Classifications

• • 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
  • A61K31/695—Silicon compounds
• • 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
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
  • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
  • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07J—STEROIDS
  • C07J63/00—Steroids in which the cyclopenta(a)hydrophenanthrene skeleton has been modified by expansion of only one ring by one or two atoms
  • C07J63/008—Expansion of ring D by one atom, e.g. D homo steroids
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
  • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

• Betulin is a pentacyclic triterpenoid derived from the outer bark of paper birch trees (Betula paperifera). It can be present at concentrations of up to about 24% of the bark of white birch. Merck Index, twelfth edition, page 1236, 1996.
• Lupeol is a related compound also found in birch bark and in other plant sources. Lupeol is present at concentrations of about 1.5-3% of birch bark and at up to about 8.2%> in Canavalia ensiformis, a plant widespread in the humid tropics of Asia, India, and Africa.
• AUobetulin is another triterpenoid found in birch bark. A typical pulp mill that processes birch produces enough bark waste to allow for the inexpensive isolation of significant quantities of these triterpenoids.
• betulin and related compounds have been shown to have anti-viral activity against herpes simplex virus. Carlson et al., U.S. Patent No. 5,750,578.
• Bacteria are very common pathogens of humans. Among the bacterial species that cause serious disease are the gram negative bacterium Escherichia coli and gram positive bacteria of the genus Staphylococcus. Staphylococcus aureus is the most serious pathogen of the Staphylococcus bacteria. It is estimated to causes 13% of the 2 million hospital infections each year, and result in 80,000 deaths in the United States. Staphylococcal infections occur most commonly in persons weakened by poor health or immunodeficiency.
• Antibiotic resistance of bacteria is a growing problem. New agents active against bacteria are needed. A need particularly exists for agents that will act against a range of species, including gram-negative and gram-positive species. Ideally, new agents would also be inexpensive to manufacture. New anti-bacterial agents would be less expensive to manufacture if they were abundant natural products or were easily synthesized from abundant natural products.
• the present invention provides a compound of formula (I):
• R j is hydrogen or hydroxy
• R 2 is a direct bond, carbonyl, oxy, thio, carbonyl oxy, oxy carbonyl, (C 6 - C ⁇ 0 )aryl, or (C r C 6 )alkyl;
• O P(OH) 2 OP(O)(OH)- 3 (C r C 5 )alkanoyl.
• Si(R) 3 wherem each R is H, phenyl or (C r C 6 )alkyl, C(O)N(R) 2 , benzyl, benzoyl, tetrahydropyran-2-yl, l-[(C r C 4 )alkoxy](C,-C 4 )alkyl, or a glycoside;
• the present invention also provides a compound of formula (II):
• R 3 is hydrogen, halo, carboxy, mercapto, (C 1 -C 6 )alkyl, (C 3 - C 8 )cycloalkyl, or -O-Y;
• R 4 and R 5 are each independently hydrogen, (C,-C 6 )alkyl or hydroxy(C r
• R 6 is hydrogen or is absent when the adjacent — is a bond;
• R 7 is hydrogen or (C r C 6 )alkyl;
• R 8 is hydrogen, (C r C 6 )alkyl or hydroxy(C j -C 6 )alkyl and
• R 9 and R 10 are each independently hydrogen or (C,-C 6 )alkyl; each of the bonds represented by — is independently absent or is present;
• R a is (C r C 6 )alkyl or aryl
• R b , R c , and R d are each independently hydroxy, (C r C 6 )alkoxy, hydroxy(C 2 -C 6 )alkoxy, adamantyloxy, adamantyl(C 1 -C 6 )alkoxy, norbornyloxy, lJ-di(hydroxymethyl)-2-hydroxyethoxy, carboxy(C 1 -C 6 )alkoxy, 2,3- epoxypropyloxy, benzyloxy, (C 3 -C 8 )cycloalkyloxy, NR x R y , or aryloxy;
