Design and Synthesis of Potent and Multifunctional Aldose Reductase

Article pubs.acs.org/jmc

Design and Synthesis of Potent and Multifunctional Aldose Reductase Inhibitors Based on Quinoxalinones Xiangyu Qin, Xin Hao, Hui Han, Shaojuan Zhu, Yanchun Yang, Bobin Wu, Saghir Hussain, Shagufta Parveen, Chaojun Jing, Bing Ma, and Changjin Zhu* Department of Applied Chemistry, Beijing Institute of Technology, Zhongguancun South Street, 100081 Beijing, China S Supporting Information *

ABSTRACT: Quinoxalin-2(1H)-one based design and synthesis produced several series of aldose reductase (ALR2) inhibitor candidates. In particular, phenolic structure was installed in the compounds for the combination of antioxidant activity and strengthening the ability to fight against diabetic complications. Most of the series 6 showed potent and selective effects on ALR2 inhibition with IC50 values in the range of 0.032−0.468 μM, and 2-(3-(2,4-dihydroxyphenyl)-7-fluoro-2-oxoquinoxalin-1(2H)yl)acetic acid (6e) was the most active. More significantly, most of the series 8 revealed not only good activity in the ALR2 inhibition but also potent antioxidant activity, and 2-(3-(3-methoxy-4-hydroxystyryl)-2-oxoquinoxalin-1(2H)-yl)acetic acid (8d) was even as strong as the well-known antioxidant Trolox at a concentration of 100 μM, verifying the C3 p-hydroxystyryl side chain as the key structure for alleviating oxidative stress. These results therefore suggest an achievement of multifunctional ALR2 inhibitors having both potency for ALR2 inhibition and as antioxidants.

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INTRODUCTION In the past several decades, the enzyme aldose reductase (ALR2, EC 1.1.1.21), which is a member of the aldo−keto reductase superfamily, has received much attention from researchers all over the world, because the development and progression of chronic diabetic complications including neuropathy, nephropathy, cataracts, retinopathy, accelerated atherosclerosis, and increased cardiovascular risk are confirmed to be quite related to the activation and/or overexpression of ALR2.1a,b Therefore, ALR2 may play a critical role in preventing or treating these complications which are a lifethreatening risk for diabetic patients. ALR2 is the first rate-determining enzyme in the polyol pathway and catalyzes the reduction of glucose to sorbitol in the presence of NADPH as a cofactor (Figure 1). Sorbitol is in turn converted into fructose with accompanied reduction of NAD+ by sorbitol dehydrogenase. Under normal circumstances, glucose is predominantly converted to glucose-6-phosphate by hexokinase and then enters the glycolytic pathway, whereas only a small amount of glucose is metabolized through the polyol pathway due to a relatively low affinity of ALR2 for this substrate. During hyperglycemia, the polyol metabolic pathway is activated and the increased flux of glucose through the polyol pathway triggers the accumulation of sorbitol, which mainly happens in tissues demonstrating insulin-independent uptake of glucose, such as lens, kidney, retina, and peripheral nerves.1c With the increase of concentration of sorbitol, the activity of © XXXX American Chemical Society

Figure 1. Polyol pathway of glucose metabolism and pathogenesis of diabetic complications.

sorbitol dehydrogenase was not enhanced. Sorbitol has strong polarity so it is difficult to be eliminated by penetrating the cell membrane, eventually leading to osmotic imbalance, cell swelling, and membrane permeability changes, mainly in the lens. Simultaneously, the dramatic reduction of NADPH and NAD+ gives rise to changes in cellular redox potentials and damages the activity of enzymes such as nitric oxide synthase (NOS) and glutathione reductase. These changes result in cellular oxidative stress, as a result of the imbalance between Received: September 26, 2014

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DOI: 10.1021/jm501484b J. Med. Chem. XXXX, XXX, XXX−XXX

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Journal of Medicinal Chemistry

Figure 2. Structures of aldose reductase inhibitors (ARIs).

diabetic complications. Obviously, ALR2 inhibitors (ARIs) restrain the abnormal accumulation of sorbitol and may indirectly inhibit oxidative stress. They therefore have potential as therapeutic drugs for diabetic complications and also offer new effective strategies for anti-inflammatory and anticancer treatment.8 So far, a great number of ARIs have been developed in the past few decades, and some typical ARIs are shown in Figure 2.9 Currently, epalrestat (Figure 2) is the only ARI available for the therapy in Japan and more recently in China and India. Most of the ARIs that appeared to be promising have yet to be clinically successful mainly due to pharmacokinetic drawbacks, adverse side effects, or low in vivo efficacy. The side effects are the result of a lack of selectivity relative to aldehyde reductase (ALR1, EC 1.1.1.2), which plays a detoxification role in specifically metabolizing toxic aldehydes such as hydroxynonenal (HNE), 3-deoxyglucosone, and methylglyoxal.10 ALR2 and ALR1 share a high degree of homology in their primary and secondary structures, substrate specificities, and kinetic mechanisms.11 Moreover, the reason for the low efficacy is still under study, and it is speculated that only inhibiting the accumulation of sorbitol with ARIs is not enough to prevent and treat pathological changes in different tissues. It will be a feasible strategy to directly suppress oxidative stress and simultaneously inhibit the accumulation of sorbitol for increasing efficacy. Thus, antioxidants that inhibit ROS or oxidative stress are also important candidates for the treatment of diabetic complications. Some flavonoids of natural antioxidants were found to have aldose reductase inhibitory activity but failed to be further developed probably because they were not strong enough in ALR2 inhibition. Therefore, as our efforts in the design of a new type of aldose reductase inhibitors, both the inhibition of aldose reductase and direct antioxidation are planned to be combined into an organic whole. The resulting multifunctionality will greatly increase the potential to make an effective drug for diabetic complications. We have recently developed several groups of ARIs, of which quinoxaline derivatives with a substituted phenoxyl or benzyl

increased production of reactive oxygen species (ROS) and reduced intracellular antioxidant defense. In addition, a series of stress reactions occurring downstream of the polyol pathway, including the formation of advanced glycation end products (AGEs), protein kinase C (PKC) isomer, mitogen-activated protein kinase (MAPK), and poly-ADP-ribose polymerase (PARP), also lead to oxidative stress and the corresponding series of inflammation.2 In fact, recent studies found that the involvement of aldose reductase in the inflammatory signals induced various oxidants, which may cause tissue damage, pathological changes, and corresponding colon cancer.3 Additionally, the oxidative stress may convert aldose reductase to the oxidized form and concomitantly passivate the binding activity of enzyme to ALR2 inhibitors (ARIs).4 These oxidative stress reactions along with the polyol pathway represent important pathogenesis of diabetic complications.5 ROS is an important component of free radicals which are highly reactive with other molecules due to their unpaired electrons. It is commonly produced during cellular metabolism and functional activities and has important roles in cell signaling, apoptosis, gene expression, and ion transportation.6 However, it gives rise to oxidative stress in higher concentration, and the accumulation of excess of ROS results in the oxidative degradation of vital biomolecules such as lipids, proteins, and DNA. Cells are normally able to defend themselves against ROS damage through the use of intracellular enzymes to keep the homeostasis of ROS at a low level. However, during times of environmental stress and body disorder, ROS levels increase dramatically and then cause significant cellular damage in the body. Thus, oxidative stress significantly contributes to the pathogenesis of inflammatory disease, cardiovascular disease, cancer, diabetes, cataracts, Alzheimer’s disease, and aging.7 Antioxidants may help to prevent cellular damages from oxidative stress and also lower the risk of chronic diseases. Accordingly, the polyol pathway-linked accumulation of sorbitol and series of oxidative stress are leading causes of B


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Figure 3. Synthetic quinoxalin-2(1H)-one derivatives.

styrenes was carried out using the synthetic procedure described by Azzam and co-workers.12 The same hydrolysis method was applied for the preparation of the desired compounds 8 from 7. To distinguish between the carbon− carbon double bond and carbon−carbon single bond as spacers, compounds 9 and 11 were further synthesized through the Pd/ C hydrogenation reaction. Subsequently, compounds 10 were obtained smoothly from 9. Unfortunately, the acid form of esters 11 could not be obtained by hydrolysis probably due to the instability of the desired products. Finally, 2 reacted readily with a variety of substituted phenthiols, compared with anilines and phenols in previous research, to install thiophenyl side chains at the C3 position,9j leading to methyl esters 12, and then hydrolysis with lithium hydrate yielded the corresponding 2-(3-thiophenyl-2-oxoquinoxalin-1(2H)-yl)acetic acid derivatives 13.

group as C3 side chain (Figure 2) showed distinguished activity.9i,j The present study will focus on the further optimization of the C3 side chain and the introduction of a phenolic hydroxyl group to the aromatic ring of the side chain to obtain the multifunctionality composed of antioxidant and ALR2 inhibitory activities.

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CHEMISTRY All compounds including 4, 6, 8, 10, and 13 (Figure 3) described in the present study have been obtained by synthesis starting from substituted 3-chloro-quinoxalin-2(1H)-ones (1a− g) prepared in our previous work,9j as shown in Scheme 1. Compounds 1 were alkylated at the N1 position with methyl bromoacetate to form methyl esters 2 as key intermediates for generating various target compounds. Different phenyl groups were readily attached to the C3 position of the quinoxalin2(1H)-one core in 2 by Suzuki coupling in the presence of 3 mol % of Pd(OAc)2, 7 mol % of triphenylphosphine, and 2 equiv of Cs2CO3 to construct the C3 side chains and therefore to provide compounds 3 in good yields. Hydrolysis of 3 with lithium hydroxide gave desired carboxylic acids 4 as ARI candidates. To introduce the polyphenolic hydroxyl group, resorcinol was coupled with 2 by catalysis with AlCl3, and the reaction proceeded well, providing the corresponding 2,4dihydroxyphenyl-substituted quinoxalines 5a−h in good to excellent yields. 5i−k bearing the C3 heteroarene substituents were prepared in the same way. The ester groups in the N1 side chain of 5 were hydrolyzed with lithium hydrate to yield 6. For increasing the length of the C3 side chain of the core structure, the Heck coupling reaction was also applied in the study, and the preparation of intermediates 7 from 2 with corresponding

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RESULTS AND DISCUSSION Inhibition of Enzymes. All of the designed compounds (Figure 3) were tested for their potential inhibition against ALR2 isolated from rat lenses. To evaluate their selectivity for the ALR2 inhibition, they were also tested for their inhibitory activity against ALR1, isolated from rat kidneys. The results are expressed as IC50 (μM) or the percentage of enzyme inhibition (%) and summarized in Table 1. Compounds 14, 15, and 16 reported previously9i,13 were retested along with the newly synthesized compounds for the purpose of SAR studies (Table 1). All compounds investigated in the present study include three series according to the spacer length between the C3 position of the quinoxaline core and the aromatic ring of the C3 C


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Journal of Medicinal Chemistry Scheme 1.

