Optical Properties, Thermal Stability, and Biological Activity of a Non-Centrosymmetric Hybrid Compound 3,4-Diaminopyridinium Perchlorate (3,4-DAPP)

1. Introduction

Current research on organic-inorganic hybrid materials has gained much importance in recent years due to the manufacturability and diversity of physicochemical properties of various materials, which could be used in a wide range of applications, such as optics[1,2], photovoltaic [3], catalysis [4] and other fields [5,6]. This research has mainly focused on the optical properties of hybrid materials due to their strong room-temperature emission. Moreover, by substituting halogen or metal sites through theappropriate choice of organic components, it is conceivable to govern the particular structure of the inorganic-organic sheets, which makes it possible to control the physical properties of these compounds. In almost all of the considered materials, aminopyridines are bioactive N-heterocyclic tertiary amines, particularly relevant since they significantly increase the amount of acetylcholine produced in response to conduct nerve impulses as well as electronic depolarizations of tetrodotoxin-blocked motor nerve terminals [7]. In addition, these heterocyclics show great promise as potassium channel blockers, especially giventheir high potency, high specificity of potassium channels (K⁺), and effectiveness in pharmacological dissection of the ionic channels of excitable membranes [8]. Due to their ability to improve nerve transition, aminopyridines have been offered as treatments for multiple diseases such as sclerosis [9], myasthenia gravis [10], Alzheimer's[11], anesthesia, and muscle relaxation [12]. This feature makes it widely used and has been created using compounds derived from heterocyclic molecules, amongst one of these derivates we chose to work with 3,4-diamonopyridines and perchloric acid. As a part of our ongoing work on similar structures [13,14,15], we report the chemical preparation, the structural study, the spectroscopic measurement, the thermal behavior,and finally the biological test of the title compound.

2. Materials and methods:

2.1. Synthesis

The crystals of 3,4- diaminopyridinium perchlorate with the formula (C₅H₈N₃)ClO₄ were obtained due to a solution of concentrated HClO₄acid (Sigma Aldrich 92%) and the organic molecule 3,4-DAP (3,4-diaminopyridine) (Sigma Aldrich 98%) in stoichiometric ratio 1/1. The reaction was carried out in the presence of water (10ml) and maintained at room temperature. The reaction sequence for the synthesis is reported in the following equation:

After several days of evaporation, transparent prism crystals appeared in the solution then isolated and dried in the air.

2.2. Spectroscopic Measurements

The sample’s infrared spectrum was recorded at ambient temperature using a Perkin-Elmer FT-IR 100 spectrometer in the region 4000-400 cm^-1. The RAMAN spectrum was recorded using a Horiba JobinYvon triple monochromator spectrophotometer (Lab RAM HR 800) in the range of 50 to 4000 cm^-1. Both spectra were recorded at the same resolution (50 cm^-1) with 3 cm⁻¹of spectral steps.

2.3. X-ray Data Collection and Structure Determination

In order to define the crystal structure, a suitable crystal of (C₅H₈N₃)ClO₄ was selected and characterized on a diffractometerBrukerApexII, Kappa CCD type, equipped with graphite-monochromated Mo K_α radiation ($\lambda =$0.71073 Å), and it was maintained at 200 K during data collection. The crystal structure was solved using the WingX software [16],using the SHELXT 2018.2 [17] structure solution program and refined with the SHELXL-2018.3 refinement package.The positions of all H-atoms were fixed geometrically by the appropriate instructions HFIX.The crystallographic data as well as some details of the structure refinement results are provided in Table 1.

