WO2000037465A2 - Derives de swainsonine, procedes de preparation et utilisations comme agents therapeutiques - Google Patents

Derives de swainsonine, procedes de preparation et utilisations comme agents therapeutiques Download PDF

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WO2000037465A2
WO2000037465A2 PCT/CA1999/001210 CA9901210W WO0037465A2 WO 2000037465 A2 WO2000037465 A2 WO 2000037465A2 CA 9901210 W CA9901210 W CA 9901210W WO 0037465 A2 WO0037465 A2 WO 0037465A2
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compound
formula
compounds
aryl
alkyl
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PCT/CA1999/001210
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WO2000037465A3 (fr
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Francois D. Tropper
Cheng Guo
Rajan N. Shah
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Glycodesign Inc.
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Priority to AU17643/00A priority patent/AU1764300A/en
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Publication of WO2000037465A3 publication Critical patent/WO2000037465A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/04—Ortho-condensed systems
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • 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
    • A61P37/00—Drugs for immunological or allergic disorders

Definitions

  • the present invention relates to novel 8a substituted derivatives of swainsonine, processes for their preparation, and their use as therapeutic agents. BACKGROUND OF THE INVENTION
  • Carbohydrate structures present on human tumor cells have been associated with cancer invasion and metastasis (Dennis et. al, Science 236: 582, 1987; Demetriou et. al., J. Cell Biol. 750:383, 1995). These structures include the GlcNAc ⁇ (Tl-6) branched N- and O-linked carbohydrate side chains of cell-surface glycoproteins.
  • the Golgi enzymes required for their synthesis are ⁇ (Tl-6) N-acetylglucosaminyltransferase V (i.e. GlcNAc-TV) and core 2 ⁇ (Tl-6) -V-acetylglucosaminyltransferase (i.e.
  • GlcNAc-T GlcNAc-T
  • These enzymes are up-regulated in human carcinomas (Fernandes et al., Cancer Res. 51 :718-723, 1991), a phenomenon that has been associated with the activation of the ras signaling pathway (Dennis et al. Science 236:582-585, 1987; Dennis et al Oncogene 4:853-860, 1989)).
  • overexpression of GlcNAc-TV in epithelial cells results in morphological transformation and tumor formation in mice (Demetriou et al, J. Cell Biol. 130:383-392, 1995).
  • GlcNAc-TV as well as enzymes supplying acceptor substrates to GlcNAc-TV (i.e. GlcNAc-TI, ⁇ -mannosidase II and core 2 GlcNAc-T of the C inked pathway) are targets for anti-cancer pharmaceuticals.
  • Swainsonine is an indolizidine alkaloid found in Australian Swainsona canescens (Colegate et al, Aust J Chem 32:2257-2264, 1979), North American plants of the genera Astragalus and Oxytropis (Molyneux RJ and James LF, Science 215: 190-191, 1981), and also the fungus Rhizoctonia leguminicola (Schneider et al. Tetrahedron 39;29-31, 1983). Swainsonine's ability to inhibit ⁇ -mannosidase II activity appears to be responsible for its interesting immunomodulating and cancer suppression activity.
  • Swainsonine is believed to function as an enzyme inhibitor because it can mimic the glycosylium cation intermediate generated during the hydrolytic cleavage of mannopyranosides. (Goss, P.E. et al, Clin. Cancer Res. 1: 935-944, 1995).
  • Swainsonine has positive effects on cellular immunity in mice (reviewed in Humphries M.J. and Olden , Pharmacol Ther. 44:85-105, 1989, and Olden et al, Pharmacol Ther 50:285- 290, 1991)).
  • swainsonine has been shown to alleviate both chemically-induced and tumor-associated immune suppression (Hino et al, J. Antibiot. (Tokyo) 38:926-935, 1985), increase NK cell (Humphries et al. Cancer Res. 48:1410-1415, 1988), and LAK cell activities (Yagita M and Saksela E, Scand. J. Immunol.
  • SW has also been shown to be hemorestorative in mice following treatment with both cycle-specific and nonspecific chemotherapeutic agents (Oredipe et al, J. Natl. Cancer Inst. 83: 1 149-1 156, 1991).
  • the present invention relates to a compound of the formula I
  • R, R 2 and R 3 are each independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, or Ri and R 2 together form a carbocyclic or heterocylic ring;
  • W and W" and W are each independently hydrogen, hydroxyl, alkoxy, thiol, thioalkyl, thioaryl, halo or amino, or W and W' together form a carbocyclic or heterocyclic ring;
  • X represents alkyl, alkenyl, alkynyl, cycloalkyl, alkoxy, or aryl, and salts and optically active and racemic forms of a compound of the formula I.
  • the present invention also provides a process for the preparation of a compound of the formula I as defined herein.
  • the compounds of the invention have valuable pharmacological properties and they provide antimicrobial, cancer suppressing effects, hemorestorative, chemoprotective, radioprotective, and immunomodulatory properties. Therefore, the invention contemplates a pharmaceutical composition comprising a compound of the formula I as an active agent.
  • the invention further relates to a method for stimulating the immune system, stimulating hematopoietic progenitor cell growth, treating proliferative disorders or microbial or parasitic infections, or conferring protection against chemotherapy and radiation therapy in a patient comprising administering an effective amount of a compound of the formula I of the invention.
  • the invention also relates to the use of a compound of the formula I in the preparation of a medicament for stimulating the immune system, stimulating hematopoietic progenitor cell growth, or conferring protection against chemotherapy and radiation therapy, and/or for treating proliferative disorders, and microbial or parasitic infections.
  • the present invention also relates to the use of a compound of the formula I which is esterified at free hydroxyls as a prodrug.
  • Figure 1 is a schematic diagram of a reaction for preparing a compound of the invention.
