Challenges of Aufheben to Promote Druglikeness: Chemical Modification Strategies to Improve Aqueous Solubility and Permeability

Abstract

A key challenge in drug discovery is to endow candidate bioactive molecules with druglike properties. However, the optimization of critical, often-conflicting physicochemical properties through chemical modifications is particularly difficult, and the term Aufheben has been adopted to describe the simultaneous preservation and modification of apparent opposites to achieve improvement. This perspective examines strategies to reconcile conflicting parameters, such as lipophilicity/hydrophilicity, druglikeness/flatness, and druglikeness/molecular weight, in order to improve the aqueous solubility and membrane permeability of drug candidates. We review and categorize numerous molecular design strategies that have been investigated to address these challenges and highlight some recent successful examples.

1. Significance

• Medicinal chemistry requires reconciliation of apparently conflicting physicochemical parameters to achieve druglike properties.

• We summarize molecular design strategies to simultaneously improve aqueous solubility and membrane permeability of drug candidates.

• Recent examples of successful strategies to enhance druglikeness of small molecules as well as beyond rule of five molecules, especially cyclic peptides and PROTACs, are provided.

2. Introduction

Druglikeness is a qualitative measure of a candidate compound’s suitability for development as a drug, especially its likely oral bioavailability, and is evaluated in terms of the molecular structure and physical properties, such as molecular weight (MW), lipophilicity, and numbers of hydrogen bond donors (HBDs) and acceptors (HBAs). For example, the original Rule of Five (Ro5) predicts that compounds with a MW ≤ 500, LogP (the common logarithm of partition coefficient) ≤ 5, HBDs ≤ 5, and HBAs ≤ 10 are likely to be orally bioavailable. These physicochemical parameters of Ro5 are recognized as being associated with acceptable values of both aqueous solubility in the intestine and intestinal membrane permeability. More recently, however, drugs with parameters beyond Ro5, designated as bRo5, have been successfully developed. Although various definitions for bRo5 molecules exist, we define bRo5 here as compounds with a MW ranging from 500 to 3000, thereby excluding biopharmaceuticals such as protein, antibody, and nucleic acid drugs. In such cases, it is particularly challenging to achieve an appropriate balance of the aqueous solubility and permeability. The identification of numerous important druglike parameters often leads medicinal chemists to contend with the conflicting relationships among them. Achieving a delicate balance among these interdependent properties is crucial for successful drug candidates, yet it presents a significant challenge in molecular design.

The importance of both aqueous solubility and permeability is reflected in the Biopharmaceutics Classification System (BCS), which classifies drugs with the aim of identifying compounds with minimal absorption-related risks. The BCS classifies compounds into four categories. Class 1 compounds exhibit high permeability and high solubility, ensuring efficient absorption after oral administration, whereas Class 4 compounds are characterized by both low solubility and low permeability. Class 2 exhibits low solubility–high permeability, while Class 3 shows high solubility–low permeability.

Achieving adequate permeability of orally administered drugs requires an optimal level of lipophilicity to facilitate absorption across the lipid membranes. In the early stage of drug development, the parallel artificial membrane permeability assay (PAMPA) and Caco-2 assay are commonly employed for in vitro permeability prediction. Apparent permeability (P _app) values are classified as poor (<1.0 × 10^–6 cm/s), moderate (1–10 × 10^–6 cm/s), or good (>10 × 10^–6 cm/s) according to the literature. However, high lipophilicity, together with low aqueous solubility, has become an increasingly common characteristic of hits, leads, development candidates, and even marketed drugs. This trend may be attributed to druggable binding pockets tending to have hydrophobic environments.

Aqueous solubility is also a critical physicochemical property for small-molecule drug candidates. The dose number (Do) is a parameter used to evaluate the practical adequacy of drug solubility. It is calculated as the dose divided by an uptake volume of 250 mL and the drug’s thermodynamic solubility: Do = (dose/uptake volume [250 mL]) /thermodynamic solubility. A drug candidate is considered sufficiently soluble when the Do is less than 1. Orally administered drugs must exhibit sufficient aqueous solubility to dissolve in the gastrointestinal tract, as their absorption via passive diffusion depends on the concentration gradient between the intestinal lumen and the bloodstream, a factor influenced by solubility. Furthermore, the efficacy evaluation and risk assessment of poorly soluble compounds present significant challenges. Overall, drug candidates must balance apparently contradictory physicochemical properties, namely, lipophilicity and hydrophilicity (aqueous solubility). Medicinal chemists often struggle with the conflicting relationship between water solubility and membrane permeability. Here, we adopt the term Aufheben, used by Hegel to describe the dialectical synthesis or combination of a thesis and its antithesis, to denote both preserving and modifying opposing physicochemical characteristics to achieve a simultaneous improvement of aqueous solubility and membrane permeability.

First, we consider the solubility in water. The Gibbs-free energy of dissolution (ΔG _sol ) is determined by the following equation: ΔG _sol = ΔH _sol – TΔS _sol. Dissolution is thermodynamically favorable when ΔG _sol < 0. The enthalpy of dissolution (ΔH _sol) and entropy of dissolution (ΔS _sol) are the key driving factors at a given temperature (T). The dissolution of a solid molecule in water generally proceeds through three conceptual steps, as illustrated in Figure . Releasing a molecule from its crystal lattice (step 1) requires a substantial amount of enthalpy (ΔH ₁) and an increase in entropy (ΔS ₁) due to the disruption of ordered packing. This step is largely governed by the crystal packing efficiency and the intermolecular forces within the solid. The melting point of a solute crystal is a key indicator of ΔH ₁ reflecting the energy required to overcome these lattice forces. Differential scanning calorimetry (DSC) is an experimental technique used to measure the melting point, enthalpy of fusion (ΔH _fus), and entropy of fusion (ΔS _fus), which directly reflect the stability of the crystalline state and the energy barrier to dissolution. Next, creating a void in water to accommodate the solute (step 2) involves an increase in both the enthalpy (ΔH ₂) and entropy (ΔS ₂) of the water, as water molecules must rearrange. The free solute occupies the cavity in water (step 3), leading to favorable interactions (e.g., hydrogen bonding, van der Waals forces) between the solute and water molecules. This step typically results in a decrease in both the enthalpy (ΔH ₃) and entropy (ΔS ₃) as the system becomes more ordered around the solute. Thus, the overall solubility of a solid solute in water is influenced by several factors: the crystallinity of the solute, its molecular size, and its ability to interact favorably with water. The total enthalpy of dissolution is ΔH _sol = ΔH ₁ + ΔH ₂ + ΔH ₃, and the total entropy of dissolution is ΔS _sol = ΔS ₁ + ΔS ₂ + ΔS ₃. The long-standing principle of solvation, often encapsulated in the phrase “like dissolves like”, can be thermodynamically explained by a significant decrease in ΔH ₃ and ΔS ₃ primarily due to strong hydrogen bonding between the solute and water molecules. Furthermore, ΔH _sol and ΔS _sol can be experimentally obtained by measuring the mole fraction solubility (X) at different temperatures and applying the Van’t Hoff equation: lnX = – ΔH _sol/ RT + ΔS _sol/R, where R represents the ideal gas constant.

1.

Schematic illustration of the three steps involved in drug dissolution.

A molecule with higher MW necessitates a larger void in water, thereby requiring more hydrogen bonds to be disrupted leading to a large increase of ΔH ₂ and ΔS ₂. To overcome this, large molecules can achieve both solubility and permeability through environment-responsive conformational changes. These molecules adopt an open conformation in aqueous environments, exposing their polar functional groups to enhance solubility. Conversely, in lipophilic environments, they transition to a closed conformation, masking their polar groups via intramolecular interactions, such as intramolecular HB, thereby facilitating permeability. This behavior is commonly termed molecular chameleonicity (Figure ).

2.

Molecular chameleonicity.

In modern drug discovery and development, solubility is evaluated in various ways, depending on the stage of development. In silico prediction is utilized during the early stage of drug discovery and can help prioritize the synthesis of hit compounds identified through high-throughput screening. However, accurately predicting aqueous solubility remains challenging, in part because of the variability in data sources used for predictionaqueous solubility data are often not comparable, with marked differences in reported values and evaluation methods. Kinetic solubility is typically measured starting with a solution of a compound in DMSO, with precipitation detected after a short incubation period using turbidity or UV absorption measurement. This method is advantageous during the early stages of drug discovery due to its high throughput, suitability for automation, and minimal compound consumption. However, the kinetic solubility does not take account of the crystalline form of the compound. For example, compounds in an amorphous state can be up to 100 times more soluble than crystalline forms.

In contrast, thermodynamic solubility (equilibrium solubility) refers to the solubility of the most stable crystalline form of a compound in equilibrium with the solvent. The most stable form is invariably the form with the highest melting point. The significance of the thermodynamic solubility assessment becomes greater during the late discovery stage and early development of candidate drugs. While formulation technologies can enhance the dissolution rate and induce a temporary or apparent increase in solubility, they cannot permanently change the compound’s inherent solubility. Given sufficient time, any undissolved solute will revert to its most stable crystal form under the given conditions, and the solubility will ultimately converge to the true thermodynamic solubility. , In addition, the amorphous form tends to spontaneously recrystallize during storage, and it is challenging to predict the time required for nucleation and crystal growth. Thus, it is preferable to generate drug candidates (active pharmaceutical ingredients) with sufficient aqueous solubility through medicinal chemistry efforts.

As illustrated in Figure , the solubility of a solid solute in water depends on several factors: the crystallinity of the solute, its molecular size, and its hydration. Among these, the hydration step (step 3) is a promising target to improve aqueous solubility. Reducing hydrophobicity (LogP _ow), distribution coefficient (LogD), or topological polar surface area (tPSA) by means of chemical modifications, such as the introduction of hydrophilic group(s), is a classical and widely adopted strategy for improving aqueous solubility. For instance, the replacement of a phenyl ring with heterocyclic rings has been extensively studied. , Lipophilic substituent constants (π), as defined by Hansch et al., are valuable guides in molecular design for modifying the hydrophilicity of parent molecules. However, this “like dissolves like” strategy is not universally effective, as the introduced hydrophilic group(s) may interfere with the target protein–drug interaction. More critically, a reduction in lipophilicity often results in a trade-off between the solubility increase and permeability decrease. This inherent limitation of the like dissolves like approach poses a significant challenge.

The solubility of a solid solute in water is also influenced by the crystal packing of the solute (Figure , step 1) and compounds that exhibit poor solubility due to strong crystal packing interactions are often referred to as “brick dust”. Over the past decade or two, various strategies aimed at improving the aqueous solubility of traditional pharmaceutical compounds by disrupting intermolecular interactions have been reported. In parallel, bRo5 molecules, including cyclic peptides and PROTACs (proteolysis targeting chimeras), have emerged as promising therapeutic modalities. These molecules are generally less soluble and less permeable than traditional pharmaceutical compounds due to their higher MW (Figure , step 2). However, examples of bRo5 molecules with high solubility and permeability have been reported, and their structural and physicochemical characteristics are beginning to be elucidated.

Here, we review recent progress in endowing molecules with druglikeness, focusing on strategies to increase aqueous solubility and membrane permeability. Several excellent reviews on aqueous solubility − and druglike physicochemical properties have been published recently, and this perspective differs from them in three main respects focusing on Aufheben. First, we highlight examples of strategies to achieve simultaneous improvements in seemingly contradictory pairs of physicochemical properties, such as increasing both lipophilicity and hydrophilicity (aqueous solubility) through chemical modifications that weaken intermolecular interactions. Increasing evidence suggests that such chemical modifications can enhance not only aqueous solubility but also permeability. Second, we present examples illustrating how to optimize the aqueous solubility of flat molecules, which are often considered to lack druglike properties. These first two differentiating points, which primarily focus on methodologies for more traditional pharmaceutical compounds, are detailed in Section . Third, we examine strategies to improve the membrane permeability and aqueous solubility of bRo5 molecules. This third aspect is elaborated upon in Section .

3. Improvement of Aqueous Solubility and Permeability of Small Molecules by Disrupting Intermolecular Interactions (Aufheben of Lipophilicity and Aqueous Solubility)

As the aqueous solubility of a compound is influenced by the molecular packing in the solid state (Figure , step 1), a possible strategy to improve the aqueous solubility involves disruption of the tight crystal packing of molecules. In other words, molecular modifications that weaken the intermolecular interactions in the most stable crystal form of a compound can enhance its thermodynamic solubility. The melting point of a compound is closely related to its crystal lattice and crystal packing energy, making it a useful parameter for assessing crystal packing, along with crystal density. In 1980, Yalkowsky proposed a general solubility equation, derived through semiempirical analysis, to describe the relationship between aqueous solubility and melting point: Log­[solubility (M)] = 0.5 – (LogP) – 0.01­[[melting point­(°C)] – 25]. This equation was primarily based on data for rigid, polycyclic, and halogenated aromatic compounds. In 2009, Lovering et al. proposed that increasing molecular saturation, quantified by the fraction of sp³-hybridized carbons (Fsp³) (defined as the number of sp³ carbons divided by the total carbon count of a compound), is an approach to improving clinical success. They also demonstrated through database analysis that Fsp³ positively correlated with solubility and negatively correlated with the melting point. It is worth noting, however, that the precise relationships between aqueous solubility and the melting point of pharmaceutical compounds remained a lacuna in the literature at that time. Regarding the relationship between molecular structures and the aqueous solubility of pharmaceutical compounds, we previously proposed that disruption of molecular planarityincluding not only increased saturation but also an increase in the dihedral angle of planar compounds and molecular bendingdecreases melting points and increases thermodynamic aqueous solubility, as shown by matched molecular pair analyses. Furthermore, we demonstrated that the introduction of hydrophobic substituents also improves thermodynamic aqueous solubility, realizing part of the phenomenon of “Aufheben”. In the past decade or two, various chemical modification strategies have been developed to improve the aqueous solubility of complex pharmaceutical compounds by disrupting intermolecular interactions, and as will be discussed later, these approaches can increase aqueous solubility even in cases where hydrophobicity is simultaneously increased.

3.1. Case Studies On Modifying Flatland (Aufheben of Flatland and Druglikeness)

3.1.1. Disruption of Molecular Planarity by ortho-Substitution of Biaryl Groups

As the medicinal chemistry toolbox has evolved, numerous bioactive molecules possessing biaryl groups have been reported, likely for two reasons: (1) they serve as versatile scaffolds, and (2) they can be easily synthesized by means of Suzuki coupling reactions. Notably, the AstraZeneca screening collection contained 2% biphenyl fragments in 1990, but this had increased to 12% of all registered compounds by 2014. However, biaryl molecules often exhibit low solubility due to their tight crystal packing and are often regarded as undruglike.

