WO1995006628A1 - Traitement et prophylaxie d'affections parasitaires ou bacteriennes - Google Patents

Traitement et prophylaxie d'affections parasitaires ou bacteriennes Download PDF

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WO1995006628A1
WO1995006628A1 PCT/DK1994/000332 DK9400332W WO9506628A1 WO 1995006628 A1 WO1995006628 A1 WO 1995006628A1 DK 9400332 W DK9400332 W DK 9400332W WO 9506628 A1 WO9506628 A1 WO 9506628A1
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Prior art keywords
chalcone
prop
dimethylethyl
butoxy
enyloxy
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PCT/DK1994/000332
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English (en)
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WO1995006628B1 (fr
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Arsalan Kharazmi
Søren Brøgger CHRISTENSEN
Chen Ming
Thor Grundtvig Theander
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Statens Seruminstitut
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Priority to AU76090/94A priority Critical patent/AU7609094A/en
Publication of WO1995006628A1 publication Critical patent/WO1995006628A1/fr
Publication of WO1995006628B1 publication Critical patent/WO1995006628B1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C49/00—Ketones; Ketenes; Dimeric ketenes; Ketonic chelates
    • C07C49/76—Ketones containing a keto group bound to a six-membered aromatic ring
    • C07C49/84—Ketones containing a keto group bound to a six-membered aromatic ring containing ether groups, groups, groups, or groups
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/12—Ketones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/34—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C45/00—Preparation of compounds having >C = O groups bound only to carbon or hydrogen atoms; Preparation of chelates of such compounds
    • C07C45/61—Preparation of compounds having >C = O groups bound only to carbon or hydrogen atoms; Preparation of chelates of such compounds by reactions not involving the formation of >C = O groups
    • C07C45/67—Preparation of compounds having >C = O groups bound only to carbon or hydrogen atoms; Preparation of chelates of such compounds by reactions not involving the formation of >C = O groups by isomerisation; by change of size of the carbon skeleton
    • C07C45/673—Preparation of compounds having >C = O groups bound only to carbon or hydrogen atoms; Preparation of chelates of such compounds by reactions not involving the formation of >C = O groups by isomerisation; by change of size of the carbon skeleton by change of size of the carbon skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C45/00—Preparation of compounds having >C = O groups bound only to carbon or hydrogen atoms; Preparation of chelates of such compounds
    • C07C45/61—Preparation of compounds having >C = O groups bound only to carbon or hydrogen atoms; Preparation of chelates of such compounds by reactions not involving the formation of >C = O groups
    • C07C45/67—Preparation of compounds having >C = O groups bound only to carbon or hydrogen atoms; Preparation of chelates of such compounds by reactions not involving the formation of >C = O groups by isomerisation; by change of size of the carbon skeleton
    • C07C45/68—Preparation of compounds having >C = O groups bound only to carbon or hydrogen atoms; Preparation of chelates of such compounds by reactions not involving the formation of >C = O groups by isomerisation; by change of size of the carbon skeleton by increase in the number of carbon atoms
    • C07C45/72—Preparation of compounds having >C = O groups bound only to carbon or hydrogen atoms; Preparation of chelates of such compounds by reactions not involving the formation of >C = O groups by isomerisation; by change of size of the carbon skeleton by increase in the number of carbon atoms by reaction of compounds containing >C = O groups with the same or other compounds containing >C = O groups
    • C07C45/74—Preparation of compounds having >C = O groups bound only to carbon or hydrogen atoms; Preparation of chelates of such compounds by reactions not involving the formation of >C = O groups by isomerisation; by change of size of the carbon skeleton by increase in the number of carbon atoms by reaction of compounds containing >C = O groups with the same or other compounds containing >C = O groups combined with dehydration
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to the use of a particular class of aromatic compounds, in particular bis-aromatic ⁇ , ⁇ -unsaturated ketones, most of which are novel com- pounds, for the treatment or prophylaxis of a number of serious conditions caused by microorganisms or parasites, in particular protozoa such as Leishmania, Plasmodia, and Coccidia such as Eimeria, and intracellular bacteria, including Legionella and Mycobacteria.
  • the invention also relates to the novel bis-aromatic ⁇ , ⁇ -unsaturated ketones and methods of preparing them, as well as to pharmaceutical and anti- parasitic compositions.
  • Parasitic diseases among these malaria and leishmaniasis, are, on a world basis, among the most important diseases. Most of the effective drugs against the diseases have many side effects for which reason it is not possible to maintain the treatment or prophylaxis of specific diseases for years.
  • Leishmania cause a broad spectrum of diseases ranging from the cutaneous healing skin lesions caused by L. major to a fatal visceral form of the disease called kala azar caused by L. donovani (Manson-Bahr, 1987). Leishmaniasis are widespread in many parts of the world with highest pre ⁇ valence in Africa, Asia, and Latin America (WHO, 1989). Recently an increasing number of AIDS patients are becoming infected with Leishmania (Brenguer, 1989; Flegg 1990).
  • Plasmodium falciparum Malaria, another parasitic disease, is also a serious health problem. Human malaria is caused by four species of the protozoan genus, Plasmodium. The species Plasmodium falciparum is the most dangerous, causing acute severe infections that are often fatal, especially in young children and immigrants entering endemic areas.
  • Coccidial protozoa such as Eimeria tenella are some of the most important parasites causing disease in poultry resulting in significant economic loss.
  • a class of aromatic com- pounds said class comprising compounds containing an alkylating site, show a re ⁇ markable capability of effectively suppressing the growth of parasitic protozoa and intracellular bacteria, which compounds at the same time can be so chosen that they are tolerable to animal cells such as human cells.
  • This valuable selective activity of such alkylating aromatic compounds seems to be based on their capability of interfer- ing with oxygen metabolism in the parasites by destroying their mitochondria, at concentrations at which the compounds, while thus being harmful to the microorgan ⁇ isms, do not affect the mitochondria of the animal cells.
  • the present invention in its broadest aspect, relates to the use of an aromatic compound which contains an alkylating site, and which is capable of alkylating the thiol group in N-acetyl-L-cysteine at physiological pH, for the prepara- tion of a pharmaceutical composition or a medicated feed, food or drinking water for the treatment or prophylaxis of a disease caused by a microorganism or a parasite in an animal, including a vertebrate, such as a bird, a fish or a mammal, including a human,
  • microorganism or parasite being selected from
  • tissue and blood protozoa such as Leishmania, Trypanosoma, Toxoplasma, Plasmodium, Pneumocystis, Babesia and Theileria; intestinal protozoan flagellates such as T ⁇ chomonas and Giardia; intestinal protozoan Coccidia such as Eimeria, Isospora, Cryptosporidium; Cappilaria, Microsporidium, Sarcocystis, Trichodina, Trichodinella,
  • Dacthylogurus Pseudodactylogurus, Acantocephalus, Ichthyophtherius, Botrecephalus; and intracellular bacteria, in particular Mycobacterium, Legionella species, Listeria, and Salmonella.
  • aromatic compound may in many cases advantageously be used in the form of a prodrug of the aromatic compound, and it will be understood that the present broadest aspect of the invention encompasses the use of such prodrugs.
