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WO1995021614A1 - Nouveaux inhibiteurs du trafic des proteines - Google Patents

Nouveaux inhibiteurs du trafic des proteines Download PDF

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Publication number
WO1995021614A1
WO1995021614A1 PCT/US1995/001656 US9501656W WO9521614A1 WO 1995021614 A1 WO1995021614 A1 WO 1995021614A1 US 9501656 W US9501656 W US 9501656W WO 9521614 A1 WO9521614 A1 WO 9521614A1
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Prior art keywords
compounds
bfa
compound
synthesis
stirred
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PCT/US1995/001656
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English (en)
Inventor
Manfred Weigele
Mallory F. Loewe
Christopher S. Poss
Tsvetelina Lazarova
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Ariad Pharmaceuticals, Inc.
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Publication of WO1995021614A1 publication Critical patent/WO1995021614A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D313/00—Heterocyclic compounds containing rings of more than six members having one oxygen atom as the only ring hetero atom
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D225/00—Heterocyclic compounds containing rings of more than seven members having one nitrogen atom as the only ring hetero atom
    • C07D225/04—Heterocyclic compounds containing rings of more than seven members having one nitrogen atom as the only ring hetero atom condensed with carbocyclic rings or ring systems

Definitions

  • BFA BACKGROUND Brefeldin A
  • Penicillium decumbens Singleton, V. L., et al., Nature 181:1072- 1073 (1958)
  • BFA has a molecular weight of 280.37 (C16H24O4) and reportedly has a wide range of biological activities, including antifungal, antiviral and antitumor effects. See Betina, Folia Microbiol. 37(1):3-11 (1992) for a recent review. At the cellular level, BFA has dramatic effects on the secretory pathway and protein trafficking iri mammalian cells. (Pelham, H. R.
  • BFA has been shown to also inhibit protein transport in fungi, such as Candida albicans (Arioka, M., et al., J. Gen. Microbiol., 137:1253-1262 (1991)) and inhibit the presentation of endogenous and exogenous protein antigens by MHC class II- restricted T-cells (Adorini, ., et al., Nature, 246:63-66 Quly 1990)).
  • BFA has also been shown to have selective cytotoxic activity against human tumor cell lines (Ishii, S., et al., J. Antibio , XLI 1877-1878 (1989)).
  • BFA also inhibits virus replication by interfering with the intracellular transport and maturation of viral proteins. Inhibition, as defined herein, means a significant reduction in virus particle replication, as well as complete abrogation of virus particle replication.
  • Enveloped viruses such as herpes viruses (including Herpes Simplex) and Human Immunodeficiency Virus (HIV) require the host cell secretory apparatus for transport and processing of envelope (membrane) glycoproteins during the course of virus assembly and maturation.
  • BFA has also been shown to inhibit infectious viral particle formation by preventing the transport of envelope glycoprotein to the cell surface as r ⁇ uired for assembly of mature, infectious viral particles.
  • BFA has a short biological half-life. It is rapidly deactivated in vivo via conjugation with glutathione by glutathione S-transferase and subsequently transported out of the cell (Bruning, A., et al., J. Biol. Chem., 267:7726-7732 (1992)).
  • Compounds having some or all of the biological activities of BFA combined with an extended useful biological half -life and /or improved overall therapeutic profiles would be valuable for the treatment of viral, bacterial, fungal and other diseases, as anti-cancer agents, as immunosuppresive agents and as detoxifying agents.
  • This invention concerns novel compounds related structurally to Brefeldin A; methods of synthesizing these compounds; use thereof as antiviral, antifungal, detoxification and antiproliferative agents (e.g., antitumor agents and agents to treat genital warts); pharmaceutical compositions which contain these compounds as active components; and pharmaceutical methods involving administration of these compounds to mammals, preferably human patients, in need thereof.
  • These compounds block, or inhibit, the transport of proteins from the endoplasmic reticulum (ER) and through the Golgi apparatus in a cell and are also useful as experimental research reagents.
  • This invention encompasses compounds of the formula:
  • Y is a trans xmsaturated 2- carbon unit or a fused cyclopropyl ring, i.e., Y is
  • Z is an unsaturated 2-carbon unit (cis or trans), or a hydroxy- or fluoro- substituted saturate ⁇ d ⁇ 2-carbon unit, i.e., Z is
  • Illustrative compounds in which J is O and Y is a cyclopropyl ring include the following:
  • the cyclopropyl ring junctions may have either R,R or S,S stereochemical configuration.
  • esters referred to above include among others compounds of this invention in which one or more hydroxyl goups bear acyl moieties of the formula R ⁇ CO- where each Rl is independently selected and may be alkyl, aryl or heteroaryl.
  • R 1 may be lower alkyl (i.e., upto 6 carbons), optionally substituted with one or more halogen atoms, preferably fluorine.
  • R 1 is aryl, it preferably contains a hydroxy, alkoxy or amino substituent, preferably in the para or ortho position.
  • compounds of this invention include, among others, compounds in which Rl is
  • R* is -(CH2)n-L .
