EP0634996A1

EP0634996A1 - 1,4 naphthoquinone derivtives with anti-protozoal and anti-parasitic activity

Info

Publication number: EP0634996A1
Application number: EP93907992A
Authority: EP
Inventors: Alan Thomas Hudson; Clive Leonard Yeates
Original assignee: Wellcome Foundation Ltd
Current assignee: Wellcome Foundation Ltd
Priority date: 1992-04-06
Filing date: 1993-04-05
Publication date: 1995-01-25
Also published as: ZA932458B; FI944657A; NO943737D0; SK120994A3; GB9207517D0; WO1993020044A1; HU9402850D0; CN1083048A; MX9301962A; CZ242994A3; KR950700874A; NO943737L; JPH07505388A; CA2133744A1; FI944657A0; HUT68937A; IL105322A0; AU3899293A

Abstract

Compounds of general formula (II) wherein R3 is C¿1-35? hydrocarbyl group optionally substituted by one to five substituents, selected from halo, C1-6alkoxy, hydroxy, amino, and mono- or di-C1-4alkyl-amino; and either the dotted line represents a double bond between the 2 and 3 positions of the naphthyl ring; R?1 and R4¿ each represent = O; and R2 represents a group (a) wherein n is 2 or 3, R?6 and R7¿ which may be the same or different, each represent a hydrogen atom, a C¿1-6?alkyl group, optionally substituted by hydroxy, C1-6alkoxy or a group (b) wherein R?9¿ is a C¿1-6?alkyl group optionally substituted by an amino group optionally substituted by one or two alkyl groups, or R?6 and R7¿ may each be a group (c) wherein R8 is a C¿1-6?alkyl group, A is an oxygen atom or a group (d) wherein R?5¿ represents a hydrogen atom, a C¿1-6?alkyl group optionally substituted by hydroxy, C1-6 alkoxy or an amino group optionally substituted by one or two C1-6alkyl groups or R?5¿ is a group (e) wherein R10 is a C¿1-6?alkyl group or R?5¿ is a group (f) wherein p is 2 or 3 and R?11 and R12¿ are as hereinbefore defined for R?6 and R7¿ or, when m is 1 and n is 2 R5 may be linked to R6 so that (g) forms a piperazine ring; or the dotted line represents double bonds between the 1, 2 and 3, 4 positions of the naphthyl ring, and R?1, R2 and R4¿ each represent a group (h) (wherein n, A, R?6 and R7¿ are as hereinbefore defined); and physiologically acceptable salts thereof.

Description

NAPHTHOQUINONE DERIVATIVES WITH ANTI-PROTOZOAL AND ANTI-PARASTIC ACTIVITY

The present invention relates to naphthoquinones and their use in chemotherapy. More specifically the invention is concerned with novel carbonate and carbamate derivatives of hydroxynaphthoquinones, processes for their preparation, pharmaceutical formulations thereof and their use in the chemotherapy of certain pro ozoal and parasitic infections.

Parasitic protozoal infections are responsible for a wide variety of diseases of medical and veterinary importance, including malaria in man and various coccidioses in birds, fish and mammals. Many of the diseases are life-threatening to the host and cause considerable economic loss in animal husbandry. Parasitic protozoa include the Apicomplexa, such as species of Eimeria, Cryptosporidium, Toxoplasma, and Plasmodium. Another parasitic organism of increasing concern is Pneumocvstis carinii. which can cause an often-fatal pneumonia in immunodeficient or immunocompromised hosts, including those infected with HIV. The classification of this organism is unclear and there is still uncertainty as to whether it is a protozoan or a fungus.

A wide range of naphthoquinones is known in the art. Such compounds have been variously described as having antimalarial, anticoccidial and antitheilerial activity. Some compounds have also been described as possessing activity against external parasites. Thus, Fieser et. al. J. Amer. Chem. Soc. 1948, 22,, 3156-3165 (and references cited therein) describes a large number of 2-substituted-3-hydroxy-l,4-naph- thoquinones as having antimalarial activity. A number of these compounds have also been described in U.S. Patent Specification No. 2 553 648. Further classes of 2-substituted-3-hydroxy-l,4-naphthoqui- nones having activity as antimalarial, anticoccidial and/or antitheilerial agents are described i U.S. Patents Nos. 3 367 830, and 3 347 742, U.K. Patent Specification No. 1553424, and European Patent Specifications Nos. 2 228, 77551, 77550 and 123,238. European Patent Application No. 362996 discloses, for the treatment and/or prophylaxis of infections caused by Pneumocvs is carinii, 1,4-naphthoquinones of formula:

where R is an optionally substituted, C_ non-aromatic hydrocarbon residue and R is inter alia a group -0C0R^C, OR , SR or NR^eR , which compounds are said to be believed to act as pro-drugs of compounds wherein R is a hydroxyl group.

It has now been found that a 1,4-naphthoquinone substituted at the 2-position by a carbamate group exhibits good activity in vivo against malaria in mice infected with Plas odium oelii.

Thus, in a first aspect the present invention provides a compound of general formula (II)

wherein R is C. . hydrocarbyl group optionally substituted by one to five substituents, selected from halo, C. ..alkoxy, hydroxy, amino, and mono-or di-C. ,alkyl-amino; and either the dotted line represents a double bond between the 2 and 3 positions of the

1 4 2 naphthyl ring; R and R each represent = 0; and R represents a group

f> 7 wherein n is 2 or 3, R and R which may be the same or different, each represent a hydrogen atom, a C. - alkyl group, optionally substituted by hydroxy,

\ 9 9

C. _fialkoxy or a group C-R wherein R is a C. -alkyl group optionally substituted by an amino group optionally substituted by one or two alkyl groups, or R and R may each be a group

-C- S0-R8 wherein R8 is a C. - alkyl group, A is an oxygen atom or

R⁵

\ 5 a group —N- herein R represents a hydrogen atom, a C, ,alkyl group optionally substituted by hydroxy, C. , alkoxy or an amino group optionally substituted by one or two C. _fialkyl groups or R 5

R is a C₁ ,alkyl group or R is a

2 or 3 and R and R are as 7 S hereinbefore defined for R and R or, when m is 1 and n is 2 R

R⁵

6 I may be linked to R so that N(CH„)„N forms a piperazine ring; or the dotted line represents double bonds between the I, 2 and 3, 4 positions of the naphthyl ring, and

1 ° 4 R , R and R each represent a group

(wherein n, A, R and R are as hereinbefore defined) ;

and physiologically acceptable salts thereof.

