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EP0996432A2 - Biologisch-wirksame 1,3-bis-aromatische-prop-2-en-1-on, 1,3-bis-aromatische-propan-1-on, and 1,3-bis-aromatische-prop-2-yn-1-on - Google Patents

Biologisch-wirksame 1,3-bis-aromatische-prop-2-en-1-on, 1,3-bis-aromatische-propan-1-on, and 1,3-bis-aromatische-prop-2-yn-1-on

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Publication number
EP0996432A2
EP0996432A2 EP98929241A EP98929241A EP0996432A2 EP 0996432 A2 EP0996432 A2 EP 0996432A2 EP 98929241 A EP98929241 A EP 98929241A EP 98929241 A EP98929241 A EP 98929241A EP 0996432 A2 EP0996432 A2 EP 0996432A2
Authority
EP
European Patent Office
Prior art keywords
dihydrochalcone
compound according
methoxy
hydrogen
oxy
Prior art date
Legal status (The legal status 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 status listed.)
Withdrawn
Application number
EP98929241A
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English (en)
French (fr)
Inventor
Arsalan Kharazmi
Soeren Broegger Christensen
Simon Feldbaek Nielsen
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
LICA Pharmaceuticals AS
Original Assignee
Statens Serum Institut SSI
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Filing date
Publication date
Application filed by Statens Serum Institut SSI filed Critical Statens Serum Institut SSI
Publication of EP0996432A2 publication Critical patent/EP0996432A2/de
Withdrawn legal-status Critical Current

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    • C07—ORGANIC CHEMISTRY
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    • C07C255/01—Carboxylic acid nitriles having cyano groups bound to acyclic carbon atoms
    • C07C255/32—Carboxylic acid nitriles having cyano groups bound to acyclic carbon atoms having cyano groups bound to acyclic carbon atoms of a carbon skeleton containing at least one six-membered aromatic ring
    • C07C255/40—Carboxylic acid nitriles having cyano groups bound to acyclic carbon atoms having cyano groups bound to acyclic carbon atoms of a carbon skeleton containing at least one six-membered aromatic ring the carbon skeleton being further substituted by doubly-bound oxygen atoms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/12—Ketones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A61K31/135—Amines having aromatic rings, e.g. ketamine, nortriptyline
    • A61K31/136—Amines having aromatic rings, e.g. ketamine, nortriptyline having the amino group directly attached to the aromatic ring, e.g. benzeneamine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
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    • C07C217/02—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C217/04—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C217/06—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one etherified hydroxy group and one amino group bound to the carbon skeleton, which is not further substituted
    • C07C217/14—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one etherified hydroxy group and one amino group bound to the carbon skeleton, which is not further substituted the oxygen atom of the etherified hydroxy group being further bound to a carbon atom of a six-membered aromatic ring
    • C07C217/18—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one etherified hydroxy group and one amino group bound to the carbon skeleton, which is not further substituted the oxygen atom of the etherified hydroxy group being further bound to a carbon atom of a six-membered aromatic ring the six-membered aromatic ring or condensed ring system containing that ring being further substituted
    • C07C217/22—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one etherified hydroxy group and one amino group bound to the carbon skeleton, which is not further substituted the oxygen atom of the etherified hydroxy group being further bound to a carbon atom of a six-membered aromatic ring the six-membered aromatic ring or condensed ring system containing that ring being further substituted by carbon atoms having at least two bonds to oxygen atoms
    • C—CHEMISTRY; METALLURGY
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    • C07C217/00—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton
    • C07C217/02—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton
    • C07C217/04—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C217/28—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having one amino group and at least two singly-bound oxygen atoms, with at least one being part of an etherified hydroxy group, bound to the carbon skeleton, e.g. ethers of polyhydroxy amines
    • C07C217/30—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having one amino group and at least two singly-bound oxygen atoms, with at least one being part of an etherified hydroxy group, bound to the carbon skeleton, e.g. ethers of polyhydroxy amines having the oxygen atom of at least one of the etherified hydroxy groups further bound to a carbon atom of a six-membered aromatic ring
    • C07C217/32—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having one amino group and at least two singly-bound oxygen atoms, with at least one being part of an etherified hydroxy group, bound to the carbon skeleton, e.g. ethers of polyhydroxy amines having the oxygen atom of at least one of the etherified hydroxy groups further bound to a carbon atom of a six-membered aromatic ring the six-membered aromatic ring or condensed ring system containing that ring being further substituted
    • C07C217/36—Compounds containing amino and etherified hydroxy groups bound to the same carbon skeleton having etherified hydroxy groups and amino groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having one amino group and at least two singly-bound oxygen atoms, with at least one being part of an etherified hydroxy group, bound to the carbon skeleton, e.g. ethers of polyhydroxy amines having the oxygen atom of at least one of the etherified hydroxy groups further bound to a carbon atom of a six-membered aromatic ring the six-membered aromatic ring or condensed ring system containing that ring being further substituted by carbon atoms having at least two bonds to oxygen atoms
    • C—CHEMISTRY; METALLURGY
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    • C07C225/00—Compounds containing amino groups and doubly—bound oxygen atoms bound to the same carbon skeleton, at least one of the doubly—bound oxygen atoms not being part of a —CHO group, e.g. amino ketones
    • C07C225/02—Compounds containing amino groups and doubly—bound oxygen atoms bound to the same carbon skeleton, at least one of the doubly—bound oxygen atoms not being part of a —CHO group, e.g. amino ketones having amino groups bound to acyclic carbon atoms of the carbon skeleton
    • C07C225/14—Compounds containing amino groups and doubly—bound oxygen atoms bound to the same carbon skeleton, at least one of the doubly—bound oxygen atoms not being part of a —CHO group, e.g. amino ketones having amino groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being unsaturated
    • C07C225/16—Compounds containing amino groups and doubly—bound oxygen atoms bound to the same carbon skeleton, at least one of the doubly—bound oxygen atoms not being part of a —CHO group, e.g. amino ketones having amino groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being unsaturated and containing six-membered aromatic rings
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    • C07C225/22—Compounds containing amino groups and doubly—bound oxygen atoms bound to the same carbon skeleton, at least one of the doubly—bound oxygen atoms not being part of a —CHO group, e.g. amino ketones having amino groups bound to carbon atoms of six-membered aromatic rings of the carbon skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C233/00—Carboxylic acid amides
    • C07C233/01—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms
    • C07C233/30—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by doubly-bound oxygen atoms
    • C07C233/33—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by doubly-bound oxygen atoms with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by a carbon atom of a six-membered aromatic ring
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    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C45/00—Preparation of compounds having >C = O groups bound only to carbon or hydrogen atoms; Preparation of chelates of such compounds
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    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C45/00—Preparation of compounds having >C = O groups bound only to carbon or hydrogen atoms; Preparation of chelates of such compounds
    • C07C45/41—Preparation of compounds having >C = O groups bound only to carbon or hydrogen atoms; Preparation of chelates of such compounds by hydrogenolysis or reduction of carboxylic groups or functional derivatives thereof
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    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C45/00—Preparation of compounds having >C = O groups bound only to carbon or hydrogen atoms; Preparation of chelates of such compounds
    • C07C45/45—Preparation of compounds having >C = O groups bound only to carbon or hydrogen atoms; Preparation of chelates of such compounds by condensation
    • C07C45/455—Preparation of compounds having >C = O groups bound only to carbon or hydrogen atoms; Preparation of chelates of such compounds by condensation with carboxylic acids or their derivatives
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    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C45/00—Preparation of compounds having >C = O groups bound only to carbon or hydrogen atoms; Preparation of chelates of such compounds
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    • C07C45/67—Preparation of compounds having >C = O groups bound only to carbon or hydrogen atoms; Preparation of chelates of such compounds by reactions not involving the formation of >C = O groups by isomerisation; by change of size of the carbon skeleton
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    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
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    • C07C45/61—Preparation of compounds having >C = O groups bound only to carbon or hydrogen atoms; Preparation of chelates of such compounds by reactions not involving the formation of >C = O groups
    • C07C45/67—Preparation of compounds having >C = O groups bound only to carbon or hydrogen atoms; Preparation of chelates of such compounds by reactions not involving the formation of >C = O groups by isomerisation; by change of size of the carbon skeleton
    • C07C45/673—Preparation of compounds having >C = O groups bound only to carbon or hydrogen atoms; Preparation of chelates of such compounds by reactions not involving the formation of >C = O groups by isomerisation; by change of size of the carbon skeleton by change of size of the carbon skeleton
    • C—CHEMISTRY; METALLURGY
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    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
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    • C07C45/61—Preparation of compounds having >C = O groups bound only to carbon or hydrogen atoms; Preparation of chelates of such compounds by reactions not involving the formation of >C = O groups
    • C07C45/67—Preparation of compounds having >C = O groups bound only to carbon or hydrogen atoms; Preparation of chelates of such compounds by reactions not involving the formation of >C = O groups by isomerisation; by change of size of the carbon skeleton
    • C07C45/68—Preparation of compounds having >C = O groups bound only to carbon or hydrogen atoms; Preparation of chelates of such compounds by reactions not involving the formation of >C = O groups by isomerisation; by change of size of the carbon skeleton by increase in the number of carbon atoms
    • C07C45/72—Preparation of compounds having >C = O groups bound only to carbon or hydrogen atoms; Preparation of chelates of such compounds by reactions not involving the formation of >C = O groups by isomerisation; by change of size of the carbon skeleton by increase in the number of carbon atoms by reaction of compounds containing >C = O groups with the same or other compounds containing >C = O groups
    • C07C45/74—Preparation of compounds having >C = O groups bound only to carbon or hydrogen atoms; Preparation of chelates of such compounds by reactions not involving the formation of >C = O groups by isomerisation; by change of size of the carbon skeleton by increase in the number of carbon atoms by reaction of compounds containing >C = O groups with the same or other compounds containing >C = O groups combined with dehydration
    • C—CHEMISTRY; METALLURGY
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    • C07C49/00—Ketones; Ketenes; Dimeric ketenes; Ketonic chelates
    • C07C49/76—Ketones containing a keto group bound to a six-membered aromatic ring
    • C07C49/80—Ketones containing a keto group bound to a six-membered aromatic ring containing halogen
    • C07C49/813—Ketones containing a keto group bound to a six-membered aromatic ring containing halogen polycyclic
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    • C07C49/76—Ketones containing a keto group bound to a six-membered aromatic ring
    • C07C49/84—Ketones containing a keto group bound to a six-membered aromatic ring containing ether groups, groups, groups, or groups
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to the use of l,3-bis-aromatic-prop-2-en-l-ones, 1,3-bis-aromatic- propan-1-ones, and l,3-bis-aromatic-prop-2-yn-l-ones (in short bis-aromatic compounds) for the preparation of pharmaceutical compositions for the treatment or prophylaxis of a number of serious diseases including i) conditions relating to harmful effects of inflammatory cytokines, ii) conditions involving infection by Helicobacter species, iii) conditions involving infection by viruses, iv) neoplastic disorders, and v) conditions caused by microorganisms or parasites.
  • the invention also relates to novel chalcones and dihydrochalcones having advantageous substitution patterns with respect to their effect as drug substances, and to methods of preparing them, as well as to pharmaceutical compositions comprising the novel chalcones.
  • WO 93/17671 and WO 95/06628 which are assigned to the same applicant as the present application, describe the use of particular classes of oxygenated chalcones for the treatment or prophylaxis of conditions caused by microorganisms or parasites, in particular protozoa such as Leishmania, Plasmodia, and Coccidia such as Eimeria, and intracellular bacteria including Legionella and Mycobacteria.
  • FIG. 1 The minimal inhibitory concentrations (MIC) as given in ⁇ g/ml of licochalcone A, PH74 and PH98 are shown against 20 different strains of Helicobacter pylori.
  • FIG. 1 The minimal inhibitory concentrations (MIC) as given in ⁇ g/ml of PH104, PH105, PH135 and PH136 are shown against 20 different strains of Helicobacter pylori.
  • FIG. 1 Separation of concentrated sample on Hiload superdex 200 column.
  • B Separation of fraction 1 from Hiload column on MonoQ column.
  • FIG. 4 SDS-Page of the purification of a fumarate reductase from Leishmania donovani.
  • a and D Molecular weight marks.
  • B Concentrated extract of membrane associated proteins.
  • C Approximately 90% pure fumarate reductase (peak 21 from MonoQ column). The electrophoresis was performed with a 15% gel on a Bio-Rad mini protein II apparatus. The gel was stained with colloidal comassie.
  • Figures 5-17 Effect of various oxygenated chalcones on the activity of fumarate reductase and other enzymes of the mitochondrial electron transport chain.
  • FIG. 1 Effect of hcochalcone A on Leishmania major fumarate reductase. Digitonin treated promastigotes was incubated with hcochalcone A at 28°C 30 min, and the remaining enzyme activity was measured. Data are mean ⁇ SEM from 6 different experiments.
  • FIG. 7 Effect of SBC-35ma on Leishmania major fumarate reductase. KCL-solubilized fumarate reductase was incubated with hcochalcone A at 28°C 30 min, and the remaining enzyme activity was measured. Data are mean ⁇ SEM from 6 different experiments.
  • FIG. 8 Effect of SBC-24ma on Leishmania major fumarate reductase. KCL-solubilized fumarate reductase was incubated with SBC24ma at 28°C 30 min, and the remaining enzyme activity was measured. Data are mean ⁇ SEM from 6 different experiments.
  • FIG. 11 Effect of hcochalcone A on Leishmania major NADH dehhydrogenase. Membrane rich fraction of parasite was incubated with hcochalcone A at 28°C 30 min, and the remaining enzyme activity was measured. Data are mean ⁇ SEM from 6 different experiments.
  • FIG. 12 Effect of hcochalcone A on Leishmania major succinate-cytochrome c reductase. Membrane rich fraction of parasite was incubated with hcochalcone A at 28°C for 5 min, the remaining enzyme activity was measured. Data are mean ⁇ SEM from 4 different experiments.
  • Figure 13 Effect of hcochalcone A on Leishmania major NADH-cycochrome c reductase. Membrane rich fraction of parasite was incubated with hcochalcone A at 28°C for 5 min, the remaining enzyme activity was measured. Data are mean ⁇ SEM from 4 different experiments.
  • Figure 14 Effect of hcochalcone A on human MNC succinate dehydrogenase. Membrane rich fraction of parasite was incubated with hcochalcone A at 28°C for 5 min, the remaining enzyme activity was measured. Data are mean ⁇ SEM from 4 different experiments.
  • FIG. 15 Effect of hcochalcone A on human MNC NADH dehydrogenase. Membrane rich fraction of parasite was incubated with hcochalcone A at 28°C for 5 min, the remaining enzyme activity was measured. Data are mean ⁇ SEM from 4 different experiments.
  • FIG. 16 Effect of hcochalcone A on J774 cells succinate dehydrogenase. Membrane rich fraction of parasite was incubated with hcochalcone A at 28°C for 5 min, the remaining enzyme activity was measured. Data are mean ⁇ SEM from 4 different experiments.
  • FIG. 1 Effect of hcochalcone A on J774 cells NADH dehydrogenase. Membrane rich fraction of parasite was incubated with hcochalcone A at 28°C for 5 min, the remaining enzyme activity was measured. Data are mean ⁇ SEM from 4 different experiments.
  • FIG. 18 Effects of oral administratoin of PH135 (A) and PH 104(B) on LPS-induced TNF- production in the plasma of BALB/c mice.
  • V 1 PH135 50 mg/kg, lh before, the same time, and lh after LPS injection.
  • O V control.
  • FIG. 19 Effect of intraperitoneal administration of PH135 and PH104 on LPS-induced TNF- ⁇ production in the plasma of BALB/c mice.
  • FIG. 20 Effect of oral administration of SBC-24mbc on LPS-induced septic shock in BALB/c mice.
  • SBC-24m.bc was suspended in 0.2% Na-CMC and 0.01% tween 80 and was given to mice by oral in a dose of 50 mg/kg at lh before, 2h after, 8h after, and 24h after LPS injection.
  • FIG. 21 Effect of oral administration of SBC-24mbc on LPS-induced septic shock in BALB/c mice.
  • SBC-24mbc was suspended in 0.2% Na-CMC and 0.01% tween 80 and was given to mice by oral in a dose of 50 mg/kg.
  • FIG 22 Effect of i.p. administration of SBC-24mbc on LPS-induced septic shock in BALB/c mice.
  • SBC-24mbc was dissolved in lOO ⁇ l of DMSO and then suspended in sterile pyrogen-free salin and was given to mice by i.p. route in the doses of 30 mg and lOmg/kg/time, at lh before, same time and 2h after LPS-injection.
  • Control 1 received 200 ⁇ l of 10% DMSO in sterile pyrogen- free salin.
  • Control 2 received 200 ⁇ l of sterile pyrogen-free salin.
  • FIG. 23 Dose response study of SBC-24mbc and chlorpromazine (CPZ) by i.p. route on LPS- induced septic shock in BALB/c mice.
  • SBC-24mbc was dissolved in 100 ⁇ l of DMSO and then suspended in sterile pyrogen-free salin and was given to mice by i.p. route in the doses of 10 mg, 5 mg, and 2.5 mg/kg/time, at lh before, same time, and 2h after LPS-injection.
  • Control received 200 ⁇ l of 10% DMSO in sterile pyrogen-free salin.
  • CPZ was dissolved in sterile pyrogen-free salin and was given to mice by i.p. route in the dose of 4 mg/kg, at the same time as LPS-injection.
  • FIG. 24 Effect of oral administration of PH135 and PH104 on LPS-induced septic shock in BALB/c mice.
  • PH135 and PH104 were micronized in 0.2% Na-CMC and 0.01% Tween 80 and were given to mice by oral administration in a dose of 50 mg/kg at lh before, the same time, and lh after LPS injection.
  • FIG. 25 Effect of oral administration of PHI 35 and PH104 on LPS-induced septic shock in BALB/c mice.
  • PH135 and PH104 were micronized in 0.2% Na-CMC and 0.01% Tween 80 and were given to mice by oral administration in a dose of 16.6 mg/kg at lh before, the same time, and lh after LPS injection.
