What makes molecules aggregate?

The propensity for some small molecules to form aggregates in water has bedeviled fragment-finding efforts for decades. Indeed, the phenomenon was not fully recognized until early this century. Although plenty of tools are available for detecting aggregates, I still see too many papers that omit these crucial quality controls. As annoying as aggregation can be in activity assays, in certain cases it could actually be useful for formulating drugs. There has been speculation that the good oral bioavailability of venetoclax is due to aggregation. But despite computational methods to predict aggregation, the structural features of molecules that cause them to aggregate are still not well understood. In a new open-access Nature Comm. paper, Daniel Heller and collaborators at Memorial Sloan Kettering Cancer Center and elsewhere provide some answers.

 

The researchers had previously published an article describing how indocyanine green (ICG) could be used to stabilize and visualize aggregates, and they applied the same technique to examine the aggregation potential of a small set of fragments. Benzoic acid and 2-napthoic acid did not aggregate, while 4-phenylbenzoic acid did. Intrigued, the researchers tested a set of 14 4-substituted biphenyl fragments and found that those containing both a hydrogen bond donor and acceptor, such as acids, sulfonamides, amides, and ureas, could aggregate, while those containing only donors (aniline) or acceptors (nitrile) did not.

 

Fourier transform infrared spectroscopy was used to examine the stretching region of the carbonyl of 4-phenylbenzoic acid in various states: in an aqueous aggregate, in solution in either t-butanol or DMSO, or in the solid state. Interestingly, the aggregate most resembled the solid state, consistent with close-packed self-assembly as opposed to free in solution.

 

From all this, the researchers hypothesized that a combination of aromatic groups and hydrogen bond donors and acceptors was necessary for aggregation. However, having these features does not mean aggregation is inevitable. Neither 3-phenylbenzoic acid nor 2-phenylbenzoic acid formed aggregates, with the former precipitating while the latter remained completely soluble. These three phenylbenzoic acid isomers behave very differently despite the fact that they have the same calculated logP values, and the suggestion is that the latter two molecules are less able to form pi-pi stacking interactions that lead to stable aggregation.

 

Next the researchers examined the approved drug sorafenib, which had previously been shown to aggregate. This was confirmed, and the aggregates were characterized with a battery of biophysical methods including dynamic light scattering, transmission electron microscopy, and X-ray scattering, along with molecular dynamics simulations. The conclusion is that sorafenib forms amorphous aggregates whose assembly is driven by a combination of pi-pi stacking and hydrogen-bonding. A series of sorafenib analogs was synthesized, and those that could not form strong intermolecular hydrogen bonds were less prone to aggregation.

 

All of this is fascinating from a molecular assembly viewpoint and will help to explain and predict which compounds are likely to aggregate, for better or for worse. But as of now, experimental assessment is still best practice for any new compound.