WO2007131488A2

WO2007131488A2 - Use of quinoline derivatives as anti-protozoal agent and in combination preparations

Info

Publication number: WO2007131488A2
Application number: PCT/DE2007/000879
Authority: WO
Inventors: Wolfgang Bohne; Uwe Gross
Original assignee: Georg-August-Universität Göttingen Stiftung Öffentlichen Rechts (Universitätsmedizin)
Priority date: 2006-05-15
Filing date: 2007-05-15
Publication date: 2007-11-22
Also published as: DE102006022908A1; WO2007131488A3

Abstract

The invention relates to the use of particular quinoline derivatives as anti-protozoal medicaments for human or veterinary application. The invention more particularly relates to the use of said quinoline derivatives for prophylaxis and/or treatment of a disease caused, in particularly, by protozoa belonging to the Apicomplexa or Flagellata groups. The invention also relates to combination preparations comprising said quinoline derivatives and at least one further anti-protozoal agent and the use thereof for the prophylaxis and therapy of protozoal infections.

Description

Use of quinolone derivatives as anti-protozoan agents and in combination preparations

The present invention relates to the use of certain quinolone derivatives as anti-protozoan pharmaceuticals in humans and animals. More specifically, the present concerns

Invention, the use of these quinolone derivatives for prophylaxis and / or

Treatment of a disease caused by protozoa, in particular by

Protozoa belonging to the Apicomplexa or Flagellata. Finally, the present invention is directed to combination preparations containing the quinolone derivatives according to the invention and at least one further antiprotozoal active ingredient and their use in the prophylaxis and therapy of

Infections by protozoa.

State of the art

Protozoan infections are a common cause of potentially life-threatening diseases. About one-third of the world's population lives in areas threatened by malaria. Malaria is caused, for example, by Plasmodium falciparum, the causative agent of malaria tropica. According to World Health Organization (WHO) estimates, up to 500 million people are infected with malaria each year; Of these, over one million die each year from the consequences of the disease. Although extensive efforts have been made to eradicate malaria, malaria is a serious endemic disease in many areas of Africa, Asia, Latin America and Oceania. In Africa alone, 20 to 30% of all hospitalizations are due to malaria. In fact, there is even a dramatic increase in malaria cases worldwide. Probably due to global warming, we have been experiencing an expansion of endemic areas for several years. For example, malaria cases have recently been reported from parts of Florida and northeastern Australia. This spread continues to be increasingly due to malaria becoming increasingly resistant to common malaria drugs.

Human malaria is caused by one of the four species of the protozoan Plasmodium: Plasmodium falciparum, Plasmodium malariae, Plasmodium vivax and Plasmodium ovale, with Plasmodium falciparum being the most abundant and virulent. The transmission to humans takes place via a certain mosquito species, the Anopheles mosquito. Malaria is next to HIV and the

Tuberculosis is the leading cause of fatalities caused by infectious diseases worldwide.

In Germany, malaria is no longer autochton today, the cases occurring here are almost invariably introduced. Every year between 700 and 1000 diseases are reported to the Robert Koch Institute. Despite intensive care, the disease is fatal in about 1 to 3% of cases in this country, because the malaria is diagnosed too late or not diagnosed and treated.

As mentioned, the malaria pathogens are increasingly developing resistance to common malaria medications. For example, chloroquine used to be used therapeutically as well as prophylactically. However, initial chloroquine resistance developed in plasmodia. Meanwhile, resistance to other malaria medications such as Pyrimethamine, mefloquine, halofantrine and other antimalarials.

However, not only is the development of resistance a problem in the prophylaxis and treatment of malaria, but also the limitation of the use of such drugs due to their side effects caused in humans or animals. Chloroquine, for example, has a very narrow therapeutic range, even a triple overdose is potentially lethal. Quinine therapy is often associated with fever, confusion, dyspnoea, and arrhythmias, and rapid intravenous administration may even result in potentially lethal side effects. Another standard agent sulfadoxine-pyrimethamine (Fansidar) is no longer authorized in Germany since 1992 because of its photosensitizing effect and the risk of toxic epidermolysis. Other products, such as tetracycline derivatives have harmful effects on the growth of bones and teeth and therefore can not be used especially in infants and pregnant women. Mefloquine, the most important malaria drug in the developed world, causes neuropsychiatric side effects; Serious side effects occur at curative doses with a probability of 1 in 250. Because of the neuropsychiatric effects, for example, a malaria prophylaxis with mefloquine in aviation pilots is not possible. Therefore, the side effects of malaria drugs are often a reason for premature discontinuation of therapy or prophylaxis. This has the consequence that the development of resistance is further promoted.

Thus, there continues to be a high demand for new active substances or combinations of active substances for combating parasitic infections, in particular malaria.

Another example of protozoan-induced infectious diseases is toxoplasmosis. It is caused by Toxoplasma gondii, a protozoan of the coccidia subclass. It is estimated that 25 to 30% of the world's population are chronically infected with toxoplasma. In Germany, seroprevalence correlates fairly closely with age, with approximately 50% of 50-year-olds being latently infected. Infections with toxoplasmas are usually latent and therefore unnoticed, but in particular in primary infection during pregnancy in about 50% of cases diaplazentar can pass to the child and infect the brain and / or eyes of the child. In immunocompromised individuals, toxoplasmosis occurs as an opportunistic disease, eg in AIDS patients as life-threatening cerebral toxoplasmosis. Transplant recipients with therapeutically induced immunosuppression can also be critically ill with toxoplasmosis. Toxoplasmosis also plays an important role in veterinary medicine. So it comes after infection during pregnancy, for example in sheep to a high rate of spontaneous abortions.

In addition to Toxoplasma gondii, other coccidia-related protozoa also play an important role as pathogens for humans and animals. Called here are the genera Eimeria, Isospora, Sarcocystis, Hammondia, Neospora and Cryptosporidium. Particularly noteworthy as animal pathogens are Eimeria (infections in poultry), Neospora (infections in dogs) and Cryptosporidium (infections in calves). Coccidoses can cause great losses, especially in poultry rearing. In order to avoid this, prophylactic coccidiosis agents are used in the working area. The cost of anti-coccidia in poultry farming is estimated at around US $ 300 million. However, as described above for malaria, resistances to the means used have developed, and thus these remedies are no longer usable after a short time.

