Synthesis, Investigation of Anticancer and Antioxidant Activity of Certain 2,4-Diaryl Substituted Benzodiazepine and Benzothiazepines Derived From Vanillin

Introduction

The development of new and safer anticancer agents is one of the fundamental goals in medicinal chemistry. Cancer has been constant skirmish globally with a lot of development in cures and preventive remedies. Cancer is considered to be one of the most burdensome diseases because of the inborn characteristics of cancer cells to proliferate uncontrollably, avoid apoptosis, invade and metastasize.¹ Cancer incidences in Indian population is unnerving and is estimated that 2.25 million people are living with disease. The drift of cancer is increasing across the world and thus it is the leading cause of deaths in economically developed countries and second leading cause of deaths in developing countries.² Globally, lung cancer cases and deaths are increasing, of which non-small cell lung cancer is contributing about 85% of all lung cancer cases.³ In 2018, GLOBOCAN estimated 2.09 million new cases and 1.76 million deaths, making lung cancer the most recurrent fatal cancer.⁴ Most of the presently available anticancer drugs are toxic but are considered tenable only because truly safe drugs are unavailable. Thus, more effective, selective and safer anticancer agents are urgently needed to manage this scenario more effectively. Free radicals play an important role as the DNA damage by the carcinogenic molecule is said to be free radical mediated. Therefore, compounds having free radical scavenging activity would be an additional edge in the cancer therapy.

Benzodiazepines and benzothiazepines are entities which are being synthesized in many of their derivative forms since past few years. These entities received much attention by various researchers in the recent years since they display a fascinating array of pharmacological properties such as anticancer, antioxidant, antibacterial, anti-inflammatory and antitubercular actions.^5-9 Chalcones are well known to manifest promising biological activities and are used for the construction of various class of heterocyclic compounds.¹⁰ So, in the present study it was planned to synthesize novel 2, 4-diaryl substituted benzodiazepines and benzothiazepines incorporating 3-methoxy-4-hydroxy benzene scaffold as possible anticancer and antioxidant agents.

Materials and Methods

The chemicals used were of laboratory reagent grade and used without further purification. Vanillin, substituted acetophenones, o-aminothiophenol and 2,2-diphenyl-1-picrilhydrazyl (DPPH) were purchased from Lobachemie Ltd and Himedia, laboratories Pvt. Ltd, Mumbai, India. o-phenylenediamine, ascorbic acid were purchased from SD Fine chemicals, Mumbai, India. Eagle’s Minimal Essential Medium (Eagle’s MEM) (Himedia, laboratories Pvt. Ltd, Mumbai, India),Fetal Bovine Serum (Thermo Fisher Scientific Waltham, USA) and 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide, MTT (Sigma Chemicals Co., St. Louis USA) were also purchased. Non-small cell lung cancer cell line A549 was used to screen the compounds for anticancer activity. Progress of the reaction and purity of the compounds were ascertained by thin layer chromatography using aluminium sheets precoated with silica gel 60 F254 (Merck, Darmstadt, Germany) and various solvent combinations as mobile phase; the spots were visualized by iodine vapours. All yields refer to crude product. Melting points were determined using open capillary apparatus (Veego VMP-DS, Mumbai, India) and are uncorrected. The infrared (IR) spectra were recorded on Fourier transforms Infrared spectrophotometer (Bruker Alpha-t, Massachusetts, USA) by KBr pallet technique. The ultraviolet spectra were recorded on UV spectrophotometer (UV 1601 Kyoto, Japan). Proton nuclear magnetic resonance (1HNMR) spectra were recorded in DMSO-d6 on 400 MHz spectrophotometer, (Agilent California, USA). Chemical shifts were measured on delta scale in parts per million (ppm) downfield to tetramethylsilane. Peak multiplicities were indicated as s (singlet), d (doublet), dd (doublet of doublet), t (triplet) and m (multiplet).¹³C nuclear magnetic resonance spectra were recorded at 50 MHz. The mass spectra were recorded on LC/MS (Waters-SynaptG2, Massachusetts, USA). The absorbance of MTT assay was read on Synergy TM HT Microplate reader (Bio-Tek Instruments Inc., Winooski, USA).

General procedure for synthesis of chalcone (1a-j):

A mixture of Vanillin (0.01 mol) and substituted acetophenones (0.01 mol) were dissolved in ethanol [95 %, 5ml] and was passed with dry hydrogen chloride gas for 30-45m. while stirring, during which the reaction mixture became highly coloured and viscous. The mixture was stirred further for 1 h and left overnight at room temperature and then poured into crushed ice. The excess of HCl was neutralized carefully with sodium bicarbonate if necessary. The solid separated was filtered and washed with ice cold water until excess of acid is removed off and recrystallised from ethanol.¹¹

3-(4-hydroxy-3-metoxyphenyl)-1- phenylprop-2- en-1-one (1a):

Prepared from acetophenone as dark brown powder; Mol. formula: C₁₆H₁₄O₃ ; Mol. weight: 254.29; yield: 98%; Mp: 107-1090C; Rf (n-hexane: ethylacetate 5:1 V/V):0.30; λmax (ethanol, nm): 294; IR (KBr cm^-1): 3432 (OH), 3062 (Ar-CH), 2935 (Ali-CH), 1674 (C=O), 1597 (Ar-C=C), 1116 (assym C-O-C).

