Synthesis and In Silico Docking of New Pyrazolo[4,3-e]pyrido[1,2-a]pyrimidine-based Cytotoxic Agents

To explore a new set of anticancer agents, a novel series of pyrazolo[4,3-e]pyrido[1,2-a]pyrimidine derivativeshave been designed and synthesized viacyclocondensation reactions of pyrazolo-enaminone with a series of arylidenemalononitriles; compound 5 was obtained from 5-amino-4-cyanopyrazole. The structures of the target compounds were investigated by spectral techniques and elemental analysis (IR, UV–Vis, 1H NMR, 13C NMR and ESI-MS). All compounds were evaluated for their in vitro cytotoxicity employing a panel of different human tumor cell lines, A375, HT29, MCF7, A2780, FaDu as well as non-malignant NIH 3T3 and HEK293 cells. It has been found that the pyrazolo-pyrido-pyrimidine analog bearing a 4-Br-phenyl moiety was the most active toward many cell lines with EC50 values ranging between 9.1 and 13.5 µM. Moreover, in silico docking studies of the latter with six anticancer drug targets, i.e., DHFR, VEGFR2, HER-2/neu, hCA-IX, CDK6 and LOX5, were also performed, in order to gain some insights into their putative mode of binding interaction and to estimate the free binding energy of this bioactive molecule.


Introduction
At present, cancer still constitutes a tremendous frightful disease for numerous patients worldwide. This disease is regarded as one of the foremost causes of mortality, thus endangering the health and life of humans. Furthermore, cancer cases surpassed 14 million persons in 2012, and it is expected to affect 22 million persons in 2030 [1]. Regardless of the state of development and economic prosperity in all countries worldwide, the number of persons suffering from malignant melanoma, colon, breast, human ovarian carcinoma or pharynx carcinoma increased during the last decade. Under these circumstances, besides surgery and radiotherapy, chemotherapy is the most currently used treatment to cure the various types of cancers [2]. Nowadays, chemotherapy applying a set of drugs acting by different mechanisms is one of the most promising techniques being applied to treat cancer.
Thus, in spite of all progress to treat and cure cancer patients, there is still need to search for and to develop novel anticancer agents holding superior efficacy but giving rise to minimal side effects. Therefore, the identification of new chemical entities that are more reliable and efficient remains a major challenge for medicinal chemists. As a consequence, the synthesis of small molecules still represents a potent and effective strategy to supply novel chemical entities for cancer therapy.
In this context, heterocyclic compounds, especially those holding more than one non-carbon ring atom have played a key role in the field of medicinal chemistry [3,4]. Furthermore, the previously reported results from literature reveal that the combination 2 of 17 of two or more bioactive heterocyclic pharmacophores into the same molecule appears to be an effective tool for designing new chemical entities that have improved activity. Thereby, pyrazole-fused pyrimidines constitute a promising versatile class of heterocyclic scaffolds having always attracted much interest from chemists owing to their outstanding potential, such as antitumor [5], anti-inflammatory [6], anticancer and anti-5-lipoxygenase [7], antibacterial [8], antitubercular [9] and especially cytotoxic activities ( Figure 1A-D) [10][11][12][13].
anticancer activities especially in cases wherethe NH2 group shows conjugation when it is tethered with a pyrimidine (Figure 2A,B) [21,22] or the CN group linked in a conjugate way with a pyridine ( Figure 2C,D) [23,24].
In addition, a review of the literature revealed that several strongly cytotoxic chemical scaffolds contain in their structures amine ( Figure 2E,F) or cyano ( Figure 2G,H) groups directly linked to a heterocycle [25,26]. This particular structural linkage gave the kick-off to several research teams to design and synthesize amino-pyrimidine and cyano-pyridine scaffolds due to this notable anticancer activity against a wide range of cell lines.
