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(11) Patent Number: KE 36
(45) Date of grant: 21/04/1998

 (51) Int.Cl.6: A 61K 31/47, C07D 215/44, 215/46
(21) Application Number: 1995/ 000029
(22) Filing Date: 03/06/1995
(30) Priority data: 1928/94-0 17/06/1994 CH
(86) PCT data PCT/EP95/02123 03/06/1995
WO 19/950352    28/12/1995
(73)    Owner:
F.HOFFMANN-LA ROCHE AG; of , Grenzacherstrasse 124, CH-4002 Basle, Switzerland
(72) Inventor: LEUPIN, Werner; and HOFHEINZ, Werner;
(74)    Agent/address for correspondence: JANE W. WANYAGA & CO. P.o.Box Nairobi 28275 Kenya
(57) Abstract:
Disclosed are N,N-bis(quinolin-4-y1)-diamine derivatives of general formula (I) wherein RI signifies halogen or trifluoromethyl, R2 signifies hydrogen or halogen, A signifies cyclohexane-1,3-diyi, 2-methyl-cyclohexane-1,3-diyl, cyclohexane-1,4-diyl, dicyclohexylmethane-4,4'diyl, cyclopentane-1,3-diyl, phenylene-1,4, phenylene-1,3, phenylene-1,2, ethane-1,2-diy1 or ethyne-1,2-diy1 and is and m can be the same or different and signify 0, 1 or 2, as well as their pharmaceutically acceptable salts. These products are useful as agents for preventing malaria and for treating it, especially where the pathogens are resistant to chloroquine.
The invention is concerned with novel diamines, namely N,N1-bis(quinolin-4-y1)-diamine derivatives of the general formula wherein 121 signifies halogen or trifluoromethyl, R2 signifies hydrogen or halogen, A signifies cyclohexane-1,3- diyl, 2-methyl-cyclohexane-1,3-diyl, cyclohexane-1,4-diyl, dicyclohexylmethane-4,4'-diyl, cyclopentane-1,3-diyl,
phenylene-1,4, phenyiene-1,3, phenylene-1,2, ethene-1,2-diyl or ethyne-1,2-diyl and n and m can be the same or different and signify 0, 1 or 2, as well as their pharmaceutically acceptable salts.

These novel compounds have very good activities against not only chloroquine-sensitive, but also against chloroquine resistant malaria pathogens. For this reason they are very well suited for preventing malaria and for treating it, especially where the pathogens are resistant to chloroquine.
 Objects of the present invention are the novel bis-quinoline derivatives of general formula I as well as pharmaceutically usable salts thereof per se and as pharmaceutically active substances, the manufacture of these compounds and salts, medicaments containing a compound of formula I or a pharmaceutically acceptable acid addition salt thereof, the production of such medicaments and the use of the compounds of formula I and their pharmaceutically acceptable salts in the control or prevention of illnesses, especially of malaria, and, respectively, for the production of such medicaments.
In formula I R1 preferably signifies chlorine, A preferably signifies cyclohexane-1,3-diyl, 2-methyl-cyclohexane-1,3-diyi, cyclohexane-1,4-diyl, dicyclohexylmethane-4,41-diyl, cyclopentane-1,3-diyl, phenylene-1,4, phenylene-1,3 or 5 phenylene-1,2 and n and m preferably signify 0 or 1.
Compounds of formula I in which R1 signifies chlorine, A signifies ethene-1,2-diy1 and n and m signify 1 are also preferred.
Particularly preferred compounds of general formula I are: (7-Chloroquinolin-4-y1)-[2-(7-chloroquinolin-4-yl-aminomethyl)-benzyl]-amine, (7-chloroquinolin-4-y1)-[3-(7-chloroquinolin-4-yl¬aminomethyl)-benzA-amine (7-chloroquinolin-4-y1)-[4-(7-chloroquinolin-4-yl-aminomethyl)-benzyl]-amine,
±-trans-N,N'-bis-(7-chloroquinolin-4-yI)-cyclohexane-1,4-diamine, cis-N,N1-bis-(7-chloroquinolin-4-y1)-cyclohexane-1 ,4- diamine, cis-N,N'-bis-(7-chloroquinolin-4-y1)-cyclohexane-1,3- diamine,
z    ±-trans-N,N'-bis-(7-chloroquinolin-4-yI)-cyclohexane-1,3- diamine, cis,cis-NN-bis-(7-chloroquinolin-4-y1)-2-methyl¬cyclohexane-1,3-diamine, N,N'-bis-(7-chloroquinolin-.4-y1)-phenylene-1,3-diamine,  cis-NX-bis-(7-chIoroquinolin-4-y1)-cyclopentane-1,3- diamine, (7-chloroquinolin-4-y1)43-(7-chloroquinolin-4-yl-aminomethyl)-cyclohexylmethylj-amine, N,N'-bis(7-chloroquinolin-4-y1)-dicyclohexylmethane-4,4'- 55 diamine and N,N'-bis-(7-chloroquinolin-4-y1)-2-butene-1,4-diamine.
The novel compounds of formula I can be manufactured in accordance with the invention by
a)    reacting a compound of the general formula

R1                                      N

wherein R1 and R2 have the significance set forth above and X signifies a leaving group,
in with a diamine of the general formula
                                H2N - (CH2)n - A - (CH2)m - NH2    III
Wherein n, m and A have the significance set forth above, 35 or an acid addition salt thereof, and
b)    if desired, converting the compound obtained into a pharmaceutically acceptable acid addition salt.
 Bis-quinoline derivatives of general formula I are manufactured according to variant a) of the process in accordance with the invention by reacting a corresponding compound of general formula II with the corresponding diamines of general formula III. α,α`-Diamino-o-xylene, α,α-diamino-m-xylene, a,α  diamino-p-xylene, ±-trans-cyclohexane-1,4-diamine, cis-cyclo-hexane-1,4-diamine, cis-cyclohexane-1,3-diamine, ±-trans-cyclohexane-1,3-diamine, cis,cis-2-methyl-cyclohexane-1,3-diamine, phenylene-1,3-diamine, cis-cyclopentane-1,3-diamine,1,3-bis-(aminomethyl) cyclohexane, 4,4'-diamino-dicyclohexy1-
methane or trans-2-butene-1,4-diamine can be used, for example, as the diamine. In place of the free diamines, their acid addition salts can also be used.
Suitable compounds of formula II are, for example, 4, 7- dichioroquinoline or 4-chloro-7-trifluoromethyl-quinoline.