  • DETAILED DESCRIPTION OF THE INVENTION I. COMPOUNDS OF THE INVENTION
  • alkyl refers to a branched or linear hydrocarbon radical, typically containing from 1 through 20 carbon atoms, preferably 1 through 10 carbon atoms, more preferably 1 to 6 carbon atoms.
  • alkyl groups include but are not limited to methyl, ethyl, 1-propyl, 2-propyl, 1 -butyl, 2-butyl, tert- butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, and the like.
  • alkenyl alone or in combination, refers to an unsaturated branched or linear group typically having from 2 to 20 carbon atoms and at least one double bond.
  • Examples of such groups include but are not limited to ethenyl, 1-propenyl, 2-propenyl, 1-butenyl, 1,3-butadienyl, 1- hexenyl, 2-hexenyl, 1-pentenyl, 2-pentenyl, and the like.
  • alkynyl refers to an unsaturated branched or linear group having 2 to 20 carbon atoms and at least one triple bond. Examples of such groups include but are not limited to ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, and the like.
  • cycloalkyl refers to cyclic hydrocarbon groups and includes but is not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.
  • aryl refers to a monocyclic or polycyclic group, preferably a monocyclic or bicyclic group.
  • An aryl group may optionally be substituted as described herein. Examples of aryl groups and substituted aryl groups are phenyl, benzyl, p- nitrobenzyl, p-methoxybenzyl, biphenyl, and naphthyl.
  • alkoxy alone or in combination, refers to an alkyl or cycloalkyl linked to the parent molecular moiety through an oxygen atom.
  • alkoxy groups are ethoxy, ethoxy, propoxy, vinyloxy, allyloxy, butoxy, pentoxy, hexoxy, cyclopentoxy, cyclohexoxy, and the like.
  • halo or halogen, alone or in combination, means fluoro, chloro, bromo, or iodo.
  • amino refers to a chemical functional group where a nitrogen atom (N) is bonded to three substituents being any combination of hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl, or aryl with the general chemical formula -NR 4 R 5 where R, and R 5 can be any combination of hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl, or aryl.
  • R, and R 5 can be any combination of hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl, or aryl.
  • one substituent on the nitrogen atom can be a hydroxyl group (-OH) to give an amine known as a hydroxylamine.
  • amino groups are amino (-NH2), methylamine, ethylamine, dimethylamine, 2-propylamine, butylamine, isobutylamine, cyclopropylamine, benzylamine, allylamine, hydroxylamine, cyclohexylamino (-NHCH(CH2)s), piperidine (-N(CH2)5) and benzyla ino (-NHCH2C6H5).
  • thioalkyl refers to a chemical functional group where a sulfur atom (S) is bonded to an alkyl.
  • examples of thioalkyl groups are thiomethyl, thioethyl, and thiopropyl.
  • thioaryl alone or in combination, refers to a chemical functional group where a sulfur atom (S) is bonded to an aryl group with the general chemical formula -SR ⁇ where Re is an aryl group which may be substituted.
  • thioaryl groups and substituted thioaryl groups are thiophenyl, para-chlorothiophenyl, thiobenzyl, 4-methoxy-thiophenyl, 4-nitro- thiophenyl, and para-nitrothiobenzyl.
  • the term "carbocyclic” refers to molecular rings where the framework is constructed by joining carbon atoms solely and includes but is not limited to any stable 3- to 7- membered monocyclic or bicyclic or 7- to 14-membered bicyclic or tricyclic or up to 26-membered polycyclic carbon ring, any of which may be saturated, partially unsaturated, or aromatic.
  • Examples of carbocyclic rings include substituted or unsubstituted cycloalkyl, monocyclic unsaturated hydrocarbons, and aryl as described herein, including but not limited to benzene and napthalene.
  • Heterocyclic rings are molecular rings where one or more carbon atoms have been replaced by hetero atoms (atoms not being carbon) such as for example, oxygen (O), nitrogen (N) or sulfur (S), or combinations thereof.
  • heterocyclic rings include ethylene oxide, tetrahydrofuran, thiophene, piperidine (piperidinyl group), pyridine (pyridinyl group), and caprolactam.
  • a carbocyclic or heterocyclic group may be optionally substituted at carbon or nitrogen atoms with for example, alkyl, phenyl, benzyl or thienyl, or a carbon atom in the heterocyclic group together with an oxygen atom may form a carbonyl group, or a heterocyclic group may be fused with a phenyl group.
  • R may be substituted with for example one or more of the following: alkyl, alkoxy, hydroxyl, aryl, cycloalkyl, alkenyl, alkynyl, thiol, thioalkyl, thioaryl, amino, or halo.
  • alkyl alkoxy, hydroxyl, aryl, cycloalkyl, alkenyl, alkynyl, thiol, thioalkyl, thioaryl, amino, or halo.
  • the term "one or more” used herein preferably refers to from 1 to 3 substituents, most preferably 1 to 2 substituents.
  • , R 2 , and R 3 are the same and represent hydrogen; W, W', and W" are the same and represent hydroxyl; Rj, R 2 , and R 3 are the same or different and represent alkyl, alkenyl, alkynyl, or aryl, preferably alkyl or aryl, most preferably alkyl; R] and R 2 together form a carbocyclic or heterocyclic ring; W, W', and W" represent hydroxyl, alkoxy, thiol, thioalkyl, thioaryl, halo, or amino; W and W' together form a carbocyclic or heterocyclic ring; or X represents alkyl, alkenyl, alkynyl, aryl, cycloalkyl or alkoxy, preferably alkyl or alkoxy, more preferably alkyl.
  • Preferred compounds of the formula I of the invention are those where Ri, R 2 , and R 3 represent hydrogen, and W, W ⁇ and W" represent hydroxyl, and X represents methyl, ethyl, phenyl, benzyl, or methoxy.
