Abstract
Heterocyclic compounds play a crucial role in the drug discovery process and development due to their significant presence and importance. Here, we report a comprehensive analysis of α-aminophosphonates containing pyridine (3a–g), prepared according to a clear-cut, uncomplicated procedure. The phosphonates are thoroughly characterized using various methods, such as elemental analysis, mass spectrometry, proton and carbon NMR, and FT-IR. The molecular docking interactions between the phosphonate and DRP-1 target protein observed that compound 3d had the top-ranked binding energy towards DRP-1 with a value equal to − 9.54 kcal/mol and this theoretically proves its inhibitory efficacy against DRP-1 arbitrated mitochondrial fission. Besides, the anticancer characteristics of compound 3d showed the best IC50 against HepG-2, MCF-7, and Caco-2 which confirmed our results towards suppressing DRP-1 protein (in-silico), and it elucidated no cytotoxic effects against human normal cell line (WI-38). Further, its pharmacokinetics were observed theoretically using ADMET. Moreover,compound 3d investigated the most potent antimicrobial ability against two pathological fungal strains, A. flavus and C. albicans, and four bacterial strains, E. coli, B. subtillis, S. aureus, and P. aregeunosa. Additionally, compound 3d clarified a powerful antioxidant scavenging activity against DPPH and ABTS free radicals (in-vitro). Furthermore, Density functional theory (DFT) was used to study the molecular structures of the synthesized compounds 3a–g, utilizing 6–311++G(d,p) as the basis set and to learn more about the molecules’ reactive sites, the energies of the molecular electrostatic potential (MEP), the lowest unoccupied molecular orbital (LUMO), and the highest occupied molecular orbital (HOMO) were observed. Theoretically, FT-IR and Nuclear magnetic resonance (NMR) measurements are calculated for every compound under investigation to show how theory and experiment relate. It was found that there was an excellent agreement between the theoretical and experimental data. Conclusively, all novel synthesized phosphonates could be used as pharmaceutical agents against pathogenic microbial strains and as anticancer candidates by inhibiting DRP-1-mediated mitochondrial mitophagy.
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Introduction
The development of heterocyclic chemistry and its effective application in medicinal chemistry transformed the process of finding new drugs. Organophosphorus compounds have attracted intensely expanding interest and exciting applications in agricultural and industrial fields during the past few decades as an important class in organic synthesis, medicinal chemistry, and biological activities [1,2,3]. The α-aminophosphonates, also known as bio isosteres of amino acids, are extensively explored as fascinating organophosphorus derivatives [4]. They possess promising biological and pharmacological potentials as antioxidants [5, 6], antibiotics [7, 8], antiviral [9, 10], antitumoral [9, 11], and anti-inflammatory [6].
Furthermore, the pyridine moiety is crucial in finding novel medications. It is present in many medically relevant chemicals utilized in the pharmaceutical industry, such as anesthetics, prodrugs to cure neuronal damage, and anti-cancer, and anti-inflammatory medications for some brain illnesses [12]. Notably, the pyridine derivatives with amino, chloro, and trifluoromethyl groups have been frequently reported for their anticancer activity [13]. As a result, numerous heterocyclic nuclease 2-amino pyridine derivatives have demonstrated strong pharmacological effects [14,15,16,17,18]. As per the above summary, certain pharmaceutical companies employed pyridine and phosphonates as efficacious medications, including, Vismodegib was approved by the US Food and Drug Administration (FDA) for the treatment of basal cell carcinoma (BCC) [19], Crizotinib, a small-molecule kinase inhibitor, which the (we) FDA approved for the treatment of patients with locally advanced or metastatic non-small-cell lung cancer [20], Fostamatinib, is a spleen tyrosine kinase inhibitor used to treat chronic immune thrombocytopenia [21], and Risedronate, which is an effective and well-tolerated therapy for the treatment of postmenopausal osteoporosis [22], Fig. 1.
Biomedical drugs containing pyridine and phosphonic acidscaffolds
It is well-established that mitochondrial dynamics plays a significant part in age-related disorders, such as cancer. Nevertheless, studies on mitochondrial dynamics and cancer are still in their early stages of discovery. Organelles called mitochondria are involved in various essential cellular processes, including the production of adenosine triphosphate (ATP), the processes by which cells divide and differentiate, the anabolic and catabolic functions of cells, and the breakdown of cells. In response to physiologic or stress-related stimuli, mitochondria modify their structure and function, which are closely related [23]. The important chemicals and mechanisms that influence or cause some of these structural and functional alterations have been the subject of extensive research in recent years. Mitochondrial fission and fusion occur in both normal cells and dysregulated cells, like cancer cells. These events may be the most significant of these structural alterations. Enough mitochondria are produced via mitochondrial fission to facilitate the division and growth of cells. In addition to producing new organelles, mitochondrial fission serves as a quality control mechanism by removing defective mitochondria via a process known as mitophagy. On the other hand, when mitochondria must rely on oxidative phosphorylation or respond to stress stimuli, they must fuse to produce the maximum amount of ATP. In these situations, they manifest as elongated, healthy organelles that complement the malfunctioning mitochondria [24].
The essential part of the mitochondrial fission machinery is dynamin-related protein 1 (Drp-1), a member of the dynamin family of guanosine triphosphatases (GTPases) [25]. Dynamin-related protein 1 has been connected to the emergence of several cancerous tumors. Dynamin-related protein 1 was involved in alterations to cellular metabolism, metastases, sustaining cell cycle, and proliferation. It was also associated with the oncolytic phenotype. Also, Drp-1 affects metabolic regulation in addition to a variety of other cell functions, including apoptosis, mitophagy and mitochondrial biogenesis, cell division, and transformation [26]. Drpitor1a [27], which is a pyridine derivative, is a potent DRP-1 inhibitor that prevents mitochondrial fission, Fig. 1. Another important DRP-1 inhibitor is Mitochondrial division inhibitor 1 (mdivi-1) [28], which contains di chloro substituents that decrease cancer cell proliferation by inducing mitochondrial fusion and altering oxygen consumption, Fig. 1. Mdivi-1 is the currently accepted gold standard FDA inhibitor of Drp-1, discovered by Cassidy-Stone et al. [29]. However, there was many limitations have been raised regarding the mdivi-1 [30, 31]. Therefore, there is an urgent need to find more accessible, nontoxic, potent, and specific Drp-1 GTPase inhibitors to be targeted for cancer treatment. We consider that the presence of pyridine group and dichloro groups in the skeleton of α-aminophosphonates may play a vital role in inhibition of dynamin-related protein 1 (DRP-1).
