AP111A

AP111A - Medicaments.

Info

Publication number: AP111A
Application number: APAP/P/1989/000136A
Authority: AP
Inventors: Victoria Susan Latter; Winston Edward Gutteridge; Alan Thomas Hudson
Original assignee: The Wellcome Foundation Ltd
Priority date: 1988-08-16
Filing date: 1989-08-15
Publication date: 1991-02-23
Also published as: US4981874A; GB8819477D0; AP8900136A0

Abstract

The present invention relates to the use of known and novel naphthoquinones in the treatment and prophylaxis of pneumocystis carinii infections, and for the manufacture of medicaments for the treatment of prophylaxis of p.CArinni infections, novel formulations containing said compounds, and novel naphthoquinones of formula (vi): wherein r11 and r12 each represent -0 and the dotted line represents a double bond between the 2 and 3 positions of the quinone ring, in which case r13 represents a group -0cor5; a group or6 or sr6; or a group nr7r8; or the dotted line represents double bonds at the 1,2 and 3,4 positions of the quinol ring and r11, r12 and r13 each represents a group -ocor14, wherein r14 epresents an optionally substituted c1-10alkyl group.

Description

MEDICAMENTS

The present invention relates to the treatment and prophylaxis of Pneumocystis carinii infections. More particularly the invention is concerned with the use of naphthoquinones in the treatment and prophylaxis of Pneumocystis carinii infections, the use of said compounds for the manufacture of medicaments for the treatment and prophylaxis of P.carlnii infections, and novel formulations containing said compounds.

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Pneumocystis carinii is a parasite which has a natural habitat in lung tissue. In a host with a normal immune system P.carlnii is not considered to be pathogenic. However, vhen the immune system is defective P.carlnii is liable to cause pneumonia. There is a variety of circumstances in which the Immune system may be defective or deficient. Thus, for example immune system deficiency is common in immature or premature infants (neonates). It may also result from suppression by certain drugs, which may be deliberate e.g. in certain patients receiving organ transplants, or unavoidable e.g. as a side-effect of cancer t

chemotherapy. Disordered growth of one or more constituent parts of the Immune system, e.g. as in certain forms of cancer, may also result In immunode f i c i ency.

Immune deficiency may furthermore be caused by viral infections, including human immunodeficiency virus (HIV). It has been reported (Hughes, W.T. (1987) Treatment and Prophylaxis of Pneumocystis carinii pneumonia, Parasitology Today 3(11) 332-335) that at least 60% of patients with

A 1 apo η n 11 1

JB/JJ/PA0839FF/28th July, 1989.

acquired immunodeficiency syndrome (AIDS) suffer from Pneumocystis carinii pneumonia.

In this specification the term immunocompromised host will be used to describe hosts with a deficient or defective immune system.

Without treatment, Pneumocystis carinii pneumonia is almost always fatal in immunocompromised hosts. The most widely used treatments for this condition are trimethoprim-sulphamethoxazole (cotrimoxaole) and pentamidine. However, both of these treatments have been reported to be only around 50-70% effective in AIDS patients and to produce a much higher than usual incidence of adverse reactions (about 50%) (Wofsy, C.B. Antimicrobial Agents Annual, 1986, Vol 1, p377-400). There is thus a need for new agents, especially for the prophylaxis of P.carinii pneumonia.

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 e

possessing activity against external parasites. Thus, Fieser et al. J. Amer. Chem. Soc. 1948, 70. 3156-3165 (and references cited therein) describes a large number of 2-substituted-3-hydroxy-l,4-naphthoquinones 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-1,4-naphthoquinones having activity as antimalarial, anticoccidial and/or antitheilerial agents are described in 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 and 77550.

JB/JJ/PAO839FF/28th July, 1989.

European Patent Application No. 123239 discloses synergistic combinations of anti-protozoal naphthoquinones and 4-pyridinols or alkanoic esters thereof, which are said to be especially useful for the treatment or prophylaxis of malaria.

European Patent No. 123,238 discloses 2-substituted-3-hydroxy 1,4-naphthoquinones of formula (I)

2 wherein either R is hydrogen and R ia selected from g alkoxy, aralkoxy,C^_g alkyl-C^g alkoxy, phenyl substituted by one or two groups selected from halogen and C^_g alkyl, halogen and perhalo-C^ g alkyl or R³ 2 and R are both g alkyl or phenyl, and n is zero or 1, and physiologically acceptable salts thereof. Compounds of formula (I) wherein n is zero are said to be active against the human malaria parasite Plasmodium falciparum and also against Elmerla species such as E.tenella and E.acervullna. which are causitive organisms of coccidiosis. Compounds of formula (I) where n is 1 are said to be active against protozoa of the genus Thelleria. in particular T.annulata and T.parva.

C c

c

Q <3

JB/JJ/PA0839FF/28th July, 1989.

We have now found chat a variety of naphthoquinones are active in vivo against Pneumocystis carinli pneumonia infections in rats. Activity has also been demonstrated in an in vitro preparation of P, carinli.

In a first aspect the present invention provides a naphthoquinone for use in the treatment and/or prophylaxis of Pneumocystis carlnil infections (e.g. P,carinil pneumonia) in mammals (including humans).

In another aspect the present invention provides the use of a naphthoquinone for the manufacture of a medicament for the treatment and/or prophylaxis of Pneumocystis carlnil infections in mammals (including humans).

According to a further aspect the present invention provides a method of treating and/or preventing Pneumocystis carinli infections in a mammal which comprises administering to a mammal (including a human) susceptible to infection with P.carinli pneumonia an effective amount of a naphthoquinone .

Prevention of P.carinil infections is particularly important in an immunocompromised host, as discussed hereinabove. In the case of immunosuppression resulting from HIV infection, prophylaxis may be required by those diagnosed as seropositive for HIV, and those with progressive generalised lymphadenopathy (PGL) or AIDS-related complex (ARC) as well as patients suffering from AIDS.

JB/JJ/PA0839FF/28th July, 1989.

Naphthoquinones for use according to the present 1,4-naphthoquinones of the general formula (II):	invention include
	0 ii r³
	Sr ^r4 0	(II)

wherein

R is ^2.-35 non-aromatic hydrocarbon residue optionally substituted by one or more substituents, selected from halo, galkoxy, hydroxy, phenyl, phenyl-C^ ^alkoxy and phenyl-C^ ^alkyl, each such phenyl group or moiety being optionally substituted by one or more groups selected from C^^alkoxy, C^^alkyl, C^^alkoxy-C^^alkyl, hydroxy, halogen, halo-C^^alkyl, amino, and mono-or di-C^ ^alkyl-amino; and

C

C c

D

R is hydroxy; halogen;

a group CCOR³, wherein R³ is a ^alkyl group, a cycloalkyl group, a C^^alkoxy group, or a phenyl or naphthyl group, each such R^ group being optionally substituted e.g. by amino, mono-or di-C^ ^alkylamino, carboxy or hydroxy;

JB/JJ/PA0839FF/28th July, 1989.

