesized compoundChange in bodyweight

When compared to untreated normal (Group I), animals in Group II (disease control) that were induced with DMBA exhibited a substantial (p < 0.05) increase in body weight. The cisplatin treatment of Group III considerably stopped the body weight change caused by DMBA. Body weight was monitored in Groups IV and V for the compound 5b, 5 g, and  h at a dose of 30 mg/kg and 60 mg/kg body weight, respectively (p < 0.05). Table 7 shows the impact of compounds 5b,5 g, and5 h on changes in body weight and related outcomes in Table 8. The tumor volume changes in day 0,7,14 shown in Fig. 7.

Table 7 Change in body weight of subjected mice for the in vivo studyTable 8 Effect of compounds in mice and increased tumor volume (mm3)Fig. 7

Progression of tumor volume in mice

Statistical analysis

Statistical significance was determined by one-way analysis of variance (ANOVA), followed by Dunnett’s multiple comparison test (Graph Pad Prism, version 6, Graph Pad Software Inc., La Jolla, USA). The values with p < 0.05 were considered significant.