WO1994003164A1 - Inhibition and treatment of infection by enveloped virus with calix(n) arene compounds - Google Patents

Inhibition and treatment of infection by enveloped virus with calix(n) arene compounds Download PDF

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Publication number
WO1994003164A1
WO1994003164A1 PCT/US1993/007366 US9307366W WO9403164A1 WO 1994003164 A1 WO1994003164 A1 WO 1994003164A1 US 9307366 W US9307366 W US 9307366W WO 9403164 A1 WO9403164 A1 WO 9403164A1
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compound
group
hsv
virus
calix
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PCT/US1993/007366
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French (fr)
Inventor
Kou M. Hwang
You Mao Qi
Su-Ying Liu
William Choy
Jen Chen
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Genelabs Technologies, Inc.
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Priority claimed from US07/928,108 external-priority patent/US5441983A/en
Application filed by Genelabs Technologies, Inc. filed Critical Genelabs Technologies, Inc.
Priority to AU48033/93A priority Critical patent/AU4803393A/en
Publication of WO1994003164A1 publication Critical patent/WO1994003164A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C59/00—Compounds having carboxyl groups bound to acyclic carbon atoms and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups
    • C07C59/40—Unsaturated compounds
    • C07C59/42—Unsaturated compounds containing hydroxy or O-metal groups
    • C07C59/52—Unsaturated compounds containing hydroxy or O-metal groups a hydroxy or O-metal group being bound to a carbon atom of a six-membered aromatic ring
    • 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
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/18—Sulfonamides
    • 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
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • 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
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • 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
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/255—Esters, e.g. nitroglycerine, selenocyanates of sulfoxy acids or sulfur analogues thereof
    • 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
    • A61K31/66—Phosphorus 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
    • A61K31/74—Synthetic polymeric materials
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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
    • A61K31/74—Synthetic polymeric materials
    • A61K31/795—Polymers containing sulfur
    • 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
    • A61K31/74—Synthetic polymeric materials
    • A61K31/80—Polymers containing hetero atoms not provided for in groups A61K31/755 - A61K31/795
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    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C309/00—Sulfonic acids; Halides, esters, or anhydrides thereof
    • C07C309/01—Sulfonic acids
    • C07C309/02—Sulfonic acids having sulfo groups bound to acyclic carbon atoms
    • C07C309/03—Sulfonic acids having sulfo groups bound to acyclic carbon atoms of an acyclic saturated carbon skeleton
    • C07C309/07—Sulfonic acids having sulfo groups bound to acyclic carbon atoms of an acyclic saturated carbon skeleton containing oxygen atoms bound to the carbon skeleton
    • C07C309/09—Sulfonic acids having sulfo groups bound to acyclic carbon atoms of an acyclic saturated carbon skeleton containing oxygen atoms bound to the carbon skeleton containing etherified hydroxy groups bound to the carbon skeleton
    • C07C309/11—Sulfonic acids having sulfo groups bound to acyclic carbon atoms of an acyclic saturated carbon skeleton containing oxygen atoms bound to the carbon skeleton containing etherified hydroxy groups bound to the carbon skeleton with the oxygen atom of at least one of the etherified hydroxy groups further bound to a carbon atom of a six-membered aromatic ring
    • C—CHEMISTRY; METALLURGY
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    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C309/00—Sulfonic acids; Halides, esters, or anhydrides thereof
    • C07C309/01—Sulfonic acids
    • C07C309/02—Sulfonic acids having sulfo groups bound to acyclic carbon atoms
    • C07C309/24—Sulfonic acids having sulfo groups bound to acyclic carbon atoms of a carbon skeleton containing six-membered aromatic rings
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    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C309/00—Sulfonic acids; Halides, esters, or anhydrides thereof
    • C07C309/01—Sulfonic acids
    • C07C309/28—Sulfonic acids having sulfo groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton
    • C07C309/41—Sulfonic acids having sulfo groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton containing singly-bound oxygen atoms bound to the carbon skeleton
    • C07C309/43—Sulfonic acids having sulfo groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton containing singly-bound oxygen atoms bound to the carbon skeleton having at least one of the sulfo groups bound to a carbon atom of a six-membered aromatic ring being part of a condensed ring system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C309/00—Sulfonic acids; Halides, esters, or anhydrides thereof
    • C07C309/01—Sulfonic acids
    • C07C309/28—Sulfonic acids having sulfo groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton
    • C07C309/44—Sulfonic acids having sulfo groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton containing doubly-bound oxygen atoms bound to the carbon skeleton
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    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C309/00—Sulfonic acids; Halides, esters, or anhydrides thereof
    • C07C309/01—Sulfonic acids
    • C07C309/28—Sulfonic acids having sulfo groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton
    • C07C309/57—Sulfonic acids having sulfo groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton containing carboxyl groups bound to the carbon skeleton
    • C07C309/58—Carboxylic acid groups or esters thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C309/00—Sulfonic acids; Halides, esters, or anhydrides thereof
    • C07C309/63—Esters of sulfonic acids
    • C07C309/72—Esters of sulfonic acids having sulfur atoms of esterified sulfo groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton
    • C07C309/73—Esters of sulfonic acids having sulfur atoms of esterified sulfo groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton to carbon atoms of non-condensed six-membered aromatic rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C311/00—Amides of sulfonic acids, i.e. compounds having singly-bound oxygen atoms of sulfo groups replaced by nitrogen atoms, not being part of nitro or nitroso groups
    • C07C311/22—Sulfonamides, the carbon skeleton of the acid part being further substituted by singly-bound oxygen atoms
