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Synthesis, Anti-Schistosomal Activity and Molecular Modeling of Two Novel 8-Hydroxyquinoline Derivatives Ahmad F. Eweas1,2,*, Gamal Allam3,4, Abdelaziz S.A. Abu-Elsaad3,4, Ibrahim A. Maghrabi1 and Abdul Hamid ALGhamdi4 1
College of Pharmacy, Taif University, Taif, Saudi Arabia; 2Medicinal Chemistry Dept., National Research center, Dokki, Cairo, Egypt; 3College of Medicine and Medical Sciences, Taif University, Taif, Saudi Arabia; 4Department of Zoology, Faculty of Science, Beni-Suef University, Beni-Suef, Egypt Abstract: Schistosomiasis is one of the world’s greatly neglected tropical diseases, and its control is largely dependent on a single drug, praziquantel (PZQ). Here, two novel 8-hydroxyqinoline-5-sulfonamido derivatives, compounds 3 and 4, have been synthesized, characterized and tested as anti-schistosomal agents in vitro. Exposure of adult Schistosoma (S.) mansoni adult worms to 200 μg/mL concentrations of either compound 3 or 4 reduced the motor activity and caused their death within 24 hour (h). However, adult worms incubated in a medium containing 50 μg/mL of either compound 3 or 4 showed decrease motor activity and dead after 120 and 144 h of incubation, respectively. Different concentrations of both compounds induced partial tegumental alterations in 40-50% of tested worms. Additionally, 50 μg/mL concentration of compound 3 caused 58% and 75% reduction in egg production; however compound 4 caused 51% and 60% reduction in oviposition after 48 and 72 h of incubation, respectively. Furthermore molecular docking of both new compounds was carried out using Molsoft ICM pro 3.5-0a to investigate the binding affinity and binding mode to thioredoxin glutathione reductase enzyme (TGR), a potential drug target for anti-schistosomal activity. The docking results revealed moderate to high affinity of both compounds towards TGR. It is suggested that the in vitro schistosomicidal effects of our novel 8-hydroxyqinoline-5-sulfonamido derivatives may be related to the inhibition of TGR activity in S. mansoni. Our results indicate the potential schistosomicidal effects of both 8-hydroxyqinoline derivatives.
Keywords: 8-hydroxyquinoline, Anti-schistosomal, Molecular docking, Thioredoxin-glutatione reductase (TGR). INTRODUCTION Human schistosomiasis is one of the most significant and neglected tropical diseases in the world. This parasitic disease ranks second after malaria in terms of its public health importance [1]. Different approaches have been used to control schistosomiasis transmission including educational programs, mass chemotherapy, biological and chemical control for the snail intermediate host. However, despite these control programs, the disease still affects more than 200 million people worldwide and approximately 800 million people remain under infection risk [2]. Moreover, schistosomiasis continues to spread to new geographic areas due to environmental changes that result from the development of water resources, growth and migration of populations [3]. The therapy for schistosomiasis is based on a single drug, praziquantel (PZQ), that is administered to millions of people yearly [4, 5]. They are rapidly re-infected and must be retreated on an annual or semiannual basis. If praziquantelresistant parasites develop, treatment for schistosomiasis will be in a crisis state [6, 7]. Furthermore, the use of such drug for treatment is complicated due to the difficulties and expenses involved in a long-lasting maintenance of these programs [8]. These limitations, in combination with a *Address correspondence to this author at the College of Pharmacy, Taif University, Taif, Saudi Arabia; E-mail:
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2211-35/13 $58.00+.00
