WO1999048371A1 - Nouveaux inhibiteurs de vih integrase et traitement du vih fonde sur des combinaisons de medicaments contenant des inhibiteurs d'integrase - Google Patents

Nouveaux inhibiteurs de vih integrase et traitement du vih fonde sur des combinaisons de medicaments contenant des inhibiteurs d'integrase Download PDF

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WO1999048371A1
WO1999048371A1 PCT/US1999/006700 US9906700W WO9948371A1 WO 1999048371 A1 WO1999048371 A1 WO 1999048371A1 US 9906700 W US9906700 W US 9906700W WO 9948371 A1 WO9948371 A1 WO 9948371A1
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acid
hiv
dicaffeoyl
inhibitor
tartaric acid
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PCT/US1999/006700
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W. Edward Robinson, Jr.
Peter J. King
Manfred G. Reinecke
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The Regents Of The University Of California
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Priority to EP99915065A priority Critical patent/EP1063888A1/fr
Priority to AU33668/99A priority patent/AU3366899A/en
Publication of WO1999048371A1 publication Critical patent/WO1999048371A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca

Definitions

  • the present invention concerns the medical area of treatment of viral infections and more particularly includes novel inhibitors of HIV integrase and combination drug therapies including integrase inhibitors.
  • HIV human immunodeficiency virus
  • AIDS acquired immunodeficiency syndrome
  • HIV is a member of a class of infectious agents known as retroviruses.
  • the infectious form of HIV, a virion is a particle that consists of a viral genome composed of RNA that is surrounded by proteins encoded by the genome. Infection occurs when an HIV virion enters a susceptible host cell, such as a T lymphocyte within the bloodstream. At this point, one of the viral proteins that comprised the virion, reverse transcriptase (RT), synthesizes a double-stranded DNA copy of the HIV RNA genome. The resulting HIV DNA enters the cell nucleus as part of a stable complex with other virion proteins.
  • RT reverse transcriptase
  • This complex contains all the necessary molecular apparatus for integration of the HIV DNA into the host cell's nuclear DNA. Integration, wherein the HIV DNA is covalently inserted into the host cell's genomic DNA, is absolutely required for productive HIV infection. It is only after integration that the HIV DNA can serve as the template for the production of HIV proteins and RNA that will comprise progeny virions. Among these viral proteins is the HIV protease, the activity of which is necessary for proper formation of new virions. This process, from viral entry to new virion production, is termed viral replication. Upon release from an infected host cell, the newly produced virions are capable of infecting more, previously uninfected host cells. It is through successive rounds of HIV replication and productive host cell infection that HIV disease spreads throughout numerous host cells and ultimately progresses to AIDS disease.
  • HIV treatment come from the use of combination drug therapies.
  • These therapies consist of the simultaneous administration of multiple drugs, which potently and selectively target different elements of the HIV life cycle to disrupt or forestall productive HIV infection and progression to AIDS.
  • the potency of a drug refers to its capacity to act efficaciously at as low a dose as possible, preferably at levels well below those that result in significant amounts of cell death or other signs of cellular toxicity.
  • Selectivity describes the propensity of the drug to act upon a specific target such as unique kind of viral protein. Both of these attributes are of practical importance in the design of therapeutic regimens as they determine the dose, and thus the frequency, cost, and degree of side effects that may occur with use.
  • synergism refers to greater than additive effects that are observed when drugs are taken together as opposed to separately.
  • combinations of protease inhibitors and RT inhibitors have demonstrated additive to synergistic effects in vitro, which were similar to their in vivo effects.
  • a practical consequence of such synergism is that in some cases the dosages of the drugs used in combination may be reduced while maintaining a desired level of therapeutic effect, thus reducing cost, dosage frequency, or the occurrence of undesirable side-effects, or all of these.
  • one the most potent combinations in current clinical use consists of two different RT inhibitors in conjunction with an HIV protease inhibitor.
  • the RT inhibitors selectively target the activity of HIV reverse transcriptase and serve to reduce the rate at which HIV DNA can be produced from HIV RNA, an event that occurs in the early stages of infection within a given host cell.
  • the protease inhibitor selectively acts on the HIV protease to interfere with the production of viable virions in the latter stages of infection within a given cell.
  • the use of such combination therapies has become more prevalent, and has been facilitated by the increasing variety of antiretroviral drugs that have been approved for use in humans.
  • nucleoside analogue and non- nucleoside analogue HIV RT inhibitors As well as four HIV protease inhibitors approved for use by the US Food and Drug Administration (FDA).
  • FDA US Food and Drug Administration
  • Several more HIV protease inhibitors are likely to be approved within the next year or so.
  • combination drug therapies against HIV has proven to be effective in many patients, the current drug regimens are far from ideal. Adherence to these combination regimes is remarkably difficult in terms of patient compliance, and the drug combinations are quite expensive. Their use has been further hampered because many HIV infected individuals have been on single drug therapies in the past and are currently infected with HIV viruses that are resistant to one or more inhibitors, thereby greatly reducing the effectiveness of the combination drug therapy.
  • Access to a larger variety of antiviral agents mitigates these difficulties to some degree, providing new alternative combinations when a given regimen proves ineffective; furthermore, access to a broader spectrum of selectivity in antiviral agents would greatly increase the number of possible therapeutic combinations.
  • Such a broadened spectrum of selectivity could be achieved if HIV proteins other than RT and the viral protease were the targets of therapeutic drugs.
  • the potential for synergism between drugs with three or more targets should be even greater than is currently found with dual target combinations. Such synergism could lead to greater reductions in dosages with a concomitant reduction in cost and potentially the degree of undesirable side effects.
