Combating oxidative stress as a hallmark of cancer and aging: Computational modeling and synthesis of phenylene diamine analogs as potential antioxidant

Abstract

The cross talk between the over expression of oxygen-free radicals is known as reactive oxygen species (ROS) that is associated with the excessive telomerase activity (TA). Telomerase activity is an invariable finding where human telomerase (hTERT) has been implicated in tumor oxidative stress and redox-mediated malignancy. The hTERT over expression is a novel tumor marker and is promising as a novel class of therapeutic weapons to fight against cancer.

A new series of phenylene diamines were designed, synthesized, and evaluated for their in vitro antioxidant as an indicator of inhibiting the oxidative stress tumor. Compounds 3b and 7b proved to be the most active antioxidants with high percentage ABTS inhibition ranged from 89.40% to 88.59% respectively. Molecular modeling studies indicated that the crest configuration of phenylene diamine nucleus with substitutions of trimethoxy benzamido functional proved to be crucial for enhancing the free radical scavenging activity. Molecular modeling exploration indicated the proper binding selectivity of the 3b and 7b to the 3KYL pocket with promising hTERT inhibitors as a hallmark of cancer.

1. Introduction

Reactive oxygen species (ROS) are generated by normal cellular processes, environmental stresses, and UV irradiation. ROS react with cellular components, damaging DNA, carbohydrates, proteins, and lipids causing cellular and tissue injury. The generation of ROS is a consequence of aerobic life and is unavoidable. ROS represent a constant source of assaults upon our genetic material which can be either enhanced or partly reduced by nutritional, hormonal and environmental influences (Sebastián et al., 2009; Liu et al., 2010; Salmon et al., 2004).

At the normal level, non-cancer cells are able to protect themselves against oxidative DNA damage, via natural antioxidant defense mechanisms to guard against prolonged, excessive levels of ROS by scavenging excessive levels of ROS with enhancement of DNA repair because ROS can react chemically with different proteins and other elements of a cell, altering their normal function. Overproduction and cumulative production of ROS through either endogenous or exogenous insults are harmful to living organisms and are common for many types of cancer cell that are linked with altered redox regulation of cellular signaling pathways. Oxidative stress can induce a cellular redox imbalance causing serious damage to a cell and shut off immune functions leading to the development and progression of oxidative DNA damage inducing oxidative stress tumor. ROS are known not only to attack DNA, but also additional cellular components such as proteins and lipids, leaving behind reactive species that can, in turn, couple to DNA bases leading to DNA lesion that serves as a biomarker of carcinogenesis (Jomova et al., 2010).

Antioxidants are believed to be important in health maintenance through the modulation of oxidative processes in the body (Mosca et al., 2002). Oxidative damage with the unregulated production of ROS has been implicated in a growing list of clinical disorders such as atherosclerosis, rheumatoid arthritis, stroke, cataract, Parkinson’s disease, Alzheimer’s disease and cancer (Valko et al., 2006). Mechanisms responsible for the ROS mediated injury to cells and tissues mainly include lipid peroxidation, protein oxidation, and oxidative DNA damage as a cascade of tumor evolution that is accompanied with over expression of hTRT (Shukla et al., 2011). This background has stimulated interest in the possibility of design and synthesis of a new antioxidant to serve as a tool to selectively inhibit the oxidative DNA damage and control the progression of the tumors that were mediated by over expression telomerase enzyme (Płoszaj et al., 2010).

1.1. Telomeres and telomerase

Telomerase is a cellular RNA-dependent DNA polymerase enzyme containing two essential components, a telomerase reverse transcriptase catalytic subunit (TERT, Gene ID 7015) and a telomerase RNA template (TR or TERC, Gene ID 7012). Telomerase serves to maintain the tandem arrays of telomeric TTAGGG repeats at eukaryotic chromosome ends and so counter the process of replication-associated telomere shortening. The crystallographic structure of the hTERT without ligand at the active site coded (3DU6) was used to explore the binding affinity of the newly designed analogs at the active site (Gillis et al., 2008). However most of the designed compounds showed promising recognition due to the high level of hydrophilic characters with the presence of the core phenylene diamine scaffold. Recently, Skordalakes et al. revealed the main conserved residues at the hTERT binding to RNA–DNA ligands at the active site that give hand to intensely explore the probability of comparing and competing hTERT selective inhibitors at the ligand binding site (Mitchell et al., 2010).

