Multifunctional aldose reductase inhibitors based on 2H-benzothiazine 1,1-dioxide

Abstract

A series of benzothiazine derivatives were designed and synthesized for the development of drug candidates for diabetic complications. A number of derivatives having a phenolic hydroxyl-substituted N2-aromatic side chain and a C4-acetic acid head group on the core structure were found to be potent and selective aldose reductase inhibitors. 8a with a phenolic 4-hydroxyl at N2-styryl side chain was proved to be the most active with an IC₅₀ value of 0.094 μM. All compounds with the N2-styryl side chain showed good antioxidant activity using the DPPH radical scavenging test, and among them, compounds with phenolic hydroxyl-substituted N2-styryl were potent both in activities of ALR2 inhibition and antioxidation. The results suggest a success in the development of multifunctional aldose reductase inhibitors based on the benzothiazine framework.

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Introduction

Diabetes mellitus (DM) is a chronic disease characterized by hyperglycaemia and the development of chronic diabetic complications, such as neuropathy, nephropathy, cataracts and retinopathy.^1,2 It is a major health threat to the global public and the situation continues to worsen rapidly,³ as over 171 million people worldwide were affected by this disease by the year 2000 and this number is expected to increase steadily to 366 million by 2030.^4,5 People suffering with diabetes bear a much higher economic and physical burden. Increasing evidence^6,7 demonstrates all forms of diabetes are vulnerable to chronic diabetic complications, which is the major distress to diabetic mellitus patients. In the recent several decades, a number of studies^6,8,9 have indicated that the enzyme aldose reductase (ALR2, EC, 1.1.1.21) plays a crucial role in the development of chronic diabetic complications and inflammation processes. Aldose reductase is always present in the cytoplasm of most human cells and catalyses the NADPH-dependent reduction of a series of carbonyl compounds, especially glucose.¹⁰ This enzyme is a member of the aldo-keto reductase (AKR) superfamily and together with sorbitol dehydrogenase composes the polyol pathway (Fig. 1), which is the main pathogenesis of diabetic complications.^11–14 During this glucose metabolism pathway, the ALR2 firstly catalyses the NADPH-dependent reduction of glucose to sorbitol, and sorbitol dehydrogenase in turn converts sorbitol into fructose with concomitant reduction of NAD⁺. Under normal glycaemic conditions, the ALR2 has a low activity to this pathway of glucose metabolism, and the most glucose is predominantly converted to glucose-6-phosphate by hexokinase and then enters the glycolytic pathway.^1,6 However, at high glucose concentration, especially in diabetics, the activity of the aldose reductase is motivated and about 33% of the total glucose is metabolized through the polyol pathway in tissues such as lens, kidney, retina, and peripheral nerves demonstrating insulin-independent uptake of glucose.^1,6,13 The increased intracellular glucose through polyol pathway results in its increased enzymatic conversion to sorbitol with concomitantly reduction in NADPH, and segmental sorbitol is oxidized to fructose by the enzyme sorbitol dehydrogenase with the reduction of NAD⁺ to NADH. Unfortunately, the sorbitol is a polyalcohol with strong polarity, and is difficult to penetrate through cell membrane. Therefore, the intracellular accumulation of sorbitol leads to osmotic imbalance, cell swelling, and membrane permeability changes, mainly in lenses. In addition, the abnormal decrease of NADPH and NAD⁺ result in changes in cellular redox potentials and the activity of enzymes such as nitric oxide synthase (NOS) and glutathione reductase.¹ These changes eventually give rise to the cellular oxidative stress, as a consequence of the imbalance between increased production of radical oxygen species (ROS) and reduced intracellular antioxidant defense.^1,6 Furthermore, the abnormal enhancement of levels of glycating agents such as fructose (end-product of the polyol pathway), dihydroxyacetone phosphate, glyceraldehyde-3-phosphate and methylglyoxal, increases the accumulation of advanced glycation-end products (AGEs)^15,16 and the progress of hereditary fructose intolerance.¹ In addition, the increased AGEs in return can lead to other pathological changes in the functions of intracellular proteins and result in further accumulation of ROS.¹

Fig. 1 Polyol pathway of glucose metabolism.

All those ALR2-regulated changes rooted in the polyol pathway give rise to multiple pathogenic mechanisms which become the basis for the development of diabetic complications. Therefore, inhibiting the activity of the ALR2 and further cutting off the polyol pathway will be an efficient therapy to prevent and delay the development of diabetic complications.

In the recent few decades, many series of structurally different ARIs^16–22 have been developed (Fig. 2), and some of them present excellent inhibitory activity in vitro and in vivo. However, Epalrestat is the only ARIs available for the therapy in Japan and recently in China and India. Most of the ARIs that showed great potential have not yet passed through the clinical trials mainly because of adverse side effects, low in vivo efficacies, or pharmacokinetic drawbacks. Accordingly, the side effects are believed to be mainly due to the absence of ALR2 selectivity,^23,24 which is often determined by measuring the activity against aldehyde reductase (ALR1, EC 1.1.1.2), an enzyme closely related to ALR2. The enzyme ALR1 plays a crucial role in detoxification process, as it particularly catalyzes toxic aldehydes such as methylglyoxal, 3-deoxyglucosone, and hydroxynonenal (HNE),^4,25 which arise in large quantities from pathological conditions connected with oxidative stress. Thus, inhibiting the activity of ALR1 may lead to some undesirable side effects, and the design and synthesis of more structurally diverse ARIs and subsequently the ascertainment of selectivity to targeted candidates that block ALR2, specifically are the main approaches to solve this problem. Additionally, the mechanism of low efficacy is still indeterminate,²⁶ however it is certain that the process of diabetic complications and oxidative stress are complementary to each other, thus molecules with both ALR2 inhibitory activity and anti-oxidative properties could be more effective than compounds with either ALR2 inhibition or antioxidant property alone,^6,13,15,27 and this may be a new therapeutic strategy. Accordingly,²⁸ introduction of anti-oxidative property to ARIs may be a promising way to develop new efficient ARI drug candidates for the treatment of diabetic complications.

