Synthesis , Antibacterial , Anthelmintic and Anti-Inflammatory Studies of Novel Methylpyrimidine Sulfonyl Piperazine Derivatives

Uma síntese estratégica de novas piperazinas sulfonil metilpirimidina envolvendo acoplamento Suzuki foi desenvolvida e as atividades farmacológicas dos compostos foram avaliadas. As reações foram realizadas pelo método convencional e apresentaram boas transformações dos grupos funcionais e elevados rendimentos. As estruturas dos novos compostos sintetizados foram estabelecidas por espectroscopia no infravermelho (IR), ressonância magnética nuclear (NMR) de H e de C, e cromatografia líquida-espectrometria de massas (LC-MS) e análise elementar. Os compostos foram testados quanto à atividade antibacteriana in vitro contra as cepas bacterianas Escherichia coli e Staphylococcus aureus, à atividade antihelmíntica utilizando Pheretima posthuma e à atividade anti-inflamatória envolvendo o modelo de edema de pata de rato induzido pela carragena. Provou-se que alguns compostos são farmacóforos potentes.


Introduction
Suzuki-Miyaura coupling involves the cross coupling of organic halides and organic boron compounds, it is a strong synthetic tool for carbon-carbon bond formation via borylation. 1 This coupling procedure is used to synthesize different varieties of biologically necessary intermediates.Delicate and effective amidation of carboxyl acids with amines is the most elementary and necessary reaction in organic synthesis.In nature, amide bond is one of the most important functional group that constitutes the backbone of the biologically important peptides and is found in several natural products and also in the majority of pharmaceutically fascinating compounds containing amide bond. 2,3Pyrimidine, piperazine and sulfonamide derivatives represent a category of pharmacologically interesting compounds having diverse biological activities.Intensive research has been carried out on the synthesis and analysis of pharmacological activities of these derivatives.Substituted sulfonamide derivatives are important category of pharmacophores that have a wide spectrum of pharmaceutical accomplishments: as antimalarial, 4 anti-microbial, 5 anti-bacterial, 6,7 anti-cancer, 8 anti-fungal, 9 anti-helmintic, 9 anti-oxidant, 10 anti-HIV, 11 anti-tumor, 11 antiplasmodial, 12 anti-neoplastic, 13 anti-proliferative, 14 activities and additionally known to act as 5-HT 6 , 5-HT 7 receptor antagonists, 15,16 A2B and CXCR3 Vol. 25, No. 6, 2014   antagonists, 17,18 11β-HSD, 19 histone deacetylase (HDAC) inhibitors, 20 β-secretase (BACE1) inhibitors 21 and dual PI3K/mTOR inhibitors. 22wing to our interest in developing new biologically active pyrimidine sulfonyl piperazines, our group has previously reported the new series of substituted methylpyrimidine piperazine methanones and their biological activities. 23n this article, we are intended to report the synthesis of methylpyrimidine sulfonyl piperazines.Literature survey revealed the paucity of information on these functionalities incorporated in one molecule and their collective biological significance.Prompted by these facts, it was designed and synthesized a new series of molecules having all the three nuclei in a single molecule as shown in Scheme 1 and studied their communal pharmacological impact as potent antibacterial, anthelmintic and anti-inflammatory agents.

Experimental
Melting points (mp) reported were determined in open capillary and are uncorrected.Purification of the newly synthesized sulfonyl piperazine methanone derivatives was made by column chromatography using silica gel 60-120 mesh size and petroleum ether/ethyl acetate (7:3) as solvent system.Reactions were monitored by thin layer chromatography (TLC), visualization was done using UV light (254 nm) chamber.Characterization was done by Fourier transform infrared (FTIR) spectra recorded on a Jasco FT-IR spectrometer. 1 H and 13 C nuclear magnetic resonance (NMR) spectra were recorded at 400 and 100 MHz, respectively, on a JEOL ECX NMR spectrometer using CDCl 3 as solvent and tetramethylsilane (TMS), respectively, as internal standard.Liquid chromatogram with mass spectrometry detection (LC-MS) was obtained on a Waters Alliance 2795 separation module and Waters Micromass LCT mass detector and elemental analysis (C, H, N and S) was performed on an Elementarvario Micro cube analyzer.

