US20060154904A1 - Betulinol derivatives as anti-HIV agents - Google Patents

Betulinol derivatives as anti-HIV agents Download PDF

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US20060154904A1
US20060154904A1 US11/224,430 US22443005A US2006154904A1 US 20060154904 A1 US20060154904 A1 US 20060154904A1 US 22443005 A US22443005 A US 22443005A US 2006154904 A1 US2006154904 A1 US 2006154904A1
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Brij Saxena
Premila Rathnam
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Cornell Research Foundation Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • 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
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders

Definitions

  • the present invention relates generally to methods of treating HIV-1 infection in a subject and methods of inhibiting HIV-1 activity in a cell using betulinol derivative compounds.
  • HIV Human Immunodeficiency Virus
  • RT reverse transcriptase
  • AZT AZT (3′-azido-3′-deoxythymidine)
  • protease inhibitors fusion inhibitors.
  • Common HIV drug therapy includes a cocktail drug regiment, which may utilize, for example, nucleoside analogs like AZT, 2′,3′-dideoxyinosine, and 2′,3′-dideoxycytidine. These drugs act through the inhibition of the HIV reverse transcriptase activity and/or by a mechanism of oligonucleotide chain termination.
  • Betulin, or betulinol is one of the more plentiful triterpenes, constituting up to twenty-four percent of the outer bark of the white birch ( Betula alba ) and as much as thirty-five percent of the outer bark and about five percent of the inner bark of the Manchurian white birch ( Betula platyphylla ) (Hirota et al., J.S.C.I. Japan 47:922 (1944)). Betulin also occurs in a free state in the bark of yellow and black birch (Steiner, Mikrochemie, Molisch - Festschrift, p.
  • Birch tree cortex-extracted betulinol was first mentioned as an antiseptic in 1899. Subsequently, compounds singled out from extracts of Hyptis emory and Alnus oregonu, identified as pentacyclic styrenes and their derivatives, were shown to inhibit carcinosarcoma growth (Sheth et al., J. Pharm. Sci. 61:1819 (1972); Sheth et al., J. Pharm. Sci. 62:139-140 (1973)). It has been suggested that betulinic acid is the main anti-tumor agent in the mixture of terpenoids (Tomas et al., Planta Medicina 54:266-267 (1988); Ahmat et al., J.
  • Betulinol (lup-20(29)-ene-3.beta., 28-diol) is commercially available (e.g., Sigma Chemical Co., St. Louis, Mo.) and is described for example, in “Merck 1212, ” The Merck Index, 11th ed. (1989), and Simonsen et al., The Terpenes Vol. TV, Cambridge U. Press, pp. 187-328 (1957).
  • Betulinol has been shown to have anti-viral activity, including anti-herpes virus activity (U.S. Pat. No. 5,750,578 to Carlson et al.) and anti-HIV activity (U.S. Pat. No. 6,172,110 to Lee et al.; Sun et al., J. Med. Chem. 41:4648-4657 (1998)). Certain betulinol derivatives have also been investigated with regard to potential for anti-viral activity.
  • Betulonic acid and derivatives thereof (Hashimoto et al., Bioorg. Med. Chem. 5:2133-2143 (1997); Sun et al., J. Med. Chem. 41:4648-4657 (1998)), betulinic acid and derivatives thereof, dihydrobetulinic acid and derivatives thereof (Hashimoto et al., Bioorg. Med. Chem. 5:2133-2143 (1997); Sun et al., J. Med. Chem. 45:4271-4275 (2002); Kashiwada et al., Bioorg. Med. Chem. Lett. 11:183-185 (2001); Kashiwada et al., J. Med. Chem.
  • Betulin diacetate and betulonic acid have been shown to exhibit a low therapeutic index (Sun et al., J. Med. Chem. 41:4648-4657 (1998)).
  • certain betulinic acid derivatives such as betulonic acid, have been found to be cytotoxic, interfering with the proliferation of cells (Hashimoto et al., Bioorg. Med. Chem. 5:2133-2143 (1997)).
  • no current anti-HIV agent with the exception of ⁇ -interferon, has any effect on release of virus from a chronically infected cell. Thus, the search for new anti-HIV compounds remains timely and important.
  • Betulinol derivatives in general, and betulonic acid in particular, are soluble in a number or organic solvents such as ethanol and DMSO.
  • betulonic acid and the known betulinol derivatives are generally insoluble in aqueous environment or other pharmaceutically acceptable solvents.
  • Good solubility in an aqueous environment is an important property for a pharmaceutical agent. Absent this property, administration of the pharmaceutical agent to mammals can be difficult and biological activity in such mammals (including humans) may be impeded or entirely absent. Due to their limited solubility in aqueous solutions, the use of terpenoids such as betulinol and it derivatives as pharmaceuticals has been limited. To be effective as a pharmaceutical agent, especially for oral ingestion, water soluble betulinol derivatives would be desirable.
  • the present invention is directed to overcoming these and other deficiencies in the art.
  • One aspect of the present invention relates to a method of treating HIV-1 infection in a subject. This method involves administering to a subject with HIV-1 infection a therapeutically effective amount of a compound having the formula where
  • R 1 is selected from the group consisting of —CH 3 , ⁇ O, —OH, —OCH 3 , —OC(O)CH 3 , —NNH-2,4-Dinitrophenyl Hydrazine (“DNP”), and ⁇ S and
  • R 3 is selected from the group consisting of H and C 1 -C 5 alkyl
  • n is an integer from 1 to 12;
  • Z is H or a protective group
  • Another aspect of the present invention relates to a method of treating HIV-1 infection in a subject.
  • This method involves administering to a subject with HIV-1 infection a therapeutically effective amount of a compound having the formula where
  • R 1 is selected from the group consisting of —CH 3 , ⁇ O, —OH, —OCH 3 , —OC(O)CH 3 , —NNH-2,4-DNP, and ⁇ S and
  • n is an integer from 1 to 12;
  • Z is H or a protective group
  • a further aspect of the present invention relates to a method of treating HIV-1 infection in a subject. This method involves administering to a subject with HIV-1 infection a therapeutically effective amount of a compound having the formula where
  • Y 1 and Y 2 are independently selected from the group consisting of —CH 3 , ⁇ O, —OH, —OCH 3 , —OC(O)CH 3 , —NNH-2,4-DNP, and ⁇ S;
  • R 3 is selected from the group consisting of H and C 1 -C 5 alkyl
  • Z is H or a protective group
  • n is an integer from 1 to 12,
  • Yet another aspect of the present invention relates to a method of treating HIV-1 infection in a subject.
  • This method involves administering to a subject with HIV-1 infection a therapeutically effective amount of a compound having the formula where
  • Y 1 and Y 2 are independently selected from the group consisting of CH 3 , ⁇ O, —OH, —OCH 3 , —OC(O)CH 3 , —NNH-2,4-DNP, and ⁇ S;
  • Z is H or a protective group
  • n is an integer from 1 to 12,
  • Yet a further aspect of the present invention relates to a method of treating HIV-1 infection in a subject.
  • This method involves administering to a subject with HIV-1 infection a therapeutically effective amount of a compound having the formula where
  • Y 1 , Y 2 , Y 3 , and Y 4 are independently selected from the group consisting of —CH 3 , ⁇ O, —OH, —OCH 3 , —OC(O)CH 3 , —NNH-2,4-DNP, and ⁇ S;
  • R 3 is selected from the group consisting of H and C 1 -C 5 alkyl
  • n is an integer from 1 to 12;
  • Z is H or a protective group
  • Still another aspect of the present invention relates to a method of treating HIV-1 infection in a subject.
  • This method involves administering to a subject with HIV-1 infection a therapeutically effective amount of a compound having the formula where
  • Y 1 , Y 2 , Y 3 , and Y 4 are independently selected from the group consisting of —CH 3 , ⁇ O, —OH, —OCH 3 , —OC(O)CH 3 , —NNH-2,4-DNP, and ⁇ S;
  • n is an integer from 1 to 12;
  • Z is H or a protective group
  • Still a further aspect of the present invention relates to a method of treating HIV-1 infection in a subject.
  • This method involves administering to a subject with HIV-1 infection a therapeutically effective amount of a compound having the formula wherein
  • BA is a compound having the formula: wherein
  • Y is selected from the group consisting of —CH 3 , ⁇ O, —OH, —OCH 3 , —OC(O)CH 3 , —NNH-2,4-DNP, and ⁇ S;
  • Q is BA, a leaving group, or H
  • R 3 is H or C 1 -C 5 alkyl
  • n is an integer from 1 to 12;
  • n 1 to 6
  • Another aspect of the present invention relates to a method of treating HIV-1 infection in a subject.
  • This method involves administering to a subject with HIV-1 infection a therapeutically effective amount of a compound having the formula where
  • BA is a compound having the formula: where
  • Y is selected from the group consisting of —CH 3 , ⁇ O, —OH, —OCH 3 , —OC(O)CH 3 , —NNH-2,4-DNP, and ⁇ S;
  • Q is BA, a leaving group, or H
  • n is an integer from 1 to 12;
  • n 1 to 6
  • a further aspect of the present invention relates to a method of inhibiting HIV-1 activity in a cell. This method involves providing a cell infected with HIV-1 and contacting the cell with a compound having the formula where
  • R 1 is selected from the group consisting of —CH 3 , ⁇ O, —OH, —OCH 3 , —OC(O)CH 3 , —NNH-2,4-DNP, and ⁇ S and
  • R 3 is selected from the group consisting of H and C 1 -C 5 alkyl
  • n is an integer from 1 to 12;
  • Z is H or a protective group
  • Yet another aspect of the present invention relates to a method of inhibiting HIV-1 activity in a cell. This method involves providing a cell infected with HIV-1 and contacting the cell with a compound having the formula where
  • R 1 is selected from the group consisting of —CH 3 , ⁇ O, —H, OCH 3 , —OC(O)CH 3 , —NNH-2,4-DNP, and ⁇ S and
  • n is an integer from 1 to 12;
  • Z is H or a protective group
  • Yet a further aspect of the present relates to a method of inhibiting HIV-1 activity in a cell.
