Structures of methisazone, 9, and the seven isatin-β-thiosemicarbazones (1-7) identified in a bioinformatics screen as having activity that is potentiated, rather than inhibited by expression of the multidrug transporter P-gp. 1 is being treated as a lead compound to understand the mechanism of action of the compounds. An overlay of the seven NSC compounds identified in the bioinformatics screen demonstrates the common structural features associated with them.

Scheme showing general synthetic steps in the preparation of isatin-β-thiosemicarbazones. (a) General synthetic method used for the synthesis of isatin-β-thiosemicarbazones, the example here being 1. Equimolar quantites of isatin (or a substituted isatin) and a thiosemicarbazide (here 4-(4-methoxyphenyl)-3-thiosemicarbazide) are stirred in hot ethanol, a few drops of glacial acetic acid are added, and the solution is refluxed for a short period that on cooling resulted in the precipitation of the final product. (b) The phenylthiosemicarbazide is generally reported to be synthesized by the reaction of hydrazine with the substituted phenylisothiocyanate of choice, such as p-methoxyphenyisothiocyanate shown here, yielding 4-(4-methoxyphenyl)-3-thiosemicarbazide. (c) In our hands the reaction between hydrazine and p-halogen phenylisothiocyanates such as p-fluorophenylisothiocyanate did not proceed as expected, but resulted (d) in an insoluble precipitate that was unable to undergo further reaction with isatin. It was identified by mass spectrometry to be a 2:1 combination of thiocyanate and hydrazine (mw = 338 gmol⁻¹) that formed even when hydrazine was added drop-wise to the excess thiocyanate. Using a t-Boc protected form of hydrazine, t-butylcarbazate, which is effectively monofunctional hydrazine, the t-Boc protected thiosemicarbazide could be produced, from which the thiosemicarbazone was recovered under acidic conditions.

Pharmacophores that contribute cytotoxicity and selectivity a). The pharmacophore features required for cytotoxicity, indicated by the activity of compounds tested against the parental KB-3-1 cell line. Pink sphere = A, H-bond acceptor, with the sphere located at the projected point of a hydrogen bond donor. Green sphere = H, hydrophobic group. Ring = aromatic ring. Blue sphere = X, thiosemicarbazone functional group. The pharmacophore sites are underlaid with the structure of 10 to aid in relating the pharmacophore features to structural features of the active compounds. b). The pharmacophore features required for selectivity against the P-gp expressing KB-V1 cell line: AH₁H₂H₃R₁R₂X. Pink sphere = A, H-bond acceptor, with the sphere located at the projected point of a hydrogen bond donor. Green sphere = H, hydrophobic group. Ring = aromatic ring. Blue sphere = X, thiosemicarbazone functional group. Yellow sphere = Exclusion sphere. The pharmacophore sites are underlaid with the structure of 1 to aid in relating the pharmacophore features to structural features of the active compounds.

Pharmacophores predict cytotoxicity and selectivity a). Scatter plot and comparison of the KB-3-1 cytoxicity QSAR model applied to thirteen active thiosemicarbazones. The training set correlation is characterized by one partial least-square (PLS) factor (SD = 0.12, R² = 0.96). The test set correlation is characterized by one PLS factor (Root mean-square error (RMSE) = 0.16, q² = 0.85, Pearson-R = 0.95). Experimental and calculated pIC₅₀ values are shown for the QSAR training and test set. B). Scatter plot and comparison of the KB-V1 cytoxicity QSAR model applied to twelve active thiosemicarbazones. The training set correlation is characterized by one PLS factor (SD = 0.11, R² = 0.91). The test set correlation is characterized by one PLS factor (RMSE = 0.29, q² = 0.42, Pearson-R = 0.70). Experimental and calculated pIC₅₀ values are shown for the QSAR training and test set.

Refined pharmacophore predicting sensitivity of KB-V1 cells a). The pharmacophore for activity against KB-V1 cells with overlay of compounds that match three of seven pharmacophore features (AH₁X). b). Pharmacophore for activity against KB-V1 cells with overlay of compounds that satisfy all pharmacophore features (AH₁H₂H₃R₁R₂X). The compounds that meet these requirements are the isatin-β-thiosemicarbazones and compound 21.

The pharmacophore for activity against KB-V1 cells was tested against the 60 compounds previously identified in a bioinformatic analysis for compounds that were predicted to be selectively active against P-gp expressing cells. The activities of two available compounds, the thiosemicarbazone 27, and 30, are given. a). Querying the structural database for compounds that satisfy at least AH₁X (X = thiosemicarbazone) identified the seven NSC isatin-β-thiosemicarbazones shown in Figure 1 from which the pharmacophore was partially derived, and the three remaining thiosemicarbazones in the set: 27, 28 and 29, shown over the site features demonstrating the good fit of structural features of these molecules with the pharmacophore. b). Querying the structural database for compounds that satisfy at least three out of seven sites, including AH₁R₂, but not the thiosemicarbazone feature (X), gave a further 13 non-thiosemicarbazone NSC compounds from our dataset that appear to satisfy the structural criteria for selectively killing KB-V1 cells. Two examples are shown; 30 and 31.