249

CHAPTER V

SYNTHESIS, CHACTERISATION AND

BIOLOGICAL ACTIVITY OF NOVEL

PYRAZOLINE DERIVATIVES.

250

INTRODUCTION

PYRAZOLES

Pyrazole refers both to the class of simple aromatic ring organic

compounds of the heterocyclic series characterized by a 5-

memberedring structure composed of three carbon atoms and two

nitrogen atoms in adjacent positions and to the unsubstituted

parent compound. Being so composed and having pharmacological

effects on humans, they are classified as alkaloids although they are

not known to occur in nature.

Pyrazoles are produced synthetically through the reaction of α,

β-unsaturated aldehydes with hydrazine and subsequent

dehydrogenation. Hydrogenation is a chemical reaction in which

unsaturated bonds between carbon atoms are reduced by

attachment of a hydrogen atom to each carbon.

NN

H

NH2NH

2O

It may also be prepared by the union of diazomethane with

acetylene and by warming the acetal of propargyl aldehyde with an

aqueous solution of hydrazine sulphate. It crystallizes as colourless

needles, which is very stable and behaves as a weak base. It does

not combine with the alkyl iodides. On oxidation with potassium

permanganate the C-alkyl-derivatives give carboxylic acids, whilst

251

the N-phenyl derivatives frequently split off the phenyl group

(especially if it be amidated) and have it replaced by hydrogen. On

reduction, the pyrazoles with a free NH group are scarcely affected,

whilst the N-phenyl derivatives give pyrazolines, or by the use of very

strong reducing agents the ring is ruptured and

trimethylenediamine derivatives are formed. They yield substitution

derivatives with the halogens, bromine being the most effective. The

chloro-derivatives are most readily prepared from the pyrazolones by

the action of phosphorus oxychloride.

The pyrazole carboxylic acids may also be obtained by

condensing 1, 3-diketone or oxymethylene ketone carboxylic esters

with hydrazines, or the diazo fatty esters with acetylene

dicarboxylic esters. The dihydro pyrazoles or pyrazolines are less

stable than the pyrazoles and are more like unsaturated

compounds. They are weak bases, which are only soluble in

concentrated acids. On reduction, they yield pyrazolidines, or the

ring is broken; and when oxidized they form blue or red colouring

matters. The carboxylic acids show a remarkable behavior on

heating, the nitrogen is entirely eliminated, and trimethylene

carboxylic acids are obtained. Pyrazoline is a colourless liquid,

which boils at 144 °C. It may be prepared by the action of

diazomethane on ethylene.

252

The pyrazolones (ketodihydropyrazoles) first prepared from the

elimination of the elements of alcohol from the hydrazones of 0-

ketonic acids; or on the oxidation of the pyrazolidinones with ferric

chloride. Three types are possible with the formulae: H2C CO I; NH

HC: N Pyrazol-5-one. They form salts with both acids and bases,

and yield benzylidine and isonitroso derivatives. Pyrazolone is

obtained by the condensation of hydrazine with formyl acetic ester.

It is a colourless crystalline solid, which melts at 164 °C. 1-Phenyl-

3-methyl pyrazole-5-one, is antipyrine. The isomeric 1-phenyl-5-

methylpyrazol-3-one is formed by condensing acetoacetic ester

with acetophenyl hydrazine in the presence of phosphorus oxy

chloride, or by the action of ferric chloride on the corresponding

pyrazolidone, which is produced by condensing phenyl hydrazine

with an O-halogen butyric acid. When methylated it yields

isoantipyrine, an isomer of antipyrine, which is more poisonous.

Pyrazolidines are tetrahydro pyrazoles. The N-phenyl

derivative, from sodium phenyl hydrazine and trimethylene

bromide, is oil, which readily oxidizes to phenyl pyrazoline on

exposure. Isomeric compounds may arise here when phenyl

hydrazine is used, the keto-group taking either the 3 or 5 position;

thus with ß-iodopropionic acid 1-phenylpyrazolidine-5-one is

formed, whilst potassium 1,3-iodopropionate gives the 3-

compound. Isomers of this type may be distinguished by the fact

253

that the pyrazolidine-5-one compounds are basic, whilst the 3-

compounds are acidic. The simplest member of the series,

pyrazolidine-5-one, is a liquid that is formed by the action of

hydrazine on acrylic acid. The 3.5-pyrazolidones are the cyclic

hydrazides of the malonic acid series.

Thiopyrazoles have been obtained by the action of an

aqueous or alcoholic solution of the methyl chloride or iodide of

phenyl methyl chloro pyrazole on a solution of an alkaline

hydrosulphide into which carbon bisulphide has been passed; or

by the action of sodium thiosulphate on antipyrine hydrochloride

or a similar compound1.

Pyrazoles are used for their analgesic, anti-inflammatory,

antipyretic, antiarrhythmic, tranquilizing, muscle relaxing,

psychoanaleptic, anticonvulsant, monoamineoxidase inhibiting,

antidiabetic, antimicrobial2 and antibacterial activities.

Structurally related compounds are pyrazoline and pyrazolidine.

NN

NN

H

NN

H

H

The above three represent heterocyclic nomenclature to

pyrazolines require that nitrogen atoms to be numbered one and

two in each structure. Substituted 1-pyrazolines are numbered to

produce the lower locations, or in the case of complicated

254

structures are produce the simplest name consistent with clarity of

meaning. Numbering of the 2-pyrazolins begins with the amino

nitrogen and pyrazolines are numbered to obtain for the double

bond the lower of the two possible numbers. Thus, here this

structure will be referred as:

NN

1

2

34

5

The pyrazoles, pyrazolines and the pyrazolidines form an

interesting class of compounds showing diverse biological

activities. The pyrazoline and pyrazolidines can be considered as

the hydrogenated, compounds of pyrazoles. Many pyrazoles and

pyrazolines surveyed in the literature have been screened against

various microorganism and their pharmacological activities also

compared.

PYRAZOLINES: CHEMISTRY AND METHOD OF SYNTHESIS

There are different methods that have been reported for the

synthesis of pyrazolines.

Rao et al3 synthesized pyrazolines through cycloaddition of

diazomethane to chalcone, which in turn were obtained by crossed

aldol condensation of appropriately substituted benzaldehyde and

acetophenones. Earlier reporters4,5 has suggested two alternative

structures (2) and (3) for these pyrazolines.