  • This method involves providing a cell infected with HIV-1 and contacting the cell with a compound having the formula where
  • Y 1 and Y 2 are independently selected from the group consisting of —CH 3 , ⁇ O, —OH, —OCH 3 , —OC(O)CH 3 , —NNH-2,4-DNP, and ⁇ S;
  • R 3 is selected from the group consisting of H and C 1 -C 5 alkyl
  • Z is H or a protective group
  • n is an integer from 1 to 12,
  • Still another aspect of the present invention relates to a method of inhibiting HIV-1 activity in a cell. This method involves providing a cell infected with HIV-1 and contacting the cell with a compound having the formula where
  • Y 1 and Y 2 are independently selected from the group consisting of —CH 3 , ⁇ O, —OH, —OCH 3 , OC(O)CH 3 , —NNH-2,4-DNP, and ⁇ S;
  • Z is H or a protective group
  • n is an integer from 1 to 12,
  • Another aspect of the present invention relates to a method of inhibiting HIV-1 activity in a cell. This method involves providing a cell infected with HIV-1 and contacting the cell with a compound having the formula where
  • Y 1 , Y 2 , Y 3 , and Y 4 are independently selected from the group consisting of —CH 3 , ⁇ O, —OH, —OCH 3 , —OC(O)CH 3 , —NNH-2,4-DNP, and ⁇ S;
  • R 3 is selected from the group consisting of H and C 1 -C 5 alkyl
  • n is an integer from 1 to 12;
  • Z is H or a protective group
  • a further aspect of the present invention relates to a method of inhibiting HIV-1 activity in a cell. This method involves providing a cell infected with HIV-1 and contacting the cell with a compound having the formula where
  • Y 1 , Y 2 , Y 3 , and Y 4 are independently selected from the group consisting of —CH 3 , ⁇ O, —OH, —OCH 3 , —OC(O)CH 3 , —NNH-2,4-DNP, and ⁇ S;
  • n is an integer from 1 to 12;
  • Z is H or a protective group
  • Yet another aspect of the present invention relates to a method of inhibiting HIV-1 activity in a cell.
  • This method involves providing a cell infected with HIV-1 and contacting the cell with a compound having the formula wherein
  • BA is a compound having the formula: wherein
  • Y is selected from the group consisting of —CH 3 , ⁇ O, —OH, —OCH 3 , —OC(O)CH 3 , —NNH-2,4-DNP, and ⁇ S;
  • Q is BA, a leaving group, or H
  • R 3 is H or C 1 -C 5 alkyl
  • n is an integer from 1 to 12;
  • n 1 to 6
  • Yet a further aspect of the present invention relates to a method of inhibiting HIV-1 activity in a cell.
  • This method involves providing a cell infected with HIV-1 and contacting the cell with a compound having the formula where
  • BA is a compound having the formula: where
  • Y is selected from the group consisting of —CH 3 , ⁇ O, —OH, —OCH 3 , —OC(O)CH 3 , —NNH-2,4-DNP, and ⁇ S;
  • Q is BA, a leaving group, or H
  • n is an integer from 1 to 12;
  • n 1 to 6
  • Still another aspect of the present invention relates to a method of treating and/or inhibiting HIV-1 infection in a human.
  • This method involves administering to a human infected with HIV-1 a therapeutically effective amount of a compound having the formula where
  • n is an integer from 1 to 12 and
  • Z is H or a protective group
  • Still a further aspect of the present invention relates to a method of treating and/or inhibiting HIV-1 infection in a human.
  • This method involves administering to a human infected with HIV-1 a therapeutically effective amount of a compound having the formula where
  • Z is H or a protective group
  • Another aspect of the present invention relates to a method of treating and/or inhibiting HIV-1 infection in a subject.
  • This method involves administering to a subject infected with HIV-1 a therapeutically effective amount of a compound having the formula: where
  • BA is a compound having the formula: where
  • Y is selected from the group consisting of —CH 3 , ⁇ O, —OH, —OCH 3 , —OC(O)CH 3 , —NNH-2,4-dinitrophenyl hydrazine, and ⁇ S;
  • X is selected from the group consisting of
  • each R 4 is independently selected from the group consisting of H, CH 3 , CH 2 —CH 3 , NH 2 and OH;
  • Z is H, a protective group, or BA
  • n is an integer from 1 to 8;
  • n is an integer from 1 to 6;
  • a further aspect of the present invention relates to a method of treating HIV-1 infection in a subject. This method involves administering to a subject infected with HIV-1 a therapeutically effective amount of a compound having the formula: where
  • W is H, OX, or CH 2 —OX
  • each X is independently H, a sugar, or BA, and wherein at least 1 X is BA;
  • BA is a compound having the formula: where
  • Y is selected from the group consisting of —CH 3 , ⁇ O, —OH, —OCH 3 , —OC(O)CH 3 , —NNH-2,4-dinitrophenyl hydrazine, and ⁇ S; and
  • Yet another aspect of the present invention relates to a method of treating HIV-1 infection in a human.
  • This method involves administering to a human infected with HIV-1 a therapeutically effective amount of a compound having the formula: where
  • W is H, OX, or CH 2 —OX
  • each X is independently H, a sugar, or BA, and wherein at least 1 X is BA;
  • BA is a compound having the formula: where
  • Y is selected from the group consisting of —CH 3 , ⁇ O, —OH, OCH 3 , —OC(O)CH 3 , —NNH-2,4-dinitrophenyl hydrazine, and ⁇ S;
  • Yet a further aspect of the present invention relates to a method of treating HIV-1 infection in a subject.
  • This method involves administering to a subject infected with HIV-1 a therapeutically effective amount of a compound having the formula: where
  • each X is H or a compound of the formula:
  • Y is selected from the group consisting of —CH 3 , ⁇ O, —OH, —OCH 3 , —OC(O)CH 3 , —NNH-2,4-dinitrophenyl hydrazine, and ⁇ S;
  • n is an integer from 1 to 8;
  • p is 0 or 1
  • n is an integer from 1 to 8;
  • Still another aspect of the present invention relates to a method of treating HIV-1 infection in a human. This method involves administering to a human infected with HIV-1 a therapeutically effective amount of a compound having the formula: where
  • each X is H or a compound of the formula:
  • Y is selected from the group consisting of —CH 3 , ⁇ O, —OH, —OCH 3 , —OC(O)CH 3 , —NNH-2,4-dinitrophenyl hydrazine, and ⁇ S;
  • n is an integer from 1 to 8;
  • p is 0 or 1
  • n is an integer from 1 to 8;
  • At least one X is not H, or a pharmaceutically acceptable salt thereof under conditions effective to treat the human for HIV-1 infection.
  • Still a further aspect of the present invention relates to a method of treating HIV-1 infection in a subject.
  • This method involves administering to a subject infected with HIV-1 a therapeutically effective amount of a compound having the formula: where
  • R is a C 1 to C 5 alkyl
  • n is an integer between 5 and 1000;
  • BA is a compound having the formula: where
  • Y is selected from the group consisting of —CH 3 , ⁇ O, —OH, —OCH 3 , —OC(O)CH 3 , —NNH-2,4-dinitrophenyl hydrazine, and ⁇ S; and
  • Another aspect of the present invention relates to a method of treating HIV-1 infection in a human. This method involves administering to a human infected with HIV-1 a therapeutically effective amount of a compound having the formula: where
  • R is a C 1 to C 5 alkyl
  • n is an integer between 5 and 1000;
  • BA is a compound having the formula: where
  • Y is selected from the group consisting of —CH 3 , ⁇ O, —OH, —OCH 3 , —OC(O)CH 3 , —NNH-2,4-dinitrophenyl hydrazine, and ⁇ S; and
  • the betulinol derivatives of the present invention are particularly effective against Human Immunodeficiency Virus and, in particular, HIV-1. Moreover, the betulinol derivatives of the present invention produce anti-HIV-1 activity superior to anti-HIV-1 activity known in the art for other betulinol derivatives. In addition, the compounds of the present invention provide this superior anti-HIV-1 activity without affecting the proliferation of cells.
  • FIGS. 1 A-C show chromatograms of betulonic acid and its derivatives with their corresponding retention time. Close examination of these chromatograms reveals that the conjugated betulonic acid monomer and dimer gave neat chromatograms.
  • FIG. 2A is a chromatogram that included parts of MS spectra of betulonic acid after internal calibration, and included calibrant signals: m/z 365.3016, 423.3434, 481.3853, 539.4272, and 597.4690 are the calculated masses for poly(propylene glycol) bis(2-aminopropyl ether).
  • m/z 365.3016, 423.3434, 481.3853, 539.4272, and 597.4690 are the calculated masses for poly(propylene glycol) bis(2-aminopropyl ether).
  • [M+H] + and [M+NH 4 ] + ions m/z 455 and 472 for betulonic acid
  • FIG. 2B shows the MS spectra of m/z 544, 471, and 455 for betulonic acid.
  • FIG. 3A -C are MS spectra of monomer ester showing that it is essentially a single compound, appearing as the m/z 697 singly protonated ion ( FIG. 3A ). From a higher resolution, more slowly recorded ESI-MS scan ( FIG. 3B ), the monoisotopic molecular mass of the neutral compound is computed as 696.5 ⁇ 0.2 Da. The singly protonated positive ion of this compound is rather labile. As shown in the product ion spectrum of FIG. 3C , the collision induced decomposition (CID) of the m/z 697 ion has two efficient pathways, one involving the loss of a 56 Da neutral, the other the loss of a 100 Da neutral. The fragmentation requires relatively low collision energy (10 volts). As a consequence, the m/z 641 and 597 ions also show up in the ordinary mass spectrum of FIG. 3A under source conditions where average stability molecules would not fragment.
  • CID collision induced decomposition
  • FIGS. 4 A-B show the MS spectra of dimer ester.
