Synthesis , Spectral , Anti-Liver Cancer and Free Radical Scavenging Activity of New Azabicyclic Thienoyl Hydrazone Derivatives

To exploit the potential biological activities of azabicyclic based, seven 2r, 4c-diaryl-3-azabicyclo [3.3.1] nonan-9-one-2’-thienoyl hydrazone were synthesized. The structural elucidation and stereochemistry of these compound assigned by FT-IR, 1H, 13C and 2D NMR spectral data. The Structural Activity Relationship (SAR) of the target compounds were examined for their in vitro antiproliferative, antioxidant and antimicrobial activities. The initial screen was treated against human liver cancer cell lines (HepG2) with IC50 values determined by MTT assay. Fluoro substitution at para position of phenyl ring compound 12 showed more antiproliferative activity against HepG2 at half maximum inhibitory concentration (IC50 = 3.76 μg/mL) than other target hydrazones. The mechanism of the antitumor action of active compound 12 was investigated through Hoechst stain 33342 analyses. It indicated that the compound inhibited HepG2 cancer cells proliferation by triggering apoptotic cell death. The Free radical scavenging activity of all synthesized compounds were evaluated with DPPH•, OH• and 2 O − radicals. The compounds 11 (IC50 rang 3.78 4.31 μg/mL) and 15 (IC50 rang 4.61 5.16 μg/mL) were exhibited higher free radical scavenging activity than standard BHT drug. Besides, all the target compounds were screened for their in vitro antibacterial and antifungal activity against a spectrum of microbial organisms by using twofold dilution method. These studies proved that halogen substituted compounds 12, 13 and 14 were showed excellent inhibitory potency at lowest minimum inhibitory concentration (MIC) range of 6.25 25.5 μg/mL. Nevertheless, multiple mechanisms regulating the antioxidant and anticancer effects of the hybrid molecules need to be further investigations.


Introduction
At present, human liver cancer is a major reason for death worldwide.The proportion of people suffering from cancer is estimated to continue growing largely because of the aging of population in most countries [1].Reactive oxygen, nitrogen and sulphur (NOS) are important for an organism's vital activities such as the regulation of biological active compounds.Extreme production of reactive oxygen species causes oxidative stress, chronic diseases and cancer.NOS containing molecules are well known to directly interact with all types of biological molecules, proteins, DNA and lipids [2]- [4].The shared adverse effects are termed oxidative stress.Oxidative stress has involved in the various hallmark abilities of cancer [2]- [4].Research over the past numerous decades has established that antioxidants play a protective role in multistage carcinogenesis [3] [4].Generally, a living organism has protective enzymatic and non-enzymatic antioxidant mechanisms against Reactive Oxygen Species (ROS) induced oxidative damage.Recently, significant attention has focused on identifying synthetic antioxidants that aim several signaling pathways are abnormal in cancer.
The 3-azabicyclo [3.3.1]nonane pharmacophore is present in a wide variety of naturally occurring diterphenoid/nor diterphenoid alkaloids and biological activities [5]- [7].2, 4-Diaryl-3-azabicyclo [3.3.1]nonan-9-one derivatives are showed excellent antitumor [8] and antimicrobial [9] activities.Correlation between cytotoxicity and antioxidant capacity of natural/synthetic compounds [10], we are discovered strong cytotoxicity with poor antioxidant properties of synthesized halogen substituted such as F, Cl and Br compounds may be due to the pro-oxidant effects.Although potent antioxidant often possess strong pro-oxidant activity, found low cytotoxicity in synthetic compounds with electron donating functional groups (-CH 3 , -OCH 3 and CH(CH 3 ) 2 ) present on the aryl rings attached to azabicyclo [3.3.1]nonane-9-ones in line with the observations of Lee et al. [11] Though, multiple mechanisms regulating the antioxidant and cytotoxic effects of the hybrid molecules necessitate to be further investigations.
The half maximum inhibitory concentration (IC 50 ) has measured the effectiveness of synthesized compounds in inhibiting specific biological functions of anticancer activity and free radical scavenging activity.This quantitative measurement indicates how much of a particular drug is need to inhibit a given biological microorganisms by half.It is generally used as a measure of antagonist drug potency in pharmacological research work.The IC 50 values of drug (synthesized target compounds) can be determined by constructing a dose response and investigate the effect of different concentrations of antagonist on reversing agonist activity.IC 50 values can be measured for a given antagonist by determining the concentration required to inhibit half of the maximum biological response of the agonist.Hepatocellular carcinoma (HepG2) is derived from the liver tissue of human.This cell lines obtained from National centre for cell sciences (NCCS), Pune, India and it is a best suitable cell lines to evaluate the cytotoxicity of synthesized compounds.The use of electron donating and withdrawing substituent at aromatic compounds with regards to biological activity has established [12] and several interesting investigations are made through piperidine based heterocyclic compounds and exhibited antimicrobial, antioxidant, antithrombic, calcium antagonist, hypotensive and neuroleptic activities [13]- [16].Hydrazone are noteworthy molecule containing extremely reactive azomethyne (-CONHNC<) are one useful in drug development [17].2-Thiophenecarboxylic acid hydrazide has involved significant attention due to the biological activity which has been widely recognized and practically applied in a number of drugs [18] [19].