3-(4-hydroxy-3-metoxyphenyl)-1- (4-nitrophenyl) prop-2-en-1-one (1b):

Prepared from 4-nitro acetophenone as greenish grey powder; Mol. formula: C₁₆H₁₃NO₅ ; Mol. weight: 299.38; yield: 74%; Mp: 170-1710C; Rf(n-hexane: ethylacetate 5:1 V/V):0.18; λmax (ethanol, nm): 388; IR (KBr cm^-1): 3521 (OH), 3101 (Ar-CH), 3016 (Ali-CH), 1647 (C=O), 1519 (Ar-C=C), 1122 (assym C-O-C), 1569 (N-O).

1-(4-bromophenyl)-3-(4-hydroxy3methoxyphenyl)prop-2-en-1-one (1c):

Prepared from 4-bromo acetophenone as brown powder; Mol. formula: C₁₆H₁₃BrO₃ ; Mol. weight: 333.18; yield: 94%; Mp: 101-103⁰ C; Rf (n-hexane: ethylacetate 5:1 V/V):0.30; λmax (ethanol, nm): 534; IR (KBr cm^-1): 3467 (OH), 3033 (Ar-CH), 2953 (Ali-CH), 1672 (C=O), 1598 (Ar-C=C), 1117 (assym C-O-C), 696 (C-Br).

1-(4-chlorophenyl)-3-(4-hydroxy-3methoxyphenyl) prop-2-en-1-one (1d):

Prepared from 4-chloro acetophenone as dark brown powder; Mol. formula: C₁₆H₁₃ClO₃ ; Mol. weight: 288.73; yield: 67%; Mp: 100-102⁰ C; Rf(n-hexane: ethylacetate 5:1 V/V):0.31; λmax (ethanol, nm):535; IR (KBr cm^-1): 3457 (OH), 3028 (Ar-CH), 2966 (Ali-CH), 1669 (C=O), 1592 (Ar-C=C), 1092 (assym C-O-C), 786 (C-Cl).

3-(4-hydroxy-3-methoxyphenyl)-1-(4-iodophenyl) prop-2-en-1-one (1e):

Prepared from 4-iodo acetophenone as reddish brown powder; Mol. formula: C₁₆H₁₃IO₃ ; Mol. weight: 380.18; yield: 55%; Mp: 115-117⁰ C; Rf(n-hexane: ethylacetate 5:1 V/V):0.64; λmax (ethanol, nm): 370; IR (KBr cm^-1): 3347 (OH), 3067 (Ar-CH), 2933 (Ali-CH), 1676 (C=O), 1577 (Ar-C=C), 1121 assym (C-O-C), 670 (C-I).

4-(3-(4-hydroxy-3-methoxyphenyl)acryloyl) benzonitrile (1f):

Prepared from 4-cyano acetophenone as yellowish orange powder; Mol. formula: C₁₇H₁₃NO₃ ; Mol. weight: 279.30; yield: 97%; Mp: 228-2300 C; Rf(n-hexane: ethylacetate 5:1 V/V):0.29; λmax (ethanol, nm): 382; IR (KBr cm^-1): 3345 (OH), 3068 (Ar-CH), 2991 (Ali-CH), 1657 (C=O), 1574 (Ar-C=C), 1117 (assym C-O-C), 2239 (C≡N).

3-(4-hydroxy-3-methoxyphenyl)-1-(4-hydroxyphenyl )prop-2-en-1-one (1g):

Prepared from 4-hydroxy acetophenone as orange powder; Mol. formula: C₁₆H₁₄O₄ ; Mol. weight: 270.28; yield: 60%; Mp: 240-2410 C; Rf(n-hexane: ethylacetate 5:1 V/V):0.24; λmax (ethanol, nm): 362; IR (KBr cm^-1): 3733 (OH), 3019 (Ar-CH), 2973 (Ali-CH), 1635 (C=O), 1515 (Ar-C=C), 1122 (assym C-O-C). 

3-(4-hydroxy-3-methoxyphenyl)-1-(p-tolyl) prop-2-en-1-one (1h):

Prepared from 4-methyl acetophenone as brown powder; Mol. formula: C₁₇H₁₆O₃ ; Mol. weight: 268.31; yield: 90%; Mp: 128-130⁰ C; Rf(n-hexane: ethylacetate 5:1 V/V):0.28; λmax (ethanol, nm): 382; IR (KBr cm^-1): 3508 (OH), 3046 (Ar-H) 2972 (Ali-CH), 1673 (C=O), 1583 (Ar-C=C), 1122 (assym C-O-C).

3-(4-hydroxy-3-methoxyphenyl)-1-(4-methoxyphenyl) prop-2-en-1-one (1i):

Prepared from 4-methoxy acetophenone as creamy yellow powder; Mol. formula: C₁₇H₁₆O₄ ; Mol. weight: 284.31; yield: 47%; Mp: 160-161⁰ C; Rf(n-hexane: ethylacetate 5:1 V/V):0.15; λmax (ethanol, nm): 364; IR (KBr cm^-1): 3371 (OH), 3071 (Ar-CH), 2967 (Ali-CH), 1650 (C=O), 1597 (Ar-C=C), 1118 (assym C-O-C).

