Novel phthalimide based analogues: design, synthesis, biological evaluation, and molecular docking studies

Abstract Pyrazolylphthalimide derivative 4 was synthesized and reacted with different reagents to afford the target compounds imidazopyrazoles 5-7, pyrazolopyrimidines 9, 12, 14 and pyrazolotriazines 16, 17 containing phthalimide moiety. The prepared compounds were established by different spectral data and elemental analyses. Additionally, all synthesized derivatives were screened for their antibacterial activity against four types of Gram + ve and Gram-ve strains, and for antifungal activity against two fungi micro-organisms by well diffusion method. Moreover, the antiproliferative activity was tested for all compounds against human liver (HepG-2) cell line in comparison with the reference vinblastine. Moreover, drug-likeness and toxicity risk parameters of the newly synthesized compounds were calculated using in silico studies. The data from structure-actvity relationship (SAR) analysis suggested that phthalimide derivative bearing 3-aminopyrazolone moiety, 4 illustrated the best antimicrobial and antitumor activities and might be considered as a lead for further optimization. To investigate the mechanism of the antimicrobial and anticancer activities, enzymatic assay and molecular docking studies were carried out on E. coli topoisomerase II DNA gyrase B and VEGFR-2 enzymes.


Introduction
Nowadays, the most serious public health problems in the world are cancer and infectious diseases [1][2][3] . The evidence of multi-drug resistant microbial pathogens due to extensive use of antibiotics has been appeared and stimulated the search for discovery of new safer, potent, and resistance-free antimicrobial agents 4,5 . Moreover, the research for novel, selective and more potent antitumor agents is still a vital challenge for biologists and medicinal chemists 6,7 .
Bearing in mind our program in the synthesis of biologically active heterocyclic compounds [27][28][29][30][31][32] and the molecular pharmacophores (I-V) outlined in Figure 1 and their structural requirements [33][34][35][36] , some phthalimide derivatives were designed after exploring molecular hybridization approaches with pyrazole, imidazo[1,2-b]-pyrazole, pyrazolopyrimidine, pyrazolo-triazine moieties ( Figure 1). All the newly prepared phthalimide derivatives were subjected for evaluation of both antimicrobial and anticancer activities with the study of their Drug-Likeness and Toxicity parameters. Furthermore, in-vitro enzyme assay of the most potent derivative was performed against E. coli topoisomerase II DNA gyrase B and VEGFR-2 enzymes, followed by molecular docking studies to get a distinct insight about the interactions and binding mode in the active sites of these enzymes. and 13C-NMR spectra were recorded in DMSO-d 6 at 400, 500 MHz on JEOL and Broker NMR spectrometer (d, ppm) using TMS as an internal standard. Mass spectra were obtained on JEOL JMS600 H Root mass spectrometer at 70 ev. Elemental analyses were carried out by the Micro analytical Center of Cairo University, Giza, Egypt. The antimicrobial and anticancer activities were carried out in the Medical Mycology Laboratory of the Regional Center for Mycology and Biotechnology of Al-Azhar University, Cairo, Egypt.

Biological activity
Antimicrobial activity All bacterial and fungal strains were received from the culture collection of the Regional Center for Mycology and Biotechnology (RCMB), Al-Azhar University, Cairo, Egypt. All target derivatives were screened in-vitro opposite to various kinds of bacteria, Grampositive bacteria (Streptococcus pneumoniae and Bacillus subtilis) and Gram-negative bacteria (Pseudomonas aeruginosa and Escherichia coli) and for their Antifungal activities against Aspergillus fumigatus and Candida albicans, respectively. Ampicillin and gentamycin were used as standard antibacterial drugs while amphotericin B was used as reference antifungal drug. The diameter of inhibition zone (mm) was measured for the biological activity using the diffusion technique 37 . The promising compounds were further investigated to evaluate their antimicrobial activity expressed in terms of minimum inhibitory concentration (MIC) using the modified agar well diffusion method 37 .