a

(a) BrCH2CO2CH3, K2CO3, CH3CN, 65 °C,2 h, 78−88%; (b) C6H5B(OH)2, Cs2CO3, Pd(OAc)2, Ph3P, dioxane, H2O, 60 °C, 75−85%; (c) LiOH, H2O, THF, rt, 2 h, then 0.1 N HCl, 75−86%; (d) resorcinol or other heteroarenes, AlC3, CH2ClCH2Cl, 80 °C, 78−90%; (e) styrene, Pd(OAc)2, P(o-tolyl)3, Et3N, DMF, 100 °C, 62−75%; (f) H2, 10% Pd/C, CH3OH, EtOAc, rt, 6 h, 86−90%; (g) H2, 10% Pd/C, CH3OH, EtOAc, 45 °C, 36 h, 42−49%; (h) PhOH or PhSH, DMF, K2CO3, 75 °C, 24 h, 78−88%. a

side chain (Table 1). For the first series including compounds 4 and 6, the aromatic rings are directly attached to the C3 position. For the second, compounds 13 and 14 have a spacer of one atom. Then for the third, compounds 8, 10, 15, and 16 possess a two-atom length spacer. Compounds 4 show a significant effect of substitutions in the aromatic ring of the core structure and the C3 phenyl ring on the ALR2 inhibition. Comparison of 4b−f with unsubstituted compound 4a revealed that the different substitutions at either the C7 position of the core or the para position of the C3 side chain or at both all boosted the potency although these compounds generally had only moderate activity in the ALR2 inhibition. However, interestingly, further introduction of a phenolic hydroxyl group to the ortho position of the C3 phenyl ring in compound 4f leading to 6a resulted in a great enhancement in the activity. Consequently, more optimizations of 6a formed the structural series of 6b−i. Of these compounds, 2-(3-(2,4dihydroxyphenyl)-2-oxoquinoxalin-1(2H)-yl)acetic acids 6b−g

were specified to have excellent potential in the ALR2 inhibition in the present study, and 6e was the most active compound with an IC50 value of 0.032 μM. Significantly, all compounds in this series have a halogen atom at the C6 or C7 position of the quinoxaline core and double phenolic hydroxyl substituents at the phenyl ring of the C3 side chain. Analysis of the structure−activity relationship (SAR) regarding the halogen substituents at the C6 and C7 positions indicated that the substitutions largely increased the inhibitory activity by comparing 6b−g with compound 6a. Of compounds 6b−g, the fluoro substituent was the most effective, and chloro and bromo substituents in turn decreased the inhibitory activity slightly. Also, it is likely that the C7-halogen substitutions were more effective than the C6 substitutions. However, the bulky substituent of benzyl groups at the C7 position leading to compounds 6h was less effective than the 7-halogen substitutions. Besides, instead of the C3 phenyl side chains, introduction of large groups to the C3 position of the D


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Journal of Medicinal Chemistry Table 1. Enzyme Inhibition Activity of Quinoxalinone Derivatives

substituent compd

R1

R2

X

IC50 (μM)a for ALR2

inhib (%)b of ALR1

(% inhibALR2)/(% inhibALR1)

4a 4b 4c 4d 4e 4f 6a 6b 6c 6d 6e 6f 6g 6h 6i 6j 6k 13a 13b 13c 13d 13e 13f 13g 13h 13i 13j 13k 14d 15d 16d 8a 8b 8c 8d 10a 10b epalrestat

H H 7-F 7-Cl 4-fluorophenyl H H 6-F 6-Cl 6-Br 7-F 7-Cl 7-Br 4-fluorobenzyl H 7-Br 7-Cl H H H 7-F 7-Cl 7-Br 6-Br H 7-F 7-Cl 7-Br H H H H 7-F H H H 7-F

H 4-F 4-F 4-F 4-F 4-OH 2,4-(OH)2 2,4-(OH)2 2,4-(OH)2 2,4-(OH)2 2,4-(OH)2 2,4-(OH)2 2,4-(OH)2 2,4-(OH)2 3-indolec 3-indolec 2-benzothiophenec H H 4-Br 4-Br 4-Br 4-Br 4-Br 4-Cl 4-Cl 4-Cl 4-Cl H H H 4-OH 4-OH 4-OCH3 3-OCH3, 4-OH 4-OH 4-OH

− − − − − − − − − − − − − − − − − O S S S S S S S S S S CH2 CHCH CH2−CH2 CHCH CHCH CHCH CHCH CH2−CH2 CH2−CH2

5.981 (5.893−6.069) 3.380 (3.239−3.521) 0.874 (0.768−0.979) 1.516 (1.340−1.691) 2.131 (1.990−2.272) 2.592 (2.416−2.767) 0.397 (0.344−0.449) 0.063 (0.027−0.098) 0.095 (0.059−0.130) 0.139 (0.103−0.174) 0.032 (0.014−0.049) 0.069 (0.044−0.093) 0.091 (0.062−0.121) 3.34 (3.164−3.515) 0.639 (0.603−0.674) 0.368 (0.340−0.396) 0.238 (0.217−0.259) 0.468 (0.439−0.496) 0.421 (0.392−0.449) 0.296 (0.271−0.321) 0.191 (0.169−0.212) 0.326 (0.297−0.354) 0.467 (0.435−0.498) 0.319 (0.301−0.339) 0.273 (0.248−0.297) 0.056 (0.041−0.070) 0.158 (0.129−0.186) 0.395 (0.359−0.430) 1.112 (0.981−1.243) 0.820 (0.770−0.870) 0.143 (0.125−0.161) 0.182 (0.146−0.217) 0.153 (0.128−0.177) 4.181 (4.005−4.356) 0.419 (0.366−0.471) 0.798 (0.762−0.833) 0.652 (0.620−0.683) 0.084 (0.059−0.112)

25.4 31.8 35.8 23.6 41.0 34.6 36.6 42.5 46.5 38.1 46.2 44.5 35.9 11.6 32.3 17.5 53.3 6.6 32.2 12.8 45.5 14.1 24.0 8.7 42.4 29.5 17.3 10.2 32.8 32.0 22.0 42.4 36.2 21.8 32.6 12.2 23.0 73.6

− − − − − − − − − − − − − − − − − 12.5 2.7 6.9 1.9 6.1 3.7 10.2 1.9 3.1 5.1 8.3 − − − − − − − − − −

a

IC50 (95% CL) values represent the concentration required to decrease enzymatic activity by 50%. Values were determined from dose−response curves generated using at least three concentrations, each performed in triplicate. bInhibitory effects were evaluated at a concentration of 10 μM. Data are from single experiments. cR2 was directly connected with the C3 position of 2. dCompounds prepared previously.9i,13

substitutions of 13c and 13h giving 13d and 13i, respectively, increased significantly the activity. However, C7-chloro, C7bromo, and C6-bromo substitutions were not significantly beneficial, and some of them even showed a negative effect on the ALR2 inhibition. Nevertheless, 13a, 13g, and even 13k demonstrated low ALR1 inhibition and therefore good selectivity. In the longer C3-spacer series (8, 10, 14, and 15), mixed results of the ALR2 inhibition were observed. The carbon− carbon single bond spacer had a positive effect on the activity compared with the double bond spacer (15 and 16), but hydrogenation of the C3-double bonds of 8a,b both resulted in a decrease in activity (10a,b). In addition, C7-fluoro

quinoxaline core leading to compounds 6i−k resulted in moderate activity similar to that of 6a. However, it is worthy to note that 6j formed from the combination of C3-indole and C7-bromo substituents revealed much less inhibition of ALR1, indicating a good inhibition selectivity for ALR2. Compounds 13a−k all showed good activity in the ALR2 inhibition. Of them, 13i was the best inhibitor with an IC50 value of 0.056 μM and was more potent than epalrestat. SAR study indicated that the O-spacer and the S-spacer of the C3 side chains had similar potency toward ALR2 inhibition (13a and 13b, in Table 1). Also, p-bromo and p-chloro substitutions on the C3-phenthiol ring displayed a similarly beneficial effect when comparing 13c and 13h with 13b. Further C7-fluoro E


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lost much of the radical scavenging activity (16.2% and 14.7%) compared with their parent compounds (71.8% and 67.2%). Also, a sharp drop of the activity was observed for 8c formed from blocking the p-hydroxyl of 8a with methyl, very similar to the observation in the ALR2 inhibition as described above. Coincidentally, the natural product resveratrol, a well-known excellent antioxidant, also has the p-hydroxystyryl structure.15 Particularly, it is interesting to find that the introduction of methoxyl group to the meta position of the C3 p-hydroxystyryl ring of 8a (IC50 = 51.5 μM) largely boosted the activity from 71.8% to 95.4% (8d, IC50 = 24.1 μM) at a concentration of 100 μM, and this antioxidant potency is comparable to that of Trolox at this concentration level. Despite all that, there was an observable difference in the kinetics of the radical scavenging reaction with the DPPH between 8a and Trolox. The reaction rate with 8a is slower than that with Trolox, and the steady state was reached after 240 min, while for Trolox it was achieved after 30 min (reported time of 20 min by Silva and coworkers16). Inhibition of Lipid Peroxidation. To further confirm the antioxidant activity of the synthetic compounds described above, the effect of the compounds on hydroxyl radicaldependent lipoperoxidation induced in rat brain homogenate by the oxidant system Fe(III)/ascorbic acid was also examined. In membranes, relative antioxidant reactivity is probably different from that in a homogeneous system, because additional factors have a role, such as location of the antioxidant and radicals, and antioxidant reactivity is ruled to an extent by the partition ratios between water and lipophilic compartments.9g Agreeably, the antioxidation results from the test of DPPH radical scavenging were perfectly replicated by the method of suppressing lipid peroxidation with the compounds. As shown in Figure 5, the tested compounds inhibited the

substitution of the quinoxaline core only slightly increased the activity (8a,b and 10a,b). Blocking the phenolic p-hydroxyl group of the C3 side chain with a methyl group almost blocked the ALR2 inhibition (8a and 8c), and introduction of a methoxyl group next to the p-hydroxyl group also reduced the activity (8a and 8d). SAR study regarding the spacer length of the C3 side chains suggests that the ALR2 inhibition increases as the spacer length increases, by comparing 4a, 14, 15, and 16. This observation was also confirmed by the comparison of 4f and 8a. DPPH Radical Scavenging Activity. The antioxidant properties of the synthesized compounds containing the phenolic hydroxyl group were also investigated in the present study, and 6-hydroxy-2,5,7,8-chroman-2-carboxylic acid (Trolox) was employed as a positive control. The radical scavenging potential of the active ARIs 4f, 6a−g, 8a−d, and 10a,b was assessed in vitro by using the model reaction with the stable free radicals of 2,2-diphenyl-1-picrylhydrazyl (DPPH), according to the modified method, which was first employed by Blois.14 The scavenging activity data was expressed as DPPH radical scavenging rate (%), and results are summarized in Figure 4. In this homogeneous system of the methanol solution

Figure 4. DPPH radical scavenging activity.

of DPPH (0.025 mg/mL), antioxidant activity was derived from an intrinsic chemical reactivity toward radicals. According to the obtained data, antioxidant activity was detected for all of the tested compounds although it was lower than that of the positive control Trolox. As shown in Figure 4, the series of 8a− d were significantly active in the DPPH radical scavenging and much more potent than the other series, while the series of 6a− g were less active although they possessed excellent ALR2 inhibition. Of tested compounds, 8d showed the best DPPH radical scavenging activity, and the DPPH radical scavenging rate with the compound was 95.4%, 68.8%, and 39.6% at a concentration of 100 μM, 50 μM, and 10 μM, respectively, giving an IC50 value of 24.1 μM (Table 1, Supporting Information). SAR study of 4f and 8a shows that the two compound structures are different just in the spacer of the C3 side chain but 8a possessing the C3-vinyl spacer had an activity of 71.8% at a concentration of 100 μM in the DPPH radical scavenging, almost 1 order of magnitude greater than that of 4f (Table 1, Supporting Information). Therefore, the p-hydroxystyryl moiety may be seen as a structural indication of the DPPH radical scavenging activity in the present study. Apparently, it was further demonstrated by the effect of hydrogenation of the double bond spacer in compounds 8a,b on the DPPH radical scavenging activity in which the hydrogenation products 10a,b formed from 8a,b, respectively,

Figure 5. Inhibition of lipid peroxidation.

production of thiobarbituric acid reactive substances (TBARS), an index of lipid peroxidation, with different degrees of efficacy at 100 μM. Compounds 8a,b and 8d showed appreciable antioxidant properties, much more potent than other tested compounds, further demonstrating the importance of the phydroxystyryl structure for the quinoxaline derivatives in the antioxidant activity. Given the good activity of compounds 8a,b and 8d in the ALR2 inhibition, the findings from the antioxidant tests suggest that the compounds may be excellent leading structures for drug candidates in the treatment of diabetic complications. It should be also noted at this point that the antioxidant activity is considered as an important indication for the ability of compounds to suppress the oxidative stress resulting from the formation of advanced glycation end products (AGE stress) and the activation of protein kinase C (PKC stress), which are F


