WO2000050404A1 - 4-aminoquinolines as antimalarials - Google Patents

Abstract

The present invention relates to 4-aminoquinolines and derivatives thereof. More particularly the present invention relates to 4-aminoquinolines and derivatives thereof for use in the treatment and prophylaxes of malaria. The compounds are compounds of Formula (IV) where: W is C1 or CF3; X is N or N=O; R is selected from: i) R2-NH-R1-Z-where: Z is NH, S, O or CH2; R1 is a C1 to C4 alkyl group; and R2-NH- is a group resistant to dealkylation in vivo; or ii) (a) where: Z and R1 are as described in (i) above, and (b) is a group resistant to dealkylation in vivo; or, iii) R4-Ar-Z- where: Z is as described in (i) above; Ar is a substituted or unsubstitued aromatic ring structure; and R4 is a planar or non planar, alkyl or cycloalkyl substituent on the 5' position of the aromatic ring Ar.

Description

4-AMINOQUINOLINES AS ANTIMALARIALS

The present invention relates to 4-aminoquinolines and derivatives

thereof. More particularly the present invention relates to 4-aminoquinolines

and derivatives thereof for use in the treatment and prophylaxes of malaria.

4-aminoquinolines have the basic structure identified below:

Formula 1

A large number of chemical entities derived from this structure are

known and many have been considered as antimalarials.

The most well known 4-aminoquinolines are cMoroquinine, 7-chloro-4-

(4-diethyl amino-1-methylbutylamino) quinoline, which has the structure

identified below:

Formula π

and amodiaquinine, 7-chloro-4-(3'diethylaminomethyl-4'- hydroxyanilino)

quinoline, which has the structure identified below:

Formula III

Amodiaquine differs chemically from chloroquine in that it contains a 4- hydroxy anilino function in its side chain. Both amodiaquine and chloroquine do however have four carbon atoms between the secondary and tertiary nitrogen atoms. The presence of a basic side chain (dialkylamino) has previously been considered an essential element in giving both chloroquine and amodiaquine their antimalarial activity.

Despite extensive research over the last 30 years, Pharmacol Ther. Vol

77, No. 1 pp 29-58, 1998, few effective 4-aminoquinolines for use in the

treatment and prophylaxes of malaria have been discovered.

The applicants have developed some new 4-aminoquinolines which

appear from initial studies to present particularly good efficacy against

chloroquine sensitive and resistant strains of Plasmodium falcipaπwi. These 4- aminoquinolines have the general structure identified below:

Formula IV

where:

W is Cl or CF₃;

X is N or N=0;

R is selected from: i) R₂ - NH - R, - Z - where:

Z is NH, S, O or CH₂;

R_t is a Cj to C₄ alkyl group; such as, for example, methyl, ethyl, propyl or butyl, most preferably methyl or ethyl; and

R₂ -NH- is a group resistant to dealkylation in vivo (by, for example, P450

oxidation); such as, for example, a secondary amine such as, for example, a

tert-butyl amino group; or

where:

Z and R, are as described in (i) above, and

R₃ N -

is a group resistant to dealkylation in vivo; such as, for example, a cyclic

amine, such as, for example those given below:

Formula V

CH

Formula VI

and

Formula VII

or,

where:

Z is as described in (i) above;

Ar is a substituted or unsubstitited aromatic ring structure; such as, for

example, a benzyl ring; and

R-j is a planar or non planar, alkyl or cycloalkyl substituent on the 5' position of

the aromatic ring Ar; such as, for example, a C,-C₄ alkyl group, a secondary

alkyl group, a tertiary alkyl group or a cycloalkyl group, such as, for example, a methyl, ethyl, propyl, iso-propyl, s-butyl, t-butyl or cyclohexyl group.

Preferably the aromatic ring is a six membered ring and includes substituents, most preferably a hydroxyl group, preferably in the 4' position, and a group which is less susceptible to metabolism than a diethyl amino group, preferably in the 3' position, such as, for example, a 3' tert nitrogen group, such as, for example, a N-tert butyl amino methyl group.

Preferred R groups of the formula (iii) R₄ - Ar - Z - are exemplified by the general formula shown below: Formula Vffl

where: Z is NH or a group less susceptible to undergo oxidation to a quinone-imine

derivative, such as, for example, S, 0, or CH₂;

« is a lipid solubilizing group, for example, a planar alkyl group, such as, for

example, methyl, ethyl or propyl, or a non planar group, for example, t-butyl,

isopropyl, s-butyl or cyclohexyl; and

Y is a group less susceptible to metabolism than a 3' diethyl amino group, such

as, for example, a N-tert-alkyl amino alkyl group, such as, for example, that given below:

Formula IX

According to a first aspect of the present invention there is provided a compound of Formula IV

where:

W is Cl or CF₃;

X is N or N=O; R is selected from:

i) R₂ - NH - R, - Z -

where:

Z is NH, S, O or CH₂;

R, is a C, to C₄ alkyl group; and

R₂ -NH- is a group resistant to dealkylation in vivo; or

ii) R₃ N-R,-Z- where:

Z and R, are as described in (i) above, and

is a group resistant to dealkylation in vivo; or, iii) R₄-Ar-Z- where:

Z is as described in (i) above;

Ar is a substituted or unsubstitited aromatic ring structure; and

R₄ is a planar or non planar, alkyl or cycloalkyl substituent on the 5' position of the aromatic ring Ar.

