EP0757695A1 - Composes substitues par guanidino - Google Patents

Composes substitues par guanidino

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Publication number
EP0757695A1
EP0757695A1 EP95916540A EP95916540A EP0757695A1 EP 0757695 A1 EP0757695 A1 EP 0757695A1 EP 95916540 A EP95916540 A EP 95916540A EP 95916540 A EP95916540 A EP 95916540A EP 0757695 A1 EP0757695 A1 EP 0757695A1
Authority
EP
European Patent Office
Prior art keywords
compound
propylguanidine
dideoxy
hexoside
erythro
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
EP95916540A
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German (de)
English (en)
Inventor
Sumanas Rakhit
Abdelmalik Slassi
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Allelix Biopharmaceuticals Inc
Original Assignee
Allelix Biopharmaceuticals Inc
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Filing date
Publication date
Application filed by Allelix Biopharmaceuticals Inc filed Critical Allelix Biopharmaceuticals Inc
Publication of EP0757695A1 publication Critical patent/EP0757695A1/fr
Ceased legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/02—Acyclic radicals, not substituted by cyclic structures
    • C07H15/04—Acyclic radicals, not substituted by cyclic structures attached to an oxygen atom of the saccharide radical
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C279/00—Derivatives of guanidine, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups
    • C07C279/16—Derivatives of guanidine, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of guanidine groups bound to carbon atoms of rings other than six-membered aromatic rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D211/00—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings
    • C07D211/04—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D211/06—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/02—Acyclic radicals, not substituted by cyclic structures
    • C07H15/04—Acyclic radicals, not substituted by cyclic structures attached to an oxygen atom of the saccharide radical
    • C07H15/08—Polyoxyalkylene derivatives

