Synthesis of (±)-Pisonivanone and Other Analogs as Potent Antituberculosis Agents

Abstract

A new class of alkylated chalcones and flavanones was synthesised and screened for antituberculosis, antixoidant, and cytotoxic activities. The desired compounds were synthesised using methyl substituted 2-hydroxyacetophenone as a key intermediate. The acetophenone derivative having methyl substitution was prepared in turn from methtylated phloroglucinol by formylation (by Vilsmeier-Haack reaction), followed by reduction with Wolf-Kishnner approach, and finally acetylation was involved. Among 17 compounds, compound 5 and compound 4a inhibited M. tuberculosis at minimum inhibitory concentration (MIC) in the range between 25?µg/mL and 50?µg/mL. The remaining other 15 compounds also potently inhibited M. tuberculosis at MIC in range between 50?µg/mL and 100?µg/mL. Some of these compounds also showed moderate antioxidant and cytotoxic activities.

1. Introduction

A survey of the literature revealed that many flavonoids benefit human health [1]. Among these large class of compounds some of the alkylated flavonoids isolated from plants also act as antimalarial [2], antioxidant [3], anticancer [4–6], anti-inflammatory [7], and antimicrobial agents [8] like unsubstituted flavonoids. Recently antitubercular chromones and flavonoids [9] having methyl substitution were isolated from Pisonia aculeate, which belongs to Nyctaginaceae family. Among these the titled compound pisonivanone was reported as a potent antituberculosis compound (MIC = 12.5?µg/mL). So in order to develop the activity profile of the methyl substituted flavonoids, an attempt was made to synthesize pisonivanone for the first time and other new chalcones and flavanones. These synthesized compounds were screened for some biological activities like antioxidant, cytotoxic, and antituberculosis. The scheme for the synthesis of Pisonivanone and other analogs was given below (Scheme 1).

Scheme 1 

Reagents and conditions: (i) KOH/EtOH, 24 h. (ii) EtOH, H2SO4, 60°C. 24 h.

2-Hydroxy-3-methyl-4,6-dimethoxy acetophenone (1) was synthesised in different ways [10]. In the present work, we aimed to synthesise the key intermediate acetophenone (1) from phloroglucinol using regular conventional methods like methylation [11], formylation [12], Wolf-Kishner reduction, and acetylation [12] with better yields (Scheme 2). All the intermediates were confirmed by comparing the spectral data and melting points with the literature.

Scheme 2 

Reagents and conditions: (i) DMS/Acetone, K2CO3, 70°C/6 h, (ii) DMF/POCl3, 0°C/1 h, (iii) NH2NH2·H2O/KOH, ethylene glycol, 80–90°C for 2 h followed by heating at 130 to 140°C for 2 h, (iv) CH3COCl/AlCl3, MDC. 0°C/2 h.

Chalcones are versatile molecules and intermediates for synthesis of different heterocyclic compounds [13]. Chalcones have been synthesised in different ways [14, 15]. The chalcones (3a–h) were synthesised by Claisen-Schmidt reaction. In this reaction, condensation was between acetophenone (1) and substituted aromatic aldehydes (2a–h) in the presence of aqueous KOH and ethanol at room temperature.

Flavanones (Compound 4a–h) were prepared on cyclisation of chalcones (Compounds 3a–h, resp.) in the presence of alcoholic H₂SO₄. Initially, chalcones were dissolved in alcoholic H₂SO₄ and refluxed at 60°C for 24?hrs. After cooling, alcohol was removed under vacuum. When ice cold water added to the reaction, mass pale reddish colour solids were separated. These solids were extracted with ethyl acetate. This ethyl acetate layer dried over sodium sulphate. This ethyl acetate layer was concentrated and washed with diethyl ether to remove adherent chalcone.

Finally, the targeted compound 5 (pisonivanone) was synthised from Compound 4a on demethylation. Pyridinium hydrochloride was added to Compound 4a, at inert conditions in 1?:?10 ratio and refluxed for 1 hour at 200°C. Then reaction mixture poured in ice cold water and extracted with ethyl acetate. This ethyl acetate layer dried over sodium sulphate and on column chromatography with ethyl acetate, and hexane pure pisonivanone was separated (yellow crystalline solid, yield 55%) (see Scheme 3).

Scheme 3 

2. Discussions

Based on the literature, all these synthesised Compounds 3a–h, 4a–h, Compound-5 (pisonivanone) were screened for their biological activities like antitubercular, antioxidant, and cytotoxicity. All these methyl substituted flavonoids were showed potent antitubercular activity against M. tuberculosis strain (H37Rv). Possibly the C-8 methyl group in basic skeleton of the molecules plays an important role in the antimycobacterial activity. Among 17 compounds Compound 5 showed minimum inhibitory concentration (MIC) at 25?µg/mL and Compound 4a inhibited M. tuberculosis at minimum inhibitory concentration (MIC) = 50?µg/mL. The remaining other 15 compounds also potently inhibited M. tuberculosis at MIC in range between 50?µg/mL and 100?µg/mL. These results suggest that the methyl substituted flavonoids may consider as potent antituberculosis agents, and there is a scope to improve the activity further by preparing different possible analogs. The results given in Tables 2 and 3 for chalcones and flavanones showed the compounds have moderate antioxidant and cytotoxic activities.