  • the most intense peak in the ESI mass spectrum is the singly protonated ion at m/z 1261.
  • the low level impurities with ion at m/z 581, 627, 639, and 683 are present. Since the sensitivity for the structure is low, 40 ⁇ M concentration solution was used to observe a strong m/z 1261 peak.
  • FIG. 4B the predominant fragmentation process, as it was in monomer ester, is the loss of a 100 Da neutral, presumably in the form of isobutylene +CO 2 .
  • very weak, product ions those at m/z 1204 and 734 are significant, because they can be interpreted as a loss of C 4 H 8 and a loss of a betulonic acid residue, respectively.
  • FIG. 5 is a graph showing the standard solubility curve of betulonic acid concentration versus peak area.
  • FIG. 6 is a graphic representation of the HIV inhibitory effect of various betulinol derivatives.
  • FIG. 7 is a graphic representation of % inhibition of HIV infection by certain betulinol derivatives.
  • FIG. 8 is a graphic representation of % inhibition of HIV infection by varying doses of certain betulinol derivatives.
  • FIG. 9 is a graphic representation of % inhibition of HIV infection by varying doses of betulinol.
  • FIG. 10 is a graphic representation of HIV inhibitory effect of varying doses of 28-acetoxy betulin.
  • FIG. 11 is a graphic representation of % inhibition of AZT versus betulonic acid of H9 cells infected with HIV-IIIB.
  • FIG. 12 is a graphical representation of cell viability of H9 (lymphoma) cells in the presence of AZT versus betulonic acid.
  • FIG. 13 is a graphical representation of the anti-HIV activity of betulinol derivatives in CEM (CD4+T) cells.
  • FIG. 14 shows the results of AZT resistance for patients suffering from HIV.
  • FIG. 15 shows the results of AZT resistance for patients suffering from HIV.
  • the present invention relates to methods of treating HIV-1 infection in a subject. These methods involve administering to a subject with HIV-1 infection a therapeutically effective amount of a betulinol derivative compound, or pharmaceutically acceptable salt or derivative thereof, under conditions effective to treat the subject for HIV-1 infection.
  • Betulinol and betulinol derivatives are compounds having the general chemical structure of Formula I where
  • R 1 is selected from the group consisting of —CH 3 , ⁇ O, —OH, —OCH 3 , —OC(O)CH 3 , —NNH-2,4-DNP, and ⁇ S and
  • R 2 is selected from the group consisting of —H, —CH 3 , —CHO, —CH 2 OH, —CH 2 OCH 3 , —CH 2 OC(O)CH 3 , COCH 3 , —COOH, and —CH ⁇ NNH-2,4-DNP.
  • Betulinol can be isolated from the outer layer of the bark of the white birch tree Betula alba by sublimation (Lowitz, Crell's Annalen 1:312 (1788) and Mason, Silliman's Am. J, 20:282 (1831), which are hereby incorporated by reference in their entirety) or by extraction with an alcohol, such as ethanol (Hunefeld, J. Prakt. Chem. 7:53 (1836) and Hess, Poggendorff's Annalen 46:319 (1839), which are hereby incorporated by reference in their entirety).
  • alcohol such as ethanol
  • Other sources of betulinol and methods for its isolation and purification have been described in, for example, Sheth et al., J. Pharm. Sci.
  • betulinol is isolated from the non-saponifiable substance of floral soap.
  • the crushed initial leaf wood and components of a sulfate boiling procedure (NaOH, Na 2 SO 4 , Na 2 S 2O 3 , Na 2 SO 3 ) are lodged to a boiling pot in a batch or continuous process.
  • lignin the component of wood
  • Crude cellulose is derived from the pulping liquor which is composed of lignin, cellulose, and black buck.
  • Black buck is a composition of black buck with salts of tall acid and non-saponifiable substances.
  • the crude cellulose is used in paper production, whereas the sulfate soap is separated from the black buck by centrifugation or by a settling process. Treatment of the sulfate soap with sulfuric acid produces tall oil. The non-saponifiable substances are separated as crude betulinol. Recrystallization of the crude betulinol, such as from acetone, ethyl acetate, isopropanol, butanol, ethanol, and the like, yields pure betulinol. The black buck residue present after centrifugation or settling can be advantageously recycled.
  • Betulinol derivative compounds of Formula I are synthesized by standard methods that are well known in the art. For example, detailed instructions on how to synthesize and prepare compounds of Formula I are set forth in U.S. Pat. No. 6,890,533, to Bomshteyn et al., which is hereby incorporated by reference in its entirety.
  • the structure of betulinol is based on a 30-carbon skeleton of four, six-member rings and one five-member E-ring containing an a-isopropyl group.
  • the structural component of betulinol has a primary and a secondary hydroxyl group at C-3 and C-28. Betulinol has three sites, at carbon 3, 20, and 28, where chemical modification can occur to yield derivatives. Synthetic schemes for the preparation of betulinol derivative compounds are described in the Examples below.
  • betulinol derivatives can be made more water soluble by conjugation to one or more members of a group of solubility enhancing compounds.
  • the conjugate has a significantly greater solubility in aqueous solutions but retains a high level of biological activity including, for example, activity against HIV-1 infection. This is particularly important since the chemistry required for making therapeutic agents more soluble often causes the biological activity of the therapeutic agent to be reduced or in some cases, entirely lost.
  • Conjugated and immunoconjugated betulinol derivative compounds useful in the methods of the present invention can also be synthesized from betulinol. Synthetic schemes for the preparation of conjugated and immunoconjugated betulinol derivative compounds are described the Examples below.
  • One aspect of the present invention relates to a method of treating HIV-1 infection in a subject. This method involves administering to a subject with HIV-1 infection a therapeutically effective amount of a compound having the formula where
  • R 1 is selected from the group consisting of —CH 3 , ⁇ O, —OH, —OCH 3 , —OC(O)CH 3 , —NNH-2,4-DNP, and ⁇ S and
  • R 3 is selected from the group consisting of H and C 1 -C 5 alkyl
  • n is an integer from 1 to 12;
  • Z is H or a protective group
  • n is an integer from 2 to 8.
  • the above compound has a structure where:
  • protective group Z is H or is selected from the group consisting of butyloxycarbonyl and carbobenzoxy.
  • Z is butyloxycarbonyl.
  • the above method employs a conjugated betulinol derivative monomer compound, which can be made by a method that involves reacting a reactant compound of the formula with a betulinol derivative compound of the formula where
  • R 2 is a carbonyl containing group
  • Another aspect of the present invention relates to a method of treating HIV-1 infection in a subject.
  • This method involves administering to a subject with HIV-1 infection a therapeutically effective amount of a compound having the formula where
  • Y 1 and Y 2 are independently selected from the group consisting of —CH 3 , ⁇ O, —OH, —OCH 3 , —OC(O)CH 3 , —NNH-2,4-DNP, and ⁇ S;
  • R 3 is selected from the group consisting of H and C 1 -C 5 alkyl
  • Z is H or a protective group
  • n is an integer from 1 to 12, or a pharmaceutically acceptable salt or derivative thereof, under conditions effective to treat the subject for HIV- 1 infection.
  • n is an integer from 2 to 8.
  • the above compound has a structure where:
  • the above method employs a conjugated betulinol derivative dimer compound, which can be made by a method that involves reacting a reactant compound of the formula with a compound of the formula under conditions effective to make the conjugated betulinol derivative dimer compound.
  • a further aspect of the present invention relates to a method of treating HIV-1 infection in a subject. This method involves administering to a subject with HIV-1 infection a therapeutically effective amount of a compound having the formula where
  • Y 1 , Y 2 , Y 3 , and Y 4 are independently selected from the group consisting of —CH 3 , ⁇ O, —OH, —OCH 3 , —OC(O)CH 3 , —NNH-2,4-DNP, and ⁇ S;
  • R 3 is selected from the group consisting of H and C 1 -C 5 alkyl
  • n is an integer from 1 to 12;
  • Z is H or a protective group
  • n is an integer from 2 to 8.
  • the above compound has a structure where:
  • the above method employs a conjugated betulinol derivative tetramer compound, which is made by a method that involves reacting a reactant compound of the formula with a compound of the formula under conditions effective to make the conjugated betulinol derivative tetramer.
  • Another aspect of the present invention relates to a method of treating HIV-1 infection in a subject.
  • This method involves administering to a subject with HIV-1 infection a therapeutically effective amount of a compound having the formula wherein
  • BA is a compound having the formula: wherein
  • Y is selected from the group consisting of —CH 3 , ⁇ O, —OH, —OCH 3 , —OC(O)CH 3 , —NNH-2,4-DNP, and ⁇ S;
  • Q is BA, a leaving group, or H
  • R 3 is H or C 1 -C 5 alkyl
  • n is an integer from 1 to 12;
  • n 1 to 6
  • n is an integer from 2 to 8.
  • the above compound has a structure where:
  • the above method employs a conjugated betulinol derivative polymer compound, which is made by a method that involves polymerizing a monomer of the formula under conditions effective to make the conjugated betulinol derivative polymer compound.
  • Yet a further aspect of the present invention relates to a method of treating and/or inhibiting HIV-1 infection in a human.
  • This method involves administering to a human infected with HIV-1 a therapeutically effective amount of a compound having the formula where
  • n is an integer from 1 to 12 and
  • Z is H or a protective group
  • the above compound is administered as a tablet in a dosage range of 1 mg-500 mg.
  • a further aspect of the present invention relates to a method of treating and/or inhibiting HIV-1 infection in a human.
  • This method involves administering to a human infected with HIV-1 a therapeutically effective amount of a compound having the formula where
  • Z is H or a protective group
  • the above compound is administered as a tablet in a dosage range of 1 mg-500 mg.
  • conjugated betulinol derivative compounds include conjugated betulinol derivative monomers, dimers, tetramers, and polymers.
  • Compounds herein referred to as immunoconjugated betulinol derivative compounds are also suitable for carrying out the methods of the present invention.
