CA1181009A - Treatment of diarrhoea - Google Patents

Abstract

ABSTRACT

Compounds A - B - C wherein A is 2imidazolidinimino.or N-alkyl derivative thereof, B is oxygen, sulphur, CR1R2 wherein R1 and R2 are hydrogen or alkyl, or NR3 wherein R3 is hydrogen or alkyl, and C is a 5 - or 6 - membered carbo- or heterocyclic group having a nitrogen containing substituent and up to two other substituents, are useful in treatment of diarrhoea or scours. Certain compounds A - B - C are novel.

Description

This application is a divisional of application S.N. 381,322 filed 8 July 1981 and is directed to pharmaceutical compositions containing known compounds of formula (I~ whereas the parent application is directed to the preparation of novel compounds or formula (I) and the compounds so produced.
TXEATMENT ~F DIARRHOEA
This invention relAtes to the treatment of diarrhoea in animals including man, to formulations for use in such treatment, to a class of novel compounds having activity useful in such treatment and to processes for their production.

Diarrhoea ~also referred to as scours in cat~le, sheep and pigs) can be a severe disease particularly in young animals and can often result in death. Diarrhoea is also common amongst human travellers and those expo~ed to low standards of hygiene. The diarrhoea frequently involves colonisation of the small intestine with enteropatho~enic strains of E. coli which produce heat stable and/or heat labile enterotoxins. Related enterotoxins are produced by other enteropathogens, for example, cholera, and also cause diarrhoea. These enterotoxins stimulate fluid secretion in the gut lumen and hence cause diarrhoea. The associated fluid loss may lead to loss of condition, reduced weight gain of livestocX
and often to death.

It ha~ now been dis~overèd that a ertain class of compounds have activity useful in treating diarrhoea. In particular, these compounds inhibit small-intestinal secretion whilst having less CNS activity than a-asonists of similar structure.
Accordingly, the present invention provides a method for treating diarrhoea in animals including man, which method comprises administering an effective, non-to~ic !9 amount of a compound of formula (I):

A - B - C (I) wherein A is a 2-imida701inyl group optionally substituted on a nitrogen atom with Cl 4 alkyl group;
B is an oxygen or sulphur atom; CR R , wherein R
and R2 are the same or different and each is selected from Cl 4 alkyl and hydrogen; or NR3, wherein R3 is selected from Cl ~ alkyl and hydrogen; and C is a 5 or 6 membered carbo- or heterocyclic group having a nitrogen-containing substituent and optionally also substituted by one or two groups, each selected from halogen, (Cl 4) alkyl, (Cl ~3 alkoxy and hydroxy; or an N-acyl derivative and/or an acid addition salt thereoE, to an animal, including man, sufering from diarrhoea.

It will be appreciated that certain oi the compounds of formula (I) may exist in distinct tautomeric forms. All such forms are useful in, and encompassed byt the present invention.

Any one or more of the available nitrogen atoms of a compound of formula (I) may be acylated. It will be appreciated that not all the nitrogen atoms may be available for acylation, thus when B is, for instance, a group NR3 and R i5 an alkyl moiety, only one of the nitrogen atoms of the imidazoline moiety may be acylated. However, if B
is a group NR3 and R3 is hydrogen, both nitrogens of the imidazolinyl moiety may be acylated, or one of these nitrogens and the nitrogen atom of B may be acylated~

Suitable nitrogen containing substituents include groups of Eormula:

- X - R

wherein X is methylene or a bond; and R4 is amino, mono- or di(Cl 4~ alkyl amino, amidino, guanidinyl or 2-imidazolinyl optionaliy substituted on a nitrogen atom with a (Cl 4) alkyl group.

Suitably A is 2-imidazolinyl or an acyl derivative thereof.

Suitably B is NH.

Suitably C is a substituted phenyl group having a nitrogen-containing substituent in the ortho or para position with respect to B.

Suitable acyl moieties are Cl 3 alkyl carbonyl, tri-haloacetyl,benzoyl and phenacetyl groups.

Suitahle salts are mono- and di-hydrochlorides, -hydrobromides, hydroiodides, nitrates, sulphates, citrates, ~ tartrates and pamoates.

Preferably the nitrogen containing substituent on C
is amino, guanidinyl or aminomethyl.

When C is a substituted phenyl group it is preexred that there is a substituent in the position ortho to the group B. It is also preferred that the nitrogen-containing substituent is located in the para-position.

Preferred compounds within formula (I) include compounds of formula (II~ as hereinafter defined and p~amino clonidine and salts thereof.

