Urea and Thiourea Derivatives of Dipeptides Conjugated Piperazine Analogue as A New Class of AGE’s Inhibitors: Synthesis and Molecular Docking Studies

 

D. M. Suyoga Vardhan, H. K. Kumara, H. Pavan Kumar, D. Channe Gowda*

Department of Studies in Chemistry, University of Mysore, Manasagangotri,

Mysore – 570 006, Karnataka, India

*Corresponding Author E-mail: dchannegowda@yahoo.co.in

A novel class of urea and thiourea derivatives of dipeptides conjugated 2,3-dichlorophenyl piperazine were synthesized and evaluated for their AGE’s inhibition capacity. Antiglycation assay was performed by assessing fluorescence spectrum (exitation 370 nm), and change in fluorescence intensity (to emission 440 nm), based on AGEs were monitored by using spectrofluorimeter. Our results indicate that fluoro containing dipeptide-PZN derivatives have shown excellent curative potential. Interestingly, compounds 6, 7, 14, 15, 22, 23, 30 and 31 have shown excellent potency with IC₅₀ values 13.5 ± 0.77, 8.0 ± 0.25, 14.5 ± 0.92, 7.8 ±0.44, 9.1 ± 0.41, 3.1 ± 0.45, 9.2 ± 0.80 and 2.5 ± 0.55, compared to the standard, rutin 41.9 µM. From our studies, we may draw certain conclusions like Lys containing dipeptides may serve as good antiglycating agents. On the other hand, it’s felt worthy to consider substitutions particularly at para position for the increase in potency. The IC₅₀ values of the compounds were compared with the glide scores from the molecular studies. It was observed that the main interaction force of compounds with the active site was hydrophobic. The IC₅₀ values and glide scores have exhibited better correlation. Thus, this synthetic novel urea and thioureas of dipeptide containing compounds may lead potent antiglycating agents.

 

 

1. INTRODUCTION:

Hyperglycemia, a condition characterized by an abnormal excess of sugar in blood, has been linked to the onset of type 2 diabetes mellitus and associated cardiovascular complications including hypertension.^1,2 Although a few synthetic antidiabetic drugs are available to combat the impaired insulin secretion, insulin resistance and hyperglycemia that characterize type 2 diabetes mellitus, some of these drugs can have side effects at high doses.^3-5

 

A major focus of current antidiabetic research is the development of antiglycating agents which are safe and free from negative side effects. The Advanced Glycation Endproducts (AGEs) are a class of compounds which are generated during protein glycation.⁶ This process starts with the formation of an initial adduct between the carbonyl group of reducing sugar and the –NH₂ group of a protein, a Maillard reaction. Accumulation of AGEs is associated with aging, diabetes, Alzheimer’s disease, renal failure and many other chronic diseases.⁷Peptides have occupied a prominent position in pharmaceutical chemistry in recent times for several reasons. The first and most important is that peptides allow the creation of peptide antibodies in animals without the need to purify the protein of interest.⁸ Many biologically important peptide sequences contain proline. The conformational restrictions imposed by proline motifs in a peptide chain appear to imply important structural or biological functions as can be deduced from their often remarkably high degree of conservation as found in many proteins and peptides, especially cytokines, growth factors, G-protein-coupled receptors, V3 loops of the HIV envelope glycoprotein gpl2O, and neuro- and vasoactive peptides.⁹

 

Heterocyclic molecules are of biological interest due to their potential physical and chemical properties.¹⁰ Nitrogen heterocycles are found in the majority of clinically approved small molecule pharmaceuticals. A few privileged heterocyclic scaffolds, such as pyridines, piperidines, piperazines, pyrrolidines, imidazoles, pyrazoles, indoles, β-lactams, etc., dominate in most cases. There is general recognition of the need for the development of both new heterocyclic scaffolds, as well as approaches to modify existing scaffolds in novel ways to confer desirable biological and pharmacological properties.¹¹ Among these the piperazine compounds occupy a unique position in pharmaceutical chemistry.^12-18 On the other hand, urea’s and thioureas which are of considerable industrial importance, and are linked to a series of biological activities including herbicidal activity,¹⁹ inhibition of nitric oxide,²⁰ antimicrobial,^21,22 anti-HIV,²³ antiviral,²⁴ HDL elevating,²⁵ analgesic,²⁶ anti-inflammatory,²⁷ antimalarial,²⁸ and antiglycation and urease inhibition properties.^29-31

 

Encouraged by above all overture and in continuation of our research work, we further turned our attention to prepare novel compounds with enhanced antiglycating activity by incorporating halogens at one position of the phenyl ring of urea/thiourea derivatives of dipeptide containing proline conjugated to heterocycle. The preliminary aim of this study is to synthesize the title compounds and study their biological significance for the development of new inhibitors of protein glycation.

