EP1259482A2 - Sulfur containing compounds - Google Patents

Sulfur containing compounds

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Publication number
EP1259482A2
EP1259482A2 EP01909377A EP01909377A EP1259482A2 EP 1259482 A2 EP1259482 A2 EP 1259482A2 EP 01909377 A EP01909377 A EP 01909377A EP 01909377 A EP01909377 A EP 01909377A EP 1259482 A2 EP1259482 A2 EP 1259482A2
Authority
EP
European Patent Office
Prior art keywords
ssch
compound
disulphide
compounds
mmol
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP01909377A
Other languages
German (de)
French (fr)
Inventor
Richard Francis Langler
Felix J. Baerlocker
Linda Z. c/o Ontario Cancer Institute Penn
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University Health Network
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University Health Network
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Filing date
Publication date
Priority claimed from CA002299247A external-priority patent/CA2299247A1/en
Application filed by University Health Network filed Critical University Health Network
Publication of EP1259482A2 publication Critical patent/EP1259482A2/en
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • C07C323/12—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 being acyclic and saturated
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/10—Antimycotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C313/00—Sulfinic acids; Sulfenic acids; Halides, esters or anhydrides thereof; Amides of sulfinic or sulfenic acids, i.e. compounds having singly-bound oxygen atoms of sulfinic or sulfenic groups replaced by nitrogen atoms, not being part of nitro or nitroso groups
    • C07C313/02—Sulfinic acids; Derivatives thereof
    • 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
    • C07C323/22—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 doubly-bound oxygen atoms bound to the same carbon skeleton
    • 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
    • C07C323/64—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 sulfur atoms, not being part of thio groups, bound to the same carbon skeleton
    • C07C323/65—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 sulfur atoms, not being part of thio groups, bound to the same carbon skeleton containing sulfur atoms of sulfone or sulfoxide groups bound to the carbon skeleton

Definitions

  • a process for preparing a compound of the formula PhS0 2 CH 2 SSCH 3 wherein Ph is phenyl which comprises reacting a transition metal oxidant with dimethyl disulphide to obtain an ⁇ -ester disulphide compound of the formula CH 3 SSCH 2 OC(0)CH 2 CH 3 which compound is further reacted with the sodium salt of p-toluenesulfmic acid in either aqueous acetonit ⁇ le or aqueous acetone to obtain the title compound or the compound is further reacted with potassium p- toluenesulfonate to yield the title compound
  • Another novel compound is of the formula CISCH 2 OC(0)CH 2 CH 3 , which is useful as an intermediate
  • Another part of the invention comprises antifungal agents comprising as active ingredients a therapeutically effective amount of at least one compound of the formula
  • R is CH 3 , CH 2 CH 3 , C 6 H 5 , o-CH 3 0 2 C(C 6 H 4 ), o-CH 3 S0 2 (C 6 H 4 ), p-CH 3 S0 2 (C 6 H 4 ), o- N0 2 (C 6 H 4 ), m-N0 2 (C 6 H 4 ), p-N0 2 (C 6 H 4 ), or CH 2 0 2 CCH 2 CH 3
  • X is S0 2 (C 6 H 4 )CH 3 -p, S0 2 CH 3 , S0 2 C 6 H 5 , S0 2 CH 2 CH 3 , H, 0 2 CCH 3 , 0 2 CCH 2 CH 3 , or
  • antifungal agents as described above wherein the active ingredient is selected from the group of compounds consisting of o-CH 3 OC(0) (C 6 H 4 ) SSCH 3 , [o-CH 3 S0 2 (C 6 H 4 )S] 2 , [p-CH 3 S0 2 (C 6 H 4 )S] 2 , m-0 2 N (C 6 H 4 ) SSCH 3 , o-0 2 N (C 6 H 4 ) SSCH 3 , p- 0 2 N (C 6 H 4 ) SSCH 3 , p- CH 3 (C 6 H 4 ) S0 2 CH 2 SSCH 3 , CH 3 S0 2 CH 2 SSCH 3 ,
  • Another aspect of the invention provides the use as described above wherein the agent is for the treatment of aspergillosis
  • Leukemic cells were seeded in a 24-well plate (Nunc, Naperville, IL) at 25x10 4 cells/mL Cells were then exposed in triplicate to solvent control or the approximate MTT50 concentration of compounds F (10 ⁇ g/mL), G (10 ⁇ g/mL) or I (5 ⁇ g/mL) The final solvent volume was 2 4 ⁇ L/mL of media for trypan blue exclusion assay The compound was replenished after 48 h of treatment Cell counts were evaluated using a 1 1 dilution of cell suspension in trypan blue (Gibco-BRL, Mississauga, Ontario, Canada) Viable and nonviable cells were counted using a hemocytometer Cells which excluded trypan blue were counted as viable whereas stained cells were counted as nonviable Trypan blue exclusion graphs shown are a representative experiment
  • Adherent cell lines were plated at 35 x 10 4 cells/60 cm 2 dish the day before exposure to compound Suspension cells were seeded at 25 x 10 4 cells/mL in a 6 well dish (Falcon) the day of exposure to compound Mononucleated, T-cell depleted normal bone marrow cells were seeded at 50 x 10 4 cells/mL in a 6 well dish the day of exposure to compound Cells were exposed to solvent control or approximate MTT50 and MTT30 concentrations of compound After 48 h of exposure to the compound, cells were harvested and fixed with a final concentration of 4% formaldehyde Fixed cells were stored at -20°C in 70% ethanol for no more than 5 days Approximately 10 5 cells were pelleted and labeled with 0 02 mM Biotin-dUTP and 12 5 U TdT enzyme in a 1x reaction buffer (200 mM potassium cacodylate, 25 mM Tns-HCI, 25 ⁇ g/mL bovine serum albumin, pH 6 6),
  • Figure A-1 illustrates a MTT Assay with Leukemic cell lines, WI38 and normal bone marrow
  • Figure A-2 illustrates Fixed Propidium Iodide Profiles
  • Figure D-2 illustrates Fixed Propidium Iodide Profiles
  • Figure E-1 illustrates a MTT Assay with Leukemic cell lines, WI38 and normal bone marrow,
  • FIG. E-2 illustrates Fixed Propidium Iodide Profiles
  • Figure F-1 illustrates a MTT Assay with Leukemic cell lines, WI38 and normal bone marrow,
  • Figure G-1 illustrates a MTT Assay with Leukemic cell lines, WI38 and normal bone marrow,
  • Figure H-2 illustrates a Trypan Blue Exclusion Assay
  • FIG. 1-3 illustrates Fixed Propidium Iodide Profiles
  • Figure J-1 illustrates a MTT Assay with Leukemic cell lines, WI38 and normal bone marrow;
  • FIG. 1 illustrates Fixed Propidium Iodide Profiles
  • Figure K-1 illustrates a MTT Assay
  • Figure L-1 illustrates a MTT Assay
  • Figure L-2 illustrates a MTT Assay with Normal Bone Marrow
  • Figure M-1 illustrates a MTT Assay
  • Figure M-2 illustrates a MTT Assay with Normal Bone Marrow
  • FIG. M-3 illustrates Fixed Propidium Iodide Profiles
  • Figure 0-1 illustrates a MTT Assay
  • Figure 0-2 illustrates a MTT Assay with Normal Bone Marrow
  • step (B) Impure sulphide methanesulfonate (3 8 g) from step (A) was dissolved in chloroform (75 ml), and the solution added dropwise to 10% sulfu ⁇ c acid (104 ml)
  • Me 2 SO (2 ml) was added to powdered sodium hydroxide (0.10 g, 2.5 mmol) and the mixture stirred vigorously.
  • 4-Th ⁇ apentane-l-th ⁇ ol 4,4-d ⁇ ox ⁇ de (0.38 g, 2.5 mmol) in Me 2 SO (4 ml) was added to the reaction mixture which was stirred for 5 mm.
  • Dimethyl disulphide (0.69 g, 7.3 mmol) in Me 2 SO (4 ml) was added and the reaction mixture stirred at room temperature for 24 h.
  • Hydrochloric acid (2.5%, 150 ml) was added and the resultant mixture washed with diethyl ether (three 100 ml aliquots).
  • Oily PhS0 2 CH 2 SSCH 3 (obtained in 58% yield) has i r 1325, 1155 cm 1 H n m r (270 MHz) ⁇ 2 49, s, 3H, 4 22, s, 2H, 7 60, t, 2H 7 70, t, 1 H 7 96, d, 2H 13 C n m r ⁇ 23 68, 64 32, 128 96, 129 30, 134 23, 137 62 m/z 234 (5%, M + ), 125 (28%), 93 (100%)
  • Oily CH 3 S0 2 CH 2 SSCH 3 8 9 (obtained in 53% yield) had i r 1320, 1 145 cm 1 1 H n m r (270 MHz) ⁇ 2 60, s, 3H, 3 05, s, 2H, 4 16, s, 2H 13 C n m r ⁇ 23 61 , 39 33, 61 38 m/z 172 (10%, M + ), 93 (100%)
  • the ⁇ -sulfone disulphide (4) (0.20 g, 0.81 mmol) was dissolved in a solution of thiophenol (0.19 g, 1.7 mmol) in dry methylene chloride (10 ml). Dry py ⁇ dine (0.1 ml) was added and the reaction mixture stirred at ambient temperature for 2 h 10 mm. The solvent was evaporated and the residue chromatographed on silica gel (10 g) employing chloroform (5 ml fractions) for elution. Fractions 3 and 4 were combined and dissolved in chloroform (100 ml).
  • the sulphide ⁇ -thioacetate (8) (1.0 g, 4 7 mmol) and hydrogen peroxide (30%, 1.1 g) were added to 1 ,4-d ⁇ oxan (25 ml) and the reaction mixture refluxed for 0.5 h. The solvent was evaporated and chloroform (150 ml) added. The chloroform solution was dried (MgS0 ), filtered and concentrated.
  • the disulphide propionate (2) (2.0 g, 12.0 mmol) and potassium p-toluenethiosulfonate (2.7 g, 11.9 mmol) were dissolved in 1 4 water/acetone (30 ml) and the reaction heated at 50°C for 2 h. Chloroform (200 ml) was added and the resultant mixture extracted with water (100 ml). The organic layer was dried (MgS0 4 ), filtered and the solvent evaporated. The residue was chromatographed on silica gel (200 g) employing 1.1 chloroform/light petroleum (100 ml fractions) for elution.
  • benzenesulfonyl chlorides can generally be reduced (L ⁇ AIH 4 ) to the corresponding mercaptans (Fong, H. O., Hardstaff, W. R., Kay, D. G., Langler, R F., Morse, R. G., and Sandoval, D. N., Can. J.
  • arylate thiolate anions Ginige, K A , Goehl, J E , and Langler, R F , Can J Chem , 1996, 74, 1638, Baum, J C , Bolhassan, J , Langler, R F , Pujol, R J , and Raheja, R K , Can J Chem , 1990, 68, 1450
  • dimethyl sulfoxide or hexamethylphosphoramide e g see Scheme 8
  • Powdered sodium hydroxide (0.24 g, 6 mmol) was suspended in dimethyl sulfoxide (8 ml) and a solution of o-mercapto methyl benzoate (1.0 g, 5.9 mmol) in dimethyl sulfoxide (5 ml) added.
  • the reaction mixture was stirred for 5 mm and a solution of dimethyl disulphide (1.7 g, 18 mmol) in dimethyl sulfoxide (7 ml) added.
  • the reaction mixture was stirred for 24 h at ambient temperature.
  • the high-boiling distillation fraction (b p 80-140°C/1 6 Torr) contained a mixture of the disulphide benzoate (4) (2.1 g, 9 8 mmol) and dibenzoyi anhydride (2.1 g)
  • the distillation residue was determined to contain the disulphide benzoate (4) (1.5g, 7.0 mmol) and dibenzoyi anhydride (9 1 g). Distillation, at atmospheric pressure, of the condensate from the cold trap, furnished a mixture which contained methyl methanethiosulfonate (0 7 g).
  • Methyl thioglycollate (1 896 g, 15 8 mmol) was added to dry methylene chloride (10 ml)
  • CISCH 2 OC(0)C 2 H 5 (2 44 g 15 6 mmol) was dissolved in dry methylene chloride and the resultant solution added to the reaction mixture
  • Dry pyridme (2 5 ml) was added and the reaction mixture stirred at ambient temperature for 24 h
  • Compound Q is a potent antifungal compound. It's preparation is described in the preprint "New Antifungal Disulphides: Approaching Submicrogram Toxicity", F.J.
  • Phenacyl chloride (1 004 g) was added to dry pyridme (4 ml) Thiolacetic acid (0 560 g) was dissulved in dry pyridme (6 ml) and added to the reaction mixture The reaction flask was fitted with a drying tube and the reaction mixture heated at 800°C for 1 5 h Chloroform (200 ml) was added and the resultant mixture extracted with 5% hydrochloric acid (150 ml), followed by 2 5% sodium hydroxide (100 ml) The organic layer was dried (MgS0 4 ), filtered and concentrated The product was chromatographed on silica gel (5 g) employing petroleum ether (400 ml) for elution Evaporation of the solvent afforded an orange oil
  • Phenacyl thiolacetate (4 157 g) was added to the reaction vessel dropwise Cooling and stirring continued for another 30 mm Diethyl ether (205 ml) and water (200 ml) were added and the reaction mixture cooled for another 30 mm Iodine (3 590 g) was added in small portions over 30 mm Saturated sodium thiosulfate solution (16 ml) was added
  • Phenacyl disulphide (1 603 g) and dimethyl disulphide were combined and a portion of the DMSO solution (1 ml) was added The reaction mixture was stirred at ambient temperature for 8 days 2 5% Hydrochloric acid (70 ml) was added The resultant mixture was extracted with diethyl ether (three - 50 ml aliquots) The combined organic layers were dried (MgS0 4 ), filtered and concentrated
  • the pharmaceutical composition is preferably administered in unit dosage forms
  • the pharmaceutical composition of the present invention may be administered orally, parenterally (e g intravenously), locally (e g transdermally), or rectally
  • dosage forms suited for respective routes of administration should be selected
  • Oral administration may be carried out using solid or liquid unit dosage forms such as bulk powders, powders, tablets, dragees, capsules, granules, suspensions, solutions, syrups, drops, subhngual tablets, etc
  • Bulk powders may be manufactured by comminuting the active substance into a finely divided form
  • Powders may be manufactured by comminuting the active substance into a finely-divided form and blending it with a similarly comminuted pharmaceutical carrier, e g an edible carbohydrate such as starch or mannitol
  • a cor ⁇ gent, a preservative, a dispersant, a coloring agent, a perfume, etc may also be added
  • Capsules may be manufactured by filling said finely-divided bulk powders or powders, or granules described below for tablets, in capsule shells such as gelatin capsule shells Preceding the filling operation, a lubricant or a fluidizing agent, such as colloidal silica, talc, magnesium stearate, calcium stearate or solid polyethylene giycol, may be blended with the powders Improvement in the efficacy of the drug after ingestion may be expected when a disintegrator or a solubilizer, such as carboxymethylcellulose, carboxymethylcellulose calcium, low-substitution-degree hydroxypropylcellulose, roscarmellose sodium, carboxymethylstarch sodium, calcium carbonate or sodium carbonate, is added
  • Soft capsules may be provided by suspending said finely divided powders in vegetable oil, polyethylene giycol, glycerin, or a surfactant and wrapping the suspension in gelatin sheets Tablets may be manufactured by adding an excipient to said powders, granulating or slugging the mixture, adding a disintegrator and/or a lubricant, and compressing the
  • a powdery mixture may be prepared by mixing said finely divided powders with said diluent or a base Where necessary, a binder (e g carboxymethylcellulose sodium, methylcellulose, hydroxypropylmethylcellulose, gelatin, poiyv ylpyrrolidone, poiyvmyl alcohol, etc ), a dissolution retardant (e g paraffin), a reabsorption agent (e g quaternary salts), and an adsorbent (e g bentonite, kaolin, dicalcium phosphate, etc ) may be added.
  • a binder e g carboxymethylcellulose sodium, methylcellulose, hydroxypropylmethylcellulose, gelatin, poiyv ylpyrrolidone, poiyvmyl alcohol, etc
  • a dissolution retardant e g paraffin
  • a reabsorption agent e g quaternary salts
  • an adsorbent e g bentonite, kaolin,
  • the unit dosage formulation for oral administration may be microencapsulated This formulation may be coated or embedded in a polymer, wax or other matrix to provide a prolonged action or sustained release dosage form
  • Parenteral administration may be carried out using liquid unit dosage forms for subcutaneous, intramuscular, or intravenous injection, e g solutions and suspensions
  • Such unit dosage forms may be manufactured by suspending or dissolving a predetermined amount of the compound of the invention in an injectable nontoxic liquid vehicle, for example an aqueous vehicle or an oily vehicle, and sterilizing the resulting suspension or solution
  • a nontoxic salt or salt solution may be added Moreover, stabilizers, preservatives, emulsifiers, etc may also be added
  • Rectal administration may be carried out by using suppositories manufactured by dissolving or suspending the compound in a low-melting water-soluble or watennsoluble
  • SUBSTTTUTE SHEET solid carrier such as polyethylene giycol, caccao butter, semisynthetic oil (e g Witepsol®), a higher ester (e g my ⁇ styl palmitate) or a mixture thereof

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Abstract

This invention is directed to novel and known sulfur containing compounds and pharmaceutically acceptable salts thereof that have utility as antifungals and as antiproliferative agents against mammalian cells, in particular cancer cells and most particularly leukemia-derived cells. The invention provides a method for synthesizing certain of the sulfur containing compounds that is more efficient than previously known methods.

