One-Stop Metabolite Shop

Late-Stage Chemical Synthesis

Chemical Synthesis of Drug Metabolites

The newest tool in our one-stop metabolite shop is the addition of late-stage chemical synthesis methods for cost effective access to both phase 1 drug metabolites and phase 2 conjugates, in particular glucuronides and sulfates. Chemical synthesis at Hypha complements the biosynthetic methods to provide a comprehensive service for clients to access all types of drug metabolites under the One-Stop Metabolite Shop umbrella. A combination of internal chemical synthesis at Hypha, and project management undertaken at preferred and highly trusted chemistry CROs, enables Hypha’s One-Stop Metabolite Shop to provide access to difficult-to-access metabolites from mg to kg-scale.  

Late-stage glucuronidation and sulfation

We have developed late-stage chemical methods for synthesis of all types of glucuronides, as well as glucosidated and sulfated metabolites. Reactions are fully scalable and are a proven and cost-effective way to access conjugated metabolites synthesised using these methods.

Late-stage chemical screen for the synthesis of O-, N-, N-carbamoyl and acyl glucuronides

A proprietary screen consisting of multiple sets of robust and diverse conditions has been developed at Hypha for late-stage synthesis of O-, N– and acyl glucuronides of client drug compounds. Glucuronides are produced direct from the parent compound using tailored deprotection strategies compatible with acyl glucuronides and sensitive N-glucuronides.

Using this screen, the structure of the client’s parent drug compound does not need to be revealed. The methods are readily scalable and metabolites can be purified by chemists at Hypha.

We are also experts in purification, structure elucidation of the metabolites synthesised, and can provide Certificates of Analysis.

Case Studies

Provision of N- and O-glucuronides of a client drug

cenobamate metabolites case study image

A US pharma client required >200 mg of three metabolites of a drug; an N-glucuronide (M1), an indirect O-glucuronide (M2b) and a hydroxylated metabolite (M8b). As part of this project, multiple components of Hypha’s one-stop metabolite shop were employed, including chemical synthesis, microbial biotransformation as well as purification and structure elucidation by NMR.

M1, a major N-glucuronide, was accessed using chemical synthesis after identifying the best conditions in the chemical screens, resulting in the purification of a gram of M1. Key to successful synthesis were the mild deprotection conditions used in the late-stage chemical glucuronidation procedure.

In addition to the N-glucuronide, an indirect O-glucuronide, M2b, and its aglycone, M8b, were also needed. To make M8b, the position of the hydroxyl group first had to be identified. To achieve this, Hypha chemists purified a small amount of M2b from human urine supplied by the client, and elucidated the structure of the conjugate using cryoprobe NMR spectroscopy. Then, knowing the position of hydroxylation from the structure of the phenolic glucuronide, 100s of mgs of M8b were synthesised.

In order to access large amounts of M2b, a different approach was needed as this glucuronide was not amenable to chemical synthesis due to instability and formation of side products. Instead, M2b was successfully made through microbial biotransformation of the aglycone M8b. Following a screen to determine the best microbial catalyst, 560 mg of M8b was fed to one of Hypha’s biotransforming bacterial strains, from which 254 mg of M2b was purified. Metabolites were supplied to the client along with certificates of analysis.

Development of alternative late-stage acyl glucuronidation method

A European pharma client required 5-10 mgs of an acyl glucuronide. The drug compound was initially profiled in the standard chemical glucuronidation and microbial biotransformation screens. However, we encountered some challenges in production of acyl migration isomers via the microbial route, and low yields due to extensive hydrolysis back to the parent drug in the four chemical glucuronidation methods. The problem was quickly solved by development of an alternative two-step late-stage chemical synthesis route which was more suitable for this hydrolytically-unstable acyl glucuronide, to achieve a 30% isolatable yield of the target acyl glucuronide. After purification and confirmation of the structure by NMR, 30.5 mg was delivered to the client together with a certificate of analysis.

Late-stage chemical sulfation of an intermediate drug metabolite

A sulfated metabolite was required by a US client which was produced in humans through an aryl hydroxylated intermediate. Hypha used microbial biotransformation to produce the intermediate metabolite followed by late-stage chemical synthesis to make and purify hundreds of milligrams of both unlabelled and a deuterated O-sulfated metabolite.

Our other one-stop shop solutions for accessing and characterising metabolites

Hypha’s One-Stop Metabolite Shop enables synthesis, purification and characterization of all the main types of mammalian phase 1 and 2 metabolites.

Mammalian biotransformation

Panels of liver S9s / microsomes

Recombinant enzymes

PolyCYPs, hrCYPs, AOX, FMOs

Purification & structure elucidation

COAs, acquisition / interpretation of NMR data

Resources

Explore our library of resources comprising brochures, case studies, posters and publications about the work we do.

Hypha’s One-Stop Metabolite Shop enables synthesis, purification and characterization of all the main types of mammalian phase 1 and 2 metabolites. We use chemical synthesis, microbial biotransformation, mammalian tissue fractions (multiple species of S9s and microsomes) plus recombinant enzymes such as PolyCYPs, and human recombinant CYPs, AOX1 and FMOs 1 to 5.

We’ve observed an increase in requests for synthesis of N-glucuronides over the last couple of years. We speculate that this may be due to the increasing use of N-heterocyclic chemistry in the design of new small molecule drugs, and pan company strategies to reduce CYP metabolism. The situation is further complicated by the high interspecies variability in formation of some N-glucuronides, especially aliphatic tertiary amines and aromatic N-heterocycles. UGT1A4 and UGT2B10 are key enzymes responsible for N-glucuronidation reactions in humans, rates of which can be much higher than in other animals. To compound this, synthesis of N-glucuronides is not always straightforward, and can be further muddied by metabolite stability issues, complicating interpretation of data.

Access to multiple metabolites needed to support clinical development is not always straightforward, and can sometimes mean that more than one technique needs to be applied to fulfil requirements. In one such project, a US pharma client required > 200 mg of three metabolites of a drug; an N-glucuronide (M1), an indirect O-glucuronide (M2b) and a hydroxylated metabolite (M8b). As part of this project, multiple components of Hypha’s one-stop metabolite shop were employed, including chemical synthesis, microbial biotransformation as well as purification and structure elucidation by NMR.

Test tubes with dripper
251A3171-DE01-48EB-AA27-BD82BC5AC10DCreated with sketchtool.

We worked with Hypha discovery to synthesize metabolites via a chemical approach in enough quantities for pharmacology studies for one of our products. Hypha has well understood our request and has provided a very good quality of service in time, in quantity and quality with the needed level of regular communication. We will work certainly with them in the future and will recommend them.

Dr. Laure Navarre, Director Process Chemistry

Poxel, France

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