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Figure 6. Docking of inhibitors 8a into the active site of ALR2. (a) Docking of compound 8a into the ALR2 active site. The protein structure is shown in ribbon and tube representation with selected residues labeled and shown in line representation; ligand 8a and NADP are shown as stick models. The docked pose of 8a is shown in cyan (C), red (O), and blue (N). Hydrogen bonds are shown as yellow dashed lines. (b) Surface representation of protein residues in the docking of 8a into the active site of ALR2.

although two phenolic hydroxyl groups were present in the compounds. However, 8a,b and 8d were not only sufficient to inhibit ALR2 but also effective for DPPH radical scavenging and suppressing lipid peroxidation, and even 8d was as strong as Trolox at a concentration of 100 μM in the tests of antioxidant activity. This verifies significant efficiency for alleviating oxidative stress by the compounds and then suggests success in the development of multifunctional ARIs having ALR2 inhibition and antioxidant activity. These results allowed us to evaluate the detailed effects of C3 side chains and substituents at the quinoxaline core on the inhibitory activity against ALR2 and the antioxidant activity. SAR analysis combined with docking studies concludes that the introduction of o-hydroxyl to the p-hydroxyphenyl C3 side chain of 4f greatly increased the inhibitory activity (6a) against ALR2, and particularly the insertion of a vinyl spacer into the C3 side chain in 4f more greatly enhanced the inhibitory activity but also achieved strong antioxidant functionality (8a). As a result, the C3 p-hydroxystyryl side chain was specified as the key structure in the quinoxaline compounds 8a,b and 8d, which could represent significant leads for the discovery of multifunctional ARIs.

implicated in the development of the long-term complications of diabetes.5a,17 Molecular Docking. Molecular docking of compound 8a possessing good activities both in the ALR2 inhibition and antioxidant reactions was studied to understand mechanistic details and SARs in the ALR2 inhibition by the compound. The lidorestat-bound conformation of ALR2 (PDB code: 1Z3N)9b chosen from several conformations was used for the docking study owing to the structural similarity of the synthetic compound and the ligand. The docking behavior of 8a with the ALR1−NADP+−fidarestat complex (PDB code: 3H4G)18 was also investigated. As shown in Figure 6, docking results reveal that compound 8a was tightly bound in the active site of ALR2. The carboxylate group was well inserted in the anion binding site by hydrogen-bonding to Tyr48 (2.62 Å) and His110 (3.15 Å) side chains and engaging in a stabilizing electrostatic interaction with the positively charged nicotinamide moiety of the cofactor NADP (N−O = 4.07 Å). Further, the oxygen atom of the C2-carbonyl group of the 8a core structure formed an additional hydrogen bond with the side chain of Trp111 (3.10 Å), indicating an important role of the carbonyl in the tight binding of the inhibitor with ALR2. Moreover, the p-hydroxyphenyl ring of the C3 side chain of 8a formed a stable stacking interaction with the indole ring of the Trp111 side chain and was well placed into the specificity pocket formed by the side chains of Trp111, Leu300, Phe122, Cys303, Thr113, Phe115, and Trp79.19 In addition, the quinoxalinone core matched very well the hydrophobic pocket lined by the side chains of Leu300, Trp219, Phe122, Trp20, and Trp79.

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EXPERIMENTAL SECTION

Chemistry. Melting points were recorded on an X-4 microscopic melting point apparatus and are uncorrected. All reactions were routinely checked by TLC on silica gel Merck60F254. The 1H NMR spectra were recorded on a Bruker Advance 400 spectrometer (400 MHz, Bruker (Beijing) Technologies and Services Co., Ltd.). Chemical shifts are given in δ units (ppm) relative to internal standard TMS and refer to CDCl3 or DMSO-d6 solutions. HRMS (ESI) was performed using an Agilent 6210 time-of-flight LC/MS. The following HPLC methods were used to determine the purity of acetic acid derivatives using a Hitachi D-2000 Elite HPLC system. All acetic acid derivatives tested in biological assays were >95% pure with the following method: Inertsil ODS-2 250 mm × 10 mm, 5 mm column; mobile phase: CH3CN (0.1% TFA)/CH3OH = 75/25, for 8 min; room temperature; flow rate: 1 mL min−1; detection at λ 254 nm. General Procedure for Synthesis of Methyl 2-(3-Chloro-2oxoquinoxalin-1(2H)-yl)acetate Derivatives (2). A mixture of the appropriate 3-chloro-quinoxalin-2(1H)-one 1 (20 mmol), K2CO3 (8.29 g, 60 mmol), and methyl bromoacetate (3.21 g, 21 mmol) in CH3CN (100 mL) was stirred at 65 °C for 2 h and then filtered. After evaporation of the solvent in vacuo, the residue was recrystallized from EtOAc to give desired product 2.

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CONCLUSIONS A series of quinoxalin-2(1H)-one based derivatives were designed and synthesized by focusing on the alteration of the C3 side chain as well as substitutions on the phenyl ring of the quinoxaline core structure to refine this type of ALR2 inhibitor. In particular, phenolic structure was installed in the designed compounds for a combination of antioxidant activity and ALR2 inhibition and in turn for strengthening the ability to fight against the long-term complications of diabetes. Most of the synthetic series 6 showed a potent and selective effect on the ALR2 inhibition, and 6e was identified as the most active. However, they all possessed insufficient antioxidant activity G


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7.47−7.14 (m, 3H), 5.05 ppm (s, 2H); 13C NMR (100 MHz, DMSOd6) δ 168.60, 161.17, 153.68, 134.24, 132.13, 131.72 (d, J = 7.9 Hz), 131.63, 129.20, 115.08, 114.87, 111.94, 111.70, 101.79, 101.51, 44.50 ppm; HRMS (ESI) m/z calcd for [M − H]− 315.0587, found 315.0581. 2-(7-Chloro-3-(4-fluorophenyl)-2-oxoquinoxalin-1(2H)-yl)acetic Acid (4d). Yield: 204 mg (79%); white solid; mp: 273−275 °C; purity: 97.38%; 1H NMR (400 MHz, DMSO-d6) δ 13.35 (s, 1H), 8.50−8.25 (m, 2H), 7.91 (dd, J = 8.5, 1.4 Hz, 1H), 7.80 (s, 1H), 7.44 (t, J = 10.1 Hz, 1H), 7.34 (dd, J = 8.8, 7.4 Hz, 2H), 5.08 ppm (s, 2H); 13C NMR (100 MHz, DMSO-d6) δ 168.65, 160.90, 153.56, 151.61, 135.34, 133.71, 131.85, 131.76, 131.26, 130.91, 124.09, 121.53, 115.11, 114.90, 114.50, 44.29 ppm; HRMS (ESI) m/z calcd for [M − H]− 331.0291, found 331.0300. 2-(3,7-Bis(4-fluorophenyl)-2-oxoquinoxalin-1(2H)-yl)acetic Acid (4e). Yield: 237 mg (81%); white solid; mp: 269−271 °C; purity: 98.83%; 1H NMR (400 MHz, DMSO-d6) δ 8.40 (dd, J = 8.0, 5.9 Hz, 2H), 8.06−7.60 (m, 9H), 5.24 ppm (s, 2H); 13C NMR (100 MHz, DMSO-d6) δ 168.85, 165.23, 162.20, 153.17, 151.41, 138.14, 132.65, 132.19, 131.99, 131.79, 131.22, 130.85, 129.81, 124.88, 123.91, 121.27, 115.99, 114.88, 114.65, 109.22, 44.69 ppm; HRMS (ESI) m/z calcd for [M − H]− 391.0921, found 391.0926. 2-(3-(4-Hydroxyphenyl)-2-oxoquinoxalin-1(2H)-yl)acetic Acid (4f). Yield: 177 mg (78%); white solid; mp: 289−291 °C; purity: 99.28%; 1H NMR (400 MHz, DMSO-d6) δ 10.06 (s, 1H), 8.36−8.18 (m, 2H), 7.87 (d, J = 7.9 Hz, 1H), 7.61−7.55 (m, 1H), 7.50 (d, J = 8.4 Hz, 1H), 7.39 (t, J = 7.5 Hz, 1H), 6.88 (dd, J = 8.7, 1.0 Hz, 2H), 5.05 ppm (s, 2H); 13C NMR (100 MHz, DMSO-d6) δ 168.92, 159.84, 153.85, 151.91, 132.35 (d, J = 5.7 Hz), 132.29, 131.24, 129.89, 129.35, 126.50, 123.70, 114.84, 114.43, 43.93 ppm; HRMS (ESI) m/z calcd for [M − H]− 295.0724, found 295.0735. General Procedure for Synthesis of Methyl 2-(3-(2,4-Dihydroxyphenyl)-2-oxoquinoxalin-1(2H)-yl)acetate Derivatives (5). A mixture of 2-(3-chloro-2-oxoquinoxalin-1(2H)-yl)acetate derivatives 2 (1 mmol), an appropriate (hetero)arene (1 mmol), and AlCl3 (0.15 g, 1.1 mmol) in dichloroethane (5 mL) was stirred at 80 °C for 2 h under an atmosphere of argon. After completion of the reaction, the mixture was poured into ice-cold water (15 mL), stirred for 10 min, and then extracted with ethyl acetate (3 × 20 mL). The organic layers were collected, washed with cold water (2 × 20 mL), dried over anhydrous Na2SO4, and concentrated under vacuum. The residue obtained was purified by column chromatography to give the desired product 5. Methyl 2-(3-(2,4-Dihydroxyphenyl)-2-oxoquinoxalin-1(2H)-yl)acetate (5a). Yield: 293 mg (90%); yellow solid; 1H NMR (400 MHz, DMSO-d6) δ 13.79 (s, 1H), 10.26 (s, 1H), 8.80 (dd, J = 9.0, 2.0 Hz, 1H), 7.86 (d, J = 8.0 Hz, 1H), 7.59 (d, J = 5.0 Hz, 2H), 7.41 (dd, J = 8.0, 6.0 Hz, 1H), 6.36 (dd, J = 13.9, 5.6 Hz, 2H), 5.18 (s, 2H), 3.72 ppm (s, 3H). Methyl 2-(6-Fluoro-3-(2,4-dihydroxyphenyl)-2-oxoquinoxalin1(2H)-yl)acetate (5b). Yield: 292 mg (85%); yellow solid; 1H NMR (400 MHz, DMSO-d6) δ 13.81 (s, 2H), 10.20 (s, 1H), 8.83 (d, J = 9.0 Hz, 1H), 7.75 (d, J = 8.2 Hz, 1H), 7.54 (s, 1H), 7.23 (d, J = 8.1 Hz, 1H), 6.43−6.26 (m, 2H), 5.18 (s, 2H), 3.73 ppm (s, 3H). Methyl 2-(6-Chloro-3-(2,4-dihydroxyphenyl)-2-oxoquinoxalin1(2H)-yl)acetate (5c). Yield: 280 mg (78%); yellow solid; 1H NMR (400 MHz, DMSO-d6) δ 13.79 (d, J = 2.1 Hz, 1H), 10.26 (s, 1H), 8.81 (dd, J = 9.0, 2.0 Hz, 1H), 7.87 (d, J = 8.0 Hz, 1H), 7.60 (d, J = 5.0 Hz, 1H), 7.42 (dd, J = 8.0, 6.0 Hz, 1H), 6.36 (dd, J = 13.9, 5.6 Hz, 2H), 5.18 (s, 2H), 3.72 ppm (s, 3H). Methyl 2-(6-Bromo-3-(2,4-dihydroxyphenyl)-2-oxoquinoxalin1(2H)-yl)acetate (5d). Yield: 327 mg (81%); yellow solid; 1H NMR (400 MHz, DMSO-d6) δ 11.73 (s, 1H), 9.53 (s, 1H), 7.59−7.05 (m, 4H), 6.91−6.78 (m, 2H), 5.18 (s, 2H), 3.73 ppm (s, 3H). Methyl 2-(7-Fluoro-3-(2,4-dihydroxyphenyl)-2-oxoquinoxalin1(2H)-yl)acetate (5e). Yield: 285 mg (83%); yellow solid; 1H NMR (400 MHz, DMSO-d6) δ 13.43 (s, 1H), 10.30 (s, 1H), 8.13−6.73 (m, 4H), 6.36 (dd, J = 7.0, 2.8 Hz, 2H), 4.98 (s, 2H), 3.98 ppm (s, 3H). Methyl 2-(7-Chloro-3-(2,4-dihydroxyphenyl)-2-oxoquinoxalin1(2H)-yl)acetate (5f). Yield: 291 mg (81%); yellow solid; 1H NMR (400 MHz, DMSO-d6) δ 13.46 (s, 1H), 10.28 (s, 1H), 9.16 (d, J = 1.2

