WO2006032380A1 - Quaternary alkaloid derivatives of chelidonium majus l - Google Patents

Quaternary alkaloid derivatives of chelidonium majus l Download PDF

Info

Publication number
WO2006032380A1
WO2006032380A1 PCT/EP2005/009699 EP2005009699W WO2006032380A1 WO 2006032380 A1 WO2006032380 A1 WO 2006032380A1 EP 2005009699 W EP2005009699 W EP 2005009699W WO 2006032380 A1 WO2006032380 A1 WO 2006032380A1
Authority
WO
WIPO (PCT)
Prior art keywords
alkaloid
krs
reaction product
residue
group
Prior art date
Application number
PCT/EP2005/009699
Other languages
French (fr)
Inventor
Wassyl Nowicky
Original Assignee
Nowicky Wassili
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Nowicky Wassili filed Critical Nowicky Wassili
Priority to EP05782899A priority Critical patent/EP1833839A1/en
Priority to CA002593202A priority patent/CA2593202A1/en
Publication of WO2006032380A1 publication Critical patent/WO2006032380A1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
    • C07D491/12—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains three hetero rings
    • C07D491/14—Ortho-condensed systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/553—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having one nitrogen atom as the only ring hetero atom
    • C07F9/568—Four-membered rings

Definitions

  • the present invention is in the field of drug development and health care and relates to physiologically active alkaloid derivatives wherein the nitrogen in the alkaloid molecule is a quaternary nitrogen.
  • the invention further relates to a method of manufacture of such compounds, to compositions containing such compounds and to applications thereof for the treatment of various diseases and bodily conditions.
  • alkaloid chelidonine and compositions containing chelidonine are known in the art, as are therapeutic applications of chelidonine or some chelidonine derivatives in the treatment of various bodily conditions and diseases, including metabolic dysfunctions and tumors.
  • AT 354 644 and AT 377 988 describe processes for the preparation of phosphorus derivatives of alkaloids by reaction with carcinostatic phosphorus compounds, which are provided in a water-soluble form by conversion into their salts.
  • a disadvantage of the disclosed processes is that the conversion of the reaction products into a water-soluble salt is not complete and the predominant part of the reaction products remains water- insoluble.
  • US 5 981 512 discloses the use of the substances disclosed in AT 377 988 and AT 354 644 for the treatment of radiation damage.
  • trisd - aziridinyDphosphine sulphide (hereinafter also referred to as "thiotepa”) is soluble in organic solvents, such as benzene, ether or chloroform, it is proposed in the prior art methods to remove the unreacted tris(1 - aziridinyDphosphine sulphide from the synthesis mixture by washing the reaction products with ether.
  • WO03/041721 overcomes a drawback of previously known methods of manufacture, e.g. the requirement of purification of the final product using inflammable or even explosive organic solvents. It was found and described in WO 03/041721 that the purification could also and with even better results be accomplished using an aqueous solvent.
  • a still remaining drawback of the processes yielding biologically active alkaloid derivatives is that the resulting composition is a mixture of different components more or less contributing to the overall observed cytotoxic, tumoricide and/or cancerostatic effects.
  • the invention relates to a process for the preparation of a reaction product of alkaloids, particularly of chelidonine, oxychelidonine or methoxychelidonine, with suitable derivatizing, e.g. alkylating, agents, which process involves at least one washing step with an aqueous solvent, preferably water, after completion of the derivatizing reaction.
  • suitable derivatizing e.g. alkylating, agents
  • the process also comprises a step of converting the alkaloid derivatives into water-soluble salts, for making injectable pharmaceutical preparations of low toxicity and having a broad spectrum of therapeutic activity.
  • the present invention relates to the water-soluble reaction products, e.g. comprising alkaloid derivatives, wherein the initially tertiary nitrogen in the alkaloid molecule has been converted into a quaternary nitrogen, or else wherein an initially quaternary nitrogen has been modified with regard to one of its four ligands, and wherein the fourth ligand to the quaternary nitrogen is a Lewis base type residue, preferably selected from the group consisting of a hydroxy (-OH), sulfhydryl (-SH), sulfoxy, sulfate, phosphate, ester, thioester, ether, thioether, alkyl, aryl, aralkyl, alkoxy, aryloxy residue or a residue or decomposition product of the derivatizing agent not comprised in the aforementioned group of compounds with the proviso that said residue is not a hydrogen, methyl or ethyl residue.
  • the quaternary alkaloid preferably selected from the
  • the invention relates to pharmaceutical compositions containing at least one of the quaternary alkaloid derivatives, particularly quaternary chelidonine derivatives, obtainable in a process according to the present invention.
  • the invention further relates to the use of the reaction products comprising quaternary alkaloid derivatives as drugs for use in therapeutic applications, and to the use of said derivatives for the manufacture of pharmaceutical compositions for the therapeutic treatment of various diseases or bodily conditions.
  • the process according to the invention comprises reacting an alkaloid or a mixture of alkaloids in an organic solvent with a derivatizing, e.g. alkylating agent, preferably with a derivatizing agent having itself therapeutic activity, such as for example cytotoxic phosphoramides or phosphoric acid derivatives containing at least one aziridine group, and subjecting the reaction products to at least one washing step to remove at least part of the unreacted material.
  • the washing step is carried out with water or an equivalent aqueous solvent, e.g. a mild aqueous salt solution, which facilitates inter alia the subsequent conversion step of the poorly water-soluble or water-insoluble reaction products, i.e.
  • the derivatizing agent is a cytotoxic substance it be also water-soluble or at least that it decomposes upon contact with water into water-soluble components, in order to allow for substantial removal of unreacted alkylating agent or parts thereof from the reaction mixture by the washing step with water.
  • the present process can be used, for example, for alkylating reactions of alkaloids with the carcinostatic phosphorus containing compounds mentioned in Claim 1 of AT 377 988, the phosphorus compounds shown in Figure 3 of the present application being particularly suitable, and most particularly those having an aziridine group.
  • other derivatizing agents may be used as well such as but not limited to organic compounds, particularly cytotoxic or cytostatic, tumoricide organic compounds, having Lewis base type characteristics or comprising Lewis base type reactive groups selected from the group consisting of alky!, aryl, aralkyl, alkyloxy, aryloxy compounds or residues, respectively.
  • Preferred derivatizing agents comprise ester or ether compounds including substituted phosphate or sulfate esters, as well as hydroxy or polyhydroxy compounds such as diols or polyvalent alcohols, e.g. glycol, glycerol, sugar alcohols.
  • chelidonine as used herein shall refer likewise to either of the members selected from the group consisting of chelidonine, oxychelidonine, homochelidonine and methoxychelidonine, unless stated otherwise or unless otherwise derivable implicitly from the description.
  • a suitable organic solvent according to the present invention is any agent in which the alkaloids intended for the reaction are soluble.
  • the alkaloids can, for example, be dissolved in an organic solvent that facilitates or contributes to the alkylation reaction such as a solvent selected from the group consisting of monochloromethane, dichloromethane, trichloromethane, monochloroethane, dichloroethane and trichloroethane.
  • the alkylating reaction of the alkaloids takes place at elevated temperature, preferably at the boiling point of the solvent.
  • the resulting reaction product is converted into a water-soluble form after washing with water.
  • This can be carried out according to the process described in AT 377 988 and AT 354 644, by conversion into the water- soluble salts, in particular into the hydrochlorides, for example by passing in a strong acid in liquid or gaseous form such as HCI gas or adding an HCI solution to the organic solution of the washed reaction product,, during which or after which the hydrochlorides are precipitated.
  • the residue is preferably selected from the group consisting of a hydroxy (-OH), sulfhydryl (-SH), sulfoxy, sulfate, phosphate, ester, thioester, ether, thioether, alkyl, aryl, aralkyl, alkyloxy, aryloxy residue, and a part or decomposition product of the derivatizing agent not comprised in the aforementioned group of compounds, with the proviso that said residue is another than a hydrogen, methyl or ethyl residue.
  • the subsequent formula (I) illustrates a typical quaternary alkaloid reaction product of the present invention, exemplified with chelidonine:
  • R1 a hydroxy (-OH), sulfhydryl (-SH), sulfoxy, sulfate, phosphate, ester, thioester, ether, thioether, alkyl, aryl, aralkyl, alkyloxy, aryloxy residue or a residue or decomposition product of the derivatizing agent not comprised in the aforementioned group of compounds.
  • the water-soluble salt of the reaction product is suitable for application in injection solutions, but may likewise be used in any other galenic formulation for oral or parenteral administration, such as in the form of a gel, an ointment, a lotion, a powder, a granulate, tablet or capsule.
  • the reaction is carried out with trisd -aziridinyOphosphine sulphide (CAS No. 52-24-4), which in the pharmacopoeia is also known as thiotepa.
