Abstract:
This study was aimed at the synthesis of few xanthone derivatives containing 1,4-dioxane ring system. The reaction of 3,4-dihydroxy xanthone derivatives (3a-c) with ethylene dibromide and epichlorohydrin under approriate reaction conditions afforded xanthone derivatives containing 1,4-dioxane ring system (4a-e). The structures of the synthesized compounds were confirmed on the basis of their chemical and spectral data. This work was also extended to study the possible antihepatotoxic activity of the newly synthesized compounds against carbon tetrachloride (CCl
4)-induced hepatotoxicity in female Wistar albino rats by estimating the levels of biochemical parameters such as total proteins (TP), total albumin (TA), alkaline phosphatase (ALKP), serum glutamate oxaloacetate transaminase (SGOT) and serum glutamate pyruvate transaminase (SGPT). Compound
4b [3,4-(2′-hydroxy methyl-1′,4′-dioxano) xanthone] showed a potent antihepatotoxic activity comaparable to the standard reference drug Silybon-70, whereas other compounds exhibited moderate activity. It was also observed that the unsubstituted xanthone derivatives possessing 1,4-dioxane ring have better antihepatotoxic activity in comparision to nitro-and bromo-substituted xanthone derivatives.
Keywords: Antihepatotoxic activity; Xanthone; 1,4-Dioxane; Silymarin; Silybum marianum Original article
Synthesis and antihepatotoxic activity of some new xanthones containing
1, 4-dioxane ring system
Shah Alam Khan
1*and Bahar Ahmed
21
Department of Pharmacy, Oman Medical College, Muscat, Sultanate of Oman
2
Antihepatotoxic Research Laboratory, Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Jamia Hamdard, New Delhi-110062, India *Corresponding author: Tel: 00968-97245361 Fax: 00968-24504820 E-mail address: [email protected], [email protected]
Introduction
Liver disease is a leading cause of death in many countries which occurs due to alcohol consumption, malnutrition, continuous exposure to environmental pollutants, hepatotoxic drugs, chemicals toxins and infections [1]. It results in various disorders such as acute viral hepatitis, chronic viral hepatitis and liver cirrhosis.†The different medical, surgical and therapeutic methods available at present for the treatment of liver diseases are inadequate with generally poor results. Therefore, it is essential to search for newer drugs for the treatment of liver diseases. It is well known that some naturally occuring medicinal plants are potent hepatoprotective drugs and are widely used in alternative system of medicine through out the world to cure liver ailments [2]. Silymarin isolated from the seeds of Silybum marianum, commonly known as Milk thistle [3], has been used as a potent antihepatotoxic agent against a variety of toxicants in modern medicine [4]. Silymarin is a complex mixture of three flavolignan isomers namely, silybin, silydianin and silychristin [5]. Among the isomers,
silybin is the major and the most active component and represents about 60-70%, followed by silychristin (20%) and silydianin (10%) [6]. Silybin contains 1, 4-dioxane ring system in its structure, whereas the other isomers i.e. silychristin and silydianin do not possess 1, 4-dioxane ring and they do not display significant activity [7]. We, therefore, proposed that 1, 4-dioxane unit plays an important part in exhibiting antihepatotoxic activity and thus incorporated 1, 4-dioxane ring system in chalcones and various heterocyclic compounds like coumarins, flavones and oxadiazoles [7,8] and studied the pharmacological role of 1, 4-dioxane on the liver. We got the favourable results which further encouraged us to prepare some new heterocyclic chemical compounds containing 1, 4-dioxane ring system in search of effective and safer antihepatotoxic agents.
Xanthones (9-H-xanthene-9-ones) are heterocyclic compounds with the dibenzo-γ-pyrone framework which does not occur in nature [9]. However, a number of xanthone derivatives are secondary plant metabolites and have been isolated from natural sources [10-12].
