Synthesis, characterization, and application of [1-methylpyrrolidin-2-one-SO3H]Cl as an efficient catalyst for the preparation of α-aminophosphonate and docking simulation of ligand bond complexes of cyclin-dependent kinase 2

GRAPHICAL ABSTRACT ABSTRACT A sulfonic acid functionalized ionic liquid was designed, synthesized and successfully used as a Brønsted acid catalyst for the one-pot synthesis of α-aminophosphonates containing benzothiazole at room temperature under solvent-free conditions in excellent yields. The advantages of this method are the reusability of the catalyst, high conversion, short reaction time, and simple experimental procedure. A computer modeling and docking simulation of ligand bond complexes of cyclin-dependent kinase 2 are presented. The results indicate that diethyl ((4-(dimethylamino) phenyl) ((6-nitrobenzo[d]thiazol-2-yl) amino)methyl)phosphonate was found to be the best selective inhibitor of cyclin-dependent kinase 2.


Scheme . Synthesis of acidic ionic liquid (AIL).
Recently, we have introduced a new category of ionic liquids that were successfully employed as catalysts to prepare α-haloketones 35 aryl iodides and azides, 36 2-aryl-1Hphenanthro[9, 10-d]imidazoles, 37 and α-aminophosphonates. 38 An important principle of green chemistry is replacing the use of common and hazardous organic solvents needed for chemical transformations using solvent-free protocols. 39 Solvent-free conditions offer several distinct advantages such as clean reaction profile, enhanced reaction rates, high selectivity and higher yield.
The cyclin-dependent kinases (CDKs), a family of prolinedirected serine/threonine kinases, play a key role in the regulation of the cell cycle in eukaryotic cells. 40 In addition to the positive regulatory role of cyclins and CAK, many negative regulatory proteins (CDK inhibitors, CKIs) have been detected. Since deregulation of cyclins and/or alteration or absence of CKIs has been associated with many cancers, there is strong interest in CDKs inhibitors that could play an essential role in the discovery of a new family of antitumor agents. The development of computational methods as another tool for predicting the properties of chemical compounds has been subject of intensive studies.
Herein, we report the synthesis of sulfonic acid functionalized 1-methylpyrrolidin-2-one chloride [1-methylpyrrolidin-2-one-SO 3 H]Cl (AIL) as a new Brønsted acidic ionic liquid from inexpensive and commercially available starting materials (Scheme 1) and its characterization using FT-IR, 1 H NMR spectroscopy, thermal gravimetric analysis (TGA), and differential thermal gravimetric analysis (DTA). Furthermore, we report herein a highly efficient, cost effective, and much milder one-pot multicomponent protocol for the synthesis of α-aminophosphonate derivatives via the condensation of various aldehydes, 2-aminobenzothiazole derivatives and diethyl phosphite using a homogeneous and recyclable ionic liquid catalyst at room temperature under solvent-free conditions in excellent yields (Scheme 2). In this study, the Auto-Dock 4.0 package was employed for docking synthetic compounds to cyclindependent kinase 2 (PDB ID: 1 GIH). 41 2-Aminobenzothiazole derivatives were synthesized and their cyclin-dependent kinase 2 inhibitory activities together with the SAR (structure-activity relationships) studies were evaluated.

