Structure Modification Converts the Hepatotoxic Tacrine into Novel Hepatoprotective Analogs

The liver is responsible for critical functions such as metabolism, secretion, storage, detoxification, and the excretion of various compounds. However, there is currently no approved drug treatment for liver fibrosis. Hence, this study aimed to explore the potential hepatoprotective effects of chlorinated and nonchlorinated 4-phenyl-tetrahydroquinoline derivatives. Originally developed as tacrine analogs with reduced hepatotoxicity, these compounds not only lacked hepatotoxicity but also displayed a remarkable hepatoprotective effect. Treatment with these derivatives notably prevented the chemically induced elevation of hepatic indicators associated with liver injury. Additionally, the compounds restored the activities of defense antioxidant enzymes as well as levels of inflammatory markers (TNF-α and IL-6), apoptotic proteins (Bax and Bcl2), and fibrogenic mediators (α-SMA and TGF-β) to normal levels. Histopathologic analysis confirmed the hepatoprotective activity of tetrahydroquinolines. Furthermore, computer-assisted simulation docking results were highly consistent with those of the observed in vivo activities. In conclusion, the designed tacrine analogs exhibited a hepatoprotective role in acute liver damage, possibly through their antioxidative, anti-inflammatory, and antifibrotic effects.


INTRODUCTION
The liver is a crucial organ in the human body and is essential for homeostatic maintenance. 1 Most hepatotoxic substances destroy hepatocytes, increasing tissue lipid peroxidation and oxidative stress. 2Hepatic fibrosis is associated with apoptosis, leading to excess extracellular matrix (ECM) deposition. 3,4epatic stellate cell (HSC) activation is a primary driver of ECM protein secretion. 3,4Activated HSCs are proliferative and fibrogenic, rapidly producing ECM components such as transforming growth factor-beta (TGF-β), tumor necrosis factor-alpha (TNF-α), 3,4 and apoptotic proteins (Bax and Bcl2).
Liver injury leads to fibrosis, causing specific changes in all of the liver cells.Studies show that activated HSCs contribute to ECM degradation through oxidative stress via reactive oxygen species (ROS), leukocyte chemotaxis through chemokine and cytokine production, and hepatocyte growth factor (HGF) production.Oxidative stress plays a role in the development of liver fibrosis. 5The injured liver can have various sources of oxidative stress, one of which is the cytochrome P450 2E1 enzyme that is found in hepatocytes.Infiltrating lymphocytes contribute to inflammation.The inflammatory response of hepatocytes is essential for the progression of hepatic fibrosis.Hepatocytes undergo injury and programmed cell death (apoptosis).Quiescent stellate cells are activated and produce ECM proteins, forming the fibrous scar. 6When activated, HSCs lose retinoids, express new receptors such as trans-forming growth factor-β (TGF-β) receptors, and produce new proteins like α receptors and actin.They also proliferate and synthesize ECM proteins. 7he compounds tested in this study were not primarily designed with the aim of reversing liver injury.They were initially designed in a previous study conducted in our laboratory to inhibit the cholinesterase enzyme for Alzheimer's disease treatment.This former study was based on the structural modification of tacrine (compound I, Figure 1), which is a reversible, noncompetitive acetylcholinesterase inhibitor (AChEI) known to possess hepatotoxic side effects. 8,9ccordingly, the design was aimed at modifying tacrine to produce potent molecules that had minimal hepatotoxicity.
Tacrine-induced acute hepatotoxicity has been extensively studied in the literature. 8,10,11Watkins et al., 8 showed that individuals who were administered tacrine had levels of alanine transaminase (ALT) higher than normal.Lou et al. 11,12 found that tacrine caused considerable liver impairments.However, there has been less research on structural modifications of Figure 1.Chemical structure of tacrine (I), chlorinated tacrine (II), 4aryltetrahydroquinoline (THQ; III) as a basic nucleus for the target compounds 1a, 1b, 2a, and 2b.The chemical structures of compounds 1a and 1b include hydroxide and cyanide substitutes, while compounds 2a and 2b include amino and cyanide substitutes.Letter b refers to the chloride group.investigation induced a significant improvement in the level of basic liver parameters compared with those in the group treated with silymarin, a known hepatoprotective agent, and those in the healthy control group.