Definitions

  • the present invention relates to anti-viral guanidino-substituted compounds. More particularly, the invention relates to the use of guanidino-substituted compounds as anti-HTV agents.
  • anti-viral agents are modelled to inhibit viral replication within an infected cell. Viral replication may be prevented by down-regulation or inhibition of a protein required in the viral replication pathway, or by interference with the translocation of proteins and viral nucleic acid within the cell. Effective anti-viral agents specifically target steps within the viral replication pathway thereby inhibiting or hindering viral replication within infected host cells while having a minimal cytotoxic effect on the host.
  • many anti-viral agents are specific inhibitors to virus-specified enzymes and proteins, such as viral DNA and RNA polymerases, virus-specific thymidine kinase and cleavage enzymes for viral capsid protein. Nucleoside analogues, for example, have been developed which target particular enzymes in the viral replication pathway by mimicking a natural substrate of the enzyme.
  • Cytotoxicity is a common obstacle in developing anti-viral agents, particularly at the concentrations required to attain effective antiviral activity, due to a lack of viral specificity.
  • anti-viral agents which are considered to be efficacious, i.e. agents having a high level of viral toxicity and a low level of cytotoxicity. Accordingly, there is a need to develop anti ⁇ viral agents having an effective level of anti-viral activity while exhibiting minimal cytotoxicity.
  • Rj and R 3 are independently -(W)--(R 6 ) b -Z, wherein a and b are independently 0 or 1; W is NH, O or S;
  • R ⁇ is C 1-8 alkylene in which one or two non-adjacent, intervening carbon atoms thereof is optionally replaced with nitrogen or oxygen; and Z is H, OH or -NH-C(NH)-NH 2 ; provided that when b is 0 then Z is H; R 2 is selected from H and OH; and 4 and R 5 are independently C 1-8 alkylene in which one of the carbon atoms thereof is optionally replaced with nitrogen or oxygen.
  • the present invention provides an anti-HIV composition which includes a compound as defined by formula (I), or a salt, hydrate or solvate thereof, in admixture with an acceptable carrier.
  • the present invention provides a method of inhibiting
  • composition comprising a therapeutically effective amount of a compound as defined by formula (I), is administered to a mammal.
  • FIGURE 1 illustrates the chemical process for preparing a guanidino-substituted compound of the present invention
  • FIGURE 2 graphically represents the Tar-binding affinity of compounds in accordance with the present invention
  • FIGURES 3-6 illustrate the chemical processes for preparing specific guanidino- substituted compounds of the present invention.
  • anti-HIV 11 refers to those compounds which inhibit replication of a member of the HIV family of retroviruses as determined using conventional cell culture assays, such as the well- established P24 antigen assay in which the anti-HIV nature of a compound is indicated by inhibition of viral P24 synthesis following treatment of virally infected cells with the given compound, relative to a virally infected, untreated control.
  • a binding assay model indicative of HIV inhibition in cell culture, can be used to determine the anti-HIV nature of a compound.
  • HIV is meant to encompass members of the HIV retroviral family such as HIV-1, HIV-2, SIV-1 and HTLV-1, the various strains thereof, such as the LAV, Ada, Ada-M, NL4-3, JR-FL, HXB2 and IIIB strains of HIV-1, and other viruses related thereto.
  • the present invention provides a guanidino-substituted compound, or a salt, hydrate or solvate thereof, which is generally defined by formula (I):
  • X is selected from C, O, N and S;
  • Ri and R 3 are independently -(W)--(R ⁇ ) b -Z, wherein a and b are independently 0 or 1;
  • Z is H, OH or -NH-C(NH)-NH 2 ; provided that when b is 0 then Z is H; R 2 is selected from H and OH; and R, and R 5 are independently C ⁇ -8 alkylene in which one of the carbon atoms thereof is optionally replaced with nitrogen or oxygen.
  • alkyl, alkylene, alkoxy, alkyleneoxy, alkoxyalkyl, alkylamino and aminoalkyl refer to those groups comprising linear or branched chains having from 1-8 atoms in length.
  • R, and R 3 are independently the group -(W)--(R 6 ) b -Z, wherein a and b are independently 0 or 1; W is NH, O or S; Re is C,. g alkylene in which one or two non-adjacent, intervening carbon atoms thereof is optionally replaced with nitrogen or oxygen; and Z is H, OH or -NH-C(NH)-NH 2 ; provided that when b is 0 then Z is H.
  • W, R ⁇ , Z, a and b are selected such that R, and R 3 independently represent a group selected from H, OH, NH 2 , and linear or branched alkyl, alkoxy, alkoxyalkyl, alkylamino and aminoalkyl.
  • R, and R 3 independently represent a group selected from H, OH, NH 2 , and linear or branched alkyl, alkoxy, alkoxyalkyl, alkylamino and aminoalkyl.
  • the term "intervening" with respect to the alkylene chain of ⁇ means that the first and last carbon atoms of the chain are not to be replaced with nitrogen or oxygen.
  • alkyl, alkylene, alkoxy, alkyleneoxy, alkoxyalkyl, alkylamino and aminoalkyl refer to those groups comprising linear or branched chains having from 1-8 atoms in length.
