WO2000014070A1

WO2000014070A1 - Antimalarial compounds

Info

Publication number: WO2000014070A1
Application number: PCT/GB1999/002812
Authority: WO
Inventors: Paul Michael O'neill; Brian Kevin Park; Stephen Andrew Ward
Original assignee: Ufc Limited
Priority date: 1998-09-08
Filing date: 1999-09-08
Publication date: 2000-03-16
Also published as: GB9914763D0; AU5750699A

Abstract

A compound comprising general formula (1), wherein the NR1R2 subtituent is any one of FAQ4, FAQ5 or FAQ7. The compound of the present invention may be used as an active therapeutic substance and preferably as an anti-malarial compound.

Description

ANT1MALARIAL COMPOUNDS

This invention relates to novel fluorinated 4-aminoquinoline

amodiaquine analogues as potential drugs against chloroquine resistant

plasmodium falciparum malaria.

Introduction

Malaria is a disease of immense importance in the tropical and sub¬

tropical areas of the world. It is estimated that forty percent of the world's

population are exposed to the threat of malaria and that there are an

estimated 2 million deaths associated with the disease annually.¹ At present

one of the major concerns in the treatment of malaria is the widespread

resistance² of the malarial parasite Plasmodium Falciparum to chloroquine

( 1 ). As a result there is a need for existing antimalarial drugs such as

amodiaquine (2) to be reassessed and used optimally. Amodiaquine has

been shown to be effective against resistant strains of the malarial parasite,

but its use has been limited by its toxic side-effects.

Drug-Metabolism Design

Our approach to the development of safer antiparasitic drugs involves

defining the precise chemical mechanisms responsible for toxicity of a given

agent. Having established the groups responsible for toxicity and efficacy

it can be determined whether it is possible to improve the therapeutic ratio

of the drug by chemical modification. For such an approach to be successful

it is essential that the structural features involved in toxicity can be

dissociated from those required for activity. To illustrate this approach we

can consider the antimalarial amodiaquine. Amodiaquine is a 4-

aminoquinoline antimalarial which is effective against chloroquine resistant

strains³. However, clinical use of the drug has been severely restricted

because of associations with hepatotoxicity⁴ and agranulocytosis⁵.

Paracetamol (4-hydroxyacetanilide) contains a p-hydroxyanilino moiety

which is believed to undergo P-450 catalysed oxidation to a chemically

reactive quinoneimine (Scheme 1 ). Amodiaquine also contains this

functionality and might be expected to undergo enzymic oxidation to a

reactive metabolite. Studies at Liverpool have shown that in the rat

amodiaquine is excreted in bile exclusively as the 5' thioether conjugates

(glutathione and cysteinyl). This observation indicates that the parent drug

undergoes extensive bioactivation in vivo to form amodiaquine quinoneimine

(AQQI) or semi-quinoneimine (AQSQI) with subsequent conjugate addition of glutathione.⁶ Formation of one of these reactive species in vivo and

subsequent binding to cellular macromolecules could affect cell function

either directly or by immunological mechanisms. Indeed IgG antibodies

which recognise the 5'-cysteinyl group have been detected in patients with

adverse reactions to amodiaquine.^7,8

BINDING TO CELLULAR PROTONS

HEPATOTOXICITYand AGRANULOCYTOSIS

AMODIAQUINE QUINONE IMINE (AQCH)

OH

P-450[O] BINDING TO CELLULAR π= MACROMOLECULES

HEPATOTOXJCΓΓY

NHCOCH3 NCOCH₃

N-ACE Y P- BENZOQUINONE IMINE (NAPQI)

Scheme 1 Oxidative Pathways for Amodiaquine and Paracetamol

The facile bioactivation of amodiaquine in vivo prompted us to

investigate chemical substitution of a type that would decrease the

propensity of the aromatic ring to undergo oxidation. Our initial studies

demonstrated that fluorine substitution at the 2',6'-positions and the 4'-

position produced analogues with significantly higher oxidation potentials

than the parent drug. Furthermore metabolism studies revealed that fluorine

substitution at the 2', expositions and the 4'-positions (FAQ1 , 3) also produced analogues that were most resistant to bioactivation to potentially

toxic quinoneimines.