1-(4-aminophenyl)-3-(4-hydroxy-3-methoxyphenyl) prop-2-en-1-one (1j):

Prepared from 4-amino acetophenone as yellowish orange powder; Mol. formula: C₁₆H₁₅NO₃ ; Mol. weight: 269.30; yield: 48%; Mp: 220-222⁰ C; Rf(n-hexane: ethylacetate 5:1 V/V):0.32; λmax (ethanol, nm): 315; IR (KBr cm^-1): 3478 (NH), 3301(OH), 3046 (Ar-CH), 3008 (Ali-CH), 1662 (C=O), 1581 (Ar-C=C), 1173 (assym C-O-C).

General procedure for synthesis of benzodiazepines and benzothiazepines (2a-j) and (3a-j)

A mixture of substituted chalcones 1a-j (0.01mol) and o-phenylenediamine (0.01 mol) / o-aminothiophenol (0.01mol) were dissolved in (10-20 ml) N,N-Dimethylformamide / methanol and 5ml glacial acetic acid was used as catalyst and refluxed for 45-50 h. The resulting mixture was allowed to stand for 10-15 m and mixture was poured into ice-cold water and then it was neutralized with sodium bicarbonate solution. The sticky nature was removed by treatment with brine solution if necessary. The solid obtained was washed successively with ice-cold water and recrystallised from aqueous alcohol.^12-13

2-methoxy-4-(4-phenyl-1H-1,5-benzodiazepin-2-yl) phenol (2a):

Prepared from acetophenone as brown amorphous powder; Mol. formula: C₂₂H₁₈N₂O₂ ; Mol. weight: 342.40; yield: 76.7%; Mp: 107-109⁰C; Rf(choloroform: ethylacetate 3:1 V/V):0.61; λmax (ethanol, nm): 493; IR (KBr cm^-1): 3345.25 (OH), 3064.15 (Ar-CH), 3155.85, 1598.95 (NH, C=N Diazepine), 1510.90 (Ar-C=C), 1264.85 (assym C-O-C); ¹ H NMR: δ 11.9, 6.52 (s, 2H, Diazepine NH, C=CH), 9.4 (s, 1H, OH), 6.42-8.4 (m, 12H, Ar-H), 3. (s, H, OCH3)

2-methoxy-4-(4-(4-nitrophenyl)-1H-1,5- benzodiazepin-2-yl) phenol (2b):

Prepared from 4-nitro acetophenone as yellow powder; Mol. formula: C₂₂H₁₇N₃O₄ ; Mol. weight: 387.40; yield: 85.4%; Mp: 144-146⁰ C; Rf (choloroform: ethylacetate 3:1 V/V):0.28;λmax (ethanol, nm): 313; IR (KBr cm^-1): 3355.79 (OH), 3067.89 (Ar-CH), 3215.79, 1594.60 (NH,C=N, Diazepine), 1511.48 (Ar-C=C), 1268.61 (assym C-O-C), 1342.86 (N-O); ¹H NMR: δ 12.63, 6.58 (s, 2H, Diazepine NH, C=CH), 9.5 (s, 1H, OH), 6.69-8.60 (m, 11H, Ar-H), 3.84 (s, H, OCH3); MS: m/z 387(M+).  

4-(4-(4-bromophenyl)-1H-1,5-benzodiazepin-2-yl) -2-methoxyphenol (2c):

Prepared from 4-bromo acetophenone as dark brown amorphous powder; Mol. formula: C₂₂H₁₇BrN₂O₂ ; Mol. weight: 421.28; yield: 62.7%; Mp: 176-178⁰ C; Rf (choloroform: ethylacetate 3:1 V/V):0.63; λmax (ethanol, nm): 307; IR (KBr cm-1): 3330.03 (OH), 3015.25 (Ar-CH), 3261.60, 1620.40 (NH, C=N Diazepine), 1515.55 (Ar-C=C), 1265.26 (assym C-O-C), 585.35 (C-Br) ; ¹H NMR: δ 12.52, 6.42 (s, 2H, Diazepine NH, C=CH), 9.3 (s, 1H, OH), 6.96-8.2 (m, 11H, Ar-H), 3.74 (s, H, OCH₃ ). 

4-(4-(4-chlorophenyl)-1H-1,5-benzodiazepin-2-yl) -2-methoxyphenol (2d):

Prepared from 4-bromo acetophenone as dark brown amorphous powder; Mol. formula: C₂₂H₁₇BrN₂O₂ ; Mol. weight: 421.28; yield: 62.7%; Mp: 176-178⁰ C; Rf (choloroform: ethylacetate 3:1 V/V):0.63; λmax (ethanol, nm): 307; IR (KBr cm^-1): 3330.03 (OH), 3015.25 (Ar-CH), 3261.60, 1620.40 (NH, C=N Diazepine), 1515.55 (Ar-C=C), 1265.26 (assym C-O-C), 585.35 (C-Br) ; ¹H NMR: δ 12.52, 6.42 (s, 2H, Diazepine NH, C=CH), 9.3 (s, 1H, OH), 6.96-8.2 (m, 11H, Ar-H), 3.74 (s , H, OCH₃). 

4-(4-(4-iodophenyl)-1H-1,5-benzodiazepin-2-yl) -2-methoxyphenol (2e):

Prepared from 4-iodo acetophenone as brown amorphous powder; Mol. formula: C₂₂H₁₇IN_{2 O2} ; Mol. weight: 468.28; yield: 64.4%; Mp: 110-112⁰C; Rf (choloroform: ethylacetate 3:1 V/V):0.8;λ max(ethanol, nm): 487; IR (KBr cm^-1): 3240.28 (OH), 3035.15 (Ar-CH), 3255.35, 1640.48 (NH, C=N Diazepine), 1575.68 (Ar-C=C), 1268.46 (assym C-O-C), 575.65 (C-I); ¹H NMR: δ 12.69, 6.52 (s, 2H, Diazepine NH, C=CH), 9.4 (s, 1H, OH), 6.86-8.97 (m, 11H, Ar-H), 3.76 (s, H, OCH₃ ). 