General Procedure for Synthesis of Methyl 2-(3-Phenyl-2oxoquinoxalin-1(2H)-yl)acetate Derivatives (3). A round-bottom flask was charged with a stir bar, the appropriate 2 (1 mmol), the corresponding phenylboronic acid (1.1 mmol), Cs2CO3 (0.65 g, 2 mmol), Pd(OAc)2 (11.22 mg, 0.05 mmol), and PPh3 (39.35 mg, 0.15 mmol). H2O (1 mL) was added followed by dioxane (10 mL). The reaction flask was equipped with a reflux condenser and heated to 100 °C for 18 h under an atmosphere of nitrogen. Upon cooling to room temperature, the reaction solution was added to a separatory funnel, the aqueous layer was removed, and the organic layer was washed with water (3 × 20 mL). The organic layer was dried over MgSO4, filtered, and concentrated by rotary evaporation under reduced pressure to provide the crude residue which was purified by silica gel chromatography to afford desired product 3. Methyl 2-(3-Phenyl-2-oxoquinoxalin-1(2H)-yl)acetate (3a). Yield: 250 mg (85%); white solid; 1H NMR (400 MHz, DMSO-d6) δ 8.26 (d, J = 8.0 Hz, 2H), 7.95 (d, J = 8.6 Hz, 2H), 7.36−7.13 (m, 5H), 5.20 (s, 2H), 3.73 ppm (s, 3H). Methyl 2-(3-(4-Fluorophenyl)-2-oxoquinoxalin-1(2H)-yl)acetate (3b). Yield: 237 mg (76%); white solid; 1H NMR (400 MHz, DMSO-d6) δ 8.34 (dd, J = 7.0, 5.8 Hz, 2H), 8.00−7.74 (m, 2H), 7.48 (d, J = 8.6 Hz, 2H), 7.34 (dd, J = 8.9, 7.2 Hz, 2H), 5.16 (s, 2H), 3.72 ppm (s, 3H). Methyl 2-(7-Fluoro-3-(4-fluorophenyl)-2-oxoquinoxalin-1(2H)yl)acetate (3c). Yield: 247 mg (75%); white solid; 1H NMR (400 MHz, DMSO-d6) δ 8.33 (dd, J = 8.6, 5.9 Hz, 2H), 7.98 (dd, J = 8.8, 6.0 Hz, 1H), 7.63 (dd, J = 11.0, 2.5 Hz, 1H), 7.33 (d, J = 9.2 Hz, 3H), 5.14 (s, 2H), 3.72 ppm (s, 3H). Methyl 2-(7-Chloro-3-(4-fluorophenyl)-2-oxoquinoxalin-1(2H)yl)acetate (3d). Yield: 270 mg (78%); white solid; 1H NMR (400 MHz, DMSO-d6) δ 8.34 (dd, J = 7.0, 5.8 Hz, 1H), 7.93 (dd, J = 8.6, 1.7 Hz, 1H), 7.84 (s, 1H), 7.48 (d, J = 8.6 Hz, 2H), 7.35 (dd, J = 8.9, 7.2 Hz, 2H), 5.16 (s, 2H), 3.72 ppm (s, 3H). Methyl 2-(3,7-Bis(4-fluorophenyl)-2-oxoquinoxalin-1(2H)-yl)acetate (3e). Yield: 304 mg (75%); white solid; 1H NMR (400 MHz, CDCl3) δ 8.36 (dd, J = 6.6, 3.1 Hz, 2H), 8.03 (d, J = 8.3 Hz, 2H), 7.66−7.58 (m, 3H), 7.55−7.40 (m, 4H), 5.17 (s, 2H), 3.81 ppm (s, 3H). Methyl 2-(3-(4-Hydroxyphenyl)-2-oxoquinoxalin-1(2H)-yl)acetate (3f). Yield: 238 mg (77%); white solid; 1H NMR (400 MHz, DMSOd6) δ 10.05 (s, 1H), 8.27 (d, J = 8.8 Hz, 2H), 7.57−7.41 (m, 4H), 6.88 (d, J = 8.8 Hz, 2H), 5.17 (s, 2H), 3.72 ppm (s, 3H). General Procedure for Synthesis of Methyl 2-(3-Phenyl-2oxoquinoxalin-1(2H)-yl)acetic Acid Derivatives (4). A mixture of 3 (0.7 mmol) and saturated aq LiOH (5 mL) in THF (4 mL) was stirred at rt for 2 h. Upon completion, the alkaline suspension was acidified with 0.1 N HCl to pH 3. The resulting precipitate was collected by filtration, washed with H2O, dried in vacuo, and recrystallized from CH3OH to give desired final product 4. 2-(3-Phenyl-2-oxoquinoxalin-1(2H)-yl)acetic Acid (4a). Yield: 195 mg (81%); white solid; mp: 237−239 °C; purity: 99.92%; 1H NMR (400 MHz, DMSO-d6) δ 8.26 (d, J = 7.9 Hz, 2H), 7.93 (dd, J = 8.6, 6.2 Hz, 2H), 7.16−7.05 (m, 5H), 5.09 ppm (s, 2H); 13C NMR (100 MHz, DMSO-d6): δ 168.75, 158.58, 153.80, 137.15, 132.51, 131.90 (d, J = 8.0 Hz), 130.17, 129.21, 129.13, 128.33, 126.41, 123.62, 114.57, 43.71 ppm; HRMS (ESI) m/z calcd for [M − H]− 279.0775, found 279.0781. 2-(3-(4-Fluorophenyl)-2-oxoquinoxalin-1(2H)-yl)acetic Acid (4b). Yield: 172 mg (78%); white solid; mp: 275−277 °C; purity: 98.51%; 1 H NMR (400 MHz, DMSO-d6) δ 8.37 (dd, J = 8.3, 6.0 Hz, 2H), 7.93 (d, J = 7.9 Hz, 1H), 7.65 (d, J = 7.7 Hz, 2H), 7.57 (d, J = 8.4 Hz, 1H), 7.44 (t, J = 7.5 Hz, 1H), 7.36 (t, J = 8.7 Hz, 1H), 5.09 ppm (s, 2H); 13 C NMR (100 MHz, DMSO-d6) δ 168.83, 162.20, 153.74, 151.51, 132.65, 132.19, 131.99 (d, J = 2.9 Hz), 131.79 (d, J = 8.6 Hz), 130.85, 129.81, 123.93, 115.09, 114.88, 114.65, 109.22, 44.00 ppm; HRMS (ESI) m/z calcd for [M − H]− 297.0681, found 297.0685. 2-(7-Fluoro-3-(4-fluorophenyl)-2-oxoquinoxalin-1(2H)-yl)acetic Acid (4c). Yield: 193 mg (82%); white solid; mp: 269−271 °C; purity: 99.36%; 1H NMR (400 MHz, DMSO-d6) δ 8.33 (dd, J = 8.3, 5.9 Hz, 2H), 7.96 (dd, J = 8.7, 6.2 Hz, 1H), 7.59 (dd, J = 10.9, 2.1 Hz, 1H), H


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2H), 6.43−6.32 (m, 2H), 5.06 ppm (s, 2H); 13C NMR (100 MHz, DMSO-d6): δ 169.88, 162.11, 161.75, 158.72, 153.11, 139.69, 137.26, 132.34, 130.19, 126.33, 125.97, 117.32, 115.51, 111.85, 108.37, 45.28 ppm; HRMS (ESI) m/z calcd for [M − H]− 329.0579, found 329.0583. 2-(6-Chloro-3-(2,4-dihydroxyphenyl)-2-oxoquinoxalin-1(2H)-yl)acetic Acid (6c). Yield: 218 mg (78%); orange solid; mp: >300 °C; purity: 99.81%; 1H NMR (400 MHz, DMSO-d6) δ 13.33 (s, 1H), 10.62 (s, 1H), 8.22 (s, 1H), 7.98−7.73 (m, 2H), 7.62 (s, 1H), 6.95 (m, 2H), 5.09 ppm (s, 2H). 13C NMR (100 MHz, DMSO-d6) δ 171.20, 165.58, 164.88, 156.17, 155.85, 135.65, 133.48, 133.00, 131.76, 130.42, 128.93, 118.98, 112.82, 110.07, 105.62, 47.32 ppm; HRMS (ESI) m/z calcd for [M − H]− 345.0284, found 345.0285. 2-(6-Bromo-3-(2,4-dihydroxyphenyl)-2-oxoquinoxalin-1(2H)-yl)acetic Acid (6d). Yield: 227 mg (76%); orange solid; mp: >300 °C; purity: 99.56%; 1H NMR (400 MHz, DMSO-d6) δ 13.35 (s, 1H), 10.65 (s, 1H), 8.23 (s, 1H), 8.02−7.26 (m, 4H), 6.97 (d, J = 8.0 Hz, 1H), 5.08 ppm (s, 2H); 13C NMR (100 MHz, DMSO-d6) δ 168.63, 163.03, 162.33, 153.22, 152.31, 133.10, 132.00, 131.33, 131.26, 130.82, 129.41, 115.71, 110.26, 107.54, 103.08, 44.48 ppm; HRMS (ESI) m/z calcd for [M − H]− 388.9779, found 388.9782. 2-(7-Fluoro-3-(2,4-dihydroxyphenyl)-2-oxoquinoxalin-1(2H)-yl)acetic Acid (6e). Yield: 215 mg (81%); orange solid; mp: >300 °C; purity: 97.31%; 1H NMR (400 MHz, DMSO-d6) δ 13.24 (s, 1H), 10.57 (s, 1H), 7.91 (dd, J = 11.6, 8.0 Hz, 2H), 7.72 (s, 1H), 6.93−6.84 (m, 2H), 6.57 (d, J = 8.1 Hz, 1H), 5.10 ppm (s, 2H); 13C NMR (100 MHz, DMSO-d6) δ 168.62, 162.51, 162.27, 161.83, 153.84, 151.37, 133.68, 133.46, 132.74, 129.82, 111.98, 111.74, 110.33, 107.34, 103.06, 44.88 ppm; HRMS (ESI) m/z calcd for [M − H]− 329.0579, found 329.0572. 2-(7-Chloro-3-(2,4-dihydroxyphenyl)-2-oxoquinoxalin-1(2H)-yl)acetic Acid (6f). Yield: 231 mg (82%); orange solid; mp: >300 °C; purity: 99.80%; 1H NMR (400 MHz, DMSO-d6) δ 13.48 (s, 1H), 10.45 (s, 1H), 8.73 (s, 1H), 7.88 (dd, J = 8.6, 2.2 Hz, 2H), 7.77 (s, 1H), 6.40 (d, J = 8.3 Hz, 2H), 5.06 ppm (s, 2H); 13C NMR (100 MHz, DMSO-d6) δ 168.66, 162.88, 162.15, 153.72, 152.29, 134.17, 132.93, 128.97, 128.51, 124.13, 114.48, 110.26, 107.48, 103.07, 44.78 ppm; HRMS (ESI) m/z calcd for [M − H]− 345.0284, found 345.0288. 2-(7-Bromo-3-(2,4-dihydroxyphenyl)-2-oxoquinoxalin-1(2H)-yl)acetic Acid (6g). Yield: 240 mg (85%); orange solid; mp: >300 °C; purity: 97.56%; 1H NMR (400 MHz, DMSO-d6) δ 13.52 (s, 1H), 10.29 (s, 1H), 8.75 (s, 1H), 7.89 (s, 1H), 7.80 (d, J = 8.6 Hz, 1H), 7.55 (d, J = 8.6 Hz, 1H), 6.46−6.25 (m, 2H), 5.06 ppm (s, 2H); 13C NMR (100 MHz, DMSO-d6) δ 168.67, 162.94, 162.20, 153.71, 152.42, 133.10, 132.93, 129.08, 128.79, 127.01, 117.28, 110.26, 107.50, 103.07, 44.65 ppm; HRMS (ESI) m/z calcd for [M − H]− 388.9779, found 388.9772. 2-(7-(4-Fluorobenzyl)-3-(2,4-dihydroxyphenyl)-2-oxoquinoxalin1(2H)-yl)acetic Acid (6h). Yield: 175 mg (79%); orange solid; mp: >300 °C; purity: 98.96%; 1H NMR (400 MHz, DMSO-d6) δ 13.81 (s, 1H), 10.20 (s, 1H), 8.83 (d, J = 9.0 Hz, 1H), 7.75 (d, J = 8.2 Hz, 1H), 7.54 (s, 1H), 7.33 (dd, J = 8.2, 5.7 Hz, 2H), 7.23 (d, J = 8.1 Hz, 1H), 7.11 (t, J = 8.9 Hz, 2H), 6.43−6.27 (m, 2H), 5.00 (s, 2H), 4.07 ppm (s, 2H); 13C NMR (100 MHz, DMSO-d6) δ 168.77, 162.81, 161.80, 153.96, 143.61, 132.79, 131.98, 131.51, 130.49 (d, J = 7.9 Hz), 128.65, 128.07, 127.52, 124.78, 115.17 (d, J = 21.0 Hz), 114.49, 110.38, 107.28, 103.06, 65.02, 44.40 ppm; HRMS (ESI) m/z calcd for [M − H]− 419.1049, found 419.1056. 2-(3-(3-Indolyl)-2-oxoquinoxalin-1(2H)-yl)acetic Acid (6i). Yield: 210 mg (81%); yellow solid; mp: 288−290 °C; purity: 98.57%; 1H NMR (400 MHz, DMSO-d6) δ 11.82 (s, 1H), 9.04−8.71 (m, 2H), 7.95 (d, J = 7.6 Hz, 1H), 7.61−7.32 (m, 4H), 7.32−7.18 (m, 2H), 5.12 ppm (s, 2H); 13C NMR (100 MHz, DMSO-d6) δ 169.13, 153.50, 150.41, 136.31, 133.10 (d, J = 20.4 Hz), 130.91, 128.56 (d, J = 21.2 Hz), 128.35, 126.23, 123.61, 122.94, 122.62, 121.09, 114.29, 111.92, 111.18, 43.91 ppm; HRMS (ESI) m/z calcd for [M − H]− 318.0884, found 318.0886. 2-(7-Bromo-3-(3-indolyl)-2-oxoquinoxalin-1(2H)-yl)acetic Acid (6j). Yield: 220 mg (80%); yellow solid; mp: >300 °C; purity:

Hz, 1H), 7.87 (dd, J = 13.6, 12.6 Hz, 2H), 7.44 (d, J = 8.6 Hz, 1H), 6.57−5.99 (m, 2H), 5.16 (s, 2H), 3.75 ppm (s, 3H). Methyl 2-(7-Bromo-3-(2,4-dihydroxyphenyl)-2-oxoquinoxalin1(2H)-yl)acetate (5g). Yield: 340 mg (84%); yellow solid; 1H NMR (400 MHz, DMSO-d6) δ 13.49 (s, 1H), 10.28 (s, 1H), 8.74 (d, J = 9.0 Hz, 2H), 7.73 (dd, J = 10.4, 1.7 Hz, 2H), 6.53−6.07 (m, 2H), 5.15 (s, 2H), 3.73 ppm (s, 3H). Procedure for Synthesis of Methyl 2-(7-(4-Fluorobenzyl)-3-(2,4dihydroxyphenyl)-2-oxoquinoxalin-1(2H)-yl)acetate (5h). A mixture of 2-(3-chloro-2-oxoquinoxalin-1(2H)-yl)acetate 2 (1 mmol), resorcinol (1 mmol), and AlCl3 (0.15 g, 1.1 mmol) in dichloroethane (5 mL) was stirred at 80 °C for 2 h under an atmosphere of argon. After completion of the reaction, the mixture was poured into ice-cold water (15 mL), stirred for 10 min, and then extracted with ethyl acetate (3 × 20 mL). The organic layers were collected, washed with cold water (2 × 20 mL), dried over anhydrous Na2SO4, and concentrated under vacuum to obtain the residue. Under argon atmosphere, zinc powder (0.62 g, 9.54 mmol) was suspended in tetrahydrofuran (dry, 5 mL), 1,2-dibromoethane (0.015 mL, 0.174 mmol) and trimethylsilyl chloride (0.07 mL, 0.552 mmol) were added at 60 °C, and the mixture was stirred with heating for 30 min. A solution of the corresponding benzyl bromide (4.76 mmol) in tetrahydrofuran (5 mL) was added dropwise at 60 °C. The mixture was stirred under heating for 3 h to give a benzylzinc bromide solution. A mixture of the residue obtained above, Pd(PPh3)4 (69.36 mg, 0.06 mmol), benzylzinc bromide solution (5.04 mL), and tetrahydrofuran (dry, 10 mL) was stirred at 60 °C for 3 h. After the reaction mixture was cooled, it was quenched with saturated ammonium chloride solution (20 mL). After extraction with THF (3 × 10 mL), the combined organic layers were dried by anhydrous Na2SO4.The mixture was filtered, followed by evaporation of the solvent in vacuo. The residue was purified by column chromatography on silica gel to afford 5h. Yield: 236 mg (65%); yellow solid; 1H NMR (400 MHz, DMSO-d6) δ 13.73 (s, 1H), 10.20 (s, 1H), 8.79 (d, J = 8.9 Hz, 2H), 8.00−6.91 (m, 6H), 6.55−6.17 (m, 2H), 5.16 (s, 2H), 4.07 (s, 2H), 3.72 ppm (s, 3H). Methyl 2-(3-(3-Indolyl)-2-oxoquinoxalin-1(2H)-yl)acetate (5i). Yield: 283 mg (85%); white solid; 1H NMR (400 MHz, DMSO-d6) δ 11.84 (s, 1H), 9.15−8.53 (m, 2H), 7.96 (d, J = 7.8 Hz, 2H), 7.77− 7.00 (m, 5H), 5.23 (s, 2H), 3.73 ppm (s, 3H). Methyl 2-(7-Bromo-3-(3-indolyl)-2-oxoquinoxalin-1(2H)-yl)acetate (5j). Yield: 320 mg (78%); white solid; 1H NMR (400 MHz, DMSO-d6) δ1H NMR (400 MHz, DMSO) δ 11.88 (s, 1H), 8.85 (s, 1H), 7.88 (dd, J = 8.6, 2.3 Hz, 2H), 7.66−7.36 (m, 3H), 7.25 (dd, J = 5.4, 2.5 Hz, 2H), 5.20 (s, 2H), 3.73 ppm (s, 3H). Procedure for Synthesis of Methyl 2-(3-(2-Benzothiophene)-2oxoquinoxalin-1(2H)-yl)acetate (5k). Procedure for synthesis of 5k is the same as that for compounds 4. Yield: 212 mg (85%); white solid; 1 H NMR (400 MHz, DMSO-d6) δ 8.39 (s, 1H), 8.05−7.68 (m, 2H), 7.62 (d, J = 8.3 Hz, 2H), 7.44−7.20 (m, 4H), 5.23 (s, 2H), 3.73 ppm (s, 3H). General Procedure for Synthesis of 2-(3-(2,4-Dihydroxyphenyl)-2oxoquinoxalin-1(2H)-yl)acetic Acid Derivatives (6). A mixture of 5 (0.7 mmol) and saturated aq LiOH (5 mL) in THF (4 mL) was stirred at rt for 2 h. Upon completion, the alkaline suspension was acidified with 0.1 N HCl to pH 3. The resulting precipitate was collected by filtration, washed with H2O, dried in vacuo, and recrystallized from CH3OH to give desired final product 6. 2-(3-(2,4-Dihydroxyphenyl)-2-oxoquinoxalin-1(2H)-yl)acetic Acid (6a). Yield: 210 mg (80%); orange solid; mp: 297−299 °C; purity: 98.62%; 1H NMR (400 MHz, DMSO-d6) δ 13.83 (s, 1H), 10.34 (s, 1H), 8.83 (d, J = 9.0 Hz, 2H), 7.67−7.47 (m, 3H), 6.48−6.29 (m, 2H), 5.08 ppm (s, 2H); 13C NMR (100 MHz, DMSO-d6) δ 168.77, 162.99, 162.01, 153.87, 152.14, 132.92, 131.90, 129.78 (d, J = 21.6 Hz), 129.56, 127.36, 124.05, 114.63, 110.34, 107.38, 103.09, 44.43 ppm; HRMS (ESI) m/z calcd for [M − H]− 311.0673, found 311.0678. 2-(6-Fluoro-3-(2,4-dihydroxyphenyl)-2-oxoquinoxalin-1(2H)-yl)acetic Acid (6b). Yield: 272 mg (83%); orange solid; mp: >300 °C; purity: 99.06%; 1H NMR (400 MHz, DMSO-d6) δ 13.47 (s, 1H), 10.28 (s, 1H), 8.81 (s, 1H), 7.81 (s, 1H), 7.60 (dd, J = 9.3, 4.9 Hz, I


