EP0222839A1 - Antimalarial compounds - Google Patents

Abstract

Compounds having antimalarial activity have the general formula (1), wherein X represents a halogen group or an alkyl group substituted for halogen; Y represents CH or N; and R1 and R2, which may be identical or different, represent hydrogen, aminoalkyl, mono- or dialkylaminoalkyl, mono- or diaminoalkyl (substituted for the alkyl), mono- or di (cycloalkyl) aminoalkyl, mono- or diaminoalkyl (substituted for the aryl), or alkyl substituted with a nitrogen-containing heterocyclic group or with a heterocyclic group substituted for mono- or dialkyl, provided that R1 and R2 are not both hydrogen; and that R2 is not diethylaminomethyl when X is chlorine, Y is CH and R1 is hydrogen; or when X is bromine, Y is N and R1 is hydrogen. The invention also relates to processes for the preparation of these compounds.

Description

"ANTIMALARIAL COMPOUNDS"

The present invention relates generally to having compounds having anti alarial activity, to processes for the preparation thereof, to pharmaceutical compositions containing these compounds and to methods of treatment using the compounds or compositions.

In particular, the present invention relates to a class of mono- and di-Mannich bases, derived from 4-(7'- substituted -1' , 5'- naphthyridin-4*-yla ino) phenols and 4-(7'- substituted- quinolin -4'-ylamino) phenols which have been found to exhibit antimalarial activity against a number of species of Plasmodia, particularly the principal human malarial parasite Plasmodium falciparum.

In work leading to the present invention, the inventors have synthesised and tested a series of

4 1,8-naphthyridines, N -substituted 2-methoxy(and

2-hydroxy)-1,5-naphthyridin-4-amines and

4 N -substituted 7-bromo- 1,5-naphthyridin-4-amines for antimalarial activity against Plasmodium vinckei vinckei in mice (Barlin, G.B., and Tan, W.-L.,

Aust.J.Chem., 1984, 37, 1065; 1984, 37, 2469; and

1985, 38, 459). It has now been found that a series of mono- and di-Mannich bases derived from 4-(7*-substituted-1' ,5'-naphthyridin-4^•- ylamino) phenols and 4-(7*-substituted-quinolin-4'-ylamino) phenols are able to produce complete cures in the P.vinckei vinckei - mouse model, and also have significant activity against P.falciparu when compared with the activities of the established antimalarial drugs chloroquine, mefloquine, and amodiaquine, including activity against both chloroquine-sensitive and chloroquine-resistant isolates of P.falciparum.

According to a first aspect of the present invention, there are provided compounds of the general formula (I) :

wherein X represents a halogen or halogen-substituted alkyl group;

Y represents CH or N; and R 1 and R2, which may be the same or different, represent hydrogen, aminoalkyl, mono- or di-alkylaminoalkyl, mono- or di-(substituted alkyl)aminoalkyl, mono- or di-(cycloalkyl)aminoalkyl, mono- or di-(aryl)aminoalkyl, mono- or di-(substituted aryl)aminoalkyl, or alkyl substituted by a nitrogen-containing heterocyclic or mono- or di-alkyl- substituted heterocyclic group; provided that R1 and R2 are not both hydrogen; and that R 2 is not diethylaminomethyl when X is chloro, Y is CH and R is hydrogen; or when X is bromo, Y is N and R is hydrogen.

Preferred subgroups of compounds within the general formula I are compounds in which X represents halogen and Y represents N, and compounds in which X represents halogen substituted alkyl and Y represents CH.

Preferably X represents a bromo, chloro or fluoro group, or a trifluoromethyl group.

Preferably also, R 1 and/or R2 represgnt aminomethyl, mono- or di-alkylaminomethyl, mono- or di-(substituted alkyl)aminomethyl, mono- or di-(cycloalkyl)aminomethyl, mono- or. di-(aryl)aminomethyl, mono- or di-(substituted aryl)aminomethyl, or methyl substituted by a nitrogen-containing heterocyclic or mono- or di-alkyl substituted heterocyclic group.

Particular preferred groups are represented by R 1 and/or R2 aarree::

-CH₂NEt₂

-CH NPr,

Particularly preferred compounds of this invention are the di-Mannich bases, i.e. compounds in which both R 1 and R2 are other than hydrogen.

In another aspect, this invention provides a process for the preparation of compounds of the general formula (I) , which comprises reaction of a compound of the general formula (II) :

II in which X and Y are as described above, with an appropriate amine or nitrogen-containing heterocyclic compound in the presence of formaldehyde.

Preferably, the compound of general formula II is refluxed with the appropriate amine or heterocyclic compound in ethanolic formalin. Where a moderate amount of the amine or heterocyclic compound is used, the mono-Mannich base is prepared. Excess amounts of the amine or heterocyclic compound lead to preparation of the di-Mannich bases.

The compounds of the general formula II in which X is bromo and Y is N may be prepared from 3-bromo-8-chloro-l,5-naphthyridine (Barlin, G.B., and Tan, W.-L., Aust.J.Chem.1985, 3_8, ⁴⁵⁹) ^bY heating with aqueous methanolic p-aminophenol hydrochloride. Similarly, the compound of the general formula II in which X is triflugromethyl and Y is CH may be prepared from 4-chloro-7-trifluoromethyl.quinoline (Snyder, H.R. , et.al., J. m.Chem.Soc. , 1947, 6_9, 371) by refluxing with aqueous methanolic p-aminophenol hydrochloride. Other compounds of the general formula II may be prepared in an analogous manner.

Alternatively, the compounds of the general formula I may be prepared by reaction of a compound of general formula III:

III in which X and Y are as described above, and Z represents halogen, with an aminophenol of the general formula IV:

IV

in which R 1 and R2 are as described above.

Preferably, this reaction is performed under reflux in aqueous methanol.

The compounds of the present invention have exhibited antimalarial activity when screened by in vivo evaluation for activity against P.vinckei vinckei in mice following preliminary examination of each compound for toxicity and for safe dosage levels prior to the antimalarial studies. In addition, in subsequent in vitro tests against both chloroquine-sensitive and chloroquine-resistant strains of .falciparum a number of compounds of this invention have been more effective than chloroquine, against the sensitive strain, and approximately as effective as mefloquine and amodiaquine against the resistant strain. Certain compounds of this invention have also shown activity in vivo against P.berghei in mice.

Accordingly, the present invention also provides a pharmaceutical composition for use in treatment of malaria in an animal, including a human, which comprises an effective amount of a compound of the general formula I, together with a pharmaceutically acceptable carrier or diluent therefor.

In another aspect, there is provided a method of treating malaria in an animal, including a human, which comprises administering to the animal an effective amount of a compound of the general formula I.

Further details of preferred compounds in accordance with the present invention, and of the process for the preparation thereof, are given in the following Examples. In these Examples, solids and oils for analysis were dried at 100°C/20mm Hg unless otherwise specified, and melting points were taken in Pyrex capillaries. All structures were confirmed by n.m.r. spectral analysis.