  • trisd -aziridinyOphosphine sulphide CAS No. 52-24-4
  • thiotepa aziridinyOphosphine sulphide
  • Various other synonyms for this compound are known in the art.
  • Suitable derivatizing agents may be selected from the group of compounds disclosed in Fig.3.
  • an extract of alkaloids optionally the total alkaloids of Chelidonium majus L.
  • an organic solvent is reacted with tris(1 -aziridinyl)-phosphine sulphide (thiotepa) and the resulting reaction product, optionally present as a mixture of compounds, is then washed at least once with water. Since thiotepa decomposes in water, the unreacted remainder of thiotepa present in excess after the reaction can be removed from the organic phase by this measure.
  • the organic solution containing the intermediate reaction product i.e.
  • the compound formed between alkylating agent and alkaloid is washed several times and each time is saturated with water.
  • the washing step may be repeated as required, i.e. until the excess of the highly toxic thiotepa has been substantially or completely removed from the reaction product.
  • some water-soluble undesired or even toxic alkaloids which contribute to adverse reactions in medical applications or which might even cause cirrhosis of the liver are at least partially, preferably substantially or completely removed with the aqueous phase from the reaction mixture.
  • the final reaction product is a mixture of compounds typically comprising a main portion of unreacted alkaloids together with a substantial amount of derivatized alkaloids comprising reaction products of thiotepa with the alkaloids, and low amounts of degradation products of thiotepa.
  • the reaction product usually comprises a mixture of about 60 to 70% of unreacted Chelidonium alkaloids with about 30 to 40% of reacted, i.e. derivatized, alkaloids.
  • Tertiary alkaloids represent the main part of the starting components of an alkaloid extract obtained from Chelidonium majus L., whereas quaternary alkaloids may be present at low amounts, such as for instance berberine.
  • the following alkaloids may be contained as starting components in the synthesis mixture: chelidonine, protopine, stylopine, allocryptopine, ⁇ - homochelidonine, chelamidine, chelamine, L-sparteine, chelidimerine, dihydrosanguinarine, sanguinarine, dihydrosanguinarine, oxysanguarine, oxychelidonine, methoxychelidonine, chelerythrine, dihydrochelerythrine, chelilutine, chelirubine, oxychelidonine, methoxychelidonine, corysamine and berberine.
  • the resulting intermediate reaction product may comprise compounds, wherein one molecule of the derivatizing agent is linked to one, two or three chelidonine molecules.
  • it may comprise reacted alkaloid derivatives, wherein an alkaloid molecule, e.g.
  • a chelidonine molecule is linked at its quaternary nitrogen to an alkyl, aryl, aralkyl, alkyloxy, aryloxy residue, to a sulfoxy, sulfate, phosphate, ester, thioester, ether or thioether residue, or to a residue or decomposition product of the derivatizing agent not comprised in the aforementioned group of compounds. Still further reaction compounds may be present in the reaction mixture after termination of the derivatizing reaction.
  • the subsequent washing step(s) with water and/or the acidic treatment for making water-soluble salts of the reaction products may convert at least part of the reaction products to compounds wherein to the quaternary alkaloid nitrogen a hydroxy (-OH) or sulfhydryl (-SH) group is linked.
  • reaction products obtained from the reaction of the total alkaloids of Chelidonium majus L. with the alkylating agents according to the present invention show a better spectrum of therapeutic activities than the reaction products obtained from an analogous process wherein the washing step has been carried out with an organic solvent, e.g. diethylether.
  • organic solvent e.g. diethylether.
  • At least some compounds present in the reaction products of the present invention, particularly the quaternary chelidonine derivatives selectively accumulate in cancerous tissues and destroy cancer cells by apoptosis but - in contrast to most known cytostatic agents - without also attacking healthy cells.
  • compositions comprising a said reaction product, thus allowing for therapeutic and even prophylactic use of such compositions in individuals at increased risk of developing cancer due to, e.g. hereditary disposition. They are simple to handle and have not been shown to cause significant adverse reactions when administered in therapeutic doses.
  • reaction products obtained from the reaction of one or more of the alkaloids of Chelidonium majus L. with a derivatizing agent such as thiotepa or another one of those disclosed in Fig.3 exhibit biological activity in that they interfere with the regulation of the metabolism in various beneficial ways, which activity has proven to render them suitable for application in the prevention or therapy of metabolic diseases, such as osteoporosis, but also in the prophylactic or therapeutic treatment of rheumatic diseases, various allergies, bacterial, fungal and viral infections including herpes infections and sleeping disease, epilepsy, multiple sclerosis, scars, various skin diseases, skin tumors, postoperative wounds, and radiation damage.
  • Another beneficial activity of the present products is their strengthening, stimulating and/or restoring effect on a depressed or otherwise disturbed immune system of the human body.
  • the resulting reaction product obtained according to the method of the present invention exhibits therapeutic qualities and activities that are at least comparable to those of the reaction product resulting from the alkylation reaction of the total Chelidonium alkaloids according to Example 1 .
  • physiologically active reaction products of the present invention may be formulated into various galenic forms using customary pharmaceutical excipients, in particular for manufacturing pharmaceutical compositions or medicaments in the form of solutions, for example injection or infusion solutions, or for ointments, gels, creams, lotions, compress or suspensory bases, powders, granulates, tablets or capsules, and the like.
  • Fig.1 shows an HPLC diagram with a characteristic total alkaloid composition of the roots of Chelidonium majus L.
  • Fig.2 shows the HPLC fingerprint of a preparation according to Example 1 .
  • Fig.3 shows selected phosphorus derivatives suitable as derivatizing reagents.
  • Fig.4 shows a nuclear magnetic resonance spectrum of the reaction product
  • Fig.5 shows a UV spectrum of the reaction product U-KRS.
  • Fig.6 shows a UV spectrum of chelidonine hydrochloride.
  • Fig.7 shows a first section of a mass spectrum of the reaction product U-
  • Fig.8 shows a second section of a mass spectrum of the reaction product
  • Fig.9 shows a mass spectrum of chelidonine hydrochloride.
  • A) Extraction of the alkaloids a. 25 g of an alkaloid salt mixture are suspended in water and transferred to a separating funnel. After the addition of 100 ml of dichloromethane, the separating funnel is shaken. The organic phase is then separated off and is filtered into a glass bottle. b. 1 N NaOH (pH 8-9) is added to the aqueous phase until turbidity occurs. After the addition of 100 ml of dichloromethane, the mixture is shaken. The organic phase is then separated off and is combined with the dichloromethane phase from step a. This process is repeated, for example 3 times. The organic phases are filtered and combined. c. The aqueous phase is adjusted to a pH of 10 by adding NaOH.
  • the washed solution is transferred to a glass beaker, stirred and saturated with HCI gas, a hydrochloride complex being precipitated.
  • the precipitated product is filtered off and is washed with diethyl ether, dried and then dissolved in water.
  • an LD 50 value of 485 mg/kg was determined from the reaction product according to Example 1 .
  • Studies in mice and rats showed that the product according to the invention modulates the hormone regulation of the thymus and induces the synthesis of substances having thymosin-like activity in animals whose thymus has been removed. This effect is dose- dependent.
  • the preparation increases the number of T-lymphocytes in the peripheral blood by up to 50% (4.04 ⁇ 0.43 x 10 9 /l before the treatment, 6.24 ⁇ 0.73 x 10 9 /l after the treatment), modulates the humoral immune response to penetrating antigen and the natural killer cell activity of the spleen cells (198.20 ⁇ 17.69% compared with 71 .50 ⁇ 9.10% in the control group) and enhances the interferon liberation potential of the white blood corpuscles in animal experiments. The results of the animal experiments are confirmed by clinical observations. Thus, the improvement in the immune parameters was also observed in cancer patients. Doses of about 5 mg of the preparation from Example 1 per 70 kg body weight can be used for prophylactic and immunological applications. For cancer treatment, 5 mg of the preparation per 20 kg body weight are preferably administered.
  • Example 2 HPLC fingerprints The determination was carried out by ion pair reverse-phase chromatography in the gradient mode and with spectral measurement using a DAD detector at 285 nm. At the same time, chromatograms were prepared using reference alkaloids. In addition, an HPLC-MSD analysis was carried out, which showed that there were no peaks apart from those of the alkaloids.
  • the HPLC diagrams of Figures 1 and 2 were obtained on the basis of the following experimental data: Chromatography parameters:
  • Extract before reaction ( Figure 1 ): 25 mg of alkaloids are dissolved in 40 ml of methanol by ultrasonics, made up to 50 ml and filtered through a membrane filter.
  • Reaction product ( Figure 2): The reaction product is converted into the hydrochloride salt, dissolved in water in a concentration of 1 mg/ml and adjusted to a pH of between 2.5 and 6.5.
  • the precipitate comprised slightly yellowish hygroscopic crystals having a rather narrow melting point of 205 - 207 °C (indicating a fairly well crystallized product) and exhibiting light-blue fluorescence upon irradiation with UV-light at 366 nm. Traces of yellow, orange and red fluorescent bands were also visible.