Chemicals Equipment Techniques for Structural Elucidation
2-Thiophenecarboxylic acid hydrazide, substituted aldehydes and solvents were purchased from Sigma-Aldrich ® , Himedia ® and Merck ® , Germany, were used directly without further recrystellization.Reaction process and purity were monitored by thin layer chromatography (TLC).Physical properties of melting point were determined in an Electro thermal 9100 instrument in open capillaries and are uncorrected.FT-IR analysis has been recorded in an IR-470 SHIMADZU spectrometer (Shimadzu, Tokyo, Japan.) by making pellet of compound with KBr Scheme 1. Synthesis of 2r, 4c-3-azabicyclo [3.3.1]nonan-9-one-2'-thienoyl hydrazones (9)(10)(11)(12)(13)(14)(15).and values are expressed as v max cm −1 . 1 H, 13 C and 2D NMR spectra were recorded at ambient temperature on a Bruker AMX-400 NMR spectrometer operating at 400.13MHz for 1 H and 100.62 MHz for 13 C.Chemical shift (δ) unit expressed in parts per million (ppm) with respect to TMS.Sample was prepared in CDCl 3 solvent (10 mg in 0.5 mL).Elemental analysis for all synthesized compounds was performed on an Elementor Vario EL-III CHNS analyzer.

Cell Culture and Preservation
Human liver cancer cell lines (HepG2) were obtained from the National centre for cell sciences (NCCS).Pune, India.The cells cultivated in 0.5 mg MTT (3, [4, 5-dimethythiazole-2-yl] 2, 5-diphenyl tetrazolium bromide)/ML. of serum free DMEM (Dulbecco's modified Eagle's medium) at 37˚C with 5% CO 2 and 95% air in a CO 2 incubator.The viability of cells was measured by MTT assay (Mosmann, 1983) [24] using the HepG2 cell lines.The cell lines were treated with various concentrations of test compounds (3.12, 6.25, 12.50, 25 and 50 µg/mL) for 24 h.The 50% inhibitory concentration values (IC 50 ) of all experiments of test compounds were done using three replicates.Cytotoxicity was detected by using Graph pad prism 5 software.

Apoptosis Detection of Nuclear Morphology
Cells were stained with 0.5 mL of Hoechst 33342 solution (3.5 µg/mL) and incubated for 30 min at 37˚C incubator.The Hoechst dyes can also be obtained from molecular probs.H342 is a "vital" DNA stain that binds preferentially to A-T base pains.Cells should be approximately 1.2 × 10 6 mL in buffered media, pH 7.2.It is also useful to include 2 percent fetal half cell serum (FCS) to maintain the cell, and measured by fluorescence microscope at 490 -520 nm [25].Apoptotic cell were defined on the mass of nuclear morphology changes such as chromatin condensation and fragmentation.Early apoptotic cells show navy blue fluorescence.

