CHAPTER - 1

RESEARCH ENVISAGED

The development o f new drugs has been responsible for decreasing human morbidity and mortality more than any other scientific endeavour during our lifetime. Drugs can be discovered In a number o f ways. Sometimes they are found by serendipity. M ost frequently, they are developed as part o f an organized effort to discover new ways to treat specific diseases. With the general acceptance o f the molecular basis o f disease it has been realized that the disease process can be understood at the chemical level, and consequently the disease can be Interrupted chemically. This has led to the mechanistic approach for the development o f drugs to treat diseases.

In recent years a large number of pharmacologically important heterocyclic compounds have been synthesized and are being widely used as drugs. The heterocyclics having quinazolinone nucleus have also been synthesized In good numbers, however there still exists much scope for synthesizing new compounds in this series. A survey o f literature shows that quinazolinones possess varied type of biological activities such as analgesic, anticonvulsant, anthelmentic, CNS depressant and anti-inflammatory activities.

In the quinazolinone nucleus, 2‘“‘ and 3 positions are the target for substitution with other moieties. A comparative study o f the structure o f morphine, pethidine and methadone reveals that the only common feature In all these compounds is the presence of tertiary nitrogen atom and quaternary carbon in relationship to it.

It was therefore considered worthwhile to incorporate both these features in an appropriate molecule in order to study better pharmacological actions o f quinazolinone nucleus.

1.0 QUINAZOLINES

The name quinazoline (German chinazolin) is today universally used to denote the 1,3- benzodiazine ring system (I). It was first proposed by Weddige' at the University o f Leipzig in 1887 on observing that his compounds were isomeric with the known cinnoline (II) and quinoxaline (III) derivatives.

Quinazoline (I) Cinnoline (II) Quinoxaline (III)

On the basis o f the physical and chemical properties, compounds containing the quinazoline nucleus fall into three distinct classes:

(I) The simplest class of quinazolines comprises those, which are unsubstituted in the heterocyclic ring. When they carry a substituent in the benzene ring and not in the heterocyclic ring they are termed as benzene' substituted quinazolines. The simplest member o f this class is quinazoline, first prepared in 1903 by Gabriel^ by the mild alkaline ferricyanide oxidation o f 3.4- dihydroquinazoline.

aq. KOH

K3Fe{CN)e

(II) The second distinct class o f compounds containing the quinazoline nucleus is composed of those compounds, which have a hydroxyl group in the 2 or 4 position in the quinazoline ring, adjacent to a heterocyclic nitrogen atom. Also considered in this class are tliose compounds having a functional group easily derived from or converted to a hydroxyl group in these positions. These include alkoxy, aryloxy, chloro, amino, mercapto, mercapto ethers, seleno and any others which may fall into this chemical category.

Those quinazolines having a hydroxyl group in the 2 or 4 position are tautomeric with the corresponding ketodihydroquinazolines. Thus 4-hydroxyquinazoline, tautomeric with 4-keto-3,4-dihydroquinazoline, is commonly named 4(3)-quinazolone or simply 4-quina2olone. Likewise, the 2-hydroxyquinazpline is named 2(l)-quinazolone or 2-quinazolone and 2,4- dihyroxyquinazoline, tautomeric with 2,4-diket6-l,2,3,4-tetrahydro quinazoline, is commonly called benzoyleneurea.

4(3)-Quinazolone Benzoyl urea

2(l)-Quinazolone

Because of this tautomerism, tlie quinazolones are high melting, insoluble solids, extremely stable to heat, light and air, and resistant to chemical oxidation, reduction, hydrolysis, and substitution on the benzene ring. Some of these derivatives have found use in the dye and pharmaceutical, fields.

(Ill) The third class of compounds containing the quinazoline nucleus comprises the hydrogenated quinazolines. Most important o f this series are the 3,4-dihydroquinazolinones.

1.1 PREPARATION OF 3,4-DIHYDRO-4-OXOQUINAZOLINES

3,4-Dihydro-4-oxoquinazolines are easily prepared by a variety of methods and can be converted to any of the common derivatives o f quinazoline (quinazoline, dihydroquinazoline and tetrahydroquinazoline). They are intermediates in the syntheses of many functional derivatives o f the quinazolines including chloroquinazolines, alkoxyquinazolines, mercaptoquinazolinones and their ethers. As such they are valuable intermediates.

a) NiementomkVs Synthesis:

The most common synthesis of 3,4-dihydro-4-oxoquinazolines is a. reaction, which was first described by Niementowski in 1895’\ When anthranilic acid is heated in ain open container with cxcess formamide at 120°, water is expelled and a nearly quantitative conversion to 4- oxoquinazoline is achieved.

OOH

+ HCONHj1202hr HjO

It has been carried out with a variety of substituted anthranilic acids to give the corresponding benzene substituted- 4-oxoquinazoline. As a rule a higher temperature or longer reaction time is required for substituted anthranilic acids. It has been adapted to many aliphatic amides’ to give alkyl substituents in die 2 position. A higher temperature is required, and the yields generally decrease as the molecular weight o f the amide is increased. Yields with benzamide are extremely low, but Slierrill and co-workers'* have obtained a 50% yield o f 2- phenyl-4-quinazolone by replacing benzamide with thiobenzamide or ethylimidobenzoate.

o z a

§C -N H ,

135-160H,S + HjO

A study of the Niementowski reaction has been made by Meyer and Wagner^. Working with 3- phenyl- 4-oxoquinazoIine, they demonstrated that the 4-oxoquinazoline was a strong enough base to form an anthranilate salt during the reaction, as well as formate, benzoate, phenylacetate and salicylate when heated with coiresponding acids. This prevented complete reaction, and addition, o f two molar equivalents of anthranilic acid raised the yields from 40% to 73%. A variation o f the Niementowski synthesis involves condensation o f an anthranilic acid amide with ethyl orthoformate as shown by the following formulas*:

CH (OCjHj),

OCCHjCHjOHlj/hsa

b) Cyclizaiioit o f o - Amidobenzamides:

When ammonium o-formamidobenzoate is heated to 220° over an open flame, water is evolved and 3,4-dihydro-4-dxoquinazoline is formed’. Similarly ammonium 2-acetamido-4- nitorbenzoate gives 3,4-dihydro-2-methyl-7-nitro-4-oxoquinazoline®.

The cyclization imist involve the prior formation of the amide as in the Niementowski reaction. If alcoliolic methylamine is used instead of ammonia, tiie 3-metliyi derivative is formed. 3,4- Dihydro-2-metliyl-4-oxoquinazoline®, 3,4-diiiydro-2-metiiyl-8-nitro-4-oxoquina- zoline and its 3- methyl derivative'”’ 3,4-dihydro-4-oxo-2-phenyl quinazoline and its 3-methyI derivative' can be prepared from the ethylesters of the respective anthranilic acids with alcoholic amine or methylamine. These w^ould also involve initial formation of the amine before ring closure because on heating o-acetamidobenzmethylamide, 3,4-dihydro-2,3-dimethyl-4-oxoquinazoline is formed^.

^ J S ^ C O O E t

^X ^N H C O R '

al.R"NH,R -

0

^V ^N H C O R 'R.

ThenTial cyclization is also possible by heating o-amidobenzamides above their melting points until elimination of water is complete gives respective 3,4-dihydro-4-oxoquinazolines, or their 3-methyi derivatives if one of the two hydrogen atoms of the two hydrogen o f the amide nitrogen is substituted'’ The cyclization can also be carried out in the presence of sodium benzesulphonate ” and the application of vacuum during the heating facilitates the reaction'®. The yields in the cyclization are generally reasonable and it is advisable to use an oil bath rather than a naked flame so as to minimize charring. The resulting oxoquinazolines are normally stable at high temperature^’ '®.

c) From o-AminobenzonitrUe:

o - Amino benzonitriles and acid anhydrides at temperatures above 200° for 6-10 hrs give poor yields of 3,4-dihydro-4-oxoquinazolines with substituents in position "2 depending on the anhydride used, and with substituents in the benzene ring depending on the nitrile used -®- - Excellent yields, on the other hand, can be obtained by heating o-amidobenzonitriles with alkaline hydrogen peroxide at 35-40° 20. 21. 22.21 reaction must involve the formation o f the

intermediate amide, as in the usual preparation o f amides from nitriles by alkaline hydrogen peroxide^''.

NHCOR'

OH

This metliod, however, is best suited for the preparation o f oxoquinazoiines, which lack a substituent in position 3.

d) From AnthmniUc acid derivatives and Niiriles:

Heating a mixture o f anthranilic acid and acetonitrile in a sealed tube gives a low yield o f3.4-dihydro-2-methyI-4-oxoquinazoline‘ ’ ®. Bogert and Gotthelf'* argued that the water fontied in the reaction was the cause of poor yield. They repeated the preparation, heated the mixture in the presence o f acetic anhydride, and succeeded in increasing the yield of this oxoquinazoline to 45%. When the corresponding acid replaced the anhydride, little reaction occurred and anilides, amides and decarboxylation products resulted. The use of nitriles with their corresponding anhydrides or with anlu'drides derived from other acids showed that the anhydride, not the nitrile determined the nature of oxoquinazoline formed, at least when the lower numbers o f the aliphatic series were used. Thus when anthranilic acid, propionitrile, and acetic anhydride were heated in a sealed tube at 140-150° for 3 hrs, 180-190° for 5 hrs followed by 6 hrs at 200-205°, only 3,4- dihydro-2-methyl-4-oxoquinaz©line was formed. On the other hand, when anthranilic acid, isocapronitrile and isobutyric anhydride were heated, a mixture of 3,4-dihydro-2-isopropyl- and3.4-diliydro-2'isoamyl-4-oxoquinazolines was obtained. The postulated reaction scheme is shown below:

a OOH RON

H,R m

R'CO'

When the acid is used in these reactions instead of the anhydride, the nitrile usually determines which oxoquinazoline is formed, particularly with higher acids” .

The condensation of phenylnitrile with 4-nitroanthranilic acid in alcoholic sodium ethoxide containing 1% of ammonia is reported to yield 3,4-dihydro-7-nitrQ-2-phenyl-4-oxoqumazollne^®. Methyl anthranilate, powdered sodium, and methylnitrile in benzene react exothermically to give 68% yield of dianthranilide and a poor yield o f 2-o-aminobenzoylmethyl-3,4-dihydro-4- oxoquinazoline"'’.

c) From 3,1,4-Benzoxazones (Acylanthranils) and Amines:

3,1,4-Benzoxazones r „ict with amines to give 3,4-dihydro-4^oxoquinazG)lihes. In the

simplest form of this reaction anthrianilic acid .was heated With an acid anhydride in the presence

o f ammonium carbonate^”. The anhydride acylated the anthranilic acid, the resulting o- amidobenzoic acid wad dehydrated to a 3,1,4-benzoxazone which reacted with ammonium carbonate to yield 3,4-dihydro-4-oxoquinazoline. Bogert and Seil” reacted primary aliphatic amines and anilines, with 2-methyi-5-nitro-3,l,4-benzoxa2one in boiling dilute ethanol and obtained a variety o f 3-siibstituted-3,4-dihydrO“2-methyi-5-nitro-4-oxoquinazolines.

RNH,

The reaction o f benzoxazones with amines was studied in some detail by Zentmyer and Wagner^^ who showed that two steps are involved. The first step necessitates opening o f the oxazone ring because water hydrolyzes benzoxazones to o-amidobenzoic acids. The ease o f ring opening is dependent on the substituent in position 2 and is in the order o f H > Me > E t» Ph.

R'NHyStepi step 2

3,1,4-Benzoxazone (R=H) is so unstable that it hydrolyzes to the benzoic acid e\'en on standing in a stoppered flask for 24 hrs' and has to be used as soon as it is prepared. Incidentally benzoxazones (R=H, Me, Et, Pr, Ph, tolyl, /?-chlorophenyl, nitrophenyl. or 3 ’-pyridyl) are prepared in 95-99% yields by boiling the respective o-amidobenzoic acids in acetic anhydride while allowing the acetic acid formed to distil off^"' The first step is subject to the nucleophilic nature o f the amine used and, depending on R and R’, the steric nature of the reactants. Thus when R and R ’ are small groups and R’NHi a strong nucleophile the reaction proceeds to completion giving the oxoquinazoline. When R and R’ are large and R'NHa is a weak nulceophile, more severe conditions are necessary, and the product is the intermediate o- amidobenzamide. This is a very useful synthesis because even if it stops at the o- amidobenzamide stage more severe conditions can be applied to effect the second step. Moreover the steric conditions must be really large to obstruct the reaction completely. The reaction o f amines having a primary amino group directly attached to a tertiar\- carbon atom, for example 2-amino-2-methyl-1,3-propanediol and 2-methyl-3,l,4-benzoxazone’’ and the reaction of amines with 2-o-substituted phenyl-3,1,4-benzoxazones^-fail entirely.

The nature o f the substitutent in the benzene ring of the benzoxazone has some effect on the ease o f ring opening of the heterocyclic ring in step 1 and on the ease o f ring closure in step 2. Thus although 2-methyl-5-nitro-3,l,4-benzoxazone yields the respective oxoquinazoline"” on

shaking with ammonia, 6-chloro-2-methyl-3,l,4-benzoxazone gives 2-acetamide-5- chlorobenzamide under tlie same conditions^^ 3,4-Dihydro-2-niethyl-4-oxoquinazolines have also been prepared from 2-methyl-3,l,4-benzoxazoneand ammonia^'’^ ’^ '''“, aliphatic amines’®’ ’’

(conditions varying from boiling in ethanol for an hour to heating in a suitable solvent at 200° for a few hours) and aromatic amines^^’ ''*’'* ''’’. Heterocyclic amines, e.g. aminopyridines and 2-methyl-3,l,4-benzoxazone give good yields of oxoquinazolines when heated with a naked flame"” .

The conditions for preparing 2-aryl-3,4-dihydro-4-oxoquinazoline are more drastic. 2- Phenyl-3,l,4-benzoxazone requires heating with ammonia in ethanol at 240-250° for 0.75 hrs for conversion to 3,4-dihydro-2-phenyl-4-oxoquinazoline^“.

3,1,4-Benzoxazones react with dibasic amines to give mono or dioxoquinazolines depending on the ratios of reactants used.

2 + H O

Both products can be obtained with /J-phenyienediamine'^^ and hydrazine^*. 2-Alkyl-3,l,4- benzoxazones also give oxoquinazolines with substituted sulphanilamides which are weak bases. The reagents are heated to 120-140° for 1 hour, then at 160-170® for 4-7 hrs to give over 60% yields of 3,4-dih>dro-4-oxo-3’,4’-sulphamoylphenylquinazolines substituted on the sulfonamide nitrogen atom^‘ ” .

f) From Isatoic Anhydrides;

Isatoic anhydrides readily react with an equimolecular quantity o f amines to form o- aminobenzamides'^'’ and these in turn can be converted to 3-substituted-3,4-dihydro-4- oxoquinazolines by refluxing ^ •ith formic acid Ibr 3 hrs '' or ethyl orthoformate^^ for 1.5 to 6 hrs without isolating the intermediate amides. The first stage of the reaction can be followed by the evolution of carbondioxide.

NHR' HC(OEt)a

0 6 "

When using formic acid, boiling for 1.5 iirs gives tiie o-formamidobenzamides, which can be cyclized to the oxoquinazolines by further boiling with acetic anhydride containing a trace o f phosphoric acid. Methyl-, ethyl-, propyl-, isopropyl-, allyl-, butyl-, and isobutyl-3,4-dihydro-4- oxoquinazolines can be prepared in this manner.^®’

g) From A nthranilic acid o f Esters and Imidoyl chlorides;

Mumm and Hesse^* recorded the first synthesis of 3,4-dihydro-4-oxo-2,3- diphenylquinazoline in 1910 from sodium anthranilate and N-phenyl benzimidoyl chloride in dilute alcohol but the yield was low. Levy and Stephen^® studied the reaction in detail and explained that the poor yield previously obtained was due to extensive hydrolysis o f the imidoyl chloride. They found that the reaction progressed better in acetone and that the synthesis o f the oxoquinazoline followed two pathways.

c r .

ClI

Ph-CaNPh

route 1

sh-iaNPhroute 2

a .

Ph I

C O O -C (B N P hrearrang.

PhNH.

Ph

^^^s^_.CO~N-COPh

a

CONHPh

‘ NHCOPh

When equimolar quantities of ammonium anthranilate and N-phenyl benzimidoyl chloride were reacted in acetone at 20° a 35% yield of oxoquinazoline was obtained. The low yield was e.vplained by route 2. Ammonium anthranilate hydrolyzes to a large extent in acetone (hydrolysis being appreciable even at 0°) and leads to the formation o f 2-phenyl-3,1,4- benzoxazone which reacts with aniline to give o-benzamidobenzahilide which slowly cyclizes to

10

oxoquinazoline. Tlie reactants were shown previously to give 2-phenyI-3,1,4-benzoxazone when pyridine was used as solvent. By adding solid ammonium anthranilate to the dry acetone solution o f benzimidoylchloride, the dissociation is minimized and a 70% yield of oxoquinazoline is obtained. Methyl anthranilate can be used in place of the acid but in this case attack takes place on the amino group with the liberation o f HCl. In order to obtain high yields (80-90%) of the oxoquinazoline two moles o f anthranilate must be used, the second mole removes the HCl from the reaction. 2,3-Diphenyl-, 2-phenyl-3-m-toluyl-, 3-o-methoxy phenyl-2-phenyl-, 3-/?-methoxy phenyl-2-phenyl-, 3,2’-naphthyl-2-phenyl-, 3,2’,4’-xylyl-2-phenyl~, 2-/7-chlorphenyl- and2 ,l’- naphthyl-3-phenyl-3,4-dihydro-4-oxoquinazolines were prepared in this manner^®. A variant o f this method makes use o f sodium o-nitrobenzoate and benzimidoyl chlorides, which give the intermediate N-acyl-N-aryl-o-nitrobenzamide. After reduction of this with dithionite and rapid ring closure with ammonia, the oxoquinazoline can be obtained in yields higher than 70%^'’.

li) From Antliranilic acids or Esters and Imidates:

Imidates react with anthranilic acid or its methylester in much the same way as the imidoyl chlorides.

i) Grimmel, Guenther and M organ’s Synthesis: ■

The largest numbers o f 4-oxoqutnazolines have been prepared by this synthesis. It was discovered by Grimmel, Guenther and Morgan™ in 1946 and was used for the preparation o f 2,3- disubstituted-3,4-dihydro-4-oxoquinazolines. The reason for its wide applicability followed from the discovery o f the hypnotic activity of 2-alkyl-3-aryl-3,4-dihydro-4-oxoquinazolines by Gujral, Saxena and Tiwari^'’. When 3 moles of o-amidobenzoic acids are heated with 3 moles o f an amine’together with one mole o f phosphorous trichloride in toluene for 2 hrs, 2,3-disubstitued-3,4-dihydro-4-oxoquinazolines are formed in high yields. The yields are not increased by using more than one mole of phosphorus trichlorides, and phosphoryl chloride can be used in its place.

NHCOR

The reaction proceeds via the phosphazo compounds (formed from the phosphorus halide and the amine) because equivalent quantities o f phenyl phosphazoanilide and o-acetamidobenzoic acid also give an 82% yield of 3,4-dihydro-2-niethyI-4-oxo-3-phenyl quinazoline''”’''^

In addition to aromatic amines, aliphatic amines (with long and short chains)''^, N-

nicthylpyrrolidin-3-yl methylamine^'’ and P-3,4-dimethoxy phenyiethyl amine have been used succesfully’''.

II

The reaction is said to iiave failed with a-naphthylamine, 2-amino-6- ethoxybenzothiazole, ailylamine, and 2-aminopyridine*“ but successful condensations with aliylainine” and 2-aminopyridine‘’‘' using the normal conditions, have been reported. In most cases the acyl groups in the o-araidobenzoic acids were formyl, acetyl or propionyl and 5-methyl- 2-furoyiamido benzoic acid also condensed satisfactorily'^’’^. Toluene was most commonly used as solvent but xylene‘s®, pyridine''^, nitrobenzene*^’, and also phenol^’ have been used. Other condensing agents are phosphoiyl chloride'*’’ benzenesulphonyl chloride in pyridine^"’ dicyclohexylcarbodimide in tetrahydrofuran at 20° phosphoric acid in the absence o f a solvent at 185-195° ” and polyphosphoric acid at 140-160° then at 180-200° in addition to phosphorus trichloride.

j) Sen and Ray's Synthesis:

During the investigations o f the structure o f the alkaloid vasicine, De and Ray found that boiling a solution of normal or isobutj'ryl anilides with uretliane and phosphorus pentoxide in xylene gave 2-propyl and 2-isopropyl-3,4-dihydro-4-oxoquinazolines’^ They named this method of preparation after Sen and Ray who first mentioned its feasibility in 1926’ . Phosphoric acid has also been used without advantage over phosphorous pentoxide, but no reaction occurred in the absence of condensing agent’’.

k) From Indoles and related contpounds:

Alessandri reported the Beckmann-rarrangement of 3-hydroxyimino-2-phenylindole to3,4-dihydro-4-oxo-2-phenylquinazoline in 1913”*.

NOH

NHCOPh

2-phenylindole in 3% alcoholic ammonia gives after standing at 20° for 9 months, 3,4-dihydro-4-oxo-2-phenylquinazolines . Sunlight is required for this reaction which is similar to the formation of 3,4-dihydro-4-oxo-2,6-diphenylpyrimidine form 2,5-diphenylpyrrole.

The latter reaction proceeds via oxidation o f the 2, 3 doub|e bond o f the pyrrole (elemental oxygen is necessary) and the ring opening, followed by cyclization witH aminonia*”.

12

Devender Rao and co-workers synthesized dibromo substituted quinazoiinones. They syntiiesized 6,7-dibromo-2-[(aIky!/aryl sulpliono) metliyl]-3-aryI-4(3H)-quinazolinones by condensing 2-chloro methyl-4(3H)-quinazoline with appropriate thiols followed by oxidation with hydrogen peroxides. Srivastava and his co-workers reported the synthesis of 2-[2-(4- substituted phenyl)-1.2-disubstituted ethyl]-3-(siibstituted phenyI)-4(3H)-quinazolinone by the bromination o f 2-[2-(4-substituted phenyl) ethyl]-3-(substituted phenyl)-4(3H)-quinazolinone followed by condensing with secondary amines.

Husain & Gupta reported method of synthesis of various 2-(substituted phenoxymethyl)-3-(thiadiazol-2-yl)-4-quinazolones as hypoglycemic agents. Mukerji e t al synthesized and studied the pharmacological properties of acetamide and carbamidomethyl- 4(3H)-quinazolones and reported its CNS depressant properties. Soliman E A and co-workers®^ reported the synthesis of benzoxazinone and quinazollnone derivatives by reacting anthranilic acid with P-aroyl acryloyl chlorides. Synthesis of 2-methyl-3-substituted-4(3H)-quinazolinones was reported by Buyutimkin et al **. Helene et al reported the synthesis o f benzo (f) quinazolin-7, 10-dines as potential antitumpr agent.

Dezso et al *** reported Dimorth rearrangement in which l,2-dihydro-3H-quinazolone-4-

oximes have been prepared from 4-amino-1,2-dihydro quinazoiin-3-oxides. Various a-arylazo- ■N-(2-alkyl-4(3H)-oxo-3-quinazolinyl benzylidenimines have been prepared by Shanker et al by reacting 2-alkyl-3-subsituted benzalamino-4-quinazoiin-4(3H)-ones with diazonium salts. Sinha and his co-workers'^” studied the effect o f substituents on Claisen rearrangement o f allyloxyquinazolones and developed 1-substitued-l,2-dihydro-oxazolo-[3,2-a]-quinazolinr5(4H)- ones in an unambiguous route Reddy et al reported the s\ nthesis of 2-methyl-4-oxo-N-(4-oxo-2-arylthiazolidin-3-yl)-3(4H)-quinazoIine acetamides by the reaction of 2-methyI-4- oxoquinazolin-3-acetic acid arylidene hydrazides with mercaptoacetic acid.

The synthesis and anticonvulsant activity of some novel 3-aminoquinazoHnones was reported by Kornet et al''*’. Shukla and his co-workers reported the synthesis o f 2-(o-arylidene aminophenyl)-3-(5-alkyl-l,3,4-thiadiazol-2-yl) quinazolin-4-ones and also studied its anthelmintic properties. Reddy and his co-workers have done extensive work on the synthesis of quinazoiinones in conjunction with various biologically active units in their search for potent antimicrobial and pharmocodynamic compounds. Lakhan and his co-workers reported the synthesis o f various 2-[(co^dialkylaniiino)-alkylthio]-3-aryl-(or alkyl)-6,8- disubstituted-4(3H)-quinazolones, which had shown better activity against gram positive than

13

grain negative bacteria. A series of 2-methyl-3-(4-oxotliiazolidin-2-yIidenamino)-4(3H)- quinazolinoiie derivatives have been synthesized by Buyuktimkin and Servet by cyclization o f antliraniiic acid with acetic anhydride, hydrazinolysis o f the resulting benzoxazines followed by acylation with ciiloroacetyl chloride and cyclized with ammonium thiocyanate. Shah at al reported the synthesis of 2,3-disubstituted-3,l-quinazolin-4(3H)-one derivatives and its anticancer and anti HIV properties.

Joshi and Chaudhari reported the synthesis o f 2-carbethoxy-3,4-dihydro-4- oxoquinazolines and their substituted derivatives such as 2-carboxamides, 2-carboxy hydrazides and tosylhydrazides by reacting substituted anthranilic acid amides with diniethyioxalated. Substituted quinazolone-l,3,4-oxadiazoIe were synthesized and screened for their MAO and acetylcholinesterase inhibitory activity, anticonvulsant, analgesic and reserpine reversal response by Barthwal and his co-workers A number o f substituted 2-methyl-3-(3,4-dimethoxy/dihydroxy phenyl ethyl)-4-quinazolones were synthesized by condensing substituted

acetantiiranils with P-(3,4-dimethoxy phenyl) ethylamine followed by demethylation with 47% hydroiodic acid'“‘'’

Synthesis of 2-[4(3H)-oxoquinazoiin-2-yI]-5-aryl-l,3,4-oxadiazole derivatives have been reported by Reddy & Reddy by reacting 2-hydrazinocarbonyl quinazolin-4(3H)-one with aromatic acids and also by LTA oxidation of 2-ai7lidenehydrazino carbonyl quin'azolin-4(3H)- ones. A series of N(2)-(2-halomethyl-4-oxo-3,4rdihydroquinazolin-3-yI)-carbazates and 2- haIomethyl-3-hetaiyl quinazolin-4(3H)-oncs have been synthesized and reported by Felther et al

by reacting 2-halomethyI-4H-3,]-benzoxazin-4-ones with alkyl carbazates and hetarylamines respectively. 2-(l-haloalkyl)-quinazolin-4(3H)-ones as well as some 3-aj"yl derivatives o f these compounds were reported to posses pesticidal activity.

Pandey and his co-workers reported the synthesis of 2-ary!-5-[3’-(2’-methyl-6,8- substituted quinazolyl) phenyl] pyrazoles by reacting 2-methyl-3-(4’-acetyl phenyl)-6,8- substituted-4-oxoquinazolines with different aromatic aldehydes followed by condensation with hydrazine hydrate. Shiba et al reported the aminolysis o f bis benzoxazinone with primary amines to yield N, N’-bis-2,2-[(N”-aryl/amino or aminophenyl)-benzamide] isophthalamides and also reported the synthesis of N, N ’ bis-[2,2’- ( r ,3 ’,4’-oxadiazolin-2’-thion-5’-yl) phenyl] isophthalamide and N, N’ bis-[2,2’- ( r ,2 ’,4’-triazolin-2’-thion-3’-yl) phenyl] isophthalamide by condensing hydrazide with carbondisulphide and ammonium thiocyanate respectively. Synthesis of 2-phenyl-3-(2-aminobenzamido)-quinazolin-4(3H)-one as synthons have also been reported by Reddy and Reddy from 3-amino-2-pbenyl quinazolin-4(3H)-one. l,2,4-Triazino-[4,3-c]- quinazolinone and/or l,2,4-triazino-[2,3-c]-quinazolinone' derivatives were synthesized by Allimony and his co-workers via interaction of 2-methyl-4(3H)-quinazolinone and 3- arylamino-6-iodoT2-methyl quinazolinone with chloro aqetylchloride followed by cyclization with some nitrogen compounds.

14

Tyagi et al synthesized 2-metIiyl-3-[(4’-oxo-5’-niethyI)-tliiazoliclinyl]-4(3H)-

quinazoliiiones and 2-methyl-3-[5’-(substituted phenyl)-A'-triazolinyl]-4(3H)-quinazolinones from 2-metliyI-3-aryIidenamino-4(3H)-quinazolinones by cyclocondensation with thiolactic acid

and diazomethane respectively. A facile and efficient synthesis of 4-quinazolinyl-p-lactams i.e.,1 -aryl-4-[isopropylidenamino/methyl-4(3H)-oxoquinaz61in-2-yl]azetidine-2-one from 2- chloromethyl quinazoiin-4-(3H)-ones was reported by Reddy and co-workers

Holla et al have studied the synthesis of some fluorine containing arylfurylvinyl quinazolinones as possible antibacterial agents. Raghu Ram Rao and Rajesh H Bahekar reported the synthesis of some 6-aryl benzimidazo [1,2-c] quinazolinones by reacting substitutedl,3-benzoxazino-4(3H)-one with o-phenylenediamine followed by cyclization.

1.2 PROPERTIES AND REACTIONS

Reactions associated with the tautomeric nature of the quinazolones are often quite complex and generally unpredictable.

Quinazolones are- always high melting crystalline solids, insoluble in water and most organic solvents but soluble in aqueous alkali. They are generally insoluble in dilute strong acids but are some times soluble in concentrated strong acids. Simple 2- and 4-quinazolones, although insoluble in dilute acids, are soluble in 6N hydrochloric acid. 2- and 4-Quinazolones form stable monohydrochlorides, chloroplatinates, chloroaurates and picrates"’’ and their metal salts of silver, mercury, zinc, copper, sodium, and potassium have been described ’

The ring system in quinazolones is exceedingly stable to oxidation, reduction, hydrolysis and other treatment designed to break the ring. There is no report of degradation o f a quinazolone by simple chemical oxidation. After boiling with concentrated nitric acid for several hours, 4- quinazolone can be recovered quantitatively as 6-nitro-4-quinazolone'^°. Oxidation o f 2- or 4- quinazolone or a dihydroquinazolone (i.e., 2- or 4- keto-l,2,3,4-tetrahydroquinazoline) with permanganate or chromic acid give benzoyleneurea which is not attacked by thesereagents. Any alkyl or aryl groups attached to either ring during these oxidation are converted into carboxylic acids, which may be lost through decarboxylation at the higher temperature. Common laboratory reduction methods do not affect the quinazolone ring.

