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WO2004039771A1 - Diallyldisulphide compounds having antilipidemic and antioxidant activity - Google Patents

Diallyldisulphide compounds having antilipidemic and antioxidant activity Download PDF

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Publication number
WO2004039771A1
WO2004039771A1 PCT/IN2003/000250 IN0300250W WO2004039771A1 WO 2004039771 A1 WO2004039771 A1 WO 2004039771A1 IN 0300250 W IN0300250 W IN 0300250W WO 2004039771 A1 WO2004039771 A1 WO 2004039771A1
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Prior art keywords
diallyldisulphide
pharmaceutically acceptable
formula
cooh
compound
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Manisha Tiwari
Meenakshi Sharma
Ramesh Chandra
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Department of Science and Technology of Ministry of Science and Technology India
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Department of Science and Technology of Ministry of Science and Technology India
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C323/00—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups
    • C07C323/10—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and singly-bound oxygen atoms bound to the same carbon skeleton
    • C07C323/11—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and singly-bound oxygen atoms bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton
    • C07C323/16—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and singly-bound oxygen atoms bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton containing six-membered aromatic rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C321/00—Thiols, sulfides, hydropolysulfides or polysulfides
    • C07C321/12—Sulfides, hydropolysulfides, or polysulfides having thio groups bound to acyclic carbon atoms
    • C07C321/20—Sulfides, hydropolysulfides, or polysulfides having thio groups bound to acyclic carbon atoms of an unsaturated carbon skeleton containing rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C323/00—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups
    • C07C323/01—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and halogen atoms, or nitro or nitroso groups bound to the same carbon skeleton
    • C07C323/02—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and halogen atoms, or nitro or nitroso groups bound to the same carbon skeleton having sulfur atoms of thio groups bound to acyclic carbon atoms of the carbon skeleton
    • C07C323/07—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and halogen atoms, or nitro or nitroso groups bound to the same carbon skeleton having sulfur atoms of thio groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton containing six-membered aromatic rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C323/00—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups
    • C07C323/23—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and nitrogen atoms, not being part of nitro or nitroso groups, bound to the same carbon skeleton
    • C07C323/24—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and nitrogen atoms, not being part of nitro or nitroso groups, bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton
    • C07C323/29—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and nitrogen atoms, not being part of nitro or nitroso groups, bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton containing six-membered aromatic rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C323/00—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups
    • C07C323/50—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton
    • C07C323/51—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton
    • C07C323/56—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton containing six-membered aromatic rings

Definitions

  • the present invention relates to a novel diallyldisulphide analogues of general formula I exhibiting antilipidemic and antioxidant activity and pharmaceutical compositions containing these analogues.
  • the invention also provides a process of preparation of such analogues and the pharmaceutical composition containing such analogues.
  • the novel diallyldisulphide compound is represented by
  • R C 6 H
  • X H, CI, Br, F, CF 3 , CH 3 , CN,
  • Garlic is known for its medicinal and nutritional values for a very long time. Among the various potential therapeutic applications of garlic, its antilipidemic effects has been well demonstrated in studies of humans and animals [C.C.Gardner et.al, Atherosclerosis 154, 213-220, (2001); Liu et.al, Lipids 35, 197-203, (2000) and L.A. Simons et.al, Atherosclerosis 113, 219-225, (1995)]. Allicin (diallylthiosulfinate) is reported to be one of the most active constituents of garlic. Due to its unstable nature, allicin disallows its chemical modification to achieve a potent analogues.
  • Allicin readily undergoes transformation to a volatile diallyldisulphide.
  • the precursor of allicin, i.e. Alliin is known to be effective in treating hepatic diseases and fatigue. [Nagai Katsuji, Pat No. JP 56127312].
  • the main object of the invention is to provide a novel diallyldisulphide analogues exhibiting antilipidemic and antioxidant activity.
  • Another object is to provide pharmaceutical compositions containing novel diallyldisulphide compounds.
  • Yet another object of the invention is to provide an effective and economical process for the preparation of novel diallyldisulphide compounds.
  • the present invention relates to novel diallyldisulphide analogues of general formula (I), exhibiting antilipidemic and antioxidant activity.
  • the invention relates to novel active diallyldisulphide analogues which are represented by general formula I as shown hereinbelow:
  • diallyldisulphide derivatives are:
  • R C 6 H 5 ; CeH-NO ⁇ ); C 6 H 3 (NO 2 ) 2 (2,4); C-H-COOH ⁇ ); C 6 H 4 CN(4); C 6 H 3 (COOH) 2 (2,4); C 6 H 3 (CN) 2 (2,4); C 6 H 2 (NO 2 ) 3 (2,4,6); C 6 H 3 NO 2 (4)COOH(2);
  • the novel diallyldisulphide compound and its analogues are found to be less toxic than known drugs and exhibit activity equipment to or greater than known drugs.
  • the process for the preparation of diallyldisulphide compounds comprises the steps of:
  • step (c) drying the organic layer over anhydrous sodium sulphate and evaporating the organic layer to yield bis(2-hydroxyethyl)bisulphide, (d) treating the compound of step (c) under stirring with hydrohalogenic acid and cone, sulphuric acid at an ambient temperature ???? for 15 to 30 hours followed by heating for 2 to 4 hours in a steam bath,
  • step (e) separating the upper layer of step (d), washing with 10 % aqueous alkali carbonate solution, drying the organic layer over anhydrous sodium sulphate and evaporating the organic layer to yield bis (2-bromoethyl) disulphide,
  • step (f) reacting the product of step (e) with triaryl phosphine in dry dimethyl formamide at reflux temperature for 4-6 hours,
  • step (g) cooling the reaction mixtures of step (f) to room temperature and diluting with hexane to yield the required phosphonium bromide salt of bis (2- homothyl) disulphide, (h) treating the compound of step (g) with a benzaldehye or substituted benzaldehyde derivative in the presence of alkali metal alkoxide in the corresponding alcohol at reflux temperature for 15 to 30 hours, and (i) pouring the reaction mixture of step (h) onto ice and the precipitated solid is filtered, washed and dried to yield the corresponding final diallyldisulphide analogue and if desired, converting the analogues into their pharmaceutically acceptable salts.
  • the stirring in step (c) is carried out at an ambient temperature for 24 hours followed by heating for 3 hours.
  • the benzaldehyde used in step (h) is unsubstituted or substituted with suitable funcational group selected from cyano, carboxyl, nitro, trifluoromethyl, amino, methoxyl. or combination thereof.
  • the diallyldisulphide analogue is as active and two times less toxic than the standard drug.
  • the protective effective dose of the diallyldisulphide analogue is 20 mg/kg of body weight for antilipidemic activity and 20 mg/kg of body weight for antioxidant activity.
  • the acceptable additives are selected from the group of nutrients which are pharmaceutically acceptable carrier.
  • the novel diallyldisulphide analogues may be used in the form of tablets, capsules, syrup, powder, ointment, injectable, etc.
  • the diallyldisulphide analogue may be administered through routes such as oral, nasal intravenous, intra-peritoneal, intramuscular, etc.
  • the effective dose of diallyldisulphide analogues may be in the range of 20 mg/kg of body weight.
  • novel pharmaceutically acceptable salts comprising the novel diallyldisulphide analogues, said salts may be hydrochloride, hydrobromide, maleate, citrate, sodium or potassium, etc.
  • LiOEt Lithium ethoxide
  • 50ml of ethanol were taken in a dry round bottom flask.
  • the flask was flushed with Argon gas 0.779g (ImM) of the phosphonium salt (IV) in 10ml of super dry ethanol was added to the Lithium ethoxide formed by the addition of lithium in superdry ethanol 0.37g (25mM) p-nitrobenzaldehyde dissolved in 5ml of superdry ethanol and 3ml of THF was added to the reaction mixture.
  • the reaction mixture was refluxed with stirring for 24hours.
