EP0497835A1 - Procede de traitement d'hvi et d'autres virus, et composes utiles a cet effet - Google Patents

Procede de traitement d'hvi et d'autres virus, et composes utiles a cet effet

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Publication number
EP0497835A1
EP0497835A1 EP19900915950 EP90915950A EP0497835A1 EP 0497835 A1 EP0497835 A1 EP 0497835A1 EP 19900915950 EP19900915950 EP 19900915950 EP 90915950 A EP90915950 A EP 90915950A EP 0497835 A1 EP0497835 A1 EP 0497835A1
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EP
European Patent Office
Prior art keywords
amino
ile
methyl
carbonyl
hydroxy
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP19900915950
Other languages
German (de)
English (en)
Inventor
Robert Leroy Heinrikson
Alfredo Guiseppe Tomasselli
Tomi Kim Sawyer
Heinrich Josef Schostarez
Suvit Thaisrivongs
Jackson B. Hester, Jr.
John On-Ting Hui
William Gary Tarpley
Ruth Elizabeth Ten Brink
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.)
Pharmacia and Upjohn Co
Original Assignee
Upjohn Co
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Filing date
Publication date
Application filed by Upjohn Co filed Critical Upjohn Co
Publication of EP0497835A1 publication Critical patent/EP0497835A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/02—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link
    • C07K5/0227—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link containing the (partial) peptide sequence -Phe-His-NH-(X)2-C(=0)-, e.g. Renin-inhibitors with n = 2 - 6; for n > 6 see C07K5/06 - C07K5/10
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/02—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link
    • C07K5/0207—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link containing the structure -NH-(X)4-C(=0), e.g. 'isosters', replacing two amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to a method of inhibiting a retrovirus in a human cell infected with said retrovirus which comprises treating said cell with an effective amount of a retroviral proteinase inhibitory compound of formula I.
  • the present invention also provides novel compounds which are useful in this method.
  • HTV-1 human immunodeficiency virus type I
  • HTV-1 human immunodeficiency virus type I
  • AZT zidovudine
  • U.S. Patent 4,724,232 claims a method of treating humans having acquired immunodeficiency syndrome utilizing 3'-azido-3'-deoxy-thymidine (azidothymidine, AZT).
  • HIV Human immunodeficiency virus
  • infective and non-infective HIV-isolates Sequence analysis of the complete genomes from several infective and non-infective HIV-isolates has shed considerable light on the make-up of the virus and the types of molecules that are essential for its replication and maturation to an infective species (L. Ratner, et al., Nature, 313:277-284 (1985)). HIV exhibits the same gag/pol/env organization seen in other retroviruses (L.
  • Reverse transcriptase is an enzyme unique to retroviruses that catalyzes the conversion of viral RNA into double stranded DNA. Blockage at any point during the transcription process, by AZT or any other aberrant deoxynucleoside triphosphate incapable of elongation, should have dramatic consequences relative to viral replication. Much work on the RT target is in progress based, in large measure, upon the fact that nucleosides like AZT are easily delivered to cells. However, the inefficiency of phosphorylation steps to the triphosphate, and the lack of specificity and consequent toxicity, constitute major drawbacks to use of ACT and similar nucleosides having a blocked, or missing, 3'hydroxyl group.
  • the T4 cell receptor for HTV has also been targeted as an intervention point in AIDS therapy ( R.A. Fisher, et al. , Nature, 331:76-78 (1988); R.E. Hussey, et al., Nature, 331:78-81 (1988); and K.C. Deen, et al., Nature, 331:82- 84 (1988)).
  • the exterior portion of this transmembrane protein, a molecule of 371 amino acids (sCD4) has been expressed in Chinese hamster ovary (CHO) cells and Genentech ( D.H. Smith, et al., Science, 238:1704-1707 (1987)) has had a product in clinical trials since the fall of 1987.
  • CD4 based therapy the molecules can neutralize HTV by interfering with viral attachment to T4, and other cells which express CD4 on their surfaces.
  • a variant on this theme is to attach cell toxins to CD4 for specific binding and delivery to infected cells which display glycoprotein gp-120 on their surfaces ( M.A. Till, et al., Science, 242:1166-1168 (1988); and V.K. Chaudhary, et al., Nature, 335:369-372 (1988)).
  • Another therapeutic target in AIDS involves inhibition of the viral protease (or proteinase) that is essential for processing HIV-fusion polypeptide precursors.
  • the proteolytic maturation of the gag and gag/pol fusion polypeptides (a process indispensable for generation of infective viral particles) has been shown to be mediated by a protease that is, itself, encoded by the pol region of the viral genome (Y. Yoshinaka, et al., Proc. Natl. Acad. Sci. USA, 82:1618-1622 (1985); Y. Yoshinaka, et al., J. Virol., 55:870-873 (1985); Y. Yoshinaka, et al., J. Virol., 57:826- 832 (1986); and K. von der Helm, Proc. Natl. Acad. Sci., USA, 74:911-915 (1977)).
  • protease or proteinase
  • the protease consisting of only 99 amino acids, is among the smallest enzymes known, and its demonstrated homology to aspartyl proteases such as pepsin and renin ( L.H. Pearl and W.R. Taylor, Nature, 329: 351-354 (1987); and I.
