Introduction

The benzene ring provides a stable geometry, well-defined substitution vectors, an appropriate hydrophobic surface area, and aromatic interactions. It also benefits from mature synthetic and late-stage functionalization methods. Consequently, benzene rings are widely present in drug molecules and lead compounds.

However, benzene rings may also contribute to aromatic oxidation, excessive lipophilicity, insufficient aqueous solubility, tight crystal packing, and nonspecific binding. Bicyclo[1.1.1]pentane (BCP), bicyclo[2.1.1]hexane (BCH), bicyclo[3.1.1]heptane (BCHep), bicyclo[2.2.2]octane (BCO), cubane, and heteroatom-containing bridged rings provide new structural options for modulating these properties [1].

The design objectives of benzene-ring replacement comprise two components: preserving the spatial connectivity and target-recognition functions required by the parent molecule, while improving the physicochemical, metabolic, or safety properties that limit compound development. When evaluating the outcome of a replacement, activity, selectivity, solubility, membrane permeability, metabolic stability, and synthetic feasibility should be assessed together. A decision should not be based solely on three-dimensionality or on a single in vitro parameter.

1 R&D Problems Addressed by Benzene-Ring Replacement

1.1 Roles of the Benzene Ring in Drug Molecules

The benzene ring typically performs one or more of the following functions in drug molecules.

No single replacement scaffold can reproduce all the properties of a benzene ring simultaneously. BCP can provide rigidity and approximately collinear exit vectors, but it lacks aromatic π electrons. Oxa-bridged rings can reduce lipophilicity, but they also increase polarity and hydrogen-bond acceptor capacity. Spirocycles can restrict conformation, but they do not usually reproduce the substitution geometry of a benzene ring directly.

1.2 Situations Suitable for Benzene-Ring Replacement

Benzene-ring replacement is suitable for compound series in which the structural origin of a problem has already been identified, for example:

① Metabolite identification shows that the aromatic ring is the principal site of oxidation;

② Solubility decreases progressively as the number of aromatic rings increases;

③ A high calculated partition coefficient or experimentally determined distribution coefficient is associated with clearance, plasma protein binding, or off-target activity;

④ A co-crystal structure indicates that the benzene ring mainly serves as a linker and does not form critical aromatic interactions;

⑤ High molecular planarity and a stable solid-state lattice lead to insufficient thermodynamic solubility;

⑥ Local conformation needs to be restricted, or the three-dimensional orientation of substituents needs to be altered.

If the benzene ring participates in critical π interactions, modulates the electronic properties of neighboring functional groups through conjugative or inductive effects, or occupies a narrow pocket with a high degree of shape complementarity, complete saturation may reduce binding affinity. In such structures, heteroaromatic replacement, partial saturation, or retention of the aromatic ring while optimizing other regions may be prioritized.

2 Basis for Assessing Bioisosteric Replacement

The effectiveness of a bioisostere depends on the specific molecule and target. Structural similarity can only generate a replacement hypothesis; biological activity and comprehensive property data are required to validate that hypothesis.

2.1 Geometric Matching

Geometric evaluation includes four parameters:

① The distance between the core attachment atoms;

② The distance between the termini of the substituents;

③ The angle between the substituent exit vectors;

④ The dihedral angle of the substituents relative to the core scaffold.

1,3-Disubstituted BCP is commonly used as a replacement for para-disubstituted benzene. Both have approximately collinear exit vectors, but their core distances differ. The distance between the two bridgehead carbon atoms of BCP is approximately 1.8 Å, whereas the distance between the corresponding carbon atoms of para-substituted benzene is approximately 2.8 Å. The bridgehead distance of BCO is approximately 2.6 Å, making it closer to that of para-substituted benzene [4].

BCP is therefore more suitable for compound series that tolerate changes in core length. For molecules in which two pharmacophoric groups must enter distinct subpockets precisely, BCO, oxa-BCO, or other scaffolds with more closely matched distances may provide better geometric correspondence.

