Table 1.
Structure and activity of hits and early analogues.
Fig 1.
General scheme for the synthesis of final compounds.
X = I (or Br), Y = Br (or I).
Fig 2.
Illustration of the structure activity relationship derived from an exploratory set of compounds made as a combinatorial compound library and tested in vitro against S. mansoni juvenile worms for 5 days. Colour coded to show EC50 values.
Fig 3.
Guide to core structures of compounds in Table 1 (letters refer to column “Core”).
Table 2.
Structure activity relationship of more potent and stable compounds.
Fig 4.
Guide to structures in Table 2.
Fig 5.
In vitro potency against S. mansoni.
Both plots include compounds not shown in tables. A Potency improvement obtained against S. mansoni worms resulted in a very large (>1,000 fold in many cases) ratio between cytotoxicity (as measured against hepG2 or MRC5 cells) and potency. B This series has equal potency against both juvenile (21 days old) and adult (42 days old) S. mansoni worms. The Bravais-Pearson correlation r value for the fitted line shown is 0.98.
Table 3.
SAR table showing potency of compounds against different clinically relevant Schistosoma species in vitro.
Fig 6.
A. In vivo efficacy against adult worms in the mouse model. Graph shows the Mean (+standard deviation) worm recoveries following single oral dose treatment with vehicle or vehicle plus LSHTM-3520 (Aqueous formulation- see Materials and Methods) of 42-day old infections in mice (n = 6). B. Drug concentration in plasma measured at different time points after administration. Green rectangle—PK experiment, iv dose of LSHTM-3520 0.5 mg/kg co-administered with 4 other compounds, data points are mean values from 3 mice. Purple triangle—PK experiment, oral dose of LSHTM-3520 2.5 mg/kg co-administered with 4 other compounds, data points are mean values from 3 mice. Red circle—Efficacy experiment, oral dose of LSHTM-3520 200 mg/kg, data points are single values from five different mice each sampled once during the experiment (2 mice sampled at 24 h). Yellow diamond—Arithmetic scaling of the oral dose 2.5 mg/kg cassette dose experiment to simulate a dose of 200 mg/kg to show comparison with experimental measurements of 200 mg/kg oral dose.
Fig 7.
Correlations between various mouse and human in vitro properties.
A In vitro half-life in mouse hepatocytes calculated from the percentage of test compound remaining after a set incubation time (30 minutes, grey circles or 90 minutes, black circles) compared with observed clearance measured in mouse after iv administration at 0.5 mg/kg co-administered in groups of five. B In vitro half-life in mouse or human hepatocytes calculated as described for panel A (incubation time in human hepatocytes 30 minutes, squares or 90 minutes, circles). C Plasma protein binding (expressed as free fraction), measured at 10uM. Despite difficulties in accurate measurement due to very high levels of protein binding, the data is consistent with equal levels of protein binding in mouse and human plasma.
Fig 8.
In vivo efficacy against juvenile worms in the mouse model.
Graphs show the Mean (+ standard deviation) worm recoveries following single oral dose treatment with vehicle or vehicle plus drug (Aqueous formulation—see Materials and Methods) of 21-day old infections in mice (n = 6). Further details are in the Materials and Methods section.
Fig 9.
In vivo demonstration of efficacy.
Graphs show the Mean (+ standard deviation) worm recoveries following single oral dose treatment with vehicle or vehicle plus drug of infections in mice (n = 5), #—no worms recovered (ie complete cure in all mice). The formulation of drug (or control) was either Aq (Drugs were suspended in 7% Tween-80 / 3% Ethanol / double distilled water and drug dispersal was facilitated by vortexing and using a sonicating water bath) or oil (Drugs were first dissolved in DMSO, then diluted with corn oil to give a 1:19 DMSO/corn oil ratio. Panel A shows a head to head comparison between PZQ and LSHTM-3642 in vivo against adult worms with a single oral dose giving PZQ an EC50 between 50 and 100 mg/kg and LSHTM-3642 an EC50 below 6.25 mg/kg. Panel B A demonstration of good efficacy from two compounds with low in vitro EC50 values. LSHTM-3645 was used with a single oral dose, LSHTM-3686 was dosed twice, 12h apart because it has a shorter (6h) murine half-life than some of the other compounds shown. Panel C A dose response experiment showing that LSHTM-3642 has an EC50 against juvenile worms in vivo of between 12.5 & 25 mg/kg. The experiment also shows that both LSHTM-3608 and LSHTM-3645 have EC50s below 6.25 mg/kg. Panel D This experiment shows that LSHTM-3645 and LSHTM-3608 are extremely effective in clearing juvenile worm infections in vivo from a 12.5 mg/kg oral single dose. As for panel B, LSHTM-3686 was dosed twice 12h apart because of its shorter half-life in mouse.
Fig 10.
Structures of compounds shown in Table 4.
Table 4.
in vitro, physicochemical and in vivo properties of compounds pictured in Fig 10.