The effects of ortho-substitution on biaryl groups are exemplified by agonists of peroxisome proliferator-activated receptor (PPAR), a type of nuclear receptor (Table ). An initial strategy to improve the aqueous solubility of PPAR agonists was the introduction of hydrophilic groups. Unfortunately, several such modifications diminished the PPAR agonistic activity (e.g., 2a vs 2d), as many nuclear receptors recognize the hydrophobicity of the ligands. In contrast, the introduction of a hydrophobic group(s) at the ortho-position of the biaryl group increased the PPAR agonistic activity (e.g., 1a vs 1b/1c, 2a vs 2b/2c, and 2d vs 2e). All of the analogs listed in Table exhibited greater thermodynamic solubility than their parent compounds. Interestingly, an increase in dihedral angle (2c) resulted in greater solubility in phosphate buffer than a decrease in hydrophobicity (2d) in this series. Notably, 2e demonstrated a 350-fold higher solubility than 2d in phosphate buffer. Mechanistically, all compounds with a methyl group(s) introduced at the ortho-position of the biaryl moiety (1b, 2b and 2e) exhibited increased hydrophobicity, larger dihedral angle, and lower melting point compared to their parent compounds (1a, 2a, and 2d, respectively). The most soluble analogue 2e showed the lowest melting point and the largest dihedral angle in this series. When comparing 2e with 2a, two modifications (replacement of the phenyl group with a pyridine ring and introduction of methyl groups) resulted in a more than 2700-fold increase in solubility in phosphate buffer. These findings suggest that employing a combination of strategies can be effective to improve the aqueous solubility. A scatter plot (Figure ) illustrates the relationships among PPAR agonistic activity and CLogP, as well as the impact of ortho-substitution on solubility improvement.

1. Improvement in Thermodynamic Aqueous Solubility and Biological Activity by ortho-Substitution of Biaryl Group.

				thermodynamic aqueous solubility (pH 7.4) (mg mL–1)
compd	R 1	R 2	X	50% EtOH	phosphate buffer (pH 7.4)	CLogP	HLPC retention time (min)	melting point (°C)	calculated dihedral angle (°)	PPARδ EC50 (nM)
1a	H	H		0.375	<0.001	6.3	7.98	259–262	43.5	170
1b	Me	H		0.985	<0.001	6.8	9.46	241–243	52.5	11
1c	F	H		3.22	<0.001	7.1	8.72	221–223	36.1	53
2a	H	H	CH	1.35	<0.001	6.4	7.79	177–178	36.9	29
2b	Me	H	CH	9.95	<0.001	6.9	8.65	146–149	57.5	1.6
2c	F	F	CH	10.4	0.0217	7.5	7.42	177	46.2	5.7
2d	H	H	N	9.03	0.00762	4.8	3.61	152	37.4	220
2e	Me	Me	N	17.7	2.70	5.8	4.36	104–106	78.1	76

^aThermodynamic solubility in a mixture of phosphate buffer (pH 7.4) and EtOH.

^bReversed-phase column.

3.

Aqueous solubility versus CLogP. Data points are colored by pEC₅₀ ranges (6–7 (green), 7–8 (orange), 8–9 (red)). Circle symbols denote ortho-substituted compounds, while square symbols indicate non-substituted compounds.

The effects of ortho-substitution of biaryl groups are also exemplified in β-naphthoflavone 3a, which is an aryl hydrocarbon receptor agonist (Table ). The thermodynamic solubility of the ortho-methyl analog 3b (262 μg/mL) was 3-fold greater than that of 3a. Furthermore, the position of the methyl group significantly influenced the solubility: the rank order of aqueous solubility was ortho (3b) ≫ nonsubstituted (3a) ≥ meta (3c) > para (3d). Several mechanistic studies support the idea that ortho-substitution disrupts molecular planarity by increasing the dihedral angle, which leads to a lower melting point and, consequently, higher solubility. Notably, the ortho-dimethyl analog 3e exhibited a 15-fold higher solubility (1270 μg/mL) than 3a, despite its increased hydrophobicity. The solubility was also dependent on the number of methyl group(s): the rank order was dimethyl (3e) > monomethyl (3b) > nonsubstituted (3a). This trend aligns with the order of lower melting points, larger calculated dihedral angles, and lower λmax values. On the other hand, pyridine analogue 3f, which lacks a hydrogen atom, showed a higher melting point, higher λmax, and decreased dihedral angle compared to 3a. Nevertheless, 3f demonstrated an improved aqueous solubility (299 μg/mL), likely due to its reduced hydrophobicity. Interestingly, as observed in prior examples, the increase in the dihedral angle (3e) resulted in a greater solubility improvement than the reduction in hydrophobicity (3f).

2. Improvement in Thermodynamic Aqueous Solubility by ortho-Substitution.

compd	R 1	R 2	X	thermodynamic aqueous solubility (pH 7.4) (μg mL–1)	ClogP	HLPC retention time (min)	melting point (°C)	calculated dihedral angle (°)	λmax (nm)
3a	H	H	C	84.6	4.8	7.24	165–167	17.8	273
3b	H	o-Me	C	262	5.3	8.17	135–137	37.9	265
3c	H	m-Me	C	80.9	5.3	9.44	162	16.8	274
3d	H	p-Me	C	35.4	5.3	9.43	194–195	16.5	285
3e	Me	o-Me	C	1270	5.7	9.13	92	70.0	261
3f		H	N	299	3.3	4.52	187–188	0	285

^aThermodynamic solubility in phosphate buffer (pH 7.4): EtOH (1:1).

^bReversed-phase column.

Researchers at Novartis reported GNF6702 (4a) as an antiparasitic drug candidate (Table ). However, the clinical progression of 4a was hindered due to solubility-limited oral absorption. Compound 4a features a flat molecular structure and a high melting point (224 °C), characteristics contributing to its low aqueous solubility. To address this issue, a methyl group was introduced at the ortho-position of the biaryl group (4b), resulting in a reduced melting point (139 °C) and improved aqueous solubility, despite the increased lipophilicity. When compared to the free base form of 4a, the fumarate cocrystal of compound 4b afforded a 5-fold higher supersaturation in FaSSIF, achieving a concentration of 73 μM. Moreover, the fumarate salt of 4b demonstrated superior oral bioavailability in mice (46%) compared with 4a (34%). In other models, the oral bioavailability of fumarate salt 4b was 67% in rats, 44% in dogs, and 27% in monkeys.

3. Improvement in Aqueous Solubility and Oral Bioavailability by ortho-Substitution.

compd	R	aqueous solubility (pH 6.8) (μM)	CLogP	melting point (°C)	mouse F (%)
GNF6702 (4a)	H	10	–0.29	224	34
4b	Me	17	–0.086	139	46

^aSolubility in a high-throughput assay format.

^bCLogP was estimated by us, using ChemDraw version 20.0.

ortho-Substitution has remarkable potential for enhancing aqueous solubility, achieving an over 100-fold increase (Table ). Wnt/β-catenin signaling inhibitor 5a exhibited a limited kinetic solubility of 0.05 μg/mL in PBS buffer. Quantum mechanical analysis of 5a revealed a highly planar conformation among the pyrazole ring, amide group, and quinoline ring. To address this limitation, a methyl group was introduced into the pyrazole ring (5b), effectively disrupting the molecular planarity by increasing the dihedral angle. This chemical modification resulted in a 120-fold improvement in aqueous solubility compared to 5a.

4. Improvement in Aqueous Solubility by ortho-Substitution.

compd	R	kinetic aqueous solubility (pH 7.4) (μg mL–1)	CLogP	dihedral angle 1 (°)	dihedral angle 2 (°)
5a	H	0.05	3.4	171.4	178.5
5b	Me	5.9	3.3	166.6	178.7

^aKinetic solubility of solid at 25 °C for 6 h.

^bCLogP was estimated by us, using ChemDraw version 20.0.

^cQuantum mechanics analysis.

Researchers at Merck reported the interesting phenomenon that atropisomers showed greater aqueous solubility than the racemate (Table ). For example, the kinetic aqueous solubilities of enantiomeric atropisomers 6P and 6M were greater than that of racemate 6. A key observation was the higher melting point of racemate 6 (261 °C) compared to 6M (224 °C), suggesting weaker intermolecular interactions in the atropisomeric crystal structures. However, during the 24 h incubation, the solid-state forms of both atropisomers transitioned from anhydrate to hydrate form.

5. Improvement in Aqueous Solubility of ortho-Substituted Atropisomers.

	kinetic aqueous solubility (μg mL–1)
compd	PBS pH 7.4	FaSSIF pH 6.5	FaSSIF pH 5.0	CLogP	melting point (°C)	crystal density (g cm–3)
rac-6	20	54	193	1.4	261	1.354
6M	93	211	773	1.4	224	1.372
6P	95	235	741	1.4		1.374

^aKinetic solubility of solid at 37 °C for 2 h.

^bFasted state simulated intestinal fluid.

^cCLogP was estimated by us, using ChemDraw Ultra 20.0.

Single-crystal X-ray diffraction (XRD) analysis of the anhydrate forms revealed densities of 1.374 g/cm³ for 6P, 1.372 g/cm³ for 6M, and 1.354 g/cm³ for racemate 6. However, the authors noted that lattice energy and free energy are not directly correlated with the density of a specific solid-state form. They emphasized the importance of considering entropic effects, as these are reflected in both the melting point and solubility of a compound.

Effects on ortho-substitution have also been demonstrated in unfused bicyclic aryl systems. Efforts to improve the solubility of integrin antagonist 7a initially focused on introducing hydrophilic substituents (7b–7d). However, these modifications resulted in decreased inhibitory activity in a cell-based assay using vascular smooth muscle cells (VSMCs) (Table ).

6. Improvement in Aqueous Solubility and Biological Activity by ortho-Substitution of the Unfused Bicyclic Phenyl System.

Compd	R1	R2	thermodynamic water solubility (mg mL–1)	CLogP	HLPC retention time (min)	melting point (°C)	αvβ3IC50 (nM)	VSMC IC50 (nM)
7a	H	H	<0.1	1.1	8.25	252–254	1.3	190
7b	OH	H					0.44	530
7c	F	CO2H					0.77	660
7d	OH	OH					0.30	390
7f	F	H	0.6	1.7	9.73	182–184	0.36	48
7g	OMe	H	1.3	0.79	8.72	162–164	0.19	110
7h	F	OH	0.1	1.4	6.16	193–197	0.14	53

^aReversed-phase column.

^bα_vβ₃-Mediated cell adhesion assay using human vascular smooth muscle cells (VSMC) and human vitronectin.

In contrast, a second approach involving the introduction of hydrophobic substituents (7f and 7g) led to enhanced activity in both receptor-binding and VSMC assays. Furthermore, these compounds exhibited significantly increased thermodynamic aqueous solubility, with 7f and 7g being at least 6-fold and 13-fold more soluble than parent compound 7a, respectively.

As for the mechanism of the increase in solubility of 7f and 7g compared with 7a, a relationship between the rank order of aqueous solubility and the order of melting points indicates that the increase in aqueous solubility of 7f and 7g was caused by disruption of molecular planarity, leading to a decrease of intermolecular interactions despite an increase of hydrophobicity. Additionally, the single-crystal X-ray structure of 7f revealed a substantial increase in the dihedral angle between the piperidine ring and the benzoyl group, corroborating the hypothesis of disrupted molecular planarity. Interestingly, hydroxyl analogue 7h, despite its lower hydrophobicity compared with 7f, exhibited decreased solubility. This decrease is consistent with its higher melting point, suggesting that the introduced hydroxyl group may have formed new intermolecular HBs, resulting in tighter crystal packing. These findings highlight the superior impact of disrupting molecular planarity (7f and 7g) on improving aqueous solubility, compared to merely decreasing the hydrophobicity (7h).

Researchers at Bayer identified ATR kinase inhibitor 8a, which showed low kinetic aqueous solubility (<1 μg/mL), resulting in an oral bioavailability of only 14% in rats, even though 8a has a high permeability coefficient of 102 nm/s in Caco-2 cells. Moreover, the compound also raised safety concerns due to its activity in the hERG patch clamp assay in vitro (IC₅₀: 5.8 μM). To reduce intermolecular interaction and planarity, (1) the (methylsulfonyl)­phenyl group in 8a, which is capable of head-to-head interaction (vide infra in Section ), was replaced with 1-methylpyrazole (equivalent to ortho-substitution), and (2) a methyl group was introduced at the 3-position of the morpholinyl group (equivalent to ortho-substitution) in 8a. Lead compound 8a and designed 8b have an identical LogD value of 2.1, but 8b showed more than 34-fold-increased aqueous solubility. In the Caco-2 model, 8b showed a permeability coefficient of 211 nm/s, and after oral administration, the bioavailability ranged from moderate in dogs (51%) to high in rats (87%). In the hERG patch clamp assay in vitro, 8b showed no activity (IC₅₀: >10 μM). The X-ray structure of 8b showed a twist between the naphthyridine scaffold and the 1-methylpyrazolyl group (torsion angle: 54.8°) (Figure ). Additionally, the (3R)-methyl substituent of the morpholine points out the plane of the naphthyridine scaffold (Table ).

4.

Single-crystal X-ray structure of 8b.

7. Improvement in Aqueous Solubility, Oral Bioavailability, and HERG Inhibition by ortho-Substitution of the Unfused Bicyclic Aryl System.

^aSolubility in a high-throughput assay format.

Researchers in AstraZeneca recently reported interesting relationships between the number of ring atoms of N-aryl lactams and kinetic aqueous solubility. They found that measured LogD is reduced by half a log unit by the addition of methylene to the ring, and the six-membered-ring analog 9b was 120-fold more soluble than the five-membered matched pair 9a in phosphate buffer (Table ). Additional analyses of in-house-matched molecular pairs revealed that several six-membered analogs of not only N-aryl lactams but also N-aryl imidazolinones and N-aryl oxazolinones were more soluble than the five-membered matched pairs. DFT calculation revealed that the five-membered lactams lie almost planar with the aromatic ring, whereas the six-membered lactams take a nearly orthogonal conformation due to the steric hindrance between the lactam’s carbonyl group and ortho-hydrogen atom. This orthogonal conformation would not only disrupt molecular planarity (reduction of packing energy) but also decrease electron delocalization (resonance), which can result in stronger HBs. Interestingly, the ortho-substituted six-membered ring compounds (9c) are more lipophilic and less soluble than the corresponding five-membered analogs (9d) because both types of analogs possess twisted conformation by ortho-substitution.

8. Improvement in the Kinetic Aqueous Solubility of N-aryl Lactam by Changing the Lactam Ring Size.

compd	n	aqueous solubility (pH 7.4) (μM)	ACD LogD	CLogP	measured LogD	Melting onset (°C)
9a	1	0.054	1.6	3.0	4.4	285
9b	2	6.3	2.0	3.5	3.9	243
9c	1	66			1.8
9d	2	18			1.9

^aAfter 24 h, using solids obtained by evaporation of DMSO stock solutions.

The effects of the ortho-substitution of biaryl groups are also applicable to aryl groups possessing other flat substituents. Based on the melting points of substituted benzenes (vide infra in Section ), ortho-isomers bearing flat substituent(s) (e.g., Ph, CO₂H, Ac, NO₂, CONH₂, and NHAc) tend to exhibit the lowest melting points among their regioisomers. The observed difference in mean melting points between the ortho-isomers and other isomers was approximately 12 °C, which can be attributed to the increased dihedral angles resulting from the substitution pattern.