  • Particularly preferred compounds of the invention are (IS, 2R, &R, 8--R)-8a-methyl- 1,2,8- trihydroxyindolizidine, (IS, 2R,8R, 8aR)-8a-ethyl-l,2,8-trihydroxy-indolizidine, (lS, 2R, 8R, 8aR)- 8a-propyl-l, 2, 8-trihydroxyindolizidine, and (IS, 2R, $R, 8a/?)-8a-butyl- 1 , 2, 8- trihydroxyindolizidine.
  • a compound of formula I may exist in, and be isolated in, optically active and racemic forms. It is to be understood that the present invention encompasses a compound of formula I as a mixture of diastereomers, as well as in the form of an individual diastereomer, and that the present invention encompasses a compound of formula I as a mixture of enantiomers, as well as in the form of an individual enantiomer.
  • the present invention contemplates all optical isomers and racemic forms thereof of the compounds of the invention, and the formulas of the compounds shown herein are intended to encompass all possible optical isomers of the compounds so depicted.
  • the present invention also contemplates salts and esters of the compounds of the formula
  • the present invention includes pharmaceutically acceptable salts.
  • salts are meant those salts which are suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit risk ratio.
  • Pharmaceutically acceptable salts are well known in the art and are described for example, in S. M. Berge, et al, J.
  • salts with inorganic acids such as, for example, hydrochloric, hydrobromic, sulfuric, phosphoric, and like acids; with organic carboxylic acids, such as for example acetic, propionic, glycolic, lactic, pyruvic, malonic, succinic, fumaric, malic, tartaric, citric, ascorbic, maleic, hydroxymaleic, and dihydroxymaleic, benzoic, phenylacetic, 4-aminobenzoic, 4-hydroxybenzoic, anthranilic, cinnamic, salicylic, 4- aminosalicylic, 2-phenoxybenzoic, 2-acetoxybenzoic, mandelic, and like acids; and with organic sulfonic acids such as methanesulfonic acid, and p-toluenesulfonic acid.
  • inorganic acids such as, for example, hydrochloric, hydrobromic, sulfuric, phosphoric, and like acids
  • organic carboxylic acids such as for example acetic,
  • the compounds of the formula I of the present invention can be prepared by utilizing procedures and techniques well known and appreciated by one of ordinary skill in the art. By way of illustration, descriptions of some methods that may be used to prepare compounds of the formula I of the invention are set forth herein.
  • compounds of the Formula I may be synthesized in a variety of ways by adapting common synthetic organic chemistry practices to known synthetic intermediates.
  • compounds of the formula I where X is alkyl may be synthesized by protecting swainsonine acetonide (1) at the 8 position with a protecting group; converting the protected swainsonine acetonide to an N-oxide; reacting the N-oxide with trifluoroacetic anhydride; reacting the acylated N-oxide with a nucleophile under basic conditions for example, an alkyl magnesium bromide; and removing the protecting groups.
  • (IS, 2R, SR, 8aR)-8a-methyl-l, 2, 8- trihydroxyindolizidine is prepared by converting swainsonne acetonide to (IS, 2R, SR, 8aR)-8- methoxymethoyl-l,2-(isopropylidenedioxy)indolizidine by adding tetrabutyl-ammonium iodide, methoxy methyl chloride, and sodium hydride in THF; reacting (IS, 2R, 8R, 8aR)-8- methoxymethoxy-1, 2-(isopropylidenedioxy)indolizidine with hydrogen peroxide in dichloromethane-ethanol to yield (IS, 2R, SR, 8aR)-8-methoxymethoxy-l,2-
  • Reactive groups used in processes for preparing the compounds of the invention may be blocked using appropriate protective groups.
  • Appropriate blocking and deblocking schemes are known to the skilled artisan (See T.W. Greene, Protective Groups in Organic Synthesis, 2 nd Ed, T.W. Greene, and P.G.M. Wats, John Wiley & Sons, New York, 1991).
  • protective groups are selected which adequately protect the reactive groups in question during subsequent synthetic steps and which are readily removable under conditions which will not cause degradation of the desired product.
  • ethers, silyl ethers, orthoesters, acetals, ketals, and esters can be used to protect isolated hydroxyl groups.
  • suitable protective groups which may be used in the process of the invention include O-benzyl, O-para- methoxybenzyl, O-acetoxy, O-benzoyloxy, O-pivaloyl, O-allyl, methoxymethyl, isopropylidene, benzylidene, methylidene, acetylidene, 1-methoxy ethylidene, 1,3-(1,1,3,3)- tetraisopropyldisiloxanylidene, O-trimethyl silyl, O-t-butyl dimethylsilyl. Removal of the protective groups may be carried out using procedures known in the art.
  • a free hydroxyl group may be converted to an alkoxy group in blocked/deblocked compounds to produce compounds where for example one or more of W, W', and W" are alkoxy, by reacting with an alkyl halide in the presence of a base.
  • An alkoxy group may be added by dissolving a compound with a free hydroxyl in DMF and adding it to a flask under an inert atmosphere containing a base (e.g. sodium hydride) at low temperature (0°C to 10°C). After stirring for a few minutes, benzyl bromide in DMF is added dropwise at low temperature, for example 0°C to 10°C. The reaction mixture is further stirred at room temperature for 2 to 24 hours.
  • a base e.g. sodium hydride
  • a halo group for example, fluoro
  • a halo group for example, fluoro
  • DCM dichloromethane
  • a base like pyridine
  • an appropriate amount of triflic anhydride, or mesyl chloride, or tosyl chloride is added dropwise.
  • the reaction is allowed to stir at a temperature between 0°C to 25°C. Conventional work-up of the reaction mixture yields the esterif ⁇ ed compound.