Antitumor activity
The anticancer activity of all derivatives were determined against a human liver cancer cell line (HepG-2) using the 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl teterazolium bromide (MTT) assay and vinblastine was used as a standard drug following the previously reported procedure 38,39 . All experiments were carried out in triplicate.
In-vitro enzyme assay on DNA gyrase B and VEGFR-2 The in-vitro enzyme inhibition assessment for compound 4 (which exhibited the highest potency as antimicrobial and anticancer agent in comparison with the other analogs and the reference drugs) was carried out in confirmatory diagnostic unit, Vacsera, Egypt. The evaluation performed profiling of the compound 4 against E. coli DNA gyrase and VEGFR-2 kinases using Novobiocin and Staurosporine as reference drugs, respectively according to the previously reported methods 40,41 .
In silico calculations of molecular properties Molecular descriptors display the pharmacokinetic, pharmacodynamic and physicochemical effects of all synthesized targets 3-17. The lipophilicity (milogP) and topological polar surface area (tPSA) were calculated using the online software Molinspiration 42 , while the aqueous solubility, drug-likeness, drug score were calculated using the OSIRIS property explorer software 43 . Furthermore, according to Veber et al., good bioavailability 44 , is more favorable for targets having TPSA of 140 A 2 and 10 rotatable bonds. Decreased molecular flexibility, as determined by the rotatable bond number, and low polar surface area or total hydrogen bond count, which are vital predictors of good oral bioavailability, independent of molecular weight.

Molecular modeling study
Docking study was performed by downloading the Protein Data Bank (PDB) file: 1KZN for E. coli topoisomerase II DNA gyrase B 45 and 2OH4 for VEGFR-2 46 . Verification process was performed by redocking of the co-crystallized ligands into the active sites using Molecular Operating Environment software 10.2008 (MOE) 47 . Then, compound 4 was docked by MOE after preparation of the selected compound through its 3 D protonation and selecting the least energetic conformer using the same reported docking procedure 46 .

Chemistry
The target derivatives which obtained are showed in Schemes 1-3 based on the synthetic strategies. Synthesis of the precursor hydrazone 3 was achieved by diazotization of N-aminophthalimide (1) 48 , followed by coupling with ethyl cyanoacetate at room temperature in sodium acetate (Scheme 1). The spectral data confirmed that this compound exists in the hydrazine 49

Antimicrobial activity
The newly prepared targets were subjected for in-vitro antibacterial screening against Gram-positive bacteria (Streptococcus pneumonia and Bacillus subtilis) and Gram-negative bacteria (Pseudomonas aeruginosa and Escherichia coli). Also, these compounds were tested for their antifungal activity against Aspergillus fumigatus, and Candida albicans. The compounds' solutions of concentrations (1 mg/mL) were evaluated against the different microorganism's and the inhibition zone (IZ) used diameter in mm for the biologically activity (agar well diffusion method). The results are depicted in Table 1. From the screening results, we noted that compounds 3, 4, 6, 7, 9, 10, 12, 16 and 17 exhibited significant activity ranging from moderate to excellent against all tested strains except C. albicans, showed no activity. Compound 4 showed relatively equipotent inhibition zone as the reference drugs used for different strains, followed by compounds, 9, 16, 12, 17, 6, 7, 3 and 10 respectively. The pyrazolyl-N,N-dimethylformimidamide derivative 10 exhibited a weak biologically active on both of B. subtilis and A. fumigatus, while compounds 5 and 14 had no antimicrobial activities.

Structure-activity relationship (SAR) for antimicrobial activity
For compound 3 the presence of ethyl 2-cyano-2-(2-hydrazono)acetate moiety at 2-position of isoindoline nucleus improved antibacterial activities against all tested microorganisms (compound 3). On the other hand, combination of isoindoline nucleus with pyrazole moiety increased the antibacterial activities and antifungal activity against A. fumigatus (compound 4). However, the existence of N,N-dimethyl formimidamide substituent decreased the antibacterial and antifungal activities against the tested microorganisms but increased the antibacterial activity against E. coli (compound 10). In the series of substituted imidazo[1,2-b]pyrazol-7-yl)diazenyl)-isoindoline 5-7, the presence of amino and phenyl substituents at the position-2 of imidazole ring enhanced the antibacterial activities and showed no activity against the tested fungi (compound 6 and 7) . In contrast compound 5, which has oxo group at positon-2, was found to be inactive against all the tested bacteria and fungi. Furthermore, the presence of CN group at   Anticancer activity All synthesized compounds were screened for their anticancer activity against a human liver (HepG-2) cell line using the 3-(4,5dimethylthiazole-2-yl)-2,5-diphenyl teterazolium bromide (MTT) assay and vinblastine was used as a standard drug. Cytotoxic activity was depicted in Table 3. Usage of the data to draw a dose-response curve in which the concentrations of the evaluated compounds required to kill fifty percent of cell population (IC 50 ) was decided. The results are represented in Table 3 and Figure 2 showed that compound 4 is the most potent cytotoxic derivative and at the same time is relatively equipotent in activity with the reference drug Vinblastine (IC 50 (Table 3).

