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Benzopyrazines: Synthesis, Characterization and Evaluation as Aldose Reductase Inhibitors

Role of aldose reductase (ALR2) in diabetic complications such as retinopathy, nephropathy, neuropathy, and cataract etc. is well-evident. ALR2 in the first step of polyol pathway reduces glucose to sorbitol whose elevated level leads to diabetic cataract, characterize by clouding of the lens in the eye that affects vision. Inhibition of ALR2 enzyme with small molecules as inhibitor is a rapid approach for diabetic management. In the present study the synthetic route to synthesize desired benzopyrazines and a library of sixteen (16) methyl benzopyrazines were screened against aldose reductase. From the bioactivity results, the 3'-hydroxyphenyl benzopyrazine 6l was found most active (IC50 = 1.34 ± 0.07 μM) while 3'bromophenyl analogue 6i showed comparable activity for ALR2 (IC50 = 3.48 ± 0.66 μM) as compared to standard sorbinil (IC50 = 3.14 ± 0.02 μM). Both compounds (6l and 6i) showed excellent selectivity for ALR2 over aldehyde reductase (ALR1) which has important role in detoxification of toxic aldehydes. The structure of two regio-isomers were fully characterize by 1 H and 13 C NMR two dimensional NMR techniques including COSY, NOESY, HSQC, and HMBC. Regio-isomers separation was proved to be difficult in different solvent systems. Only an isomer of 3'-bromo benzopyrazine 6i' was isolated that help to assign the structure of regioisomers from NMR data. All the benzopyrazines were fully characterized by using different spectral techniques including 1 H, 13 C NMR, IR spectroscopy, and mass spectrometry.


Introduction
Diabetes mellitus (DM) is chronic lifetime condition which disrupts body's ability to use glucose from food as energy source. DM is characterized by postprandial increased level of blood glucose which is termed as hyperglycemia [1]. The diabetic complications associated with hyperglycemia are blindness, renal failure, neuropathy, and cardiac arrest etc. Usually, under normal conditions glucose metabolize via glycolytic pathway [2]. However, during hyperglycemia normal pathway of glucose metabolism gets saturated and substantial amount of glucose enters into the polyol pathway. Aldose reductase (ALR2, EC 1.1.1.21) reduces the glucose to sorbitol which further oxidized to fructose with sorbitol dehydrogenase. ALR2 is a cytosolic enzyme that belongs to the class of aldo-keto reductase (AKR) superfamily. It is the first rate limiting enzyme of polyol pathway which reduce glucose to sorbitol. Intracellular sorbitol cannot pass through the cell membrane, hence accumulates within the cell causing osmotic stress that results in diabetic cataract complication [3]. Therefore, inhibition of ALR2 is considered an intelligent approach to reduce the progression of long term diabetic complications. Aldehyde reductase (ALR1, 1.1.1.2) is another member of AKR superfamily and share more than 65% amino acids sequence and structural homology with ALR2. [4] ALR1is present in all tissues and shows substrate specificity towards toxic aldehydes, such as hydroxynonenal, 3-deoxyglucosone, and methylglyoxal, produced in different pathological conditions associated with oxidative stress as in hyperglycemia [5]. In past years, a range of different molecules has been employed as ALR2 inhibitors [6][7][8][9][10]. Mostly known compounds are carboxylic acids such as tolrestat [11] or cyclic amides like sorbini [12], minalrestat [13] etc. A carboxylic acid drug, named epalrestat [14], is currently available in market for the treatment of neuropathy. However, some limitations like low in vivo efficacy of carboxylic acid drugs, cytotoxicity and harmful side effects of cyclic amides demands new molecules as ARL2 inhibitors. Recently, [15] group reported quinoxalinone derivatives as ALR2 inhibitors ( Figure 2). Literature revealed the diverse bioactivities of quinoxaline or benzopyrazine based compounds such as antimycobacterial [16], antileishmanial, [17], anticancer [18], antimalarial activities, [19] cholinesterases, [20][21] and αglycosidase inhibitors [22] etc. Thus, in this study, we synthesized sixteen benzopyrazine derivatives and screen evaluated them against ARL2 as well as ALR1 for selectivity purpose.

