Molecular dynamics/quantum mechanics guided ...

Journal of Molecular Structure 1171 (2018) 556e563

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Journal of Molecular Structure journal homepage: http://www.elsevier.com/locate/molstruc

Molecular dynamics/quantum mechanics guided designing of natural products based prodrugs of Epalrestat Bhawna Vyas a, Shalki Choudhary b, Pankaj Kumar Singh b, Akashdeep Singh b, Manjinder Singh d, Himanshu Verma b, Harpreet Singh c, Renu Bahadur c, Baldev Singh a, Om Silakari b, * a

Department of Chemistry, Punjabi University, Patiala, Punjab, 147002, India Molecular Modeling Lab, Department of Pharmaceutical Sciences and Drug Research, Punjabi University, Patiala, Punjab, 147002, India Indian Council of Medical Research, New Delhi, India d Chitkara College of Pharmacy, Chitkara University, Rajpura, Punjab, India b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 22 February 2018 Received in revised form 7 June 2018 Accepted 7 June 2018 Available online 14 June 2018

Over activated molecular target ALR2 of polyol pathway and oxidative stress are two well established pathological indicators in diabetic complications. In present study, mutual prodrugs of Epalrestat and natural product based antioxidants were designed, synthesized and evaluated. In silico techniques such as molecular dynamic simulations and quantum mechanical approaches, considering human esterase enzyme (hCE1) as the site of hydrolytic cleavage were employed in designing of these mutual prodrugs. Geometrical parameters (distance and Burgi-Dunitz angle favorable for nucleophilic attack) and quantum mechanical parameters (HOMO-LUMO energy gap), which govern the hydrolytic cleavage of ester prodrugs by esterase enzyme were calculated. Further, on the basis of in silico analysis, mutual prodrugs were synthesized and considering the values of in silico parameters, EPL-GUA (3g) was evaluated for its in vivo antioxidant activity. Results suggested that 3g possess significant antioxidant activity intermediary to control and Epalrestat. Thus, this study has concluded that these mutual prodrugs could be optimized to develop molecules for the management of diabetic complications. © 2018 Published by Elsevier B.V.

Keywords: Epalrestat Diabetic complications Mutual prodrugs Molecular dynamics

1. Introduction In present scenario, diabetes is one of the major health issue of both the developed and developing countries. Basically Diabetes mellitus (DM) is a metabolic disorder [1], characterized by chronic hyperglycaemia which lead to development of diabetes-specific microvascular pathology in the retina (retinopathy), renal glomerulus (nephropathy) and peripheral nerves (neuropathy). This microvascular pathology, due to continuous hyperglycaemic condition, results in the development of various diabetic complications such as blindness, renal disease and devastating neuronal disorders. Additionally, hyperglycaemia is also associated with atherosclerotic macro-vascular disease affecting the blood supply to heart, brain and lower extremities, resulting in much higher risk of myocardial infarction, stroke and limb amputation. At molecular level, some major pathophysiological hypotheses

* Corresponding author. E-mail address: [email protected] (O. Silakari). https://doi.org/10.1016/j.molstruc.2018.06.030 0022-2860/© 2018 Published by Elsevier B.V.

about how hyperglycaemia causes diabetic complications have been proposed. These hypotheses include increased polyol pathway flux (including ALR2 and Sorbitol dehydrogenase), increased advanced glycation end-product (AGE) formation, activation of protein kinase C (PKC) isoforms, increased hexosamine pathway flux (GFAT) and PARP [Poly (ADPribose) polymerase]. In addition to these pathways, oxidative stress also plays a key role in the complications arising due to hyperglycemic conditions. It was reported that hyperglycemia induced oxidative stress triggers all possible molecular pathways of diabetic complications. Thus, diabetic complications are group of complex multi-factorial metabolic disorders, depicted by abnormal metabolism of carbohydrates, proteins and lipids due to hyperglycemia [2]. Complex multi-factorial disorder cannot be effectively managed with single targeted therapy because other pathological targets continue to show their deleterious effects. For such type of the complex disorders, Morphy and Rankovic [3] have suggested a unified term ''designed multiple ligands'' (DMLs), i.e. compounds that are rationally designed to exhibit two or more specific pharmacological actions. Based on this concept a DML, Benfotiamine (1),

