Copolymerization of quinazolinone derivatives with styrene, methyl methacrylate and their silver nanocomposites for bio- logical applications

Reaction of 2-mercapto-3-phenylquinazolin-4(3H)-one (MPQ) with both 4-vinyl benzyl chloride and allyl bromide furnished the reactive heterocyclic monomers 3-phenyl-2-((4-vinylbenzyl) thio) quinazolin4(3H)-one (PVTQ) and 2-(allylthio)-3-phenylquinazolin-4(3H)-one (APQ), respectively. Copolymerization of PVTQ monomer with styrene and methyl methacrylate in the presence of 2,2′-azobisisobutyronitrile (AIBN) afforded the copolymers PS-co-PPVTQ and PMMA-co-PPVTQ, respectively. Similarly, copolymerization of monomer APQ with styrene and methyl methacrylate (MMA) afforded the copolymers PS-co-PAPQ and PMMA-co-PAPQ, respectively. The resulted copolymers were characterized by using FT-IR, 1H-NMR and GPC techniques. Silver nanocomposites of PS, PMMA, PS-co-PPVTQ, PMMA-co-PPVTQ, PS-co-PAPQ and PMMA-co-PAPQ were synthesized by the addition of silver nitrate into the polymer solution. The reduction of silver ions into silver nanoparticles was performed in DMF and water. Thermogravimetric (TGA) analysis was used to determine the thermal stability of the copolymers and their silver nanocomposites. The X-ray diffraction (XRD) analysis indicated the amorphous structures of the co-polymers and confirmed the formation of silver nanoparticles. The antitumor and antibacterial activities were screened for the copolymers and enhanced by the formation of their silver nanocomposites. In vivo antitumor activity in Ehrlich Ascitic Carcinoma (EAC) mice model showed that PS-co-PPVTQ/Ag NPs, PMMA-co-PPVTQ/Ag NPs, and PMMAco-PAPQ/Ag NPs displayed promising inhibitory effects against EAC and induce apoptosis against MCF-7 cells.


