USE OF 3-ARYLPHTHALIDES AND DERIVATIVES THEREOF AS ANTI-INFLAMMATORY AGENTS

Abstract

The present invention relates to the use of 3-arylphthalides and derivatives thereof as anti-inflammatory agents. The invention relates to the use of 3-arylphthalides, in particular 3-(4,5-dihydroxy-2-(2-5 ethylthio)ethylphenyl)phthalide and 3-(2,4-dihydroxyphenyl)phthalide, as anti-inflammatory and chemopreventive agents in inflammatory processes in general, in diseases caused by underlying inflammation processes and in inflammatory processes related to the development of diabetes mellitus and its complications.

Claims

1. A compound with general formula (I): ##STR00004## wherein: R.sub.1 to R.sub.3 are selected from the list comprising hydrogen, fluorine, chlorine, bromine, iodine, a (C.sub.1-C.sub.4) alkyl group, hydroxyl, or sulfide (RSR, RC.sub.2-C.sub.4, RC.sub.1-C.sub.4), or of any of the pharmaceutically acceptable derivatives or stereoisomers thereof, for use in the prevention, alleviation, improvement, and/or treatment of an inflammatory disease.

2. The compound for use according to the preceding claim, wherein: R.sub.1 is a (2-ethylthio)ethyl group R.sub.2 and R.sub.3 are hydroxyl groups referred to as 3-(4,5-dihydroxy-2-(2-ethylthio)ethylphenyl)phthalide, with the chemical structure of formula (II): ##STR00005##

3. The compound for use according to claim 1, wherein: R.sub.1, R.sub.2 are hydroxyl groups R.sub.3 is hydrogen referred to as 3-(2,4-dihydroxyphenyl)phthalide, with the chemical structure of formula (III): ##STR00006##

4. The compound for use according to any of claims 1-3, wherein the inflammatory disease is selected from the list consisting of: diabetes mellitus, insulitis, diabetic retinopathy, diabetic nephropathy, or any of the combinations thereof.

5. A pharmaceutical composition comprising a compound as described in any of claims 1-3, for use as described in any of claims 1-4.

6. The pharmaceutical composition for use according to the preceding claim, further comprising another active ingredient.

7. A compound of formula (II): ##STR00007## or any of the pharmaceutically acceptable derivatives or stereoisomers thereof.

8. A composition comprising the compound of formula (II), or any of the pharmaceutically acceptable derivatives or stereoisomers thereof.

9. The pharmaceutical composition according to claim 8, comprising the compound of formula (II) as the only active ingredient.

10. The composition according to any of claims 8-9, for use as a medicinal product.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0025] The figures illustrate the synthesis and characterization of the compound of formula II, referred to as 3-(4,5-dihydroxy-2-(2-ethylthio)ethylphenyl)phthalide and the compound of formula III, referred to as 3-(2,4-dihydroxyphenyl)phthalide.

[0026] FIG. 1 illustrates radical bromination of phthalide (step 1) and obtaining 3-hydroxyphthalide (step 2).

[0027] FIG. 2 illustrates bromination of 2-(3,4-dimethoxyphenyl)ethanol (step 3), obtaining 4-(2-(ethylthio)ethyl)benzene-1,2-diol (step 4), and the reaction of 4-(2-(ethylthio)ethyl)benzene-1,2-diol with 3-hydroxyphthalide (step 5) for obtaining compound II.

[0028] FIG. 3 illustrates the reaction of resorcinol with 3-hydroxyphthalide (step 6) for obtaining compound III.

PREFERRED EMBODIMENT OF THE INVENTION

Synthesis of Compounds II and III

[0029] This example illustrates the synthesis and characterization of the compound of formula II, referred to as 3-(4,5-dihydroxy-2-(2-ethylthio)ethylphenyl)phthalide, and the compound of formula III, referred to as 3-(2,4-dihydroxyphenyl)phthalide, and their anti-inflammatory activities.

Materials and Methods

[0030] Column chromatography was performed with Merck silica gel (70-230 m). HPLC separations were performed in LaChrom-Hitachi equipment with LiChrospher Si-60 columns (Merck) and an L-7400 UV detector. The solvents used were of HPLC quality. NMR spectra were recorded in an Agilent 400 spectrometer using CDCl.sub.3, CD.sub.3OD, and CD.sub.3COCD.sub.3 as solvents. Chemical shifts were referenced with respect to the signal of the solvent (.sub.H 7.26 and .sub.C 77.0 for CDCl.sub.3, .sub.H 3.30 and .sub.C 49.0 for CD.sub.3OD, and .sub.H 2.04 and .sub.C 29.8 and 202.0 for CD.sub.3COCD.sup.3). COSY, HSQC, HMBC, and NOESY spectra were carried out using Agilent standard pulse sequences.