    • C07C311/29—Sulfonamides, the carbon skeleton of the acid part being further substituted by singly-bound oxygen atoms having the sulfur atom of at least one of the sulfonamide groups bound to a carbon atom of a six-membered aromatic ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C317/00—Sulfones; Sulfoxides
    • C07C317/16—Sulfones; Sulfoxides having sulfone or sulfoxide groups and singly-bound oxygen atoms bound to the same carbon skeleton
    • C07C317/22—Sulfones; Sulfoxides having sulfone or sulfoxide groups and singly-bound oxygen atoms bound to the same carbon skeleton with sulfone or sulfoxide groups bound to carbon atoms of six-membered aromatic rings of the carbon skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C65/00—Compounds having carboxyl groups bound to carbon atoms of six—membered aromatic rings and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups
    • C07C65/01—Compounds having carboxyl groups bound to carbon atoms of six—membered aromatic rings and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups containing hydroxy or O-metal groups
    • C07C65/105—Compounds having carboxyl groups bound to carbon atoms of six—membered aromatic rings and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups containing hydroxy or O-metal groups polycyclic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D257/00—Heterocyclic compounds containing rings having four nitrogen atoms as the only ring hetero atoms
    • C07D257/10—Heterocyclic compounds containing rings having four nitrogen atoms as the only ring hetero atoms condensed with carbocyclic rings or ring systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/28—Phosphorus compounds with one or more P—C bonds
    • C07F9/38—Phosphonic acids [RP(=O)(OH)2]; Thiophosphonic acids ; [RP(=X1)(X2H)2(X1, X2 are each independently O, S or Se)]
    • C07F9/3804—Phosphonic acids [RP(=O)(OH)2]; Thiophosphonic acids ; [RP(=X1)(X2H)2(X1, X2 are each independently O, S or Se)] not used, see subgroups
    • C07F9/3834—Aromatic acids (P-C aromatic linkage)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/28—Phosphorus compounds with one or more P—C bonds
    • C07F9/38—Phosphonic acids [RP(=O)(OH)2]; Thiophosphonic acids ; [RP(=X1)(X2H)2(X1, X2 are each independently O, S or Se)]
    • C07F9/3804—Phosphonic acids [RP(=O)(OH)2]; Thiophosphonic acids ; [RP(=X1)(X2H)2(X1, X2 are each independently O, S or Se)] not used, see subgroups
    • C07F9/3882—Arylalkanephosphonic acids

Definitions

  • the present invention relates to a method for inhibiting cell infection by enveloped viruses, and more particularly, to a method which employs a defined class of calix(n)arene compounds; and in a related aspect, to a method for inhibiting infection by sexually transmitted enveloped viruses.
  • the challenge in developing an effective therapy and prophylaxis for viral disease is to achieve inhibition of viral processes without producing extreme side effects and preferably without inducing viral resistance.
  • viral replication requires use of the cellular apparatus of the host, treating virus infection by inhibiting viral replication can be lethal to the infected host cells as well.
  • the virus should be destroyed or inactivated in the host prior to its invasion of host cells. This is normally accomplished, with varying degrees of success, by the host's immune system, but this mechanism requires an earlier immune response, either by a prior infection or by vaccination.
  • viruses such as Herpes Simplex viruses (HSV) are able to effectively elude a host's immune systems, and at least one virus, the human immunodeficiency virus (HIV) is known to cripple the host's immune system (Gottling) .
  • HSV Herpes Simplex virus
  • acyclovir a purine analog used in treating a variety of viral diseases, including herpes simplex virus-1 (HSV-1) and herpes simplex viru ⁇ -2 (HSV-2) inhibits viral replication at several key points, including inhibition of viral thymidine kinase and DNA polymerase, and DNA strand elongation (Elion) .
  • Ribavirin another purine analog, is the drug of choice in treating respiratory syncytial viruses (RSV) infection.
  • This compound appears to act by reducing cellular GTP levels, blocking the action of several GTP-dependent viral processes (Smith) .
  • zidovudine Azdothy idine
  • AZT zidovudine
  • AZT acts with high affinity to block viral RNA-dependent DNA polymerase (reverse transcriptase) , but does also block human DNA- polymerase and causes chain termination (Mitsuya) .
  • nucleic acid analogs include ganciclovir, vidarabine, idoxuridine, trifluridine and foscarnet (an inorganic phosphate analog) .
  • ganciclovir vidarabine
  • idoxuridine a compound that has been modified by blocking viral replication
  • trifluridine a compound that has been modified by blocking viral replication
  • foscarnet an inorganic phosphate analog
  • Interferons are glycoproteins which have complex actions including enhancement of certain immune responses as well as direct antiviral action. They are more competent in preventing infec ⁇ tion, rather than treating established viral infec ⁇ tion, and their use leads to undesirable problems including acute, serious discomfort, bone marrow suppression, viral resistance, and development of host immune response to the interferon.
  • Treatment with "anti-sense" polymers of nucleic acids is a method in which the particular viral genome is the select target.
  • the treatment provides a highly discriminating approach which would be expected to have minimal side-effects; its use as a therapeutic is hampered by problems of targeting, introduction into cells, and the quantity of material that would be required to block each strand produced.
  • Agents which bind to and interfere with host riboso al protein synthesis will block viral replica ⁇ tion. These include the toxin ricin, various plant proteins such as pokeweed anti-viral protein, alpha sarcin, and other low molecular weight compounds. The weakness with the use of these materials is their lack of selectivity.
  • additional therapy has been developed by specifically targeting the unique retroviral enzyme, reverse tran ⁇ scriptase. Non-retroviral systems do not produce or use this enzyme, but tha virus cannot replicate without it.
  • viruses including orthomyxoviruses and paramyxovirus, herpes viruses, togaviruses and retroviruses, contain a viral envelope which surrounds the viral capsid and nucleic acid.
  • the plasma membrane of the host cell is altered to include some viral-coded proteins and, as the viral nucleoprotein core exits the host cell in which it was assembled, it becomes enveloped with the modified membrane, thus forming the viral envelope. Because this structure is unique to host cells when they are virally irfectious and distinct from normal cells, it can serve as an additional target for therapeutic assault.
