Synthesis and biological evaluation of new epalrestat analogues as aldose reductase inhibitors (ARIs)
Abstract
BayliseHillman chemistry derived four series of new epalrestat analogues were synthesized. Three structural changes are introduced in these 39 new epalrestat analogues synthesized. All compounds were evaluated for their in vitro aldose reductase inhibitory (ALR) activity. Biological activity data in- dicates that compounds 6, 16, 19, 28 and 29 are potent and the activity is in the range of reference drug, epalrestat. Molecular modelling studies were performed to understand the binding interactions of these active molecules with the ALR protein. Molecular docking data indicates the key interactions of epal- restat were retained in some of the active compounds whereas some new interactions were noticed for other molecules. The modifications introduced on epalrestat have impact for developing a new-type of ALR inhibitor.
1. Introduction
Type 2 Diabetes mellitus, also known as non-insulin dependent diabetes mellitus (NIDDM) is a type of metabolic disease in which the body loses its ability to control glucose levels in normal range. This is because of either from the alteration of insulin secretion by pancreas or development of insulin resistant by different organs especially skeletal muscle [1]. This disease is recognized as a chronic disease with high morbidity and mortality, and poses an economic burden for developing countries [2]. A recent study by WHO reveals that there were approximately 200 million people globally, ranging age 20e80 years, suffering from diabetes and this figure expected to increase to 366 million by the year 2030 [3]. Diabetic patients on prolonged exposure to uncontrolled hyper- glycemia lead to several diabetic complications [4] such as retinopathy [5], neuropathy [6], cataracts [7], nephropathy [8] and cardiovascular complication [9].
Activation of several biochemical pathways has been proposed by researchers, to explain the mechanism of diabetic complications [10]. Among these, polyol pathway is the most promising one and has been studied extensively [11]. Activation of polyol pathway has been reported in diabetic condition. Aldose reductase (ALR), the first and rate limiting enzyme of the polyol pathway is responsible for the conversion of glucose into sorbitol (Fig. 1) [12]. Generally, under euglycemia condition, most of the glucose metabolized to pyruvate via glycolytic pathway and very less amount of the glucose metabolized through polyol pathway. Therefore the role of the polyol pathway in glucose metabolism is very minor. However, when glucose levels are elevated, the contribution of the polyol pathway significantly increases, and that results in accumulation of sorbitol in the cells [13]. As a result, both oxidative stress and os- motic stress increases in the cells and leads to cellular damage that contribute to diabetic complications [14]. Although several oral hypoglycemics available in the market, but they are not that effective [15]. The achievement and maintenance of euglycemia is extremely difficult for the people with advanced stages of diabetes.
Fig. 1. Schematic diagram for polyol pathway.
However, the quality of life of diabetic patients can be improved by preventing diabetic associated complications and this can be ach- ieved by the inhibition of aldose reductase (ALR) enzyme. Hence, there is a need to synthesize new molecules that can substantially delay or prevent the development of diabetic complications through inhibition of ALR enzyme [16].
Recently, ARIs has become an attractive therapeutic strategy [17] and a variety of ARIs with different pharmacophore group have been synthesized and tested for their efficacy in several diabetic complications by researchers over the last two decades (Fig. 2). Based on their chemical structure, they can be classified into three general groups: (i) carboxylic acid derivatives (such as Zopolrestat, Epalrestat, Zenarestat, Ponalrestat, Tolrestat and recently devel- oped Lidorestat); (ii) spirohydantoins (includes Sorbinil, Ranirestat, AND-138 and Fidarestat); (iii) flavonoids (such as Quercetin and Resveratrol). But most of them were failed at clinical trials because of their side effects or poor efficacy [18]. Epalrestat is the only approved drug and it is currently used for the treatment of diabetic neuropathy in Japan, China and recently in India [19].
During the past ten years, a great deal of our efforts has been devoted to BayliseHillman chemistry [20] and its applications to- wards the synthesis of new heterocyclic compounds along with their potential biological activities [21]. In the present study we describe the synthesis of new epalrestat analogues using Baylise Hillman chemistry together with their aldose reductase inhibitor activity. Furthermore, we have performed molecular modelling studies for selected compounds to study the interaction with ALR enzyme from molecular prospective.