considerable concern about the development of PZQ resistance, have motivated the scientific community to call for research and development of novel and inexpensive drugs against schistosomiasis [9-11]. A reliable alternative to PZQ does not exist at the moment. Oxamniquine, the only other drug commercially available, is expensive and is active against only one of the main three schistosome species capable of infecting humans, S. mansoni. Artemisinins, although safe, are active only against the immature stages of the parasites. A treatment strategy that relies on just a single drug is at risk given the high probability that drug-resistant parasites will emerge [12, 13]. In this context, the identification of new and effective schistosomicidal compounds is essential. 8-hydroxyquinoline derivatives are well known as powerful iron chelatores with antioxidant property. 8Hydroxyquinoline derivatives are important constituents in a variety of pharmaceutically important compound classes. They have become of interest as a new class of potent HIV-1 integrase inhibitors [14] in modeling of the inhibition of retroviral integrases [15], protein tyrosine kinase inhibitors, protozoal and retroviral co-infections [16], anti-HIV-1 agents [17], Anti-leishmanial [18] and anti-filarial agents [19]. Very few publications were reported on the anti-schistosomal activity of 8-hydroxyquinoline derivatives [18]. It was recently discovered that in S. mansoni, specialized thioredoxin (TrxR) and Glutathione reductase (GR) enzymes are absent, and instead replaced by a unique multifunctional © 2013 Bentham Science Publishers
32 Anti-Infective Agents, 2013, Vol. 11, No. 1
enzyme, thioredoxin glutathione reductase (TGR) [20]. This reliance on a single enzyme for both glutathione disulfide (GSSG) and thioredoxin (Trx) reduction suggests that the parasite’s redox systems are subject to a bottleneck dependence on TGR. Given the importance of cellular redox systems and the biochemical differences between the redox metabolism of S. mansoni and its human host, it was hypothesized that TGR could be an essential parasite protein and a potentially important drug target. The significance of S. mansoni TGR as a putative drug target was first demonstrated using an RNA interference approach, which killed 90% of treated parasites in vitro [21]. Moreover, treatment of S. mansoni with a TGR inhibitor, auranofin, resulted in 100% mortality [13]. It was demonstrated that TGR activity is inhibited by two schistosomicidal drugs used in the past to fight the infection, antimonyl potassium tartrate and oltipraz, suggesting that the enzyme is the main target of these compounds [22]. The dependence of S. mansoni on a single protein, TGR, for its protection from oxidative stress, makes it a promising drug target [23]. Therefore, TGR is an essential protein for the survival of S. mansoni and that it meets all the major criteria of an important target for anti-schistosomal chemotherapy development. MATERIALS AND METHODS Chemistry
Eweas et al.
mixture was allowed to stir at ambient temperature overnight. The solid formed was filtered off, air dried and recrystalized from methanol to afford compound 2 as yellow crystals, 78% yiled. Mp: 221-223oC. 3-amino-1-((8-hydroxyquinolin-5-yl)sulfonyl)-1h-pyrazol5(4h)-one 3 A mixture of 0.01 mol of the sulfonylhydrazide 2 and 0.01 mol of ethyl caynoacetate in ethanolic sodium ethoxide (0.46 gm of sodium in 8 ml of absolute ethanol) was allowed to stir under reflux for 10 hr. the reaction mixture was then allowed to cool down to ambient temperature. Excess ethanol was removed under reduced pressure (rotatory evaportator). The residue was dissolved in water 50 ml and 2 ml of acetic acid. The formed product was filtered off and recrystalized from dilute acetic acid to afford compound 3 colorless crystals in 69% yield, Mp: 306-7oC. 1H NMR (270 MHz, DMSO-D6) 3.5-3.7 (1H, br, -OH); 4.7 (2H, s, CH2 Pyrazolone ring); 7.4-8.5 (5H, m, quinoline protons); 8.3 (2H, br, NH2). IR signlas 3510 cm-1 (-OH strch); 3403, 3225 cm-1 (-NH2, Symm, asymm. Stretch); 1690 cm-1 (-C-N, cyclic amide of pyrazolone ring); 1623 cm-1 (C=N, stretch); HRMS (M) + calcd for C12 H 10 N 4 O 4 S 306.0423 found 306.0428 Analysis calcd. For C12H10N4O4S; C: 47.05%; H: 3.30%, N: 18.29 % Found: C: 47.82%; H: 3.70%, N: 18.67 %.