Telomeres are the natural caps of linear eukaryotic chromosomes. Telomeres are essential units that prevent the loss of genetic information. One of the crucial features that distinguishes a cancer cell from a normal somatic cell is its ability to divide indefinitely. The hTERT is inactive in most somatic cells and in contrast, approximately 85% of the most common cancers such as breast, prostate, lung, liver, pancreatic, and colon cancers compensate for telomere shortening through over expression of telomerase, allowing them to stably maintain their telomeres and grow indefinitely. As a corollary to this mechanism telomerase is considered as a target not only for cancer diagnosis but also for the development of novel therapeutic agents to suppress the hTERT expression as an ideal strategy for the development of anticancer chemotherapeutics that promote cell survival, enhance the suppression of oxidative stress resistance cascade and induce tumor cell apoptosis (Pérez-Rivero et al., 2008; Zhang and Ju, 2010; Cataldi, 2010).

1.2. Inhibitors targeting hTERT

Most antioxidant drugs control the oxidative DNA damage and in turn demonstrate anticancer reactivity through many signal pathways including selective inhibition of telomerase. The selective inhibition of telomerase can be effective in providing unique treatments that discriminate between malignant and normal cells to a high extent, avoiding many of the deleterious side-effects present with most current chemotherapeutic regimes which rely on agents that kill dividing cells indiscriminately.

The synthetic inhibitor BIBR1532, 2-((E)-3-naphthalene-2-yl-but-2-enoylamino)- benzoic acid, is a potent hTERT inhibitor where aminobenzoic acid functional is considered as an essential pharmacophore key for its reactivity (El-Daly et al., 2005), (Fig. 1).

Figure 1.

Structures of telomerase inhibitors, BIBR1532, EGCG, BROCA19 and Salophene.

It is worth mentioning that EGCG and BROCA19 were considered recently as remarkable lead compounds that performed potent antioxidant and telomerase inhibitory effects (Naasani et al., 2003; Burger et al., 2005).

Salophene metal analogs were considered as perfect scavenging agents that are able to diminish the elevated cellular ROS that were over expressed and were implicated in cancer initiation, promotion and progression, (Fig. 1) (Lange et al., 2009).

A new series of phenylene diamine analogs is designed to possess the pharmacophore functional keys to mimic the active hTERT inhibitors mainly salicylamido and the schiff base functional groups, (Fig. 1). Combining the inherent free radical scavenging activity of phenylene diamine with these pharmacophore groups was expected to produce more active new leads. Also, the thioureido functional group was known to contribute to the enhancement of the antitumor activity (Pascolo et al., 2002).

The aim of this study is to locate novel synthetic lead compounds and their in vitro testing as free radical scavenger with promising anti DNA oxidative stress damage. Compounds possessing such activity will be candidates for discriminatory treatment of cancer by selectively invading cancer cells keeping the normal cell with their regular vivacity.

2. Experimental section

2.1. General

All reagents and solvents were obtained from commercial suppliers and were used without further purification. Melting points (°C) were determined in open glass capillaries using Branstead 9001 electrothermal melting point apparatus and are uncorrected. NMR spectra were obtained on a Bruker AC 500 ultra shield NMR spectrometer (Fallanden, Switzerland) at 500.13 MHz for ¹H. The chemical shifts are expressed in δ (ppm) downfield from tetramethylsilane (TMS) as internal standard. Deuterio-chloroform (CDCl₃) and deuteriodimethyl sulfoxide (DMSO_d₆) were used as solvents. Mass spectral (MS) data were obtained on Perkin Elmer, Clarus 600 GC/MS mass spectrometers. Thin layer chromatography was performed on precoated (0.25 mm) silica gel GF₂₅₄ plates (E. Merck, Germany), compounds were detected with 254 nm UV lamp. All modeling experiments were conducted with Hyperchem 6.03 package from Hypercube and Moelgro (Heydari et al., 2008; Hyperchem, 1999).