A concrete example of pharmaceutically relevant compounds exhibiting this trend is provided by ortho-dimethylbenzamide analogs (10c and 10d) (Table ). These analogs showed improved thermodynamic aqueous solubility compared with their monomethyl counterparts (10a and 10b). The enhanced solubility was likely due to the disruption of molecular planarity, as evidenced by the over 70 °C reduction in melting points for the ortho-disubstituted analogs relative to their monosubstituted counterparts.

9. Improvement in Thermodynamic Aqueous Solubility by ortho-Substitution of Benzamide Group.

3.1.2. Disruption of Molecular Planarity by Substitution of Benzylic Positions

Researchers at AstraZeneca identified a selective N-methyl-d-aspartate antagonist 11a, which faced challenges due to its poor solubility and bioavailability (Table ). To address these issues, the α-methyl analogue 11b was developed. The molecular planarity was disrupted in this analogue, resulting in at least a 5.8-fold improvement in aqueous solubility compared with 11a. Similarly, α-methyl analogue 11b had better oral bioavailability (30%) than the unsubstituted compound 11a (5%). Mechanistically, the α-methylation resulted in a lower melting point, which in turn enhanced solubility, despite the concomitant increase in hydrophobicity.

10. Improvement in Aqueous Solubility and Oral Bioavailability by α-Substitution.

compd	R	aqueous solubility (pH 7.4) (mg mL–1)	CLogP	melting point (°C)	Caco-2 P app (nm s–1)	Rat F (%)
11a	H	0.05	1.4	277–278	200	5
11b	Me	>0.29	1.8	245–247	11000	30

^aMethanesulfonate salt.

Hypoxia-inducible factor prolyl hydroxylase domain inhibitor 12a showed good in vivo efficacy, but its bioavailability in rats was only 16% (Table ). This low bioavailability was attributed to its poor solubility in a pH 6.5 buffer (9.5 μg/mL). To address this issue, the authors introduced fluorine atoms onto the biphenyl moiety, hypothesizing that this modification would increase the dihedral angle. Indeed, the solubility of 12b in pH 6.5 buffer was increased to 49 μg/mL. Subsequently, reducing the number of aromatic rings in the structure (12c) led to a 9-fold improvement in solubility compared with 12a. Further optimization involved the introduction of a methyl group at the α-position to disrupt intermolecular interactions. Remarkably, this modification, affording compound 12e, resulted in a dramatic increase of solubility to over 1000 μg/mLmore than 13-fold higher than that of 12d. The enhanced solubility of 12e was attributed to steric hindrance between the ortho-chloro atom and the α-methyl group, which caused increased torsion of the phenyl ring. This hypothesis was supported by the difference in melting points (12d: 262–267 °C; 12e: 238–240 °C). Importantly, 12e exhibited a significantly improved pharmacokinetic profile, with an oral bioavailability in rats of 77% compared with 16% for parent compound 12a.

11. Improvement in Aqueous Solubility and Oral Bioavailability by α-Substitution.

^aCLogP was estimated by us, using ChemDraw Ultra 20.0.

During the course of the development of an inhibitor of mutant isocitrate dehydrogenase 1, the introduction of a methyl group (13b and 13c) at the α-position of 13a resulted in 12.5- and 8.9-fold improvement of solubility, respectively (Table ). , This enhancement was likely due to the disruption of molecular planarity caused by the α-methyl group despite the increase in hydrophobicity. Additionally, the pyrimidyl analog 13d showed a 16.4-fold improvement in solubility compared with 13a, with an oral bioavailability (F) of 81% in rats. A subsequent study achieved an even more pronounced effect: the introduction of a methyl group at the α-position of 13e to produce 13f led to a remarkable 116-fold improvement in aqueous solubility, again despite increased hydrophobicity.

12. Improvement in Aqueous Solubility and Oral Bioavailability by α-Substitution.

compd	R	kinetic aqueous solubility (pH 7.4) (μM)	CLogP	rat F (%)
13a	H	0.64	1.0
13b	(R)-Me	8.0	1.4
13c	(S)-Me	5.7	1.4
13d		10.5	–0.46	81
13e	H	0.295	–0.32
13f	Me	34.5	–0.014

^aCLogP was estimated by us, using ChemDraw Ultra 20.0.

3.2. Case Studies on Decreasing the Flatness of Chemical Structures

Aromatic rings are ubiquitous in bioactive molecules, with the phenyl ring being a component of approximately 45% of marketed small-molecular drugs. The high planarity of the phenyl moiety promotes molecular stacking, contributing to reduced aqueous solubility and increased metabolic susceptibility of drug molecules. Introducing sp³ carbons into the benzene ring is an effective strategy to disrupt this planarity.

3.2.1. Saturation (Escape from Flatland)

A large-scale data analysis focusing on the ratio of sp³-hybridized carbons (Fsp³) indicated a positive correlation between Fsp³ and success in drug development. In another data set of more than 1000 compounds with simple molecular structures, Fsp³ correlated positively with solubility and negatively with melting point. Furthermore, a recent study suggested that increasing Fsp³ reduces promiscuity and CYP450 inhibition. Researchers at GSK analyzed a data set of 100,000 GSK compounds and proposed the Property Forecast Index (PFI), calculated as the sum of the chromatographic LogD _7.4 value and the aromatic ring count (Chrom LogD _7.4 + #Ar). A PFI value of less than 5 was predictive of a higher probability of achieving kinetic solubility (>200 μM).

As a concrete example of matched molecular pair analysis, vanilloid receptor-1 antagonist 14a (Table ) showed insufficient thermodynamic aqueous solubility (<1 μg/mL in PBS or 0.01 M HCl). To improve its aqueous solubility, a strategy involving partial saturation of the 4-(trifluoromethyl)­phenyl ring was employed to reduce structural planarity, disrupt π-π stacking, and disrupt crystal packing. The partially saturated analogue 14b exhibited improved thermodynamic solubility (13 μg/mL in 0.01 M HCl), being at least 13-fold more soluble than 14a. The lower melting point of 14b compared with 14a supports the hypothesis that disruption of planarity contributed to its improved solubility.

13. Improvement in Aqueous Solubility by Saturation of Aromatic Ring.

Compound 15a showed poor pharmacokinetics and low solubility, which precluded further development (Table ). The authors hypothesized that the removal of two aromatic rings, that is, the oxadiazole ring and the phenyl ring of the benzoic acid moiety, would enhance the three-dimensional morphology of the compound, the aim being to weaken the crystal packing of 15a and thereby improve the dissolution rate. Indeed, trans-cyclohexyl analog 15b showed 400-fold improved aqueous solubility, greater PAMPA permeability, and increased oral bioavailability in rats. In a phase 1 clinical trial, 15b was well absorbed in both normal healthy volunteers and patients, with both the AUC and Cmax exhibiting dose-proportionality following single oral doses in the range from 0.5 to 25 mg in the fed state.

14. Improvement in Thermodynamic Aqueous Solubility and Oral Bioavailability by Saturation/deletion of Aromatic Ring.

^aCLogP was estimated by us, using ChemDraw Ultra 20.0.

Fairhurst et al. reported that 2-formylpyridinyl ureas act as reversible covalent inhibitors of fibroblast growth factor receptor 4. Chemical modification involving substitution of the phenyl moiety in compound 16a with piperidine (16b) resulted in a lower melting point (133 °C) compared to that of 16a (175 °C). This modification also improved thermodynamic water solubility, with aqueous solubilities of <2.9 μM for 16a and 4.9 μM for 16b at pH 6.8 (Table ).

15. Improvement in Aqueous Solubility by Increase of Saturation.

3.2.2. Phenyl Ring Mimetics (Aufheben of Phenyl Spacer and Druglikeness)

Several phenyl bioisosteres containing sp³ carbons have been reported. When a benzene ring substituted at the para position is replaced, the distance between the substitution sites becomes crucial. The positional relationships and surface area of various phenyl bioisosteres are summarized in Table .

16. Structural Information of para-Substituted Phenyl Bioisosteres.

^aSurface area and volume were calculated using a model of a solvent-excluded molecular surface of a compound with hydrogen atoms substituted on both sides. C–C distance is the distance between the substitution sites.

Cubane, being approximately the same size as a phenyl group, has long been recognized as being of interest in the field of phenyl bioisosteres. The distance along the body diagonal of cubane (2.68 Å) closely matches that of benzene (2.77 Å) (Table ), despite slightly longer individual C–C bond lengths (1.573 Å (sp³) vs 1.397 Å (sp²)). A pioneering report on cubanes as bioisosteres of a para-phenyl group appeared in 2016. However, with regard to druglikeness, only the LogP values were used to predict the water solubility and membrane permeability of cubane analogs. The improvement of LogP values resulting from substitution of the phenyl group is shown in Table .

17. Effect of Replacement of a Phenyl Group with Cubane on Hydrophobicity.

In a study of bioisosteres of monosubstituted phenyl, Todd et al. synthesized compounds in which the phenyl group of the antimalarial lead compound 20a was substituted with cubane 20b, bicyclo[1.1.1]­pentane (BCP) 20c, and closo-1,2-carborane 20d (Table ). Both cubane 20b and closo-1,2-carborane (20d) exhibited higher melting points and lower aqueous solubility than 20a, suggesting that cubane- and carborane-substitutions do not always improve solubility. In addition, 20b and 20d showed high clearance and short half-lives in studies with human liver microsomes (HLMs) and mouse liver microsomes (MLMs). In the case of BCP derivative 20c, there was no significant change in lipophilicity or water solubility, and the melting point was higher than that of 20a.

18. Effect of the Bioisosteric Replacement of the Terminal Phenyl Ring.

Auberson et al. compared the water solubility of relatively simple model structures in which the benzene ring (21a, 22a) was replaced with BCP (21b, 22b), bicyclo[2.2.2]­octane (BCO) (21c, 22c), or cubane (21d, 22d) (Table ). The results indicate that BCP affords superior water solubility compared with BCO, cubane, and phenyl in these cases.

19. Improvement of Thermodynamic Aqueous Solubility by Substitution of a Phenyl Group with Phenyl Isosteres.

Stepan et al. developed a γ-secretase inhibitor 23b by replacing the para-substituted fluorophenyl ring of BMS-708,163 (23a) with a BCP motif. Compared with fluorophenyl analog 23a, BCP analog 23b showed 11.6-fold and 32.7-fold improvement in thermodynamic aqueous solubility at pH 6.5 and pH 7.4, respectively. According to our mechanistic analysis, the density of a single crystal of 23b (1.468 g/cm³) was lower than that of 23a (1.528 g/cm³) suggesting that “escape from flatland” at least partially contributed to the solubility improvement. Another mechanism contributing to the solubility improvement would be the decrease in LogD _7.4 (4.70 for 23a, 3.80 for 23b). Reduction of LogD would also reduce the clearance in human hepatocytes (CL_int,app), that is, the clearance of 23b was lower than that of 23a. BCP analogue 23b also showed a significant improvement in permeability (5.52 × 10^–6 cm/s for 23a, 19.3 × 10^–6 cm/s for 23b). Compound 23b showed excellent oral bioavailability in rats (100%), as well as sufficient brain partitioning (total brain/total plasma = 0.61) (Table ).

20. Improvement in Thermodynamic Aqueous Solubility and Permeability by Bioisosteric Replacement of the Benzene Ring with BCP.

^aRRCK (Ralph Russ canine kidney) cells with low transporter activity were isolated from MDCK (Madine–Darby canine kidney) cells.

The same group also reported BCP substitution of the benzene ring in imatinib (24a), a potent inhibitor of ABL1 kinase, affording 24c (Table ). BCP analogue 24c exhibited an over 80-fold improvement in aqueous solubility (2506 μM at pH 7.4) compared to 24a (Table ). There was a significant decrease in melting point (24a: 204 °C vs 24c: 195–198 °C), suggesting that a decrease in intermolecular interactions contributed at least partially to the solubility improvement, in addition to the decrease in lipophilicity (24a: LogD _7.4 = 2.45 vs 24c: LogD _7.4 = 1.51).

21. Improvement in Thermodynamic Aqueous Solubility by Bioisosteric Replacement of the Benzene Ring with BCP.

^aRRCK cells with low transporter activity were isolated from MDCK cells.

Hirst et al. successfully incorporated the BCP moiety into known lipoprotein-associated phospholipase A2 inhibitors as a bioisosteric phenyl substituent. The BCP-containing compound 25b exhibited a 9-fold increase in kinetic solubility (74 μM) compared to compound 25a (8 μM) (Table ). Furthermore, the thermodynamic solubility in FaSSIF was determined, with analogue 25b displaying an approximately 3-fold improvement (>1000 μg/mL versus 399 μg/mL for compound 25a). This increase in solubility was accompanied by greater lipophilicity, as indicated by the measured LogD _7.4, which increased from 6.3 to 7.0. Analog 25b showed significantly enhanced permeability of 705 nm/s compared to 230 nm/s for compound 25a. Additionally, low clearance was observed for 25b in a HLM assay (1.22 mL/min/g). The authors concluded that these data lend weight to the hypothesis that disrupting molecular planarity and reducing the aromatic ring count can increase solubility and improve the overall pharmacokinetic profile.

22. Improvement in Thermodynamic Aqueous Solubility and Permeability by Bioisosteric Replacement of the Benzene Ring with BCP.

Adsool et al. developed BCP-resveratrol by replacing the phenyl group of resveratrol (26a) with BCP to alleviate the pharmacokinetic issues associated with resveratrol itself (Table ). BCP-resveratrol 26b showed 33-fold higher thermodynamic solubility than 26a. Furthermore, 26b also showed higher C _max and AUC values than 26a, indicating a favorable in vivo PK profile.

23. Improvement in Thermodynamic Aqueous Solubility and Pharmacokinetics by Bioisosteric Replacement of the Benzene Ring of Resveratrol.

Attempts were also made to further enhance the physicochemical properties of 26b through modifications of the BCP structure. Mykhailiuk et al. synthesized difluoro-substituted BCP 27c. Compared to the compound with a benzene ring (27a), 27c exhibited a similar LogD but showed higher water solubility (Table ). However, 27c showed lower water solubility than BCP 27b.

24. Improvement in Aqueous Solubility by Substitution of Benzene Ring with Difluoro-Substituted BCP.

Ratni et al. propose the use of a bridged piperidine (BP) moiety as a phenyl bioisostere. The bond angle of BP is 170°, deviating slightly from the value of 180° seen in 1,4-phenyl, BCP, and BCO, but the distance between substituent positions is 2.9 Å, which is similar to that of 1,4-phenyl (2.8 Å) rather than BCP’s 1.9 Å (Figure ). To test this alternative scaffold, they compared the activity and physicochemical properties of BP, phenyl, BCP, and BCO derivatives of four sets of pharmaceutical compounds including a clinical candidate. , In all four case studies, BP analogs showed the highest aqueous solubility, while phenyl analogs showed the lowest solubility. LogD values of BP analogs were around 0.5 lower than those of the corresponding phenyl analogs. Thus, BP appears to be an excellent alternative to phenyl, markedly improving druglike properties such as solubility. The physicochemical properties of representative examples of phenyl isosteres, including BP, are shown in Table .