It will be appreciated that the effective dose of the compounds of the formula (I) will depend in the usual way upon factors such as the severity of the diarrhoea, the weight of the sufferer, the particular drug chosen, and on similar va~iables. However, as a guide, we believe that a suitable dose will be within the range 1 ~g to 1 mg/kg a day as a single or divided dose.

Antibacterial agents such as penicillins, aminogly-cosides and sulphonamides may also be administered as part o the treatment of diarrhoea in conjunction with the present method of treatment.

It is believed that this method of treatment may usefully be combined with oral rehydration therapy such as described in US Patent No. 4,164,568 or Belgian Patent ~o~ 872,647 Compo~mds o~ formula ~I) may be produced by known methods such as those described below in relation to compounds of formula tII) and used, for instance, for producing p-amino clonidine~

p-a~ino Clonidine can be prepared by known methods, such as that described in U.K. Patent No. 1,450,250 wherein 2,6-dichloro 4-nitro aniline is treated with N-acetyl-2-imidazolidone, the acetyl moiety is removed and then the nitro group is reduced to an amino group, for instance, by the method of Rouot and Leclerc, Bull. Soc.
Chem~ Fr~, (1979), 520. Other methods for producing p-amino clonidine will be apparent to khose skilled in the art, however, it should be noted that these will usually involve the use of a 2,6 dichloro aniline derivative bearing in the 4-position a protected amino group, or a 5 precursor for an amino group, such as the nitro group mentioned above, and the step of converting such a 4-substituent to an amino group.

Salts of p-amino clonidine can be prepared by known methods. The preparation of both the hydrochloride and dihydrochloride is disclosed by Rouot and Leclerc (above3.

Certain compounds of formula (I) are novelo Accordingly, the present inv~ntion provides compounds of formula (II) C ~ N ~ R9 (II) wllerein R is hydrogen~ ~Cl 4) alkyl or acyl;
R6 is hydxogen, ~Cl ~) alkyl o~ acyl;
~7 is hydroxy, halogen, (Cl 4) alkyl, (Cl_~) alkoxy or a group R8;
R8 is an ortho or para su~stituent selected from amino, mono- or di-(Cl ~) alkyl amino, aminomethyl, mono- or di~Cl~4) alkylamino-methyl,amidino, guanidinyl or 2-imidazolidinimino optionally substituted on a nitrogen atom with (C1~4) alkyl ox acyl;
R9 is selected from hydroxy, halogen, (Cl 4) alkoxy,.
hydrogen and a group R8 when in the para position or R9 is selected from hydroxy, halogen, (Cl 4) alkyl, and (Cl 4) alkoxy when in a meta position, or R9 is selected from hydroxy, halogen, (Cl 4) alkyl, (Cl_4) alkoxy and a group R when in the ortho position, or a salt thereof, provided that when R9 is in the ortho position and is chloro, R7 is chloro, R5 is hydrogen and R6 is hydrogen, ethyl or acetyl, then R8 is other tha~ para-amino, or a methyl, ethyl or acetyl derivat~ve or salt thereof.

Prefer~bly R5 and R6 are hydrogenO

Preferably R is at the 4-position.

Preferably R7 and R are the same and are at the

2,6 positions.

Preferably R8 is amino, substituted amino or guanidinyl.

Preferred compound~ within formula (II~ include:

2,6-dimethyl-4-aminophenylamino-2-imidazoline;
2,6-dibrvmo-4-aminophenylamino-2-imida~oline.

It will be appreciated that certain compounds of formula (XI) will exist in tautomeric forms of the structure shown above.
The present invention embraces all such tautomers.
Salts of compounds of ormula (II) need not be pharma-ceutically acceptable, if they are not they may be useful in purification of com~ounds of formula (II).

The present invention also provides compounds of formula (II) for use in~treating diarrhoea.

According to the present lnvention there is also provided a process for producing a compound of ~ormula (II) which process com-~rises (a) reacting a compound of formula (IV) X ~ (IV~
R

wherein R7 to R9 are as defined with respect to formula (II) and X is a group ~ N - CN ; ~ N ~ ;
R14 Cl R ~ H2 R14 ~ R

wherein Rl is an (Cl_~) alkyl ~rou~, with a compound of ~ormula (III) H2N - C~2 - CH2 - NHR (III) wherein Rl9 is hydrogen or (Cl 4) alkyl.