3 The .C. __ hydrocarbyl group R may be a straight or branched chain

C_{1 14} (e.g. C₁_₈)alkyl or Z_ _lh (e.g. C_{2 g})alkenyl group or a C₃_₁₀

(e.g. C_ _R)cycloalkyI group, each of which may be optionally substituted by a C_ _1f. (e.g. C_ _fi)cycloalkyl group, and each of the aforesaid cycloalkyl groups optionally being substituted by a C.

(e.g. C. ,)alkyl group. Alternatively the hydrocarbyl group R may be a C, .._ (e.g.C, _R) cycloalkyl group substituted by a phenyl group which is optionally substituted by a C^ .. (e.g. C, ,) alkyl group.

The hydrocarbyl group R preferably contains from 1 to 20 carbon

3 atoms, e.g. 1 to 14 carbon atoms. Suitable groups R include

C₃_₁₀c cloalfc l-C_{1 8}-alkyl, C₁__1£)alkyl-C₃_₁₀cycloalkyl, C₁__1(Jalkyl-

C-_₁₀cycloalkyl-C., .-alkyl and C___1Q-cycloalkyl-C _1Qcycloalkyl. R may also be substituted as hereinbefore defined by one to five halo,

C, _r alkoxy, a groun S(0) R wherein m is 0,1 or 2 and R is a l-o m

C. -allyl group, hydroxy, amino, or mono - or di-C. alkyl amino substituents.

3 Preferred groups R include:

a C₁_₁₍₎alkyl group; a C cycloalkyl group (which may be optionally substituted by a straight or branched chain C. , alkyl group, a halo-CL ..alkyl group, a C. _fialkoxy group or a phenyl group, the phenyl group itself being optionally substituted by one or more substituents selected from C. , alkyl and halogen) ; and

a C. _ιnalkyl-C.. ₇cycloalkyl group, wherein the cycloalkyl moiety may be optionally substituted as defined for the aforementioned C- _ cycloalkyl group.

3 A particularly preferred class of substituents R is represented by the formula:

wherein:

q is zero or one and

13 R is a C. _lfl alkyl group.

3 Another particularly preferred class of substituents R is represented by the formula:

(

wherein: 14 15 r is zero or one, and either R is hydrogen and R is selected from halo, halo-C, _r alkvl, C, , alkoxv, aralkoxv, C, ,

1-0 " l-o - - 1-6 aallkkyvll--CC₁, _{fi c} aallkkooxxvy,, aanndd pphheennvyll ssuuhbstituted by one or two groups -O selected from halo and C, , alkyl l-o

or

14 15 R and R are both C. , alkyl or phenyl.

3 It will be appreciated that the compounds of formula (I) wherein R contains a substituted cyclohexyl group may exist as the cis or trans isomer, that is to say that the cyclohexyl ring may be cis or trans substituted by the naphthoquinone nucleus and the subsistent on the cyclohexyl ring. Both cis and trans isomers and mixtures thereof in any ratio may be used in accordance with the present invention. In general when the compound is in the form of a mixture of isomers the trans isomer will be present in an amount of about 50% or will be the predominant isomer but the use of mixtures in which the cis isomer predominates is also included within the scope of the invention. The specific ratio of isomers may be varied as required; typical^' mixtures include those in which the cis/trans isomer ratio is about 1:1,40:60 and 5:95. For use according to the present Invention the trans isomer of such compounds of formula (I) , or a mixture of the cis and trans isomers containing at least 95% e.g. 99% of the trans isomer, is preferred.

3 An especially preferred substituent R is the 4-(4-chlorophenyl)cyclo¬ hexyl group:

/

Cl

In particular in the trans form with respect to the naphthoquinone ring. " In the compounds of formula (II) A Is preferably a group -N- and R^ represents a carbamate group

When any of the groups R , R , R , R , R , R or R represents C.

1-6 alkyl, this may be straight or branched chain e.g. methyl, ethyl, isopropyl, t-butyl, isopentyl or n-hexyl. Suitably R , R and R are each hydrogen or a C- , alkyl group. Most suitably R is methyl or

6 --"'*^• η ethyl, R is hydrogen, methyl or ethyl, R is hydrogen or a group

8 8

-CO-O-R wherein R is C. , alkyl, e.g. t-butyl.

1 4 In the compounds of formula (II) R and R preferably represent -0 and the dotted line is a bond between the 2 and 3 positions of the naphthyl ring.

A preferred group of compounds of formula (II) may be represented by the formula (III)

5 6 7 wherein R , R and R are as hereinbefore defined,

and physiologically acceptable salts thereof. Furthermore the compounds of formula (III) are preferably in the form of the trans-isomer.

2-f rans-4-(4-Chlorophenyl)cyclohexyl] -3-{N-m thyl-N- [2-(methylamino)- ethylJarbamoyloxy}-1, -naphthoquinone is a preferred compound of the formula (III).

A further preferred group of compounds of formula (II) may be represented by the formula (IV) :

wherein R , R and R are as hereinbefore defined

2-f rans-(4-t-butylcyclohexyl)methyl] -3-{N-methyl-N- [2-(methylamino)- ethylJcarbamoyloxy}-l,4-naphthoquinone is a preferred compound of the formula (IV).

By the term "hydrocarbyl" group is meant an aliphatic group, e.g. a straight branched chain or cyclic alkyl, alkenyl or alkynyl group, a carbocyclic aryl, an aliphatic group substituted by a carbocyclic aryl group optionally substituted by an aliphatic group or a carbocyclic aryl group substituted by an aliphatic group.

Without wishing to be bound by theory, it is believed that the carbonate or carbamate derivatives of formula (II) are pro-drugs of the corresponding hydroxynaphthoquinone, that is, the carbamate or carbonate or group is cleaved in vivo to give the compound of formula (I) wherein R Is a hydrox 1 group. It is believed that the compounds of formula (II) will exhibit activity against parasitic protozoa, in particular those organisms against which corresponding hydroxynaphthoquinones have been found to be active, such as Plasmodium species, eg. P.falciparum: Ei eria species eg. E. enella and E.acervulina: Theileria species e.g. T.parvum and T. nnulata: Crvptσsporidium: and Toxoplasma gondii as well as the parasitic organism Pπeumocvstis carinii. and will therefore be useful in the treatment and/or prophylaxis of parasitic infections, such as those caused by parasitic protozoa, eg. malaria, coccidiosis, cryptosporidiosis, toxoplasmosis and those caused by P.carinii eg. P.carinii pneumonia (PCP) in animals, including humans.

