Introduction

In medicinal chemistry, physicochemical properties such as molecular weight, lipophilicity, and the numbers of hydrogen-bond donors and acceptors are commonly used to assess the risk of poor solubility or low membrane permeability. Lipinski’s Rule of Five (Ro5) established empirical physicochemical property ranges for classical orally administered small-molecule drugs and indicates that the risk of poor absorption or permeability generally increases as molecular weight, lipophilicity, and hydrogen-bond-related parameters increase substantially [1].

As drug discovery increasingly expands into the beyond-Rule-of-Five (bRo5) chemical space, the relationship between conventional two-dimensional physicochemical parameters and membrane permeability becomes more complex. Molecules such as macrocycles, cyclic peptides, and proteolysis-targeting chimeras (PROTACs) often have large molecular sizes and numerous polar functional groups, yet some bRo5 drugs and drug candidates still exhibit appreciable membrane permeability [4,5].

This phenomenon does not mean that classical physicochemical principles no longer apply. For conformationally complex bRo5 molecules, an additional three-dimensional consideration is required: the polarity encoded in a two-dimensional structure is not necessarily identical to the polarity that a molecule exposes to its surroundings in a given environment.

Intramolecular hydrogen bonding, conformational folding, and environment-dependent redistribution of conformational ensembles can all alter the extent to which polar groups interact with the external environment, thereby affecting the free-energy cost associated with transfer of a molecule from the aqueous phase into a lipid membrane. Topological polar surface area (TPSA), experimental polar surface area (EPSA), and molecular chameleonicity provide complementary perspectives for understanding this process.

1. Why Can Highly Polar bRo5 Molecules Still Cross Cell Membranes?

1.1 Passive Membrane Permeation Requires Transfer from an Aqueous Phase into a Lipid Phase

For drug molecules that enter cells primarily through passive diffusion, the membrane permeation process can be simplified as:

Aqueous phase → membrane–water interface → interior of the lipid bilayer → aqueous phase on the opposite side

In the aqueous phase, polar groups such as hydroxyls, amides, amines, and carbonyls can form hydrogen bonds and dipole interactions with surrounding water molecules. Once a molecule enters the interior of the lipid bilayer, the surrounding environment changes from highly polar water to a low-polarity environment dominated by hydrocarbon chains, requiring some of the original hydration interactions to be disrupted.

This process involves desolvation.

Molecules with a larger number of exposed polar groups generally form more favorable interactions with water. When such a molecule moves from the aqueous phase into the membrane interior, the free-energy cost required to disrupt these interactions may therefore increase, raising the energetic cost of passive membrane permeation.

Studies by Rossi Sebastiano et al. on bRo5 drugs and clinical candidates showed that dynamically exposed molecular polarity is closely associated with passive permeability, whereas aqueous solubility is relatively less dependent on a specific three-dimensional conformation; differences in polarity exposure resulting from conformational changes help explain the membrane permeation behavior of some highly polar bRo5 molecules [4].

1.2 Polarity Is Not the Sole Determinant of Membrane Permeability

Passive membrane permeation generally depends on multiple factors, including:

① the ability of a molecule to transfer from the aqueous phase into the lipid membrane;

② diffusion of the molecule within the membrane;

③ molecular size and shape;

④ lipophilicity;

⑤ ionization state;

⑥ molecular conformation and conformational changes.

Therefore, a high TPSA does not necessarily mean that a compound will have low membrane permeability.

For bRo5 molecules, a more informative question is:

When the molecule enters the low-polarity membrane environment, does conformational change reduce the amount of polarity exposed to the external environment?

If some polar groups can be shielded through intramolecular interactions, the desolvation penalty associated with transfer from the aqueous phase into the membrane phase may decrease, thereby increasing the likelihood of passive membrane permeation.

2. What Is the Difference Between TPSA and Three-Dimensional Polarity Exposure?

2.1 TPSA Provides a Two-Dimensional Baseline for Molecular Polarity

TPSA is calculated from the polar atoms and their bonding environments in a two-dimensional molecular structure and is a widely used polarity parameter in medicinal chemistry.