THQs Derivatives Alleviated Collagen Deposition in Liver Tissues in Comparison to the CCl 4 -Treated Rats.
Liver histology for control animals showed normal liver parenchyma with preserved cytoplasm, a prominent nucleus and nucleolus, and a portal tract.In comparison, the CCl 4 and CCl 4 + CMC.treated groups showed severe steatosis, lobular inflammation, ballooning of the hepatocytes, and extensive bridging fibrosis in the liver.The histopathological liver architecture following treatment with drugs A, B, and D showed normal liver architecture and a portal tract, whereas animals treated with drug C showed normal liver architecture and a portal tract with focal mild steatosis.The silymarin-treated group showed moderate steatosis and focal bridging fibrosis.In all treated groups, histopathologic examination revealed normal liver parenchyma and portal tract architecture, with no evidence of steatosis, ballooning, inflammation, or fibrosis (Figure 3).

Antioxidant Properties of A, B, C, and D in Reducing Hepatic Fibrosis.
As oxidative stress is one of the pathological processes linked to the development of fibrosis, we examined the antioxidant effects of THQ derivatives.In the model group, CCl 4 -induced liver injury elevated the level of CYP2E1 protein expression and liver malondialdehyde (MDA) and caused a reduction in liver glutathione (GSH) activity compared with those in the healthy control group.Our results revealed that THQ derivative treatment decreased CYP2E1 protein expression, albeit without statistical significance, dramatically decreased liver MDA levels, and markedly increased the activity of GSH compared to the CCl 4 group (Figure 4).

THQs Derivatives Enhanced the Apoptosis Regulators of CCl 4 -Treated Rats.
In liver fibrosis, apoptosis plays a critical role in the initiation and propagation of liver injury to liver fibrosis.Expression of the proapoptotic Bax gene was downregulated in the groups treated with the four drugs; however, this was statistically significant only with drugs B, C, and D. Reverse transcription polymerase chain reaction (RT-PCR) analysis showed that there was a significant upregulation in the expression of the antiapoptotic Bcl2 mRNA following treatment with drugs A, B, C, and D, resulting in a decrease in the Bax/Bcl2 ratio, as shown in Figure 4.

THQs Exert an Anti-Inflammatory Effect on CCl 4 -Treated Rats
. Inflammation is a central feature of liver fibrosis.We used the levels of TNF-α and IL-6 protein expression as an index of the severity of inflammation.CCl 4 injection caused an increase in the expression of the pro-inflammatory cytokines (TNF-α and IL-6).THQs successfully ameliorated this increase and restored the expression to normal levels (Figure 5).

THQs Derivatives Exhibited an Antifibrotic Effect in
CCl 4 -Treated Rats.Regression of fibrosis is characterized by a decline in α-SMA and TGF-β.Levels of α-SMA and TGF-β were elevated in the livers of CCl 4 -treated rats.Surprisingly, administration of THQ derivatives reduced the expression levels of these markers toward a normal level (Figure 5).

2.3.
In-Silico Prediction of the D1−3 Possible Mechanism of Action.Docking analysis using crystal structures revealed that compounds 1b, 2a, and 2b showed superior binding to the Bax receptor, where all three drug compounds formed three hydrogen bonds with the receptor, whereas compounds 1a and silymarin had inferior binding with only one hydrogen bond formed with the receptor.These results were consistent with the above-mentioned in vivo results (see Supporting Information Figure 1).
However, the binding mode to Bcl2 indicated superior binding for compounds 2a and 2b with two hydrogen bonds forming with the receptor versus only one hydrogen bond formed by compound 1b and arene−H bonding between compound 1a and the receptor (see Supporting Information Figure 2).
Analysis of binding to IL-6 showed that compounds 2a and 2b again had superior binding, forming three hydrogen bonds with the receptor, while compound 1a only formed one hydrogen bond with an arene-cation binding to the receptor, whereas only hydrophobic binding was predicted between compound 1b and the receptor (see Supporting Information Figure 3).
For TGF-β, the binding of compound 2a was superior to all the tested compounds with two hydrogen bonds present, while only one was formed with compound 2b; compound 1a formed two arene−H bonds, and compound 1b showed only one arene−H bond to the receptor (see Supporting Information Figure 4).
Finally, TNF-α binding with the tested compounds indicated that compound 2b formed one hydrogen bond and several arene−H bonds to the receptor, while compounds 1b and 2a were only bound to the receptor by one arene−H; only hydrophobic bonding was seen between compound 1a and the receptor (see Supporting Information Figure 5).

