Introduction

Alkaline phosphatase (ALPs, E.C. 3.1.3.1) belongs to a family of ectonucleotidases that catalyze the dephosphorylation of nucleosides into corresponding nucleotides. These are metalloenzymes with two Zn2+ and one Mg2+ ions in their active site for optimum activity [1]. Elevated serum level of alkaline phosphatase is an indication of liver and bone diseases such as Paget’s disease, hyperparathyroidism, vitamin D deficiency, damaged liver cells and celiac disease [2, 3]. Reduced ALP serum level may also be observed in several other pathophysiological states like hypophosphatasia, recent heart surgery, hypothyroidism, malnutrition, severe anemia, aplastic anemia, pernicious anemia, myelogenous leukemia and Wilson’s disease. Deficiency of TNAP has been found to cause hypophosphatasia in infants that results in epileptic seizure. Moreover, this deficiency also causes hypo-mineralization of teeth and bones [4].

In human genome, four genes are known to be involved in expression of these enzymes, categorized as tissue specific (TSAP) and tissue non-specific alkaline phosphatase (TNAP). The former includes intestinal alkaline phosphatase (IAP), placental alkaline phosphatase (PALP) and germ cells alkaline phosphatase (GCALP). However, TNAP is one of the four isozymes in humans that can hydrolyze phosphate groups of wide variety of biological substrates. TNAP is post-transnationally modified to bone, kidney and liver isoforms [5]. Hypophosphatasia leads to alteration of pyridoxal 5′-phosphate, pyrophosphate and phosphoethanolamine metabolism indicating the significance of alkaline phosphatase [6].

Overexpression of ALP is found to be in association with several human cancers [7, 8]. Higher tissue non-specific alkaline phosphatase level induces carbonate-substituted hydroxyapatite crystal deposition that leads to osteoarthritis; therefore, inhibition of TNAP is an auspicious way to treat rheumatoid arthritis [9, 10]. Levamisole and tetramisole containing azole ring are recognized inhibitors of TNAP but cause skin rashes and agranulocytosis that limit their therapeutic effectiveness [11].

Thiazole moiety is a heart core of various commercially available drugs and are in current clinical practices. Thiazole containing molecules are known to possess diverse biological activities including anticancer, antimicrobial, anti-inflammatory, antioxidant and anti-tubercular activities [12, 13]. Also, various azomethine derivatives exhibit antioxidant, antimicrobial, anticancer and antimalarial activities [14,15,16] (Fig. 1). In current study, both distinctive chemical moieties are gathered in single scaffold to find potent and selective inhibitors of alkaline phosphatases.

Fig. 1
figure 1

Some literature reported bioactive thiazole and azomethine compounds and structural architecture of current work

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Experimental

All the chemicals and solvents used were acquired from the standard source (Sigma-Aldrich and Merck). Distillation of solvents and starting materials was carried out by using standard procedures. Melting point was determined on Gallenkamp melting point apparatus. 1H and 13C NMR spectra were recorded on Bruker AM-300 spectrophotometer using CDCl3) as solvent. Thin layer chromatography was carried out on pre-coated silica gel plates that observe the UV to check the progress of reaction.

General procedure for the synthesis of azomethine-thiazole hybrids (3a–i)

Suitably substituted aromatic aldehyde (1.0 mmol) was added to dry ethanol (15 mL) in a two-neck round bottom flask fitted with reflux condenser and stirred for 5 min. A solution of thiosemicarbazide (1.0 mmol) in dry ethanol (20 mL) was added dropwise. The resulting mixture was heated at reflux for 3–4 h. After accomplishment of reaction (TLC), the products precipitated out which were purified by recrystallization in acetone and ethanol to afford azomethine (2a–j).

Azomethines (2a–j) (1.0 mmol) were taken in the round bottom flask containing in 25 ml dry ethanol. A solution of 3-chloroacetyl acetone (1.0 mmol) in dry ethanol was added. The reaction mixture was refluxed for 18–20 h the completion of reaction as monitored by TLC. The solid precipitated out was filtered and purified by recrystallization in ethanol to yield the azomethine-thiazole hybrids (3a–i).