2.2. Synthesis

To spotlight on the significance of the pharmacophore functional groups that were essential for selective recognition in 3KYL binding active sites and to achieve proper antioxidant selectivity, Schemes 1 and 2 were used to prepare different series of amido and thioureido-substituted phenylene diamine (2–8) (Heydari et al., 2008).

Scheme 1.

Synthesis of the target compounds 3 a,b,c–4a,b,c.

Scheme 2.

Synthesis of the target compounds 7 a,b,c–8 a,b,c.

In the present study, a series of new salicylamide phenylene diamine analogs (3a–c and 7a–c) and their corresponding benzamide (4a–c and 8a–c) were designed and synthesized containing 1,2- and 1,3-phenylene diamine scaffolds. The molecular modeling features of the designed compounds and their recognition profiles with the binding active site of telomerase enzyme were investigated using the crystallography of 3KYL enzyme with the RNA–DNA ligands.

The synthesis of the target compounds is depicted in Schemes 1 and 2. ortho-Phenylene diamine (1) was reacted with amino protected analogs of methyl 4-Boc-aminosalicylate and 3-Boc-aminobenzoyl chloride to give the corresponding 1,2-bis(4-bocaminosalicylamido)benzene and 1,2-bis(3-bocaminobenzamido)benzene, they were deprotected by acidic hydrolysis using HCl at room temperature for 1 h of stirring to afford the corresponding the amino analogs (2a,b) respectively. Schiff’s base reaction of 2a,b with 3,4 dichlorobenzaldehyde gives the corresponding 3a and 4a respectively. Condensation reaction of (2a,b) with 3,4,5-trimethoxybenzoyl chloride affords the corresponding trimethoxybenzamide derivatives of 1,2-phenylendiamines (3b and 4b). Phenylthiourea derivatives of 1,2-bis(4-aminosalicylamido)benzene and of 1,2-bis(3-aminobenzamido)benzene (3c, 4c) were prepared by reaction of two moles of phenylisocyanate with one mole of (2a,b). 1,3-Phenylene diamine (5) was reacted with methyl 4-bocaminosalicylate and 3-bocaminobenzoyl chloride to give the corresponding 1,3-bis(4-bocaminosalicylamido)benzene and 1,3-bis(3-bocaminobenzamido)benzene, the protected amido intermediates were deprotected by acidic hydrolysis using HCl at room temperature for 1 h of stirring to afford the corresponding amino analogs 6a, 6b. Schiff’s base reaction of 6a,b with 3,4 dichlorobenzaldehyde gives the corresponding 7a and 8a respectively. Condensation reaction of (6a,b) with 3,4,5-trimethoxybenzoyl chloride affords the corresponding trimethoxybenzamide derivatives of 1,2-bis -(4-aminosalicylamido/3-benzamido)benzene (7b,8b). Phenylthiourea derivatives of 1,3-bis -(4-aminosalicylamido/3-benzamido)benzene (7c, 8c) were prepared by reaction of two moles of phenylisocyanate with one mole of (6a,b).

2.2.1. N,N′-phenylenebis(4-amino-2-hydroxy benzamide) 2a,b, 6a,b^∗ (MOLEGRO., 2009).

A solution of 0.2 mol of methyl ester of 4-boc-aminosalicylic acid or 3-Boc-aminobenzoyl chloride, in ethanol was gradually added to the appropriate phenylene diamine (1,5) in ethanol. The reaction mixture was stirred and maintained at 90 °C for 3 h. The reaction mixture was treated with HCl by stirring for 1 h at room temperature, the corresponding deprotected amino derivatives were neutralized, washed with cold water and the formed precipitate was recrystallized from ethanol to give 2a, 2b and 6a,b respectively.