  • pepstatin A 100 ⁇ M
  • pepstatin A a general aspartyl proteinase inhibitor
  • European Published Application 0 357 332 discloses the use of renin inhibitors for the treatment of AIDS by inhibition of HTV protease.
  • These inhibitors may have a moiety of the formula R a 2 R 2 b -X-C(O)- at the N-terminus, wherein X is -O-, -S-, -CH-, or -NHCH-, and wherin R a 2 and R 2 b are the same or different and are hydrogen, C 1-4 alkyl, unsubstituted or substituted aryl, and unsubstituted and substituted C 3 ,7 -cycloalkyl.
  • this application does not disclose diol moieties as the transition state insert.
  • European Published Application 0 352 000 discloses retroviral protease binding peptides, having a variety of moieties as the transition state insert, including alcohols and diols, and having such substituents as t-butyloxycaibonyl, benzyloxycarbonyl and R"C(O)-, wherein R" is hydrogen or C 1-18 alkyl, at the N-terminus.
  • European Published Application 0 369 141 discloses specific peptides which are useful for inhibiting retroviral proteases. However, these peptides differ from the peptides of the present invention by having a ⁇ -alanineat the D 9 -position, statine analogs at the transition state insert or N-4-amino-2-methyl-5-pyri midinylmethyl amide at the C- terminus.
  • the present invention particularly provides:
  • C 8 is absent or a divalent moiety of the formula XL 1 , XL 2 , XL 2a , XL 2b or other amino acyl derivative;
  • D 9 is a divalent moiety of the formula XL 3 , XL 2a , XL 2b or other amino acyl derivative;
  • E 10 -F 11 is a divalent moiety of the formula XL 6 , XL 6b , XL 6c , XL 6d , XL 6e , II, III, or IV;
  • G 12 is absent or a divalent moiety of the formula XL 4 , XL 4a or other amino acyl derivative
  • R 4 at each occurrence is the same or different as is a) hydrogen
  • R 11 is -R or -R 2 ;
  • R 12 is -(CH 2 ) n -R 13 ;
  • j is one to three, inclusive
  • n is one or two
  • r is zero to five, inclusive
  • n is independently an integer of zero to five, inclusive; wherein q is an integer of one to five, inclusive; wherein u is an integer of zero to three, inclusive;
  • v is an integer of zero to four, inclusive
  • s is an integer of zero or one so that the sum of u plus v plus s is three or four; wherein t is an integer of zero to three, inclusive;
  • w is an integer of two or three;
  • x is an integer of two to seven, inclusive
  • y is an integer of zero to six, inclusive.
  • z is an integer of zero to six so that the sum of y plus z does not exceed six; wherein aryl is phenyl or naphthyl substituted by zero to three of the following:
  • -Het is a 5- or 6-membered saturated or unsaturated ring containing from one to three heteroatoms selected from the group consisting of nitrogen, oxygen, and sulfur; and including any dicyclic group in which any of the above heterocyclic rings is fused to a benzene ring or another heterocycle and the ring may be connected through a carbon or secondary nitrogen in the ring or an exocyclic nitrogen; and if chemically feasible, the nitrogen and sulfur atoms may be in the oxidized forms; and optionally substituted by zero to three of the following:
  • G 12 is present when both C 8 and D 9 are present and E 10 -F 11 is other than LPA;
  • R 5 is other than C 1 -C 6 alkyl, C 3 -C 7 cycloalkyl or aryl;
  • Z is other than N-4-amino-2-methyl-5-pyrimidinylmethyl-amide
  • X is other than benzyloxycarbonyl or butyloxycarbonyl, when E 10 -F 11 is II or IV;
  • L-Isoleucinamide N 2 -(5-amino-4-hydroxy-2-(1-methylethyl)-1-oxooctyl]-N-(2- pyridinylmethyl)-, trifluoroacetate, (S,S,S)-; or H-LVA-Ile-Amp;
  • Octanamide 5-[(3,3-dimethyl-1-oxobutyl)amino]-4-hydroxy-7-methyl-2-(1- methylethyl)-N-[2-methyl-1-[[(2-pyridinylmethyl)amino]carbonyl]butyl][2S-[1(1R*,2R- *).2R*,4R*,5R*]]- or TBA-LVA-Ile-Amp;
  • Cyclohexanehexanamide ⁇ -[(3,3-dimethyl-1-oxobutyl)amino]- ⁇ -hydroxy- ⁇ -(1- methylethyl)-N-[2-methyl-1-[[(2-pyridinylmethyl)amino]carbonyl]butyl]-, [ ⁇ S-[N(lR*,2R- *), ⁇ R*, ⁇ R*, ⁇ R*]]- or TBA-CVA-Ile-Amp;
  • Cyclohexanehexanamide ⁇ -amino- ⁇ -hydroxy- ⁇ -(1-methylethyI)-N-[[2-methyl-1- [[(2-pyridinylmemyl)]amino]carbonyl]butyI]-,dihydrochloride,[ ⁇ S-[N(1R*,2R*), ⁇ -R * , ⁇ - R*, ⁇ R*]]- or H-CVA-Ile-Amp;
  • Cyclohexanehexanamide ⁇ -(acetylamino)- ⁇ -hydroxy- ⁇ -(1-methylethyl)-N-[2- methyl-1-[[(pyridinylmethyl)amino]carbonyl]butyl], [ ⁇ S-[N(lR*,2R*), ⁇ R*, ⁇ R*, ⁇ R *]]-; or Ac-CVA-Ile-Amp;