 

2. MATERIALS AND METHODS:

Advanced Chem. Tech. (Louisville, Kentucky, USA), Sigma Chemical Co. (St. Louis, MO) and Sigma-Aldrich chemicals [All t-Butoxycarbonyl (Boc)-amino acids, 1-(3-Dimethylaminopropyl)-3-ethyl-carbodiimide.HCl (EDCI), 1-Hydroxybenzotriazole (HOBt) and Trifluoroacetic acid (TFA), Isobutyl Chloroformate (IBCF) and N-Methyl morpholine (NMM)] were used as such without further purification. Solvents and reagents used for synthesis, spectroscopic and other physical studies were of analytical grade. The completion of reaction was monitored by TLC made using silica gel coated on a glass with the solvent system chloroform/methanol/acetic acid in the ratio 98:2:3 (R_f^a) and 95:5:3 (R_f^b). The compounds on the plates were detected by iodine vapors. Melting points were determined by Super fit melting point apparatus (India) using a calibrated centigrade thermometer and are uncorrected. IR spectra were obtained in KBr optics on a Jasco spectrometer (USA) and expressed in wave numbers (CM^-1). ¹H NMR spectra were obtained on VARIAN 400 MHz instrument (USA) using DMSO-d₆ and the chemical shifts are reported as parts per million (δ ppm) using TMS as an internal standard. Mass spectra were obtained on Bruker (model HP-1100) (USA) electrospray mass spectrometer. Elemental analysis was performed by using VARIO EL III Elementar (Germany).

 

2.1 Synthesis:

Coupling of Boc-Xaa-Pro-OBzl where Xaa = Phe (I), Val (II), Tyr(2,6-Cl₂Bzl) (III) and Lys(Z) (IV):

0.005 mol of Boc-Xaa-OH [Where Xaa = Phenylalanine, Valine, Tyrosine(2,6-Cl₂Bzl) and Lysine(Z)] was dissolved in dimethylformamide (DMF) (10ml/g) in a round bottom flask followed by addition of N-methylmorpholine (NMM) (1.10 ml, 0.005 mol). At the same time, 0.005 mol of HCl-Pro-OBzl was dissolved in minimum amount of DMF in another round bottom flask and NMM was also added to it so as to neutralize the salt under ice cold conditions. Coupling reagent isobutyl chloroformate (IBCF) was then added at -15 °C to the first flask with vigorous stirring and contents of second flask were added to that. The reaction mixture was allowed to stir for overnight. The filterate was evaporated under reduced pressure and the oily residue left was poured into about 200 ml ice cold 90 % saturated KHCO₃ solution and stirred for 30 min. The product precipitated was extracted into CHCl_3. The organic layer was washed with 5 % NaHCO₃ solution (3 × 20 ml), water (2 × 20 ml) and 0.1 N HCl solution (3 × 20 ml) followed by brine (2 × 20 ml). The chloroform layer was dried over anhydrous Na₂SO₄. The solvent was removed under reduced pressure, and triturated with dry ether or petroleum ether to obtain the products I-IV.

 

Synthesis of Boc-Xaa-Pro-OH:

Each dipeptide (I-IV) (0.004 mol) was dissolved in methanol (10 ml/g) and 1N NaOH (2 eq) was added and stirred for 2 h at rt. The completion of the reaction was monitored by TLC and the solvent was evaporated, cooled, neutralized with cold 1 N HCl and extracted with CHCl₃. The organic layer was washed with 1 N HCl followed by H₂O and dried over anhydrous Na₂SO₄. The solvent was removed under pressure and triturated with ether, filtered, washed with ether and dried to obtain debenzylated peptides (V-VIII).

