New Bioprecursor Prodrugs of Sulfadiazine: Synthesis, X-ray Structure and Hirshfeld Analysis

: Sulphonamide motif is found extensively in numerous chemotherapeutic drug candidates, it acts by stopping the production of folate inside the bacterial cell. Current research has established the synthesis and characterization of new bioprecursor prodrugs of sulfadiazine. The ﬁrst prodrug, 3, was synthesized via the coupling of diazonium salt of sulfadiazine with ethyl acetoacetate in AcONa at 0 ◦ C. The second prodrug, sulfadiazine-pyrazole, 5 , was furnished via cyclocondensation of the hydrazono derivative, 3 , and 2-pyridyl hydrazine, 4 . The generated data from the X-ray analysis is interpreted and reﬁned to obtain the crystal structure of the target compound, 5 . Density functional theory (DFT) method was used to calculate the optimized geometrical parameters, electronic state (HOMO–LUMO), and the electronic properties. Moreover, Hirshfeld analysis revealed that the most important contributions to the crystal packing of the prodrug 5 are H ··· H, O ··· H and H ··· C contacts.


Introduction
Sulfonamides inhibit bacterial folate biosynthesis and have been extensively used as broad-spectrum antimicrobials for decades, making use of the different metabolic pathways of microbial and human cells. Nonetheless, bacteria invariably develop resistance to any introduced therapy and only drug combinations, currently used in clinic, can effectively combat the multidrug-resistant (MDR) [1]. Inherent and developed resistance modes of bacteria to antibiotics are problems in the design of new drugs. In the era of bacterial resistance, a prodrug strategy can be employed that requires bacterium-specific enzymes to release the active drug at the infection site. Therefore, targeting the prodrugs to a specific enzyme has potential as a selective drug delivery system in microbial chemotherapy [2,3]. Additionally, prodrugs may enhance the pharmacological activity or pharmacokinetic properties of a parent drug molecule.
The azo group/sulphonamide hybrid structure was the first well-organized chemotherapeutic agent that could be applied efficiently for the treatment of infections caused by bacteria in humans. Prontosil (I, Figure 1) was recognized as a bioprecursor to the active compound, sulfanilamide, possibly metabolized by azoreductases released either in the liver or by gut microbiota [4,5]. Azoreductases are flavoenzymes that have been distinguished in a range of prokaryotes and eukaryotes [6]. Gaffer et al. [7] explored the synthetic sized dyes were investigated for their anti-bacterial and anti-fungal activities against Gram-positive and Gram-negative bacteria, as well as a fungi (C. albicans).
Sulfadiazine (2-sulfanilamidopyrimidine) is used to a large extent for the elimination of bacteria that cause urinary tract infections. It is also used in combined therapy with pyrimethamine and folinic acid for the treatment of some parasitic diseases such as malaria and toxoplasmosis. Silver sulfadiazine is an efficient prescription for burn and wounds treatment [1]; in addition, it has been prescribed for treating bacterial infections, e.g., otitis media, encephalitis, and severe meningococcal meningitis, besides its role as a prophylactic treatment for rheumatic fever. Sulfadiazine targets the dihydropetroate synthase (DHPS), producing a bacteriostatic effect, with a wide spectrum against most Gram-positive and many Gram-negative organisms [1,8]. As a continuation to our research on sulfa drugs [9][10][11], our current research focus is on sulfadiazine [12], a model scaffold to discover new sulfadiazine bioprecursors. Herein, novel models have been designed which feature similarities between our target compounds and lead compounds, as clarified in Figure 1. They are synthesized and characterized by microanalytical analyses, 1 HNMR and 13 CNMR. Particularly, sulfadiazine-pyrazole prodrug 5 is further investigated via X-ray single crystal diffraction which provides the screening, testing, and complete data collection. Furthermore, density functional methods (DFT) will be applied to achieve a valuable understanding of the electronic and molecular properties of the target 5.

