Beyond Rule of Five – A MedChemica Review

Recent bRo5 publications

Figure 1: Picture summarising the limitations of obeying the Ro5 and the versatility obtained by going beyond (www.sciencedirect.com/science/article/abs/pii/S1367593117300480?via%3Dihub).

It’s been more than 25 years since a Pfizer chemist: Christopher Lipinski, published¹ his influential analysis of the calculated physical properties of a set of candidate drugs, leading to the well-known “rule of 5” (Ro5) designed as an aid in filtering out regions of chemical space that are less likely to see success as an orally absorbed drug. The rule states that for a compound to be a likely successful lead candidate, orally absorbed, it must obey at least 3 of the following: <5 hydrogen bond donor groups, <10 hydrogen bond acceptor groups, a molecular mass of <500 Da and calculated lipophilicity (clogP) of <5.

Practically speaking, this set of guidelines seeks to eliminate compounds that would be too polar, too big or too insoluble to be absorbed efficiently in the gut after oral dosing. While it does well to eliminate those compounds that are clearly hopeless (e.g. hydrocarbons) from certain high throughput (in silico) screening methods. If taken literally, numerous “borderline” compounds that would make fantastic orally available lead compounds would get binned too. Indeed, a large portion of currently marketed oral drugs do not comply with these rules. This has lead to somewhat of a rebellion in the industry, against the Ro5, with Lipinski himself publishing a more relaxed characterisation² of orally absorbed drugs leading to such terms as the “extended Ro5” (eRo5) and “beyond Ro5” (bRo5).

A number of our clients are interested in the potential for exploring Ro5-noncompliant space as a path that is perhaps less well-explored, investigating therapeutic modalities such as: Proteolysis Targeting Chimeras (PROTACS),^3–5 protein-protein interaction (PPI) inhibitors/actuators^2,6,7 and macrocycles.^4,8–13 Exploiting bRo5 space does offer advantages over traditional small molecule approaches, the most obvious being that with larger molecules comes the increased dependence on intramolecular interactions leading to a more defined conformation in solution. Of course, conformational analysis is important for small molecule medicinal chemistry too but in bRo5 space, you begin to be able to mimic regular secondary structure motifs found in biological macromolecules (proteins/enzymes, nucleic acids etc.) such as ɑ-helices, β-strands, reverse turns and so on². This offers the possibility of targeting complementary sites on biological targets that span large surface areas. These are targets that would traditionally be described as smooth, featureless, flat, flexible and importantly: “undruggable”. An example of where this consideration is increasingly making a difference is the targeting of PPIs. The human proteome is expected to include up to ~1,000,000 different PPIs,⁷ and is thus expected to become one of the most important sources of novel targets for drug discovery, going forward. The historical bias against Ro5-noncompliant compounds is thought to be why most high-throughput screening campaigns targeting PPIs fail¹

Figure 2: Cyclosporin, nHBD: 5, nHBA: 12, molecular mass: 1203 Da, CX clogP: 3.38. Oral bioavailability is typically ~30%, but varies depending on many factors, including formulation.

Another feature of bRo5 compounds is the greater potential for environment-dependent conformational polymorphism, otherwise known as “molecular chameleonicity”.12,13 The character of this phenomenon and its significance in the context of drug absorption is still being investigated today. Simply put, bRo5 compounds typically have the ability to dynamically shield portions of their polar surface area (something that smaller compounds typically can’t do), allowing them to assume a vastly different chemical profile under different conditions. This is crucial when it comes to drug absorption in the gut, as the compound in question may not otherwise display sufficient permeability. Chameleonicity offers a neat explanation as to how Cyclosporin (a natural product that tramples on the Ro5) shows appreciable oral bioavailability, it can curl up when in the polar environment of the gastrointestinal tract due to it’s intramolecular interactions, leading to a diminished molecular profile, good membrane permeability and observed bioavailability¹⁴.

As with most things, there is an upper limit to the chemical space characterised by bRo5 (aside from a handful of maverick natural products, such as cyclosporin, above). This has been well described by Kihlberg & coworkers⁷, based on an analysis of recently approved oral drugs, as:

With technological advances in the fields of: chemical synthesis & purification, biochemical testing and screening techniques, bRo5 compounds are increasingly seen in medicinal chemistry projects and indeed the clinic. We expect that the investigation of bRo5 space has huge potential to satisfy hitherto unmet medical need, and will continue to attract high levels of interest from big players in the industry. With that in mind, nothing comes without risk. The development of an orally administered bRo5 drug requires care and skilful navigation of inherent potential liabilities, including solubility/permeability balance, CNS penetration, metabolic stability and more. Compounds must be subjected to timely and accurate ADMET and PK/PD studies to develop structure-activity relationship data that will truly guide the project and inform the investigator about fatal issues early. One of MedChemica’s specialities is the management of this critical stage of optimisation, and overseeing biochemical testing logistics with trusted partners across the globe. If you are interested in getting tailored and effective advice for your bRo5 drug-hunting project, do get in touch!

(1) Lipinski, C. A. et al. Adv. Drug Deliv. Rev. 1997, 23 (1–3), 3–25.

(2) Lipinski, C. A. et al. Adv. Drug Deliv. Rev. 2016, 101, 34–41.

(3) Sun, X. et al. Signal Transduct. Target. Ther. 2019, 4 (1), 64.

(4) Testa, A. et al. Angew. Chem. Int. Ed. 2020, 59 (4), 1727–1734.

(5) Ermondi, G. et al. ACS Med. Chem. Lett. 2021, 12 (7), 1056–1060.

(6) Egbert, M. et al. J. Med. Chem. 2019, 62 (22), 10005–10025.

(7) Doak, B. C. et al. J. Med. Chem. 2016, 59 (6), 2312–2327. And references therein.

(9) Giordanetto, F. et al. J. Med. Chem. 2014, 57 (2), 278–295.

(10) Rosenquist, Å. et al. J. Med. Chem. 2014, 57 (5), 1673–1693.

(11) Mackman, R. L. et al. J. Med. Chem. 2018, 61 (21), 9473–9499.

(12) Tyagi, M. et al. Org. Lett. 2018, 20 (18), 5737–5742.

(13) Rezai, T. et al. J. Am. Chem. Soc. 2006, 128 (43), 14073–14080.

(14) Faulds, D. et al. Drugs 1993, 45 (6), 953–1040.

Additional recent papers