4-(2-(4-hydroxy-3-methoxyphenyl)-1H-1,5- benzodiazepin-4-yl)benzonitrile (2f):

Prepared from 4-cyano acetophenone as yellow powder; Mol. formula: C₂₃H₁₇N₃O₂ ; Mol. weight: 367.41; yield: 59.7%; Mp: 228-230⁰ C; Rf (choloroform: ethylacetate 3:1 V/V):0.72;λmax (ethanol, nm): 374; IR (KBr cm^-1): 3332.07 (OH), 2934.18 (Ar-CH), 3060.57, 1677.79 (NH, C=N Diazepine), 1509.85 (Ar-C=C), 1271.07 (assym C-O-C), 2228.67 (C≡N); ¹ H NMR: δ 12.62, 5.31 (s, 2H, Diazepine NH, C=CH),9.50 (s, 1H, OH), 6.68-8.99 (m, 11H, Ar-H), 3.85 (s,3H,OCH₃ ).

4-(4-(4-hydroxyphenyl)-1H-1,5-benzodiazepin-2-yl) -2-methoxyphenol (2g):

Prepared from 4-hydroxy acetophenone as light green powder; Mol. formula: C₂₂H₁₈N₂O₃ ; Mol. weight: 358.38; yield: 60%; Mp: 108-110⁰ C; Rf (choloroform: ethylacetate 3:1 V/V):0.43;λmax (ethanol, nm): 317; IR (KBr cm^-1): 3232.52 (OH), 3011.67 (Ar-CH), 3061.36,1652.24 (NH, C=N Diazepine), 1597.16 (Ar-C=C), 1270.54 (assym C-O-C); 1H NMR: δ 12.6, 6.59 (s, 2H, Diazepine NH, C=CH), 9.6 & 8.88 (s, 2H, OH), 6.51-7.84 (m, 11H, Ar-H), 3.70 (s, 3H, OCH₃).

2-methoxy-4-(4-(4-methylphenyl)-1H-1,5- benzodiazepin-2-yl)phenol (2h):

Prepared from 4-methyl acetophenone as black brown amorphous powder; Mol. formula: C₂₃H₂₀N₂O₂ ; Mol. weight: 356.41; yield: 73.8%; Mp: 128-130⁰ C; Rf (choloroform: ethylacetate 3:1 V/V):0.60;λmax (ethanol, nm) 482; IR (KBr cm^-1): 3228.69 (OH), 3048.94(Ar-CH), 3130.85, 1660.13 (NH, C=N Diazepine), 1525.28 (Ar-C=C), 1261.25 (assym C-O-C); ¹H NMR: δ 12.52, 5.46 (s, 2H, Diazepine NH, C=CH), 9.4 (s, 1H, OH), 6.48-8.79 (m, 11H, Ar-H), 3.75 (s,3H,OCH3), 3.42 (s, 1H, CH₃).

2-methoxy-4-(4-(4-methoxyphenyl)-1H-1,5- benzodiazepin-2-yl)phenol (2i):

Prepared from 4-methoxy acetophenone as dark brown amorphous powder; Mol. formula: C₂₃H₂₀N₂O₃ ; Mol. weight: 372.41; yield: 80%; Mp: 106-108⁰ C; Rf(choloroform: ethylacetate 3:1 V/V):0.49;λmax (ethanol, nm): 384; IR (KBr cm^-1): 3294.29 (OH), 2934.75 (Ar-CH), 3059.92, 1657.15 (NH, C=N Diazepine), 1508.90 (Ar-C=C), 1263.33 (assym C-O-C), ¹ H NMR: δ 12.6, 6.57 (s, 2H, Diazepine NH, C=CH), 6.51-8.12 (m, 11H, Ar-H), 5.32 (s, 1H, OH), 3.80 & 3.70 (s, 6H, OCH₃ ).

N-(4-(2-(4-hydroxy-3- methoxyphenyl)-1H-1,5- benzodiazepin-4-yl) phenyl)acetamide (2j):

Prepared from 4-amino acetophenone as buff amorphous powder; Mol. formula: C₂₄H₂₃N₃O₃ ; Mol. weight: 401.47; yield: 86.9%; Mp: 220-222⁰C; Rf (choloroform: ethylacetate 3:1 V/V):0.37; λmax (ethanol, nm): 307; IR (KBr cm^-1): 3225.60 (OH), 3057.09 (Ar-CH), 3111.41, 1673.12 (NH, C=N Diazepine), 1507.34 (Ar-C=C), 1271.08 (assym C-O-C),1775.01 (C=O of NHCOCH₃ ); ¹H NMR: δ 12.6, 6.89 (s, 2H, Diazepine NH, C=CH), 10.1 (s, 1H, NH, acetamido), 9.5 (s, 1H, OH), 6.52-8.19 (m, 11H, Ar-H), 3.87 (s, 3H, OCH₃), 3.7 (s, 3H, acetamido). 

