HK1140684A - Triazinone compounds for treating diseases resulting from infretation with parasitic protozoans - Google Patents

Description

Triazinones for treating diseases caused by parasitic protozoal infections

The present application is a divisional application of patent application entitled "triazineone compounds for treating diseases caused by parasitic protozoal infections" having international application date of 2000, 11/21 (international application number PCT/EP00/11837) and international application number of 00816674.9.

The technical field is as follows:

the present invention relates to triazinones for use in the treatment, in particular prophylactic treatment, of animals infected with parasites that cause abortion or cause neurological diseases. The invention relates in particular to those triazinones which are suitable for the treatment of parasitic protozoal (e.g. coccidial) diseases which lead to abortion or to neurological diseases. The invention relates in particular to triazineones suitable for the treatment of Neospora (Neospora) infections.

Background art:

triazinones (e.g., triazinediones, such as diclazuril, and triazinetriones, such as toltrazuril) are used to treat a variety of protozoal diseases in mammals, insects, and fish; see the following U.S. patents: 4,631,218, respectively; 4,933,341, respectively; 4,935,423, respectively; 5,114,938, respectively; 5,141,938, respectively; 5,188,832, respectively; 5,196,562, respectively; 5,256,631 and 5,464,837 or EP A170316. Protozoa susceptible to these triazineone compounds include parasites that infect the internal organs of birds, mammals and insects, which cause diarrhea, cachexia, nausea and vomiting. Generally, triazineone compounds act in such a way as to attack the intermediate stages of the parasite in the gut wall cells and the gut wall cells, thereby enlarging the endoplasmic reticulum surrounding the nucleus and mitochondria in the parasite cells, which may interfere with the dividing ability of the nucleus, leaving schizonts and microgametes in the juvenile stage with only a few merozoites and microgametes produced. As a result, the parasite bodies lose their ability to penetrate new mammalian cells at a later parasitic stage, thereby achieving effective prevention of parasite proliferation in the host.

Since the 70's, the neurological disorders and/or resulting abortions of animals caused by certain parasitic protozoa have remained of particular importance. Successful isolation and culture of certain protozoa of these species has proven difficult. For example, parasites in the brain or spinal fluid were not successfully isolated until late 80 s. Once it has been established that parasites infecting the brain can cause neurological diseases and parasites infecting foetuses can cause diseases that lead to abortion, there is a need for effective antiparasitic agents that can cross the blood-brain barrier and the placental barrier without toxic side effects. Only a very small number of drugs are able to cross the blood-brain barrier and the placental barrier of animals. Many drugs that cross the blood-brain barrier and/or the placental barrier of animals to effectively treat parasitic brain infections have toxic side effects and are therefore at great risk for use. Thus, no effective drug has been approved for the effective treatment of such neurological diseases and abortion-causing diseases. The following is a brief description of the diseases caused by parasites:

neospora caninum (Neospora caninum), a new species of parasite of the protozoan species, was isolated and described by bjarkas et al in 1984 from a norwegian dog. In addition to dogs and cattle, sheep, goats and horses have also found natural infections (Dubey and Rommel 1992, Dubey and Lindsay 1993). In addition to dogs and cattle, foxes, cats, sheep and mice were also successfully infected experimentally. The ultimate host for neospora caninum may be the dog (McAllister et al 1998), except for which the complete developmental cycle has not been studied in detail.

Many different cells can be used as host cells for neospora caninum, such as macrophages, neutrophils, fibroblasts, vascular endothelial cells, muscle cells, renal tubular epithelial cells, liver cells and nerve cells. However, propagation through tachyzoites occurs more readily within organelles such as muscle and nerve cells. Pathological symptoms thus appear preferentially in these tissues after natural infection. Dogs exhibited allergic and paraparesis aggravated by radiculitis from 5 to 6 weeks after natural infection. Histopathological abnormal inflammation is further found in the nervous system and occurs primarily in the brain and spinal cord. Here mainly prolonged non-suppurative inflammation, gliosis and perivascular infiltration of monocytes (macrophages, lymphocytes and some plasma cells), and in some cases also eosinophils and neutrophils. Even necrotizing lesions of the muscle that are observable with the naked eye appear. In addition to more or less pronounced atrophy, long stripes of grey colour in the longitudinal direction are also visible. These symptoms are particularly likely to occur in the hind legs. Histologically, these lesions constitute myocytitis with minor necrotic changes and non-suppurative vasculitis. Less obvious lesions are also seen in the musculature of the front leg, diaphragm and tongue. Dogs 5-12 weeks old with these symptoms must be euthanized (Dubey et al 1988). Infection with neospora can be transmitted to the next generation by repeated placental transmission. This disease does not necessarily infect all individuals in a litter. Of 6 bitches experimentally infected with neospora on day 21 of pregnancy, 1 bitch gave 3 live puppies, and 5 other newcastle-disease newborns aborted. No parasites were detected in 3 live puppies (Cole et al 1995).

Thilsted & Dubey (1989) first described neospora infection in brain tissue of aborted fetuses of a cow in New Mexico. More isolates were subsequently obtained from cattle in the USA (Conrad et al 1993, Barr et al 1993, Marsh et al 1995), Japan (Yamane et al 1996) and Sweden (Stenlund et al 1996). Neospora caninum infection is considered to be the major cause of fetal abortion in cows in california and australia (Barr et al 1990).