(E)-1-(2-(2-((2-Hydroxynaphthalen-1-yl)methylene)hydrazineyl)-4-methylthiazol-5-yl)ethan-1-one (3a)

Yield = 77%; Yellowish powder; M.P = 294–295 °C; Rf = 0.55 (methanol: chloroform, 1:9); FT-IR (ATR) in cm−1, 3162 (H–N), 2920 (H–C, Ar), 1696 (C=O), 1583 (C=N, imine), 1496 (C = C, Ar); 1H-NMR (300 MHz, CDCl3); in δ (ppm), 12.4 (s, 1H, OH), 11.7 (s, 1H, N–H), 9.14 (s, 1H, imine), 8.62 (d, 1H, J = 6.9 Hz, naphthyl-H), 7.91–7.36 (m, 4H, naphthyl-H), 7.23 (d, 1H, J = 6.9 Hz, naphthyl-H), 2.49 (s, 3H, CH3), 2.41(s, 3H, CH3); 13C-NMR (75 MHz, CDCl3) in δ (ppm), 189.0 (acetyl, C=O), 158.0 (thiazole, C=N), 133.0 (imine, C), 131.7, 129.2, 128.4, 128.2, 123.9, 123.76, 122.9, 122.8, 119.05, 119.7, 118.8, 110.0, 29.6, 17.2. Anal. Calcd. for C17H15N3O2S C, 62.7; H, 4.65; N, 12.91; S, 9.85, found; C, 62.79; H, 4.68; N, 12.95; S, 9.87.

(E)-1-(2-(2-(4-Methoxybenzylidene)hydrazinyl)-4-methylthiazol-5-yl)ethanone (3b)

Yield = 72%; Dark brown powder; M.P = 192–194 °C; Rf = 0.51 (methanol: chloroform, 1:9); FT-IR (ATR) in cm−1, 3136 (H–N), 2980 (H–C, Ar), 1667 (C=O), 1585 (C=N, imine), 1467 (C = C, Ar); 1H-NMR (300 MHz, CDCl3); in δ (ppm), 11.89 (s, 1H, N–H), 8.41 (s, 1H, imine), 7.62 (d, 2H, J = 8.1 Hz, phenyl-H), 7.23 (d, 2H, J = 8.1 Hz, phenyl-H), 3.73 (s, 3H, methoxy-H), 2.49 (s, 3H, CH3), 2.41(s, 3H, CH3); 13C-NMR (75 MHz, CDCl3) in δ (ppm), 180.0 (acetyl, C=O), 168.1 (thiazole, C=N), 135.1 (imine, C), 130.3, 128.2, 128.4, 123.7, 119.7, 118.8, 65.8, 56.4 (OMe), 29.6, 17.2. Anal. Calcd. for C14H15N3O2S C, 58.11; H, 5.23; N, 14.52; S, 11.08 found; C, 58.14; H, 5.26; N, 14.55; S, 11.12.

(E)-1-(2-(2-(3-Hydroxy-4-methoxybenzylidene)hydrazineyl)-4-methylthiazol-5-yl)ethan-1-one (3c)

Yield = 73%; Brown powder; M.P = 239–240 °C; Rf = 0.61 (methanol: chloroform, 1:9); FT-IR (ATR) in cm−1, 3222 (H–N), 3019 (H–C, Ar), 1709 (C=O), 1566 (C=N, imine), 1536 (C = C, Ar); 1H-NMR (300 MHz, CDCl3); in δ (ppm), 12.71 (s, 1H, OH), 11.89 (s, 1H, N–H), 9.65 (s, 1H, imine), 7.72 (d, 1H, J = 6.9 Hz, phenyl-H), 7.36 (s, 1H, phenyl-H), 7.27 (d, 1H, J = 6.9 Hz, phenyl-H), 3.67 (s, 3H, methoxy-H), 2.51 (s, 3H, CH3), 2.47(s, 3H, CH3); 13C-NMR (75 MHz, CDCl3) δ (ppm), 179.6 (acetyl, C=O), 155.7 (thiazole, C=N), 135.6 (imine, C), 131.7, 129.2, 128.2, 123.9, 123.7, 122.9, 122.8, 119.0, 56.3 (OMe), 68.7, 30.6, 19.2. Anal. Calcd. for C14H15N3O3S, C, 55.07; H, 4.95; N, 13.76; S, 10.50 found; C, 55.09; H, 4.98; N, 13.79; S, 10.55.