  • the present invention includes a method for treating infection by an enveloped virus.
  • the method includes administering to the site of infection a therapeutically effective dose of a calix(n)arene compound which is derivatized, at its ring positions meta to the bridge attachments to the ring, with polar substituents having terminal carboxylate, phosphate, sulfinate, or sulfonate groups.
  • the calix(n)arene has the general structure:
  • R 2 is a polar substitutent with a terminal carboxylate, phosphonate, sulfinate, or sulfonate group, including cleavable esters and amides thereof; and
  • R, is >CHR", > CR", or a mixture thereof, where R" is H or a carboxylate group.
  • R 2 has the form: (CH 2 ) m -
  • the invention contemplates novel calix(n)arene compounds of the type described above, and having sulfinate, sulfonate, phosphonate, and carboxylate terminal groups.
  • the compound may be administered orally, for treatment, for example of human immunodeficiency virus (HIV) respiratory syncytial virus (RSV) , or herpes simplex viruses HSV-1 or HSV-2.
  • HSV human immunodeficiency virus
  • RSV respiratory syncytial virus
  • HSV-1 or HSV-2 herpes simplex viruses
  • the compound may be administered by inhalation, for treatment of respiratory syncytial virus, and topically for treatment of HSV-1 or HSV-2.
  • Other modes of administration e.g., intravenous, are also contemplated.
  • calix(n)arene compound an antiviral nucleoside analog compound for treating viral infection.
  • the calix(n)arene and nucleoside analog compounds may be formulated in an ointment vehicle, for topical administration, e.g., in treating lesions due to HSV-1 or HSV-2.
  • the compounds may be formulated in liquid or tablet form for oral administration, for treatment of systemic viral infection, or in solution form for systemic administration.
  • the invention includes a method of inhibiting infection by a sexually- transmitted enveloped virus.
  • a prophylactically effective amount of a macrocyclic compound is administered topically to an area of likely sexual contact.
  • the macrocyclic compound is composed of aryl ring subunits which are connected by ring-attached bridge linkages which form a continuous chain of connected atoms making up the backbone of the macrocycle, and which contain negatively charged substituents on non-backbone atoms of the aryl subunits.
  • the negatively charged substituents include a sulfonate group, a sulfinate group, a carboxylate group, or a phosphonate group.
  • the compound includes amide or ester forms of the negatively charged substituent, which can be cleaved to a negatively charged form in vivo.
  • the ring subunits in the macrocyclic compound preferably include naphthalene subunits having 3- and 6-position sulfinate or sulfonate groups; and/or phenyl subunits having 4-position negatively charged substituents, where the bridge linkages are between the 2 ring-carbon position of one naphthalene or phenyl group, and the 7 ring-carbon group of an adjacent naphthalene group or 5 ring-carbon position of an adjacent phenyl group.
  • the macrocyclic compound includes at least four naphthalene subunits, each having polar groups at 1- and 8-position, sulfinate or sulfonate groups at 3- and 6-positions, and bridge linkages between the 2 ring-carbon position of one subunit and the 7 ring-carbon position of an adjacent subunit.
  • One preferred compound of this type has the general structure:
  • R 2 is a sulfonate group
  • R 4 is >CH 2 or ⁇ CH, or a mixture thereof.
  • the macrocyclic compound includes at least four phenyl subunits having negatively charged substituents at 4- positions, and bridge linkages between a 2 ring- carbon position of one phenyl subunit and a 5 ring- carbon position of an adjacent phenyl subunit.
  • One preferred compound of this type has the general structure:
  • n, R,, R 2 , and R, are as above.
  • compositions in accordance with the invention are administered topically for inhibiting transmission of viral infection between sexual partners.
  • the compositions may take the form of a lubricating jelly, suppository, for example, and include a vehicle in which the macrocyclic compound is carried.
  • the compositions may be applied to the surfaces of physical barrier-type devices as a prophylactic measure against infection.
  • the invention includes a lubricating jelly composition for use in inhibiting infection by sexually transmitted enveloped viruses.
  • the jelly composition includes a lubricating jelly vehicle, and dissolved in the vehicle, a macrocyclic compound such as described above.
  • the invention includes a physical-barrier type device in combination with a macrocylic compound of the type described above, for use in inhibiting infection by sexually transmitted enveloped viruses.
  • the device is coated with a lubricating jelly composition contain ⁇ ing a macrocyclic compound such as described above.
  • sexually transmitted enveloped viruses against which the compounds of the invention are prophyl- actically effective include hepatitis delta virus (HDV) , hepatitis B virus (HBV) , hepatitis C virus (HCV) , papillomavirus, herpes simplex viruses 1 and 2 (HSV-1 and HSV-2), HIV-1, HIV-2, HTLV-I and HTLV-II.
  • Figure 1 shows the general structure of a macrocyclic compound composed of naphthalene subunits, for use in the present invention
  • Figures 3A and 3B illustrate two general methods of synthesis of a macrocyclic compound like the one shown in Figure 2A;
  • Figures 4A and 4B show an unoxidized (4A) and partially oxidized (4B) macrocycle with mixed phenyl and sulfonated naphthalene subunits;
  • Figure 5 illustrates reaction methods for converting the sulfonic acid substituents of macrocyclic chromotropic acid to glycyl sulfonamide and sulfonamide groups
  • Figure 6 illustrates a reaction method for converting sulfonate groups in a macrocycle containing chromotropic acid subunits to a sulfinate salt or sulfinate methyl or aryl ester;
  • Figure 7 shows the general structure of a macrocyclic compound composed of phenyl groups, for use in the present invention
  • Figure 8 shows a non-oxidized form of the
  • Figures 9A and 9B illustrate general methods of synthesis of non-oxidized and partially oxidized forms of the Figure 8 compound
  • Figure 10 shows a reaction scheme for replacing the ring hydroxyl groups in the Figure-8 compound with acetyl groups
  • Figure 11 shows a reaction for converting sulfonic acid substituents to glycyl sulfonamide groups in a ph ⁇ nyl-subunit macrocyclic compound
  • Figure 12 shows a reaction scheme for producing a macrocylic compound like that shown in Figure 8 but with carboxylic acid-containing bridge linkages
  • Figure 13 shows a reaction scheme for replacing hydroxyl groups in the Figure-8 compound with carboxylic acid groups.