  • Treatment of this derivative with sodium benzoate in DMF is carried out immediately, which replaces the leaving group with O-benzoate with inversion of configuration at the carbon center.
  • the free hydroxyl is generated by treatment with a base (e.g. sodium methoxide) and then reblocked by a suitable leaving group such as triflate.
  • a base e.g. sodium methoxide
  • a suitable leaving group such as triflate.
  • the triflate is treated with anhydrous tetra alkyl ammonium fluorides (preferably tetra n-butyl) or potassium fluoride in a suitable solvent (e.g diethyl ether, tetrahydrofuran or crown ether).
  • a suitable solvent e.g diethyl ether, tetrahydrofuran or crown ether
  • DAST diethyl amino) sulfur trifluoride
  • the triflate may be treated either with sodium azide, phthalimide, or benzyl amine in DMF.
  • the product may be obtained with an azido or benzyl amine group, with inversion, which on reduction with palladium on carbon in a hydrogen atmosphere (azide and benzylamine), or treatment with hydrazine or methyl amine (phthalimido) gives the free amino group.
  • a thiol group may be added by nucleophilic substitution of an alkyl halide or sulfonyl ester for example using sodium sulfhydride (NaSH) or, by nucleophilic substitution of a halide or sulfonate ester using thioacetic acid to give a thioacetate group which can then be deblocked to a free thiol upon treatment with sodium methoxide in methanol by converting the same to a Bunte salt using thiosulfate (S 2 0 3 2 ) and later hydrolyzing the Bunte salt with an acid or, by treating the hydroxyl group with a fluoropyridinium salt and N,N-dimethyl thiocarbamate (Hojo: Yoshino: Mukaiyama Chem.
  • Methods for introducing a thioalkyl or a thioaryl group in a compound of the formula I are also well known to the skilled artisan.
  • a base such as l,8-diazabicyclo[5.4.0] undecene (DBU)
  • alkylating thiols with alkyl or aryl halides or sulfonate esters or, by treating a hydroxyl group with an alkyl or aryl halide in the presence of tetramethyl thiourea followed by sodium hydride Fujisaka; Fujiwara; Norisue; Kajigaeshi Bull.
  • alkyl halides or sulfonyl esters such as tosylates can be selectively reduced with lithium aluminum hydride or a variety of other metal hydride reducing agents in different solvents such as ether.
  • a large list of methods able to achieve this transformation is provided in J. March "Advanced Organic Chemistry. Reactions, Mechanisms and Structure" 4th Edition, 1992, pp 438- 446 and references cited therein.
  • alkyl or aryl groups particularly those which may contain unsaturations or other chemical functional groups such as hydroxyl, or alkoxy for example, can be further derivatized by chemical processes such as oxidation, hydroxylation, hydrolysis, alkylation, reduction, carbon- carbon chain elongation by Grignard or Wittig reactions for example to introduce new or additional functional groups in any final compound.
  • chemical processes such as oxidation, hydroxylation, hydrolysis, alkylation, reduction, carbon- carbon chain elongation by Grignard or Wittig reactions for example to introduce new or additional functional groups in any final compound.
  • Such transformations can be achieved by anyone skilled in the art of synthetic organic chemistry.
  • the products of the processes described above may be purified by conventional methods such as column chromatography.
  • a compound of the formula I with free hydroxyl groups may also be converted into esters using conventional procedures.
  • a compound of the formula I may be dissolved in DCM and pyridine. After cooling (0°C to 5°C) benzoic anhydride or benzoyl chloride in DCM and pyridine is added dropwise. The reaction is allowed to stir at room temperature for 2 to 24 hours. Conventional work-up yields the esterified derivatives.
  • Optical antipodes of the compounds of the formula I may be prepared from the corresponding racemic forms by standard resolution techniques, involving, for example, the separation of diastereomeric salts of those compounds of the formula I characterized by the presence of a basic amino group, and an optically active acid, or by synthesis from optically active precursors.
  • the compounds of the formula I are inhibitors of oligosaccharide processing and in particular are inhibitors of mannosidase.
  • General mannosidase inhibition may be tested by measuring the inhibition of Jack Bean, ⁇ -mannosidase, or lysosomal ⁇ -mannosidase.
  • Mannosidase inhibition may also be tested using an L-PHA toxicity assay.
  • the assay is based on the finding that the specific binding of the toxic plant lectin L-PHA to transformed cell lines such as MDAY-D2 tumor cells is a specific measure of inhibition of oligosaccharide processing.
  • the measurement of IC 50 in the L-PHA toxicity assay reflects the ability of the compound to enter into cells and to effect inhibition of oligosaccharide processing.
  • a compound of the invention may be tested for its ability to inhibit N-linked oligosaccharide processing by growing transformed cells in the presence of L-PHA and the compound; measuring the amount of proliferation of the cells; and determining the ability of the compound to inhibit N-linked oligosaccharide processing by comparing the amount of proliferation of the cells with the amount of proliferation observed for the cells grown in the presence of L-PHA alone.
  • Transformed cells which may be used in this assay include MDAY-D2, L1210, CHO, B16, melanoma tumor cells, and human tumor cells such as SW 480, LS174T, HT-29, WiDr, T2, MDA-231 , MCF7, BT-20, Hs578T, K562, Hs578T, SK-BR-3, CY 6T, MDA-468, H23, HI 57, H358, HI 334, HI 155, H28, H460, Hmesol, HI 87, H510A, N417, HI 46, HI 092, H82 (Restifo, N. P. et al, J. Exper. Med. 177:265-272, 1993).
  • the amount of proliferation of the cells may be measured using conventional techniques.
  • cell proliferation may be measured by measuring incorporation of labeled thymidine. More particularly, radioactively labeled thymidine may be added for about 2-5 hours, preferably 3- 4 hours and the cells can be harvested and radioactivity counted using a scintillation counter.
