Synthesis, Characterization, and In Vivo Study of Some Novel 3,4,5-Trimethoxybenzylidene-hydrazinecarbothioamides and Thiadiazoles as Anti-Apoptotic Caspase-3 Inhibitors.

The present study aims to discover novel derivatives as antiapoptotic agents and their protective effects against renal ischemia/reperfusion. Therefore, a series of new thiadiazole analogues 2a-g was designed and synthesized through cyclization of the corresponding opened hydrazinecarbothioamides 1a-g, followed by confirmation of the structure via spectroscopic tools (NMR, IR and mass spectra) and elemental analyses. The antiapoptotic activity showed alongside decreasing of tissue damage induced by I/R in the kidneys of rats using N-acetylcysteine (NAC) as an antiapoptotic reference. Most of the cyclized thiadiazoles are better antiapoptotic agents than their corresponding opened precursors. Particularly, compounds 2c and 2g were the most active antiapoptotic compounds with significant biomarkers. A preliminary mechanistic study was performed through caspase-3 inhibition. Compound 2c was selected along with its corresponding opened precursor 1c. An assay of cytochrome C revealed that there is an attenuation of cytochrome C level of about 5.5-fold, which was better than 1c with a level of 4.1-fold. In caspases-3, 8 and 9 assays, compound 2c showed more potency and selectivity toward caspase-3 and 9 compared with 1c. The renal histopathological investigation indicated normal renal tissue for most of the compounds, especially 2c and 2g, relative to the control. Finally, a molecular docking study was conducted at the caspase-3 active site to suggest possible binding modes.

1. Introduction

Thiocarbohydrazides and their derivatives appear to be ideal candidates for the synthesis of new analogues and biological activity investigation, since they are the core feature in families of compounds known to display biological activities [1,2,3,4,5,6,7,8]. In recent years, thiocarbohydrazide derivatives have been versatile and convenient precursors for the synthesis of heterocyclic compounds, such as five membered rings [9,10,11,12], six membered rings [13,14,15], seven membered rings [16,17], and fused heterocycles [18,19] as well as for the synthesis of transition metal complexes [4,20] and pharmacological studies [21]. On the other hand, nitrogen-containing heterocycles are indispensable structural units for medicinal chemists. The chemical and biological applications of 1,3,4-thiadiazole has been subjected to intense studies from different research groups. Substituted 1,3,4-thiadiazoles have attracted significant interest in medicinal chemistry due to their wide range of pharmaceutical and biological activities, including antihypertensive [22], anti-inflammatory [23], antimicrobial [24], antitubercular [25], antifungal [26], anticonvulsant [27], anticancer [28], anti-HIV [29], antileishmanial [30], antidepressant [31], and antipsychotic [32] activities. 1,3,4-Thiadiazole derivatives also exhibit interesting in vivo and in vitro antitumor activities [33,34]. The synthesis of 1,3,4-thiadiazoles usually involves multi-step procedures, such as the cyclization of thiosemicarbazide with dicyclohexyl-carbodimide (DCC) and di-(2-pyridyl)thionocarbonate (DPT) [35]. 1,3,4-Thiadiazole derivatives are also formed by oxidative cyclization of thiosemicarbazides with FeCl3 [36] or by the reaction of thiosemicarbazides and CS2. The treatment of isothiocyanates with lithiated (trimethylsilyl)-diazomethane (Me3SiCN2Li) in Et2O, 2-amino substituted-1,3,4-thiadiazoles were obtained [37,38].

For several decades apoptosis has been known to be an important vital biological process that occurs in living cells during tissue development, immune responses [39,40], and homeostasis [41]. Anti-apoptotic mediators can be abrogated in pathological states, as in cancer [42] and autoimmunity [43], or exacerbated, as in stroke [44], neurodegeneration [45], retinal cell death [46], rheumatoid arthritis [47], myocardial and liver ischemia [48,49], and inflammatory diseases such as sepsis [50], osteoarthritis (OA) [51], and asthma [52].

The caspases family is a group of apoptotic mediators, which for many years have been considered high-priority targets for designing new target compounds. The dysregulation of caspase activity leads to many severe diseases. We focused on studying caspase-3, 8, and 9, as they play a key executioner role, and their inhibition can drastically prevent apoptosis in vitro and in vivo [53].

Renal ischemia-reperfusion usually occurs due to trauma, sepsis, renal transplantation, and some vascular surgeries, and this situation may be a reason for renal failure [54]. Although various mechanisms have been reported about the pathogenesis of I/R injury, the data about the treatment are very limited. The ischemia of the kidney starts a series of incidents, including cellular dysfunction and necrosis [55]. The combination of apoptosis and necrosis results in the death of renal cells, and ischemia-reperfusion injury (IRI) is the major cause of delayed graft function in renal allografts [56] in addition to histological damages and functional disorders due to regaining blood flow after an ischemic period in the kidney [57].

Several publications have appeared in the recent years documenting that variant of heterocycles play an important role in protecting complications accompanied with renal ischemia/reperfusion. Thiadiazolo-pyrazole I exerts a protective action against ischemia via the inhibition of NF-κB that enhances the process of cell apoptosis [58] (Figure 1). In addition, other heterocycles such as triazolopyrimidine derivative II (Figure 1) were tested in a kidney ischemia-reperfusion model, in which it showed efficacy at a dose of 10 mg/kg. Moreover, compound II is protective in a model of renal fibrosis [59]. Furthermore, it was reported that melatonin III treatment on renal I/R injury had putative protective effects, whereas melatonin administration reversed the oxidant responses and improved renal function and microscopic damage. It seems likely that melatonin protects kidney tissue against oxidative damage [60] (Figure 1).

Figure 1

Structures of some heterocycles used against ischemia-reperfusion injury in the kidney.

Because of the emergence of acute renal ischemia and its ongoing impact on the global health system, there has been a growing interest for the development of novel antiapoptotic agents in order to combat this disorder. Thus, we report here our medicinal chemistry efforts, starting with the core scaffold of the caspase inhibitor that led to the design and synthesis of new thiadazole analogues as simpler and more efficient antiapoptotic compounds (Figure 2). Moreover, the aim of this study is to test the activity of the cyclized thiadiazole derivatives 2a–g in comparison to their corresponding opened hydrazine-carbothioamides 1a–g in terms of screening antiapoptotic activity, molecular modelling studies, and caspase-3, 8, and 9 inhibition assays to study the plausible mechanism of action of the newly synthesized compounds (Figure 2).

Figure 2

Design of the target compounds 1a–g and 2a–g.

