An efficient synthesis of 2-(6-methoxy-2-napthyl)-1,3-benzoxazole derivatives using IBD/LTA: Reactivity, DFT, Anticancer and Larvicidal activities

 

Jayanna N. D.¹*, Vagdevi H. M², Dharshan J. C³, Manjuraj T.², Shreedhara S. H².

¹Department of Chemistry, S. S. M. S. College, Athani, 59104, Belagaum, Karnataka, India.

²Department of Chemistry, Sahyadri Science College (Autonomous), Shimoga 577203, India.

 

ABSTRACT:

A new route have been developed in the cyclization of Schiff’s bases 2-(6-methoxy-2-naphthyl)methylene]amino}phenol derivatives using Iodobenzoxydi acetate (IBD)/Lead tetra acetate (LTA) to yield 2-(6-methoxy-2-napthyl)-1,3-benzoxazole derivatives 3a-f. IBD is the most efficient and effective and acts as oxidant in the synthesis of target molecules with better yield than LTA. The obtained products have been characterized by IR, ¹H NMR, ¹³C NMR and Mass spectral studies. The DFT-based optimization structures, bond length, bond angle, HOMO-LUMO energy gap were calculated by B3LYP/LANL2DZ level of theory. The synthesized compounds have been exhibited encouraging anticancer and larvicidal activities. The compound 3c exhibited higher mortality as compared melathione standard against Aedes aegypti mosquito larvae. The cytotoxicity assay applied to determine anticancer activity of the compounds against HT-29- Human colorectal adenocarcinoma cell line. The anticancer activity revealed that the compounds 3a, 3c, 3d and 3f were the most active compounds in the series towards HT-29- Human colorectal adenocarcinoma cell line by total lysis with minimum concentration and supported by in silico molecular docking studies.

 

 

 

INTRODUCTION:

The oxazole containing heterocyclic compounds plays a vital role in medicinal chemistry and exhibit wide range of biological activities and finds use as a starting material for the synthesis of larger bioactive structures, and is found within the chemical structures of some pharmaceutical drugs. Five-membered heterocyclic rings, such as benzoxazoles, benzothiazoles, and benzimidazoles, are present in natural products, and in synthetic pharmaceutical and agrochemical compounds¹

 

These compounds have been extensively studied for their biological and therapeutic activities, such as acathepsin S inhibitor², a HIV reverse transcriptase inhibitor³, an anticancer agent ⁴ and an orexin-1 receptor antagonist⁵. The benzoxazoles have been in the focus line for the researchers from many years due to imported as class of heterocyclic compounds⁶. Specifically 2-substituted benzoxazoles were importantly studied trusting that this position is leading for the biological activity whereas position 5 existing the intensity of activity. The oxidative intramolecular cyclization of phenolic Schiff base is well known method and the oxidants have an effect on yield in oxidative ring-closure reactions. Some reagents have been used for oxidative cyclization of Schiff bases, including lead tetra acetate. Because of the low toxicity, ready availability and easy handling and good yield, the organo hypervalent iodine reagents⁷ such as iodobenzenediacetate (IBD)⁸ have been broadly used in organic synthesis. In connection with our recent interest in catalyzed C-C bond formation, we envisioned that by the addition of the o-hydroxy phenol to the Schiff base, which generated in situ from the condensation of Substituted 2-aminophenol with 6-methoxy napthaldehyde, the Schiff base could be further oxidized into substituted benzoxazole in situ under air or oxygen atmosphere with IBD and LTA catalyst. The LTA is used one of the oxidant commonly; however, it has found that LTA is not suitable for preparation of target molecules due to the low yield factor⁹.

 

According to the Indian Cancer Society, about 1.5 million people suffer from cancer at any point of time in India. At present scenario, development of drugs with target specific predefined anticancer potential is more essential to fight against various types of cancers. Recently, the EGFR inhibitory activity has been hypothesized to possess therapeutic potential for treatment of cancer. So there is a need for rapid and efficient computational methods capable of differentiating compounds with acceptable biopharmaceutical properties¹⁰. In the current study, we have synthesized novel-2-substituted benzoxazoles using both IBD and LTA as oxidants, comparing the yield and duration for completion of reaction and subjected to anticancer and larvicidal activities to discern the potentiality of the synthesized compounds.