  • anti-HIV combination therapy Because one of the major limiting factors in anti-HIV combination therapy is the cost of the antiviral agents, such a dosage reduction could substantially increase the number of patients who could affordably be treated with combination therapy regimens. Further, increasing the number of viral targets would decrease the likelihood that viral strains could emerge that are simultaneously resistant to all therapeutic agents.
  • IN cleaves double stranded DNA and facilitates the insertion of the pre-processed HIV DNA into the cleavage site.
  • IN covalently links the HIV DNA to both cleaved ends of the host DNA. The remaining step in integration, filling of small gaps in the DNA sequences that flank the inserted HIV DNA, is probably carried out by host cell DNA repair mechanisms.
  • Candidate therapeutic compounds that selectively target the activities of IN have been widely sought.
  • the major classes of IN inhibitors that have been reported to date include DNA-binding agents, topoisomerase inhibitors, aurintricarboxylic acid and cosalene analogues, caffeic acid phenylethyl ester (CAPE), curcumin, suramin, anthraquinones, and bis-catechols and other hydroxylated aromatic compounds.
  • zidovudine and other nucleoside analogues compounds generally used to target HIV RT, have been reported to inhibit HIV IN in vitro at sufficiently high concentrations.
  • Aurintricarboxylic acid and related compounds also inhibit RT and other phosphoryltransferase enzymes.
  • the present invention includes a group of novel compounds that are demonstrated to potently and selectively inhibit HIV integrase (IN) activity in vitro and to potently inhibit HIV replication in live, cultured cells at non-toxic concentrations.
  • novel, biologically active IN inhibitors are structural analogues of the selective and potent anti-HIV IN compound, L-chicoric acid.
  • the use of IN inhibitors of this class in combination with established anti-HIV therapeutics to form synergistic combinations that inhibit HIV replication in live, cultured cells is demonstrated for the first time.
  • the use of this novel class of IN inhibitors in combination with the established classes of anti-HIV compounds, namely reverse transcriptase (RT) inhibitors and protease inhibitors, to interfere with HIV replication provides a synergistic combination therapy.
  • RT reverse transcriptase
  • Particularly interesting novel compounds include 2,3-di(3,4- dihydroxydihydroxydihydrocinnamoyl)-L-tartaric acid, 2,3-di-(3,4- dihydroxybenzoyl)-L-tartaric acid, 2,3-di-(3,4-dihydroxyphenylacetyl)-L-tartaric acid, 2,3-di-(3,4,5-trihydroxybenzoyl-L-tartaric acid, 2,3-dicaffeoyldiamidopropionic acid, 1,2,-dicaffeoyl-L-glyceric acid, bis,-3,4-dicaffeoyldiamidobenzoic acid, di-3,4- dihydroxybenzylidene succinic acid, di-3,4-dihydrodihydroxybenzylidine succinic acid, 2,3-dicaffeoyl-L-serine, bis-dicaffeoyl-L-isoserine and 1,4-dicaffeoyl-L-lys
  • Fig. la shows a structural diagram of compounds number 1, 2, and 3.
  • Fig. lb shows a structural diagram of compound number 19.
  • Fig. lc shows a structural diagram of compound number 22.
  • Fig. 2a shows a structural diagram of compound number 25.
  • Fig. 2b shows a structural diagram of compound number 26.
  • Fig. 2c shows a structural diagram of compound number 28.
  • Fig. 3a shows a structural diagram of compound number 35.
  • Fig. 3b shows a structural diagram of compound number 36.
  • Fig. 3c shows a structural diagram of compound number 37.
  • Fig. 4a shows a structural diagram of compound number 38.
  • Fig. 4b shows a structural diagram of compound number 39.
  • Fig. 4c shows a structural diagram of compound number 40.
  • Fig. 4d shows a structural diagram of compound number 41.
  • Fig. 5 is a histogram showing the anti-HIV activity of several established anti- HIV therapeutics and L-CCA against 4 different HIV molecular clones.
  • Fig. 6 is a graph of the anti-HIV activity, as measured by RT release, of various drug combination regimens against HIN LAI . Each point is the mean of triplicate infections; error bars indicate standard deviations.
  • Fig. 7 is a graph of the anti-HIV activity, as measured by RT release, of various drug combination regimens against HIV LAI . Each point is the mean of triplicate infections; error bars indicate standard deviations.
  • Fig. 8 is a graph of the anti-HIV activity, as measured by RT release, of various drug combination regimens against the clinically isolated strain HIV R45 . Each point is the mean of triplicate infections; error bars indicate standard deviations.
  • Fig. 9 is a graph of the anti-HIV activity, as measured by RT release, of various drug combination regimens against the clinically isolated strain HIV R19 . Each point is the mean of triplicate infections; error bars indicate standard deviations.
  • Fig 10 is a diagram of Scheme 1 showing the steps in the chemical synthesis of some of the compounds disclosed in the present invention.
  • Fig 11 is a diagram of Scheme 2 showing the steps in the chemical synthesis of some of the compounds disclosed in the present invention.
  • Fig 12 is a diagram of Scheme 3 showing the steps in the chemical synthesis of some of the compounds disclosed in the present invention.
  • Fig 13 is a diagram of Scheme 4 showing the steps in the chemical synthesis of some of the compounds disclosed in the present invention.
  • Fig 14 is a graph of HIV NL4 . 3 passaged in the presence (squares) or absence
  • Fig. 15 is a schematic diagram of the cloning strategy used to analyze mutations in HIV IN.
  • Fig. 16a is a graph showing the relative resistance of 4 molecular clones of
  • Fig. 16b is a graph showing the relative resistance of 4 molecular clones of HIV to the IN inhibitor L-chicoric acid.
