WO2017025980A2 - Novel benzothiazole derivatives with enhanced biological activity - Google Patents

Novel benzothiazole derivatives with enhanced biological activity Download PDF

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Publication number
WO2017025980A2
WO2017025980A2 PCT/IN2016/000207 IN2016000207W WO2017025980A2 WO 2017025980 A2 WO2017025980 A2 WO 2017025980A2 IN 2016000207 W IN2016000207 W IN 2016000207W WO 2017025980 A2 WO2017025980 A2 WO 2017025980A2
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derivatives
novel
benzothiazole
cystine
biological activity
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PCT/IN2016/000207
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French (fr)
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WO2017025980A3 (en
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Kalpana Chauhan
Bhawana Kumari
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Shoolineuniversity Of Biotechnology And Management Sciences
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/542—Carboxylic acids, e.g. a fatty acid or an amino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/549—Sugars, nucleosides, nucleotides or nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D277/00—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings
    • C07D277/60—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings condensed with carbocyclic rings or ring systems
    • C07D277/62—Benzothiazoles
    • C07D277/68—Benzothiazoles with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached in position 2
    • C07D277/82—Nitrogen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H13/00—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids
    • C07H13/02—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids
    • C07H13/08—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids having the esterifying carboxyl radicals directly attached to carbocyclic rings

Definitions

  • the present invention relates to the field of synthetic organic chemistry. More specifically It relates to novel bioconjugates of beuzothiaaole with increased sotabiliiy and bioavailability and having powerful antibacterial, antifungal and aotica&eer activities.
  • Benz.otMazole is an aromatic heterocyclic compound with the chemical formula C7H5NS, It is a colorless and slightly viscous liquid. Though hemothiazofc is not widely used in its native form, many of its derivatives are found in commercial products or in nature,
  • BemsotMazole has considerable place in research ar s especially in synthetic as well as in pharmaceutical chemistry because of its potest and significant pharmacological activities. However, it suffers from the limitation that its bioavailability is exceptionally low due to its poor absorption and rapid metabolism in the fiver and intestinal wall Further, it is highly hydrophobic which leads to lo bioavailability, in present invention, this limitation has been overcome is an innovative manner by preparation of novel derivatives using cysiim and glucosamine. The derivatives have e hanced bloactivity md better solubility as compared to other derivatives of benzothtazole.
  • Beiizothiazole is an aromatic, heterocyclic compound with the chemical formula C?3 ⁇ 4NS. it is a colorless, slightly viscous liquid. Although the parent compound, benzotbiazole is not widely used, many of its derivatives are found in commercial products or in nature.
  • Benzothiazole consists of a 5-membered 1, 3» thiazole ring fused to a benzene ring, The nine atoms of the bi-cycie and the attached substituents are coplanar.
  • Benzothiazoleis prepared by treatment of 2-aminobenzenethiol with acid chlorides.
  • Amino acids These are biologically important organic compounds composed of amine (- NH 2 ) and carboxylic acid (-COOH) functional groups, along with a side-chain specific to each amino acid.
  • the key elements of an amino acid are carbon, hydrogen, oxygen, and nitrogen, though other elements are found in the side-chains of certain amino acids.
  • Cystine is the amino acid formed by the oxidation of two cystine molecules that are covalently linked via a disulfide bond. This sulfur compound has the formula - (SCH 2 CH(NH 2 )C0 2 H) 2 . It is a white solid that is slightly soluble in water. Human hair and skin contain approximately 10-14% cystine by mass. Cystine serves as a substrate for the cystine-glutamate transport system. This transport system, which is highly specific for cystine and glutamate, is used to increase the concentration of cystine inside the cell. In this system, the anionic form of cystine is transported in exchange for glutamate. Cystine is quickly reduced to cysteine. Cysteine prodrugs, e.g. acetyl cysteine, increase glutamate are release into the extracellular space.
  • Glucosamine (C 6 H 13 N0 5 ) is an amino sugar and a prominent precursor in the biochemical synthesis of glycosylated proteins and lipids. Glucosamine is part of the structure of the polysaccharides chitosan and chitin, which compose the exoskeletons of crustaceans and other arthropods, as well as the cell walls of fungi and many higher organisms. Glucosamine is one of the most abundant monosaccharides. It is produced commercially by the hydrolysis of crustacean exoskeletons or, less commonly, by fermentation of a grain such as corn or wheat. Glucosamine appears to be safe for use as a dietary supplement though its effectiveness has not been established for any condition. In the US it is one of the most common non-vitamin, non-mineral, dietary supplements used by adults.
  • Bioconjugates are chemical strategies to form a stable covalent link between two molecules, at least one of which is a biomolecule. Synthetically modified biomolecules can have diverse functionalities, such as tracking cellular events, revealing enzyme function, determining protein biodistribution, imaging specific biomarkers, and delivering drugs to targeted cells. Bioconjugation is a crucial strategy that links these modified biomolecules with different substrates.
  • Pharmacological profile It refers to the study of therapeutic activity of a compound or composition in terms of safety and efficacy in a biological system.
  • Antibacterial activity It refers to anything that destroys bacteria or suppresses their growth or ability to reproduce. Heat, chemicals and antibiotic drugs - all have antibacterial properties.
  • Biological activity In pharmacology, biological activity or pharmacological activity describes the beneficial or adverse effects of a drug on living matter. When a drug is a complex chemical mixture, this activity is exerted by the substance's active ingredient (also called API or 'active pharmaceutical ingredient' but can be modified by the other constituents which are called 'excipients'.
  • active ingredient also called API or 'active pharmaceutical ingredient' but can be modified by the other constituents which are called 'excipients'.
  • Bioavailability In pharmacology, bioavailability (BA) is a subcategory of absorption and refers to 'the fraction of an administered dose of unchanged drug that reaches the systemic circulation '. It is one of the principal pharmacokinetic properties of drugs. By definition, when a medication is administered intravenously, its bioavailability is 100%.
  • Antifungal composition/medication An antifungal medication is a pharmaceutical fungicide used to treat and prevent mycoses such as athlete's foot, ringworm, candidiasis (thrush), serious systemic infections such as crypto-coccal meningitis, and others.
  • Escherichia coli also known as E. coli is a Gram-negative, anaerobic (growing in absence of oxygen), rod-shaped bacterium of the genus Escherichia that is commonly found in the lower intestine of warm-blooded organisms (endotherms). Most E. coli strains are harmless, but some serotypes can cause serious food poisoning in their hosts and are occasionally responsible for product recalls due to food contamination. The harmless strains are part of the normal flora of the gut and can benefit their hosts by producing vitamin K2 (menaquinone) and preventing colonization of the intestine with pathogenic bacteria.