• R e is H, aryl or (C r C 6 )alkyl
• R f is hydrogen, (C j -C ⁇ alkyl, (C 1 -C 6 )alkanoyl, phenyl or benzyl;
• R g and R h are each independently selected from the group consisting of hydrogen, (C 1 -C 6 )alkyl, hydroxy(C r C 6 )alkyl, adamantyl, adamantyl(C 1 -C 6 )alkyl, amino(C 1 -C 6 )alkyl, aminosulfonyl, (C r C 6 )alkanoyl, aryl and benzyl; or R b and R,. together with the nitrogen to which they are attached form a pyrrolidino, piperidino, or morpholino radical; and
• R x and R y are each independently hydrogen, (C r C 6 )alkyl. (C C 6 )alkanoyl, aryl or benzyl; wherein each aryl of Y, R a -Rj , R g -R h , R x , and R y may optionally be substituted by 1, 2, or 3 aminosulfonyl, carboxy, NR j R j (C 1 -C 6 )alkyl, (C,- C 6 )alkoxy, hydroxy, halo, nitro, cyano, mercapto, carboxy, hydroxy(C r C 6 )alkyl, halo(C 1 -C 6 )alkyl, trifluoromethoxy, (C 1 -C 6 )alkanoyl, (C r C 6 )alkoxycarbonyl, (C r C 6 )alkylthio, or (C ] -C 6 )alkano
• the present invention also provides a method of inhibiting or killing a bacterium, the method comprising contacting the bacterium with an effective antibacterial amount of a triterpene of formula (I):
• Rj is hydrogen or hydroxy
• R 2 is a direct bond, carbonyl, oxy, thio, carbonyl oxy, oxy carbonyl, (C 6 - C 10 )aryl. or (C r C 6 )alkyl;
• the present invention also provides a method of inhibiting or killing a bacterium, the method comprising contacting the bacterium with an effective antibacterial amount of a triterpene of formula (II):
• R 3 is hydrogen, halo, carboxy, mercapto, (C r C 6 )alkyl, (C 3 -C 8 )cycloalkyl, or -O-Y;
• R 4 and R 5 are each independently hydrogen, (C 1 -C 6 )alkyl or hydroxy(C r C 6 )alkyl; R 6 is hydrogen or is absent when the adjacent — is a bond;
• R 7 is hydrogen or (C,-C 6 )alkyl
• R a is (C,-C 6 )alkyl or aryl
• R b , R c , and R d are each independently hydroxy, (C r C 6 )alkoxy, hydroxy(C 2 -C 6 )alkoxy, adamantyloxy, adamantyl(C ⁇ -C 6 )alkoxy, norbornyloxy, lJ-di(hydroxymethyl)-2-hydroxyethoxy, carboxy ⁇ -C ⁇ alkoxy, 2,3- epoxypropyloxy, benzyloxy, (C 3 -C 8 )cycloalkyloxy, NR i y , or aryloxy;
• R e is H, aryl or (C r C 6 )alkyl
• R f is hydrogen, (C C 6 )alkyl, (C r C 6 )alkanoyl, phenyl or benzyl;
• R g and R h are each independently selected from the group consisting of hydrogen, (C j -C ⁇ alkyl, hydroxy(C 1 -C 6 )alkyl, adamantyl, adamantyl(C,-C 6 )alkyl, aminosulfonyl, (C,-C 6 )alkanoyl, aryl and benzyl; or R b and R- together with the nitrogen to which they are attached form a pyrrolidino, piperidino, or morpholino radical; and
• R_. and R y are each independently hydrogen, (C r C 6 )alkyl, (C r C 6 )alkanoyl, aryl or benzyl; wherein each aryl of Y, R a -R d , R g -R h , R ⁇ > and R y may optionally be substituted by 1, 2, or 3 aminosulfonyl, carboxy, NR j R,- (C r C 6 )alkyl, (C j - C 6 )alkoxy, hydroxy, halo, nitro, cyano, mercapto, carboxy, hydroxy(C C 6 )alkyl, halo(C r C 6 )alkyl, trifluoromethoxy, (C 1 -C 6 )alkanoyl, (C j -C ⁇ alkoxycarbonyl, (C r C 6 )alkylthio, or (C j -C 6 )alkanoy
• R is hydrogen or hydroxy
• R 2 is a direct bond, carbonyl, oxy, thio, carbonyl oxy, oxy carbonyl, (C 6 - C 10 )aryl, or (C r C 6 )alkyl;
• the present invention also provides a method of inhibiting or killing a bacterium, the method comprising contacting the bacterium with an effective antibacterial amount of a triterpene of formula (I):
• R ! is hydrogen or hydroxy
• R 2 is a direct bond, carbonyl, oxy, thio, carbonyl oxy, oxy carbonyl, (C 6 -
• the invention provides novel compounds of formula (I) and formula (II), intermediates for the synthesis of compounds of formula (I) and formula (II), as well as methods of preparing compounds of formula (I) and (II).