It will be appreciated that the amount of a compound of formula (II) or its salt required for use in the treatment or prophylaxis of the above-mentioned diseases will depend inter alia on the particular compound administered, the route of administration, the age and weight of the mammal (e.g. human) to be treated and the nature and severity of the condition being treated. In general, a suitable dose for administration to man for the treatment of malaria is in the range of O.lmg to 200mg per kilogram bodyweight per day, for example from lmg/kg to lOOmg/kg, particularly 10 to 40 mg/kg. It will be appreciated that for administration to neonates, lower doses may be required.

For prophylactic treatment a compound of formula (II) or a salt thereof may also be given less frequently, e.g. as a single dose on alternate days, once or twice per week or once or twice per month. The dosage for prophylatic treatment will depend inter alia on the frequency of administration, and, where a depot preparation or controlled release formulation is used the rate of release of the active ingredient. Thus for once-weekly administration a suitable prophylactic dose is in the range 0.1 to 100 mg/kg,e.g. 0.5 to 50 mg/kg particularly 5 to 50 mg/kg. It should be understood that for consistency the dosages referred to above are calculated In terms of the compound of formula (II) per se. and may require adjustment in the event a salt is employed.

The present invention thus further provides a method for the treatment •and/or prophylaxis of parasitic infections e.g. parasitic protozoal Infections such as malaria, or infections caused by P.carinii. in mammals e.g. humans, which comprises administering to a mammal suffering from or susceptible to said infection, an effective amount of a compound of formula (II) or a physiologically acceptable salt thereof.

There is also provided a compound of formula (II) or a physiologically acceptable salt thereof for use in therapy, e.g. in the treatment and/or prophylaxis of parasitic diseases as hereinbefore defined.

The invention also provides the use of a compound of formula (II) or a physiologically acceptable salt thereof for the manufacture of a medicament for the treatment and/or prophylaxis of parasitic infections as hereinbefore defined.

For use according to the present invention a compound of formula (II) or a physiologically acceptable salt thereof is preferably presented as a pharmaceutical formulation. In general such pharmaceutical formulations will comprise a compound of formula (II) or a physiologically acceptable salt thereof together with one or more pharmaceutically acceptable carriers therefor and optionally other therapeutic and/or prophylactic ingredients. The carrier(s) must be acceptable in the sense of being compatible with the other ingredients of the formula and not deleterious to the recipient thereof.

The present invention, therefore, further provides a pharmaceutical formulation comprising a compound of formula (II) or a pharmaceuti¬ cally acceptable salt thereof together with a pharmaceutically acceptable carrier therefor. There is also provided a method for the preparation of a pharmaceut¬ ical formulation comprising bringing into association a compound of formula (II) or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier therefor.

A compound of formula (II) or its salt may conveniently be presented as a pharmaceutical formulation in unit dosage form. A convenient unit dose formulation contains a compound of formula (II) or a physiologically acceptable salt thereof in an amount of from 10 mg to lg-

Pharmaceutical formulations include those suitable for oral, topical (including dermal, buccal and sublingual) , rectal and parenteral (including subcutaneous, intradermal, intramuscular and intravenous) administration. The formulation may, where appropriate, be conveniently presented in discrete dosage units and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing into association a compound of formula (II) or a physiologically acceptable salt thereof with liquid carriers or finely divided solid carriers or both and then, if necessary, shaping the product into the desired formulation.

Pharmaceutical formulations suitable for oral administration wherein the carrier is a solid are most preferably presented as unit dose formulations such as boluses, capsules or tablets each containing a predetermined amount of the active ingredient(s) . A tablet may be made by compression or moulding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient(s) in a free-flowing form such as a powder or granules optionally mixed with a binder, lubricant, inert diluent, lubricating agent, surface-active agent or dispersing agent. Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered active ingredient(s) with any suitable carrier. Tablets may be optionally coated and, if uncoated, may optionally be scored. Capsules may be prepared by filling the active Ingredient(s) , either alone or In admixture with one or more accessory ingredients, into the capsule shells and then sealing them In the usual manner. Cachets are analogous to capsules wherein the active ingredient(s) together with any accessory Ingredient(s) is (are) sealed in a rice paper envelope. A compound of formula (II) or a physiologically acceptable salt thereof may also be formulated as dispersible granules, which may for example be suspended in water before administration, or sprinkled on food. The granules may be packaged e.g. in a sachet. Formulations suitable for oral administration wherein the carrier is a liquid may be presented as a solution or a suspension in an aqueous liquid or a non-aqueous liquid, or as an oil-in-water liquid emulsion, e.g. a syrup, elixir, emulsion or a draught. A syrup may be made by adding the active ingredient(s) to a concentrated aqueous solution of a sugar, for example sucrose, to which may also be added any accessory ingredients which may include flavourings, agents to retard crystallisation of the sugar or agents which increase the solubility of other ingredients such as polyhydric alcohols for example glycerol or sorbitol.

Formulations for oral administration include controlled release dosage forms e.g. tablets wherein the active ingredient(s) is (are) formulated in an appropriate release - controlling matrix, or coated with a suitable release - controlling film. Such formulations may be particularly convenient for prophylactic use.

Pharmaceutical formulations suitable for rectal administration wherein the carrier is a solid are most preferably presented as unit dose suppositories. Suitable carriers include cocoa butter and other materials commonly used in the art. The suppositories may be conveniently formed by admixture of the active ingredient(s) with the softened or melted carrier(s) followed by chilling and shaping in moulds.

Pharmaceutical formulations suitable for parenteral administration include sterile solutions or suspensions of the active ingredient(s) in aqueous or oleaginous vehicles. Injectible preparations may be adapted for bolus injection or continuous infusion. Such preparations are conveniently presented in unit dose or multi-dose containers which are sealed after introduction of the formulation until required for use. Alternatively, the active ingredient(s) may be in powder form eg. freeze-dried which is constituted with a suitable vehicle, such as sterile, pyrogen-free water, before use.

A compound of formula (II) or a physiologically acceptable salt thereof may also be formulated as a long-acting depot preparation, which may be administered by intramuscular injection or by implantation e.g. subcutaneously or intramuscularly.Depot preparations may include, for example, suitable polymeric or hydrophobic materials, or ion-exchange resins. Such long-acting formulations are particularly convenient for prophylactic use.

