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Fast in vitro methods to determine the speed of action and the stage-specificity of anti-malarials in Plasmodium falciparum.

Le Manach C, Scheurer C, Sax S, Schleiferböck S, Cabrera DG, Younis Y, Paquet T, Street L, Smith P, Ding XC, Waterson D, Witty MJ, Leroy D, Chibale K, Wittlin S - Malar. J. (2013)

Bottom Line: This has the advantage that initial results can be achieved within 4-7 working days, which helps to distinguish between fast and slow-acting compounds relatively quickly.The results obtained for the anti-malarials chloroquine, artesunate, atovaquone, and pyrimethamine are consistent with previous observations, suggesting the methodology is a valid way to rapidly identify fast-acting anti-malarial compounds.Another advantage of the approach is its ability to discriminate between static or cidal compound effects.

View Article: PubMed Central - HTML - PubMed

Affiliation: Parasite Chemotherapy Unit, Swiss Tropical and Public Health Institute, Basel, Switzerland. sergio.wittlin@unibas.ch.

ABSTRACT

Background: Recent whole cell in vitro screening campaigns identified thousands of compounds that are active against asexual blood stages of Plasmodium falciparum at submicromolar concentrations. These hits have been made available to the public, providing many novel chemical starting points for anti-malarial drug discovery programmes. Knowing which of these hits are fast-acting compounds is of great interest. Firstly, a fast action will ensure rapid relief of symptoms for the patient. Secondly, by rapidly reducing the parasitaemia, this could minimize the occurrence of mutations leading to new drug resistance mechanisms.An in vitro assay that provides information about the speed of action of test compounds has been developed by researchers at GlaxoSmithKline (GSK) in Spain. This assay also provides an in vitro measure for the ratio between parasitaemia at the onset of drug treatment and after one intra-erythrocytic cycle (parasite reduction ratio, PRR). Both parameters are needed to determine in vitro killing rates of anti-malarial compounds. A drawback of the killing rate assay is that it takes a month to obtain first results.

Methods: The approach described in the present study is focused only on the speed of action of anti-malarials. This has the advantage that initial results can be achieved within 4-7 working days, which helps to distinguish between fast and slow-acting compounds relatively quickly. It is expected that this new assay can be used as a filter in the early drug discovery phase, which will reduce the number of compounds progressing to secondary, more time-consuming assays like the killing rate assay.

Results: The speed of action of a selection of seven anti-malarial compounds was measured with two independent experimental procedures using modifications of the standard [3H]hypoxanthine incorporation assay. Depending on the outcome of both assays, the tested compounds were classified as either fast or non-fast-acting.

Conclusion: The results obtained for the anti-malarials chloroquine, artesunate, atovaquone, and pyrimethamine are consistent with previous observations, suggesting the methodology is a valid way to rapidly identify fast-acting anti-malarial compounds. Another advantage of the approach is its ability to discriminate between static or cidal compound effects.