Materials and Equipments
All materials and instruments are given in Supplementary materials.

Synthesis of (E)-4-(2-
Synthesis of compound 3 was performed according to the method reported in the literature as described in Supplementary materials [12]. Then, a solution of the hydrazino 3 (1.0 mmol) in absolute EtOH (10 mL), 2-hydrazinopyridine 4 (1.2 mmol) was added, Sulfadiazine (2-sulfanilamidopyrimidine) is used to a large extent for the elimination of bacteria that cause urinary tract infections. It is also used in combined therapy with pyrimethamine and folinic acid for the treatment of some parasitic diseases such as malaria and toxoplasmosis. Silver sulfadiazine is an efficient prescription for burn and wounds treatment [1]; in addition, it has been prescribed for treating bacterial infections, e.g., otitis media, encephalitis, and severe meningococcal meningitis, besides its role as a prophylactic treatment for rheumatic fever. Sulfadiazine targets the dihydropetroate synthase (DHPS), producing a bacteriostatic effect, with a wide spectrum against most Gram-positive and many Gram-negative organisms [1,8].
As a continuation to our research on sulfa drugs [9][10][11], our current research focus is on sulfadiazine [12], a model scaffold to discover new sulfadiazine bioprecursors. Herein, novel models have been designed which feature similarities between our target compounds and lead compounds, as clarified in Figure 1. They are synthesized and characterized by microanalytical analyses, 1 HNMR and 13 CNMR. Particularly, sulfadiazine-pyrazole prodrug 5 is further investigated via X-ray single crystal diffraction which provides the screening, testing, and complete data collection. Furthermore, density functional methods (DFT) will be applied to achieve a valuable understanding of the electronic and molecular properties of the target 5.

Materials and Equipments
All materials and instruments are given in Supplementary materials.

X-ray Structure Analyses
The accomplished utilizing of 5 was determined using the method described in Supplementary data [13][14][15][16]. The crystal data are given in Table 1. Analysis of the crystal packing was accomplished utilizing using Crystal Explorer 17.5 program [17].

Chemistry
The facile synthetic protocol was performed for preparation of new sulfadiazine prodrug 5 (Scheme 1). Reaction of the sulfadiazine 1 with the solution of NaNO 2 to accomplish the diazotization step at 0 • C at pH = 2-3 afforded the corresponding diazonium salt 2 as a clear solution. The coupling of 2 with the previously prepared carbanion salt of ethylacetoacetate at 0 • C gave the desired hydrazono-ethylacetoacetate 3. Confirmation of the correct structure 3 was accomplished by the spectroscopic analysis where 1 HNMR provided two separate peaks at δ H : 11.72 and 11.53 ppm correspond to two N-H protons, as well as two non-homotopic protons signals at 4.26 and 1.22 ppm representing two types of ethyl protons. Furthermore, the singlet signal at δ H : 2.38 revealed to CH 3 CO; 13 CNMR spectra of 3 showed two different types of C=O at δ C : 194.5 and 162.7 beside two different signals at δ C 25.7, 14.1 ppm revealed to carbons of two non-homotopic CH 3 . Cyclocondensation of the prodrug 3 with 2-hydrazinopyridine 4 was accomplished by heating in absolute EtOH affording the desired sulfadiazinepyrazolo prodrugs derivative 5. Confirmation of the correct structure of the sulfonamide 5 was accomplished by the spectroscopic analysis where 1 HNMR gave characteristic signals at δ H : 13.03 and 11.81 ppm revealed to two different sets of NH protons beside one signals in the aliphatic region at δ H : 2.23 ppm representing to CH 3 also 13 C NMR of 5 showed 3 types of carbonyl carbons at δ C : 158.9, as well one signal in aliphatic region at δ C 12.2 ppm corresponded to CH 3 carbons.
HNMR provided two separate peaks at δH: 11.72 and 11.53 ppm correspond to two N-H protons, as well as two non-homotopic protons signals at 4.26 and 1.22 ppm representing two types of ethyl protons. Furthermore, the singlet signal at δH: 2.38 revealed to CH3CO; 13 CNMR spectra of 3 showed two different types of C=O at δC: 194.5 and 162.7 beside two different signals at δC 25.7, 14.1 ppm revealed to carbons of two non-homotopic CH3. Cyclocondensation of the prodrug 3 with 2-hydrazinopyridine 4 was accomplished by heating in absolute EtOH affording the desired sulfadiazinepyrazolo prodrugs derivative 5. Confirmation of the correct structure of the sulfonamide 5 was accomplished by the spectroscopic analysis where 1 HNMR gave characteristic signals at δH: 13.03 and 11.81 ppm revealed to two different sets of NH protons beside one signals in the aliphatic region at δH: 2.23 ppm representing to CH3 also 13 C NMR of 5 showed 3 types of carbonyl carbons at δC: 158.9, as well one signal in aliphatic region at δC 12.2 ppm corresponded to CH3 carbons. Scheme 1. Synthesis of the target prodrugs 3 and 5.