3. Experimental

3.1. Materials and Methods

Phloroglucinol and other chemicals used were of AR grade (Merck). Melting points were taken in open glass capillary by using Remi melting point apparatus and were uncorrected. ¹H NMR spectra were recorded on Brucker 400?MHz and ¹³C NMR was on Brucker 100?MHz spectrophotometers. Mass spectra recorded on LC-MS Agilent LC-1100 series.

3.1.1. Synthesis of 2,4,6-Trimethoxy Phloroglucinol

A solution of anhydrous phloroglucinol (50?g, 0.396?mole) in dry acetone (700?mL) is refluxed for 6?h at 60°C under anhydrous conditions with dimethyl sulphate (112.10?mL, 148?g, 1.17?mole) in presence of potassium carbonate (280?g, 2.02?mole). The acetone layer was filtered and distilled. The residue on column chromatography with hexane and ethyl acetate (95?:?5) trimethoxy phloroglucinol separated. (% of yield 94, white solid, M.P—52–54°C.)

3.1.2. Synthesis of 2,4,6-Trimethoxy Benzaldehyde

Dimethyl formamide (36.8?mL, 1.3?eq, 0.4797?mole) is added to trimethoxy phloroglucinol (62?g, 0.369?mole, 1?eq) and cooled (0°C). Phosphorous oxychloride (153.33?mL, 65.63?gm, 1.16?eq) is added drop wise to this cooling mixture slowly under anhydrous conditions. The mixture is allowed to stand for 1?h at room temperature. The reaction mixture poured into crushed ice and basified with 8?N KOH. The filtered solid dissolved in ethyl acetate and treated with brine. The ethyl acetate layer dried over sodium sulphate and concentrated. (% of yield—91.6, yellow viscous solid, M.P—119–120°C.)

3.1.3. Synthesis of 3-Methyl-2,4,6 Trimethoxy Phloroglucinol

2,4,6-Trimethoxy benzaldehyde is dissolved in ethylene glycol and hydrazine hydrate is added. Powdered KOH portion wise added to the reaction mixture and heated to 80–90°C for 2?h then foaming is formed. The reaction mixture heated to 135–145°C for another 2?h. Then the reaction mixture is poured into ice cold water, acidified with dil HCl, and extracted with hexane. Hexane layer is washed with sodium carbonate, water, and brine. This hexane layer dried over sodium sulphate and concentrated. (% of yield 80, colour less liquid.)

3.1.4. Synthesis of 2-Hydroxy-3-methyl-4,6-dimethoxy Acetophenone (1)

A solution of 3-methyl-2,4,6 Trimethoxy phloroglucinol (55?g, 0.302?mole) in absolute ether (450?mL) is added to a cold solution of anhydrous aluminum chloride (86.39?g, 0.64?mole, 2.2?eq) in absolute ether (100?mL). The cooled (0–5°C) and stirred solution is treated with acetyl chloride (25.21?mL, 0.319?mole, 1.2?eq), added dropwise during 1?hr. The mixture is stirred for 4?hr more. The whole reaction is done under anhydrous condition. The mixture is left overnight at room temperature and treated in cold with crushed ice. It is acidified with 5?N HCl and extracted with ethyl acetate. This ethyl acetate layer dried over sodium sulphate and concentrated. (% of yield 85, light green color crystals, M.P—90–92°C.)

3.1.5. General Method for Synthesis of Chalcones (Compound 3a–h)

To a solution of acetophenone (0.5?g, 0.00238?mole) and substituted benzaldehydes (2a–h) (1?eq) in ethanol (10?mL) is added aqueous potassium hydroxide (0.599?g, 4.5?eq in 0.5?mL water). The mixture is stirred at room temperature for 24?h. To this ice cold water added and separated solid was filtered. The separated solid was dissolved in ethyl acetate, died over sodium sulphate, and concentrated under vacuum.

3.1.6. Spectral Data of Synthesized Compounds

Compound 3a. It is having the molecular formula C₁₉H₂₀O₅ which showed (M+H)⁺ at 329 in LC-MS. So the molar mass of the Compound 3a is 328?g/mol. Yield—85%, M.P—157–160°C.

¹H NMR (CDCl₃, 400?MHz, d in ppm): 3H, s, 3.991 (Ar-OCH₃), 3H, s 3.932 (Ar-OCH₃), 3H, s, 3.917 (Ar-OCH₃), 1H, s, 14.045 (Ar-OH), 2H, d, 7.98 (Ar¹-H), 1H, dd, 7.71 (Ar¹-H), 1H m 7.4 (Ar¹-H), 1H, d, 7.05 (Ar¹-H), 1H, d, 7.1 (C=C–H), 1H, d, 6.3 (C=C–H).

¹³C NMR (CDCl₃, 100?MHz, d in ppm): 192.72 (C=O), 7.32 (ArCH₃), 56.1 (Ar-OCH₃), 55.8 (Ar-OCH₃), 55.69 (Ar¹-OCH₃), 163.6, 162.9, 161.5 (Ar-C-O), 158.32 (Ar¹-C), 87.65 (Ar-C), 105.6, 104.2 (Ar-C), 120.87, 111.88 (C=C), 137.28, 131.93, 128.96, 127.77, 123.30 (Ar¹-C).