6 6 a group OR or SR , wherein R is an optionally substituted C^_^alkyl, ^Qcycloalkyl, phenyl or naphthyl group as defined for r5; or

8 7 8 a group NR R , wherein R and R each independently represent 7 8 hydrogen or C^^alkyl, or the group NR R represents a 5-7 membered saturated heterocyclic ring, which may optionally contain a further heteroatom selected from nitrogen, oxygen or sulphur;

and physiologically acceptable salts and other physiologically functional derivatives thereof.

A non-aromatic hydrocarbon residue R may be a straight or branched chain C₁₁₄ (e.g. C_{l g})alkyl or C₂₁₄ (e.g. C₂__g)alkenyl group or a C_{3 χθ} (e.g. C^ g)cycloalkyl group, each of which may optionally carry a θ (e.g. C₃__g)cycloalkyl group, and each of the aforesaid cycloalkyl groups optionally carrying a C_11Q e.g. (C^^Jalkyl group. The non-aromatic hydrocarbon residue R^ preferably contains from 1 to 20 carbon atoms, e.g.

to 14 carbon atoms. Suitable residues R include C, _1ncycloalkyl-C- 3-1U 1^-8 alkyl, __1Qalkyl- C₃ __1Qcycloalky1, _1Qalkyl-C_{3 1Q}cyclo-alkyl-__1Qalky1 and C₃ cycloalkyl-C₃ ^pcycloalkyl.

Compounds of formula (II) containing an acidic hydroxy or carboxy group, 4 such as compounds wherein R is hydroxy, may form salts with bases, and compounds (II) containing a basic amino group may form salts with acids. Suitable base salts include inorganic base salts such as alkali metal (e.g.

JB/JJ/PAO839FF/28th July, 1989.

sodium and potassium) sales and alkaline earth metal (e.g. calcium) salts; organic base salts e.g. phenylethylbenzylamine, dibenzylethylenediamine, ethanolamine and diethanolamine salts; and amino acid salts e.g. lysine and arginine. Suitable acid addition salts include those formed from hydrochloric, hydrobromic, nitric, perchloric, sulphuric, citric, tartaric, phosphoric, lactic, glutamic, oxalic, aspartic, pyruvic, acetic, succinic, fumaric, maleic, oxaloacetic, isethionic, stearic, phthalic, methanesulphonic, p-toluene sulphonic, benzenesulphonic, lactobionic and glucuronic acids .

Without wishing to be bound by theory, it is believed that compounds of formula (II) wherein is a group - OCOr\ 0r\ SR^ or NR^R® may act as pro-drugs or bioprecursors which are converted in vivo either by the host or the parasite to a compound of formula (II) wherein R^ is hydroxy. Such compounds will be referred to hereinafter as physiologically functional derivatives. Such compounds may also however possess intrinsic biological activity.

The invention includes within its scope the use of isomers of compounds of formula (II) and mixtures of such isomers. The compounds of formula (II) may exist in a tautomeric form in which the hydroxyl group donates its proton to one of the oxo groups and the use of such tautomeric forms is included within the scope of this invention. However, it is believed that the stable form is that shown in formula (II).

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JB/JJ/PA0839FF/28th July, 1989.

A preferred group of compounds for use according to the invention is that of formula (III):

(ΙΠ) wherein R is a _1Qalkyl group;

a Cj .j cycloalkyl group (which may be optionally substituted by a straight or branched chain alkyl group, a halo-C^ _galkyl group, a C^galkoxy group or a phenyl group, the phenyl group itself being optionally substituted by one or more substituents selected from ^Cy.g alkyl and halogen); or a C, ^aj-kyl-Cj ^cycloalkyl group, wherein the cycloalkyl moiety may be optionally substituted as defined for the aforementioned cycloalkyl group; and physiologically acceptable salts and other physiologically functional derivatives thereof.

JB/JJ/PA0839FF/28th July, 1989.

Another group of compounds which may be used invention is that of formula (IV) according to the present

wherein R is an alkyl group of from 1 to 10 carbon atoms and m is 0 or 1, and physiologically acceptable salts * and other physiologically functional derivatives thereof.

In the compounds of formula (IV) is suitably a straight-chain alkyl group, preferably methyl.

A further group of compounds which may be used according to the present invention is that of formula (I) as hereinbefore defined, and physiologically acceptable salts and other physiologically functional derivatives thereof.

Vuuuucn

Preferred compounds of formula (I) for use according to the present 1 2 invention include those wherein n is zero, R is hydrogen and R is phenyl substituted by one or two groups selected from halogen and & alkyl, preferably halogen.

JB/JJ/PA0839FF/28th July, 1989.

Particularly preferred compounds for use according to the present invention include:

2- (4-i-butylcyclohexyl) - 3-hydroxy-1,4-naphthoquinone

2- (4-£-butylcyclohexylmethyl) -3-hydroxy-1,4-naphthoquinone

2-(4-(4- chlorophenyl) cyclohexyl ] - 3 - chloro -1,4 -naphthoquinone and physiologically acceptable salts and physiologically functional derivatives thereof.

An especially preferred compound for use according to the present invention is 2-(4-(4-chlorophenyl)cyclohexyl]-3-hydroxy-l, 4-naphthoquinone, represented by formula (V):

JB/JJ/PAO839FF/28th July, 1989.

Thus, in a preferred aspect the present invention provides the compound of formula (V) and physiologically acceptable salts and other physiologically functional derivatives thereof for use in the treatment and/or prophylaxis of Pneumocystis carinil infections (e.g. P.carlnli pneumonia) in mammals (including humans).

In another preferred aspect the present invention provides the use of the compound of formula (V) and physiologically acceptable salts and other physiologically functional derivatives thereof for the manufacture of a medicament for the treatment and/or prophylaxis of Pneumocystis carinii infections in mammals (including humans).

According to a further preferred aspect the present invention provides a method of treating and/or preventing Pneumocystis carinii infections which comprises administering to a mammal (including humans) susceptible to infection with P.carinii pneumonia an effective amount of the compound of formula (V), or a physiologically acceptable salt or other physiologically functional derivative thereof.

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It will be appreciated that the compounds of formula (I) wherein R^ is hydrogen, and the compounds of formulae (IV) and (V) 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 substituent on the cyclohexyl ring (in the case of formula (V) the chlorophenyl group). 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

AP 0 n n 1 1 1

JB/JJ/PA0839FF/28th July, 1989.

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 5:98. For use according to the present invention the trans isomer of the compound of formula (V), or a mixture of its cis and trans isomers containing at least 95% e.g. 99% of the trans isomer, is preferred.

Physiologically functional derivatives of the compound of formula (V) include those of formula (VI)

wherein

12

R and R each represent -0 and the dotted line represents a double bond 13 between the 2 and 3 positions of the quinone ring, in which case R represents a group -OCOR^; a group OR^ or SR^; or a group NR?r\ wherein 5 6 7 8

R , R , R* and R° are as hereinbefore defined; or the dotted line represents double bonds at the 1,2 and 3,4 positions of the quinol ring and rH, r!2 and R^ each represents a group -OCOR¹^, wherein R^ represents an optionally substituted ^alkyl group.