The so-called protozoa (plasmodia and coccidia) are members of the so-called Apicomplexa, a parasite group with a common developmental origin, which are characterized by a predominantly intracellular lifestyle. Another important group of parasitic protozoa that frequently cause infectious diseases are the flagellates. Of particular importance to humans are infections with Trypanosoma cruzi, the causative agent of Chagas' disease, infections with Trypanosoma brucei, the pathogen of sleeping sickness and infections with Leishmania, the causative agents of leishmaniasis.

From the above it is clear that there is still a high demand for new active substances or combinations of active ingredients for combating infections caused or accompanied by parasitic protozoa.

The present invention has therefore set itself the task of new substances with activity against parasitic protozoa, such as Apicomplexa and To provide flagellates. These new compounds are said to be associated with little side effects, be effective against protozoa and be effective in the lowest possible doses. The aim is to increase the therapeutic scope of these pharmaceutical agents, in particular to the treatment of problematic patient groups, such as children, patients with immunodeficiency, pregnant women, etc. expand.

Brief description of the invention

It has now surprisingly been found that certain quinolone derivatives according to the general formula I

show an anti-protozoal effect,

wherein Ri is a hydrogen, OH, a C ₁ -C ₆ alkyl group, a C ₃ -C ₇ cycloalkyl group, a C ₁ -C ₆ alkenyl group, a C ₁ -C ₆ alkoxy group or a C ₁ -C ₆ alkenyloxy group, these groups may be optionally substituted;

R ₂ is a C ₄ -C _2O alkyl group, a C ₄ -C ₂ O alkenyl group, a C ₄ -C _2O alkoxy group or a C ₄ -C _2O alkenyloxy group, these groups may optionally be substituted, or 4- (4-halophenyl ) -cyclohexyl, 4-phenylcyclohexyl, which may optionally be substituted; R ₃ is 4- (4-halophenyl) cyclohexyl, 4-phenylcyclohexyl, which may optionally be substituted, or hydrogen or OH;

R ₄ is each independently a C1-C1 ₀ alkyl, alkenyl, alkoxy, alkenyloxy, amino, or diamino group which may be optionally substituted, piperazin-1-yl, 4-methylpiperazin-i-yl, 4- (C 1 -C ₄ alkyl) piperazin-1-yl, OH or halogen;

n is an integer from 0 to 4; or pharmacologically acceptable salts, solvates or hydrates thereof.

The present invention is based on the finding that an alkyl chain, an alkenyl chain, an alkoxy radical or an alkenyloxy radical at the 2-position of the quinolone derivative according to the formula I exhibits advantageous anti-protozoal properties. In particular, this chain is an alkyl chain at position 2 of the quinolone derivative. It should also be emphasized that in contrast to previous quinolone derivatives at position 3 no carboxyl group is present.

The present invention is further directed to the use of the quinolone derivatives according to the invention according to the formula I in accordance with the invention

Compound with already known anti-protozoal drugs, such as already known malaria drugs or toxoplasmosis preparations as combined preparations.

By the combined use of these at least two components, it is possible to use dosages of these drugs, which do not achieve a therapeutic effect on single administration, so-called sub-anti-protozoan doses. This has the advantage that the single active ingredients otherwise used in high dosage and the side effects associated with the high dosage can be reduced. These second anti-protozoal agents are preferably selected from the fluoroquinolones, artemisinin, chloroquine, proguanil, mefloquine, quinine, doxycycline, halofantrine, primaquine, sulfadoxine, tetracycline and in particular

Active agents which interfere with the respiratory chain or folic acid metabolism, e.g. Pyrimethamine, atovaquone, proguanil.

Finally, the present invention discloses novel pharmaceutical compositions comprising at least one quinolone derivative according to the general formula I and at least one further anti-protozoan active ingredient.

These two agents, the at least one quinolone derivative and the at least another anti-protozoan active ingredient, are for example included in sub-anti-protozoan doses, ie in doses that show no administration of the drug alone anti-protozoal effect, ie in non-therapeutic amounts in monotherapy.

Brief description of the figures

Figures 1a, 1 b, and 1c show studies on the inhibition of Toxoplasma gondii by 1-hydroxy-2-dedecyl-4 (1 H) -quinolone (HDQ). Figure 1a shows the concentration-dependent influence of HDQ on the number of parasites per parasitophorous vacuole. It can be clearly seen that the effective concentrations of HDQ are in the nanomolar range. Figure 1b shows the amount of reacted substrate of the enzyme beta-galactosidase determined by photometric determination as a function of the amount of added HDQ. Again, the effect of the HDQ in the nanomolar range is clearly visible. Figure 1c shows the concentration-dependent influence of HDQ on the replication of Plasmodium falciparum. Shown is the parasitemia depending on the concentration of HDQ.

Figure 2 depicts the inhibition of Leishmania mexicana growth by HDQ. As a measure, the parasite count in 0.01 μl of media is determined as a function of the HDQ concentration. Again, there is an inhibition of growth in the concentrations of HDQ used.

Figure 3 shows the effectiveness of various 2-alkyl-1-hydroxy-4 (1H) quinolones. Figure 3a shows the time of destruction of a cell lawn by the protozoa at different concentrations of the various quinolone derivatives. C5, C6, etc. denotes the length of the alkyl group at position 2 according to formula (I) which corresponds to the radical R ₂ . Figure 3b shows the results for the C8 derivative in Plasmodium falciparum and Figure 3c for the C12 derivative. It is clear that the IC50 value is lower for the C12 derivative than for the C8 derivative. Figures 4A and 4B show the combined effect of HDQ with known antiprotozoal drugs. As a measure of effectiveness, either the destruction of the cell lawn is used as in Figure 3 (4A) or, as in Figure 1, the number of parasites per parasitophorous vacuole (FIG. 4B).