  • the in vitro tests involve establishing the inhibition of the multiplication of the protozoa or bacteria on the one hand and the animal cells on the other hand by de ⁇ termining the inhibition of the uptake of radiolabelled precursors as an indication of the inhibition of the growth of the parasite or the animal cells in the presence of the test compound in the concentration in question (see Example 4 herein).
  • the tests involve a particularly suitable assay for assessing the tolerability of the aromatic alkylating compounds to animal cells, that is, an assay based on the assessment of the reduction caused by the compound on the thymidine uptake by lymphocytes of the animal in the Lymphocyte Proliferation Assay (LPA) which is the assay described in greater detail in Example 4.
  • LPA Lymphocyte Proliferation Assay
  • the alkylating site may be a carbon-carbon double bond conjugated with a carbonyl group
  • the carbonyl group is a ketonic carbonyl group which is further conjugated with an aromatic ring, such as a phenyl group.
  • the phenyl group may carry electron-donating groups, confer what is discussed above, in particular one or several hydroxy groups or derivatives thereof. In the case of hydroxy groups, these may be masked in order to prevent metabolism, confer the detailed discussion further below.
  • the masking groups are preferably chosen from groups from which the free phenol may be released in the body, either enzymatically or non-enzymatically.
  • the aromatic alkylating compound is one which, in a concentration in which it causes less than 50% reduction, preferably less than 40% reduction, and more preferably less than 20% reduction, of the thymidine uptake by human lymphocytes in the Lymphocyte Proli ⁇ feration Assay using phytohemagglutinin (PHA), meets at least one of the criteria a)- d) defined in claim 2.
  • the compounds to be used according to the invention are com ⁇ pounds which meet all of the criteria a) to d), because this is an indication of a broad- spectred activity and selectivity.
  • compositions prepared according to the invention are human leishmaniasis caused by Leishmania donovani, L. infantum, L. aethiopica, L. major, L. tropica, L. mexicana complex, or L. braziliensis complex or human malaria caused by Plasmodium falciparum, P. ovale, P. vivax, or P. malariae as well as parasitic diseases in livestock, such as Babesia in cattle, or a parasitic disease in birds, such as a disease caused by Coccidia such as Eimeria tenella in poultry such as chicken or turkey, or a parasitic disease in fish, such as Pseudodactylogurus or Trichodina.
  • the aromatic compound is preferably one which contains an aromatic ring attached to the alkylating site.
  • the compound in particular one which has electron-donating groups attached to an aromatic ring.
  • the alkylating site is typically a double or triple bond conjugated with a carbonyl group which carbonyl group optionally is further con ⁇ jugated with an aromatic ring such as a phenyl group, the aromatic ring attached to the alkylating site preferably containing at least one electron-donating group such as an oxygen, nitrogen or sulphur function such as hydroxy, alkoxy (e.g. methoxy), amino, alkylamino, dialkylamino, mercapto, or alkylthio. It is preferred that the electron-donating group(s) is/are attached to the aromatic ring in a position next to and /or most remote relative to the position through which the aromatic ring is attached to the alkylating site.
  • an aromatic ring such as a phenyl group
  • the aromatic ring attached to the alkylating site preferably containing at least one electron-donating group such as an oxygen, nitrogen or sulphur function such as hydroxy, alkoxy (e.g. methoxy), amino
  • Plasmodium falciparum The important human malaria parasites with which hundreds of millions of humans are infected, are Plasmodium falciparum, P. ovale, P. vivax, and P. malariae.
  • Plasmodium falciparum is the most important human parasite and the number one parasite killer of civilization.
  • the malaria parasites show widespread resistance against almost all available antimalarial drugs. For this reason, the fact that a new class of antimalarial drugs, chemically unrelated to the known antimalarial drugs has been provided, is a feature of the invention which is of great importance.
  • Another important aspect of the invention is that malaria parasites resistant against Chloroquine, the most commonly used antimalarial drug, show very high degree susceptibility to the compounds described herein. Malaria disease will not be brought under control without drugs which are effective against the drug resistant strains of the parasite.
  • Another important aspect of the invention is the antileishmanial activity of the compounds defined above.
  • Visceral leishmaniasis caused by Leishmania donovani or L. infantum, inflicts several million people in the world, and this disease recently appears to be a major problem for AIDS patients coming in contact with Leishmania parasites, combined with large scale clinical resistance in endemic areas such as India (which is announced "alarming" by the World Health Organization).
  • Other major diseases are diseases caused by other species of Leishmania, such as L. aethiopica, L. major, L. tropica, L. mexicana complex, and L. braziliensis complex. Some of these species cause severe disfiguring and morbidity in millions of humans in Central and South America and many parts of Africa.
  • the invention relates to the use of an aromatic compound which is a bis-aromatic ⁇ , ⁇ -unsaturated ketone of the general formula I as defined in claim 15 or 16.
  • the bis-aromatic ⁇ , ⁇ -unsaturated ketones act by selectively destroying the cells of the microorganisms or cells of multicellular parasites; as will appear from the below discussion and the examples herein, the bis-aromatic ⁇ , ⁇ -unsaturated ke ⁇ tones in appropriate concentration ranges will selectively kill the microorganisms or the multicellular parasites by destroying the cells of the microorganisms or cells of the multicellular parasites while showing a high degree of tolerance for the host cells which are subjected to exposure to the compounds.
  • the mechanism of action is via interference of the 0 2 -meta- bolism of the microorganism or parasite in question in that the bis-aromatic ⁇ , ⁇ -un ⁇ saturated ketone inhibits or interferes with the O ---metabolism of the mitochondria (where applicable) of the microorganism such as the parasite or the O 2 -metabolism of the bacteria itself.
  • the mitochondria of humans have been found to be able to tolerate the compounds in question in the same concentrations which will inhibit or kill the microorganism or the multicellular parasite. It is this remarkable selectivity of certain classes of bis-aromatic ⁇ , ⁇ -unsaturated ketones which constitutes the basis of this aspect of the present invention.
  • the invention opens up the possibility of controlling parasitic diseases not only by admini ⁇ stration to the animals, including humans, as therapy or prophylaxis, but also by kil ⁇ ling the parasite in its vector by spraying or otherwise applying an aromatic com ⁇ pound of the type defined above, such as a bis-aromatic ⁇ , ⁇ -unsaturated ketone, in the infected areas so that the vector will take up the compound, whereby the parasite will be subjected to the compound.
  • an aromatic com ⁇ pound of the type defined above such as a bis-aromatic ⁇ , ⁇ -unsaturated ketone
  • one aspect of the invention relates to a method for controlling transmission of parasitic diseases caused by parasites which have part of their life cycles in a vector, comprising applying an aromatic compound as defined above, such as a bis-aromatic ⁇ , ⁇ -ketone of the general formula I, to a locus which is a habitat of the vector so as to eradicate the parasites.
  • the parasites will, in this case, in particular be Leishmania, Plasmodium, or Trypanosoma, and the eradication of the parasite will, depending on the vector's tolerance to the compound, take place with or without concomitant eradication of the vector.