2. Results and discussion
2.1. Chemistry
In view of the fact, that the rhodanine (thiazolidine) function- ality is essential for ARI activity (Fig. 2, epalrestat), we focused on the synthesis of new epalrestat analogues carrying other functional groups. In this context, to increase the efficacy of epalrestat we mainly modified epalrestat in three domains: (i) substituted phenyl, furyl, thienyl or naphthyl ring in place of phenyl ring, (ii) nitrile group in place of methyl, and (iii) kept the rhodanine moiety intact but sometimes replaced the acetic acid group by benzoic acid, phenyl acetic acid and hydrogen moieties for the study (Fig. 3). The stereochemistry of two double bonds kept intact.
A series of new epalrestat analogues were synthesized as out- lined in Scheme 1 and Table 1. Aromatic aldehydes (2ae2l) with suitable substitution were reacted with acrylonitrile (3) in the presence of catalyst DABCO, under solvent free conditions [22] leading to the formation of BayliseHillman adducts (4ae4l). The BayliseHillman adducts (4ae4l) were oxidized using ionic liquid [23a] [Hmim] HSO4 and NaNO3 to obtain corresponding [E]-a- cyanocinnamaldehyde derivatives (5ae5l) with control of double bond stereochemistry [23]. The [E]-a-cyanocinnamaldehyde de- rivatives (5ae5l) were treated with rhodanine compounds under Knoevenagel condensation conditions to obtain final compounds (6e44) with control of double bond stereochemistry [24]. Rhoda- nine compounds 1a and 1b (Fig. 4) were obtained commercially whereas 1c and 1d (Fig. 4) were prepared as per the published procedure [25]. X-ray crystal structure is determined for compound 39, which helped us in further defining stereochemistry of two double bonds (Fig. 5). The new epalrestat analogues synthesized are grouped in to four series (Scheme 1, Table 1 and Chart 1).
Accordingly, we first examined the aldehyde (5a) with the rhodanine acetic acid (1a) as a choice of substrates for the study using various organic and inorganic bases under different solvent systems (Table 2). After many trials, we finally settled with an efficient procedure for the synthesis of epalrestat analogues (6a) in good yield (81%), when the reaction was carried out in acetic acid as a solvent and NH4OAc as a base (Table 2). Encouraged by this suc- cessful result, we examined all other aldehydes with various rho- danine compounds (1ae1d) and the results are summarized in Table 1.
2.2. Biological activity
2.2.1. Aldose reductase inhibitory activity
Thus synthesized 39 epalrestat analogues were taken forward to understand their ability towards aldose reductase inhibition. The enzyme aldose reductase converts aldehydes to alcohols in the presence of NADPH. Based on this property of aldose reductase, we used a kinetic based assay for the evaluation of inhibition of aldose reductase activity with the synthesized compounds (6e44). For this assay we used DL-glyceraldehyde as an aldehyde which is reduced to glycerol, in the presence of NADPH by aldose reductase enzyme. NADPH is a cofactor that is being oxidized to NADPþ. Therefore we measured the absorbance of unchanged NADPH, at different time intervals and at 340 nm. Absorbances of respective blanks were taken before adding NADPH [31].
All the synthesized compounds 6e44 were evaluated in vitro for their ability to inhibit aldose reductase activity using epalrestat as reference compound and results are presented in Fig. 6. Among the all 39 compounds, 6, 16, 19, 28 and 29 exhibited more than 50% inhibition and were comparable to standard drug epalrestat while the rest of the compounds exhibited less than 50% inhibition. The IC50 values were determined for those compounds displaying greater than 50% inhibition and corresponding data presented in Table 3 and Fig. 7. Among the all tested compounds, 29 found to be the most potent aldose reductase inhibitor with an IC50 value of 0.22 mM compared to the standard, epalrestat which has IC50 value 0.4 mM. Other compounds 6, 16, 19 and 28 exhibited excellent inhibitory activities with IC50 values of 0.75, 0.67, 0.69 and 0.86 mM respectively.
The 39 analogue compounds synthesized were grouped as se- ries 1 to 4 (Chart 1, Table 1). Under the series 1 (compound 6e17,Chart 1, Table 1) compound 6 and 16 are found to be comparable with standard epalrestat (Table 3) and the remaining compounds 7e15 and 17 are moderately active. Considering the epalrestat structure, the methyl group is replaced by an electron withdrawing nitrile group on the olefin chain did not make much difference in compound 6, in terms of its activity. The phenyl group having various substitutions (7e13, Chart 1, Table 1) did reflect in lowering inhibition activity. Replacing the phenyl group by naphthyl (com- pound 14) or with thiophene (compound 17) also provided reduced ARI activity. Compound 16 has furan ring and exhibited excellent activity comparable with epalrestat. All these compounds 6e17 carry rhodanine acetic acid end group as epalrestat. Compounds in series 2 (18e28, Chart 1, Table 1) carry three changes introduced.
