AP102A - Compounds for use for breaking resistance to antimalarials - Google Patents

Abstract

Racemic and optionally active substituted phenylacetonitriles of the formula 1 where r1, r2 and r3 are hydrogen, halogen, c1-4 alkyl, c1-4-alkoxy trifluoromethyl, or two adjacent substituents. R1and r2 or r3 to another form -ch2 -ch -ch2 -ch2 or -ch=ch-ch=ch, r4 is alkyl or cycloalkyk having up to 15 carbon atoms or phenyl, r5 is hydrogen or c1-4-alkyl, are each 2,3 or 4 and up m and n is alkyl having up to 20 carbon atoms, c1-5-cycloalkyl or the group where r7, r8, and r9 are hydrogen, halogen, c1-c4, -alkyl, c1-c4-alkoxy, trifluoromethyl, or two adjacent substituents r2 and r8 or r9 together form -ch2-ch2-ch2- or -ch=ch-ch=ch;as well as the salts thereof with physiologically tolerated acids which can be used to prepare drugs to break resistance to antimalarials such as chloroquine, hydrochloroquinine, uinine, pyrimethamine, mefloquine and primaquine in malaria patients.

Description

The present invention relates to the use of substituted phenylacetonitriles for breaking resistance to antimalarials.

The development of resistance to chemotherapy, especially including multiple resistance, continues to be a great problem in medicine. This may result in proven drugs becoming completely useless, and often no satisfac10 tory alternative is available. The ways in which resistance develops are still substantially unexplained.

Resistance cells are able to pump medicaments, which have penetrated into them, to the outside again or else to detoxify them chemically (Spectrum der Wissen15 schaft, October 1988, page 30/31).

The observation that verapamil is able to break resistance in cancer cells (JP-82-83 624/1983) and

Plasmodia (Proc. Nat. Acad. Sci. USA, 83, (1987), 7310, Science, 235, ( 1987), 899) has therefore led rapidly to clinical uses in oncology. In this connection the known cardiovascular activity of verapamil proved tc· be a side effect interfering with utilizability in practice. The high doses which are necessary entail special clinical precautions (intensive care unit) for use, or prevent them altogether.

It is furthermore known that the potent calcium antagonists verapamil and nifedipine also reduce the resistance of malaria pathogens to chemotherapeutics (WO 88/03802) .

We have now found that certain substituted phenylacetonitriles can be used advantageously for breaking resistance.

The present invention relates to the use of those

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I, where

R¹, R² and R³

R⁴

Rl

R2 I CN R5

Xy- C-(CH₂) -N-(CHj -R6

Ti. ·

R J are hydrogen, halogen, C_x.*alkyl, C..,alkoxy, trifluoromethyl, or two adjacent substituents R¹ and R² or R³ together form -CH₂-CH₂-CH₂-CH₂- or -CH=CH-CH=CH-, is saturated or unsaturated alkyl or cycloalkyl having up to 15 carbon atoms or phenyl, is hydrogen or Cx.₄-alkyl, m and n are each 2, 3 or 4, and is saturated or unsaturated alkyl having up to 20 carbon atoms, C^-cycloalkyl or the group

Rfl

I V

R9 where R⁷, R⁸ and R⁹ are identical or different and are hydrogen, halogen, Οχ-C,-alkyl, Cx-C4-alkoxy, trifluoromethyl, or two adjacent substituents R⁷ and R⁸ or R⁹ together form -CH2-CH₂-CH₂-CH₂- or -CH=CH-CH=CH-, as well as the salts thereof with physiolgically tolerated acids, which

a) have an EC₅₀ above 10⁷ in the test for a calciumantagonistic action described in Advances in Myocardiology Vol. 4 (1983) on page 506,

b) have an ED₂₀ above 1.0 mg/kg in the test for an action lowering blood pressure, and

c) have a threshold dose above 2.0 mg/kg in the test for an AV-blocking action, for the preparation of drugs for breaking resistance to antimalarials. -----AP 0 0 0 1 0 2

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Over 10'⁷ means that the figure is greater than IO'⁷, for example IO’⁶.

