Int J Pharm Pharm Sci, Vol 6, Issue 7, 59-63Original Article
SYNTHESIS, CHARACTERIZATION, ANTI-MICROBIAL, ANTI-CANCER, AND ANTI-OXIDANT ACTIVITY OF NOVEL 1-(NAPHTHALEIN 2-YL OXY)(PHENYL)(METHYL) THIOUREA MANNICH BASE AND ITS METAL COMPLEXES
M.SIVAKAMI1, B.NATARAJAN1*, M.VIJAYACHANDRASEKAR2, S. RAM KUMAR PANDIAN3 AND KRISHNAN SUNDAR4
1,1*&2Department of Chemistry, SRM University, Kattankulathur, Tamilnadu, India, 3,4Department of Biotechnology, Kalasalingam University, Tamilnadu, India.
Email: sivakamisudhasankar@gmail.com
Received: 08 June 2014 Revised and Accepted: 09 Jul 2014
ABSTRACT
Objective: Mannich bases of 2-naphthol are predominantly popular in metal-mediated and ligand-accelerated catalysis of enantioselective carbon-carbon bond formation. Since these compounds have multiple centres for chelation with metal ions, they are likely to be potent inhibitors of metallo-enzymes. A number of pharmaceutical and agricultural agents have naphthalein framework. Our present study focuses on the synthesis of Mannich base derived from the condensation of 2-naphthol, benzaldehyde and thiourea and its metal complexes and their biological activities.
Methods: The ligand 1-(naphthalein-2-yloxy)(phenyl)(methyl)thiourea (BNBTU) was synthesized by Mannich condensation reaction between 2- naphthol, benzaldehyde and thiourea in 1:1:1 molar ratio. Mn(II), Co(II), Ni(II), Cu(II) and Zn(II) complexes of the new Mannich base BNBTU have also been synthesized.
Results: The anti-bacterial activity of the ligand and all the metal complexes leads to the conclusion that most of the complexes were found to have activities against E.coli and B. subtilis. The cytotoxic effects of the newly synthesized ligand have been found good inhibition activity against the cancer cell line. Further the ligand and the metal complexes have been screened for their fungicidal and anti-oxidant properties and they are found to be significantly active.
Conclusion: The ligand 1-(naphthalein-2-yloxy )(phenyl)(methyl) thiourea (BNBTU) has shown as one of the novel ligand and its coordination with transition metals exhibited enhanced biological activity.
Keywords: Mannich base, Spectral studies, Antimicrobial activity, Anti-cancer activity.
INTRODUCTION
The field of coordination chemistry has grown from a readily defined and limited area into the most active research field especially in drug designing due to their applications in pharmaceutical chemistry. Metal complexes of Mannich bases have been studied extensively in recent times, due to the selectivity of ligands towards various metal ions [1-3]. Mannich bases when form complexes with transition metals can exhibit enhanced microbiological activities due to the presence of metal atoms or other variations of structural factors. Transition metal ions play important role in metabolic activities of living organisms [4].
Metal chelates of Mannich bases having both sulphur and nitrogen as potential donors have been increased much interest in biochemistry because of their versatile use as antibacterial [5],anticancer [6], analgesic, anti-inflammatory [7], anticonvulsant [8], antimalarial [9], antiviral [10], antioxidant [11] and CNS depressant activities [12].
The extensive use of Mannich base metal complexes in biological field lies in the fact that the synthesis of Mannich base ligand introduces the basic function which can provide a molecule soluble in aqueous solvents and they can easily be transformed into a number of compounds. Many research articles are available in the literature for the synthesis of Mannich bases and their metal complexes using 2-naphthol [13,14], benzaldehyde and substituted benzaldehydes [15].
These informations have given a thrust for the synthesis of a new Mannich base from 2-naphthol, benzaldehyde and thiourea using Mannich reaction. Metal complexes of the newly formed Mannich base with manganese(II),cobalt(II),nickel(II),copper(II),and zinc(II) have also been synthesized. All these metal complexes are characterized using different physicochemical techniques.