JB/JJ/PA0839FF/28th July, 1989.

Compounds of formula (VI) are believed to be novel and form a further aspect of the present invention.

Compounds of formula (VI) have been found to exhibit activity in vitro against the parasite Plasmodium falciparum and in vivo against the parasite Plasmodium yoelli as illustrated hereinafter. These compound may therefore be useful in the treatment and/or prophylaxis of malaria.

A preferred compound of formula (VI) is 2-acetoxv-3-ί trans-4-(4-chlorophenyl) cyclohexyl]-1,4-naphthoquinone. This compound has the advantage of improved water·solubility as compared with the parent compound of formula (V).

A further preferred compound of formula (VI) is 2-ftrans-4-(4-chlorophenyl) cyclohexyl]-1,3,4-triacetoxynaphthalene. This compound is colourless, in contrast to the compound (V), which is yellow, and may therefore have advantages in terms of its formulation and presentation.

Further derivatives of the compound (V) which may be used in accordance with the present invention are those of formula (VII)

Cl δρ ο η n 11 1 (VII)

JB/JJ/PA0839FF/28th July, 1989.

wherein X is a halogen atom, e.g. a chlorine, bromine or iodine atom, preferably a chlorine atom.

The compound of formula (VIX) wherein X is chlorine has previously been described as an intermediate e.g. in the preparation of the compound of formula (I) but no biological activity has been ascribed to it. In a further aspect therefore the present invention provides a compound of formula (VII) for use as a medicament, e.g. an antiprotozoal agent, or a medicament for the treatment of Pneumocystis carinli infections.

The synthesis of compounds of formulae (I) to (VII) may be effected by methods already known and described in the chemical literature (for example the patent specifications listed hereinbefore) or by analogous methods. In particular novel compounds of formula (VI) may be prepared by the following methods which form a further aspect of this invention:

(a) reaction of a compound of formula (V) or (VII), with a compound 13 serving to introduce the required group R , and where appropriate the _O11 a groups R and R ;

(b) reaction of a compound of formula (VIII):

(VIII)

JB/JJ/PA0839FF/28th July, 1989.

wherein R is as defined above with a donor compound serving to introduce the 4-(4-chlorophenyl)cyclohexyl group.

12

With regard to process (a) compounds (VI) wherein R and R represent - 0 13 5 and R represents a group OCOR may be prepared by esterification of the compound (V). Esterification may be effected in conventional manner using the appropriate acid R^COOH or acid derivative e.g. an acid anhydride, acid chloride or an activated ester such as an alkylhaloformate e.g. an 11 12 alkylchloroformate. To prepare a compound of formula (VI) wherein R , R and R^ each represent a group-OCOl(\ the esterification is carried out in the presence of a reducing agent, e.g. zinc.

Compounds of formula (VI) wherein R^ is _a group OR^ or SR^ may be prepared from a compound (VII) wherein X is a halogen atom. Thus for example the group 0r6 may be Introduced by reaction with the appropriate alcohol, e.g. methanol or ethanol in the presence of sodium, and the group SR^ may be introduced by reaction with the corresponding thiol, R^SH.

«

7 8

Compounds of formula (VI) wherein R is -NR R may be prepared by reduction of the corresponding compound wherein R^ i_s azido, e.g. using lithium aluminium hydride in tetrahydrofuran, followed where necessary and/or desired by alkylation of the resulting amino group. The azido compound may be prepared from a compound of formula (VII) wherein X is halogen, by reaction e.g. with sodium azide.

Compounds of formula (VII) may be prepared for example in an analogous manner to process (b) described below.

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JB/JJ/PA0839FF/28th July, 1989.

With regard to process (b) , a suitable donor compound is the corresponding cycloalkane carboxylic acid which may undergo oxidative decarboxylation. For Instance persulphate with a catalyst, such as silver ions, is convenient for the purpose, (c.f.Jacobson, N., et al.. Annalen, 1972, 763.

135 and Acta Chem. Scand, 1973, 22. 3211). Conveniently ammonium persulphate can be used as the oxidising agent, and the catalyst is silver nitrate. Further details of this process are described in EPA 123238. The compound of formula (VIII) used as starting material may be prepared from the corresponding 3-halo compound using methods analogous to process (a).

Hereinafter naphthoquinones active against P.carinil. including compounds more particularly described by formulae (I) to (VI), and their physiologically acceptable salts and other physiologically functional derivatives will be referred to as the naphthoquinone. It will be appreciated that the amount of the naphthoquinone required for use in the treatment or prophylaxis of P.carinii will depend inter alia on the activity of the particular compound, the route of administration, the age and weight of the patient and the severity of the condition being treated. In general, a suitable dose for administration to man for the treatment of P.carinii pneumonia may be in the range of O.lmg to 200mg per kilogram bodyweight per day, for example from lmg/kg to lOOmg/kg, particularly 10 to 50 mg/kg. For administration by inhalation the dose may conveniently be in the range of 0.1 to 20 mg/kg/day, e.g. 0.5 to 10 mg/kg/day. It will be appreciated that for administration to neonates, lower doses may be required.

JB/JJ/PA0839FF/28th July, 1989.

η

For prophylactic treatment the naphthoquinone 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 prophylactic treatment will depend inter alia on the activity of the naphthoquinone, 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 could be in the range 0.05 to 100 mg/kg,e.g. 0.05 to 50 mg/kg particularly 5 to 50 mg/kg.

Suitable dosages of a compound of formula (VI) for the treatment or prophylaxis of malaria in man are also within the ranges given above for the treatment and prophylaxis of P.carinii pneumonia.

For use according to the present invention the naphthoquinone is preferably presented as a pharmaceutical formulation.

Pharmaceutical formulations comprise the naphthoquinone or a physiologically acceptable salt or other physiologically functional derivative 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 naphthoquinone may conveniently be presented as a pharmaceutical formulation in unit dosage form. A convenient unit dose formulation

AP 0 0 0 1 1 1

JB/JJ/PA0839FF/28th July, 1989.

contains the naphthoquinone in an amount of from 10 mg to 3g e.g. 10 mg to

U·

Pharmaceutical formulations include those suitable for oral, topical(including dermal.buccal and sublingual), rectal or parenteral (including subcutaneous, intradermal, intramuscular and intravenous), nasal and pulmonary administration e.g. by inhalation. 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 the naphthoquinone 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 naphthoquinone. 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 naphthoquinone 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 an inert liquid diluent. Tablets may be optionally coated and, if uncoated, may optionally be scored. Capsules may be prepared by filling the naphthoquinone, 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 naphthoquinone

JB/JJ/PA0839FF/28th July, 1989.

together with any accessory ingredient(s) is sealed in a rice paper envelope. The naphthoquinone compound 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.

