Design, synthesis, molecular modelling, and biological evaluation of novel substituted pyrimidine derivatives as potential anticancer agents for hepatocellular carcinoma

Abstract New anticancer agents are highly needed to overcome cancer cell resistance. A novel series of pyrimidine pyrazoline-anthracene derivatives (PPADs) (4a-t) were designed and synthesised. The anti-liver cancer activity of all compounds was screened in vitro against two hepatocellular carcinoma (HCC) cell lines (HepG2 and Huh-7) as well as normal fibroblast cells by resazurin assay. The designed compounds 4a-t showed a broad-spectrum anticancer activity against the two cell lines and their activity was more prominent on cancer compared to normal cells. Compound 4e showed high potency against HepG2 and Huh-7 cell lines ((IC50=5.34 and 6.13 μg/mL, respectively) comparable to that of doxorubicin (DOX) activities. A structure activity relationship (SAR) has been investigated and compounds 4e, 4i, 4m, and 4q were the most promising anticancer agents against tested cell lines. These compounds induced apoptosis in HepG2 and Huh-7 cells through significant activation of caspase 3/7 at all tested concentrations. In conclusion, 4e could be a potent anticancer drug.


Introduction
Cancer (called tumour) is the result of uncontrolled growth and spread of abnormal cells 1 . Cancer is considered the leading cause of death worldwide based on the world health organisation (WHO) statistics. The most common types of cancer are lung, stomach, liver, colorectal, and female breast cancers. Hepatocellular carcinoma (HCC), one of the most common cancer types, is a primary malignancy of the liver and occurs predominantly in patients with underlying chronic liver disease. The mortality rate of HCC has significantly increased worldwide. In 2018, the American Cancer Society estimated that new liver cancer patients in USA to be over 40,000 cases 2 . One of the most common risk factors for developing HCC is the chronic viral infection with hepatitis C virus (HCV) or hepatitis B virus (HBV) or both 3 . There are many therapeutic strategies for targeting HCC. This includes chemotherapy, radiotherapy, and immunotherapy. These types of treatment are often accompanied by high systemic toxicity and sometimes drug resistance. Therefore, new therapeutic agents are highly needed to overcome this tumour drug resistance 4 .
The medicinal chemistry importance of the three pharmacophores, i.e. pyrimidine, pyrazoline and anthracene, encouraged us to design novel series of compounds hybridised with the three moieties. The hybridisation of these pharmacophores in a single molecule ( Figure 4) is expected to result in a hybrid molecule that possesses anticancer activity. In this study, we report the design,  synthesis and anticancer activity of a novel hybrids of pyrimidine derivatives (4a-t) bearing pyrazoline and anthracene pharmacophores against both human liver cancer cell lines (HepG2 and Huh-7) and normal fibroblast cells by resazurin assay. In addition, apoptosis and effects on caspase-3 and -7 activation against human liver cancer cell lines HepG2 and Huh-7 were also performed for the most active derivatives. Structure and activity relationship (SAR) and possible mechanisms of action of these compounds were also investigated.

Instruments
All melting points were determined with Electro-thermal IA 9100 apparatus (Shimadzu, Tokyo, Japan) and were uncorrected. FT-IR spectra were recorded as potassium bromide pellets on a Perkin-Elmer 1650 spectrophotometer (PerkinElmer, Waltham, MA), Faculty of Science, Cairo University, Cairo, Egypt. 1 HNMR and 13 C-NMR spectra were recorded in DMSO-d 6 13  General procedure for the preparation of compounds (3a-d). A mixture of 9-acetylanthracene (0.01mol) and the appropriate aldehyde (0.01 mol) in 50 mL 10% ethanolic KOH solution was stirred at room temperature for 24 h. The solution was cooled, poured on ice/ water acidified with dil. HCl. The produced precipitate was filtered off, dried and crystallised from ethanol to give compounds 3a-d. General procedure for the preparation of compounds (4a-t). A mixture of compound 2a-e (4 mmol), the appropriate propenone 3a-d (4 mmol) and potassium hydroxide (0.2 g, 5 mmol) in absolute ethanol (30 mL) was refluxed for 72 h. The reaction mixture was poured on water, neutralised with 2 N hydrochloric acid and the residue was filtered off. The crude product obtained was crystallised from ethanol.