255

R

RHC=CH-C

R

RO

R

R

NHN

C

O R

R

R

R

NN

C

O R

R

(1)

(2)(3)

1

2

3

1

1

2

2

3

3

Mustafa et al6 were report that 5-arylcryloyl-6-hydroxy-4, 7-

dimethoxy-benzofuran react with hydrazine hydrate in ethanol to

give hydrazones which on boiling with acetic acid yield the

corresponding pyrazolines.

Aziz et al7 reported the reaction between hydrazine hydrate

and 5-aryl, acryloyl-6-methoxy-benzofuran was shown to yield the

corresponding 2-pyrazoline via., 1.4 addition shown to yield the

corresponding 2-pyrazoline via., 1.2 addition in acetic acid, the

isomerization of 1, 4 addition product to 1, 2 addition product in

boiling acetic acid was also reported by these workers.

Wadodkar et al8,9 by taking account of above synthesis is

reported that 2-hydroxy chalcone (1) reacts with hydrazine

hydride, but the products were identified as 5-aryl-3-(2-hydroxy

256

phenyl)-2-pyrazoline(2).The formation of pyrazoline is possible only

by 1,2 addition.

On boiling in acetic acid this pyrazoline did not undergo

isomerization as reported by Musfate et al10 5-aryl-1-acetyl-3- (2-

hydroxy phenyl) 2-pyrazoline (3).

It was been also reported that hydroxyl chalcone (4) with

hydrazine hydrate in dimethyl formamide or hyrazone

hydrochloride and aqueous sodium acetate or sodium hydroxide in

ethanol were similar to (5) and not the corresponding isomer of (6).

Further it has been also reported that hydroxyl chalcone failed to

react with hydrazine hydrochloride in ethanol alone, even on

prolonged refluxing (6 to 8 h).

OHR

RC-CH=CH

O

RNNO

HbHa

HxR

R

H

RNNOH

Hb

Ha

HxR

R

Ac

R

3 3

3

1

2

1

2

1

2

4 5 6

Hydroxyl chalcone reacts with hydrazine hydrochloride in dimethyl

formamide (soft base) to give (7), which remains unchanged on prolonged boiling

in acetic acid.

C-CH=CH

OHR

R

O

NNOHR

R Hb

HaNH2NH

2.HCl

DM

1

2 2

1

7

257

Osman et al11 has reported that substituted cinnamolyl

chloride (8) reacts with 2-aryl-5H-oxazolo, (4,5b) phenoxazoline (9)

in dioxane in the presence of triethylamine to give (10). 5-

substituted benzalacetyl oxazolo phenoxazine derivative (10) react

easily with hydrazine hydride and Phenyl hydrazine to give

corresponding pyrazolines.

It has been reported that interaction of compound (10) with

hydrazides under suitable conditions gave a variety of pyrazolines.

Hydrazine hydrate itself interacts with (10) in dioxane giving

unstable pyrazolines (11). But when this was treated with glacial

acetic acid, then stable monoacetyl pyrazoline was obtained (12).

258

CH=CHCOCl

N

O

H

N

OAr

N

O

COCH=CH

N

OAr

NH2NH

2H

2O

GLACIAL ACETIC ACID

R

N

O

N

OAr

N N

CO

CH3

N

O

N

OAr

N NC

6H

5

RR

+

9

10

11 12

8

Phenyl hydrazine reacts with (10) in the presence of base

catalyst. The reaction was carried out in diaxone in presence of

piperidine giving N phenyl pyrazoline (12).

Sachchar et al12 synthesized fluorinated heteroaryl

pyrazolines, in this the fluorinated hetero aryl chalcone reacts with

phenyl hydrazine to yield phenyl hydrazone, which immediately

rearranges to the corresponding isomeric, 1-phenyl-3- (substituted

fluro phenyl)-5-heteroaryl pyrazolines(13) in presence of glacial

acetic acid. In this case it was reported that mixture was refluxed

259

in an oil bath at 1200-130oc for 3 to 4 hours. After cooling the

resultant mass was diluted with water and the solid obtained was

recrystallized from ethanol. These derivatives when placed on filter

paper and exposed to bromine vapour tuned green and have

identified as pyrazolines.

CH3COOH

C6H

5NH

2NH

2NN

R

CH=CH-C R

RO

R

120-130o

+

13

1

1

260

Review of Literature

PYRAZOLES:

Dadiboyena et al13 synthesized pyrazole derivatives (14) and

reported their anti bacterial and anti inflammatory activity.

Porter et al14 synthesized Tetrahydroisoquinoline amide (15)

substituted phenyl pyrazoles as selective Bcl-2 inhibitors.

O

N

NH2

NN

Cl

O

N

15

Wahab et al15 synthesized and evaluated antimicrobial

activity of 1-(benzofuran-2-yl)-4-nitro-3-aryl butan-1-ones and 3-

(benzofuran-2-yl)-4,5-dihydro-5-aryl-1- 4-(aryl)-1,3-thiazol-2-yl]-

1H-pyrazoles(16), (17) and (18).

NN

R

NH

CH3

14

261

N NO

N

S

Ar

N NO

NH2

S

OAr

NO2

16 17 18

Prakash et al16 synthesized and evaluated antibacterial

activity of some new 2,3-dimethoxy-3-hydroxy-2- (1-phenyl-3-aryl-

4-pyrazolyl) chromanones(19).

.

O

OOH

OMe

OMeN

N

Ar

O

OOH

Ph

N

N

Ar

Ph

IBD

MeOH

19

Gupta et al17 Synthesized N-aryl-5-amino-4-cyanopyrazole

derivatives (20) as potent xanthine oxidase inhibitors.

R

NN NH

2

CN

HC(OEt)3,Ac

2O

NH3

R

NN

N

N

NH2

20

Kralj et al18 reported a simple synthesis of 4-(2-aminoethyl)-

5-hydroxy-1H-pyrazole (21).

262

N O N O

NMe2

NN

NH2

OH

COPh COPh

21

Pyrazoline as antimicrobial and anti bacterial:

Sachchar et al19 synthesized several 1-phenyl-3-(substituted

fluro phenyl)-5-heteroayl-2-pyrazolines (22) and noted their anti

microbial as well as anti bacterial actions especially against

bacteri-B anthrasis.