  • Immunoconjugated betulinol derivative compounds, including monomers, dimers, tetramers, and polymers are prepared by attaching an antibody directly to a conjugated betulinol derivative compound.
  • Another aspect of the present invention relates to a method of treating HIV-1 infection in a subject by administering to a subject with HIV-1 infection a therapeutically effective amount of a compound having the formula where
  • R 1 is selected from the group consisting of —CH 3 , ⁇ O, —OH, —OCH 3 , —OC(O)CH 3 , —NNH-2,4-DNP, and ⁇ S and
  • n is an integer from 1 to 12;
  • Z is H or a protective group
  • a further aspect of the present invention relates to a method of treating HIV-1 infection in a subject. This method involves administering to a subject with HIV-1 infection a therapeutically effective amount of a compound having the formula where
  • Y 1 and Y 2 are independently selected from the group consisting of —CH 3 , ⁇ O, —OH, —OCH 3 , —OC(O)CH 3 , —NNH-2,4-DNP, and ⁇ S;
  • Z is H or a protective group
  • n is an integer from 1 to 12,
  • Yet another aspect of the present invention relates to a method of treating HIV-1 infection in a subject.
  • This method involves administering to a subject with HIV-1 infection a therapeutically effective amount of a compound having the formula where
  • Y 1 , Y 2 , Y 3 , and Y 4 are independently selected from the group consisting of —CH 3 , ⁇ O, —OH, —OCH 3 , —OC(O)CH 3 , —NNH-2,4-DNP, and ⁇ S;
  • n is an integer from 1 to 12;
  • Z is H or a protective group
  • Yet a further aspect of the present invention relates to a method of treating HIV-1 infection in a subject.
  • This method involves administering to a subject with HIV-1 infection a therapeutically effective amount of a compound having the formula where
  • BA is a compound having the formula: where
  • Y is selected from the group consisting of —CH 3 , ⁇ O, —OH, —OCH 3 , —OC(O)CH 3 , —NNH-2,4-DNP, and ⁇ S;
  • Q is BA, a leaving group, or H
  • n is an integer from 1 to 12;
  • n 1 to 6
  • a preferred type of antibody for making the above immunoconjugated betulinol derivative compounds and for use in the above methods is a gammaglobulin.
  • IgG, IgA, IgE, and IgM subclasses are particularly preferred.
  • Some representative immunoglobulins are monoclonal or polyclonal antibodies to human or animal tumor associated antigens; human B- and T-cell antigens; human Ia antigens; viral, fungal and bacterial antigens; and cells involved in human inflammatory or allergic reactions.
  • Preferred subjects for treating HIV-1 infection in accordance with the methods of the present invention include, without limitation, any mammal, preferably a human.
  • a therapeutically effective amount of a betulinol derivative compound is preferably administered to the subject to treat the subject for AIDS.
  • the administering step is carried out to prevent AIDS in the subject infected with HIV-1.
  • treating means amelioration, prevention, or relief from the symptoms and/or effects associated with HIV-1 infection, and includes the prophylactic administration of a conjugated or immunoconjugated betulinol derivative compound, or a pharmaceutically acceptable salt or derivative thereof, to substantially diminish the likelihood or seriousness of the condition.
  • the relative activity, potency, and specificity of the compound may be determined by a pharmacological study in animals, for example, according to the method of Nyberg et al., Psychopharmacology 119:345-348 (1995), which is hereby incorporated by reference in its entirety.
  • the differential metabolism among patient populations can be determined by a clinical study in humans, less expensive and time-consuming substitutes are provided by the methods of Kerr et al., Biochem. Pharmacol. 47:1969-1979 (1994) and Karam et al., Drub Metab. Discov. 24:1081-1087 (1996), which are hereby incorporated by reference in their entirety.
  • the potential for drug-drug interactions may be assessed clinically according to the methods of Leach et al., Epilepsia 37:1100-1106 (1996), which is hereby incorporated by reference in its entirety, or in vitro according to the methods of Kerr et al., Biochem. Pharmacol. 47:1969-1979 (1994) and Turner et al., Can. J. Physio. Pharmacol. 67:582-586 (1989), which are hereby incorporated by reference in their entirety.
  • a prophylactic or therapeutic dose of the compound will vary with the nature and severity of the condition to be treated and the route of administration.
  • the dose, and perhaps the dose frequency, will also vary according to the age, body weight, and response of the individual subject.
  • the total daily dose of compound may be administered in single or divided doses.
  • the compound should be administered in an effective amount.
  • exemplary doses of betulinol derivatives for oral administration that provide an effective amount of the betulinol derivative typically range from about 1 mg per unit dose to 2,000 mg per unit dose and more typically from about 10 mg per unit dose to 500 mg per unit dose.
  • the dosage is in the range of 1.0 to 200 mg/kg/day and the preferred dosage range is 1.0 to 50 mg/kg/day.
  • Any suitable route of administration may be employed and, may include, without limitation, oral, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, or intranasal administration.
  • the agent may be administered alone or with suitable pharmaceutical carriers, and can be in solid or liquid form, such as tablets, capsules, powders, solutions, suspensions, or emulsions.
  • Dosage forms include, for example, tablets, troches, dispersions, suspensions, solutions, capsules, powders, solutions, suspensions, emulsions, and patches.
  • compositions of the present invention include at least one betulinol derivative compound, a pharmaceutically acceptable salt or derivative thereof, or combinations thereof.
  • Such compositions may include a pharmaceutically acceptable carrier, and optionally, other therapeutic ingredients or excipients.
  • pharmaceutically acceptable salt thereof refers to salts prepared from pharmaceutically acceptable, non-toxic acids including inorganic acids and organic acids, such as, for example, acetic acid, benzenesulfonic (besylate) acid, benzoic acid, camphorsulfonic acid, citric acid, ethenesulfonic acid, fumaric acid, gluconic acid, glutamic acid, hydrobromic acid, hydrochloric acid, isethionic acid, lactic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, mucic acid, nitric acid, pamoic acid, pantothenic acid, phosphoric acid, succinic acid, sulfuric acid, tartaric acid, and p-toluenesulfonic acid.
  • inorganic acids and organic acids such as, for example, acetic acid, benzenesulfonic (besylate) acid, benzoic acid, camphorsulfonic acid, citric acid, ethenesul
  • compositions may be conveniently presented in unit dosage form, and may be prepared by any of the methods well known in the art of pharmacy.
  • Preferred unit dosage formulations are those containing an effective dose, or an appropriate fraction thereof, of the active ingredients.
  • compositions of the present invention may include a pharmaceutically acceptable carrier.
  • the carrier may take a wide variety of forms, depending on the forms preparation desired for administration, for example, oral or parenteral (including intravenous).
  • any of the usual pharmaceutical media may be employed, such as, water, glycols, oils, alcohols, flavoring agents, preservatives, and coloring agents in the case of oral liquid preparation, including suspension, elixirs and solutions.
  • Carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders and disintegrating agents may be used in the case of oral solid preparations such as powders, capsules and caplets, with the solid oral preparation being preferred over the liquid preparations.
  • Preferred solid oral preparations are tablets or capsules, because of their ease of administration. If desired, tablets may be coated by a standard aqueous or nonaqueous technique. Oral and parenteral sustained release dosage forms may also be used.
  • Oral syrups, as well as other oral liquid formulations, are well known to those skilled in the art, and general methods for preparing them are found in any standard pharmacy school textbook. For example, chapter 86, of the 19th Edition of Remington: The Science and Practice of Pharmacy, entitled “Solutions, Emulsions, Suspensions and Extracts,” describes in complete detail the preparation of syrups (pages 1503-1505, which are hereby incorporated by reference in their entirety) and other oral liquids.
  • sustained release formulations are well known in the art, and Chapter 94 of the same reference, entitled “Sustained-Release Drug Delivery Systems,” describes the more common types of oral and parenteral sustained-release dosage forms (pages 1660-1675, which are hereby incorporated by reference in their entirety). Because they reduce peak plasma concentrations, as compared to conventional oral dosage forms, controlled release dosage forms are particularly useful for providing therapeutic plasma concentrations while avoiding the side effects associated with high peak plasma concentrations that occur with conventional dosage forms.
  • the solid unit dosage forms can be of the conventional type.
  • the solid form can be a capsule, such as an ordinary gelatin type containing the betulinol derivative and a carrier, for example, lubricants and inert fillers, such as lactose, sucrose, or cornstarch.
  • these betulinol derivatives can be tableted with conventional tablet bases, such as lactose, sucrose, or cornstarch, in combination with binders, like acacia, cornstarch, or gelatin, disintegrating agents, such as cornstarch, potato starch, or alginic acid, and lubricants, like stearic acid or magnesium stearate.
  • compositions may also be administered in injectable dosages by solution or suspension of these materials in a physiologically acceptable diluent with a pharmaceutical carrier.
  • a pharmaceutical carrier include sterile liquids, such as water and oils, with or without the addition of a surfactants, adjuvants, excipients, or stabilizers.
  • sterile liquids such as water and oils
  • Illustrative oils are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, or mineral oil.
  • water, saline, aqueous dextrose and related sugar solutions, and glycols, such as propylene glycol or polyethylene glycol are preferred liquid carriers, particularly for injectable solutions.
  • the pharmaceutical compositions in solution or suspension may be packaged in a pressurized aerosol container together with suitable propellants, for example, hydrocarbon propellants like propane, butane, or isobutane, and with conventional adjuvants.
  • suitable propellants for example, hydrocarbon propellants like propane, butane, or isobutane, and with conventional adjuvants.
  • the pharmaceutical compositions may also be administered in a non-pressurized form, such as in a nebulizer or atomizer.
  • the present invention also relates to methods of inhibiting HIV-1 activity in a cell using betulinol derivative compounds.