Figure 1 Structures of a) silybin, b) silychristin and c) silydianin
O O O OCH3 OH CH2OH HO OH O OH O O OCH3 OH HO OH O OH CH2OH OH O HO OH O OH c) OH OCH3 O OH O a) b)
These oxygenated heterocyclic derivatives bear a structural relationship to flavonoids and coumarins and possess diverse pharmacological properties such as antioxidant [13], anti-inflammatory [14], antimicrobial [15], antifungal [16], diuretic and monoamine oxidase (MAO) inhibitory activity [17]. Literature survey reveals that xanthone derivatives have not been paid much attendtion for their antihepatotoxic activity though they have marked antioxidant activity. Hence the present study was undertaken to synthesize few compounds having different nature and positions of substituents like-NO2, -Br on the xanthonic nucleus as well as -CH
2OH side chain on postion-2 of dioxane ring and to study their antihepatotoxic potential. In this paper, we report the synthesis of five new xanthone derivatives containing 1, 4-dioxane ring system (4a-e) and their antihepatotoxic activity in carbon tetrachloride-intoxicated rats. The aspect of structure activity relationship (SAR) of the synthesized compounds is also tentatively discussed.
Materials and Methods
All chemicals were purchased from E. Merck (Germany) and S. D. Fine Chemicals (India) and were of analytical reagent grade. All the kits were the products of Biosystems (Spain) and Sigma Chemical Company (USA). Silybon-70 tablets (a standardized extract of silymarin) manufactured by Micro Labs India, were purchased from the local Pharmacy. All melting points were determined in open capillaries and were uncorrected. Purity of the compounds was checked on thin layer chromatography (Silica gel G) plates using iodine vapours as visualizing agent. The IR (KBr) spectra were recorded on a Hitachi IR- 270-300 spectrometer (cm-1). 1H NMR spectra were recorded on 300 MHz (Bruker model DRX-300 NMR spectrometer) in CDCl
3 and DMSO-d6 using TMS as an internal reference (chemical shift in
δ ppm) and mass spectra on Jeol JMS DX-303 spectrometer and are presented as m/z.
Synthesis of 3, 4-dihydroxy xanthone (3a) [18]
A mixture of POCl3 (30 mL) and fused ZnCl2 (13 g) were added to salicylic acid (1a) (4.14 g, 30 mmol) and pyragallol (2) (6.3 g, 50 mmol). It was then heated
over sand bath for 2.5 h at 70°C. After cooling it was poured in crushed ice where upon a brown solid appeared. It was purified by silica gel column chromatography using petroleum ether : ethyl acetate (4:1) as eluent to obtain the crystals of 3a; Rf: 0.81 (petroleum ether : ethyl acetate, 3:2); yield: 66%; m.p. 241-242°C (Ref [19] 238-240°C); IR (KBr): Vmax 3380 (OH), 1670 (C=O), 1491 (C=C), 1154, 1062 (C-O), 983, 759 and 628; 1H-NMR (CDCl3): δ 6.557 (d,1H, J=8.4 Hz, H-1), 6.68 (d, 1H, J=8.3 Hz, H-2), 7.0 (brm, 2H, H-7,8), 7.474 (brm, 2H, H-5,6), 10.19 (brs, 1H, OH), 10.32 (brs, 1H, OH); MS (70 ev): m/z 228 (M+, C 13H8O4) (7.4), 200 (2.3), 192 (5.5), 150 (5.7), 121 (100), 92 (16.2).
Synthesis of 5, 7 dinitro 3, 4-dihydroxy xanthone (3b)
It was prepared by the above procedure using 3, 5 dinitro salicylic acid (1b) (2.01, 10 mmol); Rf: 0.87 (benzene:ethyl acetate, 8:2); yield: 81%; m.p. 116-118° C; IR (KBr): Vmax 3300 (OH), 3040 (Ar-CH), 1740 (C=O), 1590, 1560, 1470, 1400 (NO
2), 1270, 1170, 860, 750, 640; 1H-NMR (CDCl3): δ 6.35(d, 2H, J=8.4Hz, H-1, 2), 6.89 (d, 1H, J=2.5 Hz, H-6), 7.01 (d, 1H, J=2.5 Hz, H-8), 9.60 (brs,1H, OH), 10.43 (brs,1H, OH).
