WO2010034512A1 - Compounds with antimalarial activity - Google Patents

Compounds with antimalarial activity Download PDF

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WO2010034512A1
WO2010034512A1 PCT/EP2009/006960 EP2009006960W WO2010034512A1 WO 2010034512 A1 WO2010034512 A1 WO 2010034512A1 EP 2009006960 W EP2009006960 W EP 2009006960W WO 2010034512 A1 WO2010034512 A1 WO 2010034512A1
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compound
nmr
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usnic acid
compounds
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Luisella Verotta
Diego Monti
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Universita' Degli Studi Di Milano
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
    • C07D409/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings
    • C07D409/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings linked by a chain containing hetero atoms as chain links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
    • A61P33/06—Antimalarials
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
    • C07D307/77—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D307/91—Dibenzofurans; Hydrogenated dibenzofurans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D493/00—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system
    • C07D493/12—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system in which the condensed system contains three hetero rings
    • C07D493/20—Spiro-condensed systems
    • 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

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  • the present invention relates to antimalarial compounds and pharmaceutical compositions thereof. More precisely, new usnic acid derivatives, conjugation products of usnic acid with biologically active molecules and pharmaceutical compositions thereof are described, which are useful for the prophylaxis and therapy of infections from protozoa of the Plasmodium genus.
  • Usnic acid 2,6-Diacetyl-7,9-dihydroxy-8,9b-dimethyldibenzofurane- l,3(2H,9bH)-dione, is a dibenzofurane derivative produced by several lichen species. Its anti-bacterial, anti-fungal, anti-viral, anti-inflammatory properties are known, as well as the consequent uses in the cosmetic and pharmaceutical field, particularly as active ingredient or preservative in creams, deodorants, tooth pastes, sunscreen products, or as dietary supplement. The use of usnic acid in combinatorial synthesis is described in S. Tomasi et al. (Solid-Phase Synthesis of Polyfunctionalized Natural Products: Application to Usnic Acid, a Bioactive Lichen Compound. J.
  • Object of the present invention are derivatives of usnic acid active against Plasmodium falciparum, particularly conjugation products between usnic acid and biologically active molecules such as antimalarial drugs, whose pharmacological action is potentiated through the protraction of the therapeutic effect or the improvement of the pharmacokinetic profile.
  • the products of the invention are endowed with a dual activity, being active both in the hepatic and in the erythrocytic phase of the parasite life-cycle.
  • n 2-10, preferably 2-5, more preferably 2-3;
  • R' and R" are selected independently from hydrogen and C]-C 3 alkyl, more preferably hydrogen or methyl, while R is selected from:
  • n is as above defined.
  • the compounds of the invention have shown a very good inhibitory activity of the growth of both chloroquine-sensitive or resistant strains of Plasmodium falciparum and Plasmodium yoelii in both hepatic and erythrocytic stages.
  • compositions containing at least one compound of the invention together with pharmaceutically acceptable excipients.
  • compositions may be administered through the oral or parenteral route, preferably in the form of solid preparations, such as tablets, capsules, powders, granulates, semisolid preparations such as creams, ointments, or liquid preparations such as solutions, syrups, drops, preferably in the form of injectable solutions.
  • the dose of the active principle will depend on the form and administartion route, on the solubility and on other pharmacokintetic parameters of the compound.
  • a dose between 0.1 and 1.000 mg, preferably between 0.1 and 500 mg of active ingredient for administration unit is generally acceptable.
  • the daily amount of active principle and the length of the treatment may vary depending upon the severity of the disease and the characteristics of the patient to be treated.
  • the compounds and compositions of the invention are used for the prophylactic and therapeutic treatment of infections caused by several parasites of the Plasmodium genus, such as P. yoelii, P. falciparum, P. berghei, P. vivax, P. ovale, P. malaria or P. knowlesi.
  • the compounds and compositions of the invention may also be used in combination with known antibiotic agents which show antimalarial activity in order to potentiate their action.
  • Antibiotic agents which can be combined with the compounds of the invention preferably include tetracycline, oxytetracycline, demeclocycline, erythromycin, azithromycin, ciprofloxacine, norfloxacine, doxycycline and tigecycline.
  • Example 10 Toxicity and pharmacological activity
  • the cell line HepG2/CD81 was used to evaluate the toxicity and antimalarial activity of the compounds in the hepatic stage of Plasmodium yoelii (265 BY).
  • Toxicity assay toxicity was determined using the colorimetric MTT (tetrazolium) assay (Mosmann, 1983). After incubation of HepG2/CD81 cells for 48 hours with different concentrations of the compounds 100 ⁇ l of MTT solutions were introduced in each well. This solution was prepared as follows: 5 mg of MTT in 1ml of water, IOX dilution in calf foetal-serum free culture medium. The final concentration of the solution was 0.5 mg/ml.