N N

RR

R = furyl, thienyl,pyridylR1= F,Cl,OH.CH3

1

22

Many other authors also reported the synthesis and

antibacterial activity of some new pyrazoline derivatives.20-24

Stirrewiberg et al25 reported antibacterial as well as

insecticidal action of some new pyrazoline derivatives (23).

R

NN

R

C

O

NH R

R

23

1

2

3

263

Desai et al26 synthesized several pyrazoline derivatives of

phenothiazines and reported their antibacterial activity and

tuberculostatic activity of some compounds.

Ead et al27 reported the synthesis of two novel series of

pyrazoles and 2-pyrazolines with their antibacterial activity.

Pyrazolines as antifungal:

Sachchar et al28 extended their studies on several pyrazoline

derivatives and reported them as antifungal. Aspergillus niger and

Helmithosporium sativum were employed as fungi for the testing of

fungicidal activity.

Ritcha et al29 synthesized 3, 5 dimethyl -4- nitro pyrazole

and 1, 3, 5 trimethyl-4-nitrosopyrazole compounds and have

reported them as anti fungal agents. Their fungicidal activity

increased by increasing the size of hydrocarbon side group was

also reported by them.

Mitra et al30 reported that 5-pyrazolone and its derivatives 4-

acetyl-2-pyrazoline-5-one and 2-pyrazoline-5-thione (24) were

associated with significant fungicidal activity against the rice born

pathogen Pyricularia oryzae and brown leaf spot pathogen

Helminthosporium oryzae31-33

264

NN O

CO S

N

R

S

24

Mitra et al34 have synthesized 4-N (aryl) amino methyl-2-

pyrazoline-5-one by Mannich reaction of 2-pyrazoline-5-one

derivative. The same authors also reported cobalt II complex with

2-pyrazoline-5-one derivative as having anti fungal activity.

Sadasiva et al35 synthesized several hydroxyl aryl-pyrazole

(25) and tested them for antibacterial as well as anti-fungal activity

where none of the compounds were found to possess anti-bacterial

activity. But all compounds were found to possess anti-fungal

activity. Antifungal activity has been assessed by employing

Drechslera prostrate (Drechs) and Alternatia alternate (Keissler).

All compounds tested could inhibit the spore germination at 30-

mcg/ml upto a maximum level.

N N

OH

R

R

1

25

R=alkyl or aryl R1=H or phenyl

265

Nayak et al36 synthesized several 4,4‟-bis-5-pyrazoline and 4,

4‟,unsaturated products and found their fungicidal activity against

rice blast pathogen Pyricularia oryazae and brown leaf spot

pathogen, Helmithosporium oryzae.

Tiwari et al37 synthesized several 1-acetyl/aroyl-3-methyl-4-

substituted anilido -5-aryl pyrazolines and 3-methyl-4-substituted

anilido-5-aryl pyrazolines and 3-methyl-4-substituted anilido-5-

aryl isoxazolines, and tested against Cephalosporium sacchari,

Helmithosporium oryzne, and saprolegina parasitica, acellya Orion

all the compounds showned remarkable activity.

Mohanthy et al38 reported that 1-phenyl-5-aryl-1-2-

pyrazoline 3-4-thiazolidine-2-one derivative exhibited antifungal

properties.

The fungicidal activity of the compounds was determined by

poisoned food technique at various concentrations. It was also

reported that all compounds inhibit the growth of the fungus

Aspergillus flavours.

Pyrazoline as antiviral:

Sachachar et al39 extended their study on several phenyl1-3-

(substituted fluro phenyl)-5-hetero aryl-2-pyrazoline derivative and

found antiviral activity against Sunn hemp rosette virus (SRV).

266

Pyrazoline as antiserotonin and anti-oedima:

Another derivative Dipyrone was prepared which showed a

strong antiserotonin and anti-oedima activities in rats40 but found

to be clinically disappointing in the treatment of rheumatic

arthritis41. Other compounds like 4-(N-Nicotinyl amino) derevative

was also found to be less toxic and clinically effective in various

inflammation diseases42

Frangnly et al43 have reported anti-inflammatory activity of

some new pyrazoles pyrazolines and 4(3H)-quinazolines.

Derivative of 3, 5-pyrazolindinedione as anti inflammatory:

After the discovery of the 5-pyrazoline derivative as anti-

inflammatory agents, modifications were made on the basic

structure and this resulted in the synthesis of a new compound,

phenyl butazone, which was found to be a potent inhibitor of

inflammation.

A metabolic product of phenyl butazone was found to be

less toxic and equally potent as the parent compound. The result

was the synthesis of oxyphenbutazone.

Modification of the parent compound to increases the acidity

of the molecule resulted in the synthesis of a new derivative sulfin

267

pyrazole44 it was enhanced uricosuric activity and is potent against

gout.

N

N

R

O

O

R

C6H

5

1

2

26

R1 R2

Phenyl butazone -C6H5 -C4H9

Oxyphen butazone -C6H4-OH (P) -C4H9

Sulfin pyrazone -C6H5 -CH2-CH2-S-C6H5

Pyrazole derivative as anti-inflammatory agent:

Coli et al45 reported that pyrazole derivative itself has anti-

inflammatory activity (27) e.g. Benzylame (Tantum)

NN

CH2-Ph

OCH2R

27

R = -CH2-CH2-N (CH3)2, -COCH3

268

Sarangam et al46 have synthesized number of derivative of

pyrazole –(3,4-d) pyrimidine-4-6 diones (28) and screened them for

C.N.S. depression properties and anti-inflammatory activity. It was

also reported that some derivatives showed anti-inflammatory

properties equivalent to aspirin.47

H

N

N

O

ON

N

R

R

1

28

R1= Phenyl, o-Tolyl R2 = Phenyl, o-Tolyl, o-Anisyl

Pyrazole derivatives as antidiabetic:

Froesch et al48 has reported antidiabetic activity in the 5-

methyl pyrazole-3-carbaxilic acid (29).

CH3

NN

COOH

H

29

Pyrazole derivative as vasodilator:

Brunner et al49 have reported vasodilator activity in pyrazole

derivative (30).

269

N

NN

N

CH3

30

Pyrazole derivative as hypoglycemic and hypotensive agent:

Smith et al50 reported that 3, 5-dimethyl pyrazole and 3-

methyl pyrazole-5-carboxilic acid exhibited hypoglycemic activity

(31).