  • the aromate is preferably phenyl such as illustrated of the examples herein, but it is reasonable to contemplate that any of the aromate types mentioned in claim 15 can be the Ar 1 or Ar 2 of the bis-aromatic ⁇ , ⁇ -unsaturated ketone, considering that such aro ⁇ matic rings will affect the electron density in the unsaturated ketone similarly to the two phenyl rings, and that such aromates will also give possibilities for charge trans ⁇ fer complexes and lipophilic interactions with the target molecule, such as do the two phenyl rings.
  • interesting compounds are defined in greater detail in claim 17.
  • the aromate may carry other substituents which either will not to any substantial extent detract from the useful effect and selectivity of the bis-aromatic ⁇ , ⁇ - unsaturated ketones, or will enhance these properties or relevant properties related to the use and utility of the bis-aromatic ⁇ , ⁇ -unsaturated ketones, e.g., their solubility (such as when the bis-aromatic ⁇ , ⁇ -unsaturated ketones carry a nitrogen-containing basic group or a carboxyl group which can form water-soluble salts with pharmaceuti- cally acceptable counter ions).
  • the pre ⁇ ferred ones are generally those in which A is O, mainly because of their excellent pro ⁇ perties with respect to activity and selectivity/tolerability, such as will appear from the results reported herein.
  • the oxygen atom in the form of oxy in many biologically active compovmds may, with greater or lesser retention of, and indeed in certain cases with enhancement of, the biological activity, be replaced with bioisosteric groups, such as -S-, -NH-, and -N(Ci_. 6 alkyl)- as mentioned above.
  • the bis-aromatic ⁇ , ⁇ -unsaturated ketone has the general formula II as defined in claim 18 and 19.
  • the substituent or substituents on the phenyl groups is/are selected from methyl, ethyl, propyl, isopropyl, tert.-butyl, prop-2-enyl, 1,1-dimethylpropyl, l,l-dimethylprop-2-enyl, 3-methylbutyl, and 3-methylbut-2-enyl.
  • the host animals to be treated are primarily vertebrates such as birds, fish and mammals, including humans. It is evident that with respect to some of the microorganisms and multicellular parasites mentioned above, the host to be treated is defined once the microorganism or multicellular parasite is given. Thus, for example, when the microorganism is Leishmania, the hosts to be treated are humans or dogs; when the microorganism is Theileria, the animals to be treated are cattle, sheep and goats; when the microorganism is Eimeria, the animals to be treated are chickens and turkeys.
  • Mitochondria are oval-shaped organelles, typically about 2 ⁇ m in length and 0.5 ⁇ m in diameter, located intracellulary in all organisms except bacteria. Mito- chondria have two membrane systems, an outer membrane and an extensive, highly folded inner membrane, hence there are two compartments in mitochondria: the intermembrane space between the inner membrane and the outer membrane, and the matrix, which is bounded by the inner membrane.
  • Mitochondria are the organelles involved in the O 2 -metabolism of the cell.
  • Oxidative phosphorylation is the process in which ATP is formed as electrons are transferred from NADH or FADH 2 to O 2 by a series of electron carriers. This is the major source of ATP in aerobic organisms.
  • Oxidative phosphorylation is carried out by respiratory assemblies located as an integral part of the inner mitochondrial membrane. The outer membrane is quite permeable to most small molecules and ions. From B. Inoue et al, /. Toxicol. Sci.
  • licochalcone A is an important potential antiparasitic, in particular, antimalarial and antileishmanial drug.
  • the surprising effect and selectivity found is not limited to licochalcone A, but is characteristic of the class of bis-aromatic ⁇ , ⁇ -unsaturated ketones discussed herein and, for the reasons given above, is believed to apply more broadly to the aromatic compounds defined above.
  • aromatic compounds such as the bis-aromatic ⁇ , ⁇ -un ⁇ saturated ketones defined herein interfere with the O 2 -metabolism of the cytoplasmic membrane corresponding to the interference with the O 2 -metabolism of the mitochondria of higher developed organisms, thereby destroying the bacteria.
  • Peliminary experiments involving oral administration of licochalcone A to mice and rats and injection of licochalcone A to mice indicate that in animals such as mam ⁇ mals, the bis-aromatic ⁇ , ⁇ -unsaturated ketones which possess a free phenolic hydroxy group will be eliminated from the blood stream already after the first passage to through the liver. This is in accordance with what is known about the metabolism of other phenolic compounds.
  • an interesting aspect of the invention is constituted by compounds in which the phenolic hydroxy group or groups or bioiso ⁇ steric other group or groups AZ are masked, in other words, the so-called prodrugs, that is, compounds which are readily decomposed under conditions prevailing in the animal body to liberate the free groups which are associated with the active forms of the drugs.
  • the prodrugs used according to the invention are, e.g., compounds of the general formula I or II in which Z is a group which is readily decomposed under conditions prevailing in the animal body to liberate the group AH.
  • Z is a group which is readily decomposed under condi ⁇ tions prevailing in the animal body to liberate the group OH.
  • prodrug forms suitable in connection with particular substitu- ents in drugs is based upon the fact that certain types of groups will tend to be decom ⁇ posed in the animal body in accordance with various decomposition pathways.
  • the groups (A), (D), and (E) are groups which will be decomposed by esterases to result in the corresponding free group such as the hydroxy group.
  • the group (B) will be subjected to removal of one of the methyl groups in the liver, and the group thus formed will be relatively readily decomposable in plasma.
  • the oxy-containing groups (C) are groups which are rela ⁇ tively labile under acidic conditions and, as thus, are adapted to be decomposed, e.g., under the conditions under which Leishmania amastigotes exist in the human body, that is, in macrophages.
  • the prodrug group Z will be one which pre ⁇ vents the active molecule from being converted, in the liver, to a form which, from a practical point of view, will be inactive and quickly will be eliminated from the animal body, such as the forms where free phenolic OH groups are sulphated in the liver or are coupled to glucuronic acid in the liver.
  • Z is a group selected from the groups (A)-(E) as defined above.
  • Examples of particularly preferred groups Z are pivaloyl, pivaloyloxymethyl and N,N-dimethylcarbamoyl.
  • the administration route of the aromatic compound as defined above may be of any suitable route which leads to a concentration in the blood corresponding to a therapeutic concentration by the oral route, the parenteral route, the cutaneous route, the nasal route, the rectal route, the vaginal route and the ocular route. It should be clear to a person skilled in the art that the administration route is dependant on the compound in question, particularly, the choice of administration route depends on the physico- chemical properties of the compound together with the age and weight of the patient and on the particular disease and the severity of the same.
  • the aromatic compounds as defined above may be contained in any appropriate amount in a pharmaceutical composition, and are generally contained in an amount of about 1- 95% by weight of the total weight of the composition.
  • the composition may be in form of, e.g., tablets, capsules, pills, powders, granulates, suspensions, emulsions, solutions, gels including hydrogels, pastes, ointments, creams, plasters, drenches, delivery devices, suppositories, enemas, injectables, implants, sprays, aerosols and in other suitable form.
  • the pharmaceutical compositions may be formulated according to conventional pharmaceutical practice, see, e.g., "Remington's Pharmaceutical Scien ⁇ ces" and "Encyclopedia of Pharmaceutical Technology".
  • compositions according to the present invention may be formulated to release the active compound substantially immediately upon administration or at any substantially predetermined time or time period after administration.
  • the latter type of compositions are generally known as controlled release formulations.