Synthesis of 5, 7-dibromo 3,4-dihydroxy xanthone (3c)
It was prepared by the above procedure using 3,5 dibromo salicylic acid (1c) (3.16, 10 mmol); Rf: 0.71 (benzene:ethyl acetate, 8:2); yield: 76%; m.p. 185-187° C; IR (KBr): Vmax 3400 (OH), 3060 (Ar-CH), 1700 (C=O), 1630, 1590, 1560, 1500 (C=C), 1460, 1340, 1300, 1200, 1150, 1060, 940, 860, 760 (Br), 650 (Br); 1H-NMR (DMSO-d6): δ 6.37(d, 1H, J=8.4Hz, H-1), 6.91 (d, 1H, J=8.3 Hz, H-2), 7.75 (d, 1H, J=2.7 Hz, H-6), 7.89 (d, 1H, J=2.7 Hz, H-8), 8.72 (brs, 1H, OH), 9.60 (brs,1H, OH).
Synthesis of 3,4-(1′,4′-dioxano) xanthone (4a)
A solution of KOH (1.84 g, 33 mmol) in water (15 mL) was added to the mixture of 3a (2.28 g, 10 mmol) and 1,2-dibromo ethane (4.23 g, 22.5 mmol) in water (10 mL) with stirring. After 20 h at reflux, the solvent and excess of 1, 2-dibromo ethane were removed under
vacuum. The residue was taken up in chloroform and the insoluble material was filtered off, the organic layer was dried over anhydrous Na2SO4 and evaporated to get the yellowish solid, which was recrystallized from methanol; Rf: 0.47 (benzene: methanol, 4:1); yield: 33%; m.p. 197-198°C; IR (KBr): Vmax 2497 (CH2), 1654 (C=O), 1560, 1508 (C=C), 1260, 1190, 1082 (C-O), 898, 789, 621; 1H-NMR (DMSO-d6): δ 4.19 (m, 4H, 2 x CH2), 6.56 (d, 1H, J=8.4Hz, H-1), 6.69 (d, 1H, J=8.3 Hz, H-2), 7.02 (brm, 2H, H-7, 8), 7.57 (brm, 2H, H-5, 6); MS (70 eV): m/z 254 (M+ C15H10O4) (100), 226(4.5), 194 (9.6), 175 (18.1), 121 (84.2), 93 (20.1).
Synthesis of 3, 4-(2′-hydroxy methyl, 1′, 4′-dioxano) xanthone (4b)
2.28 g (10 mmol) of compound 3a was dissolved in an aqueous ethanol [30 mL of alcohol (95%) in 17.1 mL of water] containing sodium hydroxide (0.5 g). To this epichlorohydrin (8.0 mL, 9 mmol) was added and the resulting solution was heated under reflux at 75°C for about 2 h with stirring. The solution was then further stirred for 3 h at room temperature. Ice-cold water was added to the reaction mixture. The oily fraction that settled down at the bottom of the flask was separated from the aqueous layer and concentrated to get the semi-solid crude product; Rf: 0.63 (benzene: ethyl acetate, 9:1); yield: 47%; 1H-NMR (DMSO-d 6): δ 3.73 (ddd, 1H, J=2.1, 7.8, 2.4 Hz, Ha-3″), 3.76 (ddd, 1H, J=2.4, 7.9, 2.1 Hz, Hb-3″), 3.85 (brm, 1H, W2=5.2, -CH-CH2OH), 4.09 (ddd, 1H, J=2.3, 7.4, 2.1 Ha-CH2OH), 4.175 (ddd, 1H, J= 2.3, 9.0, 2.4 Hb-CH2OH), 6.50 (d, 1H, J=8.4Hz, H-1), 6.61 (d, 1H, J=8.0 Hz, H-3), 7.02 (brm, 2H, H-7,8), 7.28 (brm, 2H, H-5,6); MS (70 eV): m/z 284 (M+ C 16H12O5) (22.3), 256 (7.0), 254 (3.9), 226 (5.8), 205 (4.9), 121 (58.6), 93 (10.3).