(E)-1-(2-(2-Benzylidenehydrazineyl)-4-methylthiazol-5-yl)ethan-1-one (3d)

Yield = 83%; Light green powder; M.P = 266–268 °C; Rf = 0.55 (ethyl acetate: n-hexane, 4:6); FT-IR (ATR) in cm−1, 3120 (H–N), 2960 (H–C, Ar), 1715 (C=O), 1578 (C=N, imine), 1484 (C = C, Ar); 1H-NMR (300 MHz, CDCl3); in δ (ppm), 11.71 (s, 1H, N–H), 9.75 (s, 1H, imine), 7.66 (m, 4H, phenyl-H), 2.43 (s, 3H, CH3), 2.33 (s, 3H, CH3); 13C-NMR (75 MHz, CDCl3) in δ (ppm), 180.0 (acetyl, C=O), 165.0 (thiazole, C=N), 136.3 (imine, C), 130.3, 128.7, 128.2, 123.9, 123.76, 122.9, 122.8, 33.1, 18.0. Anal. Calcd. for C13H13N3O2S, C, 56.71; H, 4.76; N, 15.26; S, 11.65, found; C, 56.73; H, 4.79; N, 15.29; S, 11.68.

(E)-1-(4-Methyl-2-(2-(4-nitrobenzylidene)hydrazineyl)thiazol-5-yl)ethan-1-one (3e)

Yield = 86%; Light orange powder; M.P = 286–287 °C; Rf = 0.53 (ethyl acetate: n-hexane, 4:6); FT-IR (ATR) in cm−1, 3122 (H–N), 2990 (H–C, Ar), 1697 (C=O), 1580 (C=N, imine), 1517 (C = C, Ar); 1H-NMR (300 MHz, CDCl3); in δ (ppm), 12.09 (s, 1H, N–H), 8.41 (s, 1H, imine), 7.52 (d, 2H, J = 8.1 Hz, phenyl-H), 7.23 (d, 2H, J = 8.1 Hz, phenyl-H), 2.62 (s, 3H, CH3), 2.51(s, 3H, CH3); 13C-NMR (75 MHz, CDCl3) in δ (ppm), 175.0 (acetyl, C=O), 160.1 (thiazole, C=N), 151.3, 138.0 (imine, C), 130.3, 129.0, 128.4, 124.5, 120.7, 118.5, 30.1, 18.4. Anal. Calcd. for C13H12N4O3S, C, 51.31; H, 3.97; N, 18.41; S, 10.54, found; C, 51.34; H, 3.99; N, 18.46; S, 10.57.

(E)-1-(4-Methyl-2-(2-(3-nitrobenzylidene)hydrazineyl)thiazol-5-yl)ethan-1-one (3f)

Yield = 77%; Yellow powder; M.P = 262–264 °C; Rf = 0.57 (ethyl acetate: n-hexane, 4:6); FT-IR (ATR) in cm−1, 3204 (H–N), 2980 (H–C, Ar), 1703 (C=O), 1588 (C=N, imine), 1489 (C = C, Ar); 1H-NMR (300 MHz, CDCl3); in δ (ppm), 11.34 (s, 1H, N–H), 8.63 (s, 1H, imine), 7.77 (t, 1H, J = 6.3 Hz, phenyl-H), 7.61 (s, 1H, phenyl-H), 7.23 (d, 1H, J = 6.3 Hz, phenyl-H), 7.11 (d, 1H, J = 6.3 Hz, phenyl-H),2.77 (s, 3H, CH3), 2.63(s, 3H, CH3); 13C-NMR (75 MHz, CDCl3) in δ (ppm), 172.1 (acetyl, C=O), 163.3 (thiazole, C=N), 160.6, 135.1 (imine, C), 147.3, 130.6, 130,1, 129.09, 128.9, 125.6, 124.6, 120.7, 118.5, 31.1, 17.6. Anal. Calcd. for C13H12N4O3S, C, 51.31; H, 3.97; N, 18.41; S, 10.54, found; C, 51.36; H, 3.99; N, 18.45; S, 10.56.