  • Branched groups preferably comprise branched moieties having from 1-3 carbon atoms, e.g. methyl, ethyl or propyl alkyl branches. More preferably, however, Rj and R 3 are linear containing from 1-6 atoms, i.e.
  • an alkyl group such as methyl, ethyl, propyl, butyl, pentyl or hexyl
  • an alkoxy group such as methoxy, ethoxy, propyloxy, butyloxy, pentyloxy, or hexyloxy
  • an alkoxyalkyl group such as methoxy methyl, ethoxy methyl, methoxyethyl, propyloxy methyl, or butyloxymethyl
  • an alkylamino group such as methylamino, ethylamino, propylamino, butylamino or pentylamino
  • an aminoalkyl group such as aminomethyl, aminoethyl, aminopropyl, aminobutyl or aminopentyl.
  • W, Re, Z, a and b are selected such that R, is the group -O-R ⁇ -H or -O-- NH-C(NH)-NH 2 herein ⁇ s C-. 8 alkylene in which one or two non-adjacent, intervening carbon atoms are optionally replaced with nitrogen or oxygen.
  • R t is methoxy, ethoxy, -O-ethylene- O-ethylene-O-ethyl, or -O-propylene-NH-C(NH)-NH 2 and most preferably methoxy or ethoxy.
  • W, R ⁇ , Z, a and b are selected such that
  • R 3 represents H, OH or the group -O-R 6 -NH-C(NH)-NH 2 wherein ⁇ is as previously defined.
  • R 3 is H, OH or -O-propylene-NH- C(NH)-NH 2 and most preferably R 3 is H.
  • R 4 and R 5 are divalent groups independently selected from linear or branched C 1-8 alkylene in which one of the carbon atoms thereof is optionally replaced with nitrogen or oxygen.
  • R 4 and R 5 are linear C ⁇ . 8 alkylene in which a carbon atom is replaced with oxygen.
  • R 4 is C,. g alkylene in which the carbon atom adjacent to the ring is replaced with oxygen to form an alkyleneoxy group such as methyleneoxy, ethyleneoxy, propyleneoxy, butyleneoxy or pentyleneoxy.
  • R 5 is C ⁇ -8 alkylene in which the carbon atom that is beta to the ring (i.e. the second carbon from the ring) is replaced with oxygen.
  • R 5 is preferably one of methyleneoxy-methylene, ethyleneoxy-methylene, propyleneoxy-methylene, butyleneoxy-methylene, pentyleneoxy-methylene or heptyleneoxy-methylene.
  • R is propyleneoxy and R 5 is propyleneoxy-methylene.
  • X may be any one of a carbon, oxygen, sulfur or nitrogen atom.
  • X is an oxygen atom
  • the substituents R 1 -R 5 represent groups that, combined with the oxygen-containing ring, form a substituted sugar, e.g. a substituted pyranose such as a pyranoside.
  • particular compounds in accordance with formula (I) include: methyl-4,6-di-O-[n-propylguanidine]-2,3-dideoxy-D-erythro-hexoside;
  • Such guanidino-substituted sugar compounds may be made using suitable sugar-based starting materials, such as, for example, a protected glucal.
  • suitable sugar-based starting materials such as, for example, a protected glucal.
  • the glucal is reacted with the appropriate alcohol to yield an enopyranoside substituted at position 1 by the deprotonated alcohol.
  • the enopyranoside is then prepared for substitution at positions 4 and 6 by alkylation at these positions, followed by a series of reactions which introduce the desired guanidino-substituted groups at these positions. Included within these process steps is the hydrogenation of the double-bond between C 2 and C 3 of the enopyranoside to yield a fully saturated hexoside.
  • Figure 1 illustrates the process steps for the synthesis of the compound, methyl-
  • W is NH
  • a protected glucal such as tri-O-acetyl-D-glucal.
  • a protected glucal is reacted with an N-protected alkyl-amine (Y-NH-Pr 1 ) to give the corresponding N-glucosylated compound wherein Y is alkyl, guanidino substituted alkyl, hydroxy-alkyl or alkoxy-alkyl; Pr 1 is a suitable amino protecting group such as Boc or Cbz and Pr 2 is a suitable hydroxyl protecting group such as acetyl.
  • the reaction is carried out in an aprotic solvent such as benzene, toluene or dichloromethane in the presence of a Lewis acid such as SnCl 4 , A1C1 3 , ZnCl or BF 3 in a temperature range of approximately -40 °C and +20 °C.
  • a Lewis acid such as SnCl 4 , A1C1 3 , ZnCl or BF 3 in a temperature range of approximately -40 °C and +20 °C.
  • the amino protecting group Pr 1 is removed with a suitable reagent allowing the N- glucosylated compound then to be substituted at the 4 and 6 positions, as previously described, ie. alkylation followed by a series of reactions which introduce the desired guanidino-substituted groups at these positions.
  • appropriate heterocyclic starting materials can be used to prepare corresponding substituted piperidine-based compounds in which X is nitrogen, cyclohexane-based compounds in which X is carbon and tetiahydrothiopyran-based compounds in which X is sulfur.
  • X is sulfur
  • 5-thio-D- glucose Aldrich catalogue, item no. 85,986-9
  • 1-deoxynojirimycin may be used, the synthesis of which is described in Shankar et al, Tetrahedron Letters, 1993, 34(45):7171.
  • N and S- containing homologues of the oxygen-containing sugar compounds listed above are prepared from these starting compounds using the following steps: 1) protection at the 4 and 6 positions; 2) deoxygenation followed by reduction at positions 2 and 3; 3) oxidation followed by alkylation at position 1 to yield the desired R j substituent; 4) deprotection at positions 4 and 6 followed by alkylation with N— Boc-aminoalkylbromide, hydrolysis of the N-Boc group to obtain free amino, transformation of the free amino to bis-Boc-guanidino and hydrolysis to yield free guanidino.
  • Steps 1) to 4) may also be used to obtain homologues wherein X is carbon from a suitable starting compound such as quebrachitol (Aldrich catalogue, item no. 36,060-0) or inositol.