Formulations for oral administration include controlled release dosage forms e.g. tablets wherein the naphthoquinone is formulated in an appropriate release - controlling matrix, or is 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 naphthoquinone with the softened or melted carrier(s) followed by chilling and shaping in moulds.

ΔΡ0 0 0 1 1 1

Pharmaceutical formulations suitable for parenteral administration include sterile solutions or suspensions of the naphthoquinone 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

JB/JJ/PA0839FF/28th July, 1989.

introduction of the formulation until required for use. Alternatively, the naphthoquinone may be in powder form which is constituted with a suitable vehicle, such as sterile, pyrogen-free water, before use.

The naphthoquinone 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.

Formulations suitable for pulmonary administration via the buccal cavity are presented such that particles containing the naphthoquinone and desirably having a diameter in the range 0.5 to 7 microns are delivered into the bronchial tree of the recipient. Such formulations may be in the form of finely comminuted powders which may conveniently be presented in a pierceable capsule, for example of gelatin, for use in an inhalation device, or as a self-propelling formulation (also referred to as an aerosol «

formulation) comprising the naphthoquinone, a suitable liquid or gaseous propellant and optionally other ingredients such as a surfactant and/or a solid diluent. Suitable liquid propellants include propane and the chlorofluorocarbons, and suitable gaseous propellants include carbon dioxide. Suitable surfactants include sorbitan trioleate (which is available for example under the trade name Arlacel 85), Polysorbate 20 and oleic acid. Self-propelling formulations may also be employed wherein the active ingredient is dispensed in the form of droplets of solution or

JB/JJ/PA0839FF/28th July, 1989.

supension. The self-propelling formulation typically contains from 0.05 to 20mg/ml e.g. 0.1 to 5mg/ml of the active ingredient.

Such self-propelling formulations are analogous to those known in the art and may be prepared by established procedures. Suitably they are presented in a container provided with either a manually-operable or automatically functioning valve having the desired spray characteristics; advantageously the valve is of a metered type delivering a fixed volume, for example, 25 to 100 microlitres, upon each operation thereof.

As a further possibility the naphthoquinone may be in the form of a solution or suspension for use in an atomiser or nebuliser whereby an accelerated airstream or ultrasonic agitation Is employed to produce a fine droplet mist for inhalation. Such solutions or suspensions may comprise, in addition to the naphthoquinone and solvent(s), optional ingredients such as surfactants. Suitable surfactants include those described above for self-propelling formulations. The solution or suspension typically contains from 0.05 to 20mg/ml e.g. 0.1 to 5mg/ml of the naphthoquinone. When a suspension of the naphthoquinone is employed, this compound is preferably in finely divided form, e.g. in micronised form.

Formulations suitable for nasal administration include presentations generally similar to those described above for pulmonary administration. When dispensed such formulations should desirably have a particle diameter in the range 10 to 200 microns to enable retention in the nasal cavity; this may be achieved by, as appropriate, use of a powder of a suitable particle size or choice of an appropriate valve. Other suitable

AP 0 0 0 1 1 1

JB/JJ/PA0839FF/28th July, 1989.

formulations include coarse powders having a particle diameter in the range 20 to 500 microns, for administration by rapid inhalation through the nasal passage from a container held close up to the nose, and nasal drops comprising 0.2 to 5% w/v of the naphthoquinone in aqueous or oily solution or suspension.

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.

None of the references listed hereinabove contains an invitation to administer the compounds of formula (II) by the nasal or pulmonary route or any suggestion that the said compounds, if administered in such a manner, would be effective in the treatment of the conditions therein taught; the said disclosures likewise contain no description of any formulation suitable for administration by the nasal or pulmonary route.

Pharmaceutical formulations of the compounds of formula (II) adapted for administration by the nasal or pulmonary route are believed to represent novel formulations and thus form a further feature of the present invention.

JB/JJ/PA0839FF/28th July, 1989.

Novel compounds of formula (VI) may also be formulated In the manner described above for use In the treatment and/or prophylaxis of malaria and such formulations form a further aspect of the present invention.

The above naphthoquinones may also be used in accordance with the present invention in combination or concurrently with other therapeutic agents, for example agents used in the treatment of immunocompromised patients, including anticancer agents such as interferons e.g. alpha-interferons; antiviral agents such as azidothymidine (AZT,zidovudine), immunostimulants and immunodulators. The naphthoquinone may also be administered in combination with a 4-pyridinol compound, as described in EPA 123,239 e.g.

3,5-dichloro-2,6-dimethylpyridinol (meticlorpindol).

The following non-limiting examples illustrate inter alia the following aspects of the present invention:

the use of naphthoquinones in the treatment and prophylaxis of P.CaylhU infections;

novel pharmaceutical formulations;

novel compounds of formula (VI).

I I I 0 0 0 dV

JB/JJ/PA0839FF/28th July, 1989.

Example 1 - Preparation of compound (V)

2- ί trans-4- (4-Chlorophenvl)cvclohexyl1 -3-hydroxy-l ,4-naphthoquinone

a) 4- (4-ChlQrophenvl)cvclohexane-1-carboxyllc Acid

Acetyl chloride (30g) and finely powdered aluminium chloride (60g) were stirred together In carbon disulphide (120 ml) and then cooled to -50°C, in a CX^/oxitol bath. Cyclohexene (30 g), previously cooled to -50°C, was added dropwise during 10 minutes while maintaining the temperature of the reaction mixture at below -20°C. The mixture was stirred at -50°C for a further 60 minutes and the solvent then decanted to leave a gummy orange complex. A little chlorobenzene was added as the material warmed to ambient temperature; the remainder of the chlorobenzene (total 300 ml) was then added, the so-obtained solution heated at 40°C for 3 hours with stirring, poured onto a mixture of ice and concentrated hydrochloric acid and the organic layer separated, washed with 2M hydrochloric acid, 2M sodium hydroxide «

and water, dried over anhydrous sodium sulphate and evaporated to dryness. The product was distilled in vacuo. the fraction boiling at 140-154°C (0.1 mm Hg) collected, diluted with an equal volume of petroleum ether (40-60), cooled to -6°C and a continuous stream of nitrogen gas bubbled through, and the separated colourless solid recovered.