Computational chemistry advancements and knowledge of biological and biochemical structures and reactions have come together recently to create a comprehensive companion [32]. It has significantly advanced our knowledge of molecular characteristics, structure, and reaction selectivity [32]. In addition, it provides us with essential details about the compounds we are studying, such as total energy, dipole moment, electronic energy, binding energy, bond lengths, HOMO, and LUMO. This information’s high worth rises when it aligns with experimental results. It also aids in our comprehension of how the molecule will probably behave during reactions [33]. Density functional theory (DFT) is the most promising approach for applying computer-based calculations to study the electrochemical characteristics of various substances [6, 11, 33, 34].
Considering these, we attempted to create a novel class of α-aminophosphonates with pyridine nucleus and investigated their antimicrobial, antioxidant, and anticancer impact via elucidatingtheir inhibitory role on DRP-1 mediated mitochondria fission (in-silico and in-vitro). Additionally, using the DFT technique, the effect of modifications to the electrical and molecular structures on the biological activity of freshly synthesized compounds was examined. Further, their bioavailability and drug-likeness were examined using ADMET.
Experimental
Materials and instrumentation
The chemical and instrument data are all contained in the supplementary file (Section S1).
Synthesis of α-aminophosphonate compounds 3a–g
Triethyl phosphite was added together with anhydrous lithium perchlorate LiClO4 (10 mol%) to a stirred mixture of 3,5-dichloropyridin-2-amine 1 (0.01 mol) and suitable aldehyde derivatives 2a–g (0.0012 mol) in dry dichloromethane CH2Cl2. TLC results showed that the reaction was finished after 72–84 h of stirring at room temperature. After that, CH2Cl2 was evaporated, and cold methanol was used to precipitate the α-aminophosphonates. The precipitate was removed by filtering, and then recrystallize from ethanol producing fresh α-aminophosphonates 3a-g in a very good yield.
Diethyl(((3,5-dichloropyridin-2-yl)amino)(4-hydroxy-3-methoxyphenyl)methyl)phosphonate, 3a
Yield (1.11 gm, 85%); 72 h; yellow solid; M.p.: 66–68 °C; TLC: Ethyl acetate: Petroleum ether 1:3); Rf: 0.28; IR: ν/cm−1: 3248 (NH), 3029 (CH–Arom.), 2925 (CH-Aliph), 1254 (P=O), 1086 (P–O–C), 761 (P–CH). 1H NMR (500 MHz, DMSO-d6): δ = 3.66 (s, 3H, OCH3), 1.18 (t, 6H, J = 6.0 Hz, 2xCH3), 3.81–4.02 (m, 4H, 2xCH2), 6.46 (s, 1H, CH–P),6.66(1H, OH, exchangeable with D2O), 7.89 (s, 1H, Hpyridine), 9.74 (s, 1H, Hpyridine), 7.69 (s, 1H, Harom), 6.92 (d, 1H, J = 8.5 Hz, Harom), 7.35 (d, 1H, J = 9.0 Hz, Harom), 10.210 (s, 1H, NH, exchangeable with D2O) ppm. 13C NMR (125 MHz, DMSO-d6): δ 14.23 (2CH3), 62.24 (2CH2), 68.74 (CH–P), 56.35 (O–CH3), 153.49 (C=N pyridine), 154.95 (C–NH), 111.11–148.78 (Ar–C) ppm. MS (EI) m/z: 436 (M+, 3%, C17H21Cl2N2O5P), 298 (M−3, 50%, C13H12Cl2N2), 161(100%, C5H4Cl2N2), 136 (M−2, 28%, C4H11O3P). Anal. Calcd. For C17H21Cl2N2O5P (434.06): C, 46.91; H, 4.86; N, 6.44; P, 7.12. Found;C, 46.88; H, 4.81; N, 6.38; P, 7.11.
Diethyl (((3,5-dichloropyridin-2-yl) amino)(4-hydroxy phenyl) methyl)phosphonate, 3b
Yield (0.97 gm, 80%); 80 h; buff solid; M.p.: 56–58 °C; TLC: Ethyl acetate: Petrolum ether 1:3); Rf: 0.25; IR: ν/cm−1: 3248 (NH), 3029 (CH–Arom.), 2925 (CH–Aliph), 1254 (P=O), 1086 (P–O–C), 761 (P–CH). 1H NMR (500 MHz, DMSO-d6): δ = 1.20 (t, 6H, J = 6.5 Hz, 2xCH3), 3.97–4.03 (m, 4H, 2xCH2), 6.09 (s, 1H, CH–P), 6.47(1H, OH, exchangeable with D2O), 7.89 (s, 1H, Hpyridine), 9.76 (s, 1H, Hpyridine), 6.89 (d, 2H, J = 7.5 Hz, Harom), 7.72 (d, 2H, J = 9.5 Hz, Harom), 10.56 (s, 1H, NH, exchangeable with D2O) ppm.13C NMR (125 MHz, DMSO-d6): δ 27.72 (2CH3), 58.38 (2CH2), 69.91 (CH–P), 155.04 (C=N pyridine), 163.81 (C–NH), 114.29–142.14 (Ar–C) ppm. MS (EI) m/z: 403 (M−, 7%, C16H19Cl2N2O4P), 270 (M+1, 8%, C12H10Cl2N2O), 161(100%, C5H4Cl2N2), 136 (M−2, 28%, C4H11O3P); Anal. Calcd. For C16H19Cl2N2O4P (404.05):C, 47.43; H, 4.73; N, 6.91; P, 7.64.Found;C, 47.40; H, 4.69; N, 6.85; P, 7.62.