Introduction
Synthesize of multifunctional macromolecular material showed a considerable interest for their potential usage in different polymer fields [1]. The radical homo-polymerization of benzocyclobuten-4-yl-acrylate and its copolymerization with styrene or n-butyl acrylate were carried out to produce the corresponding linear polymers, thermal analysis of the thermosetting systems suggests that they have potential use as high temperature macromolecular materials [2]. Co-polymerization of methyl methacrylate (MMA), glycidyl methacrylate (GMA) and the fluorinated monomer furnished the fluoropolymers showed high thermal stability, hydrophobicity, antifouling effect and antibacterial activity against Escherichia coli and Staphylococcus aureus [3]. Synthesis of 2,4,6-Trichlorophenyl methacrylate (TClPhMA) by the reaction of methacryloyl chloride with 2,4,6-trichlorophenol was copolymerized with monomers including styrene, 1,4-divinylbenzene and 2-hydroxymethyl methacrylate by bulk polymerization using benzoyl peroxide. These copolymers have potential application in polymer optical fiber technology due to their low refractive indices and high thermal resistance [4].
Anionic polymerization of p-(2,2′-diphenylethyl) styrene (DPES) monomers was achieved to afford well-defined block copolymer known as PDPES-b-PS. Radical polymerization of MMA in the presence of DPES effectively afforded a graft copolymer composed of a polystyrene backbone and PMMA branches [5]. Furthermore, the homopolymers of 2,4-dichlorophenyl methacrylate and its copolymers with 8-quinolinyl methacrylate (8-QMA) were prepared. The presence of 2,4-dichlorophenyl methacrylate led to inhibition of microorganism growth [6]. Phenylethyl acrylate (PEA) and 2-Phenylethyl methacrylate (PEMA) were synthesized by reacting 2-Phenyl ethanol with acryloyl-and methacryloyl chloride, respectively. Copolymers of PEA and PEMA with methyl acrylate and N-vinyl pyrrolidone (NVP) of different compositions were prepared and tested on leather for their pressure sensitive adhesive property [7]. By free radical polymerization, 8-acryloxyquinoline (8-AQ) monomer was prepared through the reaction of 8-hydroxyquinoline with either acryloyl chloride or acrylic acid in the presence of triethylamine or N, N-dicyclohexylcarbodiimide. The antimicrobial activity of all compounds demonstrated moderate to good activity against the tested strains providing wider possibilities for the synthesis of pharmacologically active polymers [8]. Improved polymerizations of monomers bearing electron transporting substituents based on 2,5-diphenyloxadiazole and 2,3-diphenylquinoxaline attached directly to a vinyl group were reported, these materials have been incorporated into light emitting polymer devices in which hole conduction properties are provided by 4-vinyltriphenylamine groups for usage in optimized devices [9].
Preparation and structural analysis of poly (styrene-co-acrylic acid) composite nanospheres (PSA) and silver nanoparticles (Ag-NPs) loaded PSA was reported, the Ag-NPs were well-dispersed on the surfaces of PSA composite nanospheres by in situ chemical reduction of AgNO3 [10]. A modified method based on in situ chemical reduction was developed to prepare mono-dispersed polystyrene/silver (PS/Ag) composite microspheres. With the addition of [Ag(NH3)2] + to the PS dispersion, [Ag(NH3)2] + complexions were absorbed and reduced to Ag-NPs on the surfaces of PS-PDA microspheres to form PS/Ag composite microspheres [11]. The green PS/AgNPs nanocomposite exhibited antimicrobial activity against Gram-negative and Gram-positive bacteria that showed a potential application in food packaging [12]. Additionally, it has been reported that of silver polystyrene sulfonate polyelectrolyte showed promising biomedical applications [13]. The PS/Ag composite microspheres were prepared successively by addition of [Ag (NH3)2]+ complex ions to the templates dispersion, adsorbing to the surfaces of templates, and then reduction of [Ag(NH3)2]+ complex ions to Ag-NPs. These PS/Ag composite microspheres showed potent catalytic properties [14]. This study aimed to synthesis copolymers of quinazolinone derivatives with styrene, methyl methacrylate and their silver nanocomposites and evaluate their antibacterial and anticancer activities. The importance of the present work lies in the possibility that the polymers and their conjugates might be more efficacious antibacterial and antitumor agents, which could be helpful in designing more potent drugs for therapeutic use.
Compound MPQ (5.0 g, 19.7 mmol) was dissolved in methanol (30 mL) and 0.79 g (19.7 mmol) of NaOH, to which 2.99 g (3.94 mmol) of 4-vinyl benzyl chloride was added. The reaction mixture was stirred at r.t. for 2 h (tlc) and the resulted solid was separated by filtration and crystallized from methanol, m.p. 160-162 ᵒC and yield 69 %. FTIR ( Figure  1A  2.2.7. Copolymerization of compound APQ with styrene and/or methylmethacrylate to form the copolymers PS-co-PAPQ and PMMA-co-PAPQ. Compound APQ (0.5 g, 1.70 mmol), 1 mL of St (8.74 mmol) and/or MMA (9.40 mmol) and 0.01 g (0.061 mmol) of AIBN were heated for 5 h till solidification. The resulted solids were dissolved in CHCl3 (30 mL) and precipitated by pouring on a large amount of methanol to furnish the PS-co-PAPQ and PMMA-co-PAPQ, respectively. PS, PMMA, PS-co-PPVTQ, PMMA-co-PPVTQ, PS-co-PAPQ and/or PMMA-co-PAPQ (0.5 g, 90 wt%) were dissolved in DMF (20 mL) to which 0.056 g (10 wt%) of silver nitrate was added. The reaction mixtures were stirred at r.t for 2 h followed by heating at 90 °C for 6 h. until brown color was observed. The reaction mixtures were cooled, and the products were precipitated by pouring the mixtures on a large amount of methanol, filtered off and dried in vacuum oven at 40 °C for 2 days.