2.2 Matching of Target-Recognition Features

Saturated bridged rings can mimic part of the volume and substitution directions of a benzene ring, but they cannot reproduce delocalized π electrons. Before replacement, it is necessary to determine whether the region surrounding the benzene ring contains:

① Aromatic interactions involving phenylalanine, tyrosine, or tryptophan;

② Cation–π interactions involving a protonated amine, arginine, or lysine;

③ Edge interactions between the aromatic ring and amide, carboxylic acid, or sulfur-containing groups;

④ Tight shape complementarity between the benzene ring and a planar hydrophobic pocket.

When the para-substituted benzene ring of imatinib was replaced with BCO or 2-oxa-BCO, some physicochemical properties improved, but neither analogue showed significant ABL1 kinase inhibitory activity. This result indicates that the pharmacological role of this benzene ring cannot be explained solely by geometric distance and hydrophobic volume [4].

2.3 Integrated Functional Matching

An effective replacement must satisfy requirements at three levels:

① Spatial function: key pharmacophoric groups maintain appropriate distances and orientations;

② Recognition function: major binding interactions are retained or compensated for;

③ Property function: the targeted physicochemical or metabolic parameters are improved without introducing more serious liabilities.

Benzene-ring replacement should therefore be regarded as multiparameter structural optimization rather than a simple scaffold exchange.

3 Six Key Variables Affecting Replacement Outcomes

3.1 Exit Vectors and Spatial Occupancy

Exit vectors determine the directions in which substituents extend in three-dimensional space. Benzene and bridged-ring scaffolds with the same nominal substitution pattern may differ substantially in exit distance, exit angle, and local substituent conformation.

BCP has two compact and approximately collinear bridgehead exit vectors. BCO has a longer bridgehead distance. Different substitution positions and stereoisomers of BCH and BCHep can provide angled or non-collinear exit vectors and may serve as candidate mimetics for specific ortho-, meta-, or polysubstituted benzene arrangements [1].

During design, binding conformations before and after replacement should be compared preferentially, rather than two-dimensional structural drawings. When a co-crystal structure is available, key pharmacophoric atoms can be directly superimposed. In the absence of structural data, conformational searches, molecular docking, and established structure–activity relationships can be combined to define the range of acceptable geometric changes.

3.2 Aromatic Interactions and Electronic Properties

The π electrons of a benzene ring affect target binding and also influence the electronic properties of neighboring functional groups. After replacing a benzene ring with a saturated scaffold, the following parameters may change:

① π–π and cation–π interactions may be lost;

② Resonance transmission through the aromatic ring may be interrupted;

③ The acid–base properties of adjacent carboxylic acids, amines, or amides may change;

④ The molecular dipole moment and local electrostatic potential may be redistributed;

⑤ The interaction pattern with polar residues in the target may change.

Heteroatom-containing bridged rings can introduce new dipolar and hydrogen-bonding interactions through oxygen or nitrogen, but the positions and directions of these interactions differ from those associated with the π electrons of a benzene ring. Heteroatom incorporation therefore represents a redesign of the recognition pattern rather than a direct reproduction of aromatic interactions.

3.3 Conformational Preorganization

Bridged bicyclic and spirocyclic scaffolds can reduce conformational freedom around nearby rotatable bonds, bringing the unbound molecule closer to its bound conformation and thereby reducing the conformational entropy penalty associated with binding.

The effect of conformational restriction depends on the conformation being fixed. If the scaffold directs key pharmacophoric groups away from the active conformation, increased rigidity will reduce the molecule’s conformational adaptability. Therefore, when evaluating a high proportion of sp³-hybridized carbon atoms, expressed as the fraction of sp³-hybridized carbons (Fsp³), the active conformation and exit vectors should also be considered. Fsp³ should not be treated as an independent optimization objective.

The primary role of a spirocycle is to alter local conformation and substitution directions. Some spirocycles can mimic the spatial connectivity of a benzene ring, but they are more commonly used for conformational restriction, modulation of crystal packing, and shielding of metabolic sites [7].

3.4 Lipophilicity, Solubility, and Membrane Permeability

For a compound to move from the crystalline state into an aqueous phase, it must overcome lattice interactions and achieve stable hydration. Aqueous solubility is therefore influenced by both solid-state and solution-phase properties.

Saturated three-dimensional scaffolds may reduce molecular planarity and weaken regular crystal packing. Heteroatom-containing bridged rings may additionally increase the dipole moment and hydration capacity. Hydrocarbon bridged rings, however, do not necessarily reduce lipophilicity, and larger hydrocarbon cage structures may increase the hydrophobic surface area.