Fig. 2 Structures of some ARIs.

In recent years, we have developed several groups of ARIs,^16,18,21 most of them endowed with potential inhibitory activity were without the antioxidant activity. In the present study, a series of multifunctional ARIs based on 2H-benzothiazine 1,1-dioxide were developed by combination of antioxidant activity in designed compounds.

Results and discussion

Chemistry

The designed compounds with a carboxylate substituent at C4 position and a variety of aromatic substituents at N2 position of the benzothiazine framework were obtained by the syntheses starting from 2H-benzothiazine-4(3H)-one 1,1-dioxide 1 prepared in our previous work.^29–31 As shown in Scheme 1, compound 1 was alkylated at the C4 position with methyl 2-(triphenylphosphoranylidene)-acetate to form methyl ester 2 as the key intermediate to prepare desired compounds 5–8. Different benzyl groups were attached to the N2 position of 2 forming compounds 4 in high yields, and then hydrolysis of 4a–c with lithium hydroxide gave 5a–c. Then, demethylation of the methoxyl at 4b,c with AlCl₃ or BBr₃ followed by hydrolysis of the esters with hydroxide gave compounds 6a–c. On the other hand, different styryl groups were attached to the N2 position of compound 2 by the C–N coupling reaction in the presence of CuI forming the intermediates 3a–d, which were in turn hydrolyzed to provide corresponding compounds 7a–d. Treatment of 3b–d with BBr₃ or AlCl₃ resulted in partial or complete demethylation of methoxyl groups at N2 side chain, and then hydrolysis of the demethylation products led to the preparation of 8a–f.

Scheme 1 (a) Ph₃P=CHCOOCH₃, PhCH₃, reflux; (b) substituted bromostyrene, CuI, Cs₂CO₃, N,N′-dimethyl-1,2-ethanediamine, dioxane, 100 °C; (c) substituted Bn-Br, K₂CO₃, CH₃CN, 70 °C; (d) LiOH, H₂O, THF, rt, 2 h, then 0.1 N HCl; (e): (i) BBr₃ or AlCl₃, dry dichloromethane; (ii) LiOH, H₂O, THF, rt, 2 h, then 0.1 N HCl.

Biological activity

Two series of compounds were obtained and examined for their biological activities. The one series (5–6) contains N2-benzyl side chain while the other series (7–8) has N2-styryl side chain. As our previous work suggested,²² both groups of C4-acetic acid and N2-benzyl in the 2H-benzothiazine 1,1-dioxide core structure were typical side chains in the present ARI design which resulted in the preparation of compounds 5–6. Besides, given that the styryl group had impact in addition to benzyl group on potent ARIs based on quinoxalinone,¹³ N2-styryl side chain was an option in the present study leading to compounds 7–8. Furthermore, phenolic hydroxyl often endowed with antioxidant activity and therefore was employed in the present design in order to incorporate antioxidant property into the ARIs.

Inhibition of enzymes

All newly synthesized compounds 5–8 shown in Scheme 1 were evaluated for their inhibitory activity against ALR2 extracted from rat lenses, and their selectivity for ALR2 inhibition was tested by the identification of inhibitory activity against ALR1 isolated from rat kidneys.³² The test results are summarized in Table 1.

Table 1 Biological activity of 2H-benzothiazine 1,1-dioxide derivatives

Compd	Substituent		IC50 (μM)ALR2^a	Inhib. (%)ALR1^b	Activity ratio	DPPH sca. %
	R	X			(ALR2/ALR1)	100 μM	50 μM	10 μM
a IC50 values represent the concentration required to produce 50% enzyme inhibition.b The inhibitory effect was estimated at a concentration of 10 μM.c The inhibitory effect was estimated at a concentration of 1 μM.d Not tested.
5a	H	CH2	9.952(9.524–10.380)	19.35	2.58^b	5.6	^d	^d
5b	4-OCH3	CH2	7.821(7.197–8.445)	20.13	3.23^b	7.3	^d	^d
5c	3,5-(OCH3)2	CH2	5.365(4.826–5.904)	25.1	2.95^b	8.1	^d	^d
6a	4-OH	CH2	1.655(1.567–1.743)	18.26	4.87^b	75.2	48.6	27.9
6b	3-OH, 5-OCH3	CH2	4.321(3.785–4.857)1	15.32	4.96^b	23.2	^d	^d
6c	3,5-(OH)2	CH2	2.286(2.057–2.515)	11.36	7.31^b	68.5	43.3	25.4
7a	H	CH=CH	1.682(1.417–1.946)	14.29	6.16^b	87.4	68.4	40.5
7b	4-OCH3	CH=CH	1.124(1.065–1.183)	17.52	5.37^b	88.6	69.3	43.2
7c	3,4-(OCH3)2	CH=CH	1.352(0.928–1.775)	12.18	7.39^b	86.7	65.1	38.5
7d	3,4,5-(OCH3)3	CH=CH	3.214(2.568–3.859)	13.41	5.82^b	85.3	64.2	33.5
8a	4-OH	CH=CH	0.094(0.075–0.113)	20.49	11.53^c	91.8	69.8	45.8
8b	3-OCH3, 4-OH	CH=CH	0.206(0.152–0.259)	18.61	10.13^c	92.6	71.6	48.2
8c	3,4-(OH)2	CH=CH	0.126(0.108–0.140)	13.27	13.21^c	94.6	72.5	50.1
8d	3,4-(OCH3)2, 5-OH	CH=CH	0.568(0.503–0.633)	21.81	9.84^c	87.3	67.1	36.9
8e	3,5-(OH)2, 4-OCH3	CH=CH	0.356(0.343–0.369)	12.27	12.39^c	88.5	68.7	41.3
8f	3,4,5-(OH)3	CH=CH	0.284(0.256–0.312)	15.69	12.62^c	92.3	70.5	46.8
Epalrestat			0.091(0.072–0.110)	50.3	3.81^c	^d	^d	^d
Trolox			^d	^d	^d	96.2	89.6	78.2

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Compounds in the series 5–6 with N2-benzyl side chain showed activity in the ALR2 inhibition with IC₅₀ values ranging from 1.655 μM to 9.952 μM. Structure–activity relationship study regarding the series indicated that the methoxyl or phenolic hydroxyl substituents on the N2 benzyl obviously improved the inhibitory activity when comparing 5a with the remaining compounds. The most improvement benefit was provided by the phenolic hydroxyl group particularly at the para-position of the N2 benzyl ring (6a).