In one embodiment the R group is:

(i) R₂ - NH - Rj - Z - in which:

Z is NH;

Rj is a Ci - C₄ alkyl group; and R₂ is a secondary amine.

In another embodiment the R group is:

(ii) Rf - R, - Z - in which:

Z is NH;

R, is a C, - C₄ alkyl group; and

R N - is a cyclic amine.

In another embodiment the R group is: Formula VIII

in which:

Z is NH;

R₄ is a planar or non planar alkyl group; and Y is a N - tert alkyl alkyl amino group.

According to a second aspect of the present invention there is provided a

pharmaceutical preparation comprising, as an active ingredient, a compound of the invention.

According to a third aspect of the present invention there is provided a compound of the invention, or a pharmaceutically acceptable salt thereof, for

use in the manufacture of a medicament.

According to a fourth aspect of the present invention there is provided a

compound of the invention, or a pharmaceutically acceptable salt thereof for use

in the manufacture of a medicament for use in the treatment or prophylaxes of

malaria.

According to a fifth aspect of the present invention there is provided a

compound of the invention, or a pharmaceutically acceptable salt thereof for use in the manufacture of a medicament for use in the treatment or prophylaxes of chloroquine resistant strains of malaria.

In one embodiment the preferred compounds of the invention are based on chloroquine and have the general formula identified in Formula IV above:

These include compounds of the formulae identified below: Formula X

Formula XI

Formula XU

Formula Xffl

Formula XIN

and Formula XXIII

In another embodiment the preferred compounds of the invention are based on

amodiaquine and have the general formula identified below:

Formula XV

where W, X, Y, Z and R₄ are as previously defined

These include compounds of the formulae identified below:

Formula XVI

For ula XVH

Formula XVTII

Formula XTX

Formula XX

Formula XXI

Formula XXH

The invention will now be described, by way of example only with

reference to the following examples and test data.

Chloroquine based derivation

The first series of compounds investigated contained the structural

entities of chloroquine known to be effective against the parasite; that is, the basic quinoline ring and a substiment for example Cl or CF₃ in the 7-position. This series also excluded one or more entities thought to confer drug resistance (for example, the metabolisable diethylamino sidechain), in an attempt to produce a series of antimalarials which may circumvent the parasite's mechanism of resistance. The synthetic route for producing these compounds was straightforward containing 1-2 steps. A 4,7-dichloroquinoline precursor was subjected to nucleophilic substitution with the basic sidechain to give the desired product. Slater, A.F.G., Pharmacol. Ther., 1993,57, 203-235. Details are given below:

O 00/50404 - 1 5 - PCT/GBOO/00678

7-Chloro^l(2^,-piperidyl)€thyllaπ-inoquinoline (formula XI)

To a stirred solution of 4,7-dichloroquinoline (l.Og, 5mmol) in ethanol (10 ml) was added (2φiperidyl)ethyl--mine (0.9g, 7mmol). The reaction was heated at reflux for 18 hours, after which time the solvent was removed in va<nιo. The resultant solid was purified by column chromatography (silica gel) using chchtoromethanemethanol (4: 1) as eluent to give the required product as a light brown solid (65%) ms (EI)[M+H*], m ₂ 290; Η-mnr (200MHz): δ, 1.45(2H, m, CH2X 1.6(4H, m, 2CH2),2.5(4H, m, 2CH2) 2.7(2H, m, CH2), 3.2-3.4(2H, m, CH2), 6.3(1H, d, ArH), 7.3-7.4(2H, dd, ArH), 7,65(1H, d, AiH), 8.5(1H, d, ArH); Anal. (C₁₆H₂,N₃C1) C, H, N.

7^htoπ»-4-l(2^,-pyιτoIidy-)ethyl|am oquinoline (formate XII)

To a stirred solution of 4,7-dicUoroquinoline (l.Og, S mol) in ethanol (10 ml) was added (2-pyrrolidyl)ethylaπ-ine (0.8g, 7mmol). The reaction was heated at reflux for 18 hours, after which time the solvent was removed in vacuo. The resultant solid was purified by column chromatography (silica gel) using ώchloiomethane methanol (4: 1) as eluent to give the required product as an off-white solid (71%) ms (EI)[M+H*], m 2 276; ^lH-mnr (200MHz): δ, 1.5(4H, m, 2CH2),2.4(4H, , 2CH2) 2.7(2H, m, CH2), 3.2-3.4C2H, m, CH2), 6.3(1H, d, ArH), 7.3-7.4(2H, dd, AiH), 7,65(1H, d, ArH), 8.5(1H, d, ArH); AnaL (CjsH.βNsCl) C, H, N.

7- J-Joro^[(2'-nιorphι-li-ao)ethyll-u- ---oq-^ (formula XHI)

To a stirred solution of 4,7-dichloroquinoline (l.Og, 5mmoI) in ethanol (10 ml) was added (2-moιpholino)ethylamine (0.91 g, 7mmoI). The reaction was heated at reflux for 18 hours, after which time the solvent was removed in vacuo. The resultant solid was purified by column chromatography (silica gd) using dic--doτomethane/mema-ιol (4:1) as eluent to give die required product as a fawny solid (72%) ms (EIXM+Hf], m/z 292; ^lH-mnr (200MHz): δ, 1.9(4H, m, 2CH2λ2.6(4H, m, 2CH2) 2.8(2H, m, CH2), 3.4(2H, m, CH2), 6.3(1H, d, ArH), 73-7.4(2H, dd, ArH), 7,65(1H, d, ArH), 8.5(IH, d, ArH); AnaL (Ctj-H.iNaOCl) C, H, N.