Structure-activity relationship (SAR) for cytotoxic activity
It was noticed that there is a great similarity between SAR for antimicrobial activity and that for cytotoxic activity. Substitution at p-    50 : compound concentration which inhibit cell proliferation by 50%. b positive control, SEM: mean of the standard error; each value is the mean of three values.  In-vitro enzyme assay on DNA gyrase B and VEGFR-2 The antimicrobial and the cytotoxic results revealed that analog 4 exhibited the highest activity among other analogs. So, subsequent mechanistic studies were supplied through investigating the binding affinity of representative active derivative 4 to E. coli DNA gyrase B and VEGFR-2 kinases assaying their effects using suitable positive controls, Novobiocin and Staurosporine, respectively. From Table 4, it was observed that compound 4 represented a nearly equipotent IC 50 value with Novobiocin as DNA gyrase B inhibitor (IC 50 ¼0.34 ± 0.63 and 0.28 ± 1.45 mM, respectively). On the other hand, it exhibited excellent and two folds the inhibitory activity of Staurosporine towards VEGFR-2 (IC 50 ¼0.09 ± 1.30 and 0.17 ± 1.02 mM, respectively).

In silico calculations of molecular properties
Drug-Likeness parameters: Molecular descriptors illustrate the pharmacokinetic, pharmacodynamic and physicochemical properties of the compounds 3-17 exhibiting good oral bioavailability of these derivatives theoretically. The calculation results shown in Table 4 revealed that most of the compounds follow the Lipinski rules of the five 51,52 , revealing that there would not be problems with oral bioavailability of these compounds theoretically. Expected poor intestinal absorption was accompanied with molecules having TPSA values around 140A˚2 or more. Thus, all compounds (except 9, 12, 14, 16 and 17) have represented a TPSA less than 140A˚2, exhibiting a good permeability of the drug in the cellular plasma membrane. It has been shown that for the compound to have a reasonable probability of being well absorbed, miLogP value must be in the range of À0.4 to þ5 44 . On this basis, all the synthesized compounds were found to have miLogP values under the acceptable criteria and they are expected to have good oral absorption (Table 5). Also, compounds 9 and 12 have shown very high percentage of absorption (%ABS), that is a parameter of good bioavailability via oral administration but the rest of compounds have a reasonable probability of absorption. Molecules with more than 10 rotatable bonds may have problems with bioavailability 44 . All the tested compounds have 2-5 rotatable bonds and they might not have problems with bioavailability. Furthermore, all nOHNH values (H-bond donors) are in the range of 1-4 indicating their solubility in cellular membranes. All compounds having one or zero violation of Lipinski's rule are expected not to have problems with bioavailability (Table 5), while those violating more than one may have problems with bioavailability 53 . The toxicity risk assessment (TRA) indicators, including irritant, tumorigenic, mutagenic and reproductive effects are the tools for the risks of toxicity. This assessment proposed that compounds 5, 6, 7, 9, 12 and 14 did not show any toxicity risk profile. However, compounds 16 and 17 showed the low of mutagenic and high tumorigenicity effects, respectively. Also, compounds 3, 4 and 10 have shown the high of irritancy and low reproductive effects, respectively ( Table 6). The absorption and distribution characteristics of a compound were significantly affected by its aqueous solubility. It is well known that low solubility is accompanied with bad absorption and the general aim is to be away from the poorly soluble compounds. So, there are more than 80% of the drugs on the market having solubility values greater than À4. Table 6 showed those compounds 3, 4, 5, 6 and 10 exhibiting solubility values above À4 and they are suggested to have good aqueous solubility which significantly influences their absorption and distribution characteristics. Drug-likeness with a positive value points that the derivative consists of fragments involved in most applicable drugs. The drug score merge the risk of toxicity, solubility, lipophilicity, drug-likeness and molecular weight into a single numerical value which can be applied to foretell a global value for each derivative as a potential new drug candidate 54 . The data shown in Table 6 indicated that all compounds have displayed   (Table 6). Finally, it could be observed that compounds 5 and 6 have potential as new drug candidates, but the rest of the series have drug scores from low to moderate values comparing with the reference drugs used.