The vast majority of calves infected with neospora seem to be aborted at 3-9 months of age. In these fetuses, a large number of tachyzoites were first discovered. The presence of cysts has only been demonstrated in young calves to date. Infected calves died 3-17 days after birth. The condition was similar to that seen in dogs. Ataxia occurs, knee reflexes are extremely reduced, and lameness occurs in the hind limbs (in some cases all limbs). Histological observations were similar to those seen in dogs: non-suppurative meningitis and myelitis are the main symptoms. As in dogs and other animals, monocyte infiltration and necrosis can be found in brain tissue, particularly in perivascular regions. In particular, focal parasites, mainly tachyzoites or pseudocysts with tachyzoites, are found in nerve tissue and in muscle cells in few cases, but in many cases in a smaller number. Notably, in sharp contrast to Toxoplasma infections, neospora infections can be repeatedly transmitted from dam to dam, which has been demonstrated in dogs and cattle (Bjerkas et al 1984, Dubey and Rommel 1992, Dubey et al 1988).

Neospora caninum, isolated from dogs and cattle to date, was not found to be different, whether by morphological ultrastructural sectioning, protein analysis, or by subsequent sequence comparison of rRNA or ITS1 sequences in molecular biology (Holmdahl and Mattsson 1996). The popularity and economic significance

Since the discovery of neospora caninum in 1984, such parasitic protozoa have been isolated worldwide to date (reviewed in Dubey/Lindsay). In california, it is estimated that the proportion of abortions in cows due to neospora caninum infection is particularly high. In 468 aborted fetuses, 45.5% of lines were due to neospora caninum infection (Dubey and Lindsay 1993). Neospora specific DNA was detected in switzerland from brain tissue of 29% aborted bovine fetuses; accordingly, the annual economic loss in Switzerland is estimated to be 1020 million Switzerland Falang (Gottstein/Bern), and the annual loss in Australia is 1 hundred million Australian (Johnson/Sydney). The peak onset of the disease occurs in the winter months from 12 to 2 months of the year in california, as is also the case in new zealand (Thornton et al 1991). Here abortion due to neospora caninum infection occurred in 5-7 months. To date, no effective method for treating, particularly prophylactically treating, neospora caninum infection has been disclosed.

Equine protozoal Encephalomyelitis (EPM) is a neurological disease that occurs primarily in young horses that are subjected to great stress (e.g., elite racehorses and purebred performance horses), and is therefore a disease of substantial economic significance in the horse economy. EPM was first established as a disease in the 70's, and no successfully cultured E.neurona was isolated from a horse with EPM until 1991 (Sarcocystis neurona). In 1997, a Neospora protozoan, Neospora textilis (Neospora hugesi), was isolated from the brain tissue of a horse with EPM. Accordingly, it is presently believed that EPM is likely to be caused either only by this newly identified organism, only by sarcocystis neurona, or by infection of both protozoa at the same time. The most common symptoms caused by EPM are asymmetric ataxia, fainting and spasticity. This disease can be like any neurological disease state, can have an extremely acute onset, or can manifest as a chronic disease. The initial phase of a chronic attack is often latent until the late stage of the disease is difficult to diagnose and can lead to death. The only clinical symptoms in the lightest cases may be hip paralysis or a mild wheezing. Most severely ill horses are unable to swallow or stand. It is now known that in most severely ill horses, parasites (e.g. Sarcocystis neurona) infect and cause severe damage to brain tissue. The clinical symptoms of EPM are caused by direct damage to the nervous system (brain and spinal cord) by parasites as well as brain damage due to infiltration of inflammatory cells, edema, and nervous necrosis with merozoites and schizonts in the Central Nervous System (CNS). There is currently no effective precaution to control EPM. The combination of trimethoprim and sulfonamides has been used in humans, but this therapy is expensive and requires multiple repeated administrations.

A further parasite belonging to this group of coccidia, Toxoplasmagnedii, has been known for a long time. Toxoplasma gondii was originally isolated from the visceral and muscular tissues of cats. The ultimate host for this parasite is a cat that can hold Toxoplasma gondii for extended periods of time, during which the Toxoplasma oocysts can be transmitted to other animals, including cattle, sheep, pigs and humans. The infection of sheep, cattle and humans with Toxoplasma gondii can lead to abortion-causing diseases and diseases that mainly affect the central nervous system. More recently, toxoplasma has been more associated with abortion and malformations in infected dams (seronegative during pregnancy prior to infection). Hosts other than cats, such as cattle, sheep, pigs and humans, do not produce oocysts, but express and blunt-colonize their development, muscle and brain tissues are affected by them, and the clinical symptoms of this disease caused by express and blunt-colonize are neurological syndromes and fetal miscarriage with fetal malformations. Toxoplasma seropositivity was reported for 60% of cats. Furthermore, there is no treatment, especially effective prevention, for toxoplasmosis.

The use of triazineone compounds, such as diclazuril, toltrazuril or toltrazuril-sulfone (recently new name punazuril), for treatment, in particular prophylactic treatment, without unacceptable side effects on animals at risk of coccidial (in particular coccidia of the family Sarcocystidae) infections has not been disclosed or taught in previous literature, including the literature mentioned at the outset. It is therefore an object of the present invention to provide an effective treatment, in particular prophylactic treatment, for animals at risk of the above-mentioned parasitic infections.

The invention content is as follows:

surprisingly, it has now been found that by treatment, in particular prophylactic treatment, with triazinones, a complete prevention of parasitic protozoal infections is achieved.

Accordingly, the present invention relates to a method for the therapeutic, in particular prophylactic, treatment of an animal at risk of parasitic diseases, either neurological diseases or diseases which can lead to foetal abortion, characterized in that a pharmaceutically effective dose of a triazineone-type drug is administered to said animal. Such animals include, but are not limited to, horses, cattle, cats, dogs, pigs, sheep, birds, insects, and humans, for example.