7-Clϋore^(2^,-^errt>βtylJUiιi-io)ed»yll--nι--αo^ (formula XIV)

To a stirred solution of 4,7κticl-loiOqu-noline (l.Og, 5mmol) in ethanol (10 ml) was added (2-teribut ----αino)emyiamine (0.81 g, 7-nmol). The reaction was heated at reflux for 18 hours, after which time die solvent was removed in vacuo. The resultant solid was purified by column chromatography (silica gel) using dichloromethane/n-tethanol (4:1) as eluent to give the required product as a pale yellow solid (62%) ms (EIXM+rTj, m z 278; 'H-n-mr (200MHz): δ, 1,1(9H, s, tBu),

2.8-3.0(2H, m, CH2), 3.2-3.4(2H, m, CH2), 6.3(IH, d, ArH), 7.3-7.4(2H, dd, ArH), 7,65( 1 H, d, ArH), 8.45(1H, d, ArH); Anal. (C.₅H₂₀N₃CI) C, H, N.

It is this inexpensive and simplistic synthesis which renders these compounds

commercially feasible. These compounds (Formulae X to XlVand XXIII)

exhibited a 3 fold higher antimalarial activity than chloroquine (Formula II)

against chloroquine resistance strain of P. falciparum.

The results obtained for these compounds are illustrated in Table 1

below.

TABLE 1

WO 00/50404 - 1 7 PCT/GBOO/00678

Amndiaquinine based derivation

A number of 4-aminoquinoline Mannich base derivatives were synthesised, in which:-

i) the 3'-die ylamino function of amodiaquine (AQ) was replaced by a V-tert-

butylamino group, and

ii) an aliphatic hydrocarbon entity was incorporated into the 5'- position of the 4'-

hydroxyanilino side chain.

Seven alkyl Mannich base derivatives were screened and found to be active against

both chloroquine sensitive and resistant strains of Plasmodium falciparum in vitro. The propyl

and isopropyl alkyl derivatives were found to be the most active and consequently these

derivatives were tested against a non sensitive strain of Plasmodium berghi in vivo. These

derivatives were found to be 3 times more active than AQ, irrespective of the route of administration (oral or intaparential).

The applicants have synthesised a new series of Mannich base derivatives where the

diethylamino function of AQ has been replaced by a N-terr-butyl-i-nino group to prevent the

side chain being metabolised to metabolites which display cross resistance. In addition, to

evaluate the importance of lipophilicity with respect to antimalarial activity, a series of alkyl

substituents were placed in the 5'- position of the 4'-hydroxyanilino side chain. The ability of

each compound within the series (Formulae XVI - XXII) to arrest the growth of CQ sensitive

and resistant strains of Plasrnodia in vitro and in vivo were measured to see if these

derivatives circumvented the mechanism of drug resistance.

Chemistry

The alkyl substituted Mannich base compounds were prepared using a modified

procedure of Kesten et al (1987) as depicted in Fig.l . All the 2-alkylphenols were WO 00/50404 - 1 8 - PCT/GBOO/00678

commercially available, with the exception of the o-cyclohexylphenol. Friedels-Craft

alkylation of phenol with cyclohexyl chloride, using ferric chloride as the catalyst, gave a

mixture of the o- and yclophenol isomers (Abdurasuleva, AR. haidarova, T.; Veber,

N.V. Condensation of phenol with cyclohexyl chloride in the presence of catalyic amounts of

iron and ferric chloride. Chem Abs. 1965,62,p7667f.) which could be readily separated by

column chromatography. The synthetic route of 7-chloro-4-[3'-(alkyl)-4^,-hydroxyl-5'-[(tert-

butyla--rdno)methyl]aminoquinoline (Formulae XVI - XXII) employs multiple steps from the

substituted 2-alkyl phenol. In general, the 2-alkylphenols were diazitised and without

isolation, the diazo group was converted to an amino function using sodium dithionite. However, the 2-alkylaniiines readily decomposed when exposed to air, consequently without purification the amino substituent was acetylated to give 3-(alkyl)-4-hydroxylacetanilide. These Compounds were purified and allowed to react with excess N-tert-butylamine and aqueous formaldehyde in ethanol to give 5'-(alkyl)-4'-hydroxyl-3'-[(tert- butylamino)methyl]acetanilide. Acid hydrolysis of the acetanilides followed by treatment with the commercially available precursor, 4,7-dichloroquinoline in ethanol gave the target compounds in an overall yield of approximately 50%.

Alkylation of phenol with cyclohexyl chloride proved to be the most difficult step in

the preparation of the compound of formula XXII. The favoured isomer during the alkylation-

of phenol using ferric chloride as the catalyst was the />-cyclohexylphenol isomer. Higher

temperatures only enhanced the yield of the /7-isomer (above 190 °C), while lower

temperatures (below 120 °C) decreased the overall yield of both isomers. Best results were

observed using a temperature range between 160-180 °C, yielding 13% and 35% of the o- and

•-isomer respectively.