  • Antimalarial activity was evaluated on the hepatic stages of P. yoelii in HepG2-A16/hCD81-EGFP cells. Toxicity was evaluated with the MTT assay using HepG2-A16/hCD81-EGFP cells.
  • the artemisinin derivative of Example 6 showed an even better activity at nanomolar concentrations, comparable to that of artemisinin itself.
  • HepG2/CD81 cells were seeded in a 96-well plate (25.000 cells for well) in DMEM culture medium added with L-glutamine (2 mM), penicillin (50 ⁇ g/mL), neomycin (100 ⁇ g/mL), streptomycin (50 ⁇ g/mL) and calf foetal serum (10%). After 24 hours, cells were infected with P. yoelii sporozoites (20.000 sporozoites/well) in 50 ⁇ l of the same culture medium. At the same time the test compounds were added at the final concentrations in an equivalent volume.
  • the plates were then centrifuged for 5 minutes at 2.000 rpm in order to accelerate the sedimentation of the sporozoites, then they were incubated at 30 0 C.
  • the culture media with the test compounds were changed 3 and 24 hours after the infection. After a 48-hour contact with the test compounds, cultures were fixed with cold methanol. Cultures infected with Plasmodium were marked with a primary antibody directed against Heat Shock Protein 70 (HSP70) of the parasite (30 min.
  • HSP70 Heat Shock Protein 70

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  • Health & Medical Sciences (AREA)
  • Tropical Medicine & Parasitology (AREA)
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  • Chemical Kinetics & Catalysis (AREA)
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Abstract

New usnic acid derivatives, conjugation products of usnic acid with biologically active molecules and pharmaceutical compositions thereof are described, which are useful for the prophylaxis and therapy of infections from protozoa of the Plasmodium genus.

Description

COMPOUNDS WITH ANTIMALARIAL ACTIVITY
The present invention relates to antimalarial compounds and pharmaceutical compositions thereof. More precisely, new usnic acid derivatives, conjugation products of usnic acid with biologically active molecules and pharmaceutical compositions thereof are described, which are useful for the prophylaxis and therapy of infections from protozoa of the Plasmodium genus.
Technical background
Usnic acid, 2,6-Diacetyl-7,9-dihydroxy-8,9b-dimethyldibenzofurane- l,3(2H,9bH)-dione, is a dibenzofurane derivative produced by several lichen species. Its anti-bacterial, anti-fungal, anti-viral, anti-inflammatory properties are known, as well as the consequent uses in the cosmetic and pharmaceutical field, particularly as active ingredient or preservative in creams, deodorants, tooth pastes, sunscreen products, or as dietary supplement. The use of usnic acid in combinatorial synthesis is described in S. Tomasi et al. (Solid-Phase Synthesis of Polyfunctionalized Natural Products: Application to Usnic Acid, a Bioactive Lichen Compound. J. Comb. Chem. 2006, 8, 11-14). Other derivatives of usnic acid are described in GB 800,114. Usnic acid is not active against Plasmodium falciparum, as reported in the publication "In vitro antimalarial activity of hyperforin, a prenylated acylphloroglucinol. A structure activity study" (L. Verotta, G. Appendino, E. Bombardelli, R. Brun.
Bioorg & Med Chem Lett 2007, 17(6), 1544-1548. doi: 10.1016/j.bmcl.2006.12.100.
Description of the invention
Object of the present invention are derivatives of usnic acid active against Plasmodium falciparum, particularly conjugation products between usnic acid and biologically active molecules such as antimalarial drugs, whose pharmacological action is potentiated through the protraction of the therapeutic effect or the improvement of the pharmacokinetic profile. Of particular relevance, the products of the invention are endowed with a dual activity, being active both in the hepatic and in the erythrocytic phase of the parasite life-cycle.
The compounds of the invention are characterized by the following general formula (I):
Figure imgf000003_0001
(I) wherein represents a single or double bond and R is a residue selected from:
Figure imgf000004_0001
Figure imgf000004_0002
\ (CH2)n— SO3H \ (CH2)n— S-S (CH2)^1-CH3 >— (CH2)n-
NH
wherein n=2-10, preferably 2-5, more preferably 2-3;
R' and R", independently from one another, represent H, linear or branched, saturated or unsaturated C1-Cs alkyl, in particular prenyl -CH2-CH=C(CHs)2 or geranyl -CH2-CH=C(CH3)(CH2)2CH=C(CH3)2:
Figure imgf000004_0003
prenyl
Figure imgf000004_0004
geranyl their enantiomers and pharmaceutically acceptable salts.
