PI 3 K Inhibitors of Novel Hydrazide Analogues Linked 2-Pyridinyl Quinazolone Scaffold as Anticancer Agents

Department of Chemistry, Faculty of Science, Menoufiya University, Menoufiya, Egypt Department of erapeutical Chemistry, National Research Centre, Dokki, Cairo 12622, Egypt Pharmaceutical Chemistry Department, Drug Exploration & Development Chair (DEDC), College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia Department of Pharmaceutical Chemistry, Faculty of Pharmacy (Girls), Al-Azhar University, Cairo 11754, Egypt Department of Pharmacognosy, National Research Centre, Dokki, Cairo 12622, Egypt


Introduction
Recently, cancer is considered as the second leading reason for dying.It is characterized by fast, uncontrolled, and pathological proliferation of abnormal cells.Almost 12 million deaths will be from cancer in 2030, according to statistics from the World Health Organization (WHO) [1,2].
e phosphatidylinositol-3-kinase (PI3K) signaling pathway is a crucial signaling pathway, which plays a substantial role in the regulation of numerous cellular processes, such as cell growth, proliferation, differentiation, motility, and survival.Disturbance in regulation of this signal transduction way is related to the development of many cancers [3,4].erefore, a great attention in the treatment of malignant tumors has been regarded to the compounds that inhibit the PI3K signal transduction pathway.In addition, quinazolinone derivatives constitute an important class of biologically active compounds.is heterocycle core has been associated with a broad spectrum of pharmacological properties as an anticancer agent [5].Idelalisib derivatives (Figure 1) are known to contain quinazoline rings and are effective in the treatment of different kinds of cancer diseases via inhibition on PI3K.Furthermore, substituted quinazoline derivatives displayed various medicinal applications including anticholinesterase [6], anticonvulsant [7], anti-HIV [8], antidiabetic [9], anti-inflammatory [10], antihypertensive [11], antimalarial [12], antimicrobial [13], dihydrofolate reductase inhibition [14], antitubercular [15], antitumor [16], cellular phosphorylation inhibition [17], and kinase inhibitory activities [18].Considering the much broader range of pharmacological effects, a vast number of synthetic approaches have been developed to prepare various quinazolinone compounds [19,20].Among them, quinazolin-4(3H)-one derivatives represent an attractive scaffold for designing interesting anticancer drugs [21,22], and they have attracted more interest because of their inhibitory activity for PI3K [23].In our current study, we incorporate di erent substitutions at p-3 of a quinazolin-4(3H)-one sca old to develop novel anticancer agents targeting PI3Ks.

Experimental
2.1.Chemistry 2.1.1.Materials and Methods.Electrothermal capillary apparatus was used for recording the melting points of all products and are uncorrected.Elemental analyses were found within ±0.4% of the theoretical values.Jasco FT/IR-6100 was used for measuring infrared spectra using potassium bromide pellets.Joel 270 MHz and Jeolsx 500 MHz spectrometers were used for recording NMR spectra down eld from TMS as a reference.Jeol JMS-AX 500 was used to determine mass spectra at 70 eV.TLC silica gel F 254 precoated sheets followed the reactions of all the new products.
e resulting solid was washed with water and crystallized from ethyl alcohol.

4
Journal of Chemistry e excess concentrated solvent was poured into ice, and the precipitate created was washed with water and crystallized from ethyl alcohol. (
Additionally, treatment of product 8 with various reagents, namely, thiosalicylic acid, thioglycolic acid, acetyl chloride, or chloroacetyl chloride via di erent cyclocondensation reactions yielded the corresponding 4-oxobenzo[e] [1,3]thiazine, 4-oxothiazolidine, and 4-oxoazetidine derivatives 9-12, respectively (Scheme 3).e obtained products were proved with di erent spectral data.Compound 9 as an example from this group of compounds indicated in its IR spectrum bands for hydroxyl, amine, and carbonyl functions at 3388, 3210, 1689, and 1674 cm −1 , respectively.In addition, the hydroxyl and amine protons appeared in the 1 H NMR spectrum as two singlets at δ 9.15 and 11.40 ppm.Furthermore, MS indicates the molecular formula (C 38 H 25 N 5 O 5 S) of product 9 by appearance of the molecular ion peak at m/z 627.

Journal of Chemistry
Furthermore, reaction of compounds 3a and b with 3chloro-2,4-pentadione, ethyl 2-bromopropanoate, phenacyl bromide, or chloroacetic acid a orded the corresponding thiazole derivatives 13-16.Compound 16a as an example indicated the existence of bands corresponding to amine and carbonyl absorptions at 3138, 1702, and 1685 cm −1 in the IR spectrum.In addition, 1 H NMR spectrum characteristics of new singlet found at δ 4.07 ppm con rms methylene thiazole ring protons, besides another singlet was found at δ 10.21 ppm due to the amine proton.Also, the MS displayed the molecular ion peak at m/z 337 con rming the molecular formula C 16 H 11 N 5 O 2 S of product 16a.Synthesis of derivatives 17a-d was accomplished by reaction of 16a and 16b with vanillin or 3,4,5-trimethoxybenzaldehyde, respectively (Scheme 4).
e existence of bands at 3490, 3130, 1702, and 1676 cm −1 in the IR spectrum of 17a as an example conrmed the existence of -OH, -NH, and CO functionalities, respectively.Furthermore, 1 H NMR spectrum clearly indicates the formation of ole nic CH by existence of singlet at δ 8.11 ppm, besides two other singlets at δ 9.96 and 11.25 ppm con rmed the presence of hydroxyl and amine protons.Moreover, 13 C NMR and MS spectrum revealed the carbons at their expected regions and the molecular formula of the target product.

