Introduction

Synthesis of organophosphorus compounds has been over emphasised due to their eminent role in biology and pharmaceutical areas (Esalah and Husein 2008). Among them α-aminophosphonates popped up abundantly due to their isosteric structure with α-amino acids (Kafarski and Lejczak 1991). Owing to this, α-aminophosphonates have formed diverse applications as antibiotics (Lajczak et al. 1986), anti-thrombotics (Meyer and Barlett 1998), inhibitors of HIV protease (Stowasser et al. 1992; Ananda et al. 2017), anti-cancer agents (Bhattacharya et al. 2013), anti-inflammatory (Damiche and Chafaa 2017), anti-tubercular (Mulla et al. 2014), catalytic antibodies, (Hirschmann et al. 1994; Smith et al. 1994), anti-oxidants (Gundluru et al. 2016), anti-microbials (Sreekanth et al. 2018), plant growth regulators (Maier 1990; Forlani et al. 2013), herbicides (Chea et al. 2016), anti-viral agents (Xie et al. 2017; Xu et al. 2006) and carriers of hydrophilic organic molecules across the phospholipid membranes (Antipin et al. 1999).

Nowadays to manage the inflammation caused by the response of tissues to destructive pathogens, we are using non-steroidal remedies, which are alternative to the steroidal therapeutics are being used. Even though non-steroidal anti-inflammatory drugs are acting fine on inflammation, they have some drawbacks such as gastrointestinal irritation and bleeding. These problems prompted the researchers to synthesise new compounds having anti-inflammation properties with reduced side effects.

Five-membered heterocyclic compounds bearing both sulfur and nitrogen atoms are privileged essential organic structural scaffolds in compounds used as medicine. Due to their high potency towards therapeutic properties, tremendous efforts have been to synthesis thiazole bearing derivatives. These structural motifs are having numerous therapeutic properties such as anti-inflammatory (Ugwu et al. 2018), antihypertensive α-blocking (Bakr et al. 2008), anti-leishmanial (Sharma et al. 2013), neuroprotective agents in Parkinson’s and Alzheimer’s diseases (Anzini et al. 2010), Some of these organic structural scaffolds are used in radiodiagnostics to measure the size of the tumours in radiotherapy (Tzanopoulou et al. 2010), along with transition metals like Re and Tc. Recently it has been found that this structural motif combined with pyrimidine is having good herbicidal activities (Zuo et al. 2016), are also found use in semiconducting materials and organic light-emitting diodes (Kudrjasova et al. 2014).

Synthesis of α-amino phosphonates by the Kabachnik–Fields reaction, a vast number of catalytic protocols were established such as uncatalyzed conditions (Tibhe et al. 2010), Metal triflates (Qian and Huang 1998; Firouzabadi et al. 2004), InCl3 (Ranu et al. 1999), SmI2 (Xu et al. 2003), pentafluorophenylammoniumtriflate (PFPAT) (Malamiri and Khaksar 2014), Mg(ClO4)2 (Bhagat and Chakraborti 2007), BiCl3 (Zhan and Li 2005), LiClO4 (Azizi and Saidi 2003) have been employed. Most of the catalysts gave satisfactory to high yields, but they showed disadvantages like cost expensive, toxic and hygroscopic nature of catalysts, requirement of stoichiometric quantities of catalysts and long reaction times. Development of metal free, environmentally benign and economically feasible synthetic protocols for several organic transformations is the need of the hour. In this context, an elegant corrosive and recyclable catalyst called Caffeine hydrogen sulfate (CHS) (Shalini et al. 2018; Agarwal et al. 2019) has been used for the synthesis of α-(2-aminothiazole) alkylphosphonates by the reaction of 2-aminothiazole with various aldehydes and dialkyl phosphites under MW irradiation conditions. The MW irradiation (Gundluru et al. 2016; Kandula et al. 2018) offered new method to energising the reaction mixture as it involves the direct transfer of energy to the substrate molecules and will enhance the rate of the reaction, by rapid kinetic excitation of molecules and increases the product yield. This serves an ideal platform for the one-pot three components of Kabachniks–Fields reaction. In addition, in vitro antioxidant, anti-inflammatory and molecular docking studies were also carried out for the synthesised compounds (4a–j) to establish their bio activities.

Experimental

All the chemicals, reagents and solvents were procured from Aldrich S.D. Fine Chem. Ltd, Boisar, India and Qualigens, Mumbai, India and used without further purification. MWI was carried out in a microwave oven, catalyst system (CATA-4R). The purity and progress of the reaction was confirmed by thin layer chromatography on pre-coated silica gel plates purchased from Merck. Visualization is done under UV light chamber. Melting points were determined in an open capillary tube on EZ Melt Automated Melting Point Apparatus. IR spectra were recorded as neat samples on Bruker Alpha-Eco ATR-FTIR interferometer with single-reflection sampling module equipped with Zn–Se crystal; the data were expressed in reciprocal centimetres (cm−1). 1H, 13C and 31P NMR spectra were recorded on Jeol Resonance spectrometer operating frequency at 400, 100 and 161.9 MHz respectively. NMR data were recorded in CDCl3 and tetramethylsilane was used as a standard for both 1H and 13C-NMR and 85% H3PO4 is used as a standard for 31P NMR. The high-resolution mass spectra are recorded on micromass Q-TOF micromass spectrometer using electrospray ionisation.