  • a carbon-carbon double bond is introduced at position 5 prior to the protection step 1) by established techniques such as the classical Wittig reaction or organometallic chemistry.
  • diastereomers of compounds of the present invention may be separated using techniques well- established in the art, for example, silica gel column chromatography, and utilized equally in anti-HIV compositions according to the present invention.
  • Example 2 describes the separation of diastereomers of the compound 1,4,6-tri-O- propylguanidine-2,3-dideoxy-hexoside to its and ⁇ anomers. Moreover, each of these anomers exhibits similar anti-HIV activity as set out in Example 11.
  • Acid addition salts of the present compounds are formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as acetic acid propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like.
  • inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like
  • organic acids such as acetic acid propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fuma
  • the preparation of an acid addition salt comprises admixing a compound of formula (I) with the requisite organic or inorganic acid.
  • aqueous solution of the salt is treated with base, e.g. sodium carbonate or potassium hydroxide, to liberate the free base which is then extracted using an appropriate solvent such as ether.
  • base e.g. sodium carbonate or potassium hydroxide
  • the free base is then separated from the aqueous portion, dried and treated with the appropriate acid to yield the desired alternative salt compound.
  • specttoscopy is used to confirm the identity of a compound including, for example, infra-red, ultra-violet, nuclear magnetic resonance (NMR), electron spin resonance (ESR), and mass specttoscopy (MS).
  • RP-HPLC Reverse phase high-pressure liquid chromatography
  • Fractionation of components in the product mixture using liquid chromatography is generally accomplished by running a linear gradient, e.g. a mobile phase comprising an increasing percentage of organic solvent such as acetonitrile, in an aqueous buffer usually containing a small amount of an ion- pairing agent such as 0.1 % trifluoroacetic acid (TFA), through alkylated silica columns, e.g. Q-, C 8 -, or C 18 - silica.
  • ion- pairing agent such as 0.1 % trifluoroacetic acid (TFA)
  • alkylated silica columns e.g. Q-, C 8 -, or C 18 - silica.
  • methods such as ion- exchange chromatography may be used to purify the product.
  • compositions are useful to prepare anti-HIV compositions, according to an aspect of the invention.
  • These compositions comprise an effective amount of a guanidino-substituted compound in admixture with an acceptable carrier.
  • an effective amount is meant to encompass amounts of the anti-HIV compound sufficient to prevent or cause a reduction in HIV replication.
  • Such compositions have use in preventing growth of HIV, i.e. by inhibiting viral replication, both in vitro as well as in vivo.
  • an anti-HIV compound in accordance with the invention is combined with a carrier suitable for the in vitro application for which it is to be used.
  • a carrier suitable for the in vitro application for which it is to be used.
  • the selected anti-HIV compound will preferably be combined with a carrier such as an aqueous solvent.
  • the compound will require combination with an organic carrier such as DMSO.
  • the compound may be used as a media supplement and is subsequently combined with a growth media appropriate for the cells to be cultured.
  • the concentration of anti-HIV compound will vary depending on the compound itself and on the cell culture to be treated.
  • anti-HIV compositions for in vivo administtation i.e. for treating infected individuals, are also contemplated.
  • Such compositions comprise a therapeutically effective amount of an anti-HIV compound together with a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable means acceptable for use in the pharmaceutical and veterinary arts, i.e. non-toxic and not adversely affecting the anti-viral activity of the compounds of the present invention.
  • terapéuticaally effective amount means an amount of the compound sufficient to cause a reduction in the replication of EfTV in the infected individual without causing adverse effects. Such reduction is most properly revealed by assaying virus titer in serum samples derived from the individual before and after treatment.
  • compositions for in vivo administtation include conventional carriers generally selected for combination with sugar-based drugs such as diluents, excipients and the like.
  • compositions in accordance with the present invention will depend on the administrable form required to treat the infection.
  • the compounds are formulated for administration by injection, either sub-cutaneously or intravenously, and are accordingly provided as aqueous solutions in sterile and pyrogen-free form and optionally buffered or made isotonic.
  • the compounds may be administered in sterile water or, more desirably, in saline or 5% dextrose solution.
  • the compounds herein designated as preferred compounds are substantially water- soluble. Water solubility of these and other compounds of the invention may be enhanced, if desired, by incorporating a solubility enhancer, such as cetyltrimethylammonium bromide or chloride.