  • the present invention generally related to integrase inhibitors having the general structural formula (formula(I)):
  • R, and R 3 are selected from hydrogen, OR 6 pest NR 6 and aralkyl groups, optionally substituted with between one and three substituents selected from hydroxyl, halo, lower alkoxy, lower alkylcarbonyloxy and lower alkoxycarbonyloxy groups;
  • R and R 5 are selected from hydrogen, COOR 7 and CONHR 7 ;
  • R 2 and R 4 are hydrogen or may combine with each other to form a cycloalkyl ring or with R, and R 4 , respectively, to form aromatic rings optionally substituted with from one to three substituents selected from OR 6 and NR 6 groups;
  • R 6 is selected from
  • X is a saturated or unsaturated, acyclic or cyclic, straight or branched, chiral or achiral hydrocarbyl group with from 0 to 10 carbon atoms
  • n is between 0 and 4
  • m is between 0 and 3.
  • R 7 is selected from hydrogen, alkyl and aralkyl groups
  • R 8 is selected from hydrogen, hydroxy, halo, lower alkoxy, lower alkycarbonyloxy and lower alkoxycarbonyloxy or a cyclic carbonate group when the hydroxy groups are on adjacent carbons;
  • R and R 5 are COOR 7 or CONHR 7
  • the groups R,, R 2 and R 3 , R 4 may combine to form an arylidene group, optionally substituted from 1 to 3 substituents selected from hydroxy, halo, lower alkoxy, lower alkycarbonyloxy and lower alkoxycarbonyloxy.
  • a preferred group of compounds of formula (I) is that wherein, R and R 5 are COOR 7 and CONR 7 R ⁇ _; R 2 and R 4 are hydrogens; R, and R 3 are aralkyl or R,, R, and R 3 , R 4 may combine to form an arylidene group, optionally substituted with up to 3 hydroxy, alkylcarbonyloxy or alkoxycarbonyloxy groups or OR 6 or NR 6 ; R 6 is
  • X is saturated or unsaturated hydrocarbyl group with from 0 to 4 carbon atoms
  • R 7 and R are same or different and may be hydrogen, lower alkyl and alkenyl or aralkyl or R 7 and R-, may combine to form a heterocychc ring, optionally substituted with one or more lower alkyl with from 1 to 5 carbon atoms, aralkyl and hydroxy groups
  • R 8 represents up to 3 hydroxy, alkylcarbonyloxy or alkoxycarbonyloxy groups and n is 0. -13-
  • Examples include:
  • R, R 2 , R 4 and R 5 are hydrogens or R 2 and R 4 may combine to form a cycloalkyl ring or with R ] and R 2 may form an aromatic ring substituted with up to 3 OR 6 or NR 5 groups; R, and R 3 are OR 6 or NR 6 , where R 6 , X, Y, R 8 and m are described as earlier and n is 0-4.
  • Examples include:
  • R is COOR 7 and CONR 7 R ⁇ ,; R perpetrat R 4 and R 5 are hydrogens; R 2 and R 3 are OR 6 and NR 6 ; R 6 , X, Y, R 7 , R 8 , Rg and m are as defined earlier and n is 0-4.
  • Examples include:
  • Figs. 1-4 The chemical structures of the compounds are illustrated in Figs. 1-4.
  • Fig. la the D- and meso- isomers of L-chicoric acid are identical in this illustrative format.
  • Pharmacological data about the compounds that constitute the invention can be found in Table 1.
  • Table 1 lists the compounds by name, with a chemical identification number keyed to the drawings, LD 5 , ED 50 , and IC 50 .
  • the first compound in Table 1 L-chicoric acid, a previously described DCTA, is included for reference.
  • LD 5 the 5% lethal dose, is a measure of the toxicity of compound to living, cultured cells. It is the concentration at which cell growth is inhibited by 5%.
  • ED 50 the 50% effective dose
  • IC 50 the 50% inhibitory dose
  • disintegration is a term used to refer to the reverse of integration.
  • the biological role of IN is to catalyze the insertion of viral DNA into the double-stranded, nuclear DNA of a host cell, while leaving flanking free viral 5' ends.
  • IN is a true enzyme, it can also catalyze the reverse reaction, namely, excision of viral-like DNA from a double-stranded DNA substrate where free viral-like 5' ends occur.
  • the use of an appropriately designed DNA substrate allows for IN dependent disintegration to be measured. Not all of the possible enzymatic functions of IN are completely understood; therefore, it is not warranted to state that -16-
  • in vitro measurement of disintegration inhibition is a complete measure of IN inhibition. Nonetheless, the compounds presented herein generally show a strong correlation between in vitro potency as measured by inhibition of disintegration, and biological potency as measured by inhibition of HIV replication in living, cultured cells. Furthermore, for those compounds in which other measures of IN activity (3'- end processing and strand transfer) have been measured there has been a 1 : 1 correlation between inhibition of these steps and disintegration.
  • L-chicoric acid L-chicoric acid
  • Pairwise combinations of L-CCA with individual RT inhibitors or a protease inhibitor were tested in living cell assays against three different molecular clones of HIV.
  • the molecular clones were HIV NL4 _ 3 clone 7 ., which has silent mutations in the gene for IN and shows an increased susceptibility to the RT inhibitor dideoxycytidine (DDC), HIV NL4 . 3 M185V which has a methionine to valine mutation in the gene for RT and shows partial resistance to DDC and another RT inhibitor 2',3'-dideoxyinosine (DDI), and HIV NL4 .