'fwo reactions o f quinazolones have been the source of extensive investigation. These are the alkylation of quinazolones with alkyl halides and sulfates, and the cholrination o f quinazolones to 2 - and 4-chloroquinazolines with phosphorous chlorides,

15

2-Aryl-3-aroylaminoquinazolone undergoes a complex rearrangement when warmed with dilute aqueous alkali to give substituted triazoles

r j[ I r j| /C,H,^ ^ C O O H

“ p .

h.c ~ os= n

HjO

HsC“ CS®N

Koelsch'^*' has reported an unusual reaction o f 3-phenyl-4-quinazolone with benzyl magnesium

chloride in which the chief product isN- (P, P’-diphenylisopropyl) anthranilanilide.

o

l-CeHj

(CHjCgHg)However, when the quinazolone carries a substituent in the 2 position, attack by the Grignard apparently occurs at the carbonyl group in the 4 position’ ’ with l,2-dimethyl-4-quinazolone a similar reaction with Grignard reagent occurs'^®. However, reaction o f 2,3-diphenyl-4- quinazolone with phenylmagnesiumbromide results in the formation o f 2,4,4-triphenyi-3,l-

CjHjMaBr

s' eNis a C — “OM gBr

'V n—CjHjNlgBr

-H,0

1.3 APPLICATIONS OF QUINAZOLINES:

Alkaloids: Several alkaloids containing quinazoline nucleus have been described. Among them are vasicine (peganine), rutecarpine and evodiamine. The most interesting quinazoline alkaloid is

16

febrifugine, a substance o f higli aiitimalarial activity, but of such high toxicity as to preclude its practical use. The alitaloid, first described as dichroine, was isolated from the roots of Dichroa febrifitga Lour, long used as febrifuge in china under the name of Ch’ang Shan. A number o f quinazoline analogs o f common quinoline and isoquinoline alkaloids and medicinals have been prepared. In no case has notable activity o f the type displayed by the parent compound been reported for the q u i nazol i ne analog.

Medicinal: There have been only scattered reports of the investigation o f die m edical properties o f quinazoline derivatives. These are mainly compounds prepared as antimalarial drugs or as substituted sulfanilamide. Biological data on such compounds are scanty.

As antimalarial drugs, great many quinazoline analogs of the 4-aminoquinoline (chloroquine) series have been made and two analogs of the 8-aminoquinoIine (plasmochin) series have been reported. The former is rather easily prepared by reaction o f the appropriate amine with a 4-chloroquinazoline or a 4-thioquinazolone or its metliyl ether.

A number of N-quinazolylsulfonamides have been reported. 4-Aminoquinazoline prepared by amination o f 4-chloroquinazoline, and 2-aminoquinazoline, from o-amino benzaldehyde and guanidine chloride in pjridine to give the corresponding N‘*-acetyl-N‘- quinazolinyl sulfanilamides. Hydrolysis in acid or alkali gave the “sulfaquinazolines”. They were reported to be “too insoluble to exhibit chemotherapeutic activity” ' ® and showed no activity against avian malaria in initial evaluations. N'-Tetrahydroquinazolylsulfonamides prepared from these are reported to have slight protective action in mice infected with p-hemolytic streptococcus*''®.

1.4 BIOLOGICAL EVALUATIONS

It is evident from the literature that afloqualone [6-amino-2-fluoromethyl-3- (o-tolyl)- 4(3H)-quinazoIinone] and fluproquazone [4-(/7-fluoro phenyl)-l-isopropyl-7-methyl-2(lH)- quinazolinone] are reported to possess centrally acting muscle relaxant'^'"'” and analgesic

antipyretic'’’ activities respectively and the medicines'^® derived from quinazolone nucleus viz., quinethazone (1) and metalozone (II) are clinically used as diuretic similar to that o f

benzothiadiazines and methaqualone (111) a relative hypnotic drug.

■ (Ml)

17

Anti-inflammatory activity has been observed among 5-aryl-2- ( I ’-pyrazolyl)- quinazolines'^^, 3-N, N-dialkylamino methyl-5- (2-alkyl-l, 3-quinazolin-4-yloxymethyl)-2-thio-l,3,4-oxodiazoles'''‘’, and hexahydro-1- (2-alkyl-4-(3H)-oxo-3-quinazolinyI)-3-aryl-2-thioxo pyrimidine-4,6-dines’'". Presence of a chiorophenyl group at the side chain nitrogen in the

compounds o f the latter series enhances the anti-inflammatory activity''*^ Among various a -

aryl/heteroalkyl azobenzal aniline derivatives prepared, it was observed that the compounds having quinazolinone substituent shov/ed maximum anti-inflammatory activity.

Four novel series of 4(3H)-quinazolinone derivatives have been synthesized by cyclization of the key intermediates 3-aryI-2-(3-aryl-3-oxopropenyi)'4-(3H)-quinazolinones with different reagents and evaluated for anti-inflammatory activity'‘'^ Ei-Feksy et al., reported the synthesis and anti-inflammatory properties of some novel thiazolidinones and imidazolidinones derived from 4-(3-phenyi-4(3H)-quinazolinon-2-yl)-3-thiosemicarbazone. Some o f the synthesized compounds showed superior activity than phenylbutazone and they were also devoid o f any ulcerogenic activity''*''.

Two approaches were used by Santagati and co-workers’^ to yield 4-quinazolinone by appropriately substituted anthranilates and secondly through benzoxazin-4-ones intermediates and screened for their anti-inflammatory activity. These compounds have showed significant anti­inflammatory effect in an experimental ocular inflammation model and lowered the prostaglandin Ea (PGE2) production with respect to control group. 3-Cyclohey.yl-6-chloroqiiinazolin-4(3H)-one and 3-cyclohexyl quinazoline derivatives were reported as the most active compounds which significantly reduced PGE2 levels more than the reference drug Tolmetin''‘ Bekhit and Khali! reported the synthesis o f 4(3H)-quinazolinone derivatives by cyclization of the intermediate 3- aryl-2-(6-aryl'2-cycIohexen- I-on-5-yl)-4(3H)-quinazolinones with hydrazine, phenylhydrazine, hydroxylamine and thiourea and the synthesized compounds were screened for their anti­inflammatory activity*''®.

Recently, quinazolinofarmazans were synthesized by Kalsi et al''*’ by attaching formazan moiety at position 3 and the arylaldehydic substituent at position 2 of the quinazolinone nucleus. It was observed that quinazolinoforniazans possessing a methyl substituent at position 2 o f the quinazolinone nucleus possess higher anti-inflammatory activity as compared to the corresponding compounds with a substituted aryl-ethenyl moiety.

IO-Substituted-6-aryl aminomethyl-12H quino-[2,l-b]-quinazplin-5(6H),12-diones and

5,6-dihydro-5-substituted-12H-quino-[2,I-b]-quinazolin-5,12(6H)-diones Have.been synthesized and evaluated for anti-inflammatory activity by Saxena and co-workefs‘'"‘. 10-Iodo-6-(3-methoxy

phenyl aminomethyl-12H quino-[2,l-b]-quinazoIiii-5(6H),I2-diones showed 58 % anti-

18

inflammatoiy activity (100 mg/kg p.o) as compared to phenylbutazone which showed 50 % anti­

inflammatory activity (100 mg/kg p.o).

A series of (2-aryI-2,3-dihydro-4(lH)-quinazolon-l-yl)-alkyl substituted cyanoguanidines and ureas with histamine, cimetidine or roxatidine partial structure have been prepared and tested for H r and H2 antagonism at the isolated ileum and the isolated right atrium of the guinea pig3-[3-(l-piperidinyl methyl) phenoxy] propyl cyanoguanidines and ureas were reported to be more potent H2 antagonists than cimetidine and all the compounds showed weak H] antagonists.

Gravier et al., reported the synthesis of 4-quinazolinones and their anti-platelet activity invitro with respect to aggregation induced by ADP, collagen, arachidonic acid and the platelet serotonin release reaction. Some o f the synthesized compounds showed inhibiting power similar to that o f acetylsalicylic acid under the same conditions and even greater when ADP induced aggregation. Reduction of the 4-quinazolinone derivatives to their 1,2,3,4-tetrahydroquinazoline derivatives showed an increase in platelet inhibitory action except when ADP is the inductor'^®. 2-{4-[l-(2-chlorophenyl)-piperazinyl]}-methy^2,3-dihydroimidazo-[l,2-c]-quinazoIin-5(6H)-one

inhibited platelet aggregation and ATP release induced by arachidonic acid, collagen, PAF and U46619 in washed rabbit platelets and in human platelet rich plasma. It also significantly suppressed the platelet aggregation and ATP release challenged by epinephrine and ADP. The data indicates that anti-platelet effect of quinazolinone was due to the direct inhibition o f phosphoinositides breakdown'^'.

Structural and molecular modeling o f derivatives of the anticonvulsant methaqualone leads to the discovery of unsaturation in the 2-substituent produced active but less toxic compounds. Duke and Codding developed 2-aryIethanone derivatives and studied its SAR. All the synthesized compounds showed different profile of anticonvulsant activity. Each compound was observed as the neamine tautomer containing an intramolecular hydrogen bond between the ethanone and the amine N atom, and the molecular conformations are same. From the study, they concluded that recognition o f the anticonvulsants arises from specific binding o f an ortho substituent on the N (3) phenyl substituent, rather than form bonding of a particular conformation or tautomeric form adopted by the compound containing an ortho substituent, and that such

recognition was characteristic o f a broad range of anticonvulsant drugs'^^. A series of 3-(/>- suifamoyl phenyl)-4(3H)-quinazolinone derivatives were synthesized by condensation of sulfanilamide with various 4H-3,l-benzoxazinones and evaluated for anticonvulsant activity against pentetrazol-induced seizures.'” Some of the compounds showed significant anticonvulsant effect. A number o f l-(3-phenyl-4(3H)-quinazolinone-2-yI -mercaptbacetyl)-4- substituted thiosemicarbazide and 2-(3-phenyl-4(3H)r-quina2olinone-2-yl-

mercaptoacetyihydra2ano)-3-substituted-4-thiazolidone derivatives'^'* and quina;zolinone

19

thiazolidiiie derivatives'^^ have been synthesized and screened for their anticonvulsant activity

against MES induced seizures.

A series of l-deoxy-l-(a-substituted-3amino/iTiethylamino-4-quinazolone)-D-fructose

have been synthesized by Nautiyal et al’ *' and jsvaiuated the effect o f aldose and amine linkage on central nervous systems. These compounds showed higher anticonvulsant and analgesic activity

as compared to the reference compound.

Hepatoprotective efficacy o f synthetic quinazolines have been studied by Aruna and co- workers'^’ by evaluating their inhibitory effects on CCI4 induced microsomal lipid peroxidation and scavenging o f hydroxyl radical formation and the compounds showed comparable protective potential with standard liver protective compound namely Silymarin.

Synthesis of 2-aryl-5-[3’-(2’-methyI-6,8-dubstituted quinazolyl)-phenyl]-pyrazole derivatives have been reported to possess antiviral activity against plant and animal viruses by Pandey and c o - w o r k e r s F e w of the compounds showed maximum activity against plant viruses’ in-vitro and showed moderate antiviral activity against chick embryo system. Nevv 2,3- disubstitued quinazolinone derivatives have been synthesized and were evaluated for their anti­

inflammatory activity by Tyagi et a l." ’ 2-Methyl-3[5’-(3,6-dichlorophenyl)-<"-triazolinyl]-

4(3H)-quinazolinone showed 51% anti-inflammatory activity as compared to phenylbutazone

(ED50 dose).

Anti -HIV activity of several l-[2-phenyl-4(4H)-oxo-3-quinazolinyl]-2-methyl-4-

arylidene-5-oxoimidazolines, 2-phenyl-3-(arylamino)-4(4H)-oxoquinazolines, and N'-2-methyl- 4(4H)-oxo-3-quinazoHnyl-N^-arylthioureas has been reported by Desai and co-workers'^’ . Inhibition of H IV ' reverse transcriptase by 6-chloro-4(s)cyclopropyl-3,4-dihydro-4-[(2-pyrldyl)- ethynyl]-quinazolin-2(lH)-one have been reported by Carroll and co-workers'*”.

Several sulfonate esters containing quinazolinone derivatives that are substituted or fused with aryl, hetaryl, or heteryl phenyls ring systems were synthesized by Habib et al'*' by prior preparation of the 2-[4’-(benzensulfonyl)-phenyl]-l,4(H)-benzoxazin-4-one derivative and subsequent reaction with several nitrogen neucleophiles and evaluated for their efficacy as new bactericidal and/or fungicidal agents. Most of the compounds showed high antimicrobial activity

as compared to standard chemotherapeutic agents. Several other quinazolinone and bis-

quinazoline derivatives were synthesized and screened for antimicrobial activity'*"''®‘\

, A series o f 2-[(N,N-disubstituted thiocarbomylthib)“methyl]-quinazolinpnea have been synthesized by reaction of the potassium salts of disubstituted ditlTioc?rbamic acids and the

respective 2-bromomethyl-4(3H)-quinazolin6ne, 4 or 3-aryl-2-chloromethyl-4(3H)-

20

quifiazolinoiies and tested against 23 pathogenic fungi at 2 and 5 % concentration invitro. Some o f tile synthesized ditiiiocarbamate derivatives showed broad spectrum antifungal activity as compared to Tolnaftate, the clinically used thiocabramate compound and also exhibited comparable activity to Clotrimazole against Candida species and F. solani

3'Substituted-4(3H)-quinazolinones were synthesized and studied for structure activity relationship by Bartoli et al'®*. These compounds displayed higher invitro activities against Filam entons fungi and shorter half-life.

A series of new 3-(3-phenylisoxazol-5-yI)/ 3-[(3-phenylisoxazol-5-yl)*amino]-substituted

4(3H)-quinazolinone derivatives have been synthesized and reported to possess antineoplastic activity. Most active quinazolinones showed ICjq values in the range o f 16 - 30nM against Raji (human burkitt limphoina), K-562 (human-chronic myelogeneous leukemia) and U937 (human histiocytic limphoma) cell lines'*’®. Baek and co-workers reported the synthesis and invitro inhibitory activity of 5-substituted quinazolinone derivatives. Compounds containg OH and COOH as R substituents were most effective, indicating that hydrogen bonding may contribute to the increased inhibitory activity and showed high cytotoxicity against tumor cells in culture'®’. A series of derivatives of 2,3-dihydro-2-aryl-4(lH)-quinazolinone (DHQZ) with known anti­tumor activity were re-evaluated in the National Cancer Institute by Hamel et al'®*. The design, synthesis and biological evaluation of a new class of inhibitors of thymidylate synthetase (TS) has been studied'®’'^'. The synthesis and pharmacological activity of isoindolo-[l,2-b]-quinazolin- l2(10H)-ones and isoindolo-[2,l-a]-benzimidazoles have been studied by Megalla and co­workers

Several quinazoline derivatives containing substituted thiosemicarbazido and S-methyl isothiosemicarbazido groups at the 2-position and at both the 2 & 4‘'’ positions have been synthesized and evaluated for antitoxoplasmosis effect” ’’.

New 6-chloro-2,3-dihydro-4(lH)-quinazolinones have been synthesized and evaluated for gastrointestinal prokinetic and antiemetic activities in comparison witli structurally related benzamides and 6-chloro-2,3-dihydro-(l H)-l,3-benzoxazin-4-ones'’‘'.

2-[4-(3-Tert butyl amino)-2-hydroxy propoxy]-phenyl-3-methyl-6-methox>'-4(3H)-

quinazolinone was evaluated as a selective P-1 adrenoceptor ligand for Positron Emission

Tomography (PET) by Valette and co-workers'’^ A series of 2-alkyl and 2-aryl substituted 8-h)droxy, 8-methoxy and 8-methyl quinazolin-4(3H)-ones have been synthesized and evaluated for poly (ADP-ribdse) polymerase (PARP) inhibitory activity in permeabilized L 1210 murine leukemia cells. 8-Methoxy and 8-methyl quinazolinones were synthesized by acylation o f 3- substituted anthranilamides with appropriate acid chloride followed by base catalyzed cyclization.

21

2-Phenyl quinazolinones were marginally less potent than the corresponding 2-methyi quinazolinones, but the introduction of an electron withdrawing group or electron donating group 4 ’-substituted on the 2-aryl ring invariably increased potency. Among the synthesized compounds, 8-hydroxy or 8-niethyI substituent enhanced inhibitory activity in comparison to an 8-niethoxy group’’®. Recently, Molnar-Kiniber et al., and Duplainer et al., have reported a quinazolinone derivative, Nitraqazone to possess potent phosphodiesterase inhibitory activity that is potentially useful in treatment of asthma. Raghu Ram Rao and co-workers studied bronchodilatory action of 6-aryl benzimidazo-[I,2-c]-quinazoline.

Several other 2-substituted-4(3H)-quinazolinone derivatives reported to possess various biological properties like calcium antagonist poly (ADP-ribose) synthetase inhibitor'*^,immunotropic Non-steroidal anti-inflaminatoiy antimicrobial anticonvulsant

5-HT2A antagonist'®\ H2-receptor antagonist anti-hypertensive thromboxane

synthetase inhibitor^””, cardiotonic^”', vasodilator^®^, analgesic^“ , anti-allergic^“‘', selective (3'- adrenoceptor antagonist^®^ antiviral^“ , CNS depressant"' , anti-fibrillatory and antiphologistic"®* agents.

In the quinazolinone nucleus 2"‘' and 3"* positions are the main target for substitution with other moieties. A comparative study of the structure of morphine, pethidine and methadone

reveals that only common feature in all these compounds is the presence of nitrogen atom and

quaternary carbon in p-relationship to it“ ’. Using the concept o f quatemary carbon and a tertiary

nitrogen atom in P-relationship to each other, as an optimal and essential feature o f potent

analgesic, we synthesized a series o f new 2-substiuted-6 ,8-dibromo quinazolinone derivatives and screened for the following biological and pharmacological properties:

« Analgesic activity:

1. Acetic acid induced writhing method2. Tail immersion method

• Anticonvulsant activity

• Toxicity studies:

1. Determination of LD50 and ED50

2. Gastrointestinal toxicity

• Antibacterial activity.

22

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25

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FLAVONOIDS

31

RESEARCH ENVISAGED

In the earlier part o f the thesis, the synthesis o f the compounds were based on tiie dibromo anthranilic acid nucleus which was suitably exploited to give various quinazolinone derivatives and related compounds. Flavones are an important group o f natural occurring compounds attributed with innumerable physiological and biological properties. It was therefore envisaged to synthesis some such compounds which may incorporate the dibromo amino group in one o f their ring and hence these studies were undertaken. In one scheme, reascetophenone was synthesized which was converted into the 4-methyl-5-hydroxy-6-acetyl coumarin. Attempts to prepare its ester followed by diketone with N-acetyl dibromo anthranilic acid did not succeed and hence, some simple acetophenones viz. resacetophenone and o-hydroxy acetophenone were selected for preparing the desired compounds via their ester and the p-diketone derivatives. However, these attempts did not succeed. The details are given below. Finally anotlier route was planned where o-nitro benzoic acid ^ •as made to react with o-hydroxy acetophenone to give the corresponding ester which was converted to the P-diketone derivative followed by conversion to the flavone derivative. The nitro group was then reduced in the usual m annerto give the 2-amino derivative. It was then brominated for synthesizing the desired 3 ’, 5’ dibromo compound. However the final product was characterized as the penta bromo derivative.

32

2.0 FLAVONOIDSFlavonoids, are a large group of naturally occurring benzopyrone derivatives, which to

some extent resemble coumarins. According to Geissman et al’ (1952), flavonoids are the compounds whose structure is based on Cc - Cj - Cg skeleton. Flavonoids have been known to possess marked physiological activity and high specificity o f function and are widely distributed in plants as water-soluble glycosides.

In the flavonoids, two benzene rings (A and B) are linked by propane bridge in the form of A-C^-C^-C'-B (I) except in isoflavonoids and neoflavonoids. In isoflavonoids the linkage is in A-C'^-C^(B)-C‘ (II) fashion e.g., diadzem, genistein and orobal and in neoflavonoids linkage is in the form o f A-C^ (B)-C^-C' (III) e.g., dalbergin. The basic skeleton o f flavonoids can be derived

from variable nature o f heterocycle, which is derived from pyran (IV) or pyrylium (V) or y- pyrone (VI).

(I) (II) (III)

0 0 0(IV) (VI) (V)

Variations in the structure of the flavonoids depends upon the various levels of saturation and oxidation of 1-benzopyran which occurs when the cyciization takes place between the third carbon (Ci) o f the chain and OH group of the ring A-ortho to this chain leading to the formation o f chromane (Vll), 2H-chromene (VIII), 4H- chromene (IX), 4-chromarione (X) and chromone

33

C O 0 0 C O a ; ) 0 0(VII) (VIII) (IX) (X) (XI)

2.1 SYNTHESIS OF FLAVANONES:

Flavonones, isomeric with the chalcones are obtained from latter by acid or alkali catalyzed ring closure. Ring closure is favoured by an OH group at 6 position of the chalcone.

The 6-, 8-methyl flavones, strobopin (XII) and cryptostrobin (XIII) are synthesized in a single step by condensation of 2'hydrox.y-4,6-dibenzyloxy-3-methylacetophenone or 2,4- dihydroxy-6-methoxy-5-methylacetophenone with benzaldehyde in ethanolic potassium hydroxide, followed by cleavage o f the protecting group^.

(XII) R’ = Me, R2 = H (Strobopin)(XIII) R' - H, R- = Me (Cryptostrobin)

Polyhydroxy flavonones were synthesized in a one step process by reacting appropriate hydroxy acetophenones and hydroxy benzaldehydes in the presence of boric acid, in a mixed solvent system \

Aromatic aldehydes when condensed with malonitrile gave RCH;C(CN)2, which when treated with pholoroglucinoi give corresponding flavanones''. Flavonoids have also been synthesized from corresponding benzopyrans using a free radical process involving the BujSnH/AlBN system in refluxing benzene'*. Synthesis of 6-prenyl flavanones has also been reported’ and is based on Claisen rearrangement and cyclization reaction.

34

2.2 SYNTHESIS OF FLAVONES:

Flavones can be synthesized from chalcones or flavanones or simple precursors by condensations.

(i) Dcliydrogenation o f chalcones and flavanones:

Nadkarni et al*, have reported the synthesis of flavones from chalcones (o-hydroxy chalcones) by acetylation and addition o f bromine, followfed by reacting the dibromides with ethanolic potassium or sodium hydroxide. Partially acetylated flavonories such as naringenin(XIV), hesperetin (XV), and homoeriodyctiol (XVI) have been converted to respective flavones, apigenin (XVII), diosmetin (XVIII) and chrysoeriol (XIX) by the reaction of benzoyl peroxide catalyzed bromination with N-bromo succinimide in carbon tetrachloride followed by acid hydrolysis’.

(XIV) R ' = Ac, = H(XV) R ' = Me, R^ = OAc(XVI) R' = A c, R2 =OM e

(XVII) r ‘, r - = H(XVIII) R ' = Me, R^ = OH(XIX) R ‘ = H, = OMe

Flavones have been synthesized in good yields by oxidising o-hydroxy chalcones and flavonones in presence of selenium dioxide®’®. Methyl ethers o f luteolin (XX) and 6- hydroxyluteolin (XXI) and poly hydroxy flavones with completely disubstituted A ring was prepared by dehydrogenation with selenium dioxide. Bose et al’”, have reported a cyclization and simultaneous dehydrogenation of 2’,4’-dihydroxychalcones to the corresponding flavones by heating with palladium on charcoal.

OH

( X X ) R = H (XXi) R = OH

35

Ficine, a novel flavonoidal alkaloid was isolated from Ficus pantoniana'^, which was synthesized'’ by reaction of 1,3,5-triniethoxybenzene with y-amino butyric acid followed,by basification, and reduction to give l-methyl-2-(2’,4’,6’-trimethoxy phenyl)-pyrrolidine, Tlie pyrrolidine was subjected to Friedel Crafts acylation to form a hydroxy acetophenone, which was converted to chalcone then to flavone and finally dehydrogenated to give the flavone, ficine. Flavonones are converted to flavones by a treatment with Ti(N02)3'‘ or Ti(0 3SC6H4Me-4)3 and Ti(N0.i)3 in ethylcyanide or methylcyanide respectively’'’. Flavonone undergo facile dehydrogenation with Ti(oAc)3 in acetic acid or methylcyanide to give falvones'^. 3- Benzylflavones were synthesized either by the reaction of flavonone with aromatic aldehydes or by the conversion of 3-arylidene flavonones, both catalyzed by piperidine'*.

(ii) Cyclization of P-diketones:

R-CO-CH2-CO-R’ is P-diketone, in which R is o-hydroxy phenyl and R’ is alkyl, aryl, OH etc. 0-Methyl dibenzoyl methanes with hydrogen iodide undergo simultaneous demethylation and ring closure to give flavones.

a) Claisen condensations:

o-Hydroxy acetophenone and ethyl propionate give the diketone, which were cyclized by acetic acid and hydrochloric acids to 2-ethyIchromene'®. This condensation can be efTected in pyridine or xylene or by means o f sodium hydride’’.

b) Baker-V enkataranm an rearrangement:

In this method hydroxyl group o f o-hydroxy acetophenone are acylated with aromatic acid chloride in acetone-potassium carbonate or pyridine at oil bath temperature and the resulting

esters are converted into diketone with potassium hydroxide in pyridine or with sodium hydride'®. Finally, the ring closure is performed in ethanolic sulfuric acid in glaojal acetic acid and anhydrous sodium acetate or simply by heating the diketone in vacuum yielding a flavone'.

An o-aroyloxy acetophenone, formed by base catalysis is first cyclized to a compound o f the 2-hydroxyflavone, type, which then rearranges to o-hydroxy dibenzoyl methane derivative. Under acid catalysis, cyclization again results in a 2-hydroxy flavone. Song and Ahn'® reported the synthesis o f dibenzoyl methanes as intermediates for flavone synthesis by a modified Baker- Venkataraman rearrangement.

The aldol condensation of a-chloro-2-hydroxy acetophenone with aromatic aldehydes in aqueous alcohol containing 5-10% sodium hydroxide at room temperature yields the corresponding flavones'®“.

c) Kostanecki synthesis^”:

This is a general method for synthesizing flavones and consists in condensing the ester o f an alkylated salicylic acid with an acetophenone in the presence of sodium metal. For flavone this reaction is carried out with methyj o-methoxy benzoate and acetophenone.

d) Allan Robinson condensation^':

Flavones are obtained in one step by the condensation o f o-hydroxy acetophenones with the anhydride o f an aromatic acid in the presence of the salt of the same acid or in the presence of triethylamine or pyridine as catalyst at oil bath temperatures'. Side product such as o-aryloxy acetophenones and o-hydroxy dibenzoyl methanes are also formed in this synthesis and are not isolated.

To avoid side reactions the temperature o f the oil bath should not be much higher than that of the melt. If a partial demetliylation occurs during the Allan Robinson acylation it Is accompanied by a simultaneous ring isomerization^'".

In a variant o f these two methods, flavones that are partially or completely alkylated in the side phenyl can be prepared by melting together phloroglucinol and benxyloxy or methoxy benzyl acetic acid ethyl esters^l

Marder and co-workers^^ reported the solution phase combinatorial synthesis o f flavone derivatives and evaluated their affinity for the central benzodiazepine receptor. Am ong the synthesized compounds 6-bromo-3’-fluoroflavone, 6,3Ndlchloroflavpne, 6-brom o-3’- chloroflavone, and 6-chloro-3’-bromoflayone were found to possess high affinity benzodiazepine receptor ligands.

36

37

2.3 BIOLOGICAL ACTIONS

From the literature, it was reported that flavonolds are used in the treatment o f capillary fragility, retinal haemorrhage, hypertension, diabetic retinopathy, rheumatic fever, arthritis, radiation disease, frostbite, histamine and anaphylactic shock and in experimental cancer.

Flavonoids structurally resemble nucleosides, isoalloxazine and folic acid, and these form the basis of many current hypotheses for their physiological action. The structure and features o f flavonoids considered to be o f importance in biological functions are:

(i) presence o f an extended conjugated resonating system with a carbonylchromphore

(ii) presence of aromatic hydroxyl groups and(iii) their molecule shape.

Presence of a conjugated resonating system in the flavonoids is responsible for the presence o f important jjigments in the plants. The aromatic hydroxyl groups lead to the ability o f flavonoids are to interact with certain enzyme systems where as the molecular shape, particularly in isoflavonoids is responsible for physiological activities, due to their similarity in the structure to the animal hormones.

a) Anti-inflammatory activity:

A pharmaceutibal composition consisting of neomycin and a topical anti-inflammatory flavonoid in a suitable carrier has been patented for the treatment o f acne^‘‘. Another patent was taken un xanthorhamin as anti-inflammatory agent, isolated from seeds of Rhammis infecotria and recommended for use in ophthalmology, particularly for topical treatment o f collyria and in rheumatoid conditions. The flavonoids from Astragallus had a significant anti-exudative effect on experimental model^®.

Kim et al ®, have reported that 5,7-dihydroxy flavonols having hydroxyl groups in B ring and a isoflavone showed broad inhibitory activities (14-52%) against croton oil or arachidonic acid induced ear edema by oral or topical administration'*. The anti-inflammatory activity and its mechanism witli respect to flavonoids have been studied by Ferrandiz et al^’ and reported that Inhibition o f arachidonic acid metabolism was one o f the mechanism by which flavonoids exert their anti-inflammatory effects.

Flavone and its 2’-,4 ’-,3’- and 6’-hydroxy derivatives have also been reported"® to produce dose related anti-inflammatory effects in both acute and ehrpnie models o f inflamriiation

38

in rats and the study indicates that hydroxylation favors the anti-inflammatory activity o f the flavone nucleus more than does methoxylation. Anti-inflammatory activity o f flavonoids were studied by Panthong e t al“ who reported that structural features necessary fo r activity are the presence o f methoxy groups at Cs and C7 of the flavonoid molecule. Hsin-kaw and co-workers^° studied inhibitory effects of 2 ’- and 3’ - hydroxy chalcones and 2,5’ - dihydroxy chalcpnes on the activation o f mast cells and neutrophile.

Flavonoids affect the inflammatoiy process of the mammalian system by inhibiting nitric oxide production. Nitric oxide produced by inducible nitric oxide synthase is one o f the inflammatory mediators, the effects o f various naturally occurring flavonoids on nitric oxide production in LPS activated RAW 264.7 (macrophage cell line) cells were evaluated^’. Flavonoids such as apigenin, wogonin, luteolin, tectorigenin and quercetin inhibited nitric oxide production measured by nitrite formation. Among 26 flavonoids, apigenin, wogonin and luteolin was found IC50 values o f 23, 17 and 27 micro M respectively and AMT, a synthetic selective inducible nitric oxide synthase inhibitor was shown ICso value o f 0.09 micro M. From the study, Kim and Co-workers^' concluded that C-2,3 double bond was important and the potency o f inhibition was dependent on the substitution patterns of the flavonoid molecules and their inhibitory activity was due to reduction o f inducible nitric oxide synthase enzyme expression.

2 ’-Hydroxy-3,4-dimethoxy-3’,4’-dimetliyl chalcone and 2’-hydroxy-3’,4’,3,4-tetra methoxy chalcone and their corresponding flavones, 3’,4’-dimethoxy-7,8-dimethyl flavone and 3’,4’,7,8-tetra methoxy flavone were prepared^^ by reacting 3,4-dimethoxy cinnamic acid with the respective phenol and evaluated for their anti-inflammatory properties.

b) Anti-microbial activity;

The presence o f enone function in the molecule confers antibiotic property to the

chalcone. This property is enhanced by substitution at the a-(nitro and bromo) and (3-(bromo and hydroxyl) positions^^ Antibiotic activity is also associated with the C=C bond of the chalcone molecules. Addition o f cystein to chalcones hampers activity owing to reduction with SH group- -’.

Quercetin, among the naturally occurring flavonoids was found to completely inhibit the

growth o f S. aureus at concentration of 100 p.g/ml’'*. Fistenedin chloride at low concentrations, iinhMtedihe growth of S. albus, S. aureus, B. substilis and C, albicans.

