Unlocking Undruggable Targets: Ring Expansion Strategies for Natural Product-Derived Medium-Sized Rings

Chloe Mitchell Jan 09, 2026 176

This article provides a comprehensive guide for researchers on overcoming the synthetic challenges of constructing 8- to 11-membered medium-sized rings from natural products.

Unlocking Undruggable Targets: Ring Expansion Strategies for Natural Product-Derived Medium-Sized Rings

Abstract

This article provides a comprehensive guide for researchers on overcoming the synthetic challenges of constructing 8- to 11-membered medium-sized rings from natural products. It begins by exploring the foundational importance and unique challenges of these scaffolds in drug discovery, particularly for targeting 'undruggable' proteins. It then details core methodological approaches, including ring-expansion of polycyclic precursors and C-H functionalization-initiated strategies. The guide further addresses critical troubleshooting aspects, such as managing transannular strain and reaction selectivity, and validates these strategies through cheminformatic analysis of the novel chemical space they access. The synthesis of these concepts underscores ring expansion as a transformative strategy for converting complex natural products into diverse, drug-like screening libraries with significant biomedical potential.

The Why and Wherefore: The Critical Role and Synthetic Challenge of Medium-Sized Rings in Drug Discovery

The Therapeutic Potential and Chemical Space of Medium-Sized Rings

Medium-sized rings (MSRs), typically defined as 8- to 11-membered carbocycles and heterocycles, occupy a unique chemical space with significant yet underexplored therapeutic potential. Their conformational rigidity bridges the flexibility of macrocycles and the flatness of small rings, enabling selective, high-affinity binding to challenging biological targets. Their study is central to a broader thesis on ring expansion strategies for accessing natural product-derived MSR analogs.

Table 1: Therapeutic Areas and Targets for Medium-Sized Rings

Therapeutic Area Example Targets Known MSR-Based Modulators (Examples) Key Advantage of MSR Scaffold
Oncology KRAS, SHP2, Bcl-2, Kinases Dihydrocortistatin A (KRAS), Certain constrained peptides Disrupting large protein-protein interfaces (PPIs)
Infectious Diseases Viral proteases, Bacterial ribosomes Cyclic lipopeptides (e.g., Anidulafungin) Metabolic stability, membrane permeability
Neuroscience GPCRs, Ion channels, Neuropeptide receptors Ziconotide (ω-conotoxin MVIIA) Precise topology for subtype selectivity
Metabolic Disorders PPARs, FXR Some synthetic agonists/antagonists Optimal pre-organization for nuclear receptors

Chemical Space Analysis & Synthesis Strategies

Accessing the underexplored chemical space of MSRs is non-trivial due to transannular strain and entropic penalties during cyclization. Ring expansion from smaller, natural product-derived cores is a pivotal strategy.

Table 2: Key Ring Expansion Strategies for MSR Synthesis

Strategy Description Typical Ring Size Accessed Key Challenge
Fragmentation/Rearrangement C-C bond cleavage in a fused system followed by recombination. 8-10 Controlling regioselectivity.
Oxy-Cope / Aza-Cope [3,3]-sigmatropic rearrangement of divinylcycloalkanes. 8-9 Achieving correct precursor geometry.
Ring-Closing Metathesis (RCM) Using Grubbs catalysts to form large rings. 7-11 Mitigating dimerization/oligomerization; E/Z selectivity.
Photoinduced Expansion Light-mediated radical or electrocyclic ring opening/expansion. 8-11 Product stability, wavelength specificity.
Tandem Cyclization/Expansion Simultaneous formation and enlargement of a ring. Varies Complexity of reaction design.

G NP Natural Product (Small Ring Core) RE1 Ring Expansion (Fragmentation/Rearrangement) NP->RE1 RE2 Ring Expansion (Oxy-Cope/RCM) NP->RE2 MSR1 Medium-Sized Ring (8-11 membered) RE1->MSR1 RE2->MSR1 LIB Focused Library MSR1->LIB SCR Screening & SAR LIB->SCR LEAD Optimized Lead SCR->LEAD

Diagram Title: Ring Expansion Workflow from Natural Products

Application Notes & Detailed Protocols

Protocol 3.1: Ring-Closing Metathesis (RCM) for 9-Membered Lactam Synthesis

Objective: Synthesize a 9-membered lactam core via RCM, a key expansion step from a linear diene precursor. Thesis Context: This protocol exemplifies a direct cyclization approach to access MSR heterocycles, relevant for mimicking natural product pharmacophores.

Materials (Research Reagent Solutions):

  • Grubbs Catalyst 2nd Generation (G-II): Ruthenium carbene complex; drives RCM of dienes with high functional group tolerance.
  • Dichloroethane (DCE), degassed: Anhydrous, aprotic solvent suitable for metathesis reactions.
  • Linear Diene Precursor: Contains terminal alkene groups and protected amine/carbonyl for lactam formation.
  • Tetrahydrofuran (THF), anhydrous: For workup and dilution.
  • Silica Gel (60-200 mesh): For flash column chromatography purification.
  • Ethyl Acetate & Hexanes: For chromatography mobile phase.

Procedure:

  • In a flame-dried Schlenk flask under argon, dissolve the linear diene precursor (1.0 equiv, ~0.2 mmol) in degassed DCE (0.005 M concentration).
  • Add Grubbs II catalyst (0.05-0.10 equiv) in one portion under positive argon flow.
  • Heat the reaction mixture to 40°C and monitor by TLC (or LC-MS) for consumption of the starting material (typically 4-16 hours).
  • Upon completion, cool to room temperature and add a few drops of ethyl vinyl ether to quench the catalyst. Stir for 30 minutes.
  • Concentrate the mixture under reduced pressure. Purify the crude residue by flash chromatography on silica gel (gradient: 10% to 40% EtOAc in hexanes) to isolate the 9-membered cyclic product.
  • Characterize the product by ( ^1 \text{H} ) NMR, ( ^{13}\text{C} ) NMR, and HRMS. Key diagnostic: appearance of characteristic vinyl proton signals from the newly formed alkene (~5.3-5.6 ppm) and significant shifts in alkyl chain protons due to cyclization.
Protocol 3.2: Biological Evaluation: Inhibition of a PPI in an Oncology Pathway

Objective: Assess the activity of synthesized MSR compounds in a cell-based assay measuring disruption of the KRAS-PDEδ interaction, a representative challenging PPI. Thesis Context: This functional assay validates the thesis that MSRs obtained via expansion can modulate biologically relevant, druggable interfaces.

H KRAS KRAS (GTPase) PPI KRAS-PDEδ Complex KRAS->PPI PDED PDEδ (Chaperone) PDED->PPI Memb KRAS Mis-Localization (Cytosolic) PPI->Memb Required for Membrane Localization MSR MSR Compound Inhib Inhibition of Complex Formation MSR->Inhib Inhib->PPI  Disrupts Inhib->Memb Leads to Sig Attenuated Downstream Signaling (e.g., p-ERK↓) Memb->Sig Out Reduced Cell Proliferation Sig->Out

Diagram Title: MSR Inhibition of KRAS-PDEδ PPI Pathway

Materials (Research Reagent Solutions):

  • HEK293T Cells: Model cell line for transfection and pathway analysis.
  • Dual-Luciferase Reporter Assay System: Measures downstream pathway activity (Firefly) with internal control (Renilla).
  • KRAS and PDEδ Expression Plasmids: For validating direct target engagement in co-immunoprecipitation.
  • Anti-KRAS & Anti-PDEδ Antibodies: For Western blot and co-IP analysis.
  • CellTiter-Glo Luminescent Cell Viability Assay: Quantifies proliferation changes.

Procedure:

  • Reporter Gene Assay: Seed HEK293T cells in 96-well plates. Co-transfect with a SRF-RE reporter plasmid (Firefly luciferase, responsive to downstream KRAS/ERK signaling) and a Renilla luciferase control plasmid.
  • 24h post-transfection, treat cells with test MSR compounds (1-100 µM range) and positive/negative controls for 18-24 hours.
  • Lyse cells and measure Firefly and Renilla luciferase activity using the Dual-Luciferase kit. Calculate normalized Firefly/Renilla ratio.
  • Data Analysis: Express data as % inhibition of pathway activation relative to DMSO control. Generate dose-response curves to calculate IC(_{50}) values.
  • Validation (Co-Immunoprecipitation): In parallel, lyse treated cells expressing tagged KRAS and PDEδ. Immunoprecipitate KRAS and probe the blot for PDEδ to quantify complex disruption.
  • Proliferation Assay: Treat relevant cancer cell lines (e.g., MIA PaCa-2) with active MSR compounds for 72h. Measure viability using CellTiter-Glo. Link pathway inhibition to functional phenotype.

Table 3: Example Data from a Hypothetical MSR Series in KRAS-PDEδ Assay

Compound ID Core Size Synthesis Route Reporter IC(_{50}) (µM) Co-IP % Inhibition @10µM Cell Viability IC(_{50}) (µM)
MSR-08 8-membered lactam Fragmentation >50 15% >50
MSR-09 9-membered lactam RCM (Protocol 3.1) 8.2 ± 1.5 78% 12.4 ± 2.1
MSR-10 10-membered ether Oxy-Cope 15.7 ± 3.2 45% 25.9 ± 4.8
Dihydrocortistatin A (Ref) 9-membered amine Natural Product 0.15 ± 0.05 95% 0.8 ± 0.2

The Scientist's Toolkit: Essential Research Reagents

Table 4: Key Research Reagent Solutions for MSR Studies

Item Function & Relevance
Grubbs & Hoveyda-Grubbs Catalysts (G-II, HG-II) Essential for ring-closing metathesis (RCM) to form 8-11 membered rings with efficiency.
Degassed, Anhydrous Solvents (DCM, DCE, Toluene) Critical for moisture-/oxygen-sensitive reactions like RCM and metal-mediated expansions.
Chiral HPLC/UPLC Columns (e.g., CHIRALPAK) For separation and analysis of enantiopure MSRs, as chirality is often crucial for activity.
SPR/Biacore Instrumentation & Chips For label-free, quantitative analysis of MSR binding kinetics to purified protein targets.
CETSA (Cellular Thermal Shift Assay) Kits To confirm target engagement of MSR compounds in a cellular context.
Membrane Permeability Assay Kits (e.g., PAMPA) To evaluate the often-optimized passive permeability of conformationally constrained MSRs.
Stable Isotope-Labeled Building Blocks (¹³C, ¹⁵N) For mechanistic studies of ring expansion reactions and for advanced NMR structural analysis of MSRs.
Cryo-Electron Microscopy Grids & Reagents For determining high-resolution structures of MSR compounds bound to their protein targets.

Within the broader thesis on ring expansion strategies for natural product-derived medium-sized rings, defining and overcoming the inherent synthetic hurdles is paramount. Medium-sized rings (typically 8-11 membered) occupy a unique and underexplored region of chemical space that is highly relevant for drug discovery [1] [2]. Their distinct three-dimensional architectures, which blend conformational flexibility with defined spatial constraints, make them excellent candidates for modulating challenging biological targets, including those deemed "undruggable" [1] [3]. Naturally occurring medium-sized rings are found in numerous bioactive compounds, underscoring their biological precedence and utility [1].

However, their synthesis represents a long-standing challenge in organic chemistry. Traditional direct cyclization strategies, effective for smaller and larger rings, often fail due to the unique thermodynamic and kinetic barriers posed by medium-sized rings [2] [4]. The primary hurdles are transannular strain and entropic barriers. Transannular strain arises from unfavorable non-bonded interactions between atoms across the ring, leading to increased enthalpy [1] [5]. Concurrently, the reduced degrees of freedom during the cyclization of a linear precursor result in a large, unfavorable entropic penalty [2]. This combination makes the transition state for ring closure energetically disfavored. Consequently, medium-sized rings are severely underrepresented in commercial screening libraries and among approved pharmaceuticals [4]. This application note details the quantitative analysis of these barriers and provides practical ring-expansion protocols to circumvent them, enabling the systematic exploration of this valuable chemical space.

Quantitative Analysis of Synthetic Barriers

The challenges in forming medium-sized rings can be quantified through experimental and computational data, which inform the design of successful synthetic strategies.

Table 1: Experimental Yield Comparison: Direct Cyclization vs. Ring Expansion

Ring Size Target Compound Class Direct Cyclization Typical Yield Ring Expansion Strategy Ring Expansion Typical Yield Key Advantage of Expansion
8-membered Lactams (e.g., Benzolactams) <10% (Low) Amidyl Radical Migration & C-C Cleavage [2] 45-82% Avoids high-dilution, redox-neutral
9-membered Benzannulated Ethers/Lactones Low, mixture of oligomers Oxidative Dearomatization-Rearomatization (ODRE) [1] 51-90% Biomimetic, from stable bicyclic precursors
10-membered Polycyclic Steroid Derivatives Difficult via macrolactonization C-H Oxidation / Beckmann Rearrangement [4] 65-85% (2 steps) Leverages pre-existing ring scaffold
11-membered Functionalized Macrocycles Moderate, requires high dilution Tandem Umpolung ODRE Strategy [1] 60-88% High functional group tolerance

Table 2: Transannular Strain and Conformational Analysis

Ring Size Representative System (e.g., Cycloalkane) Estimated Transannular Strain Energy (kcal/mol) [5] Characteristic Non-Bonded Interaction Dominant Conformation(s) Implications for Synthesis & Reactivity
8-membered Cyclooctane ~9.7 H---H (1,5- and 1,6-interactions) Boat-Chair (BC) High strain favors ring-expansion from less strained fused systems.
9-membered Cyclononane ~12.6 Multiple H---H interactions [3^3]-Crown, Twisted Severe strain and multiple conformers complicate predictions.
10-membered Cyclodecane ~12.8 Severe H---H (1,6-interactions) Boat-Boat-Boat, Twisted "Preferred" conformation still involves significant internal strain.
11-membered Cycloundecane ~11.5 Reduced relative to C10 All-anti (extended) Lower strain allows for more flexible scaffolds in drug design.

Core Experimental Protocols

The following protocols exemplify modern strategies to overcome transannular and entropic barriers via ring expansion.

Protocol 1: Electrochemical Dehydrogenative Ring Expansion for Medium-Sized Lactams [2]

  • Principle: This method bypasses entropic penalties by using a rigid, fused bicyclic precursor. An electrochemically generated amidyl radical triggers a 1,5-hydrogen atom transfer (HAT) and subsequent C–C bond cleavage, leading to ring expansion.
  • Materials: Benzocyclic ketone substrate, primary amide, tetrabutylammonium hexafluorophosphate (NBu4PF6) electrolyte, anhydrous dichloroethane (DCE), carbon felt electrodes, undivided cell, constant current power supply.
  • Step-by-Step Procedure:
    • Cell Setup: In an undivided electrochemical cell equipped with a carbon felt anode and cathode, combine the benzocyclic ketone (1.0 equiv), primary amide (1.2 equiv), and NBu4PF6 (0.1 M) in anhydrous DCE.
    • Electrolysis: Perform constant current electrolysis at 5 mA under a nitrogen atmosphere at room temperature. Monitor reaction completion by TLC or LCMS (typically 3-5 hours, 2.0 F/mol charge passed).
    • Work-up: Once complete, quench the reaction by diluting with saturated aqueous NH4Cl. Extract the aqueous layer three times with ethyl acetate.
    • Purification: Combine the organic layers, dry over anhydrous Na2SO4, filter, and concentrate in vacuo. Purify the crude residue by flash column chromatography on silica gel to obtain the expanded medium-ring lactam.

Protocol 2: Sequential C–H Oxidation/Beckmann Rearrangement of Steroids [4]

  • Principle: This two-phase diversification strategy first installs a reactive ketone handle via site-selective C–H oxidation on a complex natural product core, then expands the ring via Beckmann rearrangement, mitigating the strain of direct cyclization.
  • Materials: Steroid substrate (e.g., DHEA, Estrone), Oxone (potassium peroxomonosulfate), acetonitrile, hydroxylamine hydrochloride, pyridine, phosphorus oxychloride (POCl3), sodium acetate buffer (pH 4.5).
  • Step-by-Step Procedure: Phase 1: Site-Selective C–H Oxidation to Ketone
    • Dissolve the steroid substrate (1.0 equiv) in acetonitrile/water (4:1).
    • Add Oxone (3.0 equiv) in one portion at 0°C.
    • Stir the reaction mixture, allowing it to warm to room temperature over 12 hours.
    • Quench with saturated Na2S2O3, extract with EtOAc, dry (Na2SO4), and concentrate. Purify via flash chromatography to isolate the ketone intermediate. Phase 2: Ring Expansion via Beckmann Rearrangement
    • Dissolve the ketone intermediate (1.0 equiv) in pyridine.
    • Add hydroxylamine hydrochloride (2.0 equiv) and heat to 80°C for 4 hours to form the oxime. Cool and concentrate.
    • Re-dissolve the crude oxime in dichloromethane. Cool to 0°C and add POCl3 (1.5 equiv) dropwise.
    • Stir at 0°C for 1 hour, then warm to room temperature and stir for an additional 3 hours.
    • Carefully quench the reaction by pouring into cold, saturated sodium acetate buffer (pH 4.5).
    • Extract with DCM, dry the combined organic layers (Na2SO4), and concentrate. Purify the residue by preparative HPLC to yield the medium-ring lactam-expanded steroid.

Protocol 3: Biomimetic Oxidative Dearomatization-Ring Expansion (ODRE) [1]

  • Principle: Inspired by biosynthesis, this method starts with a planar, aromatic or phenol-derived bicyclic system. Oxidative dearomatization generates a strained, reactive intermediate that undergoes spontaneous or nucleophile-triggered C–C bond cleavage and ring expansion.
  • Materials: Phenol-derived bicyclic substrate, hypervalent iodine reagent (e.g., PhI(OAc)2), nucleophile (alcohol, carboxylic acid), anhydrous solvent (CH2Cl2, MeCN).
  • Step-by-Step Procedure:
    • Charge a flame-dried flask with the bicyclic phenol substrate (1.0 equiv) and dissolve in anhydrous dichloromethane under N2. Cool to -40°C.
    • Add the hypervalent iodine reagent (e.g., PhI(OAc)2, 1.1 equiv) in one portion.
    • Stir at -40°C for 30 minutes to generate the dearomatized cyclohexadienone intermediate.
    • Add the desired nucleophile (e.g., a primary alcohol, 2.0 equiv). Warm the reaction mixture to 0°C and stir until complete by TLC.
    • Quench by adding saturated aqueous Na2S2O3. Warm to room temperature, separate layers, and extract the aqueous layer with DCM.
    • Dry the combined organic extracts (MgSO4), filter, and concentrate. Purify the product by flash chromatography to yield the benzannulated medium-sized ring (e.g., aryl ether, lactone).

Visualization of Strategies and Concepts

G start Linear or Small-Ring Precursor hurdle Key Hurdle: High TS Energy start->hurdle strat1 Strategy 1: Ring Expansion start->strat1 strat2 Strategy 2: Biomimetic ODRE start->strat2 strat3 Strategy 3: C-H Func. / Rearrangement start->strat3 ts High-Energy Transition State hurdle->ts problem Direct Cyclization (High ΔG‡) ts->problem mech1 Cleave C-C Bond in Fused System strat1->mech1 mech2 Oxidative Dearomatization & Rearomatization strat2->mech2 mech3 Insert Atoms via Named Rearrangement strat3->mech3 product Medium-Sized Ring (8-11 membered) mech1->product mech2->product mech3->product

Strategies to Overcome the Medium-Ring Synthetic Hurdle

G cluster_ODRE Oxidative Dearomatization-Ring Expansion (ODRE) [1] cluster_Electro Electrochemical Ring Expansion [2] Phenol Bicyclic Phenol (Stable, Planar) Ox Oxidant (e.g., PhI(OAc)₂) Phenol->Ox Dienone Dearomatized Cyclohexadienone Ox->Dienone Cleave Nucleophile Addition & C-C Bond Cleavage Dienone->Cleave ProductODRE Benzannulated Medium-Ring Ether/Lactone Cleave->ProductODRE Ketone Benzocyclic Ketone (Rigid Fused System) Electrolysis Constant Current Electrolysis Ketone->Electrolysis Amide Primary Amide Amide->Electrolysis Radical Amidyl Radical 1,5-HAT & Cleavage Electrolysis->Radical ProductElectro Medium-Sized Lactam Radical->ProductElectro StartNP Polycyclic Natural Product (e.g., Steroid) CHOx Site-Selective C-H Oxidation [4] StartNP->CHOx KetoneInt Ketone Intermediate CHOx->KetoneInt Rearr Beckmann Rearrangement KetoneInt->Rearr ProductNP Lactam-Expanded Natural Product Rearr->ProductNP

Workflow Comparison of Key Ring-Expansion Methodologies

The Scientist's Toolkit: Essential Research Reagents & Materials

Table 3: Key Research Reagent Solutions for Ring-Expansion Synthesis

Reagent / Material Function / Role in Protocol Key Consideration for Success
Hypervalent Iodine Reagents (e.g., PhI(OAc)₂, PIDA) Oxidant for dearomatization in ODRE protocols [1]. Must be fresh or properly stored (desiccated). Reactions are often moisture- and air-sensitive.
Electrochemical Setup (C felt electrodes, undivided cell, power supply) Enables redox-neutral generation of reactive radicals for bond cleavage [2]. Requires rigorous drying of solvent/electrolyte. Nitrogen atmosphere is critical for reproducibility.
Oxone (Potassium Peroxomonosulfate) Terminal oxidant for selective C–H oxidation to ketones on complex scaffolds [4]. Reaction pH and temperature control crucial for site-selectivity on polyfunctional molecules.
Phosphorus Oxychloride (POCl₃) Lewis acid catalyst for in situ activation of oximes in Beckmann rearrangements [4]. Highly moisture-sensitive and corrosive. Must be handled with care under inert atmosphere.
Strained Bicyclic Precursors (e.g., [3.2.1] or [4.3.0] systems) Substrates for ring-expansion via cleavable C–C bonds; reduce entropic penalty [1]. Synthesis often requires multi-step sequences. Conformation and stereochemistry are critical.
Tetrabutylammonium Hexafluorophosphate (NBu₄PF₆) Supporting electrolyte for electrochemical reactions [2]. Must be of high purity and thoroughly dried to prevent side reactions and ensure conductivity.
Dimethyl Acetylenedicarboxylate (DMAD) Two-carbon insertion reagent for formal [2+2] cycloaddition/ring expansion sequences [4]. A highly reactive dienophile/alkyne. Reactions can be exothermic; controlled addition is needed.

Why Ring Expansion? Overcoming the Limits of Direct Cyclization

The synthesis of medium-sized rings (8-11 membered) is a pivotal challenge in modern organic chemistry, particularly for accessing natural product-derived scaffolds with therapeutic potential. These structures are notably underrepresented in screening libraries despite their presence in numerous bioactive compounds and top-selling drugs [1]. The primary obstacle lies in the intrinsic limitations of direct end-to-end cyclization strategies. These conventional methods face significant enthalpic and entropic penalties due to transannular strain and unfavorable conformational preorganization, leading to low yields and poor functional group tolerance [1] [6].

Ring expansion strategies offer a powerful alternative by bypassing the high-energy transition states of direct cyclization. By strategically cleaving and reorganizing bonds within a pre-formed cyclic precursor, these methods provide efficient, scalable, and diversifiable routes to complex medium-sized rings. This article, framed within a broader thesis on ring expansion for natural product research, details the core limitations of direct methods, presents contemporary expansion protocols, and provides the practical toolkit for their application in drug discovery.

Direct Cyclization vs. Ring Expansion: A Comparative Analysis

The following table summarizes the fundamental challenges of direct cyclization and contrasts them with the strategic advantages offered by ring expansion approaches.

Table 1: Key Limitations of Direct Cyclization and Advantages of Ring Expansion

Aspect Direct Cyclization (Linear Precursors) Ring Expansion (Cyclic Precursors)
Thermodynamic Drive Unfavorable due to high transannular strain and loss of entropy during ring closure [1]. Leverages release of ring strain (e.g., from cyclopropanes, cyclobutanes) or aromatization as a driving force [1] [7].
Kinetic Competition High dilution required to favor intramolecular pathway over intermolecular oligomerization [6]. Intramolecular by design; avoids high-dilution conditions, enabling more scalable and concentrated reactions [6].
Conformational Preorganization Requires linear precursor to adopt a strained, pseudo-cyclic conformation. Starting cyclic structure provides inherent preorganization for the key bond-forming/breaking event.
Functional Group Tolerance Often limited by harsh conditions needed to achieve cyclization. Generally milder, more selective conditions can be employed (e.g., photochemical, enzymatic) [8] [7].
Strategic Versatility Primarily alters macrocycle perimeter. Enables skeletal editing—changing the core ring size and atom connectivity from a common precursor [8].
Library Synthesis Potential Low; each ring size often requires a bespoke linear synthesis. High; a single precursor can generate diverse ring sizes and functionalities via divergent pathways [1] [6].

Core Ring Expansion Methodologies: Application Notes and Protocols

This section provides detailed experimental protocols for three representative ring expansion strategies, highlighting their mechanism, scope, and utility in library synthesis.

Protocol 1: Conjugate Addition/Ring Expansion (CARE) Cascade for Lactam Synthesis

This versatile one-pot cascade builds medium-sized and macrocyclic lactams from smaller cyclic precursors without high-dilution conditions [6].

  • Principle: N-Acylation of a lactam forms an acryloyl imide. Conjugate addition of a primary amine triggers a ring expansion cascade via nucleophilic attack on the lactam carbonyl, culminating in ring opening and re-lactamization to form a larger ring [6].
  • Key Advantages: Operationally simple, air/moisture insensitive, excellent functional group tolerance, and highly adaptable for library synthesis.

Detailed Experimental Protocol [6]:

  • Preparation of Acryloyl Imide (3): Dissolve the starting lactam (1) (1.0 equiv) and acryloyl chloride (1.1 equiv) in dry dichloromethane (DCM, 0.1 M) under nitrogen. Add triethylamine (2.2 equiv) dropwise at 0°C. Stir the reaction mixture at room temperature for 2-3 hours (monitor by TLC). Quench with water, extract with DCM, dry the combined organic layers over anhydrous MgSO₄, filter, and concentrate in vacuo. Purify the residue by flash column chromatography to obtain acryloyl imide 3.
  • CARE Cascade Reaction: Dissolve acryloyl imide 3 (1.0 equiv) and the desired primary amine 4 (1.1 equiv) in methanol (0.5 M). Stir the reaction at room temperature for 4 hours.
  • Work-up and Purification: Concentrate the reaction mixture directly under reduced pressure. Purify the crude residue via flash column chromatography to isolate the expanded ring lactam product 6.
  • Representative Yield Data: This protocol has been successfully applied to 6-, 7-, 8-, and 9-membered ring imides, generating 10-, 11-, 12-, and 13-membered lactams, respectively. Yields for 36 diverse examples ranged from 51% to 96%, demonstrating broad applicability [6].
Protocol 2: Chemoenzymatic Skeletal Editing via C–H Oxidation-Rearrangement

This hybrid strategy combines enzymatic C–H activation with classic rearrangement chemistry for site-selective ring expansion [8].

  • Principle: An engineered cytochrome P450 enzyme performs selective oxidation of an aliphatic C–H bond within a ring system to form a ketone. This ketone is then subjected to a Baeyer-Villiger oxidation (to insert an oxygen, forming a lactone) or ketone homologation (to insert a methylene unit, expanding the ring by one carbon) [8].
  • Key Advantages: Achieves precise, late-stage skeletal editing of complex natural products at unfunctionalized C–H sites, enabling rapid exploration of structure-activity relationships (SAR).

Detailed Experimental Protocol [8]:

  • Enzymatic Oxidation:
    • Prepare a solution of the substrate natural product (e.g., 0.1 mmol) in a suitable buffer (e.g., potassium phosphate, pH 7.4).
    • Add the engineered P450 enzyme (typically 1-5 mol%) and the required cofactor regeneration system (e.g., glucose-6-phosphate/glucose-6-phosphate dehydrogenase).
    • Initiate the reaction by adding NADP⁺. Incubate with shaking at 25-30°C for 6-24 hours.
    • Extract the reaction mixture with ethyl acetate, dry over Na₂SO₄, and concentrate. Purify to obtain the ketone intermediate.
  • Baeyer-Villiger Ring Expansion:
    • Dissolve the ketone intermediate (1.0 equiv) in DCM (0.05 M). Add a peroxy acid (e.g., meta-chloroperoxybenzoic acid, mCPBA, 1.5 equiv) at 0°C.
    • Warm to room temperature and stir until complete by TLC (typically 2-12 h).
    • Quench carefully with saturated Na₂S₂O₃ solution, extract with DCM, dry, and concentrate. Purify to yield the ring-expanded lactone.
  • Application Note: The regioselectivity of the initial oxidation is controlled by the engineered P450 variant, allowing expansion at different sites of the same molecule to generate diverse analogs from a single precursor [8].
Protocol 3: Tandem Photocycloaddition-Ring Expansion to Fused Systems

This sequence constructs complex bicyclic frameworks through a light-driven cyclization followed by a strain-driven ring expansion [7].

  • Principle: An intramolecular [2+2] photocycloaddition creates a strained cyclobutane-fused intermediate. Subsequent acid-promoted hydrolytic ring opening and retro-aldol rearrangement relieve the strain, yielding a larger, fused ring system [7].
  • Key Advantages: Rapidly builds molecular complexity and fused ring architectures that are difficult to access by other means.

Detailed Experimental Protocol [7]:

  • Synthesis of Cyclic Vinylogous Ester (1): Combine cyclopentane-1,3-dione (2.0 equiv), 1,3-propanediol (1.0 equiv), and p-toluenesulfonic acid (p-TSA, 0.02 equiv) in benzene (0.25 M). Reflux using a Dean-Stark apparatus for 20 hours to remove water. After standard aqueous work-up (quench with NaHCO₃, extraction with DCM), purify by column chromatography to obtain 1 (67% yield).
  • Photochemical [2+2] Cycloaddition: Dissolve compound 1 in methanol (0.01 M) in a quartz tube. Irradiate with a 254 nm UV lamp under an inert atmosphere for 24-48 hours. Monitor by TLC. Concentrate the mixture and purify by column chromatography to afford the strained tetracyclic product 2 in 86% yield.
  • Acid-Promoted Ring Expansion: Dissolve photoproduct 2 in acetonitrile/water (2:1 v/v, 0.05 M). Add p-TSA (5.0 equiv). Reflux the reaction mixture for 40 hours. Cool, concentrate, and purify by column chromatography to obtain the fused [5.3.0] bicyclic triketone 4 in 75% yield.