Figure 5 shows a comparison of the anti-parasitic effects of HDQ and moxifloxacin. Again, the complete destruction of the cell lawn is used as a measure of the effectiveness of the drugs.

Detailed description of the invention

It has surprisingly been found that certain quinolone derivatives have excellent anti-protozoal activity against a wide variety of protozoa, e.g. Show Apicomplexa and Flagellata. The quinolone derivative is a quinolone derivative according to the formula I:

wherein Ri is a hydrogen, OH, a CI-C _Ö alkyl group, a C ₃ -C ₇ cycloalkyl group, a CrCβ alkenyl group, a Ci-Cβ alkoxy group or a Ci-Cβ alkenyloxy group, these groups may be optionally substituted;

R ₂ is a C ₄ -C ₂₀ alkyl group, a C ₄ -C ₂₀ alkenyl group, a C ₄ -C ₂₀ alkoxy group or a C ₄ -C ₂₀ alkenyloxy group, these groups may optionally be substituted, or 4- (4-halophenyl) cyclohexyl, 4 -phenylcyclohexyl, which may be optionally substituted; R ₃ is 4- (4-halophenyl) cyclohexyl, 4-phenylcyclohexyl, which may optionally be substituted, hydrogen or OH;

Each R ₄ independently is a C 1 -C 10 alkyl, alkenyl, alkoxy, alkenyloxy, amino, or diamino group which may optionally be substituted, piperazin-1-yl, 4-methyl-piperazin-i-yl, 4- (C 1 -C ₄ alkyl) piperazin-1-yl, OH or halogen;

n is an integer from 0 to 4; or pharmacologically acceptable salts or solvates or hydrates thereof for the preparation of an anti-protozoan pharmaceutical composition for the prophylaxis and / or treatment of an infection with protozoa and consequent secondary and concomitant diseases.

Preference is given to compounds in which the radical R 1 represents a hydrogen, an OH group or a methyl radical.

Furthermore, the radical R preferably represents a cycloalkyl group, preferably a C unsubstituted or substituted ₃ -C ₇ cycloalkyl group such as a cyclopropane group, possible substituents include alkyl, alkoxy, halogen, trifluoromethyl, nitro, acyl, carboxyl, Sulfonic acid residues and the like.

Also preferred are compounds in which R _{2 is} a C ₄ -C ₂₀ alkyl group, more preferably R _{2 is} a CΘ-CU alkyl group, in particular a C-io-Cu alkyl group, such as a Cn, Ci ₂ , C ₁₃ or Cu alkyl group. Furthermore, as R _{2 is} a C ₆ -C ₄

Alkoxy group, such as a C ₁₀ -C 14 alkoxy group and especially a Cn,

Ci ₂ , C ₁₃ or Cu alkoxy group. These alkyl or alkoxy groups may be in the substituted and / or unsubstituted state.

In a further preferred embodiment, R _{3 is} a hydrogen, OH or CH ₃ .

It is furthermore preferred for n to be an integer 0, 1 or 2. Finally is furthermore, the radical R ₄ independently of one another preferably denotes a C ₁ -C 10 -alkyl group, a 4- (4-halophenyl) -cyclophenyl group or a halogen, such as fluorine, chlorine, bromine or iodine.

In a preferred embodiment, n = 1 and R ₄ is a halogen atom located at the 6-position of the quinolone derivative of the formula 1.

In a further embodiment, the radical R ₄ is absent, ie n is 0. A particularly preferred embodiment is a quinolone derivative in which R ₁ is OH, R ₂ is a C 6 -C 12 -alkyl group, R ₃ is a hydrogen and n is 0.

In a most preferred embodiment, the quinolone derivative is 1-hydroxy-2-dodecyl-4 (1) quinolone (HDQ).

In the present application, "alkyl" means a straight-chain or branched-chain or cyclic alkyl radical The number of carbon atoms in the alkyl radical is called R for the respective radical This alkyl radical may optionally be substituted.

In general, when a group or radical is optionally substituted, are possible substituents are independently one or more of inter alia -C ₆ alkyl, -C ₆ alkenyl group, CIC _east alkoxy (eg methoxy, ethoxy, etc.), OH, halogen (eg Fluorine, chlorine, bromine, iodine), trifluoromethyl, oxo groups,

Amino, nitro, substituted or unsubstituted acyl radicals, substituted or unsubstituted aryl radicals, unsubstituted or substituted heteroaryl radicals, carboxyl radicals, sulfonic acid radicals and the like are suitable, these may optionally be substituted again, wherein R _{3 is} not substituted with a

Has carboxyl.

Alkenyl includes straight-chain or branched-chain and cyclic alkenyl groups having the number of carbon atoms mentioned for the respective radicals; these may optionally be substituted. The term "alkoxy" is understood as meaning straight-chain or branched-chain or cyclic alkoxy radicals with correspondingly stated number of carbon atoms, these alkoxy radicals being optionally substituted.

"Alkoxy" or "alkenyloxy" denotes a straight-chain or branched-chain or cyclic alkyloxy or alkenyloxy radical having an appropriately stated number of carbon atoms. These alkoxy or alkenyloxy groups may optionally be substituted.

The compounds according to the invention allow, for example, for double bonds or chiral groups, the occurrence of spatial isomers. The use of the compounds according to the invention comprises all spatial isomers both as pure substances and in the form of their mixtures. Furthermore, the compounds according to the invention also comprise the corresponding tautomeric forms of the compounds according to the invention, as described e.g. especially for the inventive 1-hydroxy-2-alkyl-4 (1 H) quinolone are conceivable.

By acyl is meant residues derived from an acid. Examples include: alkanoyl, alkenoyl, etc. The aryl radical includes groups such as phenyl, ToIyI, XyIyI, naphthyl and the like, Aroyle, aralkanoyl, Aralkenoyl etc, which may optionally be substituted.