When vinyl group was used as the spacer instead of the methylene group in the N2 side chain leading to series 7–8 (IC₅₀ = 0.094–3.214 μM), remarkable enhancement effect on the ALR2 inhibition was found by comparison of compounds 7a,b and 8a with their counterparts in the series 5–6, respectively. 8a having phenolic para-hydroxyl in the N2 side chain was the most potent inhibitor in the present study while 8c with phenolic 2,4-dihydroxyl in the side chain was also excellent.

As observed in series 5–6, the methoxyl and especially phenolic hydroxyl substituent at the para-position of the N2-styryl ring greatly elevated the inhibitory activity since 8a and 7b (IC₅₀ = 0.094 and 1.124 μM) were much active than 7a (IC₅₀ = 1.682 μM). However, more methoxyl substitution at the styrene ring in 7b reduced the ALR2 inhibition (7b > 7c > 7d) whereas combinations of methoxyl and phenolic hydroxyl in the styrene ring (8a–f) could keep the inhibitory activity at IC₅₀ levels of 0.126 to 0.568 μM much higher than that of series 7. It is interesting to further find that the release of free phenolic hydroxyl group from the methoxyl blockage resulted in an increase in the inhibitory activity (8c > 8b, and 8f > 8e > 8d). This hydroxyl-dependent elevation in the ALR2 inhibition suggests a fundamental importance of the phenolic hydroxyl in these series of ARIs.

These results indicated that longer spacer of the N2 side chain and phenolic hydroxyl substitution may be effective way for the design of more potent ARIs based on the benzothiazine framework.

Compounds 7–8 all showed much low activity in the ALR1 inhibition indicating their good selectivity for the ALR2 inhibition.

The antioxidant activity

The scavenging of stable free radicals of 2,2-diphenyl-1-picrylhydrazyl (DPPH) was used to evaluate potential antioxidant properties of the synthesized compounds,³³ and 6-hydroxy-2,5,7,8-chroman-2-carboxylic acid (Trolox) was employed as a positive control. Phenolic hydroxyl-containing compounds 6a–c revealed obvious antioxidant activity with DPPH radical scavenging rates ranging from 23.2 to 75.2% at the concentration of 100 μM, whereas removal or block of the hydroxyl (5a–c) resulted in loss of activity (Table 1).

Compounds in series 7–8 having the N2-styryl side chain were all outstanding at antioxidation. Of them, 8c containing phenolic 3,4-dihydroxyl groups in the N2-styryl was the most active with the DPPH radical scavenging rate of 94.6% at the concentration of 100 μM, and 8a,b with 4-hydroxyl and 3-methoxy-4-hydroxyl, respectively, were also excellent. Noticeably, it was found that the antioxidant ability of the compounds is phenolic hydroxyl-independent resulting from series 7a–d that had great and same level of antioxidant activity in spite of their blocked phenolic hydroxyls (7b–d) and the absence of hydroxyl (7a). Nevertheless, phenolic hydroxyl substitution in the N2-styryl side chain could further make compounds (8a–f) stronger in the antioxidant activity as comparing 8a–f with their counterparts in 7a–d, respectively, and the 3,4-dihydroxyl (8c) produced the most effect.

Molecular modelling

The compound 8a endowed with best activity of the ALR2 inhibition and excellent antioxidant activity was studied by molecular docking performed on the human ALR2/NADP⁺/IDD594 complex (PDB entry code 1US0). The result shows that the compound fitted well to the active site of ALR2 (Fig. 3), in which the carboxylate group formed tight hydrogen bonds with Trp 111 (2.89 Å) and His 110 (2.54 Å). The 4-hydroxyl oxygen atom of the N2-side chain formed an additional hydrogen bond with the side chain of Tyr 309 (2.94 Å) explaining the key role of the 4-hydroxyl in the enhancement of the ALR2 inhibition. The phenyl ring of N2 side chain was well paralleled to the indole ring of Trp 111 forming a stacking interaction. On the other hand, the docking behavior of 8a with the ALR1–NADP⁺–fidarestat complex (PDB code: 3H4G) was also conducted. It was found that the benzyl side chain appeared to be mismatched, floating out from the active cleft of ALR1, and this could explain the selectivity of 8a for ALR2 listed in Table 1.

Fig. 3 Docking of 8a into the active site of ALR2. (a) The protein structure is shown in ribbon and tube representation with selected residues labeled and shown in line representation, ligand 8a is shown as stick models. The docked pose of 8a is shown in cyan (C), red (O), blue (N), and yellow (S). Hydrogen bonds are shown as blue dashed lines. (b) Protein residues are in surface representation.

Conclusions

Design of ARIs based on benzothiazine 1,1-dioxide resulted in the formation of a series of novel candidates, of which the series 8a–f with N2 styryl side chain was potently active in the ALR2 inhibition with IC₅₀ values at submicromolar level, but less active in the ALR1 inhibition with inhibition percentages no more than 25.1% at concentration of 10 μM, indicating good selectivity of the compounds for ALR2. 8a bearing the phenolic 4-hydroxyl at the N2 styryl side chain was found to be the most potent ARI with IC₅₀ value of 0.094 μM. In addition, DPPH radical scavenging potency was tested in order to verify the antioxidant activity of the designed compounds. All compounds of series 7–8 with the N2 styryl side chain displayed good antioxidant activity by the test of DPPH radical scavenging, and 8c containing 3,4-dihydroxyl groups in the N2-styryl was identified as a distinguished antioxidant even comparable with Trolox. 8c was also excellent in the ALR2 inhibition. Structure–activity relationship studies on the compounds suggest that the phenolic hydroxyl-substituted N2 styryl besides the carboxylate head group is the key structure for the potent ARIs, and the vinyl spacer of the N2 side chain is essential for the antioxidant ability of ARIs.

