The Anti-Breast Cancer Activity of Dihydroartemisinin-5-methylisatin Hybrids Tethered via Different Carbon Spacers

Sixteen dihydroartemisinin-5-methylisatin hybrids 6a–c and 7a–m tethered via different carbon spacers were assessed for their antiproliferative activity against MCF-7, MDA-MB-231, MCF-7/ADR and MDA-MB-231/ADR breast cancer cell lines as well as cytotoxicity towards MCF-10A cells to investigate the influence of the length of carbon spacers on the activity. The preliminary results illustrated that the length of the carbon spacer was the main parameter which affected the activity, and hybrids tethered via the two-carbon linker showed the highest activity. Amongst the synthesized hybrids, the representative hybrid 7a (IC50: 15.3–20.1 µM) not only demonstrated profound activity against both drug-sensitive and drug-resistant breast cancer cell lines, but also possessed excellent safety and selectivity profile. Collectivity, hybrid 7a was a promising candidate for the treatment of both drug-sensitive and drug-resistant breast cancers and worthy of further preclinical evaluations.


Introduction
Breast cancer remains a worldwide public health dilemma and is the most common type of fatal ailment in females around the world [1,2]. Over 2.3 million new cases and 685,000 deaths from breast cancer occurred in 2020, and it is predicted to increase to over 3 million new cases and 1 million deaths by 2040 [3,4]. Diverse therapeutic strategies, inclusive of surgery, chemotherapy, endocrine therapy, targeted therapy, and immunotherapy, have been developed for the treatment of breast cancer. However, drug resistance has become a tremendous obstacle in overcoming recurrence and metastasis and is responsible for most breast cancer fatalities [5,6]. Moreover, current available anti-breast cancer agents could lead to some adverse events such as cardiotoxicity, bronchospasm, sleep disorders, and angioedema [7,8]. Hence, there is a critical need to develop novel anti-breast cancer agents to address drug resistance and side effects.
Dihydroartemisinin (DHA), a derivative of artemisinin (ART), demonstrated many advantages compared to ART, including high water solubility, easy absorption, wide distribution, rapid excretion and metabolism, high efficiency, and low toxicity [9,10]. DHA could exert anticancer effects through various mechanisms, such as excessive reactive oxygen species (ROS) production, proliferation inhibition, inducing apoptosis, repression of tumor metastasis and angiogenesis, promotion of immune function, guide of autophagy, and endoplasmic reticulum (ER) stress [11,12]. Accordingly, DHA exhibits profound anticancer activities on a wide range of cancer types including breast cancer both in vitro and in vivo, representing a useful template for the discovery of novel anticancer chemotherapeutics.
Isatin (Indoline-2,3-dione or indole-1H-2,3-dione), an endogenous substance, is characterized by a borad of biological properties such as low toxicity, mutagenicity, and genotoxicity in vivo [13,14]. Isatin is a highly "privileged motif" for the target-based design and development of anticancer agents since its derivatives are potential inhibitors of lysine-specific histone demethylase 1 (LSD1), β-carbonic anhydrase, topoisomerase II, tyrosine kinase, proteases, phosphatases, tubulin, and epidermal growth factor receptor (EGFR) [15,16]. Notably, many isatin derivatives have been proved with promising activities against both drug-sensitive and multidrug-resistant breast cancer, and several isatin-based compounds have already been approved for cancer therapy [17,18]. Hence, isatin moiety has also been considered as a useful pharmacophore against breast cancer.
Hybridization of DHA and isatin into one molecule could obtain novel anti-breast cancer candidates with broader spectrum, higher efficiency, lower toxicity, as well as multiple mechanisms of action that could overcome drug resistance [19][20][21][22]. DHA-isatin hybrids have the potential in the treatment of breast cancer. As a continuous program to seek for more effective anti-breast cancer candidates, a series of DHA-5-methylisatin hybrids tethered via different carbon spacers were designed, synthesized, and the antiproliferative activities against drug-sensitive MCF-7 and MDA-MB-231 breast cancer cell lines as well as their multidrug-resistant counterparts MCF-7/ADR and MDA-MB-231/ADR were also evaluated in this study.
Isatin (Indoline-2,3-dione or indole-1H-2,3-dione), an endogenous substance, is characterized by a borad of biological properties such as low toxicity, mutagenicity, and genotoxicity in vivo [13,14]. Isatin is a highly "privileged motif" for the target-based design and development of anticancer agents since its derivatives are potential inhibitors of lysine-specific histone demethylase 1 (LSD1), β-carbonic anhydrase, topoisomerase II, tyrosine kinase, proteases, phosphatases, tubulin, and epidermal growth factor receptor (EGFR) [15,16]. Notably, many isatin derivatives have been proved with promising activities against both drug-sensitive and multidrug-resistant breast cancer, and several isatinbased compounds have already been approved for cancer therapy [17,18]. Hence, isatin moiety has also been considered as a useful pharmacophore against breast cancer.
Hybridization of DHA and isatin into one molecule could obtain novel anti-breast cancer candidates with broader spectrum, higher efficiency, lower toxicity, as well as multiple mechanisms of action that could overcome drug resistance [19][20][21][22]. DHA-isatin hybrids have the potential in the treatment of breast cancer. As a continuous program to seek for more effective anti-breast cancer candidates, a series of DHA-5-methylisatin hybrids tethered via different carbon spacers were designed, synthesized, and the antiproliferative activities against drug-sensitive MCF-7 and MDA-MB-231 breast cancer cell lines as well as their multidrug-resistant counterparts MCF-7/ADR and MDA-MB-231/ADR were also evaluated in this study.
As can be seen from Table 1, most of the synthesized hybrids (IC 50 : 15.3-99.9 µM) were active against MCF-7, MDA-MB-231, MCF-7/ADR, and MDA-MB-231/ADR breast cancer cell lines. The SARs illustrated that the carbon spacers between DHA and isatin moieties were the main parameter influenced the activity, and the relative contribution order was two-carbon > four-carbon > three-carbon. Hydrogen bond donors at C-3 position of isatin moiety could enhance the activity, and the relative contribution order was NOH > NNHCSNH 2 > NNHCONH 2 . Additionally, alkyloxime at C-3 position of isatin moiety could improve the activity to some extent, while benzoxime decreased the activity.    The RI values of hybrid 7a were 1.03 and 1.23 respectively, proving its potential to overcome drug resistance. Moreover, hybrid 7a (IC 50 : >100 µM) was non-toxic towards normal MCF-10A breast cells, and the SI values were >5.15 and >6.53, revealing its excellent safety and selectivity profiles. It seems that the hydrogen bond donors especially NOH at C-3 position of isatin moiety showed higher SI values.

Experimental Section
3.1. Chemistry 3.1.1. General 1 H NMR and 13 C spectra were determined on a Mercury-400 spectrometer (Varian, Palo Alto, CA, USA) in DMSO-d 6 or CDCl 3 using tetramethylsilane (TMS) as an internal standard. Electrospray ionization (ESI) mass spectra and high-resolution mass spectra (HRMS) were obtained on a Q-Tap mass spectrometer (MDSSCIEX, Concrod, CA, USA) and AccuTOF CS JMS-T100CS mass spectrometer (JEOL, Tokyo, Japan), respectively. Unless otherwise noted, the reagents were obtained from commercial supplier and used without further purification.