In Vitro Antibacterial and Antifungal Activity
Minimum inhibitory concentration (MIC) in µg/mL values were found out by two-fold serial dilution method [26].Bacterial strain namely Staphylococcus aureus.Escherichia coli, Klebsiella pheumoniae, pseudomonas aeruginosa and Salmonella typhi and fungal strain namely Cryptococcus neoformans, Candida albicans, Rizopus sp, Aspergillus nigar and Aspergillus flavus, were obtained from Faculty of medicine, Annamalai University, Annamalai Nagar, 608002, India.Seeded broth (broth containing microbial sporus) was prepared in NB from 24 hours old bacterial cultures on nutrient ager (Himedia, Mumbai) at 37˚C while fungal strains were cultures in sabourauds dextrose broth (SDB).Testing was performed at pH 7.4 ± 0.2 for bacteria (NB) and at a pH 5.6 for fungi (SDB).Plating techniques were used to determine the colony forming units (cfu) of the seeded broth in the adjusted range of 10 4 to 10 5 cfu/mL and 1.1 to 1.5 × 10 2 cfu/mL were the final inoculums size for antibacterial and antifungal assay, respectively.The respective test compounds 9-15 were dissolved in dimethyl sulphoxide (DMSO) to obtain 1 mg/mL stock solution.The growth of the microbes in the test medium was measured on the turbidity of the culture after 24 hours of bacterial incubation and 72 -96 hours of fungal incubation.

Statistical Analysis
All investigated data were presented as the percentage of Mean ± SEM (standard error of mean) of at least three individual experiments.Statistical analysis was performed with One-way ANOVA test analysis using the prism 5 statistical software package (graph pad software.USA).Differences were considered as being significant probability at p < 0.005.

Chemistry
The structural elucidation and stereochemistry of target compounds 9-15 were assigned by IR and NMR spectral data.Their purities were checked by elemental analysis Table 1.For compound 9, proton NMR 1 H and 13 C NMR signal were assigned unambiguously using two-dimensional spectra.

Numbering of Atoms
Figure 1 shows the numbering pattern of the bicyclo [3.3.1]nonane part.The ipso carbons of the phenyl rings at C-2 and C-4 are designated as C-2" and C-4", respectively.The other carbons of the phenyl ring at C-2 are denoted also o, m and p carbons and those of the phenyl ring at C-4 were denoted as oʹ, mʹ, pʹ, carbons.Protons are numbered accordingly.Thiophene ring sulpfur atom was denoted as 1'.For illustrated, the benzyl proton at C-2 was denoted as H-2, C-4 was denoted as H-4 and so on.The methylene protons in the cyclohexane ring are  assigned as axial and equatorial protons assuming chair conformation for the cyclohexanone ring.C-7 protons are denoted as H-7a and H-7e.

IR Spectral Analysis
Hydrazones formation were assigned by C=N stretching frequency at present in the range of 1485 -1516 cm −1 .The bicyclic NH stretching frequency was in the range 3300 -3309 cm −1 and the amide (C=O) stretching frequency was in the range 1633 -1641 cm −1 .The amide NH stretching frequency was in the range 3024 -3163 cm −1 , the adsorption band in the region of 2845 -3041 cm −1 were attributed to aromatic, thienoyl ring and aliphatic C-H stretching frequencies [8].due to special interaction between the nitrogen (>CONH-) of thienoyl hydrazone analogue and one of the bridgehead proton.Outstanding to this interaction, partial charges created between them [27].As a result, the bridge head α-carbon acquires slight negative (-ve) charge and the attached proton gets slight (+ve) charge as shown in Figure 4. From this, the deshielded signal can be unambiguously assigned to syn α(H-5) bridgehead proton whereas shielded signal was assigned to anti α(H-1) proton.Among the two observed signal 1.59 ppm and 1.88 ppm were deshielded signal assigned to H-6e and H-8e protons and the shielded one was assigned to H-6a and H-8a protons.Furthermore, the signals for C-7 protons were magnetically nonequivalent and observed at 2.87 and 1.41 ppm respectively for H-7a and H7e.H-7a proton were deshielded due to deshieding creates vanderwaals interaction.Therefore H-7a proton was highly deshielded than H-7e proton 1 H-1 H COSY correlation chemical shift of compound 9 shown in Figure 5, H-2 and H-4 proton have also shown a cross peak with doublet at 7.64 ppm which suggest that the signal have four protons integral is due to ortho protons of phenyl ring.All the aromatic and thienoyl ring protons which were collectively resonate between 7.13 to 8.16 ppm.All these surveillance were consistent with twin-chair (CC) conformation for this compound 9.For all other target compounds the 1 H signal were assigned by comparison with compound 9.

