Design , Synthesis and in Vitro Evaluation of Antimicrobial and Anticancer Activity of Some Novel α , β-Unsaturated Ketones and their Corresponding Fused Pyridines

Objective: This study aimed synthesis of fused pyridine due to the importance of these heterocycles as antimicrobial and anticancer. Method: Some novel substitutedpyrido[3,2-c]pyran-6-one,pyrido[2,3-c]isoxazole,pyrazolo[4,3-c] pyridine, pyrido[4,3-c]pyrimidine, pyrido[3,2-c]pyrimidine[2,3-b]triazine-2-thione and pyrido[3,2-c]pyrano[2,4-d] pyrimidine-2thione derivatives have been reported to possess various pharmacological activities like antimicrobial and antitumor. Results: A novel series of azoles and azines were designed and prepared via reaction of 3-(arylidene)-1-methy-4piperidonewith some electrophilic and nucleophilic reagents. The structures of target compounds were confirmed by elemental analyses and spectral data. Conclusion: It could be concluded that the tested compounds 14a,e, 13a, 11b and 17d1 have highest antibacterial activity. Compounds 11b, 13a, 14c,e,g, 16e, 17d1 have antifungal activity than antibiotic standard (Nystatin) used. Compound 13a and 25a have shown good anticancer agent.