Its advantages are clear:

① no three-dimensional molecular conformation is required;

② it can be calculated rapidly;

③ it allows straightforward comparison across large numbers of compounds;

④ it has substantial practical value in early-stage drug property assessment.

However, TPSA itself is essentially independent of molecular conformation.

When the same molecule changes from an extended conformation to a folded conformation, its atomic composition and two-dimensional connectivity remain unchanged, and therefore its TPSA remains the same.

At the same time, however, the three-dimensional orientations of polar groups and the extent to which they are exposed to the surrounding solvent may change substantially.

2.2 The Same TPSA Can Correspond to Different States of Polarity Exposure

Using the same flexible bRo5 molecule as an example, different conformations may exhibit different three-dimensional characteristics:

TPSA therefore reflects the potential polarity encoded in the two-dimensional topology of a molecule, but it cannot directly represent the solvent accessibility of polar groups in a specific three-dimensional conformation.

This difference may be relatively minor for small molecules with limited conformational variability. For larger and more flexible bRo5 molecules, however, conformational changes may have a substantially greater effect on polarity exposure [4].

3. How Do Intramolecular Hydrogen Bonding and Conformational Folding Alter Polarity Exposure?

3.1 Intramolecular Hydrogen Bonds Can Shield Polar Sites

Intramolecular hydrogen bonding (IMHB) is a common mechanism by which external polarity exposure can be reduced.

When a hydrogen-bond donor (HBD) and a hydrogen-bond acceptor (HBA) are independently exposed to the aqueous phase, both can form hydrogen bonds with water.

If the three-dimensional conformation of the molecule places the HBD and HBA at an appropriate distance and orientation, they may form an intramolecular hydrogen bond:

HBD···HBA

After an IMHB forms, the ability of the corresponding donor and acceptor to form hydrogen bonds with the external solvent may decrease, thereby reducing the apparent contribution of these polar sites to the external environment.

Goetz et al. investigated IMHB in drug-like molecules using supercritical fluid chromatography combined with nuclear magnetic resonance experiments. Their results showed that structures capable of forming IMHB can display different experimental polarity characteristics, providing a methodological basis for high-throughput investigation of polarity shielding [2].

It should be noted that the presence of a potentially favorable HBD–HBA geometric relationship in a two-dimensional structure does not demonstrate that an IMHB is continuously formed in solution. The stability of an IMHB is also influenced by conformational energy, interatomic distance and orientation, solvent polarity, and the ionization state of the molecule.

3.2 Conformational Folding Can Reduce Solvent-Accessible Polarity

For large, flexible molecules, polarity shielding often involves more than a single IMHB and may also be accompanied by three-dimensional folding of the entire molecule.

During folding, the following may occur:

① polar groups become oriented toward the molecular interior;

② HBDs and HBAs form intramolecular interactions;

③ hydrophobic regions form intramolecular contacts;

④ the overall molecular conformation becomes more compact;

⑤ the solvent-accessible polar region decreases.

Rossi Sebastiano et al. found that, within a set of bRo5 compounds, lower solvent-accessible three-dimensional polarity was associated with higher passive permeability. Dynamic IMHBs, molecular flexibility, and intramolecular interactions between aromatic regions may all contribute to regulation of polarity exposure [4].

This can be represented as a continuous physical process:

Conformational change → altered spatial arrangement of polar groups → altered solvent-accessible polarity → changes in hydration and membrane partitioning → altered passive permeability

However, the formation of IMHBs or compact conformations does not necessarily result in increased membrane permeability. Conformational changes may simultaneously affect solubility, ionization state, molecular size, and diffusion within the membrane. Their overall effects therefore still need to be evaluated using experimental data.

