University of Nigeria Research Publications

MBAH, Chika John

Aut

hor

PG/Ph.D/86/4768

Title

Synthesis and Anti-Inflammatory Properties of Aminomethyl Derivatives of 2 – Naphthol

Facu

lty

PHARMACEUTICAL SCIENCES

Dep

artm

ent

Pharmaceutical Chemistry

Dat

e

January, 1990

Sign

atur

e

SYNTHESIS AND ANTI-INFLAMMATORY

PROPLRTIES OF AMINOMETHYL DERIVATIVES

OF 2-NAPHTHOL

MBAH, CHIKA JOHN

(REG. NO. ~ ~ / ~ h . ~ / 8 6 / 4 7 6 8 )

DEPARTMENT OF PHARMACEUTICAL CHEMISTRY

FACULTY OF PHARMACEUTICAL SCIENCES

UNIVERSITY OF NIGERIA

NSUKKA

JANUARY 1990.

SYNTHESIS AND ANTI-INFLAMMATORY

PROPERTIES OF AMINOMETHYL DERIVATIVES

OF 2-NAPHTHOL

MBAH, CHIKA JOHN

(REG, NO. ~ ~ / P h . D / 8 6 / 4 7 6 8 )

A RESEARCH PROJECT UNDERTAKEN I N PARTIAL

FULFILMENT OF THE REQUIREMENTS FOR

THE AWARD OF DEGREE OF DOCTOR OF

PHILOSOPHY ( p h . D ) , UNIVERSITY OF

NIGERIA, NSUKKA.

S U P E R V I S O R

PROF- P. I. AKUBUE

HEAD OF DEPARTMENT OF PHARMACOLOGY

FACULTY OF PHARMACEUTICAL SCIENCES

UNIVERSITY OF NIGERIA, NSUKKA-

T H I S T H E S I S I S APPROVED FOR THE AWARD OF DOCTORATE

DEGREE I N THE DEPARTMENT OF PHARMACEUTICAL CHEMISTRY,

FACULTY OF PHARMACEUTICAL S C I E N C E S , UNIVERSITY OF

NIGERIA, NSUKKA-

THb WORK EMBODIED I N THE T H E S I S I S ORIGINAL AND HAS

NOT BEEN SUBMITTED I N PART OR I N FULL FOR ANY OTHER

DIPLOMA OR DEGREE OF T H I S UNIVERSITY OR ANY OTHER

UNIVERSITY,

SINGH PROF, P.I. AKUBUE (SUPERVISOR) HEAD OF DEPARTMENT HEAD OF DEPARTMENT OF PHARMACOLOGY

. OF PHARMACEUTICAL CHLMISTRY FACULTY OF PHARMACEUTICAL SCIENCES UNIVERSITY OF NIGERIA UNIVERSITY OF NIGERIA, NSUKKA- NSUKKA.

ACKNOLILEDG EMENTS iii,

To my g e n e r a l l y l o v e d and most r e v e r e d s u p e r v i s o r ,

P r o f , P O I . Akubue, who d e s p i t e h i s p r o f e s s i o n a l commitments

w i t h i n and o u t s i d e t h e c o u n t r y , g a v e h i s v e r y e f f i c i e n t

g u i d a n c e t h r o u g h o u t t h e d u r a t i o n of t h i s p r o j e c t , To him,

I wish t o e x p r e s s m y s i n c e r e g r a t i t u d e ,

I a l so wish t o e x p r e s s my s p e c i a l t h a n k s t o

P r o f , A. K a r , t h e c o - s u p e r v i s o r who g a v e h i s i n v a l u a b e

s u p e r v i s i o n on t h e C h e m i s t r y a s p e c t of t h i s p r o j e c t , but

Icft t h e Depar tment b e f o r e t h e p r e s e n t a t i o n of t h i s t h e s i s ,

I am i n d e b t e d t o D r . GOB. Okide of t h e Depar tment of

P h a r m a c e u t i c a l C h e m i s t r y who a s t h e i n t e r n a l c o - s u p e r v i s o r

c o n t r i b u t e d immensely t o t h i s p r o j e c t and a l s o i n f i n d i n g

. t i m e t o r e a d t h r o u g h t h e work,

My s p c x i a l t h a n k s g o t o D r . (Mrs.) A, S i n g h , t h e

Head o f Depar tment of P h a r m a c e u t i c a l C h e m i s t r y , fo r h e r

encouragemen t t h r o u g h o u t t h e d u r a t i o n of t h e p r o j e c t .

I* ;wish a l s o t o t h a n k D r , P.Alcah, o f t h e Pharmacology

Depar tment and D r , R , Awachie, of t h e P h y s i c s Depar tment ,

who s u p p l i e d t h e p h l o g i s t i c a g e n t and micrometer u s e d i n

t h i s r e s e a r c h , r e s p e c t i v e l y . G r a t e f u l acknowledgement

a l s o g o e s t o D r , O.V. Ekechukwu, M r . A, Olukayode and

D r . U. Okoro for t h e i r aides.

F i n a l l y , I wi sh t o thank Messrs, Ogbu, C., Eze,

G,E,, Agada, C., and t h e e n t i r e s t a f f of P h a r m a c e u t i c a l

C h e m i s t r y Depar tment ,

DEDICATION

TO MY LATE FATHER,

TO MY MOTHER AND

TO REV. SR. REBECCA.

PAGE

ABSTRACT

TABLE OF CONTENTS

CHAPTLR ONE; IN'1'HODUCTION

CHAPTER TWO: THEORETICAL ASPECTS

CHAPTER THREE: RLSULTS AND DISCUSSION ..a

CHAPTER FOUR : LXPCKIMLNTHL

RE;F LRENCES

iii

ii.

LIST OF TABLES

ANTI-INFLAMMATORY MEASUREMENT DATA . ANTI-INFLAMMATORY ACTIVITY OF AMINOMETHYL DERIVATIVES OF 2-NAPHTHOL -

SOLUBILITY DETERMINED VALUES OF THE DERIVATIVES o

APPARENT PARTITION COEFFICIENT DETERMINED VALUES OF THE DERIVATIVES m

HYDROPHOBIC SUBSTITUENT CONSTANTS 0 0

POTENTIOMETRIC DETLRMINATION DATA OF THE DERIVATIVES - m

APPARENT DISSOCIATION CONSTANT VALUES - 0

REGRESSION LINE OF LOGARITHM OF BIOLOGICAL RESPONSE ON LOGARITHM OF PARTITION COEFFICIENT .a m o

LEAST SUUI\KE PARABOLA OF LOGARITHM OF BIOLOGICAL RESPONSE ON LOGARITHM OF PARTITION COEFFICIENT.

COMPARISON OF CALCULATED AND OBSERVED ANTI-INFLAMMATORY ACTIVITIES OF AMINOMETHYL DERIVATIVES OF 2-NAPHTHOL m -

PAGE

83

ABSTRACT

The usefulnesq of non-steroidal anti-inflammatory

agents in the management of rheumatoid &iseases cannot be

over-emphasised. As this disease is characterised by

chronic inflammation, it is usual to test compounds for

anti-inflammatory activity in animals and then hope that

the active ones should be clinically useful as anti-rheumatic

drugs,

From the literature, non-steroidal anti-inflammatory

agents have covered a wide range of chemical structures

aryl acetic, aryl-propionic, arylbutyric and anthranilic '

acid derivatives, Other compounds including the

benzothiazines are also known to be potent agents.

This work involved the synthesis of some aminomethyl

derivatives of 2-naphthol and screenin$ them as potential

anti-inflammatory agents. These derivatives from the point

of view of thsir electronic and stereochemical characteristics

are considered to be similar to the known bis(2-hydroxynaphthy1)-

methane derivatives, earlie;. L'G+UL l e d iu i l ave pronounced anti-

inflammatory activity, The physicochemical properties of

these compounds were .also determined in an effort to

correlate observed biological activity and chem,ical

structure.

prior to this study, little was known about the

pharmacological ropert ties of thes.e aminomethyl derivatives of

2-naphthol from the point of view of their potential anti-

inflammatory activity. Also there are limited reports on the

physicochemical properties of these derivatives.

The .anti-inflammatory activity was tested by inducing

oedema on the rat right hind paw by subplantar injection of a

1% suspension of carrageenin in 0.9% saline. The development

of the oedema in treated and control rats was followed by

m,easuring the changes in foot thickness of the h i x i paw using

a micrometer. The. solubilities in water, apparent partition

, coefficients in 1-octanol/phosphate buffer (pH 7.4) and

apparent dissociation constants in dioxane-water (2:l) at

room temperature (30 - + ~ O C ) were determined. -1

Of the compounds synthesized, bis(2-hydroxynaphthyl-

methy1)piperazine was found to be the most active, about 1.8

times the potency'of aspirin. Dimethylaminomethyl-2-naphthol

exhibited activity that was comparable to aspirin. The later

derivative also showed the highest solubility, about 27 times

the solubility of 2-naphthol.

The N-methylanilinomethyl derivative was the least soluble,

about 0.37 times that of 2-napthol. Half-neutralisation

method was utilised to compute the dissociation constants.

CHAPTER ONE

Rheumatoid a r t h r i t i s i s a systemic connective

t i s sue disorder which a f fec t s predominantly the synovial

joints. It i s one of the disorders referred t o a s 1

rheumatoid disease. The disease a l so incl udesankylosing

spondyllt is , gout, rheumatic fever, systemic lupus

ergthematous, psoriasis , o s t eoa r th r i t i s and polyar te ' r i t i s

nodosa. A t the onset of rheumatoid a r t h r i t i s , variable

symptoms t ha t manifest include symmetrical per lpher a 1

po lya r th r i t i s (acute or chronic ), severe ear ly morning

s t i f fness , a r t h r i t i s of atypical d is t r ibut ion

(e.g. monoarthritis), tenosynovitis and/or carpal tunnel

syndrome, systemic symptoms l i k e fat ique , weight los s

and fever, acute onset of pe r i ca rd i t i s and pleurisy with

fever.

Other non-art icular features include subcutaneous

nodules and c i rcu la t ing antiglobulins (rheumatoid factor ).

The disease i s characterised by chronic inflammation of

the joint lining. Inflammation, a response t o injured

c e l l s , takes place i n the damaged

inflammatory response immediately

overlaps i n i t i a l s t ab i l i sa t ion by

plugging of the wound cavity with

tissue2. This ,

follows and often

haemostatis and

blood clot. The main

s igns of inflammation a r e - redness and heat due t o

d i l a t a t i o n of small blood vessels normally i nv i s ib l e t o

the naked eye, swelling r e su l t i ng from f l u i d passing

through the walls of small blood vesse l s i n t o the space .

around them and pain caused by d i r e c t s t imulat ion of

nerve endings.

Substances produced by damaged and migrating c e l l s

a s a r e s u l t of in jury a r e responsible for mediating these

changes3 and they a re - histamine, serotonin, ka l l i k r e in ,

and bradykfnii (k inins 1, slow react ing substances,

prostaglandins, antigen-ant ibody complex and i n t r a c e l l u l a r

enzymes (lysosomes). Ia tra~e l i i i i ar c y c l i c adenosine

monophosphate (cycl ic -AMP ) and guanosine monophosphat e

(cyclic-GMP) a r e involved i n the re lease of a mediator l i k e

histamine. Prostaglandins which appear t o ac t v ia

i n t r ace l lu l a r cyclic-AMP and GMP, might theref ore be 2 expected t o a f f e c t the r e l e a se cS other m e d i h t o r s .

Also immunologic mediators such a s rheumatoid f ac to r ,

lymphokines and complements have been implicated t o be of 4 great po t en t i a l importance i n inflammation. In most

inflammatory react ions white blood c e l l s migrate cut of

blood vesse l s i n t o the surrounding t i s s u e , where they

ingest any bacteria invading the ,round and a l so remove

necrot ic t i s sue . The injury may be brought about by

physical agents (e.g. excessive heat or cold, mechanical

trauma, u l t r a -vo i l e t or ion i s ing r ad i a t i on ), chemical agentss\

( inorganic and organic , tox ins of var ious bac te r i a ),

i n t r a c e l l u l a r r e p l i c a t i o n of v i ru se s (myxoviruses ) and

hypersens i t iv i ty reac t ions ( i . e . reac t ion of antibody or

s ens i t i s ed lymphocytes with ant igenic m u t e r i ~ l ~ such as .

invasive bacter l a or inhaled dust ). .

A t i s s u e injury of short dura t ion i s ca l l ed acute

inflammation (exudative ) and i t involves engorgement of

t he small blood vas se l s i n the v i p f r l i of ir; j s ~ j r , chznges

i n t h e blood flow through them and escape of p ro t e in and

leucocytes from the engorged ves se l s i n t o the t i s s u e space.

The other type of inflammation, c a l l ed chronic inflammation,

involves t i s s u e i r i :wy fo r a prolonged perlod. It is usual ly

accompanied by the production of new f ib rous t i s sue . , Chemical agents t h a t can re l i eve t he pain and s t i f f n e s s

associa ted with a l l types of a r t h r i t i s and s o f t t i s s u e ;I rheumatism are ca l l ed analges ic anti-inflamrnat ory a g e i t s .

These the rapeu t ic agents have been broadly c l a s s i f i e d into

t h r e e ca tegor ies : 1

( 1 ) LONG-ACT ING DRUGS

The ac t i on of these agents on the j o i n t s i s

accompanied by improvement i n ext ra-ar t i cu l a r f ea tu r e s of

the disease, reduction i n erythrocyte sedimentation r a t e

6 (ESR) and rheumatoid factor t i t r a . They are indicated

i n pers is tent ly active disease i n adul ts a f t e r six months

of treatment with optimal non-steroidal anti-inflammatory

kherapy , progressive disease (e i ther w i t h developing

deformities, r e s t r i c t i o n of joint motion or new erosions

on radiographs), excessive requirement of cor t icosteroids

and i n troublesome extra-art icular features. A s they are

continued for long periods and sometimes for l i f e , they

potent ia l ly have serf ous side-ef fect s. These long-act ing

drugs include :

(a ) Penicillamine: 2-amino-3-methyl-3-mercapto-1-b~tar;efc

acid (l), i

Penicillamine i s effect ive i n rheumatic a r t h r i t i s ,

palindromic rheumatism, some types of juvenile chronic

a r t h r i t i s ( S t i l l ' s d isease) , juveni le rheumatoid

a r t h r i t i s , but because of i t s s ide-effects i t i s best

reserved for those pa t ien ts who cannot be managed with

analgesic anti-inflammato~y drugs. It is found t o be 1

i n e f f e c t i v e juveni le ankylosing spondyli t is , p s o r i a t i c

a r thropathy or any o ther inflammatory a r t h r i t i s . This i

or a l l y a c t i v e c h e l a t ing agent, a c t s by lowering r hsumatoid

f a c t o r t i t re, ESR and improves t h e e x t r a - a r t i c u l a r f e a t u r e s

such a s tenosynovit i s and lymphadenopathy. These changes

r each maximum a f t e r 4 - 6 weeks bf traatment. I n v i t r o ,

it has the a b i l i t y t o depolymerise and i n a c t i v a t e p r o t e i n s

through its sulphurdryl group. The s ide -e f fec t s include

e a r l y r a sh , l o s s of t a s t e , nausea and vomit t ing, pancytopenia,

t hr ombocyt openia and pr ote inur i a . ( b ) Gold: i s one of the o l d e s t ' long-acting drugs f o r

t h e t reatment of rheumatoid a r t h r i t i s , and was introduced

with t h e mistaken b e l i e f t h a t rheumakoid a r t h r i t i s had

some l i n k with t u b e r c u l o s i s , and t ha t gold was good f o r

tuberculosis. The two gold compounds i n major use a r e gold 7

sodium thiomalate (2 ) and aurothi&lucose (3 ) . These

p repara t ions a r e water so luble and a r e administered

intramuscular ly.

8 9 Sut ton et a 1 and Walz e t a 1 , described t h e a n t i - a r t h r i t i c

propert ies of a se r i e s of phosphine gold co-ordination

compounds such a s triethylphosphine ( (a-g1ucopyranosy)thio)

gold (4.), which showed the unique --property of ' being

absorbed on o ra l a h i n i s t r a t ion.

The therapeutic indications of gold are similar t o those

of pencillamine. The side e f f e c t s include resh, p r o t e i m ~ i s ,

nephrot i c syndrome, thrombocytopenia and pancyt openia.

(c ) Azathiopr ine : 6-C (l-methyl-4-nitroimidazol-5-yl ) t h iol-

Purine . ( 5 ) , i s the most widely used immumosuppre sive drug

10 i n the therapy of rheumatoid a r t h r i t i s . The d r u g , l i k e

penicillamine i s more effect ive than gold i n retarding

the progress of radiological changes i n rheumatoid

a r t h r i t i s . The theurapeutic indications are the same a s

for penicillamine and may be par t icular ly useful for pa t ien ts

who f a i l t o respond t o penicillamine, since they a l so f a i l

t o respond t o gold. Azathioprine i s also useful i n

pa t ien ts with pers is tent ly active or progressive p ro r i a t i c

arthropathy i n connective t i s sue disorders and polymyositis.

The s ide-ef f e c t s include leucopenla, thrornbocytopenia,

pancytopenia, nausea, diarrhoea and other g a s t r o - i n t e ~ t i n a l

problems.

(d ) Chloroquine: 7-chloro-4ff 4-(diethylamino)-1-

which has t h e same type of a c t i o n i n rheumatoid

a r t h r i t i s a s gold and penicil larnine, and i s perhaps the

most suitable for p a t i e n t s w i t n less severe but

p e r s i s t e n t l y a c t i v e d i sease which i s no t adequately

c o n t r o l l e d by anti-inflammatory drugs. The occular

t o x i c i t y and t h e slow onset of beneficial e f f e c t s a re #

bel ieved t o outweigh t h e the rapeu t i c mer i t s of the 7 drug . Retinopathy seems t o be t h e main s ide -e f fec t .

( 0 ) Levamisole(7) i s an anthelmintic which a l so has

actions resembling those of penicill'amine in rheumatoid 10 31 arthr i t i s . Chang and Lombardino , reported t h a t this

immunost imul ?tory agent, when administered a s an a n t i -

inflammatory agent, c l i n i c a l symptoms of rheumatoid

ar thr i t i s improve i n many patients after three months of

therapy.

(2) CORTICOSTEROIDS

Corticosteroids and corticotrophin(ACTH) are potent

anti-inflammatory drugs and achieve profound suppression -!

of symptoms i n rheumatic a r t h r i t i s and other inflammatory

disease S. 67 l2 They do not have the addit ional e f fec t s of

penicillamine, such a s improvement i n extra-art icular

features of rheumatoid a r thr i t i s ,and there i s no evidence

tha t they a l t e r the course of the disease. Corticosteroids

nust therefore be regarded a s n non-specific or symptomatic

therapy i n rheumatoid a r t h r i t i s .

Unfortunately, the d ~ z c z t_n_str src. ~ ~ y ~ : i . w d to sus t a in

adequate symptomatic r e l i e f often cause Cushingls

syndrome. Also doses which a t f i r s t control the disease

are often insuff ic ient a t a l a t e r stage. Corticosteroids

i n rheumatic diseases s i l~u ld be reserved for specific

indications par t icular ly when other ant i-inflammatary

agents have proved unsuccessful. These indications

include polymyalgia rheumatica and temporal a r t e r f t i s ,

which require systemic cort icosteroid therapy because

cor t icbsteroids are the only drugs which consistently

re l ieve the symptoms. Prednisolone(8) i s the drug of

choice i n these diseases. Diseases l i k e polyrnyositis,

dermatomyositis and po lya r t e r i t i s nodosa a l so requlre

systemic corticosteroids.

(2) CORTICOSTEROIDS

Cort icosteroids and corticotrophin(ACTH) a re potent

anti-inflammatory drugs and achieve profound suppression 1

of symptoms i n rheumatic a r t h r i t i s and other inflammatory

diseases. 6 s They do not have the addit ional e f fec t s of

penicillamine, such as improvement i n extra-art icular

features of rheumatoid a r thr i t i s ,and there i s no evidence

tha t they a l t e r the course of the disease. C o r t i c ~ s t e r o i d s

must therefore be regarded a s 3 non-specific or symptomatic

therapy i n rheumatoid a r t h r i t i s .

adequate symptomatic r e l i e f often cause Cushingl s

syndrome. Also doses which a t f i r s t control the disease

are often insuff ic ient a t a l a t e r stage. Corticosteroids

i n rheumatic diseases should be reserved for specific

indications par t icular ly when other anti-inflammatary

agents have proved unsuccessful. These indications

include polymyalgia rheumatics and temporal a r t e r i t i s , . which require systemic cort icosteroid therapy because

cor t icbsteroids are the only drugs which consistently

re l ieve the symptoms. Prednisolone(8) i s the d r u g of

choice i n these diseases. Diseases l i k e polymyositis,

dermatomyositis and po lya r t e r i t i s nodosa a l so require

systemic cort icosteroids.

C o r t i c o s t e r o i d s act by p r e v e n t i n g a r a c h i d o n i c acid release

11 p r o s t a g l a n d i n and c o l l a g e n a s e p r o d u c t i o n .

( 3 ) NO'bl-STEROIDAL ANTI-IEIFLAIVIMATORY DRUGS (NSAIDS)

These a g e n t s are grouped i n t o acidic compounds o f t h e

a r y l c a r b o x y l i c or e n o l i c t y p e and non-acid compounds. 13-14

A l l of the NSAIDS have similar p r o f i l e of a c t i v i t y , viz -* ant i -oedema, a n t i - p y r e t i c and a n a l g e s i c effects. None of

t h e r e c e n t NSAIDS are c l i n i c a l l y c l e a r l y superior to h i g h

d o s e s of a s p i r i a which is one of the e a r l y MSAIDS used

t h e r a p e u t i c a l l y . Some of t h e recent MAIDS are less t o x i c

and are used i n p a t i e n t s who are n o t c o n t r o l l e d by a s p i r i n .

A l l t h e s e a g e n t s l i k e a s p i r i n have g a s t r o - i n t e s t i n a l tract

side-effects, t h u s t h e s e a r c h for a more potent a n d safer

d r u g c o n t i n u e s . D e s p i t e t h e i r chemical s i m i l a r i t y , i n d i v i d u a l

r e s p o n s e s to d i f f e r e n t g roup members can show marked v a r i a t i o n ,

i n d i c a t i n g t h a t f a i l u r e to respond t o one drug d o e s n o t i n v a l i -

date a l l other group members.

A r y l C a r b o x y l i c a c i d types :

( A ) S a l i c y c l i c Acid D e r i v a t i v e s :

(i 1

A s p i r i n

A c e t y l S a l i c y c l i c a c i d ( ~ s ~ i r i n l ( 9 )

i s used a s a n a n a l g e s i c and an t i - in f l ammatory agen t . 15

S i d e - e f f e c t s associated w i t h i t i n c l u d e nausea , g a s t r o -

i n t e s t i n a l b l e e d i n g , t i n n i t u s ( l e a d i n g o c c a s i o n a l l y to d e a f n e s s )

and dyspeps ia .

(ii) Sodium S a l i c y l a t e ( 10)

T h i s i s used c l i n i c a l l y i n r h e u m a t i c f e v e r , rheumato id

7,15 a r t h r i t i s and o t h e r fo rms of muscu la r and a r t i c u l a r rheumatism.

(iii) Chol ine S a l i c y l a t e ( 11)

I t is used i n cases of j u v e n i l e rheumat ic a r t h r i t i s i n which

a s p i r i n d i d no t g i v e adequa te c o n t r o l or caused g a s t r i c

i r r i t a t i o n . 15

( i v ) Trihydroxybenzoic a c i d s ( l 2 ) and (13)

I t was found t h a t 2,3,5-trihydroxybenzoic~acid(12) w a s

i n a c t i v e , wh i l e 2,3,6 d e r i v a t i v e ( 13) was 10 t i m e s more a c t i v e

t han sodium s a l i c y l a t e . 15

(v) 5-(p-f1uotophenyl)acetyl s a l i c y c l i c acid ( F l u f e n i s a l ) ( l 4 )

This i s approximately 4 t i m e s a s a c t i v e as a s p i r i n i n animal

test and has a prolonged d u r a t i o n of a c t i o n and causes minimal

g a s t r i c i r r i t a t i o n s . 7

n = 0, 2, 6, 8,- 1 0 and 14-

Members i n which the f a t t y ac id s a r e of intermediate

chain l eng th (C 4 2 ) did not cause g a s t r i c l e s ions and 4 12

have e s s e n t i a l l y a l l the systemic a c t i v i t y of a sp i r in . 16

The low degree of g a s t r i c u lce ra t ion might be due t o l i t t l e

or no l i b e r a t i o n of a s p i r i n i n the stomach due t o lack of

hydrolysis. Liberat ion of a s p i r i n occurs only a f t e r

absorption.