(b) reacting a compound of formula (V) H - N ~ ~ (V) R15 ~ R8 wherein R - R9 are as defined with respect to formula (II), and R15 is hydrogen or a group R6 as hereinbefore defin~d with respect to formula (II), with a compound of formula (VI) ~C - R18 (VI) wherein R20is hydrogen, alkyl or, when R17 and R18 are oxo, ~yl, R16 and R17 together form a bond and R17 is 16 ~Cl 4) alkylthio or benzylthio or R is hydrogen, and R17 and Rl~ together form an oxo group;

or (c) converting the corresponding compound with a nitro substituent in the ortho or para position to the required co~pound of formula (II);

and optionally thereater forming an acyl derivative and/or a salt thereof.

Process (a) may be conducted according ~o the process descrlbed in UK Patent No. 1,229,995 when Cl X i5 - N ~ ; according to the process described in Netherlands Patent No. 6,411,516 (ChemO Abs. 63, P18102 g) when X is - N ~ ; according to the process R14 ~H2 .

described in Chem. Abs., 85~ P94364b when X is N - CN , and according to the process described in l6 Chem. Abs., 85, P5633g when SR].4 X is - N - C = N

R14 R7 ~ R9 v~`~

Pxocess (b~ may be conducted according to the process described in German Offenlegungschrift 2,505,297 or Ne~herlands Patent No. 7,404,472 when R17 and R ~ together form an oxo group, and according to the process described in Chem. Abs.~ 84 P59465t when R16 and R 7 together form a bond.

Process (c) may be conducted according to the process describea in Rouot & Leclerc ~suPra) or in European Patent Application No. 12,~22, published 9 ~Julv l~S0, corresponding to Canadian P. 1,112,648 German Offenlegungsschrift No.
2,806,811 and U.K. Patent No~ 1,180,766.
The starting materials may be produced by conventional methods, as may the salts and acyl derivatives of compounds of formula (II), The present invention further provides a pharmaceutical compositinn comprising a compound of formula ~I~ and a pharmaceutically acceptable carrier therefor.

As used herein the term 'pharmaceutical composition includes compositions suitable for human and~or animal use and 'pharmaceutically acceptable' includes veterinarily acceptable, Pharmaceutical compositions of compounds of formula (I) wilL, of course, be adapted for administration to hurnans or the animaLs to be t~eated.

Thus For example the composition may be a shaped composition, such as a bolus, tablet or capsule . In such cases the pharmaceutically acceptable carrier wiLl be chosen from the usual r~nge of lubric~nts, dispersants, binders, fillers and the like. When these shaped compositions are for administration to cattle and pigs, often they will

3~ weight at least 1 g, on occasions at least 2 g For a~ninistration to humans, especially young ones, the drug may suitably be presented as a syrup lncluding suitable colouring and/or flavouring agents. Such syrups are conveniently presented in unit or multi-dose containers.

For veterinary use the composition may also be a dis-persion or a solution of a compound of formula ~I) (hereinafter referre~ to as 'the drug') in a suitable vehicle for use with an oral doser (this is a well known item of farm equipment, basically comprising a liquid reservoir, a mouthpiece adapted for insertion into animal mouths, and a pump mechanism whereby unit doses can be ejected from the reservoir through the mouthpiece). Conveniently the vehicle will be an oil or water based cream to ensure homoyeneity of the unit doses administered. Alternatively, the drug may be administered as an aqueous solution using an oral doser.

The invention, therefore, also provides an oral doser containing a multi-dose of the drug in a veterinarily acceptable vehicle.

The drugs of the invention may also be added to the animal feed or drinking water. Thus the invention also provides animal feed or animal drinking water containing a compound of formula (I). It will be convenient to formulate these animal feed and drinking water compositions with a multi-dose of the drug so that the animal takes in an appropriate quantity of the drug along with its diet. It will also be convenient to present the composition of the invention as pre-mixes for addition to the feed or drinking water.

c:~

With young humans or animals, a particularLy useful technique is to blend their milk with the drugs of this invent ion .

The compositions of the invention may also be form-ulated for injection. In such cases the drug chosen is suitably dissolved in water for injection.

Often it will be appropriate to include in the compositions a further medicine such as an antibacterial agent, for example an antibiotic such as amoxycillin or neor~lycin or a sulphonamide such as sulfadoxin.