All starting materials and reagents were purchased from Sigma-Aldrich, BDH and Fluka and used without further purification. All solvents were either of analytical grades or dried and distilled immediately prior to use. All of the reactions were performed using oven-dried glassware. Melting points were measured on a Stuart-SMP10 melting point apparatus and are uncorrected. TLC was performed using Merck precoated Silica gel 60 F254 aluminum sheets (20 20 cm, layer thickness 0.2 mm) and Merck precoated Silica gel RP-C18 F254 aluminum sheets (20 20 cm, layer thickness 0.2 mm) and spots were visualized by UV (254 nm), KMNO4 solution and/or charring with H2SO4-EtOH (5% v/v). All reaction products were stored refrigerated under 4 o C. NMR spectra were recorded with JEOL ECA-500, JEOL EX-270, Chemical shifts are expressed in parts per million (ppm) and reported either relative to an internal tetramethylsilane standard (TMS = 0.0). or relative to solvent peaks.
A mixture of 0.01 mol of the sulfonyl hydrazide 2 and 0.015 mol of ethyl acetoacetate was heated under reflux for 7 hr. The reaction mixture was then allowed to cool to ambient temperature. The formed solid was filtered off, air dried and recrystallized form methanol to afford the titled compound 4 as pale yellow crystals in 73% yield, Mp: 315-317oC; 1H NMR (270 MHz, DMSO-D6) 2.4 (3H, s -CH3); 3.5-3.7 (1H, br, -OH); 4.7 (2H, s, CH2 Pyrazolone ring); 7.4-8.5 (5H, m, quinoline protons). IR signlas 3570 cm-1 (-OH stretch); 3434 cm-1 (-NH stretch); 1705 cm-1 (C=O of pyrazolone ring); 1626 cm-1 (C=N, pyrazolone ring); HRMS (M)+calcd for C13H11N3O4S 305.047 found 305.051; Analysis calcd. For C13H11N3O4S; C: 51.14%; H: 3.63%, N: 13.76 % Found: C: 51.47%; H: 4.07%, N: 13.60 %.
8-hydroxyquinoline-5-sulfonyl chloride 1 [24]
Molecular Docking Studies
8-hydroxyquinoline (14.5 gm, 0.01 mol) was added to (43 ml, 0.5 mol) of chlorosulfonic acid over a period of 1 hr during stirring maintaing temperature blow 15oC. The reaction mixture was left for 2 hr at room temperature during stirring. The mixture was then poured over crushed ice. The pale yellow solid formed was filtered off, washed with cold water till acid free then washed with diethylether, air dired to give 1 as a pale yellow solid, 72% yield. Mp: 265-267oC.
All docking studies were performed using ‘Internal Coordinate Mechanics (Molsoft ICM 3.5-0a).
8-hydroxyquinoline-5-sulfonohydrazide 2 [25] Compound 1 (2.43 gm, 0.01 mol) was added to a mixture of Hydrazine hydrate 99% (0.01 mol) in methyl alcohol (15 ml) during stirring at ambient temperature. The reaction
1-((8-hydroxyquinolin-5-yl)sulfonyl)-3-methyl-1Hpyrazol-5(4H)-one 4
Preparation of Small Molecule Two novel 8-hydroxyquinoline derivatives were synthesized and tested as anti-schistosomal agents and compiled using ChemDraw. 3D structures were constructed using Chem 3D ultra 12.0 software [Molecular Modeling and Analysis; Cambridge Soft Corporation, USA (2010)], and then they were energetically minimized by using MOPAC (semi-empirical quantum mechanics), Jop Type with 100 iterations and minimum RMS gradient of 0.01, and saved as MDL MolFile (*.mol).