  • Figure 14 shows a reaction scheme for producing a calix(n)arene from a para-tert-butyl-precursor
  • Figure 15 shows a reaction scheme for preparing a calix(n)arene having para-carboxyl substituents
  • Figure 16 shows a reaction scheme for preparing a calix(n)arene having carboxyl substituents linked to the para position by a methylene linker
  • Figure 17 shows a reaction scheme for preparing a calix(n)arene like that of Figure 16 but where the carboxyl substituents are linked to the para position by an ethylene linker;
  • Figure 18 shows a reaction scheme for preparing a calix(n)arene having para-phosphonate substituents;
  • Figure 19 shows a reaction scheme for preparing a calix(n)arene having a phosphonate substituent linked to the para position (C-4) by a methylene group
  • Figure 20 shows a reaction scheme for preparing a p-2-bromoethyl-0-tosyl-calix(n)arene as a precursor for preparing other calix(n)arene derivatives;
  • Figure 21 shows a reaction scheme employing the bromoethyl-calix(n)arene of Figure 20 to prepare a calix(n)arene having a phosphonate group linked to C- 4 by an ethylene group;
  • Figure 22 shows a reaction scheme for preparing a calix(n)arene derivative having a sulfonate group linked to C-4 by a methylene group
  • Figure 23 shows a reaction scheme for preparing a calix(n)arene derivative having a sulfonate group linked to C-4 by an ethylene group
  • Figure 24 shows a reaction scheme for preparing a calix(n)arene having a chlorine atom at the methylene bridge (R 4 in Fig. 7) for introduction of other substituents at the methylene bridge;
  • Figure 25 shows a reaction scheme for preparing calix(n)arenes like that of Figure 12, starting from the cyclized precursor from Figure 24;
  • Figure 26 shows a reaction scheme for preparing a calix(n)arene having a carboxymethyl group attached to the bridge methylene (structure LVIII) , and a general approach for preparing a variety of calix(n)arenes having selected R groups at the methylene bridge (structure LX) , using organocuprate reagents;
  • Figure 27 shows reaction schemes for preparing a number of calix(n)arenes having 3-sulfonylpropyloxy groups attached to calix(n)arenes at various ring positions;
  • Figure 28 shows a reaction scheme for preparing a macrocyclic compound having alternating phenyl and naphthyl rings;
  • Figures 29A and 29B are plots of HSV viral yields, as a function of drug dose, for the macrocyclic compounds KY-1 (29A) and KY-42 (29B) ;
  • Figure 30 shows the inhibition of 3 H-labeled HSV-1 binding to cells by the compound KY-1;
  • Figure 31 is a plot of the inhibition in plaque formation of HSV-1 virus when the virus is exposed to the compound KY-1 before (open squares) , (ii) after (closed squares) , and during (closed circles) incubation with Vero cells;
  • Figure 32A shows SDS-PAGE autoradiogra s of HSV- 1 proteins in the presence (lane A) and absence (lane B) of mercaptoethanol, and of HSV-2 proteins in the presence (lane C) and absence (lane D) of mercaptoethanol, all with bound radiolabeled KY-1, and stained marker proteins (lane E) ;
  • Figure 32B shows SDS-PAGE autoradiograms of radiolabeled KY-1 compound bound to HSV-1 glycoproteins gD (lanes A and B) , gB (lanes C and D) , and gC (lanes E and F) ;
  • Figures 33 (A-D) show plots of effects of topical Y-l on epithelial damage (33A) , conjunctivitis (33B) , ulceris (33C) , and stromal disease (33D) subsequent to ocular application of HSV-1 in rabbits;
  • Figures 34A and 34B show the drop in HSV-1 (34A) and H
  • An “enveloped virus” means a virus containing a proteinaceous viral envelope which surrounds the viral capsid.
  • enveloped viruses include orthomyxoviruses and paramyxovirus, herpes viruses, togaviruses and retroviruses.
  • the plasma membrane of the host cell is altered to include some viral-coded proteins and, as the viral nucleoprotein core exits the host cell in which it was assembled, it becomes enveloped with the modified membrane, thus forming the viral envelop.
  • a "sexually transmitted enveloped virus” is an enveloped virus which is known or suspected to be transmitted by sexual contact.
  • Specific examples include hepatitis delta virus (HDV) , hepatitis B virus (HBV) , hepatitis C virus (HCV) , papillomavirus, herpes simplex viruses 1 and 2 (HSV-1 and HSV-2) , HIV-1 (also known as HTLV-III) , HIV-2, HTLV-I and HTLV-II.
  • aryl ring subunit is a single ring or fused ring structure containing at least one aromatic ring, i.e., a 5- or 6-member ring with 6 pi electrons necessary for aromaticity. Examples include benzene, naphthalene, fused ring structures, such as tetralin, and heterocyclic structures, including fused-ring structures, such as quinoline, isoquinoline, and indole.
  • a "macrocyclic compound composed of aryJL ring subunits” is a cyclic compound formed by linking ring atoms in aryl ring subunits to form a cyclic chain.
  • a “calix(n)arene” or “calixarene compound” is a macrocyclic compound having a skeletal structure of the form:
  • n is preferably 4-10, and in a preferred embodiment, is 4, 6 or 8.
  • An exemplary structure of a partially oxidized calix(n)arene is shown in Figure 9B.