• the invention also provides compounds of formula (I) and (II) that are useful as intermediates for the synthesis of other useful compounds.
• the invention provides for the use of compounds of formula (I) and formula (II) for the manufacture of medicaments useful for the treatment of bacterial infections in a mammal, such as a human.
• halo is fluoro, chloro, bromo, or iodo.
• Alkyl, alkoxy, alkenyl, etc. denote both straight and branched groups; but reference to an individual radical such as "propyl” embraces only the straight chain radical, a branched chain isomer such as "isopropyl” being specifically referred to.
• Aryl denotes a phenyl radical or an ortho-fused bicyclic carbocyclic radical having about nine to ten ring atoms in which at least one ring is aromatic. It will be appreciated by those skilled in the art that compounds useful in the invention having a chiral center may exist in and be isolated in optically active and racemic forms.
• the present invention encompasses any racemic, optically- active, polymorphic, or stereoisomeric form, or mixtures thereof, of a compound useful in the invention, which possess the useful properties described herein, it being well known in the art how to prepare optically active forms (for example, by resolution of the racemic form by recrystallization techniques, by synthesis from optically-active starting materials, by chiral synthesis, or by chromatographic separation using a chiral stationary phase) and how to determine antibacterial activity using the standard tests described herein, or using other similar tests which are well known in the art.
• (C r C 6 )alkyl can be methyl, ethyl, propyl, isopropyl, butyl, iso-butyl, sec-butyl, pentyl, 3-pentyl, or hexyl; partially unsaturated (C 2 -C 6 )alkyl or (C 2 -C 6 )alkenyl can be vinyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 3- butenyl, 1,-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1- hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, or 5-hexenyl; (C,-C 5 )alkanoyl can be vinyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 3- butenyl, 1,-pentenyl, 2-pentenyl, 3-
• 2-carboxybenzoyl refers to the group
• 3-carboxy-3-methylbutanoyl refers to the group -
• amino acid comprises the residues of the natural amino acids (e.g. Ala, Arg, Asn, Asp, Cys, Glu, Gin, Gly, His, Hyl, Hyp, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Nal) in D or L form, as well as unnatural amino acids (e.g.
• the term also comprises natural and unnatural amino acids bearing a conventional amino protecting group (e.g.
• acetyl or benzyloxycarbonyl as well as natural and unnatural amino acids protected at the carboxy terminus (e.g. as a (C 1 -C 6 )alkyl, phenyl or benzyl ester or amide; or as an -methylbenzyl amide).
• suitable amino and carboxy protecting groups are known to those skilled in the art (See for example, T.W. Greene, Protecting Groups In Organic Synthesis; Wiley: New York, Third Edition, 1999, and references cited therein).
• An amino acid can be linked to the remainder of a compound of formula I or II through the carboxy terminus, the amino terminus, or through any other convenient point of attachment, such as, for example, through the sulfur of cysteine.
• peptide describes a sequence of 2 to 25 amino acids (e.g. as defined herein) or peptidyl residues.
• the sequence may be linear or cyclic.
• a cyclic peptide can be prepared or may result from the formation of disulfide bridges between two cysteine residues in a sequence.
• a peptide can be linked to the remainder of a compound of formula I or II through the carboxy terminus, the amino terminus, or through any other convenient point of attachment, such as, for example, through the sulfur of a cysteine.
• a peptide comprises 3 to 25, or 5 to 21 amino acids.
• Peptide derivatives can be prepared as disclosed in U.S. Patent Numbers 4,612,302; 4,853,371; and 4,684,620.