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Related in: MedlinePlus

Synthetic route for compound 3. Reagents and Conditions: (i) trichloroacetyl isocyanate (1 eq), THF, 0°C to r.t, 2 h, 91%; (ii) bromine (3.8 eq), acetic acid, 0°C to 80°C, 14 h, 65%; (iii) ammonia, CH3OH, 0°C to r.t., 30 min, 76%; (iv) t-BuOK, DMF, RT, 14 h, 99%; (v) POCl3,N,N-dimethyl aniline (0.5 eq), 130°C, 14 h 88%; (vi) 3-dimethylaminopropylamine (1 eq), Na2CO3 (2 eq), EtOH, r.t., 14 h, 72%; (vii) 2 M methyl amine in THF (10 eq), dioxane, sealed tube, 100°C, 14 h, 83%; (viii) phenylboronic acid (1.1 eq), Pd(PPh3)2Cl2 (0.05 eq), aq. 1 M K2CO3 (1.05 eq), DMF, 90°C, 14 h, 63%.
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Figure 2: Synthetic route for compound 3. Reagents and Conditions: (i) trichloroacetyl isocyanate (1 eq), THF, 0°C to r.t, 2 h, 91%; (ii) bromine (3.8 eq), acetic acid, 0°C to 80°C, 14 h, 65%; (iii) ammonia, CH3OH, 0°C to r.t., 30 min, 76%; (iv) t-BuOK, DMF, RT, 14 h, 99%; (v) POCl3,N,N-dimethyl aniline (0.5 eq), 130°C, 14 h 88%; (vi) 3-dimethylaminopropylamine (1 eq), Na2CO3 (2 eq), EtOH, r.t., 14 h, 72%; (vii) 2 M methyl amine in THF (10 eq), dioxane, sealed tube, 100°C, 14 h, 83%; (viii) phenylboronic acid (1.1 eq), Pd(PPh3)2Cl2 (0.05 eq), aq. 1 M K2CO3 (1.05 eq), DMF, 90°C, 14 h, 63%.

Mentions: Compounds 1 and 2 were synthesized using the experimental procedures previously described [13,14]. Compound 3 was obtained from a 7-step synthesis from commercially available reagent 4 (Figure 2). Reaction of 4 with trichloroacetyl isocyanate in THF, followed by bromination gave compound 5 in 91% yield. Subsequent treatment with ammonia in methanol afforded intermediate 6, which cyclized under basic conditions. Subsequent chlorination with POCl3 gave key dichloro intermediate 7. Two consecutive N-substitution reactions with 3-dimethylaminopropylamine under basic conditions and methyl amine respectively gave intermediate 8, which underwent a final Suzuki cross-coupling reaction with phenylboronic acid to give the desired compound 3 as a white solid (Gonzalez Cabrera D et al.: 2,4-Diamino-thienopyrimidines as orally active antimalarial agents. Manuscript submitted). All three compounds were analysed by HPLC prior to biological experiments and were found to be >98% pure.


Fast in vitro methods to determine the speed of action and the stage-specificity of anti-malarials in Plasmodium falciparum.

Le Manach C, Scheurer C, Sax S, Schleiferböck S, Cabrera DG, Younis Y, Paquet T, Street L, Smith P, Ding XC, Waterson D, Witty MJ, Leroy D, Chibale K, Wittlin S - Malar. J. (2013)

Synthetic route for compound 3. Reagents and Conditions: (i) trichloroacetyl isocyanate (1 eq), THF, 0°C to r.t, 2 h, 91%; (ii) bromine (3.8 eq), acetic acid, 0°C to 80°C, 14 h, 65%; (iii) ammonia, CH3OH, 0°C to r.t., 30 min, 76%; (iv) t-BuOK, DMF, RT, 14 h, 99%; (v) POCl3,N,N-dimethyl aniline (0.5 eq), 130°C, 14 h 88%; (vi) 3-dimethylaminopropylamine (1 eq), Na2CO3 (2 eq), EtOH, r.t., 14 h, 72%; (vii) 2 M methyl amine in THF (10 eq), dioxane, sealed tube, 100°C, 14 h, 83%; (viii) phenylboronic acid (1.1 eq), Pd(PPh3)2Cl2 (0.05 eq), aq. 1 M K2CO3 (1.05 eq), DMF, 90°C, 14 h, 63%.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
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Figure 2: Synthetic route for compound 3. Reagents and Conditions: (i) trichloroacetyl isocyanate (1 eq), THF, 0°C to r.t, 2 h, 91%; (ii) bromine (3.8 eq), acetic acid, 0°C to 80°C, 14 h, 65%; (iii) ammonia, CH3OH, 0°C to r.t., 30 min, 76%; (iv) t-BuOK, DMF, RT, 14 h, 99%; (v) POCl3,N,N-dimethyl aniline (0.5 eq), 130°C, 14 h 88%; (vi) 3-dimethylaminopropylamine (1 eq), Na2CO3 (2 eq), EtOH, r.t., 14 h, 72%; (vii) 2 M methyl amine in THF (10 eq), dioxane, sealed tube, 100°C, 14 h, 83%; (viii) phenylboronic acid (1.1 eq), Pd(PPh3)2Cl2 (0.05 eq), aq. 1 M K2CO3 (1.05 eq), DMF, 90°C, 14 h, 63%.
Mentions: Compounds 1 and 2 were synthesized using the experimental procedures previously described [13,14]. Compound 3 was obtained from a 7-step synthesis from commercially available reagent 4 (Figure 2). Reaction of 4 with trichloroacetyl isocyanate in THF, followed by bromination gave compound 5 in 91% yield. Subsequent treatment with ammonia in methanol afforded intermediate 6, which cyclized under basic conditions. Subsequent chlorination with POCl3 gave key dichloro intermediate 7. Two consecutive N-substitution reactions with 3-dimethylaminopropylamine under basic conditions and methyl amine respectively gave intermediate 8, which underwent a final Suzuki cross-coupling reaction with phenylboronic acid to give the desired compound 3 as a white solid (Gonzalez Cabrera D et al.: 2,4-Diamino-thienopyrimidines as orally active antimalarial agents. Manuscript submitted). All three compounds were analysed by HPLC prior to biological experiments and were found to be >98% pure.