  • the , group attached to the same aryl ring has the form ⁇ CR; i.e., the group is double-bonded to the aryl ring, as illustrated in Fig. 9B.
  • positions of bridge attachments to the ring in a calixarene compound refer to ring positions 2 and 6 in each ring of the compound.
  • the "non-bridge positions" in a calixarene compound refer to ring positions l, 3, 4, and 5 in each ring of the compound.
  • ring position meta to the bridge attachments in a calixarene compound refer to ring position 4 in each ring of the compound.
  • a "polar substituent” refers to a radical R whose octanol/water partition coefficient is less than 1.
  • a "polar substituent having a terminal carboxylate, phosphonate, sulfonate, or sulfinate refers to R having the form -C0 2 " or R'-C0 2 " (carboxylate) , -P0 3 " or R'-P0 3 " (phosphonate) , -S0 3 ' or R'-S0 3 " (sulfonate) , -S0 2 " or R'-S0 2 " (su finate) , where R' is a linear chain 1-4 atoms in length which is effective to link the associated carboxylate, phosphonate, or sulfonate group to the phenyl ring of calixarene.
  • a “carboxylate” group includes the carboxylic acid group -C0 2 * , carboxylate salts, and carboxylic acid esters and amides which are cleavable in vivo.
  • a carboxylic acid ester has the general form -C0 2 -R, where R is an unsubstituted lower alkyl or a substituted alkyl group, and a carboxylic acid amide has the general form CONRR' f where NRR' is a secondary or tertiary amine, i.e., R and R' are each H or lower substituted or unsubstituted lower alkyl groups.
  • a carboxylic acid ester or amide is cleavable in vivo if it is hydrolysed by serum esterases or amidases, respectively, to the corresponding carboxylic group.
  • a "phosphonate” group includes the phosphonic acid group -P0 3 '2 , including phosphonate salts, and phosphonic acid esters and amides which are cleavable in vivo.
  • a phosphonic acid ester has the general form -P0 3 RR' where R and R' are lower alkyl groups, or substituted lower alkyl groups, and a phosphonic acid amide has the general form PO(OR) (NRR') , PO(NRR') 2 , where R and R' are each H or a lower alkyl group.
  • a phosphonic acid ester or amide is cleavable in vivo if it is hydrolysed by serum phosphatases or phosphoamidases, respectively, to the corresponding sulfonic acid group.
  • a "sulfonate” group includes the sulfonic acid group -S0 3 ', including sulfonate salts, and sulfonic acid esters and amides which are cleavable in vivo.
  • a sulfonic acid ester has the general form -S0 3 R, where R is an unsubstituted lower alkyl or substituted lower alkyl group, and a sulfonic acid amide has the general form S0 2 NRR', where R and R' are each H or a lower alkyl group.
  • a sulfonic acid ester or amide is cleavable in vivo if it is hydrolysed by serum esterases or sulfoamidases, respectively, to the corresponding sulfonic acid group.
  • a "sulfinate" group includes the sulfinic acid group -S0 3 ", including sulfinate salts, and sulfinic acid esters and amides which are cleavable in vivo.
  • a sulfinic acid ester has the general form -S0 2 R, where R is an unsubstituted lower alkyl or substituted lower alkyl group, and a sulfinic acid amide has the general form SONRR', where R and R' are each H or a lower alkyl group.
  • a sulfinic acid ester or amide is cleavable in vivo if it is hydrolysed by serum esterases or sulfoamidases, respectively, to the corresponding sulfinic acid group.
  • sulfonate includes the corresponding acid forms and any pharmaceutically acceptable salts thereof.
  • a "negatively charged substituent” refers to a substituent which is negatively charged under physiological conditions.
  • exemplary groups which can impart negative charge include sulfonate groups (-S0 3 ' ) , sulfinate groups (-S0 2 ”) , phosphonate groups (-P0 3 2 " ) , and carboxylate groups (-C0 2 ') .
  • the negatively charged substituent has the form -X-S0 3 ", - X-S0 2 ', -X-P0 3 ", or -X-C0 2 ", where X is a linear chain 1-4 atoms in length which is effective to link the associated carboxylate, phosphonate, sulfinate, or sulfonate group to the phenyl ring of a calixarene.
  • X is a linear chain 1-4 atoms in length which is effective to link the associated carboxylate, phosphonate, sulfinate, or sulfonate group to the phenyl ring of a calixarene.
  • a “lower alkyl group” is a linear or branched alkyl group containing 1-5 carbon atoms.
  • a “substituted lower alkyl group” is a lower alkyl group having one or more substitutions at its carbon atoms.
  • Aryl-Subunit Macrocyclic Compounds This section describes the synthesis of two general types of aryl macrocyclic compounds which are useful in the method of the invention. Methods for synthesizing the first type of compounds (naphthylene subunits) are discussed in section A below, and methods for preparing compounds in accordance with the second type (phenyl subunits) are described in sections B and C From the synthetic routes given in sections A-C, it will be apparent how macrocycles composed of mixed subunits, e.g., both naphthalene and phenyl subunits can be prepared. The synthetic methods are also generally applicable to macrocycles composed of heterocyclic subunits, particularly those having sulfonate or sulfinate substituents.
  • Figure 1 shows the general structural formula of a macrocyclic compound composed of substituted naphthalene subunits, for use in the present ⁇ invention.
  • One exemplary compound of this type is shown in non-oxidized (I) and partially oxidized (II) form in Figures 2A and 2B, respectively.
  • the compound is a tetramer of chromotropic acid (1,8- dihydroxy-3,6-disulfonic acid naphthalene) subunits linked by methylene or methine (>CH 2 or ⁇ CH) bridges (R 4 ) .
  • R 2 as noted, is a sulfonate or sulfinate group.
  • R 3 is H or an uncharged or negatively charged substituent, subject to the activity constraints discussed below. Preferably, R 3 is H.