 

EXPERIMENTAL:

Chemistry:

The electro-thermal melting point apparatus was used for recording melting points and are uncorrected. ¹H NMR and ¹³C NMR spectra were recorded on Bruker 400 MHz spectrometer in IISc, Bangalore, and Karnataka, India. The chemical shifts are shown in δ values (ppm) with tetramethylsilane (TMS) as an internal standard. LC-MS were obtained using C 18 column on Shimadzu, LCMS 2010A, Japan. The FT-IR spectra of the compounds were taken in KBr pellet (100mg) using Shimadzu Fourier Transformed Infrared (FT-IR) Spectrophotometer.

 

General procedure for the synthesis of 2-(6-methoxy-2-naphthyl) methylene] amino} phenol derivatives 2a:

6-Methoxy-2-napthaldehyde (0.01mol) amino phenols 1a (0.01mol) and catalytic amount of acetic acid was taken in methanol and refluxed for 5 h. the reaction is monitored by using TLC upto completion. Then the reaction mass was cooled and filtered to get solid products 2a. The compounds 2a-f were synthesized by same method.

 

 

General procedure for the synthesis of 2-(6-methoxy-2-napthyl)-1,3-benzoxazole derivatives 3a:

Method A: Compounds 2a(0.01mol) and Iodobenzoxydiacetate (0.01mol) was taken in methanol and stirred at room temperature for 2h. The progress of reaction was monitored by TLC. The obtained solid mass was filtered off to get 3a. The compounds 3a-f were synthesized by same method.

 

Method B: Compounds 2a (0.01mol) and Lead tetraacetate (0.01mol) were taken in methanol and stirred at room temperature for 4 h. The progress of reaction was monitored by TLC. The reaction mass was filtered to get the compounds 3a. The compounds 3a-fwere synthesized by same method and represented in Scheme 1.

 

Spectral data:

2-(6-methoxy-2-naphthyl)-1,3-benzoxazole (3a). Anal. Calcd for (%) C₁₈H₁₃NO₂(275.3 gm/mole):C 78.53, H4.76, N5.09,Found: C78.52, H4.75, N 5.10;IR (KBr) cm^-1: 3013 (OCH₃), 1471 (C=C); ¹H NMR (DMSO-d6) ppm: δ 3.91 (s, 3H, OCH₃)_, δ 7.2-8.2 (10H, Ar H);¹³CNMR (DMSO-d6):164.10, 158.49, 149.05, 142.32, 136.41, 135.0, 130.96, 128.47, 128.26, 128.04, 126.99, 125.56, 122.93, 121.20, 119.93, 110.61, 107.02 (17 Ar-C), 55.90 (methoxy);M276.1.

 

2-(6-methoxy-2-naphthyl)-5-methyl-1,3-benzoxazole (3b). Anal. Calcd for (%) C₁₉H₁₅NO₂(289.32 gm/mole):C78.87, H5.23, N4.84, Found: C78.88, H5.24, N4.82;IR (KBr) cm^-1: 3116 (CH3), 3013 (OCH₃), 1480 (C=C); ¹H NMR (DMSO-d6) ppm: δ 2.4 (s, 3H, CH₃), δ 3.91 (s, 3H, OCH₃)_, δ 7.2-8.7 (9H, Ar H);¹³CNMR (DMSO-d6):163.21, 159.44, 148.97, 142.39, 136.47, 134.67, 131.07, 128.43, 128.22, 127.96, 126.76, 124.57, 121.96, 120.22, 119.94, 110.65, 106.70 (17 Ar-C), 55.89 (methoxy), 21.48 (methyl); M290.2.

 

5-chloro-2-(6-methoxy-2-naphthyl)-1,3-benzoxazole (3c). Anal. Calcd for (%) C₁₈H₁₂ClNO₂(309.74 g/mole):C69.80, H3.90, N4.52 Found: C69.82, H3.89, N4.51;IR (KBr) cm^-1: 3086 (OCH₃), 1480 (C=C)773 (C-Cl); ¹H NMR (DMSO-d6) ppm: 3.92 (s, 3H, OCH₃)_,  7.27-8.75 (9H, Ar H);¹³CNMR (DMSO-d6):164.67, 159.65, 149.55, 143.50, 136.74, 131.18, 129.48, 128.49, 128.32, 125.76, 124.56, 121.33, 120.32, 119.79, 112.62, 106.72, (17 Ar-C), 55.92 (methoxy); M310, M⁺² 311.