  • Controlled release formulations may also be denoted “sustained release”, “prolonged release”, “programmed release”, “time release”, “rate-controlled” and/or “targeted release” formulations.
  • controlled release In general, two basically different strategies can be applied in order to obtain a controlled release formulation in which the rate of release outweighs the rate of metabolism of the compound in question.
  • the principle aims at changing the properties of the active drug substance by converting the substance into a masked form.
  • the compounds of the above formulae in which Z is one of the groups (A)-(E) are representatives of this strategy.
  • controlled release is obtained by appropriate selection of various formulation parameters and ingredients, including, e.g. various types of controlled release compositions and coatings (formulation-method).
  • controlled release compositions comprising the aromatic compounds defined above, such as bis-aromatic ⁇ , ⁇ -unsaturated ketones, according to the invention, e.g., by using a prodrug of the compound in question and then formulating according to the principles mentioned above.
  • every pharmaceutical composition is an actual drug delivery system, since upon administration it presents the active drug substance to the body of the organism.
  • the bis-aromatic ⁇ , ⁇ -ketones are preferably administered in an amount of about 0.1- 30 mg per kg body weight per day, such as about 0.5-15 mg per kg body weight per day.
  • the compound in question may be administered orally in the form of tablets, cap- sules, elixirs or syrups, or rectally in the form of suppositories.
  • Parenteral administra ⁇ tion of the aromatic compound defined above, such as the bis-aromatic ⁇ , ⁇ -unsatu ⁇ rated ketone is suitably performed in the form of saline solutions of the ketones (or salts thereof) or with the compound incorporated into liposomes.
  • an acid addition salt of a basic compound of the formula I that is, a compound of the formula I in which either an aromatic ring or a substituent contains a basic nitrogen atom
  • a solubilizer such as ethanol
  • Oral administration for compositions adapted for oral administration for systemic use, the dosage is normally 1 mg to 1 g per dose administered 1-4 times daily for 1 week to 12 months depending on the disease to be treated.
  • the dosage for oral administration for the treatment of parasitic diseases is normally 1 mg to 1 g per dose administered 1-2 times daily for 1-4 weeks, in particular the treat ⁇ ment of malaria is to be continued for 1-2 weeks whereas the treatment of leishmaniasis will normally be carried out for 3-4 weeks.
  • the dosage for oral administration for the treatment of bacterial diseases is normally 1 mg to 1 g per dose administered 1-4 times daily for 1 week to 12 months; in particular, the treatment of tuberculosis will normally be carried out for 6-12 months.
  • the dosage for oral administration of the composition in order to prevent diseases, in particular, parasitic diseases is normally 1 mg to 75 mg per kg body weight per day.
  • the dosage may be administered once or twice daily for a period starting 1 week before the exposure to the disease until 4 weeks after the exposure.
  • compositions adapted for rectal use for preventing diseases a somewhat higher amount of aromatic compounds, such as bis-aromatic ⁇ , ⁇ -unsaturated ketones or derivatives thereof is usually preferred, i.e. from approxi- mately 1 mg to 100 mg per kg body weight per day.
  • parenteral administration For parenteral administration a dose of about 0.1 mg to about 50 mg per kg body weight per day is convenient. For intravenous administra ⁇ tion a dose of about 0.1 mg to about 20 mg per kg body weight per day administered for 1 day to 3 months is convenient. For intraarticular administration a dose of about 0.1 mg to about 20 mg per kg body weight per day is usually preferable.
  • parenteral administration in general a solution in an aqueous medium of 0.5-2% or more of the active ingredients may be employed.
  • Percutaneous administration For topical administration on the skin a dose of about 1 mg to about 5 g administered 1-10 times daily for 1 week to 12 months is usually preferable.
  • antileishmanial drugs to be combined with the compounds defined herein may be mentioned pentavalent antimony-sodium gluconate and allopurinol.
  • antimalarial drugs to be combined with the compounds defined herein may be mentioned chloroquine and derivatives thereof, quinine, proguanil, cycloguanil, mefloquine, pyrimethamine and artemisinin.
  • an antituberculous drug such as isoniazide, ethambutol, pyrazinamide, and rifampicin.
  • additional antimycotic drugs to be combined with the compounds defined herein may be mentioned amphotericin B, muconarcidol, griseofulvin, and miconazol.
  • additional antibabesial drugs to be combined with the compovmds defined herein may be mentioned quinuronium sulfate, pentamidine isethionate, imidocarb or diminazene.
  • additional anticoccidial drugs to be combined with the compounds defined herein may be mentioned fulfonamides, amprocid and coccidiostatic agents such as inomycins, in particular monensin and salinomycin.
  • One general advantage of the compounds defined herein are their broad-spectered character, which makes it possible to use the compovmds as sole medication in cases where the host to be treated is infected with, or suspected to be infected with, more than one of the bacteria and parasites discussed herein, or to use them as supplements to known antibacterial agents and antiparasitic agents in order to reduce the dose of the conventional antibiotics or antiparasitic agents, thus reducing the risk of side effects, in addition to the above-mentioned advantages with respect to reduction of drug resistance development.
  • the broad-spectered character of the compounds of the general formula I is of great advantage, and may be further augmented by combina ⁇ tion with more than one antibacterial or antiparasitic agent, such as combination with both another antileishmanial agent and another antimalarial agent. It is justified to presume that also the other aromatic compounds defined herein will show the same valuable broad-spectered character.
  • This reaction which is a condensation reaction, is suitably carried out under acid or base catalyzed conditions.
  • a review of such processes may be found in Nielsen, A.T., Houlihahn, W.J., Org. React. 16, 1968, p 1-444. In particular the method described by Wattanasin, S. and Murphy, S., Synthesis (1980) 647 has been found to be very successful.
  • the reaction may suitably be carried out in protic organic solvents, such as lower alcohols (e.g. methanol, ethanol, or tert.butanol), or lower carboxylic acids (formic, glacial acetic, or propionic acid), or in aprotic organic solvents such as ethers (e.g.
  • the catalyst may be selected from sodium, lithium, potassium, barium, calcium, magnesium, aluminum, ammonium, or quaternary ammonium hydroxides, lower alkoxides (e.g.
  • Primary aromatic amines such as aniline, free secondary amines such as dimethyl amine, diethyl amine, piperidine, or pyrrolidine as well as basic ion exchange resins may also be used.
  • Acid catalysts may be selected from hydrogen chloride, hydrogen bromide, hydrogen iodide, sulfuric acid, sulfonic acids (such as paratoluenesulfonic or methanesulfonic acid), lower carboxylic acids (such as formic, acetic or propionic acid), lower halogenated carboxylic acids (such as trifluoroacetic acid), Lewis acids (such as BF 3 , POQ 3 , PCI 5, or FeCl 3 ), or acid ion exchange resins.
  • sulfonic acids such as paratoluenesulfonic or methanesulfonic acid
  • lower carboxylic acids such as formic, acetic or propionic acid
  • lower halogenated carboxylic acids such as trifluoroacetic acid
  • Lewis acids such as BF 3 , POQ 3 , PCI 5, or FeCl 3
  • acid ion exchange resins such as BF 3 , POQ 3 , PCI 5, or FeCl 3
  • a drawback of the base catalyzed condensation is the poor yield obtained if the aromatic ring in which the ketone or the aldehyde or bort is substituted with one or more hydroxy groups.