In vitro cytotoxicity activity
All compounds 2a-e, 3a-d, and PPAD's 4a-t were subjected to a screening system for evaluation of their anticancer activity against cell line of human cancer, namely liver (HepG2 and Huh-7) cancer and normal fibroblast cells obtained from the German Cancer Research Center (DKFZ), following the Resazurin Cell Growth Inhibition Assay 45 in comparison to the standard treatment using DOX (see Supplementary).

Data analysis
Results are represented as M ± SEM of at least three independent experiments. Data analysis has been done using graph pad prism and Sigma Plot 11 software.

Molecular modelling procedure
The modelling experiment described in his study was performed by using the Discovery Studio version 4.5 (Accelrys Inc., San Diego, CA) software as previously described 13 . The required pdb coordinates were downloaded from the Brookhaven website (www.rcsb.org). The hydrogen atoms were then added to both the small molecule and the DNA structure. The atom and bond types as well as the protonation state for the small molecule and the binding site were checked and corrected when needed. Water molecules were deleted. This was followed by minimising the complex with the Discovery Studio (DS) force field by using the default parameters. The binding mode of the designed compound and that of the DOX in bound to part of the DNA structure will be discussed later.

Anticancer evaluation
This study presents the synthesis and antiproliferative activity of compounds having pyrimidine, pyrazoline, and anthracene pharmacophores. All compounds 2a-e, 3a-d, and PPAD's 4a-t were investigated for their in vitro anti-HCC activity against HepG2, Huh-7 cell lines, and normal fibroblast cells and the anticancer drug DOX was used as standard treatment. The cytotoxic activities of tested compounds were measured as IC 50 (in lg/mL) value (the dose that reduces cell growth to 50%) ( Table 1). The results showed that the tested compounds exhibited good to moderate anti-proliferative activities against the tested cell lines compared to normal cells. Compounds 2a-e, 3a-d were found to show moderate activity against the two cell lines. Regarding the activity of PPAD's 4a-t against HepG2 cell line, the results showed that compound 4f (IC 50 ¼4.2 lg/mL which is equivalent to 7.2 lM) possessed the highest degree of cytotoxicity, 4e (IC 50 ¼5.34 lg/mL which is equivalent to 9.6 lM) was almost equipotent to DOX (IC 50 ¼5.43 lg/mL which is equivalent to 10.1 lM) and 4q (IC 50 ¼6.85 lg/mL which is equivalent to 11.8 lM) was quite less potent than DOX ( Figure 5). The anticancer activity of the tested PPAD's against HepG2 cell line had the following descending order: To shed the light on the SAR of these series of compound. The data suggest that the promising compounds emerging were those substituted with fluorine atom (-F) in both R and R ' such as 4e or -F and N, N(CH 3 ) 2 such as 4f and 4q. Compounds substituted with both (R) and (R ' ) were more effective than those having only R ' substituent as in 4b-d against HepG2 cell line. On the other hand, testing the compounds against Huh-7 cell line, all the tested PPAD's 4a-s showed anticancer activities with IC 50 values ranging from 4.78 to 16.75 lg/mL. Interestingly, compounds 4e and 4g (IC 50 ¼6.13 which is equivalent to 11.1 lM and 4.78 lg/mL which is equivalent to 7.6 lM, respectively) showed activity against Huh-7 cell line higher than DOX. Compound 4i (IC 50 ¼6.47 lg/mL which is equivalent to 11.25 lM) showed activity comparable to DOX ( Figure 5). However, compounds 4m, 4r, 4t, 4f, 4p, and 4q had significant activity (IC 50 ¼7.50, 7.61, 7.42, 8.45, 8.14, and 8.66 lg/ mL, respectively). The activity of the tested compounds against Huh-7cell line had the following descending order: The data presented in this study revealed that all the tested PPAD's 4a-t had broad spectrum anticancer activity against all screened HCC cell lines compared to normal cells. Some of our PPAD's exhibited potent antiproliferative activity against HepG2 and Huh-7 cell lines. The cytotoxicity of all compounds was more prominent on cancer cells compared to normal ones (Table 1). These results suggest that pyrimidine pyrazoline-anthracene backbone is an interesting anticancer pharmacophore. Moreover, some PPAD's were even more potent than the standard drug Dox. The most potent compound in this study was 4e. This could be explained by the electronegative effect of the fluorine atom on the pyrimidine and pyrazoline -anthracene backbone. PPAD's 4e, 4i, 4m, and 4q were the most promising anticancer agents against all the tested cell lines ( Figure 6). Interestingly, PPAD's 4e showed high potency against HepG2 and Huh-7 cell lines.
Regarding the structure SAR of compounds (Figure 7), there was a consistent relation between the lipophilicity and/or electronic property of the substituent groups R and R ' groups and the antiproliferative activity. Concerning the nature of substituent groups R and R ' , the order of activity of tested compounds against tested cell lines was the following: (F > N(CH 3 ) 2 >3,4,5-(OCH 3 ) 3 > 3-Indolyl > H). The introduction of a more electronegative and a lipophilic substituent to phenyl rings of the pyrimidine and pyrazoline enhance potency against HepG2 and Huh-7 cancer cell lines as shown in the fluorine substituted PPAD's 4e. However, a drop in potency against the same cell lines was observed with unsubstituted phenyl ring or 3-indolyl substituted PPAD's as in 4a-d. The sensitivity of the tested cell lines to PPAD's 4a-t was in the following descending order: (HepG2 > Huh-7).