Material and Methods
All the starting materials including acetophenone derivatives, 4-methylbenzene-1,2-diamine, selenium dioxide were purchased from Sigma-Aldrich and were used without purification unless otherwise stated. HPLC grade dioxane and distill water were used as solvents. Silica gel 60 aluminium-backed plates having 0.063-0.200 mm as the stationary phase were used for thin layer chromatography (TLC) and flash silica was used for column chromatography. Analytical grade solvents include diethyl ether, ethyl acetate (EtOAc), hexane, and pentane were used as eluents. For TLC spot visualization, UV light (254 nm) or basic potassium permanganate or vanillin solution were used. KBr discs were used for IR spectra recording on a Bruker Vector-22 spectrometer. NMR spectra were recorded on Avance Bruker AM spectrometers 300, 400 or 500 MHz in the appropriate deuterated solvent at 25°C. All the chemical shifts were recorded on the δ-scale (ppm) using residual solvents as an internal standard (DMSO; 1 H 2.50, 13 C 39.43 and CHCl 3 ; 1 H 7.26, 13 C 77.16). Mass spectra (ESI + ) were recorded at Finnigan-MAT-321A, Germany by using electrospray (ES+), electron impact (EI+ EI+) or FAB (Fast atom bombardment) techniques.

General Procedure for the Synthesis of 6/7-methyl-2-arylbenzopyrazines 6(a-o)
In a typical experiment, an oven dried round-bottomed flask was charged with acetophenone (240 mg, 2 mmol), dioxane/water (10:1) and selenium dioxide (222 mg, 2 mmol) at room temperature. The resulting solution was stirred at 100°C for 3-4 h until no starting material was left over TLC plate. The reaction mixture was cold to room temperature and filtered. Filtrate was concentrated on a rotary evaporator under reduced pressure. Water (30 mL) was added and stirred at 100°C and then treated with activated charcoal. The corresponding dicarbonyl compound 4 was collected as white solid and used without purification in the next reaction. [23]. Compound 4 was treated with 4-methylbenzene-1,2-diamine 5 (146 mg, 1.2 mmol) at room temperature for 0.5-1 h until the complete consumption of dicarbonyl compound 4 as monitored by TLC analysis. The reaction mixture was diluted with water (40 mL) water and extracted with ethyl acetate (25 mL × 3). The combined organic layers were dried over anhydrous MgSO 4 , filtered and evaporated on rotary evaporator. The crude material was purified by silica gel column chromatography (EtOAc / Hexane 1:3 to 1:1). The benzopyrazines 6(a-o) were obtained in variable yields.

Isolation and Purification of Aldehyde (ALR1) and
Aldose (ALR2) Reductase Following the previously described procedure [25], aldose reductase (ALR2) extraction and purification; from the local slaughter house calf eyes were removed from a freshly slaughtered animal. Sample was kept frozen until used. Lenses were carefully removed from calf eyes. 100-200 g of lenses was homogenized in cold distilled water. Homogenate was centrifuged at 10,000 rpm at 0-4°C for 20 min to remove insoluble material. The supernatant was precipitated with 40% saturated ammonium sulfate, followed by 50% and 75% salt saturation. The pellet from the last step, possessing pure ALR2 activity, was dissolved in 0.05 M NaCl and dialyzed against 4 L of 0.05 M NaCl overnight and stored in small aliquots in liquid nitrogen container. [26] Aldehyde reductase (ALR1) isolation and purification; from local slaughter house calf kidneys were obtained which were then dissolved in 3 volumes of 10 mM sodium phosphate buffer having pH 7.2, containing EDTA dipotassium salt (2 mM), sucrose (0.25 M) and βmercaptoethanol (2.5 mM). A knife homogenizer was used to homogenize the solution. The homogenate centrifuged at 12,000 rpm at 4°C for 45 min to remove insoluble materials. The supernatant was collected and precipitated with 40%, 50% and finally 75% powdered ammonium sulfate saturation. The precipitate collected after saturation with 75% ammonium sulfate was re-dissolved in potassium phosphate buffer and dialyzed against the same buffer overnight and stored in liquid nitrogen container until used [27].