Changes in solubility can be broken down into the following factors:

A reduction in lipophilicity is not equivalent to an improvement in membrane permeability. If polarity and hydrogen-bonding capacity increase excessively, passive diffusion may decrease and the risk of efflux transport may increase. Solubility, partitioning behavior, and permeability should therefore be evaluated jointly within the same compound series.

3.5 CYP450 Oxidation and Metabolic Soft-Spot Migration

The net reaction of aromatic-ring hydroxylation mediated by cytochrome P450 (CYP450) can be represented in simplified form as:

Ar—H + O₂ + NADPH + H⁺ → Ar—OH + H₂O + NADP⁺

Some aromatic oxidation processes may generate arene oxides or related reactive intermediates, followed by rearrangement, nucleophilic addition, or glutathione trapping. The specific pathway depends on aromatic substitution, electronic properties, and the CYP isoform involved.

The effects of benzene-ring replacement on metabolism include:

① Removal of the original aromatic hydroxylation site;

② Alteration of the molecular binding pose within the CYP active site;

③ Changes in overall lipophilicity and the tendency to bind to the enzyme;

④ Changes in local C—H bond reactivity;

⑤ Migration of the metabolic soft spot to the bridged ring, side chain, or a position adjacent to a heteroatom.

The C—H bonds of some highly strained cage scaffolds have relatively high bond strengths, which may help suppress certain oxidative pathways. Actual clearance, however, remains jointly influenced by whole-molecule conformation, lipophilicity, and enzyme-binding mode [5,6].

Metabolic stability should be evaluated using human and animal liver microsomes, hepatocyte clearance, and metabolite identification. An extended half-life only indicates a reduction in the overall rate of compound depletion; it does not directly prove that the original metabolic pathway has disappeared, nor does it exclude the formation of new metabolites.

3.6 Synthetic Accessibility and Stereochemistry

Benzene rings benefit from abundant commercially available building blocks and mature cross-coupling methods. The structural advantages of bridged-ring scaffolds can only be realized through synthetic routes that are reproducible, scalable, and compatible with analogue generation.

Synthetic accessibility primarily includes:

① Whether the desired substitution pattern can be constructed modularly;

② Whether regioisomers and stereoisomers are involved;

③ Whether the functional groups tolerate subsequent reactions;

④ Whether sites for late-stage functionalization are available;

⑤ Route length, overall yield, and purification difficulty;

⑥ The feasibility of gram-scale or larger-scale preparation.

Methods for bridgehead functionalization of BCP are now relatively well developed, whereas bridge substitution and the preparation of chiral BCP derivatives remain more challenging. Cubane was historically used mainly as a mimic of monosubstituted or para-substituted benzene, partly because 1,2- and 1,3-disubstituted cubanes were difficult to access and because some metal-catalyzed conditions could induce skeletal valence isomerization. New photochemical and copper-mediated coupling methods have expanded the available substitution patterns and late-stage modification capabilities of cubane [6].

4 Properties and Suitable Applications of Representative Scaffolds

5 Design Principles Revealed by Representative Studies

5.1 Replacement of para-Substituted Benzene with BCP: Property Improvements Can Translate into In Vivo Exposure

In a series of γ-secretase inhibitors, researchers replaced a central para-fluorophenyl ring with BCP. The BCP analogue retained similar enzyme-inhibitory activity, while improving aqueous solubility and passive membrane permeability. It also achieved approximately fourfold increases in both peak concentration and area under the concentration–time curve in mice [2].

This case involved three structural conditions:

① The replaced benzene ring mainly served as a linker and hydrophobic space-filling unit;

② The change in BCP exit vectors remained within the tolerance of the target;

③ The improvement in physicochemical properties translated into enhanced oral absorption and in vivo exposure.

This result cannot be extrapolated directly to all para-substituted benzene rings. The corresponding replacement in imatinib caused a substantial loss of activity, demonstrating that the same scaffold can produce entirely different outcomes in different binding modes.