• Glycosides are formed by reacting mono-, di- and polysaccharides with 1-2 hydroxyl groups of the compound of formula (I) or formula (II), including glucose, glucuronic acid, mannose, galactose, sorbase, ribose, maltose, sucrose, modified cellulosics, dextrans, modified starches and the like. These derivatives can advantageously exhibit improved water solubility over betulin itself. See,
• Polyethyleneimine refers to the group (-NHCH 2 CH 2 -) X [- N(CH 2 CH 2 NH 2 )CH 2 CH 2 -] y . Polyethyleneimine can be attached to a compound of formula I or II through either of the nitrogen atoms marked with hash marks. "Poly(ethylene glycol)” refers to the compound H(OCH 2 CH 2 ) n OH. It can be attached to a compound of formula I or II through the terminal hydroxyl group.
• partially unsaturated refers to a linear or branched hydrocarbon having one or more carbon-carbon double bonds.
• stable compound is meant herein a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and formulation into an efficacious antibacterial agent.
• bacteria or "bacteria” refers to any prokaryotic organism.
• a specific value for the bond between carbons 1 and 2 is a single bond.
• Another specific value for the bond between carbons 1 and 2 is a double bond.
• a specific value for R is hydrogen.
• Another specific value for R is hydroxy.
• a specific value for R 2 is a direct bond.
• a specific value for R 5 is oxy or a direct bond.
• R is hydrogen or hydroxy
• R 2 is a direct bond
• R 4 is 2-carboxybenzoyl, 2-amino-3 -methylbutanoyl, 3-carboxypropenoyl, aminoacetyl, 4-carboxybutanoyl, (carboxymethoxy)acetyl, 3 -(3 ,4- dihydroxyphenyl)propenoyl, carboxymethylenethioacetyl, 3-
• Another specific group of compounds of formula (I) is betulin; betulin- 3,28-diglycine; betulin-28-glycerol oxalate; betulin-28-glycine; betulin-28- oxalate; betulin arabinose galactan; betulin-3,28didiglycolate; betulin-3,28- diglycine; betulin-3-maleate; betulin-3,28- di-(L-Glutamic acid r-benzylester) ester; bemlin-3,28-di-L-alanine; betulin-3,28-di-L-proline; betulin-3,28- dioxalate; betulin- l-ene-2-ol; betulin-3,28-diphenylalanine; betulin-3-28- dioxalate-polyethylene amine; betulin-3,38-diphosphate; betulin-3-caffeate; betulin-3,28-
• Another specific group of compounds of formula (I) is betulin; betulin- 28-glycerol oxalate; betulin-28-oxalate; betulin arabinose galactan; betulin-3,28- didiglycolate; betulin-3,28- diglycine; betulin-3,28-di-(L-glutamic acid ⁇ - benzylester) ester; betulin3,28-di-L-proline ester; betulin-3,28-dioxalate; betulin- l-ene-2-ol; betulin-3,28-dioxalate-polyethylene amine; betulin-3,28- diphosphate; betulin-3-caffeate; betulin-28-diglycolate; betulin-28-glutarate; betulin-28-maleate; betulin-28-phthalate; betulin-3,28-dithiodiglycolate; betulin- 3,28-diglutarate
• Another specific group of compounds of formula (I) is betulin-3-caffeate; betulin-28-diglycolate; betulin-3,28-diglutarate; betulin-3,28-diglycine; betulin- 3,28-didiglycolate; betulin-3,28-dimaleate; betulin-3,28-diphosphate; betulin- 3,28-diphthalate; betulin-3,28-di-L-valine; lupeol; lupeol-3 -amine; lupeol-3- (3',3'-dimethyl)succinate; lupeol-3 -maleate; lupeol-3 -phosphate; lupeol-3- thiodiglycolate; lupenone; lupenon-l,2-ene-2-ol; or lupenon-3-oxime.