Diethyl (((3,5-dichloropyridin-2-yl)amino)(2-methoxyphenyl)methyl)phosphonate, 3c
Yield (1.04 gm, 83%); 72 h; pale yellow solid; M.p.: 73–75 °C; TLC: Ethyl acetate: Petrolum ether 1:3); Rf: 0.23;IR: ν/cm−1: 3248 (NH), 3029 (CH–Arom.), 2925 (CH–Aliph), 1254 (P=O), 1086 (P–O–C), 761 (P–CH). 1H NMR (500 MHz, DMSO-d6): δ = 3.75 (s, 3H, OCH3), 0.97–1.18 (m, 6H, 2xCH3), 3.83–3.99 (m, 4H, 2xCH2), 5.93 (d, 1H, J = 6 Hz, CH–P),7.23 (s, 1H, Hpyridine), 8.04 (s, 1H, Hpyridine), 6.48–7.90 (m, 4H, Harom),10.34 (s, 1H, NH, exchangeable with D2O) ppm.13C NMR (125 MHz, DMSO-d6): δ 17.11 (2CH3), 62.61 (2CH2), 63.89 (CH–P), 56.02 (O–CH3), 144.64 (C=N pyridine), 155.06 (C–NH), 111.12–136.66 (Ar–C) ppm. MS (EI) m/z: MS (EI) m/z: 419 (M+1, 9%, C17H21Cl2N2O4P), 280 (M−2, 100%, C13H12Cl2N2O), 177 (100%, C6H6Cl2N2), 136 (M−2, 87%, C4H11O3P); Anal. Calcd. For C17H21Cl2N2O4P (418.06): C, 48.70; H, 5.05; N, 6.68; P, 7.39.Found;C, 48.68; H, 5.03; N, 6.62; P, 7.35.
Diethyl (((3,5-dichloropyridin-2-yl)amino)(2-hydroxynaphthalen-1-yl)methyl)phosphonate, 3d
Yield (1.16 gm, 85%); 76 h; dark yellow solid; M.p.: 145–147 °C; TLC: Ethyl acetate: Petrolum ether 1:1); Rf: 0.30; IR: ν/cm−1: 3248 (NH), 3029 (CH–Arom.), 2925 (CH–Aliph), 1254 (P=O), 1086 (P–O–C), 761 (P–CH). 1H NMR (500 MHz, DMSO-d6): δ = 1.00, 1.200 (2 × t, 6H, J = 8 Hz, 2xCH3), 3.84–4.02 (m, 4H, 2xCH2), 5.72 (d, 1H, J = 9 Hz, CH–P),7.73 (s, 1H, Hpyridine), 8.91 (s, 1H, Hpyridine), 7.19 (d, 1H, J = 9.5 Hz, Harom), 7.39 (d, 1H, J = 8.2 Hz, Harom), 7.56–8.15 (5H, s, 1H, OH, m, 4H, Harom), 10.78 (br, 1H, NH, exchangeable with D2O) ppm.13C NMR (125 MHz, DMSO-d6): δ = 16.20 (2CH3), 60.22 (2CH2), 70.03 (CH–P), 144.17 (C=N pyridine), 164.23 (C–NH), 112.78–139.14 (Ar–C) ppm. MS (EI) m/z: 455 (M+1, 5%, C20H21Cl2N2O4P), 312 (M+3, 8%, C10H15Cl2N2O3P), 177 (3%, C6H6Cl2N2), 161(27%, C5H4Cl2N2), 146 (M+2, 38%, C10H8O), 136(M−2, 18%, C4H11O3P); Anal. Calcd. For C20H21Cl2N2O4P (454.06):C, 52.76; H, 4.65; N, 6.15; P, 6.80. Found;C, 52.73; H, 4.61; N, 6.12; P, 6.77.
Diethyl (((3,5-dichloropyridin-2-yl)amino)(4-(dimethylamino)phenyl)methyl)phosphonate, 3e
Yield (1.06 gm, 82%); 79 h; red solid; M.p.: 70–72 °C; TLC: Ethyl acetate: Petroleum ether 1:3); Rf: 0.24; IR: ν/cm−1: 3248 (NH), 3029 (CH–Arom.), 2925 (CH-Aliph), 1254 (P=O), 1086 (P–O–C), 761 (P–CH). 1H NMR (500 MHz, DMSO-d6): δ = 1.20 (t, 6H, J = 7 Hz, 2xCH3), 3.97–4.03 (m, 4H, 2xCH2), 6.47 (s, 1H, CH–P),7.89 (s, 1H, Hpyridine), 9.63 (s, 1H, Hpyridine), 6.72 (d, 2H, J = 8.6 Hz, Harom), 7.63 (d, 2H, J = 8.7 Hz, Harom), 2.99 (s, 6H, N(CH3)2) ppm. 13C NMR (125 MHz, DMSO-d6): δ = 16.72 (2CH3), 61.88 (2CH2), 72.06 (CH–P), 44.57 (N–(CH3)2), 144.62 (C=N pyridine), 154.73 (C–NH), 108.70–136.47 (Ar–C) ppm. MS (EI) m/z: 432 (M+1, 3%, C18H24Cl2N3O3P), 295 (M−1, 2%, C14H15Cl2N3), 177 (3%, C6H6Cl2N2), 161(M, 9%, C5H4Cl2N2), 148 (M+2, 100%, C5H3Cl2N), 136 (M−2, 18%, C4H11O3P); Anal. Calcd. For C18H24Cl2N3O3P (431.09): C, 50.01; H, 5.60; N, 9.72; P, 7.17. Found;C, 49.97; H, 5.56; N, 9.70; P, 7.12.
Diethyl (((3,5-dichloropyridin-2-yl)amino)(thiophen-3-yl)methyl)phosphonate, 3f
Yield (1.22 gm, 82%); 76 h; light brown solid; M.p.: 88–90 °C;TLC: Ethyl acetate: Petroleum ether 1:3); Rf: 0.25; IR: ν/cm−1: 3248 (NH), 3029 (CH–Arom.), 2925 (CH–Aliph), 1254 (P=O), 1086 (P–O–C), 761 (P–CH). 1H NMR (500 MHz, DMSO-d6): δ = 1.06–1.21 (m, 6H, 2xCH3), 3.91–3.99 (m, 4H, 2xCH2), 5.18 (d, 1H, J = 6.5 Hz, CH–P), 7.71 (s, 1H, Hpyridine), 8.07 (s, 1H, Hpyridine), 7.89 (s, 1H, Hthiophene), 6.47–7.44 (m, 2H, Harom)ppm. 13C NMR (125 MHz, DMSO-d6): δ = 13.56 (2CH3), 57.45 (2CH2), 63.81 (CH–P), 144.70 (C=N pyridine), 154.09 (C–NH), 99.80–136.79 (Ar–C) ppm. MS (EI) m/z: 393 (M−1, 3%, C14H17Cl2N2O3PS), 256 (M−1, 7%, C10H8Cl2N2S), 161 (M, 23%, C5H4Cl2N2), 137 (M−1, 25%, C4H11O3P). Anal. Calcd. For C14H17Cl2N2O3PS (394.01): C, 42.55; H, 4.34; N, 7.09; P, 7.84. Found;C, 42.53; H, 4.28; N, 7.03; P, 7.79.
