Synthesis, Leishmanicidal and anticancer activity of 4-(2-keto-1-benzimidazolinyl) piperidine and 5-chloro-1-(4-piperidyl)-2-benzimidazolinone and their derivatives

In the current study a series of 4-(2-keto-1-benzimidazolinyl) piperidine have been newly synthesized and activities of 5-chloro-1-(4-piperidyl)-2-benzimidazolinone derivatives that is already reported have been tested and compared. Furthermore synthesized derivatives have been characterized by physical and spectral methods (HR-EIMS, HR-FABMS, 1 H-NMR, 13 C-NMR, UV, and FT-IR). All the derivatives were tested for leishmanicidal and anticancer activities. Results demonstrated that among all the derivatives compounds 4, 9, and 10 exhibited signicant cytotoxic effects on HeLa cells and therefore tested at 10, 50 and 100 µM concentrations. Among all the eleven derivatives eight derivatives were found having leishmanicidal activity. Compounds 3, 4 and 10 exhibited signicant activity with IC 50 values in range of 7.06 ± 0.17 µg/mL − 84.6 ± 0.6 µg/mL. Furthermore, an attempt was made to develop a preliminary structure-activity relationship. Compound 4 found to be most active with IC 50 = 7.06 ± 0.17 µg/mL against leishmaniasis. Compounds 4 and 10 possess both leishmanicidal and anticancer activities. From this study we identied a new class of compounds having potent leishmanicidal activity and anticancer activity.


Introduction
There are many similarities exist between cancerous cells and parasitic cell. It leads to the idea that drugs use to combat parasites can be used against cancer or vice versa (Klinkert & Heussler, 2006). Cancer remains one of the greatest challenges. It causes around 13% of all deaths worldwide and it is constantly increasing, particularly in the developed countries. Scientists have to explore either alternative mode of action to treat the disease or design newer and better drug entities to deal with the di culties and sufferings of cancer patients ( Leishmaniasis affects 12 million people around the world with an annual death rate of approximately 80,000 people. It is a widespread parasitic disease caused by protozoan parasite of the genus Leishmania. It causes a broad spectrum of clinical manifestations ranging from self-healing cutaneous lesions to the fatal visceral forms. Clinical manifestations depend on the Leishmania species involved and ranges from a life-threatening systemic infection (visceral, VL) to self-limiting or chronic skin sores (cutaneous, CL), or dreaded metastatic complications that can cause facial dis gurement (mucosal, MCL). The clinical features of VL generally include prolonged and irregular fever, often associated with rigor and chills, hepato-splenomegaly, lymphadenopathy, progressive anemia, weight loss and hyper gamma globulinemia (mainly IgG from polyclonal B cell activation) and concomitant hypo-albuminemia. African and IndianVL patients may present with a secondary form called post kala-azar dermal leishmaniasis (Saha, Mukhopadhyay, & Chatterjee, 2011). Several drugs are available for treatment of leishmaniasis, pentavalent antimonials compounds are drugs used in rst line chemotherapy. As second line amphotericin B and pentamidine isothionate may be used, but current treatment is unsatisfactory due to the route of administration, unaffordable cost and toxic side-effects. That's why scientists are currently working to develop new, effective and affordable molecules as leishmanicidal agents. Fuertes et al reported that anticancer agents may constitute in the near future a good source of lead compounds against leishmaniasis and other parasitic diseases. On the other hand, it is also likely that some antiparasitic drugs may be used for the treatment of certain types of tumors (Carballeira et  In the present study 11 derivatives of 4-(2-keto-1-benzimidazolinyl) piperidine and 5-chloro-1-(4piperidyl)-2-benzimidazolinone derivatives have been synthesized and tested for leishmanicidal and anticancer activity. Numbers of analogues have showed potential for cancer activity.
2 Results And Discussion

Synthesis
We report here the synthesis of 4-(2-Keto-1-benzimidazolinyl)piperidine (KBIP) derivatives 1-6 by reaction of KBIP with a variety of acetophenones represented as R as shown in Table 1