Inspired by the above findings, and in continuation of our previous work on the synthesis of novel fused-pyrimidine scaffolds [27][28][29], we aimed to design and synthesize new pyrazolo [4,3-e]pyrido[1,2-a]pyrimidine derivatives. Hereby, we report for the first time the cytotoxic activity of these newly synthesized compounds toward five human tumor cell lines, A375, HT29, MCF7, A2780, FaDu as well as non-malignant NIH 3T3 and HEK293 cells. Moreover, their structure-activity relationship (SAR) was investigated.  Pyrido-pyrimidines have, for a long time, attracted the interests of both biological and synthetic researchers alike due to their various biological therapeutic properties such as anticonvulsant [14], antitumor [15], anti-proliferative [16], antifungal [17], adenosine kinase inhibition [18] and cytotoxic activities ( Figure 1E-G) [19,20].
A review of the literature has shown in several cases that amino and cyano functions can be involved in some interesting interactions with target enzymes. Indeed, various constructed heterocyclic compounds gained much attention owing to their cytotoxic and anticancer activities especially in cases wherethe NH 2 group shows conjugation when it is tethered with a pyrimidine (Figure 2A,B) [21,22] or the CN group linked in a conjugate way with a pyridine ( Figure 2C,D) [23,24].
In addition, a review of the literature revealed that several strongly cytotoxic chemical scaffolds contain in their structures amine ( Figure 2E,F) or cyano ( Figure 2G,H) groups directly linked to a heterocycle [25,26]. This particular structural linkage gave the kick-off to several research teams to design and synthesize amino-pyrimidine and cyano-pyridine scaffolds due to this notable anticancer activity against a wide range of cell lines.
Inspired by the above findings, and in continuation of our previous work on the synthesis of novel fused-pyrimidine scaffolds [27][28][29], we aimed to design and synthesize new pyrazolo [4,3-e]pyrido[1,2-a]pyrimidine derivatives. Hereby, we report for the first time the cytotoxic activity of these newly synthesized compounds toward five human tumor cell lines, A375, HT29, MCF7, A2780, FaDu as well as non-malignant NIH 3T3 and HEK293 cells. Moreover, their structure-activity relationship (SAR) was investigated.
In detail, the use of aminopyrazoles as heterocyclizing agents has received considerable attention in recent years. As a result, these molecules have proven to be the building blocks of choice in the preparation of several heterocyclic compounds, they in fact exhibit high reactivity due to the presence of a primary amine function and of a nitrile function in the alpha position. These two functions are capable of reacting as a nucleophilic and electrophilic agent, respectively, and thus subsequently undergo the cyclization reaction. This is why we chose to synthesize this type of amino-cyanopyrazole 3. Mechanistically, the reaction begins with the attack by the free doublet of the primary amine of phenylhydrazine on the ethylenic quaternary carbon of the ether function in the ethoxyalkylidene 1, releasing an ethanol molecule, then a second nucleophilic attack on the nitrile function by the secondary amine function of the phenylhydrazine leads to the expected pyrazole compound 3 (Scheme 2). Scheme 1. Synthetic pathway for the synthesis of pyrazolo-pyrido-pyrimidine derivatives 7a-l.