Molecular Modeling Study.
After in vitro evaluation, it was thought worthy to study the interaction of the promising compounds 9, 12, and 16a with PI3K using MOE 2008.10 program [30].e coordinates of the PI3K structure were obtained from the crystal structure of PI3K with its inhibitor (PDB ID: 2WXG) [31].
e root mean square difference (RMSD) between the top docking pose and original crystallographic geometry of cocrystallized ligand SW13 was 0.9 Å. e data of docking scores and interactive amino acid residues with the screened compounds are depicted in Table 4.
Molecular docking was performed to predict the binding forms and direction of the most active derivatives 9, 12, and 16a at the active site of the ATP binding site of PI3K.e distinctive binding pattern of SW13 to PI3K active site would be discussed and compared to the tested compounds.SW13 engages in hydrogen bonding with Asp787 and Tyr813.Additionally, the phenolic OH group of SW13 serves as a hydrogen bond donor to the DFG Asp911 at the start of the activation loop.
e amino group and N-3 of pyrazolopyrimidine moiety establish hydrogen bonds to the hinge residues Glu826 and Val828.
e quinazolinone moiety of SW13 is sandwiched into the induced hydrophobic specificity pocket between Trp760 and Ile777 on the one side and two P-loop residues Met752 and Pro758 on the other side [30].
From the docking results, it was observed that the protonated nitrogen atom of amide groups in compounds 9 and 12 formed hydrogen bonds with the sidechain of Asp911 as H-donors (distance: 1.98, 1.30 Å, respectively) (Figures 2  and 3).In compound 9, the arene cation interacts between the phenolic ring and Lys755.Oxygen of quinazolin-4(3H)one moiety was linked to the sidechain of Lys779 via the H-bond acceptor (distance: 2.47 Å).Furthermore, the    10 Journal of Chemistry phenolic OH formed the H-bond donor with the backbone of Asp897 (distance: 1.75 Å) (Figure 2).In compound 12, two H-bonds appeared as H-donors between hydrogens of the phenolic OH and the sidechains of Asp893 and Asn898 (distance: 2.10, 3.27 Å, respectively).Additionally, the backbone of Gly913 established the H-bond acceptor with the oxygen of the methoxy group (distance: 3.36 Å) (Figure 3).
It was noticed that quinazolin-4(3H)-one derivatives bearing thiazolidin-2-ylidenamino moieties at position-3 with free NH exhibited remarkable increase in the cytotoxic activity which may be due to formation of the H-bond donor with the sidechain of Asp911.Among this series, the excellent potency of 16a may be due to unsubstitution on C-3 adjacent to the carbonyl group of thiazoilidinone moiety which facilitates formation of a characteristic H-bond acceptor between oxygen of the CO group and the sidechain of Lys779 (Figure 4).

Conclusions
In summary, we have designed and synthesized novel twenty-five-quinazolin-4(3H)-one-based derivatives incorporating different moieties and evaluated their cytotoxic activities against HePG-2, MCF-7, and HCT-116 cancer cell lines.Among them, compound 9 (IC 50 � 60.29 ± 1.06 μM) emerged as the most active member against HePG2, as it was equipotent with doxorubicin (IC 50 � 69.60 ± 1.50 μM).Also, compounds 12 and 16a displayed excellent activity against HePG2 (IC 50 � 104.94 ± 2.46 and 126.40 ± 1.83 μM, respectively).Kinase inhibition assay against PI3K and docking studies were performed using the MOE 2008.10 program to justify the biological activities of the synthesized compounds.All active compounds could interact with a key amino acid Asp911 with a characteristic hydrogen bond.Compounds 9 and 16a as the most active compounds could interact by extra hydrogen bonds with Lys779 signifying for their strongest PI3K inhibitory activities.So the applicability of quinazolin-based hybrids containing the benzo[e] [1,3] thiazine function has the potential to be developed into anticancer active agents.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.

Figure 2 :Figure 3 :
Figure 2: e suggested binding form of derivative 9 docked in the active position of PI3K showing (a) 2D and (b) 3D ligand-receptor interactions, respectively (H bonds are clari ed as arrows; C atoms are colored gray; N atoms are colored blue; O atoms are colored red).

Figure 4 :
Figure 4: e suggested binding form of derivative 16a docked in the active position of PI3K showing (a) 2D and (b) 3D ligandreceptor interactions, respectively (H bonds are clarified as arrows; C atoms are colored gray; N atoms are colored blue; O atoms are colored red).

Table 1 :
Cytotoxic activity of the newly synthesized compounds against human carcinoma cell lines at 100 μM.
a Concentration of test compounds and positive control (doxorubicin) was 100 μM; b untreated cells in DMSO and its final concentration in the cells was less than 0.2%.

Table 2 :
IC 50 of highly cytotoxic active derivatives against human cancer cell lines.

Table 3 :
Inhibitory activities of compounds 9

Table 4 :
Docking results of the synthesized compounds 9, 12, and 16a with PI3K in comparison with the ligand SW13.