 

5,7-dichloro-2-(6-methoxy-2-naphthyl)-1,3-benzoxazole (3d). Anal. Calcd for (%) C₁₈H₁₁Cl₂NO₂(344.19 gm/mole):C62.81, H3.22, N4.07Found: C62.83, H3.86, N4.53;IR (KBr) cm^-1: 3020 (OCH₃), 1463 (C=C)775 (C-Cl); ¹H NMR (DMSO-d6) ppm: δ 3.92 (s, 3H, OCH₃)_, δ 7.27-8.75 (8H, Ar H);¹³CNMR (DMSO-d6):164.65, 159.61, 149.53, 143.480, 136.72, 130.78, 129.44, 128.43, 128.31, 125.71, 124.52, 121.35, 120.31, 119.78, 112.61, 106.74, (17 Ar-C), 55.91 (methoxy);M/z 344.2,M⁺² 346, M⁺⁴ 348.

 

2-(6-methoxy-2-naphthyl)-5-nitro-1,3-benzoxazole (3e). Anal. Calcd for (%) C₁₈H₁₂N₂O₄(320.29 gm/mole):C67.50, H3.78, N8.75 Found: C67.10, H3.8, N8.77;IR (KBr) cm^-1: 3011 (OCH₃), 1551 (-NO₂), 1471 (C=C); ¹H NMR (DMSO-d6) ppm: δ 3.91 (s, 3H, OCH₃)_, δ 7.25-8.71 (9H, Ar H);¹³CNMR (DMSO-d6):163.23, 159.42, 148.95, 142.36, 136.45, 134.65, 131.06, 128.42, 128.21, 127.94, 126.76, 124.56, 121.98, 120.20, 119.95, 110.67, 106.72 (17 Ar-C), 55.88 (methoxy);M/z 320.

 

2-(6-methoxy-2-naphthyl)-6-nitro-1,3-benzoxazole (3f). Anal. Calcd for (%) C₁₈H₁₂N₂O₄(320.29 g/mole):C67.50, H3.78, N8.75 Found: C67.10, H3.8, N8.77;IR (KBr) cm^-1: 3015 (OCH₃), 1598 (-NO₂), 1474 (C=C); ¹H NMR (DMSO-d6) ppm: δ 3.92 (s, 3H, OCH₃)_, δ 7.24-8.74 (9H, Ar H);¹³CNMR (DMSO-d6):163.22, 159.41, 148.93, 142.34, 136.44, 134.63, 131.07, 128.43, 128.22, 127.96, 126.77, 124.58, 121.96, 120.23, 119.93, 110.64, 106.70 (17 Ar-C), 55.87 (methoxy);M/z 320.

 

RESULTS AND DISCUSSION:

The 6-methoxy-2-napthaldehyde condensed with different o-amino phenols formed corresponding Schiff’s bases. The Schiff bases were cyclized by using lead tetraacetate and iodobenzoxydiacetate separately (Scheme-1). The difference in the percentage of yield and time taken for the completion of reaction using both reagents was observed (Table 1). The lead tetraacetate products were obtained in low yield as compared to the yield obtained by using iodobenzoxydiacetate. Nearly 20% of yield difference was observed and the reaction completed within 2h, when IBD catalyst used and it takes more time (4h) in LTA. The purity of the compounds was checked by TLC. Spectral data of the newly synthesized compounds 3a-f were in full accordance with their proposed structures. The absence of stretching frequency for C=O group in IR spectrum and absence of aldehydic proton of 6-methoxy-2-napthaldehyde at δ 10 value and achieve new peak for -N=CH proton in ¹H NMR confirmed the formation of Schiff bases. The cyclization of compounds 2a-f with ease of the reaction by stirring the reaction mass i.e Schiff bases 2a-f with catalyst in methanol at room temperature achieved. Here both -N=CH and –OH protons disappeared and gives the cyclized benzoxazole moiety. The characterizations of the final molecules 3a-f were based on the careful comparison of IR, ¹H NMR,¹³C NMRand mass spectral data and represented in supplementary file S1 to S8. The physicochemical parameters of the synthesized compounds were mentioned in Table 1.

 

Table 1: Physical data of compounds 3a-f

 

Computational Studies:

Molecular geometries of the singlet ground state of the derivatives 3a, 3c and 3d were fully optimized in the gas phase at the DFT/B3LYP 6-31G (d, p) and DFT/B3LYP LANL2DZ basic sets¹¹. The optimized geometry of the studied molecules, HOMO (highest occupied molecular orbital) and LUMO (lowest unoccupied molecular orbital) frontier molecular orbitals were visualized with supporting software chemcraft with chem 3D and Gaussian view.