In detail, the use of aminopyrazoles as heterocyclizing agents has received considerable attention in recent years. As a result, these molecules have proven to be the building blocks of choice in the preparation of several heterocyclic compounds, they in fact exhibit high reactivity due to the presence of a primary amine function and of a nitrile function in the alpha position. These two functions are capable of reacting as a nucleophilic and electrophilic agent, respectively, and thus subsequently undergo the cyclization reaction. This is why we chose to synthesize this type of amino-cyanopyrazole 3. Mechanistically, the reaction begins with the attack by the free doublet of the primary amine of phenylhydrazine on the ethylenic quaternary carbon of the ether function in the ethoxyalkylidene 1, releasing an ethanol molecule, then a second nucleophilic attack on the nitrile function by the secondary amine function of the phenylhydrazine leads to the expected pyrazole compound 3 (Scheme 2). The use of TCAA (pka = 0.77) facilitates the formation of an electrophilic site (C=O) of the acetylacetone 4, and therefore makes it easy to attack the primary amine's free In detail, the use of aminopyrazoles as heterocyclizing agents has received considerable attention in recent years. As a result, these molecules have proven to be the building blocks of choice in the preparation of several heterocyclic compounds, they in fact exhibit high reactivity due to the presence of a primary amine function and of a nitrile function in the alpha position. These two functions are capable of reacting as a nucleophilic and electrophilic agent, respectively, and thus subsequently undergo the cyclization reaction. This is why we chose to synthesize this type of amino-cyanopyrazole 3. Mechanistically, the reaction begins with the attack by the free doublet of the primary amine of phenylhydrazine on the ethylenic quaternary carbon of the ether function in the ethoxyalkylidene 1, releasing an ethanol molecule, then a second nucleophilic attack on the nitrile function by the secondary amine function of the phenylhydrazine leads to the expected pyrazole compound 3 (Scheme 2). The use of TCAA (pka = 0.77) facilitates the formation of an electrophilic site (C=O) of the acetylacetone 4, and therefore makes it easy to attack the primary amine's free doublet of the starting building block 3,which gives a non-isolable intermediate I 2 . The crucial role of TCAA can be seen again through the facilitation of the dehydration made by a second nucleophilic attack of the free doublet of the same amine function on the same electrophilic site, which gives the isolable intermediary 5 (pathway 1). These conditions imply the failure to obtain compound 5 (pathway 2) (Scheme 3).
doublet of the starting building block 3,which gives a non-isolable intermediate I2. The crucial role of TCAA can be seen again through the facilitation of the dehydration made by a second nucleophilic attack of the free doublet of the same amine function on the same electrophilic site, which gives the isolable intermediary 5 (pathway 1). These conditions imply the failure to obtain compound 5′ (pathway 2) (Scheme 3).

Scheme 3. Plausible mechanistic pathway for the synthesis of compound 5.
A putative mechanism (Scheme 4) for the formation of 7a-l has been depicted in Scheme 2. Thereby, the reaction sequence starts with a nucleophilic attack onto the double bond of the enaminone; this Michael addition forms intermediate I3, which subsequently undergoes a proton transfer from one carbon to another one followed by an intramolecular cyclization to afford the non-isolable intermediate I5. Upon tautomerization, a primary amine is formed, which attacks the nitrile function of the pyrazole moiety leading finally to target compound 7. A putative mechanism (Scheme 4) for the formation of 7a-l has been depicted in Scheme 2. Thereby, the reaction sequence starts with a nucleophilic attack onto the double bond of the enaminone; this Michael addition forms intermediate I 3 , which subsequently undergoes a proton transfer from one carbon to another one followed by an intramolecular cyclization to afford the non-isolable intermediate I 5 . Upon tautomerization, a primary amine is formed, which attacks the nitrile function of the pyrazole moiety leading finally to target compound 7.

Biological Evaluation
The synthesized compounds were evaluated for their cytotoxic activity by a photometric rhodamine B assay (SRB) on different human cancer cell lines, malignant melanoma (A375), colon (HT29), breast (MCF-7), human ovarian carcinoma (A2780) and pharynx carcinoma (FaDu); for comparison, non-malignant mouse embryonic fibroblasts (NIH 3T3) and human embryonic kidney cells (HEK293) were included; doxorubicin (DX) was used as a positive control [31,32]. The results are summarized in Table 1.The EC 50 values in µM from SRB assays were determined after 72 h of treatment, and the values are averaged from three independent experiments performed each in triplicate, confidence interval, CI = 95%; mean ± standard mean error).

Biological Evaluation
The synthesized compounds were evaluated for their cytotoxic activity by a photometric rhodamine B assay (SRB) on different human cancer cell lines, malignant melanoma (A375), colon (HT29), breast (MCF-7), human ovarian carcinoma (A2780) and pharynx carcinoma (FaDu); for comparison, non-malignant mouse embryonic fibroblasts (NIH 3T3) and human embryonic kidney cells (HEK293) were included; doxorubicin (DX) was used as a positive control [31,32]. The results are summarized in Table 1.The EC50 values in µM from SRB assays were determined after 72 h of treatment, and the values are averaged from three independent experiments performed each in triplicate, confidence interval, CI = 95%; mean ± standard mean error). The intermediates 3 and 5 were found to be non-cytotoxic (EC50 > 30 µM) against all the five cancer cell lines. However, many of the target compounds (7a-h) showed noteworthy cytotoxic effects for all tested human tumor cell lines.