Clearly the compositions of the invention will contain sufficient drug to enable this effective dose to be admini-stered in convenient manner. Thus by way of example useful dosage units of the composition may contain 1 ~g to 50 mg of the drug, more suitably 20 ~g to ~0 mg. Of course, it will be appreciated that many preferred compositions of the invention are in multi-dose form, as for the therapy of animals, it is often most desirable to be able rapidly to treat a number of animals~ Such multi-dose compositions will contain by way of example, at least 1 mg of the drug.
Depending on the exact nature of the said multi-dose composition, often it will contain at least 50 mg of the drug, and on occasions as much as 1 g.

si~ ical ~valuatiol- of p-ami~o Clonidine dihydrochloride The following tests were carried out:

1~ Mice Infant mice are separated from their mothers shortly before use. Animals up to 15 days of age are suitable for use but normally animals 7 - 9 days of age are used~ Groups of animals are dosed with the compound 45 mins prior to oral challenge with 0.05 -0.10 ml of culture filtrate prepared from an entero-pathogenic strain of _ coli. Control animals receive drug vehicle 45 mins prior to challenge with a similar amount of culture filtrate. The compounds are administered orally. Animals are killed two hours later and the entire intestine removed. The ratio of gut weight to remaining bodyweight ~GW/BW) is determined from each animal and the increase in this ratio is determined by subtraeting 0.06 (GW/BW for untr~ated mice) from the GW/BW of the animal. Drug treated animals axe compared with untreated controls. If the compound has had an effect in inhibiting the 1uid secretion caused by the enterotoxin(s) present in the culture filtrate then the gut weight/bodyweight ratio should be reduced in the treated animals. The percentage fluid inhibition is determined from the formula:

100- Mean increase in GW/BW ratio in treated animals X 100 i Mean increase in GW/BW ratio in control animals _ Results are given in the table below, as are comparative results for the known anti-diarrhoeal, Loperamide.

p-Amino_clonidine dihydrochloride __ _ , _ ~_ Dose (mg/kg~ % fluid inhibition .

0.1 48 0.01 36 0.001 29 _ The calculated ED50 of p-amino Clonidine di-hydrochloride in the mouse is 0.25 mg/kg.

peramide _. _ _ Dose (mg/kg~ % fluid inhibition _ _ . _ ~ _ _ _ _

4 45 1 _ 1~
-- _ _ ~ LO~g - 16 ~

These results clearly demonstrate the high level of activity of the p-amino Clonidine dihydrochloride in the reduction of fluid secretion caused by challenge with an enteropathogenic strain of E. coli~

The activity of the p-amino Clonidine dihydrochloride is highlighted by comparison with I,operamide, which as can be seen from the Table, is considerably less active than the p-amino Clo~idine dihydrochloride in the mouse test.

2. Calf Thiry-Vella Intestinal Loop Model In vivo tests were conducted using male castrate calves, each with two surgically prepared Thiry-Vella intestinal loops prepared as described by R.J. ~ywater, ., B0, 565, (1970).

The loops are washed with saline and then a saline bolus is left in the loops for 30 minutes to establish a basal absorptive rate. After 30 minutes the fluid in the loops is removed and measured. Heat stable E. coli enterotoxin from E. coli strain pl6 is added to the loop infusate which is then returned to the loops.

After a further 30 minutes the content of the loops is measured once more and at this time dxug is added to the test loop perfusate.

Measurements of absorption or secretion from the loops are made every 30 minutes over the subsequent 2~ hours.
The results are expressed in terms of fluid (ml) secreted into or absorbed from the loops after each 3G minute period. A
minimum of six observations are undertaken with each experimental compound. Perfusion periods in which drugs are present in a loop are alternated with perfusion periods in which drug is absent from the same loop.
Calf-Thiry Vella Intestinal Loop Model _ _ _. _ , _ _ 10Time (min) *Secretion *Secretlon (after toxin (ml/30 min~ (ml/30 min) administration) Control1 ~g/kg Control 1 ygJkg p-Amino compound of Clonidine~ __~ ___ -30-o -3 -3 3 3 0-304 27 25 ~7 28 30-~0 196** 18 12 60-90 122** 15 8 90-120 110** 10 7 120 ~50 11-1** 11 7 153-180 11-1** 9 4 , . _ .
* A negative value denotes absorption of fluid ** Denot~s significantly different fluid secretion from control (P<0.05 students t test) f Drug administered 30 min. after toxin ~ A~ministere~as the dihydrochloride Toxicitv The drugs have been found to havea satisfactory ~rapeutic ratio.