Visualizing Ring Expansion Strategies

G cluster_direct Direct Cyclization Path cluster_expansion Ring Expansion Path Start Synthetic Objective: Medium-Sized Ring DC Linear Precursor Start->DC Conventional Approach RE Cyclic Precursor (Smaller Ring) Start->RE Proposed Strategy DC_Challenge Major Challenges DC->DC_Challenge High-Dilution Conditions DC_Fail Low Yield Oligomerization DC_Challenge->DC_Fail Disfavored RE_Strategy Expansion Strategy RE->RE_Strategy RE_Success Target Medium-Sized Ring (High Yield) RE_Strategy->RE_Success Strain-Driven or Functionalization

Diagram 1: Strategic choice between direct cyclization and ring expansion

G Substrate Acryloyl Imide (e.g., 7-Membered) Int1 Michael Adduct Substrate->Int1 Amine Int2 Tetrahedral Intermediate Int1->Int2 Intramolecular Attack Product Expanded Lactam (e.g., 11-Membered) Int2->Product Ring Opening Amine Primary Amine R-NH₂ Arrow1 1. Conjugate Addition Arrow2 2. N-Acylation & Cleavage Arrow3 3. Re-Lactamization

Diagram 2: Mechanism of the conjugate addition/ring expansion cascade

G cluster_enzymatic Enzymatic Step cluster_chemical Chemical Rearrangement NP Complex Natural Product Substrate P450 Engineered P450 Enzyme NP->P450 Site-Selective C-H Oxidation Ketone Ketone Intermediate P450->Ketone BV Baeyer-Villiger Oxidation Ketone->BV KH Ketone Homologation Ketone->KH Lactone Ring-Expanded Lactone BV->Lactone Insert O KetoneExp Ring-Expanded Ketone KH->KetoneExp Insert CH₂

Diagram 3: Chemoenzymatic skeletal editing workflow for ring expansion

The Scientist's Toolkit: Key Reagents & Materials

Table 2: Essential Research Reagents for Ring Expansion Methodologies

Reagent/Material Primary Function Representative Use Case
Acryloyl Chloride Introduces the Michael acceptor moiety for conjugate addition. Synthesis of acryloyl imide precursors in the CARE cascade [6].
Engineered P450 Enzymes Catalyze regioselective oxidation of unactivated aliphatic C–H bonds. Generating ketone handles for chemoenzymatic skeletal editing [8].
Primary Amines (R-NH₂) Act as nucleophiles to initiate ring expansion cascades; major source of diversity. Core component in CARE reactions; varying R-group builds library diversity [6].
mCPBA (meta-Chloroperoxybenzoic acid) Common peroxy acid for Baeyer-Villiger oxidation. Converting ketone intermediates to lactones, thereby expanding the ring [8].
UV Photoreactor (254 nm) Provides high-energy UV light to drive [2+2] photocycloadditions. Key equipment for constructing strained cyclobutane intermediates in tandem expansions [7].
p-Toluenesulfonic Acid (p-TSA) Mild Brønsted acid catalyst for acetal formation/cleavage and rearrangement. Used in both substrate synthesis (acetal formation) and acid-promoted ring expansion steps [7].
Palladium Catalysts (e.g., Pd(dppf)Cl₂) Catalyze cross-coupling reactions for post-expansion functionalization. Enabling Suzuki-Miyaura coupling to diversify side chains on expanded ring lactams [6].

The persistent challenges in pharmaceutical discovery, characterized by high attrition rates and a lack of first-in-class therapies, have been attributed to the structural simplicity and limited three-dimensionality of conventional screening libraries [9]. In response, the Complexity-to-Diversity (CtD) strategy has emerged as a powerful design principle. This approach exploits the inherent structural and stereochemical complexity of natural products (NPs) as ideal starting platforms, applying synthetic transformations—particularly ring distortion and expansion reactions—to rapidly generate diverse, complex, and novel molecular scaffolds that remain within biologically relevant chemical space [9] [10] [11].

This article frames the CtD strategy within the specific context of advancing ring expansion methodologies for synthesizing medium-sized rings (8-11 members) and macrocycles. These ring systems are prized in medicinal chemistry for their ability to target challenging protein interfaces but are notoriously difficult to access via traditional end-to-end cyclization due to transannular strain and entropic penalties [6]. Ring expansion offers a strategic solution, building complexity from smaller, more readily accessible cyclic precursors [6] [12]. Herein, we detail key application notes and experimental protocols that demonstrate how contemporary ring expansion tactics are operationalizing the CtD paradigm, enabling the systematic exploration of underexplored chemical territory derived from natural product architectures.

Ring Expansion Methodologies as Central CtD Tactics

Ring expansion reactions are cornerstone transformations in CtD, enabling dramatic scaffold remodeling from a single complex starting material. The following table summarizes key modern ring expansion strategies relevant to the synthesis of NP-derived medium-sized rings.

Table 1: Key Ring Expansion Strategies in Complexity-to-Diversity Synthesis

Strategy Core Transformation Ring Size Access Key Advantage for CtD Representative Citation
Conjugate Addition/Ring Expansion (CARE) Cascade Michael addition followed by lactam ring expansion. 8- to 14-membered lactams. One-pot, functional group tolerant; enables rapid library synthesis from a common imide precursor. [6]
Tandem Photocycloaddition-Ring Expansion Intramolecular [2+2] photocycloaddition followed by acid-promoted retro-aldol ring opening. Fused 5,7- and other bicyclic systems. Uses light to generate high-energy intermediates; rapidly builds polycyclic complexity. [7]
Electrocatalytic [3+2] Ring Expansion Electrochemical insertion of small molecules (e.g., CO₂, acetone) into epoxides. 5-membered heterocycles (dioxolanes, oxazolines). Green chemistry conditions; uses electrons as redox reagents; leverages CO₂. [12]
Dearomatization/Carbonylation Cascade Palladium-catalyzed carbonylation and intramolecular indole dearomatization. Spirocyclic and fused polycyclic scaffolds. Converts flat arenes into 3D architectures; introduces multiple stereocenters. [11]

These methodologies exemplify how ring expansion serves as a critical driver for scaffold diversity. For instance, the CARE cascade can generate a library of 67 novel macrocyclic and medium-sized ring lactams from simple lactam precursors, demonstrating exceptional functional group compatibility and yielding compounds primed for further side-chain or ring elaboration [6] [13]. Similarly, the divergent synthesis of pseudo-natural products (PNPs) from a common indole-based intermediate via dearomatization cascades showcases how a single complex starting point can yield 154 PNPs across eight distinct structural classes [11].

Application Notes & Detailed Experimental Protocols

Application Note: Generating a Lactam Library via CARE Cascade

Objective: To rapidly synthesize a diverse library of functionalized macrocyclic and medium-sized ring lactams for biological screening [6]. Concept: The CARE cascade converts readily available acryloyl imides into larger lactams. The process begins with a conjugate addition of a primary amine, which triggers a ring expansion. This is followed by sequential side-chain and ring elaboration steps to maximize diversity from a limited set of advanced intermediates [13]. Outcome: A library of 67 novel compounds was synthesized and screened for antibacterial activity. While no potent antibacterial agents were identified, the study validated the efficiency of the approach for generating screening collections in an underexplored area of chemical space [6].

Part A: Initial CARE Reaction to Form Core Lactam Scaffold

  • Starting Material Preparation: Synthesize acryloyl imide 3 (1.0 equiv) by N-acylation of the corresponding parent lactam 1 with acryloyl chloride under standard conditions.
  • CARE Cascade:
    • Charge a round-bottom flask with acryloyl imide 3 (1.0 equiv, 0.2 mmol scale) and primary amine 4 (1.1 equiv).
    • Add anhydrous methanol (0.5 M concentration relative to 3) under a nitrogen atmosphere.
    • Stir the reaction mixture at room temperature for 4 hours. Monitor reaction completion by TLC or LC-MS.
    • Concentrate the mixture under reduced pressure and purify the residue by flash column chromatography (SiO₂, appropriate eluent gradient) to isolate the ring-expanded lactam product 6/7.
  • Yield & Scope: This one-pot protocol typically yields 60-95% for 10-membered rings and similar medium-sized systems. The reaction is general for a wide range of functionalized amines (e.g., containing boronic esters, aryl halides, protected amines).

Part B: Side-Chain Elaboration via Suzuki-Miyaura Cross-Coupling

  • Reaction Setup: In a microwave vial, combine CARE product containing an aryl halide handle (e.g., 11j, 1.0 equiv), arylboronic acid (1.5 equiv), and Pd(dppf)Cl₂·CH₂Cl₂ (5 mol%).
  • Add Solvents and Base: Add a degassed mixture of 1,4-dioxane and aqueous Na₂CO₃ (2.0 M, 3.0 equiv).
  • Heating: Seal the vial and heat at 50°C for 18 hours.
  • Work-up and Purification: Cool to room temperature, dilute with ethyl acetate, wash with water and brine. Dry the organic layer over anhydrous Na₂SO₄, concentrate, and purify by flash chromatography to yield biaryl product 13a-e.

Part C: Ring Elaboration via Boc-Deprotection and N-Functionalization

  • Boc Cleavage: Treat the Boc-protected lactam (e.g., 13d, 1.0 equiv) with 4 M HCl in 1,4-dioxane (10-15 vol) at room temperature for 1 hour.
  • Intermediate Work-up: Concentrate the reaction mixture in vacuo to obtain the crude amine hydrochloride salt.
  • N-Acylation (Representative Procedure): Redissolve the crude salt in dry CH₂Cl₂ (0.1 M). Cool to 0°C and add Et₃N (3.0 equiv), DMAP (0.1 equiv), and the desired acid chloride (1.2 equiv). Warm to room temperature and stir for 18 hours.
  • Purification: Quench with saturated NaHCO₃ solution, extract with CH₂Cl₂, dry, concentrate, and purify by flash chromatography to yield the difunctionalized final product 14a-c.

Objective: To construct three-dimensional spiroindolylindanone PNP scaffolds from planar indole precursors. Key Reaction: Palladium-catalyzed intramolecular carbonylation and indole dearomatization. Procedure:

  • Substrate Preparation: Synthesize bromoarene-tethered indole substrate 1a (1.0 equiv).
  • Catalyst System: In a sealed tube, combine 1a, Pd(OAc)₂ (10 mol%), XantPhos (12 mol%), and Na₂CO₃ (2.0 equiv).
  • CO Surrogate Addition: Add N-formylsaccharin (2a, 1.5 equiv) as a solid, safe CO source.
  • Solvent and Conditions: Add dry DMF (0.1 M). Purge the reaction mixture with nitrogen, seal the tube, and heat at 100°C for 16-24 hours.
  • Work-up and Purification: Cool, dilute with ethyl acetate, filter through a Celite pad, and wash thoroughly. Concentrate the filtrate and purify the residue by automated flash chromatography to afford the dearomatized spirocyclic product A1 in high yield (up to 86%).
  • Downstream Diversification: The product (A1) can be further diversified via reduction of the indolenine moiety with Hantzsch ester/PPTS or functionalization to create distinct PNP classes (B, C, etc.).

The Scientist's Toolkit: Key Research Reagent Solutions

Table 2: Essential Reagents for Ring Expansion-based CtD Synthesis

Reagent/Catalyst Function in CtD/ Ring Expansion Specific Example from Protocols
Acryloyl Imides Act as electrophilic precursors in CARE cascades; ring size dictates product ring size. Compound 3a (6-membered) yields 10-membered lactams [6].
Functionalized Primary Amines Serve as conjugate donors in CARE; side-chain introduces diversity and synthetic handles. Amines with boronic esters or aryl halides used in [6].
Pd(dppf)Cl₂·CH₂Cl₂ Catalyst for Suzuki-Miyaura cross-coupling; enables side-chain elaboration post-ring expansion. Used for biaryl formation in Part B of Protocol 1 [6].
N-Formylsaccharin Safe, solid CO surrogate for carbonylation reactions; crucial for generating carbonylative palladium intermediates. Key to the dearomatization cascade in Protocol 2 [11].
Pd(OAc)₂ / XantPhos Catalytic system for Pd-catalyzed dearomatization/carbonylation; ligand stabilizes intermediate. Used in the synthesis of spiroindolylindanones [11].
Hantzsch Ester Hydride source for selective reduction; used to reduce imines/indolenines to access new stereocenters. Converts class A PNPs to indoline-containing class B PNPs [11].

Cheminformatic Validation & Biological Relevance

The success of a CtD campaign is measured by the structural novelty and biological relevance of the generated collection. Cheminformatic analyses, such as Principal Moments of Inertia (PMI), are employed to quantify shape diversity. For example, a CtD library derived from quinine exhibited excellent spread across rod-like, disk-like, and spherical shape space, confirming significant three-dimensionality [10].

The biological relevance is affirmed through phenotypic screening. The diverse PNP collection synthesized via dearomatization [11] yielded bioactive compounds from four different structural classes, inhibiting distinct targets like Hedgehog signaling and tubulin polymerization. This demonstrates that CtD strategies, particularly those employing ring expansion, can efficiently navigate chemical space to discover novel bioactive chemotypes with complex, NP-like features.

Ring expansion strategies provide a robust and versatile synthetic engine for the Complexity-to-Diversity paradigm. By starting from natural product-inspired complexity and applying transformations like CARE cascades and dearomatative ring expansions, researchers can systematically access novel, three-dimensional, medium-sized ring systems that populate underrepresented regions of chemical space.

The future of this field lies in integrating these synthetic methodologies with predictive analytics and machine learning. The vast chemical data in resources like PubChem (containing >119 million compounds) [14] can be mined to inform the design of new ring expansion reactions and predict the biological properties of novel scaffolds. Furthermore, combining CtD with DNA-encoded library technology or automated synthesis platforms will accelerate the generation and screening of these complex, diverse libraries, ultimately enhancing the discovery of next-generation therapeutics and chemical probes.

Visual Appendix: Experimental Workflow Diagrams

care_workflow start Parent Lactam (1) step1 N-Acylation (Acryloyl Chloride, Base) start->step1 imide Acryloyl Imide (3) step1->imide step2 CARE Cascade (1) Add Primary Amine (4) (2) MeOH, RT, 4h imide->step2 core Ring-Expanded Lactam (6/7) (Core Scaffold) step2->core branch1 Side-Chain Elaboration Path core->branch1 Contains Synthetic Handle branch2 Ring Elaboration Path core->branch2 Contains Protected Amine sc1 e.g., Suzuki Coupling (on aryl halide handle) branch1->sc1 sc2 e.g., O-Acylation (on alcohol handle) branch1->sc2 re1 Boc Deprotection (4M HCl/dioxane) branch2->re1 final_sc Elaborated Compound (e.g., 13a-e) sc1->final_sc sc2->final_sc re2 N-Functionalization (Acylation, Sulfonylation, etc.) re1->re2 final_re Difunctionalized Compound (e.g., 14a-n) re2->final_re

Diagram 1: Workflow for library synthesis via CARE cascade and elaboration.

dearomatization_workflow int Common Divergent Intermediate (e.g., 1a) pathA Pd-Catalyzed Cascade N-Formylsaccharin (CO) XantPhos, Na₂CO₃, DMF int->pathA classA Class A Spiroindolylindanone (A1) pathA->classA pathA2B Reduction Hantzsch Ester, PPTS classA->pathA2B pathA2E C-H Arylation Methyl 2-bromobenzoate Pd/XantPhos classA->pathA2E classB Class B Spiro-indoline-indanone pathA2B->classB pathB2C Amine Functionalization classB->pathB2C classE Class E Indoline-indanone- isoquinolinone pathA2E->classE classC Class C N-Functionalized Derivatives pathB2C->classC

Diagram 2: Divergent synthesis of PNP classes from a common intermediate.

Medium-sized rings, defined as cyclic structures containing 8 to 11 members, occupy a unique and underexplored niche in medicinal chemistry [15]. They offer a compelling blend of structural pre-organization and conformational flexibility, making them theoretically ideal for engaging challenging biological targets with high affinity and selectivity. This profile suggests significant potential for targeting protein-protein interactions and other "undruggable" spaces [2] [16].

However, a stark paradox exists between their therapeutic potential and their representation in approved therapeutics. Analysis of recent FDA approvals reveals a significant disparity: of 17 macrocyclic and medium-sized ring drugs approved in the past five years, only two (Lurbinectedin and Lefamulin) feature a core medium-sized ring scaffold [15]. This underrepresentation is primarily attributed to formidable synthetic challenges. Classical macrocyclization strategies face severe thermodynamic and kinetic penalties for medium rings due to entropic losses upon ring closure and destabilizing transannular strain [15] [2].

This application note frames the benchmarking of medium-sized rings within the critical research thesis that ring expansion strategies are essential to overcome these synthetic barriers and unlock the latent potential of this chemical space. By benchmarking current success and providing detailed protocols for ring expansion, this document aims to equip researchers with the tools to translate the unique properties of medium-sized rings into viable clinical candidates.

Quantitative Benchmarking: Macrocycles vs. Medium-Sized Rings

A quantitative analysis of approved drugs and clinical-stage candidates highlights the current landscape and the opportunity for medium-sized rings.

Table 1: Benchmarking Approved Macrocyclic and Medium-Sized Ring Drugs (Past 5 Years) [15]

Drug Name (Brand) Ring Type Ring Size (Members) Primary Indication Target / Mechanism of Action
Lurbinectedin (Zepzelca) Medium 7 (embedded in larger system) Small Cell Lung Cancer Binds DNA minor groove, inhibits transcription
Lefamulin (Xenleta) Medium 13 (pleuromutilin core) Bacterial Pneumonia Inhibits bacterial ribosome (50S subunit)
Voclosporin (Lupkynis) Macrocycle 23 (cyclic peptide) Lupus Nephritis Calcineurin inhibitor (immunosuppressant)
Terlipressin (Terlivaz) Macrocycle 12 (cyclic peptide analog) Hepatorenal Syndrome Vasopressin receptor agonist
Lorlatinib (Lorbrena) Macrocycle 12 ALK+ NSCLC ALK/ROS1 tyrosine kinase inhibitor
Examples of other macrocycles (total of 15) Macrocycle ≥12 Various (Oncology, Infection) Various

Table 2: Analysis of Compound-Target Pairs in ChEMBL (Illustrative Subset) [17] This dataset facilitates comparison between drugs, clinical candidates, and bioactive compounds.

Dataset Metric Full Dataset Filtered Subset (BF100cdtd_dt)*
Total Compound-Target Pairs 614,594 583,398
Drug-Target Pairs (Known Interaction) 5,109 2,639
Clinical Candidate-Target Pairs (Known Interaction) 3,932 2,619
Estimated Proportion with Medium-Sized Rings Very Low (<1%) Very Low (<1%)

*Subset filtered for targets with ≥100 compounds with measured activity and ≥1 known drug/clinical candidate interaction [17].

Key Benchmarking Insights:

  • Severe Underrepresentation: Medium-sized rings constitute a negligible fraction of successful drugs and annotated compound-target pairs in major databases, despite their attractive physicochemical profile.
  • Synthetic Origin Dominates: The two approved medium-sized ring drugs are derived from synthetic or semi-synthetic processes, underscoring the difficulty of accessing this chemotype from simple linear precursors.
  • Target Diversity: Approved macrocycles address a wide range of targets (enzymes, receptors, ion channels, protein-protein interactions), proving the versatility of large-ring scaffolds [17]. This establishes a performance benchmark for medium-sized rings to match.

Detailed Experimental Protocols for Ring Expansion Synthesis

Overcoming the medium-sized ring synthesis challenge requires moving beyond direct cyclization. The following protocols detail innovative ring expansion strategies that are more efficient and generalizable.

Protocol 1: Oxidative Dearomatization-Ring Expansion-Rearomatization (ODRE) for Benzannulated Medium Rings

Principle: This biomimetic, diversity-oriented strategy transforms readily available bicyclic phenols into polycyclic intermediates that undergo controlled fragmentation and expansion to form benzannulated medium-sized rings (9-11 members) [2].

Materials:

  • Substrate: Bicyclic phenol compound (e.g., 3 from Scheme 2a in [2]).
  • Oxidant: (Diacetoxyiodo)benzene (PIDA) or Phenyliodine bis(trifluoroacetate) (PIFA).
  • Nucleophile: Acid, phenol, primary alcohol, tertiary alcohol (for functionalization).
  • Solvent: Anhydrous Dichloromethane (DCM) or Acetonitrile (MeCN).
  • Conditions: Inert atmosphere (N₂ or Ar), anhydrous.

Procedure:

  • Oxidative Dearomatization: Dissolve the bicyclic phenol (1.0 equiv) in anhydrous DCM (0.05 M) under N₂ at -78°C. Add the hypervalent iodine oxidant (PIDA or PIFA, 1.1 equiv) in one portion. Stir for 30-60 minutes, allowing the formation of a polycyclic cyclohexadienone intermediate.
  • Ring Expansion: In a separate flask, prepare the desired nucleophile (e.g., a carboxylic acid, 1.5 equiv). Warm the reaction mixture from Step 1 to 0°C. Slowly add the nucleophile via syringe. The reaction proceeds via cleavage of the scissile bond (highlighted in red in the precursor) and capture by the nucleophile, effecting the ring expansion.
  • Rearomatization & Work-up: Allow the reaction to warm to room temperature and stir until completion (monitor by TLC). Quench by adding a saturated aqueous solution of sodium thiosulfate. Extract the aqueous layer with DCM (3x). Combine the organic layers, dry over anhydrous MgSO₄, filter, and concentrate under reduced pressure.
  • Purification: Purify the crude product via flash column chromatography to yield the functionalized benzannulated medium-ring product.

Protocol 2: Electrochemical Oxidative Ring Expansion to Medium-Sized Lactams

Principle: This sustainable method uses electrochemical oxidation to generate amidyl radicals from amides, which subsequently induce migratory ring expansion of fused benzocyclic ketones to form 8-11 membered lactams [2].

Materials:

  • Reactants: Benzocyclic ketone (e.g., tetralone derivative, 1.0 equiv), primary amide (1.5 equiv).
  • Electrolyte: Tetrabutylammonium hexafluorophosphate (NBu₄PF₆), 0.1 M.
  • Solvent: Acetonitrile (MeCN)/water mixture.
  • Equipment: Undivided electrochemical cell, carbon cloth electrodes (anode and cathode), potentiostat.

Procedure:

  • Cell Setup: In an undivided electrochemical cell equipped with carbon cloth electrodes, combine the benzocyclic ketone, primary amide, and NBu₄PF₆ electrolyte in a 9:1 mixture of MeCN/H₂O (0.1 M wrt ketone).
  • Electrolysis: Apply a constant current (e.g., 5 mA/cm²) under air atmosphere at room temperature. The reaction typically requires 2.5-3.0 F/mol of electricity. Monitor reaction progress by TLC or LCMS.
  • Mechanistic Workflow: The electrochemical oxidation generates a nitrogen-centered radical from the amide. This adds to the ketone, followed by a 1,2-alkyl shift/ring expansion via C–C bond cleavage, culminating in a rearomatization step to deliver the medium-ring lactam.
  • Work-up & Purification: Upon completion, turn off the current. Dilute the reaction mixture with water and extract with ethyl acetate (3x). Dry the combined organic layers over Na₂SO₄, filter, and concentrate. Purify the residue via flash column chromatography.

Visualization of Synthesis and Benchmarking Workflows

G NP1 Linear or Small-Cycle Precursor NP2 Key Challenge: Transannular Strain & Entropy NP1->NP2 faces NP4 Ring Expansion Strategy (e.g., ODRE, Electrochemical) NP1->NP4 via transformation NP3 Direct Cyclization (High Dilution, Low Yield) NP2->NP3 leads to NP2->NP4 overcome via NP5 Strained or Activated Polycyclic Intermediate NP4->NP5 NP6 Controlled Bond Cleavage & Insertion/Migration NP5->NP6 NP7 Medium-Sized Ring Scaffold (8-11 membered) NP6->NP7 NP8 Functionalization & Library Synthesis NP7->NP8 NP9 Diverse Medium-Ring Compound Library NP8->NP9

Diagram 1: Strategic Workflow for Medium-Sized Ring Synthesis

G ND1 FDA-Approved Drugs (Past 5 Years) ND2 Macrocycles (≥12 members): 15 drugs ND1->ND2 ND3 Medium-Sized Rings (8-11 members): 2 drugs ND1->ND3 ND7 Very Low Representation of Medium-Ring Chemotype ND3->ND7 exemplifies ND4 ChEMBL Compound-Target Pairs Dataset [17] ND5 >600,000 Total Pairs ND4->ND5 ND6 ~5,100 Known Drug-Target Pairs ND4->ND6 ND5->ND7 analysis shows ND6->ND7 ND9 Benchmarking Gap = Synthetic Opportunity ND7->ND9 defines ND8 Thesis: Ring Expansion Enables Access ND9->ND8

Diagram 2: Benchmarking Analysis Revealing a Critical Gap

The Scientist's Toolkit: Essential Reagents & Materials

Table 3: Research Reagent Solutions for Ring Expansion Chemistry

Reagent / Material Function & Role in Protocol Key Considerations & Alternatives
Hypervalent Iodine Reagents(e.g., PIDA, PIFA) Protocol 1: Serve as selective oxidants for the dearomatization step, generating key reactive dienone intermediates [2]. Must be handled under anhydrous conditions. PIFA is often more reactive. Alternative: Metal oxidants (e.g., PhI(OAc)₂ with BF₃·Et₂O).
Electrochemical Cell & Potentiostat Protocol 2: Provides controlled, reagent-free oxidation to generate reactive radical species, enabling sustainable ring expansion [2]. Undivided cells simplify setup. Carbon electrodes are cost-effective. Constant current mode is typical.
Supporting Electrolyte(e.g., NBu₄PF₆) Protocol 2: Dissolves in organic solvent to provide necessary ionic conductivity for efficient electrolysis. Should be electrochemically stable in the operating window. NBu₄BF₄ is a common alternative.
Orthogonally Protected Amino Acids & Solid-Phase Resin Cyclic Peptide Synthesis: For constructing linear precursors to peptide-based macrocycles/medium rings, as used in DOTA-TATE synthesis [15]. Fmoc/t-Bu strategy is standard. Resin choice (Wang, Rink) depends on C-terminal functionality.
Coupling Agents(e.g., HATU, DIC/Oxyma) Cyclic Peptide Synthesis: Activates carboxylic acids for amide bond formation during linear chain assembly and cyclization [15]. Minimizes racemization. DIC/Oxyma is preferred for difficult couplings.
Radical Initiators / Traps(e.g., AIBN, NFSI) General Ring Expansion: May be used in alternative photochemical or thermal radical ring expansion methodologies. AIBN requires heat. NFSI is a good source of N-centered radicals under milder conditions.

Building the Scaffolds: Core Methodologies for Ring Expansion of Natural Product Cores

The synthesis of medium-sized rings (8-11 membered) represents a significant and persistent challenge in organic chemistry, primarily due to unfavorable enthalpic and entropic factors associated with direct cyclization methods [1]. These strained systems, however, are privileged scaffolds in bioactive natural products and are crucial for targeting "undruggable" protein interfaces in modern drug discovery [2]. To circumvent traditional synthetic bottlenecks, ring-expansion strategies via the strategic cleavage of C–C and C=C bonds in pre-formed polycyclic systems have emerged as a powerful and efficient platform [1]. This approach aligns with broader thesis research focused on leveraging the inherent complexity of natural product-derived frameworks to efficiently access underexplored chemical space.

This methodology operates on a top-down "complexity-to-diversity" principle [1]. Instead of constructing a medium ring from simple linear precursors, a synthetically tractable, smaller polycyclic system—often inspired by a natural product core—is first assembled. A strategic bond within this congested framework is then selectively cleaved, triggering a ring-expanding rearrangement. This biomimetic strategy mirrors biosynthesis, where nature often builds complexity through the rearrangement of simpler, fused-ring precursors [18]. Techniques such as oxidative dearomatization, electrochemical generation of radical intermediates, and epoxide-driven rearrangements enable the selective scission of strong, unactivated bonds, providing direct access to medium-sized rings that are otherwise inaccessible [2] [19]. This article details the application notes and protocols for key ring-expansion reactions via C–C and C=C bond cleavage, providing researchers with practical methodologies to advance the synthesis of natural product-derived medium-ring libraries.

Quantitative Analysis of Ring Expansion Methodologies

The following tables summarize the key quantitative data and characteristics of prominent ring-expansion strategies via bond cleavage, providing a comparative overview for method selection.

Table 1: Key Ring-Expansion Methodologies via Bond Cleavage

Method & Core Transformation Key Cleavage Bond Typical Ring Size Formed Representative Yield Range Primary Advantages Key Limitations
Oxidative Dearomatization-Ring Expansion (ODRE) [2] C(sp²)-C(sp³) in cyclohexadienone 8-11 membered benzannulated rings 45-85% Broad scope of nucleophiles; builds molecular complexity rapidly. Primarily limited to phenolic substrates; can form isomer mixtures.
Electrochemical Dehydrogenative Expansion [2] C–C bond via amidyl radical migration 8-11 membered lactams 60-92% Oxidant-free, mild conditions; good functional group tolerance. Requires specialized electrochemical equipment.
Conjugate Addition/Ring Expansion (CARE) Cascade [6] C–N bond in imide (via addition) 10-14 membered lactams 51-95% (av. ~75%) One-pot, high-yielding, air/moisture insensitive; excellent for library synthesis. Restricted to lactam products; requires N-acryloyl imide precursor.
Epoxide C–C Bond Cleavage [19] C–C bond α to epoxide Variable (acyclic & cyclic) Highly variable Overrides typical C–O cleavage; access to unique oxygenated scaffolds. Often requires precise substitution patterns for selectivity.
Biomimetic Oxidative Cleavage [1] C=C bond in bridged polycycle Medium-ring lactones/lactams Good to excellent Mild, functional-group-tolerant; versatile for macrocycle synthesis. Requires synthesis of specific alkene-bridged polycyclic precursor.

Table 2: Physicochemical Profile of Medium-Sized Rings vs. Common Drug-like Rings

Property Typical Medium-Sized Ring (8-11 membered) [1] [6] Typical 5/6-Membered Aromatic Ring (Drug-like) [20] Implications for Drug Discovery
Fraction of sp³ Carbons (Fsp³) High Low Increased 3D complexity improves selectivity and reduces aromatic metabolic pathways.
Molecular Shape Pseudocyclic, flexible conformations Flat, rigid Better suited for targeting shallow, featureless protein-protein interaction surfaces.
Synthetic Accessibility Low (historically) Very High Ring-expansion strategies directly address this synthetic challenge [1].
Presence in Screening Libraries Severely underrepresented [1] Highly overrepresented [20] Novel biological mechanisms of action are more likely.
Representation in Approved Drugs <2% [1] >70% [20] Vast, underexplored chemical space for new lead discovery.

Detailed Experimental Protocols

Protocol 1: Biomimetic Oxidative Dearomatization-Ring Expansion (ODRE) for Benzannulated Medium Rings

This protocol describes the synthesis of benzannulated medium-sized rings via an oxidative dearomatization/ring expansion/rearomatization sequence, inspired by the Tan group's work [1] [2].

Materials:

  • Substrate: Bicyclic phenol compound (e.g., 1.0 equiv).
  • Oxidant: Hypervalent iodine reagent, such as (diacetoxyiodo)benzene (PIDA, 1.1 equiv).
  • Nucleophile: A wide variety can be used (e.g., carboxylic acids, phenols, primary/secondary alcohols, 2.0 equiv).
  • Solvent: Anhydrous dichloromethane (DCM) or acetonitrile (MeCN).
  • Additive: For reactions with alcohol nucleophiles, scandium(III) triflate (Sc(OTf)₃, 0.2 equiv) is used as a Lewis acid catalyst.
  • Work-up: Saturated aqueous sodium thiosulfate (Na₂S₂O₃), saturated aqueous sodium bicarbonate (NaHCO₃), brine.
  • Purification: Silica gel for column chromatography.

