Synthesis and biological evaluation of new nanosized aromatic polyamides containing amido- and sulfonamidopyrimidines pendant structures

Background Antibiotics are biocides or products that inhibit the growth of microorganisms in the living cells and there are extensive works directed to develop efficient antimicrobial agents. The sulfonamide-containing polymers have great potential to resist gram-positive or gram-negative bacterial and fungal attacks. As a therapeutic agent, the sulfonamides have been reported as antitumor and antimicrobial agents against bacteria, being more potent against gram positive rather than gram negative strains. Design of new classes of inhibitors bearing fluorescent tails, as therapeutic and imaging agents, is currently an active area of research. Here, we describe the synthesis of a new family of polyamides based on chlorophenyl-3,5-diaminobenzamides, methyl substituted pyrimidinoamido-3,5-diamino- benzamides and methyl substituted pyrimidinosulfonamido-3,5-diaminobenzamides and evaluation of their thermal, optical and antimicrobial properties. Results We report the synthesis of a new series of nanosized polyamides containing bioactive pendent structures. The spherical nanosized polymer particles are soluble in many organic solvents and exhibited emissions ranging from blue to orange wavelength depending on the nature of the signaling unit. Pyrimidine- and p-chloroaromatic containing polymers exhibited higher bioactivity than that contain the sulfonamide group. The amidopyrimidine polymers exhibited remarkable antifungal and antibacterial activity and thus, these types of polymers are promising candidates for biomedical applications. Conclusions The SEM analysis indicated that most of the polyamides were organized as well defined nano sized spheres, but in certain derivatives small amount of aggregated nanospheres were also observed. Thermal analyses were studied up to 700 °C and results showed comparable thermal behavior. The optical results revealed that polymeric series (A) exhibited orange emission, series (B) showed green emission while series (C) exhibited yellow and blue emissions. Benzene/pyridine structure interchange resulted in red shifted peaks attributed to the localized lone pair of electrons on a nitrogen atom which offer a greater electron affinity and better electron-transporting properties. The amido- and sulfonamide pyrimidine containing polymers exhibited the most potent antimicrobial activity. Relative to the reference Gentamicin, the polymer 54 exhibited comparable antibacterial activity against gram negative bacteria. Analogues 52 and 57 exhibited remarkable antibacterial activities compared to the references used. Thus, these polyamides are likely to be promising broad spectrum antibacterial agents and deserve further investigation at the molecular level.Graphical abstract: The synthesis and characterization of a new series of nanosized polyamides containing chloroaromatic (A), pyrimidinoamido- (B) and pyrimidosulfonamido- (C) pendent structures as promising candidates for biomedical applications is described. Electronic supplementary material The online version of this article (doi:10.1186/s13065-015-0123-2) contains supplementary material, which is available to authorized users.


Background
Polymer-drug structures are currently known constructs that chemically combine the bioactive part with a specific region of the polymer to ensure its delivery to the targeted intracellular compartment [1]. Several synthetic approaches have been published to bond the polymer and drug either in the polymer backbone or in the sidechain [2][3][4]. The development of longer term bioactive antimicrobial polymers is a research area focused on solving microorganism's contamination problems [5]. Polymer modifications to achieve such activity include incorporation of known antimicrobial compounds such as hydantoins, glycolylureas, imidazolidinones and oxazolidinones [6][7][8]. In addition, several studies have reported the incorporation of a pyrimidine ring into the polymeric backbone. Incorporation of pyrimidine nuclei modify the polymer's solubility and processability due to the possibility of protonation and/or alkylation of the lone pair. Moreover, the electronegative nitrogen atoms offers many substituted pyrimidine structures through direct substitution reactions [9][10][11][12].
Sulfonamides have been used in therapeutics for many years [13,14]. The sulfonamide derivatives have been reported to show substantial antitumor activity in vitro and/or in vivo [15][16][17][18], HIV protease inhibitors [19,20] and cell entry [21]. The polysulfonamide is an active agent that shields the toxic polycations. The copolymers possess higher activity toward fungi than against bacteria and being more gram positive rather than gram negative as it is common. A novel strategy for cancer treatment based on a new class of inhibitors bearing fluorescent tails is currently an active research area for use therapeutic and imaging agents for poorly responsive tumors to classical chemo-and radiotherapies. For instance, a bioactive novel fluorescent fluoro poly(amide-sulfonamide) s possessed distinctive structure as well as unique properties were reported [22].

