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Heterocyclic replacements for benzene: Maximising ADME benefits by considering individual ring isomers
European Journal of Medicinal Chemistry 124 (2016) 1057e1068
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European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Heterocyclic replacements for benzene: Maximising ADME benefits by considering individual ring isomers Timothy J. Ritchie a, *, Simon J.F. Macdonald b, ** a b
TJR-Chem, Via Alberto 34C, 21020 Ranco (VA), Italy Fibrosis Discovery Performance Unit, GlaxoSmithKline Medicines Research Centre, Gunnels Wood Road, Stevenage, SG1 2NY, UK
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 September 2016 Received in revised form 11 October 2016 Accepted 14 October 2016 Available online 15 October 2016
The impact of replacing a mono-substituted benzene (phenyl) ring with thirty three aromatic and nine aliphatic heterocycles on nine ADME-related screens (solubility, lipophilicity, permeability, protein binding CYP450 inhibition and metabolic clearance) was assessed using matched molecular pair analysis. The results indicate that the influence on the ADME profile can differ significantly depending on the ring identity and importantly on the individual regioisomers that are possible for some rings. This information enables the medicinal chemist to make an informed choice about which rings and regioisomers to employ as mono-substituted benzene replacements, based upon the knowledge of how such replacements are likely to influence ADME-related parameters, for example to target higher solubility whilst avoiding CYP450 liabilities. © 2016 Elsevier Masson SAS. All rights reserved.
Keywords: Benzene Heteroaromatic Heteroaliphatic Regioisomer ADME
1. Introduction Benzene and heteroaromatic rings are ubiquitous in drug discovery, with benzene and pyridine being the two most commonly found aromatic systems in marketed drugs [1]. However, too many aromatic rings, particularly carboaromatic rings in a molecule increase the likelihood of encountering poor ADME profiles, due to suboptimal physicochemical properties [2,3]. Reducing lipophilicity and increasing solubility are often significant challenges in lead optimisation; adding polar functionality can achieve this, but the increase in the number of heteroatoms may limit membrane permeability and oral bioavailability and introduce other liabilities such as hERG inhibition in the case of positively ionisable amines [4]. An alternative approach is to replace a benzene ring with a heterocycle, and over the last decade or so in the patent literature, more heteroaromatic rings are being incorporated into new chemical entities at the expense of carboaromatic rings [5]. But with many ring types and multiple regioisomers available, it is not always easy to foresee how such changes will simultaneously impact ADME-related parameters such as solubility, lipophilicity,
* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (T.J. Ritchie), simon.jf.macdonald@ gsk.com (S.J.F. Macdonald). http://dx.doi.org/10.1016/j.ejmech.2016.10.029 0223-5234/© 2016 Elsevier Masson SAS. All rights reserved.
protein binding and CYP450 inhibition other than in general terms. The developability of various heterocyclic rings with respect to solubility, protein binding and CYP450 inhibition has been published but the exact substitution pattern and specific regioisomers of the rings were not considered in this study [6]. We felt that combining a comprehensive set of ring replacements with data from multiple ADME readouts would generate valuable quantitative information that would be extremely useful to medicinal chemists who wish to replace a mono-substituted benzene ring and would like to know what to expect when a particular heteroaromatic or heteroaliphatic ring is employed. More specifically, by understanding the impact on ADME parameters across a broad range of heteroring isomers, one can identify approaches where chemists can achieve the maximum benefit of employing individual rings and heteroatoms. Notwithstanding synthetic considerations, shifting ring heteroatoms incurs no molecular weight penalty or change in heteroatom count, but may, for example improve solubility whilst avoiding issues such as increased CYP450 inhibition. Knowing how specific heteroring isomers impact ADME parameters would also assist chemists involved in library design and building block selection, where the most attractive rings and isomers could be prioritised. In order to understand how particular heteroatomic arrangements in rings modulate ADME-related parameters, we have used matched molecular pairs to identify examples from the
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GlaxoSmithKline compound collection where a mono-substituted benzene ring (i.e. a phenyl substituent) has been replaced by a mono-substituted heterocyclic ring, taking into account both the ring identity and all regioisomers that are possible by varying the position of the heteroatom(s) in the ring structure. The resulting pairs were combined with measured data from multiple ADMErelated assays, namely kinetic aqueous solubility, lipophilicity (log D7.4), permeability, protein binding, CYP450 inhibition (3 isoforms) and clearance (two species), to determine how each ADME readout changed for each ring relative to mono-substituted benzene. In order to maximise the impact the ring replacements would have in the ADME assays and thus more easily differentiate the behaviour of the ring types and regioisomers, we chose mono-substituted rings in order to expose the ring heteroatoms as much as possible. In this study, we have not taken any account of how these changes might impact biological activity. If the mono-substituted benzene ring under consideration is interacting with a target predominantly via a hydrophobic interaction, the replacement with a polar heterocycle may result in a loss of binding affinity; however it may still be possible to introduce polar atoms in particular positions, which may be tolerated by the binding pocket. There has been a number of insightful MMP analyses that discuss the impact on ADME parameters of replacing a benzene ring with a heterocycle; these have either focussed on a limited number of heterocycles (often pyridine), or have reported data from only one or two assays. For example, Gleeson et al. highlighted the important fact that the three isomers of pyridine behave differently with respect to their impact on CYP450 3A4 inhibition and solubility, and by considering these separately obtained a much clearer and statistically robust picture of the effects than from a generic phenyl-topyridyl transformation [7]. Haubertin et al. identified benzene replacements such as pyridine, pyrimidine and pyrazine that reduce protein binding [8] and Dossetter et al. investigated to what extent a comprehensive series of heteroaromatic cores modulated in vitro human microsomal metabolic stability when used as replacements for 1,2-, 1,3- and 1,4-disubstituted benzenes [9]. 2. Outline of article After a brief methodology section, the ADME profiles of monosubstituted benzene ring replacements that include one or no heteroatoms will be discussed, followed by aromatic rings containing two nitrogen atoms and then aromatic rings containing nitrogen and oxygen or sulfur. Further sections will then explore the role of nitrogen-containing rings and CYP450 3A4 inhibition, the impact of mono-substituted benzene ring replacements on developability properties such as permeability and protein binding, and finally metabolic clearance. Lastly, the best and worst rings identified in this study are discussed. 3. Methodology A set of SMIRKS [10] queries were generated to search for matched molecular pairs where a mono-substituted benzene is transformed into a monosubstituted heteroaromatic, heteroaliphatic or carboaliphatic ring, taking into account all regioisomers that are possible. In this way, the following mono-substituted benzene-to-heteroring pairs, in order of abundance were identified: pyridine (3 isomers; 5661 pairs), cycloalkanes (cyclohexane, cyclopentane, cyclobutane) (3468), thiophene (2 isomers; 2190), furan (2 isomers; 1182), tetrahydropyran (3 isomers; 683), thiazole (3 isomers; 645), pyrimidine (3 isomers; 637), pyrazole (4 isomers; 531), tetrahydrofuran (2 isomers; 365), imidazole (4 isomers; 330), pyrazine (1 isomer; 269), pyrrole (3 isomers; 146), oxazole (3 isomers; 137), pyridazine (2 isomers; 120), isoxazole (3 isomers; 96)
and oxetane (60). The identity of the attachment atom was not specified in the queries, but analysis of the results indicated that the most common attachment atom in the data set was an aliphatic carbon (40.5%), followed by an aromatic carbon (26.2%), nitrogen (15.1%), carbonyl carbon (11.7%), sulfur (3.9%) and oxygen (2.6%). Data from nine high-throughput ADME-related screening assays were then collected for the matched pairs, so that the change in each assay could be determined for each mono-substituted benzene-to-ring transform. The ADME readouts were kinetic aqueous solubility [11], chromatographically determined log D7.4 values [12], artificial membrane permeability [13], human serum albumin plasma protein binding [14], CYP450 inhibition assays (2D6, 3A4 and 2C19 isoforms) [15], and in vitro metabolic clearance (where the compound is incubated in a human or rat liver microsome preparation and its depletion measured over time) [16]. Due to the lack of pairs with sufficient screening data, triazoles, oxadiazoles, thiadiazoles, isothiazoles and lactams were omitted from the study, as well as strongly ionisable rings such as tetrazoles and cyclic amines such as piperidine and morpholine etc. Data were also excluded in the case where assay results were outside the dynamic range of the screens (i.e. reported as greater than or less than a particular value). For each mono-substituted benzene ring replacement, statistical measures, such as mean change, standard deviation and standard error of the mean were calculated for each screening assay, as well as the number of matched molecular pairs and the number of chemical series (as defined by Bemis-Murkco frameworks) that were represented. Results from the log D and CYP450 assays (pIC50 values) were used directly, whereas a log10 transformation was carried out on the results from the solubility (mol/L), permeability (m/s) and clearance (ml/min/g tissue) assays. A logit transformation was applied to the % protein binding data. It should be noted that the dynamic range varies considerably between the various assays: the chromatographic log D measurements covers the widest range of values (16.6 log units), followed by CYP450 3A4 (7.1), rat clearance (6.1), 2D6 (6.1), 2C19 (5.4), solubility (5.2), protein binding (4.2), human clearance (3.4) and permeability (3.3). A paired t-test (for normally distributed values) or a signed-rank test (for non-normally distributed values) were used [17] to determine whether the observed changes were significantly different from zero. Due to the potential errors surrounding data analysis of matched pairs with limited examples [18], it was decided that where there were less than 10 matched pairs available, these rings are not shown in the plots described in the results section, although the mean values and pair counts are included in the data tables for completeness. It should also be noted that changes of ±0.2e0.3 log units may fall within the experimental error associated with the screening assay protocols, even if they are statistically significantly different from zero. Statistical values for the change in each ADME screen for each ring regioisomer are provided in the Supporting Information. The majority of rings were connected to the rest of the molecule in the most part via a carbon atom, with relatively few examples with heteroatom-linked rings that have the potential to modulate ring properties such as basicity and lipophilicity. However, one third of pyridazines and pyrimidines and half of the oxetanes were N-linked. Although there were some differences in the ADME data for these N-linked rings relative to the carbon-linked exemplars, they were not significantly different. 4. Results 4.1. Rings with one or no heteroatoms e the benefits of cyclic ethers The most conservative mono-substituted benzene replacement is the isosteric thiophene ring [19], of which two isomers are