Procedure:

  • Reaction Setup: In a flame-dried round-bottom flask under an inert atmosphere (N₂ or Ar), dissolve the bicyclic phenol substrate (1.0 equiv) in anhydrous DCM (0.1 M concentration).
  • Oxidative Dearomatization: Cool the solution to 0°C. Add the hypervalent iodine oxidant (PIDA, 1.1 equiv) in one portion. Stir the reaction mixture at 0°C for 15-30 minutes, monitoring by TLC for consumption of the starting material. This step generates a reactive polycyclic cyclohexadienone intermediate.
  • Ring Expansion: In a separate vial, pre-mix the desired nucleophile (2.0 equiv) with Sc(OTf)₃ (0.2 equiv) if using an alcohol. Add this mixture directly to the cold reaction solution.
  • Rearomatization & Completion: Allow the reaction to warm to room temperature and stir for 2-12 hours. The ring expansion proceeds via nucleophilic attack followed by C–C bond cleavage and rearomatization.
  • Work-up: Quench the reaction by adding a saturated aqueous solution of Na₂S₂O₃ (to reduce excess iodine species). Transfer to a separatory funnel, dilute with DCM, and wash sequentially with Na₂S₂O₃, saturated NaHCO₃, and brine. Dry the organic layer over anhydrous MgSO₄, filter, and concentrate under reduced pressure.
  • Purification: Purify the crude residue by silica gel column chromatography using an appropriate eluent system (e.g., hexanes/ethyl acetate) to obtain the benzannulated medium-ring product.

Key Notes: This method is highly versatile for nucleophiles but is primarily applicable to phenolic substrates. The use of the umpolung variant, where an electron-rich arene attacks an electrophilic side chain, can circumvent this limitation and prevent undesired termination pathways [2].

Protocol 2: Electrochemical Oxidative Ring Expansion for Medium-Sized Lactams

This protocol outlines an electrochemical method for synthesizing medium-ring lactams via amidyl radical-induced C–C bond cleavage, as reported by Liu et al. [2].

Materials:

  • Substrate 1: Benzocyclic ketone (e.g., tetralone derivative, 1.0 equiv).
  • Substrate 2: Primary amide (1.5 equiv).
  • Electrolyte: Tetrabutylammonium hexafluorophosphate (Bu₄NPF₆, 0.1 M).
  • Electrodes: Graphite rod (anode), platinum plate (cathode).
  • Solvent: Anhydrous acetonitrile (MeCN).
  • Setup: Undivided electrochemical cell, potentiostat/galvanostat.
  • Work-up: Water, ethyl acetate.
  • Purification: Silica gel for column chromatography.

Procedure:

  • Cell Preparation: In an undivided electrochemical cell equipped with a graphite rod anode and a platinum plate cathode, combine the benzocyclic ketone (1.0 equiv), the primary amide (1.5 equiv), and Bu₄NPF₆ electrolyte (0.1 M final concentration) in anhydrous MeCN (0.05 M wrt ketone).
  • Electrolysis: Place the cell in a cooling bath to maintain a temperature of 25°C. Apply a constant current (e.g., 5 mA/cm²) and stir the reaction mixture vigorously. Monitor the reaction progress by TLC or LC-MS. The typical charge required is 3.0 F/mol.
  • Mechanism: The process involves anodic oxidation of the amide to generate a nitrogen-centered radical, which adds to the ketone. Subsequent homolytic cleavage of a C–C bond, facilitated by rearomatization, expands the ring to form the lactam.
  • Work-up: Upon completion, turn off the current and pour the reaction mixture into water. Extract the aqueous layer three times with ethyl acetate. Combine the organic extracts, wash with brine, dry over Na₂SO₄, filter, and concentrate.
  • Purification: Purify the crude product by silica gel column chromatography.

Key Notes: This method is a sustainable alternative to chemical oxidants. The use of an undivided cell simplifies the setup. The scope is effective for the synthesis of 8- to 11-membered benzolactams.

Protocol 3: Conjugate Addition/Ring Expansion (CARE) Cascade for Lactam Library Synthesis

This protocol describes a robust, one-pot cascade reaction to generate diverse medium and macrocyclic lactam libraries, based on the work of Unsworth and colleagues [6].

Materials:

  • Substrate: N-Acryloyl imide (derived from a cyclic lactam, 1.0 equiv).
  • Nucleophile: Primary amine (1.1 equiv), diverse functionalized amines can be used.
  • Solvent: Methanol (MeOH, 0.5 M).
  • No specialized atmosphere required. The reaction is air- and moisture-insensitive.
  • Work-up: None required for direct purification, or standard aqueous work-up.
  • Purification: Silica gel column chromatography or preparative HPLC.

Procedure:

  • Reaction: In a vial or round-bottom flask, combine the N-acryloyl imide (1.0 equiv) and the primary amine (1.1 equiv) in MeOH (0.5 M concentration). Stir the reaction mixture at room temperature.
  • Monitoring: The cascade reaction typically completes within 4 hours. Monitor by TLC or LC-MS. The sequence involves conjugate addition of the amine to the acryloyl group, followed by spontaneous ring expansion via C–N bond cleavage in the imide.
  • Work-up and Purification: After confirmation of completion, the product can often be isolated directly by concentration and purification, due to the cleanliness of the reaction. Optionally, the mixture can be concentrated and then partitioned between water and an organic solvent (e.g., ethyl acetate). The organic layer is dried (MgSO₄) and concentrated.
  • Purification: Purify the crude material by silica gel chromatography or preparative HPLC to obtain the ring-expanded lactam.

Key Notes: This is a highly practical method for library synthesis. The acryloyl imide precursors are readily prepared from commercially available lactams. The reaction tolerates a wide array of functional groups (e.g., boronic esters, halides, protected amines) on the primary amine, allowing for immediate downstream diversification via cross-coupling or functional group interconversion [6].

Mechanism and Workflow Visualization

G cluster_ODRE Oxidative Dearomatization-Ring Expansion (ODRE) cluster_CARE Conjugate Addition/Ring Expansion (CARE) Cascade Title Mechanistic Pathways for Strategic Bond Cleavage & Ring Expansion O1 Bicyclic Phenol O3 Oxidative Dearomatization O1->O3 O2 Hypervalent Iodine (PIDA) O2->O3 O4 Cyclohexadienone Intermediate O3->O4 O5 Nucleophile Addition & C-C Bond Cleavage O4->O5 + NuH O6 Medium-Sized Benzannulated Ring O5->O6 C1 N-Acryloyl Imide C3 Conjugate Addition C1->C3 C2 Primary Amine C2->C3 C4 Tetrahedral Intermediate C3->C4 C5 C-N Cleavage & Ring Expansion C4->C5 C6 Medium/Macrocyclic Lactam C5->C6 Start

Diagram 1: Two dominant mechanistic pathways for strategic bond cleavage and ring expansion.

G Title Workflow for Medium-Ring Library Synthesis via Strategic Cleavage S1 Natural Product Inspiration / Scaffold Design S2 Synthesis of Polycyclic Precursor S1->S2 S3 Strategic Bond Cleavage Reaction S2->S3 S4a Oxidative (ODRE) S3->S4a C-C Cleavage S4b Radical (Electrochemical) S3->S4b C-C Cleavage S4c Polar (CARE, Epoxide) S3->S4c C-N/C-C Cleavage S5 Ring-Expanded Core Scaffold S4a->S5 S4b->S5 S4c->S5 S6 Late-Stage Diversification S5->S6 S7 Biological Evaluation S6->S7 S8 SAR & Lead Optimization S7->S8 S8->S6 Feedback Loop

Diagram 2: A generalized workflow for constructing and screening libraries of medium-sized rings.

The Scientist's Toolkit: Research Reagent Solutions

Table 3: Essential Reagents and Materials for Ring Expansion via Bond Cleavage

Item Function & Role in Strategic Cleavage Example/Note
Hypervalent Iodine Reagents Serve as mild, selective oxidants for the dearomatization of phenolic substrates, generating key cyclohexadienone intermediates for ODRE sequences [1] [2]. (Diacetoxyiodo)benzene (PIDA), (Bis(trifluoroacetoxy)iodo)benzene (PIFA).
Lewis Acid Catalysts Activate electrophiles (e.g., cyclohexadienones, epoxides) to facilitate nucleophilic attack or control the regioselectivity of C–C bond cleavage [2] [19]. Scandium(III) triflate (Sc(OTf)₃), Boron trifluoride diethyl etherate (BF₃·OEt₂).
Electrochemical Setup Provides a green alternative to chemical oxidants, enabling the generation of radical intermediates (e.g., amidyl radicals) for bond homolysis and rearrangement under mild conditions [2]. Requires a potentiostat, undivided cell, graphite anode, and supporting electrolyte (e.g., Bu₄NPF₆).
Functionalized Amine Building Blocks Act as nucleophiles in CARE cascades and provide points of diversity for late-stage functionalization (LSF) after ring expansion. Amine handles are crucial for library synthesis [6]. Amines containing aryl halides, boronic esters, protected amines (Boc, Cbz), and alkenes/alkynes.
Cross-Coupling Catalysts Enable the diversification of ring-expanded scaffolds bearing halogen or boron handles, a critical step for exploring structure-activity relationships (SAR) [6]. Palladium catalysts such as Pd(dppf)Cl₂, Pd(PPh₃)₄, and associated bases/ligands.
Silica-Bound Scavengers Used for high-throughput purification of libraries generated via methods like CARE, where products are often amine-free, but excess starting amines need removal. Silica-bound isocyanate or aldehyde cartridges to scavenge primary amines.
Chiral Auxiliaries/Catalysts Impart stereocontrol during the ring-expansion step or in subsequent transformations, essential for synthesizing enantiopure natural product analogs. Chiral phosphoric acids, N,N'-dioxide ligands for scandium, chiral phase-transfer catalysts.

Introduction: ODRE Sequences in Ring Expansion Research Within the broader thesis on ring expansion strategies for accessing natural product-derived medium-sized rings (8- to 11-membered), the Oxidative Dearomatization-Ring Expansion-Rearomatization (ODRE) sequence represents a cornerstone biomimetic tactic [21]. This approach directly addresses the historical synthetic inaccessibility of benzannulated medium rings—privileged scaffolds in bioactive natural products that are critically underrepresented in synthetic screening libraries and approved drugs [21] [2]. By mimicking biosynthetic proposals invoked for alkaloids like protostephanine, the ODRE strategy circumvents the enthalpic and entropic penalties of direct cyclization, offering a flexible and efficient pathway to diverse chemotypes [21] [2]. This document provides detailed application notes and experimental protocols for implementing ODRE chemistry, serving as a practical guide for its use in diversity-oriented synthesis (DOS) and natural product-inspired drug discovery.

Core Principles and Strategic Advantages

The ODRE strategy transforms simple, readily synthesized bicyclic phenols into architecturally complex benzannulated medium rings. Its power lies in its biomimetic logic and operational simplicity, which overcomes traditional cyclization challenges such as transannular strain and unfavorable conformational entropy [2].

Mechanistic Logic: The sequence is driven by the powerful thermodynamic gain of rearomatizing a phenol ring adjacent to the scissile bond. Oxidative dearomatization first creates a reactive polycyclic cyclohexadienone. Subsequent activation of this intermediate triggers cleavage of a strategically chosen bond (the "scissile bond"), with the ensuing ring expansion being propelled by the concurrent reformation of a stable aromatic system [21].

Biomimetic Foundation: The approach is inspired by proposed biosynthetic pathways for alkaloids such as protostephanine and members of the erythrina family, where a similar ring-expanding rearomatization of a polycyclic intermediate is hypothesized [21] [22]. This biological precedent validates the chemical feasibility and efficiency of the transformation.

Synthetic Utility: The key advantage of ODRE over classical ring-closing metathesis or macrolactonization is its avoidance of the high-dilution conditions and substrate-specific yield fluctuations typical of medium-ring cyclizations [21]. It enables the modular assembly of diverse linkages (aryl ethers, diaryl ethers, lactones, biaryls) found in natural products from a common precursor platform [21] [2].

Diversity-Oriented Application: As a central tactic in complexity-to-diversity (CtD) and biomimetic synthesis, ODRE allows for the rapid generation of libraries that occupy chemical space overlapping with medium-ring natural products but distinct from flat, drug-like molecules, making it ideal for probing undruggable targets [2].

ODRE Reaction Mechanism

G Start Bicyclic Phenol Precursor Step1 1. Oxidative Dearomatization Start->Step1 Int1 Polycyclic Cyclohexadienone (Oxidized Intermediate) Step2 2. Activation & Bond Cleavage Int1->Step2 Int2 Activated Intermediate (Scissile Bond Elongated) Step3 3. Ring Expansion & Rearomatization Int2->Step3 End Benzannulated Medium-Ring Product Step1->Int1 Oxidant (e.g., PhI(OAc)₂) Step2->Int2 Reagent R-X (e.g., TMSOTf) Step3->End Driven by Aromatization

Detailed Experimental Protocols

Protocol 1: Standard ODRE Sequence for Benzannulated Medium-Ring Synthesis

This protocol details the synthesis of a model benzannulated 9-membered ring lactone from a bicyclic phenol precursor [21].

1. Synthesis of Bicyclic Phenol Precursor (Compound 4, [21])

  • Objective: Prepare gram-scale quantities of phenol 4 (C1-methyl substituted 6-hydroxytetralone derivative).
  • Procedure:
    • Alkylation: To a stirred solution of 6-hydroxytetralone (10.0 g, 57.1 mmol) in dry DMF (100 mL) under N₂, add K₂CO₃ (15.7 g, 114 mmol) and methyl iodide (4.3 mL, 68.5 mmol). Heat at 60°C for 12 h.
    • Work-up: Cool to RT, pour into ice-water (500 mL), and extract with EtOAc (3 × 150 mL). Wash combined organics with brine, dry over MgSO₄, filter, and concentrate.
    • Purification: Purify the crude residue by flash column chromatography (SiO₂, hexanes/EtOAc 4:1) to yield the C1-methylated tetralone.
    • Side Chain Installation (3-4 subsequent steps per [21]): Perform a series of alkylation, reduction, and cyclization steps as described in the supplementary information of [21] to construct the second ring and the alkoxyethyl side chain, ultimately yielding bicyclic phenol 4.

2. Oxidative Dearomatization to Polycyclic Cyclohexadienone (Compound 5, [21])

  • Reaction Setup: In a flame-dried round-bottom flask, dissolve phenol 4 (5.0 g, 15.8 mmol) in anhydrous CH₂Cl₂ (80 mL) under argon. Cool to 0°C.
  • Addition: Add (diacetoxyiodo)benzene (PhI(OAc)₂, 6.1 g, 19.0 mmol) in one portion.
  • Stirring: Stir the reaction mixture at 0°C for 1 h, then allow to warm to room temperature and stir for an additional 3 h (monitor by TLC).
  • Work-up: Quench by adding saturated aqueous Na₂S₂O₃ solution (50 mL). Separate layers and extract the aqueous layer with CH₂Cl₂ (2 × 50 mL). Combine organic layers, wash with brine, dry over Na₂SO₄, and concentrate.
  • Purification: Purify the crude product by flash chromatography (SiO₂, hexanes/EtOAc 3:1) to obtain tricyclic cyclohexadienone 5 as a white solid. Expected Yield: 70-85%.

3. Ring-Expanding Rearomatization to Medium-Ring Lactone

  • Critical Note: This step must avoid conditions that promote the competing dienone-phenol rearrangement [21].
  • Activation: In a dried flask under argon, dissolve cyclohexadienone 5 (1.0 g, 3.0 mmol) in anhydrous acetonitrile (15 mL). Add 2,6-di-tert-butylpyridine (0.14 mL, 0.66 mmol) as a proton scavenger.
  • Ring Expansion: Cool to -40°C. Add trimethylsilyl trifluoromethanesulfonate (TMSOTf, 0.65 mL, 3.6 mmol) dropwise. Stir at -40°C for 30 min, then gradually warm to 0°C over 2 h.
  • Quench & Rearomatization: Carefully add the reaction mixture to a stirred solution of saturated aqueous NaHCO₃ (30 mL) and EtOAc (30 mL). Stir vigorously for 30 min at RT to ensure complete desilylation and rearomatization.
  • Work-up: Separate layers, extract the aqueous layer with EtOAc (2 × 30 mL). Combine organics, wash with brine, dry over Na₂SO₄, and concentrate.
  • Purification: Purify via flash chromatography (SiO₂, hexanes/EtOAc 2:1) to afford the benzannulated 9-membered ring lactone. Expected Yield: 50-70%.

This advanced protocol employs an umpolung strategy to access substrates beyond phenols and prevent alternative termination pathways.

1. Preparation of Umpolung Precursor with Tertiary Alcohol

  • Objective: Synthesize a substrate where an electron-rich aromatic ring is tethered to an electrophilic side chain bearing a tertiary alcohol.
  • Key Step - Installation: Following the methodology in [2], perform a key coupling reaction (e.g., nucleophilic attack of an aryl metal species onto a keto-epoxide) to install the tertiary alcohol moiety adjacent to the future scissile bond.

2. Tandem Oxidative Activation and Ring Expansion

  • Reaction Setup: Dissolve the umpolung precursor (1.0 mmol) in a mixture of CH₂Cl₂ and hexafluoroisopropanol (HFIP) (9:1 v/v, 10 mL total) in a dry vial.
  • Oxidation/Activation: Add a hypervalent iodine reagent such as phenyliodine bis(trifluoroacetate) (PIFA, 1.2 mmol) at 0°C.
  • Tandem Cyclization: Stir the reaction mixture at 0°C for 10 minutes, then allow to warm to room temperature and stir for 1-3 hours. The reaction proceeds via initial oxidation to form a cationic tricyclic intermediate, which undergoes immediate ring expansion driven by rearomatization and the collapse of the tertiary alcohol to a ketone.
  • Work-up & Purification: Quench with aqueous Na₂S₂O₃, extract with EtOAc, dry, concentrate, and purify by chromatography. This method yields ketone-containing medium rings (e.g., haloaryl, aryl ether, heteroaromatic) without olefin isomer mixtures or solvent adducts [2].

This protocol uses an engineered enzyme system to perform enantioselective oxidative dearomatization, providing chiral intermediates for asymmetric ODRE sequences.

1. Enzyme Preparation

  • Biocatalyst: Use the flavin-dependent monooxygenase SorbC (wild-type or expressed in E. coli) [23].
  • Cofactor Regeneration: Employ a glucose/glucose oxidase system or direct NADPH recycling to maintain FADH₂ levels.

2. Substrate Engineering & Biocatalytic Reaction

  • Positioning Group Strategy: For substrates poorly accepted by wild-type SorbC, install a cleavable ester-based "positioning group" (e.g., a lipophilic chain) at the C1 position to mimic the native substrate's interaction with the enzyme's hydrophobic tunnel [23].
  • Reaction Conditions: Incubate the engineered phenol substrate (0.1-1.0 mmol) with SorbC (0.1 mol%) and the cofactor regeneration system in a phosphate buffer (pH 7.5, 50 mM) at 30°C for 12-24 h with gentle shaking.
  • Monitoring & Work-up: Monitor conversion by HPLC or TLC. Terminate by extracting with EtOAc (3x volume). Dry the combined organic layers and concentrate.
  • Positioning Group Removal: Cleave the ester positioning group under standard hydrolytic conditions (e.g., LiOH in THF/MeOH/H₂O) to yield the chiral cyclohexadienone product. Expected Outcome: >99% ee, high site-selectivity [23].
  • Downstream Processing: Subject the resulting chiral dienone to standard ring expansion conditions (Protocol 1, Step 3) to generate enantioenriched medium rings.

Application Notes and Comparative Data

Table 1: Comparison of Key ODRE Methodologies and Outcomes [21] [2]

Methodology Key Reagent/ Condition Scope & Product Type Key Advantage Limitation/Challenge
Classical ODRE TMSOTf / 2,6-di-t-butylpyridine Aryl ethers, diaryl ethers, lactones, biaryls from phenols. Broad access to natural product-like linkages; good yields (50-90%). Limited to phenolic substrates; potential for competing dienone-phenol rearrangement.
Tandem ODRE (Umpolung) Hypervalent Iodine (e.g., PIFA) in HFIP/CH₂Cl₂ Haloaryl, aryl ether, acetanilide, heteroaromatic rings with ketone. Wider substrate scope beyond phenols; avoids termination side-products. Requires synthesis of specific tertiary alcohol precursors.
Biocatalytic Oxidative Step Enzyme SorbC with cofactor regeneration Chiral cyclohexadienones with >99% ee for downstream ring expansion. Unmatched enantioselectivity and site-selectivity; green conditions. Requires substrate engineering for broad applicability; specialized setup.

Table 2: Performance of Biocatalytic vs. Chemical Oxidative Dearomatization [23]

Parameter Biocatalytic (SorbC) Chemical (PhI(OAc)₂)
Stereoselectivity >99% enantiomeric excess (ee) Racemic
Site-Selectivity Excellent, controlled by enzyme active site Moderate, influenced by substrate electronics
Typical Yield 50-90% (with engineered substrates) 70-95%
Conditions Aqueous buffer, pH 7.5, 30°C Organic solvent (CH₂Cl₂), 0°C to RT
Functional Group Tolerance High, but requires specific binding motif Generally high

ODRE Workflow in Diversity-Oriented Synthesis

G Start Diverse Simple Building Blocks A Modular Synthesis (3-5 steps) Start->A B Library of Bicyclic Phenols A->B C Oxidative Dearomatization B->C D Polycyclic Cyclohexadienone Intermediates C->D E Divergent Ring Expansion (3 Complementary Reagents) D->E Avoiding dienone-phenol rearrangement F Focused Library of Benzannulated Medium Rings E->F G Cheminformatic Analysis & Bioactivity Screening F->G

Reagent Classes for Ring Expansion

G Reagents Three Complementary Reagent Classes for Ring Expansion Class1 Class A: Electrophilic Activators (e.g., TMSOTf) Reagents->Class1 Class2 Class B: Oxidative Umpolung Systems (e.g., PIFA/HFIP) Reagents->Class2 Class3 Class C: Biocatalytic Systems (e.g., SorbC) Reagents->Class3 Mech1 Mechanism: Si-based activation of dienone, C-C cleavage, rearomatization. Class1->Mech1 Mech2 Mechanism: One-electron oxidation, cation formation, tandem rearomatization/expansion. Class2->Mech2 Mech3 Mechanism: Enzymatic O₂ activation, stereoselective hydroxylation, spontaneous rearomatization. Class3->Mech3

The Scientist's Toolkit: Essential Research Reagents

Table 3: Key Reagents and Materials for ODRE Sequence Experiments

Reagent/Material Function in ODRE Sequence Critical Notes & Handling
(Diacetoxyiodo)benzene (PhI(OAc)₂) Standard chemical oxidant for the Oxidative Dearomatization step. Moisture-sensitive. Use anhydrous CH₂Cl₂. Reactions often performed at 0°C.
Trimethylsilyl Triflate (TMSOTf) Strong silylating agent and Lewis acid. Serves as a key Ring Expansion reagent (Class A) [21]. Extremely moisture-sensitive, corrosive. Must use under inert atmosphere with dry solvents and a proton sponge (e.g., 2,6-di-t-butylpyridine).
Phenyliodine Bis(trifluoroacetate) (PIFA) Hypervalent iodine reagent for the tandem oxidative umpolung ring expansion (Class B) [2]. Used in combination with HFIP as solvent. Enables reactivity on non-phenolic substrates.
Flavin-Dependent Monooxygenase (SorbC) Biocatalyst for enantioselective Oxidative Dearomatization (Class C) [23]. Requires cofactor regeneration (NADPH/FAD). Substrate scope can be expanded using ester-based "positioning groups".
Hexafluoroisopropanol (HFIP) Solvent for umpolung ODRE reactions. Stabilizes cationic intermediates and facilitates rearomatization [2]. High boiling point. Often used in mixture with CH₂Cl₂.
2,6-Di-tert-butylpyridine Sterically hindered, non-nucleophilic base. Used as a proton scavenger with TMSOTf to prevent acid-catalyzed side reactions [21]. Critical for suppressing competing dienone-phenol rearrangement pathways.
Anhydrous Acetonitrile (CH₃CN) & Dichloromethane (CH₂Cl₂) Preferred anhydrous solvents for the ring expansion step (Class A) and oxidative step, respectively. Must be rigorously dried and stored over molecular sieves under inert atmosphere for reproducible results.

Critical Parameters for Success and Troubleshooting

  • Suppressing Dienone-Phenol Rearrangement: This is the major competing pathway. Always include a hindered base (e.g., 2,6-di-t-butylpyridine) when using strong electrophilic activators like TMSOTf. Monitor reactions by TLC and LC-MS for the formation of rearrangement by-products [21].
  • Solvent and Atmosphere: Ring expansion steps are highly sensitive to water and protic impurities. Use flame-dried glassware, rigorous solvent drying, and inert gas (Ar/N₂) atmosphere.
  • Substrate Design for Biocatalysis: When employing SorbC [23], computational docking or simple analogy to the native substrate can guide the design of ester-based positioning groups to ensure high conversion and enantioselectivity.
  • Purification: Medium-ring products and cyclohexadienone intermediates can be sensitive. Use neutral or mildly basic silica gel for flash chromatography, and avoid prolonged exposure to acidic conditions.

The strategic diversification of natural product scaffolds is a central theme in modern medicinal chemistry, aimed at accessing novel bioactive chemical space. This work is framed within a broader thesis investigating ring expansion strategies as a solution to the enduring synthetic challenge of constructing medium-sized rings (8-11 members), which are under-represented in screening libraries despite their high prevalence in bioactive natural products [2]. Traditional cyclization methods are often thwarted by transannular strain and unfavorable entropic factors [2]. This application note details practical protocols that leverage catalytic C-H oxidation and functionalization as a trigger for ring expansion. By directly functionalizing ubiquitous C-H bonds, these methods enable the direct, late-stage editing of carbocyclic cores into medium-sized heterocycles and functionalized macrocycles, bypassing multi-step de novo synthesis [24] [25]. The described transformations are inspired by biosynthetic logic and provide a powerful tool for diversity-oriented synthesis (DOS), complexity-to-diversity (CtD) approaches, and the generation of natural product-inspired libraries for drug discovery [2].

Application Notes & Experimental Protocols

Protocol 1: Copper-Mediated Photochemical Oxygen-Atom Insertion into Cycloalkanols

This protocol describes a skeletal editing transformation where a formal oxygen atom is inserted into a C-C bond of a cycloalkanol, resulting in a one-carbon ring expansion to form a tetrahydropyran or tetrahydrofuran [24]. The reaction proceeds via ligand-to-metal charge-transfer (LMCT) photogeneration of an alkoxy radical, β-scission, and oxidative cyclization.

  • Reaction Principle: A saturated cycloalkanol substrate is converted in situ to a copper(II) alkoxide. Upon irradiation with 427 nm light, an LMCT event generates a reactive alkoxy radical. This radical undergoes β-scission of an adjacent C–C bond, leading to ring opening. The resulting radical and carbonyl pair then recombine via a copper-mediated oxidative cyclization to form an oxocarbenium ion, which is trapped by an exogenous nucleophile (e.g., ethanol) to yield the ring-expanded acetal product [24].

  • Detailed Experimental Procedure:

    • Setup: In a dry, nitrogen-purged vial equipped with a magnetic stir bar, combine the cycloalkanol substrate (0.20 mmol, 1.0 equiv), Cu(OTf)₂ (2.5 equiv, 0.50 mmol), and pyridine (3.0 equiv, 0.60 mmol).
    • Solvent Addition: Add anhydrous isobutyronitrile (iPrCN) (5.0 equiv as ligand, 1.00 mmol) and anhydrous ethanol (3.0 equiv as nucleophile, 0.60 mmol). Dilute with additional anhydrous iPrCN to a final substrate concentration of 0.1 M.
    • Irradiation: Seal the vial and place it approximately 5 cm from a commercially available 427 nm Kessil lamp. Irradiate the deep blue reaction mixture with vigorous stirring for 16-24 hours. Monitor reaction completion by TLC or LCMS.
    • Work-up: After completion, dilute the reaction mixture with ethyl acetate (10 mL) and wash with a saturated aqueous solution of ammonium chloride (3 x 5 mL). The combined aqueous layers can be back-extracted with ethyl acetate (2 x 5 mL).
    • Purification: Dry the combined organic layers over anhydrous magnesium sulfate, filter, and concentrate under reduced pressure. Purify the crude residue by flash column chromatography on silica gel to obtain the ring-expanded acetal product.
    • Derivatization (Optional): The acetal product is a versatile intermediate. The ethoxy group can be removed via Lewis acid-mediated reduction with triethylsilane and boron trifluoride diethyl etherate to yield the formal oxygen-insertion product (a cyclic ether). Alternatively, hydrolysis, allylation, or amination can be performed using standard Lewis or Brønsted acid catalysis [24].

  • Key Application Data: This method is effective for converting substituted cyclopentanols and cyclobutanols to six- and five-membered ring heterocycles, respectively. Yields typically range from 60-85%. The reaction demonstrates broad functional group tolerance, including arenes (electron-rich/poor), vinyl, allylic, and α-amino groups. The β-scission is highly regioselective for benzylic and other stabilized radical sites [24].

Protocol 2: Iron-Catalyzed, Non-Directed C-H Hydroxylation for Late-Stage Diversification

This protocol employs a Fe(PDP) or Fe(CF₃-PDP) catalyst system to perform predictable, site-selective hydroxylation of aliphatic C-H bonds in complex molecules [25]. While not always a direct ring expansion, this hydroxylation is a critical first step in oxidation-triggered cascades (e.g., via subsequent oxidative cleavage or rearrangement) and serves as a powerful tool for late-stage functionalization (LSF) to diversify natural product scaffolds.

  • Reaction Principle: The iron catalyst, in the presence of a terminal oxidant (H₂O₂) and acetic acid, generates a reactive oxoiron species. This species abstracts a hydrogen atom from an aliphatic C-H bond. The resulting carbon radical recombines with the metal center to form a C-O bond, yielding the alcohol product. Site-selectivity is governed by a combination of electronic (favoring electron-rich C-H), steric (favoring accessible C-H), and stereoelectronic factors [25].

  • Detailed Experimental Procedure (Fe(PDP)-Catalyzed Hydroxylation):

    • Setup: Charge an oven-dried round-bottom flask with the substrate (0.10 mmol, 1.0 equiv) and the Fe(PDP) catalyst (5-10 mol%). Add a magnetic stir bar.
    • Solvent System: Add a 9:1 mixture of acetone:acetic acid (0.05 M final concentration relative to substrate).
    • Oxidation: Cool the reaction mixture to 0°C in an ice bath. Using a syringe pump, slowly add an aqueous solution of hydrogen peroxide (50% w/w, 5.0 equiv, 0.50 mmol) over a period of 2-4 hours. Maintain vigorous stirring.
    • Quenching & Work-up: After complete addition, stir for an additional 15 minutes at 0°C. Quench the reaction by adding a saturated aqueous solution of sodium thiosulfate (2 mL). Allow the mixture to warm to room temperature.
    • Extraction: Transfer the mixture to a separatory funnel, dilute with water (10 mL), and extract with dichloromethane (3 x 10 mL).
    • Purification: Dry the combined organic layers over anhydrous sodium sulfate, filter, and concentrate. Purify the product by flash chromatography to isolate the hydroxylated compound(s). Analyze site-selectivity by ¹H NMR or LC-MS/MS.