  • Figure 26 Effect of oral administration of hcochalcone A on protection of C57 BL/6 mice from cerebral malaria infected with P. berghei K173 strain. Mice received 50 mg/kg of hcochalcone A once daily over 5 days.
  • Figure 28 Observed and predicted antileishmanial activities of the selected chalcones.
  • the compounds included in the model are designated with O, and vahdation set is marked with T.
  • Figure 30 Observed and predicted lymphocyte suppressing activities of the selected chalcones.
  • the chalcones used for calculating the model are depicted by O, and the vahdation set is marked with T.
  • Figure 31 The size of the coefficients for each of the five variables in eight of the ten positions, which can be substituted (position 5' and 6' only contribute to a minor extent).
  • Figure 32 Antileishmanial activity. Stereo plot of negative coefficients (0.0005) for the three probes methyl (top) water (middle) and ammonium ion (bottom).
  • Figure 33 Antileishmanial activity. Stereo plot of positive coefficients (0.0005) for the three probes methyl (top) and ammonium ion (bottom).
  • Figure 34 Lymphocyte suppressing activity. Stereo plot of negative coefficients (0.0005) for the three probes methyl (top) and water (bottom).
  • Figure 35 Lymphocyte suppressing activity. Stereo plot of positive coefficients (0.0005) for the three probes methyl (top) water (middle) and ammonium ion (bottom).
  • Figure 36 Antiplasmodium activity. Plot of negative coefficients (0.0005) for the methyl probe.
  • Figure 37 Antiplasmodium activity. Plot of positive coefficients (0.0005) for the methyl probe.
  • the present invention is based on the teachings disclosed in the above-mentioned pubhshed international patent applications.
  • studies performed by the inventors have shown that several oxygenated chalcones altered the ultrastructure of Leishmania major promastigote and amastigote mitochondria in a concentration-dependent manner without damaging the organelles of macrophages or the phagocytic function of these cells.
  • Studies on the function of the parasite mitochondria showed that Licochalcone A inhibited the respiration of the parasite in a concentration-dependent manner, as shown by inhibition of O2 consumption and CO2 production by the parasites.
  • hcochalcone A inhibited the activity of the parasite mitochondrial dehydrogenase.
  • the target molecule for the compounds appears to be parasite-specific mitochondrial enzyme fumarate reductase.
  • This enzymes does not exist in mammalian cells and, accordingly, substances having an activity against this enzymes are potential drug candidates for diseases or conditions relating to fumarate reductase such as, e.g., Leishmanasis and diseases related to Helicobacter species such as, e.g., gastric ulcer.
  • One aspect of the invention relates to a method for the preparation and purification of leishmania fumarate reductase and the use thereof in a molecular modelling model for predicting potential substances capable of inhibiting the activity of this enzyme.
  • the compounds exert activity against Helicobacter pylori and it has been demonstrated that they also have antiviral and/or antineoplastic activities as well as an inhibiting effect against inflammatory cytokines. In the following is given an overview over the biological activities.
  • the present invention relates to the use of bis-aromatic compounds like chalcones and related compounds for the preparation of a pharmaceutical composition for the treatment or prophylaxis in humans of conditions involving infection by Helicobacter species, such as H. pylori, in the human gastric mucosa, as well as a method of treating such conditions using a chalcone or a related compound defined with the general formula I.
  • Hehcobacter pylori is a microaerophilic spiral shaped organism which is found in the stomach and generally appears to have an exclusive habitat in the human gastrointestinal mucosa. It has been estimated that this bacterium will have infected the gastric mucosa of more than 60% of adult humans by the time they are 60 years old. Moreover, Hehcobacter pylori has been implicated as a contributing factor in a number of pathological conditions, including acute (type B) gastritis, gastric and duodenal ulcers, and gastric adenocarcinoma.
  • Hehcobacter pylori has recently been found to posses the enzyme fumarate reductase and it is therefore contemplated that the mechanism of action involved in the activity against different strains of Hehcobacter pylori is based on an inhibition of this enzyme by chalcones and related substances. As described in the examples, several oxygenated chalcones exhibited MIC (minimum inhibiting concentrations) values equal to or less than 16 ⁇ g/ml against a large number of H. pylori strains. Furthermore, preliminary experiments have shown that several chalcones inhibit the activity of H. pylori fumarate reductase. H. pylori fumarate reductase is an essential enzyme in H.
  • H. pylori fumarate reductase as a target for bis- aromatic compounds like chalcones and related substances makes these compounds unique and important anti-Helicobacter agents.
  • the present invention i.a. relates to the use of a bis-aromatic compound for the preparation of a pharmaceutical composition for the treatment or prophylaxis of conditions involving infection by Hehcobacter species, wherein the bis-aromatic compound has an MIC value in the Helicobacter pylori Assay of at the most 200 ⁇ g/ml, such as at the most, e.g., 100 ⁇ g/ml, 75 ⁇ g/ml, 50 ⁇ g/ml, 40 ⁇ g/ml, 30 ⁇ g/ml, 20 ⁇ g/ml, or 10 ⁇ g/ml.
  • the compound for the use according to the invention only has minor (preferably no) side effects, thus, it is preferred that the compound, at the MIC in the Hehcobacter species Assay, shows a reduction of the thymidine uptake by human lymphocytes in the Lymphocyte Proliferation Assay, as defined herein, using phytomemagglutinin (PHA), of less than 50%, preferably less than 40%, especially less than 30%, in particular less than 20%.
  • PHA phytomemagglutinin
  • the ratio between the MIC value in the Hehcobacter species Assay and the ICso value in the Lymphocyte Proliferation Assay for the compound in question should be less than 2, such as less than, e.g., 1.5, 1.2., 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, or 0.2.
  • the bis-aromatic compounds used according to the invention as well as the novel compounds according to the invention can either be used as the sole active substance or in combination with other drug substances.
  • the bis-aromatic compound - where used in the treatment or prophylaxis of conditions involving infection by Hehcobacter species - advantageously can be administered in combination with the known anti-Helicobacter combination of amoxycillin, metronidazole and bismuth or, alternatively, the bis-aromatic compound can substitute any one of amoxycillin, metronidazol or bismuth in the combination.
  • the present invention relates also to the use of chalcones and related substances of the general formula I for the preparation of a pharmaceutical composition for the treatment and/or prophylaxis of a disease caused by a virus in a mammal, including a primate such as a human.
  • the present invention claims that bis-aromatic compounds like chalcones and related substances can exhibit antiviral effects in infected cells.
  • a very important feature by the chalcones and related substances is that it has been found that they are substantially harmless to mammalian cells in concentrations at which they effectively exert the antiviral effect. This selectivity is very important and surprising.
  • the chalcones and related substances are effective against viruses selected from the group consisting of: retrovira such as human immunodeficiency virus (HIV type I and type II; the HIV virus was previously known or referred to as LAV, HTLV-III or ARV); parvovira; papovavira, such as papilloma virus; andenovira; herpes vira such as Epstein- Barr virus, cytomegalovirus, herpes simplex vira (HSV 1 and HSV 2), varicella, herpex zoster virus, hepatitis A, hepatitis B; poxvira such as vaccinia, smallpox, molluscum contagiosum, cowpox, and monkey pox virus; hepadnavira; picornavira such as rhinovira and enterovira; reovira such as rotavirus and orbivirus; arbovira such as toga-, flavi-
  • retrovira
  • the present invention also relates to the use of a bis-aromatic compound for the preparation of a pharmaceutical composition for the treatment or prophylaxis of conditions involving infection by viruses, wherein the bis-aromatic compound has an IC50 value in the Virus plaque formation and/or virus cytophatic Assay of at the most 50 ⁇ g/ml, such as at the most, e.g. 40 ⁇ g/ml, 30 ⁇ g/ml, 20 ⁇ g/ml, 10 ⁇ g/ml, or 5 ⁇ g/ml.
  • the compound for the use according to the invention only has minor (preferably no) side effects, thus, it is preferred that the compound, at the IC50 concentration in the Virus plaque formation and /or virus cytophatic Assay, shows a reduction of the thymidine uptake by human lymphocytes in the Lymphocyte Proliferation Assay, as defined herein, using phytomemagglutinin (PHA), of less than 50%, preferably less than 40%, especially less than 30%, in particular less than 20%.
  • PHA phytomemagglutinin
  • the ratio between the ICBO value in the Virus Assay and the IC50 value in the Lymphocyte Proliferation Assay for the compound in question should be equal to or less than 1, such as less than, e.g., 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, or 0.2.
  • the compound or mixture of compounds for use according to the invention are claimed to be potent antineoplastic candidates.
  • the compound or mixture of compounds for use as antineoplastic compounds may be further defined as a bis-aromatic compound like a chalcone or a related substance which, when tested according to a standard test system for testing potential anticancer drugs, demonstrates an antineoplastic effect.
  • a standard test could be, e.g., a systematic protocol established by the National Cancer Institute (NCI) involving the testing of a compound against a standard cell line panel containing 60 human tumor cell lines.
  • NCI National Cancer Institute
  • the protocol and the established statistical means for analysing the results obtained by the standardised testing are well described in the literature, see, e.g., Boyd M. R.: Principles & Practice of Oncology, PPO Updates, Volume 3, No.
  • the bis-aromatic compounds like chalcones and related compounds are effective in the treatment or prevention of neoplastic disorders such as neoplastic disorders selected from the group consisting of epithehal neoplasms and non-epithehal and mixed neoplasms.
  • neoplastic disorders selected from the group consisting of epithehal neoplasms and non-epithehal and mixed neoplasms.
  • relevant neoplasms based on a histogenetic classification.
  • Epithehal neoplasms surface papilloma carcinoma, basal-cell carcinoma, transitional cell carcinoma, papillary carcinoma, apudomas, nesidiocytoma, clear-cell carcinoma, choriocarcinoma, and trabecular carcinoma glandular adenoma adenocarcinoma spheroidal cell carcinoma, cystadenocarcinoma, papillary adenocarcinoma, and mucous or colloid carcinoma
  • Neuro-ectodermal glial cells ghomas, astrocytoma, ohgodendroghoma, ependymoma and anaplastic variants nerve cells ganglioneuroma neuroblastoma medulloblastoma retinoblastoma melanocytes pigmented naevus malignant melanoma meninges meningioma malignant meningioma nerve sheaths schwannoma neurofibrosarcoma neurofibroma
  • Haemopoietic leukaemias and lymphoreticular acute leukaemias monocytic leukaemia, myeloblastic leukaemia (AML), lymphoblastic leukaemia (ALL) and chronic leukaemia, chronic mycloid leukaemia (CML), chronic lymphocytic leukaemia (CLL), hairy cell leukaemia other myeloproliferative disorders, myelomatosis, myelofibrosis Cell or Tissue Benign Malignant Type lymphomas Hodgkin's disease, non-Hodgkin's lymphomas, and histiocytic lymphomas
  • results are given showing that the compounds tested in a concentration of 5 ⁇ g/ml or higher exhibited potent inhibitory activity against two human cell lines CEM and HL60.
  • the present invention also relates to the use of a bis-aromatic compound for the preparation of a pharmaceutical composition for the treatment or prophylaxis of neoplastic disorders, wherein the l,3-bis-aromatic-prop-2-en-l-one, 1,3-bis-aromatic-propan-l-one, or 1,3- bis-aromatic-prop-2-yn-l-one has an IC_o value in the Anti-cancer Assay of at the most 100 ⁇ g/ml, such at the most, e.g., 75, 60, 50, 40, 30, 20, or 10 ⁇ g/ml.
  • Cytokines have been shown to play an essential role in the regulation of the immune response. Some of them such as interleukin-1 (IL-1), IL-2, IL-4, IL-10, IL-12 and interferon-gamma (IFN- ⁇ ) specify whether cell-mediated or humoral immunity will develop against a given antigen or in a given infection. This is reflected in the activation of the T-helper type 1 (Thl) or type 2 (Th2) cells. The outcome of a cytokine-mediated response could be either a protective/beneficial or a pathogenic/harmful effect.
  • IL-1 interleukin-1
  • IL-2 interleukin-2
  • IL-4 interferon-gamma
  • IFN- ⁇ interferon-gamma
  • TNF- ⁇ is a cytokine produced primarily by activated monocytes/macrophages. TNF- ⁇ is synthesised as a pro-protein consisting of 233 amino acids (26 kDa). The pro-protein is cleaved by a specific metalloprotease called TNF- ⁇ -converting enzyme (TACE) to a monomer of 157 amino acids (17 kDa).
  • TACE TNF- ⁇ -converting enzyme
  • TNF- ⁇ plays a key role in the cytokine network with regard to the pathogenesis of many infectious and inflammatory diseases. TNF- ⁇ was purified and the gene cloned in the mid 1980s.
  • TNF- ⁇ effects are transmitted via crosslinking of the membrane-bound TNF receptors (TNFRI & TNFRII) on the target cell. • Knock out mice for either TNF- ⁇ itself or the TNF- ⁇ receptors are shown to resistant to endotoxin shcok or protected from obesity-induced insulin resistance.
  • TNF- ⁇ tumor necrosis factor a
  • TNF- ⁇ has been shown to be a key mediator in the endotoxin -induced toxic shock and plays a key role in the pathogenesis of septic shock.
  • High TNF- ⁇ -serum levels have been-demonstrated after hpopolysaccharide (LPS) administration to animals and human volunteers, or in septic shock patients (Michie et al. N. Eng. J. Med. 318: 1481, 1981; Waage et al. J. Exp. Med. 169: 333, 1989).
  • LPS hpopolysaccharide
  • Administration of anti-TNF antibodies has been shown to protect against the lethal effect of LPS and live bacteria in a variety of animal models (Beutler et al.
  • Anti-TNF antibodies have been shown also to be effective in a variety of animal models of human diseases, including cerebral malaria (Grau et al. Science 237: 210, 1987), bacterial meningitis (Saukkonen et al. J. Exp. Med. 171: 439, 1990), bleomyein lung toxicity (Piquet et al. J. Exp. Med. 170: 655, 1989), liver ischemia/reperfusion damage (Colletti et al. J. Clin. Invest. 85: 1936, 1990), and graft-versus-host reaction (Shalaby et al. Transplantation 47: 1057, 1989), indicating an involvement of TNF in the pathogenesis of these diseases.
  • TNF might be an important target for pharmacological action, and inhibitors of its synthesis or bioactivity might be useful in the therapy of these diseases.
  • Anti-TNF antibodies have generated a lot of interest and at present several clinical studies are underway to assess the beneficial effect of these antibodies.
  • the commonly used antiinflammatory compounds such as glucocorticoids and particularly non- steroid antiinflammatory drugs are also being considered as potential anti-TNF drugs.
  • problems with these drugs both in terms of their efficiency and their side- effects. For example it has been shown that some animal models of endotoxic shock are resistant to glucocorticoid therapy (Bone et al. N. Eng. J. Med. 317: 653, 1987). Tenidap, an antirheumatic agent, has been shown to be effective against TNF- ⁇ , but because of its toxicity further development of this drug has been abandoned.
  • a substance like a bis-aromatic compound capable of affecting a inflammatory cytokine is a potential drug candidate for the treatment or prophylaxis of all the diseases or conditions mentioned above; in more general terms, these diseases or conditions encompass immune diseases, including infectious diseases, allergic diseases, auto-immune diseases, immune defects, and graft-related or transplantation related diseases.
  • immune diseases including infectious diseases, allergic diseases, auto-immune diseases, immune defects, and graft-related or transplantation related diseases.
  • the inflammatory response in all these diseases plays an important pathogenic role and, therefore, a substance affecting inflammatory cytokines is also a potential drug candidate for the treatment and/or prevention of inflammatory diseases in general, including auto-immune diseases.
  • the above-mentioned diseases or disorders relevant in the present context include inflammatory rheumatic diseases, degenerative rheumatic diseases, metabohc diseases of the bones, rheumatic manifestations of general medical diseases and hereditary connective tissue disorders.
  • relevant diseases or conditions are arthritic disorders, including infectious arthritis, osteoarthritis, rheumatoid arthritis and other rheumatoid diseases such as Juvenile Arthritis, Systemic Lupus Erythematosis, Sj ⁇ gren's Syndrome, Progressive Systemic Sclerosis, Polymyositis, Dermatomyositis, Ankylosing Spondilitis, Reiter's Syndrome, Reynaud's Syndrome, Psoriatic Arthritis, Relapsing Polychondritis, Relapsing Pannicuhtis, Crohn's Disease, Ulcerative Cohtis, Hereditary Complement Deficiencies, Collagen Vascular Diseases, rheumatological manifestations associated with bacterial and viral endocarditis or myocarditis and other rheumatological manifestations such as anaemia of chronic disorders, mixed connective tissue disease, vascuhtis, polyarthritiis nodosa, nephritis,
  • inflammatory cytokines may play an important role also include the wide range of diseases well known in the art to be associated with The Major Histocompabihty Complex such as juvenile diabetes, Reiter's syndrome, gluten- sensitive enteropathy.
  • allergic disorders wherein inflammatory cytokines may be involved are asthma bronchiale and atopic dermatitis. Also virus induced neoplastic disorders such as Burkitt's lymfhoma are believed to be associated with cytokine interaction.
  • cytokines are beheved to play an important pathogenic role, and accordingly, such diseases may be prevented or treated by use of substances capable of regulating cytokine production and function.
  • the Cytokine Inhibition Assay described herein has been used to determine the useful effect of the compounds described herein.
  • the present invention also relates to the use of a l,3-bis-aromatic-prop-2- en-l-one, 1,3-bis-aromatic-propan-l-one, or l,3-bis-aromatic-prop-2-yn-l-one for the preparation of a pharmaceutical composition for the treatment or prophylaxis of conditions relating to harmful effects of inflammatory cytokines, wherein the l,3-bis-aromatic-prop-2-en-l-one, 1,3-bis- aromatic-propan-l-one, or l,3-bis-aromatic-prop-2-yn-l-one has an ICsovalue in the Cytokine inhibition Assay of at the most 100 ⁇ g/ml, such at the most, e.g., 75, 60, 50, 40, 30, 20,
  • the compound for the use according to the invention only has minor (preferably no) side effects, thus, it is preferred that the compound, at the ICso concentration in the Cytokine inhibition Assay, shows a reduction of the thymidine uptake by human lymphocytes in the Lymphocyte Proliferation Assay, as defined herein, using phytomemagglutinin (PHA), of less than 50%, preferably less than 40%, especially less than 30%, in particular less than 20%.