  • L-Histidinamide L-phenylalanyl-N-[2-hydroxy-5-methyl-1-(2-methylpropyl)-4- [[[2-methyl-1-[[(2-pyridinylmethyl)amino]carbonyl]butyl]amino]carbonyl]hexyl]-, [1S- (1R*,2R*,4R*(1R*,2R*)]]- or H-Phe-His-LVA-Ile-Amp;
  • L-Valinamide L-valyl-N-[2-hydroxy-5-methyl-1-(2-methylpropyl)-4-[[[2-methyl-1- [[(2-pyridinylmethyl)amino]carbonyl]butyl]amino]carbonyl]hexyl][1S-[1R*,2R*,4R*(1- R*,2R*)]]-, diacetate (salt); or H-Val-Val-LVA-Ile-Amp; or
  • Octanamide 5-[[2-(acetylammo)-3-memyl-1-oxobutyl]amino]-4-hydroxy-7-methyl- 2-(1-methylethyl)-N-[2-methyl-1-[[(2-pyridinylmethyl)amino]carbonyl]butyl]-, [2S- [N(1R*,2R*),2R*,4R*.5R*(R*)]]-, monoacetate (salt); or Ac-Val-LVA-Ile-Amp;
  • L-.alpha.-Asparagine N/u 2/d-[N-[(1,1-dimethyIethoxy)carbonyl]-L-phenylala- nyl]-N-[2-hydroxy-5-methyl-1-(2-methylpropyl)-4-[[2-methyl-1-[[(2-pyridinylme- thyl)amino]carbonyl]butyl]amino]carbonyl]hexyl]-,[1S-[1R*,2R*,4R*(1R*,2R*)]]- monoacetate (salt) or BOC-Phe-Asp-LVA-Ile-Amp;
  • L-Histidinamide N-[(1 , 1-dimethylethoxy)carbonyl]-L-phenylalanyl-N-[3,3- difluoro-2-hydroxy-4-[[2-methyl-1-[[(2-pyridinylmethyl)amino]carbonyl]butyl]amino]-4- oxo-1-(phenylmethyl)butyl]- or CH 3 -C(O)-O-CH(benzyl)-C(O)-His-LVA-Ile-Amp;
  • L-Histidinamide N-[(1,1-dimethylethoxy)carbonyl]-L-phenylalanyl-N-[4-[[[2- [(2,6-diamino-4-pyrimidinyl)amino]ethyl]amino]carbonyl]-2-hydroxy-5-methyl-1-(2- methylpropyl)hexyl]-,[1S-(1R*,2R*,4R*)]- or BOC-Phe-His-LVA-(2,6-diamino -4-pyrime- dinyl)amino-ethylamino;
  • phenyl-HC CH-(CH 2 ) n -O-C(O)-,
  • n O to five, inclusive.
  • amino acyl derivatives any of the naturally occurring amino acids such as: glycine, alanine, valine, leucine, isoleucine, phenylalanine, lysine, proline, tryptophan, methionine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, arginine, ornithine, and histidine, and synthetic derivatives thereof. These compounds may be in the L or D configuration and are well known and readily available to those skilled in the art.
  • the compounds of the present invention are effective and potent inhibitors of HIV protease.
  • the compounds of the present invention have also been found to inhibit HIV protease in cell cultures, as described below. Therefore, the compounds of formula I inhibit retroviral proteinases and thus inhibit the replication of the virus. They are useful for treating patients infected with human immunodeficiency virus (HTV) which results in acquired immunodeficiency syndrome (AIDS) and related diseases.
  • HTV human immunodeficiency virus
  • AIDS acquired immunodeficiency syndrome
  • novel compounds of the present invention have low to moderate renin inhibitory activity but are surprisingly and unexpectedly potent retroviral protease inhibitors.
  • the peptides of the present invention are also useful as novel human retroviral protease inhibitory peptide analogs. Therefore, the peptides of the present invention inhibit retroviral proteases and thus inhibit the replication of the vims. They are useful for treating human patients infected with a human retrovirus, such as human immunodeficiency virus (strains of HIV-1 or HIV-2) or human T-cell leukemia viruses (HTLV-I or HTLV-II) which results in acquired immunodeficiency syndrome (AIDS) and/or related diseases.
  • a human retrovirus such as human immunodeficiency virus (strains of HIV-1 or HIV-2) or human T-cell leukemia viruses (HTLV-I or HTLV-II) which results in acquired immunodeficiency syndrome (AIDS) and/or related diseases.
  • the capsid and replicative enzymes (i.e. protease, reverse transcriptase, integrase) of retroviruses are translated from the viral gag and pol genes as polyproteins that are further processed by the viral protease (PR) to the mature proteins found in the viral capsid and necessary for viral functions and replication. If the PR is absent or nonfunctional, the virus cannot replicate.
  • the retroviral PR such as HIV-1 PR, has been found to be an aspartic protease with active site characteristics similar to those exhibited by the more complex aspartic protease, renin.
  • human retrovirus includes human immunodeficiency virus type I, human immunodeficiency virus type II, or strains thereof, as well as human T cell leukemia virus 1 and 2 (HTLV-1 and HTLV-2) or strains apparent to one skilled in the art, which belong to the same or related viral families and which create similar physiological effects in humans as various human retroviruses.