B. Vyas et al. / Journal of Molecular Structure 1171 (2018) 556e563

has been developed by Stracke et al., 2001 that simultaneously blocks three targets of diabetic complications and successfully preventing diabetic retinopathy in rats [4]. Because of the encouraging results in animal studies, the status of this drug has been shifted from preclinical to clinical investigational drug. However, there is still no multi-targeted drug available in the market for the effective management of diabetic complications. Designing effective DMLs by current lead-discovery strategies is challenging to medicinal chemists because this designing often lead to generation of large complex molecules with low ligand efficiency, poor oral bioavailability and generates non-specificity. Moreover, DMLs usually cannot be designed for more than three targets because of specificity issues. Under such conditions concept of mutual prodrugs becomes more effective than DMLs. A mutual prodrug consists of two pharmacologically active agents coupled together so that each acts as a promoiety for the other agent and vice versa. After biotranformation, two components of mutual prodrug can display their effect with same original potencies. Epalrestat (2) is an aldose reductase inhibitor, official in Japanese pharmacopoeia, and available for the treatment of diabetic neuropathy, only in the markets of Japan, India and China [5,6]. It is a heterocyclic thiazolidine group containing acetic acid derivative and have number of serious side effects including nausea, vomiting, diarrhea, cutaneous reactions i.e. erythema, bullae and skin blistering and generalized gastric discomfort. Gastric irritation is one of the major side effect that limit the use of this drug, which is hypothesized to be due to free COOH (carboxylic acid) group present in it. Thus it was thought worthwhile to design ester mutualprodrug of Epalrestat by masking COOH group with natural antioxidant containing alcoholic functionality. These prodrugs are expected to target two pathological indications of diabetic complications i.e. ALR2 of polyol pathway and unifying mechanism of oxidative stress (Fig. 1). Rationale behind designing of mutual ester prodrugs is based on the presence of a class of human carboxylesterase enyzmes in the liver and gut. Human carboxylestrase can be subdivided into four major subclasses but only human carboxylestrase I (hCE1) are reported to play significant role in the hydrolysis of ester containing drug molecules. The crystal structure of hCE1 enzyme has been solved and well documented in the literature [7]. Exhaustive work has also been reported in the literature encompassing the cleavage of ester containing prodrugs such as cocaine at the catalytic domain of hCE1, disclosing the mechanism of action for this enzyme [8]. Moreover ab-initio, density functional theory (DFT) and semi empirical QM/MD techniques play significant role in the modern computational chemistry to deduce the catalytic mechanism of various drugs [9]. Among these techniques, DFT is one of the most successful and advanced technique to calculate the energy of electronic structures with correlation effect [10,11]. DFT is a universally accepted quantum mechanical method which have better

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features over conventional ab-initio and semi-empirical methods used in quantum chemistry [12]. Thus, in our work, considering the catalytic site of hCE1, we employed DFT technique for the calculation of energy and electron density of whole system. Further, DFT was employed for the calculation of quantum mechanical parameters (HOMO-LUMO energy gap), geometrical parameters (distance and Burgi-Dunitz angle) and molecular dynamic simulations to uncoil the mechanism of cleavage and design ester based prodrugs of Epalrestat and natural product based antioxidants [13]. 2. Material and methods 2.1. HOMO-LUMO calculations and molecular dynamic simulations The essential step in the catalytic action of hCE1 involve nucleophilic attack of hydroxyl group of serine on the carbonyl of ester group of mutual prodrugs, which could be easily explained on the basis of frontier molecular orbital approach. In this approach, the flow of electrons is determined on the basis of difference in energy between HOMO of one reactant and with LUMO of another reactant. To determine the susceptibility of prodrugs to be attacked by hCE1, initially, serine containing hydroxyl group was sketched and energy was minimized in ‘ligprep’ program using OPLS_2005 force-field. The HOMO and LUMO calculations were performed for €dinger both serine and prepared mutual prodrugs using Schro software. The DFT method was employed to calculate the HOMOLUMO energies of all structures. In the DFT calculations, Local density approximation (PWC) functional was used. No solvent was considered in the calculations along with DN basis set with SCF density convergence of 1.0 e4. To calculate the burgi-dunitz angle and distance between the structural units of enzyme and prodrugs, molecular dynamic simulations were performed. The mutual prodrugs docked complexes of enzyme were utilized to perform the molecular dynamic simulations for the time frame of 10 ns and with a time step of 1fs. The OPLS_2005 force-field was employed to carry out the simulation €dinger. For the simulation study in the Desmond module of Schro studies, initially the solvent model was built by solvating the system using TIP3P water model with an orthorhombic water box of 10 10 10 A_ 3 dimensions. The system was neutralized by adding Naþ as counter ions and salt concentration was fixed at 0.15 M. Nose-Hoover chain thermostat was used to carry out the NPT equilibrium at 310 K and Martyn-Tobias-Klein was employed for maintaining pressure of 1 bar. 2.2. Chemistry Epalrestat, employed in the study was procured as gift sample from pharmaceutical industry i.e. Microlabs Ltd., Bangalore. Drugs procured as gift sample were authenticated through melting point

Fig. 1. Structures of benfotiamine and Epalrestat.