In a preferred embodiment of the invention, R' and R" are selected independently from hydrogen and C]-C3 alkyl, more preferably hydrogen or methyl, while R is selected from:
Figure imgf000005_0001
wherein n is as above defined. The compounds of the invention have shown a very good inhibitory activity of the growth of both chloroquine-sensitive or resistant strains of Plasmodium falciparum and Plasmodium yoelii in both hepatic and erythrocytic stages.
Therefore, another object of the present invention are pharmaceutical compositions containing at least one compound of the invention together with pharmaceutically acceptable excipients.
Compositions may be administered through the oral or parenteral route, preferably in the form of solid preparations, such as tablets, capsules, powders, granulates, semisolid preparations such as creams, ointments, or liquid preparations such as solutions, syrups, drops, preferably in the form of injectable solutions. The dose of the active principle will depend on the form and administartion route, on the solubility and on other pharmacokintetic parameters of the compound. A dose between 0.1 and 1.000 mg, preferably between 0.1 and 500 mg of active ingredient for administration unit is generally acceptable. The daily amount of active principle and the length of the treatment may vary depending upon the severity of the disease and the characteristics of the patient to be treated.
The compounds and compositions of the invention are used for the prophylactic and therapeutic treatment of infections caused by several parasites of the Plasmodium genus, such as P. yoelii, P. falciparum, P. berghei, P. vivax, P. ovale, P. malaria or P. knowlesi.
The compounds and compositions of the invention may also be used in combination with known antibiotic agents which show antimalarial activity in order to potentiate their action. Antibiotic agents which can be combined with the compounds of the invention preferably include tetracycline, oxytetracycline, demeclocycline, erythromycin, azithromycin, ciprofloxacine, norfloxacine, doxycycline and tigecycline.
Description of the Figure
Dose-response curve of the compound of Example 1 , PS4, relative to the infection of exo-erythrocyte forms (EEF) of Plasmodium yoelii in cultures of murine primary hepatocytes. Activity is expressed as the inhibiting concentration 50%.
EXAMPLES
General procedure for the preparation of the compounds of formula (I) Commercially available D-(+)-usnic acid (1 mol) was refluxed in EtOH or EtOH-THF with the appropriate amine (or aminoacid) (1 eq/mol) until disappearance of the starting material. The reaction mixture was taken to dryness and the crude material crystallized, to give the appropriate derivative. Example 1. Preparation of the compound of formula (I) wherein R'=R"= H and R
Figure imgf000007_0001
(PS4)
0.402 g (1.81 mmol) of 7-chloro-4-(aminoethyl)amino-quinoline were added to a suspension of D-usnic acid (UA) (0.624 g, 1.81 mmol) in a mixture of absolute alcohol-THF 1 : 1(16 ml) and heated at 500C under N2 for 5 h. The mixture was stirred at room temperature for other 12h. The solid which was obtained by concentration in vacuo was crystallized from diisopropyl ether-EtOH 9: 1 giving 0.892 g of a white solid. (90% yield), mp: 214-216°C.
1H NMR 400 MHz (CDCl3): δ 1.64 (3H, s, CH3-13), 2.08 (3H, s, CH3-16), 2.59 (3H, s, CH3-15), 2.64 (3H, s, CH3-18), 3.74 (2H, m, H-12'), 3.84 (2H, m, H-Il'), 5.72 (IH, s, H-4), 6.14 (IH, brs), 6.43 (IH, d, J=5.4 Hz H-3'), 7.32 (lH,dd, J= 2, 8.9 Hz, H-6'), 7.87 (IH, d, J=8.9 Hz, H-5'), 7.94 (IH, d, J =2 Hz, H-8'), 8.54 (IH, d, J =5.4 Hz, H-2'), 11.68 (IH, brs), 13.24 (IH, brs), 13.62 (IH, brs).
13C NMR 400 MHz (CDCl3): δ 7.41 (C-16), 18.38 (C-15), 30.82 (C-18), 32.21 (C-13), 42.39 (t), 56.92 (C-12), 99.02 (C-4), 101.25 (C-7), 102.39 (C-119, 104.98 (C-2), 108.25 (C-9), 117.42 (s), 121.82 (d), 126.24 (d), 128.57 (d), 135.57 (s), 148.79 (s), 149.82 (s), 151.64 (d), 155.52 (C-6), 158.12 (C-IO), 163.56 (C-8), 174.19 (s), 175.49 (C-5), 190.52 (C-3), 198.30 (C-I), 200.89 (2C, C-14+17).