 The reaction is conveniently effected under a protective gas atmosphere in a temperature range of 100-2000C and in a solvent, with phenol, N-methyl-2-pyrrolidone, N,N-dimethylacetamide, ethoxyethanol or acetonitrile being preferred. The presence of a tertiary amine such as triethylamine influences the reaction favorably, but is not essential. When the acid addition salt of a diamine is used, the presence of a tertiary amine such as triethylamine is required. The reaction time can vary between 2 and 28 hours.
The conversion into a pharmaceutically acceptable acid addition salt according to process variant b) is conveniently effected by adding an acid. Hydrochloric acid, methanesulphonic acid or acetic acid is especially preferred because of the physiological compatibility of the corresponding salts.
Convenient solvents which are especially suitable are: water, methanol, ethanol, isopropanol, diethyl ether, N, N-dimethyl formamide or dioxan.
The diamines and quinoline derivatives which are required as starting materials for process variant a) are commercial products or can be prepared according to methods known per se.
As mentioned earlier, the bis-quinoline derivatives of general formula I in accordance with the invention and their pharmaceutically usable salts have extremely valuable pharmacological properties.
The following Tables 1-3 show their activity against not so only chloroquine-resistant, but also chloroquine-sensitive malaria pathogens.
Test method for the determination of the activity against Plasmodium falciparum in vitro
The preparations are tested on intraerythrocytary stages of Plasmodium falciparum from asynchronous cultures according to the method of Desjardin et al. (Desjardins, R.E. et al: Quantitative assessment of antimalarial activity in vitro by a semiautomated microdilution technique. Antimicrob. Agents Chemother. 16, 710 -718, (1979)).
The culture medium consists of RPMI 1640 with the addition of 25 mM HEPES, 25 mM NaHCO3, 100 µg/m1 neomycin and 105 human serum (A+) Human-A+ erythrocytes are used as the Plasmodium falciparum host cells. The parasites are maintained at 37°C in an atmosphere of 3% O2, 4% CO2, 93% N2 and 95% relative humidity.
In order to determine the activity, the preparations are dissolved in dimethyl sulphoxide, pre-diluted in the culture medium to a suitable starting concentration and subsequently is titrated-out on to microtitre plates in the 2nd stage over 6-7 steps. After the addition of the parasite culture (0.7% parasitemia in 2.5% erythrocyte suspension) the test plates are incubated under the conditions given above for 48 h -72 h. The parasite growth in the different preparation concentrations is determined using [G-3H]-hypoxanthine incorporation compared to untreated control cultures on the same test plates. The 50% growth inhibition (IC30) is calculated according to logit regression analysis from the resulting dosage-activity curve.
The preparations are tested on at least one chloroquine resistant and one chloroquine-sensitive Plasmodium falciparum strain. Additional sensitive and resistant strains are included for further characterization.
Test method for the determination of the activity against Plasmodium berghei in vivo
The preparations are tested on mice infected with malaria pathogens (Plasmodium berghei). Male albino mice (IBM: MORO (SPF), FUELLINSDORF) weighing about 25 g are used as the test animals. They are kept in climatized rooms at 21-22°C in groups of 5 animals per cage. They receive ad libitum a diet feed with a low PABA content (NAFAG FUTTER 0 No. 9009 PAB-45, PABA content 45 mg/kg) and drinking water. On the first day of the test (DO) the test animals are infected with Plasmodium berghei (strain ANKA). For this there is used heparinized blood of a donor mouse with about 30% parasitemia, which is diluted with physiological saline such that it contains 108 parasitized erythrocytes per ml. 0.2 ml of this suspension is injected intravenously (i.v.) into the mice to be treated and into the control mice. In untreated control animals the parasitemia normally reaches 30-40% on the third day after the infection
3.0 (D+3) and the test animals die between days +5 and +7.
The substances to be tested are dissolved or suspended in distilled water or in a mixture of 7% Tween 80, 3% alcohol (96%) and water. Usually, 0.25 ml of this solution or suspension is administered once subcutaneously and perorally to groups of 5 test animals. Treatment is effected 24 hours after the infection. 10 control animals are treated in the same manner with solvent or suspension medium per test.
All substances are tested in a first test in a single dosage of 10 mg/kg. Only those substances which in this test (10 mg/kg) have shown a parasitaemia reduction of 90% is used for the titration. Suitable dilutions of the test substance can be used to obtain an accurate titration of the activity.

48 hours after the treatment (D+3) blood smears are prepared from all animals using blood from tail veins and are stained with giemsa. The average erythrocyte infection rate (parasitemiea in %) in the control groups as well as in the groups which have been treated with the test compounds is determined by counting under a microscope. The difference in the average values of the infection rates of control group (100%) and treated groups is calculated and expressed as a percentage reduction (GI%). The ED50 or ED90 is determined mathematically by means as of the JMP programme (nonlinear fit). The ED50 (ED90) in mg/kg is that dose which after single administration reduces the average erythrocyte infection rate by 50% (90%) in comparison to the control group.


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