Chemical synthesis of novel aminopyrimidin-4-yl-1H-pyrazole derivatives as spleen tyrosine kinase (SYK) inhibitors

Abstract

Spleen tyrosine kinase (SYK) plays a pivotal role in immunoreceptor signalling pathways implicated in various cancers and autoimmune diseases. Inhibition of SYK has shown therapeutic potential by attenuating immune-mediated damage and suppressing tumour growth. This study reports the synthesis, characterization, and evaluation of a novel series of aminopyrimidin-4-yl-1H-pyrazole derivatives (31, 35, 36, 37, 39, 41, 44, 47 and 49) as selective SYK inhibitors. The chemically synthesized molecules were structurally confirmed using ¹H, ¹³C NMR and mass spectrometry techniques. Further, molecular docking studies revealed differential binding affinities with the SYK active site, highlighting molecule 44 as the lead candidate, displaying the lowest docking score and strongest interactions. Subsequent 200 ns molecular dynamics simulations confirmed the enhanced stability of 44 in complex with SYK, evidenced by consistent hydrogen bonding, minimal root-mean square deviation (RMSD), and compact protein folding relative to other ligands. Furthermore, binding free energy calculations corroborated the favourable energetic profile of 44. Also, principal component and free energy landscape analyses showed that 44 maintained a single, stable conformational state with limited flexibility. On the other hand, in vitro enzyme inhibition assays employing a luminescence-based kinase activity system demonstrated that several compounds, particularly 44, exhibited potent concentration and time-dependent inhibition of SYK inhibition activity. Kinetic characterization revealed that 44 functions via a non-competitive inhibition mechanism, suggesting allosteric binding outside the ATP substrate site. These integrative in silico and in vitro findings establish 44 as a promising lead molecule for SYK-targeted therapies, combining potent binding affinity, conformational stability, and effective enzyme inhibition. This work advances the design of novel small-molecule SYK inhibitors with potential applications in cancer and autoimmune disease treatment.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-026-38719-w.

Introduction

Acute myeloid leukemia (AML) is a diverse and aggressive cancer characterized by uncontrolled proliferation, enhanced survival, and poor differentiation of hematopoietic stem and progenitor cells¹. In an effort to find novel treatment approaches, a study combining chemical, proteomic, and genomic methods initially identified spleen tyrosine kinase (SYK) activity and oncogenic characteristics in AML^2,3. Activated immunoreceptors use SYK as a key signal transducer in a variety of downstream processes, including as phagocytosis, differentiation, and proliferation, which vary according on the type of cell⁴. Since SYK controls cellular events brought on by the B-cell receptor with high intrinsic activity and mediates important signal transduction pathways after immune cell receptor activation, it has been considered a desirable target for the treatment of several illnesses, including asthma and arthritis. Thus, in the pathophysiology of autoimmune disorders, pharmacological inhibition of SYK’s catalytic function is anticipated to have pleiotropic anti-inflammatory effects due to its central role in the transmission of antigen receptor signals that are essential for autoantibody production and the various innate immune effector functions⁵. Furthermore, previous research has shown that SYK can be a desirable target for anticancer therapy since it slows the growth of tumors and triggers apoptosis when its expression is silenced^6–10. In numerous cancer types, including B-cell lymphocytic leukemia, breast cancer, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, pancreatic cancer, lung cancer, prostate cancer, retinoblastoma, ovarian cancer, and small cell lung cancer, it has been demonstrated that SYK inhibition promotes apoptosis^11–15. Consequently, there has been an increasing interest in creating SYK small-molecule inhibitors that can be utilized to treat cancer and autoimmune illnesses^16–19.

Fostamatinib disodium, the pro-drug used to treat adults with persistent immune thrombocytopenia (ITP), has been investigated for its ability to target SYK. A phase II trial revealed that the SYK inhibitor was active therapeutic approach for NHL (Non-Hodgkin Lymphoma) and CLL (Chronic Lymphocytic Leukemia)²⁰. Although fostamatinib’s phase III clinical study for rheumatoid arthritis yielded poor results, there is still a need for additional SYK inhibitors and innovative approaches to treating cancer and autoimmune illnesses²¹. A significant number of pharmaceutical corporations and academic institutions have been active in the development of small-molecule SYK inhibitors, demonstrating the high level of interest, in addition to the growing number of worldwide applications in recent years. Table 1 depicts the previously reported promising SYK inhibitors²². in a recent study, Sovleplenib is reported as a selective SYK inhibitor with a significant preclinical test evaluating its anti-inflammation potential²³. In another report, discovery of lanraplenib exhibited human pharmacokinetic properties. Further, lanraplenib is presently under clinical trials in multiple autoimmune indications²⁴. Furthermore, the selectivity of 2-aminopyrimidine-based spleen tyrosine kinase (SYK) inhibitors improves due to several structural traits that enable specific interactions with unique residues in the SYK ATP-binding pocket. This quality sets them apart from other kinases. A key factor in selectivity involves the use of rare amino acid residues in the SYK hinge region, specifically Pro455 and Asn457. These residues create a unique binding environment not usually found in other kinases. Strengthening hydrogen bond interactions with these residues gives 2-aminopyrimidine scaffold SYK inhibitors high selectivity²⁵. Molecular docking and molecular dynamics studies show that, besides forming typical hydrogen bonds with hinge residue Ala451, selective SYK inhibitors also interact with residues in the glycine-rich loop (Lys375, Ser379) and the DFG motif (Asp512). These interactions boost affinity and selectivity²⁶. They help distinguish SYK from other kinases that have more conserved ATP-binding sites. The structural rigidity and specific conformational setup of the 2-aminopyrimidine-based inhibitor scaffold, which includes defined atropisomerism, can further enhance selectivity. This setup favours a bioactive conformation that fits the SYK binding pocket very well and reduces off-target binding to other kinases²⁷. Selective SYK inhibitors, like GS-9973, were created by adjusting their chemical structure to reduce off-target activities seen with earlier inhibitors such as R406. This adjustment improves the therapeutic window and kinase selectivity profiles²⁸. In summary, the main structural features that boost the selectivity of 2-aminopyrimidine-based SYK inhibitors include strong and optimized hydrogen bonding with rare hinge residues Pro455 and Asn457, additional stabilizing interactions with glycine-rich and activation loop residues, and conformational features that encourage selective binding unique to SYK.

Table 1.

Significant SYK inhibitors reported in the literature.

Given the critical importance of designing and developing small molecules as SYK inhibitors with minimal off-target effects, firstly we examined the interactions of the proposed and synthesized chemical molecules along with their some of the derivatives to identify those with significant interactions with SYK through molecular docking and molecular dynamics studies. We have then synthesized potent novel aminopyrimidin-4-yl-1 H-pyrazole derivatives and assessed there in vitro SYK inhibitory potential. The in silico findings were subsequently validated experimentally by measuring SYK kinase activity and inhibition using ATP-dependent luminescence detection.