Synthesis

[0031] The synthesis of compounds of formula II and III is established through an electrophilic aromatic substitution of a phthalide derivative suitably functionalized with aromatic compounds 4-(2-(ethylthio)ethyl)benzene-1,2-diol and resorcinol (benzene-1,3-diol), respectively.

[0032] The synthesis method starts with the commercial compound phthalide as the starting product. Phthalide has a benzylic position (C-3) that can be functionalized by means of a radical bromination process which leads to obtaining phthalide 3-bromoderivative. Suitable treatment of this bromoderivative in an alkaline medium leads to obtaining 3-hydroxyphthalide which, in acidic conditions, produces a phthalidyl ion that acts as an electrophile in an electrophilic aromatic substitution reaction with the corresponding aromatic 4-(2-(ethylthio)ethyl)benzene-1,2-diol derivatives or resorcinol. Under these conditions, compounds II and III are obtained with a good yield and purified by means of silica gel column chromatography and HPLC. This method can be described with high-performance selective transformations as shown in the following schemes of synthesis:

A) Obtaining 3-Hydroxyphthalide (FIG. 1)

Step 1: Radical Bromination of Phthalide

[0033] Phthalide (1.02 g, 7.57 mmol), NBS (1.52 g, 8.56 mmol), and benzoyl peroxide as a catalyst (51 mg, 0.19 mmol) are introduced in a 50 mL flask dissolved in 25 mL of CCL.sub.4. The reaction is left under reflux for 4 h and after this time the reaction is filtered and the solvent is evaporated at reduced pressure. The residue is then dissolved in 20 mL of water and extracted with CH.sub.2Cl.sub.2 (320 mL). The organic phases are combined, dried on anhydrous MgSO.sub.4, and the solvent is evaporated at reduced pressure, obtaining 1.60 g of 3-bromophthalide (7.52 mmol, quantitative).

[0034] 3-bromophthalide characterization: .sup.1H-NMR (CDCl.sub.3, 399.945 MHz): 7.94 (1H, bd, 7.5 Hz, H-7), 7.78 (1H, ddd, 7.3, 7.0, 1.1 Hz, H-6), 7.64 (1H, bd, 7.5 Hz, H-4), 7.62 (1H, bd, 8.1 Hz, H-5), 7.40 (1H, s, H-3). .sup.13C-NMR (CDCl.sub.3, 100.576 MHz): 167.3 (s, C-1), 148.8 (s, C-3a), 135.2 (d, C-5), 130.9 (d, C.sub.0-6), 125.9 (d, C-7), 124.1 (s, C-7a), 123.5 (d, C-4), 74.6 (d, C-3).

Step 2: Obtaining 3-hydroxyphthalide

[0035] A solution of 3-bromophthalide (500 mg, 2.35 mmol) in 25 mL of distilled H.sub.2O is treated with 200 mg of 85% KOH (3.5 mmol) and the mixture is heated under reflux for two hours. After this time, the reaction is left to reach room temperature and treated with KHSO.sub.4 (170 mg). The obtained solution is extracted with AcOEt (325 mL). The combined organic phases are dried on anhydrous MgSO.sub.4 and the solvent is evaporated at reduced pressure obtaining 409 mg of a yellow oil. This oil is purified by means of a silica gel chromatographic column (1.517 cm, hexane/AcOEt (6:4)), obtaining 304 mg of 3-hydroxyphthalide (2.02 mmol, Y=86%).

[0036] 3-hydroxyphthalide characterization: .sup.1H-NMR (CD.sub.3COCD.sub.3, 399.945 MHz): 7.84 (1H, bd, 8.0 Hz, H-7), 7.80 (1H, ddd, 7.5, 7.4, 1.0 Hz, H-5), 7.72 (1H, bd, 7.7 Hz, H-4), 7.66 (1H, bdd, 7.4, 7.4 Hz, H-6), 6.75 (1H, s, H-3). .sup.13C-NMR (CD.sub.3COCD.sub.3, 100.576 MHz): 169.1 (s, C-1), 147.9 (s, C-3a), 135.0 (d, C-5), 131.3 (d, C-6), 127.7 (s, C-7a), 125.3 (d, C-1), 124.3 (d, C-4), d 98.7 (d, C-3).