( v i i ) 5-(4, ~-Dihydro-2-phenyl-3H-Benzy-indol-31 ~ s a l i c y c l l c

ac id (Fendosal ) (16 )

It i s r e p o r t e d to p o s s e s s pru;u~~+ci ar la lgts ic effect and

i t s potency i s s i m i l a r to t h a t of a s p i r i n . 17

S t u d i e s o n numerous s a l i c y l a t e d e r i v a t i v e s \ . ... . .

s y n t h e s i z e d w i t h t h e aim of d e c r e a s i n g t h e s i d e - e f f e c t s o f

s a l i c y c l i c and a c e t y l s a l i c y c l i c a c i d s , showed t h a t t h e

s i m p l e s t a c t i v e an t i - in f l ammatory compound i n t h i ; series

i s s a l i c y c l i c ac id . S t r u c t u r e - a c t i v i t y r e l a t i o n s h i p

i n d i c a t e s t h a t t h e c a r b o x y l g roup i s n e c e s s a r y for a c t i v i t y ,

t h e hydroxyl group must be a d j a c e n t t o t h e c a r b o x y l group

and h a l o g e n a t e d d e r i v a t i v e s a r e a c t i v e , b u t t o x i c ,

(B) Aryl Acetic Acid D e r i v a t i v e s :

Acetic a c i d d e r i v a t i v e s are w e l l r e p r e s e n t e d among

a l l t h e chemica l c l a s s e s t h a t have been s t u d i e d f o r

p o t e n t i a l n o n - s t e r o i d a l an t i - in f l ammatory a c t i v i t y . %'hey

c o n s i s t of p h e n y l a c e t i c a c i d , c a r b o c y c l i c and h e t e r o c y c l i c

acetic a c i d d e r i v a t i v e s .

( v i i i ) 3-chloro-4-propenoxy-phenylacetic a c i d

This compound has been used in the treatmcnk of . .

osteoarthritis and other degenerative anti-inflammatory

diseases. l8 Better tolerance of the drug was also reported.

2.6-dicloroanlinophenylacetic ac'id ( 17b)

( Diclof enac)

The compound is used clinically in the treatment of . rheumatoid arthritis, osteoarthritis and other inflammatory

( ix) 4-n-Butoxy-phenylacetohydroxamic acid

It is reported to be clinically effective in rheumatoid

arthritis and osteoarthritis. 19-20

( x ) 2-(p-cNorophenyl)thiazole-4-acetic a c i d

(Fenclozic ac id ) (19 )

This drug has been withdrawn frog c l i n i c a l study because

of hepatotoxici ty.

( x i ) 1-(p-chlorobenzoyl' )-5-methoxy-2-methylindole-3-acetic *

acid ( Indome t hacin ) (20)

" The compound has prominent anti-inflammatory and a n t i -

&retic properties.21 It is used c l i n i c a l l y for the above

purposes. The anti-inflatilulc~"i~.y av t i v i i y of indomethacin .:

prompted a search for t h i s a c t i v i t y i n related compounds.

This search resulted i n the following analogues:

( a ) ~ulindac:~is-~-~luoro-2~eth~l-1-~ ( P m e t h y l s u l f i n y l )

benzylidene] indene -3 -acetic ac id (21 )

Splindac i s active vivo only, because it i s metabolised

t o the sulphide. The ac t iv i ty of the sulphide is equal t o

tha t of indom t hac in.

(b ) Tolmet in: l -Methyl -~-p- to luoylpyrro le-2-~_~et ic acid (22 )

It i s about 0.4 t imes the potency of indomethacin i n t he

carrageenin oedema assay. The compomd is used c l i n i c a l l y

f o r t he treatment of rheumatoid a r t h r i t i s .

( c ) Cimetacin:Z-cinnamoy1-~-methoxy-2-methylindole-~-

a c e t i c ac id(23)

I ts anti-inflammatory a c t i v i t y was rourlCI t o be l e s s than

. t h a t of indomethacin. The compound was obtained by

r e p l a c i n g t he p-chlorobenzoyl moiety . ... . with p-cinnamoyl moiety.

(d ) Clometacin t 3-(P-chlorobenzoyl)-6-methoxy-2-methylindole-1-

acetic a& (24)

.

19 +

T h i s i s t h e p o s i t i o n a l isomer o f i n d o m e t h a c i n , a n d was f o u n d

22 t o b e much less p o t e n t t h a n i n d o m e t h a c i n .

T h i s i s a d i r e c t a n a l o g u e o f i n d o m e t h a c i n whe re a n a z i d o

f u n c t i o n r e p l a c e s t h e c h l o r o s u b s t i t u e n t . It i s q u i t e p o t e n t

23 a n d less t o x i c i n mice t h a n i n d o m e t h a c i n .

The p r e l i m i n a r y pharmacology a n d s t r u c t u r e - a c t i v i t y

r e l a t i o n s h i p s o f a series o f r e l a t i v e l y non - tox i c o-benzami-

dophenoxy and o-benzarnidophenyl a l k a n o i c a c i d s have been

24 p u b l i s h e d . One of t h e s e open-cha in i n d o m e t h a c i n a n a l o g u e s ,

c l a m i d o x i c a c i d (26) w i t h a n t i - i n f l a m m a t o r y a c t i v i t y e q u i v a l e n t

t o p h e n y l b u t a z o n e i n t h e c a r r a g e e n i n r a t foot oedema test

h a s been c l i n i c a l l y e v a l u a t e d and f o u n d p o t e n t .

Other acetic ac id d e r i v a t i v e s include:

( x i i ) Metazinic a c i d ( 2 7 )

This i s e f f e c t i v e i n ankylosing s p o n d y l i t i s , rheumatic

a r t h r i t i s and os teoa r th r i t i s .

(xiii ) Isoxepac (28 )

@cH2cooH

This i s a potent anti-inflammatory agent with an ED of 50

6mg/kg i n t h e r a t foo t oederna A change of t h e

2-posi t ion of a c e t i c ac id group t o p o s i t i o n 3, gave another

potent anti-inflammatory agent called oxepinac.

t o be a potent anti-inflammatory agent. The agent i s

r e l a t i v e l y well t o l a r a t e d i n animals but i s t o x i c t o man. 26

(x iv ) 4-(p-chloropheny1)-2-phenyl-5-thiazole a c e t i c ac id

This was reported t o be a potent anti-inflammatory agent and Q'7

a l s o an e f f e c t i v e analges ic i n rodents.''

( c ) Arsl Propionic Acid Derivat ives:

The number rf compounds in this group has continued t o

increase , following the in t roduc t ion of t h e lead compound,

Ibuprofen.

(XV) 2-(p-isob~tylphenylj-~ro~ionic ac id (Ibuprofen) (31)

This agent i s more useful i n m i l d e ~ c a s e s of inflammatory

or degenerative a r t h r i t i s . 28

, ... . .

( x v i ) 6-Methoxy-o( -methyl=2=naphthalene a c e t i c ac id

(Naproxen) (32)

. . (32

It i s the most potent compound i n the s e r i e s of

2-naphthylacetic ac id and i s 11 times the anti-inflammatory

a c t i v i t y of phenylbutazone 29 -3 0 or a s p i r i n .

~ ~ ~ r o x o l ( 3 3 ) , t h e corresponding ca rb ino l , was b i o l o g i c a l l y

equivalent . 29

(33

( x v i i ) (5) -m-phenoxyhydratropic ac id (Fenprofen) (34)

This i s used c l i n i c a l l y a s anti-inflammatory agent and has a

shor t ha l f - l i f e . 31

( x v i i i ) 2-(p-Chloropheny1)- o( -methyl-5-benzoxazolacet i c

ac id (Benozapr ofen ) (3 5 ).

It i s fond t o be 5 t imes .more ,potent than phenylbutazone.

Has long h a l f - l i f e of 37 hours i n man.

( x i x ) P-)l-oxo-2-isoindolinyl )hydratropic ac id . (Indoprofen) (36)

This is. 19 times more potent than phenylbutazone, with

h a l f - l i f e of 3 hours i n man.

(xx) 6-Chloro-o( -methylcarbazole-2-acetic ac id (Carprofen) (37)

This compound i s as potent a s indomethacin i n t h e r a t foot . oedema, but l e s s potent i n the r a t adjuvant a r t h r i t i s .

(xxi ) 5-Benzoyl-2-methyl-24hiopheneacet i c ac id

(Tr iaprofenic acid ) (38)

The drug i s used f o r rheumatoid a r t h r i t i s , o s t e o a r t h r i t i s

and musculoskeletal d isorders . r-f - . - . I . - 7 , -: -.-, ,,, - -

- .

(xxii) 4,5-Bis(p-chlorophenyl-2-oxazolyl.)thiopropionic a c i d

(Tioxaprofen) ( 3 9 )

It is a potent anti-inflammatory agent in rat foot oedema.

(xxiii) 2-(2-fiuoro-4.-biphenylyl)propionir a c i d

The anti-inflammatory potency was found to be equal to

Ibuprofen. . (xxiv) 3-(4-biphenylyl-carbony1)propionic acid (41 )

The compound was f o m d potent in inflammatory diseases.

(xxv) 6-Benzoyl- C( -methyl-2-phenylacetic acid (42 )

8

C l i n i c a l l y found t o have a c t i v i t y e q u a l t o indomethac in.

(xxvi) 2-chloro-4-pyrrolino- 4 -methyl-2-phenylacetic

Found e f f e c t i v e i n rheumatoid a r t h r i t i s and o s t e o a r t h r i t i s .

(D) Arvl Butyric Acid Derivat ive

(xxvil) 2-(4-Isobutylphenyl )butyr i c a c i d (44 )

32 Car re te ro e t a l , found t h e compound t o possess an

a c t i v i t y comparable t o ibup1.cfc11, :*:tii i ~ s ulcerogenic

p o t e n t i a l lower than t h a t of phenylbut azone and Ibuprofen.

( x x v f i i ) 4-biphenylyl-3 -hydroxylbutyr ic a c i d ( 4 5 ) , .

The compound was found t o be s i x t imes t h e potency of

33 indomethac i n .

(E ) N - ~ r y l a n t h r a n i l i c Acid ~ e r i v a t i ves (Fenamates ):

The compounds i n t h i s c l a s s a r e d e r i v a t i v e s of

a n t h r a n i l i c ac id , t h e amine analogue of sa l i cyc l i c '

acid.

(xxix) N-2,3-xylyl-anthranilic ac id (Mefenarnic ac id ) (46 )

(46)

This i s used c l i n i c a l l y i n pain management and rheumatoid

a r t h r i t . i s . 33 The drug i s one and a ha l f times a s potent

a s a s p i r i n and f i v e t imes l e s s potent than phenylbutazone.

(xxx ) N-(o( ,&, 6 -tr if luoro-m-tolyl ) a n t h r a n i l i c ac id

(Flufenamic ac id ) (47)

(47)

This agent i s used f o r rheumatoid d i sease , and found t o

be h a l f a s potent a s phenylbutazone. and about four t imes

more potent than a s p i r i n i n c l i n i c a l s tudies. 33

(xxxi) . N-(2,64ichloro-m-tolyl ) a n t h r a n i l i c a c i d

(Meclofenamic acid ) (48)

(48

This congener of the fenamates i s found t o be twenty five

times , the anti-inflammatory potency of mefenamic acid and

i s c l i n i c a l l y used.

(xxxii ) N-Nitroso der ivat ive of flyfenamic acid (49 )

It i s reported t o have twice t he potency of flufenamic

acid. 13

( x x x i i i ) 2, ( 3 - ( t r i f l ~ o r o m e t h ~ l )an i l lno )nicotinic acid

(Niflumic Acid ) (50)

T h i s has been reported t o h v e zzt:', . ir ,fLzmstory

ac t i v i t y . l3 Other analogues of fenamatas t h a t have been

shown t o possess anti-inflammatory a c t i v i t y are ,

8-tr ifluoromethylphenothiazine -1-carboxylic ac id (51 ),

and pyrimidine i s o s t e r of niflurnic acid (52)

The s t r u c t u r a l requirements fo r optimal a c t i v i t y of

a r y l and heteroaryl alkanoic ac ids , so f a r s tudied, - !

32 include , a carboxyl group separated by one carbon atom

from a flat aromatic nucleus, t h e carboxyl group dev ia t ing

considerably from the plane of nucleus, a f r e e hydrogen

atom i n t h e @--carbon, op t iona l s u b s t i t u t i o n a t p o s i t i o n

2 or 3 by a small group, preferably a halogen and a

l i p o p h i l i c s u b s t i t u t i o n , para, t o t h e ac id residue. I

TRE ENOLIC ACJD TYPES:

This c l a s s of non-steroidal anti-inflammatory agents

can be divided i n t o pyrazolones and oxicams.

Pyrazolone s :

The compounds a r e further divided i n t o two ca tegor ies ,

v i z 5-pyrazolone and pyrazol idinedione de r iva t ives . The

5-pyrazolones include :

(b) 2,3-dimethg1~-44imethylamino-l-phenyl-pyrazolin-~-one

(Aminopyr ine ) (54 )

(54)

( c ) 2,3-dimethyl-l~phenyl-3-pyrazolin-~-one-N-methylamino

methanesulphonate sodium (Dipyrone ) ' ( 5 5 )

The three 5-pyrazolones have analges ic , a n t i-pyret i c and

anti-rheumatic a c t i v i t y s imi lar t o asp i r i n and sodium 7915 sa l i cy la tes . Their use i s r e s t r i c t e d , because of t h e i r

s e r i s ~ s side effects, such as agranulocytosis and

hyper sensitivity reactions. The 3,5-pyrazolidinedione

der i v a t ive s are :

Oxyphenylbutazone i s a metabol i te of phenylbutazons and

found t o be of equa l p 0 t e n ~ y t o phenylbuta~one , but i s

s l i g h t l y l e s s toxic.. Other metabol i tes of phenylbutazone

used a s anti-inflammatory agents a r e , y-hydr~xyphen~lbutazone ( 5 8 )

and f -ketophenylbutazone (kebuzone 1, formed by convert ing

1-hydroxy group t o ke tonic group. ~ h e n ~ l b u t a z o h e , because

of i t s t o x i c e f f e c t s , has a limited r o l e i n t h e therapy of

rheumatoid a r t h r i t i s . It i s used pr imar i - ly ?v? t h e r s t i e f

'of acu te exacerbat ions of o s t e o a r t h r i t i s .

Oxicams

This category of anti-inflammatory agents a r e i n

c l i n i c a l use. Since t h e assignment of the name sudoxican

t o an anti-inflammatory e n o l i c ac id d e r i v a t i v e of

1,2=benzothia~ine,~~ the term oxicam has been a p p l i e d to

. - s e v e ~ a l ant i-inflarnmatcry en07 1 c . ,,< ,. ~ 5 : ~ ' ~ - . v c \ l : ' ~ ~ i i l

3-(N-aryl )-carboxamide s of 4-hydroxy-1,2-benzothiazine -1,

1 -d iox ide .

Oxicains are a c i d i c . compounds with the pka v a l u e s of 6.3

and. 6.3 for sudoxicam and piroxicam respectively. 14

These oxicams include: . (i) 4-hydroxyl-2-methyl-~-2-thiazoyl-2~-1,2-benzothiazine-

3 -carboxamide-1, l - d i o x i d e (Sudoxicam) (59 )

(ii) 4-hydroxyl-2-methyl-N-2-pyridyl-2H-1,2-benzothiazine-

3-carboxamide-1, l-dioxide (Piroxicam) (60)

The potency was found to be less than piroxicam or

sudoxicarn.

(iv) 4-hydroxyl-2-methyl-N-(Phenyl d e r i v a t i v e s ) of

1, 2-benzothiazine-3-carboxamide-1, 1-dioxide (62 ) .

Compound (62a) was found t o be twice a s potent a s

phenylbutazone, while compound (62b) was 1.5 times a s

ac t ive a s indomethacin-

Several other oxicams and compounds r e l a t ed t o

oxicams have been synthesized and screened for a n t i -

inflammatory ac t iv i t y . The more potent oxieams aTe

administered t o man a t the lowest dose of any non-steroidal

anti-inflammatory agent A t 2Gmg once da i ly , sudoxicam and

piroxicam are e f f ec t ive i n rheumatoid a r t h r i t i s ,

o s t e o a r t h r i t i s , ankylosing spondyl i t i s , acu te musculoskeletal

condit ions, gout and i n various s t a t e s 'of pain.

Apparently, some l e v e l of anti-inflammatory a c t i v i t y

i s re ta ined i n a wide var ie ty of 4-hydroxy-I.,

2-bsnzothiazine-3 -carboxamide -1, l-dioxide without regard

t o whether a benzo, naphtho, thieno, benzothieno or

indole r i ng i s fused t o thiazine.

3 7

Miscellaneous types

A s a consequence of t h e wide-spread search fo r a n t i -

iiiflammatory agents , several novel s t r u c t a n c s with a

v a r i e t y of a c t i v i t y p r o l i f e s have been discover6d. These

include :

(i ) Dioxoisoquinoline-4-carboxanilides (63 )

These a c i d i c compounds have been repor ted t o have a n t i -

inflammatory a c t i v i t y .36 Compound (63a ) and (63b) were

approximately equipotent t o phenylbutazone and ( 6 3 c ) i s

t h e major human metabol i te of (63a).

The 'compound (64) possesses both anti-inflammatory and

anti-coagulant proper t ies . The ant i-inflarnva to ry

a c t i v i t y i s 1.3 t imes t h e a c t i v i t y of phenylbutazone. -\

' ( i i i ) 1-(4-methoxy-6-methylpyrirnidiny1)-3-methyl-4-

methoxyimidazole (Mepir i z o l e ) ( 6 5 )

This has been repor ted t o possess , . . , . twice t h e , a n t i -

inflammatory potency of' aminopyrine. 13

( i v ) Thieno-(2,j-c )pyr i d i n e d e r i v a t i v e (66 )

The above compound (03) has been repzrted t o possess

weak anti-inflammatory aiid analge s i c activity . 38

(v 4 -Butyl-2-(p-chlorophenyl) -1-(p-methoxy benzylidene )- . .

3,5-pyrazolidlnedione (67)

The anti-inflammatory activity of this compound is

reported t o be comparable t o phenylbutazone. 40

The compound has been observed t o possess anti-inflammatory,

anti-pyretic and ana lges ic ac E iv i t i e s comparable t o

phenylbutazone. 33

( v i i ) .1-(4-chloropheny1)-4(2-(3-(2-pyridyl )acryloxy )e thy l ) -

piperazine ($9 )

The anti-inflammatory , ant i-pyret i c and analgesic

a c t i v i t i e s of t h i s compound are comparable t o

phenylbutazone - (viii) Benzarone (70) - has both anti-inflammatory and

f ibrinolyt ic a c t i v i t i e s .

The summary of the acidic non-steroidal anti-

inflammatory agents and the miscellaneous t y p e s so far

discussed i s shown i n the modified chart (1 ) earl ier

described by Mowat. 5

N O N S TEROI9AL A N T I -1NFL4i.FATORY DRUGS 1

Family \1/

Carboxylic \I/

Enolic Acids niscellancous

~ c e t i c acids ~ a l i c y c l i c Propionic acids Fenamic acids Pyrazolones 0xi&ns, Mepirizole

etg ~ e n o x a ~ r o f en Flufenamic acid Azapropazone Proxicam Proquazone

Aspirin Fenbuf en Mefanamic acid Feprazone Sudoxicam Flumizone

A l o x i p r i n Fenoprof en

Benorylate Flutiprof en

Di f lun isa l Ibufrof en

Sa l sa l a t e Naproxen

Phenylbuta- Isoxicam Epirazole zone

Tinoridine

Oxypheny-

butazone

Phenyl ace t i c acids Carboxylic and Heterocyclic

SUB.cLASS cog ace t i c acids e.g

D i c l of enac Indomethacin

Fenclofenac Sulindac

Chart 1: Classif icat ion of t on-steroidal Anti-inflamatory Agentso

1.2 MCHANISMS OF A C T I O N O F NON-STEROIDAL ANTI- INFLAMMATORY AGENTS

The mechanisms of a c t i o n of anti-inflammatory

d rzgs t h a t a r e non-steroidal have been extens ive ly . . . .

s tud ied with the hope t h a t t h e knowledge of t h e mode of

a c t i o n would help i n t h e design of new and b e t t e r drugs .

Experimental a c t i v i t i e s aimed a t producing B

unifying biochemical concept , t o account fo r a n t i -

inflammatory a c t i v i t y have l e d t o a v a r i e t y of

suggest ions regarding poss ib le modes of a c t ion.

( i ) Uncoupling of oxidase phosphorylation:

Various anti-inflammatory drugs have t h e a b i l i t y

i n v i t r o t o i n h i b i t the syn thes i s of compounds conta in ing - high energy phosphate bonds without simultaneously

depressing t h e oxygen consumption of l i v i n g c e l l s . This

d i s s o c i a t i o n of phosphorylation and ox ida t ion i s termed

uUncouplinglt.

winter", sllggested t h a t uncoupling deprive

inflamed areas of energy dependent processes , needed

t o support t h e a c t i v e metabolising a c t i v i t i e s sus ta in ing

42 the inflammation. Whitehouse , claimed uncoupling of

oxidase phosphorylat ion t o be t he mode of a c t i o n of

s a l i c y l a t e s , phenylbutazone and indomethacin.

(ii) S t a b i l i s o t i o n of lysosomes:

The hypothesis t h t ~ntil~.ir:flamr:ato~!? ac-tfvity

should be r e l a t e d t o lysosomal s t a b i l i s a t i o n i s based

on t h e presumed capaci ty of lysosomal enzymes t o produce

t i s s u e &mags with r e s u l t a n t inflammation, r e l e a s e of

chemical media tws . a t t r a c t i o n of leucocytes and a l l

o the r sequences r e l evan t t o inflammatory d i sease , . whether acu te or chronic. Skidmore and W h i t e h ~ u s e ~ ~ ,

suggested t h a t the i n t e r a c t i o n of an t i - in f l ammat~ry

a c i d s with t h e lysyl B-amino group i n serum albumin *or

h i s t id fne decarboxylase, i n competit ion with pyr idcxa l

phosphate t o be t h e mode of a c t i o n of these agents.

Pollock and ~ r o w n ~ ~ , on t h e i r s t u d i e s on the acute

inflammatory response, showed some con t rad ic to ry r e s u l t s

t o the above hypothesis. Thus evidence a t t r i b u t i n g t h e

anti-inflammatory a c t i v i t y of s a l i c y l a t e s , pyrazolone s,

indomethacin e t c . t o s t a b i l i s a t ion of lysosomes seem

r a t h e r fragmentary.