  • the reaction mixture was poured into ice, the final compound (V) precipitated out.
  • the compound was filterted and dried.
  • LiOEt Lithium ethoxide
  • 50mM 0.0174g of Lithium ethoxide (LiOEt) (25mM) and 50ml of ethanol were taken in a dry round bottom flask.
  • the flask was flushed with Argon gas 0.779g (ImM) of the phosphonium salt (IV) in 10ml of super dry ethanol was added to the Lithium ethoxide formed by the addition of lithium in superdry ethanol 0.6g (50mM) p-aminobenzaldehyde was dissolved in 5ml of superdry ethanol and 3ml of THF was added to the reaction mixture.
  • the reaction mixture was refluxed with stirring for 24hours. After completion, the reaction mixture was concentrated under vacuum.
  • LiOEt Lithium ethoxide
  • 50mM 0.0174g of Lithium ethoxide (LiOEt) (25mM) and 50ml of ethanol were taken in a dry round bottom flask.
  • the flask was flushed with Argon gas 0.779g (ImM) of the phosphonium salt (IV) in 10ml of super dry ethanol was added to the Lithium ethoxide formed by the addition of lithium in superdry ethanol 0.68g (50mM)
  • p- methoxybenzaldehyde was dissolved in 5ml of superdry ethanol.
  • the reaction mixture was concentrated under vacuum.
  • the residue was dissolved in water and extracted with chloroform (2 X 50ml).
  • the organic layer was dried over anhydrous Na 2 SO 4 and cone, under reduced pressure.
  • Example IV Conversion of compound IV of example I to phenyl substituted diallyldisulfide (VIII).
  • BIOLOGICAL ACTIVITY Antilipidemic activity assay Data pertaining to this activity is depicted in Table I.
  • GROUP-1 Orally administered equivalent amount of saline, treated as control.
  • GROUP-2 20mg/kg.b.wt of the Diallyl thiosulphinate fraction of garlic (Allicin extract) was orally administered for 5days.
  • GROUP-3 20mg/Kg.b.wt of Lovastatin was orally administered to the rats.
  • GROUP-6 20mg/Kg.b.wt of Bis [4-methoxy-phenyl allyl] disulphide was orally administered.
  • Rats 150-200g were divided into eight groups. Five rats in each group.
  • GROUP-8 Orally administered equivalent amount of saline, treated as control GROUP-9 Rats were fed with 5% cholesterol in their diet for one week.
  • GROUP- 11 Rats were fed with 5% cholesterol in diet for one week, along with the oral administration of Lovastatin (20mg/Kg.b.wt.).
  • GROUP-12 Rats were fed with 5% cholesterol in diet for one week, along with the oral administration of Bis[4-nitro phenyl allyl] "disulphide (20mg/Kg.b.wt.).
  • GROUP-13 Rats were fed with 5% cholesterol in diet for one week, along with the oral administration of Bis[4-amino phenyl allyl] "disulphide (20mg/Kg.b.wt).
  • GROUP- 14 Rats were fed with 5% cholesterol in diet for one week, along with the oral administration of Bis [4-methoxy phenyl allyl] disulphide (20mg/Kg.b.wt.).
  • GROUP-15 Rats were fed with 5% cholesterol in diet for one week, along with the oral administration of Bis[phenyl allyl] "disulphide (20mg/Kg.b.wt.)
  • Tissue preparation At the end of the experimental period, after overnight starvation animals were anesthetized using chloroform. Blood was drawn from retroorbital sinus using capillary tubes, into dried test tubes, which were kept at an angle of 45° for ten minutes at room temperature and then for an hour at 4°C. After that the blood was centrifuged at 800 x g for 10 min. to get serum separated from cellular clot. The animals were immediately dissected to remove their tissues, which were washed in ice-cold saline (0.85% NaCl), and extraneous material was removed.
  • HMG-CoA reductase activity was determined by the method of Venugopala Rao et al (1975). Equal volumes of the 10% tissue homogenate (i.e. lg of tissue/lOml of saline arsenate sol.) and diluted perchloric acid (50ml of HCIO ⁇ of water) were mixed. This was allowed to stand for 5 min. and centrifuged (600g, 10 min.).
  • TLC Thin Layer Chromatography
  • the glyceride was saponified with 0.5 ml of 0.1 N Alcoholic KOH at 70°C for 20 minutes. 200 ⁇ l of 0.4 N H 2 SO was added and placed in boiling water bath for 10-15 min. After that 50 ⁇ l of 0.05 N sodium metaperiodate was added. Excess NalO was neutralized with 0.1 ml of 0.5 M NaAsO 2 . A brown coloration developed after the addition of 5 ml of 0.108% chromotropic acid in 50% H SO 4 . Again heated in boiling water bath for 30-40 minutes. Then the samples were cooled and 3 ml of distilled water were added. Readings were taken at 570 nm. Ref.: VanHandel, E. and Zilversmit, D.B., J. Lab. Clin. Invest. 50,152 (1957). Antioxidant acitvity assay: Data pertaining to this activity is depicted in Table II. 6e. Reduced glutathione
  • Reduced glutathione was determined by the method of Jollow et al (1974). 1.00 ml of Post Mitochondrial Supernatant (PMS) obtained after centrifugation at 10,000 x g. was precipitated with 1.0 ml of sulfosalicylic acid (4%). The samples were kept at 4°C for at least one hour and then subjected to centrifugation at 1200xg for 15 minutes at 4°C.
  • PMS Post Mitochondrial Supernatant
  • the assay mixture contained 0.1 ml of filtered aliquot, 2.7 ml phosphate buffer (0.1 M, pH 7.4) and 0.2 ml DTNB [5.5"-Dithiobis(2-nitro-bezoic acid)] (40 mg/10 ml of phosphate buffer, 0.1 M, pH 7.4) in a total volume of 3.0 ml.
  • the yellow color developed was read immediately at 412 nm. Ref.: Jollow J.D., Mitchell R.J., Zampaglione N, and Gillette R.J; Pharmacology, 11, 151- 169 (1974).
  • Glutathione reductase activity was assayed by the method of Carlberg and Mannervik (1975) as modified by Mohandas et al (1984).
  • the assay system consisted of 1.65 ml phosphate buffer (0.1 M, pH 7.6), 0.1 ml NADPH (0.1 mM), 0.1 ml EDTA(0.5 mM), 0.05 ml oxidized glutathione (1 mM) and 0.1 ml PMS (10% w/v) in a total volume of 2.0 ml.
  • Glutathione reductase activity was calculated in terms of NADPH oxidized/min/mg protein using molar extinction coefficient of 6.22 x lO ⁇ M ⁇ cm "1 . Ref.: Carlberg J, and Mannervik B., J. Biol. Chem. 250, 14, 5475-5480 (1975). 6g. Catalase activity
  • Catalase activity was assayed by the the method of Claiborne (1985).
  • the assay mixture consisted of 1.95 ml phosphate buffer (0.05 m,pH 7.0), 1.0 ml hydrogen peroxide (0.019M), and 0.05 ml PM (10% w/v) in a total volume of 3.0 ml. Changes in absorbance were recorded at 240 nm. Catalase activity was calculated in terms of nmol H 2 O 2 consumed/min/mg protein.
  • Lipid Peroxidation level was measured by the method of Yagi et.al. 0.05ml of the blood was taken in 1.0 ml of physiological saline and centrifuged at 800g for lOmin. 0.5ml of supernatant (0.02ml of serum) was added in 0.5ml of 10% phosphotungstic acid and mixed. After standing at room temperature for 5min, the mixture was centrifuged at 800g for lOmin.The sediment was suspended in 4.0ml of distilled water, and 1.0ml of TBA reagent was added. The reaction mixture was heated for 60min.at 95° in an oil bath. After cooling with tap water, 5.0 ml of n-butanol was added and the mixture was shaken vigorously. After centrifugation at 800g for 15min, the n-butanol layer was taken for fluorometrically measured at 553nm with 515nm excitation. Tetraethoxypropane was used as standard.