Molecular modeling study
Based on the kinase assessment observations and the previous literatures illustrated the important correlation between phthalimide analogs and E. coli topoisomerase II DNA gyrase B as antibacterial target 34,35 and VEGFR-2 as anticancer core 32 , we decided to investigate the possible interactions and binding modes of compound 4 with the active sites of those enzymes. Docking simulations were performed using the X-ray crystallographic structures for DNA gyrase B (PDB ID: 1KZN) 44 with the natural inhibitor clorobiocin and for VEGFR-2 (pdb code: 2OH4) 45 with the original ligand GIG. The cocrystallized ligands clorobiocin and GIG were redocked into the pocket sites of DNA gyrase B and VEGFR-2 and revealed docking score energies À11.4, À13.7 kcal/mol at RMSD value (root mean square deviation) equal 9.3, 8.5, respectively. The energy is minimized for compound 4 in 3 D picture, and then it saved in a molecular data base (MDB) file to be docked into the active sites of the two enzymes. It showed score energies lower than the cocrystallized ligands (À12.3, À15.6 kcal/mol) with DNA gyrase B and VEGFR-2, respectively. The binding map of compound 4 in the pocket of DNA gyrase B was explained through two stable hydrogen bonding interactions between the two carbonyl groups of phthalamide scaffold and the sidechains of Asn46 and Thr165 (distance: 3.12 and 2.66 Å). Furthermore, the essential amino acid Asp73 located in the motif II of N-terminal loop provided a hydrogen bond with the NH 2 proton of pyrazolone moiety (distance: 1.64 Å) (Figure 2).
Docking study results of compound 4 inside the ATP binding site of VEGFR-2 revealed that the carbonyl group of phthalimide moiety formed H-bond acceptor with the backbone of Asp1044 oriented in the C-terminal domain (distance: 2.18 Å). Furthermore, the two amino protons of pyazolone ring formed H-bond donors with the side chain of Glu883 located in the N-terminal lobe and the backbone of Phe1045 inserted in the C-terminal domain (distance: 1.97, 1.93 Å, respectively) ( Figure 3).
Finally, the docking analysis was agreed with the previous antimicrobial, anticancer and enzyme inhibitory activities and could explain how the phthalimide moiety played a pivotal role in stability in the ATP binding sites of both enzymes through its carbonyl groups. Also, the amino group of pyrazolone ring contributed considerably to the strength of binding interactions.

Conclusion
A new imidazopyrazole, pyrazolopyrimidine and pyrazolo-1,2,4triazine derivatives containing phthalimide moiety were prepared and in vitro antimicrobial and anticancer were reported. Compound 4 was the most active compound against Gram positive bacteria (S. pneumoniae and B. subtilis), Gram negative bacteria (P. aeruginosa and E. coli) and fungi (A. fumigatus). Also, compound 4 was the most potent compound in cytotoxic assay against hepatic cancer cell line (HepG-2) in comparison with the standard drug vinblastine. Drug-likeness and Toxicity risk parameters of the newly synthesized compounds were calculated using in silico studies. The promising results motivated us to perform enzyme assay and docking simulations to gain insight into the plausible mechanism of antibacterial and cytotoxic activities of target compound 4 as DNA gyrase and VEGFR-2 inhibitors. The obtained findings may open up new possibilities for developing a new class of phthalimide drugs with antimicrobial and cytotoxic activity.

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