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determination, IR, NMR and Mass spectral analysis. The antioxidants compounds used in the study including five terpenes (Menthol, Eugenol, Vanillin, Thymol, Guaiacol) were procured from Loba Chemie Ltd. Mumbai, Flavone (7-hydroxyflavone & Chrysin), Coumarin (Umbelliferone) and Sesamol were procured from Sigma Aldrich (New Delhi, India). The chemical reactions were monitored by thin layer chromatography (TLC) using pre-coated aluminium plates (Merck, Mumbai, India) visualized in UV chamber at short as well as long wavelengths. The synthesized compounds were purified by column chromatography using silica gel (100e200 mesh). The melting points were recorded by open sulfuric acid bath and uncorrected. 1H NMR spectra were recorded on Bruker AC 300 NMR Spectrometer (400 MHz), chemical shifts were reported in parts per million using tetramethylsilane as internal standard with multiplicities (br, broad; s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; dd, double doublet) and number of protons in the solvent specified. The coupling constants (J) were expressed in Hz. FTIR spectra were recorded on FT-IR Perkin-Elmer 1710 series. IR spectra were recorded as KBr pellets. High resolution mass spectra were recorded on Micromass Q-TOF micro mass spectrometer (Waters, MA, USA) in positive mode of electrospray ionization

(þESI). The solvents and reagents were dried prior to use when required, over KOH or anhydrous Na2SO4 or fused CaCl2.

2.2.1. General method employed for the synthesis of Epalrestatantioxidant prodrugs In a 250 ml round bottom flask, 12.43 mmols of drug (Epalrestat) was taken and dissolved in 30 ml of dried dichloromethane (DCM). Then 14.8 mmol of different alcohols were added and stirred for 15 min. The reaction mixture was cooled at 0 C with crushed ice. At 0 C, 3.05 g (14.83 mmol) of N,N-dicyclohexylcarbodiimide (DCC) and 0.017 g (1.4 mmol) of 4-dimethylaminopyridine (DMAP) were added. The reaction mixture was stirred at room temperature for 24 h. After 24 h, reaction was monitored by pre-coated TLC. The dicyclohexylurea (DCU) precipitates formed in the reaction was removed by filtration and the filtrate was washed with distilled water, 5% Glacial acetic acid, 1% sodium hydroxide solution and again washed with distilled water, further dried over sodium sulphate. The solvent was then evaporated and crude product obtained was recrystallized with alcohol or column chromatography to obtain pure crystals (Scheme 1).

Scheme 1. Synthetic scheme for the synthesis of Epalrestat-antioxidant prodrugs.


2.3. Biological evaluation 2.3.1. Material Male wistar rats (200e300 g) were procured from CCS University of Veterinary Sciences (Hissar) and were housed in cages under controlled conditions with 12 h light/dark cycle in the animal house, Punjabi University, Patiala. The animals were given standard laboratory pallet chow diet (Kissan Feed Ltd., Chandigarh, India) and water ad libitum. The animals were randomly allocated in groups at the beginning of all the experiments. All the compounds and reference drugs were administered per oral (p.o.) suspended in 0.5% carboxymethyl cellulose solution (CMC). Dose of the test compounds was selected on the basis of various literature reports. The study was approved by institutional animal ethical committee under CPCSEA guidelines. 2.3.2. In vivo antioxidant activity 2.3.2.1. Selection of dose. Dose of the test compounds was selected on the basis of various literature reports for the Epalrestat studies [14,15]. For in vivo antioxidant activity, five groups composed of six animals were employed. 2.3.2.2. Determination of blood glucose levels. Blood glucose concentration (mmol/L) was determined by glucose peroxidase method using commercially available kit (Reckon diagnostics Pvt. Ltd. Vadodra, India). Blood samples were collected from the animals. 2.3.2.3. Induction of diabetes. Diabetes was induced in rats by intraperitoneal (i.p) injection of streptozotocin (STZ) at a dose of 55 mg/kg b. w dissolved in distilled water (1 ml/kg b. w.) [16]. Seven days after the injection, blood glucose level was measured. Each animal with a blood glucose concentration level >17 mmol/L was considered to be diabetic and used in the experiments as shown in Table 3. To prevent the hypoglycemia during the first 24 h following STZ administration, 5% glucose solution was orally given to the diabetic rats. In all experiments, rats were fasted for 16 h prior to STZ injection. 2.3.2.4. Preparation of tissue homogenate. The glandular part of excised stomach was homogenized in ice cold phosphate buffer (pH 7.4) tissue homogenizer (PT194, Remi, New Delhi, India) for 2 min. The homogenate was centrifuged at 5000 rpm for 10min. The supernatant was again centrifuged at 15,000 for 15 min and the clear supernatant was used for the estimation of glutathione (GSH) levels [17], catalase (CAT) activity [18] and lipid peroxidation in terms of thiobarbituric acid reactive substances (TBARS) levels [19]. 3. Result and discussion 3.1. Molecular analysis of Epalrestat prodrug cleavage via human carboxylestrase 1 (hCE1) Human carboxylestrase (hCE) is class of enzyme reported for the hydrolysis of ester based prodrugs, a well-known example include cocaine. This class of enzyme can be further sub-divided into four classes, however only two major classes (hCE1 and hCE2) are reported to have any significant role in human beings. Out of these two, hCE1, due to its small catalytic domain, cleave only small alcohol unit containing ester prodrugs while hCE2 is reported to possess a comparatively bigger catalytic domain. The catalytic function of both hCE1 and 2 involves a triad system of Ser, His and Glu in the catalytic site. Out of these three, OH of serine attack on the electrophilic carbonyl of the ester prodrugs, while rest two residues stabilize the complex, thereby assisting in the catalytic