In-vitro Assay of Aldose and Aldehyde Reductase
Inhibition Following the procedure previously described, [28] the activity of aldose reductase (ALR2) was determine by observing the change in the absorbance at 340 nm. Sorbinil [29] and valproic acid [30] were used as positive control for ALR2 and ALR1, respectively The reaction mixture contained 100 mM potassium phosphate buffer (pH 6.2), 20 µL of the inhibitor, 0.1 mM NADPH and enzyme preparation in a total volume of 300 µL. The resulting mixture was incubated for 10 min at 37 º C. The reaction was initiated by the addition of 90 µL of 10 mM sodium DL-glyceraldehyde as a substrate. To correct the oxidation of NADPH, reference was used which contained all the above mentioned reagents except substrate [31].
The activity of ALR1 for synthetic compound was determined by monitoring the change in UV absorption by NADPH at 340 nm. The reaction mixture contained 100 µM potassium phosphate buffer (pH 6.2), 20 µL of the inhibitor, 0.1 mM NADPH and enzyme preparation in a total volume of 300 µL. The resulting mixture was incubated for 10 min at 37°C. The reaction was initiated by the addition of 90 µL of 10 mM sodium D-glucoronate as a substrate. One unit of enzyme was defined as the amount of enzyme required to catalyze the oxidation of 1.0 mM NADPH under the above mentioned conditions. Appropriate blanks were used for corrections. [32]

Chemistry
The synthetic route to synthesize desired benzopyrazines has been present in Figure 3. In the first step, the α-methyl group of acetophenone was oxidized to carbonyl group by using selenium dioxide. The dicarbonyl intermediate 4 was then reacted without purification with 4-methylbenzene-1,2diamine at room temperature to afford corresponding benzopyrazine (Figure 3). Following the optimized method, a range of novel benzopyrazines has been prepared [29]. 1 H NMR spectra of the resulting products showed two regioisomers in 1:1 to 1:0.5 ratio due to difference in reactivity of C-2 keto and C-3 aldehyde moieties in intermediate 4. The structure of two regio-isomers were fully characterize by 1 H and 13 C NMR two dimensional NMR techniques including COSY, NOESY, HSQC, and HMBC. Regio-isomers separation was proved to be difficult in different solvent systems. Only an isomer of 3'-bromo benzopyrazine 6i' was isolated that help to assign the structure of regioisomers from NMR data. Table 1 shows the data of isomeric mixture (6n/6n′) and Table 2 for isomer 6i′. Table 1 shows the data of 3′nitrophenyl benzopyrazine regio-isomers (6n/6n′). The only difference between both structures (I and II) is the position of methyl group (C-6/7) at phenyl ring B. The characteristic signals in both benzopyrazine isomers are C-5, C-6/7, C-8, and C-9/10. The structure I shows a broad singlet (brs) for C-5 proton at δ 7.94 ppm while in structure II brs for C-8 proton appears at δ 8.03 ppm. The HMBC spectrum shows correlation of C-5/I proton with C-7, C-9, and C-11 and C-8/II proton with C-6, C-10, and C-11. In NOESY spectrum, H-5/I correlations with H-7, C-11 and H-8/II with H-6, C-11 are observed. On the other hand, C-8/I and C-5/II protons show doublets (d) at δ 8.09 and δ 8.04 ppm, respectively, in both isomers. HMBC correlations of C-8/I proton with C-10 and C-5/II proton with C-9 are observed. COSY spectrum shows correlations of H-8/I with H-7 and H-5/II with H-6. NOESY spectrum also shows correlations of H-8/I with H-7 and H-5/II with H-6. Characteristic signals of C-7/I and C-6/II further confirm the structures of regio-isomers. A combined triplet of doublet (td) appears for both protons at δ 7.46 ppm. HMBC spectrum shows correlation of C-7/I proton with C-5, C-9, C-11 and C-6/II with C-8, C-10 and C-11. In NOESY spectrum, H-7/I shows correlation with H-8 and H-11 and H-6/II with H-5 and H-11. Further details of correlation data has been summarized in the Table 1. Moreover, 1 H, 13 C and correlations data of isolated 2-(3′bromophenyl)-7-methylbenzopyrazine 6i′ isomer has been summarized in Table 2 which corresponds to isomer (II) data in Table 1.

Conflict of Interest
There is no conflict of interest any involved in this article.

Supporting Information
The supporting information includes the experimental data and NMR spectra of benzopyrazines.