Medicinal Chemistry Organic Analytical Biological Aspect New Drugs Biology Essay

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The primary objective of medicinal chemistry is the design and discovery of new compounds that are suitable for use as drugs. This process involves a team of workers from a wide range of disciplines such as chemistry, biology, biochemistry, pharmacology, mathematics, medicine and computing, amongst others.

The discovery or design of a new drug not only requires a discovery or design process but also the synthesis of the drug, a method of administration, the development of tests and procedures to establish how it operates in the body and a safety assessment. Drug discovery may also require fundamental research into the biological and chemical nature of the diseased state. These and other aspects of drug design and discovery require input from specialists in many other fields and so medicinal chemists need to have an outline knowledge of the relevant aspects of these fields.

Medicinal chemistry is concerned with the organic, analytical and biological aspect of new drugs. The practice of it is devoted to the discovery and development of new drugs. The drug design is an integrated developing discipline which pretends an era of "tailored drug" involves the biological study of compound on the basis of molecular interaction, in terms of molecular structure or its physiochemical properties. The current trend in the drug design is to develop new clinically effective agents through the structural modification of a lead moiety.

Medicinal chemistry is a discipline firmly rooted in synthetic organic chemistry has very close links to structural chemistry, computational chemistry, and molecular biology at the discovery interface, to structural biology, toxicology and pharmacology at the development interface and to medicine at clinical interface. Thus, medicinal chemistry has occupied the central position and will continue to play a crucial role in the new drug discovery process.

The lead moiety is a new active compound which is typically found by screening many compounds for the desired biological properties. These lead can come from natural sources, such as plants, animals, or fungi. More often, it can come from synthetic sources such as historical compound collections and combinatorial chemistry.

Once a lead compound has been discovered for a particular therapeutic use the next step is to optimize the lead compound. An important method of lead optimization is optimization by bioisostearism. Bioisostears are substitute group that have similar physical and chemical properties and hence similar biological activity pattern. Bioisostearic replacement may help to decrease toxicity or to change the pharmacokinetic profile.

The final step involves the rendering the lead compounds suitable for use in clinical trials. This involves the optimization of the synthetic route for bulk production, and the preparation of a suitable drug formulation.

Basic Nucleus :

Benzodiazepine:-

A benzodiazepine ("BZD") is a psychoactive drug whose core chemical structure is the fusion of a benzene ring and a diazepine ring.

Diazepine

The first benzodiazepine, Chlordiazepoxide was discovered accidently by Leo Sternbach in 1955, and made available in 1960 by Hoffamann-La Roche, which has also marketed Diazepam since 1963.

In general, benzodiazepines are safe and effective in the short term, although cognitive impairments and paradoximal effects such as aggression or behavioral disinhibition occasionally occur. Long-term use is controversial due to concerns about adverse psychological and physical effects, increased questioning of effectiveness and because benzodiazepines are prone to cause tolerance, physical dependence and upon cessation of use, a withdrawal syndrome.

1.2 Chemistry :-

Left: The 1,4-benzodiazepine ring system. Right: 5-phenyl-1H-benzo[e][1,4]diazepin-2(3H)-one forms the skeleton of many of the most common benzodiazepine pharmaceuticals, such as Diazepam (7-chloro-1-methyl substituted).

A pharmacophore model of the benzodiazepine binding site on the GABAA receptor.White sticks represent the carbon atoms of the benzodi-azepine Diazepam while green represents carbon atoms of the nonbenzodiazepine CGS9896. Red and blue sticks are oxygen and nitrogen atoms that are present in both structures. The red spheres labeled H1 and H2/A3 are respectively hydrogen bond donating and accepting sites in the receptor while L1, L2, and L3 denote lipophilic binding sites.

Benzodiazepine drugs are substituted 1,4-benzodiazepines. Different benzodiazepine drugs have different side groups attached to this central structure. The different side groups affect the binding of the molecule to the GABAA receptor and so modulate the pharmacological properties.

1.3 Nomenclature of Benzodiazepine : -

The 1,2-, 1,3- 1,4- and 1,5-benzdiazepines are the parent compounds. The 1,2-and 1,4-diazepines are of great importance, especially the 1,4-benzodiazepine derivatives 2,3-dihydro-1,4-benzodiazepine,1,5-benzodiazepine and 1,4 benzodiazepine.

1.4 General Synthesis :-

1,5-Benzodiazepine obtained by condensation of 1,2-diaminoarenes with 1,3-diketones.

1.5 Some important examples 1, 5 Benzodiazepine derivatives :-

Sr No.

Derivatives

Use

1.

Clozapine

Aniconvulsant Drug ,

5 HT2 Antagonist used in treatment o f Shrizophenia

.

2.

Olazepine

Aniconvulsant Drug ,

5 HT2 Antagonist used in treatment of Shrizophenia

3.

Nevirapine

Antiviral Drug

4.

Pirenzepine

Antimuscarinic Drug

1.6 Mechanism of action

Benzodiazepines work by increasing the efficiency of a natural brain chemical, GABA, to decrease the excitability of neurons. This reduces the communication between neurons and therefore has a calming effect on many of the functions of the brain.

Schematic diagram of the (α1)2(β2)2(γ2) GABAA receptor complex that depicts the five-protein subunits that form the receptor, the chloride (Cl-) ion channel pore at the center, the two GABA active binding sites at the α1 and β2 interfaces and the benzodiazepine (BZD) allosteric binding site at the α1 and γ2 interface.