Our target compound 7 holds five cyclic rings, A, B, C, D and E ( Figure 3). The values of EC50 differ by the group linked to cycle E. Interestingly, the highest activity in A375 cancer cell was determined for compound 7e with a bromine substituent attached to the  The intermediates 3 and 5 were found to be non-cytotoxic (EC 50 > 30 µM) against all the five cancer cell lines. However, many of the target compounds (7a-h) showed noteworthy cytotoxic effects for all tested human tumor cell lines.
Our target compound 7 holds five cyclic rings, A, B, C, D and E ( Figure 3). The values of EC 50 differ by the group linked to cycle E. Interestingly, the highest activity in A375 cancer cell was determined for compound 7e with a bromine substituent attached to the aryl moiety E in the para position. The next molecule in this series was the methylsubstituted compound 7b, also exhibiting good cytotoxicity (EC 50 = 12.9 ± 1.6 µM), while nitro-substituted compound 7g was less cytotoxic (EC 50 = 16.2 ± 2.0 µM), followed by 7f, 7a and 7c, respectively. On the other hand, the topmost activity in colon cancer cells (HT29) was again determined for 7e holding an EC 50 value of 13.3 ± 1.8 µM, followed by 7b, 7f (Ar = 4-F-Ph) and 7g with EC 50 values of 17.5 ± 1.8, 24.7 ± 4.4, 25.9 ± 3.4 µM, respectively. Toward MCF-7, it is apparent that the same derivative 7e exhibited the highest activity with an EC 50 value of 9.2 ± 0.7µM, and compounds 7b and 7g (EC 50 = 12.2 ± 1.3 and 15.7 ± 2.2 µM, respectively) also exhibited good activities.
be due to the fact that DX acts in two ways in cancer cells: on the one hand, DX produces free radicals and thus damages cellular membranes, and on the other hand, DX interacts with DNA and disrupts topoisomerase-II-mediated DNA repair. Conjugation of our compounds with functional groups that have a particularly high affinity for cellular membranes could lead to more cytotoxic compounds. This would also represent a possibility to achieve higher tumor-cell/non-tumor cell selectivity.
Since the SRB assays showed 7e as the most active compound, molecular docking was performed to establish some structure-activity relationships (SARs). The increase in the activity of 7e might be due to the presence of an electron-withdrawing group attached to the E ring. Today, researchers increasingly use the in silico techniques and methods, including the molecular docking, to predict potential interactions of tested compounds for their cytotoxic activity with the key targets in cancer cells. Hereby, literature has shown that six key proteins (MCL-1, CDK6, HER-2/neu, LOX5, USP7 and DHFR) [33] or (DHFR, VEGFR2, HER-2/neu, hCA-IX, CDK6 and LOX5) [32] were used in the in silico docking studies of the most potent compounds in order to investigate their predicted anticancer potential. In this study, to develop a new generation of more potent multitargeted anticancer agents and motivated by previous research on that topic [28,32,34], we took advantage of the reported structure-activity relationships (SARs) of pyrazo- The highest cytotoxicity for A2780 cancer cell lines was found again for compound 7e (EC 50 = 9.1 ± 1.6 µM). In this series, compounds 7g and 7b (EC 50 = 14.5 ± 2.2 and 14.6 ± 2.2 µM, respectively) showed noteworthy activity as compared to other analogs, and their EC 50 values ranged between 18.0 ± 3.0 and 27.5 ± 8.1µM. Unfortunately, compound 7e also showed significant cytotoxic effects for the non-malignant cell lines. Our compounds are significantly less cytotoxic than "gold standard"doxorubicin (DX). This may be due to the fact that DX acts in two ways in cancer cells: on the one hand, DX produces free radicals and thus damages cellular membranes, and on the other hand, DX interacts with DNA and disrupts topoisomerase-II-mediated DNA repair. Conjugation of our compounds with functional groups that have a particularly high affinity for cellular membranes could lead to more cytotoxic compounds. This would also represent a possibility to achieve higher tumor-cell/non-tumor cell selectivity.