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ppm; HRMS (ESI) m/z calcd for [M − H]− 339.0787, found 339.0784. 2-(3-(4-Methoxystyryl)-2-oxoquinoxalin-1(2H)-yl)acetic Acid (8c). Yield: 163 mg (75%); yellow solid; mp: 278−280 °C; purity: 96.59%; 1 H NMR (400 MHz, DMSO-d6) δ 7.88 (d, J = 7.9 Hz, 1H), 7.51 (t, J = 7.3 Hz, 1H), 7.36 (t, J = 7.6 Hz, 1H), 7.29−7.18 (m, 2H), 7.08 (d, J = 8.3 Hz, 1H), 6.84 (d, J = 8.5 Hz, 2H), 5.05 (s, 2H), 3.78 ppm (s, 3H); 13C NMR (100 MHz, DMSO-d6) δ 168.34, 159.71, 157.96, 154.26, 133.79, 133.10, 132.32, 130.15, 129.78, 129.36, 123.70, 115.24, 114.21, 106.41, 44.42 ppm; HRMS (ESI) m/z calcd for [M − H]− 335.1037, found 335.1029. 2-(3-(3-Methoxy-4-hydroxystyryl)-2-oxoquinoxalin-1(2H)-yl)acetic Acid (8d). Yield: 158 mg (77%); orange solid; mp: 265−267 °C; purity: 99.12%; 1H NMR (400 MHz, DMSO-d6) δ 9.56 (s, 1H), 8.02 (d, J = 16.1 Hz, 1H), 7.83 (d, J = 7.3 Hz, 1H), 7.56 (t, J = 7.3 Hz, 1H), 7.51 (d, J = 3.7 Hz, 1H), 7.47 (d, J = 4.0 Hz, 1H), 7.39 (t, J = 7.5 Hz, 1H), 7.33 (d, J = 7.4 Hz, 1H), 7.18 (d, J = 8.2 Hz, 1H), 6.84 (d, J = 8.1 Hz, 1H), 5.05 (s, 2H), 3.87 ppm (s, 3H); 13C NMR (100 MHz, DMSO-d6) δ 169.32, 154.56, 152.24, 148.81 (d, J= 66.0 Hz), 138.69, 133.23, 132.55, 130.14, 129.50, 127.98, 124.34, 122.63, 119.06, 116.26, 115.01, 111.36, 56.11, 44.31 ppm; HRMS (ESI) m/z calcd for [M − H]− 351.0986, found 351.0985. General Procedure for Synthesis of Methyl 2-(3-(4-Hydroxyphenethyl)-2-oxoquinoxalin-1(2H)-yl)acetate Derivatives (9). To a suspension of 10% palladium on carbon (0.33 g) in methanol (10 mL) was added a solution of 2 (1 mmol) in EtOAc (10 mL). The mixture was stirred at room temperature for 6 h under a hydrogen atmosphere. The catalyst was filtered off through Celite pad, and the filtrate was concentrated and purified via flash chromatograph to obtain the pure product 9. Methyl 2-(3-(4-Hydroxyphenethyl)-2-oxoquinoxalin-1(2H)-yl)acetate (9a). Yield: 287 mg (85%); white solid; 1H NMR (400 MHz, DMSO-d6) δ 9.16 (s, 1H), 7.82 (d, J = 7.9 Hz, 1H), 7.58 (t, J = 7.4 Hz, 1H), 7.51 (d, J = 8.0 Hz, 1H), 7.38 (t, J = 7.5 Hz, 1H), 7.06 (d, J = 8.3 Hz, 2H), 6.66 (d, J = 8.4 Hz, 2H), 5.12 (s, 2H), 3.71 (s, 3H), 3.06 (t, J = 7.7 Hz, 2H), 2.92 ppm (t, J = 7.7 Hz, 2H). Methyl 2-(7-Fluoro-3-(4-hydroxyphenethyl)-2-oxoquinoxalin1(2H)-yl)acetate (9b). Yield: 279 mg (78%); white solid; 1H NMR (400 MHz, DMSO-d6) δ 9.21 (s, 1H), 7.79 (dd, J = 7.9, 1.3 Hz, 1H), 7.54 (s, 1H), 7.35 (dd, J = 15.6, 7.7 Hz, 2H), 7.07 (d, J = 8.5 Hz, 2H), 6.73−6.60 (m, 2H), 5.09 (s, 2H), 3.78 (s, 3H), 3.03 (t, J = 7.9 Hz, 2H), 2.87 ppm (t, J= 7.9 Hz, 2H). General Procedure for Synthesis of 2-(7-Fluoro-3-(4-hydroxyphenethyl)-2-oxoquinoxalin-1(2H)-yl)acetic Acid (10). A mixture of 9 (0.7 mmol) and saturated aq LiOH (5 mL) in THF (4 mL) was stirred at rt for 2 h. Upon completion, the alkaline suspension was acidified with 0.1 N HCl to pH 3. The resulting precipitate was collected by filtration, washed with H2O, dried in vacuo, and recrystallized from CH3OH to give desired final product 10. 2-(3-(4-Hydroxyphenethyl)-2-oxoquinoxalin-1(2H)-yl)acetic Acid (10a). Yield: 230 mg (80%); white solid; mp: >300 °C; purity: 99.75%; 1H NMR (400 MHz, DMSO-d6) δ 9.25 (s, 1H), 7.91 (d, J = 7.9 Hz, 1H), 7.73 (t, J = 7.4 Hz, 1H), 7.61 (d, J = 8.0 Hz, 1H), 7.52− 7.34 (m, 5H), 4.89 (s, 2H), 3.09 (t, J = 7.7 Hz, 2H), 2.94 ppm (t, J = 7.7 Hz, 2H); 13C NMR (100 MHz, DMSO-d6) δ 159.75, 157.86, 153.26, 132.79, 132.11, 131.72, 129.65, 128.87, 128.64, 122.35, 115.24, 114.21, 106.41, 55.42, 35.95, 31.25 ppm; HRMS (ESI) m/z calcd for [M − H]− 323.1037, found 323.1039. 2-(7-Fluoro-3-(4-hydroxyphenethyl)-2-oxoquinoxalin-1(2H)-yl)acetic Acid (10b). Yield: 242 mg (78%); white solid; mp: >300 °C; purity: 99.57%; 1H NMR (400 MHz, DMSO-d6) δ 9.20 (s, 1H), 7.80 (dd, J = 8.8, 6.2 Hz, 1H), 7.21−7.00 (m, 3H), 6.73−6.60 (m, 2H), 4.64 (s, 2H), 3.02 (t, J = 7.8 Hz, 3H), 2.88 ppm (t, J = 7.8 Hz, 3H); 13 C NMR (100 MHz, DMSO-d6) δ 159.39, 155.46, 153.79, 132.69, 131.86, 131.48, 129.59, 129.18, 128.86, 123.15, 115.09, 114.83, 48.59, 35.67, 30.95 ppm; HRMS (ESI) m/z calcd for [M − H]− 341.0943, found 341.0951. General Procedure for Synthesis of Methyl 2-(3-(4-hydroxyphenethyl)-2-oxo-3,4-dihydroquinoxalin-1(2H)-yl)acetate Derivatives (11). To a suspension of 10% palladium on carbon (0.36 g) in methanol (10 mL) was added a solution of 2 (1 mmol) in EtOAc (10

98.13%; 1H NMR (400 MHz, DMSO-d6) δ 11.88 (s, 1H), 8.91 (s, 1H), 7.94 (dd, J = 8.5, 2.6 Hz, 2H), 7.72−7.48 (m, 3H), 7.33 (dd, J = 5.5, 2.7 Hz, 2H), 5.18 ppm (s, 2H); 13C NMR (100 MHz, DMSO-d6) δ 169.35, 153.48, 136.56, 133.81, 132.30, 130.31, 126.68, 126.37, 123.17 (d, J = 23.3 Hz), 122.93, 121.43, 117.22, 112.20, 111.31, 67.24, 44.69 ppm; HRMS (ESI) m/z calcd for [M − H]− 395.9989, found 395.9994. 2-(7-Chloro-3-(2-benzothiophene)-2-oxoquinoxalin-1(2H)-yl)acetic Acid (6k). Yield: 220 mg (80%); yellow solid; mp: >300 °C; purity: 98.13%; 1H NMR (400 MHz, DMSO-d6) δ 8.51 (s, 1H), 7.94 (dd, J = 8.5, 2.6 Hz, 2H), 7.81−7.63 (m, 3H), 7.45 (dd, J = 8.0 Hz, 2H), 5.10 ppm (s, 2H); 13C NMR (100 MHz, DMSO-d6) δ 168.61, 152.57, 140.95, 139.84, 135.48, 130.90, 129.79, 126.69, 125.48, 124.85, 124.45, 122.37, 114.70, 44.41 ppm; HRMS (ESI) m/z calcd for [M − H]− 369.0106, found 369.0101. General Procedure for Synthesis of Methyl 2-(3-Styryl-2oxoquinoxalin-1(2H)-yl)acetate Derivatives (7). A mixture of 2-(3chloro-2-oxoquinoxalin-1(2H)-yl)acetate derivatives 2 (1 mmol), Pd(OAc)2 (6.73 mg, 0.03 mmol), and P(o-tolyl)3 (21.31 mg, 0.07 mmol) was stirred at room temperature under argon for 10 min. Add alkene (1.5 mmol), dry Et3N (0.30 g, 3 mmol), and DMF(1 mL). The reaction mixture was heated at 100 °C in a capped heavy-walled glass tube in an oil bath for 18 h. After the completion of the reaction, the mixture was poured in water and extracted with 50 mL of CH2Cl2 three times. The organic layer was collected and dried over MgSO4. After filtration and evaporation of CH2Cl2, the mixture was subjected to column chromatography to give the desired product 7. Methyl 2-(3-(4-Hydroxystyryl)-2-oxoquinoxalin-1(2H)-yl)acetate (7a). Yield: 235 mg (70%); yellow solid; 1H NMR (400 MHz, DMSO-d6) δ 9.96 (s, 1H), 7.97 (t, J = 23.0 Hz, 1H), 7.84 (d, J = 8.0 Hz, 1H), 7.67−7.30 (m, 5H), 6.84 (d, J = 8.5 Hz, 1H), 5.16 (s, 2H), 3.72 ppm (s, 3H). Methyl 2-(7-Fluoro-3-(4-hydroxystyryl)-2-oxoquinoxalin-1(2H)yl)acetate (7b). Yield: 235 mg (70%); yellow solid; 1H NMR (400 MHz, DMSO-d6) δ 9.15 (s, 1H), 7.82 (s, 1H), 7.57 (t, J = 7.2 Hz, 2H), 7.51 (d, J = 8.0 Hz, 1H), 7.38 (t, J = 7.6 Hz, 1H), 6.66 (d, J = 8.3 Hz, 2H), 5.12 (s, 2H), 3.71 ppm (s, 3H). Methyl 2-(3-(4-Methoxystyryl)-2-oxoquinoxalin-1(2H)-yl)acetate (7c). Yield: 227 mg (65%); yellow solid; 1H NMR (400 MHz, DMSO-d6) δ 8.03 (d, J = 16.4 Hz, 1H), 7.85 (d, J = 8.0 Hz, 1H), 7.71 (d, J = 7.7 Hz, 2H), 7.63−7.33 (m, 2H), 7.01 (d, J = 7.5 Hz, 2H), 5.16 (s, 2H), 3.81 (s, 3H), 3.72 ppm (s, 3H). Methyl 2-(3-(3-Methoxy-4-hydroxystyryl)-2-oxoquinoxalin-1(2H)yl)acetate (7d). Yield: 226 mg (62%); yellow solid; 1H NMR (400 MHz, CDCl3) δ 8.09 (s, 1H), 7.89 (d, J = 7.7 Hz, 1H), 7.58 (d, J = 16.0 Hz, 1H), 7.48 (s, 1H), 7.37 (s, 1H), 7.08 (d, J = 8.4 Hz, 1H), 6.94 (d, J = 7.9 Hz, 1H), 5.81 (s, 1H), 5.09 (s, 2H), 3.96 (s, 3H), 3.79 ppm (s, 3H). General Procedure for Synthesis of 2-(3-Styryl-2-oxoquinoxalin1(2H)-yl)acetic Acid (8). A mixture of 7 (0.7 mmol) and saturated aq LiOH (5 mL) in THF (4 mL) was stirred at rt for 2 h. Upon completion, the alkaline suspension was acidified with 0.1 N HCl to pH 3. The resulting precipitate was collected by filtration, washed with H2O, dried in vacuo, and recrystallized from CH3OH to give desired final product 8. 2-(3-(4-Hydroxystyryl)-2-oxoquinoxalin-1(2H)-yl)acetic Acid (8a). Yield: 128 mg (78%); orange solid; mp: 290−292 °C; purity: 99.74%; 1 H NMR (400 MHz, DMSO-d6) δ 9.93 (s, 1H), 7.97 (dd, J = 16.2, 3.6 Hz, 1H), 7.81 (d, J = 8.0 Hz, 1H), 7.64−7.30 (m, 4H), 6.82 (dd, J = 8.5, 3.8 Hz, 2H), 5.03 ppm (s, 2H); 13C NMR (100 MHz, DMSO-d6) δ 168.89, 159.18, 154.11, 151.78, 137.80, 132.74, 132.06, 129.66, 129.09, 127.00, 123.91, 118.19, 115.97, 114.52, 43.75 ppm; HRMS (ESI) m/z calcd for [M − H]− 321.0881, found 321.0887. 2-(7-Fluoro-3-(4-hydroxystyryl)-2-oxoquinoxalin-1(2H)-yl)acetic Acid (8b). Yield: 150 mg (75%); orange solid; mp: 296−298 °C; purity: 99.75%; 1H NMR (400 MHz, DMSO-d6) δ 13.63 (s, 1H), 8.77 (dd, J = 9.0, 1.3 Hz, 1H), 7.88 (d, J = 8.0 Hz, 1H), 7.80−7.57 (m, 2H), 7.46 (s, 1H), 6.53−6.19 (m, 2H), 5.08 ppm (s, 2H); 13C NMR (100 MHz, DMSO-d6) δ 168.92, 159.17, 152.76, 152.39, 139.08, 134.69, 134.06, 131.64, 131.09, 127.00, 123.91, 118.19, 115.97, 114.52, 43.73 J