Bromine (2.8ml) was added to a solution of sodium hydroxide (6.2g) in water (42 ml) at 0°C. The above-obtained substituted

JB/JJ/PA0839FF/28th July, 1989.

hexahydroacetophenone (3.1g) was dissolved in dioxan (15 ml) and the cold hypobromite solution then added, keeping the reaction mixture at below 20°C. The reaction mixture was stirred at ambient temperature for 6 hours then allowed to stand overnight. Sodium metabisulphite was added to destroy excess hypobromite, the mixture cooled and then acidified to give a colourless solid. The solid was filtered off, washed with water, dried and recrystallised from ethanol to give

4-(4-chlorophenyl)cyclohexane-1-carboxylic acid, m.p. 254-256°C.

b) 2-(4-(4-chlorophenvl) c vclohexvl1 - 3-chloro-1.4-naphthoquinone

A mixture of 2-chloro-l,4-naphthoquinone (3.95g, 0.02 mol),

4-(4-chlorophenyl)cyclohexane-l-carboxylic acid (4.9g, 0.02 mol) and powdered silver nitrate (1.05g, 0.0062 mol) was heated to reflux with vigorous stirring in 40 ml of acetonitrile. A solution of ammonium persulphate (12.Og, 0,0525 mol) in 50 ml of water was added dropwise over 1 hour. The mixture was refluxed for 3 hours then cooled in ice for 30 mins, after which it was filtered, and the residual sticky solid extracted twice with boiling chloroform to remove inorganic material. The chloroform was removed by evaporation to leave a yellow-brown solid (ca 2.7g). This was dissolved in 40 ml of boiling acetonitrile; a little insoluble material was removed by filtration. On cooling, the title compound separated as yellow crystals, (550 mg) m.p. 172-175°C.

AP 0 0 0 1 1 1

JB/JJ/PA0839FF/28th July, 1989.

mult.,

NMR, 6H(d₆-DMSO) 8.05 (2H, mult., 0-naphth), 7.85(2H, o-naphth), 7.30 (4H, s., PhH), 3.30 (1H, br.t., CH), 2.67 (1H, br.t., CH), 1.2-2.4 (8H, mult., 4xCH₂).

c) 2-f4-(4-chlorophenyl)cyclohexyl1-3-hydroxy-l.4-naphthoquinone

The product of stage (b) was suspended In 10 ml of boiling methanol and 0.55g of potassium hydroxide in 5.5 ml of water was added dropwise over 15 mins. The mixture was refluxed until a dark red solution formed, (after ca. 6 hrs) when 2 ml of concentrated hydrochloric acid was cautiously added dropwise. The mixture was cooled and filtered, and the solid residue washed thoroughly with water. The water washings were re-acidified and filtered. The combined solid residues (500 mg) mp 200-209°, were recrystallised from acetonitrile to give the title product as the trans-Isomer (300 mg) m.p. 216-219°C.

Example 2 e

-Me thoxy-3 -[trans-4-(4-chlorophenyl)eye1ohexy1]-1,4-naphthoquinone

Sodium (0.3g, 0.013mol) was dissolved in 20ml of methanol and the compound of Example 1(b) (1.5g, 0.004mol) was added. The mixture was warmed to reflux for 4 hours, then evaporated under reduced pressure. The dark red solid which remained was partitioned between water and chloroform. The chloroform layer was washed with ice cold dilute sodium hydroxide, folltwed by water and was then dried and evaporated to give a yellow solid (900mg). This was recrystallised from acetonitrile to give the impure product

JB/JJ/PAO839FF/lst August, 1989.

- 27 (800mg) mpll7-120°, which was further recrystallised from ethanol to give the title compound (600mg) mpl2O-122°.

NMR, 6H (d,-DMSO) 8.0 (2H, mult., 0-naphth), 7.85 (2H, mult, a-naphth) 7.35 o (4H, s, PhH), 4.0 (3H, s, OMe), 3.1 (1H, br.t., CH), 2.6 (1H, br.t., CH),

1.5-2.2 (8H, m, 4xCH₂).

Example,J

2-Amino-3- f trans-4- (4-chlorophenyl)cyclohexyl) -1,4-naphthoquinone

a) 2-azIdo-3-ί trans - 4-(4-chlorophenyl)cyclohexyl)-1,4-naphthoquinone

A solution of sodium azide (0.42g, 0.006mol) in 6ml of water was added to a suspension of the compound of Example 1(b) (l.lg, 0.003mol) in 15ml of ethanol. The mixture was heated to reflux with stirring and then a further 15ml of ethanol and 6ml of water were added. Heating under reflux was continued for 4 hours followed by cooling in a «

refrigerator for 1 hour. The resulting yellow crystals were filtered off and washed with water and ethanol to give the impure title compound (0.9g) mpl30-135°. This material was used in the next stage without further purification.

b) 2-Amino-3-f trans-4-(4-chlorophenyl)cyclohexyl]-1,4-naphthoquinone

The impure product of stage (a) (0.9g) was dissolved in dry tetrahydrofuran (THF) and added dropwise to a suspension of lithium

AP ο ηn 1 1 1

JB/JJ/PA0839FF/lst August, 1989.

aluminium hydride (2.0g) in THF. The mixture was stirred at room temperature for 1 hour and then 2.0ml of water was added dropwise with caution. A current of air was passed through the mixture for 1 hour and then 0.7g of sodium hydroxide in 6ml of water was added. The mixture was filtered and washed with THF. The filtrate was evaporated to dryness leaving an amorphous orange material which was triturated with SVM whereupon orange crystals formed. These were filtered off, washed well with SVM and dried to yield a first crop of product (200mg) mp210-215°. The reaction was repeated to give a further crop of product (300mg) mp2OO-21O°. The two crops were combined and chromatographed, eluting with chloroform, to give the title compound (350mg) mp212-215°.

NMR,	6H (dg-DMSO)	8.0	(2H,	mult., naphth) 7.8	(2H, mult.,	naphth),
7.35	(4H, q, PhH),	6.7	(2H,	s, NH₂), 2.6-3.0 (2H,	mult., CH),	1.5-2.4
(8H,	mult., CH₂).

Example 4

2-f trans-4-(4-Chlorophenyl)cyclohexyl]-3-(3-dimethylaminopropoxy)-1,4naphthoquinone hydrochloride

Sodium (0.10g,4.5mmol) was dissolved in 3-dimethylamino-propan-l-ol (1.55g, 5eq) and cooled and the compound of Example 1(b) (1.15g, 3ramol) was added. The mixture was stirred at room temperature for 1 hour, following which acetic acid (1ml) was added and the mixture diluted with toluene

JB/JJ/PA0839FF/lst August, 1989.

(30ml). The solution was washed with water (4 x 20ml) dried (MgSO^) and evaporated in vacuo to a purple semi solid. This was taken up in acetone and a mixture of diethyl ether and hydrochloric acid added until the purple solution turned orange. The solution was evaporated in vacuo to dryness and washed several times with toluene to give a yellow/beige solid, which was recrystallised from methanol/toluene (1:99) to give the title compound as a yellow/green crystalline solid (0.92g) mpl91-194°.

NMR, 6H (CDC1₃) 8.06 (2H, mult., 0-naphth), 7.74 (2H, mult., a-naphth),

7.25 (4H, mult., PhH), 4.28 (2H, t., OCH₂ ), 3.48 (2H, t., NCH₂ ), 2.95 (6H, s., 2xMe), 3.17 (1H, br.t., CH), 2.67 (1H, br.t., CH), 2.54 (2H, mult., CH₂) 1.4-2.3 <8H, mult., 4xCH₂).