By the term "aryl" or "aryl radical" as used herein, e.g. is used as a substituent, fall aromatic single-ring and multi-ring systems with a corresponding number of hydrogen atoms depending on the further substitution, spiro compounds are also covered by this expression. Suitable aryl radicals are e.g. -phenyl, 1H-indene, 9H-fluorenes, naphthalenes, anthracenes and phenanthrenes.

By the term "heteroaryl" or "heteroaryl" fall aryl ring systems in which one or more of the carbon atoms of the skeleton of the ring system have been replaced by a heteroatom, such as O, N, S. Suitable heteroaryls are:

Pyrrole, furan, thiophene, indolizine, indole, isoindole, coumarone, thionaphthene, pyrazole, Imidozole, oxazole, isooxazole, thiazole, isothiazole, triazole, tetrazole, thiadiazole, pyridine, pyran, thiopyran, quinoline, isoquinoline, pyridazine, pyrazine and triazine.

The quinolone derivatives of the invention have an anti-protozoal effect, i. they are for the prophylaxis and / or treatment of a disease caused by infections with protozoa, in particular flagellates such. Leishmania

Trypanosomes, lamblia, trichomonas or apicomplexa, e.g. Plasmodia or

Toxoplasma, Eimeria babesia, Isospora; Theileria, Neospora, Cryptosporidia, etc. and resulting secondary and comorbidities in humans and animals, such as mammals, suitable. According to the invention can also

Combinations of two or more of the quinolone derivatives of the invention

Formula I can be used.

The individual active compounds can be used as free bases, or acid or as different salt forms. In addition, pharmaceutically acceptable prodrugs of the individual drugs, e.g. be used in the form of organic or inorganic esters. The compounds may be present as hydrates or solvates of the active compounds or their salts.

As used herein, the term "pharmaceutically-acceptable salts" refers to salts that are toxicologically-safe for human or animal administration. These organic or inorganic salts may be selected from a group including hydrochlorides, hydrobromides, hydroiodides, sulfates, Bisulfates, nitrates, citrates, tartrates, bitatrates, phosphates, hydrogen phosphates, dihydrogen phosphates, carbonates, bicarbonates, malates, maleates, fumarates, succinates, acetates, thermophthalates, laurates, palmitates, pamoates and pectinates.

The term "sub-anti-protozoic dosage" for an individual to be treated, as used herein, refers to a single dose

Use of the anti-protozoan drug no therapeutic anti-protozoan

Produces an effect when given to an individual, such as a human or a human Animal is administered. This term encompasses both direct administration and controlled release of the drugs. It is clear that the sub-antiprotozoal dosage of an active ingredient depends on the route of administration or the route of administration. Suitable sub-anti-protozoan dosages of the active ingredients can be determined by a person skilled in the art. In any case, a sub-antiprotozoal dosage is below a dosage of the active ingredient as recommended for use alone of the active ingredient as therapeutically effective.

Any route of administration may be used to administer the composition of the invention to a human or animal. The pharmaceutical compositions can be prepared in liquid or solid form for enteral or parenteral administration. Of these, all the usual forms of administration are suitable, for example tablets, capsules, dragees, syrups, solutions, suspensions, powders or freeze-dried powders. But they can also be in the form of pastes, potions, granules, jars, medicated feed or drinking water, especially in relation to the treatment of animals. Parenteral administration is e.g. in the form of injection (intramuscular, subcutaneous, intravenous, intraperitoneal, intraocular) or by implants.

Suitable formulations are solutions such as injection solutions, oral solutions, concentrates for oral administration after dilution, solutions for use on the skin or in body cavities, infusion formulations, gels, emulsions and suspensions, solid preparations such as powders, premix or concentrates, granules, pellets, Tablets, capsules; Aerosols, inhalants, active substance-containing moldings.

Injection solutions are prepared by dissolving the active ingredient in a suitable solvent and possibly adding additional solubilizers, acids, bases, buffer salts, antioxidants and / or preservatives. The solutions are sterile filtered and bottled. These other additives are common additives used in the pharmacological field. injection solutions may also be reconstituted directly before use from freeze-dried powder with addition of a suitable solvent.

Semi-solid preparations may be administered orally or dermally. For the preparation of solid preparations, the active compounds are mixed with suitable excipients, if appropriate with the addition of auxiliaries, and brought into the desired form. Suitable carriers are all physiologically compatible solid inert substances, such as inorganic and organic substances.

Carriers may further be diluents or excipients as commonly used in pharmaceutical preparations. As additional auxiliaries, preservatives, antioxidants, dyes and binders, etc. are possible.

According to a further embodiment of the present invention, the quinolone derivatives according to the invention are used together with known anti-protozoan active compounds for the preparation of a pharmaceutical composition. These active ingredients may also be in the form of a pharmaceutically acceptable salt as hydrates or solvates.

It has been found according to the invention that in a combination of a quinolone derivative according to the formula I with a known active substance, such as pyrimethamine for the treatment of a toxoplasmosis infection, a synergistic effect can be observed between the quinolone derivative of the present invention and the pyrimethamine. This synergism has already been achieved with sub-antiprotozoal doses of the quinolone derivative and pyrimethamine. That is, by reducing the dosage of the individual drugs due to the synergistic effect between the quinolone derivative and the second known anti-protozoan drug, the side effects caused by these substances can be significantly reduced. The use of a combination therapy by means of the pharmaceutical compositions of the present invention further offers the advantage of enhancing the anti-parasitic activity of the individual substances. Thus, due to the reduction of the doses and the duration of therapy, the toxicity of the individual substances can be reduced while maintaining the anti-protozoal efficacy. Also a positive effect on the avoidance of the emergence of resistances is to be expected.

When using the combined preparations, i. of the pharmaceutical compositions containing both at least one quinolone derivative according to the formula

Contain I as well as at least one other anti-protozoan agent, they may be present in a single pharmaceutical formulation. Alternatively, the active ingredients in the form of separate pharmaceutical formulations simultaneously but also be applied sequentially. If the active substances are solids, they can be processed by conventional methods into solid pharmaceutical preparations in which e.g. both active ingredients mixed together and with conventional carrier or

Excipients, for example, compressed into tablets. But it is also possible that

Active ingredients separated from each other in a ready for sale packaging unit for

To provide, wherein the packaging unit contains the two active ingredients in separate pharmaceutical formulations.