Experimental

Materials and methods

The chemicals and reagents used in this study were procured from the commercial sources and used without further purification. The ¹H and ¹³C NMR spectra were recorded on Bruker AVANCE NEO 400 MHz (Germany) and Varian Mercury 300 MHz spectrometer (USA) spectrometer. The mass spectra (ESI) were recorded on Shimadzu LCMS-SQ-2020-N series (Shimadzu Scientific Instruments, Japan) with ESI source and ionization.

Chemical synthesis

Synthesis of ethyl 1-(2-chloropyrimidin-4-yl)-1H-pyrazole-4-carboxylate (2)

To a solution of 2,4-dichloropyrimidine (50 mg, 0.21 mmol, 1 eq) in DMF (2 ml) under argon atmosphere, ethyl 1H-pyrazole-4-carboxylate (30 mg, 0.25 mmol, 1.2 eq) and K₂CO₃ (58 mg, 0.42 mmol, 2 eq) was added. The reaction mixture was heated at 80 °C for 4 h (The reaction was monitored by TLC). The reaction mixture was quenched with ice cold water and extracted with ethyl acetate. The combined organic layer washed with water and brine solution, dried over sodium sulphate, and concentrated to get the crude. Which was purified by MPLC to get the title compound ethyl 1-(2-chloropyrimidin-4-yl)-1H-pyrazole-4-carboxylate 2. Yield: 43 g (50%); ¹H NMR (400 MHz, CHLOROFORM-d) δ 9.06 (d, J = 0.6 Hz, 1H), 8.70 (d, J = 5.6 Hz, 1H), 8.16 (d, J = 0.7 Hz, 1H), 7.90 (d, J = 5.6 Hz, 1H), 2.98 (q, J = 5.9 Hz, 2 H), 1.26 (t, 3 H); LCMS m/z = 254 (M + 2 H).

Synthesis of ethyl 1-(2-((3,5-dimethylphenyl)amino)pyrimidin-4-yl)-1H-pyrazole-4-carboxylate) (3)

To a solution of ethyl 1-(2-chloropyrimidin-4-yl)-1H-pyrazole-4-carboxylate (50 mg, 1 eq) in DMF (2 ml) under argon atmosphere, 2,4-dimethylaniline (55 mg, 1.2 eq) and K₂CO₃ (70 mg, 2 eq) was added. The reaction mixture was heated at 100 °C for 4 h (the reaction was monitored by TLC). The reaction mixture was quenched with ice cold water and extracted with ethyl acetate. The combined organic layer washed with water and brine solution, dried over sodium sulphate, and concentrated to get the crude. Which was purified by MPLC to get the title compound ethyl 1-(2-((3,5-dimethylphenyl)amino)pyrimidin-4-yl)-1H-pyrazole-4-carboxylate (3) as off white solid. Yield: 27 g (40%); ¹H NMR (400 MHz, CHLOROFORM-d) δ = 8.54 (d, J = 8 Hz, 1H), 8.51 (d, J = 2 Hz, 1H), 8.12 (d, 1H), 7.37 (m, 2 H), 7.33 (d, J = 5.1 Hz, 1H), 7.11–7.10 (d, J = 2 Hz, 1H ), 6.77–6.75 (d, J = 8 Hz, 1H), 4.29–4.35 (m, 2 H), 2.36 (s, 6 H), 1.26–1.28 (t, 3 H); LCMS m/z = 338 (M + H).

Synthesis of 1-(2-((3,5-dimethylphenyl)amino)pyrimidin-4-yl)-1H-pyrazole-4-carboxylic acid (4)

To a solution of ethyl 1-(2-((3,5-dimethylphenyl)amino)pyrimidin-4-yl)-1H-pyrazole-4-carboxylate (50 mg, 1 eq) in THF: H₂O (3:1 ml) under argon atmosphere, lithium hydroxide was added and stirred the reaction mixture at 25 °C for 24 h (the reaction was monitored by TLC). The white precipitate was observed which was filtered and dried to get the title compound 1-(2-((3,5-dimethylphenyl)amino)pyrimidin-4-yl)-1H-pyrazole-4-carboxylic acid (4). Yield: 23 g (50%); ¹H NMR (400 MHz, DMSO-d₆) δ = 12.75 (s, 1H), δ 8.54 (d, J = 8 Hz, 1H), 8.51 (d, J = 2 Hz, 1H), 8.12 (d, 1H), 7.37 (m, 2 H), 7.33 (d, J = 5.1 Hz, 1H), 7.11–7.10 (d, J = 2 Hz, 1H ), 6.77–6.75 (d, J = 8 Hz, 1H), 2.36 (s, 6 H); LCMS m/z = 310 (M + H).

Synthesis of N-benzyl-1-(2-((3,5-dimethylphenyl)amino)pyrimidin-4-yl)-1H-pyrazole-4-carboxamide (31)

To a solution of 1-(2-((3,5-dimethylphenyl)amino)pyrimidin-4-yl)-1H-pyrazole-4-carboxylic acid (20 mg, 1 eq) in NMP (2 ml) under argon atmosphere, the benzyl amine (7.68 mg, 0.077 mmol, 1.2 eq, EDC.HCl (25 mg, 0.096 mmol, 1.5 eq) and DIPEA (16.51 mg, 0.1 mmol, 2 eq) was added and stirred the reaction mixture at rt for 12 h (the reaction was monitored by TLC). The reaction was quenched with ice cold water and extracted with ethyl acetate, washed with brine solution, dried over sodium sulphate and concentrated to get the crude compound, which was purified by MPLC to get the title compound as N-benzyl-1-(2-((3,5-dimethylphenyl)amino)pyrimidin-4-yl)-1H-pyrazole-4-carboxamide (31). Yield: 15 mg (60%); ¹H NMR (400 MHz, DMSO-d₆) δ = 9.74 (s, 1H), 9.06–9.02 (m, 2 H), 8.61 (d, J = 5.2 Hz, 1H), 8.28 (s, 1H), 7.39 (d, J = 7.6 Hz, 2 H), 7.36–7.33 (m, 4 H), 7.28–7.25 (m, 2 H), 6.68 (s, 1H), 4.47 (d, J = 6.0 Hz, 2 H), 2.26 (s, 6 H) (Fig. S1); ¹³C NMR (400 MHz, DMSO-d6) δ 160.97, 160.80, 159.59, 156.51, 142.41, 139.63, 139.38, 137.48, 128.29, 128.18, 127.25, 126.81, 123.76, 121.15, 117.19, 98.88, 42.06, 21.21 (Fig. S2). LCMS m/z = 399 (M + H) (Fig. S3).

Compound 35: 1-(2-((3,5-dimethylphenyl) amino) pyrimidin-4-yl)-N-ethyl-1 H-pyrazole-4-carboxamide.