EXAMPLE 1

(a) 4-(7'-Bromo-1' -5'-naphthyridin-4'-ylamino)phenol

(la) 3-Bromo-8-chloro-l,5-naphthyridine (2.0g) , p-aminophenol hydrochloride (1.2g), water (40.0ml) and methanol (20.0ml) were heated with stirring in an oil bath at 100° for 2h. The methanol was then evaporated under reduced pressure and the remaining aqueous solution was adjusted to pH 8 with ammonium hydroxide. The yellow precipitate which formed was filtered off, washed with water, dried and recrystallized from methanol to give 4-(7'-bromo-1' ,5'-naphthyridin- 4'-ylamino)phenol (2.5g) , m.p. 245-247°. (Found: C,53.6; H,3.2; N,13.2. C₁₄H₁₀BrN₃O requires C,53.2; H,3.2; N,13.3%). (b) 4-(7'-Bromo-1' ,5'-naphthyridin-4'-ylamino)-2- dimethylaminomethylphenol (TN83) . 4-(7*-Bromo-1' ,5'-naphthyridin-4*-ylamino)phenol

(0.5g), formalin (2.0ml; 36%), and ethanolic dimethylamine (1.0ml; 33%) in ethanol (10.0ml) were refluxed with stirring for 20h. The reaction mixture was evaporated under reduced pressure and the residue purified by t.l.c. (silica; methanol) to give an oil

(0.25g) .

This oil was treated with ethanolic hydrogen bromide and the product recrystallized from ethanol to give yellow crystals of

4-(7*-bromo-1* ,5*-naphthyridin-4'-ylamino)-2- dimethylaminomethylphenol dihydrobromide (0.3g), m.p. >305° (dec). (Found: C, 38.4; H,3.7; N,10.4. C₁₇H₁₉Br₃N₄ requires C, 38.2; H, 3.6; N, 10.5%).

EXAMPLE 2

4-(7'-Bromo-1' ,5'-naphthyridin-4'-ylamino)-2-(N,N- dipropylaminomethyl)phenol (TN 84) .

4-(7*-Bromo-1' ,5'-naphthyridin-4'-ylamino)phenol

(0.5g), dipropylamine (0.48g) , formalin (2.0ml; 36%) and ethanol (10.0ml) were refluxed with stirring for 20h and the mixture worked up as described above. The produce was purified by t.l.c. (alumina; chloroform then silica; ethanol) and recrystallized from light petroleum (b.p. 60-80°) to give yellow crystals of 4-(7*- bromo-1' ,5'-naphthyridin-4'-ylamino)-2-(N,N-di- propylaminomethy1)phenol (0.15g), m.p. 138-139°.

(Found: C, 58.7; H, 5.9; N, 13.2. C₂₁H₂₅BrN₄0 requires C, 58.7; H, 5.9; N, 13.1%). EXAMPLE 3

4-(7'-Bromo-1' ,5'-naphthyridin-4'-ylamino)-2- pyrrolidin-1"-ylmethylphenol (TN 87) .

4-(7*-Bromo-1' ,5'-naphthyridin-4'-ylamino)phenol

(0.5g), pyrrolidine (0.15g), formalin (2.0ml; 36%) and ethanol (10.0ml) were refluxed with stirring for lOh and worked up as described above. The crude produce was purified by t.l.c. (silica; methanol) and the oil (0.27g) was treated with ethanolic hydrogen bromide and the solid recrystallized from ethanol to give 4-(7*- bromo-1' ,5*-naphthyridin-4'-ylamino)-2- pyrrolidin-l"-ylmethylphenol dihydrobromide, m.p. 318°

(dec). (Found: C, 41.0; H, 3.8; Br, 42.8; N, 9.6. C_lgH₂-Br₃N₄0 requires C, 40.7; H, 3.8; Br, 42.7; N, 10.0%) .

EXAMPLE 4

4-(7'-Bromo-1' ,5.'-naphthyridin-4'-ylamino)-2,6-bis-

(dimethylaminomethyl) henol (TN 78) .

4-(7'-Bromo-1' ,5'-naphthyridin-4'-ylamino)phenol

(0.5g), formalin (10ml; 36%) and ethanolic dime hyla ine (30ml; 33%) were refluxed with stirring for 20h. The product was isolated as described above and purified by t.l.c (alumina; chloroform) to give 4-(7'-bromo-1' ,5'-naphthyridin-4'-ylamino)-2,6-bis-

(dimethylaminomethyl)phenol (0.5g) as a yellow oil.

(Found: C, 55.4; H, 5.8; N, 16.0. C_2QH₂₄Br ₅0 requires C, 55.8; H, 5.6; N, 16.3%)

The tripicrate was prepared in and recrystallized from, ethanol. It had m.p. 152-153°. (Found: C,41.0; H, 3.1; N, 17.1. C_2QH₂₄BrN₅0.3(CgH^C^) requires C, 40.8; H, 3.0; N, 17.5%). EXAMPLE 5

4-(7'-Bromo-1' ,5'-naphthyridin-4'-ylamino)-2,6-bis-

(diethylaminomethyl)phenol (TN 77) .

4-(7'-Bromo-1' ,5*-naphthyridin-4*-ylamino)phenol (0.5g) , formalin (5.0ml; 36%) , diethylamine (5.0ml) and ethanol (10.0ml) were refluxed with stirring for 20h. The product was purified by t.l.c (silica; methanol) to give a yellow oil (0.7g).

A sample of this oil with ethanolic picric acid gave a yellow precipitate which was recrystallized from ethanol to yield

4-(7'-bromo-1* ,5'-naphthyridin-4'-ylamino)-2,6- bis(diethylaminomethyl)phenol tripicrate, m.p. 191-193°. (Found: C, 42.6; H, 3.4; N, 16.4. C₂₄H₃₂Br ₅0.3(CgH₃N₃0₇) requires C, 43.0; H, 3.5; N, 16.7%) .

EXAMPLE 6

4-(7'-Bromo-1' ,5'-naphthyridin-4'-ylamino)-2,6-bis- ( ,N-dipropylaminomethy1)phenol (TN 79) .

4-(7*-bromo-1' ,5*-naphthyridin-4'-ylamino) henyl (0.5g), formalin (5.0ml; 36%), dipropylamine (5.0ml) and ethanol (10.0ml) were refluxed with stirring for 20h. The 4-(7*-bromo-1' ,5*- naphthyridin-4'-ylamino)-2,6-bis(N,N-dipropyl- aminomethyl)phenol (0.5g) was isolated as a yellow oil after t.l.c. (alumina; ethanol) . (Found: C, 61.8; H, 7.5; N, 12.7. C₂gH₄₀BrN-.O requires C, 62.0; H, 7.4; N, 12.9%) .

The tripicrate was prepared in, and recrystallized from, ethanol. It had m.p. 176-178°. (Found: C, 45.0, H, 4.0; N, 15.7.

^C28^H40^BrN5⁰__H^C6^H3 3^O7. requires C, 44.9; H, 4.0; N, 15.9%) . EXAMPLE 7

4-(7'-Bromo-1' ,5'-naphthyridin-4'-ylamino)-2,6-bis-

(pyrrolidin-l"-ylmethyl)phenol (TN 80).

4-(7'-Bromo-1* ,5*-naphthyridin-4'-ylamino)phenol (0.5g), formalin (5.0ml; 36%), pyrrolidine (5.0ml) and ethanol (10.0ml) were refluxed with stirring for 20h. Excess reagents were distilled and the product purified by t.l.c (silica; methanol) to give as a yellow oil 4-(7'-bromo-1,-5'-naphthyridin-4'- ylamino)-2,6-bis(pyrrolidin- l"-ylmethyl)phenol (0.64g). (Found: C, 59.9; H, 6.2; N, 14.0. ^C24^H28^BrN5° ^re<3^{uires c}' 59.8; H, 5.9; N, 14.5%⁾.

