Pyrido-pyrimido-thiadiazinones: green synthesis, molecular docking studies and biological investigation as obesity inhibitors

Obesity is a global public health concern brought on by a combination of excessive dietary intake, inactivity and genetic predisposition. Pyridopyrimidines have received considerable interest in the development of obesity and diabetes. Mannich reaction was applied on 2-thioxo-pyridopyrimidinone derivative with a variety of aromatic and heterocyclic aromatic amines and formaldehyde by grinding at 25°C in the presence of HCl to obtain 18 fused thiadiazinones and two bis-fused thiadiazinone derivatives. Additionally, in silico docking was performed to investigate the mode of actions of the 20 synthesized compounds against fat mass and obesity-associated (FTO) protein. The ligand molecules are nicely docked to the target FTO with binding energies (ΔGbind) ranging from −11.6 to −8.0 kcal/mol. Electrostatic potential map of semiempirical optimized compound 16 was performed using Gaussian 03. Interestingly, two bis-fused thiadiazinone derivatives showed marked amelioration of adiposity in the high-fat diet model especially in hepatic and adipose tissues.


Introduction
Obesity is an international public health threat which caused by a combination of excess of food intake with lack of genetic susceptibility and physical activity [1][2][3]. In addition, the expression fat mass and obesityassociated (FTO) gene is correlated with regulation of food intake. By altering the m6A level of hormones associated to eating, FTO protein contributes to the development of obesity [2,3]. Therefore, FTO protein was chosen as a therapeutic target for identification of anti-obesity agents using molecular docking approach.
The wide range of biological and pharmacological activities of pyridopyrimidines, including their application as anti-inflammatory, anti-folate, antiviral, antibacterial and anticancer agents, has garnered considerable attention in recent years [4][5][6][7][8][9]. Also, pyridopyrimidines are used as inhibitors for hepatitis B virus, PDE IV and tyrosine kinase [9][10][11]. Furthermore, they also play a crucial role in the metabolism of fatty acids, carbohydrates and amino acids [12][13][14][15][16]. As a result, they are implicated in the treatment of diseases such as type 2 diabetes and obesity [17]. They have received considerable interest in the development of therapeutics for a variety of human diseases, including obesity, diabetes, metabolic syndrome, microbial infections and cancer [18,19], as shown in Figure 1.
The Mannich reaction is a crucial step in the synthesis of numerous bioactive compounds and is frequently utilized in the production of secondary and tertiary amine derivatives [6,20,21]. A variety of prospective pharmacological properties, such as antifungal, antitubercular, anticancer, neuroprotection and antibacterial activity, are displayed by N-Mannich bases generated from NH-heterocycles and related compounds [22][23][24][25].
Bioactive chemicals have been created via multicomponent reactions (MCRs), which combine several reactants in a single vessel [26][27][28][29][30][31][32][33]. They are crucial for creating structural diversity and libraries of drug-like compounds in industrial, synthetic, green and medicinal chemical applications [34]. Therefore, synthetic organic chemists are paying close attention to the discovery of new MCRs and/or the improvement of established MCRs for the synthesis of novel molecules [35]. An increasing consensus is focused at carrying out organic reactions in solvent-free reaction conditions due to rigorous environmental issues and green chemistry [36]. These reactions are growing in popularity as a result of their numerous benefits, including cost savings, decreased energy usage, sped-up reaction times, significant reactor size reductions and reduced capital expenditure, making the reactions cleaner, safer and simpler in this way. The explicit statement made by Sheldon [37] that "The ideal solvent (diluent) is water, while the best solvent is none at all" is significant. Additionally, solvent-free processes' environmental factor (E Factor) makes them excellent candidates for clean technology [38].
In this vein, we have been working on a programme for the past 10 years that aims to multicomponent one pot synthesize some new heterocyclic structures of biological interest [28][29][30][31][32][39][40][41][42][43][44][45][46][47] to identify new drug candidates which can be used as anti-obesity inhibitors, through interaction with the target protein, as represented in Figure 2. Additionally, the molecular docking studies and biological activity of the tested compounds were performed. Finally, the physiochemical and pharmacokinetics parameters of the best docked derivatives are calculated using free website tools.