5.

Distance between substituent positions of phenyl, BCP, BCO, and BP.

25. Improvement in Aqueous Solubility by Substitution with Phenyl Isosteres, BCP, BCO, and BP.

^aLYSA (lyophilization solubility assay) assay (after evaporation of DMSO solution, the compounds were dissolved in phosphate buffer (pH 6.5) overnight).

Zhong et al. synthesized compounds by replacing a portion of the biphenyl structure of an HCV NS5A inhibitor with a cyclohexylphenyl or bicyclo­[2,2,2]­octylphenyl motif. Compounds such as cyclohexylphenyl analog 29b and BCO analog 29c exhibited favorable oral bioavailability in rats, and their liver/plasma concentration ratios were also satisfactory (Table ).

26. SAR on the Bridge of HCV NS5A Inhibitors.

^aThe solubility data were collected by using μSOL Explorer with UV detection at 254 nm. ^b. 5 mg/kg, po administration in rats.

There have also been reports on bioisosteres of meta- and ortho-disubstituted phenyl groups, such as oxabicyclo[2.1.1]­hexanes, bicyclo[3.1.1]­heptanes (BCHep), 3-azabicyclo[3.1.1]­heptanes, and [2.2.0]­bicyclohexanes. Table summarizes the bond angles and bond lengths of the bioisosteres of para- and meta-disubstituted phenyl and meta-phenyl structures along with BCP.

27. Structural Properties of Bioisosteres of meta-Disubstituted Phenyl Groups.

Mykhailiuk et al. developed oxabicyclo[2.1.1]­hexanes as analogs of BCP. Adding an oxygen atom to BCP (30c and 30d) results in a slightly different geometry but still provides better kinetic solubility than that of BCP analog 30b (Table ). The distance between the substituted positions was slightly longer than that of BCP but slightly shorter than that of meta-disubstituted benzene (Table ). The angle γ, representing the deviation between the exit vectors in oxabicyclo[2.1.1]­hexanes (152–154°), falls between the values for para-disubstituted phenyl (180°) and meta-disubstituted benzene (120°). This suggests that oxabicyclo[2.1.1]­hexanes are promising bioisosteres not only for para-substituted phenyl, but also for meta-substituted benzene, and represent a useful tool for improving physicochemical properties.

28. Oxabicyclo­[2.1.1]­hexanes as para-Phenyl and meta-Phenyl Isosteres.

Anderson and Uchiyama independently developed practical syntheses of disubstituted BCHep as meta-substituted benzene mimetics. The internal C–C distance between the substituted positions was slightly shorter (2.1 Å) than that of meta-disubstituted benzene (2.4 Å). The angle γ, representing the deviation between the exit vectors in BCHep (119°), is almost the same as that of the meta-disubstituted benzenes (120°). BCHep analogs of sonidegib (31b) and URB597 (32b) showed remarkably similar physicochemical properties including CLogP, tPSA, and aqueous solubility (Table ). Interestingly, the permeability of BCHep-substituted 31b in Caco-2 cells was improved. These results indicated that the BCHep scaffold is suitable as a meta-benzene isostere.

29. BCHep as a meta-Phenyl Isostere.

Mykhailiuk et al. reported 3-azabicyclo[3.1.1]­heptanes as meta-substituted pyridine isosteres (Table ). The internal C–C distance between the substituted positions (2.1 Å) and the angle γ (124–126°) representing the deviation between the exit vectors in 3-azabicyclo[3.1.1]­heptanes were similar to those of substituted pyridine (2.4 Å and 125°, respectively). Azabicyclo[3.1.1]­heptane analog 33b showed 12.6-fold improved kinetic solubility and a decrease of LogD compared to the pyridine analog rupatadine (33a).

30. Improvement in Aqueous Solubility by Substitution with meta-Pyridine Isostere 3-Azabicyclo[3.1.1]­heptane.

Fessard and Brown reported [2.2.0]­bicyclohexanes ([2]-ladderanes) as meta-substituted benzene isosteres. The internal C–C distance between the substituted positions was slightly longer (2.6 Å) than that of the meta-disubstituted benzene (2.4 Å). Based on matched molecular pair analysis of simple compounds (34a vs 34b and 35a vs 35b), the authors concluded that the introduction of [2.2.0]­bicyclohexane analogs does not significantly impact physicochemical properties, including LogP, aqueous solubility, and permeability (Table ).

31. [2.2.0]­Bicyclohexane as a meta-Phenyl Isostere.

^aLYSA assay (after evaporation of their DMSO solution, the compounds were dissolved in phosphate buffer (pH 6.5) for 16 h).

There are also reports of ortho-disubstituted phenyl bioisosteres. Myknailiuk et al. reported bicyclo­[2,1,1]­hexanes as bioisosteres of ortho-disubstituted benzenes consisting of sp³ carbons (Table ). Despite the difference in planarity (angle θ) between the bicyclo­[2,1,1]­hexane core (3D) and ortho-substituted phenyl (2D) (angle θ: bicyclo­[2,1,1]­hexanes = 44.9–78.0° vs ortho-substituted phenyl = 7.5–8.3°), they share similarities in other characteristics. The distance between the substituents (d) in the ortho-benzene of telmisartan (36a) is 3.10 Å, and the d value in the corresponding bicyclo­[2,1,1]­hexanes core 36b is estimated to be close to that value, 3.22 Å. They also found that the bicyclo­[2,1,1]­hexane-type model compound 37b had 1.2-fold higher water solubility while showing slightly higher LogD than the corresponding ortho-disubstituted phenyl compound 37a (Table ).

32. Difference of Angle and Inter-substituent Distance between ortho-Substituted Phenyl and Bicyclo­[2,1,1]­hexanes.

33. Solubility and Lipophilicity of the Bicyclo­[2,1,1]­hexane Analog.

Baran et al. reported 1,2-disubstituted BCP as ortho- or meta-substituted phenyl mimetics. According to their matched molecular pair analysis, the kinetic solubility of the BCP 31c, 38b, 39b–d, 40b, and 43b increased compared to the corresponding drugs, which are neutral at physiological pH, that is, sonidegib (31a) (15- to 20-fold), boscalid (38a) (24- to 34-fold), tolvaptan (39a) (about 1.6- to 5-fold), axitinib (40a) (over 67-fold), and telmisartan (43a) (over 16-fold) (Table ). The authors noted that not much increase in solubility was achieved for drugs that are charged at physiological pH (meclizine and lomitapide (42a)). Interestingly, neutral phthalylsulfathiazole mimetics (41b–c) did not show solubility improvement, probably due to the high solubility of the parent drug (41a). They also examined LogD, permeability (RRCK), and hepatocyte stability. Unfortunately, many of the mimetics did not show improved permeability compared to the corresponding parent drugs. On the other hand, sonidegib mimetic 31c showed over 40-fold higher permeability than sonidegib (31a). All BCP were separated in an enantiopure form by supercritical fluid chromatography, and the absolute stereochemistry of (−)-40 was confirmed to be S by single-crystal X-ray crystallography.

34. Physicochemical and Metabolic Profile of 1,2-Disubstituted BCP Analogs.

3.2.3. Bridged Heterocycles

There is increasing evidence that bridging basic heterocycles can lead to enhanced solubility due to not only reduced planarity but also increased basicity. Researchers at AstraZeneca reported that bridging morpholines and piperazines across the ring with one-carbon tethers reduces lipophilicity. For example, inhibitors of interleukin-1 receptor associated kinase 4 bearing bridged morpholines 44c and 44d exhibit lower LogD _7.4 than the parent morpholine 44a, whereas monomethyl analog 44b shows increased LogD (Table ). Bridged morpholine 44d was found to be more basic than 44a. Conformational analysis revealed that the bridges introduce a more rigid structure, which significantly reduces the total surface area of the molecules, thereby decreasing the lipophilicity. Additionally, the solvent accessibility of the morpholine oxygen atom is increased in the one-carbon-bridged analogs compared to both the monomethyl analogs and morpholine itself. The lower lipophilicity due to the one-carbon bridges can have multiple benefits, especially in cases of high susceptibility to metabolism or strong hERG inhibition, where these bridges may offer useful design solutions. Several matched molecular pairs (45–47) where one-carbon bridges resulted in increased pK _aH, reduced LogD, reduced clearance in rats, and weaker hERG inhibition have been reported (see Figure ).

35. Decrease in LogD by Bridging Morpholino Group.

6.

Decrease in LogD and increase in pK _aH by bridging piperazines.

Another study found that the addition of a one-carbon bridge to piperazine (48b and 48c) and replacement with spirocyclic derivatives (48d and 48e) decreased LogD and increased basicity compared to 48a (Table ). The bridges introduce a more rigid structure, leading to a reduced total surface area and decreased lipophilicity.

36. Decrease in LogD by Bridging Piperazine or Replacement with Spirocyclic Rings.

^aSolubility in phosphate buffer pH 7.4 from a solution in DMSO after 20–24 h.

In a separate report, the addition of a one-carbon bridge to piperazine (49a) led to a significant increase in aqueous solubility. The one-carbon-bridged analogue 49b showed an over 1460-fold increase in aqueous solubility compared to 49a, accompanied by a 60 °C decrease in melting point. This result suggests that disruption of planarity caused by the bridge at least partially contributes to the improvement in solubility. Interestingly, the tPSA of 49b remains the same as that of 49a, while the ClogP of 49b is lower. Other analogs 49c and 49d also exhibit lower melting points, likely due to disrupted planarity. Replacement with a spirocyclic analogue 49i resulted in the lowest melting point in this series, likely due to bending of the molecular structure (vide infra in Section ). Another spirocyclic analogue 49j showed over 480-fold improvement in aqueous solubility compared to 49a. This improvement is likely attributed to a combination of factors, including a lower melting point caused by molecular bending and disruption of planarity as well as a decrease of LogP due to the presence of a secondary amino group. Compound 49j (2 mg/kg) showed 27.4% oral bioavailability in mice, with similar AUC and Cmax values to 49a (25 mg/kg), assuming linear pharmacokinetics (Table ).

37. Improvement in Aqueous Solubility by Bridging Piperazine or Replacement with Spirocycles.

^aMice were orally dosed with 25 mg/kg.

^bMice were orally dosed with 2 mg/kg.

3.3. Case Studies on Decreasing Intermolecular HBs (Aufheben of Hydrophobicity and Solubility)

While Ro5 predicts that the limited number of HBDs and HBAs is a good indicator of oral bioavailability, there are notable examples where druglikeness was improved by both a decrease in intermolecular HBs (Section ) and an increase in intramolecular HBs (Section ). This suggests that modern medicinal chemistry can benefit from more precise molecular design strategies that distinguish between intermolecular and intramolecular HBs.

Thalidomide (50) has low aqueous solubility due to the high melting point, probably caused by strong intermolecular HB in the solid state. Removal of the HBD by the introduction of a methyl group on the imide (50b) resulted in lower melting point and lower crystal density, leading to increasing aqueous solubility, even though the hydrophobicity of methyl analog 50b is higher (Table ).

38. Improvement in Aqueous Solubility by Decreasing Intermolecular HB.

compd	R	thermodynamic aqueous solubility (pH 6.4) (μg mL–1)	CLogP	melting point (°C)	crystal density (g mL–1)
50a	H	52	0.53	275	1.48
50b	Me	276	1.2	159	1.43

^aCLogP was estimated by us, using ChemDraw Ultra 20.0.

Upon oral administration, the cannabinoid receptor antagonist 51a was inactive in vivo, whereas methyl analog 51b was active in vivo (Table ). Both 51a and 51b were poorly soluble in water (<1 mg/mL at pH 7). However, 51a was moderately soluble in ethanol at reflux, while 51b dissolved readily under the same condition. Single-crystal X-ray structural analyses and melting point measurements revealed that 51b showed a less dense crystal packing than 51a. In the case of 51a, the two NH hydrogens form intramolecular HBs with both the dihydropyrazole moiety and the sulfonyl group. Additionally, 51a forms intermolecular HBs between the SO₂ oxygen atom and the amidine hydrogen atom of a neighboring molecule of 51a. Introducing a methyl group at the amidine position (51b) disrupted these intermolecular HBs, leading to a reduced crystal density and melting point. Consequently, the higher aqueous solubility and dissolution rate of 51b likely contributed to its improved in vivo oral activity by enhancing dissolution in the gastrointestinal tract.

39. Improvement in Solubility by Decreasing Intermolecular HB.

compd	R	kinetic solubility	CLogP	melting point (°C)	crystal density (g cm–3)
51a	H	moderate	4.3	235	1.535
51b	Me	easy	4.8	170	1.481

^aSolubility in EtOH at reflux intramolecular HBs are shown as red dotted lines. Intermolecular HBs are indicated by arrows.

Single-crystal structural analysis of androgen receptor antagonist 52a revealed an intermolecular HB between the amide carbonyl group and the hydroxyl group of a neighboring molecule of 52a, resulting in tight crystal packing and low aqueous solubility (Figure ). To improve the solubility, the amide carbonyl group in the hydantoin moiety was removed to disrupt the intermolecular HB. Removal of this amide carbonyl (52b) resulted in a 13-fold increase of solubility over 52a. The melting point of 52b was also lower than that of 52a, despite the higher CLogP. These results suggest that the reduction in crystal packing density due to the removal of the intermolecular HBAs was a key factor in the enhanced solubility of 52b, despite the increased hydrophobicity.

7.

Improvement in solubility by decreasing intermolecular HB.

Cyclic pyrrolidinyl analog 53c exhibited an approximately 3-fold improvement in aqueous solubility over the linear analog 53a (Table ). This improvement may be attributed to a decrease in intermolecular HB caused by a reduction in the number of HBAs, which is also consistent with the lower melting point of 53c relative to that of 53a. Another possible explanation is that the relatively bulky pyrrolidine moiety in 53c disrupts the coplanarity between the amide group and the phenyl ring, further contributing to the solubility enhancement. In contrast, azetidinyl analogue 53b showed lower aqueous solubility, despite having a lower CLogP, because of its higher melting point compared to 53c.

40. Improvement in Thermodynamic Aqueous Solubility by Decreasing Intermolecular HB.

3.4. Case Studies on Increasing Intramolecular HBs (Aufheben of HB and Permeability)

Although Section details several concrete examples of decreasing intermolecular HBs, aligning with the principles of Ro5, it is also important to note that an increase in intramolecular HBs improved both the solubility and permeability.

Introduction of a nitrogen atom (54b) at the ortho position of the 2-naphthamide analog of neurokinin receptor antagonist 54a resulted in the formation of an intramolecular HB (Table ). Isoquinoline analog 54b showed improved aqueous solubility and permeability. Furthermore, 54b exhibited a lower CLogP than that of 54a, suggesting that the reduction in hydrophobicity also contributed to the enhancement of aqueous solubility.