 

Boc-Phe-Pro-OH (V):

Yield = 92.0%; R_f^a = 0.61; R_f^b = 0.75; M.P. = 97-100°C (Lit. 100°C)³²; ¹H NMR (DMSO-d₆, δ ppm): Boc = 1.41 (9H, s); Phe = 3.42-3.53 (2H, d, -^βCH₂); 4.86-4.91 (1H, m, -^αCH); 6.54-7.40 (5H, m, Ar-H); Pro = 3.92-3.97 (1H, t, -^αCH), 1.68-2.22 (4H, m, -^{β, γ}CH₂), 3.40-3.51 (2H, m, -^δCH₂); Mass = 367.40 (M⁺+1); Elem. Anal. = calcd. for C₁₉H₂₆N₂O₅: C: 62.97; H: 7.23; N: 7.73; Found: C: 62.95; H: 7.20; N: 7.73

 

Boc-Val-Pro-OH (VI)

Yield = 94.0%; R_f^a = 0.56; R_f^b = 0.68; M.P. = 80-82 °C (Lit. 83 °C)³³; ¹H NMR (DMSO-d₆, δ ppm): Boc = 1.42 (9H, s); Val = 1.03 (6H, d, -(CH₃)₂), 1.88-1.90 (1H, m, -^βCH), 3.53-3.64 (1H, m, -^αCH); Pro = 3.69-3.73 (1H, t, -^αCH), 1.90-2.35 (4H, m, -^{β, γ}CH₂), 2.97-3.52 (2H, m, -^δCH₂); Mass = 315.30 (M⁺+1); Elem. Anal. = calcd. for C₁₅H₂₆N₂O₅: C: 57.31; H: 8.34; N: 8.91; Found: C: 57.32; H: 8.32; N: 8.88

 

Boc-Tyr(2,6-Cl₂Bzl)-Pro-OH (VII)

Yield = 89.2%; R_f^a = 0.56; R_f^b = 0.69; M.P. = 95-98 °C; ¹H NMR (DMSO-d₆, δ ppm): Boc = 1.43 (9H, s); Tyr = 3.66-3.76 (2H, d, -^βCH₂); 4.81-4.87 (1H, t, -^αCH); 5.17 (2H, s, side chain -CH₂); 6.70-7.90 (7H, m, ArH); Pro = 3.35-3.48 (1H, t, -^αCH), 1.68-1.88 (4H, m, -^{β, γ}CH₂), 2.91-3.13 (2H, m, -^δCH₂); Mass = 538.41 (M⁺+1); Elem. Anal. = calcd. for C₂₆H₃₀Cl₂N₂O₆: C: 58.11; H: 5.63; N: 5.21; Found: C: 58.12; H: 5.62; N: 5.28

 

Boc-Lys(Z)-Pro-OH (VIII)

Yield = 89.2%; R_f^a = 0.56; R_f^b = 0.64; M.P. = Gum; ¹H NMR (DMSO-d₆, δ ppm): Boc = 1.43 (9H, s); Lys = 1.70-1.72 (2H, m, -^δCH₂); 1.85-1.91 (2H, m,-^γCH₂); 1.93-2.01 (2H, m, -^βCH₂); 3.18-3.25 (2H, m, -^€CH₂); 4.06 (1H, m, -^αCH); 8.13 (1H, s, -NH); 5.15 (2H, s, benzyl -CH₂); 7.18-7.43 (5H, m, ArH); Pro = 4.05-4.10 (1H, t, -^αCH), 1.67-2.25 (4H, m, -^{β, γ}CH₂), 3.72-3.78 (2H, m, -^δCH₂); Mass = 478.51 (M⁺+1); Elem. Anal. = calcd. for C₂₄H₃₅N₃O₇: C: 60.36; H: 7.39; N: 8.80; Found: C: 60.32; H: 7.32; N: 8.88

 

General procedure for the coupling of Piperazine derivative with peptides (1-4):