Antimalarial activity The capacity of AQ (Formula III), N-ter/-butyl amodiaquine (TBAQ,) and the 5 '-alkyl

substituted Mannich side chain derivatives (Formulae XVI - XXII) to arrest the growth of CQ

sensitive and CQ resistant strains of P.falciparum were determined and arc shown in Table 2

below

Table 2. IC₅₀ Values for inhibition of growth of P. falciparum in vitro by alkyl Mannich base

Number of experiments

Not significant (p>0.05)

^c Significantly different p 0.05) WO 00/50404 - 20 - PCT/GBOO/00678

As reported previously, both AQ and TBAQ arrested parasite growth in the two

different strains with different efficacies. (Bray, P.G.; Hawley, S.R.; Mungthin, M.; Ward,

S.A. Physiochemical properties correlated with drug resistance and the reversal of drug

resistance in Plasmodium falciparum. Mole. Pharmacol. 1996,50,1559-1566). The marked

difference in the potency of AQ against CQ sensitive (HB3) and CQ resistance strain (Kl) is

illustrated by the calculated resistance factor of 5.35 (Table 2). All 5'-alkyl derivatives

examined in this study displayed antimalarial activity, although some members were more

effective than others. A general correlation between the size and shape of the 5 '-alkyl

substituent of the 4'-hydroxyanilino side chain and the efficacy of these drugs against both

strains of the parasite was observed. The introduction of large non-planar substituents, such as

N-terf-butyl (Formula XXI) or cyclohexyl (Formula XXII) group substantially decreased antimalarial activity. While alkyl groups which contained a 3 carbon backbone with no greater

than one branch, as seen with compounds (Formula XVIII to Formula XX) gave optimum

antimalarial activity.

In addition, the two best derivatives in vitro (Formula XVIII and Formula XIX) were

assessed for their ability to arrest the growth of NS strain of P. berghi compared to AQ in

vivo and are shown in Table 3 below.

Table 3. ED₅₀ Values for inhibition of growth of P. falciparum in vivo by alkyl Mannich base

derivatives

Two different routes of drug administration were used (oral and intaparential) to

determine whether or not antimalarial activity was influenced by variable drug absorption.

Discussion

All of the 7 alkyl substituted Mannich base derivatives prepared (Formulae XVI -

XXII) inhibited growth of both the CQ sensitive (HB3) and the CQ resistant (Kl) parasites by

varying degrees in vitro (Table 2). It was found that the replacement of the diethylamino

function of AQ with N-rert-butylamine group of the 4'-hydroxyanilino side chain (TBAQ) led

to a substantial increase in antimalarial activity against both strains. There was a 1.5-fold

increase in activity of TBAQ against CQ sensitive strain and almost a 4-fold increase in

activity against the CQ resistant strain. This result was in agreement with previous studies

(Hawley, S. R.; Bray, P. G.; O'Neill, P. M.; Park, B. K.; Ward, S. A. The role of drug accumulation in 4-aminoquinoline antimalarial potency. Biochem. PharmacoL 1996, 52, 723-

733. O'Neill, P. M.; Willock, D. J.; Hawley, S. R.; Bray, P. G.; Storr, R C; Ward, S. A.; Park, B. K. Synthesis, Antimalarial activity and molecular modeling of Tebuquine Analogues. J. Med.

Chem 1997, 40, 437-448.) Earlier studies have suggested that the enhanced potency of TBAQ

may be due to inherent differences in the level of accumulation between AQ and TBAQ. The

cellular accumulation ratio (CAR) for TBAQ was found to be 5 to 9 times greater in CQ

sensitive isolates compared to AQ and 2 to 3 fold greater in CQ resistant strains.

A similar decrease in the level of cross resistance as seen between AQ and TBAQ was

observed between AQ and the new alkyl derivatives (Formulae XVI - XXII). The data in table

2 suggest that the shape/length of the S'-alkyl substituent has a marked effect on drug activity.

Activity increased as the the length of the alkyl group increased with optimium activity

observed with the propyl and isopropyl derivatives. Replacement of the linear alkyl group

containing only one branch with a more bulkier, non-planar group such as N-terf-butyl or cyclohexyl substituent markedly reduced the efficacy of these derivatives against both strains

compared to AQ or TBAQ. All the 5 '-alkyl Mannich base derivatives displayed less cross

resistance than AQ, with resistant factors between 1.5 and 3.7 compared to 5.3 for AQ. In fact,

the 5 '-propyl and 5'-isopropyl substituted derivatives (Formula XVIII and Formula XDQ

were 2-fold more active than TBAQ and 7-fold more active than AQ against CQ resistant

(Kl) isolate. The reason for the greater activity for these two derivatives is not known.

However, given the structural similarities between TBAQ and the new derivatives (Formulae

XVI - XXII), it is probable that the 5 '-alkyl derivatives and TBAQ accumulate to a similar

levels in the CQ resistant parasite, resulting in an increased activity.