3. Pi~lets (a) Inhibition of Diarrhoea Induced by Enterotoxin Administration 2-4 day old piglets were dosed with the compound orally 45 mins prior to oral challenge with 25 ml of culture filtrate prepared from an enteropathogenic strain of E coli.
Control animals received drug vehicle 45 mins prior to challenge with a similar volume of material. Animals were observed for diarrhoea over a 6 hour period and the severity of scour scored on a 0-3 basis for each animal at hourly intervals. After 6 hours observation some piglets were given a second dose of drug (or placebo) and observations were continued for a further six hoursO The percentage inhibition in treated animals was determined as:

lOO rMean score of scour in treat~d animals x lool LMean score of scour in control animals J

(b) Inhibition of Diarrhoea Resulting fxom E~coli Infect_on Colostrum deprived piglets were infected with 5 ml of an overnight culture containing 1O8-lO9 viable E~ coli P16/ml.
The following day, piglets were matched by weight and scour score and then treated and observed for 6 hours in a similar manner to toxin challenged piglets, as described above.

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Formulations of th~ druas Example 1 p-Amino Clonidine bolus 0~2 ~

soluses of the following composition were prepared:

p-Amino Clonidine dîhydrochloride o 2mg Microcrystalline cellulose500 mg Corn starch 250 mg Magnesium stearate 25 mg Lactose, anhydrousto 2500 mg The ingredients were passed through a 30 mesh stainless steel screen and blended in a suitable blender.
The resultant compression mix was compressed directly on a tabletting machine to give tahlets each containing 0.2 mg p-amino Clonidine dihydrochloride.

Example 2 p-Amino Clonidine Oral Doser 0.1 m~

1 Kg of the following composition was pxepared:
~ by wt.
p-Amino Clonidine dihydrochloride o.
Aluminium stearate 6.0 Sunflower oil to 100 The aluminium stearate was dispersed with stirring in a portion of the sunflower oil heated to 115C.
The dispersion was added to the rest of the sunflower oil heated to 140C. The gel was stirred at 130C
for 15 minutes and then allowed to cooi without stirring to room temperature. The milled p-Amino Clonidine dihydrochloride was dispersed in the cooled gel base and then passed through a colloid mill to produce a fine, homogenous dispersion. The dispersion was filled into plastic bottles fitted with a dosing pump.

Example 3 ~mino Clonidine Injection 0.~.5 mq/ml 1 Litre of the following composition was prepared:
% w/v p-Amino Clonidine dihydrochloride 0.025 Sodium chloride 0.5 Water for injections to 100 The p-amino Clonidine dihydrochloride and sodium chloride were dissolved in the water for injections and the solution was filtered and filled into glass ampoules. The ampoules were sterilised by autoclaving.

Example 4 p-Amino Clonidine Premix A premix of the following composition was prepared:
% by wt.
p-Amino Clonidine dihydrochloride 1.0 I,imestone flour to 100 The ingredients were mixed together in a ribbon blender to give a homogenous mixture. The premix was mixed into animal feed at the rate of 1 kg per metric ton to provide a concentration of 10 g of p-amino Clonidine dihydrochloride per metric ton.

Example 5 -Amino Clonidine Soluble Powder P

1 Kg of the following composition was prepared:
~ by wt.
p-Amino Clonidine dihydrochloride o 1 Lactose to 100 The p-Amino Clonidine dihydrochloride and lactose were sieved and mixed together in a suitable blender to give a homogenous powder. The powder was filled into jars. The powder was used at the rate 0.5 y per gallon of drinking water to medicate pigs.

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Exam~le 6 Oral Rehydration Formulation contain _c~ p-amino Clonidine l kg of the following composition was prep~red by ~nixing together the ingredients in dry powder form:

Glycine 10.3 Dextrose (anhydrous) 67.6 Sodium Chloride 14.3 Potassium Dihydrogen Phosphate 6.8 Citric Acid 0.8 Tri-potassium Citrate 0.2 p-amino Clonidine dihydrochloride 0.002 60 g of the composition was then dissolved in 2 litres of water and fed to diarrhoeic calves.

Example 7 In the formulation of Example 6 p-amino Clonidine dihydrochloride was replaced by 2-(4-amino-2,6-dimethyl-ph~nylamino)-imidazoline dihydrochloride.

Example 8 The following formulation may be prepared by the method set out below:

p-amino Clonidine dihydrochloride 0.01 % w/v Bentone*~8 (1) 1.5 % w/v (ie 1.5 gtlOO ml) Propylene Carbonate 0.6 ~ w/v Pharmasorb*(2) 10 % w/v Phosphoric Acid (3) 0.1 ~ w/v Ampicillin Trihydrate 6.0 ~ w/v as free acid Soya-Bean Oil to 100 ~

(1) Bentone*38 is dimethyl dioctadecyl 125 hectorite, [Mg8Lisil2o3o(o~)6~ [(C~3~2N(C18 37)2 (2) Pharmasorb*is a brand of activated Attapulgite, (3) The phosphoric acid is present in the minor proportion to balance the alkalline pH of the Bentone.