Results and discussion
The structure of Brønsted acidic ionic liquid (AIL) was identified by studying its IR, 1 H NMR, TG, and DTA spectra. The IR spectrum of AIL showed a broad peak at 2750-3500 cm −1 related to OH of the SO 3 H group. The C=O function was observed at 1695 cm −1 . Moreover, two bands observed at 1088 and 1282 cm −1 correspond to the vibrational modes of the N-SO 2 motive.
The 1 H NMR spectrum of AIL showed one signal at δ = 13.10 for the acidic hydrogen atom of SO 3 H. The pH value for the AIL was determined using a 0.1 mol L −1 solution of AIL, which was titrated with 0.114 mol L −1 solution of NaOH. A solution of potassium hydrogen phthalate (KHP) was prepared at concentration 0.100 M and 25.00 mL of KHP solution was used to standardize the NaOH solution. The pH of the solution was measured using a calibrated glass electrode pH meter. The pH value of the AIL is 3.1.
Thermal gravimetric (TG) and differential thermal gravimetric (DTA) analysis of AIL were studied between 25 and 600°C with a temperature increase rate of 10°C · min −1 in an argon atmosphere ( Figure S2). The TG and DTA values of the catalyst showed two weight losses. The first was observed at 90°C and corresponds to H 2 O loss, while the second weight loss was observed above 300°C. Therefore, AIL could be applied as catalysts below 300°C.
The application of this AIL was studied in a new one-pot method for the synthesis of α-aminophosphonates in the presence of a catalytic amount of the AIL under solvent-free conditions (Scheme 2).
In order to optimize the reaction conditions, the reaction of 4-dimethylaminobenzaldehyde (1 mmol), 2aminobenzothiazole (1 mmol) and diethyl phosphite (1 mmol) was carried out using different quantities of AIL at room temperature. As can be seen in Table 1, maximum yield was Scheme . Synthesis of α-aminophosphonates by AIL.
obtained with 10 mol% of the catalyst under solvent-free conditions (Table 1, entry 5). A low yield (30%) was obtained when the reaction was carried out in the absence of AIL at room temperature under solvent-free conditions for 24 h (Table 1, entry 1). In order to examine the scope of this process, several aromatic aldehydes and 2-aminobenzothaizole derivatives were allowed to react under, the optimized conditions, and the results are shown in Table 2.
The synthesized compounds (4a-j) gave satisfactory elemental analyses and their molecular structures were confirmed by FTIR and 1 H, 13 C and 31 P NMR spectroscopy. The IR spectra showed the expected absorption bands at 2985-3305 and 1225-1236 cm −1 , which were attributed to NH and P = O stretching vibrations, respectively. 42 The 1 H NMR spectra of 4a, recorded in CDCl 3 solution, exhibited the signal of CHP proton as a doublet at 5.36 ppm, 43 and in the 13 C NMR spectrum the corresponding 13 C NMR signal appeared as a doublet at 55.6 ppm with 1 J PC = 155 Hz. The values are typical for proton and carbon atoms from a CHP fragment. 43 The methyl protons from the ethoxy groups give rise to two triplets due to the nonequivalence of these groups. The POCH 2 proton signals appear as three multiplets at about 3.80-4.23 ppm. The NH resonance was observed as a broad signal, which in some derivatives overlapped with other signals.
A plausible mechanism is shown in ( Figure S1). The mechanism involves the activation of the carbonyl group of the aldehyde by AIL followed by the nucleophilic addition of the amine to afford the imine 44 by the removal of water. The activated imine further reacts with the dialkyl phosphonate leading to formation of the corresponding α-aminophosphonates.
The recyclability and recovery of the AIL catalyst was investigated for the synthesis of α-aminophosphonates by the one-pot three-component condensation of 2-aminobenzothiazole and 4-dimethylaminobenzaldhyde with diethyl phosphite as model substrates at room temperature under solvent-free conditions for 1 h, As shown in (Figure S3), the catalyst could be reused at least 4 times with only a slight reduction in activity.

Materials and methods
Inhibitor molecules were drawn with Chem Draw 8.0. The geometry was optimized through by Hartree-Fock method with Basic Set 3-21 G. 45 The 1 GIH molecular model (from the PDB) was used in the simulated docking studies. Input protein structures were prepared by adding hydrogen atoms and removing non-functional water molecules. The Auto-dock software version 1.5.6 was used for the molecular docking process. 41 The Lamarckian Genetic Algorithm method was used for the global optimum binding position search. 46 The torsion angles of the inhibitors were identified, hydrogens were added to the macromolecule, bond distances were edited and solvent parameters were added to the enzyme 3D structure. Partial charges were calculated using Gasteiger's method. 47 Docking parameters were as follows: population size of 150, maximum number of energy evaluation ranges of 250,000, maximum number of generations of 27,000, mutation rate of 0.02, cross-over rate of 0.8, other docking parameters were set to the software its default values. After docking, the Inhibitors were ranked according to their docked energy as implemented in the Auto-Dock program. A residue THR14 in the active site was also chosen due to its possible specific hydrogen bonds. This residue (THR 14) was set as flexible residue, while the others residues were kept as rigid residues.
A number of 100 cycles of calculation were used in order to get a final binding position as accurate as possible. The docking procedure was run and the maximum negative Final Docking Energy (FDE) was calculated (Table S1). The following parameters were set during docking simulation: population size, 150; max steps, 100, and central of a grid size with 13, 17, 36, and 40, 40, 40 points in X, Y, and Z axes, respectively. Both were with grid spacing of 0.0375 nm and were centered at the experimentally determined binding sites.

