The Magic Methyl and Its Tricks in Drug Discovery and Development

The small, monovalent, and lipophilic methyl group (-CH3) is versatile and of great importance in the design or optimization of bioactive compounds, whether in terms of pharmacodynamic or pharmacokinetic properties. Its role in drug design and hit-to-lead optimization processes is broad, including the displacement of water molecules during molecular recognition (realization of hydrophobic interactions); participation in van der Waals interactions; modulation of physicochemical properties such as LogP and aqueous solubility; and control of the conformational properties of a given scaffold. The control of the number of conformations in a given system by methylation correlates with the strategy of conformational restriction. Other drug design strategies, such as bioisosterism and homologation, can also benefit from methyl group insertion. During the drug discovery process, controlling conformational behavior can not only favor the adoption of a bioactive conformation, generating a potency gain for target modulation, but can also help break planarity and symmetry, resulting in increased aqueous solubility while increasing lipophilicity. Other uses of the methyl group include modulating metabolic reactions by preventing their occurrence through stereoelectronic effects, serving as a metabolic point to prevent the formation of toxic metabolites, or modulating the metabolic profile to make molecules more amenable to metabolism. This plethora of effects mediated by the methyl group is commonly referred to as the "methyl effect", the "methylation effect", or the "magic methyl" effect. Previous works have reviewed this topic. This review provides key recent examples to highlight how the rational use of the methyl effect has evolved since the last review by the authors' group.

This review surveys the past decade of medicinal chemistry examples where strategic methyl insertion modulates pharmacodynamic and pharmacokinetic properties. Covered areas include: discovery of the EZH2 inhibitor tazemetostat via methylation pattern optimization; PI3K/mTOR inhibitors using a 2-methyl-imidazo[4,5-c]quinoline scaffold; selective κ-opioid receptor antagonists where 4-methylation of the piperidine ring boosts affinity; cannabinoid receptor modulators (oxazolo[5,4-d]pyrimidines, adamantanyl thiophenes, and CB1R ago-PAM derivatives) showing large affinity/potency gains from methylation and diastereoselective effects; histamine H1R antagonists where N-methylation increases binding; fragment-based PPAT inhibitors where C5-methylation and benzylic methylation yield 15–30-fold potency boosts; methyl scanning for AURKB phenocopying leads improving MECP; NK3R antagonists with methyl additions achieving low-nanomolar potency; cereblon ligands (PDHU derivatives) where ortho-methyl improves Kd and enables potent BRD4 degraders; phenotypic discovery of combretastatin A-4/NAH analogs where N-methylation affects microtubule behavior and cytotoxicity; multitarget NAH/PDE4-A2A hybrids and other PDE4 series where methylation greatly improves PDE4 potency; ROCK inhibitors where N-methylation improves ROCK1/2 potency; ligands of TLR4/MD-2 where methamphetamine (methyl-bearing) binds while amphetamine does not; macrocyclic ghrelin agonist ulimorelin where macrocycle methylation stabilizes bioactive conformation and PK; pan-genotype HCV NS3/4A protease inhibitors where methylation and deuteration improve potency, distribution, and stability; Class I HDAC macrocycles showing methyl number/placement impacts potency; trypanocidal benznidazole analogs with 2-methylimidazole modifications; antibacterial furoxan-sulfonylhydrazones where methylation is key; bis-(3-indolyl)methane phosphonate anticancer agents where 5-methyl improves antiproliferative potency; and case studies on PK/physicochemical optimization including increased aqueous solubility via NAH N-methylation, improved plasma stability via gem-dimethyl lactones, reduced hERG liability and improved metabolic stability via methylation, and mitigation of oxidative soft spots via methyl substitution.