The formulations may be formulated for administration by the oral, parenteral, topical, dermal, inhalatory route, wherein one of the active ingredients may be administered by a route of administration, while the at least two different active agent may be administered via the same or a different route of administration.

The further anti-protozoan agent may be selected from the group consisting of fluoroquinolones, artemethers, chloroquine, proguanil, mefloquine, lumefantrine, quinine, doxycycline, halofantrine,

Primaquine, sulfadoxine, tetracycline and, in particular, active substances included in the Engage respiratory chain or folic acid metabolism, such as pyrimethamine, atovaquone, cycloguanil.

As shown in the experiments below, the quinolone derivatives according to the invention in long-term experiments show a stronger inhibitory effect with increasing length of the aliphatic side chain at the position 2 of the formula I, the best values have been obtained with C12 alkyl and C14 alkyl radicals. As the solubility in an aqueous medium decreases with increasing chain length, known solubilizers are used according to the invention or the substituents R ¹ , R ³ and / or R ⁴ are selected such that the solubility of the compounds is increased without significantly lowering the activity. Optionally, for example, a pegylation of the compounds can be made to increase the solubility, as is generally known from the literature.

Furthermore, the quinolone derivatives of the formula I according to the invention have the advantage that, in comparison to the anti-parasitic fluoroquinolones, e.g. Ciprofloxacin or moxifloxacin allow a faster inhibition of protozoan proliferation.

Finally, the quinolone derivatives of the formula I according to the invention and, in particular, in combination with other anti-protozoan active compounds make it possible to reduce the dosages and thus to reduce the side effects. As a result, risk groups, such as immunocompromised individuals, pregnant women and children can be subjected to prophylaxis and therapy.

Furthermore, the use of the quinolone derivatives according to the invention alone or in combination with other anti-protozoal agents in methods for the treatment of individuals infected with protozoa is possible. These methods involve administering the quinolone derivatives of Formula I in a therapeutically effective amount for a sufficiently long period of time. The dosages may vary depending on the route of administration. For a treatment with a Combination preparation according to the present invention, the administration routes for the at least two active ingredients may be the same or different.

The methods of treating infections with protozoa are suitable for humans and animals, in particular for humans and domestic animals, such as e.g. Dogs, cats, poultry, cloven-hoofed animals and horses.

The present invention is further illustrated by the following examples. It is clear that the invention is not limited to these examples and the details described therein.

example 1

1-Hydroxy-2-dodecyl-4 (1H) quinolone (HDQ) inhibits replication of the Apicomplexa: Toxoplasma gondii and Plasmodium falciparum

In the present experiment, it is shown that the quinolone derivatives according to the invention are capable of inhibiting various apoplexy-containing protoplasts in their replication. For this purpose, various concentrations of the active ingredients were added to infected human host cells.

A. Inhibition of Toxoplasma gondii

Cell culture conditions for this and the following Examples (2-4) were as follows: Human foreskin fibroblasts were used as host cells for the obligate intracellular parasite Toxoplasma gondii. The cell culture medium for growth of the human fibroblasts consisted of Dulbecco ^'s MEM (DMEM) with 10% fetal

Calf serum (FCS) and penicillin / streptomycin (1: 100 = 100 U / ml). The humane

Fibroblasts were cultured in 24-well dishes to which glass coverslips were previously added. Confluent cell lawns from human fibroblasts were incubated with 2x10 ⁴

T. gondii of strain RH per well infected. Just before the infection became one

Medium change, with the new medium replacing 10% FCS only 1% FCS contained. Immediately after infection with T. gondii, the drugs to be tested (eg HDQ) were added. The 1-hydroxy-2-alkyl-4 (1H) quinolone derivatives were all dissolved in DMSO at a concentration of 2.5 mM. All further dilutions were made in cell culture medium. Untreated controls were supplemented with the same amount of DMSO as in the drug mixtures. The cell cultures were cultured in an incubator under 5% CO ₂ at 37 ° C.

To investigate the concentration-dependent influence of 1-hydroxy-2-dodecyl-4 (1H) quinolone (HDQ) on the replication rate of Toxoplasma gondii, fibroblasts were infected with T. gondii and spiked with different concentrations of HDQ (final concentrations: 0.001, 0.01, 0.1, 1, 10 μM). 24 h after infection, the cultures were fixed with 4% paraformaldehyde (in PBS) for 10 min and then permeabilized with 0.25% Triton-X100 (in PBS) for 15 min. The parasites were detected by an indirect immunofluorescence test. For this purpose, the fixed cells, which were on glass coverslips, were first incubated with 1% bovine serum albumin (BSA) in PBS for 1 h at room temperature (RT) in order to block unspecific binding sites. Thereafter, the samples were incubated with an anti-Toxoplasma rabbit antiserum (diluted 1: 1000 in PBS / 1% BSA) for 1 h at RT. After washing 3 times in PBS for 2 minutes each, the samples were incubated with a Cy3-conjugated anti-rabbit IgG (1: 500 in PBS / 1% BSA) for 1 h at RT. After washing 3 times in PBS, the samples were fixed with Mowiol on slides and the samples were evaluated in a fluorescence microscope.

As a measure of parasite replication, the average parasite number per parasitophorous vacuole was determined in each batch by counting 100 parasitophorous vacuoles. Figure 1A shows the average number of T. gondii per parasitophorous vacuole at different HDQ concentrations. As can be clearly seen, the effective concentrations are in the nanomolar range and are thus therapeutically extremely interesting. In another test system, this replication rate of T. gondii was also examined under the influence of HDQ. For this purpose, a confluent cell monolayer of human fibroblasts in 24-well dishes containing 1x10 ⁴ beta-galactosidase-expressing T. gondii infected (McFadden DC, Seeber F, Boothroyd JC. Use of Toxoplasma gondii expressing beta-galactosidase for colorimetric assessment of drug activity in vitro Antimicrob Agents Chemother 1997 Sep; 41 (9): 1849-53). After 4 h of incubation at 37 ° C, the cell lawn was washed 2x with DMEM to remove residual extracellular parasites. Subsequently, the cells were incubated for the further course of the experiment in a cell culture medium that consisted of DMEM without phenol red, as well as 1% FCS, penicillin / streptomycin and the various concentrations of HDQ (1, 0.1, 0.01, and 0.001 μM). In addition, this medium contained 100 μM CPRG, a substrate that is converted by the beta-galactosidase activity of the parasites. As a measure of the parasite replication was the amount of reacted substrate, which was determined by photometric determination of OD ₅₄ O and OD ₆ 3o 30h after infection.