  • This inconvinience can be overcone by masking the phenolic group by T. Hidetsugu et al. European patent application 0370 461 (1989). Deprotection is easily performed by mineral acids such as hydrochloric acid.
  • the reaction may be carried out at temperatures in the range of 0-100°C, typically at room temperature. Reaction times may be from 30 min to 24 hours.
  • compounds of the formula I in which W is -C ⁇ C- may be prepared by reacting an activated derivative of a carboxylic acid of the general formula
  • the activated derivative of the carboxylic acid IV may be an activated ester, an anhydride or, preferably, an acid halogenide, in particular the acid chloride.
  • the reaction is normally carried out using the catalysts described by Tohda, Y. et al. cited above, namely copper(I)iodide/triphenylphosphine-palladium dichloride.
  • the reaction is suitably carried out in tiiethylamine, a mixture of tiiethylamine and pyridine or tiiethylamine and toluene under a dry inert atmosphere such as nitrogen or argon.
  • the reaction is generally carried out at reduced temperature such as in the range from -80°C to room temperature, the reaction time typically being from 30 minutes to 6 hours.
  • a hydroxy group on Ari and /or Ar 2 may be protected in the form of the methoxymethyl ether, N,N-dimethylcarbamoyl ester, or allyl ether.
  • the protecting group may be removed after the reaction in a manner known per se.
  • a chlorinating agent e.g. NCS or t-butylhypochlorite
  • NCS t-butylhypochlorite
  • the chlorination and the formation of the isoxazoline ring can be performed by a one-pot method. Solvents like methylenechloride, chloroform are most commonly used. By reducing the formed isoxazoline in an aqueous medium, the reduced product will be hydrolyzed to a ⁇ -hydroxyketone of the general formula E, .
  • Ar 1 , Ar 2 , X and Y are as defined above, and optionally substituting the hydroxy group with another leaving group such as halide, alkoxy, tosyloxy, or trifluoromethanesulfonoxy, such other leaving group being introduced in a manner known per se.
  • another leaving group such as halide, alkoxy, tosyloxy, or trifluoromethanesulfonoxy, such other leaving group being introduced in a manner known per se.
  • T can be aliphatic, alicyclic or aromatic, to give the chalcone structure of the general formula (I).
  • the Wittig reaction is known as an exceedingly useful method for the synthesis of alkenes.
  • the aldehyde may be aliphatic, alicyclic or aromatic; it may contain double or triple bonds; it may contain various functional groups, such as OH, OR, NR 2 , aromatic nitro or halo, acetal or even ester groups. Double or triple bonds conjugated with the carbonyl also do not interfere; the attack is directed towards the carbonyl carbon atom.
  • the reaction is suitably carried out in aprotic organic solvents such as ethers (e.g. tetrahydrofuran, dioxan, or diethyl ether) or DMSO or mixtures of these.
  • ethers e.g. tetrahydrofuran, dioxan, or diethyl ether
  • DMSO DMSO
  • the Wittig reaction is used for the preparation of chalcone in 84% yield (Bestmann, H.J., and Kratzer, O.).
  • the hydroxy group may, if desired, be substituted with another leaving group such as alkoxy, tosyloxy, trifluoromethanesulfonoxy or acyloxy in a manner known per se.
  • another leaving group such as alkoxy, tosyloxy, trifluoromethanesulfonoxy or acyloxy in a manner known per se.
  • Another route for the synthesis of compounds of the general formula I is by reacting derivatives of cinnamic acid which is based on the fact that cirmamic acid can react with aromatic compounds (e.g. phenols and benzene).
  • aromatic compounds e.g. phenols and benzene
  • the reaction is best carried out in the presence of BF 3 (Starkov, S. P. et al, Khim. Tekhnol., 20, 1149 (1977)) or polyphosphoric acid (Reichel, L., and Proksch, G., Justus Liebigs Ann. Chem., 745, 59 (1971)). With the former agent, a high preference for para-acylation is observed.
  • BF 3 Starkov, S. P. et al, Khim. Tekhnol., 20, 1149 (1977)
  • polyphosphoric acid Reichel, L., and Proksch, G., Justus Liebigs Ann. Chem., 745, 59 (1971)
  • reaction is preferably carried out in the presence of A1C1 3 as a catalyst (Rasschaert, A. et al, Bull. Soc. Chim. Beiges., 75, 449 (1966).
  • the two latter methods are examples of the Friedel-Craft acylation.
  • the reagents (compounds L) used are not only acyl halides or the acids, but also anhydrides.
  • the reaction can be carried out with only very small amounts of catalyst, often just a trace and sometimes without any catalyst at all.
  • Ferric chloride, iodine, zinc chloride, and iron are the most common catalysts used.
  • Proton acids can be used as catalyst when the reagent (compound L) is in its acid form.
  • the reaction is often carried out in a non-aromatic fully saturated solvent or in an aromatic solvent with deactivating groups, which prevents acylation, such as, for example, nitrobenzene.
  • the reaction is carried out at wide varying temperatures depending on the nature of the reacting compounds.
  • prodrugs of the general formula I can be made directly by processes as described below, the desired prodrug groups being in place in the relevant reagents in question, or free AZ groups in which Z is hydrogen, in particular hydroxy groups, can be converted to the corresponding groups in which Z is one of the groups (A) to (E) as defined above.
  • (Acyloxy)alkyl- ⁇ -ethers such as those of the general formulas (IIDa, IIEa, IIEb, I'Db, I'Eb, mDa, illEa) can be prepared by reacting the corresponding phenols of the general formulas (Ila, lib, Ilia) with the appropriate (acyloxy)alkyl- ⁇ -halide.
  • reaction is most often carried out using acetone or butanone as solvents.
  • a weak base like potassium carbonate may be added as an acid scavenger.
  • the halogen should be iodine in order to avoid formation of pivalic esters of phenol (Sloan, K.B., and Koch, S. A. M., /. Org. Chem. 48 (1983) 3777-3783.)
  • Carboxylic esters of the phenols of the general formula I may be prepared by reacting the corresponding phenols (e.g. Ha, lib, ⁇ ia) with an activated ester (including the ⁇ -halomethylesters), an anhydride or, preferably, an acid halogenide, in particular the acid chloride.
  • the reaction is performed in an aprotic organic solvent such as lower aliphatic ketones like acetone, butanone, aliphatic ethers like tetrahydrofuran, diethylether, or dioxane or a liquid amine like pyridine.
  • an acid scavenger such as potassium or sodium carbonate, an tertiary aliphatic amine such as triethylamine, or pyridine.
  • N,N-Dimethylcarbamic esters of the phenols of the general formula I (I'Bb, IIIBa) may be prepared by reacting the corresponding phenols of the general formula I (Ila, lib) with an activated derivative of N,N-dimethylcarbamic acid such as an activated ester or, preferably, an acid halide, in particular the acid chloride.
  • an activated derivative of N,N-dimethylcarbamic acid such as an activated ester or, preferably, an acid halide, in particular the acid chloride.