1-(2,3-Dichlorophenyl) piperazine. HCl (PZN) was synthesized as previously reported method³⁴. To Boc-Xaa-Pro-OH (0.01 mol) dissolved in acetonitrile (10 mL/g of compound) and cooled to 0°C was added NMM (1.10 mL, 0.01 mol). To this EDCI (1.917 g, 0.01 mol) was added and stirred while maintaining the temperature at 0 °C. After stirring the reaction mixture for 10 min at this temperature, HOBt (1.531 g, 0.01 mol) in DMF (15 mL) was added slowly. The reaction mixture was stirred for an additional 10 min and a pre-cooled solution of 2,3-dichlorophenyl piperazine. HCl (2.68 g, 0.01 mol) and NMM (1.10 mL, 0.01 mol) in DMF (25 mL) was added slowly. After 20 min, pH of the solution was adjusted to 8 by the addition of NMM and the reaction mixture was stirred over night at room temperature. Acetonitrile was removed under reduced pressure and the residual DMF was poured into about 500 mL ice-cold 90% saturated KHCO₃ solution and stirred for 30 min. The precipitated compound was extracted into chloroform and washed sequentially with 5% NaHCO₃ solution (3 × 20 mL), water (3 × 20 mL), 0.1N cold HCl (3 × 20 mL) followed by brine. The organic layer was dried over anhydrous Na₂SO₄, the solvent was removed under reduced pressure, triturated with ether, filtered and dried to get the conjugates 1-4.

 

tert-butyl (1-(2-(4-(2,3-dichlorophenyl) piperazine-1-carbonyl) pyrrolidin-1-yl)-1-oxo-3-phenylpropan-2-yl) carbamate (1)

Yield = 90.0%; R_f^a = 0.58; R_f^b = 0.65; M.P. = Gum; ¹H NMR (DMSO-d₆, δ ppm): Boc = 1.43 (9H, s); Phe = 3.41-3.51 (2H, d, -^βCH₂), 4.59-4.91 (1H, m, -^αCH), 6.52-7.40 (5H, m, Ar-H); Pro = 3.79-3.94 (1H, t, -^αCH), 1.68-2.20 (4H, m, -^{β, γ}CH₂), 3.38-3.49 (2H, m, -^δCH₂);PZN = 3.26-3.37 (4H, m, -CH₂); 3.42-3.46 (4H, m, -CH₂); 6.52-7.40 (3H, m, Ar-H). Mass = 576.48 (M⁺+1); Elem. Anal. = calcd. for C₂₉H₃₆N₄O₄: C: 60.52; H: 6.30; N: 9.73; Found: C: 60.55; H: 6.31; N: 9.75

 

tert-butyl (1-(2-(4-(2,3-dichlorophenyl) piperazine-1-carbonyl) pyrrolidin-1-yl)-3-methyl-1-oxobutan-2-yl) carbamate (2)

Yield = 88.8%; R_f^a = 0.48; R_f^b = 0.57; M.P. = Gum; ¹H NMR (DMSO-d₆, δ ppm): Boc = 1.43 (9H, s); Val = 1.06 (6H, d, -(CH₃)₂), 1.88-1.91 (1H, m, -^βCH), 4.34 (1H, s, -^αCH); Pro = 4.02 (1H, t, -^αCH), 1.68-2.24 (4H, m, -^{β, γ}CH₂), 3.74-3.78 (2H, m, -^δCH₂);PZN = 3.34-3.42 (4H, m, -CH₂); 3.47-3.50 (4H, m, -CH₂); 7.14-7.28 (3H, m, Ar-H). Mass = 528.45 (M⁺+1); Elem. Anal. = calcd. for C₂₅H₃₆Cl₂N₄O₄: C: 56.92; H: 6.88; N: 10.62; Found: C: 56.90; H: 6.82; N: 10.68

 

Tert-butyl (3-(4-((2,6-dichlorobenzyl) oxy) phenyl)-1-(2-(4-(2,3-dichlorophenyl) piperazine-1-carbonyl) pyrrolidin-1-yl)-1-oxopropan-2-yl) carbamate (3)

Yield = 86.2%; R_f^a = 0.56; R_f^b = 0.68; M.P. = Gum; ¹H NMR (DMSO-d₆, δ ppm): Boc = 1.43 (9H, s); Tyr = 3.72 (2H, d, -^βCH₂); 4.81-4.89 (1H, m, -^αCH); 5.15 (2H, s, side chain -CH₂); 6.98-7.92 (7H, m, ArH); Pro = 3.48 (1H, t, -^αCH), 1.68-1.88 (4H, m, -^{β, γ}CH₂), 2.94-3.16 (2H, m, -^δCH₂); PZN = 3.36-3.48 (4H, m, -CH₂); 3.62-3.70 (4H, m, -CH₂); 6.98-7.92 (3H, m, Ar-H). Mass = 751.52 (M⁺+1); Elem. Anal. = calcd. for C₃₆H₄₀Cl₄N₄O₅: C: 57.61; H: 5.37; N: 7.46; Found: C: 57.62; H: 5.32; N: 7.48