; Several flavonoids were found to exhibit prophylactic action against fixed rabies virus in mice. O f these, quercetin and quercitrin showed significant and rutin promising activities^^ Flavones containg bromine at 3-position are reported to be antibacterial in nature^*’. Ward and co­workers” studied antimicrobial activities o f 3-methylene flavones.

39

The compound 2 ’-hydroxy chalcone sulfonic acid possesses weak antifungal activity whereas carboxy and sulfonic acid derivatives o f chalcone showed antifungal action^®. Some heterocyclic analogs o f chalcone like furan and 8-hydroxy quinoline type compounds have also been reported to exhibit antifungal activity^®. Substituted isoflavones, carrying a double bond in the oxygenated ring, possess antiviral activity higher than that of the corresponding isoflavones'*®.

Tuncbilek et al"', reported the synthesis o f 2-(4’-formyl phenyl)-4H,l-benzopyran-4-one o-substituted oxime and evaluated their invitro antibacterial and antifungal activities. 3- (Substituted phenacyl)-5-[4’-(4H,4-oxo-l-benzopyran-2-yl)-benzylidene]-2,4-thiazolidenedione derivatives were synthesized**^ by knovenagal reaction from 4’-f\avoTTie carboxaldehyde and 3- substituted phenacyl-2,4-thiazolidinedines and studied its invitro antimicrobial property.

Synthesis and physico-chemical properties of 3-substituted phenacyl thiazolidine-2,4- diones and 3-substituted phenacyl-5-[2-(phenyl-4H,4-oxo-1-benzopyran-6-yI)-methyl enyl]- thiazoiidine-2,4-diones were reported by Ayhan-Kilcigil et al'* by knovenagal reaction from flavone-6-carboxaldehyde and 3-(substituted phenacyl)-thiazolidine-2.4-diones. These compounds were also evaluated for their antibacterial and antifungal activities. Ferte and co- workers'*'' reported the synthesis of 28 flavonoid derivatives containing N-benzyl piperazine as a side chain and evaluated their ability to modulate multidrug resistance (MDR) invitro. Most o f the compounds potentiated doxorubicin cytotoxity on resistant K562/DOX cells at ’5|j,M and increased the intracellular accumulation o f JC-1, a fluorescent molecule recently described as a probe of /j-glycoprotein mediated MDR.

c) C ardiovascular activity:

The unsubstituted parent compound, flavone exerts coronary dilatory activity. Its combination with rutin and isoquercetin is useful in the treatment o f arteriosclerosis. Quercetin and rutin have been used as effective constituents for treatment o f capillary fragility and phlebosclerosis. Hesperidin and eriodyctiol were introduced as therapeutic agents to increase capillary resistance. Studies show an inverse correlation between dietary flavonoid intake and mortality from coronary heart disease, which is explained in part by the inhibition o f low density lipoprotein oxidation and reduced platelet aggregability'*^.

Anti-hypertensive activity of (3-phenyl-7-flavonoxy)-propanolamines has been studied"**

through depletion of myocardial norepinephrine rather than p-adrenoceptor inhibition although

tliese compounds are structurally similar to classical |3-adrenergic blocking agents. Removal o f the 3-phenyl group decreased the CNS side effects. Flavodilol was found to be most effective o f all compounds in the series o f 7-flavonoxypropanolamines'” :

40

New 1,4-diliydropyridine (1,4-DPH) flavonoid derivatives, were synthesized by Tuiicbilek and Ertan''® from 3 ’ or 4 ’-formyl flavone, followed by converting the aldehydes group to 1,4-DPH moiety by Hentzsch metliod and tested for their calcium channel blocker activity. The structure activity relationship of several flavonoids (flavonols, flavoiies, flavonones and catchins) were evaluated for their positive inotropic effect (PIE) and intrinsic activity on guinea pig papillary muscle'''^ Amongst the compounds tested, quercetin showed the m ost potent intrinsic activity and produces the strongest inotropic responses. The relative order of potency of tested flavonoids was found to be quercetin > morin = kaempferol = HEPTA > luteolin = apigenin > natsudaidain = fistein = galangin. 3-Hydroxy flavone, flavone, glycosides of quercetin ( rutin and hyperin), flavonones (naringenine) and catechines did not show any PIE. From the study, it was concluded that the presence of hydroxy group at C -4’, an alpha, beta unsaturated ketone on the C ring and a responsible lipophilic moiety in the molecule are required for the PIE''®.

Derivatives o f known lipid peroxidation inhibitors, probucol and butylated hydroxy toluene were synthesized and reported by Lewin et al^“ and evaluated their ability to inhibit the copper sulfate or endothelial cell induced lipid peroxidation o f human low. density lipoprotein

(LDL) invitro. Most of the flavones were active in the range of 0.1 - 1 /^M.

d) Diuretic activity:

The diuretic effect of myrcetin and kaempferol was observed in rabbits and the potency was increased with an increase in number o f hydroxyl groups^'. Flavone glycoside e.g., quercetin, rutin, kaempferol-3-rhamno glucoside and luteolin were also found to inhibit diuretic activity^^.

e) Anti-allergic activity:

Flavonoids are found to have anti-allergic activity which is associated with possible conversion o f flavones to 2’-hydroxy chalcones’"’. Flavonoid aglycones showed a stronger activity for histamine release inhibition than glycoside antigen induced histamine release from IgE-sensitized RBL-2H3 cells '*.

f) Anti-HIV activity and Hypoglycemic activity;

Flavonoids with hydroxy groups at C5 and Cq and with a C2 - Cj double bond were more potent inhibitors o f HIV growth and the presence of substituents (hydroxyl and halogen) in the B ring increased toxicity and/or decreased activity. Chrysin a flavone has been reported to be the most promising compound among a group of natural and synthetic flavonoids as inhibitors of HIV replication in H9 cell” .

41

A series o f new hydroxy benzoic acid and hydroxy cinnamic acid flavon-3-yl esters were synthesized and evaluated for their anti-human immunodeficiency virus (HIV) type-1 integrase (anti-IN) and anti-reverse transcriptase (RT) activity in enzyme assays and anti H IV -1 anti­proliferative and anti-topoisomerase activity in cell based assays^*. Among the synthesized compounds two gallic acid flavon-3-yl esters showed a notable IN inhibition (IC50 values were8.3 and 9.1 |iiM respectively) and two caffeic acid flavon-3-yl esters showed moderate IN

inhibition (IC50 values were 75 and 60 p-M respectively) in enzyme assay method. Replacement of hydroxyl groups resulted in loss of potency and caffeic acid 3’,4’-dichIoro flavon-3-yl ester also inhibited the RT activity but it was not active on human topoisomerses^®.

Hayashi and co-workers^^ isolated 5,6,7-trimethoxy flavone (TMF) from Callicapra japonica and subjected to anti-viral assays. It showed relatively high inhibitory effects on herpes simplex virus type I (HSV-1), human cytomegalovirus and poliovirus. TMF and acyclovir was found to be synergistic in their anti'-HSV activities.

Mishra et aP® have reported the correlation between the structures o f some flavonoids and hypoglycemic activity. Isorhamnetin-3-rhamnosyl galactoside isolated from Dodonea viscosa showed 15% blood lowering effect at 200 mg/kg (bw) in experimental model. Bozdag et al” reported the synthesis and hypoglycemic activity of flavonyl-3’-sulphonylurea derivatives by reacting several isocyanates with flavone-3-sulfphonamide. The compounds were tested for their insulinotropic activities in INS-1 cells and some of the compounds were found to increeise insulin release.

g) Anti-tumor activity;

Flavone-2’-carboxylic acid have been synthesized and evaluated for direct jnvitro toxicity towards four tumor cell lines and also for their ability to stimulate mouse peritoneal macrophages in culture to become tumoricidal (indirect toxicity)®. All the compounds have showed remarkable increase of indirect cytotoxicity, in particular, a compound with an F atom in the position 7 o f the flavone increased significantly the macrophageslytic properties.

Flavone-8-acetic acid substituted with 6-methyl, 2-phenyl and 2-heterocyclic group respectively have been synthesized and their anti-tumor activity was evaluated invitro against a panel o f human muraine tumor cell lines and invivo against MAC ISA^'’ ’’*' . Compounds with6-methyl substituent showed comparable invitro activity to flavone acetic acid, while 2- heterocylcic substituted compounds showed significant invivo activity. There was no clear-cut relationship between invitro and invivo activity in 2 phenyl substituted flavone acetic acid.

5,4’-Diamino flavone exhibited potent arid specific growth inhibitory activity against the estrogen receptor (ER) positive human breast cancer cell lines MCF-7. When 5,4’-diamiho flavone incubated with S-9 mix, its metabolites were observed and addition of Sr9 m ix to the

42

medium caused drastic decrease in activity. 6-, 8-, and 3’- derivatives o f 5,4’-diamino flavone were synthesized by Akama and co-workers*^ and studied for their antitumor activity. Among the synthesized compounds, 5,4’-diamino-6,8,3’-trifluro flavone showed strong growth inhibitor activity against MCF-7 cells ever in presence of S-9 mix and it completely suppressed tlie growth of MCF-7 inoculated in nude mice by oral administration, and the effect was more potent than 5,4’-diamino flavone. In addition to ER positive breast cancer cells, 5,4’-diamino flavone 6,8,3’- trifluro flavone was found to have growth inhibitory activity against the panel o f human cancer cell lines including a part o f ER negative breast, endometrial, ovarian and liver cancer.

New coumarin-, flavanoi- and flavonon-acetic acid derivatives were synthesized and evaluated for their cytotoxicity on human colon carcinoma cell line (LoVo) through evaluation o f neutral red uptake®^. All the synthesized compounds showed significant reduction o f lysosomal neutral red uptake at 5 X 10 (-5) M concentration.

Many other flavonoids are found active over a wide range o f enzymes. The inhibitory activities o f flavonoids on hyaluronidase®'’, histidine decarboxylase*’’’ xanthine oxidase and choline acetylase have been known since long. Recent studies have found that C4 substituted flavonoids stimulate the action of indole acetic acid^®. The stimulating effect increased considerably on addition o f C7 hydroxyl group. On contrary, an inhibition of enzyme activity occurred with flavonoids containing hydroxyl groups at C3 and C4™. Flavonoids were found to inhibit liver COMT invitro to various degrees. The glycosides being weaker inhibitors than corresponding genins’’. cAMP phosphodiesterase inhibitors have also been reported in flavonoids’ ’ Flavonoids demonstrate moderate to good Angiotensin I converting enzyme inhibitory activity in invitro tests’'*. The enzyme inhibitory activities of several flavonoids in experimental models have been studied on cytochrome P 450,’ aromatase,’® liver sialidase,’’ monoxygenase,’® phosphorylasekinase in rabbit muscles,™ rat hepatic arylhydrocarbon hydroxylase,®® and Kellis and Vickery®' reported inhibitory action o f flavone on human estrogen synthetase (aromatoase). Welton and co-workers®^ studied the effect o f flavonoids in arachidonic acid metabolism. Flavonoids were also found to have the best relative antioxidant efficiency®^ when tested along with coumarins and cinnamic acid when compared to that o f alpha tocopherol.®'*

43

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DISCUSSION

47

3.0 DISCUSSION

In the present studies quite a large number of new 2-methyI-3-substituted a!ylidenimino-6,8-dibromo-3,4-dihydroquinazolin-4-one derivatives, 2-(substituted phenyl)-3-(substituted aryIidenimino)-6,8-dibromo-l,2,3,4-tetrahydro quinazolin-4-one derivatives, 2’-amino penta bromo flavone and its intermediates and a few acetanilide derivatives were syntliesized by suitably exploiting 3,5-dibromoantIiranilic acid which was prepared by brominating anthranilic acid. The structures o f the newly synthesized compounds were established by 'HN M R and Mass spectra.

3.1 SYNTHESIS OF 2-METHYL-3-(SUBSTITUTED ARYLII>ENIMINO)-6,8- DIBROMO-3,4-DIHYDRO QUINAZOLIN-4-ONE DERIVATIVES AND SOME OTHER COMPOUNDS:

These compounds were synthesized form anthranilic acid which w as brominated to give the dibromo derivative. On refluxing witli acetic anhydride it gave the 2-methyl dibromo benzoxazinone. On treatment with hydrazine hydrate it gave the N-acetyl dibromo anthranil hydrazide derivative which on reacting witli appropriate aromatic aldehydes gave the corresponding atylidenimino derivatives. These compounds were evaluated for their biological actions. Relevant details including structure elucidation (based on NM R and Mass spectral date) are given below.

All the compounds showed very similar fragmentation pattern in their mass spectra, in which fragments arising from the dibromo anthranilic acid unit dominated. Fragments arising from the aryl ring corresponding to the aldehyde were very weak. Fragments are shown below. Wherever halogens like chlorine or bromine were present they appeared as isotopic clusters of peaks corresponding to the expected molecular weights.

48

Help was also taken o f '^CMR in establishing an ambiguity, which existed between the two possible structures of a compound (III and Ilia). It was difficult to resolve by 'H NM R alone. Details o f the work carried out are given below.

Synthesis o f 3,5-dibromo anthranilic acid (I):

A solution of bromine in glacial acetic acid was added to a hot solution o f anthranilic acid in glacial acetic acid in small portions with continuous shaking. The contents were cooled and a solid, which separated out, was filtered and washed with glacial aqetic acid. I t was boiled repeatedly with water five times and filtered each time while hot. The insoluble residue was crystallized from ethanol to give a crystalline TLC pure compound (I) m.p. 228- 3.0°. Its structure was confirmed on the basis o f 'H NMR, which showed the tw o protons H-

NH,------- ^gl. acetic acid L. II

Br '

(I)A and H-B located at 5 7.86 and 8 7.82 as two meta coupled doublets J = 2.8.

Synthesis o f 6,8-dibromo-2-methyl-3, l-benzoxazin-4-onei (II) from (I);

A mixture of (I) and acetic anhydride was refluxed and the excess of acetic anhydride was distilled off under reduced pressure. Clusters of colorless needles (II) deposited on cooling which were filtered and washed witli petroleum ether, iti.p. 174-76°. The compound was TLC pure. Its structure was established on the basis of ‘H N M R spectrum.

The NMR showed a singlet located at 5 2.5, which could arise from the methyl group. In the aromatic region there were located two meta coupled doublets at 5 8.14 and a t 8 8.24 which could arise from the proton H-B and H-A, These data are satisfactory for the cyclic structure II or an open chain structure Il-a assigned to the compound. Since an ambiguity was seen between structures U and l l-a , hence recourse was taken to ''^CMR specti-ometry.

49

Bi v , ^ S ^ O O H

lla

The ' ’C NMR spectrum o f the compound confinned the cyclic structure of the compound. The assignmehts have been shown in the following Table I;

Carbon atom no.

5 Value

C-2 157.618

C-4 129.453C-5 141.504*C-6 119.630

C-7 141.504

*C-8 120.196C-9 143.251

*C-10 121.807

c=o 161.694

* Values interchaiigableTable 1

A further information to the above structure was obtained by mass spectral data. The mass spectrum o f the compound showed the molecular ion peak located at m/z 317, analyzing for the molecular formula C9H5Br20jN. The other important peaks could be located at m/z 277 and m/z 249. The fragmentation patter is shown below:

m‘ 317m /z 277

- C O

m /z 249

50

Attempted synthesis of 6,8>dibromo-2-metIiyl-3-aiiiino-3,4-dibydro quinazolin-4-one (Ilia) from (II) which resulted in the formation of N-acetyl-3, 5-dibro'moanthranil hydrazide (III):

To a solution o f II in ethanol was added hydrazine hydrate and stirred for 2 hrs. A solid mass, which separated out, was filtered and crystallized from a mixture o f ethanol and diclilorometliane to give a colorless TLC pure crystalline compound m.p. 230-32°.

X X XBr

hydrazine hydrate

(II)

i^CONHNHj

NHCOCHj

The NMR spectrum o f the compound showed a singlet located at 5 2.75 arising from the CH3

group, the protons o f the NH'j group appeared as a broad singlet at 5 6.3. The two protons of

the dibromo benzene ring could be picked up as two meta coupled doublets located at 5 8.1 and 5 8.3. These data are in favor o f both the structure (III) or (Ilia).

However, the '^CMR data favored structure III only. The C-13 assignments have been given in the following Table II:

Carbon atom no.

5 Value

C-1 117.506C-2 142.950

C-3 122.471C-4 127.586C-5 121.809

C-6 138.747

C =0 156.862

C =0 158.198

t a b le l l

51

Synthesis of 3,S-dibromo>N'-acetyl-anthranilic acid (IV) from (II):

A solution o f II in sodium hydroxide (5%) was boiled for 2 minutes and acidified with con. HCl. A solid mass which separated out was filtered, washed with water, dried and crystallized from ethanol to give compound IV m.p. 214-16° which was TLC pure.

(Ill) (IV)

Its structure was confirmed on the basis o f '^C NMR spectrum. The assignments are shown in the following Table III.

Carbon atom no.

5 Value

C-1 118.560C-2 146.392*C-3 132.000C-4 131.804*C-5 134.754C-6 137.150

CONH 165.425COOH 168.605

* Values interchangableTable III

The above structure was further supported on the basis of mass spectral data! The mass spectrum of the compound showed the molecular ion peak located m/z 337, analysing for the molecular formula C9H 9BrjN302. The fragmentation pattern has been shown below;

52

Synthesis o f methyI-3, S-dibromo-N-acetyl an thranila te (V) from (TV):

To a solution o f IV in dry acetone was added dimetliylsulphate and anhydrous potassium carbonate and tlie contents refluxed for 4 hrs. The reaction mixfure was then filtered and the filtrate concentrated and poured onto crushed ice. A solid mass which separated out was filtered, washed with water, dried and crystallized from ethanol to give colorless crystalline compound V m.p. 136-38° which was TLC pure. Its structure was established on the basis o f NMR spectral data.

NHCOCH3

Bt ^O O H .Ir acetone^ A » K O O H . ■"“ W .ulptm*

Br Br(IV) (V)

The NMR spectrum of the compound showed a singlet at 6 2.14 and another singlet at 5 3.8 arising from the protons of the methyl groups o f N-acetyl function and ester function. The two protons namely H-A and H-B could be seen as two meta coupled doublets located at 5

7.88 and S 7.83 respectively. Tlie NH proton could be seen as a singlet located at 8 8.07. These data are satisfactory for the above structure o f the compound. A further proof to tlie above structure was obtained by mass spectral data. The mass spectrum o f the compound showed the molecular ion peak located at m/z 349 analyzing for the molecular formula CioH9Br2N 0 3 . The other important peaks were located at m/z 306, 274 and 195. The fragmentation pattern has been shown below:

V YCO O CH ,

^^^N H C O C H jBr

IW+ m/z 349

X bm /z 195

m /z 306

T KBr

m /z 274

Synthesis of N-acetyl-3,5-dibroinoanthranil hydrazide (HI) from (V):

To a solution of V in ethanol was added hydrazine hydrate and refluxed for 8 hrs. The reaction mixture was cooled to room temperatui-e and poured onto crushed ice; A solid mass, which separated out, was filtered, dried and crystallized from ethanol to give colorless crystals o f 111 m.p. 230-32°. It was TLC pure and compared with the product prepared from II

on page no. 50. Its structure was established on the basis of NMR spectrum.

= Y Y “ °“-^ .^ N H C O C H , ^^^N H C O C H g

Br . Br

(V) (III)The NMR spectrum o f the compound showed a singlet located at 6 2.75 arising from the CH3

group, the protons o f the NH2 group appeared as a broad singlet at 5 6.3. The two protons o f

the dibromo benzene ring could be picked up as two meta coupled doublets located at 5 8.1

and 5 8.3. These data are satisfactory for the structure assigned to the compound.

Attempted synthesis o f 2-metliyI-3- (benzyIidenimino)-6|3-dibromo-3,4-dihydro quinazolin-4-one (Via) which resulted in the formation of 2-(ben3^1idenimino)-4,6- dibromo-1-acetyI anthranil hydrazide (VI) from (III);

To a solution o f III in ethanol containing a few drops o f con. HCl was added benzaldehyde and the contents refluxed for 3 hrs. Excess o f solvent was then distilled off. On cooling a product, separated out, which was filtered and crystallized from ethanol to give a crystalline compound VI m.p. 208-10°, which was TLC pure and showed a yellowish green fluorescence under UV light. It was characterized on the basis of NM R spectral data.

53

(VI)The NMR spectrum o f the compound showed a singlet at 8 2.63 integrating for 3 protons,

which may be due to N-acetyl function. In the aromatic region tlie aldimine proton could be

located at 5 8.89, while the Uvo protons of the brominated ring appeared as two doublets at 5

8.06 and 5 8.2. The protons of the phenyl ring could be seen as two multiplets located at 6

7.52 and 5 7.8 integrating for 3 protons and 2 protons respectively. These data are

satisfactory for the above structure assigned to the compound.

A furtlier support to the above structure was obtained by mass spectral data, which showed the molecular ion peak located at m/z 419 analyzing for the molecular formula Ci6H||Br2N 3 0 . The other significant peaks were located at m/z 317 and m/z 77, which could be accounted for by the following ions A and B, th e fragmentation pattern is shoN\Ti below.

54

^tX^NHCOCHj

iwt 437Molacuiar Ion peak not observed

-HjO

■ ' O Q T ' ^ -

o(B)

m/Z 77

Br

m iz 419

^-"^N C O C H j

(A) m/Z 317

Attempted synthesis of 2-methyl-3- (3% 4’-dimethoxy benz\iideniinino)'6,8-dibroino-3,4- dihydro quinazolin-4-one (Vila) which resulted in the formation of 2-(3’,4’-dimethoxy benzylidenimino)-4,6-dibromo-l-acetyl anthranil hydrazide (VII) from (III);

A solution of III and veratraldehyde in ethanol containing two drops o f con. HCl was refluxed while maintaining reaction conditions and processing methods as described above. Finally a TLC pure compound VII m.p. 236-38° was obtained. Its structure was established on the basis of NMR spectral data. The NMR spectrum of the compound showed two singlets located at S 2,68 and 5 3.97, the former arising from the CH3 group of NHCOCH3 function and the latter could be due to the two methoxyl functions of the veratiyl ring, which suggested an open chain amide structure of the compound. In the aromatic region there could be seen an ortho coupled doublet located at 5 6.96, a double doublet at 5 7.33 and a meta coupled doublet at 5 7.57 which could be due to the protons H-A, H-B and H-C respectively. There were another two doublets located at 5 8.12 and 5 8.3 arising from the two protons of the dibromo anthranilic acid ring. Further there was a singlet located at 8 8.72 arising from the aldimine group. These data are in favor of the structure VII only, ruling out the possibility for structure Vila.

55

Synthesis of 2-methyI-3- (4’-methoxy benzylideniinino)-6,8-dibromo-3,4-dihydro quinazoIin-4-one (VIII) from (HI):

A solution o f III and jp-anisaldehyde in ethanol, containing a few drops of con. HCl was refluxed for 3 hrs. The reaction mixture was processed as mentioned above to give a .TLC pure compound VIII m.p. 248-50°.

P~anlsaldehyde “

‘•‘hanol/con . H C l*

Br

(III) (VIII)Its structure was confirmed on the basis of NMR data. The NMR spectrum o f the compound showed two singlets in the aliphatic region located at 5 2.6 and 5 3.9, which could arise from the CHa group and the methoxyl function. In the aromatic region there were two doublets forming an A2 B2 pattern located at 5 7.02 and 5 7.8, arising from the protons ortho to the OCH3 and meta to the OCH3 group of the p-substituted phenyl ring. There were two meta coupled doublets located at 5 8.37 and S 8.25 arising from H-A and H-B protons. There was located a singlet at 6 8.77 arising from the aldimine proton. Tliese data are in favor o f the proposed structure of the compound.

Attempted synthesis of 2-methyI-3- (4’-diethylamino benzylidenimino)-'6,8-dibromo-3,4- dihdyro quinazolin-4-one (IX) from (III):

4-Diethylamino benzaldehyde was condensed with III under reaction conditions as stated earlier. On usual processing, followed by crystallization from ethanol and dichloromethane a crystalline compound IX m.p. 136-38'’ was obtained, which was found to be a complex mixture and could not be purified. Hence further studies were abandoned.

^ X ^ N H C O C H j ethanol/Con. HCl

Br Br

(III) (IX)

Synthesis of 2-methyl-3- [(3’, 4’-methylenedioxy) benzylideniminol-6,8-dibromo-3,4- dihydro quinazolin-4-one (X) from (in):

A mixture of III, piperonal and ethanol was reifluxed for 3 hrs in presence o f con. HCl. The contents were processed as usual to give a TLC pure compound i^.p; 262-64°. It was characterized on the basis of NMR data,

56

Bs^,^^S.^CONHNH,piperonal

NHCOCHj « ‘*>®"“ '/ C o n . HCI

(III) (X)The NMR spectrum o f the compound showed a singlet integrating for three protons at 5 2.67 arising from the methyl group. There was another singlet located at 5 6.08, which could arise from the protons of the methylene dioxy group. In the aromatic region there could be seen a doublet centred at 5 6.91, a double doublet centred at S 7.24 and another doublet located at S7.52 arising from the proton H-5, H-6 and H-2 respectively. The protons o f the dibromo benzene ring appeared as doublets 5 8.1 and 5 8.3, while the aldimine proton could be located as a singlet at 5 8.77. These data are satisfactory for the structure assigned to the compound.

Synthesis o f 2-methyI-3-(4’-dimethyIainino benzylidenimino)-6,8'dibroino-3,4-dihydro quinazoIln4~one (XI) from (m ):

Compound III was condensed with 4-dimethylamino benzaldehyde and the reaction mixture processed as usual. It gave a TLC pure compound XI m.p. 244-46°. It was characterized on the basis o f NMR data.

0

U ^ N H C O C H , - ‘•’-O '/ C o n . HCI

(III) (XI)The NMR spectrum o f the compound showed two singlets integrating for 3 protons and 6

protons located at 5 2.7 and 5 3.09, which could be accounted for by the methyl group o f the heterocyclic ring and protons of the N-dimethyl amino group respectively. In the aromatic region there could be seen two doublets forming an A2B2 pattern and located at S 6.72 and 5 7.75, which accounted for the protons o f the para substituted phenyl ring. The protons o f the dibromo benzene ring appeared as two doublets located at 5 8.10 and 5 8.3, while the aldimine proton appeared as a singlet at 8 8.4. These data are satisfactory for the structure assigned to the above compound.

Synthesis of 2-methyl-3-(4’-chloro benzylideniminp)-6,8-dibromo-3,4-dihydro quinazolin-4-onc (XII) from (III):

On condensing III with 4-chloro benzaldehyde in the usuial \vay and work up o f the reaction mixture a T IX pure compound XIll m.p. 234-36° was obtained; Us structure was established on the basis o f NMR data.

57

Br B ,

(III) (XII)The NMR spectrum o f the compound showed two doublets forming an A2B2 pattern arising from the protons of the p-substituted phenyl ring. There were two meta coupled doublets located at 5 8.13 and 5 8.36 which could arise from the two protons of the dibromo benzene ring. The aldimine proton could be located as a singlet 5 8.99. These data are satisfactory for the above structure o f the compound.

Synthesis of 2-methyl-3-(2’,4’-dichloro benzylidenimino)-6,8-dibromo-3,4-dihydro quinazolin-4-one (XIII) from (III):

Compound III was condensed with 2,4-dichloro benzaldehyde and the reaction mixture worked up in the usual way to give a TLC pure compound (XIII) m.p. 232-34°, which was characterized on the basis of NMR data.

NHCOCH,

Br

ethanol / Con. HCI

(III) (XIII)

The NMR spectrum of the compound showed a singlet at 8 2.7arising from the protons of the CH3 group. The protons H-B and H-A of the dibromo benzpne ring appeared as two m eta coupled doublets located at 5 8.15 and 5 8.25. Tlie proton H-D appeared as an ortlio meta coupled doublet located at 6 7.45, while the proton H-E could be seen as a meta coupled doublet located at 5 7.5. The proton H-C appeared as an ortho coupled doublet located at 6

8.19 and the aldimine proton was seen as a singlet located at 5 9.4. These data are satisfactory for the structure assigned to the above compound.

Synthesis of 2-methyI-3- (3’,4’,5’-trimethoxy benzylideniniino)-6,8-dibromo-3,4-dihydro quinazolin-4-one (XIV) from (III). '

Compound HI and 3,4,5-trimethoxy benzaldehyde weris condensed together as per the procedure outlined above. Usual processing o f the resactipn mixture giave a compoMnd (XIV) m.p. 234-36°, which was TLC pure. It was characterized pn the basis of NMR data,

58

XX athanol/ Con. HCI NHCOCH3

Br Br

(III) (XIV)The NMR spectrum o f the compound showed a singlet located at 5 2.69 which could arise from the protons of the CH3 group. There were two closely spaced singlets located at S 3.94 integrating for 9 protons, which could arise from the three OCH3 groups. In the aromatic region there was a singlet located at 5 7.14, which accounted for the two protons o f the trimethoxy phenyl ring. The two protons of the dibromo ring appeared as two meta coupled doublets located at 5 8.13 and 5 8.5, while the aldimine proton could be seen as a singlet located at 5 8.78. These data are satisfactory for the above structure assigned to the compound.

Synthesis of 2-methyl-3- (3’-methoxy-4’-hydroxy benzyiidenimino)-6,8-dibroino-3,4- dihydro quinazolin-4-one (XV) from (HI).

A mixture o f III, 3-methoxy-4-hydroxy benzaldehyde, con. HCI and ethanol was refluxed for 3 hrs. The contents were processed as usual to give a TLC pure compound (XV) m.p. 256-58° (decomp) which was characterized on the basis of NMR spectral data.

n B A

B'V j . ^ ' n .Y^CONHNHj 3 -m e th o x |^ 4 -Jiydroxy

i L ________ ethanol / Con. HCI‘ N H C O C H 3

Br Br

(III) (XV)

The NMR spectrum o f the compound showed a doublet located at 5 6.3, a doublet at 5 7.3, and another doublet at 5 7.5 which could arise from the protons H-A, H-B, and H-C. The protons o f OCH3 group appeared as a singlet located at S 3.49, while the protons o f dibromo benzene ring could be seen as two meta coupled doublets located at 5 8.19 atad 5 8.23 and the aldimine proton appeared as a singlet located at 8 8 .6 6 . These data are in accord with tlie structure assigned to the above compound.

Synthes^ of 2-niethyl-3-(2’,6’-dichloro benzylidenimino)-6,8-dibromo-3,4-dihydro quinazolin-4-one (XVI) from (III).

To a solution of III in ethanol was added 2,6-dichlorpbenzaldeihyde alongwith few drops o f con. HCI and the contents refluxed for 3 hrs. The reaction mixture yyas processed as described above and crystallized from ethanol to give a crystalline compQund (XVI) m.p. 266-68°, which was found to be TLC pure. It was characterized by NMR speetral studies.

^xS^NHCOCHj

(HI)The NMR spectrum of the compound showed two protons of the dibromo benzene ring as two meta coupled doublets located at S S. 13 and 8 8.42, diere was a singlet at 5 2.74 integrating

for 3 protons and arising from the CHj group. The proton H-4 appeared as a triplet at 5 7.37

and the two protons H-3,5 could be located at 8 7.40 as a doublet, while the aldimine proton could be seen as a singlet at 8 9.7. These data are satisfactoiy for the structure assigned to the above compound.