The parasites responsible for the infection or pathogenesis are coccidia of the family sarcocystidae, which may manifest as neurological diseases, or diseases that lead to abortion. For purposes of illustration, but not limitation, it may be selected from the group consisting of Sarcocystis species, neospora species and Toxoplasma species. Typically selected from the group consisting of Sarcocystis neurona, neospora megaterium, neospora caninum and Toxoplasma gondii, particularly the Sarcocystidae family of the genus Neosporosis. Protozoal infections or protozoal diseases include, but are not limited to, EPM, neosporosis and toxoplasmosis.

In the practice of the present invention, the treatment, particularly prophylactic treatment, of an animal suffering from a parasitic infection or disease caused by a protozoan as described herein results in the prevention of symptoms and diseases associated therewith. Generally, the symptoms of these diseases include paralysis, ataxia, paralysis, abortion, neonatal weakness, and other related symptoms. A typical course of treatment is about 1 to 30 days, preferably 1 to 20 days, particularly preferably 1 to 10 days, and particularly preferably 1 to 6 days. Of course, the regimen for such treatment, particularly prophylactic treatment, may be once daily, twice or several times daily, every other day or once weekly, depending on the circumstances and the type of pathogenic parasite.

The preferred dose is 1 to 500mg of active substance per kg of body weight of the animal to be treated, the particularly preferred dose is 10 to 200mg/kg, and the particularly preferred dose is 20 to 150 mg/kg.

Without wishing to be bound by a particular theory of invention, it is believed that the unforeseen effects of such treatments, particularly prophylactic treatments, described herein are due to the ability of triazinones to cross the brain-blood barrier or the placental barrier. We believe that the compounds of the invention readily cross the brain-blood barrier and penetrate into the placenta, killing protozoa in situ in the brain and in the spinal fluid of the spinal cord.

To date, there has been no economical, simple to administer drug for effective prevention of these diseases without unacceptable side effects (such as toxicity and mutagenicity to animals). These triazinones, particularly, but not exclusively, the toltrazuril class of compounds will now be described hereinafter. The present disclosure and claims also include other triazinones that are useful in the sense of toltrazuril compounds.

The triazinetrione-toltrazuril compounds which can be used according to the invention have the structure shown in formula (I)

Wherein

R¹Is haloalkylthio, haloalkylsulfinyl or haloalkylsulfonyl,

R²is hydrogen, alkyl, alkoxy, alkoxyalkyl, alkylmercapto, halogen, haloalkyl or optionally substituted sulfamoyl, e.g. dialkylsulfamoyl, and

R³and R⁴Which may be identical or different, represent hydrogen, alkyl, alkenyl or alkynyl, X represents O or S, and their physiologically acceptable salts.

Furthermore, triazinetrione-toltrazuril compounds of the following formula (I) in particular have been found to be useful according to the invention, wherein,

R¹represents halo (C)₁-C₄) Alkylthio, halo (C)₁-C₄) Alkylsulfinyl or halo (C)₁-C₄) An alkyl sulfonyl group, a carboxyl group,

R²represents hydrogen, (C)₁-C₄) Alkyl, (C)₁-C₄) Alkoxy, halogen, (C)₁-C₄) Alkoxy (C)₁-C₄) Alkyl, (C)₁-C₄) Alkyl mercapto, (C)₁-C₄) Dialkylaminosulfonyl or halo (C)₁-C₄) Alkyl radicals, and

R³and R⁴May be the same or different and represents hydrogen, (C)₁-C₄) Alkyl or (C)₁-C₄) Alkenyl, X represents O or S.

The triazinedione diclazuril compounds which can be used in the invention have the structure of formula (Ia)

Wherein

R^1a、R^2aAnd R^3aEach independently of the others represents hydrogen, halogen, trifluoromethyl, C₁-C₆Alkyl radical, C₁-C₆Alkoxy radical, C₁-C₆Alkylthio or C₁-C₆An alkyl sulfonyl group, a carboxyl group,

R^4aand R^5aEach independently of the others represents hydrogen, halogen, trifluoromethyl or C₁-C₆Alkyl radicals, and

r represents hydrogen, C₁-C₆Alkyl radical, C₃-C₆Cycloalkyl or phenyl, which may optionally bear up to 3 substituents independently selected from: halogen, trifluoromethyl, C₁-C₆Alkyl radical, C₁-C₆Alkoxy radical, C₁-C₆Alkylthio or C₁-C₆An alkylsulfonyl group.

Preference is given to using compounds of the formula (Ia) in which

R^1aAnd R^2aEach independently of the others represents hydrogen, halogen, trifluoromethyl or C₁-C₆An alkyl group, a carboxyl group,

R^3arepresents hydrogen, and is selected from the group consisting of,

r represents hydrogen, C₁-C₆Alkyl, phenyl or halophenyl, and

R^4aand^5aeach independently of the others represents hydrogen, halogen, trifluoromethyl or C₁-C₆An alkyl group.

Particular preference is given to using compounds of the formula (Ia) in which

R^1aRepresents a 4-halogen atom or a salt thereof,

R^2aand R^3aRepresents hydrogen, and is selected from the group consisting of,

r represents hydrogen or methyl, and

R^4aand R^5aEach independently of the others represents hydrogen, halogen or trifluoromethyl, where R^4aAnd R^5aAt the 2-and 6-positions of the phenyl group to which they are attached.

Compounds which are particularly preferably used are, in particular

The diclazuril is used as a raw material,

toltrazuril and

pena Zhuli.

The use of toltrazuril and puna zuril is particularly preferred.