DISCUSSION
Liver fibrosis is one of the main causes of illness and mortality worldwide, with chronic liver damage being the most prevalent complication, which can progress to cirrhosis within 1 to 10 years. 12Thus, slowing fibrosis progression might be a viable treatment strategy for patients.To this end, this study aims to assess whether THQ derivatives possess an antifibrotic and hepatoprotective effect against the CCl 4 -induced hepatotoxicity and fibrosis model in rats.THQ derivatives showed no signs of toxicity, as evidenced by measurements of the different hepatic function biomarkers such as ALT, AST, STB, SA, STG, and SC, as well as kidney function biomarkers such as urea and creatinine.Additionally, the oral administration of THQ did not exhibit any decrease in the body weights of the treated rats, suggesting a safe use of THQ.
We compared the potential effects of THQ derivatives and silymarin on the liver injury model in rats.Silymarin served as a reference substance since it is a hepatoprotective and antifibrotic agent. 23Most studies were conducted using CCl 4 -induced acute liver damage, a well-known murine model of hepatic injury and fibrosis. 24,25 The ALT, AST, and bilirubin are employed clinically to assess hepatocyte damage, while the determination of total protein is widely used to assess the synthetic activity of the liver. 26This study showed that CCl 4 administration was associated with a significant increase in ALT and AST and a decreased level of total protein concentration.However, THQ derivative treatment resulted in a significant decrease in hepatocyte damage indices and preserved the synthetic activity of the liver.
In the present study, hematoxylin and eosin, and Masson's Trichrome stainings were performed to assess the effect of THQ derivatives treatment on the CCl 4 -induced liver injury. 27Cl 4 -treated group administration led to severe steatosis, lobular inflammation, ballooning of the hepatocytes, and extensive bridging fibrosis in the liver.Histological examination revealed that silymarin was able to mitigate the extent of liver fibrosis induced by CCl 4 to a moderate level of steatosis and focal bridging fibrosis.On the other hand, THQ derivative treatment significantly ameliorated the histopathologic findings pathognomonic of CCl 4 damage and normalized liver architecture and portal tract; compounds 1a, 1b, and 2b are more effective than compound 2a.
To investigate whether the improvement of liver function and architecture induced by THQ derivative treatment was associated with antioxidant activity, we measured oxidative stress in the liver.We examined the level of reduced GSH and malondialdehyde (product of lipid peroxidation; MDA) as well as key biochemical markers, including the CYP2E1 metabolic enzyme. 28CCl 4 administration significantly elevated the level of CYP2E1 protein expression, a member of the cytochrome P450 mixed-function oxidase system that is implicated in a variety of pathological conditions. 29It is considered the major isozyme involved in carbon tetrachloride bioactivation, leading to the generation of toxic intermediates and excessive amounts of ROS. 30 There was a trend toward a reduction in CYP2E1 expression with THQ derivative treatment; however, this did not reach statistical significance, and therefore, it is difficult to implicate this reduction in the hepatoprotective mechanism of action for these compounds.
Additionally, cellular antioxidant mechanisms, such as GSH, were markedly compromised by the CCl 4 administration. 31SH is a nonenzymatic antioxidant and intracellular redox homeostasis regulator that is expressed in all cell types. 32epletion of cellular GSH in the liver is thought to be a major cause of CCl 4 toxicity. 33The THQ derivative treatment markedly reversed the antioxidant mechanism as compared to the CCl 4 -only group.This may have played a key role in these compounds' hepatoprotective action against CCl 4 toxicity.Here, the restoration of GSH levels may be due to THQ hepatoprotection against CCl 4 toxicity.It is worth noting that compounds 1a and 2a had the most profound effects.
Furthermore, CCl 4 administration significantly enhanced the oxidative stress and the concentration of MDA, a well-known product of lipid peroxidation and index of oxidative stress, 34 an increase in which has been linked to CCl 4 -induced tissue damage.The significant rise in MDA levels in the investigated liver was reduced by the administration of THQ derivatives, with the superiority of compounds 1b, 2a, and 2b over compound 1a.This action may be related to the ability of THQ derivatives to scavenge hydroxyl radicals 35−40 and therefore reduce lipid peroxidation.
The antioxidant activity of THQ derivatives may be attributed to their ability to scavenge ROS to some extent, possibly due to the formation of a hydrogen bond between an electronegative atom such as amine or hydroxy and a lone pair of nitrogen atoms present on an unstable free radical. 41o investigate whether the improvement of liver function and architecture induced by THQ derivative treatment was associated with anti-inflammatory activity, we measured the pro-inflammatory cytokines TNF-α and IL-6. 42In response to oxidative stress caused by CCl 4 -induced hepatotoxicity, the expression of pro-inflammatory cytokines such as TNF-α and IL-6 is induced. 35,36TNF-α initiates a chain reaction of cytokines that drives the inflammatory response. 43TNF-α is a pleiotropic proinflammatory cytokine that is rapidly generated by macrophages in response to tissue damage. 44It promotes oxidative metabolism and nitric oxide generation in phagocytes by stimulating the release of cytokines from macrophages.TNF-α can also cause direct cytotoxicity and has been linked to increased apoptosis. 45,46TNF-α is also a key player in the pathogenesis of liver fibrosis through the stimulation of Kupffer cells. 47NF-α levels have been shown to increase steadily during CCl 4 -induced hepatotoxicity, 47 leading to fibroblast proliferation and collagen synthesis. 45,48However, the THQ derivative treatment, especially compounds 1b, 2a, and 2b, led to a marked reduction in the TNF-α level.