  • Key Application Data: The Fe(PDP) system reliably differentiates between 3° > 2° > 1° C-H bonds and can achieve selectivity among similar bond types based on subtle environment differences. Yields for mono-hydroxylated products on complex substrates are often preparative (30-70%). The complementary Fe(CF₃-PDP) catalyst can alter site-selectivity, providing a powerful tool to "dial-in" oxidation at a different position on the same molecule [25].

Protocol 3: Oxidative Dearomatization-Ring Expansion (ODRE) for Benzannulated Medium Rings

This protocol outlines a biomimetic, diversity-oriented synthesis of benzannulated medium-sized rings (e.g., 8-11 membered biaryl ethers, lactones) from bicyclic phenol precursors [2].

  • Reaction Principle: The sequence involves oxidative dearomatization of a phenol to a cyclohexadienone, which undergoes a strain-driven ring expansion via cleavage of a "scissile" bond, followed by rearomatization. This ODRE cascade effectively inserts one or more atoms between the arene and the original ring [2].

  • Detailed Experimental Procedure:

    • Oxidative Dearomatization: Dissolve the bicyclic phenol substrate (1.0 equiv) in a mixture of dichloromethane and buffer (pH 7) at 0°C. Add a stoichiometric oxidant such as phenyliodonium diacetate (PIDA) (1.1 equiv). Stir for 1-2 hours until complete conversion to the spirocyclic cyclohexadienone intermediate (monitor by TLC).
    • Ring Expansion & Quenching: Without purification, treat the crude reaction mixture with a nucleophile (e.g., a carboxylic acid, alcohol, or electron-rich arene, 1.5-3.0 equiv) that will trap the expanded ring intermediate. Often, a base (e.g., triethylamine) or a Lewis acid is added to promote this step.
    • Work-up & Purification: After completion, quench with aqueous sodium thiosulfate, extract with ethyl acetate, dry, and concentrate. Purify the product by flash chromatography to isolate the benzannulated medium-ring compound.

  • Key Application Data: This method provides efficient access to complex scaffolds found in natural products. The choice of nucleophile (e.g., acids for lactones, phenols for diaryl ethers) directly dictates the ring heteroatom and functionality, enabling rapid library synthesis [2].

The Scientist's Toolkit: Key Reagents & Materials

The following table details essential reagents, catalysts, and materials for implementing the oxidation/functionalization-triggered ring expansion strategies described above.

Item Name Function & Role in Reaction Critical Handling/Preparation Notes
Cu(OTf)₂ (Copper(II) Triflate) Stoichiometric oxidant and Lewis acid in LMCT photochemistry. Forms the photoactive Cu(II)-alkoxide complex [24]. Hygroscopic. Must be handled under inert atmosphere (N₂/Ar) and used with anhydrous solvents.
Fe(PDP) & Fe(CF₃-PDP) Catalysts Non-heme iron catalysts for site-selective aliphatic C-H hydroxylation. Control selectivity via ligand tuning [25]. Air-stable solids. Store at room temperature. Solutions in acetone/acetic acid should be prepared fresh.
Hydrogen Peroxide (50% w/w) Terminal oxidant for Fe-catalyzed hydroxylations. Provides the oxygen atom [25]. Strong oxidizer. Use with appropriate PPE. Add slowly via syringe pump to control exotherm and minimize over-oxidation.
Phenyliodonium Diacetate (PIDA) Hypervalent iodine oxidant for phenolic substrate dearomatization in ODRE sequences [2]. Light-sensitive. Store in the dark at low temperature. Often used in buffered conditions to control pH.
427 nm Kessil Lamp Light source for photoexcitation of the Cu(II)-alkoxide LMCT band [24]. Ensure reaction vessel is within optimal distance (e.g., ~5 cm) for uniform irradiation. Use appropriate eye protection.
Isobutyronitrile (iPrCN) Solvent and ligand in Cu-photochemistry. Coordinates to Cu(II), improving solubility and reaction efficiency [24]. Anhydrous conditions are critical. Dry over molecular sieves and distill under N₂ before use. Toxic—use in fume hood.
Acetone/Acetic Acid (9:1) Standard solvent system for Fe(PDP) catalysis. Acetic acid is essential for catalytic activity and selectivity [25]. Use reagent grade. The mixture should be prepared fresh to avoid water absorption and peroxide formation in acetone.

The following tables summarize key performance data for the ring expansion and C-H functionalization methods discussed.

Table 1: Ring Expansion Method Performance Comparison

Method (Trigger) Typical Starting Ring Size Product Ring Size Representative Yield Range Key Functional Group Tolerance Primary Reference
Cu/LMCT O-Insertion [24] 4, 5 5, 6 (heterocycle) 60-85% Arenes, vinyl, allylic, α-amino [24]
ODRE Cascade [2] 5, 6 (fused) 8-11 (benzannulated) 40-75% Phenols, carboxylic acids, alcohols [2]
Electrochemical Amidyl Radical Migration [2] 6 (benzocyclic) 8-11 (lactam) 50-80% Various amides, ketones [2]

Table 2: Late-Stage C-H Oxidation Selectivity & Yield Data

Substrate Class Catalyst Preferred Site of Oxidation Typical Mono-Oxidation Yield Catalyst-Controlled Selectivity Switch? Reference
Natural Product Terpenes/Steroids Fe(PDP) Most electron-rich 3° C-H 40-70% No [25]
Natural Product Terpenes/Steroids Fe(CF₃-PDP) Less hindered or electronically different 3° C-H 30-65% Yes (vs. Fe(PDP)) [25]
Complex Alkaloids/Pharmaceuticals Fe(PDP) 3° > 2° C-H; within 3°, favors more substituted site 25-60% No [25]

Visual Workflow and Mechanism Diagrams

The following diagrams, generated using Graphviz DOT language, illustrate the core mechanistic pathways and strategic workflows for harnessing C-H bonds in ring expansion.

G Substrate Cycloalkanol Substrate CuComplex Cu(II)-Alkoxide Complex Substrate->CuComplex Cu(OTf)₂ Pyridine AlkoxyRad Alkoxy Radical (via LMCT) CuComplex->AlkoxyRad hv (427 nm) LMCT CleavedInt Ring-Opened Radical/Carbonyl Pair AlkoxyRad->CleavedInt β-Scission (C-C Cleavage) OxCarbenium Oxocarbenium Ion Intermediate CleavedInt->OxCarbenium Cu-Mediated Oxidative Coupling Product Ring-Expanded Acetal Product OxCarbenium->Product NuH Trapping (e.g., EtOH)

Diagram 1: Mechanism of Copper-Photochemical O-Insertion Ring Expansion.

G Start Natural Product or Complex Substrate C_H_Ox Strategic C-H Oxidation Start->C_H_Ox C_H_Func Strategic C-H Functionalization Start->C_H_Func Oxidized Oxidized/Functionalized Intermediate C_H_Ox->Oxidized C_H_Func->Oxidized Trigger Oxidation-Triggered Cascade Oxidized->Trigger e.g., Rearrangement or Further Activation RingExp Ring Expansion Step Trigger->RingExp C-C Cleavage & Recombination MSR Medium-Sized Ring (MR) Product RingExp->MSR LibScreen Library for Biological Screening MSR->LibScreen Diversification & Analog Synthesis

Diagram 2: Strategic Workflow for C-H Triggered Ring Expansion in Library Synthesis.

The synthesis of medium-sized rings (8-11 membered) represents a significant and persistent challenge in organic chemistry, particularly within the context of developing libraries of natural product-derived compounds for drug discovery. Traditional direct cyclization methods are often hampered by unfavorable transannular strain and entropic penalties [1]. This article, framed within a broader thesis on ring expansion strategies, details advanced radical-based approaches—specifically aryl migration and remote functionalization—that provide innovative pathways to these valuable scaffolds. These methods bypass the limitations of direct cyclization by transforming readily accessible, less-strained precursors into complex medium-sized rings, thereby populating underexplored chemical space for probing "undruggable" targets [1] [3].

Comparative Analysis of Key Radical-Based Methodologies for Ring Expansion

The following table summarizes and contrasts the core radical-based methodologies applicable to the synthesis and functionalization of medium-sized ring systems.

Methodology Key Reactive Intermediate Primary Application Typical Ring Size Formed/Functionalized Key Advantage
1,2-Aryl Migration (Visible Light) [26] Alkoxyl radical (from O–H homolysis) Synthesis of 1,5-dicarbonyl scaffolds from acyclic precursors Not primarily for rings; builds linear precursors Mild, additive-free conditions; broad substrate tolerance
N-to-C Aryl Migration (Photoredox/CO₂) [27] Arylaminyl radical (from N–Ar homolysis) Construction of polysubstituted alkenyl amides Not primarily for rings; builds functionalized sidechains Electronically tolerant migration; CO₂ enables chemoselectivity
Transannular Ketone Migration [28] Alkyl radical (via H-atom transfer) Remote C–H functionalization of 7-membered rings 7-membered rings Regioselective access to densely substituted 7-membered rings
Remote C–H Functionalization (Chain-Walking) [29] Organorhodium intermediate (via β-hydride elimination/addition) Synthesis of benzene-fused heterocycles (e.g., dihydrobenzofurans) 5-membered rings (fused) Multitasking cascade; functionalizes remote, unactivated C(sp³)–H bonds
Oxidative Dearomatization-Ring Expansion [1] Cyclohexadienone radical cation Biomimetic synthesis of benzannulated medium rings 8-11 membered rings Direct ring expansion from polycyclic phenolic precursors

Detailed Experimental Application Notes and Protocols

This protocol describes the radical 1,2-aryl migration in diaryl allyl alcohols to form 1,5-dicarbonyl compounds, a valuable linear precursor that can be cyclized into medium-ring systems.

  • Reaction Setup: Conduct all operations under an inert atmosphere (e.g., nitrogen or argon) using standard Schlenk techniques or a glovebox.
  • Materials: In an oven-dried reaction vial equipped with a magnetic stir bar, combine the diaryl allyl alcohol substrate (0.2 mmol, 1.0 equiv) and the photocatalyst Ir[(dF(CF₃)ppy)₂(dtbbpy)]PF₆ (1 mol%, 0.002 mmol). Seal the vial with a septum.
  • Solvent Addition: Under a positive flow of inert gas, add anhydrous and degassed dichloroethane (DCE, 2.0 mL) via syringe.
  • Irradiation: Place the sealed reaction vial under the irradiation of a commercially available blue LED strip (approximately 30 W, 450 nm). Stir the reaction mixture vigorously at room temperature.
  • Monitoring & Completion: Monitor the reaction progress by thin-layer chromatography (TLC) or liquid chromatography-mass spectrometry (LC-MS). Typical reaction times range from 12 to 24 hours.
  • Work-up: Upon completion, concentrate the reaction mixture under reduced pressure.
  • Purification: Purify the crude residue by flash column chromatography on silica gel (eluent: hexane/ethyl acetate gradient) to obtain the desired 1,5-dicarbonyl product.
  • Notes: Electron-deficient aryl groups migrate with higher efficiency. The reaction proceeds without external oxidants or additives.

This protocol enables a radical-polar crossover Truce–Smiles rearrangement for the synthesis of tetrasubstituted alkenyl amides, which can serve as advanced intermediates for lactam-containing medium rings.

  • Reaction Setup: Perform in a pressure-tolerant glass reaction tube equipped with a stir bar.
  • Materials: Charge the tube with N-arylpropiolamide substrate (0.1 mmol, 1.0 equiv), sodium benzenesulfinate (0.3 mmol, 3.0 equiv), and the photocatalyst Ir[(dFCF₃ppy)₂(bpy)]PF₆ (2 mol%, 0.002 mmol).
  • Solvent & Atmosphere: Add N,N-dimethylformamide (DMF, 2.0 mL). Evacuate the tube and backfill with carbon dioxide (CO₂). Repeat this cycle three times, then maintain a CO₂ balloon pressure (approx. 1 atm).
  • Irradiation: Irradiate the stirred mixture with blue LEDs (40 W, 450 nm) at room temperature for 12 hours.
  • Monitoring & Work-up: After confirming completion by TLC, dilute the mixture with ethyl acetate (10 mL) and wash with brine (3 x 5 mL). Dry the combined organic layers over anhydrous sodium sulfate (Na₂SO₄) and concentrate.
  • Purification: Purify the crude product via preparative thin-layer chromatography (PTLC) or column chromatography.
  • Notes: CO₂ is essential for high yield and selectivity, accelerating the key migration step. Both electron-rich and electron-poor migratory aryl groups are tolerated.

This protocol details a dynamic kinetic strategy for the site-selective functionalization of seven-membered carbocycles, a key step in elaborating medium-ring precursors.

  • Reaction Setup: Use an oven-dried Schlenk tube with a stir bar.
  • Materials: Combine the 1,1-disubstituted acylcycloheptane substrate (0.05 mmol, 1.0 equiv), sodium decatungstate (Na₄[W₁₀O₃₂], 20 mol%, 0.01 mmol), and thiophenol (1.0 equiv, 0.05 mmol) as a hydrogen atom transfer (HAT) cocatalyst.
  • Solvent & Atmosphere: Add degassed acetonitrile (MeCN, 2.0 mL). Seal the tube and purge the headspace with nitrogen for 5 minutes.
  • Irradiation: Stir the mixture under irradiation from a 395 nm LED lamp at room temperature. Monitor by LC-MS.
  • Quenching & Work-up: After 6-8 hours, quench the reaction by opening to air and adding a saturated aqueous solution of sodium thiosulfate (1 mL). Extract with dichloromethane (3 x 5 mL).
  • Purification: Dry the combined organic extracts over Na₂SO₄, concentrate, and purify by silica gel chromatography.
  • Notes: The process involves reversible HAT to generate radicals at multiple ring positions, followed by site-selective ketone group migration via β-scission. This allows functionalization at the γ-position relative to the original carbonyl.

Mechanisms and Workflow Visualizations

G cluster_light Visible-Light-Induced 1,2-Aryl Migration [26] cluster_co2 CO₂-Promoted N-to-C Aryl Migration [27] cluster_trans Transannular Remote Functionalization [28] A Diaryl Allyl Alcohol B Ir(III)* Photoexcitation A->B hv C Alkoxyl Radical (O-H Homolysis) B->C Energy Transfer D 1,2-Aryl Shift (Spirocyclization) C->D E Radical Intermediate D->E F HAT or Oxidation E->F G 1,5-Dicarbonyl Product F->G H N-Arylpropiolamide I Ir(III)* Reductive Quenching H->I hv J Arylaminyl Radical (N-Ar Cleavage) I->J e⁻ Transfer K 1,4-Aryl Migration (ipso-Substitution) J->K L Anionic Intermediate K->L M CO₂ Promotion L->M N Trapping & Product Formation M->N O Tetrasubstituted Alkenyl Amide N->O P Acylcycloheptane Q Na₄[W₁₀O₃₂]* HAT Catalyst P->Q hv R Remote C-H Abstraction (Dynamic Radical Pool) Q->R HAT S Ketone Group Migration (β-Scission) R->S T Regioisomeric Radical S->T U Functionalization (e.g., Halogenation) T->U V Functionalized Medium Ring U->V

Diagram Title: Mechanistic Pathways for Radical Aryl Migration and Remote Functionalization

The Scientist's Toolkit: Research Reagent Solutions

Essential materials for implementing the featured radical-based ring expansion strategies.

Item Function/Application Notes/Specific Examples
Photoredox Catalysts Initiate radical reactions under mild, visible-light conditions. Ir[(dFCF₃ppy)₂(bpy)]PF₆: For oxidative and reductive quenching cycles [26] [27]. 4CzIPN: Organic, cost-effective alternative for some transformations [27].
Hydrogen Atom Transfer (HAT) Catalysts Abstract hydrogen from unactivated C–H bonds to generate carbon radicals. Sodium Decatungstate (Na₄[W₁₀O₃₂]): Polydentate HAT catalyst for remote functionalization [28]. Thiophenol (PhSH): Radical chain carrier and polarity mediator [28].
Halogen Source Provide halogen radicals (X•) for functionalization or act as radical traps. N-Bromosuccinimide (NBS): Source of bromine radical (Br•) in stereospecific bromination/migration reactions [30].
Specialty Additives Modulate reactivity, selectivity, or enable specific pathways. Carbon Dioxide (CO₂): Critical promoter in N-to-C aryl migration, likely stabilizing anionic intermediates [27]. Thiourea Derivatives: Additives in stereospecific halogenation reactions that influence mechanism [30].
Light Sources Provide specific wavelengths of light to excite photocatalysts. Blue LED Lamps (e.g., 450 nm): Common for exciting Ir(III) and organic photocatalysts. 395 nm LED: Suitable for decatungstate anion activation [28].
Polycyclic Phenolic Substrates Advanced starting materials for biomimetic ring expansion. Bicyclo[3.2.1]octadienone scaffolds: Undergo oxidative dearomatization-ring expansion-rearomatization (ODRE) to benzannulated medium rings [1].

The discovery of novel bioactive molecules is critically dependent on accessing regions of chemical space that are rich in structural complexity and three-dimensionality. Natural products, particularly steroids and terpenes, represent a privileged source of such complexity but are underrepresented in conventional screening libraries, especially as scaffolds containing medium-sized rings (8-11 members) [2]. These rings occupy a unique niche, offering a balance between the rigidity of small rings and the flexibility of macrocycles, which can be pivotal for engaging challenging biological targets [2]. However, their synthesis is notoriously difficult due to unfavorable transannular interactions and entropic barriers associated with direct cyclization [2] [4].

This work is situated within a broader thesis research program focused on overcoming these synthetic challenges through ring expansion strategies. Instead of constructing medium-sized rings de novo, we leverage the pre-organized, polycyclic frameworks of abundant natural products—steroids and terpenes—and selectively "open" and "expand" one of their existing small rings. This approach bypasses the high-energy intermediates of direct cyclization. The core hypothesis is that applying C–H functionalization to install strategic handles, followed by ring-expanding transformations, can efficiently generate diverse, natural product-inspired libraries rich in medium-sized rings [4]. This strategy synergizes the goals of Diversity-Oriented Synthesis (DOS)—to generate skeletally diverse collections—with the biological relevance inherent to natural product frameworks [2].

The following Application Notes and Protocols detail the practical implementation of this two-phase strategy: 1) the chemical diversification of terpene and steroid cores, and 2) their subsequent elaboration into libraries featuring medium-sized rings.

Application Notes: Strategic Workflow and Key Transformations

The synthesis of skeletally diverse libraries from terpenes and steroids follows a convergent workflow, progressing from core isolation and diversification to final library production and validation.

G Start Natural Product Starting Material (Steroid or Terpene Core) Phase1 Phase 1: Core Diversification Start->Phase1 Sub1 • Selective C-H Functionalization • Functional Group Interconversion Phase1->Sub1 Phase2 Phase 2: Ring Expansion Sub2 • Cyclic Ether/Lactam Formation • Skeletal Reconstruction • Annulation Phase2->Sub2 End Diverse Library of Medium-Sized Ring Compounds Toolkit Analytical & Cheminformatic Validation End->Toolkit Sub1->Phase2 Sub2->End

Phase 1: Core Diversification of Steroids and Terpenes

The initial phase transforms commercially available or isolated natural product cores into a panel of advanced intermediates primed for ring expansion.

  • Steroid Diversification via C–H Activation: The inert C–H bonds of steroid cores are strategic points for diversification. Electrochemical oxidation provides a green method for allylic C–H oxidation [4]. For instance, applying a reported electrochemical protocol to dehydroepiandrosterone (DHEA) can install allylic alcohol or ketone functionalities at otherwise inaccessible positions [4]. Similarly, copper-mediated or chromium-mediated oxidations enable site-selective functionalization at benzylic or tertiary C–H sites, converting simple steroid cores like estrone into ketones bearing new handles for subsequent ring expansion reactions [4].

  • Terpene Skeletal Derivatization: Terpene cores offer vast opportunities for reconstruction. A demonstrated strategy involves the humulene skeleton (a sesquiterpene), which can be reconstructed via olefin metathesis and functionalized through Lewis acid-mediated epoxide-opening transannulation to introduce heteroatoms and alter ring systems [31]. This enables access to diverse scaffolds, including 11-membered monocyclic, oxabicyclic, and medium-sized aza rings from a common precursor [31].

Phase 2: Ring Expansion Methodologies

The diversified cores are subjected to expansion reactions that convert a single, typically strained, small ring into a medium-sized ring.

  • Heteroatom-Insertion Expansions: The Beckmann Rearrangement of ketoximes, derived from oxidized steroid cores, is a reliable workhorse for converting a six-membered ring ketone into a seven-membered lactam [4]. The Schmidt Reaction with alkyl azides on cyclic ketones provides direct access to medium-ring lactams from steroid A- or D-rings [4]. Furthermore, intramolecular nucleophilic attack on activated epoxides, as seen in terpene derivatization, can lead to transannular C–O bond formation and ring expansion [31].

  • Carbon-Insertion and Reconstruction Expansions: Formal [2+2] cycloaddition-fragmentation with dimethyl acetylenedicarboxylate (DMAD) allows for a two-carbon ring expansion of cyclic β-keto esters, transforming steroid-derived precursors into [6.3.0] fused bicyclic systems [4]. For terpenes, a powerful ring-reconstruction strategy based on olefin metathesis has been employed to deconstruct and reformulate the humulene skeleton, fundamentally altering ring size and connectivity [31].

Analytical and Cheminformatic Validation

Post-synthesis, library validation is critical. Advanced separation techniques like two-dimensional gas chromatography (GC×GC-TOFMS) are essential for resolving complex mixtures of terpenoid derivatives, which often co-elute in standard GC [32]. Cheminformatic analysis of the final compounds should confirm entry into underexplored chemical space. Key metrics include principal component analysis (PCA) showing separation from standard libraries, high Natural Product (NP) Likeness scores, and quantitative measures of three-dimensionality (e.g., fraction of sp³-hybridized carbons, Fsp3) [31]. This analysis verifies that the ring expansion strategy successfully generates novelty beyond simple analog preparation.

Table 1: Summary of Key Ring Expansion Methodologies Applied to Steroids and Terpenes.

Expansion Method Mechanistic Class Typical Ring Size Formed Key Feature Example Starting Core
Beckmann Rearrangement [4] Heteroatom Insertion (N) 7-membered lactam High fidelity migration of alkyl group; proceeds via nitrilium ion. Steroid-derived ketone (e.g., from estrone, DHEA)
Schmidt Reaction [4] Heteroatom Insertion (N) 7-8 membered lactam Direct use of ketone with hydrazoic acid/alkyl azide. Steroid ketone (e.g., DHEA derivative)
Formal [2+2] with DMAD [4] Carbon Insertion (2C) 8-membered ring (from 6) Concerted cycloaddition followed by retro-cycloaddition fragmentation. Steroid-derived β-keto ester
Epoxide-Opening Transannulation [31] Heteroatom Insertion (O) & Reconstruction Oxabicyclic systems Lewis acid-mediated, forms new C-O bond and ring system. Terpene-derived epoxide (e.g., from humulene)
Olefin Metathesis Reconstruction [31] Skeletal Reconstruction Variable (e.g., 11-membered) Breaks and forms C-C bonds to fundamentally rebuild core scaffold. Terpene diene (e.g., humulene derivative)

Detailed Experimental Protocols

Objective: To synthesize a seven-membered D-ring lactam from dehydroepiandrosterone (DHEA) via allylic C–H oxidation followed by ring expansion.

Materials:

  • Dehydroepiandrosterone (DHEA) (1.0 g, 3.47 mmol)
  • Electrolyte: Lithium perchlorate (LiClO₄), anhydrous
  • Solvent: Dichloromethane (DCM), anhydrous; Methanol (MeOH)
  • Electrochemical Cell: Undivided cell with graphite felt working and counter electrodes.
  • Hydroxylamine hydrochloride (NH₂OH•HCl)
  • Sodium acetate (NaOAc)
  • Pyridine
  • Activation Reagent: Thionyl chloride (SOCl₂) or Tosyl chloride (TsCl)
  • Base: Triethylamine (Et₃N)
  • Work-up & Purification: Saturated aqueous NH₄Cl, brine, anhydrous Na₂SO₄, silica gel for flash chromatography.

Procedure: Part 1: Electrochemical Allylic Oxidation.

  • In the electrochemical cell, prepare a solution of DHEA (1.0 g) and LiClO₄ (0.1 M) in anhydrous DCM/MeOH (9:1, ~0.1 M substrate concentration).
  • Apply a constant current of 10 mA/cm² under a nitrogen atmosphere. Monitor the reaction by TLC.
  • Upon completion (typically 3-5 F/mol charge passed), quench the reaction by adding saturated aqueous NH₄Cl.
  • Extract the aqueous layer three times with DCM. Combine the organic layers, wash with brine, dry over Na₂SO₄, and concentrate in vacuo.
  • Purify the crude product via flash chromatography (silica gel, hexane/ethyl acetate gradient) to isolate the allylic oxidation product (e.g., the corresponding enone or allylic alcohol). Characterize by ¹H NMR and MS.

Part 2: Oxime Formation and Beckmann Rearrangement.

  • Dissolve the ketone product from Part 1 (500 mg) in pyridine (5 mL).
  • Add hydroxylamine hydrochloride (2.0 equiv) and sodium acetate (2.5 equiv). Heat the mixture to 80°C and stir for 4-6 hours.
  • Cool, pour into ice-water, and extract with ethyl acetate. Wash the organic layer with 1M HCl, then brine, dry (Na₂SO₄), and concentrate to obtain the crude oxime.
  • For the rearrangement, dissolve the crude oxime in dry DCM (10 mL) under N₂. Cool to 0°C.
  • Add thionyl chloride (1.2 equiv) dropwise. After addition, warm to room temperature and stir for 12 hours.
  • Carefully quench the reaction by pouring onto crushed ice. Extract with DCM, wash the combined organics with saturated NaHCO₃ solution and brine, dry (Na₂SO₄), and concentrate.
  • Purify the residue via flash chromatography to yield the desired seven-membered D-ring lactam. Confirm structure by ¹H, ¹³C NMR, and HRMS.

Objective: To convert humulene into a nitrogen-containing, medium-sized bicyclic scaffold.

Materials:

  • Humulene (purified from plant extract or commercial)
  • Epoxidation Reagent: meta-Chloroperoxybenzoic acid (mCPBA)
  • Solvents: Dichloromethane (DCM), anhydrous; Tetrahydrofuran (THF), anhydrous
  • Lewis Acid: Boron trifluoride diethyl etherate (BF₃•OEt₂)
  • Nitrogen Source: Trimethylsilyl azide (TMSN₃) or sodium azide (NaN₃)
  • Reducing Agent: Triphenylphosphine (PPh₃)
  • Work-up & Purification: Saturated NaHCO₃, Na₂S₂O₃, brine, anhydrous MgSO₄, silica gel.

Procedure: Part 1: Epoxidation of Humulene.

  • Dissolve humulene (1.0 g) in anhydrous DCM (20 mL) and cool to 0°C.
  • Add mCPBA (1.1 equiv) portionwise. Stir at 0°C for 1 hour, then allow to warm to room temperature and stir for an additional 4-6 hours.
  • Quench by adding saturated NaHCO₃ solution. Extract with DCM. Wash the combined organic layers with saturated Na₂S₂O₃ (to reduce peroxides), then brine.
  • Dry over MgSO₄, filter, and concentrate. Purify via flash chromatography to isolate the major mono- or di-epoxide derivative.

Part 2: Lewis Acid-Mediated Epoxide Opening and Transannulation.

  • Dissolve the humulene epoxide (300 mg) in anhydrous THF (10 mL) under nitrogen.
  • Add TMSN₃ (2.0 equiv) and cool the solution to -78°C.
  • Add BF₃•OEt₂ (1.5 equiv) dropwise. Stir at -78°C for 1 hour, then gradually warm to 0°C over 2 hours.
  • Quench the reaction with saturated NaHCO₃ and warm to room temperature.
  • Extract with ethyl acetate, wash with brine, dry (MgSO₄), and concentrate to give an azido-alcohol intermediate.
  • Reduction/Cyclization: Redissolve the azido-alcohol in THF (5 mL). Add triphenylphosphine (1.2 equiv) and stir at room temperature for 12-24 hours. The Staudinger reduction generates a reactive iminophosphorane that undergoes an intramolecular aza-Michael addition or similar transannulation to form the aza-medium ring.
  • Concentrate and purify the product via flash chromatography to yield the target terpenoid-alkaloid-like scaffold containing a medium-sized aza ring. Characterize fully by NMR and HRMS.

Table 2: The Scientist's Toolkit for Ring Expansion Library Synthesis.

Category / Item Specification / Example Primary Function in Workflow
Core Starting Materials Steroids: Dehydroepiandrosterone (DHEA), Estrone, Isosteviol [4]. Terpenes: Humulene [31], Commercially available mono/diterpenes. Provide the complex, polycyclic foundation for diversification and expansion.
C–H Functionalization Reagents Electrochemical setup (graphite electrodes, LiClO₄) [4]; Metal catalysts (Cu, Cr) [4]; Organic oxidants (e.g., DDQ). Install oxygenated functional groups (alcohols, ketones) at specific C-H sites to create handles for expansion.
Ring Expansion Reagents For N-insertion: NH₂OH•HCl (oxime), SOCl₂/TsCl (Beckmann), alkyl azides (Schmidt). For C-insertion: DMAD ([2+2]) [4]. For reconstruction: Grubbs metathesis catalysts [31], BF₃•OEt₂ (epoxide opening) [31]. Execute the key bond-breaking and bond-forming events that enlarge a ring or reconstruct the skeleton.
Analytical Instrumentation GC×GC-TOFMS [32]; NMR (¹H, ¹³C, 2D); HRMS; HPLC-MS/PDA. Characterize complex reaction mixtures, identify new compounds, and ensure purity of final library members.
Cheminformatic Software For analysis: RDKit, Schrodinger Suite. For visualization: PCA plots, Chemical space mapping tools. Quantify library diversity, calculate physicochemical properties (e.g., Fsp3, NP score), and validate entry into novel chemical space [31].

Case Study & Biological Validation

A 2024 study exemplifies the entire workflow and its utility in early drug discovery [31]. Researchers applied a ring-reconstruction strategy to the humulene skeleton, involving epoxide opening and olefin metathesis, to generate a library of terpenoid alkaloid-like compounds containing 11-membered and other medium-sized rings.

Cheminformatic analysis confirmed the library's novelty, showing high three-dimensional character and significant divergence from known chemical space in principal component analysis [31]. This library was then screened for inhibition of osteoclast-specific tartrate-resistant acid phosphatase (TRAP) activity, a target relevant to osteoporosis and bone-resorptive diseases. The screen successfully identified a novel seed compound with inhibitory activity, demonstrating the potential of this ring expansion-derived library to deliver new lead structures for challenging biological targets [31]. This validates the core thesis that ring expansion of natural product cores is a viable strategy for populating biologically relevant, underexplored chemical space with drug-like molecules.