( i i i ) Cell membrane and c e l l migration e f f e c t s :

Actions on c e l l membranes may b~ r e l a t e d t o the

e f f e c t s of t h e compounds upon p r o t e i n s t a h i l . i s u t i o n ,

l igand i n t e r a c t i o n s , c e l l u l a r migrat ion and c e l l

permeabili ty. According t o Mizushima .and ~ u z u k i ~ ~ ,

t h e thermal denatura t ion of p r o t e i n

c a n be i n h i b i t e d by a number of a n t i - i n f 1w111d Lory compounds

w i t h a n o r d e r of a c t i v i t y r o u g h l y i n agreement w i t h t h e i r

i n v i v o potency. I n h i b i t i o n of n e u t r o p h i l chemotaxis , r e d u c t i o n -- i n l e u c o c y t e m i g r a t i o n and impairment of p h a r g o c y t o s i s have

a l s o been c o n s i d e r e d to be t h e mode of a c t i o n o f a n t i -

in f l ammatory a g e n t s ,

( i v ) I n h i b i t i o n of enzymes:

Proteases from polymorphonuclear l e u c o c y t e s arae i n v o l v e d

i n t i s s u e d e s t r u c t i o n , complement a c t i v a t i o n and s t i m u l a t i o n

of lymphocytes. I t was t h e r e f o r e though t t h s t i n h i b i t i o n .of

t h e s e p r o t e o l y t i c enzymes migh t modify some of t h e m a n i f e s t a -

t i o n s of i n f lemmation. PlorsdorfQ6, tested the a b i l i t y of

an t i - in f l ammatory d r u g s t o i n h i b i t t h e breakldown of r a t paw

homogenates by exogenous enzymes a n a round that t h e potency

of s e v e r a l compounds i n t h e a s s a y p a r a l l e l e d t h e i r a n t i -

inf lammatory a c t i v i t y .

( v ) F i b r i n o l y t i c a c t i v i t y :

Glena and ~ e k h a r ~ ~ , p o i n t e d o u t t h a t a l l s u b s t a n c e s which

promote c l o t t i n g i n v i t r o produce inflammation i n vivo. -- T h i s o b s e r v a t i o n is s u p p o r t e d by the work o f Gryglewski and

48 Gryglewska , which showed t h a t s e v e r a l ant i - inf lammatory

a c i d s have demons t ra ted f i b r i n o l y t i c a c t i v i t y i n v i t k o a t

- 49 had r e l a t i v e l y h i g h c o n c e n t r a t i o n s . A l s o , F e a r n l e y et al.

earlier suggested the c l i n i c a l a p p l i c a t i o n of

f i b r i n o l y t i c agen i s i n rheumatoid a r t h r i t i s . The

f i b r i n o l y t i c a c t ion of these anti-inflammatory agents

i s considered t o be a s a r e s u l t of i n h i b i t i o n of a

plasma f a c t o r respons ib le fo r the a c t i v a t i o n of the

formation of f ibr ins .

(vl) Sulphurdryl-disulphide s t a b i l l s a t ion:

Cerber et , found t h a t anti-inflammatory

drugs acce le ra te the r e a c t i o n between SH groups and

5,5-dithio-bis(2-nitrobenzoic a c i d ) , (DTNB, Ellman's

r eagen t ) and suggested t h i s a s a poss ib le b a s i s of their

the rapeu t i c a c t i v i t y . The r e a c t i o n of DTNB i n plasma

was presumably a measure of SH groups and an increase i n

r a t e of interchange might be a t t r i b u t e d t o drug-induced

changes i n conformation of t h e plasma prote ins .

Butler et ai,51 repor ted a marked reduct ion i n

t h e r a t e of SS-SH interchange in r a t with adjuvant

a r t h r i t i s , and t h i s reduct ion was correc ted when a n t i -

inflammatory agent was administered. The s ign i f i cance

of these snd other s t u d i e s i n d i c a t e t h a t t h e r a t e of

r e a c t i o n of DTNB i n plasma depends not only upon t h e

concent ra t ion of SH groups, but a l s o p a r t l y upon the

concent ra t ion of f r e e DTNB. 5 ,S-di thio-bis(2-ni t ro-

benzoic ac id ) like anti-inflammatory drugs compete with

i t fo r binding s i t e s , t h e r a t i o of f r e e t o bound DTNB

increases . Therefore, t h e increase seen in t h e r a t e of.

r e a c t i o n (i.e. t h e SS-SH interchange ) i s not necessa r i ly

due t o an increase i n concent ra t ion of r e a c t i v e SH groups,

but t o an increase i n concentrat ion of f r e e DTNB.

Bradykinin i s a nonapept ide l i b e r a t e d when

pzukaases ( K a l l i k r e i n s ) i n t i s s u e e x t r a c t s l i k e

pancreas and s a l i v a r y glands a c t on plasma q globul ins 2

which a r e r e f e r r e d t o a s kininogens. The pept ide i s a l s o

re l eased due t o the sur face a c t i v a t i o n of one of' t he

plasma c l o t t i n g f a c t o r s - It p lays an important r o l e

i n generat ing t h e l o c a l phenomena of inf lammat ion

p a r t i c u l a r l y t h e v a s o d i l a t a t i o n , oedema and pain.

Recent evidence suggests t h a t bradykinin a c t i v a t e s

t h e pros taglandin system and i t i s these pros taglandins

t h a t a r e d i r e c t l y a lges ic . Anti-inflammatory agents

antagonise the r e l e a s e of bradykinin from i t s precursor ,

by i n t e r f e r r i n g i n t h e r eac t ion bet teen t h e enzyme and

thu disulphide l inkages wi th in t h e precursor.

( v i i i ) Antagonism of pros taglandins synthesj s :

Among t h e mechanisms of act ion of anti-inflammatory

agents so f a r discussed, the i n h i b i t i o n of prostaglandin

syn thes i s has received s p e c i a l a t t e n t i o n .

Prostaglandins a r e , l i p i d ac ids ex t rac ted from seminal

f l u i d and t h e ves icu la r gland. They a r e divided i n t o two

c l a s s e s according t o whether they were e x t r a c t a b l e by

e t h e r ( the E s e r i e s ) or not ( t h e F s e r i e s ) . Prostaglandins

a r e r e a d i l y synthesized by incubat ing a honlogenate of

sheep ves icu la r glands with polyunsaturated f a t t y a c i d s

such a s arachidonic and dihomo- l( -1inolenic acid.

Although they perform severa l funct ions i n the body, t h e

most c lear -cut func t ion i s i n connection with pa in fu l

responses. The e f f e c t s a r e pe r iphera l and seem t o be due.

t o s e n s i t i z i n g pa in endings t o the a c t i o n of bradykinin

and poss ib ly other l o c a l l y r e l eased agents*

The non-steroidal anti-inflammatory agents l i k e a s p i r i n ,

indomethacin and naproxen e t c . have been found t o

i n h i b i t t h e syn thes i s of pros taglandins by i n h i b i t i n g

cyclo-oxygenase (enzyme) which i s necessary i n convert ing

t h e arachidonic ac id t o the endoperoxides and the11 t o

prostaglandins.

. n LL, Other probable mechariisuls ol" & t L ~ l ~1 L I K S ~ non-

s t e r o i d a l anti-inflammatory agents include i n h i b i t i o n

of p l a t e l e t and ery throcyte aggregation, and reduct ion

i n hydroxyproline excret ion. The excre t ion of

hydroxyproline i n ur ine has been found t o correspond

with the a c t i v i t y and extent of the a r t h r i t i c process .

The exc re t ion depends on the a c t i v i t y of protocol lagen

p ro l ine hydroxylase, whlch has been found t o increase

i n rheumatoid synovial t i s s u e . #

49

1.3 IDENTIFICATION OF AN'L'I -Ii\lFiAi44BT(Ji;Y AGFWl 'S ,

A good number of t e s t s have been designed f o r

measuring t h e anti-inflammatory a c t i v i t y of compounds.

These methods of eva lua t ion can be v i t r o or - i n -- vivo.

The in v i t r o method involves,

S t a b i l j m t i o n of human or animal e ry th rocy tes from

haemolysls induced by hypot'onic so lu t ions or by

heat 53 -54 . I n h i b i t i o n of p l a t e l e t and ery throcyte aggregat ion, 1

induced by macr o-molecule s. 55

Measurement of the a c c e l e r a t i o n of d isu lphide

interchange between serum sulphurdry1 groups and

SO,% 5,s-dithio-bis (2-nitrobenzolc ac id )

Measurement of the f i b r i n o l y t i c a c t i v i t y a t a

r e l a t i v e l y high concent ra t ion of the a n t i -

inflammatory agent. 49 This method i s supported

by a c l i n i c a l note suggt.st:f:r-!? the a ~ j ~ , I ' A . . ' - 4 . ' A,. c k u . , . ~ I I bf

f i b r i n o l y t i c agents i n rheumatoid a r t h r i t i s . S 7

So f a r , no s i n g l e v i t r o assay has been genera l ly

accepted as t h e most promising prel iminary

screening.

The vivo mothods include:

( a ) Foot oedema i n h i b i t i o n t e s t ;

( b ) Adjuvant a r t h r i t i s a z c z y -

(c ) Granuloma i n h i b i t i o n t e s t .

The foo t oedema i n h i b i t i o n t e s t , i s t h e most v ide ly used

primary t e s t t o screen new non-steroidal anti-inflammatory

age'nts. This method, f i r s t described by Winter e t a l , 58

involves the induct ion of oedema by the subcutaneous

i n j e c t i o n of any oedema inducing agents l i k e carrageenin,

formalin( l$) , brewer yeas t powder (2.5$), undiluted f r e s h . egg white and sero tonin c r e a t i n i n e su lphate complex

containing 1 0 g of sero tonin i n t o the p l a n t a r surface of

t h e a l b i n o r a t hind paw. The most commonly used

p h l o g i s t i c agent i s carrageenin (1% i n normal s a l i n e ) .

With t h i s inflammatory agent , t h e responses a r e more

r e l a t e d t o t h e doses of t h e t e s t drugs t h a n is the case

wi th any o the r p h l o g i s t i c agent. The anti-inflammatory

a c t i v i t y i s evaluated through e i t h e r changes i n paw

weight or volume or changes i n paw diameter.

Increasing r e l i a n c e i s being upon t h e

adjuvant-induced a r t h r i t i s assay i n t h e r a t , a s i t

manifes ts both an acu te inflammatory response and

51

. disseminated secondary les ion . 59-60

An improved q u a n t i t a t i v e method t h a t employs t h e dog

has .been reported,61 i n which the pressure exer ted b; t h e

dog paw i s measured when it s tands without r e s t r a i n t ,

using dolor imeter a f t e r i n j e c t ion of an inflammatory

agent i n t o t h e corresponding knee jo in t .

The granuloma i n h i b i t ion met hcd Involvs s t h e i m p l z n t a t i z n

of p e l l e t s of co t ton wool, c e l l u l o s e sponge or s imi lar

ma te r i a l under t h e abdominal sk in of t h e r a t and l e f t f o r

7 days. Granulation t i s s u e forms around and i n t o the

p e l l e t s . The animals a r e k i l l e d a f t e r 7 days, and t h e

p e l l e t s , together with the granula t ion t i s s u e a r e removed

and d r i ed t o a constant weight. Thus t h e weight

d i f f e rence between t h i s and t h e weight got a f t e r a n t i -

inflammatory agent was administered i s used a s a measure

of t h e anti-inflammatory a c t i v i t y .

OBJECTIVE

The a v a i l a b l e an t I-inflammatory d r u g s have not

s a t i s f i e d a l l t h e q u a l i t i e s des i red by the medicinal

chemist , t h e need s t i l l e x i s t s f o r the development of

new and b e t t e r drugs with l e s s t o x i c e f f e c t s than the

a c l d i c non-steroidal a n t i - i c f l u ; ; . , ~ ~ t ~ ~ ; l z g m t s. This has

prompted i n t e n s i v e re sea rch f o r non-acidic non-steroidal

anti-inflammatory agents with a v i e v t o reducing these

t o x i c e f f e c t s of t h e e x i s t i n g drugs.

It i s i n t h e search f o r b e t t e r non-acidic non-

s t e r o i d a l an t i-inflamn~tory ;rzent s t h a t Poupel in et a1 62 -- 7

found b i s (2-hydroxy-1-naphthyl )methane d e r i v a t i v e s (71) ) t o

possess potent anti-inflammatory p r o p e r t i e s and pos tu la ted

t h e mode of a c t i o n t o be due t o complexation or c h e l a t i o n t

within the b io log ica l system. Complexat ion or che la t ion

w i t h copper i n t h e blood has been suggested t o be t h e mode

of a c t i o n of meny c l i n i c a l l y used anti-inflammatory

agents.63-64 This observat ion has va l ida ted t h e e a r l i e r

r e p o r t of ~ n d e r ~ o o d , ~ ' t h a t serum copper l e v e l s increase

markedly i n a r t h r i t i c disease. -!

Prior t o t h e r e p o r t of Poupelin e t a l , t h e parent

compound 2-naphthol, was known t o be a potent a n t i s e p t i c '

and a l s o an anthe lmint ic agent. 66 Several d e r i v a t i v e s

of 2-naphthol have been repor ted t o have a i l t i -bac te r i a l ,

ant i -fungal and anthelmint i c p roper t i e s . 67-70

Oxytocic a c t i v i t y has a l s o been observed i n piperidinomethyl

and pyrrolidinomethyl d e r i v a t ivc?s of 2 - n a p h t o l . 71 -72

The goal of t h e present study i s t o synthesize a

s e r i e s of non-acidic 2-naphthol aminomet hyl d e r i v a t i v e s

of type(P2) , which have s i m i l a r e l e c t r o n i c and I

stereochemical c h a r a c t e r i s t i c s t o t h e potent

b i s (2-hydroxy-1-naphthyl )methane d e r i v a t i v e s and then

screen them r o r anti-inflammatory a c t i v i t y , using a

standard procedure. The d e r i v a t i v e s a r e a l s o considered

amenable t o complexation or c h e l a t i o n due t o t h e

presence of lone p a i r of e l e c t r o n s on t he t e r t i a r y

. ni t rogen and the hydroxyl group.

I n order ,to c o r r e l a t e t h e observed anti-inflammatory

e f f e c t with the chemical s t r u c t u r e s , t h e physicochemical

p r o p e r t i e s y& s o l u b i l i t y , p a r t i t i o n c o e f f i c i e n t and

d i s s o c i a t i o n constant of t h e synthesized aminornethyl

d e r i v a t i v e s of 2-naphthol were determined.

(71) (72)

7 3 R- and R are e i t h e r alkyl or a r y l groups.

CHAPTER TWO

. THEORETICAL ASPECTS

The c h e m i s t r y o f Mannich r e a c t i o n , first i n t r o d u c e d by

Mannich h d s been t h e s u b j e c t o f i n v e s t i g a t i o n s by an ever-

i n c r e a s i n g number of r e s e a r c h e r s 73-74 and t h e y have p rov ided

e x c e l l e n t coverage on p r a c t i c a l l y t h e e n t i r e c h e m i s t r y of

t h e r e a c t i o n . The a p p l i c a t i o n s o f t h e Mannich reac t ' ion , t h e

problems o f o r i e n t a t i o n and mechanism, and i t s r e a c t i v i t y

have been i n v e s t i g a t e d . 73*75. The r e a c t i o n ( o t h e r w i s e known

a s dminomethyla t ion) i n v o l v e s t h e c o n d e n s a t i o n o f a n amino

compound ( p r i m a r y or secondary amine) , a n a l d e h y d e (formal-

dehyde o r paraformaldehyde o r any o t h e r a ldehyde) and a compound

. c a p a b l e of s u p p l y i n g one o r more a c t i v e hydrogen atoms.

A v a s t v a r i e t y of a l i p h a t i c , a r o m a t i c and h e t e r o c y c l i c sys tems

v i z pheno l s , n a p h t h o l s , qu inones , a l d e h y d e s , k e t o n e s , a l c o h o l s , 7

. steroids, cc l rboxyl ic a c i d s , esters, arnides and i m i d e s have been

obse rved t o undergo t h i s t y p e of r e a c t i o n . 74 For a l k y l k e t o n e s ,

t h e rnost w i d e l y and s u c c e s s f u l l y used r e a c t i o n c o n d i t i o n

i r t v o i v e s r t t f i u x i r ~ y t h e s u b s t r a t e , amine h y d r o c h l o r i d e and

paraformaldehyde (or aqueous formaldehyde) i n a l c o h o l i c s o l v e n t s

f o r s e v e r a l hours .

T h e p h e n o l s r e q u i r e a s h o r t t i m e r e f l u x ( u p t o s e v e r a l h o u r s )

o r a l l o w e d to s t a n d fer a l o n g e r t i m e ( u p to few d a y s ) .

Aqueous formaldehyde and t h e m i n e base a r e o f t a n used.

The carboxylic ac id d e r i v a t i v e s r e a c t with t h e amine and

aqueous formaldehyde a t room temperature, using water or

a lcohol a s solvent. The r e a c t i o n of he te rocyc l i c compounds

w i t h amines and formaldehyde very o f t e n occur a t room

temperature, though sometimes brief heating i s required.

Primary nlnirles can r e a c t t o give va r ious products, but v l t h

secondary amlnos, only a single product is posslblo. 76 -77

The r e a c t i o n can be acid-catalysed 78-79 or base-cetaly sed, 77

depending on the substra+t>.. . The choice of t h e solvent i s

of g rea t importance and the commonly used so lven t s include

water, a c e t i c ac id , ~soamyla lcoho l and toluene. Solvents

w i t h e levated boi l i .hg: p i n t s than e thanol can be used t o speed

up a slow r e a c t i o n , though they possess t h e p o t e n t i a l of

hastening t h e e l imina t ion of t h o amine from t h e product and

t h u s complicate the process. 89-81

Mannich r e a c t i o n in t roduces a bas ic function which can render

t h e molecule solub'le i n aqueous so lvents when it i s transformed

i n t o aminium s a l t s . The bases a r e very r e a c t i v e and can

e a s i l y be transformed i n t o numerous other compounds. The

r e a c t i v i t y of t h e bases accounts f o r their var ious

a p p l i c a t i o n s u.

( a ) Easy modif icat ion of s t r u c t u r e s f o r s t r u c t u r e -

a c t i v i t y r e l a t i o n s h i p s t u d i e s e. g. c y t o s t a t i c 82 -83 , a n t i -

and card io tonica5 a c t i v i t i e s .

( b ) Intermediates f o r t h e syn thes i s of o ther compounds 86

( c ) As medicinal agents egg. Mannich bases of hydantoin 87-88 9

91 O9 s ~ l p h o n a r n i d e s ~ ~ and succinimides b a r b i t u r i c a c i d s , ( d ) A s d y e s and surface-act ive agents.

With few 8xcept ions, aminomethylat ion of phenols occurs a t

t h e p o s i t i o n o r t h o t o hydroxyl group, even if t h e para

p o s i t ion f s unoccupied. 92

H N R R

f-4 CHO $VRR X

(74)

or x2 = H, for monosubstituted

XI, x2 # H, for d i subs t i tu ted .

The inf luence of func t iona l subst i t u e n t s on t h e o r i e n t a t i o n

of t h e Mannich r e a c t i o n i n phenols i s complex and t h e r e a c t i o n

condi t ions p l a y a:i important r o l e . Thus, 2-(2-hydroxypheny1)-

benzimidazole ( 7 5 ) i s aminomethylated a t the phenolic benzene

n u c l e u s rcithtx thdn dt the h e t e r o c y c l i c NH group, while i n

s d l i c y l a m i d e s (,,7.6~ r e a c t i o n o c c u r s f i r s t a t t h e amide group

and o n l y subsequent ly a t t h e p o s i t i o n o r t h o to t h e p h e n o l i c

hydroxyl group 9 3

94 A l s o Nob le s et d l t o b s e r v e d t h a t 4-acetamidophenol (-77)

was o n l y aminomethylated a t t h e p o s i t i o n o r t h o to t h e hydroxyl

group a f f o r d i n g Mannich bases of the t y p e ( ,78) . T h e s e are

s t a r t i n g m a t e r i a l s for t h e s y n t h e s i s of c e r t a i n q u i n o l i n e

d e r i v a t i v e s p o s s e s s i n g a n t i - m a l a r i a l a c t i v i t y .

HNRR 0 NCHO . @*.R

Bis-aminomethylation has been observed in certain phenols and

bisphenols, the arrow indicating the positions of attack.

Aminomethylation reactions on 0-and N-heterocyclic compounds

have also been carried out on hydroxyindoles (74'1, hydroxy-

quinolines ( ,801, h y d r o x y b e n z o ~ b l f u r a n s ( ~ ~ ) , hydroxycournarins(.82)

and hydroxychromones (83 1 .

xl, x2 and x3 a r e e i t h e r H or alkyl group. The products

obtained frow t I ! c nminomethylat ion of compounds (79-83)

i n d i c a t e t h a t :

( i ) For binuclear phenols having t h e hydroxy group i n a

pos i t iono( t o r i n g fus ion , the r e a c t i o n always proceeds t o

a f f o r d t h e product of or tho a t t a c k , even i f the para p o s i t i o n

i s f r e e , whereas t h e p - subs t i tu t ion i s obtained o f t e n i n good

y i e l d , when the o-posi t ion (p t o ring f u s i o n ) i s occupied 92995 . ( i i ) In t h e case of binuclear phenols having the hydroxy

group i n a p o s i t i o n B t o the r i n g fus ion , the r e a c t i o n always

proceeds i n t h e o-posi t ion o( t o r i n g fus ion , even i f t h e

0-@-position i s unoccupied; but when t h e 0-4 -pos i t ion i s

occupied, t h e r e a c t i o n occurs a t the f r e e 0-Q-position.

The 0-e-position of 5 and 6-hydroxyindoles has been found

96 i n a c t i v e i n t h e Mannich reac t ion . ,

97 Monti e t a 1 , observed a l s o t h a t r e a c t i o n usual ly occurs a t

t h e 0-o( -pos i t ion , but if t h i s i s occupied, a t t a c k occurs

a t p o s i t i o n 3 01. when t h i s i s occupied, it then occurs a t

t h e he te rocyc l i c NH.

The Mannich condensat ion of histamine (84) w i t h 3-hydroxypyr i-

dine-4-aldehyde(85] proceeds with c y c l i z a t i o n t o give

pyrido(3,4-6)imidazole der i ~ a t i v e ~ ~ ( 8 6 )

The reaction of 4,s-dihydroxypyrimidine ( 8 7 ) with formaldehyde

and primary amine (ratio 2 : l ) affords 8-hydroxy-3-methyl-2,

3-dihydro-4~-pyrimido-( 4.5-e)-1, 3-oxazinesg9 (88)

Mannich bases of phenols and related compounds have the

ability to undergo substitution of the amino group by a

group X

o HX R - CH2 - NHR -HNHR

R CH2X

1 0 0 The s u b s t i t u t i o n c a n be H - a l k y l a t i o n ,

(e.g R - CH2NRR + H2 3 R - CH3), N - a l k y l a t i o n 101-102 - HNRR 9

103-104 1 + R ~ S H S - a l k y l a t i o n (e.g R -CH2-NRR

2 - HNRR) RCHZS-R , and

S e v e r a l r e s e a r c h e r s have i n v e s t i g a t e d t h e mechanism o f t h i s

Mannich r e a c t i o n , 739106 p o s t u l a t i n g t h a t t h e r e a c t i o n l e a d s

t o p r o d u c t s of t h e t y p e R-cH~-N( , b e a r i n g an u n s y m m e t r i c a l l y

s u b s t i t u t e d m e t h y l e n e g r o u p c o n n e c t i n g t h e s u b s t r a t e r e s i d u e ( R )

w i t h t h e amino g r o u p (-N<),

The c o n d e n s a t i o n r e a c t i o n o c c u r s i n two s t e p s :

( i) t h e r e a c t i o n o f t h e amine w i t h fo rma ldehyde to g i v e

c o n d e n s a t i o n p r o d u c t ,

(ii) t h e a t t a c k o f

Thus, t h e p r o b a b l e

i s as shown below:

t h e s u b s t r a t e , R-H.

mechanism of r e a c t i o n of the p r e s e n t s t u d y

OH 1 - C - H

I H

2' amine

2.2 PHYSICO-CHEMICAL PROPERTIES

The e a r l y d e s c r i p t i o n s o f drug a c t i o n d id n o t go much

beyond r e f e r e n c e to a t o n i c o r t o x i c e f f e c t , a n d s i n c e t h e

t i m e o f E h r l i c h a n d Lang ley , t h e c o n c e p t o f r e c e p t i v e s u b s t a n c e s

o r r e c e p t o r s h a s been u s e d w i t h g r e a t e r f r e q u e n c y i n e x p l a i n i n g

d r u g e f f e c t s .