The Bentone was dispersed in the soya-bean oil, and when thoroughly distributed, the propylene carbonate was added with high speed mixing, followed by colloid milling to produce the base. Into this base was first mixed the phosphoric acid, and then the pharmasorb and the penicillin, and the resultant suspension was then passed through a colloid mill once more.

* TRADE MARK

Ex~mple 9 Example 8 was repcated, but using amoxycillin trihydrate in place of the ampicillin trihydrate.

Example 10 The following formulation was prepared by the method of Example 8:

p-amino Clonidine dihydrochloride O.Ol % w/v Sunflower Oil 1.75 % w/v Propylene Carbonate0.6 ~ w/v Pharmasorb* 10.0 % w/v Ampicillin Trihydrate5.O ~ w/v Sun~lower Oil to 100 (1) Benton~ 27 is dimethyl benzyl octadecyl ammonium hectoriete, ~Mg8LiSil2030(0H~6] [(CH3)2 ( 18 37 2 6 5 Example 11 Example 10 was repeated, but using amoxycillin trihydrate in place of the ampicillin trihydrate.

Example 12 2-(4-amino-2 6-dibromo hen limino)imidazolidine P

A solution of concentrated hydrochloric acid ~2.8 ml, 0.028 M) in aqueous ethanol (50~, lS ml) was added dropwise to a stirred heated mixture of 2-(2,6-dibromo-4-nitro phenylimino)imidazolidine (4 g, 0.011 M) and iron powder (1.9 g, 0.034 M~ in aqueous ethanol (50~). The mixture was heated under reflux for 3 hours after which time it was filtered hot and evaporated to a low volume. This residual solution was acidified with dilute hydrochloric acid and the resulting solid was removed. The solution was then basified with dilute aqueous sodium hydroxide yielding a solid which w~s collacted. This solid was triturated with hot methanol which was then evapora~ed ~o lea~e a brown solid (3.2 g). This solid was chromatographed on alumina and eluted with a methanol in methylene chloride gradient.
The fractions containing the required compound were combined and evaporated affording 2-(4-amino~2,6-dibromo-phenylimino)imidazoline dihydrochloride, mp 7320C (1.6 g).

Analysis calculated for CgH12Br2C12N4 Theory: C 26.56; H 2.97; N 13.77 Found : C 27.07; H 3.02; N 13.79 Example 13 ~-(4-amino-2,6-dimethylphenylimino)imidazolidine The 2-(2,6-dimethyl-4-nitrophenylimino)imidazolidine (8.75 g) was reduced with iron and hydrochloric acid in aqueous ethanol according to the process of Example 12.
The product was chromatographed on a silica column and eluted with a methanol in methylene chloride gradient. The fractions containing the required compound were evaporated and the residue was treated with ethanolic hydrochloric acid to give 2-(4 amino-2,6 dimethylphenylimino)imidazolidine dihydrochloride. An analytical sample was recrystallised as the mono-hydro-iodide salt, mp 252-253C.

Example 14 2-(4-amino methyl 2,6-dichlorophenylimino)imidazolidine Borane methyl sulphide complex (10.7 mll 0.107 M) was added slowly to 2-~4-carboxamido-2,6-dichlorophenylimino~
imidazolidine (8.0 g, 0.029 M) in dry tetrahydrofuran (160 ml) under nitrogen with stirring. The reaction mixture was heated under reflux for 3 hours and cooled to room temperature.
Methanol was added until the mixture became clear and stirring was continuad for 12 hours. Hydrogen chloride gas was bubbled into the mixture for 15 min and then the mixture was heated under reflux for 1 hour. It was then evaporated and the residue was taken up in water. The solution was basified with sodium hydroxide solution and the resulting oily solid was collected and crystallised from acetone. This solid was dissolved in dilute hydrochloric acid, filtered and basified with sodium hydroxide solution yielding the 2-(4-aminomethyl-2 6-dichlorophenylimino)imidazolidine. An analytical sample was recrystallised as khe mono hydro-iodide salt, mp 205 206.5 C.

