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 anti-proliferative, antioxidant and antimicrobial activities. The initial screen was treated against human liver cancer cell lines (HepG2) with IC 50 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 (IC 50 = 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 , and radicals. The compounds 11 (IC 50 rang 3.78 - 4.31 μg/mL) and 15 (IC 50 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.
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 [
The 3-azabicyclo [3.3.1] nonane pharmacophore is present in a wide variety of naturally occurring diterphenoid/nor diterphenoid alkaloids and biological activities [
The half maximum inhibitory concentration (IC50) 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 IC50 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. IC50 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 [
In this paper, we report synthesis of seven 2r, (Scheme 1) 4c-Diaryl-3-azabicyclo [3.3.1] nonan-9-one-2’- thienoyl hydrazones highlights the in vitro antioxidant, antitumor and antimicrobial activities against three kinds of free radicals, human liver hepatocellular carcinoma (HepG2) cell lines, bacteria and fungi strain organisms, respectively.
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 (9-15).
and values are expressed as vmax cm−1. 1H, 13C and 2D NMR spectra were recorded at ambient temperature on a Bruker AMX-400 NMR spectrometer operating at 400.13MHz for 1H and 100.62 MHz for 13C. Chemical shift (δ) unit expressed in parts per million (ppm) with respect to TMS. Sample was prepared in CDCl3 solvent (10 mg in 0.5 mL). Elemental analysis for all synthesized compounds was performed on an Elementor Vario EL-III CHNS analyzer.
First 2r, 4c-Diaryl-3-azabicyclo [3.3.1]nonan-9-ones (1-7) were prepared by the condensation of appropriate cyclohexanone (1equiv), respective aromatic aldehyde (2equiv) and Ammonium acetate (1.5equiv) dissolved in ethanol was kept in water bath by maintaining the bath temperature at 60˚C - 75˚C with continuous stirring using literature procedure [
2r, 4c-Diphenyl-3-azabicyclo [3.3.1] nonan-9-one-2’-thienoyl hydrazone 9
IR (KBr) (cm−1): 3034.03, 2924.09, 2854.65 (C-H stretching), 3302.13(-NH-), 1641.42 (C=O stretching), 1510.26 (>C=N stretching), 1H NMR (CDCl3, δppm): 10.214 (s, 1H, HN-C=O), 4.490 (s, 1H, H-2a), 4.386 (s, 1H, H-4a), 3.342 (s, 1H, H-5e), 2.867 (d, 1H, H-7a), 2.76 (s, 1H, H-1e), 1.885 (s, 2H, H-8e, H-6a), 1.590 (m, 3H, H-8a, H-6e and ring NH) 1.417 (s, 1H, H-7e), 8.166 - 7.135 (m, 13H, aromatic and thienoyl ring protons). 13C NMR (δppm): 65.47 (C-2), 63.60 (C-4), 46.09 (C-1), 38.48 (C-5), 28.56 (C-8), 27.34 (C-6), 21.56 (C-7), 142.39 (C-2ʹ), 141.31 (C-4ʹ), 163.56 (C-9), 161.81 (HN-C=O), 141.31 - 126.35 (aromatic and thienoyl ring carbons).
2r, 4c-Bis (p-methylphenyl)-3-azabicyclo [3.3.1] nonan-9-one-2’-thienoyl hydrazone 10