EXAMPLE 8

4-(7'-Bromo-1' ,5'-naphthyridin-4'-ylamino)-2,6-bis-

(piperidin-1"-ylmethyl)phenol (TN 81) .

4-(7*-Bromo-1' ,5'-naphthyridin-4'-ylamino)phenol

(0.5g), formalin (5.0ml; 36%), piperidine (5.0ml) and ethanol (10.0ml) were refluxed with stirring for 20h. Workup was as described above to give 4-(7'-bromo-1* ,5'-naphthyridin-4'-ylamino)-2,6-bis-

(piperidin-1"-ylmethyl)phenol (0.6g) as a yellow oil which became a semi-solid. (Found: C, 61.5; H, 6.5; N, 13.5. C₂₆H₃₂Br ₅0 requires C, 61.2; H, 6.3; N, 13.7%) .

EXAMPLE 9

4-(7'-Bromo-1' ,5'-naphthyridin-4'-ylamino)-2,6-bis-

(morpholin-4"-ylmethyl)phenol (TN 82)

4-(7'-Bromo-1,5'-naphthyridin-4'-ylamino)phenol (0.5g), formalin (5.0ml; 36%), morpholine (5.0ml) and ethanol (10.0ml) were refluxed as described above. The product was purified by t.l.c. (alumina; chloroform) to give as a yellow oil

4-(7'-bromo-1' ,5'-naphthyridin-4*-ylamino)-2,6- bis(morpholin-4"-ylmethyl)phenol (0.56g). Found: C,

56.4; H, 5.7; N, 13.2. C₂₄H₂gBrN₅0₃ requires C,

56.0; H, 5.5; N, 13.6%) .

The dipicrate was prepared in and recrystallized from ethanol. It had m.p. 216-218°. (Found: C, 44.5; H, 3.5; N, 15.6. C₂₄H₂gBr ₅0₃.2(CgH₃N₃0₇) requires C, 44.5; H, 3.5; N, 15.8%) .

EXAMPLE 10

4-(7'-Chloro-1' ,5'-naphthyridin-4'-ylamino)-2- diethylaminomethylphenol('5-azaamodiaquine') (TN 88)

4,7-Dichloro-l,5-naphthyridine (McCaustland, D.J., and Cheng, CC. J.Heterocycl. Chem. 1970, 7, 467.) (0.2g), 4-amino-2- diethylaminomethylphenol dihydrochloride (0.27g) water (15.0ml) and methanol (5.0ml) were heated with stirring in an oil bath at 100° for 2h. The_^ methanol was then evaporated under reduced pressure and the ^"aqueous solution adjusted with ammonium hydroxide to pH 7-8. The yellow precipitate was collected, washed, dried, and recrystallized from cyclohexane to give 4-(7'-chloro- 1' ,5*-naphthyridin-4*-ylamino)2-diethylaminomethyl¬ phenol (0.28g), m.p. 167-169°. (Found: C, 64.0; H, 6.0; N, 15.5. C₁₉H₂₁C1N₄0 requires C, 64.0; H, 5.9; N, 15.7%).

EXAMPLES 1 - 10 Toxicity Testing

The naphthyridines were tested for acute toxicity in mice by intraperitoneal injection in normal saline or peanut oil. Each test chemical was injected in a single dose of 200 mg/kg of body weight [except for 4-(7'-bromo-1' ,5'-naphthyridin-4'-ylamino)-2,6-bis- (diethylamino and pyrrolidin-1"-yDmethylphenol which, due to toxicity at 200 mg/kg, were run at 100 mg/kg] to three mice. No apparent ill effects were observed and all mice survived to and beyond 4 days in the above tests and in control experiments with normal saline and peanut oil.

Preliminary Antimalarial Screen

This was carried out as described previously. (Barlin, G.B. and Tan, E.-L., Aust.J.Chem. , 1985, 38, 459; 1984, 3_7_, 2469.) Each test chemical was given at a dosage of 200 mg/kg of body weight except for 4-(7'-bromo-1' ,5'-naphthyridin-4'-ylamino)-2,6-bis- (diethyla ino and pyrrolidin-1"-yl)methylphenol which were at 100 mg/kg). The results are shown in Table 1.

TABLE 1 :

Times given are those after injection of the chemical under test.

Time: h, hours; d, days; w, weeks; Oh denotes pretreatment.

Mean percentage of parasite- infected red cells Comp- Sol=r Dose ound vent^A (mg/ Oh 9h 24h 48h 3d 6d 8d 9d 14d 19d 4w kg)

(la)^J PO 200 16 34 62 88 B

TN83 NS 200 27 30 9 <1 <1 11 8 4 <1 <1 <1

TN84 PO 200 16 27 16 3 5 26 14 5 <1 <1 <1

TN87 NS 200 19 26 3 <1 <1 25 28 18 <1 <1 <1

TN78 PO 200 18 24 5 <1 <1 <1 <1 <1 <1 <1 <1

TN77 NS 100 10 9 <1 <1 <1 <1 <1 <1 16 <1 <1

TN79 PO 200 22 32 7 <1 <1 <1 <1 <1 <1 <1 <1

TN80 O 100 11 11 1 <1 <1 <1 <1 <1 <1 <1 <1

TN81 PO 200 17 13 1 <1 <1 <1 <1 <1 63 D

TN82 PO 200 . ¹⁵ 15 <1 <1 <1 <1 <1 <1 16 E

TN88 PO 200 14 12 1 <1 <1 3 15 21 <1 <1 <1

NS - - 27 45 63 88 F

PO - - 19 42 66 85 F

Chloroquine G

NS 40 28 30 4 <1 <1 10 71 82^H

A PO, peanut oil; NS, normal saline.

B All three mice dead.

C Dihydrobromide.

D Two mice dead, parasitaemia of third mouse <1%.

E One mouse dead, parasitaemia of remaining two mice <1%.

F Two of the three mice dead at 3 days.

G Diphosphate.

H All mice dead at 10 days.

J 4-(7*-bromo-1' ,5*-naphthyridin-4'-ylamino)phenol. EXAMPLE 11

2-Diethylaminomethyl-4-(7'-trifluoromethylquinolin-4'- ylamino)phenol (TN105)

4-Chloro-7-trifluoromethylquinoline (Snyder, H.Q., et al., J.Amer.Chem.Soc. , 1947, 6J9, 371) (0.5g) and 4-amino-2-diethylaminomethylphenol dihydrochloride (0.75g) in a mixture of methanol (15.0ml) and water (5.0ml) were refluxed with stirring for 2h and the methanol evaporated under reduced pressure. After chilling, the yellow precipitate was collected, washed well with water, and recrystallized from aqueous ethanol to give 2-diethylaminomethyl-4-(7'-trifluoro¬ methylquinolin-4'-ylamino)phenol (0.5g), m.p. 210-212°. (Found: C, 64.3; H.5.7; N.10.7. ^C21^H22^F3^N3° ^re<3^{uires c}' 64.8; H,5.7; N, 10.8%⁾.