Example 15 2-(2~6-dichloro-4-diethylaminomethylphenylimino)imidazolidine Lithium aluminium hydride (3.4 g, 0.08 M) was added portionwise to 2-(4-die~hylcarboxamido-2,6-dichlorophenyl imino)imidazolidine (3~4 g, 0.01 M) in ether (350 ml) with stirring. After 2.5 hours ethyl acetate was added followed by methanol. The mixture was then evaporated and dilute sodium hydroxide was added to the residue. The residue was extracted with methylene chloride and the extract was evaporated to give the required compound as an oil (3.3 g~
which was recrystallised as 2-(2,6-dichloro-4-diethylamino-methylphenylimino)imidazolidine dihydrochloride, MP ~72-274C.

Example 16 2-(2,6-dichloxo-4(2-imidazolidinimino)phenylimino)imidazolidine A mix~ure of 2-(4-amino-2,6-dichlorophenylimino) imidazolidine (3.7 g, 0.015 M) and l-acetyl-2-imidazolidone (2.2 g, 0.02 M) in phosphoryl chloride (25 ml) was heated under xeflux for 3 days. After cooling to room temperature the phosphoryl chloride was evaporated to give an oily residue. The residue was basified with sodium hydroxide (50~) and the insoluble solid was filtered off. This solid was hydrolysed with dilute sodium hydroxide in methanol.
After evaporation of the mixture the product was extracted into methylene chloride. Evaporation of the extract gave a gum which crystallised giving 2-(2,6-dichloro-4-(2-imidazoli-dinimino~henylimino)imidazolidine dihydroiodide, MP 298-300C.

Example 17 2-[2,6-dichloro-4-(1,3-dimethylguanidino~phenylimin~3imidazolidine Phosphoryl chloride (4.4 ml, 0.047 M) was added with cooling to dimethylurea (4 g, 0.045 M) in tetrahydrofuran (60 ml). After stirring at room temperature for 5 hours the mixture was added to 2-(4-amino-2,6-dichlorophenylimino) imidazolidine (3=0 g, 0.012 ~) in tetrahydrofuran (60 ml). The mixture was refluxed for 18 hours and then evaporated to give an oily solid. The residue was ex-tracted into 2M HCl, washed with methylene chloride and then basi-fied. Extraction of the alkaline mixture with methyl chlorideyielded a gum on evaporation of the extract. The gum was taken up in ethanol and the required guanidino compound (300 mg) crystal lised. The product was recrystallised as 2- C2,6-dichloro-4-(1,3-dimethylguanidino~phenylimin~ imida~olidine dihydroiodide ~MP 320 C).
Analysis calculated for C12H18N6C12I2 Theory: C 25.24; H 3.18; N 14O72 Found : C 24.91; H 3.47; N 14.92 Example 18 2Q 2(2,6-dichloro-4-~uanidino phenylimino) imidazolidine, dihydro-chloride A solution of cyanamide (0.44,0~ ) in water (1 ml~ was add~d to 2-(4-amino-2,6-dichlorophenylimino) imidazolidine, mono-hydrochloride (2 g, 0.007M) in hydrochloric acid (2M, 3.5 ml) and ethanol ~5 ml). Th~s mixture was heated under reflux for 24 hours and then evaporated to give an oily solid. This solid was taken up in the minimum amount of hot ethanol and left standing. The ~ 30 -resulting solid was recrystallised from ethanol to give 0~35 g, MP 2~3-285C.

Example 19 2-2(2/6-dichloro-4-(1,3-die~hylguanidino) phen l imino) imidazolidine 2-(4-amino-2,6-dichlorophenylimino) imidazolidine was reacted with diethyl urea as described for the dimethyl urea in Example 17, affording the free base MP 216-21~ C. This compound was treated with ethanolic hydrochloric acid to give the dihydro-chIoride, MP 274-276C.

Exampl _20 2-(4-(1,3-diallylguanidino?-2,6-dichlorophenylimino) imidazolidine 2-(4-ami*o-~,6-dichlorophenylimino) imidazolidine was reacted with diallyl urea as described for the dimethyl urea in Example 17, affording the free base MP 185-187Co This compound was treated with ethanolic hydrochloric acid to give the dihydro-chloride MP ?64-265 C.