Description

TITLE
Sulfur Containing Compounds RELATED APPLICATIONS
This application claims the benefit of the priority of the filing dates of US Provisional Patent Application No 60/185,189 filed February 25, 2000 and Canadian Patent Application No 2,299,247, filed February 25, 2000 FIELD OF THE INVENTION
This invention is directed to novel and known sulfur containing compounds and pharmaceutically acceptable salts thereof that have utility as antifungal agents and as antiproliferative agents against mammalian cells in particular cancer cells and most particularly leukemia-derived cells The invention provides a method for synthesizing certain of the sulfur containing compounds that is more efficient than previously known methods BACKGROUND OF THE INVENTION There is an enormous need worldwide for novel, safe, effective therapeutics in the clinical treatment of cancers The majority of chemotherapeutics presently available are less than ideal as they show non-specific, genotoxic killing of both normal as well as tumour cells Recent success stories suggest natural products research will uncover new molecules to help fight the cancer problem (Cardenas, M E , Sanfπdson, A , Cutler, N S , and Heitman, J (1998) Signal-transduction cascades as targets for therapeutic intervention by natural products Trends Biotechnol 16 427-33, Marks, P A , Richon, V M and Rifkind, R A (2000) Histone deacetyiase inhibitors mducers of differentiation or apoptosis of transformed cells J Natl Cancer Inst 92 1210-6) For example, the taxanes (taxol), derived from the bark of the yew tree, have emerged as effective anti-tumour agents in a wide variety of malignancies (Vaishampayan, U , Parchment, R E , Jasti, B R , and
Hussain, M (1999) Taxanes an overview of the pharmacokinetics and pharmacodynamics Urology 54, 22-9, Walsh, V , and Goodman, J (1999) Cancer chemotherapy, biodiversity, public and pπvate property the case of the anti-cancer drug taxol Soc Sci Med 49, 1215- 25) Enormous experimental effort has led to the identification of organosulfur compound (OSCs) as the active components of medicial plants such as garlic, onions, the leaves of the Mahogany tree, yet this knowledge is primarily applied to nutritional aspects of cancer prevention strategies and not directly to acute treatment protocols (Fukushima, S , Takada, N , Hon, T , and Wanibuchi, H (1997) Cancer prevention by organosulfur compounds from garlic and onion J Cell Biochem Suppl 27, 100-5, Jiao, D , Smith, T J , Yang, C S , Pittman, B , Desai, D , Amin, S , and Chung F L (1997) Chemopreventive activity of thiol conjugates of isothiocyanates for lung tumoπgenesis Carcinogenesis 18, 2143-7 Reddy, B S , Rao, C V , Rivenson, A , and Kelloff, G (1993) Chemoprevention of colon carcinogenesis by organosulfur compounds Cancer Res 53, 3493-8, Wargovich, M J (1987) Diallyl sulphide, a flavor component of garlic (Allium sativum), inhibits dimethylhydrazine-induced colon cancer Carcinogenesis 8, 487-9, Wargovich, M J , Imada O , and Stephens, L C (1992) Initiation and post-initiation chemopreventive effects of diallyl sulphide in esophageal carcinogenesis Cancer Lett 64, 39-42, Wargovich, M J , Woods, C , Eng V W , Stephens, L C and Gray, K (1988) Chemoprevention of N- nitrosomethylbenzylamine-induced esophageal cancer in rats by the naturally occurring thioether, diallyl sulphide Cancer Res 48, 6872-5) It appears that the knowledge gained from natural products research is not widely and immediately exploited for the treatment of cancer primarily because of technical limitations For instance, only a very low yield of active metabolite can be recovered from typical natural sources, the extraction procedure often leads to mixtures of structural relatives of varying specific activity compared to the target natural product, there can be environmental issues associated with harvesting medicinal plants, and mining the active component is often labour-intensive, difficult to quality assure and is expensive to produce in large quantities Numerous studies have shown that OSCs have antiproliferative potential (Jogia, MK, Andersen, RJ, Mantus, EK and Clardy, J (1989) Dysoxysulfone, a sulfur rich metabolite from the Fijian medicinal plant Dysoxylum Richn Tetrahedron Letters 30 4919-4920, Block, E, DeOrazio, R and Thiruvazhi, M (1994) Simple total synthesis of biologically-active pentathiadecane natural- products, 2,4,5,7,9-pentathιadecane 2,2,9,9-tetraoxιde (dysoxysulfone), from Dysoxylum- Richu, and 2,3,5,7,9-pentathιadecane 9,9-dιoxιde, the misidentified lenthionme precursor Se-3 from Shiitake mushroom (Lentinus-Edodes) J Org Chem 59 2273-2275, Perchellet, JP, Perchellet, EM and Belman, S (1990) Inhibition of DMBA-induced mouse skin tumoπgenesis by garlic oil and inhibition of two tumor-promotion stages by garlic and onion oils Nutr Cancer Λ 183-93) Despite the documented biological activity of OSC (Block, E The organosulfur chemistry of the genus Allium - implications for the organic chemistry of sulfur Angew Chem Int Ed. Engl 31 1135-1178, 1992, Lea, MA Organosulfur compounds and cancer Adv Exp Med Biol 401 147-54, 1996), the key structural features that directly contribute to their antiproliferative activity remains unclear
There is also a need for effective antifungal agents that are readily biodegradable and which can be used against a wide variety of pathogens in both animals and humans There are known sulfur containing compounds which have been found to be useful as antifungal agents For example, U.S. Patents No. 5,648,354 issued July 15, 1997 to Bierer, et al and No. 5,580,897 issued December 3, 1996 to Bierer, et al and No. 5,583,235 issued December 10, 1996 to Bierer, et al disclose novel 1 ,2-dιthιιn compounds having such utility In U.S. Patent No. 5,698,564 issued December 16, 1997 to Katsuyama, et al there are described diphenyl disulphide compounds having an inhibiting activity against the production of lnterleukιn-1 β (IL-1 β) or the release of Tumor Necrosis Factor α (TNFα), which are useful in the treatment or prophylaxis of diseases such as chronic rheumatism and sepsis The literature contains a number of papers disclosing antifungal compounds of the type described herein Baerlocher, Felix Jacob, et al Aust J Chem 1999, 52, 167-172 entitled Structure-Activity Relationship for Selected Sulfur-Rich Antifungal Compounds reported that the inhibition of fungal growth correlates with the presence of both sulfone and disulphide functional properties The disclosures of this paper are incorporated herein by reference
In Langler, Richard Francis, et al Aust J Chem published Feb 2000, and entitled A New Synthesis for Antifungal -Sulfone Disulphides, there is described the preparation of several new α-sulfone disulphides using an α-ester disulphide precursor These α-sulfone disulphides were all shown to be fungitoxic against Aspergillis niger and Aspergillis flavus The disclosures of this paper are incorporated herein by reference
In 1989, Andersen et al reported the isolation and structure proof of dysoxysulfone (CH3S02CH2SCH2SSCH2S02CH3) (see Tetra Lett 30, 4919 (1989)) Sample size was limited and the compound showed some antibiotic activity In 1989, Block et al described a synthesis for dysoxysulfone, which provided larger amounts (see Block et al , J Org Chem 59, 2273 (1994)) They showed that dysoxysulfone and some related natural products were active against Candida albicans, a P388 muπne leukemia cell line, Staphylococcus aureus, Bacillus subtilis, a human adrenocarcinoma cell line and a human ovarian carcinoma cell line U.S. Patent No. 4,643,994 granted to Block et al described sulfur compounds like 3 as antithrombotic agents
RS02CH2CH=CHSSR' 3 The patent describes the use of these compounds against "bacteria and fungi", as well as for "flavor enhancers in foods" In Block's patent, compounds like 2 of the formula RS02CH2SSR' were inadvertently included In 1987, Block et al obtained a Certificate of Correction, which withdrew compounds like 2 from the patent The following publications are relevant to the present invention Langler, Richard Francis, et al A New Synthesis for Antifungal α-Sulfone Disulphides Aust J Chem , 1999, 52, 1119-1121 Wong, W Wei-Lynn, et al Novel Synthetic Organosulfur Compounds Induce
Apoptosis of Human Leukemic Cells Anticancer Research 20 1367-1374 (2000)
Baerlocher, Felix Jakob, et al Structure-Activity Relationships for Selected Sulfur- Rich Antifungal Compounds Aust J Chem , 1999, 52, 167-172
Baerlocher, Felix Jakob, et al Antifungal Thiosulfonates Potency with Some Selectivity Aust J Chem , 2000, 53, 399-402
Baerlocher, Felix Jakob, et al New and More Potent Antifungal Disulphides Aust J Chem , 2000, 53, 1-5
"New Antifungal Disulphides Approaching Submicrogram Toxicity", F J Baerlocher,, M O Baerlocher, C L Chaulk, R F Langler and E M O'Brien, Sulfur Lett , - in press The disclosures of these references are incorporated herein by reference SUMMARY OF THE INVENTION
In one aspect of the invention, there are provided novel sulfone disulphides of the general formula RS02CH2SSR1 I wherein R is phenyl or lower alkyl, and R1 is lower alkyl or phenyl
This invention provides specific novel sulfone disulphides of this general formula selected from the group consisting of C6H5S02CH2SSCH3, CH3S02CH2SSCH3, and CH3S02CH2SSC6H5 Another aspect of the invention provides a process for preparing a sulfone disulphide of the general formula I as described above, which comprises reacting a transition metal oxidant or a peroxyanhydπde oxidant with a symmetrical dialkyl or arylalkyl disulphide to obtain an α-ester disulphide compound of the formula
RSSCH2OC(0)R1 wherein R and R1 are as defined above, which compound is further reacted with a sulfinic acid salt to obtain the title compound
The invention also provides compounds of the general formula RSSCH2OC(0)R1, wherein R and R1 may be the same or different and each is selected from the group of substituents comprising lower alkyl or phenyl Preferably, lower alkyl is methyl or ethyl The invention also provides a process for making the compounds of the formula defined above, which comprises reacting a transition metal oxidant or a peroxyanhydπde oxidant with a symmetrical dialkyl or arylalkyl disulfide to obtain the α-ester disulfide compound
In yet another aspect of the invention, there is provided a process for preparing a compound of the formula PhS02CH2SSCH3, wherein Ph is phenyl which comprises reacting a transition metal oxidant with dimethyl disulphide to obtain an α-ester disulphide compound of the formula CH3SSCH2OC(0)CH2CH3 which compound is further reacted with the sodium salt of p-toluenesulfmic acid in either aqueous acetonitπle or aqueous acetone to obtain the title compound or the compound is further reacted with potassium p- toluenesulfonate to yield the title compound
Another aspect of the invention provides a process for preparing compounds of the formula RS02CH2SSR1.wherein R and R1 may be the same or different and each is selected from lower alkyl and phenyl, which comprises reacting a transition metal oxidant or a peroxyanhydπde oxidant with a symmetrical dialkyl or arylalkyl disulfide to obtain the α- ester disulfide compound RSSCH2OC(0)R1.wherein R and R1 are as defined above, which compound is further reacted with a sulfinic acid salt to obtain the required compound
Another novel compound is of the formula CISCH2OC(0)CH2CH3, which is useful as an intermediate Another part of the invention comprises antifungal agents comprising as active ingredients a therapeutically effective amount of at least one compound of the formula
RSSCHX I
R1 wherein R1 is H or CH3,
R is CH3, CH2CH3, C6H5, o-CH302C(C6H4), o-CH3S02(C6H4), p-CH3S02(C6H4), o- N02(C6H4), m-N02(C6H4), p-N02(C6H4), or CH202CCH2CH3, and X is S02(C6H4)CH3-p, S02CH3, S02C6H5, S02CH2CH3, H, 02CCH3, 02CCH2CH3, or
C02CH3, and a pharmaceutically acceptable carrier
Another aspect of the invention provides antifungal agents comprising as active ingredient at least one antifungally active compound of the formulae RS02CH2SSR1 wherein R is lower alkyl or phenyl and R1 is lower alkyl or phenyl, and
RC(0)OCH2SSR1 wherein R and R1 are as defined above optionally together with pharmaceutically acceptable carriers In yet another aspect of the invention, there is provided antifungal agents as described above wherein the active ingredient is selected from the group of compounds consisting of o-CH3OC(0) (C6H4) SSCH3, [o-CH3S02 (C6H4)S]2, [p-CH3S02 (C6H4)S]2, m-02N (C6H4) SSCH3, o-02N (C6H4) SSCH3, p- 02N (C6H4) SSCH3, p- CH3 (C6H4) S02CH2 SSCH3, CH3S02CH2SSCH3,
CH3CH2C(0)OCH2SSCH3, CH3S02CH2SSPh, CH3S02CH2SSCH2CH3, CH3SSCH2OC(0)CH3, CH3SSCH2OC(0)CH2CH3,
CH3SSCH2OC(0)Ph, and PhSSCH2OC(0)CH2CH3, optionally together with a pharmaceutically acceptable carrier Another aspect of the invention provides an antiproliferative agent active against mammalian cells comprising as active ingredient at least one compound of the formulae
RS02CH2SSR1 wherein R is lower alkyl or phenyl and R1 is lower alkyl or phenyl, and RC(0)OCH2SSR1 wherein R and R1 are as defined above, and excluding those compounds disclosed herein that do not exhibit such activity, and optionally together with conventional pharmaceutically acceptable ingredients
Another aspect of the invention provides an antiproliferative agent active against mammalian cells comprising as active ingredient at least one compound selected from the group of compounds consisting of p-CH3(C6H4)S02CH2SSCH3l
PhSSPh,
CH3S02CH2SSPh, CH3S02CH2CH2SSCH3,
CH3SSCH2C(0)OCH3, and
CH3SSCH2OC(0)CH3, and optionally together with conventional pharmaceutically acceptable ingredients
In yet another aspect of the invention, there is provided the use of at least one compound of the formulae
RS02CH2SSR (I) wherein R is lower alkyl or phenyl and R1 is lower alkyl or phenyl, and
RC(0)OCH2SSR1 (II) wherein R and R1 are as defined above, and excluding those compounds disclosed herein that do not exhibit such activity in the preparation of an antifungal agent for the treatment of micoses
Another aspect of the invention provides the use as described above wherein the agent is for the treatment of aspergillosis
The invention also provides the use of at least one compound of the formulae RS02CH2SSR1 (I) wherein R is lower alkyl or phenyl and R1 is lower alkyl or phenyl, and
RC(0)OCH2SSR1 (II) wherein R and R1 are as defined above, and excluding those compounds disclosed herein that do not exhibit such activity, in the preparation of an antiproliferative agent active against mammalian cells for the treatment of cancer
In yet another aspect of the invention, there is provided the use of at least one compound selected from the group of compounds as described above in the preparation of a medicament for the treatment of cancer DETAILED DESCRIPTION OF THE INVENTION
The following correlates the chemical structures and the chemical names of the compounds discussed in this application
Chemical Structure Chemical Name CH3S02CH3 dimethyl sulfone
CH3SSCH3 dimethyl disulphide CH3SCH2SCH2SCH3 2,4,6-trιthιaheptane CH3SO2CH2SCH3 2,4-dιthιapentane 2,2-dιoxιde CH3SO2CH2SCH2SO2CH3 2,4,6-trιthιaheptane 2,2,6,6-tetraoxιde CH3SCH2SSCH3 2,3,5-trιthιahexane
CH3S02CH2SSPh 5-phenyl-2,4,5-tπthιapentane 2,2-dιoxιde
CH3S02CH2SSCH2CH3 2,4,5-trιthιaheptane 2,2, -dioxide
CH3S02CH2CH2SSCH 2,3,6-trιthιaheptane 6, 6, -dioxide
CH3S0 CH2CH2CH2SSCH3 2,3,7-tπthιaoctane 7, -dιoxιde CH3SSCH2C(0)OCH3 methyl 3,4-dιthιopentanoate
CH3SSCH2OC(0)CH3 2,3-dιthιabutyl acetate
CH3SSCH2CH2OC(0)CH3 3,4-dιthιapentyl acetate p-CH3(C6H4)S02CH2SSCH3 α-p-toluenesulfonyl dimethyl disulphide
C6H5S02CH2SSCH3 α-phenylsulfonyl dimethyl disulphide CH3S02CH2SSCH3 -methylsulfonyl dimethyl disulphide p-CH3(C6H4)S02CH2SH α-p-toluenesulfonyl methyl mercaptan
CH3CH2C(0)OCH2SSCH3 2,3-dιthιabutyl propionate
C6H5SSCH3 phenyl methyl disulphide
C6H5SSPh diphenyl disulphide o-CH3OC(0)(C6H4)SSCH3 methyl-o(methyld ith ιo)benzoate
[o-CH3S02(C6H4)S]2 o-methylsulfonylphenyl disulphide
[p- CH3S02(C6H4)S]2 p-methylsulfonylphenyl disulphide -02N(C6H4)SSCH3 methyl m-nitrophenyl disulphide o-02N(C6H4)SSCH3 methyl o-nitrophenyl disulphide p-02N(C6H4)SSCH3 methyl p-nitrophenyl disulphide p-CH3S02(C6H4)0S02CH3 p-methylsulfonylphenyl methanesulfonate p-02N(C6H4)OS02CH3 p-nitrophenyl methanesulfonate
CH3SSCH2OC(0)CH3 2,3-dιthιabutyl acetate
CH3SSCH2OC(0)CH2CH3 2,3-dιthιabutyl propionate CH3SSCH2OC(0)Ph 2,3-dιthιabutyl benzoate
PhSSCH20C(0)CH2CH3 1-phenyl-1 ,2-dιthιapropyl propionate
(CH30C(0)CH2S)2 dimethyl 3,4-dιthιaadιpate
CH3OC(0)CH2SSCH2OC(0)C2H5 methyl 3,4-dιthιa-5-propιonoxypentanoate
(C2H5C(0)OCH2S)2 2,3-dιthιabutane-1 ,4-dιpropιonate C2H5OC(0)CH2SSCH2S02(C6H4)-p-CH3 1-p-toluenesulfonyl-4-propιonoxy-2 3-dιthιabutane (C6H5)C(0)CH2SSCH3 phenacyl methyl disulphide
Biological Testing Anti-fungal Activity
Sulfur compounds were tested for antifungal activity against pure cultures of Apergillus niger and Aspergillus flavus supplied by Ward's Natural Science Ltd (St Catharines, Ontario, Canada) They were maintained on Sabouraud Dextrose Agar
For the test, 6 agar plugs (5 mm diameter) were cut from a 5-8 day old colony and homogenized in distilled, sterilized water (2 ml) A portion of this suspension (0 5 ml) was transferred aseptically to a Petπ plate with Sabouraud Dextrose Agar (15 ml) and spread evenly over the entire surface Each plate was supplied with four evenly spaced paper disks (7 mm, Whatman Number 1 filter paper) containing the test compound (0 25 50 and 100 μg respectively) Each test compound was applied to the disks as a solution (50 mg compound/10 ml acetone) Control disks were treated with neat acetone (20 μl) Test plates with fungal homogenates and disks were incubated at 18-20°C for 2 days For each test four replicate plates were used The diameter of the clear zone surrounding the disk was taken as the indicator of antifungal activity
Reasonable levels of antifungal activity can be gauged from the result of (CH3SCH2S)2 which is a known antifungal natural product which shows clear zone diameters of 2 5 and 2 3 mm against A niger and A flavus, respectively, at a dose of 100 μg/disk
Compounds (8) and (9) show activity against Aspergillus niger and Aspergillus flavus comparable in magnitude to that reported for Dysoxysulfone (1 ) against Staphylococcus aureus, Bacillus subtilis and Candida albicans (Block, E , DeOrazio, R , and Thiruvazhi, M , J Org Chem , 1994, 59, 2273) It would appear that the inhibition of fungal growth correlates with the presence of both sulfone and disulphide functional groups
Qualitatively, the observed toxicity of the compounds in Table 4 is in complete accord with the earlier proposal (Baerlocher, F J , Langler, R F , Fredeπksen, M U , Georges, N M., and Witherell, R D , Aust J Chem, 1999, 52, 167) that activated antifungal disulphides (those with a reasonable leaving group attached to the α-carbon) will have pronounced antifungal activity Quantitatively, the first and last compounds in Table 4 are the most potent fungitoxic disulphides described to date
Toxicity testing against A. niger and A. flavus revealed that (5) is considerably more potent than any other compound disclosed herein. The simple disulphides (4) and (5) are completely inactive against A. niger and A. flavus at a dose of 100 μg/disk.
Compounds (4) and (5) in Table 6 are novel compounds.
Compounds (2), (3) and (4) of Table 7 are novel compounds. A novel method for preparing compounds (2) and (4) is described in detail in the section entitled "Preparatory Methods". Compound (3) is prepared via a known method and this is detailed in the aforementioned section. Compound (1 ) is a known compound and the literature contains many references to standaard mathods that can be used to produce it.
Amphotericin B was the most potent of the 3 commercial antifungals tested (included Nystatin and Griseofulvin).
Compound (4) had no measureabie fungitoxicity against A. niger at 100 μg/disk.