It has also been found that

(a) The compounds of formula I can be prepared: by the compounds of the formula II

Wherein

R¹、R²、R³And X has the above-mentioned meaning

With isocyanates of substituted carboxylic acids of the formula III

Wherein

R⁵Represents a halogen atom, an alkoxy group or an aryloxy group, and optionally isolating the resulting substituted 1, 3, 5-triazine derivative of the formula IV

Wherein

R¹、R²、R³And X has the above-mentioned meaning

And optionally with a compound of formula V

A-Z (V)

Wherein

A represents alkyl, alkenyl or alkynyl, and

b represents halogen;

or

(b) Compounds of formula I were prepared as follows: a compound of formula II (wherein R¹，R²，R³And X hasAs defined above) with a di (chloroformyl) amine of the formula VI, optionally in the presence of an acid acceptor

Wherein

R⁶Represents an alkyl group, and is represented by,

or

(c) To obtain a compound of formula I (wherein the substituent R²、R³And R⁴And X has the above-mentioned meaning, and R¹Is haloalkylsulfinyl or haloalkylsulfonyl) by reacting a compound of formula VII with a suitable amount of a suitable oxidizing agent

Wherein

R²、R³And R⁴Have the meanings mentioned above, and

R¹' is haloalkylthio.

If N- [ 3-chloro-4- (4 '-trifluoromethylthiophenoxy) -phenyl ] -N' -methylurea and isocyanate chloroformate are used in process variant (a), the reaction scheme can be represented by the following equation:

if N- [ 3-ethoxy-4- (4' -trifluoromethylthiophenyl) -phenyl ] -thiourea and N-methyl-bis- (chloroformyl) amine are used as starting materials in process variant (b), the reaction scheme can be represented by the following reaction scheme:

a compound of the formula I (wherein R is R) obtained according to the process (a) or (b)¹Represents haloalkylthio, X represents 0) which can be oxidized to the corresponding haloalkylsulfinyl or haloalkylsulfonyl derivative according to process variant (c). If hydrogen peroxide is used as the oxidizing agent, the process scheme can be represented by the following equation:

in the formulae I, II, IV, V, VI and VII, R²、R³、R⁴、R⁶Or A represents a linear or branched alkyl group, preferably having 1 to 6, especially 1 to 4 carbon atoms. By way of example, optionally substituted methyl, ethyl, n-propyl and isopropyl and n-, isobutyl and tert-butyl groups may be mentioned.

In formulae I, II, IV, V and VII, R³、R⁴Or alkenyl represented by A is a linear or branched alkenyl group, which preferably has 2 to 6, particularly 2 to 4, carbon atoms. Mention may be made, by way of example, of optionally substituted vinyl, propen-1-yl, propen-2-yl and buten-3-yl groups.

In formulae I, II, IV, V and VII, R³、R⁴Or alkynyl represented by A is a straight-chain or branched alkynyl group preferably having 2 to 6, particularly 2 to 4, carbon atoms. By way of example, mention may be made of optionally substituted ethynyl, propyn-1-yl, propyn-2-yl and butyn-3-yl.

In formulae I, II, III, IV and VII, R²Or R⁵The alkoxy groups represented are linear or branched alkoxy groups,it preferably has 1 to 6, in particular 1 to 4, carbon atoms. By way of example, optionally substituted methoxy, ethoxy, n-propoxy and isopropoxy, and n-butoxy and isobutoxy may be mentioned.

In the formulae I, II, III, IV, V and VII, R²、R⁵Halogen as defined for Z is preferably fluorine, chlorine, bromine and iodine, especially chlorine and bromine.

In the formulae I, II, IV and VII, R¹The haloalkylthio group represented is preferably one having 1 to 4, particularly 1 or 2 carbon atoms, and preferably having 1 to 5, particularly 1 to 3, halogen atoms of the same or different species, wherein the halogen atoms are preferably fluorine, chlorine and bromine, particularly fluorine and chlorine. Mention may be made, by way of example, of trifluoromethylthio, monochlorodifluoromethylthio, bromomethylthio, 2, 2, 2-trifluoroethylthio and pentafluoroethylthio.

In formulae I, II and IV, R¹The haloalkylsulfinyl group represented is a haloalkylsulfinyl group which preferably has 1 to 4, particularly 1 or 2, carbon atoms and preferably has 1 to 5, particularly 1 to 3, identical or different halogen atoms, preferably fluorine, chlorine and bromine, particularly fluorine and chlorine. Mention may be made, by way of example, of trifluoromethylsulfinyl, chlorodifluoromethylsulfinyl, bromomethylsulfinyl, 2, 2, 2-trifluoroethylsulfinyl and pentafluoroethylsulfinyl.

In formulae I, II and IV, R¹The haloalkylsulfonyl group represented is a haloalkylsulfonyl group preferably having 1 to 4, particularly 1 to 2 carbon atoms, and preferably having 1 to 5, particularly 1 to 3, halogen atoms of the same or different species, preferably fluorine, chlorine and bromine, particularly fluorine and chlorine. Mention may be made, by way of example, of trifluoromethylsulfonyl, chlorodifluoromethylsulfonyl, bromomethylsulfonyl, 2, 2, 2-trifluoroethylsulfonyl and pentafluoroethylsulfonyl.

In formulae I, II and IV, R²The optionally substituted sulfamoyl group represented bySelected is one of the following groups:

SO₂NH₂，SO₂NH-CH₃，SO₂N(CH₃)₂，SO₂NH-C₂H₅，SO₂-N(C₂H₅)₂，

in formula III, R⁵The aryloxy group represented preferably represents a monocyclic carbocyclic aryloxy group or a bicyclic carbocyclic aryloxy group, in particular phenoxy.