 

The optimized geometry of the title derivatives, bond lengths, bond angles and dihedral angles corresponding to the optimized geometry of the title compound have been obtained using the DFT/B3LYP method. The energies of frontier molecular orbitals are important properties in several chemical and pharmacological processes. The HOMO measures the electron donating ability to cede an electron, LUMO as an electron acceptor represent the ability to receive an electron. Thus, the higher the E_HOMO is, the greater the electron donating capacity will be, and the lower the E_LUMO is, the smaller the resistance to accept electrons will be^12,13. In addition, the energy gap reflects the biological activity of molecules. The calculated values of HOMO and LUMO energies and HOMO–LUMO band gap of the compounds 3a, 3c and 3d are schematized in Figures 1 and 2.

 

Figure 1 Optimized geometry of derivatives 3a, 3c and 3d

 

Figure 2 Standard bond length and bond angles of derivatives 3a, 3c and 3d

 

The HOMO electron density are located on the benzoxazole group for derivative 3a, 3c and for derivative 3d the electron density is shifted to quinoline group due to two chlorine atoms. The LUMO electron density for derivative 3a and 3c is mainly situated on the quinoline group and for derivative 3d the electron density is distributed on chlorine atoms of benzoxazole. The values of the energy separation between the HOMO and LUMO are 6.124, 6.251 and 5.655 eV for the derivative 3a, 3c and 3d respectively. The decrease in the HOMO-LUMO energy gap explains the potential for charge transfer interactions taking place within the molecule, which may be responsible for the bioactivity of the molecules¹⁴ and represented in Figure 3.

 

Figure 3 Energy levels of the HOMO-LUMO and energy band gap of 3a, 3c and 3d

 

BIOLOGICAL EVALUATION:

Anticancer activity:

Cell Lines and Cell Culture:

Cell line-HT-29 -Human colorectal adenocarcinoma are obtained from Maratha Mandal Dental College, Belgaum, Karnataka. Human colon cancer cell (HT-29) lines were maintained in MEM medium. The media were supplemented with 10% (v/v) heat-inactivated fetal bovine serum, 100 U/mL penicillin and 100μg/mL streptomycin. Cells were cultured in a humidified atmosphere and incubated at 37°C in 5% CO₂.

 

Cytotoxicity and Anti-Proliferation Test:

Human colon cancer cell (HT-29) cell line was used as normal cell control for this study¹⁵. This cell line belongs to Human adenocarcinoma colorectal cells. HT-29 cell lines were seeded in 96-well plates (1 × 105 cells/well) and incubated in DMEM, at 37°C in 5% CO₂ for 24 hrs. The cells were pretreated with 10, 20, and 30μg/mL of each compound for 24 hrs and incubated in RPMI 1640, at 37°C in humidified atmosphere of 5% CO₂ for 72 h. After 4 h of incubation, the supernatant solution was discarded and 200μL of DMSO was added to each well to terminate the reaction. The absorbance was measured at 550nm using an enzyme-linked immunosorbent assay (ELISA) plate reader (Bio-Tek, Winooski, VT, USA). For the treated cells, viability is expressed as the percentage of control cells.

 

The compounds 3a, 3c, 3d and 3f from the series showed potent anticancer activity towards Human colon cancer cell (HT-29). Even at low concentration i.e10 µGmore than 75% of cells were destroyed and in some cases 100% lysis observed for same concentration.

 

Table 2 Anticancer activity of compounds 3a-f

 

 

 

 

Only compounds 3b and 3e were marked no lysis at concentration i.e10 µG, but possess moderate activity at slightly higher concentration. The results were tabulated in the Table 2 (Figure 4). The study further strengthened by the molecular docking analysis. The binding energies of the active molecules greater for the active compounds 3a, 3c, 3d and 3f.

 

Figure 4. Anticancer activity of compounds 3a-f

 

Larvicidal activity

The compounds 3a-f was screened to insecticidal activity to know the capability of the compounds against Aedes aegypti mosquito larvae¹⁶.

 

Table 3 Larvicidal activity of Compounds 3a-f

 

Figure 5 Larvicidal activity of compounds 3a-f

 

The melathione is used as standard drug in this study and the mortality was checked in two intervals. Insecticidal activity of the compounds was screened against second and third instar larvae of Aedes aegypti mosquito. The insecticidal effect was determined by counting the number of dead larvae after 24 h. The dead larvae were recognized when they failed to move after probing with a needle in siphon or cervical region. Each test was repeated thrice; the percentage of larval mortality was determined using the formulae- % mortality = A/B × 100, where A is number of dead larvae, B is number of larvae introduced. The compounds 3c, 3d and 3e showed higher mortality at observed concentration as compared with the standard drug melathione. The results were recorded in the Table 3 (Figure 5).

