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  • br Chemistry All the title molecules were

    2022-01-21


    Chemistry All the title molecules were generally synthesized using the procedures shown in Scheme 1, Scheme 2, Scheme 3 [17,18,23,24]. The key 2-chloropyrimidine intermediate 11 was prepared according to our previously reported method via subsequent formylation, reduction, and nucleophilic substitution reactions [17,18]. Commercially available 4-nitrobenzac -etic between (12) was reacted with morpholine in the presence of the activating reagent ethyl(dimethylaminopropyl) carbodiimide (EDC) to prepare the N-morpholine formamide derivative 13. After reducing the nitro group in compound 13 with Fe-NH4Cl, the amino derivative 14 was synthesized. Finally, the title molecule 7a is produced by substituting the chloride atom at the C-2 position of the pyrimidine intermediate 11 with an amino group in compound 14. For the synthesis of the title molecule 7b-e, 4-nitrophenol was reacted with α-bromoacetate to prepare the intermediates 16a-c in the first step. Then, 16a-c was reacted with morpholine to produce between compounds 17a-c. Also, compounds 17a-c were conveniently converted to the title molecule 7b-e via reduction and nucleophilic substitution reactions. To synthesize the title molecule 7f, 4-nitrofluoro was reacted with 2-mercapto acetate. The ester group in compound 19 was then hydrolyzed to produce intermediate 20. Compound 20 was reduced to amino-substituted 21, which was coupled with intermediate 11 in the presence of trifluoroacetic acid (TFA) to prepare the title molecule 7f. All the title molecules were evaluated for their biological activity against FAK kinase by using the ADP-Glo™ assay (Promega, Madison, WI, USA) [25,26]. As shown in Table 1, these newly synthesized compounds significantly inhibited FAK enzymatic activity at concentrations ranging from 2.58 to 75.8 nM. Two typical molecules 7a and 7f exhibited equal inhibitory potency to TAE226 (6.79 nM), with IC50 values of 5.17 and 2.58 nM, respectively. The kinase-based test result revealed that the bulky cyclopropyl group is less beneficial than the small methyl group at the formamide part. For instance, the methyl-substituted compound 7b (7.28 nM) had almost 10 times higher anti-FAK activity than compound 7c (75.8 nM), which possesses a cyclopropyl substituent. Furthermore, introducing an additional methyl (7d), or ethyl (7e) group into the C-2 aniline side chain is also unfavorable. The exemplary compounds 7d (38.0 nM) and 7e (25.6 nM) have remarkably reduced inhibitory potency compared with 7b. However, when replacing the oxygen group in the aniline side chain at the C-2 position of the pyrimidine core with a sulphur atom (7f), or removing the oxygen atom (7a), the anti-FAK activity of the desired compounds slightly improved. For the title molecules, their ClogP values could produce significant effect on the capability to penetrate cancer cell memberance, and thus affecting their anti-proliferative activity of cancer cells. Generally, the ClogP values is more high, the activity against cancer cell is more strong [[27], [28], [29]]. Accordingly, we also explored their ClogP values by using ChemDraw Ultra software®, and found that compound 7a, not only displayed strong inhibitory potency to FAK enzymatic activity, but also has the highest ClogP value in this class of inhibitors. Overall, this work led to the identification of two potent FAK inhibitors, namely 7a and 7f, which are worthy of further biological evaluation. To investigate the effects of these newly synthesized molecules on the proliferation of cancer cells in vitro, several stubborn cancer cell lines were used for biological evaluation, including pancreatic cancer (PC) cell lines (AsPC-1, BxPC-3, PANC-1 cells), and mutant H1975 cells (a gefitinib-resistant non-small cell cancer lung (NSCLC) cell line) and MCF-7/ADR cells (Adriamycin-Resistant human breast carcinoma cells). In addition, two human normal human cell lines LO2 (normal human hepatocyte cells) and HPDEC (pancreatic ductal epithelial cells) were also used to test their cytotoxicity. The novel FAK inhibitor TAE226 was also tested as a positive control. As shown in Table 2, most of these compounds displayed strong inhibitory potency against these stubborn cancer cells at drug concentrations lower than 10 μM. Compared with TAE226, this class of compounds have significantly enhanced anticancer activity. In particular, compound 7a, which has an IC50 value of 0.105 μM against AsPC-1, and 0.090 μM against BxPC-3 cells, was the strongest inhibitor against PC cell lines. In contrast, compound 7c bearing a cyclopropyl substituent was ineffective in blocking the proliferation of PC cells, with IC50 values of more than 10 μM. This finding suggests that the bulky group at the C-2 aniline side chain (compound 7c) is less favorable than the small methyl substituent. Possibly, the cyclopropyl group produced steric hindrance in the FAK binding pocket, thus reducing the binding affinity. Generally, their in vitro activity against PC cells in accordance with the inhibitory potency to FAK kinase. In spite of its similar inhibitory potency to TAE226 against FAK kinase, compound 7a exhibited improved anti-PC cells activity possibly because of its higher ClogP (5.319) value than TAE226 (4.965) (Table 1). Or possibly for the reason that most of the kinases have many subtypes (Type 1, Type 2, Type 3, etc.), such as PYK2 and FAK, which have 73% similarity in the catalytic domains [30,31], compound 7a may interfere with other subtypes or other tumor signal transduction pathways, while TAE226 has no. These further studies on the mechanism of action of kinase will be performed in our future work.