  • the UV spectrum ( Figure 5) exhibits absorption maxima at 148, 155, 160, 205, 230 and 282 nm, very much like the UV spectrum of chelidonine ( Figure 6), which differs therefrom solely in that the peak at 230 nm of U- KRS occurs at 240 nm with chelidonine. This indicates that the nitrogen in U-KRS is quaternary, while in chelidonine it is tertiary.
  • Example 4 Selective inhibition of in vitro cell growth by the anti ⁇ tumor drug U-KRS
  • Human endothelial cells were isolated from umbilical veins by collagenase treatment.
  • the culture medium for endothelial cells was M199 supplemented with 15% heat inactivated fetal calf serum, 200 ⁇ g/ml endothelial cell growth factor and 90 ⁇ g/ml heparin.
  • Fluorescence microscopy Cells were grown in 35mm dishes and incubated with 100 ⁇ g/ml U-KRS for 30-60 min. The culture medium was aspirated, the cells were washed twice with PBS. Coverslips were mounted on the cells and fluorescence was excited using a confocal laser scanning microscope equipped with an argon laser source. The emitted light was detected in a photomultiplier channel. The signals were imaged on a video monitor using the MRC 600 image processing software.
  • U-KRS In a range from 20-40 ⁇ g/ml U-KRS about 55% inhibition of cell growth with endothelial cells was measured. This concentration killed the human osteosarcoma cell line. Hybrids of the two cell types showed nearly the same sensitivity as normal cells. 2. Because of its autofluorescence U-KRS can be detected intracellularly. A laser scanning microscope showed a high uptake of U-KRS in malignant cells.
  • mice Two to five control animals were each injected with 50 ⁇ l of an Ehrlich mouse ascites tumor suspension i.p. which was 8 d old, freshly taken from a fully grown donor animal. The control group was not further treated. Test group was injected with 10mg U-KRS/ kg animal weight in the abdominal area immediately after the tumor implantation.
  • mice implanted with the ascites tumor either after intraperitoneal or after subcutaneous administration of U-KRS showed a longer survival time than the implant animals which were not otherwise treated.
  • Example 6 Modification of antinociceptive action of morphine by U- KRS in rodents.
  • results The results indicated that simultaneous administration of U-KRS and morphine modified the action of the narcotic analgesic drug. They produced antinociceptive action in the tail-flick test in rats, evident as an increase in the latency time.
  • Example 7 Induction of bimodal programmed cell death in malignant cells by the derivative U-KRS.
  • the K562 erythroleukaemia cell line was used, and U-KRS produced in pure crystallized form and dissolved in water at a concentration of 1 .2 mg/ml.
  • the DNA content of K562 cells exposed to various concentrations of U- KRS were analyzed using propidium iodide and flow cytometry.
  • Example 8 Influence of U-KRS on DNA, RNA and protein synthesis in malignant cells
  • 3 H labeled thymidine, 0.5/vCi in 20 ⁇ l medium; uridine, 0.5 ⁇ Ci in 20 ⁇ I medium and leucine, 1 .O ⁇ Ci in 20 ⁇ l medium were placed for 2-4 h into four wells with different U-KRS concentration.
  • the cell lines, guinea pig hepatocytes, C1 L hepatocytes, human tonsil cells, murine lymphomas, murine myeloma, Yoshida cells, two HeLa strains, EsB-, EB, lymphomas, ZAC/1 , P815 were grown 24 h at 37°C in 96 microtiter wells. WiDr cells were incubated in a somewhat different schema for 6 and 24 h at U-KRS concentrations of 1 , 4, 8 and 14 ⁇ g/ml U-KRS.
  • Tumor cells were taken from human tumor xenografts and serially transplanted into nude mice. These cells were used in a colony-forming assay in vitro. Tumor cells were incubated continuously for at least one week with several concentrations of the drug U-KRS. This was done with six different types, and the colony formation was scored for each tumor. The drug effects were reported as percent T/C (Test/Control)
  • Example 1 1 Induced G2/M Arrest and Apoptosis in Human Epidermoid Carcinoma Cell Lines by U-KRS
  • U-KRS treatment affects cell cycle distribution and induces apoptosis in A431 and ME180 cells.
  • Example 12 Antimetastatic effect of U-KRS and its influence on the oxygen and energy metabolism of mice with melanoma B-16
  • Metastasizing melanoma B-16 was transplanted to the right shin muscle of each mouse. On the 10 th day after the tumor transplantation, the animals were divided into two groups. The first group was given U-KRS to sinus venosus of the eye in a dose of 1 mg/kg in the volume of 0.05 ml: 5 injections once in two days. The second group was given sterile physiological solution to sinus venosus in the same regime.
  • HAV Hepatitis A, B and C, E.coli, Influenza
  • osteoporosis polyarthritis
  • psoriasis and other diseases or bodily conditions.
  • Example 13 Enhancement of macrophage tumoricidal activity by U- KRS
  • BALB/c mice were maintained by brother/sister matings in the laboratory.
  • the tumor D1 DMBA/3 was routinely transplanted in BALB/c by s. c. injection.
  • the tumor became apparent five days after implantation.
  • mice receiving U-KRS did not show any deleterious drug-related side effects.
  • Example 14 ⁇ n vitro effects of U-KRS on the phenotype of normal human lymphocytes
  • the cells were isolated on Ficoll-Paque density gradient centrifugation. Viability of cells was determined by 0.1 % trypan blue staining, and found to be 95%.
  • Lymphocyte subpopulation was quantitated by immunofluorescence using monoclonal antibodies against total T-cells, T-helper cells and T- suppressor cells. Subsequently, cells were treated with FITC/conjugated rabbit F/ab/2 fragments anti-mouse IgG, washed in PBS and mounted on slides using polyvinyl-alcohol and glycerol. In control preparations, PBS or normal mouse serum was used instead of monoclonal antibodies.
  • Example 15 Mitogenic properties of U-KRS on human peripheral blood monocytes
  • the blood was diluted with an equal volume of PBS containing 1 mM EDTA, pH 7.5, and was layered over Histopaque 1077.
  • the tubes were centrifuged at 2000rmp for 30 min.
  • the interface layers containing lymphocytes were collected and washed three times with RPMI tissue culture medium
  • U-KRS had a potent synergic effect on PHA mitogenesis resulting in significantly higher cell stimulation indices than those of PHA alone.
  • Example 16 Modulation of immune effector cell cytolytic activity and tumor growth inhibition in vivo by U-KRS
  • Tumor cells mastocytoma P815 and the AKR leukemia AKIL cell lines were maintained in DMEM medium supplemented with 9.0% fetal calf serum containing penicillin and streptomycin.
  • U-KRS is an effective biological response modifier augmenting, by up to 48-fold, the lytic activity of splenic lymphocytes obtained from alloimmunized mice.
  • the lytic activities of IL-2 treated spleen cells and peritoneal exudate lymphocytes were also significantly increased by the addition of U-KRS to the cell mediated lysis assay medium.
  • Example 17 Influence of U-KRS on immunological blood parameters in vitro and in vivo
  • U-KRS stimulates different subsets of the hematopoietic and immunological systems.
  • reticulocytosis is induced as a possible sign of stimulation of certain stem cells or of general activation of the erythropoietic system.
  • Example 18 Inhibitory effect of U-KRS on ovalbumine antigenicity and antiovalbumine IgE antibody response in mice
  • U-KRS ovalbumine-induced sensitization mice.
  • U-KRS was introduced into the mice in the mixture with antigen (ovalbumine) and adjuvant (alum) and inhibited the sensitization of mice, reflected in lower anti-OA IgE antibody response and decreased antigen-induced histamine release from mast cells isolated from peritoneal cavities of sensitized mice.
  • the effect of U-KRS on the antigenicity of ovalbumine (OA) in anaphylaxis was tested in heterologous passive cutaneus anaphylactic (PCA) reaction on rats.
  • PCA passive cutaneus anaphylactic
  • U-KRS was administered intraperitoneal ⁇ in a dose of 30mg/kg every other day for six months to female rats with ovariectomy-induced early osteoporosis. Administration of U-KRS was started on the second day after the surgical operation. At the end of the long-term treatment with U-KRS each rat was tested for the strength of both humeri and some parameters of rat femur were measured.
  • Example 20 Influence of U-KRS preparation on influenza viruses and the bacteria E.coli and S. aureus
  • Influenza viruses of the APR8/HON1 /34 strain were cultured on 10-day- old hen embryos;
  • E.coli bacteria derived from current clinical material and the strain 209P of S. aureus were employed. U-KRS preparation of the series 290614.
  • Example 21 Biological activity of U-KRS with respect to influenza virus
  • the virus was injected at 1 , 10 and 100 EID 50 per embryo.
  • U-KRS was dissolved de novo in Hanks solution.
  • mice of 1 6/2Og body weight Materials and methods CBA/J male mice of 1 6/2Og body weight. Short-term whole-body gamma-irradiation of mice at doses ranging from 6.0Gy to 7.5Gy was performed. Long-term irradiation with the cumulative dose of 8.75 Gy was performed using the CEGO device.
  • U-KRS was administered intraperitoneal ⁇ at doses of 0.1 , 1.4 and 12mg/kg body weight.
  • U-KRS The effect of U-KRS on cell survival was tested at concentration ranging from 0.2 ⁇ g/ml. The exposure times were 1 , 3 and 24h, after which the cells were washed with phosphate/buffered saline and fresh medium was added.
  • a group of 5 adults suffering from CFS at differing intensities was treated with 5mg doses (5ml ampoules) of UKR-S 1 to 3 times a week by oral delivery or intravenous injection.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Hematology (AREA)
  • Diabetes (AREA)
  • Obesity (AREA)
  • Biomedical Technology (AREA)
  • Neurology (AREA)
  • Neurosurgery (AREA)
  • Communicable Diseases (AREA)
  • Oncology (AREA)
  • Dermatology (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