In formula III, R⁵Aryloxy in the meaning preferably denotes phenoxy. Most of the ureas or thioureas of formula II which are used as starting materials have not been known to date but can be conveniently prepared by the following known methods: (a) or the substituted 4-aminodiphenyl ether and corresponding substituted isocyanate or substituted isorhodanic ester react in an inert solvent, and the temperature is controlled between 0 ℃ and 100 ℃; or the order is reversed, (b) ammonia or a substituted amine is reacted with the corresponding substituted isocyanate diphenyl ether or 4-isothiocyanate diphenyl ether under the same conditions, or (c) a substituted 4-hydroxyphenyl urea or 4-hydroxyphenyl thiourea is reacted with an activated haloaromatic compound in an aprotic solvent such as dimethyl sulfoxide, dimethylformamide or hexamethylphosphoric triamide in the presence of a base such as sodium hydride, potassium hydroxide, potassium carbonate or the like at a temperature controlled at 20 ℃ to 150 ℃.

With a suitable choice of solvent, the reaction product will generally crystallize out on cooling of the solution. However, these urea compounds can also be prepared from amines and isocyanates as described in the following references: chemie (Houben-Weyl), IV edition, volume VIII, page 157-158.

Certain bis (chloroformyl) -amines which can be used according to the invention in process (b) are known (see: Synthesis 1970, pages 542 to 543), and those hitherto unknown compounds can be prepared in an analogous manner by chlorination of cyclodialyldithioethers in inert organic solvents, preferably carbon tetrachloride.

Possible diluents for the reaction of the urea or thiourea compounds of the formula II with the carboxylic acid isocyanates of the formula III (process variant (a)) or with the bis (chloroformyl) amines of the formula VI (process variant (b)) and also for the reaction of the 1, 3, 5-triazine derivatives of the formula IV with the compounds of the formulae a to Z are all organic solvents which are inert towards these reactions.

In addition to pyridine, preferred are aromatic compounds such as benzene, toluene and xylene, halogenated aromatic compounds such as chlorobenzene and dichlorobenzene, and ethers such as tetrahydrofuran and dioxane.

Hydrochloric acid which may be formed during the reaction is selected in gaseous form or is combined with an organic or inorganic acid acceptor. Preferred acceptors of these acids include tertiary organic bases, such as trialkylamines, for example triethylamine, aromatic N-hetero (mono or di) cyclic amines, for example mono-or bicyclic pyridylazacycloalkylamines, for example diazabicyclononene, diazabicycloundecene and the like, or inorganic bases, such as alkali metal carbonates, alkali metal oxides, alkali metal hydroxides, alkaline earth metal carbonates, alkaline earth metal oxides or alkaline earth metal hydroxides.

The reaction temperature of the above reaction step may vary within a wide range. Generally, the reaction is carried out at a temperature of from about 0 ℃ to about 150 ℃, with a preferred temperature range being from about 20 ℃ to about 100 ℃.

The above reaction step may be carried out at atmospheric pressure or at elevated pressure. Generally at atmospheric pressure.

The oxidation used for the treatment of trifluoromethylthio compounds of the general formula (I) in which Y represents the oxygen to give the corresponding sulfinyl or sulfonyl compound, is carried out according to process variant (c)The agent may in each case be H₂O₂Glacial acetic acid, H₂O₂Acetic anhydride, H₂O₂Methanol, peracids such as m-chloroperbenzoic acid, and nitrates of chromic acid, potassium permanganate, sodium periodate, ammonium cerium (IV), and nitric acid.

The preparation of compounds of formula (Ia) is described in detail in EP A170316 and US4,631,278. These compounds can be prepared by the methods described therein.

The resulting compounds of formula (I) or (Ia) may be converted into the corresponding addition salts, for example by reaction with an inorganic or organic base.

When practicing the present invention, the triazineone compounds can be formulated into any desired dosage form for administration to an animal. Formulations suitable for oral administration are preferred and may be solutions, suspensions, tablets, capsules, peptones, creams, gels, or powders, granules or pills. Other modes of administration are parenteral, topical, intramuscular, intramucosal, or other modes of administration known to those skilled in the art. Topical administration in the form of a pour-on is likewise preferred.

A particularly effective method of treating parasitic protozoa, particularly neospora, is to use the compounds of formulae (I) and (Ia) in combination with live or dead vaccines. In this case, a synergistic effect is also observed.

The compositions and formulations are prepared by mixing the ingredients in a suitable apparatus, such as a mixer or other suitable apparatus.

The specific implementation mode is as follows:

the present invention is described in more detail by the following examples, which are intended to be illustrative, but not limiting.

Examples

Neospora caninum infection

Clinical, microscopic, immunohistochemical, and molecular biological parameters are the basis for the diagnosis of neospora caninum and the identification of toxoplasma gondii. Clinically, acroparalysis and repeated transplacental infection can be seen. Tachyzoites and cysts can be seen from muscle and nerve tissue by microscopic examination. Only a few cysts with a clearly thickened wall are visible in the nerve tissue. Tachyzoites produced in cell culture were detected in an indirect immunofluorescence assay (IFAT), titers of 1: 200 being considered specific. Once clinical symptoms appear, this titer can rise to 1: 20000. For the molecular biological diagnosis of neospora caninum, a rapid and unambiguous determination can be made by in vitro amplification of the I TS1 region (internal transcription spacer 1 region) (Holmdale and Mattsson 1996).

Description of in vitro test systems

Culture of VERO host cells

Neospora caninum is an obligate intracellular parasite. The use of VERO cells (kidney cells of African green-long-tailed monkeys, ATCC No.: CCL 81 Vero) as an adjuvant makes it possible to culture such parasites under conditions which can be standardized and defined. Vero cells were grown in the following medium: 87% RPMI 1648(ICN, 12-602-54) 10% FCS (fetal calf serum, ICN, 29-101-49) 1% 200mM L-glutamine (ICN, 15-801-13) 1% sodium bicarbonate (ICN, 16-883-49) 1% penicillin/streptomycin (ICN, 16-700-49). Using 25cm²(Falcon, B769031) and 75cm²(Falcon, B769051) tissue culture flasks were maintained sterile and passaged. In CO₂Vero cells were grown in incubator (Heraeus) at 37 ℃ with 5% CO₂Under gas conditions until a single cell layer is formed.

