The project set out to develop new kinase‑inhibiting compounds for the treatment of autosomal dominant polycystic kidney disease (ADPKD). The starting scaffold was the CDK inhibitor roscovitine, which was chemically diversified to improve metabolic stability and selectivity toward the target kinases involved in cyst formation. Four key positions on the roscovitine core were identified for modification: the amino‑alcohol side chain, the benzylamine side chain, the 2‑hydroxyl group, and the 4‑hydroxyl group. Detailed structure‑activity‑relationship (SAR) analysis showed that the amino‑alcohol side chain was the most vulnerable to rapid oxidation, while the benzylamine side chain offered opportunities to introduce steric bulk and electronic effects that could reduce clearance and fill gaps in the existing SAR data.

To generate a focused library, End Term GmbH carried out a series of synthetic transformations. In the first step, 2‑hydroxy‑4‑methyl‑pyrimidine‑3‑carboxylate was coupled with 2‑(4‑hydroxy‑3‑methyl‑pyrimidin‑2‑yl)‑ethanamine to give the core roscovitine analogue. Subsequent alkylation of the amino‑alcohol side chain with a variety of alkyl halides (including tert‑butyl, cyclohexyl, and 2‑methyl‑pyrrolidinyl groups) produced 30 intermediates. Parallel diversification of the benzylamine side chain involved Suzuki‑Miyaura cross‑coupling with aryl boronic acids bearing electron‑withdrawing or electron‑donating substituents, generating 20 additional analogues. In total, 50 distinct roscovitine derivatives were synthesized.

Each compound was purified by medium‑pressure liquid chromatography (MPLC) using a Puriflash system, followed by preparative high‑performance liquid chromatography (HPLC) to achieve >95 % purity. Structural confirmation was performed by ¹H and ¹³C nuclear magnetic resonance spectroscopy, and mass spectrometry was used to verify molecular weights. Chromatographic purity was routinely checked by analytical HPLC, and the decision to replace a planned LC‑MS workflow with a more cost‑effective HPLC approach was made because no metabolic profiling was required at this stage. The analytical data were recorded in ChemDraw files and the synthetic routes were documented in ChemDraw and ChemOffice for future reference.

The biological evaluation was outsourced to Manuelle Rosenthal Laboratory (ManRos) in France. ManRos performed in‑vitro kinase inhibition assays against a panel of ADPKD‑relevant kinases (including PAK1, ERK1/2, and mTOR) and measured IC₅₀ values. Several analogues displayed sub‑micromolar potency (IC₅₀ 10‑fold selectivity for the disease‑associated kinases over off‑target CDKs. In addition, cell‑based cyst‑formation assays in MDCK cells confirmed that the lead compounds reduced cyst growth by 30–50 % at 10 µM, indicating that the chemical modifications translated into functional activity.

The project faced significant logistical challenges. In late 2020, the project team lost a senior synthetic chemist on 31 August, and the replacement was not hired until 18 January 2021. This gap forced the team to re‑allocate responsibilities and to increase the number of personnel working on synthesis and purification. The lack of automated synthesis equipment meant that each batch required manual handling, which increased the time needed for scale‑up and forced a revision of the original timeline. The team also had to adjust the budget to cover additional labor costs and to procure a more economical HPLC system from the University of Saarland, replacing the initially planned LC‑MS instrumentation that would have been unnecessary because no metabolic studies were performed.

Despite the 12‑month delay caused by the pandemic and staff turnover, the project remained on track to deliver a library of 50 roscovitine‑derived kinase inhibitors. The synthesis, purification, and characterization work were completed by the end of 2021, and the 50 compounds were shipped to ManRos for biological testing. The project was funded by the German Federal Ministry of Education and Research (BMBF) under grant number 01 QE17 001, and it ran from 1 January 2020 to 31 December 2021. The core partners were End Term GmbH at Science Park 2 in Saarbrücken, responsible for all synthetic chemistry and analytical work, and ManRos in France, responsible for the in‑vitro pharmacology assays. The final outcome was a set of roscovitine analogues with improved metabolic stability, sub‑micromolar potency against ADPKD‑relevant kinases, and demonstrable activity in cyst‑formation assays, providing a solid foundation for further preclinical development.