  • the reaction is carried out in an aprotic organic solvent such as lower aliphatic ketones like acetone, butanone, aliphatic ethers such as tetrahydrofuran, diethylether, or dioxane, or a liquid amine such as pyridine, or a liquid nitrate such as acetonitrile.
  • an acid scavenger such as potassium or sodium carbonate, a tertiary aliphatic amine such as triethylamine or pyridine
  • the N,N-dimethylcarbamoyl esters may be prepared by condensing the carbamoylated phenolic benzaldehydes or phenolic acetophenones with the appropri- ate acetophenones or benzaldehydes, respectively.
  • alkoxymethoxy ethers of the general formula I are most conveniently prepared by condensing the appropriate ethers of the phenolic benzaldehydes or the phenolic acetophenones with the appropriate acetophenones or benzaldehydes, respectively. They may, however, be prepared by reacting the phenolic chalcones with the appropriate alkyl- ⁇ -alkylhalomethyl halide.
  • the reaction may be carried out in an aprotic organic solvent like a lower aliphatic ketone, such as acetone or butanone, or an ether, such as tetrahydrofuran, dioxane or dioxolane or a liquid nitrate such as acetonitrile.
  • the reaction may be performed in the presence of a acid scavenger such as an inorganic or organic base.
  • the base may be potassium or sodium or quaternary ammonium carbonate, or hydroxide.
  • Fig. 1 shows the effect of licochalcone A on the parasitic load of the footpad of the mice infected with Leishmania major as described in Example 5.
  • Fig. 2 Five Syrian golden male hamsters weighing 50-70 g which were infected with L. donovani by intiacardial injection of 2 x IO 7 stationary phase promastigotes. One day later the animals were injected intraperitoneally with 10 mg/kg body weight licochalcone A (100 ⁇ l in saline) for 6 days. The animals were sacrificed on day 8 and parasite load in the spleen and liver was determined by determining the growth of promastigotes from the spleen and the liver using 3H-thymidine uptake by promastigotes.
  • Licochalcone A was isolated from Chinese licorice root of Glycyrrhiza species rich in licochalcone A by bioassay-guided fractionation, the bioassay being the L. major growth test described in Example 4.
  • This example illustrates the preparation of benzaldehydes used as starting materials.
  • This example illustrates the preparation of bis-aromatic ⁇ , ⁇ -unsaturated ketones.
  • i3C NMR data (50 MHz, CD 3 CN-DMSO-d 6 , ⁇ ) 187.5, 138.8, 157.8, 117.5, 161.1, 99.2, 162.9, 106.8, 13.0, 131.7, 116.2, 164.0, 116.2, 131.7, 56.4, 56.2.
  • the following chalcones are prepared: 3,5-dimethoxy-4'-hydroxychalcone, 3,5-diethoxy-4'-hydroxychalcone, 3,5-di-n-propoxy-4'-hydroxy chalcone, 3,5-diisopropoxy-4'-hydroxy chalcone, 3,5-di-n-butoxy-4'-hydroxy)chalcone, 3,5-di-t-butoxy-4'-hydroxy)chalcone,
  • Licochalcone A and analogues thereof, prodrugs thereof and a heterocyclic chalcone Drugs. Licochalcone A and analogues thereof, prodrugs thereof and a heterocyclic chalcone.
  • BMNC Human blood mononuclear cells
  • PHA mitogen phytohaemagglutinin
  • PPD tubercu ⁇ lin
  • Table 4.1 The effect of chalcones on L. major promastigotes from 4-days cultures, on P. falciparum growth in vitro, and on human lymphocyte proliferation response to PHA.
  • the upper figures are percentage inhibition of human promastigotes
  • the mid ⁇ dle figures are inhibition of malaria parasites
  • the lower figures are inhi ⁇ bition of lymphocytes. When standard deviation is given, more than five experi ⁇ ments have been performed.
  • the data in Table 4.1 indicate the importance of oxygenation in the, e.g. 2,4-, 3,4- or 4'- hydroxy-3,5-position for obtaining compounds which preferentially inhibit thymidine uptake into the parasites.
  • the in vitro results reported above confirm the hypothesis that the unsaturated ⁇ , ⁇ -position is of importance for the activity. This is substanti ⁇ ated by a very low activity shown by l-(4-hydroxyphenyl)-3-(2,4-dimethoxy)phenyl-2- propan-1-one.
  • the pattern shown by the results indicates that one of the mechanisms of action might be an alkylation of the target biomolecule by the ⁇ , ⁇ -unsaturated ketones.
  • licochalcone A is able to react with a thiol-containing peptide, by which reaction a nucleophilic thiol group is added to the ⁇ , ⁇ -double bond.
  • the principle is well known from the anti-cancer activity of ⁇ -methylene sesquiterpene lactones.
  • One such ⁇ -methylene sesquiterpene lactone has been tested in the in vitro model and has been found to be extremely active against Leishmania parasites in vitro, but at the same time also to show extreme toxicity on human lymphocytes (data not shown).
  • the substituents in the chalcone skeleton contribute to the selectivity of these compounds.
  • mice BALB/c female mice aged eight weeks old were used throughout.
  • mice received s.c. injections (in 0.05 ml of PBS) in the left hind footpad with lxlO 7 stationary phase promastigotes.
  • Footpad lesions were measured and expressed as footpad thickness increase (in mm).
  • the footpad thickness of mice was measured before infection and every 3 days after 7 days of infection. From 7 days of infection, mice received licochalcone A injections i.p. once a day. After 42 days of licochalcone A injection, some of the mice were killed and the footpads, spleens and livers removed. The parasite loads in the footpads and livers were estimated by a modification of the method described by Liew et al. using 3- H-thymidine uptake. The results were expressed as cpm. The footpads, spleens and livers impression was also estimated.
  • Table 5.1 Effect of licochalcone A on the parasitic load of the footpad of the mice in ⁇ fected with L. major. The results are from 2 mice from each group and are given as mean xlO 3 cpm of 3H-thymidine uptake.
  • Table 5.2 Effect of licochalcone A on the parasitic load of the liver of the mice infected with L. major. The results are from 2 experiments and are given as mean xlO 3 cpm of 3H-thymidine uptake.
  • Fig. 1 shows the effect of licochalcone A on footpad thickness increase (swelling) in BALB/c mice infected with L. major, expressed in mm.
  • Licochalcone A was dissolved in 20 ⁇ l of 99% (v/v) ethanol, and the 980 ⁇ l of medium 199 was added, and the resulting mixture was stored at -20°C.
  • Animals were intracardially inoculated with 2 x 10 7 L. donovani promastigotes in 0.1 ml medium 199 (Day 0). One hour later, one of the animals was killed. The liver and the spleen were weighed. The liver and the spleen impression smears were made. After air-drying, the impression smears were fixed with water-free methanol and stained with Giemsa. Five of the animals were treated (i.p.) with licochalcone A (10 mg/kg body weight two times per day) from Day+1 to Day+7. Another five animals were treated with 0.85% NaCl. The animals were killed on Day+8.
  • the liver and spleen were weighed, and the liver and the spleen impression smears were made. The number of the parasite in the liver and the spleen were counted under micro ⁇ scope. The spleen of the animals were cut into very small pieces, cultured in 15 ml of the culture medium at 26°C overnight and the parasite load was determined by 3H- thymidine uptake as described in Example 4 herein.