Diethyl (((3,5-dichloropyridin-2-yl)amino)(2-hydroxyphenyl)methyl)phosphonate, 3g
Yield (0.99 gm, 81%); 77 h; light beige solid; M.p.: 100–102 °C; TLC: Ethyl acetate: Petroleum ether 1:1); Rf: 0.23; IR: ν/cm−1: 3248 (NH), 3029 (CH–Arom.), 2925 (CH–Aliph), 1254 (P=O), 1086 (P–O–C), 761 (P–CH). 1H NMR (500 MHz, DMSO-d6): δ = 1.05–1.23 (m, 6H, 2 × CH3), 3.80–4.02 (m, 4H, 2 × CH2), 6.45 (s, 1H, CH–P), 6.91–8.48 (5H, s, 1H, OH, m, 4H, Ar–H),7.89 (s, 1H, Hpyridine), 9.44 (s, 1H, Hpyridine),10.22 (br, 1H, NH, exchangeable with D2O) ppm. 13C NMR (125 MHz, DMSO-d6): δ = 16.92 (2CH3), 66.17 (2CH2), 67.56 (CH-P), 154.68 (C=N pyridine), 166.47 (C–NH), 114.07–154.68 (Ar–C) ppm. MS (EI) m/z: 404 (M, 2%, C16H19Cl2N2O4P), 265 (M−3, 48%, C12H10Cl2N2O), 165(M+4, 14%, C5H4Cl2N2), 146 (M, 100%, C5H3Cl2N) 138 (M, 15%, C4H11O3P). Anal. Calcd. For C16H19Cl2N2O4P (404.05): C, 47.43; H, 4.73; N, 6.91; P, 7.64. Found;C, 47.40; H, 4.68; N, 6.85; P, 7.60.
Antimicrobial activities
The produced compounds’ antimicrobial activity was evaluated using a panel of two gram-positive bacteria (Bacillus subtilis (#MTCC NO 441) and Staphylococcus aureus (#MTCC NO 96). as well as two Gram-negative bacteria (Pseudomonas aeruginosa (#MTCC NO 1688), Escherichia coli(#MTCC NO 452)). Two fungi [Candida albicans(#MTCC NO 183) and Aspergillus flavus(#MTCC NO 1344)] were used to assess the compounds' anti-fungal properties. Paper discs of Whatman filter paper with a standard size of 5 cm were made, dissolved in DMSO, and then sterilized in an autoclave. A solution of 1 mg/mL was prepared for each drug separately. Paper discs soaked in the desired concentration of the complex solution were aseptically placed in Petri dishes containing nutrient agar media (20 g + 3 g of beef extract + 5 g of peptone) seeded with bacterial and fungal strains. After 24 h of incubation at 36 °C, the inhibitory zones in the petri dishes were measured. There were three copies of every treatment. Using the same protocol as previously described, the antifungal Clotrimazole and the common standard antibiotic Ciprofloxacin were also tested for antibacterial activity at the same concentrations and solvent combinations[35].
Antioxidant activity using DPPH and ABTS
With slight modifications, the DPPH and ABTS methods were used to test the new phosphonate’s free radical scavenging activity [36]. Briefly, one milliliter of a 0.1 mM DPPH in methanol solution and ABTS in distilled water were added to the newly synthesized compounds. For 30 min, the mixture was left in the dark. The synthetic compounds’ ability to scavenge free radicals was compared to that of L. Ascorbic acid. At 517 nm for DPPH and 734 nm for ABTS, optical density was recorded, and the concentration inhibition was computed.
The percentage of inhibition is equal to [(A control − A test)/A control] × 100, where A control represents the control absorbance and A test represents the test sample absorbance. It was established what sample concentration (IC50) produced 50% inhibition. Every experiment was conducted in triplicate, and the mean ± SE IC50 values were reported.
Antitumor activity (in-vitro)
Cell lines
Mammary gland (MCF-7;# ATCC HTB-22), colorectal adenocarcinoma (Caco-2; # ATCC HTB-37), hepatocellular carcinoma (HepG-2; #ATCC HB-8065), and human lung fibroblast (WI-38; # ATCC CCL-75). The ATCC cell line was obtained via the Holding Company for Biological Products and Vaccines (VACSERA), Cairo, Egypt.
MTT assay
The MTT test was utilized to ascertain the phosphonate's inhibitory effects on cell growth utilizing the aforementioned cell lines. The basis of this colorimetric assay is the transformation of yellow tetrazolium bromide (MTT) by mitochondrial succinate dehydrogenase in living cells into a purple formazan derivative. 10% fetal bovine serum was added to RPMI-1640 media used to cultivate cell lines. At 37 °C in an incubator with 5% CO2, 100 units/mL of penicillin and 100 µg/mLof streptomycin were introduced as antibiotics. The cell lines were seeded at 1.0 × 104 cells/well in a 96-well plate and kept at 37 °C for 24 h with 5% CO2. Following incubation, the cells were subjected to several concentrations of newly synthesized phosphonates and left for 48 h incubation. 20 µL of a 5 mg/mL MTT solution was added and incubated for 4 h after the drug treatment lasted for 48 h. To dissolve the purple formazan produced in each well, 100 µL of dimethyl sulfoxide (DMSO) was applied. Using a plate reader (EXL 800, USA), the colorimetric test is measured and recorded at the absorbance of 570 nm. (A570 of treated samples/A570 of untreated sample) X 100 was used to compute the relative cell viability as a percentage [37,38,39].
Theory of calculations
All quantum chemistry calculations were performed using the Gaussian 09W program packages developed by Frisch and colleagues [40]. The structure of the molecules is optimized using DFT with Beck's three-parameter exchange functional and nonlocal correlation functional, Lee–Yang–Parr, B3LYP [41,42,43] utilizing the 6-311G++(d,p)basis set. HOMO (highest occupied molecular orbital), LUMO (lowest unoccupied molecular orbital), and MEP (m olecular electrostatic potential) charge density distributions were visualized using Gaussian view 05 software [44].
Docking (in-silico)
The newly synthesized α-aminophosphonates were docked pertaining to dynamin-related protein 1 (DRP-1) (PDB: 1zws) (https://www.rcsb.org/structure/1ZWS) target mitochondrial fission and mitophagy protein. The target protein was taken out of the protein data bank, polarized then, native legend, and all water molecules were removed. To get more stable findings, a 3D drawing of the ligand molecules was made using Chemdraw Ultra 8.0. The ligand molecules and target proteins underwent energy reduction by the application of the MM2 force field prior to the docking procedure. Molecular docking was computed using the Molegro virtual docker programme (MVD) (http://www.molegro.com/mvd-product.php, 17–2–2021). Target protein and α-aminophosphonates interacted, as shown by the Discovery Studio® Visualizer 2016 program [45].