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Anti-Infective Agents, 2013, Vol. 11, No. 1
Generation of Ligand and Enzyme Structures The crystal structure of target enzyme thioredoxinglutathione reductase TGR (2V6O) is was retrieved from the Protein Data Bank (http://www.rcsb.org/pdb/welcome.do). All bound waters ligands and cofactors were removed from the enzyme. Docking Using Molsoft ICM 3.4-8C Program 1) Convert our PDB file into an ICM object: This conversion involves addition of hydrogen bonds, assignment of atoms types, and charges from the residue templates. 2) To perform ICM small molecule docking a) Setup docking project • Set project name • Setup the receptor
and supplemented with penicillin (100 UI mL1), streptomycin (100 μg mL1), and 10% heat-inactivated fetal calf serum. After washing, one pair of adult worms was transferred to each well of a 24-well Falcon plates containing 2 mL of the same medium and incubated at 37°C in a humid atmosphere containing 5% CO2 prior to use. At 24 h after incubation, 8-Hydroxyquinoline derivatives were dissolved in 1% DMSO and used at concentrations of 50, 100, and 200 μg/mL. The control worms were assayed in RPMI 1640 medium with 1% DMSO as a negative control group and in 10 μg/mL PZQ as a positive control group. All experiments were carried out in triplicates and were repeated at least four times. Worm motor activity, tegumental alterations, mortality rate, egg output (oviposition), and egg development were monitored on daily basis for 6 days using an inverted microscope and a stereomicroscope (Nikon) [28, 29]. Statistical Analysis
• Review and adjust binding site • Make receptor maps b) Start docking simulation 3) Display the result: ICM stochastic global optimization algorithm attempts to find the global minimum of the energy function that include five grid potentials describing interaction of the flexible ligand with the receptor and internal conformational energy of the ligand, during this process a stack of alternative low energy conformations is saved (Table 1). All inhibitors were compared according to the best binding free energy (minimum) obtained among all the run. In Vitro Studies with S. mansoni The John Bruce Egyptian strain of S. mansoni was maintained by passage through Biomphalaria alexandrina snails and MF1 mice [26]. After 8 weeks, S. mansoni adult worms (male and female) were recovered under aseptic conditions from mice previously infected with 100 cercariae by perfusion of the livers and mesenteric veins [27]. The worms were washed in Roswell Park Memorial Institute (RPMI) 1640 medium (Invitrogen), kept at pH 7.5 with HEPES 20 mM, Table 1.
33
The statistical tests were performed with the SPSS (version 16) software. Significant differences were determined by one-way analysis of variance (ANOVA) and applying Tukey’s test for multiple comparisons with a level of significance set at P < 0.05. RESULTS AND DISCUSSION Chemistry 8-hydroxy5-quinolinsulfonyl chloride 1 was prepared according to the literature method [24] via the reaction of 8hydroxyquinoline with chlorosulfonic acid. 1 was then allowed to react with hydrazine hydrate 99% in methyl alcohol, stirring at room temperature to afford the corresponding sulfonyl hydrazide 2 (Scheme 1). Compound 2 underwent cyclization reactions with ethyl cyanoacetate, and ethyl acetoacetate (Scheme 2) to afford the corresponding sulfonamido-imidazol(one) derivatives 3 and 4. The structures of the aforementioned products were supported by their correct elemental analysis, IR and 1H NMR spectra. The Mechanism of formation of N-(3-amino-5-oxo-4,5dihydro-1H-pyrazol-1-yl)-8-hydroxyquinoline-5-sulfonamide 3
The Docking Energy Scores of Compounds 3 and 4 with the Amino acid Residues Forming Hydrogen Bonds in Comparison with Reference Ligand Praziquantel Cpd. No.
Docking score (Kcal/mol)
No. of Hydrogen Bonds
Amino Acid Residues Forming Hydrogen Bonds in Ao
Ligand (Praziquantel)
-70.41
-------
-----------------------
6
T472 hn -- m M n3 : 1.76 A Fad h06 -- m M o2 : 2.34 A Fad h31 -- m M o2 : 1.17 A Fad h31 -- m M o3 : 1.89 A Fad h07 -- m M o2 : 2.37 A A470 o -- m M h6 : 2.36 A
5
N468 hn -- m M n1 : 2.41 A N468 hn -- m M o1 : 1.64 A V469 hn -- m M o1 : 2.47 A G483 hn -- m M o2 : 2.39 A Y466 o -- m M h6 : 1.55 A
3
4
-67.78
-62.13
34 Anti-Infective Agents, 2013, Vol. 11, No. 1
Eweas et al.
