Design, synthesis and pharmacological evaluation of novel Artemisinin-Thymol

Abstract A molecular hybridization of natural products is a new concept in drug discovery and having critical roles to design new molecules with improved biological properties. Hybrid molecules display higher biological activities when compared to the parent drugs. In the present study, two natural products (thymol and artemisinin (ART)) are used for the synthesis of new hybrid thymol-artemisinin. After characterization, the cytotoxic activity of ART-thymol was tested against different cancer cell lines and non-cancerous human cell line. ART-Thymol show the cytotoxic effect with EC50 values 70,96μM for HepG2, 97,31μM for LnCap, 6,03μM for Caco-2, 77,98μM for HeLa and 62,28μM for HEK293 cells, respectively. Moreover, ART-Thymol was checked for drug-likeness, and the kinase inhibitory activity. ART-Thymol is investigated by using molecular docking. The results of qPCR was indicated CDK2 and P38 were inhibited by ART-Thymol. These results improved that thymol-artemisinin may be new candidates as an anticancer agents. Graphical abstract


Introduction
Artemisinin (ART) is a member of lactone and natural product extracted from Artemisia annua (wormwood plant) (Klayman 1985, Kong andTan 2015). It was discovered by Chinese scientist Youyou Tu, while it was played a very important role for the treatment of different diseases since thousands of years, especially as a sedative and antipyretic (Kong andTan 2015, Miller andSu 2011). Last decades, ART molecule has been gained big importance due to its amazing biological properties (Yang et al. 2020). ART is known as the most effective miracle drug for the treatment of malaria which causes more than half a million deaths according to the World Health Organization (WHO) data (Su and Miller 2015). ART has been not only used clinically as antimalarial and antiviral (White 2004), but also displayed strong anticancer activity in vivo and in vitro (Chen et al. 2020, Efferth et al. 2001, Li et al., Meshnick et al. 1996, Zhang 2020). In addition, ART and its derivatives have been used to treatment of Leishmania (Renslo andMcKerrow 2006, Sen et al. 2007) Schistosoma (Efferth et al. 2008), and Toxoplasma (Nagamune et al. 2007).
Interestingly, there are three important milestones of ART; the discovery (1972), Nobel Prize (2015) and Covid-19 (2020). After its discovery, a variety of new biological properties of ART have been reported (Kong and Tan 2015). Moreover, pharmaceutically active semisynthetic derivatives including artesunate (Efferth et al. 2008), artermer and dihydroartemisinin were synthesized and apply for the treatment of different diseases ( Figure 1) (Messori et al. 2006). Last years, scientists have focused on the exploring the anticancer properties of new natural compounds, so anticancer activity ART are tested in vitro and in animal models (Hou et al. 2008, Tan et al. 2011. It was investigated that artesunate displayed inhibitory effects against breast, melanoma, leukemia, ovarian, colon, prostate and renal cancer cells (Efferth et al. 2001, Firestone andSundar 2009). Moreover, artesunate was to be high efficiency to central nervous systems (Zuo et al. 2016). Dihydroartemisinin was also examined as anti-cancer agents for osteosarcoma, pancreatic, leukemic, and lung cancer cells (Chaturvedi et al. 2010, Singh andLai 2001). Third milestones for ART could be COVID-19 which has infected more than 6 million individuals worldwide with more than 3.58 hundred thousand patients have died because of the infection (Gibson et al. 2020, Wu andMcGoogan 2020). It was reported that commercially available anti-viral drugs like chloroquine, hydroxycholoroquine, quinine and mefloquine are used to treatments of this viral pneumonia of SARS-CoV-2 (Nittari et al. 2020). Therefore, ART and derivatives can be seen as one of the biggest candidates for the inhibition and treatment of SARS-CoV-2 as an antiviral.
On the other hand, there are some disadvantages of ART; low solubility in water and short half-life after administration. Therefore, ART based new antimalarial drug candidates have been designed and synthesized with higher stability, higher efficiency and lower toxicity by using hybridization (Ha et al. 2016, O'Neill and Posner 2004, Pepe et al. 2020. Last years, a molecular hybridization has been to be an emerging tool for drug discovery , Pepe et al., 2020. ART based hybrid molecules combining with quinoline (Capci et al. 2019), isoquinoline , quine, chloroquine (Pepe et al., 2020), thymoquinone (Gruber et al. 2018), ferrocene (de Lange et al. 2018), tamoxifen (Frohlich et al. 2020) were synthesized and found remarkable antiviral activities than currents. In addition, it is now well-reported that ART hybrids display higher pharmacological activity against different kinds of cancer types (Efferth et al. 2003, Lai and Singh 1995, Woerdenbag et al. 1993. In the light of preliminary studies, we designed and synthesized novel hybrid molecule consisting of two natural products; Artemisinin and Thymol. Thymol is a monoterpene phenol obtained from the family Lamiaceae (Thymus, Lavandula, Origanum, Ocimum and Monarda genera). Thymol has been used as antioxidant, anti-inflammatory, antiseptic, antibacterial, antifungal, antitumor, anticancer, antinociceptive agents (, Marchese et al. 2016, Salehi et al. 2018. Thymols are also very important essential oils in the food industry. By using hybridization methodologies, ART and thymol hybrid may be novel candidates for the treatments of cancer and/or viral diseases. In the present study, the cytotoxic effect of new ART-Thymol hybrid molecule was determined in different cancer lines, investigated drug-likeness properties and inhibitory activity by using molecular docking (F ıgure 2).