GABA controls the excitability of neurons by binding to the GABA-A receptor . The GABAA receptor is a Protein complex located in the synapses of neurons. All GABAA receptors contain an ion channel that conducts chloride ions across neuronal cell membrane sand two binding sites for the neurotrasmitter gamma-aminobutyric acid (GABA), while a subset of GABAA receptor complexes also contain a single binding site for benzodiazepines. Binding of benzodiazepines to this receptor complex promotes binding of GABA, which in turn increases the conduction of chloride ions across the neuronal cell membrane. This increased conductance raises the membrane potential of the neuron resulting in inhibition of neuronal firing. In addition, different GABAA receptor subtypes have varying distributions within different regions of the brain and therefore control distinct neuronal circuits. Hence, activation of different GABAA receptor subtypes by benzodiazepines may result in distinct pharmacological actions. In terms of the mechanism of action of benzodiazepines, their similarities are too great to separate them into individual categories such as anxiolytic or hypnotic. For example, a hypnotic administered in low doses will produce anxiety relieving effects, whereas a benzodiazepine marketed as an anti-anxiety drug will at higher doses induce sleep.

Once bound to the benzodiazepine receptor, the benzodiazepine ligand locks the benzodiazepine receptor into a conformation in which it has a greater affinity for the GABA neurotrasmitter. This increases the frequency of the opening of the associated chloride ion channel and hyperpolarizes the membrane of the associated neuron. The inhibitory effect of the available GABA is potentiated, leading to sedatory and anxiolytic effects. Furthermore, different benzodiazepines can have different affinities for BzRs made up of different collection of subunits. For instance, those with high activity at the α1 are associated with stronger hypnotic effects, whereas those with higher affinity for GABAA receptors containing α2 and/or α3 subunits have good anti-anxiety activity.

The benzodiazepine class of drugs also interact with peripheral benzodiazepine receptors. Peripheral benzodiazepine receptors are present in peripheral nervous system tissues, glial cells and to a lesser extent the central nervous system. These peripheral receptors are not structurally related nor coupled to GABAA receptors. They modulate the immune system and are involved in the body response to injury. Benzodiazepines also function as weak adenosine reuptake inhibitors. It has been suggested that some of their anticonvulsant, anxiolytic and muscle relaxant effects may be in part mediated by this action.

1.7 Urea :-

Urea is an organic compound with the chemical formula (NH2)2CO. The molecule has two amine (-NH2) groups joined by a carbonyl (C=O) functional group.

Urea serves an important role in the metabolism of nitrogen-containing compounds by animals and is the main nitrogen-containing substance in the urine of mammals. It is solid, colourless, and odourless. It is highly soluble in water and non-toxic. Dissolved in water it is neither acidic nor alkaline. The body uses it in many processes, most notably nitrogen excretion.

The terms urea used for a class of chemical compounds sharing the same functional group RR'N-CO-NRR', namely a carbonyl group attached to two organic amine residues. Examples include carbamide peroxide, allantoin, and hydantoin. Ureas are closely related to biurets and related in structure to amides, carbamates, carbodiimides, and thiocarbamides.

1.8 Some important Urea derivatives :-

Sr No.

Derivatives

Use

1.

Phenaceamide

Anticonvulsant

Agent

2.

Carbamazepine

Anticonvulsant

Agent

3.

Lomustine

Antineoplastic

Agent

4.

Cholrpropamide

Hypoglycemic

Agent

1.9 Convulsion ( Epilepsy ):-

Epilepsy is a very common disorder, characterised by seizures, which take various forms and result from episodic neuronal discharges, the form of the seizure depending on the part of the brain affected. Epilepsy affects 0.5-1% of the population. Often, there is no recognisable cause, although it may develop after brain damage, such as trauma, infection or tumour growth, or other kinds of neurological disease, including various inherited neurological syndromes.

THE NATURE OF EPILEPSY

The characteristic event in epilepsy is the seizure, which is associated with the episodic high-frequency discharge of impulses by a group of neurons in the brain. What starts as a local abnormal discharge may then spread to other areas of the brain. The site of the primary discharge and the extent of its spread determine the symptoms that are produced, which range from a brief lapse of attention to a full convulsive fit lasting for several minutes, as well as odd sensations or behaviours. The particular symptoms produced depend on the function of the region of the brain that is affected. Thus involvement of the motor cortex causes convulsions; involvement of the hypothalamus causes peripheral autonomic discharge, and involvement of the reticular formation in the upper brain stem leads to loss of consciousness.

TYPES OF EPILEPSY

The clinical classification of epilepsy defines two major categories, namely Partial and Generalised seizures, although there is some overlap and many varieties of each.

PARTIAL SEIZURES

Partial seizures are those in which the discharge begins locally and often remains localised. The symptoms depend on the brain region or regions involved, and include involuntary muscle contractions, abnormal sensory experiences or autonomic discharge, or effects on mood and behaviour, often termed psychomotor epilepsy.

GENERALISED SEIZURES

Generalised seizures involve the whole brain, including the reticular system, thus producing abnormal electrical activity throughout both hemispheres. Immediate loss of consciousness is characteristic of generalised seizures. Two important categories are Tonic-clonic seizures and Absence seizures. A tonic-clonic seizure consists of an initial strong contraction of the whole musculature, causing a rigid extensor spasm and an involuntary cry. Respiration stops, and defecation, micturition and salivation often occur. Absence seizures occur in children; they are much less dramatic but may occur more frequently (many seizures each day) than tonic-clonic seizures. The patient abruptly ceases whatever he or she was doing, sometimes stopping speaking in mid-sentence, and stares vacantly for a few seconds, with little or no motor disturbance.

