In Vitro Bioactivities of Isoindolin-1-3-Phosophonate Compounds

In this work an ecient synthesis of isoindolin-1-one-3-phosphonates under catalyst- and solvent-free conditions was reported to afford the desired compounds in excellent yields with potent pharmacological properties. The synthetic method involves the preparation of isoindolin-1-one-3-phosphonates by a ‘one-pot’ three-component reaction of 2-formylbenzoic acid with primary amines and dimethyl phosphite under solvent and catalyst free-conditions. All new compounds were characterized by by 1 H NMR, 13 C NMR, FT-IR and elemental analysis techniques. The synthesized compounds were screened for their antimicrobial activities against gram-positive bacterial strains (Micrococcus luteus, Listeria monocytogenes, Staphylococcus aureus and Bacillus cereus), a gram-negative bacterial strain (Salmonella typhimurium) and a fungus (Candida albicans). Compound 4a was found to be the most active against antimicrobial against L. M. luteus, L. monocytogenes and C. albicans with an inhibition zone of 35, 22 and 38 mm respectively. They were additionally also investigated for their anti-parasitical activities against Leishmania major promastigotes and amastigotes and Toxoplasma gondii in vitro. The compounds 4a,b are the most active against L. major amastigotes and promastigotes with EC50 < 1 µM. Cytotoxicity investigations of the isoindolin-1-one-3-phosphonates were conducted in two human cancer cell lines, MDA-MB-231 and MCF-7 all the compounds gave anticancer activity < 1.5 µM. We can conclude that 4a is a good drug candidate for all the biological assays, further studies for SAR detection and in vivo evaluation are highly recommended.

Treatment of isoindolin-1-one-3-phosphonate 4b,c with concentrated HCl in dioxane at 80 °C for 72 h led to production of isoindoline-1-one-3-yl phosphonic acid 5b,c ( Table 2). Table 2: Isoindolin-1-one-3-yl-phosphonic acids 5b-c prepared The structures of the synthesized products were deduced by IR, 1 H, 13 C, and 31 P NMR spectroscopy and elemental analyses. The IR spectrum of compound 4b exhibited strong absorption bands at 1681 (C=O), 1588 (C=C, acyclic), 1248 (P=O), and 1046 (P-O-C). In the 1 H-NMR spectrum of 4b, the disappearance of one OH peak of 1 and the presence of a signal for the PCHN proton that appears as a doublet at δ 5.3 ppm with a strong coupling constant with the close phosphorous atom indicated that the synthesis of compound 4b was accomplished. The signals of aromatic protons were observed between δ 7.08-7.68 ppm. In the 13 (C 10 ) and PCHN (C 7 ) carbons, respectively, also con rmed that the cyclisation had occurred.
The presence of the phosphonate group is clearly evidenced by the 31 P NMR spectrum that exhibits a singlet at 18 ppm. 1 H, 13 C and 31 P NMR spectra of derivatives 4a-f show very similar patterns of signals for the PCHN group which are very characteristic of the isoindolin-1-one-3-phosphonate structure.
3. Biological Activities a-Antimicrobial activity of isoindolin-1-one-3-phosphonates 4a-f The in vitro antimicrobial activities of the isoindolin-1-one-3-phosphonate compounds 4a-f were evaluated for in vitro antimicrobial activity by the well diffusion method [59]. All products were screened for activity against gram-positive bacteria (M. luteus, L. monocytogenes, S. aureus and B. cereus) and gram-negative bacteria (S. typhimurium). As shown in Table 3, all compounds exhibit considerable activity against the tested microorganisms except 4c. These results differ according to the compound structure and type of bacteria tested. A simple inspection of Table 3 indicates that the highest antibacterial activity against M. luteus LB 141107 was reported by 4a and 4b with inhibition zone (IZ) 35 mm and 24 mm respectively followed by 4d with IZ 18 mm. Also, 4f is the most active against S. Typhimurium ATCC 14028 with IZ 20 mm. Additionally, 4a was found to be the most active against L. monocytogenes with an inhibition zone of 22 mm. The other antibacterial results showed inhibition zones of less than 20 mm. As shown in Table 4, only 3 compounds possess antifungal activity against C. albicans-4a, 4b and 4d with inhibition zones of 38, 25 and 22 mm, respectively. The results obtained show that the different molecules have antimicrobial activity. 4a and 4b can be considered as good antimicrobial drug candidates. Isoindoline derivatives were found to have good and potent antimicrobial activity for a long time [60]. This supports our ndings. Further studies for structure activity relation (SAR) is recommended to indicate the reason for these varying biological activity. b-Anti-leishmanial activities of isoindolin-1-one-3-phosphonate compounds 4a-f Table 5 shows that all compounds except 4e had anti-leishmanial activity against L. major promastigotes in vitro with half maximal effective concentration (EC 50 ) less than 2.5 μM; two of them (4b and 4a) had EC 50 less than 1 μM, and their EC 50 values were 0.4 and 0.8 μM with Selectivity index (SI) of 12.9 and 11.9, respectively. Additionally, 4a and 4b were the most active against L. major amastigotes with EC 50 values of 0.7 and 0.8 μM and SI of 13.5 and 6.4, respectively. All compounds had a very good SI in the range of 3.5 to 41.2 with the exception of 4c against L. major amastigote. According to its SI value 4a can be considered a good antileishmanial drug candidate. The variation observed in these results may be due to the structure biology relation. Further studies for structure activity relation (SAR) is recommended to indicate the reason for these varying biological activity. However, in general, synthesized isoindoline derivatives exhibited potent biological activities [61]. Additionally, phosphonium compounds were found to have potent anti-leishmanial activity [62]. These results can agree with these suggestions. c-Anti-toxoplasmal activities of isoindolin-1-one-3-phosphonate compounds 4a-f Table 6 indicates that all compounds possess different levels of anti-toxoplasmal activity in vitro. Only 4b and 4a had EC 50 values of less than 4 μM (3.2 and 3.8 μM, respectively). The others had less potent antitoxoplasmal activity with EC 50 values in the range of 11.9 to 33.9 μM and SI in the range of 1.4 to 3.8.
The following results indicate that isoindolin-1-one-3-phosphonates are suitable candidates for antitoxoplasmal drug discovery. There have not been previous investigations of isoindoline against T. gondii, but previous investigations found that some isoindoline derivatives have less potent activity against Plasmodium [63][64]. These published results agree with our nding. Further studies for structure activity relation (SAR) is recommended to indicate the reason for these varying biological activity.