Whilst the compounds of the invention may be formulated in any known manner, for example as described above, in view of their properties they are particularly suited for formulation, for example as aqueous solutions which may be used for injection. 2- rtrans-4-(4-Chlorophen- yl)cyclohexyl] -3-{N-methyl-N-[2-(methylamino)ethyl]carbamyloxy}-1,4- naphthoquinone has improved water solubility over the parent hydroxynaphthoquinone.

It should be understood that in addition to the aforementioned carrier ingredients the pharmaceutical formulations for the various routes of administration described above may include, as appropriate one or more additional carrier ingredients such as diluents, buffers, flavouring agents, binders, surface active agents, thickeners, lubricants, preservatives (including anti-oxidants) and the like, and substances included for the purpose of rendering the formulation isotonic with the blood of the intended recipient.

The compounds of the present invention may be administered in combination or concurrently with other therapeutic agents, for example other antimalarial agents, such as 4-amlnoquInolines eg. chloroquine and amodiaquine; 8-aminoquino- lines eg. primaquine; chloroquine; mefloquine; quinine; quinldine; artemesinin; artesumate; artemether; halofantrine; dihydrofolate reductase inhibitors eg. pyrimethamine; sulphonamides eg. sulphadox- ine: proguanil; chloroproguanll; dapsone, hydroxynaphthoquinones; or mixtures eg. pyrimethamine/sulphadoxine; pyrimethamine/dapsone; and pyrimethamine/sulfalene; antibacterial agents such as trimethopri - sulphamethoxazole mixtures; anticoccidial agents such as monensin, halofuginone, arprinocid, amprolium, dinitolmide, robenidine or salinomycin; or antibiotics such as cllndamycin, tetracycline, doxycycline, or spiromycin; or agents active against Pneumocvstis carinii such as pentamidine or Eflornithine.

When compovmds of formula (II) are used in combination with a second therapeutic agent the dose of each compound may vary from that required when the compound Is used alone. Appropriate dosages can be readily determined by those skilled in the art.

Compounds of formula (II) may be prepared by reaction of a compound of formula (V)

wherein R is as hereinbefore defined and X is halo, with a compound of the formula (VI)

wherein n, A, R and R are as hereinbefore defined; provided . that when it is required to prepare a compound wherein one or more of R and R is hydrogen, the corresponding compound is prepared with a group -C0-0-R in place, instead of hydrogen and this group is then removed.

The reaction may conveniently be effected in the presence of a solvent which is inert to the reagents. Solvents which may be employed include aromatic hydrocarbons e.g. benzene or toluene; halogenated hydrocarbon e.g. chloroform or dichloromethane; dipolar aprotic solvents e.g. dimethylformamide or hexamethylphosphoric triamide; ethers e.g. tetrahydrofuran or dioxan; pyridine; acetonitrile; trimethylphosphate and triethylphosphate. The reaction temperature may conveniently be in the range of from -80 C to 100 C, preferably 0°C to 30°C.

The reaction may advantageously be effected in the presence of a base which may be an organic base, for example pyridine, 4-dimethylamino- pyridine, a tertiary amine such as triethylamine, or 1,8-diazabicy- clo[5,4,0]-7-undecene (DBU) , or an inorganic base, for example an alkali metal carbonate such as potassium carbonate or sodium hydrogen carbonate.

1 2 4 To prepare a compound of formula (II) wherein R , R and R each represent a carbonate or carbamate group the reaction should be effected after reduction of the quinone nucleus, which may be effected in conventional manner.

Salt formation can also be effected by methods well known in the art, by reacting a compound of formula (II) with an appropriate acid. o

Removal of the protecting group -C0-0-R is conveniently carried out

Q by acid, for example when R is tertiary butyl hydrogen chloride in ether. The compounds of the formula (V) may conveniently be prepared in situ from the corresponding hydroxynaphthoquinone, for example when X is chloro by the reaction of the hydroxynaphthoquinone with phosgene in an Inert solvent, as defined above, in the presence of the base used to attach the carbamate side chain. This reaction Is carried out between -50 C and 50 C and suitably at between -10 C and 10 C.

Compounds of formula (IV) may be prepared according to known methods for the synthesis of hydroxynaphthoquinone derivatives, such as are described in US Patents Nos. 2,553,648; 3,367,830; and 3,347,742; UK Patent No. 1,553,424; European Patents Nos. 2,228; 77,551; 77,550; and 123,238 and European Patent Application No. 362,996. Thus for example a compound of formula (IV) may be prepared by reaction of a 2-halo

(e.g. 2-chloro)-l,4-naphthoquinone with a compound serving to include

3 3 the,required R group, followed by alkaline hydrolysis. Where R is an optionally substituted cyclohexyl or optionally substituted cyclohexylmethyl group it may be introduced by reaction with the appropriately substituted cyclohexylcarboxylic acid or cyclohexylacetic acid.

The invention will now be further Illustrated by the following non- limiting examples:-

In vivo Activity against Plasmodium voelii in mice.

Test Compounds

Compound A: 2- \trans-4-(4-Chlorophenvl)cyclohexyl1 -3-{N-methyl-N- [2-(methylamino)ethyl]carbamoyloxy)-1,4-naphthoquinone hydrochloride mo- nohydrate.

Compound B: 2-f rans-4-(4-Chlorophenyl)cyclohexyl]-3-{N-[2-(methyl amino)ethyl]carbamoyloxy)-1,4-naphthoquinone hydrochloride monohy- drat . Compound C: 2- ftrans-4-(4-Chlorophenyl)cyclohexyl] -3-(N-methyl-N- [3-(methylamino)propyl]carbamoyloxy) -1, -naphthoquinone.

Compound D: trans-2- [4-(4-Chlorophenyl)cyclohexyl] -3- [piperazine-N- carbonyloxy] -1,4-naphthoquinone hydrochloride dihydrate.

Compound (I) : 2- \trans-4-(4-Chlorophenyl)cyclohexyl]-3-hydroxy-1,4- naphthoquinone.

Compounds A, B, C, and (I) were administered orally and intravenously, in the manner described below.

METHOD

The test method is a modified version of the 4-day suppressive test. The YM strain of P.voelii was maintained in mice by twice weekly passage. Infected blood was collected and diluted with normal saline to give an inoculum of 3x10 parasitized enythrocytes/ml. On the morning of Day 1 the test animals (male CD-I mice weighing 18-20g) were infected intravenously via the tail vein with 0.1ml of inoculum/mouse.