Thiosulfonates as Antifungal Agents
In exploring the development of new, potent antifungal disulphides (Baerlocher, F.J., Langler, R.F., Frederiksen, M.U., Georges, N.M., and Witherell, R.D., Aust. J. Chem., 1999, 52, 167, Langler, R F , MacQuarπe, S L , McNamara, R A , and O'Connor, P E , Aust J Chem , 52,1119 (1999), Baerlocner F J , Baerlocher, M O , Langler, R F , MacQuame, S L and Marchand, M E , Aust J Chem , 53 1 (2000), and Baerlocher, F J , Baerlocher, M O , Chaulk, C L , Langler, R F , and O'Brien, E M , Sulphur Letters - in press, the view that fungitoxicity is likely to be associated with biochemical sulfenylations accomplished by the present disulphides was adopted Thus, enhancing toxicity has meant making disulphides which are progressively more electrophilic at disulphide sulfur As an example phenyl methyl disulphide exerts no observable fungitoxicity at 100 μg/disk (Baerlocher, F J Baerlocher M O , Langler, R F , MacQuame S L , and Marchand, M E , Aust J Chem , 53 1 (2000)) while o-nitrophenyl methyl disulphide (2) has pronounced toxicity at a dose of 10 μg/disk (see Table 8) Since thiosulfonates are well-known sulfenylating agents for mercaptide aπions (see p 323 of Bere, C M , and Smiles, S , J Chem Soc , 1924, 125, 2361 ), selected thiosulfonates have been prepared and tested as potential fungitoxins
Dimethyl disulphide (10) shows no antifungal behaviour, while the two closely related thiosulfonates (5) and (11 ) have measureable toxicity (vide Table 8) Methyl methanethiosulfonate (11 ) is the first compound examined which is effective against only one of the test fungi Note that the enhanced toxicity of (5) is not unexpected since, up to a chain length of nine carbons, longer unbranced alkyl groups are known to enhance pharmacological effects (Silverman, R B , The Organic Chemistry of Drug Design and Drug Action' p 16 (Academic Press San Diego 1992))
For the series of compounds (4), (12), (13), the disulphide was inactive and the thiosulfonates antifungal Interestingly, the thiosulfonate (4) killed only A flavus in contrast to (11 ) which selectively inhibited the growth of A niger
Compounds (14) - (16) also demonstrated the superior antifungal potency of thiosulfonates relative to simple disulphides (see Table 1 ) but for this set, no selectivity for either of the fungi was observed
Compound (17) (Table 8) was the first of the potent second-generation antifungal disulphides to be considered here Perhaps surprisingly, the closely realted thiosulfonate (18) is not antifungal at all Similar results (dimmshed fungitoxicity for the thiosulfonates relative to the disulphide) were obtained for the set of compounds (6), (7), (8) It now appears that thiosulfonates tend to have moderate fungitoxicity - enhanced relative to inactive disulphides and diminished relative to more potent antifungal disulphides The results in Table 8 open up the possibility that there might be a pharmacological equivalent to the well-known Reactivity Selectivity Principle in organic chemistry (Lowry, T H , and Richarson, K S , 'Mechanism and Theory in Organic Chemistry' 3rd Ed , p 148 (Harper and Row New York 1987)) The pharmacological equivalent might assert that structural modifications which decrease the potency of a particular agent may be associated with enhanced selectivity in toxicity within a set of closely related organisms The interest in antifungal disulphides was encouraged by the recent observations (Pfaller, M , and Wenzell, R , Eur J Clm Microbiol Infect Dis 1992 11 287, Debono, M , and Gordee, R S , Ann Rev Microbiol , 1994, 48, 471 , and Sternberg, S , Science, 1994, 266, 1632) that fungal infections frequently prove to be lethal for immunocompromised patients It has recently been learned that (19), a disulphide related to the potent aryl disulphide fungitoxins (e g (7) and (17) in Table 8), has been patented for use in inhibiting the production of lnterleukιn-1 β and Tumor Necrosis Factor α (Katsuyama K Anga M Saito Y Hatanaka S , and Takahashi, T , U S Patent 5,698,564 (1997))
Finally, it has been reported earlier, that the α-ester disulphide (20) (see Table 8) had no antifungal activity (Baerlocher, F J , Langler, R F , Frederiksen, M U , Georges, N M , and
Witherell, R D , Aust J Chem , 1999, 52, 167) Nonetheless, when tested, it showed promise as a lead compound in fighting leukemia (Wong, W W L , MacDonald, S , Langler, R F , and Penn, L Z , Anticancer Research 20 1367) The related thiosulfonate (9) shows clearly enhanced antifungal activity In terms of currently-available sulfur-rich antifungal compounds, thiosulfonates show intriguing selectivity Anti-cancer Activity Summary of Results
The following tables group the tested compounds according to their demonstrated activities
Compounds P and Q are novel compounds Compound Q is compound (5) in Table 6 A new method for preparing compound Q is described in detail in the section entitled "Preparatory Methods"
MATERIALS AND METHODS
For references pertaining to methods, please refer to Wong, W W-L , Macdonald, S , Langler, R F , Penn, L Z Anticancer Res 20 1367, the disclosures of which are incorporated herein by reference The number of times each experiment was performed is listed in individual compound data sets Cell culture
All cell lines were assayed as asynchronously growing cells Leukemic cell lines, OCI-AML-2, OCI-AML-3 (referred to hereafter as AML-2 and AML-3, respectively), NB- 4, KK, B1 , G2, and W1 were cultured in alpha-minimal essential medium (α-MEM) (Princess Margaret Hospital Media Services) supplemented with 10% fetal bovine serum (FBS) (Sigma, St Louis, MO) Non-transformed, diploid fibroblast lines WI38 and IMR90, were cultured in α-MEM and MEM F-15, respectively supplemented with 10% FBS Media for IMR90 cells was also supplemented with 1 5 g/L bicarbonate and 1 mM pyruvate Breast tumour cell lines, MDA-231 , SK-BR-3, MCF-7, ZR-751 , and melanoma tumour cell lines, WM9, WM983, WM793, 1232, were grown in α-MEM supplemented with 10% fetal bovine serum Prostate tumour cell lines, DU145 and PC-3, were grown in RPMI 1640 media supplemented with 10% FBS All cell lines were cultured in the presence of penicillin/streptomycin Mononucleated cells were isolated from normal bone marrow using Ficoll hypaque and then T-cell depleted T-cell depletion was performed by incubating the mononucleated bone marrow cells in α- MEM at a concentration of 2 x 107 cells/mL with 10% absor FBS and 10% (v/v) sheep red blood cells for 4 to 16 h Absor FBS was prepared beforehand by heating FBS at 56°C for 1 h, incubating with sheep red blood cells at 0 5% (v/v) for 1 h and then filtering the FBS with a 0 2 μM filter The T-cell depleted, mononucleated cells were recovered using Ficoll hypaque and were maintained in α-MEM supplemented with
14
SUBSTTTUTE SHEET (RULE 26) 20% FBS and 10% 5637 conditioned media 5637 conditioned media was previously harvested from confluent 5637 cells after a 3 day incubation Normal bone marrow from bone marrow transplant donor was collected following informed consent according to institutional guidelines Organosulfur compound preparation for in vitro testing
Approximately 20 mg of each compound was dissolved in 5 mL of ACS grade acetone (Sigma) using a 5 mL volumetric flask Stock solutions of the compounds were stored in the dark at -20°C Compounds were diluted prior to each experiment MTT Assay Adherent cells were seeded at 67 x 103 cells/mL in a 96 well plate (Falcon,
Mississauga, Ontario) the day prior to exposure to compound Suspension cells were seeded at 27 x 104 cells/mL in a 96 well plate the day of exposure to compound Mononucleated, T-cell depleted normal bone marrow cells were seeded at 67 x 104 cells/mL in a 96 well plate the day of exposure to compound Compounds and solvent control were added to cells and assayed in triplicate or sextuplet Following 48 h of incubation at 37°C with 5% C02, 40 μL of a 5 mg/mL solution of 3-[4,5-dιrπethylthιazol-2- yI]-2,5-dιphenyltetrazolιum bromide (MTT) substrate (Sigma) in Dulbecco's phosphate- buffered saline (D-PBS) was added. After 4 h of incubation at 37°C with 5% C02, the resulting violet formazan precipitate was solubilized by the addition of 80 μL of a 0.01 M HCI, 10% sodium dodecyl sulfate (SDS, Sigma) solution overnight at 37°C with 5% C02 The plates were then analyzed using the BioRad Benchmark Microplate Reader (BioRad Laboratories, Hercules, California) at 570 nm to determine the optical density of the samples. MTT data was analyzed using Prism 3.0 (GraphPad Software, Inc., San Diego, California) by the Chou-Talalay method MTT graphs shown are a representative experiment.
Trypan Blue Exclusion Assay
Leukemic cells were seeded in a 24-well plate (Nunc, Naperville, IL) at 25x104 cells/mL Cells were then exposed in triplicate to solvent control or the approximate MTT50 concentration of compounds B, C, F, G or H at 10 μg/mL, or I at 5 μg/mL The final solvent volume was 2.4 μL/mL of media for trypan blue exclusion assay. The compound was replenished after 48 h of treatment. Cell counts were evaluated using a 1 :1 dilution of cell suspension in trypan blue (Gibco-BRL, Mississauga, Ontario, Canada) Viable and nonviable cells were counted using a hemocytometer. Cells which excluded trypan blue were counted as viable whereas stained cells were counted as nonviable Trypan blue exclusion graphs shown are a representative experiment Fixed Propidium Iodide (PI) Staining
Adherent cell lines were plated at 35 x 104 cells/60 cm2 dish the day before exposure to compound Suspension cells were seeded at 25 x 104 cells/mL in a 6 well dish (Falcon) the day of exposure to compound Mononucleated, T-cell depleted normal bone marrow cells were seeded at 50 x 104 cells/mL in a 6 well dish the day of exposure to compound Cells were exposed to solvent control or approximate MTT50 and MTT30 concentrations of the compound After 48 h of exposure to the compound, cells were harvested fixed in 80% ethanol for 1 h on ice and labeled with 50 μg/mL propidium iodide (Sigma) Approximately 106 cells were analyzed using a XL-MCL flow cytometer (Coulter Corporation Miami, Florida) and a FACScalibar cytometer (Becton Dickinson, San Jose, CA) Profiles shown are a representative Tdt-mediated dUTP-biotm nick end-labeling (TUNEL)
Adherent cell lines were plated at 35 x 104 cells/60 cm2 dish the day before exposure to compound Suspension cells were seeded at 25 x 104 cells/mL in a 6 well dish (Falcon) the day of exposure to compound Mononucleated T-cell depleted normal bone marrow cells were seeded at 50 x 104 cells/mL in a 6 well dish the day of exposure to compound Cells were exposed to solvent control or approximate MTT50 and MTT30 concentrations of the compound After 48 h of exposure to the compound, cells were harvested and fixed with a final concentration of 4% formaldehyde Fixed cells were stored at -20°C in 70% ethanol for no more than 5 days Approximately 105 cells were pelleted and labeled with 0 02 mM Biotin-dUTP and 12 5 U TdT enzyme in a 1x reaction buffer (200 mM potassium cacodylate, 25 mM Tπs-HCI, 25 μg/mL bovine serum albumin, pH 6 6), 2 5 mM CoCI2 and 0 01 mM dTTP (Roche Molecular Biochemicals, Laval, QC, Canada) for 45 mm at 37°C Samples were washed and incubated in 200 μL of 1 1000 fluorescein isothiocyanate (FΙTC)-conjugated avidin
(Sigma) in 4x SSC, 5% skim milk powder and 0 05% Tween-20 (Sigma) Following 1 h of mixing at room temperature, the samples were washed and resuspended in 500 μL of D-PBS containing 2 5 μg/mL DNase-free RNase (Boehnnger Mannheim) and 10 μg/mL PI Following a 30 mm incubation at room temperature, cells were analyzed using a XL-MCL flow cytometer (Coulter Corporation, Miami, Florida) and a FACScalibar cytometer (Becton Dickinson, San Jose, CA) Profiles shown are representative of one experiment
MATERIALS AND METHODS For references pertaining to methods, please refer to Wong, W W-L , Macdonald,
S , Langler, R F , Penn, L Z Anticancer Res 20 1367, the disclosures of which are incorporated herein by reference The number of times each experiment was performed is listed in individual compound data sets Cell culture
All cell lines were assayed as asynchronously growing cells Leukemic cell lines, OCI-AML-2, OCI-AML-3 (referred to hereafter as AML-2 and AML-3, respectively), NB- 4, KK, B1 , G2, and W1 were cultured in alpha-minimal essential medium (α-MEM) (Princess Margaret Hospital Media Services) supplemented with 10% fetal bovine serum (FBS) (Sigma, St Louis, MO) Non-transformed, diploid fibroblast lines WI38 and IMR90, were cultured in α-MEM and MEM F-15, respectively supplemented with 10% FBS Media for IMR90 cells was also supplemented with 1 5 g/L bicarbonate and 1 mM pyruvate Breast tumour cell lines, MDA-231 , SK-BR-3, MCF-7, ZR-751 , and melanoma tumour cell lines, WM9, WM983, WM793, 1232, were grown in α-MEM supplemented with 10% fetal bovine serum Prostate tumour cell lines, DU145 and PC-3, were grown in RPMI 1640 media supplemented with 10% FBS All cell lines were cultured in the presence of penicillin/streptomycin Mononucleated cells were isolated from normal bone marrow using Ficoll hypaque and then T-cell depleted T-cell depletion was performed by incubating the mononucleated bone marrow cells in α- MEM at a concentration of 2 x 107 cells/mL and supplemented with 10% absor FBS (FBS heated at 56°C for 1 h, incubated with sheep red blood cells at 0 5% (v/v) for 1 h and then filtered with a 0.2 μM filter) and 10% sheep red blood cells for 4 to 16 h The T-cell depleted, mononucleated cells were recovered using Ficoll hypaque and were maintained in α-MEM supplemented with 20% FBS and 10% 5637 conditioned media 5637 conditioned media was previously harvested from confluent 5637 cells after a 3 day incubation Normal bone marrow from bone marrow transplant donor was collected following informed consent according to institutional guidelines Organosulfur compound preparation for in vitro testing Approximately 20 mg of each compound was dissolved in 5 mL of ACS grade acetone (Sigma) using a 5 mL volumetric flask Stock solutions of the compounds were stored in the dark at -20°C Compounds were diluted prior to each experiment MTT Assay
Adherent cells were seeded at 67 x 103 cells/mL in a 96 well plate (Falcon, Mississauga, Ontario) the day prior to exposure to compound Suspension cells were seeded at 27 x 104 cells/mL in a 96 well plate the day of exposure to compound Mononucleated, T-cell depleted normal bone marrow cells were seeded at 67 x 104 cells/mL in a 96 well plate the day of exposure to compound Compounds and solvent control were added to cells and assayed in triplicate or sextuplet Following 48 h of incubation at 37°C with 5% C02, 40 μL of a 5 mg/mL solution of 3-[4,5-dιmethylthιazol-2- yl]-2,5-dιphenyltetrazolιum bromide (MTT) substrate (Sigma) in Dulbecco's phosphate- buffered saline (D-PBS) was added After 4 h of incubation at 37°C with 5% C02, the resulting violet formazan precipitate was solubi zed by the addition of 80 uL of a 0 01 M HCI, 10% sodium dodecyl sulfate (SDS, Sigma) solution overnight at 37°C with 5% C02 The plates were then analyzed using the BioRad Benchmark Microplate Reader (BioRad Laboratories, Hercules, California) at 570 nm to determine the optical density of the samples MTT data was analyzed using Prism 3 0 (GraphPad Software, Inc , San Diego, California) by the Chou-Talalay method (Chou TC and Talalay P Quantitative analysis of dose-effect relationships the combined effects of multiple drugs or enzyme inhibitors Adv Enzyme Reg 22 27-55, 1984, Chou, TC (1991 ) in Synergism and antagonism in chemotherapy (T C Chou and D C Rideout, eds ), pp 61-102, Academic Press, Inc , San Diego) MTT graphs shown are a representative experiment Trypan Blue Exclusion Assay
Leukemic cells were seeded in a 24-well plate (Nunc, Naperville, IL) at 25x104 cells/mL Cells were then exposed in triplicate to solvent control or the approximate MTT50 concentration of compounds F (10 μg/mL), G (10 μg/mL) or I (5 μg/mL) The final solvent volume was 2 4 μL/mL of media for trypan blue exclusion assay The compound was replenished after 48 h of treatment Cell counts were evaluated using a 1 1 dilution of cell suspension in trypan blue (Gibco-BRL, Mississauga, Ontario, Canada) Viable and nonviable cells were counted using a hemocytometer Cells which excluded trypan blue were counted as viable whereas stained cells were counted as nonviable Trypan blue exclusion graphs shown are a representative experiment
Fixed Propidium Iodide (PI) Staining
Adherent cell lines were plated at 35 x 104 cells/60 cm2 dish the day before exposure to compound Suspension cells were seeded at 25 x 104 cells/mL in a 6 well dish (Falcon) the day of exposure to compound Mononucleated, T-cell depleted normal bone marrow cells were seeded at 50 x 104 cells/mL in a 6 well dish the day of exposure to compound Cells were exposed to solvent control or approximate MTT50 and MTT30 concentrations After 48 h of exposure to the compound, cells were harvested, fixed in 80% ethanol for 1 h on ice and labeled with 50 μg/mL propidium iodide (Sigma) Approximately 106 cells were analyzed using a XL-MCL flow cytometer (Coulter Corporation, Miami, Florida) and a FACScalibar cytometer (Becton Dickinson, San Jose, CA) Profiles shown are a representative Cell viability counts and PI staining were performed as previously described (Dimitroulakos, J, Nohynek, D, Backway, KL, Hedley, DW, Yeger, H, Freedman, MH, Minden, MD and Penn, LZ Increased sensitivity of acute myeloid leukemias to lovastatin-induced apoptosis A potential therapeutic approach Blood 93 1308-18, 1999) Tdt-mediated dUTP-biotm nick end-labeling (TUNEL)
Adherent cell lines were plated at 35 x 104 cells/60 cm2 dish the day before exposure to compound Suspension cells were seeded at 25 x 104 cells/mL in a 6 well dish (Falcon) the day of exposure to compound Mononucleated, T-cell depleted normal bone marrow cells were seeded at 50 x 104 cells/mL in a 6 well dish the day of exposure to compound Cells were exposed to solvent control or approximate MTT50 and MTT30 concentrations of compound After 48 h of exposure to the compound, cells were harvested and fixed with a final concentration of 4% formaldehyde Fixed cells were stored at -20°C in 70% ethanol for no more than 5 days Approximately 105 cells were pelleted and labeled with 0 02 mM Biotin-dUTP and 12 5 U TdT enzyme in a 1x reaction buffer (200 mM potassium cacodylate, 25 mM Tns-HCI, 25 μg/mL bovine serum albumin, pH 6 6), 2 5 mM CoCI2 and 0 01 mM dTTP (Roche Molecular Biochemicals, Laval, QC, Canada) for 45 mm at 37°C Samples were washed and incubated in 200 μL of 1 1000 fluorescein isothiocyanate (FΙTC)-conjugated avidin (Sigma) in 4x SSC, 5% skim milk powder and 0 05% Tween-20 (Sigma) Following 1 h of mixing at room temperature, the samples were washed and resuspended in 500 μL of D-PBS containing 2 5 μg/mL DNase-free RNase (Boehrmger Mannheim) and 10 μg/mL PI Following a 30 mm incubation at room temperature, cells were analyzed using a XL-MCL flow cytometer (Coulter Corporation, Miami, Florida) and a FACScalibar cytometer (Becton Dickinson, San Jose, CA) Profiles shown are representative of one experiment BRIEF DESCRIPTION OF THE DRAWINGS