The results are shown in Figure 1 B. As can be seen from both FIGS. 1A and 1B, a significant inhibitory effect already takes place in the single-digit nanomolar range.

B. Concentration-Dependent Influence of 1-Hydroxy-2-dodecyl-4 (1H) quinolone on the Replication of Plasmodium falciparum

For inhibition experiments of HDQ on Plasmodium falciparum, the isolate FCBR (Raether et al.1989, Parasitol Res.75: 619-26) was used. The culture medium consisted of RPM1 1640 medium containing 10% human plasma and human blood group A erythrocytes (Rhesus positive). (Trager, W., and Jensen, JB (1976) Science 193, 673-675). The culture bottles were gassed with the following gas mixture: 90% nitrogen, 5% oxygen, 5% CO ₂ . Synchronization of the plasmodium cultures was carried out by the sorbitol method (Lambros and Vanderberg, 1979; J Parasitol 65: 418-20). For the determination of the inhibitory effect of HDQ (1-hydroxy-2-dodecyl-4 (1H) quinolone), ring stages of P. falciparum in a total volume of 10 ml medium with different HDQ concentrations (1 μM, 100 nM, 10 nM, 1 nM) in T25 bottles. The initial parasitemia was 2%. Untreated controls were supplemented with the same amount of DMSO as in the drug mixtures. After 48 h, a blood smear of each batch was performed and the respective parasitemia after Giemsa staining determined by counting. As can be seen in Figure 1C, the growth of Plasmodium is also inhibited in nanomolar concentrations of HDQ.

Example 2

1-Hydroxy-2-dodecyl-4 (1H) quinolone (HDQ) inhibits the replication of Leishmania mexicana

In the present experiment it is shown that the quinolone derivatives according to the invention are capable of inhibiting the replication of the flagellate-bearing protozoa Leishmania mexicana. For this purpose, different concentrations of HDQ were added to L. mexicana cultures.

The promastigote stage of Leishmania mexicana was cultured in T25 cell culture flasks with 5 ml of medium (Mattei DM, Goldenberg S, and Morel C (1977); FEBS Letters 74: 264-268). For passage, every 3-4 days -1x10 ⁶ parasites were added to 5 ml of fresh medium containing 10% FCS. For the inhibition experiments, Leishmania HDQ was added to the fresh medium immediately after passage of the indicated concentrations. The HDQ stock solution had a concentration of 2.5 mM and was dissolved in DMSO. Controls given the same amount of DMSO without HDQ were run in parallel. Incubation was carried out the number of parasites per ml medium at room temperature (-20 ⁰ C) were determined 72 after passaging Neubauer counting chamber h by counting in one and are shown in Figure 2. As can be seen in Figure 2, Leishmania mexicana replication is inhibited by HDQ in a concentration-dependent manner. The effective concentrations here are in the single-digit micromolar range. The control value (0 μM) is from the DMSO control containing the same amount of DMSO as the 10 μM HDQ batch.

Example 3:

In the following, various 1-hydroxy-2-alkyl-4 (1 H) quinolones were tested as active substances against protozoa from the group of Apicomplexa, Toxoplasma gondii and Plasmodium falciparum.

The active substances were 1-hydroxy-2-alkyl-4 (1H) quinolones, the alkyl chain at position 2 being the following:

C ₅ = - (CHz) ₄ -CH ₃

C ₈ = - (CH ₂ J ₇ -CH ₃

C ₁₂ = - (CH ₂ J ₁₁ -CH ₃ (1-hydroxy-2-dodecyl-4 (1 H) quinolone, HDQ)

3 A. Inhibition of Toxoplasma gondii

Derivatives with alkyl side chains of length C5, C6, C8, C12 and C14 were tested for their growth inhibitory effect on T. gondii. The cell culture and infection conditions for this experiment were carried out as described in Example 1. The concentrations of the active ingredients used were: 250 nM, 50 nM, 10 nM and 2 nM. In this long-term study, the time span that the host cells survived after infection with T. gondii was determined. For this purpose, each approach was examined once a day under the phase contrast microscope on the morphology of the fibroblast cell lawn. The time at which the cell lawn was completely destroyed by parasite growth is shown in Figure 3A. In untreated cell cultures, the cell lawn was destroyed after 3 days by the multiplying parasites. The asterisked preparations still had intact turf fibroblast turf at the end of the experiment on day 12, ie 250 nM HDQ was sufficient for survival of the host cells in this long-term experiment. During the entire duration of the experiment, no medium change was carried out, the addition of the active ingredients thus took place only once at the start of the test. As can be seen, quinolone derivatives having longer chain alkyl groups at position 2 are more active in their long-term anti-parasitic activity, ie the compounds can be used at lower concentrations. Most effective were the C12 and C14 compounds. For the C6 and C8 compounds higher concentrations are necessary.

