Macrocyclization via Ring-Closing Metathesis in Drug Discovery & Process Chemistry

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Macrocyclic molecules, containing 12 or more atoms in a ring system, are increasingly prevalent in drug discovery, with over 67 macrocyclic drugs approved so far and several notable examples of clinical success highlighted on Drug Hunter recently, including MK-0616, repotrectinib, and pacritinib. By imparting conformational restraint and reducing entropy of binding, macrocyclization can enable more potent and selective binding to targets, while simultaneously reducing rotatable bonds and hence susceptibility to metabolism or efflux, among other favorable outcomes.

While most biologically active macrocycles are peptides or natural product-derived, ring-closing metathesis (RCM) reactions have contributed to greater accessibility of unnatural macrocycles in medicinal chemistry. RCM is now used in over half of macrocyclic kinase inhibitor syntheses, for example. This minireview highlights examples of ring-closing metathesis being applied by industry scientists to the synthesis of macrocycles during drug discovery and scale-up campaigns.

Recent Applications of Ring-Closing Metathesis in Drug Discovery and Development

A selection of representative macrocycles in drug discovery where ring-closing metathesis has been applied to their synthesis are shown below, highlighting chemically diverse macrocycles from programs that have reached clinical development. These include:

simeprevir, the first commercialized drug prepared using an RCM reaction on-scale

glecaprevir, for which a 41 kg enabling route involving RCM was developed

pacritinib, a kinase inhibitor approved in 2022 for treatment of myelofibrosis

AMG 176, an Mcl-1 inhibitor in clinical development for hematologic malignancies

danoprevir, a Roche HCV protease inhibitor clinical candidate

MK-0616, a clinical Merck PCSK9 inhibitor, and compound 44 a lead en route

Ring-Closing Metathesis Catalysts Employed in Drug Discovery and Process Chemistry

Challenges for the scale-up of RCM reactions include catalyst poisoning by input impurities, creation of dimer impurities during the reaction, and difficulty of scale-up due to dilute reaction concentrations. These challenges are often solved by evaluation of various catalysts and tuning effective concentrations of key species in solution, as illustrated by the examples below. Interestingly, among the six case studies, none use the well-known Hoveyda-Grubbs II (HG-II) catalyst in the largest scale synthesis disclosed, though several employ HG-II derivatives, highlighting the rapid evolution of catalytic methods and distinct considerations for process chemistry.

Process Route to Simeprevir Using A Ruthenium-Indenylidene Complex

One of the most famous examples of RCM in drug discovery is in the discovery and development of simeprevir (TMC435), an HCV NS3/4A protease inhibitor discovered and developed by Medivir and Janssen. In the process route, the Hoveyda-Grubbs I catalyst that was used in an earlier generation of the compound’s synthesis was replaced with an easy-to-prepare ruthenium-indenylidene complex, which is also resistant to thermal deactivation.

The Enabling Route to ABT-493/Glecaprevir

A good example of the details that can be involved in RCM optimization comes from AbbVie and Enanta’s glecaprevir. Glecaprevir, or ABT-493, was approved in combination with pibrentasvir (Mavyret) for the treatment of Hepatitis C (HCV) in 2017. The enabling route toward this 18-membered macrolide and NS3/4A protease inhibitor, used on a 41 kg scale likely to support FIH studies, relied on a key ring-closing metathesis (RCM). This reaction required extensive optimization to overcome the many synthetic challenges including high catalyst loading, byproduct contamination, large-scale column chromatography, and identifying the optimal reaction time, temperature, and concentration for the desired mass balance.

Among catalysts, 10 mol% of Zhan 1B afforded the most product (82%) with the least amount of starting material (4%), cis-product (10.4%) or dimer impurity (3.1%). Like nitro-containing Grela’s catalyst, the Zhan 1B catalyst’s electron-withdrawing sulfonamide increases catalyst initiation rate. A further advantage of the Zhan catalyst is that it is recyclable, which can reduce the costs associated with ruthenium catalysts. Unlike the Grubbs or Hoveyda-Grubbs catalysts, the Zhan catalysts can be recovered and recycled by simple precipitation or filtration, since Zhan Catalyst-1B is soluble in dichloromethane, dichloroethane, chloroform, ether, and other solvents, but insoluble in methanol, ethanol, and other alcohols.

The dimer impurity could be converted to product over the course of the reaction, but was never completely eliminated, while acetic acid as an additive did not reduce the levels of the undesired cis-product. The best impurity profile was achieved with toluene as the solvent (0.02 M) at 40 °C. Slow addition of separate solutions of the diene substrate and catalyst over 6 h minimized the dimer impurity, while sparging with nitrogen helped to push the reaction to completion by removing the ethylene gas generated during the RCM. Upon reaction completion, imidazole and Filtrol were added, followed by two silica gel plugs (2 then 6-7 g silica gel per g starting material) to remove the dimer impurity and catalyst byproducts. The remaining dimer impurity was removed following saponification and four crystallizations from 2-Me-THF and heptanes. Despite the >50% yield improvement over the initial process (57% over 3 steps), the laborious purification process was not ultimately chosen for the commercial production of glecaprevir, but did afford 41 kg of API to support preclinical and early clinical activities.