A s a n o u t g r o w t h o f t h e r e c e p t o r c o n c e p t o f d r u g a c t i o n ,

o n e f i n d s i n c r e a s e d e m p h a s i s o n t h e i m p o r t a n c e of p h y s i c a l -

c h e m i c a l p r o p e r t i e s o f t h e d r u g and t h e r e l a t i o n of such

p r o p e r t i e s to b i o l o g i c a c t i o n . The phys ico-chemica l p r o p e r t i e s

of t h e d r u g m o l e c u l e t h a t may have b e a r i n g on t h e b i o l o g i c

e f f e c t i n c l u d e p r o p e r t i e s l i k e s o l u b i l i t y , p a r t i t i o n c o e f f i c i e n t ,

d e g r e e o f i o n i s a t i o n , s u r f a c e a c t i v i t y , isosterism, i n t e r m o l e c u l a r

f o r c e s , o x i d a t i o n - r e d u c t i o n p o t e n t i a l s , i n t e r a t o m i c d i s t a n c e s

be tween f u n c t i o n a l g r o u p s and s t e r e o c h e m i s t r y ,

Drug m o l e c u l e s t h a t r e a c h t h e si te o f a c t i o n , must have t o

cross v a r i o u s membranes a n d i n t e r a c t w i t h a melange o f i n t e r -

c e l l u l a r and i n t r a c e l l u l a r f l u i d s and b i o p o l y m e r s b e f o r e

r e a c h i n g t h e s i te o f a c t i o n , Under t h e s e complex c o n d i t i o n s ,

t h e phys i co -chemica l p r o p e r t i e s o f t h e d r u g mus t c o n t r i b u t e

f a v o u r a b l y to a b s o r p t i o n a n d d i s t r i b u t i o n phenomena i n o r d e r

t o i n c r e a s e t h e d r u g c o n c e n t r a t i o n a t t h e a c t i v e site. ' ~ l s o ,

t h e phys ico-chemica l p r o p e r t i e s o f t h e d r u g mus t e n s u r e a

s p e c i f i c o r i e n t a t i o n o n t h e r e c e p t o r s u r f a c e s o t h d t a c h d i n

of e v e n k s i s i n i t i d t e d t h d t l e d d s to t h e o b s e r v e d d r u g e f f e c t .

Drug r n o l e c u l e s t h e r e f o r e , t h d t do n o t have t h e r e q u i s i t e

phys ico-chern icd l p r o p c r t i c s c o n t r i b u t i n g t o b o t h a c t i v e s i te

c r c c c s s i b i l i t y dnd f ~ v o u r c r u l e d r u g - r e c e p t o r i n t e r ~ c t i v n s lrwy

Iwvc: rr111y 111i I I ~ I I I ~ I ~ L,dulol~Lt; ~ i c l i u ~ r ur. Iru LuL~rlly vu ld u l (11 L ~ C L .

11) v i c w ul L t ~ c : i1111)ur LJI ILU ui buluLll1 ky , j~clrti llurr c.uc?ll i-

c i e n t and d i s s o c i a t i o n c o n s t a n t i n t h e a b s o r p t i o n , d i s t r i b u t i o n

dnd c l i m i n d t i o n o f d r u g s w i t h i n t h e b i o l o g i c a l s y s t e m , t h e

p r e s e n t s t u d y c o n s i d e r s i t i r n p o r t d n t t o d e t e r m i n e t h e s e

p r o p e r t i e s o n t h e s y n t h e s i z e d compounds a n d h e n c e e v a l u a t e

how w e l l t h e y correldte w i t h t h e observed a n t i - i n f l a m m d t o r y

~ c t i v i t y .

The term s o l u b i l i t y i s d e k i n e d i n q u a n t i t a t i v e terms a s t h e

c o n c e n t r a t i o n o f s o l u t e i n a s a t u r a t e d s o l u t i o n a t a c e r t a i n

t e m p e r a t u r e , a n d i n a ~ u a l i t a t i v e way, i t may be s t a t e d a s

t h e s p o n t d n e o u s i n t e r a c t i o n o f two or more s u b s t a n c : e s t o form

a homogenous m o l e c u l a r d i s p e r s i o n . 107 The s o l u b i l i t y o f a

compound d e p e n d s upon t h e p h y s i c a l a n d c h e m i c a l p r o p e r t i e s o f

t h e s o l u t e a n d t h e s o l v e n t , a s w e l l a s upon s u c h f a c t o r s a s

t e m o e r a t u r e , p r e s s u r e , t h e pH o f t h e s o l u t i o n and t o a lesser

e x t e n t t h e stdte of s u b d i v i s i o n o f t h e s o l u t e . The s o l u b i l i t y

o f a d r u g mdy oe e x p r e s s e d i n a number of ways. T h e U.S.

1db 12 pharmacopoeia dnd t h e B r i t i s h N a t i o n a l Fo rmula ry , l i s t e d 8 .

t n e s o l u b i l i t y o f d r u q s ds the number of m i l l i l i t r e s o i

s o l v e n t i n which one grdln of s o l u t e w i l l d i s s o l v e . I t i s dlso

q u d n t i t d t i v e l y expressed i n ~ n o l d l i t y , m o l a r i t y dnd p e r c e n t d y e .

For S U ~ Y ~ L ~ I K ~ S wIw:;t: : i u l ~ b . L l i t i ~ ~ rir'e n o t d e f i n i t e l y known,

t h e vcrluc:, dx-e tlr~cribcd i n thc of f i c i d l I ' h ~ i r ~ r r ~ ~ c e u t i c a r l corrrperrdirl

by t h e u s e of c e r t d i n g e n e r a l terms y& v e r y s o l u b l e ( less t h a n

1 p d r t of s o l v e n t r e q u i r e d f o r 1 p a r t o f s o l u t e ) ; f r e e l y s o l u b l e

(1-10 p a r t s of s o l v e n t r e q u i r e d f o r 1 p a r t o f s o l u t e ) ; s o l u b l e

(10-30 p a r t s o f s o l v e n t r e q u i r e d f o r 1 p d r t o f s o l u t e ) ;

s p a r i n g l y s o l u b l e ( 3 0 t o 100 p r t s of s o l v e n t r e q u i r e d f o r 1

p a r t o f s o l u t e ) ; s l i g h t l y s o l u b l e ( 1 0 0 t o 1000 p a r t s o f s o l v e n t

r e q u i r e d f o r 1 p d r t o f s o l u t e ) ; very s l i g h t l y s o l u b l e ( 1 0 0 0

to 10 ,000 p a r t s o f s o l v e n t r e q u i r e d f o r 1 p a r t o f s o l u t e ) a n d

p r a c t i c a l l y i n s o l u b l e or i n s o l u b l e (more t h a n 1 0 , 0 0 0 p a r t s of

s o l v e n t r e q u i r e d f o r 1 p a r t o f s o l u t e ) . 107

lU9 h i d e r e t a 1 , i n t h e i r s t u d y , r e p o r t e d t e c h n i q u e s s u i t a b l e - f o r a c c u r a t e d e t e r m i n a t i o n o f s o l u b i l i t i e s of s o l i d compounds

i n l i q u i d s dnd t h e mutudl s o l u b i l i t i e s o f t w o l i q u i d s . T h e

s o l u b i l i t y o f a d r u g i s d u e to t h e p o l a r i t y o f t h e s o l v e n t ,

t h a t i s , t o i t s d i p o l e moment. P o l a r s o l v e n t s d i s s o l v e i o n i c

s o l u t e s and o t h e r p o l a r s u b s t a n c e s . H i l d e b r a n d a n d S c o t t , 110

h a v e shown t h a t a c o n s i d e r a t i o n o f d i p o l e moments a l o n e i s

n o t s u f f i c i e n t t o e x p l a i n t h e s o l u b i l i t y of p o l a r s u b s t a n c e s

i n wdter , b u t r d t h e r t h e a b i l i t y o f t h e solute t o form hydrogen

bonds i s GI fdr more i n f l u e n t i a l f d c t o r t h a n i s t h e p o l d r i t y a s

reflected i n a h i y h d i p o l e nrornent. Non-polar ~ o l v e n t s d i u u o l v t ~

non-poldr s o l u t e s t h r o u g h i n d u c c d dipole interdctiunb dnd ttrs

' s o l u t e m o l e c u l e s are k e p t i n s o l u t i o n by t h e weak Vander Waals - London t y p e o f forces. Thus t h e s o l u b i l i t y of a s u b s t a n c e may

be p r e d i c t e d o n l y i n a q u d l i t d t i v e way i n most cases a n d o n l y

a f te r c o n s i d e r a t i o n o f p o l a r i t y , dielectric c o n s t a n t , associa-

t i o n , s o l v a t i o n , i n t e r n a l p r e s s u r e s , acid-base r e a c t i o n s .

When s l i g h t l y s o l u b l e e l e c t r o l y t e s dre d i s s o l v e d t o form

s d t u r a t e d s o l u t i o n s , t h e s o l u b i l i t y is d e s c r i b e d b y a s p e c i a l

c o n s t a n t , known a s t h e s o l u b i l i t y p r o d u c t , ( K ) o f t h e compound. SP

Many i m p o r t a n t d r u g s b e l o n g t o t h e class o f weak acids and bases

a n d t h e y react w i t h s t r o n g a c i d s a n d bases and , w i t h i n d e f i n i t e

r a n g e s o f pH, e x i s t a s i o n s which are o r d i n a r i l y s o l u b l e i n

water. The s o l u b i l i t y of weak e l e c t r o l y t e s i s s t r o n g l y i n f l u e n c e d

b y t h e pH of the s o l u t i o n , and r e p r e s e n t i n g a weak ac id a s HB

and t h e s o l u b l e i o n i s e d form a s B-, t h e e q u i l i b r i a i n a s a t u r a t e d

s o l u t i o n of t h i s s l i g h t l y s o l u b l e weak e l e c t r o l y t e are:

.. (1)

B- . ( 2 )

s i n c e t h e c o n c e n t r a t i o n o f t h e u n i o n i s e d form i n s o l u t i o n ,

H B s o l u t i o n i s e s s e n t i a l l y c o n s t a n t , t h e e q u i l i b r i u m c o n s t a n t

f o r t h e s o l u t i o n o f e q u a t i o n (11, i s

CHt;JSOiution I

tHBl solid

The c o n s t a n t f o r t h e a c i d - b a s e e q u i l i b r i u m , o f e q u a t i o n

D e l e t i n g t h e s u b s c r i p t , f l s o l u t i o n N , M87 s o l u t i o n 8 becomes CHI33

T h e t o t a l s o l u b i l i t y S , of CHB] c o n s i s t o f t h e c o n c e n t r a t i o n

o f t h e u n d i s s o c i d t e d a c i d [ H B ~ solution a n d t h e c o n j u g a t e base

o r i o n i s e d form [u-3

S u b s t i t u t i n g So, f o r [He] from e q u a t i o n ( 3 ) a n d t h e e x p r e s s i o n

f rom e q u a t i o n ( 5 ) , for 6-1, y i e l d s

T h i s e q u a t i o n , ( 8 1 , h a s b e e n e x p r e s s e d i n v a r i o u s forms by

Krebs a n d Speakman, 111, A l b e r t , H i g u c h i a n d Kos tenbaude r . et a l . 114 - . When t h e e l e c t r o l y t e i s weak and does n o t d i s s o c i a t e

apprec iab ly , the s o l u b i l i t y o f t h e acid i n water or dcldic

s o l u t i o n s i s ,

lkluj

P u t t i n g t h e s o l u b i l i t y equcr t ion ( 7 1 , i n t o t h e logcrr i thm form,

we h a v e

109 ( S - so) - 104 ka l o g S, - l o g k303 a n d f i n a l l y S - So

pHp - PKa * 109 m m (9) So

where pH t h e pH below which t h e drug s e p a r a t e s f rom s o l u t i o n s P

as t h e u n d i s s o c i a t e d acid.

S = i n i t i a l molar c o n c e n t r a t i o n o f compound added.

So = t h e mo la r s o l u b i l i t y o f t h e u n d i s s o c i a t e d a c i d .

E q u a t i o n (9) h a s been used t o d e t e r m i n e thepKa o f d r u g s .

Ana logous d e r i v a t i o n c a n be used t o o b t a i n t h e e q u a t i o n f o r t h e

s o l u b i l i t y of a weak base as a f u n c t i o n of t h e pH o f a s o l u t i o n .

The e x p r e s s i o n i s

where S = c o n c e n t r a t i o n of t h e d r u g i n i t i a l l y added a s t h e

salt-

S, = molar s o l u b i l i t y o f t h e f ree base i n water.

PHD P t h e pH above which t h e d r u g b e g i n s t o p r e c i p i t a t e f rom

s o l u t i o n a s t h e free base . Gorman a n d Hall, i n t h e i r s t u d y

of t h e r e l a t i o n s h i p be tween s o l u b i l i t y a n d d i e l e c t r i c c o n s t a n t

of t h e s o l v e n t f o u n d t h ~ t t h e s o l u b i l i t y o f secobarbital, when

e x p r e s s e d a s t h e l o g a r i t h m o f t h e mole f r e t i o n , wds a l i n e a r

f u n c t i o n o f t h e d i e l e c t r i c c o n s t a n t o f t h e s o l v e n t b l e n d -

However, t h e s l o p e o f t h e l i n e , r e p r e s e n t i n g t h e c h a n g e i n

t h e s o l u b i l i t y w i t h d i e l e c t r i c c o n s t a n t was d i f f e r e n t w i t h

e a c h s o l v e n t b l e n d used. T h i s phenomenon o f s o l v e n t b l e n d i s

c a l l e d co - so lvency , and t h e s o l v e n t s which i n c o m b i n a t i o n ,

i n c r e a s e t h e s o l u b i l i t y of t h e s o l u t e are called co - so lven t s .

The a d d i t i o n o f d l c o h o l to d o u f f e r e d a q u e o u s s o l u t i o n o f a

w e a k e l e c 4 r o l y t e ( i ) i n c r r d s e s t h e s o l u b i l i t y of t h e

u n i o n i s e d s p e c i e s by a d j u s t i n g t h e p o l a r i t y o f t h e s o l v e n t t o

a more f a v o u r a b l e v a l u e (ii) b e i n g less p o l a r t h a n water, i t

d e c r e a s e s t h e d i s s o c i a t i o n o f a weak e l e c t r o l y t e and t h e

s o l u b i l i t y of t h e d r u g goes down as t h e d i s s o c i a t i o n c o n s t a n t

i s d e c r e a s e d (pK, i s i n c r e a s e d ) .

I n c o n t i n u a t i o n w i t h t h e c d l c u l a t i o n o f s o l u b i l i t y a s a

f u n c t i o n o f pH, S t o c k t o n a n d J o h n s o n , H i g u c h i e t a l , 117 P

h a v e s t u d i e d t h e e f f e c t s of a l c o h o l c o n c e n t r a t i o n o n t h e

d i s s o c i a t i o n c o n s t a n t o f s u l p h a t h i a z o l e . A l s o Edmonson a n d

Goycrn, i n v e s t i g u t r d t h e b f r r c t s of d l c o h o l o n t h e s o l u b i l i t y

of p h e n o b d r b i t a l , dnd found t h d t t h e pK*, 7.41, i s r a i s e d to

7.92 i n d h y u r o d l c o h o l i c s o l u t i o n c o n t a i n i n g 30 p e r c e n t by

volume of a l c o h o l .

F u r t h e r m o r e , t h e s o l u b i l i t y So, o f u n i o n i s e d p h e n o b a r b i t a l

i s i n c r e a s e d f rom 0.12 p e r c e n t o r 0,005 moldr i n water to 0.64

p e r c e n t o r 0.0276 rnolur i n a 30 p e r c e n t a l c o h o l i c s o l u t i o n .

T h e i r r e s u l t s t h e r e f o r e i n d i c a t e t h a t a l t h o u g h t h e a d d i t i o n

o f d l c o h o l i n c r e d s e s t h e pK,, i t a l so i n c r e a s e s t h e s o l u b i l i t y

of t h e u n i o n i s e d form o f t h e d r u g o v e r t h a t f o u n d i n water

s u f f i c i e n t l y , so t h a t t h e pH may b e r e d u c e d somewhat b e f o r e

p r e c i p i t a t i o n o c c u r s , O t h e r f a c t o r s t h a t c a n a f f e c t s o l u b i l i t y

i n c l u d e the size and s h a p e o f t h e drug . S o l u b i l i t y i n c r e u s e s

w i t h d e c r e a s i n g p a r t i c l e s i z e a c c o r d i n g to t h e e q u a t i o n ,

l o g - 3 HTr

where S = s o l u b i l i t y o f t h e f i n e p a r t i c l e s

S, = s o l u b i m i t y of t h e s o l i d c o n s i s t i n g of r e l a t i v e l y

l a r g e p a r t i c l e s

1 = s u r f ace t e n s i o n o f t h e p a r t i c l e s ( e x t r e m e l y

d i f f i c u l t to o b t a i n w i t h s o l i d s )

V molar volume (vo lume i n un3 p e r mole o f p a r t i c l e s )

r = f i n a l r a d i u s o f t h e p a r t i c l e s i n cm.

R r t h e g d s c o n s t a n t

T = t h e a b s o l u t e t e m p e r d t u r e

The e q u a t i o n i s v e r y o f t e n a p p l i c a b l e to so l id or l i q u i d p a r t i c l e s

i n s u s p e n s i o n s o r emuls ions . The c o n f i g u r a t i o n o f a molecu le

and t h e k i n d of drrangerrlent i n t h e c r y s t a l a l s o have some in -

f l u e n c e on s o l u b i l i t y . A synunet r ica l p a r t i c l e may be less

s o l u b l e t h a n arb unsym~ne t r i cd l one. T h i s i s due to t h e fac t

t h e p d r t i c l e s of the c r y s t x l l i n e s o l u t e . The impor tance o f

s o l u b i l i t y to a phcrrmcieuticcil chcrnist; l i e s on i t s i n f l u e n c e

on a b s o r p t i o n , d i s t r i b u t i o n ( t r a n s p o r t a t i o n ) and e l i m i n a t i o n .

F o r a b s o r p t i o n to o c c u r , the drug must be i n s o l u t i o n (disso-

l u t i o n p r o c e s s ) . A b s o r p t i o n r e s u l t s i n d rug p a s s i n g i n t o t h e

b lood stxearn and from t h e b lood stream most d r u g s are dis tr ibuted b

~ t i s s u e s . T h i s d i s t r i b u t i o n i s a dynamic p r o c e s s w i t h d rug

p a s s i n g i n t o t i s s u e and back i n t o blood. From t h e b lood , t h e

dr,ug i s also f i l t e r e d or s e c r e t e d i n t o t h e u r i n e , with subse- ' 8 .

q u e n t e x c r e t i o n .

T h e rate w i t h which a substance y v e s i p t o s o l u t i o n when l

i t i s d i s s o l v e d i n o l i q u i d i s g i v e n by Noyc - Whitney e q u a t i o n , 119 ?

dc = d i s s o i u t i o n ra te where

K = d i s s o l u t i o n r a t e c o n s t a n t

S = surface a r e a of t h e d i s s o l v i n g so l id

Cs = s a t u r a t i o n s o l u b + & i t y o f , t h e sol$.d in t h e

d i f f u s i o p l layer

c - c o n c e n t r a t i o n o f the s o l u t e i n t h e bu lk , , s o l u t i o n a t

time, t,

Under s i n k c o n d i t i o n , ( i m e m C s 7 ' l c ) or ( i f c i s less t h a n

I S d p q r c e n t o f Cs ( s o 4 u b i l i t y of t h e d r ~ g ) , , e q u a t i o n ( 1 2 1 , i s

s impljJJ@ to

T h i s e q u a t i o n h d s been of gredt i m p o r t a n c e i n thq q u a n t i t a t i v e 1

d e t e r m i n a t i o n o f d i s s o l u t i o n r a te of d r u g m o l e c u l e s m

2 . 2 - 2 APPHHLNT PAHTITIL;ON COEFFICIENT

I t i s a s o l u b i l i t y r a t i o which i s a measu re of t h e

120-121 d i s t r i b u t i o n o f a s o l u t e be tween t w o i m m i s c i b l e p h a s e .

F o r mos t p u r p o s e s , t h e s e p h a s e s are o r g a n i c s o l v e n t a n d water

( o r b u f f e r ) . T r a d i t i o n a l l y , l - o c t a n o l i s u s e d a s o r g a n i c

p h a s e , b e c a u s e such s y s t e m s have been shorn t o be extremely useful

f o r c o r r e l a t i n g and m o d e l l i n g b o t h d i s s o l u t i o n and t r a n s p o r t

phenomena, 122 M a t h e m a t i c a l l y e x p r e s s e d , i t becomes

where Co = c o r b c e n t r a t i o n i n o r g a n i c p h a s e ;

Cw = c o n c e n t r a t i o n i n a q u e o u s phase.

& q u a t i o n (14) dssurnes t h a t t h e compound, i f i t i s a n i o n i s a b l e

m o l e c u l e s u c h a s acid o r a base, is f u l l y non- ion i sed u n d e r the

c o n d i t i o n o f t h e measurement , T h i s u s u a l l y r e q u i r e s t h e u s e

o f drr acidic dqueous phdse i o r dn d c i d i c compound or of a n

a l k a l i n e p h d s e for a base. However, i t may be desirable to

medsure t h e part^ t i o n c o e f f i c i e n t u n d e r c o n d i t i o n s where t h e

compound i s p d r t i a l l y i o n i s e d f o r example b y u s i n g a pH 7-4

b u f f e r , I n t h i s cire, the ratic is teiriiied the a p p a r e n t p a r t i t i o r i

c o e t f i c i e n t to d i s t i n g u i s h i t from t h e real p d r t i t i o n c o e f f i c i e n t

for t h e t o tally non- ion i sed compound. The a p p d r r n t p d r t i t i o n

c o e f f i c i e n t ( P i s r e l a t e d t o t h e p r o p o r t i o n of d r u g p r e s e n t JPP

i n s o l u t i o n a s t h e non- ion i sed s p e c i e s , which i n t u r n d e p e n d s

upon t h e pH o f t h e a q u e o u s phdse used ,

where f U - f r a c t i o n o f m o l e c u l e s p r e s e n t a s t h e n o n - i o n i s r d

a c i d .

F o r monobas ic acid,

F o r mono a c i d i c base,

Hence f o r a n a c i d P a P

=PP 1 + 10 PH - PK,

a n d fo r a base P . . *PP

where P = a b s o l u t e (real) p a r t i t i o n c o e f f i c i e n t .

I t i s i m p o r t a n t to m e n t i o n t h a t p a r t i t i o n c o e f f i c i e n t w i l l be

c o n s t a n t o n l y when i t t r u l y r e f l e c t s t h e s p e c i e s t h a t i s i n

e q u i l i b r i u m between t h e p h a s e s , t h a t i s r a t i o o f t h e - c o n c e n -

t r a t i o n o f t h e s p e c i e s common to b o t h s o l v e n t s . F o r i n s t a n c e ,

a s i t u a t i o n , where t h e s o l u t e e x i s t s p a r t l y o r w h o l l y a s

a s s o c i a t e d m o l e c u l e s i n o n e of t h e p h a s e s o r d i s s o c i a t i n g i n t o

i o n s i n e i t h e r o f t h e l i q u i d p h a s e s , t h e d i s t r i b u t i o n law

app l i e s o n l y to the c o n c e n t r d t i o n of the species common to

b o t h phase , namely t h e monomer o r s i m p l e m o l e c u l e s of t h e

s o l u t e , T a k i n g a n acid, HA, f o r example,

( i ) when t h e r e i s no d i s s o c i a t i o n or a s s o c i a t i o n i n e i t h e r o f ,- L

t h e p h a s e s , e q u a t i o n ( 14) a p p l i e s 1.e K = ejw

where K = t r u e d i s t r i b u t i o n ( p a r t i t i o n ) c o e f f i c i e n t

C ( H A ) = molar c o n c e n t r a t i o n of the s i m p l e acid 0

m o l e c u l e s i n t h e o r g a n i c phase .

GkbJw = m o l a r c o n c e n t r a t i o n o f t h e u n d i s s o c i a t e d acid i n t h e

water phase.