  • a cell infected with HIV-1 is provided and contacted with a compound having the formula where
  • R 1 is selected from the group consisting of —CH 3 , ⁇ O, —OH, —OCH 3 , —OC(O)CH 3 , —NNH-2,4-DNP, and ⁇ S and
  • R 3 is selected from the group consisting of H and C 1 -C 5 alkyl
  • n is an integer from 1 to 12;
  • Z is H or a protective group
  • n is an integer from 2 to 8.
  • the above compound has a structure where:
  • Another aspect of the present invention relates to a method of inhibiting HIV-1 activity in a cell. This method involves providing a cell infected with HIV-1 and contacting the cell with a compound having the formula where
  • R 1 is selected from the group consisting of —CH 3 , ⁇ O, —OH, —OCH 3 , —OC(O)CH 3 , —NNH-2,4-DNP, and ⁇ S and
  • n is an integer from 1 to 12;
  • Z is H or a protective group
  • a further aspect of the present relates to a method of inhibiting HIV-1 activity in a cell. This method involves providing a cell infected with HIV-1 and contacting the cell with a compound having the formula where
  • Y 1 and Y 2 are independently selected from the group consisting of —CH 3 , ⁇ O, —OH, —OCH 3 , —OC(O)CH 3 , —NNH-2,4-DNP, and ⁇ S;
  • R 3 is selected from the group consisting of H and C 1 -C 5 alkyl
  • Z is H or a protective group
  • n is an integer from 1 to 12,
  • n is an integer from 2 to 8.
  • the above compound has a structure where:
  • Yet another aspect of the present invention relates to a method of inhibiting HIV-1 activity in a cell. This method involves providing a cell infected with HIV-1 and contacting the cell with a compound having the formula where
  • Y 1 and Y 2 are independently selected from the group consisting of —CH 3 , ⁇ O, —OH, —OCH 3 , —OC(O)CH 3 , —NNH-2,4-DNP, and ⁇ S;
  • Z is H or a protective group
  • n is an integer from 1 to 12,
  • Yet a further aspect of the present invention relates to a method of inhibiting HIV-1 activity in a cell.
  • This method involves providing a cell infected with HIV-1 and contacting the cell with a compound having the formula where
  • Y 1 , Y 2 , Y 3 , and Y 4 are independently selected from the group consisting of —CH 3 , ⁇ O, —OH, —OCH 3 , —OC(O)CH 3 , —NNH-2,4-DNP, and ⁇ S;
  • R 3 is selected from the group consisting of H and C 1 -C 5 alkyl
  • n is an integer from 1 to 12;
  • Z is H or a protective group
  • n is an integer from 2 to 8.
  • the above compound has a structure where:
  • Another aspect of the present invention relates to a method of inhibiting HIV-1 activity in a cell. This method involves providing a cell infected with HIV-1 and contacting the cell with a compound having the formula where
  • Y 1 , Y 2 , Y 3 , and Y 4 are independently selected from the group consisting of —CH 3 , ⁇ O, OH, OCH 3 , OC(O)CH 3 , —NNH-2,4-DNP, and ⁇ S;
  • n is an integer from 1 to 12;
  • Z is H or a protective group
  • a further aspect of the present invention relates to a method of inhibiting HIV-1 activity in a cell. This method involves providing a cell infected with HIV-1 and contacting the cell with a compound having the formula wherein
  • BA is a compound having the formula: wherein
  • Y is selected from the group consisting of —CH 3 , ⁇ O, —OH, —OCH 3 , —OC(O)CH 3 , —NNH-2,4-DNP, and ⁇ S;
  • Q is BA, a leaving group, or H
  • R 3 is H or C 1 -C 5 alkyl
  • n is an integer from 1 to 12;
  • n 1 to 6
  • n is an integer from 2 to 8.
  • the above compound has a structure where:
  • Yet another aspect of the present invention relates to a method of inhibiting HIV-1 activity in a cell. This method involves providing a cell infected with HIV-1 and contacting the cell with a compound having the formula where
  • BA is a compound having the formula: where
  • Y is selected from the group consisting of —CH 3 , ⁇ O, —OH, —OCH 3 , —OC(O)CH 3 , —NNH-2,4-DNP, and ⁇ S;
  • Q is BA, a leaving group, or H
  • n is an integer from 1 to 12;
  • n 1 to 6
  • contacting can be carried out as desired, including, but not limited to, contacting cells in culture in a suitable growth medium.
  • mice, rats or other mammals are injected with compounds.
  • betulinol derivative compounds are also amenable to the methods of the present invention.
  • betulinol derivatives with increased solubility may be achieved by a variety of other methods besides preparation of the above-described conjugates and immunoconjugates.
  • increased solubility of a betulinol derivative compound is achieved by attaching a solubilizing agent at C28 or C3 of the betulin derivative.
  • Preferred solubilizing agents include, without limitation, polyethylene glycol (PEG) or miniPEGs. PEG chemistry is well known and can be used to attach the PEG to the betulin derivative.
  • preferred betulinol derivative compounds with increased solubility are achieved by employing hydrophilic amino acids.
  • the hydrophilic basic amino acids (Lys, Arg, or His) can attach to the betulinol derivatives.
  • the other highly hydrophilic amino acids (Glu, Asp, Gln, or Asn) may also be used.
  • Di-peptides for example, Lys-Lys, Lys-His, Lys-Arg, Arg-His, Lys-Glu, Arg-Gln, Lys-Gln, and tri-peptides may be used to enhance the solubility of the betulinol derivative.
  • di-peptides and tri-peptides may include amino acids that are mildly hydrophilic in character (Tyr, Trp, Ser, Thr, and Gly). The coupling of these peptides may occur in a hindered position, allowing the hydrophilic portion of the peptides to remain available for solvation.
  • the coupling of peptides can be carried out in a manner similar to that disclosed herein for coupling lysine to a betulinol derivative with the addition of, for example, a reaction step that serves to protect the active group on the other residue(s).
  • amino acids or peptides containing amino acid residues having a primary or secondary amine i.e., Arg and His
  • Arg and His amino acid residues having a primary or secondary amine
  • polyamines such as spermidine, putrescine, and spermine may be attached to the betulinol derivative to increase solubility. These compounds are attached through a primary or secondary amine group.
  • Carbohydrate moieties including (1) monosaccharides (e.g. glucose, galactose, fucose, and fructose), (2) disaccharides (e.g., sucrose and maltose), and (3) aminosugars (e.g., glucosamine, galactosamine, 2-amino-2-deoxy-glucurouic acid, 2-amino-2-deoxy-glucose, 2-amino-2-deoxy-3-O-â-D-glucopyranurosyl-D-galactose, galactonojirimycin, gluconojirimycin, and derivatives thereof) may also be attached to betulinol derivatives to increase solubility.
  • monosaccharides e.g. glucose, galactose, fucose, and fructose
  • disaccharides e.g., sucrose and maltose
  • aminosugars e.g., glucosamine, galactos
  • Cyclodextrins including, for example, 2-amino-2-deoxy-3-O- ⁇ -D-glucopyranurosyl-D-galactose, ⁇ -cyclodextrin (six glucose residues); ⁇ -cyclodextrin (seven glucose residues); and ⁇ -cyclodextrin (eight glucose residues) may be attached to the betulin derivative to increase solubility.
  • the coupling of a carbohydrate to the betulinol derivative can be done by the methods known in the art of carbohydrate chemistry.
  • Increased solubility of betulinol derivative compounds may also be achieved by attaching a betulinol derivative to each of 4 glycine chains having around 2-3 glycine molecules of a sugar molecule (or 2 betulonic acid groups in the case of lysine), leaving one OH group open to attach to an antibody.
  • glycine chains are used due to increased solubility in organic solution and to avoid hinderance.
  • BA O is BA-amino acid-O
  • BA is a compound having the formula where
  • Y is selected from the group consisting of —CH 3 , ⁇ O, —OH, —OCH 3 , —OC(O)CH 3 , —NNH-2,4-DNP, and ⁇ S and
  • * is a binding site to facilitate attachment of BA to the exemplary structure.
  • BA O is BA-amino acid-BA
  • BA is a compound having the formula where
  • Y is selected from the group consisting of —CH 3 , ⁇ O, —OH, —OCH 3 , —OC(O)CH 3 , —NNH-2,4-DNP, and ⁇ S;
  • p is an integer from 1 to 10;
  • n is an integer from 1 to 6;
  • n 1 to 6.
  • the whole compound is water-soluble. Also, there will be no toxicity since the entire compound is biocompatible.
  • a long chain with NH 2 and COOH groups may be employed, alternating with an OH group at the end. These structure are achieved by attaching a betulinol derivative compound to NH 2 groups. The OH group can then be used to attach to an antibody.
  • An exemplary structure is as follows: where BA is a compound having the formula where
  • Y is selected from the group consisting of —CH 3 , ⁇ O, —OH, —OCH 3 , —OC(O)CH 3 , —NNH-2,4-DNP, and ⁇ S;
  • n is an integer from 1 to 6.
  • Another aspect of the present invention relates to a method of treating and/or inhibiting HIV-1 infection in a subject.
  • This method involves administering to a subject infected with HIV-1 a therapeutically effective amount of a compound having the formula: where
  • BA is a compound having the formula: where
  • Y is selected from the group consisting of —CH 3 , ⁇ O, —OH, —OCH 3 , —OC(O)CH 3 , —NNH-2,4-dinitrophenyl hydrazine, and ⁇ S;
  • X is selected from the group consisting of
  • each R 4 is independently selected from the group consisting of H, CH 3 , CH 2 —CH 3 , NH 2 and OH;
  • Z is H, a protective group, or BA
  • n is an integer from 1 to 8;
  • n is an integer from 1 to 6;
  • Yet another aspect of the present invention relates to a method of treating HIV-1 infection in a subject.
  • This method involves administering to a subject infected with HIV-1 a therapeutically effective amount of a compound having the formula: where
  • W is H, OX, or CH 2 —OX
  • each X is independently H, a sugar, or BA, and wherein at least 1 X is BA;
  • BA is a compound having the formula: where
  • Y is selected from the group consisting of —CH 3 , ⁇ O, —OH, —OCH 3 , —OC(O)CH 3 , —NNH-2,4-dinitrophenyl hydrazine, and ⁇ S; and
  • Yet a further aspect of the present invention relates to a method of treating HIV-1 infection in a human.
