Anti-proliferative activity, molecular genetics, docking analysis, and computational calculations of uracil cellulosic aldehyde derivatives

In this study, the oxidation of microcrystalline cellulose using NaIO4 to yield the corresponding cellulose aldehyde utilized microwave irradiation as a green tool, the obtained cellulosic aldehyde was confirmed through spectral analysis and it has an active site to react with the synthesized uracil acetamide to afford the corresponding arylidene cellulosic MDAU(4), the latter compound which can easily due to presence of active CH=group behind a cyano group react with nitrogen nucleophile’s and cyclized with hydrazine hydrate to give pyrazole cellulosic MDPA(5). The spectral analysis of the obtained cellulosic derivatives was confirmed with FT-IR, NMR, and SEM. Additionally, a neutral red uptake analysis has been used to investigate the cytotoxic activity of the cellulosic compounds MDAC(2), MDAU(4), and MDAP(5) against the cancer cells A549 and Caco2. After 48 h, Compound MDAU(4) had a stronger inhibitory effect on the growth of A549 and Caco2, compared to control cells. Then, using QRT-PCR, the expression levels of the genes β-Catenin, c-Myc, Cyclin D1, and MMP7 in A549 cells were examined. By reducing the expression levels of the Wnt signaling cascade genes (β-Catenin, c-Myc, Cyclin D1, and MMP7) when administered to A549 cells, compound MDAU(4) was shown in this investigation to be a viable candidate compared to lung cancer. Additionally, docking simulation was used to explore the uracil cellulosic heterocycles attached to different proteins, and computational investigations of these compounds looked at how well their physical characteristics matched the outcomes of their experiments.


Synthesis of cellulose aldehyde MDAC(2)
Using a microwave device, cellulose was oxidized to various degrees of oxidation with NaOI4 (20 ml).At 3, 1, and 1.5 min, the precursor was transferred to the microwave to finish the oxidation process.The oxidized product was filtered, EtOH washed, and allowed to dry for a whole night at room temperature.Using 1.5 g of cellulose and 20 ml of distilled water, aldehyde concentration was determined 26 , through the basic response of Schiff.NH 2 OH.HCl is used when aldehyde groups transform into oximes.Dialdehyde cellulose, 0.3 g, was dispersed in 20 ml of pH-5 NaOH solution-containing water, then, NH 2 OH.HCl (0.72 mol/l) was added at pH 5 for 4 hours, the mixture was stimulated at 40 °C.To titrate the emitted HCl, a 1.0 M aqueous NaOH solution was used.The volume of alkali solution consumed during the titration was recorded (in liters), and the amount of NaOH consumed when the pH value of the solution reached 5.0 was validated.The volume of 1.0 M sodium hydroxide solution consumed was measured as Vc (in liters), using the same concentration of cellulose solution at pH 5.0 as a blank.The following equation is used to designate the aldehyde content (% w/w) 27,28 .
where C NaOH = 1.0 M, m is the dry weight of DAC (0.3 g) used in the experiment, and M molecular weight of the repeating unit of cellulose (162).

Gene expression analysis
Quantitative real-time PCR RNA was isolated from A549 cells (3 × 10 4 cells/well) and treated for 48 h using RNA easy mini Kit (Qiagen, USA) then the concentration and purity of total extracted RNA were determined using NanoDrop one micro-volume UV spectrophotometer (Thermos Fisher Scientific, USA).RNA of each treatment was converted to first-strand cDNA according to manufacturer instructions using Revert Aid First Strand cDNA Synthesis Kit (Thermo Scientific, USA).Specific primer sequences are listed in Table 1.Expression levels of β-Catenin, c-Myc, Cyclin D1, and MMP7 genes were normalized concerning β-actin transcript using Maxima SYBR Green qPCR Master Mix (2X) (Thermo Scientific, USA) and calculated by 2 −ΔΔCT method 59 .The reaction conditions were as follows: 95 °C for 10 min, 95 °C for 15 s, 60 °C for 30 s and 72 °C for 30 s with a total of 40 cycles of amplification.DNA Technology Detecting Thermocycler DT Lite 4S1 was used for gene expression quantitation.

Statistical evaluation
Outcomes are expressed as Mean ± SEM.Statistical analyses of the data are achieved utilizing Sigma plot ver.125.A student t-test was used to analyses of the data and find the examined compounds' significant differences.Changes were considered significant when P < 0.05.