 

Benzyl tert-butyl (6-(2-(4-(2,3-dichlorophenyl) piperazine-1-carbonyl) pyrrolidin-1-yl)-6-oxohexane-1,5-diyl) dicarbamate (4)

Yield = 89.2%; R_f^a = 0.56; R_f^b = 0.64; M.P. = Gum; ¹H NMR (DMSO-d₆, δ ppm): Boc = 1.43 (9H, s); Lys = 1.70-1.72 (2H, m, -^δCH₂), 1.85-1.91 (2H, m,-^γCH₂), 1.93-2.01 (2H, m, -^βCH₂), 3.18-3.25 (2H, m, -^€CH₂); 4.06 (1H, m, -^αCH); 8.13 (1H, s, -NH); 5.15 (2H, s, benzyl -CH₂); 7.18-7.43 (5H, m, ArH); Pro = 4.05 (1H, t, -^αCH), 1.65-2.21 (4H, m, -^{β, γ}CH₂), 3.71-3.76 (2H, m, -^δCH₂);PZN = 3.34-3.42 (4H, m, -CH₂); 3.47-3.50 (4H, m, -CH₂); 7.14-7.28 (3H, m, Ar-H). Mass = 691.65 (M⁺+1); Elem. Anal. = calcd. for C₃₄H₄₅Cl₂N₅O₆: C: 59.13; H: 6.57; N: 10.14; Found: C: 59.12; H: 6.52; N: 10.18

 

General procedure for the synthesis of ureido and thioureido derivatives (5-36)

Each time, Boc-Xaa-Pro-PZN (0.15g) was stirred with 1.5 mL of TFA for 45 min at room temperature. After completion of the reaction (monitored by TLC), TFA was removed under vacuum, triturated with dry ether, filtered, washed with ether and dried to obtain TFA.H-Xaa-Pro-PZN.

 

Further, TFA.H-Xaa-Pro-PZN (0.001 mol) was dissolved in DMF (10 mL/g of compound), cooled to 0 °C and NMM (0.10 mL, 0.001 mol) was added. To this solution respective substituted phenyl isocyanates and isothiocyanates (0.0012 mol) was added drop-wise while maintaining the temperature at 0°C. The reaction mixture was stirred for 8h slowly warming to room temperature. DMF was evaporated under high vacuum and the residue was poured into about 50 mL ice-cold 90% saturated KHCO₃ solution and stirred for 30 min. The precipitate was extracted into chloroform and washed sequentially with 5% NaHCO₃ solution (2 × 10 mL), water (2 × 10 mL), 0.1N citric acid (2 × 10 mL) followed by brine. The organic layer was dried over anhydrous Na₂SO₄. The solvent was removed under reduced pressure, triturated with hexane filtered and dried under vacuum to obtain urea/thiourea derivatives (5-36).

 

3. PHARMACOLOGY:

3.1 Antiglycation assay (in vitro)³⁵:

Sodium phosphate buffer (pH 7.4) was prepared by mixing Na₂HPO₄ and NaH₂PO₄ (67mM) containing sodium azide (3mM), phosphate buffer saline (PBS) which was prepared by mixing NaCl (137mM) + Na₂HPO₄ (8.1mM) + KCl (2.68mM) + KH₂PO₄ (1.47mM) and pH 10 was adjusted by adding NaOH (0.25mM), while BSA (10 mg/mL) and anhydrous glucose (50 mg/mL) solutions were prepared in sodium phosphate buffer.

 

In a 96-well plate assay, each well contains 60μL of reaction mixtures (20μL bovine serum albumin [10 mg/mL + 20μL of glucose anhydrous (50 mg/mL) + 20μL of test sample]. Glycated control contains 20μL BSA + 20μL glucose + 20μL sodium phosphate buffer, while blank control contains 20μL BSA and 40μL sodium phosphate buffer. Reaction mixture was incubated at 37 °C for 7 days. After incubation, 6μL of TCA (100%) was added into each well and centrifuged (15,000 rpm) for 4 min at 4°C. After centrifugation, the pellets were rewashed with 60μL of TCA (10%). The supernatant containing glucose, inhibitor and interfering substance was removed and pellet containing AGE and BSA were dissolved in 60μL PBS. Assessment of fluorescence spectrum (excitation 370nm), and change in fluorescence intensity (excitation 370 nm to emission 440nm), based on AGEs were monitored by using spectrofluorimeter (Varioskan, Germany) and % inhibition was calculated using the following formula:

 

                                Fluorescence of Sample

% Inhibition =1-    -------------------------------- X 100

                                Fluorescence of glycated Sample

 

3.2 Molecular docking studies:

Molecular docking was performed with the help of maestro 9.3.5 version of the Schrodinger software suite, 2011. The 3D crystallographic structure of proteins (PDB ID: 1N5U) was retrieved from Protein Data Bank (www.rcsb.org/pdb). The protein structures were pre-processed and refined by Protein Preparation Wizard. Further, it was minimized by OPLS-2005 (Optimized Potential for Liquid Simulations) force field until the root mean square deviation (RMSD) reached the value of 0.3 Å. The ligands were optimized by LigPrep program using the OPLS-2005 force field to generate lowest energy state of ligands. The molecular docking studies of the ligand and protein were carried out by GLIDE. The best fit ligands with the target protein were ranked based on G score.

 

4. RESULTS AND DISCUSSION:

4.1 Synthesis and Characterization:

Dipeptides were synthesized by reacting Boc-Pro-OBzl with various amino acids like phenyl alanine, valine, tyrosine and lysine using IBCF as coupling agent. These dipeptides were debenzylated using methanol and 1 N cold NaOH. Further the debenzylated peptides were conjugated to 2,3-dichlorophenyl piperazine using EDCI/HOBt as coupling agent and NMM as base. Boc group of the conjugates was removed using TFA and converted to urea and thiourea derivatives using substituted phenyl isocyanates and phenyl isothiocyanates respectively (Scheme). Yield of the compounds obtained were >80% and characterized by IR, ¹H NMR, mass and elemental analysis. The physical and spectroscopical data of the synthesized compounds are provided in Tables 1 and 2. The stretching frequencies at ν ~1624 cm^-1 (C=O) and ν ~2021 cm^-1 (C=S) indicates the formation of urea and thiourea derivatives respectively. Further, the appearance of peaks at δ ~8.65 (s, 1H) and δ ~8.32 (s, 1H) for urea derivatives and at δ ~9.75 (s, 1H) and δ ~8.21 (s, 1H) for thiourea derivatives in ¹H NMR confirmed the structures. The high-resolution mass spectra (HRMS) and elemental analysis data were found to be in good agreement with the structures assigned.

 

Scheme. Synthesis of ureido and thioureido derivatives of dipeptide conjugated heterocycle

 

Table 1: Physical and mass data of synthesized urea/thiourea derivatives (5-36)