  • PHA phytomemagglutinin
  • the ratio between the ICeo value in the Cytokine inhibition Assay and the IC50 value in the Lymphocyte Proliferation Assay for the compound in question should be equal to or less than 1, such as less than, e.g., 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, or 0.2.
  • hydrocarbyl is intended to mean a group which in itself comprises carbon and hydrogen atoms exclusively.
  • hydrocarbyl groups mentioned herein comprise 1-12 carbon atoms (C1 12), and more specifically 1-6 carbon atoms (Ci- ⁇ ).
  • Hydrocarbyl groups may be straight-chained, branched or cychc in their basis structure, when ignoring any substituents. It should be understood that hydrocarbyl groups may also consist of, e.g. a straight-chained part and a cychc part as, e.g. in a cyclohexyl-ethyl group.
  • Hydrocarbyl groups within the meaning of the present invention may be saturated or may contain one or more unsaturated bonds selected from double bonds and triple bonds, thus, hydrocarbyl groups may be based on alkanes, alkenes, alkadienes, alkatrienes, alkynes, etc.
  • C1-12 hydrocarbyl are methyl, ethyl, propyl, isopropyl, cyclopropyl, butyl, ter.butyl, secbutyl, isobutyl, cyclobutyl, 1-methylbutyl, 1, 1-dimethylpropyl, pentyl, cyclopentyl, hexyl, cyclohexyl, 3-methylbutyl, dodecyl, vinyl, prop-2-enyl (allyl), butenyl, pentenyl, 3- methylbut-2-enyl, l,l-dimethyl-prop-2-enyl, 1-methylpentyl, 1-ethylbutyl, hexenyl, heptenyl, octenyl, decaenyl, butadienyl, pentadienyl, hexadienyl, heptadienyl, hexatrienyl,
  • hydrocarbyl may be substituted with one or more substituents, preferably substituents selected from hydroxy, carboxy, halogen such as fluoro, chloro, bromo or iodo, amino, and amino which is optionally alkylated with one or two Ci- ⁇ alkyl groups (i.e. -NH(C ⁇ - ⁇ - alkyl) and -N(C ⁇ -6-alkyl) 2 );
  • halogen used in the description and claims is intended to coer fluoro, chloro, bromo and iodo.
  • alkyl is used in its normal meaning, cf. IUPAC.
  • Examples of "Ci ⁇ -alkyl” are methyl, ethyl, propyl, isopropyl, pentyl, cyclopentyl, hexyl, cyclohexyl, and examples of "Ci- .-alkyl” are methyl, ethyl, propyl, isopropyl, cyclopropyl, butyl, isobutyl, _er.butyl, cyclobutyl.
  • Si ⁇ -alkyl are methyl, ethyl, propyl, isopropyl, cyclopropyl, butyl, isobutyl, _er.butyl, cyclobutyl.
  • C ⁇ -3-alkyl covers methyl, ethyl, propyl, isopropyl, cyclopropyl.
  • alkoxy is intended to mean “alkyl-oxy”
  • acyl is intended to mean
  • the present invention is based on the finding that various bis-aromatic compounds possess highly interesting biological effects.
  • Especially promising compounds are bis- aromatic compounds of the general formula I
  • the general formula comprises 1,3-bis-aromatic propan-1-ones, 1,3-bis-aromatic prop-2-en-l-ones, 1,3- is-aromatic propyn-1-ones, as well as ⁇ - and/or ⁇ -substituted analogues thereof.
  • the compounds may be substituted in the ⁇ - and/or ⁇ -position relative to the keto group; thus, the substituents R may independently designate hydrogen, cyano, nitro, nitroso, amino, and halogen such as fluoro, chloro, bromo, or iodo, RH, and AW, among which hydrogen, Ci-s alkyl, cyano, and halogen such as fluoro, chloro, bromo, or iodo are the most preferred.
  • the unsubstituted variants are the most common, it is contemplated that bis-aromatic compounds in which one or both of the ⁇ - and/or ⁇ -position(s) R is/are substituted with e.g. methyl or ethyl are of great value with respect to the relevant activity and selectivity /tolerability.
  • the groups Ar 1 and Ar 2 in the general formula I each designate an aromate selected from phenyl and 5- or 6-membered unsaturated heterocychc rings containing one, two or three heteroatoms selected from oxygen, sulphur, and nitrogen, such as furanyl, thiophenyl, pyrrolyl, imidazolyl, isoxazolyl, oxazolyl, thiazolyl, pyrazolyl, pyridinyl, or pyrimidinyl, etc.
  • Preferred compounds are compounds of the chalcone and the dihydrochalcone types.
  • the aromate may carry other substituents which either will not to any substantial extent detract from the useful effect and selectivity of the bis-aromatic compounds, or will enhance these properties or relevant properties related to the use and utility of the bis-aromatic compounds, e.g., their solubility (such as when the bis-aromatic compounds carry a nitrogen- containing basic group or a carboxyl group which can form water-soluble salts with pharmaceutically acceptable counter ions).
  • the aromates may be substituted with one or more substituents selected from halogen; cyano; nitro; nitroso; carboxy and C1 12, preferably C1 6, straight, branched or cychc aliphatic hydrocarbyl which may be saturated or may contain one or more unsaturated bonds selected from double bonds and triple bonds, which hydrocarbyl may be substituted with one or more substituents selected from hydroxy, carboxy halogen such as fluoro, chloro, bromo or iodo, amino, and amino which is optionally alkylated with one or two Ci- ⁇ alkyl groups; preferably such optional further substituents on the aromates Ar 1 and Ar 2 are selected from halogen; cyano; nitro; and Ci- ⁇ , straight, branched or cychc aliphatic hydrocarbyl which may be saturated or may contain a double bond, which hydrocarbyl may be substituted with one or more substituents selected from hydroxy, halogen such as fluoro, chloro, brom
  • Suitable examples of such groups are methyl, ethyl, propyl, isopropyl, butyl, isobutyl, secbutyl, tertbutyl, pentyl, 1-methylbutyl, 1,1-dimethylpropyl, hexyl, 1-methylpentyl, 1-ethylbutyl, cyclopentyl, cyclohexyl, prop-2-enyl, l,l-dimethyl-prop-2-enyl, 3-methylbutyl, and 3-methylbut-2-enyl.
  • each of the two aromates may carry one or more further substituents AW. It is believed that the compound should carry at least one substituent AW (i.e. m and n are not both zero).
  • the substituents independently designate a group ARH, AH, or a group AZ, wherein each A independently is selected from -0-, -S-, -NH-, or -NRH-, preferably from -0- and -NRH-, RH designates Ci ⁇ straight, branched or cyclic aliphatic hydrocarbyl which may be saturated or may contain one or more unsaturated bonds selected from double bonds and triple bonds, which hydrocarbyl may be substituted with one or more substituents selected from hydroxy, halogen such as fluoro, chloro, bromo or iodo, amino, and amino which is optionally alkylated with one or two Ci- ⁇ alkyl groups, and Z designates (when the compound is a prodrug) a masking group which is readily
  • the preferred ones are generally those in which at least one A is -0-, preferably each A is O, mainly because of their expected excellent properties with respect to activity and selectivity/tolerability.
  • the oxygen atom in the form of oxy in many biologically active compounds may, with greater or lesser retention of, and indeed in certain cases with enhancement of, the biological activity, be replaced with bioisosteric groups, such as -S-, -NH-, and -NRH- as mentioned above.
  • RH independently designate Ci- ⁇ straight, branched, or cychc ahphatic hydrocarbyl which may be saturated or may contain a double bond, which hydrocarbyl may be substituted with one or more substituents selected from hydroxy, halogen such as fluoro, chloro, bromo or iodo, amino, and amino which is optionally alkylated with one or two Ci ⁇ alkyl groups, preferably RH designates Ci- ⁇ straight, branched, or cychc ahphatic hydrocarbyl which may be saturated or may contain a double bond.
  • RH Especially relevant examples of the group RH are methyl, ethyl, propyl, isopropyl, butyl, isobutyl, secbutyl, .er.butyl, pentyl, 1-methylbutyl, 1,1-dimethylpropyl, hexyl, 1-methylpentyl, 1-ethylbutyl, cyclopentyl, cyclohexyl, prop-2-enyl, l, l-dimethyl-prop-2-enyl, 3-methylbutyl, and 3-methylbut-2- enyl.
  • m designates the number of further substituents AW on the aromate Ar 1 and m is 0, 1, 2, 3 or 4, i.e. aromate in the 1-position relative to the keto group carries 0-4 substituents of the type AW in addition to any of the substituents defined above.
  • the number of substituents in that particular aromate (which preferably is phenyl) is 1, 2, 3, or 4, in particular 2, 3, or 4.
  • the symbol n designates the number of further substituents AW on the aromate Ar 2 , i.e. the aromate in the 3-position relative to the keto group, and is 0, 1, 2 or 3, preferably 1, 2, or 3, in particular 2 or 3.
  • the sum n + m is at least 2, preferably at least 3, in particular at least 4.
  • the prodrugs used according to the invention are, e.g., compounds of the general formula I, II or Ila in which Z is a group which is readily decomposed under conditions prevailing in the animal body to hberate the group AH.
  • Z is a group which is readily decomposed under conditions prevailing in the animal body to hberate the group OH.
  • prodrug forms suitable in connection with particular substituents in drugs is based upon the fact that certain types of groups will tend to be decomposed in the animal body in accordance with various decomposition pathways.
  • R* and R** each independently designate hydrogen or C ⁇ -3 alkyl
  • R" designates C ⁇ -6 alkyl or an optionally substituted aromate Ar 1 or Ar 2 as defined above and in claim 15,
  • the groups (A), (D), (E), and (F) are groups which will be decomposed by esterases to result in the corresponding free group such as the hydroxy group.
  • the group (B) will be subjected to removal of one of the methyl groups in the hver, and the group thus formed will be relatively readily decomposable in plasma.
  • the oxy-containing groups (C) are groups which are relatively labile under acidic conditions and, as such, are adapted to be decomposed in the human body, that is, in macrophages.
  • the prodrug group Z will be one which prevents the active molecule from being converted, in the liver, to a form which, from a practical point of view, will be inactive and quickly will be eliminated from the animal body, such as the forms where free phenohc OH groups are sulphated in the hver or are coupled to glucuronic acid in the hver.
  • Preferred prodrug groups Z are pivaloyl, pivaloyloxymethyl, N,N-dimethylcarbamoyl, and C ⁇ -6 acyl.
  • substituents AW which contain -O- (but taking into consideration that the oxygen atom could be replaced with the a bioisosteric group), this substituent could be called “an oxy-functional substituent". While it is presumed that the activity of the oxy-functional substituent is related to the substituent in the "free" form, that is, to hydroxy when A is -0-, to thiolo when A is -S-, and to amino or monoalkylamino when A is - NH- or -NRH-, very interesting results obtained with bis-aromatic compounds of the formula I where AW is alkenyloxy raise the intriguing question whether the active form in theses cases is the alkenyloxy-substituted form, or whether the alkenyloxy group is converted to a hydroxy group, maybe in the environment where the compound is expressing its biological action, before the bis-aromatic compound exerts it action.
  • the present invention also relates to the general use of the novel chalcones and dihydrochalcones as drug substances, as well as to pharmaceutical compositions comprising such novel compounds.
  • the compounds of the general formula I are known, whereas many of the compounds of the general formula I are novel compounds.
  • the known compounds may be isolated or synthesised in accordance with methods known from the literature or methods analogous thereto.
  • the novel compounds may, hkewise, be produced by methods known per se or methods which are analogous to such methods. Examples of excellent methods for preparing compounds of the l,3-bis-aromatic-prop-2-enone or the l,3-bis-aromatic-prop-2-ynone types are given in the following. Further examples of methods for the preparation of the compound used according to the present invention are described in WO 95/06628 and WO 93/17671 and in the references cited therein.
  • aldehyde a benzaldehyde in the case where Ar 2 is phenyl
  • This reaction which is a condensation reaction, is suitably carried out under acid or base catalysed conditions.
  • a review of such processes may be found in Nielsen, A.T., Houlihahn, W. J., Org. React. 16, 1968, p 1-444. In particular the method described by Wattanasin, S. and Murphy, S., Synthesis (1980) 647 has been found to be very successful.
  • the reaction may suitably be carried out in protic organic solvents, such as lower alcohols (e.g. methanol, ethanol, or tert.butanol), or lower carboxylic acids (formic, glacial acetic, or propionic acid), or in aprotic organic solvents such as ethers (e.g.
  • the catalyst may be selected from sodium, hthium, potassium, barium, calcium, magnesium, aluminum, ammonium, or quaternary ammonium hydroxides, lower alkoxides (e.g.
  • Primary aromatic amines such as aniline, free secondary amines such as dimethyl amine, diethyl amine, piperidine, or pyrrohdine as well as basic ion exchange resins may also be used.
  • Acid catalysts may be selected from hydrogen chloride, hydrogen bromide, hydrogen iodide, sulfuric acid, sulfonic acids (such as paratoluenesulfonic or methanesulfonic acid), lower carboxyhc acids (such as formic, acetic or propionic acid), lower halogenated carboxylic acids (such as trifluoroacetic acid), Lewis acids (such as BF3, POCI3, PCl ⁇ , or FeCl ⁇ ), or acid ion exchange resins.
  • sulfonic acids such as paratoluenesulfonic or methanesulfonic acid
  • lower carboxyhc acids such as formic, acetic or propionic acid
  • lower halogenated carboxylic acids such as trifluoroacetic acid
  • Lewis acids such as BF3, POCI3, PCl ⁇ , or FeCl ⁇
  • acid ion exchange resins such as BF3, POCI3, PCl ⁇ , or FeCl ⁇
  • a drawback of the base catalysed condensation is the poor yield obtained if the aromatic ring in which the ketone or the aldehyde or both is substituted with one or more hydroxy groups.
  • This drawback can be overcome by masking the phenohc group as described by T. Hidetsugu et al. European patent apphcation 0370 461 (1989). Deprotection is easily performed by mineral acids such as hydrochloric acid.
  • the reaction may be carried out at temperatures in the range of 0-100°C, typically at room temperature. Reaction times may be from 30 min. to 24 hours.
  • the activated derivative of the carboxyhc acid may be an activated ester, an anhydride or, preferably, an acid halogenide, in particular the acid chloride.
  • the reaction is normally carried out using the catalysts described by Tohda, Y. et al. cited above, namely copper(I)iodide/- triphenylphosphine-paUadium dichloride.
  • the reaction is suitably carried out in triethylamine, a mixture of triethylamine and pyridine or triethylamine and toluene under a dry inert atmosphere such as nitrogen or argon.
  • the reaction is generally carried out at reduced temperature such as in the range from -80°C to room temperature, the reaction time typically being from 30 minutes to 6 hours.
  • pubhshed methods for preparing ⁇ -methylchalcones include cycloaddition of diazomethane to chalcones followed by pyrolysis (C.B Rao et al. Ind. J. Chem. 1986, 25B, 400-403), self- condensation of acetophenones catalysed by various catalysts to give ⁇ -methylchalcones with identical substitution pattern at the two aromatic rings (L.J. Mazza et al. Synthesis 1980, 41-44; N.O. Calloway et al. J. Am. Chem. Soc. 1937, 59, 809; R.E. Lyle et al. J. Am. Chem. Soc. 1953, 75, 5959-5961; H.
  • ⁇ -alkylated chalcones are, e.g., obtained by condensation of an arylketone with a benzaldehyde.
  • the yields are poor especially when larger arylketones are used as starting material.
  • a number of catalysts including alkali hydroxides (T. SzeTl J. Prakt. Chem. 1962, 17, 346-348), piperidinium acetate (M.L. Edwards et al. J. Med. Chem. 1990, 33, 1948-1954), and hydrochloric acid (R.D. Abell J. Chem. Soc. 1953, 2834-2836) have not increased the yields.
  • a further drawback is the difficult availability of the arylketones used as starting materials.
  • the present invention now provides general and straightforward methods for the preparation of ⁇ - and ⁇ -hydrocarbyl substituted chalcones as well as the dihydro analogues thereof.
  • ⁇ - and ⁇ -hydrocarbyl substituents are introduced by a simple route, resulting in high yields from readily available starting materials.
  • claims 306 and 311 and the claims dependent thereon see claims 306 and 311 and the claims dependent thereon.
  • the key starting material in the novel method for the preparation of ⁇ -hydrocarbyl substituted chalcones is the compound of the general formula 2 wherein Ar 1 and Ar 2 are as defined above, i.e aromates, preferably phenyl.
  • the compound 2 can easily be synthesised in high yields from the compounds 1 by conjugated addition of a cyanide such as hydrogen cyanide, potassium cyanide, or sodium cyanide, preferably sodium cyanide by methods known per se, e.g. as described in "Advanced Organic Chemistry” by Jerry March, 3 rd ed. (especially chapter 15, Reaction 5-25) and references cited therein. It is noted that the compounds of the general formula 1 can be synthesised by methods described elsewhere herein.
  • the compound of the general formula 3, wherein R p may be selected from Ci- ⁇ straight, branched, and cychc aliphatic hydrocarbyl which may be saturated or may contain one or more unsaturated bonds selected from double bonds and triple bonds, which hydrocarbyl may be substituted with one or more substituents selected from Ci- ⁇ alkoxy, hydroxy, halogen such as fluoro, chloro, bromo, or iodo, amino, amino which is optionally substituted with one or two Ci- ⁇ alkyl groups, preferably Ci- ⁇ straight, branched, or cyclic alkyl such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, cyclopentyl, and cyclohexyl, may be prepared by treatment of the compound 2 with a strong base such as NaH or a basic salt of a dialkylamine, e.g.
  • a strong base such as NaH or a basic salt of a dialkyl
  • hydrocarbyl hahdes are hydrocarbyl fluoride, hydrocarbyl chloride, hydrocarbyl bromide, hydrocarbyl iodide, among which the bromide and iodide seem to be the most relevant.