  • Lyoprotectants such as mannitol, sucrose or lactose and buffer systems, such as acetate, citrate and phosphate may also be included in the formulation, as may bulking agents such as serum albumin.
  • the compounds of the present invention may be formulated for administtation by routes other than injection.
  • Oral dosage forms such as tablets, capsules and the like, formulated in accordance with standard pharmaceutical practise, may be employed, as well as inhalable aerosol formulations.
  • a pharmaceutical composition appropriate for treatment can readily be established in appropriately controlled trials, and will correspond to an amount of anti-viral compound that affords effective results against HIV replication without causing any harmful or deleterious side effects to the host being treated. In such trials, inhibition of HIV replication can be monitored using the well-established P24 antigen assay. It is anticipated that an effective treatment regimen for HTV- infected patients will involve the systemic administtation of dosage sizes in the range of from 1 ⁇ g to about 10 mg per kg, e.g., between about 0.01 mg/kg to about 5 mg/kg. It will be appreciated, however, that effective dosage sizes will vary according to the route of administtation, the frequency of administtation, and, of course, with the particular individual to be treated.
  • the present anti-HIV compounds may be used as a diagnostic tool.
  • a biological sample including for example, blood, urine or saliva, may be analyzed for the presence of HTV.
  • an aliquot of the biological sample to be analyzed is combined with a culture of cells normally killed by the virus, e.g. HIV-sensitive cells such as HeLa-CD 4 , MT4, CEM-T4, Hut-78, PBMC or PBL cells, or differentiated monocytes/macrophages, to provide a conttol.
  • HIV-sensitive cells such as HeLa-CD 4 , MT4, CEM-T4, Hut-78, PBMC or PBL cells, or differentiated monocytes/macrophages
  • Another aliquot of the biological sample is combined with a culture of the HIV-sensitive cells along with an anti-HIV compound to provide a test sample.
  • the conttol and test samples are incubated under conditions suitable for cell growth, and are subsequently analyzed for growth of the cells using methods well-established in the art. If the biological sample is HIV-infected, the cells of the conttol will be killed by the virus. In contrast, the cells in the test sample will continue to grow, or will at least grow to a greater extent than those of the conttol. The presence of the anti-HIV compound will inhibit, or at least hinder, HIV replication in the test sample thereby allowing at least some growth of the HIV-sensitive cells to continue.
  • methyl-4,6-di-O-acetyl-2,3- dideoxy-o;, ⁇ -hex-2-enopyranoside was prepared as follows. To a stirred solution of tri-O-acetyl-D-glucal (obtained from Aldrich) (6 g, 22.038 mmol) in dry benzene (20 ml) and 2.2 mL of dry methanol, 1.5 ml of boron trifluoride-ether was added dropwise. The reaction mixture was kept under nitrogen for 45 min., and was then diluted with ethyl acetate, washed twice with saturated aqueous sodium bicarbonate and water, and dried over MgSO 4 .
  • the reaction mixture was stirred at room temperature until all starting material was consumed as indicated by TLC (approx. 2 hrs.). The mixture was then diluted with ethyl acetate and washed several times with water. The desired dialkylated intermediate (crude) was obtained following drying of the organic phase over MgSO 4 , and evaporation of remaining solvent. Purification of the product was conducted on silica gel using ethyl acetate: hexane (50:50) as the eluant to provide the title compound (60-70% yield).
  • Methyl-4,6-di-O-[bis-Boc-n-propylguanidine]-2,3-dideoxy-o;, ⁇ -D-erythro- hexoside was then prepared from the product of E) as follows. To a stirred solution of methyl-4,6-di-O-propylamine-2,3-dideoxy-o., ⁇ -D-erythro-hexoside (100 mg, 0.359 mmol) in dry DMF (1.5 ml) was added 1.5 ml of dry triethylamine. Following 15 minutes of stirring at room temperature, a solution of bis-Boc- thiourea (198.5 mg, 0.719 mmol) in 0.5 mL of dry DMF was added dropwise.
  • the reaction mixture was stirred at room temperature under argon atmosphere for 20 hr. The mixture was then diluted with ethyl acetate, quenched with water and washed twice with brine. The organic phase was dried over MgSO 4 and the solvent was evaporated. The crude product was purified on silica gel using ethyl acetate:hexane (40:60) as the eluant to render the product as a white foam (60% yield).
  • aqueous layer was extracted three times with ethyl acetate, and the combined organic layers were washed with brine (2X), dried over magnesium sulfate, filtered and evaporated to dryness to give a yellow oil.
  • the oil was purified by flash chromatography (eluant was ethyl acetate: hexane, 10%, followed by ethyl acetate: hexane, 80%) to yield 4,6-0- benzylidene-X-methyl-D-glucopyranoside as a white solid (mp was 163-164°C; yield was 75%).
  • Step (C) of Example 1 was followed using the 2,3-di-O-benzyl-methyl-o.-D- glucopyranoside product of step (C) above to yield 4,6-di-O-[l'-N-Boc- propylamine]-2,3-di-O-benzyl-methyl-Q!-D-glucopyranoside (55% yield) as a colorless oil.
  • Step (E) of Example 1 was followed using the 4,6-di-O-[l'-N-Boc- propylamine]-2,3-di-O-benzyl-methyl- ⁇ -D-glucopyranoside product of step (D) above to yield 4,6-di-O-[n-propylamine]-2,3-di-O-benzyl-methyl- ⁇ ;-D- glucopyranoside as a viscous oil (85% yield).