The mechanism of accumulation of the 4-aminoquinolines in the malarial parasite remains quite controversial. Nevertheless most hypotheses are based on a central theme, of

accumulation being dependant on the ability of drug to bind to an intraparasitic receptor, whether or not that be firee haem and/or an active exchanger. Whatever the accumulation

mechanism, it is apparent from the antimalarial activity data presented here, that antimalarial activity of the new Mannich base derivatives (Formulae XVI - XXII) is dependent on more

than one factor. The inverted bell shape curve of a plot of antimalarial activity versus decreasing planarity of alkyl substituent suggests that the activities of these derivatives

are dependent on; 1) drug lipophilicity and/or 2) substituents in the 5 '-position of the 4'-

hydroxyanilino side chain. The incorporation of a methyl or ethyl substituent in the 5'-

position of the 4 '-hydroxy anilino side chain slightly increased antimalarial activity compared

to TBAQ, which suggests that the introduction of a small (C C^) 5 '-alkyl substituent hinders

the drug binding to a receptor. Conversely, when the substituent is replaced with an alkyl

group containing a 3 carbon chain (Formula XVIII to Formula XX), the increase in

lipophilicity may be able to overcome the decreased affinity for a receptor, as shown by the WO 00/50404 - 2 3 - PCT/GBOO/00678

increased efficacy of these derivatives. Moreover, when the 5 '-alkyl substituent is replaced by

a non-planar group, such as a N-te/7-butyl (Formula XXII) or a cyclophenol group (Formula

XXI), the data indicates that the increase in drug lipophilicity is not significant to overcome

steric hinderance, leading to the observed decrease in antimalarial activity.

In addition to the in vitro investigations, the two most active derivatives (Formula

XVIII and XIX) in vitro were tested for their antimalarial activity compared to AQ against a

NS strain of P.Berghi in vivo. There was a significant difference (P>0.05) between the 2

different routes of --dministrauon (oral and intraparential) for all three drugs investigated (AQ,

Formula XVIII and Formula XIX). The drugs were almost 2-fold more active when

administered orally compared to an intraparential route of administration.

It is possible that the increased efficacy of these drugs compared to AQ is at least in part due to their increased bioavailability. The metabolism of AQ has been widely

investigated. Although it has a high absorption from the gut, due to a large first pass effect, AQ has a low bioavailability. It has been shown that AQ rapidly undergoes de-ethylation to the bis-deethylated metabolite and is ultimately excreted into the urine. In addition, further

studies have shown that AQ is excreted into rat bile exclusively as 5'-thio-ether glutathione

and subsequently into the intestines to be dither excreted into the faeces or undergo

enterohepatic recirulation. However both of these two processes of metabolic clearance have

been avoided by the introduction of 5'-alkyl substituent and the N-terf-butylamino group.

Experimental Section

All the o-alkyl phenols, with exception of o-cyclohexylphenol were obtained from

Aldrich chemical Co. Gillingham, Dorset, England. Merck iesegel 60 F254 precoated silica WO 00/50404 - 2 4 - PCT/GBOO/00678

plates for TLC were obtained from BDH, Poole, Dorset, U.K. Column chromatography was

carried out on Merck 938S silica gel.

Proton NMR spectra were recorded using Perkin Elmer R34 (220 Mhz) NMR

spectrometer. The NMR solvent was deuterated chloroform unless otherwise stated in text,

and tetramethylsilane was used as an internal reference. Full details are given for derivatives

Formulae XVI - XXII. Mass spectra were recorded at 70 eV using VG7070E mass

spectrometer. The samples were introduced using direct insertion probe. In the text the

molecular ion (M+) is given followed by peaks corresponding to major fragment losses.

Melting points were performed using a Gallenkamp melting point apparatus and are reported

uncorrected.

Chemistry of the scheme, illustrated in Fig.l. σ-Cyclohexylphenol. The

condensation of phenol with cyclohexyl chloride was performed using a modified procedure of Abdurasuleva et al (1965) (Abdurasuleva, A R; Khaidarova, T.; Veber, N. V. Condensation of phenol with cyclohexyl chloride in the pressence of catalytic amounts of iron and ferric

chloride. Chem Abs. 1965, 62, p7667f.). A mixture of phenol (0.5 mol) and cyclohexyl

chloride (0.1 mol) and ferric chloride (5.5 nu ol) were heated (170 °C) with continuous

stirring for 2 h. After cooling to room temperature, sodium hydroxide was added (20%, 20 ml)

and stirred. To facihtate the precipitation of the sodium salt of the product, diethyl ether was

added (50 ml). The salt was collected by filtration and dissolved in diluted hydrochloric acid

(1M, 50 ml) and the solution extracted with dichloromethane to give a mixture of p- and o-

cyclohexylphenol (7.5 g, 42 %). The two isomers were separated using a silica column and

dichloromethane as the mobile phase (o-cyclohexylphenol, Rf = 0.48, 1.8 g, p-

cyclohexylphenol, Rf = 0.80, 5.7 g). 3-(Alkyl)-4-hydroxylacetanilide. All the acetanilide derivatives were prepared from

the method of Kesten et al (1987). (Kesten, S. J.; Johnson, J.; Werbel, L. M. Synthesis and

Antimalarial Effects of 4-[~<7-chloro-4-quinolinyl)amino]-2-[(die l-unmo)methyl]-6-

alkylphenols and their N-oxides. J. Med. Chem. 1987, 30, 906-911.) Yields and melting point

data were all comparable to the literature values.