General
Melting points were determined with an electrothermal melting point apparatus and are not corrected. Infrared spectra (IR, KBr pellets; 3 mm thickness) were recorded on a Perkin-Elmer Infrared Spectrophotometer (FT-IR 1650). All spectra were recorded within the wave number range of 600-4,000 cm −1 at 25 °C. 1 H-NMR and 13 C-NMR spectra were recorded using the JEOL 500 mHZ spectrometer operating in DMSO-d 6 and expressed on the δ scale ppm. Absorption spectra were measured with a UV 500 UV-vis spectrometer at room temperature (r.t) in DMSO with a polymer concentration of 2 mg/10 ml. Inherent viscosities (η inh ) were measured at a concentration of 0.5 g/100 dL in DMSO at 30 °C by using an Ubbelohde Viscometer. Differential thermo gravimetric (DTG) analyses were carried out in the temperature range from 20 to 700 °C in a steam of nitrogen atmosphere by a Shimadzu DTG 60H thermal analyzer. The experimental conditions were: platinum crucible, nitrogen atmosphere with a 30 ml/min flow rate and a heating rate 20 °C/min. Thermo gravimetric (TGA) analyses were carried out using SDTQ600-V20.5-Build-15. (DTG), (TGA) and elemental analyses were performed at the Microanalytical Unit, Cairo University. The morphologies of polymer nanoparticles were observed by Scanning Electron Microscope (SEM) (JEOL-JSM5300), at the E-Microscope Unit; Faculty of Science, Alexandria University. The samples were sonicated in de-ionized water for 5 min and deposited onto carbon coated copper mesh and allowed to air-dry before the examination. The antimicrobial activities were carried out using diffusion agar techniques and the evaluation of cytotoxicity against HepG-2, HCT-116 and MCF-7 cell lines were performed at the antimicrobial unit and the regional center of mycology and biotechnology, Al-Azhar University, Cairo.

Preparation of polymer 36
Following the general method described above, isophthaloyl dichloride reacted with 3,5-diamino-N-phenylbenzamide 13 to give the polyamide 36. The following data were recorded: Yield: 0. 8

Preparation of polymer 38
Following the general method described above, isophthaloyl dichloride reacted N- (3-

Preparation of polymer 42
Following the general method described above,

Preparation of polymer 43
Following the general method described above,

Preparation of polymer 45
Following the general method described above, pyridine 2,6-dicarbonyl dichloride reacted with N- (4- Isophthaloyl chloride (2.20 mmol) was treated with a solution of the appropriate diamine 24-26 (2.20 mmol) following the general procedure described above.

Preparation of polymer 53
Following the same described method, isophthaloyl dichloride was treated with N- (4-

Preparation of polymer 54
Following the same described method given above, isophthaloyl dichloride was reacted with N- (4-

Polymer particles synthesis (general Method)
The appropriate, readily available isophthaloyl dichloride, pyridine 2,6-dicarbonyl dichloride or the prepared one 60 (0.5 mmol) and the appropriate diamine 13-17, 24-26, 33-35 or 61-66 (0.5 mmol) were separately dissolved in dioxane (50 mL). Distilled water (10 mL) was added to the diamine and the entire solution was added to the acid chloride. The resulted turbid solution was ultrasonicated at 42 kHz in a water bath for a period of 30 min. The polymer colloidal solution was extracted by centrifugal separation for 15 min. at 6,000 rpm and the resulted precipitate were carefully washed with methanol and water to purify the product of any unreacted monomer. The polymer samples were then dried in a vacuum oven at 60 °C for 10 h. The synthesis of the nanosized aramides particles 36-45, 46-51 and 52-57 was the next task. Different solution techniques are known in the literature for the preparation of nanosized particles, including emulsion, interfacial polycondensations or nanoprecipitation method [23][24][25][26][27][28][29]. The basic principle of the latter method is based on the interfacial deposition of a polymer from solvent/non-solvent phases. Generally, the current series were prepared by ultrasonication of 0.5 mmol of the appropriate diamine with 0.5 mmol of the acid chloride in a total of 115 ml dioxane solution containing distilled water (15 ml) followed by centrifugal separation at 6,000 rpm for 30 min. The presence of water is necessary for controlling the particle shape and as a reaction accelerator. As judged by SEM micrographs, Figs. 2 and 3, most polyamides were obtained as well-separated spherical nanosized forms, nevertheless, there were some degree of aggregation for those polymers containing pyridine and pyrimidine pendant groups. The aggregate formation could be attributed to the molecular H-bond