ADMET pharmacokinetic characteristics
To study the pharmacokinetics and drug-likeness prediction of the newly synthesized compounds, the Swiss Institute of Bioinformatics’ online tool SwissADME (http://www.swissadme.ch/) was utilized. A 2D structural model of the chemical was converted into SMILES using SwissADME's SMILES generator. The SMILES data analysis was then used to determine the compound’s ADMET characteristics [46].
Statistical analysis
Using GraphPad Prism software 6 (San Diego, CA) The values were derived based on the experimental data and expressed as mean ± SE.
Results and discussion
Chemistry
Synthesis and spectroscopic characterization
3,5-dichloropyridin-2-amine 1, different aromatic aldehydes 2a-g, and triethyl phosphite were combined in a lithium per chlorate LiClO4 catalyst-aided process to create a sequence of Diethyl (((3,5-dichloropyridin-2-yl) amino)(Aryl) methyl) phosphonates (3a–g), Fig. 2.
Synthetic pathway of α-aminophosphonates 3a–g
FT-IR, 1H NMR, 13C-NMR spectroscopy, and Mass spectra, as well as corrected elemental analysis (Experimental portion), were used to confirm the structures of the studied α-aminophosphonates, 3a–g.
The FT-IR spectra of compounds, 3a–g were characterized by the following absorption bands, Fig.S1. A band is identified as the stretching vibration of the P=O group at 1289.74–1236.38 cm−1. υ (P–O–C) is responsible for a band that appeared at 1082.14–1022.98 cm−1, while υ (P–CH) is absorbed around 745.55–729.50 cm−1.At 3296.12–3138.99 cm−1, the CH aromatic stretching band is absorbed, and at 3167.72–2927.22 cm−1, the CH aliphatic bands are visible. At 637.21–625.74 cm−1, the stretching vibration C–Cl is absorbed. The bands between 1662.38 and 1565.18 cm−1 are thought to be the result of C=C stretching. NH/OH groups are finally absorbed between 3492.74 and 3386.05 cm−1.
The 3a–g derivatives of α-aminophosphonate exhibit the following signals in their 1H-NMR (DMSO) spectra: The triplet signal at 0.98–1.23 ppm is caused by the 2 × CH3 aliphatic protons. A multiplet signal is generated by the 2 × CH2 aliphatic protons between 3.78 and 4.03 ppm. Signals between 5.18 and 6.46 ppm is produced by the CH aliphatic protons connected to the phosphonate group. In the 6.46–9.75 ppm range, the aromatic protons are reverberated as multiples. Finally, the NH group is responsible for the broad singlet signal around 10.25 ppm (Figs. S2–S9 supplementary materials). 13C-NMR (DMSO) analysis of the investigated substances shows the following signals: δ 63.81–72.06 (P–CHaliph.), 13.56–27.72 (2 CH3), 57.45–66.17 (2 CH2), 144.17–155.04 (C=Npyridine), 154.73–166.47 (C-NH), and 99.80–154.68 ppm (CArom.) (Figs. S10-–16 supplementary materials). Morever, the full product’s mass spectrum revealed a peak at m/z that corresponded to the molecular ion (Figs. S17–S23, supplementary materials). Elemental microanalysis findings in agreement with corresponding theoretical values (Experimental part) were used to characterize the studied substances. Each spectrum confirms the structure of the synthesized compounds.
The proposed molecular pathway for the synthesis of α-aminophosphonate analogs was illustrated in Fig. 3, which involves two primary stages [47, 48]. (a) the in situ production of Schiff base through Lewis acid (Lithium perchlorate, LiClO4) catalyst-induced formyl group activation; By nucleophilically adding a (aminopyridine) nitrogen lone pair to the electrophilic carbon of the activated carbonyl group of -CHO, this promoted the condensation reaction between aminopyridine and aromatic aldehyde; (b) phosphorus atom (triethyl phosphite) nucleophilically attacking the electrophilic carbon of the imine moiety (> C=N–), which is followed by ethanol being released by phosphonium intermediates interacting with water to generate certain α-aminophosphonate analogs, Fig. 3.
The probable mechanism for the LiClO4-Catalyzed Kabachnic-Fields Synthesis of Ethyl α-aminophosphonates 3a–g
Biological investigation
Antimicrobial activity
Because of careless antibiotic use and insufficient infection control, resistant bacteria have grown to pose a serious threat to both public health and the world economy. Therefore, to stop the spread of antimicrobial resistance (AMR), it is imperative to carry out in-depth research and create a new class of antimicrobial compounds [45, 49]. Newly synthesized phosphonates were assessed for their in-vitro antibacterial activity using a standard agar well diffusion method against Gram-positive bacteria S. aureus and B. subtilis, Gram-negative bacteria E. coli and P. aeruginosa, and fungal C. albicans and A. flavus. Based on our findings, it was determined that compound 3d had the strongest antibacterial and antifungal effects, preventing the growth of every germ under investigation compared with standard FDA approved drugs. This compound produced the biggest inhibitory zones with activity index equal to (42.3% for E. coli, 60.9% for P. aeruginosa, 62.5% forS. aureus, 52.2%forB. subtilis, 81.5% for C. albicansand 72% forA. flavusrespectively). All other phosphonateshad from moderate as compounds 3a, 3f and 3c to weak as compounds 3b, 3e and 3g biocidal effect against bacterial and fungal strains, Fig. 4.
The antimicrobial activity of the synthesized compounds 3a–g by disc diffusion method
Antioxidant activity
The stable DPPH and ABTS radical scavenging activity assays were utilized to determine the antioxidant potential of these novel phosphonates by measuring the change in absorbance generated, as shown in Fig. 5. The results demonstrated that the antioxidant activity increased with the concentration of these substances. Compound 3d showed a higher DPPH IC50 value (20.04 ± 0.14 µM) compared to the conventional L-Ascorbic acid (IC50 = 16.81 ± 0.10 µM), according to our findings. The ABTS cation radical scavenging activity was examined using the decolorization test at various doses of phosphonates and compound 3d exhibited the best IC50 with value equal to 29.14 ± 0.18 μM, compared to the standard L-Ascorbic acid which gave an IC50 value of 29.47 ± 0.17 μM. Furthermore, utilizing both DPPH and ABTS scavenging assays, compounds 3a and 3f shown a moderate impact, whereas compounds 3b, 3c, 3e, and 3g demonstrated a weak capacity to quench free radicals.