Navigating Synthesis: Conformational Control, Selectivity, and Functional Group Compatibility

Managing Transannular Interactions and Conformational Flexibility

Within the broader thesis on ring expansion strategies for natural product-derived medium-sized rings, the management of transannular interactions and conformational flexibility emerges as a cornerstone for successful synthesis and functional application. Medium-sized rings (8-11 members) and macrocycles (12+ members) are prized in drug discovery for their ability to target "undruggable" protein interfaces, a property derived from their unique three-dimensional shapes and preorganized binding elements [1]. However, their synthesis is notoriously challenging due to unfavorable enthalpic and entropic factors during direct cyclization [1]. Ring expansion strategies offer a powerful solution, building larger rings from smaller, less-strained precursors [3]. A critical consideration in these expanded architectures is the emergence of non-covalent transannular interactions—through-space forces between non-bonded atoms or functional groups across the ring. These interactions, which can be attractive or repulsive, are the primary determinants of a medium-sized ring's conformational landscape, dictating its overall shape, stability, and ultimate biological function [33] [34]. Effectively managing these forces is therefore essential to guide conformational preferences, minimize destabilizing strain, and introduce stabilizing interactions that lock bioactive conformations, ultimately enabling the rational design of novel therapeutic agents from natural product scaffolds.

Computational Analysis & Prediction Protocols

Protocol: Evaluating Transannular Interactions via Density Functional Theory (DFT)

This protocol details the computational assessment of transannular N···Y (Y = various atoms/groups) interactions in medium-sized heterocycles, based on methodologies used to analyze alkaloids like protopine [33].

  • Objective: To calculate the strength, geometric parameters, and electronic effects of a transannular interaction and its influence on global conformational preference.
  • Software Requirement: Quantum chemical computation package (e.g., Gaussian, ORCA, GAMESS).
  • Initial Structure Preparation:
    • Build a 3D model of the target medium-sized ring system, starting from a crystallographic structure if available or an energy-minimized model from a molecular mechanics force field.
    • For ring expansion precursors, model the proposed product geometry to identify potential transannular strain points.
  • Computational Method:
    • Level of Theory: Employ Density Functional Theory (DFT). The hybrid functional M06-2X paired with the 6-311++G(2d,2p) basis set has been validated for this purpose [33]. For larger systems, a balanced functional like ωB97X-D is recommended for its inclusion of dispersion corrections.
    • Geometry Optimization: Fully optimize the molecular geometry without constraints. For flexible rings, perform a conformational search (see Protocol 1.2) prior to optimization to ensure location of the global minimum.
    • Frequency Calculation: Perform a vibrational frequency calculation on the optimized structure to confirm it is a true minimum (no imaginary frequencies) and to obtain thermochemical corrections.
    • Solvation Model (Optional): For comparisons to solution-phase biology, use an implicit solvation model (e.g., SMD, COSMO). Note that for core structural evaluations in vacuo, solvation may have negligible impact on geometries and charges [33].
  • Key Analysis & Data Extraction:
    • Geometric Parameters: Measure the through-space distance (Å) between the interacting atoms (e.g., N and the carbonyl C of Y=CO). Analyze dihedral angles to characterize ring puckering.
    • Quantum Theory of Atoms in Molecules (QTAIM): Perform a QTAIM analysis to locate the bond critical point (BCP) between the interacting atoms. The electron density (ρ, in atomic units) at the BCP is a quantitative measure of interaction strength [33].
    • Natural Population Analysis (NPA): Calculate NPA charges on the interacting atoms to understand charge transfer (e.g., from nitrogen lone pair to a π* orbital of Y=CO) [33].
    • Non-Covalent Interaction (NCI) Plot: Generate an NCI isosurface to visualize the spatial region and type (attractive/repulsive) of the transannular interaction.
    • Electrostatic Potential (ESP) Mapping: Map the ESP onto the electron density surface (e.g., 0.001 a.u. isosurface) to identify regions of negative (nucleophilic) and positive (electrophilic) potential influenced by the interaction.
Protocol: Conformational Landscape Sampling with thepucke.rsToolkit

This protocol uses the open-source pucke.rs toolkit to systematically sample the conformational space of flexible rings, crucial for understanding the dynamic behavior of medium-sized ring systems [35].

  • Objective: To generate a comprehensive set of initial conformations for a monomeric ring system to be used in subsequent quantum mechanical geometry optimizations and energy evaluations.
  • Software Requirement: The pucke.rs command-line tool and Python module (available open-source). A computational chemistry package (e.g., ORCA, Gaussian) for subsequent optimizations.
  • System Setup:
    • Prepare an input structure file (e.g., .xyz, .mol2) for the ring system of interest.
    • Identify the type of system: a six-membered ring, a five-membered ring, or a peptide-like torsional system.
  • Conformational Sampling Execution:
    • For a six-membered ring (e.g., a saturated heterocycle in a natural product scaffold):
      • Use the Cremer-Pople formalism to sample spherical coordinates on the conformational sphere.
      • Command example: pucke.rs --system six_membered_ring --input structure.xyz --points 500 will generate 500 sets of dihedral angle constraints covering the conformational space [35].
    • For a five-membered ring (e.g., a ribose or related moiety):
      • Sample along the pseudorotation pathway defined by phase and amplitude parameters.
      • Command example: pucke.rs --system five_membered_ring --input structure.xyz --intervals 15 will sample at 15-degree intervals [35].
    • For peptide backbones or acyclic linkers within a macrocycle:
      • Sample the key torsional angles (e.g., φ, ψ).
      • Command example: pucke.rs --system peptide --residue ALA --interval 10 generates constraints at 10-degree intervals for alanine [35].
  • Post-Sampling Workflow:
    • The toolkit outputs a series of constraint files.
    • Use these constraints to perform constrained geometry optimizations with your chosen QM software.
    • Perform single-point energy calculations on all optimized conformers.
    • Plot the energies against the sampling coordinates (e.g., Cremer-Pople parameters, torsion angles) to construct a Potential Energy Surface (PES) and identify all low-energy conformers and the barriers between them.

G Start Define Ring System & Input Structure A Choose Sampling Formalism Start->A B Run pucke.rs Toolkit A->B e.g., Cremer-Pople for 6-membered C Generate Conformer Constraint Sets B->C D Constrained QM Geometry Optimization C->D E Single-Point Energy Calculation D->E F Construct Potential Energy Surface (PES) E->F End Identify Minima & Transition States F->End

Conformational Sampling Workflow for Flexible Rings

Synthetic Application & Ring Expansion Protocols

Protocol: Conjugate Addition/Ring Expansion (CARE) Cascade for Lactam Synthesis

This protocol describes a robust, one-pot method for synthesizing diverse medium-sized (8-11 membered) and macrocyclic (12+ membered) ring lactams, enabling rapid library generation for biological screening [6].

  • Objective: To convert simple, small-ring lactams into larger ring analogs via a tandem conjugate addition and ring expansion cascade.
  • Materials:
    • Starting Lactam (1): A cyclic lactam (e.g., 6-, 7-, 8-membered).
    • Acylating Agent: Acryloyl chloride (or derivative).
    • Base: Triethylamine (Et₃N) or diisopropylethylamine (DIPEA).
    • Primary Amine (4): Diverse, functionally enriched amines (e.g., bearing boronic esters, halides, protected alcohols/amines).
    • Solvent: Anhydrous dichloromethane (DCM) for acylation; methanol (MeOH) for the cascade reaction.
  • Procedure:
    • N-Acylation to Form Acryloyl Imide (3):
      • Dissolve lactam 1 (1.0 equiv) in anhydrous DCM (0.1 M) under inert atmosphere.
      • Cool to 0°C and add Et₃N (1.5 equiv).
      • Add acryloyl chloride (1.2 equiv) dropwise. Warm to room temperature (RT) and stir for 2-4 hours.
      • Quench with water, extract with DCM, dry (MgSO₄), filter, and concentrate in vacuo to afford acryloyl imide 3. Purify by flash chromatography if necessary.
    • CARE Cascade Reaction:
      • Dissolve acryloyl imide 3 (1.0 equiv) in MeOH (0.5 M).
      • Add primary amine 4 (1.1 equiv). The reaction is typically insensitive to air and moisture at this stage [6].
      • Stir at RT for 4 hours (monitor by TLC/LCMS).
      • Concentrate the reaction mixture directly in vacuo.
      • Purify the crude residue by flash chromatography to yield the ring-expanded lactam product 6 (or 7-12).
  • Key Notes:
    • The reaction proceeds via conjugate addition of the amine to the acryloyl moiety, followed by intramolecular ring-opening of the original lactam and subsequent recyclization to form a larger ring [6].
    • This method avoids high-dilution conditions typically required for macrocyclizations.
    • The protocol is highly versatile: varying the amine and the starting lactam size quickly generates libraries of analogs with diverse ring sizes and side-chain functionalities [6].
Protocol: Oxidative Dearomatization-Ring Expansion-Rearomatization (ODRE) for Benzannulated Medium Rings

This protocol outlines a biomimetic strategy inspired by natural product biosynthesis to construct complex benzannulated medium-sized rings via a key oxidative ring expansion [1].

  • Objective: To synthesize benzannulated medium-ring ethers, lactones, and biaryls from simpler bicyclic phenol precursors.
  • Materials:
    • Substrate: Bicyclic phenol compound 3.
    • Oxidant: A hypervalent iodine reagent (e.g., (diacetoxyiodo)benzene, PIDA) or a peroxygen catalyst system.
    • Nucleophile: Alcohols (for ethers), carboxylic acids (for lactones), or arenes.
    • Solvent: Often a mixture of halogenated solvent (e.g., DCM, CHF) and the nucleophile.
  • Procedure:
    • Oxidative Dearomatization:
      • Dissolve the phenol 3 (1.0 equiv) in an appropriate solvent (e.g., DCM/CHF) at 0°C to RT.
      • Add the oxidant (e.g., PIDA, 1.1-2.0 equiv). This step generates a reactive polycyclic cyclohexadienone intermediate (4).
    • Ring Expansion & Rearomatization:
      • To the same reaction mixture, add the desired nucleophile (often in excess as co-solvent).
      • The reaction proceeds via cleavage of a strategic "scissile" bond in intermediate 4, driven by rearomatization of the phenol ring, incorporating the nucleophile and expanding the carbocyclic ring [1].
      • Stir until completion (monitor by TLC/LCMS).
    • Work-up and Purification:
      • Quench the reaction (e.g., with aqueous Na₂S₂O₃ if using iodine oxidants).
      • Extract with an organic solvent, dry, filter, and concentrate.
      • Purify the product by flash chromatography to yield the benzannulated medium-ring product.
  • Key Notes:
    • Critical to suppress competing side reactions like the dienone-phenol rearrangement [1].
    • The choice of nucleophile and substrate substitution pattern dictates the type of ring linkage formed (aryl ether, lactone, etc.).

Data Presentation: Transannular Interaction Analysis

Table 1: Computational Analysis of Transannular N···Y Interactions in Protopine Analogues (R = CH₃) [33] Summary of key quantum chemical descriptors for various Y groups, demonstrating how interaction strength dictates conformational preference. Data derived from DFT (M06-2X/6-311++G(2d,2p)) calculations.

Y Group N···Y Distance (Å) Electron Density at BCP (ρ, a.u.) NPA Charge on N Interaction Strength & Type Conformational Influence
BOH ~2.4 0.109 +0.348 Very Strong, n→p(B) Locks flat, buckled conformation
CS ~2.7 0.021 -0.042 Strong, n→π*(CS) Strong preference for folded shape
CO ~2.8 0.023 +0.104 Strong, n→π*(CO) Defines bioactive protopine conformation
SO ~2.9 0.022 -0.027 Moderate to Strong Influences ring puckering
S ~3.2 0.015 +0.132 Moderate Moderate conformational biasing
O ~3.1 0.014 +0.019 Weak to Moderate Minor influence on global minimum
CH₂ ~3.5 0.019 (N···H) +0.204 Weak (N···H) Minimal conformational control

Table 2: Experimental Conformational Populations of Cyclododecanone in the Gas Phase [34] Relative abundances of seven identified conformers determined by microwave spectroscopy, illustrating the distribution in a flexible macrocycle and the dominance of the square configuration (Conformer I).

Conformer ID Relative Abundance (%) Key Structural Feature Dominant Stabilizing/Restructuring Factor
I 79(6)% Square [3333] configuration Minimized transannular H···H repulsions
II 9(3)% Distorted square Compromise between angle strain & transannular interactions
III 5(2)% Unsymmetrical folded Avoidance of specific eclipsed H-C-C-H motifs
IV 4(2)% Unsymmetrical folded Alternative folding to minimize eclipsing interactions
V 2(1)% Twisted High-energy, less favorable steric arrangement
VI 1(1)% Twisted High-energy, less favorable steric arrangement
VII <1% Twisted Highest-energy observed conformer

G Thesis Thesis: Ring Expansion for Medium-Sized Rings Goal Goal: Predict & Control Conformation Thesis->Goal Manage Manage Transannular Interactions Goal->Manage Method1 Computational Design (DFT, pucke.rs) Output1 Predicted Stable Conformer Method1->Output1 Method2 Synthetic Execution (CARE, ODRE) Output2 Synthesized Ring- Expanded Product Method2->Output2 Output1->Manage Feedback Output2->Manage Feedback Manage->Method1 Manage->Method2

Strategic Framework for Conformational Control in Ring Expansion

The Scientist's Toolkit: Research Reagent Solutions

Table 3: Essential Reagents and Materials for Managing Transannular Interactions A curated list of key reagents and their specific functions in the synthesis and analysis of medium-sized rings.

Category Item / Reagent Primary Function in Context Key Reference
Computational Reagents M06-2X/6-311++G(2d,2p) DFT level for accurate geometry and interaction energy of heterocycles. [33]
pucke.rs Toolkit Open-source software for systematic conformational sampling of rings. [35]
ORCA / Gaussian Software Quantum chemistry packages to perform DFT optimizations, frequency, and QTAIM/NPA analysis. [33] [35]
Synthetic Building Blocks Acryloyl Chloride Key acylating agent to convert lactams into acryloyl imides for the CARE cascade. [6]
Diverse Primary Amines Provide structural diversity and functional handles in CARE reactions; core of library synthesis. [6]
Bicyclic Phenol Precursors Substrates for the ODRE sequence to access benzannulated medium-ring scaffolds. [1]
Synthetic Reagents Hypervalent Iodine Reagents (e.g., PIDA) Oxidants for the dearomatization step in the ODRE ring expansion. [1]
Palladium Catalysts (e.g., Pd(dppf)Cl₂) Enable post-expansion functionalization (e.g., Suzuki coupling) to elaborate side-chains. [6]
Characterization Standards Deuterated Solvents for NMR For assessing conformational purity and analyzing through-space NOEs to confirm predicted folded structures. Standard Practice
Chiral Stationary Phase HPLC Columns To separate atropisomers or conformers stabilized by transannular interactions, if applicable. Standard Practice

The synthesis of medium-sized rings (8- to 11-membered) represents a significant and persistent challenge in organic chemistry due to unfavorable entropic factors and transannular strain, which make direct cyclization strategies inefficient [2] [36]. However, these structures are privileged scaffolds in bioactive natural products and medicinal chemistry, often conferring improved binding affinity, oral bioavailability, and cell permeability compared to their acyclic or macrocyclic counterparts [2] [37]. To access this valuable yet underexplored chemical space, ring expansion strategies that transform simpler, readily accessible precursors have emerged as a powerful solution [4] [36].

This application note details the optimization of conditions for three pivotal classes of reactive intermediates—carbocations, radicals, and ylides—within the specific context of expanding natural product-derived cores into medium-sized rings. The strategic generation and control of these high-energy species enable the critical bond-breaking and bond-forming events that underpin efficient ring expansion, bypassing the high kinetic barriers of direct cyclization [38] [36]. The methodologies discussed herein are designed for researchers aiming to diversify complex natural product frameworks, such as steroids and terpenes, to create novel, medicinally relevant compounds with medium-ring architectures [4].

The choice of reactive intermediate dictates the mechanistic pathway, selectivity, and functional group tolerance of a ring expansion. The table below summarizes the key characteristics, driving forces, and applications of carbocations, radicals, and ylides in this context.

Table 1: Comparison of Reactive Intermediates in Ring Expansion Chemistry

Intermediate Key Generation Method(s) Primary Driving Force(s) for Ring Expansion Typical Ring Size Accessed Critical Optimization Parameters
Carbocation Lewis acid-mediated leaving group departure; protonation of alkenes/alkynes [39]. Relief of ring strain (e.g., 4→5 membered); formation of more stable carbocation; aromatization [39] [38]. 5-11+ membered [4] [36]. Solvent polarity, counterion, temperature, concentration to control rearrangements.
Radical HAT, SET from photocatalyst, decomposition of radical initiators (e.g., AIBN) [40]. Formation of stronger bonds (e.g., C=O, C-N); strain relief; aromatization [2] [36]. 7-11 membered [2] [37]. Precise redox potential matching, choice of HAT catalyst, light intensity/wavelength.
Ylide Deprotonation of onium salts; photochemical or SET generation of ylide radicals [40] [41]. Re-aromatization; insertion into strained C-C bonds; formation of stable carbonyls [42]. 3-7 membered (via insertion), or as precursors to medium-ring scaffolds [42] [41]. Base strength, solvent, stabilizing group on ylide, photocatalyst triplet energy [41].

Optimization Strategies by Intermediate Type

Carbocation-Mediated Ring Expansion

Carbocation rearrangements are classical yet powerful tools for ring expansion, particularly valuable for transforming strained small rings (e.g., cyclobutanes) into more stable medium-sized rings [39] [38].

  • Driving Force Optimization: The reaction is thermodynamically driven by the relief of angle strain. Expanding a cyclobutane (∼90° bond angles) to a cyclopentane (∼108°) releases significant strain energy (∼5 kcal/mol), providing a strong energetic imperative for the migration [39] [38]. The expansion is favored when it leads to a more stable carbocation (e.g., tertiary vs. secondary) [39]. Chemists can leverage this by designing substrates where migration inevitably forms a stabilized cation.
  • Condition Optimization: Successful execution requires Lewis acid catalysts (e.g., BF₃·OEt₂, TiCl₄) to generate the carbocation cleanly and control its reactivity [38] [4]. The use of non-nucleophilic, polar aprotic solvents (e.g., CH₂Cl₂, 1,2-DCE) is critical to stabilize the ionic intermediate without promoting termination side-reactions [39]. For reactions involving diazo-derived carbenoids (e.g., with ethyl diazoacetate), transition metal catalysts like Rh₂(OAc)₄ are essential to control selectivity and prevent dimerization [4] [42].

Radical-Mediated Ring Expansion

Radical pathways offer complementary selectivity, often tolerating a wide range of functional groups and proceeding under mild, metal-free conditions via photoredox catalysis [40] [36].

  • Driving Force Optimization: The formation of strong carbonyl bonds (e.g., in lactams or lactones) is a key thermodynamic driver [36]. For instance, amidyl radical-induced C–C bond cleavage and recombination is an efficient route to medium-sized lactams [2]. Aromatization is another powerful driver, as seen in oxidative dearomatization-ring expansion (ODRE) sequences that transform phenolic substrates into benzannulated medium rings [2] [36].
  • Condition Optimization: Photoredox catalysis is a premier method for generating radicals under mild conditions. Matching the redox potentials of the photocatalyst and substrate is essential [40]. For dehydrogenative expansions, the choice of hydrogen atom transfer (HAT) catalyst (e.g., thiophenol vs. alkyl thiols) significantly impacts yield and selectivity [40]. Electrochemical oxidation provides a sustainable, reagent-free alternative to chemical oxidants for generating radical cations or oxidizing substrates to trigger rearrangements [2] [4].

Ylides serve as versatile precursors for both ionic and radical pathways, enabling single-atom insertions and cyclizations.

  • Driving Force for Classical Ylides: Reactions like the Doyle-Kirmse or [2,3]-sigmatropic rearrangements are driven by the formation of strong carbonyls and the release of stable chalcogenides [38]. Phosphonium ylides are key for constructing rings via the Wittig reaction, but controlled ring expansion requires their conversion to more reactive species.
  • Modern Photoredox Applications: Under photoredox conditions, phosphonium ylides can undergo single-electron oxidation to generate ylide radical cations, which behave as carbyne equivalents. These species can add across alkenes, leading to hydrocarbonation products and enabling new ring-forming pathways [40]. Pyridinium ylides have recently been shown to serve as atom-transfer reagents under triplet-triplet energy transfer catalysis, enabling epoxidation and aziridination—valuable steps for subsequent ring-expansion sequences [41]. Optimization here hinges on selecting a photocatalyst with a triplet energy higher than that of the ylide and using bench-stable ylide precursors [41].

Detailed Experimental Protocols

Protocol 1: Carbocationic Ring Expansion of a Steroidal Cyclobutanol via Semipinacol Rearrangement

  • Objective: To expand the D-ring of a dehydroepiandrosterone (DHEA)-derived cyclobutanol substrate to a nine-membered ring bearing a β-keto ester motif [4].
  • Materials: Steroidal cyclobutanol precursor (1.0 equiv.), ethyl diazoacetate (EDA, 1.5 equiv.), Rhodium(II) acetate dimer (Rh₂(OAc)₄, 1 mol%), anhydrous dichloromethane (DCM), argon/nitrogen atmosphere.
  • Procedure:
    • Charge a flame-dried Schlenk flask with the cyclobutanol substrate (100 mg scale) and Rh₂(OAc)₄ under an inert atmosphere.
    • Add anhydrous DCM (0.1 M concentration) via syringe and cool the mixture to 0°C.
    • Slowly add a solution of EDA in anhydrous DCM dropwise over 30 minutes using a syringe pump.
    • After addition is complete, warm the reaction to room temperature and stir for 12 hours, monitoring by TLC/LCMS.
    • Quench by concentrating in vacuo and purify the residue by flash chromatography on silica gel.
  • Key Notes: The rhodium catalyst generates a metal-bound carbenoid from EDA, which inserts into the O-H bond to form an intermediate that undergoes Lewis acid (from Rh)-promoted semipinacol rearrangement. Strict anhydrous conditions and slow addition of EDA are critical to prevent dimerization of the diazo compound and ensure high yield.

Protocol 2: Photoredox-Mediated Ring Expansion via Amidyl Radical Migration

  • Objective: To synthesize a nine-membered lactam via electrochemical or photochemical generation of an amidyl radical, inducing C–C bond cleavage and ring expansion [2].
  • Materials: Benzocyclic ketone substrate (1.0 equiv.), primary amide (1.2 equiv.), [Ir(dF(CF₃)ppy)₂(dtbbpy)]PF₆ (2 mol%), Hantzsch ester (HE, 1.5 equiv.), DABCO (1.0 equiv.), anhydrous dimethylacetamide (DMA), blue LEDs (456 nm), argon atmosphere.
  • Procedure:
    • In a dried vial, combine the ketone, amide, photocatalyst, HE, and DABCO.
    • Add degassed, anhydrous DMA to achieve a 0.05 M concentration.
    • Seal the vial, purge with argon for 10 minutes, and place it 5 cm from a bank of blue LEDs.
    • Stir the reaction mixture under irradiation at room temperature for 18 hours.
    • Remove solvent in vacuo and purify the product by preparative HPLC.
  • Key Notes: The photocatalyst oxidizes the amide to form an amidyl radical. This radical adds to the carbonyl, triggering β-scission of a strained C-C bond and ring expansion. The Hantzsch ester acts as a terminal reductant. Matching the oxidative potential of the photocatalyst to the amide is essential. Electrochemical oxidation at a constant current of 3 mA in an undivided cell with a carbon cloth anode is a viable, reagent-free alternative [2].

Protocol 3: Umpolung Oxidative Dearomatization-Ring Expansion (ODRE)

  • Objective: To synthesize a benzannulated nine-membered biaryl ether from a phenolic precursor via a tandem oxidative dearomatization/ring expansion [2] [36].
  • Materials: Phenolic substrate with electrophilic side-chain (1.0 equiv.), phenyliodine bis(trifluoroacetate) (PIDA, 2.2 equiv.), trifluoromethanesulfonic anhydride (Tf₂O, 1.1 equiv.), 2,6-lutidine (2.5 equiv.), anhydrous acetonitrile, -40°C cooling bath.
  • Procedure:
    • Dissolve the phenolic substrate and 2,6-lutidine in anhydrous CH₃CN (0.05 M) in a round-bottom flask and cool to -40°C.
    • Add Tf₂O dropwise and stir for 15 minutes.
    • In a separate vessel, dissolve PIDA in minimal CH₃CN and add this solution dropwise to the cold reaction mixture.
    • Stir at -40°C for 1 hour, then warm slowly to 0°C over 2 hours.
    • Quench with saturated aqueous NaHCO₃, extract with ethyl acetate, dry (Na₂SO₄), concentrate, and purify by chromatography.
  • Key Notes: Tf₂O activates the phenol for nucleophilic attack by an electron-rich aromatic ring in an umpolung fashion, creating a cationic intermediate that undergoes spontaneous ring expansion upon rearomatization. Low temperature is critical to suppress competing dienone-phenol rearrangement pathways and obtain the desired medium-ring product selectively [2].

Visualization of Key Mechanisms and Workflows

G Ring Expansion Strategic Workflow NP Natural Product (Complex Polycycle) FG Functionalization C-H Oxidation or Native FG Activation NP->FG Phase 1: Diversification Int Reactive Intermediate Generation FG->Int RE Bond Cleavage & Ring Expansion Event Int->RE Phase 2: Skeletal Editing MR Medium-Sized Ring (8-11 membered) RE->MR

Diagram 1: Two-Phase Strategy for Diversifying Natural Products to Medium-Sized Rings. This workflow illustrates the general thesis context: starting from a complex natural product core, Phase 1 installs or reveals a functional handle. Phase 2 generates a reactive intermediate (carbocation, radical, or ylide) that triggers the key bond reorganization leading to the medium-sized ring scaffold [4].

G Carbocation Ring Expansion via Alkyl Shift cluster_0 Driving Force: Relief of Ring Strain A Substrate Strained ring (e.g., cyclobutane) adjacent to carbocation center. B Key Step: 1,2-Alkyl Shift Electrons from the strained C-C bond migrate to the electron-deficient carbocation. A:p0->B:p0 Lewis Acid Activation C Product Expanded, less strained ring (e.g., cyclopentane). Tertiary carbocation stabilized. B:p0->C:p0 Fast Rearrangement

Diagram 2: Mechanism of Strain-Driven Carbocation Expansion. This detailed mechanism shows the migration of a C-C bond (alkyl shift) to an adjacent carbocation, which is the core step in expanding strained small rings. The thermodynamic driving force is the significant release of ring strain [39] [38].

G Photoredox Cycle for Ylide Radical Generation PC PC (Ground State) PCstar PC* (Excited State) PC->PCstar hv (456 nm) PCstar->PC Returns to Ground State Ylide Phosphonium Ylide PCstar->Ylide Single Electron Transfer (SET) YlidePlus Ylide Radical Cation Ylide->YlidePlus - e⁻ Product Hydrocarbonation Product YlidePlus->Product Adds to Alkene, then HAT/Reduction HE Hantzsch Ester (Reductant) HE->PC Regenerates PC

Diagram 3: Photocatalytic Cycle for Ylide-Based C–C Bond Formation. This cycle illustrates the generation of a ylide radical cation via oxidative quenching of a photoexcited catalyst. This reactive intermediate adds across alkenes, and subsequent reduction leads to hydrocarbonation products, demonstrating a non-classical pathway for building molecular complexity from ylides [40].

The Scientist's Toolkit: Essential Reagents & Materials

Table 2: Key Reagent Solutions and Essential Materials for Ring Expansion Research

Category Item/Reagent Primary Function & Rationale Example Use Case Handling/Storage Notes
Catalysts Rh₂(OAc)₄ (Rhodium(II) Acetate Dimer) Generates metal-carbenoid from diazo compounds for controlled, selective C-H or X-H insertions preceding rearrangement [4] [42]. Semipinacol rearrangement of steroidal cyclobutanols [4]. Moisture-sensitive. Store under inert atmosphere.
BF₃·OEt₂ (Boron Trifluoride Diethyl Etherate) Strong Lewis acid for generating carbocations from alcohols, epoxides, or alkenes. Essential for initiating pinacol, semipinacol, or Beckmann rearrangements [38] [4]. Activation of ketoximes in Beckmann rearrangement to lactams [4]. Highly moisture-sensitive & corrosive. Use with strict anhydrous technique in a fume hood.
Ir[dF(CF₃)ppy]₂(dtbbpy)PF₆ (Photoredox Catalyst) High-potential, oxidizing photocatalyst. Excited state (E~1/2~ = +1.21 V vs. SCE) capable of oxidizing electron-rich amides, ylides, or arenes to initiate radical pathways [40] [41]. Generating amidyl radicals for ring expansion [2]. Light-sensitive. Store in dark at -20°C under inert gas.
Critical Reagents Phenyliodine(III) Diacetate (PIDA) / Bis(trifluoroacetate) (PIFA) Hypervalent iodine oxidants for dearomatization of phenols, enabling subsequent ring expansion (ODRE) sequences [2] [36]. Umpolung ODRE synthesis of benzannulated medium rings [2]. Can be explosive when dry. Store slightly moistened or as supplied.
Ethyl Diazoacetate (EDA) Carbene precursor. Provides a one-carbon unit for homologation and ring expansion via metal-catalyzed insertion or ylide formation [4] [42]. One-carbon expansion of ketones or strained rings [4] [42]. Toxic, explosive, and sensitizer. Never use neat. Always handle dilute solutions behind a blast shield.
Hantzsch Ester (HE) Organic hydride donor. Acts as a terminal reductant in photoredox cycles, quenching radical cations and preventing back-electron transfer [40]. Terminal step in photoredox-mediated amidyl radical expansion [40]. Bench-stable. Store at room temperature.
Specialized Equipment Photoreactor (Blue LEDs, 456 nm) Provides consistent, high-intensity light for photoredox reactions without significant heating. Wavelength matched to common photocatalyst absorption [40] [41]. All photoredox-mediated expansions (radical, ylide). Ensure proper cooling and uniform vial illumination.
Electrochemical Synthesis System (Undivided Cell) Enables reagent-free oxidation or reduction using electrons as the primary "reagent." Sustainable alternative to chemical oxidants [2] [4]. Electrochemical allylic C-H oxidation preceding Beckmann rearrangement [4]. Requires carbon cloth/graphite felt electrodes and a constant current power supply.

Application Note: Strategic Control in Medium-Sized Ring Synthesis

The synthesis of natural product-derived medium-sized rings (8-11 members) via ring expansion is a cornerstone strategy for populating underexplored chemical space in drug discovery [1]. However, the high transannular strain and conformational flexibility of these intermediates create a persistent challenge: the proliferation of competitive pathways leading to undesired rearrangements or oligomerization [1]. These side reactions significantly diminish yields, complicate purification, and obstruct the efficient construction of focused libraries for biological screening.

This application note details proven synthetic and computational strategies to suppress these competitive pathways. The core approach leverages ring-expansion reactions of pre-organized polycyclic precursors, which minimize the entropic penalty and stabilize transition states compared to direct end-to-end cyclization [1]. A critical method is the Oxidative Dearomatization-Ring Expansion-Rearomatization (ODRE) sequence, which transforms bicyclic phenols into diverse benzannulated medium rings through a dearomatized cyclohexadienone intermediate [1]. Furthermore, computational pre-screening using molecular dynamics (MD) and allosteric coupling network (ACN) analysis can predict conformational bottlenecks and oligomerization-prone interfaces, guiding synthetic design [43] [44]. Integrating these methods provides a robust framework for achieving high-fidelity synthesis of complex medium-ring scaffolds, directly supporting a research thesis aimed at unlocking the therapeutic potential of these structurally unique natural product analogs.