  • Staphylococcus aureus It is a gram-positive, coccal (round shaped) bacterium that is frequently found in the human respiratory tract and on the skin. It is positive for catalase and nitrate reduction.
  • S. aureus is not always pathogenic, it is a common cause of skin infections (e.g. boils), respiratory disease (e.g. sinusitis) and food poisoning. Disease associated strains often promote infections by producing potent protein toxins and expressing cell-surface proteins that bind and inactivate antibodies.
  • MRSA pathogenic S. aureus
  • Candida albicans is a diploid fungus that grows both as yeast and filamentous cells and a causal agent of opportunistic oral and genital infections in humans and Candida onychomycosis, an infection of the nail plate.
  • Systemic fungal infections (fungemias) including those by C. albicans have emerged as important causes of morbidity and mortality in immunocompromised patients (e.g., AIDS, cancer chemotherapy, organ or bone marrow transplantation).
  • C. albicans biofilms may form on the surface of implantable medical devices.
  • hospital-acquired infections by C. albicans have become a cause of major health concerns.
  • Minimum inhibition concentration It is the lowest concentration of an antimicrobial that will inhibit the visible growth of a microorganism after overnight incubation. Minimum inhibitory concentrations are important in diagnostic laboratories to confirm resistance of microorganisms to an antimicrobial agent and also to monitor the activity of new antimicrobial agents. A MIC is generally regarded as the most basic laboratory measurement of the activity of an antimicrobial agent against an organism. Zone of inhibition (ZI): If an antibiotic stops the bacteria from growing or kills the bacteria, there will be a zone around that area where the bacteria have not grown enough to be visible. This is called a zone of inhibition. The size of this zone depends on how effective the antibiotic is at stopping the growth of the bacterium.
  • Molecular docking is a method which predicts the preferred orientation of one molecule to a second when bound to each other to form a stable complex. Knowledge of the preferred orientation in turn may be used to predict the strength of association or binding affinity between two molecules using scoring functions. Characterization of the binding behaviour plays an important role in rational design of drugs as well as to elucidate fundamental biochemical processes.
  • Benzothiazole is already reported as inhibitor of bacterial type II topoisomerase DNA gyrase (Gyr A/GyrB) and topoisomerase IV (par C/par E). Furthermore, Type II topoisomerase is found to be well defined target for drug discovery. These enzymes are homologous (structurally and functionally) and are willing to dual targeting by single ligand. This has encouraged investigation for new types of compounds with novel mechanism of action against type II topoisomerase.
  • Receptor protein tyrosine kinase plays an important role in signal transduction pathways that control cell division and segregation.
  • Benzothiazole also reported inhibitor against epidermal growth factor receptor tyrosine kinase (EGFR-TK) and has been identified as important in cancer development.
  • EGFR-TK epidermal growth factor receptor tyrosine kinase
  • US20070088017 Al Discloses use of The present invention does not disclose benzothiazole derivative for treatment of the derivative of prior art. Rather it diabetes. discloses a novel benzothiazole derivative with increased biological activity.
  • Alessia Catalano et. al. (2013) The present invention does not disclose Discloses use of 6-substituted-2- the derivative of prior art rather it aminobenzothiazole derivatives as a disclose a novel benzothiazole potential antimicrobials. derivative with increased biological activity. (Alessia Catalano et. al.,2- Aminobenzothiazole derivatives: Search
  • the primary object of the present invention is to provide novel derivatives of benzothiazole with improved biological activity as antibacterial, antifungal and anticancer agents.
  • Another object of the present invention is to disclose novel bioconjugates of benzothiazole derivatives with cystine.
  • Yet another object of the present invention is to disclose the novel compounds of benzothiazole- 2-yl-imino benzoic acid derivatives and its glucosamine conjugates.
  • a further object is to disclose a new category of antibacterials and anti-cancerous agents which are benzothiazole derivatives with broad-spectrum antimicrobial activity.
  • the present invention discloses novel bioconjugate of benzothiazole derivatives with cystine and glucosamine and their method of synthesis. Their method of synthesis is easy and eco-friendly, avoiding the use of hazardous materials or reactions.
  • the compounds offer technical advantages of increased solubility, potency, selectivity and biological activity.
  • the novel compounds are active in-vitro as antimicrobial (E. coli, S. aureus and C. albicans) and show anticancer (Hep-2 and caco cell lines) activity.
  • Formula, physico-chemical data and biological activity of the novel derivatives are as given in Table 1 to Table 9.
  • Figure 12 5-acetamido-3,4,6-trihydroxytetrahydro-2H-pyran-2-yl)methyl 4 - (((4- methoxybenzo[d]thiazol-2-yl)imino)methyl)benzoate (Me-glu-03) 13.
  • Figure 13 4-(((6-nitrobenzo[i/
  • the present invention discloses novel derivatives of benzothiazole with cystine and glucosamine conjugates, having powerful antibacterial, antifungal and anticancer activities.
  • Method of synthesis of the compounds is also disclosed.
  • the compounds offer technical advantages of increased solubility, potency, selectivity and biological activity.
  • the novel compounds are active against bacterial and fungal species and also cancerous cells. Their method of synthesis is easy and eco-friendly, avoiding the use of hazardous materials or reactions.
  • the mixture was subjected to microwave irradiation at 60% power for 2-3 minute.
  • the product was purified by extensive washing with hot water and ethanol. 5. The purified product was dried and recrystallized from ethanol.
  • the mixture was irradiated at power of 40% for 10 min under simultaneous cooling with airs.
  • the mixture was irradiated in microwave at power of 40% for 10 min under simultaneous cooling with airs.
  • test organisms S. aureus, E. coli, C. albicans (from IMTECH Chandigarh).
  • This broth suspension of the test bacterium was flooded on the surface of a solid medium (Muller Hinton agar) and potato dextrose agar for fungus.
  • Extracts were added in the wells along with the control (solvent: DMSO).
  • MIC was determined by broth micro dilution method i.e. CLSI M7-A7 (The Clinical and Laboratory Standards Institute) Culture (bacteria and fungus) was added and plate was incubated for 24 h.
  • Resazurin was used as indicator and added after 24 hour incubation and kept for 2-4 hour.
  • the assay was done using 96 well plates in Mueller Hinton broth.