The breaking of resistance applies, in particular, to the following antimalarials: chloroquine, hydrochloroquine, quinine, pyrimethamine, mefloquine and primaquine, as well as combinations of these substances.

The following substances are particularly suitable for breaking resistance to antimalarials:

a. 1-(3-Methoxyphenyl) -3-aza-7-cyano-7- (3,4,5-trimethoxyphenyl)-8-methylnonane hydrochloride

b. (-)-(S)-l,7-Bis(phenyl)-3-methylaza-7-cyano-8methylnonane hydrochloride c . (+)-1-(3,4-Dimethoxyphenyl)-3-methylaza-7-cyano-7( ),4,5-trimethoxyphenyl)-8-methylnonane hydrochloride

d. ( /.) -1-Cyclohexyl-3-methylaza-7-cyano-7 - (3,4,5tiimethoxyphenyl)-8-methylnonane hydrochloride

e. l-Cyclohexyl-3-methylaza-7-cyano-7-phenyl-8-methylnoiane hydrochloride f . 7-4ethylaza-ll-cyano-ll-(3,4,5-trimethoxyphenyl)-12methyltridecane hydrochloride

g. 1,7-Bis(phenyl)-3-aza-7-cyano-8-methylnonane hydrochloride

h. (R) — 1— (3-Methoxyphenyl)-3-methylaza-7-cyano-7-(3,4dimethoxyphenyl)-8-methylnonane hydrochloride 2isopropyl-valeronitrile i . l-Cyclohexyl-3-methylaza-7-cyano-7,7-bis-(3,4dimethoxyphenyl)-heptaneamidosulfonates j · ( + ) - (R) -1,7-Bis (pheny1)-3-methylaza-7-cyano-8methylnonane hydrochloride

k. R-Verapamil

Of these substances, b, c, d, e and k are preferred .

The abovementioned substances can, if desired, be in the form of their salts with physiological acids. Suitable and preferred physiologically tolerated acids are: hydrochloric, sulfuric, phosphoric, acetic, citric,

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e.

malonic, salicylic, maleic, fumaric, succinic, ascorbic, malic, methanesulfonic, lactic, gluconic, glucuronic, sulfamic, benzoic and tartaric acids.

The compounds of the formula I can be adminis5 tered together with or separate from the antimalarials.

The compounds I are usually administered orally, whereas the antimalarials (= active substance) are given orally or parenterally (for example i.v., i.p.).

The ratio of compound I to active substance 10 depends on the disease to be treated, the medical condition of the patient and the active substance used. As a rule, the ratio is about 1:1 to 500:1, preferably 0.1:1 to 10:1. In this connection, the compounds I are usually administered in an amount of from 2C to 2,000 mg per patient and day on oral dosage and 10 to 300 mg on intravenous or 20 to 500 mg on intraperitoneal dosage per patient and day. The active substances are administered in the amount which is also intended for dosage of these substances alone and which can be found, for example, in the Rote Liste 1988 and the scientific data sheets mentioned therein.

The substances can be in the form of tablets, capsules or coated tablets for oral administration or of an injection solution for parenteral (i.v., i.p·, i.m.) administration. Solutions can also be infused. The administration forms are prepared in a known manner by conventional methods .

The test for a calcium-antagonistic reaction described in Advances in Myocardiology, Vol. 4 (1983) on page 506 is carried out as follows:

Two test contractions were induced with calcium in spiral strips of aorta (20 mm long, 2 mm wide), depolarized with K’, from Sprague-Dawley rats (body weight 250-300 g, under ether anesthesia). The preload was 1 g;

relaxation was achieved within about 1 h in tyrode solution at 37'C, through which was bubbled a mixture of 95% O₂ and 5% CO₂ (pH 7.3-7.4). The strips were rinsed

three times for 5 min in Ca-free tyrode solution containing 0.2 mM NaEDTA. The Ca-free strips were depolarized with tyrode solution in which 100 mM Na* had been replaced by K*. After 10 min, the first contraction was generated with 0.5 mM Ca²\ 15 min later the strips were washed for min with Ca-free tyrode solution containing 0.2 mM NaEDTA and again depolarized. 15 min before the second test contraction generated with calcium (0.05 mM) the drugs were added. The tension was recorded isometrically.