  • Step (F) of Example 1 was followed using the free base product of step (E) above to yield 4,6-di-O-[bis-Boc-propylguanidine]-2,3-di-O-benzyl-methyl- ⁇ -D- glucopyranoside (yield was 64%).
  • Step (D) of Example 1 was followed using the product of step (F) above to yield 4,6-di-O-[Bis-Boc-propylguanidine]-methyl-o;-D-glucopyranoside as a white foam (55% yield).
  • Step (G) of Example 1 was followed using the product of step (G) above to yield the title compound.
  • step (A) To a mixture containing the liquid product (3g, 20.3 mmol) of step (A), N,N-dimethylformamide (50 mL) and benzaldehyde dimethyl acetal (15 mL, 101.3 mmol) was added a catalytic amount of p-toluenesulfonic acid (771 mg, 4.05 mmol). The reaction mixute was stirred at room temperature for 24 hours, diluted with ethyl acetate, and quenched with water (100 mL). The aqueous layer was extracted with ethyl acetate (3X). The combined organic layer was washed successively with saturated aqueous NaHNO 3 and brine, and dried over magnesium sulfate. The solvent was evaporated off, and the remaining was purified by chromatography on silica gel (eluant was ethyl acetate: hexane, 50%) to yield a colorless liquid (yield was 75%).
  • step (C) This step corresponded to step (C) as described in Example 1.
  • step (C) To a solution of the product (1 g, 3.06 mmol) of step (C) in THF was added 15 mL of aqueous 1 N HCL dropwise. The reaction mixture was stirred overnight at room temperature and then diluted with ethyl acetate. Solid NaCl (5 g) was added to the mixture. The aqueous layer was extracted with ethyl acetate
  • Ethyl-4,6-di-O-[l'-N-Boc-propylamine]-2,3-dideoxy-D-erythro-hex-2- enopyranoside 3 (lg, 2.046mmol) was dissolved in 15ml of ethyl acetate. To the solution was added 80mg of 10% Pd/C suspended in 5ml of ethyl acetate. The reaction mixture was stirred at room temperature under a balloon of hydrogen for 2hrs, and then filtered through celite. The filtrate was concentrated and passed through a short silica gel column using an ethyl acetate hexane (50:50) eluant. The desired hydrogenated compound was obtained as a colorless syrup (approx. 100%).
  • Ethyl-4,6-di-O-[l '-N-Boc-propylamine]-2,3-dideoxy-D-erythro-hexoside 4 (950mg, 1.936mmol) was dissolved in 9.6ml of 3N HCl in ethyl acetate and stirred at room temperature for 2hrs. The solution was removed under vacuum and the resulting oil was triturated with dry ether followed by evaporation. The crude salt obtained was converted to the corresponding free base by passage through an ion exchange resin column (Amberlite IRA-400 OH) using pure methanol as solvent to leave the free base 5 as a colorless syrup (83.8%).
  • the title compound was prepared as outlined in Figure 5.
  • the synthesis was essentially the same as that described for the compound of example 1 with the exception that in the step A, 2-(2-ethoxy-ethoxy)ethanol was used in place of MeOH to give the intermediate (ethoxy-ethoxy-ethyl)-4,6-di-O-acetyl-2,3-dideoxy- a , ⁇ -D-erythro-hex-2-enopyranoside.
  • the ability of a compound to inhibit formation of the HIV Tat-TAR binding interaction provides evidence of the anti-HIV properties of a compound. This inhibition can be assessed using an electtophoretic gel mobility shift assay.
  • Inhibition of Tat-TAR binding by compounds in accordance with the present invention was examined by conducting a series of binding reactions in which the concentrations of Tat and 32 P-labelled TAR remained constant, while the concenttation of the anti-HIV compound being tested was varied. The products of the binding reaction were then separated using non-denaturing polyacrylamide gel electtophoresis. Bands corresponding to free TAR and the Tat-TAR complex were identified by autoradiography and cut from the gel. The amount of RNA in each band was determined by liquid scintillation counting and data were analyzed to determine the IC 50 value for the anti-HIV compound.
  • the TAR substrate for the Tat-TAR binding experiments having the nucleotide sequence, 5'-GGAGAUCUGAGCCUGGGAGCUCUCUCC-3' (SEQ ID NO:l), was synthesized by the method described in Ogilvie et al , 1988, Proc. Natl. Acad. Sci. USA, 85:5764, the contents of which are incorporated herein by reference.
  • the inhibition assays were carried out in a reaction volume of 20 ⁇ l that contained 10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 1 mM DTT, 1 mM EDTA, 0.5 U/ml RNAsin (Promega), 0.09 mg/ml BSA, 5% (v/v) glycerol, 50 nM TAT (obtained from ABT) and 0.1 nM RNA (2000-5000 cpm).
  • the compound described in Example 1 above was added at concentrations ranging from 0.1 -
  • the IC 50 value was determined using the program Grafit (Leatherbarrow, 1990), and the results of this determination are graphically illustrated in Fig. l.
  • the IC 50 of the compound of example 1 was determined to be 0.051 ⁇ M.
  • Example 11 Tat-TAR Competition Binding of anomers of 1.4.6-tri-O-fn- propylguanidine "
  • Example 10 The binding assay described in detail in Example 10 was also used to determine the affinity of the stereoisomers of example 2 for the TAR substrate. The following data was obtained:
  • the binding assay described in detail in example 10 was used to determine the affinity of the compounds of examples 3-9 for the TAR substtate.
  • the IC 50 value calculated for each compound tested is shown in the following table.