2.2.1.1. N,N′-(1,2-phenylene)bis(4-amino-2-hydroxybenzamide) 2a

2a: Yield: 85% (ethanol); Mp: 225 °C; ¹H NMR (CDCl₃) δ in ppm: δ 5.50 (s, 2H, exchangeable-H, OH), 6.10 (brs, 4H, exchangeable-H, NH₂), 7.80–8.00 (m, 10H, Ar-H), 9.30 (s, 2H, NH). MS m/e (379.14, 22%). Anal. (C₂₀H₁₈N₄O₄) C, H, N.

2.2.1.2. N,N′-(1,3-phenylene)bis(4-amino-2-hydroxybenzamide) 6a

6a: Yield: 80% (ethanol); Mp: 115 °C; ¹H NMR (CDCl₃) δ in ppm: δ 5.30 (s, 2H, exchangeable-H, OH), 6.20 (brs, 4H, exchangeable-H, NH₂), 7.70–7.80 (m, 10H, Ar-H), 9.20 (s, 2H, NH). MS m/e (380.14, 3.1%). Anal: (C₂₀H₁₈N₄O₄) C, H, N.

2.2.1.3. N,N′-(1,3-phenylene-bis(4-aminobenzamide) 2b

Yield: 80% (ethanol); Mp: 110 °C; ¹H NMR (CDCl₃) δ in ppm: 5.80 (brs, 4H, exchangeable-H, NH₂), 7.80–7.90 (m, 10H, Ar-H), 9.00 (s, 2H, NH). MS m/e (379.14, 22%). Anal. (C₂₀H₁₈N₄O₄) C, H, N

2.2.2. N,N′-bis(4-((E)-(3,4-dichlorobezyledinyl-imino)-2-hydroxy benzamido)benzene 3a, 7a

A solution of 0.2 mol of 3,4dichlorobezaldehyde in absolute ethanol was gradually added to the appropriate aminobenzamide (2a,6a) in acidic solution of ethanol. The reaction mixture was stirred under reflux for 5 h. The reaction mixture was evaporated, the residue was washed, neutralized with diluted aqueous NaOH and the formed precipitate was recrystallized from ethanol to give 3a and 7a respectively.

2.2.2.1. 1,2-Bis(4-((E)-(3,4-dichlorobezyledinylimino)-2-hydroxybenzamido)benzene 3a

3a: Yield: 75% (HCCl₃); Mp: 190 °C; ¹H NMR (CDCl₃) δ in ppm: δ 2.50 (s, 2H,CH=), 5.50 (s, 2H, exchangeable-H, OH), 7.80–8.00 (m, 16H, Ar-H), 8.80 (brs, 2H, exchangeable-H, NH). MS m/e (694.07, 77.9%). Anal. (C₃₄H₂₆C_l4N₄O₄) C, H, N.

2.2.2.2. 1,3-Bis(4-((Z)-(3,4-dichlorobezyledinylimino)-2-hydroxybenzamido)benzene 7a

7a: Yield: 65% (HCCl₃); Mp: 245 °C; ¹H NMR (CDCl₃) δ in ppm: δ 2.40 (s, 2H,CH=), 5.20 (s, 2H, exchangeable-H, OH), 7.10–7.50 (m, 16H, Ar-H), 9.00 (brs, 2H, exchangeable-H, NH). MS m/e (698.06, 47.7%). Anal. (C₃₄H₂₆C_l4N₄O₄) C, H, N.

2.2.3. N,N′-bis(4-(3,4,5-trimethoxybezamido)-2-hydroxybenzamido)benzene 3b,7b

To a stirred solution of 2a or 6a (0.01 mol) in absolute ethanol (50 ml), 0.02 mol of 3,4,5-trimethoxybezoyl chloride in acetone (50 ml) was added. The reaction mixture was heated under reflux for 9 h, the separated solids were filtered, dried and recrystallized from ethanol to afford 3b, 7b respectively.