Compound 3b. It is having the molecular formula C₁₉H₂₀O₅ which showed (M+H)⁺ at 329 in LC-MS. So the molar mass of the compound 3b is 328?g/mol. Yield—80%, M.P—125–130°C.

¹H NMR (CDCl₃, 400?MHz, d in ppm): 1H, d, 14.03 (Ar-OH), 1H, d, 7.8 (Ar¹-H), 1H, d, 7.7 (Ar¹-H), 1H, s, 5.99 (Ar-H), 1H, s, 7.29 (Ar¹-H), 1H, m, 7.33 (Ar¹-H), 1H, d, 7.21 (C=C–H) 1H, d 6.9 (C=C–H), 3H, s, 3.9 (Ar¹OCH₃), 3H, s, 3.9 (Ar-OCH₃), 3H, s, 3.8 (Ar-OCH₃), 3H, s, 2.03 (Ar-CH₃).

¹³C NMR (CDCl₃, 100?MHz, d in ppm): 193.00 (O=C), 164.27, 163.65 (Ar-C-OCH₃), 161.13 (Ar-OH), 159.93 (Ar¹-OCH₃), 141.72 (C=C–Ar), 128.35 (C=C–C=O), 86.43 (Ar-C), 106.40 (Ar-C-CH₃), 106.22 (Ar–C–C=O), 137.1, 129.80, 120.87, 115.51, 113.63 (Ar¹-C), 55.84, 55.49 (Ar-OCH₃), 55.28 (Ar¹-OCH₃), 7.23 (Ar-CH₃).

Compound 3c. Compound having the molecular formula C₂₁H₂₄O₇ is showed molecular ion peak at 389.2 (Neg) in LC-MS. Molecular weight of the compound 3c is 388?g/mol. Yield—86%, M.P—200–205°C.

¹H NMR (CDCl₃, 400?MHz, d in ppm): 1H, s, 14.09 (Ar-OH), 3H, s 3.93 (Ar¹-OCH₃), 6H, s 3.9 (Ar¹-OCH₃), 3H, s 3.9 (Ar-OCH₃), 3H, s 3.8 (Ar-OCH₃), 3H 2.03 (Ar-CH₃), 1H, s 5.99 (Ar-H), 2H, s 6.83 (Ar¹-H), 1H d 7.7 (C=C–H), 1H, d 7.69 (C=C–H).

¹³C NMR (CDCl₃, 100?Hz, d in ppm): 192.75 (O=C), 164.29, 163.57, (Ar-C-OCH₃), 161.02 (Ar-OH), 153.44 (Ar¹-OCH₃), 141.97 (Ar¹-OCH₃), 141.97 (Ar¹-OCH₃), 140.21 (C=C–Ar), 127.35 (C=C–C=O), 86.43 (Ar-C), 106.40 (Ar-C-CH₃), 106.24 (Ar–C–C=O), 131.24, (Ar¹-C), 105.72 (Ar¹-C), 105.72 (Ar¹-C), 60.95, 56.18 (Ar-OCH₃), 55.79, 55.49 55.28 (Ar¹-OCH₃), 7.23 (Ar-CH₃).

Compound 3d. It is having the molecular formula C₁₈H₁₇O₄Cl which showed molecular ion (M−H)⁻ peak in negative mode at 331 in LC-MS. So the molar mass of the compound 3d is 332?g/mol. Yield—70%, M.P—185–190°C.

¹HNMR (400MHz, d in ppm, CDCl₃): 1H, s,14.00 (Ar-OH), 3H, s, 3.95 (Ar-OCH₃), 3H,s, 3.980 (Ar-OCH₃), 3H,s, 2.037 (Ar-CH₃), 1H,s, 5.92 (Ar-H), 2H,d 7.37 (Ar¹-H), 2H,d, 7.53 (Ar¹-H), 1H d, 7.7 (C=C-H), 1H,d,7.8 (C=C-H).

¹³CNMR (100MHz, d in ppm, CDCl₃): 192.73 (C=O), 164.28, 163.76 (Ar-C-OH₃), 161.11 (Ar-C-OH), 86.40 (Ar-C), 106.32 (Ar-C-CH₃), 106.24 (Ar-C-C=O), 140.33 (-C=C-Ar), 128.50 (-C=CH), 135.74 (Ar¹-Cl), 134.22 (Ar¹-C),129.39 (Ar¹-C-H), 128.50 (Ar-C-H), 137.45(CH=C), 127.85 (C=C-H).55.85, 55.52 (Ar-O-CH₃), 7.24 (Ar-CH₃).

Compound 3e. It is having the molecular formula C₁₈H₁₇O₄F which showed molecular ion peak in positive mode (M+H)⁺ at 317 in LC-MS. So the molar mass of the compound 3e is 316?g/mol. Yield—75%, M.P—165–170°C.

¹H NMR (400?MHz, d in ppm, CDCl₃): 1H,s, 14.01 (Ar-OH), 1H,s, 5.985 (Ar-H), 3H,s, 3.98 (Ar-O-CH₃), 3H,s, 3.89 (Ar-O-CH₃), 3H,s, 2.03 (Ar-CH₃), 1H,d, 8.00 (C=C-H), 1H,d, 7.84 (C=C-H), 1H,m, 7.5 (Ar¹-H), 1H,m, 7.3 (Ar¹-H), 1H,m, 7.14 (Ar¹-H), 1H, m, 7.09 (Ar¹-H).