  • Patients to be treated would be those individuals: 1) infected with one or more strains of a human retrovirus as determined by the presence of either measurable viral antibody or antigen in the serum and 2) in the case of HIV, having either a symptomatic AIDS defining infection such as i) disseminated histoplasmosis, ii) isopsoriasis, iii) bronchial and pulmonary candidiasis including pneumocystic pneumonia iv) non- Hodgkin's lymphoma or v) Kaposi's sarcoma and being less than sixty years old; or having an absolute CD4 lymphocyte count of less than 200/m 3 in the peripheral blood.
  • a symptomatic AIDS such as i) disseminated histoplasmosis, ii) isopsoriasis, iii) bronchial and pulmonary candidiasis including pneumocystic pneumonia iv) non- Hodgkin's lymphoma or
  • HIV human immunodeficiency virus
  • HTLV-III or LAV human immunodeficiency virus
  • AIDS human acquired immunodeficiency disease syndrome
  • HIV-I protease This enzyme, HIV-I protease, has been classified as an aspartyl protease and has a demonstrated homology to other aspartyl proteases such as renin, L.H. Pearl, et al., Nature 329:351 (1987); I. Katoh, et al.. Nature 329:654 (1987). Inhibition of HIV-I protease blocks the replication of HIV and thus is useful in the treatment of human AIDS, E.D. Clerq, J. Med. Chem. 29: 1561 (1986). Inhibitors of HIV-I protease are useful in the treatment of AIDS.
  • Pepstatin A a general inhibitor of aspartyl proteases, has been disclosed as an inhibitor of HIV-I protease, S. Seelmeier, et al, Proc. Natl. Acad. Sci. USA, 85:6612 (1986).
  • Other substrate derived inhibitors containing reduced bond isosteres or statine at the scissle position have also been disclosed, M.L. Moore, et al., Biochem. Biophys, Res. Commun. 159:420 (1989); S. Billich, et al., J. Biol. Chem. 263: 17905 (1988); Sandoz, D.E. 3812-576-A.
  • the peptides of the present invention are useful for treating diseases caused by retroviruses, such as human acquired immunodeficiency disease syndrome (AIDS).
  • AIDS human acquired immunodeficiency disease syndrome
  • the peptides of the present invention are also useful for treating non-human animals infected with a retrovirus, such as cats infected with feline leukemia virus.
  • a retrovirus such as cats infected with feline leukemia virus.
  • viruses that infect cats include, for example, feline infectious peritonitis virus, calicivirus, rabies virus, feline immunodeficiency virus, feline parvovirus (panleukopenia virus), and feline chlamydia.
  • Exact dosages, forms and modes of administration of the peptides of the present invention to non-human animals would be apparent to one of ordinary skill in the art, such as a veterinarian.
  • the compounds of formula I of the present invention are prepared as described in the publications listed in Table I below, all of which are incorporated by reference herein, or are prepared by methods analogous thereto, which are readily known and available to one of ordinary skill in the art of peptide synthesis.
  • the compounds of the present invention can occur in several diastereomeric forms, depending on the configuration around the asymmetric carbon atoms. All such diastereomeric forms are included within the scope of the present invention.
  • the stereochemistry of the amino acids corresponds to that of the naturally occurring amino acids.
  • the present invention provides for compounds of formula I or pharmacologically acceptable salts and/or hydrates thereof.
  • Pharmacologically acceptable salts refers to those salts which would be readily apparent to a manufacturing pharmaceutical chemist to be equivalent to the parent compound in properties such as formulation, stability, patient acceptance and bioavailablility.
  • the compounds of the present invention are useful for treating patients infected with human immunodeficiency virus (HIV) which results in acquired immunodeficiency syndrome (AIDS) and related diseases.
  • HIV human immunodeficiency virus
  • the compounds of formula I are administered by oral, nasal, transdermal and parenteral (including i.m. and i.v.) routes in doses of 1 ⁇ g to 100 mg/kg of body weight.
  • the compounds of this invention into appropriate pharmaceutical dosage forms.
  • the dosage forms include oral formulations, such as tablets or capsules, or parenteral formulations, such as sterile solutions.
  • Solid compositions are prepared by mixing the compounds of this invention with conventional ingredients such as talc, magnesium stearate, dicalcium phosphate, magnesium aluminum silicate, calcium sulfate, starch, lactose, acacia, methyl cellulose, or functionally similar pharmaceutical diluents and carriers.
  • Capsules are prepared by mixing the compounds of this invention with an inert pharmaceutical diluent and placing the mixture into an appropriately sized hard gelatin capsule.
  • Soft gelatin capsules are prepared by machine encapsulation of a slurry of the compounds of this invention with an acceptable inert oil such as vegetable oil or light liquid petrolatum.
  • Syrups are prepared by dissolving the compounds of this invention in an aqueous vehicle and adding sugar, aromatic flavoring agents and preservatives.
  • Elixirs are prepared using a hydroalcoholic vehicle such as ethanol, suitable sweeteners such as sugar or saccharin and an aromatic flavoring agent.
  • Suspensions are prepared with an aqueous vehicle and a suspending agent such as acacia, tragacanth, or methyl cellulose.