  • Minimum effective antibacterial concentration (100 ⁇ g/mL) was chosen.
  • IZ maximum inhibition zone
  • Fungus C. albicans MIC value ranges from 12.5 to 3.125 ⁇ g/mL
  • the synthesized compounds were screened against E. coli and S. aureus for anti-microbial activity which were grown in nutrient broth (NB) medium overnight in incubator shaker at 37 °C.
  • Nutrient broth solution was prepared by dissolving 1.3 g of solid NB in 100 mL of distilled water in a 250 mL flask. The flask containing the NB, and all apparatus were autoclaved before use.
  • test tubes were taken for each sample.
  • 5.0 mL NB and 1.0 mL of bacterial culture were added. This test tube was considered as a control in which the normal bacterial growth took place.
  • test tube 5 mL of NB, 1.0 mL bacterial culture and sample solution (200 and 500 ⁇ g/mL) were added. Same concentration of sample with 10 mL of NB was also required as reference without the addition of culture. These tubes were considered as reference of the samples.
  • IZ maximum inhibition zone
  • Fungus C. albican MIC value ranges from 12.5 to 1.25 ⁇ g/mL
  • cervix carcinoma cell line Hep 2
  • caco cell line was quantitated by the ability of metabolic active cell to trim down the yellow MTT [3-(4,5-Dimethylthiazol-2- yl)-2,5-Diphenyltetrazolium Bromide] to purple formazone product.
  • Hep 2 and caco cell line were cultivated at 37 °C in an atmosphere of 5% C0 2 in Dubecco's modified eagle's minimal medium.
  • the %cell viability was calculated by using following formula.
  • %cell viability [abs57o of treated cell/absj7o of control cell] x lOO
  • the pharmacological antimicrobial mechanism was evaluated via docking study.
  • Novelty discloses novel benzothiazole derivative with glucosamine and cystine, having potent antibacterial, antifungal and anticancer properties. Derivatives having good antibacterial activity are depicted in Fig. 3 to Fig. 14.
  • the inventive step of the invention lies in developing novel benzothiazole bio- conjugates using cystine and N-acetyl-D-glucosamine derivatives which have antibacterial, antifungal and anti-cancer activity.
  • novel derivatives of present invention are easy to synthesize and can be manufactured at industrial level. They have considerable economic value as antibacterial, antifungal and anti-cancer agents.

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Abstract

The present invention discloses novel conjugates of benzothiazole derivatives with cystine and glucosamine and their method of synthesis. Their method of synthesis is easy and eco-friendly, avoiding the use of hazardous materials or reactions, The compounds offer technical advantages of increased solubility, potency, selectivity and biological activity. The novel compounds show antibacterial, antifungal and anticancer activities. Formula, physico-chemical data and biological activity of the novel derivatives are as given in Tables 1 to 9.

Description

I
NOVEL ENZOTHIAZOLE DERIVATIVES WITH ENHANCED BIOLOGICAL
ACTIVITY
FIELD OF INVENTION
The present invention relates to the field of synthetic organic chemistry. More specifically It relates to novel bioconjugates of beuzothiaaole with increased sotabiliiy and bioavailability and having powerful antibacterial, antifungal and aotica&eer activities.
BACKGROUND OF THE INVENTION
Benz.otMazole is an aromatic heterocyclic compound with the chemical formula C7H5NS, It is a colorless and slightly viscous liquid. Though hemothiazofc is not widely used in its native form, many of its derivatives are found in commercial products or in nature,
BemsotMazole has considerable place in research ar s especially in synthetic as well as in pharmaceutical chemistry because of its potest and significant pharmacological activities. However, it suffers from the limitation that its bioavailability is exceptionally low due to its poor absorption and rapid metabolism in the fiver and intestinal wall Further, it is highly hydrophobic which leads to lo bioavailability, in present invention, this limitation has been overcome is an innovative manner by preparation of novel derivatives using cysiim and glucosamine. The derivatives have e hanced bloactivity md better solubility as compared to other derivatives of benzothtazole.
Terms and Definitions
L Befi¾othla¾0te: Beiizothiazole is an aromatic, heterocyclic compound with the chemical formula C?¾NS. it is a colorless, slightly viscous liquid. Although the parent compound, benzotbiazole is not widely used, many of its derivatives are found in commercial products or in nature.
Figure imgf000002_0001
2. Bes¾>tM¾¾ok straetare and preparation; Benzothiazole consists of a 5-membered 1, 3» thiazole ring fused to a benzene ring, The nine atoms of the bi-cycie and the attached substituents are coplanar. Benzothiazoleis prepared by treatment of 2-aminobenzenethiol with acid chlorides.
C6H4(NH2)SH + RC(0)C1→ C6H4(NH)SCR + HC1 + H20
3. Amino acids: These are biologically important organic compounds composed of amine (- NH2) and carboxylic acid (-COOH) functional groups, along with a side-chain specific to each amino acid. The key elements of an amino acid are carbon, hydrogen, oxygen, and nitrogen, though other elements are found in the side-chains of certain amino acids.
4. Cystine: Cystine is the amino acid formed by the oxidation of two cystine molecules that are covalently linked via a disulfide bond. This sulfur compound has the formula - (SCH2CH(NH2)C02H)2. It is a white solid that is slightly soluble in water. Human hair and skin contain approximately 10-14% cystine by mass. Cystine serves as a substrate for the cystine-glutamate transport system. This transport system, which is highly specific for cystine and glutamate, is used to increase the concentration of cystine inside the cell. In this system, the anionic form of cystine is transported in exchange for glutamate. Cystine is quickly reduced to cysteine. Cysteine prodrugs, e.g. acetyl cysteine, increase glutamate are release into the extracellular space.
5. Glucosamine: Glucosamine (C6H13N05) is an amino sugar and a prominent precursor in the biochemical synthesis of glycosylated proteins and lipids. Glucosamine is part of the structure of the polysaccharides chitosan and chitin, which compose the exoskeletons of crustaceans and other arthropods, as well as the cell walls of fungi and many higher organisms. Glucosamine is one of the most abundant monosaccharides. It is produced commercially by the hydrolysis of crustacean exoskeletons or, less commonly, by fermentation of a grain such as corn or wheat. Glucosamine appears to be safe for use as a dietary supplement though its effectiveness has not been established for any condition. In the US it is one of the most common non-vitamin, non-mineral, dietary supplements used by adults.