B) Obtaining Compound II (FIG. 2)

Step 3: Bromination of 2-(3,4-dimethoxyphenyl)ethanol

[0037] 2-(3,4-dimethoxyphenyl)ethanol (0.6 g, 3.30 mmol) and PPh.sub.3 (1.22 g, 4.66 mmol) dissolved in 25 mL of CH.sub.2Cl.sub.2 are introduced in a 50 mL flask cooled in an ice bath. CBr.sub.4 (1.17 g, 3.52 mmol) is then added and after 5 minutes the reaction is left to react at room temperature until the starting product disappears. The reaction mixture is vacuum-concentrated and the product is purified by means of silica gel column chromatography (322 cm, hexane/AcOEt (7:3)), obtaining 2-(3,4-dimethoxyphenyl)ethyl bromide in a quantitative manner.

[0038] 2-(3,4-dimethoxyphenyl)ethyl bromide characterization: 1H-NMR (CDCl.sub.3, 399.945 MHz): 6.82 (1H, bd, 8.0 Hz, H-5), 6.76 (1H, dd, 8.2, 1.9 Hz, H-6), 6.72 (1H, bd, 1.9 Hz, H-2), 3.88 (3H, s, OMe), 3.86 (3H, s, OMe), 3.55 (2H, t, 7.8 Hz, H-1), 3.10 (2H, t, 7.4 Hz, H-2). .sup.13C-NMR (CDCl.sub.3, 100.576 MHz): 148.9 (s, C-3), 148.0 (s, C-4), 131.5 (s, C-1), 120.6 (d, C-6), 111.9 (d, C-2), 111.3 (d, C-5), 39.1 (t, C-2), 33.2 (t, C-1).

Step 4: Obtaining 4-(2-(ethylthio)ethyl)benzene-1,2-diol

[0039] NaEtS (342 mg, 4.07 mmol) is added under inert atmosphere to a solution of 2-(3,4-dimethoxyphenyl)ethyl bromide (98 mg, 0.40 mmol) in 2 mL of dimethylformamide. The reaction mixture is brought to reflux for 3 h and after this time the solution is cooled to 0 C., treated with a 5% HCl solution, and extracted with AcOEt (35 mL). The combined organic phases are dried on anhydrous MgSO.sub.4 and evaporation is performed under reduced pressure, leading to a residue which is purified by means of silica gel column chromatography (1.517 cm, hexane/AcOEt (8:2)), obtaining 63 mg of the product 4-(2-(ethylthio)ethyl)benzene-1,2-diol (0.32 mmol, Y=80%).

[0040] 4-(2-(ethylthio)ethyl)benzene-1,2-diol characterization: .sup.1H-NMR (CDCl.sub.3, 399.945 MHz): 679 (1H, d, 8.4 Hz, H-6), 6.73 (1H, d, 1.7 Hz, H-3), 6.63 (1H, dd, 80, 1.9 Hz, H-5), 2.76-2.72 (4H, m, H-1 and H-2), 2.57 (2H, c, 7.2 Hz, CH.sub.2CH.sub.3), 1.25 (3H, t, 7.6 Hz, CH.sub.2CH.sub.3). .sup.13C-NMR (CDCl.sub.3, 100.576 MHz): 143.6 (s, C-3), 142.0 (s, C-4), 133.7 (s, C-1), 120.8 (d, C-6), 115.6 (d, C-5), 115.4 (d, C-2), 35.5 (t, C-2), 33.3 (t, C-1), 26.0 (t, CH.sub.2CH.sub.3), 14.7 (c, CH.sub.2CH.sub.3).

Step 5: Reacting 4-(2-(ethylthio)ethyl)benzene-1,2-diol with 3-hydroxyphthalide (Obtaining Compound II)

[0041] 5 mL of a solution of H.sub.2SO.sub.4/H.sub.2O (1:1) are added to a flask with 100 mg (0.67 mmol) of 3-hydroxyphthalide and left under stirring for 10. 4-(2-(methylthio)ethyl)benzene-1,2-diol (196 mg, 0.99 mmol) is then added and left under stirring until the reaction monitoring by means of thin layer chromatography indicates the disappearance of 3-hydroxyphthalide. The reaction is neutralized with NaOH and the solution is extracted with CHCl.sub.3 (310 mL). The organic phases are dried on anhydrous MgSO.sub.4 and the solvent is evaporated at reduced pressure. The reaction crude is purified by means of a chromatographic column (217 cm, hexane/AcOEt (6:4)), obtaining 130 mg of compound II (0.39 mmol, Y=60%).