Molecular docking studies
Dockage of uracil cellulosic heterocycles using the MOE program 30 to confirm the biological action and communication with them concluded different binding proteins such as Crystal structure of EGFR kinase domain G719S mutation in complex with Iressa (PDBID:2ito) 31 , Crystal Structure Analysis of the FGF10-FGFR2b Complex (PDBID:1nun) 32 , Bcl2-inhibitor complex (PDB ID: (5jsn) 33 and Crystal structure of the Skp1-FBXO31-cyclin D1 complex (PDB ID: (5vzu) 34 ; correspondingly.For separate proteins, 9 dissimilar docking simulations were operate utilized default limits 3,35 .

DFT optimization
Uses of the gas phase of DFT/B3LYP/6-311(G) level utilized Gaussian 09 program 36 was used for the geometry optimization 37,38 .The majority benefit of DFT methods is that they afford a substantial growth in computational accurateness lacking increasing computation time.All chemicals were imagined via Gauss-View 39 .Physical limitations were evaluated using the E HOMO and E LUMO values, while other limitations were retrieved from files 40,41 .

Chemistry part
Oxidation of microcrystalline cellulosic ring MCEC(1) with sodium hypochlorite utilized Microwave irradiation MDAC(2) in excellent yield, the formation of cellulose aldehyde MDAC(2) was confirmed through previous studies 28,42 and determination of cellulose aldehyde content, the reactivity of cellulose aldehyde MDAC(2) was so reactive which has methyl group which easily of attack with any active methylene group, as displayed in

IR characterization
FT-IR of the novel synthesized cellulosic heterocycles was cellulose aldehyde MDAC(2), arylidine MDAU(4), and Pyrazole cellulose MDAP(5) as displayed in Figure 2. The compound MDAC(2) showed a characteristic absorption band of C=O at 1650 cm −1 represents to two aldehyde group, and these bands can be so small or hidden in hydrated form but there is hemiacetal group which confirms the presence of aldehyde group.Furthermore, the presence of an adsorption band at 800-750cm −1 conforms to the hemiacetal bond which is a typical peak of aldehyde 26,28 .Moreover, the MDAU(4) showed a characteristic beak of the presence of three NH bands in the range 3352-3174 cm −1 due to uracil moiety, and the C≡N group showed bands at 2250 cm −1 , carbonyl group at 1650 cm −1 and more CH stretching vibration at 2990 cm −1 for uracil and glycoside ring, also the MDAP(5) showed a wide range in OH vibration in 3455-3400 cm −1 , and more bands for NH and NH 2 which take the wide range in 3364-3175 cm −1 for amino pyrazole and uracil rings and cyano group is absent and there is more bending vibration for MDAU(4) and MDAP(5) for CH aliphatic of glucose and uracil and change the vibration of aldehyde cellulose.

SEM analysis
Additionally, SEM examination of cellulose aldehyde presented the tight bundles crossed to each other and it changed the morphology of cellulose due to the oxidation process as shown in Fig. 3A Furthermore, the reaction of MDAC(2) with uracil acetamide showed cracks on the surface of cellulose due to presence of more hydrogen bonds of NH and OH groups as showed in Fig. 3B and these cracks which bonded to each other's again when reacted NH 2 NH 2 to gave the pyrazole displayed its surface as the bundles collect to its self MDAP(5) as seen in Fig. 3C.

Anti-cancer activity
Uracil pyrazole cellulose compound was investigated on the cell growth from two tumor cells (A549 and Caco2) at (6.25, 12.5, 25, 50, 100, and 200 µM) concentrations via neutral red uptake analysis which is constructed on the ability of viable cells to include and bind the supravitally dye neutral red in the lysosomes.Doxorubicin (Dox) was used as a standard drug with IC 50 values of 4.8 and 22.5 µM against tested cells (A549 and Caco2 respectively).The use of DMSO as a solvent had an insignificant effect on the viability of A549 and Caco2 cells when preserved for 48 h.Compared with control values, all heterocycles meaningfully affected cell growth inhibition.Data in Table 2 and Fig. 4 exposed that the cytotoxic activity of the heterocycles was in the descendant order of synthesized heterocycles MDAU(4) > MDAC(2) > MDAP(5) against A549 cancer cells.At 48 h, compounds