Physical properties of the polymers Solubility
The polyamides are readily soluble in polar aprotic solvents such as NMP, DMAc, DMF and DMSO while insoluble in boiling alcoholic or halogenated solvents. The observed solubility of the pyridine-containing polyamide compared to that polyamides containing phenylene moiety could be attributed to the dipole-dipole interaction of polymer-solvent system. The pyrimidine-containing polymers showed inferior (lower) solubility may be due to the presence of pyrimidine structural that aggravate macromolecule hydrogen. The presence of the sulfonamide group leads to increase solubility due to their effective contribution to the cohesive energy which counteracting their influence in the increment in the main chain-main chain distance.

Inherent viscosity
The inherent viscosity (η inh ) of the polymers, as a suitable criterion for evaluation of molecular weight, was measured at a concentration of 0.5 g/100 mL in DMSO at 30 °C. The η inh of phenylene-containing polyamides 36-40 were in the range 0.14-1.48 dL/g while their analogues 41-45 were in the range 0.47-1.44 dL/g indicating low to moderate molecular weights in this series. The η inh of the amido-and sulfonamido-pyrimidine containing polymers 46-51, 52-57, respectively, were closely similar in the range 0.24-1.61 dL/g. Noteworthy, no significant change in inherent viscosity was noticed on phenylene/pyridine replacement.

Optical properties
The optical properties of the polyamides 36-45 The optical properties of polyamides series containing chloroaromatic pendent moiety and 36-45 were investigated by UVvis and photoluminescence spectroscopy in DMSO using concentration of 2 mg/10 ml. The values of molar extinction coefficients were in the range 14,640-23,530 M −1 cm −1 . The PL spectra were measured at 290 nm excitation.
wavelength using polymer concentration of 10 −4 . Table 1 compiles the optical data of this polymer series and several interesting points are concluded: • Relative to the unsubstituted polyamide, all substituted polymers showed slightly shifted absorption peaks due to the electronic effect of the substituent that increase the electron density, thereby leading to a relatively large energy band gap for π-π * transitions. • The fluorescence emission spectra of all polyamides exhibited two emission peaks at 346 nm and 580 nm. • The orange emission observed for all polyamides at 580 nm could be attributed to the substituent' electronic effect. • Pyridine containing polyamide 41 exhibited slightly blue shifted absorption band relative to its phenylene analogue 36 while its emission showed a red shifted emission peak at 420 nm. Compared to a benzene ring, pyridine has a greater electron affinity and better electron-transporting properties.  Table 2. From the UV-vis spectral data given in Table 2 several remarks are found: • Pyrimidine containing polyamide 46 exhibited a blue shifted absorption peak at 267 nm relative to its phenylene analogue 36. • Introduction of one methyl substituent led to a new absorption at 312 nm while the presence of two methyl substituents red-shifted the peak to 331 nm.   Table 3. From the UV-vis spectral data several remarks are found: • All polymers in this series exhibited yellow emissions at 572 nm. No change upon phenylene/pyridine exchange except polymer 56 in which replacement red shifted the absorption. Furthermore, methyl substitution red-shifted the absorption bands. • This series of polyamides exhibited red-shifted absorptions and emission peaks relative to their pyrimidine-containing polymers. This could be attributed either to the sulfonamide's electronic effect or the increase of molecular polarizability which reduce the energy level separation.