Characterization
All melting points were uncorrected and performed by the open capillary melting point apparatus. FT-IR spectra were recorded on Bruker, Tensor 27FT-IR spectrophotometer with frequency range 4000cm -1 to 400cm -1 (Central Lab, Tanta University, Egypt) with KBr pellets and 4 cm-1 resolutions. Elemental analyses were determined on Heraeus (Microanalysis Center, Cairo University, Giza, Egypt). 1H-NMR spectra were measured on a Varian mercury VX-300 (300 MHz) NMR spectrometer (Nuclear Magnetic Resonance lab., Faculty of science, Cairo University, Cairo, Egypt). The mass spectra (MS) were recorded on GCMS/QP 1000 Ex mass spectrometer at 70 eV (Microanalysis Center, Cairo University, and the National Research Centre, Dokki, Giza, Egypt). The TGA data were obtained by using shimadzu thermal analyzer system at a heating rate of 5 ᵒC/min, sample weight of 5-6 mg under nitrogen (20 mL/min) flow. The range investigated from 30-400 ᵒC (The Central Lab, Tanta University, Egypt). The XRD measurements were carried out using GRN, APD 2000 PRO X-Ray diffraction, equipped with a Ni-filtered Cu-Kα radiation (λ= 1.54 Å) at a scanning rate of 0.05°/s and divergent slit 0.3 (Central Lab. Tanta University, Egypt).

Determination of antibacterial activity
For antibacterial screening, a stock solution of 5 mg of quinazolinone derivatives, their copolymers and copolymers/Ag NPs were dissolved in 1 mL of dimethyl sulfoxide (DMSO) and graded quantities of the test compounds were incorporated in specified quantity of molten sterile agar (nutrient agar, 28 g/L, pH=7.4). The media were sterilized in autoclave before experiment and poured into a Petri dish to give a depth of 3-4 mm and allowed to solidify. Suspension of the microorganism was prepared to contain approximately 5 × 10−5 cfu/mL. The culture plates were seeded with test organisms. Uniform wells were created, filled with 100 µL of the test compounds and incubated at 37 °C for 24 h. DMSO was used as a control with zero inhibition zone [17,18].

In vivo anticancer studies using Ehrlich Ascetic Carcinoma (EAC) model
Female Swiss albino mice weighting 20 ± 2 g were obtained from National Research Center (NRC, Cairo, Egypt). Mice were kept for a week before starting the experiment for adaptation at the Animal Facility, Zoology Department, Faculty of Science, Tanta University. The experimentation, transportation and care of the animals were performed and handled in compliance with the ethical guidelines approved by the animal care and use committee, Faculty of Science, Tanta University (ACUC-SCI-TU-103), Egypt and according to the National Institutes of Health guide for the care and use of Laboratory animals (NIH Publications No. 8023, revised 1996). EAC cells were collected in sterile 0.9% saline from the tumor bearing mice. The viable and dead EAC cells were counted using trypan blue method and the number was adjusted at 2×10 6 cells/mouse for inoculation. Total viable cells were calculated as following: Mean number of unstained cells × dilution × 10 4 / ml. Eighty female Swiss albino mice were divided into ten groups (n = 8). All groups were inoculated i.p with 2×10 6 (EAC) cells/mouse and after 24 hours, Gp2 had treated with Cisplatin reference anticancer drug (2 mg/kg), Gp3-10 had treated with PS-co-PPVTQ, PMMA-co-PPVTQ, PS-co-PAPQ, PMMA-co-PAPQ, PS-co-PPVTQ/Ag NPs, PMMA-co-PPVTQ/Ag NPs, PS-co-PAPQ/Ag NPs, and PMMA-co-PAPQ/Ag NPs (10 mg/kg), respectively, daily for six consecutive days. On day 14, all groups were sacrificed to assess the tumor volume, count, and live/dead cells.