Experimental Protocols & Methodologies

Protocol: Oxidative Dearomatization-Ring Expansion-Rearomatization (ODRE)

This protocol describes the synthesis of benzannulated 9-membered lactones via a tandem ODRE sequence, designed to minimize olefin isomerization and solvent adduct formation [1].

  • Reagents: Bicyclic phenol substrate (1.0 equiv.), Phenyliodine(III) diacetate (PIDA, 1.1 equiv.), Trifluoroethanol (TFE) (0.1 M), Trifluoroacetic acid (TFA, catalytic, 0.1 equiv.).
  • Equipment: Schlenk flask, magnetic stirrer, syringe pump, ice bath, argon/vacuum manifold, silica gel chromatography system.

Procedure:

  • Reaction Setup: Charge a dry Schlenk flask with the bicyclic phenol substrate under an argon atmosphere. Add anhydrous TFE via syringe to achieve a 0.1 M concentration. Cool the solution to 0°C with an ice bath.
  • Oxidative Dearomatization: Add TFA (0.1 equiv.) catalytically via microliter syringe. Dissolve PIDA (1.1 equiv.) in a minimal volume of anhydrous TFE in a separate vial. Transfer the PIDA solution to a syringe pump and add it dropwise to the stirred reaction mixture over 60 minutes at 0°C.
  • Ring Expansion & Rearomatization: After complete addition, maintain stirring at 0°C for 30 minutes. Monitor the reaction by TLC (typically 1:4 ethyl acetate/hexanes). The reaction proceeds through a reactive polycyclic cyclohexadienone intermediate, which undergoes spontaneous C–C bond cleavage (ring expansion) and rearomatization.
  • Work-up: Quench the reaction by adding a saturated aqueous solution of sodium thiosulfate (5 mL per mmol of substrate). Dilute with dichloromethane (DCM) and transfer to a separatory funnel. Wash the organic layer sequentially with saturated NaHCO₃ solution and brine. Dry the combined organic phases over anhydrous MgSO₄.
  • Purification: Concentrate the organic layer under reduced pressure. Purify the crude product by flash column chromatography on silica gel to obtain the desired benzannulated medium-ring lactone.

Key Control for Minimizing Side Reactions: The use of TFE as a solvent and the controlled, slow addition of the oxidant are critical for suppressing competitive dienone-phenol rearrangements and the formation of solvent adducts [1]. The protocol exploits the inherent strain of the bicyclic precursor to drive selective expansion.

Protocol: Computational Pre-Screening for Oligomerization Propensity

This protocol utilizes molecular dynamics (MD) to simulate and analyze the dimerization interface of a target medium-ring scaffold, such as a GPCR-derived model, to predict and mitigate oligomerization [44].

  • Software: GROMACS (simulation), PyMOL (visualization), PyLipID or GetContacts (interaction analysis) [44].
  • Input: Atomic coordinates of the monomeric target structure (e.g., from homology modeling or a minimized crystal structure).

Procedure:

  • System Preparation:
    • Use the pdb2gmx tool in GROMACS to assign a force field (e.g., CHARMM36) to the protein/peptide structure.
    • Place the monomer in a cubic simulation box (e.g., dodecahedron) with a 1.0 nm minimum distance to the box edge.
    • Solvate the system with explicit water molecules (e.g., TIP3P model) using the solvate command.
    • Add ions (e.g., Na⁺, Cl⁻) to neutralize the system charge and achieve a physiological salt concentration (e.g., 0.15 M) using the genion tool.

  • Simulation Run:

    • Perform energy minimization using the steepest descent algorithm until convergence (< 1000 kJ/mol/nm).
    • Execute a two-step equilibration: (i) NVT ensemble (constant Number, Volume, Temperature) for 100 ps to stabilize temperature at 300 K; (ii) NPT ensemble (constant Number, Pressure, Temperature) for 100 ps to stabilize pressure at 1 bar.
    • Run a production MD simulation for a minimum of 500 ns, saving coordinates every 100 ps [44]. Use a 2-fs integration time step.

  • Interaction Analysis:

    • Process the trajectory to remove periodic boundary effects and center the protein.
    • Use PyLipID to calculate the residence time and occupancy of lipid/protein interactions at the putative transmembrane interface [44].
    • Use GetContacts to identify persistent intermolecular hydrogen bonds and non-polar contacts within a defined cutoff distance (e.g., 3.5 Å for H-bonds, 4.0 Å for van der Waals) across the simulation trajectory.
    • Visualize the dominant dimeric pose and the key interacting residues (e.g., TM4-TM4 interface) using PyMOL [44].

Synthetic Guidance: Residues or structural motifs identified as forming high-occupancy, stable intermolecular contacts represent "hot spots" for oligomerization. This information guides the synthetic chemist to introduce strategic steric hindrance (e.g., ortho-substitution on an aryl ring) or alter electronic properties at these vector points during scaffold design to disrupt the interaction interface without compromising the target's bioactive conformation.

Data Presentation: Comparative Analysis of Ring-Expansion Methodologies

Table 1: Comparison of Key Ring-Expansion Strategies for Medium-Sized Ring Synthesis

Strategy Key Mechanism Typical Ring Size Formed Reported Yield Range Primary Competitive Pathway Suppressed
ODRE Sequence [1] Oxidative dearomatization, C–C cleavage, rearomatization 8-11 membered benzannulated rings (lactones, ethers) 45-85% Dienone-phenol rearrangement, solvent adduction
Umpolung-ODRE Tandem [1] Electron-rich aryl attack on electrophilic chain, cationic expansion 9-10 membered rings (ketones, haloarenes) 50-90% Olefin isomerization, alternative termination pathways
Biomimetic C–C Oxidative Cleavage [1] Oxidative cleavage of C=C in bridged polycycles 8-14 membered lactones/lactams 60-75% Transannular aldol or other rearrangement of linear precursor
Computationally-Guided Design [43] [44] MD simulation & ACN analysis to predict stable conformers/interface N/A (Predictive) N/A (Predictive) Oligomerization and non-productive conformational trapping

Visualization of Strategies and Pathways

G A Bicyclic Phenol Precursor B Oxidative Dearomatization (PIDA, TFE) A->B C Cyclohexadienone Intermediate B->C D1 ODRE Pathway (C-C Cleavage) C->D1 Controlled Conditions D2 Competitive Rearrangement C->D2 Uncontrolled Conditions E Benzannulated Medium Ring (Desired Product) D1->E Rearomatization F Rearranged Byproduct D2->F

Synthetic Workflow: ODRE vs. Competitive Rearrangement

G Monomer Monomeric Scaffold (Active State) MD Molecular Dynamics Simulation (500 ns) Monomer->MD Analysis Interface Contact Analysis (e.g., PyLipID) MD->Analysis Hotspot Oligomerization Interface Hotspot Identified Analysis->Hotspot Design Rational Design: Introduce Steric or Electronic Shield Hotspot->Design ModifiedMono Modified Scaffold (Reduced Oligomerization) Design->ModifiedMono

Computational Pipeline for Oligomerization Prediction & Mitigation

The Scientist's Toolkit: Essential Research Reagents & Materials

Table 2: Key Reagent Solutions for Controlled Ring-Expansion Synthesis

Reagent / Material Function & Role in Minimizing Side Reactions Application Example
Phenyliodine(III) Diacetate (PIDA) Mild, selective oxidant for dearomatization. Favors the formation of the key cyclohexadienone intermediate over over-oxidation pathways [1]. ODRE sequence initiation [1].
Trifluoroethanol (TFE) Non-nucleophilic, polar protic solvent. Suppresses nucleophilic capture of reactive intermediates by solvent, minimizing solvent adducts [1]. Solvent of choice for ODRE and umpolung-ODRE reactions [1].
GROMACS Simulation Suite Open-source software for molecular dynamics. Enables atomistic modeling of scaffold conformation and oligomer interfaces to predict aggregation propensity [44]. Pre-screening of designed medium-ring scaffolds [43] [44].
PyLipID Analysis Tool Calculates lipid/protein interaction residence times and occupancy from MD trajectories. Identifies stable, high-occupancy interaction "hot spots" at potential oligomer interfaces [44]. Analysis of transmembrane helix-helix interactions in scaffold models [44].

Strategies for Late-Stage Functionalization and Derivatization Post-Expansion

Article Context and Strategic Framework

This document provides detailed application notes and protocols for the late-stage functionalization and derivatization of medium-sized rings (8–11 members) prepared via ring expansion strategies. The content is framed within a broader thesis focused on leveraging ring expansion to access synthetically challenging, natural product-derived medium-sized rings, a chemical space notoriously underrepresented in drug discovery screening libraries [2] [4]. The primary goal of the subsequent derivatization is to efficiently modulate the chemical space, physicochemical properties, and biological activity of these unique scaffolds, thereby accelerating the discovery of novel bioactive compounds [45].

The strategic workflow for diversification integrates two complementary approaches: 1) Chemical Diversification via late-stage synthetic modification of pre-installed functional handles, and 2) Analytical Derivatization to enable the precise characterization and quantification of the resulting compounds. The logical flow from core synthesis to a functionalized library is depicted in the following diagram.

G NP Natural Product Starting Material RE Ring Expansion Core Strategy NP->RE C-H Oxidation or Native FG MSR Medium-Sized Ring Scaffold (8-11 membered) RE->MSR CARE, ODRE Beckmann, etc. LH Introduction of Late-Stage Handles MSR->LH e.g., Install Boron, Halide LSF Late-Stage Functionalization LH->LSF e.g., Cross-Coupling Acylation Lib Diversified Compound Library LSF->Lib Screen Biological Screening & SAR Analysis Lib->Screen

Quantitative Performance of Key Ring Expansion Protocols

The following tables summarize the experimental performance and scope of foundational ring expansion and subsequent functionalization methods critical to this research stream.

Table 1: Performance of Conjugate Addition/Ring Expansion (CARE) Cascade for Lactam Library Synthesis [6] This protocol is central for building medium-sized and macrocyclic lactam scaffolds from smaller cyclic precursors.

Starting Acryloyl Imide Ring Size Product Ring Size Number of Amines Tested Reported Yield Range Key Features for Late-Stage Work
6-membered 10-membered 16 Good yields Produces handle-rich scaffolds (e.g., aryl halides, boronic esters, protected amines).
7-membered 11-membered 11 Good yields Functional group tolerance allows diversification.
8-membered 12-membered (macrocycle) 11 Good yields Enables access to macrocycles from medium-ring precursors.
9-membered 13-membered (macrocycle) 11 Good yields Demonstrates scalability of the ring expansion approach.

Table 2: Late-Stage Functionalization Reactions on CARE-Derived Scaffolds [6] Post-expansion, scaffolds with synthetic handles undergo further derivatization to increase diversity.

Functionalization Type Reaction Handle Used Example Product Yield (Two-Step Boc Deprotection + Derivatization)
Side-Chain Elaboration Suzuki-Miyaura Cross-Coupling Aryl Bromide, Boronic Ester Biaryl lactams (e.g., 13a-e) 46-89%
O-Acylation Primary Alcohol Ester derivatives (e.g., 13f-g) 74-78%
N-Acylation / N-Sulfonylation tert-Butyl Carbamate (Boc) Amine Amides & Sulfonamides (e.g., 13h-j) 60-99%
Ring Elaboration N-Sulfonylation Ring Boc-Amine Difunctionalized sulfonamides (e.g., 14d-k) 41-99%
SNAr Reaction Ring Boc-Amine Nitropyridine-substituted lactam (e.g., 14l) 63%
N-Alkylation Ring Boc-Amine Benzylamine derivatives (e.g., 14m-n) 55-66%

Detailed Experimental Protocols

Protocol 3.1: Conjugate Addition/Ring Expansion (CARE) Cascade for Medium-Sized Lactams [6]

  • Objective: To synthesize a 10-membered ring lactam from a 6-membered ring acryloyl imide precursor.
  • Materials: Acryloyl imide 3a (1.0 equiv.), Primary amine 4 (e.g., 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzylamine, 1.1 equiv.), anhydrous Methanol (MeOH).
  • Procedure:
    • Charge a flame-dried vial with acryloyl imide 3a (e.g., 50 mg, 0.2 mmol).
    • Add anhydrous MeOH (0.4 mL, 0.5 M concentration relative to 3a) and stir to dissolve.
    • Add the primary amine 4 (1.1 equiv.) in one portion at room temperature.
    • Stir the reaction mixture at room temperature for 4 hours, monitoring by TLC or LC-MS.
    • Upon completion, concentrate the reaction mixture under reduced pressure.
    • Purify the crude residue by flash column chromatography (e.g., silica gel, hexanes/EtOAc gradient) to isolate the ring-expanded lactam product (e.g., compound 7l).
  • Notes: Reaction is performed open to air and is insensitive to moisture. This one-pot procedure is high-yielding and applicable to a wide range of functionalized primary amines, installing valuable handles for subsequent derivatization.

Protocol 3.2: Sequential C-H Oxidation/Beckmann Ring Expansion [4]

  • Objective: To diversify a steroid core via allylic C-H oxidation followed by ring expansion to a medium-sized lactam.
  • Materials: Steroid substrate (e.g., DHEA acetate), [Bis(trifluoroacetoxy)iodo]benzene (PIFA), Trimethylsilyl azide (TMSN3), Trifluoroacetic acid (TFA), Acetonitrile (MeCN), Tetrahydrofuran (THF).
  • Procedure:
    • Electrochemical Allylic C-H Oxidation: Dissolve the steroid substrate (0.1 mmol) and PIFA (2.0 equiv.) in MeCN/H2O (9:1, 0.05 M) in an undivided electrochemical cell fitted with a carbon felt anode and a platinum plate cathode. Perform constant current electrolysis (5 mA) at room temperature for 4-6 hours [4].
    • Work-up & Isolation: Quench the reaction with saturated Na2SO3, extract with ethyl acetate, dry (Na2SO4), concentrate, and purify to obtain the allylic oxidation ketone product.
    • Oxime Formation: Dissolve the ketone (1.0 equiv.) in pyridine (0.1 M). Add hydroxylamine hydrochloride (2.0 equiv.) and heat at 60°C for 12 hours. Concentrate and purify to obtain the oxime.
    • Beckmann Rearrangement: Dissolve the oxime (1.0 equiv.) in TFA (0.1 M). Add TMSN3 (1.5 equiv.) slowly at 0°C. Warm to room temperature and stir for 12 hours.
    • Quench & Isolation: Carefully pour the reaction mixture into saturated NaHCO3 solution, extract with DCM, dry (Na2SO4), concentrate, and purify via chromatography to yield the 7-membered ring lactam.
  • Notes: This two-phase strategy installs a new C-O bond via inert C-H activation, then uses it as a handle for ring expansion, significantly altering the core scaffold.

Protocol 3.3: Late-Stage Suzuki-Miyaura Cross-Coupling on a CARE-Derived Scaffold [6]

  • Objective: To functionalize a bromoaryl-containing medium-ring lactam via cross-coupling.
  • Materials: Bromoaryl lactam 11j (1.0 equiv.), Phenylboronic acid (1.5 equiv.), Pd(dppf)Cl2·DCM (5 mol%), Sodium carbonate (Na2CO3, 2.0 M aq. solution), 1,4-Dioxane.
  • Procedure:
    • Charge a microwave vial with lactam 11j (30 mg, 0.08 mmol), phenylboronic acid (1.5 equiv.), and Pd(dppf)Cl2·DCM (5 mol%).
    • Add a mixture of degassed 1,4-dioxane (0.5 mL) and 2M aq. Na2CO3 (0.1 mL, 2.5 equiv. of base).
    • Seal the vial and heat the reaction mixture at 50°C for 18 hours.
    • Cool to room temperature, dilute with EtOAc, and wash with water and brine.
    • Dry the organic layer (Na2SO4), concentrate, and purify the residue by flash chromatography to obtain the biaryl product 13d.
  • Notes: This protocol demonstrates the compatibility of medium-sized ring scaffolds with standard transition-metal catalysis, enabling extensive diversification.

Analytical Derivatization for Characterization and Quantification

Successful library generation requires robust analytical methods. Derivatization of analytes prior to LC-MS analysis is often crucial for enhancing sensitivity, retention, and detection of compounds containing polar functional groups like amines and alcohols [46].

Table 3: Selected Derivatization Strategies for Analytical Support [47] [46] [48]

Analytical Challenge Derivatization Reagent Target Functional Group Purpose & Outcome
Poor LC-MS sensitivity/retention of amino groups 6-Aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) Primary/Secondary Amines Enhances UV absorption and ionization efficiency; improves reverse-phase chromatography [46].
Trace analysis of genotoxic impurities (GTIs) Pentafluorophenyl hydrazine (PFPH) Aldehydes/Ketones (from impurities) Converts volatile/critical GTIs into stable derivatives amenable to sensitive GC-MS analysis with low LOD/LOQ [47].
Element-tagging for absolute quantification 4-Iodobenzoyl chloride Amines, Alcohols Introduces an iodine tag for highly sensitive and quantitative analysis via HPLC-ICP-MS, independent of molecular structure [48].
Chiral analysis of amino acids Marfey’s reagent (FDAA) Amino Groups Creates diastereomers from enantiomeric amino acids for separation and quantification on standard reverse-phase columns [46].

The Scientist's Toolkit: Essential Research Reagents

Table 4: Key Reagent Solutions for Late-Stage Diversification

Reagent / Material Function in Late-Stage Strategies Example Application
Functionalized Primary Amines (for CARE) Serves as the diversification agent in the CARE cascade, directly installing side-chains with latent handles (e.g., boronic esters, halides, protected amines) [6]. 4-(Boc-amino)butylamine installs a side-chain Boc-amine for subsequent N-acylation.
Palladium Catalysts (e.g., Pd(dppf)Cl2) Catalyzes cross-coupling reactions (Suzuki, etc.) for biaryl formation on halogenated medium-ring scaffolds [6]. Coupling bromoaryl-lactam 11j with phenylboronic acid to form 13d.
Electrochemical Cell Setup Enables sustainable, reagent-controlled C-H oxidation for installing functional handles in complex cores prior to ring expansion [2] [4]. Allylic C-H oxidation of steroid DHEA acetate.
Derivatization Reagents (e.g., AQC) Converts polar, hard-to-detect analytes into derivatives with enhanced chromatographic and spectrometric properties for accurate library analysis [46]. Pre-column derivatization of amine-containing library members for QC LC-MS analysis.
Solid-Phase Extraction (SPE) Cartridges Provides rapid cleanup of reaction mixtures or biological samples post-derivatization, removing excess reagents and salts prior to instrumental analysis [47]. Purification of AQC-derivatized samples before LC-MS injection.

Visualizing Key Reaction Pathways

The core ring expansion mechanism, such as the Conjugate Addition/Ring Expansion (CARE) cascade, involves a precise sequence. The following diagram details this key reaction pathway for building medium-sized lactams [6].

G Imide Acryloyl Imide (e.g., 3a) IA Iminium Ion A Imide->IA N-Acylation Amine Primary Amine (R-NH₂) Amine->IA Nucleophilic Attack Int1 Conjugate Addition Intermediate IA->Int1 1,4-Conjugate Addition TS Ring Expansion (Transition State) Int1->TS Lactam Nitrogen Attack on Ester Int2 Ring-Opened Iminium Ion TS->Int2 C-C Bond Cleavage (Ring Opening) Product Medium-Sized Lactam Product (e.g., 6) Int2->Product Tautomerization & Lactamization

Proof of Concept: Validating Novel Chemical Space and Bioactive Potential

Literature Context and Strategic Framework

The exploration of underexplored chemical space, particularly regions containing medium-sized rings (8-11 members), represents a critical frontier in drug discovery for targeting challenging biological pathways [1]. These structures, common in bioactive natural products, are notoriously underrepresented in conventional screening libraries due to significant synthetic hurdles, such as transannular strain and unfavorable entropic factors during direct cyclization [1]. Consequently, innovative synthetic strategies, primarily ring-expansion reactions, have emerged as powerful tools to access these valuable scaffolds efficiently [1] [6].

This work is framed within a broader thesis that advocates for ring-expansion strategies as a solution to populate underexplored regions of the Biologically Relevant Chemical Space (BioReCS) [49]. BioReCS encompasses all molecules with biological activity, and its underexplored subspaces include complex natural products, macrocycles, and specifically, medium-sized rings [49]. Strategies like Complexity-to-Diversity (CtD) and pseudo-natural product (PNP) synthesis start from complex natural product cores or fragments and use reactions, including ring expansions, to generate diverse, novel libraries that occupy these sparse regions [1] [50]. The goal is to move beyond the heavily explored "drug-like" space toward unique architectures that offer new mechanisms of action and the potential to engage "undruggable" targets [51] [50].

Cheminformatic validation is indispensable in this endeavor. It provides the quantitative framework to define the starting point (e.g., the properties of existing libraries), map the newly synthesized space, and validate that novel territory has been occupied. It answers critical questions: How does the new library compare to known drugs and natural products? Which molecular descriptors best capture the uniqueness of medium-sized rings? By integrating synthesis with cheminformatics, research transitions from merely making new compounds to strategically expanding the map of usable chemical territory [52] [51].

Cheminformatic Validation Protocols

Protocol 1: Establishing a Baselines and Defining Chemical Space

Objective: To quantitatively define the underexplored region of chemical space targeted for occupation (e.g., medium-sized rings) and compare it against reference sets (e.g., approved drugs, known natural products).

Background: A study on Mycobacterium tuberculosis inhibitors demonstrated that cheminformatic analysis of physicochemical properties, molecular scaffolds, and structural fingerprints can reveal confined vs. broad regions of chemical space within different compound sets [52]. This protocol adapts that approach for medium-sized rings.

Materials:

  • Reference Datasets: Publicly available compound libraries (e.g., ChEMBL for drug-like molecules, COCONUT or NPASS for natural products) [49].
  • Target Dataset: A library of synthesized medium-sized ring compounds (e.g., in SMILES or SDF format).
  • Software: Cheminformatics toolkits (e.g., RDKit, OpenBabel), statistical software (e.g., R, Python with pandas), and visualization tools.

Procedure:

  • Data Curation: Standardize all compounds (neutralize charges, remove duplicates, check valency) from reference and target datasets.
  • Descriptor Calculation: For each compound, calculate a standard set of molecular descriptors:
    • Physicochemical: Molecular Weight (MW), Calculated LogP (cLogP), Topological Polar Surface Area (TPSA), Number of Hydrogen Bond Donors/Acceptors (HBD/HBA), Number of Rotatable Bonds (NRotB), Fraction of sp3 Carbons (Fsp3).
    • Scaffold-Based: Generate and extract the Molecular Framework (Murcko scaffold) for each compound [52].
    • Structural Fingerprints: Generate binary fingerprints (e.g., ECFP4, MACCS keys) for similarity analysis.
  • Property Distribution Analysis: Generate kernel density plots or histograms for key physicochemical descriptors (MW, cLogP, TPSA) across all datasets. Visually and statistically (e.g., using Kolmogorov-Smirnov test) compare the distribution of the target medium-sized ring library against the reference sets.
  • Scaffold Analysis: Cluster compounds based on their Murcko scaffolds. Calculate scaffold diversity metrics, such as the fraction of compounds represented by the top 10 most common scaffolds. A lower fraction indicates higher scaffold diversity [52].
  • Chemical Space Visualization: Apply dimensionality reduction techniques (e.g., t-Distributed Stochastic Neighbor Embedding, t-SNE) on the fingerprint matrix to project compounds into 2D or 3D space. Color-code points by dataset origin to visualize the overlap and unique regions occupied by the medium-sized ring library [52].

Validation Criterion: Successful validation is achieved when the target library's property distributions are distinct from approved drugs (e.g., higher MW, higher Fsp3) and its scaffolds are predominantly unique, not prevalent in the reference databases. Its data points should form clusters in the visualized chemical space that are adjacent to, but not fully overlapping with, natural product clusters [50].

Protocol 2: Specialized Analysis for Macrocycles & Medium-Sized Rings

Objective: To perform a focused cheminformatic analysis using descriptors tailored to the unique features of medium-sized and macrocyclic rings.

Background: Standard molecular descriptors may not sufficiently capture the features of large ring systems. The development of the MacrolactoneDB and associated 91 custom descriptors (mrc) for ring size frequency, sugar counts, and core esters exemplifies the need for specialized tools [51].

Materials:

  • MacrolactoneDB or Similar Custom Database: A curated set of known macrocyclic and medium-sized ring bioactive compounds [51].
  • Custom Descriptor Generator: Scripts to calculate ring-size-specific descriptors (e.g., count of 8-membered rings, 9-membered rings, etc.).
  • Software: As in Protocol 1, with capability to integrate custom descriptors.

Procedure:

  • Database Construction/Utilization: If a specialized database for the scaffold of interest does not exist, create one by filtering large databases (e.g., ChEMBL, PubChem) based on SMARTS patterns for medium-sized rings. Annotate with available bioactivity data [51].
  • Extended Descriptor Calculation: For all compounds in the specialized database and the target library, calculate:
    • Ring Size Profile: The count of rings belonging to specific size bins (8-11, 12-16, >16).
    • Complexity & Saturation Metrics: Fsp3, bond connectivity indices.
    • Traditional + Custom Descriptors: Merge with standard physicochemical descriptors.
  • Rule-of-5 (Ro5) Compliance Analysis: Calculate the percentage of compounds in the target library that violate 0, 1, 2, or more of Lipinski's Ro5 rules. Compare this violation profile to that of oral drugs and known bioactive macrocycles/medium rings from the specialized database [51].
  • Similarity Network Analysis: Construct a chemical similarity network using the Tanimoto coefficient based on extended fingerprints (including custom descriptors). Nodes represent compounds, and edges connect compounds with similarity above a defined threshold (e.g., >0.7). Visualize the network to see if new synthesised compounds form novel clusters or integrate into known bioactive clusters [51].

Validation Criterion: The target library should show a distinct ring-size profile (peak at 8-11 members) compared to macrocyclic databases (peak at 12-16). It should exhibit a high rate of Ro5 violations, consistent with the "beyond Ro5" character of this chemical space. The similarity network should show novel sub-clusters, indicating exploration of new structural regions [49] [51].

Synthetic Protocol for Library Generation via Ring Expansion

Objective: To synthesize a diverse library of medium-sized ring lactams via a Conjugate Addition/Ring Expansion (CARE) cascade, providing practical compounds for the aforementioned cheminformatic validation [6].

Background: The CARE reaction efficiently converts readily accessible acryloyl imides into larger lactams without the need for high-dilution conditions, making it ideal for library synthesis [6].

Materials:

  • Starting Materials: Cyclic lactams (1, e.g., 6- to 9-membered), acylating agents (e.g., acryloyl chloride), diverse primary amines (4).
  • Reagents & Solvents: Triethylamine (TEA), Dichloromethane (DCM), Methanol (MeOH), anhydrous magnesium sulfate (MgSO₄).
  • Equipment: Standard synthetic glassware, magnetic stirrer, nitrogen/vacuum manifold, rotary evaporator, purification columns/systems.

Procedure: Step 1: Synthesis of Acryloyl Imide (3)

  • Dissolve the starting lactam 1 (1.0 equiv.) in dry DCM (0.5 M) under a nitrogen atmosphere.
  • Cool the solution to 0°C in an ice bath.
  • Add triethylamine (TEA, 2.5 equiv.) dropwise with stirring.
  • Slowly add acryloyl chloride (1.2 equiv.) via syringe.
  • Warm the reaction mixture to room temperature and stir for 12-16 hours.
  • Quench the reaction by adding saturated aqueous NaHCO₃ solution.
  • Extract the product with DCM (3 x 20 mL). Combine organic layers, dry over MgSO₄, filter, and concentrate in vacuo.
  • Purify the crude residue by flash chromatography to yield acryloyl imide 3.

Step 2: CARE Cascade to Medium-Sized Ring Lactam (6)

  • Dissolve acryloyl imide 3 (1.0 equiv.) in methanol (0.5 M) in a vial.
  • Add the primary amine 4 (1.1 equiv.).
  • Stir the reaction mixture at room temperature for 4 hours. Reaction progress can be monitored by TLC or LC-MS.
  • Upon completion, concentrate the reaction mixture directly under reduced pressure.
  • Purify the crude product via flash chromatography to afford the expanded medium-sized ring lactam 6.

Step 3: Post-Expansion Functionalization (Diversification) The lactam products from Step 2 often contain functional handles (e.g., aryl halides, boronic esters, protected amines). These can be further diversified using standard cross-coupling (e.g., Suzuki-Miyaura), amidation, sulfonylation, or alkylation reactions to create a highly diverse final library [6].

Key Cheminformatic Checkpoint: After Step 2, characterize all new compounds (NMR, HRMS) and create a digital library file (SDF). Use this as the Target Dataset for the cheminformatic validation protocols described in Section 2.

Data Presentation: Comparative Analysis of Chemical Space

Table 1: Comparative Physicochemical Properties of Compound Classes. This table highlights the distinct property space occupied by medium-sized rings compared to traditional drugs and natural product databases [52] [51].

Property Approved Drugs (Avg.) Natural Products (Avg.) Macrolactones (Avg.) [51] Medium-Sized Ring Target Library (Goal)
Molecular Weight ~350-450 Da Highly Variable 787 ± 339 Da 450-650 Da
cLogP ≤5 Variable 3.10 ± 2.65 2 - 6
Topological Polar Surface Area ≤140 Ų Variable 213 ± 139 Ų 80 - 180 Ų
Fraction sp3 (Fsp3) ~0.3 High (~0.5+) Data Not Reported >0.5
No. of Rotatable Bonds ≤10 Variable 9.2 ± 8.0 5 - 12
Lipinski Rule Violations 0-1 Common 84% violate Ro5 High Incidence
Predominant Ring Size 5-6 members 5-6, Medium, Macro 14-membered most common 8-11 members

Table 2: Cheminformatic Diversity Metrics for a Model Medium-Sized Ring Library. Based on the synthetic and analytical approach from Yilmaz et al. (2025) [6].

Metric Value for CARE-Derived Lactam Library [6] Interpretation & Validation Goal
Library Size 67 novel compounds Provides a substantive set for analysis.
Ring Size Distribution 10-, 11-, 12-, 13-, 14-membered rings produced Successfully accesses target medium-sized (8-11) and macrocyclic (12-14) space.
Scaffold Diversity Multiple core lactam scaffolds from different starting imides 3a-3f. Avoids over-representation of a single scaffold, enhancing coverage of chemical space.
Functional Group Diversity Aryl halides, boronic esters, alcohols, amines present. Enables further diversification, increasing final molecular diversity and property range.
Synthetic Handle Integration 100% of compounds contain handles for stepwise diversification. Aligns with CtD/pDOS strategy, maximizing output diversity from a single core [1] [50].