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function. Thus, as most of the selected natural product based antioxidants for the study were having monocyclic phenolic structure, hCE1 enzyme was selected to analyze the cleavage of the prodrugs. Further to analyze hydrolytic cleavage of the designed prodrugs by nucleophilic substitution using hCE1 enzyme, suitable parameters were considered and selected. As the mechanism of catalysis involves transfer of electrons from protein residues to the prodrugs in the catalytic domain, quantum mechanics approaches such as HOMO-LUMO difference can provide suitable insight into the exact mechanism. The highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) of a molecule are called the frontier orbitals. It was Fukui, who initially studied the prominent role played by HOMO and LUMO in governing chemical reactions. It has been revealed by recent investigation that the gap in energy between the HOMO and LUMO is an important stability index. A large gap implies high stability and small gap implies low stability. The high stability in turn indicates low chemical reactivity and small gap indicates high chemical reactivity. The energy and symmetry type of, and the charge distribution in HOMO, and the energy and symmetry type of LUMO are known to determine the structures of molecules. The energy and symmetry types of such frontier orbitals are also found to be the principal factor for determining the occurrence and non-occurrence of chemical reactions [20]. Since the hydrolytic cleavage is mediated by nucleophilic attack, calculation of burgi-dunitz angle (BD angle) along with the distance becomes imperative. The BD angle is an angle that defines the geometry of “attack” of a nucleophile on a trigonal unsaturated center in a molecule. The angle was named after crystallographers HansBeat Bürgi and Jack D. Dunitz. The BD angle primarily depends on the molecular orbital shapes, occupancies of the unsaturated center and secondarily on the molecular orbitals of the nucleophile. The reason for the alteration in the BD angle can be attributed to the requirement of maximum overlapping between the HOMO of the nucleophile and LUMO of the unsaturated trigonal center of the electrophile [21]. Thus, for each prodrug, all the three parameters were calculated by performing molecular dynamic simulations for complexes of prodrugs and crystallized structure of hCE1 (PDB ID: 1YA8), selected on the basis of resolution. Molecular dynamics (MD) studies were performed using Desmond which operates on the OPLS_2005 force-field, for the time period of 10 ns [22,23]. The simulations were performed to obtain stable conformations of enzyme with prodrugs, which were further utilized for the calculations of reactivity parameters. Additionally, to derive significant conclusion, the obtained values for all three parameters of each prodrug were compared with values of cocaine, a well-documented substrate for hCES. Initially, following quantum mechanics approach frontier molecular orbital (FMO) energy difference, in eV, was calculated. The HOMOprotein-LUMOprodrug gap was calculated by considering HOMO of serine residue of the esterase enzyme and LUMO of each prodrug, (Fig. 2). The values mentioned in Table 1 clearly indicate that reaction between serine and synthesized prodrugs possess smaller energy gap than the reaction of serine and cocaine, as standard, thus quantum mechanics approach favors the cleavage of synthesized prodrugs more efficiently than cocaine. Additionally, to calculate the Burgi-Dunitz angle (aBD) and distance between the nucleophilic eOH of serine and electrophilic carbonyl of prodrugs, molecular dynamic simulation was employed for a period of 10 ns. As the literature suggests, nucleophile positioned at an obtuse angle with range of 100 ± 10 towards plane of electrophilic center is favorable for the electron transfer. Thus, after 10 ns the esterase-prodrug complexes were stabilized, comparisons were made on the basis of calculated aBD and distance of hydroxyl group of serine from the carbonyl of ester group of prodrugs. The

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3.2. Chemistry Total nine Epalrestat-antioxidant prodrugs were synthesized using Steglich esterification. Different well known antioxidants having alcoholic/phenolic hydroxyl group including terpenes (Menthol, Eugenol, Vanillin, Thymol, Guaiacol), Flavones (7hydroxyflavone & Chrysin), Coumarin (Umbelliferone) and Sesamol were selected to conjugate with -COOH of Epalrestat to develop mutual prodrugs having ALR2 inhibitory activity along with antioxidant property. The antioxidants used to synthesize these prodrugs are given in Scheme 1 along with their structures (Scheme 1).

Fig. 2. HOMO eLUMO gap representation.

Table 1 Calculated HOMO and LUMO energies for all the designed prodrugs along with HOMO-LUMO gap with serine. S.no

Ligands

HOMO

LUMO

HOMO#-LUMO$(eV)

a) b) c) d) e) f) g) h) i) j) k)

3a 3b 3c 3d 3e 3f 3g 3h 3i Cocaine Serine

0.164 0.165 0.153 0.159 0.167 0.199 0.157 0.164 0.166 0.157 0.186

0.126 0.124 0.079 0.121 0.077 0.129 0.119 0.125 0.126 0.078 0.069

- 0.060 - 0.062 - 0.107 0.065 0.109 0.057 0.067 0.061 0.060 - 0.108 e

HOMO# ¼ Serine. LUMO$ ¼ Molecules (i-j).