In vitro cytotoxic effect by MTT assay
To determine the anticancer activity of the synthesized copolymers/nanocomposites in vitro, MTT assays were used to on MCF-7 cells, the tested polymers were dissolved in dimethyl sulfoxide (DMSO) and diluted with saline to the appropriate concentration. Different concentrations (from 200 to 3.125 μg/mL) of these compounds were applied in triplicate to the MCF-7 cells (at 70-80% confluent in Dulbecco's modified Eagle medium), and the wells were incubated, then, 10 μL of a 12 mM MTT stock solution [5 mg/mL MTT in sterile phosphate buffered saline (PBS)] was added to each well. This was followed by incubation for 4 h at 37 °C. The MTT solution was removed, and the purple formazan crystal formed at the bottom of the wells was dissolved with 100 μL of DMSO for 20 min. Cisplatin (Cis) was used as a positive standard. The absorbance at 570 nm was read on an enzyme-linked immunosorbent assay reader (StatFax-2100, Awareness Technology, Inc.). The concentration of thecompounds inhibiting 50% of cells (IC50) was calculated with the sigmoidal curve.

Statistical analysis
Data expressed as means ± S.D. that were analyzed by One-way analysis of variance (ANOVA) was used to assess the significant differences among treated groups. p < 0.05 was considered as a significant value for all statistical analyses.

Synthesis, FTIR, 1 H-NMR and MS Characterizations
The FITR spectra of compounds PVTQ and APQ showed C=O at 1691, 1678, C=C at 1609 and 1606 cm -1 , respectively ( Figure 1A   FITR spectrum of copolymer PMMA-co-PAPQ showed stretching aromatic CH at 3001, aliphatic CH at 2952 and C=O at 1730 cm -1 that may be referred to the overlapped carbonyl groups of PMMA and quinazolinone moieties as shown in Fig 1B. 1 H-NMR spectrum displayed a broad CH2CH protons at 0.765-0.945 (protons d and e), a broad CH2C protons at 1.835 (protons c), a singlet CH3C protons at 2.501 (protons b), a singlet CH3O protons at 3.320 (protons a), a doublet SCH2 protons at 3.403-3.562 (protons f) and a multiplet aromatic protons at 6.348-8.252 ppm (Figure 3).

Thermogravimetric Analysis (TGA)
Thermal degradation and the thermal stability of copolymers PS, PMMA, PS-co-PPVTQ, PMMA-co-PPVTQ, PS-co-PAPQ, PMMA-co-PAPQ and their Silver nanocomposites were investigated using TGA and DTG as shown in Figures. 4, 5 and Table 1. T10 and T50 represent the temperatures at which weight change can be detected with 10 % and 50 % of weight loss, respectively. Tp represents the temperature of maximum rate of degradation. DTG curves showed that the thermal degradation of PS, PS/Ag NPs, PS-co-PPVTQ, PS-co-PAPQ and PS-co-PAPQ/Ag NPs occurred in one-step degradation at Tp indicating random scission of the polymer chains. The thermal degradation of PS-co-PPVTQ/Ag NPs occurs in two-step degradations with close temperatures indicating  random scission decomposition of hetero moieties followed by the polymer chains. This may be due to the coordination binding of silver nanoparticles with the hetero atoms in the polymer chains.
According to decomposition temperatures at T10, T50 and Tp of PS, PS/Ag NPs, they nearly have the same thermal stability. PS-co-PPVTQ, PS-co-PAPQ and PS-co-PPVTQ/Ag NPs decomposition temperatures at T10 and T50 are lower than the corresponding values of PS. Thus, we can conclude that PS-co-PPVTQ, PS-co-PAPQ and PS-co-PPVTQ/Ag NPs are less thermally stable than PS. In addition, PS-co-PAPQ/Ag NPs decomposition temperatures at T10 and T50 are higher than the corresponding values of PS and PS-co-PAPQ. Thus, PS-co-PAPQ/Ag NPs is more thermally stable than PS and PS-co-PAPQ. This may be due to the presence of hetero moieties through the polymer chain and the distribution and bonding of silver nanoparticles along the polymer hetero atoms [19]. The DTG curves showed that the thermal degradation of PMMA, PMMA/Ag NPs and PMMA-co-PAPQ/Ag NPs occurred in two-steps degradation. The first degradation is attributed to the decomposition of PMMA unsaturated chain ends and the second degradation is related to random scission of the polymer chains [20]. Additionally, the thermal degradation of PMMA-co-PPVTQ, PMMA-co-PAPQ and PMMA-co-PPVTQ/Ag NPs occurred in one-step degradation indicating random scission of the polymer chains. According to decomposition temperatures at T10 and T50 of PMMA, PMMA/Ag NPs, they nearly have the same thermal stability. PMMA-co-PPVTQ, PMMA-co-PAPQ, PMMA-co-PPVTQ/Ag NPs and PMMA-co-PAPQ/Ag NPs decomposition temperatures at T10 and T50 are higher than the corresponding values of PMMA and PMMA/Ag NPs. Thus, they are more thermally stable than PMMA and PMMA/Ag NPs. This may be due to the presence of hetero moieties through the polymer chain, the distribution and bonding of silver nanoparticles along the polymer hetero atoms.

