Cytokines are important proteins in cell signaling and help to control inflammation in the body. They are released by cells and affect the behavior of other cells, and sometimes the releasing cell itself. There are many types of cytokines including chemokines, interferons, interleukins, lymphokines, and tumor necrosis factor (TNF-α) that are produced by a broad range of cells, including immune cells like macrophages, B lymphocytes, T lymphocytes and mast cells, as well as endothelial cells, fibroblasts, and various stromal cells. The most essential and master kind of cytokines is tumor necrosis (TNF-α) [1] [2] .

It is well known that TNF-α has an essential role in processes such as immunomodulation, fever, inflammatory response, inhibition of tumor formation, and inhibition of viral replication. In addition, TNF-α and its receptors are ubiquitously expressed in developing organs and they regulate the survival, proliferation, and apoptosis of embryonic stem cells (ESCs) and progenitor cells [1] [2] .

However, overexpression of this key pro-inflammatory cytokine (TNF-α), together with other mediators play a central role in the pathogenesis of autoimmune inflammatory responses in rheumatoid arthritis (RA), inflammatory bowel disease (IBD), Crohn’s disease (CD), and ankylosing spondylitis (AS) [3] - [5] .

Recently, TNF-α received much attention in the therapy of these diseases especially the rheumatoid arthritis (RA) which represents one of the most prevalent autoimmune diseases worldwide and characterized by inflammatory arthritis and extra-articular involvement.

In which the immune system can significantly damage the joints and other tissues, ultimately leading to irreversible joint deformity [6] [7] .

Accordingly, extensive efforts are employed for TNF-α inhibitors for the treatment of this disease and others [8] .

One approach is anti-TNF-α biologics (such as infliximab, adalimumab, etanercept, golimumab, and certolizumab pegol) have markedly improved the outcome of the management of autoimmune inflammatory diseases [9] . However, a considerable proportion of patients do not respond to this anti-TNF-α treatment [10] . Moreover, anti-TNF-α biologics are expensive, and are associated with some adverse side effects.

Recent studies indicate that specifically targeting of one of TNF-α receptors may represent a more effective and safer treatment for autoimmune disorders [11] .

A third option is inhibiting TNF-α converting enzymes (TACE) which is a member of the reprolysin family of the metzincin superfamily that also includes matrix metalloproteinases (MMPs) identified by its ability to cleave the transmembrane form of TNF-α at its physiological processing site [12] . Some inhibitors of MMPs have also shown inhibitory activity of TACE, suggesting some similarities between the active sites of these enzymes. Accordingly, TACE inhibitors have been derived based on the knowledge of the MMP inhibition [13] [14] .

Thus, due to the previous diseases that can be caused by the over release of TNF-α, there is arising need for new anti-TNF-α compounds with high efficacy and minimum side effects [15] - [18] .

We presume that organophosphonates can be a candidate for this purpose due to their extensive biological activity. For instance, they possess anti-inflammatory [19] [20] , herbicidal [21] , and antitumor activity [22] . Moreover, they showed protective properties against UV radiation [23] , and other utilities [24] .

This work is an extension of previous reports in which organophosphate compounds prepared by us and were found to be active as anti-TNF- beside others that showed MMPs inhibition [25] [26] . So, three different compounds of 2-amino-1-cyclopentenylphosphonates class 1a-c which were prepared by the reaction of diethyl 5-chloro-1-pentynylphosphonate with benzylamine, propylamine and heptylamine respectively were explored as TNF-α inhibitors as shown in Scheme 1 [27] .

In summary, two 2-aminocyclopentenylphosphonate compounds demonstrated promising TNF-α inhibition activity, particularly diethyl (2-(benzylamino)cyclopent-1-en-1-yl)phosphonate which showed proximate activity compared to bude-sonide medicine.

Motivated by these in vitro findings, we plan to extend our research to in vivo studies and to synthesize other phosphonate compounds and explore them for this purpose.

Chemical experimental section: [27]

Procedure for the synthesis of 1a: To 0.238 g (1 mmol) of diethyl 5-chloro-1-pentylphosphonate was added to 2 mmol (0.214 g) of benzylamine in a 25 ml round-bottom flask. After stirring for 3 hrs r.t., the reaction mixture was washed with 0.1 N NaHCO₃ solution, extracted with (2 × 20 ml CH₂Cl₂), and separated on silica gel column (10% methanol: 90% dichloromethane), and was analyzed by GCMS, elemental analysis, and NMR spectroscopy in 88% isolated yield.

¹H NMR (300 MHz, Acetone d): 1.88 (t, 6H, J_HH = 7.2 Hz), 1.93 (qn, 2H, J_HH = 7.2 Hz), 2.97 (dt, 2H, J_HH = 7.8 Hz, ³J_PH = 2.4 Hz), 3.41 (t, 2H, J_HH = 6.9 Hz), 3.87 (qn, 4H, J_HH = 7.2 Hz), 4.42 (s, 2H), 7.20-7.38 (overlap, 5H). ³¹P NMR (121.4 MHZ, Acetone d): δ 27.85; ¹³C NMR (75.5 MHz, Acetone d): δ 16.1 (d, ³J_PC = 6.9 Hz), 21.3, 32.1, 49.5, 52.6, 60.0 (d, ²J_PC = 5.2 Hz), 127.3, 127.4, 128.8, 137.2, 164.5 (d, ²J_PC = 20.1 Hz); MS(EI):m/z (%) 309 (8.5), 264 (2.1), 207 (1.1), 172 (100), 144 (6.0), 104 (10.3), 91 (80.1), 77 (1.2), 65 (21.3), 41 (10.3); Anal. Calcd for C₁₆H₂₄NO₃P: C, 62.12; H, 7.82; N, 4.53; P, 10.01. Found: C, 62.01; H, 7.89; N, 4.49; P, 10.09.