41. Improvement in Aqueous Solubility and Permeability by Increasing Intramolecular HB.

compd	X	kinetic aqueous solubility (pH 6.4) (μg mL–1)	CLogP	PAMPA (10–6 cm s–1)	Caco-2 (10–6 cm s–1)
54a	CH	1.57	4.28	5.96	14.65
54b	N	2.80	3.14	15.5	25.20

Diastereomers 55a and 55b illustrate the remarkable impact of stereochemistry on physicochemical and biological properties (Table ). cis-C8,C9 diastereomer 55b exhibited 87-fold higher aqueous solubility than the trans-C8,C9 diastereomer 55a. Mechanistic studies revealed that cis- 55b had a LogD value of 3.9, which is 0.6 units lower than that of trans- 55a. The most stable neutral conformations of amines 55a and 55b are both rigid due to a strong intramolecular HB between the amide N–H and the tertiary amine (Figure ). However, differences in strain energy were observed: neutral cis- 55b was 3.1 kcal/mol higher in free energy than trans- 55a, and its cationic form was 1.2 kcal/mol higher, resulting in a total energy difference of 1.9 kcal/mol. Further investigations using ¹H NMR spectroscopy supported these findings. In line with the increase in its LogD value, 55a showed enhanced permeability in Caco-2 cells, and cis- 55b demonstrated a significant increase in permeability in cells with a pH of 7.4 on the apical side compared to pH 6.5, whereas trans- 55a showed no pH-dependent permeability changes. The results underscore the importance of preparing and evaluating pure stereoisomers in drug discovery or chemical probe projects, as stereochemistry can have profound effects on physicochemical, pharmacokinetic, and pharmacodynamic properties.

42. Difference of Aqueous Solubility between Diastereomers.

compd	R 1	R 2	kinetic aqueous solubility (pH 7.4) (μM)	LogD 7.4	pK aH (N34, pyridine)	pK aH (N15, 3′-amine)	Caco-2 P app pH 6.5/7.4 (10–6 cm s–1)	Caco-2 P app pH 7.4/7.4 (10–6 cm s–1)
55a	H	Me	1	4.5	2.76	6.08	63.8	63.9
55b	Me	H	87	3.9	2.97	7.16	21.8	27.5

^aDetermined at pH 7.4 in PBS containing 1% DMSO.

8.

(a) The most stable conformations of the neutral forms of 55a and 55b. (b) Newman projections looking down the C10–C9 bond (left) and the C9–C8 bond (right) with gauche interactions highlighted with red arrows. Their stable conformations shown in panel (a) were generated using maestro (Schrodinger), and the images were generated by UCSF Chimera software.

3.5. Removal of Head-to-Head Interactions

Disruption of the intermolecular interaction between methylsulfonyl groups has been shown to improve solubility. For example, 56a exhibited very low solubility (0.03 μM) and a high melting point (201–202 °C), despite lacking HBDs (Table ). Single X-ray crystal structure analysis revealed that 56a adopts a flat conformation, with efficient molecular stacking facilitated by strong polar interactions between sulfonyl groups and methyl group hydrogens (Figure ). These head-to-head interactions align the molecules, resulting in a tight crystal lattice.

43. Improvement in Aqueous Solubility by Decreasing Intermolecular Interaction of the Sulfonyl Group.

compd	aqueous solubility (pH7.4) (μM)	LogD 7.4	melting point (°C)	crystal density (g cm–3)
56a	0.03	3.2	201–202	1.361
56b	23	3.4	147–148	1.277

^aShaken for 24 h.

9.

Head-to-head interaction of sulfonyl groups.

A search of the Cambridge Crystallographic Database indicated that 18% of methyl sulfone moieties are associated with head-to-head molecular interactions; another 18% are involved in ladder-like interactions, and 23% are implicated in both. In line with this analysis, replacing the (methylsulfonyl)­phenyl group in 56a with a pyridyl group (56b) resulted in a 760-fold improvement in solubility, accompanied by a slight increase in LogD _7.4. Single-crystal X-ray crystal analysis of 56b confirmed the absence of head-to-head interactions and the network of polar interactions associated with the methyl sulfone group. This led to a reduced crystal density, a lower melting point, and improved solubility.

Compound 57a exhibited low solubility in both the Japanese Pharmacopoeia first fluid (JP1, pH 1.2) and FeSSIF (Table ). Substituting the methylsulfonyl group in 57a with a dimethylcarbamoyl group (57b) improved the solubility by 24-fold in JP1 and 4-fold in FeSSIF, despite an increase in CLogP.

44. Improvement in Aqueous Solubility by Decreasing Intermolecular Interaction of Sulfonyl Group.

^aJapanese Pharmacopoeia first fluid for dissolution test adjusted to pH 1.2.

3.6. Bending Molecular Structure by Changing the Position of Substituents

Sections and discussed strategies to improve druglikeness by modulating molecular planarity, which refers to a molecule’s flatness or thickness (akin to the dimension perpendicular to the plane of depiction). In contrast, this section focuses on molecular linearity or bending, which describes a different axis of the molecular shape. Bending characterizes a molecule’s deviation from a straight line (i.e., its linearity or width), distinct from flatness. There’s increasing evidence that bending molecular structures is a strategy to improve druglikeness. The melting points of C18 cis-fatty acids decrease as the number of cis-double bonds increases. This is attributed to the introduction of bent molecular structures that disrupt crystal packing, which leads to lower melting points. Therefore, modifying linear molecular structures to create bent conformations is expected to be a promising strategy to enhance solubility by reducing intermolecular interactions.

For example, repositioning the tetrahydropyrimidylamino group from the 4-position of the piperidine ring in integrin antagonist 7a to the 3-position (7i) led to a substantial increase in aqueous solubility (at least 35-fold) compared to 7a (Table ). Notably, this improvement was achieved without alteration of the molecular weight. Compound 7i also exhibited greater hydrophobicity and a lower melting point compared to 7a, highlighting the role of bent molecular shapes in improving solubility (Figure ).

45. Improvement in Aqueous Solubility by Bending the Molecular Structure.

compd	thermodynamic water solubility (mg mL–1)	CLogP	HLPC retention time (min)	melting point (°C)
7a	<0.1	1.1	8.25	252–254
7i	3.5	1.1	12.2	181–184

^aReversed-phased column.

10.

The overlay of 7a (gray) and 7i (cyan). Their stable conformations were generated using maestro (Schrodinger), and the images were generated by UCSF Chimera software.

Poor solubility of the retinoic acid receptor agonist 58a was attributed to its high lipophilicity and linear chemical structure (Table ), and bent analogs (58b–58d) showed dramatically increased solubility. The meta-substituted analog 58b showed 22-fold higher solubility in a phosphate buffer/EtOH (7:3) mixture compared to 58a and was also soluble (0.42 μg/mL) in a mixture of phosphate buffer and EtOH (9:1). The ortho-substituted analog 58c showed 240-fold higher solubility and was also soluble in phosphate buffer alone (4.0 μg/mL). The most pronounced improvement was observed for 58d, with an 890-fold increase in solubility compared to 58a. As for the mechanism, the order of lower melting points (58d < 58c < 58b < 58a) was the same as the order of higher solubility in a mixture of phosphate buffer and EtOH (7:3) (58d > 58c > 58b > 58a). As regards hydrophobicity, the LogP values of 58b and 58d were almost the same as that of 58a. These results indicate that the increased aqueous solubility of 58b and 58d is mainly due to loosening of the crystal structures due to bent molecular shapes.

46. Improvement in Thermodynamic Aqueous Solubility by Bending the Molecular Structure.

		thermodynamic aqueous solubility (pH 7.4) (μg mL–1)
compd	position	7:3	9:1	10:0	LogP	melting point (°C)	λmax (nm)
58a	para	8.4	<0.1	<0.1	4.8	151	280
58b	meta	190	0.42	<0.1	4.8	130.5–132.5	272
58c	ortho	2000	30	4.0	4.5	100.5	256
58d		7500	2.7	<0.1	4.7	80.9–81.1	<220

^aThermodynamic aqueous solubility in a mixture of phosphate buffer (pH 7.4) and EtOH.

Bent analogs of 2-nitroimidazopyrazinone, such as meta-phenylpyridines 59c and 59d, also showed significantly improved solubility compared to their para-phenylpyridine counterparts 59a and 59b (Table ).

47. Improvement in Aqueous Solubility by Bending the Molecular Structure.

compd	meta or para	R	kinetic aqueous solubility (pH 1) (μM)	ALogP	HLPC retention time (min)
59a	para	OCF3	<1	3.9	2.94
59b	para	Me	7.8	2.3	2.69
59c	meta	OCF3	16	3.5	2.87
59d	meta	Me	27	1.8	2.61

^aA solution of the compound in DMSO was aliquoted into 0.1 M HCl (pH 1) and shaken for 24 h at room temperature.

^bReversed-phase column.

Substitution of a piperazine ring with a homopiperazine ring is another effective strategy to improve solubility. Compound 60a exhibited limited aqueous solubility (44 μM) (Table ), but replacing its N-methyl piperazine group with N-methyl homopiperazine (60b) resulted in a 23-fold increase in solubility (Table ). This improvement was attributed to increased basicity and reduced LogD of 60b, as well as a lower melting point (205–216 °C for 60b vs 240–258 °C for 60a), suggesting that bending the molecular structure resulted in reduced intermolecular interaction, which in turn at least partially contributed to the improved solubility.

48. Improvement in Aqueous Solubility by Bending the Molecular Structure.

compd	n	kinetic aqueous solubility (pH 7.4) (μM)	LogD	pK aH	melting point (°C)
60a	1	44	3.3	7.4	240–258
60b	2	990	2.6	8.3	205–216

^aSolubility in phosphate buffer pH 7.4 from a solution in DMSO after 20–24 h.

Substitution of the benzene ring of 61a with pyrazole rings (61b and 61e) led to 3-fold and 12-fold improvements in solubility, respectively, accompanied by reductions in CLogP and melting point (Table ). Furthermore, substituting the 3-position methyl group of quinolone in 61b with an ethyl group (61d) decreased the melting point and increased solubility, despite a concomitant increase in CLogP. This result was likely due to disruption of the planarity and increased entropy.

49. Improvement in Aqueous Solubility by Bending the Molecular Structure.

Azobenzenes are a group of photoswitchable molecular machines that can exist in either trans or cis form. trans-Azobenzenes have planar conformations that are thermodynamically more stable and can be generated by visible light irradiation or spontaneously by thermal isomerization. On the other hand, irradiation of azobenzenes with UV light generates the cis isomers. cis-Azobenzene adopts a bent conformation with its phenyl rings twisted about 55° out of the plane of the azo group. Therefore, we hypothesized that cis-azobenzenes would possess better aqueous solubilization than the corresponding trans isomers as a result of weaker intermolecular interactions. Indeed, the aqueous solubilization of azobenzene 62a could be controlled reversibly by irradiation with UV and visible light (Figure ), and it varied depending on the UV irradiation wavelength and intensity. The solubilization of compound 62b in phosphate buffer was increased by up to 20-fold by exposure to UV irradiation, compared to that without irradiation.

11.

Improvement in aqueous solubilization of azobenzene 62a by cis-isomerization.

3.7. Disruption of Molecular Symmetry

For simple compounds, higher molecular symmetry (quantified by the symmetry number, σ) is generally associated with higher melting point, , though the relationship between molecular symmetry and physicochemical properties in more complex pharmaceutical compounds remains underexplored. The estrogen receptor antagonist cyclofenil (63a), for example, exhibits poor aqueous solubility (<1 μg/mL in 0.067 M phosphate buffer) due to its symmetric molecular structure (σ = 2; point group: C₂v, considering conformational isomerization of the cyclohexyl group) (Table ). To improve the solubility, the molecular symmetry was disrupted by introducing alkyl groups, yielding desymmetric analogs 63c, 63d, and 63e (point group: Cs) and (R)-63f (point group: C₁). These modifications significantly enhanced the aqueous solubility, despite the associated increase in hydrophobicity. For example, 63c showed a 17.3-fold improvement in solubility over its symmetric isomer 63b, and (R)-63f was 7.7-fold more soluble than symmetric 63g. These increases in solubility were correlated with reductions in both the melting point and crystal density, suggesting that disruption of intermolecular interactions contributed to the observed improvement. Additionally, (R)-63f possesses a chiral center, further breaking molecular symmetry. Interestingly, the presence of chiral centers has been associated with a higher success rate in progressing from discovery to clinical testing and eventual drug approval. This study underscores the importance of incorporating chirality into molecular design to achieve better druglike properties, including enhanced solubility and membrane permeability.

50. Improvement of Thermodynamic Aqueous Solubility by Disruption of Molecular Symmetry.

compd	R 1	R 2	R 3	R 4	R 5	R 6	thermodynamic aqueous solubility (pH 6.8) (μg mL–1)	LogP	melting point (°C)	crystal density (g cm–3)	dihedral angle (°)
63a	Me	Me					7.76	4.91	137.5	1.268	55.3
63b	Et	Et					0.622	6.02	139.0
63c	Me	n-Pr					10.8	6.00	69.0	1.236
63d			Me	H	H	H	10.3	5.26	114.0		54.4
63e			H	Me	H	H	23.6	5.12	99.9		66.1
(R)-63f			H	H	Me	H	27.8	5.34	92.0	1.227
63g			H	H	H	Me	3.61	5.41	137.0	1.243

^aSolubility in a mixture of phosphate buffer (pH 6.8) and EtOH (6:4).

^bThe most stable forms were estimated with Spartan’18.

3.8. Introduction of an Out-of-Plane Substituent at the meta Position of a Phenyl Group

An analysis of the melting points of substituted benzene regioisomers (161 sets) revealed no significant difference in melting points between ortho- and meta-isomers when general substituents were considered. However, ortho-isomers bearing flat substituents tended to exhibit the lowest melting points, as discussed in Section . This indicates that the rule that meta-substitution leads to improved solubility should exist. To explore this idea, we defined a plane substituent as the one possessing an extension in two dimensions, collected and analyzed the dihedral angles of substituents bearing an sp² atom from the Cambridge Structural Database, and divided them into plane and out-of-plane substituents (plane substituents: Ph, CO₂H, Ac, NO₂, CONH₂, and NHAc; out-of-plane substituent groups: Me, Et, t-Bu, F, Cl, Br, CF₃, CN, NH₂, OH, OMe, SO₂NH₂, and SO₂Me). Among compounds with out-of-plane substituents in the above 161 sets, meta-isomers were observed to have the lowest melting points among the regioisomers. We then validated this trend in pharmaceutical compounds with more complex structures using lead compounds with out-of-plane substituents such as 64a, 65a, and the androgen receptor antagonist bicalutamide eutomer 66i. Across six of eight sets of pharmaceutical compounds bearing disubstituted benzene rings with out-of-plane substituents (Table ), the meta-isomers showed the lowest melting points and the highest thermodynamic aqueous solubility among the isomers. These findings highlight that meta-substitution with out-of-plane groups is a promising strategy to interfere with crystal packing, thereby improving the aqueous solubility of pharmaceutical compounds.