Since the SRB assays showed 7e as the most active compound, molecular docking was performed to establish some structure-activity relationships (SARs). The increase in the activity of 7e might be due to the presence of an electron-withdrawing group attached to the E ring.
Today, researchers increasingly use the in silico techniques and methods, including the molecular docking, to predict potential interactions of tested compounds for their cytotoxic activity with the key targets in cancer cells. Hereby, literature has shown that six key proteins (MCL-1, CDK6, HER-2/neu, LOX5, USP7 and DHFR) [33] or (DHFR, VEGFR2, HER-2/neu, hCA-IX, CDK6 and LOX5) [32] were used in the in silico docking studies of the most potent compounds in order to investigate their predicted anticancer potential. In this study, to develop a new generation of more potent multitargeted anticancer agents and motivated by previous research on that topic [28,32,34], we took advantage of the reported structure-activity relationships (SARs) of pyrazolo-pyrido-pyrimidine analogs. Thus, in order to assess the ability of the top-ranked active compounds to inhibit six anti-cancer drug targets (DHFR, VEGFR2, HER-2/neu, hCA-IX, CDK6 and LOX5), we carried out docking study for complexes consisting of compound 7e and the target proteins. This ligand fits very well in the active site binding cavity of all target enzymes (Figure 4). lo-pyrido-pyrimidine analogs. Thus, in order to assess the ability of the top-ranked active compounds to inhibit six anti-cancer drug targets (DHFR, VEGFR2, HER-2/neu, hCA-IX, CDK6 and LOX5), we carried out docking study for complexes consisting of compound 7e and the target proteins. This ligand fits very well in the active site binding cavity of all target enzymes (Figure 4). The choice of these six proteins among the eight proposed above was based on the importance of the interactions shown by the docked compound with the residues forming the active site of each target enzyme, while, toward MCL-1 and USP7 enzymes, the ligand 7e did not show interesting interactions, and especially the absence of hydrogen bonds ( Figure 5). The obtained results showed excellent interaction with all the above targets as compared to their respective co-crystallized ligands ( Table 2). The choice of these six proteins among the eight proposed above was based on the importance of the interactions shown by the docked compound with the residues forming the active site of each target enzyme, while, toward MCL-1 and USP7 enzymes, the ligand 7e did not show interesting interactions, and especially the absence of hydrogen bonds ( Figure 5).
lo-pyrido-pyrimidine analogs. Thus, in order to assess the ability of the top-ranked active compounds to inhibit six anti-cancer drug targets (DHFR, VEGFR2, HER-2/neu, hCA-IX, CDK6 and LOX5), we carried out docking study for complexes consisting of compound 7e and the target proteins. This ligand fits very well in the active site binding cavity of all target enzymes (Figure 4). The choice of these six proteins among the eight proposed above was based on the importance of the interactions shown by the docked compound with the residues forming the active site of each target enzyme, while, toward MCL-1 and USP7 enzymes, the ligand 7e did not show interesting interactions, and especially the absence of hydrogen bonds ( Figure 5). The obtained results showed excellent interaction with all the above targets as compared to their respective co-crystallized ligands ( Table 2). The obtained results showed excellent interaction with all the above targets as compared to their respective co-crystallized ligands ( Table 2). Considering interactions with all the targets under observation, ligand 7e fits well inside the pocket; it displayed crucial hydrogen bonds by its (CO) and (NH 2 ) groups; thereby in the case of the most favorable complex of compound 7e with DHFR, four H-bonds were formed: three between the amine function