MECHANISM OF ACTION OF ANTIEPILEPTIC DRUGS

Three main mechanisms appear to be important in the action of antiepileptic drugs:

Enhancement of GABA action

Inhibition of sodium channel function

Inhibition of calcium channel function.

Other mechanisms include inhibition of glutamate release and block of glutamate receptors. Many of the current antiepileptic drugs were developed empirically on the basis of activity in animal models. Their mechanism of action at the cellular level is not fully understood. As with drugs used to treat cardiac dysrhythmias the aim is to prevent the paroxysmal discharge without affecting normal transmission. It is clear that properties such as use-dependence and voltage-dependence of channel-blocking drugs are important in achieving this selectivity, but our understanding remains fragmentary.

Enhancement of GABA action

Several antiepileptic drugs (e.g. Phenobarbital

and Benzodiazepines) enhance the activation of GABAA receptors, thus facilitating the GABA-mediated opening of chloride channels. Vigabatrin (see below) acts by inhibiting the enzyme GABA transaminase, which is responsible for inactivating GABA, and Tiagabine inhibits GABA uptake; both thereby enhance the action of GABA as an inhibitory transmitter Gabapentin.

Inhibition of sodium channel function

Several of the most important antiepileptic drugs (e.g. Phenytoin, carbamazepine

,valproate, Lamotrigine

) affect membrane excitability by an action on voltage-dependent sodium channels , which carry the inward membrane current necessary for the generation of an action potential. Their blocking action shows the property of use-dependence; in other words, they block preferentially the excitation of cells that are firing repetitively, and the higher the frequency of firing, the greater the block produced. This characteristic, which is relevant to the ability of drugs to block the high-frequency discharge that occurs in an epileptic fit without unduly interfering with the low-frequency firing of neurons in the normal state, arises from the ability of blocking drugs to discriminate between sodium channels in their resting, open and inactivated states. Depolarisation of a neuron (such as occurs in the PDS described above) increases the proportion of the sodium channels in the inactivated state. Antiepileptic drugs bind preferentially to channels in this state, preventing them from returning to the resting state, and thus reducing the number of functional channels available to generate action potentials.

Inhibition of calcium channels

Several antiepileptic drugs have minor effects on calcium channels , but only Ethosuximide

specifically blocks the T-type calcium channel, activation of which is believed to play a role in the rhythmic discharge associated with absence seizures. Gabapentin acts on L-type calcium channels by binding to the α2δ subunit , but whether this is important for its antiepileptic properties is uncertain.

BENZODIAZEPINES

Diazepam , given intravenously or rectally, is used to treat status epilepticus, a life-threatening condition in which epileptic seizures occur almost without a break. Its advantage in this situation is that it acts very rapidly compared with other antiepileptic drugs. With most benzodiazepines , the sedative effect is too pronounced for them to be used for maintenance therapy. Clonazepam

and the related compound Clobazam are claimed to be relatively selective as antiepileptic drugs. Sedation is the main side effect of these compounds, and an added problem may be the withdrawal syndrome, which results in an exacerbation of seizures if the drug is stopped abruptly.

1.10 ANTICANCER ACTIVITY:

The medical term for "cancer" or "tumor" is neoplasm, which means a relatively autonomous growth of tissue. Tumor is a general term indicating any abnormal mass or growth of tissue.

The difference between benign and malignant neoplasm is that benign tumors do not metastasize, whereas malignant tumors do metastasize. A metastasis is a secondary growth originating from the primary tumor.

Types of Cancer

Cancers are divided into two general categories according to their embryological origin. In the early embryo of multicellular organism before organs begin to form cells arrange themselves into three layers-ectoderm, mesoderm and endoderm. Mesodermal cells form bone, muscle, cartilage and related tissues.

Sarcoma

A cancer that arises from mesodermal tissue is called a sarcoma.

Carcinoma

A cancer that arises from ectodermal and endodermal cells is called a carcinoma.

Carcinosarcoma

A highly malignant tumor with the appearance of both a carcinoma and a sarcoma is termed a carcinosarcoma.

Teratoma

A cancer derived from all three embryonic layers is called teratoma

Blastoma

Blastoma is used to indicate certain types of tumors that have a primitive appearance resembling embryonic structure.

2.LITRATURE REVIEW :-

1.A.R.Saundane et al . Synthesis and antimicrobial activity of some Indole derivatives containing Pyrazoline and Benzodiazepines system. The synthesized compounds were screened for their antimicrobial activity against Bacillus substilis, Staphylococcus and Escheria coli.

2. Bhuwan C.Joshi et al ., Synthesis of Pyrido (2,3-b) (1,4) and (2,3-B) (1,5)-Benzodiazepine and their derivatives. The synthesized compounds were characterized by elemental and spectral (IR, 1H NMR, and Mass) analysis.

3. Vijay Kumar Tirlapur et al ., Synthesis and antimicrobial activity of some new Benzodiazepine and Benzothiazepines. The synthesized compounds were screened for their antimicrobial activity.