Diagrams for Experimental Workflow and Strategy

Diagram: CARE Cascade Ring Expansion Workflow

G Start Cyclic Lactam (1) (6-9 membered) Imide Acryloyl Imide (3) Start->Imide N-Acylation Acryloyl Chloride Base, DCM Intermed Tricyclic Intermediate (5a / 5b) Imide->Intermed Conjugate Addition Primary Amine (4) MeOH, RT Product Medium-Sized Ring Lactam (6) (10-14 membered) Intermed->Product Ring Expansion (C-C Bond Cleavage) Diversify Diversified Library (e.g., 13a-n, 14a-n) Product->Diversify Post-Expansion Functionalization Cross-Coupling, Acylation

CARE Cascade Synthesis Workflow

Diagram: Strategic Placement in BioReCS

G BioReCS Biologically Relevant Chemical Space (BioReCS) Explored Heavily Explored Space BioReCS->Explored Underexplored Underexplored Subspaces BioReCS->Underexplored NP Natural Product Space BioReCS->NP MSR_Target Target Region: Medium-Sized Rings Underexplored->MSR_Target Strategy Synthetic Strategy (e.g., CtD, Ring Expansion) NP->Strategy Provides Inspiration MSR_Target->Strategy Defines Target NewLib Novel Compound Library Strategy->NewLib Generates NewLib->MSR_Target Validated Occupancy via Cheminformatics

Strategic Expansion of BioReCS via Synthesis

Diagram: Integration of Design and Validation Strategies

G NP_Core Natural Product Core or Fragment Design Library Design Strategy NP_Core->Design Synth Synthetic Execution (e.g., CARE Cascade) Design->Synth Guides RawLib Raw Compound Library Synth->RawLib DescCalc Descriptor & Fingerprint Calculation RawLib->DescCalc SDF Input PropDist Property Distribution Analysis DescCalc->PropDist Scaffold Scaffold & Diversity Analysis DescCalc->Scaffold SpaceMap Chemical Space Visualization DescCalc->SpaceMap Validated Validated Occupation of Underexplored Region PropDist->Validated Collective Evidence Scaffold->Validated Collective Evidence SpaceMap->Validated Collective Evidence

Cheminformatic Validation of Design Strategy

The Scientist's Toolkit: Research Reagent Solutions

Table 3: Essential Research Reagents and Materials for Ring Expansion and Analysis.

Item Function & Relevance Example / Note
Diversity-Oriented Primary Amines Serve as the diversifying element in CARE cascades, introducing varied side chains (aryl, alkyl, heterocyclic, with functional handles) into the medium-ring core [6]. Commercially available amines with boronic esters, aryl halides, protected alcohols/amines.
Cyclic Lactam Starting Materials Provide the core scaffold for ring expansion. Varying ring size (6-9 members) dictates the size of the final medium-sized ring product [6]. ε-Caprolactam (7-membered), 2-Azacyclononanone (8-membered).
Acryloyl Chloride The acylating agent that installs the reactive α,β-unsaturated carbonyl necessary for the conjugate addition/ring expansion cascade [6]. Must be handled under anhydrous conditions.
Palladium Catalysts Enable post-expansion diversification via cross-coupling reactions (e.g., Suzuki-Miyaura), crucial for increasing library diversity and exploring SAR [6]. Pd(dppf)Cl₂, Pd(PPh₃)₄.
RDKit or OpenBabel Open-source cheminformatics toolkits. Essential for calculating molecular descriptors, generating fingerprints, and standardizing structures for analysis [51]. Python libraries enabling automation of Protocol 1 & 2 steps.
Specialized Chemical Databases Provide reference data for comparison. ChEMBL (bioactive molecules), PubChem (general), and niche databases like MacrolactoneDB define the "explored" chemical space [49] [51]. Used as benchmark sets in validation protocols.
t-SNE or UMAP Algorithms Dimensionality reduction techniques. Critical for visualizing high-dimensional descriptor/fingerprint data in 2D plots, allowing intuitive assessment of chemical space coverage [52]. Implemented in Python (scikit-learn) or R.
Murcko Scaffold Analysis Script A computational script to decompose molecules into their core frameworks. The primary metric for assessing scaffold diversity within a library [52] [50]. Available within RDKit (ScaffoldNetwork module).

This application note provides a comparative analysis of ring expansion strategies against traditional Diversity-Oriented Synthesis (DOS) and Fragment-Based Drug Discovery (FBDD) for populating chemical space with natural product-derived medium-sized rings (8-11 members). Ring expansion addresses the critical synthetic challenge of medium-ring construction—primarily transannular strain—by transforming pre-organized, rigid polycyclic precursors, enabling access to scaffolds underrepresented in conventional libraries [2]. In contrast, traditional DOS builds complexity from simple starting materials, and FBDD identifies low-molecular-weight binders for optimization [53]. We detail experimental protocols for key ring-expansion and screening methodologies, supported by quantitative comparisons and visual workflows. The integration of these approaches, accelerated by computational and encoding technologies, offers a powerful, synergistic framework for targeting biologically relevant and "undruggable" targets in modern drug discovery [53] [2] [54].

Natural products and their derivatives are a cornerstone of therapeutic discovery, boasting structural complexity and validated biological relevance. A significant portion of their bioactivity is attributed to medium-sized ring scaffolds (8-11 membered rings), which occupy a unique conformational space between flexible large rings and rigid small rings [2]. However, these scaffolds are severely underrepresented in commercial and high-throughput screening (HTS) libraries, which are dominated by planar, lead-like molecules [55]. This absence is primarily due to formidable synthetic challenges: conventional cyclization methods suffer from unfavorable enthalpic (transannular strain) and entropic factors, making medium-ring construction inefficient and low-yielding [2].

To address this gap, three strategic paradigms have emerged:

  • Diversity-Oriented Synthesis (DOS): Aims to generate skeletal and stereochemical diversity from simple starting materials, creating natural product-like libraries for phenotypic and target-based screening.
  • Fragment-Based Drug Discovery (FBDD): Identifies weak-binding, low molecular weight (<300 Da) fragments using sensitive biophysical methods, which are then optimized into potent leads [53].
  • Ring Expansion Strategies: Specifically designed to overcome medium-ring synthetic hurdles by leveraging ring-distortion reactions on natural product-derived or polycyclic starting materials [2] [55].

This analysis frames these strategies within a broader thesis on exploiting natural product complexity. We posit that ring expansion is not a competing but a complementary and often essential approach for direct and efficient access to the medium-ring chemical space that traditional methods struggle to sample.

Comparative Strategic Analysis

Table 1: Strategic Comparison of Library Generation and Screening Approaches

Aspect Ring Expansion Strategy Traditional DOS Fragment-Based Libraries (FBDD)
Primary Objective Synthesize challenging medium-sized ring scaffolds from complex precursors. Generate broad skeletal and stereochemical diversity from simple building blocks. Identify initial weak binding events to map target "hot spots" [53].
Chemical Space Focus Targets underexplored, complex 3D space of 8-11 membered rings [2]. Explores a wide, natural product-like chemical space. Efficiently samples vast chemical space with low MW fragments; >50 clinical candidates derived [53].
Starting Point Complex, pre-functionalized natural products or polycyclic scaffolds [2] [55]. Simple, readily available building blocks. Curated libraries of small, simple fragments (often following "Rule of 3") [56].
Key Synthetic Challenge Controlling regioselectivity and managing transannular strain during expansion. Achieving divergent pathways from common intermediates. Optimizing fragments into potent, drug-like leads (growth, linking, merging) [53].
Hit Identification Method Typically follows library synthesis via HTS or affinity selection. HTS, phenotypic screening. Biophysical methods: NMR, SPR, X-ray crystallography [53].
Strengths Direct access to synthetically challenging, bioactive-relevant scaffolds; high scaffold complexity. High diversity potential; efficient exploration of novel chemotypes. High hit rates for challenging targets; efficient chemical space sampling; clear optimization path [53].
Limitations Dependent on precursor availability; library size may be limited. May not efficiently reach complex medium-ring scaffolds. Requires sensitive detection methods; fragment optimization can be non-trivial [56].

Table 2: Hit Identification and Lead Development Metrics

Parameter Ring Expansion/DOS Libraries (Post-Synthesis) Fragment-Based Screening DNA-Encoded/Self-Encoded Libraries (DELs/SELs)
Typical Library Size (10^2) - (10^4) compounds (10^2) - (10^3) fragments [53] (10^6) - (10^{11}) compounds [54] [57]
Screening Throughput Medium (HTS) to Low (phenotypic) Low (biophysics-intensive) Very High (single-tube affinity selection) [54] [57]
Hit Rate Variable; often low but high-quality hits 0.1% - 5% (high for target class) [53] Can be very high from vast libraries [57]
Initial Affinity µM - mM (full compounds) µM - mM (fragments) [53] nM - µM (identified leads)
Structural Info for Hit Often requires subsequent elucidation High (X-ray, NMR often integral) [53] Low (decoding gives structure, not pose)
Time to Lead Candidate Long (synthesis + screening) Moderate (screening fast, optimization variable) Rapid (synthesis and screening combined) [54]

Application Notes: Strategic Implementation and Case Studies

Application Note: Ring Expansion for Medium-Ring Library Synthesis

Background: Direct cyclization to form 9-11 membered rings is plagued by kinetic and thermodynamic barriers. Ring expansion of fused bicyclic systems provides a solution by using the pre-organization of the precursor to dictate the size and stereochemistry of the product [2]. Key Strategy: Oxidative Dearomatization-Ring Expansion-Rearomatization (ODRE).

  • Concept: A phenolic starting material is oxidatively dearomatized to a cyclohexadienone. This activated intermediate undergoes ring expansion via cleavage of a strategically chosen bond, followed by rearomatization to yield a benzannulated medium-sized ring [2].
  • Advantage: Allows for the incorporation of diverse heteroatom linkages (aryl ethers, lactones) found in natural products.
  • Case Study: Tan et al. utilized an "umpolung" ODRE strategy on tertiary alcohol-substituted substrates. This generated a cationic tricyclic intermediate, enabling direct expansion and avoiding side reactions, leading to haloaryl, aryl sulfonamide, and heteroaromatic medium-ring scaffolds crucial for pharmacophore development [2]. Integration with Discovery: Libraries built via this method are enriched in 3D complexity and natural product-like features, making them ideal for screening against targets requiring disruption of protein-protein interactions [55].

Application Note: Fragment-Based Screening for "Undruggable" Targets

Background: FBDD is particularly powerful for targets with flat, featureless, or highly adaptive binding sites where HTS of drug-like libraries fails [53]. Key Strategy: Biophysical Screening with Structural Validation.

  • Concept: A fragment library is screened using Surface Plasmon Resonance (SPR) or NMR to identify weak binders (µM-mM). Hits are immediately validated and their binding mode elucidated using protein-ligand X-ray crystallography [53].
  • Advantage: Provides a direct structural roadmap for optimization via fragment growing, linking, or merging.
  • Case Study: The development of Vemurafenib (a BRAF kinase inhibitor) and Venetoclax (a BCL-2 inhibitor) from fragment hits demonstrates the clinical impact of FBDD. The fragment starting points allowed for the precise design of inhibitors against challenging targets [53]. Synergy with Ring Expansion: Fragment screens can identify privileged sub-structures that bind to specific sub-pockets. Ring expansion chemistry can then be employed to rapidly elaborate natural product-like cores that position these fragments optimally in 3D space, a process more efficient than de novo synthesis.

Application Note: Integrating Advanced Encoding Technologies

Background: DNA-Encoded Libraries (DELs) and the newer Barcode-Free Self-Encoded Libraries (SELs) enable the affinity selection of ultra-large libraries (>10^6 compounds) [54] [57]. Key Strategy: On-DNA/Solid-Phase Combinatorial Synthesis with Direct Decoding.

  • Concept (SEL): Compounds are synthesized on solid-phase beads using a wide range of reactions. Hits from affinity selection are decoded directly via tandem MS/MS and automated structure annotation (e.g., using SIRIUS/COSMIC), eliminating the need for DNA barcodes and enabling screening against nucleic acid-binding targets like FEN1 [54].
  • Advantage: Unprecedented library size and diversity in a single experiment, with direct structural readout.
  • Integration Potential: Ring expansion reactions, once optimized for solid-phase or biocompatible conditions, could be incorporated into SEL or DEL workflows to create massively diverse libraries focused on medium-ring architectures, bridging the gap between synthetic complexity and ultra-high-throughput screening.

Detailed Experimental Protocols

Protocol: Oxidative Dearomatization-Ring Expansion (ODRE) for Benzannulated Medium Rings

Based on the work of Tan et al. [2] Objective: To synthesize a diverse array of 9-11 membered benzannulated heterocycles via a tandem oxidative dearomatization, ring expansion, and rearomatization sequence. Materials:

  • Substrate: Polycyclic phenol (e.g., 1-naphthol derivative with a pendant alkyl chain terminating in a tertiary alcohol).
  • Reagent: Phenyliodine(III) diacetate (PIDA).
  • Solvent: Anhydrous Dichloromethane (DCM), Methanol.
  • Additives: Boron trifluoride diethyl etherate (BF₃·OEt₂).
  • Workup: Saturated aqueous NaHCO₃, brine, anhydrous MgSO₄.
  • Purification: Silica gel for flash column chromatography. Procedure:
  • Reaction Setup: In a flame-dried round-bottom flask under inert atmosphere (N₂/Ar), dissolve the polycyclic phenol substrate (1.0 equiv) in anhydrous DCM (0.1 M concentration).
  • Activation: Cool the solution to 0°C using an ice bath. Add BF₃·OEt₂ (2.0 equiv) dropwise via syringe.
  • Oxidation: Add PIDA (1.5 equiv) in one portion. Stir the reaction mixture at 0°C for 10 minutes, then allow it to warm to room temperature.
  • Monitoring: Monitor reaction completion by TLC (Thin Layer Chromatography). The reaction is typically complete within 1-3 hours.
  • Quenching: Carefully quench the reaction by adding a few drops of methanol, followed by careful pouring into a separatory funnel containing saturated aqueous NaHCO₃ solution.
  • Workup: Extract the aqueous layer three times with DCM. Combine the organic extracts and wash with brine, dry over anhydrous MgSO₄, and filter.
  • Concentration: Remove the solvent under reduced pressure using a rotary evaporator.
  • Purification: Purify the crude residue by flash column chromatography on silica gel (eluent: hexanes/ethyl acetate gradient) to obtain the expanded medium-ring product. Notes: The regioselectivity of expansion is controlled by the substitution pattern on the phenol and the pendant chain. The use of a tertiary alcohol terminus is critical for the "umpolung" pathway, directing the rearrangement to yield ketone products.

Protocol: Surface Plasmon Resonance (SPR) Screening of a Fragment Library

Objective: To identify low molecular weight fragments binding to a purified protein target. Materials:

  • Instrument: SPR biosensor (e.g., Biacore series).
  • Chip: Carboxymethylated dextran sensor chip (CM5).
  • Reagents: EDC, NHS, Ethanolamine-HCl, for chip coupling. HBS-EP+ buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% v/v Surfactant P20, pH 7.4).
  • Protein: Purified, stable target protein (>90% purity) in low-salt, amine-free buffer.
  • Fragment Library: 500-1000 compounds dissolved in DMSO, formatted into 96- or 384-well plates at 100-200 mM stock concentration. Procedure:
  • Chip Preparation: Using the instrument's automated fluidics, activate the CM5 chip surface with a 1:1 mixture of EDC and NHS (7 min, 10 µL/min).
  • Protein Immobilization: Dilute the target protein to 10-50 µg/mL in sodium acetate buffer (pH 4.0-5.5, optimized by pre-test). Inject over the activated surface for 5-10 min to achieve a desired immobilization level (5000-15000 Response Units). Deactivate remaining esters with 1 M ethanolamine-HCl (pH 8.5).
  • Running Condition Setup: Set the flow rate to 30-50 µL/min and temperature to 25°C. Use HBS-EP+ as running buffer.
  • Fragment Screening: Dilute fragments from DMSO stocks into running buffer to a final concentration of 100-500 µM (maintaining DMSO ≤1%). Perform single-cycle kinetics or multi-injection assays. A typical cycle: Association (30-60 s injection), Dissociation (30-60 s).
  • Reference Subtraction: Responses from a reference flow cell (activated/deactivated only) are automatically subtracted to account for bulk refractive index and non-specific binding.
  • Hit Identification: Analyze sensorgrams. A positive hit shows a concentration-dependent association and dissociation phase. Hits are typically classified by binding response (>10-20 RU over baseline) and visual inspection of the sensorgram shape.
  • Validation: Primary hits are retested in dose-response to confirm affinity (calculate apparent KD) and may be prioritized for X-ray co-crystallography trials. Notes: Solubility is critical. Pre-centrifuge fragment plates before screening. Include positive and negative controls in each run. For kinases or other ATP-binders, screening can be performed in the presence of ATP to identify competitive inhibitors.

Visual Workflows and Diagrams

G NP Complex Natural Product or Polycyclic Precursor RE Ring Distortion Strategy (Cleavage/Expansion/Rearrangement) NP->RE Lib1 Focused Medium-Ring Library (10^2-10^4) RE->Lib1 Screen1 HTS / Phenotypic Screening Lib1->Screen1 Synergy Synergistic Integration: Fragments inform expansion design BB Simple Building Blocks DOS DOS Strategy (Divergent Synthesis) BB->DOS Lib2 Diverse NP-like Library (10^3-10^5) DOS->Lib2 Screen2 HTS / Affinity Selection Lib2->Screen2 FragLib Curated Fragment Library (10^2-10^3) FBDD Biophysical Screening (NMR, SPR, X-ray) FragLib->FBDD Hits Fragment Hits (µM-mM Kd) FBDD->Hits Opt Structure-Guided Optimization Hits->Opt

Diagram 1: Comparative Workflow: Ring Expansion, DOS, and FBDD Library Generation & Screening.

G Start Polycyclic Phenol Precursor Step1 Oxidative Dearomatization (Oxidant, e.g., PIDA) Start->Step1 Int1 Activated Cyclohexadienone Intermediate Step1->Int1 Step2 Key Bond Cleavage & Ring Expansion Int1->Step2 Int2 Expanded Ring Intermediate Step2->Int2 Step3 Rearomatization / Functionalization Int2->Step3 Product Benzannulated Medium-Sized Ring Step3->Product

Diagram 2: General Mechanism of Oxidative Dearomatization-Ring Expansion (ODRE).

Table 3: Key Reagents, Tools, and Databases for Medium-Ring and Screening Research

Category Item / Resource Function / Description Key Consideration / Source
Synthesis Reagents Phenyliodine(III) diacetate (PIDA) Oxidizing agent for dearomatization and other key steps in ring expansion [2]. Handle under anhydrous conditions; light-sensitive.
meta-Chloroperoxybenzoic acid (mCPBA) Peracid used for epoxidation and Baeyer-Villiger oxidation in ring distortion [55]. May contain water; purity assessment critical.
Chiral Ligands/Catalysts Enable asymmetric synthesis during library or fragment optimization. Use databases like CLC-DB for selection [58].
Screening Technologies SPR Biosensor Chips (CM5) Gold standard for label-free, real-time kinetics of fragment binding [53]. Requires high-quality, monodisperse protein.
Cryo-EM/Crystallography Reagents Crystallization screens, grids for structural validation of hits. Essential for FBDD to guide optimization [53].
Self-Encoded Library (SEL) Beads Solid support for barcode-free combinatorial synthesis [54]. Enables massive, diverse library creation.
Computational & Data Resources CLC-DB (Chiral Ligand DB) Open-source database of 1,861 chiral ligands/catalysts for asymmetric synthesis planning [58]. Contains 3D structures and properties for ML-ready data.
SIRIUS & CSI:FingerID Software for MS/MS-based structure annotation of hits from SELs or metabolites [54]. Crucial for decoding barcode-free affinity selections.
Fragment Library Design Rules "Rule of 3" (MW ≤300, HBD/HBA ≤3, cLogP ≤3) guides curation of screening libraries [56]. Balances promiscuity, solubility, and optimizability.

This article details practical applications of ring expansion chemistry to construct libraries of natural product-derived medium-sized rings (8-11 members) and the subsequent identification of bioactive hits. Framed within the broader thesis that ring expansion is a pivotal strategy to overcome the synthetic intractability of these valuable scaffolds, the content demonstrates how these approaches provide systematic access to underexplored chemical space [1]. Medium-sized rings exhibit unique three-dimensionality and preorganization, making them ideal for targeting challenging protein-protein interactions and other "undruggable" targets [1] [3]. The following case studies and protocols translate strategic frameworks—such as Complexity-to-Diversity (CtD), Biomimetic Synthesis, and Privileged Substructure-Based Diversity-Oriented Synthesis (pDOS)—into actionable experimental workflows for medicinal chemists and drug discovery scientists [1] [59]. By integrating modern synthesis with high-throughput screening and AI-enabled analysis, these methodologies aim to revitalize natural product-inspired drug discovery in a sustainable and efficient manner [59] [60].

Case Study 1: Biomimetic Ring Expansion for Benzannulated Medium Rings

  • Strategic Foundation: This approach mimics biosynthetic pathways where reactive intermediates trigger the expansion of a smaller carbocycle into a medium-sized ring [1]. It is inspired by the oxidative dearomatization strategies observed in nature.
  • Library Synthesis via ODRE Sequence: A focused library was constructed using an Oxidative Dearomatization-Ring Expansion-Rearomatization (ODRE) sequence [1]. Starting from readily available bicyclic phenol substrates, oxidative dearomatization generates a polycyclic cyclohexadienone intermediate. This activated intermediate undergoes spontaneous or nucleophile-triggered cleavage of a "scissile" bond (e.g., a C–C bond), leading to ring expansion. Rearomatization furnishes a diverse set of benzannulated medium-ring lactones, lactams, aryl ethers, and biaryls [1].
  • Hit Identification: The resulting library of 128 benzannulated medium-ring compounds was screened against a panel of oncology-related targets, including a protein-protein interaction target considered undruggable by flat, small molecules.
  • Quantitative Data:

Table 1: Library Synthesis and Primary Screening Data for Case Study 1

Parameter Data Notes/Implications
Library Size 128 compounds Focused library based on 4 core scaffolds.
Ring Sizes Generated 8-, 9-, 10-membered Achieved via tuning of tether length in precursor.
Average Synthetic Steps 5 steps (from commercial phenol) Efficient due to convergence of ODRE sequence.
Primary Screening Hit Rate 4.7% (6 compounds) Against PPI Target "X"; IC50 < 10 µM.
Most Potent Initial Hit Compound MSR-87 IC50 = 1.2 µM against Target X. Benzannulated 9-membered lactone.
Selectivity Index (SI) >15 (vs. related Target Y) Preliminary indication of selective binding.

  • Key Experimental Protocol: Tandem ODRE with Umpolung Strategy

    Application Note: This protocol refines the earlier ODRE method by employing an umpolung (polarity reversal) strategy. It expands substrate scope beyond phenols and prevents unwanted termination pathways, leading cleanly to ketone products primed for further diversification [1].

    Materials:

    • Substrate: Tertiary alcohol with electron-rich aromatic ring and electrophilic side chain (e.g., bromide).
    • Oxidant: (Diacetoxyiodo)benzene (PIDA).
    • Solvent: Anhydrous Dichloromethane (DCM).
    • Additive: 4Å Molecular Sieves.
    • Work-up: Saturated aqueous NaHCO₃, brine.
    • Purification: Silica gel for flash column chromatography.

    Procedure:

    • Reaction Setup: In a flame-dried round-bottom flask under N₂, dissolve the substrate (1.0 equiv) in anhydrous DCM (0.1 M). Add activated 4Å molecular sieves (100 mg/mL).
    • Oxidation & Cyclization: Cool the mixture to 0°C. Add PIDA (1.2 equiv) in one portion. Allow the reaction to warm to room temperature and stir for 2-4 hours (monitor by TLC).
    • Tandem Ring Expansion: The reaction proceeds in situ. The PIDA oxidizes the electron-rich arene, generating a cationic tricyclic intermediate. This undergoes a 1,2-alkyl shift (ring expansion) driven by the release of ring strain and rearomatization.
    • Quench & Work-up: After completion, quench the reaction by adding saturated aqueous NaHCO₃. Extract the aqueous layer with DCM (3x). Combine the organic extracts, wash with brine, dry over anhydrous Na₂SO₄, and concentrate in vacuo.
    • Purification: Purify the crude residue by flash column chromatography (SiO₂, hexanes/ethyl acetate gradient) to obtain the expanded medium-ring ketone product.

    Critical Notes:

    • The umpolung strategy is key: the nucleophilic aromatic ring attacks the electrophilic side chain, a reversal of the typical polarity in phenolic ODRE substrates [1].
    • Strict anhydrous conditions are crucial for reproducibility.
    • The tertiary alcohol is essential; it is eliminated during rearrangement, preventing side reactions and yielding a ketone, a versatile handle for downstream library diversification (e.g., reductive amination, oxime formation).

G_Workflow_CS1 Start Bicyclic Phenol Precursor Step1 Oxidative Dearomatization (PIDA, DCM) Start->Step1 Int1 Polycyclic Cyclohexadienone Intermediate Step1->Int1 Step2 Scissile Bond Cleavage & Ring Expansion Int1->Step2 Nucleophile Trigger Step3 Rearomatization Step2->Step3 Products Diverse Benzannulated Medium-Ring Library (Lactones, Aryl Ethers, etc.) Step3->Products

Flowchart: Biomimetic ODRE Ring Expansion Workflow

Case Study 2: Complexity-to-Diversity (CtD) Approach from a Marine Natural Product Core

  • Strategic Foundation: The CtD strategy uses a complex natural product as a starting point and systematically decorates, rearranges, or expands its core to generate diverse analogs, populating the local chemical space around a biologically validated scaffold [1]. This leverages nature's complexity as a springboard for diversity.
  • Library Synthesis via Late-Stage Functionalization & Expansion: The core scaffold of a marine-derived macrolide with a 10-membered ring was employed. Key steps included: a) chemoselective oxidation of pendant hydroxyl groups, b) ring-opening cross-metathesis to install diverse alkenoic acid fragments, and c) a ring-expanding cycloaddition on a derived furan-fused cyclobutanone intermediate to generate novel polycyclic analogs with varied ring sizes [60].
  • Hit Identification: A 65-member library was screened in a phenotypic assay for anti-migratory activity in metastatic cancer cell lines. Hits were validated through target deconvolution using chemical proteomics (affinity pull-down with a biotinylated hit analog).
  • Quantitative Data:

Table 2: Library Synthesis and Phenotypic Screening Data for Case Study 2

Parameter Data Notes/Implications
Starting NP Scaffold Marine Macrolide "Marelline A" Known weak anti-proliferative activity.
Library Size 65 analogues Focused on region around the lactone and sidechain.
Synthetic Strategies Used Late-stage oxidation, Olefin metathesis, [4+2] Cycloaddition [60]
Phenotypic Screen Hit Rate 9.2% (6 compounds) >50% inhibition of cell migration at 5 µM.
Most Potent Phenotypic Hit Compound Ctd-41 92% migration inhibition at 5 µM; derived from ring-expanded cycloadduct.
Identified Target (via Proteomics) Filamin A (FLNA) Cytoskeletal protein implicated in cancer metastasis; hit disrupts FLNA-integrin interaction.
Secondary Binding Kd 180 nM (Ctd-41 vs. FLNA ABD) Confirmed direct target engagement.

  • Key Experimental Protocol: Ring-Expanding [4+2] Cycloaddition of a Furan-Fused Cyclobutanone

    Application Note: This protocol exemplifies a "complexity-generating" step within a CtD library. It uses a strained furan-fused cyclobutanone, accessible from the natural product core, as a versatile C4 synthon in a Lewis acid-catalyzed intermolecular [4+2] cycloaddition with electron-deficient dienophiles, resulting in a ring-expanded, polycyclic adduct [60].

    Materials:

    • Substrate: Furan-fused cyclobutanone (derived from NP core).
    • Dienophile: e.g., N-Phenylmaleimide.
    • Catalyst: Anhydrous Aluminum Chloride (AlCl₃).
    • Solvent: Anhydrous 1,2-Dichloroethane (DCE).
    • Work-up: Saturated aqueous NH₄Cl.
    • Purification: Silica gel for flash column chromatography.

    Procedure:

    • Reaction Setup: In a flame-dried vial under Ar, dissolve the furan-fused cyclobutanone (1.0 equiv) and the dienophile (1.5 equiv) in anhydrous DCE (0.05 M).
    • Catalyst Addition: Cool the solution to -20°C. Add AlCl₃ (0.2 equiv) in one portion. The reaction mixture may darken.
    • Cycloaddition: Allow the reaction to warm slowly to room temperature over 12 hours. Monitor by TLC/LC-MS. The strained cyclobutanone acts as a 4π component.
    • Quench & Work-up: Quench the reaction by careful addition of cold, saturated aqueous NH₄Cl. Extract with DCM (3x). Combine organic layers, dry over Na₂SO₄, and concentrate.
    • Purification: Purify the crude product via flash chromatography to obtain the bridged, ring-expanded polycyclic adduct.

    Critical Notes:

    • The regioselectivity is governed by the catalyst and the electronics of the dienophile.
    • The strain in the cyclobutanone is the driving force for the reaction, enabling reactivity not seen with simple furans.
    • This single step adds significant topological complexity and alters the macrocycle's conformation, which was crucial for the bioactivity observed in this case study [60].

Case Study 3: Privileged Substructure-Based DOS (pDOS) for Medium-Ring Heterocycles

  • Strategic Foundation: pDOS utilizes "privileged substructures"—molecular frameworks recurrent in drugs and known to interact with multiple target families—as starting points for library synthesis [1]. This strategy biases the library toward biologically relevant chemical space from the outset.
  • Library Synthesis: A tetrahydrofuran (THF)-containing polyketide-like scaffold, a privileged motif found in many bioactive macrolides, was chosen [60]. A build/couple/pair strategy was implemented: 1) Build: Synthesize stereodefined THF fragments with orthogonal protecting groups. 2) Couple: Iteratively couple fragments via Suzuki and esterification reactions to form linear precursors. 3) Pair: Employ a ring-closing metathesis (RCM) followed by a controlled ring expansion via Baeyer-Villiger oxidation to convert a 9-membered lactone to a 10-membered lactone, accessing different conformational spaces.
  • Hit Identification: A 96-compound library was screened against a panel of three therapeutically relevant G Protein-Coupled Receptors (GPCRs). Hits were confirmed through dose-response assays and counter-screens for agonist/antagonist activity.
  • Quantitative Data:

Table 3: Targeted Screening Data for pDOS Library (Case Study 3)

Target GPCR Number of Hits (IC50 < 10 µM) Most Potent Compound Activity (IC50 / EC50) Functional Activity
GPCR-A 8 pDOS-22 320 nM (Antagonist) Full antagonist in cAMP assay.
GPCR-B 3 pDOS-67 5.1 µM (Agonist) Partial agonist (45% efficacy).
GPCR-C 1 pDOS-14 8.7 µM (Antagonist) Weak antagonist.
Selectivity Summary pDOS-22 showed >30-fold selectivity for GPCR-A over GPCR-B/C.

  • Key Experimental Protocol: Ring-Closing Metathesis (RCM) for Medium-Ring Lactone Formation

    Application Note: RCM is the pivotal cyclization step in this pDOS pipeline for forming the medium-ring lactone core. The challenge lies in the favored formation of smaller rings; thus, careful optimization of catalyst, dilution, and substrate geometry (pre-organization via H-bonding) is required to favor the 9-membered ring closure [1].

    Materials:

    • Substrate: Linear diene with terminal olefins and a free carboxylic acid.
    • Catalyst: Grubbs 2nd Generation catalyst (or Hoveyda-Grubbs II).
    • Solvent: Anhydrous, degassed Dichloromethane (DCM) or Toluene.
    • Additive: Ti(OiPr)₄ (optional, for substrate pre-organization).
    • Quench: Ethyl vinyl ether.
    • Purification: Silica gel for flash column chromatography.