  • n is an integer from 2 through about 6 and L is -CO2H, SO3H, PO3H, amino, alkylamino, dialkylamino or trialkylamrnonium and pharmaceutically acceptable salts thereof.
  • the alkyl groups may be the same or different and may be substituted or unsubstituted and may be straight-chain, branched or cyclic.
  • alkyl substituents include saturated straight-chain, cyclic or branched hydrocarbon moieties, preferably of one to about twelve carbon atoms, including methyl, ethyl, n- propyl, i-propyl, cyclopropyl, n-butyl, i-butyl, t-butyl, cyclobutyl, cyclopropylmethylene, pentyl, hexyl, heptyl, octyl and so forth, and may be optionally substituted with one or more substituents such as lower alkoxy, carboxy, amino, phenyl, aryl, mercapto, halo (fluoro, chloro, bromo or iodo), azido and cyano.
  • substituents such as lower alkoxy, carboxy, amino, phenyl, aryl, mercapto, halo (fluoro, chloro, bromo or iodo), azi
  • R 2 is alkyl, preferably lower alkyl; n is 0, 1 or 2; and m is 1 or 2.
  • R2 is methyl or ethyl and n is 2.
  • R ⁇ is methyl
  • compounds of this invention include, among others, the following:
  • Such lactams have the general formula described above in which J is -NW- where W is -H, alkyl, aryl, arylalkyl, acyl or alpha aminoacyl. Further examples of suitable W moieties are disclosed below.
  • W is H:
  • W is a substituted or unsubstituted, straight chain or branched lower alkyl group.
  • R' is -H or a substituted or unsubstituted, straight chain or branched, preferably lower, alkyl group.
  • the -COCHR'NH2 moiety may be provided by any D- or L-amino acid, including but not limited to any of the naturally occurring amino acids.
  • R" is H or a branched or straightchain, substituted or unsubstituted alkyl group, preferably lower alkyl-
  • R" is a side-chain of an alpha amino acid, naturally occurring or otherwise.
  • R" may be H (glycine), -CH3 (alanine), -CH2phenyl (phenylalanine), and so forth.
  • R ⁇ is -H, -OH, -alkoxy, -aryloxy, -arylalkoxy or -O-acyl and Y is a carbon- carbon double bond (trans) or a fused cyclopropyl group, as well as their esters and the individual diastereoisomers and their diastereomeric mixtures.
  • Y is a carbon- carbon double bond (trans) or a fused cyclopropyl group, as well as their esters and the individual diastereoisomers and their diastereomeric mixtures.
  • R3 is OH, i.e., hemiacetals.
  • hemiacetals are illustrated by the following structure, where Z in this case is a -C — C- unit and Y is as previously defined:
  • R3 is-alkoxy, -aryloxy, -arylalkoxy or -O-acyl (i.e., -O-CO-U).
  • R3 is-alkoxy, -aryloxy, -arylalkoxy or -O-acyl (i.e., -O-CO-U).
  • Y is as previously defined and U is alkyl, aryl, aryloxy or acyl:
  • R 4 is alkyl, alken l, aryl, arylalkyl, -OR 6 or NR 7 R 8 ; where R 6 is alkyl, alkylene, aryl or arylalkyl and R 7 and R 8 are the same or different and are H, alkyl, alkylene, aryl or arylalkyl;
  • This invention thus encompasses three subclasses of compounds corresponding to formulas 1, 2 and 3:
  • the compounds of this invention can be used as biological reagents to inhibit the intracellular transport of proteins from the ER of a cell through the compartments of the Golgi apparatus, and, ultimately, to the cell surface.
  • the compounds of this invention can be used to inhibit maturation of viral membrane glycoproteins in a cell infected with a virus by inhibiting the transport of viral membrane glycoproteins from the ER of the cell through the compartments of the Golgi apparatus, and, ultimately, to the cell surface, as required for the assembly of mature, infectious virus particles.
  • These compounds can likewise be used to inhibit the transport, and thus the otherwise concomitant processing and presentation, of antigens by antigen presenting cells.
  • compositions which contain as active components compounds described herein which are effective for one or more of the indications noted herein and which can be administered to an individual in need thereof.
  • a compound of this invention can be combined with a physiologically compatible carrier for administration to an individual infected with a fungus or virus, or to an individual harboring a tumor.
  • the compounds of this invention may act, at least in part, by inhibiting the transport of proteins critical, for example, to the maturation, intracellular replication and/or infectivity of virus, to the growth and/or proliferation of tumor cells, or to fungal growth.
  • the compounds of this present invention can typically be produced synthetically from BFA.
  • BFA can be prepared by fermentation followed by product recovery from the culture medium as described in detail in Harri, E., et al., Helv. Chim. Acta, 46:1235 (1963). Alternately, BFA can be synthesized using standard laboratory methods. (Baudouy, R., et al., Tetrahedron Letters, 34:2973- 2976 (1977); LeDrian, C, et al., J. Am. Chem. Soc.,104:5473-5483 (1982); Kitahara, T. and Mori, K., Tetrahedron, 40:2935-2944 (1984)). BFA can then be transformed to produce the compounds described herein as described in the Examples which follow.