Example 5

2-Acetoxy-3-f trans-4-(4-chlorophenyl)cyclohexyl]-1,4-naphthoquinone

The compound of Example 1(c) (1.5g, 0.004mol) was suspended in acetic anhydride (3ml) and a few drops of concentrated sulphuric acid were added with vigorous stirring. A further quantity of acetic anhydride (3ml) was then added, the mixture stirred for 30 minutes and then poured into 15ml of water causing a vigorous reaction. The resulting mixture was cooled in ice and filtered to give pale yellow crystals which were washed with water and dried to give the impure product (1.6g) mpl49-152°. This was recrystallised from 100ml of SVM to give the title compound (1.3g) mp!58-160°.

C

c.

JB/JJ/PA0839FF/lst August, 1989.

NMR, 6H (dg-DMSO) 8.1 (2H, m, 0-naphth), 7.9 (2H, a, a-naphth), 7.35 (4H, s, PhH), 3.1 (1H, br.t., CH), 2.6 (1H, br.t., CH), 2.5 (3H, s, COHe),

1.5-2.0 (8H, in, CH₂).

Example 6

2-Ethoxycarbonyloxy-3-f trans-4-(4-chlorophenyl)cyclohexyl]-1,4 naphthoquinone

The compound of Example 1(c) (l.lg, 0.003mol) and pyridine (0.24g,

0.003mol) was stirred in 5ml of toluene and cooled In a water bath while

4ml of ethyl chloroformate was added dropwise over a period of 15 minutes. The mixture was stirred for a further 30 minutes then poured into a mixture of ethyl acetate and water. The organic layer was separated, dried and evaporated to a yellow solid which was recrystallised from chloroform/petrol to give the impure product (850mg) mpl45-149°. This material was dissolved in chloroform, washed several times with ice cold 0.1N sodium hydroxide and then water. The organic layer was dried and evaporated to give product (450mg) mpl47-9 . The reaction was repeated on the same scale, the product combined with the aforementioned material and then recrystallised from chloroform/petrol to give the title compound (1.3g) mpl53-155°.

NMR, 6H (dg-DMSO) 8.1 (2H, m, 0-naphth), 7.9 (2H, m, a-naphth), 7.4 (4H, s, PhH), 4.3 (2H, q, OCHp, 3.1 (1H, br.t., CH), 2.6 (1H, br.t,, CH) , 1.5-2.0 (8H, m, CH₂), 1.4 (3H, t, Me).

JB/JJ/PA0839FF/lst August, 1989.

Example 7

2- ίtrans-4-(4-Chlorophenyl)cyclohexyl]-3-(4-dimethylaminobenzoyloxy)1,4-naphthoquinone

The compound of Example 1(c) (2.0g, 5.4mmol) in dry toluene (50ml) containing dry pyridine (0.44g, leq) was stirred at around room temperature. 4-Dimethylaminobenzoyl chloride (lg, leq) in dry toluene (25ml) was added dropwise over 15 minutes. The mixture was stirred at around room temperature for 1 hour, heated at reflux for 10 hours, left standing for 38 hours, and then refluxed for a further 7 hours. The mixture was then cooled, washed with water, sodium bicarbonate solution and again with water, dried (MgSO^) and evaporated in vacuo to a yellow solid which was recrystallised from ethanol to give the title compound (1 mpll7-121° (shrinks above 113°).

NMR, 5H (CDCip 8.1, 6.75 (4H, mult., 0-naphth + o-Me₂N-Ph), 7.72 mult., a-naphth), 7.18 (4H, mult., PhH), 6.72 (2H, d, 0-Me₂N-Ph), 3.18 br.t., CH), 3.14 (6H, s., 2xMe), 2.52 (1H, br.t., CH), 1.4-2.2 (8H, mult., 4xCH₂).

Example 8

2- f trans-4-(4-Chlorophenyl)cyclohexyl)-1,3,4-triacetoxynaphthalene

The compound of Example 1(c) (l.Og) and zinc dust (l.Og) was stirred at room temperature for 24 hours in acetic anhydride (6ml) with one drop TEA.

• 25g) (2H, (1H,

C c

c.

a

JB/JJ/PA0839FF/lst August, 1989.

The reaction mixture was filtered and added to water (50ml) and stirred for one hour. The resulting white precipitate was filtered, washed with water (4 x 20ml) and dried to give the title compound (0.4g) mpl77-179°.

NMR, 6H (d,-DMSO) 7.87 (2H, mult., 0-naphth), 7.63 (2H, mult., α-naphth), o

7.35 (4H, s., Ph.H), 2.95 (1H, br.t., CH), 2.64 (1H, br.t., CH) , 2.62 (3H,

s., OAc), 2.49 (3H, s., OAc), 1.4-2.3 (8H, mult., 4xCH₂).

Example 9

The following examples illustrate conventional pharmaceutical formulations which may be employed in accordance with the present invention:A. Injectable solution

A solution for intramuscular

Compound of Example 1 Dimethyl sulphoxide

Sorbitan monooleate

Corn oil injection may be prepared by mixing:9.5 parts by weight «

19.0 parts by weight

4.5 parts by weight 67.0 parts by weight

100.0

JB/JJ/PA0839FF/lst August, 1989.

B. Injectable solution

Compound of Example 1	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 1	25.0	®g
Lactose BP	48.5	“g
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

a «3

JB/JJ/PA0839FF/lst August, 1989.

D. Oral suspension

Compound of Example 1	50	mg
Avtcel RC 591	75	mg
Sucrose syrup	3.5	ml
Me thylhydroxybenzoate	5	mg
Colour	0.01% w/v
Cherry flavour	0.1	% v/v
Tween 80	0.2	% v/v
Water	to 5	ml

E. Injectable suspension

Compound of Example 1	100 mg
Polyvinyl pyrrolidone (PVP)	170 mg
Tween 80	0.2% v/v
Methylhydroxybenzoate	0.1% w/v
Water for Injection	to 3 ml

F. Capsule

Compound of Example 1

Starch 1500

Magnesium stearate filled into a hard gelatin capsule

100 mg 150 mg

2.5 mg

JB/JJ/PA0839FF/lst August, 1989.

Example 10.

The following examples illustrate novel pharmaceutical formulations according to the present invention.

A. Suspensions for Nebulisation

a) Compound of Example 1, sterile Water for Injections

1.0 mg to 10.0 ml

Disperse the naphthoquinone in the Water for Injections previously sterilised in a sterile container. Fill in to sterile glass ampoules, 10 ml/ampoule under aseptic conditions, and seal each ampoule by fusion of the glass.

b) The following suspension was prepared:

Αρη η n111

Compound of Example 1, micronised Polysorbate 20

Water for Injections to

l.Og

0.1% w/v ml

The Polysorbate 20 was dispersed in the water for injections, followed by the compound of Example 1. This suspension was filled into sterile glass ampoules, lOml/ampoule under aspetie conditions, and the ampoules sealed by fusion of the glass.