¹³CNMR (100?MHz, d in ppm, CDCl₃):193.04(C=O), 106.42 (Ar-C-CH₃),164.34 (Ar-O-CH₃), 86.44 (Ar-C-H),163.80 (Ar-O-CH₃),162.97 (Ar-C-OH),106.21 (Ar-C-C=O),55.77,55.53 (Ar-OCH₃), 7.26 (Ar-CH₃),161.25,160.45 (Ar¹-C-F),123.78,123.90 (Ar¹-C),131.18,131.09 (Ar¹-C), 130.79, 130.72 (Ar¹-C), 129.76, 129.73 (Ar¹-C), 116.33, 116.11 (Ar¹-C), 134.46(HC=C), 124.38(HC=C).

Compound 3f. It is having the molecular formula C₁₈H₁₇O₄Br which showed molecular ion peak in positive mode (M+H)⁺ at 378 in LC-MS. So the molar mass of the compound 3f is 377?g/mol. Yield—60%, M.P—175–180°C.

¹H NMR (CDCl₃, 400?MHz, d in ppm): 1H, s 13.99 (Ar-OH), 3H, s 2.032 (Ar-CH₃), 3H, s 3.93 (Ar-OCH₃), 3H, s 3.89 (Ar-OCH₃), 1H, d, 7.68 (Ar-H), 2H, d, 7.5 (Ar¹-H), 2H, d, 7.44 (Ar¹-H), 1H, d, at 7.85 (O=C–CH=C), 1H, d, 5.98 (C=C–H).

¹³C NMR (CDCl₃, 100?MHz, d in ppm): 192.70 (Ar–C=O), 7.23 (Ar-CH₃), 106.26 (Ar-C-CH₃), 164.31 (Ar-C-OCH₃), 86.42 (Ar-C-H), 163.78 (Ar-C-OCH₃), 106.33 (Ar–C–C=O), 161.12 (Ar-C-OH), 55.84 (Ar-OCH₃), 55.50 (Ar-O-CH₃), 134.68 (Ar¹-C<), d at 129.60 (Ar¹-C-H), 128.65 (Ar¹-C-H), 132.08 (Ar¹-C-Br), 129.60 (Ar¹-C-H), 124.04 (Ar¹-C-H), 140.32 (O=C–CH=C), 128.65 (C=C–H).

Compound 3g. It is having the molecular formula C₂₀H₂₂O₆ which showed molecular ion peak in negative mode (M−H)⁻ at 357.2 so the molar mass of the compound 3g is 358.2?g/mol. Yield—75%, M.P—130–135°C.

¹H NMR (CDCl₃, 400?MHz, d in ppm): 1H, s 14.18 (Ar-OH), 3H, s 2.03 (Ar-CH₃), 6H, s 3.9 (Ar-OCH₃), 1H, s, 5.98 (Ar-H), 1H, d, 7.21 (O=C–CH=C), 1H, d, 6.89 (C=C–H), 2H, m, 7.7 (Ar¹-H), 1H, s 7.11 (Ar¹-H), 3H, s 3.92 (Ar¹-OCH₃), 3H, s, 3.89 (Ar¹-OCH₃).

¹³CNMR (100MHz, d in ppm, CDCl₃): 192.85 (Ar-C=O), 106.39 (Ar-C-CH₃), 164.27 (Ar-C-OCH₃), 163.43 (Ar-C-OCH₃), 160.99 (Ar-C-OH), 86.46 (Ar-C-H), 106.20 (Ar-C-C=O), 55.98 (Ar-OCH₃)55.89 (Ar-O-CH₃), 125.92 (Ar¹-C<), 110.65 (Ar¹-C-H), 111.31 (Ar¹-C-H), 122.52 (Ar¹-C-H), 151.07 (Ar¹-C-OCH₃), 149.22 (Ar¹-C-OCH₃), 55.79 (Ar¹-OCH₃), 55.47 (Ar¹-OCH₃), 142.18(CH=C), 128.52 (C=C-H).7.25 (Ar-CH₃)_.

Compound 3h. It is having the molecular formula C₁₈H₁₆O₄F₂ which showed molecular ion peak in negative mode (M−H)⁻ at 333.2 in LC-MS. So the molar mass of the compound 3h is 334.2?g/mol. Yield—60%, M.P—135–140°C.

¹H NMR (CDCl₃, 400?MHz, d in ppm): 3H, s, 2.03 (Ar-CH₃), 3H, s 3.95 (Ar-OCH₃), 3H, s 3.90 (Ar-H), 1H, s, 7.78 (Ar-OCH₃), 1H, s 13.94 (Ar-OH), 1H, d, 6.89 (O=C–CH=C), 1H, d, 7.21 (C=C–H), 1H, m, 7.7 (Ar¹-H), 1H, m 7.3 (Ar¹-H), 1H, m, 7.1 (Ar¹-H).