2-methoxy-4-(4-phenyl-2,3-dihydro-1, 5benzothiazepin-2-yl)phenol (3a):

Prepared from acetophenone as brown solid powder; Mol. formula: C₂₂H₁₉NO₂S; Mol. weight: 361.42; yield: 94.4%; Mp: 78-80⁰ C; Rf (choloroform: ethylacetate 3:1 V/V):0.74;λmax (ethanol, nm): 299; IR (KBr cm^-1): 3360.15 (OH), 3034.25 (Ar-CH), 1636.46 (C=N), 1523.24 (Ar-C=C), 750.29 (C-S), 1260.59 (assym C-O-C); ¹ H NMR: δ: 8.62 (s, 1H, OH), 6.22-8.12 (m, 12H, Ar-H), 3.99 (t, 1H, CH Thiaz), 3.64 & 3.72 (dd, 2H, CH2Thiaz), 3.82 (s, 3H, OCH3); MS: m/z 362.06 (M++1). 

2-methoxy-4-(4-(4-nitrophenyl)-2,3dihydro-1, 5-benzothiazepin-2-yl)phenol (3b):

Prepared from 4-nitro acetophenone as yellow black powder; Mol. formula: C₂₂H₁₈N₂O₄S; Mol. weight: 406.46; yield: 93.9%; Mp: 118-120⁰ C; Rf (choloroform: ethylacetate 3:1 V/V):0.77;λmax(ethanol, nm): 296; IR (KBr cm-1): 3370.38 (OH), 3065.88 (Ar-CH), 1596.59 (C=N), 1596.59 (Ar-C=C), 758.38 (C-S), 1261.51 (assym C-O-C), 1516.26 (N-O);¹ H NMR: δ: 8.98 (s, 1H, OH), 6.69-8.14 (m, 11H, Ar-H), 5.17 (t, 1H, CH Thiaz), 2.90 & 3.35 (dd, 2H, CH₂ Thiaz), 3.65(s, 3H, OCH₃ ).

4-(4-(4-bromophenyl)-2,3-dihydro1,5benzothiazepin-2-yl)-2-methoxyphenol (3c):

Prepared from 4-bromo acetophenone as brown amorphous powder; Mol. formula: C₂₂H₁₈BrNO₂S; Mol. weight: 440.35; yield: 86%; Mp: 120-121⁰ C; Rf (choloroform: ethylacetate 3:1 V/V):0.86;λmax (ethanol, nm): 294; IR (KBr cm^-1): 3369.62 (OH), 3059.85 (Ar-CH), 1676.47 (C=N), 1583.24 (Ar-C=C), 753.99 (C-S), 1266.69 (assym C-O-C), 541.89 (C-Br); ¹H NMR: δ: 8.77 (s, 1H, OH), 6.40-7.88 (m, 11H, Ar-H), 4.69 (t, 1H, CH Thiaz), 3.55 & 3.66 (dd, 2H, CH₂Thiaz), 3.75 (s, 3H, OCH₃ ); MS: m/z 440.35(M+). 

4-(4-(4-chlorophenyl)-2,3-dihydro1,5benzothiazepin-2-yl)-2-methoxyphenol (3d):

Prepared from 4-chloro acetophenone as orange powder; Mol. formula: C₂₂H₁₈Cl NO₂S; Mol. weight: 395.90; yield: 97%; Mp: 138-140⁰ C; Rf (choloroform: ethylacetate 3:1 V/V):0.77;λmax (ethanol, nm): 512; IR (KBr cm^-1): 3367.16 (OH), 3059.47 (Ar-CH), 1675.76 (C=N), 1590.56 (Ar-C=C), 750.39 (C-S), 1266.85 (assym C-O-C), 632.71 (C-Cl); ¹H NMR: δ: 8.8 (s, 1H, OH), 6.40-7.98 (m, 11H, Ar-H), 4.69 (t, 1H, CH Thiaz), 3.4 & 3.75 (dd, 2H, CH₂Thiaz), 3.67 (s, 3H, OCH₃).

4-(4-(4-iodophenyl)-2,3-dihydro1,5benzothiazepin-2-yl)-2-methoxyphenol (3e):

Prepared from 4-iodo acetophenone as dark brown powder; Mol. formula: C₂₂H₁₈INO₂S; Mol. weight: 487.35; yield: 97.3%; Mp: 110-112⁰ C; Rf (choloroform: ethylacetate 3:1 V/V):0.92;λmax (ethanol, nm): 307; IR (KBr cm^-1): 3364.25 (OH), 3056.83 (Ar-CH), 1673.67 (C=N), 1586.46 (Ar-C=C), 753.71 (C-S), 1268.9 (assym C-O-C), 549.45 (C-I); ¹ H NMR: δ: 8.6 (s, 1H, OH), 6.54-7.87 (m, 11H, Ar-H), 4.58 (t, 1H, CH Thiaz), 3.7 & 3.48 (dd, 2H, CH₂Thiaz), 3.86 (s, 3H, OCH₃ ).

4-(2-(4-hydroxy-3-methoxyphenyl)-2,3dihydro1, 5-benzothiazepin-4-yl)benzonitrile (3f):

Prepared from 4-cyano acetophenone as yellow powder; Mol. formula: C₂₃H₁₈N₂O₂S; Mol. weight: 386.46; yield: 97%; Mp: 108-110⁰ C; Rf (choloroform: ethylacetate 3:1 V/V):0.82; λmax (ethanol, nm): 321; IR (KBr cm^-1): 3362.79 (OH), 3059.64 (Ar-CH), 2227.24 (C≡N)1659.31 (C=N), 1600.54 (Ar-C=C), 758.66 (C-S), 1270.95 (assym C-O-C); ¹H NMR: δ: 8.98 (s, 1H, OH), 6.64-8.24 (m, 11H, Ar-H), 5.2 (t, 1H, CH Thiaz), 3.75 & 3.85 (dd, 2H, CH₂Thiaz), 3.64 (s, 3H, OCH₃).