Atilaw et al. studied a VHL-based PROTAC using nuclear magnetic resonance experiments combined with conformational analysis and found that its three-dimensional conformation was strongly environment-dependent. In polar media, the molecule generally favored more extended conformations with greater polarity exposure; in chloroform, which was used to mimic the low-polarity environment of the membrane interior, more compact and folded conformations were populated. Intramolecular hydrogen bonding, shielding of amide groups, and other intramolecular interactions collectively reduced solvent-accessible three-dimensional polar surface area and molecular size, providing a structural basis for the molecular chameleonic behavior of this compound. [Atilaw et al., 2021] [9]

The figure above shows representative conformations of PROTAC 1 and changes in polarity exposure under environments of different polarity. The original study showed that PROTAC 1 can adopt more extended conformations with greater polarity exposure in polar environments, whereas in low-polarity environments it can form more compact folded conformations accompanied by intramolecular hydrogen-bond formation and a reduction in solvent-accessible three-dimensional polar surface area. The figure presents representative results for this specific PROTAC and does not imply that all bRo5 molecules undergo the same conformational changes. Image adapted from Atilaw Y, et al. ACS Med Chem Lett. 2021;12:107–114, CC BY. [9]

4. Molecular Chameleonicity Arises from Environment-Dependent Changes in Conformational Ensembles

4.1 Flexible bRo5 Molecules Usually Exist as Multiple Interconverting Conformations

Flexible molecules in solution generally do not remain in a single three-dimensional structure for extended periods. Instead, they exist as multiple interconverting conformations.

Together, these conformations constitute a conformational ensemble.

Different conformations have different free energies and therefore different population fractions within the conformational ensemble. When the solvent environment changes, intramolecular and intermolecular interactions also change, altering the relative stabilities of different conformations.

In more polar environments, exposed polar groups can form favorable interactions with the solvent, and some more open conformations may therefore have higher populations.

In less polar environments, the external environment provides less stabilization for exposed polar groups. Compact conformations that can form IMHBs, reduce polarity exposure, or establish favorable intramolecular contacts may therefore become relatively more stable.

This behavior is manifested as an environment-dependent redistribution of the conformational ensemble.

NMR combined with NAMFIS analysis further showed that PROTAC 1 does not simply switch between a single “open state” and a single “folded state.” Instead, it forms conformational ensembles with different compositions and population distributions in different media. In CDCl₃, the major conformations are generally more folded, whereas in DMSO and DMSO/water systems, more extended conformations are observed. These findings indicate that environmental changes alter the relative stability and population distribution of different conformations rather than inducing a single, well-defined conformational transition. [Atilaw et al., 2021] [9]

The figure above shows the major solution conformational ensembles of PROTAC 1 in media of different polarity. The figure displays major conformations with population fractions ≥10%. Green indicates predominantly linear conformations, light brown indicates folded conformations with one turn, and gray indicates folded conformations with two turns. Intramolecular hydrogen bonds are represented by black dashed lines on a yellow background. The conformational ensembles were obtained from NMR experimental data combined with NAMFIS analysis. Adapted from Atilaw Y, et al. ACS Med Chem Lett. 2021;12:107–114, CC BY. [9]

4.2 Molecular Chameleonicity Does Not Simply Mean “the Ability to Fold”

Molecular chameleonicity refers to the ability of a molecule to respond to changes in its surrounding environment by adjusting its conformational ensemble and thereby altering the physicochemical properties it presents to that environment.

Using nuclear magnetic resonance (NMR), Danelius et al. studied the macrocyclic drugs roxithromycin, telithromycin, and spiramycin and found that the conformational ensembles adopted by these molecules in nonpolar environments were generally more compact and displayed lower polarity exposure than those observed in polar environments [5].

The following three concepts should therefore be distinguished:

A molecule may form a stable, compact conformation and exhibit low polarity exposure. However, if its conformation and properties are not sensitive to environmental changes, this alone is insufficient to conclude that the molecule exhibits pronounced molecular chameleonicity.

In 2023, Garcia Jimenez et al. developed the chromatographic parameter Chamelogk, which experimentally characterizes this environment-responsive behavior through changes in compound behavior under different chromatographic conditions. The method was applied to studies of macrocycles, nonmacrocyclic bRo5 molecules, and PROTACs [6].