Yield: 70%; ¹H NMR (400 MHz, DMSO-d₆) δ = 8.54 (d, J = 8 Hz, 1H), 8.51 (d, J = 2 Hz, 1H), 8.38(t, 1H), 8.12 (d, 1H), 7.37 (m, 2 H), 7.33 (d, J = 5.1 Hz, 1H), 7.11–7.10 (d, J = 2 Hz, 1H ), 6.77–6.75 (d, J = 8 Hz, 1H), 4.29–4.35 (m, 2 H), 2.36 (s, 6 H), 1.26–1.28 (t, 3 H) (Fig. S4); ¹³C NMR (400 MHz, DMSO-d6) δ 160.93, 160.52, 159.58, 156.52, 142.25, 139.64, 137.47, 127.98, 123.76, 121.47, 121.15, 117.21, 98.84, 33.46, 21.20, 14.75 (Fig. S5). LCMS m/z = 337 (M + H) (Fig. S6).

Compound 36: 1-(2-((3,5-dimethylphenyl) amino) pyrimidin-4-yl)-1 H-pyrazole-4-carboxamide.

Yield: 65%; ¹H NMR (400 MHz, DMSO-d₆) δ = 9.73 (s, 1H), 8.96 (s, 1H), 8.58 (d, J = 5.2 Hz, 1H), 8.22 (s, 1H), 7.96 (s, 1H), 7.38 (s, 2 H), 7.29–7.25 (m, 2 H), 6.68 (s, 1H), 2.28 (s, 6 H) (Fig. S7); ¹³C NMR (400 MHz, DMSO-d6) δ 162.58, 160.91, 159.60, 156.59, 142.75, 139.63 137.47, 128.28, 123.76, 121.37, 117.22, 98.96, 21.22 (Fig. S8). LCMS m/z = 309 (M + H) (Fig. S9).

Compound 37: 1-(2-((3,5-dimethylphenyl) amino) pyrimidin-4-yl)-N-(pyridine-3-ylmethyl)-1 H-pyrazole-4-carboxamide.

Yield: 45%; ¹H NMR (400 MHz, DMSO-d₆) δ = 9.75 (s, 1H), 9.11 (t, J = 5.7 Hz, 1H), 9.01 (s, 1H), 8.61 (d, J = 5.2 Hz, 1H), 8.56 (s, 1H), 8.47 (d, J = 3.6 Hz, 1H), 8.29 (s, 1H), 7.74 (d, J = 8.0 Hz, 1H), 7.39 (d, J = 10.0 Hz, 3 H), 7.25 (d, J = 5.2 Hz, 1H), 6.67 (s, 1H), 4.47 (d, J = 5.6 Hz, 2 H), 2.27 (s, 6 H) (Fig. S10); ¹³C NMR (400 MHz, DMSO-d6) δ 160.98,159.58, 156.49, 148.84, 148.15, 142.40, 139.61, 137.48, 135.16, 134.83, 128.21, 123.77, 123.47, 120.93, 117.20, 98.88, 21.21 (Fig. S11). LCMS m/z = 400 (M + H) (Fig. S12).

Compound 47: 1-(1-(2-((3,5-dimethylphenyl) amino) pyrimidin-4-yl)-1 H-pyrazole-4-carbonyl) piperidine-4-carbonitrile.

Yield: 35.5%; ¹H NMR (400 MHz, DMSO-d₆) δ = 8.54 (d, J = 8 Hz, 1H), 8.51 (d, J = 2 Hz, 1H), 8.12 (d, 1H), 7.37 (m, 2 H), 7.33 (d, J = 5.1 Hz, 1H), 7.11–7.10 (d, J = 2 Hz, 1H ), 6.77–6.75 (d, J = 8 Hz, 1H), 3.49–3.43 (m, 4 H), 2.55 (s,1H ) 2.45–2.42 (m, 4 H), 2.27 (s, 6 H) (Fig. S22); ¹³C NMR (400 MHz, DMSO-d6) δ 161.54, 160.93, 159.56, 156.49, 143.21, 139.63, 137.42, 127.76, 123.73, 121.85, 119.25, 117.17, 98.85, 30.66, 28.11, 25.36, 21.23 (Fig. S23). LCMS m/z = 402 (M + H) (Fig. S24).

Synthesis of tert-butyl (2-(1-(2-((3,5-dimethylphenyl)amino)pyrimidin-4-yl)-1H-pyrazole-4-carboxamido)ethyl)carbamate (4a)

To a solution of 1-(2-((3,5-dimethylphenyl)amino)pyrimidin-4-yl)-1H-pyrazole-4-carboxylic acid (20 mg, 1 eq) in NMP (2 ml) under argon atmosphere, the tert-butyl (2-aminoethyl)carbamate (7.68 mg, 0.077 mmol,1.2 eq, EDC.HCl (25 mg, 0.096 mmol, 1.5 eq) and DIPEA (16.51 mg, 0.1 mmol, 2 eq) was added and stirred the reaction mixture at rt for 12 h (the reaction was monitored by TLC). The reaction was quenched with ice cold water and extracted with ethyl acetate, washed with brine solution, dried over sodium sulphate and concentrated to get the crude compound, which was purified by MPLC to get the title compound as tert-butyl (2-(1-(2-((3,5-dimethylphenyl)amino)pyrimidin-4-yl)-1H-pyrazole-4-carboxamido)ethyl)carbamate. Yield: 14.5 mg (50%); ¹H NMR (400 MHz, DMSO-d6) δ = 9.77 (s, 1H), 8.95 (d, J = 0.4 Hz, 1H), 8.76 (t, J = 5.4 Hz, 1H), 8.60 (d, J = 5.2 Hz, 1H), 8.32 (d, J = 0.4 Hz, 1H), 7.94 (s, 3 H), 7.40 (s, 2 H), 7.26 (d, J = 5.2 Hz, 1H), 6.68 (s, 1H), 3.50 (t, J = 6.1 Hz, 2 H), 2.98 (q, J = 5.9 Hz, 2 H), 2.29 (s, 6 H);1.45 (s, 9 H); LCMS m/z = 456 (M + H).

Compound 4g: Yield: 65%; ¹H NMR (400 MHz, DMSO-d₆) δ = 9.74 (s, 1H), 8.96 (s, 1H), 8.70 (t, J = 5.5 Hz, 1H), 8.60 (d, J = 5.2 Hz, 1H), 7.82 (s, 2 H), 7.39 (s, 2 H), 7.26 (d, J = 5.2 Hz, 1H), 6.68 (s, 1H), 3.30 (q, J = 6.4 Hz, 2 H), 2.89–2.81 (m, 2 H), 2.28 (s, 6 H), 1.83–1.76 (m, 2 H); 1.45 (s, 9 H); LCMS m/z = 465 (M + H).