Bottom Line: This has the advantage that initial results can be achieved within 4-7 working days, which helps to distinguish between fast and slow-acting compounds relatively quickly.The results obtained for the anti-malarials chloroquine, artesunate, atovaquone, and pyrimethamine are consistent with previous observations, suggesting the methodology is a valid way to rapidly identify fast-acting anti-malarial compounds.Another advantage of the approach is its ability to discriminate between static or cidal compound effects.

View Article: PubMed Central - HTML - PubMed

Affiliation: Parasite Chemotherapy Unit, Swiss Tropical and Public Health Institute, Basel, Switzerland. sergio.wittlin@unibas.ch.

ABSTRACT

Background: Recent whole cell in vitro screening campaigns identified thousands of compounds that are active against asexual blood stages of Plasmodium falciparum at submicromolar concentrations. These hits have been made available to the public, providing many novel chemical starting points for anti-malarial drug discovery programmes. Knowing which of these hits are fast-acting compounds is of great interest. Firstly, a fast action will ensure rapid relief of symptoms for the patient. Secondly, by rapidly reducing the parasitaemia, this could minimize the occurrence of mutations leading to new drug resistance mechanisms.An in vitro assay that provides information about the speed of action of test compounds has been developed by researchers at GlaxoSmithKline (GSK) in Spain. This assay also provides an in vitro measure for the ratio between parasitaemia at the onset of drug treatment and after one intra-erythrocytic cycle (parasite reduction ratio, PRR). Both parameters are needed to determine in vitro killing rates of anti-malarial compounds. A drawback of the killing rate assay is that it takes a month to obtain first results.

Methods: The approach described in the present study is focused only on the speed of action of anti-malarials. This has the advantage that initial results can be achieved within 4-7 working days, which helps to distinguish between fast and slow-acting compounds relatively quickly. It is expected that this new assay can be used as a filter in the early drug discovery phase, which will reduce the number of compounds progressing to secondary, more time-consuming assays like the killing rate assay.

Results: The speed of action of a selection of seven anti-malarial compounds was measured with two independent experimental procedures using modifications of the standard [3H]hypoxanthine incorporation assay. Depending on the outcome of both assays, the tested compounds were classified as either fast or non-fast-acting.

Conclusion: The results obtained for the anti-malarials chloroquine, artesunate, atovaquone, and pyrimethamine are consistent with previous observations, suggesting the methodology is a valid way to rapidly identify fast-acting anti-malarial compounds. Another advantage of the approach is its ability to discriminate between static or cidal compound effects.

Show MeSH
Related in: MedlinePlus