• a specific group of compounds of formula (II) is 3- ⁇ -acetoxy-19 H- 19,28 lactone oleanan; allobetulin; allobetulin-3-succinate; allobetulin-3-glycine ester; allobetulin lactone; allobetulin lactone-3 -acetate; allobetulin lactone-3- phosphate; allobetulin-3-L-alanine; allobetulin-3-L-valine; allobetulin-3-L- proline; allobetulin-3 -succinate; allobetulin-3-diglycolate; allobetulin-3- glutarate; allobetulin-3 -phthalate; allobetulin-3 -methylenamine; allobetulin-3- ethanolamine; allobetulin-3 -ethanolamine hydrochloride; allobetulin-3-glycolate; allobetulin-3- glutarate; allobetulin-28-glutarate; allobe
• Another specific group of compounds of formula (II) are 3- ⁇ -acetoxy- 19 ⁇ H-19,28 lactone oleanan; allobetulin; allobetulin-3 -glycine ester; allobetulin lactone-3 -phosphate; allobetulin-3 -succinate; allobetulin-3-ethanolamine; allobetulin-3-glutarate; allobetulin-28-glutarate; allobetulin-3 -methylamine HCl; allobetulin-3-phosphate; allobetulon; allobetulon lactone- l-ene-2-ol; 3- allobetulon-l-ene-2-succinate; orursolic acid.
• a specific method of the invention is the method of treating a mammal afflicted with a bacterial infection comprising administering to the mammal an effective anti-bacterial amount of a compound of formula (I) or formula (II), wherein the bacterial infection is caused by Escherichia coli, Staphylococcus sp.,
• Another specific method of the invention is the method of treating a mammal afflicted with a bacterial infection comprising administering to the mammal an effective anti-bacterial amount of a compound of formula (I) or formula (II), wherein the bacterial infection is caused by Staphylococcus aureus.
• Another specific method of the invention is the method of inhibiting or killing a bacterium or bacteria comprising contacting the bacterium with an effective antibacterial amount of a triterpene of formula (I) or formula (II), wherein the bacterium is Escherichia coli, Staphylococcus sp., Enterococcus faecalis, or a combination thereof.
• Another specific method of the invention is the method of inhibiting or killing a bacterium or bacteria comprising contacting the bacterium with an effective antibacterial amount of a triterpene of formula (I) or formula (II) wherein the bacterium is Staphylococcus aureus.
• Another specific method of the invention is the method of inhibiting or killing a bacterium or bacteria comprising contacting the bacterium with an effective antibacterial amount of a triterpene of formula (I) wherein the contacting is in vivo.
• Another specific method of the invention is the method of inhibiting or killing a bacterium or bacteria comprising contacting the bacterium with an effective antibacterial amount of a triterpene of formula (I) wherein the contacting is in vitro .
• Another specific method of the invention is the method of inhibiting or killing a bacterium or bacteria comprising contacting the bacterium with an effective antibacterial amount of a triterpene of formula (II) wherein the contacting is in vivo.
• Another specific method of the invention is the method of inhibiting or killing a bacterium or bacteria comprising contacting the bacterium with an effective antibacterial amount of a triterpene of formula (II) wherein the contacting is in vitro.
• lu ⁇ enon-l,2-ene-2-ol has a double bond between carbons 1 and 2.
• the other compounds in Table 1 have a single bond at that position.
• Processes for preparing compounds of formula (I) and formula (II) are provided as further embodiments of the invention and are illustrated by the following procedures in which the meanings of the generic radicals are as given above unless otherwise qualified.
• the compounds of formula (I) or formula (II) can be prepared from convenient starting materials, employing procedures (e.g., reagents and reaction conditions) known to those of skill in the art.
• suitable reagents and reaction conditions are disclosed, e.g., in Advanced Organic Chemistry, Part B: Reactions and Synthesis, Second Edition, Carey and Sundberg (1983); Advanced Organic Chemistry, Reactions, Mechanisms, and Structure, Second Edition, March (1977); Greene, T.W.; Wutz, P.G.M. Protecting Groups In Organic Synthesis, Second Edition, 1991, New York, John Wiley & sons, Inc.; and Comprehensive Organic Transformations, Second Edition, Larock (1999). Additionally, specific exemplary procedures are shown in the examples herein below.
• salts are organic acid addition salts formed with acids which form a physiological acceptable anion, for example, tosylate, methanesulfonate, acetate, citrate, malonate, tartarate, succinate, benzoate, ascorbate, -ketoglutarate, and ⁇ -glycerophosphate.
• Suitable inorganic salts may also be formed, including hydrochloride, sulfate, nitrate, bicarbonate, and carbonate salts.