  • L-CCA shows synergistic effects when paired with the RT inhibitor DDC and when paired with the protease inhibitor Nelfinavir, against all three molecular clones of HIV. L-CCA also shows synergism when paired with the RT inhibitor ZDV against two of the three HIV clones.
  • ZDV zidovudine
  • L-CCA L-ch ⁇ cor ⁇ c acid
  • PI protease inhibitor (nelfinavir)
  • VSS Very Strong Synergism
  • Fa x 100 is the percent protection. Values for 20%, 40%, 60%, 80%, and 90% are indicated. f ls the combination index. It was calculated using CalcuSyn for Windows and is one representative experiment from a minimum of three. Each experimental value
  • Fa x 100 is the percent protection
  • Values for 20%, 40%, 60%, 80%, and 90% are indicated.
  • f l the combination index. It was calculated using CalcuSyn for Windows and is one representative experiment from a minimum of three. Each experimental value was determined in triplicate. Values in parenthesis are 1.96 x SD as estimated in the computer program.
  • Fa x 1 00 is the percent protection
  • Ms the combination index. It was calculated using CalcuSyn for Windows and is one representative experiment from a minimum of three. Each experimental value was determined in triplicate. Values in parenthesis are 1 .96 x SD as estimated in the computer program.
  • Figs. 8 and 9 show that similar results were obtained against two clinically isolated HIV strains, HIV R45 and HIV R19 .
  • a triple combination including L-CCA, zidovudine and protease inhibitor had greater anti-HIV replication activity than a dual combination of ZDV -24-
  • the lead compound L-CCA has demonstrable compatibility and additive to synergistic effects with compounds from the established classes of anti-HIV agents.
  • the novel, analogue compounds of L-CCA presented generally show equivalent or improved anti-HIV effects consistent with similar potency and selectivity shown when evaluated individually.
  • the translation of such in vitro synergistic effects to in vivo effects has already occurred with combinations of established anti-HIV compounds.
  • the addition of this new class, as represented by the novel compounds introduced here, of IN inhibitors with demonstrable anti-HIV replication activity provides a new avenue for the pursuit and development of clinically valuable anti-HIV therapeutics.
  • Each novel compound was pursued using the following steps: 1) chemical synthesis, 2) chemical characterization, 3) biological characterization with living cells, including a) determination of LD 5 by cell toxicity assay, b) determination of ED 50 by anti-HIV assay, and c) assessment of selectivity against IN by ED 50 determination against the L-chicoric acid (L-CCA) resistant strain, HIV NL4 . 3clonel . D4 , 4) biochemical characterization, i.e., determination of IC 50 with the disintegration assay, and 5) determination of pharmacological compatibility of anti-HIV activity when used in combination with members of the established classes of anti-HIV therapeutics, i.e., determination of capacity to act synergistically. This entire course of assessment is not presented for all compounds. Representative examples of these steps and relevant data illustrated by figures and tables are presented below.
  • analogues of the DCTA, L-CCA were synthesized. Exemplary synthetic schemes for a few of these compounds are shown in Figs. 1-4 and described below. The length of the side chains, spatial arrangement of the phenolic hydroxyl groups, size and structure of the central molecular core structure, and number of free carboxyl groups were all varied. The effects of these changes were assayed against HIV-1 IN in the disintegration reaction as well as against HIV-1 replication and cell growth in tissue culture.
  • L-(l), D-(2)- and meso-(3) DCTA were synthesized by acylation of the bis(di- phenylmethyl) tartrates (la, 2a and 3a) with the protected caffeoyl chloride (35a) to give the fully-blocked compounds (20, 2b and 3b) from which the phenol and carboxyl blocking groups were sequentially removed via (21, 2c and 3c) (Fig. 10, Scheme 1).
  • a compound with a single carboxyl group, dicaffeoylglyceric acid (19) was prepared by acylation with (35a) and deprotection via 19a (Fig. 11, Scheme 2).
  • Direct acylation of L-tartaric acid with the acid chlorides of the phenol protected acids 22a, 28a and 26a gave 22b, 28b and 26b from which the phenol blocking groups were removed (Fig. 12, Scheme 3).
  • HPLC used a C-18-10 ⁇ m, 250mm x 4.6mm analytical column or a C18-10 ⁇ m, 250 x 22mm preparative column eluted with either methanol-water or acetonitrile-water mixtures containing 1% acetic acid with UV detection at 254 nm.
  • the following general synthetic procedures refer to both Schemes 1-4 and the description of the preparation of the specific compounds that follows. No attempt was made to optimize yields.
  • Procedure C Formation of Diphenylmethyl Esters: A solution of 1.25eq diphenyldiazomethane per COOH group in chloroform was added to the carboxylic acid in MeOH-CHCl 3 .
  • Procedure E Direct Acylation of Alcohol Groups: The alcohol was reacted with a slight excess of the acid chloride without solvent in an oil bath at 130-140 °C for 15 min and the residue chromatographed on silica gel.
  • Procedure F Solution Acylation of Alcohol Groups: The acyl chloride was reacted with the alcohol in anhydrous benzene with pyridine or triethylamine as a catalyst for 2.5hr at room temperature. The reaction mixture was successively washed with IN HC1, saturated NaHCO 3 and water and the organic layer evaporated to give the ester which was purified on a silica gel column.
  • Procedure G Direct Acylation of L-Tartaric Acid: A modification of Scarpati's method (Tetrahedron 4:43-48 (1958)) involves heating L-tartaric acid with an excess of an acyl chloride in an oil bath at 135-160°C for 10-30min followed by hydrolysis of the intermediate anhydride with 80% HOAc on a steam bath for 30 min. The residue from removal of the solvent below 40°C under reduced pressure was partitioned between water and ether and the latter dried and evaporated to give a crude product which was purified by chromatography on silica gel or Sephadex LH-20.