5'-(-vlethyl>-4'-hydroxyl-3'-I(ter/-butylamino) methyl] acetanilide. (Precursor to

Formula XVT). 3-(Methyl)-4-hydroxylacetanilide (5 mmol) was subjected to a Mannich reaction with N-tβrt-butylamine (10 mmol) and aqueous formaldehyde (10 mmol) in ethanol. After refluxing for 48 h the solvent was removed under reduced pressure and the residue dissolved in dichloromethane (10 ml). The organic solution was extracted with dilute hydrochloric acid (0.1 M, 2 x 20 ml). This solution was basified (pH 9 - 10) and extracted with dichloromethane (3 x 20 ml). The combined extracts were washed with water (1 x 20 ml), dried (MgSO₄) and the solvent evaporated under reduced pressure to give the product as a crude oil. Recrystallisation was achieved using toluene/light petroleum (40 - 60 °C) (20/80 v/v) to afford a light brown solid, sufficiently pure for the next reaction (65%, mp 245 - 247

°C). 1H NMR dl.l (s, 12H, f-butyl, -CH₃), 1.9 (s, 3H, NH-C(O)-CH₃), 2.2 (3H, -CH₃), 3.7 (s, 2H, Ar-CH₂-NH-C(O)-CH₃), 6.8 (s, 1H, Ar), 7.0 (s, 1H, Ar), 8.1 (s, 1H, NH-C(O)-.

The synthesis of the precursors to formula XVII to XXII were completed by a similar

procedure as above, by selecting the appropriate acetanilide.

7-Chloro-4-[5*-(methyl)-4'-hydroxyl-3^,-[(/ert-butylamino)methyll aminoquiαoline

(Formula XVI). A solution of 5'-(Methyl 4'-hydroxyl-3'-[(tert-butylamino) (5.0 g, 23 WO 00/50404 „ PCT/GBOO/00678

- 2 6 -

mmol) in hydrochloric acid (25 ml of 6 N HC1) was heated under reflux for lh. This solution

was concentrated by reduced pressure and then coevapourated with ethanol. The residue was

dissolved in ethanol (30 ml) and 4,7-dichloroquinoline (4.5 g, 23 mmol) was added and the

solution heated under reflux for 2 h. The solution was concentrated by reduced pressure to

give a viscous residue which was poured into ice cold ammonium hydroxide (5 %, 200 ml).

The sticky solid which separated was dissolved in dichloromethane (100 ml) and separated

from the basic solution. The organic solution was washed with water (100 ml), dried (MgS0₄)

and evaporated to dryness under reduced pressure to give the crude product This solid was

recrystallised twice from aqueous ethanol to give an analytical product (48%, mp 209 - 210

°C). 1H NMR dl.l (s, 12H, t-butyl, -CH₃), 2.2 (3H, -CH₃), 3.9 (s, 2Η, Ar-CH₂-NH-C(0)- CH₃), 6.5 (br s, IH, -NH-), 6.6 (d, J = 5 Hz, H-3), 6.7 (s, IH, Ar), 7.0 (s, IH, Ar), 7.4 (dd, J =

9 Hz and 2 Hz, IH, H-6), 7.8 (dd, J = 9,2 Hz, IH, H-5), 7.9 (d, J = 2 Hz, IH, H-8), 8.4 (d, J =

5Hz, IH, H-2). MS m/z 369.5 (M + 1). Anal. (C₂ιH₂₄N₃OCl) C, H, N.

7-Chloro-4-[5'-(ethyl)-4^,-hydroxyl-3'-[(/ert-butylamino)methylJaminoquinol-j-ιe

(Formula XVH). A yellow solid (51%); mp 195 - 196 °C [from aqueous ethanol]. ^lH NMR d

1.1 (s, 12H, /-butyl, -CH₂CH₃), 2.6 (q, J = 7 Hz/-CH₂CH₃), 3.7 (s, 2H, Ar-CH₂-NH-), 6.5 (br

s, IH, -NH-), 6.6 (d, J = 5 Hz, H-3), 6.8 (s, IH, Ar), 6.9 (s, IH, Ar), 7.4 (dd, J = 9,2 Hz, IH,

H-6), 7.8 (dd, J = 9,2 Hz, IH, H-5), 7.9 (d, J = 2 Hz, IH, H-8), 8.4 (d, J = 5Hz, IH, H-2). MS

m/z 383.5 (M + 1). Anal. (C₂₂H₂₆N₃OCl) C, H, N.

7-Chloro-4-[5'-(propyl)-4'-hydroxyl-3'-[(rert-butylamino)methyl] aminoquinoline

(Formula XVIII). off white solid (54%); p 168 - 169 °C [from aqueous ethanol]. Η NMR

d 0.9 (t, J = 7 Hz, 3H, -CH₂CH₃ ) 1.1 (s, 9Η, t-butyl), 1.6 (m, 2H, -CH₂-CH₂-CH₃), 2.6 (q, J = WO 00/50404 - 2 7 - PCT/GBOO/00678

7 Hz, - CH₂-CH₂-CH₃), 3.7 (s, 2H, Ar-CH₂-NH-), 6.5 (br s, IH, -NH-), 6.6 (d, J = 5 Hz, H-3),

6.8 (s, IH, Ar), 6.9 (s, IH, Ar), 7.4 (dd, J = 9 Hz and 2 Hz, IH, H-6), 7.8 (dd, J = 9 Hz and 2

Hz, IH, H-5), 7.9 (d, J = 2 Hz, IH, H-8), 8.4 (d, J = 5 Hz, IH, H-2). MS m/z 398.5 (M + 1).