Synthesis o f 2-methyI-3-(3’-ethoxy-4’-hydroxy beiizy]idenimino)-6,8-dibromo-3,4- dihydro quinazolin'4-one (XVII) from (III).

3-Ethoxy-4-hydroxy benzaldehyde and compound 111 was condensed as per the procedure described in other similar experiments. The reaction mixture was processed in the usual way to give a TLC pure compound (XVII) m.p. 218-20°. It was characterized on the

basis of NMR data.

590 Cly 3

Br

NHCOCHj

(III) (XVII)In the NMR spectrum of the compound the presence o f an aldimine proton at 8 8.66 as a singlet explained successful condensation. In the aliphatic region there was a triplet located at

8 1.58 and a quartet at 8 4.23 which could arise from the CH3 and CH2 groups of the ethoxy

function respectively. A singlet at 8 2.67 accounted for the methyl group. The two meta

coupled doublets located at 8 8.12 and 8 8.3 could arise from the protons o f the dibromo

benzene ring. The proton H-B appeared as an ortho coupled doublet located at 8 7.01 while

the proton H-A appeared as an ortho meta coupled doublet located at 8 7.33. The proton H-C

could be seen as a meta coupled doublet located at 8 7.53. These data are in complete accord with the proposed structure o f the above compound.

Synthesis of 2-methyl-3-(3’hydroxy benzylidenimino)-6^8-dibroino-3,4-dihydr6

quinazolin-4-onc (XVIII) from (III):

A mixture of HI, 3-hydroxy benzaldehyde, con,, HCl and ethanol was refluxed for 3 iirs. The reaction mixture was worked up as usual to give a TLC pure compound (XVIII) m.p. 246-48°. The NMR data supported the expected structure of the compound.

60

Br

(III)

Tlie NMR spectrum o f the compound showed a singlet located at 5 2.75 arising from the protons o f the methyl group attached to the heterocyclic ring system. The aldimine proton

could be picked up at 5 8.9, while the two meta coupled protons of the dibromo benzene ring appeared as two meta coupled doublets located at 5 8.12 and 8 8.34. The protons of the 3-

hydroxy phenyl ring appeared as a multiplet integrating for four protons centred at 5 7.31. Tliese data are satisfactory for the structure assigned to tlie above compound.

Synthesis of 2-methyl-3-(2’-hydroxy quinazolin-4-one (XIX) from (HI):

benzylidenimino)-6,8-dibromo-3,4-'dihydro

To a solution o f III in ethanol was added 2-hydroxy benzaldehyde and refluxed for 3 hrs in presence of con. HCl. On usual workup, a solid mass was obtained, which was crystallized from ethanol to give TLC pure compound (XIX) m.p. 242-44°, It was characterized on the basis of NMR data.

'N H CO CH ,

2 - hydroxy benaldehyda ^

ethanol / Con. HCl

(III) (XIX)

The NMR spectrum o f the compound showed a singlet located at 5 9.1 arising from the

aldimine proton, which inferred successful condensation. There was a singlet located at 6

10.6 arising from the phenolic hydroxyl group. The two protons of the dibromo benzene ring

appeared as two meta coupled doublets located at 8 8.1 and 8 8.3. The protonis of the salicyl

ring could be picked up as two doublets located at 8 7.08 and 8 7-38 arising from the protons, H-3’ and H-6 ' respectively, while the protons H-5’ and H-4’ could be seen as two triplets

located at 8 7.01 and 5 7.48. A singlet located at 8 2.7 integrating for 3 protons could arise from the CH3 group. All these data are in complete accord with the proposed structure.

61

Synthesis of 2-methyl-3-(thienyUdenimino)-6,8-dibromo-3,4-(lihydro quinazolin-4-one(XX) from (III):

Thiophen-2-aIdehyde was condensed with III and processed further as mentioned in earlier cases to give compound XX as TLC pure pink colored crystals m.p. 242-44'’. It was characterized on the basis o f NMR studies.

^ ^ ^ y ^ N H C O C H , ethanol/Con. HOI V

Br

(III)

The NMR spectrum o f the compound showed a singlet located at 6 2.7 arising from the CH3

group. In the aromatic region there could be seen two doublets located at 5 8.1 and 5 8.37 arising from the protons o f the dibromo benzene ring. A singlet located at 5 9.15 indicated successful formation of the expected compound, while the protons o f the thiophene ring appeared as a doublet located at S 7.19 arising from the proton H-4’ and the protons H-5’ and H-3’ gave as doublets located at 5 7.56 and 6 7.72, respectively.

Synthesis of 2-methyl-3-(a-methyIbenzyIideni mino)-6,8-dibromo>3,4-dihydroquinazolin-4-one (XXI) from (111):

A mixture of III and acetophenone was refluxed for 96 hrs and cooled to room temperature. It was poured onto crushed ice. A solid mass, which separated out, was filtered and crystallized from ethanol to give XXI as TLC pure colorless crystals m.p. 260-62°. The structure was confirmed on the basis o f NMR spectral data.

■ X X

CONHNH.acetophenone

A /1 2 0 “N H CO C H ) '

(III) (XXI)The NMR spectrum o f the compound showed two singlets located at 5 2.23 and 5 2.7 arising from the CHj groups o f the acetophenone moiety and the N-acetyl function. The two protons o f the dibromo benzene ring could be located at S 8.13 and 8 8.39 as two meta coupled doublets while the protons of the phenyl ring could be seen as a multiplct eind a doublet located at 6 7.5 and 5 8.02 arising from the protons H-3,4,5 atid H-2,6 ; These data are satisfactory for the structure assigned to the compound, The mass spectrum also gi:\'e support to the above structure.

62

Tlie mass spectrum of the compound showed the molecular ion peak at m/z 433 (though o f low intensity) analyzing for the molecular formula Ci7Hi3Br2N3 0 . Another significant peak could be picked up at m/z 314, Both the ions are shown below:

Synthesis of 2-methyI-3-(cyclohexylidenimino)-6,8-dibromo-3,4-dihydro qainazolin-4- one (XXII) from (HI):

A mixture o f III and cyclohexanone was refluxed for 96 hrs. The reaction mixture was processed as described above. A viscous mass so obtained was extracted with diethyl ether and dried over anhydrous sodium sulfate. After filtering off the inorganic salt, the solvent was evaporated off and the residue crystallized from petroleum ether to give TLC pure colorless fine crystals of compound (XXII) m.p. 142-44°. It was characterized on the basis of NMR data.

Bn,,_^IV^C0NHNH2

'NHC0CH3uCBr

cyclohexanone

A/120“

(III) (XXII)

The NMR spectrum of the compound showed a singlet for the methyl group o f the heterocyclic ring located at 8 2.66. The protons of the three methylenes o f the cyclohexane ring were located as a multiplet at 5 1.55, while the remaining protons of the two methylenes ortho to the double bond position appeared as a multiplet centred at 6 2.68. In the aromatic region the two meta coupled doublets at 8 8.1 and 5 8.4 may be accounted by the protons of the dibromo benzene ring. These data are satisfactory for the structure assigned to the compound.

Attempted synthesis of 2-methyl-3-(a-methyl-2’-hydroxy dibromo-3,4-diliydro quinazoIin-4-one (XXIII) from (III);

benzylidenimino)-6,8-

A mixture o f 111 and 2-hydroxy acetophenone was heated at 4 2 0 ° for 9 6 hrs. The reaction mixture was processed as usual to give a solid mass,which was crystallized from

63

ethanol to give a crystalline compound. Its TLC and m.p. 230-32° matched with III indicating that the desired compound XXIII could not be syntliesized.

o

‘ NHCOCH,XX,Br

(III)

2- hydroxy acotophenom

A/120®

(XXIII)

Attempted synthesis of 2-raethyl-3-(a-methyl-2’, 4’-dihydroxy benzylidenimino)-6,8- dibromo-3,4-dihydro quinazoIin-4-one (XXIV) from (III):

Compound III was heated with resacetophenone at 120° for 96 hrs. The reaction mixture was processed as usual to give a brown colored compound, which was found to be a complex mixture on TLC examination and could not be purified, Hence, ftirther studies were abandoned.

Br

resacetophenone

NHCOCH3 A /1 2 0 “

X OH

Br

(III) (XXIV)

Synthesis of 2 -( l’,2’,4’-triazolin-5’-thione-3’-yI)-4,6-dibromo acetanilide (XXV) from

(HI):

A mixture of III and ammonium thiocyanate was heated at 210° for 1 hr. On usual workup, a solid mass was obtained which was crystallized from a mixture o f ethanol and dichloromethane to give XXV as yellow crystals m.p. 270-72° (decomp) which was found to be TLC pure. NMR data confirmed the formation o f desired compound XXV.

B C s^^S s^C O N H N H j

^ y ^ N H C O C H j

Br

(111)

NH^SCN

A / 120“

(XXV)

The NMR spectrum o f the compound showed two meta coupled doublets located at 5 8.13

and 5 8,2 arising from the two protons of tlie dibromo benzene ring. T he protons of the N-

acetyl group appeared as a singlet located at 5 23, while NH proton could be seen as a singlet located at 5 12.6 these data are in accord with the structure assigned to the aboye compound.

64

A further proof to the above structure was obtained by mass spectral studies. The mass spectrum of the compound showed the molecular ion peak at m/z 390 analyzing the molecular formula CioHgBr20N4S. The fragment ion pattern has been shown below:

Synthesis of 2 -(l’,3’,4’-oxadiazolin-2’-thion-5’-yl)-4,6-dibrorao acetanilide (XXVI) from (III):

To a suspension of III in ethanolic potassium hydroxide was added carbondisulphide dropwise at room temperature with continuous stirring followed by refluxing for 4 hrs, The solvent was evaporated off and the residue neutralized with con. HCI. A solid mass, which separated was filtered and crystallized from a mixture of ethanol and dicliloromethane to give TLC pure colorless crystalline compound (XXVI) m.p. >300°. It was characterized on the basis of NMR spectral data.

s x .Br

(III)

►NHCOCH,

(XXVI)

The NMR spectrum of the compound showed a singlet located at 5 2.38, which could arise from the metliyl group o f N-acetyl function. The two protons of the dibromo benzene ring appeared as two meta coupled doublets located at 8 8.14 and 8 8.3, while the SH proton could be seen located at 5 12.61. These data are satisfactory for the structure assigned to the compound. A further proof to the above structure was obtained by the mass spectral data.

65

NHCOCH,

tw- m /Z 391

HCOCHj^ Q C „ hc„ck

m /Z 312

B r

m/Z 315

The mass spectrum of the compound showed the molecular ion peak analyzmg for C]oH7Br202N3S located at m/z 391. There could be picked up another peak at m/z 312 arising by the loss of one bromine mass unit. A peak at m/z 315 could be due to the cyanogen ion. These fragmentation have been shown above.

Synthesis of 2-(<?-nitro phenyI)-5-(2’-acetamido-3’,5’-dibromo phenyl)-!,2,4-oxadiazole (XXVII) from (HI);

A mixture of III, 2-nitrc benzoic acid and phosphorus oxychloride was refluxed for 5 hrs. On usual workup followed by basification with sodium bicarbonate solution, a solid mass separated out, which was crystallized from ethanol to give compound (XXVII) as brownish-orange colored amorphous powder m.p. 298-300°. It was TLC pure. It was characterized on the basis o f NMR data.

■ p cCONHNH,

NHCOCH,

Br

(III) (XXVII)

The NMR spectrum o f the compound showed a singlet located at 8 2.3, which could arise from the protons ofNHCOCH3 group. In the aromatic region there could be seen a multiple

at 5 7.85 arising from the proton H-A, there was another multiplet centred a t 5 8.12 arising

from the protons H-B and H-C. There was another multiplet located at 5 8.45 arising from H D. The two doublets located at S 8.26 and 5 8.47 could arise from the two protons o f th< dibromo ring. These data are satisfactory for the above structure assigned to the compound.

66

Synthesis of l-(2~acctami<!o-3,S'dibromo benzoyl)-3-inethyU3-hydroxypyrazolidin-5-one(XXVIII) from (III);

Compound III was refluxed with ethylacetoacetate at 120° for 16 hrs. On usual workup, followed by crystallization, a TLC pure compound (XXVIII) m.p. 216-18° was obtained. It was characterized on the basis o f NMR spectral studies.

J ll athyl acBtoa

NHCOCH] A /1 2 0 '

B r

(III) (XXVIII)

The NMR spectrum o f the compound indicated the formation o f pyrazolidinone derivative. It showed two singlets located at 8 2.39 and 8 2.57 each integrating for 3 protons arising from the methyl group attached to the pyrazolidinone ring and the methyl group of the N-acetyl

system. The protons H-A and H-B could be seen as two meta coupled doublets located at 5

8.35 and 5 8.15. The NH proton was located at 8 9.60, The protons of the -CHa group o f the

pyrazolidinone ring system appeared as two doublets centred at 5 3.71. These data are in complete agreement with the structure assigned to the compound.

Attempted synthesis o f 2-methyI-3-(carboethoxy methylainino)-6,8-dibromo-3,4'dihydro quinazolin'4-one (XXIX) from (III):

o

chloroethylacotate v >

^ H K c o c H .Br ■ Br

(111) (XXIX)Its synthesis was undertaken by the following four different routes, with unsuccessful

results.

M ethod-A :

To a solution o f III in dry pyridine was added anhydrous potassium carbonate and chloroethylacetate. The reaction mixture was refluxed for 24 hrs and processed. A solid mass so obtained was crystallized from ethanol to give a colorless crystalline compound

which was the unreacted hydrazide derivative only.

67

M ethod - B:

A mixture of III and chloroethylacetate was refluxed at of 120° for 12 hrs. On usual workup, the final products was found to be the hydrazide derivative only.

M ethod - C;

A mixture of III, sodium iodide and chloroethylacetate was heated at 120° for 8 hrs, however the end product was found to be the hydrazide derivative only.

M ethod - D;

To a hot mixture of III, sodium bicarbonate and water was added chlorothylacetate in small portions with continuous stirring during a period o f 15 min. Stirring was continued for another 8 hrs followed by refluxing for 6 hrs. The reaction mixture was processed, which gave the unreacted hydrazide only and hence further studies were abandoned.

Attempted synthesis of 2-methyl-3-[4’-(4”-methoxy phenyI)-3’-ch lo ro-r-azetid inon-2’- yl]-6,8-dibromo-3,4-dihydro quinazolin-4-one (XXX) from (VUI):

o ___ o_ ^ I* ji 'i-------v^n—

OCH, ,CICOCHjjCl/ N(CJHg)3dry 1,4 dloxan

H,

(VIII) (XXX)

Its synthesis was undertaicen by two methods A & B, however, the desired product could not be obtained.

M ethod - A;

To a solution o f Vlll in 1,4-dioxan was added, chiorolacetylchloride and triethylamine. The reaction mixture was kept overnight and then refluxed for 8 hrs. On usual workup, followed by crystallization from ethanol, a colorless crystalline compound m.p. 252* 54° obtained, was characterized as triethylamine hydrochloride on TLC examinationalongwith an authentic sample with and hence further studies were abandoned.

68

Method - B:

To a solution o f VIII in dry tetrahydrofuran, chlorolacelylchloride and sodium hydride was added and the contents refluxed for 6 hrs. The reaction mixture was left overnight and the solvent evaporated off to dryness. After cooling to room temperature, it was extracted with ether, followed by washing with NaHCOa until there was no effervescence. It was finally washed with water. The ethereal layer was dried over sodium sulfate and after filtering off the inorganic salt, ether was evaporated off to dryness, which gave the unreacted compound VIII, and hence further‘studies were not carried out.

Attem pted synthesis of 2-methyl-3-[4’-(3” ,4”-dimethoxy phenyI)-3’-chIoro-l’- azetidinon-2’-yl]-6,8-dibromo-3,4-dihydro quinazolin-4>one (XXXI) from (VH):

A mixture o f VII, dry 1,4-dioxan, chloroacetylchloride and triethylamine was refluxed for 4 hrs and thereafter poured into a large volume of petrol while hot, when a solid mass separated out which was filtered and crystallized from methanol to give a brown colored compound. It was found to be unreacted VII and hence further studies were abandoned.

OCHj

VII XXXI

Attem pted synthesis of 2-[3’,4’-dimethoxy styi7l)-3-(4”-methoxy benzylidenimino)-6,S- dibromo-3,4-dihydro quinazoIin-4-one (XXXII) from (VIII):

Its synthesis was i

69

Method — A:

A mixture o f VIII, verataldehyde, piperidine or triethylamine, and ethanol / 1,4- dioxan / dry tetrahydrofuran (as given in experimental details) respectively was refluxed for 6 hrs. On usual vi'orkup, a solid mass obtained was found to be the starting compound VIII.

Method - B:

A mixture of VIII, veratraldehyde, sodium hydride and dry tetrahydrofuran was refliixed for 4 hrs. On usual workup a residue was obtained, which was found to be a mixture on TLC examination and could not be purified inspite o f several attempts and hence further studies were not carried out.

70

3.2 SYNTHESIS OF 2-(SUBSTITUTED PHENYL)-3- (SUBSTITUTED ARYLIDENIMINO)- 6,8-DIBRMO-l,2,3,4- TETRAHYDRO QUINAZOLIN-4-ONE DERIVATIVES.

The compounds of this series were synthesized by suitably exploiting the dibromo anthanil hydrazide derivative, which was prepared form dibromo anthranilic acid via its methyl ester. Tlie hydrazide derivative on condensing with appropriate aromatic aldehydes gave the corresponding title compounds, which were duly characterized on the basis o f spectral studies (NMR and mass spectra). The mass spectra of all the compounds showed the molecular ion peaks corresponding to the molecular formula except a few. All the compounds showed two fragmentations from the molecular ion (i) corresponding to loss o f aryl moiety from C-2 (ii) corresponding to loss o f Ar-CH=N unit from the hydrazone moiety. These ions underwent further fragmentation like loss of CO, HCN, A r or CON. However, not all tlie above peaks were present in every compound. The fragmentation is shown below. In the fragments wherever halogen, like chlorine or bromine were present, they appeared as isotopic clusters corresponding to the expected intensities. These compounds were evaluated for tlieir biological actions.

/ Br \ CO> / - hcn

R " H C N

'

71

Synthesis o f methyl-3,5-dibromo anthranilate (XXXIII) from (I):

A mixture of I, anhydrous potassium carbonate, dry acetone and dimethylsulfate was refluxed for 4 hrs. The reaction mixture was cooled and, the inorganic salt filtered off. The filtrate was concentrated and poured onto crushed ice. A solid mass, which separated out, was filtered and crystallized from methanol to give a TLC pure colorless crystalline compound(XXXIII) m.p. 78-80'*, Its structure was established on the basis of NMR spectral data.

x cBr

OHK2CO3; dry acetone /,

dimethyl sulphate

Br

(I) (XXXIII)

In the NMR spectrum o f the compound, the two protons o f the dibromo benzene ring appeared as two doublets located at S 7.68 and 5 7.9, tlie protons o f the NH2 group appeared as a broad singlet at 5 6.3, while the protons o f CH3 group of the methyl ester moiety appeared as a singlet located at 5 3.9. These data are satisfactory for the proposed structure of the compound.

Synthesis o f 3,5-dibromo anthranil hydrazlde (XXXIV) from (XXXIII);

A mixture of XXXIII, hydrazine hydrate and ethanol was refluxed for 24 iirs and was cooled down to room temperature, A solid mass, which separated out, was filtered and crystallized from methanol to give TLC pure colorless crystal o f the desired compound (XXXIV) m.p. 190-92°. Its structure was established on the basis of NMR spectral data.

(XXXIII) (XXXIV)

The NMR spectrum of the compound showed two meta coupled doublets located at 5 7.7 and 68.1 arising from the two protons of the dibromo benzene ring. There were two more singlets at 86.52 and 8 9.5 which may be due to the NH and NHj protons. These data are satisfactory for the hydrazide structure of the compound.

72

To a solution o f XXXIV in etlianol containing catalytic amount of HCl was added p~ anisaldehyde and the contents refluxed for 3 hrs. The reaction mixture was cooled to room temperature. A solid mass, which separated out, was filtered and ciystallized from methanol to give a TLC pure crystalline compound (XXXV) m.p. 156-58°. Its structure was established on the basis of NMR data.

Synthesis of 2-(4’-methoxy phenyl)iJl-(4”-metlioxy bcnzylideniinino)-6^dibromo-l,2^,4-tetra hydro quinazolin-4-one (XXXV) from (XXXIV);

Br

CONHNHjanisaldehyde

.......'3ethanol/Con. HCl

OCH3

(XXXIV) (XXXV)The NMR spectrum o f the compound showed two singlets arising from the protons of the two methoxyl groups located at 5 3.77 and 5 3.83. In the aromatic region tlie two singlets located at 5 5.3 and S 6.29 could be due to CH and NH protons. The aldimine proton appeared as a singlet at 8 9.04, while the two aromatic protons of the dibromo ring appeared as two ortho coupled doublets at 8 7,6 and 5 8.03 and the protons o f the two /?-anisyl rings appeared as four doublets forming an A2B2 pattern and located at 8 6,86 and 8 6.90 as one set and at S 7.33 and 8 7.55, the other set. These data are satisfactory for the assigned structure.

A further support to the above structure was obtained by the mass spectral data. The mass spectrum o f the compound showed the molecular ion peak located at m/z 543. There was another peak located at m/z 408. Some other significant peaks were located at m/z 302 and 133. The fragmentation pattern has been shown below:

cS n

73

A mixture of XXXIV, ethyl vanillin, con. HCl and ethanol was refluxed, for 3 hrs. The reaction mixture was processed as usual to give TLC pure crystalline compound (XXXVI) m.p. 118-20°. Its structure was established on the basis o f NMR spectral data.

Synthesis of 2-(3’-ethoxy-4’-liydroxy pIienyl)-3-(3”-ethoxy-4”-hydroxy benzylidenimino) -6,8-dibromo>l^,3,4-tetrahydro quinazoUn-4-one (XXXVI) from (XXXIV):

X C .Br

(XXXIV) (XXXVI)The NMR spJectrum of the compound showed the presence of two methyl groups and two CH2

groups arising from tlie two ethoxy functions located at 6 1.42 and S 4.07 respectively. Tlie protons H-5”, 2’, 5’, 6’ appeared as a multiplet centred at 6 6.97 while the proton H-6” could be seen as a double doublet located at 8 7.06 and the proton H-2” appeared as meta coupled doublet located at 5 7.19. The protons H-B and H-A appeared as two meta coupled doublets located at 5

7.6 and 5 8.02. The aldimine proton was located as a singlet at 5 8.9. The lone CH proton o f the heterocyclic ring system could be picked up at 6 5.29 while the NH proton was located at 5 6.2. These data are satisfactory for the structure assigned to the compound. This structure was further supported by the mass spectral data.

Tlie mass spectrum of the compound showed the molecular ion peak located at m/z 603. The other diagnostic peaks could be picked up at m/z 292, 166 and 137. The fragmentation pattern has been shown below.

+

74

A mixture o f XXXIV, veratraldehyde and ethanol was refliixed in presence o f con. HCl. The reaction conditions and processing methods were kept the same as described earlier to give a crystalline compound (XXXVII) m.p. 176-78° which was found to be TLC pure. Its structure was confirmed on the basis o f NMR spectral data.

Synthesis of 2-(3’,4’-dimethoxy phenyl)-3-(3”,4”,-dimethoxy benzylidenimino)-6,8-dibronio-1,2,3,4-tetrahydro quinazoIin-4-one (XXXVII) from (XXXIV);

■ «

CONHNH,

(XXXIV)The NMR spectrum of the compound showed two multiplets located at 5 3.85 and 5 3.9 integrating for 12 protons, which could arise from 4 X OCH3 groups. The lone CH protons o f the heterocyclic ring gave a signal at 5 5.34 while NH proton gave a signal, at S 6.2. The proton H-B

and H-A appeared as two doublets at 5 7.62 and 5 8.02, while the aldimine proton could be seen as a singlet located at 5 9.02. The protons of the two veratryl rings gave signals as multipiet located at 5 6.8, 6.9, 7.08, 7.42 and 7.62 which could arise from the protons, H-5”, 5’; H-2” or 2 '; H-6” or 6’; H-6’ or 6” and H-2’ or 2”. These data are satisfactory for the structure assigned to the compound. This structure was further confirmed by the mass spectra! data.

The mass spectrum of the compound showed the molecular ion peak at m/z 605 analysing for the molecular formula C25H23Br205N3. It showed the following fragmentation pattern.

75

Attempted synthesis of 2-(4’-diethylamino phenyI)-3-(4”-diethyIamino benzylidenimino)-6,8-dibromo-i;Z,3,4-tetrahydro qainazolin-4-ane (XXXVIIl) from (XXXIV):

To a solution of XXXIV in ethanol was added 4-diethyIamino benzaldehyde and con.HCl. The reaction mixture was refluxed for 3 hrs and processed as described in earliei* cases. Asolid mass so obtained was crystallized to give a TLC pure compound m.p. 236-38°, which on thebasis of NMR spectral data was found to be the starting compound XXXIV itself.

0

■Br

p-dlethylamlne Br< benzaldehydo

I

(XXXIV) (XXXVIII)

Attempted synthesis of 2-(4’-dimethyIamino phenyl)-3-(4”-dimethylamino benzylidenimino)-6,8-dibromo-l^,3,4-tetrahydro quinazolin-4-one (XXXIXa) which resulted in the formation of 2-(4’-dimethylaniino benzylidenimino)-l'(4”-dimethylamino benzylidenimino)-4,6-dibromo anthranil hydrazide (XXXIX) from (XXXTV):

To a solution o f XXXIV in ethanol was added 4-dimethylamino benzaldehyde and catylic amount o f con. HCl. The reaction conditions and procs^ssing method were the same as described in earlier cases. UV fluorescent and TLC pure fluffy crystals m.p. 244-46°so obtained were characterized on the basis o f NMR spectral data as (XXXIXa)

0CONHNH, /^dlmethylamine

benzaldehyde

Br

(XXXIV)

(XXXIX)

The NMR spectrum of the compound showed a singlet located at 5 3.08, arising from the - 4 X NCH.i groups. There were two doublets located at 8 6.7 and 6 7.7 forming an A 2B2 pattern, which

could arise from the two /^'substituted phenyl rings. The two doublets located at 5 8.14 and 5 8.44 could arise from the protons H-B and H-A while the proton of the CONH group appeared as

76

a singlet at 6 8.42 and the two aldimine protons also appeared as a singlet at 8 9.03. These data favoured the open chain structure of the compound as shown above.

Synthesis o f 2-(2’-thienyl)-3-(2’-thienylidenimino)'6,S-dibroino-l ^ ,4-tetrahydro quinazolin-4-one (XL) from (XXXIV):

A mixture of XXXIV, thiophen-2-aldehyde and ethanol, containing a few drops o f con. HCl was refluxed for 3 hrs. Usual workup of the reaction mixture followed by crystallization from ethanol gave TLC pure colorless crystalline compound (XL) m.p. 180-82°. Its structure was established by NMR studies.

o

thiophen- z - aldehydo

ethanol f Con. HCI

(XXXIV) (XL)

The NMR spectrum o f the compound showed a singlet at 5 9.67, which could arise from the

aldimine proton o f the condensed product. The two doublets at 8 5.51 and 5 6.49 could arise from the N H and CH protons. There were located multiplets arising from the protons of the two

thiophene rings; 5 6.89 (H-4 or H-4’); 5 7.03 (H-4' or H-4); 5 7.08 (H-5 or H-5’); 5 7.20 (H-5’ or

H-5); 6 7.36 (H-3 or H-3’); 5 7.42 (H-3’ or H-3). Tlie two meta coupled doublets located at 5

7.70 and 5 7.99 could arise from the two protons o f the dibromo benzene rjng. These data are satisfactory for the structure assigned to the compound. The mass spectral data further supported the structure assigned.

The mass spectrum of the compound showed the molecular ion peak located at ni/z 497. The two other significant peaks could be picked up at m/z 457 and 440. These fragments are shown below;

77

Synthesis of 2-(4’-chloro plicnyI)-3-(4”-chIoro tetrahydro quinazoIin-4-one (XLI) from (XXXIV):

benzyIideniinmo)-6,8-dibromo-l,2^,4-

To a solution o f XXXIV in ethanol was added p-cliloro benzaldehyde and con. HCl in catali'tic amount. The contents were then refluxed for 3 hrs. On cooling, a solid separated out, which was filtered and crystallized from methanol to give a crystalline compound (XLI) m.p. 178-80°. It was found to be TLC pure. Its structure was confirmed by NMR studies.

-N H ,

P“C*’*°'~°b8n2aldehytle ethanol/ Con. HCl

(XXXIV) (XLI)The NMR spectrum of the compound showed two signals at 6 5.37 and 8 6.31 arising from the CH proton o f the heterocyclic ring and NH proton. There were 3 doublets forming an A2B2

system located at 6 7.3 and one more doublet forming an A2B2 system located at 5 7..5S arising from the two para substituted phenyl rings. There were two doublets located at 8 7.6 and 5 8.01, which could arise from the protons H-B and H-A. There was a singlet located at 5 9.2 arising from the aldimine proton. These data are satisfactory for the structure assigned to the compound. A further support to the above structure was obtained by the mass spectral studies. The mass spectrum o f the compound showed the molecular ion peak located at m/z 551. The other important peaks and fragmentation patterns are given below.

78

To a solution of XXXIV in etlianol was added piperanol and con. HCl. The reaction mixture was refliixed for 3 hrs. On usual workup, a solid mass was obtained which was crystallized from ethanol to give a TLC pure crystalline compound (XLII) m.p. 208-10°. Its structure was confirmed on the basis of NMR data.

Synthesis of 2-[(3 ’,4’-methylenedioxy)-phenylI-3-[(3”,4”-methylenedioxy)-benzyIi(!en-imino] -6,8-dibromo-i;2,34-tetrahydro quinazolin-4-one (XLII) from (XXXIV):

vc*Br

(XXXIV) (XLII)

The NMR spectrum of the compound showed two singlets located at 8 5.28 and 5 6.22, which could arise from NH and CH groupings of heterocyclic ring. The protons o f the two-methylene

dioxy groups showed two singlets at 8 5.94 and 8 5.99. The aldimine proton was located as a

singlet at 8 9.06. The two protons of the dibromo benzene ring showed two meta coupled

doublets located at 6 7.67 and 5 8.02. The protons of one methylene dioxy ring namely H-2”, H-

5”, and H-6” gave signals at 5 7.24 (d), 8 6.78 (d) and 8 7.04 (dd) and the protons o f the other

methylene dioxy ring gave signals at 8 6.73 (d) H-5’, 5 6.88 (d) H-2’ and 8 6.88 (dd) H-6’. These data are in conformity with the above structure assigned to tlie compound.

Synthesis o f 2-(3’-methoxy-4’-hydroxy phenyl)-3-(3”-methoxy-4”-hydroxy benzyliden- imino)-6,8-dibromo-l,2,3,4-tetrahdyro quinazolin-4-one (XLIII) from (XXXIV):

A mixture o f XXXIV, vanillin and ethanol was refluxed for 3 hrs and worked up further to give a TLC pure crystalline compound XLIII m.p. 216-18°. Its structure was confirmed on the basis o f NMR data.