SO2Cl
SO2NHNH2 b
a N
N OH
N OH
OH 2
1 Scheme (1). Regents and conditions; a = CISO3H, stirring 15oC; b = N2H4.H2O, 99% stirring rt.
O O2 S
N N
a N SO2NHNH2
NH2
3
OH
O N OH
O2S
N N
b N OH
CH3
4
Scheme (2). Reagents and conditions; a = ethyl cyanoacetate NaOEt/Et2OH; b = Ethyl acetoacetate.
is explained by first removal of EtOH molecule under the influence of sodium ethoxide followed by cyclization of the intermediate to afford the 5-iminopyrazolidin-3-one derivative which undergoes amine imine tautmorism to afford the more favorable 3-amino-1H-pyrazol-5(4H)-one derivative 3 Fig. (1). Molecular Docking Study In S. mansoni, thiol redox homeostasis is completely dependent on the enzyme thioredoxin-glutathione reductase (TGR), which directs the NADPH reducing equivalents to both GSH and thioredoxin [12, 22, 30]. Characterization of the parasite TGR revealed enzymatic properties that differed from those of mammalian TGR, TrxR, and GR. TGR was also identified as a multifunctional oxidoreductase with a remarkably wide substrate specificity, capable of directly reducing peroxides, selenium-containing compounds, and several important low-molecular-weight antioxidants, as well as Trx, 5,5'-dithiobis 2-nitrobenzoic acid (DTNB), reduces glutathione disulfide (GSSG), and glutathione-hydroxyethyl disulfide (GSH-HED) [21]. This dependence of S. mansoni on TGR for its protection from oxidative stress, makes it a promising drug target [23]. Based on these finding we decided to dock our new 8-hydroxyquinoline derivatives against thioredoxin glutathione reductase (TGR) and compare it with the reference drug praziquantel. Molecular docking studies were carried out using Mol soft ICM 3.5-0a. The aim of the flexible docking calculations is the prediction of correct binding geometry for each binder.
The scoring functions and hydrogen bonds formed with the surrounding amino acids of the receptor TGR are used to predict tested compounds binding modes. We evaluated the new compounds 3 and 4 through molecular modeling and docking techniques against TGR crystal structure which was downloaded from PDB website PDB id (2V6O). Fig. (2) shows binding mode of the reference drug (Praziquantel) into its binding site of TGR while Figs. (3, 4) show binding modes of compounds 3 and 4 respectively. Praziquantel “the reference drug” docking results to TGR reveals docking score of G= -70.41 Kcal/mol and no hydrogen bonding to the active site, on the other hand compound 3 docking result against TGR shows docking score of G=-67.78 Kcal/mol. Compound 3 also showed 6 receptor-ligand H-bonds (Table 1). While compound 4 docking results shows docking score of G=-62.13 Kcal/mol and 5 receptor-ligand H-bonds. In Vitro Studies Effect of 8-hydroxyquinoline derivatives on adult schistosomes survival The survival of 56-day-old adult worms of S. mansoni was assessed in vitro by incubation with different concentrations of new 8-hydroxyquinoline derivatives. The effect of 8hydroxyquinoline derivatives on the mortality rate of both male and female adult worms was analyzed with respect to the concentration and incubation time. All worms died at a concentration of 200 μg/mL of both compounds 3 and 4 after 24 h. The lethal effect of both derivatives was also noted at a
Synthesis, Anti-Schistosomal Activity and Molecular Modeling
Anti-Infective Agents, 2013, Vol. 11, No. 1
O H N
H
O HN
EtO +
C
RSO2NH
NaOEt
N
HN
C
N
RSO2
R= O O R
S O
N
O
OH
O N
R N
NH2
S
N
O
3
Fig. (1). Mechanism of formation of compound 3.
Fig. (2). Binding mode of the reference ligand Praziquantel into its binding site of TGR.
Fig. (3). Binding mode of compound 3 into its binding site of TGR.
N H
NH
35
36 Anti-Infective Agents, 2013, Vol. 11, No. 1
Eweas et al.