The invention relates to alkaloid reaction products obtainable in a process wherein alkaloids are reacted with a derivatizing agent, preferably thiotepa or another one of the compounds listed in Fig.3, whereafter unreacted derivatizing agent and other water-soluble compounds are removed from the reaction mixture by washing with water or a suitable aqueous solvent, whereafter the reaction mixture is subjected to a treatment with strong acid, preferably hydrogen chloride (HCI), to precipitate a water soluble salt of the reaction products. The precipitated reaction products comprise at least one quaternary alkaloid derivative and are suitable as drugs for prophylactic or therapeutic application, particularly in the treatment of immunological or metabolic dysfunctions, and cancer.

Description

QUATERNARY ALKALOID DERIVATIVES OF CHELIDONIUM MAJUS L
FIELD OF THE INVENTION
The present invention is in the field of drug development and health care and relates to physiologically active alkaloid derivatives wherein the nitrogen in the alkaloid molecule is a quaternary nitrogen. The invention further relates to a method of manufacture of such compounds, to compositions containing such compounds and to applications thereof for the treatment of various diseases and bodily conditions.
STATE OF THE ART
The alkaloid chelidonine and compositions containing chelidonine are known in the art, as are therapeutic applications of chelidonine or some chelidonine derivatives in the treatment of various bodily conditions and diseases, including metabolic dysfunctions and tumors.
DE 2 028 330 and US 3 865 830 disclose the preparation of thiophosphoramide-isoquinoline adducts by reacting selected alkaloids of Chelidonium majus L. with tris(1 -aziridinyl)phosphine sulphide in an organic solvent.
AT 354 644 and AT 377 988 describe processes for the preparation of phosphorus derivatives of alkaloids by reaction with carcinostatic phosphorus compounds, which are provided in a water-soluble form by conversion into their salts. A disadvantage of the disclosed processes is that the conversion of the reaction products into a water-soluble salt is not complete and the predominant part of the reaction products remains water- insoluble.
US 5 981 512 discloses the use of the substances disclosed in AT 377 988 and AT 354 644 for the treatment of radiation damage.
The compounds described in said patents have different cytostatic and carcinostatic activity. Mixtures of alkaloids, in particular of the total alkaloids of Chelidonium majus L., have proved therapeutically particularly promising, the pharmacological activity of which has been demonstrated in several studies on cancer treatment. Unreacted reagent is removed from the synthesis mixture following completion of the reaction. Since trisd - aziridinyDphosphine sulphide (hereinafter also referred to as "thiotepa") is soluble in organic solvents, such as benzene, ether or chloroform, it is proposed in the prior art methods to remove the unreacted tris(1 - aziridinyDphosphine sulphide from the synthesis mixture by washing the reaction products with ether.
The method disclosed in WO03/041721 overcomes a drawback of previously known methods of manufacture, e.g. the requirement of purification of the final product using inflammable or even explosive organic solvents. It was found and described in WO 03/041721 that the purification could also and with even better results be accomplished using an aqueous solvent.
A still remaining drawback of the processes yielding biologically active alkaloid derivatives is that the resulting composition is a mixture of different components more or less contributing to the overall observed cytotoxic, tumoricide and/or cancerostatic effects.
There was a need therefore to unveil and identify the most important bioactive alkaloid derivative components present in the reaction mixture after alkylation of an alkaloid extract from the herb Chelidonium majus L., and to find a way to make pharmaceutical compositions containing one or more clearly defined bioactive ingredients from the group of quaternary alkaloid derivatives, particularly of quaternary alkaloid derivates obtained by alkylation of one or more alkalkoids as naturally occurring in Chelidonium majus L.
BRIEF DESCRIPTION OF THE INVENTION
In one aspect the invention relates to a process for the preparation of a reaction product of alkaloids, particularly of chelidonine, oxychelidonine or methoxychelidonine, with suitable derivatizing, e.g. alkylating, agents, which process involves at least one washing step with an aqueous solvent, preferably water, after completion of the derivatizing reaction.
The process also comprises a step of converting the alkaloid derivatives into water-soluble salts, for making injectable pharmaceutical preparations of low toxicity and having a broad spectrum of therapeutic activity.
In another aspect the present invention relates to the water-soluble reaction products, e.g. comprising alkaloid derivatives, wherein the initially tertiary nitrogen in the alkaloid molecule has been converted into a quaternary nitrogen, or else wherein an initially quaternary nitrogen has been modified with regard to one of its four ligands, and wherein the fourth ligand to the quaternary nitrogen is a Lewis base type residue, preferably selected from the group consisting of a hydroxy (-OH), sulfhydryl (-SH), sulfoxy, sulfate, phosphate, ester, thioester, ether, thioether, alkyl, aryl, aralkyl, alkoxy, aryloxy residue or a residue or decomposition product of the derivatizing agent not comprised in the aforementioned group of compounds with the proviso that said residue is not a hydrogen, methyl or ethyl residue. In a preferred embodiment the quaternary alkaloid derivatives are of a nature such as to selectively accumulate in target tissues, particularly in cancerous tissues.
In another aspect the invention relates to pharmaceutical compositions containing at least one of the quaternary alkaloid derivatives, particularly quaternary chelidonine derivatives, obtainable in a process according to the present invention.
The invention further relates to the use of the reaction products comprising quaternary alkaloid derivatives as drugs for use in therapeutic applications, and to the use of said derivatives for the manufacture of pharmaceutical compositions for the therapeutic treatment of various diseases or bodily conditions.
Further embodiments of the present invention are laid down in the claims.
DETAILED DESCRIPTION OF THE INVENTION
The process according to the invention comprises reacting an alkaloid or a mixture of alkaloids in an organic solvent with a derivatizing, e.g. alkylating agent, preferably with a derivatizing agent having itself therapeutic activity, such as for example cytotoxic phosphoramides or phosphoric acid derivatives containing at least one aziridine group, and subjecting the reaction products to at least one washing step to remove at least part of the unreacted material. In a preferred embodiment, the washing step is carried out with water or an equivalent aqueous solvent, e.g. a mild aqueous salt solution, which facilitates inter alia the subsequent conversion step of the poorly water-soluble or water-insoluble reaction products, i.e. quaternary alkaloid derivatives, into water-soluble compounds, e.g. salts. It is preferred that in case the derivatizing agent is a cytotoxic substance it be also water-soluble or at least that it decomposes upon contact with water into water-soluble components, in order to allow for substantial removal of unreacted alkylating agent or parts thereof from the reaction mixture by the washing step with water.
The present process can be used, for example, for alkylating reactions of alkaloids with the carcinostatic phosphorus containing compounds mentioned in Claim 1 of AT 377 988, the phosphorus compounds shown in Figure 3 of the present application being particularly suitable, and most particularly those having an aziridine group. However, other derivatizing agents may be used as well such as but not limited to organic compounds, particularly cytotoxic or cytostatic, tumoricide organic compounds, having Lewis base type characteristics or comprising Lewis base type reactive groups selected from the group consisting of alky!, aryl, aralkyl, alkyloxy, aryloxy compounds or residues, respectively. Preferred derivatizing agents comprise ester or ether compounds including substituted phosphate or sulfate esters, as well as hydroxy or polyhydroxy compounds such as diols or polyvalent alcohols, e.g. glycol, glycerol, sugar alcohols.
The term chelidonine as used herein shall refer likewise to either of the members selected from the group consisting of chelidonine, oxychelidonine, homochelidonine and methoxychelidonine, unless stated otherwise or unless otherwise derivable implicitly from the description. A suitable organic solvent according to the present invention is any agent in which the alkaloids intended for the reaction are soluble. The alkaloids can, for example, be dissolved in an organic solvent that facilitates or contributes to the alkylation reaction such as a solvent selected from the group consisting of monochloromethane, dichloromethane, trichloromethane, monochloroethane, dichloroethane and trichloroethane.
The alkylating reaction of the alkaloids takes place at elevated temperature, preferably at the boiling point of the solvent.
The resulting reaction product is converted into a water-soluble form after washing with water. This can be carried out according to the process described in AT 377 988 and AT 354 644, by conversion into the water- soluble salts, in particular into the hydrochlorides, for example by passing in a strong acid in liquid or gaseous form such as HCI gas or adding an HCI solution to the organic solution of the washed reaction product,, during which or after which the hydrochlorides are precipitated. It appears that by this acidic treatment most of the alkylating agent is split off from an intermediate reaction compound formed between the alkaloids and the alkylating agent, leaving behind modified alkaloid derivatives, wherein the initially tertiary nitrogen atoms have been converted into quaternary nitrogens, wherein to the quaternary nitrogen a hydrogen residue or a residue originating either from the alkylating agent, from the washing solution or from the strong acid used for conversion into water-soluble salts is bound as a fourth ligand. The residue is preferably selected from the group consisting of a hydroxy (-OH), sulfhydryl (-SH), sulfoxy, sulfate, phosphate, ester, thioester, ether, thioether, alkyl, aryl, aralkyl, alkyloxy, aryloxy residue, and a part or decomposition product of the derivatizing agent not comprised in the aforementioned group of compounds, with the proviso that said residue is another than a hydrogen, methyl or ethyl residue. For a better understanding, the subsequent formula (I) illustrates a typical quaternary alkaloid reaction product of the present invention, exemplified with chelidonine:
Figure imgf000006_0001
R1 = a hydroxy (-OH), sulfhydryl (-SH), sulfoxy, sulfate, phosphate, ester, thioester, ether, thioether, alkyl, aryl, aralkyl, alkyloxy, aryloxy residue or a residue or decomposition product of the derivatizing agent not comprised in the aforementioned group of compounds.
From an elementary analysis of one of the reaction products precipitated according to the present invention (see Example 3) it appears - without being bound by theory - that at least a part of the alkylating agent or decomposition compounds of the alkylating agent, obtained by the acidic treatment of the reaction mixture after the alkylating and water-washing step, may be occluded to some extent in the crystals of the water-soluble precipitate or are somehow strongly attached to the crystals, thus withstanding purification of the precipitate by washing with organic solvents such as ether and dichloromethane. Nevertheless it could be proved that the reaction product is still fully functional even in the presence of such accompanying substances.