Arya et al51 synthesized several pyrazolidine derivatives and

reported the hypotensive activity. (32)

NN

CH3

CH3

H

NN

CH3

H

HOOC

31

NN

H

C

O

CH2

CH2

NN CH3

CH3

32

270

Pyrazolidone derivative as inhibitor:

Sweeny et al52 reported that pyrazofurin which is a natural

antibiotic was very effective in inhibiting pyrimidine biosynthesis.

Chasin et al53 discovered a potent new compound pyrazole

pyridine derivative (33) that was 60 times more potent as an

inhibitor of rat brain PDE than theophyllin.

N

C2H

5OOC

NH-N=C-(CH3)

NN

33

Novinson et al54 reported a series of pyrazole pyrimidine

derivatives (34,35) and reported them to be inhibitor of PDE from

rabbit lung and beef heart.

NN

NR

R

NNN

N

R

R

EtOOC

H

1

34 35

1

Many compounds containing the pyrazoline nucleus have

been found to show different biological activities.55-58

271

METHODOLOGY

NH2

CH3

CH3

CH3

ONNH

2NH

2.H

2O

NH2

ON

C

O

R

NH2

ON

CH2CO

C2H

5OH

C2H

5OH

CH2

C O

O

C

CH2CONHNH

2

CH CH

N

N

R

+

R1

R

PZ1-14

reflux

Scheme IV

R1

CLH

CLE

CH1-14

24hrs

272

Preparation of Caprolactam hydrazide (CLH):

A mixture of t-Butyl (s)-3-amino benzocaprolactum (CLE,2.9gm,

0.01 mol) and 99% of hydrazine hydrate (0.05 mol) in dry ethanol

(50 ml) was refluxed for 24 Hrs. The solution upon cooling gave a

solid mass that was filtered and recrystallized from chloroform.

(Yield) 58%, MP: 170 0C.

Synthesis of chalcones (CH1-14):

A solution of acetophenone (12gm, 0.1mol) and benzaldehyde

(11gm, 0.1mol) were prepared in 100ml of ethanol separately. The

acetophenone solution was then added to benzaldehyde solution.

The mixture was stirred for 5mins. Then a solution potassium

hydroxide (10%) was added dropwisely until turbidity appears.

Then mixture was stirred for 12hrs. The precipitate obtained was

filtered, washed with water and recrystallized from ethanol.

Following the same procedure other chalcones were also prepared.

Synthesis of pyrazolines:

A mixture of benzocaprolactam hydrazide (2.5gm, 0.01 mol)

and chalcone (2.0gms, 0.01mol) was refluxed for 6hrs in dry

ethanol (50ml) in the presence of 1ml of glacial acetic acid. Upon

cooling the precipitate obtained was filtered, washed with ether

and recrystallized from the ethanol. Following the same procedure

remaining compounds of the series were prepared. The physical

chacteristics of compounds are complied in the table no.17.

273

Table No.17: Chacterisation data of pyrazoline derivatives (PZ1-14):

S.No

.

Code Substituents Molecular

Formula

M.P.

(oC)

Yield

(%) R1 R

01. PZ1 H H C26H26O2N4 152 70

02. PZ2 H 4-N-Dimethylamino- C28H31N5O2 128 72

03. PZ3 4-Chloro- H C26H25O2N4Cl 145 68

04. PZ4 4-Chloro- 4-Chloro- C26H24O2N4Cl2 116 64

05. PZ5 H 4-Methoxy- C27H28O3N4 180 70

06. PZ6 4-Bromo- 4-Methoxy- C27H28O3N4Br 195 66

07. PZ7 4-Chloro- 4-N-Dimethylamino- C28H30O2N5Cl 204 68

08. PZ8 H 2-Nitro- C26H25O4N3 166 72

09. PZ9 2-Chloro- 2-Nitro- C26H24O4N3Cl 175 66

10. PZ10 4-Bromo- H C26H25O2N4Br 188 74

11. PZ11 H 2-Chloro- C26H25O2N4Cl 126 79

12. PZ12 4-Chloro- 4-Methoxy- C27H27O3N4Cl 180 75

13. PZ13 H 4-Chloro- C26H25O2N4Cl 168 73

14. PZ14 4-Chloro- 2-Chloro- C26H24O2N4Cl2 90 60

NH2

ON

CH2CO N

N

R1

R

274

SPECTRAL DATA OF SYNTHESIZED COMPOUNDS:

Compound CLH:

(3-Amino-2-oxo-2,3,4,5-tetrahydro-benzo[b]azepin -1-yl)-acetic acid hydrazide

IR (KBr)cm-1 :

3364 and 3308(NH and NH2), 3060(Ar.C-H str.), 2970, 2943, 2881,

2835 {Ali.C-H str. of CH2 groups(CH2,N-CH2)asymmetric and

symmetric}, 1744(ring C=O), 1668(C=O of CONH2), 1602(C=N),

1602, 1491, 1456(C=C ring str.), 1395, 1366(C-H bending).

1H NMR:

δ 9.2(1H,s,NH of CONH), 6.87-7.64(4H,m,Ar-H), 4.74-3.0 (6H,m,2H

of NH2 of NHNH2+2H of ring C-NH2 + 2H of CH2 of N-CH2), 2.5(1H

of C-H of ring CH-NH2;merged with solvent peak), 2.23-

2.07(2H,s,ring CH2), 1.74(2H,s,ring CH2)

NH2

ON

CH2CONHNH

2

275

13C NMR of compound CLH:

N

NH2

O

O

NH

NH2

57

68 1 2

4

3 11

12

9

10

Sl.No Ppm Values Assignment

1 27.866 1

2 38.623 2

3 49.456 3

4 51.062 4

5 122.722 5

6 126.084 6

7 127.299 7

8 128.875 8

9 135.918 9

10 141.513 10

11 167.537 11

12 174.639 12

276

277

278

279

Compound PZ1:

3-Amino-1-[3-(3,5-diphenyl-4,5-dihydro-pyrazol-1-yl)-2-

oxo-propyl]-1,3,4,5-tetrahydro-benzo[b]azepin-2-one

IR (KBr)cm-1 :