(ii) when t h e r e i s d i s s o c i d t i o n i n a q u e o u s p h a s e , the e q u a t i o n

below a p p l i e s , 123

w h e r e K, d i s s o c i a t i o n c o n s t a n t o f t h e a c i d

= hydrogen i o n c o n c e n t r a t i o n ( c a l c u l a t e d from t h e

pH o f t h e a q u e o u s p h a s e )

Cw c o n c e n t r d t i o n o f t h e d c i d i n a q u e o u s phase .

K = t r u e d i s t r i b u t i o n ( p a r t i t i o n ) c o e f f i c i e n t

C = c o n c e n t r u t i o n o f t h e a c i d i n t h e water p h a s e ( o r t h e

orgoln ic phase) b e f o r e t h e d i s t r i b u t i o n i s c a r r i e d o u t .

cr plot oi Ka + C'3~f7

dguinat [ t i j~+) , y i u l d s t~ s t r a i y h t l i n e cw k + l K, with d s l o p e d = - a n d a n i n t e r c e p t b = - C C

~l t e r n d t i v e l y, the t r u e d i s t r i b u t i o n c o n s t a n t c o u l d be obto l ined

a c c o r d i n g to e q u a t i o n (14) by a n a l y s i s o f o r g a n i c phdse and o f

t h e water p h a s e d t a s u f f i c i e n t l y l o w pH (pH 2.3), where t h e

a c i d would e x i s t c o m p l e t e l y i n t h e u n i o n i s e d form. The a d v a n t a g e

of e q u a t i o n ( 1 5 ) i s t h a t t h e o r g a n i c p h a s e need n o t be a n a l y s e d ,

o n l y t h e hydrogen i o n c o n c e n t r a t i o n a n d Cw, t h e t o t h l c o n c e n t r a -

t i o n relrraining i n t h e a q u e o u s p h a s e a t e q u i l i b r i u m need b e

d e t e r m i n e d .

. ( i i i ) When t h e r e i s a s s o c i a t i o n i n o r g a n i c p h a s e , f o r example

b e n z o i c a c i d d i s t r i b u t e d be tween benzene dnd a c i d i f i e d w a t e r ,

t h e e q u a t i o n below a p p l i e s , 124

a s s o c i a t e d s i m p l e m o l e c u l e s

m o l e c u l e s

a n d t h e e q u i l i b r i u m c o n s t a n t e x p r e s s i n g t h e d i s soc i a t i an of

a s s o c i a t e d m o l e c u l e s i n t o s i ~ r ~ p l e m o l e c u l e s i n t h i s s o l v e n t i s

where HA^^' = t o t d l ino la r c v n c e n t r c i t i o n o f t h e s o l u t e i n

t h e o r g a n i c lclyer. I n t h i s s y s t e m , n = 2, h e n c e e q u a t i o n ('18) I I

becomes A

(k ido = conskcint &-

A knowledge o f p a r t i t i o n c o e f f i c i e n t i s i m p o r t a n t to t h e

p h a r m a c e u t i c a l ctiemist, f o r t h e p r i n c i p l e i s i n v o l v e d i n

s e v e r a l a i e u s of c u r r e n t p n a r m a c e u t i c a l i n t e r e s t , T h e s e i n c l u d e

p r e s e r v a t i o n o f o i l - water s y s t e m s (e.g e m u l s i o n s , creams

e t c ) , d r u g a c t i o n a t n o n - s p e c i f i c s i tes a n d t h e d b s o r p t i o n and

d i s t r i o u t i o n o f d r u g s t h r o u g h o u t t h e body,

Rdi-in dnd Conn, 125 f o u n d t h a t t h e p r e s e r v a t i v e o r bacterio-

s tdtic a c t i o n o f b e n z o i c a c i d a n d s i m i l a r a c i d s i s d u e e n t i r e l y

t o t h e u n d s s o c i ~ t e d d c i d dnd n o t t o t h e i o n i c form. T h i s

miirked p r e s e r v d t i v e a c t i o n o f u n d i s s o c i a t e d b e n z o i c a c i d &s

compared w i t h t h e i n e f f e c t i v e n e s s o f t h e b e n z o a t e i o n i s p re -

sumably d u e t o t h e r e l a t i v e e a s e of t h e u n i o n i s e d m o l e c u l e a n d

c o n v e r s e l y t h e d i f f i c u l t y of t h e i o n to p e n e t r a t e l i v i n g membrane.

I n l i n e w i t h t h i s r e p o r t , Uean e t a l , rn

showed t h a t b a c t e r i a

i n o i l - wdter sys tems a r e g e n e r a l l y l o c a t e d i n t h e aqueous

p h a s e and a t t h e o i l - w a t e r i n t e r f a c e , hence t h e p r e s e r v a t i v e I

a c t i o n of b e n z o i c a c i d o r any wedk a c i d i s v e r y l a r g e l y due to

t h e c o n c e n t r d t i o n of t h e u n d i s s o c i a t e d a c i d i n aqueous phdse,

I n t h e i r e x t e n s i v e s t u d y of t h e u s e o f t h e p a r t i t i o n

c o e f f i c i e n t i n r e l a t i n g p h y s i c a l p r o p e r t i e s t o b i o l o g i c e f f e c t ,

Hdnsch and ~ n d e r s o n , showed t h a t t h e d e p r e s s a n t e f f e c t s of

b o t h homologous and non-homologous series o f a l i p h a t i c a l c o h o l s ,

arnines, esters, k e t o n e s , u r e t h a n e s , s u l p h i d e s , ca rbamates ,

pheno l s , r e s o r c i n o l s and 5 , s - d i s u b s t i t u t e d b a r b i t u r a t e s i n c r e a s e

w i t h a n i n c r e a s e i n t h e i r r e s p e c t i v e p a r t i t i o n c o e f f i c i e n t s .

They a l s o found t h a t a t v a l u e s g r e a t . e r t h a n l o g a r i t h m of p a r t i -

t i o n c o e f f i c i e n t , l o g P , (i.e l o g P o f t h e compound producing

t h e maximal d e p r e s s a n t e f f e c t ) , t h e r e i s a n obse rved d e c r e a s e

i n t h e b i o l o g i c e f f e c t . This cu t , -o f f p o i n t i s c o n s t r u e d as

a loss of d e p r e s s a n t d rug i n non-act ive s i te l i p i d areas

t o g e t h e r w i t h the p o t e n t i a l c o m p e t i t i v e effects of metabolism

and e x c r e t i o n i n whole animal s t u d i e s . Hansch and F u j i t a , 128

have i n d i c a t e d t h a t a p o t e n t i a l mechanism for the n o n s p e c i f i c

d rug effect o f t h e s e series of compounds may be r e l a t e d t o t h e

i n h i b i t i o n of e l e c t r o n t r a n s p o r t i n o x i d a t i v e metabolism.

The s i g n i f i c a n c e of p a r t i t i o n c o e f f i c i e n t i n t h e a b s o r p t i o n

a n d d i s t r i b u t i o n of d r u g s t h r o u g h o u t t h e body l i e s i n t h e

f a c t t h a t body membrdnes are m a i n l y l i p o i d i n n a t u r e and

c o n s e q u e n t l y dre more e a s i l y p e n e t r d t e d by l i p o p h i l i c m o l e c u l e s

t h a n by h y d r o p h i l i c m o l e c u l e s . A s t h e p a r t i t i o n c o e f f i c i e n t

be tween o r g a n i c s o l v e n t dnd wdter i s a n i n d e x o f t h e l i p i d

s o l u b i l i t y of a conlpound, t h e r e i s o f t e n a r e l d t i o n s h i p be tween

this dnd t h e rdte of t r d n s f e r of a m o l e c u l e across a b i o l o g i c d l

mernbrdne. This p r i n c i p l e of t h e pH - p a r t i t i o n h y p o t h e s i s

a l so d p p l i e s to d b s o r p t i o n i n t h e mouth, transfer o f d r u g s f rom

or i n t o v d r i o u s o t h e r t i s s u e s or o r g a n s , i n c l u d i n g t r a n s f e r

f r o m t h e blood stream i n t o t h e s a l i v a , t h e e x c r e t i o n o f d r u g s

v i a - sweat, t h e s e c r e t i o n o f d r u g s i n t o m i l k and t h e t r a n s f e r

of d r u g s across t h e c o r n e a , t h e p l a c e n t a , a n d i n t o and o u t o f

c e r e b r o s p i n a l f l u i d and t h e b r a i n , I n a series o f compounds

c o n s i s t i n y of diitercnt s u b s t i t u e n t g r o u p s o n a co~nrnon n u c l e u s ,

t h e c o n t r i b u t i o n o f a p a r t i c u l a r s u b s t i t u e n t (the s u b s t i t u e n t

c o n s t a n t , TT) i s d e f i n e d as , 129

= l o g Px - l o g P,,

w h e r e PH 5 p a r t i t i o n c o e f f i c i e n t o f t h e p a r e n t compound.

Px = p a r t i t i o n c o e f f i c i e n t of the d e r i v a t i v e ,

Hansch e t a l , - 130-131 i n a n o t h e r s t u d y showed t h e a d v a n t a g e

o f u s i n g p a r t i t i o n c o e f f i c i e n t s i n c o n n e c t i o n w i t h t h e H a m m e t t

e q u a t i o n to r a t i o n a l i s e t h e s u b s t i t u e n t effect on t h e growth-

promot ing a c t i v i t y of t h e phenoxyace t i c a c i d s and t h e bacteri-

c i d a l a c t i o n of ch lo romyce t in d e r i v a t i v e s on v a r i o u s bacteria.

They found t h a t s u b s t i t u e n t c o n s t a n t , n, p a t t e r n e d a f t e r t h e

H d r n l n e t t c o n s t a n t , 6, wds u s e f u l i n e v a l u a t i n g t h e l i p o -

h y d r o p h i l i c c h a r d c t e r o f a molecule upon which b i o l o g i c a l

a c t i v i t y i s h i g h l y dependent . Hdnsch and F u j i t a , 128 u s i n g t h e

e l e c t r o n i c s u b s t i t u r n t c o n s t a n t d, and a s u b s t i t u e n t ( l i p o p h i l i c )

c o n s t a n t , n , showed t h e e f f e c t of subst i tuents on t h e b i o l o g i c a l

a c t i v i t y of benzoic a c i d s on mosquito l a r v a e , p h e n o l s on gram-

p o s i t i v e and gram-negative b a c t e r i a , phenyl e t h y l phospha te

i n s e c t i c i d e s on h o u s e f l i e s , t h y r o x i n e d e r i v a t i v e s on r o d e n t s ,

d i e t h y l a m i n o e t h y l b e n z o a t e s on g u i n e a p i g s and c a r c i n o g e n i c

compounds o n mice.

T h i s i s a n e q u i l i b r i u m c o n s t d n t i n which t h e e s s e n t i a l l y

c o n s t a n t c o n c e n t r a t i o n o f the s o l v e n t i s i n c o r p o r a t e d , F o r a

weak a c i d ( H A ) ,

whe re aHt - a c t i v i t y of hydrogen i o n ;

a ~ - - a c t i v i t y of the a n i o n a n d

a = a c t i v i t y of t h e weak a c i d . HA

a t aOH- For a weak base, Kb = B

a BOH

F o r water, K" .n a + aOH0 H (23)

These c o n s t a n t s are c a l l e d thermodynamic c o n s t a n t s , and t h e y

are t r u e c o n s t a n t s d e p e n d e n t o n l y upon t e m p e r a t u r e . 332

The c o n c e n t r a t i o r l c o n s t a n t , KC, e.9.

F o r a weak acid Kc = [H fa-3

when combined w i t h e q u a t i o n (21) a n d - l o g yi = 0.5C9 Z: fi

w h e r e = a c t i v i t y c o e f f i c i e n t

Z = v a l e n c y o f t h e i o n . I = t h e i o n i c s t r e n g t h .

A t v e r y d i l u t e s o l u t i o n , t h e c o n c e n t r a t i o n c o n s t a n t , Kc

a p p r o a c h e s t h e thermodynctirric c o n s t a n t Ka.

The most w i d e l y u s e d method f o r t h e d e t e r m i n a t i o n of a c i d i t y

c o n s t a n t s i s p o t e n t i o m e t r i c pH t i t r a t i o n , The t e c h n i q u e s u s e d

i n c a r r y i n g o u t t h e t i t r d t i o n are s t a n d a r d and are w e l l c o v e r e d

by A l b e r t dnd S e r j e d n t . 133 T h i s i n v o l v e s a medsurernent o f

pH w i t h a g l d s s e l e c t r o d e - s a t u r a t e d calomel electrode cel l -

The p H meter s h o u l d be a c c u r a t e l y calibrated w i t h a t leas t t w o

s t a n d a r d b u f f e r s , t h e b u f f e r s a n d t h e s o l u t i o n s to be t i t r a t e d

s h o u l d a l l be k e p t a t a c o n s t a n t t e m p e r a t u r e , a l l s o l u t i o n s

s h o u l d be c a r b o n d i o x i d e free, t h e t i t r a t i o n s h o u l d be carried o u t

u n d e r n i t r o g e n , and t h e pH s h o u l d be read f o r a l a r g e number o f

t i t r a t i o n a d d i t i o n s . A s pH i s a measu re of a c t i v i t y , t h e s e

e x p e r i m e n t a l measu remen t s lead t o e v a l u a t i o n of a q u a n t i t y t h d t

i s n e i t h e r Ka n o r Kc. I t i s i n s t e a d a c o m p o s i t e q u a n t i t y

2 c a l l e d t h e a p p a r e n t d i s s o c i a t i o n c o n s t a n t , K a , d e f i n e d by

F o r many p u r p o s e s , K,' i s s u f i i c i e n t l y close to Ka a n d i f L o t h do

n o t d i f f e r by more t h a n 0.01 - 0.02 u n i t , i t i s u n n e c e s s a r y t o

correct t h e K: v e l u e . T h e r e a r e a l a r g e number o f p o s s i b l e ways

i n wh ich t h e e x p e r i m e n t a l dd ta may be e v a l u a t e d -

The f i r s t ' i s t h e h a l f - n e u t r a l i s a t i o n method. 134 T h i s method

i s b a s e d o n t h e Henderson - H a s s e b a l c h a q u a t i o n ,

Ka = S t o i c h i o m e t r i c d i s s o c i a t i o n c o n s t a n t . The e q u a t i o n

u t i l i s e s t h e p r i n c i p l e t h d t when t h e c o n c e n t r a t i o n o f t h e b a s e

( c o n j u g a t e bdse, i f a c i d i s b e i n g t i t r a t e c i ) equals t h e concen-

t r d t i o n o f a c i d , t h e s u b s t a n c e i s h a l f - n e u t r a l i s e d , A t this

p o i n t t h e pH of t h e s o l u t i o n a s r e a d on t h e pH meter- e q u a l s

t h e pka o f the drug. Goldman and Meites, 13' i n t h e i r s t u d y o n

how d i l u t i o n a n d hydrogen- ion e f f e c t i n t r o d u c e e r r o r s on t h e

a s su rnp t ion t h a t pH - pKa a t h a l f n e u t r a l i s a t i o n , d e r i v e d t h e

f o l l o w i n g e q u a t i o n t o locate t h e p o i n t a t which pH a c t u a l l y

d o e s e q u a l pK, d u r i n g p o t e n t i o m e t r i z t i t r a t i o n ,

fpK, I d e g r e e of n e u t r a l i s d t i o n when pH e q u a l s pK,.

Kw L t h e i o n i c p r o d u c t of water

53 r = - 6 . i.e r d t i o o f t h e c o n c e n t r a t i o n b e i n g t i t r a t e d to t h e

=b c o n c e n t r a t i o n o f t h e t i t r a n t , E q u a t i o n (281 , tries to e x p a l i n

t h a t fpK, w i l l c o i n c i d e w i t h h a l f n e u t r a l i s a t i o n o n l y when K,

k! K . S u b s t a n t i a l errors w i l l r e s u l t f r o m t a k i n g pK, to be

4 e q u d l t o pH a t f - 4 i f K . a i s w i d e l y d i f f e r e n t f rom (K,) , a n d t h i s i s t r u e if t h e c o n c e n t r a t i o n o f t h e r e a g e n t s are

small , The v a l u e o f r , a f f e c t s f p k a , i n t h e s e n s e t h a t i f t h e

c o n c e n t r a t i o n s of t h e t i t r a n t a n d t h e s o l u t i o n t o be t i t ra ted

a r e equal , t h e r e i s a t e n d e n c y f o r t h e fpk, t o d r o p t o 0,364.

This i s d u e t o a g r e d t e r d i l u t i o n c f k e c t and g i v e s d good

i l l u s t r d t i o n o f t h e p r i n c i p l e b e h i n d t h e usual instructians

c d l l i n g f o r a c o n c e n t r a t e d s o l u t i o n of t h e t i t r d n t . The s e c o n d

method, , i s j u s t a v a r i a t i o n of t h e " h a l f - n e u t r a l i s a t i o n "

method, T h i s assumes t h a t t h e pka is t a k e n t o be e q u a l t o t h e

pH a t t h e p o i n t of i n f l e c t i o n o f t h e s i g m o i d t i t r a t i o n c u r v e ,

4 Aga in , t h i s method o n l y g i v e s a correct pka when k, = ( K w ) , o t h e r w i s e f , ( t h e d e g r e e o f n e u t r a l i s a t i o n a t t h e i n f l e c t i o n

p o i n t ) p r e c e d e s fpka and t h e d i v e r g e n c e i n c r e a s e s as acid

s t r e n g t h i n c r e a s e s . P a k e a n d Ddvis , 136 i n d n a t t e m p t t o

overcome some of t h e errors i n h e r e n t i n u t i l i s i n g t h e h a l f -

n e u t r a l i s a t i o n method, p a r t i c u l a r l y t h a t due t o t h e f a c t t h a t

t h e c o n c e n t r a t i o n of hydrogen i o n i s n o t n e g l i b l e , a s compared

t o t h e c o n c e n t r a t i o n o f ac id or base f o r f a i r l y s t r o n g acids

(pka 2-41 a n d base (pka 10 - 121, i n t r o d u c e d a d i f f e r e n t

n o n - l i n e a r method f o r d e t e r m i n i n g d i s s o c i a t i o n c o n s t a n t s .

T h i s t h i r d method i n v o l v e s t h e u s e o f hydrogen - i o n b i n d i n g

c u r v e s t o d e t e r m i n e t h e a p p a r e n t d i s s o c i a t i o n c c n s t a n t s ,

The method e n t a i l s t i t r a t i n g i d e n t i c a l vo lumes of s a m p l e

s o l u t i o n and b l a n k w i t h a s t r o n g t i t r a n t o v e r the pH r a n g e

2 - 12. The c u r v e s of

t h e s e two t i t r d t i o n s were p l o t t e d on a graph hav ing pH a s t h e

a b s c i s s a and t h e amount o f t i t r a n t added a s t h e o r d i n a t e .

A t h i r d c u r v e , t h e d i f f e r e n c e c u r v e , was t h e n drawn w i t h t h e . a . . . , 8 ' ' .,...,! , .,, , , . - , + t -

u n i t s o f t h e o r d i n a t e b e i n g t h e d i f f e r e n c e s i n mil l i l i t res o f . ,. L .,

t i t r c l n t r e q u i r e d f o r t h e sa~riple and b lank t o r e a c h t h e same pH.

P r e v i o u s l y prepdred t r d n s p d r r e n t masks on which t h e s t a n d a r d

c u r v e has been drdWn dnd tho p o i n t of i n f l e c t i o n marked are t h e n

f i t t e d t o t h u difference curve . S i n c e "any t i t r ~ b l e group produces

a n i n f l e c t i o n having t h e same shape r e g a r d l e s s of i t s p o s i t i o n

o n t h e pH scale, t h e a p p a r e n t d i s s o c i a t i o n c o n s t a n t w i l l be t h e

pH a t t h e p o i n t of i n f l e c t i o n n . Garrett, i n h i s a n a l y s i s of

t h e t h e o r e t i c a l b a s i s f o r t h e P a r k e and Davis method, showed

t h a t f o r d i s s o c i a t i o n c o n s t a n t s i n t h e r a n g e 4 - 10, t h e

p r o c e d u r e h a s l i t t l e a d v a n t a g e o v e r t h e t r a d i t i o n a l t e c h n i q u e of

d e t e r m i n i n g a p p a r e n t pK1s a t h a l f - n e u t r a l i s a t i o n . H e a l s o no ted

t h a t f o r d i s s o c i a t i o n c o n s t a n t s i n t h e r a n g e 2 - 4, t h e t e c h n i q u e

i s i n v a l i d , e s p e c i a l l y f o r estimates o f s t o i c h i o m e t r y d u e to

e r r o c s a r i s i n g i n t h e s u b s t r a c t i o n of volumes. J o s e p h , and

Druckrey, 139 t rdns fo rmed t h e s igmoid form of t i t r a t i o n c u r v e s

based on t h e mass a c t i o n law i n t o a l i n e a r form by l o g a r i t h m i c

method, I n t h i s f o u r t h method, t h e l o g a r i t h m i c t r a n s f o r m a t i o n

h a s t h e advan tage of e a s e i n p l o t t i n g and i n v i s u a l i n g t h e r a t io

o f u n i o n i s e d t o i o n i s e d c o n c e n t r a t i o n . Al though l i n e a r l o g a r i t h m i c

p l o t s are e a s y t o c o n s t r u c t and h e l p f u l i n v i s u a l i z i n g t h e ra t io

acid t o base c o n c e n t r d t i o n s , t h e y o f f e r no improvement i n t h e

a c c u r a c y o f d i s s o c i a t i o n c o n s t a n t d e t e r m i n a t i o n s as compared to

t h e f i r s t t h r e e methods l is ted u n d e r n o n l i n e a r r e l a t i o n s h i p s .

The l a s t method to be c o n s i d e r e d , i s t h e n o n - l o g a r i t h m i c l i n e a r

t i t r d t i o n c u r v e method, w h i c h seems to p r o d u c e t h e most reliable

r e s u l t s . H o f s t e c , 140 d e m o n s t r a t e d t h e u s e of n o n - l o g a r i t h m i c

l i n e a r t i t r d t i o n c u r v e s f o r t h e d e t e r m i n a t i o n of t h e d i s s o c i a t i o n

c o n s t a n t a n d t h e t i t r a t i o n end p o i n t of a m o n o p r o t i c s p e c i e s .

H e d e r i v e d h i s e q u a t i o n o n t h e h e i s of t h e e l e c t r o n e u t r a l i t y

o f t h e s o l u t i o n , I n u s i n g t h e e l e c t r o n e u t r a l i t y e q u a t i o n , (291,

i t i s n e c e s s d r y to know t h e i o n i c s t r e n g t h of t h e s o l u t i o n so

t h a t hydrogen and h y d r o x i d e i o n a c t i v i t i e s mdy be c o n v e r t e d t o

c o n c e n t r a t i o n s , s i n c e i t i s t h e sum o f t h e c o n c e n t r a t i o n s o f t h e

v a r i o u s c h d r g e d s p e c i e s w h i c h must b a l a n c e , For t h e t i t r d t i o n

o f a w e a k d c i d (HA) w i t h a s t r o n g base, BOH

Li'J + Cs9 = K.3 + [~H-I ,. (29) w h e r e CH+J = c o n c e n t r a t i o n of t h e g i v e n s p e c i e s .