Fig. (4). Binding mode of compound 4 into its binding site of TGR.
concentration of 100 μg/mL, and all worms died after 72 and 96 h by incubation with compounds 3 and 4, respectively. The 50 μg/mL concentration of compound 3 caused the death of all worms after 120 h of incubation. However, the similar concentration of compound 4, resulted in 100% mortality after 144 h of incubation. In general, male worms were susceptible to both derivatives than female ones. The differences between S. mansoni male and female worms in terms of susceptibility have also been reported in cases in which PZQ [31], ginger extract [32] and essential oil of Ageratum conyzoides [33] were employed. In contrast, the worms remained viable in the negative control group (DMSO 1% plus RPMI 1640 medium) until the end of the incubation period (144 h), whereas PZQ at 10 μg/mL, the positive control, caused the death of all the parasites within 24 h (Table 2). Subsequently, the effect of new 8-hydroxyquinoline derivatives on the motor activity of the worms was examined, and a dose-dependent reduction of activity was observed in both compounds 3 and 4. A significant decrease in motor activity was observed in adult parasites after 24 h of exposure to both derivatives at a concentration of 200 μg/mL. However, the 100 μg/mL concentration of both compounds 3 and 4 resulted in 100% reduction in motor activity of male worms after 24 and 48 h, respectively. Whereas, female worms showed 100% reduction in motor activity after incubation with compound 3 and 4 for 48 and 72 h, respectively. In contrast, worms incubated with 50 μg/mL of compounds 3 and 4 showed 100% reduction in motor activity after 72 and 120 h, respectively. On other hand, all the adult worms incubated in a medium containing 10 μg/mL PZQ became immediately contracted, and their motor activities were significantly reduced (Table 2). In addition to the mortality rate and changes in the motor capacity of S. mansoni adults, the results highlighted the effect of 8-hydroxyquinoline derivatives on the parasite’s tegument. As shown in Table 2, only partial morphological alterations of the tegument occurred in a dose-dependent manner. No extensive tegumental alterations were observed in all worms incubated with either compound 3 or 4. In con-
trast, 10 μg/mL PZQ group had tegumental alteration in all the worms and severe tegumental alteration were more pronounced in male than in female adults. Meanwhile, no tegumental changes in adult worms were observed in the negative control group. From the aforementioned data both 8-hydroxyquinoline derivatives have a potent schistosomicidal activity, especially at higher concentrations (100 and 200 μg/mL). The exact mechanism by which 8-hydroxyquinoline derivatives (compounds 3 and 4) exert their in vitro schistosomicidal effect is not clear. However, there are two possible mechanisms; first: both compounds 3 and 4 have a high binding energy (G= -67.78 and -62.13 Kcal/mol, respectively, as mentioned previously) with an essential protein, TGR, for the survival of S. mansoni. Such binding could inhibit the activity of TGR. This assumption supported by the previous studies that showed silencing of TGR expression lead to death of S. mansoni in vitro within 4 days [21]. Moreover, treatment of S. mansoni with a TGR inhibitor resulted in 100% mortality of parasite in vitro [13]. This finding explains why compound 3, which has a high binding energy with TGR, is more effective in killing of S. mansoni adult worms than compound 4. Second: It is well documented that 8-hydroxyquinoline is a potent chelator to various metals, especially iron [34, 35]. The Fungicidal and bactericidal action of 8-hydroxyquinoline derivatives has been attributed in part to its ability to chelate essential trace metals [36, 37]. Iron (Fe) is an important trace element found in nearly all organisms, and is used as a cofactor in many biological reactions. Schistosomes require Fe for development and maintain their life [38]. Lipid solubility of the neutral iron-8hydroxyquinoline complex ensures penetration of epithelial cells and distribution within the cells [39]. It was shown that iron bound to 8-hydroxyquinoline causes oxidative damages (via free radical lipid peroxidation) in living cells [39,40] as well as DNA strand breakage in cultured cells [41]. Therefore, our new 8-hydroxyquinoline derivatives could exert its schistosomicidal effect through inhibition of TGR activity and Fe chelation.