Antibacterial activity
In vitro antibacterial activity of the sulfonyl piperazine methanone derivatives (11a-j) was studied against Gram positive Staphylococcus aureus (NCIM-5022) and Gram negative Escherichia coli (NCIM-5051) bacterial strains.All the bacterial strains were procured form CSIR-National Chemical Laboratory (NCL) (Pune, India).Agar well diffusion method was incorporated for the study, 24 broth cultures of bacterial strains were incubated for 24 h and were uniformly smeared on sterile nutrient agar medium in each petri plates using sterile L-shaped glass rod.Five uniform wells with 6 mm diameter were bored using cork borer to accommodate 50 µL of solution in each well.Samples were dissolved in dimethylsulfoxide (DMSO), a negative control which showed no zone of inhibition and ciprofloxacin (10 µg 50 µL -1 ) was taken as standard drug, a positive control was purchased from Himedia (Mumbai, India).Concentrations of 2.5, 5, 10 and 20 µg 50 µL -1 were used to assess the dose dependent activity of test samples.Sterile micropipette tips were used to load the wells with appropriate amount of sample, control and standard.Then, the plates were incubated at 37 °C for 36 h.After the incubation period, the diameter of the zone of inhibition of each well was measured in mm; the experiment was performed in triplicates.

Anthelmintic activity
Anthelmintic activity of compounds (11a-j) was done using P. posthuma (Indian Earthworm), worms were maintained under normal vermicomposting medium with adequate supply of nourishment and water for about 3 weeks.Adult earthworms of approximately 4 cm in length and 0.2-0.3cm in width were chosen for experiment.Different concentrations 50 and 100 mg of samples were evaluated as per the standard method reported. 25Five groups, each with six earth worms, were taken.Each P. posthuma was washed separately with normal saline before the initiation of experimental procedure and was placed into a 20 mL of normal saline.Group I earthworms were placed in 20 mL saline in a clean petri plate and group II earthworms were placed in 20 mL saline containing standard drug piperazine citrate (50 mg mL -1 ).Similarly, group III to XXII earthworms were placed in a 20 mL saline containing 50 and 100 mg mL -1 of test samples, respectively.Observation was done keeping time taken for paralysis and the time taken for death as objective and was documented in minutes.Paralysis time was analyzed based on behavior of the worms with no revival body state in normal saline medium.Death was concluded based on total loss of motility with faded body color. 26

Anti-inflammatory activity
Effect of compounds (11a-e) on carrageenan induced paw edema was studied on albino Wistar rats of either sex which were obtained from Sree Venkateshwara Enterprises Bangalore.The animals were acclimatized, maintained under standard laboratory condition for a week.Given free access to UV purified and filtered water and standard pelleted feed procured from M/S Pranava Agro Industries Sangli, Maharastra, ad libitum.All the studies conducted were approved by the Institutional Animal Ethics Committee (IAEC) of Sree Siddaganga College of Pharmacy, Tumkur (Ref No. SSCPT/IAEC.Clear/141/2012-13).The standard was indomethacin from Research Lab Fine Chem Industries, (B.No. 99550509, Mfg date: May 2009, exp.date: May 2014).Test compounds (100 mg kg -1 body weight) and indomethacin (10 mg kg -1 body weight).The dose selected in the present study is done on the previously published literature which dealt with pyrimidine, piperazine and Vol. 25, No. 6, 2014   sulfonamide as moieties in molecules exhibiting anti-inflammatory activity. 27,28Test compounds were made it into suspension by using 1% carboxy methyl cellulose (vehicle) and administered through oral route.
Carrageenan induced rat paw edema 29 experiment was carried out on test system (albino Wistar rats) weighing between 150-180 g randomly divided into seven groups of six animals each and was fasted overnight.Group I served as carrageenan inducer (control) and received vehicle, group II standard indomethacin (10 mg kg -1 body weight) through oral route.Group III to group VII were administrated with test compounds (11a-e) in the dose of (100 mg kg -1 body weight) and after administering samples/ indomethacin/vehicle, test systems were kept under clinical sign observation for 30 min and suspension of carrageenan (0.1 mL of 1% kg m -3 ) was injected into the sub-plantar region of right hind paw of each test system.The paw volume was measured by using digital plethysmometer (IITc Life science, USA), immediately after injection, again at 30, 60, 120 and 180 min intervals.

Statistical analysis
The data of antibacterial and anthelmintic were expressed as mean ± standard error (SE) of triplicates and six Pheretima posthuma in each group.The difference in values at p ≤ 0.01 was considered as statistically significant.The analysis of variance (ANOVA) was performed using ezANOVA (version 0.98) software to determine the mean and standard error of the inhibition zone in antibacterial activity and standard error of paralysis and death time of earthworms.Anti-inflammatory data were expressed as mean ± standard error of the mean (SEM) of seven groups of six albino Wistar rats in each group, the difference in values at p < 0.05, p < 0.01 and p < 0.001 when compared with carrageenan control using ANOVA followed by Dunnett multiple comparison test on Graph Pad Prism 5.