  • parenteral solutions are prepared by dissolving the compounds of this invention in water and filter sterilizing the solution before placing in a suitable sealable vial or ampule.
  • Parenteral suspensions are prepared in substantially the same way except a sterile suspension vehicle is used and the compounds of this invention are sterilized with ethylene oxide or suitable gas before it is suspended in the vehicle.
  • Patients to be treated would be those individuals: 1) infected with one or more than one strain of a human immunodeficiency virus as determined by the presence of either measurable viral antibody or antigen in the serum and 2) having either a symptomatic AIDS defining infection such as i) disseminated histoplasmosis, ii) isoporiasis, iii) bronchial and pulmonary candidiasis including pneumocystis pneumonia, iv) non-Hodgkin's lymphoma, or v) Kaposi's sarcoma and being less than sixty years old; or having an absolute CD4 lymphocyte count of less than 200/mm 3 in the peripheral blood.
  • Treatment would consist of maintaining an inhibitory level of the compounds of this invention in the patient at all times and would continue until the occurrence of a second symptomatic AIDS defining infection indicates alternate therapy is needed.
  • the HTV-1 protease has been expressed in E. coli, isolated, characterized and used to determine the inhibitory constants (K i ) of potential inhibitory compounds as follows:
  • the synthetic peptide H-Val-Ser-Gln-Asn-Tyr-Pro-Ile-Val-OH serves as the substrate for the measurement of HTV-1 protease activity.
  • This peptide corresponds to the sequence from residue 128 to 135 in the HIV gag protein. Cleavage of the synthetic peptide, as well as the gag protein, takes place at the Tyr-Pro bond.
  • HTV-1 protease activity is measured at 30°C in 50 mM sodium acetate, pH 5.5, containing 10% glycerol, 5% ethylene glycol, 0.1 % Nonidet P-40 and 2.8 mM substrate in a total volume of 50 ⁇ l.
  • the screening tests are performed with primary human lymphocytes. Thereby, undesired testing of transformed cell lines is avoided in which host cell and virus may have undergone processes of mutual adaptation. Performance of cell culture in serum containing media closely mimics the in vivo situation.
  • the dose of the test compound causing half maximal suppression of virus replication is determined.
  • the screening system is standardized and automated to a high degree.
  • test compound Effects of the test compound on cell proliferation are determined by lymphocyte proliferation assays. Starting with a 100 micromolar solution, the test compound is 10 fold serially diluted. One tenth of the concentration of the test compound causing half maximal inhibition of cellular proliferation is employed for all subsequent testing.
  • Peripheral human lymphocytes are isolated by density gradient centrifugation. After stimulation by mitogen the cells are infected with a standardized preparation of
  • the infected cells are cultured in the presence of the test compound for four days. Individual cultures are established to measure viral replication two, three and four days following infection.
  • Untreated cells and AZT-treated cells are included as controls in parallel with the test compounds under investigation.
  • the amount of viral core protein p24 synthesized and released by the infected cells is determined in the supernatant by capture-ELISA technique on days two, three and four. By comparing with a standard preparation, the amount of protein produced by the virus infected cells will be calibrated.
  • the total amount of viral RNA synthesized by the infected lymphocytes is determined by a special nucleic acid hybridization technique on days two, three and four of culture. By including a standard preparation of HRV-RNA the amount of synthesized RNA will be quantified.
  • test compound shows antiviral effects in the primary screening, all steps of the primary screening will be repeated.
  • viability of HRV-infected cells will be determined in parallel with assays for viral p24 and RNA.
  • concentration dependency of the test compound action will be measured.
  • CV-1 cells were seeded at 2 x 10 -5 cells/well in 24 well Costar dishes and infected 6 to 12 hours later with vVK-1 at 5 PFU/cell (V. Karacostas, et al., "Human Immunodeficiency Virus-Like Particles Produced by a Vaccinia Virus Expression Vector (retrovirus/AIDS/virus assembly/reverse transcriptase, " Proc. Natl. Acad. Sci., USA, in press. 1989).
  • the test compounds were dissolved in DMEM containing 2.5% fetal bovine serum and added to triplicate wells immediately after virus addition.
  • the culture medium was removed, the monolayer washed with 1 ml of PBS and the cells lysed by the addition of 0.1 ml of loading buffer (62.5 mM Tris-Hcl pH 6.8, 2.3 % SDS, 5 % B-mercaptoethanol, 10% glycerol).
  • the cells lysates were collected individually, placed in boiling water for 3 minutes, and then 0.025 ml of each is subjected to electrophoresis on 12% SDS-polyacrylamide gels.
  • the proteins were electroblotted onto nitrocellulose and analyzed by Western blotting.
  • the primary antibodies were sheep anti-Pr24 and sheep anti-Pr17 and the secondary antibody in both cases was alkaline-phosphatase conjugated rabbit-anti sheep IgG (all obtained from Kirkegaard & Perry Laboratories, Gaithersburg, MD).
  • Pr55 to the mature viral structural proteins Pr24 and Pr17 in the above cells infected with the recombinant vaccina virus expressing the HIV-1 gag-pol genes.
  • the HIV-1 like particles released from inhibitor-treated cells contained almost exclusively Pr55 and other gag precursors, but not Pr24.