The accompanying drawings are used to illustrate the invention only and should not be used to limit the scope of the claims The letter prefix to each of the Table or Figure numbers corresponds to a respective compound The Tables and Figures have corresponding labels Thus, Compound A data can be found in Table A-1 which corresponds to Figure A-1 In the accompanying Figures,
Figure A-1 illustrates a MTT Assay with Leukemic cell lines, WI38 and normal bone marrow, Figure A-2 illustrates Fixed Propidium Iodide Profiles,
Figure B-1 illustrates a MTT Assay with Leukemic cell lines, WI38 and normal bone marrow,
Figure B-2 illustrates Trypan Blue Exclusion Assay, Figure B-3 illustrates Fixed Propidium Iodide Profiles, Figure B-4 illustrates TUNEL Profiles,
Figure C-1 illustrates a MTT Assay with Leukemic cell lines, WI38 and normal bone marrow,
Figure C-2 illustrates a Trypan Blue Exclusion Assay, Figure C-3 illustrates Fixed Propidium Iodide Profiles, Figure C-4 illustrates TUNEL Profiles,
Figure D-1 illustrates a MTT Assay with Leukemic cell lines, WI38 and normaj bone marrow,
Figure D-2 illustrates Fixed Propidium Iodide Profiles, Figure E-1 illustrates a MTT Assay with Leukemic cell lines, WI38 and normal bone marrow,
Figure E-2 illustrates Fixed Propidium Iodide Profiles,
Figure F-1 illustrates a MTT Assay with Leukemic cell lines, WI38 and normal bone marrow,
Figure F-2 illustrates a Trypan Blue Exclusion Assay,
Figure F-3 illustrates Fixed Propidium Iodide Profiles,
Figure F-4 illustrates TUNEL Profiles,
Figure G-1 illustrates a MTT Assay with Leukemic cell lines, WI38 and normal bone marrow,
Figure G-2 illustrates a Trypan Blue Exclusion Assay,
Figure G-3 illustrates Fixed Propidium Iodide Profiles,
Figure G-4 illustrates TUNEL Profiles,
Figure H-1 illustrates a MTT Assay with Leukemic cell lines, WI38 and normal bone marrow,
Figure H-2 illustrates a Trypan Blue Exclusion Assay,
Figure H-3 illustrates Fixed Propidium Iodide Profiles,
Figure H-4 illustrates TUNEL Profiles,
Figure 1-1 illustrates a MTT Assay with Leukemic cell lines, WI38 and normal bone marrow,
Figure I-2 illustrates a Trypan Blue Exclusion Assay,
Figure I-3 illustrates Fixed Propidium Iodide Profiles,
Figure I-4 illustrates TUNEL Profiles,
Figure J-1 illustrates a MTT Assay with Leukemic cell lines, WI38 and normal bone marrow;
Figure J-2 illustrates Fixed Propidium Iodide Profiles,
Figure K-1 illustrates a MTT Assay,
Figure K-2 illustrates a MTT Assay with Normal Bone Marrow,
Figure L-1 illustrates a MTT Assay, Figure L-2 illustrates a MTT Assay with Normal Bone Marrow,
Figure M-1 illustrates a MTT Assay,
Figure M-2 illustrates a MTT Assay with Normal Bone Marrow,
Figure M-3 illustrates Fixed Propidium Iodide Profiles,
Figure N-1 illustrates a MTT Assay, Figure N-2 illustrates a MTT Assay with Normal Bone Marrow,
Figure N-3 illustrates Fixed Propidium Iodide Profiles,
Figure N-4 illustrates TUNEL Profiles,
Figure 0-1 illustrates a MTT Assay,
Figure 0-2 illustrates a MTT Assay with Normal Bone Marrow,
20
SUBSTTTUTE SHEET (RULE 26) Figure P-1 illustrates a MTT Assay,
Figure P-2 illustrates a MTT Assay with Normal Bone Marrow, Figure P-3 illustrates Fixed Propidium Iodide Profiles, Figure P-4 illustrates TUNEL Profiles, Figure Q-1 illustrates a MTT Assay,
Figure Q-2 illustrates a MTT Assay with Normal Bone Marrow, Figure Q-3 illustrates a MTT Assay with Breast/Prostate/Melanoma cell lines, Figure Q-4 illustrates Fixed Propidium Iodide Profiles, Figure Q-5 illustrates TUNEL Profiles Table A-1
Table A-2
Notes
No anti-proliferative properties
Table B-1
Table B-2
Trypan Blue Exclusion Assays n=3
21
SUBSTTTUTE SHEET (RULE 26) Table B-3
Table B-4
Notes:
No anti-proliferative activity
Table C-1
Table C-2
22
SUBSTTTUTE SHEET (RULE 26) Table C-3
Notes: Anti-proliferative activity
Table D-1
Table D-2
Notes: Anti-proliferative activity
Table E-1
23
SUBSTTTUTE SHEET (RULE 26) Table E-2
Notes:
Anti-pro! iterative activity
Table F-1
Table F-2
Trypan Blue Exclusion Assays n=3
Table F-3
Table F-4
24
SUBSTTTUTE SHEET (RULE 26) Notes:
Anti-proliferative activity
Table G-1
Cell line MTT50 ! Number of uM 1 Experiments
Leukemic
• AML-3 114(>25ug/mL) ! 6
• KK 114(>25ug/mL) I 6
Normal
• WI38 114(>25ug/mL) 16
• Normal bone marrow 114(>25ug/mL) f 3
Table G-2
Trypan Blue Exclusion Assays n=3
Table G-3
Table G-4
Notes:
No anti-proliferative activity
Table H-1
Table H-2
Trypan Blue Exclusion Assays n=3
Table H-3
Table H-4
Notes: Anti-proliferative activity Table 1-1
Table 1-2
Trypan Blue Exclusion Assays n=3
Table 1-3
26
SUBSTTTUTE SHEET (RULE 26) Table 1-4
Notes: Anti-proliferative activity
Table J-1
Table J-2
Notes:
No anti-proliferative activity
Table K-1
27
SUBSTTTUTE SHEET (RULE 26) Table K-2
Table L-1
Table L-2
28
SUBSTTTUTE SHEET (RULE 26) Table M-1
Table M-2
Table M-3
29
SUBSTTTUTE SHEET (RULE 26) Table N-1
Table N-2
Table N-3
30
SUBSTTTUTE SHEET (RULE 26) Table N-4
Table 0-1
Table 0-2
31
SUBSTTTUTE SHEET (RULE 26) Table P-1
Table P-2
Table P-3
Table P-4
Table Q-1
Table Q-2
Table Q-3
Table Q-4
34
SUBSTTTUTE SHEET (RULE 26) Table Q-5
DISCUSSION
Initially, the efficacy of ten synthetic OSCs was explored for their antiproliferative activity against mammalian cells This was expanded to include seven more compounds On the basis of activity (MTT assays) the OSCs were separable into three distinct groups, Group I (compounds A, B, G, J), Group II (compounds F, H) and Group III (compounds C, D, E, I) The trypan blue exclusion results, in combination with the MTT activity results indicate that compounds C, D, E, F, H and I were cytotoxic to the leukemic cells in a dose- dependent manner Exposure to compounds F and H led to an accumulation of leukemic cells in the G2/M phase of the cell cycle prior to the cells undergoing apoptosis By contrast, compounds C, D, E and I trigger the leukemic cells to undergo apoptosis in all the phases of the cell cycle as determined by fixed PI and TUNEL Interestingly, the viability of non-transformed human WI38 fibroblasts was not affected following exposure to compounds A, B, G, J, F and H yet decreased in response to compounds C D, E and I The common structural feature of all the active antileukemic OSCs analyzed in this study was the presence of a disulphide Indeed, many biologically active natural products of the genus allium are disulphides or closely related thiosulfinates or thiosulfonates (Block, E The organosulfur chemistry of the genus Allium - implications for the organic chemistry of sulfur Angew Chem Int Ed Engl. Z 1135-1 178, 1992) Clearly in this study, however, the sulfone disulphide F and disulphide ester H exhibit specificity towards transformed cell lines
A handful of investigators have explored the effects of naturally-occurring OSCs, such as S-allylmercaptocysteine (SAMC) or diallyl disulphide (DADS) on the growth of tumor cell lines Sigounas et al (Sigounas, G, Hooker, JL, Li, W, Anagnostou, A and Steiner, M S- allylmercaptocysteme, a stable thioallyl compound, induces apoptosis in erythroleukemia cell lines Nutr Cancer 28 153-9, 1997, Sigounas, G, Hooker, J, Anagnostou, A and Steiner, M S-allylmercaptocysteine inhibits cell proliferation and reduces the viability of
35
SUBSTTTUTE SHEET (RULE 26) erythroleukemia, breast, and prostate cancer cell lines Nutr Cancer 27 186-91 , 1997) have shown that SAMC reduces viability of erythroleukemia, breast and prostate cancer cell lines The effects of compounds F and H appear to be similar to SAMC and DADS Both SAMC and DADS have been shown to growth arrest cells in the G2/M phase of the cell cycle prior to apoptosis in a time and dose dependent manner (Sigounas, G, Hooker, JL, Li, W, Anagnostou A and Steiner, M S-allylmercaptocysteine, a stable thioallyl compound, induces apoptosis in erythroleukemia cell lines Nutr Cancer 28 153-9, 1997, Sundaram, SG and Milner, JA Diallyl disulphide inhibits the proliferation of human tumor cells in culture Biochim Biophys Ada 1315 15-20, 1996, Sundaram, SG and Milner, JA Diallyl disulphide induces apoptosis of human colon tumor cells Carcinogenesis 17 669- 73, 1996, Knowles, LM and Milner, JA Depressed p34cdc2 kinase activity and G2/M phase arrest induced by diallyl disulphide in HCT-15 cells Nutr Cancer 30 169-74, 1998) However, unlike compound F and H, SAMC does not appear to be tumor-specific as it has been shown to inhibit proliferation of non-transformed cells (Sigounas, G, Hooker, JL, Li, W, Anagnostou, A and Steiner, M S-allylmercaptocysteine, a stable thioallyl compound, induces apoptosis in erythroleukemia cell lines Nutr Cancer 28 153-9, 1997) Compared to these natural compounds, Group II and Group III OSCs in this study were approximately 2 and 10 fold more potent, respectively, suggesting the specific activity of natural OSCs can be increased by structure-activity analysis of synthetic derivatives It appears that the structural criteria of OSCs for their antifungal and antitumor activities are separable and distinct Comparison of the antifungal (Baerlocher, FJ, Langler, RF, Frederiksen, MU, Georges, NM and Witherell, RD Structure-activity relationships for selected sulfur-rich antifungal compounds Aust J Chem 52 167-172, 1999) and antileukemic activity of the synthetic OSCs reveals compounds A, B, G and J do not possess antiproliferative activity on either fungal or mammalian cells By contrast compounds C, E and I possess antifungal and anti-mammalian cell activity, which is consistent with the notion that disulphides are general toxins (Rice, WG, Turpin, JA, Schaeffer, CA, Graham, L, Clanton, D, Buckheit, RW, Jr , Zaharevitz, D, Summers, MF, Wallqvist, A and Covell, DG Evaluation of selected chemotypes in coupled cellular and molecular target-based screens identifies novel HIV-1 zinc finger inhibitors J Med Chem 39 3606-16, 1996) Finally, compounds D, F and H do not possess antifungal activity but can inhibit mammalian cell growth Moreover, compound F and H are distinct from compound D as these compounds possess tumor-specific activity Unlike other natural products currently being explored as therapeutics, these compounds are relatively simple and inexpensive to synthesize The discussion under the heading "Summary of Results" describes these results as well as the results for compounds K to Q Preparatory Methods
The methods described herein encompass both novel and known methods of preparation The preparation of novel compound 4 in Table 6 represents a novel method Compound Q is a novel compound and its method of preparation is also novel The method for preparing compound 2 in Table 7 is new, but compound 3 is made by a known method The same is true for compound 4 of Table 7 and for compound P The following description provides an overview of various aspects of the methods described herein It is believed that the person skilled in the art could readily apply the various methods for preparing the compounds of this invention to produce additional compounds having the same basic structure The additional detail found in the specific methods described provides instruction for the detail of such methods This is particularly true for any group of compounds or specific compound mentioned herein, the preparation for which is not described
From the beginning of this program to make biologically active organosulfur compounds, α-substituted disulphides have been targeted for synthesis Initially, singly -substituted dimethyl disulphides i e 1 were chosen for construction
CH3SSCH2W 1
A) Conditions for disproportionations in dimethyl disulphide which afford unsymmetπcal methyl disulphides smoothly have been optimized This chemistry affords methyl disulphides even when reactions proceed through the metastable intermediates RSCH2S" (see eq. [1] for an example) CH3SNa (cat )
(1) CH3SCH2SSCH2SCH3 > CH3SCH2SSCH3
(CH3S)2 (78%)
B) Well-known, base-catalyzed condensations of sulfenyl chlorides and mercaptans have been exploited for the construction of unsymmetπcal disulphides Symmetrical disulphides have traditionally been used to make sulfenyl chlorides, so that only one sulfenyl chloride is produced (see eq [2])
CH2CI2
[2] RSSR + S02CI2 > RSCI + CISR
In a number of cases, symmetrical disulphides (e g (p-02N(C6H4)S)2 or (CH3OC(0)CH2S)2) will not react with our chlorinating agent It has now been established that the corresponding methyl disulphides cleave smoothly with S02Cl2/CH2CI2 (see eq [3] for an example)
CH2CI2 [3] CH3SSCH2C(0)OCH3 + S02CI2 > CISCH2C(0)OCH3 + CH3SCI
37
SUBSTTTUTE SHEET (RULE 26) Methanesulfenyl chloride is very volatile and is completely removed when the solvent is evaporated Hence, we have established that unsymmetπcal methyl disulphides are useful precursors for the preparation of homogeneous sulfenyl chlorides
C) Now described are new transition metal oxidations of dialkyl disulphides which furnish, the virtually unknown α-ester disulphides in which the ester group is attached to the disulphide framework by an oxygen atom (see eq [4] for an example)
C2H5C02H
[4] CH3SSCH3 + KMn04 > CH3SSCH2OC(0)C2H5
These α-ester disulphides show, inter alia, antifungal and antileukemic properties
D) It has been established that α-ester disulphides serve as effective precursors for the preparation of α-sulfonyl disulphides which also show antifungal and antileukemic properties (see eq [5] for an example)
(5] CH3SSCH2OC(0)C2H5 + p-CH3(C6H4)S02Na — > CH3SSCH2S02(C6H4)CH3-p
E) In a powerful conjunction of B) and D), the sulfenyl chloride ester 2 has been prepared
CISCH2OC(0)C2H5
2
This versatile intermediate permits nucleophihc attack at both S and at C It is a synthetic equivalent for 3
+S-CH2 + 3
3 permits general access to unsymmetπcal disulphides, through α-ester disulphides (see eq [6])
[6] CISCH2OC(0)C2H5 + RSH > RSSCH2OC(0)C2H5
T Nu RSSCH2Nu
Infrared spectra were recorded on a Perkin-Elmer 710B grating spectrophotometer for chloroform solutions unless otherwise specified 1H n m r spectra (60 MHz) were obtained
38
SUBSTTTUTE SHEET (RULE 26) on a Vaπan EM360L instrument H n m r (270 MHz) and 13C n m r spectra were obtained on a JEOL JNM-GSX 270 Fourier-transform n m r system Unless otherwise specified, all n m r spectra were obtained for (D)chloroform solutions with tetramethylsilane as internal standard Mass spectra were obtained on a Hewlett-Packard 5988A g l c /m s system Melting points were determined on a Gallenkamp MFB-595 capillary melting point apparatus and are uncorrected Preparation of Compounds of Table 3 Previously Prepared Compounds
Compounds (4) and (6) were prepared as described in Ahem, T P , Hennigar, T L , MacDonald, J A , Morrison, H G , Langler R F , Satyanarayana, S , and Zawarotko, M J , Aust J Chem , 1997, 50, 683 and compound (8) was prepared as described in Ahem, T P , Langler, R F , and McNeil R L , Can J Chem , 1980, 58, 1996 Compound (13) was prepared as described earlier (Georges, N M Johnson, M D , Langler R F , and Verma, S D , Sulfur Lett 22, 141 (1999) Compounds (5) (Robson, P , Speakman, P H R , and Stewart, D G , J Chem Soc C, 1968, 2180, Bohme, H , and Heller P Chem
Ber , 1953, 86, 785) and (7) (Dubs, P , and Stuessi, R , Helv Chim Ada, 1978, 61 , 2351 ) have been prepared by earlier workers Preparation of 2,4-Dithiapentane 2,2-Dioxιde (5)
(A) A solution of 2,4-dιthιapentane (0 98 g, 9 0 mmol) and hydrogen peroxide (1 03 g, 30%) in 1 ,4-dιoxan (24 ml) was refluxed behind a safety shield for 0 5 h and the solvent evaporated
(B) Potassium permanganate (0 74 g, 4 7 mmol) was covered with water (4 2 ml) and tetrahydrofuran (17 ml) Crude product from part (A) was dissolved in water (7 ml) and tetrahydrofuran (28 ml) and added to the reaction mixture The reaction mixture was stirred at ambient temperature for 1 h and filtered through a Celite filter pad The filtered solution was added to solid sodium thiosulfate (35 g) and the mixture stirred for 0 5 h The solid was filtered off and the organic solvent evaporated affording a wet residue The residue was extracted with chloroform (three 100 ml aliquots) The organic layers were combined, dried (MgS04), filtered and concentrated The crude product was chromatographed on silica gel (70 g) employing chloroform elution (50 ml fractions) Fractions 14-16 were combined and concentrated affording clean 2,4-dιthιapentane 2,2-dιoxιde (5) (0 13 g, 0 9 mmol, 10%) as an oil I r 1310, 1160 cm 1 1H n m r (60 MHz) δ 2 43, s, 3H, 3 06, s, 3H, 3-85, s, 2H m/z 140 (9%, M+), 61 (100) Preparation of 2,3,5-Trithiahexane (7) Sodium metal (0 163 g, 7 0 mmol) was dissolved in methanol (10 ml), the solvent evaporated and the sodium methoxide dried in vacuum Dimethyl sulfoxide (Me2SO) (3 ml) was added and the resultant suspension stirred vigorously for 5 h
Dimethyl disulphide (3 7 g, 38 8 mmol), 3,5-dιthιahexan-2one (Ahern T P , Haley, M F , Langler, R F and Trenholm, J E Can J Chem , 1984, 62, 610) (1 0 g, 7 7 mmol)
39
SUBSTTTUTE SHEET (RULE 26) and Me2SO (2 ml) were added The reaction mixture was stirred at ambient temperature for 2 days Hydrochloric acid (2 5%, 100 ml) was added and the resultant mixture extracted with diethyl ether (three 100 ml aliquots) The combined organic layers were concentrated and the extraction procedure was repeated The combined organic layers were dried (MgS04) and filtered, and the solvent was evaporated
The crude product was chromatograpfied on silica gel (10 g) employing light petroleum (10 ml fractions) for elution Fractions 3-7 were combined and the residue was distilled furnishing 2,3,5-tnthιahexane (7) (0 45 g, 3 2 mmol, 42%), b p 106°C/18 Torr 1H n m r (270 MHz) δ 2 23, s, 3H, 2 50, s, 3H, 3 85, s, 2H 13C n m r δ 15-15, 2342, 44 22 m/z 140 (26%, M+), 61 (100)
Preparation of 2,4,5-Trithiaheptane 2,2-Dioxide (9)
Ethanethiol (1 4 g, 22 4 mmol) was dissolved in dry pyπdine (50 ml) and CH3S02CC12SOCH3 (Ahern, T P , Langler, R F , and McNeil R L , Can J Chem , 1980 58, 1996) (1 2 g, 4 5 mmol) added The reaction mixture was stirred at ambient temperature for 24 h Chloroform (200 ml) was added and the resultant mixture washed with 5% hydrochloric acid (100 ml aliquots) until the aqueous layer remained acidic The organic layer was dried (MgS04), filtered and concentrated The residue was chromatographed on silica gel (150 g) employing chloroform elution (100 ml fractions) Fractions 8-1 1 were combined and concentrated affording clean sulfone disulphide (9) (0 45 g, 2 4 mmol, 53%) Recrystallized 2,4,5-trιthιaheptane 2,2-dιoxιde (methanol) had m p 34 6-36 3°C (Found C, 25 6, H, 5 4 C4H10O2S3 requires C, 25 8, H 5 4%) I r 1325, 1150 cm'1 H n m r (270 MHz) δ 1 37, t, 3H, 2 91 , q, 2H, 3 04, s, 3H, 4 09, s, 2H 13C n m r δ 14-17, 33.31 , 39 21 , 62 40 m/z 186 (13%, M+ ), 107 (67), 79 (100) Preparation of 3, 6-Dithiaheptan-2-one