Compound 4h: Yield: 60%; ¹H NMR (400 MHz, DMSO-d₆) δ = 9.74 (s, 1H), 8.96 (s, 1H), 8.70 (t, J = 5.5 Hz, 1H), 8.60 (d, J = 5.2 Hz, 1H), 7.82 (s, 2 H), 7.39 (s, 2 H), 6.68 (s, 1H), 3.30 (q, J = 6.4 Hz, 2 H), 3.4 (m, 1H),3.1(m,1H),2.89–2.81 (m, 4 H), 2.89–2.81 (m, 4 H), 2.28 (s, 6 H); 1.45 (s, 9 H); LCMS m/z = 506 (M + H).

Compound 4i: Yield: 55%; ¹H NMR (400 MHz, DMSO-d₆) δ = 9.74 (s, 1H), 8.96 (s, 1H), 8.70 (t, J = 5.5 Hz, 1H), 8.60 (d, J = 5.2 Hz, 1H), 7.82 (s, 2 H), 7.39 (s, 2 H), 6.68 (s, 1H), 2.89–2.81 (m, 8 H), 2.28 (s, 6 H); 1.45 (s, 9 H); LCMS m/z = 478 (M + H).

Synthesis of N-(2-aminoethyl)-1-(2-((3,5-dimethylphenyl)amino)pyrimidin-4-yl)-1 H-pyrazole-4-carboxamide hydrochloride (39)

To a stirred solution of tert-butyl (2-(1-(2-((3,5-dimethylphenyl)amino)pyrimidin-4-yl)-1H-pyrazole-4-carboxamido)ethyl)carbamate (50 mg in 5 ml DCM and was added HCl (Dioxane) (0.102 ml, 0.408 mmol) at 0 °C and the reaction mixture was stirred for 2 h at 25 °C. The reaction mixture was concentrated under reduced pressure to get the crude. The obtained crude was washed with diethyl ether and pentane to afford the N-(2-aminoethyl)-1-(2-((3,5-dimethylphenyl)amino)pyrimidin-4-yl)-1H-pyrazole-4-carboxamide hydrochloride as white solid (39). Yield: 29 mg (70%); ¹H NMR (400 MHz, DMSO-d6) δ = 9.77 (s, 1H), 8.95 (d, J = 0.4 Hz, 1H), 8.76 (t, J = 5.4 Hz, 1H), 8.60 (d, J = 5.2 Hz, 1H), 8.32 (d, J = 0.4 Hz, 1H), 7.94 (s, 3 H), 7.40 (s, 2 H), 7.26 (d, J = 5.2 Hz, 1H), 6.68 (s, 1H), 3.50 (t, J = 6.1 Hz, 2 H), 2.98 (q, J = 5.9 Hz, 2 H), 2.29 (s, 6 H) (Fig. S13); ¹³C NMR (400 MHz, DMSO-d6) δ 161.38,160.81, 159.46, 156.53, 142.44, 139.54, 137.50, 128.42, 123.87, 121.11, 117.30, 98.90, 36.54, 21.23 (Fig. S14). LCMS m/z = 352 (M + H) (Fig. S15).

Compound 41: N-(3-aminopropyl)-1-(2-((3,5-dimethylphenyl)amino)pyrimidin-4-yl)-1 H-pyrazole-4-carboxamide hydrochloride.

Yield: 70%; ¹H NMR (400 MHz, DMSO-d₆) δ = 9.74 (s, 1H), 8.96 (s, 1H), 8.70 (t, J = 5.5 Hz, 1H), 8.60 (d, J = 5.2 Hz, 1H), 7.82 (s, 2 H), 7.39 (s, 2 H), 7.26 (d, J = 5.2 Hz, 1H), 6.68 (s, 1H), 3.30 (q, J = 6.4 Hz, 2 H), 2.89–2.81 (m, 2 H), 2.28 (s, 6 H), 1.83–1.76 (m, 2 H) (Fig. S16); ¹³C NMR (400 MHz, DMSO-d6) δ 161.06, 160.82, 159.48, 156.55, 142.37, 139.57, 137.50, 128.16, 123.84, 121.20, 117.28, 98.90, 36.75, 35.76, 27.34, 21.23 (Fig. S17). LCMS m/z = 366 (M + H) (Fig. S18).

Compound 44: N-(2-aminocyclohexyl)-1-(2-((3,5-dimethylphenyl) amino) pyrimidin-4-yl)-1 H-pyrazole-4-carboxamide hydrochloride.

Yield: 80%; ¹H NMR (400 MHz, DMSO-d₆) δ = 9.74 (s, 1H), 8.96 (s, 1H), 8.70 (t, J = 5.5 Hz, 1H), 8.60 (d, J = 5.2 Hz, 1H), 7.82 (s, 2 H), 7.39 (s, 2 H), 6.68 (s, 1H), 3.30 (q, J = 6.4 Hz, 2 H), 3.4 (m, 1H),3.1(m,1H),2.89–2.81 (m, 4 H), 2.89–2.81 (m, 4 H), 2.28 (s, 6 H) (Fig. S19); ¹³C NMR (400 MHz, DMSO-d6) δ 161.68, 161.09, 159.52, 156.55, 143.12, 142.66, 139.60, 137.54, 128.92, 123.80, 121.43, 120.94, 117.24, 98.82, 53.20, 50.65, 47.18, 31.36, 27.42, 24.15, 21.22 (Fig. S20). LCMS m/z = 406 (M + H) (Fig. S21).

Compound 49: (1-(2-((3,5-dimethylphenyl) amino) pyrimidin-4-yl)-1 H-pyrazol-4-yl)(piperazin-1-yl) methanone hydrochloride.

Yield: 78%; ¹H NMR (400 MHz, DMSO-d₆) δ = 9.74 (s, 1H), 8.96 (s, 1H), 8.70 (t, J = 5.5 Hz, 1H), 8.60 (d, J = 5.2 Hz, 1H), 7.82 (s, 2 H), 7.39 (s, 2 H), 6.68 (s, 1H), 2.89–2.81 (m, 8 H), 2.28 (s, 6 H) (Fig. S25); ¹³C NMR (400 MHz, DMSO-d6) δ 160.94, 160.35, 159.57, 156.2, 142.52, 139.66, 137.49, 128.06, 123.74, 121.41, 117.16, 98.81, 50.38, 32.19, 23.54, 21.18 (Fig. S26). LCMS m/z = 378 (M + H) (Fig. S27).

Molecular docking and MD simulations

Molecular docking

Molecular docking was performed using AutoDock 4.2 to predict the binding interactions between the target protein and selected synthesized molecules^31–33. The three-dimensional crystal structure of the protein was retrieved from the Protein Data Bank (PDB) and prepared by removing water molecules and heteroatoms, followed by the addition of polar hydrogens and assignment of Kollman charges. Molecular structures were processed by adding Gasteiger charges and defining rotatable bonds. Both protein and synthesized molecules were saved in PDBQT format using AutoDockTools. A grid box was generated covering the entire protein for blind docking with a spacing of 1.0 Å, and the grid parameter file (GPF) was prepared. Docking simulations were performed using the Lamarckian Genetic Algorithm with multiple runs to ensure reliable conformational sampling and docking parameter files (DPF) were created accordingly. AutoGrid was used to pre-calculate the interaction energies, and AutoDock executed the docking runs to generate possible binding poses.