EXAMPLE 12

(a) 4-(7'-Trifluoromethylquinolin-4'-ylamino)phenol

(12a) 4-Chloro-7-trifluoromethylquinoline (0.5g) , p-aminophenol hydrochloride (0.41g), methanol (15ml)_, and water (5.0ml) were refluxed with stirring for 2h, and the methanol evaporated under reduced pressure. After chilling, the yellow precipitate was collected, suspended in water 30 (ml) , and adjusted with ammonium hydroxide to pH 7-8, and then recrystallized from aqueous ethanol to give 4-(7'-trifluoromethyl¬ quinolin-4'-ylamino)phenol (0.6g), m.p. 253-254°. (Found: C, 61.3; H, 3.9; N, 8.9.

^C16^H11^F3^N2°-^^H2^{0 re<}3^uires C, 61.3; H, 3.9; N, 8.9%) . (b) 2,6-Bis(dimethylaminomethyl)-4-(7'-trifluoro¬ methylquinolin-4*-ylamino)phenol (TN 107) 4'-(7'-Trifluoromethylquinolin-4'-ylamino)phenol (0.3g) , formalin (5.0ml) and ethanolic dimethyla ine (15.0ml, 33%) were refluxed with stirring for 20h. The solution was evaporated to dryness and the oily residue was subjected to t.l.c (alumina; ether) to give an oil which slowly crystallized and was recrystallized from aqueous ethanol to give yellow crystals of 2,6-bis(dimethylaminomethyl)-4-(7'-trifluoro¬ methylquinolin-4'-ylamino)phenol (0.32g) , m.p. 86-88°. (Found, for a sample dried at 40°/0.2mmHg: C, 63.5; H, 6.2; N, 13.5.

^C22^H25^F3^N4° ^re(3^uires C, 63.2; H, 6.0; N, 13.4%) .

EXAMPLE 13

2,6-Bis(diethylaminomethyl)-4-(7'-trifluoromethyl¬ quinolin-4'-ylamino) henol (TN 108)

4-(7'-Trifluoromethylquinolin-4'-ylamino)phenol

(0.3g), diethylamine (5.0ml) and formalin (5.0ml) in ethanol (10.0ml) were refluxed as above. The crude * product was subjected to t.l.c. (alumina; chloroform) and recrystallized from a mixture of methanol and light petroleum (b.p. 60-80°) to give yellow crystals of 2,6-bis(diethylaminomethyl)-4-(7'-trifluoromethyl¬ quinolin-4'-ylamino)phenol (0.31g), m.p. 169-170°.

⁽Found: C, 65.8; H, 7.1; N, 12.0. ^C26^H33^F3^N4° requires C, 65.8; H, 7.0; N, 11.8%). EXAMPLE 14

2,6-Bis(dipropylaminomethyl)-4-(7*-trifluoromethyl¬ quinolin-4'-ylamino)phenol (TN 110)

4-(7*-Trifluoromethylquinolin-4'-ylamino)phenol

(0.3g), dipropylamine (5.0ml), formalin (5.0ml) and ethanol (10.0ml) were refluxed as above. The crude product was subjected to t.l.c (alumina, chloroform; then silica, ether) and the yellow oil crystallized on standing to give 2,6-bis(dipropylaminomethy1-4-

(7'-trifluoromethylquinolin-4'-ylamino)phenol (0.20g) , m.p. 113-114°. (Found, for sample dried at 80°/0.2mmHg: C, 67.9; H, 7.8; N, 10.5. C^H^F^O requires C, 67.9; H, 7.8; N, 10.6%).

EXAMPLE 15

2,6-Bis(pyrrolidin-1"-ylamino)-4-(7'-trifluoromethyl¬ quinolin-4'-ylamino)phenol (TN 109)

4-(7'-Triflupromethylquinolin-4'-ylamino)phenol (0.3g), pyrrolidine (5.0ml), formalin (5.0ml) and ethanol (10.0ml) were refluxed as above, and excess pyrrolidine was removed by distillation in a vacuum. The residue was triturated with water (50ml) , and the crude product subjected to t.l.c. (alumina, methanol; then alumina, ether) to give as a yellow oil, 2,6-bis(pyrrolidin-1"-ylmethyl)-4-(7'-trifluoromethyl¬ quinolin-4'-ylamino)phenol (0.23g). (Found: C, 64.1; H, 6.4; N, 10.9. C^H.gF.N.O.l.lH 0 requires C,63.7; H, 6.4; N, 11.4%) . EXAMPLE 16

2.6-Bis(piperidin-1"-ylmethyl)-4-(7'-trifluoromethyl¬ quinolin-4'-ylamino) henol (TN 112)

4-(7*-Trifluoromethylquinolin-4'-ylamino)phenol (0.3g) , piperidine (5.0ml), formalin (5.0ml) and ethanol (10.0ml) were treated as above. The crude product was purified by t.l.c. (alumina; chloroform) and recrystallized from cyclohexane to give 2,6-bis- (piperidin-1"-ylmethyl)-4-(7'-trifluoro ethylquinolin- 4'-ylamino) henol (0.31g), m.p. 170-171°. (Found: C, 67.5; H, 6.9; N, 11.3. C_2gH₃₃F_{3 4}0 requires C, 67.5; H, 6.7; N, 11.2%) .

EXAMPLE 17

2,6-Bis(morpholin-1"-ylmethyl)-4-(7'-trifluoromethyl¬ quinolin-4'-ylamino)phenol (TN 111) .

4-(7*-Trifluoromethylquinolin-4'-ylamino)phenol (0.3g), morpholine (5.0ml), formalin (5.0ml) and ethanol (10.0ml) were treated as above to give, after t.l.c. (alumina; chloroform) and recrystallization from aqueous ethanol, yellow needles of 2,6-bis- (morpholin-1"-ylmethyl)-4-(7'-trifluoromethylquinolin- 4'-ylamino)phenol (0.29g), m.p. 198-200°. (Found: C, 62.4; H, 5.9; N, 11.4. C₂gH_2gF_{3 4}0₃ requires C, 62.1; H, 5.8; N, 11.15%) .

EXAMPLES 10 - 17 Toxicity Testing

Each quinoline used in the antimalarial screening was tested for acute toxicity in three mice by intraperitoneal injection, each with a single dose in normal saline or peanut oil, at a dose of 100 mg/kg of body weight. No apparent ill effects were observed. and all mice survived to and beyond 6 days in the above tests, and in control experiments with normal saline and peanut oil.

Preliminary Antimalarial Screen

This was carried out as described previously. Each test chemical was given at a dosage of 100 mg/kg of body weight and blood counts were made at 9, 24, 48, and 72h and thereafter as shown in Table 2.

TABLE 2 :

Times given are those after injection of the chemical under test.

Time: h, hours; d, days; w, weeks; Oh denotes pre reatmen .

Mean percentage of parasite-infected red cells

A* Oh 9h 24h 48h 3d 6d 8d 9d lOd lid 12d 14d 15d 4w

12a 7 10 10 51 80 B

TN105 11 5 <2 <1 <1 C D 27 52 67 83 11 7 <1

TNI07 6 3 1 <1^E <1 <1 <1 <1 <1 <1 <1 <1 <1 <1

TNI08 6 14 6 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1

TNI10 11 6 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1

TNI09 8 4 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1

TNI12 5 2 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1

TNI11 18 12 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1

NS 25 35 65 86 F

PO 20 23 58 ^*85 E r^~

Chloroquine (NS)

12 19 2 <1 <1

A* Solvent - peanut oil (PO) ; dose of compounds in PO was 100 mg/kg. NS, normal saline.

