## 3 Results and Discussion
### Inhibition Studies  
The binding and inhibition potency of the compounds were tested against purified recombinant Tdp1. Binding experiments were conducted using thermal shift assay [71] and an intrinsic fluorescence binding assay that we previously developed [35; 45; 49]. Thermal shift assay measures the stability of proteins against thermal degradation. If a molecule binds to the protein, it may be stabilized or destabilized by the binding of the molecule, thus leading to a shift of the protein melting temperature. The intrinsic fluorescence binding assay measures the fluorescence intensity of tryptophan residues of a protein. The active site of Tdp1 is close to several tryptophan residues. Binding of Tdp1 inhibitors may change the environment of these tryptophan residues, thus leading to quenching of fluorescence intensity. However, no significant changes in the melting temperature of Tdp1 or quenching of Tdp1 fluorescence intensity were observed (Supplementary Figures S1-S6). This may be due to the low solubility of our compounds (1 mM or less) in aqueous buffer. As our binding assays typically required at least tens of \(\mu\)M of Tdp1, we reasoned that the lack of changes in melting temperature and intrinsic fluorescence intensity are likely due the low ligand-to-protein ratio needed in our experiments, which did not allow complete formation of Tdp1-inhibitor complex. Therefore, the inhibition potency of our compounds **44a,b-47a,b**; **50a,b**; **51** were tested against Tdp1 by using a biosensor assay previously developed [29]. The biosensor is 16-mer single-stranded oligonucleotide containing a 5'-FAM (5(6)-carboxyfluorescein) fluorophore donor and a quenching 3'-BHQ1 (Black Hole Quencher-1). Because BHQ1 and FAM are located within the Forster radius, the fluorescence is quenched. The mechanism of action is based on the Tdp1 enzyme's ability to selectively cleave substituents from the 3'-end of DNA [72], thus deriving the Tdp1 activity by real-time fluorescence detection [29; 32]. Table 1 shows the Tdp1 inhibition potency of the compounds **44a,b-47a,b**; **50a,b**; **51**. Furamidine **11**, a commercially-available Tdp1 inhibitor, was used as a positive control. Compounds **46b** and **47b** were not investigated in Tdp1 inhibitory activity tests due to their instability. The compounds synthesized can be divided in to two groups: (1) amides and thioamides of 1- and 2-adamantane carboxylic acids (**44a,b**; **45a,b**; **46a; **47a**); (2) derivatives of citronellic acid (**50a,b**; **51**). Comparing inhibitory activity of the first group (Table 1) and compounds synthesized previously [49] it can be concluded that amides **44-47** are less active than their ester counterparts are. For example, in the cases of derivatives **29** (ester), **44** (amide), **48** (thioamide), which are structural analogues, the following IC\({}_{50}\) values were obtained: 0.92, >75 and 3.3 \(\mu\)M, respectively. Note that derivatives of 2-adamantane carboxylic acid were found to be more active compared to compounds obtained from 1-admantane carboxylic acid. Thus, amides **44a** and **44b** were not active, whilst **45a** and **45b** showed Tdp1 50% inhibition at 5.2 and 2.5 \(\mu\)M, respectively (Table 1). Comparing amide **45a** and thioamide **47a** activity it can be concluded that, for 2-adamantane carboxylic acid derivatives, oxygen atom replacement by sulphur also led to activity increasing, with IC\({}_{50}\) being 5.2 \(\mu\)M for **45a** and 0.64 \(\mu\)M for **47a**. Similar trends were observed for citronellic acid derivatives **50a,b**; **51**. For example, 1-aminoadamnatane derivative **50a** was not active whilst 2-aminoadamantane **50b** activity was found to be slightly higher and the best IC\({}_{50}\) value was obtained for thioamide **51** (2.3 \(\upmu\)M), also a 2-aminoadamantane derivative.