Thioacetic S-acid (49 8 g, 655 mmol) and 4-chloro-2-thιabutane (Fong, H O , Hardstaff, W R , Kay, D G , Langler, R F , Morse, R G , and Sandoval, D N , Can J Chem , 1979, 57, 1206) (40 0 g, 363 mmol) were added to dry pyπdine (550 ml), and the reaction mixture was refluxed for 2 h Dichloromethane (2 7 litres) was added and the resultant mixture extracted with 5% hydrochloric acid (600 ml portions) until the aqueous layer remained acidic The organic layer was dried (MgS04), filtered and the solvent distilled off at atmospheric pressure The residue was rectified at reduced pressure affording 3,6-dιthιaheptan-2-one (32 1 g, 214 mmol, 59%), b p 80°C/43 Torr I r (liquid film) 1690 cm 1 1H n m r (270 MHz) δ 2 17, s, 3H, 2 35, s, 3H, 2 67, t, 2H, 3 10, t, 2H 13C n m r δ 15 37, 28 72, 30 65, 33 86 m/z 150 (9%, M+), 74 (55), 61 (40), 43 (100) Preparation of 3-Thiabutane-l-thiol
3,6-Dιthιaheptan-2-one (32 1 g, 214 mmol) was dissolved in methanol (900 ml) and sodium hydroxide (1 1 1 g, 277 mmol) in water (500 ml) added The reaction mixture was stirred at ambient temperature for 1 h Water (1 litre) and 10% hydrochloric acid (400 ml)
40
SUBSTTTUTE SHEET (RULE 26) were added The resultant mixture was extracted with methylene chloride (five 800 ml aliquots) The bulk of the solvent was distilled off at atmospheric pressure The residue was dissolved in methylene chloride (500 ml) and the resultant solution washed with 5% sodium hydroxide solution (three 200 ml portions) The aqueous layer was strongly acidified (concentrated hydrochloric acid) and extracted with dichloromethane (three 200 ml aliquots) The combined organic layers were dried (MgS04) and filtered, and the solvent was distilled off at atmospheric pressure Crude 3-thιabutane-l-thιol was rectified at reduced pressure (10 8 g, 100 mmol, 47%), b p 120°C/160 Torn I r (liquid film) 2580 cm 1 1H n m r (270 MHz) δ 1 75, t, 1 H, 2 15, s, 3H, 2 73, s, 2H, 2 75, s, 2H 13C n m r δ 15 28, 24 13, 38 14 m/z 108 (100%, M+), 61 (95) Preparation of 2,3,6-Trithiaheptane
Me2SO (50 ml) was added to powdered sodium hydroxide (2 6 g, 65 mmol) and the reaction mixture stirred to produce a homogeneous solution 3-Thιabutane-l-thιol (3 0 g, 27 mmol) in Me2SO (20 ml) was added to the reaction mixture which was stirred at room temperature for 5 mm Dimethyl disulphide (6 5 g, 69 1 mmol) in Me2SO (30 ml) was added and the resultant mixture stirred at ambient temperature for 24 h Hydrochloric acid (2 5%, 1 litre) was added and the resultant mixture washed with diethyl ether (three 1 litre aliquots) The combined organic layers were concentrated and the extraction procedure was repeated The concentrate was covered with 2 5% sodium hydroxide solution (1 litre) and the resultant mixture extracted with diethyl ether (three 1 litre portions). The combined organic layers were dried (MgS04) and filtered, and the solvent was evaporated The residue was distilled at reduced pressure giving 2,3,6-tπthιaheptane as a colourless oil (2 4 g, 15 5 mmol, 24%), b p 80°C/2 Torr 1H n.m.r (270 MHz) δ 2.15, s, 3H, 2 43, s, 3H, 2-83, m, 2H, 2 92, m, 2H 13C n m r δ 15 54, 23.45, 33 54, 37 24 m/z 154 (1 %, M+ ), 75 (100) Preparation of 2,3,6-Trithiaheptane 6-Oxide
2,3,6-Trιthιaheptane (1 0 g, 6 4 mmol) was dissolved in 1 ,4-dιoxan (45 ml) and hydrogen peroxide (0 37 g, 30%) in 1 ,4-dιoxan (5 ml) added The reaction mixture was refluxed behind a safety shield for 30 mm The solvent was evaporated and the residue chromatographed on silica gel (100 g) employing chloroform (100 ml fractions) for elution Fractions 9-18 were combined and concentrated affording clean 2,3,6-tπthιaheptane 6-oxιde as a colorless oil (0 54 g, 3 1 mmol 48%) I r (liquid film) 1040 cm 1 1H n m r (270 MHz) δ 2 42, s, 3H, 2.63, s, 3H, 3 03, m, 2H, 3 09, m, 211 13C n m r δ 22 87, 29 55, 38 60, 53 49 m/z 106 (100%), 79 (95) Preparation of 2,3,6-Trithiaheptane 6.6-Dioxide (10)
2,3,6-Trιthιaheptane 6-oxιde (0 54 g, 3 1 mmol) was dissolved in acetone (40 ml) and the reaction mixture cooled to 0°C Anhydrous magnesium sulfate (3 8 g) in acetone (10 ml) was added and the reaction mixture stirred at ambient temperature Potassium permanganate (0 50 g) was added in three portions at half-hour intervals Upon
41
SUBSTTTUTE SHEET (RULE 26) completion of the addition, the reaction mixture was filtered through a Celite pad and the solvent evaporated The residue was chromatographed on silica gel (50 g) employing 1 1 methylene chloride/light petroleum (50 ml fractions) for elution Fractions 7-11 were combined and concentrated yielding clean (10) (0 51 g, 2 7 mmol, 87%) 2,3,6-Trιthιaheptane 6,6-dιoxιde had b p 158-162°C/1 -4 Torr (Found C, 25 9. H, 5 6
C4H1002S3 requires C, 25 8, H, 5 4%) I r (liquid film) 1310, 1 145 cm 1 1H n m r (270 MHz) δ 2 44, s, 3H, 2 99, s, 3H, 3 08, m, 2H, 3 43 m, 2H 13C n m r δ 22 80, 28 67, 41 45, 54 17 m/z 186 (21 %, M+ ), 106 (80), 79 (100) Preparation of S-4-Thiapentyl Thioacetate 4,4-Dioxide (A) 4-Thιapentan-l-ol (Langler, R F , Maπni, Z A , and Spalding, E S Can J
Chem , 1979, 57, 3193 (5 3 g, 49 5 mmol) and tπethylamine (4 9 g, 48 5 mmol) in dry pyπdine (75 ml) were cooled with an ice/salt/water bath Methanesulfonyl chloride (5 8 g, 50 8 mmol) was added dropwise over 15 mm The reaction mixture was stirred at ambient temperature for 3 days Chloroform (100 mi) was added and the resultant mixture washed with 10% hydrochloric acid (50 ml portions) until the aqueous layer remained acidic The organic layer was dried (MgS04), filtered and concentrated The residue was rectified at reduced pressure affording impure sulphide methanesulfonate (3 30 g, b p 110-120°C/2 Torr)
(B) Impure sulphide methanesulfonate (3 8 g) from step (A) was dissolved in chloroform (75 ml), and the solution added dropwise to 10% sulfuπc acid (104 ml)
During the addition, solid potassium permanganate (13 7 g) was also added in small portions Upon completion of the additions, the reaction mixture was stirred at ambient temperature for 2 days The reaction mixture was cooled with an ice/water bath and sodium bisulfite added in small portions until the reaction mixture was decolorized The layers were separated and the aqueous layer was extracted with chloroform (three 100 ml aliquots) The combined organic layers were concentrated affording impure sulfone methanesulfonate (3 7 g)
(C) Thioacetic S-acid (1 3 g) in dry pyndine (30 ml) was added to impure sulfone methanesulfonate (3 7 g) from step (B) and the reaction mixture stirred at room temperature for 2 days Chloroform (200 ml) was added and the resultant mixture washed with 2 5% hydrochloric acid (100 ml aliquots) until the aqueous pH remained acidic The organic layer was dried (MgS04), filtered and concentrated Crude sulfone thioacetate was chromatographed on silica gel (250 g) employing 1 1 light petroleum/chloroform (100 ml fractions) for elution Fractions 53-72 were combined and concentrated yielding clean S-4-thιapentyl thioacetate 4,4-dιoxιde (0 93 g, 4 7 mmol, 8% from 4-thιapentan-1-ol) Recrystallized (methanol) sulfone thioacetate had m p 61 8-62 7°C (Found C, 36 8, H, 6 1 CβH1203S2 requires C, 36 7, H, 6 2%) I r (KBr) 1690, 1300, 1160, 1130 cm 1 1H n m r (270MHz) δ 2 15, quin, 2H, 2 36, s, 3H, 2 92, s, 3H, 3 04, m, 4H m/z 196 (6%, M+ ), 116 (39), 43 (100)
42
SUBSTTTUTE SHEET (RULE 26) Preparation of 4-Thiapentane-1 -thiol 4,4-Dioxide
S-4-Thιapentyl thioacetate 4,4-dιoxιde (0 25 g, 1.2 mmol) was dissolved in methanol (25 ml) and a solution of sodium hydroxide (0.08 g, 2 0 mmol) in water (125 ml) added. The reaction mixture was stirred at ambient temperature for 1 h Water (25 ml) and 10% hydrochloric acid (6 ml) were added, and the resultant mixture was extracted with chloroform (four 50 ml aliquots) The combined organic layers ware dried (MgS04), filtered and concentrated giving 4-thιapentane-l-thιol 4,4-dιoxιde (0 13 g, 0.8 mmol, 67%). The thiol was subjected to a bulb-to-bulb distillation (bath. 200°C; pressure: 1 5 Torr) l.r (liquid film) 2600, 1310, 1 145 cm"1 1H n.m.r. (270 MHz) δ 1.45, t, 1 H; 2.17, quin, 2H, 2.72, q, 2H; 2.95, s, 3H, 3 20, t, 2H. 13C n.m r δ 23.21 ,. 26.37, 40 88, 52.85. m/z 154 (36%, M+'), 74 (100), 41 (76) Preparation of 2,3,7-Trithiaoctane 7,7-Dioxide (II).
Me2SO (2 ml) was added to powdered sodium hydroxide (0.10 g, 2.5 mmol) and the mixture stirred vigorously. 4-Thιapentane-l-thιol 4,4-dιoxιde (0.38 g, 2.5 mmol) in Me2SO (4 ml) was added to the reaction mixture which was stirred for 5 mm. Dimethyl disulphide (0.69 g, 7.3 mmol) in Me2SO (4 ml) was added and the reaction mixture stirred at room temperature for 24 h. Hydrochloric acid (2.5%, 150 ml) was added and the resultant mixture washed with diethyl ether (three 100 ml aliquots). The combined organic layers were concentrated and the extraction procedure was repeated. Sodium hydroxide solution (2.5%, 150 ml) was added to the residue and the resultant mixture extracted with diethyl ether (three 100 ml portions). The combined organic layers were dried (MgS04) and filtered, and the solvent was evaporated. The crude product was chromatographed on silica gel (40 g) employing 1. 1 methylene chloride/light petroleum (40 ml fractions) for elution. Fractions 12-24 were combined and concentrated furnishing clean sulfone disulphide (11) (0.15 g, 0.7 mmol, 30%). After recrystal zation (methanol),
2,3,7-trithiaoctane 7,7-dιoxιde (1 1 ) had m.p. 34.8-35.3°C (Found: C, 30 1. H 6 1. C5H1202S3 requires C, 30.0; H 6.0%). l.r 1305, 1140 cm"1. 1H n.m.r (270 MHz) δ 2.31 , quin, 2H; 2.42, s, 3H; 2.84, t, 2H; 2.94, s, 3H; 3.17, t, 2H. 13C n.m.r. δ 21.50, 23.00, 35.55, 40.85, 52.87 m/z 200 (13%, M+'), 121 (100), 79 (76), 41 (78). Preparation of Methyl 3,4-Dithiapentanoate (12)
Sodium hydride (1.8 g, 76.5 mmol) was suspended in Me2SO (30 ml) and methyl thioglycolate (5.2 g, 49.2 mmol) in Me SO (30 ml) was added. After 3 mm, dimethyl disulphide (13.0 g, 138 mmol) in Me2SO (90 ml) was added and the reaction mixture stirred at room temperature for 24 h Hydrochloric acid (2.5%, 1 litre) was added to the reaction mixture and the resultant mixture extracted with diethyl ether (three 1 litre aliquots). The combined ether layers were concentrated and the extraction procedure was repeated. Sodium hydroxide solution (2.5%, 1 litre) was added to the residue and the resultant mixture extracted with diethyl ether (three 1 litre aliquots). The combined organic layers were dried (MgS04), filtered and concentrated The residue was rectified at reduced
43
SUBSTTTUTE SHEET (RULE 26) pressure affording methyl 3,4-dιthιapentanoate (12) (1 26 g, 8 2 mmol, 17%) The disulphide ester (M) had b p 108-110°C/18 Torr (Found C, 31 4, H, 5 4 C4H802S2 requires C, 31 6, H, 5 3%) I r (liquid film) 1740 cm 1 1H n m r (270 MHz) δ 2 47, s, 3H, 3 50, s, 2H, 3 77, s, 3H 13C n m r δ 23 0I, 40 67, 52 54, 170 20 m/z 152 (39%, M+ ), 93 (48), 45 (100)
Pteparation of 3,4-Dithiapentan-1-ol
Me2SO (30 ml) was added to sodium hydroxide (2 64 g, 65 4 mmol) A solution of 2-mercaptoethanol (5 0 g, 64 6 mmol) in MezSO (30 ml) was added and the reaction mixture stirred for 3 mm Dimethyl disulphide (18 g, 281 mmol) in Me2SO (90 ml) was added and the reaction mixture stirred at ambient temperature for 1 week Hydrochloric acid (2 5% 1 litre) was added and the resultant mixture washed with diethyl ether (three I litre aliquots) The combined organic layers were concentrated and the extraction procedure was repeated The residue was dissolved in diethyl ether (1 litre) and extracted with 2 5% sodium hydroxide solution (1 litre) The organic layer was dried (MgS04) and filtered, and the solvent evaporated The residue was rectified at reduced pressure furnishing clean 3,4-dιthιapentan-1-ol (2 1 g, 16 9 mmol, 26%) The disulphide alcohol had b p 155-160°C/80 Torr I r (liquid film) 3250 cm 1 1H n m r (270 MHz) δ 2 43, s, 3H, 2 87, t, 2H, 3 08, br s, 1 H, 3 88, t 2H 13C n m r δ6 23 19, 40 28, 60 26 m/z 124 (63%, M+*), 80 (100), 45 (99) Preparation of 3,4-Dithiapentyl Acetate (14)
3,4-Dιthιapentan-l-ol (1 0 g, 8 6 mmol) was added to acetyl chloride (20 ml), and the reaction mixture refluxed for 0 - 5 h The solvent was evaporated and the residue rectified at reduced pressure affording clean disulphide acetate (14) (0 44 g, 2 6 mmol, 30%) 3,4-Dιthιapentyl acetate (14) had b p 135-140°C/18 Torr (Found C, 36 4, H, 6 4 C5H10O2S2 requires C, 36 1 , H, 6 1 %) I r (liquid film) 1740 cm 1 1H n m r (270 MHz) δ
2 08, s, 3H, 2 43, s, 3H, 2 93, t, 2H, 4 34, t, 2H m/z 166 (2%, M+ ), 87 (51 ) 43 (100)
The following information and data were published on the website of CSIRO in February, 2000 in the paper mentioned earlier and entitled A New Synthesis for Antifungal α-Sulfone Disulphides Compounds (10) and (1 1 ) were prepared as shown in Test results as shown in Table
3 for compounds 8, 9 and 13 appear to establish that the sulfone and disulphide functionalities need be attached to a common carbon for significant fungicidal capacity
Scheme 1
Reaction of the disulphide propionate (2) with the sodium salt of p-toluenesulfinic acid in either aqueous acetonitrile or aqueous acetone leads to smooth displacement of the propionate group (see Scheme 2).
CHr- — SOjNa ♦
Scheme 2
Sequential reaction of (4) with thiophenoxide ions and acetyl chloride produces the thioacetate (6) as depicted in Scheme 3. H, (0)CI
CH3(θ)CSCH2Sθ2 _ ~~ CH3
(6)
Scheme 3
45
SUBSTTTUTE SHEET (RULE 26) An unambiguous synthesis (see Scheme 4) of the sulfone thioacetate corresponding to (6) proved that (6) is not a thioacetate sulfinate ester
(7)
HSC(0)CH3/ pyπdiπe
(8)
Scheme 4
Clearly, sulfinate anion attack on (2) has occurred exclusively with the sulfur atom under our reaction conditions (see Scheme 2)
Schemes 3 and 4 not only establish that (4) is the target α-sulfone disulphide but also establish that (5) is an α-mercapto sulfone
Typically, preparation of the disulphide ester (2) produces disulphide contaminated with the corresponding sulphide ester (3) (see Scheme 1 ) Fractional distillation usually leaves a small amount of sulphide ester (3) contaminating distilled disulphide ester (2) It seemed likely that preparation of a sulfone disulphide [e g (4), Scheme 2] from (2) would also produce the corresponding sulfone disulphide which would be difficult to remove However, neither the sulphide propionate (3)3 nor the sulphide acetate (9)3 react with sulfinate anions in warm aqueous acetonitrile (see Scheme 5)
(9)
CH3SCH2OC(0)CH2CH3 + CH3— -S02Na -^ζ-
(3)
Scheme 5
Thus, α-sulfone disulphides (1 , R' = CH3), prepared as shown in Scheme 2, are readily purified Several other α-sulfone disulphides (1 ) have been prepared from (2) and tested for fungitoxic activity Antifungal test results for these α-sulfone disulphides and both disulphide propionate (2) and the α-mercaptosulfone (5) are presented in Table 4
In connection with a related synthetic problem the disulphide ester (2) was reacted with potassium p-toluenethiosulfonate This reaction (see Scheme 6) produced the α- sdlfone disulphide (4)
CH3
Scheme 6
Apparently the reagent is transformed under the reaction conditions, into the potassium salt of p-toluenesulfinic acid Preparation of Compounds of Table 4 Previously Prepared Compounds
Compound (2) was prepared as described in Georges, N M , Johnson, M D , Langler, R F , and Verma, S D , Sulfur Lett , 1999, 22, 141 Preparation of α-Sulfone Disulphides (1) (R' = CH3)
Table 4 α-sulfone disulphides were prepared in the manner described below for (4) A solution of sodium p-toluenesulfmate (2 4 g, 13 4 mmol) and the disulphide propionate (2) (2 0 g, 12 0 mmol) in 1 4 water/acetone (30 ml) was immersed in a constant temperature bath at 50°C for 2 h Chloroform (150 ml) was added and the resultant mixture washed with water (100 ml) The organic layer was dried (MgS04), filtered and the solvent evaporated The residue was chromatographed on silica gel (200 g) employing 1 1 chloroform/light petroleum (100 ml fractions) for elution Fractions 13-28 were combined and concentrated furnishing the α-sulfone disulphide (4) (1 6 g, 6 4 mmol, 53%) Recrystallized (methanol) α-sulfone disulphide (4) had m p 46 2-48 6°C (Found C, 44 1 , H, 5 1 C9H1202S3 requires C, 43 5, H, 4 9) I r 1342, 1148 cm 1 1H n m r (270 MHz) δ 2 46, s, 3H, 2 50, s, 3H, 4 20, s, 2H, 7 38, d, 2H, 7 83, d, 2H 13C n m r δ 21 70, 23 69, 64 38, 128 98, 129 92, 134 67, 145 36 m/z 248 (6%, M+ ), 139 (56%), 93 (100%)
Oily PhS02CH2SSCH3 (obtained in 58% yield) has i r 1325, 1155 cm 1H n m r (270 MHz) δ 2 49, s, 3H, 4 22, s, 2H, 7 60, t, 2H 7 70, t, 1 H 7 96, d, 2H 13C n m r δ 23 68, 64 32, 128 96, 129 30, 134 23, 137 62 m/z 234 (5%, M+ ), 125 (28%), 93 (100%)
Oily CH3S02CH2SSCH3 8 9 (obtained in 53% yield) had i r 1320, 1 145 cm 1 1H n m r (270 MHz) δ 2 60, s, 3H, 3 05, s, 2H, 4 16, s, 2H 13C n m r δ 23 61 , 39 33, 61 38 m/z 172 (10%, M+ ), 93 (100%)
47
SUBSTTTUTE SHEET (RULE 26) Conversion of (4) into the a-Mercatosulfone (5)
The α-sulfone disulphide (4) (0.20 g, 0.81 mmol) was dissolved in a solution of thiophenol (0.19 g, 1.7 mmol) in dry methylene chloride (10 ml). Dry pyπdine (0.1 ml) was added and the reaction mixture stirred at ambient temperature for 2 h 10 mm. The solvent was evaporated and the residue chromatographed on silica gel (10 g) employing chloroform (5 ml fractions) for elution. Fractions 3 and 4 were combined and dissolved in chloroform (100 ml). The chloroform layer was washed with 2.5% sodium hydroxide (two 50 ml portions), dried (MgS04), filtered and the solvent evaporated. G.l.c./m.s. established the presence of phenyl methyl disulphide and diphenyl disulphide in these fractions. Column fraction 5 furnished a mixture (0.06 g) of unchanged (4) and the α-mercaptosulfone (5). Fractions 6-9 were combined and concentrated yielding clean α-mercaptosulfone (5) (0.09 g, 0.44 mmol, 54%). Recrystallized (5)(methanol) had m.p. 86.4-87 4°C (Found: C, 47.3; H, 5.0. C8H10θ2S2 requires C, 47.5; H, 5.0). l.r 2500, 1330, 1 170 cm"1 1H n.m.r. (270 MHz) δ 2.21 , t, 1 H; 2.46, s, 3H, 3.94, d, 2H; 7.38, d, 2H; 7.83, d, 2H. 13C n.m.r. δ 21.68, 49.43, 129.08, 129.85, 133.67, 145.37.
Conversion of (5) into the Sulfone α-Thioacetate (6)
The α-mercaptosulfone (5) (0.06 g, 0.29 mmol) was covered with acetyl chloride (10 ml) and the reaction mixture refluxed for 1 h. The solvent was evaporated and the residue chromatographed on silica gel (5 g) employing 1 :4 light petroleum/methylene chloride (5 ml fractions) for elution. Fractions 6-8 were combined and concentrated giving the sulfone thioacetate (6) (0.024 g, 0.10 mmol, 34%). (Found: C, 49.3%; H, 5.1 %. C10H12O3S2 requires C, 49.2; H, 5.0). l.r. 1720, 1335, 1 165 cm"1. 1H n.m.r. (270 MHz) δ 2.30, s, 3H; 2.45, s, 3H; 4.44, s, 2H; 7.34, d, 2H; 7.81 , d, 2H. 13C n.m.r. δ 21.70, 29.94, 52.09, 128.93, 129.71 , 134.20, 145.39, 190.41. m/z 244 (1 %, M+ ), 150 (31 %), 43 (100%). Conversion ofp-Tolyl Methyl Sulphide into the Sulphide Thioacetate (8)
(A) A solution of p-tolyl methyl sulphide (5.0 g, 36.2 mmol) in dry methylene chloride (50 ml) was refluxed and a solution of sulfuryl chloride (5.0 g, 37.3 mmol) in dry methylene cfιloride(50 ml) added dropwise over 20 mm. The solvent was evaporated and the residue rectified at reduced pressure yielding p-tolyl chloromethyl sulphide (7) (2.6g, 15.3 mmol, 42%), b.p. 138-142°C/18 Torr. 1H n.m.r. (270 MHz) δ 2.33, s, 3H; 4.88, s, 2H; 7.15, d, 2H, 7.40, d, 2H. 13C n.m.r. δ 21.11 , 51.83, 129.48, 129.97, 131.67, 138.34.