Chemistry
2-Thioxo-pyridopyrimidinone derivative 1 [48] reacts with the aromatic amines 2a-m and two equivalents of formaldehyde 3 via the Mannich reaction conditions; grinding at 25°C in the presence of HCl to afford fused thiadiazinone derivatives 4a-m, respectively (Scheme 1).
Finally, compounds 1 (2 equivalents) and 3 (4 equivalents) were allowed to react with one equivalent

Docking study
In the present study, FTO protein is associated reproducibly with human body mass. Therefore, FTO protein (PDB ID: 3LFM) was chosen as a therapeutic target for identification of anti-obesity agents. Herein, molecular docking approach [49][50][51][52][53][54][55][56][57][58] of the newly synthesized compounds 4-18 was performed for identifying new obesity inhibitors. The molecular docking software PyRx was used to calculate the binding affinity of the prepared compounds to the active site pocket of the target. The 3D binding modes of intermolecular interactions between the compounds with highest affinities 16 and 18 with the target are represented in Figure 3. Table  1 showed the docking affinities ( G bind ) of the best docked complexes. The ligand molecules were successfully docked to the active site residues of the target through a network of non-covalent interactions such as H-bonding and/or π stacking as shown in Figure 1. Upon the docking simulation, it has been shown that all compounds are nicely docked to the target FTO, with binding energies ( G bind ) ranging from −11.6 to −8.0 kcal/mol.
The ligand molecule 16 with binding energy (−11.6 kcal/mol), docked to the target protein through one arene-cation interactions and one hydrogen bonds with the residues Lys216 and Leu91 at 5.90 and 1.90 Å, respectively. In addition, compound 18 docked to the target through one hydrogen bond with the residue Asn101 at 2.98 Å.
Redocking of 3-methylthymidine (co-crystalized ligand) was performed to validate the docking process (RMSD < 2 Å) and the obtained results showed similar fitness to the docked compounds. In addition, the electrostatic potential map of semiempirical was performed to investigate the molecular interactions of  the best docked compound 16 to the target. The 3D electrostatic potential map reflects the arene-cation interaction between phenyl moiety of compound 16 and the lysine residue, as shown in Figure 4. Further, by applying Lipinski rule of five (Ro5) to these ligand molecules, the best compounds 16 and 18 obeyed Ro5, as tabulated in Table 2. Furthermore, the absorption rates of these compounds exceeded 99%, indicating that these compounds possess excellent absorption and distribution properties. Moreover, the results have presented that they are non-toxic and non-carcinogenic. These results shed light on bis-fused thiadiazinones 16 and 18 as potent anti-obesity drug candidates.

Histopathological findings of specific organs (liver and adipose tissue)
The liver of control animal showed normal hepatic parenchyma. The hepatocytes were arranged in cordslike manner around the central vein and with normal portal structures. Each hepatocyte was separated with hepatic sinusoids with few Kuffer's cells. The liver of the adiposity group showed marked granular hepatic cell swelling with marked intrahepatic fatty changes associated with clear and round vacuoles mostly. The sinusoids showed moderate congestion with prominent Kuffer's cell. The diseased groups treated with bisfused thiadiazinones 16 and 18 demonstrated marked decrease in vascular congestion and hepatic steatosis ( Figures 5 and 6). The adipose connective tissue of control animal showed normal small adipocytes separated with mar ked connective tissue septa. The diseased groups showed marked enlarged distend adipocytes with fat and most of them showed loss of their membrane. The diseased groups treated with bis-fused thiadiazinones 16 and 18 showed marked decrease in the fat cells of the adipocytes with decrease in their diameter. Quantitative estimation of the diameter of the fat cells showed marked increase in their diameter in adiposity group in comparison with control groups (P < 0.05). While the treated groups with bis-fused thiadiazinones 16 and 18 revealed marked decrease in the diameter of the fat cells in comparison with adiposity group (P < 0.05) ( Figures  7 and 8).

Chemistry
On an electrothermal device, all melting points were calculated without being adjusted. GCMS-Q1000-EX Shimadzu spectrometers were conducted to record the mass spectra of the samples on the ionizing voltage at 70 eV. On the BRUKER 400 FT-NMR system spectrometer, 1 H-and 13 C-NMR spectra (500 MHz for 1 H-NMR) were captured in DMSO solutions. On KBr discs, IR spectra were captured using a Shimadzu FT-IR 8201 PC spectrophotometer.