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possible, followed by other aromatic rings with one heteroatom namely pyrrole (three isomers), furan (two isomers) and pyridine (three isomers). The pyridines reduce log D significantly (p < 0.05,
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Tukey-Kramer method [20]) more than the pyrroles, due to the available nitrogen lone pair, which does not contribute to the aromatic sextet, but in terms of increasing solubility both ring types
Fig. 1. A comparison of the impact on solubility, lipophilicity and CYP450 3A4 inhibition when a mono-substituted benzene ring is replaced with heterocycles containing a single heteroatom or a cycloalkyl ring. Points are coloured by the observed change in CYP450 3A4 pIC50 (red ¼ increase; white ¼ no change; green ¼ decrease, relative to monosubstituted benzene). THF ¼ Tetrahydrofuran; THP ¼ Tetrahydropyran. Error bars indicate the standard error of the mean. Points further to the right and lower in the plots indicate rings that exhibit larger average increases in solubility and decreases in log D. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Table 1 The impact on nine ADME-related screening assays when a mono-substituted benzene ring is replaced with the three regioisomers of pyridine and two representative cyclic ethers. Values quoted are the average change observed in each assay: Aqueous solubility (SOL), artificial membrane permeability (PERM) and human or rat clearance (Cl) data are on a logarithmic scale (i.e. a change of þ1.00 would indicate a 10-fold increase), and a logit transformation is applied to the human serum albumin (HSA) % protein binding data; CYP450 data are pIC50 values.Values in italicised bold type indicate changes that are not statistically different from zero based on a paired t-test (for normally distributed values) or a signed-rank test (for non-normally distributed values). Cells are colour-coded with a gradient (change >1 red (detrimental); change ¼ 0 white; change <1 green (beneficial)), except for solubility and permeability where the colours are reversed. Where fewer than 10 matched pairs were available, the values are stated but greyed out and the cells are not colour-coded.
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behave similarly. Cyclic ethers such as oxetane, tetrahydrofuran and tetrahydropyran are useful solubilising motifs in medicinal chemistry [21] and have advantages over the aromatic rings that contain a single heteroatom, in part due to their higher hydrogen-bond basicities [22]. Fig. 1 shows the change in log solubility and log D when a mono-substituted benzene ring is replaced with these rings: in general, the increase in solubility for each ring mirrors the concomitant reduction in lipophilicity, although the correlation between the change in solubility and the change in lipophilicity is weak (r ¼ 0.36, r2 ¼ 0.13, n ¼ 13,695). Tetrahydrofuran and tetrahydropyran isomers outperform the aromatic thiophenes, furans and pyrroles in terms of lowering lipophilicity and increasing solubility, and benefit also from reducing CYP450 3A4 inhibition. This latter behaviour is in contrast to the increase in 3A4 inhibition that is observed when mono-substituted benzene is replaced with the 3- and 4-isomers of pyridine. Although it has been reported that there is generally no bias in the distribution of pyridine isomers in chemical collections and reagents [23], the 2-isomer appears to be more useful in that it lowers log D and increases solubility without the 3A4 liability of the 3- and 4-isomers. In comparison, the average values seen when replacing mono-substituted benzene with a cycloalkyl group does not improve ADMET properties, with cyclohexane and cyclopentane actually increasing log D. For the cyclic ethers, it is possible to modulate solubility and lipophilicity to a
significant extent by modifying the environment of the single oxygen atom in terms of ring angle and/or position in the ring relative to the attachment atom. The changes in log D seen with the cyclic ethers and furans are in line with the solvent accessible polar surface area (PSA) associated with the oxygen atom (calculated in MOE 2014.09 [24] using the lowest energy conformer for phenylsubstituted cyclic ethers and furans using the MMFF94x forcefield [25] and LowMode MD conformational search method), with more solvent-exposed oxygen atoms resulting in a larger reduction in log D. Topological 2-D PSA calculations [26] are not useful in this case as the oxygen atom in all the cyclic ethers are treated identically by this algorithm. In the current data set, pyridine is a very common monosubstituted benzene replacement; cyclic ethers less so, presumably due to the not insignificant synthetic considerations of these rings and the chiral nature of some of the tetrahydrofuran and tetrahydropyran moieties. However their ADME profiles would suggest that these rings should be considered as useful monosubstituted benzene or even pyridine replacements: Pyridines and cyclic ethers significantly increase solubility and lower log D, and it is interesting to compare the most soluble ethers with the isomers of pyridine across all the ADME assays being studied in order to give a complete picture of the similarities and differences observed (Table 1). 4-Tetrahydropyran and 3-tetrahydrofuran have
Fig. 2. Impact on log solubility and lipophilicity when a mono-substituted benzene ring is replaced with an aromatic ring containing two nitrogen atoms. Pyridine isomers are included as a reference set (cf. Fig. 1). Points are coloured by the observed change in CYP450 3A4 pIC50 (red ¼ increase; white ¼ no change; green ¼ decrease, relative to monosubstituted benzene). Error bars indicate the standard error of the mean. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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generally similar profiles to the pyridines but differ in that they lack the CYP450 liability seen particularly in the 4-pyridine isomer. It is also interesting to note that the ethers and pyridines reduce permeability to the same extent relative to mono-substituted benzene but the ethers are significantly (p < 0.05) less protein bound than the pyridines. As a general trend, the reduction of lipophilicity associated with the replacement of mono-substituted benzene with a more polar heterocycle decreases artificial membrane permeability and plasma protein binding, and is discussed in more detail later for the complete set of ring replacements. In addition, 4-tetrahydropyran reduces log D significantly (p < 0.05) less than 4-pyridine (1.40 and 1.92 units respectively), in line with their hydrogen bond basicity strengths [22] but has significantly (p < 0.05) higher solubility (þ0.62 and þ0.42 log units respectively), possibly due to its higher sp3 character [27]. 3Tetrahydrofuran appears to reduce clearance the most but more pairs would help to confirm this trend. 4.2. Aromatics rings containing two nitrogen atoms as monosubstituted benzene replacements There are fourteen permutations possible to replace a monosubstituted benzene with a heteroaromatic ring containing two nitrogen atoms, employing the 5-membered rings imidazole (4
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regioisomers) or pyrazole (4), or the 6-membered rings pyrazine (1), pyrimidine (3) or pyridazine (2). However, due to prototropic tautomerism between the 4- and 5-isomers of imidazole via a hydrogen shift, data for these forms were combined, leaving 3 isomers rather than 4. For the same reason, data for the 3- and 5isomers of pyrazole were also merged. Whilst it is not surprising that replacing a mono-substituted benzene ring with these polar heterocycles results in lower lipophilicity and higher solubility, the magnitude of the changes observed differ considerably depending on the type of ring and regioisomer (Fig. 2 and Table 2). In general, imidazoles lower log D to the largest extent, due to their higher basicity, although how this increases solubility varies, with the more sterically hindered 2-isomer having a significantly lesser impact than its 1- and 4/5-isomers. 1-Pyrazole, which lacks a free NH group, reduces log D significantly (p < 0.05) less than the other pyrazole isomers but has a similar impact on solubility; this relative difference is not seen in the case of 1-imidazole, presumably due to the fact that the other nitrogen is more exposed in the latter case. In the six-membered ring congeners, a mono-substituted benzene-topyridazine transformation reduces log D more than pyrimidine and pyrazine, although again the behaviour varies depending on the regioisomer. Thus the specific position of the two nitrogens relative to the attachment atom can have an influence on solubility over and above that due to the decrease in lipophilicity.
Table 2 The impact on nine ADME-related screening assays when a mono-substituted benzene ring is replaced with aromatic heterocycles containing two nitrogen atoms. For a description of the assays and colouring scheme see Table 1. *Due to prototropic tautomerism between the 4- and 5-imidazoles, and the 3- and 5-pyrazoles, the data for these isomers were merged. The asterisk indicates the alternative position of the hydrogen atom in these two cases.