IR (KBr) (cm−1): 3041.74, 2956.87, 2922.16 (C-H stretching), 3307.92 (-NH-), 1633.71 (C=O stretching), 1516.05 (>C=N stretching), 1H NMR (CDCl3, δppm): 10.005 (s, 1H, HN-C=O), 4.476 (s, 1H, H-2a), 4.364 (s, 1H, H-4a), 3.278 (s, 1H, H-5e), 2.901 (s, 1H, H-7a), 2.751 (s, 1H, H-1e), 1.899 (s, 2H, H-8e, H-6a), 1.663 (m, 3H, H-8a, H-6e and ring NH) 1.442 (s, 1H, H-7e), 2.457 (d, 6H, p-
2r, 4c-Bis (p-methoxylphenyl)-3-azabicyclo [3.3.1] nonan-9-one-2’-thienoyl hydrazone 11
IR (KBr) (cm−1): 3032.10, 2926.01, 2845 (C-H stretching), 3302.13 (-NH-), 1639.49 (C=O stretching), 1510.26 (>C=N stretching), 1H NMR (CDCl3, δppm): 9.933 (s, 1H, HN-C=O), 4.447 (s, 1H, H-2a), 4.337 (s, 1H, H-4a), 3.231 (s, 1H, H-5e), 2.885 (bs, 1H, H-7a), 2.716 (s, 1H, H-1e), 1.902 (s, 2H, H-8e, H-6a), 1.774 - 1.567 (m, 3H, H-8a, H-6e and ring NH) 1.452 (s, 1H, H-7e), 3.879 (s, 6H, p-
2r, 4c-Bis (p-fluorophenyl)-3-azabicyclo [3.3.1] nonan-9-one-2’-thienoyl hydrazone 12
IR (KBr) (cm−1): 3024.38, 2920.23, 2848.86 (C-H stretching), 3300.20 (-NH-), 1633.71 (C=O stretching), 1510.26 (>C=N stretching), 1H NMR (CDCl3, δppm): 10.311 (s, 1H, HN-C=O), 4.437 (t, 2H, H-2a and H-4a are merged together), 3.283 (s, 1H, H-5e), 2.849 (d, 1H, H-7a), 2.737 (s, 1H, H-1e), 1.915 (d, 2H, H-8e, H-6a), 1.758 - 1.676 (m, 3H, H-8a, H-6e and ring NH), 1.462 (d, 1H, H-7e), 8.244 - 7.144 (m, 11H, aromatic and thienoyl ring protons). 13C NMR (δppm): 64.14 (C-2), 62.97 (C-4), 46.04 (C-1), 38.38 (C-5), 28.94 (C-8), 27.05 (C-6), 21.11 (C-7), 143.38 (C-2ʹ), 141.40 (C-4ʹ), 163.43 (C-9), 160.99 (HN-C=O), 136.34 - 115.25 (aromatic and thienoyl ring carbons).
2r, 4c-Bis (p-chlorophenyl)-3-azabicyclo [3.3.1] nonan-9-one-2’-thienoyl hydrazone 13
IR (KBr) (cm−1): 3041.74, 2924.09, 2854.65 (C-H stretching), 3309.85 (-NH-), 1633.71 (C=O stretching), 1485.19 (>C=N stretching), 1H NMR (CDCl3, δppm):10.579 (s, 1H, HN-C=O), 4.489 (s, 1H, H-2a), 4.419 (d, 1H, H-4a), 3.437 (s, 1H, H-5e), 2.779 (d, 2H, H-7a and H-1e are merged together), 1.904 - 1.596 (m, 5H, H-8e, H-6a, H-8a, H-6e and ring NH), 1.447 (s, 1H, H-7e), 8.20 - 7.202 (m, 11H, aromatic and thienoyl ring protons). 13C NMR (δppm): 64.12 (C-2), 62.86 (C-4), 45.85 (C-1), 38.25 (C-5), 28.95 (C-8), 27.21 (C-6), 21.43 (C-7), 143.34 (C-2ʹ), 140.66 (C-4ʹ), 164.01 (C-9), 161.51 (HN-C=O), 140.30 - 126.42 (aromatic and thienoyl ring carbons).
2r, 4c-Bis (p-bromophenyl)-3-azabicyclo [3.3.1] nonan-9-one-2’-thienoyl hydrazone 14
IR (KBr) (cm−1): 3039.81, 2966.52, 2922.16 and 2854.65 (C-H stretching), 3300.20 (-NH-), 1639.49 (C=O stretching), 1512.19 (>C=N stretching), 1H NMR (CDCl3, δppm): 10.519 (s, 1H, HN-C=O), 4.465 (s, 1H, H-2a), 4.395 (s, 1H, H-4a), 3.471 (s, 1H, H-5e), 2.798 (m, 2H, H-7a, H-1e are merged together), 1.905 - 1.759 (m, 5H, H-8e, H-6a, H-8a, H-6e and ring NH are merged together), 1.441 (s, 1H, H-7e), 8.229 - 7.206 (m, 11H, aromatic and thienoyl ring protons). 13C NMR (δppm): 64.16 (C-2), 62.92 (C-4), 42.78 (C-1), 38.20 (C-5), 28.95 (C-8), 27.04 (C-6), 21.42 (C-7), 140.75 (C-2ʹ), 140.04 (C-4ʹ), 164.58 (C-9), 163.31 (HN-C=O), 134.80 - 121.23 (aromatic and thienoyl ring carbons).