  • Wistar rats (administered 5% cholesterol in their diet)

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Abstract

The present invention relates to a novel diallyldisulphide derivative having formula I as shown herebelow and their pharmaceutically acceptable salts; R-CH = CH - CH2-S-S-CH2-CH = CH-R (Formula I)wherein R=C6HnXp[when n = 3 to 5, p = 2 to 0; X=H, Cl, Br, F, CF3, CH3, CN, COOH, NO2, NH2, OH, OCmH2m+1(m = 1 to 8)]

Description

DIALLYLDISULPHIDE COMPOUNDS HAVING ANTILIPIDEMIC AND ANTIOXIDANT ACTIVITY
Technical Field
The present invention relates to a novel diallyldisulphide analogues of general formula I exhibiting antilipidemic and antioxidant activity and pharmaceutical compositions containing these analogues. The invention also provides a process of preparation of such analogues and the pharmaceutical composition containing such analogues. The novel diallyldisulphide compound is represented by
R-CH=CH-CH2-S-S-CH2-CH=CH-R
Wherein R=C6H„Xp[when n = 3 to 5, p = 2 to 0; X= H, CI, Br, F, CF3, CH3, CN,
COOH, NO2, NH2, OH, OCmH2m+1(m = 1 to 8)]
Formula 1 Background Art
Garlic is known for its medicinal and nutritional values for a very long time. Among the various potential therapeutic applications of garlic, its antilipidemic effects has been well demonstrated in studies of humans and animals [C.C.Gardner et.al, Atherosclerosis 154, 213-220, (2001); Liu et.al, Lipids 35, 197-203, (2000) and L.A. Simons et.al, Atherosclerosis 113, 219-225, (1995)]. Allicin (diallylthiosulfinate) is reported to be one of the most active constituents of garlic. Due to its unstable nature, allicin disallows its chemical modification to achieve a potent analogues. Allicin readily undergoes transformation to a volatile diallyldisulphide. The precursor of allicin, i.e. Alliin is known to be effective in treating hepatic diseases and fatigue. [Nagai Katsuji, Pat No. JP 56127312].
There has been also report of various active sulphur compounds from garlic, namely, Alliin, Allicin, Ajoene, diallyldisulphide, diallyldisulphide, allylmercaptan and S- allylcysteine [J.G. Dausch et.al, Preventive Medicine 19, 346-361 (1990); Y. Itokawa et.al, J. Nutr. 103, 88-92, (1973) and R. Gebhardt and H.Beck, Lipids 31, 1269-1276 (1996)]
This opens up the avenue for the synthesis of analogues of diallyldisulphide moiety with greater stability and activity in the reduction of serum lipids and this is of immense interest in the development of agents to combat cardiovascular risk factors. Objects of the Invention
The main object of the invention is to provide a novel diallyldisulphide analogues exhibiting antilipidemic and antioxidant activity.
Another object is to provide pharmaceutical compositions containing novel diallyldisulphide compounds.
Yet another object of the invention is to provide an effective and economical process for the preparation of novel diallyldisulphide compounds. Summary of the Invention
Accordingly, the present invention relates to novel diallyldisulphide analogues of general formula (I), exhibiting antilipidemic and antioxidant activity.
R-CH=CH-CH2-S-S-CH2-CH=CH-R
Formula I Wherein R=C6HnXp[when n = 3 to 5, p = 2 to 0; X= H, CI, Br, F, CF3, CH3, CN,
COOH, NO2, NH2, OH, OCmH2m+1(m = 1 to 8)]
Description of the Invention
Accordingly, the invention relates to novel active diallyldisulphide analogues which are represented by general formula I as shown hereinbelow:
R-CH=CH-CH2-S-S-CH2-CH=CH-R
Formula I Wherein R=C6H„Xp[when n = 3 to 5, p = 2 to 0; X= H, CI, Br, F, CF3, CH3, CN,
COOH, NO2, NH2, OH, OCmH2m+1(m = 1 to 8)]
The preferred diallyldisulphide derivatives are:
R-CH = CH - CH2-S-S-CH2-CH = CH-R
(Formula I)
Wherein R=C6H5; CeH-NO^); C6H3(NO2)2(2,4); C-H-COOH^); C6H4CN(4); C6H3(COOH)2(2,4); C6H3(CN)2(2,4); C6H2(NO2)3(2,4,6); C6H3NO2(4)COOH(2);
C6H3NO2(2)COOH(4); C6H4(CF3)(4) or C6H4CF3(2)
The novel diallyldisulphide compound and its analogues are found to be less toxic than known drugs and exhibit activity equipment to or greater than known drugs. The process for the preparation of diallyldisulphide compounds comprises the steps of:
(a) treating 2-mercapto ethanol with liquid bromine in presence of a suitable aqueous inorganic base in a halogenated hydrocarbon solvent at a temperature of about 0°C,
(b) separating the organic phase and extracting the aqueous phase with a halogenated hydrocarbon solvent,
(c) drying the organic layer over anhydrous sodium sulphate and evaporating the organic layer to yield bis(2-hydroxyethyl)bisulphide, (d) treating the compound of step (c) under stirring with hydrohalogenic acid and cone, sulphuric acid at an ambient temperature ???? for 15 to 30 hours followed by heating for 2 to 4 hours in a steam bath,
(e) separating the upper layer of step (d), washing with 10 % aqueous alkali carbonate solution, drying the organic layer over anhydrous sodium sulphate and evaporating the organic layer to yield bis (2-bromoethyl) disulphide,
(f) reacting the product of step (e) with triaryl phosphine in dry dimethyl formamide at reflux temperature for 4-6 hours,
(g) cooling the reaction mixtures of step (f) to room temperature and diluting with hexane to yield the required phosphonium bromide salt of bis (2- homothyl) disulphide, (h) treating the compound of step (g) with a benzaldehye or substituted benzaldehyde derivative in the presence of alkali metal alkoxide in the corresponding alcohol at reflux temperature for 15 to 30 hours, and (i) pouring the reaction mixture of step (h) onto ice and the precipitated solid is filtered, washed and dried to yield the corresponding final diallyldisulphide analogue and if desired, converting the analogues into their pharmaceutically acceptable salts. In one embodiment of the invention, the stirring in step (c) is carried out at an ambient temperature for 24 hours followed by heating for 3 hours.
In an another embodiment of the invention, the benzaldehyde used in step (h) is unsubstituted or substituted with suitable funcational group selected from cyano, carboxyl, nitro, trifluoromethyl, amino, methoxyl. or combination thereof. In another embodiment of the invention, the diallyldisulphide analogue is as active and two times less toxic than the standard drug.
In still another embodiment of the invention, the protective effective dose of the diallyldisulphide analogue is 20 mg/kg of body weight for antilipidemic activity and 20 mg/kg of body weight for antioxidant activity.
In yet another embodiment of the invention, the acceptable additives are selected from the group of nutrients which are pharmaceutically acceptable carrier. In still another embodiment of the invention, the novel diallyldisulphide analogues may be used in the form of tablets, capsules, syrup, powder, ointment, injectable, etc. In a further embodiment of the invention, the diallyldisulphide analogue may be administered through routes such as oral, nasal intravenous, intra-peritoneal, intramuscular, etc.
In an embodiment of the invention, the effective dose of diallyldisulphide analogues may be in the range of 20 mg/kg of body weight. Yet another embodiment of the invention relates to novel pharmaceutically acceptable salts comprising the novel diallyldisulphide analogues, said salts may be hydrochloride, hydrobromide, maleate, citrate, sodium or potassium, etc.
The preparation of diallyldisulphide derivatives starting from 2-mercaptoethanol is represented by Scheme I. The details of the reaction conditions employed are referred to in Examples 1 to 4.