  • the reaction is typically performed in an anhydrous aprotic solvent such as in dimethylformamide, lower ahphatic ketones like acetone and butanone, ahphatic ethers like tetrahydrofuran or diethylether, at a temperature between -100 and 0°C, preferably between - 100 and -50°C, and in the case of tetrahydrofuran preferably between -90 and -70°C such as around -78°C.
  • a relatively low reaction temperature is preferred in order to suppress by-product formation. Dry tetrahydrofuran seems to be especially suited as a solvent as in addition to non- reactivity toward the strong base and the hahde it remains soluble at -78°C.
  • the compound of the general formula 4 may be obtained by elimination of HCN from the ⁇ , ⁇ - disubstituted species 3.
  • the elimination conditions may be estabhshed by addition of a strong base such as NaH or a basic salt of a dialkylamine, e.g. hthium diisopropylamide, to compound 3.
  • Addition of a strong base may be done either with or without prior isolation and optionally also purification of the compound 3, with due consideration, of course, to the solvent selected. Good results have been obtained with isolation of the compound 3.
  • the elimination reaction is typically performed in an inert organic solvent such as benzene, toluene, o-, m-, or /j-xylene at a temperature between room temperature and the boiling point of the organic solvent, preferably at the boiling point of the organic solvent or at a temperature of 50-120°C.
  • an inert organic solvent such as benzene, toluene, o-, m-, or /j-xylene
  • ⁇ -alkyl substituted chalcones With the above method it is possible to prepare ⁇ -alkyl substituted chalcones. Furthermore, the ⁇ -hydrocarbyl substituted dihydrochalcone analogues thereof may be prepared from the corresponding chalcone by the methods for hydrogenation described herein.
  • the present invention also provides a novel method for the preparation of ⁇ -hydrocarbyl substituted chalcones.
  • the key starting material in this method is the compound of the general formula 5, wherein Ar 1 and Ar 2 are as described above, and where X2 is selected from cyano and halogen such as fluoro, chloro, bromo, or iodo.
  • X2 is cyano
  • the compound 5 is identical to compound 2 and can be prepared as described above, and where X2 is halogen, the compound 5 may be prepared from 1 by regioselective hydrohalogenation methods known per se, see e.g. "Advanced Organic Chemistry” by Jerry March, 3 rd ed. (especially Chapter 15, reaction 5-1).
  • the compound 6, wherein R is selected from Ci-e straight, branched, and cychc alkyl may be prepared by reacting 5 with a trialkylsilyl hahde such as .ertbutyldimethylsilyl chloride, in the presence of a strong base, e.g. NaH.
  • a trialkylsilyl hahde such as .ertbutyldimethylsilyl chloride
  • a preferred silyl enol-ether is the teributyldimethyl enol ether since this leads to stable compounds which can be isolated and purified.
  • the silylation reaction is performed in an anhydrous aprotic solvent such as dimethylformamide, lower ahphatic ketones like acetone, butanone, ahphatic ethers like tetrahydrofuran or diethylether, preferably tetrahydrofuran, at a temperature between 0°C and the boiling point of the solvent, preferably at room temperature.
  • the alkylation reaction is performed in the presence of a dried fluoride-donating agent such as HF, LiF, KF, RbF, CsF, preferably CsF (which can be almost completely dried), or a fluoride salt of mono-, di-, tri-, or tetraalkylammonium such as (C2Hs)3NHF and
  • a dried fluoride-donating agent such as HF, LiF, KF, RbF, CsF, preferably CsF (which can be almost completely dried
  • a fluoride salt of mono-, di-, tri-, or tetraalkylammonium such as (C2Hs)3NHF and
  • the reaction is performed in an anhydrous aprotic solvent as described for the reaction between the compound 5 and the trialkylsilyl halide at a temperature between 0°C and the boiling point of the solvent, preferably at room temperature.
  • a temperature between 0°C and the boiling point of the solvent preferably at room temperature.
  • dimethylformamide is used.
  • the compound 8 may be prepared by subjecting the compound 7 to elimination conditions whereby HX2, e.g. HCN, is eliminated.
  • the elimination conditions may comprise addition of a strong base such as NaH, a basic salt of a dialkylamine like hthium diisopropylamide, or an alkoxide hke sodium ethoxide, to compound 7 either with or without isolation and optionally purification of the compound 7.
  • the reaction is performed in an inert organic solvent such as benzene, toluene, o-, m-, or p-xylene at a temperature between room temperature and the boiling point of the organic solvent, preferably at the boiling point of the organic solvent.
  • an inert organic solvent such as benzene, toluene, o-, m-, or p-xylene
  • aromatic methyl ketones of the formula (WA) -Ar 1 "CO-CH3 an acetophenone in the case where Ar 1 is phenyl
  • aromatic aldehydes of the formula HCO-Ar 2 -(AW) n a benzaldehyde in the case where Ar 2 is phenyl
  • aromatic carboxyhc acids of the formula (WA)m-Ar 1 -COOH benzoic acids where Ar 1 is phenyl
  • ethyne derivatives of the formula H-C ⁇ C-Ar 2 -(AW)n phenylethynes where Ar 2 is phenyl
  • the administration route of the aromatic compound as defined above may be of any suitable route which leads to a concentration in the blood corresponding to a therapeutic concentration.
  • the following administration routes may be applicable although the invention is not limited thereto: the oral route, the parenteral route, the cutaneous route, the nasal route, the rectal route, the vaginal route and the ocular route.
  • the administration route is dependant on the compound in question, particularly, the choice of administration route depends on the physico-chemical properties of the compound together with the age and weight of the patient and on the particular disease and the severity of the same.
  • aromatic compounds as defined above such as the bis-aromatic ⁇ , ⁇ -unsaturated ketones or derivatives thereof, may be contained in any appropriate amount in a pharmaceutical composition, and are generally contained in an amount of about 1-95% by weight of the total weight of the composition.
  • the composition may be presented in a dosage form which is suitable for the oral, parenteral, rectal, cutaneous, nasal, vaginal and/or ocular administration route.
  • the composition may be in form of, e.g., tablets, capsules, pills, powders, granulates, suspensions, emulsions, solutions, gels including hydrogels, pastes, ointments, creams, plasters, drenches, dehvery devices, suppositories, enemas, injectables, implants, sprays, aerosols and in other suitable form.
  • compositions may be formulated according to conventional pharmaceutical practice, see, e.g., "Remington's Pharmaceutical Sciences” and
  • compositions according to the present invention may be formulated to release the active compound substantially immediately upon administration or at any substantially predetermined time or time period after administration.
  • the latter type of compositions are generally known as controlled release formulations.
  • controlled release formulation embraces
  • Controlled release formulations may also be denoted “sustained release”, “prolonged release”, “programmed release”, “time release”, “rate-controlled” and/or “targeted release” formulations.
  • Controlled release pharmaceutical compositions may be presented in any suitable dosage forms, especially in dosage forms intended for oral, parenteral, cutaneous nasal, rectal, vaginal and/or ocular administration.
  • TI is defined as the ratio of median lethal dose (LD 50 ) t median effective dose (ED50)],
  • ii) has a narrow absorption window in the gastro-intestinal tract. In such cases, it is important that the intact dose of the aromatic compound reaches the site of absorption in order to avoid a substantially uniform distribution of the compound administered in the whole gastrointestinal tract,
  • iii) has a very short biological half-live so that frequent dosing during a day is required in order to sustain the plasma level at a therapeutic level
  • the principle aims at changing the properties of the active drug substance by converting the substance into a masked form.
  • the compounds of the above formulae in which Z is one of the groups (A)-(E) are representatives of this strategy.
  • controlled release is obtained by appropriate selection of various formulation parameters and ingredients, including, e.g. various types of controlled release compositions and coatings (formulation-method).
  • the first strategy comprises use of the prodrug principle, i.e. converting the active drug substance into a per se inactive derivative which, upon administration to the organism, within the body of the organism by an enzymatic or non-enzymatic process releases the active drug substance so that the drug substance can exert its therapeutic effect.
  • the prodrug it is possible to obtain a prodrug which releases the active drug substance with a controlled rate so that it thereby is possible to extend the effect of the drug in the body.
  • the other strategy comprises the use of the active drug substance per se and then formulate the active drug substance together with appropriate excipients into a pharmaceutical composition which upon administration of the composition to the organism releases the active substance in a controlled manner.
  • suitable excipients include single or multiple unit tablet or capsule compositions, oil solutions, suspensions, emulsions, microcapsules, microspheres, nanoparticles, liposomes, delivery devices such as those intended for oral, parenteral, cutaneous, nasal, vaginal or ocular use.
  • every pharmaceutical composition is an actual drug dehvery system, since upon administration it presents the active drug substance to the body of the organism.
  • Formulations for oral use include tablets which contain the active ingredient in admixture with non-toxic pharmaceuticaUy acceptable excipients. These excipients may be, for example,
  • inert diluents or fiUers such as sucrose, sorbitol, sugar, mannitol, microcrystalline cellulose, starches including potato starch, calcium carbonate, sodium chloride, lactose, calcium phosphate, calcium sulfate or sodium phosphate;
  • ceUulose derivatives including microcrystalline ceUulose, starches including potato starch, croscarmellose sodium, alginates or alginic acid;
  • binding agents for example, sucrose, glucose, sorbitol, acacia, alginic acid, sodium alginate, gelatin, starch, pregelatinized starch, microcrystalline ceUulose, magnesium aluminum silicate, carboxymethylceUulose sodium, methylceUulose, hydroxypropyl methylceUulose, ethylceUulose, polyvinylpyrrolidone or polyethylene glycol; and
  • lubricating agents including ghdants and antiadhesives, for example, magnesium stearate, zinc stearate, stearic acid, silicas, hydrogenated vegetable oils or talc.
  • pharmaceuticaUy acceptable excipients can be colorants, flavouring agents, plasticizers, humectants, buffering agents etc.
  • the tablets may be uncoated or they may be coated by known techniques, optionaUy to delay disintegration and absorption in the gastrointestinal tract and thereby providing a sustained action over a longer period.
  • the coating may be adapted to release the active drug substance in a predetermined pattern, e.g; in order to achieve a controUed release formulation (see below) or it may be adapted not to release the active drug substance until after passage of the stomach (enteric coating).
  • the coating may be a sugar coating, a film coating (e.g.
  • hydroxypropyl methylceUulose methylceUulose, methyl hydroxyethylceUulose, hydroxypropylceUulose, carboxymethylceUulose, acrylate copolymers (Eudragit E®), polyethylene glycols and/or polyvinylpyrrolidone) or an enteric coating (e.g. based on methacrylic acid copolymer (Eudragit® L and S), ceUulose acetate phthalate, hydroxypropyl methylceUulose phthalate, hydroxypropyl methylceUulose acetate succinate, polyvinyl acetate phthalate, shellac and/or ethylceUulose).
  • a time delay material such as, e.g., glyceryl monostearate or glyceryl distearate may be employed.
  • solid tablet compositions as mentioned above may be provided with a coating adapted to protect the composition from unwanted chemical changes, e.g. chemical degradation, prior to the release of the active drug substance.
  • the coating may be apphed on the solid dosage form in a similar manner as that described in "Aqueous film coating” by James A. Seitz in “Encyclopedia of Pharmaceutical Technology", Vol 1, pp.337-349 edited by Swarbrick, J. & J. C. Boylan, Marcel Dekker, Inc., New York, 1988.
  • Formulations for oral use may also be presented as chewing tablets, or as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, potato starch, lactose, microcrystalline ceUulose, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oU medium, for example, peanut oil, hquid paraffin, or ohve oil.
  • an inert solid diluent for example, potato starch, lactose, microcrystalline ceUulose, calcium carbonate, calcium phosphate or kaolin
  • oU medium for example, peanut oil, hquid paraffin, or ohve oil.
  • Powders and granulates may be prepared using the ingredients mentioned above under tablets and capsules in a conventional manner using, e.g., a mixer, a fluid bed apparatus or a spray drying equipment.
  • ControUed release compositions for oral use may, e.g., be constructed to release the active drug substance by controlling the dissolution and/or the diffusion of the active drug substance.
  • Dissolution or diffusion controUed release can be achieved by appropriate coating of a tablet, capsule, peUet or granulate formulation of the aromatic compounds defined above, such as the bis-aromatic ⁇ , ⁇ -unsaturated ketones, or by incorporating the compound in question in, e.g., an appropriate matrix.
  • aromatic compounds defined above such as the bis-aromatic ⁇ , ⁇ -unsaturated ketones
  • a controUed release coating may comprise one or more of the coating substances mentioned above and/or, e.g., sheUac, beeswax, glycowax, castor wax, carnauba wax, stearyl alcohol, glyceryl monostearate, glyceryl distearate, glycerol palmitostearate, ethylceUulose, acrylic resins, dl-polylactic acid, ceUulose acetate butyrate, polyvinyl chloride, polyvinyl acetate, vinyl pyrrolidone, polyethylene, polymethacrylate, methylmethacrylate, 2-hydroxymethacrylate, methacrylate hydrogels, 1,3-butylene glycol, ethylene glycol methacrylate and/or polyethylene glycols.
  • sheUac beeswax, glycowax, castor wax, carnauba wax, stearyl alcohol, glyceryl monostearate, gly
  • the matrix material may comprise, e.g., hydrated metylcellulose, carnauba wax and stearyl alcohol, carbopol 934, sdicone, glyceryl tristearate, methyl acrylate-methyl methacrylate, polyvinyl chloride, polyethylene and/or halogenated fluorocarbon.
  • a controlled release composition of the aromatic compound defined above may also be in the form of a buoyant tablet or capsule, i.e. a tablet or capsule which upon oral administration floats on top of the gastric content for a certain period of time.
  • a buoyant tablet formulation of the compound in question can be prepared by granulating a mixture of the drug, excipients and 20-75% w/w of hydrocolloids, such as hydroxyethylceUulose, hydroxypropylceUulose and hydroxypropylmethylceUulose. The obtained granules can then be compressed into tablets. On contact with the gastric juice, the tablet can form a substantially water-impermeable gel barrier around its surface. This gel barrier takes part in maintaining a density of less than one, thereby aUowing the tablet to remain buoyant in the gastric juice.
  • Powders, dispersible powders or granules suitable for preparation of an aqueous suspension by addition of water are also convenient dosage forms.
  • Formulation as a suspension provides the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives.
  • Suitable dispersing or wetting agents are, for example, naturally-occurring phosphatides, as e.g. lecithin, or condensation products of ethylene oxide with e.g. a fatty acid, a long chain aliphatic alcohol or a partial ester derived from fatty acids and a hexitol or a hexitol anhydrides, for example, polyoxyethylene stearate, polyoxyethylene sorbitol monooleate, polyoxyethylene sorbitan monooleate etc.
  • naturally-occurring phosphatides as e.g. lecithin
  • condensation products of ethylene oxide with e.g. a fatty acid, a long chain aliphatic alcohol or a partial ester derived from fatty acids and a hexitol or a hexitol anhydrides for example, polyoxyethylene stearate, polyoxyethylene sorbitol monooleate, polyoxyethylene sorbitan monooleate etc.
  • Suitable suspending agents are, for example, sodium carboxymethylceUulose, methylceUulose, sodium alginate etc.
  • the pharmaceutical composition may also be administered parenterally by injection, infusion or implantation (intravenous, intramuscular, intraarticular, subcutaneous or the hke) in dosage forms, formulations or e.g. suitable dehvery devices or implants containing conventional, non- toxic pharmaceuticaUy acceptable carriers and adjuvants.
  • compositions for parenteral use may be presented in unit dosage forms, e.g. in ampoules, or in vials containing several doses and in which a suitable preservative may be added (see below).
  • the composition may be in form of a solution, a suspension, an emulsion, an infusion device or a dehvery device for implantation or it may be presented as a dry powder to be reconstituted with water or another suitable vehicle before use.
  • the compositions may comprise suitable parenteraUy acceptable carriers and/or excipients or the active drug substance may be incorporated into microspheres, microcapsules, nanoparticles, liposomes or the like for controUed release.
  • the composition may, in addition, conveniently comprise suspending, solubihsing, stabilising, pH-adjusting agents and/or dispersing agents.
  • the pharmaceutical compositions according to the invention may comprise the active drug substances in the form of a sterile injection.
  • the suitable active drug substances are dissolved or suspended in a parenterally acceptable hquid vehicle.
  • acceptable vehicles and solvents that may be employed are water, water adjusted to a suitable pH by addition of an appropriate amount of hydrochloric acid, sodium hydroxide or a suitable buffer, 1,3-butanediol, Ringer's solution and isotonic sodium chloride solution.
  • the aqueous formulation may also contain one or more preservatives, for example, methyl, ethyl or n-propyl p-hydroxybenzoate.
  • controUed release parenteral compositions may be in form of aqueous suspensions, microspheres, microcapsules, magnetic microspheres, oU solutions, oil suspensions, emulsions or the active drug substance may be incorporated in biocompatible carrier(s), liposomes, nanoparticles, implants or infusion devices.
  • Biodegradable/bioerodible polymers such as polyglactin, poly-(isobutyl cyanoacrylate), poly(2- hydroxyethyl-L-glutamine) and poly(lactic acid).
  • Biocompatible carriers which may be used when formulating a controlled release parenteral formulation are, e.g., carbohydrates such as dextrans, proteins such as albumin, lipoproteins or antibodies.
  • Non-biodegradable as, e.g., polydimethylsiloxane
  • biodegradable such as, e.g., poly (cap rolactone), poly(lactic acid), poly(glycohc acid) or poly(ortho esters).
  • suitable dosage forms for a composition include suppositories (emulsion or suspension type), and rectal gelatin capsules (solutions or suspensions).
  • the active drug compounds are combined with an appropriate pharmaceuticaUy acceptable suppository base such as cocoa butter, esterified fatty acids, glycerinated gelatin, and various water-soluble or dispersible bases like polyethylene glycols and polyoxyethylene sorbitan fatty acid esters.
  • an appropriate pharmaceuticaUy acceptable suppository base such as cocoa butter, esterified fatty acids, glycerinated gelatin, and various water-soluble or dispersible bases like polyethylene glycols and polyoxyethylene sorbitan fatty acid esters.
  • additives like, e.g., enhancers or surfactants may be incorporated.
  • typical dosage forms include nasal sprays and aerosols for inhalation.