(B) Thioacetic S-acid (0.4 g, 5.8 mmol) was dissolved in dry pyπdine (25 ml) and p-tolyl chloromethyl sulphide (1.0 g, 5.8 mmol) added. The reaction mixture was stirred at ambient temperature for 23 h. Chloroform (100 ml) was added and the resultant mixture extracted with 5% HCI (50 ml aliquots) until the aqueous pH remained acidic The organic layer was extracted with 2.5% NaOH (50 ml), dried (MgS04), filtered and the solvent evaporated. The residue was rectified at reduced pressure furnishing the sulphide thioacetate (7) (0.8 g, 3.7 mmol, 64%), b.p. 138-140°C/1.7 Torr. l.r. (liquid film) 1700 cm"1 1H n.m.r. (270 MHz) δ 2.29, s, 3H; 2.33, s, 3H; 4.29, s, 2H; 7.12, d, 2H; 7.32, d, 2H. 13C
48
SUBSTTTUTE SHEET (RULE 26) n.m.r. δ 21.11 , 30.39, 34.75, 129.82, 130.62, 131 46, 137 69. 194.34. m/z 212 (55%, M+ ),
124 (100%), 91 (55%), 43 (65%).
Conversion of (8) into the Sulfone a-Thioacetate (6)
The sulphide α-thioacetate (8) (1.0 g, 4 7 mmol) and hydrogen peroxide (30%, 1.1 g) were added to 1 ,4-dιoxan (25 ml) and the reaction mixture refluxed for 0.5 h. The solvent was evaporated and chloroform (150 ml) added. The chloroform solution was dried (MgS0 ), filtered and concentrated. The residue was chromatographed on silica gel (100 g) employing chloroform (100 ml fractions) for elution Fractions 4 and 5 were combined and concentrated affording clean sulfone α-thioacetate (6) (0 34 g, 1.4 mmol, 30%) The product was identical to material described under "Conversion of (5) into the Sulfone α- Thioacetate (6)" by i.r., 1H n.m.r. (270 MHz) and 13C n.m.r Reaction of the Disulphide Ester (2) with Potassium p-Toluenethiosulfonate
The disulphide propionate (2) (2.0 g, 12.0 mmol) and potassium p-toluenethiosulfonate (2.7 g, 11.9 mmol) were dissolved in 1 4 water/acetone (30 ml) and the reaction heated at 50°C for 2 h. Chloroform (200 ml) was added and the resultant mixture extracted with water (100 ml). The organic layer was dried (MgS04), filtered and the solvent evaporated. The residue was chromatographed on silica gel (200 g) employing 1.1 chloroform/light petroleum (100 ml fractions) for elution. Fractions 17-22 were combined and concentrated affording the α-sulfone disulphide (4) (0.38 g, 1.5 mmol, 13%) which was identical to (4) described under "Preparation of α-Sulfone Disulphides (1 ) (R' = CH3)" by i.r., 1H n.m.r. (270 MHz) and 13C n.m.r. spectroscopy.
The next targets selected were (3; X = o-S02CH3) and (3, X = p-S02CH3). Given that benzenesulfonyl chlorides can generally be reduced (LιAIH4) to the corresponding mercaptans (Fong, H. O., Hardstaff, W. R., Kay, D. G., Langler, R F., Morse, R. G., and Sandoval, D. N., Can. J. Chem., 1979, 57m 1206), we elected to prepare the appropriate methylsulfonyl-substituted benzenesulfonyl chlorides (14) and (15) (Gmige, K A., Goehl, J.E., and Langler, R. F., Can. J. Chem., 1996, 74, 1638). Lithium aluminum hydride reduction, followed by attempted thiomethylation, as shown in Scheme 7, gave none of the target methyl disulphides, but instead furnished the symmetrical disulphides (7) and (8).
Scheme 7
49
SUBSTTTUTE SHEET (RULE 26) The disuifone disulphides (7) and (8) are fungitoxic (see Table 5) The next set of target disulphides included (3, X = o-N02), (3, X = m-N02) and (3, X = p-N02) Each of these molecules contains a most powerful electron withdrawing group and so, were expected to be potent antifungal disulphides The results for those compounds ((9), (10) and (1 1 ) in Table 1 ) are in complete accord with expectations
Appropriately substituted nitrophenyl or methylsulfonylphenyl aromatics can undergo smooth nucleophilic aromatic substitutions which arylate thiolate anions (Ginige, K A , Goehl, J E , and Langler, R F , Can J Chem , 1996, 74, 1638, Baum, J C , Bolhassan, J , Langler, R F , Pujol, R J , and Raheja, R K , Can J Chem , 1990, 68, 1450) in dimethyl sulfoxide or hexamethylphosphoramide (e g see Scheme 8)
Scheme 8
Preparation of Compounds of Table 5
The test results (Table 5) for compounds (12) and (13) demonstrate that potential nucleophilic aromatic substitutions which arylate by displacement of the entire disulphide linkage do not inhibit fungal growth Previously Prepared Compounds
Compound (12) was prepared as described in Ginige, K A , Goehl, J E , and Langler, R F , Can J Chem , 1996, 74, 1638 and compound (13) was prepared as outlined in Baum, J C , Bolhassan, J , Langler, R F , Pujol, R J , and Raheja, R K , Can J Chem , 1990, 68, 1450 Preparation of Phenyl Methyl Disulphide
Sodium metal (0 02 g, 0 69 mmol) was dissolved in methanol (1 ml) and thiophenol (0 1 ml) added The solvent was evaporated and the sodium thiophenate dried in vacuo The thiophenate salt was dissolved in dimethyl sulfoxide (10 ml) A portion of the resultant solution (1 ml) was added to a mixture of diphenyl disulphide (1 98 g, 9 1 mmol) and dimethyl disulphide (12 ml) The reaction mixture was stirred at ambient temperature for 8 days
2 5% Hydrochloric acid (70 ml) was added and the resultant mixture washed with diethyl ether (three - 50 ml aliquots) The organic layers were combined, dried (MgS04), filtered and the solvent evaporated The concentrate was rectified at reduced pressure affording phenyl methyl disulphide (2 38 g, 15 2 mmol, 84%), b p 82 - 86°C/1 8 Torr 1H
50
SUBSTTTUTE SHEET (RULE 26) n.m.r (270 MHz) δ 7 51 , d, 2H, 7 30, t, 2H, 7 20, t, 1 H, 2.40, s, 3H 13C n.m r δ 22.82, 126.77, 127 47, 128 96, 136.83. m/z 156 (100%, M+ ), 141 (65%) and 109 (57%). Preparation of Methyl o-Mercaptobenzoate o-Mercaptobenzoic acid (9.9 g, 64 2 mmol) was dissolved in methanol (300 ml) and concentrated sulfuπc acid (0.5 ml) added. The reaction mixture was refluxed for 72 h. Chloroform (300 ml) was added and the resultant mixture extracted with water (two - 100 ml aliquots) and 1 % sodium hydroxide (two - 100 ml aliquots) The combined aqueous layers were added to chloroform (75 ml), ice (100 ml) and concentrated hydrochloric acid (8 ml). Chloroform (150 ml) was added, the layers separated, the organic layer dried (MgS0 ) and filtered The solvent was evaporated and the residue rectified at reduced pressure affording clean o-mercapto methyl benzoate (6.9 g, 41.0 mmol, 64%), b.p. 103 - 108 C/2.7 Torr. l.r. (liquid film) 1710 cm"1. 1H n.m.r. (270 MHz) δ 7.99, d, 1 H, 7.29, d, 1 H; 7.14, m, 2H, 4.68, s, 1 H, 3.90, s, 3H 13C n.m.r. δ 167.09, 138.27, 132.45, 131.65, 130.86, 125.74, 124.62, 52.19 m/z 168 (21 %, M+ ), 136 (100%), 108 (35%). Preparation of o-Carbomethoxyphenyl Methyl Disulphide (6)
Powdered sodium hydroxide (0.24 g, 6 mmol) was suspended in dimethyl sulfoxide (8 ml) and a solution of o-mercapto methyl benzoate (1.0 g, 5.9 mmol) in dimethyl sulfoxide (5 ml) added. The reaction mixture was stirred for 5 mm and a solution of dimethyl disulphide (1.7 g, 18 mmol) in dimethyl sulfoxide (7 ml) added. The reaction mixture was stirred for 24 h at ambient temperature.
2.5% Hydrochloric acid (150 ml) was added to the reaction and the resultant mixture extracted with diethyl ether (three - 100 ml aliquots). The organic layers were combined and concentrated. 2.5% Hydrochloric acid (150 ml) was added to the concentrate and the resultant mixture washed with diethyl ether (three - 100 ml aliquots) The organic layers were combined and concentrated. 2.5% (W/V) Sodium hydroxide solution (150 ml) was added to the residue and the resultant mixture extracted with diethyl ether (three - 100 ml aliquots). The combined organic layers were dried (MgS04), filtered and the solvent evaporated. The crude product was chromatographed on silica gel (100 g) employing 3.2 chloroform/light petroleum (100 ml fractions). Fractions 3 and 4 were combined and concentrated and the product rectified at reduced pressure giving clean (6) (0.09g, 0.4 mmol, 7%), b.p. 141 - 142°C/2.1 Torr (Found: C, 50.5; H, 4.6. C9H10O2S2 requires C, 50.4, H, 4.7). l.r. (liquid film) 1705 cm"1. 1H n.m.r. (270 MHz) δ 8.15, d, 1 H, 8.04, d, 1 H; 7.58, t, 1 H; 7.25, t, 1 H; 3.93, s, 3H; 2.40, s, 3H. 13C n.m.r. δ 166.80, 141.30, 132.92, 131.57, 126.90, 125.09, 52.29, 21.99. m/z 214 (35%, M+ ), 167 (100%), 152 (37%), 136 (41 %) Preparation of o-Chlorophenyl Methyl Sulphide
Sodium metal (0.80 g, 34 mmol) was dissolved in methanol (80 ml) and a solution of o- chlorothiophenol (5.1 g, 35 mmol) in methanol (10 ml) added. The reaction mixture was cooled with an ice/water bath and a solution of methyl iodide (5.0 g, 35 mmol) in methanol (10 ml) was added dropwise The reaction mixture was stirred at ambient temperature for 24 h Water (100 ml) was added and the resultant mixture extracted with chloroform (three - 100 ml aliquots) The organic layers were combined, dried (MgS0 ) and the solvent evaporated The residue was distilled at reduced pressure yielding o-chlorophenyl methyl sulphide (4 7 g, 29 7 mmol, 85%), b p 80 - 86°C/3 0 Torr 1H n m r (270 MHz) δ 7 33, d, 1 H, 7 22, t, 1H, 7 12, d, 1 H, 7 07 t, 1H, 247, s, 3H 13C n m r δ 137 70,131 75, 129 69, 129 34, 127 18, 125 46, 15 13 m/z 160 (33%), 158 (100%, M+ ), 145 (24%) 143 (66%) Preparation of o-Chlorophenyl Methyl Sulfone o-Chlorophenyl methyl sulphide (4 1 g, 25 9 mmol) in chloroform (86 ml) was added dropwise to 10% sulfunc acid (120 ml) Simultaneously, potassium permanganate (13 9 g) was added in small portions The double addition took 45 mm Upon completion of the addition, the reaction mixture was stirred at ambient temperature for 1 h The reaction mixture was cooled in an ice/water bath and sodium bisulfite added until the reaction mixture became colorless The layers were separated and the aqueous layer extracted with chloroform (three - 100 ml portions) The combined organic layers were dried (MgS04), filtered and the solvent evaporated Crude chlorosulfone was recrystallized (methanol) affording clean chlorosulfone (4 0 g, 21 0 mmol, 81 %), m p 93 5 - 95 1 °C I r 1330, 1 165 cm 1 1H n m r (270 MHz) δ 8 15, d, 1 H, 7 58, m, 2H, 7 48, m 1 H, 3 29, s, 3H 13C n m r δ 138 03, 134 79, 131 91 , 130 84, 127 52, 42 73 m/z 192 (7%), 190 (20%, M+ ), 113 (33%), 111 (100%) Preparation of o-Methylsulfonylphenyl Benzyl Sulphide
Sodium metal (0 24 g, 10 4 mmol) was dissolved in methanol (5 ml) and benzyl thiol (1 3 ml) added The solvent was evaporated and sodium benzyl thiolate dried in vacuo The sodium benzyl thiolate was dissolved in dimethyl sulfoxide (50 ml) and o-chlorophenyl methyl sulfone (2 0 g, 10 6 mmol) added The reaction mixture was stirred at ambient temperature for 19 h 10% Hydrochloric acid (200 ml) was added and the product filtered off Dried sulfone sulphide (2 0 g) was recrystallized (methanol) furnishing o- methylsulfonylphenyl benzyl sulphide (1 6 g, 5 8 mmol, 55%), m p 132 0 - 133 4°C I r 1315, 1155 cm 1 1H n m r (270 MHz) δ 8 07, d, 1 H, 7 50, m, 2H, 7 37, m, 6H, 4 24, s, 2H, 3 17, s, 3H 13C n m r δ 139 47, 137 15, 136 08, 133 59, 131 05, 130 10, 128 93 128 70, 127 67, 126 42, 42 05, 39 28 m/z 278 (3%, M+ ), 91 (100%) Preparation of o-Chlorosulfonylphenyl Methyl Sulfone (14) o-Methylsulfonylphenyl benzyl sulphide (1 0 g, 3 5 mmol) was suspended in glacial acetic acid (35 ml) and water (3m!) Cl2 (ca 200 ml/mm) was bubbled into the reaction mixture for 45 mm Ice/water cooling was employed, as necessary, to maintain the reaction temperature below 30°C Chloroform (100 ml) was added and the resultant mixture extracted with 2 5% (W/V) sodium hydroxide (three - 50 ml aliquots) The organic layer was dried (MgS04), filtered and the solvent evaporated The sulfone sulfonyl chloride was recrystallized (dry carbon tetrachloπde) yielding o-chlorosulfonylphenyl methyl sulfone (0 64 g, 2 5 mmol 71 %), m p 140 4 - 142 2°C I r 1380, 1330, 1155 cm 1 1H n m r (270 MHz) δ 8 42, t, 2H, 7 95 m, 2H, 3 41 , s, 3H 13C n m r δ 142 80, 139 08,135 99, 134 64, 133 18, 131 72, 45 12 Preparation of the Disulfone Disulphides (7) and (8) Both sulfone disulphides were prepared as described below for di-o- methylsulfonylphenyl disulphide (7) Note that the preparation of p-chlorosulfonylphenyl methyl sulfone has been described earlier
(A) Lithium aluminum hydride (1 2 g, 31 5 mmol) was added to tetrahydrofuran (20 ml) A solution of o-chlorosuifonylphenyl methyl sulfone (2 0 g, 7 8 mmol) in tetrahydrofuran (80 ml) was added dropwise over 25 mm The reaction mixture was refluxed for 1 h After cooling to ambient temperature the following chemicals were added sequentially in a dropwise manner ethyl acetate (20 ml), methanol (10 ml), water (10 ml), 1 % hydrochloric acid (40 ml) and concentrated hydrochloric acιd(12 ml) Chloroform (250 ml) was added and the resultant mixture washed with water (two - 150 ml aliquots) The organic layer was dried (MgS04), filtered and concentrated affording crude phenyl methyl sulfone (0 33 g) Phenyl methyl sulfone was recrystallized (methanol) and shown to be identical to authentic material by m p , mixture m p , i r and 1H n m r (60 MHz)
The aqueous layer from the extraction procedure was acidified (12 ml of concentrated hydrochloric acid) and the resultant mixture extracted with chloroform (three - 100 ml aliquots) The combined organic layers were dried (MgS04), filtered and the solvent evaporated affording crude oily o-mercaptophenyl methyl sulfone (0 6 g)
(B) Sodium metal (0 25 g, 10 7 mmol) was dissolved in methanol (25 ml) and methanethiol (250 ml) bubbled into the solution The solvent was evaporated and the sodium methanethiolate dried in vacuo The sodium methanethiolate was dissolved in dimethyl sulfoxide (15 ml) and a solution of oily o-mercaptophenyl methyl sulfone (1 9 g, 10 mmol), dimethyl disulphide (3 0 g, 31 mmol) and dimethyl sulfoxide (5 ml) added The reaction mixture was stirred at ambient temperature for 20 h 2 5% Hydrochloric acid (150 ml) was added and the resultant mixture extracted with diethyl ether (three - 100 ml aliquots) The organic layers were combined, dried (MgS04), filtered and the solvent evaporated Crude disulfone disulphide (7) was recrystallized from methanol (175 ml)
Clean disulfone disulphide (7) (0 69 g, 1 8 mmol, 46% from the sulfonyi chloride) had m p 225 - 227°C (Found C, 45 0, H, 3 8 C14H1404S4 requires C, 44 9, H, 3 8) I r (KBr) 1300, 1140 cm 1 1H n m r (DMSO-d6, 270 MHz) δ 8 06, d, 1 H, 7 86, m, 2H, 7 65, t, 1 H, 344, s, 3H 13C n m r (DMSO-d6) δ 138 22, 135 67, 134 92, 130 04, 127 90, 127 64, 42 52 m/z 374 (21 %, M+ ), 296 (11 %), 234 (15%), 188 (100%)
Clean disulfone disulphide (8) (22% from the sulfonyi chloride) had m p 183 - 185°C (Found C. 45 2, H, 3 9 C14H1404S4 requires C, 44 9, H, 3 8) I r (KBr) 1308, 1155 cm 1 1H n m r (DMSO-d6, 270 MHz) δ 7 93, d, 4H, 7 80, d, 4H, 3 22, s, 6H 13C n m r (DMSO-d6) δ 141 61 , 139 52, 128 05, 126 45, 43 33 m/z 374 (31 %, M+ ), 234 (18%), 188 (100%)
53
SUBSTTTUTE SHEET (RULE 26) Preparation of the Nitrophenyl Disulphides (9), (10) and (11)
The nitrophenyl methyl disulphides were prepared from the appropriate symmetrical dι(nιtrophenyl) disulphides as described below for the para-nitro case
Sodium metal (0 018 g, 0 78 mmol) was dissolved in methanol (10 ml) and methanethiol (20 ml) bubbled into the solution The solvent was evaporated and the sodium methanethiolate dried in vacuo
The sodium methanethiolate was dissolved in dimethyl sulfoxide (10 ml) Dι(p- nitrophenyl) disulphide (2 0 g, 6 6 mmol) was added to dimethyl sulfoxide (2 ml) and a portion of the methanethiolate solution (1 ml) added Dimethyl disulphide (12 ml) was added and the reaction mixture stirred at ambient temperature for 8 days The reaction mixture became homogeneous after stirring for 24 h
2 5% Hydrochloric acid (70 ml) was added and the resultant mixture extracted with diethyl ether (three - 50 ml aliquots) The organic layers were combined, dried (MgS04), filtered and rotary evaporated The residue was chromatographed on silica gel (50 g) employing light petroleum (twelve - 50 ml fractions) followed by 1 1 light petroleum/chloroform (50 ml fractions) for elution Fractions 13 - 22 were combined and rectified at reduced pressure affording p-nitrophenyl methyl disulphide (11 ) (1 2 g, 5 9 mmol, 45%), b p 146 - 149°C/0 9 Torr (11 ) crystallized on standing and after recrystallization (methanol) had m p 42 9 - 44 3°C Clean o-nitrophenyl methyl disulphide (10) (31 %) had m p 49 - 51 °C (Found C, 41 9,
H, 3 3 C7H7N02S2 requires C, 41 8, H, 3 5) I r 1524, 1340 cm 1 1H n m r (270 MHz) δ 8 29, t, 2H, 7 71 , t, 1 H, 7 37, t, 1 H, 2 43, s, 3H 13C n m r δ 137 25, 134 10, 126 79, 126 27, 126.09, 21 89 m/z 201 (14%, M+ ), 136 (100%), 122 (41 %)
Clean m-nitrophenyl methyl disulphide (9) (74%) had b p 152 - 158°C/2 Torr (Found C, 42 0, H, 3 6 C7H7N02S2 requires C, 41 8, H, 3 5) I r 1530, 1350 cm 1 1H n m r (270 MHz) δ 8 39, s, 1 H, 8 04, d, 1 H, 7 80, d, 1 H, 7 52, t, 1 H, 2 49, s, 3H 13C n m r δ 148 78, 140 10, 132 26, 129 79, 121 37, 121 07, 22 85 m/z 201 (100%, M+ ), 140 (48%)
Clean p-nitrophenyl methyl disulphide (11 ) (45%) had C, 41 9, H, 3 5 C7H7N02S2 requires C, 41 8, H, 3 5 I r 1515, 1340 cm 1 1H n m r (270 MHz) δ 8 20, d, 2H, 7 66, d, 2H, 2 48, s, 3H 13C n m r δ 146 43, 125 73, 124 15, 22 71 m/z 201 (100%, M+ ), 140 (56%) Preparation of Compounds of Table 6
An older example of α-ester disulphide preparation which applied a Pummerer reaction to a thiosulfinate (Saito, I , and Fukui, S , J Vitammol (Kyoto), 1966, 12, 244) is illustrated by the following reaction scheme (see Scheme 9)
Scheme 9
The foregoing reaction appears to proceed through the intermediacy of an acetoxy sulfonium ion. Therefore the direct reaction of a disulphide with dibenzoyi peroxide should produce a benzoyloxy sulfonium ion and, from there, an α-ester disulphide Relative to prior disclosed reaction schemes, dibenzoyi peroxide provided a significant improvement in the yield of an α-ester disulphide as shown in Scheme 10
CH3SSCH3 + (phC(0)θ)2 Q^a » CH3SSCH2OC(0)Ph + CH3Sθ2SCH3
(4) 26% 22%
+ (PhCO)20 47%
Scheme 10 Although antifungal testing on (4) showed it to be very potent (see Table 6), it was less fungitoxic than several other disulphides, including (3), that have been described earlier (Baerlocher, F.J., Langler, R.F , Frederiksen, M.U., Georges, N M., and Witherell, R.D., Aust. J. Chem., 1999, 52, 167; Langler, R.F., MacQuame, S.L., McNamara, R.A., and O'Connor, P.E., Aust. J. Chem. 52, 1119 (1999); and Baerlocher, F.J., Baerlocher, M.O., Langler, R.F., MacQuame, S.L., and Marchand, M.E., Aust. J. Chem. 53, 1200). In order to examine a structural variant of (4), the α-ester disulphide PhSSCH2OC(0)CH2CH3 (5) was synthesized. Unfortunately, potassium permanganate/propionic acid oxidation of phenyl methyl disulphide only furnishes (5) in 0.9% yield. Consequently, an alternative two-step synthesis from (3) was developed (vide Scheme 11 )
CH3SSCH2OC(0)CH2CH3 + S02CI2 CISCH2OC(0)CH2CH3 (3) (6)
PhSH / CH2CI2 pyridine
PhSSCH2OC(0)CH2CH3 (5) 38%
Scheme 11 Compound (2) and (3) are known compounds Methods of preparing these compounds are also known, these methods having been described in Georges, N M Johnson, M D., Langler, R F , and Verma, S D , Sulfur Lett. 22, 141 (1999) Preparation of Disulphide Benzoate (4) Five parallel reactions were conducted as follows A solution of dimethyl disulphide
(1 '3 g, 13 8 mmol) and dibenzoyi peroxide (5 1 g, 21 0 mmol) in chloroform (60 ml) was refluxed behind a safety shield for 24 h
Upon completion of the reflux period, the five runs were combined and chloroform (250 ml) was added The resultant solution was extracted with 2 5% sodium hydroxide solution (two - 125 ml aliquots) The organic layer was dried (MgS04), filtered and the solvent evaporated. The crude product was rectified at reduced pressure affording a low-boiling fraction (2.0 g, b p. 25-80°C/1 9 Torr). A portion of the low-boiling fraction (1 0 g) was chromatographed on silica gel employing 1 1 chloroform/light petroleum (100 ml fractions) for elution Methyl methanethiosulfonate (0 08 g) was obtained Based on g I c /m.s , the high-boiling distillation fraction (b p 80-140°C/1 6 Torr) contained a mixture of the disulphide benzoate (4) (2.1 g, 9 8 mmol) and dibenzoyi anhydride (2.1 g) In the same manner, the distillation residue was determined to contain the disulphide benzoate (4) (1.5g, 7.0 mmol) and dibenzoyi anhydride (9 1 g). Distillation, at atmospheric pressure, of the condensate from the cold trap, furnished a mixture which contained methyl methanethiosulfonate (0 7 g).