DPPH anti-oxidant assay

In-vitro antioxidant activity of newly synthesised compounds (4a–j) was performed by DPPH method (Matsubara et al. 1991), which determines the free radical inhibitory ability of synthesised compounds by scavenging the very stable 2, 2-diphenyl-1-picrylhydrazyl (DPPH) free radical in methanol. Stock solution of DPPH (1.3 mg/mL) in methanol was prepared. Stock solution of DPPH 100 μL was added to 3.0 mL of methanol and absorbance was recorded at 516 nm. The various concentrations of synthesised compounds (25, 50, 75 and 100 μL) in methanol were prepared. All sample solutions 1.0 mL each is diluted with 3.0 mL with methanol and 100 μL of stock solution of DPPH was added. Test tubes were kept for 30 min in dark to complete the reaction. After 30 min, absorbance of each test tube was recorded at 516 nm on UV-VIS spectrophotometer against methanol as a blank. The effective concentration of sample required to scavenge DPPH radical by 50% (IC50 value) was obtained by linear regression analysis of dose-response curve plotting between percentage inhibition and concentrations. The DPPH free radical scavenging activity was calculated using the following formula:

$${\mathrm{DPPH}}\,{\mathrm{Scavenging}\;(\% )} = \frac{{({\mathrm{A}}_{{\mathrm{cont}}} - {\mathrm{A}}_{{\mathrm{test}}})}}{{{\mathrm{A}}_{{\mathrm{cont}}}}} \times {\mathrm{100}}$$

Where; Control is absorbance of a DPPH solution without compound; Test is the absorbance of the test compound with DPPH. The degree of discoloration indicates the free radical scavenging efficiency of the compound. Ascorbic acid was used as the standard free radical scavenger reference compound.

Ferric reducing antioxidant power assay

The reactive principle of chemicals in which iron reacts with a colorimetric probe to produce a blue product was used to quantitate Ferric reducing antioxidant power assay (Raquel et al. 2000). An aliquots of different concentration of test compounds (25, 50, 75 and 100 μL) was mixed with 90 μL water and 900 μL ferric reducing antioxidant power (FRAP) reagent (2.5 mL of 20 mmol/L of 2, 4, 6-tri(2-pyridinyl)-1,3,5-triazine), in 40 mmol/L of HCl, 2.5 mL of 20 mmol/L of ferric chloride, 25 mL of 0.3 mol/L of acetate buffer (pH 3.6) and incubate at 37 °C for 30 min. After incubation, the absorbance values were recorded at 593 nm with ultraviolet-visible (UV-vis) spectrophotometer. The antioxidant activity was expressed as the amount of extract required to reduce 1 mmol of ferrous ions.

$${\mathrm{FRAP}}\,{\mathrm{Scavenging}}\,(\% ) = \frac{{({\mathrm{A}}_{{\mathrm{cont}}} - {\mathrm{A}}_{{\mathrm{test}}})}}{{{\mathrm{A}}_{{\mathrm{cont}}}}} \times {\mathrm{100}}$$

Where; control is absorbance of a FRAP solution without compound; test is the absorbance of the test compound with FRAP. The degree of discoloration indicates the free radical scavenging efficiency of the compound. Ascorbic acid was used as the standard free radical scavenger reference compound.

Cell line studies

Maintenance of cell lines

RAW 264.7 murine macrophage cell lines were procured from NCCS Pune. RAW macrophages were plated into T75 flasks and cultured in Dulbecco’s Modified Eagle Medium (DMEM) with high glucose, stable glutamine and sodium pyruvate, supplemented with 10% foetal bovine serum heat-inactivated (65 °C for 20 min) and 1% penicillin-streptomycin antibiotics (100 IU/mL penicillin and 100 μg/mL streptomycin) at 37 °C in 5% CO2 atmosphere.

Toxicity studies

The study was conducted in accordance with OECD guidelines (Testing of Chemical Number 423). Healthy RAW 264.7 murine macrophage cell lines were used in this investigation. Cell lines were treated with different concentrations (i.e., 100 µg/mL and 200 µg/mL) of title compounds and observed for 48 h from the time of administration. The Median non-toxic concentration (MNTC) and cytotoxic concentration (CC50) were determined (Fig. 3). The minimum concentration of the test compounds which has not shown any toxic effect on healthy RAW 264.7 cell lines were selected for further anti-inflammatory analysis.

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay

Cell viability was measured based on the formation of purple formazan, which is metabolised from colourless MTT (Sigma-Aldrich) by mitochondrial dehydrogenases, enzymes that are only active in live cells. RAW 264.7 cells (5 × 105cells/ml) were seeded in a 96-well plate. The cells were pre-treated with various concentrations of synthesised compounds for 1 h and then stimulated by 10 µg/ml LPS for 24 h. Following the incubation with title compounds and LPS, the cultured media was replaced with fresh media and the cells were incubated with 0.5 mg/ml MTT solution for 3 h. The supernatant was then discarded and the formazan blue, which was formed in the cells, was dissolved with dimethyl sulfoxide (Sigma-Aldrich). The optical density was measured at 540 nm with an ELISA plate reader. Percentage of inflammation was calculated by subtracting the percentage of cell viability over control cells (Park et al. 2009).