MATERIALS AND METHODS
All the reagents and solvents used for the synthesis of ligand and its metal complexes were of the highest available purity and used as such received. Micro elemental data were obtained with Perkin-Elmer 2400 analyzer and were found within ± 0.5%. The IR spectra were recorded as KBr pellets on Perkin- Elmer 1000 unit instrument. Absorbance in UV-Visible region was recorded in DMF solution using UV-Visible spectrometer.
The 1H&13C NMR of the ligand was recorded on a Bruker instrument employing TMS as internal reference and DMSO – DMF as solvent. The mass spectral study of the ligand was carried out using LC mass spectrometer.
Magnetic susceptibility measurements at room temperature were made by using a Guoy magnetic balance. Electrical conductivity of metal complexes were measured at room temperature in approximately 10-3M ethanol solution using a Systronics direct reading digital conductivity meter -304 with dip type conductivity cell.
RESULTS AND DISCUSSION
Synthesis of the Mannich base
The ligand 1-(naphthalein-2-yloxy)(phenyl)(methyl) thiourea (BNBTU) was synthesized by Mannich condensation reaction between 2- naphthol, benzaldehyde and thiourea in 1:1:1 molar ratio. 2-naphthol (5g, 0.1mmol), was mixed with benzaldehyde (3.68g, 0.1mmol) followed by adding thiourea (2.5g, 0.1mmol) in DMF solution at room temperature with constant stirring for 24hrs.
After 2 weeks, a light brown colored solid mass was obtained and then washed and dried at 60oC in an air oven and recrystallized from ethanol. The yield of the compound was obtained as 87%. (Fig1)
Synthesis of metal complexes
All the metal complexes of BNBTU were prepared by slow addition of hot methanolic solution of the metal salt with hot ethanolic solution of the ligand in the 1:1 molar ratio. The insoluble metal complexes were formed after 2 weeks. It was washed with methanol and ethanol to remove unreacted metal salt or ligand and then dried in an air oven at 60oC. From the analytical measurements (Table 1) and spectral data, the proposed structures of the metal complexes are shown in figure 2.
Fig. 1: Synthesis of Mannich base BNBTU (1)
Fig. 2: Proposed structures of the metal complexes.
UV-Vis SPECTROSCOPIC STUDIES
The electronic spectra of the metal complexes was recorded for their solution in DMSO in the range of 180-1800nm which is listed in Table 2. The UV-Vis spectrum of Manganese chloride metal complex shows absorption bands at 18021 cm-1, 24967 cm-1, 28305 cm-1 for 6A1g→4T1g6, A1g→4Eg +4A1g, 6A1g→4T2g transitions respectively. The μeff value was found to be 5.31B.M which suggests octahedral geometry.[16-20]. The electronic spectrum of Manganese sulphate complex exhibits four absorption bands at 17980 cm-1, 25227cm-1, 29235cm-1 and 30268cm-1 for 6A1g→4T1g (G),A1g→4Eg,4A1g(G), 6A1g→4Eg(D), 6A1g→4T1g(P) transitions respectively. The μeffvalue of 5.96B.M points to a high spin octahedral geometry.[21]
The Cobalt chloride complex shows four absorption bands at 3860cm-1, 6745cm-1, 14846 cm-1,29096 cm-1 assigned for 4A2(F)→4T2(F), 4A2(F)→4T1(F), 4A2(F)→4T1(P) and charge transfer transition. The μeff value was found to be 4.52B.M which agrees with tetrahedral geometry.The Nickel chloride complex exhibits absorption bands at 3994 cm-1,8457 cm-1,15082 cm-1due to, 3T1g(F)→3T2g(F), 3T1g(F)→3A2g(F), 3T1g(F)→3T2g(P) transitions respectively. The charge transfer transition bands for the chloro compound occur at 25396 and 28473cm-1. The μeff value was found to be 3.86 B.M suggesting tetrahedral geometry[22]. The Nickel nitrate complex shows absorption bands at 7435 cm-1,12566 cm-1 and26317 cm-1 for the transitions 3A2g→3T2g, 3A2g→3T1g(F), 3A2g→3T1g (P) respectively. The μeff value was found to be 3.36 B.M suggestive of distorted octahedral geometry[23].