Oral (p.o. )

Test compounds A, B and (I) were formulated by ball milling in 0.25% celacol with stainless steel balls. The total requirement was formulated at the beginning of a test and thereafter stored at 4 C.

Each test compound was administered orally (p.o.) to four or more usually five groups of mice, each group receiving a different dose level (dilution) . A further group of mice was administered celacol only, as a control (ie. a total of 30 mice are used to obtain each ED_c-.) . The mice were dosed orally 24 hours post-infection.

Intravenous (i.v.. I.v. formulations of test Compounds A and B were prepared by dissolving the compound in water immediately before use, and administered via the tail vein as a single dose 24 hours post-infection.

On the morning of day 5 smears of tail blood were prepared from each mouse and the parasitaemia counted. Data were analysed to yield ED_ςn values by the best fit to a slgmoidal dose response curve. The results are presented in Table 1 below.

Results

Table 1

Dosing Schedule ED₅₀ mg/kg

1 x p.o. 1 x I.v.

0.16 0.12

0.15 0.12

0.2 0.27

0.4 0.29

0.4

In vivo Activity against Pneumocvstis carinii in mice

Drugs tested

Septrin - Wellcome Paediatric Suspension containing 200 mg sulfamethoxazole/40 mg trimethoprim per 5 ml Atovaquone-2- [trans-4-(4-chlorophenyl)cyclohexyl] -3-hydroxy-1,

4-naphthoquinone Test compound-2- [trans-4-(4-chlorophenyl)cyclohexyl] -3- {N-methyl-

N- [2-(methylamino)ethyl] carbamoyloxy) -1,4-naphtho- quinone hydrochloride monohydrate

Method

Pneu ocvstis carinii (PC)-free female SCID (C.B-17/lcr-scid/scid) mice

(20-25g) were started on dexamethasone (2m/L in drinking water) , 7 days prior to intratracheal infection with single dose of

4 cryo-preserved mouse-derived PC (dose:-2 x 10 cysts equivalent to about 10 trophozoites) . Drugs were evaluated for prophylaxis by continuous daily oral dosing of groups of 10 mice with each drug from

1 day post infection until day 42. Trials were terminated and all the mice killed 24 h after the last dose. The lungs were removed and an impression smear prepared from a representative portion of lung selected at random. Lung smears were stained for P.carinii cysts using an im unofluorescent antibody test kit (Detect IF P. carinii,

Shield Diagnostics Ltd.) Slides were scored for the intensity of P. carinii infection by scanning the entire smear at lOOx magnification and counting the highly fluorescent cysts. Smears were assigned one of the following scores:

0 no cysts/smear +1 = 1 to 5 cysts/smear +2 = 6 to 50 cysts/smear +3 - 51 to 250 cysts/smear

+4 >250 cysts/smear

Drug formulation

Septrin was diluted into drinking water; the dose was calculated on the basis that each mouse would drink a minimum of 2.5 ml/day. Atovaquone was administered as a suspension in 0.25% celacol. The test compound was dissolved in distilled water and the solution administered within 1 hour of preparation. Results

The results which are presented in Table 2 below, were based on two independent blind examinations, and are expressed as an average infection score of the 10 mice in each group. Data are also expressed as a percentage of the control (dosed daily p.o. with distilled water) .

Treatment Group

Control

Septrin (250/50 mg/kg/day)

Atovaquone (100 mg/kg/day p.o

Test, compound (100 mg.kg.day

Formulation Examples

The following examples illustrate, with particular reference to the compound of Example 2, pharmaceutical formulations which may be employed in accordance with the present invention. It will be appreciated that other compounds of formula (II) may be formulated in similar manner.

A. Iniectable solution

A solution for intramuscular injection may be prepared by mixing:-

Compound of Example 2 9.5 parts by weight

Dimethyl sulphoxide 19.0 parts by weight

Sorbitan monooleate 4.5 parts by weight

Corn oil 67.0 parts by weight

100.0 Iniectable solution

Compound of Example 2 5 parts by weight N-methyl-pyrollidone 48.3 parts by weight Tween 80 2 parts by weight Span 80 4.7 parts by weight Miglyol 812 40 parts by weight

100.0

Tablet

Compound of Example 2 25.0 mg

Lactose BP 48.5 mg

Microcrystalline Cellulose BP 10.0 mg

("Avicel pH 101") Low-substituted Hydroxypropyl; 10.0 mg

Cellulose BP ("LHPC LH-11") Sodium Starch Glycollate BP 3.0 mg

("Explotab") Povidone BP ("K30") 3.0 mg Magnesium Stearate BP 0.5 mg

100.0 mg

IV Solution Freeze Dried

Compound of Example 2 50mg Water for Injections to 10ml

Dissolve the naphthoquinone in the Water for Injections. Sterilise by filtration. Fill into glass vials and freeze dry. Reconstitute shortly before use with Water for Injections. E. Capsule

Compound of Example 2 100 mg

Starch 1500 150 mg

Magnesium stearate 2.5 mg filled into a hard gelatin capsule

F. Aerosol Formulations

a) Compound of Example 2, micronised 1.0 mg Aerosol propellant to 5.0 ml

Suspend the micronised compound in the aerosol propellant. Fill this suspension into preformed aerosol cannisters, 5 ml/cannister under pressure, through the valve orifice.

b) Compound of Example 2, micronised 1.0 mg Arlacel 85 0.1% w/v Aerosol propellant to 5 ml

Disperse the Arlacel 85 in the aerosol propellant and then add compound of Example 4. Fill the suspension into preformed aerosol cannisters, 5ml/cannister under pressure, through the valve orifice.

G. Powder Inhalation

Compound of Example 2, micronised 1.0 mg Lactose 29.0 mg

Triturate and blend the micronised compound with the lactose. Fill the resulting powder blend into hard gelatin capsule shells, 30 mg per capsule.

tert-butyl N-methyl-N-f -(methylamino)ethyl carbamate This was prepared from N,N' -dimethylethylenediamine following the procedure of W.S. Saari, J.E. Schwering, P.A. Lyle, S.J. Smith and E.L. Engelhardt. J.Med.Che . vol 33, 97, 1990.