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The impact on membrane permeability and protein binding for these ring types is discussed later; no correlation was found between the change in log D and the change in membrane permeability or protein binding. In terms of CYP450 inhibition there are also significant differences (Fig. 4): imidazoles are well known to inhibit CYP450 enzymes, which is the case here although each isomer exhibits a different profile across the 3A4, 2D6 and 2C19 isoforms. The 2-isomer however, appears to have lower CYP450 inhibition than mono-substituted benzene across all three isoforms, due to the increased steric hindrance of the nitrogen atom. Pyrimidines can exhibit 3A4 inhibition depending on the isomer, with the 4- and 2-isomers showing the most and least inhibition respectively. Rings with the nitrogen atoms adjacent (pyrazoles and pyridazines) do not inhibit 3A4 or they reduce inhibition relative to mono-substituted benzene, highlighting that subtle modifications in atom position can have a significant impact on ADME profiles. Further discussion of CYP450 inhibition follows below. Due to lack of pairs, it is not possible to assess the impact of the majority of these rings as mono-substituted benzene replacements in terms of clearance, however 2-pyrimidine appears to reduce both human and rat clearance (Table 2). 4.3. Rings containing nitrogen, oxygen and sulfur As expected thiazoles lower log D less than oxazoles, particularly in the 2-isomers; The 5-substituted isomers of oxazole and thiazole exhibit increased CYP450 3A4 inhibition relative to monosubstituted benzene (Fig. 3 and Table 3) due to the exposed nitrogen atom, but this effect appears to be ameliorated in the 5-
isoxazole with the caveat that the number of pairs are small, again highlighting the benefit of rings with adjacent heteroatoms. 4.4. CYP450 inhibition of nitrogen-containing aromatic rings It is to be expected that monosubstituted nitrogen-containing heteroaromatic rings are more likely to inhibit CYP450 enzymes when a nitrogen acceptor atom is more accessible to the enzyme i.e. not ortho to the attachment atom. However the analysis results indicate that this behaviour is heavily dependent on the type of ring and regioisomer: Fig. 4 describes the percentage of ring transforms that exhibit changes in 3A4 inhibition, from a strong increase (>1.5 log units in red), a smaller increase (0.3e1.5, orange), little or no change (0.3 to 0.3, yellow), a small decrease (1.5 to 0.3, pale green) and strong decrease (<1.5, dark green). The 3A4 isoform was chosen as more drugs interact with this enzyme and more data for the ring replacements were available from this screen. Where less than 10 pairs were available for particular ring isomers, these data are not shown. This presentation allows the differences between rings and isomers to be differentiated in more detail than just comparing mean values. For example, 5-oxazole and 4-pyrimidine both increase 3A4 inhibition by the same amount (0.65 and 0.64 log units respectively) but the percentage of pairs that exhibit a strong (>1.5 log units) increase differs (11% and 37% respectively). Rings with an exposed nitrogen atom flanked by a CH group tend to inhibit 3A4 relative to mono-substituted benzene, with 1imidazole, 4-pyrimidine and 4-pyridine having the highest percentages showing a strong (>1.5 log units) increase, and the highest mean changes. As drawn, 4-imidazole does not have an exposed
Fig. 3. Impact on log solubility and lipophilicity when a mono-substituted benzene ring is replaced with an aromatic ring containing nitrogen and oxygen or sulfur. Pyridine isomers are included as a reference set (see Fig. 1). Points are coloured by the observed change in CYP450 3A4 pIC50 (red ¼ increase; white ¼ no change; green ¼ decrease; grey ¼ no data; relative to mono-substituted benzene). Error bars indicate the standard error of the mean. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Fig. 4. Impact on CYP450 3A4 pIC50 when a mono-substituted benzene ring is replaced with a nitrogen-containing aromatic ring. Top panel: 5-membered rings; bottom panel: 6-membered rings. The coloured bars indicate the percentage of the matched pairs that exhibit a strong increase (>1.5 log units, red), a smaller increase (0.3e1.5, orange), little or no change (0.3 to 0.3, yellow), a small decrease (1.5 to 0.3, pale green) and strong decrease (<1.5, dark green). The values above the bars indicate the mean change in 3A4 inhibition for each ring type: values with an asterisk indicate changes that are significantly different from zero (p < 0.05). *Due to prototropic tautomerism between the 4- and 5-imidazoles, and the 3- and 5-pyrazoles, the data for these isomers were merged. The asterisks indicate the alternative position of the hydrogen atom in these two cases. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
nitrogen acceptor atom but does inhibit 3A4, presumably due to resonance to give the 5-isomer. Less inhibition can clearly be seen in rings with adjacent nitrogens (4-pyrazole, 3- and 4-pyridazine); it has been suggested that an additional acceptor atom protruding towards the binding nitrogen decreases the risk of binding to the haem complex [28]. It is expected that rings containing nitrogens
that are adjacent to a substituent should exhibit lower 3A4 inhibition due to steric hindrance and this is indeed the case for 2pyridine, 2-imidazole, 2-pyrimidine and 2-oxazole. 2-Pyrazine and 5-pyrimidine are noteworthy in that they have exposed nitrogens (in a position analogous to 3-pyridine), but exhibit less 3A4 inhibition. No correlation was observed between the change in 3A4
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Table 3 The impact on nine ADME-related screening assays when a mono-substituted benzene ring is replaced with aromatic heterocycles containing nitrogen and oxygen or sulfur. For a description of the assays and colouring scheme see Table 1.
inhibition and the change in lipophilicity (log D) associated with replacing mono-substituted benzene with the heteroaromatic rings. As stated in the introduction, this analysis has focused on monosubstituted heterocycles in order to maximise the impact that the rings will have on the ADME screens. This has highlighted several rings with exposed nitrogen atoms that do not inhibit CYP450 enzymes as well as some that are particularly prone to exhibit increased CYP450 inhibition. Since the incorporation of steric bulk close to aromatic nitrogens is a well known tactic to reduce such inhibition, it was decided to assess the extent to which methylation could attenuate the effect, and whether by doing so other ADME parameters would be affected. When monosubstituted benzene is replaced with 4-pyridine, 3A4 inhibition increases significantly by 0.87 ± 0.83 log units, n ¼ 542, with 2D6 and 2C19 inhibition also increasing (Table 4). In contrast, when mono-substituted benzene is replaced with 3-methyl-4-pyridine, there is little change in 3A4 inhibition (0.08 ± 0.45, n ¼ 31). In fact, the data in Table 4 shows that methylation adjacent to the pyridine
nitrogen also reduces the 2D6 and 2C19 inhibition liability without impacting other ADME parameters such as solubility and clearance. It is likely therefore that replacing mono-substituted benzene with a nitrogen containing heterocycle with an appropriately placed methyl group may attenuate the CYP450 inhibition without affecting the behaviour in other ADME-related screens. 4.5. Protein binding and permeability Replacing mono-substituted benzene with a heterocycle (or aliphatic carbocycle) modulates protein binding (to human serum albumin) in a way that reflects the change in lipophilicity of the ring, with the correlation coefficient (r) between the change in log D and the change in protein binding being 0.50 (r2 ¼ 0.33, n ¼ 6608). There is little correlation between the change in log D and the change in permeability (r ¼ 0.28, r2 ¼ 0.08, n ¼ 2053), although there is a weak correlation between protein binding and permeability (r ¼ 0.39, r2 ¼ 0.15, n ¼ 1892). The relationship between protein binding and membrane permeability is shown in
Table 4 The effect of replacing mono-substituted benzene with 4-pyridine or 3-methyl-4-pyridine on CYP450 pIC50 and other ADME-related parameters. For a description of the assays and colouring scheme see Table 1.