  • the active ingredients are dissolved or dispersed in a suitable vehicle.
  • the pharmaceuticaUy acceptable vehicles and excipients and optionaUy other pharmaceutically acceptable materials present in the composition such as dUuents, enhancers, flavouring agents, preservatives etc. are aU selected in accordance with conventional pharmaceutical practice in a manner understood by the persons skiUed in the art of formulating pharmaceuticals.
  • compositions may also be administered topicaUy on the skin for percutaneous absorption in dosage forms or formulations containing conventionaUy non-toxic pharmaceutical acceptable carriers and excipients including microspheres and hposomes.
  • the formulations include creams, ointments, lotions, liniments, gels, hydrogels, solutions, suspensions, sticks, sprays, pastes, plasters and other kinds of transdermal drug delivery systems.
  • the pharmaceuticaUy acceptable carriers or excipients may include emulsifying agents, antioxidants, buffering agents, preservatives, humectants, penetration enhancers, chelating agents, gelforming agents, ointment bases, perfumes and skin protective agents.
  • emulsifying agents are naturally occurring gums, e.g. gum acacia or gum tragacanth, naturally occurring phosphatides, e.g. soybean lecithin and sorbitan monooleate derivatives.
  • antioxidants examples include butylated hydroxy anisole (BHA), ascorbic acid and derivatives thereof, tocopherol and derivatives thereof, butylated hydroxy anisole and cysteine.
  • preservatives are parabens, such as methyl or propyl p-hydroxybenzoate and benzalkonium chloride.
  • humectants examples include glycerin, propylene glycol, sorbitol and urea.
  • penetration enhancers examples include propylene glycol, DMSO, triethanolamine, N,N- dimethylacetamide, N,N-chmethylformamide, 2-pyrrolidone and derivatives thereof, tetrahydrofurfuryl alcohol and Azone®.
  • chelating agents examples include sodium EDTA, citric acid and phosphoric acid.
  • gel forming agents are Carbopol, ceUulose derivatives, bentonite, alginates, gelatin and polyvinylpyrrolidone.
  • ointment bases are beeswax, paraffin, cetyl palmitate, vegetable oUs, sorbitan esters of fatty acids (Span), polyethylene glycols, and condensation products between sorbitan esters of fatty acids and ethylene oxide, e.g. polyoxyethylene sorbitan monooleate (Tween).
  • compositions mentioned above for topical administration on the skin may also be used in connection with topical administration onto or close to the infected parts of the body which is to be treated.
  • the compositions may be any suitable medicated mass adapted for direct apphcation or for introduction into relevant orifice(s) of the body, e.g. the rectal, urethral, vaginal or oral orifices.
  • the compositions may simply be apphed directly onto thejnfected part, e.g. the mucosa. In certain cases it might be apphed by means of special drug dehvery devices such as dressings or alternatively plasters, pads, sponges, strips or other forms of suitable flexible material.
  • a controUed release percutaneous and/or topical composition may be obtained by using a suitable mixture of the above-mentioned approaches.
  • the active drug substance is present in a reservoir which is totally encapsulated in a shallow compartment molded from a drug-impermeable laminate, such as a metallic plastic laminate, and a rate-controUing polymeric membrane such as a microporous or a non-porous polymeric membrane, e.g., ethylene-vinyl acetate copolymer.
  • a drug-impermeable laminate such as a metallic plastic laminate
  • a rate-controUing polymeric membrane such as a microporous or a non-porous polymeric membrane, e.g., ethylene-vinyl acetate copolymer.
  • the active drug substance is only permitted to be released through the ratecontrolling polymeric membrane.
  • the active drug substance may either be dispersed in a sohd polymer matrix or suspended in an unleachable, viscous hquid medium such as silicone fluid.
  • the adhesive polymer is preferably a polymer which is hypoaUergenic and compatible with the active drug substance.
  • a reservoir of the active drug substance is formed by directly dispersing the active drug substance in an adhesive polymer and then - by, e.g., solvent casting - spreading the adhesive containing the active drug substance onto a flat sheet of substantiaUy drug-impermeable metallic plastic backing to form a thin drug reservoir layer.
  • a matrix dispersion-type system is characterized in that a reservoir of the active drug substance is formed by substantiaUy homogeneously dispersing the active drug substance in a hydrophilic or lipophihc polymer matrix and then, the drug-containing polymer is molded into disc with a substantiaUy weU-defined surface area and controUed thickness.
  • the adhesive polymer is spread along the circumference to form a strip of adhesive around the disc.
  • a microreservoir system may be considered as a combination of the reservoir and matrix dispersion type systems.
  • the reservoir of the active substance is formed by first suspending the drug sohds in an aqueous solution of water-soluble polymer and then dispersing the drug suspension in a hpophilic polymer to form a multiphcity of unleachable, microscopic spheres of drug reservoirs.
  • compositions for administration to the eye are provided.
  • Formulations for administration to the eye may be presented in the form of eye drops, lotions, ointments or dehvery devices.
  • the composition comprises the active drug substance in combination with pharmaceuticaUy inert vehicles or the active drug substance is incorporated in a suitable carrier system.
  • Pharmaceutically inert vehicles and/or excipients for the preparation of eye drops include, e.g., buffering agents such as boric acid or borates, pH adjusting agents to obtain optimal stability or solubility of the active drug substance, tonicity adjusting agents such as sodium chloride or borates, viscosity adjusting agents such as hydroxypropyl ceUulose, methylceUulose, polyvinylpyrrolidone, polyvinyl alcohols or polyacrylamide, oily vehicle such as vehicles comprising arachis oU, castor od and or mineral od.
  • Emulsions and suspensions of the active drug substance may also be presented in form of eye drops.
  • the composition may furthermore comprise stabilizing, dispersing, wetting, emulsifying and/or suspending agents.
  • Eye lotions and eye ointments may comprise pharmaceuticaUy acceptable carriers and/or excipients such as those used in an eye drop composition or in other relevant topical composition such as, e.g., ointments, creams and lotions.
  • an aqueous eye drop composition may be prepared by dissolving the active drug substance (or preferably a water-soluble salt or prodrug thereof) in sterile water in a specific concentration, optionaUy adjusting pH to a suitable pH by adding an appropriate amount of an appropriate buffer solution or hydrochloric acid or sodium hydroxide, optionaUy adding a preservative such as phenethanol, optionally adding a viscosity increasing agent such as methylceUulose, and subject the resulting solution to filtration foUowed by sterilization e.g. by autoclaving or by membrane filtration.
  • the aromatic compounds defined above such as the bis-aromatic ⁇ , ⁇ - unsaturated ketones or derivatives thereof, may be most valuable for controlling parasites in e.g. cattle, birds and fish. This may be carried out, e.g. by adding the compound in question to the feed or the drinking water of the animals, or when the animals to be treated are fish, the compound in question may also be added to the fish water.
  • the bis-aromatic ⁇ , ⁇ -ketones are preferably administered in an amount of about 0.1-50 mg per kg body weight per day, such as about 0.5-25 mg per kg body weight per day.
  • the compound in question may be administered orally in the form of tablets, capsules, ehxirs or syrups, or rectally in the form of suppositories.
  • Parenteral administration of the aromatic compound defined above, such as the bis-aromatic ⁇ , ⁇ -unsaturated ketone is suitably performed in the form of saline solutions of the ketones (or salts thereof) or with the compound incorporated into hposomes or in oil emulsions and cubic structures in od.
  • an acid addition salt of a basic compound of the formula I that is, a compound of the formula I in which either an aromatic ring or a substituent contains a basic nitrogen atom
  • a solubihser such as ethanol
  • compositions adapted for oral administration for systemic use the dosage is normally 2 mg to 1 g per dose administered 1-4 times daily for 1 week to 12 months depending on the disease to be treated.
  • the dosage for oral administration for the treatment of parasitic diseases is normally 1 mg to 1 g per dose administered 1-2 times dady for 1-4 weeks, in particular the treatment of malaria is to be continued for 1-2 weeks whereas the treatment of leishmaniasis will normaUy be carried out for 3-4 weeks.
  • the dosage for oral administration for the treatment of bacterial diseases is normaUy 1 mg to 1 g per dose administered 1-4 times dady for 1 week to 12 months; in particular, the treatment of tuberculosis will normaUy be carried out for 6-12 months.
  • the dosage for oral administration of the composition in order to prevent diseases is normaUy 1 mg to 75 mg per kg body weight per day.
  • the dosage may be administered once or twice daily for a period starting 1 week before the exposure to the disease untU 4 weeks after the exposure.
  • Rectal administration For compositions adapted for rectal use for preventing diseases, a somewhat higher amount of aromatic compounds, such as bis-aromatic ⁇ , ⁇ -unsaturated ketones or derivatives thereof is usuaUy preferred, i.e. from approximately 1 mg to 100 mg per kg body weight per day.
  • Parenteral administration For parenteral administration, a dose of about 0.1 mg to about 50 mg per kg body weight per day is convenient.
  • For intravenous administration a dose .of about 0.1 mg to about 20 mg per kg body weight per day administered for 1 day to 3 months is convenient.
  • intraarticular administration a dose of about 0.1 mg to about 20 mg per kg body weight per day is usuaUy preferable.
  • a solution in an aqueous medium of 0.5-2% or more of the active ingredients may be employed.
  • Percutaneous administration For topical administration on the skin, a dose of about 1 mg to about 5 g administered 1-10 times daily for 1 week to 12 months is usuaUy preferable.
  • pentavalent antimony-sodium gluconate examples of other antimalarial drugs to be combined with the compounds defined herein may be mentioned chloroquine and derivatives thereof, quinine, proguanil, cycloguanil, mefloquine, pyrimethamine and artemisinin.
  • an antituberculous drug such as isoniazide, ethambutol, pyrazinamide, and rifampicin or anti-Helicobacter drugs such as bismuth, metranidazole and amoxicUhn.
  • additional antimycotic drugs to be combined with the compounds defined herein may be mentioned amphotericin B, muconarcidol, griseofulvin, and miconazol.
  • additional antibabesial drugs to be combined with the compounds defined herein may be mentioned quinuronium sulfate, pentamidine isethionate, imidocarb or diminazene.
  • additional anticoccidial drugs to be combined with the compounds defined herein may be mentioned fulfonamides, amprocid and coccidiostatic agents such as inomycins, in particular monensin and salinomycin.
  • One general advantage of the compounds defined herein are their broad-spectered character, which makes it possible to use the compounds as sole medication in cases where the host to be treated is infected with, or suspected to be infected with, more than one of the bacteria and parasites discussed herein, or to use them as supplements to known antibacterial agents and antiparasitic agents in order to reduce the dose of the conventional antibiotics or antiparasitic agents, thus reducing the risk of side effects, in addition to the above-mentioned advantages with respect to reduction of drug resistance development.
  • the broad-spectered character of the compounds of the general formula I is of great advantage, and may be further augmented by combination with more than one antibacterial or antiparasitic agent, such as combination both with another antUeishmanial agent and another antimalarial agent. It is justified to presume that also the other aromatic compounds defined herein will show the same valuable broad-spectered character.
  • QSAR Quantitative Structure Activity Relationship
  • the QSAR analysis methodology is exceUently suited for the types of compounds used according to the present invention, especiaUy for the group of chalcones ((E)-l,3-diphenyl-prop-2-en-l-ones), for the group of dihydrochalcones (1,3-diphenyl-propan-l-one), and for the group of dehydrochalcones (l,3-diphenyl-prop-2-yn-l-one) used according to the present invention.
  • the QSAR model is equaUy apphcable for aU variants of the compounds defined herein, also, e.g., for the ⁇ , ⁇ -substituted variants of the compounds of the general formula I.
  • the influence of the substitution pattern on the two aromatic nuclei of the compounds used according to the invention can be elucidated by synthesis of chalcones covering substituents having different properties for variables such as hydrophobic parameters, e.g. lipophilicity ( ⁇ ) and partition coefficient Qog P), electronic parameters, e.g. ionisation constants (pKa), charge, and Hammet values ( ⁇ ) such as the electron distribution in the two aromatic rings ( ⁇ m and ⁇ P ), and steric parameters, e.g.
  • MR molecular refractivity
  • Van der Waals volume testing the activity of the compounds in a relevant assay, and performing a QSAR analysis on the data thus obtained, (see, e.g., Skagerberg, B., Bonelh, D., Cruciani, G., Ebert, C, Principal Properties for Aromatic Substituents. A Multivariate Approach for Design in QSAR. Quant. Struct. -Act. Relat., 1989, 8, 32-38.).
  • An optimum way of selecting compounds for the QSAR analysis is to use a statistical design based on the so-caUed Principal Component Analysis (PCA) for reduction of the number of variables to be processed.
  • PCA Principal Component Analysis
  • the principal components which summarise the information in the selected variables are linear combinations of the selected variables, possibly multiphed by a "loading" factor.
  • the principal components may be determined using suitable chemical/statistical software, such as, e.g., SIMCA P 2.1TM, SIMCA Umetri AB, Umea Sweden.
  • variable lipophilicity which can be relatively broadly represented by, e.g., including compounds having a hydroxy group and a iert-butyl group, respectively, as a substituent in a specific position.
  • the number of principal components and the number of places where substitution can be performed determine the necessary number of individual compounds to be included in the analysis.
  • the compounds defined herein which are chalcones there are, in principle, 12 possible substitution sites, which means that the number of compounds should preferably be at least 2 to the power of principal components multiphed by 12.
  • the number of compounds to be included in the QSAR should preferably be at least 48.
  • the compounds defined herein which are 1,3-diphenylpropenynes there are, in principle, 10 positions that may be substituted, and the number of compounds to scan this spectre of possibilities in the QSAR wiU preferably be at least 40, assuming that the number of principal components is two also here.
  • FinaUy in the compounds defined herein which are 1,3-diphenylpropanones, there are, in principle, 14 positions that might be substituted, which means that the number of compounds to be included in the QSAR should preferably be at least 56, stiU presuming that there are two principal components.
  • the preferred minimum number of compounds included can be smaUer in accordance with the calculations described above. Whde the above explanation with respect to the number of compounds to be included in the QSAR analysis represent the so-caUed complete factor design approach, it is also possible to use smaller numbers of compounds according to the so-called fractional factor design approach or the so- caUed D-optimal design.
  • the QSAR analysis is based on the set of compound selected according to the statistical design described above.
  • the relevant biological activity is determined, typicaUy in vitro. It is preferred that each of the compounds in the set actuaUy possesses a measurable biological activity in order to obtain the most precise prediction of the biological activity for compounds outside the set of compounds. It is also preferred that the range of biological activity represented by the set of compounds is a broad as possible. Thus, these considerations may necessitate exchange of some of the compounds which were suitable according to the statistical design described above. Ease of synthesis is another factor which should be taken into consideration in the selection of the compounds.
  • results of the QSAR analysis are given as predicted IC50 values and as a representation indicating sites in which increase of the variable in question, such as lipophilicity or hydrophihcity, wiU increase or decrease, respectively, the affinity to the target involved in the biological effect in question (whether the target molecule is known or not).
  • this representation is an indication of the range of values to be aimed at in each particular site.
  • the presentation is a stereo presentation showing regions in which increase of the variable in question will increase or decrease, respectively, the affinity to the target.
  • the person skiUed in the art can select, for each position, substituents which wUl have properties increasing or decreasing the IC50 value.
  • the selection may typicaUy be performed using data avaUable for the relevant variable in standard chemical tables or databases. (See Example 9).
  • the method points to a number of appropriate "suggested" substituents in each site, which means that the stereographic representation can be said inter alia to represent, to the person skiUed in the art, a relatively well-defined class of suggested compounds of increased or decreased (as the case may be) biological effect with respect to the target in question.
  • the person skiUed in the art can construct one or more compounds and subject these constructed compounds to the QSAR analysis. Those constructed compounds which are predicted by the QSAR analysis to have a desired low IC50 with respect to the desired activity against the disease-related target can then be synthesised and tested for their actual activity.
  • Representative compounds thus synthesised and tested and found to have improved actual activity can then, if desired, be included in the panel of compounds to constitute the base of a new QSAR analysis, and in this iterative manner, better and better compounds - with respect to the desired activity in question - can be identified.
  • a desirable low activity in a particular assay can be a desired property of a compound, such as, e.g., indicating low toxicity.
  • the QSAR analysis lends itself excellently to the prediction of IC50 values for the activity of the compounds defined herein in the Lymphocyte Proliferation assay.
  • the QSAR analysis is an exceUent tool for predicting the relevant biological activity of subclasses of the compounds. This is most valuable in the screening for lead compounds.
  • the synthesis of the compounds defined herein is relatively simple and efficient, it is important that the synthesis can be limited to a smaUer number of compounds which can then be used as a basis for guiding, through the QSAR analysis, the skiUed person towards compounds having a high hkehhood of showing more valuable properties. Once attractive candidate compounds have been identified using the QSAR analysis, it is easy to synthesise and test the compound.
  • the QSAR analysis can be used to predict one or several properties of compounds.
  • one valuable utilisation of the QSAR analysis would be to predict both the biological activity of compounds against the target to be controlled and (in another QSAR analysis) the biological activity against the human system itself, in other words, the tolerabihty or toxicity data of compounds.
  • AU of the above-discussed assays relating to targets to be controUed can be used in connection with QSAR analysis.
  • the value of the parameter Q 2 indicates the predictivity of the particular QSAR model in the particular context.
  • Q 2 is defined as one minus the summation of the second powenof the difference between the measured and the predicted IC50 values of the panel of compounds divided by the summation of the second power of the difference between the measured IC50 values and the mean IC50 value of the compounds included. It is preferred that Q 2 is at least 0.5, preferably at least 0.6 and more preferably at least 0.7 or, even more preferably, at least 0.8.
  • the set of basis compound (or simply "a set of compounds”) is designed taking into consideration both hydrophobic, electronic, and steric parameters, e.g. lipophilicity ( ⁇ ), Hammet values ( ⁇ ) such as the electron distribution in the two aromatic rings ( ⁇ m and ⁇ P ), and molecular refractivity (MR).
  • hydrophobic, electronic, and steric parameters e.g. lipophilicity ( ⁇ ), Hammet values ( ⁇ ) such as the electron distribution in the two aromatic rings ( ⁇ m and ⁇ P ), and molecular refractivity (MR).