  • L-chicoric acid L-DTCA or L-CCA
  • Dicaffeoyl-D-tartaric acid (D-chicoric acid, D-DCTA) (2).
  • Dicaffeoyl-meso-tartaric acid (meso-chicoric acid, meso-DCTA) (3).
  • meso- Tartaric acid was converted by Procedure C to bis(diphenylmethyl) meso-tartrate
  • Procedure I converted 25a to the acid chloride which reacted with bis(diphenylmethyl) L-tartrate (la) according to Procedure F to give bis(diphenylmethyl) bis(3,4,5-trimethoxycarbonylbenzoyl) L-tartrate (25b), gum: ⁇ NMR (C): 7.62 (s, 4H), 7.30-7.36 (m, 20H), 6.96 (s, 2H), 6.08 (s, 2H), 3.94 (s, 6H), 3.92 (s, 12H); 13 CNMR (C): 164.0, 162.5, 152.4, 151.5, 143.6, 138.4, 138.1, 128.6, 128.54, 128.50, 128.3, 128.2, 127.5, 126.8, 126.5, 122.3, 79.3, 71.6, 56.4, 56.1.
  • Procedure I converted 26a to the acid chloride which reacted by Procedure G to give bis(3,4- dimethoxycarbonylbenzoyl)-L-tartaric acid (26b), gum; ⁇ NMR (C+M): 8.04 (dd, 8.4, 1.9, 2H), 8.07 (bs, 2H), 7.46 (d, 8.4, 2H), 6.00 (s, 2H), 5.78 (bs, 2H), 3.93 (s, 3H), 3.92 (s, 3H); l3 CNMR (C+M): 167.5, 163.5, 152.7, 152.4, 146.3, 142.0, 128.5, 127.4, 124.6, 123.0, 71.8, 55.6 (x2).
  • V2 (21mer): 5'-ACTGCTAGAGATTTTCCACAT-3';
  • Oligonucleotide was gel purified by the manufacturer. Oligonucleotide
  • Tl was labeled at the 5'-end using T4 polynucleotide kinase and [ ⁇ - 32 P] ATP (3000 Ci/mmol, Amersham).
  • the substrate for assaying disintegration activity was prepared by annealing the labeled Tl strand with oligonucleotides T3, V2, and V1/T2.
  • the DNA substrate 0.1 pmol was incubated with 1.5 pmol recombinant IN for 60 minutes at 37°C in a buffer containing a final concentration of 20 mM HEPES pH 7.5, 10 mM DTT, 0.05% Nonidet P-40, and 10 mM MnCl 2 .
  • reaction mixture 1 ⁇ l of inhibitor at various concentrations in solvent or solvent alone was added. The reaction was stopped by the addition of EDTA to a final 18 mM concentration. Reaction products were heated at 90°C for 3 minutes before analysis by electrophoresis on a 15% polyacrylamide gel with 7M urea in Tris-borate-EDTA buffer. All reactions were performed at enzyme excess and reactions were stopped within the linear range of the reaction. Although it has been suggested by one group that the inhibitory effects of bis-catechols are metal ion dependent, all reactions were performed in the presence of MnCl 2 rather than MgCl 2 .
  • HIV NL4 . 3 plasmid (a gift from Dr. P. Krogstad, UCLA, Los Angeles, CA), was transfected in HeLa cells using Lipofectin ® (Gibco/BRL). Excess DNA was removed by washing and cells were co-cultured with H9 cells for 18 hours. The H9 cells were removed and re-cultured in growth medium. When the culture was 100% positive for HIV antigens by indirect immunofluorescence, the virus was inoculated onto H9 cells and incubated at 37°C for several weeks in the presence of 2 ⁇ M L-chicoric acid. When this culture was 100% positive the virus was isolated and one aliquot was passaged in a similar manner in 4 ⁇ M L-chicoric acid. Finally, virus was cultured in the presence of 8 ⁇ M L-chicoric acid and the resultant virus filter-clarified, aliquoted, and stored at -70°C.
  • HIV NL4 . 3 following culture in 8 ⁇ M L-chicoric acid, was tested for resistance to the anti-HIV activity of the compound using a cytopathicity-based first described by Montefiori et al. This assay takes advantage of the lytic nature of T-cell tropic clones of HIV and decreased cell viability in this assay has been shown to correlate well with HIV replication.
  • the fifty-percent effective dose (ED 50 ) of L-chicoric acid against HIV NL4.3 control virus was 400 nM while HIV NL4 . 3 passaged in the presence of 8 ⁇ M L-chicoric acid was completely resistant to the compound ( Figure 14). Cloning and sequencing of virus from the HIV NL4 . 3 passaged in the presence of 8 ⁇ M L- chicoric acid was undertaken to determine the molecular basis for resistance to L- chicoric acid and to isolate a resistant HIV clone.
  • INS oligonucleotide primers
  • INX Core 1, 5'- cagctgtgataaatgtcagcta-3' (nt3721-3741), Core 2: 5'-ccatttgtactgctgtcttaa-3' (nt4122- 4142), INSPF: 5'-gcaatttcaccagtactacagt-3' (nt3962-3983), and INSPR: 5'- gtagggaatgccaaattcctg-3' (nt4016-4036). Manual sequence analysis was confirmed by automated DNA sequencing. -36-
  • Sequencing the integrase genes from both drug-resistant and control HIV NL4 . 3 demonstrated several mutations.
  • Control virus contained two silent mutations at nucleotides 3832 and 4009. These silent mutations are believed to arise from a discrepancy in the published sequences of HIV NL4 . 3 and were likely not a result of passage of HIV in the absence of inhibitor.