Synthesis of 5, 7-dinitro 3, 4-(2′ hydroxy methyl-1′, 4′-dioxano) xanthone (4c)
This compound was synthesized by the usual method; Rf: 0.71 (benzene: ethyl acetate, 9:1); yield: 43.6%; IR (KBr): Vmax 3305 (OH), 1673 (C=O), 1508 (C=C), 1461 (NO 2), 1340, 1243, 1109, 966, 850, 789, 69; 1H-NMR (DMSO-d 6): δ 4.07 (dd, 2H, J=1.8, 7.2 Hz, 3′-CH2), 4.51 (dd, 2H, J=2.1, 8.4 Hz, CH2OH), 4.76 (brm, 1H, 2′-CH), 8.37 (brm, 2H, H-6, 8), 8.51 (d, 2H, J=8.4 Hz, H-1,2).
Synthesis of 5, 7-dibromo 3, 4-(1′, 4′-dioxano) xanthone (4d)
This compound was synthesized by the usual method; Rf: 0.72 (benzene: ethyl acetate, 9:1); yield: 39%; m.p. 111-112°C; 1H-NMR (DMSO-d6): δ 3.68 (m, 2H, 2′-CH2), 4.18 (m, 2H, 3′-CH2), 6.37 (d, 1H, J=8.4 Hz, H-1), 6.91 (d, 1H, J=8.3 Hz, H-2), 7.75 (d, 1H, J=2.7 Hz, H-6), 7.89 (d, 1H, J=2.7Hz, H-8); MS (70 eV): m/z 412 (M+ C15H8O4Br2) (3.6), 384 (4.1), 352 (6.3), 279 (5.8), 175 (14.1), 93 (15.3).
Synthesis of 5, 7-dibromo 3, 4-(2′ hydroxy methyl, 1, 4′-dioxano) xanthone (4e)
This compound was also synthesized by the usual method; Rf: 0.59 (benzene: ethyl acetate, 9:1); yield: 46%; IR (KBr): Vmax 3245 (OH), 1674 (C=O), 1571, 1510 (C=C), 1349, 1240, 1204, 1002 (C-O), 808, 761 (Br), 649; 1H-NMR (DMSO-d 6): δ 3.64 (d, 1H, W2=3.6Hz, 2′-CH), 3.95 (ddd, 2H, J=5.1,8.4, 4.5 Hz, -CH 2OH), 4.22 (ddd, 2H, J=7.3, 3.2, 7.0Hz, 3′-CH2), 6.50 (d, 1H, J=8.4Hz, H-1), 6.93 (d, 1H, J=8.7 Hz, H-2), 7.48 (d, 1H, J=2.7Hz, H-6), 7.92 (d, 1H, J=2.7Hz, H-8). Antihepatotoxic activity Experimental animals
The antihepatotoxic studies of compounds (4a-e) were carried out on the female Wistar albino rats (150-200 g). The study was approved by an institutional animal ethics committee and ethical norms were strictly followed during all the experimental procedures. The rats were bred in a colony in the Central Animal House of Jamia Hamdard. They were fed with a standard pellet diet (Gold Mohar, Lipton India Ltd., Calcutta) and water ad libitum. Before and during the experiment, the rats were kept in a standard environmental conditions (25-28°C and 12 h light/dark cycle).
Experimental design
The animals were divided into eight groups of five animals each in all the sets of experiments. Carbon
tetrachloride (CCl4) mixed with liquid paraffin (1:1) was used as a hepatotoxic agent. The drugs were administered for seven days after CCl4 administration, in the form of an aqueous suspension made from carboxymethyl cellulose as per the following treatment schedule.