For the sulphur complex of Nickel it appears at 3892 cm-1,8405 cm-1,15281 cm-1 due to 3T1g(F)→3 T2g(F), 3T1g(F)→3A2g(F),3 T1g(F)→3 T2g(P) transitions respectively. The charge transfer transition bands occur at 26885 cm-1. The μeff value was found to be 5.31B.M in agreement with tetrahedral geometry.
The copper nitrate complex registers absorption bands at 9204 cm-1,10388 cm-1, 11915cm-1 due to 2B1g→2A1g, 2B1g→2B2g, 2Eg→2T2g(F) transitions respectively. The charge transfer transition bands occur at 24062 and 32008 cm-1 suggesting pseudo tetrahedral geometry.The spectra of Zn(II) complexes exhibited bands assigned to L→M charge transfer. They are diamagnetic as expected from the molar conductivity data and magnetic susceptibility measurements. Octahedral structure is assigned for sulphate complex of zinc and tetrahedral structure is assigned for chloride and nitrate complexes of zinc. The molar conductance data indicate that the isolated metal complexes show 1:1 stoichiometry and non-ionic behaviour [24]. The non-electrolytic behaviour of the metal complexes suggests that the anions of the salts have been coordinated with the metal ions.
Table 1: Physical characterization and Analytical data of the ligand BNBTU and its metal complexes
| Compound | Color | Yield % |
Found (Calculated %) | ||||||
| C | H | N | O | S | Cl | M | |||
BNBTU -1 (C18H16N2OS) |
Light Brown | 87 | 69.95 (70.00) |
5.31 (5.28) |
8.98 (9.01) |
5.15 (5.19) |
10.25 (10.40) |
- | - |
MnCl2.2H2O.BNBTU - 2a (C18H20Cl2MnN2O3S) |
Dark Brown | 80 | 45.83 (45.87) |
4.33 (4.29) |
5.87 (5.92) |
10.16 (10.20) |
6.98 (6.92) |
14.92 (14.98) |
11.94 (11.88) |
MnSO4.2H2O. BNBTU - 2b (C18H20MnN2O7S2) |
Dull White | 92 | 43.53 (43.60) |
3.98 (4.02) |
5.48 (5.52) |
22.10 (22.15) |
12.64 (12.68) |
- | 10.95 (11.00) |
CoCl2. BNBTU -3a (C18H20Cl2CoN2O3S) |
Dark Green | 95 | 44.82 (44.88) |
4.50 (4.45) |
5.85 (5.91) |
10.07 (10.12) |
6.52 (6.56) |
14.72 (14.75) |
12.38 (12.43) |
NiSO4. BNBTU - 4a (C18H12Ni N2O5S2) |
Light Green | 88 | 46.85 (46.88) |
3.37 (3.42) |
6.02 (6.05) |
17.22 (17.27) |
13.86 (13.90) |
- | 12.60 (12.67) |
Ni(NO3)2.2H2O BNBTU - 4b (C18H16Ni N4O7S) |
Green | 94 | 42.62 (42.71) |
2.98 (3.06) |
11.85 (11.87) |
23.91 (23.98) |
6.65 (6.71) |
- | 12.23 (12.28) |
Ni Cl2. BNBTU - 4c (C18H16 Cl2Ni N2OS) |
Dull Green | 92 | 49.25 (49.30) |
3.44 (3.48) |
6.28 (6.32) |
3.56 (3.61) |
7.13 (7.17) |
16.11 (16.19) |
13.25 (13.30) |
Cu(NO3)2.BNBTU - 5a (C18H16 CuN4O7S) |
Brown | 90 | 42.19 (42.25) |
3.15 (3.19) |
11.82 (11.86) |
23.05 (23.10) | 6.58 (6.64) |
- | 13.21 (13.16) |