comparison depicts that majority of the prodrugs (3b, 3c, 3d, 3f, 3g, 3h, 3i) maintained the angle close to burgi-dunitz angle (80 e110 ) (Fig. 3), while in two cases (3a, 3e), prodrugs failed to acquire the desired angle range (40 e80 ). Similarly, comparison of the distances between the eOH group of serine of esterase and carbonyl of prodrugs indicated that only three prodrugs 3b, 3d and 3g possess distance within 4 Å (Fig. 4). As it is evident from cocaine, details available in literature, the distance should lie below 4 Å for favorable nucleophilic attack, thus on the basis of distance only three prodrugs as expected to interact favorably with hCE1. In conclusion, both the energy gap (obtained from quantum mechanics approach) and burgi-dunitz angle are in coherence with each other and thus favor the cleavage of prodrugs. However, distance as a parameter does not favor the cleavage of many of prodrugs. One of the reasons for this could be the size of phenolic/ alcoholic unit in the prodrugs. All the prodrugs with monocyclic aromatic alcoholic unit fits well into the catalytic cleft and thus possess favorable distance. On the other hand, prodrugs with bicyclic aromatic alcohol unit, which is bulky or alicyclic (3a), with different conformations (Table 2), does not properly occupy the catalytic cleft leading to increase in distance between attacking nucleophile (serine) and electrophile (ester of prodrugs).

3.2.1. General procedure for the synthesis of ester prodrugs of Epalrestat The Epalrestat based ester prodrugs (3a-3i) were synthesized with stirring of Epalrestat (1) and different alcohol/phenols (2a-2i) in the presence of DMAP and DCC in anhydrous dichloromethane according to a reported procedure with slight modifications (Scheme 1) [24]. During the reaction, precipitated DCU (dicyclohexylurea) was filtered off and the filtrate evaporated using rotary evaporator. The residue was taken up in DCM solution which was further washed twice with 0.5 N HCl and with saturated solution of NaHCO3 and then dried over MgSO4. The solvent was removed by evaporation and the product was recrystallized. Obtained product was further purified on silica gel column using CHCl3 as eluent. The structures of prodrugs (3a-3i) were characterized on the basis of their analytical and spectroscopic data. In 1H NMR spectra the aromatic protons of all the compounds appeared downfield at d7.0e7.8 and the protons of -CH2 of the prodrugs appeared at d4.3e5.5. The five aromatic protons appeared in the range of d7.0e7.8 which indicated the presence of Epalrestat molecule in the structure of prodrugs. In prodrugs 3b and 3h, proton peaks of aldehyde and hydroxyl groups shifted downfield at d9.0 and 12.6 respectively. The compounds were characterized through IR, 1H NMR, and MS (þESI) spectral analysis. The optimized reaction conditions, melting point and percent yield of each compound are mentioned below. [2-Isopropyl-5-methylcyclohexyl-2-((Z)-5-((E)-2-methyl-3phenylallylidene)-4-oxo-2-thioxothiazolidin-3-yl) acetate](3a) Epalrestat, 3.54 g; Menthol 2.30 g; DCC; DMAP; in 100 ml of DCM Stirring time 24 h, pale yellow crystal, yield 90%,m.p 233e236 C. IR cm1:2845 (s, C-H Ar, str), 1708 & 1751 (s, C]O str), 1622 (C]C str), 1397 (m, C-H, -CH3, bend), 1318 (s, C-N str), 1187 (m, C-O str). 1H NMR (CDCl3): 7.25e7.36 (5H, m, ArH), 7.05 (1H, d, CH), 7.01 (1H, s, CH), 4.74 (2H, s, -CH2), 4.64e4.79 (1H, m, CH), 2.19 (3H, d, -CH3), 1.93e1.96 (1H, m, CH), 1.79e1.83 (1H, m, CH), 1.58e1.61 (2H, s, -CH2), 1.49 (2H, m, CH) 1.28e1.31 (1H, s, CH), 0.92e0.98 (2H, m, -CH2) 0.77e0.84 (9H, m, -CH3), 0.67e0.69 (2H, m, CH2). 13C NMR (100 MHz, CDCl3): 193.12, 167.18, 165.47, 144.02, 140.12, 139.89, 135.97, 133.30, 129.51, 128.63, 128.54, 121.36, 49.13, 46.93, 45.00, 40.88, 34.07, 31.35, 29.67, 25.51, 21.93, 20.71, 16.16, m/z 458.23 [MH]þ [4-Formyl-2-methoxyphenyl-2-((Z)-5-((E)-2-methyl-3phenylallylidene)-4-oxo-2-thioxothiazolidin-3-yl) acetate] (3b) Epalrestat, 3.54 g; Vanillin 2.24 g; DCC; DMAP; in 100 ml of DCM, Stirring time 24 h,pale yellow crystals, yield75%,m.p 223e226 C. IR cm1:2835 (s, C-H str), 1705 & 1765 (s, C]O str), 1620 (m, C]C str), 1390 (m, C-H, -CH3, bend), 1318 (s, C-N str), 1180 (m, C-O str). 1H NMR: (CDCl3): 9.8 (1H,s, CHO), 7.5 (1H, m, ArH), 7.4 (2H, m, ArH), 7.3 (5H, m, ArH), 7.2 (1H, s, CH), 7.0 (1H, s, CH), 5.0 (2H, d, CH2), 3.8 (3H, s, OCH3), 2.1 (3H, s, CH3). 13C NMR (100 MHz, CDCl3): 192.04, 166.22, 164.29, 152.97, 151.18, 146.22, 143.43, 137.38, 135.79, 135.48, 134.90, 133.00, 131.65, 129.63, 126.63, 124.46, 123.84, 123.33, 120.02, 119.76, 112.19, 56.16, 15.73, 44.64, m/z 476.11