• salts may be obtained using standard procedures well known in the art, for example by reacting a sufficiently basic compound such as an amine with a suitable acid affording a physiologically acceptable anion.
• a sufficiently basic compound such as an amine
• a suitable acid affording a physiologically acceptable anion.
• Alkali metal (for example, sodium, potassium or lithium) or alkaline earth metal (for example calcium) salts of carboxylic acids can also be made.
• the compounds of formula I or II can be formulated as pharmaceutical compositions and administered to a mammalian host, such as a human patient in a variety of forms adapted to the chosen route of administration, i.e., orally or parenterally, by intravenous, intramuscular, topical or subcutaneous routes.
• the present compounds maybe systemically administered, e.g., orally, in combination with a pharmaceutically acceptable vehicle such as an inert diluent or an assimilable edible carrier. They may be enclosed in hard or soft shell gelatin capsules, may be compressed into tablets, or may be incorporated directly with the food of the patient's diet.
• a pharmaceutically acceptable vehicle such as an inert diluent or an assimilable edible carrier.
• the active compound may be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.
• Such compositions and preparations should contain at least 0.1% of active compound.
• the percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 60% of the weight of a given unit dosage form.
• the amount of active compound in such therapeutically useful compositions is such that an effective dosage level will be obtained.
• the tablets, troches, pills, capsules, and the like may also contain the following: binders such as gum tragacanth,. acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, fructose, lactose or aspartame or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring may be added.
• a liquid carrier such as a vegetable oil or a polyethylene glycol.
• any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed.
• the active compound may be incorporated into sustained-release preparations and devices.
• the active compound may also be administered intravenously or intraperitoneally by infusion or injection.
• Solutions of the active compound or its salts can be prepared in water, optionally mixed with a nontoxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
• the pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredient which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes.
• the ultimate dosage form should be sterile, fluid and stable under the conditions of manufacture and storage.
• the liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof.
• the proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants.
• the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, buffers or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
• Sterile injectable solutions are prepared by incorporating the active compound in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter sterilization.
• the preferred methods of preparation are vacuum drying and the freeze drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile-filtered solutions.
• the present compounds may be applied in pure form, i.e., when they are liquids. However, it will generally be desirable to administer them to the skin as compositions or formulations, in combination with a dermatologically acceptable carrier, which may be a solid or a liquid.
• Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina and the like.
• Useful liquid carriers include water, alcohols or glycols or water-alcohol/glycol blends, in which the present compounds can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants.
• Adjuvants such as fragrances and additional antimicrobial agents can be added to optimize the properties for a given use.
• the resultant liquid compositions can be applied from absorbent pads, used to impregnate bandages and other dressings, or sprayed onto the affected area using pump-type or aerosol sprayers.
• Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses or modified mineral materials can also be employed with liquid carriers to form spreadable pastes, gels, ointments, soaps, and the like, for application directly to the skin of the user.
• compositions which can be used to deliver the compounds of formula I or II to the skin are known to the art; for example, see Jacquet et al. (U.S. Pat. No. 4,608,392), Geria (U.S. Pat. No.
• Useful dosages of the compounds of formula I or II can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art; for example, see U.S. Pat. No. 4,938,949.
• the concentration of the compound(s) of formula I or II in a liquid composition will be from about OJ-25 wt-%, preferably from about 0.5-10 wt-%.
• concentration in a semi-solid or solid composition such as a gel or a powder will be about OJ-5 wt-%, preferably about 0.5-2.5 wt-%.
• the amount of the compound, or an active salt or derivative thereof, required for use in treatment will vary not only with the particular salt selected but also with the route of administration, the nature of the condition being treated and the age and condition of the patient and will be ultimately at the discretion of the attendant physician or clinician.
• a suitable dose will be in the range of from about 0.5 to about 100 mg/kg, e.g., from about 10 to about 75 mg/kg of body weight per day, such as 3 to about 50 mg per kilogram body weight of the recipient per day, preferably in the range of 6 to 90 mg/kg/day, most preferably in the range of 15 to 60 mg/kg/day.
• the compound is conveniently administered in unit dosage form; for example, containing 5 to 1000 mg, conveniently 10 to 750 mg, most conveniently, 50 to 500 mg of active ingredient per unit dosage form.