Synthesis of 1-methyl-3-(4-chlorobenzylidene or 2theinylidene)-2,3,4,5 tetrahydro pyridineoxitne 9a,b.
To a mixture of the same number of gram of 3-(arylidene)-l-methyl-4-piperidone la,b and hydroxyl amine hydrochloride, five time (by volume) of absolute ethanol and pyridine were added the stirred reaction mixture was refluxed for 6-hours.After that the ethanol and pyridine were evaporated, the residue was washed with water and the oxime was obtained as solid, that was profiled by recrystallisation from ethanol.
Claisen condensation of this hydroxy pyridine with ethyl formate, to give condensation product was isolated as the cyclic hemiacetal 2-(4chlorobenzylidene)-8-hydroxy-4-methyl-2,3,4,5tetrahydro pyrido [3,2-c] pyran-6-one (3a).Ring chain tautomerism has been described for 2-hydroxy pyran-4one, shown to exist in solution in an equilibrium between dicarbonyl, keto-enol and the cyclic hemiacetal form.The IR spectrum of compound 3a displayed the characteristic absorption band for the pyran ring carbonyl 1680 cm -1 .One proton singlet in a low magnetic field  3.90 ppm was observed in NMR for 3a, which could be ascribed as hydroxy proton.Treatment Hydroxy chalcones 7awas obtained by reacting compound 2a with4-chlorobenzaldehyde under base catalysed Aldol condensation.Compound 7a was first heated at 100°C for 15 min.with dimethyl sulfoxide and small amount of sulfuric acid, then a catalytic amount of iodine was added the mixture was heated at 100°C for 2h.The major product obtained in 63% yield after purification was characterized as 7-(methylthio)-8-(4-chlorophenyl)-2-(4-chloro-benzylidene)-4-(methyl)-2,334,5-tetrahydro pyrido[3,2-c]pyran-6-one (8a).The structure of 7a and 8a were confirmed on the basis of its elemental analysis and spectral date.Reasonable mechanism 21 for these reactions is that shown in (Figure 2).The starting chalcones isomerises in the presence of concentrated sulfuric acid to the compound 7a which undergoes iodination and reaction with dimethyl sulfoxide to give 7b which converted to7cby loss of methyl iodide, further iodination and dehydrohalogenation leading to 8a.
The synthesis of the oxime 9a,b was carried out by the reaction of α,β-unsaturated cyclic ketones containing anexocyclic double bond 3-(4chlorobenzylidene or 2-thenylidene)-1-methyl-4piperidone 1a,b with hydroxylaminehydrochloride in the presence of pyridine and absolute ethanol as solvent .The conversion of the oximes into the isoxazole derivatives was carried out by heating with iodine and potassium iodide, for eight hours, in a THF-water solution containing sodium bicarbonate, it was noticed that shorter reaction times led to substantially higher yields in the isoxazole synthesis 10b.On the other hand, it was observed that the use of twice the amount of iodine and potassium iodide gave higher yields.On the basis of the results reported by Meisenheimer [21]it could be deduced that the oximes obtained from α,βunsaturated ketones cyclized' to isoxazolines probably according to (Scheme 2), but that they cannot be transformed directly into the isoxazole derivatives without the presence of an oxidizing agent.The formation of isoxazole derivatives by cyclization of unsaturated oxime in the presence of iodine and sodium bicarbonate explained by Büchi and Vederas 21 as shown in (Figure 3) through any of the two intermediatesa and which lead to c and d by internal nucleophilic substitution unsaturated system in conjugation with aromatic substituent on theβ-carbon atom and so the groups on the aromatic ring should have some sort of influence in the cyclization reaction.On the contrary, in adipolar intermediate ion like b, the carbon atom in β of the oximate is positive enough to be attacked easily by an ionic oxygen of this attack involves the cyclic transition state, the substituents should not have an appreciable influence.The Unsaturated Mannich ketones 11a-c have been prepared from the corresponding 3-(arylidene)-lmethyl piperidone la,b, secondary amines (pyrrol or morpholine), formaldehyde and ethanol as solvent in presence of HC1 as a catalyst.The high chemical shifts of their methylene hydrogen δ 2.70 ppm is due to isotopic neighboring effect of the near living carbonyl, influence one of the hydrogens.
The designed target compounds depicted in (Scheme 3)were obtained by reacting the starting material l-methyl-4-piperidone with variety of aromatic aldehydes under aldol condensation conditions to produce the α,β-unsaturated ketone analogues 14a,c-e,f.
Compounds 14a,d,e were subjected to cycloaddition condensation reaction using thiosemicarbazides under acidic conditions yielded only one diastereoisomer of 3-H, 3a-Hcis 15a,d,e which have been separated.The IR spectrum of isolated products showed in each case the absence of carbonyl bond and revealed the presence of two bonds in the regions of 3422-3268 cm -1 due to the NH2 protons, two singlet signal at δ 6.31, 3.48 ppm, for pyrazole -3H and pyridine-3H, in addition to multiplets at 7.66-7.32and2.50 ppm, corresponding to aromatic and methylene protons In order to obtain potentially antibacterial substance, compounds 15a,d,e were further utilized for another cyclocondensation reaction using 4-nitrophencylbromid in refluxing ethanol containing catalytic amount of piperidine to afford the 7arylidene-3-aryl-2-[3-(4-nitrophenylthiazole) 3,3a,4, 5,6,7-hexahydro-5-methyl-2H-pyrazolo [4,3-c]  On the other hand, when the cyclization performed between α,β-unsaturated ketones l4d,e and semicarbazide afforded the mixture of 3-H,3a-Hcis and trans diasteroisomer,7-arylidene-3-aryl-2-carbomoyl, 3,3a,4,5,6,7-hexahydro-5-methyl-2H-pyrazolo[4,3-c] pyridine (17d,e).The structure and relative configuration of the compounds 17d,e have been determined by J H-NMR and 13 C-NMRspectroscopic method.The vicinal H-3, H-3a coupling is 9.30 and 11.50 Hz for isomer , while the value are in accordance with expected ratio 3 Jcis> 3 J trans [22]difference is far two small for affirm differentiation of cis or trans configuration in the case of single compounds without their counter parts moreover, because of broadened signals ,it was not possible to determine the value of this coupling constant for all compounds and in most cases the splitting was between the two values (about 10-11 Hz) measured for the 17d1 and 17d2 On the outlay, the 13 C-NMR field effect arising in the cis isomers is affirm base to identify the C-3, C-3a configurations
The mass spectrum of the product furnished a significant proof the amidine structure 18a.The prominent peaks at m/z=(401), (386) and (385) were produced by loss of neutral molecules of NH3, HN=C=NH and the H-N=C-NH2 radical from the molecular ion (m/z=401).In addition, the second most abundant peak at m/z (CHN) + was a clue that indicated a pyrazolinium species.All cycloadducts 18a-,c gave satisfactory elemental and the spectra data the 1 H-N MR of each compound showed a singlet at  2.50-2.49ppm assignable to the proton at position 3a-H.
Furthermore, refluxing equimolar amounts of 14a,f with hydrozonylhalides and triethylamine for 6 h. in Chloroform gave after work up in each case only one spirocycloadducts.All cycloadducts 19a,f gave satisfactory elemental analyses and spectral data the 1 H-NMR spectrum of each compound showed a singlet signal at 5.50 ppm assignable to proton at position 4 satisfactory elemental analyses and spectral data the 1 H-NMR spectrum of each compound showed a singlet signal at 5.50 ppm assignable to proton at position 4. Compounds 14a,e on reaction with guanidine sulfate yielded 8-arylidene-2-amine or imido-4-aryl-4,4a,5,6,8-hexahydro-6-methyl-2H-pyrido[4,3c]pyrimidine (20a,e).Mechanistically, in presence of base, guandine sulphate is converted to free base which can act as a bidentate nucleophile and facilitate Michael type addition with α,β-unsaturated system or internal condensation with carbonyl group of 14 followed by cyclization will result in the unstable dihydropyrimidine derivatives 20a,e.Structural determination of 20ewasconfirmed on the basis of elemental analysis and spectral data.Its IR spectrum showed amino stretch at 3445,3330 cm' 1 and 1 H-NMR spectrum showed in addition to the aromatic signals, singlet integration for two protons at  5.15ppm which was attributed to the 2amino group protons and doublet atδ6.23ppmintegrating for7H-pyrimidine ring the reaction of 20e with aroylisothiocynates gave two compounds which were separated 21e, 22e, were the intermediate 20'eis not isolated , but it through dehydrocyclization provides a simple and more facile route for the synthesis of biologically active 1,3,5-triazine system21e.This is due to the behaviour of 2-amino pyrimidine molecule in amino-imino form which facilitates condensation and dehydration.Compound 22ewas also obtained in 20-25% yields.The formation of 22e can be explained through elimination of thiocyanic acid from 20 ' 'e.
The IR spectrum of 22e showed the absorption bands corresponding to NH 3388 cm -1 and carbonyl 1660 cm -1 .The 1 H-NMR spectrum of 21e,22e showed a resonance at 6.33 and 6.80ppm corresponding to H-4 of pyrimidine ring.The mass spectrum of 21e furnished a significant proof of the structure 21e.The prominent peaks atm/z (257),(11.5%)and (83) were produced by loss of natural molecules of NH3 and H2S.