Molecular dynamics

Molecular dynamics (MD) simulations were conducted using GROMACS version 2024.2, a high-performance molecular simulation toolkit³⁴. The protein–protein complexes obtained from HADDOCK were subjected to energy minimization using the steepest descent algorithm. The system was then equilibrated under NVT (constant number of particles, volume, and temperature) and NPT (constant number of particles, pressure, and temperature) ensembles. Simulations were carried out using an appropriate force field (AMBER ff99SB-ILDN) and explicit solvent models like SPC water in a periodic dodecahedron box³⁵. Long-range electrostatics were treated using the Particle Mesh Ewald (PME) method. The production run was conducted for 100 ns, during which key structural and energetic parameters, such as root mean square deviation (RMSD), root mean square fluctuation (RMSF), radius of gyration (Rg), and hydrogen bonding, were calculated to assess the stability and dynamics of the complex.

Binding energy estimation

The binding free energy of the complex was estimated using the Molecular Mechanics/Poisson–Boltzmann Surface Area (MM/PBSA) method^36,37. Post-processing of the MD trajectories was performed to extract representative snapshots for energy calculation. Calculations were carried out using tools such as gmx_mmpbsa, and binding energy values were averaged over multiple frames to ensure statistical reliability. The results provide an estimate of the binding affinity and energetic contributions of each component of the complex.

Principal component analysis (PCA) and free energy landscape (FEL) analysis

PCA was used to analyze trajectory’s high amplitude concerted motion leveraging the eigenvectors derived from the mass-weighted covariance matrix of protein atomic fluctuations. The cosine content (ci) of each principal component (pi) in the covariance matrix was computed to construct the free energy landscape via PCA analysis. GROMACS’s built-in tools, “g_covar” for generating the covariance matrix with the protein backbone as a reference structure for rotational fitting, and “g_anaeig” for analyzing and visualizing the eigenvectors, were employed. Principal components exhibiting smaller cosine content values, typically below 0.2, tend to produce qualitatively superior results, often showing a single basin³⁸. The Free Energy Landscape (FEL) was constructed by utilizing the cosine contents of the first two projection eigenvectors (referred to as PC1 and PC2) that were below 0.2.

Measurement of SYK kinase activity and Inhibition using ATP-dependent luminescence detection

The enzymatic inhibition screening for SYK (Spleen tyrosine kinase) was performed using a luminescence-based kinase assay system employing the Kinase-Glo Max reagent (Promega), a sensitive and quantitative ATP detection method based on luciferase-mediated luminescence^39,40. All assays were performed in duplicate to ensure reproducibility and statistical confidence. Initially, key reagents were thawed to room temperature, including 5x kinase assay buffer, ATP solution (500 µM), and the synthetic peptide substrate Poly-Glu, Tyr (10 mg/mL), known for its robust tyrosine kinase substrate properties⁴¹. A master reaction mixture was prepared for all wells by combining 10 µL of 5x kinase assay buffer, 1 µL of ATP, 1 µL of Poly-Glu, Tyr substrate, and 13 µL of nuclease-free water per well, for a total volume of 25 µL. This master mix was aliquoted uniformly to wells in a white 96-well microplate to minimize background fluorescence and optimize luminescence signal detection⁴². Further, 5 µL of inhibitor solutions were added to wells marked as “Test Inhibitor.” For the “Positive Control” wells (using Dasatinib as a known SYK inhibitor) and “Blank” wells, the same volume of inhibitor buffer without inhibitor was added to maintain equal reaction volumes and buffer conditions across wells. To control for background signal, “Blank” wells received 20 µL of 1x kinase assay buffer instead of enzyme or substrate. SYK enzyme stocks were handled with care due to enzyme sensitivity to freeze-thaw cycles, which can reduce enzymatic activity. Upon thawing on ice, enzyme aliquots were gently spun to collect all content, diluted fresh to 1 ng/µL in 1x kinase assay buffer, and immediately used to initiate reactions. Remaining enzyme stocks were stored at -80 °C in aliquots to avoid repeated freeze-thaw cycles. Reactions were initiated by adding 20 µL of diluted SYK enzyme to each well (except blanks) and incubating the plate at 30 °C for 45 min, conditions optimized for SYK catalytic activity. Post-incubation, 50 µL of Kinase-Glo Max reagent was added to each well under minimal light exposure to preserve reagent stability. Plates were covered with aluminium foil and incubated at room temperature for 15 min, allowing the luciferase reagent to convert remaining ATP into a luminescent signal proportional to residual ATP concentration.

Luminescence was measured on a microplate reader with appropriate integration settings to capture relative light units (RLU). Since kinase activity consumes ATP, an inverse correlation was expected between enzymatic activity and luminescence signal. To further investigate inhibitors’ mechanisms, time-dependent inhibition assays were performed at the lowest active concentration for selected inhibitors (39 and 44). Enzyme and inhibitor were pre-incubated, and kinase activity was measured at multiple time points to explore possible slow-binding or irreversible inhibition kinetics^43,44. Additionally, enzyme kinetic studies were conducted varying substrate concentration to elucidate inhibitor binding mode with respect to substrate. Non-competitive inhibition patterns would suggest allosteric binding, while competitive inhibition would suggest ATP or substrate binding site engagement^25,45.

Results and discussions

Chemical synthesis

As stated in the introduction section, we determined the interactions of the molecules that have to be synthesized against SYK using molecular docking and molecular dynamics studies and the potent molecules were synthesized by following the synthesis approach as depicted in Scheme 1. The chemical synthesis of compounds 31, 35, 36, 37 and 47 was performed through SNAr reaction on 2,4-dichloropyrimidine using K₂CO₃ in DMF solution at 80 °C to displace 4-Cl with ethyl-1 H-pyrazole-4-carboxylate to yield an intermediate 2 and then again performed SNAr reaction on intermediate 2 to displace the 2-Cl with 2,4-dimethylaniline to yield an intermediate 3 using the same condition at 100 °C. Further, the hydrolysis was carried out on 3 using LiOH in THF and H₂O at room temperature to yield an intermediate 4. Acid amine coupling on 4 was performed using EDC.HCl, DIPEA, NMP, at room temperature to obtain compound 5. The similar reaction (acid amine coupling) was performed to get remaining compounds using inputs b, c, d and e.

Scheme 1.

Chemical synthesis route of compounds 31, 35, 36, 37 and 47.

On the other hand, as shown in Scheme 2, acid amine coupling on intermediate 4 was performed with the tert-butyl-(2-aminoethyl) carbamate using HATU, DIPEA, DMF, at room temperature to obtain an intermediate 4a and performed boc-deprotection using HCl, DCM, at 25 °C to get the desired product 39. Further, using the same conditions on input of 4 g, 4 h and 4i, compounds 41, 44 and 49 were isolated.