Anti-Proliferation Activity in Treated HeLa Cells
Anti-proliferation activity in treated HeLa cells has been carried out according to the literature protocol(Wang, Yang, Petrenko, & Torchilin, 2010). Among 1-11 synthesized derivatives few compounds exhibited anti-cancer potential at a 100 uM concentration (Table-3). Concentration dependent decline in cellular viability was observed during study. The growth inhibition of HeLa cell line was observed at all concentrations after exposure to these synthesized compounds. Later the percent inhibition was calculated, which revealed greater than 50% inhibition at 100 µM concentration. The IC 50 values of these compounds were calculated using the ED50V10 Excel add-in. The experiment was conducted three times in duplicate. Compounds 4, 9 and 10 exhibited signi cant cytotoxic effect on HeLa cells and tested at 10, 50 and 100 µM concentrations (Table 4 -5). Cytotoxic effect of compounds 4, 9 and 10 were observed after 24 h treatment showing IC 50 of 57.619, 56.594 and 27.235 µM, respectively. The error bars represent the SEM from the mean signi cantly different in treated cells compared to untreated control cells (p < 0.05). The cell titer blue assay was used to assess the metabolic activities of cancer cells. The viable cells showed enhanced uorescence whereas the cells experiencing the cytotoxic effect demonstrated a reduced uorescence. Varying concentrations of the three potent compounds were employed on HeLa cells for 24 h to achieve the IC 50 drug response, which represented the concentration of drug necessary to induce a reduction of 50% in growth. The compounds 4, 9 and 10 exhibited a strong cytotoxic action on human cervical cancer cells, even at the lower concentrations of 10 and 50 µM. It can be elicited that the synthesized compounds 4, 9 and 10demonstrated a decline in cellular functions of human cervical cancer cell as observed by a decrease in uorescence in a concentration dependent manner (Wang et al., 2010).

Leishmanicidal Activity
Numbers of attempts have been made to discover potent antileishmanial drugs with less toxicity. The aim of present study is to investigate the in-vitro leishmanicidal activity of benzimidazolinyl piperidine derivatives in comparison with the standard leishmanicidal drugs amphotericin B (IC 50 = 0.29 ± 0.05 µg/mL) and pentamidine (IC 50 = 5.09 ± 0.09 µg/mL). All synthesized derivatives 1-11 of 4-(2-keto-1benzimidazolinyl)piperidine (I) and 5-chloro-1-(4-piperidyl)-2-benzimidazolinone (II) were evaluated for their leishmanicidal activity, (Saied Saify et al., 2014). Results showed most of them are active againstextracellular promastigotes of Leishmania major. Results are presented in table-6 along with their IC 50 values. Comparing SAR of different Benzimidazolinyl Piperidine derivatives, it can be concluded that the slight changes in structure, functional groups and their position play important role in making compound active or inactive. Among 1-11 synthesized derivatives eight compounds were found active. Compounds 3, 4 and 10 exhibited signi cant activity with IC 50 values in range of (7.06 ± 0.17 µg/mL to 84.6 ± 0.6 µg/mL), Compounds 1 and 7 showed good activities while 2, 8 and 9 exhibited low activity. Furthermore, an attempt was made to develop a preliminary structure-activity relationship for the synthetic compounds. Compound 4 found to be most active with IC 50 = 7.06 ± 0.17 µg/mL against leishmaniasis. Compound 10 also showed signi cant result with IC 50 = 23.24 ± 0.035 µg/mL there is a 3 times reduction in leishmanicidal activity in compound 10 due to the chloro group substituent present at benzimidazolinone ring. Both the derivatives possesses adamantane ring indicated that it is responsible for signi cant leishmanicidal activity. Compound 3 also showed signi cant activity possibly due to the presence of two electron donating methoxy group substituents present at ortho and meta positions of phenyl ring. Compound 1 and 7 also showed good activity. These compounds are phenacyl halide derivative of benzimidazolinyl piperidine. They have uorine group at para position of benzene ring both possesses comparable leishmanicidal activity. Compounds 8 and 9 showed weak activity. On the other hand Compound 5, 6, 11 and parent I showed devoid of activity against leishmaniasis. Parent molecule I found inactive while interestingly II exhibited moderate activity. Generally all newly synthesized derivatives showed promising activity. These investigations suggested that compounds 4 and 10 may serve as lead compound in search of anti-leishmanial drugs. It has been reported that anticancer agents may constitute in the near future a good source of lead compounds against leishmaniasis and other parasitic diseases (Perez et al., 2008). On the other hand, it is also likely that some antiparasitic drugs may be used for the treatment of certain types of tumors. Due to their drug-like properties, this series of compounds can potentially serve as templates for future drug-optimization and drug-development efforts for use as therapeutic agents.