Fig. 2. Chemical structures of some known aldose reductase inhibitors (ARIs).
Fig. 3. Structural modification of epalrestat.
The methyl group on the olefin chain is replaced by nitrile, did not make much difference. The rhodanine acetic acid is replaced by just rhodanine, not carrying acetic acid end group did reflected that, it is not mandatory in exerting ARI activity as evidenced in compounds
19 and 28. The compounds given in Fig. 2, quercetin (phenolic hydroxy), sorbinil, and ADN-138 are ARIs and do not carry car- boxylic group. Further the compound 19 has a substituent at the para position of phenyl group. The compound 28 indicates that replacing the phenyl group by thiophene residue did not affect much in the ARI activity. The observation made in the compounds of series 2 (18e28, Chart 1, Table 1) not carrying acetic acid end group, but still exerting the ARI activity (compound 19 and 28) is new and interesting in terms of understanding the small molecules and macromolecular interactions leading to its functioning. Series 3 compounds (29e39) have the special feature of replacing acetic acid by benzoic acid. Out of 11 compounds of series 3, only one compound 29 showed excellent activity. This is also an interesting observation that benzoic acid end group can also exert excellent ARI activity. The compounds of series 4, 40e44 (Chart 1, Table 1) carrying phenyl acetic acid end group did not contribute to ARI activity and all these five compounds 40e44 are moderately active compare to epalrestat standard. The structural changes introduced in making 39 epalrestat analogues revealed new and interesting structure activity relationships.
Fig. 4. Various rhodanine compounds used to make new ARIs.
Fig. 5. The molecular structure of 39 with the atom-numbering scheme. Displacement ellipsoids are drawn at 30% probability level and H atoms are shown as small spheres of arbitrary radius. The molecule crystallizes with DMSO solvent in 1:1 stoichiometric ratio.
2.2.2. Docking
In order to gain insight about proteineligand interactions of newly synthesized analogues in the active site of aldose reductase enzyme, molecular docking study was performed. The aldose reductase enzyme complexed with tolrestat (PDB ID: 2FZD) was selected [26]. Geometries of five active molecules were optimized by PM3 method using Gaussian 09 software [27]. The docking simulations in the active site of aldose reductase enzyme were done with aid of FlexX software [28] as it was observed the reproduction of binding interactions (Y48, H110, W111) of co-crystal with enzyme when tolrestat is docked into the active site of it. All the active compounds (6, 16, 19, 28 and 29) along with epalrestat (reference compound) were docked into the same active site of tolrestat (2FZD) and the results are summarized in Table 4. Among the five active compounds, compound 6, 16 and 29 were exhibited same interactions (Y48, H110, W111) as co-crystal ligand in anionic site and within these series, compound 16 and 29 exhibited addi- tional interactions with S302 (Fig. 8). The binding poses of these compounds are same as epalrestat in the anionic site of the enzyme. The common feature in these molecules is the presence of carboxylic acid functional group at the rhodanine terminal. Unlike epalrestat, compounds 19 and 28 adopted different poses and exhibited interactions with other residues of enzyme (Table 4). Both these compounds possess the free amide group in rhodanine.
3. Conclusion
In this paper we described the synthesis, biological activity, and structureeactivity relationships of a series of epalrestat analogues. BayliseHillman adducts were used in the synthesis of these newer epalrestat analogues. These are four series of compounds with free amide, N-acetic acid, N-benzoic acid and N-phenyl acetic acid. X-ray crystal structure helped us to define the stereochemistry of two double bonds. Compounds 6, 16, 19, 28 and 29 found to be active and the IC50 values of these compounds are in the same magnitude of the reference compound, epalrestat. Molecular docking studies gave an idea about the binding of these inhibitors, which in turn helped us to understand the role of acetic acid group. The present study demonstrated structural modification on epalrestat and showed five hit compounds from four series of compounds. These results of these bindings help us to design newer molecules/ana- logues towards developing potent ALR inhibitors.
Fig. 7. Concentration dependent aldose reductase inhibitory activity of most active new epalrestat analogues.