    Procedure:

    • Substrate Pre-organization (Optional): Dissolve the linear diene substrate (1.0 equiv) in degassed DCM (0.001 M – high dilution is critical). Add Ti(OiPr)₄ (1.5 equiv) and stir for 30 mins at RT. This Lewis acid can chelate the carbonyl oxygen, potentially pre-organizing the chain for macrocyclization.
    • Metathesis Reaction: Add the Grubbs II catalyst (0.05-0.10 equiv) to the stirring solution. Heat to 40°C if using toluene.
    • Reaction Monitoring: Monitor closely by TLC and LC-MS (reaction times: 4-24h). The high dilution minimizes dimerization/oligomerization.
    • Quench: Upon completion, cool to RT and quench by adding excess ethyl vinyl ether (0.5 mL) and stirring for 30 minutes. This deactivates the catalyst.
    • Concentration & Purification: Concentrate the reaction mixture in vacuo and purify the residue directly by flash chromatography to obtain the cyclized medium-ring lactone.

    Critical Notes:

    • Dilution is paramount. A concentration of 0.001-0.0005 M is typical for 9-membered ring formation.
    • Catalyst choice is key. Grubbs II and Hoveyda-Grubbs II are often optimal for challenging RCM.
    • The presence of an internal H-bond donor (like the free acid) or the use of Lewis acids can pre-organize the linear chain, significantly improving cyclization yields by reducing the entropic penalty [1].

The Scientist's Toolkit: Essential Research Reagent Solutions

Table 4: Key Reagents, Tools, and Platforms for Ring Expansion & Screening Research

Category Item / Solution Function & Application Note Example/Provider
Specialized Synthesis (Diacetoxyiodo)benzene (PIDA) / Hypervalent Iodine Reagents Key oxidants for biomimetic dearomatization and oxidative ring expansion reactions [1] [60]. Sigma-Aldrich, TCI Chemicals
Grubbs & Hoveyda-Grubbs Catalysts Essential for ring-closing metathesis (RCM) to form medium-ring cyclic alkenes and lactams [1]. Materia, Sigma-Aldrich
Chiral Lewis Acids (e.g., Ti(OiPr)₄ with chiral ligands) Induce asymmetry in cycloadditions and control stereochemistry during ring-forming steps.
Library Synthesis & Management DNA-Encoded Library (DEL) & Self-Encoded Library (SEL) Platforms Enable synthesis and screening of ultra-large libraries (>10⁶ compounds). SEL overcomes DEL limitations with nucleic acid targets [60]. Companies developing SEL tech [60]
Automated Nanoscale Synthesis Workstations Enable "direct-to-biology" synthesis and screening, rapidly generating microgram quantities of hundreds of analogues for immediate phenotypic assay [60]. Echo Acoustic Liquid Handlers, etc.
Screening & Hit ID High-Content Imaging & Cell Painting Assay Phenotypic screening platform that quantifies morphological changes; uses reference cytotoxic compounds to identify and filter nuisance compounds early [60]. Broad Institute Cell Painting protocol
Chemical Proteomics Kits (e.g., desthiobiotin-based pull-down) For target deconvolution of phenotypic hits from CtD and other libraries. Isolates binding proteins from cell lysates. Thermo Fisher Scientific, Promega
Data Analysis & AI Genome Mining Tools (AntiSMASH, DeepBGC) Identifies cryptic biosynthetic gene clusters in microbial genomes to discover novel NP scaffolds for inspiration [59]. Public bioinformatics platforms
AI/ML for Molecular Generation & Property Prediction Designs novel macrocyclic structures, predicts synthetic accessibility, and forecasts ADMET properties prior to synthesis [59] [60]. Various commercial & academic AI platforms
Meta-Analysis Software (e.g., R metafor, RevMan) Statistically combines bioactivity data from multiple similar studies to derive more robust structure-activity relationships (SAR) [61] [62]. Open-source and Cochrane tools

Strategic Visualization: Integrating Synthesis and Screening

G_Integrated_Strategy NP Natural Product Inspiration S1 Core Scaffold Definition NP->S1 S2 Diversity-Oriented Synthesis (DOS) Strategy S1->S2 Synth1 Biomimetic Ring Expansion S2->Synth1 Synth2 Complexity-to-Diversity (CtD) Elaboration S2->Synth2 Synth3 Privileged Substructure Assembly (pDOS) S2->Synth3 Lib Focused Library of Medium-Sized Rings Synth1->Lib Synth2->Lib Synth3->Lib Screen1 Primary HTS (Target/Phenotypic) Lib->Screen1 Screen2 Hit Validation (Dose-Response) Screen1->Screen2 Screen3 Selectivity & Counter-Screens Screen2->Screen3 Screen4 Mechanistic Studies (Target ID, MOA) Screen3->Screen4 Hit Confirmed Bioactive Hit with SAR & Target Data Screen4->Hit

Diagram: Integrated Roadmap from NP-Inspired Design to Bioactive Hit

Assessing Drug-Likeness and Developability of Medium-Ring Scaffolds

This document provides application notes and detailed experimental protocols for assessing the drug-likeness and developability of medium-sized ring (8-11 membered) scaffolds. The content is framed within a broader thesis on ring expansion strategies for natural product-derived medium-sized rings research [2]. The synthesis of medium-sized rings is recognized as a significant challenge in medicinal chemistry due to issues like transannular strain and unfavorable enthalpic and entropic factors associated with direct cyclization [2] [1]. Consequently, ring expansion reactions—transforming smaller, more accessible cyclic precursors into larger rings—have emerged as a pivotal strategy for constructing these underrepresented yet valuable chemotypes [2].

Medium-sized rings occupy a unique chemical space, often mimicking the structural complexity and three-dimensionality of bioactive natural products [2] [1]. Their exploration is essential for discovering novel therapeutic agents, particularly for targeting protein-protein interactions and other "undruggable" targets [63] [1]. This work integrates advanced synthetic methodologies with modern computational and biophysical assessment tools to establish a robust pipeline for transforming novel medium-ring scaffolds into viable lead candidates.

The following tables summarize core data on synthetic strategies, computational assessment tools, and key developability parameters relevant to medium-ring scaffolds.

Table 1: Overview of Ring Expansion Strategies for Medium-Ring Synthesis This table classifies key synthetic methodologies for accessing medium-sized rings, highlighting their utility for generating diverse, drug-like scaffolds.

Strategy Category Key Description & Mechanism Representative Ring Sizes Formed Key Advantages for Drug Discovery Reported Yield Ranges
C–C Bond Cleavage [2] Oxidative cleavage of C=C or C–C bonds in polycyclic precursors (e.g., bridged systems). 8-11 membered lactones, lactams High functional group tolerance; access to diverse lactam/lactone cores. 45-92% [2]
ODRE Sequence [2] [1] Oxidative Dearomatization-Ring Expansion-Rearomatization of phenolic substrates. Benzannulated 8-10 membered rings (aryl ethers, biaryls) Biomimetic; generates natural product-like aromatic ring linkages. 40-85% [2]
Tandem Umpolung ODRE [2] [1] Modified ODRE using an umpolung strategy with electron-rich arenes. 9-11 membered rings with haloaryl, sulfonamide groups Broad substrate scope beyond phenols; avoids side-product formation. 50-90% [2]
Electrochemical Expansion [2] Amidyl radical migration-induced C–C bond cleavage via electrochemical oxidation. 8-11 membered bridged lactams Green chemistry principles; atom-economical; mild conditions. 60-88% [2]
Multi-Component Reaction (MCR) [63] Use of reactions like Groebke-Blackburn-Bienaymé to build complex, rigid scaffolds. Fused polyheterocycles (imidazopyridines) Rapid derivatization and library synthesis; high complexity and rigidity. Not specified

Table 2: Computational Tools for Scaffold Assessment and Hoping This table compares molecular representation and modeling methods used to assess and evolve medium-ring scaffolds for drug-like properties.

Tool/Method Name Type / Category Key Principles & Application Reported Performance Metric Ref.
WHALES Descriptors [64] [65] Holistic 3D Molecular Representation Encodes shape, partial charge, and atom distribution. Used for scaffold hopping from complex NPs to simpler mimetics. Identified novel CB1/CB2 modulators (35% hit rate); Superior scaffold-hopping ability (SDA% = 89±8). [64] [65]
AnchorQuery [63] Pharmacophore-Based Database Screening Screens a 31M+ synthesizable MCR library for scaffolds fitting a target pharmacophore. Successfully identified novel GBB-based molecular glue scaffolds for 14-3-3/ERα. [63]
FSCA (Flexible Scaffold Approach) [66] Structure-Based Cheminformatics Designs polypharmacological ligands by fitting a flexible core into distinct binding poses for different targets. Designed IHCH-7179, a dual 5-HT1AR agonist/5-HT2AR antagonist with in vivo efficacy. [66]
MEVO Framework [67] Generative AI / 3D Pharmacophore Evolution VQ-VAE and diffusion model for pocket-aware, pharmacophore-guided molecule generation and optimization. Designed KRASG12D inhibitors with predicted affinity comparable to known high-activity inhibitors. [67]
3D-QSAR & Pharmacophore Modeling [68] Ligand-Based Modeling Identifies key functional groups for target binding (e.g., α-glucosidase) to guide scaffold design. Model quality: q²=0.571, r²=0.926 for CoMFA; GoldScore fitness of 60.57 for designed inhibitor. [68]

Table 3: Key Experimental Assays for Developability Assessment A summary of orthogonal biophysical and cellular assays used to profile medium-ring compound developability.

Assay Type Specific Technique Measured Parameter Throughput Key Utility for Medium-Rings Ref.
Binding Affinity Surface Plasmon Resonance (SPR) Kinetic constants (Ka, Kd), binding specificity. Medium Label-free measurement of target engagement for novel PPI stabilizers. [63]
Binding Affinity Time-Resolved FRET (TR-FRET) Binding equilibrium (IC50/Kd) in a homogeneous format. High Ideal for screening ring-expansion library compounds for target binding. [63]
Complex Stabilization Intact Mass Spectrometry (MS) Direct observation of protein-ligand complex formation. Low-Medium Critical for validating molecular glue mechanism of action. [63]
Cellular Target Engagement NanoBRET (in live cells) Stabilization of protein-protein interactions in a physiological context. Medium Confirms cellular permeability and activity for medium-ring scaffolds. [63]
Cellular Efficacy Phenotypic Screening (unbiased) Functional readout (e.g., cell viability, gene expression). Varies Identifies bioactivity for novel medium-ring chemotypes against undruggable targets. [2] [1]

Detailed Experimental Protocols

Protocol 3.1: Biomimetic Oxidative Dearomatization-Ring Expansion (ODRE) for Benzannulated Medium-Rings

Adapted from Tan et al. for synthesizing diverse 8-10 membered benzannulated rings [2] [1].

Objective: To synthesize a library of medium-sized ring scaffolds via a biomimetic, diversity-oriented ring expansion.

Materials:

  • Starting Material: Bicyclic phenol compound (e.g., 3, 100 mg scale).
  • Oxidant: (Diacetoxyiodo)benzene (PIDA) or Hypervalent iodine(III) reagent.
  • Nucleophiles: Diverse set (e.g., carboxylic acids, phenols, primary alcohols, tertiary alcohols).
  • Solvent: Anhydrous Dichloromethane (DCM) or Trifluoroethanol (TFE).
  • Conditions: Inert atmosphere (N₂ or Ar), dry glassware.

Procedure:

  • Reaction Setup: In a flame-dried round-bottom flask under inert atmosphere, dissolve the bicyclic phenol 3 (1.0 equiv.) in anhydrous DCM (0.1 M concentration).
  • Oxidative Dearomatization: Cool the solution to 0°C. Add the hypervalent iodine oxidant (e.g., PIDA, 1.2 equiv.) in one portion. Stir the mixture at 0°C for 30 minutes, monitoring by TLC. The formation of a polycyclic cyclohexadienone intermediate (4) is expected.
  • Ring Expansion: To the same reaction vessel, add the desired nucleophile (e.g., a carboxylic acid, 2.0 equiv.). Allow the reaction to warm to room temperature and stir for 4-16 hours.
  • Work-up: Quench the reaction by adding a saturated aqueous solution of sodium thiosulfate (10 mL). Extract the aqueous layer with DCM (3 x 15 mL). Combine the organic layers, dry over anhydrous MgSO₄, filter, and concentrate under reduced pressure.
  • Purification: Purify the crude residue via flash column chromatography (silica gel, hexanes/ethyl acetate gradient) to isolate the benzannulated medium-ring product.
  • Analysis: Characterize products using ( ^1H/^{13}C ) NMR, HRMS, and determine yields.

Notes: The choice of nucleophile dictates the ring linkage (e.g., lactone, aryl ether). The umpolung variant of this protocol uses non-phenolic substrates with an electrophilic side chain and a tertiary alcohol to drive the expansion [2] [1].

Protocol 3.2: Computational Scaffold Hoping Using WHALES Descriptors for Medium-Ring Mimetics

Adapted from the prospective discovery of cannabinoid receptor modulators [64] [65].

Objective: To identify synthetically accessible, drug-like mimetics of a complex medium-ring natural product scaffold using a holistic molecular similarity search.

Software/Tools: Python/R environment with RDKit; WHALES descriptor calculation script [64] [65]; access to a commercial compound database (e.g., ZINC, Enamine REAL).

Procedure:

  • Query Preparation:
    • Select a medium-ring natural product as the query (e.g., a cannabinoid).
    • Generate a low-energy 3D conformation using a force field (e.g., MMFF94) and calculate atomic partial charges (e.g., using the Gasteiger-Marsili method) [65].
  • Descriptor Calculation for Query:
    • For the query molecule, compute the weighted atom-centered covariance matrix (Sw(j)) for each non-hydrogen atom j (Equation 1) [65].
    • Compute the Atom-Centered Mahalanobis (ACM) distance matrix from Sw(j) (Equation 2) [65].
    • For each atom, derive the remoteness (row average of ACM) and isolation degree (column minimum of ACM) values [65].
    • Generate the final 33-dimensional WHALES descriptor vector by calculating the deciles, minimum, and maximum of the atomic remoteness, isolation degree, and their ratio [64] [65].
  • Database Screening:
    • Calculate WHALES descriptors for all compounds in a large database of commercially available or synthetically feasible molecules.
    • Compute molecular similarity (e.g., using Euclidean or Manhattan distance) between the query's WHALES vector and all database entries.
    • Rank the database compounds by descending similarity (or ascending distance).
  • Hit Selection & Analysis:
    • Inspect the top-ranked compounds (e.g., top 100-500). Prioritize those with:
      • High similarity score.
      • Simplified scaffold complexity compared to the natural product query.
      • Favorable calculated drug-like properties (e.g., Rule of 3 for fragments, Rule of 5 for leads).
    • Visually inspect 2D/3D structures to confirm scaffold hop.

Validation: Selected virtual hits should be procured or synthesized and tested in relevant biological assays to confirm the transfer of bioactivity.

Protocol 3.3: Orthogonal Biophysical Assessment of Molecular Glue Function for Medium-Ring Scaffolds

Adapted from the characterization of 14-3-3/ERα molecular glues [63].

Objective: To rigorously characterize the binding affinity, mechanism, and cellular activity of a medium-ring scaffold designed to stabilize a protein-protein interaction.

Part A: Binding Affinity via TR-FRET

  • Materials: Recombinant proteins, Europium (Eu)-cryptate donor streptavidin, XL665 acceptor streptavidin, biotinylated peptide, black low-volume 384-well plate.
  • Procedure:
    • Pre-incubate the target protein with a biotinylated phosphopeptide (mimicking the client protein) for 30 minutes.
    • Serially dilute the medium-ring test compound in assay buffer (e.g., PBS with 0.01% BSA).
    • In the assay plate, mix the protein/peptide complex with donor and acceptor streptavidin reagents. Add the compound dilution.
    • Incubate for 1-2 hours at RT protected from light.
    • Read the TR-FRET signal (excitation: ~337 nm, emission: 665 nm and 620 nm) on a compatible plate reader.
    • Calculate the ratio of acceptor (665 nm) to donor (620 nm) emission. Fit dose-response curves to determine the EC₅₀ or IC₅₀ for stabilization or inhibition.

Part B: Direct Binding Analysis via Surface Plasmon Resonance (SPR)

  • Materials: SPR instrument (e.g., Biacore), CMS sensor chip, recombinant target proteins, HBS-EP+ buffer.
  • Procedure:
    • Immobilize one protein partner (e.g., 14-3-3) on the sensor chip surface via amine coupling.
    • Use the second protein/peptide partner (e.g., phospho-ERα peptide) as the analyte. Pre-mix the analyte with a range of concentrations of the medium-ring compound.
    • Inject the mixture over the immobilized surface and monitor the binding response (Response Units, RU).
    • Analyze sensograms. A molecular glue will typically show cooperative binding, evidenced by a significant increase in analyte binding response (RUmax) and/or a decrease in dissociation rate (kd) in the presence of the compound compared to analyte alone [63].

Part C: Cellular Target Engagement via NanoBRET

  • Materials: HEK293T cells, plasmids for NanoLuc-fused protein and HaloTag-fused partner protein, HaloTag NanoBRET 618 ligand, furimazine substrate, test compounds.
  • Procedure:
    • Co-transfect cells with the two fusion protein constructs.
    • Seed transfected cells into a 96-well plate. The next day, add the HaloTag ligand and incubate.
    • Add serial dilutions of the test compound and incubate (e.g., 4-6 hours).
    • Add the NanoLuc substrate (furimazine) and immediately measure BRET (donor: 450 nm, acceptor: 618 nm).
    • Calculate the BRET ratio. An increase in ratio indicates stabilization of the PPI by the compound in live cells.

Visualization of Workflows and Relationships

G NP Natural Product with Medium Ring Synth Ring Expansion Synthesis NP->Synth Bioinspiration Lib Medium-Ring Scaffold Library Synth->Lib Generates Comp Computational Assessment & Hoping Lib->Comp Input for Comp->Synth Feedback Loop Des Designed Mimetic Scaffold Comp->Des Outputs Exp Experimental Developability Assays Des->Exp Validated by Exp->Comp Feedback Loop Lead Optimized Lead Candidate Exp->Lead Optimizes to

Diagram 1: Integrated Pipeline for Medium-Ring Scaffold Discovery and Optimization (Max Width: 760px)

G cluster_comp Computational Design Phase cluster_exp Experimental Validation Phase Start Initial Scaffold (Medium-Ring) Rep3D 3D Conformer Generation Start->Rep3D DescCalc Descriptor Calculation (e.g., WHALES) Rep3D->DescCalc Similarity Similarity Search or Generative Design DescCalc->Similarity NewScaff Novel, Drug-like Scaffold Hop Similarity->NewScaff Select DB Large Compound Database DB->Similarity SAR Biophysical Profiling (SPR, TR-FRET, MS) NewScaff->SAR Cell Cellular Assay (e.g., NanoBRET) NewScaff->Cell PKPD Early DMPK Assessment NewScaff->PKPD Data Integrated Data & SAR SAR->Data Cell->Data PKPD->Data Data->Similarity Iterative Refinement

Diagram 2: Iterative Computational-Experimental Workflow for Scaffold Assessment (Max Width: 760px)

The Scientist's Toolkit: Research Reagent Solutions

Table 4: Essential Materials and Reagents for Medium-Ring Scaffold Research

Item / Reagent Category Function in Research Example / Supplier Note
Hypervalent Iodine(III) Reagents Synthetic Chemistry Key oxidants for initiating ring expansion reactions like ODRE. (Diacetoxyiodo)benzene (PIDA), Phenyliodine bis(trifluoroacetate) (PIFA).
Chiral Bicyclic Phenol Precursors Building Blocks Advanced starting materials for synthesizing enantiopure, natural product-like medium rings. Custom synthesis required; often derived from biomass or via enzymatic resolution.
Enamine REAL / ZINC20 Database Computational Screening Source of billions of synthetically feasible, purchasable compounds for virtual screening and scaffold hoping. Primary resource for WHALES similarity searches or MCR library screening (AnchorQuery).
Recombinant PPI Pairs with Tags Biophysics Purified proteins for biophysical assays (SPR, TR-FRET, MS) to assess molecular glue mechanism. e.g., 14-3-3σ and biotinylated phospho-ERα peptide [63]; requires recombinant expression systems.
NanoBRET PPI Systems Cell Biology Validated kits for measuring protein-protein interaction stabilization in live, physiologically relevant cellular environments. Promega; requires fusion protein constructs (NanoLuc and HaloTag).
Crystallography Plates & Conditions Structural Biology For obtaining ternary complex structures (Protein-Protein-Scaffold), essential for rational optimization. Commercial sparse matrix screens (e.g., from Hampton Research, Molecular Dimensions).
AI/ML Modeling Software Suite Computational Chemistry Integrated platforms for running descriptor calculations, molecular docking, dynamics, and generative AI models. Software like OpenEye Toolkits, Schrödinger Suite, or open-source (RDKit, PyMOL, AutoDock).

The pursuit of therapeutics for "undruggable" targets—proteins characterized by flat, featureless interaction surfaces, shallow binding pockets, or intrinsically disordered structures—represents a frontier in modern drug discovery [69] [70]. Key target classes include small GTPases (e.g., KRAS), transcription factors (e.g., p53, Myc), phosphatases, and the extensive network of protein-protein interactions (PPIs) that govern cellular signaling [69] [71]. Historically, the lack of deep, hydrophobic pockets amenable to traditional small-molecule inhibition rendered these targets intractable. However, a paradigm shift is underway, driven by strategies such as covalent inhibition, allosteric modulation, and the development of macrocyclic peptides and proteolysis-targeting chimeras (PROTACs) [69] [71].

Concurrent advancements in synthetic chemistry are providing the crucial molecular scaffolds needed to implement these strategies. Within this context, ring expansion strategies for constructing natural product-derived medium-sized rings (typically 8-11 members) emerge as a critical enabling methodology [1] [3]. These rings occupy a unique and underexplored region of chemical space, offering conformational pre-organization and the ability to present functional groups over large surface areas, making them ideal for targeting expansive PPI interfaces [1] [72]. This article details the application of these synthetic strategies within a drug discovery pipeline, providing specific protocols and data frameworks for targeting undruggable domains.

The Druggability Landscape: Quantitative Analysis of PPI Targets and Modalities

The clinical translation of PPI modulators validates the feasibility of targeting undruggable domains. The following table summarizes key approved and clinical-stage therapeutics, highlighting the diversity of target classes and modalitiess [69] [73].

Table 1: Approved and Late-Stage PPI Modulators for Oncological Indications

Target PPI / Protein Modulator Name Modality Indication (Status) Key Mechanism
Bcl-2 (Anti-apoptotic PPIs) Venetoclax Small Molecule CLL, AML (Approved) Inhibits Bcl-2, promoting apoptosis [73].
KRASG12C Sotorasib, Adagrasib Covalent Small Molecule NSCLC (Approved) Covalently binds mutant Cys12, locking KRAS in inactive state [69] [71].
MDM2-p53 Idasanutlin, Brigimadlin Small Molecule Various cancers (Phase II/III) Disrupts MDM2-p53 interaction, stabilizing p53 [73].
CCR5-CCL5 (GPCR interface) Maraviroc Small Molecule HIV (Approved) Allosteric inhibitor of CCR5, blocking viral entry [71] [73].
IL-6 Receptor Tocilizumab, Sarilumab Monoclonal Antibody Rheumatoid Arthritis, Cytokine Storm (Approved) Binds IL-6R, inhibiting pro-inflammatory signaling [71].

The synthesis of modulators for such targets often requires access to non-traditional chemical space. Ring expansion methodologies are pivotal for constructing the core scaffolds of potential inhibitors, as summarized below.

Table 2: Ring Expansion Methodologies for Medium-Sized Ring Synthesis

Methodology Key Transformation Substrate Class Ring Size Formed Primary Advantage
Oxidative Dearomatization-Ring Expansion (ODRE) [1] [72] C–C bond cleavage & expansion of bicyclic phenols Bicyclic Cyclohexadienones 8-11 Biomimetic; generates diverse benzannulated medium rings.
C–C Bond Cleavage of Bridged Polycycles [1] Oxidative cleavage of alkene-bridged systems Bicyclic Alkenes 9-12 High functional group tolerance; produces lactones/lactams.
Photoredox-Catalyzed Radical Relay [72] C–N cleavage and radical addition Unstrained Cyclic Amines 8-9 Access to medium-sized lactams from simple precursors.
Aryne Insertion [72] σ C–N bond insertion Bicyclic Aziridines 8-membered N,O-heterocycles Rapid assembly of oxazocine scaffolds.

Core Experimental Protocols

Protocol: Hot Spot Analysis via Alanine Scanning Mutagenesis and Computational Prediction

Objective: To identify energetically critical residues ("hot spots") at a PPI interface for focused inhibitor design [74]. Workflow:

  • Complex Structure Preparation: Obtain a high-resolution structure (X-ray, cryo-EM, or high-confidence AlphaFold Multimer prediction) of the target protein complex [71] [75].
  • In Silico Alanine Scanning: Use computational tools like FoldX or Robetta Alanine Scanning to mutate each interfacial residue to alanine in silico and calculate the predicted change in binding free energy (ΔΔG) [74].
  • Hot Spot Identification: Residues with a predicted ΔΔG ≥ 2.0 kcal/mol are classified as potential hot spots [74].
  • Experimental Validation (Optional but Recommended):
    • Cloning & Mutagenesis: Generate plasmid constructs for wild-type and individual alanine mutants of the target protein.
    • Protein Expression & Purification: Express and purify proteins using standard systems (e.g., E. coli, HEK293).
    • Binding Affinity Measurement: Determine binding constants (KD) for wild-type and mutant complexes using biophysical assays such as Isothermal Titration Calorimetry (ITC) or Surface Plasmon Resonance (SPR). A significant increase in KD (weaker binding) for a mutant confirms a true hot spot.

Objective: To synthesize a diverse library of medium-sized ring scaffolds inspired by natural products for HTS against undruggable targets. Reaction Scheme: Oxidative Dearomatization → Ring Expansion → Rearomatization. Procedure:

  • Starting Material: Dissolve bicyclic phenol substrate 3 (1.0 equiv, 0.1 mmol) in anhydrous dichloromethane (DCM, 2 mL) under nitrogen at -78°C [1].
  • Oxidative Dearomatization: Add phenyliodine bis(trifluoroacetate) (PIFA, 1.1 equiv) dropwise. Stir at -78°C for 30 minutes to form polycyclic cyclohexadienone intermediate 4 [1].
  • Ring Expansion & Nucleophile Quench: In one pot, add a nucleophile (Nu-H, 2.0 equiv; e.g., carboxylic acid, phenol, alcohol) and warm the reaction mixture to 0°C. Stir for 2-6 hours, monitoring by TLC/LC-MS. The reaction proceeds via cleavage of the scissile bond (highlighted in red in intermediate 4) and expansion [1].
  • Work-up: Quench the reaction with saturated aqueous NaHCO₃, extract with DCM, dry over MgSO₄, and concentrate in vacuo.
  • Purification: Purify the crude product, benzannulated medium ring 5, by flash column chromatography. Note: This biomimetic, diversity-oriented synthesis allows the incorporation of various ring heteroatoms and functional groups, generating libraries with high three-dimensional complexity [1] [72].

Objective: To identify small-molecule or macrocyclic disruptors of a target PPI in a cellular context. Procedure:

  • Construct Design: Create plasmids encoding the two target proteins fused to the complementary fragments of NanoLuc luciferase (LgBiT and SmBiT).
  • Cell Assay Setup: Seed HEK293T cells in 384-well white-walled plates. Co-transfect with the two fusion constructs using a transfection reagent.
  • Compound Addition & Incubation: 24 hours post-transfection, add compounds from the screening library (e.g., medium-sized ring library) and positive/negative controls. Incubate for 4-16 hours.
  • Signal Detection: Add a cell-permeable luciferase substrate. Measure luminescence on a plate reader. Inhibition of the PPI reduces complementation and lowers luminescent signal.
  • Data Analysis: Normalize signals to controls. Compounds showing >50% inhibition at screening concentration are considered primary hits for downstream validation (dose-response, counterscreens).

Visualization of Key Concepts and Workflows

Diagram 1: KRAS signaling pathway and therapeutic inhibition strategies.

G Start Bicyclic Phenol Precursor (3) Step1 Step 1: Oxidative Dearomatization (PIFA, -78°C) Start->Step1 Intermediate Polycyclic Cyclohexadienone (4) Step1->Intermediate Step2 Step 2: Nucleophile Addition & Ring Expansion (Warm to 0°C) Intermediate->Step2 Product Benzannulated Medium-Sized Ring (5) Step2->Product Library Diverse Compound Library for HTS Product->Library Iteration with diverse Nu-H

Diagram 2: Workflow for ring expansion synthesis of screening library.

The Scientist's Toolkit: Essential Research Reagents & Materials

Table 3: Key Reagent Solutions for Undruggable Target Research

Reagent / Material Function / Application Key Consideration
Stabilized Alpha-Helix Mimetics (e.g., hydrocarbon-stapled peptides) To target PPIs mediated by helical interfaces; enhances cell permeability and proteolytic stability [70]. Optimize staple position/length to maintain affinity while improving pharmacokinetics.
DNA-Encoded Chemical Library (DEL) For ultra-high-throughput screening (uHTS) of billions of compounds against purified target proteins [69]. Requires target immobilization and stringent off-DNA hit validation.
Cryo-EM Grids & Vitrobot For high-resolution structure determination of large, flexible protein complexes and PPI interfaces [71]. Sample homogeneity and ice thickness are critical for success.
AlphaFold-Multimer & RosettaFold Computational tools for accurate prediction of protein complex structures to guide rational design [71] [75]. Predictions are models; experimental validation of key interfaces is essential.
PROTAC Linker Toolbox Contains heterobifunctional linkers (E3 ligase ligand + target binder) for constructing degraders of undruggable proteins [69] [76]. Linker length and composition dramatically influence degradation efficiency and selectivity.
NanoBiT / Split-Luciferase System For sensitive, real-time monitoring of PPIs in live cells for HTS and mechanism-of-action studies [75]. Fusion tag position can affect protein function and interaction dynamics.
Isothermal Titration Calorimetry (ITC) Gold-standard for label-free measurement of binding affinity (KD), stoichiometry (n), and thermodynamics (ΔH, ΔS) [73]. Requires high protein solubility and concentration; low throughput.
Molecular Glue Screening Library Collections of small molecules known or predicted to induce novel PPIs, often leading to target degradation [75]. Discovery is largely serendipitous; phenotypic screening is a primary route.

Conclusion

Ring expansion strategies represent a paradigm shift for accessing the valuable yet synthetically elusive chemical space of medium-sized rings. By reframing the synthesis of these 8- to 11-membered systems from a problem of difficult cyclization to one of strategic bond cleavage and rearrangement of natural product-derived polycycles, chemists can now systematically populate screening libraries with unprecedented three-dimensional complexity. As demonstrated, methodologies ranging from biomimetic rearrangements and C-H activation cascades to radical aryl migrations provide robust, generalizable toolkits for diversification. The resulting compounds occupy a distinct and validated region of chemical space, poised to interact with challenging biological targets like protein-protein interfaces. The future of this field lies in further increasing enantioselective control, developing even broader substrate-agnostic platforms, and directly applying these diverse libraries to phenotypic and target-based screens against the most intractable diseases, ultimately translating unique molecular shape into novel therapeutic function.

References