• the active ingredient should be administered to achieve peak plasma concentrations of the active compound of from about 0.5 to about 75 ⁇ M, preferably, about 1 to 50 ⁇ M, most preferably, about 2 to about 30 ⁇ M.
• This may be achieved, for example, by the intravenous injection of a 0.05 to 5% solution of the active ingredient, optionally in saline, or orally administered as a bolus containing about 1-100 mg of the active ingredient. Desirable blood levels maybe maintained by continuous infusion to provide about 0.01-5.0 mg/kg/hr or by intermittent infusions containing about 0.4- 15 mg/kg of the active ingredient(s).
• the desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per day.
• the sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced administrations; such as multiple inhalations from an insufflator or by application of a plurality of drops into the eye.
• the compounds of the invention maybe also be useful as pharmacological tools for the further investigation of the mechanism of their antibacterial action.
• the compounds of the invention can also be administered in combination with other therapeutic agents that are effective to treat bacterial infections, or to inhibit or kill a bacteria.
• the system used to name the compounds of the invention will be clear to one of skill in the art based on the following examples. Names generally consist of the base structure, e.g., betulin, allobetulin, or lupeol, followed by a substituent.
• betulin-28-succinate with the structure shown in Example 1, consists of a succinic acid molecule esterified to the hydroxyl at carbon 28 of betulin. If no number is given for the substituent, the substitent is attached to the hydroxyl at carbon 3 on the base structure.
• Betulin- l-ene-2-ol is a compound of formula (I), wherein the bond between carbons 1 and 2 is a double bond, R j is hydroxyl, R 2 and R 3 together are hydroxymethyl, and R 4 and R 5 together are oxo.
• Uvaol is a compound of formula (II), wherein R 10 is methyl, Re, is hydrogen, R 8 is methyl, R 7 is hydrogen, R ⁇ is hydroxymethyl, R 6 is absent and the bond between carbons 12 and 13 is double, R 3 is hydrogen, R 4 and R 5 are methyl, R 2 is hydrogen, and R j is hydroxy.
• Oleanolic acid has the same structure as uvaol, except it has a carboxy at R ⁇ instead of hydroxymethyl.
• the structure of hederin hydrate is disclosed at page 871 of the Aldrich Chemical Co. 2000- 2001 catalog.
• the structure of other named compounds can be found in standard sources such as the Merck Index.
• "Betulin arabinose galactan” refers to betulin in a solution of arabino-galactan.
• betulin-3, 28-diglycine is the same compound as betulin-3,28-diglycine ester.
• Betulin-28-succinate m 0.200 g
• Betulin-3 ,28-disuccinate m 0.200 g 38 H 58 0 8
• Betulin-3,28-didiglycolate m 0.200g 38 H 58 O 10
• Betulin-3 ,28 -diglutarate m 0.300 g
• Betulin-3 ,28-dimaleate m 0.300g 38 H 54 O 8
• Betulin-28-maleate m 0.300g 34 H 52 O 5
• Betulin-3 ,28-diphthalate m 0.300g 6 H 58 O 8
• Betulin was isolated from paper birch (B. papyrifera) bark. Shredded, dry bark (500 g) has been extracted with chloroform on a Soxhlet apparatus for 10 hours. The extract was evaporated and then was left overnight at 5-7°C. Crystals were filtered and washed with hexane and then dried in oven to give 94.5 g of crude Betulin. Double crystallization from chloroform and then mixture of chloroform-isopropyl alcohol(4:l) gives 64-68 g of pure Betulin mp. 258-259°C [lit mp 256-261°C].
• Betulinic aldehyde (1.5 g) was dissolved in 45 ml of ethyl acetate and then was placed in a 100 ml heatable column. 0.6 ml of distilled water and 23 mg of ABIN was added to the solution. Oxygen has been bubbled through the mixture at 50-60°C for 6 hours with periodic addition of ABIN (5 mg per hour). Evaporation of solvent and following crystallization from MeOH gives 1.42 g of white crystals mp. 288-291°C [lit.