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Fig. 5. The relatively non-polar carbocyclic rings, thiophenes, furans and pyrroles that decrease log D by less than one log unit have very little impact on protein binding and membrane permeability relative to mono-substituted benzene. The majority of the more polar rings reduce permeability by 0.3e0.5 log units, and decrease protein binding by 0.2e0.7 log units, although there are some outliers: 4/5-imidazole and 4-pyrimidine decrease protein binding by a similar degree (around 0.5 log units) but show significantly different changes in permeability (0.77 and 0.11 log units respectively), presumably due to the increased polarity of the imidazole. 2-Tetrahydropyran is another example where protein binding is decreased (0.48 log units) without impacting permeability too much (0.22 log units). Rings that exhibit the largest mean reduction in protein binding are 3-pyridazine and the cyclic ethers 3-oxetane and 3-tetrahydropyran. 4.6. Metabolic clearance Human and rat clearance data from liver microsomes were only available for a subset of rings (Fig. 6); where less than ten matched pairs were available, data for these rings were not included. Replacing mono-substituted benzene with a heterocycle (or aliphatic carbocycle) modulates clearance (in human and rat liver microsomes) to some extent in line with the lipophilicity of the ring, although the overall correlations between the change in log D and the change in rat/human clearance are weak (rat: r ¼ 0.40,
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r2 ¼ 0.16, n ¼ 454; human: r ¼ 0.30, r2 ¼ 0.09, n ¼ 356). Thus the relatively lipophilic carbocyclic rings, together with the isomers of thiophene and furan, tend to increase clearance although the magnitude of the effect is relatively small (0.1e0.3 log units); monosubstituted furans and thiophenes have the potential to form reactive metabolites [29]. More polar rings tend to reduce clearance, particularly in the case of 2-pyrimidine (rat and human) and 5-pyrimidine (rat). The replacement of mono-substituted benzene with 4-tetrahydropyran results in a significant (p < 0.05) decrease in clearance relative to cyclohexane, again highlighting the beneficial effect of incorporating a polar oxygen atom into a cycloalkane. Although it might be expected that rings that strongly inhibit CYP450 3A4 may block their own metabolism and as a consequence have lower clearance, there was no correlation found between the change in 3A4 inhibition and change in human or rat clearance for this data set. In general, the heterorings behave similarly in rat and human microsomes, with the exception of 5-pyrimidine, which reduces rat clearance but not human clearance. Although the number of 5-pyrimidine pairs are relatively low (15 for human; 16 for rat), they consist of multiple chemical frameworks (13 for human; 14 for rat) with many of the same compounds having been tested in both assays, suggesting that this is a real difference. 4.7. ‘Best’ and ‘worst’ rings With the data in hand, it is possible to highlight particular rings
Fig. 5. Impact on human serum albumin (HSA) protein binding and artificial membrane permeability when a mono-substituted benzene ring is replaced by a heterocycle or carbocycle. Points are sized and coloured by change in log D: large, red points represent rings that increase log D; smaller, green points represent rings that decrease log D. Error bars indicate the standard error of the mean. Cyclic ethers are represented by squares; other rings by circles. The black line indicates a 1:1 relationship between the x- and y-axis. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Fig. 6. Impact on human and rat clearance when a mono-substituted benzene ring is replaced by a heterocycle or carbocycle. The x-axis is sorted by the change in clearance with rings that reduce clearance the most towards the right. Bars indicate the mean change in human (orange) and rat (green) clearance. Error bars indicate the standard error of the mean. An asterisk indicates that the observed change is significantly different from zero (p < 0.05). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
that appear to be the most useful as mono-substituted benzene replacements, although what is considered the ‘best’ ring to use will ultimately depend on the requirements of a particular chemistry project, and which ADME properties are being targeted for improvement. However, it is clear that 3-pyridazine, 2-pyrimidine and 2-imidazole offer significant advantages to mono-substituted benzene across the majority of the ADME readouts particularly with respect to increasing solubility and lowering lipophilicity with a concomitant reduction in CYP450 inhibition (Table 5 top panel). The 2-pyrimidine also decreases clearance significantly (p < 0.05) in both human and rat microsomes. As stated above, some cyclic ethers such as 4-tetrahydropyran also have attractive profiles, and the 2-tetrahydropyran appears to reduce protein binding without decreasing permeability. Perhaps this offers a challenge to chemists to invent more efficient synthetic methodologies to facilitate the preparation of such derivatives. Some useful reviews of synthetic methodologies employed in the preparation of tetrahydrofuranand tetrahydropyran-containing natural products have been published [30,31] and a synthetic summary of four-membered ringcontaining spirocycles, which includes many oxetane examples, is also useful in this regard [32]. There are examples in the literature where reports of solubility-enhancing groups such as bicyclo[1.1.1] pentane [33] have prompted the investigation of new synthetic methods, which have allowed such motifs to become more readily available [34]. Some representative rings that offer little or no advantages, or suffer from liabilities, as replacements for mono-substituted benzene are shown in Table 5 bottom panel. Cyclopentane (and other cycloalkyls rings) and 2-thiophene exhibit ADME profiles very similar to mono-substituted benzene; although these are popular replacements in the data set, they do not appear to lead to improvements in ADME profiles over mono-substituted benzene. 1imidazole and 4-pyrimidine, in contrast to their 2-isomers, suffer
from increased CYP450 inhibition across all three isoforms, whereas the CYP450 liability of 4-pyridine resides mainly in the 3A4 isoform. Whilst these monosubstituted rings have CYP450 issues, as seen with the 4-pyridine it is likely that ring methylation ortho to the exposed nitrogen atom will attenuate the CYP450 inhibition without compromising the behaviour in the other ADME screens. 5. Conclusions Some general conclusions can be drawn from the analysis of the ADME profiles of ring types and regioisomers of the data set described above: Replacing a mono-substituted benzene with a heterocycle has a substantial effect on lipophilicity (up to three orders of magnitude reduction) but a lesser effect on ADME parameters (generally up to one order of magnitude increase or decrease). This is a reflection of the dynamic range of the chromatographic log D assay, which spans 16.6 log units, which is considerably wider than the other assays (3.3e7.1 log units). Rings such as cycloalkanes, thiophenes, furans and pyrroles are popular mono-substituted benzene replacements and make up 42% of this data set. Whilst they may contribute to modulating the binding interactions with target proteins, in reality they make little difference to ADME profiles when used as monosubstituted benzene replacements, and can be considered more as isosteres. Although more difficult to synthesise, cyclic ethers improve all ADME properties apart from permeability and have the most attractive profiles of the rings containing one heteroatom. As expected, replacing mono-substituted benzene with polar heterocycles lower lipophilicity and increase solubility, but the
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Table 5 Examples of rings that have good overall profiles (top panel) and similar or worse profiles (bottom panel) as mono-substituted benzene replacements. For a description of the assays and colouring scheme see Table 1.
impact on these parameters varies considerably (log D by 1.0e3.0 log units; solubility by 0.1e0.7 log units) depending on the type of ring and the regioisomer. In some cases, subtle rearrangements of the heteroatom(s) in the same aromatic ring system can result in significant differences in behaviour. When mono-substituted benzene is replaced with a heterocycle, there is a trend for protein binding, permeability and clearance to decrease in line with the decrease in lipophilicity observed, although the overall correlations are relatively weak (r2 ¼ 0.1e0.3). Although common dogma suggests that aromatic heterocycles with exposed nitrogen atoms are likely to inhibit CYP450 enzymes, based on this data set this is not always the case and certain nitrogen-containing rings can reduce CYP450 inhibition relative to mono-substituted benzene. Examples of nitrogencontaining aromatic heterocycles that significantly improve ADME profiles without incurring any CYP450 liability are 3pyridazine, 2-pyrimidine and 2-imidazole. In the case of rings such as 4-pyridine that exhibit increases in CYP450 inhibition
relative to mono-substituted benzene, methylation adjacent to the ring nitrogen can reduce the CYP450 liability without negatively impacting other ADME parameters. In summary, the results described in this study enable the medicinal chemist to make an informed choice about which rings can be employed as mono-substituted benzene replacements, based upon the knowledge of how such replacements are likely to influence ADME-related parameters, such as solubility, permeability, protein binding, CYP450 inhibition and clearance. Chemists involved with compound library generation and building block preparation can also benefit by using the information to select or prioritise rings and isomers that have the best ADME profiles relative to mono-substituted benzene.
Acknowledgements The authors thank Stephen Pickett for his comments and detailed reviewing of the manuscript, Richard Hatley, John
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Pritchard, Tim Barrett, Ashley Hancock, Pan Procopiou and Gemma White for their useful comments and discussion, and the journal referees for their constructive comments and suggestions. TJR thanks GlaxoSmithKline for funding this analysis. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.ejmech.2016.10.029. References [1] R.D. Taylor, M. MacCoss, A.D.G. Lawson, J. Med. Chem. 57 (2014) 5845. [2] T.J. Ritchie, S.J.F. Macdonald, Drug Discov. Today 14 (2009) 1011. [3] T.J. Ritchie, S.J.F. Macdonald, R.J. Young, S.D. Pickett, Drug Discov. Today 16 (2011) 164. [4] C. Jamieson, E.M. Moir, Z. Rankovic, G. Wishart, J. Med. Chem. 49 (2006) 5029. [5] N. Schneider, D.M. Lowe, R.A. Sayle, M.A. Tarselli, G.A. Landrum, J. Med. Chem. 59 (2016) 4385. [6] T.J. Ritchie, S.J.F. Macdonald, S. Peace, S.D. Pickett, C.N. Luscombe, Med. Chem. Commun. 3 (2012) 1062. [7] M.P. Gleeson, G. Bravi, S. Modi, D. Lowe, Bioorg. Med. Chem. 17 (2009) 5906. [8] D.Y. Haubertin, P. Bruneau, J. Chem. Inf. Model. 47 (2007) 1294. [9] A.G. Dossetter, A. Douglas, C. O'Donnell, Med. Chem. Commun. 3 (2012) 1164. [10] SMIRKS e A Reaction Transform Language: http://www.daylight.com/ dayhtml/doc/theory/theory.smirks.html, Daylight Chemical Information Systems, Inc., Laguna Niguel, CA. (accessed 10 10 2016). [11] S.N. Battachar, L.A. Deschenes, J.A. Wesley, Drug Discov. Today 11 (2006) 1012. [12] K. Valko, Anal. Chem. 69 (1997) 2022. [13] D.F. Veber, S.R. Johnson, H.Y. Cheng, B.R. Smith, K.W. Ward, K.D. Kopple, J. Med. Chem. 45 (2002) 2615.