OH-CH2-CH2SH ► HOCH2-CH2-S-S-CH2-CH2OH
(I)
Br [Ph3P+CH2CH2-S-S-CH2CH2P+Ph3]Br- BrCH2-CH2-S-S-CH2-CH2Br
(HI) (II)
R-CH=CH-CH2-S-S-CH2-CH=CH-R
(IV)
Wherein R=C6HnXp[when n = 3 to 5, p = 2 to 0; X= H, CI, Br, F, CF3, CH3, CN, COOH, NO2, NH2, OH, OCmH2m+1(m = 1 to 8)]
Scheme I The invention is further illustrated with reference to the following Examples and should not be construed to limit the scope of the invention:
Example 1 Synthesis of P-nitrophenyl substituted diallyldisulphide Step (a): Conversion of 2-mercaptoethanol(I) to Bis[2-hydroxy ethyl] Disulphide(II)
Hαα^CH^H E KH-Q( inH --Hp2) f HC ^^^O^O^OH
i »
Procedure
40ml Dichloromethane, 3ml (40mM) 2-Mercaptoethanol(I) and 10% aqueous KHCO3 were added to a round bottom flask. The flask was immersed in ice, and 2ml Bromine (99%) 40mM in Dichloromethane was added to the reaction vessel. The organic phase was separated and the aqueous phase was extracted with dichloromethane. The organic phases were combined and dried with anhydrous Na2SO4. The solvent was evaporated yielded the pure disufide (lg, 33.8%) II. Y Yiieelldd l lgg % % Yield 33.8%
T.L.C.
Solvent system
20:80 :: Ethyl acetate : Hexane Rf
IR (thin film) (Major peaks)
3330.0 cm"1 OH str.
2925.8cm"1 C-H str.
696.0cm"1 C-S str.
NMR 1H NMR (300MHz; CDC13): δ 2.88[t, 2x2H(a), J=1.05Hz]; 2.99[s, 2xlH (OH)]; 3.89 [t, 2H(b), J=1.05Hz) Step (b): Conversion of (II) to Bis[2-bromoethyl] Disulfιde(III)
H2SO4
HO-CH2-CH2-S-S-CH2-CH2-OH -*. Br-CH^CH^S-S-CrirCrirBr
48%HBr 111 Procedure
70ml of 48% HBr was taken in a 500ml 3 necked flask, fitted with a stirrer, condenser & dropping funnel. The flask was cooled in an ice bath and 46ml of cone. H SO was added to HBr with stirring. To the resulting ice cold solution, 2g of compound II was added dropwise. The reaction mixture was left on stirring for 24hours at room temperature. After this the reaction mixture was heated for 3hours on a steam bath. Two layers separated out, 10ml of dichloromethane was added. The upper layer was taken, washed with water, 10% Na2CO3 solution and then dried over anhydrous Na2SO4. The dichloromethane was evaporated using a rotavapor and the desired compound III was obtained. Yield -2g %Yield 55%
T.L.C.
Solvent System 20:80:: Ethyl acetate:Hexane
IR (thin film) (Major peaks)
2922 cm"1 Csp3 - H str.
617 cm"1 C-S str.
563 cm"1 C-Br str.
444 cm"1 -S-S- str. NMR
1HNMR (300MHz; CDC13): δ 3.10 [t, 2x2H(a), J = 1.3Hz); 3.62[t, 2x2H(b), J=1.3Hz)
Step (c): Conversion of (III) to the analogous Phosphonium bromide (IV)
BrO O ββO O βr "^ "^ - / -°W«-ββ<»1-*°
III IV Procedure
2.8g (0.01M) of Compound III and 5.5g (0.02M) of Triphenyl phosphine (PPh3) were taken in a round bottom flask. 15ml of dry dimethylformamide (DMF) was added to the reactants. The reaction mixture was refluxed with stirring for 5hours. The mixture was allowed to cool to room temperature and 15ml of hexane was added. The desired phosphonium salt precipitated out. The solution was filtered, washed with hexane and dried. White crystals of compound IV were obtained. Yield-5.6g % Yield 71.7
Melting point of white crystals of compound IV-130°C
T.L.C.
Solven System 20:80:: Ethyl acetate: Hexane
Rf 0.35
IR
IR (KBr pellets)
3053 cm"1 -C3p2-H str.
691 cm"1 -C-S- str.
NMR Spectroscopy
1H NMR (300MHz; CDC13): δ 2.61 (t, 2x2H(a), J=0.29Hz); 2.86-3.10 [m, 2x2H(b)];
7.40-7.50 (m, 24H, aromatic protons); 7.50-7.63(m, 6H, aromatic protons)
Step (d): Conversion of IV to the p-nitrophenyl substituted
Figure imgf000008_0001
[Ph3p -CH^c^-s-s-α α pphj
IV
BOH/ LiOEt
Figure imgf000008_0002
Figure imgf000008_0003
diallyldisulfide (V)
Procedure
0.0174g of Lithium ethoxide (LiOEt) (25mM) and 50ml of ethanol were taken in a dry round bottom flask. The flask was flushed with Argon gas 0.779g (ImM) of the phosphonium salt (IV) in 10ml of super dry ethanol was added to the Lithium ethoxide formed by the addition of lithium in superdry ethanol 0.37g (25mM) p-nitrobenzaldehyde dissolved in 5ml of superdry ethanol and 3ml of THF was added to the reaction mixture. The reaction mixture was refluxed with stirring for 24hours. The reaction mixture was poured into ice, the final compound (V) precipitated out. The compound was filterted and dried.
Yield 200 mg %yield 60.6
5 Melting point of dry compound V-140°C
T.L.C.
Solvent System 20:80 :: Ethyl acetate: Hexane
Rf 0.4
IR
LR KBr pellet
3055 cm"1 -C3p2-H str.
1583 cm"1 -N = O str.(anti) 5 1433 cm"1 -N = O str. (sym)
690.5 cm"1 -C-S- str. ,
NMR H NMR (CDC13 + DMSO): δ 4.75 [d, 2x2H(a), J=1.10Hz]; 7.4-7.6[m, 2x4H(b, c
&aromatic protons)]; 7.6-7.8 (m, 2x2H aromatic protons) 0
Example II:
Conversion of compound IV of example I to p-aminophenyl substituted diallyldisulfide (VI)
[Ph3P -CH2-CH2-S-S-CH2-CH2- PPh3] + Br b a a b Br
IV
EtOH / LiOEt
Figure imgf000009_0001
Figure imgf000009_0002
VI Procedure
0.0174g of Lithium ethoxide (LiOEt) (25mM) and 50ml of ethanol were taken in a dry round bottom flask. The flask was flushed with Argon gas 0.779g (ImM) of the phosphonium salt (IV) in 10ml of super dry ethanol was added to the Lithium ethoxide formed by the addition of lithium in superdry ethanol 0.6g (50mM) p-aminobenzaldehyde was dissolved in 5ml of superdry ethanol and 3ml of THF was added to the reaction mixture. The reaction mixture was refluxed with stirring for 24hours. After completion, the reaction mixture was concentrated under vacuum. The residue was dissolved in water and extracted with chloroform (2 X 50ml). The organic layer was dried over anhydrous Na2SO and cone, under reduced pressure. The yellow solid obtained was finally recrystallized from chloroform-hexane (75:25). The compound was filtered and dried. Yield 220 mg %yield 65.7
Melting point of dry compound VI-150°C
T.L.C.
Solvent System 20:80 :: Ethyl acetate: Hexane
Rf 0.514.
IR
IR KBr pellet
3435.97 cm"1 =N - H str.(anti)
3054.34 cm"1 -C3p2-H str.