2.2.3.1. 1,2-Bis(4-(3,4,5-trimethoxybezamido)-2-hydroxybenzamido)benzene 3b

3b: Yield: 70% (EtAc); Mp: 105 °C; ¹H NMR (CDCl₃) δ in ppm: δ 3.85 (s, 18H,CH₃), 5.35 (s, 2H, exchangeable-H, OH), 7.10–7.80 (m, 14H, Ar-H), 9.10 (brs, 4H, exchangeable-H, NH). MS m/e (767.25, 45.6%). Anal. (C₄₀H₃₈N₄O₁₂) C, H, N.

2.2.3.2. 1,3-Bis(4-(3,4,5-trimethoxybezamido)-2-hydroxybenzamido)benzene 7b

7b: Yield: 60% (EtAc); Mp: 125 °C; ¹H NMR (CDCl₃) δ in ppm: δ 3.80 (s, 18H,CH₃), 5.00 (s, 2H, exchangeable-H, OH), 7.20–7.50 (m, 14H, Ar-H), 9.25 (brs, 4H, exchangeable-H, NH). MS m/e (768.26, 40.6%). Anal. (C₄₀H₃₈N₄O₁₂) C, H, N.

2.2.4. N,N′-Bis(4-(phenylthioureido)-2-hydroxy benzamido)benzene 3c,7c

To a stirred solution of 2a or 6a (0.01 mol) in absolute ethanol (50 ml), 0.02 mol of phenylisocyanate in ethanol (50 ml) was added. The reaction mixture was heated under reflux for 5 h, the separated solids were filtered, dried and recrystallized from methanol to afford 3c, 7c respectively.

2.2.4.1. 1,2-Bis(4-(phenylthioureido)-2-hydroxy benzamido)benzene 3c

3c: Yield: 90% (Methanol); Mp: 280 °C; ¹H NMR (CDCl₃) δ in ppm: δ 4.10–4.25 (brs, 4H, exchangeable-H, NH), 5.35 (s, 2H, exchangeable-H, OH), 7.00–7.50 (m, 20H, Ar-H), 9.10 (brs, 2H, exchangeable-H, NH). MS m/e (649.16, 40.66%). Anal. (C₃₄H₂₈N₆O₄S₂) C, H, N.

2.2.4.2. 1,3-Bis(4-(phenylthioureido)-2-hydroxy benzamido)benzene 7c

3c: Yield: 85% (Methanol); Mp: 175 °C; ¹H NMR (CDCl₃) δ in ppm: δ 4.00–4.15 (brs, 4H, exchangeable-H, NH), 5.15 (s, 2H, exchangeable-H, OH), 6.80–7.50 (m, 20H, Ar-H), 9.00 (brs, 2H, exchangeable-H, NH). MS m/e (650.16, 10.5%). Anal. (C₃₄H₂₈N₆O₄S₂) C, H, N.

2.2.5. N,N′-bis(3-((E,Z)-(3,4-dichlorobezyledinylimino)benzamido) benzene 4a, 8a

A solution of 0.2 mol of 3,4dichlorobezaldehyde in absolute ethanol was gradually added to 0.2 mol of the appropriate aminobenzamido derivatives (2b,6b) in acidic solution of ethanol. The reaction mixture was stirred under reflux for 7 h. The reaction mixture was evaporated, the residue was washed, neutralized with diluted aqueous NaOH and the formed precipitate was recrystallized from ethanol to give 4a and 8a respectively.

2.2.5.1. 1,2-Bis(3-((E)-(3,4-dichlorobezyledinylimino)-benzamido)benzene 4a

4a: Yield: 65% (ethanol); Mp: 220 °C; ¹H NMR (CDCl₃) δ in ppm: δ 2.80 (s, 2H,CH=), 7.20–7.50 (m, 18H, Ar-H), 8.80 (brs, 2H, exchangeable-H, NH). MS m/e (662.04, 47.6%). Anal. (C₃₄H₂₂C_l4N₄O₂) C, H, N

2.2.5.2. 1,3-Bis(3-((E)-(3,4-dichlorobezyledinylimino)-benzamido)benzene 8a

8a: Yield: 75% (ethanol); Mp: 160 °C; ¹H NMR (CDCl₃) δ in ppm: δ 2.90 (s, 2H,CH=), 7.25–7.70 (m, 18H, Ar-H), 9.00 (brs, 2H, exchangeable-H, NH). MS m/e (660.00, 5.2%). Anal. (C₃₄H₂₂C_l4N₄O₂) C, H, N.