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European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Heterocyclic replacements for benzene: Maximising ADME benefits by considering individual ring isomers Timothy J. Ritchie a, *, Simon J.F. Macdonald b, ** a b
TJR-Chem, Via Alberto 34C, 21020 Ranco (VA), Italy Fibrosis Discovery Performance Unit, GlaxoSmithKline Medicines Research Centre, Gunnels Wood Road, Stevenage, SG1 2NY, UK
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 September 2016 Received in revised form 11 October 2016 Accepted 14 October 2016 Available online 15 October 2016
The impact of replacing a mono-substituted benzene (phenyl) ring with thirty three aromatic and nine aliphatic heterocycles on nine ADME-related screens (solubility, lipophilicity, permeability, protein binding CYP450 inhibition and metabolic clearance) was assessed using matched molecular pair analysis. The results indicate that the influence on the ADME profile can differ significantly depending on the ring identity and importantly on the individual regioisomers that are possible for some rings. This information enables the medicinal chemist to make an informed choice about which rings and regioisomers to employ as mono-substituted benzene replacements, based upon the knowledge of how such replacements are likely to influence ADME-related parameters, for example to target higher solubility whilst avoiding CYP450 liabilities. © 2016 Elsevier Masson SAS. All rights reserved.
Keywords: Benzene Heteroaromatic Heteroaliphatic Regioisomer ADME
1. Introduction Benzene and heteroaromatic rings are ubiquitous in drug discovery, with benzene and pyridine being the two most commonly found aromatic systems in marketed drugs [1]. However, too many aromatic rings, particularly carboaromatic rings in a molecule increase the likelihood of encountering poor ADME profiles, due to suboptimal physicochemical properties [2,3]. Reducing lipophilicity and increasing solubility are often significant challenges in lead optimisation; adding polar functionality can achieve this, but the increase in the number of heteroatoms may limit membrane permeability and oral bioavailability and introduce other liabilities such as hERG inhibition in the case of positively ionisable amines [4]. An alternative approach is to replace a benzene ring with a heterocycle, and over the last decade or so in the patent literature, more heteroaromatic rings are being incorporated into new chemical entities at the expense of carboaromatic rings [5]. But with many ring types and multiple regioisomers available, it is not always easy to foresee how such changes will simultaneously impact ADME-related parameters such as solubility, lipophilicity,
* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (T.J. Ritchie), simon.jf.macdonald@ gsk.com (S.J.F. Macdonald). http://dx.doi.org/10.1016/j.ejmech.2016.10.029 0223-5234/© 2016 Elsevier Masson SAS. All rights reserved.
protein binding and CYP450 inhibition other than in general terms. The developability of various heterocyclic rings with respect to solubility, protein binding and CYP450 inhibition has been published but the exact substitution pattern and specific regioisomers of the rings were not considered in this study [6]. We felt that combining a comprehensive set of ring replacements with data from multiple ADME readouts would generate valuable quantitative information that would be extremely useful to medicinal chemists who wish to replace a mono-substituted benzene ring and would like to know what to expect when a particular heteroaromatic or heteroaliphatic ring is employed. More specifically, by understanding the impact on ADME parameters across a broad range of heteroring isomers, one can identify approaches where chemists can achieve the maximum benefit of employing individual rings and heteroatoms. Notwithstanding synthetic considerations, shifting ring heteroatoms incurs no molecular weight penalty or change in heteroatom count, but may, for example improve solubility whilst avoiding issues such as increased CYP450 inhibition. Knowing how specific heteroring isomers impact ADME parameters would also assist chemists involved in library design and building block selection, where the most attractive rings and isomers could be prioritised. In order to understand how particular heteroatomic arrangements in rings modulate ADME-related parameters, we have used matched molecular pairs to identify examples from the
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GlaxoSmithKline compound collection where a mono-substituted benzene ring (i.e. a phenyl substituent) has been replaced by a mono-substituted heterocyclic ring, taking into account both the ring identity and all regioisomers that are possible by varying the position of the heteroatom(s) in the ring structure. The resulting pairs were combined with measured data from multiple ADMErelated assays, namely kinetic aqueous solubility, lipophilicity (log D7.4), permeability, protein binding, CYP450 inhibition (3 isoforms) and clearance (two species), to determine how each ADME readout changed for each ring relative to mono-substituted benzene. In order to maximise the impact the ring replacements would have in the ADME assays and thus more easily differentiate the behaviour of the ring types and regioisomers, we chose mono-substituted rings in order to expose the ring heteroatoms as much as possible. In this study, we have not taken any account of how these changes might impact biological activity. If the mono-substituted benzene ring under consideration is interacting with a target predominantly via a hydrophobic interaction, the replacement with a polar heterocycle may result in a loss of binding affinity; however it may still be possible to introduce polar atoms in particular positions, which may be tolerated by the binding pocket. There has been a number of insightful MMP analyses that discuss the impact on ADME parameters of replacing a benzene ring with a heterocycle; these have either focussed on a limited number of heterocycles (often pyridine), or have reported data from only one or two assays. For example, Gleeson et al. highlighted the important fact that the three isomers of pyridine behave differently with respect to their impact on CYP450 3A4 inhibition and solubility, and by considering these separately obtained a much clearer and statistically robust picture of the effects than from a generic phenyl-topyridyl transformation [7]. Haubertin et al. identified benzene replacements such as pyridine, pyrimidine and pyrazine that reduce protein binding [8] and Dossetter et al. investigated to what extent a comprehensive series of heteroaromatic cores modulated in vitro human microsomal metabolic stability when used as replacements for 1,2-, 1,3- and 1,4-disubstituted benzenes [9]. 2. Outline of article After a brief methodology section, the ADME profiles of monosubstituted benzene ring replacements that include one or no heteroatoms will be discussed, followed by aromatic rings containing two nitrogen atoms and then aromatic rings containing nitrogen and oxygen or sulfur. Further sections will then explore the role of nitrogen-containing rings and CYP450 3A4 inhibition, the impact of mono-substituted benzene ring replacements on developability properties such as permeability and protein binding, and finally metabolic clearance. Lastly, the best and worst rings identified in this study are discussed. 3. Methodology A set of SMIRKS [10] queries were generated to search for matched molecular pairs where a mono-substituted benzene is transformed into a monosubstituted heteroaromatic, heteroaliphatic or carboaliphatic ring, taking into account all regioisomers that are possible. In this way, the following mono-substituted benzene-to-heteroring pairs, in order of abundance were identified: pyridine (3 isomers; 5661 pairs), cycloalkanes (cyclohexane, cyclopentane, cyclobutane) (3468), thiophene (2 isomers; 2190), furan (2 isomers; 1182), tetrahydropyran (3 isomers; 683), thiazole (3 isomers; 645), pyrimidine (3 isomers; 637), pyrazole (4 isomers; 531), tetrahydrofuran (2 isomers; 365), imidazole (4 isomers; 330), pyrazine (1 isomer; 269), pyrrole (3 isomers; 146), oxazole (3 isomers; 137), pyridazine (2 isomers; 120), isoxazole (3 isomers; 96)
and oxetane (60). The identity of the attachment atom was not specified in the queries, but analysis of the results indicated that the most common attachment atom in the data set was an aliphatic carbon (40.5%), followed by an aromatic carbon (26.2%), nitrogen (15.1%), carbonyl carbon (11.7%), sulfur (3.9%) and oxygen (2.6%). Data from nine high-throughput ADME-related screening assays were then collected for the matched pairs, so that the change in each assay could be determined for each mono-substituted benzene-to-ring transform. The ADME readouts were kinetic aqueous solubility [11], chromatographically determined log D7.4 values [12], artificial membrane permeability [13], human serum albumin plasma protein binding [14], CYP450 inhibition assays (2D6, 3A4 and 2C19 isoforms) [15], and in vitro metabolic clearance (where the compound is incubated in a human or rat liver microsome preparation and its depletion measured over time) [16]. Due to the lack of pairs with sufficient screening data, triazoles, oxadiazoles, thiadiazoles, isothiazoles and lactams were omitted from the study, as well as strongly ionisable rings such as tetrazoles and cyclic amines such as piperidine and morpholine etc. Data were also excluded in the case where assay results were outside the dynamic range of the screens (i.e. reported as greater than or less than a particular value). For each mono-substituted benzene ring replacement, statistical measures, such as mean change, standard deviation and standard error of the mean were calculated for each screening assay, as well as the number of matched molecular pairs and the number of chemical series (as defined by Bemis-Murkco frameworks) that were represented. Results from the log D and CYP450 assays (pIC50 values) were used directly, whereas a log10 transformation was carried out on the results from the solubility (mol/L), permeability (m/s) and clearance (ml/min/g tissue) assays. A logit transformation was applied to the % protein binding data. It should be noted that the dynamic range varies considerably between the various assays: the chromatographic log D measurements covers the widest range of values (16.6 log units), followed by CYP450 3A4 (7.1), rat clearance (6.1), 2D6 (6.1), 2C19 (5.4), solubility (5.2), protein binding (4.2), human clearance (3.4) and permeability (3.3). A paired t-test (for normally distributed values) or a signed-rank test (for non-normally distributed values) were used [17] to determine whether the observed changes were significantly different from zero. Due to the potential errors surrounding data analysis of matched pairs with limited examples [18], it was decided that where there were less than 10 matched pairs available, these rings are not shown in the plots described in the results section, although the mean values and pair counts are included in the data tables for completeness. It should also be noted that changes of ±0.2e0.3 log units may fall within the experimental error associated with the screening assay protocols, even if they are statistically significantly different from zero. Statistical values for the change in each ADME screen for each ring regioisomer are provided in the Supporting Information. The majority of rings were connected to the rest of the molecule in the most part via a carbon atom, with relatively few examples with heteroatom-linked rings that have the potential to modulate ring properties such as basicity and lipophilicity. However, one third of pyridazines and pyrimidines and half of the oxetanes were N-linked. Although there were some differences in the ADME data for these N-linked rings relative to the carbon-linked exemplars, they were not significantly different. 4. Results 4.1. Rings with one or no heteroatoms e the benefits of cyclic ethers The most conservative mono-substituted benzene replacement is the isosteric thiophene ring [19], of which two isomers are