2.4. Hirschfeld Surface Calculations

Hirschfeld surface analysis serves as an effective tool for studying the solid-state behavior of molecules as well as defining the various intermolecular interaction types in the studied compound. Molecular Hirschfeld surfaces are constructed using the distribution of electrons calculated as the sum of spherical atom electron densities [18,19]. The identification of contacts is made possible via the normalized contact distance (d_norm) relating to the distances from the surface to the nearest core inside and outside the surface (d_i and d_e, respectively) and the van der Waals radii (vdw) of the atom given by the equation below:

$${\mathrm{d}}_{\mathrm{n}\mathrm{o}\mathrm{r}\mathrm{m}}={\displaystyle \frac{d_i-r_i^{vdw}}{r_i^{vdw}}}+{\displaystyle \frac{d_e-r_e^{vdw}}{r_e^{vdw}}}$$

where r_i^vdw and r_e^vdw are the Van der Waals radiations of the corresponding inner and outer surface atoms, respectively. Hirschfeld surface was mapped using a red, blue, and white color scheme. The red region indicates the proximity of the hydrogen bond donors to the acceptors and a negative d_norm value. The blue region indicates the distance of the hydrogen bond from the donor to the acceptor and a positive d_norm value. The white region indicates the contact distance, which is equal to zero, and indicates the normalized contact distance of hydrogen bond donors to acceptors.

2.5. Thermal Analysis

Thermogravimetric analysis (TGA) was carried out using a multi-module 92 SETA RAM analyzer from room temperature up to 500 °C, with a heating rate of 10°C/min under Oxygen gas. Also, theDifferential Scanning Calorimetric (DSC) of the title compound was performed on crystals (m = 4.4 mg)using a Perkin-Elmer analyzer, in a temperature range of -50 to 300°Cwith a heating rate of the order of 5°C/min under constant N₂ flow.

2.6. Optical Study

The absorption spectrum of UV/vis was captured at room temperature using a HELIOS OMEGA v8. 01 spectrometer with a wavelength range of 200-600 nm and H₂O was used as a reference. Using a Perkin Elmer LS-55 spectrometer, the photoluminescence spectrum was recorded over the 200-800 nm range and exited with 340 nm radiation.

2.7. Computational Details:

All calculations were performed by using the Gaussian 09 program [20]. The density functional theory at the B3LYP level optimized the ground-state geometric structure, employinga 6–31G+ (d) basis set. Frequency calculationswere performed to confirm that the geometry is at a global minimum and has no imaginary frequencies.The electronic transitions were calculated using Density Functional Theory(DFT) at the same level. The theoretical absorption spectrum simulation (GAUSSIAN spread model at an HWHM of 20 nm) and the isodensity molecular orbitals generation were performed using the GABEDIT 2.1.0 software [21].

2.8. Determination of In-Vitro Antibacterial Activity by Agar Well Diffusion Method

Six pathogenic bacteria including 3 gram-positive bacteria, Staphylococcus aureus, Bacillus cereus and Bacillus subtilis and 3 gram-negative bacteria, namely Pseudomonas aeruginosa, Enterococcus faecalis,and Salmonella typhimurium were used in this assay.

A volume of 100 µl suspension, having 10⁶ CFU/ml of each bacterial strain, was dispensed on a nutrient agar medium. Wells of approximately 10 mm was bored using a well cutter. A volume of 100 µl of 3,4-DAPP samplefrom the concentrations of 50 and 100 mg/ml were added to the wells. The plates were then incubated at 37 °C for 24 h. The antibacterial activity was assayed by measuring the diameter of the inhibition zone formed around the well. The 3,4-DAP was used as a negative control and the gentamycin antibiotic was used as a positive control [22].

3. Result and Discussion

3.1. Crystal Structures

The 3,4-diaminopyridinium perchlorate (C₅H₈N₃)ClO₄ is built up of ClO₄^- tetrahedra and 3,4-diaminopyridine (3,4-DAP) cations.This compound crystallized in a monoclinic system, with a non-symmetric space group P2₁, two units per cell (Z=2) and the following lattice parameters: a= 5.1261 (9), b= 9.3003 (17), c=8.6659 (16) Å, β=102.595 (7)° and V= 403.2 (12) ų (Table 1). Figure 1 depicts the asymmetric unit, showcasing a protonated cation-anion par linked by a hydrogen bond.

The crystal structure displays significant inversion symmetry breaking, as supported by a Flack parameter of 0.48, indicating an asymmetric arrangement of chiral molecules. Furthermore, the cumulative intensity distribution curve suggests that the crystal data are more likely acentric than twinned or centric, providing additional evidence. The arrangement of the crystal structure is organized with parallel inorganic segments interspersed by organic chains aligning along the $\overrightarrow{b}$-axis, as illustrated in Figure S1.