¹³C NMR (CDCl₃, 100?MHz, d in ppm): 7.78 (Ar-CH₃), 106.30 (Ar-C-CH₃), 163.86 (Ar-C-OCH₃), 86.42 (Ar-C-H), 164.31 (Ar-C-OCH₃), 106.27 (Ar–C–C=O), 161.12 (Ar-C-OH), 55.86 (Ar-OCH₃), 55.47 (Ar-O-CH₃), 192.69 (Ar–C=O), 129.0 (Ar¹-C<), 124.99 (Ar¹-C-H), 125.06 (Ar¹-C-H), 133.06 (Ar¹-C-F), 132.98 (Ar¹-C-F), 117.8 (Ar¹-C-H), 139.26 (CH=C), 126.81 (C=C–H).

3.1.7. General Method for Synthesis of Flavanones (Compound 4a–h)

To the previously obtained chalcones (3a–h) (0.2?g) was refluxed in alcoholic sulphuric acid (10%, 10?mL) for 24?h. The alcohol was distilled off in vacuo and the residue is diluted with water. The separated product was dissolved in ethyl acetate and dried over sodium sulphate. The ethyl acetate layer was concentrated the residue was washed with ether to remove any adherent chalcone to obtain pure flavanones (4a–h).

Compound 4a. It is having the molecular formula C₁₉H₂₀O₅ which showed molecular ion peak in positive mode (M+H)⁺ at 329 in LC-MS. So the molar mass of the compound 4a is 328?g/mol. Yield—70%, M.P—172–175°C.

¹H NMR: (CDCl₃, 400?MHz, d in ppm): 1H, s, 6.27 (Ar-H), 1H, m, 7.19 (Ar¹-H), 2H, m, 6.7 (Ar¹-H), 1H, d, 6.8 (Ar¹-H), 1H, d, 4.82 (O-C-H), 6H, s, 3.86 (Ar-O-CH₃), 3.71 (3H, s, Ar¹-O-CH₃), 3H, s, 2.16 (Ar-CH₃), 3.06 and 2.88 (1H, d and 1H, d O=C–CH₂).

¹³C NMR: (CDCl₃, 100?MHz, d in ppm): 7.81 (Ar-CH₃), 106.36 (Ar-C-CH₃), 163.56 (Ar-C-OCH₃), 88.64 (Ar-C-H), 160.12 (Ar-C-OCH₃), 105.98 (Ar–C–C=O), 160.52 (Ar-C-O), 55.92 (Ar-OCH₃), 55.08 (Ar-O-CH₃), 189.83 (Ar–C=O), 129.73 (Ar¹-C<), 118.01 (Ar¹-C-H), 159.90 (Ar¹-C-OCH₃), 111.58 (Ar¹-C-H), 140.99 (Ar¹-C-H), 113.63 (Ar¹-C-H), 55.62 (Ar¹-OCH₃), 45.75 (O=C–CH₂), 78.28 (>C-H).

Compound 4b. It is having the molecular formula C₁₉H₂₀O₅ which showed molecular ion peak in positive mode (M+H)⁺ at 329 in LC-MS. So the molar mass of the compound 4b is 328?g/mol. Yield—60%, M.P—166–170°C.

¹H NMR: (CDCl₃, 400?MHz, d in ppm): 1H, s, 6.125 (Ar-H), 2H, s, 7.04 (Ar¹-H), 1H, dd, 6.8 (Ar¹-H), 1H, dd, 6.98 (Ar-H), 1H, dd, 5.3 (O-C-H), 3H, s, 3.93 (Ar-O-CH₃), 3H, s, 3.93 (Ar-O-CH₃), 3H, s, 3.83 (Ar¹-O-CH₃), 3H, s, 2.06 (Ar-CH₃), 2.9, and 2.8 (1H, dd and 1H, dd, O=C–CH₂).

¹³C NMR: (CDCl₃, 100?MHz, d in ppm): 7.81 (Ar-CH₃), 106.3 (Ar-C-CH₃), 161.12 (Ar-C-OCH₃), 88.64 (Ar-C-H), 160.52 (Ar-C-OCH₃), 105.98 (Ar–C–C=O), 163.56 (Ar-C-O), 55.62 (Ar-OCH₃), 56.06 (Ar-O-CH₃), 189.83 (Ar–C=O), 140.99 (Ar¹-C<), 129.97 (Ar¹-C-H), 159.90 (Ar¹-C-OCH₃), 118.01 (Ar¹-C-H), 111.58 (Ar¹-C-H), 113.6 (Ar¹-C-H), 55.27 (Ar¹-OCH₃), 45.75 (O=C–CH₂), 78.28 (>C-H).

Compound 4c. It is having the molecular formula C₂₁H₂₄O₇ which showed molecular ion peak in positive mode (M+Na)⁺ at 411.2 in LC-MS. So the molar mass of the compound 4c is 388.2?g/mol. Yield—65%, M.P—164–170°C.

¹H NMR: (CDCl₃, 400?MHz, d in ppm): 1H, s, 6.13 (Ar-C-H), 3H, s, 2.06 (Ar-CH₃), 2H, s, 6.69 (Ar¹-C-H), 1H, dd, 5.3 (Ar¹-O-C-H), 3H, s, 3.94 (Ar-OCH₃), 3H, s, 3.91 (Ar-OCH₃), 3H, s, 3.8 (Ar¹-OCH₃), 6H, s, 3.88 (Ar¹-OCH₃), 1H, dd at d 2.97 and 1H, dd at d 2.86 (–O=C–CH2–).