2. Results and Discussion

2.1. Chemistry

When equimolar amounts of 3,4,5-trimethoxybenzylidenethiocarbonohydrazide (3) [61] and 2-substituted isothiocyanates 4a–g were stirred in dry DMF at room temperature, compounds (E)-N-(substituted)-2-(2-(3,4,5-trimethoxybenzylidene)hydrazinecarbonothi- oyl)hydrazinecarbothioamides 1a–g were obtained in good yields (Scheme 1).

Scheme 1

Synthesis of N-substituted-2-(2-(3,4,5-trimethoxy-benzylidene)hydrazinecarbonothioyl)hydrazinecarbothioamides 1a–g and (E)-N-substituted-2-(2-(3,4,5-trimethoxy benzylidene) hydrazinecarbonothioyl)hydrazinecarbothioamides 2a–g.

The structure of the obtained products was elucidated by NMR, IR, and mass spectra, in addition to elemental analysis. For example, the 1H NMR spectrum of 1c displayed five different broad signals with the integral ratio 1:1:1:1:1 centered at δ = 11.87, 10.13, 9.43, 7.97, and 7.90 ppm (exchangeable with D2O) due to 4NH, 3NH, 6NH, cyclopropyl-NH, and CH=N, respectively. The 1H NMR spectrum of 1c clearly indicated the presence of the cyclopropyl group, which appeared as two multiplets resonated at δ 0.60–0.65 and 3.02–3.06 ppm due to 2CH2 and CH, respectively. The three methoxy groups appeared at δ = 3.69 and 3.83 ppm as two methoxy proton sets, which were overlapped. The presence of the cyclopropyl group was also evident from the 13C-DEPT NMR spectrum exhibiting a positive signal at δ = 27.18 ppm (CH) and negative signals at δ = 6.28 ppm due to (2CH2). The formation of 1a–g was evident by both the presence of a signals at δ = 182.54 and 179.26 ppm attributed to the C=S group in the 13C NMR spectrum. Distinctive 13C NMR carbon signals of 1c were appeared at δ = 153.07 (Ar-C-O), 142.93 ppm for (CH=N), 141.84, 139.10, 129.35 (Ar-C), 104.87 (Ar-CH), 56.08, and 60.05 ppm (OCH3).

Synthesis of 5-(2-(3,4,5-trimethoxybenzylidene)hydrazinyl)-1,3,4-thiadiazol-2-amines 2a–g, was established via the heterocyclization of (E)-N-substituted-2-(2-(3,4,5-trimethoxybenzylidene)hydrazinecarbonothioyl)hydrazinecarbothioamide 1a–g during gentle heating in absolute ethanol. Accordingly, the assigned compounds 2a–g were obtained as the only obtained products in 89–94% yields (Scheme 1). The isolated compounds 2a–g (Scheme 1) were characterized by their IR, 1H NMR, 13C NMR, and mass spectral data in addition to elemental analysis. As for example, the IR spectrum of 2c was characterized by the presence of a broad NH at ν = 3305-3250, and a band at ν = 1618 cm−1 that attributed to the C=N stretching. The bands attributed to C=S stretching vibrations were not observed in the IR spectra of 2a–g. The chemical shifts obtained from the 1H NMR spectrum of 2c support the proposed structure. Resonance attributed to a propyl group was detected at δ = 0.73–0.88 (2 CH2-cyclopropyl) and 3.07–3.08 ppm (CH-cyclopropyl). The 1H NMR spectrum clearly showed a singlet signal at δ = 7.96 due to CH=N, whereas two single broad bands with exchangeable D2O were observed at δ = 8.32 and 11.56 ppm for the NH-propyl and NH-hydrazine, respectively. 13C NMR spectroscopic data of representative compounds 2a–g, which were obtained using the DEPT technique at 100 MHz, also supported the carbon framework by discrimination of CH2, CH, and quaternary carbons. The 13C NMR spectrum of 2c showed downfield signals at δ = 166.64, 160.05, 153.07, 142.04, 104.77, and 26.95 ppm attributed to thiadiazole-C2, thiadiazole-C5, Ar-C-O, CH=N, Ar-CH, and CH-cyclo-propyl, respectively. The upfield signal resonated at δ = 6.78 ppm due to (CH2-cyclopropyl).

The E-form of compounds 2a–g was proved from X-ray structure analysis of (E)-N-phenyl-5-(2-(3,4,5-trimethoxybenzylidene)hydrazinyl)-1,3,4-thiadiazol-2-amine (2d) (Figure 3). Tables of X-rays are shown in Figures S1-S7 in Supplementary Data.

Figure 3

Molecular Structure of 2d in the crystal (one crystallographic independent molecule is shown, displacement parameters are drawn at 30% probability level).

2.2. Evaluation of Biological Activity

This part describes the antiapoptotic effect of the novel target compounds 2a–g in comparison to their intermediates 1a–g on decreasing tissue damage induced by I/R in kidneys of rats in a dose equivalent to 30 mg N-acetylcysteine (NAC) [62] as an antiapoptotic reference [63]. Recently, it was reported that NAC was used as an antiapoptotic reference in estimating the apoptotic inhibition of new quinolone-based heterocycles, such as pyrazole and triazole [64]. The rats were subdivided into sham, I/R, NAC, and treated with tested compounds groups, and each group comprises six rats (weighing 140–150 g). The tested compounds were administrated intra-peritoneal (i.p) an hour before ischemia (0.5 h) and reperfusion (0.5 h), after which the organs were cut (Figure 4). This study is supported by biochemical, histological, and morphometric studies using different types of biomarkers.

Figure 4

Photos of kidneys for groups of control (A), I/R (B), reference (NAC) (C), and treated with compounds 1c (D) and 2c (E), as concentrations were measured in serum to detect renal injury due to ischemia/reperfusion (I/R).

Some of these biomarkers increased, and others decreased, indicating antiapoptotic effects, as illustrated in Table 1.

Table 1

The expression of the biomarkers during apoptosis for kidneys.

* Biomarker	Effect during Anti-Apoptosis in Kidney
EP [65], Renal NOx [63], TAC [57]	Increase
Creatinine [56], Urea [66], MDA [60]	Decrease

* EP = epinephrine, MDA = manoldialdahyde, NOx = total nitrite/nitrate, TAC = total antioxidant capacity.