The water-soluble salt of the reaction product is suitable for application in injection solutions, but may likewise be used in any other galenic formulation for oral or parenteral administration, such as in the form of a gel, an ointment, a lotion, a powder, a granulate, tablet or capsule.
In one embodiment of the invention, the reaction is carried out with trisd -aziridinyOphosphine sulphide (CAS No. 52-24-4), which in the pharmacopoeia is also known as thiotepa. Various other synonyms for this compound are known in the art.
Other suitable derivatizing agents may be selected from the group of compounds disclosed in Fig.3.
In a further embodiment of the invention, an extract of alkaloids, optionally the total alkaloids of Chelidonium majus L., in an organic solvent is reacted with tris(1 -aziridinyl)-phosphine sulphide (thiotepa) and the resulting reaction product, optionally present as a mixture of compounds, is then washed at least once with water. Since thiotepa decomposes in water, the unreacted remainder of thiotepa present in excess after the reaction can be removed from the organic phase by this measure. Preferably, the organic solution containing the intermediate reaction product, i.e. the compound formed between alkylating agent and alkaloid, is washed several times and each time is saturated with water. The washing step may be repeated as required, i.e. until the excess of the highly toxic thiotepa has been substantially or completely removed from the reaction product. In addition, some water-soluble undesired or even toxic alkaloids which contribute to adverse reactions in medical applications or which might even cause cirrhosis of the liver are at least partially, preferably substantially or completely removed with the aqueous phase from the reaction mixture. By means of the Ames test, it was shown that the reaction products prepared according to the present invention, are not mutagenic.
When starting with a total alkaloid extract from Chelidonium majus L. the final reaction product is a mixture of compounds typically comprising a main portion of unreacted alkaloids together with a substantial amount of derivatized alkaloids comprising reaction products of thiotepa with the alkaloids, and low amounts of degradation products of thiotepa. As a result of the synthesis process, the solubilities of the alkaloids change. The reaction product usually comprises a mixture of about 60 to 70% of unreacted Chelidonium alkaloids with about 30 to 40% of reacted, i.e. derivatized, alkaloids.
Tertiary alkaloids represent the main part of the starting components of an alkaloid extract obtained from Chelidonium majus L., whereas quaternary alkaloids may be present at low amounts, such as for instance berberine. The following alkaloids may be contained as starting components in the synthesis mixture: chelidonine, protopine, stylopine, allocryptopine, α- homochelidonine, chelamidine, chelamine, L-sparteine, chelidimerine, dihydrosanguinarine, sanguinarine, dihydrosanguinarine, oxysanguarine, oxychelidonine, methoxychelidonine, chelerythrine, dihydrochelerythrine, chelilutine, chelirubine, oxychelidonine, methoxychelidonine, corysamine and berberine.
After termination of the derivatizing reaction water-soluble quaternary alkaloids such as unreacted portions of berberine are substantially removed from the reaction mixture by the washing step with water, while water- insoluble unreacted alkaloids and derivatized alkaloid reaction products remain in the organic solvent. Depending on the nature of the organic solvent and/or of the derivatizing agent used for the derivatizing reaction, the resulting intermediate reaction product may comprise compounds, wherein one molecule of the derivatizing agent is linked to one, two or three chelidonine molecules. In addition, it may comprise reacted alkaloid derivatives, wherein an alkaloid molecule, e.g. a chelidonine molecule, is linked at its quaternary nitrogen to an alkyl, aryl, aralkyl, alkyloxy, aryloxy residue, to a sulfoxy, sulfate, phosphate, ester, thioester, ether or thioether residue, or to a residue or decomposition product of the derivatizing agent not comprised in the aforementioned group of compounds. Still further reaction compounds may be present in the reaction mixture after termination of the derivatizing reaction. Moreover, the subsequent washing step(s) with water and/or the acidic treatment for making water-soluble salts of the reaction products may convert at least part of the reaction products to compounds wherein to the quaternary alkaloid nitrogen a hydroxy (-OH) or sulfhydryl (-SH) group is linked.
The reaction products obtained from the reaction of the total alkaloids of Chelidonium majus L. with the alkylating agents according to the present invention show a better spectrum of therapeutic activities than the reaction products obtained from an analogous process wherein the washing step has been carried out with an organic solvent, e.g. diethylether. At least some compounds present in the reaction products of the present invention, particularly the quaternary chelidonine derivatives, selectively accumulate in cancerous tissues and destroy cancer cells by apoptosis but - in contrast to most known cytostatic agents - without also attacking healthy cells. This results in the good tolerance of a therapy with pharmaceutical compositions comprising a said reaction product, thus allowing for therapeutic and even prophylactic use of such compositions in individuals at increased risk of developing cancer due to, e.g. hereditary disposition. They are simple to handle and have not been shown to cause significant adverse reactions when administered in therapeutic doses.
The reaction products obtained from the reaction of one or more of the alkaloids of Chelidonium majus L. with a derivatizing agent such as thiotepa or another one of those disclosed in Fig.3 exhibit biological activity in that they interfere with the regulation of the metabolism in various beneficial ways, which activity has proven to render them suitable for application in the prevention or therapy of metabolic diseases, such as osteoporosis, but also in the prophylactic or therapeutic treatment of rheumatic diseases, various allergies, bacterial, fungal and viral infections including herpes infections and sleeping disease, epilepsy, multiple sclerosis, scars, various skin diseases, skin tumors, postoperative wounds, and radiation damage. It was also found that they are at least to some extent effective in the treatment of pain, particularly in combination with another pain relieving medicament, and in the prophylaxis or therapy of chronic fatigue syndrome and related pathological conditions of the human body. Another beneficial activity of the present products is their strengthening, stimulating and/or restoring effect on a depressed or otherwise disturbed immune system of the human body.
Surprisingly, it was found that when starting with commercially available chelidonine, homochelidonine, oxychelidonine or methoxycheli- donine as the sole alkaloid source, the resulting reaction product obtained according to the method of the present invention (see for instance Example 3) exhibits therapeutic qualities and activities that are at least comparable to those of the reaction product resulting from the alkylation reaction of the total Chelidonium alkaloids according to Example 1 . The physiologically active reaction products of the present invention may be formulated into various galenic forms using customary pharmaceutical excipients, in particular for manufacturing pharmaceutical compositions or medicaments in the form of solutions, for example injection or infusion solutions, or for ointments, gels, creams, lotions, compress or suspensory bases, powders, granulates, tablets or capsules, and the like.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig.1 shows an HPLC diagram with a characteristic total alkaloid composition of the roots of Chelidonium majus L.
Fig.2 shows the HPLC fingerprint of a preparation according to Example 1 . Fig.3 shows selected phosphorus derivatives suitable as derivatizing reagents. Fig.4 shows a nuclear magnetic resonance spectrum of the reaction product
U-KRS.
Fig.5 shows a UV spectrum of the reaction product U-KRS. Fig.6 shows a UV spectrum of chelidonine hydrochloride.
Fig.7 shows a first section of a mass spectrum of the reaction product U-
KRS. Fig.8 shows a second section of a mass spectrum of the reaction product
U-KRS at a higher resolution. Fig.9 shows a mass spectrum of chelidonine hydrochloride.
The following examples are set forth to further illustrate the invention.
Example 1
A) Extraction of the alkaloids: a. 25 g of an alkaloid salt mixture are suspended in water and transferred to a separating funnel. After the addition of 100 ml of dichloromethane, the separating funnel is shaken. The organic phase is then separated off and is filtered into a glass bottle. b. 1 N NaOH (pH 8-9) is added to the aqueous phase until turbidity occurs. After the addition of 100 ml of dichloromethane, the mixture is shaken. The organic phase is then separated off and is combined with the dichloromethane phase from step a. This process is repeated, for example 3 times. The organic phases are filtered and combined. c. The aqueous phase is adjusted to a pH of 10 by adding NaOH. After the addition of dichloromethane, the mixture is shaken. The organic phase is then separated off, filtered and mixed with the other organic phases. The aqueous phase is now adjusted to a p H of 13 with NaOH and the extraction is repeated with dichloromethane. d. The combined organic phases are evaporated to give an oily, brown material. B) Reaction with tris(1-aziridinyl)phosphine sulphide:
The alkaloid residue is dissolved in dichloromethane, and tris(1 - aziridinyDphosphine sulphide is added. The mixture is refluxed at 8O0 C for 2 hours. After cooling to room temperature, the reaction mixture is clarified. Filtration is then carried out and the filtrate is washed several times, for example 3 times or more, with 250 ml of water in a separating funnel. C) Reaction with HCI
The washed solution is transferred to a glass beaker, stirred and saturated with HCI gas, a hydrochloride complex being precipitated. The precipitated product is filtered off and is washed with diethyl ether, dried and then dissolved in water.
In rats, an LD50 value of 485 mg/kg was determined from the reaction product according to Example 1 . Studies in mice and rats showed that the product according to the invention modulates the hormone regulation of the thymus and induces the synthesis of substances having thymosin-like activity in animals whose thymus has been removed. This effect is dose- dependent. The preparation increases the number of T-lymphocytes in the peripheral blood by up to 50% (4.04 ± 0.43 x 109/l before the treatment, 6.24 ± 0.73 x 109/l after the treatment), modulates the humoral immune response to penetrating antigen and the natural killer cell activity of the spleen cells (198.20 ± 17.69% compared with 71 .50 ± 9.10% in the control group) and enhances the interferon liberation potential of the white blood corpuscles in animal experiments. The results of the animal experiments are confirmed by clinical observations. Thus, the improvement in the immune parameters was also observed in cancer patients. Doses of about 5 mg of the preparation from Example 1 per 70 kg body weight can be used for prophylactic and immunological applications. For cancer treatment, 5 mg of the preparation per 20 kg body weight are preferably administered.