Determination of nitrite production

Nitrite accumulation (NO2) in cell culture media was determined by the Griess method (Pekarova et al. 2009). Briefly, 1 × 106 cells were seeded in a T75 flask, allowed to adhere overnight, and then treated as previously mentioned in MTT assay. At the end of the different incubations in the CO2 incubator at 37 °C with 5% CO2, the different cell supernatants were collected, the samples (1 mL) were mixed with an equal volume of Griess reagent (1 mL of 1:1 0.1% naphthyl-ethylenediamine and 1% sulfanilamide in 5% phosphoric acid) in a tube, and incubated in the dark for 10 min at room temperature. Then the absorbance of the reaction mixture was measured at 540 nm on a microplate reader (Thermo Scientific Multiskan EX). The NO2 concentration was determined using a sodium nitrite (NaNO2) standard curve (working range: 0.1–6.25 μM). Each treatment was carried out in triplicates and the final results were expressed as µmol NO2 -/mg protein.

Statistical analysis

All experiments were repeated at least three times. Results are expressed as the means ± standard deviation (SD) of three experiments. Statistical calculations were performed using prism Graph Pad 6.0.

Results and discussion

An elegant one-pot three-component Kabachnik–Fields reaction of 2-aminothiazole (1), various aldehydes (2a) and dialkyl phosphite (3a–b) in presence of caffeine hydrogen sulfate (CHS) as a simple reusable catalyst under MW irradiation conditions has been accomplished (Scheme 1). The synthesis involves by the simple workup procedures and offers moderate to excellent product yields.

Scheme 1
scheme 1

Synthesis of α-amino phosphonates

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To optimise the experimental conditions required for this reaction, initially the reaction is run with 2-aminothiazole (1), 4-nitrobenzaldehyde (2a) and dimethyl phosphite (3a) at ambient temperature under neat conditions in the absence of catalyst for 10 h (Table 1). There was no reaction (Table 1, Entry 1) and followed by increased the temperature to 100 °C for 10 h. The reaction, when run with several solid catalysts like AlCl3, FeCl3, p-TSA, ZnCl2, NiBr2, CrCl3, CeCl3.7H2O, Pd(OAc)2 (Table 1, Entry 2–9) by varying the temperature from ambient to 100 °C furnished a mixture of 60% of product and some amount of reactants and imine. To our delight, later when CHS (Table 1, Entry-10) is used as a catalyst the reaction proceeded even at ambient temperature and offered 70% product in just 0.5 h. Later there was no enhancement of yield of the product, even after extended the time and raising the temperature to 100 °C (Table 1).

Table 1 Optimisation of catalyst for the synthesis of compound (4a)
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To enhance the yield of the Kabachnik–Fields product (4a) the concentration of CHS catalyst is varied (Table 2, Entry 1-4). We have noticed 5 mol% of CHS is sufficient to get a 93 % yield of the product (Table 2, Entry 5). Further increasing quantity of the catalyst there is no notable improvement in the yield of the product. The reaction is screened to check the effect of the solvent on outcome of the product with 5 mol% of catalyst by using various solvents polar and non-polar solvents (Table 2, Entry 5–12). Out of all solvents, the reaction in methanol (Table 2, Entry 9) offered 93% of yield of the product, but it is a time-consuming process (20 min), but the same reaction in solvent-free condition offered the product within short time (10 min).

Table 2 Optimisation of the amount of catalyst and effect of solvent (4a)a
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To optimise microwave irradiation for the reaction of 2-aminothiazole (1), 4-nitrobenzaldehyde (2a) and dimethyl phosphite (3a) were reactants with 5 mol% of CHS as a catalyst, we have employed microwave protocol from 100 to 600 W and the results are listed in Table 3. The reaction at 400 W afforded excellent conversion in less time and offered 97% yield (Table 3, Entry 3). The reaction at 500 W (Table 3, Entry 4) affords low conversion, due to the decomposition of the substance in the reaction mixture. Hence 400 W microwave irradiation is optimised for the reaction for it effective completion.

Table 3 Effect of microwave energy in watts
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After optimisation of experimental conditions, the scope of the Kabachiniks-Fields reaction with 5 mol% of caffeine hydrogen sulfate as the catalyst, the reaction of 2-aminothiazole and commercially available aldehydes contains both electron withdrawing as well as electron donating groups and dialkyl phosphites was studied and the results are presented in Table 4.

Table 4 Synthesis of the α-substituted thiazolyamine methyl phosphonates (4a–j)a
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On the basis of previous reports and literature, a reasonable mechanism has been proposed for this reaction (Scheme 2). The aldehyde is activated initially by the catalyst CHS and carbonyl carbon (2) renders to more electrophilic, this facilitates the nucleophilic addition of the amine (1) leads to form an imine. Further imine is activated by CHS by co-ordinating with the imine nitrogen and drawing π-electrons of the imine. Consequently the imine carbon present in Schiff’s base becomes electrophilic. This situation facilitates the nucleophilic addition of phosphate (3) to the imine leads to the formation aminophosphonates (4) with the release of CHS.