  • An exceUent example of a set of 24 chalcones which represent the variables lipophilicity ( ⁇ ), the electron distribution in the two aromatic rings ( ⁇ m and ⁇ P ), molecular refractivity (MR), and Van der Waals volume is a set comprising
  • a corresponding set of dihydro analogues e.g. the dihydro analogues of the compounds (l)-(24) may be prepared by the method described herein and used in the QSAR Model, and in the case where ⁇ - and/or ⁇ -substituted chalcones are relevant, a variant of the set of compounds, e.g. the compounds (l)-(24), where either further ⁇ - and/or ⁇ -substituted compounds or ⁇ - and/or ⁇ - substituted analogues of the compounds of the set are prepared and included in the QSAR Model.
  • the compounds according to the invention or the compounds to be used according to the invention are compounds which, according to QSAR analysis, are predicted to have an ICeo below a certain defined value with respect to the disease target to be controUed, and/or, according to QSAR analysis, are predicted to have an IC ⁇ o above a certain defined value with respect to assays for testing the toxicity of the compounds.
  • Another aspect of the invention relates to a method for selecting bis-aromatic compounds which have an IC ⁇ o below a certain defined value with respect to the disease target to be controUed, and/or, according to QSAR analysis, are predicted to have an IC ⁇ o above a certain defined value with respect to assays for testing the toxicity of the compounds, the method comprising estabhshing a panel of compounds of the molecular type in question and having a variability of variables suitable for the QSAR analysis, including for the estabhshment of Principal Components, testing the panel of compounds in the relevant assay, subjecting the compounds with their thus estabhshed assay data to QSAR analysis, constructing compounds which, according to the output of the QSAR analysis, would be likely to have an improved activity in the assay in question, synthesising and testing the compounds constructed and optionaUy including one or more of the thus synthesised and tested compounds with their data in a further QSAR analysis, etc.
  • the skiUed person wiU based on the disclosure herein, and based on the suitability to QSAR analysis demonstrated herein for relevant compounds, be able to identify classes of or individual compounds according to the invention which have a superior biological effect and a low toxicity.
  • the QSAR analysis described above is based on the biological effects of the bis-aromatic compounds discussed herein.
  • Another approach is the receptor-based method (the drug design method) described in the foUowing.
  • the present invention also provides a method for the isolation and purification of fumarate reductase, it is believed that knowledge about this specific receptor, in combination with the drug design method described below, can be used for identification of novel bis-aromatic compound, which have superior pharmacological profiles.
  • knowledge about this specific receptor in combination with the drug design method described below, can be used for identification of novel bis-aromatic compound, which have superior pharmacological profiles.
  • a known hgand e.g. a natural substrate or a bis-aromatic compound known to inhibit fumarate reductase-hgand interaction
  • the bis-aromatic compound can be any compound which has one of the above mentioned effects on the interaction between fumarate reductases and known ligands and thereby action of the fumarate reductases.
  • EspeciaUy interesting bis-aromatic compounds are those which are hkely to interact with the substrate binding part of the fumarate reductase, but interaction with other sites in the fumarate reductases may also cause the desired inhibitory effect. This can be the result of direct steric blocking of the normal binding between the substrate and the fumarate reductase, but it may also be an effect of a conformational change in the fumarate reductase.
  • a method of identifying bis-aromatic compounds to be used in the method of the invention is disclosed below.
  • the interaction between the bis-aromatic compound and the fumarate reductase may be a covalent as weU as a non-covalent binding to the fumarate reductase by the bis-aromatic compound.
  • an aspect of the invention relates to a method for identifying a potentially therapeutically useful bis-aromatic compound capable of interacting with an fumarate reductase, thereby inhibiting binding between substrate and fumarate reductase, the method comprising at least one of the foUowing steps:
  • a bis-aromatic compound which has been established in vitro to inhibit the interaction between a fumarate reductase and a given known ligang, the experimental animal being inoculated with parasites or bacteria before, simultaneously with or after the administration of the bis-aromatic compound, and electing as a bis-aromatic compound suitably capable of interacting with a fumarate reductase, a bis-aromatic compound preventing and/or curing and/or alleviate disease caused by the parasites and bacteria.
  • an analogue of a fumarate reductase denotes any compound which have the abUity of binding at least one known hgand of the fumarate reductase in a manner corresponding to the binding between fumarate reductase and bis-aromatic compound.
  • Such an analogue of the fumarate reductase can be a truncated form of the intact fumarate reductase or it can be a modified form of the fumarate reductase which may e.g. be coupled to a probe, marker or another moiety.
  • the analogue of the fumarate reductase can be an isolated, but partiaUy of fuUy functional, active site of the fumarate reductase or a synthetic compound which mimics such an active site.
  • the immobilisation mentioned above may be simple non-covalent binding to an adhering surface or a host or receptor molecule such as an antibody, or covalent binding to a spacer molecule such as a polymer or a peptide.
  • the known hgand being bound to fumarate reductase or an analogue thereof can be detected in a number of ways, e.g. by the known hgand being labelled, or by means of a labeUed agent (such as an antibody) capable of reacting with the known ligand, or by means of a refractive index based determination of the extend of binding, such as the Pharmacia BiaCore® assay.
  • a labeUed agent such as an antibody
  • fumarate reductase or the analogue thereof being bound to the known hgand may be detected by fumarate reductase or the analogue thereof being labeUed, by means of a labeUed agent (e.g. antibody) capable of reacting with fumarate reductase or the analogue thereof, or by means of a refractive index based determination of the extend of binding, such as the Pharmacia BiaCore® assay.
  • a labeUed agent e.g. antibody
  • step lc) fumarate reductase or the analogue thereof being bound to the known ligand may be detected by separation of hgand-fumarate reductase complexes (e.g. by ultracentrifugation, ultrafiltration, hquid chromatography, such as size exclusion chromatography, or electrophoresis).
  • separation of hgand-fumarate reductase complexes e.g. by ultracentrifugation, ultrafiltration, hquid chromatography, such as size exclusion chromatography, or electrophoresis.
  • step lc) The determination of binding energy in step lc) is preferably performed in a microcalorimetric system using the weU-known technique of microcalorimetry.
  • the above-indicated steps serve 3 purposes.
  • the types of assays in step 1) are intended to shed hght over the ability of the candidate bis-aromatic compound of interacting with the fumarate reductase.
  • the label could be a radioactive label, a fluorescent or light absorbing label, an enzyme such as horse-radish peroxidase, a hgand such as biotin, or any other conventional labelling system known to the person skiUed in the art.
  • radioactivity may be measured in a hquid-scintiUation counter, a gamma counter, or any other convenient detection system for radioactivity
  • enzyme-labels are detected by the presence or absence of a specific substrate for the enzyme (optical density assessment, chemical reactivity of the remaining substrate or of the product etc.)
  • fluorescent labels may be detected by fluorescence microscopy or simple measurement of the fluorescent emission
  • light-absorbing labels may be detected by measurement of absorbtion of hght of a characteristic wavelength
  • biotin may be detected by its binding to streptavidin.
  • the separation of high molecular complexes by ultracentrifugation or ultrafiltration in 1) may be detected by one of the components of the complex being labeUed as described above; it is thus possible to detect the ratio between bound and unbound known hgand, but the detection step may also rely on the binding of antibodies to one of the components of the complex, and the subsequent detection of this antibody.
  • Any conventional chromatographic technique may be employed (HPLC, FPLC, size exclusion, etc)
  • the separation by electrophoresis may e.g. be performed by capillary electrophoresis.
  • the assay(s) in step 2) relate to the effects of the candidate bis-aromatic compound on parasitic activity in vitro.
  • the demonstration of a reduction in growth rate of the parasites of course cannot be contributed to the effect of interaction with fumarate reductases only, but a demonstration of this kind should provide a good estimate of the potential therapeutical usefulness of such a bis-aromatic compound.
  • the determination of growth rate may be performed by thymidine uptake in leishmania parasites or CFU in H. pylori, ATP, or by any other convenient detection system known to the person skiUed in the art.
  • the animal study in step 3) is performed in order to demonstrate the potential therapeutic usefulness of the candidate bis-aromatic compound in vivo. Further, such animal -studies should also establish the a priori values regarding effective dosage and toxicity before the candidate bis- aromatic compound finaUy is tested in human beings in controUed clinical trials.
  • the animal studies should also provide information regarding the convenient formulation of the bis-aromatic compound in a pharmaceutical preparation as weU as the preferred route of administration, as it is possible to obtain, from the animal model, data for absorbtion of the bis-aromatic compound as well as data for the metabohsm and excretion of the bis-aromatic compound.
  • the experimental animal is preferably a mouse, a rat, a cat, a dog, a monkey, a horse, a cow, a pig, or a chicken.
  • suitable capable of interacting with an fumarate reductase is intended to indicate that a bis-aromatic compound, apart from being capable of interacting with a fumarate reductase, is also capable of exerting effects in an in vivo system, i.e. that the bis-aromatic compound in addition to its binding capabUity also exhibits compatibility with a biological system, i.a. a patient.
  • the foUowing condition should be fulfilled:
  • the compound should exert a significant effect on hgand/fumarate reductase interaction (or on an interaction in an equivalent system which correlates weU to hgand/fumarate reductase interaction), the significant effect being one which with no doubt can be attributed to the interaction between the bis-aromatic compound and the fumarate reductase and which is not an unspecific interaction between the fumarate reductase and the bis-aromatic compound (due to e.g. radical changes in the physical and chemical environment when the bis-aromatic compound is added).
  • One way of excluding unspecific interactions as the reason for the exerted effect is to use at least one control which is a chemically comparable bis-aromatic compound (with respect to molecular mass, charge/polarity and gross 3-dimensional conformation (globular, fibrUlar etc.). If the control does not result in substantially the same effect in the assay as the bis-aromatic compound, it can be concluded that the bis-aromatic compound must be regarded as an assay-positive bis-aromatic compound.
  • the invention also relates to a method for identifying and/or designing a bis-aromatic compound, X, capable of interacting with a fumarate reductase, e.g. binding to the fumarate reductase, with a predicted binding energy equal to or better than a predetermined threshold value, the method comprising
  • step 4 untU the predicted binding free energy determined between the resulting bis- aromatic compound, X, and the site in the fumarate reductase is equal to or better than the predetermined threshold value.
  • Step 7) further estabhshes that the candidate bis-aromatic compound stands good chances of being therapeutically useful.
  • predicting the binding free energy is meant to imply that the binding free energy is determined by calculation rather than by performing experimental work determining the actual binding free energy.
  • One (theoretical) way of predicting binding free energy is by performing free energy perturbation (FEP) calculations on the interacting bis-aromatic compounds, but because of the vast amount of calculations such an approach would have as a result it is preferred that the empirical approximative method described below is employed.
  • FEP free energy perturbation
  • the term “better than” is intended to mean that the binding free energy has a value which is higher than the binding free energy which has been chosen as the threshold value, meaning that the ⁇ G is numericaUy higher than the threshold value selected. Or in other words: The term is intended to mean that the binding between the bis-aromatic compound and the fumarate reductase is more favourable energeticaUy than the situation were the bis-aromatic compound and the fumarate reductase are suspended independently in solution.
  • ⁇ V vdw ⁇ s > defined as ⁇ V vdw ⁇ . s >B - ⁇ V vdw ⁇ s >A, between the contribution from non-polar interactions to the potential energy between the chemical bis- aromatic compound X and its surroundings (denoted s) in two states, one state (A) being where the chemical bis-aromatic compound is surrounded by solvent, the other state (B) being where the chemical bis-aromatic compound, bound to a fumarate reductase or an analogue thereof, is surrounded by solvent, and
  • the symbol ⁇ > means molecular dynamics average.
  • the index X-s means compound-solvent (or compound-surrounding), the letter "X" denoting the chemical bis-aromatic compound X.
  • NormaUy the bis-aromatic compound X wiU function as an inhibitor of the binding between the fumarate reductase and known hgands, but as discussed herein, it is also a possibility that the compound or drug wiU affect the fumarate reductase in such a way that the binding between known hgands and the fumarate reductase is enhanced.
  • an analogue of a fumarate reductase should be understood, in a broad sense, any compound which mimics (with respect to binding characteristics) an interesting part of a fumarate reductase (e.g. the substrate binding part(s)), and the interaction of which with a chemical bis-aromatic compound or a group or plurality of chemical bis-aromatic compounds, e.g. drug candidates, is to be studied.
  • the analogue may simply be any other chemical compound regarded as capable of interacting with the chemical bis-aromatic compound in a manner which mimics the binding between the fumarate reductase and a known substrate in vivo, but most often the analogue wiU be a relatively large molecule, in other words a macromolecule such as a protein or an ohgonucleotide, which is relatively large compared to the chemical bis-aromatic compound; although the chemical bis-aromatic compound interacting with the analogue, of course, in itself be a macromolecule.
  • fumarate reductase or analogue thereof is preferably the fumarate reductase or an analogue thereof which exhibits at least one interesting binding characteristic relevant for the assembly of pill
  • ⁇ GAB is the free energy difference between B and A
  • ⁇ the corresponding reorganisation energy
  • ⁇ > ⁇ denotes an average evaluated near the minimum of the potential i.
  • this result may be exploited in the foUowing manner: For each solvation process, i.e.
  • the electrostatic contribution to the binding free energy can be approximated by ⁇ G el bmd ⁇ x ⁇ V e -s> (where the ⁇ now refers to the difference between protein and water) and thus obtained from two MD simulations of the solvated bis-aromatic compound and of the bis-aromatic compound-protein complex.
  • ⁇ Gbind 1 /2 ⁇ V el ⁇ . s > + ⁇ V vdw ⁇ . s > (1)
  • the parameter ⁇ being determined by empirical calibration.
  • c is a constant reflecting extrapolation to zero size of the chemical bis-aromatic compound, that is, where the regression line is distinctly offset from origin when moving towards zero size of the chemical bis-aromatic compound.
  • the parameter c may also be used to correct for possible systematic errors due to e.g. the neglect of induced polarisation, possible force field deficiencies etc.
  • c wiU normaUy assume a value between - 10 and 10 kcal/mol, typicaUy between -3 and 3 kcal/mol, such as between -2 and 2 kcal/mol, e.g. between -1 and 1 kcal mol.
  • c can suitably be set to zero, as the extent of deviation will be of minor importance for the usefulness of the predicted values.
  • the solvent used in the above method is suitably and most often an aqueous solvent like water
  • any other suitable solvent including, e.g., methanol, ethanol, acetone, acetonitrile, chloroform, hexane, etc., or mixtures thereof or combinations of such solvents or mixtures thereof with water.
  • the selection of the solvent wiU be of httle importance to the predicted values as long as the solvent is one which is able to dissolve or solvate the receptor molecule and the bis-aromatic compound (in the present context this means that a sufficient amount of fumarate reductase or analogue thereof can be homogeneously mixed with the solvent without precipitation so as to aUow the determination of binding energies by some suitable method), but there may be cases where it is advantageous to modify the solvent environment (e.g. by modulating the ionic strength) in which the interaction of the bis-aromatic compound and the receptor molecule is to take place.
  • the environment in which the interaction between the chemical bis-aromatic compound, such as a drug, and a fumarate reductase or an analogue thereof is to take place in the actual use of the drug is the human body, it might be particularly suitable to imitate e.g. human plasma as the solvent.
  • the inventors of the present invention wiU by the use of X-ray crystaUography elucidate the mechanism of binding between fumarate reductase and its natural or synthetic ligands thereby identifying an essential part of a defined active site responsible for the binding between hgands and their fumarate reductases, and thus providing a method to enable drug design of fumarate reductase inhibiting anti-parasitic and bacterial compounds.
  • PLIM is a Protein Ligand Interaction ModeUer that constructs putative hgands for a protein using thermodynamic criteria. It calculates the energy of interaction between the protein and sample probes that are successively placed at different points on a regular grid around the molecule. For each position and orientation the interaction energy between the probe and the atoms of the protein is calculated. The energies are stored, and the best positions for a particular probe are written out (the basic calculations are described by Goodford (1985) and Boobbyer (1989) and implemented in the commercially avadable program GRID; the PLIM implementation is somewhat different in that the energy values are converted to discrete points that are associated with the chemical probe, enabling easy output to e.g. data base searching programs).
  • the program then buUds up the hgand by incorporating selected probe atoms at positions of energy minima on the grid.
  • the user selects which atoms and groups should be used as probes, and which criteria should be used to determine those that wiU be incorporated into the hgand.
  • the energy is calculated as the sum of electrostatic, Van der Waals and hydrogen-bonding contributions as described herein.
  • the PLIM runs result in a number of suggested positions and orientations of favourable chemical groups in the region near the active site. These groups which have physical properties like charge, hydrogen bonding directionality and extended atom radia, wiU hereafter be denoted "site points".
  • a search for potential hgands is then made by searching a database for known molecular structures that match the positions of these groups of site points, using PLIM_DBS.
  • the core of PLIM_DBS is an algorithm for subgraph isomorphism (cf. UUman (1976) and Brint (1987)), where three sitepoints are represented as a distance matrix ("the pattern matrix").
  • the program looks for this distance pattern in the distance matrix formed from every entry in the database. If the pattern is found, the entry is superimposed on to the sitepoints and if the corresponding atom types match the entry and its orientation is saved in a hit-list.
  • surface complementarity can be used, i.e. only entries which are matching the protein surface with respect to hydrophobic and steric properties are saved.
  • PLIM_DBS is thus a database searcher which hunts through a coUection of 3-dimensional molecule coordinate sets, looking for entries that contain a certain pattern of atoms. This pattern is specified in terms of atom type, and of spatial position and orientation; for instance a search may be made for compounds containing an sp3 carbon atom that is 4.2 A from a sp2 oxygen and 5.1 A from a hydroxyl group that in turn is 5.6 A from the oxygen.
  • the strictness of the search can be adjusted by the user by varying the tolerance on the distance criteria and the atom-type matching, determining, for instance, whether a sp2 carbon that is a httle more than 4.2 A from an oxygen should be considered as a hit. Those hits that are found are then ranked according to a score that reflects how weU the target atoms superimpose on the real molecule, and also on how complementary the molecular surface of the compound is to that of the binding pocket of the protein.