  • Pentanoic acid 5-[[1-(cyclohexylmethyl)-2-hydroxy-5-methyl-4-[[[2-methyl-1- [[(2-pyridinylmethyl)amino]carbonyl]butyl]amino]carbonyl]hexyl]amino]-4-[(1H-indol-2- ylcarbonyl)amino]-5-oxo-,[1S-[1R*(R*),2R*,4R*(1R*,2R*)]]or1H-indol-2-yl-carbonyl- Glu-CVA-Ile-Amp;
  • L-Asparaginamide 1-(naphthoxy)acetyl-N-[2-hydroxy-5-methyl-1-(2-methyl- propyl)-4-[[[2-methyl-1-[[[2-(N-oxido)pyridinylmethyl]amino]carbonyl]butyl]amino]carb- onyl]hexyl]-N-alpha-methyl-, [1S-[1R*,2R*,4R*(1R*,2R*)]]- or NOA-Asp-CVA-Ile- Amp;
  • Aco is acetyloxy
  • Amp is 2-(aminomethyl) pyridine
  • Amp-NO is (2-pyridylmethyl) amino, pyridine N-oxide
  • Boc is t-butoxycarbonyl
  • BOP is benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophos- phate
  • Bz is benzyl
  • Cbz is benzyloxycarbonyl
  • CcD is the moiety of formula X wherein R 1 is cyclohexyl, R 2 is ⁇ -hydroxy, R 4 is ⁇ -hydroxy and R 3 is ⁇ -CH 2 -cyclohexyl;
  • CCD is the moiety of formula X wherein R 1 is cyclohexyl, R 2 is ⁇ -hydroxy R 3 is ⁇ -CH 2 -cyclohexyl and R 4 is ⁇ -hydroxy;
  • CDCl 3 is deuteriochloroform
  • Celite is a filter aid
  • CVA is Cha ⁇ [CH(OH)CH 2 ]Val of formula X wherein R 1 is cyclohexyl, R 2 is hydrogen, R 3 is ⁇ -isopropyl and R 4 is ⁇ -hydroxy;
  • chpVA is the moiety of formula X wherein R 1 is cycloheptyl, R 2 is hydrogen, and R 3 is ⁇ -isopropyl, and R 4 is ⁇ -hydroxy;
  • CLA is the moiety of formula X wherein R 1 is cyclohexyl, R 2 is hydrogen, R 3 is - ⁇ -isobutyl, and R 4 is ⁇ -hydroxy;
  • CLD is the moiety of formula X wherein R 1 is cyclohexyl, R 2 is ⁇ -hydroxy, R 4 is ⁇ -hydroxy and R 3 is ⁇ -isobutyl;
  • CPD is the moiety of formula X wherein R 1 is cyclohexyl, R 2 is ⁇ -hydroxy, R 4 is ⁇ -hydroxy and R 3 is ⁇ -benzyl;
  • CVD is the moiety of formula X wherein R 1 is cyclohexyl, R 2 is ⁇ -hydroxy, R 3 is ⁇ -isopropyl and R 4 is ⁇ -hydroxy;
  • CVD is the moiety of formula X wherein R 1 is cyclohexyl, R 2 is ⁇ -hydroxy,
  • R 4 is ⁇ -hydroxy, and R 3 is ⁇ -isopropyl
  • DANS is dansyl or 5-dimethylaminonaphthalenesulfonyl
  • DCC is dicyclohexylcarbodiimide
  • DEPC diethylphosphoryl cyanide
  • EtOAc is ethyl acetate
  • g is grams
  • ⁇ -Glu is ⁇ -glutamic acid
  • Gly is glycine
  • N-MeHis is N ⁇ -methyl histidine
  • HOBT is 1-hydroxybenzotriazole
  • HPLC high performance liquid chromatography
  • IVA is isovaleryl
  • LCA is the moiety of formula X wherein R 1 is isopropyl, R 2 is hydrogen, R 3 is - ⁇ -CH 2 -cyclohexyl and R 4 is ⁇ -hydroxy;
  • LFA is the difluoro version of LVA as described more fully in PCT Pub. No. WO86/06379 (6 November 1985), and is the moiety of formula IV wherein R 1 is isopropyl;
  • LLA is the moiety of formula X wherein R 1 is isopropyl, R 2 is hydrogen, R 3 is - ⁇ -isobutyl, and R 4 is ⁇ -hydroxy;
  • LID is the moiety of formula X wherein R, is isopropyl, R 2 is ⁇ -hydroxy, R 4 is ⁇ -hydroxy and R 3 is ⁇ -isobutyl;
  • LLd is the moiety of formula X wherein R t is isopropyl, R 2 is ⁇ -hydroxy, R 4 is ⁇ -hydroxy, and R 3 is ⁇ -isobutyl;
  • LLD is the moiety of formula X wherein R 1 is isopropyl, R 2 is ⁇ -hydroxy, R 4 is ⁇ -hydroxy, and R 3 is ⁇ -isobutyl;
  • LPA is the moiety of formula X wherein R 1 is isopropyl, R 3 is hydrogen, R 3 is - ⁇ -benzyl and R 4 is ⁇ -hydroxy;
  • LVA is Leu ⁇ (CH(OH)CH 2 )Val with the S configuration at C4 (the hydroxyl- bearing carbon atom) of the formula X wherein R 1 is isopropyl, R 2 is hydrogen, R 3 is ⁇ -isopropyl and R 4 is ⁇ -hydroxy;
  • LVD is the diol version of LVA as described more fully in PCT Pub. No. WO87/05302 (11 September 1987) and is the moiety of formula X wherein R 1 is isopropyl, R 2 is ⁇ -hydroxy, R 4 is ⁇ -hydroxy and R 3 is ⁇ -isopropyl;
  • LVDA' is the moiety of formula X wherein R 1 is isopropyl, R 2 is ⁇ -hydroxy, R 4 is ⁇ -hydroxy, and R 3 is ⁇ -isopropyl;
  • Mba is 2S-methylbutylamine
  • Me is methyl
  • ml is milliliter
  • MPLC medium pressure liquid chromatography
  • MS is mass spectroscopy
  • NOA is (1-naphthyloxy)acetyl
  • Ph is phenyl
  • Phe is phenylalanine
  • POA is phenyloxyacetyl
  • PPD is the moiety of formula X wherein R 1 is phenyl, R 2 is ⁇ -hydroxy, R 4 is ⁇ -hydroxy and R 3 is ⁇ -benzyl;
  • Pro is proline
  • 2-Py-Ala is D,L-2-pyridyl-alamine.