Scheme 2
scheme 2

Possible reaction mechanism for the synthesis of α-aminophosphonates

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Reusability of catalyst

Reusability of catalyst is very important from the economical and industrial point of view. To analyse the activity and reusability of the CHS, we studied this reaction by examining up to six cycles shown in Fig. 1. The activity of catalyst decreased in the subsequent runs. After each run, we have recovered the catalyst by the addition of DCM to the reaction mixture and the insoluble catalyst was recovered by centrifugation, it was washed twice with DCM and dried in oven at 60 °C for next use.

Fig. 1
figure 1

recyclability of catalyst

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The chemical structures of the all the synthesised compounds (4a–j) were characterised by IR, 1H-NMR, 13C-NMR and 31P NMR spectroscopy. In FT-IR spectra the following stretching frequencies were observed, (a) -NH stretching frequencies at 3285–3250 cm−1, (b) –P = O stretching vibration at 1280–1240 cm−1, (c) -P-O-C stretching vibrations at 1048–1006 cm−1, (d) –P–C stretching vibration at 780–740 cm−1. In 1H-NMR spectra the chemical shift for N–H proton was found in the range between 6.25–5.60 ppm. The signals for P–CH appeared in the range from 5.50 to 4.70 ppm as a doublet. 13C-NMR chemical shift appeared in the range from 13.20–168.30 ppm. 31P NMR chemical shifts appeared in the region of 23.78–21.10 ppm.

General procedure for the synthesis of compound (4a)

A mixture of 2-aminothiazole (1 mmol), 4-nitrobenzaldehyde (1 mmol), dimethyl phosphite (1.0 mmol) and CHS (5 mol %) were charged in 25 ml conical flask and reaction was monitored at 400 W in microwave. The progress of the reaction was monitored by TLC analysis by using ethyl acetate: hexane (8:2) as eluents. After completion of reaction, the resulting reaction mixture was treated with DCM and filtered to remove the catalyst for reuse. After separation of the catalyst, it was washed with 20 ml of DCM and dried under oven for reuse. Further the filtrate was quenched with 10 ml of water followed by 5 ml of brine solution dried over anhydrous magnesium sulfate and concentrated by using rotary evaporator. Later the pure products obtained were purified by recrystallisation from ethanol. The same procedure is adopted for synthesis of remaining compounds.

Spectral characterisation

Dimethyl ((4-nitrophenyl)(thiazol-2-ylamino)methyl)phosphonate 4a

White solid; Yield 97%, Mp; 125–127 °C; FT-IR (cm−1): vmax 3282 (NH), 1274 (P = O), 1018 (P-O-C), 785 (P-C); 1H-NMR (CDCl3, 400 MHz) δ (ppm): 8.29 (1H, d, J = 12.0 Hz, Ar-H), 8.12 (2H, d, J = 8.0 Hz, Ar-H), 7.97 (1H, d, J = 8.0 Hz, Ar-H), 6.98 (1H, d, J = 4.0 Hz, Ar-H), 6.98 (1H, d, J = 8.0 Hz, Ar-H), 5.73 (1H, d, J= 24.0 Hz, P-CH), 3.82 (3H, d, J= 12.0 Hz, -OCH3), 3.61 (3H, d, J = 12.0 Hz, -OCH3); 13C-NMR (CDCl3, 100 MHz) δ (ppm): 161.55 (C-4), 145.92 (C-11), 136.30 (C-8), 133.67 (C-6), 132.67 (C-13 & C-9), 123.46 (C-12 & C-10), 113.29 (C-7), 68.99 (C-1), 54.03 (d, J = 7.0 Hz, C-21), 53.69 (d, J= 8.0 Hz, C-22); 31P-NMR (CDCl3, 161.8 MHz) δ (ppm): 23.78 Hz; HRMS (ESI) m/z Calcd. for C12H14N3O5PS [M + H]+ 344.3018 found 344.3010.

Dimethyl ((4-chlorophenyl)(thiazol-2-ylamino)methyl)phosphonate 4b

White solid; Yield: 94%, Mp; 128–130 °C; IR (cm−1): vmax 3283 (NH), 1277 (P = O), 1013 (P-O-C), 749 (P-C); 1H-NMR (CDCl3, 400 MHz) δ (ppm): 7.38 (2 H, d, J= 8.0 Hz, Ar-H), 7.37 (2H, d, J= 8.0 Hz, Ar-H), 7.09 (1H, d, J = 4.0 Hz, Ar-H), 6.89 (1H, d, J = 4.0 Hz, Ar-H), -NH is exchanged with D2O, 4.90 (1H, dd, J1 = 8.0 Hz & J2 = 4.0 Hz, -PCH), 4.33 (3H, d, J= 8.0 Hz, -OCH2CH3), 4.21 (3H, d, J= 8.0 Hz, -OCH2CH3); 13C-NMR (CDCl3, 100 MHz) δ (ppm): 163.21 (C-4), 136.1 (C-6), 135.70 (C-7), 132.90 (C-17), 128.25 (C-15 & C-19), 125.25 (C-16 & C-18), 113.01 (C-7), 65.60 (d, J = 10.0 Hz C-1), 53.40 (d, J= 15.0 Hz, C-14& C-13): 31P-NMR (CDCl3, 161.8 MHz) δ (ppm): 21.15; HRMS (ESI) m/z Calcd. for C12H14ClN2O3PS 333.7468 [M + H]+ found 333.7460.