51. Improvement in Thermodynamic Aqueous Solubility by Introduction of a Out-of-plane Substituent.

compd	R	thermodynamic aqueous solubility (pH 7.4) (μg mL–1)	LogP	melting point (°C)
64a	o-Me	5.2	4.6	142.6
64b	m-Me	21.4	4.7	112.0
64c	p-Me	19.1	4.8	121.3
65a	o-OMe	0.0255	4.3	132.1
65b	m-OMe	0.275	4.1	90.9
65c	p-OMe	0.0473	4.1	142.1
66a	o-Me	12.6	2.3	139
66b	m-Me	18.8	2.2	125
66c	p-Me	11.8	2.2	161
66d	o-Et	19.3	2.8	117
66e	m-Et	49.2	2.7	48
66f	p-Et	7.9	2.7	139
66g	o-F	27.2	1.8	157
66h	m-F	4.1	2.0	194
66i	p-F	14.6	1.9	184
66j	o-Cl	13.6	2.1	145
66k	m-Cl	29.3	2.5	113
66l	p-Cl	5.4	2.4	173
66m	o-Br	8.4	2.3	187
66n	m-Br	56.3	2.6	131
66o	p-Br	11.6	2.5	163
66p	o-OMe	69.9	1.8	145
66q	m-OMe	30.6	2.1	125
66r	p-OMe	14.7	2.0	155
66s	H	7.0	1.9	181

^aThermodynamic aqueous solubility in a mixture of phosphate buffer (pH 7.4) and EtOH (7:3).

^bThermodynamic aqueous solubility in phosphate buffer (pH 7.4).

4. Improvement in Aqueous Solubility and Permeability of bRo5 Molecules (Aufheben of MW and Druglikeness)

Recent advancements in biology and medical sciences have led to the identification of novel drug targets for which traditional drug discovery approaches, such as screening for enzyme inhibitors, are ineffective, and new strategies have been developed to tackle these challenging targets. However, the designed molecules, including macrocycles and bifunctional molecules such as PROTACs, often lie in the bRo5 chemical space due to their high MW and structural complexity. This shift has underscored the importance of studying factors influencing the druglikeness of bRo5 molecules, particularly their aqueous solubility and membrane permeability. This section summarizes key findings on the solubility and permeability of bRo5 molecules and discusses guidelines for developing druglike bRo5 compounds.

4.1. Solubility and Permeability of bRo5 Molecules

According to the solute dissolution model shown in Figure , water forms a cavity to accommodate solute dispersion during the second step of the process. The formation of this cavity incurs a free energy penalty primarily due to the entropy loss of water. This penalty is particularly pronounced for larger solutes (i.e., high-MW molecules), making cavity formation less favorable compared to that of smaller solutes. Consequently, as the MW of a compound increases, its solubility tends to decrease. Similarly, membrane permeability inversely correlates with MW. These characteristics have been validated through multiple analyses of physicochemical parameters using proprietary pharmaceutical company data and academic databases. − However, the MW is not the sole determinant of solubility and permeability. The solute dissolution model encompasses multiple steps beyond cavity formation, making MW only one of several contributing factors, and thus, the solubility of high-MW molecules can be improved by leveraging other parameters. For example, Gleeson’s analysis of solubility assay data for 44,584 molecules at GlaxoSmithKline demonstrated that ionization state significantly influences solubility. Additionally, Tolls et al. investigated the relationship between molecular size and aqueous solubility using C10- to C19-alkanes, highlighting the impact of molecular conformation. Their free energy simulations of solvation revealed that folded conformations are energetically more favorable than all-trans conformations as folding reduces cavity size and minimizes the associated free energy penalty. Thus, molecular designs that modulate ionization states and promote favorable folding can enhance the solubility of bRo5 drugs. Regarding permeability, Gleeson et al. observed a complex relationship between ionization state and permeability, mediated by lipophilicity. While neutral and basic molecules exhibit similar permeability, acidic and zwitterionic molecules are generally less permeable. Interestingly, zwitterionic molecules with CLogP < 3 are less permeable than acidic molecules, whereas highly lipophilic zwitterionic molecules (CLogP > 5) are more permeable. These findings suggest that factors other than MW, such as lipophilicity and ionization state, also play crucial roles in determining the permeability of high-MW molecules.

Although hydrophilicity and ionization state also affect the solubility and permeability of small molecules, molecular design for optimal solubility/permeability in bRo5 molecules differs significantly due to their more three-dimensional structures. In the following sections, we summarize key studies and discuss parameters that should be considered in designing bRo5 molecules with favorable druglikeness.

4.2. Lessons from Orally Available bRo5 Drugs

Over the past decade, increasing numbers of orally available bRo5 drugs, encompassing both naturally occurring molecules and synthetic compounds, have been reported. Analyses of these successful cases from various perspectives have provided valuable guidelines for designing bRo5 molecules with favorable druglikeness. This section highlights the lessons learned so far.

4.2.1. 3D Polarity: a Determinant of Solubility of bRo5 Molecules

In the hydration step of the solute dissolution model (Figure ), polarity emerges as a critical factor for improving solubility. Whitty et al. analyzed 20 clinically approved, orally available macrocycles and proposed that achieving good solubility of high-MW bRo5 drugs requires tPSA of more than 0.2 Ų per unit of MW. However, while predicting the solubility of bRo5 compounds might appear straightforward based on polarity parameters such as tPSA, actual polarity depends on the compound’s conformation in water. Kihlberg and colleagues demonstrated that the difference between tPSA and maximum molecular 3D PSA increases with MW. Notably, their analyses showed that the coefficient of determination (r ²) for LogS vs 3D PSA was higher than that for LogS vs tPSA, underscoring the importance of incorporating conformational data when assessing solubility.

Jerhaoui et al. developed orally available Mcl-1 protein–protein interaction (PPI) inhibitors with macrocyclic structures and MW exceeding 650 (Table ). Beginning with the poorly soluble Mcl-1 PPI inhibitor 67 (AMG176), the authors optimized the compound to obtain 71a, which exhibited significantly improved solubility. Interestingly, during this optimization process, a single stereochemical change was found to influence the solubility. For example, 70b showed markedly lower solubility and higher lipophilicity than its epimer 70a. Similar stereochemistry-dependent effects were observed in comparisons between 71a and 71b and between 71c and 71d. Molecular dynamics (MD) simulations of 71c and 71d revealed that stereochemical changes altered the conformation, reducing the polar fraction of the total solvent-accessible surface area in 71d compared to 71c. These findings highlight the role of specific 3D conformations in water in determining the aqueous solubility.

52. Structures, Activities, and Properties of the Mcl-1 Inhibitors.

4.2.2. Chameleonicity: a Property Explaining Why bRo5 Drugs Can Be Both Soluble and Permeable

The cellular membrane is a hydrophobic environment that favors lipophilic molecules. This characteristic was highlighted in a comprehensive permeability analysis of over 200 macrocycles conducted by Kihlberg et al., which established a strong positive correlation between lipophilicity and cell permeability. Conversely, parameters related to polarity, such as the number of HBDs/HBAs and charge, were found to restrict the permeability.

Naturally occurring cyclic peptides and macrocycles with diverse scaffolds often exhibit good passive permeability and solubility despite their large MW, typically violating Ro5. Cyclosporine A (CsA), for instance, is a clinically used immunosuppressive drug that is orally available. Extensive physicochemical and computational studies on CsA and CsA-inspired synthetic cyclic peptides have provided insights into the reasons for their membrane permeability. A key property underlying their behavior is conformational flexibility, which enables these compounds to extend their structures in aqueous environments to expose polar functional groups (HBDs and HBAs) for hydration while folding to shield these groups in the hydrophobic cellular membrane. − This behavior, termed “molecular chameleonicity”, arises from environment-dependent intramolecular HB switching. In lipophilic environments, such as the lipid bilayer, intramolecular HBs reduce the number of exposed HBDs and HBAs. This widely accepted mechanism has been shown to apply not only to cyclic peptides but also to bRo5 synthetic clinical candidates, macrocycles, antiviral drugs, and PROTACs.

Several research groups have explored the prediction and evaluation of bRo5 molecular permeability and druglikeness through chameleonicity theory. For instance, Ermondi et al. employed chameleonicity as a guideline for designing bRo5 drugs based on physicochemical properties. They investigated model molecules by comparing experimental and computational predictions of chameleonicity. Experimentally, changes in capacity factors in a PLRP-S column system eluted with varying organic phase ratios were used to assess chameleonic behavior, reflecting changes in lipophilicity due to environment-dependent conformational shifts. Computationally, differences in conformational sampling between water and CHCl₃ were analyzed, and strong agreement was observed between experimental and predicted results. Price et al. introduced a high-throughput chameleonicity descriptor, the experimental PSA (EPSA)-to-tPSA ratio (ETR). EPSA, determined by a supercritical fluid chromatography (SFC)-based method, leverages a low dielectric constant mobile phase that promotes intramolecular HB formation. Thus, the ETR (EPSA per tPSA) quantifies a compound’s ability to reduce its effective polarity due to external influences. Their analysis of a data set using ETR established simple MW and ETR thresholds for good permeability: ETR ≤0.8 for MW 500–800 and ≤0.6 for MW 800–1000. Lokey et al. proposed lipophilic permeability efficiency (LPE) as a robust metric for quantifying membrane permeability. For compounds with AlogP values below 4, they observed a strong correlation between the experimental decadiene–water distribution coefficient (LogD _dec/w) and PAMPA, even distinguishing stereoisomers. They further established a linear relationship between AlogP, which is known to correlate with aqueous solubility, and LogD _dec/w. Based on these findings, the authors derived the equation: LPE = 1.06 × AlogP – 5.47. A higher LPE value indicates a greater membrane permeability.

Interestingly, structural modifications of drugs often result in relatively consistent changes in LPE. For example, methylation of a solvent-exposed amide NH increased the LPE by approximately +1.0. This allows for the estimation of changes in physicochemical properties based on the ΔLPE values.

Theoretical studies on the membrane permeability of bRo5 molecules have increasingly attracted the attention of computational chemists and chemoinformaticians. Numerous studies have simulated solvent-dependent conformational flexibility of bRo5 molecules, particularly cyclic peptides, and computational predictions of druglikeness for these compounds have been explored. − ,

4.2.3. N-Methylated Amide Bond and Ester Bond: Strategies for Improving ADME of bRo5 Molecules

N-methylated amide bonds and ester bonds are frequently found in naturally occurring cyclic peptides and are commonly employed to enhance the permeability of not only small molecules (see Section ), but also cyclic peptide-based bRo5 drugs. , It has been shown that the number of N-methylations does not correlate with the permeability of cyclic peptides. However, N-methylation at the peptide backbone often induces conformational changes, as N-alkylated amide bonds, such as those in proline-containing peptides and peptoids, are known to promote cis-trans isomerization. , This conformational impact of N-methylation has been demonstrated to enhance both the permeability and chameleonicity of cyclic peptides. ,,,

The Lokey group investigated the effects of N-methylation and β-branching using the scaffold of sanguinamide A (72a, Table ) as a model, which features two amide NH groups involved in intramolecular HB. Comparisons of the solubility and permeability of sanguinamide A (72a) and its four N-methylated derivatives revealed position-dependent effects (Table ). Specifically, methylation at the 3-position (73c) significantly enhanced permeability, while the solubility increased only 2-fold. In contrast, methylation at the 2-position (73a), where the NH group is involved in intramolecular HB, markedly improved solubility but reduced permeability. This outcome is consistent with the disruption of intramolecular HB by N-methylation, which alters the conformation, increases the number of solvent-exposed amide bonds, and disrupts chameleonicity driven by intramolecular HB. The substantial solubility improvement arising from 2-position methylation also suggests that the closed form of the molecule predominates in aqueous environments, raising questions about the extent to which chameleonicity influences druglikeness. In contrast, N-methylation at the 3-position, which does not involve intramolecular HB, may reduce the 3D PSA without inducing significant conformational changes.

53. Impact of Sanguinamide A N-methylation on the Physicochemical Properties and Druglikeness.

compd	R 1	R 2	R 3	R 4	thermodynamic aqueous solubility (μM)	ALogP	HLPC retention time (min)	PAMPA (10–6 cm s–1)	Caco-2 (10–6 cm s–1)	LHSA (Å)	-ΔGH20 (kcal mol–1)
72a	H	H	H	Me	107	2.86	2.77	5.3	1.3	221	24.8
73a	Me	H	H	Me	801	3.07	1.48	0.5	0.2	228	24.3
73b	H	Me	H	Me		3.07	1.79	1.3	0.4
73c	H	H	Me	Me	172	3.07	3.03	11.0	7.4	340	23.6
73d	H	H	Me	H	296	2.61	2.24	9.1	6.8

^aThermodynamic solubility in PBS.

^bLargest hydrophobic surface area.

^cDesolvation energy.

Similarly, the Fairlie group demonstrated that N-methylation of sanguinamide A induces position-dependent changes in conformation and the largest hydrophobic surface area (LHSA), with permeability positively correlating with LHSA (Table ). They proposed that a larger hydrophobic surface is associated with a lower desolvation energy (Figure a) compared to that of a polar surface (Figure b), resulting in higher passive membrane permeability.

12.

Models of water networks. (a) Methylation of cyclic peptide increases the hydrophobic surface (gray) having minimal interactions with water, lower desolvation energy, and higher passive membrane permeability. (b) Hydrophobic surface (gray) punctuated by polar atoms (red and blue) that interact with water, raising the desolvation energy and reducing passive membrane permeability.

According to Lokey et al., N-methylation at positions involved in intramolecular HB should be avoided to enhance the permeability. Rational methods for identifying solvent-exposed amide NH groups are therefore needed. To address this, Craik et al. investigated amide NMR chemical shift temperature coefficients (ΔδNH/ΔT). They found a correlation between ΔδNH/ΔT and hydrogen–deuterium (H-D) exchange rates in model cyclic hexapeptides. Backbone amide NHs with ΔδNH/ΔT values below −4.6 ppb/K typically exhibited rapid H-D exchange rates, indicating their lack of involvement in intramolecular HB. Based on this finding, the authors performed an N-methylation scan of backbone amide NHs in a model cyclic hexapeptide (Table ). N-methylation at NHs with ΔδNH/ΔT values below −4.6 ppb/K (e.g., 74h) significantly improved the Caco-2 permeability, demonstrating the utility of this method for predicting solvent-exposed NHs. Using this approach, they identified an orally available cyclic peptide with an oral bioavailability (F) of 33%.