4. Sanjay Kumar et al ., An efficient synthesis of 1,5-Benzodiazepine catalyzed by GaCl3 under solvent free condition. The synthesized compounds were screened for their pharmacological activity like antianxiety, anticonvulsant, sedative, hypnotics and antidepressive property.

5. Urika Rosenstrom et al ., Design and synthesis of new benzodiazepine-based turn mimetic incorporated in Ang 11. The synthesized compounds were screened for their antimicrobial activity.

6.B.Basavaraju et al ., Synthesis, characterization and antimicrobial studies of Transition metal complexes of methyl-quinolino(3,2-b)(1,5)-Benzodiazepine derivatives .The synthesized compounds were screened for their antimicrobial activity.

7. Ajoy K.Banerjee et al ., Synthesis and characterization of Iodine in organic synthesis of 1,5-Benzodiazepines derivatives. The synthesized compounds were characterized by elemental and spectral (IR, 1H NMR, and Mass) analysis.

8. M.S.Balakrishna et al ., A simple and new method for the synthesis of 1,5-Benzodiazepine derivatives on a solid surface. The synthesized compounds were characterized by elemental and spectral (IR, 1H NMR, and Mass) analysis.

9. Shivaji S.Pandit et al ., Synthesis and characterization of (1,5)-benzodiazepine derivatives from cyclocondensation of O-phenylene diamine by using LaCl3 7 H2O as a catalysed. . The synthesized compounds were characterized by elemental and spectral (IR, 1H NMR, and Mass) analysis.

10. Raviraj A.Kusanur et al ., Synthesis of spiro (Indolo-1,5-Benzodiazepines) from 3-acetyl coumarins for use as possible antianxiety agent. The synthesized compounds were screened for their antimicrobial and antianxiety activity in mice. The synthesized compounds were characterized by elemental and spectral (IR, 1H NMR, and Mass) analysis.

11. M.A. Pasha et al ., An Expeditious synthesis of 1,5-Benzodiazepine derivatives catalyzed by P-toluenesulfonic acid. The synthesized compounds were characterized by elemental and spectral (IR, 1H NMR, and Mass) analysis.

12. Shaabari et al ., A fast and efficient method for the synthesis of 1,5-Benzodiazepine derivatives under solvent free condition. The synthesized compounds were characterized by elemental and spectral (IR, 1H NMR, and Mass) analysis.

13. Jyoti R. Kavali et al ., 1,5-Benzodiazepine derivatives of 3-arylsydnones: Synthesis and antimicrobial activity of 3-aryl-4-[2,-aryl-2,,4,,6,,7,- tetrahydro-(1, H)-1,,5,-benzodiazepine-4,-yl] sydnones.

14. Mario Di Braccio et al .,1,5-Benzodiazepines. Part XII. Synthesis and biological evaluation of tricyclic and tetracyclic 1,5-benzodiazepine derivatives as nevirapine analogues.

15. Xin Zhou et al ., An efficient synthesis of 1,5-benzodiazepine derivatives catalyzed by boric acid.

16. D. Shobha et al ., Room temperature synthesis of 1,5-benzodiazepine and its derivatives using cage type mesoporous alumino-silicate catalysts

17. Harjyoti Thakuria et al ., A one-pot synthesis and self-assembled superstructure of organic salts of a 1,5-benzodiazepine derivative.

18. Minothora Pozarentzi et al ., An efficient method for the synthesis of 1,5-benzodiazepine derivatives under microwave irradiation without solvent.

19.Arun Singh et al., Studies on new ion-exchange resin for effluent treatment of metal industries.

20.K. Sucheta et al ., Microwave induced solvent-free synthesis of substituted 1,5- Benzodiazepine derivatives.

21. Sébastien Fortin et al ., Synthesis, antiproliferative activity evaluation and structure reactivity relationships of novel aromatic urea and amide analogues of N-phenyl-N0-(2-chloroethyl)ureas.

22. Koneni V. Sashidhara et al ., Rare dipeptide and urea derivatives from roots of Moringa oleifera as potential anti-inflammatory and antinociceptive agents .

23. Jung-Min Hwang et al ., A phenolic acid phenethyl urea compound inhibits lipopolysaccharide-induced production of nitric oxide and pro-inflammatory cytokines in cell culture.

24. Bedia Koc¸yi˘git Kaymakcet al ., Synthesis and biological evaluation of new N-substituted-N_-(3,5- di/1,3,5-trimethylpyrazole-4-yl)thiourea/urea derivatives.

3. AIM AND OBJECTIVES

Molecular modification of a promising lead compound is still a major line of approach for the discovery of new drug. Molecular modification involves substituting, elimination, or adding new moieties to a parent lead compound, there by making gradual changes in the physico-chemical properties of the parent compound and thus biological activity of the compound.

It is clear from the literature review that a number of benzodiazepine derivatives are known for the antibacterial, antifungal, anticancer, anticonvulsant, sedatives, hypnotic's activities and analgesic properties.

The present studies were performed with the following objectives:

Synthesis of new series 1,5- benzodiazepine derivatives with urea which also

anticonvulsant so total drug Anticonvulsant activity is drastically increased

Characterization of newly synthesized compounds by spectral methods viz.

infrared spectra (IR spectra), Nuclear magnetic resonance spectra (¹H NMR

spectra) and Mass spectra ( EIMS )

Screening of the anticonvulsant and anticancer activity of the newly synthesized compounds using Mice and for anticancer activity Hep-2 cell line were used .