B All mice dead in 4 days.

C Parasitaemia of one mouse 75%, other two mice <1%.

D One mouse dead, parasitaemia of other two 15%.

E One mouse dead.

F Mice dead at 3 days.

G Diphosphate: dose 40 mg/kg.

H 4-(7'-trifluoromethylquinolin-4'-ylamino)phenol. EXAMPLE 18

4-(7'-Bromo-1' ,5'-naphthyridin-4'-ylamino)-2,6-bis-

(4"-methylpiperazin-1"-ylmethyl)phenol (TN 117)

4-(7'-Bromo-1* ,5'-naphthyridin-4'-ylamino)phenol

(see Example 1(a)) (0.2g) , formalin (2.0ml), N-methyl- piperazine (2.0ml) and ethanol (10.0ml) were refluxed with stirring for 20h. Excess amine was distilled off under vacuum and the oily residue purified by t.l.c

(alumina; chloroform) to give a yellow oil (0.17g).

(Found: C, 54.9; H, 6.2; N, 16.8. C_2gH₃₄Br ₇0.1.8- H₂0 requires C, 54.5; H, 6.6; N, 17.1%).

EXAMPLE 19

4-(7'-Bromo-1* ,5'-naphthyridin-4'-ylamino)-2,6-bis-

(2"-methylpiperidin-1"-ylmethyl)phenol (TN 118).

4-(7'-Bromo-1' ,5'-naphthyridin-4'-ylamino)phenol

(0.2g), formalin (2.0ml), 2-methylpiperidine (2.-0ml) and ethanol (10ml.) were treated as above. Traces of 2-methylpiperidine were removed by triturating with water (50ml) before the oily residue was purified by t.l.c. (alumina? chloroform) to give a yellow oil

(0.2g). (Found: C, 62.9; H, 6.9; N, 12.8. C_2gH₃gBr ₅0 requires C, 62.5; H, 6.7; N, 13.0%).

EXAMPLE 20

4-(7'-Bromo-1' ,5'-naphthyridin-4'-ylamino)-2,6-bis- (3",5"-dimethylpiperidin-1"-ylmethyl)phenol (TN 116) . 4-(7'-Bromo-1' ,5'-naphthyridin-4'-ylamino)phenol (0.2g), formalin (2.0ml), 3,5-dimethylpiperidine (2.0ml) and ethanol (10.0ml) were treated as above, and the oily residue purified on t.l.c. (alumina; chloroform, then silica; methanol) to give a yellow oil (0 .12g) . (Found: C , 63 . 1 ; H , 7 .2 ; N , 12 .1. ^C30^H40^BrN5° ^re<ϊ^{uires c} - 63 .6 ; H , 7 .1 ; N, 12.4% ) .

EXAMPLE 21

4-(7'-Bromo-1' ,5*-naphthyridin-4'-ylamino)-2-

(dimethylaminomethyl)-6-(pyrrolidin-1"-ylmethyl)phenol

(TN 115) 4-(7'-Bromo-1* ,5'-naphthyridin-4'-ylamino)-2-

(pyrrolidin-1"-ylmethyl)phenol (see Example 5).

(0.15g), formalin (2.0ml) and ethanolic dimethylamine

(10ml; 33%) were refluxed with stirring for lOh. The reaction mixture was evaporated to dryness in a vacuum to leave an oil which was purified by t.l.c. (alumina; chloroform) to give a yellow oil (O.llg)., (Found: C, 57.5; H, 5.8; N, 14.9. C₂₂H₂gBrN_gO requires C, 57.9; H, 5.7; N, 15.3%) .

EXAMPLE 22

(a) 4-(7'-Chloro-1' ,5'-naphthyridin-4'-ylamino)phenol A mixture (c1:1.5) of 4,7-dichloro-l,5-naphthy- ridine (McCaustland, D.J. and Cheng, CC. , supra) (0.66g), and p-aminophenol hydrochloride (0.72g), in methanol (20.0ml) and water (20.0ml) was refluxed with stirring for 2h. The methanol was then evaporated under reduced pressure and the aqueous solution adjusted to pH 8 with ammonium hydroxide. The yellow precipitate was collected, washed with water and recystallized from methanol to give 4-(7'-chloro-1' ,5'-naphthyridin- 4'-ylamino)phenol (0.7g), m.p. 240-241°. (Found: C, 61.9; H, 3.8; N, 15.1. C₁₄H₁₀ClN₃O requires C, 61.9; H, 3.7; N, 15.5%). (b) 4-(7'-Chloro-1' ,5'-naphthyridin-4'-ylamino)-2,6- bis(di ethylaminoethyl) henol (TN 122)

4-(7'-Chloro-1' ,5*-naphthyridin-4*-ylamino)phenol (0.2g), formalin (2.0ml) and ethanolic dimethylamine (20.0ml; 33%) were treated as above to give a yellow oil (0.22g). (Found: C, 61.2; H, 6.4; N, 17.7.

^C20^H24^C1 5° _J-^H2° ^re<?^{uires c}' 60.8; H, 6.4; N, 17.7%) .

EXAMPLE 23

4-(7'-Chloro-1' ,5'-naphthyridin-4'-ylamino)-2,6-bis-

(diethylaminomethyl)phenol (TN 124)

4-(7'-Chloro-1' ,5*-naphthyridin-4'-ylamino)phenol

(0.2g), formalin (2.0ml), diethylamine (2.0ml) and ethanol (10.0ml) were treated as above, and the residue purified by t.l.c. (silica; methanol) to give a yellow oil which slowly crystallized (0.19g)> m.p. 106-107°. (Found: C, 65.0; H, 7.5; N, 15.7. ^C24^H32^C1N5° ^re<3^uij:es C, 65.2; H, 7.3; N, 15.8%⁾.

EXAMPLE 24

4-(7'-Chloro-1' ,5'-naphthyridin-4'-ylamino)-2,6-bis-

(dipropylaminomethyl)phenol (TN 125)

4-(7*-Chloro-1* ,5*-naphthyridin-4'-ylamino) henol

(0.2g) , formalin (2.0ml), dipropylamine (2.0ml) and ethanol (10.0ml) were refluxed with stirring for 40h

(the reaction was incomplete at 20h) . The solution was evaporated to dryness under reduced pressure and the residue purified on t.l.c. (silica; methanol) to give a yellow oil (0.20g). (Found: C, 66.4; H, 8.1; N, 13.8. C_2gH₄₍-ClN₅0 ,H₂0 requires C, 66.3; H, 8.1; N, 13.8%) . EXAMPLE 25

4-(7'-Chloro-1' ,5'-naphthyridin-4'-ylamino)-2,6-bis-

(pyrrolidin-1"-ylmethyl)phenol (TN 123).