4. Experimental section
4.1. Chemistry
General: All commercially available chemicals were used without further purification. Melting points were determined on a Mel-Temp apparatus and are uncorrected. IR spectra were recorded using a Thermo Nicolet Nexus 670 FTIR spectrometer. The NMR spectra were recorded on Brucker Avance 300 magnetic resonance spectrometer at 300 MHz for 1H and 75 MHz for 13C respectively, using TMS as internal standard. The chemical shifts are expressed as d values in parts per million (ppm) and the coupling constants (J) are given in hertz (Hz). ESI-MS was obtained on Thermo-Finnigan MAT- 1020B instrument. Elemental analyses were carried out with a Perkin Elmer 2400 Series II elemental analyzer. Column chroma- tography was performed on silica gel (60e120 mesh, Acme, India).
Fig. 6. Aldose reductase inhibitory activity of new epalrestat analogues (1 mM in DMSO). Standard Drug: epalrestat (1 mM in DMSO).
4.2. General procedure for the synthesis of BayliseHillman adducts
Aromatic aldehydes (2ae2l) (10 mmol), acrylonitrile (3) (20 mmol) and DABCO (30 mol% with respect to aldehyde) were mixed and allowed to stir at room temperature until completion of the reaction (10e12 h). Upon completion, the reaction mixture was diluted with water (15 mL) and extracted with diethylether (3 25 mL). The combined organic layers were dried over Na2SO4, concentrated under reduced pressure and purified by column chromatography using 10% EtOAc in hexane as eluent to afford pure BayliseHillman adducts (4ae4l) in 80e90% yield. The spectro- scopic and analytical data of all the synthesized compounds were in good agreement with those reported in the literature [22].
4.3. General procedure for the synthesis of [E]-a- cyanocinnamaldehyde (5ae5l) [23a]
A stirred solution of BH adduct (4ae4l) (3 mmol) and NaNO3 (3 mmol) in 3 mL of [Hmim] HSO4 was heated at 80 ◦C for 1e2 h.The reaction progress was monitored by TLC. Upon completion, the reaction mixture was cooled to RT and extracted with ethylacetate (3 15 mL). The combined organic layers were dried over Na2SO4, filtered and evaporated under reduced pressure. The resulting crude product was purified by column chromatography using 10% EtOAc in hexane as eluent to afford pure [E]-a-cyanocinnamalde- hyde derivatives (5ae5l). The characterization data of the newly synthesized compounds were given below.
4.4. General procedure for the synthesis of epalrestat analogues (6e44)
A mixture of rhodanine (3 mmol), substituted [E]-a-cyano- cinnamaldehyde (3 mmol) and anhydrous ammonium acetate (3 mmol) were taken in glacial acetic acid (10 mL). The reaction mixture was heated to 120 ◦C in an oil bath for 3e4 h. The reaction was monitored by TLC. Upon completion, the reaction mixture was cooled, filtered and washed with water. Finally it was recrystallized from methanol to yield pure compounds.
4.5. Aldose reductase inhibition assay protocol
Rat kidney tissue was utilized as a source of aldose reductase enzyme. Rat kidney tissue was homogenized in ice cold potassium phosphate buffer (50 mM, pH 7.4). The homogenate was centrifuged at 12,000 rpm at 40 ◦C for 30 min. The resulting supernatant was stored at 80 ◦C and used as a source of aldose reductase enzyme for further assay. The in-vitro assay of aldose reductase enzyme inhibition was performed according to the method described by Hayman and Kinoshita [31]. Assay mixture containing 0.1 M potassium phosphate buffer pH 6.2, 0.9 M DL-Glyceraldehyde (SigmaeAldrich, USA), 2 M lithium sulphate (Acros Organics), mercapto ethanol (Biomatik), distilled water and 2 mM NADPH (SigmaeAldrich, USA) was prepared. Test compound is incubated along with the enzyme for 20 min at 37 ◦C. Reaction mixture devoid
of NADPH was added followed by incubation for 5 min at 37 ◦C, and used as a respective blank whose absorption has been recorded. The reaction was then initiated by addition of NADPH. The change in the absorbance at 340 nm due to NADPH oxidation was measured with the help of Lambda 25 UVeVisible Spectropho- tometer. The inhibitory activity of test compounds was evaluated based on the decrease in the absorbance of NADPH at 340 nm and quantified using formula Inhibition% ¼ 100-[T/C] × 100, where T ¼ absorbance of test reaction and C ¼ absorbance of control reaction. The therapeutic drug epalrestat was used as reference aldose reductase inhibitor. The inhibitory concentration 50% (IC50) was calculated by nonlinear regression. The doseeresponse curve was obtained by plotting the percentage inhibition versus the concentrations.