Zn SO4.BNBTU - 6a (C18H17ZnN3O5S2) |
Dull White | 86 | 41.81 (41.87) |
3.67 (3.72) |
9.05 (9.12) |
17.30 (17.36) |
13.84 (13.92) |
- | 14.02 (14.09) |
Zn Cl2.BNBTU - 6b (C18H15Cl2ZnN3OS) |
Brown | 88 | 42.61 (42.67) |
3.55 (3.59) |
9.82 (9.86) |
3.74 (3.79) |
7.43 (7.49) |
16.72 (16.78) |
15.40 (15.50) |
Zn(NO3)2.BNBTU - 6c (C18H20ZnN4O9S) |
Brown | 90 | 40.35 (40.40) |
3.65 (3.68) |
10.28 (10.32) |
26.80 (26.87) |
5.94 (6.00) |
- | 12.15 (12.19) |
Table 2: Molar Conductance (in DMF), magnetic susceptibility, assigned transitions with λmax and Geometry of the metal complexes
| Compound | Λm (ohm-1 cm2 mol-1) |
μeff (B.M) |
λmax (cm-1) |
Transition Assignment | Geometry |
| MnCl2. 2H2O BNBTU - 2a | 72 | 5.31 | 18021 24967 28305 |
6A1g→4T1g 6A1g→4Eg + 4A1g 6A1g→4T2g |
Octahedral |
| MnSO4. 2H2O BNBTU - 2b | 64 | 5.96 | 17980 25227 29235 30268 |
6A1g→4T1g(G) 6A1g→4Eg,4A1g(G) 6A1g→4Eg(D) 6A1g→4T1g(P) |
High spin Octahedral |
CoCl2. BNBTU - 3a |
57.5 | 4.52 | 3860 6745 14846 29096 |
4A2(F)→4T2(F) 4A2(F)→4T1(F) 4A2(F)→4T1(P) CT |
Tetrahedral |
NiSO4. BNBTU - 4a |
96 | 3.15 | 3892 8405 15281 26885 |
3T1g(F)→3 T2g(F) 3T1g(F)→3A2g(F) 3T1g(F)→3 T2g(P) CT |
Tetrahedral |
| Ni(NO3)2. 2H2O BNBTU - 4b | 66 | 3.36 | 7435 12566 26317 |
3A2g→3T2g 3A2g→3T1g(F) 3A2g→3T1g (P) |
Distorted octahedral |
NiCl2. BNBTU - 4c |
45 | 3.86 | 3994 8457 15082 25396,28473 |
3T1g(F)→3 T2g(F) 3T1g(F)→3A2g(F) 3T1g(F)→3 T2g(P) CT |
Tetrahedral |
| Cu(NO3)2. BNBTU - 5a | 93 | 2.26 | 9204 10388 11915 24062, 32008 |
2B1g→2A1g 2B1g→2B2g 2Eg→2T2g(F) CT |
Pseudo-Tetrahedral |
FT-IR ANALYSIS
The coordination mode or bonding sites of the ligand and the metal complexes were investigated with the characteristic absorption bands of the free ligand and the metal complexes. (Table 3) The IR spectrum of the ligand BNBTU show a broad band in the region of 3377 & 3275cm-1 due to νNH stretching and aromatic C-H stretching vibrations. Aromatic C-C and C-H bending were observed as sharp bands at 1462 & 813 cm-1. The characteristic C=S stretching frequency of thiourea for the ligand was appeared in the region of 733 cm-1. In the spectra of the metal complexes, the νNH stretching frequency was found to be decreased thus showing the coordination of nitrogen atom of thiourea with the metal ion. At the same time, the C=S stretching frequency of thiourea is almost same in all the metal complexes thus confirming the non-coordination of sulphur atom of thiourea with the metal complexes. In all the complexes, BNBTU behave as a bidentate ligand coordinating through oxygen of naphthalein ring and nitrogen of thiourea.