A mixture of 4-(7'-chloro-1' ,5'-naphthyridin-4'- ylamino) henol (0.2g), formalin (2.0ml), pyrrolidine

(2.0ml) and ethanol (10.0ml) was treated as above, and the residue purified by t.l.c. (silica; methanol) to give a yellow oil (0.21g). (Found: C, 64.6; H, 6.6; N, 15.4. C₂₄H_2gCl ₅0 _jJ-UO requires C, 64.5; H, 6.5; N, 15.7%) .

EXAMPLE 26

2,6-Bis(4"-methylpiperazin-1"-ylmethyl)4-(7'-tri- fluoromethylquinolin-4'-ylamino)phenol (TN 121) .

4-(7'-Trifluoromethylquinolin-4'-ylamino)phenol (see Example 12(a)) (0.2g) , formalin (2.0ml),

N-methyIpiperazine (2.0ml) and ethanol (10.0ml) were treated as above.. The product was purified by column (alumina; chloroform) and thin-layer chromatography (silica; methanol) to give a yellow oil which crystallized m.p. 163-164°. (Found: C, 62.9;

H, 7.2; N, 15.7. C₂gH₃₅F₃NgO.j^O requires C, 62.6;

H, 6.8; N, 15.6%) .

EXAMPLE 27

2,6-Bis(2"-methylpiperidin-1"-ylmethyl)-4'-(7'-tri- fluoromethylquinolin-4'-ylamino)phenol (TN 119)

A mixture of 4-(7'-trifluoromethylquinolin-4'- ylamino)phenol (0.2g) , formalin (2.0ml) , 2-methyl¬ piperidine (2.0ml) and ethanol (10.0ml) was treated as above. The oily residue was purified by t.l.c. (alumina; chloroform) to give a solid which was recrystallized from aqueous ethanol to give yellow crystals (0.18g) , m.p. 102-104°. (Found: C, 68.5; H, 7.3; N, 10.4. C₃₍₎H₃₇F₃N₄0 requires C, 68.4; H, 7.1; N, 10.6%).

EXAMPLE 28

2,6-Bis(3",5"-dimethylpiperidin-l"-ylmethyl)-4-(7'- trifluoromethylquinolin-4'-ylamino)phenol (TN 120)

4-(7'-Trifluoromethylquinolin-4'-ylamino)phenol (0.2g), formalin (2.0ml), 3,5-dimethylpiperidine (2.0ml) and ethanol (10.0ml) were treated as above.

The product was purified by t.l.c. (alumina; chloroform) to give a yellow oil (0.19g). (Found:

C, 67.0; H, 7.5; N, 9.7. C₃₂H₄₁F₃N₄O.H₂0 requires

C, 67.1; H, 7.6; N, 9.8%) .

EXAMPLES 18 - 28 Toxicity Testing

Each 1,5-naphthyridine and quinoline used for antimalarial screening was tested for acute toxicity in mice by intraperitoneal injection in normal saline or peanut oil. Each test chemical was injected in a single dose of 100 mg/kg of body weight [except for 2,6-bis(4"-methylpiperazin-1"-ylmethyl)-4-(7'-tri- fluoromethylquinolin -4'-ylamino)phenol which, due to the death of one mouse at a dose of 100 mg/kg, was run at 50 mg/kg] to three mice.

No apparent ill effects were observed, and all mice survived to and beyond 30 days in the above tests, and in control experiments with normal saline and peanut oil.

Preliminary Antimalarial Screen

This was carried out as described previously. Each test chemical was given at a dosage of 100 mg/kg of body weight except for 2,6-bis(4"-methylpiperazin- 1"-ylmethyl)-4-(7'-trifluoromethylquinolin-4'- ylamino)phenol which was at 50 mg/kg, and blood counts were made at 9, 24, 48 and 72h and thereafter as shown in Table 3.

TABLE 3.

Times given are those after injection of the chemical under test.

Time: h, hours; d, days; Oh denotes pretreatment.

Mean percentage of parasite-infected red cells Oh 9h 24h 48h 4d 4d 5d 6d 7d 8d 9d lOd lid 12d 14d 19d

TN 117* ^"

10 4 1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 3^A

TN 118* _R

9 3 <1 <1 <1 <1 <1 <1 <1 <1 1 14 28 55

TN 116*

14 20 13 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1

TN 115*

9 6 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1

TN 122*

13 11 1 <1 <1 <1 <1 <1 <1 <1 <1 3 5 19 ^C

TN 124*

4 1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1

TN 125*

10 7 2 1 <1 <1 <1 2 6 17 21 27 7 3 2 <1^U TN 123*

9 5 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1

TN 121** „

7 4 2 <1 <1 <1 <1 <1 <1 5 14 33 40 * TN 119*

18 18 4 2 <1 <1 <1 <1 <1 <1 <1 <1 <1 3 26 ^G TN 120*

12 12 6 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 <1 Normal saline „

11 19 28 57 Peanut oil „

C (normal saline - dose 40 mg/kg)

2 <1 <1 <1 2 2 <1 <1 <1 <1 <1 <1 <1

' Solvent used throughout (except where stated) - peanut oil; Dose iθO mg/kg. ** Dose 50 mg/kg. A Parasitaemia of one mouse 12% at 14 days. This mouse dead at 19 days, parasitaemia of other two mice <1%. B Two mice dead at 14 days, parasitaemia of remaining mouse 8% at 14 days and <1% at 19 days. C One mouse dead at 14 days, parasitaemia of other two average 11%. D Parasitaemia rose in one mouse only, to 80%, then declined. E One mouse dead at 19 days, parasitaemia of other two mice <1%. F Two mice dead at 12 days, parasitaemia of other mouse <1% at 19 days. G All mice dead at 19 days. H One mouse dead at 3 days, all mice dead at 4 days. I Diphosphate.

EXAMPLE 29

Mono- and di-Mannich bases derived from 4-(7'-bromo or 7'-chloro-1' ,5'-naphthyridin-4*- ylamino) henol and 4-(7'-trifluoromethylquinolin-4'- ylamino)phenol were assayed for antimalarial activity (using an in vitro radioisotopic technique) against three isolates of-Plasmodium falciparum. The results are shown in Tables 4, 4A and 5. Many compounds from these two series had an -_-C_5Q value (concentration of compound causing 50% inhibition of H-hypoxanthine incorporation) comparable to or better than those of mefloquine and amodiaquine, for both a chloroquine- sensitive isolate (FCQ-27) and a chloroquine-resistant isolate (Kl) . At least two compounds, 2,6-bis- (piperidin-1' '-ylmethyl)-4-(7'-trifluormethylquinolin- 4'-ylamino)phenol (TN112) and 2,6-bis(3" ,5"-dimethyl- piperidin-1"-ylmethyl)-4-(7'-trifluoromethylquinolin- 4'-ylamino)phenol (TNI 120), showed significant superior activity to the three antimalarials against these isolates. Some toxicity studies on the more active compounds have also been carried out and the results, together with those for chloroquine and amodiaquine for reference, are recorded in Table 6. The 1,5- naph hyridines TN78 and particularly TN79 were significantly less toxic than amodiaquine (or chloroquine) . The 7-trifluoromethylquinolines TN108 and TN112 were, on the evidence available, also less toxic than amodiaquine and chloroquine.

Details of the materials and methods used in the testing against P.falciparum in vitro are set out below.