2r, 4c-Bis (p-isopropylphenyl)-3-azabicyclo [3.3.1] nonan-9-one-2’-thienoyl hydrazone 15
IR (KBr) (cm−1): 3026.31, 2958.80, 2864.29, 2926.01 (C-H stretching), 3304.06 (-NH-), 1635.64 (C=O stretching), 1514.12 (>C=N stretching), 1H NMR (CDCl3, δppm): 10.625 (s, 1H, HN-C=O), 4.505 (s, 1H, H-2a), 4.419 (d, 1H, H-4a), 3.472 (s, 1H, H-5e), 2.990 (d, 3H, H-7a and p-isopropyl
The antioxidant capacity was measured by the 2, 2-Diphenyl-1-picrylhydrazyl (DPPH•) explained by Blois [
Free radical scavenging activity (%) =
where Ac is the absorbance of control and As is absorbance of sample. BHT (Butylated hydroxytoluene) was used as standard drug. Which was calculated based on its concentration of compound required to reduce free radicals by 50% (IC50) as follows.
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% CO2 and 95% air in a CO2 incubator. The viability of cells was measured by MTT assay (Mosmann, 1983) [
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 × 106 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 [
Minimum inhibitory concentration (MIC) in µg/mL values were found out by two-fold serial dilution method [
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.
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
Comp ound | Molecular Formula | Molecular weight | Melting Point ˚C | Yield % | Elemental analysis found* (calculated) in % | |||
---|---|---|---|---|---|---|---|---|
C | H | N | S | |||||
9 | C25H25N3OS | 415.55 | 216 | 93 | 72.52 (72.26) | 6.32 (6.06) | 10.42 (10.11) | 7.52 (7.72) |
10 | C27H29N3OS | 443.60 | 220 | 89 | 73.35 (73.10) | 6.34 (6.59) | 9.68 (9.47) | 7.43 (7.23) |
11 | C27H29N3O3S | 475.60 | 204 | 88 | 68.37 (68.18) | 6.21 (6.15) | 8.76 (8.84) | 6.58 (6.74) |
12 | C25H23 F2N3OS | 451.53 | 224 | 78 | 66.56 (66.50) | 5.32 (5.13) | 9.33 (9.31) | 7.47 (7.10) |
13 | C25H23Cl2N3OS | 484.44 | 210 | 81 | 61.69 (61.98) | 4.58 (4.79) | 8.57 (8.67) | 6.59 (6.62) |
14 | C25H23Br2N3OS | 573.34 | 217 | 82 | 52.34 (52.37) | 4.28 (4.04) | 7.36 (7.33) | 5.89 (5.59) |
15 | C31H37N3OS | 499.71 | 209 | 89 | 74.56 (74.51) | 7.32 (7.46) | 8.77 (8.41) | 6.37 (6.42) |
*The measured values for C, H, N and S were within ±0.4% of the theoretical values.
assigned as axial and equatorial protons assuming chair conformation for the cyclohexanone ring. C-7 protons are denoted as H-7a and H-7e.
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 [
For compound 9 the 1H NMR signals were unambiguously assigned based on the observed correlation in the two dimensional (2D) NMR spectra. A broad and down field singlet at 10.21 ppm was characteristic of the NH amide group. Signal broadening is due to the faster exchange of NH proton with solvent moisture than the resonance time scale. NH proton of the bicyclic ring and H-8a & H-6e protons were merged together and appeared as multiplets at 1.59 ppm. However, signal that appeared at 2.76 and 3.34 ppm should be due to its bridgehead protons H-1e and H-5e, respectively. Two signals appeared at 4.49 and 4.39 ppm corresponds to each one proton integral. Furthermore, these two signal strong NOE shown in
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 [
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 1H-1H COSY correlation chemical shift of compound 9 shown in
In 13C 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 1H-13C COSY (HSQC) correlations shown in
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).