Scheme 2.

Chemical synthesis route of compounds 39, 41, 44 and 49.

Figure 1 depicts the molecular structures of the target molecules synthesized through Schemes 1 and 2. The molecular structures of final targets are confirmed through ¹H and ¹³C NMR and HPLC chromatograms with mass spectral data (Fig. S1). These target molecules were further used to assess the SYK inhibition efficacy through in vitro and in silico studies.

Fig. 1.

Molecular structure of final target molecules synthesized through Scheme 1 and Scheme 2, used for SYK inhibition studies.

Molecular docking results

The molecular docking studies revealed varying binding affinities of the molecules with the target protein, as indicated by their docking scores. Among the tested compounds, 44 exhibited the most favourable binding energy (–7.22 kcal/mol) (Table 2), suggesting the strongest interaction and highest affinity for the binding pocket. This was followed by 47 (–6.06 kcal/mol) and 37 (–5.89 kcal/mol), both of which also demonstrated relatively strong binding potential compared to the other molecules. The remaining molecules, including 31, 35, 39, 41 and 49, showed moderate binding energies ranging from − 5.07 to − 5.76 kcal/mol, indicating weaker interactions with the protein. Overall, the results highlight 44 as the most promising candidate, with 37 and 47 also showing potential for further evaluation (Fig. 2). These top-scoring molecules could be prioritized for additional analyses, such as binding interaction profiling and molecular dynamics simulations, to confirm their stability and potential as lead molecules.

Table 2.

Docking results of spleen tyrosine kinase (SYK, PDB ID: 4XG2) with synthesized molecules.

Molecule code	Binding affinity (Kcal/mol)
31	-5.76
35	-5.31
37	-5.89
39	-5.36
41	-5.07
44	-7.22
47	-6.06
49	-5.76

Fig. 2.

Docking Results of spleen tyrosine kinase (SYK, PDB ID: 4XG2) with three ligands, where (A) 44, (B) 47 and (C) 37.

Molecular dynamics simulation results

The molecular dynamics simulation results provide insights into the stability and conformational behaviour of the protein–ligand complexes. As shown in the RMSD plot brown trace (Fig. 3A), the protein backbone RMSD remains stable throughout the 200 ns simulation, fluctuating within a narrow range of approximately 0.20–0.35 nm. After an initial equilibration phase during the early part of the trajectory, the RMSD reaches a stable plateau with only minor transient deviations, which is typical for a well-equilibrated protein system. Importantly, no continuous drift or large-scale conformational instability is observed. The RMSD plots (Fig. 3A) further supported this observation, showing that the red complex exhibited the lowest deviations (~ 0.5 nm) after 50 ns, suggesting strong stability and minimal conformational changes. The green complex stabilized around 0.8–1.0 nm, indicating moderate stability, while the blue complex showed higher deviations (~ 1.2–1.4 nm), suggesting greater flexibility and conformational rearrangements.

Fig. 3.

(A) RMSD (Root Mean Square Deviation), (B) RMSF (Root Mean Square Fluctuation) and (C) Radius of gyration (Rg) of the three molecules, where blue (44) and red (47) and green (37) and brown Protein SYK (PDB ID: 4XG2).

RMSF analysis (Fig. 3B) highlighted that most residues exhibited fluctuations below 0.5 nm, with higher peaks observed in the terminal and loop regions (residues 2500–3200), which are naturally more flexible. Importantly, the core residues remained stable across all systems, confirming that ligand binding did not compromise the protein’s structural integrity. Collectively, these findings suggest that all three complexes are stable; however, the red complex demonstrates superior conformational stability and stronger interactions compared to the green and blue complexes.

The radius of gyration (Rg) analysis (Fig. 3C) revealed that all complexes maintained structural compactness throughout the 200 ns simulation. Although initial fluctuations were observed during the first 50 ns, particularly indicating structural adjustments upon ligand binding, the systems gradually stabilized, with Rg values converging between 1.75 and 1.85 nm. This indicates that the overall protein folding remained intact, confirming the structural stability of the complexes.

Based on the hydrogen bonding analysis, 44, Fig. 4(A) demonstrates the most rigid and ordered structure, maintaining the highest average of approximately 52–53 hydrogen bonds with exceptionally minimal fluctuation. This extreme stability suggests a highly packed and optimized molecular environment, such as a stable protein core. In contrast, 37 displays a lower yet still very stable average of 44–45 hydrogen bonds, indicating a well-ordered and equilibrated structure but with slightly less density in its bonding network than 44 (Fig. 4A). The molecule 47 (Fig. 4B), which has the lowest average of approximately 36–37 hydrogen bonds and exhibits significant fluctuations throughout the simulation. This pattern of frequent bond breaking, and reforming is characteristic of a flexible region, likely solvent-exposed or comprising a flexible loop, where thermal motion and competing water interactions prevent a stable network from forming. The comparative results indicate a spectrum of flexibility across the ligand, where 44 represents a highly rigid state, 37 (Fig. 4C), a stable but less densely bonded intermediate, and 47 a dynamic and flexible environment crucial for processes requiring molecular mobility.

Fig. 4.

Hydrogen bond analysis of three ligands, where (A) 44, (B) 47 and (C) 37.

The molecular occupancy analysis of spleen tyrosine kinase (SYK, PDB ID: 4XG2) (Table 3) reveals the contribution of specific amino acid residues to molecular stabilization within the binding pocket. Among the residues, TRP183 showed the highest occupancy at 9.49%, suggesting that its aromatic side chain plays a significant role in hydrophobic stacking or stabilizing π–π interactions with the ligand. SER124 was observed with occupancies of 6.76% and SER24 with 5.85%, indicating its recurrent involvement in polar or hydrogen bonding interactions, although with moderate contribution. LYS103, with an occupancy of 6.61%, likely participates through electrostatic interactions or hydrogen bonding due to its positively charged side chain. Overall, the data highlight that ligand binding is supported by a combination of aromatic, polar, and charged interactions, with none dominating independently. Instead, the synergistic contribution of these residues ensures molecule stabilization, suggesting that effective molecular design for SYK should account for interactions with both aromatic residues like TRP183 and polar/charged residues such as SER124, SER24 and LYS103.

Table 3.

Molecular occupancy analysis of key interacting residues in spleen tyrosine kinase (SYK, PDB ID: 4XG2).

Spleen tyrosine kinase (SYK) (4XG2)	Ligand	Occupancy
SER124	37	6.76%
TRP183	44	9.49%
SER24	47	5.85%
LYS103		6.61%

The table lists residues involved in ligand interactions along with their respective occupancy values, indicating the percentage contribution of each residue to ligand binding stability.