Example 2: HPLC fingerprints The determination was carried out by ion pair reverse-phase chromatography in the gradient mode and with spectral measurement using a DAD detector at 285 nm. At the same time, chromatograms were prepared using reference alkaloids. In addition, an HPLC-MSD analysis was carried out, which showed that there were no peaks apart from those of the alkaloids. The HPLC diagrams of Figures 1 and 2 were obtained on the basis of the following experimental data: Chromatography parameters:
Column: LiChrospher 60 RP Select B, 5 μm, 125 x 24 mm ID Eluent: A) 200 ml (acetonitrile) + 800ml (water) + 1 .5 g
(hexanesulphonic acid) + 0.3 ml (85% phosphoric acid) B) 900 ml (acetonitrile) + 100 ml (water) + 1 .5 g (hexanesulphonic acid) + 0.3 ml (85% phosphoric acid) Gradient: 5 min isocratically 100% A; up to 40% B in 24 min up to 100% B in 1 min 5 min 100% B;
5 min equilibration with 100% A Detection: UV light at 285 nm Eluate flow rate: 1 ml/min, stop after 35 min.
Injected volume: 10 μl Sample preparation:
Extract before reaction (Figure 1 ): 25 mg of alkaloids are dissolved in 40 ml of methanol by ultrasonics, made up to 50 ml and filtered through a membrane filter.
Reaction product (Figure 2): The reaction product is converted into the hydrochloride salt, dissolved in water in a concentration of 1 mg/ml and adjusted to a pH of between 2.5 and 6.5.
Example 3
Commercially available, purified chelidonine (Sigma) was subjected to reaction with tris(1 -aziridinyl)phosphine sulphide ( = thiotepa) according to the conditions described in Example 1 . After termination of the alkylation reaction, the subsequent washing step and the conversion step using HCI gas, the final product was further processed as follows: 340 g of the HCI-treated hence water-soluble reaction product were dissolved in water and concentrated close to saturation and allowed to rest in a refrigerator at 80C. After some hours, spontaneous precipitation occurred and 264 mg precipitate (hereinafter termed U-KRS) was collected. The precipitate comprised slightly yellowish hygroscopic crystals having a rather narrow melting point of 205 - 207 °C (indicating a fairly well crystallized product) and exhibiting light-blue fluorescence upon irradiation with UV-light at 366 nm. Traces of yellow, orange and red fluorescent bands were also visible. The product did not move when subjected to thin layer chromatography but remained at the starting position (Rf = 0), except for the traces which at least moved to give an Rf > 0.1 . From the nuclear magnetic resonance (NMR) spectrum (Figure 4) it gets clear that U-KRS contains aromatic rings comparable to those contained in the chelidonine molecule. The UV spectrum (Figure 5) exhibits absorption maxima at 148, 155, 160, 205, 230 and 282 nm, very much like the UV spectrum of chelidonine (Figure 6), which differs therefrom solely in that the peak at 230 nm of U- KRS occurs at 240 nm with chelidonine. This indicates that the nitrogen in U-KRS is quaternary, while in chelidonine it is tertiary.
Further analytical details can be derived from the mass spectrograms presented in Figure 7 and Figure 8 (U-KRS) and Figure 9 (chelidonine), and from the result of an elementary analysis of U-KRS revealing the following composition of matter (Tab. 1 ):
Table 1 : Elementary composition of U-KRS in % of total mass
Figure imgf000013_0001
Figure imgf000014_0001
The following examples show various applications of the compound U- KRS resulting from the procedure described in Example 3.
Example 4: Selective inhibition of in vitro cell growth by the anti¬ tumor drug U-KRS
Material and methods Cell culture was performed in Roux bottles at 37-37.5° C in a humidified atmosphere containing 8% CO2 . Confluent cultures were detached by a solution of 0.01 % trypsin and 0.2% EDTA in phosphate buffered saline (PBS) and split in a ratio ranging from 1 :5 to 1 : 25.
Human endothelial cells were isolated from umbilical veins by collagenase treatment. The culture medium for endothelial cells was M199 supplemented with 15% heat inactivated fetal calf serum, 200 μg/ml endothelial cell growth factor and 90 μg/ml heparin.
Fluorescence microscopy Cells were grown in 35mm dishes and incubated with 100 μg/ml U-KRS for 30-60 min. The culture medium was aspirated, the cells were washed twice with PBS. Coverslips were mounted on the cells and fluorescence was excited using a confocal laser scanning microscope equipped with an argon laser source. The emitted light was detected in a photomultiplier channel. The signals were imaged on a video monitor using the MRC 600 image processing software.
Results
1. In a range from 20-40 μg/ml U-KRS about 55% inhibition of cell growth with endothelial cells was measured. This concentration killed the human osteosarcoma cell line. Hybrids of the two cell types showed nearly the same sensitivity as normal cells. 2. Because of its autofluorescence U-KRS can be detected intracellularly. A laser scanning microscope showed a high uptake of U-KRS in malignant cells.
Example 5: U-KRS as Anticancer Agent - Oxygen Consumption
Material and methods
In vivo experiments in mice. Two to five control animals were each injected with 50μl of an Ehrlich mouse ascites tumor suspension i.p. which was 8 d old, freshly taken from a fully grown donor animal. The control group was not further treated. Test group was injected with 10mg U-KRS/ kg animal weight in the abdominal area immediately after the tumor implantation.
Results
Mice implanted with the ascites tumor, either after intraperitoneal or after subcutaneous administration of U-KRS showed a longer survival time than the implant animals which were not otherwise treated..
The measurement of oxygen consumption of an ascites tumor suspension by means of an electrode in vitro brought about a brief increase in consumption after the addition of U-KRS, followed however, by a rapid drop different from that of the control suspension not mixed with U-KRS
Example 6: Modification of antinociceptive action of morphine by U- KRS in rodents.
Material and methods
Animals: Experiments were performed on male Albino Swiss mice and male Wistar rats. Treatment: U-KRS was given i.p. in doses starting from 20 mg/kg for mice and 25 mg/kg for rats.
Experimental procedures: In each experiment the four groups of animals were injected with 1 ) placebo, 2) morphine, 3) U-KRS, 4) U-KRS and morphine.
Results: The results indicated that simultaneous administration of U-KRS and morphine modified the action of the narcotic analgesic drug. They produced antinociceptive action in the tail-flick test in rats, evident as an increase in the latency time.
The present results show that U-KRS given simultaneously with morphine changes susceptibility of experimental animals to nociceptive reaction in the described tests. The present results suggest that U-KRS can interact with analgesic drugs which are used in cancer.
Example 7: Induction of bimodal programmed cell death in malignant cells by the derivative U-KRS.
Material and methods The K562 erythroleukaemia cell line was used, and U-KRS produced in pure crystallized form and dissolved in water at a concentration of 1 .2 mg/ml.
The DNA content of K562 cells exposed to various concentrations of U- KRS were analyzed using propidium iodide and flow cytometry.
Results
The results of this study show that U-KRS induces bimodal cell death programs, the first of which, apoptosis, is mediated by quinidine sensitive Ca2 + -dependent K+ channels; the second modality, blister cell death, is mediated by preventing microtubule formation and thus inducing polyploidy.
Example 8: Influence of U-KRS on DNA, RNA and protein synthesis in malignant cells
Material and methods
3H labeled thymidine, 0.5/vCi in 20μl medium; uridine, 0.5 μCi in 20μI medium and leucine, 1 .OμCi in 20μl medium were placed for 2-4 h into four wells with different U-KRS concentration. Prior to that, the cell lines, guinea pig hepatocytes, C1 L hepatocytes, human tonsil cells, murine lymphomas, murine myeloma, Yoshida cells, two HeLa strains, EsB-, EB, lymphomas, ZAC/1 , P815 were grown 24 h at 37°C in 96 microtiter wells. WiDr cells were incubated in a somewhat different schema for 6 and 24 h at U-KRS concentrations of 1 , 4, 8 and 14μg/ml U-KRS.
Results Fluorometric evaluations show stronger affinity of U-KRS to elements of the cancer cell nucleus that to other cancer cell areas. Fluorescence phenomena may clearly show the strong and the strong and quick affinity exerted by U-KRS in tumor and metastasis areas. No toxic effects are seen in normal cells treated in doses which are 100 percent growth inhibitory to cancer cell lines tested to date.
Example 9: Influence of U-KRS on human xenografts
Material and methods Tumor cells were taken from human tumor xenografts and serially transplanted into nude mice. These cells were used in a colony-forming assay in vitro. Tumor cells were incubated continuously for at least one week with several concentrations of the drug U-KRS. This was done with six different types, and the colony formation was scored for each tumor. The drug effects were reported as percent T/C (Test/Control)
Results •
Many different kinds of tumors are sensitive to U-KRS correlating to the variety tested by U-KRS. There the tumoricidal effects seem to be dependent on the regeneration ability of the immune apparatus, which stimulation and modulation may be accomplished by U-KRS.
Example 10: Influence of U-KRS on human malignant cell lines
Material and methods
Four different malignant cell lines were used: 1 ) mouse sarcoma; 2) female mammary carcinoma; 3) human colon carcinoma; 4) human melanoma.
U-KRS and PP9AA02 derivatives were added to the culture media. After irradiation, 200 cells were plated per slide and incubated for one week, then stained and counted. Results
The results presented here indicated that U-KRS and PP9AA02 derivatives act on human malignant cell lines synergistically as cytotoxic substances.
Example 1 1 : Induced G2/M Arrest and Apoptosis in Human Epidermoid Carcinoma Cell Lines by U-KRS
Material and methods Primary human keratinocytes were isolated from neonatal skin specimens. Epidermal sheets were trypsinized and single cell suspensions collected by centrifugation.
Results U-KRS inhibits cell cycle progression in a dose-dependent manner.
U-KRS treatment affects cell cycle distribution and induces apoptosis in A431 and ME180 cells.
Expression of the cyclins, CDKs and CDK inhibitor p27 changes after treatment with U-KRS
Example 12: Antimetastatic effect of U-KRS and its influence on the oxygen and energy metabolism of mice with melanoma B-16
Material and methods The experiment was carried out on 133 C57B/6 male mice.
Metastasizing melanoma B-16 was transplanted to the right shin muscle of each mouse. On the 10th day after the tumor transplantation, the animals were divided into two groups. The first group was given U-KRS to sinus venosus of the eye in a dose of 1 mg/kg in the volume of 0.05 ml: 5 injections once in two days. The second group was given sterile physiological solution to sinus venosus in the same regime.
Results
The study was showing that a day after the first intravenous injection on U-KRS the indices of the oxygen regime in the muscular tissue noticeably improved. The rate of pθ2 level increased up to the maximum during the oxygen inhalation and the rate of pθ2 decreased from the maximum to the initial level after cessation of inhalation. In animals of the experimental group certain indices of the oxidative phosphorylation of the liver mitochonodria also improved a day after the preparation administration. It is known that under progression of the malignant process the oxygen and metabolism is inhibited. In mice which were given 5 injections of U-KRS such inhibition is less pronounced. In the animals of the control group the level of oxygen tension in muscular tissue and the rate of O2 delivery to it were statistically higher. Generalizing the data obtained it is possible to conclude that U-KRS in mice with B/16 melanoma improves the delivery of oxygen to tissues as well as inhibits the destructive effect of the malignant process on the organism bioenergetics.