  • Example 4 which can be synthesized by reacting BFA with a slurry of trimethylsulfox- onium iodide, dimethyl sulfoxide and pentane-washed sodium hydride.
  • An illustrative procedure is provided in Example 4.
  • the method of synthesis described herein results in a mixture of two diastereomers. Under the conditions described in the examples which follow, the 2R, 3R-diastereomer is the major product.
  • the two diastereomers can be separated by standard laboratory methods.
  • the apex of the cyclopropyl ring can be further modified to include one or two, halogens (e.g., fluoride), or, alternately, one, or two, unbranched alkyl groups.
  • esters of the compounds disclosed herein can be prepared prior to or following final deprotection of the lactones. See e.g. USSN 08/207,319 and 08/207,496, supra..
  • Flouro and di-fluoro compounds of this invention can be prepared from the corresponding hydroxy (with stereochemical inversion) and keto compounds, respectively, (typically with protection of other hydroxy groups and subsequent deprotection) using diethylaminosulfur trifluoride (DAST).
  • DAST diethylaminosulfur trifluoride
  • the biological activity of these compounds can be evaluated and compared using conventional in vitro assays for inhibition of protein trafficking between the endoplasmic reticulum (ER) and the Golgi apparatus and specifically for antiviral, antitumor, immunosuppressive and antifungal activity as discussed in further detail below.
  • ER endoplasmic reticulum
  • the inhibitory activity of the compounds with respect to protein transport can be evaluated in a cell-free system as described in Orci, L., et al., Cell, 64:1183- 1195 (1991).
  • secretory proteins such as membrane glycoproteins
  • Golgi apparatus membranes can be isolated and incubated with cytosol, ATP, an ATP regenerating system and the compounds to be tested as described in Orci, L., et al, Cell, 64:1183-1195 (1991).
  • the activity of our compounds in inhibiting protein transport may also be evaluated using a Guanine Nucleotide Exchange Factor (GEF) assay as described in detail in the Examples.
  • GEF Guanine Nucleotide Exchange Factor
  • the GEF assay is based on assays described in
  • cytosolic proteins are specifically associated with the Golgi apparatus.
  • One such protein, ⁇ -COP is rapidly released from the Golgi upon treatment with BFA. This release occurs within 20 seconds of BFA treatment and is complete in 1-2 minutes. Upon removal of BFA, ⁇ -COP rapidly reassociates with the Golgi apparatus. (Klausner, R.D., et al., J. Cell Biol. 116:1071-1080 (1992)).
  • ADP-ribosylation factor ADP-ribosylation factor
  • ARF ADP-ribosylation factor
  • ARF association with the Golgi is, in turn, dependent on binding the guanine nucleotide, GTP.
  • a component of Golgi membranes specifically catalyzes the exchange of GTP onto ARF.
  • BFA prevents the assembly of ⁇ -COP onto the Golgi membrane by inhibiting the GTP- dependent interaction of ARF with the Golgi membrane.
  • This immature virion is transported through the endoplasmic reticulum (ER) and undergoes subsequent de-envelopment, with release of the immature virus particle in proximity to the Golgi apparatus. Subsequent maturation of the immature virion occurs at the Golgi apparatus, which involves a second envelopment of these immature capsids by membrane proteins derived from the Golgi apparatus, containing fully processed viral glycoproteins. The resulting mature, infectious enveloped virus particle is released from the cell by fusion of the outer membrane of the virion envelope with the plasma membrane of the host cell, or, alternately, can be transported via transport vesicles to the cell surface, where membrane fusion results in presentation of viral glycoproteins on the cell surface. BFA does not affect protein synthesis at the translational level, but blocks the post-translational processing and export of viral glycoproteins to the Golgi apparatus, thus, inhibiting viral replication by preventing the formation and /or release of mature, infectious virus particles.
  • ER endoplasm
  • the compounds of this invention can be tested for specific antiviral activity as described in Example 7 as well as by other conventional antiviral assay methods. See e.g. Whealey et al, supra; Johnson et al, J Virol 43(3): 1102-1112 (1982); Sidwell et al, Nucleotides and Nucleosides 8:833-836 (1989) and Chen et al, J Virol 65(3): 1427-1439 (1991).
  • HSV-1 Herpes Simplex Virus type 1
  • BFA can be used as a control, as can clinically relevant or other known positives, such as IUDR (iodouracyl deoxyribocyte) which can be used as an antiviral, positive control.
  • Compounds can be evaluated with respect to specific antifungal, anticancer, immunosuppressive or other pharmaceutically relevant activities using conventional materials and methods. See e.g. Arioka, J. Gen. Microbiol., 137:1253-1262 (1991) (evaluation of antifungal activity); Ishii et al., J. Antibiot. XLI 1877-1878 (1989) (evaluation of cytotoxic /antitumor activity); Sun et al, US Patent 5,206,249 (27 April 1993)(evaluation of in vitro growth inhibitory activity on cultured leukemia cells); and Yoshida et al., Experimental Cell Research 192:389-395 (1991)(evaluation of anti-toxin activity).