Experimental

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 Merck 60F254. The ¹H and ¹³C NMR spectra were recorded on a Bruker Avance 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. 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.

Procedure for synthesis of acetate 2

A mixture of 2H-benzothiazine-4(3H)-one 1,1-dioxide 1 (1.183 g, 6 mmol) and methyl (triphenylphosphoranylidene) acetate (3.009 g, 9 mmol) dissolved in toluene (45 mL) was stirred at 100 °C overnight and cooled to room temperature subsequently. After evaporation of the solvent, the residue was purified by column chromatography (ethylacetate–hexane; 1 : 4) to provide the product 2.

Methyl 2-(1,1-dioxido-2H-benzo[e][1,2]thiazin-4-yl)acetate (2). White solid (1.23 g, 81%), purity: 95.18%, mp: 173–175 °C; ¹H NMR (400 MHz, [D₆]DMSO): δ = 7.92 (s, 1H), 7.89 (m, 1H), 7.72 (m, 1H), 7.61 (m, 1H), 6.25 (m, 1H), 4.23 (d, 2H), 3.65 ppm (d, 3H); ¹³C NMR (100 MHz, [D₆]DMSO) δ = 168.46, 138.14, 137.11, 136.13, 134.47, 133.09, 132.36, 125.34, 117.65, 68.45, 45.31 ppm.

General procedure for synthesis of acetates 3

A mixture of the appropriate 2 (1 mmol), the substituted bromostyrene (1.5 mmol), Cs₂CO₃ (3 mmol), CuI (0.5 mmol), and N,N′-dimethyl-1,2-ethanediamine (7 mmol) in dried dioxane (10 mL) charged in round-bottom flask under nitrogen protection was stirred at 100 °C until reaction finished. The resulting mixture was cooled to room temperature, filtered, and solvent evaporated in vacuo to obtain a crude mixture, which was purified by column chromatography (ethylacetate–hexane; 1 : 3) to provide the product 3.

Methyl 2-(1,1-dioxido-2-styryl-2H-benzo[e][1,2]thiazine-4-yl)aceta-te (3a). White solid (0.30 g, 85%); purity: 96.13%; mp: 261–262 °C; ¹H NMR (400 MHz, [D₆]DMSO): δ = 7.82 (m, 1H), 7.66–7.35 (m, 8H), 6.78 (m, 1H), 6.43 (s, 1H), 6.10 (d, J = 8 Hz, 1H), 4.28 (d, J = 8 Hz, 2H), 3.65 ppm (d, 3H); ¹³C NMR (100 MHz, [D₆]DMSO) δ = 171.06, 141.51, 138.21, 135.28, 133.64, 133.15, 132.76, 132.32, 130.27, 130.21, 129.11, 128.34, 128.12, 126.26, 126.15, 125.38, 116.35, 65.53, 45.11 ppm.

Methyl 2-(2-(4-methoxystyryl)-1,1-dioxido-2H-benzo[e][1,2]thiaz-in-4-yl)acetate (3b). White solid (0.32 g, 84%); purity: 98.21%; mp: 289–290 °C; ¹H NMR (400 MHz, [D₆]DMSO): δ = 7.82 (m, 1H), 7.66–7.40 (m, 5H), 6.95 (m, 2H), 6.78 (d, J = 8 Hz, 1H), 6.45 (s, 1H), 6.10 (m, 1H), 4.15 (s, 2H), 3.65 ppm (d, 3H); ¹³C NMR (100 MHz, [D₆]DMSO) δ = 171.12, 157.14, 142.21, 135.82, 134.37, 133.19, 132.16, 131.32, 129.28, 128.54, 127.13, 126.58, 124.24, 123.18, 119.34, 118.25, 115.51, 55.23, 55.21, 45.21 ppm.

Methyl 2-(2-(3,4-dimethoxystyryl)-1,1-dioxido-2H-benzo[e][1,2]th-iazin-4-yl)acetate (3c). White solid (0.33 g, 80%); purity: 97.80%; mp: 301–302 °C; ¹H NMR (400 MHz, [D₆]DMSO): δ = 7.81 (m, 1H), 7.66–7.40 (m, 3H), 7.12–6.89 (m, 3H), 6.78 (d, J = 8 Hz, 1H), 6.43 (s, 1H), 6.13 (s, 1H), 4.43 (m, 2H), 4.01–4.03 (m, 9H) ppm; ¹³C NMR (100 MHz, [D₆]DMSO) δ = 171.16, 158.04, 158.03, 142.01, 135.71, 134.21, 132.19, 131.47, 129.11, 128.04, 127.23, 126.08, 124.54, 123.31, 119.12, 118.94, 115.15, 55.81, 55.80, 45.31, 45.08 ppm.

Methyl 2-(1,1-dioxido-2-(3,4,5-trimethoxystyryl)-2H-benzo[e][1,2]-thiazin-4-yl)acetate (3d). White solid (0.39 g, 82%); purity: 98.18%; mp: 203–205 °C; ¹H NMR (400 MHz, [D₆]DMSO): δ = 7.81 (m, 1H), 7.68–7.40 (m, 3H), 6.78 (m, 2H), 6.69 (d, J = 8 Hz, 1H), 6.38 (s, 1H), 6.12 (s, 1H), 4.42 (m, 2H), 3.83–4.01 ppm (m, 12H); ¹³C NMR (100 MHz, [D₆]DMSO) δ = 171.16, 158.04, 158.03, 158.03, 137.71, 135.01, 133.91, 130.12, 128.21, 128.04, 127.13, 126.58, 124.47, 123.01, 119.12, 118.94, 115.15, 55.81, 55.81, 55.80, 45.31, 45.12 ppm.