Culture of ED cells

ED cells (horse skin cells, ATTC No. ccl 57) were grown in the following medium: 87% EMEM (ICN, 12-106-54) 10% FCS (fetal calf serum, ICN, 29-101-49) 1% 200mM L-Glutamine (ICN, 15-801-13), 1% NEA (non-essential amino acids, Gibco, 11140-,16-700-49). Using 25cm²(Falcon, B769031) and 75cm²(Falcon, B769051) tissue culture flasks were maintained sterile and passaged. Growth of ED cells in incubator (Hereus) at 37 ℃ without CO₂Gas conditions until a single cell layer is formed.

Cell passage

When the culture has formed a well-fused cell layer, the host cells are passaged, i.e., distributed onto new cell culture dishes. If the medium contains 10% FCS, it is passaged twice a week. The medium was decanted, the cell layer washed with 5ml of pancreatin-ethylenediaminetetraacetic acid (EDTA) (ICN, 16-891-49), and then 5ml of pancreatin-EDTA at 37 ℃ in CO₂Incubate for 5-10 minutes until the cells are detached from the bottom of the dish. The pancreatin-EDTA suspension of the cells was centrifuged at 1500r/min for 5 minutes with 1-2ml of pre-warmed FCS (Varifuge3.0, Heraeus). The supernatant was discarded and the sediment was dissolved in 15ml of medium (92% RPMI1640, 5% FCS, 1% L-glutamine, 1% sodium bicarbonate, 1% penicillin/streptomycin). For each tissue culture 3 new flasks were made and 5ml of cell suspension was added to each flask, i.e.the distribution was 1: 3.

Cryopreservation of cells in liquid nitrogen

The cells in the flasks were frozen in C541 medium (50% RPMI1640, 40% Fetal Calf Serum (FCS), 10% dimethyl sulfoxide (DMSO, Merck 9578)) or in C2 medium (86% RPMI1640, 10% DMSO, 2% FCS, 1% L-glutamine, 1% penicillin/streptomycin). Cells were treated with trypsin beforehand (see above), and the separated cells were centrifuged, resuspended in 3ml of C541 freezing medium or C2 medium, and transferred to a 2m l freezing tube. The final storage was in liquid nitrogen at-196 ℃ where the cells could be stored permanently. Before final storage in liquid nitrogen, the cryostraw was placed in a Styropor box with a wall thickness of 1cm, and the cryostraw and the cell layer on its inner wall were slowly frozen to-80 ℃. The Styropor box can continuously cool the content at the rate of 1-2 ℃ per minute, so that the water in the cells is lost due to the osmosis action of the cells. This is crucial to the viability of the cell.

Thawing of frozen cells

The thawing of the cryovial taken out of the liquid nitrogen on a water bath at 37 ℃ is as rapid as possible. The cell suspension was pipetted into 10ml of medium and then divided into two equal 250ml tissue culture flasks. The cell culture conditions were 37 ℃ and CO₂The concentration was 5%. After 24 hours, the cell culture medium was changed to remove DMSO present in the freezing medium. Infection of cell cultures with neospora caninum

The following neospora caninum isolates were used to infect the cell cultures: NC-1 (isolate of canine DUBEY, (1988)) and NC Swe B-1/generation 9 (isolate of cattle, Swedish national veterinary institute, Uppsala, Sweden). The infectious agent used was either an infected cell culture stored in liquid nitrogen (see above) or a purified tachyzoite of the infected culture (see below).

Isolation of tachyzoites from cell cultures by Sephadex

Neospora caninum was purified from infected VERO monolayer cells or ED monolayer cells under sterile conditions. First, the infected cell culture was scraped from the bottom of the culture flask with a small spatula (Tec No Mara, 3010) and then aspirated into a 10ml disposable syringe using a 23 gauge cannula (Luer 23 Gx1, 0.6X 25 mm). During this process the host cell and the tissue encapsulation contained therein are mechanically disrupted. The cell suspension was then centrifuged at 1500r/min for 7 minutes (Varifuge3.0, Heraeus), the supernatant discarded, and the sediment resuspended in exactly 2.5ml of physiological phosphate buffer (PBS: 1mM PO4, 12mM NaCl, 0.87mM KCl, pH 7.4). Using Sephadex column (PD-10)^TMSephadexG-25M, Pharmacia Biotech, 17-0851) for further purification. The Sephadex column was equilibrated first with 25ml PBS, loaded with 2.5ml PBS sample and then eluted with 5ml PBS. Tachyzoites move rapidly through the column and are found in the first 3ml of eluate, whereas high molecular weight cell debris and cell membranes elute from the Sephadex column with lag time. For removing undesired organelles and free host cellsCell DNA, samples 1500r/min centrifugal 7 minutes, discard the supernatant, 40ml PBS will settle 3 times washing.