Tables S8–S11 (Supplementary Data) indicate that I/R, 1b, 1g, 2b and 2e groups displayed significant increases in serum creatinine and urea and significant decreases in EP when compared to control and NAC groups. Meanwhile 1a, 1c, 1d, 1e, and 1f groups displayed significant decreases in creatinine and urea and significant increases in EP when compared to the I/R group. The same table lists that groups I/R, 1b, 1c, 1f, 1g, 2b, 2c, 2e, and 2f had a significant increase in MDA along with a significant decrease in NOx and TAC when compared to control and NAC groups. Groups 1a, 1d, and 1e showed significant improvements in oxidative stress parameters when compared to the I/R group. On the other hand, groups 1c and 1f showed significant results in MDA, NOx and TAC when compared to I/R group.

2.2.1. Inhibition Effect on Serum Creatinine, Urea, and Renal MDA

The present study was performed to investigate the validity of serum creatinine levels as an indicator of postischemic renal dysfunction in mice [65]. The results show that the cyclized compounds 2c and 2g had excellent serum creatinine, serum urea, and renal MDA inhibition effects with conc 1.05 and 2.1 mg/dL (creatinine); 37.41 and 57.28 mg/dL (urea); and 54.55 and 84.01 nmol/gm (tissue) (renal MDA), which were respectively better than their corresponding opened intermediates 1a and 1g of conc 1.8 and 3.41 mg/dL (creatinine); 58.87 and 95.69 mg/dL (urea); and 85.48 and 154 nmol/gm (tissue) (renal MDA) and better than those, respectively, in comparison to NAC (3.18 mg/dL for creatinine, 92.17 mg/dL for urea, and 153.1 for renal MDA) (Figure 5).

Figure 5

Concentrations of (A) serum creatinine (mg/dL); (B) serum urea (mg/dL); and (C) renal MDA (nmol/gm tissue) in the kidneys of I/R rats treated with cyclized thiadiazols derivatives 2a–g and their corresponding opened hydrazinecarbothioamides 1a–g in comparison with NAC (reference).

Compounds 2e and 2a showed moderate inhibition to creatinine and urea when compared to their corresponding opened intermediates 1e and 1a, respectively. Compounds 2b, 2d, and 2f exhibited fewer inhibition effects to all biomarkers (creatinine, urea, and MDA) than their corresponding opened intermediates 1b, 1d, and 1f (Figure 5 and Tables S8–S11 in the Supplementary Data).

2.2.2. Activation Effect on Serum Epinephrine, Renal NOx, and Serum TAC

The assay of activation parameters (serum epinephrine, renal NOx, and serum TAC) was conducted for the treated groups of opened 1a–g and cyclized compounds 2a–g in comparison with sham and NAC, and the results are outlined in Tables S8–S11 (supplementary data). Both cyclized compounds 2c and 2g had excellent epinephrine activation effects with 44.51 and 27.39 ng/mg protein, respectively, in comparison with their corresponding opened intermediates 1a and 1g of conc 26.58 and 14.05 ng/mg protein, as illustrated in Figure 6 and Tables S8–S11 (Supplementary Data).

Figure 6

Concentrations of (A) serum epinephrine (ng/mg protein); (B) renal NOx (nmol/gm tissue); and (C) serum TAC (mmol/L) in the kidneys of I/R rats treated with cyclized thiadiazols derivatives 2a–g and their corresponding opened hydrazinecarbothioamides 1a–g in comparison with NAC (reference).

Moreover, compounds 2c and 2g had excellent renal NOx and serum TAC activation effect with conc 133.2 and 85.57 nmol/gm tissue (NOx) and 3.14 and 1.56 mmol/L (TAC), respectively, relative to their corresponding opened intermediates 1a and 1g of conc 81.78 and 56.37 nmol/gm tissue (NOx). Meanwhile, these activation biomarkers were moderately increased in the model of 2b when compared to the opened form 1a; however, the cyclized forms of 2d and 2f showed lesser activation in comparison with the opened forms of 1d and 1f treated groups, as illustrated in Tables S8–S11 (Supplementary Data).

2.2.3. Caspases-3, 8, and 9 Inhibition and Selectivity

The above findings indicate remarkable antiapoptotic activity of our target compounds and suggest that the inhibition of apoptotic caspases may be the suggested target mechanism. Caspases can delay the apoptosis condition and thus implicating a potential role in drug screening efforts [20]. Cell-based caspase-3, and proapoptotic caspase-8 and 9 can cleave the synthetic substrate to release free AFC (7-amino-4-(trifluoromethyl)coumarin) as well as selectivity of caspase-3 and cell-permeable fluorescent reporters can be quantified by fluorometry. The compounds that are to be screened can directly be added to the reaction, and the level of inhibition of caspase-3 activity can be determined by a comparison of the fluorescence intensity in samples with and without the testing inhibitors. Therefore, the mechanism of the caspase-3, 8, and 9 inhibition activity and selectivity in MOLT-4 Cell Line was investigated.

To outline the mechanism of the most active antiapoptotic cyclized thiadiazol derivative 2c and its relative opened hydrazinecarbothioamides 1c, whether through the inhibition of the intrinsic or the extrinsic pathways or both, their effects on caspase-8 and caspase-9 were also evaluated in comparison with the control and the NAC-treated group, as shown in Table S12 (Supplementary Data). Moreover, to investigate caspase-3 inhibition as well as selectivity being the suggested mechanism, the cyclized thiadiazol derivative 2c and its relative opened hydrazinecarbothioamide 1c were further studied for their effects on caspase-3, 8, and 9 and compared to the reference NAC.

Results indicate that compound 2c showed downregulation in the level of active caspase-3 and caspase-9 with conc = 69.77 and 17.58 ng/mL (fold change = 3.1, 3.41), which was better than 1c with conc = 163.1 and 50.6 ng/mL (fold change = 1.33, 1.19), respectively, and better when compared to NAC (conc = 135.2, 46.83 ng/mL, respectively), as shown in (Table S12, Supplementary Materials).

Moreover, the effects of compound 2c and 1c on caspases-8 were also evaluated, revealing a slight increase in the levels of caspases-8 with conc = 35.57 and 20.56 ng/mL, relative to those of NAC, respectively, and compared to those of the untreated control (Figure 7) and (Table S12, Supplementary Materials).

Figure 7

Effects of compounds 1c, 2c, and NAC on Conc (ng/mL) of the active caspases-3, 8, and 9.