General procedure for synthesis of acetates 4

A mixture of the appropriate 2 (1 mmol), substituted benzyl bromide (1.5 mmol) and K₂CO₃ (3 mmol) in acetonitrile (10 mL) was stirred at 65 °C for 3 hours. After the reaction, the resulting mixture was cooled to room temperature, filtered and solvent evaporated in vacuo to obtain a crude mixture. The residue was purified by column chromatography (ethylacetate–hexane; 1 : 3) to provide the product 4.

Methyl 2-(2-benzyl-1,1-dioxido-2H-benzo[e][1,2]thiazin-4-yl)aceta-te (4a). White solid (0.25 g, 78%); purity: 96.42%; mp: 257–259 °C; ¹H NMR (400 MHz, [D₆]DMSO): δ = 7.82 (m, 1H), 7.53 (m, 3H), 7.33 (m, 1H), 7.26 (m, 3H), 6.14 (s, 1H), 6.06 (s, 1H), 4.42 (s, 2H), 3.89 (s, 2H), 3.59 ppm (s, 3H); ¹³C NMR (100 MHz, [D₆]DMSO) δ = 171.06, 142.94, 138.01, 134.30, 133.57, 133.50, 132.66, 132.55, 130.97, 130.25, 129.1, 126.36, 126.24, 125.18, 116.97, 65.51, 45.12, 45.01 ppm.

Methyl 2-(2-(4-methoxybenzyl)-1,1-dioxido-2H-benzo[e][1,2]thiaz-in-4-yl)acetate (4b). White solid (0.31 g, 83%); purity: 96.12%; mp: 240–241 °C; ¹H NMR (400 MHz, [D₆]DMSO): δ = 7.83 (m, 1H), 7.65 (m, 3H), 7.15 (m, 2H), 6.86 (m, 2H), 6.06 (s, 1H), 4.68 (s, 2H), 4.34 (s, 2H), 3.83–3.94 ppm (m, 6H); ¹³C NMR (100 MHz, [D₆]DMSO) δ = 171.06, 158.94, 141.01, 138.30, 133.47, 132.90, 132.66, 131.55, 130.97, 130.55, 129.14, 126.16, 125.64, 125.18, 113.17, 65.51, 64.52, 45.31, 45.08 ppm.

Methyl 2-(2-(3,5-dimethoxybenzyl)-1,1-dioxido-2H-benzo[e][1,2]t-hiazin-4-yl)acetate (4c). White solid (0.35 g, 86%), purity: 95.35%, mp: 212–213 °C; ¹H NMR (400 MHz, [D₆]DMSO): δ = 7.72 (m, 1H), 7.49 (m, 3H), 7.32 (m, 2H), 6.98 (d, 2H), 5.16 (s, 2H), 4.43 (d, 2H), 3.75–3.82 ppm (m, 9H); ¹³C NMR (100 MHz, [D₆]DMSO) δ = 171.06, 158.94, 158.91, 139.12, 134.17, 133.29, 132.16, 131.05, 130.57, 130.12, 129.13, 126.08, 125.54, 125.18, 113.17, 65.51, 64.52, 64.45, 45.09, 44.31 ppm.

General procedure for synthesis of acids 5a–c

A mixture of the appropriate 4 (0.5 mmol), tetrahydrofuran (5 mL) and saturated aqueous lithium hydroxide (5 mL) was stirred at room temperature for 6 h. 0.1 N HCl solution was add to the mixture and adjusted the solution pH to acidic. The reaction was quenched with water, transferred to a separatory funnel and organics extracted three times with ethyl acetate (3 × 30 mL). The combined organic layers were dried with anhydrous MgSO₄, filtered and solvent evaporated in vacuo to obtain the desired products 5a–c.

2-(2-Benzyl-1,1-dioxido-2H-benzo[e][1,2]thiazin-4-yl)acetic acid (5a). White solid (0.12 g, 75%); purity: 95.43%; mp: 278–279 °C; ¹H NMR (400 MHz, [D₆]DMSO): δ = 10.96 (s, 1H), 7.82 (m, 1H), 7.53 (m, 3H), 7.33 (m, 1H), 7.26 (m, 3H), 6.14 (s, 1H), 6.06 (s, 1H), 4.42 (s, 2H), 3.89 ppm (s, 2H); ¹³C NMR (100 MHz, [D₆]DMSO) δ = 171.06, 142.94, 138.01, 134.30, 133.57, 133.50, 132.66, 132.55, 130.97, 130.25, 129.1, 126.36, 126.24, 125.18, 116.97, 65.51, 45.01 ppm.

2-(2-(4-Methoxybenzyl)-1,1-dioxido-2H-benzo[e][1,2]thiazin-4-yl)acetic acid (5b). White solid (0.15 g, 81%); purity: 96.43%; mp: 243–244 °C; ¹H NMR (400 MHz, [D₆]DMSO): δ = 10.67 (s, 1H), 7.83 (m, 1H), 7.65 (m, 3H), 7.15 (m, 2H), 6.86 (m, 2H), 6.06 (s, 1H), 4.68 (s, 2H), 4.34 (s, 2H), 3.83 ppm (s, 3H); ¹³C NMR (100 MHz, [D₆]DMSO) δ = 171.06, 158.94, 141.01, 138.30, 133.47, 132.90, 132.66, 131.55, 130.97, 130.55, 129.14, 126.16, 125.64, 125.18, 113.17, 65.51, 64.52, 45.31 ppm.

2-(2-(3,5-Dimethoxybenzyl)-1,1-dioxido-2H-benzo[e][1,2]thiazin-4-yl)acetic acid (5c). White solid (0.16 g, 83%), purity: 92.43%, mp: 213–214 °C; ¹H NMR (400 MHz, [D₆]DMSO): δ = 10.32 (s, 1H), 7.72 (m, 1H), 7.49 (m, 3H), 7.32 (m, 2H), 6.98 (d, 2H), 5.16 (s, 2H), 4.43 (d, 2H), 3.75 ppm (d, 6H); ¹³C NMR (100 MHz, [D₆]DMSO) δ = 171.06, 158.94, 158.91, 139.12, 134.17, 133.29, 132.16, 131.05, 130.57, 130.12, 129.13, 126.08, 125.54, 125.18, 113.17, 65.51, 64.52, 64.45, 44.31 ppm.