(E)-1-(2-(2-(4-Hydroxybenzylidene)hydrazineyl)-4-methylthiazol-5-yl)ethan-1-one (3g)

Yield = 82%; Light green powder; M.P = 297–298 °C; Rf = 0.53 (ethyl acetate: n-hexane, 4:6); FT-IR (ATR) in cm−1, 3123 (H–N), 2985 (H–C, Ar), 1687 (C=O), 1605 (C=N, imine), 1509 (C = C, Ar); 1H-NMR (300 MHz, CDCl3); in δ (ppm), 12.74 (s, 1H, OH), 11.53 (s, 1H, N–H), 8.19 (s, 1H, imine), 7.66 (d, 2H, J = 8.1 Hz, phenyl-H), 7.39 (d, 2H, J = 8.1 Hz, phenyl-H), 2.88 (s, 3H, CH3), 2.61(s, 3H, CH3); 13C-NMR (75 MHz, CDCl3) δ (ppm); 178.1 (acetyl, C=O), 164.1 (thiazole, C=N), 160.2, 135.6 (imine, C), 130.3, 129.0, 128.8, 124.7, 120.7, 119.5, 30.5, 17.1. Anal. Calcd. for C13H13N3O2S C, 56.71; H, 4.76; N, 15.26; S, 11.65, found; C, 56.74; H, 4.78; N, 15.29; S, 11.68.

(E)-1-(2-(2-(4-(Ethynyl(methyl)amino)benzylidene)hydrazineyl)-4-methylthiazol-5-yl)ethan-1-one (3h)

Yield = 86%; Green powder; M.P = 255–256 °C; Rf = 0.61 (methanol: chloroform, 1:9); FT-IR (ATR) in cm−1, 3122 (H–N), 2988 (H–C, Ar), 1670 (C=O), 1577 (C=N, imine), 1496 (C = C, Ar); 1H-NMR (300 MHz, CDCl3); in δ (ppm), 11.53 (s, 1H, N–H), 8.63 (s, 1H, imine), 7.69 (d, 2H, J = 8.1 Hz, phenyl-H), 7.57 (d, 2H, J = 8.1 Hz, phenyl-H), 2.88(s, 3H, CH3), 2.71(s, 3H, CH3), 2.60(s, 3H, CH3), 1.8 (s, 1H, CH); 13C-NMR (75 MHz, CDCl3) in δ (ppm), 179.1 (acetyl, C=O), 171.8, 167.1 (thiazole, C=N), 139.6 (imine, C), 130.3, 129.0, 128.8, 125.7, 120.78, 119.5, 81.51 72.78, 67.6, 40.1, 30.5, 17.1. Anal. Calcd. for C16H16N4OS C, 61.52; H, 5.16; N, 17.93; S, 10.26, found; C, 61.55; H, 5.18; N, 17.96; S, 10.27.

(E)-1-(2-(2-(3,4,5-Trimethoxybenzylidene)hydrazineyl)-4-methylthiazol-5-yl)ethan-1-one (3i)

Yield = 77%; Light green powder; M.P = 247–248 °C; Rf = 0.41(ethyl acetate: n-hexane, 4:6); FT-IR (ATR) in cm−1, 3130 (H–N), 2977 (H–C, Ar), 1698 (C=O), 1587 (C=N, imine), 1513 (C = C, Ar); 1H-NMR (300 MHz, CDCl3); in δ (ppm), 12.19 (s, 1H, N–H), 8.72 (s, 1H, imine), 7.77 (s, 1H, phenyl-H), 7.22 (s, 1H, phenyl-H), 3.73(s, 9H, methoxy), 2.47 (s, 3H, CH3), 2.44 (s, 3H, CH3); 13C-NMR (75 MHz, CDCl3) in δ (ppm), 176.0 (acetyl, C=O), 165.1 (thiazole, C=N), 136. (imine, C), 130.3, 128.9, 128.8, 123.7, 121.7, 119.9, 56.50, 56.4, 23.6, 15.1. Anal. Calcd. for C16H19N3O4S C, 55.00; H, 5.48; N, 12.03; S, 9.18 found; C, 55.04; H, 5.52; N, 12.07; S, 9.20.