Scheme :-

4. EXPERIMENTAL WORK

Synthesis of 1,5-Benzodiazepine derivatives:

MATERIAL REQUIRED:

Urea

Benzyl Chloride

Methanol

Ethanol

Conc.HCl

Sodium nitrite

Ethyl acetoacetate

Sodium acetate

Benzaldehyde

Furfuraldehyde

Vanillin

p-dimethyl amino benzaldehyde

Salicylaldehyde

Glacial acetic acid

O-Phenylene diamine

Ether

Method:

All the synthetic work was done by using laboratory grade reagents and solvents.

The solvents and reagents were purified and dried according to the procedure given in Vogel's textbook of practical organic chemistry.

TLC's were performed to monitor the reactors, the reaction and to determine the purity of the products. Further the compounds were purified by recrystallization using suitable solvents.

The melting point of the synthesized compounds was determined by open capillary method and is uncorrected.

The solubility of synthesized compound was determined by using different organic solvents

FTIR spectra was recorded on Shimadzu FTIR spectrometer using KBr pellets technique and expressed in cm-1.

Absorbance was recorded on Double beam spectrophotometer using methanol as a solvent.

¹H NMR spectra was recorded on BRUKER 300MHZ NMR spectrophotometer. Using (CDCl3+DMSO) as solvent

Mass spectra was recorded on Shimadzu spectrophotometer using (CDCl3+DMSO) as solvent

5. METHODOLOGY

In this present work an attempt was made to synthesize some new 1,5-Benzodiazepine derivatives with Urea and to study the Anticonvuvalsant Activity and Anticancer Activity exerted by theses compounds.

All the reaction was carried out under prescribed laboratory condition. Solvents and reagent used were of laboratory grade and were purified by distillation and crystallization techniques whenever necessary and their melting points were checked with available literature. The synthesized compounds were purified by recrystallization and their melting points were determined by open capillary method and were uncorrected. The purity of the compounds was confirmed by TLC methods.

The IR spectra were recorded on "SHIMADZU" spectrophotometer using a thin film supported on KBr pellets.

¹H NMR spectra were recorded on "BRUKER "300MHZ NMR spectrophotometer. The spectra were obtained in CDCl3+DMSO and chemical shift value are reported as values in ppm relative to TMS as internal slandered.

The Mass spectra were recorded on EIMS-2010A "SHIMADZU".

Experimental Procedure:-

Step 1) Synthesis of 1-benzylurea :-

Benzyl Chloride ( 0.1 M ,1.26 ml ) and Urea (0.1 M ,6.06 gm ) solution were refluxed for 3-hours at 100°-110°C. Then reaction mixture was cooled, a polymeric product was washed and the separated product was purified by recrystalisation from ethanol.

Step 2) (R,E)-ethyl 2-(benzylcarbamoyldiazenyl)-3-oxobutanoate : -

First step product 1-benzylurea of (0.1M,15.018 gm ) was dissolved in 30 ml of glaical acetic acid warm slightly to dissovle , then mixture is cooled to room temperature and 20ml conc HCl added and after it was cooled to 0-5 oC in ice-salt bath, cold aq. solution of 6.9ml sodium nitrite in 8 ml of water was added drop wise to the above solution, diazonium salt so formed was filtered into cold mixture of 13.5ml of ethyl acetoacetate and 4gm of Sodium acetate in 25 ml of ethanol. The resulting solid was filtered and washed with water and it was recrystalysed from ethanol.

Step 3) (E)-4-benzyl-1-(2-oxobut-3-enyl)semicarbazide : -

Step 2 product (R,E)-ethyl 2-(benzylcarbamoyldiazenyl)-3-oxobutanoate (0.1M ,3.05 gm),was added in different aromatic aldehyde in ethanol (20ml) and aq 4% sodium hydroxide Solution(4%). The mixture was stirred for 24hrs at room tempt. The contents were poured on crushed ice and neutralized with (25ml) of10% hydrochloric acid. The product was filtered, dried and recrystalysed from ethanol.

Step 4) (E)-1-(((1E,3Z)-5H-benzo[b][1,5]diazepin-2-yl)methyl)-4-

benzylsemicarbazide:-

Step 3 product (E)-4-benzyl-1-(2-oxobut-3-enyl)semicarbazide ( 0.1 M ) in ethanol (25ml),add few drops of glacial acetic acid and added O-Phenylene diamine(0.01M) in ethanol (20ml) was refluxed for 6 hours at 110 0C. The contents were cooled and poured on crushed ice. The isolated products was recrystalized from ethanol.

Physical data of Intermediates in the reaction:

Table 1: Physical data of 1-benzylurea

Comp.

Molecular formula

Molecular weight

% Yield (w/w)

Melting point(oc)

Rf value*

1

C8H10N2O.

150.18

78.14

310-320

0.72

*Mobile phase-Conc HCl : Formic Acid: (5:5)

Table 2: Physical data of (S,Z)-ethyl 2-(benzoylcarbamoyldiazenyl)-3-

oxobutanoate :-

Comp.

Molecular formula

Molecular weight

% Yield (w/w)

Melting point(oc)

Rf value*

1

C14H17N3O4.

291.3

65.14

220-230

0.88 *Mobile phase-Conc HCl : Formic Acid: (5:5)

Table 3: Physical data of (Z)-4-benzoyl-1-(2-oxobut-3-enyl)semicarbazide derivatives (3a-3e) :-

Comp.

R

Mol. formula

Mol. Wt.