  • Bioactivity can be further evaluated in conventional animal model systems including anti-viral, anti-fungal, antitumor, immunosuppression and detoxification assays involving experimental animal models, e.g. using rats, mice, rabbits, guinea pigs, sheep or non-human primates. Numerous animal models for such studies, as well as animal models for determining biological half-life, pharmacokinetics and toxicology, are well known in the art. In vivo toxicity can be readily evaluated with conventional toxicity assays as well as by the method described in Example 8.
  • the effectiveness of the compounds of this invention in controlling viral infection can be evaluated in any of the conventional assay systems. See e.g. Stanberry, "Pathogenesis of Herpes Simplex Virus Infection and Animal Models for its Study” and Renegar, Laboratory Animal Science 42(3):222.
  • HSV infection can be evaluated using guinea pig and mouse model systems that are art-recognized models used in the study of genital herpes.
  • the guinea pig model system is described in detail in Stanberry, L.R., et al., J. Infect. Diseases, 153:1055-1061 (1986), and Bourne, N., et al., Antimicrob. Agents and Chemo.,
  • Compounds of this invention which prevent, inhibit or reduce the severity of viral infection (e.g. an infection by a virus such as a Herpes Simplex virus), fungal infection (e.g. an infection by a fungus such as Candida albicans), tumors or tumor growth or the effect of toxic substances or which have an immunosuppressive effect may be used in pharmaceutical compositions and methods for treatment or prevention in a mammal in need thereof.
  • viral infection e.g. an infection by a virus such as a Herpes Simplex virus
  • fungal infection e.g. an infection by a fungus such as Candida albicans
  • tumors or tumor growth or the effect of toxic substances or which have an immunosuppressive effect may be used in pharmaceutical compositions and methods for treatment or prevention in a mammal in need thereof.
  • Mammals include rodents such as mice, rats and guinea pigs as well as dogs, cats, horses, cattle, sheep, non-human primates and humans.
  • the preferred method of such treatment or prevention is by administering to a mammal an effective amount of a compound of this invention to prevent, alleviate or cure said disease or disorder.
  • An effective amount of a compound of this invention is an amount of one or more compounds of this invention which inhibits one or more of protein transport from the endoplasmic reticulum, viral replication, fungal growth, tumor cell growth and pathological effect(s) of a toxin, or which results in immunosuppression, as the case may be.
  • Such effective amounts can be readily determined by evaluating the compounds of this invention in conventional assays well-known in the art, including assays described herein.
  • the invention provides methods of treating, preventing and /or alleviating the symptoms and /or severity of a disease or disorder referred to above by administration to a subject a compound of the invention in an amount effective therefor.
  • the subject will be an animal, including but not limited to animals such as cows, pigs, chickens, etc., and is preferably a mammal, and most preferably human.
  • a compound of this invention e.g., encapsulation in liposomes, microparticles, microcapsules, etc.
  • One mode of delivery of interest is via pulmonary administration, as detailed more fully infra.
  • Other methods of introduction include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural and oral routes.
  • a compound of this inventions may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local.
  • preferred routes of administration are oral, nasal or via a bronchial aerosol or nebulizer.
  • compositions comprise a therapeutically (or prophylacticaUy) effective amount of a compound of this invention, and a pharmaceutically acceptable carrier or excipient.
  • a carrier includes but is not limited to saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the carrier and composition can be sterile. The formulation should suit the mode of administration.
  • the composition/if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
  • the composition can be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder.
  • the composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides.
  • Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc.
  • the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings.
  • compositions for intravenous administration are solutions in sterile isotonic aqueous buffer.
  • the composition may also include a solubilizing agent and a local anesthetic to ease pain at the side of the injection.
  • the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.
  • the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline.
  • an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
  • Administration to an individual of an effective amount of one or more of the compounds described herein can also be accomplished topically by administering the compound(s) directly to the affected area of the skin of the individual.
  • the compounds are administered or applied in a composition including a pharmacologically acceptable topical carrier, such as a gel, an ointment, a lotion, or a cream, which includes, without limitation, such carriers as water, glycerol, alcohol, propylene glycol, fatty alcohols, triglycerides, fatty acid esters, or mineral oils.
  • topical carriers include liquid petroleum, isopropyl palmitate, polyethylene glycol, ethanol (95%), polyoxyethylene monolaurate (5%) in water, or sodium lauryl sulfate (5%) in water.
  • Other materials such as anti- oxidants, humectants, viscosity stabilizers, and similar agents may be added as necessary.
  • the compounds of this invention may be disposed within devices placed upon, in, or under the skin.
  • Such devices include patches, implants, and injections which release the compound into the skin, by either passive or active release mechanisms.
  • genital infection with HSV is characterized by herpetic lesions on the external genital skin.
  • latent infection is established.
  • One possible mechanism for the maintenance of latency involves the migration of virus from recurrent lesions back to sensory ganglia, where a new set of neurons are infected and become a source of latent virus responsible for recurrent disease. (Stanberry, L.R., et al., J. Infect. Dis., 153:1055-1061 (1986)).
  • administration of an antiviral agent which inhibits the formation of mature infectious virus particles would be useful to prevent migration of HSV and reasonably prevent establishment of a latent HSV infection.