3237(weak broad peak of –NH2), the disappearance of peaks at

3363, 3308 for NH and NH2 of CONHNH2 support the formation

pyrazoline. 2937, 2862 (Ali. C-H str. of CH2 groups), 1744(ring C=O

properly resolved). The change in the nature of the broad peak at

1667(C=O of side chain) may be due to fermi resonance,

1600(C=N), 1558, 1491, 1458(C=C ring str.), 1398(C-N), 758(mono

substituted phenyl rings),

1H NMR :

The disappearance of the signals at 9.2 for NH of CONH and 2H of

NH2 of CONHNH2 of the hydrazide confirm the formation of

pyrazoline. Other important of signals of pyrazoline PZ1 are δ 8.0-

7.0(14H,m, Ar-H), 5.0(2H,s,NH2 of CHNH2 of ring),4.6-

4.2(2H,dd,2H of pyrazoline), 4.0-3.2(6H,m,4H of ring 2xCH2 + 2H

of N-CH2). The C-H of -CHNH2 of ring perhaps merged with solvent

peak.

NH2

ON

CH2CO N

N

280

281

282

Compound PZ2:

3-Amino-1-{3-[5-(3-dimethylamino-phenyl)-3-phenyl-4,5-dihydro pyrazol-1-yl]-2-oxo-propyl}-1,3,4,5-tetrahydro-benzo[b]azepin-2-one

IR(KBr)cm-1 :

3398(NH2), The characteristic absorption peaks for NH and NH2 of

CONH2 of hydrazide have disappeared. 2922,2204(Ali. C-H str.of

CH2and CH3 groups), 1740(ring C=O), 1670 (C=O of side chain),

1599(C=N), 1566, 1521, 1490(C=C ring str.), 1444,1361(C-H

bending of CH2 and CH3 groups), 769,815(substituted benzene).

1H NMR:

Disappreance of signal for NH of CONH of hydrazide. δ

7.8-7.2(13H,m,Ar-H), 6.6(2H,dd,NH2), 4.8 and 4.2(2H,d,2H of

pyrazoline), 3.2(2H,s,2H of N-CH2),3.0-2.6(5H,m,2H of ring + 1H of

CHNH2 + 2H of CH2 of pyrazoline), 2.1(6H,s, 6H of (CH3)2-N),

1.8(2H,s,2H of CH2 of ring)

Mass Spectrum: The molecular ion peak at m/z 482

corresponding to the molecular weight of the compound is

obtained. In addition, it also gives M+1 and M+2 peaks. Incedently

the molecular ion peak at m/z 482 it self is a base peak. Other

prominent fragment ion peaks are obtained at m/z

468,397,380,351,308,289,266,252,217,161,144,132,106,91, 78.

NH2

ON

CH2CO

NN

N

CH3

CH3

283

284

285

286

CompoundPZ3 :

NH

2

ON

CH2CO N

N

Cl

3-Amino-1-{3-[3-(3-chloro-phenyl)-5-phenyl-4,5-dihydro-pyrazol

-1-yl]-2-oxo-propyl}-1,3,4,5-tetrahydro-benzo[b]azepin-2-one

IR(KBr)cm-1:

3358(NH2), 3059 (Ar, C-H str.), 2929, 2860 (Ali C-H str of CH2 and

–NCH2 groups). The carbonyl peaks at near 1740 clearly

resolved for ring C=O.1670(C=O of side chain), 1599(C=N), 1529,

1491, 1460(C=C ring str.), 1400 (C-N), 1359, 1300 (C-H bending of

CH2), 833, 760 (substituted phenyl rings), 588(C-Cl).

1H NMR:

The signal for NH of CONH at δ 9 is not observed. δ 8.4-

6.7(13H,m,Ar-H), 4.9(2H,d,2H of NH2), 4.2 and 4.6(2H,dd,2H of

pyrazoline), 4.3(2H,s,2H of N-CH2), 2.8-2.6 and 2.2-1.8(6H,m,6H of

ring (CH2)2 and C-H +1H of pyrazoline) Due to the poor solubility of

compound the signals are not clearly appeared. How ever the

disappearance of characteristic hydrazide peaks in the NMR

spectra of pyrazoline supports the formation of pyrazoline. The

structure is further confirmed by its mass spectrum.

287

Mass:

The molecular ion peak of the compound is observed at m/z 471

corresponding to the molecular weight of the compound. In

addition it gives M+1, M+2 peaks. Indecently this is the base peak.

Further the fact that the peak heights of M+1 and M+2 peaks in

the ratio 3:1 confirm the presence of Cl in the compound. The

compound under investigation contains chlorine atom in its

molecule.

288

289

290

291

Compound PZ4:

NH2

ON

CH2CO

ClN

N

Cl

3-Amino-1-{3-[3,5-bis-(3-chloro-phenyl)-4,5-dihydro-pyrazol

-1-yl]-2-oxo-propyl}-1,3,4,5-tetrahydro-benzo[b]azepin-2-one

IR (KBr)cm-1 : 3358(NH2), 3059 (Ar. C-H str.), 2929, 2860(Ali. C-H

str. of CH2 and –NCH2 groups). The carbonyl peaks at near 1740

clearly resolved for ring C=O. 1670(C=O of side chain), 1599(C=N),

529, 491, 460(C=C ring str.), 1400(C-N), 1359,1300 (C-H bending

of CH2), 833, 760(substituted phenyl rings), 588(C-Cl).

1H NMR: The disappearance of peak for NH of CONH of hydraide

supports the formation of pyrazoline. δ 8.2-7(12H,m, Ar-H),

4.9(2H,d,2H of NH2), 4.4-4.6 and 4.2-4.0(2H,dd,2H of pyrazoline),

4.3(2H,s,2H of N-CH2), 2.8-1.8(6H,m,4H of 2xCH2 + 1H of CH-NH2

+ 1H of pyrazoline).

Mass Spectrum: The molecular ion peak of the compound is

observed at m/z 505 corresponding to the molecular weight of the

compound. This is also a base peak. M+, M+1, M+2 peaks are also

observed. The peak heights of M+1and M+2 peaks are in the ratio

3:1.This suggests presence of Cl and confirms the formation and

structure of the compound.