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Journal of Medicinal Chemistry mL). The mixture was stirred at 45 °C for 36 h under a hydrogen atmosphere. The catalyst was filtered off through Celite pad and the filtrate was concentrated and purified via flash chromatograph to obtain the pure product 11. Yield: 140 mg (42%); white solid; 1H NMR (400 MHz, CDCl3) δ 7.27 (s, 1H), 7.22 (d, J = 8.3 Hz, 2H), 7.05 (s, 1H), 6.94 (d, J = 8.4 Hz, 2H), 6.91 (dd, J = 8.0, 2.9 Hz, 2H), 5.37 (d, J = 17.5 Hz, 1H), 4.52 (d, J = 17.5 Hz, 1H), 4.19 (dd, J = 7.8, 5.7 Hz, 1H), 4.02 (s, 3H), 2.80 ppm (t, J = 8.1 Hz, 2H). General Procedure for Synthesis of Methyl 2-(3-Thiophenyl-2oxoquinoxalin-1(2H)-yl)acetate Derivatives (12). A mixture of K2CO3 (139 mg, 1 mmol) and phenol or phenthiol (1.1 mmol) in DMF (10 mL) was stirred at 85 °C for 12 h. Then 2-(3-chloro-2oxoquinoxalin-1(2H)-yl)acetate derivative 2 (1 mmol) was added. The reaction mixture was stirred at 75 °C for 12 h and cooled to room temperature. Then H2O (100 mL) was added, and the residue was filtered off and washed with H2O to give the resulting residue, which was purified by column chromatography to provide the desired product 12. Methyl 2-(3-Phenoxy-2-oxoquinoxalin-1(2H)-yl)acetate (12a). Yield: 226 mg (73%); white solid; 1H NMR (400 MHz, DMSO-d6) δ 7.39 (m, 9H), 4.96 (s, 2H), 3.71 ppm (s, 3 H). Methyl 2-(3-Thiophenoxy-2-oxoquinoxalin-1(2H)-yl)acetate (12b). Yield: 254 mg (78%); white solid; 1H NMR (400 MHz, DMSO-d6) δ 7.63 (d, J = 7.8 Hz, 2H), 7.53 (t, J = 8.0 Hz, 1H), 7.31 (m, 6H), 5.06 (s, 2H), 3.73 ppm (s, 3H). Methyl 2-(3-(4-Bromothiophenoxy)-2-oxoquinoxalin-1(2H)-yl)acetate (12c). Yield: 302 mg (75%); white solid; 1H NMR (400 MHz, DMSO-d6) δ 7.73 (dd, J = 8.4, 1.9 Hz, 2H), 7.58 (dd, J = 8.5, 1.9 Hz, 2H), 7.52 (d, J = 5.7 Hz, 2H), 7.35−7.27 (m, 2H), 5.17 (s, 2H), 3.72 ppm (s, 3H). Methyl 2-(7-Fluoro-3-(4-bromothiophenoxy)-2-oxoquinoxalin1(2H)-yl)acetate (12d). Yield: 301 mg (71%); white solid; 1H NMR (400 MHz, DMSO-d6) δ 7.73 (dd, J = 8.5, 1.8 Hz, 2H), 7.58 (dd, J = 8.5, 1.7 Hz, 2H), 7.55−7.48 (m, 4H), 5.13 (s, 2H), 3.74 ppm (s, 3H). Methyl 2-(7-Chloro-3-(4-bromothiophenoxy)-2-oxoquinoxalin1(2H)-yl)acetate (12e). Yield: 311 mg (71%); white solid; 1H NMR (400 MHz, DMSO-d6) δ 7.70 (dd, J = 8.4, 2.0 Hz, 2H), 7.60 (dd, J = 8.4, 1.8 Hz, 2H), 7.53−7.46 (m, 4H), 5.12 (s, 2H), 3.74 ppm (s, 3H). Methyl 2-(7-Bromo-3-(4-bromothiophenoxy)-2-oxoquinoxalin1(2H)-yl)acetate (12f). Yield: 334 mg (69%); white solid; 1H NMR (400 MHz, DMSO-d6) δ 7.90 (s, 1H), 7.76−7.61 (m, 4H), 7.43 (d, J = 19.5 Hz, 2H), 5.15 (s, 2H), 3.74 ppm (s, 3H). Methyl 2-(6-Bromo-3-(4-bromothiophenoxy)-2-oxoquinoxalin1(2H)-yl)acetate (12g). Yield: 342 mg (72%); white solid; 1H NMR (400 MHz, DMSO-d6) δ 7.81 (s, 1H), 7.69−7.55 (m, 4H), 7.31 (d, J = 19.5 Hz, 2H), 5.11 (s, 2H), 3.70 ppm (s, 3H). Methyl 2-(3-(4-Chlorothiophenoxy)-2-oxoquinoxalin-1(2H)-yl)acetate (12h). Yield: 252 mg (70%); white solid; 1H NMR (400 MHz, DMSO-d6) δ 7.73 (s, 1H), 7.58 (dd, J = 8.5, 1.9 Hz, 2H), 7.52 (d, J = 5.7 Hz, 2H), 7.43−7.32 (m, 2H), 5.16 (s, 2H), 3.70 ppm (s, 3H). Methyl 2-(7-Fluoro-3-(4-chlorothiophenoxy)-2-oxoquinoxalin1(2H)-yl)acetate (12i). Yield: 257 mg (68%); white solid; 1H NMR (400 MHz, DMSO-d6) δ 7.73 (s, 1H), 7.58 (dd, J = 8.5, 1.7 Hz, 2H), 7.55−7.48 (m, 4H), 5.13 (s, 2H), 3.74 ppm (s, 3H). Methyl 2-(7-Chloro-3-(4-chlorothiophenoxy)-2-oxoquinoxalin1(2H)-yl)acetate (12j). Yield: 296 mg (75%); white solid; 1H NMR (400 MHz, DMSO-d6) δ 7.81 (s, 1H), 7.65 (d, J = 10.9 Hz, 1H), 7.40−7.20 (m, 5H), 5.16 (s, 2H), 3.70 ppm (s, 3H). Methyl 2-(7-Bromo-3-(4-chlorothiophenoxy)-2-oxoquinoxalin1(2H)-yl)acetate (12k). Yield: 311 mg (71%); white solid; 1H NMR (400 MHz, DMSO-d6) δ 7.74 (s, 1 H), 7.68 (dd, J = 6.6 Hz, J = 2 Hz, 2H), 7.59−7.32 (m, 4H), 5.17 (s, 2H), 3.74 ppm (s, 3H). General Procedure for Synthesis of 2-(3-Thiophenyl-2-oxoquinoxalin-1(2H)-yl)acetic Acid 13. A mixture of 12 (0.7 mmol) and saturated aq LiOH (5 mL) in THF (4 mL) was stirred at rt for 2 h. Upon completion, the alkaline suspension was acidified with 0.1 N HCl to pH 3. The resulting precipitate was collected by filtration, washed with H2O, dried in vacuo, and recrystallized from CH3OH to give desired final product 13.

2-(3-Phenoxy-2-oxoquinoxalin-1(2H)-yl)acetic Acid (13a). Yield: 182 mg (81%); white solid; mp: 249−251 °C; purity: 98.11%; 1H NMR (400 MHz, DMSO-d6) δ 7.37 (m, 9H), 5.07 ppm (s, 2H); 13C NMR (100 MHz, DMSO-d6) δ 168.82, 153.38, 152.28, 149.95, 148.28, 131.61, 129.74, 127.99, 127.30, 125.64 (d, J = 8.4 Hz), 123.91, 121.77, 114.59, 109.22, 44.09 ppm; HRMS (ESI) m/z calcd for [M − H]− 295.0724, found 295.0728. 2-(3-Thiophenoxy-2-oxoquinoxalin-1(2H)-yl)acetic Acid (13b). Yield: 210 mg (77%); white solid; mp: 260−262 °C; purity: 98.47%; 1H NMR (400 MHz, DMSO-d6) δ 7.63−7.29 (m, 9H), 5.06 ppm (s, 2H); 13C NMR (100 MHz, DMSO-d6) δ 168.57, 158.41, 151.83, 135.17, 129.54, 129.34, 129.10, 127.97, 123.98 (d, J = 8.1 Hz), 114.77, 103.80, 43.92 ppm; HRMS (ESI) m/z calcd for [M − H]− 311.0496, found 311.0501. 2-(3-(4-Bromothiophenoxy)-2-oxoquinoxalin-1(2H)-yl)acetic Acid (13c). Yield: 236 mg (81%); white solid; mp: 266−268 °C; purity: 95.16%; 1H NMR (400 MHz, DMSO-d6) δ 7.73 (d, J = 8.5 Hz, 2H), 7.62−7.42 (m, 4H), 7.31 (s, 1H), 5.06 ppm (s, 2H); 13C NMR (100 MHz, DMSO-d6) δ 168.71, 159.52, 153.54, 149.81, 148.28, 131.60, 129.55, 128.08, 127.35, 123.98, 123.69 (d, J = 8.4 Hz), 116.35 (d, J = 23.3 Hz), 114.58, 44.15 ppm; HRMS (ESI) m/z calcd for [M − H]− 388.9601, found 388.9605. 2-(7-Fluoro-3-(4-bromothiophenoxy)-2-oxoquinoxalin-1(2H)-yl)acetic Acid (13d). Yield: 220 mg (78%); white solid; mp: 250−252 °C; purity: 98.50%; 1H NMR (400 MHz, DMSO-d6) δ 7.73 (d, J = 5.6 Hz, 2H), 7.55 (dd, J = 20.0, 7.5 Hz, 4H), 7.15 (s, 1H), 5.02 ppm (s, 2H); 13C NMR (100 MHz, DMSO-d6) δ 168.65, 158.52, 152.54, 149.37, 148.82, 131.98, 129.34, 128.80, 126.35, 123.36, 123.69 (d, J = 8.3 Hz), 117.35, 113.68, 44.07 ppm; HRMS (ESI) m/z calcd for [M − H]− 406.9507, found 406.9510. 2-(7-Chloro-3-(4-bromothiophenoxy)-2-oxoquinoxalin-1(2H)-yl)acetic Acid (13e). Yield: 250 mg (82%); white solid; mp: 284−286 °C; purity: 95.77%; 1H NMR (400 MHz, DMSO-d6) δ 7.78−7.64 (m, 3H), 7.58 (d, J = 8.3 Hz, 2H), 7.46 (d, J = 8.5 Hz, 1H), 7.33 (s, 1H), 5.01 ppm (s, 2H); 13C NMR (100 MHz, DMSO-d6) δ 168.86, 153.08, 152.28, 149.79, 148.82, 131.94, 129.74, 127.96, 127.53, 125.71 (d, J = 8.3 Hz), 123.15, 121.57, 114.33, 109.96, 44.19 ppm; HRMS (ESI) m/z calcd for [M − H]− 422.9211, found 422.9206. 2-(7-Bromo-3-(4-bromothiophenoxy)-2-oxoquinoxalin-1(2H)-yl)acetic Acid (13f). Yield: 234 mg (79%); white solid; mp: 299−301 °C; purity: 99.19%; 1H NMR (400 MHz, DMSO-d6) δ 7.80 (s, 1H), 7.74 (d, J = 8.3 Hz, 2H), 7.47−7.33 (m, 4H), 5.01 ppm (s, 2H); 13C NMR (100 MHz, DMSO-d6) δ 168.46, 158.66, 151.94, 140.85, 136.92, 134.70, 132.49, 131.38, 129.68 (d, J = 27.5 Hz), 129.41, 126.95, 126.55, 121.73, 117.56, 109.21, 44.41 ppm; HRMS (ESI) m/z calcd for [M − H]− 466.8706, found 466.8701. 2-(6-Bromo-3-(4-bromothiophenoxy)-2-oxoquinoxalin-1(2H)-yl)acetic Acid (13g). Yield: 242 mg (82%); white solid; mp: >300 °C; purity: 95.58%; 1H NMR (400 MHz, DMSO-d6) δ 7.81 (s, 1H), 7.69− 7.55 (m, 4H), 7.31 (d, J = 8.4 Hz, 2H), 5.03 (s, 2H); 13C NMR (100 MHz, DMSO-d6) δ 168.58, 154.09, 151.38, 149.69, 132.64, 131.13, 130.76, 130.53, 129.25, 124.21, 118.13, 116.74, 115.60, 44.21 ppm; HRMS (ESI) m/z calcd for [M − H]− 466.8706, found 466.8708. 2-(3-(4-Chlorothiophenoxy)-2-oxoquinoxalin-1(2H)-yl)acetic Acid (13h). Yield: 232 mg (80%); white solid; mp: 279−281 °C; purity: 98.17%; 1H NMR (400 MHz, DMSO-d6) δ 7.73 (s, 1H), 7.58 (dd, J = 8.5, 1.9 Hz, 2H), 7.52 (d, J = 5.7 Hz, 2H), 7.43−7.32 (m, 2H), 5.16 (s, 2H); 13C NMR (100 MHz, DMSO-d6) δ 168.76, 158.02, 152.14, 136.94, 134.17, 132.38, 131.08, 129.38, 128.09, 127.91, 124.03, 123.77, 122.75, 121.32, 114.43, 43.79 ppm; HRMS (ESI) m/z calcd for [M − H]− 345.0106, found 345.0113. 2-(7-Fluoro-3-(4-chlorothiophenoxy)-2-oxoquinoxalin-1(2H)-yl)acetic Acid (13i). Yield: 227 mg (78%); white solid; mp: 240−342 °C; purity: 99.56%; 1H NMR (400 MHz, DMSO-d6) δ 7.83 (s, 1H), 7.60 (dd, J = 8.2, 1.8 Hz, 2H), 7.53−7.42 (m, 4H), 5.13 ppm (s, 2H); 13C NMR (100 MHz, DMSO-d6) δ 168.57, 161.32, 152.68, 151.52, 149.84, 133.16, 132.63, 129.26 (d, J = 10.3 Hz), 126.73, 124.22, 117.97, 111.42, 101.90, 44.15 ppm; HRMS (ESI) m/z calcd for [M − H]− 363.0012, found 363.0010. 2-(7-Chloro-3-(4-chlorothiophenoxy)-2-oxoquinoxalin-1(2H)-yl)acetic Acid (13j). Yield: 240 mg (76%); white solid; mp: >300 °C; K