  • Topical administration of a compound of this invention directly to the areas of the skin affected with the herpetic lesions would be an attractive method of administration.
  • anti-viral application of a pharmaceutical agent see Whitley et al, "Acyclovir: A Decade Later", New England Journal of Medicine pp. 782-789 (10 September 1992).
  • a compound of this invention can be formulated in neutral or salt forms.
  • Pharmaceutically acceptable salts include those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2- ethylamino ethanol, histidine, procaine, etc.
  • the effective dose of compounds of this invention will typically be in the range of about 0.01 to about 50 mg/kgs, preferably about 0.1 to about 10 mg/kg of mammalian body weight, administered in single or multiple doses.
  • the compounds of this invention may be administered to patients in need of such treatment in a daily dose range of about 1 to about 2000 mg per patient.
  • the amount of a compound of this invention which will be effective in the treatment or prevention of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques.
  • in vitro or in vivo assays may optionally be employed to help identify optimal dosage ranges.
  • Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.
  • the precise dosage level of our compounds, as the active component(s) should be determined by the attending physician or other health care provider and will depend upon well known factors, including route of administration, biological activity of the particular compound, and the age, body weight, sex and general health of the individual; the nature, severity and clinical stage of the disease; and the use (or not) of concomitant therapies.
  • the invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention.
  • a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention.
  • Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceutical or biological products, which notice reflects approval by the agency of manufacture, use or sale for human ad-ministration.
  • a compound of this invention is administered by pulmonary administration, e.g. via aerosolization.
  • This route of administration may be particularly useful for treatment or prophylaxis of bronchial or pulmonary infection or tumors.
  • Pulmonary administration can be accomplished, for example, using any of various delivery devices known in the art (see e.g., Newman, S.P., 1984, in Aerosols and the Lung , Clarke and Davia (eds.), Butterwarths, London, England, pp. 197-224; PCT Publication No. WO 92/16192 dated October 1, 1992; PCT Publication No.
  • nebulizers including but not limited to nebulizers, metered dose inhalers, and powder inhalers.
  • Various delivery devices are commercially available and can be employed, e.g., Ultravent nebulizer (Mallinckrodt, Inc., St. Louis, Missouri); Acorn II nebulizer (Marquest Medical Products, Englewood, Colorado), Ventolin metered dose inhaler (Glaxo Inc., Research Triangle Park, North Carolina); Spinhaler powder inhaler (Fisons Corp., Bedford, Massachusetts) or Turbohaler (Astra).
  • Such devices typically entail the use of formulations suitable for dispensing from such a device, in which a propellant material may be present.
  • Ultrasonic nebulizers tend to be more efficient than jet nebulizers in producing an aerosol of respirable size from a liquid (Smith and Spino, "Pharmacokinetics of Drugs in Cystic Fibrosis," Consensus Conference, Clinical Outcomes for Evaluation of New CF Therapies, Rockville, Maryland, December 10-11, 1992, Cystic Fibrosis Foundation).
  • a nebulizer may be used to produce aerosol particles, or any of various physiologically acceptable inert gases may be used as an aerosolizing agent.
  • Trimethylsulfoxonium iodide (0.258 gram) was added to a 10 mL flask containing a stir bar, nitrogen inlet, 2.5 mL dimethyl sulfoxide (DMSO) and pentane washed NaH. The resulting slurry was stirred at room temperature until gas evolution ceased (15 minutes). Brefeldin A (0.107 gram) was added at once and stirring was continued for an additional hour at ambient temperature. The reaction mixture was quenched with water (4 mL) and extracted with ethyl acetate (4x 50 mL). The combined organic extract was washed with water (2x 25 mL), brine (25 mL) and dried over sodium sulfate. The solvent was removed under vacuum and the residue chromatographed through a silica gel column, eluted with ethyl acetate, to separate isomers.
  • DMSO dimethyl sulfoxide
  • the minor S,S-isomer was isolated as an oil.
  • the S,S-isomer structure was consistent with the following ⁇ H/l ⁇ C NMR data: lH NMR (CDCI3) ppm 0.75(m,lH) 1.08(d, 2H, CH3) l.l-1.2(m,lH) 1.25-1.4(m, 5H) 1.55(m, IH) 1.7(m, IH) 1.8-2.2(m, 8H) 2.3(m, IH) 3.7(m, IH) 4.2(m, IH) 4.8(d, IH) 5.3(m, 2H)
  • Examples 1, 2 and 3 may be carried out using Compound II (R,R, S,S or a mixture of the two) in place of BFA to yield the corresponding 12-hydroxy and triene compounds bearing a 2,3 cyclopropyl group.
  • Example 6 Guanine Nucleotide Exchange Factor Assay Recombinant myristoylated ADP-ribosylation factor (ARF) is purified from Escherichia coli co-expressing the human ARF-1 gene and N- myristoyltransferase as described in Weiss, O., et al., J. Biol. Chem., 264:21066-
  • ARF ADP-ribosylation factor
  • Golgi membranes from rat livers are obtained by sucrose gradient centrifugation as described in Balch, W. E., et al., Cell, 39:525-536 (1984).