1H NMR Data (DMSO/TMS, 500.3MHz):
The 1H NMR spectra of the ligand shows a singlet at 3.383δ due to CH proton of aldehyde. The multiplet between 7.079-7.772 δ corresponds to aromatic protons. The singlet for one proton at 9.716 δ is assigned to amide –NH (7a).
13C NMR Data (DMSO/TMS, 125.7 MHz):
The number of signals of sharp peaks represents the number of carbons of the ligand which are not chemically equivalent. 134.56-108.61 (aromatic carbon atoms), 155.24(bridge head carbon), 183.85(thio carbon) (7b).
LC Mass Data: Calculated for BNBTU C18H16N2OSm/z=308.40; Found 310.15 (M+2) (7c).
Anti-bacterial activity of Mannich base
The minimal inhibitory concentration of ligand BNBTU was found to be 300µg for E.coli and B.subtilis. This is well marked with the reduction A600 with the increase in concentration of drug in the medium. The activity was higher rate at high concentration, at low concentrations survival of bacteria was observed. The inhibitory effect was proved with well-diffusion method and cleared zone of inhibition was observed with Mannich base shown in Table 4. Among nine metal complexes five metal complexes have shown good activity. (Table 4) The ligand BNBTU had shown more activity compared with other metal complexes. The effect of metal complexes as anti-bacterial agents has been discussed in the literature [25]. The decreased activity of other metal complexes is due to poor bioavailability as the result of decreased solubility upon complexation.
Table 3: Characteristic IR spectral data of BNBTU and its Metal Complexes
| Compound | νNH | νC=S | νC=C(b) | νC=N(st) | ν3 | ν4 | ν1 | ν2 | ν5 | νM-X | νM-O |
| BNBTU - 1 | 3377 | 733 | 1462 | 1377 | - | - | - | - | - | - | - |
MnCl2.2H2O BNBTU -2a |
3368 | 739 | 1460 | 1401 | - | - | - | - | - | 476 | 581 |
| MnSO4.2H2O BNBTU -2b | 3277 | 739 | 1466 | 1409 | 1130 | 621 | - | 815 | - | - | 512 |
| CoCl2. BNBTU - 3a | 3372 | 742 | 1506 | 1429 | - | - | - | - | - | 474 | 563 |
| NiSO4. BNBTU - 4a | 3374 | 736 | 1463 | 1405 | 1150 | 624 | - | 813 | - | - | 477 |
| Ni(NO3)2.2H2O BNBTU - 4b | 3325 | 744 | 1543 | 1384 | - | - | 1275 | 819 | 1625 | - | 476 |
| NiCl2. BNBTU - 4c | 3370 | 738 | 1465 | 1397 | - | - | - | - | - | 477 | 571 |
| Cu(NO3)2. BNBTU - 5a | 3296 | 744 | 1554 | 1384 | - | - | 1209 | 816 | 1629 | - | 474 |
ZnSO4.2H2O BNBTU - 6a |
3318 | 740 | 1504 | 1404 | 1169 | 673 | - | 814 | - | - | 476 |
| ZnCl2. BNBTU - 6b | 3401 | 742 | 1507 | 1401 | - | - | - | - | - | 476 | 561 |
| Zn(NO3)2. BNBTU - 6c | 3388 | 740 | 1499 | 1385 | - | - | 1280 | 818 | 1499 | - | 476 |
Table 4: Diameter of inhibition against bacteria in millimeter (mm) by BNBTU and its metal complexes
| Compound | E.coli | B. subtilis |
| BNBTU - 1 | 2.0±0.2 | 1.9±0.3 |
| MnCl2. 2H2O.BNBTU - 2a | 2.5±0.1 | 2.3±0.2 |
| CoCl2. BNBTU - 3a | 2.2±0.3 | 2.1±0.05 |
| NiSO4. BNBTU - 4a | 1.8±0.4 | 1.7±0.2 |
| Ni(NO3)2. BNBTU - 4b | 1.6±0.5 | 1.7±0.3 |
| Ni Cl2. BNBTU - 4c | 1.4±0.2 | 1.3±0.2 |
Cytotoxicity of Mannich base
The effect of Mannich base ligand against cancer cells was analyzed by the MTT assay. The drug was able to reduce the viability of HeLa cells in a dose-dependent manner, as shown in Fig. 4. The IC50 value of the enzyme was found to be 250 µg/ml when the cells were treated with the drug for 24 hrs.