5. What Experimental Information Does EPSA Add to TPSA?

5.1 EPSA Reflects Exposed Polarity under Defined Experimental Conditions

The EPSA method developed by Goetz et al. is based on supercritical fluid chromatography (SFC). The retention behavior of a compound in the chromatographic system is influenced by its interactions with the stationary and mobile phases and can therefore be used to characterize the polarity that the molecule presents to its surroundings [2,3].

Although EPSA contains the term “polar surface area,” it is not a direct measurement of the three-dimensional geometric surface area of a molecule. Rather, it is an experimental polarity parameter that can respond to changes in apparent polarity caused by IMHB formation, conformational shielding, and related effects.

TPSA and EPSA therefore provide information at different levels:

EPSA does not replace TPSA. Analyzing the two together makes it possible to determine whether there is a substantial difference between the polarity encoded in a molecule’s two-dimensional structure and the polarity observed experimentally.

5.2 ETR Is Used to Compare EPSA with TPSA

In 2024, Price et al. analyzed approximately 1,000 compounds with human absorption data and examined approximately 10,000 AbbVie tool compounds, including approximately 7,000 Ro5 compounds, approximately 2,000 bRo5 compounds, and approximately 1,000 PROTACs [7].

The study introduced the EPSA-to-TPSA ratio, or ETR:

ETR = EPSA / TPSA

ETR is used to compare experimental polarity with two-dimensional topological polarity on a relative basis.

Within the analytical framework established in this study, a lower ETR means that EPSA is lower relative to TPSA, suggesting that the compound displays a more pronounced polarity reduction characteristic [7].

TPSA is a calculated topological parameter, whereas EPSA is derived from chromatographic experiments. ETR is an empirical ratio established from these two parameters and is used to compare and identify compounds with distinctive polarity-reduction behavior. It does not represent a physical partitioning of the true geometric surface area of a molecule.

5.3 ETR and Molecular Chameleonicity Should Not Be Considered Equivalent

A low ETR can indicate that the experimentally observed polarity of a compound is substantially lower than its topological polarity. However, a single ETR value does not directly compare the conformational ensembles of the molecule across two or more environments.

Molecular chameleonicity, by contrast, focuses on the following question:

When the environment changes, do the conformational distribution and apparent properties of the molecule change accordingly?

ETR is therefore suitable for identifying compounds with pronounced polarity-reduction characteristics. If the objective is to further investigate environment-dependent conformational changes, ETR can be combined with NMR under different solvent conditions, conformational calculations, and experimental parameters such as Chamelogk [5–7].

TPSA, EPSA, ETR, and molecular chameleonicity therefore represent several interrelated but non-equivalent levels of analysis:

Two-dimensional topological polarity → three-dimensional polarity shielding → experimentally observed apparent polarity → environment-dependent conformational change

6. How Do These Properties Influence bRo5 Drug Design?

6.1 From Reducing Polar Groups to Modulating Polarity Exposure

One traditional structural optimization strategy for improving membrane permeability is to reduce the number of hydrogen-bond donors, hydrogen-bond acceptors, or other polar functional groups, thereby lowering TPSA.

However, polar groups in drug molecules often serve specific functions, for example:

① forming key hydrogen bonds with the target protein;

② maintaining binding selectivity;

③ modulating aqueous solubility;

④ influencing ionization state.

Directly removing these groups may improve permeability, but it may also reduce target-binding capability or alter other pharmacokinetic properties.

For some bRo5 compounds, an alternative design strategy can therefore be considered:

Retain the polarity required for pharmacological activity while using three-dimensional conformation to reduce unnecessary external polarity exposure.

This is an important implication of dynamic-polarity research for medicinal chemistry design.

6.2 Structural Modifications Should Consider Coupled Changes in Conformation and Polarity

The following structural changes may influence the conformation and polarity exposure of bRo5 molecules:

There is no single universal optimization direction for these structural modifications.