  • the result from a PLIM_DBS search is a hst of molecular structures and their atomic coordinates, superpositioned on to the sitepoints, and given a score ("goodness of fit").
  • the procedure does not try to optimize the positioning of the structures, nor does it perform any molecular mechanics or dynamics calculations.
  • Both protein and the extracted structures are treated as rigid bodies.
  • the structures from the database search are displayed in the context of the protein and its surface on a graphics system using a commonly avadable molecular modelling package. Usually the structures show some unfavourable interactions with the protein, or lack groups to fill out e.g. hydrophobic pockets.
  • the structures form the database search are regarded as templates, to be modified and improved by an organic chemist. This process also involves choosing compounds which are easy to synthesize, which is of particular interest if the synthesis capacity is limited.
  • the templates may then be modified using a commercially avaUable olecule 3D builder (e.g. MacMimic).
  • a commercially avaUable olecule 3D builder e.g. MacMimic
  • Each template gives rise to a compound class.
  • Each modification of the class is assigned a specific number and the coordinates and a description are stored in a tree structure, e.g. using the program ARVS_JAKT developed by Symbicom AB in Sweden.
  • the design should be performed in a coUaboration between protein structure experts and organic chemists, in order to provide the best tools possible for the chemists who will actually synthesize the compounds.
  • the bis-aromatic compound A is likely to be a bis-aromatic compound which is capable of binding to the selected active site.
  • the above-indicated steps are especially preferred when the ligand is a substrate known hgand such as a natural substrate of the fumarate reductase or a part thereof.
  • BMNC Human blood mononuclear ceUs
  • IC ⁇ o values are determined as described in the examples under the heading Cytokine production by human peripheral blood mononuclear ceUs in vitro.
  • IC ⁇ o values are determined by the virus plaque formation and/or the virus cytopathic assay using standard methods weU-known in the art.
  • a WHO reference vaccine strain of L. major originally isolated from a patient in Iran and a Kenyan strain of . donovani (MHOM(/KE/85/NLB 274) were cultured in medium 199 containing 0.02 mg/ml gentamycin, 25 mM Hepes, 4 mM L-glutamine, and 20% heat inactivated fetal calf serum (FCS). Incubation was carried out at 26°C. Promastigotes were harvested on day 3 and 6 of the culture and used for the parasite growth inhibition.
  • test compounds on promastigotes was assessed by a method simUar to the one described by Pearson et al., by incubating promastigotes (3xl0 6 /ml) at 26°C for 2 hrs in the presence of a given compound or the medium alone in 96 weUs flat bottom microtiter plates.
  • mice BALB/c female mice aged eight weeks old were used throughout the experiment.
  • the WHO reference vaccine strain of L. major originally isolated from a patient in Iran was cultured as described in the Leishmania promastigotes Assay above.
  • mice received s.c. injections (in 0.05 ml of PBS) in the left hind footpad with 1x10 ' stationary phase promastigotes.
  • Footpad lesions were measured and expressed as footpad thickness increase (in mm).
  • the footpad thickness of mice was measured before infection and every 3 days after 7 days of infection. From 7 days of infection, mice received injections of the test compound (dissolved in 20 ⁇ l of 99% (v/v) ethanol, and then 980 ⁇ l of medium 199 was added and the resulting mixture was stored at -20°C before use) i.p. once a day. After 42 days of injections, some of the mice were kUled and the footpads, spleens and livers removed. The parasite loads in the footpads and livers were estimated by a modification of the method described by Liew et al. using 3-H-thymidine uptake. The results were expressed as cpm. The footpads, spleens and hvers impression was also estimated.
  • L. donovani promastigotes were used. Animals were intracardiaUy inoculated with 2 x 10 7 L. donovani promastigotes in 0.1 ml medium 199 (Day 0). One hour later, one of the animals was kdled. The hver and the spleen were weighed. The hver and the spleen impression smears were made. After air-drying, the impression smears were fixed with water-free methanol and stained with Giemsa.
  • mice Five of the animals were treated (i.p.) with the test compound (dissolved in 20 ⁇ l of 99% (v/v) ethanol, and the 980 ⁇ l of medium 199 was added, and the resulting mixture was stored at -20°C.) (10 mg/kg body weight two times per day) from Day+1 to Day+7. Another five animals were treated with 0.85% NaCl. The animals were kiUed on Day+8. The hver and spleen were weighed, and the hver and the spleen impression smears were made. The number of the parasite in the liver and the spleen were counted under microscope.
  • the spleen of the animals were cut into very smaU pieces, cultured in 15 ml of the culture medium at 26°C overnight and the parasite load was determined by 3 H-thymidine uptake as described above.
  • mice BALB/c or CFlfemale mice aged eight weeks were used.
  • the Plasmodium sp. causing malaria in humans can only infect certain primates. Therefore it has not been possible to determine whether a given test compound inhibits parasite multiphcation of human malaria parasites in vivo.
  • Plasmodium sp. that infect rodents. These systems have earher been used to test the ability of drugs to inhibit malaria infections in vivo. In the experiments described below mice were infected with either P. yoelii YM strain or P. berghei K173 and were compared to the outcome of infection in untreated control animals and in animals treated with the test compound.
  • the parasites were maintained by passage through BALB/c or CFlmice, and the animals were infected by injection of infected erythrocytes obtained from mice with a parasitemia of approximately 40%.
  • the animals were injected intraperitoneaUy with either 1 x 10 6 parasitized erythrocytes diluted in 0.9% NaCl and in a final volume of 0.2 ml.
  • the day of infection was termed day 0.
  • the outcome of infection was assessed microscopically by examination of Giemsa stained blood films.
  • the load of infection (the parasitemia) was calculated as the percentage of infected erythrocytes of the total number of erythrocytes.
  • Legionella pneumophila serogroup 1 and 3 Legionella micdadei (L. detroit, L. bari, L. F 1433). Eight Legionella pneumophila serogroups 1-7 and one strain of each of L. bozemanii, L. dumoffii, L. gormanii, L. micdadei, L. feelei, L. wadswort-hii, L. longbeacheae. Staphylococcus aureus ATCC 25923 was the control strain. The Legionella strains were subcultured on buffered charcoal yeast extract with alfa-ketoglutarate (B CYE- ⁇ ), and the rest of the strains were subcultured on 10% horse blood agar for 48 hours and 24 hours, respectively.
  • B CYE- ⁇ buffered charcoal yeast extract with alfa-ketoglutarate
  • Macrodilution rows were made with buffered yeast extract with alfa-ketoglutarate (BYE- ⁇ ) with 2 ml aliquots in vials, containing various concentrations of the test compound. Suspensions of Legionella species and the other pathogens and commensals were made in BYE- ⁇ . AU the chlution rows were inoculated to give a final concentration of 10 5 CFU/ml. After incubation at 37°C for 2 and 24 hours, respectively, ahquots of 10 ⁇ l were taken from aU dilution steps and plated onto BCYE-x agar plates (aU Legionella species) and to 10% horse blood agar (aU non-Legionella strains).
  • 63 strains of mycobacteria were used. The bacteria were grown in Dubos broth media before susceptibility testing. The test compound was dissolved in dimethyl sulfoxide (DMSO) and diluted in distiUed water to the desired concentration.
  • DMSO dimethyl sulfoxide
  • Susceptibility testing was performed radiometrically by using a BECTEC 460-TB apparatus in a confined atmosphere (5% CO2). Bacterial growth was measured as a function of the ability of the bacteria to catabohze 14 C-labeUed palmitic acid in the BECTEC 7H12B TB medium during growth, which resulted in the release of 14 C-labeUed CO2. The growth was expressed as a numerical value called the growth index (GI) which ranged from 1 to 999.
  • GI growth index
  • the 7H12 vials were inoculated with 0.1 ml of an appropriately chluted Dubos broth culture to give a final inoculum of about 5xl0 4 colony-forming units (CFU) per ml together with 0.1 ml of different concentrations of the test compound.
  • the final concentrations of the test compound ranged from 1.25 ⁇ g/ml to 80 ⁇ g/ml.
  • the final inoculum was determinated by culturing 0.1 ml from the control vial onto one Lowenstein-Jensen slant.
  • the vials were incubated under stationary conditions at 35°C and growth was monitored by dady GI determination for 7 days. At day 7, 0.1 ml from each vial with a GI reading ⁇ 30 was cultured onto one Lowenstein-Jensen slant. Colony counts were enumerated after incubation at 35°C for 3 weeks.
  • MIC Minimal inhibitory concentration
  • MMC Minimal bactericidal concentration
  • the experiment was carried out in coUaboration with Korn og Foderstof Kompagniet (KFK) at KFK's Experimental Station (Fors ⁇ gsgard, Sdr. Forumvej 18, DK-6715 Esbjerg, Denmark).
  • the test compound was mixed manually with chicken feed one week before use. 2.6 g of the test compound was mixed with 1 kg rye flour. The mixture was then mixed with ten kg chicken feed.
  • the oocysts were washed and resuspended in 30 ml saline to give a concentration of 15 x 10 6 /30 ml.
  • a volume of 0.1 ml (50,000 oocysts) was given to each chicken.
  • the experimental set-up consisted of 4 groups of 14- days old chickens. During the first 14 days of life, all the chickens received chicken feed_ containing no coccidiostatic agents. The first 3 groups were given 50,000 E. tenella oocysts per chicken by oral administration on day 14. Feeding the chickens with the feed preparations described above started one day before infection with the parasite (day 13). The treatment continued for 14 days according to the set-up shown in the table below.
  • Parasite load in the intestine or number per smear may be determined at the end of the experiment.
  • the number of oocysts in 10 viewfields may be counted at 100 x enlargement and the average of 10 fields were used.
  • Oocysts index may calculated as: Oocysts in infected animal/field x 100
  • the QSAR Model is based on a predetermined number of compounds selected by a statistical design and their corresponding biological activities (e.g. the IC ⁇ o values from an in vitro assay).
  • the low energy conformations of the compounds are predicted by using MacroModel 4.5 (F. Mohamadi, N.G.J. Richards, W.C. Guida, R. Liskamp, M. Lipton, C. Caufield, G. Chang, T. Hendrickson, W.C. StiU., MacroModel - An Integrated Software System for Modeling Organic and Bioorganic Molecules Using Molecular Mechanics. J. Comput. Chem. 1990, 11, 440-476.
  • the MacroModel software is avaUable from W.C. StiU, Department of Chemistry, Columbia University, New York, NY 10027, U.S.A.).
  • the interaction energies are calculated by GRID [version 15 (P.
  • GOLPE rejects variables having a total sum of squares (SS) lower than lO 7 .
  • the number of variables were further reduces by D-optimal preselection or region selection before applying variable selection.
  • Variables were selected according to their position in the weight space, using a D-optimal design criterion. The variables containing most information and having least correlation were selected. The number of variable were reduced by no more than 50% each time, untU the R 2 value starts to decrease.
  • Region selection A number of seeds (1920) were selected using a D-optimal design criterion in the weight space. Structural difference between different molecules in the series wdl be reflected in groups of variables, and therefore groups were generated around each seed in the 3D-space. Variables with a distance of no more than 1A to the seeds are included in the groups. If two neighbour groups (distance smaller than 2A) contained the same information, the groups were coUapsed. The groups were used in the variable selection procedure replacing the original variables. The effect of the groups on the predictivity were evaluated and groups instead of individual variables were removed from the data file.
  • the effect of the variables on the predictivity was evaluated using a Fractional Factor Design (FFD) procedure.
  • FFD Fractional Factor Design
  • a number of reduced models (twice the number of variables) were build removing some of the variables according to the FFD design.
  • the effect of dummy variables (20%) on the predictivity were calculated and only if a variable had a positive effect on the predictivity larger than the effect on the average dummy variable, the variable was included in the final model.
  • the models were vahdated using random groups. Molecules were assigned in a random way to 5 groups of equal size. Reduced models were build keeping out on group at a time. The formation of the groups was repeated 10 times.
  • NMR spectra were recorded on a Bruker AC-200F spectrometer. Splitting pattern are described as singlet (s), doublet (d), triplet (t), quartet (q) and broad (b). * indicates that signals with simUar shifts might be interchanged.
  • Mass spectra were recorded on a JEOL AX505W mass spectrometer. Melting points were determined on an Electrothermal melting point apparatus, and were not corrected. All moisture sensitive reactions were performed under nitrogen using oven-dried glassware. Solvents were dried before use: Tetrahydrofurane was freshly distUled from sodium/benzophenone, toluene was distUled and stored over sodium. HPLC grade dimethylformamide was dried and stored over 4 A molecular sieves.
  • Trifluoroacetic acid (770 ⁇ l, 10.0 mmol) was slowly added to a stirred suspension of Licochalcone A (0.507 g, 1.5 mmol) in dichloromethane (5 ml) and triethylsUane (240 ⁇ l, 1.5 mmol). Water was added after stirring for 2 h.
  • 2-methoxy-4,4'-dihydroxy-5-propylchalcone was synthesised according to procedure C using crude 2-methoxy-4-(2-tetrahydropyranyl)oxy-5-propylbenzaldehyde (1.26 g) and 4-(2- tetrahydropyranyl)oxyacetophenone (S. Sogawa et al. J. Med. Chem. 1993, 36, 3904-3909) (0.48 g, 2.2 mmol) as startmg materials: Orange-brown crystals (0.32 g, 41 % overaU yield based on 2,4-dihydroxy-5-propyl-benzaldehyde), mp: 126.5°C-128.0°C.
  • 2-methoxy-4,4'-dihydroxy-5-hexylchalcone was synthesised according to procedure C using crude 2-methoxy-4-(2-tetrahydropyranyl)oxy-5-hexylbenzaldehyde (0.61 g) and 4-(2- tetrahydropyranyl)oxyacetophenone (S. Sogawa et al. J. Med. Chem. 1993, 36, 3904-39Q9) (0.10 g, 0.45 mmol) as starting materials: An orange amorphous powder (0.19 g, 51 % overaU yield based on 2,4-dihydroxy-5-hexylbenzaldehyde).
  • 3-methyl-2,4-dihydroxybenzaldehyde was synthesised according to procedure D using 3-methyl- 2,4-dihydroxytoluene (1.48 g, 11.9 mmol) as starting material: White crystals (1.46 g, 80 %), mp: 149.6°C-150.3°C. ⁇ NMR (CDsOD) ⁇ 9.61 (s, OH, CHO), 7.30 (d J 8,6 Hz, H6), 6.47 (d J 8,6 Hz, H5), 4,94 (b, OH), 2.04 (s, CHs).
  • 2-methoxy-3-methyl-4,4'-dihydroxychalcone was synthesised according to procedure C using crude 2-methoxy-3-methyl-4-(2-tetrahydropyranyl)oxybenzaldehyde (0.69 g) and 4-(2- tetrahydropyranyl)oxyacetophenone (S. Sogawa et al. J. Med. Chem. 1993, 36, 3904-3909) (0.38 g, 1.7 mmol) as starting materials: Yellow crystals (0.38g, 54 % overall yield based on 3-methyl- 2,4-dihydroxybenzaldehyde), mp: 185.4°C-186.7°C.
  • 2,4-dihydroxy-6-methylbenzaldehyde was synthesised according to procedure D using 2,4- dihydroxy-6-methyltoluene (2.9 g, 20.8 mmol) as startmg material: Orange crystals (65 %), mp: 173.7°C-177.3°C. ⁇ NMR (CDsOD) ⁇ 10.2 (s, OH, CHO), 6.18 (d J 2 Hz, H5), 6.08 (d J 2 Hz, H3), 4.95 (s, OH) 2.47 (s , CHs).
  • 2-methoxy-4,4'-dihydroxy-6-methylchalcone was synthesised according to procedure C using crude 2-methoxy-4-(2-tetrahydropyranyl)oxy-6-methylbenzaldehyde (1.74 g) and 4-(2- tetrahydropyranyl)oxyacetophenone (S. Sogawa et al. J. Med. Chem. 1993, 36, 3904-3909) (0.80 g, 3.6 mmol) as startmg materials: YeUow crystals (0.73 g, 51 % overall yield based on 2,4- dihydroxy-6-methylbenzaldehyde), mp: 197.0°C-197.9°C.
  • 4,4'-dimethylaminochalcone was synthesised according to procedure E using 4- dimethylaminobenzaldehyde (0.12 g, 0.8 mmol) and 4-dimethylaminoacetophenone (H. Gilman et al. J. Am. Chem. Soc. 1933, 55, 1265-1270) (0.13 g, 0.8 mmol) as starting materials: Orange crystals (0.18 g, 76 %), mp: 149.8-150.7°C.
  • 4,4'-dinitro-chalcone was synthesised according to procedure F using 4-nitrobenzaldehyde (0.46 g, 3.0 mmol) and 4-nitroacetophenone (0.50 g, 3.0 mmol) as startmg materials: YeUow crystals (0.35 g, 40 %), mp: decompose.
  • 4-nitro-4'-dimethylaminochalcone was synthesised according to procedure E using 4- nitrobenzaldehyde (0.17 g, 1.1 mmol) and 4-dimethylaminoacetophenone (H. Gilman et al. J. Am. Chem. Soc. 1933, 55, 1265-1270) (0.20 g, 1.1 mmol) as startmg materials: Orange crystals (0.29 g, 80 %), mp: 198.5-199.5°C.
  • 3,5-dimethoxy-2'-butoxychalcone_ was synthesised according to procedure E using 3,5- dimethoxybenzaldehyde (2.0 g, 12.0 mmol) and 2'-butoxyacetophenone (E.R. Bockstacler et al. J. Am. Chem. Soc. 1949, 71, 3760-3766) (2.3 g, 12.0 mmol) as starting materials: YeUow crystals (3.8 g, 92 %), mp: 69.5-70.1°C.
  • 3,5-dimethoxy-2'-dimethylaminochalcone was synthesised according to procedure E using 3,5- dimethoxybenzaldehyde (1.5 g, 8.9 mmol) and 2'-dimethylaminoacetophenone (H. Rupe et al. Berichte, 1901, 34, 3523-3524) (1.45 g, 8.9 mmol) as starting materials: YeUow crystals (1.9 g, 66 %), mp: 159.8-160.5°C.