  • the conditions for carrying out the above assay will be selected having regard to the nature of the compound and the cells employed. For example, if the transformed cells are MDAY- D2 tumor cells a concentration of about 1-4 x 10 4 cells, preferably 2 x 10 4 may be used.
  • the MDAY-D2 cells are generally cultured for about 10 to 30 hours, preferably 18 to 20 hours, followed by addition of L-PHA at a concentration of about 10-50 ⁇ g/ml, preferably 20-30 ⁇ /ml, more preferably 25 ⁇ g/ml.
  • L-PHA assay may be used to assay for inhibition of oligosaccharide processing (i.e. Golgi ⁇ -mannosidase II) in viable cells.
  • MDAY-D2 tumor cells are inoculated into 96 well micro-test plates at 2 x 10 4 cells/well, containing serial dilutions of the compound to be tested in MEM plus 10% FCS. The cells are cultured for 18-20 hours, followed by the addition of L-PHA at 25 ⁇ g/ml for an additional 24 hours.
  • Cell proliferation is measured by adding 0.5 ⁇ Ci/well of 3 H-thymidine for 3-4 hours, harvesting onto glass fibre disks using a Titertek harvester, and counting the disks in a liquid scintillation counter.
  • the apparent IC 50 values for the test compounds are the drug concentrations showing 50% protection from L-PHA toxicity; that is 50% 3 H-thymidine incorporated compared with cells grown in the absence of L-PHA.
  • the ability of the compounds of the formulae I in which the free hydroxyls have been esterified, to be converted into more active compounds in cells can be measured by performing the L-PHA toxicity assay in the presence of an esterase inhibitor such as diethyl p-nitrophenyl phosphate.
  • the esterase inhibitor diethyl p-nitrophenyl phosphate can be added to MDAY-D2 cells in the above described assay method about 4 hours prior to the ⁇ -mannosidase inhibitors.
  • An increase in IC 50 in the L-PHA toxicity assay in the presence of diethyl p-nitrophenyl phosphate indicates that the compound requires activation by esterases and would accordingly be useful as a prodrug.
  • the compounds of the formula I have valuable pharmacological properties and they provide immunostimulatory, antimicrobial and anti-cancer effects.
  • the compounds are useful in the prevention, treatment, and prophylaxis of tumor growth and metastasis of tumors.
  • the anti-metastatic effects of the compounds of the invention may be demonstrated using a lung colonization assay.
  • melanoma cells treated with a compound may be injected into mice and the ability of the melanoma cells to colonize the lungs of the mice may be examined by counting tumor nodules on the lung after death. Suppression of tumor growth in mice by the compound administered orally or intravenously may be examined by measuring tumor volume. Examples of protocols for confirming the activities of the compounds of the invention are included in the Example section.
  • the compounds of the invention may be especially useful in the treatment of various forms of neoplasia such as leukemias, lymphomas, melanomas, adenomas, sarcomas, and carcinomas of solid tissues in patients.
  • the composition may be useful for treating malignant melanoma, pancreatic cancer, ovarian cancer, cervico-uterine cancer, cancer of the kidney, stomach, lung, rectum, breast, bowel, gastric, liver, thyroid, head and neck such as unresectable head and neck cancers, lymphangitis carcinamatosis, cervix, salivary gland, leg, tongue, lip, bile duct, pelvis, mediastinum, urethra, bronchogenic, bladder, esophagus and colon, non-small cell lung cancer, and Kaposi's Sarcoma which is a form of cancer associated with HIV- infected patients with Acquired Immune Deficiency Syndrome (AIDS).
  • the compounds may also be used for other anti-pro
  • the compounds of the formula I may be used to stimulate bone marrow cell proliferation (hemorestoration), in particular following chemotherapy or radiotherapy.
  • the myeloproliferative activity of a compound of the formula I may be determined by injecting the compound into mice, sacrificing the mice, removing bone marrow cells and measuring the ability of the compound to stimulate bone marrow proliferation by directly counting bone marrow cells and by measuring clonogenic progenitor cells in methylcellulose assays.
  • the compounds of the invention are immune modulators and in particular they have immunostimulatory properties. Therefore, the compounds of the formula I may be used in cases where a patient has been immunocompromised such as patients infected with HIV or hepatitis C, or other viruses or infectious agents including bacteria, fungi, and parasites, in patients undergoing bone marrow transplants, and in patients with chemical or tumor-induced immune suppression.
  • the compounds also have an antiviral effect in particular on membrane enveloped viruses such as retroviruses, influenza viruses, cytomegaloviruses and herpes viruses.
  • the compounds of the invention may also be used in the treatment of inflammation. In particular, they may be useful in the treatment of arthritis and asthma.
  • the compounds may render carbohydrate structures on neutrophils unable to bind selectins. Selectins present at the site of damage interact with the carbohydrate structures on neutrophils in such a way that the neutrophils roll along the epithelial wall, stick, infiltrate, and cause tissue damage.
  • the compounds of the invention may be used to protect against the lethality of various chemotherapeutic agents as well as against lethal doses of irradiation.
  • the compounds may also be used in the prevention of tumor recurrence after surgery i.e. adjuvant therapy.
  • the term "patient” refers to a warm-blooded animal such as a mammal which is afflicted with a particular disease state or condition as described herein.
  • animals within the scope of the meaning of the term are dogs, cats, rats, mice, horses, bovine cattle, sheep, and humans.
  • the compounds may be converted using customary methods into pharmaceutical compositions.
  • the pharmaceutical compositions contain the compounds either alone or together with other active substances.
  • Such pharmaceutical compositions can be for oral, topical, rectal, parenteral, local, inhalant, or intracerebral use. They are therefore in solid or semisolid form, for example, pills, tablets, creams, gelatin capsules, capsules, suppositories, soft gelatin capsules, liposomes (see for example, U.S.