  • This method involves administering to a human infected with HIV-1 a therapeutically effective amount of a compound having the formula: where
  • W is H, OX, or CH 2 —OX
  • each X is independently H, a sugar, or BA, and wherein at least 1 X is BA;
  • BA is a compound having the formula: where
  • Y is selected from the group consisting of CH 3 , ⁇ O, —OH, —OCH 3 , —OC(O)CH 3 , —NNH-2,4-dinitrophenyl hydrazine, and ⁇ S;
  • Still another aspect of the present invention relates to a method of treating HIV-1 infection in a subject.
  • This method involves administering to a subject infected with HIV-1 a therapeutically effective amount of a compound having the formula: where
  • each X is H or a compound of the formula:
  • Y is selected from the group consisting of —CH 3 , ⁇ O, —OH, —OCH 3 , —OC(O)CH 3 , —NNH-2,4-dinitrophenyl hydrazine, and ⁇ S;
  • n is an integer from 1 to 8;
  • p is 0 or 1
  • n is an integer from 1 to 8;
  • Still a further aspect of the present invention relates to a method of treating HIV-1 infection in a human.
  • This method involves administering to a human infected with HIV-1 a therapeutically effective amount of a compound having the formula: where
  • each X is H or a compound of the formula:
  • Y is selected from the group consisting of —CH 3 , ⁇ O, —OH, —OCH 3 , —OC(O)CH 3 , —NNH-2,4-dinitrophenyl hydrazine, and ⁇ S;
  • n is an integer from 1 to 8;
  • p is 0 or 1
  • n is an integer from 1 to 8;
  • At least one X is not H, or a pharmaceutically acceptable salt thereof under conditions effective to treat the human for HIV-1 infection.
  • Another aspect of the present invention relates to a method of treating HIV-1 infection in a subject.
  • This method involves administering to a subject infected with HIV-1 a therapeutically effective amount of a compound having the formula: where
  • R is a C 1 to C 5 alkyl
  • n is an integer between 5 and 1000;
  • BA is a compound having the formula: where
  • Y is selected from the group consisting of —CH 3 , ⁇ O, —OH, —OCH 3 , —OC(O)CH 3 , —NNH-2,4-dinitrophenyl hydrazine, and ⁇ S; and
  • a further aspect of the present invention relates to a method of treating HIV-1 infection in a human. This method involves administering to a human infected with HIV-1 a therapeutically effective amount of a compound having the formula: where
  • R is a C 1 to C 5 alkyl
  • n is an integer between 5 and 1000;
  • BA is a compound having the formula: where
  • Y is selected from the group consisting of —CH 3 , ⁇ O, —OH, —OCH 3 , —OC(O)CH 3 , —NNH-2,4-dinitrophenyl hydrazine, and ⁇ S; and
  • Betulinol is isolated from the non-saponofiable fraction of the crude sulfate soap prepared by boiling the outer bark of the white birch tree in NaOH, Na 2 SO 4 , Na 2 SO 3 , and Na 2 S 2 O 3 at 110-120° C. Betulinol is then crystallized by using solvents such as acetone, ethyl acetate, isopropanol, butanol, ethanol, etc.
  • solvents such as acetone, ethyl acetate, isopropanol, butanol, ethanol, etc.
  • the chemical structure of betulinol is:
  • Betulinol is a non-steroidal, lupeol-derived, pentacyclical, lupan-row alcohol of the group of styrenes.
  • Betulinol also known as betulin
  • the structure of betulinol is based on a 30-carbon skeleton of four, six-member rings and one, five-member E-ring containing an a-isopropyl group.
  • the structural component of betulinol has a primary and a secondary hydroxyl group at C-3 and C-28.
  • Betulinol has three sites (C-3, C-20, and C-28) where chemical modification can occur to yield derivatives. With the availability of betulinol and its ability to react with various other organic compounds, eleven derivatives of betulinol were synthesized as shown in Scheme 1.
  • the alkylated betulinol derivatives can be prepared in a variety of ways. Keto-derivatives can be acquired by treating betulinol with suitable oxidizing reagent, such as Jone's reagent or pyridinium chlorochromate (PCC) (Kim et al., Synthetic Communications 27:1607-1612 (1997); Komissarova et al., Chemistry of National Compounds 38:58-61 (2002); Ito et al., J. Nat. prod. 64:1278-1281 (2001) which are hereby incorporated by reference in their entirety).
  • oxidizing reagent such as Jone's reagent or pyridinium chlorochromate (PCC)
  • PCC pyridinium chlorochromate
  • betulinol is first dissolved in acetone, and then oxidized with oxidizing reagent at 0° C.
  • Betulin acetate derivatives betulin diacetate, 3-acetoxy betulin, and 28-acetoxy betulin were prepared by acylation reaction (Kim et al., Bioorg. Med. Chem. Lett., 8:1707-1712 (1998); Hiroya et al., Bioorg. Med. Chem. 10:3229-3236 (2002), which are hereby incorporated by reference in their entirety).
  • a dry pyridine solution of betulinol was treated with anhydrous acetic anhydride and stirred for 6 hrs.
  • Betulin dimethyl ether was prepared by alkylation. To a solution of NaOH and betulinol in dry tetrahydrofuran, iodomethane was added and the resulting mixture was refluxed for 40 hrs. Distilled water was added drop wise to stop the reaction. Betulin dimethylether was obtained after column chromatography. Chlorocarbonyl betulin was obtained by treatment with oxalyl chloride (Sun et al., J. Med.
  • This betulone aldehyde was dissolved in 17 mL CH 3 CN—H 2 O containing 877 mg of NaH 2 PO 4 .H 2 O and cooled to 0-5° C. 220 ⁇ L of 30% of aqueous H 2 O 2 and 200 mg of NaClO 2 dissolved in 16 mL water were added in tandem. The mixture was brought up to room temperature and stirred for one hour. The reaction was quenched by the addition of 380 mg of Na 2 S 2 O 5 . The betulonic acid was extracted with 300 mL ethyl acetate. The organic extract was washed with water and brine, and dried by 100 mg of Na 2 SO 4 . The organic solution was filtered through filter paper and the filtrate was evaporated.
  • FIGS. 1 A-C show a typical chromatogram of betulonic acid and its derivatives with their corresponding retention time. Close examination of these chromatograms reveals that the conjugated betulonic acid monomer and dimer, gave neat chromatograms.
  • Betulonic acid and its derivatives were subsequently subjected to spectroscopic analysis in order to resolve their molecular structure.
  • the Electrospray mass spectrometric analysis confirmed their pentacyclic styrene nature of these compounds.
  • the samples were mass analyzed on a Micromass Quattro II triple quadrupole instrument with electrospray (ES) ionization in the positive mode. Samples were introduced by continuous infusion at a rate of 5 ⁇ L/min as a nominal 200 ⁇ M concentration solution in a 75:25:2 (v/v) acetonitrile-water-acetic acid. When necessary, product ion spectra were obtained by maintaining argon gas in the collision chamber of the instrument at a pressure of 4 ⁇ 10 ⁇ 3 mBar.
  • FIG. 2A included parts of MS spectra of betulonic acid after internal calibration, and included calibrant signals: m/z 365.3016, 423.3434, 481.3853, 539.4272, and 597.4690 are the calculated masses for poly(propylene glycol) bis(2-aminopropyl ether).
  • m/z 365.3016, 423.3434, 481.3853, 539.4272, and 597.4690 are the calculated masses for poly(propylene glycol) bis(2-aminopropyl ether).
  • [M+H] + and [M+NH 4 ] + ions m/z 455 and 472 for betulonic acid
  • FIG. 2B shows the MSMS spectra of m/z 544, 471, and 455 for betulonic acid.
  • the MS spectra of monomer ester show that it is essentially a single compound, appearing as the m/z 697 singly protonated ion ( FIG. 3A ). From a higher resolution, more slowly recorded ESI-MS scan ( FIG. 3B ) the monoisotopic molecular mass of the neutral compound is computed as 696.5 ⁇ 0.2 Da. The singly protonated positive ion of this compound is rather labile.
  • the collision induced decomposition (CID) of the m/z 697 ion has two efficient pathways, one involving the loss of a 56 Da neutral, the other the loss of a 100 Da neutral. The fragmentation requires relatively low collision energy, 10 volts. As a consequence, the m/z 641 and 597 ions also show up in the ordinary mass spectrum of FIG. 3A under source conditions where average stability molecules would not fragment.
  • the MS spectra of dimer ester is shown in FIGS. 4 A-B.
  • the most intense peak in the ESI mass spectrum is the singly protonated ion at m/z 1261.
  • the low level impurities with ion at m/z 581, 627, 639, and 683 are present. Since the sensitivity for the structure is low, 40 ⁇ M concentration solution was used to observe a strong m/z 1261 peak.
  • the predominant fragmentation process, as it was in monomer ester is the loss of a 100 Da neutral, presumably in the form of isobutylene +CO 2 .
  • very weak, product ions those at m/z 1204 and 734 are significant, because they can be interpreted as a loss of C 4 H 8 and a loss of a betulonic acid residue, respectively.
  • N ⁇ -butyloxycarbonyl-N ⁇ -benzyloxycarbonyl-Lysine(Boc-Lys (Cbz)-OH), having the formula (4) was obtained from Sigma-Aldrich. Both amines on C-2 and C-6 were protected by butyloxycarbonyl (Boc) and carbobenzoxy (Cbz), respectively.
  • Boc-Lys(Cbz)-OMe (5) was dissolved in 40 mL MeOH:Ethyl acetate. 100 mg of Palladium on active carbon (Pd/C) was added to the solution. The solution was stirred under hydrogen for 2 hrs. The organic solution was filtered through Celite and washed with 10 mL MeOH. The filtrate was evaporated under reduced pressure to yield Boc-Lys-OMe of formula (6) as a white solid, which was used directly for conjugation with betulonic acid (3).
