X. Yang, et al.
Bioorganic&MedicinalChemistryLetters30(2020)127557
Fig. 2. Stereoview of the binding site and nearby residues from compound CE17 in EGFR-TK (PDB: 1M17).
significantly improved when the central methine chain is lack of the
rigid cyclohexene ring. Finally, surprisingly, the IC50 of compound
CE17 reduced approximately 14-fold (IC50 = 0.177 μM) compared with
the parent drug erlotinib (IC50 = 2.628 μM) in A549 cells, indicating
that the trifluoromethyl might enhance the antiproliferative activity of
above conjugates.
addition, the erlotinib moiety of compound CE17 successfully enters
into the active hydrophobic cavity (a hydrophobic pocket with the
surrounding residues like Leu-694, Met-742, Leu-768 and Leu820, and
the N-1 of the quinazoline accepts an H-bond from the Met-769 amide
nitrogen),23 while the heptamethine cyanine dye moiety locates at the
outside of the cavity. Thus, above results might interpret the possible
binding mode between compound CE17 and EGFR in tumor cells.
In conclusion, we designed and synthesized a series of novel hep-
tamethine carbocyanine dyes-erlotinib conjugates. Most compounds
exhibited superior antiproliferative activity against A549, H460, H1299
and MDA-MB-231 cell lines compared with the parent drug erlotinib.
Meanwhile, representative compounds (CE8 and CE17) exhibited weak
cytotoxicity on human normal mammary epithelial MCF-10A cells.
Moreover, the conjugate CE17 also showed higher EGFR-TK inhibition
activity (IC50 = 0.124 μM) than erlotinib (IC50 = 5.182 μM) in A549
cell line. Together with the result of the antiproliferative activity assay
and EGFR-TK inhibition assay, erlotinib conjugates probably enhanced
their antitumor activity by synergistic targeting effect. Moreover, the
molecular docking results were consistent with the suppression of
tumor cells. Therefore, the conjugation of tumor cell targeting hepta-
methine dyes and EGFR-TKI will be an attractive approach for the an-
titumor drug design.
To verify whether the combination of EGFR-TKIs with the hepta-
methine cyanine dye will improve their tumor cell targeting and the
transmembrane ability, we investigated the cytotoxicity effect of re-
presentative compounds (CE8 and CE17) on MCF-10A cells (human
normal mammary epithelial cells). As shown in Table 2, the compounds
CE8 and CE17 demonstrated relatively weak cytotoxicity on MCF-10A
cells, suggesting the in vitro tumor cells selectivity of the representative
compounds could be enhanced.
As a result, the activities of most compounds are more excellent,
which demonstrates that integrating heptamethine cyanine dye with
EGFR-TK inhibitor perhaps enhance the antiproliferative effect by the
synergistic targeting to tumor cells.
Since the direct test of the inhibitory effect of above compounds on
EGFR-TK makes little sense, we chose to investigate the cancer cells
EGFR-TK inhibiting effect of above compounds in vitro, so as to better
reflect the transmembrane effect of the target compounds. A549 cells
were selected to determine the EGFR-TK inhibiting activity of com-
pounds CE12 and CE17 with the best antiproliferation activity in A549
cell. Therefore, the inhibition of intracellular EGFR-TK activity was
assessed by human EGFR ELISA Kit (Boster Biological Technology,
China, EK0327). According to the instructions of the kit, after treating
with the concentration of 0.1 μM, 5 μM, 50 μM, 200 μM, and 1000 μM
of compounds CE12, CE17 and erlotinib 30 h, A549 cells were lysed.
The protein level was determined by the bicinchoninic acid (BCA)
method, and the EGFR activity of the cells was measured by the ELISA
method. The absorbance (OD value) at 450 nm was measured using a
microplate reader (US Synergy-HT). The results are shown in Table 3.
compound CE17 exhibited more excellent EGFR-TK inhibition than
compound CE12 and erlotinib, which is consistent with the MTT re-
sults. Therefore, the antiproliferative activity and EGFR-TK inhibitory
activity of compound CE17 are superior to erlotinib, implicating that
the antitumor effect is probably associated with the increased in-
tracellular concentrations of target conjugates due to their enhanced
targeting and transmembrane ability to tumor cells.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgments
The authors appreciate the financial supports from the National
Natural Science Foundation of China (Grant No. 81903463), the
Natural Science Foundation of Liaoning Province (Grant No.
20180540032), the Career Development Support Plan for Young and
Middle-aged Teachers in Shenyang Pharmaceutical University (Grant
No. ZQN2018018) and China Postdoctoral Science Foundation (Grant
No. 2020M673042).
Finally, in order to better understand and verify the potential action
mode of compounds with EGFR-TK, the molecular docking was con-
ducted. Compound CE17 was chosen to dock with EGFR-TK (PDB:
1 M17). As exhibited in Fig. 2, there is an H-bond interaction between
Appendix A. Supplementary data
Supplementary data (synthetic procedures and NMR spectra of
compounds CE1-CE17) to this article can be found online at https://
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