Binding free energy results

Molecule 37 (Fig. 5C), achieves the highest and least favourable energy state, with its moving average stabilizing around − 24.81 kcal/mol. While it shows some initial fluctuation, the energy plateaus into a steady, stable trajectory, indicating the system has reached a well-equilibrated state, albeit in a relatively high-energy conformation. In contrast, 44 (Fig. 5A), samples a much broader and more favourable energy landscape, with its moving average converging to approximately − 29.76 kcal/mol. The trace shows significant variability, with deep energy wells followed by rapid increases, suggesting the system is dynamically exploring different conformational states. However, the overall trend and moving average indicate a stable convergence to a low-energy basin, making it the most energetically favourable of the three systems. Molecule 47 (Fig. 5B) exhibits behaviour that is intermediate to the other two. Its final energy state, with a moving average around − 32.16 kcal/mol, is lower than 37 but not as low as the deepest points sampled by 44. The trajectory is characterized by moderate fluctuations and a noticeable shift to a lower energy regime near the 3000-frame mark, after which it stabilizes. This suggests a distinct conformational change or relaxation event occurred during the simulation, leading to a more stable and favourable energy state for the remainder of the production run. Overall, the energy data correlates well with the hydrogen bonding analysis, where greater stability and lower energy are linked to a more rigid and highly bonded network, while dynamic systems explore a wider range of energies.

Fig. 5.

Binding free energy results of three ligands, where (A) 44, (B) 47 and (C) 37.

Principal component analysis (PCA) and free energy landscape (FEL) results

Based on the free energy landscapes (FELs) projected onto the first two principal components (PC1 and PC2), the three systems exhibit distinct conformational preferences and stabilities. Molecule 44 possesses the most stable and favourable free energy minimum among the three, with a deeply stable basin at 0 kJ/mol. The confined, low energy well indicates that the system occupies a single, highly stable conformational state with minimal flexibility, which is consistent with its previously observed rigid hydrogen bonding network and low potential energy. In addition, 37 (Fig. 6C), also displays a stable conformational free energy basin, centered at 0 kJ/mol with a slightly higher overall landscape maximum (13.5 kJ/mol) compared to 44 (12.9 kJ/mol) (Fig. 6A). This indicates that while 37 is structurally stable, its conformational space is marginally less favourable and may have a higher energy barrier for transitioning to other states, reflecting its intermediate energetic and dynamic properties observed in prior analyses.

Fig. 6.

Free energy landscape (FEL) results of three ligands, where (A) 44, (B) 47 and (C) 37.

In contrast, 47 (Fig. 6B), shows a notably higher maximum free energy (14.4 kJ/mol) across its landscape, suggesting the presence of higher-energy conformational substates or greater frustration. The broader or more complex topography implies that this system samples a wider range of conformations with less depth in its primary energy minimum. This aligns with its previously noted flexibility, dynamic hydrogen bonding, and the conformational shift observed in its potential energy trajectory, highlighting its functional adaptability at the cost of thermodynamic stability.

SYK Inhibition activity analysis

The SYK inhibition assay using the kinase Glo Max luminescence system demonstrated that all tested molecules affected kinase activity in a concentration-dependent manner, as depicted in Fig. 7. The results showed a non-classical biphasic inhibition pattern for most inhibitors, characterized by two distinct phases of enzyme activity alteration as the inhibitor concentration increased.

Fig. 7.

SYK enzyme activity of inhibitors in a concentration-dependent manner.

From Table 4, it is evident that molecules 39 and 44 exhibited significant enzyme inhibition even at the lowest tested concentration of 2.5 µM, with enzyme activities reduced to 86.75 U/min/ml and 84.75 U/min/ml, respectively. In contrast, other molecules showed inhibition mainly at higher concentrations, but none as significantly as 39 and 44 at the low concentration level. The reason for significant activity of molecules 39 and 44 is mainly due to the fact that both are having two carbon atoms attached to the amine group, which is highly polar in nature. In contrast, the other molecules are structurally different from 39 to 44.

Table 4.

Different inhibitors’ effect on SYK enzyme activity at different concentrations.

Inhibitor	Activity (U/min/ml)	Concentration (µM)
31	589	5
39	86.75	2.5
41	48	40
44	84.75	2.5
35	308.25	20
37	310.75	20
47	225.75	20
49	433.25	20

Time-dependent kinetics performed using molecules 39 and 44 at 2.5 µM revealed distinct inhibition profiles (Fig. 8). Molecule 44 displayed a progressive decline in SYK activity over the 45-minute incubation, consistent with classical time-dependent inhibition. Conversely, 39 exhibited a biphasic response with an initial decrease followed by partial rebound or plateauing of activity, indicating a more complex interaction.

Fig. 8.

Time-dependent SYK activity by molecules 39 and 44 at 2.5 µM concentration.

Further enzyme kinetic analysis revealed that molecule 44 acts in a non-competitive manner concerning substrate Poly-Glu, Tyr, indicating that the inhibitor does not bind to the substrate-binding site. This is supported by enzyme velocity plots showing dissociation from classical Michaelis-Menten competitive kinetics with inhibition constant changes that are indicative of allosteric or alternative site binding. In summary, the data highlights two categories of inhibition: typical time-dependent inhibition seen with molecule 44, and biphasic inhibition seen with molecule 39. As molecule 44 shows the normal pattern of inhibition with time dependency, for further studies, molecule 44 should be considered.

Predictive mechanism of SYK Inhibition

The biphasic inhibition pattern observed in many of the tested inhibitors is notable and suggests a complex underlying mechanism of SYK enzyme regulation. This biphasic behaviour implies that inhibitors may be engaging with multiple binding sites or enzyme conformations. Initially, inhibitors bind to an active or regulatory site to suppress activity, but at higher concentrations, binding to additional allosteric sites or changes in enzyme conformation could lead to partial reactivation or altered enzymatic properties. This dual-binding characteristic may reflect SYK’s structural flexibility and multiple allosteric pockets, a known feature in kinases that underlies their intricate regulatory behaviour^46–48.