In addition, these compounds demonstrated impressive antimalarial

activity against the chloroquine resistant K1 strain in vitro and in vivo

against Plasmodium Berghei.

These initial studies⁹ demonstrated that introduction of fluorine can

influence the critical balance between drug activation and drug detoxification

by reducing the process of oxidative bioactivation.

This dissociation of activity and toxicity by chemical modification was

most striking for des-hydroxy-4-fluoroamodiaquine (FAQ1 ) which does not

undergo bioactivation to a reactive metabolite in vivo but has similar

antimalarial activity to amodiaquine.

Resistance Considerations

In terms of drug resistant parasites, a disadvantage with amodiaquine

and fluoroamodiaquine (FAQ1 ) is that both derivatives undergo N-de-

ethylation as a principle means of metabolism in vivo in rodent models.⁹³

The de-ethylated metabolites are considerably less potent than the parent

drugs against chloroquine resistant strains in vitro.

Summary of Invention

This invention is concerned with the preparation of three derivatives

that have been designed, not only to prevent toxic metabolite formation in vivo, but are less likely to undergo metabolism to dealkylated metabolites

that are less potent against chloroquine resistant strains in vivo.

Statement of Invention

Therefore the present invention relates to fluorinated derivatives of

general formula 1 (below) where the NR^ substituent is as denoted in FAQ

4, FAQ 5 or FAQ 7.

General Formula 1 Substituent Code

Description of the Invention

The general synthetic route for the novel fluorinated derivatives is

shown in Scheme 2.

The first step in the sequence involves free radical bromination of 2-

fluoro-5-nitrotoluene using bromine and UV irradiation to give the benzylic

bromide (4) as a crystalline solid in 73 % yield. The resulting benzyl bromide

is then be reacted with a selected amine in a nucleophilic substitution reaction to give (5) which is then reduced using tin/HCI to give (6). The final

step in the reaction sequence involves a nucleophilic aromatic substitution

of the 4-chlorine atom in 4,7-dichloroquinoline to give the required target.

The sequence was repeated using various amines to give the target

fluorinated derivatives.

2-Fluoro-5-nitrobenzylbromide was synthesised as described in O'Neill

et al. J.Med.Chem., 1994, 37, 1 362-1370.

Scheme 2 Synthesis of Fluorinated Derivatives of the 4-Aminoquinoline

Amodiaquine.

Examples

Example 1 FAQ 1 7-Chloro-4-(4'fluoro-5'diethylaminomethylanilino)

quinoline

The benzyl bromide (4) (4 g, 0.017mol) and diethylamine (2.95 g, 0.034 mol) was heated under reflux in toluene for 4h. The hydrobromide

salt of the excess of diethylamine was filtered off and the solvent removed

under reduced pressure. The residue was dissolved in about 10 ml of dry

ether and a solution of 20% ethanolic HCI was added dropwise to give 4-

fluoro-3-(diethylamino)methylnitrobenzene as the hydrochloride salt:; ¹H

NMR (CDCIg, 200MHz) 69.2 (1 H, d, J_H._F = 6.0 Hz, Ar-H), 8.4 (1 H, dd, J_H_

_H = 9.35 Hz, J_H._F = 4.4Hz and Ar-H), 7.4 (1 H,dd,J_H._F and J_H-_H = ⁸-^{8 Hz}>- ⁴-⁵ (2H, s, CH₂N), 3.1 5 (4H, broad s, N(CH CH--) ), 1 .5 (6H, t, N(CH₂CH₃)₂) .

Without further purification this hydrochloride (2.0 g) was dissolved in cone.

