Screening of a Benzimidazole Derivative for Anthelmintic Activity with "Rule of 5" Approach

Purpose: Synthetic chemistry is an important route to develop new therapeutics for every disease. The presented research is focusing on the design, synthesis, evaluation, and Lipinski’s rule approach for a new biologically active benzimidazole derivative. Methods: The synthesis comprises the reaction between lactic acid and o-phenylenediamine (OPD) to produce a corresponding product followed by self-condensation for obtaining a complex multiple. Results: The newly produced composite was found to hold weighty anthelmintic property when we compared the same with Albendazole(Standard drug). When the concentration of the desired compound increases paralysis as well as death time found to decreases respectively. 50μg/ml of the synthesized drug gave a time for paralysis is 30.43 ± 5.33 min & time for death is 0.56 ± 5.32 min. Conclusion: The synthesized final compound showed to have anthelmintic activity and its orally active property was also predicted from the Molinspiration software using physicochemical (RO5) as well as bioactive limits. It can be used for anthelmintic drugs as an oral formulation.


INTRODUCTION
The structural variation of benzimidazole derivatives is so convenient for the expansion of molecules of therapeutic or biological interest. Benzimidazole derivatives with different substitutions on different positions have found to be sources of much pharmacological utility. They are used in the treatment of ulcer, hypertension, viral & fungal infection, cancer, and antihistaminics, etc [1]. The improved benzimidazole form has been produce for utilization as improved drugs. Presently available some important drugs, are Omeprazole Pimobendan, Mebendazole, etc. Various new scheme for the preparation of benzimidazole derivatives have been reported and synthesized with an intention of searching for its enhanced therapeutic values. Anthelmintics are the drugs that kill or removed out parasitic worm from the living body. They are also sometimes called vermicides or vermifuges.
The aim & objective of this study is to produce an innovative derivative of benzimidazole likely to have upright anthelmintic property and to explore its physicochemical properties for RO5 approach & to predict its bioactive score.

Preparation of 1-(1H-benzimidazol-2-yl) ethanol; 2-hydroxy ethyl benzimidazole
O-phenylenediamine (OPD) and lactic acid (Equi-molar fraction) were refluxed till the reactin is completed in the presence of 4N HCl. Thin layer chromatography was used to find out the end of reaction. Recrystallization of the resulting compound was carried out through use of methanol.

Preparation of 1-(1H-benzimidazol-2-yl) ethanone; 2-acetyl benzimidazole
The developed product from step-1 was weighed and taken in a RBF. Then it was oxidized in presence of K 2 Cr 2 O 7 (Potassium dichromate) and refluxed with glacial acetic acid till end of the reaction. Thin layer chromatography was used to determine the completion of the reaction. The residue was collected and kept in room temperature. Then it was neutralizedwith the help of adding ammonia solution dropwise. The resulting product was collected safely and recrystallized.

Preparation of 1,3-bis(1H-benzimid azol-2-yl)-3-hydroxybutan-1-one
The product obtained from step-2 along with 10% NaOH was added in a RBF. The reaction mixture was refluxed till the reaction was complete, on to it glacial acetic acid was added, Finally the content of RBF was filtered by filtering apparatus, the solid residue over the filter paper was dried, and then recrystallized [1].

Preparation of 1,3-bis(1H-benzimidazol-2-yl)-3-hydroxy-5-phenyl-4-pentene-1one
Resulting residue obtained from 3 rd step was allowed to condensed through aryl aldehyde. It was neutralized by means of dil. CH 3 COOH subsequently recrystallization, which gave the ultimate composite with an yields value of 70%. The respective aryl aldehyde (10 mmol) selected was added to 2-acetylbenzimidazole (1.5 g, 10 mmol) in an ethanolic solution of NaOH. It was obtained by adding 40 ml ethanol in 75 mmol sodium. It was then stirred for 5 hours. It was neutralized with 30% CH 3 COOH leading to formation of precipitate. Finally the product was separated out by filtration, and then dried afterward recrystallized with the help of C 7 H 8 (toluene).
Pheretima posthuma were divided into three (03) separate groups and each group contains six earthworms (06). Albendazole ( Standard drug) was diluted with normal saline to obtain a medium of 20 µg/ml. it was considered as the standard and kept in a petri dishes. The compound going to be screened was diluted to prepare a medium of 20 µg/ml concentration. Normal saline served as a control. The time taken for paralysis and lethal time were calculated. When the earthworms found to immobile that time is paralysis time which was noted down for the same. To determine the death condition of every single worm they were exposed to external stimuli frequently that stimulate and induce movement in earthworms if were they alive. Adult Indian earthworms were used because physiologically as well as anatomically it is similar to the intestinal worms of humans. Paralysis and death time of individual worms was measured (Table-2). Paralysis occurred when the worms did not revive even in normal saline. Earthworms were mentioned death when they lost their movement and fading of body color. Scheme:

Approach of "Rule of 5" for Orally Active Drugs [8-9]
By utilizing this rule one can predict orally active drugs from their physicochemical properties such as Molecular weight, Partition co-efficient, Hydrogen bond acceptors, Hydrogen bond donors. All the values of these properties can be obtained by the software Molinspiration cheminformatics. Lipinski's rule says that, if the values of all the properties obey the range as given in Table-1 then the compound must be active orally.

Approach for Bioactive Score [9-10]
The bioactive score can also obtain from the software Molinspiration cheminformatics. It gives the score for GPCR, Ion channel, Nuclear receptor, Protease inhibitor, Kinase inhibitor, Enzyme inhibitor, etc. The score will determine whether the drug is active or inactive. The score can be interpreted using the

RESULTS AND DISCUSSION
A unique derivative of benzimidazole was designed and prepared with a good yielding value using a simple laboratory conditions. The general properties of the final composite were as follows (Table-3).
The structure of the title compound was interpreted and confirmed on the basis of FTIR data (Fig-2). The peak obtained was as follows IR (KBr,cm  Partition co-efficient Less than or equal to five (5) 2 Molecular weight Less than or equal to five hundred (500) 3 Hydrogen bonds acceptors Less than or equal to ten (10) 4 Hydrogen bonds donors Less than or equal to five (5) Table 2. Standard values of the bioactive score [10] Parameter Value Result

Bioactive score
Less than −5.0 Not-active Between −5.0 and 0.0 Moderately active More than 0.0 Active

Bioactive score evaluation
The bioactive score of the title compound as tabulated below (Table-5). It was observed that GPCR ligand, Enzyme inhibitor & nuclear receptor ligand score is more than 0.0, hence the compound is the biologically active compound for the respective receptor. Whereas Ion channel modulator, Kinase inhibitor, Protease inhibitor score is in between 0.0 to -5.0, hence the title compound has moderately active on respective cases.

Physicochemical Properties Evaluation
According to lipinski's rule of five, all the parameters of the synthesized title compound are in the range (mentioned in Table-1). The values obtained were tabulated (Table-6). It can be concluded that the title compound will be an orally active anthelmintic drug.
The topological polar surface area (TPSA) value was found to be 94.67. A value more than 140, usually found to have less permeability in the cell membrane and value below 90, can cross BBB or effective for CNS. Hence the title compound of this research has said to appreciable activity on CNS. [11][12] Molecules violation was found to Zero (0). If the resulted values are more than 1 then it may interfere with the bioavailability. Hence the title compound will better on the aspect of bioavailability. If the rotatable bonds number is 10 or less it may results in an efficient orally active drug [13].

CONCLUSION
Heterocyclic compound, derivatives of benzimidazole have a vital place in the arena of synthetic medicinal chemistry. The attachment of a benzimidazole nucleus in the desired moiety have a significant role for beneficial pharmacologic activity. We designed a original and modest procedure for synthesis of a 1,3-Bisbenzimidazole derivative which exhibited noteworthy anthelmintic property. It was also confirmed that the synthesized compound is orally active with good bioavailability and has an effect on CNS.

DISCLAIMER
The products used for this research are commonly and predominantly use products in our area of research and country. There is absolutely no conflict of interest between the authors and producers of the products because we do not intend to use these products as an avenue for any litigation but for the advancement of knowledge. Also, the research was not funded by the producing company rather it was funded by personal efforts of the authors.

CONSENT
It is not applicable.

ETHICAL APPROVAL
It is not applicable.