  • Patent Serial No. 5,376,452 for parenteral and intracerebral uses, those forms for intramuscular or subcutaneous administration can be used, or forms for infusion or intravenous or intracerebral injection can be used, and can therefore be prepared as solutions of the compounds or as powders of the active compounds to be mixed with one or more pharmaceutically acceptable excipients or diluents, suitable for the aforesaid uses and with an osmolarity which is compatible with the physiological fluids.
  • those preparations in the form of creams or ointments for topical use or in the form of sprays should be considered; for inhalant uses, preparations in the form of sprays, for example nose sprays, should be considered.
  • the pharmaceutical compositions can be prepared by per se known methods for the preparation of pharmaceutically acceptable compositions which can be administered to patients, and such that an effective quantity of the active substance is combined in a mixture with a pharmaceutically acceptable vehicle.
  • Suitable vehicles are described, for example, in Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa, USA 1985).
  • the pharmaceutical compositions include, albeit not exclusively, the compounds in association with one or more pharmaceutically acceptable vehicles or diluents, and contained in buffered solutions with a suitable pH and iso-osmotic with the physiological fluids.
  • the compounds are indicated as therapeutic agents either alone or in conjunction with other therapeutic agents or other forms of treatment (e.g. chemotherapy or radiotherapy).
  • the compounds of the invention may be administered concurrently, separately, or sequentially with other therapeutic agents or therapies.
  • the compounds of the invention may be used in combination with anti- proliferative agents, antimicrobial agents, immunostimulatory agents, or anti-inf ammatories.
  • the compounds may be used in combination with anti-viral and or anti-proliferative agents such as a Thl cytokine.
  • Thl cytokines include interleukins-2 and 12 (IL-2, IL-12), and the interferons- , ⁇ , ⁇ (IFN- ⁇ , IFN- ⁇ , IFN- ⁇ ), and inducers thereof.
  • the compounds of the invention can be used with poly (I.C), poly (I.C.)-LC, tumor necrosis factor (TNF), or transforming growth factor (TGF).
  • the compounds may be administered to a patient being treated with a myelosuppressive agent, or a bone marrow transplant recipient.
  • the compounds can be used in combination with chemotherapeutic agents including doxorubicin, 5-fluorouracil, cyclophosphamide, and methotrexate, with isoniazid for the prevention and treatment of peripheral neuropathy, and with NSAID for the prevention and treatment of gastroduodenal ulcers.
  • a dosage range of the compounds in the composition is envisaged for administration in human medicine of from about 0.001 to 50 g kg of body weight daily.
  • the dosage is for example about .1 to 0.6 mg/kg/day, and for oral compositions the dosage is about 0.5 to 10 mg/kg/day, more preferably 1.5 to 9 mg/kg/day.
  • Amounts of drug administered to produce serum levels 10-lOOOx the IC 50 for inhibition of oligosaccharide processing in the L-PHA assay are preferably employed. It will also be appreciated that it may be necessary to deviate from the amounts mentioned and in particular to do so as a function of the body weight of the animal to be treated, the particular disease to be treated, the nature of the administration route and the therapy desired. In addition, the type of animal and its individual behaviour towards the medicine or the nature of its formulation and the time or interval at which it is administered may also indicate use of amounts different from those mentioned. Thus it may suffice, in some cases, to manage with less than the above-mentioned minimum amounts whilst in other cases the upper limit mentioned must be exceeded.
  • a compound of the invention may be used as a vaccine adjuvant to induce a potent immune response to itself and/or induce immunity to antigens, particularly antigens that are normally poor immunogens.
  • a compound of the invention may augment vaccine immunogenicity through activation of antigen presenting cells, such as monocytes or macrophages, to release cytokines that can promote T-cell help for B cell and CTL response. As a result, the compound may induce a more favorable antibody response with high titers, which appear earlier in the course of immunization and persist over time, as well as increase memory responses and CD8+ MHC Class I-restricted CTL.
  • a compound of the invention may be contained in a vaccine or it may be administered separately.
  • a compound of the invention may be used to enhance immunogenicity of antigens that induce T cell responses (e.g. T cell antigens), and in particular they may be used to enhance the immunogenicity of carbohydrate antigens associated with cancers or infectious diseases.
  • vaccines which may employ a compound of the invention to augment immunogenicity include cancer vaccines (e.g. breast cancer vaccines), and vaccines for chronic infectious diseases.
  • test compound (lS,2R,8R,8aR)-8a-methyl-l,2,8-trihydroxyindolizidine
  • a 40 ⁇ M stock Present in each determination is 10 ⁇ l diluted test compound, 25 ⁇ l of 10 mM paranitrophenyl mannopyranoside, 200 mM sodium acetate, pH 5.6 and 15 ⁇ l of purified rat liver Golgi mannosidase II. After incubating the reaction for 60 minutes at 37°C, the reaction is quenched with 50 ⁇ l of 0.5M sodium carbonate. Abso ⁇ tion is read at 405 nm.
  • the calculated IC 50 for inhibition of purified Golgi mannosidase II by the test compound is 18.214 + 4.437 ⁇ M.
  • the effects of the test compound on lysosomal mannosidase were measured by adding (lO ⁇ l) of the compound into 96 well Elisa plates followed by the addition of 200 mM sodium acetate pH 5.0 and 25 ⁇ l of 10 mM p-nitrophenyl ⁇ -D-mannospyranoside. 15 ⁇ l of lysosomal mannosidase ( about 8mM/mL) was added to each well and the plates were incubated for 60 min at 37°C. The reaction was stopped by the addition of 50 ⁇ l of 0.5M sodium carbonate and formation of p-nitrophenol was measured with a plate set at 405. The calculated IC 50 for inhibition of lysosomal mannosidase by swainsonine hydrochloride is 9.388 + 3.021 ⁇ M.
