Design, Synthesis of Analgesics and Anticancer of Some New Derivatives of Benzimidazole

This work, contains some new compounds from benzimidazole derivatives, which are synthesized by condensation of Orthophenylene diamine and Carbon disulfide resulting in 2-Mercapto-benzimidazole which is treated by alcoholic potassium hydroxide forming potassium salt of 2-mercaptobenzimidazole which reacts with different substances (alkyl chloroacetates, chloroacetic chloride, alkyl halides) also the ethoxy carbonyl methyl thiobenzimidazole reacts with different amines. In addition to chloromethyl benzimidazole which resulted from the reaction between orthophenylene diamine and chloroacetic acid, which reacted with different amines. The synthesized compound tested as analgesics and anticancer activity the new derivatives revealed moderate, strong and very strong analgesics and moderate and strong anticancer activity.


Introduction
The chemical structure of heterocyclic compounds especially the structures which have benzo diazine and benzo diazole (phthalazine, Quinoxaline, Quinazoline and benzimidazole) have many biological activities antibacterial, anticonvulsant, oral hypoglycemics, anticancer, anthelmintics, antiprotozoal, anti-inflammatory, analgesics, antioxidants, growth regulators, and other biological uses. So, we choose the benzimidazoles which are promising nucleus. It has an analgesic activity like diclofenac and also has anticancer activity as doxirubsin i.e., six membered ring International Journal of Organic Chemistry

Biological Testing
Experimental Animal Adult healthy female Wister albino rats weighting between 160 -200 gm were used for the study. The animals were housed in standard conditions (temperature 24 + 2 with 50% -60% relative humidity and a 12-hour light dark cycle). All animals had free access to water and normal diet. The study was approved by Institutional Animal Ethical Committee (IAEC) and was in accordance with the guideline of the Committee for the Purpose of Control and Supervision of Experimental Animal (CPCSEA).

Materials and Methods
The animals were handled in accordance with the criteria outlined in the   And the new synthesized drugs action can be illustrated against control, voltarin and indomethacin as the following figures ( Figure 1 to Figure 4).

Discussion
In this experiment the treatment with indomethacin produce elevation in latency time, voltarin produce a significant in-crease in latency time when compared to control animals. Compounds (12 and 13c) showed a very highly significant in-crease in latency time, while a highly significant increase in latency was obtained with treatment of compounds (3a, 5c, 7a-c, 14b), a mild significant increase in response was observed during carried out the experiment with compounds (3c, 10, 11, 13b, 14c, 15, and 5b), finally there is no significant increase Indomethacin (10 mg/kg), voltarin (7 mg/kg), tested compounds (100 mg/kg), was given orally one hour before carried out the experiment. A Significantly different from control group. Using one-way ANOVA followed by Tukey-Kramer test for multiple comparisons at P ≤ 0.05. Data are expressed as means ± SD of six mice per group. Indomethacin (10 mg/kg), voltarin (7 mg/kg) tested compounds (100 mg/kg), was given orally one hour before carried out the experiment. A Significantly different from control group. Using one-way ANOVA followed by Tukey-Kramer test for multiple comparisons at P ≤ 0.05. Figure 2. Analgesic effects of indomethacin, voltarin and tested compounds (6, 7a, 7b, 7c, 8, 9 and 10) in comparison to control non-treated mice using hot plate method.
Data are expressed as means ± SD of six mice per group. Indomethacin (10 mg/kg), tested compounds (100 mg/kg), was given orally one hour before carried out the experiment. a Significantly different from control group. Using one-way ANOVA followed by Tukey-Kramer test for multiple comparisons at P ≤0.05. Figure 3. Analgesic effects of indomethacin, voltarin and tested compounds (11, 12, 13a, 13b, 13c, 14a, 14b and 14c) in comparison to control non-treated mice using hot plate method.
in latency time for the remaining compounds when compared to non-treated animals.

In Vitro Cytotoxicity Activity
In vitro cytotoxicity against the MCF7 and HepG2 cell lines and against human normal cell (Cell culture Protocol).
Cell Line (MCF7 and HepG2) was attained from the American Type Culture Collection.