[B+I = c o n c e n t r o t i o n o f BOH added,

CA-~ = c o n c e n t r d t i o n of t h e c o n j u g a t e base,

P u t t i n g Z , as t h e a l g e b r a i c sum o i a l l t h e known i o n i c concen-

t r a t i o n s with r e s p e c t to e q u a t i o n (29), a t a n y p o i n t i n a t i t r i i t i o n

z - te+j + - [on] - 0 (30)

[A-1 c a n be o b t a i n e d by s o l v i n g two s i m u l t a n e o u s e q u a t i o n s (31)

a n d (32)

&qua t i o n ( 31) i s the cief i n i t i o n f o r t h e s t o i c h i o r n e t r i c

a i s s o c i d t i o n c o n s t d n t of a weak acid (K ) C

L q u d t i o n ( 3 2 ) i s the conscrv~tion of mdus c y u a t i o n . C: =

T h e r e f o r e , a p l o t o f Y v e r s u s zCH+J s h o u l d g i v e a s t r a i g h t

0 l i n e w i t h d s l o p e o f - - and dn i n t e r c e p t of C o w Kc

S i l l l i l d r l y , f o r tile t i t r u t i o n ol- a wedk bcrse,

0 Z - Cb - K c ( i ~ ) .o (35)

' would be a s t r a i g h t . l i n e w i t h a Thus a p l o t o f z v e r s u s - L W s l o p e ehual t o minus t h e d i s s o c i d t i o n c o n s t a n t dnd a n i n t e r -

0 c e p t o f Cb. L e s s o n a n d ~ r o w n l * ~ , pointed o u t t h a t t h e non-

l ogc i r i tl~ruic eyucl t ions o f b e n e t dnd Goyan, 142 uqudtions (34)

a n d (35 ) a r e n o t t r u l y g e n e r a l s i n c e t h e a u t h o r s worked w i t h

c o n c e n t r a t e d t i t r a n t such t h u t volume c h a n g e s c o u l d be i g n o r e d .

Thus , t h e y w o r k e d w i t h vdlurnt: c h a n g e s o f a b o u t 2 - 5% and

proposed a v a r i a t i o n of e q u a t i o n ( 3 4 ) . Accord ing t o t h e i r

d e r i v d t i o n s ,

z t = B+ + H+ - b ~ -

( S i m i l a r t o e q u a t i o n 3 0 )

where H = numbers of moles of hydrogen i o n p r e s e n t i n t h e

s o l u t i o n ,

~ 1 * - n u m b r r ~ of moles of s t r o n g base added t o t h e

s o l u t i u n .

OH- = numbers of moles of hydroxide i o n p r e s e n t i n t h e

s o l u t i o n .

The numbers o f mo les o f B' c a n be d e t e r m i n e d by m u l t i p l y i n g t h e

number o f m l o f t i t r a n t added times i t s n o r m a l i t y d i v i d e d b y

1000. However, t h e number of moles o f hydrogen a n d h y d r o x i d e

i o n s mus t be o b t a i n e d f r o m t h e e x p e r i m e n t a l pH data.

Thus ,

whe re V P volume of s o l u t i o n i n m l a t any p a r t i c u l a r p o i n t i n t h e

titrdtim !i.e c r i g i n u l volume t volume of t i t r a n t added) .

Thus , u s i n g moles a c c o r d i n g t o t h e method of Lesson a n d Brown,

f o r t h e t i t r a t i o n of a p u r e weak acid,

T h e r e f o r e , a p l o t o f Z* v e r s u s z * c H * ~ , would b e a s t r a i g h t

l i n e w i t h a slope e q u a l t o t h e n e g a t i v e r e c i p r o c a l of t h e 0

d i s s o c i a t i o n c o n s t a n t a n d a n i n t e r c e p t equal t o C,. The non-

l o g a r i t h m i c t i t r d t i o n p l o t u s i n g t h e L e s s o n a n d Brown d e r i v a t i o n

dl lows t h e r e s e d r c h e r t o u s e any c o n c e n t r a t i o n of t i t r d n t t h a t

h e wishes . The dccuracy l i e s on t h e a b i l i t y o f t h e pH meter t o

read correct v a l u e s and t h e resedrcher to c o n v e r t a c t i v i t i e s t o

c o n c e n t r a t i o n s .

According t o Benet and Goyan, the advolntages of t h i s - m e t h o d

i n c l u d e

(i) good r e p r o d u c i b l e r e s u l t s c a n be determined u s i n g

t i t r d t i o n d a t a from t h e pH r a n g e s 1.5 - 4 and 9 - 11. (ii) v e r y weak c o n c e n t r a t i o n s of a c i d s and b a s e s which might n o t

g i v e c l e a r l y d e f i n e d i n f l e c t i o n p o i n t s on a pH v e r s u s m l

t i t r a n t p l o t can g i v e a c c u r a t e L - e s u l t s when [H*_) and C O H ~ are i n c l u d e d i n t h e Z t e r m for a non- logar i thmic , ..I:... p l o t and,

t h e a d v a n t a g e s o f u s i n g low c o n c e n t r a t i o n s are I I

(a ) less problems w i l l be e n c o u n t e r e d r e s u l t i n g from t h e , 8 , , I I

p r e c i p i t a t i o n of t h e u n i o n i s e d s p e c i e s .

(b) t h e i o n i c s t r e n g t h and t h e r e f o r e t h e a c t i v i t y

c o e f f i c i e n t s c a n be better c o n t r o l l e d .

(iii) f o r wttdk crcid t h e t i t r c i t i o n nrdy start a t a pH a l r e a d y . / l a r g e r t h a n t h e pK, w i t h o u t a f f e c t i n g t h e a c c u r a c y o f , t h e ,

) . I t , , ' , I I '

d e t e r m i n a t i o n ,

( i v ) t h e l i n e drdwn i n t h e non-Aogarithmic p l o t s e r v e s a s a n .I ' 1 '

e s t i m a t e of t h e a c c u r a c y o f t h e d a t a . Any p o i n t which , ..,,,

d e v i a t e s g r e a t l y from t h e l i n e immedia te ly shows t h a t t h e ".

d a t a a t t h a t p o i n t are i n v a l i d .

(v). one o f t h e g r e a t a d v a n t a g e s o f non- logar i thmic t i t r a t i o n I a

,.' , 3 . '

c u r v e s is, i n t h e a b i l i t y t o d e t e r m i n e t h e molarity o f . .

t h e s o l u t i o n b e i n g t i t r d t e d w i t h o u t r e s o r t i n g t o e l e g d n t

d r y i n g t e c h n i q u e s o r a n e l e m e n t a l a n a l y s i s . T h i s makes

t h e method a v a l u a b l e tool when d e t e r m i n i n g t h e d + s s o c i a - . . 1 1

t i o n c o n s t a n t ,of a new m o n o p r o t i c s p e c i e s . . ,

I n summary, t h e r e f o r e , a n a p p a r e n t d i s s o c i a t i o n c o n s t d n t , i s

o n e t h d t i s d e t e r m i n e d a t h a l f - n e u t r a l i s a t i o n , i.e when t h e

c o n c e n t r a t i o n o f t i t r d n t added e q u a l s one-half t h e o r i g i n a l

c o n c e n t r a t i o n o f t h e weak a c i d o r b a s i c s u b s t a n c e , t h e pH r e a d o n

t h e meter e q u a l s t h e a p p a r e n t d i s s o c i a t i o n c o n s t a n t . The

a p p a r e n t g a i s u s u a l l y d e s i g n a t e d as pKa a n d i s a h y b r i d o f t h e

s t o i c h i o m e t r i c ( c o n c e n t r a t i o n ) pK, a n d t h e thermodynamic

( a c t i v i t y ) pKa 143 . The a p p a r e n t d i s s o c i a t i o n c o n s t a n t i s v e r y

i m p o r t d n t t o a p h a r m a c e u t i c d l c h e m i s t b e c a u s e i t c a n be u s e d t o

re la te

(i) P h y s i o l o g i c a l a c t i v i t y , f o r example

( a ) C o r r e l a t i o n o f a n t i - h i s t a m i n i c a c c i v i t y w i t h pK,. 144

( b ) Cor re l i i i t i on o f a n t i b a c t e r i a l a c t i v i t y o f s u l p h o n a m i d e s

w i t h pKa. 14 5

(ii) S o l u b i l i t y a n d r a te o f s o l u t i o n 146

( i i i ) E x t e n t o f b i n d i n g t o t h e p lasma p r o t e i n s 147

( i v ) t h e s t a b i l i t y or e x p l a i n t h e k i n e t i c s o f d e g r a d a t i o n

f o r a dissociable d r u g i n v a r i o u s b u f f e r s o l u t i o n s .

(v) w h e t h e r t h e d r u g i s i n t h e i o n i c o r non- ion ic form

( v i ) t h e complex f o r m a t i o n .

CHAPTER THREE

RESULTS AND DISCUSSION

CHAPTER THREE

RESULTS A N D DISCUSSION

Majority of the r e p o r t e d methods f o r t h e s y n t h e s i s of

Mannich bases o f p h e n o l s and r e l a t e d c o m p o u n d s ' i n v o l v e t h e

r e a c t i o n of t h e p h e n o l , t h e amhe and aqueous fo rma ldehyde 7

a t room t e m p e r a t u r e o r r e f l u x i n g f o r a s h o r t t i m e or sometimes

f o r ' s e v e r a l h o u r s , I n t h i s s t u d y , 2 -naphthol w a s r e a c t e d w i t h

aqueous fo rma ldehyde and d i f f e r e n t s e c o n d a r y arnines unde r

d i f f e r e n t e x p e r i m e n t a l c o n d i t i o n s . I n some c a s e s , t h e

r e a c t i o n w a s c a r r i e d o u t a t 50 - 60°c f o r h a l f a n h o u r , w h i I e

i n o t h e r s r e f l u x i n g w a s done f o r 4 - 6 h r s . or s e v e r a l hours.

The r e a c t i o n p r o d u c t s were o b t a i n e d i n good y i e l d s and

r e c r y s t a l l i s e d from t h e a p p r o p r i a t e solvents.

s p e c t r o s c o p y were i n good ag reemen t w i t h t h e a s s i g n e d

s t r u c t u r e s . I n the i n f r a - r e d s p e c t r a , t h e s t r e t c h i n g , . * -.. . -1

v i b r a t i o n a l a b s o r p t i o n band a t 3560 c m d e n o t e s t h e 0 - H

g r o u p , w h i l e t h e absorption band a t 1360 crn-' i s c o n s i s t e n t

w i t h t h e C-N of t h e t e r t i a r y amine. The e l e m e n t a l a n a l y s i s of

t h e compounds w a s c o n s i s t e n t w i t h t h e f o r m u l a e o f t h e prepared

compounds, I n t h e p h a r m a c o l o g i c a l a s s a y , a l l t h e compounds . were a d m i n i s t e r e d a t a d o s a g e l e v e l of 300 mg/kg body w e i g h t ,

e x c e p t compounds 1 and $1. A t 300 mg/kg body w e i g h t , t h e

. ..-. , P . I . . -t;. :\, 1,

, . + - -

two compounds produced convulsion, followed by death of

the animals within one hour of administration of the compounds,

Or! reduction of the dosage level to ,100 mg/kg body weight,

the compounds exhibited varying degrees of anti-inflammatory

effect without any behavioural or autonomic effects being

observed.

The results of the ph.armacologica1 screening shown

in tables 1 and 2, indicate: that: ~is(2-hydroxynaphthylmethyl)

piperazine {compound 121, was the most active, about 1.8

times.the potency sf aspirin. Dimethylaminomethyl derivative

(compound 11, displayed activity that was comparable. to that

of aspirin, The dibutylaminomethyl-2-naphthol(compound 3 )

was observed to possess a higher anti-inflammatory activity

than dipropylaminomethyl-2-naphthol(compound 2 ) , and the

activities of both were less to that of dimethylaminomethyl-2-

naphthol..

Among the saturated heterocyclic aminomethyl derivatives

of 2-naphthol, piperidinomethyl derivative (compound 10) was

observed to be the most active. This was followed by the

pyrrolidinomethyl derivative (compound 11) and the least

anti-inflammatory activity was exhlbi ted by the

morphilinomethyl-2-na~hthol (compound 9) .

TABLE 1

ANTI-INFLAMMATORY MEASUREMENT DATA

Compound I n i t i a l Diameter ~ i ~ a i Diameter of t h e r a t right of t h e r a t r i g h t h i n d Paw ( m m ) h i n d Paw ( m m ) .

C o n t r o l 2.41 4.23 I + 0.04

2.52 4.55 + 0.17 I

2.54 4.51 + 0.08 - Mean v a l u e = 1.94 + 0.11 -

Mean value = 0.90 + 0 - 2 2 - 3.29 4 - 5 1 + 0.07 - 2.13 3 -45 + 0.05 - 3-13 4.55 + 0.15 -

Mean v a l u e = 1.38 + 0.16 -

2.34 3 - 5 6 + 0.07 - 2.52 3.77 + '0 .02 -

Mean v a l u e = 1 - 2 2 + 0.03 -

D i f f e r e n c e

Compound I n i t i a l Diameter Final Diameter

Mean v a l u e = 1 . 4 3 ~ 0 . 1 0

Mean v a l u e = 1.14 + 0.15

Mean value = 1.46 + 0.11 -

Mean v a l u e = 1.56 + 0.12 -

Mean v a l u e = 1.82 + 0.13 -

Mean v a l u e = 1.35 + 0.13 .I

Compound

10

A s p i r i n

I n i t i a l Diameter F i n a l Diameter

Mean v a l u e = 1.17 - +' 0.07

Mean v a l u e = 1.23 - + 0 . 0 6

Mean v a l u e = 0 . 3 3 + 0.05 -

D i f f e r e n c e

1 .19

1 .O9

1 .23

Mean v a l u e = 1.05 + 0 . 1 3 -

Compound

' Control

Aspirin

96

TABLE 2

ANTI-INFLAMMATORY ACTIVITY OF AMINOMETHYL

Dose (mg/kg )

Oedema (mm+s.~) - Inhibition (%)

+SOD -

a = significantly different from the control a t P < 0.01 b = Not significantly different from the c s n t r o i at ~)0.05

S.D Standard deviation,

The r e s u l t s o f tile s o l u b i l i t y d e t e r m i n a t i o n i n t a b l e 3 ,

showed t h a t dimethylaminomethyl-2-naphthol (compound 1)

e x h i b i t e d t h e h i g h e s t s o l u b i l i t y , a b o u t 27 times t h e 7

s o l u b i l i t y of 2 -naphthol , As t h e homologous series i s b e i n g

d e s c e n d e d from d i p r o p y l a m i n o m e t h y l (compound 2 t o

d i b u t y l a m i n o m e t h y l (compound 3 ) d e r i v a t i v e s , s o l u b i l i t y was

o b s e r v e d t o d e c r e a s e and t h e v a l u e s were h i g h e r t h a n t h e

p a r e n t compound, A l l t h e s a t u r a t e d h e t e r o c y c l i c aminomethyl

d e r i v a t i v e s d i s p l a y e d h i g h e r s o l u b i l i t i e s t h a n 2 -nsph tho l ,

w i t h t h e p y r r o l i d i n o m e t h y l d e r i v a t i v e (compound 11) showing

t h e h i g h e s t v a l u e , a b o u t 5.9 t i m e s the s o l u b i l i t y of

2 -naph tho l ,

The d i c y c l o h e x y l a m i n o m e t h y l d e r i v a t i v e (compound 5 )

showed s o l u b i l i t y t h a t was s l i g h t l y a b o v e t h e p a r e n t compound.

The rest of t h e d e r i v a t i v e s were less s o l u b l e t h a n t h e p a r e n t

compound. The least s o l u b l e d e r i v a t i v e was N-methylan i l inomethyl -

2 -naph tho l (compound 81, b e i n g a b o u t 0.37 times t h a t ' o f

TABLE 3

DETERMINED VALUES O F S O L U B I L I T Y

COMPOUND

1

2

3

4

5

6

7

8

9

10

11

12

Naphthol

ABSORBANCE MOLAR SOLUBILITY

The apparent-partition coefficient study results listed

in table 4 showed the dimethylaminomethyl derivative

(compound 1) to possess the least partition coefficient

val ua, whi lu thu dlluobuty lcl~nlnomethyl der iva t ive (con\pound 4 )

gWfe the highest value. The rest of the derivatives gave

varying values for the partition coefficient depending on

their degree of lipophilicity.

The determined hydrophobic substituent constants

(n), table 5, indicate that the derivatives showed negative values except compounds 3 - 7 which exhibited positive values,

This hydrophobic substituent constant is a measure

of the substituent's contribution to solubility behaviour

in such a series of partitioning, Thus relative to

hydrogen (m = 01, if this constant is positive, it

contributes to the lipophilic character of the molecule,

if it is negative, it contributes to the hydrophily or water

solubility of the compound.

TABLE 4

APPARENT PARTITION COEFFICIENT DETERMINED VALUES

COMPOUND

1

2

3 - 4

5

6

7

8

9

10

11

12

Naph tho1

ABSORBANCE

0.493

0.126

0,076

0,045

0,058

0,095

0.070

0,132

0.115

0.128

0.302

0.347

0.107

LogP

-0.17

0.66

0.93

1.12

1.03

0.80

0.94

0.67

0.71

0.65

0-18

0-09

0.74

TABLE 5

HYDROPHOBIC SUBSTITUENT CONSTANTS ( 11)

COMPOUND

.The results of the apparent dissociation constants of

the compounds determined by potentiometric titration are

listed in tables 6 and 7. The values were evaluated by

employing the Henderson-Hasselbalch equation (ioeo the

half-neutralisation equation). The determined pKa values

were found to be in the range of 3.4 - 6.6. TABLE 6

POTENTIOMETRIC DETERMINATION DATA

,Dimethylaminomethyl Derivative (Compd. 1)

Volume (mL 1

0 00

005

1.0

lo5

200

2.5

3.0

305

4.0

4.5

5.0

505

~ipropylaminomethyl Derivative (Compd. 2)

Volume (ml)

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.8

0.9

1.0

PH

2 0 1

2.4

2.9

3.1

4.0

407

7.4

8.1

8.6 , .

8.9

Dibutylaminomethyl Derivative (compound 3 )

Volume (ml) I P"

Diisobutylaminomethy1 Derivative (Compd. 4)

Volume (ml)

0.0

0-2

0.4

006

0.8

1.0

102

1.4

1.6

1.8

2 -0

Dicyclohexyaminomethyl D e r i v a t i v e (Compd. 5 )

Volume (rnl) I pH

Dibenzylarninomethyl D e r i v a t i v e (Compd.6)

Volume

0.0

0.1

Om2

OS3

0.4

0.5

0.6

0.7

0.8

1.0

1.2

PH

3.5

3.9

4.2

4.5

4.7

5.0

5.3

6.6

6.8

7.1

7.3

N-Methylanilinomethyl Derivative (Compd. 8 )

Morphilinomethyl Derivative (Compd. 9)

Volume (ml)

0.0 0.2

0.4

0.6

PH

1.5 2.3

3.1

3.6

Volume (ml) PH

0.0

0-2

0.4

0.6

7.3

6.9

6.6

5.7

Pyrrolidinomethyl Derivative (Compd. 11)

Piperidinomethyl Derivative (Compd. 10)

Volume (ml)

Volume (ml)

0.0

0.5

1 -0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

PH

8.2

7.4

7.0

6.9

6.7

6.5

6.1

4.8

4.0

3.4

3.2

Piperazinomethyl Derivative (Compd. 12)

Vol m e I

TJ4BLE 7

APPARENT D I S S O C I A T I O N CONSTANT VALUES

Compound

1

2

3

4

5

6

7

8

9

10

11

12

Naphthol

To c o r r e l a t e t h e p h a r m a c o l o g i c a l r e s u l t s o b t a i n e d

w i t h t h e e x p e c t e d p h a r m a c o l o g i c a l a c t i v i t y of t h e s e

compounds, Hansch and F u j i t a h y p o t h e s i s , 12* was a p p l i e d .

The h y p o t h e s i s showed b i o l o g i c a l a c t i v i t y of a series of

d r u g s t o depend on 43- or l o g P , assuming t h a t t h e

e l e c t r o n i c and s teric effects a r e c o n s t a n t . Thus, logBR o r

lo+ a -KR + K'R + K* ... .........(39)

( I

where BR, i s t h e biological ' r e s p o n s e , C i s t h e molar

b i o l o g i c a l r e s p o n s e and K , K' and K~ are c o n s t a n t s , for a

g i v e n series of c o n g e n e r i c d rugs .

I n g e n e r a l , t h e r e a r e t w o ways i n which a c t i v i t y are

compared. I n o n e , a molar c o n c e n t r a t i o n of d r u g is

found which p r o d u c e s a s t a n d a r d b i o l o g i c a l r e s p o n s e ,

i s p r e f e r e d t o r e p r e s e n t r e l a t i v e Fo r t h i s c o n d i t i o n , .tf

b i o l o g i c a l a c t i v i t y , I n t h e s econd s i t u a t i o n , whe re a

f i x e d amount of compound i s a p p l i e d and v a r i a b l e r e s p o n s e s

p e r u n i t time are o b s e r v e d , t h e symbol BR is used. The

p r e s e n t s t u d y , u t i l i s e s t h e l a t t e r i n t h e a n a l y s i s .

The r e g r e s s i o n a n a l y s i s o f t h e p h a r m a c o l o g i c a l r e s u l t s

o b t a i n e d showed t h e least s q u a r e l i n e e q u a t i o n (logBR =

a l o g P + b) table 8, t o h a v e a n e g a t i v e c o r r e l a t i o n

c o e f f i c i e n t , w h i l e t h a t of t h e leas t s q u a r e p a r a b o l i c 2

e q u a t i o n (logBR = -a logP + b l o g P + c ) t ab le 9 gave . a

p o s i t i v e c o r r e l a t i o n c o e f f i c i e n t . The n e g a t i v e v a l u e

i n d i c a t e d p r a c t i c a l l y no l i n e a r r e l a t i o n s h i p be tween logBR

and logP. However, t h e r e was a v e r y good n o n - l i n e a r

r e l a t i o n s h i p i n d i c a t i n g t h e p a r a b o l i c n a t u r e o f t h e c u r v e ,

and t h i s w a s c o n f i r m e d by t h e p o s i t i v e c o r r e l a t i o n

cosf f i c i e n t c a l c u l a t e d ,

T h e r e seemed t o be a good o v e r - a l l ag reemen t be tween

t h e o b s e r v e d and p r e d i c t e d a n t i - i n f l a m m a t o r y a c t i v i t y f o r

a l l t h e compounds i n t a b l e 10, w i t h t h e l o g a r i t h m o f

p a r t i t i o n c o e f f i c i e n t s of -0,171 t o 1 - 1 2 and c o n c e n t r a t i o n

of 300 mq/kg body w e i g h t . Compounds 1 and 11 were

a d m i n i s t e r e d a t LOO mg/kg body w e i g h t , The o b s e r v e d

a c t i v i t y d i f f e r e d g r e a t l y f rom t h e p r e d i c t e d a c t i v i t y i n

N-me thy lan i l i nome thy l d e r i v a t i v e of 2-naphthol (compound 8 ) .

T h i s c o u l d be as a r e s u l t of l a c k of p r o p e r s p a t i a l

o r i e n t a t i o n , l e a d i n g t o i n a p p r o p r i a t e bond ing t o t h e

r e c e p t o r . F u r t h e r m o r e , the i n a c t i v i t y o f t h i s d e r i v a t i v e

c o u l d be a t k r i b u t e d t o m a l a b s o r p t i o n d u e t o i t s s o l u b i l i t y

problem as shown i n t a b l e 3,

I n c o r r e l a t i n g t h e r e s u l t s o f aqueous s o l u b i l i t y

d e t e r m i n e d a t 30°C(+20C) w i t h t h e c h e m i c a l s t r u c t u r e s , i t

c a n be s a i d t h a t t h e i n s u l a t i o n o f t h e r e s o n a n c e affect

between t h e f u n c t i o n a l g r o u p and a r o m a t i c r i n g by t h e

m e t h y l e n e (CH ) g r o u p p e r m i t s g r e a t e r l o c a l i s a t i o n o f t h e 2

e l e c t r o n s , t h u s r e n d e r i n g t h e compounds more p o l a r i n n a t u r e .

The r e s u l t a n t i n c r e a s e i n p o l a r i t y makes i t more p o s s i b l e

for water m o l e c u l e s t o i n t e r a c t w i t h t h e p o l a r g r o u p s ,

h e n c e r e s u l t i n g i n an i n c r e a s e of t h e aqueous s o l u b i l i t y

i n compounds 1, 2, 3, 5, 9, 10, 11 and 12. Another

i m p o r t a n t phenomenon which i s p a r t i c u l a r l y l i k e l y t o a f f e c t

t h e aqueous s o l u b i l i t y of t h e s e compounds is i n t e r m o l e c u l a r

hydrogen bonding , T h i s t y p e of bond ing h a s t h e t e n d e n c y

of r a i s i n g t h e c r y s t a l l a t t i c e e n e r g i e s o f t h e m o l e c u l e s ,

r e s u l t i n g i n l o w s o l u b i l i t y . The compounds t h e bond ing

c a n a f f e c t include 4, 6 , 7 and 8.