The antioxidant scavenging activity of all novel phosphonates 3a–g using DPPH and ABTS
Molecular Docking (in-silico)
Molecular docking has been widely used to identify novel drugs because it is an effective tool for fast and precisely estimating protein–ligand complex binding energies and biomolecular conformations. Herein, the novel phosphonate's ligands, 3a–g, were docked into DRP-1 and are well-known, appealing therapeutic target proteins for the development of anticancer drugs. Mitochondrial division and fusion are engaged in the control of intrinsic apoptosis that is mitochondrial-dependent. This process was dependent on the release of mediators of cell death, such as cytochrome c, from the mitochondria and the permeabilization of the outer membrane [29]. Due to the function of ocular atrophy 1 protein (OPA1) in maintaining cristae, which reduces the release of cytochrome c produced by MOMP, mitochondrial fusion shields cells against apoptosis [50]. Numerous apoptotic models have been linked to mitochondrial fragmentation. Drp-1 plays a part in the permeabilization of the outer mitochondrial membrane (OMM) and cytochrome c release when it forms complexes with bcl-2-associated X protein (BAX) at mitochondrial fission sites [51]. Thus, Drp-1 plays a crucial role in many other aspects of cell biology, including apoptosis and cell death. All novel phosphonate’s interactions with target Drp-1 protein were described in Table 1; Fig. 6. Our results elucidated that compound 3d exhibited the most binding energy against target Drp-1 proteinwith a value equal to − 9.54 kcal/mol. Furthermore, compounds 3a and 3f elucidated moderate inhibitory effect with binding energies equal to − 7.66 and − 7.16 K cal/mol respectively. On the other hand, compounds 3b, 3c, 3e, and 3g observed slightly weak binding energy equal to − 4.32, − 6.47, − 4.01, and − 6.20 kcal/mol respectively. Therefore, compound 3d was strongly recommended to be used as an anticancer agent via its prospective inhibitory effect on Drp-1-mediated mitochondria fission.
Molecular docking interactions between newly synthesized phosphonates 3a–g and target DRP-1 mediated mitochondrial fission protein
Anti-tumor activity
In research on new anticancer agents, the most common experimental screening method after the theoretical study was testing against a group of different cancer cell lines [52, 53]. In this study, an MTT assay was done to determine the antitumor effect of phosphonates compounds on Caco-2, HepG-2, and MCF-7 proliferation, and the cytotoxicity limit on WI-38 normal cell line after 48 h, Fig. 7. Compound 3d showed significant antitumor effects on Caco-2, HepG-2 and MCF-7 cancer cell lines with an IC50 equal to 15.47 ± 1.2, 10.23 ± 0.8, and 7.69 ± 0.5 μM, respectively. Also, compounds 3a and 3f showed remarkable antitumor effects on HepG-2 and MCF-7 cell lines with low effect on Caco-2 cells with IC50 values (17.92 ± 1.3,9.37 ± 0.8 μM) (26.79 ± 1.9,19.50 ± 1.3 μM) respectively. On the other hand, compounds 3c, 3e, and 3g showed moderate to weak impact on all panels of cancer cell lines compared with the IC50 of DOX reference chemotherapeutic drug 12.49 ± 1.1, 4.50 ± 0.2and 4.176 ± 1.3 μM, respectively. Moreover, all newphosphonates showed lower cytotoxic effects on WI-38 normal cells compared with DOX which observed highly toxic effects on normal cells with IC50 equal to 6.72 ± 0.5 μM. This signifies that compounds 3d, 3a, and 3f, in the same manner,were effective against proliferative cancer through inhibiting DRP-1 mediated mitochondrial fission and suppressing mitochondrial mitophagy that causesat the same time activation to BAX/ Cytochrome-C signaling proteins resulting in activating caspases enzymes and finally cause apoptosis without any toxic effects on normal cells. As a result of their potential inhibition of DRP-1 target protein, newly synthesized phosphonates could be exploited as therapeutic candidates for cancer therapy, according to docking and in-vitro studies.
Antitumor/cytotoxic activity of compounds 3a–g, against a panel of human tumor cells and a normal cell
ADMET in-silico drug-likeness and bioavailability features
The ADMET has to certify the drug’s effectiveness as a top candidate against any illness [54]. Physio-chemical in-silico techniques were used to compute the donor hydrogen bond, drug similarity, and partition coefficient (cLogP). Furthermore, pharmacokinetic and bioavailability analyses have been performed to do these kinds of clinical studies on recently synthesized phosphonates. The topological polar surface area (TPSA) must be less than < 140 Å2 to have superior oral bioavailability. Our results showed that the phosphonate TPSA ranged from 60.45 to 89.91. Moreover, the results showed that phosphonates had good gastrointestinal absorptions and no BBB, indicating their CNS protection. Before being taken into consideration for development, the recently synthesized candidate needs to pass a toxicity risk assessment. Phosphonates did not exhibit any mutagenic harmful effects, according to the results of the AMES toxicity analysis. Remarkably, none of the substances proved carcinogenic, prompting an in-silico analysis, the results of which are shown in Table 2, Fig. 8. Our research indicates that the best-docked phosphonate 3d, which also significantly inhibits the target DRP-1 protein, has appropriate physio-chemical, pharmacokinetic, and bioavailability in silico and seems to be a potentially useful new class of cancer therapies. It also showed no toxicity or carcinogenicity.
ADMET radar pharmacokinetics features of newly synthesized phosphonates
Structure–activity relationship analysis (SAR) of α-aminophosphonates
To our knowledge, numerous research examining the relationship between α-aminophosphonates' structure and activity (SAR relationship) have demonstrated their anti-cancer properties.Regarding the structural surface area ratio (SAR) of the molecule, Fig. 9, a consistent relationship was observed between the anti-cancer activity and the electrical characteristics and/or lipophilicity of the substitute Aromatic (Ar) groups. The sequence of the investigated compounds’ action against cancer cells is indicated in Table 3 with regard to the nature of substituent group Ar: (2-hydroxy-1-naphthyl>4-hydroxy-3-methoxy-phenyl>thiophen-3-yl>2-methoxy-phenyl>2-hydroxy-phenyl>4-(dimethylamino)phenyl>4-hydroxy-phenyl), Fig. 9. From the previous configuration, the addition of a naphthyl group (a more π-conjugated ring system) to the diphenyl phosphonates increases their anticancer activity against HepG-2, MCF-7, and Caco-2, with IC50 values of 10.23, 7.69, and 15.47, respectively. It might originate from naphthalene's significant medicinal advantages[55].Also, the presence of the 4-hydroxy-3-methoxy-phenyl group (i.e. vanillin) in the phosphonate skeleton enhances the biological activity [56].