  • mice BALB/c female mice aged eight weeks were used.
  • mice were infected withP. yoelii YM strain (Table 7.1) and were compared to the outcome of infection in untreated control animals and in animals treated with licochalcone A.
  • the parasites were maintained by passage through BALB/c mice, and the animals were infected by injection of infected erythrocytes obtained from mice with a parasitemia of approximately 40%.
  • the animals were injected intraperitoneally with either 1 x IO 6 parasitized erythrocytes diluted in 0.9% NaCl and in a final volume of 0.2 ml.
  • the day of infection was termed day 0. Assessment of effect.
  • the outcome of infection was assessed microscopically by examination of Giemsa stained blood films.
  • the load of infection (the parasitemia) was calculated as the percentage of infected erythrocytes of the total number of erythro- cytes.
  • Table 7.1 Effect of licochalcone A and analogues thereof in BALB/c mice infected with P. yoelii YM strain. Parasitemia in mice with licochalcone A or an analogue thereof. The treatment was initiated 24 hours after infection with 1 x 106 parasites/mouse, and the mice received i.p. various concentrations of each compound as indicated below for a total of 5.5 days. The symbol # indicates number of animals.
  • Licochalcone A and some of the analogues thereof are able to completely clear the infection of P. yoelii in mice.
  • mice for some of the analogues The dose range protective for the mice for some of the analogues is fairly broad. It is interesting to note that the 4'-hydroxy-, 2,4-dimethoxy-4'-hydroxy- and 4'-allyloxychalcones exhibit potent antimalarial activity in low doses. It is contem ⁇ plated that this applies generally to the classes to which these chalcones belong, that is, the classes of chalcones defined in claims 21, 24, 25, 26, 29, 32, 35, 38, 40, 41and 46 and that doses of at the most 20 mg per kg body weight per day, such as at the most 10 mg per kg body weight per day or even at the most 5 mg per kg body weight per day, will be preferred dosages for such compounds.
  • Licochalcone A was isolated as described in Example 1.
  • Legionella strains Five clinical isolates from bronchial secretions and a lung abscess: 2 Legionella pneumophila serogroup 1 and 3 Legionella micdadei (L. detroit, L. ban, L. F 1433). Eight Legionella pneumophila serogroups 1-7 and one strain of each of L. bozemanii, L. dumoffii, L. gormanii, L. micdadei, L. feelei, L. z ⁇ adswort- hii, L. longbeacheae. Staphylococcus aureus ATCC 25923 was the control strain. The Legionella strains were subcultured on buffered charcoal yeast extract with alfa- ketoglutarate (BCYE- ⁇ ), and the rest of the strains were subcultured on 10% horse blood agar for 48 hours and 24 hours, respectively.
  • BCYE- ⁇ buffered charcoal yeast extract with alfa- ketoglutarate
  • Macrodilution rows were made with buffered yeast extract with alfa-ketoglutarate (BYE- ⁇ ) with 2 ml aliquots in vials, containing various concentrations of licochalcone A. Suspensions of Legionella species and the other pathogens and commensals were made in BYE- ⁇ . All the dilution rows were inoculated to give a final concentration of 105CFU/ml. After incubation at 37°C for 2 and 24 hours, respectively, aliquots of 10 ⁇ l were taken from all dilution steps and plated onto BCYE-x agar plates (all Legionella species) and to 10% horse blood agar (all non-Legionella strains). All the BCYE-x plates were incubated for 48 hours in a humid atmosphere at 37°C and read. The inoculated 10% horse blood agar plates were incubated in a normal atmosphere at 37°C for 24 hours and read.
  • BYE- ⁇ buffered yeast
  • Licochalcone A exhibited a clear anti-legionella activity at MIC values form 1 ⁇ g/ml, in most cases from 1 to 4 ⁇ g/ml.
  • the low MIC for L. pneumophila, the human pathogen, is promising and therefore licochalcone A can be considered as a potential drug against respiratory infections.
  • the reason that the MIC was very high for the inhibition of Legionella micdadei could be that the cell wall of L. micdadei is different from the cell wall of L. pneumophila (Hebert et al, 1984) and therefore, that the uptake of licochalcone A in L. micdadei is poorer than the uptake in L. pneumophila .
  • Susceptibility testing was performed radiometrically by using a BECTEC 460-TB 15 apparatus in a confined atmosphere (5% CO 2 ). Bacterial growth was measured as a function of the ability of the bacteria to catabolize i4C-labelled palmitic acid in the BECTEC 7H12B TB medium during growth, which resulted in the release of ⁇ C-labelled CO 2 . The growth was expressed as a numerical value called the growth index (GI) which ranged from 1 to 999.
  • GI growth index
  • the 7H12 vials were inoculated with 0.1 ml of 0 an appropriately diluted Dubos broth culture to give a final inoculum of about 5xl0 colony-forming units (CFU) per ml together with 0.1 ml of different concentrations of licochalcone A.
  • the final concentrations of licochalcone A tested ranged from 1.25 ⁇ g/ml to 80 ⁇ g/ml.
  • the final inoculum was determinated by culturing 5 0.1 ml from the control vial onto one Lowenstein-Jensen slant.
  • the vials were incuba ⁇ ted under stationary conditions at 35°C and growth was monitored by daily GI determination for 7 days. At day 7, 0.1 ml from each vial with a GI reading ⁇ 30 was cultured onto one Lowenstein-Jensen slant. Colony counts were enumerated after incubation at 35°C for 3 weeks. 0
  • MIC Minimal inhibitory concentration
  • MMC Minimal bactericidal concentration
  • Eimeria tenella sporulated oocysts were obtained from the Agricultural and Food Council Institute for Animal Health, Compton Laboratory, Berkshire, England. The oocysts were washed and resuspended in 30 ml saline to give a concentration of 15 x 10 6 /30 ml. A volume of 0.1 ml (50,000 oocysts) was given to each chicken.
  • the experimental set-up consisted of 4 groups of 14-days old chickens. During the first 14 days of life, all the chickens received chicken feed containing no coccidiostatic agents. The first 3 groups were given 50,000 E. tenella oocysts per chicken by oral administration on day 14. Feeding the chickens with the feed preparations described above started one day before infection with the parasite (day 13). The treatment continued for 14 days according to the set-up shown in Table 10.1.
  • Parasite load in the intestine or number per smear may be determined at the end of the experiment.
  • the number of oocysts in 10 viewfields may be counted at 100 x en- largement and the average of 10 fields were used.
  • Oocysts index may calculated as: Oocysts in infected animal/field x 100
  • Table 10.2 Effect of licochalcone A on weight gain of chickens.
  • the groups are as shown in Table 10.1.
  • the weights are given in grams per chicken.
  • Feed consumption measured as the amount of feed consumed per kg chicken weight gain in the group receiving licochalcone A was the lowest among all the 6 groups both on 7 days of treatment and on 14 days of treatment (Table 10.3).
  • the feed consumption of the group receiving licochalcone A was even lower than the group receiving standard chicken feed containing salinomycin which is a know cocci- diostatic agent. This indicates that licochalcone A might have a growth promoting effect or another form of nutritional value.