Dimethyl ((4-bromophenyl)(thiazol-2-ylamino)methyl)phosphonate 4c

White solid; yield: 95%, Mp; 128–130 °C; IR (cm−1): vmax 3284 (NH), 1277 (P = O), 1013 (P-O-C), 749 (P-C); 1H-NMR (CDCl3, 400 MHz) δ (ppm): 7.55 (2 H, s, J = 8.0 Hz, Ar-H), 7.48 (2H, d, J = 8.0 Hz, Ar-H), 7.17 (1H, d, J = 4.0 Hz, Ar-H), 6.98 (1H, d, J = 4.0 Hz, Ar-H) –NH is exchanged with D2O, 5.20 Hz (1 H, d, J= 4.0 Hz, P-CH), 3.80 (3H, d, J= 4.0 Hz, -OCH3), 3.75 (3H, d, J= 4.0 Hz, -OCH3); 13C-NMR (CDCl3, 100 MHz) δ (ppm): 165.20 (C-4), 140.20 (C-11), 136.05 (C-8), 132.20 (C-6), 129.20 (C-13 & C-9), 125.50 (C-12 & C-10), 114.48 (C-7), 63.76 (d, J= 10.0 Hz, C-1), 53.04 (d, J = 5.0 Hz C-13), 49.56 (C-14); 31P-NMR (CDCl3, 161.8 MHz) δ (ppm): 22.10; HRMS (ESI) m/z Calcd. for C12H14 BrN2O3PS 378.2008 [M + H]+ found 378. 2010.

Dimethyl (furan-2-yl(thiazol-2-ylamino)methyl)phosphonate 4d

Brown solid; yield: 90%, Mp; 110-112 °C; FT-IR (cm−1): vmax 3278 (NH), 1256 (P = O), 1019 (P-O-C), 764 (P-C); 1H-NMR (CDCl3, 400 MHz) δ (ppm): 7.09 (1H, d, J = 12.0 Hz, Ar-H), 6.71 (1H, d, J = 12.0 Hz, Ar-H), 6.48 (1H, d, J = 8.0 Hz, Ar-H), 6.26 (1H, s, J = 8.0 Hz, Ar-H), 6.12 (1H, d, J = 4.0 Hz, Ar-H), -NH is exchanged with D2O, 5.47 (1H, d, J= 20.0 Hz, P-CH), 3.79 (3H, d, J= 8.0 Hz, -OCH3), 3.55 (3H, d, J= 8.0 Hz, -OCH3); 13C-NMR (CDCl3, 100 MHz) δ (ppm): 168.69 (C-4), 152.20 (C-9), 139.38 (C-12), 138.65 (C-6), 115.02 (C-7) 108.92 (C-11), 107.78 (C-10), 63.40 (C-1), 54.14 (C-17), 48.91 (C-18); 31P-NMR (CDCl3, 161.8 MHz) δ (ppm): 22.95; HRMS (ESI) m/z Calcd. for C10H13N2O4PS 289.2658 [M + H]+ found 289.2663.

Diethyl ((4-nitrophenyl)(thiazol-2-ylamino)methyl)phosphonate 4e

White solid; yield: 97%, Mp; 120-122 °C: FT-IR (cm−1): vmax 3284 (NH), 1242 (P = O), 1117 (P-O-C), 774 (P-C); 1H-NMR (CDCl3, 400 MHz) δ (ppm): 8.22 (2H, d, J = 12.0 Hz, Ar-H), 7.69 (2H, d, J = 8.0 Hz, Ar-H), 7.17 (1H, s, J= 12.0 Hz, Ar-H), 6.97 (1H, d, J = 12.0 Hz, Ar-H), 6.05 (1H, s, -NH), 4.90 (1H, t, J1 = 16.0 Hz & J2 = 4.0 Hz, P-CH), 4.23-3.84 (4H, m, -OCH2CH3), 1.34 (3H, t, J1 = 4.0 Hz & J2 = 8.0 Hz, -OCH2CH3), 1.19 (3H, t, J1 = 4.0Hz & J2 = 8.0 Hz, -OCH2CH3); 13C-NMR (CDCl3, 100 MHz) δ (ppm): 159.04 (C-4), 144.42 (C-11), 133.36 (C-6), 129.04 (C-13 & C-9), 119.29 (C-12 & C-10), 114.83 (C-7), 63.79 (C-4), 63.66 (t, J1 = 7.0 Hz & J2 = 28.0 Hz, C-1), 55.70 (C-18), 54.26 (C-19), 16.30 (d, J = 14.0 Hz, C-20) 14.65 (d, J= 20.0 Hz, C-24); 31P-NMR (CDCl3, 161.8 MHz) δ (ppm): 22.97. HRMS (ESI) m/z Calcd. for C14H19N3O5PS [M + H]+ 372.3558 found 372.3570.