SAR investigation
SAR examination showed the activity of the combination of heterocyclic moieties with cellulose compounds participated in variation in the cytotoxic effect of these compounds.At 48 h incubation time, The presence of dioxo tetrahydropyrimidinyl hydroxyl butenamide moiety in compound MDAU(4) is more effective and more cytotoxic than the presence of pyrimidinyl pyrazole carboxamide ring in MDAP(5) compared to the presence of dimethyl aminooxopentenenitrile ring in starting compound MDAC(2) when treated with A549 and Caco2 cells.These outcomes recognized the importance of the presence of uracil pyrazoles cellulosic compounds for anticancer activity as displayed in Fig. 6 45,46 .

Antitumor docking analysis
Moreover, the docking simulation of uracil pyrazole cellulose was improved with bond lengths in Å units via the Moe program 30,47 .The less energies were then executed to preserve the geometrical optimization and systematic inquiries with an RMS gradient of 0.01 Å. Crystal structure of EGFR kinase domain G719S mutation in complex with Iressa (PDBID:2ito) 31 , crystal structure analysis of the FGF10-FGFR2b Complex (PDBID:1nun) 32 as demonstrated which were occupied from protein data bank in Table 3 and Fig. ) and (Lys A1975, Lys A153, Arg A155, Gln A175, Tyr A177, Arg A193) which showed most interaction from OH of glycose of the cellulosic chain as presented in Fig. 7D.From the above results, we concluded that the presence of uracil acetamide attached to cellulosic moiety increased the electrostatic hydrogen interaction and presence of NH, C=O enhanced this activity, and the presence of pyrazole uracil cellulosic derivatives enhanced the biological evaluation and docking result confirmed the biological evaluation.

Molecular studies
The impact of A549 cells preserved with uracil pyrazole cellulose MDAC(2), MDAU(4), MDAP(5) on mRNA expression levels β-Catenin, c-Myc, Cyclin D1, and MMP7 genes were estimated utilizing IC 50 values of these heterocycles after 48 hours and were estimated through calculating the percentage of its expression to that of β-Actin and in comparison to control values.From earlier analysis, it is fit recognized that the expression levels of β-Catenin, Myc, Cyclin D1, and MMP7 are up-regulated in A549 cells 48,49 .The Wnt signaling pathway is a complex pathway that regulates cell growth and proliferation 50 .The abnormal excitation of the pathway due to genetic mutation or increased stability can activate the abnormal expression of downstream target genes, including, c-Myc, Cyclin, and MMP-7, which can lead to cell proliferation, inhibition of cell apoptosis, and tumor formation 51 .Canonical Wnt/β-catenin pathway stimulates gene transcription through β-catenin 48,52 .MYC protooncogene amplification is closely related to tumor formation, development, and metastasis and is highly expressed in cervical cancer, breast cancer, gastric cancer, and other tumors 41 .Cyclin D1 considerably contributed to the development of the G1 to S phase 10,53 .The previous studies showed that the MMP-7 expression has been closely associated with tumor invasion and metastasis 5,10,41,54 .The current results showed that doxorubicin decreased significantly the expression levels of β-Catenin, Cyclin D1, and MMP7 genes in A549 cells as compared to control values (Fig. 8).On the other hand, treatment of A549 cells with compounds MDAC(2) and MDAP(5) resulted in a significant reduction in levels of MMP7 and c-MYC genes in comparison with control values.Besides, compound MDAU(4) exhibited a significant reduction in levels of β-Catenin, c-Myc, Cyclin D1, and MMP7 genes in A549 cells compared to control values (Fig. 8).From obtained results, we found that compound MDAU(4) is the most promising anticancer agent against A549 cancer cells through the reduction of expression levels of β-Catenin, c-Myc, Cyclin D1, and MMP7 genes compared to control values 41,50 .