% yield (w/w)

M.P.(0C)

Rf Value*

3a

Benzaldehyde

C18H17N3O2

307.35

58.82

145

0.73

3b

Fufuraldehyde

C16H15N3O3

297.31

69.85

135

0.71

3c

C19H19N3O4

353.37

73.52

117-119

0.81

3d

C18H17N3O3

323.35

63.23

108-110

0.82

3e

p-dimethyliamino Benzaldehyde

C20H22N4O2

350.41

74.26

131

0.79

*Mobile phase-Conc HCl : Formic Acid: (5:5)

Table 4: Physical data of (Z)-1-(((1E,3Z)-5H-benzo[b][1,5]diazepin-2-yl)methyl)-4- benzoylsemicarbazide derivatives (4 a - 4 e ) :-

Compound

4a

R

Benzaldehyde

Structure

Molecular formula

C24H21 N5 O

Mol. Wt.

395.46

% yield (w/w)

58.82

M.P.(0C)

175

Rf Value*

O.62

Elemental Analysis

C

72.89 %

H

5.35 %

N

17.71 %

O

4.05 %

*Mobile phase-Conc HCl : Formic Acid: (5:5)

Compound

4b

R

Fufuraldehyde

Structure

Molecular formula

C22H19N5 O2

Mol. Wt.

385.42

% yield (w/w)

59.85

M.P.(0C)

162

Rf Value*

O.61

Elemental Analysis

C

68.56 %

H

4.97 %

N

18.17 %

O

8.30 %

*Mobile phase-Conc HCl : Formic Acid: (5:5)

Compound

4c

R

Vannilin

Structure

Molecular formula

C25H23N5O3

Mol. Wt.

441.18

% yield (w/w)

73.52

M.P.(0C)

181-190

Rf Value*

0.90

Elemental Analysis

C

68.01%

H

5.25 %

N

15.86 %

O

10.87 %

*Mobile phase-Conc HCl : Formic Acid: (5:5)

Compound

4d

R

Salicylaldehyde

Structure

Molecular formula

C24H21N5O2

Mol. Wt.

411.46

% yield (w/w)

63.23

M.P.(0C)

108-110

Rf Value*

0.82

Elemental Analysis

C

70.06 %

H

5.14 %

N

17.02 %

O

7.78 %

*Mobile phase-Conc HCl : Formic Acid: (5:5)

Compound

4e

R

p-dimethyliamino benzaldehyde

Structure

Molecular formula

C26H24N6O2

Mol. Wt.

452.51

% yield (w/w)

74.26

M.P.(0C)

181

Rf Value*

0.58

Elemental Analysis

C

69.01 %

H

5.35 %

N

18.57 %

O

7.07 %

*Mobile phase-Conc HCl : Formic Acid: (5:5)

6. Pharmacological activities:

6.1 Acute toxicity:

Animals: Swiss albino mice weighing 20 - 25 gms were used for the study. Animals were fed a standard pellet and water and maintained at 24-28OC temperature, 60 - 70% relative humidity and 12 hr day and night cycle. Animals described as fasted were deprived of food for 4 days, but had free access to water.

Procedure:

Acute oral toxicity studies were performed (Ecobichon, 1997) according to OECD (organization for economic Co-operation and department). Swiss albino male mice (n=3/each dose) were selected by random sampling technique were employed in this study. The animals were fasted for 4 hr with free access to water only. Ethanolic extract of 1,5 Benzodiazepine derivative (suspended in CMC) were administered orally at a dose of 5 mg/kg initially to separate group of mice and mortality was observed for 3 days. Here no mortality is observed at the dose of 5mg/kg ,so same procedure is repeated for 50 mg/kg then animal showing some action but no mortality is seen. Now next, same procedure is repeated for 300 mg/kg but mortality was observed in 2/3, 3/3 animals, then this dose administered was considered as toxic dose. The following general behaviors were observed for first one hour and after 24 hrs of test drug administration.

OBSERVATION CHART:

Effect of the derivative (50mg/kg) on General behaviors in mice:

S.NO

Signs & Symptoms

Drug Treatment

Animal

1

Animal

2

Animal

3

Weight & Marking

22gm

22gm

20 gm

1

Tremor

+

+

+

2

Convulsion

+

+

+

3

Straub Reaction

_

_

+

4

Pilo erection

_

+

+

5

Catatonia

_

_

_

6

Loss of righting reflux

+

+

+

7

Decreased motor activity

+

+

+

8

Increased motor activity

_

_

_

9

Sedation

+

+

+

10

Muscle relaxation

+

+

+

11

Analgesia

_

_

_

12

Ptosis

_

_

_

13

Lacrimation

_

_

_

14

Salivation

_

_

_

15

Diarrhoea

_

_

_

16

Change in skin colour

_

_

_

"-" = Absent & "+" = Present

From the study of acute toxicity it has been observed that there was no mortality after 36 hours and also there was no change in general behaviour of the animal. So the drug is safe.

6.2 Anticonvulsant activity:

Isoniazid induced convulsions test: -

Isoniazid can precipitate convulsions in patients with seizure disorders. The compound is regarded as a GABA-synthesis inhibitor (Costa et al. 1975). Clonic tonic seizures are elicited in mice which are antagonized by anxiolytic drugs.