The subsequent Examples illustrate immuno-modulating and metabolism-regulating properties of U-KRS, rendering U-KRS particularly suitable for the therapeutic treatment of allergic reactions, virus diseases
(HIV, Hepatitis A, B and C, E.coli, Influenza), osteoporosis, polyarthritis, psoriasis, and other diseases or bodily conditions.
Example 13: Enhancement of macrophage tumoricidal activity by U- KRS
Materia/ and methods
BALB/c mice were maintained by brother/sister matings in the laboratory. The tumor D1 DMBA/3 was routinely transplanted in BALB/c by s. c. injection. The tumor became apparent five days after implantation.
In vivo treatment with U-KRS was initiated five days after subcutaneous tumor implantation. Three routs of administration were employed, i.e., intravenous, intraperitoneal and subcutaneous. All three experimental groups, of at least 10 mice each, received 5.0μU-KRS in
0.15ml of PBS. This dosage was chosen based on preliminary experiments.
Results
The number of macrophages was significantly increased and the rate of tumor growth in treated mice was significantly diminished. Also, the mice showed a better immunological status thus proving the immune system strengthening and/or restoring efficacy of the applied U-KRS preparation of the present invention. The mice receiving U-KRS did not show any deleterious drug-related side effects.
Example 14: \n vitro effects of U-KRS on the phenotype of normal human lymphocytes
Materia/ and methods
The study was performed on lymphocytes isolated from peripheral blood of 10 healthy volunteers. The cells were isolated on Ficoll-Paque density gradient centrifugation. Viability of cells was determined by 0.1 % trypan blue staining, and found to be 95%.
Lymphocyte subpopulation was quantitated by immunofluorescence using monoclonal antibodies against total T-cells, T-helper cells and T- suppressor cells. Subsequently, cells were treated with FITC/conjugated rabbit F/ab/2 fragments anti-mouse IgG, washed in PBS and mounted on slides using polyvinyl-alcohol and glycerol. In control preparations, PBS or normal mouse serum was used instead of monoclonal antibodies.
Results The present study indicating the possibility of direct influence of U-KRS on T-cell subpopulations confirmed the earlier observations that U-KRS could be a good immunostimulator of cellular immunity in cancer patients and in other patients suffering from a depressed or otherwise disturbed immune system.
Example 15: Mitogenic properties of U-KRS on human peripheral blood monocytes
Material and methods Peripheral blood mononuclear cells. The blood was diluted with an equal volume of PBS containing 1 mM EDTA, pH 7.5, and was layered over Histopaque 1077. The tubes were centrifuged at 2000rmp for 30 min. The interface layers containing lymphocytes were collected and washed three times with RPMI tissue culture medium
Results It was found that even a short period of pre-treatment of the cells with
U-KRS had a potent synergic effect on PHA mitogenesis resulting in significantly higher cell stimulation indices than those of PHA alone.
Moreover it was found that a short period of PHA treatment of the cells is almost imperative for U-KRS to exert its mitogenic effects.
This study is showing a significant increase in circulating lymphocytes in patients in advanced stages of malignancies treated with U-KRS.
Example 16: Modulation of immune effector cell cytolytic activity and tumor growth inhibition in vivo by U-KRS
Material and methods
Tumor cells : mastocytoma P815 and the AKR leukemia AKIL cell lines were maintained in DMEM medium supplemented with 9.0% fetal calf serum containing penicillin and streptomycin.
Results
The present in vitro study demonstrates that U-KRS is an effective biological response modifier augmenting, by up to 48-fold, the lytic activity of splenic lymphocytes obtained from alloimmunized mice. The lytic activities of IL-2 treated spleen cells and peritoneal exudate lymphocytes were also significantly increased by the addition of U-KRS to the cell mediated lysis assay medium.
The results, taken in conjunction that U-KRS also enhances the cytolytic activity of spleen lymphocytes, indicate that the therapeutic effect of U-KRS observed in vivo is mediated by stimulating immune effector cell cytolytic activity.
Example 17: Influence of U-KRS on immunological blood parameters in vitro and in vivo
Material and methods
96 Wistar rats were used for this study. The initial age was 16 weeks for both male and female rats. U-KRS and PHA were tested in a 3Hthymidine test on T lymphocytes to evaluate the stimulation index in doses from 0.01 to 20μg/ml. Results
U-KRS stimulates different subsets of the hematopoietic and immunological systems. In this experiment reticulocytosis is induced as a possible sign of stimulation of certain stem cells or of general activation of the erythropoietic system. As no changes in the absolute leukocyte counts could be demonstrated, it may be postulated that by the action of U-KRS only strong modulating properties, e.g., a dislocation of the different subsets, happened in this experiment.
Stimulation comparable to that gained in these experiments was seen in vitro, including apoptosis in cancer cells.
Example 18: Inhibitory effect of U-KRS on ovalbumine antigenicity and antiovalbumine IgE antibody response in mice
Material and methods
The ability of U-KRS to inhibit ovalbumine-induced sensitization was tested in BALB/c and F1 (BALB/c x C57BI/6J) mice. U-KRS was introduced into the mice in the mixture with antigen (ovalbumine) and adjuvant (alum) and inhibited the sensitization of mice, reflected in lower anti-OA IgE antibody response and decreased antigen-induced histamine release from mast cells isolated from peritoneal cavities of sensitized mice. The effect of U-KRS on the antigenicity of ovalbumine (OA) in anaphylaxis was tested in heterologous passive cutaneus anaphylactic (PCA) reaction on rats.
Results
The results show that the OA prepared in the mixture with U-KRS had a decreased ability to react with anti-OA IgE antibodies raised against native OA in mice and fixed on the surface of rat mast cells in heterologous PCA reactions. The results suggest that U-KRS pre-treatment of OA may affect its antigenic property and the ability to react with anti-OA IgE antibodies raised against the native IgE molecules.
Example 19: Effect of treatment with U-KRS on early osteoporosis
Material and methods
U-KRS was administered intraperitoneal^ in a dose of 30mg/kg every other day for six months to female rats with ovariectomy-induced early osteoporosis. Administration of U-KRS was started on the second day after the surgical operation. At the end of the long-term treatment with U-KRS each rat was tested for the strength of both humeri and some parameters of rat femur were measured.
Results
The results show that the decrease in the mechanical strength of the humeral bones and some changes in the femur caused by ovariectomy were prevented by the six-month treatment with U-KRS.
Example 20: Influence of U-KRS preparation on influenza viruses and the bacteria E.coli and S. aureus
Material and methods Influenza viruses of the APR8/HON1 /34 strain were cultured on 10-day- old hen embryos;
E.coli bacteria derived from current clinical material and the strain 209P of S. aureus were employed. U-KRS preparation of the series 290614.
Results
This study confirms the existing of anti-infectious action of U-KRS preparation in the infected macroorganism. This influence is exerted through the stimulation of some elements of the host immune system due to a secondary destruction of microorganisms or cells infected by these microorganisms.
Example 21 : Biological activity of U-KRS with respect to influenza virus
Material and methods Virus of A type, Port-Chalmers 1 /73 culture, antigenic H3N2 variety.
The virus was injected at 1 , 10 and 100 EID50 per embryo. U-KRS was dissolved de novo in Hanks solution.
Results It was confirmed that U-KRS has the hampering action upon the development of the infective process. Example 22: Action of U-KRS on effects of irradiation
Materials and methods CBA/J male mice of 1 6/2Og body weight. Short-term whole-body gamma-irradiation of mice at doses ranging from 6.0Gy to 7.5Gy was performed. Long-term irradiation with the cumulative dose of 8.75 Gy was performed using the CEGO device.
U-KRS was administered intraperitoneal^ at doses of 0.1 , 1.4 and 12mg/kg body weight.
Results
The ability of U-KRS to modify the effects of irradiation was studied in
CBA/J mice using the dosage of the drug from 0.1 to 12 mg/kg. U-KRS was found to increase the survival rate of mice by 50-60 % at irradiation doses from 5.00 to 7Gy with no effect at the dose of 7.5Gy. Varying the dosage of the drug did not influence the outcome of irradiation.
The main outcome of the present study is the finding that U-KRS is capable of modifying the effects of irradiation when applied in both prophylactic and curative regimes.
Example 23: Effects of U-KRS against ionizing radiation
Material and methods
Breast carcinoma, colorectal adenocarcinomas, glioblastoma and pancreas adenocarcinomas cell lines. U-KRS preparation.
The effect of U-KRS on cell survival was tested at concentration ranging from 0.2μg/ml. The exposure times were 1 , 3 and 24h, after which the cells were washed with phosphate/buffered saline and fresh medium was added.
Results
U-KRS treatment resulted in differential time- and dose-dependent reduction in clonogenic cell survival. All four human tumor cell lines tested showed different sensitivities towards U-KRS with an up to 100-fold higher reduction of clonogenic survival as compared with human fibroblasts for 24h incubation. Examplθ 24: Effects of U-KRS in the treatment of allergies
Material and methods
A group of children aged 12 to 18 years and suffering from clinically relevant allergies caused by various agents including dust, mites, mite excrements, dog hair, cat hair and plant pollen received 1 to 3 times per week a dosis of 5mg U-KRS in 5ml PBS (5 ml ampoules) per os each over a period of 1 to 4 weeks.
Results
70% of the children reported significant reduction of the severeness of allergic reactions already after one week of treatment. After 4 weeks 90 % reported a significant relief in allergic sensitivity and/or severeness of allergic reactions towards their respective allergens.
It may be useful or in some cases even required to continue the anti¬ allergic therapy over a longer period of time in order to maintain the protective effect.
Example 24: Effects of U-KRS in the treatment of chronic fatigue syndrome (CFS)
Material and methods
A group of 5 adults suffering from CFS at differing intensities was treated with 5mg doses (5ml ampoules) of UKR-S 1 to 3 times a week by oral delivery or intravenous injection.
Results
All 5 volunteers reported an improvement of their bodily conditions and activities as well as of their subjective feeling of a decrease in their fatigue syndromes both in intensity and length already after 2 to 4 weeks of UKR-S administration. A particular advantage of the intravenous over the oral delivery form was not seen in this study, probably due to the small number of probands which does not allow for a statistically relevant evaluation of differences.