A B

r i

Br

(XXXIV)The NMR spectrum of the compound showed a singlet located at 8 8.9 arising from the aldimine

proton. There could be seen two singlets located at 8 3,7 and 8 3.8 each integrating for 3 protons, which could arise from the Uvo OCH3 groups. There were two meta coupled doublets located at 6

79

7.9 and 8 8.4 arising from the two protons o f the dibromo benzene ring, while the CH and NH

protons o f the heterocyclic ring gave signals at 5 6.30 and 6 6.70. The two protons H-B appeared as two ortho coupled doublets located at 6 6.73 and S 6.86, while the two protons H-A appeared

-as two doublets located at 5 6.79 and 6 7.07 and the two protons H-C appeared as two doublets located at 6 7.01 and 5 7.3 which satisfactorily explained the above structure assigned to the compound. These data were supported by the mass spectral studies also.

The mass spectrum of the compound showed the molecular ion peak located at m/z 575 analysing for the molecular formula m/z C23Hi9Br2N305. The other important ions could be picked up a t m/z 440, 412,278 and 151. The fragmentation pattern has been given below.

Synthesis o f 2-(3’,4’,5’-trimethoxy phenyl)-3-(3”,4”,5”-trimethoxy benzylldenimino)-6,8- dibromo-1,2,3,4-tetrahdyro quinazolin-4-one(XLIV) from (XXXIV):

A mixture of XXXIV, 3,4,5-trimethoxy benzaldehyde in ethanolic solution containing a few drops of con. HCl and ethanol was refluxed for 3 hrs. The reaction conditions and processing methods were the same as described above to give a TLC pure crystalline compound XLIV m.p.118-20°. Us structure was established on the basis of NMR spectral studies.

. ■ o>CONHNH, 3, 4,5- trfmethoxy

benzaldehyde

ethanol/ con. HCl

OCH.

(XXXIV) (XLIV)

80

The NMR spectrum o f the compound showed two singlets located at 8 3.72 and 6 3.74 integrating for 3 and 6 protons arising from the three methoxyl groups, Tliere were another two singlets integrating in the same ratio at 5 3.82 and 8 3.86 which could arise from the other set o f three methoxyl groups. There were two singlets located at 8 6.70 and 8 7.02, which could arise from the two separate pairs of the protons of tlie trimethoxy phenyl ring. H-2 of the heterocyclic ring appeared as a doublet at 6 6.36 and NH proton also appeared as a doublet at 8 7.38. The two protons of the dibromo benzene ring appeared as two meta coupled doublets at 8 7.66 and 8 7.89. There was a singlet located at 8 9.11, which could arise from the aldimine proton. These data are satisfactory for the assigned structure.

Synthesis of 2-(phenyI)-3-(benzylidenimino)-6,8-dibromo-l,2^,4-tetrahydro quinazolin-4- one (XLV) from (XXXIV);

To a solution of XXXIV and benzaldehyde in ethanol were added few drops o f con. HCI. The reaction mixture was refluxed for 3 hrs. On usual workup, a solid mass was obtained, which was crystallized from ethanol to give TLC pure crystalline compound XLV m.p. 168-70°. The structure v/as confirmed on the basis of NMR data.

CONHNH.

NH,

(XXXIV) (XLV)The NMR spectrum of the compound showed two singlets located at 8 5.41 and 8 6.36 arising from the NH and CH protons o f the heterocyclic ring. The aldimine proton could be seen as a

singlet located at 8 9.27, while the protons H-B and H-A gave two meta coupled doublets located

at 8 7.63 and 5 8.03. The two protons H-2”, 6” gave an ortho meta coupled double doublet

located at 8 7.66, while rest o f the protons o f the two phenyl rings gave a multlplet centered at 8

7.35. The aldimine proton could be seen as a singlet located at 8 9.27. These data are satisfactory for the above structure assigned to the compound. This structure was supported by the mass

spectral studies also.

The mass spectrinii of the cornpound showed the molecular ion peak located at m/z 483, which analysed for the molecular formula C2|Hi5Br2N.iO. It showed two characteristics peaks located at m/z 379 and m/z 3 i 7. The fragments are shown below;

81

Attempted synthesis o f 2-(2’-hydroxy pIienyl)-3-(2”-liydroxy benzylidenimiiio)-6,8- dibromo-l,2,3,4-tetrahydro quinazoIin-4-one (XLVIa) which resulted in the formation of an uncyclized compound (XLVI):

To a solution of XXXIV and o-hydroxy benzaldehyde in ethanol were added a few drops o f con. HCl The reaction mixture was refluxed for 3 hrs. On usual workup and crystallization from ethanol a TLC pure crystalline compound XLVI m.p. 228-30° was obtained. Its structure was established on the basis o f NMR data.

The NMR spectrum of the compound showed a singlet located at 8 8.74 arising from the aldimine

proton. There was another singlet located at 5 11.2 which could possibly arise from the proton of the second aldimine group suggesting an open chain structure. The two protons o f the dibromo ring appeared as two meta coupled doublets located at 5 7.88 and 8 7.97. The rest o f the protons

i appeared as a multiplet located between 5 6.67 and 8 7.33. These data are satisfactory for the above structure assigned to the compound.

82

Synthesis of 3,4-dihydro-4-oxo-6,8-dibromo-l,2,3-benzotriazine (XLVII) from (XXXIII):

To an ice cold solution of XXXIII in a mixture o f con. HCI and water was added dropwise and with stirring a cold solution o f sodium nitrite to give the diazonium salt solution, which was then made alkaline with concentrated ammonium hydroxide while stirring the contents. A brown colored solid mass separated out, which was filtered, washed with water and ciystallized from petroleum ether to give XLVII m.p. 188-90°. It was found to be TLC pure. The proposed structure was confirmed on the basis of NMR spectral data.

GO O CH ,NH4OH ; NaN02

Con. HCI

Br Br

(XXXIII) (XLVII) .Tlie NMR spectrum of the compound showed two meta coupled doublets located at 6 8.33 and 68.35 which could arise from the two protons H-B and H-A. The proton o f the CONH group gave rise to a broad singlet located at 5 11.76. These data are satisfactory for the above structure of the compound. A further supported to this structure was obtained by the mass spectral studies.

The mass spectrum of the compound showed the molecular ion peak located at m/z 303, analysing for the molecular formula' C7H3Br20N,i. The fragmentation pattern has been shown below.

+

> Y i i

Br

m /Z 232

- B r

x >+

m/z 153

83

3.3 SYNTHESIS OF FLAVONE DERIVATIVES

In order to prepare the desired flavones from o-hydroxy acetophenones, (XLIX) and (LI), their synthesis has been described in the following paragraphs. Synthesis of o-nitro benzoic acid (L) has also been described. (XLIX) and (LI) were condensed with (IV) to synthesize their corresponding esters, however, no progress could be made in this direction and hence o-nitro benzoic acid was condensed with o-hydroxy acetophenone to give the corresponding ester which by Baker-venkatraman reaction was converted into the corresponding beta diketone which was cyclized to give the 2’-nitro flavone. On reduction o f the nitro group, the corresponding amino derivative was obtained, which on bromination gave the 2 ’-amino penta bromo flavone. The stregy followed is discussed below.

Synthesis o f 2,4-dihydroxy acetophenone (XLVIII) from resorcinol:

Freshly fused zinc chloride was dissolved in glacial acetic acid with the aid o f heat and resorcinol was added in portions. The reaction mixture was maintained at a temperature of 150° for a period of 20 minutes and then kept at room temperature for 30 minutes, diluted with a mixture o f concentrated hydrochloric acid and water, cooled to 0-5°. An orange colored solid separated which was filtered, washed with ice cold dilute hydrochloric acid and crystallized from concentrated hydrochloric acid to give TLC pure orange crystalline compound XLVIII m.p. 144-46°.

(XLVIII)Synthesis of 2-hydroxy-4-methoxy acetophenone (XLIX) from (XLVIII):

To a solution of XLVIII in dry acetone was added dimethyl sulfate and anhydrous potassium carbonate and refluxed for 8 hrs. After completion o f the reaction, the inorganic salt was filtered off and the filtrate was concentrated, cooled to room temperature and poured onto crushed ice. A solid mass which separated was filtered, washed with water and crystallized from petrol to give TLC pure light green colored crystalline compound XLIX

m.p. 50-52°.

dryacBtone/anhyd. ^dimethyl sulphate

(XLVlli) (XLIX)

84

Synthesis o f o -nitro benzoic acid (L) from o - nitro toluene:

As o-nitro benzoic acid was not available, hence it was synthesized form o-nitro toluene. To a warm solution o f potassium permanganate in water was added o-nitro toluene and refluxed for 3 hrs. The reaction mixture was then filtered while hot. To the filtrate was added a pinch of sodium bisulfate and concentrated hydrochloric acid to remove die pink color. The reaction mixture was poured onto a mixture of concentrated hydrochloric acid and water. A fine colorless crystalline compound L m.p. 146-48° separated out which was filtered. It was TLC pure. (Lit. m.p. 148“)

CO OH

KMNO.

Con. HCl

N O ,

(L)

Synthesis o f 4-methyl-5-hydroxy-6-acetyIcoumarin (LI) from (XLVIII):

To a solution o f aluminum chloride in nitro benzene was added XLVIII and ethylacetoacetate and the contents refluxed for 3 hrs at a temperature of 120-140®. Nitro benzene was then removed by steain distillation. Dilute hydrochloric acid (10 %) was added to the residue. A solid mass which separated out was filtered and crystallized from ethanol to give brownish-green TLC pure ciystalline compound LI m.p. 164-66°.

Attempted synthesis of 2-acetyI phenyl-(2-acetamido-3,5-dibromo)-benzoate (LII) from

(IV):To an ice cold solution o f IV and o-hydroxy acetophenone in dry pyridine was added

phosphorus oxychloride and the contents stirred for 5 hrs. The reaction mixture was poured onto crushed ice. A solid ma,ss so obtained was filtered, washed with water, sodium bicarbonate solution and finally with water. It was crystallized form petrol to give a colorless crystalline compound m.p. 176-78% which was TLC pure. It did not give any color with

85

The NMR spectrum of tiie compound showed a singlet located at 5 2.5, which could arise from the methyl group. In the aromatic region there were located two meta coupled doublets at 5 8.14 and at 5 8.24 arising from the protons H-B and H-A respectively. These data are satisfactory for the structure II only, which was further confirmed by TLC and m.p. There was no evidence for the expected structure LII and hence further preparation o f p-diketone and the corresponding flavone was not carried out.

Attempted synthesis of 2-acetyi-4-methoxy phenyl-(2-acetamido-3,5-dibromo)-benzoate (LIII) from (IV):

To a stirred ice cold solution o f IV in dry pyridine was added a solution o f XLIX in dry pyridine. Phosphorous oxychloride was added to the above reaction mixture dropwise while stirring the contents for 5 hrs. On usual work up, a solid mass was obtained, which was crystallized to give TLC pure compound m.p. 172-74°. It was characterized as II. There was no evidence for the fomiation o f LIII and hence further studies to prepare the corresponding p-diketone and corresponding flavone derivative were abandoned.

HjCO

(XLIX) (IV) (LIII)

Attempted synthesis o f l-(4-methyl-6-acetylcoumarin)-N-acetyI-3,5-dibromo anthranilate (LIV) from (IV):

To an ice cold solution o f IV in di^ pyridine was added v/ith stirring a solution o f LI in dry pyridine containing phosphorous oxychloride. After usual work up, a solid mass was obtained, which was found to be a mixture on TLC examination and it could not be purified.

Hence further studies to prepare corresponding ester and p-diketone were abandoned.

(LI) (IV) (LIV)

86

Synthesis of (2’-acctyI phenyl)-2-nitro benzoate (LV) from (L);

To a stirred ice cold solution of L in dry pyridine was added o-hydroxy acetophenone. Phosphorous oxychloride was then added to the above reaction mixture dropwise and the contents stirred for 5 hrs. The reaction mixture was then poured onto crushed ice, a solid mass so obtained was filtered, washed with water followed by sodium bicarbonate solution washing and water washing again. It was crystallized from methanol to give a crystalline compound LV m.p. 126-28°, which was TLC pure. It did not give any color with alcoholic ferric chloride and was characterized on the basis of NM R data.

o c -COOH

O'(L)

The NMR spectrum of the compound showed an acetyl function as a singlet located a t 6 2.58. In the aromatic region there were multiplets centred at 5 7.40 and 6 8.1 each integrating for two protons, which could be accounted for by the protons H- A, C and H- E, D. An ortho meta coupled double doublet located at 5 7.86 could arise from the proton H- H, while three multiplets each integrating from one proton and centred at 5 7.60, 7.66 and 7.8 could be accounted for by the protons and H-G. These data are satisfactory for the abovestructure assigned to the compound as an ester.

Synthesis o f l- (2 ’-nitrophenyl)-3-(2”-hydroxy phenyl)-propan-l,3-dione (LVI) from

(LV):

To a solution of LV in dry pyridine was added a powder o f potassium hydroxide pellets and shaken mechanically for 4 hrs with occasional wanning. The reaction mixture was then diluted with water and poured onto a mixture o f concentrated hydrochloric acid and water. A light yellow solid mass separated which was filtered and recrystallized from methanol to give TLC pure crystalline compound LVI m.p. 160-62°. It gave a violet color with alcoholic ferric chloride and was characterized on the basis of NM R spectral data.

87

The NMR spectrum of the compound showed a singlet located at 5 6.5 which could arise from the olefmic proton o f the enol form. There were two triplets located at 5 6.90 and 7.48 arising from the protons H-C and H-B, while the protons H-A and H-D’ appeared as two double doublets located at 5 6.99 and 7.94. The rest o f tlie protons namely H-D, H -A ’, B ’, C ’ appeared as a complex multiplet centred at S 7.68. • These data are in accord with the proposed structure.

Synthesis o f 2-nitro flavone (LVII) from (LVI):

M ethod A:

A mixture o f LVI, freshly fused sodium acetate and glacial acetic acid was refluxed on an oil bath at a temperature o f 150-160° for 4 hrs. After refluxing, the reaction m ixture was cooled to room temperature and poured onto crushed ice. A solid mass so obtained was filtered and crystallized from petrol and acetone mixture to give a brown colored fluffy compound LVII m.p. 184-86° which was TLC pure. It did not show any color with alcoholic ferric chloride and gave a pink color with Mg/HCl.

M ethod B:

A solution o f LVI in 4% ethanolic sulfuric acid was refluxed for 24 hrs. The reaction mixture was then cooled to room temperature and poured onto crushed ice. A solid mass which separated out was filtered and crystallized from ethanol and purified by column chromatography to give a TLC pure compound LVII m.p. 184-86“. It gave a pink color with Mg/HCl and did not show any color with alcoholic ferric chloride. It was characterized on the basis o f NMR spectral data.

4 % ethanolic H g S O ^^ ^

6

5

(LVI) (LVII)1 ne inMR spectrum o f the compound showed a singlet located at 5 6.58, v/hich could arise

from the proton H-3. The protons H-6 and H-8 appeared as two muhiplets located at 5 7.35

and 5 7.4, while the protons H-3’ and H-6’ could be seen as two ortho meta coupled doublets

located at 5 8.07 and 8 8.2. The rest of the protons H-5, H-7, H-4’, and H-5’ could be picked

up as a complex multiplet located at 5 7.70. These data confidently explain that this

compound is 2’-nitroflavone.

88

Synthesis of 2 ’-amino flavone (LVIII) from (LVII):

The nitro group o f (LVII) was reduced to NH2 group as per the experimental procedure outlined in the "Text book o f Practical Organic Chemisdy" by A I Vogel to give a yellow colored crystalline compound LVIII m.p. 150-52°, which was TLC pure. It was characterized on the basis o f N M R studies.

The NM R spectrum of the compound showed a singlet located at 6 6.66 arising from H-3

characteristic o f a flavone derivative, A doublet located at 5 6.78 could arise Irom the proton

H-8, while a triplet at 6 6.85 could be accounted for by the proton H-6. The proton H-3’

appeared as a double doublet located at 8 7.30, while the protons H-7, 5’, 6 ’ appeared as a

multiplet centred at 5 7.47. The protons H-4’ and H-5 appeared as a doublet of triplet and

double doublet located at 6 7.69 and 5 8.23. These data satisfactorily explained the structure assigned to the above compound as 2’- amino flavone.

Synthesis of 2 ’-N-acetyl flavone (LIX) from (LV m ):

Acetic anhydride was added to a solution of LVIII in dry pyridine and heated for 2 hrs on a water bath. The reaction mixture was cooled to room temperature and poured onto crushed ice. A solid mass which separated was filtered and washed with water and crystallized form methanol to give a colorless crystalline TLC pure compound LIX m.p. 172- 74°. Its structure was confirmed on the basis of NMR data.

a ce tjca n h yd rld ^^

dry pyridineNHCOCH..

(LVIII) (LIX)

I h i ; NMR spectrum of the compound showed a singlet located at 5 6.55 arising from H-3.

The proton H-6 appeared as a doublet at S 7.30, while the protons H-3’ and H-8 could be seen

as a multiplet centred at 5 7.46. The other protons H-7, 5, 6’ also appeared as a multiplet

ccntred at 5 7.64. The proton H-4’ could bee seen as a triplet located at 5 7.74 and the proton

M - 5 appeared as a doublet at 5 8.20, These data are satisfactory for the structure assigned to

the compound as 2 ’-N-acetyl flavone, ■

89

Attempted synthesis o f 2 ’'am ino-3’,S’-dibronioflavonc (LXa) which resulted in the formation o f 2’-amino«3% 5% 3 ,6 ,8-penta bromo flavone (LX) from (LVIII):

A solution o f bromine in glacial acetic acid was added to an ice cold solution of LVIII in glacial acetic acid in small portions with stirring for 30 minutes. The contents were poured onto crushed ice and a solid mass, which separated out was filtered and washed tlioroughly with water. It was recrystallized form ethanol to give reddish brown amorphous powder m.p. 180-82°, which was TLC pure. Its structure as (LX) was established on the

Br

The NMR spectrum o f the compound could be interpreted for 2-amino-3\ 5’, 3, 6, 8 - penta bromo flavone based on the following evidences. There was a broad singlet located at 5 4.04 arising from the protons o f the NH2 group. The protons H-4’ and H-6’ appeared as two meta coupled doublets located at 5 7.7 and 5 8.0, while the two meta coupled doublets located at 5 7.46 and 5 8.47 could be due to tlie two protons H-7 and H-5 respectively. These data are strictly in accord with the proposed structure of the compound.

EXPERIMENTAL

4.0 EXPERIMENTAL

The reagents/chemicals/solvents used during the course of these studies were obtained from Merck (India), SD Fine and CDH Laboratories and were of the laboratory grade. The solvents were purified by distillation before their use.

The solvent systems used for Thin Layer Chromatography were:

Irrigant ‘a’: Toluene: Ethylacetate: Formic acid (5:4:1)Irrigant‘b’: Benzene: Ethanol (9:1)Irrigant ‘o’: Benzene; Ethanol (8:2)Irrigant ‘d’: Benzene (I ml: Ethanol 2 drops)Irrigant ‘e’: Bei^ene: Ethylacetate (9:1)Irrigant T : Benzene; Ethanol (1:1)Irrigant ‘g’: BenzeneIrrigant ‘h’: Petrol: Toluene; Ethylacetate (10:5;4)

Which have been accordingly mentioned at appropriate places. Silica Gel G used for Thin Layer Chromatography was o f CDH brand. Iodine chamber and UV lamps were used for visualization o f TLC spots. Whatmann filter paper (No.l, England) was used for filtration (vacuum or ordinary). " '

The C'^ and H ' NMR spectra were recorded either on 300 MHz or 400 MHz instruments. The Mass spectra were recorded on Jeol JMS-D 300 instrument. Melting points o f all the compounds were recorded in liquid paraffin bath in open capillary tubes and are uncorrected.

4.1 SY N TH ESIS O F 2-M ETH YL-3- (SUBSTITUTED A R Y L ID E N IM IN O )- 6,8-D IBR O M O -3,4-D IH Y D R O Q UINAZO LIN-4-ONE D E R IV A T IV E S A N D

SOM E O T H E R CO M PO UNDS;

Synthesis of 3,5-dibromo anthranilic acid (1):

A solution o f bromine (0.375 mole s 20 ml) in glacial acetic acid (15 ml) was added in small portions to a hot solution of anthranilic acid (0.124 mole; 17 gm) in glacial acetic acid (250 ml) with continuous shaking. The reaction mixture was cooled and a solid mass that separated out was filtered washed with glacial acetic acid and purified by boiling with water (500 ml) and filtering. This process was repeated five times to remove 5-bromo anthranilic acid and the insoluble mass was crystallized from ethanol to give a brown colored crystalline compound (I) (Yield 75%) m.p. 228-30° (Lit m.p. 232°) which was found to be

TLC pure (irrigant 'e').Spectral studies:NMR : Refer page 48.

90

3,5-Dibromo anthranilic acid (I; 0.03 mole; 8.85 gms) was refluxed for 20 minutes witli acetic anhydride (O.I mole; 10.3 gins s 9.5 ml). Excess acetic anhydride was distilled off under reduced pressure. Upon cooling the solution clusters o f colorless needles deposited, which were filtered, washed with petroleum ether and dried m.p. 174-76° (Lit m.p. 176®) (Yield 83%). It was found to be pure on TLC examination (irrigant 'g').Spectral studies:NMR : Refer page 48'^CNMR ; Refer page 49Mass : 317 (M^), m/z 277, 249

Attempted synthesis o f 6,8-dibromo-2-methyl-3-amino-3,4-dihydroquinazoIin-4-one (nia) from (II) which resulted in the formation of N-acetyl-3, 5-'dibromo anthranil hydrazide (III):

A solution o f II (0.1 mole: 31.9 gms) in absolute ethanol and hydrazine hydrate (0.12 mole; 6 gms s 5.8 ml) was stirred in cold conditions for a period of two hours. A solid mass which separated out, was filtered and Ctystallized from ethanol and dichlorometliane mixture (8:2) to give colorless fluffy ciystalline compound III which was TLC pure (irrigant 'b') m.p. 230-32° (Yield 86%). . .Spectral studies:NMR : Refer page 50'^CNMR : Refer page 50

Synthesis of 3,5-dibromo-N-acetyl anthranilic acid (TV) from (II):

Compound II (0.006 mole; 2 gms) was boiled for 2 minutes in dilute sodium hydroxide solution (5%; 5 ml). It was acidified with con. HGl while hot. A product so obtained was filtered, washed with water and dried. It was crystallized from ethanol to give TLC pure colorless crystalline compound IV m.p. 214-16“ (Yield 65%).Spectral studies:NMR : Refer page 51'^CNMR : Refer page 51Mass : 337 (MO, m/z 295, 293, 275, 240,258.

Synthesis o f methyl-3, S-dibromo -N-acetyl anthranilate (V) from (IV):

A mixture o f IV (0.1 mole; 22.7 gms), dimethyl sulfate (0.2 mole; 25.2 gms s 19 ml), anhydrous potassium carbonate (3 gms) arid dry acetone (25 ml) was refluxed for 4 hrs. The reaction mixture was cooled and filtered to remove potassium carbonate. The filtrate was concentrated, cooled and poured onto crushed ice, A solid mass, which separated was filtered, washed with water and crystallized from ethanol to give TLC pure (irrigant 'a') colorless crystalline compound (V)m.p. 136-38° (Yield 64%).

91

Synthesis of 6)8-dibromo-2*‘inethyl-3, l-bcnzoxazin-4-onc (II) from (I);

Spectral studies:NMR : Refer page 52Mass : 349 (M*), m /z 3 06,274, 195.

Synthesis of N-acetyl-3,S-dibronioanthranil hydrazide (HI) from (V);

To a solution of V (0.1 mole; 35.1 gms) in ethanol (25 ml) was added hydrazine hydrate (0.12 mole; 6 gms s 5.8 ml). The reaction mixture was refluxed for 8 hrs, cooled to room temperature and poured onto crushed ice. A product, which separated was filtered and washed with water and air-dried. It was crystallized from ethanol to give colorless crystalline fluffy compound III m.p. 230-32° (Yield 75%) which was TLC pure (irrigant 'b'),Spectral studies:NMR : Refer page 52

Synthesis o f 2-methyl-3-(substituted arylideniniino)-6,8-dibromo-3,4-dihydroquinazolin-4-ones from (ni):

General procedure:

A mixture of III and substituted benzaldehyde was dissolved in ethanol and refluxed for 3 hrs in presence o f con. .HCl (2-3 drops). Excess o f solvent was distilled o f f and the contents cooled to room temperature. A product, which separated out, was filtered and crystallized from suitable solvent to give TLC pure crystals.

Following the above procedure, the compounds mentioned below were synthesized. Experimental details regarding each compound have been given.

Attempted synthesis o f 2-methyl-3- (benzylidfinimino)-6,8-dibromo-3,4-dihydro quinazoIin-4-one (Via) which resulted in the formation of 2-(benzyIidenimino)-4,6- dibromo-1-acetyl anthranil hydrazide (VI):

92

Compound III 0.01 mole ; 3.5 gmsBenzaldehyde C-.Ol mole ; 1.06 gms = 1.02 mlEthanol 20 mlCon. HCl 2-3 dropsReaction time (reflu'xing) 3 hrsCrystallization solvent EthanolShape/nature o f the compound Colorless crystalline compoundMelting point 208-10°Percentage yield 82%TLC behavior Single spot in irrigant ‘b’ showed

flouorescene under UV light.Spectral studies:NMR Refer page 53Mass 437 (M^ molecular ion peak not observed),

m /z4 1 9 ,3 l7 ,7 7

93

Attempted synthesis of 2-methyl-3- (3% 4’-dimethoxy benzylidenimino)-6,8-dibromo-3,4- dihydro quinazoIin-4-one (V ila) which resulted in the formation of 2-(3’,4’-dimethoxy benzylidenimido)-4,6-dibromo-l- acetyl anthranil hydrazide (VII);

Compound III3,4-Dimethoxy benzaldehydeEthanolCon. HCIReaction time (refluxing) Crystallization solvent Shape/nature o f the compound Melting point Percentage yield TLC behavior

Spectral studies;NMR

0.01 mole ; 3.5 gms 0.01 mole ; 1.6 gms 20 ml 2-3 drops 3 hrsEthanol and dichloromethane mixture (8:2) Colorless crystalline compound 236-38°74%Single spot in irrigant'd'

Refer page 54

Synthesis o f 2-methyl-3- (4’-methoxy benzylidenimino)-6,8-dibronio-3,4>dihydro quinazolin-4-one (VIII):

Compound III 4-Methoxy benzaldehyde Ethanol Con. HCIReaction time (refluxing) Crystallization solvent Shape/nature o f the compound Melting point Percentage yield TLC behavior

Spectral studies:NMR

0.01 mole ; 3.5 gms0.01 mole ; 1.36 gms = 1.2 mi20 ml2-3 drops3 hrsEthanol and dichloromethane mixture (8:2) Colorless crystalline compound 248-50“78%Single spot in irrigant'd'

Refer page 55

Attempted synthesis o f 2-methyl-3- (4’-diethylamino benzylidenimino)-6,8-dibromo-3,4- dihydro quinazolin-4-one (IX) :

Compound III4-DiethyIamino benzaldehydeEthanolCon. HCIReaction time (refluxing) Crystallization solvent Shape/nature o f the compound Melting point Percentage yield TLC'behavior

0.01 mole ; 3.5 gms 0.01 mole ; 1.77 gms 20 ml 2-3 drops 3 hrsEthanol and dichloromethane mixture (8:2) Yellow colored crystalline compound 136-38° .84%Complex mixture in irrigant'd ' which could not be purified. Hence further studies were abandoned.

94

Synthesis of 2-methyI-3- [3% 4’-(methylenedioxy) benzylidenimino]-6,8-dlibromo-3,4-dihydroquina2olin-4-onc (X);

Compound III Piperanol Ethanol Con. HCIReaction time (refluxing) Crystallization solvent Shape/nature o f the compound Melting point Percentage yield TLC behavior

Spectral studies:NMR

0.01 m ole; 3.5 gms 0.01 m ole; 1.5 gms 20 ml 2-3 drops 3 hfsEtlianol and dichloromethane mixture (8:2) Colorless crystalline compound 262-64°80%Single spot in irrigant'd'

Refer page 56

Synthesis of 2- methyl-3- (4’-dimethylamino benzyIidenimino)-6,8-dibromo-3,4-dihydro quinazolin-4-one (XI):

Compound III4-Dimethylamino benzaldehydeEthanolCon. HCJReaction time (refluxing) Crystallization solvent Shape/nature o f the compound Melting point Percentage yield TLC behavior

0.01 mole ; 3.5 gms 0.01 mole ; 1.5 gms 20 ml 2-3 drops 3 hrsEthanol and dichloromethar.e mixture (8:2) Yellow colored needle shape crystals 244-46°80%Single spot in irrigant'd'

Spectral studies: NMR Refer page 56

Synthesis o f 2-m ethyl-3-(4’-chIoro quinazoIin-4-one (XII):

benzylidenim ino)-6,8-dibrom o-3,4-dihydro

Compound III 4-Chloro benzajdehyde Ethanol Con. HCIReaction time (refluxing) Crystallization solvent Shape/nature o f the compound Melting point Percentage yield TLC behavior

0.01 mole ; 3.5 gms 0.01 mole ; 1.4 gms 20 ml 2-3 drops 3 hrs EthanolColorless crystalline compound 234-36“82%Single spot in irrigant'd'

Spectral studies;NMR Refer page 57

95

Synthesis of 2-methyl-3- (2% 4’-dichioro benzylidenimiiio)-63-dibronio-3,4-dihydroquinazolin-4-one (XIII):

Compound III2,4-Dichloro benzaldehyde Ethanol Con. HClReaction time (refluxing) Crystallization solvent Shape/nature o f the compound Melting point Percentage yield TLC behavior

Spectral studies:NMR

0.01 mole ; 3.5 gms0.01 mole ; 1.75 gms20 ml2-3 drops3hrsEthanolColorless crystalline compound 232-34°72%Single spot in irrigant'd'

Refer page 57

Synthesis o f 2-methyl-3-(3’,4’,5’-trimethoxy benzyUdenimino)-6,8-dibromo-3,4-dihydro quinazoiin-4-one (XIV):

Compound III3,4,5-Trimethoxy benzaldehydeEthanolCon. HClReaction time (refluxing) Crystallization solvent Shape/nature o f tlie compound Melting point Percentage yield TLC behavior

0.01 mole ; 3.5 gms 0.01 m o le ; 2.0 gms 20 ml ■2-3 drops 3 hrs EthanolColorless crystalline compound 234-36°68%Single spot in irrigant'd'

Spectral studies: NMR Refer page 58

Synthesis o f 2-methyl-3- (3’-methoxy-4’-hydroxy benzylldenimino)-6,8-dibromo-3,4- dihydroquinazoIin-4-one (XV):

Compound III3-Methoxy-4-hydroxy benzaldehydeEthanolCon. HClReaction time (refluxing) Crystallization solvent Shape/nature o f the compound Melting poin t,Percentage yield TLC behavior

0.01 m o le ; 3.5 gms 0.01 mole ; 1.5 gms 20 ml 2-3 drops 3 hrs EthanolColorless crystalline compound 256-58° (Decomp)75%Single spot in irrigant'd'

Spcctral studies:NMR Refer page 58

96

Synthesis of 2~tnethyI-3- (2% 6’-dichloro benzylidenimino)-6,8-dibromo-3,4>dihydroquinazolin-4-onc (XVI):

Compound III 2,6-Dichlor benzaldehyde Ethanol Con. HClReaction time (refluxing) Crystallization solvent Shape/nature o f the compound Melting point Percentage yield TLC behavior

Spectral studies:NMR

0.01 m ole ; 3.5 gms0.01 mole ; 1 .7 gms20 ml2-3 drops3hrsEtlianolColorless crystalline compound 266-68°73%Single spot in irrigant'd'

Refer page 59

Synthesis o f 2-methyl-3- (3’-ethoxy-4’-hydroxy dihydroquinazolin-4-one (XVII);

benzylideniinino)-6,8-dibrmo-3,4-

Compomid III3-Ethoxy-4-hydroxy benzaldehydeEthanolCon. HClReaction time (refluxing) Crystallization solvent Shape/nature o f the compound Melting point Percentage yield TLC behavior

0.01 mole ; 3.5 gms 0.01 mole ; 1.6 gms 20 ml2-3 drops 3 hrs EthanolYellow colored amorphous powder 218-20°68%Single spot in irrigant'd'

Spectral studies:NMR Refer page 59

Synthesis o f 2-metiiyl-3- quinazoIin-4-one (XVIII):

(3’-hydroxy benzylidenimino)-6,8-dibromo-3,4-dihydro

Compound III3-Hydroxy benzaldehyde Ethanol Con. HClReaction time (refluxing) Crystallization solvent Shape/nature o f the compound Melting point Percentage yield TLC behavior

0.01 mole ; 3.5 gms 0.01 mole ; 1.2 gms 20 ml 2-3 drops 3 hrs EthanolColorless crystalline compound 246-48°70%Single spot in irrigant'd'

Spcctral studies:NMR Refer page 60

97

Compound III 2-Hydroxy benzaldehyde Ethanol Con. HClReaction time (refluxing) Crystallization solvent Shape/natare o f the compound Melting point Percentage yield TLC behavior

Spectral studies:NMR

Synthesis of 2-methy!-3-quinazolin-4-one (XIX):

(2’-hydroxy ben2ylidenimino)-6,8-dibromo-3,4-dihydro

0.01 mole ; 3.5 gms0.01 mole ; 1.2 gms = 0.8 ml20 ml2-3 drops3 hrsEthanolColorless crystalline compound 242-44'*68%Single spot in irrigant'd'

Refer page 60

Synthesis of 2-methyl-3- (thienylideniniino)-6,8-dibromo-3,4-dihydroqiunazolin-4-one (XX):

Compound III3-Hydroxy benzaldehyde Ethanol Con. HClReaction time (refluxing) Crystallization solvent Shape/nature o f the compound Melting point Percentage yield TLC behavior

Spectral studies:NMR

0.01 mole ; 3.5 gms0.01 m ole; 1.12 gms s 0.9 ml20 ml2-3 drops3 hrsEthanolB uff colored needle sliape crystals 242-44“76%Single spot in irrigant'd'

Refer page 61

Synthesis o f 2-niethyI-3- (a-substltuted quinazolin-4-one (XXI - XXIV) from (HI):

arylidenimino)-6,8-dibromo-3,4-dihydro

General Procedure:

A mixture o f 111 and substituted ketones were refluxed for 96 hrs and cooled to room temperature. Then reaction mixture was poured onto crushed ice. A solid separated w-as filtered and washed with water and recrystallized. In case where liquid was separated, it was extracted with diethyl ether three times, the combined ethereal layers were separated, washed with water, dried over anhydrous sodium sulfate. After filtering off the inorganic salt, ether was evaporated to dryness and the residue was dissolved in petroleum ether and filtered. The filtrate upon concentration and leaving at room temperature gave a crystalline compoimd, which was found to be pure on TLC examination.