General procedure for synthesis of acids 6a–c

A mixture of appropriate 4 (1 mmol) and anhydrous AlCl₃ (10 mmol) in dried dichloromethane (25 mL) was stirred at 0 °C for 0.5 h, and then heated to 45 °C overnight. The reaction was quenched with ice-cold water (20 mL), transferred to a separatory funnel and extracted the organics three times with ethyl acetate (3 × 30 mL). The combined organic layers were dried with anhydrous MgSO₄, filtered and solvent evaporated in vacuo to obtain the crude solid. The residue was purified by column chromatography. After hydrolysis reaction, the desired products 6a–c were obtained.

2-(2-(4-Hydroxybenzyl)-1,1-dioxido-2H-benzo[e][1,2]thiazin-4-yl)acetic acid (6a). Yellow solid (0.28 g, 80%); purity: 98.18%; mp: 290–291 °C; ¹H NMR (400 MHz, [D₆]DMSO): δ = 0.57 (s, 1H), 7.72 (s, 1H), 7.59–7.62 (m, 3H), 7.23 (m, 2H), 6.62 (m, 2H), 6.06 (s, 1H), 5.35 (s, 1H), 4.42 (s, 2H), 3.31 ppm (s, 2H); ¹³C NMR (100 MHz, [D₆]DMSO) δ = 171.06, 157.04, 141.01, 139.82, 134.17, 133.49, 132.56, 131.75, 130.35, 130.02, 129.33, 126.48, 125.24, 125.18, 113.17, 64.25, 45.11 ppm.

2-(2-(3-Hydroxy-5-methoxybenzyl)-1,1-dioxido-2H-benzo[e][1,2]-thiazin-4-yl)acetic acid (6b). Yellow solid (0.29 g, 78%); purity: 99.25%; mp: 277–278 °C; ¹H NMR (400 MHz, [D₆]DMSO): δ = 9.58 (s, 2H), 7.83 (m, 1H), 7.50–7.66 (m, 3H), 6.35–6.53 (m, 3H), 6.06 (s, 1H), 5.15 (s, H), 4.42 (s, 2H), 4.21 ppm (s, 2H), 3.83 (s, 3H); ¹³C NMR (100 MHz, [D₆]DMSO) δ = 171.06, 161.04, 157.01, 140.82, 135.27, 133.39, 132.96, 131.45, 130.35, 130.02, 129.33, 126.48, 125.24, 125.18, 121.21, 65.21, 64.25, 45.11 ppm.

2-(2-(3,5-Dihydroxybenzyl)-1,1-dioxido-2H-benzo[e][1,2]thiazin-4-yl)acetic acid (6c). White solid (0.27 g, 76%); purity: 98.25%; mp: 278–279 °C; ¹H NMR (400 MHz, [D₆]DMSO): δ = 9.58 (s, 1H), 7.73 (m, 1H), 7.63–7.55 (m, 3H), 7.08–6.63 (m, 3H), 6.05 (s, 1H), 5.36 (s, 2H), 4.42 (m, 2H), 4.26 ppm (m, 2H); ¹³C NMR (100 MHz, [D₆]DMSO) δ = 171.06, 158.04, 158.01, 141.82, 136.47, 133.21, 132.69, 131.12, 130.53, 129.13, 126.28, 125.24, 125.18, 121.21, 120.31, 64.51, 45.21 ppm.

General procedure for synthesis of acids 7a–d

A mixture of the appropriate 3 (0.5 mmol), tetrahydrofuran (5 mL) and saturated aqueous lithium hydroxide (5 mL) was stirred at room temperature for 6 h. 0.1 N HCl solution was add to the mixture and adjusted the solution pH to acidic. The reaction was quenched with water, transferred to a separatory funnel and organics extracted three times with ethyl acetate (3 × 30 mL). The combined organic layers were dried with anhydrous MgSO₄, filtered and solvent evaporated in vacuo to obtain the desired products 7a–d.

2-(1,1-Dioxido-2-styryl-2H-benzo[e][1,2]thiazin-4-yl)acetic acid (7a). Grey solid (0.13 g, 75%); purity: 97.25%; mp: 315–316 °C; ¹H NMR (400 MHz, [D₆]DMSO): δ = 11.38 (s, 1H), 7.81 (m, 1H), 7.66–7.35 (m, 8H), 6.78 (m, 1H), 6.43 (s, 1H), 6.10 (d, J = 8 Hz, 1H), 3.98 ppm (d, J = 8 Hz, 2H); ¹³C NMR (100 MHz, [D₆]DMSO) δ = 171.16, 142.04, 141.01, 139.82, 138.27, 135.39, 134.16, 132.51, 130.21, 130.54, 129.13, 126.48, 125.24, 125.18, 121.21, 120.21, 119.51, 44.21 ppm.

2-(2-(4-Methoxystyryl)-1,1-dioxido-2H-benzo[e][1,2]thiazin-4-yl)acetic acid (7b). Yellow solid (0.13 g, 72%); purity: 97.27%; mp: 294–295 °C; ¹H NMR (400 MHz, [D₆]DMSO): δ = 11.48 (s, 1H), 7.82 (m, 1H), 7.66–7.40 (m, 5H), 6.95 (m, 2H), 6.78 (d, J = 8 Hz, 1H), 6.45 (s, 1H), 6.10 (m, 1H), 3.95 ppm (s, 2H); ¹³C NMR (100 MHz, [D₆]DMSO) δ = 171.16, 157.04, 142.01, 135.82, 134.27, 133.39, 132.16, 131.51, 129.21, 128.54, 127.13, 126.48, 124.24, 123.18, 119.21, 118.21, 115.51, 55.21, 45.21 ppm.