Diethyl ((4-chlorophenyl)(thiazol-2-ylamino)methyl)phosphonate 4f

Yellow solid; yield: 95%, Mp; 120–122 °C (Boughabaa et al. 2018).

Diethyl ((4-bromophenyl)(thiazol-2-ylamino)methyl)phosphonate 4g

Brown solid; yield: 93%, Mp; 131–133 °C (Boughabaa et al. 2018).

Diethyl((4-hydroxyphenyl)(thiazol-2-ylamino)methyl)phosphonate 4h

White solid; yield: 90%, Mp; 128–130 °C; FT-IR (cm−1): vmax 3487 (-OH), 3290 (NH), 1267 (P = O), 1112 (P-O-C), 797 (P-C); 1H-NMR (CDCl3, 400 MHz) δ (ppm): 7.63 (2H, d, J= 8.0 Hz, Ar-H), 7.40 (2H, d, J = 8.0 Hz, Ar-H), 7.09 (1H, s, Ar-H), 6.91 (1H, s, Ar-H), 6.21(1H, s, -NH), 5.49 (1H, s, -OH), 4.85 (1H, dd, J1= 8.0 Hz & J2 = 8.0 Hz, -PCH), 4.19-3.73 (4H, m, -OCH2CH3), 1.27 (3H, t, J1 = 8.0 Hz & J2 = 8.0 Hz, -OCH2CH3), 1.12 (3H, t, J1 = 8.0 Hz & J2 = 8.0 Hz, -OCH2CH3); 13C-NMR (CDCl3, 100 MHz) δ (ppm): 160.91 (C-4), 155.45 (C-11), 137.89 (C-6), 131.46 (d, J= 7.2 Hz, C-5 & C-9), 124.03 (d, J= 6.25 Hz), 113.01 (C-12 & C-10), 109.42 (C-7), 69.83 (C-1), 64.10 (d, J= 6.25 Hz, C-18), 63.38 (d, J= 6.5 Hz, C-19), 16.33 (d, J= 6.25 Hz, C-20)), 14.13 (d, J = 26.25, C-21); 31P-NMR (CDCl3, 161.8 MHz) δ (ppm); 21.00. HRMS (ESI) m/z Calcd. for C14H19N2O4PS 343.3578 [M + H]+ found 343.3574.

Diethyl((4-hydroxy-3-methoxyphenyl)(thiazol-2-ylamino)methyl)phosphonate 4i

Yellow solid; Yield: 90%; Mp: 125-127 °C; IR (cm−1): vmax. 3436 (-OH), 3312 (-NH), 1269 (P = O), 1023 (P-O-C), 756 (P-C); 1H-NMR (CDCl3, 400 MHz) δ (ppm): 7.42 (1H, d, J= 4.0 Hz, Ar-H), 6.79 (1 H, d, J= 8.0 Hz, Ar-H), 6.56 (1H, s, Ar-H), 6.85 (1H, d, J = 4.0 Hz, Ar-H), –NH and –OH is exchanged with D2O, 4.75 (1H, d, J= 8.0 Hz, P-CH), 4.16-3.81 (4 H, m, -OCH2CH3), 3.93 (3H, s, -OCH3), 1.30 (3H, t, J1 = 8.0 Hz & J2 = 8.0 Hz, -OCH2CH3), 1.18 (3H, t, J1 = 8.0 Hz & J2 = 8.0 Hz, -OCH2CH3); 13C-NMR (CDCl3, 100 MHz) δ (ppm): 158.23 (C-2), 148.21 (C-8), 144.34 (C-9), 140.90 (C-4), 131.83 (C-6), 130.09 (C-11), 122.47 (C-11), 116.23 (C-07), 113.42 (C-10), 68.03 (d, J= 37.51 Hz, C-15) 62.40 (d, J= 61.2 Hz, C-22), 57.36 (C-24), 16.98 (d, J = 25.2 Hz, C-21), 14.78 (C-23); 31P-NMR (CDCl3, 161.8 MHz) δ (ppm): 21.78; HRMS (ESI) m/z Calcd. for C15H21N2O5PS 373.3838 [M + H]+ 373.3830.

Diethyl (furan-2-yl(thiazol-2-ylamino)methyl)phosphonate 4j

Brick red solid; yield: 92%, Mp; 112–114 °C; FT-IR (cm−1): vmax. 3283 (NH), 1277 (P = O), 1013 (P-O-C), 749 (P-C). 1H-NMR (CDCl3, 400 MHz) δ (ppm):7.63 (1H, d, J = 4.0 Hz Ar-H), 7.52 (1H, d, J = 8.0 Hz, Ar-H), 6.68 (1H, d, J = 12.0 Hz, Ar-H), 6.40 (1H, d, J= 8.0 Hz, Ar-H), 5.67 (1H, s, -NH), 5.10 (1H, d, J = 12.0 Hz, P-CH), 4.17-4.06 (4H, m, P-OCH2CH3), 1.32-1.26 (6H, m, P-OCH2CH3); 13C-NMR (CDCl3, 100 MHz) δ (ppm): 165.00 (C-4), 150.01 (C-5), 145.10 (C-12), 138.59 (C-6), 110.74 (C-7), 109.00 (C-11), 108.00 (C-11) 66.59 (d, J= 9.0 Hz, C-11), 54.16 (C-17), 54.09 (C-17), 16.14 (d, J = 24.0 Hz, C-17), 13.20 (d, J = 24.0 Hz, C-14); 31P-NMR (CDCl3, 161.8 MHz) δ (ppm): 21.10. HRMS (ESI) m/z Calcd. for C12H17N2O4PS [M + H]+ 317.3198 found 317. 3190.