2951.92 cm"1 -C3p3-H str
NMR
1H NMR (CDC13); δ 2.26(s, 2x2H,-NH2); 2.74[d, 2x2H(a), J=1.14Hz]; 7.2-7.4[m, 2x3H(b, c & aromatic protons)]; 7.4-7.6 (m, 2x2H aromatic protons), 7.85(s, 2xlH aromatic protons) Example III: Step IVc Conversion of compound IV of example I to p-methoxyphenyl substituted diallyldisulfide (VII)
[Ph3P -CH2-CH2-S-S-CH2-CH2- PPhJ Br a a b Br
IV
MeO- _VcHO BOH / LiOEt
Figure imgf000011_0001
Figure imgf000011_0002
VII
Procedure
0.0174g of Lithium ethoxide (LiOEt) (25mM) and 50ml of ethanol were taken in a dry round bottom flask. The flask was flushed with Argon gas 0.779g (ImM) of the phosphonium salt (IV) in 10ml of super dry ethanol was added to the Lithium ethoxide formed by the addition of lithium in superdry ethanol 0.68g (50mM) p- methoxybenzaldehyde was dissolved in 5ml of superdry ethanol. After completion, the reaction mixture was concentrated under vacuum. The residue was dissolved in water and extracted with chloroform (2 X 50ml). The organic layer was dried over anhydrous Na2SO4 and cone, under reduced pressure. The yellow gummy residue was chromatographed on silica gel using hexane-Ethylacetate as eluent. The compound was finally recystallized from Ethylacetate-hexane (80:20). The compound was filtered and dried. Yield 240mg %yield 77.8
Melting point of dry compound VII-155°C
Solvent System 20:80 :: Ethyl acetate: Hexane Rf 0.542 IR
LR KBr pellet 3435cm"1 O-H str. (phenolic impurity) 3047.97 cm"1 -C3p2 -H s
2923.40 cm"1 -C3p3-H str.
1654.17 cm"1 -C=C-str.
691.20 cm"1 -C-S-str.
477.83 cm"1 -S-S-str.
NMR
1H NMR (CDC13): δ 3.75(d, 2x2H,J=1.0Hz,); 3.81(s, -OCH3, 2x3H); 6.81- 6.94[m,2x2H(b, c)]; 7.45(d,2x2H aromatic protons, J=0.51Hz); 7.76 (d,2x2H aromatic protons, J=0.51Hz)
Example IV: Conversion of compound IV of example I to phenyl substituted diallyldisulfide (VIII).
[Ph3P+-CH2- CH?-S-S-CHP-CH9- PPhJ
Br a 2 a 2 b 2 3 Br
IV
EtOH / LiOEt
Figure imgf000012_0001
Figure imgf000012_0002
VIII
Procedure 0.0174g of Lithium ethoxide (LiOEt) (25mM) and 50ml of ethanol were taken in a dry round bottom flask . The flask was flushed with Argon gas 0.779g (ImM) of the phosphonium salt (IV) in 10ml of super dry ethanol was added to the Lithium ethoxide formed by the addition of lithium in superdry ethanol 0.53g (50mM) p-benzaldehyde dissolved in 5ml of superdry ethanol. The reaction mixture was refluxed with stirring for 24hours. After completion (TLC), the reaction mixture was concentrated under vaccum. The residue was dissolved in water and extracted with chloroform (2 X 50ml). The organic layer was dried over anhydrous Na2SO4 and cone, under reduced pressure. The yellow solid obtained was finally recystallized from chloroform-hexane (75:25). The compound was filtered and dried.
Yield 190 mg %yield 69.3
Melting point of dry compound VIII-220oC( Decomp.) T.L.C.
Solvent System 20:80 :: Ethyl acetate: Hexane
Rf 0.428
IR
IR KBr pellet 3435.67cm"1 O-H str. (hydroxylic impurity/moisture)
3055.83 cm"1 -C3p2-H str.
2927.08 cm"1 -C3p3 -H str.
1685.41 cm"1 -C=C-str.
691.44 cm"1 -C-S-str. 427.07 cm"1 -S-S-str.
NMR
1H NMR (CDCI3): 2.87[d, 2x2H(a), J=lHz]; 7.49-7.51 [m, 2x5H(b, c& aromatic protons]; 7.68-7.75 (m, 2x2H aromatic protons)
BIOLOGICAL ACTIVITY Antilipidemic activity assay: Data pertaining to this activity is depicted in Table I.
1. Materials and methods
Material-Garlic (Allium sativum) was bought from the local market. All other chemicals and reagents used were of highest analytical grade available commercially. U.V. spectrophotometeric studies were done using model Shimadzu UV-1601. NMR studies were done using Bruker spectro spin 300 MHZ instrument.
2. Animals
Adult male rats of Wistar strain (n=25) with body weight range 200-250g obtained from Animal House Facility at A.C.B.R.University of Delhi, were used in the present study. They were maintained in an air-conditioned room, and were provided with standard food pellets and tap water ad libitum. 3. Method of preparation of garlic clove polar and thiosulphinate fractions
After homogenization of garlic cloves in water at 10 ml/g, the thiosulphinates (Allicin extract) were extracted with two volumes of chloroform, quickly rotary evaporated at ambient temperature, and redissolved in water at 2 mg/ml. All homogenates, fraction and compounds were kept at 4°C while in use and at -20°C for storage. Planning of Experiment
Experiment on Normocholesterolemic rats
Rats (250-300g) were divided in seven equal groups. Five rats in each group.
GROUP-1 Orally administered equivalent amount of saline, treated as control. GROUP-2 20mg/kg.b.wt of the Diallyl thiosulphinate fraction of garlic (Allicin extract) was orally administered for 5days.
GROUP-3 20mg/Kg.b.wt of Lovastatin was orally administered to the rats.
GROUP-4 20mg Kg.b.wt. of Bis[4-nitro phenyl allyl] disulphide was orally administered.
GROUP-5 20mg Kg.b.wt. of Bis [4-amino phenyl allyl] disulphide was orally administered.
GROUP-6 20mg/Kg.b.wt of Bis [4-methoxy-phenyl allyl] disulphide was orally administered.
GROUP-7 20mg/Kg.b.wt. of Bis [phenyl allyl] disulphide was orally administered
Experiment on hypercholesterolemic rats Rats (150-200g) were divided into eight groups. Five rats in each group.
GROUP-8 Orally administered equivalent amount of saline, treated as control GROUP-9 Rats were fed with 5% cholesterol in their diet for one week. GROUP-10 Rats were fed with 5% cholesterol in diet for one week, along with oral administration of 20mg/Kg.b.wt. diallyl thiosulphinate fraction of garlic
(Allicin extract). GROUP- 11 Rats were fed with 5% cholesterol in diet for one week, along with the oral administration of Lovastatin (20mg/Kg.b.wt.). GROUP-12 Rats were fed with 5% cholesterol in diet for one week, along with the oral administration of Bis[4-nitro phenyl allyl] "disulphide (20mg/Kg.b.wt.). GROUP-13 Rats were fed with 5% cholesterol in diet for one week, along with the oral administration of Bis[4-amino phenyl allyl] "disulphide (20mg/Kg.b.wt). GROUP- 14 Rats were fed with 5% cholesterol in diet for one week, along with the oral administration of Bis [4-methoxy phenyl allyl] disulphide (20mg/Kg.b.wt.). GROUP-15 Rats were fed with 5% cholesterol in diet for one week, along with the oral administration of Bis[phenyl allyl] "disulphide (20mg/Kg.b.wt.)
5. Tissue preparation At the end of the experimental period, after overnight starvation animals were anesthetized using chloroform. Blood was drawn from retroorbital sinus using capillary tubes, into dried test tubes, which were kept at an angle of 45° for ten minutes at room temperature and then for an hour at 4°C. After that the blood was centrifuged at 800 x g for 10 min. to get serum separated from cellular clot. The animals were immediately dissected to remove their tissues, which were washed in ice-cold saline (0.85% NaCl), and extraneous material was removed. For biochemical studies, approximately lg of tissue was kept for estimation of lipids and the remaining was homogenized in Potter-Elvejem type glass homogenizer in 0.1 M Potassium phosphate buffer (pH-7.4) having 0.25M sacrose to give a 20% homogenate, according to the method of Hogeboom and Umbelt et al. Ref: 1. Hogeboom, G.H. Methods in Enzymology (Colowick, S.P. and N.O. Kaplar eds.); Academic Press, New York, 1, 16, (1955) 2..Umbeit, W.W., Burries, R.H. and Stauffer, S.F. Burgers Publishing Co. Minneapolis; HI Ed. 10, (1957).