  • [ 35 S]GTP is incubated at 37°C for 15 minutes.
  • the specific reactions are set up with 5 ⁇ l of 2.3M sucrose, 10 ⁇ l of 0.5 mM compound (except for the background and control reactions, in which no compound is added), 5 ⁇ l buffer, 5 ⁇ l of 16 mg/ml Ovalbumin, 5 ⁇ l of 0.6 mg/ml golgi (none in background run), 8 ⁇ l of 0.4 gm/ml Arf-1 (except none in background run) and 12.5 ⁇ l of 20 ⁇ M GTP ( 35 S), with the background and control reactions diluted with 23 ⁇ l and 10 ⁇ l, respectively, of water.
  • ARF-bound and -unbound [35s]GTP are separated with 10 kD molecular weight cutoff cellulose filters. Nonspecific binding (from background run) is subtracted. Alternatively, the ARF-bound [35s]GTP can be separated by Sephadex
  • the antiviral activity and cytotoxic microscopic effects of our compounds may be determined in the following manner.
  • Hep2 cells in RPMI/1640 medium with 5% fetal calf serum are grown to provide a confluent sheet of cells.
  • Various concentrations of the compound(s) to be tested, as well as positive and negative controls, are added.
  • the cells are then incubated at 37°C, in 5% carbon dioxide.
  • a cytotoxicity screening assay different concentrations of the compound(s) to be tested, as well as positive and negative controls, are added to the cell culture. Cytotoxicity is determined by microscopic examination on days 3 and 6.
  • the antiviral and cytotoxic effects of the compounds on Hep2 cells infected with HSV-1 can be determined as follows.
  • Hep2 cells in RPMI/1640 medium with 5% fetal calf serum are added to microtiter wells and incubated at 37°C in 5% carbon dioxide.
  • various concentrations (4 wells /concentration compound) of the compound(s) to be tested, as well as positive and negative controls are added. Typical test concentrations may run from about 0.1-50 ⁇ g/ml.
  • HSV-1 virus is added 7 hours later. The cell culture specimens are examined for evidence of viral growth and Hep2 microscopic toxicity on days 1, 2, 3, 4, 5, 6, 7 and 10 post-infection. Virus controls at dilutions of 10 ⁇ 10 to 10 ⁇ ***4 are included.
  • Example 8 In vivo Toxicity Study
  • An intraperitoneal dose response study of the compounds of this invention can be carried out in mice to provide toxicological data as follows.
  • mice Female CD-I mice (6 /group) are employed in this investigation.
  • the compound is suspended in 0.5% carboxymethylcellulose (CMC).
  • CMC carboxymethylcellulose
  • the animals are scheduled to receive the compound, intraperitoneally, at dosage levels of 50, 100 and 400 mg/kg/day for 5 consecutive days. All doses are administered in a constant volume of 20 ml/kg.
  • Another group of animals receives 0.5% CMC (20 ml/kg) and serves as a control. The animals are then observed for at least two weeks after which a necropsy is performed. Tissues are collected for histopathologic evaluation.
  • the compounds of this present invention can be produced synthetically from BFA or other starting materials.
  • BFA can be prepared by fermentation followed by product recovery from the culture medium as described in detail in Harri, E., et al., Helv. Chim. Acta, 46:1235 (1963). Alternately, BFA can be synthesized using standard laboratory methods. (Baudouy, R., et al., Tetrahedron Letters, 34:2973-2976 (1977); LeDrian, C, et al., J. Am. Chem. Soc.,104:5473-5483 (1982); Kitahara, T. and Mori, K., Tetrahedron, 40:2935-2944 (1984)). BFA can then be transformed to produce the lactams described herein by the following general strategy:
  • N-butyl lithium (2N, 2.75 mL) is slowly added to stirring, anhydrous methanol (8 mL) cooled jn a -20°C bath. After 10-30 minutes, the -20°C bath is removed and the solution allowed to come to room temperature. A methanolic solution of compound I is added and stirring is continued for 5 hours at room temperature. The solution is concentrated under reduced pressure and the residue taken up in ethyl acetate (50 mL) and water ( 20 mL). The organic layer is washed successively with saturated sodium bicarbonate water and brine. The organic layer is dried over sodium sulfate, filtered and concentrated under reduced pressure. The residue may then be column chromatographed over silica gel (ethyl acetate/ hexane (1:4)).
  • reaction solution is then poured into ethyl acetate (100 mL) and extracted sequentially with water, saturated sodium bicarbonate and brine.
  • the organic layer is dried over sodium sulfate, filtered and concentrated. This residue may be purified if desired by column chromatography over silica gel ( hexane /ET Ac, 2:1).
  • Lactams of the formula (A- VI) where W represents an alkyl group may be prepared from A-III by adaptation of the method of Example A-4 but substituting the desired amine for ammonia. Alternatively, such compounds may be prepared by the following route followed by deprotection:
  • Substituted lactams of the formula A-VII may be prepared by the following route using conventional transformation and deprotection methods:
  • Substituted lactams of the formula A-VIII may be prepared by the following route using conventional transformation and deprotection methods:
  • BFA I(A)-He Brefeldin A may be converted to the hemiacetal with DiBAL by conventional methods. See e.g. J Org Chem 47, 4750 (1982) and J Med Chem 28, 1580 (1985).