Results of molecular docking
From the results obtained 4i was found to be the best selective known inhibitor of cyclin-dependent kinase 2 because the lowest energy obtained in docking simulations of 4i was obtained with the CDK2 model ( Figure S4).
Docking studies predicted the interaction of inhibitors with protein and residues in the inhibitor-protein complex. For such interaction studies, the most important requirements are proper orientation and conformation of inhibitor which fitted to the enzyme binding site appropriately and formed protein-ligand complex. Optimal interactions and docking results are presented in Table S1.
The analysis of available interactions between small molecules and proteins can be achieved by various software packages including Auto-Dock, LigandFit/Cerius, FlexX, GOLD, Glide, and DOCK, which are widely used in screening large compound libraries, especially when seeking inhibitors of enzymes where assays are not suitable for high throughput screening. The general objective of molecular docking is to search for the energetically most agreeable conformation of a protein-ligand complex and the scoring of resultant geometries with respect to binding energy. Many docking programs consider the scoring and geometry prediction as one matter and use the scoring function to fit the complex with the lowest energy. Therefore, minimizing the root-mean-square deviation between the predicted and experimentally determined complex geometries could result in careful predictions with reasonable binding energies.
Bonding affinity of the designed molecular structures (inhibitors 1-10) was studied. Docked conformers of those were generated in Auto-Dock Tools (ADT) software. In docking process, flexible side chain of the active site pocket residues was allowed to be rotatable (THE14 in cyclin-dependent kinase 2).
The phosphonic acid moiety is considered to bind to the affected protein more strongly than the corresponding carboxylic acid because of its dianionic character. Some polyphosphates containing R-aminomethylene fragments are now industrially used in enormous quantities as antiscaling and anticorrosive agents. 48 The analysis of the number of hydrogen bonds between inhibitors and CDK2 shows that the CDK2-4i complex has a higher number of intermolecular hydrogen bonds, which indicates that it has higher affinity for CDK2 than others. Further inhibition experiments can confirm this prediction.

Conclusions
In summary, we have designed and synthesized an AIL and have characterized it by FT-IR, 1 H NMR, TGA, and DTA. We have successfully employed it as a catalyst for the onepot synthesis of α-aminophosphonates containing benzothiazole moiety in excellent yields. The application of this AIL is studied in a new one-pot method for the synthesis of αaminophosphonate derivatives under solvent-free conditions. The advantages offered by this protocol include reusability of the catalyst, high conversion, short reaction time, and simple experimental procedure. A computer modeling and docking simulation of ligand bond complexes of cyclin-dependent kinase 2 indicate that 4i has the most selective inhibitor of cyclindependent kinase 2, because it shows the lowest docked energy.

Materials and instrumentation
All reagents were purchased from Merck and used without further purification. The melting points of the products were determined with an Electrothermal Type 9100 melting point apparatus. The Fourier transform Infrared (FT-IR) spectra were recorded with an Avatar 370 FT-IR Thermal Nicolet spectrometer. The mass spectra were recorded with a 5973 Network Mass Selective Detector. The 1 H, 13 C, and 31 P NMR spectra were recorded with a Bruker DRX-400 spectrometer at 400, 100.65, and 165 MHz, respectively, using DMSO-d 6 or CDCl 3 as deuterated solvents. Chemical shifts are reported in ppm downfield from TMS as internal standard; coupling constants J are given in Hz.

Synthesis of [1-methylpyrrolidin-2-one-SO 3 H]Cl ionic liquid
A 100-mL round-bottom flask was charged with 1methylpyrrolidin-2-one (10 mmol, 0.99 g) in dry CH 2 Cl 2 (50 mL), and then chlorosulfonic acid (10 mmol, 1.15 g) was dropped into the mixture under stirring for 30 min at room temperature. Afterwards the reaction mixture was stirred for 1 h and the CH 2 Cl 2 was decanted. The residue was washed with dry CH 2 Cl 2 (3 × 20 mL) and dried under vacuum to give [

Preparation of α-Aminophosphonates 4a-4j: General procedure
The corresponding aldehyde (1.0 mmol), 2-aminobenzothiazole (1.0 mmol), diethyl phosphite (1.0 mmol), and AIL (10 mol%) were mixed together and stirred at room temperature for appropriate time ( Table 2). After the completion of the reaction as monitored by TLC, the mixture was washed with CH 2 Cl 2 (3 × 3 mL). The combined extracts were filtered and the solvent was removed under reduced pressure to afford the crude product, which was purified by recrystallization from ethylacetate/n-hexane. The catalyst, which does not dissolve in CH 2 Cl 2 , remained in the residue.
Selected 1 H, 13 C, and 31 P NMR as well as mass spectra for compound 4a-4j are presented in the Supplemental Material ( Figures S5-S36).