Entry	R	X	Rfa	Rfb	Yield (%)	MP(°C)	Mol. For.	Mass (M+ +1)
5	3F	O	0.53	0.61	91	75	C31H32Cl2FN5O3	613.50
6	4F	O	0.55	0.63	88	73	C31H32Cl2FN5O3	613.54
7	4F	S	0.51	0.59	94	58	C31H32Cl2FN5O2S	629.55
8	3Cl	O	0.49	0.58	85	72	C31H32Cl3N5O3	629.10
9	3Cl	S	0.45	0.56	84	58	C31H32Cl3N5O2S	646.10
10	4Cl	S	0.42	0.57	86	60-62	C31H32Cl3N5O2S	646.12
11	3Br	O	0.52	0.65	88	68	C31H32BrCl2N5O3	674.12; 676.15
12	3Br	S	0.50	0.62	90	55	C31H32BrCl2N5O3	690.02; 692.09
13	3F	O	0.49	0.58	92	58	C27H32Cl2FN5O3	565.45
14	4F	O	0.45	0.56	83	58-60	C27H32Cl2FN5O3	565.52
15	4F	S	0.43	0.53	86	Gum	C27H32Cl2FN5O2S	581.58
16	3Cl	O	0.47	0.58	81	Gum	C27H32Cl3N5O3	581.90
17	3Cl	S	0.45	0.55	84	Gum	C27H32Cl3N5O2S	598.02
18	4Cl	S	0.44	0.55	88	Gum	C27H32Cl3N5O2S	598.05
19	3Br	O	0.46	0.59	90	55	C27H32BrCl2N5O3	625.35; 627.42
20	3Br	S	0.44	0.57	91	Gum	C27H32BrCl2N5O2S	641.50; 643.38
21	3F	O	0.60	0.71	92	85-87	C38H36Cl4FN5O4	788.50
22	4F	O	0.63	0.70	90	90	C38H36Cl4FN5O4	788.48
23	4F	S	0.58	0.66	95	75-77	C38H36Cl4FN5O3S	804.63
24	3Cl	O	0.63	0.71	88	65	C38H36Cl5N5O4	804.85
25	3Cl	S	0.59	0.68	89	62-64	C38H36Cl5N5O3S	821.09
26	4Cl	S	0.57	0.65	86	65	C38H36Cl5N5O3S	821.12
27	3Br	O	0.61	0.67	88	82	C38H36BrCl4N5O4	849.51; 851.21
28	3Br	S	0.57	0.63	90	70	C38H36BrCl4N5O3S	865.58; 867.61
29	3F	O	0.49	0.57	80	72	C36H41Cl2FN6O5	728.63
30	4F	O	0.45	0.59	84	75	C36H41Cl2FN6O5	727.71
31	4F	S	0.42	0.55	86	60-62	C36H41Cl2FN6O4S	744.75
32	3Cl	O	0.44	0.58	81	65	C36H41Cl3N6O5	745.12
33	3Cl	S	0.45	0.55	87	58	C36H41Cl3N6O4S	761.17
34	4Cl	S	0.47	0.56	84	55-57	C36H41Cl3N6O4S	761.20
35	3Br	O	0.49	0.58	82	56-58	C36H41BrCl2N6O5	789.58; 791.12
36	3Br	S	0.41	0.53	90	54	C36H41Cl2N6O4S	805.58; 807.44

 

 

Table 2: IR and NMR values of urea and thiourea derivatives of dipeptides conjugated PZN

^b = The chemical shift values for the fragment ‘Heterocycle’ of the compounds 5-36 are almost same as obtained for compound 5

 

4.2 Bioactivity discussion:

In our previous work,²⁹ we have reported the synthesis of a new series of urea/thiourea derivatives of glycine/proline conjugated piperazine analogue as promising antiglycating agents. Further, it was found that compounds containing halogen substituents have exerted highly potent activity. Prompted by these results, the present work involves the synthesis of 32 urea/thiourea derivatives of dipeptides, Xaa-Pro [Xaa = Phe, Val, Tyr and Lys], conjugated to piperazine analogue and synthesized derivatives which contains only halogens like fluorine, chlorine and bromine as substituents. Synthesized compounds were screened for their in vitro antiglycation activity. The results were expressed in terms of IC₅₀(Table 3).

 

 

 

Table 3: Antiglycation assay of the synthesized compounds (1-36)

^aValues are mean of three determinations, the range of which are < 5% of mean in all case

 

The antiglycation activity of urea and thiourea derivatives of proline containing dipeptides conjugated to piperazine analogue was evaluated. Positive control contained flavonol quercetin-(α-L-rhamnopyranosul-(1-6))-β-D-glucopyranose) glycoside called rutin, which was used as a standard (IC₅₀ = 41.9 ± 2.9 µM). The preliminary structure-activity relationship (SAR) studies showed that the presence of urea or thiourea moiety may be responsible of antiglycation activity in this series of compounds. Furthermore, the structure-activity suggested that the antiglycation activity of a particular molecule is apparently governed by the substitution present at aromatic residues of urea and thiourea. Compound 31 with an IC₅₀ value of 2.5 ± 0.55 µM was found to be 20 folds more active than the standard, probably due to the presence of a fluoro group at para position. Interestingly, based on the activity profile (5-36) it is clearly understood that the activity is favored by the presence of electron withdrawing (particularly F) group at para position which has increased the level of inhibition of protein glycation remarkably. Compounds 6, 7, 14, 15, 22, 23, 30 and 31 having excellent IC₅₀ values 13.5 ± 0.77, 8.0 ± 0.25, 14.5 ± 0.92, 7.8 ±0.44, 9.1 ± 0.41, 3.1 ± 0.45, 9.2 ± 0.80 and 2.5 ± 0.55 proved the above statement. All other compounds, including the bromo substituents were found to be active where their IC₅₀ values were found to be lesser than the standard value 41.9 µM. This may be due to the high electron withdrawing nature of halogens (F> Cl> Br) and also due to inductive and resonance effect. This may also be due to the fact that the halogens are not involved in resonance stabilization. It was concerned to note that the compounds derived from K(z)P-PZN (29-36) dipeptides have shown excellent increase in inhibiting the protein glycation which is followed by Y(2,6-Cl₂-Bzl) P-PZN (21-28), VP-PZN (13-20) and FP-PZN (5-12) dipeptide derivatives. Between urea and thiourea analogues, compounds with thiourea moiety have shown increased activity over the former. This may probably be due to the more nucleophilic character of sulphur compared to oxygen³⁶.It was felt worthy to note that the dipeptides heterocycle conjugates 1 - 4 with IC₅₀ values 145.6 ± 0.96; 157.1 ± 0.98; 100.0 ± 0.98 and 34.2 ± 0.12 respectively were poorly active, ie., their activity was found to be more than the standard (> 41.9 µM) which might be due to absence of urea and thiourea functionalities.