  • the R 4 bridge linking the chromotropic acid derivative subunits is preferably of the form >CHR or ⁇ CR (indicating unsaturated bridges in the partially oxidized form) , where R is H or a small carbon-containing group, such as lower alkyl, alkenyl, ketone, or carboxylic acid group, or aryl group.
  • the bridge may also be of the form -CH 2 NR'CH 2 -, where R' is similarly H or a small carbon-containing group, such as a lower alkyl group.
  • the bridges in the macrocycle may be ring structures, including aryl ring structures, such as in the dimeric macrocycle shown in Figure 4, or analogous structures formed by bridging through heterocyclic rings, such as pyran or furan rings.
  • the number of subunits may vary from 4 (e.g.. Fig. 2A structure) to 8, with macrocycles containing 4, 6 and 8 subunits being preferred.
  • R, R 2 , R 3 , and R 4 substituents in Table 1 below.
  • the KY and Y number in the left and column in the table refers to the analog designation of the corresponding compound.
  • R 4 is -CH 2 -
  • Figures 3A and 3B illustrate two preferred synthetic methods for preparing macrocyclic chromotropic acid compounds.
  • the method illustrated in Figure 3A involves cyclization of a chromotropic acid derivative (including chromotropic acid itself) with an aldehyde (RCHO) to form a macrocyclic com ⁇ pound, such as the tetramer shown in Figure 2, in which the chromotropic acid subunits are linked by R- substituted methylene groups, i.e., in which R 4 is
  • KY-42 is prepared by cyclization with glyoxylic acid (Example IC) ; KY-48, in the presence of glyceraldehyde; KY-85, in the presence of benzaldehyde; KY-97, in the presence of acrolein; and KY-110, in the presence of pyruvic aldehyde.
  • glyoxylic acid Example IC
  • KY-48 in the presence of glyceraldehyde
  • KY-85 in the presence of benzaldehyde
  • KY-97 in the presence of acrolein
  • KY-110 in the presence of pyruvic aldehyde.
  • the R bridge group may be further modified after the cyclization reaction.
  • KY-193 may be prepared by brominatien of the KY-97 compound.
  • cyclization of the chromotropic acid derivatives (III) is carried out by reaction with hexamethylenetetramine, to form a 3-atom chain bridge of the type -CH 2 N(CH 3 )CH 2 - (V).
  • the cyclization reaction for the synthesis of KY-346 is given in Example 1J.
  • the -CH 2 N(CH 3 )CH 2 - bridge may be modified, after the cyclization reaction, to form a variety of N-substituted bridges of the -CH 2 N(R')CH 2 -, where R' is one of a variety of small carbon- containing groups, according to known synthetic methods.
  • FIG. 4A shows the non-oxidized (VI) and partially oxidized (VIII) forms of one compound) .
  • the chromotropic acid derivative is modified after cyclization so that the cyclized product will either contain the selected R t , R 2 , and R 4 substituent, or contain a substituent which can be readily modified to the selected substituent.
  • This approach is illustrated by the synthesis of KY-3, which has an S0 2 NH 2 R 2 substituent, as detailed in Example IB.
  • Figure 6 illustrates the conversion of the sulfonate groups of cyclized chromotropic acid to sulfinate salts (XII) and sulfinate methyl esters (XIV) .
  • the first stage of the reaction involves formation of the corresponding sulfonyl chloride derivative (IX) , as outlined above. This compound is then treated with sodium sulfite, to form the corresponding sulfinate salt (XII) . Reaction with dimethyl sulfate in the presence of sodium bicarbonate produces the corresponding sulfinate methyl ester (XIV) .
  • macrocyclic compounds with a variety of R j substituents may be prepared by modification of chromotropic acid after cyclization.
  • cyclized chromotropic acid is first converted to the diether of hexanoic acid by initial reaction of cyclized chromotropic acid with 6-bromohexanoic acid under basic reaction conditions.
  • the selected substituent is formed on the subunit naphthalene rings by derivatization of the naphthalene subunit, with subsequent subunit cyclization to form the desired macrocycle.
  • the synthetic method for forming selected-substituent macrocyclic com- pounds may include both prior derivatization of chromotropic acid and subsequent derivatization of the subunits after cyclization.
  • chromotropic acid subunits are first reacted at the R, positions, to form the dimethyl ether derivative as described above.
  • the com ⁇ pound is further derivatized at the R 2 position, also as described above, to convert the S0 3 group to the desired S0 2 NH 2 substituent.
  • the KY compounds described above can be converted readily to a variety of sulfinate and sulfonate acids and salts. Salt and acid forms can be interchanged by passage over standard cation- exchange resins, to displace one cationic counter ion for another, according to well known methods.
  • Salt and acid forms can be interchanged by passage over standard cation- exchange resins, to displace one cationic counter ion for another, according to well known methods.
  • several of the KY compounds shown in Table 1 are ammonium salts formed by cation exchange of protons in the presence of an ammonium salt, such as ammonium chloride.
  • the macrocyclic compound may produce both a metal salt and a metal chelate of the macrocyclic compound in which the metal is chelated at interior polar pocket in the compound.
  • metal cations such as the cations of Ca, Ba, Pt, Cu, Bi, Ge, Zn, La, Nd, Ni, Hf, or Pb
  • Figure 7 shows the general structural formula of a calix(n)arene compound of the type that can be used in the method of the present invention.
  • One exemplary compound of this type is shown in Figure 8, which is a tetramer of phenol parasulfonic acid subunits linked by methylene bridges (XV) .
  • XV methylene bridges
  • Figure 9A illustrates a general method for forming calix(n)arene compounds.
  • the precursor shown at the left (XVI) is a tert-butyl calix(n)arene, where n is the number of phenolic subunits (with para-position t-butyl substituents) in the macrocycle, and the bridge connections are methylene groups, t-butyl calixarenes with 4, 6, and 8, subunits are commercially available, and larger and uneven-numbered subunit calix(n)arenes can be prepared by standard purifying methods.