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possible, followed by other aromatic rings with one heteroatom namely pyrrole (three isomers), furan (two isomers) and pyridine (three isomers). The pyridines reduce log D significantly (p < 0.05,
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Tukey-Kramer method [20]) more than the pyrroles, due to the available nitrogen lone pair, which does not contribute to the aromatic sextet, but in terms of increasing solubility both ring types
Fig. 1. A comparison of the impact on solubility, lipophilicity and CYP450 3A4 inhibition when a mono-substituted benzene ring is replaced with heterocycles containing a single heteroatom or a cycloalkyl ring. Points are coloured by the observed change in CYP450 3A4 pIC50 (red ¼ increase; white ¼ no change; green ¼ decrease, relative to monosubstituted benzene). THF ¼ Tetrahydrofuran; THP ¼ Tetrahydropyran. Error bars indicate the standard error of the mean. Points further to the right and lower in the plots indicate rings that exhibit larger average increases in solubility and decreases in log D. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Table 1 The impact on nine ADME-related screening assays when a mono-substituted benzene ring is replaced with the three regioisomers of pyridine and two representative cyclic ethers. Values quoted are the average change observed in each assay: Aqueous solubility (SOL), artificial membrane permeability (PERM) and human or rat clearance (Cl) data are on a logarithmic scale (i.e. a change of þ1.00 would indicate a 10-fold increase), and a logit transformation is applied to the human serum albumin (HSA) % protein binding data; CYP450 data are pIC50 values.Values in italicised bold type indicate changes that are not statistically different from zero based on a paired t-test (for normally distributed values) or a signed-rank test (for non-normally distributed values). Cells are colour-coded with a gradient (change >1 red (detrimental); change ¼ 0 white; change <1 green (beneficial)), except for solubility and permeability where the colours are reversed. Where fewer than 10 matched pairs were available, the values are stated but greyed out and the cells are not colour-coded.
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behave similarly. Cyclic ethers such as oxetane, tetrahydrofuran and tetrahydropyran are useful solubilising motifs in medicinal chemistry [21] and have advantages over the aromatic rings that contain a single heteroatom, in part due to their higher hydrogen-bond basicities [22]. Fig. 1 shows the change in log solubility and log D when a mono-substituted benzene ring is replaced with these rings: in general, the increase in solubility for each ring mirrors the concomitant reduction in lipophilicity, although the correlation between the change in solubility and the change in lipophilicity is weak (r ¼ 0.36, r2 ¼ 0.13, n ¼ 13,695). Tetrahydrofuran and tetrahydropyran isomers outperform the aromatic thiophenes, furans and pyrroles in terms of lowering lipophilicity and increasing solubility, and benefit also from reducing CYP450 3A4 inhibition. This latter behaviour is in contrast to the increase in 3A4 inhibition that is observed when mono-substituted benzene is replaced with the 3- and 4-isomers of pyridine. Although it has been reported that there is generally no bias in the distribution of pyridine isomers in chemical collections and reagents [23], the 2-isomer appears to be more useful in that it lowers log D and increases solubility without the 3A4 liability of the 3- and 4-isomers. In comparison, the average values seen when replacing mono-substituted benzene with a cycloalkyl group does not improve ADMET properties, with cyclohexane and cyclopentane actually increasing log D. For the cyclic ethers, it is possible to modulate solubility and lipophilicity to a
significant extent by modifying the environment of the single oxygen atom in terms of ring angle and/or position in the ring relative to the attachment atom. The changes in log D seen with the cyclic ethers and furans are in line with the solvent accessible polar surface area (PSA) associated with the oxygen atom (calculated in MOE 2014.09 [24] using the lowest energy conformer for phenylsubstituted cyclic ethers and furans using the MMFF94x forcefield [25] and LowMode MD conformational search method), with more solvent-exposed oxygen atoms resulting in a larger reduction in log D. Topological 2-D PSA calculations [26] are not useful in this case as the oxygen atom in all the cyclic ethers are treated identically by this algorithm. In the current data set, pyridine is a very common monosubstituted benzene replacement; cyclic ethers less so, presumably due to the not insignificant synthetic considerations of these rings and the chiral nature of some of the tetrahydrofuran and tetrahydropyran moieties. However their ADME profiles would suggest that these rings should be considered as useful monosubstituted benzene or even pyridine replacements: Pyridines and cyclic ethers significantly increase solubility and lower log D, and it is interesting to compare the most soluble ethers with the isomers of pyridine across all the ADME assays being studied in order to give a complete picture of the similarities and differences observed (Table 1). 4-Tetrahydropyran and 3-tetrahydrofuran have
Fig. 2. Impact on log solubility and lipophilicity when a mono-substituted benzene ring is replaced with an aromatic ring containing two nitrogen atoms. Pyridine isomers are included as a reference set (cf. Fig. 1). Points are coloured by the observed change in CYP450 3A4 pIC50 (red ¼ increase; white ¼ no change; green ¼ decrease, relative to monosubstituted benzene). Error bars indicate the standard error of the mean. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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generally similar profiles to the pyridines but differ in that they lack the CYP450 liability seen particularly in the 4-pyridine isomer. It is also interesting to note that the ethers and pyridines reduce permeability to the same extent relative to mono-substituted benzene but the ethers are significantly (p < 0.05) less protein bound than the pyridines. As a general trend, the reduction of lipophilicity associated with the replacement of mono-substituted benzene with a more polar heterocycle decreases artificial membrane permeability and plasma protein binding, and is discussed in more detail later for the complete set of ring replacements. In addition, 4-tetrahydropyran reduces log D significantly (p < 0.05) less than 4-pyridine (1.40 and 1.92 units respectively), in line with their hydrogen bond basicity strengths [22] but has significantly (p < 0.05) higher solubility (þ0.62 and þ0.42 log units respectively), possibly due to its higher sp3 character [27]. 3Tetrahydrofuran appears to reduce clearance the most but more pairs would help to confirm this trend. 4.2. Aromatics rings containing two nitrogen atoms as monosubstituted benzene replacements There are fourteen permutations possible to replace a monosubstituted benzene with a heteroaromatic ring containing two nitrogen atoms, employing the 5-membered rings imidazole (4
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regioisomers) or pyrazole (4), or the 6-membered rings pyrazine (1), pyrimidine (3) or pyridazine (2). However, due to prototropic tautomerism between the 4- and 5-isomers of imidazole via a hydrogen shift, data for these forms were combined, leaving 3 isomers rather than 4. For the same reason, data for the 3- and 5isomers of pyrazole were also merged. Whilst it is not surprising that replacing a mono-substituted benzene ring with these polar heterocycles results in lower lipophilicity and higher solubility, the magnitude of the changes observed differ considerably depending on the type of ring and regioisomer (Fig. 2 and Table 2). In general, imidazoles lower log D to the largest extent, due to their higher basicity, although how this increases solubility varies, with the more sterically hindered 2-isomer having a significantly lesser impact than its 1- and 4/5-isomers. 1-Pyrazole, which lacks a free NH group, reduces log D significantly (p < 0.05) less than the other pyrazole isomers but has a similar impact on solubility; this relative difference is not seen in the case of 1-imidazole, presumably due to the fact that the other nitrogen is more exposed in the latter case. In the six-membered ring congeners, a mono-substituted benzene-topyridazine transformation reduces log D more than pyrimidine and pyrazine, although again the behaviour varies depending on the regioisomer. Thus the specific position of the two nitrogens relative to the attachment atom can have an influence on solubility over and above that due to the decrease in lipophilicity.
Table 2 The impact on nine ADME-related screening assays when a mono-substituted benzene ring is replaced with aromatic heterocycles containing two nitrogen atoms. For a description of the assays and colouring scheme see Table 1. *Due to prototropic tautomerism between the 4- and 5-imidazoles, and the 3- and 5-pyrazoles, the data for these isomers were merged. The asterisk indicates the alternative position of the hydrogen atom in these two cases.
