alkylation of the second nitrogen of the piperazine ring with the genotoxic reagent 4-chloro-1-bromobutane (Scheme 10). 64 APIs streams may contain genotoxic alkyl halides impurities when a reaction takes place in alcoholic solvent at reflux temperature in the presence of hydrogen halides or alkali halides and strong acids. These reaction conditions may be applied in cyclization, decarboxylation, sulfonyl cleavage, Narylation and API salt formation. Note that this list is not exhaustive. For instance, capecitabine is a chemotherapeutic agent used in the treatment of metastatic breast and colorectal cancers. 65 During its synthesis, D-ribose is heated under reflux in methanol in the presence of concentrated HCI and acetone Scheme 2. O-Alkylation of Isovanillin during the Synthesis of Aliskiren Using Genotoxic 1,3-Dibromopropane Scheme 3. Use of Methyl Iodide in the Preparation of Cerivastatin Scheme 4. S-Alkylation with Methyl Iodide during the Last Synthetic Step of Lamifiban Scheme 5. S-Alkylation with Methyl Iodide during the Synthesis of Eldacimibe Scheme 6. N-Methylation with Genotoxic Methyl Iodide during the Preparation of Efegatran Scheme 7. Quaternization by Means of Methyl Iodide during Milameline Synthesis Scheme 8. Direct Use of Genotoxic 2-Iodopropane in the Synthesis of Latanoprost Scheme 9. Use of Genotoxic 2-Bromopropane and Nitrogen Mustard during the Synthesis of Mazapertine

Neural stem cells (NSCs) have the potential to generate the cells of the nervous system and, when cultured on nanofiber scaffolds, constitute a promising approach for neural tissue engineering. In this work, the impact of combining nanofiber alignment with functionalization of the electrospun poly-ε-caprolactone (PCL) nanofibers with biological adhesion motifs on the culture of an NSC line (CGR8-NS) is evaluated. A five-rank scale for fiber density was introduced, and a 4.5 level, corresponding to 70-80% fiber density, was selected for NSC in vitro culture. Aligned nanofibers directed NSC distribution and, especially in the presence of laminin (PCL-LN) and the RGD-containing peptide GRGDSP (PCL-RGD), promoted higher cell elongation, quantified by the eccentricity and axis ratio. In situ differentiation resulted in relatively higher percentage of cells expressing Tuj1 in PCL-LN, as well as significantly longer neurite development (41.1 ± 1.0 µm) than PCL-RGD (32.0 ± 1.0 µm), pristine PCL (25.1 ± 1.2 µm), or PCL-RGD randomly oriented fibers (26.5 ± 1.4 µm), suggesting that the presence of LN enhances neuronal differentiation. This study demonstrates that aligned nanofibers, functionalized with RGD, perform as well as PCL-LN fibers in terms of cell adhesion and proliferation. The presence of the full LN protein improves neuronal differentiation outcomes, which may be important for the use of this system in tissue engineering applications.

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