6. Biochemical estimations 6a. HMG-CoA reductase
HMG-CoA reductase activity was determined by the method of Venugopala Rao et al (1975). Equal volumes of the 10% tissue homogenate (i.e. lg of tissue/lOml of saline arsenate sol.) and diluted perchloric acid (50ml of HCIO^ of water) were mixed. This was allowed to stand for 5 min. and centrifuged (600g, 10 min.). In case of Mevalonate estimation, 1.0 ml of supernatant was treated with 0.5 ml of freshly prepared hydroxylamine hydrochloride reagent (equal volumes of hydroxylamine hydrochloride reagent (2 mol/L) and water) and after 5 min., 1.5 ml of ferric chloride reagent was added to the same tube and the tube was vortexed. In case of HMG-CoA estimation 1.0 ml of supernatant was treated with 0.5 ml of Alkaline hydroxylamine hydrochloride reagent [equal volume of hydroxylamine hydrochloride reagent (2 mol/L of water) and sodium hydroxide solution (4.5 mol/L of water)]. And after 5 min. 1.5 ml of ferric chloride reagent was added and the tube was vortexed. Readings were taken after 10 min., at 540 nm vs. A similarly treated saline arsenate blank. The ratio of HMG-CoA/Mevalonate was taken as an index of HMG-CoA reductase activity. Ref.: Venugopala Rao A and Ramakrishnan S., Clin. Chem. 21,10, 1543-1525 (1975) 6b. Isolation of lipids For estimation of lipds the tissue was weighed. Approximately 1 g of tissue was homogenized with 10 ml of methanol, then 20 ml of chloroform was added and process continued for a further 2 min. After filtering, the solid was washed once more with chloroform (20 ml) and once with methanol (10 ml), the combined filtrates were transferred to the measuring cylinder and one quarter of the total volume of the filtrate 0.88% KC1 in water was added, the mixture was shaken thoroughly and allowed to settle. The upper layer was removed by aspiration, one quarter of the volume of the lower layer of water-methanol was added. The lower layer contains the purified lipid. 6c. Estimation of total cholesterol
For quantitative estimation of total cholesterol the method of Zlatkis et al. (1953) and Hanley (1957). A known amount of (200 μl) the sample from the total lipid in CHC13 was taken and evaporated to dryness. To the dried sample, 5 ml of 0.05% FeCl3 in CH3COOH were added. The contents were mixed using a vortex mixer, 2 ml of Cone. Sulphuric acid was added to each of the tubes, and then vortexed and allowed to stand for 20-30 minutes. The absorbance was recorded at 560 nm. For standard, Cholesterol (1 mg/ml of CH3COOH) was used. Ref.: Zlatkis, A, Zak, B. and Boyle, A.J., J. Lab. Clin. Med. 41, 486, (1953) 6d. Estimation of Triglycerides
Thin Layer Chromatography (TLC) was used to fractionate the neutral lipids silica gel G was used as an adsorbent. A known amount (~100μl) of the total lipid, in duplicate, was applied on silica gel treated plates. Tristearin (100 mg/dl) was used as a standard. The plates were developed upto 13-15 cm from the original in chamber lined with sufficient amount of saturated solvent system petroleum ether (60-80), solvent ether, and acetic acid (80:20:2). The plates were dried and the spots made visible by exploring the plates to iodine vapors in a closed chamber. The fraction was then scrapped off the plates and the glyceride concentration was estimated by the method of Van Handel and Zilversmit. The glyceride was saponified with 0.5 ml of 0.1 N Alcoholic KOH at 70°C for 20 minutes. 200 μl of 0.4 N H2SO was added and placed in boiling water bath for 10-15 min. After that 50 μl of 0.05 N sodium metaperiodate was added. Excess NalO was neutralized with 0.1 ml of 0.5 M NaAsO2. A brown coloration developed after the addition of 5 ml of 0.108% chromotropic acid in 50% H SO4. Again heated in boiling water bath for 30-40 minutes. Then the samples were cooled and 3 ml of distilled water were added. Readings were taken at 570 nm. Ref.: VanHandel, E. and Zilversmit, D.B., J. Lab. Clin. Invest. 50,152 (1957). Antioxidant acitvity assay: Data pertaining to this activity is depicted in Table II. 6e. Reduced glutathione
Reduced glutathione was determined by the method of Jollow et al (1974). 1.00 ml of Post Mitochondrial Supernatant (PMS) obtained after centrifugation at 10,000 x g. was precipitated with 1.0 ml of sulfosalicylic acid (4%). The samples were kept at 4°C for at least one hour and then subjected to centrifugation at 1200xg for 15 minutes at 4°C. The assay mixture contained 0.1 ml of filtered aliquot, 2.7 ml phosphate buffer (0.1 M, pH 7.4) and 0.2 ml DTNB [5.5"-Dithiobis(2-nitro-bezoic acid)] (40 mg/10 ml of phosphate buffer, 0.1 M, pH 7.4) in a total volume of 3.0 ml. The yellow color developed was read immediately at 412 nm. Ref.: Jollow J.D., Mitchell R.J., Zampaglione N, and Gillette R.J; Pharmacology, 11, 151- 169 (1974).
6f . Glutathione reductase activity
Glutathione reductase activity was assayed by the method of Carlberg and Mannervik (1975) as modified by Mohandas et al (1984). The assay system consisted of 1.65 ml phosphate buffer (0.1 M, pH 7.6), 0.1 ml NADPH (0.1 mM), 0.1 ml EDTA(0.5 mM), 0.05 ml oxidized glutathione (1 mM) and 0.1 ml PMS (10% w/v) in a total volume of 2.0 ml. Glutathione reductase activity was calculated in terms of NADPH oxidized/min/mg protein using molar extinction coefficient of 6.22 x lO^M^cm"1. Ref.: Carlberg J, and Mannervik B., J. Biol. Chem. 250, 14, 5475-5480 (1975). 6g. Catalase activity
Catalase activity was assayed by the the method of Claiborne (1985). The assay mixture consisted of 1.95 ml phosphate buffer (0.05 m,pH 7.0), 1.0 ml hydrogen peroxide (0.019M), and 0.05 ml PM (10% w/v) in a total volume of 3.0 ml. Changes in absorbance were recorded at 240 nm. Catalase activity was calculated in terms of nmol H2O2 consumed/min/mg protein.
6h. Glutathione-S-transferase activity
Glutathione-S-transferase activity was measured by the method of Habig et al. (1974) The reaction mixture consisted of 1.425 ml phosphate buffer (0.1 M, pH 6.5), 0.2 ml reduced glutathione (1 mM), 0.025ml CDNB (l-Chloro-2,4-dinitrobenzene) (1 mM) and 0.30 ml PMS (10% w/v) in a total volume of 2.0 ml. The changes in absorbance were recorded at 340 nm and the enzyme activity was calculated as nmol CDNB conjugate formed /min/mg protein using a molar extinction coefficient of 9.6 x 103 M^cm"1. Ref.: Habig, W.H., Pabst, M.J., and Jaokby, W.B.J. Biol. Chem. 249, 7130-7139 (1974). 6i. Lipid Peroxidation
Lipid Peroxidation level was measured by the method of Yagi et.al. 0.05ml of the blood was taken in 1.0 ml of physiological saline and centrifuged at 800g for lOmin. 0.5ml of supernatant (0.02ml of serum) was added in 0.5ml of 10% phosphotungstic acid and mixed. After standing at room temperature for 5min, the mixture was centrifuged at 800g for lOmin.The sediment was suspended in 4.0ml of distilled water, and 1.0ml of TBA reagent was added. The reaction mixture was heated for 60min.at 95° in an oil bath. After cooling with tap water, 5.0 ml of n-butanol was added and the mixture was shaken vigorously. After centrifugation at 800g for 15min, the n-butanol layer was taken for fluorometrically measured at 553nm with 515nm excitation. Tetraethoxypropane was used as standard.