  • B16F10 melanoma tumor cells are cultured for 48 hours in the presence or absence of the test compound (0.36 ⁇ g/ml) before injection of 10 5 cells into the lateral tail veins of C57BL mice. Lung nodules are counted on day 24 after injection of tumor cells as described in Dennis, JW, Cancer Res. 46:5131-5136, 1986.
  • mice are given drinking water with or without 5.0 ⁇ g/ml of the test compound 2 days before tumor cells are injected into the lateral tail vein and maintained on the test compound for periods of 1-17 days. Lung nodules are counted on day 24 after injection of tumor cells.
  • mice are provided with drinking water either with or without test compound (3.0 ⁇ g/ml) 2 days before 10 5 MDAY-D2 tumor cells are injected. Tumor diameters are measured with calipers twice weekly, then on day 15 after tumor cell injection, tumors are excised and weighed. The tumor growth rate and tumor weight on day 15 in mice given test compound supplemented drinking water and/or two i.p. injections of poly (I.C.) are compared as described in Dennis JW Cancer Res. 46:5131-5136, 1986.
  • the cells are cultured at 37°C in a 5% C0 2 atmosphere and on day 5 the cell number is determined.
  • the method is as described by Dennis, J.W. JNCI 81 :1028-1033, 1989.
  • BM Bone marrow
  • spleen cells from each are processed according to the procedures of the GIBCO-BRL Mouse Bone Marrow Stem Cell Proliferation Kit (Cat. # 3827SA, Grand Island, NY).
  • the potential colonies that form in the semi-solid medium are the CFU-GEMM, the CFU-GM, and the BFUs.
  • the plates are incubated for 10-14 days at 37°C in a humidified atmosphere of 5% C0 2 and 95% air, and colonies consisting of at least 40 cells are enumerated using an inverted microscope (20X magnification) to demonstrate stimulation of hematopoietic progenitor cell growth.
  • mice are treated with either 3 ⁇ g/ml of test compound in their drinking water or injected with 20 ⁇ g/mouse of test compound daily for 2-6 days. Proliferation is assessed by the incorporation of [ 3 H]-thymidine (5 ⁇ Ci/ l) for 18 hours at 37°C into cultures containing equal numbers of freshly isolated BM cells in complete medium. The radiolabeled cells are collected with the aid of a cell harvester onto glass filters, and radioactivity is determined using a liquid scintillation counter. Cellularity of the bone marrow is also determined by using the Coulter counter to directly count BM cells after they are flushed from the tibias and femurs. H. In vivo progenitor assay: Spleen Colony Formation Assay
  • mice (10-14 weeks old) are x-irrradiated for a total whole body exposure of 700cGY.
  • the irradiated mice are maintained on sterile drinking water (approximately 3 ⁇ g/ml) and are given antibiotics to minimize mortality from infection.
  • the number of BM stem cells is estimated by the method of Till and McCulloch (Biochim Biophys Acta 1980 Nov 26;605(4):431-59), which is based on the ability of intravenously injected progenitor stem cells to form colonies in the spleens of recipient mice previously exposed to a lethal dose of whole-body irradiation.
  • the number of CFUs is proportional to the number of pluripotent hematopoietic stem cells present in the hematopoietic graft.
  • recipient mice are sacrificed, their spleens are removed and fixed in Bouin's solution, and grossly visible colonies are counted. I. Bone marrow transplant and repopulation
  • mice Prior to transplantation with bone marrow cells, mice are pre-treated with either a lethal dose of a chemotherapeutic agent or a lethal dose of x-irradiation, as described in White et al (Cancer Communications 3:83, 1991) and Oredipe et al. (JNCI 83: 1149, 1991).
  • Mice aged 10-14 weeks, are irradiated using Phillips RT 250 x-ray machines (two opposing therapeutic 250 Kvp x- ray machines, 235 KV, 15 mA, filtration 0.25 copper and 0.55 aluminum, with a half layer of 0.99 mm copper).
  • Irradiation occurs with a dose rate of 126 cGy/min (63 cGy/min X 2) for 5 minutes and 33 seconds, for a total whole body exposure of 700 cGy. This level of irradiation exposure is within the range described as being lethal for mice.
  • animals are infused with 10 5 bone marrow cells freshly prepared from either control or test compound-treated donor mice.
  • the test compound-treated donor mice receive approximately 20 ⁇ g/ml of test compound for 6 days. Recipient mice are monitored for survival over a period of 30 to 50 days.
  • J. Thl immune response Natural Killer (NK) and iymphokine-activated killer (LAK) cell assays
  • PBMCs Human peripheral blood mononuclear cells
  • the PBMCs are seeded into six-well plates in 5 ml cultures at a concentration of 1.5 million cells per ml either alone (control) or with varying concentrations of test compound, together with 1000 International Units (IU)/ml of IL-2 for three days for the LAK assay or 1000 IU/ml interferon-alpha overnight for the NK assay.
  • IU International Units
  • the NK cell activity of the cultured PBMCs is measured in a Cr 51 release assay using the K562 cell line (NK cell-sensitive) as target cells.
  • LAK cell activity is measured using Cr 51 -labeled Daudi cell line (NK cell-resistant) as targets.
  • Drug activity against viral hepatitis may be determined by infecting mouse strains with mouse hepatitis virus-3 (MHV-3). Previous studies with MHV-3 have focused on mouse strains which develop fulminant hepatitis (Balb ⁇ cJ) or display resistance (A/J) to MHV-3 (Yuwaraj et al, 1996).
  • the CH3/HeJ strain, which develops chronic hepatitis in response to MHV-3 infection is treated with either saline or test compound (20 ⁇ g/mouse/day) alone or in combination with IFN. Before and during treatment, the levels and activation status of STATs is measured as well as serum cytokine levels, viral load and survival. L.
