Supplementary figure 2S · Interactive

Structure–activity relationship analysis of spiro compounds

Pocket 1 binding scores for the nine novel spiro[indoline-tetrazolo[1,5-c]quinazoline] derivatives, their correlation with physicochemical descriptors, and the scaffold regions that govern HDAC8 engagement.

(A) Pocket 1 binding scores of the nine novel derivatives

(B) Pearson correlation of descriptors with binding score (n = 9)

Spiro[indoline-tetrazolo[1,5-c]quinazoline] core

Position 1 of the indoline ring

Carboxamide / carbothioamide: strong enhancement

Acetamide linker: moderate enhancement

Unsubstituted: weak binding

Direct amide attachment yields a more favourable binding geometry than the acetamide linker.

Phenyl-ring substituent

Electron-withdrawing (CN, CF₃): strong enhancement

Halogen (Cl): moderate enhancement

Unsubstituted: baseline

Electron-withdrawing groups may strengthen electrostatic contacts (descriptive; not statistically significant, p = 0.39).

Tetrazolo[1,5-c]quinazoline core

Spiro junction: positions the aromatic system for π-contacts

Nitrogen heterocycles: support the Asp101 interaction

Aromatic surface: π–π stacking with Phe152, Phe208, Tyr306

Provides the aromatic contacts shared by the high-affinity compounds.

Structure–activity data

Compound	Score (kcal/mol)	Position-1 group	Phenyl substituent	MW (Da)	LogP	TPSA (Å²)	Tier	Key SAR feature
KB-282	−12.3	Carboxamide	4-Cyano	434.42	2.60	128.83	Strong	Direct carboxamide + EWG
KB-281	−11.0	Carboxamide	3-Trifluoromethyl	477.41	3.85	105.04	Strong	Direct carboxamide + EWG
KB-269	−10.5	Acetamide	4-Chloro	457.88	3.42	105.04	Strong	Acetamide linker + halogen
KB-271	−10.4	Acetamide	2-CF₃ (benzyl)	505.46^†	3.51	105.04	Strong	Acetamide linker + EWG
KB-280	−10.4	Carbothioamide	Unsubstituted	425.48	3.41	87.97	Strong	Direct carbothioamide
KB-270	−10.0	Acetamide	3-Chloro	457.88	3.40	105.04	Moderate	Acetamide linker + halogen
KB-268	−8.8	Unsubstituted	None	290.29	1.98	84.73	Lower	Parent compound
KB-272	−8.5	2-Chloroacetyl (6′)	None	366.77	2.14	93.01	Lower	Substitution at 6′ is unfavourable
KB-283	−8.5	Carboxamide	N-Propyl	375.39	2.46	105.04	Lower	Alkyl substituent reduces π-contacts

Structure–activity observations

Position-1 hierarchy: carboxamide (mean −11.65) > carbothioamide (−10.4) > acetamide (mean −9.58) > unsubstituted (−8.8); differences are descriptive given singleton groups (Kruskal–Wallis H = 4.50, p = 0.21).
Electronic effect: electron-withdrawing substituents (CN, CF₃) accompany the highest scores, though the difference versus electron-donating groups was not significant (Welch t = −0.94, p = 0.39).
Polarity penalty: binding score correlates negatively with HBA (r = −0.83, p < 0.01) and TPSA (r = −0.70, p < 0.05): higher polarity tends to weaken binding.
Geometry: direct attachment at position 1 (KB-282, KB-281) outperforms the acetamide linker; modification at 6′ (KB-272) and N-alkyl substitution (KB-283) reduce affinity.
Model fit: a three-descriptor regression (SlogP + TPSA + HBA) explains much of the variance (R² = 0.80, F = 6.55, p = 0.035); KB-282 behaves as a high-affinity outlier (Cook's D = 3.24), consistent with a non-linear relationship at the top of the series.

Strong (≤ −10.4 kcal/mol) Moderate (−10.3 to −9.0) Lower (≥ −8.9) KB-282 (highest affinity)

Sources. Binding scores: Table 2 and Supplementary S1–S2. SAR groupings: Table 3 and Supplementary S15. Physicochemical values: Table 5 and Supplementary S13/S16. Correlations and tests: Supplementary S14/S20. Panel B shows Pearson r between each descriptor and Pocket 1 score (n = 9 novel compounds): ** p < 0.01, * p < 0.05; teal = positive, red = negative association with binding strength. ^† KB-271 (MW 505.46 Da) marginally exceeds the 500 Da Lipinski threshold (one violation); all other novel compounds comply (Supplementary S19).