  • Drug-resistant HIV NL4 . 3 had the same silent mutations as well as a single G to A transition at nucleotide position 4025 leading to an amino acid change from glycine to serine at amino acid 140.
  • the integrase genes from drug-resistant HIV NL4 . 3 were cloned into the native HIV NL4 . 3 plasmid (pNL4-3). This cloning was accomplished through site- directed mutagenesis introducing several silent mutations immediately upstream and downstream of the integrase gene. These mutations generated two unique restriction sites: an upstream SacII and a downstream Xbal (Fig. 15). Introduction of these mutations allowed the entire integrase gene with only minimal upstream and downstream nucleotides to be digested and "swapped" between drug-resistant and drug-sensitive clones.
  • Amino acid 140 of integrase has not been mutated previously using site- directed mutagenesis. Furthermore, a search of the GenBank database does not indicate any naturally occurring mutations at this site. This amino acid is also highly conserved in integrases from other retroviruses, retrotransposons, and transposable elements of bacteria. Mutation at this site, from the highly conserved glycine to serine, has little effect on HIV replication but completely abrogates the anti-HIV activity of L-chicoric acid.
  • HIV Molecular clones of HIV included wild-type HIV NL4 _ 3 , HIV NL4 . 3 M184V , and HIV NL4 . 3 JF26/A7 . All three clones were a generous gift from P. Krogstad (UCLA, Los Angeles, CA). Viruses were initially transfected in adherent HeLa cells using Lipofectin ® . After 48 hours, H9 cells were added. Following 24 hours of co-culture, non-adherent cells were removed and cultured. Cells were monitored by indirect immunofluorescence and RT release as described below until the culture was 100% infected by HIV-1. Supernatant fluids were collected and clarified of cells by low- speed centrifugation followed by filtration through 0.45 ⁇ m filters.
  • Zidovudine, DDC, and 2',3'-dideoxyinosine (DDI) were purchased from Sigma Chemical Co (St. Louis, MO). All three were reconstituted to ImM stock solutions in deionized water and stored at -20°C until use.
  • Michael Melnick (Agouron Pharmaceuticals, San Diego) provided Nelfinavir.
  • Manfred Reinecke (Texas Christian University, Fort Fort Worth, TX) provided L-chicoric acid. Both were reconstituted in deionized water and stored at -70°C until use. All stocks were diluted in growth medium and filter-clarified before being tested for cell toxicity and anti-HIV activity.
  • Anti-HIV activity of compounds was determined both alone and in combination using a cytopathicity based assay as described above. This assay utilizes Finter's neutral red dye; protection from HIV-induced cell death is highly correlated with HIV-1 antigen synthesis, RT release, and the formation of infectious progeny virions. All drugs were tested at concentrations well below their toxic doses either alone or in combination. The fifty-percent effective dose (ED 50 ) was calculated for triplicate infections. Mean ED 50 (shown in Figure 5) were calculated for each drug against each HIV variant from a minimum of three experiments performed in triplicate.
  • fa is the fraction affected by the dose
  • fu is the unaffected fraction
  • D is the dose of the drug
  • D n ⁇ is the median-effect dose determined to be the x-intercept of the median-effect plot
  • m is the sigmoidicity of the dose-effect curve, determined by the slope of the median-effect plot.
  • the median effect plot is based on the following equation:
  • D is the dose of drug 1 and D 2 is the dose of drug 2 in combination that results in x% inhibition and (DJ, and (D x ) 2 are the doses of drug 1 and drug 2 alone that result in x% inhibition.
  • Tables 4-6 The results of mixed drug analyses for the pairwise combinations of L-CCA plus DDC, L-CCA plus nelfinavir, and L-CCA plus zidovudine are shown in Tables 4-6 respectively.
  • ZDV was purchased from Sigma Chemical Company (St. Louis, MO) and was reconstituted with cold deionized water to a final concentration of 1 mM.
  • Protease inhibitor (PI) AG1350 (Agouron Pharmaceuticals, La Jolla, CA) and reconstituted in 14% ethanol/37.5% DMSO/48.5% H 2 O to a final concentration of 7 mM.
  • AG1350 is slightly (less than 10-fold) less active than the recently FDA-approved PI, Viracept ® .
  • the L-CCA was dissolved in H 2 O to a final concentration of 2.1 mM.
  • HIV R19 and HIV R45 were obtained approximately three weeks after inoculation with 100 ⁇ l of freshly drawn serum onto MT-2 cells (the time required for the MT-2 cells to become infected). Previous work has indicated that such isolates of HIV are predominantly syncytium- inducing, rapid-growing, T-cell-tropic isolates of HIV.
  • Cell toxicity and anti-HIV assays were performed as reported above. Briefly, compounds were diluted 1 : 1 in growth medium, filter sterilized, and further two-fold serially diluted from 1 :8 to 1 : 1280 in triplicate wells of a microtiter plate. To each 50 ⁇ l of diluted drug, 50 ⁇ l of growth medium was added followed by 100 ⁇ l of MT-2 cell suspension (2 x 10 ⁇ cells). Cells were incubated with drug for 48 hours at 37°C, then harvested for cell viability in a neutral red dye assay as described. Similar toxicities were also seen if the cells were incubated for 72 hours prior to harvest.