HCI and reduced using an excess of tin to give the required amine as a

yellow oil. The amine was dissolved in ethanol and one equivalent of 4,7-

dichloroquinoline added. The solution was heated under reflux for 4h and

a solution of ammonia was added until precipitation of the product occurred.

The product was dried and purified by means of flash column

chromotography with 5% MeOH/95% dichloromethane as eluent:

¹H NMR (CDCI ₃, 200MHz), δ 8.51 (1 H, d, J_H. = 5.0 Hz, Ar-quin.2H),

8.07(1 H, d, J_H__H = 8.8Hz, Ar-quin. 5H), 7.83 (d, 1 H, J_H._H = 2.2HZ, Ar-quin.

8H), 7.49 (1 H, d, J_{H H} = 8.8 Hz, J_H._H = 2.2 Hz, Ar-quin. 5H), 7.40 (d, 1 H, J_H_

_F = 5Hz, ArH) 7.05-7.20 (2H, m, J_H._F= 10.5Hz, ArH), 6.75 (1 H, d, J = 5Hz,

Ar-quin. 3H), 3.7 (2H, s, CH₂N), 2.6 (4H, q, N(CH₂CH₃)₂), 1 .1 (6H, t,

N(CH,CH,),); IR (nujol mull) 2970, 1 595, 1 590, 1 560, 1 510, 1470, 1 390, 1 240, 1210, 1 100, 850 and 809 cm^"1; MS m/z 357 (M⁺'20%), 344 (35%),

342 (100%), 286 (63%), 270 (37%), 250 (81 %), 86 (40%).

Example 2 FAQ 4 7-Chloro-4-(4'fluoro-5'f-butyiaminomethylanilino)

quinoline

This derivative was synthesised as for FAQ1 , except that N-tert-

butylamine was used in the first step of the sequence. The product was

synthesised in 80% overall yield from the bromide. ¹ H NMR (CDC1₃,

200MHz), δ 8.50 (1 H, d, J_H._H = 5.50Hz, Ar-quin. 2H), 7.98 (1 H, d, J_H.

_H = 2.2Hz, Ar-quin. 5H), 7.87 (1 H, d, J_H._H = 2.2Hz, Ar-quin.8H), 7.02-7.46

(4H, m, Ar-H), 6.75 (1 H, d, J_H._H = 5.22 Hz, Ar-H) 3.79 (2H, s, CH₂N), 1 .17

(9H, s, Nt-Bu); LC-MS MeOH (25-60%) AcOH (1 %). m/z 358 (M + , 100%)

Example 3 FAQ5 7-Chloro-4-(4'fluoro-5'pyrollidinylmethylanilino) quinoline

This derivative was synthesised as for FAQ1 , except that pyrollidine

was used in the first step of the synthetic sequence. The product was

synthesised in 90% overall yield from the bromide. 'H NMR (CDC1₃,

200MHz), δ 8.53 (1 H, d, J_H._H = 4.95Hz, Ar-quin. 2H), 8.02 (1 H, d, J_H.

_H = 2.2Hz, Ar-quin. 5H), 7.87 (1 H, d, J_H._H = 2.2Hz, Ar-quin. 8H), 7.02-7.48

(4H, m, Ar-H), 6.81 (1 H, d, J_H._H = 4.40 Hz, Ar-H) 3.79 (2H, s, CH₂N), 2.71

(4H, s, 2 x CH₂, Py) 1 .91 (4H, s, 2 x CH₂, Py); LC-MS MeOH (25-60%)

AcOH ( 1 %) . m/z 356 (M + 1 ) 100%

Example 4 FAQ7 7-Chloro-4-(4'fluoro-5'-{3"-hydroxypyrollidinylmethyl aniiino) quinoline

Synthesised as for FAQ1 except that 3-hydroxypyrollidine was used

in the first step The product was synthesised in 90% overall yield from the

bromide. ¹ H NMR (CDC1₃, 200MHz), δ 8.53 (1 H, d, J_H__H = 5.50 Hz, Ar-quin.