General Experimental Conditions
All reagents used were purchased from Sigma Aldrich Company and organic solventswere of analytical grade. Watt man's lter paper was used for ltration. Thin layer chromatography (TLC) was performed on pre-coated silica gel aluminum plates (Kieselgel 60, 254, E. Merck, Germany). Chromatograms were visualized by UV at 254 and 365 nm.The synthesized derivatives were dried and stored in vacuum anhydrous condition. Melting points of products were noted on Buchi 434 melting point apparatus and are uncorrected. Hitachi U-3200 spectrophotometer used to record Ultraviolet (UV) spectra while methanol used as solvent. Infra-Red (IR) spectra were analyzed on a Jasco 302 Fourier transform FTIR spectrophotometer by making KBR disc method. Mass spectrometry were carried out on Varian Massen spectrometer MAT 311A spectrometer Varian Massen spectrometer MAT 312, MAT 113 DMASPEC system. Nuclear magnetic resonance 1 HNMR and 13 CNMR spectral analysis were carried out at Bruker AM 300, 400 and 500 MHz spectrophotometers. Solvents used were DMSO-d 6 and MeOD. Chemical shifts (δ) were recorded in parts per million (ppm) and coupling constants J in Hertz. HeLa cells were cultured in 75 cm 2 polystyrene culture asks with canted neck and ltered caps (Corning Inc, NY, USA) in DMEM supplemented with 10% FBS and 1% (penstrep) agent. Cells were consistently provided a warm and humidi ed environment of 37°C and 5% CO 2 . Cells were grown to 80% con uence before every experiment. The cancer cells were washed with PBS and were harvested with a solution of trypsin-EDTA whilst in a logarithmic phase of growth.
The cell plating was performed in a 96-well plate, when cells were 80% con uent. After 24 h the drug treatment was performed. Next day cells were exposed to the uorescent dye, celltiter blue (CTB), the indicator compound in the CTB is blue colored resazurin, which is oxidized by the viable cells into red colored resoru n compound. Following 2 and 4 hour of incubation at 37°C, the uorescence is calculated in a spectro-uorometer at 544/590 nm excitation and emission wavelength of a lter respectively (Wang et al., 2010). All experiments were repeated as a minimum of three times. Data from the experiments was assembled and the statistical signi cance between control and treated groups was evaluated by Student's t-test. The signi cance level (p-value) of p < 0.05 was considered signi cant.
In vivo biological potential of eleven derivatives 1-11 were evaluated by using Cervical (HeLa) Cancer cell line. Compounds 4, 9, and 10 exhibited signi cant cytotoxic effect on HeLa cell line. They exhibited signi cant cytotoxic action on human cervical cancer cells, even at the lower concentrations of 10 and 50 µM. It can be elicited that the synthesized compounds 4, 9, and 10 demonstrate a decline in cellular functions of human cervical cancer cell as observed by a decrease in uorescence in a concentration dependent manner. Due to their promising results, they can be selected for further studies as lead molecule.
In vitro Leishmanicidal potentialof benzimidazolinyl piperidine derivatives 1-11 was evaluated using modi ed NNN biphase and RPMI 1640 mediums. Most of the synthesized compounds showed leishmanicidal potential. Compounds 3, 4, and 10 exhibited signi cant Leishmanicidal activities. These investigations suggest that compounds 4 and 10 may serve as lead compound in search of better leishmanicidal drugs.