Synthesis and Antimicrobial Evaluation of Novel Carbazole Based β-diketones and its Pyrazole Derivatives

Novel 9-ethyl-9H-carbazole-3-carboxylic acid derivatives including ester, β-diketone and pyrazole were prepared and characterized by FT-IR, 1H NMR, 13C NMR and mass spectroscopic techniques. All synthesized compounds evaluated for their in vitro antimicrobial activities against four bacteria (Escherichia coli, Pseudomonas putide, Bacillus subtilis, and Streptococcus lactis) and three fungi (Aspergillus niger, Penicillium sp and Candida albicans). Among the compounds tested, 3a, 3b, 3c, 4a, 4b, 4c, 5a and 5b exhibited pronounced antibacterial activity as compared with standard drug ampicillin. Notably, carbazole based pyrazole derivatives 5a and 5b showed potent antifungal activity against C. albicans comparable to reference drug greseofulvin.


INTRODUCTION
ISTINGUISHABLE interest of synthetic organic chemists have attracted considerable attention to carbazole frame because of its derivatives that can be easily reformed by introducing various functional groups. [1]These distinct characteristics results in the broad potential applications of carbazole-based derivatives as industrially and pharmacologically important products (Figure 1). [2]any recent literatures have reported that carbazole derivatives exhibit a variety of biological activities such as antimicrobial, [3][4][5] antiviral, [6] anticancer, [7] anti-inflammatory, [8] antimala-rial, [9] antipsychotic [10] and are used in the treatment of obesity. [11]he carbazole carboxylic acid derivatives are significant intermediate because the carboxylic group is one of the active functional group which display an important role in transformation of biological function, these compounds combining low toxicity with high antitumor activity. [12]Functionalized β-diketones are clinically important molecules showing antibacterial, [13] antiviral, [14] insecticidal, [15] antioxidant, [16] potential prophylactic antitumor [17] and pharmacophore of HIV-1 Integrase (IN) inhibitors. [18]The synthesis of β-diketones containing carbazole fragment and their complexes have already been reported, whereas β-diketone containing carbazole fragments still remain unknown, though such βdiketones should be very important and promising for use in optoelectronic materials. [19][24] Insight the literature, carbazole based pyrazole derivatives possesses potent antibacterial and antifungal activities. [25]In continuation of our studies in synthesizing various biologically active compounds, [26,27] in this study, we have synthesized and characterized the novel carbazole assembled esters, β-diketones and pyrazoles derivatives from 9-ethyl-9H-carbazole-3-carboxylic acid and evaluated for in vitro antibacterial and antifungal activities.
The structures of 3(a-e), 4(a-e) and 5(a-e) were confirmed by FT-IR, 1 H and 13 C NMR, and mass spectra.For example, the infrared spectra of 3(a-e) shows an intense absorption band at around 1735 cm −1 for -O−CO group occurs at higher frequencies than that of normal ketones because force constant of the carbonyl bond is increased by the electron attracting nature of adjacent oxygen atom and the 1 HNMR spectrum of 3a contained characteristic singlet at δ 2.55 ppm for CO-CH3 which confirmed the esterification of 9-ethyl-9H-carbazole-3-carboxylic acid 2, aromatic protons resonated in the region δ 7.33−9.01ppm.In the 13  The infrared spectra of 4(a-e) shows a strong and characteristic band for 1,3-diketone linkage at 1677−1590 cm −1 and 2979 cm −1 for -OH stretching.The representative 1 HNMR spectrum of 4b shows disappearance of a singlet at around δ 2.55 ppm (corresponding to CO-CH3) but it displayed two sharp singlets due to two protons at δ 16.27 ppm and δ 12.48 ppm, which confirm the presence of enolic proton (since enol form in β-diketone is more stable) and phenolic -OH adjacent to the carbonyl group respectively. 13C NMR spectra showed a singlet at δ 203.07 ppm due to ketonic carbon and at δ 168.47 ppm due to enolic carbon confirming the keto-enol tautomerism in β-diketone 4b.The negative test for ester, the presence of characteristic 1 HNMR and 13 C NMR peaks are consistent with the structure 4b and aromatic carbon signals of compounds 4(a-e) observed in the region of δ 109.25−142.53ppm.The mass spectrum of 4b displayed a molecular ion peak at m/z 392 [m+1].The infrared spectrum of 5a showed the appearance of absorption band at 3373, 3246 and 1455cm -1 corresponding to NH, OH and C=N functional group respectively.Also, its 1 HNMR spectrum supported its structure, as it revealed the pyrazole ring protons at δ 7.26 and two broad signals at δ 12.61 and 8.80 ppm assignable to OH and NH protons, respectively.The 13

Antibacterial and Antifungal Evaluation
Antimicrobial activity of newly synthesized compounds 3, 4 and 5 was evaluated against two gram negative (E. coli, P. putide), two gram positive (B.subtilis, S. lactis) bacterial strains, and three (A.niger, Penicillium sp, C. albicans) fungal strains using Ampicillin and Greseofulvin as a standard drugs respectively.The inhibition zone diameter (mm) and minimal inhibitory concentration (MIC) values of all synthesized compounds were noted in Table 1.Graphical representations Figure 2   3a