CCl 4 administration is associated with elevated IL-6 levels, a well-known pro-inflammatory factor that plays a critical role in inflammatory and immunological illnesses, 49 IL-6 is generated by macrophages and monocytes and is vital to the liver's local inflammatory response. 50THQ derivatives, especially compounds 1b, 2a, and 2b, surprisingly lowered the levels of IL-6, thereby dampening the inflammatory response in the liver.−53 Additionally, decreased ROS production by THQ culminates in the suppression of cytokine activation, thereby attenuating the expression of pro-inflammatory mediators such as TNF-α and IL-6. 54ince CCl 4 -induced hepatic injury is associated with extensive apoptosis, 55 we evaluated the antiapoptotic effect of the THQ derivatives by assessing the levels of the apoptosisrelated proteins (Bax and Bcl2). 56Apoptosis is programmed cell death that is distinguished by membrane blebbing, cell shrinkage, chromatin condensation, and nuclear fragmentation.Pro-and antiapoptotic proteins interact with one another during apoptosis to protect cellular integrity. 57The B-cell lymphoma-2 (Bcl-2) protein family controls mitochondrial dysfunction 58 and includes antiapoptotic (Bcl2) and proapoptotic (Bax) effector proteins. 57,59Loss of the electrochemical gradient over the inner mitochondrial membrane results in the uncoupling of oxidative phosphorylation, leading to the formation of superoxide free radicals.This is a key action of Bcl-2 family proteins implicated in mitochondrial changes associated with apoptosis. 60In addition, cytochrome c (Cyt c) is released into the cytosol from mitochondria.Cyt c triggers proteolytic processing and the activation of cell death proteases known as caspases, which are essential for hepatocyte apoptosis. 61Overexpression of Bcl-2 prevents Cyt c release and caspase activation, whereas induction of Bax promotes these alterations. 62As a result, the ratio of Bax to Bcl2 is a crucial index of apoptotic potential.Apoptosis is induced by the upregulation of Bax and the downregulation of Bcl2. 55n the mitochondria of animals with CCl 4 -induced hepatic injury, the expression of Bcl-2 is downregulated, whereas the expression of Bax is upregulated, resulting in an elevated Bax/ Bcl-2 ratio. 63Treatment with THQ derivatives caused downregulation of Bax protein and upregulation of Bcl2 protein, decreasing the Bax/Bcl2 ratio, suggesting that these compounds, especially 1b, 2a, and 2b, are endowed with antiapoptotic activity. 36,40,64t has been shown previously that heterocyclic compounds bearing quinoline in their structure have antiapoptotic activity. 65Electronegative atoms impart moderate antitumor activity to compounds. 66ince CCl 4 damage eventually leads to liver fibrosis, we sought to assess the potential antifibrotic effect of the tested compounds by measuring the levels of the fibrosis markers α-SMA and TGF-β.Hepatic fibrosis is an energetic process that initiates hepatocyte necrosis and then involves the activation of inflammatory cells, such as macrophages, HSC activation and proliferation, and the release of fibrogenic mediators. 3TGF-β family signaling pathways are master regulators of several cellular activities, such as proliferation, differentiation, migration, and cell death, that are necessary for tissue and organic homeostasis. 51TGF signaling is implicated in all stages of liver disease, from liver damage through inflammation and fibrosis to cirrhosis and cancer.TGF-β has cytostatic and apoptotic actions on hepatocytes, which then promote liver differentiation during embryonic development and physiological liver regeneration. 67TGF-β is the most significant profibrotic cytokine and promotes the production of type I procollagen. 48The CCl 4 -treated group showed a marked increase in TGF-β levels, possibly contributing to the CCl 4induced hepatic injury and fibrosis. 25,68THQ derivatives treatment lowered TGF-β levels in our study, with compounds 1b, 2a, and 2b being more superior than 1a in their antifibrotic effects.This suggests that the hepatoprotective effect of the compounds under investigation may be partly due to their antifibrotic activity by lowering the TGF-β level. 37,40,51he administration of CCl 4 was associated with an increase in the expression of α-SMA, 69 a distinct actin isoform found in vascular smooth muscle cells but lacking functionality in skeletal muscle fibrogenesis. 70However, α-SMA is important for the mobility and contraction of myofibroblasts 71 during hepatic fibrogenesis, so they may reach the damaged liver tissue.Currently, α-SMA is believed to adjust myofibroblast contractility and signal the nucleus to control collagen formation.Thus, α-SMA can communicate with the nucleus to control collagen synthesis. 72Moreover, stimulation of HSCs is an essential phase in the progression of liver fibrosis. 35The activation of HSCs is crucial to the occurrence of liver fibrosis. 73Elevation of α-SMA levels acts as a phenotypic marker, indicating HSC phenotypic transition into myofibroblast-like cells.In the current study, THQ derivatives treatment reduced α-SMA expression as compared with that in the control group, with compounds 1b and 2a being more superior than the others, suggesting that THQ derivatives may inhibit fibrosis by blocking the activation and differentiation of HSCs into myofibroblasts. 53,74This reduction in α-SMA is probably a consequence of lowering TGF-β activity. 75he antifibrotic effect of THQ derivatives is related to the substitution pattern of the 4-phenyl group, which was found to be critical for potent TGF-β inhibitory activity.Considerable hydrophobic interactions were present in this part of the molecule for potent TGF-β inhibitory activity. 75,76