Results and Discussion
The synthetic pathway for the synthesis of compounds (11a-j) is illustrated in Scheme 1.Initial borylation of (1) with (2), in presence of Pd(dppf)Cl 2 , methylene chloride, potassium acetate and 1,4-dioxane and water afforded (3).Treatment of (3) with (4) in 1,4-dioxane:water using potassium carbonate and catalytic quantity of dikis, underwent Suzuki coupling to give (5).Upon hydrolysis of (5), it gave (6) and it was further condensed with (7) in presence of triethylamine and dehydrating agent T3P ® to get (8) a key intermediate.Deprotection of N-boc group from (8) was done by using trifluroacetic acid to get (9)   a pre-final product.Compound (9) revealed to be the important intermediate which was reacted with appropriate substituted aryl/alkyl/hetro-substituted sulfonyl chlorides to get pyrimidine sulfonyl piperazine derivatives (11a-j) in good yield as reported in Table 1.
Initial investigation of the antibacterial screening data revealed that all tested compounds (11a-j) showed activity against E. coli and P. aeruginosa, data given in Table 2. Test samples upon detailed investigation for activity at different concentrations of 2.5, 5, 10 and 20 µg 50 µL -1 against E. coli and P. aeruginosa, respectively, given in Table 3. Minimum inhibitory concentrations (MIC) obtained by micro dilution method were observed to be 10 µg 50 µL -1 .Data reveal that the tested samples are active at and above the concentration level 10 µg 50 µL -1 which is the minimum inhibitory concentration.Further the compounds were screened at 10 and 20 µg 50 µL -1 against E. coli and P. aeruginosa, respectively and it is evident that all the tested compounds showed activity against tested strains.Evaluation of anthelmintic activity using P. posthuma showed comparably moderate results with that of standard drug piperazine citrate, given in Table 4. Earthworms belonging to control group showed paralysis time at 142.67 ± 1.45 min and death time at 168.00 ± 1.53 min.For the sample tests (11a-j) upon detailed anthelmintic activity study at different concentrations 15, 25, 35 and 50 mg mL -1 , shown in Table 5, it was observed activity at concentration of 50 mg mL -1 and above.Further testing of samples at 50 mg mL -1 and 100 mg mL -1 concentrations showed the time (in min) of paralysis and death as reported.Investigation of anthelmintic activity revealed that methylpyrimidine sulfonyl piperazines were potent anthelmintic agents.
Increase in paw volume was observed in all time intervals after the administration of carrageenan (1%, 0.1 mL).Samples (11a-e) were administered orally to test  systems at dose of 100 mg kg -1 body weight, significantly (p < 0.05, p < 0.01 and p < 0.001) inhibited the carrageenan (control) induced paw edema.Treatment with standard drug indomethacin also significantly (p < 0.001) inhibited the carrageenan induced paw edema.At 30 min interval, no significance was observed for all the tested compounds against carrageenan induced paw edema.At 60 min interval, for compounds 11a, 11b, 11d and 11e, it was moderately significance (p < 0.01), in addition at 120 min interval for compounds 11a and 11c, it was also showed moderate significance (p < 0.01).Finally, results obtained at 180 min interval were comparably significance (p < 0.001) to standard drug indomethacin (Figure 1).
Values are expressed in mean ± SEM for five animals in each group.ANOVA followed by Dunnett multiple comparison tests.Values are statistically p < 0.05, p < 0.01 and p < 0.001 when compared with carrageenan control.
Successful design and synthesis of methylpyrimidine sulfonyl piperazines with different R group (alkyl, cycloalkyl, substituted hetero and aromatic rings) in the final molecule in search of better antibacterial, anthelmintic and antifungal agents were achieved.Further evaluation of the antibacterial activity against E. coli and P. aeruginosa bacterial strains, anthelmintic activity against P. posthuma model and antiinflammatory activity studied on carrageenan induced rat paw edema model was successfully carried out.The results show new group of sulfonamides with a promising activity.

Conclusion
Synthesis of pyrimidine sulfonyl piperazine derivatives resulted in potent antibacterial, anthelmintic and anti-inflammatory agents.Compounds 11b, 11c, 11f, 11g, 11h, 11i and 11j were proven to be good antibacterial and anthelmintic agents.Compounds 11a and 11c were proven to be good anti-inflammatory agents.Further studies for better therapeutic activities can be observed by functional group modifications and detailed toxicity studies will be taken up in future.

Table 1 .
Physical and analytical data of methylpyrimidine sulfonyl piperazine derivatives 11a-j