  • I(A)-e may be prepared from BFA by adaptation of the method of Kraus, G.A. et al, J Org Chem 55, 1105 (1990) and J Org Chem 45, 4820 (1980).
  • THP protecting groups may be added to BFA as described in J Org Chem 44, 1439 (1979)
  • the di-THP BFA lactone may be converted into the methyl ester of the open-chain alcohol using methyl lithium as described in JCS Chem Comm 695, 1986, followed by removal of the THP groups with MeOH/TsOH.
  • Other esters may be produced by conventional methods such as ester exchange using organolithium reagents containing the desired OR ⁇ group, as exemplified below.
  • Di-THP-(C-2(a)) may be readily acetylated (e.g., J Am Chem Soc 100, 8272 , 1978) and deprotected to yield C-2(b).
  • Di-THP-(C-2(a)) may alternatively be acylated with any desired -(CO)R ⁇ moiety using any number of conventional coupling approaches and then deprotected:
  • Di-THP-2(a) may be converted into the desired urethane using conventional procedures such as condensation of the alcohol with the appropriate isocyanate (see e.g. J Org Chem 49, 720, 1984) and then deprotected, as exemplified above.
  • Di-THP-2(a) may be converted into the desired sulfonate using conventional procedures such as condensation of the alcohol with the appropriate sulfonyl chloride (see e.g. J Am Chem Soc 92, 553, 1970) and then deprotected, as exemplified above.
  • Di-THP-(C-2(d)) may be converted into the iodide, C-2(e), using conventional procedures such as treatment with sodium iodide /acetone (see e.g. J Chem Soc 92, 3650, 1950) and then deprotected, as exemplified above.
  • Iodo compounds such as C-2(e) may be converted into the corresponding alkyl compounds with zinc and acetic acid as illustrated above (see e.g. Org Syn Coll Vol 2, 320, 1943).
  • Protected tosylates such as C-2(d)-di-THP, as well as unprotected tosylates such as C-2(d), may be subjected to elimination conditions to form the alkenes, and then (in the former case) de-protected to yield compounds such as C-2(g). See e.g., J Chem Soc C 1115, 1967. (I) Synthesis of Compound C-2(h)
  • Di-THP-C-2(a) may be converted into the desired silyl derivative using conventional procedures such as condensation of the alcohol with the appropriate silyl chloride (see e.g. Can J Chem 53, 2975, 1975) and then deprotected, as exemplified above.
  • Di-THP-2(a) may be converted into the methyl ether with methyl iodide
  • Di-THP-C-2(a) may be converted various ethers using the appropriate bromide and conventional procedures, (as illustrated above with benzyl bromide)(see e.g. Bull Chem Soc Japan 60 1529, 1987) and then deprotected, as exemplified above.
  • Di-THP-C-2(a) may be converted into the diphenylmethyl ether by the method of example 1(K) above, using diphenylmethylbromide in place of benzylbromide, followed by deprotection.
  • Chlorides can be prepared as illustrated above, using, e.g. the procedures of Can J Chem 46, 86, 1968.
  • Di-THP-C-2(a) may be converted into the keto derivative using conventional procedures (see e.g. Tett Lett 3363, 1968) and then deprotected, as exemplified above.
  • esters may be produced from compounds such as di-THP-2(j) by ester exchange using organolithium reagents containing the desired OR-*- group, followed by de-protection, as exemplified in the synthesis of Compound C-2(n) [and C-2(o) and C-2(q), below]. See J Chem Soc, Chem Comm 695, 1986.
  • esters such as di-THP-2(j) may be produced using conventional methods, including the use of aluminum reagents, as exemplified above (see e.g. Tett Lett 4171, 1977), followed by deprotection.
  • Keto compounds may be prepared by conventional methods, including the route identified above. See e.g. Org React 18 1, 1970.
  • Allylic ethers including those in which Rl is methyl or benzyl, for example can be prepared from di-THP-2(j) by the route identified above. See e.g. J Am Chem Soc 107 1777, 1985.
  • the 2,3-dihydro derivative (III) can be synthesized using either of the two following methods.
  • 2,3-dihydro BFA may be used in place of BFA in the syntheses decribed herein.
  • Example C-3 Use of alternative protecting groups Other protecting groups for the hydroxyl groups at carbons 6 and 7 (per
  • Example D-2 Attachment of R 2 0-(CH2CH2 ⁇ ) n -(CH2)m-E to C7 position of
  • R 2 0-(CH2CH2 ⁇ )n-(CH2)m-E compounds may be attached to the C7 position of BFA by conventional methods (See e.g. Corey and Wollenberg, Tet Lett, No. 51, 4701-4704 (1976)) as depicted below:
  • Example D-3 Further syntheses BFA analogs which retain the 7-hydroxyl group may be used (with conventional protection and de-protection as appropriate) in place of BFA to produce analogous compounds containing the R 2 0-(CH2CH2)n-(CH2)m-0- group at position 7. The resulting compounds may also be used in accordance with this invention.