A t t h i s j u n c t u r e , i t i s w o r t h m e n t i o n i n g t h a t t h e

l i v i n g cell surfaces a re imrnersed i n e x t r a c e l l u l a r f l u i d s

and t h e s e compounds r e a c h i n g t h e cell s u r f a c e must do so

b y means of t r a n s p o r t t h r o u g h t h e e x t r a c e l l u l a r f l u i d s .

Thus compounds i n s o l u b l e i n t h e e x t r a c e l l u l a r f l u i d s and

a lso n o t s o l u b i l i s e d by enzyme or o t h e r c h e m i c a l a c t i o n

c a n n o t be t r a n s p o r t e d e f f e c t i v e l y b y t h e s e f l u i d s t o t h e

cell s u r f a c e and n o r m a l l y c a n be e x p e c t e d t o show n o

s i g n i f i c a n t a c t i v i t y . The p h a r m a c o l o g i c a l r e s u l t s

o b t a i n e d t e n d to v a l i d a t e t h e s o l u b i l i t y i n f l u e n c e on

b i o l o g i c effect, i n t h a t compound 8, which p roved t o be t h e

least a c t i v e , a l so e x h i b i t e d t h e lowest aqueous s o l u b i l i t y

v a l u e , w h i l e compound 1 2 which h a s t h e g r e a t e s t a c t i v i t y

a l s o e x h i b i t e d s u f f i c i e n t aqueous s o l u b i l i t y as shown i n

t a b l e 3.

F u r t h e r m o r e , t h e c o n v u l s i o n , followed by t h e d e a t h

of t h e a l b i n o r a t s o b s e r v e d a f t e r o n e hour o f o r a l admini-

s t r a t i o n of 300 mg/kg body w e i g h t of compounds 1 and 11

c o u l d be s a i d t o be a s a r e s u l t of h i g h c o n c e n t r a t i o n o f

t h e s e compounds i n t h e blood, r e s u l t i n g p r o b a b l y from t h e i r

h i g h d i s s o l u t i o n rates w h i c h a r e d i r e c t l y p r o p o r t i o n a l t o

s o l u b i l i t y , To a s c e r t a i n f u r t h e r , t h e a c c u r a c y of t h e

s o l u b i l i t y s t u d y , t h e h y d r o p h o b i c s u b s t i t u e n t c o n s t a n t ( T T )

was c a l c u l a t e d . T h e s e n v a l u e s shown i n t a b l e 5 , seem t o

a g r e e w i t h t h e s o l u b i l i t y d e t e r m i n a t i o n , i n t h a t t h e

s u b s t i t u e n t g roup , d ime thy laminomethy l , which g a v e t h e

g r e a t e s t n e g a t i v e n v a l u e (-0.911, e x h i b i t e d t h e h i g h e s t

aqueous s o l u b i l i t y ,

A t a p h y s i o l o g i c a l p H of 7.4, t h e h y d r o p h o b i c

s u b s t i t u e n t c o n s t a n t v a l u e o f d ime thy laminomethy l ( -0 .91) ,

i n d i c a t e s t h a t a b o u t 8 times more o f t h e d e r i v a t i v e is

d i s t r i b u t e d i n t h e aqueous p h a s e t h a n i n o c t a n o l . The same

e x p l a n a t i o n c a n h o l d f o r o t h e r d e r i v a t i v e s , s u c h as

compounds 2 , 8, 9 , 10, 11 and 1 2 a s shown i n t a b l e 5. To

c o r r e l a t e t h e e x p e r i m e n t a l l y d e t e r m i n e d p a r t i t i o n

c o e f f i c i e n t s w i t h t h e b i o l o g i c effect , e a r l i e r r e p o r t s on

i t s i m p o r t a n c e was c o n s i d e r e d ,

C o l l a n d e r , 147 showed t h a t t h e rate of movement of a

g r e a t v a r i e t y of o r g a n i c compounds t h r o u g h c e l l u l a r m a t e r i a l

(e.g. N i t e l l a ce l l ) i s d i r e c t l y p r o p o r t i o n a l t o t h e

l o g a r i t h m of t h e i r p a r t i t i o n c o - e f f i c i e n t s be tween an 1 3 0

o r g a n i c s o l v e n t and water, Hansch et a 1 , i n s t e a d of

u s i n g P , d e v e l o p e d a c o m p a r a t i v e s u b s t i t u e n t c o n s t a n t n ,

which i s a free e n e r g y change r e s u l t i n g i n moving a

d e r i v a t i v e from o n e p h a s e t o a n o t h e r . An i d e a l n v a l u e

(To) f o r a- s u b s t i t u e n t is t h a t i n which Px i s 1, f o r

v a r i o u s c e l l u l a r p h a s e s , T h i s i m p l i e s t h a t a m o l e c u l e

w i t h i n t h e cell would have maximum freedom of movement when

Px f o r t h e cell p h a s e s is 1, An i n c r e a s e or d e c r e a s e f rom

u,, w i l l r e s u l t i n a slower r a t e of movement of t h e

m o l e c u l e i n t h e p a r t i t i o n - d i f f u s i o n p r o c e s s by which t h e

s i t e of a c t i o n is a t t a i n e d ,

Fe rguson , 148 e x p l a i n e d t h e f a l l off i n a c t i v i t y w i t h

i n c r e a s e i n P , a f t e r r e a c h i n g a maximum, as s u b s t i t u e n t s

are i n t r o d u c e d i n t o t h e p a r e n t compound t o b e a s a r e s u l t

o f a d e c r e a s e i n t h e thermodynamic a c t i v i t y f o l l o w i n g t h e

i n c r e a s e i n t h e p a r t i t i o n c o e f f i c i e n t . I n h i s e x p l a n a t i o n ,

i t w a s assumed t h a t t h e compound i n s o l u t i o n q u i c k l y

e s t a b l i s h e d an e q u i l i b r i u m w i t h t h a t a t t h e s i te o f a c t i o n

i n s i d e t h e cell, and t h a t , as t h e a c t i v e s i te would be more

l i p o p h i l i c t h a n t h e s u r r o u n d i n g s o l u t i o n , an i n c r e a s e i n t h e

f at-water p a r t i t i o n co-ef f i c i e n t s h o u l d r e s u l t ( o the r

factors b e i n g h e l d c o n s t a n t ) , i n an i n c r e a s e i n t h e concen-

t r a t i o n a t t h e s i t e of a c t i o n and h e n c e g r e a t e r a c t i v i t y .

Fe rguson also n o t e d t h a t t h e r e l a t i v e thermodynamic

a c t i v i t y of a g i v e n m o l e c u l e w i l l d e c r e a s e as i t s p a r t i t i o n

c o e f f i c i e n t is i n c r e a s e d b e c a u s e i t s s o l u b i l i t y i n t h e

a q u e o u s p h a s e w i l l a p p r o a c h a l i m i t v a l u e . T h a t is as

s i A (St = molar c o n c e n t r a t i o n r e q u i r e d to p r o d u c e t h e S

desired b i o l o g i c e f f e c t and S = molar s o l u b i l i t y of the 0

compound i n t h e aqueous p h a s e ) a p p r o a c h e s u n i t y , t h e c h e m i c a l

pstential w i l l drop. As i t d r o p s to t h e p o i n t where t h e

c o n c e n t r a t i o n n e c e s s a r y t o p r o d u c e t h e b i o l o g i c a l r e s p o n s e

r e a c h e s S , t h e m o l e c u l e i s c o m p l e t e l y i n a c t i v e . 0

I n c o n t r a s t t o Fe rguson ' s exp lana t ion , Hansch et a l , 130

e x p l a i n e d t h e f a l l o f f i n a c t i v i t y w i t h i n c r e a s e i n P t o be

d u e to t h e l o n g e r t i m e needed t o f i n d t h e site of a c t i o n

a s p, i n c r e a s e s beyond t h e i d e a l v a l u e . They assumed

b i o l o g i c a l a c t i v i t y t o f o l l o w a normal Gauss i an t y p e o f

d i s t r i b u t i o n w i t h r e s p e c t t o l o g P or n, o t h e r f a c t o r s

b e i n g h e l d c o n s t a n t and r e l a t e d to t h e c o e f f i c i e n t A as

f o l l o w s : 2

A .; f (II) = ae -in -no) 9

o w . . ...(40) b

w h e r e A i s t h e p r o b a b i l i t y o f a m o l e c u l e r e a c h i n g t h e s i te I of a c t i o n i n a g i v e n t i m e i n t e r v a l , a and b are c o n s t a n t s .

S u b s t i t u t i o n of e q u a t i o n ( 4 0 ) i n t o e q u a t i o n (411 ,

r a t e of b i o l o g i c a l r e s p o n s e - d ( r e s p o n s e ) = ACK,r .* . . (41) d t

2 - ( T F - n o ) C K ~ , w . w . ( 4 2 )

e q u a t i o n ( 4 2 ) i s g o t . d ( r e s p o n s e 1 = ae

Where C = e x t r a c e l l u l a r mo la r c o n c e n t r a t i o n of t h e compound

b e i n g t e s t e d ,

A s most b i o l o g i c a l r e s u l t s o b t a i n e d i n a d e f i n i t e

t i m e i n t e r v a l a r e r e p o r t e d i n terms of a , c o n s t a n t e q u i v a l e n t

r e s p o n s e (e ,g . LDSO, EDs0, % i n h i b i t i o n , i s o t o x i c

c o n c e n t r a t i o n ) d ( r e s p o n s e ) / d t c a n b e r e p l a c e d w i t h a

c o n s t a n t . To do t h i s , l o g a r i t h m of e q u a t i o n ( 4 0 ) i s t a k e n

and a f t e r r e a r r a n g e m e n t , e q u a t i o n ( 4 3 ) i s . o b t a i n e d .

where C = c o n c e n t r a t i o n of t h e s u b s t a n c e ( u n d e r i n v e s t i g a t i o n )

i n moles/litre, p r o d u c i n g a g i v e n e f f e c t i n a d e f i n i t e

t i m e i n t e r v a l .

The Hammekt e q u a t i o n , 14' w h i c h a p p l i e s t o e i t h o r t h e

e q u i l i b r i u m or r a t e c o n s t a n t s i s f o r m u l a t e d as l o g kx = &6

w h e r e 6 = l o g % - "H

T h e 6 i s a c o n s t a n t d e p e n d e n t upon t h e e l e c t r o n i c c h a r a c t e r i s t i c s o f

t h e s u b s t i t u e n t x r e l a t i v e t o H, The more e l e c t r o n - a t t r a c t i n g a I s u b s t i t u e n t is by e i t h e r r e s o n a n c e or i n d u c t i o n , t h e more positive I

i s i t s 6 v a l u e ( r e l a t i v e t o H, as 0,OO). e i s a c o n s t a n t related t o i t h e r e a c t i o n o f t h e p a r e n t m o l e c u l e u n d e r g o i n g t h e r e a c t i o n and 1

j a p p r o p r i a t e l y c a l l e d r e a c t i o n c o n s t a n t . The real s i g n i f i c a n c e o f Q I is t h a t i t m e a s u r e s t h e s e n s i t i v i t y of t h e r e a c t i o n t o t h e

I \ I i i

e l e c t r i c a l e f f e c t s of s u b s t i t u e n t s i n o r t h o , p a r a or meta p o s i t i o n s ,

A l a r g e e c o n s t a n t , p o s i t i v e or n e g a t i v e means a h i g h s e n s i t i v i t y

t o s u b s t i t u e n t i n f l u e n c e s . kx and kH a r e t h e o b s e r v e d b i o l o g i c

effect of t h e d e r i v a t i v e and t h e p a r e n t m o l e c u l e r e s p e c t i v e l y , Kx

and kH a r e t h e d i s s o c i a t i o n c o n s t a n t s o f t h e d e r i v a t i v e and t h e

p a r e n t m o l e c u l e r e s p e c t i v e l y ,

A p l o t o f kx a g a i n s t KX g i v e s a s t r a i g h t l i n e , i n which is

€he s l o p e o f t h e l i n e . S u b s t i t u t i o n o f e q u a t i o n ( 4 4 ) i n t o ( 4 3 )

y i e l d s e q u a t i o n (451,

no is t h e i d e a l v a l u e f o r a s u b s t i t u e n t s u c h t h a t t h e sum' of

t h e many free e n e r g y c h a n g e s i n t h e p e n e t r a t i o n p r o c e s s i s a minimium,

S i n c e t h i s i s a c o n s t a n t f o r a g i v e n t y p e of p a r e n t m o l e c u l e

i n a p a r t i c u l a r b i o l o g i c a l s y s t e m , e q u a t i o n ( 4 5 ) r e d u c e s to

e q u a t i o n ( 4 6 ) and t h e c o n s t a n t s i n t h i s e q u a t i o n a r e d i f f e r e n t

f rom t h o s e o f ( 4 5 ) . 2

Log+ = -K n + ~ ' n + p6+ KO .......(46)

The above e q u a t i o n t h e r e f o r e enables p rob lems i n v o l v e d i n

d i s e n t a n g l i n g s teric, a l o c t r o n i c and p e n e t r a t i o n charac ter i s t ics

c o n f e r r e d on a b i o l o g i c a l a c t i v e m o l e c u l e s by s u b s t i t u e n t s to be

s o l v e d , It h a s been o b s e r v e d t h a t h i g h l y a c t i v e m o l e c u l e s are

o b t a i n e d m a i n l y w i t h s u b s t i t u e n t s h a v i n g p o s i t i v e b v a l u e s w h i l e

a c t i v i t y found w i t h s u b s t i t u e n t s w i t h n e g a t i v e 6 v a l u e s c o u l d

n i c e l y be a c c o u n t e d for by t h e i r i d e a l T/ v a l u e s ,

R e l a t i n g t h e s e e a r l i e r r e p o r t s t o t h e p h a r m a c o l o g i c a l

r e s u l t s o b t a i n e d , i t seems t h a t l i p o p h i l i c i t y h a s l i t t l e

c o n t r i b u t i o n to t h e o b s e r v e d a c t i v i t y as t h e most a c t i v e of t h e

compounds (compound 1 2 ) showed a h i g h d e g r e e o f h y d r o p h i l i c i t y ,

As a l l t h e d e r i v a t i v e s showed p o s i t i v e s i g m a ( 6 ) v a l u e s , t h e y

are by i m p l i c a t i o n e x p e c t e d t o e x h i b i t a n t i - i n f l a m m a t o r y

a c t i v i t y .

The c o r r e l a t i o n of t h e a p p a r e n t d i s s o c i a t i o n c o n s t a n t s

w i t h t h e p h a r m a c o l o g i c a l a c t i v i t y , showed t h e m o s t a c t i v e

compound (compound 1 2 ) to have a pK, v a l u e of 5 .8 . The

o t h e r derivatives w i t h pK, values above or be low t h i s ,

showed less a c t i v i t y , a s i n d i c a t e d i n t a b l e 7 .

TABLE 8

f1uqrc:~r;lun lint: of l o q U 1 4 on l o q P and the

l i n e a r carrelati o n co-ef f icient between tha:wt V a r i a b l e s

The r e g r e s s i o n l i n e of logBR on logP , i s given by t h e l e a s t

s q u a r e l i n e e q u a t i o n ,

where Y and X r e p r e s e n t logBR and logP r e s p e c t i v e l y , a. and

al a r e c o n s t a n t s o b t a i n e d by s o l v i n g t h e normal e q u a t i o n s ,

P u t t i n g t h e v a l u e s i n t a b l e 8 i n t o t h e above e q u a t i o n s and

s o l v i n g s i m u l t a n e o u s l y , t h e e q u a t i o n o f a least square

l i n e f i t t i n g t h e d a t a i s o b t a i n e d . Thus

17.99 = a 1 2 + a 7.61 (from e q u a t i o n 48) 0 1

9.73 = a 7.61 + a 6.59 ( f r o r n e q u a t i o n 4 9 ) 0 1

S u b s t i t u t i o n i n t o e q u a t i o n (481, gives a. = 2.103

. 0 Y = 2.103 - 0.9516x.

The linear correlation co-efficient between these

v a r i a b l e s is given by

TABLE 9

Least square parabola of loqBR on loqP and the non-linear correlation co-efficient between

these variables

The regression equation of the least square parabola fitting

the above data is

2 Y = a + a l X + a X

0 2

where Y and X represent L O ~ B R and logP r e s p e c t i v e l y , a 0'

a and a are cons tant s . The normal equat ions are: 1 2

17.99 - 12ao + 7.61al + 6.59a (from equat ion 52) 2

9.73 = 7.61a + 6.59a + 5.87a2 (from equation 53) 0 1

7.868 = 6.59a + 5.867a + 5.46a2 (from equation 54) 0 1

Reducing to a. and substract ing:

S o l v i n g s i m u l t a n e o u s l y , .

a 1.995, a = 0.797 and a2 = -1.824. 0 1

a. , g o t by s u b s t i t u t i n g a l and a2 i n t o e q u a t i o n ( 5 2 ) .

Thus t h e e q u a t i o n of a least s q u a r e p a r a b o l a f i t t i n g t h e

2 d a t a i s Y = 1.995 + 0.799X - 1.824X . .... ....(55)

his i s t h e same as,

logBR -1.824 logp2 + 0.799 logP + 1.995.

The n o n - l i n e a r c o r r e l a t i o n c o - e f f i c i e n t between t h e s e

2 where explained v a r i a t i o n = (Yest- y ) and

2 t o t a l v a r i a t i o n = 2 ( ~ - Y )

'est got from s u b s t i t u t i n g d i f f e r e n t v a l u e s of X i n t o

e q u a t i o n ( 5 5 )

The v a l u e o t r was found to be 0.96.

TABLE 10

Comparison of Calculated and Observed Anti-inflammatory A c t i v i t i e s of Aminomethyl Derivatives of 2-naphthol:

Compound LogP

-0.171

0.66

0.93

1.12

1.03

0.80

0.94

0.67

0. 'Jl

0.65

0.18

0.092

Lo@ Calc.

1.80

1.71

1.16

1.10

1.02

1.46

1.13

1 . 51 1.64

1.74

1.85

2.05

O t h e r phys i coche rn i ca l p a r a m e t e r s t h a t c o u l d be u s e f u l i n

s t r u c t u r e - a c t i v i t y r e l a t i o n s h i p i n c l u d e ,

i) S t e r i c s u b s t i t u t i o n c o n s t a n t ( T a f t s u b s t i t u t i o n c o n s t a n t 6 ' ) :

The steric e f f e c t s of s u b s t i t u e n t s on b i o l o g i c a l a c t i v i t y ,

150 can be e v a l u a t e d i f K u t t e r and Hansch e q u d t i o n i n v o l v i n g

taf t s t c r i c prarc-ter, Es is er~~ployed .

LOCJBR =-an2 + bn + CE, + d + e 6 . . ( 5 6 )

r e a c t i u r l centre iind cdn be c a l c u l a t e d u s i n g Ck;;-rrtonls

151 h y p o t h e s i s .

i i ) The P a r a c h o r t

McGowan, 152 e x p l a i n e d t h a t t h e p r o c e s s o f s o l u t i o n i n

water i n v o l v e s t h e f o r m a t i o n o f a c a v i t y i n t h e s o l v e n t

f o l l o w e d by o c c u p a t i o n by t h e s o l u t e . The e n e r g y c h a n g e s

i n v o l v e d are E, a n d E,, r e s p e c t i v e l y . T h e r e f o r e E, - E, r e p r e s e n t s t h e f r e e e n e r g y of s o l u t i o n . I f a s a t u r a t e d

s o l u t i o n i s c o n s i d e r e d t o be o n e i n e q u i l i b r i u m , t h e n

where Cs a n d C1 are molar c o n c e n t r a t i o n s o f s a t u r a t e d

s o l u t i o n a n d p u r e l i q u i d r e s p e c t i v e l y .

As E, - E, d e p e n d s o n d i f f e r e n c e be tween t h e i n t e r n a l

p r e s s u r e o f s o l u t e a n d s o l v e n t , t h e m o l a r volume of

s o l u t e , e q u a t i o n ( 5 7 ) c a n be r e - w r i t t e n as l o g C1 =

l o g Cs + KV, where V = molar volume and K i s a c o n s t a n t a t

c o n s t a n t t e m p e r a t u r e .

C1 = 1 0 0 0 D , where D = d e n s i t y a n d M - m o l e c u l a r we igh t . M

As t h e m o l a r volume i s n o t a s u i t a b l e p a r a m e t e r b e c a u s e of i t s

v a r i a t i o n w i t h t e m p e r a t u r e and t h e c a l c u l a t i o n d e p e n d i n g h i g h l y

o n m o l e c u l a r s t r u c t u r e , p a r a c h o r ( P I overcomes t h e s e

d i f f i c u l t i e s .

P a r a c h o r u s e s s u r f a c e t e n s i o n , a p r o p e r t y d e p e n d e n t on

i n t e r m o l e c u l a r a t t r a c t i o n , t h u s i t is d e f i n e d as,

P = ~ r ~ * ~ 5 V r 0.25 i - 9 where r = s u r f a c e t e n s i o n , D and d a r e I D-d

d e n s i t i e s of l i q u i d and v a p o u r r e s p e c t i v e l y . ;

V E molar volume,

P a r a c h o r h a s been used s u c c e s s f u l l y t o estimate p a r t i t i o n

c o - e f f i c i e n t s (or 11) of o r g a n i c compounds be tween o r g a n i c

s o l v e n t s and water and s o l u b i l i t i e s o f o r g a n i c compounds i n

w a t e r . 1 5 3

( i i i ) D i p o l e Moment ( E l e c t r o n i c P a r a m e t e r ) :

The permanent d i p o l e moment ( P ) of a d i a t o m i c m o l e c u l e i s 0

d e f i n e d as t h e c h a r g e d i f f e r e n c e be tween a toms on e i t h e r end

of t h e bond m u l t i p l i e d b y t h e d i s t a n c e be tween them. 1 5 4

155 Cammarata e t a1 , r e v i e w e d t h e c o r r e l a t i o n o f permanent

d i p o l e s w i t h Hamrnett f u n c t i o n s f o r s e v e r a l g r o u p s o f compounds

by t h e e q u a t i o n , u = ail + b, where a and b are c o n s t a n t s .

D i p o l e moment h a s been used i n t h e d e t e r m i n a t i o n o f c h e m i c a l

s t r u c t u r e and also h a s been used i n , c o r r e l a t i n g t h e i n h i b i t i o n

of n e u r a m i d a s e by 1-phenoxymethyl-3, 4 - d i h y d r o i s o q u i n o l i n e , 1 5 6

Chromatographic Parameter:

Green and Marcinkiewicz, 157 using reversed-phase tankless flat-

bed chromatography showed that RM, which was defined by Bate-

Smith and Westall, 158

as RM - lug (1) -1 is an additive and R f

constitutive property just like logP. They introduced the

term ARIvl for the effect of a substituent such as CH3 upon

the Rf value of a parent compound. ARN is related to by

the equation = RDI + b, where a and b are constants.

Bowen et al, 15 9 used RM to estimate partition co-efficient

while Biagi et al, 160

applied it in the correlation of

biological activities of penicillins.

SUMIWRY :.

A number of aminomethyl derivatives of 2-naphthol were

synthesized and screened for anti-inflammatory activity by

the in-vivo method. -- Physicochemical properties like solubility, partition co-

efficient and dissociation constant were determined and

correlated with the observed anti-inflammatory activity.

Regression analysis was used to compare the observed anti-

inflammatory activity with the predicted activity.

The study gave rise to agents that produced some degree

of anti-inflammatory activity greater than or equal to that

of aspirin, yi-z ~i s (2 -hydroxy-1 -naphthy lmethy lbp iperaz ine

and dirnet hy lar~~ir~ornethyl-2-naphthol.

CHAPTER FOUR

EXPERIMENTAL

Melting points were determined with a Gallenkamp

electrically heated block and are not corrected. Infrared

and ultravoilet spectra were recorded on Unicam Sp-1000 and

Unicam Sp-8000 spectrophotometers respectively.