C29H26ClN3O6CaIc. C 63.56, H 4.78, N 7.67; Found C 63.55, H 4.79, N 7.68. Example 2. Preparation of the compound of formula (I) wherein R'=R"= H and R
Figure imgf000008_0001
(PS5)
0.079 g (0.60 mmol) of L-asparagine were added to a suspension of D-usnic acid (UA) (0.207 g, 0.60 mmol) in absolute ethanol (15 ml) and refluxed under N2 for 4 h. The mixture was stirred at room temperature for further 15 h. The solid which was obtained by concentration in vacuo was crystallized from diisopropyl ether-EtOH 9: 1 giving 0.217 g (79% yield) of a yellow solid, mp: 221-223°C.
1H NMR 400 MHz (DMSO-d6): δ 1.65 (3H, S9CH3-13), 1.96 (3H, s, CH3-Io), 2.60 (3H, s, CH3-15) 2.62 (3H, s, CH3- 18), 2.73 (2H, m, H-3'), 4.96 (IH, m, H-2'), 5.85 (IH, s, H-4), 12.21 (IH, s), 13.28 (2H, s). 13C NMR 400 MHz (DMSO-d6): δ 7.93 (C-16), 18.85 (C-15), 31.42 (C-18), 32.18 (C-13), 37.39, 53.39, 56.88 (C-12), 101.24 (C-7), 102.33 (C-Il), 102.85 (C-4), 105.66 (C-2), 107.11 (C-9), 156.27 (C-3), 158.08 (C-IO), 163.08 (C-8), 171.04, 172.1, 173.45, 175.14 (C-5), 189.13 (C-3), 198.06 (C-I), 201.20 (2C, C14+17). C22H22N2O9 CaIc. C 57.64, H 4.84, N 6.11; Found C 57.65, H 4.83, N 6.1. Example 3. Preparation of the compound of formula (I) wherein R'=R"= H and R
Figure imgf000009_0001
(PS8)
0.157 g (0.87 mmol) of L-tyrosine were added to a suspension of D-usnic acid (UA) (0.300 g, 0.87 mmol) in absolute ethanol (10 ml) and 3 ml of water and refluxed under N2 for 6 h. The mixture was stirred at room temperature for further 15 h. The solid obtained by concentration in vacuo was crystallized from diisopropyl ether-EtOH 9:1 giving 0.406 g of a yellow solid. (92%). mp: 183-185°C.
1H NMR 400 MHz (DMSO-d6): δ 1.63 (3H, s, CH3- 13), 1.97 (3H, s,
CH3-Io), 2.36 (3H, s, CH3-15), 2.62 (3H, s, CH3-I8), 2.98 (IH, m, H-3'A),
3.04 (IH, m, H3'B), 4.93 (IH, m, H-2'), 5.85 (IH, s, H-4), 6.64 (2H, d, J= 8.4 Hz, Ar 5'+9'), 6.96 (2H, d, J=8.4 Hz, Ar 6'+8', d, 2H), 12.11 (IH, s), 13.28
(IH, brs), 13.38 (IH, s).
13C NMR 400 MHz (DMSO-d6): δ 7.79 (C-16), 19.47 (C-15), 31.67
(C- 18), 32.70 (C-13), 39.19, 57.10 (C- 12), 58.41 (s), 101.24 (C-7), 101.70
(C-I l), 103.05 (C-4), 105.65 (C-2), 107.21 (C-9), 116.03 (d), 126.16 (s), 131.35 (d), 157.31 (C-6), 158.60 (s), 163.02 (C-10), 171.58 (s), 173.40 (s),
174.96 (C-5), 189.23 (C-3), 198.06 (C-I), 201.43 (2C, C-14+17).
C27H25NO9 CaIc. C 63.90, H 4.97, N 2.76; Found C 63.91, H 4.98, N 2.75.
Example 4. Preparation of the compound of formula (I) wherein R'=R"= H and R
Figure imgf000010_0001
(PS9) 0.139 g (0.87 mmol) of tryptamine were added to a suspension of
D-usnic acid (UA) (0.300 g, 0.87 mmol) in absolute ethanol (10 ml) and refluxed under N2 for 5 h. The mixture was stirred at room temperature for further 16 h. The solid obtained by concentration in vacuo was crystallized from diisopropyl ether-EtOH 9: 1 giving 0.393 g (93%) of a yellow solid, mp: 218-2200C.
1H NMR 400 MHz (CDCl3): δ 1.69 (3H, s, CH3-B), 2.12 (3H, s,
CH3-16), 2.55 (3H, s, CH3-15), 2.69 (3H, s, CH3-18), 3.22 (2H, m, H-8'), 3.82
(2H, m, H-9'), 5.78 (IH, s, H-4), 7.17 (3H, m, H-3'+6'+5'), 7.40 (IH, d, J=8.0
Hz, Ar-7'), 7.59 (IH, d, J=7.8 Hz, Ar-4'), 8.38 (IH, brs), 11.98 (IH, brs), 13.35 (2H, s).