Procedure for the synthesis of 1b: Identical procedure for 1a except adding propylamine.

¹H NMR (300 MHz, Methanol d): δ 0.93 (t, 3H, J_HH = 7.5 Hz), 1.28 (t, 6H, J_HH = 7.2 Hz), 1.68 (m, 2H), 1.92 (qn, 2H, J_HH = 6.9 Hz), 2.83 (dt, 2H, J_HH = 7.5 Hz, ³J_PH = 2.1 Hz), 3.16 (t, 2H, J_HH = 7.2 Hz), 3.42 (t, 2H, J_HH = 6.6 Hz), 3.95 (qn, 4H, J_HH = 7.2 Hz). ³¹P NMR (121.4 MHZ, Methanol d): δ 31.78; ¹³C NMR (75.5 MHz, Methanol d): δ 10. 8, 15.7 (d, ³J_PC = 6.8 Hz), 20.8, 21.0, 32.3, 41.2, 52.4, 60.9 (d, ²J_PC = 5.4 Hz), 165.4 (d, ²J_PC = 20.9 Hz); MS(EI):m/z (%) 261 (16.7), 232 (26.5), 216 (8.8), 204 (8.8), 146 (17.6), 124 (100), 110 (42.2), 97 (68.6), 83 (68.6), 55 (29.9), 41 (55.9); Anal. Calcd for C₁₂H₂₄NO₃P: C, 55.16; H, 9.26; N, 5.36; P, 11.85. Found: C, 55.03; H, 9.20; N, 5.46; P, 11.94.

Procedure for the synthesis of 1c: Identical procedure for 1a except adding 1-heptylamine.

¹H NMR (300 MHz, Methanol d): δ 0.90 (t, 3H, J_HH = 6.6 Hz), 1.27 (t, 6H, J_HH = 7.2 Hz), 1.20-1.56 (overlap, 10H), 1.91 (qn, 2H, J_HH = 7.5 Hz), 2.81 (dt, 2H, J_HH = 7.8 Hz, ³J_PH = 2.1 Hz), 3.19 (t, 2H, J_HH = 7.8 Hz), 3.39 (t, 2H, J_HH = 7.2 Hz), 3.90 (qn, 4H, J_HH = 7.2 Hz). ³¹P NMR (121.4 MHZ, Methanol d): δ 29.94; ¹³C NMR (75.5 MHz, Methanol d): δ 13.1, 16.0 (d, ³J_PC = 7.2 Hz), 21.3, 22.4, 25.8, 26.1, 27.0, 28.6, 40.5, 45.7, 52.6, 70.0 (d, ²J_PC = 5.1 Hz), 165.8 (d, ²J_PC = 21.0 Hz); MS(EI):m/z (%) 317 (3.0), 302 (5.8), 246 (13.8), 233 (40.0), 152 (60.2), 138 (88.8), 79 (100), 83 (15.1), 55 (20.6), 41 (48.8); Anal. Calcd for C₁₆H₃₂NO₃P: C, 60.55; H, 10.16; N, 4.41; P, 9.76. Found: C, 58.90; H, 9.79; N, 4.69; P, 9.92.

Biological experimental section:

The same protocol of our previous study was used [25] .

Peritoneal cells were harvested from the peritoneum of C57BL/6 female mice 4 days after intraperitoneal injection of 1.5 ml of a 3% thioglycollate medium.

The thioglycollate medium macrophages were washed with phosphate-buffered saline, re-suspended in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal calf serum, and plated (1.2 × 10⁻⁵) in 96-microwell plates flat-bottom. Following 2 - 3 h of incubation at 37˚C, the non-adherent cells were removed by intensive rinsing. About 95% of the adherent cells were macrophages. The vinylphosphonates were first dissolved in absolute ethanol (1 mg/50 - 100 μl ethanol), and the solutions were further diluted with DMEM. For each compound, various nontoxic concentrations (as determined by MTT assay) were added to the macrophages, followed by addition of 1 μg/ml of lipopolysaccharide (LPS) for activation. The thioglycollate medium macrophages were then cultivated in a humid atmosphere with 5% CO₂ for 24 h. The supernatant fluids were harvested and kept at −20˚C until assayed for TNF-α.

TNF-α in the supernatants of the vinylphosphonates treated (8 μl/ml) LPS-activated macrophages was determined by ELISA.

The consistency system was checked by negative control in which the cells were not treated with LPS. In addition, a positive control was taken by treating the macrophages cell with budesonide (BDU) to check the extent of inhibition. The TNF-α analysis was performed via Enzyme-linked immunosorbent assay ELISA method on triplicates.