Thermal analysis
Thermal properties of the polyamides 36-45 The thermal properties of the prepared polymers were evaluated by differential thermo gravimetric (DTG) and differential thermal analysis (DTA) techniques. Thermal stability of the polymers was studied in the range 20-700 °C (char yield), Table 4. Structure-property relationship demonstrated an interesting connection between a single structure change and its thermal property. Phenylene-containing polymers 36-40 exhibited similar degradation behavior. DTA analysis revealed that the polyamide 36 exhibited an endothermic peak at 440 °C and an exothermic peak at 634 °C. The TGA exhibited degradation processes at 158 °C (8.6 % wt loss), 303 °C    Fig. 4.
The major degradation occurred in the range 400-700 °C leaving traces of the polymer as a mass residue. The introduction of the methyl substituent in the main chain of a polymer has no significant effect on the thermal stability. The DTA curve of the polyamide 46 exhibited an exothermic peak at 537 °C. The TGA curve showed successive degradation processes at 295 °C (14.2 % wt loss), 375 °C (10.3 % wt loss), 445 °C (13.4 % wt loss) and 699 °C (61.9 % wt loss), leaving 0.3 % of as a mass residue. The polyamide 47 exhibited an endothermic peak at 592 °C and another exothermic peak at 656 °C (DTA analysis). The TGA showed degradation processes at 443 °C (26.71 % wt loss), 550 °C (19.4 % wt loss) and 700 °C (52.2 % wt loss), leaving 1.8 % mass residue. The polyamide 48 exhibited an exothermic peak at 607 °C (DTA analysis). The TGA analysis showed successive degradation processes at 215 °C (11.58 % wt loss), 403 °C (16.6 % wt loss) and 597 °C (68.5 % wt loss), leaving 3.32 % as a residue.
Thermal properties of the polyamides 52-57 Thermal properties of the polyamides 52-57 are collected in Table 6    In summary, pyrimidine-containing polyamides exhibited relatively higher thermal stability compared to their sulfonamido-pyrimidine analogues. This may be explained by the feature of the supramolecular structure, namely by a high density of packing of polymeric chains, realized through a level-by-level stacking of these chains. With such stacking a strong intermolecular interaction between the amide fragments of adjacent polymeric chains is provided. However, in the case of the former polymers, the interchain interaction can occur due to specific contacts between the amide fragments of one chain and the nitrogen atoms of the pyrimidine cycles of the other chain. Owing to this fact the pyrimidine cycles serve as an additional amplifier of the interchain interaction in polyamides, thus causing the strength and thermal stability to increase. The presence of a sulfonamide group adjacent to pyrimidine, as in the case of the latter polymer series, led to decrease thermal stability. This may be attributed to the acidic nature of the hydrolyzable group that retain the high polarity and thus, the polymer degraded before melting stage. Nevertheless, the methyl substitution enhanced the thermal stability in this series.

Calculations of limiting oxygen index
Flammability of polymers is one important property which could limit their applications [30]. Despite the fact that high-performance polymeric materials offer many advantages over conventional metals, their flammability and possible release of toxic byproducts increase the fire risk and thus the introduction of flame-retardant additives are the easiest way to diminish the polymer flammability. The flame retardancy is evaluated by limiting oxygen index (LOI). The LOI is defined as the minimum oxygen concentration needed in an inert gas medium for the material to achieve burning after ignition. The LOI is a measure of the ratio of oxygen to other gases in the air surrounding a substrate. A material with an LOI of greater than 21 % but less than 28 % would be considered "slow burning" while a material with an LOI of greater than 28 % would be considered "self-extinguishing". Char yield can be used as criteria for evaluating LOI of the polymers in accordance with Van Krevelen and Hoftyzer equation [31]; LOI = 17 + 0.4CR, where CR = chars yield. The calculated LOI values of all polymers based on their char yield were less than 28, Tables 7,8.