  • 3,5-dimethoxy-2'-nitrochalcone was synthesised according to procedure F using 3,5- dimethoxybenzaldehyde (1.0 g, 6.1 mmol) and 2'-nitroacetophenone (1.0 g, 6.1 mmol) as starting materials: YeUow crystals (0.45 g, 23 %), mp: 98.4-98.9°C.
  • 3,5-dimethoxy-2'-fluorochalcone was synthesised according to procedure E using 3,5- dimethoxybenzaldehyde (2.4 g, 14.4 mmol) and 2'-fluoroacetophenone (2.0 g, 14.4 mmol) as starting materials: YeUow crystals (2.9 g, 71 %), mp: 71.6-72.5°C.
  • 3,5-dimethoxy-3'-cLimethylaminochalcone was synthesised according to procedure E using 3,5- dimethoxybenzaldehyde (2.0 g, 12.3 mmol) and 3'-dimethylaminoacetophenone (H. Rupe et al. Berichte, 1901, 34, 3523-3524) (2.0 g, 12.3 mmol) as starting materials: YeUow crystals (2.6 g, 49 %), mp: 81.8-82.6°C.
  • 2,4-dimethoxy-3'-nitrochalcone was synthesised according to procedure E using 2,4- dimethoxybenzaldehyde (0.5 g, 3.0 mmol) and 3'-nitroacetophenone (0.5 g, 3.0 mmol) as starting materials: Grey crystals (0.61 g, 64 %), mp: 88.1-88.7°C.
  • 3,5-dimethoxy-3'-fluorochalcone was synthesised according to procedure E using 3,5- dimeth oxybenzaldehyde (2.4 g, 14.4 mmol) and 3'-fluoroacetophenone (1.78 ml, 14.4 mmol) as starting materials: White crystals (2.3 g, 56 %), mp: 89.5-90.3°C.
  • 3,5-dimethoxy-4'-cyclohexylchalcone was synthesised according to procedure E using 3,5- dimethoxybenzaldehyde (0.82 g, 4.9 mmol) and 4-cyclohexylacetophenone (1.0 g, 4.9 mmol) as starting materials: Brown crystals (1.6 g, 92 %), mp: 75.5-76.2°C.
  • 3,5-dimethoxy-4'-dimethylaminochalcone was synthesised according to procedure E using 3,5- dimethoxybenzaldehyde (0.67 g, 4.0 mmol) and 4-dimethylaminoacetophenone (H. Gilman et al. J. Am. Chem. Soc. 1933, 55, 1265-1270) (0.66 g, 4.0 mmol) as starting materials: Orange crystals (0.91 g, 73 %), mp: 155.1-156.2°C.
  • 2-nitro-2', 3', 4' -trimethoxychalcone was synthesised according to procedure F using 2- nitrobenzaldehyde (0.72 g, 4.8 mmol) and 2,3,4-trimethoxyacetophenone (0.8 ml, 4.8 mmol) as starting materials: Red-brown crystals (0.22 g, 13 %), mp: 107.1-108.0°C.
  • 3-phenoxy-2',3',4'-trimethoxychalcone was synthesised according to procedure E using 3- phenoxybenzaldehyde (0.82 ml, 4.8 mmol) and 2,3,4-trimethoxyacetophenone (0.87 ml, 4.8 mmol) as starting materials: YeUow crystals (1.1 g, 58 %), mp: 63.3-64.0°C.
  • 3-dimethylamino-2',3',4'-trimethoxychalcone was synthesised according to procedure E using 3- dimethylaminobenzaldehyde (W. Cocker et al. J. Chem. Soc. 1938, 751-753) (1.0 g, 6.9 mmol) and 2,3,4-trimethoxyacetophenone (1.3 ml, 6.9 mmol) as starting materials: Orange crystals (2.1 g, 90 %), mp: 87.2-87.9°C.
  • 3-fluoro-2', 3', 4' -trimethoxychalcone was synthesised according to procedure E using 3- fluorobenzaldehyde (1.7 ml, 16.1 mmol) and 2,3,4-trimethoxyacetophenone (2.9 ml, 16.1 mmol) as starting materials: YeUow crystals (1.7 g, 34 %), mp: 79.3-80.2°C.
  • 3,5-dimethoxy-2'-hydroxychalcone was synthesised according to procedure E using 3,5- dimethoxybenzaldehyde (0.47 g, 2.8 mmol) and 2-hydroxyacetophenone (0.34 ml, 2.8 mmol) as starting materials: Orange crystals (0.27 g, 34 %), mp: 110.2-110.9°C.
  • 3,5-dimethoxy-3'-hydroxychalcone was synthesised according to procedure E using 3,5- dimethoxybenzaldehyde (0.47 g, 2.8 mmol) and 3-hydroxyacetophenone (0.38 g, 2.8 mmol) as starting materials: YeUow crystals (0.25 g, 32 %), mp: 126.2- 126.8°C.
  • 3,5,2', 3',4'-pentamethoxychalcone was synthesised according to procedure E using 3,5- dimethoxybenzaldehyde (0.79g, 4.8 mmol) and 2,3,4-trimethoxyacetophenone (0.87 ml, 4.8 mmol) as starting materials: YeUow crystals (1.4 g, 83 %), mp: 111.6-112.5°C.
  • SBC-24m4'ac 2,4-dimethoxy-4'-aUyloxychalcone 3.
  • SBC-26m4'bc 2,6-chmethoxy-4'-butoxychalcone
  • Stock solutions were made at a concentration of 1 mg/ml by dissolving 1 mg of each compound in 100 ⁇ l DMSO (dimethylsulfoxide) foUowed by addition of 900 ⁇ l of heat-inactivated fetal claf serum (HFCS). These stock solutions were dUuted with HFCS to make solutions having the foUowing concentrations: 320 ⁇ g/ml, 160 ⁇ g/ml, 80 ⁇ g/ml, 40 ⁇ g/ml and 20 ⁇ g/ml.
  • DMSO dimethylsulfoxide
  • HFCS heat-inactivated fetal claf serum
  • DUution rows were made with 3 ml ahquots in vials containing the compounds in the foUowing final test concentrations: 16 ⁇ g/ml, 8 ⁇ g/ml, 4 ⁇ g/ml, 2 ⁇ g/ml and 1 ⁇ g/ml.
  • DMSO- HFCS containing an amount of DMSO equal to the amount of test compound in the solution with 320 ⁇ g/ml compound.
  • Bacteria 20 strains of Helicobacter pylori were used in an agar plate assay. The compounds were incorporated in the agar plates at different concentrations and then they were inoculated with different strains of Helicobacter pylori foUowed by incubation at microaerophilic atmosphere at 37°C in 72 hours. The number of colony-forming units (CFU) were counted after the incubation period. The MIC values were determined as the minimal concentration required to inhibit growth of the bacteria.
  • PH104 3,5-dimethoxy-4'-cyclohexylchalcone 10.
  • PH135-2A 3,5-dimethoxy-2'fluorochalcone
  • Stock solutions were made at a concentration of 1 mg/ml by dissolving 1 mg of each compound in 100 ⁇ l DMSO (dimethylsulfoxide) foUowed by addition of 900 ⁇ l of heat-inactivated fetal claf serum (HFCS). These stock solutions were diluted with HFCS to make solutions having the foUowing concentrations: 2560 ⁇ g/ml, 1280 ⁇ g/ml, 640 ⁇ g/ml, 320 ⁇ g/ml, 160 ⁇ g/ml and 80 ⁇ g/ml.
  • DMSO dimethylsulfoxide
  • HFCS heat-inactivated fetal claf serum
  • Dilution rows were made with 3 ml aliquots in vials containing the compounds urthe foUowing final test concentrations: 128 ⁇ g/ml, 64 ⁇ g/ml, 32 ⁇ g/ml, 12 ⁇ g/ml, 8 ⁇ g/ml and 4 ⁇ g/ml.
  • As control was used DMSO-HFCS containing an amount of DMSO equal to the amount of test compound in the solution with 320 ⁇ g/ml compound.
  • Bacteria 20 strains of Helicobacter pylori were used in an agar plate assay. The same method as described above under Experiment A was employed and MIC values were determined.
  • Licochalcone A at a concentration of 16 ⁇ g/ml inhibited all 20 strains of Helicobacter pylori.
  • SBC-35m4'nic at a concentration of 16 ⁇ g/ml inhibited 12 out of 20 strains of Helicobacter pylori.
  • SBC-2ni2',3',4'mc at a concentration of 16 ⁇ g/ml inhibited 4 out of 20 strains of Helicobacter pylori.
  • Licochalcone A 20 out of 20 strains gave a MIC value equal to or less than 16 ⁇ g/ml
  • H. pylori uses fumarate reductase to generate ATP when grown under anaerobic conditions. These conditions are somewhat simUar to those in the in vivo environment of the host. Under the in vitro screening conditions it is not possible to grow H. pylori under complete anaerobic conditions. The growth conditions used in the present screening studies have been with 5% oxygen. Therefore it is possible that the concentrations required to kiU the bacteria in the in vivo environment might be lower than those in vitro.
  • Example 4 Effect of oxygenated chalcones on the activity of Helicobacter pylori fumarate reductase
  • Helicobacter pylori (007 strain) were supplied by Dr. Lief P. Andersen, Legionella pneumophilia,Taflachia. micdadei , E. coli, Seerratia and Vibrio ch ⁇ lena were supplied by Dr. Alice Friis-M ⁇ Uer.
  • Extraction of (FRD) Helicobacter pylori fumerate reductase 1) Extraction of soluble FRD: Bacteria were harvested by centrifugation at 18,000 ⁇ for 10 min at 4 °C, and were washed 2 times in an isotonic phosphate saline buffer (50 mM sodium phosphate, pH 7.2, 90 mM NaCl, 5 mM Kcl). Bacteria were lysed in 5 mM Tris-HCl, pH 7.4, for 15 minutes, and centrifuged 18,000 g for 10 minutes.
  • Membrane associated proteins were solubihzed from the peUet in 150 mM KC1, pH 7.0, in ice bath for 60 min, and then the supernatant was collected after centrifugation (18,000 g for 10 minutes) for the measurement of FRD activity.
  • NADH-FRD activity was determined as the rate of NADH-oxidation upon addition of 1 mM fumarate in the KCl-solubihzed fraction or sonicated fraction.
  • the measurement was carried out in a Shimadzu UV-190 double-beam spectrophotometer. Protein concentrations were determined using a Bio- Rad protein assay (Bio-Rad Laboratories, 200 Alfred Nobel Dr. Hercules, CA 94547, USA).
  • NADH-fumarate reductase The acitivities of NADH-fumarate reductase, succinate dehydrogenase, NADH dehydrogenase of Helicobacter pylori were investigated.
  • the presence of NADH-fumarate reductase was demonstrated in the sonicate of H. pylori and fumarate could reduce H2O2 production from NADH indicating fumarate to be an endogenous substrate for accepting electrons from NADH.
  • 2- thenoyltrifluoroacetone at concentration of 0.2 mM almost completely inhibited the activity of NADH-fumarate reductase, whereas succinate, malonate and antimycin could not inhibit it.
  • Fumarate and malonate exhibited a concentration-dependent inhibitory effect on the activity of succinate dehydrogenase, whde antimycin could no inhibit it. These date indicate that the oxidation of succinate to fumarate could be reversed by increasing the concentration of fumarate, whereas the reduction of fumarate to succinate could not be reversed by increasing the concentration of succinate. These results indicate that the fumarate to succinate pathway is predominant in H. pylori and this bacterium might use fumarate as a terminal electron acceptor.
  • Extraction of soluble FRD Parasites were harvested by centrifugation at lOOOg for 10 min, and were washed 2 times in an isotonic phosphate saline buffer (50 mM sodium phosphate, pH 7.2, 90 mM NaCl, 5 mM KC1). 1 - 8 X 1010/ml parasites were lysed in 5 mM Tris-HCl, pH 7.4, for 15 minutes, and centrifuged 4,550 g for 10 minutes.
  • Membrane associated proteins were solubihzed from the peUet in 150 mM KC1, pH 7.0, in ice bath for 30 min, and then the supernatant was collected after centrifugation (4,550 g for 10 minutes) for the measurement of FRD activity.
  • NADH-FRD activity was determined as the rate of NADH-oxidation upon addition of 1 mM fumarate in the KCl-solubilized fraction.
  • KC1 solubihzed fraction of Leishmania major promastigotes was incubated with different concentrations of various oxygenated chalcones at 28°C and the remaining FRD activity was measured. Purification of FRD:
  • AU chromatography was performed on a FPLC system. Crude extract was concentrated on a MonoQ anion-exchange chromatography column and eluted in 1.5 M NaCl, 25 mM Tris-HCI pH 8.0. Elution was submitted to size exclusion chromatography on a Hi-Load superdex-200 column and eluted in 75 mM NaCl, 25 mM Tris-HCI pH 8.0. Fractions were tested for FR activity, and submitted for anion-exchange chromatography on a MonoQ column. The enzyme was eluted in a 0.075 M NaCl gradient, peaks were tested for FR activity, desalted on a PD-10 column and purity was assessed by SDS-PAGE on a 15% gel.
  • Hiload superdex 200 column separates proteins in range 104-6x105 Da ⁇
  • NCC NADH-cytochrome c reductase
  • FRD is a key enzyme in the energy metabohsm in a number of microorganisms, including Leishmania and malaria parasites, but absent in human cells. For this reason, FRD is a potentiaUy important target for antUeishmanial drugs.
  • the Danish Toxicology Center has been in charge of aU the toxicological tests initiated by the Statens Serum Institut. The testing has primarUy been carried out at Inveresk Toxicology, United Kingdom, or Scantox, Denmark.
  • SBC-35ma (3,5-dimethoxy-4'-aUyloxychalcone) as a development candidate
  • this compound was studied in vitro using the chromosomal aberration assay with CHO ceUs and the mouse lymphoma tk assay.
  • SBC-35ma showed a clear clastogenic response in the CHO ceUs both with and without metabohc activation (S9 mix).
  • the mouse lymphoma tk assay was positive with S9 mix and negative/inconclusive without S9 mix. Due to these positive responses, a number of other compounds were studied for their clastogenic potential in a screening design of the CHO cell chromosomal aberration assay.
  • SBC-2,4m4'ac 2,4-dimethoxy- 4' aUyloxychalcone
  • SBC-4'ac 4'-allyloxychalcone
  • SBC-2,3m4'hc 2,3-dimethoxy-4'-hydroxychalcone
  • SBC-2,5m4'hc 2,5-dimethoxy-4'hydroxychalcone
  • the selected compounds were tested for inhibition of the growth of two human ceU lines CEM and HL60.
  • the ceUs were grown in the presence of different concentrations of each compound for 48 hrs, after which ceU number was determined in a Coulter counter. Based on these results the IC50 values are determined.
  • Licochalcone A 1/15000 1/50000 1/600
  • Virus input was 100 plaque forming units (PFU).
  • PFU plaque forming units
  • the objective of this study was to investigate the effect of certain oxygenated chalcones on the induction, production and secretion of inflammatory cytokines in in vitro and in vivo models and on protection of mice from LPS-induced septic shock.
  • the following test systems have been employed:
  • TNF- ⁇ by human peripheral blood mononuclear cells stimulated in vitro by LPS or SPAG. Detection in culture supernatant (ELISA).
  • TNF- ⁇ Production of TNF- ⁇ by human peripheral blood mononuclear cells stimulated in vitro by LPS or SPAG. Detection of intracellular TNF- ⁇ (Flow cytometry).
  • TNF- ⁇ Production of TNF- ⁇ in a mouse model of LPS-induced septic shock. Detection in plasma (ELISA).
  • TNF- ⁇ by human peripheral blood mononuclear cells stimulated in vitro by LPS or SPAG. Detection in culture supernatant (ELISA).
  • CeUs Peripheral blood mononuclear ceUs obtained from healthy individuals. The ceUs were separated by a conventional method (lymphoprep) and suspended in RMPI 1640 containing 5% human serum.
  • Licochalcone A, SBC-35ma (3,5-dimethoxy-4'-aUyloxychalcone), SBC-24ma (2,4- dimethoxy-4'-aUyloxychalcone) and SBC-24m.bc (2,4-dimethoxy-4'-butoxychalcone) and several other compounds as shown in the results section were synthesised either by Clauson Kaas AS, Farum, Denmark according to GMP rules or by one of the inventors.
  • Exoantigens were affinity purified from culture medium of P. falciparum essentiaUy as described previously (Jepsen & Andersen, 1981; Jakobsen et al., 1988) using as hgand a pool of IgG from clinicaUy immune African adults. Before chromatography the culture medium was centrifuged at 7000 g for 10 min, fUtered through a 0.22 ⁇ m membrane and dialysed overnight at 4°C against column buffer. LPS was purchased from Sigma Chem. Co.
  • Non-attached sites were blocked for 1 h by 200 ⁇ l per well of 2% human serum slbumin in PBS (1 h at 37°C), and the wells were washed 4 times in 2.5% NaCl, 0.1% Tween 20 (Merck, Darmstadt, Germany). This washing procedure was done after each of the foUowing incubating steps:
  • Enzyme activities were quantitated after addition of 100 ⁇ l per weU of 0.67 mg/ml 1,2- phenyldiamine hydrochloride (DAKO) dissolved in 100 mM citric acid-phosphate buffer, pH 5.0 containing 0.015% (v/v) H2O2.
  • the reactions were stopped by adding 50 ⁇ l per weU of 2.5 M H2SO4, and the optical densities are measured in an ELISA scanner at 490 nm against a test reference at 620 nm.

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EP98929241A 1997-06-26 1998-06-26 Biologisch-wirksame 1,3-bis-aromatische-prop-2-en-1-on, 1,3-bis-aromatische-propan-1-on, and 1,3-bis-aromatische-prop-2-yn-1-on Withdrawn EP0996432A2 (de)

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DK76297 1997-06-26
DK76297 1997-06-26
PCT/DK1998/000283 WO1999000114A2 (en) 1997-06-26 1998-06-26 Biologically active 1,3-bis-aromatic-prop-2-en-1-ones, 1,3-bis-aromatic-propan-1-ones, and 1,3-bis-aromatic-prop-2-yn-1-ones

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