6. Bioconjugates: Bioconjugation is a chemical strategy to form a stable covalent link between two molecules, at least one of which is a biomolecule. Synthetically modified biomolecules can have diverse functionalities, such as tracking cellular events, revealing enzyme function, determining protein biodistribution, imaging specific biomarkers, and delivering drugs to targeted cells. Bioconjugation is a crucial strategy that links these modified biomolecules with different substrates.
Pharmacological profile: It refers to the study of therapeutic activity of a compound or composition in terms of safety and efficacy in a biological system.
Antibacterial activity: It refers to anything that destroys bacteria or suppresses their growth or ability to reproduce. Heat, chemicals and antibiotic drugs - all have antibacterial properties.
Biological activity: In pharmacology, biological activity or pharmacological activity describes the beneficial or adverse effects of a drug on living matter. When a drug is a complex chemical mixture, this activity is exerted by the substance's active ingredient (also called API or 'active pharmaceutical ingredient' but can be modified by the other constituents which are called 'excipients'.
Bioavailability: In pharmacology, bioavailability (BA) is a subcategory of absorption and refers to 'the fraction of an administered dose of unchanged drug that reaches the systemic circulation '. It is one of the principal pharmacokinetic properties of drugs. By definition, when a medication is administered intravenously, its bioavailability is 100%.
Antifungal composition/medication: An antifungal medication is a pharmaceutical fungicide used to treat and prevent mycoses such as athlete's foot, ringworm, candidiasis (thrush), serious systemic infections such as crypto-coccal meningitis, and others.
Escherichia coli: also known as E. coli is a Gram-negative, anaerobic (growing in absence of oxygen), rod-shaped bacterium of the genus Escherichia that is commonly found in the lower intestine of warm-blooded organisms (endotherms). Most E. coli strains are harmless, but some serotypes can cause serious food poisoning in their hosts and are occasionally responsible for product recalls due to food contamination. The harmless strains are part of the normal flora of the gut and can benefit their hosts by producing vitamin K2 (menaquinone) and preventing colonization of the intestine with pathogenic bacteria.
Staphylococcus aureus: It is a gram-positive, coccal (round shaped) bacterium that is frequently found in the human respiratory tract and on the skin. It is positive for catalase and nitrate reduction. Although S. aureus is not always pathogenic, it is a common cause of skin infections (e.g. boils), respiratory disease (e.g. sinusitis) and food poisoning. Disease associated strains often promote infections by producing potent protein toxins and expressing cell-surface proteins that bind and inactivate antibodies. The emergence of antibiotic-resistant forms of pathogenic S. aureus (e.g. MRSA) is a worldwide problem in clinical medicine.
Candida albicans: Candida albicans is a diploid fungus that grows both as yeast and filamentous cells and a causal agent of opportunistic oral and genital infections in humans and Candida onychomycosis, an infection of the nail plate. Systemic fungal infections (fungemias) including those by C. albicans have emerged as important causes of morbidity and mortality in immunocompromised patients (e.g., AIDS, cancer chemotherapy, organ or bone marrow transplantation). C. albicans biofilms may form on the surface of implantable medical devices. In addition, hospital-acquired infections by C. albicans have become a cause of major health concerns.
Minimum inhibition concentration (MIC): It is the lowest concentration of an antimicrobial that will inhibit the visible growth of a microorganism after overnight incubation. Minimum inhibitory concentrations are important in diagnostic laboratories to confirm resistance of microorganisms to an antimicrobial agent and also to monitor the activity of new antimicrobial agents. A MIC is generally regarded as the most basic laboratory measurement of the activity of an antimicrobial agent against an organism. Zone of inhibition (ZI): If an antibiotic stops the bacteria from growing or kills the bacteria, there will be a zone around that area where the bacteria have not grown enough to be visible. This is called a zone of inhibition. The size of this zone depends on how effective the antibiotic is at stopping the growth of the bacterium.
Molecular docking study:
Molecular docking is a method which predicts the preferred orientation of one molecule to a second when bound to each other to form a stable complex. Knowledge of the preferred orientation in turn may be used to predict the strength of association or binding affinity between two molecules using scoring functions. Characterization of the binding behaviour plays an important role in rational design of drugs as well as to elucidate fundamental biochemical processes.
Benzothiazole is already reported as inhibitor of bacterial type II topoisomerase DNA gyrase (Gyr A/GyrB) and topoisomerase IV (par C/par E). Furthermore, Type II topoisomerase is found to be well defined target for drug discovery. These enzymes are homologous (structurally and functionally) and are willing to dual targeting by single ligand. This has encouraged investigation for new types of compounds with novel mechanism of action against type II topoisomerase.
Receptor protein tyrosine kinase plays an important role in signal transduction pathways that control cell division and segregation. Benzothiazole also reported inhibitor against epidermal growth factor receptor tyrosine kinase (EGFR-TK) and has been identified as important in cancer development.
S.No. Prior Patent Present invention
1. US20070088017 Al: Discloses use of The present invention does not disclose benzothiazole derivative for treatment of the derivative of prior art. Rather it diabetes. discloses a novel benzothiazole derivative with increased biological activity.
2. US20130324734 Al: Discloses 2-anilino The present invention does not dis nicotinyl linked 2-amino benzothiazole closes use of a 2-anilino nicotinyl conjugates as a potential anticancer agent linked 2-amino benzothiazole against human cell lines. conjugates rather it disclose a benzothiazole bioconjugates derivatives with biological activity.
3. US5198440 A: Discloses synergistic The present invention does not disclose combinations of 2 use of combination of derivatives (Thiocyanomethylthio) benzothiazole suggested in prior art rather it disclose and hexahydro-1, 3,5-tris(2- novel conjugates of benzothiazole hydroxyethyl)-s-triazine as an antifungal derivative.
and antibacterial agent. Rajeevna B. (2009): Discloses 2-(5- The present invention does not disclose substituted- 1 ,3,4-oxadiazole-2-yl)-l ,3- any benzothiazole derivatives of benzothiazole (3a-j) - derivatives of present invention rather it disclose benzothiazole showing anti-microbial novel bioconjugates of benzothiazole activity. derivative with increased biological activity.
(Rajeevna B. et. al., Synthesis and
Antimicrobial Activity of Some New 2- Substituted Benzothiazole Derivatives, E- Journal of Chemistry, 2009, 6(3), 775- 779)
P.Gadjose/. al. (2009): Discloses use The present invention does not disclose Eleven new 2-styrylbenzothiazole-N- the derivative of prior art rather it oxides have been prepared by aldol - type disclose a novel benzothiazole condensation reactions between 2- derivatives with increased biological methylbenzothiazole-N-oxide and para- activity.
substituted benzaldehyde different from
present invention showing biological
activity.