RCM in the Medicinal Chemistry of Pacritinib

Pacritinib, a kinase inhibitor approved in 2022 for treatment of myelofibrosis, was also synthesized with an RCM in its med. chem. route,, catalyzed by 10 mol% of either Grubbs 2nd generation catalyst or Zhan 1B catalyst. In the discovery paper, the compound was isolated as an inseparable 85:15 trans/cis mixture, and it is not clear how the drug is currently manufactured, though it likely required significant route improvement. Today, it is possible that modern catalysts enabling stereoselective formation of alkenes may help address such challenges.

Multiple Metatheses in Merck’s Macrocycles

The scope of ruthenium-carbene catalyzed RCM is impressively illustrated by the synthesis of compound 44, a Merck PCSK9 inhibitor lead en route to clinical molecule MK-0616, as well as in a synthesis of MK-0616 itself. Macrocyclic ring-forming reactions occur three times during the compounds’ synthesis, making these molecules a chemical tour de force. In both the case of compound 44’s northern ring and in the case of MK-0616’s eastern ring, Zhan 1B catalyst is used, creating isomeric mixtures that favor the desired trans isomer.

Application of RCM in a synthesis of MK-0616:

Application of RCM in a synthesis of compound 44:

M73-SIMes in a Multi-kilo Synthesis of AMG 176:

RCM reactions usually leverage dilute reaction conditions to favor intramolecular cyclization over intermolecular interactions. However, dilute reactions make for large reaction volumes, which limit production throughput, require higher solvent costs, and produce larger waste streams, among other issues. AMG 176, an Amgen Mcl-1 inhibitor and oncology drug candidate that contains a 16-membered ring, was initially synthesized using the HG-II catalyst under very dilute conditions, resulting in a challenging scale-up. Substrate modification, a change in catalyst (M73-SIMes), and creating pseudodilute conditions resulted in a 94% improvement in volumetric efficiency and an 80% reduction in catalyst loading, ultimately enabling the RCM route on a >45 kg scale.

M73-SIMes is a derivative of the Hoveyda-Grubbs precatalysts developed by Dr. Marc Mauduit and his team at Rennes, with a patent assigned to Umicore. It contains a moderately activating carbamate group that also gives it a stronger affinity for silica gel, and is air- and moisture-stable. In the case of AMG 176, M73-SIMes was chosen from a screen because it could perform at a lower loading (2-4 mol%) than the HG-II catalyst (10 mol%) used previously.

A Proprietary Catalyst in Roche’s Synthesis of Danoprevir

Some pharmaceutical research teams have elected to develop and patent their own catalysts, with impressive results. Roche Palo Alto, for example, patented a hexacoordinated Hoveyda-Grubbs II-derived amide catalyst, which they employed in patented processes for danoprevir intermediates.

This step was conducted under vacuum to bring the reaction to completion by removing the ethylene gas byproduct. Remarkably, the readiness of this substrate to cyclize due to conformational effects in the uncyclized species allowed the reaction to be run at 8 wt % substrate, or ~0.1 M! The custom catalyst was also employed at a remarkably low loading of 0.05 mol%.

New Opportunities for Metathesis

As the impressive molecules above highlight, ring-closing metathesis has aided access to macrocyclic drug candidates of remarkable complexity. The choice of catalysts employed is influenced by different factors for each project, with reaction performance, IP status, commercial quantity available, recycling potential, stability and required loading all being aspects to consider. Increasing catalyst choices will only further RCM’s applications in drug discovery and development.

Whatever the key factors leading to your choice of catalysts may be, Sinocompound can help provide a range of ruthenium catalysts for use in RCM screening and macrocycle development. Visit Sinocompound’s website to learn more about their catalyst products and services.

Further Reading:

  1. For an excellent overview of the RCM reaction in process chemistry by Bristol Myers Squibb scientists, see: Yu, M.; Lou, S.; Gonzalez-Bobes, F. Ring-Closing Metathesis in Pharmaceutical Development: Fundamentals, Applications, and Future Directions. Organic Process Research & Development 2018, 22 (8), 918–946. https://doi.org/10.1021/acs.oprd.8b00093.
  2. For a recent review on macrocycles in drug discovery, see: Jiminez, D. G.; Poongavanam, V.; Kihlberg, J. “Macrocycles in Drug Discovery-Learning from the Past for the Future.” J. Med. Chem. 2023, 66, (8), 5377-5396. https://pubs.acs.org/doi/10.1021/acs.jmedchem.3c00134

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