2.2.4. Assay of Cytochrome C Inactivation

For more proof of caspase-3 inhibition by the synthesized compounds, cell-based cytochrome C was assayed in an SR human cell line. It was reported that cytochrome C concentrations in the cell have a critical role in the inactivation of caspases and prohibiting the intrinsic apoptosis pathway [67]. Antiapoptotic proteins can inhibit apoptosis by blocking the release of cytochrome C, whereas proapoptotic members function as activators of its release. When cytochrome C levels are maintained in mitochondria the corresponding caspase-8 and 9 will not be activated that leads to the inactivation of caspase-3 and ceases the apoptosis process and therefore reserves the cell from programmed death [68].

The thiadiazole derivative 2c and its opened precursor 1c were evaluated for cytochrome C against the L-SR human cell line, and the results are listed in Figure 8. Compound 2c caused a down-expression of cytochrome C levels about 5.5-fold better than 1c (4.1-fold), compared to NAC (6.19-fold), and lower than the control. The displayed results can be a good guide for suggesting that antiapoptotic activity may be attributed to the attenuation of cytochrome C and hence inactivation of the intrinsic apoptotic pathway induced by the tested compounds (Figure 8).

Figure 8

Effects of compounds 1c, 2c, and NAC on ((A) fold change) alongside conc (ng/mL (B)) conc of cytochrome C in SR human cell line.

Structure Activity Relationship

It can be concluded from the previous results that cyclized thiadiazoles heterocycles showed antiapoptotic activity better than their corresponding opened hydrazinecarbothioamides. It is noted that thiadiazoles of 3–5 carbon chains, such as 2c and 2g which were substituted with saturated alicyclic (cyclopropyl) or aliphatic groups (pentyl), showed the highest activity, but there was activity to a lesser extent when substituted with carbons chain <3, carbons as an ethyl group (compound 2b), cyclohexyl (compound 2e), or an aromatic benzyl group (compound 2a) (Figure 9).

Figure 9

Summary of SAR of cyclized thiadiazole derivatives 2a–g and their corresponding opened hydrazinecarbothioamides 1a–g.

2.2.5. Histopathological Investigation

From the previous work, consistent results motivated us to carry out a histopathological investigation of the tested compounds in the kidney. The groups numbers described below are represented as the following: gp1 = control, gp2 = I/R, gp3 = NAC, gp4 = 2a, gp5 = 2b, gp6 = 2c, gp7 = 2d, gp8 = 2e, gp9 = 2f, and gp10 = 2g.

Renal Cortex

The Sham group showed normal organization of the renal cortex. The glomerular capillary tufts, the parietal layer of Bowman’s capsule, and the Bowman’s space were studied. The proximal convoluted tubules (PCTs) had narrow lumen and a highly acidophilic cytoplasm, whereas the distal convoluted tubules (DCTs) had a wider lumen with less acidophilic cytoplasm. Sections from the model group exhibited distorted glomeruli and severe tubular dilation. Multiple hemorrhagic areas were frequently seen among the sections. Furthermore, reference group (NAC) appeared with an amelioration of the glomerular and tubular morphology. Additionally, groups 1, 3, 5, and 6 showed apparent normal renal tissue. In few dark, dense nucleoli were detected in the previous structures. In contrast, groups 4 and 7 showed a moderate affection of glomeruli and tubules with more observable apoptotic cells (arrows) than previously mentioned groups. It was also noticed that groups 2 and 8 displayed severe damage to the glomeruli with severe tubular dilatation (d) which can be seen by during the appearance of more numerous apoptotic cells (arrows) (Figure 10).

Figure 10

Photomicrographs of renal cortex of (a) Sham group showing normal organization of the renal tissue. Notice the glomerular capillary tufts, (g) the parietal layer of Bowman’s capsule (arrow), and Bowman’s space (s). PCTs (p) have a narrow lumen and a highly acidophilic cytoplasm. DCTs (d) have a wider lumen and a less acidophilic cytoplasm. (b) Model group showing distorted glomeruli (g) and severe tubular dilation (d). Notice the hemorrhagic area (h). (c) Reference group showing amelioration of the glomerular (g) and tubular (d) morphology. (d–j) Groups 1, 3, 5, and 6 show the apparent normal renal tissue, however, a few dark and dense nucleoli (arrows) were detected. (h,i) groups 4 and 7 show moderate affection of glomeruli (g) and tubules (d). Notice the more observable apoptotic cells (arrows) than previously mentioned groups. (g,k) Groups 2 and 8 showing severe damage to the glomeruli (g) and severe tubular dilatation (d). Notice the more numerous apoptotic cells (arrows). H & E, × 400.

Supra Renal Cortex

The sham group showed an adrenal gland covered by a connective tissue capsule. Beneath the capsule, the cells of the zona glomerulosa were seen. The next wider zone was the zona fasciculata with a regular arrangement of its cells and the zona reticularis. The model group showed distorted zones with degeneration with frequent dilated arteries. The reference group showed amelioration of all zones, but vacuolations could be seen in most sections. It was noticed that groups 1, 3, 5, and 6 showed apparent normal supra renal tissue, but vacuolations could still be seen. Few dark, dense nucleoli (arrows) could be detected (arrow). Meanwhile, drugs 4 and 7 appeared with moderate thickened capsules (Figure 11).

Figure 11

Photomicrographs of the supra renal tissue of (a) Sham group showing adrenal gland covered by a connective tissue capsule (C). Beneath the capsule, the cells of the zona glomerulosa (Zg) are seen. The next wider zone is the zona fasciculata (ZF) with a regular arrangement of its cells and the zona reticularis (zr). (b) Model group showing distorted zones with degeneration (star) with a dilated artery (A). (c) Reference group showing amelioration of all zones, but vacuolations could be seen in most sections (V). (d–j) groups 1, 3, 5, and 6 showing apparent normal supra renal tissue, but vacuolations can still be seen (V). Few dark, dense nucleoli (arrows) could be detected (arrow). (h,i) Drug 4 and 7 showing moderate thicken capsule (c) with some areas appear with tissue lost (arrows). Notice the more observable apoptotic cells (arrows). (g,k) Groups 2 and 8 showing severe damage to the suprarenal tissue (D). Notice the thicken capsule (c) and the dilated blood (a). Notice the more notable apoptotic cells (arrows). H & E, × 400.

Renal Tissue

Groups 4, 6, and 8 showed renal tissue nearly approaching the normal renal structure, but a few dark, dense nucleoli could be detected. Groups 4, 6, and 8 exhibited apparent normal supra renal tissue, but vacuolations could still be detected. Few dark dense nucleoli could be detected. Meanwhile, drugs 10 and 7 displayed moderate thicken capsule. More notable apoptotic cells can be seen in them than in the previously mentioned groups. Drugs 5 and 9 showed severe damage to the suprarenal tissue with thickened capsules and interstitial hemorrhaging. More observable apoptotic cells can be seen in them than in the previously mentioned groups (Figure 12).