(P. Gadjos et. al., New Conjugated
Benzothiazole-N -oxides: Synthesis and
Biological Activity, MOLECULES
2009,14,5382-5388
;doi:W.3390/moleculesl 4125382)
Alessia Catalano et. al. (2013): The present invention does not disclose Discloses use of 6-substituted-2- the derivative of prior art rather it aminobenzothiazole derivatives as a disclose a novel benzothiazole potential antimicrobials. derivative with increased biological activity. (Alessia Catalano et. al.,2- Aminobenzothiazole derivatives: Search
for new antifungal agents, ELSEVIER,
European Journal Of Medicinal
Chemistry,64 (2013) 357-364 )
7. Yadav et.al. (2011): Discloses use of The present invention does not disclose various derivatives of benzothiazole in the various derivatives of prior art various biological activities such as rather it disclose a novel benzothiazole antimicrobial, antifungal, antidiabetic, derivative with increased biological anticancer. activity.
{Yadav et. ai, (2011), Benzothiazole:
Different Methods of Synthesis and
Diverse Biological Activities
International Journal of Pharmaceutical
Sciences and Drug Research 2011; 3(1):
01-07)
OBJECTS OF THE PRESENT INVENTION
The primary object of the present invention is to provide novel derivatives of benzothiazole with improved biological activity as antibacterial, antifungal and anticancer agents.
Another object of the present invention is to disclose novel bioconjugates of benzothiazole derivatives with cystine.
Yet another object of the present invention is to disclose the novel compounds of benzothiazole- 2-yl-imino benzoic acid derivatives and its glucosamine conjugates.
A further object is to disclose a new category of antibacterials and anti-cancerous agents which are benzothiazole derivatives with broad-spectrum antimicrobial activity. SUMMARY OF THE INVENTION
The present invention discloses novel bioconjugate of benzothiazole derivatives with cystine and glucosamine and their method of synthesis. Their method of synthesis is easy and eco-friendly, avoiding the use of hazardous materials or reactions. The compounds offer technical advantages of increased solubility, potency, selectivity and biological activity. The novel compounds are active in-vitro as antimicrobial (E. coli, S. aureus and C. albicans) and show anticancer (Hep-2 and caco cell lines) activity. Formula, physico-chemical data and biological activity of the novel derivatives are as given in Table 1 to Table 9.
DETAILED DESCRIPTION OF DRAWINGS
1. Figure 1: The general scheme of synthesis for cystine series
2. Figure 2: The general scheme of synthesis for glucosamine series
3. Figure3:2-amino-3-((2-amino-3-(4-((2-benzo[flf]thiazol-2-l)hydrazono)methyl)phenoxy)- 3-oxopropyl)disulfanyl)propanoic acid (MCYS-04)
4. Figure4:=2-amino-3-((2-amino-3-(4-((2-(4-chlorobenzo[i Jthiazol-2- l)hydrazono)methyl)phenoxy)-3-oxopropyl)disulfanyl)propionic acid (ClCYS-04)
5. Figure 5: 2-amino-3-((2-amino-3-(4-((2-7-methoxybenzo[d]thiazol-2- yl)hydrazono)methyl)phenoxy)-3-oxopropyl)disulfanyl)propionic acid (MeCYS-04)
6. Figure 6: 2-amino-3-((2-amino-3-(4-((2-(6-nitrobenzo[d]thiazol-2- yl)hydrazono)methyl)phenoxy)-3-oxopropyl)disulfanyl)propionic acid (NCYS-04)
7. Figure 7: 4-((2-(benzo[i/]thiazol-2-yl)hydrazono)methyl)benzoic acid (M-03)
8. Figure 8: 5-acetamido-3-4,6-trihydroxytetrahydro-2H-pyran-2-yl)methyl 4-((2- (benzo[i ]thiazol-2-yl)hydrazono)methyl)benzoate (M-glu-04)
9. Figure 9: 4-(((4-chlorobenzo[c?]thiazol-2-yl)imino)methyl)benzoic acid (Cl-02)
10. Figure 10: 5-acetamido-3,4,6-trihydroxytetrahydro-2H-pyran-2-yl)methyl4-(((4- chlorobenzo[(/]thiazol-2-yl)imino)methyl)benzoate (Cl-glu-03)
11. Figure 11: 4-(((4-methoxybenzo[d]thiazol-2-yl)imino)methyl)benzoic acid (Me-02)
12. Figure 12: 5-acetamido-3,4,6-trihydroxytetrahydro-2H-pyran-2-yl)methyl 4 - (((4- methoxybenzo[d]thiazol-2-yl)imino)methyl)benzoate (Me-glu-03) 13. Figure 13: 4-(((6-nitrobenzo[i/|thiazol-2-yl)imino)methyl)benzoic acid (N-02)
14. Figure 14: 5-acetamido-3,4,6-trihydroxytetrahydro-2H-pyran-2-yl)methyl4-(((6- nitrobenzo[cQthiazol-2-yl)imino)methyl)benzoate (N-glu-03)
DETAILED DESCRIPTION OF THE INVENTION
The present invention discloses novel derivatives of benzothiazole with cystine and glucosamine conjugates, having powerful antibacterial, antifungal and anticancer activities. Method of synthesis of the compounds is also disclosed. The compounds offer technical advantages of increased solubility, potency, selectivity and biological activity. The novel compounds are active against bacterial and fungal species and also cancerous cells. Their method of synthesis is easy and eco-friendly, avoiding the use of hazardous materials or reactions.
Method of synthesis of the novel derivatives
(A) Series of compounds with cystine i) Synthesis of 2-aminobenzothiazole derivatives (Cl-01, Me-01, N-01)
1. Substituted aromatic aniline (where R represents CI or OCH3 or N02 substituent on aromatic ring) (1.0 mmol), potassium thiocyanate (4.0 mmol), and Tetra-butyl ammonium tri-bromide (1.0 mmol) were taken in ethanol.
2. Subjected to microwave irradiation at power of 195 W (watt) for 5 min.
3. The resulting solid was poured in water, neutralized with 25% of ammonia solution which results precipitation.
4. The precipitate was collected by filtration and recrystallized from ethanol and water.
(ii) Synthesis of 2-hydrazino benzothiazole derivatives (M-02, ClH-02, MeH-02, NH-02)
1. A mixture of above synthesized benzothiazole derivatives i.e. M-01 or Cl-01 or Me-01 or N-01 (0.2 mol) and 80% hydrazine hydrate (0.2 mol) was added and mixed thoroughly in DMSO.