4.3 Molecular docking studies:

Molecular docking was performed on all the synthesized dipeptide-heterocyclic conjugates/urea and thiourea derivatives (1-36) to identify possible biding mode which explains the reason for their potency. In order to gain insight into the exact binding location of ligand and protein, the active molecules (5-36) including standards were subjected to molecular docking with active site of protein (PDB ID: 1N5U)

 

 

 

Figures 1 and 2 2D and 3D images of compound 31 and 23 with 1N5U.

 

The potentiality of compounds bind to the active site of protein was ranked based on glide score (Table 3). The compound forming the most stable drug receptor complex is the one having the least dock score value. The docking result of antiglycation activity reveals that the molecules (5-36) have good binding affinity towards target protein with G score ranging from -10.424 to -4.626. Among all the docked ligand most of all the compounds have shown good biding affinity. The docking results revealed that the main interaction force of the compounds with active sites was hydrophobic. The best match between docking results and biological studies were exhibited by fluoro substitution bearing compounds particularly at para position (6, 7, 14, 15, 22, 23, 30 and 31).Interestingly, compound 31 ie.,parafluoro substituted K(Z)P dipeptide conjugated PZN may be considered as a good inhibitor of protein glycation particularly with protein 1N5U with highest G score -10.424. The binding modes (2D and 3D) of compound 31 and 23 were illustrated in Figure 1 and 2 respectively. Based on the observations made on biding modes (2D and 3D) of compound 31, the NH group of thiourea moieties shown H-bond (side chain) interaction with TYR 161. And also, the benzene ring of heterocycle 2,3-dichlorophenyl piperazine has π-π stacking interaction with PHE 134 and TYR 138. The G-score of compound 31 was found to be the highest docked score and based on these results, it was concluded that the NH group of thiourea moiety, benzene ring of heterocycle were responsible for interaction with active site of protein. The IC₅₀ values of antiglycation activity and the glide scores from the molecular docking studies were found to exhibit better correlation.

 

5. CONCLUSION:

The search for anti-glycating agents is itself an important clinical issue, in that glycation is not confined to eye protein and in diabetic patient’s glycation leads to damage of several other physiologically important proteins such as collagen, albumin and importantly, Hb. Few anti-glycating agents have been reported in the literature (e.g., Carnosine) and an apparently nontoxic agent like urea and thiourea showing antiglycating properties may itself have important clinical significance. From our studies, we may draw certain conclusions like Lys and Tyr containing dipeptides may serve as good antiglycating agents. On the other hand, the results revealed that compounds containing para flouro substitution in the phenyl ring of the thiourea moiety have more binding interaction with active site of 1N5U. Furthermore, from docking study we have strong evidence that conjugation and derivatization of amino acids and peptide analogs play a significant role on the biological activities. Thus, this synthetic novel urea and thioureas of dipeptide containing compounds may lead potent antiglycating agents.

 

6. ACKNOWLEDGMENTS:

The authors gratefully acknowledge University Grants Commission (UGC) New Delhi for awarding Post-Doctoral fellowship (PDFSS), BSR faculty fellowship and DST Inspire fellowship.

 

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Received on 06.05.2017 Modified on 11.06.2017

Accepted on 20.07.2017 © AJRC All right reserved