2H), 8.07 (1 H, d, J_H._H = 2.2 Hz, Ar-quin. 5H), 7.87 (1 H, d, J_H._H = 8.80 Hz, Ar-

quin. 8H), 7.02-7.48 (4H, m, Ar-H), 6.78 ( 1 H, d, J_H._H - 4.95 Hz, Ar-H) 4.36

( 1 H, m, CHOH) 3.73 (2H, s, CH₂N), 2.15-2.87 (2H, m, py), 1 .91 (4H, s, 2

x CH₂ py); LC-MS MeOH (25-60%) AcOH ( 1 %). m/z 372 (M + 1 ) 100%

Biological Properties of the Invention

In Vitro Antimalarial Activity

Two strains of Plasmodium falciparum were used in this study: a) the

uncloned 1 strain which is known to be chloroquine resistant and b) the

HB3 strain which is sensitive to chloroquine. The IC50 measurement in this

study represents the concentration of each drug required to inhibit uptake

of radiolabelled hypoxanthine from the culture medium.

Table 1 In Vitro Antimalarial Testing of Fluorinated derivatives vs

Chloroquine Resistant K1 Plasmodium Falciparum

Drug IC50 nM Standard Error

Chloroquine 300 2.3

Amodiaquine 20 1 .3

Fluoroamodiaquine FAQ1 35 5.5 FAQ4 39 5.5

Fluoroamopyroquine FAQ5 10 2.8

FAQ7 30 2.9

Table 2 In Vitro Antimalarial Testing of Selected Fluorinated derivatives vs

Chloroquine Sensitive HB3 Plasmodium Falciparum

Drug IC50 nM Standard Error

Chloroquine 35 4.3

Amodiaquine 20 7.3

Fluoroamodiaquine FAQ1 37 4.5

FAQ4 20 2.3

Fluoroamopyroquine FAQ5 22 2

FAQ7 45 4.9

In Vivo Antimalarial Activity

FAQ 4 and FAQ 5 were selected for in vivo antimalarial activity based

on their potent in vitro potency. Amodiaquine was used as reference drug.

The NS strain of Plasmodium berghei was used to infect a total of 45 mice.

Both FAQ 4 and FAQ 5 demonstrated potent in vivo antimalarial activity.

FAQ 4 was active at 5 mg/kg with complete clearance of parasitaemia at 10

mg/kg. FAQ 5 was also active at 10 mg/kg (See Graph 1 ). The ED50 of

amodiaquine was 10 mg/kg indicating that these two new antimalarials, FAQ

4 and FAQ 5, are potent antimalarials, both in vivo and in vitro. Graph 1 Dose response of NS Berghei versus Amodiaquine FAQ 4 and

FAQ 5

percent of

control

growth

0.1 10

cone, (mg/kg)

Amodiaquine (AQ) IC50 = 10.71 (mg/kg)

FAQ 4 ED50 = 5.50 mg/kg

FAQ5 ED50 = 10.38 mg/kg

Biological Testing Protocols

In vitro

Two strains of Plasmodium falciparum from Thailand were used in this

study: a) the uncloned K1 strain which is known to be chloroquine resistant

and b) the cloned T9.96 strain which is sensitive to all antimalarials.

Parasites were maintained in continuous culture using a method derived from that of Jensen and Trager¹⁰. Cultures were maintained in

culture flasks containing human erythrocytes (2-5%) with parasitaemia

ranging from 0.1 -10% suspended in RPMI 1 640 medium supplemented with

25mM HEPES buffer, 32mM NaHCO₃ and 10% human serum (complete

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

N₂.

Antimalarial activity was assessed using an adaptation of the 48 hour

sensitivity assay of Desjardins et al^{1 1} using [³H]- hypoxanthine incorporation

as an assessment of parasite growth. Stock drug solutions were dissolved in 100% ethanol and diluted to an appropriate concentration with complete

medium (final concentrations contained less than 1 % ethanol). Assays were

performed in sterile 96 well microlite plates, each well containing 100μL of

medium which was seeded with 10μL of a parasitised red blood cell mixture

to give a resulting initial parasitaemia of 1 % with a 5% haematocrit. Control

wells (which constituted 100% parasite growth) consisted of the above,

with the omission of the drug.