• Betulin-3 ,28-dioxalylchloride:polyethylenimine (MW av 600) ratio a:b l:l;
• Betulin-3 ,28-dioxalylchloride:polyethylenimine (MW av 600) ratio a:b l:3;
• Betulin-3,28-dioxalylchloride:polyethylenimine (MW av 600) ratio a:b l:10;
• Betulin-3,28-dioxalylchloride:polyethylenimine (MW av 600) ratio a:b l:l;
• Betulin-3 ,28-dioxalylchloride:polyethylenimine (MW av 600) ratio a:b l:2;
• E. coli is a gram- negative, non-spore forming, rod-shaped, opportunistic pathogen that resides in the human digestive tract.
• S. aureus is a gram positive coccus, is usually found morphologically as a packet, and is an opportunistic human pathogen, often residing on or in the skin.
• B. subtilis is a gram positive spore-forming rod.
• Control disks impregnated with DMSO or 0.85% saline produced no zone of inhibition.
• betulin-3 ,28-diphosphate, betulin-3,28- diglutarate, and betulin-3 ,28-didiglycolate were effective (Tables la and lb). Each had a a minimum inhibitory concentration of 0.282 ⁇ M (data not shown).
• the compounds effective against S. aureus were betulin-3 ,28-dimaleate and betulin-3,28-diphthalate (Tables la and lb). Each had a minimum inhibitory concentration of 2.3 ⁇ M.
• Betulin-3 ,28-dimaleate was more effective against the laboratory strain (not methiciUin resistant) S. aureus than streptomycin.
• S. aureus was inhibited by 56 ng of betulin-3 ,28-dimaleate applied to the disk, while inhibition by streptomycin required 10 ⁇ g.
• E. coli was sensitive to betulin-3,28-diglycine, betulin-3 -caffeate, and betulin-28-diglycolate.
• S. aureus was sensitive to betulin-3, 28-di-L-valine, as well as to betulin-3 ,28-diphthalate. No compounds were found that inhibited B. subtilis.
• lupeol Derivatives of lupeol were tested for inhibition of bacterial growth using the Kirby-Bauer disk diffusion method and by growth in liquid culture.
• the disk diffusion method compounds were dissolved in DMSO and 100 ⁇ l was added to the 10 mm filter paper disk. The amount of compound applied to the disk varied.
• the amount applied for each compound was as follows: lupeol (50 ⁇ g), lupeol-3 -maleate (15 ⁇ g), lupeol-3-thiodiglycolate (15 ⁇ g), lupeol-3- dimethyl succinate (60 ⁇ g), lupeol-3 -phosphate (30 ⁇ g), lupenone (50 ⁇ g), lupenon-3-oxime (50 ⁇ g), lupeol-3-amine (50 ⁇ g), lupenon-l,2-ene-2-ol (50 ⁇ g).
• the compounds were dissolved in DMSO at 1 mg/ml and then 100 ⁇ l was added to 7 ml of sterile broth, along with a 1/200 dilution of freshly grown bacterial liquid culture.
• the strains tested were Staphylococcus aureus, Staphylococcus epidemidis, and Enterococcus faecalis.
• Tables 3 and 4. Antibacterial activities against Staphylococcus aureus, Staphylococcus epidemidis, and Enterococcus faecalis using the Kirby-Bauer disk diffusion method.
• Table 4 Antibacterial activity against Staphylococcus aureus, Staphylococcus epidermis, and Enterococcus faecilis using optical density measurements. Optical density at 600 nm of bacteria in liquid culture was measured after growth for 24 hours in the presence of the test compound at 14 ⁇ g per ml.
• the compounds shown to be active against at least one strain of bacteria in the assays above are the following: betulin-3-caffeate; beralin-3,28- diglutarate; betulin-28-diglycolate; betulin-3, 28-diglycine; betulin-3 ,28- didiglycolate; betulin-3,28-diphthalate; betulin-3,28-diphosphate; betulin-3,28- disuccinate; betulin-3,28-di-L-valine; lupeol; lupeol-3 -amine; lupeol-3-(3',3'- dimethyl)succinate; lupeol-3-maleate; lupeol-3-phosphate; lupeol-3- thiodiglycolate; lupenone; lupenon- l,2-ene-2-ol; lupenon-3-oxime.

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• 2001
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