Figure 2. Cumulative intensity distribution curve of the structure data of 3,4-DAPP.

Figure 2. Cumulative intensity distribution curve of the structure data of 3,4-DAPP.

An examination of the organic component reveals 3.4-DAP cations trapped in the spacing between perchlorate tetrahedra (Figure 3a) along the [010] orientation, linked by N-H$\cdots$O bonds ranging from 2.3533 to 2.5161 Å. Furthermore, the distance between aromatic nuclei measures 5.12 Å, surpassing the typical π-stacking distance (>3.8 Å), thus indicating the absence of π- π interaction within the structure [23].Conversely, the inorganic component exhibits four-coordination with (C₅H₈N₃)⁺ cations across a similar distance spectrum as the organic part. This form of weak hydrogen bonding creates a supramolecular crystal characterized by an infinite zero-dimension network, comprising four distinct types of networks labeled $R_4^4$(19), $R_2^1$(7), $R_4^2$(8) and $R_4^3$(17) in according with Bernstein's theory nomenclature [24] (Figure 3b). Analysis of the geometric attribution of the perchlorate tetrahedra and the computed average Baur distortion indices {ID (Cl-O)= 0.003, ID (O-Cl-O)= 0.0046, and ID (O-O)= 0.0088} (Table S1), indicates a slightly distorted tetrahedral configuration, albeit describable as regular, which correlates with the stereochemical behavior of ClO₄^- anions and their hydrogen bond interactions [25]. Detailed hydrogen bond geometries are provided in Table S2.

3.2. Vibrational Spectroscopy Measurement:

For more information about the crystal structure results, we recorded the infrared (IR) and RAMAN spectra of 3,4-DAPP at room temperature.The experimental IR, RAMAN and computed IR vibrational frequencies are listed in Table S3. The most typical features in this regard are the characteristics bands of the 3,4-diaminopyridine skeleton vibrations between 3082 and 3319 cm^-1in the IR spectra (Figure 4), which is assigned to the symmetric and asymmetric stretching modes of NH₂ group, and out of plane deformation of the same group observedat 615 and 1635 cm^-1[15].For the NH⁺ stretching and deformation vibration, they presented with the associated range of bands between 2029 - 2575 cm^-1and1355 - 1507 cm^-1, respectively. The out-of-plan deformation of the (C-C-C) and (C-N) of the aromatic ring appears between 544 - 554 cm^-1 and 594 - 607 cm^-1, respectively.Concerning the vibrational modes of the inorganic part (ClO₄^-), the tetrahedral geometry admits four deformation modes, two in plan deformation (stretching) represented by ν₁ (ClO₄^-) and ν₃ (ClO₄^-)observed in regions between 926 and 1060 cm^-1 in the IR spectra, and two out of plan deformation (wagging) represented by ν₂ (ClO₄^-) and ν₄ (ClO₄^-) observed at lower regions between 451 and 622 cm^-1 in the RAMAN spectra.[23].The strong luminescence of the studied compound is confirmed by the unique curve displayed in the RAMAN spectra (Figure S2). The experimental and computed FT-IR frequencies are both consistence (Figure 4).