4-(4-(4-hydroxyphenyl)-2,3-dihydro-1, 5benzothiazepin-2-yl)-2-methoxyphenol (3g):

Prepared from 4-hydroxy acetophenone as yellow amorphous powder; Mol. formula: C₂₃H₁₉NO₃S; Mol. weight: 377.45; yield: 74%; Mp: 150-152⁰ C; Rf (choloroform: ethylacetate 3:1 V/V):0.74;λmax (ethanol, nm): 316; IR (KBr cm^-1):3315.48 (OH), 3028.26 (Ar-CH), 1556.39 (C=N), 1566.57 (Ar-C=C), 751.28 (C-S), 1264.61 (assym C-O-C); ¹ H NMR: δ: 8.99, 9.1 (s, 2H, OH), 6.75-8.59 (m, 11H, Ar-H), 5.7 (t, 1H, CH Thiaz), 3.46 & 3.19 (dd, 2H, CH₂ Thiaz), 3.43 (s, 3H, OCH₃ ).

2-methoxy-4-(4-(4-methylphenyl)-2, 3dihydro-1,5-benzothiazepin-2-yl)phenol (3h):

Prepared from 4-methyl acetophenone as black brown amorphous powder; Mol. formula: C₂₃H₂₁NO₂S; Mol. weight: 375.48; yield: 93%; Mp: 180-182⁰ C; Rf (choloroform: ethylacetate 3:1 V/V):0.88;λmax (ethanol, nm): 295; IR (KBr cm^-1): 3362.76 (OH), 3048.65 (Ar-CH), 1655.31 (C=N), 1595.16 (Ar-C=C), 754.23 (C-S), 1263.95 (assym C-O-C); ¹H NMR: δ: 8.75 (s, 1H, OH), 6.48-8.64 (m, 11H, Ar-H), 5.43 (t, 1H, CH Thiaz), 3.46 & 3.57 (dd, 2H, CH₂Thiaz), 3.37 (s, 3H, OCH₃), 3.3 (s, 1H, CH₃). 

2-methoxy-4-(4-(4-methoxyphenyl)-2,3dihydro-1, 5-benzothiazepin-2-yl)phenol (3i):

Prepared from 4-methoxy acetophenone as dark yellow amorphous powder; Mol. formula: C₂₃H₂₁NO₃S; Mol. weight: 391.48; yield: 98.5%; Mp: 110-112⁰ C; Rf (choloroform: ethylacetate 3:1 V/V):0.66;λmax (ethanol, nm): 308; IR (KBr cm^-1): 3361.11 (OH), 3056.49 (Ar-CH), 1664.73 (C=N), 1599.58 (Ar-C=C), 757.98 (C-S), 1255.58 (assym C-O-C); ¹H NMR: δ: 8.96 (s, 1H, OH), 6.58-8.03 (m, 11H, Ar-H), 5.04 (t, 1H, CH Thiaz), 2.83 & 5.45 (dd, 2H, CH₂Thiaz), 3.68 & 3.83 (s, 6H, OCH₃); ¹³C NMR: δ 170.36, 164.07, 154.75, 149.67, 148.40, 137.58, 137.12, 137.09, 131.67, 131.24, 127.16, 124.86, 120.70, 117.36, 116.26, 112.82, 57.89, 57.77, 42.53, 39.46; MS: m/z 391.48 (m+).

4-(4-(4-aminophenyl)-2,3-dihydro-1, 5benzothiazepin-2-yl)-2-methoxyphenol (3j):

Prepared from 4-amino acetophenone as yellow crystals; Mol. formula: C₂₄H₂₂N₂O₃S; Mol. weight: 418.51; yield: 42.7%; Mp: 162-163⁰ C; Rf (choloroform: ethylacetate 3:1 V/V):0.64; λmax (ethanol, nm): 327; IR (KBr cm^-1): 3342.84 (OH), 3032.89 (Ar-CH), 1770.25(C=O of NHCOCH3), 1648.63 (C=N), 1575.14 (Ar-C=C), 755.90 (C-S), 1267.98 (assym C-O-C); ¹H NMR: δ: 10.8 (s, 1H, NH, acetamido), 9.96 (s, 1H, OH), 6.59-8.73 (m, 11H, Ar-H), 5.7 (t, 1H, CH Thiaz), 3.48& 3.76 (dd, 2H, CH₂Thiaz), 3.57 (s, 3H, OCH₃), 3.68 (s, 3H, acetamido).