Calculations of thermodynamic parameters
The thermodynamic parameters of decomposition processes of polymers, namely, activation energy ∆E enthalpy (∆H), entropy (∆S) were evaluated by employing the Horowitz-Metzger equation [32], Additional file 1: Tables S1, S2.  The order of chemical reactions (n) was calculated via the peak symmetry method by Kissinger [33]. The asymmetry of the peak, S, is calculated as follows: The value of the decomposed substance fraction, αm, at the moment of maximum development of reaction (with T = T m ) being determined from the relation (3): where S is the entropies of activation, R represents molar gas constant, Φ rate of heating (K s −1 ), K the Boltzmann constant, and h the Planck's constant [34]. The change in enthalpy (∆H) for any phase transformation taking place at any peak temperature, Tm, can be given by the following equation: ∆ S = ∆H/Tm. Based on least square calculations, the Ln ∆T versus 1,000/T plots for all complexes, for each DTA curve, gave straight lines from which the activation energies were calculated according to the reported methods [35]. The slope is of Arrhenius type and equals −E/R. The kinetic data obtained from the nonisothermal decomposition of the prepared polyamides series containing chloroaromatic pendent moiety 36-45 are given in Additional file 1: Table S1. The following trends and conclusions may be achieved: The kinetic data obtained from the nonisothermal decomposition of the polymers 46-57 are given in Additional file 1: Table S2. The following trends and conclusions may be achieved:   Antimicrobial activity of polymeric series 36-45 Chloro aromatic compounds played a vital role in the development of different medicinal agents where chlorine is electronegative, and therefore oxidizes peptide link and denatures proteins. Exposure of strains of E. coli, Pseudomonas spp. and Staphylococcus spp. to lethal doses causes a decrease in ATP production. Chlorine acts on the permeability of the external membrane of E. coli through a primary lethal phenomenon which consists in a substantial leakage of K + ions; such leakage does not occur for macromolecules. Sub-lethal doses inhibit cellular respiration due to a nonspecific oxidizing effect (bactericidal effect) [36]. Results of antimicrobial activity of polyamides 36-40, derived from isophthaloyl chloride, and the comparative activity of currently used antibacterial and antifungal agents are presented in Additional file 1: Table S3. Thus, the introduction of choro substituents clearly enhanced the antimicrobial activity against all fungi and B. subtilis with inhibition zone diameters ranging between 13.4 and 19.6 mm. Compared to other analogues, the polyamide containing p-chloro substituent showed higher activity against the tested microorganisms.
The antimicrobial activity comparative tests of the polymeric series 41-45, derived from pyridine 2,6-dicarbonyl dichloride, are presented in Additional file 1; Table S4. Polyamides containing both sulfonamide and chloro substituents showed higher antimicrobial activity against all fungi and gram positive bacteria. The presence of such bioactive groups in the backbone of the polymer play the key role in catalyzing both biological and chemical systems. Compared to their analogues 25-29, the polyamides 30-34 showed relatively higher antibacterial activities against all tested microorganisms.
Antimicrobial activity of polymeric series 46-57 Sulfonamides are chemotherapeutic agents which display various biological interactions, including inhibition of carbonic anhydrase and affecting insulin releasing in addition to their antimicrobial, antitumor and anti-inflammatory activities. The antimicrobial activity of the amido-and sulfonamido-pyrimidine containing polymers 46-57 are presented in Additional file 1: Tables S4, S5. From the screening results, the following remarks are concluded: • Pyrimidine-containing polyamides exhibited high antifungal activity than their analogues containing sulfonamidopyrimidine pendant structures. Thus, the presence of sulfonamide structures in such polymeric series considerably alters the antimicrobial activity of the polymer. The polyamides 46-48 exhibited remarkable antifungal activities against A. fumigatus and, interestingly, the observed activity were more potent than those of the reference Amphotericin B. Over 80 % of the reported Aspergillus-related cases, such as extrinsic allergic alveolitis, asthma, allergic sinusitis, chronic eosinophilic pneumonia, hypersensitivity pneumonitis, and allergic bronchopulmonary aspergillosis are most frequently caused by A. fumigatus [37].
Moreover, introduction of methyl substituents in case of the polyamides 47 and 48 produced potent antifungal polymers against S. racemosum and, the noteworthy, the activity were higher than the reference Amphotericin B and thus, the introduction of a methyl group to the pyrimidine promotes antifungal activity. S. Racemosum is well known to cause skin and soft tissue infection and fungal rhinosinusitis [38].
Sulfonamidopyrimidine-containing polyamides analogues 51-53 exhibited higher antibacterial activity against gram negative bacteria than their analogues 46-48. Thus, replacement of the amide linkage by sulfonamide linkage promoted specifically the antibacterial activity against gram negative type. Noteworthy, relative to the reference antibiotic Gentamicin, the polyamide 54 exhibited comparable antibacterial activity against gram negative bacteria. It has been reported that P. aeruginosa is the most common pathogen causing chronic infection in people with cystic fibrosis (an inherited disease that affects the lungs, digestive system and sweat glands) [39].
• Pyrimidine-containing polymer analogue 52 exhibited remarkable antibacterial activities against S. pneumoniae, a gram positive bacterium and P. aeruginosa a gram negative bacterium. In both cases, activities were more potent compared to the references antibiotics used. Thus, the polyamide 52 is likely to be a promising broad spectrum antibacterial agent.
• In general, polymers have pyrimidinoamide linkages exhibited lower activities toward gram positive bacteria than their analogues have sulfonamidopyrimidine linkage. • The polyamide analogue 57 exhibited promising antibacterial activities against both gram positive and gram negative bacteria. Interestingly, its activity as reflected by the inhibition zone diameter is higher than the reference antibiotic Gentamicin.

Antimicrobial activities' statistical analyses
The antimicrobial activity data of the most promising polymers were analyzed against their corresponding controls using SPSS software package version 18.0 (SPSS, Chicago,