Parasites

The FCQ27 line of Plasmodium falciparum (origin: Papua New Guinea, first isolated at The Walter and Eliza Hall Institute of Medical Research, Melbourne) (Chen et al.. Southeast Asian Journal of Tropical Medicine and Public Health, 1980, 1_1, 435), was obtained from Dr.G.Butcher (Royal Newcastle Hospital, Newcastle, Australia) . The Kl line (origin: Kanchanaburi, Thailand) (Thaithong and Beale, Transactions of the Rαyal Society of Tropical Medicine and Hygiene, 1981, 75_^, 271) was obtained from Dr. .Saul (Queensland Institute of Medical Research) . The FCQ2 line (origin Madang, Papua New Guinea, first isolated at The Walter and Eliza Hall Institute of Medical Research, Melbourne) (Chen et al., supra) was obtained from Dr. .Saul (Queensland Institute of Medical Research) . All isolates were maintained routinely by the in vitro culture technique of Trager and Jensen (Science, 1976, 193, 673). Stock cultures contained a 5% suspension of washed human erythrocytes, type O in RPMI 1640 medium (Flow), supplemented with 25mM HEPES-KOH pH 7.2, 32mM NaHC0₃ and 10% human serum type O. Gentamicin (40μg/ml) was added to all media. Cultures were maintained in modular incubator chambers (Flow) at 37.5°C in a gas mixture of 5% C0₂, 5% 0₂ and 90% N.,.

Drugs

Chloroquine phosphate was purchased from Sterling Pharmaceuticals (Sydney, Australia) and amodiaquine hydrochloride from Parke Davis and Co., (Sydney). Mefloquine hydrochloride was a gift from the Walter Reed Army Institute of Research. The series of 4-(7'-bromo- and 7*-chloro-1' ,5*-naphthyridine-4'- ylamino)phenols and trifluoromethylquinolin-4*-ylamino phenols were prepared in the laboratories of the John Curtin School of Medical Research, Canberra. Stock solutions of chloroquine and amodiaquine were prepared in ddw and subsequently diluted in RPMI medium. Stock stolutions of mefloquine, and the naphthyridine and the trifluoromethylquinoline compounds were prepared in 10ml of 50% ethanol:ddw. Up to 4ml of alcohol was evaporated off under a current of air and the volume returned to 10ml with ddw. The solutions were subsequently diluted in ddw and thence in RPMI medium. Control experiments demonstrated that the remaining concentration of alcohol did not affect parasite growth and development.

Isotope

3 Incorporation of [G- H]-hypoxanthine was used as the index of parasite growth and development

(Desjardins et al.. Antimicrobial Agents and

Chemotherapy, 1979, ljj_, 710). The isotope was supplied as a lyophylate (5000 mCi/ml) in ampoules containing 5.0 mCi (Amersham, U.K.). For experimental purposes a solution containing 20μCi of H-hypoxan hine per ml of 1640 medium was prepared.

Experimental

Microculture plates (Falcon, 96 well, flat bottom) were prepared as described by Desjardins et al (supra) with some modifications. A volume of 50μl of complete RPMI solution was first added to all wells. Then, leaving Row A as the control row a further 50μl of chloroquine in RPMI was added to the first three wells of Row B and serial twofold dilutions made across the plate and continued in Row C The same procedure was repeated for the test compounds in subsequent rows. A working volume of 50μl was preferred to 25μl in order to reduce replicate error.

A constant volume (175μl) of a 1.5% haematocrit of unsynchronized parasitized erythrocytes (0.2-0.4% parasitaemia) was then added to all wells except the last three of Row A to which was added 175μl of a 1.5% haematocrit of unparasitized erythrocytes. Plates were then incubated for 24h at 37.5°C in the gas mixture described above. At this time a further 25μl

3 of RPMI medium containing H-hypoxanthine (O.lμCi) was added to all wells. The total volume was then 250μl giving a 1:10 dilution of the original drug concentration in the first three wells and twofold dilutions across the row. Determination of the IC-.^

(the concentration causing 50% inhibition of the

3 incorporation of H-hypoxanthine) was based on triplicate results for eight serial dilutions from a starting concentration of 64 nmol/1 for all compounds except chloroquine when the Kl isolate was being tested. Here the starting concentration was 1024 nmol/1.

Following the addition of the isotope, plates were incubated for a further 18h before the contents were harvested on a Titertek (Flow) semi-automated cell harvester and particulate matter deposited onto glass fibre discs. Discs were washed, dried, and placed into scintillation vials containing 5ml of 5% 2,5-diphenyloxazole (PPO) scintillant in toluene and radioactivity measured on an LKB 1217 Rack Beta liquid scintillation spectrometer.

Data analysis

Results were analysed using the technique described by Sixsmith et al (American Journal of

Tropical Medicine and Hygiene, 1984, 3_3_, 772).

Computation of the concentration causing a 50%

3 reduction in the incorporation of H-hypoxanthine was calculated by interpolation using one data point above and one data point below the dpm midpoint between the parasitized and non-parasitized controls. A logarithmic transformation of both concentration and cpm values was made and the formula IC₀ = antilog

(log X_χ + [(log Y_5Q - log Y_χ ) (log X₂ - log X₁)/(log

Y₂ - log Y,)]) was employed. Y_5Q was the dpm value midway between parasitized and non-parasitized control cultures and X- , Y.. , X_ and Y₂ are the concentration and dpm values for the data points above and below the dpm midpoints. Differences between mean IC₀ values were compared using Student's t-test, with P <0.05 considered significant. Toxicity Testing

Mice were injected with a single i.p. dose of the test chemical in peanut oil (0.4ml) (except for amodiaquine hydrochloride and chloroquine diphosphate which were in 0.4ml normal saline), and were observed at 0.5h, 3h and daily thereafter to 7 days.

TABLE 4

In vitro antimalarial activity of a series of

4-(7'-bromo-1* , 5'-naphthyridin-4'-ylamino)phenols chloroquinine, amodiaquine and mefloquine against three isolates of P.falciparum

TN63^E 34.7 ± 11.0 >64

TN77 4.6 ± 2.0 20.8 ± 8.9 4 3^D

TN78 2.9 ± D

1.1 6.6 ± 2.2

TN79 2.5 ± 1.6 8.5 ± 4.0 ^3*7D

TN80 3.0 ± 0.9 9.3 ± 4.3 ⁸4.8D^U

TN81 34.8 ± 9.2 >64

TN82 24.1 ± 0.3 >64

TN83 28.8 ± 4.8 >64

TN84 >64 >64

TN87 34.7 ± 7.6 >64

TNI15 >64 >64

TN116 1.3 ± 0.8 8.4 ± 6 0.5^D

TNI17 >64 >64

TNI18 - 22.6 * ¹² B

Chloroquine 13.0 ± _6.4_B_r 325 ± 199 59.3 ± 20

Amodiaquine 2.9 + 0.3^ 11.7 + 5.5^C 2.6 ± 0.3

Mefloquine 10.1-± 5.4 4.9 ± 3.3 24.6 + 9

A Results are expressed as the mean (± SE) of three tests calculated after logarithmic transformation of data unless specified otherwise.

B Results are the mean of 10 tests.

C Results are the mean of 4 tests.

D Results from a single test.