Anal. (C₂₃H₂₈N₃OCl) C, H, N.

7-Chloro-4-[5'-(isopropyl)-4'-hydro---yl-3'-[(/-r/ -butylamino)methylJ

aminoquinoline (Formula XIX): yellow solid (52%); mp 188.5 - 190.0 °C [from aqueous

ethanol]. 1H NMR d 1.1 (m, 15H, t-butyl, 2 x - CH₃), 1.6 (m, 2H, -CH₂-CH₃), 3.7 (s, 2H, Ar-

CH₂-NH-), 6.5 (br s, IH, -NH-), 6.6 (d, J = 5 Hz, H-3), 6.7 (s, IH, Ar), 7.0 (s, IH, Ar), 7.4

(dd, J = 9 Hz and 2 Hz, IH, H-6), 7.8 (dd, J = 9 Hz and 2 Hz, IH, H-5), 7.9 (d, J = 2 Hz, IH,

H-8), 8.4 (d, J = 5Hz, IH, H-2). MS m/z 398.5 (M + 1). Anal.

C, H, N.

7-Chloro-4-[5'-(j-butyl)-4'-hydroxyl-3'-l(terr-butylamino)methyll aminoquinoline

(Formula XX): fawny solid (48%); mp 217 °C [from aqueous ethanol]. ^lH NMR d 0.9 (t, J =

7 Hz, 3H, -CH₂CH₃ ) 1.1 (m, 12H, t-butyl, -CH₃), 1.6 (m, 2H, -C /₂-CH₃), 3.0 (m, -CH(CH₃)-

CH₂-CH₃), 3.7 (s, 2H, Ar-CH₂-NH-), 6.5 (br s, IH, -NH-), 6.6 (d, J = 5 Hz, H-3), 6.7 (s, IH,

Ar), 7.0 (s, IH, Ar), 7.4 (dd, J = 9 Hz and 2 Hz, lH, H-6), 7.8 (dd, J = 9 Hz and 2 Hz, IH, H-

5), 7.9 (d, J = 2 Hz, IH, H-8), 8.4 (d, J = 5Hz, IH, H-2). MS m/z 412.5 (M + 1). Anal.

(C₂₄H₃₀N₃OC1) C, H, N.

7-Chloro-4-[5'-(r-butyl)-4'-hydroxyl-3'-[(/-butylam-no)methyll aminoquinoline

(Formula XXI): fawny solid (38%); mp 225 - 226 °C [from aqueous ethanol]. Η NMR dl .l

(s, 9H, t-butyl), 1.3 (s, 9H, t-butyl), 3.7 (s, 3H, Ar-CH_rNH-), 6.5 (br s, IH, -NH-), 6.6 (d, J =

5 Hz, H-3), 6.7 (s, IH, Ar), 7.0 (s, IH, Ar), 7.4 (dd, J = 9 Hz and 2 Hz, IH, H-6), 7.8 (dd, J = WO 00/50404 „ PCT/GBOO/00678

- 2 8 -

9 Hz and 2 Hz, IH, H-5), 7.9 (d, J = 2 Hz, IH, H-8), 8.4 (d, J = 5Hz, IH, H-2). MS m/z 412.5

(M + 1). Anal. (C₂₄H₃₀N₃OC1) C, H, N.

7-Chloro-4-[5'-(cycloheιyl)-4'-hydroxyl-3'-[(i^,ert-butylamino)methyl]

aminoquinoline (Formula XXII: fawny solid (39%); mp 189 - 190 °C [from aqueous

ethanol]. 1H NMR d 1.1 (s, 9H, t-butyl), 1.3 - 1.8 (m, 10H, cyclohexyl), 3.0 (m, IH, -CH of

cyclohexyl), 3.7 (s, 3H, Ar-CH₂-NH-), 6.5 (br s, IH, -NH-), 6.6 (d, J = 5 Hz, H-3), 6.7 (s, IH,

Ar), 7.0 (s, IH, Ar), 7.4 (dd, J = 9 Hz and 2 Hz, IH, H-6), 7.8 (dd, J = 9 Hz and 2 Hz, IH, H-

5), 7.9 ( , J = 2 Hz, IH, H-8), 8.4 (d, J = 5Hz, IH, H-2). MS m/z 438.5 (M + 1). Anal.

(C^H_jjN_jOCl) C, H, N.

Antimalarial activity. Two strains of P.falciparum from Thialand were used in this

study: (a) The uncloned Kl strain which is known to be CQ resistant and (b) the HB3 strain

which is sensitive to all antimalarials. Parasites were maintained in continous culture using the method of Trager and Jenson. (Trager, W.; Jenson, J. B. Human malaria parasites in continuous

culture. Sci. 1976, 193, 673-675). Cultures were grown in culture flasks containing human

erythrocytes (2-5%) with parasitemia in the range of 1% to 10% suspended in RPMI 1640

medium supplemented with 25 mM HEPES and 32 mM NaHCO₃, and 10% human serum

(complete medium). Cultures were gassed with a mixture of 3% O₂, 4% CO₂ and 93% N₂.