A sample of the higher-boiling distillation fraction (2 g) was chromatographed on silica gel (200 g) employing 3 1 light petroleum/chloroform (100 ml fractions) for elution Fractions 9-12 were combined and concentrated affording disulphide benzoate (4) The disulphide (4) (0.6 g) had b.p. 139°C/2 Torr (Found. C, 50.7; H, 4 6. C9H10O2S2 requires C, 50.4; H, 4 7). l.r. 1720 cm'1 1H n m.r (270 MHz) δ 2.51 , s, 3H; 5.55, s, 2H, 7.47, t, 2H, 7.60, m, 1 H; 8.07, d, 2H. 13C n.m.r δ 24 48, 73 55, 128.52, 129 51 , 129 77, 133 4, 165 76 m/z 184 (9%, M+ -CH20), 105 (100), 77 (36) Preparation of the Sulfenyl Chloride (6)
A solution of sulfuryl chloride (1.6 g, 11 9 mmol) in dry methylene chloride (10 ml) was added to a solution of the disulphide propionate (3)2 (2 0 g, 12 mmol) in dry methylene chloride (6 ml) The reaction mixture was refluxed for 0.5 h and the solvent carefully evaporated.
Crude product was distilled at reduced pressure affording impure sulfenyl chloride (6) (0.8 g). The sulfenyl chloride (6) had b.p. 94-114°C/53 Torr and was not further purified Impure sulfenyl chloride (6) had i.r 1750 cm'1 1H n m r (270 MHz) showed major signals at δ 1 18, t, 3H, 2.42, q, 2H, 5.60, s, 2H 13C n.m.r showed major signals at δ 8 84, 27.39, 72.31 , 173 76 m/z 154 (6%, M+ ) 118 (11 ), 57 (100) The sulfenyl chloride (6) was routinely stored in the freezer
56
SUBSTTTUTE SHEET (RULE 26) Preparation of the Propionate Disulphide (5)
Distilled impure sulfenyl chloride (6) (1 0 g) was added to a solution of benzene thiol (0 7 g, 6 3 mmol) and pyridme (1 ml) in dry methylene chloride (15 ml) The reaction mixture was stirred at ambient temperature for 2 h Chloroform (100 ml) was added and the resultant mixture washed with 2 5% HCI (100 ml) and then 2 5% sodium hydroxide solution (100 ml) The organic layer was dried (MgS04), filtered and the solvent evaporated The residue was chromatographed on silica gel (100 g) employing light petroleum (twenty - 100 ml fractions) followed by chloroform (100 ml fractions) Fraction 22 furnished clean disulphide propionate (5) (0 53 g 2 3 mmol) The reaction was repeated on impure sulfenyl chloride (6) (2 0 g) and the chromatographed (5) so-obtained was added to the chromatographed product from the first run The disulphide was rectified at reduced pressure affording clean (5) (1 7 g, 7 4 mmol) whose properties were in full accord with those expected Preparation of Compounds of Table 7 Preparation of (CH3OC(0)CH2SSCH2C(0)C2H5) methyl 3,4-dιthιa-5- propionoxypentanoate
Methyl thioglycollate (1 896 g, 15 8 mmol) was added to dry methylene chloride (10 ml) CISCH2OC(0)C2H5 (2 44 g 15 6 mmol) was dissolved in dry methylene chloride and the resultant solution added to the reaction mixture Dry pyridme (2 5 ml) was added and the reaction mixture stirred at ambient temperature for 24 h
Chloroform (200 ml) was added to the reaction and the resultant solution washed first with 2 5% hydrochloric acid (200 ml) then with 2 5% sodium hydroxide (200 ml) The organic layer was dried (MgS04) filtered and concentrated
The residue was chromatographed on silica gel (250 g) employing petroleum ether (200 ml fractions) for fractions 1 - 19, then chloroform (200 ml fractions) Fraction 23 was concentrated and rectified at reduced pressure affording CH3OC(0)CH2SSCH2OC(0)C2H5 (0 53 g, bp 140 - 150°C/4 5 Torr) I r 1755 cm 1 1H n m r (270 MHz) δ 1 17, t, 3H, 2 40, g, 2H, 3 58, s, 2H, 3 77, s, 3H, 5 32, s, 2H 13C n m r δ 8 84, 27 55, 41 76, 52 63, 72 65, 169 54, 173 63 m/z 224 (1 %, M1,), 194 (41 %), 57 (100%) Preparation of ((C2H5OC(0)CH2S)2) 2,3-dithiabutane-1,4-dιpropιonate
(CH3SSCH2OC(0)C2Hs) 2,3-dιthιabutylpropιonate (18 029 g, 109 mmol) was added to propionic acid (350 ml) and the solution refluxed Portions of potassium permanganate (ca 3 g each, 18 081 g in total) were added The solution would immediately turn brown upon the addition of an aliquot of permanganate but would turn white thereafter Provided the color change took place in less than 5 mm, another 3 g portion of permanganate was added. When the color change took longer than 5 mm, smaller portions (ca 0 5 g each) were added at 5 m intervals Propionic acid (ca 5 ml) was used to rinse each aliquot of permanganate into the reaction mixture When the last addition had been done (elapsed time 1 h), the reaction mixture was cooled in an ice/water bath
57
SUBSTTTUTE SHEET (RULE 26) Chloroform (500 ml) was added and the resultant mixture washed with 10% sodium hydroxide solution (six - 250 ml portions), after which the aqueous pH remained basic. The organic layer was dried (MgS04), filtered and concentrated. The residue was rectified at reduced pressure affording unchanged starting material (4 342 g, bp 161 -189°C/I Torr) and (C2H5C(0)OCH2S)2 (5.061 g, bp 1 10- 122°C/0.7 Torr).
- (C2H5C(0)OCH2S)2 had i.r. 1750 cm1 1H n.m.r. (270 MHz) δ 1.18, t, 3H, 2.42, q, 2H; 5.28, s, 2H. 13C n.m.r δ 27.5, 72 7, 173 5. m/z 208 (14%), 57 (100%) Preparation of (C2H5C(0)OCH2SSCH2S02(C6H4)CH3-p) 1-p-toluenesulfonyl-4- propionoxy-2,3-dithiabutane ((C2H5C(0)OCH2S)2) 2,3-dιtfιιabutane-1 ,4-dιpropιonate (0.507 g) was dissolved in a solution of acetone/ water (4:1 respectively, 30 ml) Sodium p-toluenesulfinate polyhydrate (0.411 g) was added and the reaction mixture heated at 49°C for 2 h. At the end of this period, the reaction mixture was dark orange in color.
Water (100 ml) was added and the resultant mixture washed with chloroform (three 50 ml portions). The combined organic layers were dried (MgS04) and concentrated. Crude product was chromatographed on silica gel (50 g), employing chloroform for elution. Fractions 10 - 12 were combined and concentrated affording the product (0.21 g). C2H5C(0)OCH2SSCH2S02(C6H4)CH3-p had i.r. 1749, 1326, 1151 cm"1. 1H n.m.r. δ 1.16, t, 3H; 2.38, q, 2H; 2.47, s, 3H; 4.24, s, 2H; 5.34, s, ZH; 7.38, d, 2H; 7.80, d, 2H. 13C n.m.r. δ 8.82, 21.70, 27.41 , 63.93, 72.38, 128.99, 129.94, 134.43, 145.49, 173.43. m/z 290 (13.7%), 57 (100%).
Preparation of Compound Q, 1-phenyl-1,2-dithiapropyl propionate, PhSSCH2OC(0)C2H5(See Table 6)
Compound Q is a potent antifungal compound. It's preparation is described in the preprint "New Antifungal Disulphides: Approaching Submicrogram Toxicity", F.J.
Baerlocher,, M.O. Baerlocher, C L. Chaulk, R.F Langler and E.M. O'Brien, Sulfur Lett., - in press, the disclosures of which are incorporated herein by reference See earlier description relative to Scheme 5 for method. Preparation ofN, 2,3,5-trithiahexane, CH3SCH2SSCH3 (See Table 3) Sodium metal (0.020 g) was dissolved in methanol (2 ml) and methanethiol (20 ml) slowly bubbled into the solution. The resultant solution was concentrated and the residue dried in vacuuo. The resulting solid was dissolved in DMSO (10 ml). A portion of this solution (1 ml) was added to a mixture of dimethyl disulphide (12 ml) and (CH3SCH2S)2 (2.00 g)(preparatιon - P. Dubs and R. Stuessi, Helv. Chim. Acta 61 , 2351 (1978)). The reaction mixture was stirred at ambient temperature for eight days.
2.5% Hydrochloric acid (70 ml) was added and the resultant mixture extracted with diethyl ether (three - 50 ml aliquots). The organic layers were combined and dried (MgS04), filtered and concentrated. The concentrate was distilled at reduced pressure yielding N (2 336 g, bp 92 - 102°C/18 Torr) N had 1H n m r (270 MHz) δ 2 22, s, 3H, 2 49, s, 3H,
3 86, s, 2H 13C n m r δ 65 12, 73 39, 94 23
Preparation of P, phenacyl methyl disulphide, PhC(0)CH2SSCH3
A) Phenacyl chloride (1 004 g) was added to dry pyridme (4 ml) Thiolacetic acid (0 560 g) was dissulved in dry pyridme (6 ml) and added to the reaction mixture The reaction flask was fitted with a drying tube and the reaction mixture heated at 800°C for 1 5 h Chloroform (200 ml) was added and the resultant mixture extracted with 5% hydrochloric acid (150 ml), followed by 2 5% sodium hydroxide (100 ml) The organic layer was dried (MgS04), filtered and concentrated The product was chromatographed on silica gel (5 g) employing petroleum ether (400 ml) for elution Evaporation of the solvent afforded an orange oil
Fractional distillation provided phenacyl thiolacetate (0 959 g, bp 137-139°C/2 4 Torr) I r 1710, 1685 cm 1 Η n m r (270 MHz) δ 2 38, s, 3H, 4 39, s, 2H, 7 46, t, ZH, 7 58, t, 1 H, 7 97, d , 2H 13C n m r δ 30 17, 36 62, 128 44, 128 74, 133 67, 135 50, 193 10, 194 08 m/z 194 (M1 , 3%), 105 (100%) B) Potassium carbonate (19 00 g) was added to methanol (147 ml) which was cooled
(0°C) and stirred 1 h Phenacyl thiolacetate (4 157 g) was added to the reaction vessel dropwise Cooling and stirring continued for another 30 mm Diethyl ether (205 ml) and water (200 ml) were added and the reaction mixture cooled for another 30 mm Iodine (3 590 g) was added in small portions over 30 mm Saturated sodium thiosulfate solution (16 ml) was added
Diethyl ether (85 ml) was added to the reaction mixture and the layers separated The organic layer was washed with distilled water (two - 200 ml aliquots The organic layer was dried (MgS04), filtered and concentrated in vacuuo The product was chromatographed on silica gel (400 9) employing chloroform for elution (100 ml fractions) Fractions 16-29 were combined and concentrated The concentrate was recrystallized from methanol /benzene which produced a crop of gummy crystals (0 551 g) A second recrystallization furnished phenacyl disulphide with the following properties I r 1675 cm 1 1H n m r δ4 20, s, 4H, 7 46, t, 4H, 7 59, t, 2H, 7 94, d, 4H 13C n m r δ 45 36, 128 74, 128 77, 133 68, 135 35, 194 30 m/z 105 (100%) C) Sodium metal (0 019 g) was dissolved in methanol (10 ml) Methanethiol (20 ml) was bubbled through the solution The solvent was evaporated and the residue dried in vaccuo DMSO (10 ml) was added to the solid and the mixture stirred for 30 m
Phenacyl disulphide (1 603 g) and dimethyl disulphide were combined and a portion of the DMSO solution (1 ml) was added The reaction mixture was stirred at ambient temperature for 8 days 2 5% Hydrochloric acid (70 ml) was added The resultant mixture was extracted with diethyl ether (three - 50 ml aliquots) The combined organic layers were dried (MgS04), filtered and concentrated
The crude product was chromatographed on silica gel (200 g) employing 3 7 chloroform/petroleum ether (100 ml fractions) for elution Fractions 12-19 were combined and concentrated The residue was rectified at reduced pressure furnishing phenacyl methyl disulphide (1 121 g, bp 144 - 1540,C/1 -7 Torr) It had i r 1690 cm 1 1H n m r δ 2 37, s, 3H, 4 09, s, 2H, 7 47, t, 2H, 7 59, t, IH), 7 97, d, 2H 13C n m r δ 22 93, 44 25, 128 7, 128 73, 133 51 , 135 23, 194 44 rn/z.198 (Ml, 15%), 105 (100%) Preparation of Compounds in Table 8 Previously Prepared Compounds
Compound (2) was prepared as described earlier (Langler, R F , MacQuame, S L , McNamara, R A , and O'Connor, P E , Aust J Chem - in press) The preparations of compounds (3), (12), (17) and (7) were outlined previously (Baerlocher, F J , Baerlocher, M O , Langler, R F , MacQuame, S L , and Marchand, M E , Aust J Chem - in press) The synthesis of compound (20) has been reported (Baerlocher, F J , Langler, R F , Frederiksen, M U , Georges, N M , and Witherell, R D , Aust J Chem , 1999, 52, 167 Syntheses for compounds (4), (13), (15) and (16) have been described in refs Oae, S , Takata, T , and Kim, Y H , Bull C S Jpn , 1982, 55, 2484, Goodndge, R J , Hambley, T W , and Haynes, R K , J Org Chem ,1988, 53, 2881 , Langler, R F , Ryan, D A , and Verma, S D , Sulfur Lett 24, 51 (2000), and Back, T G , Collins, S , and Krishna, M V , Can J. Chem , 1987, 65, 38, respectively Three Approaches to the Synthesis of Phenyl Methanethiosulfonate (4)
(A) Disulphide Oxidation Methyl phenyl disulphide (See Baerlocher, F J , Baerlocher, M O , Langler, R F ,
MacQuame, S L , and Marchand, M E , Aust J Chem - in press ) (1 0 g, 6 4 mmol) and hydrogen peroxide (30%, 1 5 g) were dissolved in glacial acetic acid (25 ml) and the reaction refluxed behind a safety shield for 0 5 h Chloroform (100 ml) was added and the resultant mixture extracted with 2 5% sodium hydroxide solution (three - 50 ml aliquots) The organic layer was dried (MgS04), filtered and the solvent evaporated The crude product was chromatographed on silica gel (100 g) employing 1 1 chloroform/light petroleum (100 ml fractions) for elution Fractions 11 and 12 were combined and concentrated affording clean phenyl methanethiosulfonate (4) (0 20 g, 1 1 mmol, 17%) Recrystallized (4) (methanol) had m.p. 88 9 - 904EC I r 1335, 1145 cm 1 1H n m r (270 MHz) δ 3 19, s, 3H, 7 54, m, 3H, 772, d, 2H 13C n m r 647 39, 127 93, 129 92, 131 67, 136 24 m/z 188 (35%, M+ ), 125 (57), 109 (100)
(B) Benzenesulfenyl Chloride From Disulphide, Then Reaction With Methanesulfinate Anions
Diphenyl disulphide (1 0 g, 4 6 mmol) was dissolved in dry methylene chloride (5 ml) and a solution of sulfuryl chloride (0 6 g, 4 6 mmol) in dry methylene chloride (5 ml) added dropwise Upon completion of the addition, the reaction mixture was refluxed for 0 5 h
A solution of sodium methanesulfinate (0 94 g, 9 2 mmol) in acetone (40 ml) and water (10 ml) was added to the reaction mixture which was then immersed in a constant temperature bath at 50EC for 1 h The workup described for part (A) furnished diphenyl disulphide (0 35 g, 35% from column fractions 2 and 3) and the thiosulfonate (4) (0 66 g, 3 5 mmol, 38%, from fractions 7 - 17)
(C) Benzenesulfenyl Chloride From Mercaptan, Then Reaction With Methanesulfinate
Anions Benzenethiol (1 Og, 9 0 mmol) was reacted with sulfuryl chloride (1 3 g, 9 7 mmol) and sodium methanesulfinate (0 96 g, 9 4 mmol) as described for diphenyl disulphide in part (B) Extractive workup and column chromatography, as described in part (B), furnished diphenyl disulphide (0 11g, from fraction 3) and the thiosulfonate (4) (0 95g, 5 0 mmol, 55%, from fractions 7 - 11 ) Preparation of Methyl Ethanethiosulfonate (5)
(A) Sodium benzenethiolate (2 1 g, 15 6 mmol) was dissolved in acetone (50 ml) and ethanesulfonyl chloride (1 0 g, 7 8 mmol) added The reaction mixture was refluxed for 1 h Chloroform (200 ml) was added and the resultant mixture washed with water (100 ml) The aqueous layer was concentrated and dried in vacuuo for 8 h, producing a mixture (1 12 g) of sodium ethanesulfmate and sodium chloride The product had 1H n m r (270 MHz, D20 + DSS) δ 1 08, t, 3H, 2 33, q, 2H 13C n m r δ 7 90, 56 44
(B) Dimethyl disulphide (0 45 g, 4 8 mmol) was dissolved in dry methylene chloride (5 ml) and a solution of sulfuryl chloride (0 65 g, 4.8 mmol) in dry methylene chloride (5 ml) added dropwise. Upon completion of the addition, the reaction mixture was refluxed for 0.5 h A solution of the mixture of sodium ethanesulfmate and sodium chloride (1 12 g) in water (10 ml) and acetone (40 ml) was added The reaction mixture was immersed in a constant temperature bath at 50EC for 1 h
Chloroform (200 ml) was added and the resultant mixture extracted with water (100 ml) The organic layer was dried (MgS04), filtered and the solvent evaporated The crude product was chromatographed on silica gel (100 g) employing 1 1 chloroform/light petroleum (100 ml fractions) for elution Fractions 7 - 9 were concentrated and combined affording oily methyl ethanethiosulfonate (5)(0.32 g, 2 3 mmol, 29%) I r 1325, 1140 cm 1 1H n m r (270 MHz) δ 1.48, t, 3H, 266, s, 3H, 334, q, 2H 13C n m r δ 8 37, 1821 , 5561 m/z 140 (75%, M+ ), 61 (47), 48 (100) Preparation of p-Nitrophenyl Methanethiosulfonate (6) p-Nitrophenyl methyl disulphide (See Baerlocher, F J , Baerlocher, M O , Langler, R F , MacQuame, S.L., and Marchand, M E., Aust. J Chem. - in press ) (1 0 g, 5 0 mmol) was dissolved in dry methylene chloride (5 ml) and a solution of sulfuryl chloride (0 67 g, 5 0 mmol) in dry methylene chloride (5 ml) added dropwise The reaction mixture was refluxed for 0 5 h A solution of sodium methanesulfinate (0 5 g, 5 0 mmol) in acetone (40 ml) and water (10 ml) was added and the reaction mixture immersed in a constant temperature bath at 50EC for 1 h
Chloroform (200 ml) was added and the resultant mixture washed with water (100 ml) The organic layer was dried (MgS04), filtered and concentrated The crude was recrystallized from methanol (8 ml) and the first crop chromatographed on silica gel (50 g) employing
61
SUBSTTTUTE SHEET (RULE 26) chloroform (50 ml fractions) for elution Fraction 4 was concentrated affording clean p- nitrophenyl methanethiosulfonate (6) (0 31 g, 1 3 mmol, 26%) The nitrothiosulfonate (6) had m p 98 - 99EC (Found C, 36 1 , H, 30 C7H7N04S2 requires C, 36 0, H, 3 0%) I r 1530, 1440, 1145 cm 1 1H n m r (270 MHz) δ 3 27 s, 3H 7 92 d, 2H 8 33, d, 2H 13C n m r δ 48 56, 124 63, 13540, 136 73, 149 56 m/z 233 (79%, M+ ), 170 (100) Preparation of p-Nitrophenyl p-Toluenethiosulfonate (8) p-Nitrophenyl methyl disulphide (7) (See Baerlocher, F J , Baerlocher, M O , Langler, R F , MacQuame, S L , and Marchand, M E , Aust J Chem - in press ) (2 5 g, 12 4 mmol) was converted into p-nitrophenyl p-toluenethiosulfonate (8) using the procedure (replace sodium methanesulfinate with sodium p-toluenesulfinate) outlined above for the preparation of (6) Crude product was not recrystallized but was chromatographed on silica gel (150 g) employing 1 1 chloroform/light petroleum (100 ml fractions) for elution Fractions 3 - 10 were combined and concentrated and the product recrystallized (methanol) Recrystallized (8) was sublimed (110EC/2 Torr/12 h) affording p-nitrophenyl p-toluenethiosulfonate (8)(1 4 g, 4 6 mmol, 37%) The thiosulfonate (8) had m p 135 - 137EC (Found C, 49 6, H, 34 daHnNO^ requires C, 50 5, H, 3 6%) I r 1525, 1345, 1145 cm 1 1H n m r (270 MHz) δ 244, s, 3H, 725, d, 2H, 7 50, d, 2H, 760, d, 2H, 8 18, d, 2H 13C n m r δ 21 73, 124 15, 127 57, 129 76, 135 77, 137 10, 140 17, 145 60, 149 38 Preparation of o-Carbomethoxyphenyl Methanethiosulfonate (18) o-Mercapto methylbenzoate (See Baerlocher, F J , Baerlocher, M O , Langler, R F ,