Bis !2- r (tert-butoxycarbonyl aminoIethyDamine

To a stirred solution of diethylenetriamine (lOg; 97 ml; Lancaster synthesis) in tetrahydrofuran (5ml) cooled to 0 C was added dropwise a solution of di-tert-butvl dicarbonate (10.5g; 48mM) in tetrahydrofuran (50 ml) . The mixture was allowed to warm to room temperature and stirred for 18 hours, filtered concentrated in vacuo and partitioned between brine and ethyl acetate. The aqueous layer was extracted once with ethyl acetate and the combined extracts were dried (MgSO, ) and concentrated .in vacuo to leave a clear oil (6.7g). Chomatography on silica gel with 1:4 methanol: chloroform gave the title compound as a clear oil (2g; 27%), NMR δH (CDC1₃) 3.15-3-3 (4H, m) , 2.75 (4H t,J5Hz), 1.45 (18 H,s).

Example 1

2-ftrans-4- (l±-Chlorophenyl)cyclohexyl] -3-(N-methyl-N-f2-(N-methyl- N- ert-butoxycarbonyl)aminoethyπcarbamoyloxy) -1.4-naphthoquinone

To a stirred suspension of 2-hvdroxy-3-ftrans-4-(4-chlorophenyl-cyclo- xyl] -1,4-naphthoquinone (3.7g; lOmmol) in dry dichloromethane (60ml), cooled to 0 C under nitrogen, was added all at once a solution of phosgene in toluene (10ml; 1.25M solution). Pyridine (0.8ml; lOmmol) was added dropwise and the solution stirred for a further 45 minutes at 0°C. A mixture of tert-butvl N-methyl-N- [2-(methylamino)ethyl] carbamate (1.85g; 10.5mmol) and pyridine (0.8ml; lOmmol) in dry dichloromethane (10ml) was added dropwise over 15 minutes and the mixture left stirring at room temperature for 18 hours. The mixture was diluted with dichloromethane (50 ml), washed with water, 1M hydrochloric acid and water, dried (magnesium sulphate) and concentrated to leave an orange oil. Chromatography on silica with 1:9 ethyl acetate:dichloromethane followed by trituration with 1:2 ether:hexane gave the title compound (3.12g; 54%), m.p. 113-115 C, NMR SH (CDC1₃) 3.0-8.2 (2H, m) , 7.65-7.8 (2H, ) , 7.1-7.3 (4H, ) , 3.4-3.7 (4H, ), 3.15 (3H, 2xs) , 3.05-3.25 (1H, ) , 3.0 (3H, 2xs) , 1.5-2.15 (9H. 2xs).

Example 2

2-Ctrans-4-r4-Chlorophenyl)cvclohexyll -3-!N-methyl-N-■ 2-(methylamino) ethylIcarbamoyloxy)-1.4-naphthoquinone hydrochloride monohvdrate

To ether saturated with HC1 (30ml) was added 2-I^"trans-4-(4-Chlorophen- yl)cyclohexyl]-3-{N-methyl-N-[2-(N-methyl-N-tert-butoxycarbonyl) mino ethyl]carbamoyloxy}-1,4-naphthoquinone (1.74g; 3mmol) and the solution stirred at room temperature for 5 hours. The precipitate was filtered off, washed with ether and dried .in vacuo to give the title compound (1.48g 92%), m.p. >140°C (decomposes), NMR 5H CDC1₃) 9.1-9.3 (1H, bs), 7.95-8.2 (2H, m),7.65-7.85 (2H, m) , 7.1-7.35 (4H, s) , 3.7-4.0 (1H, br s), 3.0-3.5 (6H, ) , 2.8 (3H, br s) , 2.65 (1H, m) , 1.75-2.15 (8H, m), 1.4-1.75 (2H, ) .

The following were prepared in a similar manner to example 1:

2-rtrans-4-(4-Chlorophenyl)cyclohexyl]-3-{N-[2-(N-methvl-N- ert-butoxv carbonylamino)ethyl]carbamoyloxy}-l,4-naphthoquinone, from tert-butvl N-methyl-N-(2-aminoethyl)carbamate (W.S. Saari et al. J.Med.Chem. vol 33, 97, 1990), m.p. 128-131°C, NMR SH (CDC1₃) 8.05-8.15 (2H, m) , 7.65-7.8 (2H, m) , 7.15-7.3 (4H, m) , 3.5 (4H, m) , 3.0-3.2 (1H, m) , 3.0 (3H, s), 2.5-2.7 (1H, ) , 1.75-2.15 (6H, ) , 1.45-1.7 (11H, m) .

2-rtrans-4-(4-Chlorophenyl)cyclohexyl]-3-{N-methyl-N-[3-(N-methyl-N- tert-b toxycarbonylamino)propyl]carbamoyloxy)-1,4-naphthoquinone, from tert-butvl N-methyl-N-[3-(methylamino)propyl]carbamate (W.S. Saari et al. J.Med.Chem. vol 33, 97, 1990), oil, NMR δR CDC _j) 8.0-8.2 (2H, m), 7.65-7.8 (2H, m) , 7.1-7.35 (4H, m) , 3.25-3.6 (4H, m) , 3.0-3.25 (4H , m) , 2. 9 ( 3H, s ) , 2. 5 -2. 7 (1H , m) , 1. 75 - 2 . 15 (8H , m) , 1 .4- 1. 7 (11H . m) .

2-[trans-4-(4-Chlorophenyl)cyclohexyl]-3-{N-ethyl-N-[2-(N-ethyl-N- tert-butoxycarbonylamino)ethyl]carbamoyloxy)-1,4-naphthoquinone, from tert-butvl N-ethyl-N- [2-(ethylamino)ethyl]carbamate (W.S. Saari et al. J.Med.Chem. vol 33, 97, 1990), oil.

2- ftrans-4-(4-Chlorophenyl)cyclohexyl] -3- fN-tert-butoxy-carbonylpiper- azine-N-carbonyloxy] -1,4-naphthoquinone, from N-tert-butoxvcarbonyl- piperazine (Carpino et al J.Or .Chem.. vol 48(5), 664, 1983), oil.

2- [trans-4-(4-Chlorophenyl)cyclohexyl] -3-{ [2-(N-methyl-N-tert-butoxy¬ carbonyl amino)ethoxy]carbonyloxy)-1, -naphthoquinone, from tert-butvl N-(2-hydroxyethyl)-N-methylcarbonate (W.S. Saari et al. J.Med.Chem. vol 33, 97, 1990), m.p. 110-111°C, NMR δH (d_g-DMS0) 7.9-8.1 (4H, m) ,

7.3 (4H, s), 4.35-4.45 (2H, t, J-4Hz)), 3.5-3.6 (2H, t, J-4Hz) , 3.0-3.15 (1H, m), 2.5-2.7 (1H, m) , 1.7-2.0 (6H, m) , 1.45-1.65 (2H, m) ,

1.4 (9H, s).