and GLY20, ASP21 and SER59 and one through the carbonyl group with THR56. The fused heterocyclic system seems to play a significant binding role through the appearance of a pi-sigma interaction with LEU22. In addition, the phenyl groups exhibit many hydrophobic interactions ( Figure 6A). Moreover, toward active site amino acids of VEGFR2 ( Figure 6B) 7e is involved in two H-bonds with SER884 and ARG1027, a pi-sigma interaction with ILE888, a pi-sulfur interaction with CYS1024, as well as alkyl with PRO821 and pi-alkyl with LYS 887 and ILE888. Furthermore, in the case of HER-2/neu, compound 7e forms some substantial H-bonds, i.e., two through its amine function and one by its carbonyl group, a pi-sigma interaction with LEU852 besides many hydrophobic interactions with the amino acid sequence VAL734, ALA751, LEU800, MET801 and CYS 805 ( Figure 6C). In the same manner, toward active site amino acids of hCA-IX, ligand 7e formed a conventional hydrogen bond by its carbonyl group with THR201, showed pi-sigma interaction with LEU199 and hydrophobic interactions with residues HIS68, HIS94, HIS119, VAL121, VAL130, VAL142 and TRP210 ( Figure 6D). Furthermore, for the complex CDK6-7e, a hydrogen bond formed between (NH 2 ) and ASP102, pi-anion with ASP104 and hydrophobic interactions with ILE19, VAL27, LEU152 and ALA162 ( Figure 6E) were found. Finally, against LOX, 7e demonstrated three H-bonds through its CO and NH 2 groups with PHE177 and LEU607 and showed some hydrophobic interactions with the amino acids sequence PHE177, HIS367, LYS409 and LEU607 ( Figure 6F).

General
A detailed description of materials and methods can be found in the Supplementary Materials file. For molecular docking, the 3D crystal structures of PDB were obtained from the RSCB protein data bank (PDB): DHFR (ID:5HQY [35]), VEGFR2 (ID:5EW3 [35,36]), HER2/neu (ID:3RCD [35,37]), hCA-IX (ID:5FL4 [35,38]), CDK6 (ID:3NUP [35,39]) and LOX5 (ID:3V99 [35,40]). All water molecules were removed, and hydrogen atoms were added before docking using Discovery Studio Visualizer D.S. (2017) (BIOVIA, San Diego, CA, USA); Gasteiger charges were also added to the system during the preparation of the receptor input file. Docking studies were performed using AutoDock 4.2 software (Scripps Research; http://autodock.scripps.edu, accessed on 7 July 2021). The structures of the compounds were drawn using ChemDraw [ver. 10.0]. The optimization of all the geometries of scaffolds was performed with ACD (3D viewer) software (http://www. filefacts.com/acd3d-viewer-freeware-info, accessed on 7 July 2021). Co-crystalized ligands in the proteins were taken as reference ligands and re-docked into the active site of proteins for energy comparison. The top-scored conformation was recorded for each compound and used for further analysis, and 2D images were captured through Discovery Studio Visualizer (2017) developed by Accelrys (BIOVIA, San Diego, CA, USA). 13 C NMR spectra (Supplementary Materials) were recorded as APT spectra showing CH and CH 3 groups as positive signals and CH 2 groups and quaternary carbons as negative signals.

Conclusions
In summary, a new class of pyrazolo [4,3-e]pyrido[1,2-a]pyrimidine derivatives was designed, synthesized, characterized and evaluated for its cytotoxic activity toward five human cancer cell lines A375, HT-29, MCF-7, A2780, FaDu, as well as non-malignant NIH 3T3 and HEK293. Compound 7e displayed a noteworthy cytotoxic effect toward all cancer cell lines. An SAR study demonstrated that fused heterocyclespyrazole, pyridine and pyrimidine and group linked to the aryl moiety E in the para position, especially the presence of a bromine substituent, seem to play a crucial role in the cytotoxic activities. In addition, the molecular docking indicated that this class of heterocyclic molecules exhibits important binding energy and interaction with interesting residues of anticancer targets such as, DHFR, VEGFR2, HER-2/neu, hCA-IX, CDK6 and LOX5.