98

Synthesis of 2>methyl-3-quinazoliu-4-one (XXI):

(a-methyl benzyIidenimino)-6,8-dibronio-3,4‘-dihydro

Compound III Acetophenone Reaction time (refluxing ) Crystallization solvent Shape/nature o f the compound Melting point Percentage yield TLC behavior

Spectral studies:NMRMass

0.001 mole ; 0.350 gms 0.025 m ole ; 3 gms s 3 ml 96 hrs at 120°EthanolColorless crystalline compound 260-62°63%Single spot in irrigant 'b'

Refer page 61 433 (M^), m/z 414

Synthesis of 2-methyl-3- (cyclohexyIidenimino)-6,8-dibromo-3,4- one (XXD):

Compound III Cyclohexanone Reaction time (refluxing) Diethyl etherAnhydrous sodium sulfale Ci7 stallization solvent Shape/nature o f the compound Melting point Percentage yield TLC behavior

Spectral studies:NMR

0.001 mole ; 0.350 gms 0.028 mole ; 2.82 gms = 3 ml 96 hrs at 120°20 ml1 gmPetroleum etherColorless fine needle shape cr\'stal 142-44“58%Single spot in irrigant 'a'

Refer page 62

Attempted synthesis o f 2-methyI-3-(a-methyl-2’-hydroxy benzylidenim ino)-6 ,8-dibromio -3,4-dihydroquinazoIin-4*one (XXIII):

Compound III2-Hydroxy acetophenone Reaction time (refluxing ) Crystallization solvent Shape/nature o f the compound Melting point Percentage yield TLC behavior

0.001 mole ; 0.350 gms 0.025 mole ; 3.39 gms = 3 ml 96 hrs at 120°EthanolColorless crystalline compound 230-32^54% ■Single spot matched with that o f the starting hydrazide in irrigant 'b'

A ttem pted synthesis of 2 -metIiyi-3-(a-methyI-2 ’,4 ’-dihydroxy benzyIidenim ino)-6 ,8 - dibrom o-3,4-dihydroquinazoIin-4-onc(XXIV):

Compound III2,4-Dihydroxy acetophenone Reaction time (refluxing)

0.001 mole ; 0.350 gms 0.002 mole ; 0.300 gms 96 hrs at 120°

Crystallization solvent EthanolShape/nature o f the compound Brown colored amorphous powderMelting pointPercentage yield 58%TLC behavior Cotnpiex mixture in irrigant ’a’, which could

not be purified. Hence, further study to characterize the compound was not carried out. .

Synthesis of 2-(l%2%4’-triazoIin-S’-thion-3’-yl)-4,6-dibromo acetanilide (XXV) from (IID:

A mixture o f III (0.003 mole; 1 gm) and ammonium thiocyanate (2 gm) was heated on an oil bath maintaining the temperature between 200-10° for an hour. The reaction mixture was cooled to room temperature and the solid mass was triturated with water (25 ml). The suspended solid mass was filtered and ctystallized from ethanol and dichloromethane mixture (7:3) to give a green colored TLC (irrigant 'a') pure ciystalline compound XXV m.p. 270-72° (decomp) (yield 70.2%).Spectral studies;NMR : Refer page 63Mass ; 390 (M^), m /z316, 274

Synthesis o f 2 - ( l’,3’,4’-oxadiazolin-2’-thion-5’-yl)-4,6-dibromo acetanilide (XXVI) from (HI):

To a suspension o f III (0.01 mole; 3.5 gms) in ethanol (50 ml) containing potassium hydroxide (1 gm) was added carbondisulphide (20 ml) dropwise at room temperature during a period o f 30 minutes with continuous shaking. The reaction mixture was refluxed for 4 hrs on a water bath. The solvent was then evaporated off and the residue neutralized with con. HCl (1 ml). A solid mass, which separated out was filtered, washed with water and dried. It was recrystallized from ethanol and'dichloromethane mixture (8:2) to give a TLC (irrigant ‘a ’) pure colorless crystalline compound XXVI (yield 83.6%) m.p. >300°.Spectral studies:NMR : Refer page 64Mass ; 391 (M -^),m/z312,315

Synthesis o f 2-tf-nitrophenyl-5-(2’-acetamido-3’,5’-dibromo phenyl)-!,2,4-oxadiazole

(XXVII) from (III):

A mixture o f 111 (0.003 mole; 1 gm) and o-nitro benzoic acid (0.003 mole; O.SOOgm) in phosphorous oxychloride (5 ml) was refluxed for 5 hrs. The reaction mixture was cooled to room temperature and poured onto crushed ice followed by basificatiori w'ith sodium bicarbonate solution (5%). A solid mass, which separated out was filtered and washed with water. It was air dried and crystallized from ethanol to yield brownish orange colored amorphous powder XXVll m.p. 298-300° (yield 62%). It was TLC pure (irrigant ‘b ’).

Spectral studies:NMR : Refer page 65

Synthesis o f l-(2-acetamido-3,5-dibromobenzoyl)-3-methyI-3-hydroxy pyrazolin-5-one(XXVIII) from (III):

A mixture o f III (0.002 mole; 0,700 gm) and redistilled ethylacetoacetate (0.038 mole; 4.9 gms s 4.9 ml) was refluxed in glacial acetic acid bath (to maintain the temperature at 120°) for 16 hrs. The reaction mixture was cooled to room temperature and poured onto crushed ice. A solid mass, which separated out, was filtered and washed with water. It was air dried and crystallized from ethanol to give TLC pure (irrigarit ‘d’) crystalline compound XXVIII m.p. 216-18° (yield 24%).Spectral studies:NMR : Refer page 66

Attempted synthesis of 2-methyl-3-(carbethoxy methyIamino)-6,8~dibromo-3,4-dihydro quinazolin-4-one (XXIX) from (III):

This synthesis was attempted by four different methods with unsuccessful result.

iMethod A:

To a solution o f III (0.001 mole; 0.350 gm) in dry pyridine (25 ml) was added anhydrous potassium carbonate (2 gm) and chloroethylacetate (0.001 mole; 0 .12 2 gm =0.1

ml). The reaction mixture was then refluxed on a water bath for 24 hrs and after cooling to room temperature was poured onto crushed ice. A solid mass, which separated out, was filtered, washed with water and dried. It was crystallized from ethanol to give colorless crystalline compound m.p. 226-28° (yield 58.4%). On TLC examination (irrigant ‘e’), the R f value of the compound matched with that o f the starting hydrazide.

Method B:

A mixture of III (0.001 mole; 0.350 gm) and chloroethylacetate (0.03 mole; 3.06 gms s3 ml) was heated in glacial acetic acid bath for 12 hrs and after cooling to room temperature was poured onto crushed ice. A semi solid mass so obtained was filteied, washed with water and crystallized from methanol. On TLC examination (irrigant ‘e ’) Rf value o f the compound matched with that o f the starting hydrazide.

Method C:

A mixture of III (0.001 mole; 0.350 gm), sodium iodide (0.001 mole; 0.150 gm s) and chloroethylacetate (0.001 mole; 0 .12 2 gms a 0.10 ml) was heated in glacial acetic acid bath for 8 hrs. After cooling to room temperature, the reaction mixture was poured onto crushed

100

lOl

ice. A product so obtained was filtered, wasiied and dried. On TLC examination (irrigant ‘e ’) Rf value of the compound matched with that of the starting hydrazide.

Method D;

A mixture o f III (0.001 mole; 0.350 gm), sodium bicarbonate (2.0 gms) and water (5 ml) was heated on a hot plate. To this hot mixture was added chioroethylacetate (6.001 mole; 0.122 gins s 0 .10 ml) in small portions with continuous stirring during a period o f fifteen minutes. Stirring was continued for another 8 hrs without heating. The reaction mixture was then refluxed for 6 hrs and cooled to room temperature. A solid mass that separated out was filtered and washed with water. Melting point and TLC pattern (irrigant ‘e ’) of the compound matched with that o f the starting hydrazide.

Attempted synthesis o f 2-methyl-3-[4’-(4”-methoxy phenyI)-3’-chIoro-l’-azfttidinon-2’- yl]-6,8-dirbomo-3,4-dihydroquinazolin-4-one (XXX) from (VIII):

The synthesis of the desired compound was carried out by two methods, however, the results were unsuccessful.

Method A: . _ .

To a solution o f 'VIII (0.001 mole; 0.450 gms) in 1,4-dioxan (25 ml) was added chloroacetyl chloride (0.017 mole; 2.0 gms s 1.8 ml) and triethylamine (0.036 mole; 3.^6 gms s 5ml) at 0-5°. The reaction mixture after leaving overnight was refluxed for 8 hrs. It was then poured onto crushed ice after cooling to room temperature. A product so obtained was filtered, washed witli water and dried. It was crystallized from ethanol to give colorless needles. Melting point and TLC examination (irrigant ‘f ’) o f crystals, matched with triethylamine hydrochloride (m.p. 252-54°),

Method B:

To a solution of VIII (0.001 mole; 0.450gms) in dry tetrahydrofuran (30 ml) was added chloroacetyl chloride (0.001 mole; 0.112 gms s 0,1 ml) and sodium hydride (0.042 mole; 1 gm). The mixture was refluxed for 6 hrs and after leaving overnight, the solvent was evaporated o ff completely. Diethyl ether (20 ml) was then added followed by dropwise addition water till the effervescence ceased. The ethereal layer was separated and the extraction repeated three times.. All the ethereal layers were combined, washed with water and dried over anhydrous sodium sulfate. After filtering off the inorganic salt, the solvent was evaporated o ff to dryness. On TLC examination (irrigant ‘a’), its Rf value corresponded

with VIII.

To a solution of VII (0.001 mole; 0.480 gms) in dry 1,4-dioxan (10 ml) was added cliloroacetyl chloride (0.005 mole; 0.56 gms sO.5 ml), trietliylamine (0.001 mole; O.lOOgms s 0.14 ml) and the contents refluxed for 4 hrs. The reaction mixture was then poured into petroleum ether while hot. A solid mass, which separated out, was filtered, washed with petroleum ether and crystallized from methanol to give brown amorphous powder. Melting point and TLC pattern (irrigant ‘a ’) o f the compound were exactly matching with VII.

Attempted synthesis o f 2-(3*,4’-dimcthoxy styryI)-3-(4”-methoxy benzyIidenimino)-6,8- dlbromo-3,4-dihydroquinazoIin-4-one (XXXII) from (VUI):

Its synthesis was carried out by two methods, however, the results were unsuccessful.

102

Attempted synthesis of 2-metliyW-[4’-(3”,4”-dimethoxy phenyI)-3’-chloro-l’-azetidinon-2’-yi]~6,8-dibroino-3,4-liydroquinazolin-4-one (XXXI) from (VII):

Method A;

To a solution o f VIII (0.001 mole; 0.450 gms) in ethanol or 1,4-dioxan (10 ml) was added veratraldehyde (0.001 mole; 0.160 gms) and piperidine (2ml). The contents refluxed for 6 hrs. The reaction mixture was concentrated to a small volume and allowed to cool to room temperature. When a solid mass separated out which was filtered and crystallized from ethanol. On melting point and TLC examination basis, it was found to match exactly with the starting hydrazide.

Method B:

To a solution o f VIII (0.001 mole; 0.450 gms) in dry tetrahydrofuran (30 ml) was added veratraldehyde (0.001 mole; 0.160 gms) and sodium hydride (0.042 mole; 1 gm). The contents were refluxed for 4 hrs. The reaction mixture was left over night and the solvent was then evaporated to dryness. After cooling to room temperature, diethyl ether (20 ml) was added, followed by dropwise addition of water until effervescence had ceased. Ethereal layer was separated and further extraction with ether was carried out three times. All the ethereal layers were combined together washed with water and dried over anhydrous sodium sulfate. After filtering off the inorganic salt, solvent was evaporated off completely. The residue was found to be a complex mixture on TLC examination (irrigant ‘a’) which could not be purified.

4.2 SYNTHESIS OF 2-(SUBSTITUTED PHENYL)-3- (SUBSTITUTED ARYLIDENIMINO)-6,8-DIBROMO- 1, 2, 3, 4 - TETRAHYDRO QUINAZOLIN-4-ONE DERIVATIVES

Synthesis of methyl-3,5-dibromo anthranilate (XXXIII) from (I);

To a solution of I (0.003 mole; 1 gm) and dimethyl sulfate (0.007 mole; 0,924 gms s 0.7 ml) in dry acetone (25 ml) was added anhydrous potassium carbonate (3 gms) and refluxed for 4 hrs. After cooling to room temperature the inorganic salt was filtered off and the filtrate was concentrated. The reaction mixture was cooled to room temperature and poured onto crushed ice. A solid mass, which separated out, was filtered washed with water and crystallized from methanol to give TLC pure (irrigant ‘d ’) colorless crystalline compound XXXIII m.p. 78-80° (yield 6 6 .8%).Spectral studies:NMR : Refer page 71

Synthesis o f 3,5-dibromo anthranii hydrazide (XXXIV) from (XXXIII):

To a solution o f XXXIII (0.01 mole; 3 gms) in ethanol (20 ml) was added hydrazine hydrate (0,04 mole; 2.3 gms s 2.3 ml) and refluxed for 24 hrs. After cooling to room temperature a solid mass, which separated, was filtered and crystallized from methanol to yield colorless crystalline compound XXXIV m.p. 190-92“ (yield 72%). It was found to be TLC pure (irrigant ‘c ’).Spectral studies;NMR ; Refer page 71

Synthesis o f 2-(substituted plienyl)-3-(substituted aiylidenimino)-6,8-dibromo-l,2,3,4- tetrhydroquinazoIin-4-one (XXXV-XXXXVII) from (XXXIV):

General Procedure;

To a solution of XXXIV (0.01 mole) in ethanol were added appropriate aldehydes (0.02 mole) and a few drops o f con. HCl. The reaction mixture was refluxed for 3 hrs and cooled to room temperature. A solid mass, which separated, was filtered and crystallized to

give TLC pure crystals.

Synthesis o f 2-(4’-roethoxy phenyl)-3-(4”-methoxy benzyIidenimino)-6,8-dibromo-1,2,3,4-tetrahydroquinazoliii -4-one (XXX\Os

Compoimd XXXIV 0.01 m o le ; 3.1 gms4-Methoxy benzaldehyde 0.02 mole ; 2.72 gms = 2,4 mlEthanol 20 mlCon, HCl 2-3 drops

103

104

Reaction time (refluxing) Crystalli2ation solvent Shape/nature o f the compound Melting point Percentage yield TLC behavior

Spectral studies:NMRMass

3 lirs MetlianolColorless crystalline compound 156-58°64%Single spot in irrigant ‘c ’

Refer page 72543 (MO, m /z408,302, 133

Synthesis o f 2-(3’-ethoxy-4’-hydroiy phenyl)-3-(3”-ethoxy-4”-hydroxy benzylidenitnino) -6,8-dibromo-l,2,3,4-tetrahydroquinazoIin-4-one (XXXVI):

Compound XXXIV3-Ethoxy-4-hydroxy benzaldehyde Ethanol Con. HCIReaction time (refluxing) Crystallization solvent Shape/nature o f the compound Melting point Percentage yield TLC behavior

Spectral studies;NMRMass

0.01 m ole ; 3.1 gms0.02 m o le ; 3.30 gms20 ml2-3 drops3 hrsMethanolYellow,colored amorphous powder 118-20°74%Single spot in irrigant ‘c’

Refer page 73605 ( M l m /z292, 166, 137

Synthesis o f 2-(3’,4’-dimethoxy phenyl)-3-(3”,4”-dimethoxy benzylidenimino)-6,8- dibromo-l,2,3,4-tetrahydroquinazolin-4-one (XXXVII):

Compound XXXIV3,4-Dimethoxy benzaldehyde Ethanol Con. HCIReaction time (refluxing) Crystallization solvent Shape/nature o f the compound Melting point Percentage yield TLC behavior

0.01 mole ; 3.1 gms 0.02 mole ; 3.30 gms 20 ml 2-3 drops 3 hrsMethanolLight yellow colored needle shape crystals 176-78°58%Single spot in irrigant‘c’

Spectral studies: NMR .Mass

Refer page 74605 ( M l m/z 466, 4.55, 164,136

105

Attempted synthesis o f 2-(4’-diethyIammo phenyl)-3-(4”-diethylaminobenzylidenimino)-6,8-dibromo-l^,3,4-tetrahydroquinazolin-4-one (XXXVIII);

Compound XXXIV 4-Diethylamino benzaldehyde Ethanol Con.HCIReaction time (refluxing)Crystallization solventShape/nature o f the compoundMelting pointPercentage yieldTLC behaviorInference

0-01 mole ; 3.1 gms0,02 mole ; 3.54 gms20 ml2-3 drops3 hrsMethanolLight yellow colored fine crystals 236-38°62.3%Single spot in irrigant ‘c’On the basis o f NMR spectral data, was found to be the starting compound XXXIV.

Attempted synthesis o f 2-(4’-dimethylamino phenyi)-3-(4”-dim ethyIainino benzylidenimino)-6,8-dibromo-l,2,3,4-tetrahydroquinazolin-4-one (XXXIXa) which resulted in the form ation o f 2-(4’-dimethylamino benzyIidenimino)-l-(4”-dim ethylani!no bnzylidenimino)-4,6-dibromo an thran il hydrazide (XXXIX):

Compound XXXIV4-Dimethylamino benzaldehyde Ethanol Con. HClReaction time (refluxing) Crystallization solvent Shape/nature o f the compound

Melting point Percentage yield TLC behavior

Spectral studies;NMR

0.01 mole ; 3.1 gms 0.02 mole ; 3.0 gms 20 ml 2-3 drops 3 hrs MethanolFluorescent yellowish green coloredcrystalline compound244-46°78%Single spot in irrigant ‘c’

Refer page 75

Synthesis o f 2-(2 ’-thienyl)-3-(2 ’-thienyIidenimino)-6 ,8-dibrom o-I,2 ,3 ,4-te trahydro quinazolin-4-one (XL):

Compound XXXIV Thiophen-2-aldehyde Ethanol Con. HClReaction time (refluxing) Crystallization solvent Shape/nature o f the compound Melting point Percentage yield TLC behavior

0.01 mole ; 3.1 gms0.02 mole ; 2 .2 gms s 1.8 mi20 ml2-3 drops3 hrsMethanolBrown colored fine crystals 180-82°70% .Single spot in irrigant ‘b

106

Spectral studies;NMRMass

Refer page 76 497(M ’"),m /z457 ,440

Synthesis o f 2-(4’-chloro phenyl)-3-(4”-chloro tetrahydro qumazolin-4-one (XLI);

benzylidenimino)>6,8<.dibronio-l,23,4-

Conipound XXXIV4-CIiIorobenzaIdehyde Ethanol Con. HCIReaction time (refluxing) Crystallization solvent Shape/nature o f the compound Melting point Percentage yield TLC behavior

Spectral studies:NMRMass

0.01 mole ; 3.1 gms0.02 mole ; 2.8 gms20 ml2-3 drops3hrsMethanolLight yellow colored needle shape crystals 178-80°59.2%Single spot in irrigant ‘b’

Refer page 77551 (M'^X m/z 441,413, 303, 275

Synthesis o f 2-[(3’,4’-methylenedioxy)-piienyl]-3-[(3”,4”-methylenedioxy)-benzyliden- iinino|-6,8-dibromo-l,2,3,4-tetrahydro quinazoIin-4-one (XLII);

Compound XXXIV Piperoiial Ethanol Con. HCIReaction time (refluxing) Crystallization solvent Shape/nature o f the compound Melting point Percentage yield TLC behavior

Spectral studies:NMR

0.01 mole ; 3.1 gms 0.02 mole ; 3.0 gms 20 ml2-3 drops 3 hrs EthanolLight yellow colored needle shape crystals 208-10°56%Single spot in irrigant ‘c’

Refer page 78

Synthesis of 2-(3’-metlioxy-4’-hydroxy phenyl)-3-(3”-methoxy-4”-hydroxy benzyliden- imino)-6,8-dibromo-l,23,4-tctrahydro quinazolin-4-one (JflLIII):

Compound XXXIV3-Methoxy-4-hydroxy benzaldehyde Ethanol Con. HCIReaction time (refluxing) Crystallization solvent

0.01 mole ; 3.1 gms 0.02 mole ; 3.0 gms 20 ml 2-3 drops 3 hrs Ethanol

107

Shape/nature o f the compound Melting point Percentage yield TLC beliavior

Spectral studies:NMRMass

Light yellow colored crystalline compound 216-18°58%Single spot in irrigant ‘b’

Refer page 78575 (M^X m/z 440,412, 278,151

Synthesis o f 2-(3’,4’,5’-trimethoxy phenyl)-3-(3”,4”,5”-trimethoxy benzylidenimino)-6,8- dibromo-l,2,3,4-tetrahydro qainazolin-4-one (XLIV);

Compound XXXIV3,4,5-trimethoxy benzaldehyde Ethanol Con. HClReaction time (refluxing) Crystallization solvent Shape/nature o f the compound Melting point Percentage yield TLC behavior

Spectral studies:NMR

0.01 mole ; 3.1 gms 0.02 mole ; 3.9 gms 20 ml 2-3 drops 3 hrs EthanolBrownish-yellow colored crystals 118-20“52%Single spot in irrigant ‘b ’

Refer page 80

Synthesis o f 2-(phenyl)-3-(benzylidenimino)-6,8-dibromo-l,2,3,4-tetrahydro quinazolin-4-one (XLV):

Compound XXXIV4-Dimethylamino benzaldehyde Ethanol Con. HClReaction time (refluxing) Crystallization solvent Shape/nature o f the compound Melting point Percentage yield TLC behavior

Spectral studies:NMRMass

0.01 mole ; 3.1 gms 0.02 mole ; 2.1 gms 20 ml 2-3 drops3 hrs EthanolLight yellow colored needle shape crystals 168-70°76%Single spot in irrigant ‘b ’

Refer page 80483 (M*),m/z 379,317

Attempted synthesis o f 2-(2’-hydroxy phenyl)-3-(i”-hydroxy benzylidenimino)-6,8- dibromo-1,2,3,4- tctrahydro quinazolin-4-one (XLVia) which resulted in the formation o f an uncyclised compound (XLVI):

Compound XXXIV 2-Hdroxy benzaldehyde

0.01 m ole; 3.1 gms 0.02 mole ; 2.4 gms

108

Ethanol Con. HClReaction time (refluxmg; Crystallization solvent Shape/nature o f the compound Melting point Percentage yield TLC behavior

Spectral studies;NMR

20 ml 2-3 drops 3h rs EthanolYellow colored crystalline compound 228-30''62%Single spot in irrigant ‘b’

Refer page 81

Synthesis of 3,4-dihydro-4-oxo-6,8-dibrom o-l,2,3-benzotriazine (XLVH) from (XXXEU):

To a cooled (0-5°) solution o f XXXIII (0.024 mole; 7.6 gms) in a mixture o f con. HCl (20m!) and w^ater (25 ml) was added dropwise and with stirring a,cold solution o f sodium nitrite (4 gms) in water (20 ml). Stirring was continued for another 5 minutes and the reaction mixture was iiiade alkaline with concentrated ammonium hydroxide with stirring. A brown solid mass which separated was filtered and washed with ice cold ethanol and crystallized from petroleum ether to give dark reddish brown crystalline compound XLVII m.p. 188-90° (yield 38.6%). It was found to be TLC pure (irrigant‘a’).Spectral studies:NMR : Refer page 82Mass : 303 (M '), m/z 275, 260, 232, 153.

109

4.3 SYNTHESIS OF FLAVONE DERIVATIVES

Synthesis o f 2,4-dihydroxy acetophenone (XLVni) from resorcinol:

Freshly fused zinc chloride (0.121 mole; 16.5 gms) was dissolved in glacial acetic acid (16 ml) by heating on an oil bath. Resorcinol (0.1 mole; 11 gms) was then added in portions. The reaction mixture was maintained at a temperature o f 150° for a period o f 20 minutes. It was then left at room temperature for 30 minutes and diluted with a mixture of concentrated hydrochloric acid and water (1:1) and cooled to 0-5°. An orange solid mass separated out which was filtered, washed witli cold dilute hydrochloric acid (5 X 20 ml) and crystallized from dilute hydrochloric acid to give an orange crystalline TLC pure compound (XLVIII) (irrigant ‘a’) m.p. 144-46° (Lit. m.p, 145-47“) (Yield 88%).

Synthesis o f 2-hydroxy-4-methoxy acetophenone (XLDQ from (XLVIII):

A mixture of XLVIII (0.05 mole; 7.6 gms), dimethyl sulfate (0.5 mole; 6.3 gms s 4.8 ml), anhydrous potassium carbonate (4 gms) and dry acetone (25 ml) was refluxed for 8

hours. The reaction mixture was cooled, and filtered to remove potassium carbonate. The filtrate v/as concentrated, cooled and poured onto crushed ice. A solid mass, which separated was filtered, washed with water and crysiallizcd from petrol to give TLC (irrigant ‘a ’) pure light green yellow colored crystalline compound XLIX, m.p. 50-52° (Yield 60%).

Synthesis of o-nitro benzoic acid (L) from o-nitro toluene;

To a solution o f potassium permanganate (0.057 mole; 9 gms) in warm water (150 ml), o-nitro toluene (0.025 mole; 3.48 gms s 3 ml) was added and refluxed for 3 hrs. It was then filtered while hot, a pinch of sodiumbisulphate and concentrated hydrochloric acid (3 ml) was added to remove the pink color. The reaction mixture was poured onto a well stirred mixture o f concentrated hydrochloric acid and Water (1:2.5). A fine colorless crystalline compound (L) separated out which was filtered and found to be TLC pure (irrigant ‘a ’) m.p. 146-48° (Yield 44%) (Lit. m.p. 148°).

Synthesis o f 4-methyl-5-liydroxy-6-acetyl coumarin (LI) from XLVIII:

To a solution o f aluminum chloride (0.023 mole; 3 gms) in nitrobenzene (10 ml), was added compound XLVIII (0.01 mole; 1.5 gms)and ethylacetoacetate (0.016 mole; 2.05 gms s2 ml). The reaction mixture was refluxed for 3 hrs at 120-140°, cooled to room temperature and water (50 ml) was added. Nitrobenzene was distilled off by stem distillation. To the residue, 10% hydrochloric acid (10 ml) was added. A solid mass separated out, which was filtered and crystallized from etlianol to give TLC (irrigant ‘a’) pure brownish-green colored ci7 stalline compound LI m.p. 164-66° (Yield 18%) which was florescence under UV light.

no

Attempted synthesis of 2-acetyl substituted phenyl'(2-acetamido-3,5-dibromo)-benzoate(L II-L IV )fron i(IV );

General Procedure:

To an ice cold solution o f IV in dry pyridine was a added solution of substituted acetophenone in dry pyridine atjd stirred magnetically under anhydrous condition. To the above reaction mixture phosphorous oxychloride was added dropwise and further stirring was continued for 5 hrs. It was then poured onto crushed ice with continuous stirring. A solid, which separated out was filtered and washed with water followed by sodium bicarbonate solution (5%) and water. It was crystallized from some suitable solvent to give TLC pure crystalline compound.

Attempted synthesis of 2-acetyl phenyl- (2-acetamido-3,S-dibromo) benzoate (Lit);

Compound IV o-Hydroxy acetophenone Dry pyridinePhosphorous oxy chloride Reaction time (stirring)Sodium bicarbonate Crystallization solvent .Shape and nature of the compound Melting point Percentage yield TLC behaviour

Spectral studies;NMR

0.001 mole ; 0.330 gms 0.002 mole ; 0.272 gms s 0.24 ml 5 ml 0.5 ml 5 hrs5% ; 10 ml PetrolCream colored crystalline compound 176-78°56%Single spot in irrigant ‘a’

Refer page 85

Attempted synthesis of 2-acetyI-4-methoxy phenyI-(2-acetamido-3,5-dibromo) benzoate (LIII):

Compound IV Compound XLIX Dry pyridinePhosphorous oxy chloride Reaction time (stirring)Sodium bicarbonateCrystaUization solventShape and nature o f the compoundMelting pointPercentage yieldTLC behaviourInference

0.001 m ole ; 0.330 gms 0.0013 mole ; 0.216 gms 5 ml 0.5 ml 5 hrs .5% ; 10 ml PetrolCream colored crystalline compound 172-74°52%Single spot in irrigant ‘a’ .It was not the desired compound and was identified as II

I l l

Attempted synthesis o f l-(4-methyl-6-acetylcouniBrin)-N-acetyl-3,S-dibronio anthranilatc (LIV):

Compound IV Compound LI Dry pyridinePhosphorous oxy chloride Reaction time (stirring)Sodium bicarbonate Crystallization solvent Shape and nature of tlie compound TLC behaviour

0.001 mole ; 0.330 gms0.0013 m o le ;0.285 gms 5 ml0.5 ml5 hrs5% ; 10 ml MethanolBrown colored semi solid Mixture o f compounds in irrigant ‘a ’ which could not be'purified. Hence further studies were abandoned.