X-ray diffraction
The X-ray diffraction patterns of PMMA-co-PPVTQ, PMMA/Ag NPs, PMMA-co-PPVTQ/Ag NPs, PS-co-PAPQ, PS/Ag NPs and PS-co-PAPQ/Ag NPs ( Figure 6). Previous study showed that silver nanoparticles exhibited peaks at 2θ angles of 38.1°, 44.3°, 64.4°, 77.4° and 81.5° corresponding to the reflections of the (111), (200), (220), (311) and (222) crystal plane of the face-centered cubic (fcc) structure of Ag [10,11]. The strong reflections of PS-co-PPVTQ and PMMA-co-PPVTQ around 2θ = 18.95° and 15.55°, respectively are assigned to their amorphous structures. XRD of PS/Ag NPs, PS-co-PPVTQ/Ag NPs, PMMA/Ag NPs, and PMMA-co-PPVTQ/Ag NPs displayed the same previous peaks that referred to amorphous structure and indicated to the formation of silver nanoparticles with the appearance of Ag reflections peaks at 32.35°, 38.10°, 43.95°, 64.25° and 77.60°. Similarly, XRD of PS-co-PAPQ/Ag NPs and PMMA-co-PAPQ/Ag NPs displayed the same previous peaks that referred to amorphous structure and indicated to the formation of silver nanoparticles with the appearance of Ag reflections peaks at 38.15°, 44.40° and 75.65°. The weak intensity of silver nanoparticles peaks may be referred to the small amount of reduced silver ions. It further confirmed that silver nanoparticles with crystallinity could be obtained successfully by reducing of AgNO3 using DMF and water on the surface of the polymers under study [21].