JB/JJ/PA0839FF/lst August, 1989.

B. Aerosol Formulations

a) Compound of Example 1, micronlsed

Aerosol propellant to

1.0 mg

5.0 ml

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

b) Compound of Example 1, micronised

Arlacel 85

Aerosol propellant to

1.0 mg 0.1% w/v ml

Disperse the Arlacel 85 in the aerosol propellant and then add compound of Example 1. Fill the suspension into preformed aerosol cannisters, 5ml/cannister under pressure, through the valve orifice.

C. Powder Inhalation

Compound of Example 1, micronised 1.0 mg

Lactose 29.0 mg

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

JB/JJ/PA0839FF/lst August, 1989.

D. Nasal Drops

Compound of Example 1 100.0 mg Methylhydroxybenzoate 10.0 mg Water for Injections to 10.0 ml

Disperse the naphthoquinone and the methylhydroxybenzoate in the Water for Injections. Fill this suspension into suitable dropper bottles, 10 ml/bottle, and close by securing the dropper nozzle and bottle cap.

AP 0 0 0 1 1 1

JB/JJ/PA0839FF/lst August, 1989.

BIOLOGICAL TEST RESULTS.

Example 11

Activity against Pneumocystis carinii

Test Compounds

A: 2-[trans-4-(4-chlorophenyl)cyclohexyl]- 3-hydroxy-1,4-naphthoquinone

B: 2-Γ cis-4-(4-chlorophenyl)cyclohexyl]- 3-hydroxy-1,4-naphthoquinone

C: 2-[4-(4-chlorophenyl)cyclohexyl]- 3-chloro-1,4-naphthoquinone

D: 2-(4-£-butylcyclohexyl)-3-hydroxy-l,4-naphthoquinone

E: 2-(4-X-butylcyclohexylmethyl)-3-hydroxy-1,4-naphthoquinone

a) Prophylaxis

Groups of 10 rats were treated with dexamethasone to allow latent Pneumocystis carinii infection to develop. Tetracycline was also administered to protect against bacterial infections. Test compound A was administered, by gavage, from day 4 of the dexamethasone treatment, at a dose of 100 mg/kg/day. Two control groups cf rats were treated with dexamethasone and tetracycline only. A further group of rats was given cotrimoxazole (trimethoprim +

JB/JJ/PA0839FF/lst August, 1989.

sulphamethoxazole, 50 + 250 mg/kg/day, orally) in place of the test compound.

At the end of the test period the animals were sacrificed and autopsies carried out. The lungs were removed and the right lung bisected. An imprint was made onto microscope slides and stained with toluidine blue. One half of the lung was placed in formalin, processed in paraffin blocks, sectioned and stained by the Gomori methanamine silver nitrate method.

After autopsy the extent of P.carlnil pneumonitis was scored under coded study as none if no organism seen; 1+ if P.carlnil cysts seen sparsely distributed with less than one per 25 high power field (h.p.f.); 2+ if focal areas of P.carlnil pneumonitis surrounded by 10 to 25 h.p.f. of normal lung and 3+ if lung diffusely and extensively involved with organisms in almost all h.p.f.s.

AP 0 0 0 1 1 1

JB/JJ/PA0839FF/lst August, 1989.

Results

No of	Early	No of with	No of rats	with
Rats	deaths	P.carinii	PCP/
	or	Pneumonitis	No of rats	tested
	cannibal-	None 1+ 2+ 3+
	isatlon

Test Compound

A		10	2*	8	0	0	0	0/8
Control	(1)	10	2	0	1	2	5	8/8
Control	(2)	10	0	0	0	2	8	10/10
TMP/SMZ		10	0	«				0/10

* one early death, believed due to gavage, one cannlbalisation.

JB/JJ/PAO839FF/lst August, 1989.

b) Prophylaxis

A further series of tests was carried out using the same general method as described above. Test compound A was administered at various dose levels, by gavage and in the diet.

The results are shown in Table 2.

δρο η n111

JB/JJ/PA0839FF/lst August, 1989.

Extent of P, carinii after prophylaxis: histopathology of lung sections (Gomori-Grocott stain)

No.of Rats No.with P. carinii Pneumonitis

JB/JJ/PA0839FF/lst August, 1989.

c) Treatment

Groups of 10 rats were treated with dexamethasone and tetracycline for 4-6 weeks, as described in experiment (a) above. Three groups of rats were treated with Test compound A beginning after 4 weeks of immunosuppression, when Pneumocystis carinii pneumonia (PCP) had developed. Another group of rats in a parallel study was treated with Test compound A after 6 weeks of immunosuppression, when PCP infection was at an advanced stage. The results are shown in Table 3.

&pn η n111

JB/JJ/PA0839FF/lst August, 1989.

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JB/JJ/PA0839FF/lst August, 1989.

d) Treatment

Groups of 15 rats were treated with dexamethasone and tetracycline for 4 weeks, as described in experiment (a) above. Test compounds (A) (E) were administered orally by stomach tube from the beginning of week 5 to the end of week 7.

In parallel with each test group of rats as a control compound, Celacol was administered to one The results are given in Table 4.

JB/JJ/PA0839FF/lst August, 1989

- 47 TABLE 4

SCORE NO. INFECTED/ %

Test GROUP 01234 NO. EXAMINED INFECTED

Compound (Dose/kg/day)

Celacol	1	0	1	2	6	9/10	90
50mg/kg	1	3	3	5	0	11/12	92
75mg/kg	2	5	2	1	2	10/12	83
lOOmg/kg	4	7	1	1	0	9/13	69

Celacol	0	8 7	0	12/15	100
25mg/kg	3	7 4	1	12/15	80
50mg/kg	1	6- 4	2	12/13	92
lOOmg/kg	4	6 2	0	8/12	67

IlUUUdV

Celacol	0	8	7	0	0	15/15	100
25mg/kg	1	8	5	1	0	14/15	93
50mg/kg	3	6	4	0	2	12/15	80
lOOmg/kg	2	6	2	4	0	12/14	86

JB/JJ/PA0839FF/lst August, 1989.

Celacol	0	8	7	0	15/15	100
25mg/kg	0	5	6	3	14/14	100
50mg/kg	0	8	5	2	15/15	100
lOOmg/kg	4	6	4	1	11/15	73

Celacol	0	8	2	3	2	15/15	100
25mg/kg	0	7	7	1	0	15/15	100
50mg/kg	1	8	4	1	0	13/14	93
lOOmg/kg	3	8	3	0	1	12/15	80

	Celacol	0	8·	2	3	2	15/15	100
E	25mg/kg	1	3	2	4	4	13/14	93
	50mg/kg	0	2	6	4	2	14/14	100
	lOOmg/kg	1	4	2	6	0	12/13	92

JB/JJ/PA0839FF/lst August, 1989.

- 49 Example 12

Antlmalarial Activity of Compounds (VI)

Test methods

Activity against Plasmodium Falciparum in vitro

The test method was modification of that described by Desjardins et al. .