Synthesis o f (2’-acetyl phenyl)-2-nitro benzoate (LV) from (L):

To an ice cold solution o f (L) (0.001 mole; 0.170 gms) in dry pyridine was added o- hydroxy acetophenone (0.002 mole; 0.272 gms = 0.24 ml) under anhydrous condition followed by dropwise addition o f phosphorous oxyohloride (0.5 ml). Stirring was continued for 5 hrs, it was poured onto crushed ice with continuous stirring. A solid mass which separated.out was filtered and washed witli water and crystallized from methanol to give a crystalline compound LV m.p. 126-28° (Yield 92%). It was found to be TLC (inigant ‘a’) and did not give any color with alcoholic ferric chloride.Spectral studies:NMR : Refer page 86

Synthesis of l-(2 ’-nitrophenyl)-3-(2’’-hydroxy phcnyl)-propan-l,3-dione (LVI) from (LV);

To solution o f LV (0.0017 mole; 0.500 gms) in dry pyridine was added powdered potassium hydroxide (4 pellets) and shaken mechanically for 4 hrs with occasional warming. The reaction mixture was diluted with water (10 ml) and poured onto a mixture of concentrated hydrochloric acid and water (1:7; 30 ml). A light yellow colored solid mass which separated out was filtered and crystallized from methanol to give TLC pure (irrigant ‘a ’) yellow crj'stalline compound LVI m.p. 160-62° (Yield 86%). It gave a violet color with alcoholic ferric chloride.Spectral studies:NMR : Refer page 87

Synthesis of 2-nitro flavone (LVII) from (LVI);

It was synthesized by the following two methods;

Method 1:

A mixture o f LVI (0.0021 mole; 0.600 gms), glacial acetic acid (20 ml) and freshly fused sodium acetate was heated on an oil bath a t a temperature o f 150-160“ for 4 hrs. The reaction mixture was cooled to room temperature and poured onto crushed ice. A solid mass, which separated out was filtered and recrystallized from petrol to give a brown colored amorphous powder. It was purified by column chromatography to give TLC pure (irrigant ‘a ’) compound LVII m.p. 184-86° (Yield 36%), It did not give any color with alcoholic ferric chloride and gave a pink color on reacting with magnesium and hydrochloric acid.

Method 2:

Compound LVI (0.0021 mole; 0.600 gms) was dissolved in 4% ethanolic sulfuric acid (20 ml) and refluxed for 24 hrs. The reaction mixture was then cooled and poured onto crushed ice. A solid mass, which separated out, was filtered and crystallized from ethanol. For further purification it was chromatographed on a column of silica gel.

A column (50 cm in lengtli and 30 mm in diameter) after plugging witli cotton was filled with petroleum ether and silica gel (50 gms) was carefully poured into the column to avoid air pockets. The crude product (700 mg) was dissolved in chloroform in a porcelain crucible and silica gel (3.0 gms) was added to it. The solvent was evaporated off on a water bath with proper mixing so that tlie compound was unifomily adsorbed on silica gel. It was added into the column and tlie column was then eluted with petroleum ether, collecting 100

ml fractions. Thirty such fractions were collected. Fractions 1-5 did not yi'eld any compound while fractions 6-28 gave a yellowish brown residue on evaporating o f the solvent, which showed two spots oil TLC examination. Later on the column was eluted with the mixture o f petrol and benzene (8:2) fraction o f 50 ml each and (5:5) fraction of 50 ml each. On evaporating o f the' solvent showed a single spot comparable with diketone on TLC examination. Furtlier the column was eluted with pure benzene and ten fractions o f 25 ml each were collected, which gave a brown colored TLC pure (irrigant ‘a’) compound (fraction2, 3, 4 and 5). Tlie column was finally eluted with alcohol and chloroform. No other compound was isolated from tliese fractions.Spectral studies:NMR : Refer page 87

Synthesis of 2 ’-amino flavone (LVIU) from (LVII):

Compound LVII (0.01 mole; 2.67 gms) was reduced with tin and hydrochloric acid as per the procedure given in the "Text book o f Practical Organic Chemistry” by Vogel A I, 3 “ edition (1955) which gave compound LVIII m.p. 150-52'’ (Yield 73.2%) which was found to

be TLC pure in irrigant Mi’.Spectral studies:NMR : Refer page 88

112

113

To a solution o f LVIII (0.001 mole; 0.237 gras) in dry pyridine (5 ml) was added acetic anhydride (1 ml) and heated for 2 hrs in water bath. The reaction mixture was cooled to room temperature and poured onto crushed ice. A solid mass separated was filtered and washed with water and dried. The compound was crystallized from methanol to give colorless crystalline compound LIX m.p. 172-74“ (Yield 83.6%) which was found to be TLC pure (irrigant ‘a’).Spectral studies:NMR : Refer page 88

Attempted synthesis o f 2 ’-amino-3’,5’-dibromo flavone (LXa) which resulted in the formation of 2 ’-amino-3%5’,3,6,8-penta bromo flavone (LX) from (LVUT):

To an ice cold solution o f LVIII (0.001 mole; 0.237 gms) in glacial acetic acid (2 ml) was added solution o f bromine (0.002 mole; 0.3 ml) in glacial acetic acid (Im l) and stirred magnetically for 30 minutes. After stirring, the reaction mixture was poured onto crushed ice, filtered and washed tlioroughly with water. The compound was crystallized from methanol to give reddish brown colored amorphous powder, m.p. 180-82° (Yield 84.3%) and was found to be TLC pure (irrigant ‘a ’).Spectral studies:NMR : Refer page 89

Synthesis of 2’-acetamido flavone (LIX) from (LVIII):

BIOLOGICAL EVALUATIONS

BIOLOGICAL EVALUATIONS

5.1 MATERIALS AND METHODS

5.1.1 TOXICITY STUDIES

(a) Determination of LDjq o f compounds VI and XLV*:

Animals: Tlie study was carried out on Swiss albino mice (either sex) weighing between 20- 25gm. The mice were grouped and maintained under standard laboratoiy conditions with natural light dark cycle. They were fed on standard pellet diet and water ad libitum. Mice were acclimatized to their environment for one week prior to experimentation.

Test compounds, their doses and routes of administration: Compounds VI and XLV were selected for the study and the compounds were administered intraperitoneally to mice in ascending and widely spaced doses in 0.5% CMC (Carboxy Metliyl Cellulose) suspension.

Experimental Procedure:

The animals were fasted overnight and divided in groups o f six animals each. Test compounds were injected intraperitoneally and the animals were observed for 2 hrs for death due to acute toxicity. The percentage o f mortality values were calculated and converted to probit values by reading the corresponding probit units from the probit table^. Graph was plotted between probit values against log doses, and LD50 value o f the compounds was read as the dose, which corresponds to probit 5.

(b) Determination o f EDso values^:

Animals: Tlie study was carried out on Swiss albino mice (either sex) weighing between 20- 25gm. The mice were grouped and maintained under standard laboratory conditions with natural light dark cycle. They were fed on standard pellet diet and water ad libitum. Mice were acclimatized to their environment for one week prior to experimentation.

Test compounds, the ir doses and routes of administration; Acetic acid (lm l/100 mg (bw) of 0.6% acetic acid) was injected intraperitoneally to each of the mice.. Test compounds VI to XXII, XXV to XXVII were given at a dose o f 25 mg/kg, 50 mg/kg, 100 mg/kg & 200 mg/kg (bw) and compounds XXXV - XLVI were given at a dose of 50 mg/kg, lOOmg/kg, 200 mg/kg

114

1. Miller L C and Trainter M L. Proc Soc Exptl Biol Med., 57,261 (1944).2. Kulkanii S K. Toxicology In “ Hand Book o f Experimental Pharmacology" 3' edition.

(1999) Vallabh Prakshan, Delhi. Page No. 165.

& 300 mg/kg (bw). The test compounds were administered intraperitoneally in tiie form o f (0.5% CMC) suspension.

Experimental Procedure:

Over night fasted mice were grouped in six each and the test compounds were administered intraperitoneally. The control group of animals was administered 0.1 ml o f 0.5% CMC suspension. After a gap o f 30 min o f the administration o f the test compounds, all the groups of mice were given the writhing agent acetic acid, in a dose o f 1 ml/lOOg (bw) intraperitoneally. The writhing produced in these animals were counted for 30 m inutes and the number o f writhing produced in the treated group was compared with those in the control

No. o f wriths in test% Protection = 1 0 0 - ( ----------------------------------)X 1 0 0

No. of Wriths in control group. The percentage o f protection was calculated and converted to probit values by reading the corresponding probit units from the probit table’. Linear regression o f probit values against log doses was computed and the EDso value o f the Compounds was read as the dose, which corresponds to probit 5 which represents 50 % response o f the activity. The results are tabulated in Table 1.

(iii) Ulcerogenic activity^:

Animals; In this experiment Wistar rats o f either sex weighing 150 - 200 gm were used and divided in groups o f six animals each and housed in propylene cages. They were fed on standard diet water ad libitum. Rats were acclimatized to their environment for a week prior to experimentation.

Test compounds, drugs and their doses: The drugs used as standard were Diclofenac 20 mg/kg (bw) and Aspirin 25 mg/kg (bw) and test compounds (VIII, XII, XVI & XX) and (XXXV, XXXVII, XXXIX, XL & XLIII) were given at a dose of 100 mg/kg (bw) and 200 mg/kg (bw) respectively. The standard and test compounds were administered through oral route in the form o f suspension (0.5% CMC).

Experimental Procedure; The animals were divided in groups and control group animals was given only 0.5% CM C suspension. Test compounds and standard drugs were administered orally daily for 7 days. The animals were fasted for 8 hrs prior to dosing and for4 hrs post dosing. Food was available at all other times, free access to water was provided through out the experiment. Four hours after the last dose, the animals were sacrificed usjng chloroform. The abdomen was opened at the midline and the stomach and the first 3-cm o f the duodenum were removed. The stomach was opened along the lesser cur\'ature and

3. Shanbhag V R, Crider A M, Gokhale R, Harpalan i A, and Dick R M . / Phartn ScL, 81 (2),

149(1992).

115

washed with d istilled w ater. The m ucus was w iped o f f and the num ber o f lesions was examined by m eans o f 2 X 2 binocular magnifier. A ll ulcers > 0.5 m m w ere counted and recorded as average num ber o f ulcers per com pound.

5.1.2 ANALGESIC ACITIVTY

Animals: The analgesic activity was carried out on Swiss albino mice (either sex) weighing between 18-25 gm housed in propylene cages. They were fed on standard diet and water ad libitum. Mice were acclimatized to their environment for a week prior to experimentation.

Experimental methods:

(i) Acetic acid induced writhing metliod^:

Test compounds, d rugs an d their doses: Tlie drugs used as standards were Aspirin and Diclofenac in a dose o f 25 mg/kg (bw) and 20 mg/kg (bw) respectively. Acetic acid (lm l/100 mg (bw) o f 0.6% acetic acid) was injected intraperitoneally to each o f the mice. Test compounds VI to XXII, XXV to XXVII were given at a doses o f 25 mg/kg, 50 mg/kg, 100 mg/kg <& 200 mg/kg (bw) and compounds XXXV to XLVI were given at a doses o f 50 mg/kg, 100 mg/kg, 200 mg/kg & 300 mg/kg (bw). The standard and test compounds were administered through intraperitoneally in the form of (0.5% CMC) suspension.

Method: Aspirin like non-narcotic analgesic activity o f test compounds was investigated by their ability to protect writhing syndrome in mice. Over night fasted mice were grouped in six each and standard drugs were administered intraperitoneally. The control group of animals was administered 0.1 ml o f 0.5% CMC suspension and the remaining groups were administered different test compounds. After a gap o f 30 min of the administration of the test compounds all the groups o f mice were given the writhing agent acetic acid, in a dose of 1

ml/lOOg (bw) intraperitoneally. The writhing produced in tliese animals were counted for 30 minutes and the number o f writhing produced in the treated group was compared with tiiose in the control group and percentage protection was calculated using the formula given below;

No. O f wriths in test

% Protection = 100 - (------------ -------- -------------)^100■No. O f Wriths in control

116

4. Gosh M N. Some Common Evaluation Techniques in Fundamentals o f Experimental Pharmacology". 2"‘* ed. (1984) Scientific Book Agency, Calcutta. Page No. 144.

(li) Tail immersion method *;

Test compounds, d rugs and th e ir doses: The drug used as standard was Pentazocine in a dose of 5 mg/kg (bw). Test compounds VI to XXII, XXV to XXVII were given a t a dose o f 100 mg/kg (bw) and compounds XXXV to XLVI were given at a dose o f 200 mg/kg (bw). The standard and test compounds were administered intraperitoneally in the form o f suspension (0:5% CMC).

Method; Morphine like narcotic analgesic activity was determined by Tail immersion method. Mice were selected by immersing the tail in hot water at temperature 55“ ± 5 ° and the basal reaction time was noted. The mice which is showing a positive response with in 3 to5 seconds time duration for withdrawal of the tail clearly out o f water was taken for the study. The selected animals were divided into groups o f six each and standard drug was injected to one group, control group o f animals was received die 0.5 % CMC suspension only and remaining groups were administered different test compounds. Immediately after the administration o f drug and at intervals of 30 min, 1 hr, 2 hrs, 3 hrs the reaction time was recorded. As the reaction time reached 15 seconds, it was considered maximum analgesia and the tail was removed from the water to avoid tissue damage. Percentage protection was calculated using the formula given belov ,

Duration o f loss of withdrawal of Tail out o f water in test group

% Protection = ( --------------- ^ ^ ----------------------- — 1) X 100Duration o f loss of withdrawal of Tail out of water in control group

5.1.3 ANTICONVULSANT ACTIVITY^:

Animals; The study was carried out on swiss albino mice o f either sex weighing between 20-25 gm. The mice were grouped and maintained under standard laboratory conditions with natural light and dark cycle. They were fed on standard pellet diet and water ad libitum. Mice were acclimatized to their environment for one week prior to experimentation.

Test compounds, their doses and routes of administrations The drug used as standard was Phenytoin i n a dose of 25 mg/kg body weight. The dose o f the test compounds was 100

5. Witkin L B, Heuber C F. Galdi F, O ’ Keefe E, Spitaletta P and Plummer A J. J P /m a c o /E x p r/ier., 133, 400 (1961).

6. Kitano Y, Usui C, Takamuna K, Hirohashi M and Nomura M. J Pharmacol Toxicol

Meitorfs. 35, 25 (1996).

117

mg/kg (bw) for compound VI to XXII and XXV to XXVII and 200 mg/kg (bw) for compound XXXV to XLVL Tlie standard and test compounds were administered intraperitoneally in the form o f suspension (0.5% CMC).

Experim ental P rocedure: Measurement o f the seizure threshold for each animal was determined by the Increasing Current Electroshock Seizure (ICES) Test method *. The animals were divided in groups of six each and standard drug was injected to one group, the control group o f animals was given only 0.5 % CMC suspension and the remaining groups were administered different test compounds. After a gap of 30 min o f administration o f test compounds, ail the groups o f mice were given electrosliock via ear electrodes (forceps style) using an electroconvulsiometer. Tlie eiectroshock consisted of a single train o f pulse with linearly increasing current intensity (initial current of 2 mA, increment of 2 mA/ 2 sec). The current at which tonic hindlimb extension occurred was recorded as the seizure threshold current (STC) for each mouse. If no tonic hindlimb extension was observed by a current o f 30 mA, eiectroshock was terminated and this cut-off current was used in the analysis. The statistical significance o f differences between control and treatment groups was determined and the results were given in Table No. 13 & 14.

5.1.4 ANTIBACTERIAL ACTIVITY’ :

The microbiological testing of the synthesized compounds was done by agar diffusion method (cup plate method) against Bacillus .^ubtilis and Escherichia coli. The medium used was meat peptone agar med ium.

Washed spores o f these organisms were added into sterile and cooled media a t 45° and these organisms were poured into plates and allowed to solidify. Stainless steel cylinder of 8-mm dia (presterilized) was used to bore the cavities. All the synthesized compounds (100 fig/ml) mentioned in table serially were placed in the cavities witli the help of micropipetes and allowed to diffuse for one hour. These plates were incubated at 37° for twenty-four hours. Solvent was poured as control, norfloxacin and erythomycin were used as reference drugs.

The plates were observed after twenty-four hours and Zone of inhibition was recorded and given in tlie Table No. 15.

; 7; British Pharmacopoeia, Pharmaceutical Press, London (1953) Page no. 796. 8;; ihdian Pharmacopoeia -11, 3'“* edition, (1985) A-88.

5.2 RESULTS AND DISCUSSION

TOXICITY STUDIES

Toxicology is the science that deals with the study o f potential harmful effects o f chemicals and drugs on living organisms. All drugs are capable o f producing harmful as well as beneficial effects. Toxicity testing of new chemical entity is essential, yet animal toxicity data may give little guidance to hazards in man. It is essential to use at least two species (usually a rodent and a non-rodent) in the evaluation o f tlie potential toxicity o f a drug because species differ in their responses to the drugs. The following studies are performed on laboratory animals for determining the toxicity profile o f a compound:1. Acute toxicity studies2. Sub acute toxicity studies3. Chronic toxicity studies

d e t e r m i n a t i o n o f LD jo:

In the determination of LD50 i.e., the dose that will kill 50% of animals, single doses of the drug are used in each animal on one occasion. Among the newly synthesized compounds, compound VI and XLV were selected for the toxicity testing considering structural similarity to other final compounds. Before the actual LDjo determination, a pilot study was made on small group of mice mainly to select the dose ranges for the subsequent study. The compound was administered intraperitoneally to pairs of mice in ascending and widely spaced doses. The treated mice were observed continuously for 4 hrs and finally overnight mortality was recorded. The dose killing one out of two mice in the experiment gave an approximate estimate of tiie LD50.

After finding out the range between tlie maximum non-lethal and minimum lethal dose, the LD30 assay was done using five dose levels within the range with six animals in each group. There are various methods used to calculate LD50 values, viz., the graphical method, arithmetical method and the statistical approach. For calculating the LDso value graphical method was employed. The percentage of mortality values were calculated and converted to probit values. Then graph was plotted between probit value against log dose and LD50 value of the compound was found as the dose, which corresponds to probit 5.

119

121

LDjo value of compound XLV:

G roupsD ose

M g/kg, ipL o g Dose D ead/Total % D ead

C o rre cted

% P r o b it

1 3000 3.4771 0/6 0 4.17' 3.270 3500 3.5440 2/6 33.3 33.3 4.57

3 4000 3.6021 4/6 66.7 66.7 5.43

4 4500 3.6532 5/6 83.3 83.3 5.96

5 5000 3.6989 6/6 100 95.83* 7.55

*Correction for 0% dead = 100 (0.25/n), and 100% dead = 100(n-0.25/n)

From the calculation, the LD50 value of compound XLV was found to be 3715 mg/kg ^bw).

120

Calculation:

LDso value o f com pound VI:

G roupsD ose

IVIg/kg, ipL o g Dose D ead/Totnl % D ead

C o rrected

% P ro b it

1 2500 3.3979 0/6 0 4.17* 3.27

2 3000 3.4771 2/6 33.3 33.3 4.57

3 3500 3.5441 3/6 50 50 5.00

4 3750 3.5740 5/6 83.3 83.3 5.96,

5 4000 3.6021 6/6 100 95.83* 7.55

♦Correction for 0% dead = 100 (0.25/n), and 100% dead = 100(n-0.25/n)

From the calculation, the LD50 value of compound VI was found to be 3162 mg/kg (bw).

The determination o f ED50 (the dose effective in producing certain expected response in 50% of the animal group) values helps in ascertaining the potency o f a drug. The

calculation o f ED50 value is done when a drug is showing graded response. But, when the

response is quantal or all or none, the EDjo value becomes LD50. These values, i.e., ED50 and

LDso are important for knowing tlie safety o f drug. The ratio between LD50 and ED50

represents therapeutic index. Greater the therapeutic index safer is the drug.

The ED50 values o f newly synthesized 2-methyl-3-substituted arylidenimino-6 ,8-

dibromo-3,4-dihydroquinazoHn-4-one derivatives (Compd. VI-XXII) and 2-substituted

phenyl -3-substituted arylidenimino-6,8-dibromo-l,2,3,4-tetrahydro quinazolin-4-one derivatives (Compd. XXXV-XLVI) and some other derivatives (Compd. XXV-XXVII) were

determined by acetic acid induced writhing analgesic method in mice and results are given in

Table No. 1

From the results, 2-metliyl-3-substitued arylidenimino-6,8-dibromo-3,4-dihydro quinazoIin-4-one derivatives were comparatively more potent than 2-substituted phenyl-3-

substitued ■ arylidenimino-6,8-dibromo-l,2,3,4-tetrahydroquinazolin-4-one derivatives. Among thfe 2-methyl-3-siibstitued arylideniniino-6,8-dibromo-3,4-diliydroquinazolin-4-one

derivatives, compound XX was most potent [ED50 17.75 mg/kg (bw) and least potency was

shown by compound XIII, ED50 101.62 mg/kg (bw). Compound VII, XII and XVI showed

EDso value o f 33.99 mg/kg (bw), 36.89 mg/kg (bw) and 37.03 mg/kg (bw), respectively. Compounds VI, X, XI, XIV, XV, XVII, XVIII and XIX EDSO values ranged from 41 to 56

mg/kg (bw) and the remaining compounds, VII, XI and XII showed an ED50 value of 85.57,

76.30 and 78.54 mg/kg (bw), respectively.

In the 2-substituted phenyl-3-substitued arylidenimino-6,8-dibromo-l,2,3,4-

tetrahydroquinazolin-4 -one derivatives (compd. XXXV-XLVI) compound XLIII was found

to have EDjo o f 57.23 mg/kg (bw) and compounds XXXV, XXXVII, XXXIX and XLII

showed ED50 o f 65.93, 75.93. 63.07 and 90.26 mg/kg (bw), respectively. All remaining

compounds in this series showed higher EDso values and compound XXXVI was least potent

[EDso 191.96 mg/kg (bw)].

Apart from the quinazolinone derivatives, the other compounds such as oxadiazole,

thiadiazole and triazole derivatives showed ED50 o f 50.4, 39.88 and 44.43 mg/kg (bw),

respectively. From the study, it was concluded that instead o f aryl moiety, alkyl substitution

in the ,2"“’ position o f quinazolinone ring increases the potency o f the molecule.

122

d e t e r m i n a t i o n o f E D so:

123

Table No. 1

EDso values of newly synthesized compounds were determined by acetic acid induced writhing analgesic test:

CompoundNo.

EDso Values in mg/kg ^

VI 52.26VII 85.56VIII 33.99

X 55.50XI 46.18XII 36.89XIII 101.62XIV 44.31XV 56.43XVI 37.03XVII 53.09XVIII ,49.42XIX 41.28XX 17.75XXI 76.30XXII 78.54XXV 50.21XXVI 39.88XXVII 44.43

CompoundNo.

EDso Values in mg/kg *

XXXV 65.93XXXVI 191.96XXXVII 75.93XXXIX 63.07

XL 185.05XLI 122.21XLII 90.26XLIII 57.23XLIV 108.34XLV 142.96XLVI 107.52

# Drugs were given through i.p route o f administration

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u l c e r o g e n ic ACTIVITY;

Gastrointestinal side effects constitute the most frequent of the adverse reactions o f Non-steroidal anti-inflammatoiy drugs. These reactions range in both severity and frequency from relatively mild to the more serious and potentially life threatening such as gastrointestinal ulceration and hemorrhage.

The ulcerogenic activity o f compounds 2-methyl-3-subsituted arylidenimino-6,8- dibromo-3,4-dihydroquinazolin-4-one derivatives (compounds VIII, XI, XII, XV & XX) and

2-substituted phenyl-3-substituted aryUdenimino-6,8-dibromo-l,2,3,4-tetrahydro quinazolin-4-one derivatives (compounds XXXV, XXXVII, XXXIX, XL & XLIII), which showed promising analgesic effect, at a dose level of 50 mg/kg (bw) and 100 mg/kg (bw), respectively

was evaluated using aspirin [25 mg/kg (bw)] and diclofenac [20 mg/kg (bw)] as standards for comparison. The average number o f ulcers found in the gastric mucosa is presented in Table

No. 2

None o f the newly synthesized compounds produced ulcers in test animals, while diclofenac and aspirin produces ulcers (average ulcers 10.0 and 12.57 respectively).

Table No. 2

Ulcer index of newly synthesized and standard compounds:

C oin pd. No. Ulcers*

vm Nil

XII Nil

XVI Nil

XX Nil

XXXV Nil

XXXVII Nil

XXXIX Nil

XL Nil

XLIII NilDiclofenac 9.5 + 2.07

Aspirin 12.0 ±1.67* Average number of ulcers >0.5mm foiined ± SD (n ■" 6)

ANALGESIC ACTIVITY;

The analgesic activity o f ail newly syntliesized compounds were evaluated by Tail immersion method and acetic acid induced writhing method using Pentazocine [5 mg/kg (bw)], Aspirin and Diclofenac [25 mg/kg & 20 mg/kg (bw)] respectively were used as standards for comparison.

(i) Acetic acid induced writhing method:(a) 2-MethyI-3~substitut£d ftr}'lideniinino-6,8-dibroino-3,4-dihydroquinazoIin -4-one

derivatives:

The newly synthesized 2-methyl-3-substituted aylidenimino-6,8-dibromo-3,4- dihydroquinazlin-4-ones (VI to XXII) were tested at a dose level of 25, 50, 100, 200 mg/kg (bw). All the test compounds showed dose dependent response in their analgesic action. The data was subjected to statistical analysis by One-way Analysis of Variance (ANOVA) followed by Dunnet’s test. All compounds showed significant differences when compared with control in all dose levels (p< 0.001). A pValue of < 0.5, < 0.1, < 0.05, < 0.01 and <0.001 were considered to be statistically not significant, slightly significant, significant, highly significantly and veiy highly significant respectively. The results are tabulated in Table No. 3-6 (Fig. 1-4).

At a dose level o f 25 mg/kg (bw)

At a dose level of 25 mg/kg (bw) all the test compounds (VI to XXII, XXV to XXVII) exliibited lower degree of analgesia i.e., less than 45% protection except compound XX, which showed 50.01% protection. The standard drug aspirin and diclofenac showed 64.52% and 81.45% protection, respectively.

The test compounds did not show any ulcerogenicity (as described in gastrointestinal toxicity in Materials & Methods section) even at higher doses i.e., 50 mg/kg (bw) while the standard drugs aspirin and diclofenac were ulcerogenic even at a dose of 25 mg/kg (b\\ ) and 20 mg/kg (bw) respectively. Hence the test compounds at all dose levels were compared with standard drugs aspirin and diclofenac 25 mg/kg (bw) and 20 mg/kg (bw) respectively b>' One Way Analysis of Variance (ANOVA) followed by Dunnet’s test.

At a dose level o f 50 mg/kg (bw)

In general, among the synthesized compounds of 2-methyl-3^substituted arylideniminO'6,8-dibromo-3,4-dihydroquinazolin-4-one derivatives (VI to XXII, XXV to XXVII), compound XX was found to be quite superior in their analgesic activity at a dose of 50 mg/kg (bw) percentage protection 80.65 with a pValue of more than 0.5 indicating that they are not statistically significantly different from aspirin. Compounds VllI, XII, XIX and

125

XXVI showed 61.29, 64.52, 59.68 and 58.07 % protection and their pValue are less than 0.5 indicating that they are not statistically significantly different from aspirin. Hence, these compounds could be considered to be equivalent in their action. Compounds XI, XV, XVI and XXV showed 54.85 % protection and tlieir pValues were less than 0.1 indicating that they are slightly significant and these compounds could be considered to be good analgesics in comparison to standard drug aspirin. Compounds XIV, XVII and XXVII showed 52.43, 51.62 and 51.62 % protection respectively, witli pValue less than 0.05, indicating that they are significantly different and better analgesic activity in comparison to aspirin. Compounds VI, X and XVII showed moderate action and their percentage protection was in the range o f 48- 50?/o with pValue less than 0.01 (p<0.01) indicating that they are highly significant and these compounds showed less analgesia when compared with that o f aspirin. Rest o f the compounds were showed poor analgesia.

In comparison with diclofenac, compound XX (80.65%) exhibited significant analgesia in comparison to standard drug diclofenac (81.45%), statistically they were not significantly (p<0.5) different from diclofenac in tlie protection and it could be considered to be equivalent in their analgesic action in comparison to diclofenac. Compounds VIII, XII, XXVI were found to exhibit moderate protection ranging from 60 to 62 % protection and tiiese compounds were statistically very highly significantly {p<0.001) different from diclofenac. Rest compounds VI, X, XI, XIV to XIX, XXV and XXVII showed 50 to 54 % protection and remaining all compounds did not showed any analgesic action.

At a dose level of 100 mg/kg (bw)

All the test compounds showed 66 to 83 % protection o f analgesia at a dose of 100 mg/kg (bw) except compound no. VII and XIII. Among the tested compounds, compound XX exhibited 83.87 % protection followed by compound XIV & XXVI, which showed 80.65 % protection and compounds XII, XVI, XIX and XXVII exhibited 79.04% protection. These compounds were statistically not significant (p<0.5) in comparison to standard drug diclofenac and it could be considered as equal in their analgesic action with diclofenac. Compounds VIII and XVIII showed 74.20 and 71.78 % protection and their pValue is less than 0.05 when compared against diclofenac indicating that they are significant different from diclofenac. Hence these compounds were considered to have better analgesic activity in comparison to diclofenac. Compounds VI, XI, XXII and XXXV showed 67.75, 66.94, 66.13, 67.5 % protection, respectively and they are statistically very highly significantly different

(p<0.001) in comparison with standard drug diclofenac.

At a dose level o f 200 mg/kg (bw)

Almost all compounds (Compounds VI, VIII, IX, XI, Xll, XIV, XVII, XVIII, XIX, XX, XXl) showed an ceiling effect (80 to 91 % protection) at dose of 200 mg/kg (bw) and

compounds Vll, X, X lll, XIV, XVI and XXI exhibited 67 to 78% protection.

126

127

Table No. 3

A n a lg e s ic activity of 2-mcthyl-3-substituted aiylidenimino-6,8-dibromo-3,4- dihydroquinazolin-4-one and some other compounds at a dose of 25 mg/kg (bw) by

acetic acid induced writhing method.

Compd. Control V I V II V III X X I XII XIII XIV X V A spirinDiclofc-

nnc

Mean 20.67 15.33 17.50 12.17 14.50 14.00 13.67 17.00 14.50 14.83 7.33 3.83

S.D 2.25 1.97 1.76 2.32 0.55 2.83 3.20 1.10 0,55 1.17 2.07 0.98

%Inhibition

25.82 15.34 41.14 29.85 32.27 33.88 17.76 29.85 28.24 64.52 81.45

Table No. 3 (contd.)