Figure 12

Photomicrographs of renal cortex of (A–C): groups 4, 6, and 8, respectively, showing renal tissue nearly approach normal; renal glomerulus (g), proximal convoluted tubules (PCT), and distal convoluted tubules (d) except for a few dark dense nucleoli (arrows) could be detected in previously structures. (D,E) (10 and 7, respectively) showing globular congestion (gc) and both tubular and globular vacuolation (V). Notice the more notable apoptotic cells (arrows) than the previously mentioned drugs. (F,G) groups (5 and 9, respectively) showing shrunken glomerulus (g), intratubular hyalin cast (c), dilated tubules (p,d) and more observable apoptotic cells (arrows). H & E, × 400.

Suprarenal Tissue

Groups 4, 6, and 8 showed apparent normal supra renal tissue, but vacuolations could still be seen. Few dark, dense nucleoli could be noticed. Additionally, groups 7 and 10 showed moderate thickened capsules. More notable apoptotic cells than previously mentioned groups could be seen here. Groups 5 and 9 showed severe damage to the suprarenal tissue. Thickened capsules and hemorrhaging were frequently seen among the sections. More observable apoptotic cells (arrows) can be seen here (Figure 13).

Figure 13

Photomicrographs of the supra renal tissue of (A–C): groups 4, 6, and 8, respectively, showing apparent normal supra renal tissue, but vacuolations could still be seen (V). Few dark, dense nucleoli (arrows) could be detected (arrow). (D,E) Groups 10 and 7, respectively, showing moderate thickened capsules (c). Notice more notable apoptotic cells (arrows) than previously mentioned drugs. (F,G) groups 5 and 9 respectively showing severe damage to the suprarenal tissue (D). Notice the thicken capsule (C) and the hemorrhage (h). Notice the more observable apoptotic cells (arrows). H & E × 400.

Morphometric Study

The renal and suprarenal tissues showed a significant increase in the mean number of apoptotic cells when compared to the control group. However, groups 1, 3, 5, and 6 showed no significant differences when compared to the reference group. Groups 7 and 10 also showed significant differences when compared to the reference group. Groups 5 and 11 showed a highly significant histopathological difference when compared to the reference group (all p value ˂ 0.005) (Figure 14).

Figure 14

Histogram showing the mean number of (A) renal apoptotic cells in all studied groups; and (B) supra renal apoptotic cells in all studied groups. Results represented by the mean ± SE (n = 8).

2.2.6. Molecular Docking Studies Using MOE® Program

The MOE® Dock program was used for performing the molecular docking for compounds under investigation. All tested compounds docked into the binding pocket of the active site of caspase-3 (PDB: 1RHJ) to investigate the docking fitness scores of bioactive conformations and their specificity for the caspase-3 enzyme. The docking reliability was validated using the known X-ray structure of caspase-3 in a complex with PZN. The ligand PZN was extracted from the complex, and the ligand NAC was re-docked to the binding site of caspase-3 (Figure 15). The top-ranked conformation of each compound was selected on the basis of docking score. The docking scores of the studied compounds are shown in Table S13 (Supplementary Data).

Figure 15

Three-dimensional representation of docking and ligand interactions of (A); NAC, (B); compound 2c, (C); superimposition of the active docked poses of 2a (red) aligned with 2c (blue) and 2e (green) inside the caspase-3 active site (PDB code: 1RHJ).

The molecular docking studies show that most of the tested compounds interacted similarly to the reference NAC, showing a hydrogen bonding interaction with amino acid residues ASP791, GLU789, and LYS259 of the capase-3 enzyme. The tested ligands were found to bind strongly to caspase-3, as inferred by the binding energy values, whose binding scores ranged from −24.58 to −39.77 kcal/mol. Figure 15 shows that the binding mode was similar to the reference NAC for compounds 2b, which coordinate with caspase-3 through a hydrogen bonding interaction with amino acid residues ASP791 and LYS259. Moreover, both compounds 2a and 2c exhibited a hydrogen bonding interaction with the amino acid residue ASP791; however, both of them showed extra binding, two with hydrophobic binding with ARG786 and one with hydrogen bonding with CYS792 amino acid residues, respectively. The superimposition of the active docked poses inside the protein binding pocket for the most active antiapoptotic compounds 2a, 2c, and 2e are shown in Figure 15. Although the docking results for compound 2f kept hydrogen bonding interactions as well as compound 2b with LYS259, compounds 2d, 2e, 2f, and 2g exhibited extra hydrophobic binding with ARG266 and thus can explain the good antiapoptotic activity for these compounds. On the other hand, both of compound 2f lacked binding with all amino acid residues as the reference; thus, this type of interaction can describe the week antiapoptotic activity for it.

3. Conclusions

New thiadiazole derivatives 2a–g were prepared through the cyclization of corresponding hydrazinecarbothioamides 1a–g and were characterized by different spectroscopic techniques. It is worth mentioning that the tested compounds 2c and 2g showed more potent activity toward the tested biomarkers in serums and tissues than other thiadiazoles. Histopathological examinations for the renal tissue treated with targeted compounds 1a–f and 2a–f indicated that compounds 2c and 5g revealed apparent normal renal cells; however, compound 2d and 2f showed dilated blood vessels with more apoptotic cells when compared with NAC. Moreover, caspase-3, 8, and 9 expression was assayed to exhibit that compound 2c provided lower expression than that of NAC, which showed more potency and selectivity toward caspase-3 than 1c. Molecular docking studies with the caspase-3 enzyme showed comparable binding scores and similar binding interactions to that of reference NAC. Interestingly, most of the tested compounds showed good binding with the enzyme, especially for compound 2c. In summary, the conversion of opened hydrazinecarbothioamides into cyclized thiadiazols improves antiapoptotic activity, especially when substituted with 3–5 carbon chains. Compounds 2c and 2g are promising antiapoptotic agents that are recommended to be further studied.