The percentage inhibition of the calcium contraction in the presence of various drug concentrations was evaluated. Linear regressions were calculated from the linear part of the concentration-effect plot. The relative activities were estimated from the distance between these linear regression lines.

The action lowering blood pressure was determined after i.v. administration on Sprague-Dawley rats under urethane anaesthesia by measuring the mean blood pressure in the carotid artery. The ED 20% was calculated from the dose-effect relations as the dose (mg/kg) which lowers the blood pressure by 20%. Furthermore, the dose (threshold dose) after which blockade of atrioventricular conduction (second degree AV block) occurs in the ECG was determined.

The use of the substituted phenylacetonitriles is advantageous for malaria control inasmuch as they display better cardiac and vascular tolerability.

PREPARATION EXAMPLES

1. 500 mg of (-)-(S)-1,7-bis(phenyl)-3-methylaza-730 cyano-8-methylnonane hydrochloride and 200 mg of chloroquine were dissolved in 250 ml of physiological saline solution, sterilized and introduced under sterile conditions into an infusion bottle.

2. Tablets of the following formulation were prepared:

( + )_i_(3_z 4-Dimethoxyphenyl)-3-methylaza7-cyano-7- ( 3,4,5-trimethoxyphenyl )-8methylnonane hydrochloride 0 0 mg

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Chloroquine di(hydrogen sulfate) 50 mg

Auxiliaries 200 mg

The efficacy of the combination therapy of malaria was determined in the following test methods.

Test method I

All the substances were tested, initially alone and then in combination with the antimalarial, on human erythrocytes infected with resistant Plasmodia (Pl. falciparum). The incorporation of radiolabeled hypoxan10 thine was used to measure the growth of the parasites.

Using the microdilution technique (Antimicrob. Agents Chemotherapy 16, (1979), 710) revealed that the substances according to the invention alone inhibited hypoxanthine incorporation not at all or only slightly. By contrast, in combination with an antimalarial a distinctly synergistic inhibition was observed. The FIC index (a mathematical representation of an isobologram) was used for evaluation. A figure of 1.0 means additive effects and a figure <0.5 means synergism. The figure obtained for racemic verapamil and chloroquine is 0.45.

[A]

FIC index = + [B]

- (ng/ml)

IC₅ob

IC₅₀ = Concentration of test substance bringing about 25 50% inhibition of the parasites.

[A] = IC₅₀ of substance A measured in the presence of a defined amount (e.g. 500 to 125,000 ng/ml) of substance B.

[B] = ic_io of substance B measured in the presence of a defined amount (e.g. 500 to 125,000 ng/ml) of substance A.

IC_50a = IC₅₀ of substance A given alone.

IC_50B = IC_J0 of substance B given alone.

The figures determined in combination with 35 chloroquine were as follows:

Test substance	FIC index
R-verapamil	0.56
b)	0.19
c)	0.53

5	d)	0.52
	g)	0.19
	h)	0.29
	k)	0.37

Thus it has been shown that chloroquine and other 10 antimalarials become highly effective again, even against resistant malaria pathogens, by addition of the substanΓ' ces according to the invention.

v- Test method II

For confirmation and further differentiation of 15 the resistance-breaking effect, mice were inoculated, following the method described in Ann. Trop. Med. Parasit., 72, (1978), 23 (cf. also Exp. Ptrasitol. 17, (1965), 89, An. Trop. Med. Parasit. 6, (1975), 155) with erythrocytes infected by parasites and, 1 day after the injection, treated on four consecutive day.j. The pirasjtemia was determined one day after the end of treatment.

1. General test conditions

Male Swiss albino mice weighing from 18 to 20 g and free of Eperythrozoon coccoides are used for all the tests.

They are housed in plastic cages with 5 mice in each cage in rooms with a controlled temperature (22* ♦ 2*C).