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The impact on membrane permeability and protein binding for these ring types is discussed later; no correlation was found between the change in log D and the change in membrane permeability or protein binding. In terms of CYP450 inhibition there are also significant differences (Fig. 4): imidazoles are well known to inhibit CYP450 enzymes, which is the case here although each isomer exhibits a different profile across the 3A4, 2D6 and 2C19 isoforms. The 2-isomer however, appears to have lower CYP450 inhibition than mono-substituted benzene across all three isoforms, due to the increased steric hindrance of the nitrogen atom. Pyrimidines can exhibit 3A4 inhibition depending on the isomer, with the 4- and 2-isomers showing the most and least inhibition respectively. Rings with the nitrogen atoms adjacent (pyrazoles and pyridazines) do not inhibit 3A4 or they reduce inhibition relative to mono-substituted benzene, highlighting that subtle modifications in atom position can have a significant impact on ADME profiles. Further discussion of CYP450 inhibition follows below. Due to lack of pairs, it is not possible to assess the impact of the majority of these rings as mono-substituted benzene replacements in terms of clearance, however 2-pyrimidine appears to reduce both human and rat clearance (Table 2). 4.3. Rings containing nitrogen, oxygen and sulfur As expected thiazoles lower log D less than oxazoles, particularly in the 2-isomers; The 5-substituted isomers of oxazole and thiazole exhibit increased CYP450 3A4 inhibition relative to monosubstituted benzene (Fig. 3 and Table 3) due to the exposed nitrogen atom, but this effect appears to be ameliorated in the 5-
isoxazole with the caveat that the number of pairs are small, again highlighting the benefit of rings with adjacent heteroatoms. 4.4. CYP450 inhibition of nitrogen-containing aromatic rings It is to be expected that monosubstituted nitrogen-containing heteroaromatic rings are more likely to inhibit CYP450 enzymes when a nitrogen acceptor atom is more accessible to the enzyme i.e. not ortho to the attachment atom. However the analysis results indicate that this behaviour is heavily dependent on the type of ring and regioisomer: Fig. 4 describes the percentage of ring transforms that exhibit changes in 3A4 inhibition, from a strong increase (>1.5 log units in red), a smaller increase (0.3e1.5, orange), little or no change (0.3 to 0.3, yellow), a small decrease (1.5 to 0.3, pale green) and strong decrease (<1.5, dark green). The 3A4 isoform was chosen as more drugs interact with this enzyme and more data for the ring replacements were available from this screen. Where less than 10 pairs were available for particular ring isomers, these data are not shown. This presentation allows the differences between rings and isomers to be differentiated in more detail than just comparing mean values. For example, 5-oxazole and 4-pyrimidine both increase 3A4 inhibition by the same amount (0.65 and 0.64 log units respectively) but the percentage of pairs that exhibit a strong (>1.5 log units) increase differs (11% and 37% respectively). Rings with an exposed nitrogen atom flanked by a CH group tend to inhibit 3A4 relative to mono-substituted benzene, with 1imidazole, 4-pyrimidine and 4-pyridine having the highest percentages showing a strong (>1.5 log units) increase, and the highest mean changes. As drawn, 4-imidazole does not have an exposed
Fig. 3. Impact on log solubility and lipophilicity when a mono-substituted benzene ring is replaced with an aromatic ring containing nitrogen and oxygen or sulfur. Pyridine isomers are included as a reference set (see Fig. 1). Points are coloured by the observed change in CYP450 3A4 pIC50 (red ¼ increase; white ¼ no change; green ¼ decrease; grey ¼ no data; relative to mono-substituted benzene). Error bars indicate the standard error of the mean. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Fig. 4. Impact on CYP450 3A4 pIC50 when a mono-substituted benzene ring is replaced with a nitrogen-containing aromatic ring. Top panel: 5-membered rings; bottom panel: 6-membered rings. The coloured bars indicate the percentage of the matched pairs that exhibit a strong increase (>1.5 log units, red), a smaller increase (0.3e1.5, orange), little or no change (0.3 to 0.3, yellow), a small decrease (1.5 to 0.3, pale green) and strong decrease (<1.5, dark green). The values above the bars indicate the mean change in 3A4 inhibition for each ring type: values with an asterisk indicate changes that are significantly different from zero (p < 0.05). *Due to prototropic tautomerism between the 4- and 5-imidazoles, and the 3- and 5-pyrazoles, the data for these isomers were merged. The asterisks indicate the alternative position of the hydrogen atom in these two cases. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
nitrogen acceptor atom but does inhibit 3A4, presumably due to resonance to give the 5-isomer. Less inhibition can clearly be seen in rings with adjacent nitrogens (4-pyrazole, 3- and 4-pyridazine); it has been suggested that an additional acceptor atom protruding towards the binding nitrogen decreases the risk of binding to the haem complex [28]. It is expected that rings containing nitrogens
that are adjacent to a substituent should exhibit lower 3A4 inhibition due to steric hindrance and this is indeed the case for 2pyridine, 2-imidazole, 2-pyrimidine and 2-oxazole. 2-Pyrazine and 5-pyrimidine are noteworthy in that they have exposed nitrogens (in a position analogous to 3-pyridine), but exhibit less 3A4 inhibition. No correlation was observed between the change in 3A4
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Table 3 The impact on nine ADME-related screening assays when a mono-substituted benzene ring is replaced with aromatic heterocycles containing nitrogen and oxygen or sulfur. For a description of the assays and colouring scheme see Table 1.
inhibition and the change in lipophilicity (log D) associated with replacing mono-substituted benzene with the heteroaromatic rings. As stated in the introduction, this analysis has focused on monosubstituted heterocycles in order to maximise the impact that the rings will have on the ADME screens. This has highlighted several rings with exposed nitrogen atoms that do not inhibit CYP450 enzymes as well as some that are particularly prone to exhibit increased CYP450 inhibition. Since the incorporation of steric bulk close to aromatic nitrogens is a well known tactic to reduce such inhibition, it was decided to assess the extent to which methylation could attenuate the effect, and whether by doing so other ADME parameters would be affected. When monosubstituted benzene is replaced with 4-pyridine, 3A4 inhibition increases significantly by 0.87 ± 0.83 log units, n ¼ 542, with 2D6 and 2C19 inhibition also increasing (Table 4). In contrast, when mono-substituted benzene is replaced with 3-methyl-4-pyridine, there is little change in 3A4 inhibition (0.08 ± 0.45, n ¼ 31). In fact, the data in Table 4 shows that methylation adjacent to the pyridine
nitrogen also reduces the 2D6 and 2C19 inhibition liability without impacting other ADME parameters such as solubility and clearance. It is likely therefore that replacing mono-substituted benzene with a nitrogen containing heterocycle with an appropriately placed methyl group may attenuate the CYP450 inhibition without affecting the behaviour in other ADME-related screens. 4.5. Protein binding and permeability Replacing mono-substituted benzene with a heterocycle (or aliphatic carbocycle) modulates protein binding (to human serum albumin) in a way that reflects the change in lipophilicity of the ring, with the correlation coefficient (r) between the change in log D and the change in protein binding being 0.50 (r2 ¼ 0.33, n ¼ 6608). There is little correlation between the change in log D and the change in permeability (r ¼ 0.28, r2 ¼ 0.08, n ¼ 2053), although there is a weak correlation between protein binding and permeability (r ¼ 0.39, r2 ¼ 0.15, n ¼ 1892). The relationship between protein binding and membrane permeability is shown in
Table 4 The effect of replacing mono-substituted benzene with 4-pyridine or 3-methyl-4-pyridine on CYP450 pIC50 and other ADME-related parameters. For a description of the assays and colouring scheme see Table 1.
