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Abstract
Synthesis of 4-aryl-6-indolylpyridine-2-carbonitriles and evaluation of their biological activity
A novel series of 4-aryl-6-indolylnicotinonitrile-2-ones (1a-e) was prepared via the one-pot multicomponent synthesis, in the presence of piperidine as a base catalyst following the microwave irradiation approach and traditional thermal heating approach, scheme 1.
The rate of reaction and the product yield obtained by the microwave irradiation approach were compared to that obtained by the conventional heating method. Under the microwave irradiation, the synthesis was achieved in (15-20) minutes, and the yield was (77-87%). While the synthesis of the same compounds took )10-18( hours under the thermal heating and afforded (44-63%) yield, which was lower than the microwave irradiation method, table 1.
Compounds (1a-e) were treated with phosphoryl chloride for (18-24) hours at 80 oC under reflux, yielding the 2-chloro-6-indolylnicotinonitrile derivatives (2a-e), scheme 3.
Refluxing the 2-chloro-6-indolylnicotinonitriles (2a-e) with ethylene-1,2-diamine for 36-48 h in ethanol using TEA as a base catalyst, yielded the corresponding 2-((2-aminoethyl)- amino)-6-indolylnicotinonitriles (3a-e), scheme 4.
All compounds were tested for their anti-proliferative potency on a panel of cancer cell lines including; (SK-OV-3), (MCF-7), and (HeLa) cells. Interestingly, among the tested compounds (3a, 3b, 3d, and 3e) were the most active in inhibiting the proliferation of the cancer cells. Interestingly, these four compounds were more potent in SK-OV-3 and MCF-7 cells compared to their activity in HeLa cells, reflecting their cell-specificity, figure 1. The IC50 values for compounds (3a, 3b, 3d, and 3e) also were determined, table 2 and figure 3.
Furthermore, compounds (3b, d, and e) were screened for their antimicrobial activities at concentration (2 mg/ mL), by using the well diffusion method against four microbial strains. The three derivatives were active against Bacillus subtilis, and Staphylococcus aureus as examples for gram positive bacteria, and Candida as an example for yeast. However, they did not show any activity against Escherichia coli as an example for gram negative bacteria, table 3.
Part 2
Grafting of 2-((2-aminoethyl)amino-6-indolyl nicotinonitrile to cellulose tosylate
Microcrystaline cellulose MCC with DP 225 was employed to prepare 6-deoxytosyl cellulose TsMCC, with DSTs 0.44. This DSTs value of TsMCC improved cellulose solubility, as it became soluble in DMF, DMA, dioxane, and DMSO, scheme 5.
2-((2-Aminoethyl)amino)-6-indolylnicotinonitriles (3b, d, and e) which had promising antimicrobial activity, were selected for the grafting of cellulose tosylate via its partial nucleophilic substitution for the tosyl group (Ts is a good leaving group). The reaction was carried out in refluxing DMF at 80oC, and TEA was used as a base catalyst, affording the corresponding novel aminocellulose derivatives (4a-c), scheme 6.
The new aminocellulose derivatives (4a-c) were evaluated for their antimicrobial activity, on four different pathogenic bacterial strains namely; Staphylococcus, and Bacillus subtilis (gram positive), Escherichia coli (gram negative), and Candida (yeast). The results suggested that, the immobilization of the active heterocyclic amines on the surface of tosyl cellulose retarded their antimicrobial performance.
Part 3
Modification of 2,3-dialdehyde carboxymethyl cellulose 2,3-DACMC by 2-aminoindolylnicotinonitriles
The regioselective oxidation of carboxymethyl cellulose (CMC) was carried out by using sodium periodate (NaIO4), yielding the corresponding 2,3-dialdehyde carboxymethyl cellulose 2,3-DACMC, scheme 7.
The 2,3-DACMC was used as a precursor for the synthesis of some new biologically active Schiff`s bases (5a-c) through its reaction with 2-((2-aminoethyl)amino)-6-indolyl nicotinonitriles (3b, d, and e) using triethylamine as a base catalyst, scheme 8.
The antimicrobial activity for the Schiff`s bases (5a-c) was evaluated against four different microorganisms including Bacillus subtilis, and Staphylococcus aureus as gram positive bacteria, Escherichia coli as gram negative bacteria, and Candida as yeast. The results revealed that, both Schiff’s bases (5a) and (5b) had promising broad spectrum promising antimicrobial activity in contrast to (3b) and (3d) which had moderate activity against gram positive bacteria and Candida only but did not show activity against gram negative E. Coli, table 4.