[0042] 3-(4,5-dihydroxy-2-(2-ethylthio)ethylphenyl)phthalide characterization: .sup.1H-NMR (CD.sub.3OD, 399.945 MHz): 7.92 (1H, d, 7.6 Hz, H-7), 7.75 (1H, ddd, 7.8, 7.8, 1.0 Hz, H-5), 7.63 (1H, dd, 7.8, 7.8z, H-6), 7.41 (1H, dd, 7.8, 0.8 Hz, H-4), 6.78 (1H, s, H-3), 6.74 (1H, s, H-3), 6.12 (1H, s, H-6), 3.00-2.84 (2H, m, ArCH.sub.2CH.sub.2S), 2.84-2.72 (2H, m, ArCH.sub.2CH.sub.2S), 2.54 (2H, c, 7.4 Hz, CH.sub.2CH.sub.3), 1.23 (3H, t, 7.4 Hz, CH.sub.2CH.sub.3). .sup.13C-NMR (CD.sub.3OD, 100.576 MHz): 172.8 (s, C-1), 151.7 (s, C-3a), 147.7 (s, C-4), 145.2 (s, C-5), 135.7 (d, C-5), 133.9 (s, C-1), 130.5 (d, C-6), 127.6 (s, C-7a), 126.1 (d, C-7), 126.0 (s, C-2), 124.7 (d, C-4), 118.4 (d, C-3), 115.7 (d, C-6), 82.0 (d, C-3), 34.6 (t, ArCH.sub.2CH.sub.2S), 33.6 (t, ArCH.sub.2CH.sub.2S), 26.9 (t, CH.sub.2CH.sub.3), 15.2 (c, CH.sub.2CH.sub.3).

C) Obtaining Compound III (FIG. 3)

Step 6: Reacting Resorcinol with 3-hydroxyphthalide (Obtaining Compound III)

[0043] 4 mL of H.sub.2O and 1 mL of dioxane are added to a flask with 100 mg (0.67 mmol) of 3-hydroxyphthalide. 250 l of 37% HCl are then added and left under stirring for 5 minutes. After this time, resorcinol (115 mg, 1.05 mmol) is added and left under stirring at room temperature until 3-hydroxyphthalide disappears. The reaction is neutralized with NaHCO.sub.3 (500 mg) and the solution is extracted with AcOEt (315 mL). The organic phases are dried on anhydrous MgSO.sub.4 and the solvent is evaporated at reduced pressure. The reaction crude is purified by means of a chromatographic column (216 cm, hexane/AcOEt (6:4)), obtaining 162 mg of 3-(2,4-dihydroxyphenyl)phthalide (0.67 mmol, Y=100%).

[0044] 3-(2,4-dihydroxyphenyl)phthalide characterization: .sup.1H-NMR (CD.sub.3COCD.sub.3, 399.945 MHz): 7.86 (1H, d, 7.8 Hz, H-7), 7.72 (1H, ddd, 7.4, 7.4, 1.2 Hz, H-5), 7.59 (1H, dd, 7.4, 7.4 Hz, H-6), 7.49 (1H, d, 7.8 Hz, H-4), 6.78 (1H, s, H-3), 6.76 (1H, d, 8.4 Hz, H-6), 6.48 (1H, d, 2.4 Hz, H-3), 6.31 (1H, dd, 8.4, 2.4 Hz, H-5). .sup.13C-NMR (CD.sub.3COCD.sub.3, 100.576 MHz): 171.1 (s, C-1), 160.2 (s, C-4), 157.7 (s, C-2), 151.8 (s, C-3a), 134.9 (d, C-5), 129.8 (d, C-6), 128.8 (d, C-6), 127.2 (s, C-7a), 124.8 (d, C-7), 123.1 (d, C-4), 1155 (s, C-1), 108.0 (d, C-5), 103.8 (d, C-3), 78.5 (d, C-3).

Anti-Inflammatory Activity in Immune Cell Lines Organospecific for Type 1 Diabetes Mellitus

[0045] Anti-inflammatory activity was measured on Bv.2 microglial cell line (innate immune system of the central nervous system), macrophages, Raw 264.7 cell line (peripheral immune system), renal tubular cell lines, MCT (renal tubule epithelials) and pancreatic beta cells, INS-1 (insulin-producing cells targeted in the process leading to diabetes mellitus). The cell lines were treated increasing doses of compound II or compound III for 24 h until the maximum concentration of 25 M for compound II and 50 M for compound III, where no cytotoxic effect was detected in any of the treated cell lines. The most potent anti-inflammatory effect was determined at the dose of 10 M in the presence of stimulation with a bacterial lipopolysaccharide (LPS) conventionally used as an inflammation inducer in immune cell lines (Bv.2 and Raw 264.7) at a concentration of 2 g.Math.mL.sup.1, or with a proinflammatory cytokine cocktail (TNF, IL1, IFN, and TFG) at the concentration of 10 ng.Math.mL.sup.1 in epithelial cell lines (MCTs and INS-1). The activation of the inflammatory process induces the secretion of nitrites (NO.sub.2) into the medium, so the induction and modulation of the inflammatory response by 3-(4,5-dihydroxy-2-(2-ethylthio)ethylphenyl)phthalide and 3-(2,4-dihydroxyphenyl)phthalide can be determined by means of the Greiss colorimetric technique.