Part 2-Biological results
Preliminary screening for the antimicrobial activity of the synthesized compounds using the agar The representative dose responses for the highly active compounds were plotted and the potencies were determined.The antimicrobial activity of the highly active compounds compared with standard antimicrobial agents showed that in case of Bacillus cereus (Tables 1 and 2 and Figure 4) the compound 14g had higher antibacterial activity amongst the tested compounds and is nearly equal to that of the standard antibiotic ceftazidime.
Staphylococcus aureus (Tables 3, 4 and Figure 4) showed higher antibacterial activity against compounds 11b and 17d1 these activities were more higher than that against the standard antibiotics, Todar 23 reported that many of the community associated Staphylococcal infections were now methicillin resistant.These organisms were uniformly resistant to penicillins and cephalosporins.

Cytotoxicity evaluation using viability assay
For cytotoxic assay, the cells were seeded in 96-well plate at a cell concentration of 1 x 10 4 cells per well in 100 μl of growth medium.Fresh medium containing different concentrations of the test sample was added after 24 h of seeding.Serial two-fold dilutions of tested chemical compound were added to confluent cell monolayers dispensed into well, flatbottomed microtiter plates (falcon, NJ, USA) using a multichannel pipette.The microtiter plates were incubated at 37°C in a humidified incubator with 5% CO2 for a period of 48 h.Three wells were used for each concentration of the sample.Control cells were incubated without test sample and with or without DMSO (the little percentage of DMSO present in the wells) was found not to affect the experiment.
After incubation of the cells for 24 hrs.at 37°C, various concentrations of sample (50,25,12.5,6.25,3.125,1.56μg) were added, and the incubation was continued for 48 hrs.and viable cells yield was determined by calorimetric method.In brief, after the end the incubation period, media were aspirated and the crystal, violet solution (1%) was added to each well for a least 30 minutes.The stain was removed and the plates were rinsed using tap water until all excess stain is removed.Glacial acetic acid (30%) was then added to all wells and mixed thoroughly, and then the absorbance of the plates were measured after gently shaken on microplate using a test wavelength of 490 nm.All results were corrected for background absorbance detected in wells without added stain.Treated samples were compared with the cell control in the absence of the tested compounds.All experiments were carried out in triplicate.The cell cytotoxic effect of each tested compound was calculated.