Antibacterial activity
Using polymeric nanoparticles and nanocomposites as controlled drug delivery systems have attracted great attention due to their high drug loading capacity and ability to release the loaded drug in controlled fashion [22]. Table 2 and Figure 7 showed the antibacterial activity against some Gram-positive and Gram-negative bacteria using agar cup plate method, ciprofloxacin was used as standard drug [23]. Compound PVTQ, copolymers PSco-PPVTQ, PMMA-co-PPVTQ, PS-co-PAPQ and PMMA-co-PAPQ showed a negative inhibition zones towards all types of bacteria under study. Compound APQ showed a high inhibition zone (20 mm) according to the standard drug (21 mm) towards Klebsiella pneumoniae and negative results for other types of bacteria. Thus, the copolymerization of compounds PVTQ and APQ with styrene and methyl methacrylate does not affect all types of bacteria under study. Addition of silver nanoparticles to PS, PS-co-PPVTQ, PMMA-co-PPVTQ, PMMA, PS-co-PAPQ and PMMA-co-PAPQ resulted in a significant increase in the inhibition zones compared with copolymers PS-co-PPVTQ, PMMA-co-PPVTQ, PS-co-PAPQ and PMMA-co-PAPQ. Inhibition zones of PS-co-PPVTQ/Ag NPs, PS-co-PAPQ/Ag NPs, PMMA-co-PPVTQ/Ag NPs, and PMMA-co-PAPQ/Ag NPs were enhanced compared with PS/Ag NPs and PMMA/Ag NPs. This may be due to the presence of heterocyclic moieties (PVTQ and APQ) in the copolymer chain and Ag NPs. This increase in inhibition zones is referred to the accumulation of Ag NPs in the bacterial cell membrane caused a change in the permeability, structural and morphological changes that resulting in cell death [12]. As the silver nanoparticles contact with the bacteria, they agglutinate to the cell wall and membrane [24]. Once bound, some silver exceeds inside and combines with phosphate moieties of DNA and RNA, while another portion adheres to the sulfurcontaining proteins on the membrane [24]. At the membrane, silver-sulfur interactions cause structural variations and pores formation in bacterial cell wall [25]. Through these pores, cellular components are freed into the extracellular fluid due to the osmotic difference. Through the cell, the presence of silver creates a low molecular weight region where DNA condenses [25]. The condensed DNA inhibits the cell's replication proteins contact with the DNA. Thus the introduction of Ag NPs inhibits replication and cause cell death [25,26].

In vivo anticancer activity using carcinoma mice model
Synthetic polymers, biopolymers, and their nanocomposites have been widely applied in different medical areas particularly in cancer therapy, owing to their desired mechanical and physical properties, manufacturability into various shapes, sterilizing capability, and ease of modification with multiple functionalities [27,28]. Development of such these materials require high standards regarding their biocompatibility, functionality, and bioactivity [29]. The synthesized copolymers and/or their silver nanocomposites in this study were evaluated as anticancer agents using EAC mice model. Each compound was injected as a concentration of 10 mg/kg daily for consecutive 6 days starting from day 0. For comparison, cisplatin (Cis) used as standard reference anticancer drug an evaluated under the same conditions, which injected as 2mg/kg for consecutive 6 days. Tumor profile including the total EAC volume, count, and viability of copolymers and/or their silver nanocomposites and Cis are shown in Table 3. It was evident that the promising compounds that displayed EAC tumor growth inhibition can be order as the following PMMA-co-PPVTQ/Ag NPs > PMMA-co-PAPQ/Ag NPs > PS-co-PPVTQ/Ag NPs > PS-co-PAPQ/Ag NPs > PMMA-co-PPVTQ > PMMA-co-PAPQ, when compared to EAC-bearing mice control. These findings were agreed with previous study evaluated the antitumor efficacy of some biocompatible polymers and nanocomposites [17,18,30,31].  (Table 4). These findings were agreed with previous studies reported the effectiveness of copolymers and/or their nanocomposites in inhibition of cancer growth, copolymer based on tri (ethylene glycol)-methyl ether methacrylate and [10 -(2-acryloxy)-3,30 -dimethyl-6-nitrospiro-(2H-1-benzopyran-2,20 -indoline)] has proven its effectiveness in the case of cancer therapy [33][34][35].

Conclusion
Some copolymers of quinazolinone derivatives with styrene and methyl methacrylate were successfully prepared and their chemical structures were elucidated using FT-IR and 1H-NMR techniques. The formation of silver nanoparticles with the prepared copolymer was performed in DMF and water using silver nitrate. The thermal stability and degradation of copolymers and their silver nanocomposites were evaluated through TGA and XRD. The synthesized copolymers showed no antibacterial activity towards the biological species so, their antibacterial activity was enhanced by the formation of silver nanoparticles on the surface of copolymers. Furthermore, in vivo, and in vitro antitumor activity in mice model showed that PS-co-PPVTQ/Ag NPs, PMMA-co-PPVTQ/Ag NPs, and PMMA-co-PAPQ/Ag NPs have the most promising inhibitory effects against tumor growth.