2. The mixture was subjected to microwave irradiation at 60% power for 2-3 minute.
3. Cooled to room temperature.
4. The product was purified by extensive washing with hot water and ethanol. 5. The purified product was dried and recrystallized from ethanol.
(iii) Preparation of 2-benzylidene-hydrazinobenzothiazole (MOH-03, ClOH-03, MeOH- 03, NOH-03)
1. Equimolar mixture of different derivatives of 2-hydrazinobenzothiazol i.e. M-02 or C1H- 02 or MeH-02 or NH-02 (0.02 mol) and appropriate p-hydroxybenzaldehyde (0.02 mol) in 10 mL of ethanol with 3-4 drops of glacial acetic acid was kept at room temperature for 5 min.
2. The mixture was then placed in microwave irradiation at power of 40% for 2-3 min.
3. Product was purified by washing and recrystallization with ethanol.
(iv) Synthesis of Bio-conjugates of 2-benzylidene-hydrazinobenzothiazoIe with cystine (MCYS-04, ClCYS-04, MeCYS-04, NCYS-04)
1. In a reaction vessel a mixture of above synthesized Schiff-bases of different derivatives i.e.
MOH-03 or ClOH-03 or MeOH-03 or NOH-03 (5.0 mmol) and corresponding amino acids cystine (2.0 mmol) with 1-2 drops of H2S0 (as catalyst) were taken.
2. The mixture was irradiated at power of 40% for 10 min under simultaneous cooling with airs.
3. Dilute the final product with hot ethanol (5 mL).
4. The mixture was allowed to stand overnight at 5 °C.
5. The precipitate was filtered and washed with dilute acid.
The general scheme of synthesis for cystine series is shown in Figure 1 and that for glucosamine series is shown in Figure 2.
All Compounds for cystine series showing good activity are:
(i) benzo[i/]thiazole-2-thiol (M-01)
(ii) 2-hydrazinylbenzo[d]thiazole (MH-02)
(iii) 4-((2-(benzo[^thiazol-2-yl)hydrazono)methyI)phenol (MOH-03)
(iv) 2-amino-3-((2-amino-3-(4-((2-benzo[ifJthiazol-2-yl)hydrazono)methyl)phenoxy)-3- oxopropyl)disulfanyl)propanoic acid (MCYS-04) (Figure 3)
(v) 4-cholorobenzol[i/]thiazol-2-amine (Cl-01)
(vi) 4-choloro-2-hydrazinylbenzo[i ]thiazole (ClH-02) (vii) 4-((2-(4-chlorobenzo[i ]thiazol-2-yl)hydrazono)methyl)phenol (ClOH-03)
(viii) 2-amino-3-((2-amino-3-(4-((2-(4-chlorobenzo[i/|thiazol-2-l)hydrazono)methyl)phenoxy)- 3-oxopropyl)disulfanyl)propionic acid (ClCYS-04) (Figure 4)
(ix) 4-methoxybenzo[i/]thiazol-2-amine (Me-01)
(x) 2-hydrazinyl-4-methoxybenzo[i/]thiazole (MeH-02)
(xi) 4-((2-4-methoxybenzo[i/|thiazol-2-yl)hydrazono)niethyl)phenol (MeOH-03)
(xii) 2-amino-3-((2-amino-3-(4-((2-7-methoxybenzo[i/]thiazol-2- yl)hydrazono)methyl)phenoxy)-3-oxopropyl)disulfanyl)propionic acid (MeCYS-04) (Figure 5)
(xiii) 6-nitrobenzo[i jthiazol-2-amine (N-01)
(xiv) 2-hydrazinyl-6-nitrobenzo[i/]thiazole (NH-02)
(xv) 4-((2-(6-nitrobenzo[i ]thiazol-2-yl)hydrazono)methyl)phenol (NOH-03)
(xvi) 2-amino-3-((2-amino-3-(4-((2-(6-nitrobenzo[i/]thiazol-2-yl)hydrazono)methyl)phenoxy)- 3-oxopropyl)disulfanyl)propionic acid (NCYS-04) (Figure 6)
Table- 1: Physico-chemical data for compounds (Cystine series)
Figure imgf000012_0001
(B) Series of compounds with N-acetyl-D-glucosamine
(i) Synthesis of 2-aminobenzothiazole derivatives (Cl-01, Me-01, N-01) 1. Substituted aromatic aniline (where R represents CI or OCH3 or N02 substituent on aromatic ring) (1.0 mmol), potassium thiocyanate (4.0 mmol), and Tetra-butyl ammonium tri-bromide (1.0 mmol) were taken in ethanol.
2. Subjected to microwave irradiation at power of 195 W for 5 min.
3. The resulting solid was poured in water, which results precipitation.
4. The precipitate was collected by filtration and recrystallized from ethanol and water.
(ii) Synthesis of benzothiazole methyl benzoic acid (Schiff-bases) (Cl-02, M-03, Me-02, N-02)
1. Equimolar mixture of different derivatives of benzothiazole (0.02 mol) and appropriate p- carboxybenzaldehyde (0.02 mol) in 5-6ml of DMF (Dimethylformamide) with 1 drop of glacial acetic acid.
2. Kept at room temperature for 5min.
3. The mixture was then placed in microwave irradiation at power of 40% for 4-5 minute.
4. Product was poured into distilled ice and purified by washing with hot water and ethanol.
(iii) Synthesis of Bio-conjugates of benzothiazole methyl benzoic acid (Schiff-bases) with N- acetyl-D-glucosamine (Cl-gIu-03, M-glu-04, Me-glu-03, N-glu-03)
1. In a reaction vessel a mixture of above synthesized Schiff-bases of different derivatives (0.02 mmol) and N-acetyl-D-glucosamine (0.04mmol) in DMF with 1-2 drops of H2S04 (as catalyst) were taken.
2. The mixture was irradiated in microwave at power of 40% for 10 min under simultaneous cooling with airs.
3. Upon reaction completion, the product was poured into distilled ice, filtered and washed with warm water.
The general scheme for the synthesis is shown in Figure 2.