CONCLUSIONS
This study showed that THQs were effective in lowering CCl 4induced acute liver damage and fibrosis in a rat model by controlling oxidative stress, lipid peroxidation, inflammation (via reduction of TNFα and IL-6 expression), and restriction of HSC activation (via regulation of TGF-β and α-SMA expression).THQ protected hepatocytes from CCl 4 -induced apoptosis in a less favorable manner by regulating the expression of the Bcl-2 family of proteins with a lower expression of proapoptotic proteins and a higher expression of antiapoptotic proteins.Histological examination revealed the beneficial effect of THQ derivatives.The results of the in silico molecular docking evaluation of the compounds against Bax, Bcl2, IL-6, TGF-β, and TNF-α were also in good agreement with the biologically obtained results.Thus, this study demonstrates that these compounds have the potential to be developed into novel therapeutics for the prevention of acute liver damage and fibrosis without deleterious effects on the kidneys.

Drug Synthesis.
All of the reagents and solvents were purchased from commercial suppliers.Compound melting points in open glass capillaries were obtained by using the Thomas−Hoover melting point equipment.Thin-layer chromatography (TLC) using silica gel-precoated aluminum sheets (Type 60 GF254; Merck; Germany) was used to monitor the reactions and determine the purity of the chemicals employed in the study.Compounds were identified by exposing spots on TLC sheets to an ultraviolet lamp at k = 254 nm for few seconds.Microanalyses for purity were performed at the regional Center for Mycology and Biotechnology, El Azhar University, and the found values were within ±0.3% of the theoretical values.

Animal Studies.
Wistar albino male rats with an age of 5−7 weeks, weighing 130−200 g, were procured from the Institute of Graduate Studies & Research Animal House.The rats were maintained in well-ventilated polypropylene cages and fed standard pellets and water at a constant temperature of (24 °C ± 1).Animals were fasting but were provided unrestricted access to water overnight before the start of the experiments.All tests were executed following the faculty's animal ethics committee's norms and regulations (Institutional Review Board; IRB).The experimental protocol was approved by the Alexandria University-Institutional Animal Care and Use Committee (AU-IACUC)-(Approval Code.06−2020− 11−24−1−84).All authors complied with the ARRIVE guidelines, and the reporting in the manuscript follows its recommendations.