Proton nuclear magnetic resonance (H-n.m.r) spectra were

recorded on a Varian Associates T-60 (60MHZ) spectrophotometer,

Chemical shifts are reported on the b scale relative to

tetramethylsilane (TMS) used as internal standard,

Thin-layer chromatography (T.L.C.) was conducted with

60 GF254 precoated silica gel plates, Iodine was used to

locate t.1.c. spots for the compounds.

2-Naphthol (5 , 0.03rnol) was dissolved in ethanol and warmed g

A mixture of dimethylamine (0.03mol) and formaldehyde

solution (37%) (0.03mol) in ethanol at the same temperature

was added slowly t o n a p h t h o l so l u t i o n w i t h c o n s t a n t -

s t i r r i n g . T h e r e s u l t i n g m i x t u r e was v i g o r o u s l y s h a k e n f o r

10 m i n u t e s and a l l o w e d t o s t a n d a t room t e m p e r a t u r e , u n t i l

p r e c i p i t a t i o n i s c o m p l e t e . The p r e c i p i t a t e was c o l l e c t e d

b y f i l t r a t i o n and r e c r y s t a l l i s e d f rom m e t h y l a t e d s p i r i t t o

g i v e l - d i m c t h y l a ~ r ~ i n o r ~ ~ u t h y l - 2 - n a p h t h o l ( 85% y i e l d ) a s f i n e 0

crrm powder, rnp bl/-b9 ( L I ~ , * ' 68-70~) U.V: Amax 345nm.

1 - R : V 3560 crn - l ( -0Hgp) , 1360crn-~ ( C - ~ g g ) , max

1610crn-~ (C = C aromatic)

H-nmr ( A c e t o n e - d 6 ) 6 2 . 3 ( s , 6H), 4 . 1 (s , Z H ) , 6.9-8.0 (m,6H)

and 1 1 - 4 ( s , HI.

To a s o l u t i o n o f 2 -naph tho l (0.03mol) i n a b s o l u t e e t h a n o l

was added a mixture of d i p r o p y l a m i n e (0.03mol) and f o r m a l d e h y d e

s o l u t i o n (0.03rnol) w i t h s h a k i n g a t room t e m p e r a t u r e . The

s o l u t i o n was b o i l e d u n d e r r e f l u x for 4 h o u r s , f o l l o w e d by

s o l v e n t r emova l u n d e r r e d u c e d p r e s s u r e and t h e r e s i d u e a l l o w e d

t o cool. The s o l i d p r o d u c t was f i l t e r e d and r e c r y s t a l l i s e d

f r o m e t h a n o l t o g i v e 1-dipropylarninomethyl-2-naphthol (67%)

as l i g h t y e l l o w powder, mop. 186-188O

A max = 34Snrn

1 . R : Vmax 3540crn-l, 1360cm-l, 1610cm-~

The p r o c e d u r e f o r compound 2 was u t i l i s e d i n the

s y n t h e s i s , u s i n g d i b u t y l a m i n e . The r e s i d u e was e x t r a c t e d

w i t h CHC13. The CHClj was e v a p o r a t e d and t h e r e s u l t i n g s o l i d

product was recrystallised from methanol t o g i v e

1-dibutylarninomethyl-2-naphthol (63% y i e l d ) as light brown

powder, m.p 105 - 1 0 7 ~ .

"*": A m a x '345nm

1.R: V max 3540 c m - l , 1360 c m - l , 1610cm-I

H-nrnr (Acetone-d6) 6 0.9 (t, 4H), 1.3-1.6 (sbr 4 ~ 1 , 2 . 6

( t , 6H), 4.2 ( s14H) , 4.95 (s, 2H), 9,6 ( s , H) and 7-8.4(m, 6H).

Anal. c a l c . for =19 H27 NO: C, 80.00; H , 9.47; N , 4.91.

Found C , 79.96; H, 9.42; N, 4.85.

The same procedure used for compound 2 was utilised for

t h e s y n t h a s i s us ing d i i sobuty lamine . The s o l i d product was

r e c r y s t a l l i s e d from t o l u e n e to give l-diisobutylaminomethyl-

2-naphthol, (71%) as grey ish powder, mop. 198 - 2 0 0 ~ .

U V : ,,, 345nm

1 . R : V 3560 crnnl, 1360crn-l, 1610crn-~ max

Anal. calc, for C H HO: C, 80 .00 H , 9.47 N , 4 .91 . 19 27

Found C , 79.94, ti, 9.27 N, 4.86.

H-nmr = None, due to s o l u b i l i t y problem.

To a s o l u t i o n of 2-naphthol (0.03mol) i n e t n a n o l was

added a mix tu r e of d icyc lohexy lamine (0.03mol) and

formaldehyde s o l u t i o n w i t h shak ing a t room temperature .

. T h e s o l u t i o n was r e f l u x e d f o r 30 minu tes , t h e s o l v e n t removed

under reduced p r e s s u r e and t h e r e s i d u e al lowed t o coo l .

The p r e c i p i t a t e d s o l i d was f i l t e r e d and r e c r y s t a l l i s e d from

a t h a n c l kc g i v e dicyclohexylaminomethy~-2-naphthol

(74% y i e l d ) , a s c o l o u r l e s s powder, m.p. 18? - 18g0 ( L i t 86

188 - 190~). ".":&max 335nrn

I o R : V 3550 cm-', 1 3 6 0 ~ m - ~ , 1610cmo1 max

H-nmr ( ~ c e t o n e - d ) b 1.25 (sb; loti), 2.4 (s,, 4H), 4.4 6

( s , 8 H ) , 4.85 (s , 2H), 7.0 - 8.4 ( m , 6H) and 9.0 (3, H ) o

The procedure for compound 5, was employed i n t h e

s y n t h e s i s u s i n g dibenzylamine. The s o l i d r e s i d u e was

r e c r y s t a l l i s e d from e t h a n o l t o g i v e l-dibenzylarninomethy1-2- 0

n a p h t h o l (82% y i e l d ) , as colourless powder, m.p 110-112 . U.V: A max

335nm, 278nm.

1 .R : "rnax 3560 c m - l , 1380 ern-l, 1610crn-~

H-nmr (Acetone-d6)& 3.65 ( s , 4H), 4.2 (s, 2H), 7.0 - 8.0

(m, 16H) and 1 2 ( s , H ) .

Anal. c a l c . for CZ5 HZ3NO: C , 84.98; H , 6.51; N , 3.97.

Found C, 84.68; H, 6.49; N , 3.85.

(7) l -Diphenylaminorne thy l -2 -naph tho l :

2-Naphthol (0.03mol) e t h a n o l i c s o l u t i o n was mixed w i t h

e t h a n o l i c s o l u t i o n of d i p h e n y l a m i n e (O.03rnol) and aqueous

f o r m a l d e h y d e (0.03rnol) and c o n t e n t o f t h e f l a s k w a s r e f l u x e d

f o r 1 4 h o u r s , t h e s o l v e n t was removed u n d e r r e d u c e d p r e s s u r e

and the residue allowed to cool. After cooling, it was

kept in the refrigerator for 24 hours and the solid filtered

and recrystallised from alcohol to give l-diphenylamino-

methyl-2-naphthol (56% yield), as light green powder, mop.

0 132 - 1 3 5

- 1 - 1 - 1 I , ll r VIIbUX ! I , 1 J I , l l ~ l U CIII

Ii-nmr (Acetone-d6)& 4.95 ( s , 2H), 7.1 - 8.45 (m, 1 6 ~ )

and 9.0 ( s , HI.

H NO: C, 84.92; H, 5-85; N, 4.31. Anal. calc. for C23 lg

Found C, 84.87; H, 5.63; N, 4.10.

The procedure for compound 2, was adopted in the

synthesis using N-methylaniline. The solid product was

r e c r y s t a l l i s e d from e t h a n o l - C H C l j t o g i v e 1-N-methylanilinomethyl-

2 - n a p h t h o l (73% y i e l d ) , as l i g h t y e l l o w powder , mop. 1 7 4 - 176O.

U.V:,( 335nm, 291nm. max

1.R: V max 3650 c m - l , 1380 cmal, 1 6 1 0 c m m l

n-nmr (Acetope-d6)6 2.8 (t, 4 H ) , 4.2 ( s , 2H) 6.5 - 7 -8

(rn, l l t i ) .

Ana l . calc. fo r C18H17NO: C, 82.12; H, 6.46; N , 5.32.

Found C, 82.00; H , 6 . 3 3 ; N, 5.26.

To a s o l u t i o n of 2 - n a p h t h o l (0 .03mol ) i n e t h a n o l r a i s e d

0 t o 6 0 was added a m i x t u r e of m o r p h i l i n e (0 .03mol) and

f o r m a l d e h y d e s o l u t i o n a t t h e same t e m p e r a t u r e . The m i x t u r e

was v i g o r o u s l y s h a k e n f o r 1 5 m i n u t e s and a l l o w e d t o cool a t

139

room temperature. The solid product was recrystallised

from ethanol to give 1-morpholinomethyl-2-naphthol

(83% yield), as colourless powder, mop. 113 - 115O (lit, lo4 115 - 116O). " 0 ':Amax 330nm, 275nm. 1 . R : V max

3560 cm-l, 1360 =in-', 1070 cm-l, 1610 ern"

H-nmr (Acetone-d6)& 2.6 (t, 4111, 3.7 (t, 4~), 4.2 s 2111,

7.0 - 8.1 (m, 6 ~ ) and 9.0 (s, H).

The procedure for the preparation is the same as

compound 9, but using piperidine. The solid product was

recrystallised from ethanol to give 1-piperidinomethyl-2-

naphthol (81% yield) as colourless powder, m.p. 89 - 91° (litlo* g4.5 - 95.501. U.V: h m a x 330nm, 275nm.

1 . R : V max

3560 cm-I, 1360 cn-', 1610 cm-'

. T h e p r o c e d u r e of s y n t h e s i s is t h e same as compound

9 but using p y r r o l i d i n e .

The s o l i d p r o d u c t was r e c r y s t a l l i s e d from e t h a n o l t o g i v e

1 -py r ro l id ino rne thy l -&naph tho l (70% y i e l d ) as c o l o u r l e s s

powder, mop. 81 - 8 3 O it^^ 173O as HCL s a l t ) .

".": A max 330nm, 275nm.

-1 1 . R : V 3 5 6 0 c m , 1360 ~ m - ' , 1610 Cmol max

The procedure utilised in the synthesis is the same

as compound 10, The solid product was recrystallised from

acetone to give bis (2-hydroxy-1-naphthyImethy1)-piperazine

(60% yield), as light yellow powder, m.p 218 - 220°,

1 . R : V max 3600 cm-l, 1320 an-' , L61Q FIII -~

Anal . talc. f o r C24H26N202: C, 77-00; H, 6.95; N, 6-17.

Found C, 76.94; H, 6-02; N, 6.01,

H-nrnr: None, due to solubility problem.

I n the H-n.rnm.r. v a l u e s ,

s = s i n g l e t , sb = broad s i n g l e t , t = t r i p l e t and

m = multiplet.

(4 .2 ) PHARMACOLOGICAL STUDY

P r o c e d u r e : 63 The i n - v i v o method o f a s s a y as d e s c r i b e d by Winter e t a 1

was a d o p t e d f o r t h e p h a r m a c o l o g i c a l s c r e e n i n g , A l b i n o r a t s i n

t h e w e i g h t r a n g e of 1 7 0 - 230g were used f o r a l l t h e e x p e r i m e n t s .

Food and t a p water w e r e w i t h h e l d d u r i n g t h e e x p e r i m e n t o n l y .

The compounds were suspended i n 0.25% gum t r a g a c a n t h and

a d m i n i s t e r e d o r a l l y by means of s tomach t u b e i n a volume of

5ml i n which t h e d o s e was c o n t a i n e d . T h i s i s t o e n s u r e u n i f o r m

h y d r a t i o n o f t h e a n i m a l s and r e d u c e t h e v a r i a b i l i t y o f oedema

p r o d u c t i o n . T h e test compounds were g i v e n 1 hour before t h e

c a r r a g e e n i n t r e a t m e n t , The same t i m e i n t e r v a l was a l l o w e d

before the a d m i n i s t r a t i o n of the v e h i c l e i n c o n t r o l a n i m a l s .

I m m e d i a t e l y a f t e r 1 h o u r , t h e d i a m e t e r of t h e h i n d paw

was measured w i t h a m i c r o m e t e r a c r o s s a s a g i t a l s e c t i o n a s

d e s c r i b e d by Newbould. 59 T h i s was f o l l o w e d by i n j e c t i o n of

0.1 m l of a 1% s u s p e n s i o n of c a r r a g e e n i n i n 0.9% s a l i n e . The

d e g r e e of t h e c a r r a g e e n i n - i n d u c e d s w e l l i n g s of t h e h i n d paws

was measu red 3 hour a f t e r t h e c a r r a g e e n i n t r e a t m e n t w i t h a

micrometer.

I n t h e p h a r m a c o l o g i c a l s c r e e n i n g , s i x a n i m a l s were used

per test d r u g dose . The same number of a n i m a l s w e r e u sed

f o r t h e r e f e r e n c e d r u g , a s p i r i n , and t h e c o n t r o l ( r e c e i v i n g

5 m l o f t h e v e h i c l e ) ,

?Ihe a n t i - i n f l a m m a t o r y measurement d a t a l isted i n table 1

are t h e v a l u e s f o r a n a v e r a g e o f 2 a n i m a l s p e r group. Each

g r o u p c o n s i s t e d of 6 a n i m a l s and t h e a s s a y was c a r r i e d o u t i n

d u p l i c d t e . The p e r c e n t i n h i b i t i o n was c a l c u l a t e d u s i n g t h e

mean t h i c k n e s s i n c r e a s e f o r e a c h g r o u p and t h e s t u d e n t ' s

"t"' W ~ S deterrnirlrd t o estimdte t h e s i g n i f a n c e of e d c h test .

R e r c e n t i n h i b i t i o n ( a n t i - i n f l a m m a t o r y e f f e c t ) =

A - B x z , I --

H 1

where A E diameter of c a r r a g e e n i n t r e a t e d paw - i n i t i a l diameter o f t h e paw.

B a diameter o f c a r r d g e e n i n treated paw a n d t h e d r u g - i n i t i a l didmeter of t h e pcrw.

To c a l c u l a t e t h e "t" tes t o f s i g n i f a n c e , t h e e q u a t i o n , 161

below a p p l i e s .

w h e r e Mx, My = c a l c u l a t e d mean v a l u e s f o r x a n d y

d e t e r m i n a t i o n s .

- S.Ex a s t a n d a r d error of t h e c o n t r o l g roup .

S.Ey = s t a n d a r d error o f t h e tes t group .

The s t a n d a r d error (S.E) = 6 1

where 6 .I s t a n d a r d d e v i a t i o n

N number o f measurements -

($03) CALIBRATION GRAPH OF 2-NAPHTHOL t I f r e = = = = = = = E E = = = = f = = = E i - G z r . t i E f ; D 1

P r o c e d u r e ; 2-Naphthol (100 mg) w a s weighed a c c u r d t e l y i n t o

a 10 r n l volunlet.ric f l a s k and d i s s o l v e d i n e t h a n o l (96%).

This volunw wds t r ~ t n s f e r e d i n t o a n o t h e r 50 m l v o l u m e t r i c

f lc l sk clnd d i . l u t , d t o vo lume w i t 1 1 d j s t i l l e d water. Grddud

cor lccntrdt ion: u f L t i i s :;Lock s o l u t i o n (O.5, 1.0, 1.5 m l s e tc)

were made up to 4 i n 1 w i t h d i s t i l l e d water, F o r each d i l u t i o n ,

0.5 m l gave t h e a b s o r b d n c e shown below,

C o n c e n t r a t i o n ( M i l l i l i t e r s )

Absorbance

C a l c u l a t i o n o f t h e m o l a r a b s c r p t i v i t y (€1, i n v o l v e s

where H r a b s o r b d n c e

C - c o n c e n t r d t i o n ( m o l e s p e r l i t e r )

L = P a t h l e n g t h ( i n a), E = mola r a b s o r p t i v i t y .

The v a l u e o f t h e molar a b s x p t i v i t y was f o u n d t o be 57.14.

T h e p lo t ; of dbsorbat~ce d g d i n s t c o n c e n t r a t i o n obeyed the

Beer - L a m b e r t g s l a w i n the c o n c e n t r a t i o n r a n g e of

12.5 - 8 7 - 5 mg%. The d e t e r m i n d t i o n was done i n d u p l i c a t e .

Concent ration Fg-1 : Cali bration graph of 2 - Naphthol

T h i s w d s determined by p l a c i n g excess (200 mg) of

e a c h arnir lomethyl d e r i v a L i v e of 2 - n d p h t h o l a n d 2 - n a p h t h o l i n

s e p d r a t e f l a s k s c o n t a i n i r r g 1 5 m l o f water and were s h a k e n

c>c vc for 24 1roul.s d L 30 ( 1 1. A L L11t: CIIJ U L wLIIGI~ Ll lc

tlltrdtc wds d i l u t e d w i t h d i s t i l l e d water t o S m l . From t h e

d i l u t e d s o l u t i o n , 0.5 ml wds used f o r a n a l y s i s .

I n t h e a n a l y s i s , t h e a b s o r b a n c e was read a t 34Snm a n d

t h e c o n c e n t r a t i o n s o f t h e compounds i n t h e i r s a t u r a t e d

s o l u t i o n s were c a l c u l a t e d by r e f e r e n c e t o t h e c a l i b r a t i o n

g r a p h o f 2 - n a p h t h o l as shown i n f i g u r e 1, T h e r e s u l t s of t h e

d u p l i c a t e d e t e r m i n a t i o n s ilre shown i n t a b l e 3.

I n t h e c a l c u l a t i o n , t h e c o n c e n t r a t i o n of cach a b s o r b a n c e

was multiplied by 10 ( d i l u t i o n f a c t o r ) t o g e t t h e e q u i l i b r i u m

c o n c e n t r a t i o n s ( s o l u b i l i t y ) o f t h e compounds.

(4.5) APPARENT PARTITION COEFFICIENT

The a p p a r e n t p a r t i t i o n c o e f f i c i e n t of 2 - n a p h t h o l a n d

t h e a m i n o m e t h y l d e r i v a t i v e s was d e t e r m i n e d i n 1 - o c t a n o l - w a t e r

systems, T h e aqueous p h a s e :*as 0.1M p h o s p h a t e b u f f e r

(pH 7.4) a n d t h e o r g a n i c p h a s e was 1 - o c t a n o l .

A c c u r a t e l y w e i g h e d compounds ( 5 0 mg o f e a c h ) w e r e

d i s s o l v e d i n a m i x t u r e o f 10 m l o f t h e a q u e o u s p h a s e and 1 5 m l

of t h e o r g a n i c p h a s e , e a c h s y s t e m b e i n g p r e v i o u s l y s a t u r a t e d

w i t h t h e o t h e r , o v e r n i g h t (1:4). S h a k i n g was d o n e a t 30 oc

( + - 20') f o r 2 h o u r s to a c h i e v e c o m p l e t e e q u i l i b r a t i o n .

After s e p a r a t i n g t h e two p h a s e s , t h e s o l u t e c o n c e n t r a t i o n

i n a q u e o u s p h a s e was d e t e r m i n e d by u s i n g 0.5 m l a l i q u o t , a n d

t h e a b s o r b a n c e r e a d i n g s were t a k e n a t 345mm. The r e s u l t s of

t h e d u p l i c a t e d e t e r m i n a t i o n s a re shown i n t ab le 4. The a p p a r e n t

p a r t i t i o n c o e f f i c i e n t s w e r e c a l c u l a t e d u s i n g e q u a t i o n , 1 6 2

(61) b e l o w

Where P = a p p a r e n t p a r t i t i o n c o e f f i c i e n t .

Co = c o n c e n t r a t i o n o f t h e compound i n t h e o r g a n i c p h a s e .

C1 = i n i t i a l c o n c e n t r a t i o n i n t h e a q u e o u s p h a s e

c" = c o n c e n t r a t i o n of t h e compound i n t h e a q u e o u s p h a s e .

"w = volume o f t h e a q u e o u s p h a s e m

"0 = volume o f t h e o r g a n i c p h a s e .

-149-

( 4 . 6 ) APPARENT DISSOCIATION CONSTANT

D i s s o c i a t i o n c o n s t a n t w a s c a l c u l a t e d from t h e d a t a

o b t a i n e d b y t i t r a t i n g f r a c t i o n a l molar s o l u t i o n s o f

a m i n o m e t h y l d e r i v a t i v e s of 2 - n a p h t h o l w i t h s t a n d a r d HCL or

NaOH s o l u t i o n s ( f o r t h e h y d r o c h l o r i c a c i d s a l t s ) . For t h e b a s e s ,

2 0 m l p o r t i o n s of 0.005M s o l u t i o n s were t i t r a t e d w i t h a n

e q u i v a l e n t v o l u m e of 0.05M HCL. After e a c h a d d i t i o n , the pH

o f t h e s o l u t i o n was r e c o r d e d by means of a n E x t c h d i g i t a l

r e s e a r c h pH meter s t a n d a r d i s e d a g a i n s t 0.05M p o t a s s i u m

h y d r o g e n p h t h a l a t e (pH 4.00) a n d 0 . 0 1 M s o d i u m b o r a t e (pH 9.18).

T h e t i t r a t i o n s were c a r r i e d out a t 300C ( + - 2 0 ~ ) . A l l s o l u t i o n s

w e r e p r e p a r e d with d i o x a n e - w a t e r ( 2 : l ) . The i o n i c s t r e n g t h was

m a i n t a i n e d a t 0.03 w i t h KCL. F o r t h e h y d r o c h l o r i c a c i d s a l t s ,

t h e s a l t was formed by a d d i n g a s t o i c h i o m e t r i c amount o f

c o n c e n t r a t e d HCL to t h e b a s i c compound i n a b s o l u t e e t h a n o l .

10 m l p o r t i o n s of 0.03 - 0.04M o f s o l u t i o n s were t i t r a t e d w i t h

' a n e q u i v a l e n t vo lume of 0.3M NaOH.

Half n e u t r a l i s a t i o n method was u s e d t o e s t i m a t e t h e a p p a r e n t

d i s s o c i a t i o n c o n s t a n t s (pK o f t h e compounds. a

For pH 4 - 10, pKa = pH - l o g 2 ........ ( 6 2 ) C - b

where b 3 concentration of the titrant (NaOH).

c = concentration of the compound to be estimated,

Equations (62) - (641, apply only for assay of weak acids

with strong bases, the acid may be neutral, positive or

negative. For the assay of weak bases, with strong acids,

the following equations apply.

pH 10, PKa = pH - log C -' a - (OH-) 0 0 0 (67) a + (OH') ..

where a = concentration of the chloride iron provided

by the titrant i.e. initial concentration x volume added total volume.

c = concentration of the compound being estimated.

i.e, initial concentration x initial volume total volume,

Absorbance Transmittance 0-1 A Rsnge

I- 'd3 I ,-

OOE SLZ G S i SZZ 002 . --- I

CHART 5-607 M.31 1. 2 . l r

A I I I I " " " " 1 ' " ' I ' " ' I " " I ' , " ' 1 " " 1 '

I --. - - -. . . . - . - 1 I 500 3 0 0 1 0 0 100 0 Wz

- -

I I , I 1 I 9 8 1 l , , , . l . , , , l . , , , l . . . , l , , , , l , , , # l , , , , l

8.0 7.0 0.C 5 .0 P P M A i 1 4 .0 3.0 2.0 7.0 0

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J. Gaddurn. P h a r m a c o l o g i c a l l y a c t i v e p e p t i d e s . I n "Gaddum's Pharmacologyu , Eds, A. S. V. Burgen and J , F , M i t c h e l l , Oxfo rd U n i v e r s i t y P r e s s , London, 1978 , P o 110-

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A. G. Mowat. A n a l g e s i c a n t i - i n f l a m m a t o r y d r u g t h e r a p y , Med. I n t , 4 ( I ) , 4 6 1 (1981)

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