2-(2-(3,5-Dimethoxystyryl)-1,1-dioxido-2H-benzo[e][1,2]thiazin-4-yl)acetic acid (7c). White solid (0.16 g, 81%); purity: 98.80%; mp: 309–311 °C; ¹H NMR (400 MHz, [D₆]DMSO): δ = 12.34 (s, 1H), 7.81 (m, 1H), 7.66–7.40 (m, 3H), 7.12–6.89 (m, 3H), 6.78 (d, J = 8 Hz, 1H), 6.43 (s, 1H), 6.13 (s, 1H), 4.43 (m, 2H), 3.83 ppm (s, 6H); ¹³C NMR (100 MHz, [D₆]DMSO) δ = 171.16, 158.04, 158.03, 142.01, 135.71, 134.21, 132.19, 131.47, 129.11, 128.04, 127.23, 126.08, 124.54, 123.31, 119.12, 118.94, 115.15, 55.81, 55.80, 45.31 ppm.

2-(1,1-Dioxido-2-(3,4,5-trimethoxystyryl)-2H-benzo[e][1,2]thiazin-4-yl)acetic acid (7d). Grey solid (0.18 g, 82%); purity: 99.18%; mp: 215–217 °C; ¹H NMR (400 MHz, [D₆]DMSO): δ = 12.39 (s, 1H), 7.81 (m, 1H), 7.68–7.40 (m, 3H), 6.78 (m, 2H), 6.69 (d, J = 8 Hz, 1H), 6.38 (s, 1H), 6.12 (s, 1H), 4.42 (m, 2H), 3.83 ppm (s, 9H); ¹³C NMR (100 MHz, [D₆]DMSO) δ = 171.16, 158.04, 158.03, 158.03, 137.71, 135.01, 133.91, 130.12, 128.21, 128.04, 127.13, 126.58, 124.47, 123.01, 119.12, 118.94, 115.15, 55.81, 55.81, 55.80, 45.31 ppm.

General procedure for synthesis of acids 8a–f

A mixture of appropriate 3 (1 mmol) and anhydrous AlCl₃ (10 mmol) in dried dichloromethane (25 mL) was stirred at 0 °C for 0.5 h, and then heated to 45 °C overnight. The reaction was quenched with ice-cold water (20 mL), transferred to a separatory funnel and extracted the organics three times with ethyl acetate (3 × 30 mL). The combined organic layers were dried with anhydrous MgSO₄, filtered and solvent evaporated in vacuo to obtain the crude solid. The residue was purified by column chromatography. After hydrolysis reaction, the desired products 8a–f were obtained.

2-(2-(4-Hydroxystyryl)-1,1-dioxido-2H-benzo[e][1,2]thiazin-4-yl)acetic acid (8a). White solid (0.30 g, 86%); purity: 98.43%; mp: 282–284 °C; ¹H NMR (400 MHz, [D₆]DMSO): δ = 12.59 (s, 1H), 7.83 (m, 1H), 7.66–7.45 (m, 5H), 6.78 (m, 1H), 6.65 (m, 2H), 6.43 (s, 1H), 6.21 (s, 1H), 5.83 (s, 1H), 4.38 ppm (m, 2H); ¹³C NMR (100 MHz, [D₆]DMSO) δ = 171.16, 157.04, 140.71, 138.01, 136.91, 135.51, 134.31, 131.24, 127.53, 126.51, 124.07, 122.05, 123.01, 119.12, 118.94, 115.15, 110.81, 45.31 ppm.

2-(2-(4-Hydroxy-3-methoxystyryl)-1,1-dioxido-2H-benzo[e][1,2]-thiazin-4-yl)acetic acid (8b). Yellow solid (0.30 g, 79%); purity: 98.79%; mp: 315–317 °C; ¹H NMR (400 MHz, [D₆]DMSO): δ = 12.29 (s, 1H), 7.83 (m, 1H), 7.64–6.99 (m, 6H), 6.75 (m, 1H), 6.45 (s, 1H), 6.11 (s, 1H), 5.83 (s, 1H), 4.38 (m, 2H), 3.88 ppm (s, 3H); ¹³C NMR (100 MHz, [D₆]DMSO) δ = 171.16, 158.04, 157.17, 138.21, 136.01, 135.15, 134.13, 131.04, 127.13, 126.61, 124.17, 122.15, 123.01, 119.12, 118.94, 115.15, 110.81, 56.1, 45.31 ppm.

2-(2-(3,4-Dihydroxystyryl)-1,1-dioxido-2H-benzo[e][1,2]thiazin-4-yl)acetic acid (8c). Yellow solid (0.26 g, 71%); purity: 98.12%; mp: 305–306 °C; ¹H NMR (400 MHz, [D₆]DMSO): δ = 12.59 (s, 1H), 7.79 (m, 1H), 7.64–6.93 (m, 6H), 6.76 (m, 1H), 6.42 (s, 1H), 6.10 (s, 1H), 5.33 (s, 2H), 4.37 ppm (m, 2H); ¹³C NMR (100 MHz, [D₆]DMSO) δ = 171.16, 157.04, 156.17, 138.01, 136.91, 135.51, 134.31, 131.14, 127.53, 126.01, 124.38, 124.12, 123.01, 119.12, 118.94, 115.15, 110.81, 45.31 ppm.

2-(2-(3-Hydroxy-4,5-dimethoxystyryl)-1,1-dioxido-2H-benzo[e][1,2] thiazin-4-yl)acetic acid (8d). Yellow solid (0.31 g, 75%); purity: 99.33%; mp: 279–281 °C; ¹H NMR (400 MHz, [D₆]DMSO): δ = 12.39 (s, 1H), 7.81 (m, 1H), 7.69–6.91 (m, 5H), 6.68 (m, 1H), 6.43 (s, 1H), 6.15 (s, 1H), 5.35 (s, 1H), 4.37 (m, 2H), 3.83 ppm (m, 6H); ¹³C NMR (100 MHz, [D₆]DMSO) δ = 171.16, 158.14, 158.17, 157.01, 136.91, 135.51, 134.31, 131.24, 127.53, 126.51, 124.07, 122.05, 123.01, 119.12, 118.94, 115.15, 110.81, 58.02, 57.51, 45.31 ppm.

