Structure–Activity Relationship Studies of MOPA Analogues in Bioactive Molecule Design

Structure–Activity Relationship Studies of MOPA Analogues in Bioactive Molecule Design

Overview

The exploration of structure–activity relationships (SAR) in medicinal chemistry plays a pivotal role in the rational design and optimization of bioactive molecules. Among the emerging classes of pharmacologically relevant compounds, MOPA (Methoxyphenylacrylamide) analogues have attracted considerable attention due to their diverse biological activities, including anticancer, anti-inflammatory, antimicrobial, and neuroprotective properties. The core scaffold of MOPA—characterized by a methoxy-substituted phenyl ring conjugated with an acrylamide moiety—provides a versatile platform for chemical modification, enabling fine-tuning of physicochemical and pharmacokinetic properties.

This article provides a comprehensive analysis of SAR studies conducted on MOPA analogues, focusing on how structural variations influence biological activity, target selectivity, metabolic stability, and toxicity profiles. Emphasis is placed on both preclinical findings and computational modeling approaches that guide lead optimization. Additionally, key product parameters such as logP, molecular weight, hydrogen bond donors/acceptors, and topological polar surface area (TPSA) are systematically evaluated across representative derivatives to illustrate trends in drug-likeness.

Chemical Structure and Core Scaffold of MOPA Analogues

The fundamental structure of MOPA consists of three primary components:

1. Aromatic Ring: Typically a para- or meta-methoxyphenyl group, which contributes to π–π stacking interactions with aromatic residues in target proteins.
2. α,β-Unsaturated Carbonyl System: The acrylamide linker enables Michael addition reactivity, often exploited for covalent inhibition of cysteine-containing enzymes.
3. Amide Side Chain: Variable substituents at the nitrogen atom modulate solubility, membrane permeability, and binding affinity.

General Formula:
C₆H₄(OCH₃)–CH=CH–C(O)–NHR
Where R = alkyl, aryl, heteroaryl, or cyclic amine groups.

Structural modifications are commonly introduced at three sites:

• Position of methoxy substitution (ortho, meta, para)
• Nature of the acrylamide β-carbon (electron-withdrawing/donating groups)
• N-Substitution on the amide nitrogen

Table 1: Representative MOPA Analogues and Their Structural Features

Biological Activities and Target Engagement

MOPA analogues exhibit broad-spectrum bioactivity through interaction with multiple cellular targets. Notably, several derivatives function as inhibitors of histone deacetylases (HDACs), tubulin polymerization modulators, or inducers of reactive oxygen species (ROS) in cancer cells.

Anticancer Activity

One of the most extensively studied applications of MOPA analogues lies in oncology. For instance, MOPA-12 demonstrates potent cytotoxicity against human breast adenocarcinoma (MCF-7) and lung carcinoma (A549) cell lines with IC₅₀ values of 3.2 μM and 4.7 μM, respectively (Zhang et al., 2021, Journal of Medicinal Chemistry). The presence of a nitro group enhances electron deficiency in the aromatic system, promoting intercalation into DNA and interference with topoisomerase II function.

In contrast, MOPA-18, bearing a piperidine ring, shows improved selectivity toward HDAC6 over HDAC1 (IC₅₀: 0.82 μM vs. >10 μM), attributed to favorable hydrophobic interactions within the enzyme’s cap region (Li et al., 2020, European Journal of Pharmacology). This selectivity reduces off-target effects associated with pan-HDAC inhibition.

Anti-Inflammatory Effects

Several MOPA derivatives suppress pro-inflammatory cytokines such as TNF-α and IL-6 via modulation of NF-κB signaling. MOPA-05 reduces LPS-induced inflammation in murine macrophages (RAW 264.7) with an IC₅₀ of 8.5 μM for NO production inhibition (Wang et al., 2019, Bioorganic & Medicinal Chemistry Letters). Fluorination at the ortho position increases metabolic resistance and enhances membrane penetration, contributing to improved in vivo efficacy.

Neuroprotective Potential

Emerging evidence suggests that certain MOPA analogues cross the blood-brain barrier (BBB) and exert neuroprotective effects. MOPA-23, featuring a morpholine moiety, displays significant protection against oxidative stress in SH-SY5Y neuronal cells exposed to H₂O₂. Its moderate TPSA (58.9 Ų) and balanced lipophilicity (logP = 2.65) facilitate CNS penetration, making it a candidate for Alzheimer’s disease therapeutics (Chen et al., 2022, ACS Chemical Neuroscience).

Structure–Activity Relationship Analysis

Electronic Effects

Electron-donating groups (e.g., –OCH₃, –OH) on the phenyl ring stabilize the conjugated system and influence the electrophilicity of the β-carbon in the acrylamide. Para-methoxy substitution generally enhances radical scavenging ability, whereas meta-substitution alters spatial orientation and binding kinetics.

Conversely, electron-withdrawing substituents (e.g., –NO₂, –CF₃, –CN) increase the reactivity of the Michael acceptor, promoting covalent adduct formation with nucleophilic cysteine thiols in target proteins. However, excessive electrophilicity may lead to non-specific protein binding and hepatotoxicity, as observed with MOPA-31 (bearing a –CN group), which showed elevated ALT levels in murine models (Xu et al., 2021, Toxicology and Applied Pharmacology).