[0046] Tables 1 and 2 show the results of the nitrite production inhibitory activity caused by compounds II and III (10 M), respectively, on LPS-stimulated Bv2 and Raw264.7 cell lines and on cytokine-stimulated MCT and INS-1 cell lines.

TABLE-US-00001 TABLE 1 Nitrite production inhibition caused by 3-(4,5-dihydroxy- 2-(2-ethylthio)ethylphenyl)phthalide (compound II). LPS or % of cytokines + reduction LPS or Compound in nitrite Baseline cytokines II (10 M) production Bv.2 1.00 0.01 3.83 0.19 1.98 0.203 48.31% Raw 264.7 1.00 0.02 6.87 0.249 1.38 0.43 79.88% MCTs 1.00 0.01 8.096 0.18 1.788 0.29 77.91% INS-1 1 0.25 4.489 0.03 3.644 0.004 18.82%

TABLE-US-00002 TABLE 2 Nitrite production inhibition caused by 3-(2,4- dihydroxyphenyl)phthalide (compound III). LPS or cytokines + % of reduction LPS or Compound in nitrite Baseline cytokines III (10 M) production Bv.2 1.00 0.01 9.37 0.92 3.47 0.27 62.92% Raw 264.7 1.00 0.01 5.41 0.39 3.60 0.42 33.38% MCTs 1.00 0.01 9.11 0.71 1.53 0.06 83.14% INS-1 0.99 0.04 4.29 0.04 1.48 0.02 65.32%

[0047] In the presence of compound II (10 M), nitrite production in LPS-stimulated Bv.2 and Raw cells decreased by 48.31% and 79.88%, respectively, and nitrite production in cytokine-stimulated MCT and INS-1 cells decreased by 77.91% and 18.82%, respectively.

[0048] In the presence of compound III (10 M), nitrite production in LPS-stimulated Bv.2 and Raw cells decreased by 62.92% and 33.38%, respectively. In the presence of compound III (10 M), nitrite production in cytokine-stimulated MCT and INS-1 cells decreased by 83.14% and 65.32%, respectively.

[0049] Therefore, compounds II and III exhibit a high in vitro anti-inflammatory activity in different cell types both of epithelial and immune origins.

Anti-Inflammatory Activity in Organotypic Cultures or Retinal and Kidney Explants from an Animal Model with Type 1 or Autoimmune Diabetes Mellitus

[0050] Anti-inflammatory activity was determined using organotypic cultures or retinal and kidney explants from a murine model which develops type 1 or autoimmune diabetes mellitus spontaneously, i.e., BioBreeding (BB) rat. This animal model of type 1 diabetes mellitus reproduces disease progression in a manner similar to that occurring in patients. Tissues, retina, and kidney are extracted, maintaining cytoarchitecture and tissue connections of each of the organs, from BB rats that are 7 weeks old, which is the time before the presence of constant hyperglycemia, which appears around weeks 9-11 of life. A prediabetic model with active inflammatory parameters that resemble those occurring during the progression of type 1 diabetes mellitus in patients is thereby obtained. The cultured explants were treated with 3-(2,4-dihydroxyphenyl)phthalide (compound III) at a dose of 20 M for 24 h.

[0051] Activation of the inflammatory process induces nitric oxide synthase, Nos2, gene expression, with messenger RNA quantification being determined by means of the quantitative PCR technique (qPCR), and can show inflammatory response modulation in retinal and kidney explants from an animal model with type 1 or autoimmune diabetes mellitus.

[0052] Table 3 shows Nos2 expression inhibition results observed by means of treatment with 3-(2,4-dihydroxyphenyl)phthalide.

TABLE-US-00003 TABLE 3 Nos2 expression inhibition caused by 3-(2,4-dihydroxyphenyl)phthalide. BB rat + 3-(2,4-dihydroxy % of reduction phenyl)phthalide of Nos2 BB rat (20 M) expression Retinal explant 1.00 0.01 0.482 0.05 51.8% Kidney explant 1.00 0.01 0.348 0.05 65.2%