Anticancer activity: 

The title compounds were evaluated for anticancer activity by MTT assay. The cells were seeded at 1×105 cells/ml in 96 well micro titer plates in Eagle’s MEM with 10% fatal bovine serum. The cells were incubated overnight at 37⁰ C in 95% humidity and 5% CO₂ for attachment. Compound concentrations (ranging from 7.81-500 μM/300μl) in serial three-fold dilutions were added in triplicates and incubated for 48h at 5% CO₂ at 37⁰ c. Thereafter, the cells were washed twice with phosphate buffer pH 7.4 solutions and treated with 20 μl of MTT (5 mg/ml in phosphate buffer pH 7.4 solution) and further incubated for 4 h. All the medium including MTT solution was aspirated from the wells. The remaining formazan crystals were dissolved in 100 μl of DMSO and the absorbance was measured at 570 nm using a 96 well micro plate reader. The cytotoxicity index was determined using the untreated cells as negative control. 5-Flurouracil was used as standard drug. The percentage of cytotoxicity was calculated using the background corrected absorbance as follows.¹¹

The percentage cell viability was calculated using excel sheet and IC₅₀ were calculated using Graph pad prism.

Antioxidant activity:

Stock solution of DPPH (0.038 mg/ml) in ethanol (95%) was prepared. The various concentrations of benzodiazepine and benzothiazepine derivatives were prepared. For all sample solutions of 0.05, 0.1, 0.2, 0.4, 0.8 ml was added 2 ml stock solution of DPPH, further it is made up to 3 ml with ethanol (95%). Test tubes were kept for 30 m in dark and absorbance was recorded at 517 nm against reagent as blank. Ascorbic acid was used as the reference compound. The experiment was carried out in triplicate. The effective concentrations of sample required to scavenge DPPH radical by 50% (IC50)was obtained by linear regression analysis between % inhibition and concentrations.^14-15

The DPPH free radical scavenging activity was calculated using the formula:

The drug concentration that scavenged 50% (IC50) of DPPH concentration was determined by plotting triplicate data points over a concentration range and calculating values using regression analysis by word excel.

Results and Discussion

Chalcones (1,3-diaryl-2-propene-1-ones) are important precursors of many biologically active compounds and serve as important intermediates for the construction of corresponding benzodiazepines and benzothiazepines. Vanillin (3-methoxy-4-hydroxy- benzaldehyde) is a pleasant-smelling naturally occurring aromatic compound used as flavouring agent in pharmaceuticals. Literature review revealed that vanillin and its derivatives possess various biological activities.^16-17 It was suitably used as precursor for the development of 3-methoxy-4-hydroxy benzene incorporated benzodiazepines and benzothiazepines. The synthetic pathway leading to the title compounds 1a-j, 2a-j and 3a-j is depicted in scheme 1.

A series of chalcones (1a-j) was synthesized according to Claisen-Schmidt reaction by condensing various substituted acetophenones with vanillin in presence of dry HCl in good yield. The presence of hydroxyl substituents in aromatic aldehyde hinders the base catalyzed reaction and Results and Discussion decrease the activity of aldehyde component because of stabilization of the resultant anion by delocalization.There for the acid catalyzed reaction was carried out.¹⁸ All the synthesized chalcones (1a-j) were recrystallized from aqueous alcohol. The UV-visible spectra of (1a-j) exhibited λ max in the range of 294 - 535 nm due to π→π * transitions typical of α-β-unsaturated ketones. The IR spectra of compound (1a-j) showed characteristic α-β-unsaturated C=O stretching at 1635 - 1676 cm-1 and C=C stretching frequencies at 1515 - 1598 cm^-1 which confirms the formation of chalcones. The reaction of chalcones with o-phenylenediamine / o-aminothiophenol represents a convenient and versatile method for the preparation of benzodiazepines / benzothiazepines. The chalcones were allowed to react in N, N-Dimethylformamide / methanol with o-phenylenediamine / o-aminothiophenol to yield 2,4-disubstituted benzodiazepines (2a-j) / benzothiazepines (3a-j) in moderate to good yield. It required refluxing for 45-50 h depending upon their reactivity in presence of catalytic amount of glacial acetic acid. All the synthesized compounds were purified by recrystallization from aqueous ethanol. The purity was checked by performing TLC and characterized on the basis of physical and spectral data. The UV spectra of (2a-j) and (3a-j) exhibited λmax in the range of 297 - 493 nm and 294 - 512 nm respectively. The IR specta of the derivatives 2a-j and 3a-j showed the presence of C=C stretching frequency at 1507.34 - 1600.54 cm^-1, aromatic C-H stretching at 2934.118 -3067.89 cm^-1 and O-H stretch at 3225.60 - 3370.38 cm^-1, assym C-O-C stretching at 1255.58 - 1271.08 cm^-1 , C=N stretch at 1594.60 - 1677.79 cm^-1 and absence of band at frequencies 1635 - 1676 cm^-1 (Characteristic of α-β-unsaturated ketone). In addition, compounds 2a-j exhibited NH stretching of benzodiazepines ring at 3059.92 - 3261.60 cm^-1 and compounds 3a-j exhibited C-S stretching of benzothiazepines at 753.38 - 758.387 cm^-1. In ¹ H NMR spectra of the compounds 2a-j and 3a-j the aromatic protons appeared at δ 6.50 - 8.99. The OH proton and OCH₃ proton derived from vanillin appeared at δ 5.32 – 9.5 and 3.64 - 3.87 respectively. Further in the compounds 2a-j, NH and CH protons of benzodiazepine ring resonated in the range of δ 10.1 – 12.63 and 6.58 - 6.89 respectively. In addition, compound 2j also showed a peak at δ 3.7 representing three protons of acetamido group. The compounds 3a-j exhibited two doublet of doublet in the range of δ 2.90 - 6.41 due to two methylene protons at C-3 of benzothiazepine ring. The proton at C-2 position of benzothiazepine showed triplet in the range of δ 4.69 - 5.2. In ¹³C NMR spectra of compound 3i, the benzothiazepine C-2, C-3, and C-4 signals appeared at δ 42.53, 39.46 and 164.07 respectively. Whereas methoxy carbons resonate at δ 57.77, 57.89 and the carbon attached to methoxy groups appeared at δ 149.67 and 170.36 respectively. The carbon attached to hydroxyl group resonated at δ 148.40. The structure of the compounds 2a-j and 3a-j were also supported by the mass spectra of representative compounds 2b, 3a, 3c and 3i which showed molecular ion peak at m/z 387.00, 362.06, 440.35 and 391.48 M⁺ , M⁺⁺¹, M⁺ and M⁺ respectively in their mass spectra confirmed their molecular weight. The above spectral information confirming the formation of benzodiazepines and benzothiazepines.