  • a t-butyl calixarene with a selected subunit number is treated with concentrated sulfuric acid, typically for about 4 to 5 hours at 75-85°C to effect substantially complete displacement of the 4-position t-butyl group by a sulfonic acid group.
  • a similar method is used for preparing a sulfonated calixarene with partially oxidized 1- position OH groups, as shown at 9B.
  • the t-butyl calixarene starting material is treated with cone.
  • reaction is effective to sulfonate the subunit rings and to partially oxidize the interior OH groups.
  • partial oxidation can lead to a conjugated calix(n)arene structure (XVIII) in which the bridge contributes delocalized electrons.
  • This conjugated structure is colored, and the development of a colored product can be used to monitor the course of the oxidation reaction. Details of the reaction are given in Example 2B.
  • the desired macrocycle can also be formed directly by reacting parasulfonic acid phenol (or precursors thereof) under suitable bridging conditions, such as described above for producing naphthalene-subunit macrocycles. This is illustrated by the reaction shown in Figure 12, for production of a macrocyle having carboxylic acid-containing bridge groups.
  • phenol parasulfonic acid is reacted with glyoxylic acid, under conditions similar to those described in Example IC, to form the cyclized structure shown (XXII) .
  • a synthesis of XXII is described in Example 2F.
  • the calix(n)arene compounds formed as above can be modified, according to general procedures outlined in Section IIA above, to achieve selected R j groups, modified sulfonyl groups, and/or addition of R 2 groups.
  • Figures 10, 11, and 13 illustrate various reaction methods for modifying the R j or R 4 group of an already formed macrocycle.
  • the sulfonated structure shown in Figure 8 is treated with acetic anhydride, to form an O-acetyl Rj group. Details of the reaction are given in Example 2C
  • Example 2G describes a similar reaction scheme for forming a toluene sulfonic acid ester at the R ⁇ position.
  • Figure 11 illustrates a general method for forming sulfonamides, such as glycylsulfonamide (XXI) of the Figure 8 compound.
  • XXI glycylsulfonamide
  • the sulfonated phenyl calix(n)arene compound (XVII) is treated with chlorosulfonic acid, to form the corresponding sulfonyl chloride analog (XX) .
  • Further reaction with a selected amine, in this case glycine gives the desired sulfonamide.
  • the R 3 is generally H, but may be an uncharged or negatively charged substituent, similar to the R 3 group described in Section IIA above.
  • the R ⁇ bridge linking the chromotropic acid derivative subunits is preferably of the form >CHR or > CR, where R is H or a small carbon-containing group, such as lower alkyl, alkenyl, ketone, or carboxylic acid group, or aryl group, as noted above, or of the form -CH 2 NR'CH 2 -, where R' is similarly H or a small carbon-containing group, such as a lower alkyl group.
  • the bridges in the macrocycle may be ring structures, including aryl ring structures, analogous to the dimeric macrocycle shown in Figure 4.
  • the number of subunits may vary from four (e.g., Figure 8 structure) to 8, with macrocycles containing 4, 6 and 8 subunits being preferred.
  • Representative calix(n)arene compounds which have been synthesized and tested for anti-viral activity are identified by their Rj, R 2 , and R 4 substituents in Table 2 below.
  • the KY and Y number in the lefthand column in the table refers to the analog designation of the corresponding compound, as in Table 1.
  • Sulfinate. Phosphonate. and Carboxylate Groups One general class of compounds which are useful in inhibiting infection by enveloped viruses, in accordance with the present invention, are calix(n)arene compounds in which the ring position meta to the bridge attachments, i.e., 4-position carrying substituent Rj in Fig 7, is substituted with a negatively charged substitutent having a terminal sulfonate, sulfinate, phosphonate, or carboxylate group.
  • Examples 2A, 2B, and 2C Methods for preparing calix(n)arene compounds in which a sulfonate group is carried at the ring 4 position are given in Examples 2A, 2B, and 2C, including compounds with different substitutions at the ring 1 position.
  • Compounds having sulfonamide group, including a group which terminates with an end terminal carboxyl group are given in Examples 2D and 2E, respectively.
  • Fig. 14 illustrates a method for converting a t- butyl calix(n)arene (XXV) to the unsubstituted compound (XXVI) , which can be used as a starting material for some of the syntheses described below. Details of the reaction are given in Example 2J.
  • Fig. 15 shows the conversion of a calix(n)arene (XXVII) carrying a p-acetyl group to the corresponding calix(n)arene with a p-carboxyl group (XXVIII) . Details are given in Example 2K.
  • Fig. 17 illustrates the synthesis of a carboxyethyl calix(n)arene (XXXIII).
  • the intermediate (XXIX) from above (Fig. 16) is treated sequentially with Mel and the sodium salt of diethylmalonate to give the diethylmalonylmethyl compound (XXXII) . Heating in acid gives the desired compound XXXIII. Details are given in Example 2M.
  • Fig. 18 illustrates the synthesis of a p- phosphonate calix(n)arene (XXXVI).
  • Example 20 Details are given in Example 20. Note that the chloromethyl intermediate is also useful in synthesis of the sulfonylmethyl calix(n)arene analog.
  • the synthesis of a p-2-bromoethyl compound useful in the synthesis of a phosphonoethyl or sulfonylethyl calix(n)arene is outlined in Fig. 20, with details given in Example 2P.
  • compound XXVI from above is allylated at the phenol hydroxyl (compound XL) , and heated to give the rearrangement product XLI.
  • Tosylation serves to protect the phenyl hydroxyl position (compound XLII) , allowing conversion to the p-hydroxyethyl derivative (XLIII) . Further reaction with triphenylphosphine dibromide gives the desired p-bromoethyl XLIV compound.