Detection of drug action with neospora caninum cell cultures

The effect of the drug was tested in 96-well plates (Falcon 3872) since the system required only a very small amount of starting material (about 1 mg). First, a monolayer of host cells (VERO or ED) of the cell culture is cultured on a cell culture plate. For this purpose, 250ml tissue culture flasks (total cell culture area 50 cm)²) In the flask, there was an uninfected monolayer of cells grown. In CO₂The cell layer of the culture was detached with 5ml of trypsin-EDTA (Gibco, 45300-. After 10 minutes of incubation, most of the cells had exfoliated. The cell suspension was transferred with a 5ml pipette into a 50ml centrifuge tube (Greiner, B769331) to which approximately 1ml of pre-heated fetal bovine serum had been added. After centrifugation at 1500r/min (Varifuge3.0, Heraeus) for 5 minutes, the supernatant was discarded and the cell cake was resuspended in 100ml of RPMI medium (95% RPMI1640, 2% FCS, 1% L-glutamine, 1% sodium bicarbonate, 1% penicillin/streptomycin). 150 μ l of this cell suspension was aspirated into each well of a 96-well plate. 100ml of medium was sufficient to fill 6 microplates. The plates loaded with the cell culture were incubated at 37 ℃ (CO)₂Concentration 5%) for 24 hours. The cultured cells were then infected with pure neospora caninum tachyzoites at a concentration of 48000 tachyzoites per well and then incubated at 37 deg.C (CO)₂Concentration 5%) for 24 hours. The test drug was weighed into a 1.5ml Eppendorf reaction tube, the drug weight being 0.5-1.5 mg. Then, DMSO was inhaled in an amount of 1ml DMSO per mg of drug, which corresponds to the dilution of the drug to 1X 10^-3g/ml. The media formulations used for the rest of the concentration dilutions were as follows: 87% RPMI1640, 10% FCS, 1% L-glutamine, 1% sodium bicarbonate, and 1% penicillin/streptomycin. In the preliminary screening, three concentrations of 10ppm, 25ppm and 50ppm were used. 24 hours after infection with neospora caninum, the diluted drug was added to the cell culture plates at 150. mu.l per well. Untreated media was used in the first row, which included both infected and uninfected controls. Then will beCell culture plates at 37 ℃ and 5% CO₂The culture was carried out for 5 days in the environment. During this period, tachyzoites multiply within the host cell, thereby disrupting the monolayer structure. If a drug is sufficiently effective, tachyzoites are destroyed and the monolayer of cells remains. An intact monolayer of cells can be detected by a protein binding assay (live cell staining). One method that may be used is staining with 0.25% crystal violet (Sigma C3886). Prior to staining, the plates were washed with 100. mu.l PBS and fixed with 100. mu.l methanol. Magnifications of 25X 10 at 4 days after the start of treatment and 5 days after infection were examined under an inverted microscope. The following evaluation protocol was used:

evaluating the visual appearance

Complete destruction of 0 ═ null monolayers

Partial destruction of 1 ═ weakly effective monolayers, visible parasite colonies

2-sufficiently potent monolayer of cells was not destroyed, and no tachyzoites for the parasite were observed

T ═ cytotoxic cell death, rounded sphere

TABLE 1 Effect of toltrazuril on Neosporosis Canine in VERO cells

Test drugs	50ppm	25ppm	10ppm
				Control of infection	0	0	0
Toltrazuril pure active compound	2	2	1
				2.5% toltrazuril solution*	2	2	1

100ml solution contained: 2.50g toltrazuril; 30.00g triethanolamine; 80.70g polyethylene glycol; the three ingredients are simply mixed together.

Evaluating the visual appearance

Complete destruction of 0 ═ null monolayers

Partial destruction of 1 ═ weakly effective monolayers, visible parasite colonies

2-sufficiently potent monolayer of cells was not destroyed, and no tachyzoites for the parasite were observed

T ═ cytotoxic cell death, rounded sphere

II in vivo Effect

There is very little information on the effect of drugs in vivo, since suitable in vivo test methods have yet to be developed. In experimentally infected mice, sulfadiazine (taken by drinking water) is only effective if administered prophylactically, i.e. before infection. In this case no parasitological symptoms are observed, thus preventing the onset of clinical symptoms. Late onset treatments have not been successful (Bjerkas et al 1984, Dubey et al 1988, Lindsay and Dubey 1989, Lindsay and Dubey 1990). In dogs, only one treatment with sulfadiazine and clindamycin was successful, that time treatment was successful since the clinical symptoms of radiculitis were observed and the start of the treatment was immediate.

Description of mouse test model

The Committee of the Bayer corporation (BAYER AG) taught by doctor Simone Eperon and Bruno Gottstein, the institute for parasites in veterinary and medical departments, university of Bertoni, designed and conducted experiments (Eperon et al 1999, Parasite Immunology 21: 225-236.)

Mouse

Female Wt C57BL/6 mice, age at treatment: 7.5 weeks.

Fast breeder of neospora caninum

The E.canis tachyzoites were passaged on VERO cells and purified by column chromatography using sterile PBS at 2X 10⁶The worm/100. mu.l concentration was prepared in several aliquots. Vital staining with trypan blue showed 97% viable tachyzoites.

Medicine for prevention (Table 2)

The purified toltrazuril active compound was prepared as a stock solution of 50mg/10ml water +100 μ l polyethylene glycol. Each 0.1ml of this solution contains 0.5mg of pure active compound per application. For a 20g mouse this corresponds to a concentration of 25 mg/kg. The medicine is continuously taken orally for 6 days by a tube feeding method, which is equivalent to the total dosage of 150 mg/mouse.

A solution formulated with 2.5% toltrazuril (100ml solution containing: 2.50g toltrazuril, 30.00g triethanolamine, 80.70g polyethylene glycol; the various ingredients were simply mixed together): 6.25ml of this 2.5% solution was diluted with 250ml of water, after which the experimental mice were allowed to take the drugs by drinking water for 6 consecutive days.

Infection with viral infection

4 hours after prophylactic administration, 2X 10 of the drug was administered to each mouse⁶The worm bodies were infected in 100. mu.l sterile PBS.

Sacrifice of experimental animals

14 days after infection, experimental mice were treated with CO₂And (6) killing. To ensure that random sequences were used for evaluation (blind), mice were sacrificed and randomly numbered. To obtain serum, blood was collected from the heart. The brain was carefully dissected out and half of it was stored at-80 ℃ for PCR analysis. The remainder were fixed with 4% paraformaldehyde/PBS for immunohistological studies (IFAT).