Article

Journal of Medicinal Chemistry purity: 97.98%; 1H NMR (400 MHz, DMSO-d6) δ 7.71 (s, 1H), 7.61 (dd, J = 23.2, 8.2 Hz, 4H), 7.45 (d, J = 8.6 Hz, 1H), 7.31 (d, J = 8.5 Hz, 1H), 5.05 ppm (s, 2H); 13C NMR (100 MHz, DMSO-d6) δ 168.42, 158.47, 155.40, 152.00, 136.91, 133.56, 132.24, 131.10, 129.55 (d, J = 9.6 Hz), 129.40, 126.55, 124.12, 116.13, 114.72, 44.27 ppm; HRMS (ESI) m/z calcd for [M − H]− 378.9716, found 378.9718. 2-(7-Bromo-3-(4-chlorothiophenoxy)-2-oxoquinoxalin-1(2H)-yl)acetic Acid (13k). Yield: 251 mg (81%); white solid; mp: 277−379 °C; purity: 99.61%; 1H NMR (400 MHz, DMSO-d6) δ 7.73 (s, 1 H), 7.65 (dd, J = 6.6 Hz, J = 2 Hz, 2 H), 7.53 (d, J = 8.8 Hz, 1 H), 7.41− 7.32 (m, 3 H), 5.18 ppm (s, 2H); 13C NMR (100 MHz, DMSO-d6) δ 168.67, 159.58, 153.82, 149.76, 148.14, 132.93, 128.97, 128.76, 126.85, 123.66 (d, J = 8.4 Hz), 120.71, 117.07, 116.68, 44.19 ppm; HRMS (ESI) m/z calcd for [M − H]− 422.9211, found 422.9215. Biology, Materials, and Methods. ALR2 and ALR1 were obtained from Wistar rats, body weight 200−250 g, supplied by Vital River, Beijing (China). STZ was from Amresco. D ,L Glyceraldehyde, sodium D-glucuronate, and NADPH were from Sigma-Aldrich. All other chemicals were of reagent grade. ALR1 and ALR2 were prepared in accordance with the method of Kinoshita20 and La Motta et al.9d Enzyme activity was assayed spectrophotometrically on a Shimadzu UV-1800 spectrophotometer by measuring the decrease in absorption of NADPH at λ 340 nm, which accompanies the oxidation of NADPH catalyzed by ALR2 and ALR1. Malondialdehyde (MDA) detection kit and total protein quantification kit (Coomassie Brilliant Blue) were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Enzyme Assays. ALR2 activity was performed at 30 °C in a reaction mixture containing 0.25 mL of NADPH (0.10 mM), 0.25 mL of sodium phosphate buffer (0.1 M, pH 6.2), 0.1 mL of enzyme extract, 0.15 mL of deionized water, and 0.25 mL of D,L-glyceraldehyde (10 mM) as substrate in a final volume of 1 mL. The reaction mixture, except for D,L-glyceraldehyde, was incubated at 30 °C for 10 min. The substrate was then added to start the reaction, which was monitored for 4 min. ALR1 activity assays were performed at 37 °C in a reaction mixture containing 0.25 mL of NADPH (0.12 mM), 0.1 mL of enzyme extract, 0.25 mL of sodium phosphate buffer (0.1 M, pH 7.2), 0.15 mL of deionized water, and 0.25 mL of sodium D-glucuronate (20 mM) as substrate in a final volume of 1 mL. The reaction mixture, except for sodium D-glucuronate, was incubated at 37 °C for 10 min. The substrate was then added to start the reaction, which was monitored for 4 min. The inhibitory activity of the newly synthesized compounds against ALR2 and ALR1 was assayed by adding 5 mL of the inhibitor solution to the reaction mixture described above. All compounds were dissolved in dimethyl sulfoxide (DMSO) and diluted with deionized H2O. To correct for the nonenzymatic oxidation of NADPH, the rate of NADPH oxidation in the presence of all the reaction mixture components except the substrate was subtracted from each experimental rate. The inhibitory effect of the synthetic compounds was routinely estimated at a concentration of 100 μM (the concentration refers to that of the compound in the reaction mixture). The compounds found to be active were tested at additional concentrations between 100 μM and 10 nM. Most dose−response curves were generated using at least three concentrations of compound with inhibitory activity between 20% and 80%, with three replicates at each concentration. The 95% confidence limits (95% CL) were calculated from t values for n − 2, where n is the total number of determinations. DPPH Assay. To investigate the antiradical activity of the tested compounds in a homogeneous system, a method based on the scavenging of the stable free radical DPPH was used. Briefly, 100 μL of a methanolic solution of various compounds with different concentrations was added to 1 mL of DPPH methanolic solution (0.025 mg/mL) and 1.9 mL of methanol solution to give final concentrations of 0.1, 0.2, and 0.01 mM for the tested compounds, respectively. After vortexing thoroughly and leaving for 30 min at room temperature, the optical density was measured at λ 517 nm using the Shimadzu UV-1800 spectrophotometer. The tested compounds and the reference compound Trolox were dissolved in methanol, and

after 240 min (steady state), the percentage of DPPH radical scavenging was determined by the equation as shown in Figure 7. The experiments were performed in triplicate.

Figure 7. Equation of percentage of DPPH radical scavenging. Lipid Peroxidation Determination. Fresh rat brain was isolated and crushed with ice-cold normal saline, and a homogenate was prepared. The homogenate was centrifuged, and the supernatant was used for biochemical analyses. The protein concentration in the supernatant was determined by the Coomassie Brilliant Blue method using a total protein quantification kit (A045; Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The MDA concentration in the homogenate was determined using a commercially available kit (A003; Nanjing Jiancheng Bioengineering Institute, Nanjing, China) based on thiobarbituric acid (TBA) reactivity. Briefly, after mixing trichloroacetic acid with the homogenate and centrifuging, a supernatant was obtained, and TBA was added. The developed red color of the resulting reaction was measured at 532 nm with a spectrophotometer. Other procedures were carried out following the manufacturer’s instructions.21 Docking Studies. Docking was performed using Molegro Virtual Docker, version 5.0. The crystal structure of human aldose reductase with bound inhibitor (lidorestat) retrieved from the RCSB Protein Data Bank (PDB code: 1Z3N) and aldehyde reductase alone (PDB code: 3H4G) were used for docking. All solvent molecules within the protein structure were removed for the docking procedure. All structural parameters of ligands such as bond orders, hybridization, explicit hydrogen atoms, and charges were assigned when necessary in Molegro Virtual Docker software. To obtain better potential binding sites in ALR2, detecting possible binding cavities was implemented, and five cavities were obtained. The cavity around the anion binding site (volume of ∼187 Å3) was used for docking calculations and further modified using side chain minimization. To obtain better potential binding sites in ALR1, detecting possible binding cavities was implemented, and five cavities were obtained. The cavity (volume of ∼247 Å3) was used for docking calculations and further modified using side chain minimization. All docking calculations were carried out using the grid-based Mol-Dock score (GRID) function with a grid resolution of 0.30 Å. The best ligand poses were chosen on the basis of the MolDock score and ReRank score. The docking calculations were performed with a dual processor Windows 7 based computer with 4 GB RAM, and each docking process took 4−6 min.

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ASSOCIATED CONTENT

S Supporting Information *

General information for the data of DPPH radical scavenging and inhibition of lipid peroxidation of all tested compounds. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +86-10-68918506. Fax: +86-10-68918506. E-mail: zcj@ bit.edu.cn. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (grant no. 21272025), the Research Fund L


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for the Doctoral Program of Higher Education of China (grant no. 20111101110042), Beijing Natural Science Foundation (no. 7142096), and the Science and Technology Commission of Beijing (China) (grant no. Z131100004013003).

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ABBREVIATIONS USED ALR2, aldose reductase; ALR1, aldehyde reductase; ARI, aldose reductase inhibitor; THF, tetrahydrofuran; DMF, N,Ndimethylformamide; DMSO, dimethyl sulfoxide; NADPH, βnicotinamide adenine dinucleotide phosphate reduced form; NADP+, β-nicotinamide adenine dinucleotide phosphate; NAD+, β-nicotinamide adenine dinucleotide; SAR, structure− activity relationship; ROS, reactive oxygen species; NOS, nitric oxide synthase; AGEs, advanced glycation end products; PKC, protein kinase C; MAPK, mitogen-activated protein kinase; PARP, poly-ADP-ribose polymerase; DNA, deoxyribonucleic acid; HNE, hydroxynonenal; Trolox, 6-hydroxy-2,5,7,8-chroman-2-carboxylic acid; DPPH, 2,2-diphenyl-1-picrylhydrazyl; TBARS, thiobarbituric acid reactive substances

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Design and Synthesis of Potent and Multifunctional Aldose Reductase

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