54. Amide Temperature Coefficient (ΔδNH/ΔT) and Caco-2 Permeability of N-methylated Peptides.

	ΔδNH/ΔT value of each position
cyclic peptide	NHa	NHb	NHc	NHd	NHe	Caco-2 P app (10–6 cm s–1)
74a	–0.93	–7.74	–8.29	–5.69	–8.01	1.09
74b	–1.80	NMe	–6.90	–1.58	–6.44	1.68
74c	–4.40	–8.02	NMe	–4.10	–7.20	7.83
74d	–0.42	–7.76	–7.34	–3.84	NMe	9.13
74e	–2.19	NMe	NMe	–0.76	–6.96	10.76
74f	–1.69	NMe	–7.21	–1.24	NMe	7.75
74g	–0.60	–8.25	NMe	–2.94	NMe	11.33
74h	–1.75	NMe	NMe	–0.23	NMe	15.92
74i	NMe	–5.24	–6.82	–7.18	–7.70	1.13
74j	NMe	–5.24	–6.04	–3.18	NMe	5.18

Ester bonds in cyclic peptides have also been recognized as structural features that enhance membrane permeability, given their prevalence in membrane-permeable natural products and their impact on conformation. In 2021, it was reported that amide-to-ester substitutions in the ligand linkages of heterobifunctional PROTAC structures improved membrane permeability (see Section ). In 2023, the collaborative group of Sando, Morimoto, and Lokey evaluated the effect of amide-to-ester substitutions on cyclic peptide permeability. Using a single-ester/N-methyl scan of amide bonds in a cyclic hexapeptide, they found that ester-substituted cyclic peptides exhibited higher permeability than their N-methylated counterparts. Their experimental and computational analyses suggested that the increased lipophilicity of ester bond-containing macrocycles contributes to this improvement. Another factor identified was the difference in conformation between ester-bonded derivatives and N-methylated analogs. While ester-bonded derivatives retained conformations similar to those of the original cyclic peptides, N-methylation altered the conformation, potentially generating new solvent-exposed amide NHs and reducing permeability.

However, ester-bond substitutions in larger cyclic peptides did not consistently enhance permeability to the same extent as N-methylation. These findings highlight the need for further studies to determine the conditions under which ester-bond substitutions are most effective.

4.2.4. Desirable Properties for bRo5 Cyclic Peptides Learned from CsA

A research group from Chugai Pharmaceuticals identified key parameters for designing druglike bRo5 cyclic peptides. They focused on the properties of CsA and evaluated the druglikeness of 553 cyclic peptides with characteristics similar to those of CsA (Table ).

55. Comparisons of Structural Features and Physicochemical Parameters of 553 Cyclic Peptides and CsA.

	553 peptides
properties	min	Max	CsA
no. of amino acids residues	8	12	11
no. of N-alkyls	0	8	7
CLogP	4.4	15.2	14.4
no. of OHs	0	1	1
MW	845	1499	1203

Their analysis revealed three desirable parameters for cyclic peptides to exhibit favorable druglikeness: (1) 9 to 11 residues, (2) 6 or more N-alkylated amino acid residues (NAAs) in the peptide backbone, and (3) a CLogP value of 12.9 or higher (Table ). Using these criteria, they constructed a second library of 11-residue cyclic peptides with nine randomized amino acids, ensuring that 65% of the peptides theoretically contained the desired number of NAAs. While CLogP values varied among peptides in the library, this was not considered problematic, as lipophilicity can be fine-tuned by modifying side-chain structures without significantly altering the conformation. Screening this library led to the discovery of the KRAS inhibitor AP8784 (75), whose optimization resulted in LUNA18 (76), a compound undergoing phase I clinical trials as of December 2024 (Tables and ).

56. Desirable Structural Features and CLogP of Cyclic Peptides for Favorable Druglikeness and the Values of AP8784 and LUNA18.

	no. of residues	no. of N-alkyls	CLogP
desirable values for favorable druglikeness	9 to 11	6 or more	12.9 or more
AP8784 (75)	11	7	12.7
LUNA18 (76)	11	8	14.5

57. PK Parameters of LUNA18 (76).

	mouse		rat		dog		monkey
route	IV	PO	IV	PO	IV	PO	IV	PO
dose (mg kg–1)	1	10	1	10	0.3	0.3	0.165	3
parameter
AUCinf (ng h mL–1)	1700	3600	3400	740	4100	2000	680	3400
CL (mL min–1 kg–1)	9.8		5.0		1.3		4.2
Vss (L kg–1)	2.6		0.96		0.99		1.2
T 1/2 (h)	5.5	3.5	5.2	3.6	14	15	8.0	8.5
C max (ng mL–1)		780		1800		190		630
T max (h)		2.0		2.0		2.0		2.3
F (%)		21		22		47		26

An integrated evaluation of the 553 cyclic peptides with CsA-like properties revealed that metabolic stability generally increased with the number of residues, whereas permeability was optimal for 8-, 9-, and 11-residue peptides but decreased for 10- and 12-residue peptides. Based on these findings, the authors identified 11 residues as the optimal number. This conclusion aligns with the theory of chameleonicity: hydrophilic open conformations in aqueous environments can suppress oxidative metabolism, while lipophilic closed conformations enhance the membrane permeability. The case of LUNA18, which exhibits both favorable solubility and permeability, further supports this hypothesis.

Finally, the authors proposed that these desirable properties could be generalized and applied to other cyclic peptides and bRo5 molecules. They translated their findings into two general criteria: (1) a CLogP-to-residue ratio of 1.17 or higher and (2) an N-alkylation ratio of 0.5 or higher. This study raises the possibility that integrated investigations of other bRo5 molecules, such as large peptide-mimetic protease inhibitors (e.g., HIV inhibitors) and heterobifunctional compounds such as PROTACs, might yield other criteria for optimal properties.

4.3. Druglikeness Studies of PROTACs

PROTACs are compounds that induce the degradation of a protein of interest (POI) by leveraging the ubiquitin–proteasome system. , PROTACs possess heterobifunctional structures, consisting of a small-molecule (or peptidic) ligand targeting the POI linked to another small-molecule (or peptidic) ligand that binds to an E3 ubiquitin ligase, thereby bringing the POI and E3 into close proximity. The success of PROTACs has established this induced-proximity-based strategy as a generalizable approach for drug design, leading to the emergence of other “TAC (targeting chimera)” technologies. Since the first report on PROTACs in 2001, they have gained recognition as a promising modality for drug discovery, with over 25 PROTACs now in clinical trials. This growing interest underscores the importance of studying the druglikeness of PROTACs. Medicinal chemistry optimizations of PROTACs have demonstrated that their maximum activity can be achieved at picomolar concentrations, despite the individual binding affinities of their warheads being in the nano- to micromolar range. , This difference is attributed to their catalytic mechanism, which allows them to function effectively at doses lower than their binding affinities might suggest. Furthermore, low doses are preferred to mitigate the hook effect, where excessive doses reduce the efficacy. However, PROTACs typically have MW ranging from 800 to 1200 Da, resulting in low aqueous solubility (often in the low micromolar range) and limited membrane permeability (<1.0 × 10^–6 cm/s), both of which are commonly classified as poor. Consequently, although improving biological activity is crucial, enhancing the bioavailability of PROTACs represents a significant challenge.

This section summarizes studies focused on the solubility and permeability of PROTACs as well as strategies for their improvement.

4.3.1. Evaluation of Physicochemical Properties of PROTACs

Extensive research has been conducted on the relationships between the physicochemical properties that govern the aqueous solubility and membrane permeability of PROTACs and the druglikeness of PROTACs. The Caron group identified key determinants of the PROTAC solubility, demonstrating a correlation between solubility and lipophilicity. They measured the physicochemical properties of 21 commercially available PROTACs and plotted experimental solubility (LogS) against other properties. They found that LogS vs BRLogD and LogS vs Marvin LogP showed promising linear correlations, with r ² values of 0.67 and 0.69, respectively.

However, plots of LogS against polarity parameters, such as Logk _W ^IAM and PSA, showed poor correlations, despite polarity being a known factor influencing the solubility of cyclic peptides (see Section ). The logarithm of the experimental chromatographic retention factor (Logk _W ^IAM), determined using an immobilized artificial membrane (IAM) column, serves as an indicator of molecular polarity but did not correlate strongly with solubility in this context.

Further support for the findings by Caron et al. comes from the work of Harling et al., who optimized RIPK2-targeting PROTACs by reducing their lipophilicity. For instance, replacing the E3 ligand moiety in 77a with a less lipophilic ligand (77b, Table ) significantly improved solubility. Additionally, substituting the POI ligand in 77b with another less lipophilic ligand (77c) resulted in a further enhancement of the solubility.

58. Structures of RIPK2 PROTACs and Their Binding Potency, Solubility, and Lipophilicity Parameters.

^aCLND, chemiluminescent nitrogen detection.

A collaborative group from Boehringer Ingelheim and the University of Dundee reported that introducing a methyl group (78b) into the linker of PROTAC 78a resulted in an over 18-fold improvement in aqueous solubility at pH 4.5, despite an increase in CLogP. Additionally, this modification led to a more than 12-fold enhancement in Caco-2 permeability (Table ). However, their analysis revealed no clear correlation between CLogP and solubility or permeability among the PROTAC analogs studied (Figure ). This finding suggests that simple lipophilicity descriptors, such as CLogP, are insufficient to fully explain the relationship between lipophilicity and druglikeness in PROTACs, and additional factors must be considered.

59. CLogP Itself Does Not Necessarily Determine the Solubility and Permeability of PROTACs.

compd	kinetic aqueous solubility (pH 4.5) (μg mL–1)	CLogP	Caco-2 P app (10–6 cm s–1)
78a	<1	8.9	<0.5
78b	18	9.2	6

^aSolubility was determined by dilution of a 10 mM DMSO solution into a buffer to a final concentration of 125 μg/ml. The mixture was incubated for 24 h, followed by filtration and analysis of the filtrate by LC-UV.

13.

Plot of solubility versus permeability of the PROTACs. There appears to be no correlation between solubility and permeability.

Kihlberg and colleagues also demonstrated that, even among a few PROTACs with different linkers, CLogP did not correlate with solubility or membrane permeability (Table ), further supporting the need for alternative descriptors. Their NMR and MD analyses revealed that the most lipophilic, yet most water-soluble, analogue 79c adopts a more elongated and loosely folded conformation in CHCl₃ compared to 79a and 79b. These findings suggest that the 3D structure of PROTACs significantly influences their druglikeness and that calculated lipophilicity parameters must account for these structural factors. Moreover, they identified a correlation between the radius of gyration (R _gyr) in CHCl₃, representing a lipophilic environment, and the solvent-accessible 3D polar surface area (SA-3D PSA). Based on these results, they concluded that molecular flexibility is a critical property, enabling PROTACs to adopt folded conformations with low SA-3D PSA, which is associated with high cell permeability.

60. Linker SAR of PROTACs Suggests Lipophilicity Does Not Correlate with Druglikeness .

										SA-3D PSA (Å2)		R gyr (Å)
cmpd	MW	HBD	HBA	Solubility (mg L–1)	CLogP	Caco-2 (nm s–1)	PAMPA (−LogP e, cm s–1)	tPSA (Å2)	NRotB	NAMFIS	MD simulation	NAMFIS	MD simulation
79a	897	3	12	56	1.50	30	6.56	210	21	209 (S), 167 (R)	218	5.40 (S), 5.16 (R)	7.84
79b	853	3	12	31	1.26	11	n.d.	218	17	232.26 (S), 245.84 (R)	277	5.44 (S), 5.60 (R)	6.94
7c	851	3	11	63	2.56	6	>7.37	209	17		300		7.96

^aRather, the actual molecular size (R _gyr ) and polarity (SA-3D PSA) in the solution appear to be crucial.

^bNumber of rotational bond.

^cNAMFIS (NMR analysis of molecular flexibility in solution), population weighted mean; S and R in brackets indicate the stereochemistry of thalidomide.

^dNot determined.

Garon et al. evaluated the physicochemical properties of several PROTACs using chromatographic techniques and identified a strong correlation (r ² = 0.85) between Caco-2 permeability (LogP _app) and an experimental molecular polarity descriptor, ΔLogk _W ^IAM. ΔLogk _W ^IAM represents the difference in Logk _W ^IAM values, where higher ΔLogk _W ^IAM indicates greater molecular polarity. Interestingly, no correlation was found between the calculated PSA values and ΔLogk _W ^IAM, suggesting that experimental polarity (ΔLogk _W ^IAM) is a better measure of PROTAC polarity. This discrepancy highlights the limitations of computed PSA values arising from conformational factors, as discussed earlier.

In a collaborative study by the Lokey and Ciulli groups, the permeability and other propertiessuch as MW, lipophilicity, and polarityof 11 PROTACs were measured and compared to identify determinants of permeability. The results suggested that physicochemical properties other than MW significantly influence the PROTAC permeability. For example, the PROTACs with the highest and lowest MWs (81 and 82c) exhibited the lowest permeability (Table ). Moreover, compounds with nearly identical properties displayed stark differences in their permeability. For instance, PROTACs 80b and 82b showed a 10-fold difference in permeability despite having almost the same properties. Similarly, a comparison of PROTACs 80a and 82a, which also have nearly identical properties, revealed that the permeability of 80a was 120-fold higher than that of 82a.

61. Structures of PROTACs and a Dimer of the VHL Ligand, Together with Physicochemical Parameters.

compd	n	MW	HBD	HBA	LogD	ALogP	LPE	PAMPA (10–6 cm s–1)
80a	2	959	4	11	–1.1	3.7	0.5	0.6
80b (MZ1)	3	1003	4	12	–1.6	3.6	0.1	0.03
81	5	1179	6	16	–3.2	0.6	1.6	0.002
82a	1	961	4	11	–3.8	3.7	–2.3	0.005
82b	2	1005	4	12	–4.3	3.6	–2.7	0.003
82c		945	4	10	–3.8	4.5	–3.1	0.002

The authors utilized LPE to explain these results. For example, in the comparisons between compounds 80a and 82a and between 80b and 82b, despite the similar AlogP values, identical numbers of HBDs/HBAs, and nearly identical MW of each pair, the membrane permeability differed by a factor of 10. However, when the difference in the LPE (ΔLPE) was calculated, both comparisons yielded a value of 2.8. It is known that the addition of one solvent-exposed amide NH decreases the LPE by approximately 1.8. Based on this information, the authors hypothesized that the significant differences in membrane permeability were due to solvent exposure of the amide NH in the penicillamine-type compounds 82a and 82b, whereas the NH remained masked in t-Leu-containing compounds 80a and 80b.

Additionally, a comparison of the linkage between the von Hippel-Lindau (VHL, an E3 ligase) ligand and the linker revealed that ester compounds 80c and 83b exhibited better PAMPA than their amide counterparts 80b and 83a. Notably, the LPE values for the ester compounds were comparable to or higher than those of the amides (Table ). Based on these findings, two design strategies were proposed for improving PROTAC permeability: (1) ether oxygen in PEG linkers may form intramolecular HB, shielding the amide NH in PROTAC molecules, and (2) replacing solvent-exposed amide bonds with ester bonds can enhance membrane permeability.