Compd. Control X V I X V II X V III XIX XX XX I XXII X X V X X V I X X V II A spirin Diclofe­

nac

Alefln 20.67 12,67 14.67 15.00 14.00 10,33 17.67 16.00 15.00 14.00 14.50 7.33 3.83

S.D 2.25 3.27 0.82 2.53 2.83 1.37 1.63 3.90 2.53 2.83 0.55 2.07 0.98

%Inliibition

38.72 29.04 27.43 32.27 50.01 14.53 22.59 27.43 32.27 29,85 64.52 81.45

128

Table No, 4

Analgesic activity of 2-methyl-3-substituted arylidenimino-6,8-dibromo-3,4- dlhydryquinazoIin-4-one and some otlier compounds at a dose of 50 rag/kg (bw) by

acetic acid induced writhing method.

CoBipd. Control VI V II vin X XI XII X III X IV X V A sp irinD iclofe­

nac

Mean 20,67 10.33 16.00 8.00 10.33 9.33 7.33 14.50 9.83 9.33 7 .3 3 3.83

S.D 2.25 0.52 3.90 1.79 3.72 2.0 7 1.37 0.55 0.98 1.03 2 .0 7 0,98

%

Inhibition50,00" 22 .5 8 6 1,2 9 '’® 50.01* 5 4 .8 5 ' 64.52>® 29.85 52.43“' 54.85° 6 4 .5 2 81.4 5

Table No. 4 (contd.)

Compd. Control XV I X V II X V III XIX X X XXI X X II XXV X X V I X X V II A sp irinD iclofe­

nac

IVIean 20.67 9,33 10 .67 10.00 8.67 4.00 17,00 13,67 9.33 8,33 10.00 7.33 3.83

S.D 2,25 4,08 1.63 1.79 2.88 0.89 1,10 1.37 1.03 2,88 1.26 2 .0 7 0.98

%

Inhibition54.85'= 4 8 ,4 0 ' 5 1 .6 2 “ 58,07'’ 80.65"

cn17.76 33.88 54.85' 59.68'’® 51.62'* 64 .52 8 1.4 5

a = p >0.5, b = p < 0.5, c = p < 0 .1, d = p < 0.05, e = p < 0.01; when compared with Aspirin

® = p < 0.5, ® = p <0,05, ® = p < 0.001; when compared with Diclofenac.

129

Table No. 5

AnaJgesic activity of 2-methyI-3-substituted arylidenimino-6,8-dibromo-3,4- dihydroquinazolin-4-one and some other compounds at a dose of 100 mg/kg (bw) by

acetic acid induced writliing method.

Compd. Control VI vri VIII X XI xn XIII XIV XV Aspirin Diclofe­nac

Mean 20 .67 6 .6 7 8.33 5.33 6 .17 6.83 4.33 10.83 4 ,0 0 7.83 7 .3 3 3.83

S.D. 2.25 2 .4 2 1.51 1.03 1.94 0.41 0.52 1.72 0 .8 9 1 .1 7 2 .0 7 0.98

%

inhibitiDn 67.75*^ 59.68 74.20*’ 7 0 .1?** 6 6 .9 4 ' 79 .0 4 “ 4 7.59 8 0 .65“ 62 ,10 6 4 .5 2 8 1 .4 5

Table No. 5 (contd.)

Compd. Control XVI XVII XVIII XIX XX XXI XXII XXV XXVI xxv ir Aspirin Diclofe­nac

MEAN 20 .67 4.33 6.33 5.83 • 4.33 3.33 5.00 7.00 6.67 4.00 4.33 7 ,3 3 3 .8 3

S,D, 2 .25 1.03 2.25 1.94 1.86 0.52 1.55 2 .3 7 1.03 0.89 0 .52 2 ,0 7 0 ,98

%

inhibition79.04" 69.36*' 7 1 .7 8

c79.04" 83.8 7“ 7 5 .8 1 '’ 6 6 .1 3 ' 6 7 .7 5 ' 80.65“ 79 .0 4 “ 64 ,5 2 8 1 .4 5

a = p < 0.5, b = p < 0.1, c = p< 0.05, d = p < 0.01, e = p < 0.001; when compared with diclofenac

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Table No. 6

Analgesic activity of 2-methyi-3-substituted arylidenimino-6,8-dlbromo-3,4-dihydroquinazolin-4-onc at a dose of 200 mg/kg (bw) by acetic acid induced

writhing method.

Compd. Control VI VII VIII XI XU xin XIV XV XVI XVII XVIII X IX XX XXI XXII

Mean 20.67 2.33 5.00 3.67 6.50 2.33 4.17 6.33 2.33 5.33 4.50 3.67 2.33 2.00 2,17 3,83 6.67

S.D, 2.25 0.52 1.67 1.03 1.22 0,52 0,75 1.63 0.52 2.16 1.52 1.03 0.52 0.89 0,41 1.17 1.37

% inhibition 88.71 75.81 82.26 68.55 88.71 79.84 69.36 88,71 74.20 78,23 82,26 88,71 90.32 89.52 81.45 67.75

(b) 2-Substituted phenyl-3-substituted arylidenimino-6,8-(libroino>l,2,3,4- tetrahydro quinazoIin-4-one derivatives;

In 2-substituted plieny[-3-substituted arylidenimino-6,8-dibromo-l,2,3,4-tetrahydro quina7olin-4one derivatives (XXXV to XLVI), compound XLIII was found to be most active compound (70% protection) followed by XXXIX (63.75% protection) at a dose of 100 mg/kg (bw). At a dose of 200 mg/kg (bw), most of tlie compounds exliibited >60% protection of analgesia except compound XXXVl and XL, which showed 51.26 % and 51.88 % protection, respectively. The results are tabulated in Table no. 7-10 (Figure 5-8).

At a dose level of 50 ing/kg (bw)

At a dose of 50 mg/kg (bw) all the test compounds showed less than 50% protection. Compounds XXXVl, XXXVII, XLI and XLII exhibited percentage protection in the range of

43 to 47 %. Rest all compounds showed negligible analgesic activity. Standard drug aspirin [25 mg/kg (bw)] and diclofenac [20 mg/kg (bw)] showed 72.50 % and 85.63 % protection of

analgesia respectively.

At a dose lleve! o f 100 mg/kg (bw)

Compound XLIII showed 70 % protection at a dose o f 100 mg/kg (bw) with a pValue

of less than 0.5 (p<0.5) indicating statistically not significant difference when compared with

aspirin. Hence both were similar in their potency, followed by compound XXXIX (63.75 %)

with a pValue o f less than 0.1, indicating that they are statistically slightly significantly

different from aspirin and could be considered to be having better analgesic activity compared

to aspirin. Compound XL, XXXVII and XXXVI exhibited 56.26, 55.63, 55.01 % protection

of analgesia and they were statistically highly significantly different from aspirin (p<0.01)

and compound XLII, XLIV, XLV and XLVI showed 50 —53 % protection and their pValues

are less than 0.001 indicating that they are very highly significantly different from aspirin.

Statistically, all compounds were very highly significantly different from diclofenac

indicating that test compounds were not as good as diclofenac in their analgesic activity except compound XLIII (p<0.01).

131

At a dose level o f 200 mg/kg (bw)

At a dose o f 200 mg/kg (bw), compounds XXXV, XXXIX, XLI, XLII, XLIII and

XLVI showed their percentage protection of analgesia in the range of 77.5 to 79.38 % and

statistically compounds XXXV and XXXIX are slightly significantly different in comparison

to standard diclofenac (p<0.1) and compound XLI, XLII, XLIII and XLVI with pValue

(p<0.05) considering better activity in comparison to diclofenac and they are significantly

different against diclofenac. Compound XXXVII showed 70% with the pValue (p<0.001)

protection followed by compounds XLV and XLIV, which has shown 66.25 and 62.50 %

protection o f analgesia respectively and statistically they are very highly significantly

different when, compared against diclofenac. At the same time, when the test compounds

were compared with aspirin almost all compounds are statistically not significantly different

from aspirin (p<0.5), except compound XXXVI. Statistically, compounds XLIV and XLV

are significantly different (p<0.05) and compound XXXVI and XL are very highly

significantly different (p<0.001) when compared with aspirin.

At a dose level o f 300 mg/kg (bw)

Except compound XL and XXXVI (54.38 and 67.5% protection respectively), all

other compounds were exhibited 75 to 93 % protection of analgesia.

Analgesic activity of2-(substituted phenyl).3-(substlfuted arylidenimino)-6,8-dibroi..„l,2,3,4-tetrahydroquinazolin-4-one derivatives at a dose of 50 mg/lcg (bw) by acetic acid

induced writiiing method

Compel. Control XXXV XXXVII XXXIX XL XLII XLIII XLIV XLV XLVI Aspirin Diclofen*ac

Mc»n 26,67 14.17 15.67 15.17 23.50 18.00 14.50 19.00 24.67 23.00 7.33 3.83

S.D. 4.13 2.56 0.52 2.56 4.28 1.79 1.52 2.10 3.50 2.10 2.07 0.98

%iolilbition

46.88 41.26 43.13 11.89 32.51 45.63 28.76 7.51 13.76 72.50 85.63

Table No. 8

Analgesic activity of 2-(substituted pheny!)-3-(substituted aryIidenimino)-6,8-dibromO'l,2,3,4-tetrahydroquinazoIin-4-one derivatives at a dose of 100 mg/kg (bw) by acetic acid

induced writhing method

Compd. XXXV XXXVI XXXVII XXXIX XL XLI XLII XLIII XLIV XLV XLVI Aspirin Diclofe­nac

Mean 12.00 20.00 11.83 9.67 11.67 16.33 13.33 8.00 13.00 13.33 12.33 7.33 3.83

S.D. 1.26 1.26 1.83 1.03 1.86 2.25 1.86 1.67 2.00 2.34 4.59 2.07 0.98

%i«Mbition 5 5 .o r 25.01 55.63“ 63.75” 56.26' 38,76 50.01^ 70.00*" 51.26'' 50.01*' 53.76'’ 72.50 85.63

a = p< 0.5, b = p < 0.1, c = p < 0.01, d = p < 0.001; when compared with aspirin

® = p < 0.01; when compared with diclofenac.

133

Table No. 9

Analgesic activity of 2-{substituted phenyl)-3-(substituted arylidenimlno)-6,8-dibromo-l,2,3,4-tetrahydroquinazolin-4-one derivatives at a dose of 200 mg/kg (bw) by acetic acid

induced writhing method

Compd. Control XXXV XXXVI XXXVII XXXIX XL X U XLII XLIII XLIV XLV XLVI Aspirin Diclofe-nac

Mean 26.67 . 5.50 13.00 8.00 5.83 12.83 6.33 6.83 6.00 9.00 10.00 6,00 7,33 3,83

S.D. 4.13 1.05 3.90 2.61 1.47 1,47 1.21 0,98 0.63 ,2.10 1,79 1.79 2.07 0,98

%inbibition

7938«i 51.26' 70.00*® 78.13® 51.88' 76,25“® 74.38*® 77,50“® 66.25" 62,50'’ 77.50“ 72.50 85.63

a = p < 0.5, b = p < 0.05, c = p < 0.001; when compared against aspirin

® = p<0.1,@ = p< 0.05, @ = p < 0.001; when compared against diclofenac

Table No. 10

Analgesic activity of 2-(substituted phenyl)-3-(substitntcd arylidenimino)-6,8-dibroino- l,2^,4-tetrahydroquinazoIin-4-one derivatives at a dose of 300 mg/kg (bw) by acetic acid

induced writhing method

Compd. Control XXXV XXXVI XXXVII XXXIX XL XLI XLII XLIII XLIV XLV XLVI

Mean 26.67 2.50 8.67 3.83 3.67 12.17. 6.33 3.67 1.83 3.00 6.50 2.83

S.D. 4.13 0.55 1.51 1.17 0,52 3.19 0.52 1.63 0.75 0.89 2.43 1.17

%inhibition 90.63 67.50 ■85.63 86.25 ,54.38 76.25 86.25 93.13 88.75 75.63 89.38

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Structure Activity Relationship;

1. Compounds with alkyl group in the 2"‘‘ position o f quinazolinone ring significantly increases the analgesic activity.

2. Replacement o f alkyl group in the 2"** position by an aryl/hetryl moiety decreases the activity however, substituent in tlie aryl group results in better analgesic activity.

3. Saturation o f ring B, decreases the analgesic activity,

4. Replacement o f benzylidenimino by a thenylidenimino group in the 3** position enhances the analgesic activity.

5. Effects o f substitution in the phenyl ring o f benzylidenimino group at 3** position in 2- methyl -3-substituted arylidenimino -6,8-dibromo-3,4-dihydro quinazolin-4-one:

• Introduction o f tertiary nitrogen atom at para position in the phenyl ring increases the

analgesic activity.

• Replacement o f hydrogen atom at 2"‘‘ or 3'^ position o f phenyl ring by an -O H group

does not alter the activity.

• In addition to -O H group at 2"'* position, -OCHj or -OCjHs group at 3" carbon atom o f the phenyl ring did not affect the activity,

• Substitution o f chlorine atom at 4”' position significantly increases the activity and introduction o f another chlorine atom in 2"‘* or 6"' position of phenyl ring decreases

the analgesic activity.

• Substitution o f methoxy group at 4' position decreases the activity, while substitution o f two m ethoxy groups (at 3" and 4“' position) or three methoxy groups (at 3' '*, 4"' & 5”’ position) generally increases the activity in the order o f 3 ,4-OCH3 > 3,4,5- O C H 3

> 4- OCH3.6. Substitution in the phenyl ring o f benzylidenamine group at 3"‘‘ position in 2-substituted

aryl quinazolinone nucleus did not affect the activity o f the compound. The order o f

activity o f the substituents is as follows; 3-OCH3-4-OH > 4-N(CH3)2 > 3 -OC 2H 5-4 -O H = 4 -OCH3 > 2-OH > 3,4,5-tri-OCHi > H > 4-Cl > 2,3-di-OCH3.

7. Replacement o f benzylidenamino group at 3"‘ position o f quinazolinone ring in 2 - substituted hetryl quinazolinone nucleus by an heterylidenamino substituent

comparatively decreases the activity.

(ii) Tail im m ersion method:

In tail immersion method, all the newly synthesized compounds exhibited a lower degree o f analgesic activity as tlieir percentage protection was lesser in comparison with Pentazocine (at a dose o f 5 mg/kg (bw) 236.06 % protection after 2 hrs) (Table No. 11 & 12).

In the 2-methyl-3-substituted arylidenimino-6,8-dibromo-3,4-dihydro quinazoiin-4- one derivatives (VI to XXII), all the compounds exhibited 20 to 80 % protection except compounds XII, XV & XVIII which showed 190.29 %, 140.24 % and 130.23 % protection, respectively at a dose o f 100 mg/kg (bw) at 1 hour after administration and the analgesia was completely reduced after 2 hrs. Compound XVII and XXI showed 108.90 % protection after 2 hrs. At 3 hrs compound XVII showed 127.07 % protection and the analgesia o f rem aining compounds was found to be completely ceased.

Acetanilide and oxadiazole derivatives (XXV to XXVII) also showed lower degree o f analgesia. Compound XVII possessing oxadiazole nucleus showed maximum percentage protection o f 81.65 at 3 hrs and compound XXV showed 163.40 % protection after I hour and compound XXVI showed 160.26 % protection after 2 hours after the administration.

At a dose o f 200 mg/kg (bw) 2-substituted phenyl-3-substituted arylidenimino-6,8- dibromo-l,2,3,4-tetrahydro quinazoIin-4-one analogs (XXXV to XLVI) did not show good activity. Compounds XXXV and XLV showed 40.14 and 50.15 % protection after I hour after administration and the activity was completely ceased after 2 hours. Compounds XXXVI, XLIV, XLVI did not show any activity even after 3 hours and compounds XXXVII, XXXIX, XL, XLI, XLII, XLIII showed 54.41, 81.65, 99.82, 136.15 and 108.90 % protection

after 3 hours after administration.

From the study, it was concluded that all compounds showed lower degree o f

peripheral analgesic activity.

135

136

Table No. 11

Analgesic activity o f 2-m ethyl-3-substituted aiylidenim ino-6,8-dibrom o-3,4-dihydro quinazolin-4-one and some other com pounds by Tail Im m ersion M ethod

CompoundsAfter 1 hr After 2 lirs After 3 lirs

Mean ± S.D. % Protection Wean ± S.D. % Protection Mean ± S.D. % Protection

VI 4.33 ±0.58 30.13 4.67 ± 0.58 27.16 3.33 ±0.58 -9.17

VII 5.67 ±0.58 70.17 4.00 ± 0.00 8.99 3.33 ±0.58 -9.17

VIII 4.67 ±1.15 40.14 5.33 ±0.58 45.32 4.33 ±0.58 18.07

X 4.67 ± 0.58 40.14 4.33 ±0.58 18.07 4.33 ±0.58 18.07

XI r3.67± 1.15 10.11 3.67 ±0.58 0.00 4.00 ± 0.00 8.99

XII 9.67 ±0.58 190.29 10.00 + 0,00 172.48 10.00 ±0.00 172.48

XIII 6.00 ±2.65 80.18 4.67 ± 2.08 27.16 3.67 ± 1.15 -0.09

XIV 6.00 ±1.73 80.18 4.33 ±1.15 18.07 3.67 ±0,58 -0.09

XV 8.00 ±1.73 140.24 5.67 ±1.15 -54,41 4.33 ± 1.53 18.07

XVI . 6.00 ± 2.65 80.18 4.67 ± 2.08 . 27.16 4.00+1.00 8.99

XVII 5.33 ±1.33 60.16 7.67 ± 2.08 108.90 8.33 ± 2.08 127.07

XVIII 7.67 ±2.08 130.23 8,00 ±2.00 117.98 8.33 ±2.08 127.07

XIX 4.33 ±0.58 30.13 4.33 ± 1.15 18.07 3.33 ±0.58 -9.17

XX 5.33;±0.58 60.16 5.00+1.00 36.24 4.00 ± 1.00 8.99

XXI 5.67 ±0.58 70.17 7.67 ±0.58 108.90 5.00 ± 1.00 36.24

XXII 4.00 ±1.00 20.12 4.33 ±1.53 18.07 • 4.00 ± 1.73 8.99

XXVI 6.67 ± 0.58 100.20 9.67 + 0.58 163.40 4.67 ±0,58 27.16

XXV 8.67±1.15 160.26 6.67 ±1.15 81.65 4.67 ±0,58 27.16

XXVII 5.00 ± 1.00 50.15 6.00 ± 1,73 63.49 6.67 ± 0.58 81.65

Pcntazocinc 4.67 ±0,58 40.14 13.67 ±0.58 272.39 11.33 ±1.53 208.81

Centre! 3.33 ± 0.58 3,67 ±0.58 3.67 ±0.58

137

Table No. 12

Analgesic activity of 2-substituted phenyl-3-substituted arylidenimino-6,8-dibromo-1,2,3,4-tetrahydro quinazolin-4-one derivatives by Tail Immersion Method

CompoundsAfter 1 hr After 2 hrs After 3 hrs

Mean ± S.D. % Protection Mean ± S.D. % Protection Mean ± S.D. % Protection

XXXV 4.67+1.53 40.14 4.00+1.00 8.99 4.33 ± 0.58 18.07

XXXVI 3.67 ± 1,15 10.11 3.67 + 0.58 -0.09 3,67 ± 0.58 -0.09

XXXVII 4.00+1.00 20.12 5.00 ±1.00 36.24 5.67 ± 0.58 54.41

XXXIX 3.67 ± 0.58 10.11 6.67±1.15 81.65 6.67 ± 1.15 81.65

XL 5.33 ±0.58 60.16 5.67 ±0.58 54.41 6.67 ± 1.15 81.65

XLI 4.33 ± 0.58 30.13 7.33 ±0.58 99.82 7.33 ± 1.53 99.82

XLII 4.67 ± 0.58 40.14 7.33 ±0.58 99,82 8.67 ± 1.15 136.15

XLIIl 6.33 ±1.15 90.19 8.00 ±1.00 117.98 7.67 ± 2.08 108.90

XLIV 3.33+0.58 0.10 - ■4.33 ± 0.58 18.07 4.33 ± 0.58 18.07

XLV 5.00 ± 1.73 50.15 5.33 ±2.08 45.32 3.67 ± 0.58 -0.09

XLVI 3.33 ±0.58 0.10 3.67±1.15 -0.O9 4.00 ± 1.00 8.99

Pcntnzocinc 4.67 ± 0.58 40.14 13.67 ±0.58 272.39 11.33 ±1.53 208.81

Control 3.33 ±0.58 3.67 ±0.58 3.67 ±0.58

ANTICONVULSANT ACTIVITY

Electroshock induced seizure is a common model o f epileptic seizure and has been used to screening of antiepileptic drugs. The increasing current electroshock seizure test

(ICES) is a new method for the assessment o f anti-convulsant and pro-convulsant activity in small number o f animals. Moreover, this test was used to evaluate the weak anticonvulsant effect of morphine. The characteristic feature o f ICES test is that it employs an increasing current delivery system. An electroshock is applied through ear electrodes, and the stimulus current is continuously increased at a constant rate from the non-convulsive level until the mouse shows convulsions. Increasing current electroshock produced serial symptoms as follows (1) vocalization and struggling (2) generalized colonic seizure with loss o f righting reflux (3) tonic flexion (4) tonic extension (5) death. Tonic extension o f the hind limb was selected as the end point.

The effects o f 2-methyl-3-substituted arylidenimino -6,8-dibromo-3,4-dihydro quinazolin-4-one derivatives (VI to XXII, XXV to XXVIl) and 2-substituted phenyl-3- substituted ary!idenimino-6,8-dibromo-l,2,3,4-tetrahydroquinazolin-4--one derivatives (XXXV to XLVI) on seizure threshold current (STC) are shown in Table no. 13 & 14.

Among the 2-methyl-3-substituted-6,8-dibromo-3,4-dihydroquinazolin-4-one

derivatives, all compounds increased STC at a dose o f 100 mg/kg (bw) and all were statistically significantly different (p<0.05) from the control group except compound XXL All compounds (VI to XXII, XXV to XXVII) were very highly significantly (p<0.001) different from the standard anti-convulsant drug, phenytoin [30 mg/kg (bw)] indicating that these compounds were not effective as standard drug except compound XVI and XIV. These compounds increased the STC as standard drug and these are not statistically significantly (p<0.5) when compared with standard drug phenytoin indicating that, the effects o f these two

compounds (XVI & XIV) are similar to that o f phenytoin.

In 2-substituted phenyl-3-substituted arylidenimino -6,8-dibroino-l,2,3,4-tetrahydro quinazolin-4-one derivatives at a dose o f 200 mg/kg (bw), compounds XXXVI and XLVI increases STC statistically indicating that they are very highly significantly (p<0.001) different from control and compounds XXXV, XXXVII, XL, XLI, XLIIl and XLV were slightly statistically significant (p<0.01). Compound XLIl showed statistically significant

(p<0.05) increase in STC and rest were not significant. All compounds were highly

statistically significantly (p<0.001) different in comparison to phenytoin.

From the study, it was concluded that quinazolinone nucleus possess a weak

anticonvulsant property.

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Table No. 13Anticonvulsant activity of 2-methyl-3-substitated arylidenimino-6,8-dibromo-3,4-

dihydroquinazoIin-4-one derivatives

S.No. Compounds

Seizure threshold cu rren t (mA)

M ean ± S.D.

1. VI 17.67±0.82**

2. r VII 15.66±1.97

3. VIII 15.33±1.03

4. X 19.33±1.03**

5. XI 17.33±2.73**

6. XII 17.33±1.03**

7. XIII 21.67±1.97**

8. XIV 25.67±1.51*

9. XV 17.33+1.03**

10. XVI 27.00±3.95*

11. XVII 17.67+3.88**

12. XVIII 16.00±0.00**

13. XIX 19.67±0.82**

14. XX 22.33±1.97**

15. XXI 14.67±1.03

16. XXII 19.33+1.03**

17. XXV 22.67+2.07**

18. XXVI 17.33+1.03**

19. XXVII 16.67+1.03**

20. Control 13.33±1.03

21. Phenytoin 27.00±1.10

* p < 0.5, ** p < 0.001 wlien compared with standard drug (Phenytoin)

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Table No. 14

Anticonvulsant activity o f 2-substitutcd phenyl -3-substituted aryiidenimino-6,8- dibromo-l,2^^tetrahydroquinazoIin-4-one derivatives

S.No. Compounds

Seizure threshold cu rren t (mA)

M ean ± S.D.

1. XXXV 15.00± 1.10**

2. XXXVI 18.67±2.07***

3. XXXVII 15.33±2.07**

4. XXXIX I3.33±1.03

5. XL 15.33±1.03**

6. XLI 15.33+1.03**

7. XLII 14.67±1.03*

8. XLIII 15.00±1.10**

9. XLIV 14.00±0.00

10. XLV 15.00±1.10**

U . XLVI 16.00±1,79***

13. Control 13.00±1.10

14. Phenytoin 27.00±1.10

* p < 0.5, ** p < 0.01, *** p < 0.001 when compared with control (n == 6)

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ANTIBACETIRAL ACTIVITY;

The antibacterial activity of the test compounds (100 fxg/ml) were studied against Staphylococcus aureus (gram positive bacteria) and Escherichia coli (gram negative bacteria) by agar diffusion method in particular cup-plate method using norfloxacin (lOng/ml) anderythromycin (lOng/ml) as a standard for comparison. The zone o f inhibition o f testcompounds and standard drugs are given in Table no. 15.

In general, all test compounds (VI to XXII, XXV to XXVII, XXXV to XLVI)showed only moderate action against S. aureus and compounds VII, XXVII, XXXV, XXXVII showed comparable antibacterial activity to erythromycin, compounds XV, XVI, XVII, XLV and XLVI did not show antibacterial activity against E. coli.

Table No. 15

Anti-bacterial activity of newly synthesized compounds

S.No. CompoundZone of inhibition (mm)

S. aureus £. coli

I. VI -hr ++

2. VII ■H" ++

3. VIII ++ ++

4. X + ++

5. XI + ++

6. XII + -H-

7. XIII + -H-

8. XIV + ++

9. XV + -

10. XVI + -

11. XVII + -

12. XVIII + ++

13. XIX + ++

14. XX + +

15. XXI + -

16. XXII + -

17. XXV ++ ++

18. XXVI ++ ++

19. XXVII ++ +++

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20. XXXV + +++21. XXXVI + +22. XXXVII + +++

23. XXXIX + +

24. XL + +

25. XLI ++ ++

26. XLII +4- ++

27. XLIII ++ -H-

28. XLIV ++ +

29. XLV ++ -

30. XLVI ++ -

31. Norfloxacin -

32. Erythromycin - ++++

+ = Poor activity (0-5 mm)= M oderate activity (6-10 mm)= Good activity (1 l-15mm)= Excellent activity (16-20 mm)

SUMMARY

SUMMARY

2-Methyl-3-(substituted arylidenimino)-6,8-dibromo-3,4-dihydro quinazolin-4-ones, 2- (substituted arylidine)-3-(substituted arylidenimino)-6,8-dibromo-I,2,3,4-tetraliydro quinazolin- 4-ones, acetanilide derivatives and substituted flavotie derivatives liave been synthesized for testing their biological actions. All the synthesized compounds have been characterized by the spectral studies.

Fifteen new 2-methyl-3-(substituted arylidenimino)-6,8-dibromo-3,4-dihydro quinazolin- 4-one derivatives were synthesized by condensing N-acetyl-3,5-dibromo anthranil hydrazide v/ith different substituted aldehydes. Substituted oxadiazole, triazole and pyrazolidine were also prepared from N-acetyl-3,5-dibromo anthranil hydrazide by reacting CS2, NH4SCN, 2-nitro benzoic acid and ethylacetoacetate respectively. All the newly synthesized compounds were screened for their analgesic, anticonvulsant and anti-bacterial actions. Besides these pharmacological activities, selected compounds were tested for toxicity studies like LD 50, EDso and ulcerogenic toxicity studies.

Compounds VI and XLV were selected for the determination o f LD50. The values are given in result and discussion section. Compounds VI to XX, XXV to XXVII and XXXV to XLVI were selected for the determination of ED50 and the results are given in Table 1. Among the test compounds VI to XX and XXV to XXVII showed good analgesic activity at lower dose in comparison to the compounds XXXV to XLVI.

Compounds VIII, XI, XIII, XV, XX and XXXV, XXXVII, XXXIX, XLIII, XLVI were selected for the evaluation o f ulcerogenicity at a dose o f 100 mg/kg (bw) and 200 mg/kg (bw) respectively, using Aspirin, 25 mg/kg (bw) and Diclofenac, 20 mg/kg (bw) as standards for comparison. None o f the test compounds produced ulcer in test animals, while diclofenac and aspirin produced ulcer and the average number o f ulcers founds in the gastric m ucosa are presented in the Table 2.

All the pharmacological data were subjected to the statistical analysis by One-way ANOVA followed by Dunnet’s test. All the test compounds showed significant difference when

compared to control.

The analgesic activity of all the synthesized compounds were screened by acetic acid induced writhing method at different doses and also by tail immersion method and the results are given in Table 3-12. All the compounds showed dose dependent response. Among the synthesized compounds o f 2-Methyl-3-(substituted aryIidenimino)-6,8-dibromo-3,4-dihydro quinazoin-4-ones, compound XX was found to be most active at low dose [50 mg/kg (bw)]. Statistically, at a doe o f 50 mg/kg (bw), compound XX was found to be quite superior in its analgesic activity when compared to aspirin [25 mg/kg (bw)]. Compounds VIII, XII, XIX and

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XXVI showed comparable analgesic activity to aspirin and compounds XI, XIV, XVI and XXV showed good analgesic activity and rest of the compounds showed moderate analgesic activity. At a dose o f 100 mg/kg (bw), all the compounds in this series showed 66 to 83 % protection. Compound XX showed 83.7 % protection followed by XIV and XXVI, which exhibited 80.65 % protection. Compounds XII, XVI, XIX and XXVII exliibited 79.04 % protection.

In the second series, 2-(substituted arylidine) -3-(substituted arylidenimino)-6,8- dibromo-l,2,3,4-tetrahydro quinazolin-4-ones (XXXV to XLVI), none of the compound showed 50 % of analgesic activity at a dose o f 50 mg/kg (bw). At a dose o f 100 mg/kg (bw) compound XLIII showed good analgesic activity followed by XXXIX in comparison to Aspirin. Statistically, all the test compounds were not as good as diclofenac in their analgesic activity. A t 200 mg/kg (bw) compounds XXXV, XXXIX, XLI, XLII, XLLIII and XLVI showed percentage protection o f analgesia in the range o f 77-80 % protection.

In the tail immersion method, onh’ few compounds showed good analgesic activity and rest showed a lower degree of analgesia.

Anticonvulsant activity o f all the test compounds was screened by ICES (Increasing Current Electroshock Seizure) method and the effects of synthesized compounds on seizure threshold current (STC) are shown in Table 13 & 14. Compounds XVI and XIV showed sim ilar anticonvulsant activity as phenytoin and the remaining compounds showed weak anticonvulsant property.

All the test compounds were tested for their antimicrobial activity against S. aureus and E. coli. The results are given in Table 15. Compounds VII, XXVII, XXXV and XXXVII showed good activity in comparison to erythromycin against gram negative bacteria {E. coli).

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