Example 21 The activity of various compounds of formula (Ij was assessed ~n mice by the method given above. The results were as follows:

N I ~ ~b ComFo~d of R R T R ~ ~ dc~se %
Exa~r~le No. 5 6 a 11 m~ Cg inhibition _ H CH3 P- 2 o~Cl Cl200 72 _ H H ~NHEt o-Cl Cl200 97 _ H H~NEIO~c o-Cl Cl2()0 57 _ H H ~NH2 ~Cl Cl 51O 78 _ H H P~ 2 H Cl H HF~CH2NEt ~Cl Cl 5QO 42 13 H H p-NH2 CH 3 CH3200 69 500 saA

12 H H IP NH2 o-Br Br200 85 . 50 ~5 _ .._ ___ 10 ..

~;ul ts ccr~tinued ~alple No. R5 R6 Ra -- Rll mg/~g inhlbitlon 17 H H ~ n. o11 Cl . 53 ~t ~r lO 37 14 H H p CH2~2 o-Cl Cl200 39 38 ~t, NH
16 H H ~<~ o Cl CllOO 29,~l f/

18 H H p-N=C (NH2) o-Cl Cl200 33 19 H H p-N= ~NHCE~2-3 2 o~Cl Cl 10 30 19 . 2EICl H H CH ) 3 2 o-Cl Cl 200 42 20 . 2HCl H H p-N=C(NHC~12 CH= CH 2 ) o -- C 1 C 1 2 0 0 4 1 . _ .
_ _ - 32a-Example 22 Rat Jejunal Perfusion Jejunal absorption or secretion of fluid was measured in ana~sthetised rats by perfusion of an isotonic solution contain-ing 14c labelled - polyethylen~ glycol essentially as described by Klipstein, Lee and Engert (Infect. Immun. 1976, 14, 1004-1010).
The effect of ST toxin alone or in the presence of drug was established by incorporating these substances in the perfusion solution averaging 14c counts over 2 hours perfusion in six rats and calculating absorption per g. of perfused jejum.

Absorption ~1 per p***
Perfusate min. per g No. of Rats (Students T) . _ . _ _ _ Control (no toxin~~1.3 20 0.001 Toxin (60 mouse units/ml)-8.0 14 __ Toxin -~ p-amino chlo~idine 40 ~g/ml 4.5 6 Mot Sing.

Toxin + p-amino chlonidine 80 ,ug/ml+0.2 7 0.001 Toxin -~
compound of Example 13 40 ,ug/ml-2.3 7 0.05 *** Absorption compared to absorption by jejunum perfused with toxin.

- 32b -

Claims (5)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A pharmaceutical composition for the treatment of diarrhoea or scours comprising an effective amount of a compound of formula (I):

A - B - C (I) wherein A is a 2-imidazolidinimino group or said group substituted on a nitrogen atom with a C1-4 alkyl group;
B is NR3, wherein R3 is selected from C1-4 alkyl and hydrogen; and C is a 5 or 6 membered carbo- or heterocyclic group having a nitrogen-containing substituent and optionally also substituted by one or two groups, each selected from halogen, (C1-4) alkyl, (C1-4) alkoxy and hydroxy;
exclusive of compounds of formula (II) (II) wherein R5 is hydrogen, (C1-4) alkyl or acyl;
R6 is hydrogen, (C1-4) alkyl or acyl;
R7 is hydroxy, halogen, (C1-4) alkyl, (C1-4) alkoxy or a group R8;
R8 is an ortho or para substituent selected from amino, mono- or di-(C1-4) alkyl amino, aminomethyl, mono- or di-(C1-4) alkylaminomethyl, amidino, guanidinyl or 2-imidazolidinimino optionally substituted on a nitrogen atom with (C1-4) alkyl or acyl;

R9 is selected from hydroxy, halogen, (C1-4) alkoxy, hydrogen and a group R8 when in the para position or R9 is selected from hydroxy, halogen, (C1-4) alkyl, and (C1-4) alkoxy when in a meta position, or R9 is selected from hydroxy, halogen, (C1-4) alkyl, (C1-4) alkoxy and a group R8 when in the ortho position, or a salt thereof, provided that when R9 is in the ortho position and is chloro R7 is chloro, R5 is hydrogen and R6 is hydrogen, ethyl or acetyl, then R8 is other than para-amino, or a methyl, ethyl or acetyl derivative;
or a pharmaceutically acceptable N-acyl derivative and/or acid addition salt of said compound of formula (I), and a pharmaceuti-cally acceptable carrier therefor.

2. A composition according to claim 1 wherein the compound of formula (I) is p-amino clonidine or an acid addition salt thereof.

3. A composition according to claim 1 wherein the carrier is feedstuff or drinking water provided for an animal.

4. A composition according to claim 1 containing about 1 µg to 50 mg of said compound of formula (I).

5. A composition as claimed in claim 4 containing about 20 µg to 20 mg of said compound.