2-(2-(3,4-Dihydroxy-5-methoxystyryl)-1,1-dioxido-2H-benzo[e][1,2]thiazin-4-yl)acetic acid (8e). Yellow solid (0.29 g, 71%); purity: 98.27%; mp: 294–295 °C; ¹H NMR (400 MHz, [D₆]DMSO): δ = 12.69 (s, 1H), 7.84 (m, 1H), 7.69–7.45 (m, 3H), 6.78 (m, 1H), 6.72 (m, 2H), 6.45 (s, 1H), 6.11 (s, 1H), 5.35 (s, 2H), 4.37 (m, 2H), 3.83 ppm (m, 3H); ¹³C NMR (100 MHz, [D₆]DMSO) δ = 171.16, 158.14, 157.09, 157.01, 137.21, 136.31, 134.28, 131.31, 127.23, 126.11, 124.12, 123.05, 123.01, 119.02, 118.94, 115.15, 110.81, 57.51, 45.31 ppm.

2-(1,1-Dioxido-2-(3,4,5-trihydroxystyryl)-2H-benzo[e][1,2]thiazin-4-yl)acetic acid (8f). Yellow solid (0.30 g, 78%); purity: 97.25%; mp: 315–316 °C; ¹H NMR (400 MHz, [D₆]DMSO): δ = 12.39 (s, 1H), 7.82 (m, 1H), 7.66–7.40 (m, 3H), 6.79 (m, 1H), 6.69 (m, 2H), 6.41 (s, 1H), 6.09 (s, 1H), 5.35 (s, 9H), 4.32 ppm (m, 2H); ¹³C NMR (100 MHz, [D₆]DMSO) δ = 171.16, 157.24, 157.05, 157.03, 137.11, 136.21, 135.56, 133.12, 128.13, 127.11, 124.17, 124.12, 121.24, 119.12, 118.94, 115.15, 110.81, 44.98 ppm.

Biology methods

ALR2 and ALR1 were obtained from Wistar rats, 200–250 g, b.w., supplied by Vital River, Beijing, China. Sodium D-glucuronate, D,L-glyceraldehyde, and NADPH were from Sigma-Aldrich. All other chemicals were of reagent grade. ALR1 and ALR2 were prepared in accordance with the method of Kinoshita³⁴ and La Motta.²⁷ Enzyme activity was assayed spectrophotometrically on Unico 4802S UV/VIS double beam spectrophotometer by measuring the decrease in absorption of NADPH at 340 nm, which accompanies the oxidation of NADPH catalyzed by ALR2 and ALR1.

Enzyme assays

The ALR2 inhibition activity was tested in a reaction mixture containing 0.25 mL NADPH (0.10 mM), 0.25 mL sodium phosphate buffer (pH = 6.2, 0.1 M), 0.1 mL enzyme extract, 0.15 mL deionized water, and 0.25 mL D,L-glyceraldehyde (10 mM) as substrate in a final volume of 1 mL. Before adding to D,L-glyceraldehyde, the reaction mixture was incubated at 30 °C for 10 min, then the substrate was added to start the reaction, which was monitored for 5 min.

The ALR1 inhibition activity was performed at 36 °C in a reaction mixture containing 0.25 mL NADPH (0.12 mM), 0.1 mL enzyme extract, 0.25 mL sodium phosphate buffer (pH = 7.2, 0.1 M), 0.15 mL deionized water, and 0.25 mL sodium D-glucuronate (20 mM) as substrate in a final volume of 1 mL. Before adding to sodium D-glucuronate, the reaction mixture was incubated at 37 °C for 10 min, then the substrate was added to start the reaction, which was monitored for 5 min.

The inhibitory activity of the newly synthesized compounds against ALR2 and ALR1 was assayed by adding 5 μL 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 of the reaction mixture components except the substrate was subtracted from each experimental rate. The inhibitory effect of the synthetic compounds was routinely estimated at 10⁻⁴ M (the concentration is referenced to that of the compound in the reaction mixture). The compounds found to be active were tested at additional concentrations between 10⁻⁴ and 10⁻⁸ M, the log(dose)–response curves were then constructed from the inhibitory data, and the IC₅₀ values were calculated by least-square analysis of the linear portion of the log(dose) versus response curves (0.912 < r² < 0.996).

DPPH assay

In order to identify the radical scavenging activity of target compounds in a homogeneous system, a method based on the scavenging of the stable free radical DPPH was employed. A 100 μL of methanolic solution of various compounds with different concentrations was added to 1 mL DPPH methanolic solution (2.5 × 10⁻⁵ g mL⁻¹) and 1.9 mL methanol solution to give final concentrations of 0.1, 0.05, 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 (Trolox) were dissolved in methanol, and after 240 min (steady state), the percentage of DPPH radical scavenging was determined by the equation as shown below. The experiments were performed in triplicate.

A_control = DPPH + methanol

A_sample = tested compound + DPPH + methanol

A_blank = tested compound + methanol

Docking studies

Docking studies were performed using Molegro Virtual Docker, version 5.0. The crystal structure of human aldose reductase with bound inhibitor IDD594 retrieved from the RCSB Protein Data Bank (PDB code: 1US0). All solvent molecules within the protein structure were removed for the docking procedure. Four binding cavities were detected to get the potential binding site in the protein. The cavity around the anion binding site (volume of 141 ų) was chosen using the grid-based Mol-Dock score (GRID) function with a grid resolution of 0.30 Å. The best ligand pose was chosen according to the Mol-Dock score and ReRank score.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (grant no. 21272025 and grant no. 21572021), the Research Fund for the Doctoral Program of Higher Education of China (grant no. 20111101110042).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra25984c

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