62. Impact of Amide-to-Ester Linkage Switching on Activity, Lipophilicity, and Membrane Permeability.

Focusing on these strategies, the collaborative groups investigated the impact of amide-to-ester conversion on PROTAC properties, including permeability (for the case of cyclic peptides, see Section ). They found that amide-to-ester substitutions in model bifunctional molecules with AlogP values between 1 and 4 improved PAMPA. However, this effect was not observed in compounds with AlogP > 4. There was also a positive correlation with MDCK cell permeability, although ester-containing compounds exhibited higher efflux ratios than their amide counterparts. Building on these results, they applied amide-to-ester conversion to known BET bromodomain-targeting PROTACs 80b (MZ1) and 83a (ARV-771). This modification led to improved PAMPA (Table ) and enhanced the BRD4 degradation activity.

Pirali et al. reported that PROTAC 84b, designed using the amide-to-ester strategy, did not exhibit improved solubility but was very stable during incubation with MLM (Table ). These findings suggest that the amide-to-ester conversion strategy may be effective not only for cyclic peptides but also for PROTACs and potentially other bRo5 drugs.

63. Impact of Amide-to-Ester Linkage Switching on the Solubility, Lipophilicity, and Metabolic Stability.

^aCalculated with ChemDraw ultra 20.0.

^bResidual substrate after 1 h of incubation with MLM in the presence of NADPH.

4.3.2. Chameleonicity/Flexibility in PROTACs

Kihlberg et al. focused on the conformational changes of PROTACs in different environments and proposed that the chameleonicity of PROTACs is a key behavior enabling their permeation through lipid bilayers. Their study demonstrated that PROTAC 86 (Figure ) adopts a higher population of closed, low-3D PSA conformations in CHCl₃ compared to DMSO–water. This finding suggests that PROTACs favor open conformations in polar environments, such as extracellular and intracellular compartments, in order to form HBs with water while adopting closed conformations in apolar environments, such as lipid bilayers, to shield hydrophilic groups. As in the case of cyclic peptides, this behavior allows PROTACs to adjust their polarity in response to the environment, facilitating membrane permeability. Since this chameleonic behavior depends on intramolecular HBs and molecular flexibility, the plasticity of linker structures is likely a critical factor influencing chameleonicity and cell permeability.

14.

Structure and properties of PROTAC 86.

The Kihlberg group further investigated the relationship between linker structure and cell permeability using three PROTACs with identical POI and E3 ligands but different linker structures, employing NMR structural analysis and MD simulations (Table in Section ). In vitro permeability analyses revealed that PROTAC 79a, which features a longer PEG-based linker, exhibited the highest cellular permeability. The study yielded three conclusions. (1) For linkers of the same length, alkyl linkers are somewhat more flexible than PEG-based linkers. (2) PEG-based linkers are more likely to adopt folded, low-3D PSA conformations than alkyl linkers, due to the inherent gauche effect of PEG structures, in contrast to the anticonformation favored by alkyl linkers. (3) Among PEG-based linkers, longer linkers are more flexible and more likely to populate folded conformations than shorter ones. These findings are consistent with the superior permeability of PROTAC 79a compared to other PROTACs in the study.

However, studies on the druglikeness of PROTACs with varying linker structures, as discussed in the following section, indicate that flexible linkers are not always optimal. Thus, chameleonicity should be considered as one of several parameters influencing the druglikeness of PROTACs, rather than a sole determinant.

4.3.3. Linkerology in PROTACs Focusing on Druglikeness

In the PROTAC design, the modification of E3 ligands and POI ligands is often constrained by limitations in linkage positions and acceptable structural variations. Consequently, the linker moiety has become a primary target for customization to improve both physicochemical properties and biological activity. This has led to an increased focus on SAR studies of linkers, termed linkerology, which seek to elucidate the relationships between linker structures and PROTAC activities or properties. Cyclic linker structures, such as piperazine moieties, have gained particular attention for enhancing the druglikeness of PROTACs following the disclosure of clinical trial PROTAC structures that frequently include cyclic linkers. Several studies have demonstrated the impact of the linker design on the solubility and permeability of PROTACs. For example, the physicochemical properties of PROTACs composed of cereblon binders, the BRD4 inhibitor JQ1, and various linkers were comprehensively evaluated (Table ). All PROTACs in this study exhibited thermodynamic aqueous solubility ranging from 32.3 to 78.5 μM, which is similar to or slightly lower than the solubility of their conjugating ligands, such as JQ1 (52.1 μM), thalidomide analogs (55.7–84.6 μM), and phenyl glutarimide (PG) analogs (76.1–148.5 μM), another class of cereblon binders. In a comparison of linkers, alkyl linkers (87a, 87b) showed a slightly higher solubility than the triazole linker (88c). As regards permeability, all PROTACs exhibited low Caco-2 permeability (around or below 2 × 10^–6 cm/s). For thalidomide-type PROTACs, oxygen-linked compounds (88a, 88c) demonstrated 7.3- and 6.7-fold higher permeability, respectively, than nitrogen-linked compounds (88b, 88d). This difference is likely due to the additional HBDs present in 88b and 88d. Among PG-type PROTACs, 4-substituted compounds (87a, 87b) showed higher permeability than 3-substituted ones (87c, 87d) and were comparable to those of the well-known thalidomide-based PROTAC dBET1 (85). Notably, two 5-substituted cereblon-based PROTACs, ARV-471 and ARV-110, are orally available, suggesting that conjugation at positions distant from the glutarimide core in cereblon binders may enhance permeability.

64. Linker Structure–Property Relationships of the PROTACs.

cmpd	n	R	X	thermodynamic aqueous solubility (μM)	Caco-2 (10–6 cm s–1)
85 (dBET1)	1	Thal		53.5	1.16
87a	1	4-PG		78.5	1.66
87b	2	4-PG		78.3	1.43
87c	1	3-PG		65.3	0.1
87d	2	3-PG		65.3	0.97
88a		Thal	O	50.8	1.72
88b		Thal	NH	32.3	0.22
88c		4-PG	O	60.4	2.14
88d		4-PG	NH	56.8	0.32

Piperazine is frequently used as a linker moiety in PROTACs with favorable druglikeness. In a study by Pirali et al., PROTAC 89 (Figure ) showed remarkable solubility (5034 μM) in 0.01 M HCl (pH 2.0).

15.

Structure of PROTAC entry 89.

In a linker SAR study conducted by Harling et al., replacing PEG-based linkers with more rigid heteroaromatic/aromatic or piperazine/piperidine tandem linkers generally reduced solubility (Table ). Their analysis revealed the following trends. (1) Increasing the number of atoms in the linker decreased solubility (90b vs 90c vs 90d). (2) Piperazine linkers exhibited better solubility than piperidine linkers, likely due to their lower lipophilicity (90c vs 90e, 90d vs 90f). (3) Solubility was not significantly affected by the type of heteroaromatic ring (90f vs 90g vs 90h vs 90i).

65. Linker Structure–Property Relationship Study Focusing on Rigid Cyclic/Aromatic Linker Structures.

Ciulli et al. improved Caco-2 permeability of PROTACs by modifying the linker structure. PROTAC 91a, which possesses an ethylene glycol linker, exhibited poor passive permeability (A–B rate of 1.1 × 10^–7 cm/s) and a high transporter-mediated efflux (B–A rate of 20.7 × 10^–6 cm/s), resulting in an efflux ratio of 190 (Table ). In contrast, PROTAC 91b, with a 4-ethylbenzyl linker, showed improved permeability (A–B rate of 8.4 × 10^–7 cm/s, B–A rate of 7.6 × 10^–6 cm/s, and efflux ratio of 9.1). Interestingly, ACBI1 (91c), featuring a 4-oxyethoxybenzyl linker, showed an even greater permeability improvement, exhibiting an A–B rate of 2.2 × 10^–6 cm/s, a B–A rate of 3.8 × 10^–6 cm/s, and an efflux ratio of 1.7. Notably, ACBI1 differs from 91b only in the addition of a single-oxygen atom. Despite the reduced CLogP due to this modification, the A–B rate significantly improved, while the B–A rate was reduced by approximately half. Although the detailed mechanisms underlying these changes remain unclear, these findings suggest that factors other than lipophilicity can play a critical role in determining permeability.

66. Impact of Linker Structure on Membrane Permeability and Efflux.

Ciulli et al. reported the discovery of PROTAC XL01126 (92, Figure ), an orally available (mouse bioavailability F: 15%) and BBB-permeable leucine-rich repeat kinase 2 degrader. This compound was identified through a comprehensive medicinal chemistry campaign. In that study, the authors investigated various linker structures, including flexible linkers (PEG, alkyl) and rigid linkers (aromatic, alkyne, and cyclic). Among the compounds examined, PROTAC XL01126, which incorporates a trans-1,4-cyclohexyl linker, exhibited the most potent activity as well as favorable pharmacokinetic profiles. This finding aligns with the observation that many orally available PROTACs feature sp³-cyclic linkers. While such linkers are less conducive to the conformational chameleonicity described earlier, this trend highlights the importance of NRotB in addition to chameleonicity in determining the druglikeness of PROTACs.

16.

Structure and druglikeness of PROTAC XL01126.

5. Conclusions

Improvement of the aqueous solubility and permeability of lead compounds remains a critical challenge in drug discovery programs, especially for bRo5 compounds, including cyclic peptides and PROTACs, which are of increasing importance in modern medicinal chemistry. Medicinal chemists must navigate and reconcile complex and often contradictory parameters, such as lipophilicity versus solubility, solubility versus flatness, and solubility/permeability versus molecular weight.

Over the past two decades, significant advancements have been made to achieve Aufheben of such parameters of druglikeness. As for the apparently contradictory physicochemical properties of lipophilicity and aqueous solubility, there is increasing evidence that chemical modification to weaken intermolecular interactions can improve not only aqueous solubility but also permeability by increasing lipophilicity. Various molecular design strategies to enhance the aqueous solubility by weakening intermolecular interactions have been proposed. In particular, advances in synthetic methods for phenyl ring mimetics have promoted the achievement of Aufheben of phenyl spacers and druglikeness. In general, structural modifications undertaken to enhance aqueous solubility typically result in either increased activity (due to a better fit within the protein pocket) or decreased activity (if the fit is compromised), and the introduction of hydrophilic substituents often interferes with protein–drug interactions. In such cases, the introduction of hydrophobic group would provide an alternative approach for improving aqueous solubility (Table ).

We have also presented concrete examples of strategies to increase the membrane permeability and aqueous solubility of bRo5 molecules. Because rationales and guidelines for adjusting PROTACs’ solubility and cell permeability are still limited, concise synthetic methodologies and an efficient assay of druglikeness have been developed. A further strategy for improving the cellular uptake of bRo5 molecules is the utilization of transmembrane proteins as enhancers. Overall, there are an increasing number of examples of bRo5 molecules that are both water-soluble and cell-permeable.

In recent years, medicinal chemistry has faced the challenge of addressing undruggable targets, in part because of the limited availability of drug targets and the expansion of modalities for intervention. While the complexity of this challenge continues to grow, medicinal chemistry has consistently demonstrated its ability to overcome contradictions and innovate. The pursuit of new opportunities for Aufhebenthe resolution of opposing propertieswill remain a driving force in shaping the future of the field.

Glossary

Abbreviations

BCHep

Bicyclo­[3.1.1]­heptanes

BCO

Bicyclo­[2.2.2]­octane

BCP

Bicyclo­[1.1.1]­pentane

BCS

Biopharmaceutics Classification System

BP

Bridged piperidine

CLND

Chemiluminescent nitrogen detection

CsA

Cyclosporine A

Do

Dose number

Fsp³

sp³-hybridized carbons

HBA

Hydrogen bond acceptor

HBD

Hydrogen bond donor

HLM

Human liver microsomes

IAM

Immobilized artificial membrane

LHSA

Largest hydrophobic surface area

LogD

Distribution coefficient

LPE

Lipophilic permeability efficiency

LYSA

Lyophilization solubility assay

MD

Molecular dynamics

MDCK

Madine–Darby canine kidney

MLM

Mouse liver microsomes

MW

Molecular weight

NAA

N-alkylated amino acid residue

NAMFIS

NMR analysis of molecular flexibility in solution

NRotB

Number of rotational bond

PAMPA

Parallel artificial membrane permeability assay

P _app

Apparent permeability

PFI

Property forecast index

PG

Phenyl glutarimide

POI

Protein of interest

PPAR

Peroxisome proliferator-activated receptor

PPI

protein–protein interaction

PROTAC

Proteolysis targeting chimeras

PSA

Polar surface area

R _gyr

Radius of gyration

Ro5

Rule of five

RRCK

Ralph Russ canine kidney

SA

Solvent-accessible

TAC

Targeting chimera

tPSA

Topological polar surface area

VSMC

Vascular smooth muscle cells

XRD

X-ray diffraction

ΔG _sol

Gibbs-free energy of dissolution

ΔH _sol

Enthalpy of dissolution

ΔS _sol

Entropy of dissolution

Biographies

Minoru Ishikawa

Minoru Ishikawa received his M.Sc. (1996) from Tokyo Institute of Technology and began his career as a Researcher at the Medicinal Chemistry Research Laboratories in Meiji Seika Kaisha, Ltd., Japan (1996–2008). During this period, he completed his Ph.D. at the University of Tokyo in 2006 under the direction of Professor Yuichi Hashimoto. In 2008, he joined the Institute of Molecular and Cellular Biosciences at the University of Tokyo, where he served as an Assistant Professor, Lecturer, and Associate Professor until 2019. Subsequently, he was appointed Professor at the Graduate School of Life Sciences, Tohoku University, a position he has held since 2019. His research focuses on medicinal and bioorganic chemistry, with particular interests in improving the physicochemical properties of drug candidates, targeted protein degradation, and exploring novel drug discovery modalities for neurodegenerative diseases.

Shusuke Tomoshige

Shusuke Tomoshige completed his Ph.D. at The University of Tokyo in 2016 under the supervision of Professor Yuichi Hashimoto. After postdoctoral training for 2 years at the University of Notre Dame, under the guidance of Professor Shahriar Mobashery, he joined Tokyo University of Science, working with Professor Kouji Kuramochi. Since September 2019, he has been an Assistant Professor at Graduate School of Life Sciences, Tohoku University. His research focuses on improving the physicochemical properties of drug candidates, the induced-proximity approach to control protein homeostasis, including degradation and aggregation, aiming at the development of novel therapeutic strategies for neurodegenerative disorders and mitochondria-related intractable diseases.

Shinichi Sato

Shinichi Sato obtained his Ph.D. from the University of Tokyo in 2011 under the supervision of Prof. Yuichi Hashimoto. He held a position as a Japan Society for the Promotion of Science (JSPS) Research Fellow for 1 year within the research group led by Prof. Carlos F. Barbas at Scripps Research Institute. Subsequently, he worked as an Assistant Professor in Prof. Hiroyuki Nakamura’s laboratory at both Gakushuin University and Tokyo Institute of Technology. He has been a Principal Investigator at the Frontier Research Institute for Interdisciplinary Sciences, Tohoku University as an Assistant Professor since 2020 and is currently an Associate Professor there. His research focuses on protein chemical modification, proximity labeling, and chemical proteomics.

The authors declare no competing financial interest.