2.2.6. N,N′-bis(3-(3,4,5-trimethoxybezamido)-benzamido)benzene 4b,8b

To a stirred solution of 2b or 6b (0.01 mol) in absolute ethanol (50 ml), 0.02 mol of 3,4,5-trimethoxybezoyl chloride in acetone (50 ml) was added. The reaction mixture was heated under reflux for 6 h, the separated solids were filtered, dried and recrystallized from ethanol to afford 4b, 8b respectively.

2.2.6.1. 1,2-Bis(3-(3,4,5-trimethoxybezamido)-benzamido)benzene 4b

4b: Yield: 60% (ethanol); Mp: 200 °C; ¹H NMR (CDCl₃) δ in ppm: δ 3.45 (s, 18H,CH₃), 7.60–7.75 (m, 16H, Ar-H), 9.00 (brs, 4H, exchangeable-H, NH). MS m/e (735.26, 45.1%). Anal. (C₄₀H₃₈N₄O₁₀) C, H, N.

2.2.6.2. 1,3-Bis(3-(3,4,5-trimethoxybezamido)-benzamido)benzene 8b

8b: Yield: 65% (ethanol); Mp: 155 °C; ¹H NMR (CDCl₃) δ in ppm: δ 3.70 (s, 18H,CH₃), 7.50–7.80 (m, 16H, Ar-H), 9.25 (brs, 4H, exchangeable-H, NH). MS m/e (736.26, 25.1%). Anal. (C₄₀H₃₈N₄O₁₀) C, H, N.

2.2.7. N,N′-bis(3-(phenylthioureido)-benzamido) benzene 4c,8c

To a stirred solution of 2b or 6b (0.01 mol) in absolute ethanol (50 ml), 0.02 mol of phenylisocyanate in ethanol (50 ml) was added. The reaction mixture was heated under reflux for 5 h, the separated solids were filtered, dried and recrystallized from methanol to afford 3c, 7c respectively.

2.2.7.1. 1,2-Bis(3-(phenylthioureido)-benzamido) benzene 4c

4c: Yield: 70% (Methanol); Mp: 195 °C; ¹H NMR (CDCl₃) δ in ppm: δ 4.00–4.15 (brs, 4H, exchangeable-H, NH), 6.80–7.40 (m, 22H, Ar-H), 9.15 (brs, 2H, exchangeable-H, NH). MS m/e (617.17, 40.6%). Anal. (C₃₄H₂₈N₆O₂S₂) C, H, N.

2.2.7.2. 1,3-Bis(3-(phenylthioureido)-benzamido) benzene 8c

8c: Yield: 60% (Methanol); Mp: 265 °C; ¹H NMR (CDCl₃) δ in ppm: δ 4.00–4.20 (brs, 4H, exchangeable-H, NH), 7.00–7.35 (m, 22H, Ar-H), 9.00 (brs, 2H, exchangeable-H, NH). MS m/e (618.17, 10.5%). Anal. (C₃₄H₂₈N₆O₂S₂) C, H, N.

2.3. ABTS antioxidant assay

ABTS [2, 2-azinobis(3-ethylbenzothiazoline 6-sulfonate] radical cation (ABTS+) scavenging activity was measured according to the described methods. (Kalim et al., 2010; Re et al., 1999) ABTS was dissolved in water to a 7 mM concentration and the ABTS radical cation was produced by adding potassium persulfate to a final concentration of 2.45 mM. The completion of radical generation was obtained in the dark at room temperature for 12–16 h. This solution was then diluted with ethanol to adjust its absorbance at 734 nm to 0.50 ± 0.02. Different compounds were prepared by dissolving 1 mg each in 0.5 ml methanol and 0.5 ml phosphate buffer. To determine the scavenging activity, 1 ml of diluted ABTS⁺ solution was added to 50 μl of tested isolated compounds (or water for the control), and the absorbance at 734 nm was measured 6 min after the initial mixing, using ethanol as the blank. The percentage of inhibition was calculated by the equation:

$$\text{Inhibition percentage}(\%\text{IP})=[\mathit{Ac}-\mathit{As}/\mathit{Ac}]\times 100$$

where Ac and As are the absorbance of the control and of the tested samples respectively.