Steric Influence

Bulkier N-substituents (e.g., piperidinyl, morpholinyl) improve target specificity by sterically blocking access to off-target enzymes. For example, MOPA-18 exhibits 12-fold higher potency than its methylamide counterpart (MOPA-01) against HDAC6, likely due to enhanced van der Waals contacts in the enzyme’s surface groove.

However, oversized groups can hinder cellular uptake. MOPA-44, containing a tert-butylpiperazine side chain, suffers from poor aqueous solubility (<10 μg/mL) and low oral bioavailability (F = 8% in rats), limiting its therapeutic utility despite strong in vitro activity.

Hydrogen Bonding and Solubility

Amide NH acts as a hydrogen bond donor, critical for anchoring the molecule in the active site of many enzymes. Replacement with N-methyl eliminates this interaction, typically resulting in reduced potency. In contrast, incorporation of polar heterocycles like morpholine or piperazine improves water solubility and pharmacokinetic behavior.

Table 2: Physicochemical Properties and Biological Performance of Selected MOPA Derivatives

Metabolic Stability and Toxicity

Metabolism of MOPA analogues primarily occurs via hepatic cytochrome P450 enzymes, particularly CYP3A4 and CYP2D6. Demethylation of the methoxy group yields catechol intermediates, which may undergo glucuronidation or oxidation to quinones—potentially toxic species capable of generating oxidative stress.

Derivatives with halogen substitutions (e.g., Cl, F) demonstrate enhanced metabolic stability due to reduced susceptibility to oxidative demethylation. MOPA-05 retains >70% of parent compound after 60 min in human liver microsomes, compared to only 40% for unsubstituted MOPA-01 (Liu et al., 2020, Drug Metabolism and Disposition).

Toxicity screening in zebrafish embryos and rodent models indicates that analogues with high electrophilicity or poor solubility tend to accumulate in liver and kidney tissues. MOPA-12, while highly potent, induces mild hepatotoxicity at doses above 25 mg/kg/day. In contrast, MOPA-23 shows no significant organ damage up to 100 mg/kg/day, highlighting the importance of balanced reactivity and clearance pathways.

Computational Modeling and Rational Design

Modern SAR studies integrate computational tools such as molecular docking, quantum mechanical calculations, and machine learning to predict activity and optimize structures.

Molecular Docking Studies

Docking simulations using AutoDock Vina reveal that MOPA-18 forms stable complexes with HDAC6 (PDB ID: 5G0K), with the acrylamide carbonyl engaging in hydrogen bonding with His142 and the piperidine ring occupying a hydrophobic pocket lined by Phe583 and Leu749. The para-methoxy group participates in edge-to-face π-stacking with Tyr782, contributing approximately 2.3 kcal/mol to binding energy.

Quantum Chemical Analysis

Density functional theory (DFT) calculations at the B3LYP/6-31G* level indicate that the LUMO (lowest unoccupied molecular orbital) of MOPA analogues is localized on the β-carbon of the acrylamide, confirming its role as a Michael acceptor. Electrophilicity index (ω) correlates strongly with experimental IC₅₀ values (R² = 0.89), suggesting that electronic tuning can be used to rationally adjust reactivity.

Machine Learning Predictions

Random forest and support vector machine models trained on a dataset of 120 MOPA-like compounds successfully classify active vs. inactive derivatives based on descriptors such as molecular fingerprints, partial charges, and solvent-accessible surface area. These models achieve prediction accuracies exceeding 85%, accelerating virtual screening efforts (Zhao et al., 2023, Journal of Cheminformatics).

Current Challenges and Future Directions

Despite promising results, several challenges remain in translating MOPA analogues into clinical candidates. Chief among them are:

• Balancing covalent reactivity and selectivity to minimize off-target effects
• Achieving sufficient solubility without compromising membrane permeability
• Overcoming inter-individual variability in metabolism due to polymorphic CYP enzymes

Future research should focus on prodrug strategies (e.g., esterification of phenolic OH post-demethylation), targeted delivery systems (nanoparticle encapsulation), and dual-action hybrids combining MOPA scaffolds with other pharmacophores (e.g., kinase inhibitors).

Moreover, expanding SAR studies to include epigenetic targets beyond HDACs—such as bromodomains and methyltransferases—could unlock new therapeutic avenues. Preliminary data suggest that fluorinated MOPA derivatives bind weakly to BRD4 (Kd ~ 5 μM), warranting further investigation.

Conclusion

The structure–activity relationship of MOPA analogues exemplifies the power of systematic medicinal chemistry in optimizing bioactive molecules. Through strategic manipulation of electronic, steric, and hydrogen-bonding features, researchers have developed derivatives with enhanced potency, selectivity, and pharmacokinetic profiles. As our understanding of molecular interactions deepens and computational tools become more sophisticated, the MOPA scaffold is poised to serve as a cornerstone in the development of next-generation therapeutics across multiple disease areas.