13 C NMR Spectral Analysis
In 13 C NMR spectral of synthesized compound 9, two downfield signals 163.56 and 161.81 ppm were assigned for C=N and C=O carbons, respectively.However, there were two resonance carbon signals at 65.47 and 63.60 ppm is respectively due to C-2 and C-4 carbons.Furthermore, the carbon concerned in the fusion part C-5 and C-1 were consigned from the downfield signal at 38.41 and 46.09 ppm, respectively.The methylene carbons signal C-6, C-7 and C-8 of cyclohexanone were assigned in the region of 27.34, 21.56, and 28.56 ppm, respectively.Aromatic, ipso and thienoyl hydrazone carbons were assigned with admiration to their 1 H- 13 C COSY (HSQC) correlations shown in Figure 6.Assignments for the other synthesized compounds 10-15 were made by comparison with compound 9. X-ray crystallographic study established that 2r, 4c-diphenyl-9t-ethynyl-3-azabicyclo [3.3.1]nonan-9t-ol exists in twin chair conformation with equatorial orientations of the aryl groups [28].Taken together, all of the above observations substantiate planned structure and twin-chair (CC) conformation of 2r, 4c-diaryl-3-azabicyclo [3.3.1]nonan-9-one-2-thienoyl hydrazones (9-15).

In Vitro Free Radical Scavenging Activity
Seven various 2-thienoyl hydrazone derivatives 9-15 were examined for their in vitro antioxidant activity against DPPH • , OH • and • 2 O − radicals.50 percent (50%) inhibitory concentration (IC 50 ) values for the free radical scavenging effect of BHT and various substituted synthetic compounds 9-15 were shown in Table 2. Free radical scavenging activity was treated with different concentration level 3.12, 6.25, 12.5, 25 and 50 µg/mL.We reported that electron-donor methoxy (OCH 3 ) substitution compound 11 (IC 50 range 3.78 -4.31 µg/mL) showed excellent antioxidant capacity compared to other compounds and standard antioxidant, a known antioxidant used as a positive control shown in

In Vitro Anticancer Activity
Human liver cancers foremost reason for human death and malignancy throughout the world, according to survey 7.6 million death in 2010 century and anticipated to reach the figure of almost 13 million death by 2030 century, about 70% cancers [29] [30].The aim of present work challenged cytotoxic activity against on a panel of human liver hapatocellular carcinoma (HepG2) [31] was treated with the innovative target compound 9-15 significantly inhibited the proliferation of cancer cell lines in various concentrations manner (3.12, 6.25, 12.5, 25

Discovery of Apoptosis by Hoechst Staining
In treated and controlled cells, apoptosis in nuclear morphology by using Hoechst 33342 (H342) were experiential and compared in the Figure 8. Fluorescence microscopy analysis was used to assess the apoptotic feature in HepG2 cancer cells induced by fluoro substituted compound 12.The control showed normal health and whole nuclei without any cytomorphological abnormalities.The HepG2 cell lines were treated with 50% inhibitory