13C NMR 400 MHz (CDCl3): δ 7.45 (C- 16), 18.50 (C-15), 22.87,
25.19, 31.10 (C-18), 32.12 (C-13), 44.46, 68.51, 101.26 (C-7), 105.11 (C-Il),
107.94 (C-2), 110.99 (C-9), 111.60, 118.48, 119.76, 122.42, 122.99, 126.70,
136.47, 155.74 (C-6), 158.57 (C-10), 163.71 (C-8), 175.01 (C-5), 189.92, 198.15(C-I), 200.72 (C-14+17).
C28H26N2O6 CaIc. C 69.12, H 5.39, N 5.76; Found C 69.1 1, H 5.38, N 5.77. Example 5. Preparation of the compound of formula (I) wherein R'=R"= H and R
Figure imgf000011_0001
(PSlO)
0.200 g (1.45 mmol) of benzoylhydrazide were added to a suspension of D-usnic acid (UA) (0.500 g, 1.45 mmol) in absolute ethanol (15 ml) and refluxed under N2 for 5 h. The mixture was stirred at room temperature for further 15 h. The solid obtained after concentration in vacuo was crystallized from diisopropyl ether-EtOH 9: 1 giving 0.590 g (88%) of a yellow solid, mp: 228-230°C.
1H NMR 400 MHz (DMSO-d6): δ 1.69 (3H, s, CH3- 13), 1.97 (3H, s, CH3-Io), 2.64 (3H, s, CH3-15), 2.70 (3H, s, CH3- 18), 5.95 (IH, s, H-4), 7.57 (2H, m, Ar 3'+7'), 7.67 (IH, m, Ar 5'), 7.94 (2H, m, Ar 4'+6'), 11.68 (2H, brs), 12.02 (IH, s), 13.38 (IH, s), 14.77 (IH, brs).
13C NMR 400 MHz (DMSO-d6): δ 8.04 (C-16), 17.91 (C-15), 30.89
(C18), 32.18 (C-13), 56.84 (C-12), 101.23 (C-7), 101-25 (C-I l), 102.79,
105.38 (C-2), 106.94 (C-9), 128.23, 129.27, 131.60, 133.16, 156.26 (C-6),
158.08 (C-IO), 163.02 (C-8), 165.35, 172.10, 173.92, 189.49 (C-3), 198.57 (C-l+14). 201.70 (C17).
C25H22N2O7 CaIc. C 64.93, H 4.80, N 6.06; Found C 64.92, H 4.81, N 6.07. Example 6. Preparation of the compound of formula (I) (artemisinin derivative) wherein R'=R"= H and R
Figure imgf000012_0001
Synthetic scheme:
Figure imgf000012_0002
Figure imgf000012_0003
To a solution of D-usnic acid (0.400 g, 1 eq) in EtOH (10 ml) at room temperature, 4-aminobutyric acid (0.120 g, 1 eq) was added. After stirring at 75°C for 4 h and at 25°C for 14 hours, the mixture was concentrated under reduced pressure. Purification by crystallization with ether/ethanol gave the desired compound (0.371 g, 78%) as a yellow solid (A).
1H NMR 400 MHz (DMSO-d6): δ 1.63 (3H, s), 1.57 (2H, m), 1.98 (3H, s), 2.44 (2H, m) 2.62 (6H, s), 3.58 (2H, m), 5.80 (IH, s), 12.01 (IH, s), 13.19 (2H, s); 13C NMR 400 MHz (DMSO-d6): δ 7.42 (CH3), 18.31(CH3), 20.09 (CH3), 21.19 (CH3), 23.16 (CH2), 34.25 (CH2), 42.76 (CH2), 56.05 (CH), 101.24 (C), 102.79 (C), 105.29 (C),128.22 (C), 129.27 (C),156.27 (C), 158.08 (C), 161.80 (C), 169.85 (C), 172.15 (C), 173.98 (C), 175.14 (C), 189.45 (C), 198.53 (C), 201.67 (C). To a solution of compound A obtained in the previous step (0.25Og,
1 eq) and dihydroartemisinin (DHA) (0.173 g, 1 eq.) in THF (10 ml) at room temperature, 2-chloro-4,6-dimethoxy-l,3,5-triazine (0.106 g, 1 eq.) and 1-methyl-morpholine (3 eq.) were added. After stirring at 25°C for 24 h, the mixture was concentrated under reduced pressure. Then the solution was diluted with 18 ml of dichloromethane and washed once with a solution of 0.5 N HCl and twice with water. Purification by column cromatography (hexane/ethyl acetate 2: 1) gave the desired compound (0.203 g, 48%) as a pale yellow solid, mp: 243-245°C.