H. M. Sakr et al. International Journal of Organic Chemistry
Data are expressed as means ± SD of six mice per group. Indomethacin (10 mg/kg), voltarin (7 mg/kg) tested compounds (100 mg/kg), was given orally one hour before carried out the experiment. a Significantly different from control group. Using one-way ANOVA followed by Tukey-Kramer test for multiple comparisons at P ≤ 0.05. Figure 4. Analgesic effects of indomethacin, voltarin and tested compounds (15, 16, 17, 5a, and 5b) in comparison to control non-treated mice using hot plate method. Data are expressed as means ± SD of six mice per group. Indomethacin (10 mg/kg), voltarin (7 mg/kg) tested compounds (100 mg/kg), was given orally one hour before carried out the experiment. a Significantly different from control group. Using one-way ANOVA followed by Tukey-Kramer test for multiple comparisons at P ≤0.05.
Cells were cultured by using DMEM augmented with 10% fetal bovine serum 10 ug/ml of insulin (Sigma), and 1% penicillin-streptomycin. All of the additional chemicals and reagents were taken from Sigma or Invitrogen. Cells were spread in a 96-well flat-bottom microliter plate at a density of 10,000 cells/well and allowed to follow for 24 hours at 37˚C in CO 2 incubator. After 24 hours of incubation. The growth medium was exchanged with a fresh medium. Cells were then supplied with different concentrations of the desired compound for 24 hours at 37˚C. After 24 hours of incubation, the growth medium was changed with a fresh medium. Subsequently [25], 10 µl of MTT working solution (5 mg/mL in phosphate buffer solution) was added to each well and the plate was hatched for 4 hours at 37˚C in incubator [26]. The medium was then extracted, and the produced formazan crystals were solubilized by adding 50 L of DMSO per well for 30 min at 37˚C in a CO 2 incubator. Lastly, the intensity of the dissolved crystals. (Purple color) was counted using the ROBONIK P2000 ELISA reader at 540 nm with a reference wavelength set at 650 nm. The suspension was moved to the cuvette of a spectrophotometer and the OD values were measured at 595 nm by using DMSO as a blank [27], measurements were achieved and the concentration needed for a 50% inhibition of viability (Cs) was determined graphically Standard Graph was plotted by taking the log. The concentration of the drug in the X-axis and relative cell viability in the Y-axis.
Systematic experimental steps were taken to set the potential cytotoxicity of the drug at different concentrations by MTT assay. It shows a decreasing absorbance at 540 nm in the cells which were treated with increasing concentration of H. M. Sakr et al. International Journal of Organic Chemistry the drug in comparison to the control cells without any treatment. There is a decreased absorbance in the cells treated with drugs suggesting cytotoxicity. MTT assay significantly helps the researchers to determine whether any of the test compounds have cell toxicity or proliferative activity. All data were measured in triplicate, and IC50 values are given as mean values USD.

Molecular Modeling
A major challenge of modern medicine is to design compounds that modulate specific enzymes while leaving related isozymes unaffected. The two notable enzymes namely Cyclooxygenase-2 (COX-2) and inducible Nitric Oxide Synthase Nonsteroidal Anti-inflammatory drugs (NSAIDs) like Indomethacin act via inhibition of COX enzyme which catalyzes the first step of the biosynthesis of prostaglandins [28].
Prostaglandins (PGs), found in most of the tissues and organs, are the arachidonic acid metabolites of the Cyclooxygenase (COX) pathway and are major mediators in the regulation of the inflammation and immune function [29]. It has been shown that the COX enzyme exists in two isoforms COX-1 and COX-2 [30]. In terms of amino acid composition, these enzymes are approximately 60% identical, and their catalytic regions are widely conserved [31] [32] [33]. COX-1 enzyme is responsible for maintaining gastric and renal integrity and COX-2 is an inducible enzyme responsible for the production of proinflammatory PGs causing inflammation and pain [34].
The COX-2 inhibitors are effective for the relief of chronic pain in elderly patients with osteoarthritis and rheumatoid arthritis [35].
Inducible Nitric Oxide Synthase (iNOS), is another inducible enzyme, that plays a significant role in the over production of nitric oxide (NO) and has been implicated in several pathophysiological states, for example; various inflammation, septic shock, vascular dysfunction in diabetes and cancer patients [36] [37] [38].
Three homologous NOS isozymes [inducible NOS (iNOS), endothelial NOS (eNOS), and neuronal NOS (nNOS)] catalyze the five-electron, two-step oxidation of L-arginine (L-Arg) to form nitric oxide which is an important biological signaling molecule and cellular cytotoxin [39]. The constitutive isozymes, eNOS and nNOS, function to produce low levels of NO predominantly for blood pressure regulation and nerve function respectively. In contrast, iNOS is induced by microbial products, such as lipopolysaccharide (LPS) and inflammatory cytokines such as interleukin-1 (IL-1), tumor necrosis factor-α (TNF-α) and interferon-γ (INF-γ) in macrophages and some other cells [40]. COX-2 and iNOS over expression has been observed in many human invasive malignant tumors, e.g.
Therefore, the modulation of iNOS and COX-2 can be a good strategy for the management of diseases accompanying the overproduction of NO and PGs.
With our long-standing interest in the transcriptional regulation-based control of inflammation, the objectives of the present study are to obtain binding and inhibitory parameters of Benzimidazole derivatives and Indomethacin (NSAID) on COX-2 and iNOS by means of (MOE) prediction of their absorption and distribution properties.

Material and Methods
Carrageenan induced paw oedema in rats the anti-inflammatory activity of Benzimidazole derivatives was determined by inducing acute in vivo and in silico Analysis Divulges the Anti-Inflammatory Activity inflammation by carrageenan in rats [44]. The Institutional Animal Ethical Committee approved protocols International Journal of Organic Chemistry that were followed for experimental analysis. All the animals were acclimatized for a week before use and were grouped in polyacrylic cages and maintained under standard laboratory conditions. The room temperature was maintained at

Pharmacokinetic Properties
The bioavailability of Benzimidazole derivatives and Indomethacin was determined using PK/DB Database. The ligand structures were manually entered or edited using PK/DB sketcher, then converted into SMILES format and searched for pharmacokinetic properties like human intestinal absorption (%HIA), human oral bioavailability (%F), plasma protein binding (%PPB), blood brain barrier (logBB) and water solubility (logS).