Name of compounds from (N-acetyl-D-glucosamine series)
(i) benzo[i/]thiazole-2-thiol (M-01) (ii) 2-hydrazinylbenzo[<flthiazole (MH-02)
(iii) 4-((2-(benzo[i/)thiazol-2-yl)hydrazono)methyl)benzoic acid (M-03) (Figure 7)
(iv) 5-acetamido-3-4,6-trihydroxytetrahydro-2H-pyran-2-yl)methyl 4-((2-(benzo[i ]thiazol-2- yl)hydrazono)methyl)benzoate (M-glu-04) (Figure 8)
(v) 4-chlorobenzo[cT]thiazol-2-amine (Cl-01)
(vi) 4-(((4-chlorobenzo[i ]thiazol-2-yl)imino)methyl)benzoic acid (Cl-02) (Figure 9)
(vii) 5-acetamido-3,4,6-trihydroxytetrahydro-2H-pyran-2-yl)methyl4-(((4- chlorobenzo[d]thiazol-2-yl)imino)methyl)benzoate (Cl-glu-03) (Figure 10)
(viii) 4-methoxybenzo[i ]thiazol-2-amine (Me-01)
(ix) 4-(((4-methoxybenzo[i¾thiazol-2-yl)imino)niethyl)benzoic acid (Me-02) (Figure 11)
(x) 5-acetamido-3,4,6-trihydroxytetrahydro-2H-pyran-2-yl)methyl 4 - (((4- methoxybenzo[i/]thiazol-2-yl)imino)methyl)benzoate (Me-glu-03) (Figure 12)
(xi) 6-nitrobenzo[</]thiazol-2 -amine (N-01)
(xii) 4-(((6-nitrobenzo[i/]thiazol-2-yl)imino)methyl)benzoic acid (N-02) (Figure 13)
(xiii) 5-acetamido-3,4,6-trihydroxytetrahydro-2H-pyran-2-yl)methyl4-(((6- nitrobenzo[i/]thiazol-2-yl)imino)methyl)benzoate (N-glu-03) (Figure 14)
Table- 2: Physico-chemical data for compounds ( -acetyl-D-glucosamine series)
Figure imgf000014_0001
(C) In vitro antibacterial and antifungal screening with Cystine derivatives family Inhibition-Zone (IZ) measurements 1. All the synthesized compounds were evaluated by the agar-well diffusion technique using different concentration i.e. 100, 140 and 200 μg/mL in DMSO.
2. The test organisms: S. aureus, E. coli, C. albicans (from IMTECH Chandigarh).
3. This was grown in Nutrient broth (NB -Liquid culture) at 37 °C for 24 h.
4. After overnight growth of culture, 1.0 mL of culture was mixed with 0.7% of soft agar medium.
5. This broth suspension of the test bacterium was flooded on the surface of a solid medium (Muller Hinton agar) and potato dextrose agar for fungus.
6. After solidification of the medium wells were bored using the wide end of sterile 1.0 mL tip.
7. Extracts were added in the wells along with the control (solvent: DMSO).
8. The Petri plates were allowed to stand for 1-2 h to allow proper diffusion of the extracts into the medium and then incubated at 37 °C for 24 h.
The results were recorded for each tested compounds as the average of three measurement of diameter of inhibition zones (IZ) of fungal and bacterial growth around the wells in millimeters (mm).
MIC (Minimum Inhibitory Concentration) by micro dilution method
1. MIC was determined by broth micro dilution method i.e. CLSI M7-A7 (The Clinical and Laboratory Standards Institute) Culture (bacteria and fungus) was added and plate was incubated for 24 h.
2. Resazurin was used as indicator and added after 24 hour incubation and kept for 2-4 hour.
Lowest concentration that prevented color change was taken as the minimum inhibitory concentration.
3. The assay was done using 96 well plates in Mueller Hinton broth.
4. Minimum effective antibacterial concentration (100 μg/mL) was chosen.
5. Dilution series of compounds were added to the wells.
All synthesized compounds were tested and found to be active. The results are presented as antimicrobial effect in terms of "maximum inhibition zone (IZ)" as below: • 25 to 15 at cone. 200 μg/mL against S. aureus
• 22 to 17 at cone. 200 μg/mL against E. coli.
• It also showed antifungal effect against C. albicans with inhibition zone of 29 to 16 at cone.
200 μg/mL.
MIC Range
• MIC value ranges from 6.25 to 25 μg/mL against S. aureus and E. coli
• Fungus C. albicans MIC value ranges from 12.5 to 3.125 μg/mL
Results are given in Table 3.
Table 3 Minimum inhibition concentration (MIC, ug/mL) and inhibition zone (IZ, mm) results for novel cystine derivatives.
Figure imgf000016_0001
Colony Forming Unit (CFU Method)
The synthesized compounds were screened against E. coli and S. aureus for anti-microbial activity which were grown in nutrient broth (NB) medium overnight in incubator shaker at 37 °C. Nutrient broth solution was prepared by dissolving 1.3 g of solid NB in 100 mL of distilled water in a 250 mL flask. The flask containing the NB, and all apparatus were autoclaved before use.
Then three test tubes were taken for each sample. In one test tube 5.0 mL NB and 1.0 mL of bacterial culture were added. This test tube was considered as a control in which the normal bacterial growth took place. In the others test tube 5 mL of NB, 1.0 mL bacterial culture and sample solution (200 and 500 μg/mL) were added. Same concentration of sample with 10 mL of NB was also required as reference without the addition of culture. These tubes were considered as reference of the samples.
10 samples from each test tube were taken and that was diluted 2, 3 or 4 times according to the optical density values of sample and culture. Dilution was done in eppendorf tube with autoclaved water/mL. All these test tube were kept in the incubator shaker at 37 °C.
Colonies were counted at 0, 24 and 48 h by seeding the aliquot of incubated sample on nutrient agar plates. To determine the CFU mL"1, the number of microorganisms growing in the plate was multiplied by dilution factor and divided by the volume used to seed the plate. To generalize the results for control and tested compounds we defined parameter for dimensionless bacterial concentration (CFU*) using equations CFU* = CFUT/ CFUc, where CFUT is the bacterial count in test set and CFUC is the bacterial count in control set respectively.