Group I (normal control) was given only distilled water. Group II (toxic control) was treated with CCl4 (1.0 mL/kg) for the first day of study to produce toxicity in the liver. Group III (Silybon-70 treated) was given a single dose of CCl4 (1.0 mL/kg) on the first day and then Silybon-70, 10 mg/kg, daily) was given for seven days. Groups IV to VIII were administered with a single dose of CCl4 (1.0 mL/kg) on the first day followed by an oral treatment with a daily dose (10 mg/kg) of xanthones
4a-e respectively for seven days. On the last day, blood
was collected directly from the retro plexus orbital of four rats from each group and serum was separated for biochemical analysis.
Biochemical analysis
The biochemical parameters such as serum glutamate oxaloacetate transaminase (SGOT) [20], serum glutamate pyruvate transaminase (SGPT) [20], alkaline phosphatase (ALKP) [21], total proteins (TP), total albumin (TA) were analyzed according to the reported methods [22].
Statistical analysis
The results of the biochemical estimations were reported as mean ± S.E.M. The statistical analysis was done by one-way analysis of variance (ANOVA) followed by Studentûs ùtû test by using 13th version of SPSS software.
Results and Discussion
The results of the synthesized compounds (Scheme 1) and their pharmacological screening have been summarized in Table 1. The compounds were obtained in average yield and were characterized by their spectral and chemical data. Single dose of CCl4 (1 mL/kg) significantly elevated the SGPT, SGOT and ALKP activities (71.52, 59.40 and 46.34 units/mL) when compared to the normal animals (36.28, 30.30 and 15.80 units/mL) respectively indicating hepatocellular damage. Treatment of the rats with the compounds under investigation
have decreased the enzyme levels in the range of 58.90-66.80 units/mL for SGOT, 47.24-54.20 units/mL for SGPT and 35.92-39.70 units/mL for ALKP, which were found to be comparable to the enzyme levels (SGOT, SGPT, ALKP) reduced by standard drug Silybon-70 (53.84, 45.80 and 28.88 units/mL, respectively). Silybon-70, a standardized extract of silymarin, is a well established hepatoprotective drug capable of decreasing the elevated levels of liver enzymes in various drug induced hepatotoxicity [23]. The most potent compound which significantly reversed the hepatotoxicity and exhibited almost similar activity comparable to standard drug Silybon-70 was found to be the compound
4b (58.90, 47.20 and 35.92 units/mL, respectively).
Other compounds also exhibited a moderate activity (61.04-66.80 units/mL, 50.68-54.20 units/mL and 37.40-39.70 units/mL, respectively).
The toxicant CCl4 also reduced the level of total protein (4.31 g/dL) and increased the level of total albumin (4.30 g/dL) in comparison to normal values (5.32 g/dL, 3.32 g/dL, respectively). The administration of test compounds enhanced the reduced level of total protein in the range of 4.49-5.15 g/dL, and decreased the elevated values of total albumin in the range of 3.75-4.00 g/dL in comparison to standard drug Silybon-70 (6.22 and 3.76 g/dL, respectively). The most potent compound 4b i. e., 3, 4 (2′-hydroxy methyl 1′, 4′-dioxano) xanthone (5.15 and 3.75 g/dL, respectively) displayed these values comparable to standard drug Silybon-70, whereas other derivatives showed inferior results.
It was also observed that compound 4b possess 2-hydroxy methyl group at position-2 of the dioxane ring of xanthone derivative, which also suggests that the presence of hydroxy methyl group at position-2 in dioxane ring might play a significant role in exhibiting the antihepatotoxic activity. This is in accordance with the view that silybin too possess the same group at the same position which might be responsible for its better absorption and bioavailability.
