Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease without any effective treatment. Protein TDP-43 is a pathological hallmark of ALS in both sporadic and familiar patients. Post-translational modifications of TDP-43 promote its aggregation in the cytoplasm. Tau-Tubulin kinase (TTBK1) phosphorylates TDP-43 in cellular and animal models; thus, TTBK1 inhibitors emerge as a promising therapeutic strategy for ALS. The design, synthesis, biological evaluation, kinase-ligand complex structure determination, and molecular modeling studies confirmed novel pyrrolopyrimidine derivatives as valuable inhibitors for further development. Moreover, compound <b>29</b> revealed good brain penetration <i>in vivo</i> and was able to reduce TDP-43 phosphorylation not only in cell cultures but also in the spinal cord of transgenic TDP-43 mice. A shift to M2 anti-inflammatory microglia was also demonstrated <i>in vivo</i>. Both these activities led to motor neuron preservation in mice, proposing pyrrolopyrimidine <b>29</b> as a valuable lead compound for future ALS therapy.