2.4. Molecular modeling calculations

Molecular modeling studies of the new analogs in complex with 3DU6 have been done to delineate features that differentiate their mode of interaction. Starting coordinate of the X-ray crystal structure of the 3KYL pdb enzyme in complex is obtained from the RCSB Protein Data Bank of Brookhaven National Laboratory (Mitchell et al., 2010). The energy minimization was carried out using the molecular mechanics force field “AMBER”. The energy-minimized structure was used for molecular dynamics studies. The new analogs were constructed from fragment libraries in the Hyperchem program (Heydari et al., 2008). The partial atomic charges for each analog were assigned with the semiempirical mechanical calculation method “AM1” implemented in Hyperchem 6.0. Conformational search was performed around all the rotatable bonds with an increment of 10° using conformational search module as implemented in HyperChem 6.0. All the conformers were minimized until the RMS deviation was 0.01 kcal/mol Å. For each of the chosen analogs, energy minimizations (EM) were performed using 1000 steps of the steepest descent, followed by conjugate gradient minimization to a RMS energy gradient of 0.01 kcal/mol Å. The docking was carried out using a flexible fitting module in MOLEGRO program (Hyperchem, 1999). Each inhibitor was geometrically optimized in the enzyme-binding pocket.

3. Results and discussion

Reactive oxygen species (ROS) function as critical second messenger in a variety of intracellular signaling pathways. Thus, a defect or deficiency in the antioxidant defense system and the excessive intracellular generation of ROS render a cell oxidatively stressed. As a consequence, direct or indirect involvement of ROS in numerous diseases has been documented. The role of free radical oxidative damage in the pathophysiology of human diseases is currently a topic of considerable interest as free radical activity has been implicated in a wide spectrum of clinical conditions, ranging from cancer to atherosclerosis, stroke and neurodegenerative diseases (Subramanian et al., 2010).

In the present study antioxidant capacity was measured in response to ascorbic acid (86.15%). The antioxidant effect of Compounds 3b and 7b showed highest antioxidant activities. Compounds 3a, 4a, 7a and 8a showed moderate antioxidant activities. Compounds 4c and 8c showed lowest antioxidant effects, (Table 1).

Table 1.

Anti-oxidant assays by the ABTS method.

	Method	ABTS
		Abs(control) − Abs(test)/Abs(control) × 100
No	Compounds	Absorbance of samples	% Inhibition
	Control of ABTS	0.491	0%
	Ascorbic-acid	0.068	86.15%
1	3a	0.242	77.59%
2	3b	0.312	89.40%
3	3c	0.354	68.63%
4	4a	0.052	80.24%
5	4b	0.154	86.35%
6	4c	0.11	66.39%
7	7a	0.067	82.07%
8	7b	0.313	88.59%
9	7c	0.069	79.42%
10	8a	0.088	70.87%
11	8b	0.426	85.94%
12	8c	0.412	36.25%

3.1. Molecular modeling study

Recently, the availability of a high resolution crystallographic structure of the human telomerase complex with RNA–DNA ligands affords the opportunity to expand the research targeting the small molecules with potential telomerase inhibition activity as a marker of inhibiting the oxidative stress cascade aiming to bring to light selective and safe cure of cancer.

Computational studies were performed to the designed compounds (2–8) to examine their degree of recognition at the binding active site with the conserved amino acids of the 3KYL telomerase enzyme in the presence of the DNA–RNA ligands.

