| dc.description.abstract | Due to their central role in regulating vital cellular processes, protein kinases are increasingly attractive targets in modern drug development. Small molecule drugs have become valuable tools for selectively modulating the activity of specific kinases, opening up for novel targeted therapies. New druggable targets are continuously emerging, and there are, as of July 2023, 76 small molecule drugs targeting kinases on the market. One promising target is the Colony Stimulating Factor 1 Receptor (CSF1R), crucial for the regulation of myeloid cell lines such as macrophages and osteoclasts. Modulating abnormal CSF1R function holds promise in treating certain cancer types, neurological disorders, and osteoporosis.
The objective of this thesis work has been to develop and evaluate 4,6-disubstituted pyrrolo[2,3-d]pyrimidines as potential therapeutic agents targeting CSF1R. Most compounds were synthesized by modification of a di-halogenated pyrrolopyrimidine precursor, allowing for regiospecific amination at C-4 and regioselective Suzuki-Miyaura cross-coupling at the C-6 positions. An alternative method for C-6 functionalization based on directed lithiation using aldehydes and ketones as electrophiles was also investigated. This resulted in the development of a lithiation addition protocol excellently suited for cyclic ketones, 2,2,2-trifluoroacetophenone, and aldehydes. Additionally, certain reduction, dehydration and hydrolysis reactions were developed and employed in post-modifications on the pyrrolopyrimidine scaffold. The pyrrole unit was protected throughout most of this functionalization chemistry, whereas a final deprotection step afforded the finished inhibitors. Substantial efforts were made in scale up reactions of promising inhibitors for use in chow and other in vivo experiments.
Inhibitor development was continuously guided by in vitro potency assessments combined with molecular docking studies. This approach led to the identification of well-defined structure-activity relationships (SAR) for inhibition of CSF1R in enzymatic studies. Promising candidates underwent further evaluations for cellular potency, cellular toxicity, ADME and pharmacokinetic properties. Our most potent inhibitors exhibit potency in enzymatic assays almost 10 times higher than PLX3397, the only CSF1R inhibitor currently on the market. Additionally, evaluation of selected inhibitors in kinase panels exhibited excellent selectivity, surpassing that PLX3397. Introduction of a saturated ring system at the C-4 position and fine-tuning metabolic stability of compounds with potent motifs at C-6 emerged as crucial aspects for ensuring favorable drug characteristics, central for further development.
Despite the excellent enzymatic potency displayed by this line of pyrrolopyrimidine-based inhibitors, maintaining cellular activity was a central issue throughout development. In some instances, ADME studies indicate permeability issues may be responsible for the observed loss of activity in cellular assays. In other cases, however, the mechanism behind this loss of activity remains unclear. Activation state experiments revealed an almost exclusive affinity of all our pyrrolopyrimidine inhibitors toward the autoinhibited form of CSF1R. This finding may explain the excellent selectivity exhibited by our inhibitors but may also have implications for cellular and in vivo efficacy.
In addition to new building blocks for further development, 63 new pyrrolopyrimidines were synthesized and evaluated as CSF1R inhibitors. Moreover, the work has contributed to a growing understanding of this class of pyrrolopyrimidine inhibitors and has opened several promising avenues for further development. | en_US |