1H NMR 400 MHz (CDCl3): δ 0.98 (3H, d, J=6.7 Hz, CH3- 19'), 1.04 (IH, m, H-I l '), 1.23 (3H, d, J=6.9, Hz CH3-17'), 1.39 (2H, m, H-12'), 1.42 (IH, m, H-10'), 1.47 (3H, s, CH3-18'), 1.49 (IH, m, H-16'), 1.62 (IH, m, H-14'), 1.65 (2H, m, H-13'), 1.69 (IH, m, H-16'), 1.71 (3H, s, CH3-13), 1.74 (2H, m, H-8'), 1.96 (IH, m, H-9'), 2.07 (2H, m, H-3'), 2.11 (3H, s, CH3-16, s), 2.36 (IH, m, H-9'), 2.62 (2H, m, H-2'), 2.64 (3H, s, CH3-15), 2.68 (3H, s, CH3-18, s), 3.57 (2H, m, H-4'), 5.46 (IH, s, H-6'), 5.80 (2H, m, H-4+H-5'), 11.81 (IH, s), 13.28 (2H, s).
13C NMR 400 MHz (CDCl3): δ 7.47 (C-16), 12.14 (q), 18.29 (C-15), 20.18 (q), 22.01 (t), 22.87 (q), 24.15 (t), 24.58 (t), 25.93 (q), 30.93 (t), 31.15 (C-18), 31.25 (C-15), 31.99 (t), 34.08 (t), 36.06 (q), 37.30 (d), 42.85 (t), 45.22 (d), 51.56 (d), 80.08 (s), 91.54 (d), 92.33 (d), 101.39 (C-7), 104.52 (C-I l), 105.12 (C-2), 108.02 (C-9), 155.91 (C-6), 158.33 (C-10), 163.19 (C-8), 169.97, 171.13 (C-5), 198.34 (C-I), 200.66 (2C, C-14+17).
C37H45NO12 CaIc. C 63.87, H 6.52, N 2.01, O 27.59 Found C 63.89, H 6.53, N 2.00, 0 27.58.
Example 7. Preparation of the compound of formula (I) (taurine derivative) wherein R'=R"= H and R
Figure imgf000014_0001
To a solution of D-usnic acid (0.400 g, 1 eq) in EtOH (10 ml) and water (6 ml) at room temperature, taurine (0.145 g, 1 eq) was added. After stirring at
75°C for 4 h and at 25°C for 14 hours, the mixture was concentrated under reduced pressure. Purification by crystallization from ether/ethanol gave the desired compound (0.420 g, 86%) as a yellow solid, mp: 235-236°C.
1H NMR 400 MHz (DMSO-d6): δ 1.63 (3H, s, CH3- 13), 1.98 (3H, s, CH3- 16), 2.62 (6H, s, CH3- 15+18), 3.53 (4H, m, CH2 l '+2'), 5.80 (IH, s, C-4), 12.01 (IH, s), 13.19 (2H, s).
13C NMR 400 MHz (DMSO-d6): δ 8.42 (CH3), 17.21(CH3), 20.09 (CH3), 21.18 (CH3), 34.85 (CH2), 42.66 (CH2), 56.03 (CH), 101.24, 102.89, 105.13, 128.22, 156.27, 158.08, 161.79, 169.85, 172.15, 173.98, 198.53, 201.67.
C20H21NO9S CaIc. C 53.21, H 4.69, N 3.10, O 31.90 Found C 53.20, H 4.70, N 3.12, O 31.91.
Example 8. Preparation of the compound of formula (I) (agmatine derivative) wherein R'=R"= H and R
Figure imgf000014_0002
265 mg (1.16 mmol) of agmatine sulfate were added to a suspension of D-usnic acid (UA) (1.16 mmol) in EtOH (15 ml)/ water 5 ml/ TEA 3 ml. The mixture was refluxed under N2 for 5 h then stirred at room temperature for further 15h. The product obtained by concentration in vacuo was washed twice with a 5% NaHCO3 solution (20 ml). The solid was crystallized from diisopropyl ether-EtOH 9: 1 giving a yellow solid. Yields 82%. mp:
231-233°C. 1H NMR 400 MHz (DMSO-d6): δ 1.55 (2H, m, H-4'),1.57 (3H, s,
H- 13), 1.76 (2H, m, H-3 '), 1.91 (3H, s, H-16), 2.55 (3H, s, H-15), 2.56 (3H, s,
H- 18), 3.04 (2H, m), 3.29 (2H, m, H-4'), 5.67 (IH, s, H-4), 7.52 (3H, brs),
8.96 (IH, brs), 12.42 (IH, brs), 13.24 (2H, s).