CONCLUSIONS
Novel 9-ethyl-9H-carbazole-3-carboxylic acid derivatives including ester, β-diketone and pyrazole were prepared investigated for their in vitro antimicrobial activities.Among the synthesized compounds, compounds 3a, 3b, 3c, 4a, 4b and 4c showed moderate to promising antimicrobial activities in comparison with standard drug.In addition to compounds 5a and 5b were identified as the most potent antibacterial and antifungal agents compared with reference compounds.As structure activity relationship  (SAR) study of all compounds were taken into account, it was observed that the introduction of carbazole moiety to β-diketone, ester and pyrazole derivatives caused enriched activities against most test organisms.The results also suggested that the antimicrobial activities of the carbazole derivatives were distinctly influenced by the aromatic substituents.Compounds 3a, 3b, 3c, 4a, 4b, 4c, 5a, 5b and 5c with electron withdrawing substituents (Cl and Br) in the  phenyl ring were more potent against most of the tested microorganisms than compounds with electron donating ones.Furthermore, compounds 3e, 4e and 5e without substituent in the phenyl ring showed satisfactory activities against all tested bacterial and fungal strains.High potency and promising antimicrobial activity of newly synthesized compounds 3(a-e), 4(a-e) and 5(a-e) suggest that these compounds could serve as good leads for further optimization and development.

EXPERIMENTAL
The recorded melting points were determined in an open capillary and are uncorrected.IR spectra were recorded on Perkin Elmer Fourier-transform infrared (FTIR) spectrometer from KBr pellets.The 1 H NMR and 13 C NMR spectra were recorded on Bruker Avance II (400 MHz) and Bruker (125 MHz) spectrometer respectively, using TMS as internal standard.Mass spectra were recorded on a Waters, Q-TOF micromass, while HRMS were scanned on Bruker impact HD (ESI-Q-TOF) spectrophotometer.The thin layer chromatography (TLC) was carried out on precoated silica gel aluminum plates to check compound purity.The substituted 2-hydroxyacetophenones are commercially available.

In Vitro Antimicrobial Assay
The antimicrobial activity was evaluated by the agar well diffusion method.The activity was determined by measuring the diameter of inhibition zone (in mm).The samples of the tested compound concentrations (50μL, 1 mg/mL) were loaded into wells on the plates.All solutions were prepared in DMSO, and pure DMSO was loaded as a control.The plates were incubated at 37 ºC for 1-5 days and then were examined for the formation of inhibition zone.Each inhibition zone was measured three times to get an average value.The test was performed three times for each bacterium culture. [31]

Minimal Inhibitory Concentration (MIC) Measurement
The potato dextrose broths and microorganisms susceptibility tests in nutrient media were used for the determination of MIC.The tested compounds stock 1000 µg/mL solutions, Ampicillin and Greseofulvin were prepared in DMSO followed by dilutions to 250−25 µg/mL concentrations.Inoculated microorganism suspensions were incubated at 37 °C for 1-5 days for MIC determination. [31].

General Procedure for Esterification of Compounds 3(a-e)
A mixture of compound 1 (1.36 g, 10 mmol) and 9-ethyl-9Hcarbazole-3-carboxylic acid 2 (2.3 g, 10 mmol) was dissolved in dry pyridine (10 mL).Cooled the flask in an ice bath and phosphorousoxychloride (1.53g, 10 mmol) was added dropwise with constant stirring while maintain the temperature between 0−10 °C.After complete addition of phosphorousoxychloride, the reaction mixture was kept overnight at room temperature, then poured over crushed ice and acidified using cold dilute HCl.The off white solid product obtained was filtered and washed with cold dill.NaHCO3 solution followed by washing with cold water.Crude product was dried and recrystallized from ethanol to obtain the desired product in pure form 3(a-e), which gave a positive test for ester.
C NMR spectra of 3(a-e) showed aromatic carbon signals in the region of δ 108.47−145.59ppm, whereas conjugated carbonyl ester appeared at δ 164.59 ppm and carbonyl carbon at δ 195.67 ppm.The mass spectrum of 3a displayed a molecular ion peaks at m/z 426 [m+1], 427[m+2] and 429 [m+4] confirmed the compound 3a contained two chlorine atoms.
C NMR spectrum of the compounds 5(a-e) showed aromatic carbon signals in the region δ 109.20−140.25 ppm.
Inhibition zone diameters were measured for stock solutions (100μg/mL). (b) Minimal inhibitory concentration (MIC) values. 1 % DMSO was used as control.NA-No activity.

Table 1 .
Antimicrobial activities(a)of the synthesized compounds 3, 4 and 5 against pathological organisms expressed as inhibition diameter zones in millimeters (mm) and (b) MIC (µg/mL, between brackets)