Caspase 3/7 assay
The caspases are a family of endoproteases that provide critical links in cell regulatory networks through controlling inflammation and cell death (apoptosis). Caspase-3 and caspase-7 cleave proteins involved in programmed cell death events 46 . It is well established that the induction of the apoptotic cascade is one of the main mechanisms of most of the currently available anticancer agents 45,47,48 . To determine the mechanism involved in the antitumor activity of our PPADs demonstrated above whether is a result of apoptosis or not, Caspase-Glo 3/7 assay was performed 47 . HepG-2 ( Figure 8) and Huh-7 cell lines ( Figure 9) were treated with the target PPAD's samples or DMSO (solvent control). Figures  8 and 9 show that PPADs 4e, 4i, 4m, and 4q stimulated caspase activity in both cell lines at all tested concentrations and caused significant increase in activation of caspase 3/7 in a dose-dependent manner. These results suggest that our compounds induced apoptosis is, in part, due to activation of caspses3/7 and apoptosis is may be the main mechanism of action. Moreover, the apoptosis induced by the tested target compounds on Huh-7 cell line was greater than its effect on HepG2 cell line. Table 1. IC 50 values in lg/mL for cytotoxic activity of the compounds against hepatocellular carcinoma cell lines and fibroblast cells.

Molecular modelling
One possible reason for the potent anticancer activity of the designed compounds is the presence of the planar aromatic tricyclic (Anthracene) ring. The anthracene containing compounds, such as DOX or the designed compounds 4a-t, are hypothesised to function primarily at the DNA level by blocking the replication and transcription processes. The binding to DNA structure is generally hypothesised to be essential for the cytotoxic activity of these compounds. To predict and understand the possible binding mode and the respective interactions of the designed compounds with the cell DNA structure, the co-crystal structure of DOX-DNA sequence d(CGATCG) complex (PDB: 1D12) was used 49 . As shown in Figure 10, the anthracene planar chromophore of compound 4e is intercalated with the DNA helix. While the rest of the structure is directed towards the minor groove of DNA forming additional van der Waals interactions.

Conclusions
A novel series of pyrimidine pyrazoline-anthracene hybrids 4a-t were designed and synthesised via a cyclocondensation reaction of 2-hydrazinopyrimdine derivatives 2a-e and the appropriate chalcones 3a-d and their spectral and elemental analyses proved chemical structures. The antiproliferative activity of all synthesised compounds was screened against both HCC cell lines (HepG2 and Huh-7) and normal fibroblast cells, using DOX as reference. PPAD's 4a-t had broad spectrum anticancer activity against two HCC cell lines compared to normal cells. Compound 4f possessed the most potent cytotoxicity against HepG2 cell line. In addition, PPAD's 4e and 4g showed decent activity against Huh-7 cell line higher than DOX. Compound 4e showed high potency against HepG2 and Huh-7 cell lines. PPAD's 4e, 4i, 4m, and 4q were the most promising anticancer agents against all the tested cell lines. Further studies on the mechanism of action demonstrated that these compounds induce apoptosis in HepG2 and Huh-7 cell lines through significant activation of caspase 3/7 at all tested concentrations. The molecular modelling study performed suggested another possible mechanism of action for these compounds. Similar to the DOX, the presence of the tricyclic planar anthracene chromophore could intercalate with the DNA helix of the cancer cell. While the rest of the structure will be directed towards the minor groove of DNA forming additional van der Waals interactions. In conclusion, the designed pyrimidine pyrazoline-anthracene scaffold is an interesting anticancer pharmacophore and considered as novel lead scaffold for any future optimisation.