Test parasite

P. yoelii NS, obtained from P. yoelii N: moderately resistant to chloroquine. Maintained by cyclic passage through Anopheles Stephensi with exposure to drugs in the mouse (60 mg/kg s.c. once ’ 35 during passage) (for details, see Ann. Trop. Med.

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2. Test of the schizontizidal action in blood

Male mice are inoculated intravenously with erythrocytes (10⁷) infected with parasites of the P. yoelii NS strain. The animals are then treated once a day on four consecutive days, starting on the day of infection. The compounds are dissolved or suspended, using *Tween 80, in sterile distilled water and administered subcutaneously. In cases where preparation of an aqueous solution proves to be extremely difficult the test substance is first dissolved in dimethyl sulfoxide. Aqueous dilutions are subsequently prepared .for use. The parasitemia is determined on the day after the final treatment. The suppression of the parasites by comparison with the untreated controls is calculated by probit analysis of the log dose/effect plot.

EXAMPLES EXAMPLE 1

Mice infected with the chloroquine-resistant

Plasmodium yoelii ssp were treated subcutane ?usly ’.vith increasing doses of chloroquine over 4 days- as desct'Joed under II.2.

One day after the treatment the parasitemia was determined and compared with that of the control. Even

TABLE 1

Daily dose mg/kg chloroquine	Number of mice	Mean control % Parasitemia parasitemia
0.3	5	—	100
1.0	5	-	79
3.0	5	-	32
10.0	5	-	14
30.0	5	-	7
60.0	5	-	7
0	10	23	100
In the	EXAMPLE 2 same test run R-verapamil	(in place of
chloroquine) was found	to have virtually no effect on the
parasitemia.

TABLE 2

Daily dose mg/kg R-verapamil	Number of mice	Mean control parasitemia	% Parasitemia
0.1	5	—	83
0.3	5	-	79
1.0	5	-	77
3.0	5	-	82
10.0	5	-	85
0	10	26

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EXAMPLE 3

Infected mice were treated as in Example 1 but now with R-verapamil and chloroquine simultaneously. The

TABLE 3a

Daily dose mg/kg R-verap. + chlor	Number of mice	Mean control parasitemia	% Parasitemia
0.1 + 30.0	5	—	5
0.3 + 30.0	5	-	4
1.0 + 30.0	5	-	4
3.0 + 30.0	5	-	3
10.0 + 30.0	5	-	3
0	10	26

TABLE 3b
Daily dose	Number	Mean control	% Parasitemia
mg/kg	of mice	parasitemia
R-verap. + chlor	•
0.1 + 60.0	5	—	3
0.3 + 60.0	5	-	2
1.0 + 60.0	5	-	2
3.0 + 60.0	5	-	0.7
10.0 + 60.0	5	-	0.4
0	10	26

The parasitemia was virtually completely suppressed with small doses of R-verapamil which are tolerated excellently.

EXAMPLE 4

Test substance d) alone had only a very weak effect on the parasitemia.

TABLE 4

Daily dose mg/kg d)	Number of mice	Mean control parasitemia	% Parasitemia
0.1	5	-	92
0.3	5	-	94
1.0	5	-	81
3.0	5	-	78
10.0	5		73

10

EXAMPLE 5

The combination of substance d) and chloroquine has a highly synergistic effect even at low doses in the test method used in Example 1. TABLE 5
Daily dose mg/kg d) + chlor.	Number of mice	Mean control parasitemia	%	Parasitemia
0.1	+ 0.3	5			47
0.3	+ 0.3	5	-		37
1.0	+ 0.3	5	-		36
3.0	+ 0.3	5	-		33
10.0	+ 0.3 0	5 10	23		14

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TABLE 6

5	Daily dose mg/kg d) + chlor.	Number of mice	Mean control parasitemia	% Parasitemia
	0.1 + 1.0	5		4
	0.3 + 1.0	5	-	2
	1.0 + 1.0	5	-	1
10	3.0 + 1.0	5	-	0.7
	10.0 + 1.0	5	-	0.03
	0	10	23
	In the	table	which follows,	the substances
15	according to the	invention are compared by way of example

witl R,S-verapamil with regard to their therapeutically relevant properties.