2- ftrans-4-(4-Chlorophenyl)cyclohexyl1-3-{N,N. -bis[2-(tert-butoxy carbonylamino)ethyl]carbamoyloxy)-1,4-naphthoquinone, from bis {2- [ (tert-butoxycarbonyl)amino]ethyl) amine, yellow oil, NMR SE (CDC1₃) 8.0-8.2 (2H,m), 7.65-7.8 (2H, m) , 7.1-7.3 (4H, m) , 3.35-3.7 (4H,m), 3.15(1H, m) , 2.62 (1H, m) , 1.8-2.1 (4H, m) , 1.45 (9H,br s), 1.4(9H,br s)

The following were prepared in a similar manner to example 2 from the corresponding tert-butoxycarbonyl protected carbonates or carbamates :

2-ftrans-4-(4-Chlorophenyl)cyclohexyl] -3-{N-[2-(methylamino)ethyl]car¬ bamoyloxy)-1,4-naphthoquinone hydrochloride monohydrate, m.p.

194-197°C, NMR δH (d,-DMS0) 9.1-9.3 (2H, bs) , 8.4-8.5 (1H, t),7.8-8.1 o

(4H, m), 7.3 (4H, s), 3.4-3.6 (2H, m) , 3.0-3.2 (3H, m) , 2.55-2.75 (4H, m), 1.4-2.15 (8H, m) . 2-ftrans-4-(4-Chlorophenvl)cyclohexyl1 -3-{N-methyl-N-[3-(methylamino) propyl] carbamoyloxy)-1,4-naphthoquinone hydrochloride monohydrate, m.p. 208-210°C, NMR δH (CDC _j) 8.1-8.2 (IH, d, J-5Hz) , 8.0-8.1 (IH, d, J-5Hz), 7.65-7.85 (2H, ) , 7.15-7.3 (4H, m) , 3.05-3.35 (7H, m) , 2.5-2.8 (5H. ), 1.45-2.1 (10H, m) .

2-[trans-4-(4-Chlorophenyl)cyclohexyl]-3-{N-ethyl-N-[2-(ethylamino) ethyl]carbamoyloxy)-1,4-naphthoquinone hydrochloride monohydrate, m.p. 197-201°C. -NMR δH (CDCL_j) 8.1-8.2 (IH, d, J-8Hz) , 8.0-8.1 (IH, d, J=8Hz), 7.65-7.8 (2H, ) , 7.1-7.3 (4H, ) , 3.0-4.1 (7H, ) , 2.5-2.7 (IH, m), 1.25-2.15 (14H, m) .

2-ftrans-4-(4-Chlorophenyl)cyclohexyl] -3-[piperazine-N-carbonyloxy]■

,o,

1,4-naphthoquinone hydrochloride dihydrate, m.p. >160 C (decomposes), NMR δH (d_g DMSO) 7.8-8.1 (4H, m) , 7.25-7.4 (4H, m) , 3.6-4.0 (4H, ) , 3.0-3.5 (5H, m), 2.5-2.7 (IH, m) , 1.7-2.05 (6H, m) , 1.4-1.7 (2H, m) .

2-ftrans-4-(4-Chlorophenyl)cyclohexyl]-3-[(2-methylaminoethoxy)carbon- yloxy]-1,4-naphthoquinone hydrochloride, m.p. 211-213°C, NMR δH (d- DMSO) 7.95-8.05 (2H, m) , 7.74-7.9 (2H, m) , 7.33 (4H, s), 4.2-4.3 (2H, m), 3.5-3.6 (2H, m) , 3.0-3.2 (IH, m) , 2.75 (3H, s) , 2.5-2.7 (IH, m) , 2.05-2.3 (2H, m), 1.8-2.0 (2H, m) , 1.4-1.7 (4H, m) .

2-ltrans-4-(4-Chlorophenyl)cyclohexyl) -3-fN.N. -bis (2-aminoethyl) car¬ bamoyloxy] -1,4-naphthoquinone dihydrochloride dihydrate, m.p. 215-218°C, NMR δH (d_g-DMSO)8.25-8.7 (4H, br) 7.85-8.15 (4H, m) , 7.3-7.45 (4H,m) 3.5-4.0(4H,m) , 3.0-3.4 (5H, m) 2.7 (IH, m) , 1.5-2.2 (8H, m)

Example 3

2-rtrans-(4-tert-Butylcvclohexyl)methyll -3-(N-methyl-N-T2-(N -methyl-N- ert-butoxycarbonylamino)ethyllcarbamoyloxy)-1.4- naphthoquinone To a stirred solution of 2- ftrans-(4- ert-butylcvclohexyDmethyl] - 3-hydroxy-1, -naphthoquinone (6.76:19mM) in dry dichloromethane (250 mL) under nitrogen, was added phosgene (20 mL; 12.5% solution in toluene), followed by pyridine (1.6 ml.20 mM) added dropwise, and the mixture was stirred for 1 hour. A solution of pyridine (1.6 mL; 20mM) and tert-butyl-N-methyl-N- [2-(methylamino)ethyl]carbamate (3.7g;21mM) was added dropwise, and the reaction mixture stirred at room temperature for a further 3.5 hours. Evaporation of the solvents in vacuo gave a yellow solid, which was chromatographed on silica, eluting with toluene and then 1:8 ethyl acetate:toluene, to give the title compound (3.19g; 31%) m.p. 117-118°C, NMR δH (CDC1₃) 8.05-8.15 (2H,m). 7.65-7.75 (2H,m), 3.45-3.6 (4H, m) , 3.2-3.1 (3H, 2xs), 2.95(3H, 2xs), 2.45-2.55 (2H, m) , 1.7-1.9 (5H, m) 1.5 (9H, 2xs) , 0.9-l.l(5H, m), 0.85(9H,s).

Example 4

2- Ttrans-(4-tert-Butylcvclohexyl)methyl1 -3-fN-methyl-N- 2 -(methyla- mino)ethyl] carbamoyloxy)-1,4-naphthoquinone hydrochloride

2-ftrans-(4-tert-butylcyclohexyl)methyl] -3-{N.methyl-N-[2- (methylamino)ethyl] ^'carbamoyloxy)-1, 4-naphthoquinone (lg) was added to ether saturated with HC1(20 ml) and the solution stirred at room temperature in the dark for 1.5 hours. The ether was evaporated in vacuo to leave a yellow oil, which was dissolved in ethyl acetate and filtered. Cyclohexane was added to the filtrate, resulting in the precipitation of the title compound as a yellow powder (0.43g) m.p.> 140 C (decomposes).

