*2.3. Cytotoxicity of Compounds* **3**–**12** *against NSCLC Cell Lines*

The in vitro cytotoxic activities of the series of semisynthesized renieramycin-type derivatives and the natural renieramycins analogs were analyzed by MTT assay against the human H292 and H460 NSCLC cell lines (Table 2). The positive controls were cisplatin and doxorubicin, which were the first-line chemotherapeutic drugs. The results showed that compound **3**, which has a primary alcohol at the C-22 position, and renieramycins **5**–**8**, which possess unique structures (1,4-quinone or 1,3-dioxole-bridged phenol at ring A, methylene or secondary alcohol at C-14 of ring D, and 1,4-quinone or 1,4-hydroquinone at ring E), exhibited weak cytotoxicity against both H292 and H460 NSCLC cell lines at the IC50 values of 72.64–183.37 nM (Table 2, entries 1 and 3–6). However, compound **4** exhibited the strongest cytotoxicity among the series of natural renieramycins at the IC50 values of 35.36 and 33.86 nM against H292 and H460 cells, respectively (Table 2, entry 2). The semisynthesized renieramycin-type derivatives (**9**–**11**) exhibited substantially high cytotoxic activity at nanomolar concentrations against both cell lines (Table 2, entries 7–9). The known semisynthesized alkaloid (**9**), which possesses bis-tetrahydroisoquinolinequinone and 4- -pyridinecarbonyl moieties, exhibited the strongest cytotoxicity among all the renieramycin derivatives in this study. The compound **9** was approximately 10-fold and 9-fold more potent than the parent compound (**4**) against H292 and H460 NSCLC cell lines, respectively [22]. Both compound **10**, the known 8-hydroquinone, and compound **11**, the renieramycin–ecteinascidin hybrid derivative, contained the 5-*O*-(4- -pyridinecarbonyl) ester. They exhibited significant cytotoxicity, similar to those of **4** and docxorubicin, at the same IC50 level. Moreover, compound **11** was 3-fold and 2-fold more potent than **7**, the parent compound, against the H292 and H460 cell lines. Interestingly, compound **12**, a renieramycin-type derivative with a hydroxyl group at C-5, lost its cytotoxic activity and inhibited both the H292 and H460 cell lines at micromolar IC50 values (Table 2, entry 10). However, compound **12** exhibited 3-fold and 2-fold stronger potency than cisplatin against the H292 and H460 NSCLC cell lines, respectively. Therefore, the presence of the 4- -pyridinecarbonyl ester and the chemical structure of ring A constitute the essential pharmacophore controlling the cytotoxic potency [15,22,25]. The cytotoxicity of the renieramycin-type derivatives having a hydroxyl group at either the C-5 or C-22 position was weak.


**Table 2.** Cytotoxicity of the natural renieramycins and semisynthesized renieramycin derivatives against non-small-cell lung cancer cell lines.