Antimlcrob. Agents and Chemotherapy, 1979, 16. 710-718. Compounds were .3 dissolved in ethanol at a concentration of 4.8x10 M and dilutions down to -4

1x10 M were made. The drug solutions were serially diluted using RPMI

1640 medium + 10% human plasma in microtitration plates. Parasitised and 3 fresh red blood cells were added, together with G- H-hypoxanthine, in RPMI 1640 medium + 10% human plasma and the cultures Incubated for 48 hours.

Cultures were then harvested, the particulate contents collected on a glass fibre filter paper and washed copiously with water. The filter papers were dried and the radioactivity measured using a scintillation counter. Infected untreated and uninfected untreated cultures were included as controls .

The results are shown in Table 5.

I I IUU UdV

Activity against Plasmodium yoelil in vivo

JB/JJ/PAO839FF/lst August, 1989.

The naphthoquinone was suspended in 0.25% (w/v) celacol in water by milling for 16-24 hours at 26°C. The suspensions were subsequently serially diluted with 0.25% (w/v) celacol in water.

At time 0, 0.1 ml of a suspension of 5x10^ P.yoelii-parasitised red blood cells/ml of phosphate saline were injected intravenously into 15-20 g mice through a tail vein. Groups of 5 mice per treatment were dosed orally at times 6, 22, 30, 46, 54, 70 and 78 hours with 0.2 ml of the drug suspension. The compound of Example 4 was also administered intravenously. Tail-blood smears were taken at 96 hours, stained with Giemsa and the percentage of red blood cells infected determined and compared to untreated, infected controls. Percent inhibition was correllated with dose to provide Εϋ^θ values. The results are shown in Table 5 below.

JB/JJ/PA0839FF/lst August, 1989.

TABLE 5	Antimalarial activity in vitro and in vivo
Compound of	In vitro In vivo
Example No:	^ic50OiM) ED₅₀ mg/kg fi£Al l.v.
1C	0.002 0.03
2	0.36 5.8
3	1.14 1.26
4	0.059 0.61 2.08 0.68
5	0.068 0.12
6	0.080 0.09
7	0.22 0.12

AP 0 0 0 1 1 1

Claims

1. The use of a naphthoquinone for the manufacture of a medicament for the treatment or prophylaxis of Pneumocystis carinii infections in mammals .

2 -[4 -(4-chlorophenyl)cyclohexyl]- 3-chloro -1,4-naphthoquinone and physiologically acceptable salts and physiologically functional derivatives thereof.

2 -(4-£-butylcyclohexylmethyl)- 3-hydroxy-1,4-naphthoquinone apo η n11 1

JB/JJ/PA0839CL/28th July, 1989.

PA0839CL

2. Use according to claim 1 wherein the naphthoquinone is a compound of general formula (II) (II) wherein

R is ₃₃ non-aromatic hydrocarbon residue optionally substituted by one or more substituents, selected from halo, ^alkoxy, hydroxy, phenyl, phenyl-C^ ^alkoxy and phenyl-C^ _galkyl, each such phenyl group or moiety being optionally substituted by one or more groups selected from ^alkoxy, _galkyl, _galkoxy-C^ _galkyl, hydroxy, halogen, halo-C^ _galkyl, amino, and mono-or di-C^ ^alkyl-amino; and • j --5'

JB/JJ/PAO839CL/28th July, 1989

PAO839CL

S3

R is hydroxy; halogen;

a group OCOR³, wherein R³ is a ^alkyl group, a cycloalkyl group, a ^alkoxy group, or a phenyl or naphthyl group, each such R³ group being optionally substituted by amino, mono-or di-C^ ^alkylamino, carboxy or hydroxy;

a group OR^ or Sr\ wherein R^ is an optionally substituted C^^alkyl, Cj^cycloalkyl, phenyl or naphthyl group as defined for R³; or

7 8 7 8 a group NR R , wherein R and R each independently represent 7 8 hydrogen or ^alkyl, or the group NR R represents a 5-7 membered saturated heterocyclic ring, which may optionally contain a further heteroatom selected from nitrogen, oxygen or sulphur;

3. Use according to claim 1 or claim 2 wherein the naphthoquinone is selected from:2-(4-t-butylcyclohexyl)-3-hydroxy-1,4-naphthoquinone

4. Use of 2-[4-(4-chlorophenyl)cyclohexyl]-3-hydroxy-l,4-naphthoquinone or a physiologically acceptable salt or other physiologically functional derivative thereof for the manufacture of a medicament for the treatment or prophylaxis of Pneumocystis carinil infections in mammals .

5. Use according to claim 4 wherein the compound is in the form of the trans isomer or a mixture of cis and trans isomers containing at least 95% of the trans isomer.

G. Use of 2-rtrans-4-(4-chloroDhenyl)cvclohexy1]-3-hydroxy-l,4naphthoqulnone for the manufacture of a medicament for the treatment or prophylaxis of Pneumocystis carlnil Infections In mammals.

A naphthoquinone as defined in any of claims 1 to 5 for use in the treatment or prophylaxis of Pneumocystis carlnil infections in mammals.

7 A method of treating or preventing Pneumocystis carinil infections which comprises administering to a mammal susceptible to infection with P. carinil an effective amount of a naphthoquinone as defined in any of claims 1 to 5.

JB/JJ/PA0839CL/28th July, 1989.

PAO839CL

8-.

Novel compounds of formula (VI)

11 12

R and R each represent -0 and the dotted line represents a double bond between the 2 and 3 positions of the quinone ring, in which case

R^ represents a group -OCOR^; a group OR^ or SR^; or a group NR^R®,

5 6 7 8 wherein R , R , R and R are as hereinbefore defined; or the dotted line represents double bonds at the 1,2 and 3,4 positions of the quinol ring and R^^, R^ and R^ each represents a group -OCOR^^, wherein R*⁴ represents a Cj.jq alkyl group optionally substituted as hereinbefore defined for R .

c c

c

O

A pharmaceutical formulation which comprises a naphthoquinone as defined in any of claims 1 to 5 together with a pharmaceutically acceptable carrier therefor, adapted for nasal administration.

A pharmaceutical fonrulation which comprises a naphthoquinone as defined in any of claims 1 to 5 together with a pharmaceutically acceptable carrier therefor, adapted for pulmonary administration.

JB/JJ/PA0839CL/28th July, 1989.

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I I . A pharmaceutical formulation which comprises a compound of formula (VI) in association with a pharmaceutically acceptable carrier therefor .

J .

yl. A process for preparing a compound of formula (VI) as defined in claim 8 which comprises:

wherein X is a halogen atom, with a compound serving to introduce 13 the required group R , and if necessary or desired the _DH u η^χ2 groups R and R ; or

JB/JJ/PAO839CL/28th July, 1989.

&7

PA0839CL (b) reaction of a compound of formula (VIII) (VIII) with a donor compound serving to 4-(4-chlorophenyl)cyclohexyl group.

introduce the