Structure–activity relationship analysis (SAR) of Diethylaminophosphonates 3a–g
Computational details
Quantum chemical calculations
Many different molecule properties, such as reactivity, shape, and binding locations, as well as molecular fragments and substituents, can be defined using quantum chemistry techniques and molecular modeling processes. Quantum chemical computations were utilized to examine the correlation between the activity of α-aminophosphonates and structural variables. The density DFT technique was used in the computational study through Beck’s three-parameter exchange functional (B3LYP) with 6-311G++(d, p) basis set implemented in the Gaussian 09 program package to optimize the molecular structures of the compounds under investigation. Figure 10 shows the optimized chemical systems with the lowest energy discovered by calculations of the studied substances.
Optimized α-aminophosphonate structures 3a–g
Quantum chemical calculations revealed that compound 3d, which contains a 2-naphthol substituent in the aryl aldehyde moiety of the α-aminophosphonates, exhibited more powerful biological activity in comparison to compound, 3g, which has a 2-OH-phenyl moiety and the remaining phosphonates. To enhance its capacity to take in electrons from the target protein, compound 3d has the highest LUMO energy (− 1.6964 eV), Table 4, and is anticipated to respond as an electrophile (electron acceptor). Furthermore, a simple method for examining donor–acceptor behavior in a chemical system is provided by hardness (η) and softness (σ). A soft molecule has a small energy gap, whereas a hard molecule has a big energy gap [57]. As a result, soft molecules will polarize more readily than hard molecules. The results shown in Table 4 demonstrate that the 3d phosphonate exhibited greater softness (0.4454 eV) than 3g (0.4233 eV). One measure of a molecule’s electron-absorbing capacity is the global electrophilicity ω index [58]. When compared to other phosphonates, compound 3d exhibits superior electrophilicity characteristics.The observed reactivity index by energy stabilization is the largest range of electronic loads (ΔNmax) obtained from the environment (donor) through the inhibitor (acceptor). Based on the calculation, compound 3d has the highest ΔNmax (1.7555e), Table 4. This indicates that it allows for charge transfer and an alternation of electron density between the compound and protein, leading to an excellent fit with the experimental data regarding the growth of biological activity.After analyzing the conversation, the computational calculations indicated that 3d, α-aminophosphonate, boosts reactivity more than the other phosphonates, which may be more advantageous for the enzyme's reactivity and agrees with the experimental data appropriately, Table 4.
The frontier molecular orbitals (FMOs)
The highest energy of matched electrons (EHOMO), and the lowest energy of unmatched electrons, (ELUMO), can be used to assess the characteristics of excitation and the electron's carrying capacity [59,60,61].They are essential to the chemical stability of the molecule [62]. The FMOs allow researchers to predict molecular interactions. Whereas the LUMO is mostly an electron acceptor, the HOMO is primarily an electron giver [63].The distinction between HOMO and LUMO determines the molecules’ chemical stability and reactivity. As seen in Fig. 11, the HOMO–LUMO orbitals, their distributions, and energy levels were estimated at the B3LYP/6-311G++(d, p) level. for every synthesized α-aminophosphonate molecule.For compound 3d, other than the ethyl ester groups, the HOMO distributes charges throughout the molecule, which can be interacted as a nucleophile (hydrogen bond donor) with the biological target, while its LUMO is selectively delocalized at the naphthyl group,which functions as an electrophile to interacte with the biological target (hydrogen bond acceptor).For compound 3e, the HOMO distributes charges over the substituted phenyl moiety containing N(CH3)2 substituent, while its LUMO distributes charges over the di chloro pyridine moiety. The HOMO of the remaining phosphonate compounds distributes charges over the molecules except for the ethyl ester group, while the LUMO distributes charges over the di chloro pyridine moiety only Fig. 11.
The calculated HOMO, LUMO of compounds 3a–g
Molecular electrostatic potential
Molecular electrostatic potential (MEP) is a three-dimensional representation of a molecule’s charge distributions. Every molecule's properties, including its dipole moment, electronegativity, partial charges, and chemical reactivity, can be connected through the use of molecular electrostatic potential [64]. Analyzing phenomena like electrophilic and nucleophilic sites, hydrogen bonding interactions, solvent effects, and more can be done with a molecular electrostatic [65, 66]. Different colors designate the zones of positive, negative, and neutral potentials. The parts that are red and yellow are linked to electrophilic reactivity and correspond to the area of high electron density [67]. White represents a zone of positive electrostatic potential, whereas blue represents low electron density and nucleophilic reactivity. On the other hand, green coloursrepresent regions of zero potential [68]. To tighten the reactive zone (attacks by electrophile and nucleophile sites) for phosphonates, molecular electrostatic potential is computed. The potential zones that are neutral, negative, and positive are represented by different colors.The red and yellow zones, which correspond to the region of high electron density, are associated with electrophilic reactivity. A zone of positive electrostatic potential is represented by white, whereas a zone of low electron density and nucleophilic reactivity is represented by blue.Green, on the other hand, denotes areas with zero potential. These regions of varying electrostatic potential can offer helpful information about many kinds of intermolecular interactions and help in predicting the molecule's chemical behavior. The MEP graphs for the produced compounds, 3a–g are shown in Fig. 12.
The calculated MEP of compounds 3a–g
The phosphonate group’s oxygen atoms contained the electron-rich regions. These locations designate the molecular segments that are appropriate for electrophilic reactions. By examining the MEP plots, one can determine the electrostatic forces that interact between the investigated compounds and the organic target, Fig. 12.