The AEI (activity enhancement index) is the quotient of the ED₉₀ for chloroquine alone and of the ED_go for addition of 10 mg/kg test substance. Thus, it is a direct measure for the increase in the effect of the antimalarial on resistant pathogens when the substances according to the invention are administered simultaneously .

The table also includes three conventional test methods for testing cardiovascular effects. The dose which results in 50% relaxation of the rat aortic strip (method 1) is determined and is a measure of the vasodilating action - lowering of blood pressure - of a substance. Method 2 investigates the action lowering blood pressure in the intact animal (rat) on intravenous administration.

Method 3 investigates the action of the test substance on the heart, also on the intact animal.

Reported in each case is the extent to which the dose of the test substance may be greater than that of

effect.

The selectivity index S in the table is the product of this factor with the AEI and thus describes the extent to which the cardiovascular effects of the test substances are weaker than those of racemic verapamil when they are used for breaking resistance to antimalarials in equieffective doses.

Compared with the dose of 240 to 480 mg of verapamil employed in blood-pressure therapy, the dose of the test substances therapeutically utilizable for breaking resistance can, on the basis of this table, be categorized as having no cardiovascular activity.

TABLE 7

Substance	AEI (10 mg/kg)	Rat aortic strips ed50	Lowering of blood pressure ed50	ECG (AV block)
rat i ED	. V.
S,R-verapamil	2.56	- 1	- 1	S	- 1	S	- 1	S
R-verapamil	4.15	1.6x	6.3	10.2x	3.5	8.9x	4.6	7.4·«
a)	5.0	19.5x	2.9	5.7x	2.0	3.9x	4.6	8,’κ
b)	4.5	1.8x	7.7	13.5	2.7	4.9x	2.15	3.9x
c)	4.4	1.7x	12.6	21.6x	3.4	5.8x	4.6	7.8x
d)	4.20	1.6x	6.0	9.8x	4.3	6.9x	2.15	3. 4x
e)	4.1	1.6x	6.6	10.5x	6.3	lOx	2.15	3.4x
f)	3.44	1.3x	5.7	7.7x	4.3	5.6x	>2.15	>2.8x
g)	4.1	1.60x	2.9	4.6x	4.3	6.9x	4.6	6.9x
h)	3.7	1.44x	11.0	15.8x	2.0	2.88x	2.15	3. Ox
i)	3.5	1.4x	-	-	9.2	12.5x	-	-
k)	3.3	1.3x	6.3	8. lx	20.0	26. Ox	2.15	2.8x

ccscr'Rca ano Asc^rJ* n'.d MY'CtR ΕΛιΟ .NVENTION ANO IN WHAT MANNER THE CAME IS TO BE PERFORMED. I/WE DECLARE THAT I/WE CLAIM IS:·

Claims (1)

1. We claim:1. The use of those racemic and optically active substituted phenylacetonitriles of the formula I

   I, where

   R¹, R² and R³

   R*

   R m and n n6 are identical or different and are hydrogen, halogen, C^alkyl, (λ.,-alkoxy, trifluoromethyl, or two adjacent substituents R¹ and R² or R³ together form -CH₂-CH₂-CH₂CH₂- or -CH=CH-CH=CH-, is saturated or unscMturated alkyl or cycloalkyl having up to 15 carbon atoms or phenyl, is hydrogen or C^-alky., are each 2, 3 or 4, and is saturated or unsatuiated alkyl having up to 20 carbon atoms, C*.₈-cycloalkyl or the group where R , R⁸ and R⁹ are identical or different and are hydrogen, halogen, Cj-C,-alkyl, Ci-C,-alkoxy, trifluoromethyl, or two adjacent substituents R⁷ and R⁸ or R³ together form -CH₂-CH₂-CH₂-CH₂- or -CH=CH-CH=CH-, as well as the salts thereof with physiolgically tolerated acids, which

   a) have an EC₅₀ above 10’⁷ in the test for a calciumantagonistic action described in Advances in Myocardiology Vol. 4 (1983) on page 506,

   b) have an ED₂₀ above 1.0 mg/kg in the test for an action lowering blood pressure, and

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