Time-dependent inhibition, as demonstrated by inhibitor 44, aligns with known mechanisms of kinase inhibition where slow-binding or covalent inhibitor-enzyme complexes form over time. This temporal dynamic distinguishes such inhibitors from classical reversible competitive inhibitors, which reach equilibrium quickly and do not exhibit increased inhibition with prolonged incubation. This kinetic profile makes inhibitor 44 potentially more effective in sustained SYK inhibition, an important feature for therapeutic kinase inhibitors that require durable target engagement^48,49. The non-competitive inhibition mode indicated by kinetic studies suggests that these inhibitors do not compete with the substrate for the active site but rather bind an alternative site, influencing enzyme function allosterically. This could be advantageous in overcoming limitations of ATP-competitive kinase inhibitors, often compromised by mutations or high intracellular ATP levels. The differential inhibition profiles among compounds highlight the structural and functional diversity in SYK inhibitor binding, emphasizing the importance of characterizing both the concentration- and time-dependent kinetics in drug discovery. Biphasic inhibitors, while more complex, may offer unique opportunities to modulate kinase function with nuanced effects, potentially allowing therapeutic fine-tuning over standard inhibitors^50,51. Inhibitors 44 and 49 share the core scaffold, but the slight structural variation, specifically the position/nature of the substituent on the aryl ring, results in a significant difference in biological activity. The superior activity of compound 44 suggests that its specific substitution pattern allows for more favourable steric or electrostatic interactions within the SYK binding pocket compared to 49, as supported by the docking studies showing optimal orientation for 44. It is further suggested that allosteric inhibitors like 44 could be particularly beneficial in later-stage disease or relapsed/refractory cases where patients have developed resistance to first-line ATP-competitive drugs. Furthermore, we suggest its potential utility in combinatory treatment regimens, where targeting both the catalytic and allosteric sites could provide a synergistic blockade of SYK signalling. Overall, these findings contribute to the understanding of SYK inhibitor mechanisms, supporting further exploration of allosteric and time-dependent inhibitor designs for improved therapeutic efficacy.

Conclusions

To sum up, we have synthesized and characterized a novel series of aminopyrimidin-4-yl-1 H-pyrazole derivatives (31, 35, 36, 37, 39, 41, 44, 47 and 49) through SNAr and acid amine coupling reactions as selective SYK inhibitors. The molecular dynamics (MD) simulation results for spleen tyrosine kinase (SYK) complexes provide a comprehensive understanding of ligand binding and stability, highlighting significant differences among the top ligands screened. The analyses reveal that molecule 44 consistently displays the most favourable interaction profile, with superior binding affinity, structural rigidity, and energetic stability compared to Ligands 37 and 47. Further, molecule 44 is identified as the most promising candidate due to its lowest docking score (-7.22 kcal/mol), highest average hydrogen bond count (52–53), and stable free energy minimum.MD trajectories demonstrate robust stability of SYK-ligand complexes, with all systems maintaining overall protein folding and compactness throughout simulation runs, but molecule 44 exhibits notably lower RMSD values and minimal conformational fluctuation, affirming strong stability. Principal component analysis and free energy landscape mapping show that molecule 44 occupies a single, highly stable conformational state with minimal flexibility, matching its rigid hydrogen bonding network. Critical residue analysis identifies TRP183 as a key stabilizing residue through hydrophobic and aromatic stacking interactions, while SER124, SER24, and LYS103 contribute synergistically via polar and electrostatic effects, reinforcing the need to consider these contacts in future SYK inhibitor design. On the other hand, in vitro enzyme inhibition assays employing a luminescence-based kinase activity system demonstrated that several compounds, particularly 44, exhibited potent concentration and time-dependent inhibition of SYK inhibition activity. Kinetic characterization revealed that 44 functions via a non-competitive inhibition mechanism, suggesting allosteric binding outside the ATP substrate site. These integrative in silico and in vitro findings establish 44 as a potential lead molecule for SYK-targeted therapies, exhibiting “potential selectivity” against SYK enzyme based on the docking scores and initial enzymatic data. Further, a broader kinase panel screening is required to fully establish the selectivity profile of the lead compounds. These integrative in silico and in vitro findings establish 44 as a promising lead molecule for SYK-targeted therapies, combining potent binding affinity, conformational stability, and effective enzyme inhibition. This work advances the design of novel small-molecule SYK inhibitors with potential applications in cancer and autoimmune disease treatment. Emerging role of SYK in various malignancies is crucial point. This work concludes that the scaffold of compound 44 represents a versatile starting point for developing therapeutics relevant to both autoimmune disorders (e.g., Rheumatoid Arthritis) and hematological cancers (e.g., lymphomas), warranting further investigation in disease-specific models.

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations

DCM

Dichloromethane

DIPEA

Diisopropylethylamine

DMF

N,N-dimethylformamide

DMSO

Di-methyl sulfoxide

EtOAc

Ethyl acetate

EDC.HCl

1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride

HATU

Hexafluorophosphate Azabenzotriazole Tetramethyl Uronium

HCl

Hydrochloric acid

H₂O

Water

K₂CO₃

Potassium carbonate

LiOH

Lithium hydroxide

MPLC

Medium pressure liquid chromatography

Na₂SO₄

Sodium sulphate

NMP

N-Methyl-2-pyrrolidone

rt

Room temperature

Rt

Retention time

TEA

Triethylamine

THF

Tetrahydrofuran

TLC

Thin-layer chromatography

AML

Acute myeloid leukemia

ATP

Adenosine triphosphate

CAS

Chemical abstracts service

DMF

N,N-dimethylformamide

DIPEA

N,N-diisopropylethylamine

EDC.HCl

1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide Hydrochloride

FEL

Free energy landscape

HATU

Hexafluorophosphate azabenzotriazole tetramethyl uronium

IC50

Half maximal inhibitory concentration

K₂CO₃

Potassium carbonate

LCMS

Liquid chromatography-mass spectrometry

MPLC

Medium pressure liquid chromatography

MD

Molecular dynamics

NMP

N-Methyl-2-pyrrolidone

NMR

Nuclear magnetic resonance

PCA

Principal component analysis

PDB

Protein data bank

PME

Particle Mesh Ewald

QSAR

Quantitative structure-activity relationship

RMSD

Root mean square deviation

RMSF

Root mean square fluctuation

Rg

Radius of gyration

SNAr

Nucleophilic aromatic substitution

SPC

Simple point charge (water model)

SYK

Spleen tyrosine kinase

THF

Tetrahydrofuran

TLC

Thin layer chromatography

Author contributions

**Rajasheker K.V.:** Conceptualization, Methodology, Investigation, Formal analysis, Writing- Original draft preparation.; **Pallavi M. S.:** Conceptualization, Methodology, Investigation, Formal analysis, Writing- Original draft preparation.; **Pallavi Singh: ** Conceptualization, Methodology, Investigation, Formal analysis, Writing- Original draft preparation.; Victor Stupin: Visualization, Project administration, validation.; Ekaterina Silina: Visualization, Project administration, validation.; **Kavitharaj Varadaraju: ** Methodology, Visualization, Project administration, validation.; **Mohammad Y. Alfaifi: ** Data curation, Software, Visualization.; **Ali A. Shati: ** Data curation, Software, Visualization.; **Serag Eldin I. Elbehairi: ** Formal analysis, Writing- Reviewing and Editing.; **Chandan Shivamallu: ** Resources, Methodology.; **Manvi: ** Software, Methodology.; **Arun P. C.:** Resources.; **Shiva Prasad Kollur: ** Conceptualization, Methodology Resources, Supervision, Writing- Reviewing and Editing.

Funding

Deanship of Research and Graduate Studies at King Khalid University through large research Project under grant number RGP.2/361/45.

Data availability

All the data generated or analysed during this study are included within the article and supporting information.

Competing interests

The authors declare no competing interests.