  • the chickens receiving Ucochalcone A did not perform the same way as those receiving standard chicken feed containing salinomycin. However, when comparing the licochalcone A group with the group receiving standard feed, it should be noted that this group received standard feed which besides a coccidiostatic agent also contains larger amounts of nutrients, vitamin, and growth promoting factors. In the above experiment, licochalcone A did not show a complete protection against E. tenella infection. This is probably due to the dosage of licochalcone A used in the ex ⁇ periment. It should also be mentioned that the experimental infection is a much stronger form of infection than the infection which will normally be encountered in practice.
  • the apolar eluent was used for the fol ⁇ lowing chalcones: 3,4,5-trimethoxy-4'-(3-methylbut-2-enyloxy)chalcone, 2,4-dimethoxy- 4'-(3-methylbut-2-enyloxy)chalcone and 2,4,6-tiimethoxy-4'-(3-methylbut-2-enyloxy)- chalcone.
  • the rate of decline of the chalcone concentration was followed by HPLC.
  • the rate constant was estimated using Grafit and assuming that the reaction followed first order kinetic; i.e.:
  • [c] is the concentration of the chalcone in question at time t
  • [c 0 ] is the concentra ⁇ tion of the chalcone in question at time zero
  • k is the rate constant
  • t the time. In all cases, very good fits between the observed concentiations and the concentrations calculated using the estimated rate constants were obtained.

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Abstract

Composés aromatiques ou leurs précurseurs contenant un site d'alkylation et capables d'alkyler le groupe thiol de la N-acétyl-L-cystéine, et en particuler les cétones bi-aromatiques α,β-insaturées, et servant à préparer des compositions pharmaceutiques ou des aliments, nutriments ou boissons médicalisés servant au traitement ou à la prophylaxie de maladies causées par des micro-organismes ou parasites, et en particulier par des protozoaires tels que les Leishmania, Trypanosoma, Toxoplasma, Plasmodium, Pneumocystis, Babesia, Theileria et des protozoaires intestinaux tels que les Trichomonas et Ciardia; des coccidies telles que les Eimeria, Isospora, Cryptosporidum, Cappilaria, Microsporidium, Sarcocystis, Trichodina, Trichodinella, Dacthylogurus, Pseudodacthylogurus, Acantocephalus, Ichthylophtherius, Botrecephalus; et des bactéries intracellulaires en particulier les Mycobacterium, les espèces legionella, les Listeria et les Salmonella. Les composés préférés présentent la formule (II): Xm-Ph-C(O)-CH=CH-Ph-Yn où chacun des groupes phényl (Ph) peut être mono ou polysubstitué, X et Y désignent ARH ou AZ, où A est O, S, NH ou N(C1-6alkyl), RH représente un hydrocarbyle aliphatique, Z est H ou un groupe masque qui se décompose en libérant AH, m est 0, 1 ou 2, et n est 0, 1, 2 ou 3, où si m est 2, les deux X sont identiques ou différents et où si n est 2 ou 3, les deux ou trois Y sont identiques ou différents, pour autant que m et n ne soient pas tous deux nuls. On peut citer comme exemples de ces composés des chalcones et notamment la licochalcone A (tirée notamment de racines du réglisse chinois de l'espèce Glycyrrhiza, p.ex. du G. uralensis ou du G. inflata), ainsi que leurs analogues hydroxy, alc(én)yle et/ou alc(én)yloxy qui agissent in vitro et/ou in vivo notamment contre le L. major et le P. falciparum.
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EP0817624A1 (fr) * 1995-03-30 1998-01-14 The Regents Of The University Of California Inhibiteurs de proteases de metazoaires parasites
WO1999000114A2 (fr) * 1997-06-26 1999-01-07 Statens Serum Institut 1,3-bis-aromatique-prop-2-en-1-ones, 1,3-bis-aromatique-propane-1-ones et 1,3-bis-aromatique-prop-2-yn-1-ones a action biologique
WO2001021164A2 (fr) * 1999-09-22 2001-03-29 Advanced Life Sciences, Inc. Compositions anti-mycobacterium, procedes de preparation et d'utilisation associes
US6673351B1 (en) 2001-03-16 2004-01-06 Astralis, Llc Compositions and methods for the treatment and clinical remission of psoriasis
US7432303B2 (en) * 1999-12-23 2008-10-07 J. Phillip Bowen Chalcone and its analogs as agents for the inhibition of angiogenesis and related disease states
WO2011097797A1 (fr) * 2010-02-10 2011-08-18 Peng Jinlian Dérivé d'un polyacide phénol-acrylique, sa préparation et son utilisation dans la préparation de médicaments
CN101648865B (zh) * 2008-08-16 2013-03-20 重庆市科学技术研究院 多酚丙烯酸衍生物及其在药物中的应用
CN115363136A (zh) * 2022-08-30 2022-11-22 河南科技大学 甘草多糖在制备改善肉鸡盲肠健康状况、空肠免疫状况、小肠形态制剂方面的应用

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US6194421B1 (en) 1995-03-28 2001-02-27 The Regents Of The University Of California Inhibitors of metazoan parasite proteases
EP0817624A1 (fr) * 1995-03-30 1998-01-14 The Regents Of The University Of California Inhibiteurs de proteases de metazoaires parasites
EP0817624A4 (fr) * 1995-03-30 1999-03-10 Univ California Inhibiteurs de proteases de metazoaires parasites
WO1999000114A2 (fr) * 1997-06-26 1999-01-07 Statens Serum Institut 1,3-bis-aromatique-prop-2-en-1-ones, 1,3-bis-aromatique-propane-1-ones et 1,3-bis-aromatique-prop-2-yn-1-ones a action biologique
WO1999000114A3 (fr) * 1997-06-26 1999-08-12 Statens Seruminstitut 1,3-bis-aromatique-prop-2-en-1-ones, 1,3-bis-aromatique-propane-1-ones et 1,3-bis-aromatique-prop-2-yn-1-ones a action biologique
WO2001021164A3 (fr) * 1999-09-22 2002-01-10 Advanced Life Sciences Inc Compositions anti-mycobacterium, procedes de preparation et d'utilisation associes
WO2001021164A2 (fr) * 1999-09-22 2001-03-29 Advanced Life Sciences, Inc. Compositions anti-mycobacterium, procedes de preparation et d'utilisation associes
US6677350B1 (en) 1999-09-22 2004-01-13 Advanced Life Sciences, Inc. Beta-fluoroethyl thiourea compounds and use
US7432303B2 (en) * 1999-12-23 2008-10-07 J. Phillip Bowen Chalcone and its analogs as agents for the inhibition of angiogenesis and related disease states
US6673351B1 (en) 2001-03-16 2004-01-06 Astralis, Llc Compositions and methods for the treatment and clinical remission of psoriasis
CN101648865B (zh) * 2008-08-16 2013-03-20 重庆市科学技术研究院 多酚丙烯酸衍生物及其在药物中的应用
WO2011097797A1 (fr) * 2010-02-10 2011-08-18 Peng Jinlian Dérivé d'un polyacide phénol-acrylique, sa préparation et son utilisation dans la préparation de médicaments
CN115363136A (zh) * 2022-08-30 2022-11-22 河南科技大学 甘草多糖在制备改善肉鸡盲肠健康状况、空肠免疫状况、小肠形态制剂方面的应用

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