3.3. Hirschfeld Surfaces and Crystal Voids

The use of Hirschfeld surface analysis is to measure and display the close connections between atoms in the crystal structure of 3,4-DAPP. The way molecules in a crystal interact with each other is shown on HS plotted over d_norm (Figure S3b), where the intensive bright red spots on the surface related to the O$\cdots$H/H$\cdots$O interactions, while the blue regions correspond to longer contacts with positive d_normvalue [26].The pie chart (Figure S3a) summarizes the considerable contributions from several intermolecular interactions on the Hirschfeld surface (HS). The integration of thecrystal packing is mainly assured by N-O$\cdots$H interactions,which constitute half of the total interactions (50%). The H$\cdots$H contacts are distributed as widely scattered points of the high density of (d_i, d_e) pairs with 19%. However, some minor intermolecularinteractions such as C$\cdots$H, N$\cdots$H, C$\cdots ,$O and others, exhibit relative contribution interactions extended between 6 and 9%.Moreover, the shape index is highly sensitive to very subtle changes in surface shape. Indeed, the relative placement of red and blue adjacent strangles on a shape index map provides insight into the interaction between π electrons. Notably, these strangles are absent from the picture (Figure S4a.), indicating the lack of π–π stacking in this compound [23]. Furthermore, the curvedness (Figure S4b) confirms this with the large flat areas of the surface which is outlined in blue.To check the mechanical stability of the crystal, a void analysis was performed to inspect the “empty” regions of the crystal structure,based on the isosurfaceof theprocrystal electron densities of the spherically symmetric atoms contained in the asymmetric unit [27]. Figure 5 is the graphical representation of voids in the crystal packing of 3,4-DAPP. The volume of the voids is 14.27 ų and the percentage of free space in the unit cell is3.5%, which indicates the absence of any large cavity in the crystal,and the molecules are packed tightly, thereby this compound is expected to have a good mechanical properties [28,29].

3.4. Thermal Analysis:

The thermal stability of the reported compound was studied using thermogravimetric analysis (TGA-DTG) and differential scanning calorimetry (DSC).The TGA-DTG curve results (Figure 6) showed that the investigated compound had thermal stability up to 285 °C with three weight loss stages. The first weight loss peak of 5.5% observed in the DTG curve at 285 °C may indicate the beginning of crystal decomposition. The second weight loss of 17.1% occurs in the temperature range of approximately 300–400 °C due to the decomposition of the organic moieties. The DTG curve shows a large weight loss of 31.8% at 387 °C, which is likely due to the decomposition of the remaining organic framework and possibly perchlorate decomposition. In addition, the DSC measurement was recordedin order to determine the exact phase transition temperatures. The diagram (Figure S4) showsone exothermic peak that appears at T = 50°C,attributed to the loss of surface water molecules, and the significant peak at T= 270 °C is attributed to the melting of the compound.

3.5. Optical Study:

Todetermine the optical band gapof 3,4-DAPP, UV-vis diffuse reflectance was carried out. Figure 7 shows the variation of the reflectance (%) of the studied compound according to a function of the incident wavelength λ. The reflectance spectrum revealsthat the material exhibits strong absorption in the UV region, particularly below 400 nm, highlighting significant electronic transitions. Beyond 600 nm, the reflectance increases and stabilizes, suggesting higher reflectivity in the visible and near-infrared regions. The first derivative of the reflectance curve highlights a critical transition at 3.58 eV, corresponding to the material’s band gap. Similarly, the absorbance spectrum (Figure S6) shows strong absorption in the UV absorption, particularly at wavelengths below 300 nm; further indicating key electronic transitions related to the band gap. As the wavelength increases, the absorbance gradually decreases, suggesting that the material becomes more transparent in the visible and near-infrared regions. The first derivative of the absorbance depicted in the inset Figure S6, indicates a significant transition estimated at 3.6 eV, which corresponds to the material’s band gap and aligns with the typical range of large band gaps found in semiconductor materials [30,31]. This wide value is consistent with its high absorbance in the UV range and lower absorbance in the visible spectrum. Generally, the most important criterion for designing an efficient optoelectronic device is a wide band gap to ensure transparency to light in a particular wavelength range.

The CIE chromaticity coordinates (x=0.4426, y=0.3718) indicate that it emits green-yellow light close to the white point on the CIE diagramshown in Figure 8, suggesting a balanced emission across the visible spectrum. This position is near the white pointindicating lower color purity, meaning the emitted color is likely a mix of wavelengths and the color is relatively desaturated, resembling a soft, light pastel shade rather than a fully vibrant hue.These analyses suggest that the materialis characterized by strong UV absorption and high reflectance in the visible spectrummaking it potentially suitable forapplications in optoelectronics, UV-sensitive devices, or coatings, that require strong UV absorption and high visible reflectivity.