Monomers of cellulosic heterocycles optimization
The Gaussian(09) program was used for uracil pyrazole heterocycle optimization 36,55,56 through DFT/B3LYP/6-311(G) level.Besides, physical features of molecular structures of MDAC(2), MDAU(4), and MDAP(5) were regarding (σ) softness 57 , (χ) electronegativities 58 , (ΔN max ) electronic charge 59 , (η) hardness, (ω) 60 electrophilicity 61 , (S) softness 62 , and (Pi) chemical potential 63,64 , from the equations (1 _ 8) which were listed in Table 5 and Fig. 10    www.nature.com/scientificreports/Moreover, the uracil heterocycles exhibited the non-planarity with DFT/B3LYP/6-311(G) level, and MDAC(2) showed total energy of it was (− 18,653.3596eV)(− 430,156.581kcal/mol) with coplanarity of two carbonyl group in glycoside ring and they were far of each other and blinded with each other with hydrogen bonds which make the band energy gap with ∆E = 3.97262 eV and this high gap gave the aldehyde stability and ability to interact again with other compound and as demonstrated in Fig. 10A,B the delocalization of electrons in HOMO and LUMO were in all MDAC(2) and indicate of reactivity of it.Furthermore, the dipole moment of MDAC(2) showed that 2.9272D can simply for energy departure and provided it capability to react once more, its electronegativity which designated the affinity of an atom to cooperate with mutual pair of electrons exhibited a high value of 3.832 eV and its hardness and softness showed low value with 1.986 eV and 0.503; respectively and it due to ability to change its electron cloud surrounding the HC=O of aldehyde group.The Pi chemical potential of MDAC (2) gives it the capability to respond and captivate more energy in dissimilar temperature varieties and they presented − 3.832 eV which gave them the capability to accumulate energy inside it.ω indicated electrophilic attractiveness and electron flow between donor and acceptor so, the MDAC(2) exhibited higher electrophilic attractiveness to captivate electrons with 3.696 eV 65,66 , Additionally, the energy of the cellulosic compound MDAU(4) is more stable than MDAC(2) with (− 37,095.0520509eV)( − 855,432.003784kcal/mol) due to the presence of the uracil moiety connected to MDAC(2) and more NH groups, which increase the stability of the molecule 35,42 .This is demonstrated in Fig. 9C,D.Additionally, FMO represents electron affinity and ionization potential.Additionally, the heterocycle's stability and reactivity were determined by the band gap energy.While heterocycles with a smaller band gap will be less stable and more reactive, those with a larger band gap will be more stable [67][68][69] , According to Fig. 9B, the HOMO-LUMO orbitals' energy planes and circulations were computed at the B3LYP/6-311G(d, p) level for the complex MDAU(4) on the glucose and uracil moiety in HOMO and LUMO due to more NH and OH groups, which provided it stability with a gap energy of 3.39029 eV.With a dipole moment of 12.1785D, this compound has a high dipole attraction that makes charge separation simple.Due to the presence of CN-CH=, which gives an atom the ability to respond again, practical MDAU(4) displayed a high value of 4.451 eV for its electronegativity, and similarly, the (eV) can easily change the electron charge with − 4.451 eV can be easily to store more energy inside it [70][71][72][73] .Due to the formation of pyrazole attached to cellulose with (− 39,037.179122eV)  ( − 900,218.50658kcal/mol), its HOMO-LUMO delocalization of charge on pyrazole and uracil rings and lack of delocalization on OH group of glycose ring, as well as the presence of NH and carbonyl of pyrazole uracil, the MDAP(5) demonstrated greater stability than MDAU(4), gave it stability, its band energy gap was 0.95948 eV, and the charge energy departure of its dipole moment was 2.5425D.As a result, the MDAP(5) displayed high electrophilic attractiveness with 6.980 eV and electronic charge ΔN max with 5.39 eV due to more NH and NH 2 , which increased the electronic charge 74,75 on it as shown in Table .5 and Fig. 10(E, F).

Conclusion
In this elucidation, the cellulosic aldehyde was combined with uracil acetamide to produce the corresponding arylidene cellulosic derivatives, which served as the active site for the nucleophilic addition to give the corresponding pyrazole cellulosic derivative.These heterocycles were then confirmed through various analyses,

Figure 4 .
Figure 4. Outcome of cellulosic compounds on A549 cells at 48 h.

Figure 5 .
Figure 5.Effect of uracil pyrazole cellulose on Caco2 cells with different concentrations.

Figure 6 .
Figure 6.Effect of cellulosic compounds on Caco2 cells at 48 h.

Table 1 .
Primer sequences used in the RT-PCR investigation.

Table 2 .
The cytotoxicity activity of heterocycles on A549 and Caco2 cells.IC 50 : Concentration required to inhibit cell viability through 50%.

Table 5 .
Computed physical parameters for synthesized cellulosic heterocycles.