292

13C NMR of compound PZ-4:

N

NH2

O

NO

N

Cl

Cl

1 2

6

22

4 21

35

20

13

13

11

11 18

14 9

9 12

12

7

19

158

10

a

PZ-4

Sl.No Ppm Values Assignment

1 20.419 1

2 21.641 2

3 27.671 3

4 36.911 4

5 37.79 5

6 50.447 6

7 118.49 7

8 122.784 8

9 127.28 9

10 127.478 10

11 128.732 11

12 128.883 12

13 129.518 13

14 130.455 14

15 130.629 15

16 133.826 16

17 134.676 17

18 135.7.1 18

19 136.075 a

20 137.974 19

21 142.971 20

22 172.463 21

23 187.995 22

293

294

295

296

297

Compound PZ6:

3-Amino-1-{3-[3-(3-bromo-phenyl)-5-(3-methoxy-phenyl)-4,5-dihydro pyrazol-1-yl]-2-oxo-propyl}-1,3,4,5-tetrahydro-benzo[b]azepin-2-one

IR (KBr)cm-1 :

The peak for NH and NH2 of hydrazide are disappeared. The

NH2 of CHNH2 perhaps merged with the broad peak appearing

between 3250 and 3064 (Ar. C-H str.), 2935, 2841(Ali C-H str. CH2

of OCH3 groups), the peak for C=O of ring around 1740 clearly

resolved. The peak at 1567 may be due to C=O of side chain. 1593

(C=N), 1510, 1458(C=C ring str.), 1421(C-N), 1396, 1332(C-H

Bending), 1031 (C-O-C) 819,738(substituted phenyl ring), 665 (C-

Br).

1H NMR:

The disappearance of signal for NH of CONH of hydrazide is

observed. δ 8.1-6.8(12H,m, Ar-H), 5.5(2H,d,2H of NH2),5.2-5.3 and

4.9-4.7(2H,dd,2H of pyrazoline), 4.2(2H,d,2H of N-CH2),

3.8(3H,s,3H of OCH3), 3.7-3.5(1H,m,1H of CH-NH2), 3.2-3.0 and

2.2-2.0(5H,m,4H of 2xCH2 of ring +1H of pyrazoline).

NH2

ON

CH2CO

OMeN

N

Br

298

Mass Spectrum:

The mass spectrum of the compound at m/z 546 corresponding to

the molecular weight of the compound. It is very surprising to see

hear that the peaks at m/z 333, 291 and 259 show 100% intensity

showing the stability of fragment ions as base peak. Other

prominent peaks are observed at m/z 475, 409, 355, 315, 277,

219, 203, 185, 144, 130, 106, 91,78.

299

300

301

302

Compound PZ7 :

NH2

ON

CH2CO

N

CH3

CH3

N

N

Cl

PZ7

3-Amino-1-{3-[3-(3-chloro-phenyl)-5-(3-dimethylamino-phenyl)-4,5-dihydro-

pyrazol-1-yl]-2-oxo-propyl}-1,3,4,5-tetrahydro-benzo[b]azepin-2-one

IR (KBr)cm-1 :

3213(NH2 of ring), peaks for NH, NH2 of hydrazide disappeared.

2935, 2820(Ali C-H str. of CH2 and CH3 groups), 1672 (C=O of side

chain), 1600(C=N), 1523, 1489, 1460(C=C ring str.), 1431(C-N),

1364, 1338(C-H bending of CH2 and CH3 groups), 812,

736(substituted phenyl ring), 523(C-Cl).

1H NMR:

The signal for NH of CONH of hydrazide at down field is

disappeared. δ 6.6-8.2(12H,m, Ar-H), 4.9-1.6(18H,m, 2H of NH2 +

2H of NCH2 + 2H of pyrazoline + 4H of 2 x CH2 of ring + 1H of

CHNH2 + 6H of N(CH3)2 + 1H of pyrazoline).

Mass Spectrum:

The molecular ion peak of the compound is observed at m/z 516

corresponding to the molecular weight of the compound. M+, M+1,

303

M+2 peaks are also observed the peak heights of in the ratio 3:1

indicating the presence of Cl. Other prominent peaks are observed

at m/z 502, 431, 380, 342, 298, 286, 235, 217, 144, 132, 106, 91,

and 77.

304

305

306

307

Compound PZ9 :

3-Amino-1-{3-[3-(2-chloro-phenyl)-5-(2-nitro-phenyl)-4,5-dihydro- pyrazol-1-yl]-2-oxo-propyl}-1,3,4,5-tetrahydro-benzo[b]azepin-2-one

IR (KBr) cm-1 :

3310(NH2), 3065(Ar.C-H str.), 2935,2864(C-H str.ofCH2

groups), 1740,1667(ring C=O and C=O of CH2-C=O), 1601(C=N),

1522,1489,1458(C=C ring str.), 1394(C-N), 1522 &1342(-NO2),

832(1,4-disubstitued phenyl ring), 592(C-Cl)

1H NMR:

δ The signal for NH of CONH (hydrazide) at downfield is

disappeared.6.6-8.8(12H,m,Ar-H), 5.0-4.1(2H,d,2H of NH2), 4.6-4.5

and 4.2-4.1(2H,dd, 2H of pyrazoline), 4.3(2H,s,2H of CH2-N), 2.9-

3.0(1H,m,1H of CH-NH2),2.6(1H,m,1H of pyrazoline), 2.2(2H,d,2H

of ring CH2), 1.8(2H,d,2H of ring CH2).

Mass Spectrum:

The molecular ion peak is observed at m/z 518 corresponding

to the molecular weight of the compound. The M+2 peak at m/z

520 also observed. The peak heights of M+ and M+2 appeared to be

NH2

ON

CH2CO

O2N

N

N

ClPZ9

308

in the ratio of 3:1 due to the presence of chlorine atom in the

molecule. The prominant fragment ion peaks are also observed at

m/z 513, 424, 399,368, 288, 256, 235, 177, 144, 132, 106, 80

etc.,

309

310

311

312

BIOLOGICAL ACTIVITY:

The antibacterial, antifungal and analgesic activities for

Pyrazoline derivatives were carried out by following same protocols

and procedures as mentioned in chapter No.III. The results are

represented in the table no.18, 19 and 20 respectively.

TABLE No.18: Antibacterial activity of synthesized

Pyrazolines (PZ1-14):

*Average triplicate ± Standard deviation Note: ‘-‘denotes no activity, 4-5 mm poor activity, 6-7 mm moderate activity, 8-

9 above good activity.