Ref: Yagi, K. (1979) Biological damage imposed by Oxygen. Methods in Enzymology. 10(5), 328-331. 6j. Estimation of Protein Protein was estimated by the method of Lowry et al (1951). 0.1 ml of tissue homogenate was diluted to 1 ml with water and 5 ml alkaline copper sulphate reagent containing sodium carbonate (2%), CuSO4 (1%) and sodium potassium tartrate (2%) was added. After 10 minutes, 0.5 ml of Folin's reagent was added, and incubate the mixture for 30 min. at room temperature, the blue color developed and the absorbance was read at 660 nm. Bovine serum albumin (0.1 mg/ml) was used as a standard.
Ref.: Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., J. Biol. Chem., 193, 265-275 (1951). Biological Activities of Bis [4-amino phenyl allyl ] disulphide(Compound VI) and Bis [4- methoxy phenyl allyl ] disulphide (Compound VII)
Effect of Compound [(VI, VLT),20mg/Kg.b.wt.], Lovastatin (20mg/kg.b.wt.) and Allicin(Garlic extract) given, for 5 days on Lipid profile of Wistar rats Cholesterol
Figure imgf000019_0001
Triglyceride
Figure imgf000019_0002
HMG-CoA Reductase
Figure imgf000019_0003
Effect of Compound [(VI, VII),20mg/Kg.b.wt.], Lovastatin (20mg/kg.b.wt.) and Allicin(Garlic extract) given for 5 days on Oxidative stress in Wistar rats Reduced Glutathione
Figure imgf000019_0004
* nmol of GSH / mg.protein Glutathione Reductase
Figure imgf000020_0001
nmol NADPH oxidised/min/ mg protein
Glutathione S-transferase
Figure imgf000020_0002
nmol of CDNB conjugate formed /min/ mg protein
Catalase
Organ Control Allicin Lovastatin Compound VI Compound VII (Mean± S.E.) (Mean± S.E.) 20mg/Kg. b.wt. 20mg/Kg. b.wt. 20mg/kg.b.wt. (Mean± S.E.) (Mean± S.E.) (Mean± S.E.)
Liver* 8.75 ± 1.38 11.8 ± 0.187 9.11 ± 1.20 8.94 ± 1.089 6.689 ± 1.326
Spleen* | 2.82 ± 0.404 .24 ± 1.089 | 4.37 ± 0.347 3.49 ± 0.67 2.14 ± 0.212
* μmol of H202 consumed/min. / g. wet weight tissue
Lipid Peroxidation
Figure imgf000020_0003
* nmol of MDA conjugate formed / ml of Serum
Biological Activities of Bis [4-nitro phenyl allyl ] disulphide(Compound V) and Bis [ phenyl allyl ] disulphide (Compound VIII)
Effect of Compound [(V, VIII),20mg Kg.b.wt.], Lovastatin (20mg/kg.b.wt.) and Allicin(Garlic extract) given for 5 days on Lipid profile of Wistar rats
Figure imgf000020_0004
Tri l ceride
Figure imgf000021_0003
HMG-CoA Reductase
Figure imgf000021_0004
Effect of Compound [(V, VIII),20mg Kg.b.wt.], Lovastatin (20mg/kg.b.wt.) and Allicin(Garlic extract) given for 5 days on Oxidative stress in Wistar rats
Reduced Glutathione
Figure imgf000021_0001
nmol of GSH / mg.protein
Gluathione reductase
Figure imgf000021_0005
* nmol NADPH oxidised/min/ mg protein Gluatathione -S-transferase
Figure imgf000021_0006
* nmol of CDNB conjugate formed /min/ mg protein
Figure imgf000021_0002
* f H O / i Lipid Peroxidation
Control Allicin Lovastatin Compound V Compound VIII (Mean± S.E.) (Mean± S.E.) (Mean± S.E.)
Serum* 244.81 ± 5.18 120.59 ± 3.99 143.84 + 16.4 127.53 ± 26.6 241.50 ± 6.34
* nmol of MDA conjugate formed / ml of Serum.
Biological Activities of Bis [4-amino phenyl allyl ] disulphide(Compound VI) and Bis
[4-methoxy phenyl allyl ] disulphide (Compound VII)
Effect of Compound [(VI, VII),20mg/Kg.b.wt.], Lovastatin (20mg/kg.b.wt.) and
Allicin(Garlic extract) given for 5 days on Lipid profile of Mild Hypercholesterolemic
Wistar rats (administered 5% cholesterol in their diet)
Figure imgf000022_0001
Figure imgf000022_0002
Figure imgf000022_0003
HMG-CoA Reductase
Figure imgf000023_0001
HMG-CoA Reductase
Figure imgf000023_0002
Effect of Compound [(VI, VII),20mg/Kg.b.wt.], Lovastatin (20mg/kg.b.wt.) and Allicin(Garlic extract) given for 5 days on Oxidative stress in Wistar rats
Reduced Glutathione
Figure imgf000023_0003
nmol of GSH / mg.protein
Glutathione Reductase
Figure imgf000023_0004
nmol NADPH oxidised/min/ mg protein
Glutathione S-transferase
Figure imgf000023_0005
* nmol of CDNB conjugate formed /min/ mg protein Catalase
Organ Hypercholesterol Allicin Lovastatin Compound VI Compound VII emic rats (Meani S.E.) 20mg/Kg. b.wt. 20mg/Kg. b.wt. 20mg/kg.b.wt. (Mean± S.E.) (Mean± S.E.) (Mean± S.E.)
Liver* 5.47 ± 0.20 6.39 ± 0.135 5.79 ± 0.97 5.67 ± 1.204 5.160 ± 1.204
Spleen* 1.12 ± 0.086 2.66 ± 0.086 2.23 ± 0.172 1.806 ± 0.087 1.49 ± 0.34 μmol of H2O2 consumed min. / g. wet weight tissue
Lipid Peroxidation
Figure imgf000024_0001
* nmol of MDA conjugate formed / ml of Serum
Biological Activities of Bis [4-nitro phenyl allyl ] disulphide(Compound V) and Bis [ phenyl allyl ] disulphide (Compound VIII)
Effect of Compound [(V, VIII),20mg/Kg.b.wt.], Lovastatin (20mg/kg.b.wt.) and Allicin(Garlic extract) given for 5 days on Lipid profile of Mild Hypercholesterolemic Wistar rats
Cholesterol
Figure imgf000024_0002
Triglyceride
Figure imgf000024_0003
Figure imgf000025_0001
Effect of Compound [(V, VIII),20mg/Kg.b.wt.], Lovastatin (20mg/kg.b.wt.) and Allicin(Garlic extract) given for 5 days on Oxidative stress in Wistar rats
Reduced Glutathione
nmol of GSH / mg.protein
Glutathione Reductase
Figure imgf000025_0003
* nmol NADPH oxidised/min/ mg protein Glutathione S-transferase
Figure imgf000025_0004
* nmol of CDNB conjugate formed /min/ mg protein Catalase
Figure imgf000025_0005
* μmol of H202 consumed/min. / g. wet weight tissue Lipid Peroxidation
Hypercholes Allicin Lovastatin Compound V Compound terolemic (Mean± S.E.) (Mean± S.E.) VIII rats
Serum* 300.08 ± 1.36 229.87 ± 3.79 248.86 ± 5.05 238.29 ± 15.80 299.63 ± 2.1 * nmol of MDA conjugate formed / ml of Serum Toxicological studies
Effect of sin le dose of s nthesized com ounds on Wistar rats.