Synthesis, Anti-Schistosomal Activity and Molecular Modeling
Table 2.
Anti-Infective Agents, 2013, Vol. 11, No. 1
37
In Vitro Effects of 8-Hydroxyquinoline Derivatives against 56-Day-Old Adult S. mansoni Motor Activity Reduction (%)
Groups
Control b
PZQ
Drug Concentration
RPMI 1640 + 1% DMSO
10 μg/mL
50 μg/mL
Compound 3
100 μg/mL
200 μg/mL
Compound 4
50 μg/mL
Incubation Period (h)
Worms with Tegumental Alteration (%)
Dead Worms (%) Slight
Significant
Partial
Extensive
M
F
M
F
M
F
M
F
M
F
24
0
0
0
0
0
0
0
0
0
0
48
0
0
0
0
0
0
0
0
0
0
72
0
0
0
0
0
0
0
0
0
0
96
0
0
0
0
0
0
0
0
0
0
120
0
0
0
0
0
0
0
0
0
0
144
0
0
0
0
0
0
0
0
0
0
24
100
100
0
0
100
100
10
20
90
80
48
100
100
0
0
100
100
10
20
90
80
72
100
100
0
0
100
100
10
20
90
80
96
100
100
0
0
100
100
10
20
90
80
120
100
100
0
0
100
100
10
20
90
80
144
100
100
0
0
100
100
10
20
90
80
24
0
0
0
0
0
0
0
0
0
0
48
0
0
20
10
0
0
0
0
0
0
72
70
60
10
30
90
70
0
0
0
0
96
100
80
0
0
100
100
20
10
0
0
120
100
100
0
0
100
100
20
20
0
0
144
100
100
0
0
100
100
20
20
0
0
24
20
0
40
10
60
20
0
0
0
0
48
50
40
0
20
100
80
10
0
0
0
72
100
100
0
0
100
100
20
20
0
0
96
100
100
0
0
100
100
20
20
0
0
120
100
100
0
0
100
100
20
20
0
0
144
100
100
0
0
100
100
20
20
0
0
24
100
100
0
0
100
100
20
20
0
0
48
100
100
0
0
100
100
20
20
0
0
72
100
100
0
0
100
100
20
20
0
0
96
100
100
0
0
100
100
20
20
0
0
120
100
100
0
0
100
100
20
20
0
0
144
100
100
0
0
100
100
20
20
0
0
24
0
0
0
0
0
0
0
0
0
0
48
0
0
0
0
0
0
0
0
0
0
72
0
0
0
0
0
0
0
0
0
0
96
0
0
40
10
20
20
0
0
0
0
38 Anti-Infective Agents, 2013, Vol. 11, No. 1
Eweas et al.
(Table 2) contd…. Motor Activity Reduction (%) Groups
Drug Concentration
100 μg/mL
200 μg/mL
Incubation Period (h)
Worms with Tegumental Alteration (%)
Dead Worms (%) Slight
Significant
Partial
Extensive
M
F
M
F
M
F
M
F
M
F
120
50
50
30
40
70
60
0
0
0
0
144
100
100
0
0
100
100
0
0
0
0
24
0
0
30
10
20
0
0
0
0
0
48
30
10
10
30
90
20
10
0
0
0
72
100
50
0
40
100
60
20
10
0
0
96
100
100
0
0
100
100
20
10
0
0
120
100
100
0
0
100
100
20
10
0
0
144
100
100
0
0
100
100
20
10
0
0
24
100
100
0
0
100
100
30
20
0
0
48
100
100
0
0
100
100
30
20
0
0
72
100
100
0
0
100
100
30
20
0
0
96
100
100
0
0
100
100
30
20
0
0
120
100
100
0
0
100
100
30
20
0
0
144
100
100
0
0
100
100
30
20
0
0
Worm motor activity and survival of the parasites was monitored under a stereomicroscope. Tegumental alterations were monitored using an inverted microscope. The effects of the compounds on motor activity and tegumental alterations of 20 adult S. mansoni worms were assessed qualitatively from four separate experiments.