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Fig. 3. Burgi-Dunitz angle calculated between eOH of serine and ester carbonyl of prodrug 3 g.

Fig. 4. Nucleophilic attack distance between eOH of serine and ester carbonyl of prodrug 3 g.

Table 2 Calculated nucleophilic attack distance in A for all the designed prodrugs along with angle between eOH and carbonyl of 3 g. Molecule

Distance

Angle

3a 3b 3c 3d 3e 3f 3g 3h 3i Cocaine

7 4 > 10 4 > 10 9 4 8 8 4

60 90±10 100±10 100±10 unstable 100±10 90±10 100±5 100±10 60

þ

[MþNa] . [4-(But-3-en-2-yl)-2-methoxyphenyl-2-((Z)-5-((E)-2-methyl3-phenylallylidene)-4-oxo-2-thioxothiazolidin-3-yl) acetate] (3c)

Table 3 Dose/Kg, Bodyweight, blood glucose levels of control, vehicle and STZ-diabetic rats. Group(n ¼ 6)

Dose/kg

Body Weight (g)

Blood Glucose (mmol/L)

Initial

Final

Initial

Final

Control Vehicle (CMC) STZ Epalrestat 3g

e 0.5% w/v 55 mg/kg, i.v. 40 mg/kg 53 mg/kg

277 ± 2.61 280 ± 2.30 290 ± 2.08 282 ± 2.82 278 ± 2.47

281 ± 1.96 279 ± 2.01 231 ± 1.78 232 ± 2.59 238 ± 3.88

6.7 ± 0.07 6.4 ± 0.16 6.3 ± 0.20 5.7 ± 0.20 7.0 ± 0.24

6.3 ± 1.7 6.6 ± 1.5 21.3 ± 0.8 18.9 ± 1.5 16.1 ± 1.0

Epalrestat, 3.54 g; Eugenol, 2.42 g; DCC; DMAP; in 100 ml of DCM Stirring time 24 h,pale yellow crystals, yield89%,m.p 181e182 C. IR cm1:2843 (s, C-H str) 1774 & 1708 (s, C]O str), 1600 (s, C]C str), 1315 (m, C-N str), 1160 (m, C-O str), 725 (m, CH2bend). 1H NMR: (CDCl3)7.5 (1H, s, CH), 7.4 (5H, m, ArH), 7.0 (1H,