(a) in vitro testing. Antimalarial activity was assessed using an adaption of the 48 h

sensitivity assay of Desjardins et al. (Desjardins, R E.; Canfield, C. J.; Haynes, J. D.; Chulay,

J. D. Quantitative Assessment of Antimalarial activity in vitro by semiautomated microdilution

technique. Antimicrob. agents. Chemother. 1979, 16, 710-718) using [³H]-hypoxanthine

incorporation as an assessment of parasite growth. Stock drug solutions were prepared in 100% dimethylsulphoxide (DMSO) and diluted to the appropriate concentration using

complete medium. Assays were preformed in sterile 96-well microtitre plates, each plate

contained 200 ml of parasite culture (2% parasitemia, 0.5% haematocrit) with or without 10

ml drug dilutions. Each drug was tested in triplicate and parasite growth compared to control

wells (which consituted 100 % parasite growth). After 24 h incubation at 37 °C, 0.5 mCi

hypoxanthine was added to each well. Cultures were incubated for a further 24 h before they

were harvested onto filter-mats, dried for 1 h at 55 °C and counted using a Wallac 1450

Microbeta Trilux Liquid scintillation and luminescence counter. IC₅₀ values were calulated by

interpolation of the probit transformation of the log dose - response curve.

(b) in vivo testing. Male, radom Swiss albino mice weighing 18 - 22 g are inoculated

intraperitoneally with 10 parasitised erythrocytes with P. berghi NS strain. Animals were

then dosed daily via two routes (intraparential and oral) for four consecutative days beginning

on the day on infection. Compounds were dissolved or suspended in the vehicle solution

consisting of methanol, phosphate buffered saline and DMSO (2:5:3). The parasitemia was

determined on the day following the last treatment and the ED₅₀ (50% suppression of parasites

when compared to vechile only treated controls) determined from a plot of log dose against

parasitemia.

Claims

A compound of Formula IN

where:

W is Cl or CF₃;

XisΝorΝ = O;

R is selected from:

i) R₂ - NH - R, - Z -

where:

ZisNH, S,OorCH₂;

R, is a C, to C₄ alkyl group; and

R₂ -NH- is a group resistant to dealkylation in vivo; or

where:

Z and R, are as described in (i) above, and

/-\

R₃N-

is a group resistant to dealkylation in vivo; or,

iii) R₄-Ar-Z-

where: Z is as described in (i) above;

Ar is a substituted or unsubstitued aromatic ring structure; and

R, is a planar or non planar, alkyl or cycloalkyl substituent on the 5' position of the aromatic rirg

Ar.

2. A compound as claimed in claim 1 wherein the R group is: (i) R₂ - NH - R, - Z - in which:

Z is NH;

R, is a C, - C₄ alkyl group; and

R₂ is a secondary amine.

3. A compound as claimed in claim 1 wherein the R group is:

/~\ (ii) R₃ N - R, - Z - in which:

Z is NH;

R, is a C, - C₄ alkyl group; and ^

R₃ N - is a cyclic amine.

4. A compound as claimed in claim 3 in which the cyclic amine is selected from the group

consisting of:

Formula V;

Formula VI; and

Formula VII

5. A compound as claimed in claim 1 wherein the R group is: Formula VIII

in which:

Z is NH;

R₄ is a planar or non planar alkyl group; and

Y is a N - tert alkyl alkyl amino group.

6. A compound as claimed in claim 5 wherein the N-tert alkyl alkyl amino group is: Formula IX

7. A compound as claimed in claim 1 having the formula:

Formula X

8. A compound as claimed in claim 1 having the formula:

Formula XI

9. A compound as claimed in claim 1 having the formula: Formula XII

10. A compound as claimed in claim 1 having the formula:

Formula XIII

1 1. A compound as claimed in claim 1 having the formula:

Formula XIN

12. A compound as claimed in claim 1 having the formula:

Formula XXIII

13. A compound as claimed in claim 5 having the formula: Formula XV

14. A compound as claimed in claim 13 having the formula:

Formula XVI

15. A compound as claimed in claim 13 having the formula:

Formula XVII

16. A compound as claimed in claim 13 having the formula:

Formula XVIII

17. A compound as claimed in claim 13 having the formula:

Formula XIX

18. A compound as claimed in claim 13 having the formula:

Formula XX

19. A compound as claimed in claim 13 having the formula:

Formula XXI

20. A compound as claimed in claim 13 having the formula:

Formula XXII

21. A pharmaceutical preparation comprising, as an active ingredient, a compound as claimed

in any of the preceding claims.

22. A compound of any of claims 1 to 20, or a pharmaceutically acceptable salt thereof, for

use in the manufacture of a medicament.

23. A compound of any of claims 1 to 20 or a pharmaceutically acceptable salt thereof for

use in the manufacture of a medicament for use in the treatment or prophylaxes of malaria.

24. A compound of any of claims 1 to 20, or a pharmaceutically acceptable salt thereof for

use in the manufacture of a medicament for use in the treatment or prophylaxes of chloroquine

resistant strains of malaria.