  • Anti-HIV assays were performed as described above. Based upon cell toxicity data, compounds were diluted in growth medium such that a final 1 :4 dilution of the sample would result in a concentration of sample that inhibited MT-2 cell growth by 5% (5% ⁇ lethal dose, LD 5 ). The compounds were then two-fold serially diluted in triplicate. To each 50 ⁇ l of diluted compound, 50 ⁇ l of HIV LAI was added and the virus-drug mixture was incubated for 1 hr at 37°C. Next, 100 ⁇ l of MT-2 cell suspension (2 x 10 ⁇ cells) was added to each well and cells were incubated for 72 hr at 37°C. Final multiplicity of infection (MOI) was 1-5. Cells were harvested to quantitate cytopathic effect using a neutral red dye assay as described. The antiviral concentration reported is the concentration of drug necessary to protect MT-2 cells -42-
  • ED 50 fifty percent effective dose
  • modified integrase inhibitors disclosed herein show similar synergistic properties. It is expected that the variations in molecular structure will prove advantageous in actual patient trials. For example, replacing ester linkages with more stable amide or even aliphatic linkages significantly improves the effectiveness of modified integrase inhibitors. A version of compound 36 in which the amide bonds are replaced with ester bonds is virtually ineffective

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Abstract

La présente invention concerne un groupe de nouveaux composés qui constituent des inhibiteurs puissants et sélectifs de l'activité de VIH intégrase (IN) in vitro, et des inhibiteurs puissants de la réplication du VIH dans des cellules vivantes de culture à des concentrations non toxiques. Les nouveaux composés renferment de l'acide 2,3 -di(3,4 -dihydroxydidihydroxydihydrocinnamoyl) -L-tartrique, de l'acide 2,3 -di-(3,4 -dihydroxybenzoyl) -L-tartrique, de l'acide 2,3 -di-(3,4 -dihydroxyphénylacétyl) -L-tartrique, de l'acide 2,3 -di(3,4,5 -trihydroxybenzoyl-L-tartrique, de l'acide 2,3 -dicaféyldiamidopropionique, de l'acide 1,2,-dicaféyl-L-glycérique, de l'acide bis,-3,4 -dicaféyldiamidobenzoïque, de l'acide di-3,4 -dihydroxybenzylidène succinique, de l'acide di-3,4 -dihydrodihydroxybenzylidyne succinique, de la 2,3 -dicaféyl-L-sé rine, de la bis-dicaféyl -L-isosérine et de la 1,4 -dicaféyl-L-lysine. Des essais impliquant des inhibiteurs d'intégrase avec la 2',3'-didésoxycytidine, la zidovudine et le nelfinavir (inhibiteur de protéase) ont montré une synergie puissante contre un virus résistant aux inhibiteurs de transcriptase inverse. Les avantages potentiels provenant de l'ajout d'inhibiteurs d'intégrase à des thérapies à combinaison de médicaments sont considérables.
PCT/US1999/006700 1998-03-27 1999-03-26 Nouveaux inhibiteurs de vih integrase et traitement du vih fonde sur des combinaisons de medicaments contenant des inhibiteurs d'integrase WO1999048371A1 (fr)

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AU33668/99A AU3366899A (en) 1998-03-27 1999-03-26 Novel hiv integrase inhibitors and hiv therapy based on drug combinations including integrase inhibitors

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WO2000063152A1 (fr) * 1999-02-22 2000-10-26 The Government Of The United States Of America As Represented By The Secretary, Department Of Health And Human Services Analogues acetyles et correspondants d'acide chicorique en tant qu'inhibiteurs de l'integrase du vih
EP1166782A2 (fr) * 2000-06-22 2002-01-02 Chi-Chiang Yang Compositions pharmaceutiques anti-retrovirales
WO2002002516A2 (fr) * 2000-06-30 2002-01-10 Thomas Jefferson University Inhibiteurs symetriques d'integrase de vih, topoisomerase de mammifere et protease de serine
KR20020005222A (ko) * 2000-06-28 2002-01-17 치-치앙 양 Ｈｉｖ 또는 레트로바이러스에 관련된 질환의 치료 또는예방용 약학적 조성물 및 조합물
WO2002026697A2 (fr) * 2000-09-27 2002-04-04 Pharmacor, Inc. Derives aromatiques a proprietes inhibitrices d'integrase de vih
FR2830251A1 (fr) * 2001-10-01 2003-04-04 Univ Claude Bernard Lyon Procede de synthese de l'acide chicorique
EP1326640A2 (fr) * 2000-09-15 2003-07-16 Virologic, Inc. Therapie antiretrovirale a base d'inhibiteurs de la transcriptase inverse
US6620841B1 (en) 1998-12-25 2003-09-16 Shionogi & Co., Ltd. Aromatic heterocycle compounds having HIV integrase inhibiting activities
EP1671535A1 (fr) 2004-12-16 2006-06-21 Stiftung Caesar Center of Advanced European Studies and Research Procédés de modification des concentrations en composés phénoliques dans des cellules végétales
WO2007089030A1 (fr) * 2006-02-01 2007-08-09 Japan Tobacco Inc. Utilisation d'acide 6-(3-chloro-2-fluorobenzyl)-1-[(2s)-1-hydroxy-3-méthylbutan-2-yl]-7-méthoxy-4-oxo-1,4-dihydroquinoléine-3-carboxylique ou d'un de ses sels pour traiter une infection rétrovirale