Molecular docking
In the present study, fat and obesity protein (FTO protein) is selected as therapeutic target for identifying potential inhibitors for obesity. All newly synthesized compounds 4-18 are subjected to molecular docking by calculating the minimum energy to inhibit the target protein. The protein structure (PDB ID: 3LFM) is downloaded from RCSB protein databank [59]. Ligand molecules are sketched using ChemDraw ultra 8.0 (in cdx format) and converted using Open Babel 2.4.1 (SDF format) [60]. All the compound structures and the target are subjected to energy minimization using universal force field [61] in Open Babel and CHARMM Force Field [62] in Discovery Studio, respectively. PyRx-Virtual screening tool [63] is used to perform the molecular docking. Electrostatic potential map of PM6 semiempirical method was performed using Gaussian 03. Finally, the computational study for prediction of ADMET and physicochemical properties of the best docked compounds are calculated through AdmetSAR (http://lmmd.ecust.edu.cn/admetsar2/) and Mol inspiration (https://www.molinspiration.com) softwares.

Experimental animal study
White male albino rats (Wistar rat) (Rattus norvegicus), with age 10 weeks and about 120-160 g body weight.
The used animals were collected from Faculty of Veterinary Medicine, Cairo University, Egypt.
Adult rats were kept in Lab Animal Experimental Facility, University of South Valley, under acclimatization for 1 week before experimentation. The animals were (3) rats per cage, kept at a temperature of 23 ± 2°C, with acceptable ventilation and a relative humidity of 50 ± 5%. The rats were fed either a regular mouse pellet diet or a high-fat diet (HFD), with consistent light/dark cycles lasting 12 h each [64]. They were allowed to drink ad libitum during the whole course of the experiment.
Animals were divided into four groups, G1 (control group), G2 (HFD group) injected with 35 mg/kg STZ, IP and supplemented with HFD composed as follow: Powdered NPD (365 mg), Lard (310 mg), Vitamin and mineral mix (60 mg), Casein (250 mg), Cholesterol (10 mg), dl-Methionine (3 mg), Yeast powder (1 mg) and Sodium chloride (1 mg) for 24 weeks. The two further groups were subjected to both STZ and HFD and treated with bis-fused thiadiazinones 16 (G3) and 18 (G4). Our protocol complies with Faculty of Science, South Valley University for the Care and Use of Laboratory Animals.

Histopathology technique
Liver and adipose samples were collected and fixed in 10% neutral buffered formalin. After dehydration and clearance, the tissues were fixed in paraffin and sectioned in 5 µm thickness. The serial sections were subjected to staining with haematoxylin and eosin [65]. Scoring of hepatic steatosis was done according to [66], wherein the amount of fat contained within each hepatocyte was graded: grade 0 (healthy, < 5%), grade 1 (mild, 5-33%), grade 2 (moderate, 34-66%) and grade 3 (severe, > 66%). The diameter of fat cell was measured using ImageJ analysis software, NIH, USA and expressed as mean ± SD.

Statistical analysis
All data were expressed as mean ± standard deviation. ANOVA test (one-way analysis of variance) was carried out with Tukey's post hoc analysis using Graph-Pad Prism 5 (GraphPad Software for Windows Inc., San Diego, CA, United States) and p value < 0.05 was considered to be statistically significant.

Conclusion
During the current investigation, we synthesized a new fused thiadiazineone derivatives 4a-m, 6, 8, 10, 12 and 14 via grinding technology starting from the corresponding thione 1 and a variety of aromatic amines or heterocyclic amines and formaldehyde, and their structural and spectral data were elucidated. Furthermore, the docking approach was carried out to investigate the mode of interactions of the compounds 4-18 against FTO protein. The ligand molecules are nicely docked to the target FTO, with docking affinities ( G bind ); −11.6 to −8.0 kcal/mol. Finally, the biological activity of the tested compounds were in line with in silico results, wherein they exhibited that bis-fused thiadiazinones 16 and 18 revealed marked amelioration of adiposity in the HFD model especially in hepatic and adipose tissues; making them as possible anti-obesity agents.

Disclosure statement
No potential conflict of interest was reported by the author(s).