After 24h incubation at 37°C, 0.5/yCi of Hypoxanthine was added to

each well. After a further 24 hours incubation the cells were harvested onto

filter mats, dried overnight, placed in scintillation vials with 4mL of

scintillation fluid and counted on a liquid scintialltion counter.

IC50 values were calculated by interpolation of the probit transformation of the log-dose response curve. Each compound was tested

against both strains to ensure reproducibility of the results.

In vivo

The standard 4-day test was used to obtain ED50 values ¹²

References

(1 ) WHO Practical Chemotherapy of Malaria 1990. Technical Report

Series No. 805. World Health Organisation, Geneva.

(2) Krogstad, D.J., Gluzman, I.Y., Kyle, D.E., Oduda, A.M.J, Martin, S.K.,

Milhous, W.K., Schleisinger, P.H., Science, 1987, 235, 1283.

(3) Watkins, W.M., Sixsmith, D.G., Spencer, H.G., Boriga, D.A., arjuki,

D.M., Kipingor, T., Koech, D.K. Lancet I, 1984, 357.

(4) Neftel, K.A., Woodtly, W., Schmid, M., Br. Med. J., 1986, 292, 721 .

(5) Lind, D.E., Levi, J.A., Vincent, P.C., Br. Med. J., 1973, 1 , 458.

(6) Harrison, A.C., Kitteringham, N.R., Clarke, J.B., Park, B.K., Biochem.

Pharmac, 1992, 43, 1421 .

(7) Clarke, J.B., Maggs, J.L., Kitteringham, N.R., Park, B.K., Int. Arch.

Allergy Immunol., 1990, 1335-1342.

(8) Maggs, J.L., Kitteringham, N.R., Park, B.K., Biochem. Pharmac,

1988, 37, 303-31 1 .

(9) O'Neill P.M., Harrison A.C., Storr R.C., Hawley S.R., Ward S.A., Park

B.K., J Med Chem, 1994, 37, 1362. (9a) O'Neill, P.M., PhD Thesis, University of Liverpool, 1995.

(10) Jensen, J.B,; Trager, W.J. Parasitol.,.1977, 63, 883-886.

(11) Desjardins, R.E.; Canfield, J; Haynes, D.; Chulay, D.J. Antimicrob.

Chemotherap, 1979, 16, 71.

(12) Peters, W., Robinson, B.L., Rossiter, J.C, Jefford, C.W. Ann. Trop.

Med. Prasitol. 1993, 87, 1.

Claims

A compound comprising general formula 1 characterised in that the

NR_tR₂ substituent is any one of FAQ4, FAQ5 or FAQ7.

General Formula 1 Substituent Code

A compound according to claim 1 either by itself or in combination

with any other compound(s) for use as an active therapeutic

substance.

A compound according to claim 1 in combination with a

pharmaceutically acceptable carrier.

A compound according to claim 1 either by itself or in combination

with any other compound(s) for use as an anti-malarial.

Use of a compound according to claim 1 in the manufacture of a

medicament for use in the treatment of malaria.

A method of manufacture of a compound comprising general formula

1 characterised by the reaction scheme 2 shown below.

NH₂ (6)

line

Reaction scheme 2

A method of manufacture according to claim 6 wherein the reactants

and/or reaction conditions used in the separate stages of the reaction

scheme are as follows:-

(i) Br₂, UV, reflux;

(ii) HR, toluene, reflux;

(iii) Sn, HCI; and

(iv) 4,7-Dichloroquinoline, EtOH, reflux.

8. A method of manufacture according to claims 6 or 7 wherein the

NR,R₂ substituent is any one of FAQ4, FAQ5 or FAQ7.