The substitution in the aromatic ring of xanthones has no significant role in exhibiting antihepatotoxic activity. However, it was observed that the unsubstituted xanthone derivatives have better activity in comparision to
nitro- and bromo-substituted xanthones. Thus it could be inferred that introduction of electron withdrawing groups like -NO2 or -Br on ring B of xanthone nucleus decreases the antihepatotoxic activity.
S.No. R 1 R2 X 4a H H H 4b H H CH2OH 4c NO2 NO2 CH2OH 4d Br Br H 4e Br Br CH 2OH
Scheme 1 Synthesis of xanthone derivatives
Conclusion
In the present study, administration of xanthone derivatives containing 1, 4-dioxane ring system significantly protected against CCl4 induced hepatotoxiciy
OH COOH R1 R2 + HO OH OH 1a:R1=R2=H 1b:R1=R2=NO2 1c:R1=R2=Br (i) POCl3 (ii) ZnCl2 (iii) 75 ÌC, 2.5 h O O OH R1 R2 OH 3a:R1=R2=H 3b:R1=R2=NO2 3c:R1=R2=Br
(i) Br-CH2CH2Br/KOH, H2O,2 h (ii) CH2 CH-CH2-Cl
O
(a) NaoH/aq EtOH/75 ÌC, 2h O O R1 R2 O O1' X 1 4 4' A B 2 3 5 7 2
Table 1 Biochemical parameters of synthesized compounds in CCl
4 induced hepatotoxicity in albino rats
TA (g/dL) TP (g/dL) ALKP SGPT SGOT Dose Treatment Groups (units/mL) (units/mL) (units/mL) (mg/kg/b.wt)
3.32 ± 0.17 5.32 ± 0.16 15.80 ± 0.37 30.30 ± 0.19 36.28 ± 1.19 ---- Normal I 4.30 ± 0.08 4.31 ± 0.48 46.34 ± 1.04 59.40 ± 0.36 71.52 ± 1.36 1 mL/kg Toxic II 3.76 ± 0.13* 6.22 ± 0.19* 28.88 ± 0.23* 45.80 ± 0.62* 53.84 ± 0.65* 10 Silybon-70 III 3.95 ± 0.08 4.93 ± 0.05* 38.56 ± 0.29* 51.40 ± 0.55 61.51 ± 0.32 10 Xanthone-4a IV 3.75 ± 0.03* 5.15 ± 0.05* 35.92 ± 0.91* 47.24 ± 0.47* 58.90 ± 0.35* 10 Xanthone-4b V 3.89 ± 0.03* 4.95 ± 0.08* 38.38 ± 0.29* 50.68 ± 0.33* 65.26 ± 1.01* 10 Xanthone-4c VI 4.00 ± 0.07* 4.75 ± 0.08* 39.70 ± 0.21* 54.20 ± 0.46 66.80 ± 0.80* 10 Xanthone-4d VII 3.94 ± 0.05* 4.49 ± 0.07 37.40 ± 0.18* 50.90 ± 0.39* 61.04 ± 0.37* 10 Xanthone-4e VIII
Values are Mean ± S.E.M. (n=5 animal per group)
SGOT=Serum glutamic oxaloacetic acid transaminase; SGPT=Serum gluatamic pyruvic transaminase; ALKP=Alkaline phosphatase; TP = Total protein; TA=Total albumin
in rats. Compound 4b having a hydroxymethyl group on the 1, 4-dioxane ring was found to be the most potent antihepatotoxic agent among the synthesized compounds. The exact mechanism by which it protects the liver is unknown however the effect could be due to the significant antioxidant activity of xanthones [13] and due to the presence of 1, 4-dioxane ring. Further detailed studies are needed to ascertain their exact mechanism of action.
Acknowledgements
Authors are thankful to the Head, Department of Pharmaceutical Chemistry for providing the necessary research facilities. Dr. Bahar Ahmed (Principal Investigator) is also thankful to CSIR, New Delhi for financing the research project along with a post of SRF, which was awarded to SAK through a selection committee.
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