Biological activity

DPPH radical scavenging activity

The antioxidant activity of all the synthesised compounds was expressed as IC50 (inhibitory concentration, 50%). In the case of dialkyl((substituted phenyl)(thiazol-2ylamino)methyl) phosphonates (4a–j) DPPH radical scavenging activity was measured with respect to ascorbic acid (IC50 42.82 µg/mL) as a control (Table 5). Out of 4a–j compound 4h showed the remarkable DPPH radical scavenging activity (IC50 of 28.06 µg/mL) when compared with that of standard. The other compounds exhibited DPPH radical scavenging activity in the following order 4i (IC50 29.48 ± 0.24 µg/mL) > 4e (IC50 30.22 ± .0.44 µg/mL) > 4b (IC50 32.06 ± 0.46 µg/mL) > 4a (IC50 34.26 ± 0.16 µg/mL) > 4c (IC50 48.32 ± 0.42 µg/mL) > 4g (IC50 54.26 ± 0.24 µg/mL) > 4j (IC50 55.32 ± 0.24 µg/mL) > 4f (IC50 61.96 ± 0.36 µg/mL) 4d (IC50 60.42 ± 0.22 µg/mL) (Fig. 2).

Table 5 The in vitro free radical scavenging activity of α-aminophosphonates (4a–j)
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Fig. 2
figure 2

Anti-oxidant activity of titled compounds

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Ferric ion reducing antioxidant power assay

The reducing power assay of the all the titled compounds (4a–j) was measured in IC50 values and ascorbic acid was taken as a standard with its IC50 value as 51.02 µg/mL. Majority of the synthesised compounds showed higher anti-oxidant activity than the control (Table 5). The compound 4i showed remarkable anti-oxidant activity with its IC50 value as 25.44 µg/mL. The remaining compounds displayed FRAP scavenging activity in the following order 4h (IC50 29.86 ± 0.66 µg/mL) > 4a (IC50 30.16 ± 0.38 µg/mL) > 4e (IC50 32.02 ± .0.64 µg/mL) > 4b (IC50 53.46 ± 0.20 µg/mL) > 4g (IC50 48.84 ± 0.36 µg/mL) > 4c (IC50 52.62 ± 0.42 µg/mL) > 4f (IC50 53.46 ± 0.20 µg/mL) 4d (IC50 54.02 ± 0.20 µg/mL) > 4j (IC50 69.12 ± 0.12 µg/mL) (Fig. 2).

Cytotoxic activity

Prior to the evaluation of anti-inflammatory effect of title compounds, cytotoxicity was determined using RAW 264.7 cell lines. The cytotoxicity (cell death) was not observed for the test compounds up to 200 μg/mL. The minimum lowest concentrations such as 25 μg/mL and 50 μg/mL were taken into consideration to test the anti-inflammatory effect against LPS-induced inflammatory cell death in RAW 264.7 cell lines respectively. The cytotoxicity of test compounds was shown in Fig. 3.

Fig. 3
figure 3

Cytotoxic effect of title compounds during the treatment with different concentration in RAW 264.7 macrophage cell lines MTT Assay

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In order to determine the anti-inflammatory effect of synthesised compounds, MTT assays were performed in RAW 264.7 cell lines. Cells were treated with test compounds with different concentrations (25 μg/mL and 50 μg/mL) followed by LPS (10 μg/mL). As demonstrated in Fig. 4, all the test compounds exhibited strong anti-inflammatory activity against LPS-stimulated inflammation in RAW 264.7 cell lines. Higher than 50% of inflammation was observed during the treatment with LPG at 10 μg/mL. Whereas significant reduction in cell death was observed during the pre-treatment with tested compounds. The percentage of inflammation was reduced on dose dependent manner during the treatment with synthesised compounds. Among all the title compounds, 4a, 4b, 4e, 4h and 4i have shown strong anti-inflammatory activity against LPS-induced cell death in RAW 264.7 cell lines.

Fig. 4
figure 4

Anti-inflammatory effects of title compounds with different concentrations during the pre-treatment in LPS-induced inflammation in RAW 264.7 macrophage cell lines

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NO production

In order to provide strength of the synthesised compounds as to be anti-inflammatory agents the effect of synthesised compounds on NO production stimulated by LPS was also investigated using RAW 264.7 cell lines. The addition of LPS to RAW 264.7 cells resulted in an increase in NO production levels (Fig. 5). However, on pre-treatment with synthesised compounds significantly suppress LPS-induced NO production in a concentration-dependent manner. Concomitant to the previous report, out of all title compounds 4a, 4b, 4e, 4h and 4i have shown strong inhibition against LPS-induced NO production in RAW 264.7 cell lines. Significantly reduced levels of NO production were observed in all compounds during pre-treatment.