The Description of X-ray Structure of 5
The X-ray structure of 5 shown in Figure 2A agreed very well with its spectral data. It crystallized in the monoclinic C2/c space group. There are eight molecules per unit cell and one molecular unit as an asymmetric formula. The unit cell parameters are a = 21.8909(2) Å, b = 11.45860(10) Å, c = 15.77880(10) Å, β = 99.0430(10)° and V = 3908.74(6) Å 3 . Selected geometric parameters are depicted in Table 2. The pyridyl and the five membered rings are connected to one another by C(5)-N(2) bond where both rings are twisted from one another by only 6.45°. On the other hand, the phenyl moiety is found twisted further from the five membered ring mean plane. The twist angle in this case is 23.30°. Scheme 1. Synthesis of the target prodrugs 3 and 5.

The Description of X-ray Structure of 5
The X-ray structure of 5 shown in Figure 2A agreed very well with its spectral data. It crystallized in the monoclinic C2/c space group. There are eight molecules per unit cell and one molecular unit as an asymmetric formula. The unit cell parameters are a = 21.8909(2) Å, b = 11.45860(10) Å, c = 15.77880(10) Å, β = 99.0430(10) • and V = 3908.74(6) Å 3 . Selected geometric parameters are depicted in Table 2. The pyridyl and the five membered rings are connected to one another by C(5)-N(2) bond where both rings are twisted from one another by only 6.45 • . On the other hand, the phenyl moiety is found twisted further from the five membered ring mean plane. The twist angle in this case is 23.30 • .

Hirshfeld Surface Analysis
The Hirshfeld calculation is a simple and accurate tool for the finding the different atom-atom contacts in the crystal structure. Hence, decomposition of the different intermolecular contacts in the crystal structure of 5 was performed using Hirshfeld calculations. The resulting Hirshfeld maps are presented in Figure 3. There are different levels of inter-molecular contacts as indicated from the presence of red, white, and blue regions in the d norm map.