D-Anti-cancer activity
As shown in Table 7, all tested compounds signi cantly affected the viability of malignant cell lines, showing a promising level of cytotoxicity with EC 50 of < 1.5 μM. However, the cytotoxicities of isoindolin-1-one-3-phosphonate compounds 4d, 4c and 4d were much stronger in MCF7 cells with EC 50 values 0.6, 0.7 and 0.7 μM, respectively. The cytotoxicity of isoindolin-1-one-3-phosphonate compounds 4c and 4f against MDA-MB-231 cells was strongest with EC 50 of 0.8 μM, followed by 4a and 4d with EC 50 of 0.9 μM. These results agree with previous results that showed that isoindoline derivatives have activity against cancer cells [65]. Further studies for structure activity relation (SAR) is recommended to indicate the reason for these varying biological activity.

Conclusion
In conclusion, we have developed an e cient three-component synthesis of isoindolin-1-one-3phosphonates. The reaction proceeds quickly and with no undesirable side reactions observed. This procedure may nd wide application in the large-scale synthesis of cyclic a-aminophosphonates. Further investigations are currently in progress to demonstrate the potential of this methodology in the diastereoselective preparation of isoindolin-1-ones bearing phosphonate functionality of biological interest. The biological activities revealed that 4a and 4b are good drug candidates for antimicrobial (better than standard antibiotic) and anti-leishmanial agents and have potent anticancer activity against both MCF7 and MDA-MB-123. However, the activity of the above compounds against normal vero cells indicates their safety (half maximal inhibitory concentration (IC 50 ) in the range between 5 -50 µg/ml) that can support and enhance our suggestion about the uses of these compounds in future as drug candidates. Further studies for structure activity relation (SAR) is recommended to indicate the reason for these varying biological activity as well as in vivo evaluation.