Chemistry
The 1 H-NMR spectra of the ART-Thymol gave aromatic peaks at 7.18 ppm (doublet), 7.03-6.98 ppm (multiple) and 6.80 ppm (singlet) belonging to Thymols. The Tymol's protons can be recognized easily from its characteristic position in aromatic region. There are two different methyl on the thymol compounds, one is shifted at 1.43 ppm as a single (CH 3 ), and other methyl give the doublet (6 protons) at 1.18 ppm. The characteristic peaks of Artesunate can be seen around 5.82 ppm as a doublet and 5.44 ppm as a singlet due to electronegative effect of the oxygen atoms on the ring. There is a singlet at 2.30 ppm came from the CH 3 where neighbors peroxide bridge. As seen in 1 H-NMR spectra, Artesunate's remain CH 3 peaks are detected around 0.96 ppm and 0.84 ppm. The 13 C-NMR spectra of hybrid compound is important to determination of carbonyl (C ¼ O) peak between thymol and artemisinin structure. There are two ester functional groups on the hybrid molecule. Carbonyl's peaks are shifted at 170.93 ppm and 170.9 ppm in 13 C-NMR spectra. Moreover, four peaks between 105 ppm and 80 ppm belong to artesunate ( Figure S1 and Figure S2).

Cell culture and growth, cytotoxicity
Cytotoxic activity of ART-Thymol was determined against different cancer cell lines and non-cancerous human cell line for the first time. All of the cells were incubated with a series of dilutions (6.25-250 lM) of ART-Thymol at 24 h as described at method part. As shown in Figure S5, ART-Thymol exhibited cytotoxic effects on all the cell lines in a dose-dependent manner. From the results, ART-Thymol showed the cytotoxic effect with EC 50 values 70,96lM for HepG2, 97,31lM for LnCap, 6,03lM for Caco-2, 77,98lM for HeLa and 62,28lM for HEK293 cells, respectively. ART-Thymol is a more potent cytotoxic agent to Caco-2 colon carcinoma cells when compared to other cell lines ( Figure S5). This is the first study to report the cytotoxic effect of newly synthesized compound namely, ART-Thymol was determined in different cancer lines. As described in methods part that compound was synthesized by using Thymol and Artemisinin which are well known cytotoxic agents, thymol has cytotoxic and anticancer activities in different cell lines. Similarly, cytotoxic activities of ART are described in various cancer cell lines. Similar to these results, our newly synthesized molecule was high cytotoxic activity. Even, that compound is more potent than thymol and artemisinin alone. EC 50 of Thymol or mostly thymol containing extracts in CaCo-2 cell line was found 293.53 lM and 250 lMSimilarly, artemisinin was showed cytotoxic activity towards colorectal cancer cells namely, HCT116 and SW480. EC 50 concentration of artemisinin was found 204 and 156 lM in these cells, respectively . On the other hand, our chimeric compound was more potent than thymol and artemisinin.