  • RIP means a compound having the formula H-Pro-His-Phe-His-Phe-Phe-Val-
  • TBA is t-butylacetyl
  • TBAP is tetra-n-butylammonium phosphate
  • TEA is triethylamine
  • TFA is trifluoroacetic acid
  • THF is tetrahydrofuran
  • TLC is thin layer chromatography
  • TsOH is p-toluenesulfonic acid
  • Tyr is tyrosine
  • (OCH 3 )Tyr is O-methyl tyrosine
  • Val is valine.
  • the wedge-shape line indicates a bond which extends above the plane of the paper relative to the plane of the compound thereon.
  • the dotted line indicates a bond which extends below the plane of the paper relative to the plane of the compound thereon.
  • L-Histidinamide N-[(1,1-dimethylethoxy)carbonyl]-L-phenylalanyl-N-[4- (cyclohexylmethyl)-2-hydroxy-1-(2-methylpropyl)-5-[[2-methyl-1-[[(2-pyridinylmethyl)- amino]carbonyl]butyl]amino]-5-oxopentyl]-, [1S-[1R*,2R*,4S*,5(1R*,2R*)]]-; or Boc- Phe-His-LCA-Ile-Amp;
  • FABMS (found): 685.4382; (78) Hydroxyacetyl-L-histidyl-5S-amino-2R-benzyl-6-cyclohexyl-3R.4R- dihydroxy-hexanoyl-L-isoleucyl-2-pyridylmethylamide or (HO)Ac-His-CPD-Ile-Amp. FAB-MS (found): 734.4248;
  • FAB-M (found): 786.4540; (89)5-Quinolinylhydroxyacetyl-L-histidyl-5S-amino-6-cyclohexyl-4S-hydroxy- 2S-isopropyl-hexanoyl-L-isoleucyl-2-pyridylmethylamide or Qoa(b)-His-CVA-Ile-Amp.
  • FAB-MS (found): 797:
  • reaction is quenched with 1N ammonium hydroxide, diluted with methanol and concentrated under reduced pressure - first under house vacuum and then under lower pressure using a vacuum pump - to give a black colored solid.
  • This material is dissolved in ethyl acetate and washed with brine (3X). The aqueous washes are back-extracted with ethyl acetate and the combined organic extracts are dried (Na 2 SO 4 ), filtered and concentrated to give a black colored solid (a mixture of starting diol, tosylate and desired product).
  • Section I The material of Section I is dissolved in 15 mL of dimethylformamide, treated with 0.32 mL of methyl bromoacetate (Aldrich), cooled to 0°C under a nitrogen atmosphere followed by treatment with 0.15 g of a 60% mineral oil emulsion of sodium hydride.
  • the resulting green colored mixture is stirred at 0°C for 1.5 h, quenched with saturated ammonium chloride, diluted with methanol and then concentrated under reduced pressure - first under house vacuum and then under lower pressure using a vacuum pump.
  • the resulting brown colored solid is suspended in 50 mL of water and extracted with ethyl acetate.
  • aqueous phase is back-washed with ethyl acetate and the combined organic extracts are dried (Na 2 SO 4 ), filtered and concentrated to give a brown colored oil (primarily a mixture of 4-[CH 3 (OCH 2 CH 2 ) 3 O]-1-naphthoxyaceticacid, methyl ester, 1,4-[CH 3 (OCH 2 CH 2 ) 3 O]-naphthalene and CH 3 (OCH 2 CH 2 ) 3 OTs).
  • the methyl ester of Section 2 (263 mg); is dissolved in 3 mL of methanol. Water (1 mL) and IN sodium hydroxide (1 mL) are added and the resulting solution is let stir at room temperature for 2 h. The solution is then poured into 10 mL of 1N sodium hydroxide and extracted 3X with ethyl ether. The combined organic extracts are discarded and the aqueous phase is acidified to pH 5 with aqueous hydrochloric acid and then extracted 3X with methylene chloride. The combined organic extracts are dried (Na 2 SO 4 ), filtered and concentrated to give 187 mg of 4-[CH 3 (OCH 2 CH 2 ) 3 O]-1- naphthoxyacetic acid as a brown colored oil. TLC analysis showed only a spot at the origin using 20% ethyl acetate/chloroform. This material is used without purification in the subsequent coupling experiment.