Part 4
Design, synthesis, and evaluation of chitosan conjugated
GGRGDSK peptides as targeting molecular transporter
The peptide of sequence thiopropionyl-GGRGDSK was prepared, by assembling the amino acids (K, S, D, G, R, G, G, and thiopropionic acid) on the rink amide resin following the fmoc/tBu solid phase peptide synthesis approach, scheme 9.
Chitosan oligosaccharide COS reacted with (Sulfo-SMCC), as a bifunctional linker in phosphate buffer solution affording COS-SMCC polymer. That was followed by coupling of the peptide to COS-SMCC plymer through the formation of stable thioether bonds, giving chitosan conjugated peptide derivative COS-SMCC-GGRGDSK, which have been evaluated as a new molecular transporter system, scheme 10.
COS-SMCC-GGRGDSK polymer had a safe cytotoxic profile, since it did not show significant toxicity on (CCRF-CEM) cell line at concentrations (0.5 mg/mL, and 1.0 mg/mL). While a mild toxicity was observed (20%) at concentration (2 mg/ mL), figure 6.
Moreover, the new carrier system was able to deliver the negatively charged, hydrophobic, phosphopeptide (F`-GpYEEI) into CCRF-CEM cell line. where the cellular uptake of (F`-GpYEEI) was enhanced by 18 folds, upon its loading with COS-SMCC-GGRGDSK at (1 mg/ mL), and 24 fold at polymer concentration (2 mg/ mL) in comparison with the unloaded form of the phosphopeptide, figure 7a, b.
Furthermore, nanoparticles formulated from COS or COS-SMCC-GGRGDSK were prepared by ionic gelation method in different weight ratios of polymer to TPP. We found that, the NPs size and the positive value of zeta potential decrease by increasing the polymer/TPP ratio, table 5.
Compounds (3b), and (3d) as models for the hydrophobic anticancer agents, figure 8, were loaded with COS and COS-SMCC-GGRGDSK NPs. The size and zeta potential of the (drug/polymer) NPs were determined, table 6.
Finally, COS and COS-SMCC-GGRGDSK NPs carring the anticancer agents (3b), and (3d) were evaluated for their anti-proliferative activity against DU-145 and CCRF-CEM cancer cell lines. Their efficacy were compared to the free (3b), and (3d), using paclitaxel (PTX) as a standard anticancer drug. The results revealed that, the cytotoxic potency of (3b), and (3d) was strongly enhanced upon their loading on COS-SMCC-GGRGDSK NPs, figure 9.
Part 5
Design, synthesis, and evaluation of (fatty acyl)CGKRK conjugated to chitosan and in vitro gene delivery
The tumor homing peptide of sequence CGKRK was synthesized by assembling the amino acids (K, R, K, G, C) on rink amide resin following the fmoc/tBu solid phase peptide synthesis approach, by employing the fmoc solid-phase synthesis. Then, the peptide was coupled to the fatty acids (palmitic, stearic and oleic) at the peptidyl N-terminal, and the corresponding palmitoyl, stearoyl and oleoyl derivatives of CGKRK peptide were obtained, schemes 11 and 12.
Chitosan was conjugated to the (fatty acyl)CGKRK peptides by coupling the free thiol group of the peptide chain, with the maleimide arm of COS-SMCC polymer, resulting in the formation of stable thioether bond, scheme 13.
The ability of COS-SMCC-(fatty acyl)CGKRK peptide to complex with siRNA was determined by SYBR green test and compared to the unmodified COS polymer, figure 11a and 11b.
The particles size and zeta potential were studied for polymer/siRNA complexes at different weight ratios of the polymers to siRNA. The data suggested that, COS-SMCC-(oleoyl)CGKRK derivative had the smallest particle size at all polymer ratios. While, The stearoyl substitution effectively enhances the assembling with siRNA, more than COS modified by (palmitoyl) or the (oleoyl)CGKRK moieties figure 12a, and 12b.
The efficacy of the polymer to protect siRNA from serum degradation was determined. The unmodified COS was able to protect siRNA more than the stearoyl, oleoyl and palmitoyl conjugates, figure 13.
Generally, the cytotoxicity profile of COS-SMCC-(palmitoyl)/(stearoyl)CGKRK derivatives in MDA-MD-231 cells were safe in comparison to COS, figure 14a.
The cytotoxic potentiality of the polymer/siRNA complexes at different ratios also was studied on the MDA-MD-231 cancer cells. Among the screened ratios, the ratio of 10:1 significantly reduced the cell viability by 35% and 22% with the oleoyl and palmitoyl conjugates respectively, figure 14b.