  • Monomer-OMe (N ⁇ -betuloniccarbonyl-Lysine Methyl Ester) was prepared as described in Chun et al., J. Org. Chem. 69:7344-7347 (2004), which is hereby incorporated by reference in its entirety. Specifically, 20 mg of monomer (7) was dissolved in anhydrous 1 mL CH 2 Cl 2 at 0° C. A solution of 11 ⁇ L trifluoroacetic acid (TFA) in 11 ⁇ L CH 2 Cl 2 was added drop wise. The reaction mixture was stirred at room temperature for 12 h. The solvent was evaporated under vacuum. The residue was triturated with petroleum ether. The organic solvent was evaporated under vacuum to obtain crude Monomer-OMe of formula (7b)
  • dimer (8) and 10.9 mg of LiOH.H 2 O were dissolved in 3 mL THF and 100 ⁇ L H 2 O. The resulting solution was stirred at room temperature until (8) was completely used up as monitored by TLC. The solution was concentrated in vacuo. The resulting solid was subjected to silica gel column chromatography to obtain 142.6 mg of Dimer-Boc of formula (8a) as white solid.
  • dimer (8) 100 mg was dissolved in anhydrous 1 mL CH 2 Cl 2 at 0° C. A solution of 31 ⁇ L TFA in 31 ⁇ L CH 2 Cl 2 was added drop wise. The reaction mixture was stirred at room temperature for 12 hrs. The solvent was evaporated under vacuum. The residue was triturated with petroleum ether. The organic solvent was evaporated under vacuum to obtain crude Dimer-OMe of formula (8b)
  • Tetramer (9) 8.3 mg of tetramer (9) and 1 mg of LiOH.H 2 O was dissolved in 300 ⁇ L MeOH and 50 ⁇ L H 2 O. The resulting solution was stirred at room temperature until (9) was completely used up as monitored by TLC. The solution was concentrated in vacuo. The resulting solid was subjected to silica gel column chromatography to obtain 3 mg of Tetramer-Boc (10) as white solid with 3.4 mg of unreacted tetramer (9). Tetramer (9), abbreviated as can be used for synthesis of pentamer-BA of formula (15) which contains six molecules of betulonic acid (3).
  • tetramer-OMe (11) could connect to betulonic acid (3) directly instead of the monomer derivative (12). This will yield tetramer-BA of formula (16), which contains five molecules of betulonic acid (3). Conjugation of (11) with betulonic acid (3) yields tetramer-BA-OMe of formula (14). The same hydrolysis on (14) to remove methyl ester generates tetramer-BA of formula (16).
  • Example 8 Preparation of Pentamer With Six Molecules of Betulonic Acid
  • pentamer A simple and direct way to prepare pentamer is to conjugate pentalysine with betulonic acid.
  • pentalysine itself, without any protecting groups, is unstable, because it is easily polymerized and cyclized.
  • the C-1a carboxyl group of pentalysine can be easily coupled with ⁇ -amine on C-2e or primary amines on C-6(a-e) of another molecule to form a polymer.
  • This polymer is composed of different numbers of amino acid groups, yielding different lengths of peptide.
  • the coupling reaction can happen in the same molecule, which connects the amino and carboxy ends of the pentalysine and cyclizes, which is illustrated as follows:
  • pentalysine derivative is needed to conjugate with betulonic acid.
  • pentalysine methyl ester of formula (17) has C-1a carboxyl protected by ester with all other amines free to conjugate.
  • pentalysine methyl ester (17) could react with six molecules of betulonic acid (3) and is catalyzed by DCC and HOBt to yield pentamer methyl ester of formula (18).
  • Subjecting the ester to hydrolysis removes the methyl protecting group to generate pentamer of formula (19), which contains six molecules of betulonic acid.
  • Pentamer (19) with six molecules of betulonic acid and free carboxyl can be used for immunoconjugation with an antibody.
  • Immunoconjugates of betulin derivatives were made by conjugation of ⁇ -globulin with lysinated betulonic acid.
  • the synthetic structural modification strategy described previously is seen as a prelude to create sites for conjugation to a monoclonal antibody.
  • monomer was conjugated with rabbit ⁇ -globulin via an activated carboxyl group (COOH).
  • COOH carboxyl group
  • Carbodiimide method is presently assessed. This bioconjugation reaction made use of different activated intermediates.
  • EDC solution 0.4 mg of EDC in 50 ⁇ L of DMF
  • NHS solution 0.4 mg of NHS in 25 ⁇ L DMF
  • the reaction was kept at room temperature for 30 min and then kept at 4° C. overnight.
  • the mixture was added slowly to 2 mg ⁇ -globulin which was dialyzed against 250 mL of 0.1 M pH 9.4 carbonate buffer 4° C. 18 h.
  • the reaction was carried out at 4° C. for overnight.
  • the reaction mixture was dialyzed against 200 mL of 0.01 M phosphate buffer pH 7.2 containing 0.015 NaCl (PBS) for 72 h with two exchange of this buffer to give the monomer-antibody conjugate.
  • the mixture was centrifuged (10,500 rpm) for 6 minutes and then precipitate was stored for cell culture.
  • Flash column chromatography (“FCC”) was performed using silica gel grade 9385 of 230-400 mesh (E. Merck). A stepwise solvent polarity gradient was employed. TLC was performed on aluminum sheets precoated with silica gel 60 (HF-254, E. Merck) to a thickness of 0.25 mm.
  • betulonic acid The standard solutions of betulonic acid were prepared as follows. 45.4 mg of betulonic acid was dissolved in 200 ⁇ L neat chloroform to yield 0.5 mol/L concentration of betulonic acid. After three times of double dilution, three different known concentrations (0.25 mol/L, 0.125 mol/L, and 0.0625 mol/L) of betulonic acid were obtained. 2.27 mg of betulonic acid was dissolved in 500 ⁇ L neat chloroform to yield 0.01 mol/L concentration of betulonic acid. Double dilution of this solution generated a 0.005 mol/L concentration of betulonic acid. 0.227 mg of betulonic acid was dissolved in 500 ⁇ L neat chloroform to yield 0.001 mol/L concentration of betulonic acid.
  • Ethanol was chosen as a biocompatible solvent for use in in vivo studies.
  • Up to 43.3 mg of betulonic acid was completely dissolved in 1 mL of neat (100%) ethanol to yield a saturated solution.
  • This solution can be diluted with culture medium to yield 0.8 mg of betulonic acid completely dissolved in 1.76 mL of culture medium containing 10% human serum with 10% ethanol concentration to generate a concentration of 1 ⁇ 10 ⁇ 3 mol/mL (0.5 mg/mL) of betulonic acid.
  • Human y-Globulin and human albumin are two major biocompatible components in human serum. Betulonic acid was dissolved in neat ethanol and diluted with PBS containing various concentrations of y-globulin and Albumin (Table 8). TABLE 8 Dilution of BA with PBS at Various Concentrations Human ⁇ - Human Betu- globulin Albumin lonic (27 mg/mL (42 mg/mL Acid Ethanol in PBS) in PBS) Con.
  • betulonic acid could be dissolved in PBS with increasing concentration of human albumin. It was completely dissolved in human albumin PBS solution with 10% ethanol to yield a 1 ⁇ 10 ⁇ 3 mole/L concentration of betulonic acid.
  • Betulonic acid dissolved in neat ethanol was diluted with neat human serum to sustain it in solution as shown in Table 9.
  • Table 9 BA Dissolved in Neat Ethanol Diluted with Human Serum Con. of Con. of Betulonic Vol. of Ethanol Vol. of Human Total volume Betulonic Acid Betulonic Acid Acid (mg) ( ⁇ L) Serum ( ⁇ L) (mL) (mol/L) (mg/mL) Observation 1.34 31 700 731 4 ⁇ 10 ⁇ 3 1.8 Clear Solution
  • Table 9 show that 1.34 mg betulonic acid was dissolved in 31 ⁇ L neat ethanol and diluted with human serum to yield a 4.2% final concentration of ethanol and a 4 ⁇ 10 ⁇ 3 mol/L concentration of drug. The compounds remained soluble and are suitable for in vivo studies. Thus, improvements in the solubility of betulonic acid and its derivatives in biocompatible medium have been achieved.
  • betulonic acid and its lysinated derivatives may be completely dissolved in neat ethanol and diluted with PBS containing 4% human albumin (similar to the concentration of albumin in human serum) to achieve a final concentration of 10% ethanol to yield a 1 ⁇ 10 ⁇ 3 mol per liter concentration of betulonic acid.
  • PBS containing 4% human albumin similar to the concentration of albumin in human serum
  • solubilized betulonic acid and monomer-Boc at a final concentration of 22% ethanol and 2 ⁇ 10 ⁇ 3 mol per liter of betulonic acid are well tolerated by mice with prostate cancer cell xenografts.
  • MOI multiplicity of infection
  • PBS phosphate buffered saline
  • DMSO phosphate buffered saline
  • TSP thrombospondin peptide
  • Two HIV isolates were used, a patient isolate (“child HIV”), and the standard CXCR4 co-receptor utilizing isolate IIIB.
  • TSP peptide (“control”) 1 ⁇ g/ml betulinol (“OL”) 1 ⁇ g/ml (in DMSO) betulonic acid (“BOA”) 1 ⁇ g/ml (in DMSO) 3-acetoxy betulin (“BL”) 1 ⁇ g/ml (in DMSO) betulin dimethyl ether (“BDE”) 1 ⁇ g/ml (in DMSO) 28-acetoxy betulin (“BU”) 1 ⁇ g/ml (in DMSO) betulone aldehyde (“AL”) 1 ⁇ g/ml (in DMSO) betulin diacetate (“BA”) 1 ⁇ g/ml (in DMSO)
  • Cultures were maintained in culture medium (RPMI-1640+10% fetal bovine serum (“FBS”)) for 4 days, the culture supernatants were then collected, lysed with Triton®-X 100 surfactant, and HIV-1 gag (p24) antigen activity assessed by a standard technique, the Antigen Capture ELISA (enzyme-linked immunosorbent assay) (Roche-NEN).