¹³C NMR: (CDCl₃, 100?MHz, d in ppm): 7.83 (Ar-CH₃), 105.91 (Ar-C-CH₃), 161.04 (Ar-C-OCH₃), 88.62 (Ar-C-H), 160.54 (Ar-C-OCH₃), 103.03 (Ar–C-C=O), 163.61 (Ar-C-O), 56.20 (Ar-OCH₃), 56.06 (Ar-O-CH₃), 189.85 (Ar–C=O), 138.01 (Ar¹-C<), 103.03 (Ar¹-C-H), 153.47 (Ar¹-C-OCH₃), 153.47 (Ar¹-C-OCH₃), 134.96 (Ar¹-C-OCH₃), 103.03 (Ar¹-C-H), 55.64 (Ar¹-OCH₃), 55.64 (Ar¹-OCH₃), 60.83 (Ar¹-OCH₃), 45.78 (O=C–CH₂), 78.42 (>C-H).

Compound 4d. It is having the molecular formula C₁₈H₁₇O₄Cl which showed molecular ion peak in positive mode (M+H)⁺ at 333.2 in LC-MS. So the molar mass of the compound 4d is 332?g/mol. Yield—60%, M.P—177–180°C.

¹HNMR (400MHz, d in ppm, CDCl₃): 3H,s, 2.04 (Ar-CH₃), 3H,s, 3.93 (Ar-OCH₃),3H,s, 3.90 (Ar-OCH₃), 1H,s, 6.128 (Ar-H), 2H,m, 2.9 (O=C-CH₂), 1H,dd, 5.38(>C<_𝐻 ), 4H, m, 7.4 (Ar¹-H).

¹³C NMR: (CDCl₃, 100?MHz, d in ppm): 7.79 (Ar-CH₃), 106.33 (Ar-C-CH₃), 160.87 (Ar-C-OCH₃), 88.69 (Ar-C-H), 160.58 (Ar-C-OCH₃), 105.33 (Ar–C–C=O), 163.67 (Ar-C-O), 55.63 (Ar-OCH₃), 56.06 (Ar-O-CH₃), 189.47 (Ar–C=O), 134.16 (Ar¹-C<), 127.21 (Ar¹-C-H), 128.8 (Ar¹-C-H), 137.87 (Ar¹-C-Cl), 128.88 (Ar¹-C-H), 127.21 (Ar¹-C-H), 45.56 (O=C–CH₂), 77.74 (>C-H).

Compound 4e. It is having the molecular formula C₁₈H₁₇O₄F which showed molecular ion peak in positive mode (M+H)⁺ at 317.3 in LC-MS. So the molar mass of the compound 4e is 316.3?g/mol. Yield—65%, M.P—169–175°C.

¹H NMR: (CDCl₃, 400?MHz, d in ppm): 3H, s, 2.04 (Ar-CH₃), 3H, s, 3.94 (Ar-OCH₃), 3H, s, 3.90 (Ar-OCH₃), 1H, s, 6.14 (Ar-H), 2H, m, 2.9 (O=C–CH₂), 1H, dd, 5.69 (>C<_H), 1H, m, 7.6 (Ar¹-H), 1H, m, 7.3 (Ar¹-H), 1H, m, 7.2 (Ar¹-H), 1H, m, 7.1 (Ar¹-H).

¹³C NMR: (CDCl₃, 100?MHz, d in ppm) 7.74 (Ar-CH₃), 106.41 (Ar-C-CH₃), 161.13 (Ar-C-OCH₃), 88.80 (Ar-C-H), 160.62 (Ar-C-OCH₃), 105.97 (Ar–C–C=O), 163.56 (Ar-C-O), 56.10 (Ar-OCH₃), 55.64 (Ar-O-CH₃), 189.50 (Ar–C=O), 115.5 (Ar¹-C<), 126.8 (Ar¹-C-H), 129.8 (Ar¹-C-H), 160.96, (Ar¹-C-F), 127.15 (Ar¹-C-H), 124.4 (Ar¹-C-H), 44.73 (O=C–CH₂), 73.14 (>C-H).

Compound 4f. It is having the molecular formula C₁₈H₁₇O₄Br which showed molecular ion peak in positive mode (M+H)⁺ at 378.1 in LC-MS. So the molar mass of the compound 4f is 377?g/mol. Yield—60%, M.P—166–170°C.

¹H NMR: (CDCl₃, 400?MHz, d in ppm): 3H, s, 2.04 (Ar-CH₃), 3H, s, 3.93 (Ar-OCH₃), 3H, s 3.90 (Ar-OCH₃), 1H, s, 6.12 (Ar-H), 2H, m, 2.8 (O=C–CH₂), 1H, dd, 5.3 (>C<_H), 2H, d, 7.5 (Ar¹-H), 2H, d, 7.3 (Ar¹-H).