Materials and methods

Transfection

COS-7 cells were used for transfection of h-TNAP and h-IAP proteins. When COS-7 cells reached to confluency level of 80–90%, serum-free DMEM (Dulbecco’s modified Eagle’s medium) containing 6 µg of plasmid and Lipofectamine (transfecting reagent) was added. Cells along with transfection mixture were incubated for 48–72 h at 37 °C in 5% CO2 incubator. After that, transfection mixture was replaced with normal growth medium. Transfected cells were washed with cold Tris-buffer saline and centrifuged the cells at 30g, at 4 °C for 5 min. Cells were washed with same ice cold buffer, and were resuspended in 10 µg/mL aprotinin. Protein was quantified and preserved in 7.5% glycerol and stored at −80 °C [17, 18].

Enzyme inhibition assay

Inhibitory potentials of thiazole derivatives were analyzed by using CDP-Star®(disodium 2-chloro-5-(4-methoxyspiro[1,2-dioxetane-3,20-(5-chlorotricyclo[3.3.1.13.7]decan])-4-yl]-1-phenyl phosphate) that is a chemiluminescent substrate. The inhibitory assay was performed after slight modification in previously reported spectrophotometric method [19]. Working enzymes were made in DEA (250 mM) buffer containing 0.005 mM ZnCl2 and 2.5 mM MgCl2. The working solutions of enzymes were 5 and 3.335 µg/mL for h-TNAP and h-IAP, respectively. Assay was preceded by adding 20 µL of enzyme followed by 10 µL of potential inhibitor in each well of 384 wells white plate. The final concentration of inhibitor in assay volume was 0.2 M. The assay mixture was incubated for 10 min at 37 °C. The reaction was started by addition of 20 µL of CDP-Star® in each well with final concentration of 105.2 and 177 µM for h-TNAP and h-IAP, respectively. Luminescence was measured at 0 min and second read was taken after 10 min of incubation by using microplate reader (BioTek FLx800, Instruments, Inc. USA). The activity of each inhibitor was compared with negative control (without any inhibitor). Inhibition potential of each compound was measured by following mentioned formula. Compounds exhibiting inhibition more than 50% were further investigated for IC50 values. All experiments were performed in triplicate format. PRISM 5.0 (GraphPad, San Diego, California, USA) was used for non-linear regression analysis for dose response curve and IC50 value determination [19].

Enzyme kinetics studies

Enzyme kinetics studies were performed to find out the mode of enzyme inhibition. Compounds 3c and 3f were selected for h-TNAP, as both the compounds were found to be most potent among the tested series. Both the compounds were used at concentration of 0, 0.1, 0.5 and 1 µM, whereas the compound 3f was used for enzyme kinetic study toward h-IAP. Here, the used compound concentrations were 0, 1.5, 3.0 and 6.0 µM. For all these mentioned compounds, the used CDP-Star substrate concentrations were 0, 12.5, 25, 50 and 100 µM.

Selection of the protein structures and preparation of ligands

Crystallographic structures of h-TNAP and h-IAP are not available; thus, homology modeled structures of h-TNAP and h-IAP were employed for molecular docking studies. The proteins and ligands were prepared prior to molecular docking studies using Molecular Operating Environment (MOE) [21]. h-TNAP and h-IAP models binding validation was done with the positive standards, Levamisole for h-TNAP and l-phenylalanine for h-IAP, used in the in vitro analysis. MOE site finder was used for the selections of binding site of these receptors, keeping the catalytic zinc ions in the center of the active site. After initial validation, molecular docking of the selected ligands was carried out [20].

Docking experiment

The homology models of h-TNAP and h-IAP previously reported were used without any alternation and modification to identify the putative binding modes of the inhibitors [21]. Molecular docking studies were performed on LeadIT (BioSolveIT GmbH, Germany) [22] through default parameters, for the selected compounds as well as the reference standards (used in in vitro assay). The most promising docked pose was selected for each ligand, and it was further analyzed by the HYDE assessment tool. The 3D interactions of the poses were examined using Discovery Studio Visualizer [23].

Results and discussion

Azomethine-clubbed thiazole were synthesized according to the route shown in Scheme 1. The first step involved the reaction of suitably substituted benzaldehydes with thiosemicarbazide in dry ethanol to afford azomethines intermediates (1a–i). Heterocyclization of azomethines (1a–i) with 3-chloroacetyl acetone (2) furnished the desired azomethine-thiazoles (3a–i) in good to excellent percentage yield and in high purity.