13C NMR 400 MHz (DMSO-d6): δ 7.42, 18.31 , 20.09, 21.19, 25.34, 31.46, 41.06, 41.84, 56.05, 76.32, 101.24, 102.79, 105.29, 128.22, 129.27,
121.61 , 156.27, 158.08, 161.80, 169.85, 172.10, 173.92, 189.49, 198.58,
201.69.
C23H28N4O6 CaIc. C 60.52, H 6.18, N 12.27 O 21.03; Found C 60.51 , H
6.19, N 12.26, 0 21.04. Example 9. Preparation of the compound of formula (I) (6-chloro-9-
[(2-aminoethyl)amino]-2-metoxy-acridine derivative) wherein R'=R"= H and R
Figure imgf000015_0001
To a solution of 6,9-dichloro-2-methoxy-acridine (2 g) in 24 ml of ethylenediamine and 3 ml of 4-methyl-morpholine were added. The mixture was stirred for 8 hours at reflux and for 12 hours at room temperature. Then the solution was diluted with 28 ml of water at 00C and an orange precipitate formed. After filtration, the orange solid was washed with 30 ml of water (1.9 g) 88%. 1.61 mmol of 6-chloro-9-[(2-aminoethyl)amino]-2-methoxy-acridine obtained in the previous step were added to a suspension of D-usnic acid (UA) (1.61 mmol) in a mixture of absolute ethanol-THF 1 : 1(16 ml) and heated at 65°C under N2 for 5 h, then the mixture was stirred at room temperature for further 12 h. The solid obtained by concentration in vacuo was crystallized from diisopropyl ether-EtOH 9:1 giving a brown solid. Yield 87%. mp: 187-189°C.
1H NMR 400 MHz (CDCl3): δ 1.67 (3H, s, H-13), 2.07 (3H s, CH3-Io), 2.65 (6H, s, CH3- 15+18), 3.73-3.90 (4H, m, CH2 15'+16'), 3.94 (3H, s, CH3- 17'), 5.78 (IH3 s, H-4), 7.25-7.44 (3H, m, Ar 2'+3'+5'), 7.87-8.15 (3H, m, Ar 7'+8'+10\ m, 3H) 11.65 (IH, brs), 11.99 (IH, s), 13.35 (IH, brs), 13.90 (IH, brs).
13C NMR 400 MHz (CDCl3): δ 7.48, 18.21, 31.21, 31.91, 42.57, 42.83, 56.20, 97.94, 100.35, 101.56, 102.77, 104.94, 108.07, 119.17, 123.99, 125.44, 126.41, 129.06, 131.95, 135.93, 146.19, 148.60, 156.08, 158.01, 163.80, 165.49, 174.17, 175.18, 187.68, 199.02, 200.71. C34H30ClN3O7 CaIc. C 65.02, H 4.81, N 6.69; Found C 65.01, H 4.82, N
6.70.
Example 10. Toxicity and pharmacological activity The cell line HepG2/CD81 was used to evaluate the toxicity and antimalarial activity of the compounds in the hepatic stage of Plasmodium yoelii (265 BY).
Toxicity assay: toxicity was determined using the colorimetric MTT (tetrazolium) assay (Mosmann, 1983). After incubation of HepG2/CD81 cells for 48 hours with different concentrations of the compounds 100 μl of MTT solutions were introduced in each well. This solution was prepared as follows: 5 mg of MTT in 1ml of water, IOX dilution in calf foetal-serum free culture medium. The final concentration of the solution was 0.5 mg/ml.
The plates were incubated for 4 hours at 37°C. The supernatant was discharged and 100 μl of DMSO-ethanol (1 : 1 V/V) were added to each well, mixing thoroughly in order to dissolve the dark-blue crystals. After a few minutes at room temperature to allow dissolution of all of the crystals the plate was read in an ELISA-plate reader (absorbance wave-length 590 nm, reference wave-length 630 nm). The results were expressed as μM based on an experimental calibration curve of the activity of the reference compound primaquine and are reported in Table. Table
Figure imgf000017_0001
Activity (inhibiting concentration 50%) and toxicity (toxic concentration 50%) of usnic acid derivatives. Antimalarial activity was evaluated on the hepatic stages of P. yoelii in HepG2-A16/hCD81-EGFP cells. Toxicity was evaluated with the MTT assay using HepG2-A16/hCD81-EGFP cells.