Structural activity relationship A. For Bacillus cereus Gram positive rods)
The compounds 14g,e have highest antibacterial activity due to presence of α,β-unsaturated ketones group presence olefin group 24 and N-methyl piperid-4-one 24 in the structure of the compounds ,all this groups affect activity of the compounds.The compound 13a have antibacterial activity due to presence of pyrazolo [3,2-c] pyridine system and olefin group 24 in the structure of the compound, all this affect activity of the compounds.

B. For Staphylococcus aureus Gram positive cocci
Compounds 11b Mannich ketone compound and 17d1 pyrazolo [3,2-c] pyridine derivative have equal higher antibacterial activity than antibiotic standard used due to the two compounds contain olefin group 24 .

Cytotoxic activity
In this study ,seven of newly synthesized compound that show cytotoxic activity against HEPG2 human liver cell line and MCF-7 human breast cell line using Vinblastine as a standard drug control.Each cell line was incubated with six concentrations (1.56-50g) for each compound and was used to create compound concentration versus survival fraction curves.The response parameter (IC50) was calculated for each cell line (Tables 7, 8).
The IC50 value corresponds to the compound's concentration causing a net 50% loss of initial cells at the end of the incubation period (48 h).
The Cytotoxic activity was measured in vitro on HEPG2 human liver cell line, and MCF-7 human breast cell line using Vinblastine (reference drug control)assay applying the method of Mosmann 25 .
The cells were propagated in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum, 1% Lglutamine, HEPES buffer and 50g/ml gentamycin.All cells were maintained at 37°C in a humidified atmosphere with 5% CO2 and were subcultured two times a week.
Cell toxicity was monitored by determining the effect of the test sample on cell morphology and cell viability.The activity of the tested compounds could be correlated to structure variation and modifications, investigating the variation in selectivity of the tested compounds over the two cell lines, it was revealed that nearly all of the compounds are nitrogen heterocyclic piperdines are an important group of compounds in the field of medicinal chemistry with interesting biological and pharmacological properties.In compound 13a pyrazolo [4,3-c]pyridine have shown good anticancer agent.Presence olefinic group in compound and aromatic ring substituted in pyrazolo ring may be increase the anticancer activity in compound 13a IC50 = 4.70 against MCF-7 human breast cell line higher activity than standard used IC50=l1.60 and against HEPG2 human liver cell line compound 13a showed activity less than standard used IC50 =4.60 and IC50 =23.50 of compound 13a.
In compound 25a presence of pyrimidine-2thion derivative have antitumor activity and Showed activity against MCF-7 human breast cell line IC50=3.75than standard, and showed activity against HEPG2 human liver cell line IC50 = 36.20 less than standard

CONCLUSION
It could be concluded that the tested compounds 14a,e have highest antibacterial activity due to presence of α,β-unsaturated ketones group and the presence of olefinic group.The compound 13a have antibacterial activity due to presence of pyrazolo [3,2-c]   pyridine system and olefinic group.Compound 11b and 17d1 pyrazolo[2,3-c] pyridine derivatives have equal higher antibacterial activity than antibiotic standard.Compounds 11b, 13a, 14c,e,g, 16e, 17d1 have antifungal activity than antibiotic standard used, .Compound 13a pyrazolo [4,3-c] pyridine have shown good anticancer agent.

Figure 2 .
Figure 2. Possible mechanisms for the formation of compounds 7a and 8a.

Figure 3 .
Figure 3.The formation of isoxazole derivatives

Figure 4 .
Figure 4. Response of a series of dose levels at the highly active compounds in the agar diffusion assay using Bacillus cereus.

Figure 5 .Figure 6 .
Figure 5. Response of a series of dose levels at the highly active compounds in the agar diffusion assay using Staphylococcus aureus