These results proved that the cytotoxic nature of the Mannich base against HeLa cells was effective. IC50 concentration was used anti-oxidant assays. The result was concurred that Mannich base unveil the dose dependent toxicity against cancer cells.
When the concentration of the ligand goes beyond 250 µg/ml, more than 50 % of the cells shown ruined structure. Based on the studies, scientists agreed that Mannich bases having the potential to inhibit the proliferation of cancer cells [26]. The ligand had shown greater effect against cancer cells than normal cells (Fig. 3).
Fig. 3: The effect of the ligand BNBTU in inhibition of the growth of cancer cells.
Effect on iron reduction as anti-oxidant
The metal complexes exhibited anti-oxidant activity as, measured by DPPH method. These assays prove that metal complexes have the ability to scavenge free radicals generated in vitro by donating hydrogen atom [27]. The metal complex at a concentration of 250 µg/ml demonstrated equal or higher activity than the standard anti-oxidants analyzed as illustrated in Table 5. Observing the outcomes from DPPH assay, it confirms that the metal complexes act as anti-oxidant agents. BNBTU coordinated with MnCl2 had shown greater anti-oxidant effect compared with other compounds.
Table 5: Anti-oxidant activity of selected metal complexes
| Compound | Anti-oxidant Activity |
| MnCl2. 2H2O.BNBTU - 2a | + |
| CoCl2. BNBTU - 3a | + |
| NiSO4. BNBTU - 4a | + |
| Ni(NO3)2. 2H2O.BNBTU - 4b | + |
| Ni Cl2. BNBTU – 4c | + |
Anti-fungal activity
The results from well-diffusion assay confirmed that the ligand and the metal complexes have the potential of inhibiting fungal growth. Samples 4 and 7 were shown the inhibition against fungal growth. The inhibition zones were measured and compared with controls. At the concentration of 400µg/ml the metal complexes potentially increase the clear zone against the growth of the fungus. This demonstrates that Mannich base and the metal complexes have the anti-fungal activity (Table 6). The antifungal activity of each compound was compared with standard drug Flucanozole. Among screened compounds, ligands with CoCl2, NiSO4, Ni(NO3)2, Cu(NO3)2 emerged as active against fungal strains. Mannich bases are physiologically active because of the molecule solubility in aqueous phase. Compared with other compounds the ligand BNBTU show cases its potential in reducing the growth of fungus [28].
Table 6: Anti-fungal activity of selected metal complexes
| Compound | A. niger | C.albicans |
| CoCl2. BNBTU - 3a | 3 | 7 |
| NiSO4. BNBTU - 4a | 5 | 5 |
| Ni(NO3)22H2O. BNBTU - 4b | 3 | 9 |
| Cu(NO3)2. BNBTU - 5a | 2 | 3 |
CONCLUSION
This paper describes the summary of Mannich reaction, its important properties and also discussed about the metal coordination and their biological importance. Based on the spectral data, the ligand behaves as bidentate through the oxygen atom of 2-naphthol and nitrogen atom of thiourea. The biological activity of the synthesized compound and the metal complexes shows marked activity against the selected micro organisms. The cytotoxic effect of the newly synthesized ligand BNBTU have been found good inhibition activity against the cancer cell line.
CONFLICT OF INTERESTS
Declared None
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