Seven various 2-thienoyl hydrazone derivatives 9-15 were examined for their in vitro antioxidant activity against DPPH•, OH• and
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 [
Compound | R | IC50 values for free radical scavenging activity(µg/mL) | ||
---|---|---|---|---|
DPPH• | OH• | |||
9 | H | 35.23 ± 0.083 | 35.65 ± 0.056 | 34.26 ± 0.010 |
10 | p-CH3 | 8.09 ± 0.074 | 7.56 ± 0.012 | 8.49 ± 0.098 |
11 | p-OCH3 | 3.78 ± 0.056 | 4.31 ± 0.063 | 4.23 ± 0.063 |
12 | p-F | 41.44 ± 0.053 | 40.46 ± 0.098 | 40.26 ± 0.057 |
13 | p-Cl | 37.92 ± 0.077 | 38.59 ± 0.0925 | 37.95 ± 0.090 |
14 | p-Br | 35.01 ± 0.084 | 37.93 ± 0.035 | 36.97 ± 0.091 |
15 | p-CH(CH3)2 | 4.61 ± 0.071 | 4.59 ± 0.039 | 5.16 ± 0.093 |
BHT | - | 5.46 ± 0.064 | 5.66 ± 0.013 | 5.86 ± 0.011 |
and 50 µg/mL.), after 24 hours of incubation were examined by means of the MTT assay. IC50 values for the cytotoxic effect of synthetic compounds were shown in
In treated and controlled cells, apoptosis in nuclear morphology by using Hoechst 33342 (H342) were experiential and compared in the
Compound | Compound concentrations (µg/mL) | Ic50 values (µg/mL) | ||||
---|---|---|---|---|---|---|
3.12 | 6.25 | 12.50 | 25.00 | 50.00 | ||
Death cells (% of Mean ± SE)b | ||||||
9 | 5.371 ± 0.029 | 12.320 ± 0.057 | 20.595 ± 0.042 | 31.372 ± 0.044 | 57.291 ± 0.024 | 42.49 |
10 | 12.277 ± 0.035 | 25.319 ± 0.015 | 34.144 ± 0.041 | 46.320 ± 0.029 | 81.963 ± 0.023 | 26.26 |
11 | 21.275 ± 0.042 | 39.513 ± 0.081 | 48.544 ± 0.037 | 80.648 ± 0.038 | 97.286 ± 0.068 | 12.92 |
12 | 39.312 ± 0.044 | 69.759 ± 0.023 | 84.536 ± 0.002 | 98.365 ± 0.013 | >100 | 3.76 |
13 | 28.202 ± 0.026 | 47.343 ± 0.060 | 79.348 ± 0.025 | 89.341 ± 0.015 | 98.114 ± 0.014 | 6.94 |
14 | 25.382 ± 0.049 | 41.325 ± 0.012 | 79.105 ± 0.073 | 89.405 ± 0.100 | 98.103 ± 0.004 | 8.10 |
15 | 14.196 ± 0.033 | 25.347 ± 0.063 | 39.173 ± 0.018 | 78.252 ± 0.014 | 93.235 ± 0.017 | 17.52 |
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%.
concentration among the efficient cytotoxic compound 12 and IC50 value is 3.76 µg/mL for 48 hours. The dark blue (navy blue) fluorescence indicated the viable cells while the bluish white fluorescence indicated the death cells. The above results of 490 - 520 nm of fluorescence microscopy using Hoechst stain control
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 [
Compounds | Bacterial strains Minimum inhibitory concentration (MIC) in µg/mL | Fungal strains Minimum inhibitory concentration (MIC) in µg/mL | ||||||||
---|---|---|---|---|---|---|---|---|---|---|
S. aureus | E. coli | K. pheumoniae | P.aeruginosa | S. typhi | C. albicans | C. neoformans | Rizopus. sp | A nigar | A. flavus | |
9 | 25 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 50 |
10 | 25 | 50 | 50 | 50 | 25 | 50 | 100 | 50 | 50 | 50 |
11 | 50 | 200 | 200 | 100 | 200 | 100 | 200 | 100 | 25 | 25 |
12 | 6.25 | 12.5 | 6.25 | 25 | 6.25 | 6.25 | 6.25 | 6.25 | 12.5 | 12.5 |
13 | 12.5 | 12.5 | 6.25 | 25 | 12.5 | 6.25 | 6.25 | 12.5 | 12.5 | 12.5 |
14 | 12.5 | 12.5 | 6.25 | 12.5 | 12.5 | 6.25 | 12.5 | 6.25 | 12.5 | 12.5 |
15 | 12.5 | 25 | 12.5 | 25 | 25 | 25 | 50 | 25 | 25 | 25 |
Standard* | 12.5 | 25 | 12.5 | 12.5 | 12.5 | 25 | 25 | 25 | 25 | 25 |
*Standard drug used: for antibacterial activity―Streptomycin, for antifungal activity―Amphotericin B.
effect of an array of 2-thienoyl hydrazones 9-15 with MIC values shown in the
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 CH3, OCH3 and CH(CH3)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.
The authors were great thankful to The Department of Chemistry, Annamalai University, Tamilnadu, India, for the recording of the NMR spectral data’s and providing all necessary facilities to found the present work successfully. We extend our gratitude to the RMMCH, Annamalai University for biological studies.
M.Manimaran,A.Ganapathi,T.Balasankar, (2015) Synthesis, Spectral, Anti-Liver Cancer and Free Radical Scavenging Activity of New Azabicyclic Thienoyl Hydrazone Derivatives. Open Journal of Medicinal Chemistry,05,33-47. doi: 10.4236/ojmc.2015.53004