Sample

Code

*Inhibition zone diameter in mm

B.subtilis B.pumilis E.coli S.aureus

50g 100g 50g 100g 50g 100

g 50g

100

g PZ1 4 6 4 7 4 5 - 5

PZ2 5 8 5 7 4 5 - 5

PZ3 5 7 - 5 4 5 - 6

PZ4 6 8 5 7 4 7 5 8

PZ5 4 6 - 6 4 6 - 5

PZ6 6 8 5 6 4 7 4 5

PZ7 5 7 5 4 4 5 4 4

PZ8 5 7 5 7 4 5 - 5

PZ9 5 8 5 7 4 5 4 6

PZ10 4 5 5 6 5 6 4 4

PZ11 6 7 5 6 5 5 5 5

PZ12 5 8 5 6 4 7 4 7

PZ13 6 7 7 8 - 5 4 5

PZ14 5 7 5 7 4 5 4 6

Ciprofloxacin 6 10 5 9 5 8 6 9

DMF - - - - - - - -

313

Table No.19: Antifungal activity of Pyrazolines (PZ1-14):

Note:- “ – “denote no activity, 06 – 07mm poor activity, 08 – 10mm moderate

activity, 11-12mm good activity.

Sample code

*Inhibition zone diameter in mm

A.Nigier C.Albicans

50g 100g 50g 100g PZ1 05 08 05 08

PZ2 06 10 06 09

PZ3 06 09 06 10

PZ4 07 11 07 11

PZ5 04 07 - 07

PZ6 06 11 05 11

PZ7 07 12 06 11

PZ8 05 10 04 10

PZ9 06 11 06 11

PZ10 05 09 05 09

PZ11 05 09 05 08

PZ12 07 12 - 10

PZ13 05 09 05 08

PZ14 05 11 06 09

Clotrimazole 09 14 08 13

DMF - - - -

314

Table No.20: Analgesic activity of Pyrazolines derivatives:

Treatment

No. of animals

Avg. Wt of Animals (gm)

Avg. Dose (mg)

Basal reaction time (secs.) after

0 min 15 min 30 min

60 min

90 min

120 min

Control gum

acacia 6 34.00 -

3.2 ±0.37

3.81 ±0.473

4.12 ±0.553

4.28 ±0.503

4.87 ±0.324

3.88 ±0.37

2 Standard Pentazoc

in 10 mg/kg

6 28.00 0.28 6.24 ±0.89

12.87 ±1.332

12.91 ±1.320

13.87 ±0.279

13.65 ±0.851

11.83 ±1.04

2

Compound PZ1

6 25.33 2.53 6.35 ±0.35

6.90 ±0.384

8.24 ±0.135

9.52 ±0.453

11.21 ±0.882

9.33 ±0.54

9

Compound PZ4

6 29.33 2.93 5..18

±0.508 8.59

±0.490 9.74

±0.260 12.69

±0.313 10.11

±0.882

9.33 ±0.54

9

Compound PZ5

6 22.00 2.20 6.61

±0.372 7.49

±0.257 8.58

±0.692 8.71

±0.740 9.92

±0.411

8.79 ±0.32

0

Compound PZ7

6 26.00 2.60

6.13

±0.826 8.39

±0.579 9.80

±0.924 11.96 ±1.10

10.584 ±0.493

9.60 ±0.44

8

Compound PZ8

6 20.66 2.06 5.87

±0.775 8.13

±0.288 9.75

±0.316 12.10

±0.919 11.10

±0.684

9.03 ±0.27

7

Compound PZ10

6 27.33 2.73 5.31

±0.374 7.57

±0.487 9.36

±0.553 11.62

±1.146 10.71

±0.666

8.07 ±0.29

8

Compound PZ11

6 27.33 2.73 6.46

±0.743 7.68

±0.349 10.4

±0.794 11.9

±0.971 10.96

±0.346

9.74 ±0.37

0

Compound PZ13

6 29.33 2.93 5.16

±0.632 7.27

±1.505 9.46

±1.368 11.87

±0.078 12.12

±0.675

10.43 ±0.61

9

315

RESULTS AND DISCUSSIONS:

A.Antibacterial activity:

All the pyrazoline derivatives synthesized during the present

investigations were screened for their antibacterial activity against

bacteria B. substils , B.pumilus, E. coil and S. aureus at

concentration 50mg and 100mg by disc diffusion method. The

Ciprofloxacin was used as standard and DMF as solvent control.

Surprisingly all the compounds of the series exhibited excellent

activity almost resembling as that of the standard at the

concentration studied. Hence the detailed toxicity study may be

more beneficial .If the highly active compounds proved to be less

toxic, may be used as topical antibacterial agents in the form of

ointments and gels.

B.Antifungal activity:

All the compounds screened for antibacterial activity were also

screened for antifungal activity against the fungi Aspergillus niger

and C.albicans at the concentration of 50 and 100 µg/ml by

following disc diffusion method. Clotrimazole an antifungal agent

was used as standard. The compounds PZ1. PZ5, PZ8 PZ10, PZ11

PZ13 and PZ14, showed moderate activity, while PZ2, PZ3, PZ4, PZ6,

PZ7 and PZ12, showed significant activity in comparison with

standard against the fungi A. niger at both concentration levels.

Similarly PZ2, PZ3, PZ4, PZ7, PZ9 and PZ14 exhibited good activity

316

in comparison with the standard against C.albicans at both

concentrations. The presence of pyrazoline moiety, substituents

particularly one having N-CO-NH2 in the ring and compact

coplanar structure may be responsible for antifungal activity of

this class of compounds.

C.Analgesic activity:

Pyrazoline series of compounds PZ1, PZ4, PZ5, PZ7, PZ8, PZ10,

PZ11 and PZ13 were screened for their analgesic activity by Eddy‟s

hot plate method. Mice of either sex were used for the study.

Pentazocin 10mg/kg body weight used as standard drug for

comparison of activity. The standard drug showed percentage

analgesic activity as 218.75.All the compounds screened showed

promising analgesic activity. The activity ranges from moderate –

equipotent-highly potent. The compounds PZ4, PZ7, PZ8, and PZ10

exhibit significant activity almost equal to that of the standard. The

compound PZ1, PZ5, PZ7 and PZ11 showed moderate activity.