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Abstract

Nouveaux composés substitués par guanidino correspondant à la formule générale (I), ou leur sel, hydrate ou solvate. Dans cette formule, X est choisi entre C, O, N et S; R1 et R3 représentent indépendamment -(W)a-(R6)b-Z, où a et b valent séparément 0 ou 1; W représente NH, O ou S; R6 représente alkylène C1-8 dont un ou deux atomes de carbone intermédiaires et non adjacents sont éventuellement remplacés par azote ou oxygène; et Z représente H, OH ou -NH-C(NH)-NH2, à condition que Z représente H quand b vaut 0; R2 est choisi entre H et OH; et R4 et R5 représentent indépendamment alkylène C1-8 dont un des atomes de carbone est éventuellement remplacé par azote ou oxygène. Des composés conformes à la formule (I) inhibent la réplication du VIH.
EP95916540A 1994-04-28 1995-04-28 Composes substitues par guanidino Ceased EP0757695A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US23412194A 1994-04-28 1994-04-28
US234121 1994-04-28
PCT/CA1995/000250 WO1995029926A1 (fr) 1994-04-28 1995-04-28 Composes substitues par guanidino

Publications (1)

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EP0757695A1 true EP0757695A1 (fr) 1997-02-12

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EP95916540A Ceased EP0757695A1 (fr) 1994-04-28 1995-04-28 Composes substitues par guanidino

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EP (1) EP0757695A1 (fr)
AU (1) AU2301295A (fr)
CA (1) CA2188452C (fr)
WO (1) WO1995029926A1 (fr)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2771092B1 (fr) * 1997-11-18 1999-12-17 Rhone Poulenc Rorer Sa Procede de preparation de derives de la classe des taxoides
US6525182B1 (en) * 2000-01-21 2003-02-25 Regents Of The University Of California Guanidinylation, guanidinoglycosides, uses, and methods of assaying their activity

Family Cites Families (2)

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Publication number Priority date Publication date Assignee Title
CA2032420A1 (fr) * 1989-12-22 1991-06-23 Akira Okuyama Derives de la phenylguanidine
US5627194A (en) * 1993-11-17 1997-05-06 Allelix Biopharmaceuticals Inc. Anti-viral guanidino-substituted compounds

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9529926A1 *

Also Published As

Publication number Publication date
CA2188452A1 (fr) 1995-11-09
CA2188452C (fr) 2000-12-05
WO1995029926A1 (fr) 1995-11-09
AU2301295A (en) 1995-11-29

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