Claims

1. Compounds of general formula (II) and physiologically acceptable salts thereof

wherein R³ is C_1-35 hydrocarbyl group optionally substituted by one to five substituents, selected from halo, C_1-6alkoxy, hydroxy, amino, and mono-or di-C_1-4alkyl-amino; and either the dotted line represents a double bond between the 2 and 3 positions of the naphthyl ring; R¹ and R⁴ each represent = O; and R² represents a group

wherein n Is 2 or 3, R⁶ and R⁷ which may be the same or different, each represent a hydrogen atom, a C_1-6 alkyl group, optionally substituted by hydroxy, C_1-6alkoxy or a wherein R⁹ is a C_1-6alkyl group optionally substituted by an amino group optionally substituted by one or two alkyl groups, or R⁶ and R⁷ may each be a group wherein R⁸ is a C_1-6 alkyl group, A is an oxygen atom or a group wherein R⁵ represents a hydrogen atom, a C_1-6alkyl group optionally substituted by hydroxy, C_1-6 alkoxy or an amino group optionally substituted by one or two C_1-6alkyl groups or R ⁵ is a group wherein R¹⁰ is a C_1-6alkyl group or R⁵ is a group p is 2 or 3 and R¹¹ and R¹² are as

hereinbefore defined for R⁶ and R⁷ or, when A is and n is 2 R⁵ may be linked to R⁶ so forms a piperazine ring;

or the dotted line represents double bonds between the 1, 2 and 3, 4 positions of the naphthyl ring, and R¹, R² and R⁴ each represent a group

wherein n, A, R⁶ and R⁷ are as hereinbefore defined.

2. Compounds as claimed in claim 1 wherein R³ is a C_1-10alkyl group; a C_5-7 cycloalkyl group (which may be optionally substituted by a straight or branched chain C_1-6 alkyl group, a halo-C_1-6alkyl group, a C_1-6alkoxy group or a phenyl group, the phenyl group itself being optionally substituted by one or more substituents selected from C_1-6 alkyl and halogen); or a C_1-10alkyl-C_5-7cycloalkyl group, wherein the cycloalkyl moiety may be optionally substituted as defined for the aforementioned C_5-7 cycloalkyl group.

3. Compounds as claimed in claim 1 or claim 2 wherein R³ is represented by the formula:

wherein: q is zero or one and R¹³ is a C_1-10 alkyl group.

4. Compounds as claimed in claim 1 or claim 2 wherein R³ is represented by the formula:

wherein: r is zero or one, and either R¹⁴ is hydrogen and R¹⁵ is selected from halo, halo-C_1-6 alkyl, C_1-6 alkoxy, aralkoxy, C_1-6 alkyl-C_1-6 alkoxy, and phenyl substituted by one or two groups selected from halo and C_1-6 alkyl or R¹⁴ and R¹⁵ are both C_1-6 alkyl or phenyl.

5. Compounds as claimed in claim 4 wherein R³ is the

4-(4-chlorophenyl)cyclohexyl group:

6. Compounds as claimed in claim 5 wherein R³ is in the trans form with respect to the naphthoquinone ring.

7. Compounds as claimed in any of claims 1 to 6 wherein A is and R² represents a carbamate group

8. Compounds as claimed in claim 7 wherein R⁵, R⁶ and R⁷ are each hydrogen or a C_1-4 alkyl group.

9. Compounds as claimed in claim 8 wherein R⁵ is methyl or ethyl R⁶ is hydrogen, methyl or ethyl and R⁷ is hydrogen or a group

-CO-O-R⁸ wherein R⁸ is t-butyl.

10. Compounds as claimed in any of claims 1 to 9 wherein

R¹ and R⁴ represent =O and the dotted line is a bond between the

2 and 3 positions of the naphthyl ring.

11. Compounds as claimed in claim 1 represented by the formula (III) wherein R⁵, R⁶ and R⁷ are as hereinbefore defined, and physiologically acceptable salts thereof.

12 Compounds as claimed In claim 11 In the form of the trans-isomer.

13. The compound 2-[trans-4-(4-Chlorophenyl)cyclohexyl]-3(N-methyl- N-[2-(methylamino)ethyl]carbamoyloxy)-1,4-naphthoquinone as claimed in claim 11 and physiologically acceptable salts thereof.

14. The hydrochloride monohydrate salt of the compound according to claim 13.

15. Compounds as claimed in claim 1 represented by the formula (IV):

wherein R⁵, R⁶ and 7³ are as hereinbefore defined and physiologically acceptable salts thereof.

16. The compound 2-[trans-(4-t-butylcyclohexyl)methyl]-3-(N-methyl- N-[2-(methylamino)ethyl]carbamoyloxy)-1,4-naphthoquinone as claimed in claim 15 and physiologically acceptable salts thereof.

17. A method for the treatment and/or prophylaxis of parasitic protozoal infections or infections caused by P. carinii, in mammals, which comprises administering to a mammal suffering from or susceptible to said infection, an effective amount of a compound of formula (II) or a physiologically acceptable salt thereof.

18. A compound of formula (II) or a physiologically acceptable salt thereof for use in therapy.

19. The use of a compound of formula (II) or a physiologically acceptable salt thereof for the manufacture of a medicament for the treatment and/or prophylaxis of parasitic infections.

20. A pharmaceutical formulation comprising a compound of formula (II) or a physiologically acceptable salt thereof together with one or more pharmaceutically acceptable carriers therefor and optionally other therapeutic and/or prophylactic ingredients.

21. A unit dose formulation containing a compound of formula (II) or a physiologically acceptable salt thereof in an amount of from 10mg to 1g.

22. A process for the preparation of compounds of formula II and physiologically acceptable salts thereof which comprises reaction of a compound of formula (V) wherein R³ is as hereinbefore defined and X is halo, with a compound of the formula (VI)

wherein n, A, R⁶ and R⁷ are as hereinbefore defined provided that when It is required to prepare a compound wherein one or more of R⁶ and R⁷ is hydrogen, the corresponding compound is prepared with a group or -CO-O-R⁸ in place, instead of hydrogen and this group is then removed.