Results

1. Serum IgG (Table 3)

Total immunoglobulin G (IgG) amounts against neospora caninum were determined by the ELISA method reported by Eperon et al (Parasite Immunology1999, 21: 225-236). The results of the treatment groups were compared to a group of uninfected and a group of infected control groups. Positive control mice were inoculated first with crude extract of neospora caninum tachyzoites and then with 10⁶Neospora caninum tachyzoite infection. The mice had particularly high neospora caninum specific IgG titers.

Mice treated with toltrazuril (the pure active compound) had a reduced concentration of neospora caninum-specific IgG compared to infected and uninfected control mice. This concentration was significantly lower in mice treated with 2.5% formulated toltrazuril solution (close to mice in the uninfected control group).

PCR analysis (Table 4)

DNA was isolated from brain tissue of infected mice and subjected to the Polymerase Chain Reaction (PCR) specific for neospora caninum as reported by Eperon et al (Parasite Immunology1999, 21: 225-236). All uninfected mice were negative for neospora caninum specific PCR detection. All mice infected with neospora caninum specific PCR assays were positive. Among the mice of the test group treated with toltrazuril pure active compound for prophylactic administration, 4/7 showed negative PCR detection. All mice treated with 2.5% of the toltrazuril solution were negative for the PCR assay.

3. Immunofluorescence assay (IFAT, Table 4)

Brain tissue samples fixed with paraformaldehyde were embedded in paraffin and dehydrated. Three sets of consecutive sagittal sections were made. One set of sections was stained with hematoxylin-eosin and two additional sets were subjected to immunofluorescent labeling assays as reported by modified Eperon et al (Parasite Immunology1999, 21: 225-236): the first antibody was a rabbit polyclonal anti-neospora caninum tachyzoite whole cell antibody diluted 1: 400 times in Phosphate Buffered Saline (PBS) containing 1% Bovine Serum Albumin (BSA). The second antibody was goat anti-rabbit FITC labeled antibody diluted 1: 100 fold in PBS with 0.5% BSA.

No lesions or abnormalities were seen in all the hematoxylin-eosin stained sections of each group, and therefore a method of immunolabeling with specific neospora caninum antibodies was used to mark the various stages of the parasite in brain tissue. All sections were studied in the evaluation. All individual stages of the parasite were counted and evaluated using the following criteria:

(-) -Wu Su Zi

Less than 10 tachyzoites

(+) -10-200 tachyzoites

(+++) -more than 200 parasites

The case of (+++) was found only in the positive control. The positive control was a knockout variant (μ MT mice) which produced no antibody and died on day 31 post-infection due to infection. IFAT tests were negative in all uninfected mice. All the toltrazuril-treated test groups were negative in the IFAT test.

TABLE 2 effects of toltrazuril against neospora caninum in mice

Sky	Date	Pure toltrazuril active compound, 25 mg/kg/day, p.o.	2.5% toltrazuril formulation, i.w.	Control of infection	Uninfected controls
						BL/6 mice		7 are	5 are	5 are	5 are
Day 0	21.06.99	25 mg/mouse	2.5％i.w.
						4h after infection	21.06.99	2×106Tachyzoite i.p.	2×106Tachyzoite i.p	2×106Tachyzoite i.p	Is not infected
P.i. 1 day.	22.06.99	25 mg/mouse	2.5％i.w.	-	-
						P.i. for 2 days.	23.06.99	25 mg/mouse	2.5％i.w.	-	-
P.i. for 3 days.	24.06.99	25 mg/mouse	2.5％i.w.	-	-
						P.i. for 4 days.	25.06.99	25 mg/mouse	2.5％i.w.	-	-
P.i. 5 days.	26.06.99	25 mg/mouse	2.5％i.w.	-	-
P.i. for 14 days.	5.07.99	Sacrifice of	Sacrifice of	Sacrifice of	Sacrifice of

After infection, p.o. ═ oral, i.w. ═ aqueous solutions

TABLE 3 anti-neospora caninum IgG in serum of individual mice and mean concentration of IgG in serum of each treatment group

TABLE 4 PCR and IFAT assays specific for neospora caninum

Test group	Mouse number*	Neosporosis PCR	IFAT
				Uninfected controls	242
Uninfected controls	228
				Uninfected controls	240
Uninfected controls	227
				Uninfected controls	233
Control of infection	239	+	+
				Control of infection	224	+	+
Control of infection	246	+	++
				Control of infection	234	+
Control of infection	222	+	+
				Toltrazuril 25mg/kg	241	+
Toltrazuril 25mg/kg	225
				Toltrazuril 25mg/kg	244
Toltrazuril 25mg/kg	220
				Toltrazuril 25mg/kg	231	+
Toltrazuril 25mg/kg	235
				Toltrazuril 25mg/kg	245	+
Toltrazuril 2.5% i.w.	230
				Toltrazuril 2.5% i.w.	238
Toltrazuril 2.5% i.w.	243
				Toltrazuril 2.5% i.w.	221
Toltrazuril 2.5% i.w.	229
				Positive control	14	+	+++

^*Mouse numbers were assigned randomly to ensure a fair assessment was made; mouse numberNot in logical order.

Claims (3)

1. Use of toltrazuril and punaizuril in the preparation of a composition for the prophylactic treatment of an animal against neospora caninum.

2. Use of toltrazuril and punaizuril in the preparation of a composition for use with a live or killed vaccine to treat an animal against a parasitic protozoan.

3. A composition comprising a triazineone-based active compound selected from toltrazuril and punanazuril and a live or killed vaccine as a combination product against parasitic protozoa.