- Strategic insertion of methyl groups can profoundly affect potency and selectivity by modulating hydrophobic contacts, conformations, and protein–ligand fit, and can also optimize ADMET properties. - EZH2/tazemetostat: Methylation pattern on a 4,6-dimethylpyridone system was critical; unmethylated analogs showed >10-fold loss in potency; dimethylated analogs were most potent, enabling FDA-approved tazemetostat. Perspectives note four methyl groups contributed to >100,000-fold activity improvement. - PI3K/mTOR: 2-methyl-imidazo[4,5-c]quinolines improved permeability while reducing heteroatoms; lead 18 showed best selectivity, in vitro/in vivo efficacy, and PK. - κ-Opioid receptor antagonists: 4-methylation of a piperidine ring (20) increased κ-OR affinity 18-fold versus unmethylated 19. - Cannabinoid system: Oxazolo[5,4-d]pyrimidine 23 (5-methyl) was a selective CB2R antagonist with low-nM affinity vs unmethylated 24. Adamantanyl thiophenes: adding one methyl (26) boosted CB2R affinity ~50-fold from weak 25; n-propyl (27) gave only ~3-fold increase. CB1R ago-PAM derivatives: an α-methyl next to nitro created diastereomers with erythro outperforming threo; first diastereoselective CB1R allosteric interaction. - H1R antagonists: N-methylation of 32 to 33 strongly increased H1R binding affinity. - PPAT inhibitors: Fragment 34 to 35 via C5-methylation delivered ~15-fold potency gain through added hydrophobic contacts; benzylic R-methyl analog 39 gave ~30-fold boost over 37/38. - AURKB phenotypic series: p-methyl analog 41 had MECP 0.625 µM; optimized 42 achieved MECP 0.019 µM with strong cytotoxic activity and AURKB loss-of-function phenotypes. - NK3R antagonists: 8-position methyl raised potency to nanomolar; adding a second methyl at pyridine C6 achieved low-nM activity (48, 49). - Cereblon ligands: PDHU (50) Kd 3.05 µM; ortho-methyl (51) Kd 1.24 µM; optimized ligand 52 Kd 0.21 µM enabled potent BRD4 degraders. - Combretastatin/NAH analogs: N-methylated LASSBio-1735 (55) showed microtubule-destabilizing behavior and superior cytotoxicity vs non-methylated/benzylated homologs. - PDE4 inhibitors: Hybrid NAH 60 (N-methyl) inhibited PDE4A1A with IC50 1.08 µM and had A2A Ki 1.5 µM, outperforming 59; GEBR series methylated 62 IC50 0.47 µM (PDE4D3) vs de-methyl 61 IC50 11 µM; LASSBio-1632 (N-methyl sulfonylhydrazone) inhibited PDE4A IC50 0.5 µM and PDE4D IC50 0.7 µM and showed anti-asthmatic effects. - ROCK inhibitors: N-methylation (70) improved ROCK1/2 inhibition 3–4 fold vs 69. - TLR4/MD-2 ligands: Methamphetamine bound MD-2 with Kd ~7.0–8.9 µM (non-enantioselective), while amphetamine had no detectable binding up to 40 µM, indicating the methyl group is essential for recognition. - Ghrelin receptor agonist: Macrocycle methylation stabilized the bioactive conformation; ulimorelin (74) was 4–5× more potent for receptor activation than hit 73 and had acceptable PK for clinical development. - HCV NS3/4A protease inhibitors: Adding two methyls (75→76) improved GT-3a activity from IC50 51 nM to 8 nM; further optimization (77) with CF3 and an additional methyl gave IC50 4.8 nM and improved in vivo distribution and metabolic stability. - Class I HDAC macrocycles: Removing a methyl (78) reduced potency to IC50 69–110 nM (HDAC1–3) vs prototype 79 (3.1–8.9 nM); adding a second methyl (80) modestly decreased potency to 11–21 nM, indicating optimal fit with a single methyl. - Trypanocidal agents: 2-methyl-4-nitro imidazole–N-arylhydrazone hybrids produced best derivative 83 with IC50 206.98 µM against trypomastigotes. - Antibacterials: Furoxan-sulfonylhydrazones identified 88 as most potent; the methyl group was a key structural feature. - Bis-(3-indolyl)methane phosphonates: 5-methyl analogs (89, 91) showed greater antiproliferative potency vs unmethylated (90, 92) in ovarian/lung lines. - Physicochemical/PK optimization: NAH N-methylation (94 vs 95) significantly increased aqueous solubility with minimal activity change; 6-position methyls on morpholin-2-one antifungals improved plasma stability, with gem-dimethyl (99) most stable; CHK1 inhibitor 101 retained potency (IC50 16.1 nM) and reduced hERG inhibition (35.5% at 10 µM) vs 100 (43.4%); MOR ligand 103 (2′-methyl indole) had ~7-fold lower hERG inhibition; PI3Kδ series introduced a "magic methyl" in linker to block oxidative metabolism, yielding 105 with favorable oral bioavailability and maintained potency (IC50 0.014 µM).

The compiled evidence demonstrates that judicious methyl insertion can substantially influence both pharmacodynamics and pharmacokinetics. By enhancing hydrophobic contacts, biasing conformations toward bioactive states, and filling complementary subpockets, methyl groups often increase potency and selectivity (e.g., EZH2/tazemetostat, κ-OR, CB receptors, HDACs). In parallel, methylation can tune ADMET properties: improving solubility by disrupting planarity/symmetry or conformationally restricting scaffolds; blocking metabolic soft spots; improving plasma stability through steric hindrance (gem-dimethyl); and mitigating off-target liabilities such as hERG inhibition. These findings validate the methyl group as a versatile and powerful medicinal chemistry tool, supporting its rational use in hit-to-lead and lead optimization across diverse targets and modalities, including fragments, macrocycles, and multitarget designs.

This review underscores the broad applicability of the methylation effect in small-molecule drug discovery and development. Highlighted by the tazemetostat case (>100,000-fold activity improvement through multiple methyl insertions), numerous examples show that appropriate methyl placement can enhance potency, selectivity, and physicochemical/PK profiles, including solubility, plasma stability, metabolic stability, and reduced hERG liability. From molecular recognition to ADMET modulation, the "magic methyl" remains a valuable lever for optimization. The curated examples aim to guide rational application of methylation strategies and deepen understanding of structure–activity relationships for new chemical entities.