Integrated SAR interpretation and strength of evidence

Since IC50 values are condition dependent, cross-group comparisons were interpreted qualitatively, and quantitative ranking was restricted to groups within the same assay format. To support SAR interpretations across study groups, the strength of evidence was assigned using predefined criteria for reproducibility and methodological alignment, as described in Table 2.

Strength of evidence | Criteria
High | Trend was observed in both studies within the same assay group (A or B) and supported by at least one additional study from another group (including C)
Moderate-high | Trend was observed in studies within the same assay group (A or B) without additional support from additional studies across groups, or from three studies from different assay formats, or from a single study supported by direct structural support such as X ray-crystallography or NMR
Moderate | Trend was observed by at least two studies from different assay groups (A+B or A+C or B+C)
Low | Trend based on a single study without structural support

Evidence was ranked as high when the trends were reproduced in both studies within the same assay group and further supported by at least one additional study from another group. Moderate-High strength was assigned when trends were reproduced within a single assay group, with no supportive evidence from other groups. Moderate strengths were assigned when trends were observed across non-aligned studies without replication within the same assay group, and low strength of evidence when the trend was observed in a single study or in non-aligned studies. SAR features, direction, evidence source, and strength are shown in Table 3. The most potent compounds identified across included studies are summarized in Table 4, whereas Figure 4 shows their chemical structures.

SAR feature | Effect on activity | Evidence strength | Evidence group & references | Comments
Free thiol zinc binding group (ZBG) | Essential for activity | High | A [41,42]B [38,43]C [39,40] | Required for Zn (II) coordination
Thiol masking | Decrease | Low | A [42] | Prevent Zn (II) coordination
Hydrophobic/steric substitution | Increase until steric ceiling | High | A [41,42]B [38,43]C [40] | Pocket filling (until steric ceiling)
Pyrrolidine ring modification/ring scaffold geometry | Increasereplacement with indoline | Low | A [42] | Excessive enlargements reduce hydrophobic contact; simplified scaffolds remain active; benzyl-ring substitutions (e.g. meta-methyl, ortho-F, ortho-OH) were weak, suggesting a constrained tolerance space
Increasereplacement with benzyl amid | Moderate-high | A [41]B [42]C [40]
Increase ring expansion: 6-member ringDecreasering expansion:7-member ring | Moderate-high | B [38,43]
Stereochemistry | D > L | Moderate-high | A [42]B [43]C [40] | Optimal carboxylate/proline placement in NDM-1 active site
R > S(at C adjacent to thiol) | Moderate-high | A [41]B [43]C [40]
R > S(at C adjacent to nitrogen) | Moderate | A [42]B [43]
α-carboxylate moiety on proline | Essential for binding | Moderate* | A [42]C [40] | Act as anchoring group in NDM-1 active site
Carboxylate position on the ring** | Para > ortho > > meta | Moderate-high | B [38]supported by structural evidence | Para carboxylate position strengthened hydrophobic interaction; meta carboxylate position weakened both hydrophobic and hydrogen bonding interactions
Carboxylate replacement carboxamide vs. glycylamide | Increase carboxamide > > glycylamide | Low | A [42] | Maintain an anionic/H-bonding anchor positioned for loop/water network interactions
Fluorination position (CF₃ placement: α vs. β) | α-CF₃ > β-CF₃ | Low | B [43] | α-CF₃ oxidizes faster (stability issue)

Group | Study | Number of synthesized derivatives | Compound | IC50, μM | Key modification
A | [41] | 17 | Compound 14a | 0.1 | Bulky aromatic
[42] | 13 | Compound (S, R)-6d | 3.5 | Ring expansion:indoline scaffold
B | [43] | 20 | Compound (2R, 2′R)-5αC | 0.3 | Trifluoromethyl substitution
[38] | 10 | Compound 11 | 4.6 | Ring expansion:tetrahydroisoquinoline substituted ring
C | [40] | 29 | Compound 22 | 1.5 | Simplified amide
[39] | 6 | D-captopril | 20.1 | Core scaffold stereochemistry

Figure 4.: Representative structures of the most potent captopril derivatives by each study group (A-C). The conserved mercaptopropionamide zinc-binding region is shown in red. The IC50 values are reported as in the original studies