E 4-(7'-bromo-1' ,5'-naphthyridin-4'-ylamino)-2- (diethylaminomethyl)phenol. TABLE 4A

In vitro antimalarial activity of a series of 4-(7 '-chloro-1 ' ,5'-naphthyridin-4'-ylamino) - phenols, chloroquine, amodiaquine and mefloquine against three isolates of P.falciparum

TN122 - 16.2 + 9.3 -

TNI23 - >64 -

TN124 2.9 ± 0.6 10.6 ± 2.2 -

TNI25 1.8 ± 0.8 16.7 + 5.3

Chloroquine 13.0 ± 6.4 325 ± 199* 59.3 ± 20

Amodiaquine 2.9 ± 0.3° 11.7 ± 5.5 2.6 ± 0.3

Mefloquine 10.1 ± 5.4 4.9 ± 3.3 24.6 ± 9

A Results are expressed as the mean (± SE) of three tests calculated after logarithmic transformation of data unless otherwise specified.

B Results are the mean of ten tests.

C Results are the mean of four tests.

TABLE 5

In vitro antimalarial activity of a series of 4-(7'-trifluoromethylquinolin-4'-ylamino)- phenols, chloroquine, amodiaquine and mefloquine against three isolates of P.falciparum

TNI05 2.1 + 0.4 5.5 + 2.7

TNI07 4.3 + 1.8 7.0 + 2.3

TNI08 1.0 + 0.5 2.6 + 0.3 1.7^E

TNI09 0.8 + 0.4 2.1 + 0.7

TNI10 1.7 + 1.6 2.0 + 0.7

TNI11 4.5 + 0.4 6.8 + 2.2

TNI12 0.5 + 0.3 0.8 + 0.7 ± 0.4^B

TNI19 0.4 + 0.2^B 6.6 + °-⁴B

TNI20 0.9 + 0.6^B 1.0 + 0.3^E

TNI21 3.1 + 0.2^B 8.6 +

Chloroquine 13.0 +

⁶'⁴D 325 + 59.3 + 20

Amodiaquine 2.9 + 11.7 + 2.6 ± 0.3

Mefloquine 10.1 + 5.4 4.9 + 3.3^B 24.6 ± 9

A Results are expressed as the mean (± SE) of 5 tests calculated after logarithmic transformation of the data unless specified otherwise.

B Results are the mean of 3 tests.

C Results are the mean of 10 tests.

D Results are the mean of 4 tests.

E Result from a single test.

TABLE 6

Toxicity Testing

Compound Dosage No.of No.of mice living at the following times after intra

(mg/kg) lXLe xn tests -peritoneal ιnτ<action 0.5h 7d

TN78 200 6 6 6

TN79 800 3 3 3

TN80 100 6 6 6

TNI08 200 6 5 5

TNI12 200 6 5 5

150 6 6 6

Amodiaquine* 200 6 1 1

150 6 6 6

Chloroquine# 100 2 0 0

* Hydrochloride.

# Diphosphate. The acute intraperitoneal LD_5Q of chloroquine in mouse is 73mg/kg.

EXAMPLE 30

In vivo evaluation of compounds TN77, TNI12 and chloroquine against the "ANKA" strain of P.bergei in mice was carried out in a single test as detailed below. Parasites

The Antwerp/Kasapa (ANKA) strain of Plasmodium bergei was isolated on 7 March, 1965 at the Prince Leopold Institute, Antwerp, Belgium from infected mosquitoes caught in the forest galleries of Kasapa, Zaire by Bafort [Vincke,I.H. and Bafort,J. (1968). Resultats de deux ans d'observation sur la transmission cyclique de Plasmodium berghei. Ann.Soc.Belg.Med.Trop. , 48, 439-454]. This strain was obtained from the School of Public Health and Tropical Medicine, Sydney, Australia. Protocol

The test mice were each given a dose of 10 million parasites of the ANKA strain of P.berghei in sodium citrate on Day 1. The parasitaemia of the mice was then allowed to reach £ 20% on Day 5. Then a dose 0.8mg/kg of TN77, TN112, and chloroquine were each given orally to three separate replicate mice for four consecutive days and the parasitaemia followed for up to 21 days.

The results are set out in Table 7.

TABLE 7

Preliminary antimalarial screening results against ANKA strain of P.berghei in mice. Results are the average for three mice with the drugs given on Days 5, 6, 7 and 8.

Drug Day 5 Day 7 Day 9 Day 11 Day 13 Day 21

Nil (control! ) 22 30 48 55 All dead

TN77 24 0 0 0 0 0

TNI12 27 0 0 0 0 0

Chloroquine 21 13 25 49 50 All dead

Claims

CLAIMS:

1. A compound of the general formula (I)

wherein X represents a halogen or halogen-substituted alkyl group;

Y represents CH or N; and R 1 and R2, which may be the same or different, represent hydrogen, aminoalkyl, mono- or di-alkylaminoalkyl, mono- or di-(substituted alkyl)aminoalkyl, mono- or di-(cycloalkyl)aminoalkyl, mono- or di-(aryl)aminoalkyl, mono- or di-(substituted aryl)aminoalkyl, or alkyl substituted by a nitrogen-containing heterocyclic or mono- or di-alkyl- substituted heterocyclic group; provided that R 1 and R2 are not both hydrogen; and

2 that R is not diethylaminomethyl when X is chloro, Y is CH and R is hydrogen; or when X is bromo, Y is N and R is hydrogen.

2. A compound according to claim 1, wherein X represents halogen and Y represents N.

3. A compound according to claim 1, wherein X represents a halogen-substituted alkyl group and Y represents CH.

4. A compound according to claim 1 wherein X represents a bromo, chloro or fluoro group, or a trifluoromethyl group.

5. A compound according to claim 1, wherein R 2 and/or R represent aminomethyl, mono- or di-alkylaminomethyl, mono- or di-(substituted alkyl)aminomethyl, mono- or di-(cycloalkyl)aminomethyl, mono- or di-(aryl)aminomethyl, mono- or di-(substituted aryl)aminomethyl, or methyl substituted by a nitrogen-containing heterocyclic or mono- or di-alkyl substituted heterocyclic group.

6. A compound according to claim 1, wherein R and/or R represent:

-CH₂NMe₂ -CH₂NEt₂ -CH₂NPr₂

7. A compound according to claim 1, wherein both R 1 and R2 are other than hydrogen.

8. A process for the preparation of a compound of the general formula (I), as defined in claim 1, which comprises reaction of a compound of the general formula (II) :

II in which X and Y are as defined in claim 1, with an appropriate amine or nitrogen-containing heterocyclic compound in the presence of formaldehyde.

9. A process according to claim 8, wherein the compound of general formula II is refluxed with the appropriate amine or heterocyclic compound in ethanolic formalin.

10. A process for the preparation of a compound of the general formula (I) as defined in claim 1 which comprises reaction of a compound of general formula

III:

Z

in which X and Y are as defined in claim 1, and Z represents halogen, wit an aminophenol of the general formula IV:

IV

in which R 1 and R2 are as defined in claim 1.

11. A process according to claim 10, wherein the reaction is performed under reflux in aqueous methanol.

12. A pharmaceutical composition for use in treatment of malaria in an animal, including a human, which comprises an effective amount of a compound of the general formula (I) as defined in claim 1, together with a pharmaceutically acceptable carrier or diluent therefor.

13. A method of treating malaria in an animal, including a human, which comprises administering to the animal an effective amount of a compound of the general formula (I) as defined in claim 1.