For example, N-methylation can improve permeability in some cyclic peptides, but the effect may vary substantially depending on the position being modified. The EPSA studies by Goetz et al. demonstrated that N-methylation at different positions can produce different effects on polarity and permeability [3].

Similarly, increasing conformational rigidity may stabilize a favorable conformation with low exposed polarity, but it may also restrict access to other necessary conformations. Therefore, when evaluating a structural modification, it is necessary to determine how that modification actually changes the conformational ensemble and experimentally measured properties.

6.3 A Continuous Evidence Chain Is Needed from Structural Parameters to Permeability Experiments

When studying a bRo5 series, data at different levels can be analyzed within a unified structure–property relationship:

Two-dimensional structure, TPSA, and ionization properties

↓

Conformational analysis and IMHB formation

↓

EPSA and ETR

↓

Passive permeability or Caco-2 cell monolayer permeability experiments

↓

Efflux transport

↓

Solubility and in vivo absorption

This analytical framework can help distinguish problems arising from different sources.

For example, if a compound has a high TPSA but also shows favorable EPSA and permeability, this may indicate effective polarity shielding. If EPSA is low but cellular permeability remains poor, molecular size, ionization, or efflux transport may need to be examined further.

The principal value of EPSA and ETR therefore lies in connecting chemical structure, three-dimensional conformation, and experimentally measured permeability, rather than replacing actual ADME experiments.

7. From Membrane Permeability to Oral Absorption: What Other Factors Need to Be Considered?

Passive membrane permeability is only one step in the process by which a drug moves from the gastrointestinal tract into the systemic circulation.

Oral absorption may also be influenced by:

① drug solubility and dissolution;

② ionization state in the gastrointestinal tract;

③ passive membrane permeability;

④ uptake and efflux transport;

⑤ intestinal wall metabolism.

After entry into the portal circulation, systemic exposure is also affected by processes such as hepatic first-pass metabolism.

Therefore, compounds with pronounced polarity shielding or molecular chameleonicity do not necessarily exhibit favorable oral absorption.

This consideration is particularly important in PROTAC research.

In 2026, Le Manach et al. systematically evaluated existing oral-design guidelines using a set of AstraZeneca PROTACs. The study did not find that predicted or experimentally determined molecular chameleonicity parameters could consistently distinguish oral absorption performance within the dataset. In contrast, lowering the efflux ratio in the human colorectal adenocarcinoma Caco-2 cell monolayer model provided more useful information for design decisions within this dataset [8].

This study demonstrates that molecular chameleonicity can help explain the three-dimensional polarity and membrane permeation behavior of bRo5 molecules. However, extrapolating from membrane permeability to oral absorption requires simultaneous consideration of other processes, including transport, solubility, and metabolism.

For bRo5 molecules, TPSA remains an important two-dimensional polarity parameter. However, when molecules possess substantial conformational freedom, three-dimensional polarity exposure and its environment-dependent changes must also be taken into account. Studies of EPSA, ETR, and molecular chameleonicity provide complementary information for understanding the relationships among conformation, polarity, and membrane permeability in these molecules. In practical drug design, these parameters still need to be evaluated together with solubility, ionization, cellular permeability, efflux transport, and in vivo pharmacokinetic data.

8. Representative Chemicals Related to the Passive Transmembrane Permeation Mechanisms of bRo5 Molecules: Classification, Product Information, and Research Applications

Table 1. Compounds Related to Macrocycle and Molecular Chameleonicity Research

Table 2. Compounds Related to the Construction of Targeted Protein Degraders

Table 3. N-Methyl Amino Acids and Building Blocks for Conformational Modulation of Peptides

Note: The products listed above are representative Aladdin products related to scientific research. Their specific applications should be determined in conjunction with product specifications, batch COAs, and the intended reaction or evaluation system. Additional information on product specifications, grades, and COAs can be searched on the Aladdin website using the “product name/CAS/catalog number.”