Fig. 5
figure 5

Inhibition of NO production by title compounds with different concentrations in LPS-stimulated RAW 264.7 macrophages

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In Silico studies

Molecular docking

The structure of 3LN1 (COX-2) and Celicoxib (CEL) were taken from Protein Data Bank. The molecules were loaded into MOE working environment followed by protonation and energy minimisation using MMFF94x force field at a cut off value of 0.05. Chem sketch Module has been used for structure drawing of the title compounds; energy minimisation was done with universal force field (UFF) and converted into pdb format as required for docking. Alpha triangle placement methodology was used for effective docking of compounds and the poses are generated by superposition of triplets of receptor site points and ligand atom triplets. For each compound, ten conformations were generated and they were refined and rescored. Interactions of the ligands with target protein were analysed using PyMol Visualizer (Vilar et al. 2008). The structures of 3LN1 and CEL were mentioned in Fig. 6.

Fig. 6
figure 6

Structures of COX-2 and Celecoxib

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All the ten title compounds were docked into the active site of the enzyme COX-2 (PDB ID: 3LN1) which formed high binding energies than the reference compound celecoxib except 4d (Table 6). Compounds 4a, 4e, 4h, 4i and 4g have shown highest binding energies with COX-2 enzyme than the rest of the title compounds. All the title compounds were actively fitted in the active site of the target gene, COX-2. Asp, Gly, Glu and Lys were the key residues of COX-2, which are involved in bond formation during the docking with title compounds. Out of all the title compounds, compound 4f have formed hydrophobic interaction with COX-2. The protein-ligand interactions of best lead titled compounds were shown in Fig. 7.

Table 6 Bonding characterisation of synthesised compounds (4a–j) and Celecoxib, (Reference drug) against COX-2
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Fig. 7
figure 7

Bonding interactions of the title lead compounds and standard with COX-2

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Structure activity relation (SAR studies)

Most of the compounds such as 4a, 4b, 4e, 4h and 4i showed better free radical scavenging activity than the control in both DPPH and FRAP assays. Out of all the titled compounds 4i and 4j showed remarkable anti-oxidant activity. Their high-antioxidant activity may be attributed, to the presence of hydroxy and methoxy groups present in the aromatic ring.

Further in MTT assay, out of all the titled compounds 4a, 4h and 4i (25 μg/mL & 50 μg/mL) showed that remarkable decrease in percentage of anti-inflammation in RAW 264.7 macrophage cell lines with respect to LPS as a standard. Again we carried out nitric oxide production in cell lines same results were repeated in the compounds 4a, 4h and 4i. This may be, due to the presence of nitro, methoxy and hydroxy groups in the aromatic ring.

Exploration of structure–bioactivity relationship of the titled compounds (4a–j) reveals that even though the basic core moiety of α-aminophosphonate remains same in all compounds, different substituent’s present in the phenyl ring and at the α-carbon excert significant effect on their biological and physical properties. The binding models signifying that the titled compounds are held in the active site by a combination of various hydrogen bonding interactions. When compare with standard celecoxib all the compounds exhibited good binding energies. Out of all the compounds compound 4e with three hydrogen bonds showed highest binding energy (−8.5 K cal/mol). In the compound 4e, hydrogen bonding interactions are present between –NH, and -NO2 groups with Glu 332, Phe 566 and Lys 293 amino acid residues with bond distance 2.5, 2.0 and 2.1 respectively. Whereas the other active compounds such as 4a, 4b, 4c, 4d, 4f, 4g, 4h and 4j showed better interactions than that of the reference with amino acids viz Asp 333, Lys 253, Asn 567, Gly 310, Gln 313, Asn 333, Phe 556 through H-bonding with bond lengths of 2.1, 2.2, 2.3, 2.5, 2.7, 2.8 and 2.9 by using –NH, -P = O, -OH and –OCH3 as the active sites present on aminophosphonates. The nitro substituted compounds 4a & 4e at the para position of the phenyl ring showed strong binding energies between these two compound 4e exhibited higher binding energy due to the presence of ethyl group on phosphorous.

Conclusion

In conclusion, we have accomplish synthesis of dialkyl((substituted phenyl)(thiazol-2ylamino) methyl)phosphonates (4a–j) by using one-pot three-component Kabachniks–Fields reaction by reacting 2-aminothiazole, different aldehydes and dialkylphosphate in presence of environmentally benign and reusable catalyst called caffeine hydrogen sulfate (CHS) as a low expensive green catalyst in solvent-free conditions under microwave irradiation. The compounds such as 4a, 4e, 4h and 4i have been established as potent anti-oxidant and anti-inflammatory agents through in vitro studies. According to the molecular docking studies among all the synthesise compounds 4a, 4e and 4i showed good binding energies −8.2, −8.5 and −8.3 respectively. On the basis of the findings, further investigations are being carried out to develop best anti-oxidant and anti-inflammatory compounds by fine tuning the structure of the compounds with respect to their bioactivity.