Drug likeness activity
For a molecule to be a potential drug, it must have good pharmacokinetic properties in biological systems, in addition to having good biological activity. The ART-Thymol was checked for drug-likeness by performed the molecular properties and bioactivity prediction using by Molinspiration tool (Table S1).
It is known that for molecules, having a bioactivity score is more than 0.0 the molecule is active, while values À0.5 to 0.0 are expected to be moderately active and if the score is less than 0.5 then it is inactive. As seen in Table S1, the kinase inhibitor score of the ART-Thymol was between À5.0 and 0.0, and ART-Thymol can be utilized as a therapeutic agent for inhibition of kinases. Therefore, kinase inhibition potential of ART-Thymol was determined by using molecular docking studies.

QPCR results
The effects of ART-Thymol on gene expression analysis of kinases namely, CDK2 and P38 were determined in Caco-2 cell line by real-time PCR with SYBR Green detection chemistry. Real Time-PCR results of mRNA levels were represented in Figure S8. CDK2 mRNA level was decreased 55% at ART-Thymol treated cells with respect to control.
(p < 0.05). Similarly, nearly 90% decrease was seen at the mRNA level of P38 as a result of ART-Thymol treatment. These results clearly showed that ART-Thymol had high potential of kinase inhibition.
One of the well-known serine/threonine kinase members is P38 mitogen-activated protein kinases (MAPK). It plays important role in cancer progression including proliferation, migration, invasion and differentiation (Martinez-Limon et al. 2020). For example, apoptosis in cancer cells inhibited by P38 (Ding and Chen 2018). Therefore, inhibition of P38 MAPK is one of the important strategies to inhibit tumorigenesis. In silico studies indicated that ART-Thymol showed stronger binding affinity to p38 MAPK among the tested kinases. This was confirmed by qPCR results. Cyclin-dependent kinase 2 (CDK2) is one of the protein kinase that is involved in regulation of cell cycle by controlling the-S and M phase progression. Overexpression of CDK2 caused loss of cell division control which is related with hyperproliferation of cancer cells (Arba et al, Roskoski 2019). Therefore, strategies including development of inhibitors for CDK2 were considered to develop safe and selective control of cancer cells. Similar to P38 MAPK, ART-Thymol showed strong binding affinity to CDK2. Moreover, ART-Thymol was caused suppression of CDK2 mRNA level. These results showed that ART-Thymol has a potent inhibitory potential for P38 MAPK and CDK2. However, further researches such as western blot, kinase activity and toxicity tests were required for the evaluation of this molecule in vivo and in vitro.

Conclusion
In the present study, new hybrid thymol-artemisinin molecule was synthesized and characterized. After isolation, we obtained in 59% yields of ART-Thymol. Then, ART-thymol was used to finding cytotoxic activity against the different cancer cell lines and non-cancerous human cell line. ART-Thymol displayed the cytotoxic effect with EC 50 values 70,96 lM for HepG2, 97,31lM for LnCap, 6,03lM for Caco-2, 77,98lM for HeLa and 62,28lM for HEK293 cells, respectively. It was observed that ART-Thymol is a more potent cytotoxic agent to Caco-2 colon carcinoma cells. Moreover, kinase inhibition potential of ART-Thymol was found by using molecular docking and qPCR studies. In silico studies indicated that ART-Thymol showed stronger binding affinity to p38 MAPK and CDK2. These were confirmed by qPCR experiment. These results improved that thymol-artemisinin may be new drug candidates for the treatments of cancer.

Disclosure statement
No potential conflict of interest was reported by the authors.