PROCEDURE :-

Six mice in each group of either sex with a weight of 18 to 22 gm are taken. Group I control animals received 30% aqueous PEG 400 only, group II received standard (diazepam 10 mg/kg) i.p. Group III-VII are treated with the test compound (4a-4e) by oral administration. The synthesized compounds (4a-4e) were suspended in 0.1% solution of SCMC and administered orally in a standard volume of 0.5 ml/20 g body weight at 50 mg kg-1 doses. 30 min after i.p. or 60 min after p.o. treatment the animals are injected with a subcutaneous dose of 300 mg/kg isoniazid (isonicotinic acid hydrazide).The occurrence of clonic seizures, tonic seizures and death is recorded.

Anticonvulsant activity was expressed as percentage of tonus and clonus mortality.

The one way ANOVA test performed by using Graph pad Insat software. The results obtained are tabulated in.

Anticonvulsant activity of compounds (4a-4e):-

Groups

Latency of clonus (min.)

% of clonus

% of tonus and clonus mortlity.

Control

3.20 ± 0.1797

100

100

Standard

8.57 ± 0.2144**

100

0

4a

4.03 ± 0.1806*

100

66.67

4b

4.52 ± 0.1366

100

83.34

4c

7.53 ± 0.1438**

100

16.57

4d

8.15 ± 0.0551**

100

16.67

4e

6.40 ± 17.84*

100

50.00

n=6; dunnets t test; * P<0.05; ** P<0.01; ***P<0.001 when compared with control.

All the compounds i.e. 4a to 4e showed a significant decrease in % of latency of clonus and % of tonus and clonus mortality. The compounds 4c, 4d,4e showed good anticonvulsant activity and compound 4a and 4b showed moderate activity.

6.3 Anticancer Activity :-

METHOLDOLOGY

MATERIALS REQUIRED IN MEM

Monolayer culture bottle of Hep 2 cells

5ml, 10ml serological pipette

Minimal essential media (MEM) with 10%, 2% foetal calf serum

TPVG (Trypsin, PBS, versene, glucose)

Discarding jar, inverted microscope, dessicator

Gloves, spirit, cotton, label pad, marker pen

MATERIALS REQUIRED IN CYTOTOXICITY ASSAY:

Monolayer culture in log phase

Drug extract (different concentrations)

MEM without FCS

0.4µ filter

5ml sterile storage vial

Tissue paper, spirit, cotton, marker pen and gloves

Micropipette and tips

Discarding jar

MINIMAL ESSENTIAL MEDIA PREPARATION:

Media is defined as a complex source of nutritional supplementation vital for the growth proliferation and maintenance of cells in vitro.

The MEM vial is dissolved in the pre sterilized Millipore distilled water and mixed well, closed and sterilized at 15lbs 121°c for 15mins. Allow ingredients in the quantity, depending on the concentration of foetal calf serum (2% or 10%) mix well by shaking. Take care avoid spills pass CO2 using sterile pipette, Shake the bottle, check Ph and adjust to 7.2 to 7.4. The MEM bottles are kept for 2 days at 37°c and checked for sterility, PH drop and floating particles they are then transferred to the refrigerator.

PREPARATION OF INGREDIENT:

Penicillin and Streptomycin: (Concentration 100IU of Penicillin and 100 µg 0f Streptomycin)

Dissolve both antibiotics in sterile Millipore distilled water, so as to give a final concentration 100 IU of penicillin and 100µg of streptomycin/ml. Mix well and distribute in 1ml aliquots. Store at -20° C Check sterility.

Fungizone (amphotericin B): (conc: 20µg/ml)

Dissolve in sterile Millipore distilled water so as to give a final concentration of 20µg/ml and distribute in 1ml aliquots in vials. Store at -20°c. Check sterility.

L.glutamine: 3%

Weigh 3g of l-glutamine accurately and dissolve in 100ml sterile Millipore distilled water and mix well. Filter through Millipore membrane filter 0.22µ and distribute in 5ml aliquots in vials. Store at -20°c. Check sterility.

4.7.5% sodium-bi-carbonate

Weigh requisite quantity of sodium-bi-carbonate (to give 7.5% solution) accurately and dissolve in 100ml of sterile Millipore distilled water. Filter through what man filter paper No.4, distribute into bottles and at 121°c, 15lbs, 15mins. Cool and store at +4°c.

5. Foetal calf serum

Bring FCS at room temperature. Inactivated at 56°c in water bath for½ hour and cool at room temperature. If floating particles are seen filter through Seitz filter. Distribute in 100ml,50ml, 20ml quantities in sterile bottles. Store at -20°c.

6. Trypsin, PBS,versene, glucose solution: (TPVG)

2% Trypsin: 100ml

Weigh 2g of trypsin accurately; dissolve in 100 ml sterile Millipore distilled water with magnetic stirrer for ½ hour. Filter through membrane filter. Store at -20°c.

0.2%EDTA (versene)

Weigh 200mg of EDTA accurately. Dissolve in 100 ml of sterile Millipore distilled water. Autoclave at 15lbs/15mins.

10% Glucose -100ml

Weigh 1g of glucose accurately. Dissolve in 100 ml of sterile Millipore distilled water and filter through Whatman filter paper and autoclave at 15lbs/15mins.

TPVG-100ml

PBS - 840ml

2%trypsin -50ml

0.2%EDTA -100ml

10%glucose -5ml

Penicillin & streptomycin -5ml

Mix all ingredients and adjust the pH to 7.4 with 0.1 N HCl or 0.1 N NaOH. Distribute in 100 ml aliquots. Store at -20°c.