Scheme 1
scheme 1

Synthetic pathway to (E)-1-(2-(2-((aryl)methylene)hydrazineyl)-4-methylthiazol-5-yl)ethan-1-ones (3a–i)

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Figure 2 shows the complete molecular structures of the synthesized azomethine-thiazole derivatives (3a–i) along with their percent yields.

Fig. 2
figure 2

Chemical structures of new azomethine-clubbed thiazoles (3a–i)

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The FT-IR analysis revealed the characteristic absorptions for N–H at 3222–3103 cm−1, C–H aromatic at 2960–3019 cm−1, C=O at 1667–1715 cm−1 and C=N at 1566–1605 cm−1. In a typical case of 3a, 1H-NMR shows singlets at δ 12.44 for OH, at δ 11.71 for N–H, at 9.14 for imine proton, besides signals at for methyl of thiazole ring at 2.49, besides the methyl of acetyl group at δ 2.41. The aromatic protons appeared in the range of 7.91–7.23 ppm. 13C-NMR signals at δ 189.0 corresponded to acetyl carbonyl, that 179.0 to C=N of thiazole, 133.0 for ipso carbon attached to acetyl. Similarly, the methyl carbons of thiazole ring and acetyl resonated at 29.6 and 17.24, respectively.

Inhibitory potential of azomethine-thiazoles for alkaline phosphatases and Structure Activity Relationship (SAR)

All the synthesized thiazole derivatives were analyzed for inhibitory potential toward h-TNAP and h-IAP and results are listed in Table 1.

Table 1 Inhibitory potential of thiazole derivatives for h-TNAP and h-IAP
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The results indicated that all derivatives are inhibitors of both isozymes of alkaline phosphatases. Compounds 3c and 3f were found to most potent for h-TNAP with IC50 value of 0.15 ± 0.01 and 0.50 ± 0.01 µM with potency is much higher than standard Levamisole (IC50 of 25.2 ± 1.9 µM) and is also evident from the molecular docking scores (i.e., −17.65 and −21.02 for compounds 3c and 3f against −12.02 for levamisole). The presence of 3-hydroxy-4-methoxybenzylidene in 3c results in most potent inhibitor of TNAP. Removal of 3-hydroxyl group in 3b leads to decrease in inhibitory potential for h-TNAP that signifies the importance of hydroxyl group at position 3 of the benzene ring. The molecular basis for the hydroxyl group activity is clarified by the docking study which showed its hydrogen bonding potential with Glu315 and Arg167. Similarly, 3d and 3g having the hydroxyl group on positions 2 and 4, respectively, also exhibited the potent inhibition (Fig. 3). Lower binding scores of −14.09 for compound 3b were observed against the binding scores of −14.26 and −15.74 for compounds 3d and 3g. Similarly, the presence of nitro (–NO2) on phenyl ring also increases the potency about 95 times for 3e (IC50: 12.30 ± 0.3 µM and binding scores of −14.52) to 3f (IC50: 050 ± 0.01 µM and binding scores of −21.02).

Fig. 3
figure 3

Influence of hydroxyl group (–OH) and nitro group (–NO2) substitutions on inhibitory potential of thiazole derivatives for h-TNAP

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Fig. 4
figure 4

Effect of substitution of aryl-ring on the inhibitory potential of thiazole derivatives for h-IAP

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For h-IAP, 3f and 3a were found to be more potent with IC50 values of 2.56 ± 0.02 and 2.59 ± 0.04 µM, respectively. Both inhibitors exhibited more potent activity as compared to l-phenylalanine (IC50: 100 ± 3.0 µM), a positive inhibitor of h-IAP. Replacement of 2-hydroxyphenyl in 3d with 2-hydroxynaphthyl in 3a caused to increase in inhibitory potency from 6.91 ± 0.1 to 2.59 ± 0.04 µM and binding scores of −18.97 to −20.99, respectively (Fig. 4). In addition, 3f 3c and 3g were found to be more selective for h-TNAP as compared to h-IAP, as shown by respective IC50 values for both isozymes.