Dose Selection.
CCl 4 at a dose of 1 mL/kg body weight/72 h, i.p. for 14 days, was used as a previous study had demonstrated that this dose leads to a significant increase in liver function indices, oxidative stress, and levels of fibrosis markers. 78he dose of THQ (25 mg/kg/day, p.o.) daily for 14 days was selected based on prior findings with similar derivatives that had previously been shown to possess preventive and therapeutic properties against hepatotoxicity. 79The dose of silymarin (SIM, 100 mg/kg/day, p.o.) was chosen based on prior research showing hepatoprotective benefits for silymarin against various types of tissue damage and CCl 4 -induced toxicity. 78.6.Sample Collection and Processing.Animals were weighed and euthanized 24−48 h after injection of the last dose using isoflurane (3% for 3 to 5 min). 80Blood was extracted through the retro-orbital puncture, allowed to clot, and then centrifuged for 10 min at 5000 rpm to extract serum.The serum was evaluated promptly for liver function enzymes.Biochemical and histological tests were conducted on the dissected liver.After cervical dislocation, the rats were euthanized, and hepatic tissue samples were collected via laparotomy.Livers were harvested and washed in ice-cold, phosphate-buffered saline.Samples were maintained at −80 °C for additional analysis of liver fibrosis, inflammatory markers, oxidative stress, and apoptosis.The relative expression of Bax and Bcl2, apoptotic progression markers, was measured using RT-PCR.Total RNA was purified from liver tissue samples using a TriFast reagent.For quality and yield determination, the ratio of absorbance at 260 and 280 nm was assessed using the NanoDrop Spectrophotometer.In a two-step RT-PCR setup, total RNA was reverse-transcribed into single-stranded complementary DNA using the QuantiTect Reverse Transcription Kit (Qiagen, USA) and a random primer hexamer.Rotor-Gene Q (Qiagen, USA) was used to examine the mRNA of genes, with β-actin serving as a housekeeping gene.cDNA amplicons were amplified using Applied Biosystems TaqMan Universal PCR Master Mix (Applied Biosystems, Foster City, CA, USA) and specific primers: Bcl-2 gene; forward; 5′-GGATCCAG-GAT AACGGAGGC-3′, reverse; 5′-CCAGATAGG CACC-CAGGGT-3′, Bax gene; forward; 5′-CTTTTGCTTCAG G G T T T C A T C C -3 ′ , r e v e r s e ; 5 ′ -T T G A G A C A C T CGCTCAGCTTCT-3′, β-actin (House-Keeping Gene); forward; 5′-CCTGGCACCCA GCACAA-3′, reverse; 5′-GCCGATCCAC ACGGAGTACT-3′.The relative expression of the target gene was computed using the 2 −ΔΔct method. 83.8.Docking Study.Molecular Operating Environment (MOE Dock 2015) software (Chemical Computing Group, Montreal, QC) was used to perform computer-assisted simulation docking experiments using an MMFF94X force field.
5.8.1.Docking Protocol.Crystal structures of Bax, Bcl2, IL-6, TGF-β, and TNF-α were obtained from the Protein Data Bank (PDB ID: 6EB6, 5WHH, 1ALU, 1TGJ, and 2AZ5, respectively) and used in the docking simulations.The ligand molecules were constructed using the builder molecule and were energy-minimized.Ligands were docked within the active site by using the MOE Dock.The lowest energy conformation was selected, and the ligand interactions such as hydrogen bonding, arene−H, and arene−arene interactions, together with other hydrophobic interactions with the receptors, were recorded.
5.9.Statistical Analysis.Statistical analysis was performed using Prism 8.0.2 (263) software (GraphPad, La Jolla, CA).Differences between groups for parametric data were analyzed using a one-way analysis of variance with Tukey's posthoc test, and nonparametric data were analyzed using Kruskal−Wallis's test with the appropriate Dunn's multiple comparisons test.

■ ACKNOWLEDGMENTS
All the authors thank the editors and anonymous experts for their comments, which have improved the study.Funding: This research was funded and supported by the Faculty of Pharmacy, Alexandria University, Alexandria, Egypt.

Figure 2 .
Figure 2. (A) Male albino rats' body weight through the 14 days of the experiment.At a 5% level of significance (*p < 0.05), no significant difference was obvious between the groups; n = 5; (B) biochemical parameters of the toxicity study for the synthetic drugs (A: 1a, B: 1b, C: 2a, and D: 2b); (C) effect of THQ-derivatives on liver histology.It showed no pathological effect on liver tissue as represented by hematoxylin and eosin and Masson Trichrome; values are expressed in mean ± SEM (n = 5).