¹³C NMR: (CDCl₃, 100?MHz, d in ppm): 7.78 (Ar-CH₃), 106.31 (Ar-C-CH₃), 160.84 (Ar-C-OCH₃), 88.73 (Ar-C-H), 160.56 (Ar-C-OCH₃), 105.89 (Ar–C–C=O), 163.64 (Ar-C-O), 56.05 (Ar-OCH₃), 55.63 (Ar-O-CH₃), 189.33 (Ar–C=O), 131.84 (Ar¹-C<), 122.24 (Ar¹-C-H), 127.51 (Ar¹-C-H), 138.41 (Ar¹-C-Br), 127.51 (Ar¹-C-H), 122.24 (Ar¹-C-H), 45.50 (O=C–CH₂), 77.77 (>C-H).

Compound 4g. It is having the molecular formula C₂₀H₂₂O₆ which showed molecular ion peak in positive mode (M+Na)⁺ at 381.3 in LC-MS. So the molar mass of the compound 4g is 359.3?g/mol. Yield—75%, M.P—179–182°C.

¹HNMR (400MHz, d in ppm, CDCl₃): 1H,s, 6.13 (Ar-C-H), 3H,s, 2.06 (Ar-CH₃), 2H,s,7.0 (Ar¹-C-H), 1H,d, 6.98 (Ar¹-C-H), 1H, dd, 5.3 (Ar¹-O-C-H), 3H,s,3.93 (Ar-OCH₃), 9H,s,3.90 (Ar-OCH₃), 2 (Ar¹-OCH3), 1H, dd and 1H, dd, 2.97 and 2.86 (-O=C-CH₂-).

¹³C NMR: (CDCl₃, 100?MHz, d in ppm) 7.83 (Ar-CH₃), 106.29 (Ar-C-CH₃), 161.17 (Ar-C-OCH₃), 88.59 (Ar-C-H), 160.52 (Ar-C-OCH₃), 106.29 (Ar–C–C=O), 160.52 (Ar-C-O), 56.07 (Ar-OCH₃), 55.99 (Ar-O-CH₃), 190.03 (Ar–C=O), 131.93 (Ar¹-C<), 109.40 (Ar¹-C-H), 118.38 (Ar¹-C-H), 149.15 (Ar¹-C-OCH₃), 149.23 (Ar¹-C-OCH₃), 111.27 (Ar¹-C-H), 55.97 (Ar¹-OCH₃), 55.63 (Ar¹-OCH₃), 45.59 (O=C–CH₂), 78.29 (>C-H).

Compound 4h. It is having the molecular formula C₁₈H₁₆O₄F₂ which showed molecular ion peak in positive mode (M+Na)⁺ at 357.2 in LC-MS. So the molar mass of the compound-4h is 334?g/mol. Yield—55%, M.P—176–180°C.

¹HNMR (400?MHz, d in ppm, CDCl₃):1H,t, 7.34 (Ar¹ -H), 2H, m, 7.2 (Ar¹-H), 1H,s, 6.1 (Ar-C-H), 1H, dd, 5.3(>C<_H), 3H,s, 3.9 (Ar-OCH₃), 3H,s, 3.9 (Ar-OCH₃), 2H,m, 2.8(O=C-CH₂), 3H,s,2.05 (Ar-CH₃).

¹³C NMR: (CDCl₃, 100?MHz, d in ppm) 7.81 (Ar-CH₃), 106.32 (Ar-C-CH₃), 160.64 (Ar-C-OCH₃), 88.32 (Ar-C-H), 163.71 (Ar-C-OCH₃), 105.85 (Ar–C–C=O), 160.59 (Ar-C-O), 56.08 (Ar-OCH₃), 55.66 (Ar-O-CH₃), 189.02 (Ar–C=O), 136.44 (Ar¹-C<), 115.21 (Ar¹-C-H), 121.13 (Ar¹-C-F), 121.84 (Ar¹-C-F), 115.02 (Ar¹-C-H), 117.6 (Ar¹-C-H), 45.54 (O=C–CH₂), 77.32 (>C-H).

Compound 5. It is having the molecular formula C₁₆H₁₄O₅ which showed molecular ion peak in negative mode (M−H)⁻ at 285.1 in LC-MS. So the molar mass of the compound 5 is 286?g/mol.

¹H NMR: (CDCl₃, 400?MHz, d in ppm): 1H, s, 9.65 (Ar-OH), 1H, s, 9.407 (Ar-OH), 1H, s 9.339 (Ar¹-OH), 1H, t 7.030 (Ar¹-OH), 1H, d 6.822 (Ar¹-OH), 1H, t 6.622 (Ar¹-H), 1H, t, 6.44 (Ar¹-H), 1H, s 6.260 (Ar-H), 1H, d, 4.636 (O-C-H), 2H, dd 3.047 (O=C–CH₂), 3H, s, 1.984 (Ar-CH₃).

¹³C NMR: (CDCl₃, 400?MHz, d in ppm) 7.81 (Ar-CH₃), 106.36 (Ar-C-CH₃), 163.56 (Ar-C-OH), 88.64 (Ar-C-H), 161.52 (Ar-C-OH), 105.98 (Ar–C–C=O), 160.52 (Ar-C-O), 189.83 (Ar–C=O), 129.73 (Ar¹-C<), d at 159.90 (Ar¹-C-OH), 113.63 (Ar¹-C-H), 140.99 (Ar¹-C-H), 111.58 (Ar¹-C-H), 118.01 (Ar¹-C-H), 44.65 (O=C–CH₂), d at 74.23 (>C-H).