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s, CH), 6.9 (1H, s, ArH), 6.7 (2H, m, ArH), 5.9 (1H, m, CH), 5.1 (4H, m, -CH2), 3.7 (3H, m, -OCH3), 3.3 (2H, d, -CH2), 2.2 (3H, d, CH3). 13C NMR (100 MHz, CDCl3): 193.01, 167.12, 164.32, 150.65, 144.15, 140.08, 139.44, 137.57, 136.98, 135.98, 133.32, 129.54, 128.68, 128.58, 122.26, 121.34, 120.63, 116.21, 112.85, 55.91, 49.19, 44.62, 40.06, 33.90, 29.70, 24.92, 16.19, m/z 480.62 [MH]þ. [2-Isopropyl-5-methylphenyl-2-((Z)-5-((E)-2-methyl-3phenylallylidene)-4-oxo-2-thioxothiazolidin-3-yl) acetate] (3d) Epalrestat, 3.54 g; Thymol 2.22 g; DCC; DMAP; in 100 ml of DCM Stirring time 24 h, pale yellow crystals, yield84%,m.p 198e201 C. IR cm1: 2875 (s, C-H str), 1704e1770 (s, C]O str), 1605 (s, C]C), 1320 (m, C-N), 1178 (m, C-O). 1H NMR (CDCl3): 7.5 (1H, s, CH), 7.3e7.2 (5H, m, ArH), 7.2 (1H, s, CH) 7.1 (1H, s, ArH), 7.0 (1H, s, ArH), 6.9 (1H, s, CH), 6.7 (1H, s, ArH) 5.0 (2H, s, CH2), 2.9 (1H, m, CH), 2.2 (3H, s, CH3), 2.1 (3H, s, CH3), 1.1 (3H, m, CH3), 1.0 (3H, m, CH3), m/z 452.25 [MH]þ. [2-oxochroman-7-yl-2-((Z)-5-((E)-2-methyl-3phenylallylidene)-4-oxo-2-thioxothiazolidin-3-yl) acetate] (3e) Epalrestat, 3.54 g; Umbelliferone, 2.42 g; DCC; DMAP; in 100 ml of DCM Stirring time 24 h, pale yellow crystals, yield77%,m.p242246 C. IR cm1: 2910 (s, C-H str), 1690e1770 (s, C]O str), 1580 (s, C]C str), 1310 (m, C-N str), 1130e1150 (m, C-O str).1H NMR (CDCl3): 7.6 (1H, s, CH), 7.5 (1H, d, CH), 7.2e7.3 (5H, m, ArH), 7.1 (2H, m, ArH), 5.0 (2H, s, CH2), 2.2 (3H, d, CH3), 0.8e0.7 (4H, m,CH2CH2), 13C NMR (100 MHz, CDCl3): 193.05, 167.04, 164.14, 160.16, 154.62, 152.51, 144.78, 142.72, 140.75, 135.88, 134.28, 133.22, 132.42, 131.32, 130.89, 129.72, 129.58, 128.83, 128.68, 128.61, 120.97, 118.10, 117.08, 116.42, 110.29, 44.79, 28.91, m/z 465.76 [MH]þ. [Benzo[d] [1,3]dioxol-5-yl 2-((Z)-5-((E)-2-methyl-3- phenylallylidene)-4-oxo-2-thioxothiazolidin-3- yl) acetate](3f) Epalrestat, 3.54 g; Sesamol, 2.04 g; DCC; DMAP; in 100 ml of DCM Stirring time 24 h, pale yellow crystals, yield55%, m. p253255 C.IR cm1: 2925 (s, C-H str), 1705e1760 (s, C]O str), 1545 (s, C]C str), 1325 (m, C-N str), 1180e1300 (m, C-O str). 1H NMR (CDCl3): 7.6 (1H, s, CH), 7.3e7.6 (5H, m, ArH), 7.1 (1H, s, CH), 6.8 (1H, m, ArH), 6.4 (2H, m, ArH) 5.9 (2H, s, CH2), 4.6 (2H, d, CH2), 3.5 (3H, m, CH3), 13C NMR (100 MHz, CDCl3): 167.11, 164.91, 156.76, 148.02, 145.71, 144.48, 142.46, 140.44, 135.93, 133.99, 133.27, 129.71, 129.56, 128.75, 128.66, 128.59, 121.16, 113.77, 107.96, 103.47, 101.80, 49.16, 44.83, 16.17, m/z 454.26 [MH]þ. [2-Methoxyphenyl-2-((Z)-5-((E)-2-methyl-3-phenylallylidene)4-oxo-2-thioxothiazolidin-3-yl) acetate] (3g) Epalrestat, 3.54 g; Guaiacol 1.83 g; DCC; DMAP; in 100 ml of DCM Stirring time 24 h, pale yellow crystals, yield92%,m.p203205 C. IR cm1: 2844 (s, C-H str), 1709 & 1775 (s, C]O str), 1568 (s, C]C str), 1315 (m, C-N str), 1166 (m, C-O str). 1H NMR (CDCl3): 7.5 (1H, s, CH), 7.3e7.4 (5H, m, ArH), 7.1e7.2 (1H, m, CH), 7.0 (2H, m, ArH), 6.9 (2H, m, ArH), 5.1 (2H,s, CH2), 3.83 (3H, s, CH3), 2.2 (3H, d, CH3), 13C NMR (100 MHz, CDCl3): 193.02, 167.12, 164.21, 150.90, 144.17, 140.10, 139.34, 135.98, 133.32, 129.54, 128.66, 126.58, 127.28, 122.57, 121.33, 120.70, 112.55, 55.93, 44.61, 29.70, 16.19, m/z 426.41 [MH]þ. 4-Oxo-2-phenyl-4H-chromen-7-yl-2-((Z)-5-((E)-2-methyl-3phenylallylidene)-4-oxo-2-thioxothiazolidin- 3-yl) acetate] (3h) Epalrestat, 3.54 g; 7-hydroxyflavone 3.52 g; DCC; DMAP; in 100 ml of DCM Stirring time 24 h, pale yellow crystals, yield 65% m.p227-228 C. IR cm1: 2910 (s, C-H str) 1710e1770 (s, C]O str), 1560 (s, C]C str), 1250 (m, C-N str), 1140e1080 (m, C-O str). 1H NMR (CDCl3): 8.2 (1H, s, ArH), 7.7 (2H, s, ArH), 7.6e7.4 (5H, m, ArH), 7.4e7.3 (4H, m, ArH), 7.3 (1H, s, ArH), 7.2 (1H, s, CH), 7.1 (1H, s, CH) 6.8 (1H, s, ArH), 4.6 (2H, d, CH2), 2.3 (3H, s, CH3), 13C NMR (100 MHz, CDCl3): 167.94, 163.74, 144.84, 132.42, 130.90, 129.59, 129.13, 128.85, 128.80, 128.63, 126.35, 68.16, 38.70, 30.74, 22.99, 19.19, 14.07, 10.96, m/z 454.26 [MH]þ. [5-Hydroxy-4-oxo-2-phenyl-4H-chromen-7-yl-2-((Z)-5-((E)-2methyl-3-phenylallylidene)-4-oxo-2thioxothiazolidin-3-yl)