3 B. Inhibition of Plasmodium falciparum

1-Hydroxy-2-alkyl-4 (1H) quinolones with alkyl side chains of length C8 and C12 (HDQ) were compared for their growth inhibitory effect on Plasmodium falciparum. The culture conditions were as described in Example 1. For the determination of the half-maximal inhibitory concentrations (IC50) of the C8 and C12 derivatives, young Plasmodium falciparum trophozoites in a total volume of 10 ml of medium with different concentrations of the 1-hydroxy-2-alkyl-4 (1H) quinolones (1 μM, 100 nM, 10 nM, 1 nM) in T25 bottles for 36 h. The initial parasitemia was 2%. Each drug concentration was tested in triplicates. 2 μM chloroquine diphosphate, which at this concentration completely inhibits Plasmodium falciparum growth, was used as a control to detect the background signal of the originally used parasites in the subsequent fluorometric growth assay. A control without inhibitors was also performed. After 36 h, the cultures were resuspended and 1 ml of 0.08% saponin (in PBS) was added to an 800 μl aliquot. The following steps of the fluorometric growth test were performed as in the following publication: Smeijsters et al. 1996; Antimicrob Agents Chemother. 40: 835-8. The samples were measured in a spectrophotometer at 340/460 nm wavelengths. The IC50 was calculated as follows: The required drug concentration at which the fluorescence signal a Value which lies exactly between that of the untreated control (maximum growth) and that of the choroquine control (maximum inhibition). As can be seen in Figures 3B and 3C, the concentration at which semi-maximal inhibition (IC50) occurs is -14 nM lower for the C12 derivative than for the -70 nM C8 derivative. As with T. gondii, the antiparasitic effect correlates with the length of the side chain.

Example 4

Combination treatment of HDQ with pyrimethamine or atovaquone

It will be shown below that a combination treatment of HDQ with pyrimethamine or atovaquone has a synergistic effect.

4A: Combination treatment with HDQ and pyrimethamine.

Cell culture conditions for this experiment were performed as described in Example 1. In this experiment, the cells were treated either once with a) pyrimethamine or b) HDQ, or c) a combination of pyrimethamine and HDQ. The concentrations of the active ingredients used were: 100 nM, 10 nM and 1 nM. In combination approach (c), each of the two single active substances was used in the concentrations indicated.

In this long-term experiment, as in Example 2A, the time period in which the fibroblasts survived after T. gondii infection was determined. For this purpose, the morphology of the fibroblast cell lawn in each batch was examined once a day under the phase contrast microscope. The time at which the cell lawn was completely destroyed by parasite growth is shown in Figure 4. The star-marked batches still had an intact lawn of fibroblasts at the end of the experiment on day 12. During the entire duration of the experiment, no medium change was carried out, the addition of the active ingredients thus took place only once at the start of the test. As can be seen clearly from Figure 4a, a synergistic effect can be observed with simultaneous administration of HDQ and pyrimethamine. A combination of 10 nM HDQ and 10 nM pyrimethamine, once added to the cell culture medium at the time of infection, effectively protected the host cells from destruction by parasite growth. On the other hand, a treatment with the 10-fold concentration of the individual substances (100 nM) led to a complete destruction of the fibroblasts within the experimental period. Pyrimethamine, in combination with sulfadioxin, is a standard therapy for toxoplasmosis infections. However, side effects may occur with this therapy. Due to the observed synergism of HDQ and pyrimethamine, potential side effects of the individual substances used should be significantly reduced, since these individual substances can be used in much lower concentrations. In particular, such combination therapies may represent an alternative strategy for the treatment of toxoplasmosis in high-risk patients, eg, during pregnancy and childhood.

4B: Combination treatment with HDQ and atovaquone

Cell culture conditions for this experiment were performed as described in Example 1. In this experiment, cells were treated either once with a) atovaquone, or b) HDQ, or c) a combination of atovaquone and HDQ. The concentrations of the active ingredients used were: 100 nM, 10 nM and 1 nM. In combination approach (c), each of the two individual active substances was used in the concentrations indicated. The evaluation was carried out after 36 h, wherein as described in Example 1, the average number of parasites per parasitophorous vacuole was determined. As can be seen in Figure 4B, the combination of HDQ + atovaquone acts synergistically, with the IC50 value well below 1 nM. Example 5:

Comparison of the anti-parasitic effect of HDQ and known fluoroquinolones.

In the following experiment the different properties of HDQ and fluoroquinolones are investigated.

A: Investigation of the active concentration of the quinolone derivative according to the invention and known fluoroquinolones

The cell culture conditions for this experiment were carried out as described in Example 1, except that the strain "NTE" was used instead of the T. gondii strain RH in this experiment In this experiment, the cells were treated either once with a) HDQ or b The final concentrations of the drugs used were for HDQ: 250 nM, 62 nM, 16 nM, 4 nM and 1 nM and for moxifloxacin: 4 μM, 1 μM, 250 nM, 62 nM, 16 nM.

In this long-term experiment, as in Example 2, the time period in which the fibroblasts survived after infection with T. gondii was determined. For this purpose, each approach was examined once a day under the phase contrast microscope on the morphology of the fibroblast cell lawn. The time at which the cell lawn was completely destroyed by parasite growth is shown in Figure 4. In untreated cells, this period was 4 days. The star-marked batches still had an intact lawn of fibroblasts at the end of the experiment on day 15. During the entire duration of the experiment, no medium change was carried out, the addition of the active ingredients thus took place only once at the start of the test. As can be seen in Fig. 5, HDQ has a much stronger anti-parasitic effect than moxifloxacin, with 62 nM HDQ having a similar long-term protective effect as 4 μM moxifloxacin.

B: Investigation of the kinetics of the inhibitory effect

Flurochinolones, e.g. Ciprofloxacin or moxifloxacin show an inhibitory effect on the replication of Toxoplasma gondii only after a time delay (Fichera et al 1997, Nature 390: 407-409). This delayed effect could be confirmed in the experiment described above. A morphological examination in the phase contrast microscope 24 hours after infection showed a limited parasite division even in the highest concentration of 4 .mu.M moxifloxacin. In contrast, the inhibitory effect of e.g. 100 nM HDQ (see Example 1) immediately on, i. the parasites are already prevented from their first cell division.

The comparative studies show that there are significant differences in both the kinetics and the active concentration between HDQ and the fluoroquinolones. It is known that the molecular effect of the fluoroquinolones, which is based on inhibition of the topoisomerase ("gyrase inhibitors"), makes the carboxyl group at position 3 and the fluoro radical indispensable, as these two residues are missing in HDQ, for example Side chains at position 2 are essential for the action of the quinplonder derivative, eg, it has been shown that the effect of the 1-hydroxy-2-alkyl-4 (1H) quinolones depends on the length of the alkyl side chain at position 2. Known gyrase inhibitors, such as Moxifloxacin and ciprofloxacin have no side chain at this position.