MAINTENANCE OF CELL LINE:

Maintenance of cells involves the following operations:

Dispersion and Sub culturing (seeding) of cells.

Preservation of cells in repository.

Revival of cells from repository.

SUBCULTURING AND MAINTENANCE OF CELL LINE:

1. Bring the medium and TPVG to room temperature for thawing.

2. Observe the tissue culture bottles for growth, cell degeneration, pH and turbidity by seeing in inverted microscope.

3. If the cells become 80% confluent it goes for sub culturing process

4. Wipe the mouth of the bottle with cotton soaked in spirit to remove the adhering particles.

5. Discard the growth medium in a discarding jar keep distance between the jar and the flask.

6. Then add 4 - 5 ml of MEM without FCS and gently rinsed with tilting. The dead cells and excess FCS are washed out and then discard the medium.

7. TPVG was added over the cells. And incubate at 37° C for 5 minutes for disaggregation. The cells become individual and it's present as suspension.

8. Add 5ml of 10% MEM with FCS by using serological pipette.

9. Gently give passaging by using serological pipette. If any clumbs is present then repeat the process.

10. After passaging split the cells into 1:2, 1:3 ratio for cytotoxicity studies for plating method

"Seeding of cells":

After homogenize take one ml of suspension and pour in to 24 well plates. In each well add 1ml of the suspension and kept in a desiccators in 5% CO2 atmosphere. After 2 days incubation observe the cells in inverted microscope. If the cells became 80% confluent.

Cytotoxicity assay:

In order to study the antitumor activity of a new drug, it is important to determine the cytotoxicity concentration of the drug. Cytotoxicity tests define the upper limit of the extract concentration, which is non-toxic to the cell line. The concentration nontoxic to the cells is chosen for antiviral assay.

After the addition of the drug, cell death and cell viability was estimated. The result is confirmed by additional metabolic intervention experiment such as MTT assay.

PERFORMANCE OF DRUG CYTOTOXICITY ASSAY

Cytotoxicity is the toxicity or damage caused to the cells on addition of drug. After the addition of the drug, cell viability is estimated by staining techniques, where by cells are treated with Trypan blue. Trypan blue is excluded by live cells, but stains dead cells blue. The results are confirmed by additional metabolic intervention experiments such as MTT assays.

Materials Required

Monolayer cultures in log phase.

Drug extract (different concentrations)

MEM without FCS

0.45  filter

5mL sterile storage vial

Tissue paper, Marker pen, Spirit, Cotton and Gloves

1 mL, 2 mL pipettes, Micropipette and tips

Discarding jar with 1 % Hypochlorite solutio

Drug Dilutions

Stock drug concentration

100 mg of drug is dissolved in 10 mL of serum free MEM giving a concentration of 10mg / 1 mL. The stock is prepared fresh and filtered through 0.45  filter before each assay. Working concentrations of drug ranging from 10mg/ml to 0.3125mg/mlare prepared as follow

Preparation of working stock of 1 mg /mL

To 4.5 ml MEM add 0.5 mL of stock to give a working concentration of 1mg/mL

Drug concentration can be prepared from the working stock in MEM without FCS. Prepare required volume of test sample for each concentration.

48hr monolayer culture of Hep2 cells at a concentration of one lakh /ml /well (10 cells / ml / well) seeded in 24 well titer plate.

2. The plates were microscopically examined for confluent monolayer, turbidity and toxicity if the cells become confluent.

3. The growth medium (MEM) was removed using micropipette. Care was taken so that the tip of the pipette did not touch the cell sheet.

4. The monolayer of cells was washed twice with MEM without FCS to remove the dead cells and excess FCS.

5. To the washed cell sheet, add 1ml of medium (without FCS) containing defined concentration of the drug in respective wells.

6. Each dilution of the drug ranges from 1:1 to 1:64 and they were added to the respective wells of the 24 well titer plates.

7. To the cell control wells add 1ml MEM (w/o) FCS.

8. The plates were incubated at 37°c in 5% CO2 environment and observed for cytotoxicity using inverted microscope.

MTT ASSAY:

MTT assay is called as (3-(4, 5-dimethyl thiazol-2yl)-2, 5-diphenyl tetrazolium bromide.MTT assay was first proposed by Mossman in 1982.

Procedure:

After incubation, remove the medium from the wells carefully for MTT assay.

In each well wash with MEM (w/o) FCS for 2 - 3 times. And add 200µl of MTT conc of (5mg/ml).

And incubate for 6-7hrs in 5% CO2 incubator for Cytotoxicity.

After incubation add 1ml of DMSO in each well and mix by pipette and leave for 45sec

If any viable cells present formazan crystals after adding solubulizing reagent (DMSO) it shows the purple color formation.

The suspension is transferred in to the cuvette of spectrophotometer and an O.D values is read at 595 nm by taking DMSO as a blank.

Cell viability (%) = Mean OD/Control OD x 100

S.no

Concentration (µg/ml)

Dilutions

Absorbance

Cell viability

1

1000

Neat

0.17

26.98

2

500

1:1

0.23

36.50

3

250

1:2

0.27

42.85

4

125

1:4

0.31

49.20

5

62.5

1:8

0.34

53.96

6

31.25

1:16

0.42

66.66

7

15.625

1:32

0.50

79.36

8

7.8125

1:64

0.59

93.65

9

Cell control

-

0.63

100

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