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Fig. 5. The relatively non-polar carbocyclic rings, thiophenes, furans and pyrroles that decrease log D by less than one log unit have very little impact on protein binding and membrane permeability relative to mono-substituted benzene. The majority of the more polar rings reduce permeability by 0.3e0.5 log units, and decrease protein binding by 0.2e0.7 log units, although there are some outliers: 4/5-imidazole and 4-pyrimidine decrease protein binding by a similar degree (around 0.5 log units) but show significantly different changes in permeability (0.77 and 0.11 log units respectively), presumably due to the increased polarity of the imidazole. 2-Tetrahydropyran is another example where protein binding is decreased (0.48 log units) without impacting permeability too much (0.22 log units). Rings that exhibit the largest mean reduction in protein binding are 3-pyridazine and the cyclic ethers 3-oxetane and 3-tetrahydropyran. 4.6. Metabolic clearance Human and rat clearance data from liver microsomes were only available for a subset of rings (Fig. 6); where less than ten matched pairs were available, data for these rings were not included. Replacing mono-substituted benzene with a heterocycle (or aliphatic carbocycle) modulates clearance (in human and rat liver microsomes) to some extent in line with the lipophilicity of the ring, although the overall correlations between the change in log D and the change in rat/human clearance are weak (rat: r ¼ 0.40,
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r2 ¼ 0.16, n ¼ 454; human: r ¼ 0.30, r2 ¼ 0.09, n ¼ 356). Thus the relatively lipophilic carbocyclic rings, together with the isomers of thiophene and furan, tend to increase clearance although the magnitude of the effect is relatively small (0.1e0.3 log units); monosubstituted furans and thiophenes have the potential to form reactive metabolites [29]. More polar rings tend to reduce clearance, particularly in the case of 2-pyrimidine (rat and human) and 5-pyrimidine (rat). The replacement of mono-substituted benzene with 4-tetrahydropyran results in a significant (p < 0.05) decrease in clearance relative to cyclohexane, again highlighting the beneficial effect of incorporating a polar oxygen atom into a cycloalkane. Although it might be expected that rings that strongly inhibit CYP450 3A4 may block their own metabolism and as a consequence have lower clearance, there was no correlation found between the change in 3A4 inhibition and change in human or rat clearance for this data set. In general, the heterorings behave similarly in rat and human microsomes, with the exception of 5-pyrimidine, which reduces rat clearance but not human clearance. Although the number of 5-pyrimidine pairs are relatively low (15 for human; 16 for rat), they consist of multiple chemical frameworks (13 for human; 14 for rat) with many of the same compounds having been tested in both assays, suggesting that this is a real difference. 4.7. ‘Best’ and ‘worst’ rings With the data in hand, it is possible to highlight particular rings
Fig. 5. Impact on human serum albumin (HSA) protein binding and artificial membrane permeability when a mono-substituted benzene ring is replaced by a heterocycle or carbocycle. Points are sized and coloured by change in log D: large, red points represent rings that increase log D; smaller, green points represent rings that decrease log D. Error bars indicate the standard error of the mean. Cyclic ethers are represented by squares; other rings by circles. The black line indicates a 1:1 relationship between the x- and y-axis. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Fig. 6. Impact on human and rat clearance when a mono-substituted benzene ring is replaced by a heterocycle or carbocycle. The x-axis is sorted by the change in clearance with rings that reduce clearance the most towards the right. Bars indicate the mean change in human (orange) and rat (green) clearance. Error bars indicate the standard error of the mean. An asterisk indicates that the observed change is significantly different from zero (p < 0.05). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
that appear to be the most useful as mono-substituted benzene replacements, although what is considered the ‘best’ ring to use will ultimately depend on the requirements of a particular chemistry project, and which ADME properties are being targeted for improvement. However, it is clear that 3-pyridazine, 2-pyrimidine and 2-imidazole offer significant advantages to mono-substituted benzene across the majority of the ADME readouts particularly with respect to increasing solubility and lowering lipophilicity with a concomitant reduction in CYP450 inhibition (Table 5 top panel). The 2-pyrimidine also decreases clearance significantly (p < 0.05) in both human and rat microsomes. As stated above, some cyclic ethers such as 4-tetrahydropyran also have attractive profiles, and the 2-tetrahydropyran appears to reduce protein binding without decreasing permeability. Perhaps this offers a challenge to chemists to invent more efficient synthetic methodologies to facilitate the preparation of such derivatives. Some useful reviews of synthetic methodologies employed in the preparation of tetrahydrofuranand tetrahydropyran-containing natural products have been published [30,31] and a synthetic summary of four-membered ringcontaining spirocycles, which includes many oxetane examples, is also useful in this regard [32]. There are examples in the literature where reports of solubility-enhancing groups such as bicyclo[1.1.1] pentane [33] have prompted the investigation of new synthetic methods, which have allowed such motifs to become more readily available [34]. Some representative rings that offer little or no advantages, or suffer from liabilities, as replacements for mono-substituted benzene are shown in Table 5 bottom panel. Cyclopentane (and other cycloalkyls rings) and 2-thiophene exhibit ADME profiles very similar to mono-substituted benzene; although these are popular replacements in the data set, they do not appear to lead to improvements in ADME profiles over mono-substituted benzene. 1imidazole and 4-pyrimidine, in contrast to their 2-isomers, suffer
from increased CYP450 inhibition across all three isoforms, whereas the CYP450 liability of 4-pyridine resides mainly in the 3A4 isoform. Whilst these monosubstituted rings have CYP450 issues, as seen with the 4-pyridine it is likely that ring methylation ortho to the exposed nitrogen atom will attenuate the CYP450 inhibition without compromising the behaviour in the other ADME screens. 5. Conclusions Some general conclusions can be drawn from the analysis of the ADME profiles of ring types and regioisomers of the data set described above: Replacing a mono-substituted benzene with a heterocycle has a substantial effect on lipophilicity (up to three orders of magnitude reduction) but a lesser effect on ADME parameters (generally up to one order of magnitude increase or decrease). This is a reflection of the dynamic range of the chromatographic log D assay, which spans 16.6 log units, which is considerably wider than the other assays (3.3e7.1 log units). Rings such as cycloalkanes, thiophenes, furans and pyrroles are popular mono-substituted benzene replacements and make up 42% of this data set. Whilst they may contribute to modulating the binding interactions with target proteins, in reality they make little difference to ADME profiles when used as monosubstituted benzene replacements, and can be considered more as isosteres. Although more difficult to synthesise, cyclic ethers improve all ADME properties apart from permeability and have the most attractive profiles of the rings containing one heteroatom. As expected, replacing mono-substituted benzene with polar heterocycles lower lipophilicity and increase solubility, but the
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Table 5 Examples of rings that have good overall profiles (top panel) and similar or worse profiles (bottom panel) as mono-substituted benzene replacements. For a description of the assays and colouring scheme see Table 1.
impact on these parameters varies considerably (log D by 1.0e3.0 log units; solubility by 0.1e0.7 log units) depending on the type of ring and the regioisomer. In some cases, subtle rearrangements of the heteroatom(s) in the same aromatic ring system can result in significant differences in behaviour. When mono-substituted benzene is replaced with a heterocycle, there is a trend for protein binding, permeability and clearance to decrease in line with the decrease in lipophilicity observed, although the overall correlations are relatively weak (r2 ¼ 0.1e0.3). Although common dogma suggests that aromatic heterocycles with exposed nitrogen atoms are likely to inhibit CYP450 enzymes, based on this data set this is not always the case and certain nitrogen-containing rings can reduce CYP450 inhibition relative to mono-substituted benzene. Examples of nitrogencontaining aromatic heterocycles that significantly improve ADME profiles without incurring any CYP450 liability are 3pyridazine, 2-pyrimidine and 2-imidazole. In the case of rings such as 4-pyridine that exhibit increases in CYP450 inhibition
relative to mono-substituted benzene, methylation adjacent to the ring nitrogen can reduce the CYP450 liability without negatively impacting other ADME parameters. In summary, the results described in this study enable the medicinal chemist to make an informed choice about which rings can be employed as mono-substituted benzene replacements, based upon the knowledge of how such replacements are likely to influence ADME-related parameters, such as solubility, permeability, protein binding, CYP450 inhibition and clearance. Chemists involved with compound library generation and building block preparation can also benefit by using the information to select or prioritise rings and isomers that have the best ADME profiles relative to mono-substituted benzene.
Acknowledgements The authors thank Stephen Pickett for his comments and detailed reviewing of the manuscript, Richard Hatley, John
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Pritchard, Tim Barrett, Ashley Hancock, Pan Procopiou and Gemma White for their useful comments and discussion, and the journal referees for their constructive comments and suggestions. TJR thanks GlaxoSmithKline for funding this analysis. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.ejmech.2016.10.029. References [1] R.D. Taylor, M. MacCoss, A.D.G. Lawson, J. Med. Chem. 57 (2014) 5845. [2] T.J. Ritchie, S.J.F. Macdonald, Drug Discov. Today 14 (2009) 1011. [3] T.J. Ritchie, S.J.F. Macdonald, R.J. Young, S.D. Pickett, Drug Discov. Today 16 (2011) 164. [4] C. Jamieson, E.M. Moir, Z. Rankovic, G. Wishart, J. Med. Chem. 49 (2006) 5029. [5] N. Schneider, D.M. Lowe, R.A. Sayle, M.A. Tarselli, G.A. Landrum, J. Med. Chem. 59 (2016) 4385. [6] T.J. Ritchie, S.J.F. Macdonald, S. Peace, S.D. Pickett, C.N. Luscombe, Med. Chem. Commun. 3 (2012) 1062. [7] M.P. Gleeson, G. Bravi, S. Modi, D. Lowe, Bioorg. Med. Chem. 17 (2009) 5906. [8] D.Y. Haubertin, P. Bruneau, J. Chem. Inf. Model. 47 (2007) 1294. [9] A.G. Dossetter, A. Douglas, C. O'Donnell, Med. Chem. Commun. 3 (2012) 1164. [10] SMIRKS e A Reaction Transform Language: http://www.daylight.com/ dayhtml/doc/theory/theory.smirks.html, Daylight Chemical Information Systems, Inc., Laguna Niguel, CA. (accessed 10 10 2016). [11] S.N. Battachar, L.A. Deschenes, J.A. Wesley, Drug Discov. Today 11 (2006) 1012. [12] K. Valko, Anal. Chem. 69 (1997) 2022. [13] D.F. Veber, S.R. Johnson, H.Y. Cheng, B.R. Smith, K.W. Ward, K.D. Kopple, J. Med. Chem. 45 (2002) 2615.
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