In the binding model, the docked compounds, 3b and 7b, at 3KYL the RNA–DNA hybrid binding site showed hydrogen bonding interactions with the critical key residues and competitive interference with DNA and RNA by its U shaped configuration that conceals the DNA–RNA strands away from the conserved amino acid residues at the primer grip of telomerase enzyme with promising selective inhibition reactivity to hTERT in the tumor cells, (Fig. 2).

Figure 2.

Molecular docking modeling of the most active free radical scavengers at the binding site of hTERT. Compounds, 3b and 7b, at 3KYL the RNA-DNA hybrid binding site showed hydrogen bonding interactions with the critical key residues and competitive interference with DNA and RNA by its U shaped configuration that conceals the DNA–RNA strands away from the conserved amino acid residues at the primer grip of telomerase enzyme including the most crucial amino acid residues namely, Gly309, Pro311 and Lys416.

3.2. Molecular dynamic study

The investigated compounds were subjected to molecular modeling study to evaluate their recognition profile at hTERT binding-pocket in the presence of the RNA–DNA ligands (Mitchell et al., 2010). The tertiary complex of hTERT, RNA and DNA was used as a reference for modeling and docking. At hTERT pocket, RNA strand formed bifurcated H-bonding with the key residues Ile196, Val197, Gly309 and Pro311, (Fig. 3) (Mitchell et al., 2010).

Figure 3.

TERT–RNA template associations. The nucleotide located at the 5′ end of the RNA template (rC1) is coordinated by Ile196 and Val197 of motif 2 and Gly309 of motif B′. rU2 interacts with Pro311 of motif B′, and rG3 coordinates the backbone of helix α15 via a water molecule (Wat18; red sphere), Ref 10.

Fig. 2 showed the binding mode and residues involved in the recognition of the most active compound 3b (86% inhibition of oxidation) docked and minimized in the hTERT concealing the conserved aminoacids from interaction with DNA or RNA strands at the binding pocket including the most crucial aminoacid residues namely, Gly309, Pro311 and Lys416.

3.3. Structure activity relationship (SAR)

3b and 7b showed an augment in the antioxidant activity due to the presence of the hydroxyl group of the two salicylamide moieties. Also the presence of trimethoxy groups at both terminal phenyl rings enhanced the antioxidant actions. Also, compounds 4b and 8b with trimethoxy groups as two terminals of the benzamido moieties improve the antioxidant actions. However the absence of 2-hydroxyl groups (4b and 8b) abates to some extent the hydrophilic recognition within the enzyme binding site. Compounds 3a, 7a and 4a, 8a that have dichlorophenyl imino groups as a substitution of salycilamido- and benzamido- showed appropriate antioxidant activities (ranged from 70% to 82%). Substitution with the phenyl group at both terminals of the substituted thiourea benzamido of 1,2 and 1,3-phenylene diamines resulted in a decrease in the antioxidant effect of 4c and 8c due to the absence of donating functional groups with the increase of hydrophobic interactions with the surrounding conserved aliphatic amino acid residues (66.39%, 36.25%, respectively). On the contrary, the phenyl thiourea group with salicylamido substituted 1,2 and 1,3-phenylene diamines (3c and 7c) resulted in moderate antioxidant activities due to the presence of the hydroxyl group at position C2, that improves the hydrophilic/hydrophobic balance with the surrounding hydrophilic amino acids leading to proper recognition at the hTERT binding site (68.63%, 79.42% respectively).

4. Conclusion

Compounds 3b,4b,7b,8b proved to be the most active free radical scavenger with percentage inhibition of oxidation range of 82–86%. Structure activity relationship studies revealed that, the type of substituent at positions 4- of both sides of 1,3-phenylene diamine with trimethoxy phenyl groups manipulates the antioxidant activity comparable to the known natural antioxidant product, ascorbic acid.

Molecular modeling study was performed for the investigated compounds to evaluate their recognition profiles at hTERT binding-pocket. It is concluded that recognition with key amino acid Gly309 and Pro311 is essential for binding and biological activities.