In Vitro Antimicrobial Activity
The in vitro antibacterial activity of synthesized compounds 9-15 were treated with bacterial strains viz., (S. aureus.E. coli, K. pheumoniae, p. aeruginosa and S. typhi) and were expressed as minimum inhibitory concentrations (MIC) in µg/mL.Streptomycin was taken as standard drug [32] and the observed MIC values were given in Table 4 Compound 12, 13 and 14 possessing para halo substitution at phenyl rings of azabicyclononane derivatives account for the superior inhibitory effect against S. aureus, K. pheumoniae and S. typhi at MIC values of 6.25 µg/mL, and p. aeruginosa at MIC values of 12.50 µg/mL when compared to standard antibiotic streptomycin.Evidenced that electron withdrawing group substituted azabicyclononane derivatives exhibited marvelous antimicrobial activities [33].Other target compound displayed reduced inhibitory effect against different bacterial strains compared to standard drug.The account of the present study also provide proof for the antifungal  effect of an array of 2-thienoyl hydrazones 9-15 with MIC values shown in the Table 4. Compound 12, 13 and 14 had enhanced inhibitory effect against all tested fungi strains and standard drug (Amphotericin B) of MIC range was 6.25 -12.5 µg/mL, and isopropyl substituted compound 15 exhibited identical potency compared to standard drug except C. neoformans strain.Other compounds exhibited mild inhibitory potency against different fungi strains compared to standard drug.

Conclusion
The report of the present study of 2-thienoyl hydrazones of 2r, 4c-diaryl-3-azabicyclo [3.3.1]nonan-9-ones in excellent yield, stereochemistry and characterized by elemental analysis and spectral data 1D and 2D NMR spectra.All of the above observations substantiate the proposed structure and twin-chair (CC) conformation of all target compounds 9-15.The compounds were evaluated various biological activities.Through the target compound with electron withdrawing such as F, Cl, Br substitution compounds were superior anticancer, antibacterial and antifungal activity.However, the electron donating substituted such as CH 3 , OCH 3 and CH(CH 3 ) 2 compounds act great antioxidant activity than electron withdrawing substituted compounds.Studies on the structural mechanisms by which the new synthetic molecules put forth its anticancer, antioxidant, antibacterial and antifungal activity are making development and will be delivered in the prospect.

Figure 7 . 2 O
Tested with compound 9 devoid of any substituent at phenyl ring at C-2 and C-4 positions of the azabicyclo [3.3.1]nonan-9-one inhibited 50% of the different free radicals at the concentration range 34.26 -35.65 µg/mL.Compound possessing electron withdrawing fluoro, chloro and bromo substitutions at the phenyl rings showed admirable in-vitro free radical scavenging effect against DPPH • , OH • and • − radicals.Therefore, antioxidant capacity of all the synthesized compounds were in the decreasing order 11 > 15 > 10 > 9 > 14 > 13 > 12.

Figure 7 .
Figure 7. Antioxidant activity of standard (BHT) drug and synthesized compounds 9-15.and 50 µg/mL.),after 24 hours of incubation were examined by means of the MTT assay.IC 50 values for the cytotoxic effect of synthetic compounds were shown in Table 3.The incorporation of electron withdrawing group such as F, Cl and Br substitution at para position of C-2, C-4 diaryl rings of the 3-azabycyclo [3.3.1]nonan-9-one compounds 12, 13 and 14 exhibit more cytotoxicity than electron donating group such as CH 3 , OCH 3 and CH (CH 3 ) 2 substitution at para position of C-2, C-4 diaryl rings of the 3-azabycyclo [3.3.1]nonan-9-one compounds 10, 11 and 15.Especially the compound 12 (IC 50 = 3.76 µg/mL) shows excellent cytotoxicity than compounds 13 and 14.We found the strong cytotoxicity with poor antioxidant property of synthesized compounds 12, 13 and 14 may be due to the pro-oxidant effect by halogens[11].The overall cytotoxicity effect of synthesized compounds were in the decreasing order 12 > 13 > 14 > 11 > 15 > 10 > 9. However several mechanisms regulating the antioxidant and anticancer activity of hybrid molecule are needed for advance research.

Figure 8 .
Figure 8.The effect of fluorescent microscope image of (a) control (b) the cell treated HepG2 cell lines with IC 50 concentration 3.76 µg/mL of the most active lead compound 12. Stained with Hoechst 33342 (H342).These cell lines detected by 490 -520 nm of fluorescent microscope excitation/emission.

*
Standard drug used: for antibacterial activity-Streptomycin, for antifungal activity-Amphotericin B.
*The measured values for C, H, N and S were within ±0.4% of the theoretical values.
bValues are expressed as the mean ± SE (standard error) from at least three independent experiments.IC50: Compound concentration required to inhibit cancer cells proliferation by 50%.