  • reaction solution is allowed to cool to room temperature and then quenched with saturated ammonium chloride, diluted with methanol and then concentrated under reduced pressure - first under house vacuum and then under lower pressure using a vacuum pump - to give a product containing mixture as a yellow solid.
  • This solid is dissolved in ethyl acetate and washed with 50 mL of water (2X). The aqueous washes are back- washed with ethyl acetate and the combined organic extracts are dried (MgSO 4 ), filtered and concentrated to give an orange colored oil.
  • TLC analysis of this oil indicates a five component mixture comprised of the desired product [5-[CH 3 (OCH 2 CH 2 ) 3 O]-1- naphthoxyaceticacid, methyl ester], an unidentified component, 1 ,5-[CH 3 (OCH 2 CH 2 ) 3 O]- naphthalene, 1,5-[CH 3 O 2 CCH 2 O]-naphthalene and 5-[CH 3 (OCH 2 CH 2 ) 3 O]-1-naphthol.
  • This mixture is chromatographed over 50 g of silica gel (63-200 ⁇ ), eluting with 20% ethyl acetate/chloroform while collecting 6 mL fractions.
  • the methyl ester of Section I (241 mg; 0.63 mmoL) is dissolved in 3 mL of methanol. Water (1 mL) and IN sodium hydroxide (1 mL) are added and the resulting solution is let stir at room temperature for 2 h. The solution is then poured into 10 mL of IN sodium hydroxide and extracted 3X with ethyl ether. The combined organic extracts are discarded and the aqueous phase is acidified to pH 5 with aqueous hydrochloric acid and then extracted 3X with methylene chloride.
  • the solution is transferred with washing with methanol to a 1-necked (24/40) 200 mL round-bottomed flask and then concentrated on a rotary evaporator - first under house vacuum and then under lower pressure using a vacuum pump - to give the crude Boc-Asn-CVA-Ile-Amp.
  • This material is suspended in a solution of 3 mL of methylene chloride and 3 mL of trifluoroacetic acid (Aldrich). The resulting yellow colored mixture is stirred at room temperature for 2 h and then concentrated under reduced pressure.

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Abstract

Procédé d'inhibition d'un retrovirus dans une cellule mammifère contaminée par ledit retrovirus, consistant à traiter ladite cellule à l'aide d'une dose efficace d'un composé de la formule (I): X-C8-D9-E10-F11-G12-Z. L'invention présente également de nouveaux composés utiles dans ce procédé.
EP19900915950 1989-10-27 1990-10-16 Procede de traitement d'hvi et d'autres virus, et composes utiles a cet effet Withdrawn EP0497835A1 (fr)

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US428291 1999-10-27

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JP (1) JPH05501879A (fr)
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US5643878A (en) * 1991-09-12 1997-07-01 Ciba-Geigy Corporation 5-amino-4-hydroxyhexanoic acid derivatives
RU2126794C1 (ru) * 1992-03-11 1999-02-27 Нархекс Лимитед Аминопроизводные оксо- или гидроксизамещенных гидразинов, способ их получения и фармацевтические композиции для ингибирования ретровирусной протеазы
US5888992A (en) * 1992-03-11 1999-03-30 Narhex Limited Polar substituted hydrocarbons
ATE253050T1 (de) 1992-03-11 2003-11-15 Narhex Ltd Aminderivate von oxo- und hydroxy- substituierten kohlenwasserstoffen
US6071895A (en) * 1992-03-11 2000-06-06 Narhex Limited Polar-substituted hydrocarbons
TW217410B (fr) * 1992-04-01 1993-12-11 Ciba Geigy
DE4215874A1 (de) * 1992-05-14 1993-11-18 Bayer Ag Dithiolanylglycinhaltige HIV-Proteaseinhibitoren vom Hydroxyethylenisostertyp
IL108459A0 (en) * 1993-02-05 1994-04-12 Opjohn Company 4-Hydroxy-benzopyran-2-ones and 4-hydroxy-cycloalkyl [B] pyran-2-ones useful for treating infections due to hiv and other retroviruses
IL110898A0 (en) * 1993-09-10 1994-11-28 Narhex Australia Pty Ltd Polar-substituted hydrocarbons
IL129871A (en) 1994-05-06 2003-11-23 Pharmacia & Upjohn Inc Process for preparing 4-phenyl-substituted octanoyl-oxazolidin-2-one intermediates that are useful for preparing pyran-2-ones useful for treating retroviral infections
ES2180600T3 (es) * 1994-10-19 2003-02-16 Novartis Ag Eteres de isosteros substrato de aspartato proteasa antivirales.

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US4613676A (en) * 1983-11-23 1986-09-23 Ciba-Geigy Corporation Substituted 5-amino-4-hydroxyvaleryl derivatives
EP0144290A3 (fr) * 1983-12-01 1987-05-27 Ciba-Geigy Ag Dérivés éthylènediamine substitués
DE3812328A1 (de) * 1988-04-14 1989-10-26 Merck Patent Gmbh Aminosaeurederivate

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Title
See references of WO9106561A1 *

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