3.2. Bioactivity Studies

3.2.1. Antituberculosis

Compounds 3a–h, 4a–h, and 5 (pisonivanone) were screened for antituberculosis on Myco bacterium tuberculosis in LJ medium by using convention method [16] as follows.

Drug Free media (Lowenstein Jensen media) base was prepared according to manufacturer’s instructions (HiMedia, Merck). The culture media was prepared by weighing 18.65 grams of the LJ medium base and dissolved in 300?mL of distilled water. Six mL glycerol (reagent grade) was added. The solution was autoclaved at 121°C for 30 minutes and cooled. 500?mL of homogenized whole egg was added and mixed. Then 6–8?mL of the medium was dispensed into a 28 × 110?mm screw capped Mac Cartney culture tubes (Universal containers). These culture tubes were inspissated at 85°C for 55 minutes. For sterility check, prepared culture media were incubated at 37°C for 48 hours and were kept in the refrigerator for storage when no bacterial contaminants were detected. All tubes were tightly capped to prevent evaporation during storage.

Drug containing media was also prepared as above. Drug containing media was prepared as given in Table 1. Stock solution was prepared by dissolving the 10?mg of drug (Compounds 3a–h, 4a–h, and Compound 5) in 5?mL of DMF. Final concentration of stock solution is considered as 2000?mg/L.

Table 1 

Media Preparation for anti tuberculosis activity.

Table 2 

Anti oxidant and cytotoxic activity of chalcones (Compounds 3a–h).

Table 3 

Anti oxidant and cytotoxic activity of Flavanones (Compounds 4a–h).

3.2.2. Drug Susceptibility Test

MTB isolate (preferably H37 Rv) were tested for drug susceptibility using the absolute concentration method on egg-based LJ medium, using a standardized inoculums (4?mg/4?mL) grown on drug free media along with media containing graded concentrations of the drug(s). All the test cultures are kept incubator at 37°C for 4–6 weeks.

The susceptibility test of antitubercular drugs was examined using five concentrations of Compounds 3a–h, 4a–h, and 5 (12.5, 25, 50, 100, and 200?mg/L).

Resistance is expressed in terms of the lowest concentration of the drug that inhibits growth, that is, minimal inhibitory concentration (MIC).

3.3. Antioxidant Activity

Superoxide Scavenging Method. Superoxide scavenging activity of the compounds 3a–h, 4a–h, 5a–e, and 6a–e was determined by the method [17] modified by [18] Kutlan et al., which depends on the light induced superoxide generation by riboflavin and the corresponding reduction of NBT. The assay mixture contained different concentration of the test substances and EDTA (6?mM containing 3?µg NaCN), NBT (50?µM) riboflavin (2?µM), and phosphate buffer 58?mM, pH 7.8 in a total volume of 3?mL. The tubes received uniform illumination for 15?min and thereafter optical density was measured at 560?nm. Consider the following: %inhibition=control−samplecontrol×100. (1) An IC₅₀ value was determined as the concentration that elicited the half maximal response.

Stastical Analysis. The data were analyzed by (single factor) for multiple groups and the significance level was chosen as 𝑃<0.05. Data is expressed as the mean (+) or (-)-SEM with a minimum of three experiments performed per each variable.

3.4. DPPH Free Radical Scavenging Activity

DPPH (1,1-diphenyl-2picryl-hydrazyl) free radical scavenging activity of the synthesised compounds 3a–h, 4a–h, and 5 was determined by the method [19] of Lamaison et al., which depends on scavenging of colored free radical (DPPH) in methanol solution by the test drugs. The reaction mixture contains DPPH and test drug in a final concentration of 3?mL. Absorption of DPPH at its adsorption maximum 516?nm is inversely proportional to the concentration of the scavenger (test drug). The activity was expressed as inhibitory concentration 50 (IC₅₀), that is, the concentration of the test solution required to give 50% reduction in absorbance of the test solution as compared to that of blank solution. Consider IC₅₀=(ODofcontrol−(ODoftest−ODtestblank))ODofcontrol×100. (2) The results obtained by both methods were given in Tables 2 and 3.

3.5. Cytotoxic Activity

Cytotoxic activity of the synthesised compounds 3a–h, 4a–h, and 5 were determined by using a known method [20, 21]. 5?mL of brine solution is taken into each test tube. Test substances were added to the tube according to their concentrations. The solutions were thoroughly mixed with the help of the cyclomixer. Then 10 shrimps were added to each test tube. Suitable conditions like temperature should be maintained for proper results. Finally by observing the lethality rate of the shrimps after 24 hours the ED₅₀ value is calculated. The ED₅₀ using probed analysis at 95% confidence limits from observed data. Replicas maintained to get accurate results (Tables 2 and 3).

Acknowledgments

The authors are thankful to Acharya Nagarjuna University, Guntur, A.P-India, and UGC, New Delhi (UGC-MRP/F. no. 39-752/2010 (SR) dated 13.01.2011) for their financial support. Also they are greatly thankful to G. V. Subba Raju, Director and C.E.O. Natsol Laboratories Pvt. Ltd., for his helpful discussions.