  • FBS fetal bovine serum
  • Results are shown in FIG. 6 .
  • Data are presented in optical density (“OD”) units, which are linear with ng/ml of p24 Ag from 0.15 to 1.5 OD, and can be converted to pg/ml of HIV-1 antigen using a standard curve.
  • OD optical density
  • betulin dimethyl ether BDE
  • 3-acetoxy betulin BL
  • 28-acetoxy betulin BU
  • BDE betulin dimethyl ether
  • BL 3-acetoxy betulin
  • BU 28-acetoxy betulin
  • the anti-HIV activity of betulonic acid and betulin diacetate has previously been disclosed, for example, in U.S. Pat. No. 6,172,110 to Lee et al., which is hereby incorporated by reference in its entirety.
  • the anti-HIV activity of betulone aldehyde has previously been disclosed, for example, in U.S. Pat. Nos. 5,869,535 and 6,225,353 to Pezzuto et al., which are hereby incorporated by reference in their entirety.
  • betulin derivatives such as, for example, betulonic acid, betulin dimethyl ether, 3-acetoxy betulin, and 28-acetoxy betulin had no effect on total cell number or cell viability.
  • betulone aldehyde (AL) showed 37% inhibition
  • betulin diacetate (BA) showed 57% inhibition.
  • betulone aldehyde (AL) and betulin diacetate (BA) were tested for dose-related effects, with doses of 0.5, 1 .5, and 2 ⁇ g/ml. Progressive increases in anti-HIV effect were shown, again without cell toxicity.
  • varying doses of the parental compound betulinol (OL) (1.3, 1.6, and 2 ⁇ g/ml) showed increasing anti-HIV effect.
  • varying doses of 28-acetoxy betulin (BU) 0.5, 1, 1.5, and 2 ⁇ g/ml
  • BU 28-acetoxy betulin
  • Doses higher than 2 ⁇ g/ml could not be used, because the concentration of the vehicle used to dissolve these agents (DMSO) would be too high for the present culture system.
  • Viral isolates standard HIV-1 lab isolate IIIB, highly sensitive to all known anti-HIV compounds, and two patient isolates obtained from Haiti, with varying degrees of anti-HIV drug sensitivity.
  • Target cells CD4+ Jurkat and CEM-SS human T lymphoblasts, were grown in culture medium (RPMI 1640 plus 10% heat-inactivated FBS).
  • Human peripheral blood mononuclear cells (“PBMC”) were derived from heparinized venous blood by density gradient centrifugation using Ficol-paque (Amersham-Pharmacia).
  • PBMCs were pre-activated with 1 ⁇ g/ml phytohemagglutinin (“PHA”) and 32 U/ml interleukin-2 (“IL-2”) for 2-3 days prior to exposure to HIV-1.
  • PHA phytohemagglutinin
  • IL-2 interleukin-2
  • HIV-1 infections were performed as previously described herein. Briefly, 2.5 ⁇ 105 target cells (cell lines or PHA-activated PBMCs) were exposed to stock virus (500 pg of HIV-1 p24 antigen) for 2 h at 37° C., washed twice with PBS, and replated with fresh medium. One half of the culture supernatants were removed from each well every 3-4 days and replaced with fresh medium. At various times after viral inoculation, HIV-1 activity was determined by antigen capture ELISA (Roche-NEN) for HIV-1 p24 gag protein in Tritono-X 100 solubilized culture supernatants, as described.
  • Drugs The reverse transcriptase inhibitor AZT and the HIV protease inhibitors ritonavir and nelfinavir were used alone, and in potential synergy experiments with compounds of Formula I. The drugs were added to target cell cultures either before or after the two hour incubation of target cells with virus. AZT was used in concentrations of 0.01-5 ⁇ M and the protease inhibitors at concentrations of 0.5-10 ⁇ M.
  • NIH National Institute of Health
  • HIV enzyme assays HIV RT was assessed by ELISA (Roche-NEN) using the purified enzyme with polyrA/T as substrate and AZT as a positive control, with varying concentrations of compounds of Formula I added. HIV protease was similarly assessed using, as substrate, a 9 amino acid synthetic peptide spanning the p17/p24 junction of HIV gag. Specific activity against this peptide is 12.1 ⁇ M/min/mg over 10 min.
  • Compounds of Formula I were evaluated for cellular effects which might indicate toxicity or non-specific anti-viral properties. Effects of varying doses of compounds of Formula I on T cell proliferation was assessed by standard methods. In addition, potential induction of apoptosis by these compounds at the anti-HIV doses used, as well as at high concentrations of compounds was assessed.
  • Apoptosis identification Levels of apoptosis were assessed by TO-PRO-3 staining (VanHooijdonk, et al., Cytometry 17:185-189 (1994), which is hereby incorporated by reference in its entirety). Briefly, cells were air dried on slides fixed in 4% paraformalydehyde for 10 min. at room temperature, washed with PBS, and treated with 70% EtOH for 15 min. at ⁇ 20° C. The slides were fixed in a 1:9 solution of acetic acid:ethanol for 1 h, washed, then treated with 2% TritonlX-100 for 2 min., followed by exposure to RNAse A for 20 min. at 4° C.
  • HIV envelope proteins Recombinant HIV-1 gp120 of CXCR4 phenotype (obtained from NIH AIDS Program, described above) and CCR5 phenotype were used.
  • T cell targets bearing HIV co-receptors and CD4 (CEM-T) or co-receptors but no CD4 (CEM-SS) were utilized.
  • CEM-T co-receptors and CD4
  • CEM-SS co-receptors but no CD4
  • Different target cells bearing CXCR4 but not CCR5 (MO7E) were also used.
  • Varying concentrations of oligomeric X4 gp160 were added for 1 h at 37° C. to target cells. The cells were then washed and incubated with 10 pg/ml of human mAb 1331A, specific for the C terminus of gp120, or with a human mAb against the HIV-1 core protein p24 as a control, both conjugated to phycoerythrin (“PE”), and fluorescence intensity assessed. Displacement of a fixed amount of oligomeric viral envelope, as detected by the human anti-gp120 mAb, by increasing amounts of compounds of Formula I were examined. Positive controls for CD4 (monoclonal antibody) CXCR4 (SDF-1, 500 to 1500 ng/ml), and CCP5 (1500 ng/ml RANTES) were included.
  • CD4 monoclonal antibody
  • CXCR4 SDF-1, 500 to 1500 ng/ml
  • CCP5 1500 ng/ml RANTES
  • Plasmid constructs, plasmid transfections and reporter assays The reporter plasmid pC15CAT (Arya et al., Science 229:69-73 (1985), which is hereby incorporated by reference in its entirety) contains sequences for SV40 regulatory genes, bacterial chloramphenical acetyl transferase (“CAT”), and the HIV-1 long terminal repeat (“LTR”).
  • the HIV-1 tat plasmid pCV-I (Arya et al., Science 229:69-73 (1985), which is hereby incorporated by reference in its entirety) contains a 1.8 kb cDNA fragment encompassing both exons of tat.
  • Electrophoretic Mobility Shift Assay This is a standard assay for assessing NF ⁇ activity.
  • Target cells were exposed to compounds of Formula I alone, in the presence of a known NF ⁇ B activator (TNF- ⁇ ), or with HIV-1 for 48 h.
  • Nuclear extracts were then prepared using a Nuclear extract kit (Sigma).
  • H9 (lymphoma) cells 1.5 ⁇ 10 5 of H9 (lymphoma) cells were plated in each culture well in 1 mL of RPMI media containing 10% FBS in the presence of 0, 2, 5, 10, and 20 mM of betulonic acid and AZT and incubated at 37° C. On day 3, the drug effects on cell viability were assessed using Trypan Blue Dye Exclusion Assay. Results are set forth in FIG. 12 . The data is presented as both living cell counts and percentage. Chemical resources were obtained through Sigma Aldrich.
  • Acute HIV infection was performed using HIV-1 isolate IIIB stock virus.
  • CEM CD4+T cells (2.5 ⁇ 10 5 target cells) were exposed to stock virus at a MOI of either 0.02 or 0.15 for 2 h at 37° C., washed twice with PBS, and replated in tissue culture microwells with 0.3 ml of fresh culture medium.
  • Compounds of Formula I dissolved in DMSO were added into the culture and were tested for anti-HIV activity with reference to thrombospondin (TSP), a known anti-HIV drug.
  • TSP thrombospondin
  • HIV activity was determined on day seven using an ELISA antigen capture assay for HIV-1 p24 (Gag) core protein (Dupont Medical Products, Boston, Mass.) with Triton X-100 solubilized culture supernatants. Inhibition was calculated as percent of the control.
  • Thrombospondin (TSP) was used at a concentration of 1 mg/mL and yielded an inhibition of 51%.
  • Compounds of Formula I were also used at a concentration of 1 ug/mL. Results are set forth in FIG. 13 .
  • betulinol derivatives like betulinol aldehyde and diacetate and their conjugates, including peptide coupled compounds (e.g., attached via a lysine, histidine, arginine) will yield significantly higher inhibition to AZT-resistant HIV strains.
  • peptide coupled compounds e.g., attached via a lysine, histidine, arginine

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US20080286291A1 (en) 2008-11-20
EP1786433A4 (de) 2008-02-27
WO2006031706A3 (en) 2006-11-23
EA200700611A1 (ru) 2008-02-28
WO2006031706A2 (en) 2006-03-23
US20060154903A1 (en) 2006-07-13
EP1786433A2 (de) 2007-05-23
US20090176753A1 (en) 2009-07-09
US8008280B2 (en) 2011-08-30
WO2006031756A3 (en) 2007-02-08
US8088757B2 (en) 2012-01-03
EP1802648A2 (de) 2007-07-04

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