acetate] (3i) Epalrestat, 3.54 g; Chrysin, 3.75 g; DCC; DMAP; in 1000 ml of DCM Stirring time 24 h, pale yellow crystals, yield72%m.p211213 C. IR cm1: 3360 (broad, OH str), 2710 (s, C-H str), 1705e1760 (s, C]O str), 1600 (s, C]C str), 1325 (m, C-N str), 1050e1260 (w, CO str). 1H NMR (CDCl3): 12.6 (1H, d, OH), 7.4e7.8 (5H, m, ArH), 7.3e7.2 (4H, m, ArH), 7.2 (1H, m, ArH), 7.1 (1H, m, CH), 7.0 (1H, s, ArH), 6.9 (1H, s, ArH), 6.8 (1H, dd, CH), 6.6 (1H, s, ArH), 5.0 (2H, d, CH2), 2.2e2.3 (3H, m, CH3), 13C NMR (100 MHz, CDCl3): 193.02, 182.83, 168.41, 167.03, 164.80, 163.82, 161.97, 156.67, 155.91, 144.75, 142.69, 140.72, 135.87, 134.25, 133.22, 132.23, 131.32, 130.91, 129.71, 129.18, 128.80, 128.60, 126.42, 109.20, 106.12, 100.85, 68.14, 44.81, 16.16, m/z 439.18 [MH]þ.

3.3. Biological evaluation Synthesized prodrugs were evaluated for in vivo antioxidant activities using reported methods.

3.3.1. In vivo antioxidant activity Since in silico analysis revealed that mutual prodrug 3g is expected to be readily cleaved considering the calculated parameters (nucleophilic attack distance, burgi-dunitz angle and HOMO-LUMO energy gap) and constraints in availability of animals, only 3 g was evaluated for in vivo oxidative stress in wistar rats. The in vivo antioxidant activity was evaluated by monitoring oxidative stress induced markers such as glutathione (GSH), catalase (CAT) and lipid peroxidation in terms of TBARS [25]. The effects of mutual prodrug 3g with respect to control group on various peripheral markers of oxidative stress are displayed in Fig. 5. The streptozotocin (STZ) decreased the CAT and GSH levels and increased the level of TBARS in comparison to control. However, 3g reversed these changes in comparison to STZ, although not equivalent to the control which suggested that the compound is a good antioxidant. Parent drug epalarestat (EPL) also reverse the changes in comparison to STZ but have less activity than most potent prodrug molecule 3g. The levels of these three oxidative markers were brought maximally close to the control values by 3g, indicating that it exhibited protective effects against oxidative stress and produced synergistic effect.

Fig. 5. Effects of various pharmacological interventions on Catalase (CAT), glutathione (GSH) and thiobarbituric acid reactive substance (TBARS) level. Values are expressed as mean ± SEM. aValues are statistically different from the control at p < 0.001. bValues are statistically different with respect to STZ at p < 0.001.


3.3.2. Statistical analysis The results of pharmacological evaluations were expressed as ±standard error of mean (SEM) of three experiments (n ¼ 3). The statistical significance was determined by one way ANOVA followed by Dunnet's test. The statistical significance was checked at p < 0.001. 4. Conclusion Failure in the management of diabetic complications still poses a big challenge for the research community. Side effects of Epalrestat limit its use and clinical applicability. This scenario is an opportune moment to utilize the concept of mutual prodrug. Results obtained from molecular dynamic simulation studies of the designed mutual prodrugs reveal the fact that monocyclic antioxidant based mutual prodrugs are an easy substrate for human esterase enzyme than bicyclic antioxidants. These results were further validated by quantum mechanical approach. Among all synthesized mutual prodrugs, on the basis of in-silico evaluation, 3g was subjected to in vivo study which showed significant free radical scavenging capacity. The reason for the improved activity of 3g over Epalrestat could be hypothesized due to synergistic effect, upon cleavage by hCE1, of both natural product based antioxidant and Epalrestat, as suggested by in silico studies. Thus, these mutual prodrugs could be considered potential candidates for the development of effective therapy against diabetic complications. Acknowledgments Authors thank the ‘Indian Council of Medical Research (ICMR)’, New Delhi, for providing fellowship as Research Associate (ICMRRA); Award No. BIC/11(12)/2015. Appendix A. Supplementary data Supplementary data related to this article can be found at https://doi.org/10.1016/j.molstruc.2018.06.030. Abbreviations ALR2 DM WHO DCM CAT GSH TBARS STZ EPL ANOVA SEM DCC DMAP DCU CMC DPPH

Aldose reductase Diabetes mellitus World Health Organization Dichloromethane Catalase Glutathione Thiobarbituric acid reactive substances Streptozotocin Epalarestat Analysis of variance standard error of mean N,N-dicyclohexylcarbodiimide 4-dimethylaminopyridine Dicyclohexylurea Carboxymethyl cellulose solution 2,2-diphenyl-picryl hydrazyl radical

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