Claims

1 . An alkaloid reaction product, comprising at least one physiologically active alkaloid derivative obtained through chemical reaction of one or more alkaloids with a derivatizing agent, characterized in that said at least one alkaloid derivative has a quaternary nitrogen to which, as a fourth ligand, a Lewis base type residue is bound, said residue selected from the group consisting of a hydroxy (-OH), sulfhydryl (-SH), sulfoxy, sulfate, phosphate, ester, thioester, ether, thioether, alkyl, aryl, aralkyl, alkyloxy, aryloxy residue or a residue or decomposition product of the derivatizing agent not comprised in the aforementioned group of compounds, with the proviso that said residue is not a hydrogen, methyl or ethyl residue.
2. The alkaloid reaction product according to claim 1 , wherein at least one alkaloid derivative is present in the form of a water-soluble salt, preferably in the form of a hydrochloride.
3. The alkaloid reaction product according to claim 1 or 2, wherein at least one alkaloid derivative is from an alkaloid occurring in the herb Chelidonium majus L., preferably from a mixture of several or all alkaloids occurring in Chelidonium majus L.
4. The alkaloid reaction product according to any one of claims 1 to 3, wherein at least one alkaloid derivative is from an alkaloid selected from the group consisting of chelidonine, homochelidonine, oxychelidonine, methoxychelidonine, protopine, allocrγptopine, stylopine, sanguinarine, chelerythrine, chelilutine, chelirubine, oxisanguinarine, dihydrosanguinarine, dihydrochelerythrine, chelamidine, chelamine, and L-sparteine.
5. The alkaloid reaction product according to any one of claims 1 to 4, wherein chelidonine, oxychelidonine, homochelidonine or methoxychelidonine is present as the sole alkaloid source.
6. The alkaloid reaction product according to any one of claims 1 to 5, further comprising at least one unreacted tertiary or, where applicable, quaternary alkaloid selected from the group consisting of chelidonine, protopine, stylopine, allocryptopine, homochelidonine, sanguinarine. oxisanguinarine, dihydrosanguinarine, chelerythrine, dihydrochelerythrine, chelilutine, chelirubine, chelamidine, chelamine, L-sparteine, oxychelidonine, methoxychelidonine, corysamine and berberine.
7. The alkaloid reaction product according to any one of claims 1 to 6, further comprising unreacted derivatizing agent and/or one or more decomposition products of the derivatizing agent.
8. The alkaloid reaction product according to claim 1 , wherein it is further characterized by at least one analytical determination selected from the group consisting of the NMR spectrum in Fig.4, the UV spectrum in Fig.5, the mass spectrum in Figures 7 and 8, and the elementary analysis in Table 1 .
9. The alkaloid reaction product according to any one of claims 3 to 6, wherein said forth ligand is a hydroxy (-OH) or sulfhydryl (-SH) group.
10. A pharmaceutical composition comprising an alkaloid reaction product according to any one of claims 1 to 9.
1 1 . The composition according to claim 10, comprising as a major or sole active ingredient a chelidonine derivative, wherein its nitrogen atom is in a quaternated form according to the subsequent formula (I),
Figure imgf000027_0001
wherein R1 is a hydroxy (-OH), sulfhydryl (-SH), sulfoxy, sulfate, phosphate, ester, thioester, ether, thioether, -alkyl, aryl, aralkyl, alkoxy, aryloxy residue or a residue or decomposition product of the derivatizing agent, with the proviso that said residue is another than a hydrogen, methyl or ethyl residue.
12. The composition according to claim 1 1 , wherein R1 is a hydroxy (-OH) or sulfhydryl (-SH) group.
13. The composition according to any one of claims 10 to 12, in the form of a water-soluble preparation for oral or parenteral delivery, preferably in the form of an aqueous injection solution or in the form of a tablet, capsule, granulate or powder for oral ingestion .
14. The composition according to any one of claims 10 to 13, as a medicament.
15. Use of the reaction product defined in any one of claims 1 to 9 for the manufacture of a pharmaceutical composition for the prophylactic or therapeutic treatment of a disease or bodily condition selected from the group consisting of bacterial, fungal and viral infections, cancer, immunological dysfunctions, metabolic dysfunctions, radiation damage, epilepsy, multiple sclerosis, skin diseases, postoperative wounds and pain.
16. Use according to claim 15, wherein the disease or condition is selected from the group consisting of sleeping disease, herpes infections, influenza virus infections, skin tumors, allergies, chronic fatigue syndrome, osteoporosis, rheumatic diseases, and scars.
17. Use of the reaction product defined in any one of claims 1 to 9 for the manufacture of a pharmaceutical composition for stimulating, strengthening, and/or restoring a depressed or otherwise disturbed immune system of the animal or human body.
PCT/EP2005/009699 2004-09-20 2005-09-09 Quaternary alkaloid derivatives of chelidonium majus l WO2006032380A1 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
EP05782899A EP1833839A1 (en) 2004-09-20 2005-09-09 Quaternary alkaloid derivatives of chelidonium majus l
CA002593202A CA2593202A1 (en) 2004-09-20 2005-09-09 Quaternary alkaloid derivatives of chelidonium majus l

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP04022299 2004-09-20
EP04022299.4 2004-09-20

Publications (1)

Publication Number Publication Date
WO2006032380A1 true WO2006032380A1 (en) 2006-03-30

Family

ID=35501129

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2005/009699 WO2006032380A1 (en) 2004-09-20 2005-09-09 Quaternary alkaloid derivatives of chelidonium majus l

Country Status (5)

Country Link
EP (1) EP1833839A1 (en)
AR (1) AR050648A1 (en)
CA (1) CA2593202A1 (en)
TW (1) TW200610763A (en)
WO (1) WO2006032380A1 (en)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015036875A1 (en) 2013-09-10 2015-03-19 Probiotical S.P.A. Compositions comprising rutin useful for the treatment of tumors resistant to chemotherapy
WO2016009256A1 (en) 2014-07-17 2016-01-21 Probiotical S.P.A. Compositions comprising melatonin and flavonoids for use in the treatment of tumours resistant to chemotherapy
WO2021073603A1 (en) * 2019-10-17 2021-04-22 Chengdu Anticancer Bioscience, Ltd. Benzophenanthridine alkaloids and their methods of use
US11096975B2 (en) 2015-03-05 2021-08-24 Probiotical S.P.A. Compositions for use in the treatment of tumors resistant to chemotherapy
CN113549085A (en) * 2021-07-16 2021-10-26 湖北科技学院 Synthetic method of natural product 6-HHC

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US2670347A (en) * 1952-01-08 1954-02-23 American Cyanamid Co Thiophosphoric acid derivatives and method of preparing the same
WO1996038154A1 (en) * 1995-06-01 1996-12-05 Nowicky Wassili Means for treating radiation injuries

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US2670347A (en) * 1952-01-08 1954-02-23 American Cyanamid Co Thiophosphoric acid derivatives and method of preparing the same
WO1996038154A1 (en) * 1995-06-01 1996-12-05 Nowicky Wassili Means for treating radiation injuries

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BRÜLLER, W: "Studies concerning the effect of Ukrain in vivo and in vitro", DRUGS EXPTL CLIN RES, vol. 18, no. 13-16, 1992, pages 13 - 16, XP009059623 *
NOLTING, D.M.: "Untersuchungen zur Zytotoxizität von Alkylanzien, Topoisomerasehemmstoffen und Ukrain auf vier Ewing-Sarkom Zelllinien", 24 March 2005, WWW.MEIND.DE, MÜNSTER, XP002362491 *
SOSNOWSKY, G. ET AL.: "In the search for new Anticancer Drugs. 17.", J.MED.CHEM., vol. 29, 1986, pages 1250 - 1256, XP002361880 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015036875A1 (en) 2013-09-10 2015-03-19 Probiotical S.P.A. Compositions comprising rutin useful for the treatment of tumors resistant to chemotherapy
US9757405B2 (en) 2013-09-10 2017-09-12 Probiotical S.P.A. Compositions comprising rutin useful for the treatment of tumors resistant to chemotherapy
WO2016009256A1 (en) 2014-07-17 2016-01-21 Probiotical S.P.A. Compositions comprising melatonin and flavonoids for use in the treatment of tumours resistant to chemotherapy
US11026922B2 (en) 2014-07-17 2021-06-08 Probiotical S.P.A. Compositions comprising melatonin and flavonoids for use in the treatment of tumours resistant to chemotherapy
US11096975B2 (en) 2015-03-05 2021-08-24 Probiotical S.P.A. Compositions for use in the treatment of tumors resistant to chemotherapy
WO2021073603A1 (en) * 2019-10-17 2021-04-22 Chengdu Anticancer Bioscience, Ltd. Benzophenanthridine alkaloids and their methods of use
CN113549085A (en) * 2021-07-16 2021-10-26 湖北科技学院 Synthetic method of natural product 6-HHC

Also Published As

Publication number Publication date
TW200610763A (en) 2006-04-01
EP1833839A1 (en) 2007-09-19
AR050648A1 (en) 2006-11-08
CA2593202A1 (en) 2006-03-30

Similar Documents

Publication Publication Date Title
AU2002311985B2 (en) Methods for inhibiting angiogenesis
US4816462A (en) Method for diagnosing and for the therapeutic treatment of tumors and/or infectious diseases of different types with alkaloid-compounds
EP1833839A1 (en) Quaternary alkaloid derivatives of chelidonium majus l
GB2110533A (en) Compositions containing alkaloid derivatives
EP1402894A1 (en) Cancerous metastasis inhibitors containing carbacyclic phosphatidic acid derivatives
NO333672B1 (en) Quaternary chelidonin and alkaloid derivatives, processes for the preparation of the same, and their use for the preparation of medicaments
WO1996014065A1 (en) Therapeutic quassinoid preparations with antineoplastic, antiviral, and herbistatic activity
JP2006520763A5 (en)
JP7311177B2 (en) Combined use of A-NOR-5α androstane drugs with anticancer drugs
CN103906751A (en) [1,3]dioxolo[4,5-g]quinoline-6(5h)thione and [1,3]dioxolo[4,5-g][1,2,4]triazolo[1,5-a]quinoline derivatives as inhibitors of the late sv40 factor (lsf) for use in treating cancer
JP2002509532A (en) Pharmaceutical compositions of arglabin and arglabin derivatives
JP2005509006A (en) Production method for reacting alkaloids and use of reaction product pharmaceutical preparation
CN1901896B (en) Use of selected amino acid-zinc complexes as anti-malarials
US20040116385A1 (en) Treating and preventing viral infections with porphyrin-based compounds
US20040077621A1 (en) Antioxidants
Hammed et al. In Vivo Study of the Anticancer Activity of Doxorubicin Loaded on a Cellulose-Based Nanocarrier System
CN1706845A (en) Thiophosphoric acid derivative of quaternary ammonium chelidonium alkaloid
WO2017034893A1 (en) Novel polygodial analogs for the treatment of cancer and other proliferative diseases
CN104619325A (en) Drug composition for treating tumors and application thereof

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KM KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NA NG NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SM SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): BW GH GM KE LS MW MZ NA SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IS IT LT LU LV MC NL PL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
NENP Non-entry into the national phase

Ref country code: DE

WWE Wipo information: entry into national phase

Ref document number: 2005782899

Country of ref document: EP

Ref document number: 2593202

Country of ref document: CA

WWP Wipo information: published in national office

Ref document number: 2005782899

Country of ref document: EP