 

Molecular docking studies:

Molecular docking work was performed with the Hex molecular modeling package version 8.0¹⁷. Docking studies of the synthesized compounds 3a-f were evaluated against Human colon cancer cell (HT-29) lines. For macromolecular docking studies, chemical structures of the synthesized 3a-f were drawn using Chem. Draw Ultra. 3D optimization was performed using Chem. Draw 3D Ultra software and stored as pdb files. Hex docking was carried out by setting suitable parameters, outlined in Table 4 and this docking score can be interpreted as the interaction energy. A more negative E-total energy value implies that a strong interaction exists between drug and receptor which leads to the inhibition of receptor activity¹⁸. Molecular docking work was performed with the Hex molecular modeling package version 8.0.

 

Table 4: Docking Scores

 

The compounds were converted to 2D and 3D energy-minimized conformations using Hex 3D Ultra 8.0 and the conformation was visualized using Acceryl Discovery Studio 3.1 Client. In the present study, an attempt was made to evaluate their anti-cancer property, so we selected Human colon cancer cell (HT-29) lines and obtained docking scores (binding interaction energy) were tabulated in Table-4. With respect to Human colon cancer cell (HT-29) lines, all the synthesized compounds 3a-f exhibited more and comparable binding interaction energy (E-total value) range from -202.76 to-231.92 kJ/mol. Among all the compounds docked, compounds 3a, 3c, 3d and 3f showed more binding energy as compared with compound of rest. The docking studies also reveal that, all the compounds exhibited the bonding with various amino acids in the active pockets (Figure 6-8).

 

 

Figure 6. 3D & 2D interaction of compound 3a with receptor 1M17

 

Figure 7. 3D & 2D interaction of compound 3b with receptor 1M17

 

Figure 8. 3D & 2D interaction of compound 3c with receptor 1M17

 

 

The estimated binding affinity of molecules 3a-f with the complex hydrogen network and other interactions with amino acids^19-22 are MET78, LEU74, ILE84, ILE166, ASN155, LYS152, ASN155, ASP150, ASN155, LEU167, ASN155, GLY170, HIS148, SER208, TYR188, ILE212, SER208, LYS152 and ASP150, which are present in active sites gives a clue about the importance of hydrogen bond formation and other interactions for effective enzyme binding. Among all the synthesized compounds docked, derivatives 3a, 3b, 3c, 3d, 3e and 3f showed more least E-total values -211.78, -225.76, -202.76, -219.61, -231. 92 and -217.05 kcal mol^-1 and had a significantly better inhibiting ability.

 

CONCLUSION:

The synthesis of benzoxazole moiety with the ease and procedural simplicity are the key aspects of the synthesis. The synthesis of 2-(6-methoxy-2-napthyl)-1,3-benzoxazole derivatives have been achieved easily via iodobenzoxydiacetate and lead tetraacetate. It was observed that, the iodobenzoxydiacetate was the effective catalyst and acted as good oxidant than lead tetraacetate in good yield at lesser time. Optimized geometries, HOMO-LUMO energy gaps of derivatives 3a, 3c and 3d showed the chemical stability at B3LYP/LANL2DZ level of theory. The result of the anticancer screening revealed that, among the title compounds, the compounds 3a, 3c and 3d and 3f showed greater cell lysis while the other compounds displayed moderate lysis towards Human colon cancer cell (HT-29) lines. The docking score of targeted receptor supporting the anticancer activity of the compounds. In case of Insecticidal activity, the compound 3c, 3d and 3e have exhibited very good mortality among the series of compounds. Considering all the data, the iodobenzoxydiacetate is the effective oxidant in the cyclization of Schiff base than lead tetra acetate and some compounds from the series emerges as potent biologically active molecules.

 

ACKNOWLEDGEMENT:

Authors are thankful to the Principal of S.S.M.S College Athani and Sahyadri science college Shivamogga for providing laboratory facilities, and also The Director, IISc Bangalore.

 

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Received on 31.05.2020                    Modified on 25.06.2020

Accepted on 10.07.2020                   ©AJRC All right reserved