3.6. DFT Analysis:

In order to reach more accurate assignments of the observed features in the context of molecular orbital, we optimized a deeper analysis for the geometry of 3,4-DAPP with the density functional theory (DFT) using a 6–31G+ basis set.The electron transition from the highest occupied molecular orbital (HOMO) to the lowest unoccupied molecular orbital (LUMO) creates a gap energy, and this latter is a critical parameter that determines the kinetic stability, chemical reactivity, optical polarizability, and chemical hardness-softness of the molecule [32].According to the analysis of the orbital contribution, the HOMO orbital is localizedmainly on the organic molecule (3,4-DAP) and partly on the perchlorate anions, while the LUMO orbital is located on the cationic part.The energies of HOMO and LUMO are -6.6957 and -2.3922eV, respectively, and the energy gap between these two orbitals is 4.3 eV (Figure 9), which is worth noting that the DFT results are appropriate as compared to the experimental value obtained by photoluminescence andsuggesting that the title compound indicate high chemical hardness and kinetically stable [33].As a matter of fact, this outcome indicates that this compound is a good candidate for a semiconducting device.

3.7. In-Vitro Antibacterial Results

The antimicrobial activity of the compound 3,4 DAPP was evaluated against gram-positive (Staphylococcus aureus, Bacillus cereus, and Bacillus subtilis) and gram-negative bacteria (Pseudomonas aeruginosa, Enterococcus faecalis and Salmonella typhimurium) by the agar well diffusion method (as shown in Figure 10). The 3,4-diaminopyridine (3,4-DAP) and gentamicin antibiotic were used as controls. The inhibition zone depends on the tested compound and the tested bacteria. Indeed, we demonstrated that 3,4-DAPP (50 mg/ml) showed an inhibition zone of about 10 mm slightly higher than 3,4-DAP (7 mm) towards Bacillus cereus. This activity disappears when we increase the concentration to (100 mg/ml) of both 3,4-DAPP and the 3,4-DAP. In addition, we demonstrated that 3,4-diaminopyridine (50 mg/ml) showed an inhibition zone of about 10 mm and 9 mm for both Staphylococcus aureus and Bacillus subtilis, respectively. These inhibition zones increased when we used the concentration of 100 mg/ml. These results prove the effectiveness of 3,4-DAP against these 2 bacteria. Unfortunately, the addition of the perchlorate group in 3,4-DAPP reduced this activity since we didn’t find any inhibition zone of 3,4 DAP against Staphylococcus aureus and Bacillus subtilis (Table 2, Figure 10). On the other hand, the 3,4-diaminopyridine (50 mg/ml) was active against Enterococcus faecalis (14 mm) and Pseudomonas aeruginosa (14 mm), this activity decreases to 8 mm and disappears with the 3,4-DAPP compound (50 mg/ml) towards these two bacteria, respectively.This result further confirms that the addition of the acid group in the newly synthesized molecule was ineffective against Enterococcus faecalis and Pseudomonas aeruginosa[34]. Moreover, both 3,4 DAPP and 3,4-diaminopyridine are inactive against Salmonella thyphi. The gentamicin used as positive control showed an important inhibition zone of about 32-40 mm (Table 2, Figure 10). Looking forward, our perspectives include expanding our analysis to test our compound against Alzheimer's disease, exploring its potential as a therapeutic agent for neurodegenerative conditions, and further understanding its mechanism of action in different biological contexts.

4. Conclusions

In this reported research, we prepared and characterized a new non-centrosymmetric hybrid compound based on aminopyridine derivate (3,4-diaminopyridin) and perchlorate salt. The crystallographic data show that this compound crystallizes in the monoclinic system with the space group P2₁ and the supramolecular structure isstabilized by weak hydrogen interactions (H$\cdots$O).In addition, the volume fraction occupied by cavities voids indicates the mechanical strength of the structure. Thermal studies (TGA) and DSC were used to confirm the composite phase transition and the high thermal stability, while the opticalproperties indicate the green-yellow luminescence characteristic.Furthermore, the molecular orbitals studied by DFT theory confirm the semiconducting properties of the studied compound. Additionally, Biological activity tests revealed that 3,4-DAPP exhibited moderate antibacterial activity, particularly against Bacillus cereus, but was less effective than 3,4-DAP against other bacteria, suggesting the perchlorate group may reduce its antimicrobial efficacy.