  • test compound Activity in patients with chronic hepatitis C
  • the response to treatment with a test compound or test compound plus interferon-alpha in patients with chronic hepatitis C can be monitored by a decrease in viral load and serum liver alanine aminotransferase (ALT) measured during treatment, for example at 3, 6, and 12 months. Improvement in liver histology can also be assessed by performing biopsies before and after treatment.
  • the test compound is administered orally, twice daily, at doses between 50 and 200 ⁇ g/kg either alone, or in combination with alpha-interferon, which is administered at doses of 1 to 3 MU three times weekly. During this time, the test compound may be administered continuously or intermittently (e.g. 2 weeks on, one week off).
  • the response in patients receiving test compound is compared to the response in patients receiving placebo or alpha-interferon.
  • Detection of hepatitis C viral RNA in serum, liver, and peripheral blood mononuclear cells is performed by the reverse transcriptase-polymerase chain reaction method (RT-PCR), using primer specific for the highly conserved, 5 '-untranslated region (UTR) for qualitative or, with appropriate internal control RNA, quantitative detection.
  • the second method is a signal amplification or branched chain DNA (bDNA) assay. Viral nucleic acids are hybridized to microtiter plates and reacted with virus-specific extender probes followed by bDNA polymers.
  • the Histologic Activity Index based on a scoring system developed by Knodell et al (Hepatology 1981, 1 :431-435), assigns grades in four categories: periportal necrosis, interlobular necrosis, portal inflammation and f ⁇ brosis.
  • a system based on grading hepatic inflammation (0-4) and staging fibrosis (0-4) can be used (Scheuer PJ, J. Hepatol 1991; 13:372-374).
  • M. Hemorestoration/Chemoprotection Cellular and animal models of hemorestoration/chemoprotection are described in Oredipe et al, 1991, supra, and White et al, 1991, supra.

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Abstract

La présente invention concerne des dérivés de swainsonine 8a substitués, des procédés destinés à leur préparation, et leurs utilisations comme agents thérapeutiques. L'invention concerne aussi des compositions pharmaceutiques contenant ces composés ainsi que leurs utilisations comme agents thérapeutiques.
PCT/CA1999/001210 1998-12-18 1999-12-17 Derives de swainsonine, procedes de preparation et utilisations comme agents therapeutiques WO2000037465A2 (fr)

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CA002352858A CA2352858A1 (fr) 1998-12-18 1999-12-17 Derives de swainsonine, procedes de preparation et utilisations comme agents therapeutiques
AU17643/00A AU1764300A (en) 1998-12-18 1999-12-17 Novel derivatives of swainsonine, processes for their preparation, and their useas therapeutic agents

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6395745B1 (en) 1997-04-15 2002-05-28 Glycodesign, Inc. Alkaloid halide salts of swainsonine and methods of use
WO2005070418A1 (fr) * 2004-01-21 2005-08-04 M N L Pharma Limited Alcaloides immunomodulateurs
CN102432609A (zh) * 2011-12-15 2012-05-02 西藏自治区农牧科学院 一种茎直黄芪中苦马豆素的微波辅助提取工艺
CN103319481A (zh) * 2013-07-05 2013-09-25 西藏自治区农牧科学院 茎直黄芪中苦马豆素的提取工艺

Citations (1)

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Publication number Priority date Publication date Assignee Title
US5633261A (en) * 1991-11-06 1997-05-27 Toronto Research Chemicals, Inc. Immunostimulating swainsonine analogs

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US5633261A (en) * 1991-11-06 1997-05-27 Toronto Research Chemicals, Inc. Immunostimulating swainsonine analogs

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J. M. MCINTOSH: "Azapropellanes as phase-transfer catalysts-II" TETRAHEDRON., vol. 38, no. 2, 1982, pages 261-6, XP002138010 ELSEVIER SCIENCE PUBLISHERS, AMSTERDAM., NL ISSN: 0040-4020 *
M.G. REINECKE ET AL.: "Peripheral synthesis of secondary medium-ring nitrogen heterocycles" JOURNAL OF ORGANIC CHEMISTRY., vol. 38, no. 19, 1973, pages 3281-87, XP002138009 AMERICAN CHEMICAL SOCIETY. EASTON., US ISSN: 0022-3263 *
M.G. REINECKE ET AL.: "The peripheral synthesis of medium-ring nitrogen heterocycles by displacement reactions" JOURNAL OF ORGANIC CHEMISTRY., vol. 37, no. 22, 1972, pages 3494-99, XP002138011 AMERICAN CHEMICAL SOCIETY. EASTON., US ISSN: 0022-3263 *
S.F. MARTIN ET AL.: "Application of reductive, single electron transfer processes to the generation and cyclization of omega-unsaturated and alpha-amino radicals" TETRAHEDRON LETTERS., vol. 29, no. 51, 1988, pages 6685-88, XP002138008 ELSEVIER SCIENCE PUBLISHERS, AMSTERDAM., NL ISSN: 0040-4039 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6395745B1 (en) 1997-04-15 2002-05-28 Glycodesign, Inc. Alkaloid halide salts of swainsonine and methods of use
WO2005070418A1 (fr) * 2004-01-21 2005-08-04 M N L Pharma Limited Alcaloides immunomodulateurs
CN102432609A (zh) * 2011-12-15 2012-05-02 西藏自治区农牧科学院 一种茎直黄芪中苦马豆素的微波辅助提取工艺
CN103319481A (zh) * 2013-07-05 2013-09-25 西藏自治区农牧科学院 茎直黄芪中苦马豆素的提取工艺
CN103319481B (zh) * 2013-07-05 2014-12-17 西藏自治区农牧科学院 茎直黄芪中苦马豆素的提取工艺

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