Table 4 CFU results of cell count at initial and after incubation with cystine series at different time against E. coli and S. aureus
S.NO Compounds CFU at 0 h CFU at 24 h CFU at 48 h
5. aureus E. coli S. aureus E. coli S. aureus E. coli
(1.0) (1.0) (l x lO2) (l x lO2) (1 * 104) (l x lO4)
1 M-cyst-04L 1.0 1.0 0.36 0.78 0.19 0.18
2 M-cyst-04H 0.86 0.77 0.31 0.56 0.17 0.16
3 Cl-cyst-04L 0.86 0.72 0.28 0.72 0.002 0.03
4 Cl-cyst-04H 0.72 0.88 0.20 0.64 0.00 0.00 5 N-cyst-04L 0.90 0.77 0.36 0.69 0.059 0.00
6 N-cyst-04H 0.78 0.941 0.26 0.50 0.049 0.050
7 e-cyst-04L 1 0.88 0.30 0.70 0.067 0.071
8 Me-cyst-04H 0.72 0.83 0.27 0.89 0.058 0.056
L = low (200 g/mL) and H = high (500 ug/mL)
(D)In vitro antibacterial screening using N-acetyl-D-glucosamine derivatives
All synthesized compounds were tested and found to be active. It showed antimicrobial effect in terms of "maximum inhibition zone (IZ)" as below:
• 25 to 17 at cone. 200 μ{*/πιΙ_- against S. aureus
• 24 to 17 at cone. 200 μg/mL against E. coli.
• It also showed antifungal effect against C. albicans with inhibition zone of 34 to 14 at cone.
200 μg/mL.
MIC Range
• MIC value ranges from 6.25 to 25 ]i lvs\L against S. aureus and E. coli
• Fungus C. albican MIC value ranges from 12.5 to 1.25 μg/mL
Results are given in Table 5.
Table 5 Minimum inhibition concentration (MIC, μg ml) and inhibition zone (IZ, mm) of the newly synthesized compounds.
Figure imgf000018_0001
10 Me-glu- 13 14 19 6.26 14 15 21 6.25 21 22 25 1.25 03
1 1 N-01 15 19 24 12.5 18 20 22 6.25 19 20 21 3.125
12 N-02 14 16 23 25 13 15 19 12.5 19 15 14 6.25
13 N-glu- 13 15 19 6.25 14 17 22 6.25 21 23 28 1.25 03
CFU results for glucosamine series in Table 6
Table 6 CFU results of cell count at initial and after incubation with Glucosamine series at different time against E. coli and S. aureus
Figure imgf000019_0001
L = low (200 μ&/ιηΙ,) and H = high (500 μg/mL)
Anticancer activity
1. Growth of cervix carcinoma cell line (Hep 2) and caco cell line was quantitated by the ability of metabolic active cell to trim down the yellow MTT [3-(4,5-Dimethylthiazol-2- yl)-2,5-Diphenyltetrazolium Bromide] to purple formazone product.
2. Compounds were prepared as 1.0 mg/mL stock solution dissolved in DMSO.
3. Hep 2 and caco cell line were cultivated at 37 °C in an atmosphere of 5% C02 in Dubecco's modified eagle's minimal medium.
4. Cells were seeded at a density of 1 *104 cells per well in 96-well tissue culture plates and grown for 40 h before adding the tested compounds.
5. Various concentrations between 1 to 25 μg/mL were added to the wells.
6. Plates enclosed well for control (healthy cell), treated cell and for DMSO.
7. Each plate has replicates for different concentrations, control and DMSO. 8. Cells were incubated for 24 h. After incubation time, 10 μΙ_, of the MTT reagent were added to each well and cells were incubated further for 4 h to allow cleavage of the MTT reagent by viable cell mitochondrial dehydrogenase.
9. Afterwards 100 μΐ, of DMSO were added to each well to solubilize formazone product for colorimetric quantification in an ELISA micro plate reader at 570 nm.
10. The %cell viability was calculated by using following formula.
%cell viability = [abs57o of treated cell/absj7o of control cell] x lOO
Table 7 Percentage of Cell viability with Hep-2 and Caco cell line against different concentration of Glucosamine and cystine bio-conjugates of benzothiazole
Figure imgf000020_0001
Mechanism Characterization via Molecular Docking
The pharmacological antimicrobial mechanism was evaluated via docking study.
1. The X-ray crystal structures of bacterial DNA gyrase B (PDB code-3TTZ, S. aureus) and cell line Tyrosine kinase (PDB-1M17) were used for docking analysis.
2. All synthesized compounds were docked into the binding sites of receptor using Auto Dock 4.2.
Virtual screening results of all compounds are expressed in terms of estimated free energy of binding in Table 8. Table 8 Free estimated binding energy of Cystine with DNA gyrase and Tyrosine kinase.
Figure imgf000021_0002
Docking results prove that lower the binding energy more will be the interaction with targets. Estimated energy ranges from -10.69 to -4.89 kcal/mol (DNA gyrase) and -1 1.52 to -5.39 kcal/mol (Tyrosine kinase) with the new synthesized cystine series. Moreover, the conjugates of cystine have more competence as lower binding energy compare to benzothiazole.
Table 9 Free estimated binding energy of Glucosamine with DNA gyrase and Tyrosine Kinase
Figure imgf000021_0001
From docking results it is proved that lower the binding energy more will be the interaction with targets. Estimated energy ranges from -9.59 to-4.89 kcal/mol (DNA gyrase) and -9.61 to -5.39 (Tyrosine kinase) with the new synthesized Glucosamine series. Moreover, the conjugates of glucosamine have more competence as lower binding energy compare to benzothiazole
Novelty, Inventive Step and Industrial Application
Novelty: The present invention discloses novel benzothiazole derivative with glucosamine and cystine, having potent antibacterial, antifungal and anticancer properties. Derivatives having good antibacterial activity are depicted in Fig. 3 to Fig. 14.
Inventive Step: The inventive step of the invention lies in developing novel benzothiazole bio- conjugates using cystine and N-acetyl-D-glucosamine derivatives which have antibacterial, antifungal and anti-cancer activity.
Industrial Application: The novel derivatives of present invention are easy to synthesize and can be manufactured at industrial level. They have considerable economic value as antibacterial, antifungal and anti-cancer agents.

Claims

! I CLAIM:
1. Novel conjugates of benzothiazole derivatives with enhanced biological activity wherein the derivatives are synth siz d by inserting cystine,
2. Novel conjugates of ben othia oie derivatives with enhanced biological activity wherein the derivatives are synthesized by inserting glucosamine.
3. The novel derivatives as claimed m claim 1 and claim 2 wherein the biological activity exhibited by the novel derivatives is antibacterial, antifungal and anticancer,
4. The novel derivatives of cystine as claimed in claim I wherein the derivatives are having physico-chemical properties as given in Table 1.
5. The novel derivatives of glucosamine as claimed in claim 2 wherein the derivatives are having physico-chemical properties as given in Table 2.
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