Hirshfeld Surface Analysis
The Hirshfeld calculation is a simple and accurate tool for the finding the different atom-atom contacts in the crystal structure. Hence, decomposition of the different intermolecular contacts in the crystal structure of 5 was performed using Hirshfeld calculations. The resulting Hirshfeld maps are presented in Figure 3. There are different levels of intermolecular contacts as indicated from the presence of red, white, and blue regions in the dnorm map. Analysis of these interactions using fingerprint plot is given in Figure 4. The fingerprint area gave the percentages of all intermolecular interactions in the crystal structure of 5. Presentation for these interactions and the percentages for all contacts in 5 is shown in Figure 5. The percentages of the H···H, H···C, N···H, and O···H interactions are 36.4, 12.2, 17.3, and 16.9%, respectively. It is worth noting that the N···H and O···H contacts have small interaction distances (Table 4). In addition, the presence of π-π stacking interactions is revealed by the presence of short C1···C15 contact (3.211 Å) and the presence of red/blue triangles in the shape index map and flat green area in curvedness ( Figure 3). Analysis of these interactions using fingerprint plot is given in Figure 4. The fingerprint area gave the percentages of all intermolecular interactions in the crystal structure of 5. Presentation for these interactions and the percentages for all contacts in 5 is shown in Figure 5. The percentages of the H···H, H···C, N···H, and O···H interactions are 36.4, 12.2, 17.3, and 16.9%, respectively. It is worth noting that the N···H and O···H contacts have small interaction distances (Table 4). In addition, the presence of π-π stacking interactions is revealed by the presence of short C1···C15 contact (3.211 Å) and the presence of red/blue triangles in the shape index map and flat green area in curvedness (Figure 3).

DFT Studies
The minimum energy structure of 5 is shown in Figure 6. Its overlay with the X-ray geometry is shown in the same figure. Generally, there is structural matching between both structures and also good correlations between the optimized and X-ray geometric parameters (Figure 7) which reveal these observations very well (Table S1, Supporting information).

DFT Studies
The minimum energy structure of 5 is shown in Figure 6. Its overlay with the X-ray geometry is shown in the same figure. Generally, there is structural matching between both structures and also good correlations between the optimized and X-ray geometric parameters (Figure 7) which reveal these observations very well (Table S1, Supporting information).

DFT Studies
The minimum energy structure of 5 is shown in Figure 6. Its overlay with the X-ray geometry is shown in the same figure. Generally, there is structural matching between both structures and also good correlations between the optimized and X-ray geometric parameters ( Figure 7) which reveal these observations very well (Table S1, Supporting information).
Natural charges of 5 shown in Figure 8 Figure 9. The molecule has a net dipole moment of 4.9143 D.   Figure 9. The molecule has a net d 4.9143 D.   Among electronic parameters which play an important role are the HOMO and LUMO levels. These molecular orbitals are shown in Figure 9. It is clear that both are distributed over the π-system of the molecule. Hence, the HOMO→LUMO excitation is mainly a π-π* transition. Their energies are calculated to be −6.1158 and −2.7671 eV, respectively, and the HOMO-LUMO gap is 3.3487 eV. As a result, the reactivity indices [21][22][23][24][25][26][27] such as ionization potential (I), electron affinity (A), hardness (η), electrophilicity index (ω) and chemical potential (µ) are calculated to be 6.1158, 2.7671, 3.3487, 2.9455 and −4.4415 eV, respectively. Among electronic parameters which play an important role are the HOMO and LUMO levels. These molecular orbitals are shown in Figure 9. It is clear that both are distributed over the π-system of the molecule. Hence, the HOMO→LUMO excitation is mainly a π-π* transition. Their energies are calculated to be −6.1158 and −2.7671 eV, respectively, and the HOMO-LUMO gap is 3.3487 eV. As a result, the reactivity indices [21][22][23][24][25][26][27] such as ionization potential (I), electron affinity (A), hardness (η), electrophilicity index (ω) and chemical potential (μ) are calculated to be 6.1158, 2.7671, 3.3487, 2.9455 and −4.4415 eV, respectively.

Conclusions
The novel bioprecursor prodrugs of sulfadiazine have been synthesized and evidenced through the elemental and spectral analyses "FT-IR, 1 HNMR, 13 CNMR, and MS". More structural elucidations of the prodrug 5 were determined via X-ray and its supramolecular structure aspects were analyzed using Hirshfeld calculations. Additionally, the natural charge distribution, dipole moment, HOMO, LUMO, and MEP map of 5 were analyzed based on B3LYP/6-31G (d,p) calculations. Its structural aspects were investigated using DFT and NBO calculations.