Surprisingly the compound PZ13 that contains chlorine on the p-

position of aryl ring has shown much superior activity. As the

compounds have shown promising analgesic activity it can be

concluded that the pyrazole moiety still serves as a promising

moiety and may provide more clinically useful compounds, if

molecule is suitably modified. Hence the further study of this

317

series of compounds particularly with reference to the toxicity

studies may result useful compound to the human kind.

318

REFERENCES

1. http://www.nationmaster.com

2. Wahab BFA, Aziz HAA, Ahmed EM. Eur J Med Chem 2008; 44:

2632.

3. Rao BCH, Raju SGV. J Ind Chem Soc 1986; 25: 400.

4. Ellis GP, Thomas IL. J Chem Soc Perkin Trans 1 1973: 2781.

5. Arndt F. Chem Ber 1925; 58: 1612.

6. Mustafa A, Hismat OH. Chem Abstr 1966; 65: 3823.

7. Aziz G, Nosseir MH. Ind J Chem 1976; 15B: 446.

8. Thakare VG, Wadodkar KH. Ind J Chem 1996; 25B: 610.

9. Wadodkar KN. Synthesis in heterocyclic compound, Ph.D,

Thesis Nagpur University, Nagpur,1977.

10. Mustafa A, Fleifel AM. J Org Chem 1959; 24: 1740.

11. Osaman AM, Maghuraby MA. J Ind Chem Soc 1977; 54:

394.

12. Sachchar SP, Singh AK. J Ind Chem Soc 1985; 62: 142.

13. Dadiboyena S, Valente EJ, Hamme AT. Tetrahedron Lett

2009; 50: 291.

14. Porter J, Payne A, Candole B, Ford D, Hutchinson B,

Trevitt G et al. Bioorg Med Chem Lett 2009; 19: 230.

15. Wahab BFA, Aziz HAA, Ahmed EM. Eur J Med Chem 2008;

44: 2632.

319

16. Prakash O, Kumar R, Sehrawat R. Eur J MedChem 2008;

44: 1763.

17. Gupta S, Rodrigues LM, Esteves AP, Campos AMFO,

Nascimento MSJ, Nazareth N et al. Eur J Med Chem 2008;

43: 771.

18. Kralj D, Groselj U, Meden A, Dahmann G, Stanovnik B, Svete

J. Tetrahedron 2007; 63: 11213.

19. Sachchar SP, Singh AK. J Indian Chem Soc 1985; 62: 142.

20. Sharma TC, Bokadia MM. J Indian Chem Soc 1980; 19: 228.

21. Nayak A, Mitra AS. J Indian Chem Soc 1980; 57: 643.

22. Tiwari N, Dwivedi B, Nizamuddin. Bol Chem Farm 1989;

128: 332.

23. Mohanty SK, Sridhar R. J Indian Chem Soc 1977; 15: 1146.

24. Mecalcon SE. Agric Chem 1947; 2: 31.

25. Sirrewiberg WE. Chem Abstr 1978; 89.

26. Desai NC, Trivadi PB, Unnadevi NK, Dave AM, Bhat KN. Ind J

Chem 1993; 32B: 760.

27. Ead HA, Hassaneen HM, Abdallah MA, Mousa HAH. Arch

Pharm Weinheim Ger 1991; 324: 35.

28. Sachchar SP, Singh AK. J Indian Chem Soc 1985; 62: 142.

29. Ritch S, Horsfall JC. Chem Abstr 1952; 56: 11543.

30. Mitra P, Nayak A. J Indian Chem Soc 1982; 59: 1005.

320

31. Burns JJ, Yu TF, Ritterband A, Perel JM, Gutman AB,

Brodie BB. J Pharmacol Exp Ther 1957; 119: 418.

32. Coli B, Silerstrint. Exp Med Therap 1957.

33. Rosmer I, Buliard M. Med Pharmacol Exp 1967; 16: 25.

34. Mitra P, Mitra AG. J Indian Chem Soc 1981; 63: 695.

35. Sadasiva M, Rao R. J Indian Chem Soc 1982; 59: 1104.

36. Nayak A, Mitra AS. J Indian Chem Soc 1980; 57: 643.

37. Tiwari N, Dwivedi B, Nizamuddin. Bol Chem Farm 1989;

128: 332.

38. Mohanty SK, Sridhar R. J Indian Chem Soc 1977; 15: 1146.

39. Sachchar SP, Singh AK. J Indian Chem Soc 1985; 62: 142.

40. Smith LI, Denyes RO. J Am Chem Soc 1936; 58: 304. John

W, Gunther P, Schmeil M. Chem Ber 1938; 71: 2637.

41. Elhabiri M, FigueiredoP, Fougerousse A, Brouillard R.

Tetrahedran Lett 1995; 36: 4611.

42. Eder H, Ansri M. Forsch 1961; 11: 1043.

43. Frangly AM, Chaban I, Khalil MA, Behkit AA. Arch Pharm

Winhim 1990; 325.

44. Burns JJ, Yii. J Pharmac Ep Therap 1957.

45. Coli B, Silerstrint. Exp Med Therap 1957.

46. Saragan S, Somashekara S. J Ind Chem Soc 1976; 53.

47. Jone RG. J Am Chem Soc 1952; 74: 488.

48. Froesch ER, Wolaogel M. Mol Pharmacol 1967; 3: 429.

321

49. Brunner HR, Eichenberger K. Experimenta 1966; 22: 208.

50. Smith DL, Forist AA, Dulin WE. J Med Chem 1965; 8: 350.

51. Arya VP, Grewal RS. Experimenta 1967; 23: 824.

52. Sweeny MJ, Davis FA, Gutowski GE, Hamill RL, Hoffman

DH, Poore GA. Cancer Res 1973; 33: 2619.

53. Chasin M, Harris DN, Phillips MB, Sidney M. Hess Bio Chem

Pharmacol 1972; 21: 2443.

54. Navinson T, Scholten H. Am Chem Soc, Abstr Med 1972;

52.

55. Kerdawy MM, Agemy A. Acta Pol Pharm 1975; 7:105.

56. Wrezicino U. Acta Pol Pharm 1975; 30: 433.

57. Gover RK, Moore JD. Phyto Pathology 1962; 52: 876.

58. Nayak A, DasS. J Indian Chem Soc 1977; 54: 485.