Anticancer Activity by MTT assay:

Compounds 2 (a, b, c, d, h, i, j) and 3(a, b, c, f, g, i, j) were evaluated for in vitro anticancer activity at different concentrations against A549 cell lines (lung carcinoma) by MTT assay. The IC50 of benzodiazepine derivatives 2 (a-j) ranged between 46.28-184.34µM. Compound 2a with no para substitution, 2 (b, c, d) with electron withdrawing substituents and 2(h, i, j) with electron releasing substituents exhibited IC₅₀ ranging between 46.28µM, 108.44-184.34µM and 49.66-89.28µM respectively. Compound 2a showed maximum anticancer activity with least IC₅₀ value of 46.28µM indicating it is highly active among the tested benzodiazepine derivatives. The IC₅₀ of benzothiazepine derivatives (3a-j) ranged between 19.20-165.22 µM. The compound 3a with no para substitution, 3(b, c, f) with electron withdrawing substituents and 3(g, i, j)with electron releasing substituents exhibited IC₅₀ ranging between 44.88µM, 92.17-165.22µM and 19.20-83.15µM respectively. Compound 3i with methoxy substituent exhibited IC₅₀ of 19.20µM indicating it is highly active among the tested benzothiazepine derivatives. All the tested compounds except compound 3i showed feeble anticancer activity against A549 cell lines exhibiting much higher IC₅₀ than standard 5-flurouracil (10.23µM). The IC50 values obtained for each compound are summarized in table no 1.

Antioxidant activity by DPPH assay

All the newly synthesized compounds 2a-j and 3a-j were screened for in vitro free radical scavenging activity at different concentrations by DPPH method. All the compounds produced concentration dependent scavenging of the free radical, DPPH. The IC₅₀ values of 2a-j and 3a-j ranged between 0.017-0.112µM and 0.023- 0.161µM respectively. In the 2a-j series, all the compounds except 2g and 2j showed excellent free radical scavenging activity with IC₅₀ value equal or less than the standard drug ascorbic acid (IC₅₀, 0.047µM). Compound 2c with bromo substituent exhibited IC₅₀ of 0.017µM indicating it is highly active among the series. Whereas, compounds 2g and 2j exhibited good activity with IC₅₀ of 0.112 and 0.075µM respectively. In the 3a-j series, compounds 3a, 3c and 3i exhibited excellent free radical scavenging activity with IC₅₀ ranging between 0.023-0.036µM vis-a-vis ascorbic acid. Compound 3i with methoxy substituent on it exhibited IC₅₀ of 0.023µM indicating it is highly active among the series. Whereas, rest of the Compounds displayed good activity with IC50 ranging between 0.067-0.161µM. Results are summarized in table no1.Anticancer and antioxidant activity results of 2a-j and 3a-j indicate that, compounds with no para substitutions and electron releasing groups at para position favoured the anticancer activity against the cell line used in the study. In contrary, antioxidant activity does not depend on the nature of substitution at para position. i.e. unsubstitution at para position, substitution at para position either by electron releasing or electron withdrawing substituents favoured the antioxidant activity. However, benzodiazepine derivatives favored the antioxidant activity than the benzothiazepine derivatives. Among the compounds evaluated in the series, compound 3i exhibited high anticancer and antioxidant activity with IC₅₀ value of 19.20µM and 0.023µM respectively. Most of the compounds that exhibited excellent to good antioxidant activity, failed to exhibit good anticancer activity.

Conclusion

In the present study, twenty compounds belonging to two series derived from vanillin chalcones having benzodiazepine and benzothiazepine ring structure were successfully synthesised by a facile protocol and characterised by UV, IR, NMR and Mass spectral data. Chalcones were synthesized according to Claisen-Schmidt reaction by condensing various substituted acetophenones with vanillin. The cyclocondensation of substituted chalcones with o-phenylenediamne / o-aminothiophenol in presence of glacial acetic acid offered the title compounds 2a-j, 3a-j. All the synthesized compounds, except (3i) exhibited weak anticancer activity against A549 cell lines when studied by MTT assay. All the compounds exhibited excellent to good free radical scavenging activity in DPPH assay. Among the compounds synthesized and screened in the study, 2-methoxy-4-(4-(4-methoxyphenyl)-2,3-dihydro-1, 5-benzothiazepin-2-yl)phenol (3i) exhibited highly active anticancer and antioxidant activity with IC50 values of 19.20 μM and 0.023 μM respectively. In conclusion, insights gained in this study would be useful for the further development of novel anticancer and antioxidant agents.

Conflict of Interest

The authors declare no conflict of interest.