MacQuame, S L , and Marchand, M E , Aust J Chem - in press ) (2 0 g, 11 9 mmol) was dissolved in dry methylene chloride (5 ml) and a solution of sulfuryl chloride (1 6 g, 11 9 mmol) in dry methylene chloride (5 ml) added dropwise The reaction mixture was refluxed for 0 5 h A solution of sodium methanesulfinate (1 2 g, 11 9 mmol) in acetone (40 ml) and water (10 ml) was added to the reaction mixture which was immersed in a constant temperature bath at 50EC for 1 h
Chloroform (200 ml) was added and the resultant mixture washed with water (100 ml) The organic layer was dried (MgS04), filtered and the solvent evaporated Crude product was chromatographed on silica gel (100 g) employing 1 1 chloroform/light petroleum (100 ml fractions) for elution Fractions 8 - 18 were combined and concentrated affording thiosulfonate (18) (2 0 g, 8 1 mmol, 68%) After recrystallization from methanol, o-carbomethoxyphenyl methanethiosulfonate (18) had m p 37 9 - 384EC (Found C, 44 4, H, 4 1 C9H10O4S2 requires C, 43 9, H, 4 1%) I r 1730, 1335, 1140 cm 1 1H n m r (270 MHz) δ 3 24, s 3H 3 95 s, 3H, 7 61 , m, 2H, 7 91, m, 2H 13C n m r δ 48 73, 52 79 127 54, 130 60 131 25, 132 35 135 73, 138 34, 168 83 m/z 167 (100%, M+ - CH3S02)
Preparation of Carbomethoxymethyl p-Toluenethiosulfonate (9)
A solution of p-nitrophenyl p-toluenethiosulfonate (8) (1 0 g, 3 2 mmol) in dimethyl sulfoxide (5 ml) was added to a solution of sodium methylthioglycollate (0 4 g, 3 2 mmol) in dimethyl sulfoxide (5 ml) and the reaction mixture stirred at ambient temperature for 2 5 h 2 5% Hydrochloric acid (200 ml) was added and the resultant mixture extracted with diethyl ether (three - 100 ml aliquots) The organic layers were combined and concentrated and the extractive procedure repeated The combined organic layers were dried (MgS04), filtered and the solvent evaporated Crude product was chromatographed on silica gel (100 g) employing chloroform (100 ml fractions) for elution Fractions 6 and 7 were combined and concentrated, yielding oily thiosulfonate (9) I r 1745, 1320, 1150 cm 1 1H n m r (270 MHz) δ 2 47, s, 3H, 3 72, s, 3H, 4 11 , s, 2H, 7 38, d, 2H, 7 82, d, 2H 13C n m r δ 21 73, 53 08, 60 93, 128 54, 129 90, 135 71 , 145 52, 162 99 m/z 228 (3%, M+ - CHsO), 155 (51 ), 91 (100) Administration - Pharmaceutical Compositions For use as a medicine, the compound of the present invention may be administered to an animal including human being either as it is or in the form of a pharmaceutical composition containing, for example, 0 01-99 5%, preferably 0 5-90%, of the compound in a pharmaceutically acceptable nontoxic, inert carrier
As the carrier, one or more of solid, semisolid, or liquid diluent, filler, and other formulation auxiliaries may be employed The pharmaceutical composition is preferably administered in unit dosage forms The pharmaceutical composition of the present invention may be administered orally, parenterally (e g intravenously), locally (e g transdermally), or rectally Of course, dosage forms suited for respective routes of administration should be selected Oral administration may be carried out using solid or liquid unit dosage forms such as bulk powders, powders, tablets, dragees, capsules, granules, suspensions, solutions, syrups, drops, subhngual tablets, etc
Bulk powders may be manufactured by comminuting the active substance into a finely divided form Powders may be manufactured by comminuting the active substance into a finely-divided form and blending it with a similarly comminuted pharmaceutical carrier, e g an edible carbohydrate such as starch or mannitol Where necessary, a corπgent, a preservative, a dispersant, a coloring agent, a perfume, etc may also be added
Capsules may be manufactured by filling said finely-divided bulk powders or powders, or granules described below for tablets, in capsule shells such as gelatin capsule shells Preceding the filling operation, a lubricant or a fluidizing agent, such as colloidal silica, talc, magnesium stearate, calcium stearate or solid polyethylene giycol, may be blended with the powders Improvement in the efficacy of the drug after ingestion may be expected when a disintegrator or a solubilizer, such as carboxymethylcellulose, carboxymethylcellulose calcium, low-substitution-degree hydroxypropylcellulose, roscarmellose sodium, carboxymethylstarch sodium, calcium carbonate or sodium carbonate, is added
Soft capsules may be provided by suspending said finely divided powders in vegetable oil, polyethylene giycol, glycerin, or a surfactant and wrapping the suspension in gelatin sheets Tablets may be manufactured by adding an excipient to said powders, granulating or slugging the mixture, adding a disintegrator and/or a lubricant, and compressing the
63
SUBSTTTUTE SHEET (RULE 26) whole composition A powdery mixture may be prepared by mixing said finely divided powders with said diluent or a base Where necessary, a binder (e g carboxymethylcellulose sodium, methylcellulose, hydroxypropylmethylcellulose, gelatin, poiyv ylpyrrolidone, poiyvmyl alcohol, etc ), a dissolution retardant (e g paraffin), a reabsorption agent (e g quaternary salts), and an adsorbent (e g bentonite, kaolin, dicalcium phosphate, etc ) may be added The powdery mixture may be processed into granules by wetting it with a binder, e g a syrup, a starch paste, gum arable, a solution of cellulose, or a solution of a high polymer, stirring to mix, drying it, and pulverizing the same Instead of granulating such powders, it is possible to compress the powders with a tablet machine and crush the resulting slugs of crude form to prepare granules The resulting granules may be protected against mteradhesion by the addition of a lubricant such as steaπc acid, a salt of stearic acid, talc or mineral oil The mixture thus lubricated is then compressed The resulting uncoated tablets may be coated with a film coating composition or a sugar coating composition The compound of the invention may be mixed with a free-flowing inert carrier and the mixture be directly compressed without resort to the above-mentioned granulation or slugging process A transparent or translucent protective coat consisted of, for example, a hermetic shellac coat, a sugar or polymer coat, or a polishing wax coat may also be applied Other oral compositions such as a solution, a syrup, and an elixir may also be provided in unit dosage forms each containing a predetermined amount of the drug substance Syrups may be manufactured by dissolving the compound in suitable flavored aqueous media, while elixirs may be manufactured using nontoxic alcoholic vehicles Suspensions may be formulated by dispersing the compound in nontoxic vehicles Where necessary, solubilizers and emulsifiers (e g ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, etc ), preservatives, and flavorants (e g peppermint oil, saccharin, etc ) may also be added
Where necessary, the unit dosage formulation for oral administration may be microencapsulated This formulation may be coated or embedded in a polymer, wax or other matrix to provide a prolonged action or sustained release dosage form Parenteral administration may be carried out using liquid unit dosage forms for subcutaneous, intramuscular, or intravenous injection, e g solutions and suspensions Such unit dosage forms may be manufactured by suspending or dissolving a predetermined amount of the compound of the invention in an injectable nontoxic liquid vehicle, for example an aqueous vehicle or an oily vehicle, and sterilizing the resulting suspension or solution For isotonizmg an injection, a nontoxic salt or salt solution may be added Moreover, stabilizers, preservatives, emulsifiers, etc may also be added
Rectal administration may be carried out by using suppositories manufactured by dissolving or suspending the compound in a low-melting water-soluble or watennsoluble
64
SUBSTTTUTE SHEET (RULE 26) solid carrier such as polyethylene giycol, caccao butter, semisynthetic oil (e g Witepsol®), a higher ester (e g myπstyl palmitate) or a mixture thereof
The invention may be varied in any number of ways as would be apparent to a person skilled in the art and all obvious equivalents and the like are meant to fall within the scope of this description and claims The description is meant to serve as a guide to interpret the claims and not to limit them unnecessarily

Claims

Claims:
1 Sulfone disulphides of the general formula RS02CH2SSR1, wherein R is phenyl or lower alkyl, and R1 is lower alkyl or phenyl
2 Sulfone disulphides as claimed in claim 1 selected from the group consisting of CeH5S02CH2SSCH3,
• CH3S02CH2SSCH3,and CH3Sθ2CH2SSCβH5
3 A process for preparing a sulfone disulphide of the general formula as claimed in Claim 1 , which comprises reacting a transition metal oxidant or a peroxyanhydnde oxidant with a symmetrical dialkyl or arylalkyl disulphide to obtain an α-ester disulphide compound of the formula RSSCH2OC(0)R1, wherein R and R1 are as defined in Claim 1 , which compound is further reacted with a sulfinic acid salt to obtain the required compound
4 Compounds of the general formula RSSCH2OC(0)R1, wherein R and R1 may be the same or different and each is selected from the group of substituents comprising lower alkyl or phenyl 5. Compounds of the general formula as set out in claim 4 wherein lower alkyl is methyl or ethyl. 6 2,3-Dιthιabutyl benzoate 7 1-Phenyl-1 ,2-dιthιapropyl propionate
8 Methyl 3,4-dιthιa-5-propιonoxypentanoate
9 2,3-Dιthιabutane-1 ,4-dιpropιonate
10. 1-p-Toluenesulfonyl-4-propιonoxy-2,3-dιthιabutane
11. Phenacyl methyl disulphide 12. A process for preparing a compound of the general formula as claimed in Claim 4, which comprises reacting a transition metal oxidant or a peroxyanhydnde oxidant with a symmetrical dialkyl or arylalkyl disulfide to obtain the α-ester disulfide compound
13 A process for preparing compounds of the formula RS02CH2SSR1 ,whereιn R and R1 may be the same or different and each is selected from lower alkyl and phenyl, which comprises reacting a transition metal oxidant or a peroxyanhydnde oxidant with a symmetrical dialkyl or arylalkyl disulfide to obtain the α-ester disulfide compound RSSCH2OC(0)R1.wherein R and R1 are as defined above, which compound is further reacted with a sulfinic acid salt to obtain the required compound
14 A process for preparing a compound of the formula PhS02CH2SSCH2CH3,whereιn Ph is phenyl which comprises reacting a transition metal oxidant with dimethyl disulfide to obtain an α-ester disulfide compound of the formula H3SSCH2OC(0)CH2CH3, which compound is further reacted with the sodium salt of p-toiuenesulfmic acid in either aqueous acetonitrile or aqueous acetone to obtain the title compound or the compound is further reacted with potassium p-toluenethiosulfonate to yield the title compound 15 A process for the preparation of α-ester disulphides and α-sulfonyl disulphides of the formula RSSCH2S02R .wherein R and R1 may be the same or different and each is selected from the group comprising lower alkyl, phenyl, phenyl substituted with ????, which comprises reacting a compound of the formula RSH, wherein R is as defined above with a compound of the formula CISCH2OC(0)CH2CH3, in the presence of a base to yield a compound of the formula RSSCH2OC(0)CH2CH3,whereιn R is as defined above, reacting this compound with a compound of the formula R1S02Na, wherein R1 is as defined above, to obtain the desired compound
16 A compound of the formula CISCH2OC(0)CH2CH3 17 Antifungal agents compπsing as active ingredient at least one antifungally active compound of the formulae RS02CH2SSR1, wherein R is lower alkyl or phenyl and R1 is lower alkyl or phenyl, and RC(0)OCH2SSR1, wherein R and R1 are as defined above together with a pharmaceutically acceptable carrier 18 Antifungal agents comprising as active ingredients a therapeutically effective amount of at least one compound of the formula
RSSCHX
R1 wherein R1 is H or CH3, R is CH3, CH2CH3, C6H5, o-CH302C(C6H4), o-CH3S02(C6H4), p-CH3S02(C6H4), o
N02(C6H4), m-N02(C6H4), p-N02(C6H4), or CH202CCH2CH3, and
X is S02(C6H4)CH3-p, S02CH3, S02C6H5, S02CH2CH3, H, 02CCH3, 02CCH2CH3, or C02CH3, and a pharmaceutically acceptable carrier 19 Antifungal agents as claimed in Claim 17 wherein the active ingredient is selected from the group of compounds consisting of o-CH3OC(0) (C6H4) SSCH3,
[o-CH3S02 (C6H4)S]2,
[p-CH3S02 (C6H4)S]2, m-02N (C6H4) SSCH3, o-02N (C6H4) SSCH3, p- 02N (C6H4) SSCH3, p- CH3 (C6H4) S02CH2 SSCH3,
C6HsS02CH2SSCH3, CH3S02CH2SSCH3,
CH3CH2C(0)0CH2SSCH3,
CH3S02CH2SSPC6Hs,
CH3Sθ2CH2SSCH2CH3,
CH3SSCH20C(0)CH3, CH3SSCH2OC(0)CH2CH3, CH3SSCH2OC(0)PC6H5, and PhSSCH2OC(0)CH2CH3, together with a pharmaceutically acceptable carrier. 20. An antiproliferative agent active against mammalian cells comprising as active
• ingredient a therapeutically effective amount of at least one antiproliferative active compound of the formulae RS02CH2SSR1 (I) wherein R is lower alkyl or phenyl and R1 is lower alkyl or phenyl, and RC(0)OCH2SSR1 (II) wherein R and R1 are as defined above, together with a pharmaceutically acceptable carrier.
21. An antiproliferative agent active against mammalian cells comprising a therapeutically effective amount of at least one compound selected from the group of compounds defined by the formula RSSCH2X, wherein X is S02(C6H4)CH3-p, S02CH3, C02CH3,
02CCH3, SCH3, 02CCH2CH3, (0)CC6H5 or CH2S02CH3; and R is CH3 or C6H5 together with a pharmaceutically acceptable carrier.
22. An antiproliferative agent active against mammalian cells comprising as active ingredient a therapeutically effective amount of at least one antiproliferative compound selected from the group of compounds consisting of
CH3(C6H4)S02CH2SSCH3
(C6H5)SS(C6H5)
CH3S02CH2SS(C6H5)
CH3SSCH2OC(0)CH3 CH3S02CH2SSCH2CH2CH2CH2CH3
CH3S02S(C6H5) o-, m- and p-N02(C6H4)SSCH3
CH3SCH2SSCH3
CH3OC(0)CH2SSCH2C(0)OCH3 (C6H5)C(0)CH2SSCH3
(C6H5)SSCH20C(0)CH2CH3
CH3S02CH2CH2SSCH3, and
CH3SSCH2C(0)0CH3, together with a pharmaceutically acceptable carrier.
23. An anti-tumour agent active against leukemic cells comprising as active ingredient a therapeutically effective amount of a compound of the formula CH3S02CH2CH2SSCH3 and a pharmaceutically acceptable carrier.
24. An antiproliferative agent active against mammalian cells comprising as active ingredient a therapeutically effective amount of at least one therapeutically effective amount of CH3SCH2SSCH3 together with a pharmaceutically acceptable carrier. An antiproliferative agent active against mammalian cells comprising as active ingredient a therapeutically effective amount of at least one therapeutically effective amount of (C6H5)C(0)CH2SSCH3 together with a pharmaceutically acceptable carrier An antiproliferative agent active against mammalian cells comprising as active ingredient a therapeutically effective amount of at least one therapeutically effective amount of (C6H5)SSCH2OC(0)CH2CH3 together with a pharmaceutically acceptable carrier An antifungal agent comprising as active ingredient a therapeutically effective amount of a compound of the formula (C6H5)SSCH2OC(0)CH2CH3 together with a pharmaceutical carrier The use of at least one of the compounds as claimed in any of claims 1 to 8 and 27 in the preparation of a medicament for antifungal treatment The use of at least one of the compounds as set out in any of claims 21 to 26 in the preparation of a medicament for the treatment of cancer
69
SUBSTTTUTE SHEET (RULE 26)
EP01909377A 2000-02-25 2001-02-26 Sulfur containing compounds Withdrawn EP1259482A2 (en)

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CA2571800A1 (en) * 2005-12-23 2007-06-23 Canadian Blood Services Nitrophenyls and related compounds and thimerosal for the inhibition of immune related cell or tissue destruction
WO2013124441A1 (en) * 2012-02-23 2013-08-29 Pancosma S.A. Use of dialkyl-thiosulfinate and/or thiosulfonate to improve the resistance of an animal infected by a pathogen
US20150238579A1 (en) * 2014-02-26 2015-08-27 Investfood, LLC Methods for assembling a product for modulating an immunomodulatory response in humans
WO2023059201A1 (en) * 2021-10-08 2023-04-13 Crop Health Vision B.V. Organosulfur compounds as plant biostimulants

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US4643994A (en) * 1984-12-17 1987-02-17 The Research Foundation Of State University Of New York Novel organic trithio oxides and method for the preparation thereof
US5583235A (en) * 1994-03-11 1996-12-10 Shaman Pharmaceuticals, Inc. Preparation of symmetrical and unsymmetrical 3,6-disubstituted-1,2-dithiins having antifungal activity

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