Effect of 8-hydroxyquinoline derivatives on the reproductive fitness of S. mansoni In order to evaluate the egg production by adult worms of S. mansoni, both 8-hydroxyquinoline derivatives were tested at 50 μg/mL, a concentration which did not cause death of worms after 48 h of incubation. As depicted in Fig. (5), compound 3 group showed a significant decrease in the number of eggs by 58% (P < 0.01) and 75% (P < 0.001) after 48 and 72 h of incubation, respectively, in comparison with the negative control group. On the other hand, compound 4 groups showed a significant decrease in the number of eggs by 51% (P < 0.01) and 60% (P < 0.001) after 48 and 72 h of incubation, respectively, in comparison with the negative control group. However, there was no significant difference in egg deposition between both compounds 3 and 4 groups and control group after 24 h incubation. The effect of new 8-hydroxyquinoline derivatives on the development of eggs produced by the adult worms was further investigated based on the presence or absence of the miracidium. As shown in the Fig. (6), compound 3 significantly (P < 0.01) reduced the percentage of developed eggs by 63%, 86% and 91% after 24 h, 47 h and 72 h of incubation, respectively, in comparison with the negative control group. Whereas, the development of eggs was inhibited by 50%, 60% and 71% after 24 h, 47 h and 72 h of incubation with compound 4, respectively, in comparison with the negative control group.
Fig. (5). In vitro effect of compound 3 and 4 on Schistosoma mansoni oviposition. Adult worm couples were incubated with 50 μg/mL of either compound 3 or 4, and at the indicated time periods, the cumulative number of eggs per worm couple was assessed and scored using an inverted microscope. Values are means ± SD (bars) of ten worm couples. *P < 0.01 and **P < 0.001 compared with control group (RPMI 1640 + 1% DMSO).
Considering the strong lethal effect of both 8hydroxyquinoline derivatives on adult schistosomes, the in vitro oviposition and maturation of deposited eggs were continually monitored to assess the sexual fitness of treated worms. From previously mentioned data, sub-lethal concentration of both compounds 3 and 4 showed a potent reduction
Synthesis, Anti-Schistosomal Activity and Molecular Modeling
in ova maturation and deposition. This effect could be attributed to chelation of Fe by 8-hydroxyquinoline derivatives. Schistosomes store abundant Fe in vitelline (eggshellforming) cells of the female system. Platyhelminths synthesise eggshells from among a range of precursors proteins belonging to tyrosine-rich proteins, the P14, P19 and P48 proteins [42]. The eggshell is formed by enzymatic oxidation of the tyrosine residues to dihydroxyphenylalanine-quinones, which then bind lysine residues of adjacent proteins [43,44] to form a rigid structure. Fe plays an important role in crosslinking and stabilization of dihydroxyphenylalanine-rich proteins [38,45,46]. 8-hydroxyquinoline and its derivatives have been reported as potent Fe chelator [37]. Consequently, depravation of Fe leads to disruption of egg formation.
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[2]
[3] [4]
[5] [6]
[7] [8] [9]
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Fig. (6). In vitro effects of compound 3 and 4 on the egg development. Quantitative analysis of the development phenotype. Adult worm couples were incubated with 50 μg/mL of either compound 3 or 4, and at the indicated time periods, the eggs were microscopically examined and scored as developed or undeveloped based on the presence or absence of the miracidium. Data are presented as mean ± SD of cumulative developed eggs of ten worm couples. Columns not sharing common superscripts denote significant differences, P < 0.01.
In conclusion, our results indicate that 8-hydroxyquinoline derivatives possess in vitro schistosomicidal activity against S. mansoni adult worms and may be novel broad-spectrum antischistosomal drug candidates. For future work, an in vivo study is required to evaluate schistosomicidal effect of both derivatives on S. mansoni adult worms and their induced pathology.
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[19]
CONFLICT OF INTEREST The author(s) confirm that this article content has no conflicts of interest.
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ACKNOWLEDGEMENTS
[21]
This work was financially supported from Taif University, Taif, Saudi Arabia by a grant No. 1-432-1222. [22]
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Accepted: March 19, 2012
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