Figure imgf000026_0001

Claims

Claims:
1. Novel diallyldisulphide derivative having formula I as shown herebelow and their pharmaceutically acceptable salts;
R-CH = CH - CH2-S-S-CH2-CH = CH-R
(Formula I) Wherein R=C6HnXp[when n = 3 to 5, p = 2 to 0; X= H, CI, Br, F, CF3, CH3, CN,
COOH, NO2, NH2, OH, OCmH2m+1(m = 1 to 8)]
2. Diallyldisulphide derivatives as claimed in claim 1, are represented by the structural formula I and pharmaceutically acceptable salts thereof.
R-CH = CH - CH2-S-S-CH2-CH = CH-R
(Formula I)
Wherein R=C6H5; C6H4NO2 (4); C6H3(NO2)2(2,4); C-K-COOH^); C6H4CN(4); C6H3(COOH)2(2,4); C6H3(CN)2(2,4); C6H2(NO2)3(2,4,6); C6H3NO2(4)COOH(2);
C6H3NO2(2)COOH(4); C6H4(CF3)(4) or C6H4CF3(2).
3. Compounds as claimed in claim 1, wherein the pharmaceutically acceptable salts are selected from hydrochloride, hydrobromide, citrate, maleate, fumarate, camphorsulphonic acid, sodium or potassium.
4. A process for the preparation of diallyldisulphide derivatives having formula I and their pharmaceutically acceptable salts thereof, the said process comprising steps of: R-CH = CH - CH2-S-S-CH2-CH = CH-R
(Formula I)
Wherein R=C6HnXp[when n = 3 to 5, p = 2 to 0; X= H, CI, Br, F, CF3, CH3, CN, COOH, NO2, NH2, OH, OCmH2m+1(m = 1 to 8)],
(a) treating 2-mercapto ethanol with bromine in presence of an aqueous inorganic base in a halogenated hydrocarbon solvent around 0°C, (b) separating the organic phase and extracting the aqueous phase with a halogenated hydrocarbon solvent,
(c) drying the organic layer over anhydrous sodium sulphate and evaporating the organic layer to yield bis(2-hydroxyethyl)bisulphide, (d) treating the compound of step (c) under stirring with hydrohalogenic acid and cone, sulphuric acid at an ambient temperature for 15 to 30 hours followed by heating for 2 to 4,
(e) separating the upper layer of step (d), washing with 10 % aqueous alkali carbonate solution, drying the organic layer over anhydrous sodium sulphate and evaporating the organic layer to yield bis (2-bromoethyl) disulphide,
(f) reacting the product of step (e) with triaryl phosphine in dry dimethyl formamide at reflux temperature for 4-6 hours,
(g) cooling the reaction mixtures of step (f) to room temperature and diluting with hexane to yield the required phosphonium bromide salt of bis (2- homothyl) disulphide, (h) treating the compound of step (g) with a benzaldehye or substituted benzaldehyde derivative in the presence of alkali metal alkoxide in the corresponding alcohol at reflux temperature for 15 to 30 hours, and (i) pouring the reaction mixture of step (h) onto ice and the precipitated solid is filtered, washed and dried to yield the corresponding final diallyldisulphide analogue and if desired, converting the analogues into their pharmaceutically acceptable salts.
5. A process as claimed in claim 1 wherein in step (a), the halogenated hydrocarbon is selected from a group consisting of carbontetrachloride, methylenechloride, dichloromethane and /or chloroform.
6. A process as claimed in claim 1 wherein in step (c), the stirring is carried out at an ambient temperature for 24 hours followed by heating for 3 hours.
7. A process as claimed in claim 1 wherein in step (d), the hydrohalogen acid is selected from a group consisting of hydrochloric, hydrobromic and hydroiodic acid, preferably hydrobromic acid.
8. A process as claimed in claim 1 wherein in step (d), the stirring at ambient temperature is carried out for 24 hours followed by heating for 3 hours.
9. A process as claimed in claim 1 wherein in step (e), the alkali carbonate used is selected from sodium or potassium carbonate, preferably sodium carbonate.
10. A process as claimed in claim 1 wherein in step (f), the triaryl phosphine is triphenyl phosphine.
11. A process as claimed in claim 1 wherein in step (h), the benzaldehyde used is unsubstituted or substituted with a functional group selected from nitro, carboxy, trifluoromethyl, cyano, amino, methoxy and simple unsubstituted benzaldehyde or combinations thereof.
12. A process as claimed in claim 1 wherein in step (h), the substituted benzaldehyde is selected from electron withdrawing or donating or combination thereof, preferably mono or disubstituted benzaldehyde.
13. A process as claimed in claim 1 in step (h), the refluxing is performed for 24 hours.
14. Use of diallyldisulphide derivatives having formula I and their pharmaceutically acceptable salts thereof for preparing a pharmaceutical composition comprising an effective amount of diallyldisulphide derivatives having formula I or their pharmaceutically acceptable salts of in combination with pharmaceutically acceptable ingredients.
15. Use as claimed in claim 14, wherein the said composition is administered to a subject such as animals, mammals, and in particular human beings.
16. Use as claimed in claim 14, wherein the said composition is administered to pathological conditions arising due to high cholesterol levels and other cardiovascular risk factors.
17. Use as claimed in claim 14, wherein ED50 value of active diallyl disulphide derivatives is in the range of hypolipidemic activity 20 mg/kg body weight and antioxidant activity 20 mg/kg body weight.
18. Use as claimed in claim 14, wherein pharmaceutically acceptable ingredient is selected from a group consisting of carrier, diluent, solvent, filter, lubricant, excipient, binder or stabilized.
19. Use as claimed in claim 14, wherein the preferred dosage of the composition is in the range of 20 mg/kg body weight.
20. Use as claimed in claim 14, wherein the diallyldisulphide derivatives may be administered systematically or orally.
21. Use as claimed in claim 14, wherein the preferred dosage is in the range of 20 mg/kg body weight.
PCT/IN2003/000250 2001-11-15 2003-07-24 Diallyldisulphide compounds having antilipidemic and antioxidant activity Ceased WO2004039771A1 (en)

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WO2006027711A3 (en) * 2004-08-26 2007-03-15 Nicholas Piramal India Ltd Prodrugs and codrugs containing bio- cleavable disulfide linkers
JP2008510795A (en) * 2004-08-26 2008-04-10 アッパラオ・サティアム Novel biocleavable linker
US7932294B2 (en) 2004-08-26 2011-04-26 Apparao Satyam Prodrugs containing novel bio-cleavable linkers
EP2266623A3 (en) * 2004-08-26 2011-10-12 Piramal Life Sciences Limited Prodrugs containing novel bio-cleavable linkers
EP2075011A3 (en) * 2004-08-26 2012-02-29 Piramal Life Sciences Limited Prodrugs containing bio-cleavable disulfide linkers
US8349901B2 (en) 2004-08-26 2013-01-08 Piramal Enterprises Limited and Apparao Satyam Prodrugs containing novel bio-cleavable linkers
US8354455B2 (en) 2004-08-26 2013-01-15 Piramal Enterprises Limited and Apparao Satyam Prodrugs containing novel bio-cleavable linkers
US8357723B2 (en) 2004-08-26 2013-01-22 Piramal Enterprises Limited and Apparao Satyam Prodrugs containing novel bio-cleavable linkers
WO2017081314A1 (en) 2015-11-12 2017-05-18 L'oreal Cationic direct dye comprising an aliphatic chain and bearing a disulfide/thiol/protected-thiol function for dyeing keratin fibres
US11096880B2 (en) 2017-06-16 2021-08-24 L'oreal Process for dyeing keratin fibres using at least one direct dye and at least one disulfide, thiol or protected-thiol fluorescent dye
US11278482B2 (en) 2017-06-16 2022-03-22 L'oreal Process for dyeing keratin materials using at least one blue, purple or green dye and at least one disulfide, thiol or protected thiol fluorescent dye

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