Vibrational spectra
Table 5 depicts the correlation between scaled wavenumbers and experimental wavenumbers of the substances under examination using DFT/B3LYP/6-311G++(d, p) method. Figure 13 presents a visual representation of the observed and computed infrared spectra. The appropriate wavenumber assignments to a given vibration are determined by comparing the calculated and experimental wavenumbers. The difference in wave number values between the observed and projected values can be explained by contrasting the solid phase data with theoretical estimates for the gaseous phase [69]. The symmetric stretching vibrations of the N–H group are responsible for the broadband (3492.74–3375.37) cm−1 that was seen in the experiment, while the calculated wide band is (3645.94–3550.62) cm−1. The typical aromatic C-H stretching is responsible for the band that is seen at (3296.12–3138.99) cm−1, whereas the calculated band is located at (3247.80–3196.07) cm−1.The observed CH aliphatic stretching band is (3150.30–2927.22) cm−1, while the calculated one is (3214.16–3013.40) cm−1. Also, the stretching vibration of the P = O group may be crucial for studying the α-aminophosphonates that are absorbed in (1289.74–1236.38) cm−1 (experimental) and at (1257.23–1252.38) cm−1 (calculated). The measured and calculated wave-wide variation υ (P–O–C) were observed at (1103.17–1022.98) cm−1 and (999.90–996.88) cm−1, respectively. Furthermore, one of the most significant phosphonate characteristic bands is υ (P–CH), which is observed at (745.55–729.50) cm−1 in experiments and (761.58–723.44) cm−1 in calculations. The band was ultimately found to be caused by υ (C–Cl) at (637.21–625.74) cm−1 (experimental) and (702.00–652.27) cm−1 (calculated). The calculated and experimental findings exhibit a high degree of accuracy, as evidenced by the linear correlation coefficient (R) value of 0.998, Fig. 14.
Comparison of the computed (red color) and experimental (black color) FT-IR spectra of the α-aminophosphonates 3a–g
Correlation charts between the experimental and computed frequencies of the chemicals under investigation
NMR spectral analysis
Modern chemistry relies heavily on Nuclear Magnetic Resonance (NMR) spectroscopy as a crucial research tool for precise molecular geometry predictions [70,71,72]. Several chemical areas have developed applications for computational NMR [73]. Part of theoretical research involves the active computation of some crucial NMR parameters, including chemical shifts, shielding constants, and nuclear spin–spin couplings [74]. GIAO (Gaussian with Gauge In-dependent Atomic Orbital) was used to obtain the theoretical chemical shifts of α-aminophosphonates [75]. based on the improved B3LYP/GIAO/6-311G technique in DMSO solvent. Experimental and computed 1H-NMR and 13C-NMR spectrum data are summarized in Tables 6 and 7, respectively. Figures 15 and 16 show a comparison of the theoretically and experimentally obtained 1H-NMR and 13C-NMR isotropic shift values, indicating a linear association with a high correlation coefficient.The results demonstrated a high degree of agreement between the GIAO-NMR method and the experimental NMR chemical shift values.
Comparison between the theoretical 1H-NMR chemical shift values and the experimental results for the α-aminophosphonates 3a–g, as measured by the B3LYP/6311G (DMSO) method
Comparison of experimental data with theoretical 13C-NMR chemical shift values obtained by B3LYP/6311G (DMSO) method for the α-aminophosphonates 3a–g
Conclusion
Diethyl(((3,5-dichloropyridin-2-yl)amino)(Aryl)methyl)phosphonate, 3a–g were synthesized and their structures were elucidated using different spectroscopic methods.The phosphonate compound which has naphthyl substituent, 3d, clarified the most potentantimicrobial and antioxidant activity among all other phosphonates. Moreover,Molecular docking revealed that compound 3d, could be a targeted anticancer agent because it has a good docking score and its nitrogen, oxygen atoms, and phenyl moieties form hydrogen bonds, and hydrophobic and electrostatic interactions with crucial residues within the binding pocket of Drp-1 target protein. Confirming the docking inhibitory results, the in-vitro anti-cancer activities, against panel of cancer cell lines were interpreted the suppressive impact of compound 3d. Also,the theoretical FT-IR data had been calculated to discover the characteristic vibration frequencies of the compounds which show a good correlation with experimental data. Moreover, the theoretical nuclear magnetic resonance (NMR) of the synthesized phosphonates was synthesized and a good correlation was obtained between theoretical and experimental data. Thus, our study recommended the usage of compound 3d as newly antimicrobialagent and a targeted inhibitory candidate against dynamin-related protein 1 (Drp-1) mitochondrial fission protein that responsible for cancer therapy. Also, we recommended further synthesis of novel α-am containg more than one phenyl and hydroxyl groups that may investigate a powerful antimicrobial and antitumor impact.
Availability of data and materials
• The microbial strains were provided from Microbial Type Culture Collection (MTCC) and all deposited codes were added two gram-positive bacteria (Bacillus subtilis (#MTCC NO 441) and Staphylococcus aureus (#MTCC NO 96). as well as two Gram-negative bacteria (Pseudomonas aeruginosa (#MTCC NO 1688), Escherichia coli(#MTCC NO 452)). Two fungi (Candida albicans(#MTCC NO 183) and Aspergillus flavus(#MTCC NO 1344)). • The cell lines were provided from the American Type Culture Collection (ATCC) via VACSERA, Cairo, Egypt, and all accession codes were added Mammary gland (MCF-7;# ATCC HTB-22), colorectal adenocarcinoma (Caco-2; # ATCC HTB-37), hepatocellular carcinoma (HepG-2; #ATCC HB-8065), and human lung fibroblast (WI-38; # ATCC CCL-75). • The datasets generated and/or analyzed during the current study are available in: Macromolecule protein structure, can be deposited in the worldwide protein data bank repository, (https://www.rcsb.org/structure/1ZWS). • the geometry of investigated compounds. The density functional theory (DFT) was utilized to simulate chemical processes and predict material characteristics using the hybrid density functional technique B3LYP combination with a 6-311G++(d, p) basis set for examined substances. The data supporting the current study's findings are available from the corresponding author upon reasonable request.
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The authors thank the Theoretical Applied Chemistry Unit (TACU), Faculty of Science, Tanta University, Tanta, for helping to bring out this research.
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All of the Authors made a significant contribution to the work reported, whether that is in the study design, analysis, writing, reviewing, interpretation, and editing of the original draft. Hend A.Hekal and Hayam A. Abd El-Salam are responsible for the synthesis and characterization of the new compounds. Also, Hend A.Hekal performed and visualized the theoretical studies with the help of the theoretical Applied Chemistry Unit. Maha M. Salem is responsible for the in vitro biological studies and the in-silico molecular docking simulation studies. All the authors gave final approval of the version to be published; and have agreed on the journal to which the article has been submitted.
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Hekal, H.A., Salem, M.M. & El Salam, H.A.A. Inhibition of DRP-1 mitochondrial mitophagy and fission by novel α-aminophosphonates bearing pyridine: synthesis, biological evaluations, and computer-aided design. BMC Chemistry 18, 174 (2024). https://doi.org/10.1186/s13065-024-01268-2
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DOI: https://doi.org/10.1186/s13065-024-01268-2