Enzyme Kinetics studies for h-TNAP and h-IAP

Lineweaver Burk plot was used to define the enzyme kinetic parameters of most potent inhibitors of h-TNAP and h-IAP. Compound 3c exhibited mixed type of inhibition for h-TNAP. Similarly, 3f was also analyzed for enzyme kinetic studies for both ALP isozymes. It was found that this molecule has competitive mode of inhibition for h-TNAP and uncompetitive mode of inhibition for h-IAP. Here, the used substrate concentrations were 0, 12.5, 25, 50 and 100 µM. (Fig. 5).

Fig. 5
figure 5

Double-reciprocal plots of the inhibition kinetics of h-TNAP by compounds 3c (a) exhibited mixed type of inhibition and 3f (b) indicating competitive mode of inhibition. For h-IAP, 3f exhibited uncompetitive type of inhibition (c)

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Molecular docking studies

Molecular docking studies against TNAP

The binding process of potent compounds in h-TNAP was validated through the binding mechanism of its positive control Levamisole (Fig. 6). In vitro analysis reckoned 3c, 3f and 3g as promising inhibitors of the TNAP protein. Zn2+483 ion and His154 were the common residues to bind with all the selected test compounds. Ser93, Arg167 and Asp320 were the other prominent residues to interact with the test compounds. Major interactions depicted were hydrogen bonding, π–π interactions, π–alkyl interactions and π–cation/anionic interactions between the residues and compounds. The compound 3c showed hydrogen bonding with Asp43, Ser93, His154, Arg167 and His437 residues, along with π–cation/anionic interactions with Zn2+483, Asp320 and π–sulfur interactions with His324 residue of the protein (Fig. 7). Compound 3f exhibited hydrogen bonding with His154, His434, Thr436 and His437 residues, along with π–cation/anionic interactions with Zn2+483, Zn2+484, Arg167, Asp320 and π–π interactions with His324 residue of the protein (Fig. 8). Compound 3g interacted through H-bonding with Gly317, Thr436 and His437 residues, along with π–cation/anionic interactions with Zn2+483, Arg167 and π–π interactions with His154, His324 and His434 residues of the protein (Fig. 9).

Fig. 6
figure 6

Levamisole docking with h-TNAP

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Fig. 7
figure 7

Compound 3c docking with h-TNAP

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Fig. 8
figure 8

Compound 3f docking with h-TNAP

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Fig. 9
figure 9

Compound 3g docking with h-TNAP

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Molecular docking studies for (h-IAP)

l-phenylalanine was docked into the h-IAP protein to find the binding site residues to validate the bindings for the test compounds. l-phenylalanine exhibited interactions with the two Zn2+ ions along with hydrogen bonding with Ser92, Arg166, His432 and His358 while π–π interactions with His317 and His320 residues (Fig. 10). In vitro analysis found 3a and 3f as potent inhibitors of the h-IAP protein, and the molecular docking provided the validation for it by imitating the interactions of l-phenylalanine. The compound 3a showed hydrogen bonding with Ser92, His153, Arg166, Thr431 and His432 residues, along with π–cation/anionic interactions with Zn2+530, Asp316 and π–sulfur interactions with His320 residue of the protein. Phe107 and His320 also exhibited π–π interactions with the compound 3a (Fig. 11). The compound 3f showed hydrogen bonding with Ser92, Arg166, His358 and His432 residues, along with π–cationic interactions with Zn2+530, Zn2+531, Arg166, Asp316, Asp357 and π–lone pair interactions with Ser429 residue of the protein (Fig. 12).

Fig. 10
figure 10

l-phenylalanine docking with h-IAP

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Fig. 11
figure 11

Compound 3a docking with h-IAP

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Fig. 12
figure 12

Compound 3f docking with h-IAP

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The docking score of the whole series against both isozymes was carried out and results are listed in Table 2.

Table 2 Docking scores of compounds (3a–3i)
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Conclusions

Newly synthesized azomethine-thiazoles were evaluated for their inhibitory potential against h-TNAP and h-IAP. Compounds 3c and 3f were found to be most potent compounds toward h-TNAP with IC50 values of 0.15 ± 0.01 and 0.50 ± 0.01 µM, respectively, while 3a and 3f exhibited maximum potency for h-IAP with IC50 value of 2.59 ± 0.04 and 2.56 ± 0.02 µM, respectively. The compounds exhibiting high potency were also subjected to the molecular docking studies which revealed significant binding interactions between the selected compounds and enzyme active pockets. Such interactions provided further confirmation of in vitro analysis.