4. Experimental Section

4.1. Chemistry

The IR spectra were recorded by ATR technique (ATR = Attenuated Total Reflection) with an FT device (FT-IR Bruker IFS 88) (Institute of Organic Chemistry, Karlsruhe University, Karlsruhe, Germany). The NMR spectra were measured in DMSO-d6 on a Bruker AV-400 spectrometer, using 400 MHz for 1H and 100 MHz for 13C. The chemical shifts are expressed in δ (ppm), versus internal tetramethylsilane (TMS) = 0 for 1H and 13C, and external liquid ammonia = 0. The description of signals includes: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, dd = doublet of doublet, and m = multiplet. Mass spectra were recorded on a FAB (fast atom bombardment) Thermo Finnigan Mat 95 (70 eV). Elemental analyses were carried out at the Microanalytical Center, Cairo University, Egypt. TLC was performed on analytical Merck 9385 silica aluminum sheets (Kieselgel 60) with Pf254 indicator. TLCs were viewed at λmax = 254 nm. The synthesized compounds were purified by several recrystallizations from the stated solvent to provide a purity of about 98–99%, confirmed by the elemental analyses and other spectroscopic techniques.

4.1.1. General Method for the Synthesis of Compounds 1a–g

3,4,5-Trimethoxybenzylidenethiocarbonohydrazide (3) was prepared according to the published literature [69]. A mixture of 3 (1 mmol) in dry DMF (30 mL) and 1 mmol of substituted isothiocyanates 4a–g was stirred at room temperature for 4–6 h (the reaction was monitored by TLC). After reaction completion, the reaction mixture was poured onto a mixture of crushed ice and water with vigorous stirring. The precipitated solid was filtered, washed with water, dried, and recrystallized from ethanol to afford compounds 1a–g. Substituted-2-(2-(3,4,5-trimethoxybenzylidene)hydrazinecarbonothioyl)hydrazinearbothioamide 1a,b,d–g were prepared according to the literature’s method [70].

(E)-N-Cyclopropyl-2-(2-(3,4,5-trimethoxybenzylidene)hydrazinecarbonothioyl)hydrazinecarbothioamide (1c). Pale yellow crystals (DMF/EtOH), 3.1 g (81%), mp 182–184 °C. IR (KBr) υ 3316–3249 (NHs), 3155 (Ar-CH), 2968 (Ali-CH), 1611, 1578 (Ar-C=C), 1530 (NH-def. and C-N str.), 1384, 1125, 932 cm−1 (C=S and C-N). 1H NMR (DMSO-d6) δ 0.60–0.65 (m, 4H, 2 CH2-cyclopropyl), 3.02–3.06 (m, 1H, CH-cyclopropyl), 3.69 (s, 3H, OCH3), 3.83 (s, 6H, 2 OCH3), 7.14 (s, 2H, Ar-CH), 7.90 (s, 1H, CH=N), 7.97 (s, 1H, NH-cyclopropyl), 9.43 (s, 1H, 6NH), 10.13 (s, 1H, 3NH), 11.87 ppm (s, 1H, 4NH). 13C NMR (DMSO-d) δ 6.28 (CH2-cyclopropyl), 27.18 (CH-cyclopropyl), 56.08, 60.05 (OCH3), 104.81 (Ar-CH), 129.35, 139.10 (Ar-C), 142.93 (CH=N), 153.07 (Ar-C-O), 179.26, 182.54 ppm (C=S). MS: m/z 383 (M+, 60), 349 (16), 284 (52), 252 (100), 210 (16), 195 (44), 180 (22), 167 (42), 99 (28). Anal. Calcd for C15H21N5O3S2 (383.49): C, 46.98; H, 5.52; N, 18.26; S, 16.72. Found: C, 46.77; H, 5.65; N, 18.39; S, 16.55.

4.1.2. General Method for the Synthesis of Compounds 2a–g

Substituted-2-(2-(3,4,5-trimethoxybenzylidene)hydrazinecarbonothioyl)hydrazine carbothioamides 1a–g (1 mmol) was dissolved in 50 mL absolute ethanol. The mixture was then heated at 60 °C for 2–4 h. Compounds 2a–g precipitated as yellow crystals, were filtered and washed with cold ethanol (20 mL), and recrystallized from the listed solvents.

(E)-N-Benzyl-5-(2-(3,4,5-trimethoxybenzylidene)hydrazinyl)-1,3,4-thiadiazol-2-amine (2a). Yellow crystals (CH3CN), 0.359 g (90%), mp 240–242 °C. IR (KBr) υ 3310–3280 (NHs), 3141 (Ar-CH), 2928 (Ali-CH), 1626, 1573 (Ar-C=C), 1527 cm−1 (NH-def. and C-N str.). 1H NMR (DMSO-d6) δ 3.67, 3.83 (s, 9H, 3OCH3), 4.43 (d, 2H, CH2), 7.13 (m, 2H, Ar-CH), 7.36–7.50 (m, 5H, Ar-CH), 8.20 (s, 1H, CH=N), 9.18 (s, 1H, NH-benzyl), 11.82 ppm (s, 1H, NH). 13C NMR (DMSO-d6) δ 45.93 (CH2), 55.22, 59.18 (OCH3), 103.89, 125.77, 126.28, 128.95 (Ar-CH), 130.58, 137.23, 138.46 (Ar-C), 141.50 (CH=N), 152.23 (Ar-C-O), 159.79 (thiadiazole-C5), 166.66 ppm (thiadiazole-C2). MS: m/z 399 (M+, 100), 307 (9), 195 (32), 149 (86), 99 (17), 91 (38). Anal. Calcd for C19H21N5O3S (399.47): C, 57.13; H, 5.30; N, 17.53; S, 8.03. Found: C, 57.05; H, 5.43; N, 17.66; S, 8.09.

(E)-N-Ethyl-5-(2-(3,4,5-trimethoxybenzylidene)hydrazinecarbonothioyl)hydrazine-carbothioamide (2b). Yellow crystals (EtOH), 0.300 g (89%), m.p. 225–227 °C. IR (KBr) υ 3306–3254 (NHs), 3140 (Ar-CH), 2930 (Ali-CH), 1617 cm−1 (C=N). 1H NMR (DMSO-d6) δ 1.07 (t, 3H, CH3), 3.48 (q, 2H, CH2), 3.68, 3.84 (s, 9H, 3OCH3), 7.16 (s, 2H, Ar-CH), 7.95, 7.97 (d, 2H, CH=N and NH-ethyl), 11.55 (s, 1H, NH). 13C NMR (DMSO-d6) δ 14.48 (CH3), 38.46 (CH2), 56.11, 60.05 (OCH3), 104.88 (Ar-CH), 129.35, 139.10 (Ar-C), 143.07 (CH=N), 153.07 (Ar-C-O), 160.00 (thiadiazole-C5), 166.64 ppm (thiadiazole-C2). MS: m/z 337 (M+, 100), 308 (15), 293 (30), 200 (55), 195 (10), 154 (40). Anal. Calcd for C14H19N5O3S (337.40): C, 49.84; H, 5.68; N, 20.76; S, 9.50. Found: C, 49.99; H, 5.56; N, 20.89; S, 9.35.