Experimental General information
Chemicals were purchased from Sigma Aldrich and used without further puri cation. All solvents were puri ed and dried with the MBraun SPS 800 solvent puri cation system. NMR spectra were recorded with a Varian System instrument (400 MHz for 1 H, and 100 MHz for 13 C) with CDCl 3 as the solvent and TMS as the internal standard signal. NMR multiplicities are abbreviated as follows: s = singlet, d = doublet, t = triplet, and m = multiplet signal. IR spectra were recorded on a 398 spectrophotometer (Perkin-Elmer). Elemental microanalysis was performed on an ElementarVario El III Carlo Erba 1108 elemental analyzer, and the values found were within ± 0.3% of the theoretical values. Melting points were determined with Ko er bench at Isste of Borj cedria (Hammam Lif, University of Carthage, Borj Cedria, Tunisia) [46].
General procedure for the synthesis of Diethyl 3-oxoisoindolin-1-yl phosphonate compounds (4a-f) 2-Formylbenzoic acid (2.5 g, 17 mmol), amine (18 mmol) and diethylphosphite (2.9 g, 21 mmol) were stirred at 80 °C for 1.0 h, and the progress of the reaction was monitored by TLC. The mixture was extracted with dichloromethane, dried with MgSO 4 , ltered and evaporated. Finally, the crude product was puri ed by column chromatography. The crude product was analyzed by 1 H, 13 C and 31 P NMR spectroscopy [46].   Synthesis of isoindolin-1-one-3-yl phosphonic acids (5a-b) A solution of isoindolin-1-one-3-yl phosphonate 4a-b (3.01 mmol) in a mixture of concentrated HCl and dioxane (1:1) (100 ml) was heated at 80 °C for 72 h, cooled and the solvent was evaporated. The crystalline residue was treated with ethanol (10 ml), ltered off, washed and dried to give compounds 5ab as solids. L. major cell isolation, culture conditions, and assays Promastigotes of L. major were isolated from a Saudi male patient in February 2016 and maintained at 26 °C in Schneider's Drosophila medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) and antibiotics in a tissue culture ask with weekly transfers. Promastigotes were cryopreserved in liquid nitrogen at concentrations of 3 × 10 6 parasites/mL. The virulence of L. major parasites was maintained by passing in female BALB/c mice by injecting hind footpads with 1x10 6 . Fifty-six days later, the amastigotes of L. major were collected from the infected animals. Then transformed to promastigote stages via culturing at room temperature using complete Schneider's medium containing FBS 10% with antimicrobial. For infection, in vitro subculturing was used for maintaining different pathogen stages [65,66].
For the evaluation of the compounds for their activity against L. major promastigotes, complete RPMI 1640 medium containing 10% FBS without phenol red-free was used for culturing the parasite in 96-wells plates to yield 10 6 organisms mL -1 (200 µl/well), the counting was conducted by hemocytometer. Then the compounds were added in concentrations of (50,25,12  Then, the effects of test compounds on parasite growth were expressed as EC 50 (effective concentration at 50%) values. EC 50 values were obtained from three independent experiments [68,69].
In vitro cytotoxicity assay For the assessing the compound cytotoxicity MTT colorimetric assay was carried out according to the method described previously [67]. Ninety six well plates were used for culturing vero cells and two human cancer cell lines-MCF7 and MDA-MB-231-at concentration of (5 × 10 3 cells/well/200 µl) for one day in complete RPMI 1640 medium containing 10% FBS and then kept in 5% CO 2 with temperature of 37 °C.
followed by washing with PBS and then incubated with the compounds for 3 days at varying concentrations (100, 33, 11, 3.7, 1.2, 0.4, 0.14 and 0.04 µg/ml). Medium in 2% FBS was used as a negative control. Thereafter, the supernatant was removed, 50 µl of RPMI 1640 medium containing 14 µl of MTT (5 mg/ml) was added, and the cells were incubated for 4 h. After that, the supernatant was removed, and 200 µl of DMSO was added to dissolve the formazan. A FLUOstar OPTIMA spectrophotometer was applied for colorimetric analysis (λ = 540 nm). Cytotoxic effects were expressed as IC 50 values (concentration that caused a 50% reduction in viable cells). IC 50 values were obtained from three independent experiments [70][71][72].

Declarations
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Figure 1
Structure of pharmacologically compounds that contain isoindolin-1-one core structure.

Figure 2
Protocol synthesis of Isoindolin-1-one-3-phosphonantes 4a-f Supplementary Files This is a list of supplementary les associated with this preprint. Click to download. SPECTRESINDEXphosphonate.docx