  • Esters
  • Q is a halide (e.g. CI or Br) or hydroxy
  • Q is a halide (e.g. CI or Br) or hydroxy
  • the 4,7-disubstituted compounds may be prepared using at least a two-fold excess of the acid chloride, anhydride or acid.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

L'invention se rapporte à de nouveaux composés rattachés structurellement à la Brefeldine A et utilisés comme agents antiviraux, antifongiques, antiproliférateurs, immunosuppresseurs et de détoxification, ainsi qu'à des compositions pharmaceutiques et des procédés basés sur ces composés.
PCT/US1995/001656 1994-02-09 1995-02-09 Nouveaux inhibiteurs du trafic des proteines WO1995021614A1 (fr)

Applications Claiming Priority (12)

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US19396194A 1994-02-09 1994-02-09
US19382094A 1994-02-09 1994-02-09
US08/193,961 1994-02-09
US08/193,820 1994-02-09
US19896194A 1994-02-18 1994-02-18
US08/198,961 1994-02-18
US20731994A 1994-03-07 1994-03-07
US20749694A 1994-03-07 1994-03-07
US08/207,319 1994-03-07
US08/207,496 1994-03-07
US30831794A 1994-09-19 1994-09-19
US08/308,317 1994-09-19

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996000726A3 (fr) * 1994-06-29 1996-02-15 Us Health Nouveaux derives de brefeldine a et leur utilite dans le traitement du cancer
WO1996040112A1 (fr) * 1995-06-07 1996-12-19 Zymogenetics, Inc. Utilisation de brefeldine a et de ses derives dans le traitement de l'hyperplasie et de troubles apparentes
EP1107789A1 (fr) * 1998-08-24 2001-06-20 The Board Of Trustees Of The Leland Stanford Junior University Compositions et procedes pour proteger des organes, tissus et cellules contre des lesions induites par le systeme immunitaire
CN104592208A (zh) * 2014-12-24 2015-05-06 浙江工业大学 7-n3-布雷菲德菌素a及其1,2,3-三氮唑类衍生物的制备与抗肿瘤应用
CN105566277A (zh) * 2016-01-25 2016-05-11 杭州科兴生物化工有限公司 一种布雷菲德菌素a及其衍生物和应用
CN114886842A (zh) * 2022-05-24 2022-08-12 吴丽莎 一种布雷非德菌素a软膏的制备方法及应用

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Publication number Priority date Publication date Assignee Title
US3535343A (en) * 1966-08-30 1970-10-20 Syntex Corp Process for the preparation of benzoic acid lactones and novel intermediates therefor

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Publication number Priority date Publication date Assignee Title
US3535343A (en) * 1966-08-30 1970-10-20 Syntex Corp Process for the preparation of benzoic acid lactones and novel intermediates therefor

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Title
DIE PHARMAZIE, Volume 47, No. 8, issued August 1992, B. PROSKA et al., "Oxidation of Brefeldin A", pages 582-584. *

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996000726A3 (fr) * 1994-06-29 1996-02-15 Us Health Nouveaux derives de brefeldine a et leur utilite dans le traitement du cancer
US5696154A (en) * 1994-06-29 1997-12-09 The United States Of America As Represented By The Department Of Health And Human Services Brefeldin A derivatives and their utility in the treatment of cancer
US5824674A (en) * 1994-06-29 1998-10-20 The United States Of America As Represented By The Department Of Health And Human Services Brefeldin A derivatives and their utility in the treatment of cancer
WO1996040112A1 (fr) * 1995-06-07 1996-12-19 Zymogenetics, Inc. Utilisation de brefeldine a et de ses derives dans le traitement de l'hyperplasie et de troubles apparentes
EP1107789A1 (fr) * 1998-08-24 2001-06-20 The Board Of Trustees Of The Leland Stanford Junior University Compositions et procedes pour proteger des organes, tissus et cellules contre des lesions induites par le systeme immunitaire
EP1107789A4 (fr) * 1998-08-24 2004-09-01 Univ Leland Stanford Junior Compositions et procedes pour proteger des organes, tissus et cellules contre des lesions induites par le systeme immunitaire
EP1964572A3 (fr) * 1998-08-24 2009-03-04 The Board Of Trustees Of The Leland Stanford Junior University Compositions et procédés de protection d'organes, tissus et cellules contre les dégâts engendrés par le système immunitaire
CN104592208A (zh) * 2014-12-24 2015-05-06 浙江工业大学 7-n3-布雷菲德菌素a及其1,2,3-三氮唑类衍生物的制备与抗肿瘤应用
CN104592208B (zh) * 2014-12-24 2017-04-12 浙江工业大学 7‑n3‑布雷菲德菌素a及其1,2,3‑三氮唑类衍生物的制备与抗肿瘤应用
CN105566277A (zh) * 2016-01-25 2016-05-11 杭州科兴生物化工有限公司 一种布雷菲德菌素a及其衍生物和应用
CN114886842A (zh) * 2022-05-24 2022-08-12 吴丽莎 一种布雷非德菌素a软膏的制备方法及应用

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