5.2 Replacement of ortho-Substituted Benzene with 2-Oxa-BCH: Oxygen Modulates Hydration and Lipophilicity

2-Oxa-BCH was used to replace the ortho-disubstituted benzene rings in fluxapyroxad and boscalid. Crystallographic analysis showed that the key exit angles of 2-oxa-BCH were close to those of ortho-substituted benzene, while the scaffold displayed a pronounced nonplanar structure.

In the fluxapyroxad series, experimental kinetic solubility in a pH 7.4 phosphate-buffered system increased from 25 μM to 155 μM. In the boscalid series, it increased from 11 μM to 152 μM. The corresponding analogues retained antifungal activity, although potency decreased under some low-concentration test conditions. Across the four tested series, 2-oxa-BCH reduced cLogP or experimentally measured logD₇.₄ by approximately 0.5–1.4 units, whereas changes in metabolic stability were structure-dependent [3].

This study indicates that heteroatom incorporation has a relatively consistent effect on lipophilicity, whereas solubility, metabolic stability, and biological activity are influenced by the structure of the entire molecule.

5.3 Replacement of para-Substituted Benzene with 2-Oxa-BCO: Improved Physicochemical Properties Do Not Guarantee Retention of Activity

The following compounds were tested as free bases, and solubility refers to experimental kinetic solubility measured in a pH 7.4 phosphate-buffered system.

The hydrocarbon BCO reduced solubility. The 2-oxa-BCO restored solubility to a level close to and slightly higher than that of the parent compound, while further reducing lipophilicity. Compared with imatinib, both bridged-ring analogues showed reduced intrinsic clearance in human liver microsomes. For the 2-oxa-BCO analogue, the microsomal half-life increased from 60 min to 87 min. Neither saturated analogue showed significant ABL1 kinase inhibitory activity [4].

These results distinguish two types of effects:

① BCO and oxa-BCO have relatively similar geometric structures;

② The oxygen atom primarily alters local polarity, dipolar characteristics, and hydration capacity;

③ The loss of activity mainly suggests that the original benzene ring possesses a recognition function that cannot be reproduced by the bridged-ring scaffolds.

5.4 Matched Molecular Pair Studies: Scaffold Effects Are Series-Dependent

Matched molecular pair (MMP) studies keep the remaining regions of the molecule relatively unchanged while altering only the benzene ring and its bridged-ring replacement, thereby reducing interference from differences in structural background.

A systematic study published in 2025 compared multiple sp³-rich bridged bicyclic scaffolds with their monosubstituted benzene counterparts. Most of the tested bridged rings improved metabolic stability. Bridgehead fluorination and incorporation of an oxygen atom into the bridge further altered metabolic and absorption, distribution, metabolism, and excretion (ADME) properties. In this model series, oxa-bridged rings showed a relatively balanced profile in terms of metabolism, solubility, and permeability. Metabolite analysis also indicated a reduction in some aromatic-ring-related reactive metabolites [5].

The conclusions of this study apply to the tested amide model series. For other chemical classes, scaffold effects still need to be established through experimental comparisons in the same molecular background.

6 Project-Level Selection Workflow for Benzene-Ring Bioisosteres

Bioisosteric replacement of a benzene ring should begin with the molecular function of the original benzene ring, its substitution topology, and the problem to be addressed. Project-level selection can be divided into three stages: functional diagnosis, design of the candidate set, and integrated decision-making.

6.1 Define the Function of the Benzene Ring and the Problem to Be Solved

Four interrelated questions should be answered before scaffold selection.

Whether aromatic oxidation represents the principal problem should preferably be determined through metabolite identification. A high microsomal clearance rate alone does not prove that the benzene ring is the primary metabolic soft spot, because clearance may also occur at a side chain, a position adjacent to a heteroatom, or another alkyl site.

High lipophilicity and low solubility should likewise not be interpreted automatically as an indication to select an oxa-bridged ring. In the tested series, 2-oxabicyclo[2.1.1]hexane reduced lipophilicity, but its effects on solubility and metabolic stability were dependent on the molecular structure.

6.2 Establish Candidate Comparison Sets According to Substitution Topology

After defining the function of the benzene ring, first-round candidate scaffolds can be selected according to the substitution topology.