Claims

claims

1. Use of a quinolone derivative according to formula I:

wherein Ri is a hydrogen, OH, a Ci-Cβ Alkyigruppe, a C ₃ -C ₇ cycloalkyl group, a CrCβ alkenyl group, a C1-C6 alkoxy group or a CrC ₆ alkenyloxy group, these groups may be optionally substituted;

4- R2 is a C4-C20 alkyl group, a C4-C20 alkenyl group, a C4-C20 alkoxy group or a C ₄ -C _2O alkenyloxy group, these groups may be optionally substituted, or 4- (4-halophenyl) -cyclohexyl, phenyl cyclohexyl, which may be optionally substituted;

R3 is 4- (4-halophenyl) cyclohexyl, 4-phenylcyclohexyl, which may optionally be substituted, or hydrogen or OH;

R ₄ is in each case independently of one another a C 1 -C ₁₀ -alkyl, alkenyl, alkoxy, alkenyloxy, amino or diamino group which may optionally be substituted, piperazin-1-yl, 4-methyl-piperazin-1-yl , 4- (C 1 -C ₄ alkyl) piperazine-1-yl, OH or halogen;

n is an integer from 0 to 4; or pharmacologically acceptable salts or solvates or hydates thereof for the preparation of an anti-protozoan pharmaceutical composition for the prophylaxis and / or treatment of an infection with protozoa and resulting secondary and concomitant diseases.

2. Use according to claim 1, wherein Ri is H, OH or CH ₃ .

3. Use according to claim 1 or 2, wherein R _{2 is} a C ₄ -C ₂₀ alkyl group.

4. Use according to one of the preceding claims, wherein R _{2 is} a C ₆ - C-I4 alkyl group, preferably a C ₁ -C 14 alkyl group and in particular a C ₁ ₂ alkyl group.

5. Use according to one of the preceding claims, wherein R _{3 is} hydrogen or OH.

6. Use according to one of the preceding claims, wherein n is O, 1 or 2.

7. Use according to one of the preceding claims, wherein R _{4 is} independently a C ₁ -C ₁₀ alkyl group or halogen.

8. Use according to one of the preceding claims, wherein n is 1 and R ₄ is a halogen atom which is located at position 6 according to formula I.

9. Use according to one of the preceding claims, wherein n is 0.

10. Use according to one of the preceding claims, wherein R _{1 is} OH, R ₂ is a C ₆ -C ₄ alkyl group, R ₃ is hydrogen and n is 0.

Use according to any one of the preceding claims, wherein the quinolone derivative is 1-hydroxy-2-dodecyl-4 (1H) -quinolone.

12. Use according to one of the preceding claims for the prophylaxis and / or treatment of a disease caused by infection with

Apicomplexa or flagellata.

13. Use according to claim 12 for the prophylaxis and / or treatment of Plasmodium, Babesia, Theileria, Toxoplasma, Eimeria, Isospora, Sarcocystis, Hammondia, Neospora, Cryptosporidium, Leishmania,

Trypanosoma, Lamblia, Trichomonas.

14. Use according to one of the preceding claims for the prophylaxis and / or treatment of malaria, toxoplasmosis, leishmaniasis, periplasmosis, coccidosis, Chagas disease and sleeping sickness and their concomitant symptoms.

Use according to any one of the preceding claims, wherein the route of administration is selected from the group consisting of oral, parenteral, topical, dermal or inhalatory administration.

16. Use of a quinolone derivative defined in any one of claims 1 to 11 and at least one further anti-protozoan active ingredient for the preparation of a pharmaceutical composition for the treatment and / or prophylaxis of infection with protozoa and resulting secondary and comorbidities.

17. Use according to claim 16, wherein the at least one further antiprotozoal active ingredient is selected from the group consisting of

Fluoroquinolones, artemethers, chloroquine, proguanil, mefloquine, Lumefantrine, quinine, doxycycline, halofantrine, primaquine, sulfadoxine, tetracycline, pyrimethamine, atovaquone, cycloguanil.

18. Use according to claim 16 or 17 for the prophylaxis and / or treatment of a disease according to one of claims 12 to 14.

19. Use of a sub-anti-protozoan dose of a quinolone derivative as defined in any one of claims 1 to 11 in combination with a sub-antiprotozoal dose of at least one second anti-protozoan agent, for the preparation of a pharmaceutical composition for

Treatment and / or prophylaxis of a protozoan infection and the resulting secondary and concomitant diseases.

Use according to any one of claims 16 to 19, wherein the composition is adapted for administration by the oral, parenteral, topical, dermal or inhalatory routes.

21. Use according to any one of claims 16 to 20 for the preparation of a combination preparation for simultaneous, separate or sequential administration in the anti-protozoan treatment and / or prophylaxis.

22. A pharmaceutical composition comprising a quinolone derivative defined in any one of claims 1 to 11 and at least one further anti-protozoan agent.

23. A pharmaceutical composition according to claim 22 further comprising a pharmaceutically acceptable carrier, diluent and / or excipient.

24. A pharmaceutical composition according to claim 22 or 23, wherein the at least one quinolone derivative in sub-anti-protozoan dose is contained and / or the at least one further anti-protozoäre drug in sub-anti-protozoäre dose is included.

25. A pharmaceutical composition according to any one of claims 22 to 24, wherein the at least one further anti-protozoic agent is selected from the group consisting of fluoroquinolones, artemethers, chloroquine, proguanil, mefloquine, lumefantrine, quinine, doxycycline, halofantrine, primaquine, sulfadoxine, Tetracycline, pyrimethamine, atovaquone, cycloguanil.

26. A pharmaceutical composition according to any one of claims 22 to 25 adapted for administration by the oral, parenteral, topical, dermal or inhalational route.