WO2008009655A3 (fr) * 2006-07-17 2008-05-29 Univ Muenster Wilhelms Utilisation médicale de dérivés d'acides n-phénylpropénoyl-aminés et de composés apparentés
US7541485B2 (en) 2005-10-13 2009-06-02 Wyeth Methods for preparing glutamic acid derivatives
US7553873B2 (en) 2005-07-11 2009-06-30 Wyeth Glutamate aggrecanase inhibitors
US8884034B2 (en) 2009-07-08 2014-11-11 Dermira (Canada), Inc. TOFA analogs useful in treating dermatological disorders or conditions
US10426780B2 (en) 2010-01-27 2019-10-01 Viiv Healthcare Company Antiviral therapy

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Cited By (29)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6620841B1 (en) 1998-12-25 2003-09-16 Shionogi & Co., Ltd. Aromatic heterocycle compounds having HIV integrase inhibiting activities
US7098201B2 (en) 1998-12-25 2006-08-29 Shionogi & Co., Ltd. Heteroaromatic derivatives having an inhibitory activity against HIV integrase
US6645956B1 (en) 1998-12-25 2003-11-11 Shionogi & Co., Ltd. Heteroaromatic derivatives having an inhibitory activity against HIV integrase
WO2000063152A1 (fr) * 1999-02-22 2000-10-26 The Government Of The United States Of America As Represented By The Secretary, Department Of Health And Human Services Analogues acetyles et correspondants d'acide chicorique en tant qu'inhibiteurs de l'integrase du vih
EP1166782A2 (fr) * 2000-06-22 2002-01-02 Chi-Chiang Yang Compositions pharmaceutiques anti-retrovirales
EP1166782A3 (fr) * 2000-06-22 2002-01-09 Chi-Chiang Yang Compositions pharmaceutiques anti-retrovirales
KR20020005222A (ko) * 2000-06-28 2002-01-17 치-치앙 양 Ｈｉｖ 또는 레트로바이러스에 관련된 질환의 치료 또는예방용 약학적 조성물 및 조합물
WO2002002516A2 (fr) * 2000-06-30 2002-01-10 Thomas Jefferson University Inhibiteurs symetriques d'integrase de vih, topoisomerase de mammifere et protease de serine
WO2002002516A3 (fr) * 2000-06-30 2002-10-24 Univ Jefferson Inhibiteurs symetriques d'integrase de vih, topoisomerase de mammifere et protease de serine
EP1326640A4 (fr) * 2000-09-15 2004-12-15 Virologic Inc Therapie antiretrovirale a base d'inhibiteurs de la transcriptase inverse
EP1326640A2 (fr) * 2000-09-15 2003-07-16 Virologic, Inc. Therapie antiretrovirale a base d'inhibiteurs de la transcriptase inverse
US6528655B1 (en) 2000-09-27 2003-03-04 Pharmacor, Inc. Aromatic derivatives with HIV integrase inhibitory properties
WO2002026697A3 (fr) * 2000-09-27 2002-05-16 Pharmacor Inc Derives aromatiques a proprietes inhibitrices d'integrase de vih
WO2002026697A2 (fr) * 2000-09-27 2002-04-04 Pharmacor, Inc. Derives aromatiques a proprietes inhibitrices d'integrase de vih
FR2830251A1 (fr) * 2001-10-01 2003-04-04 Univ Claude Bernard Lyon Procede de synthese de l'acide chicorique
WO2003029183A1 (fr) * 2001-10-01 2003-04-10 Universite De Bordeaux I Procede de synthese de l'acide chicorique
EP1671535A1 (fr) 2004-12-16 2006-06-21 Stiftung Caesar Center of Advanced European Studies and Research Procédés de modification des concentrations en composés phénoliques dans des cellules végétales
US7553873B2 (en) 2005-07-11 2009-06-30 Wyeth Glutamate aggrecanase inhibitors
US7998965B2 (en) 2005-07-11 2011-08-16 Wyeth Llc Glutamate aggrecanase inhibitors
US7541485B2 (en) 2005-10-13 2009-06-02 Wyeth Methods for preparing glutamic acid derivatives
JP2011037857A (ja) * 2006-02-01 2011-02-24 Japan Tobacco Inc レトロウイルス感染症の治療のための、６−（３−クロロ−２−フルオロベンジル）−１−［（２ｓ）−１−ヒドロキシ−３−メチルブタン−２−イル］−７−メトキシ−４−オキソ−１，４−ジヒドロキノリン−３−カルボン酸またはその塩の使用
JP2009525261A (ja) * 2006-02-01 2009-07-09 日本たばこ産業株式会社 レトロウイルス感染症の治療のための、６−（３−クロロ−２−フルオロベンジル）−１−［（２ｓ）−１−ヒドロキシ−３−メチルブタン−２−イル］−７−メトキシ−４−オキソ−１，４−ジヒドロキノリン−３−カルボン酸またはその塩の使用
WO2007089030A1 (fr) * 2006-02-01 2007-08-09 Japan Tobacco Inc. Utilisation d'acide 6-(3-chloro-2-fluorobenzyl)-1-[(2s)-1-hydroxy-3-méthylbutan-2-yl]-7-méthoxy-4-oxo-1,4-dihydroquinoléine-3-carboxylique ou d'un de ses sels pour traiter une infection rétrovirale
WO2008009655A3 (fr) * 2006-07-17 2008-05-29 Univ Muenster Wilhelms Utilisation médicale de dérivés d'acides n-phénylpropénoyl-aminés et de composés apparentés
US8884034B2 (en) 2009-07-08 2014-11-11 Dermira (Canada), Inc. TOFA analogs useful in treating dermatological disorders or conditions
US9434718B2 (en) 2009-07-08 2016-09-06 Dermira (Canada), Inc. TOFA analogs useful in treating dermatological disorders or conditions
US9782382B2 (en) 2009-07-08 2017-10-10 Dermira (Canada), Inc. TOFA analogs useful in treating dermatological disorders or conditions
US10426780B2 (en) 2010-01-27 2019-10-01 Viiv Healthcare Company Antiviral therapy
US11234985B2 (en) 2010-01-27 2022-02-01 Viiv Healthcare Company Antiviral therapy

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