All the tested compounds showed an IC50 of less than 15 μM in this assay. In particular, the chloroquine derivative of (+)-usnic acid of Example 1, PS4, showed a considerable inhibitory activity on sporozoites cultures, with an IC50 of 0.13 μM.
Activity in the erythrocytic stage
The compound of Example 1 also showed activity in the erythrocytic stage of the sensitive DlO (IC50 = 257 nM; IC50 of control chloroquine = 40 nM) or resistant W2 (IC50= 341 nM; IC50 control chloroquine - 1127 nM) strains of Plasmodium falciparum.
In the erytrocytic stage of the above sensitive or resistant strains of Plasmodium falciparum, the artemisinin derivative of Example 6 showed an even better activity at nanomolar concentrations, comparable to that of artemisinin itself.
Infection assay: HepG2/CD81 cells were seeded in a 96-well plate (25.000 cells for well) in DMEM culture medium added with L-glutamine (2 mM), penicillin (50 μg/mL), neomycin (100 μg/mL), streptomycin (50 μg/mL) and calf foetal serum (10%). After 24 hours, cells were infected with P. yoelii sporozoites (20.000 sporozoites/well) in 50 μl of the same culture medium. At the same time the test compounds were added at the final concentrations in an equivalent volume. The plates were then centrifuged for 5 minutes at 2.000 rpm in order to accelerate the sedimentation of the sporozoites, then they were incubated at 300C. The culture media with the test compounds were changed 3 and 24 hours after the infection. After a 48-hour contact with the test compounds, cultures were fixed with cold methanol. Cultures infected with Plasmodium were marked with a primary antibody directed against Heat Shock Protein 70 (HSP70) of the parasite (30 min. at 37°C) and revealed with a secondary murine anti-IgG antibody conjugated to the AlexaFluor680 chromophore (λex =680 nm; λem =700 nm) (30 min at room temperature, 1 μg/mL, sheltering from light). Cultures were then maintained in PBS at 4°C. The number of the parasites was determined using Odyssey Infrared Imaging System (LI-COR Biosciences), with excitation at 680 nm, detection at a 700 nm and resolution 21 μm The infrared fluorescence images generated by the scanner were then analyzed using Image J software in order to automatically determine the number of fluorescent spots, where each spot was corresponding to a parasite (Gego et al,. AAC, 2006). The results are shown in the Figure.

Claims

1. A compound of general formula (I):
Figure imgf000019_0001
(I) wherein represents a single or double bond and R is a residue selected from:
Figure imgf000019_0002
Figure imgf000019_0003
NH, f (CH2Jn-SO3H I (CH2)n— S— S (CH2)^-CH3 >— (CH.)n-
NH wherein n=2-10;
R' and R", independently from one another, are H, straight or branched, saturated or unsaturated (C1-C5)BIlCyI; enantiomers and pharmaceutically acceptable salts thereof.
2. A compound as claimed in claim 1, in which R' and R" are independently selected from hydrogen, (C1-C3) alkyl, prenyl, or geranyl
Figure imgf000020_0001
prenyl
Figure imgf000020_0002
geranyl
3. A compound as claimed in claim 1 and 2 wherein R' and R" are independently selected from hydrogen and methyl, whereas the R group is selected from:
Figure imgf000020_0003
wherein n is as defined above.
4. A pharmaceutical composition containing a compound of claim 1 together with pharmaceutically compatible excipients.
5. A compound as claimed in claims 1-3 or a pharmaceutical composition as claimed in claim 4, for use in the preventive or therapeutic treatment of infections caused by Plasmodium.
6. A compound or a pharmaceutical composition according to claim 5, wherein the Plasmodium is selected from P. yoelii, P. falciparum, P. berghei, P. vivax, P. ovale, P. malaria o P. knowlesi.
7. The use of a compound as claimed in claim 1, for the preparation of an antimalarial medicament.
8. The use according to claim 7, wherein said antimalarial medicament additionally contains an antibiotic agent selected from tetracycline, oxytetracycline, demeclocycline, erythromycin, azithromycin, ciprofloxacine, norfloxacine, doxycycline and tigecycline.
PCT/EP2009/006960 2008-09-29 2009-09-28 Compounds with antimalarial activity WO2010034512A1 (en)

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VEROTTA ET AL: "In vitro antimalarial activity of hyperforin, a prenylated acylphloroglucinol. A structure-activity study", BIOORGANIC & MEDICINAL CHEMISTRY LETTERS, PERGAMON, ELSEVIER SCIENCE, GB, vol. 17, no. 6, 20 February 2007 (2007-02-20), pages 1544 - 1548, XP005895360, ISSN: 0960-894X *
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