References

[1] Lipinski CA, Lombardo F, Dominy BW, Feeney PJ. Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Adv Drug Deliv Rev. 2001;46(1–3):3–26. doi:10.1016/S0169-409X(00)00129-0.

[2] Goetz GH, Farrell W, Shalaeva M, Sciabola S, Anderson D, Yan J, Philippe L, Shapiro MJ. High Throughput Method for the Indirect Detection of Intramolecular Hydrogen Bonding. J Med Chem. 2014;57(7):2920–2929. doi:10.1021/jm401859b.

[3] Goetz GH, Philippe L, Shapiro MJ. EPSA: A Novel Supercritical Fluid Chromatography Technique Enabling the Design of Permeable Cyclic Peptides. ACS Med Chem Lett. 2014;5(10):1167–1172. doi:10.1021/ml500239m.

[4] Rossi Sebastiano M, Doak BC, Backlund M, Poongavanam V, Over B, Ermondi G, Caron G, Matsson P, Kihlberg J. Impact of Dynamically Exposed Polarity on Permeability and Solubility of Chameleonic Drugs Beyond the Rule of 5. J Med Chem. 2018;61(9):4189–4202. doi:10.1021/acs.jmedchem.8b00347.

[5] Danelius E, Poongavanam V, Peintner S, Wieske LHE, Erdélyi M, Kihlberg J. Solution Conformations Explain the Chameleonic Behaviour of Macrocyclic Drugs. Chem Eur J. 2020;26(23):5231–5244. doi:10.1002/chem.201905599.

[6] Garcia Jimenez D, Vallaro M, Rossi Sebastiano M, Apprato G, D’Agostini G, Rossetti P, Ermondi G, Caron G. Chamelogk: A Chromatographic Chameleonicity Quantifier to Design Orally Bioavailable Beyond-Rule-of-5 Drugs. J Med Chem. 2023;66(15):10681–10693. doi:10.1021/acs.jmedchem.3c00823.

[7] Price E, Weinheimer M, Rivkin A, Jenkins G, Nijsen M, Cox PB, DeGoey D. Beyond Rule of Five and PROTACs in Modern Drug Discovery: Polarity Reducers, Chameleonicity, and the Evolving Physicochemical Landscape. J Med Chem. 2024;67(7):5683–5698. doi:10.1021/acs.jmedchem.3c02332.

[8] Le Manach C, Viklund J, Kwapień K, Winiwarter S. Evaluation of Oral PROTAC Guidelines: Efflux Ratio Outweighs Chameleonicity Descriptors. ACS Med Chem Lett. 2026;17(4):890–899. doi:10.1021/acsmedchemlett.6c00043.

[9] Atilaw Y, Poongavanam V, Svensson Nilsson C, Nguyen D, Giese A, Meibom D, Erdélyi M, Kihlberg J. Solution Conformations Shed Light on PROTAC Cell Permeability. ACS Med Chem Lett. 2021;12(1):107–114. doi:10.1021/acsmedchemlett.0c00556.

For more related articles, see below:

The Role of 7-Membered Nitrogen Heterocycles in Drug Discovery: Microstate Management, Conformational Bias, and Developability Trade-offs (with Research Selection Navigator and Product Tables 1–3)

PCR-single-stranded conformational polymorphisms

Single-strand conformational polymorphism and heteroduplex analysis methods for mutation detection experiments

Pyrrolidine and Its Derivative Systems: How a Five-Membered N-Heterocycle Tunes Properties and Use Cases via “Charge State–Conformation–Functional-Group Switching” (with Tables 1–4 for Selection Navigation)

Multiple Physicochemical Properties Close to Benzamide, Conformational Orientation Closer to Sulfonamide: The Dual Bioisosteric Design Value of 3-Aryl-3-aminooxetanes

Rationale for Applying Spirocyclic Building Blocks in Drug Molecule Optimization: Three-Dimensionalization, Conformational Control, and Property Trade-offs

Piperidine and Its Derivatives: Controllable Design of Charge, Conformation, and Connecting Exit with Product Navigation (Tables 1–5)