First-Round Comparison Set

The first round can generally consist of three core members:

Depending on the project problem, the following controls may be added:

① When rigidity and shape effects need to be distinguished, include a conventional cycloalkane or flexible linker;

② When the scaffold has different stereoisomers, evaluate representative isomers separately;

③ When the series is sensitive to core length, include a second bridged ring of a different size but with similar exit directions;

④ When aromatic interactions are suspected to be important, include a heteroaromatic ring or an aromatic-ring control with different electronic properties.

This comparison strategy has a clearer experimental purpose than routinely including multiple scaffolds such as BCP, BCO, and BCHep. The value of matched molecular pair studies also lies in keeping the remainder of the molecule relatively unchanged, so that observed property changes can be attributed more reliably to the core scaffold.

6.3 Make an Integrated Decision Based on Core Data

The first-round evaluation does not need to cover every parameter relevant to drug development simultaneously. Experimental data can be selected around three decision questions.

Biological activity and physicochemical properties should be evaluated separately. When the benzene ring in imatinib was replaced with 2-oxa-BCO, solubility, lipophilicity, and microsomal stability improved, but ABL1 inhibitory activity was not retained. This demonstrates that improved properties cannot replace validation of target-recognition function.

The integrated results can be handled as follows:

The object of evaluation in benzene-ring bioisosteric replacement is the complete property profile, including target function, the targeted physicochemical properties, metabolic pathways, and synthetic feasibility. No individual parameter, including microsomal half-life, Fsp³, logD, or solubility, is sufficient on its own to determine the project value of a scaffold.

7 Classification and Research Applications of Saturated Benzene Bioisosteres and Related Three-Dimensional Molecular Building Blocks

Note: Monofunctional building blocks are mainly used to introduce the corresponding bridged-ring or cage fragment. Whether they can provide an effective replacement for a specific ortho-, meta-, or para-substituted benzene arrangement still depends on the final substitution positions, stereochemistry, mode of connection, and target-bound conformation.

Table 1 Bicyclo[1.1.1]pentane Series

Table 2 Bicyclo[2.1.1]hexane, Bicyclo[3.1.1]heptane, and Heteroatom-Containing Derivatives

Table 3 Bicyclo[2.2.2]octane and Oxabicyclo[2.2.2]octane Series

Table 4 Cubane and Spirocyclic Series

References

[1] Tsien J, Hu C, Merchant RR, et al. Three-dimensional saturated C(sp³)-rich bioisosteres for benzene. Nature Reviews Chemistry, 2024, 8: 605–627. DOI: 10.1038/s41570-024-00623-0.

[2] Stepan AF, Subramanyam C, Efremov IV, et al. Application of the bicyclo[1.1.1]pentane motif as a nonclassical phenyl ring bioisostere in the design of a potent and orally active γ-secretase inhibitor. Journal of Medicinal Chemistry, 2012, 55(7): 3414–3424. DOI: 10.1021/jm300094u.

[3] Denisenko A, Garbuz P, Voloshchuk NM, et al. 2-Oxabicyclo[2.1.1]hexanes as saturated bioisosteres of the ortho-substituted phenyl ring. Nature Chemistry, 2023, 15: 1155–1163. DOI: 10.1038/s41557-023-01222-0.

[4] Levterov VV, Panasiuk Y, Sahun K, et al. 2-Oxabicyclo[2.2.2]octane as a new bioisostere of the phenyl ring. Nature Communications, 2023, 14: 5608. DOI: 10.1038/s41467-023-41298-3.

[5] Surendran S, Raju S, Kalyani N, et al. Beyond Bioisosteres: Impact of Replacing a Benzene Ring with sp³-Rich Three-Dimensional Saturated Bridged Bicyclic Match Pairs on Biotransformation, Metabolic Stability, and Other ADME Profiles. Journal of Medicinal Chemistry, 2025, 68(20): 21615–21634. DOI: 10.1021/acs.jmedchem.5c01879.

[6] Wiesenfeldt MP, Rossi-Ashton JA, Perry IB, et al. General access to cubanes as benzene bioisosteres. Nature, 2023, 618: 513–518. DOI: 10.1038/s41586-023-06021-8.

[7] Varela MT, Dias GG, de Oliveira LFN, et al. Spirocyclic compounds as innovative tools in drug discovery for medicinal chemists. European Journal of Medicinal Chemistry, 2025, 287: 117368. DOI: 10.1016/j.ejmech.2025.117368.

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