(E)-N-Cyclopropyl-5-(2-(3,4,5-trimethoxybenzylidene)hydrazinyl)-1,3,4-thiadiazol-2-amine (2c). Yellow crystals (EtOH), 0.317 g (91%), mp 210–212 °C. IR (KBr) υ 3305–3250 (NHs), 3142 (Ar-CH), 2933 (Ali-CH), 1618 cm−1 (C=N). 1H NMR (DMSO-d6) δ 0.73–0.88 (m, 4H, 2 CH2-cyclopropyl), 3.07 (m, 1H, CH-cyclopropyl), 3.70, 3.87 (s, 9H, 3 OCH3), 7.06 (s, 2H, Ar-CH), 7.96 (s, 1H, CH=N), 8.32 (s, 1H, NH-cyclopropyl), 11.56 ppm (s, 1H, NH). 13C NMR (DMSO-d6) δ 6.78 (CH2-cyclopropyl), 26.95 (CH-cyclopropyl), 56.05, 60.05 (OCH3), 104.77 (Ar-CH), 129.52, 139.01 (Ar-C), 142.04 (CH=N), 153.07 (Ar-C-O), 160.05 (thiadiazole-C5), 166.64 ppm (thiadiazole-C2). MS: m/z 349 (M+, 100), 308 (21), 276 (15), 256 (9), 195 (40), 154 (47). Anal. Calcd for C15H19N5O3S (349.41): C, 51.56; H, 5.48; N, 20.04; S, 9.18. Found: C, 51.77; H, 5.64; N, 19.89; S, 9.06.

(E)-N-Phenyl-5-(2-(3,4,5-trimethoxybenzylidene)hydrazinyl)-1,3,4-thiadiazol-2-amine (2d). Yellow crystals (EtOH), 0.362 g (94%), mp 220–222 °C. IR (KBr) υ 3312–3270 (NHs), 2996 (Ar-CH), 1621 cm−1 (C=N). 1H NMR (DMSO-d6) δ 3.69, 3.83, (s, 9H, 3 OCH3), 6.91–6.95 (s, 3H, Ar-CH), 7.28–7.32 (m, 2H, Ar-CH), 7.54–7.56 (m, 2H, Ar-CH), 7.95 (s, 1H, CH=N), 9.83 (s, 1H, NH-phenyl), 11.93 ppm (s, 1H, NH). 13C NMR (DMSO-d) δ 56.21, 60.57 (OCH3), 103.85, 117.19, 121.38, 129.41 (Ar-CH), 130.54, 138.96, 141.59 (Ar-C), 142.75 (CH=N), 153.61 (Ar-C-O), 163.22 (thiadiazole-C5), 165.12 ppm (thiadiazole-C2). MS: m/z 385 (M+, 28), 192 (40), 176 (16), 165 (32), 130 (10), 116 (26), 91 (100), 77 (36). Anal. Calcd for C18H19N5O3S (385.44): C, 56.09; H, 4.97; N, 18.17; S, 8.32. Found: C, 55.97; H, 4.92; N, 18.29; S, 8.41.

(E)-N-Cyclohexyl-5-(2-(3,4,5-trimethoxybenzylidene)hydrazinyl)-1,3,4-thiadiazol-2-amine (2e). Yellow crystals (CH3CN), 0.363 g (93%), mp 235–237 °C. IR (KBr) υ 3390–3280 (NHs), 3087 (Ar-CH), 1625 cm−1 (C=N). 1H NMR (DMSO-d6) δ 1.67 (m, 4H, 2 cyclohexyl-CH2), 1.94 (m, 6H, 3 cyclohexyl-CH2), 2.73 (m, 1H, cyclohexyl-CH), 3.68, 3.87 (s, 9H, 3 OCH3), 7.12 (s, 2H, Ar-CH), 7.90 (s, 1H, CH=N), 7.93 (s, 1H, NH-hexyl), 11.90 ppm (s, 1H, NH). 13C NMR (DMSO-d6) δ 25.31, 32.18, 35.74 (cyclohexyl-CH2), 52.81 (cyclohexyl-CH), 56.08, 60.09 (OCH3), 104.83 (Ar-CH), 130.30, 138.25, 139.13 (Ar-C), 143.03 (CH=N), 153.15 (Ar-C-O), 160.25 (thiadiazole-C5), 165.13 ppm (thiadiazole-C2). MS: m/z 391 (M+, 20), 307 (38), 281 (16), 168 (5), 156 (100). Anal. Calcd for C18H25N5O3S (391.49): C, 55.22; H, 6.44; N, 17.89; S, 8.19. Found: C, 55.35; H, 6.32; N, 18.01; S, 8.11.

(E)-N-Allyl-5-(2-(3,4,5-trimethoxybenzylidene)hydrazinyl)-1,3,4-thiadiazol-2-amine (2f). Yellow crystals (CHCl3/EtOH), 0.310 g (89%), mp 209–211 °C. IR (KBr) υ =3309–3252 (NHs), 3173 (Ar-CH), 2933 (Ali-CH), 1621 (C=N) cm−1. 1H NMR (DMSO-d6) δ 3.69, 3.83 (s, 9H, 3 OCH3), 4.21–4.24 (m, 2H, CH2N), 5.11–5.19 (m, 2H, allyl-CH2), 5.91–5.99 (m, 1H, allyl-CH), 7.07 (s, 2H, Ar-CH), 7.99 (s, 1H, CH=N), 8.62 (s, 1H, NH-allyl), 11.57 ppm (s, 1H, NH). 13C NMR (DMSO-d6) δ 45.71 (CH2N), 56.10, 60.27 (OCH3), 104.71 (Ar-CH), 115.40 (allyl-CH2), 129.21 (Ar-C), 135.13 (allyl-CH=), 139.00, 140.12 (Ar-C), 142.17 (CH=N), 153.11 (Ar-C-O), 161.22 (thiadiazole-C5), 164.41 ppm (thiadiazole-C2). MS: m/z 349 (M+, 32), 308 (58), 276 (5), 195 (45), 155 (100). Anal. Calcd for C15H19N5O3S (349.41): C, 51.56; H, 5.48; N, 20.04; S, 9.18. Found: C, 51.65; H, 5.39; N, 20.18; S, 9.07.

(E)-N-Pentyl-5-(2-(3,4,5-trimethoxybenzylidene)hydrazinyl)-1,3,4-thiadiazol-2-amine (2g). Yellow crystals (CH3OH), 0.348 g (92%), mp 214–216 °C. IR (KBr) υ 305–3272 (NH), 3146 (Ar-CH), 2929 (Ali-CH), 1613 cm−1 (C=N). 1H NMR (DMSO-d6) δ 0.88 (t, 3H, CH3), 1.33 (m, 4H, 2 CH2), 1.60 (m, 2H, CH2), 3.56 (m, 2H, CH2), 3.70, 3.86 (s, 9H, 3 OCH3), 7.04 (s, 2H, Ar-CH), 7.99 (s, 1H, CH=N), 8.50 (s, 1H, NH-pentyl) 11.50 ppm (s, 1H, NH). 13C NMR (DMSO-d6) δ 13.91 (CH3), 22.10, 28.36, 28.39, 43.40 (CH2-pentyl) 56.10, 60.01 (OCH3), 104.85 (Ar-CH), 129.99, 139.97, 140.01 (Ar-C), 143.01 (CH=N), 153.04 (Ar-C-O), 161.80 (thiadiazole-C5), 165.00 ppm (thiadiazole-C2). MS: m/z 379 (M+, 100), 336 (46), 307 (7), 286 (5), 204(8), 195 (24), 154 (9). Anal. Calcd for C17H25N5O3S (379.48): C, 53.81; H, 6.64; N, 18.46; S, 8.45. Found: C, 53.77; H, 6.70; N, 18.60; S, 8.57.

4.2. Single Crystal X-ray Structure Determination of

Single crystals were obtained by recrystallization from acetonitrile. The single crystal X-ray diffraction study was carried out on a Bruker D8 VENTURE diffractometer with a PhotonII CPAD detector at 298 K using Cu Kα radiation (λ = 1.54178 Å). Dual space methods (SHELXT) [71] were used for the structure solution, and refinement was carried out using SHELXL [72] (full-matrix least-squares on F2). Hydrogen atoms were localized by difference electron density determination and refined using a riding model (H(N) free). A semi-empirical absorption correction was applied.

2d: C18H19N5O3S, M = 385.44 g mol−1, yellow crystal, size 0.32 × 0.08 × 0.02 mm, monoclinic space group P21/n (no.14), a = 17.8658 (3) Å, b = 5.3161(2) Å, c = 19.6971 (3) Å, β = 96.408 (1) Å, V = 1860.99 (5) Å3, Z = 4, D = 1.376 mg m−3, F(000) = 808, μ = 1.80 mm−1, T = 298 K, 15205 measured reflection (2θmax = 144.8°), 3683 independent [R 0.031], 253 parameters, 2 restraint, R [for 3428 I > 2σ(1)] = 0.032, wR (for all data) = 0.092, S = 1.07, largest diff. peak and hole = 0.22 eÅ−3/−0.23 eÅ−3.

CCDC 2056821 (2d) contains the supplementary crystallographic data for this paper. These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif (deposited 18 January 2021).

4.3. Biological Evaluation

4.3.1. Materials and Methods of Biomarkers (Creatinine, Urea, Ep, MDA, TAC, NOx)

The concentration of EP was determined in serum using the spectrophotofluorometric method (Shimadzu RF-5000). Transmitter’s oxidations were conducted by adding 0.1N iodine, followed by stopping the oxidation by alkaline sulfite addition to produce a certain fluorescence. The induced fluorescence measured at a specific emission wavelength after excitation at another specific wavelength differs according to the type of transmitter [73] (see supplementary data). Determination of serum urea was assayed by using an enzymatic colorimetric urea kit.

Principle of measuring urea and ammonia in the chosen sample: The Berthelot reactionhas long been used for the measurement of urea and ammonia [74]. This method is a modification of the Berthelot reaction, in which phenol is replaced by salicylic acid. Urea in the sample was hydrolyzed by the enzyme urease to yield ammonia and carbon dioxide. The ammonium ions then reacted with a mixture of salicylate, sodium nitroprusside, and sodium hypochloride to yield a green indophenol. (See Supplementary Materials).

Assessment of Total Nitrite in Kidney: The stable oxidation end products of NO, nitrite (NO2¯), and nitrate (NO3¯) were used in vitro and in vivo as indicators of NO production. Thus, NO2¯ and NO3¯ levels were estimated as an index of NO production. Total nitrite (nitrite + nitrate) was measured after the reduction of nitrate to nitrite by copperized cadmium granules (Cd) in a glycine buffer at pH 9.7 (2.5 to 3.0 g of Cd granules for a 4 mL reaction mixture). Quantitation of NO2¯ was based on the Griess reaction, in which a chromophore with a strong absorbance at 540 nm is formed by the reaction of nitrite with a mixture of naphthylethylenediamine and sulphanilamide [75] (See Supplementary Materials).

4.3.2. Assay of Caspase-3, 8, and 9 Inhibition

The Caspase-3 Inhibitor Drug Screening Kit (Catalog #JM-K153-100; 15 B Constitution Way, Woburn, 01801-MA, USA) provides an effective means for screening caspase inhibitors using fluorometric methods. The assay utilizes the synthetic peptide substrate DEVD-AFC (AFC, 7-amino-4-trifluoromethylcoumarin). Active caspase-3 cleaves the synthetic substrate to release free AFC, which can then be quantified by fluorometry. Compounds to be screened can be directly added to the reaction, and the level of inhibition of caspase-3 activity can be determined by comparison of the fluorescence intensity in samples with and without the testing inhibitors (See Supplementary Materials).

4.3.3. Assay of Cytochrome C

Cells were collected from American Type Culture Collection to be grown in SR containing 10% fetal bovine serum at 37 °C, stimulated with the compounds to be tested for cytochrome C using Cytochrome C Human ELISA Kit (ab119521 –Cytochrome C Human ELISA Kit, Vaccera institute, Cairo, Egypt) [76] (See Supplementary Materials).

4.3.4. Histopathological Investigation

Multiple testicular specimens were stained by hematoxylin-eosin (ab245880, Abcam, Cambridge, MA, USA) using Cosentino’s score [76] for the semi-quantitation of pathological changes in different seminiferous tubules. By using Johnson’s scoring system [76], the effect of ischemia on spermatogenesis can be studied (See Supporting Materials).

4.3.5. Molecular Docking Study

The docking simulation study was carried out using the Molecular Operating Environment (MOE®) version 2014.09 (Chemical Computing Group Inc., Montreal, QC, Canada). The computational software operated under “Windows XP”, installed on an Intel Pentium IV PC with a 1.6 GHz processor and 512 MB memory. (See Supplementary Materials).