  • the p-bromoethyl calix(n)arene (XLIV) is used in the synthesis of the p-phosphonoethyl compound (XLVI) , by a reaction sequence which is analogous to that shown in Fig. 19, described above. Details of the reaction scheme shown in Fig. 21 are given in Example 2Q.
  • the intermediate p-chloromethyl calix(n)arene (compound XXXVII) used above can also be used in the synthesis of a p-sulfonylmethyl calix(n)arene
  • sulfonate groups can be converted to sulfinate groups by the methodology illustrated in Fig. 6 and exemplified in Example ID.
  • the foregoing synthetic methods can be used to produce calix(n)arene compound having at the position meta to the bridge positions (the C4 ring positions) , negatively charged substituents which terminate with sulfonate, sulfinate, phosphonate, and carboxylate groups.
  • the syntheses outlined show both direct acid-group attachment to the rings, or attachment through alkyl linkages, such as methyl and ethyl linkages. It will be appreciated from the discussion above how acid groups linked to the rings through longer alkyl groups can be prepared. Also, as detailed above, the acid groups can be converted to the corresponding salts.
  • esters and amides of the terminal acid groups in the macrocyclic compounds of the invention can be prepared.
  • Such derivative can be used as "pro-drugs", where an ester or amide is converted in vivo to the corresponding negatively charged acid group by enzyme-catalyzed hydrolysis (e.g., by an esterase) .
  • the acid esters of carboxylic acid and sulfonic acid can be prepared by standard esterification reactions in which the acid is converted to, for example, an acid chloride, then reacted with an alcohol, such as an alkyl alcohol.
  • the amides of carboxylic and sulfonic acid can similarly be formed by reaction of the acid chloride with an amine, such as an alkyl amine.
  • Preferred esters include aryl and lower alkyl carbonate esters, such as n-butyl alkyl group.
  • Preferred amides include amides of lower alkyl groups.
  • a phosphonic acid calix(n)arene to a corresponding ester or amide likewise follows conventional phosphonate esterification or amidation reaction methods.
  • One method for generating a diethylphosphonyl ester has been described above with reference to Fig. 21.
  • the present invention contemplates, for use in the method of the invention, calix(n)arene compounds which are substituted at other ring positions and at the bridge positions in the macrocycle.
  • the C3 and/or C5 ring positions may be substituted with halogens such as F of Cl.
  • Figs. 24 and 25 illustrate one method of attaching carboxyl groups to the bridge methylene in calix(n)arene.
  • the hydroxyl group of p-t-butyl calix(n)arene (XXV) is acetylated (L) , and the product is oxidized at the bridge methylene, to give the bridge ketone (LI) .
  • a variety of bridge substitutions can be produced, by the method outlined in Fig. 26, using a suitable cuprate reagent as illustrated at the left in the figure.
  • the reaction at the right in the figure shows how a calix(n)arene LIII can be converted to a compound having a carboxymethyl group attached to the bridge methylene group. Details of this reaction sequence are given in Examples 2W and 2X.
  • the final reaction product (LVIII) can be p-sulfonated or derivatized with other acid groups at the para position as above.
  • Fig. 27 shows a variety of derivatization reactions involving calix(n)arenes and propane-1,3- sulfone.
  • Fig. 28 shows the preparation of a mixed macrocyle containing alternating phenyl and naphthyl groups. The reaction method is described in Example 3B.
  • HSV-1 herpes simplex virus-1
  • HSV-2 herpes simplex virus-2
  • non-enveloped viruses including adenovirus, a double-stranded DNA virus (Rowe; Hilleman) , and rhinovirus, a single- strand RNA virus (Dick) .
  • adenovirus a double-stranded DNA virus
  • rhinovirus a single- strand RNA virus
  • aryl macrocyclic compounds including those shown in Tables 1 and 2 were examined for toxicity in cell culture, using a panel of human cell lines, as detailed in Example 4. Briefly, the selected KY- or Y- compound was added to cell cultures at a final concentration of 5, 10, 25, 50, or 100 ⁇ g/ml. Three days later the cells were washed to remove drug, and stained with a vital stain, to determine the percentage of dead cells in each culture.
  • the IC 50 drug concentration i.e., value concentration of drug which produced 50% cell death, was 50 ⁇ g/ml for KY- 143, KY-151, and KY-163, and lOO ⁇ g/ml or greater for all of the other KY compounds tested.
  • KY-1 which has a molecular weight of 1404 daltons
  • a drug concentration of 100 ⁇ g/ml is equivalent to about 66 ⁇ M.
  • compositions containing one of the compounds in Tables 1 and 2 were tested for inhibi ⁇ tion of cytopathic effects (CPE's) in cultured, HSV- infected cells.
  • CPE's cytopathic effects
  • Vero cells were infected with HSV-1 or HSV-2 and allowed to grow in culture until cytopathic effects were clearly visible.
  • the cells form an even monolayer of fibroblast-like cells.
  • HSV infection a cytopathic effect characterized by round cells in suspension is clearly evident after 24 hours, followed by clumping and lysis of infected cells after 24-72 hours.
  • Example 5 In the drug inhibition study reported in Example 5, cells were exposed to HSV-1 or HSV-2 virus and, at the same time, to a selected aryl macrocyclic compound, at a final drug concentration of 10 ⁇ g/ml. Twenty-four hours later the cells were examined for cytopathic effect. If a clear cytopathic effect was not observed with lO ⁇ g/ml of the drug, the study was repeated at a drug concentration of 20 ⁇ g/ml for some compounds.
  • Table 3 lists 50 naphthalene-subunit macrocycles which were tested in this assay.
  • a "+” symbol in the second column indicates that the compound was effective in inhibiting cytopathic effects at 10 or 20 ⁇ g/ml.
  • a "-” symbol indicates that CPE was observed at 10 or 20 ⁇ g/ml.
  • the compounds used in Table 3 were further tested for activity against HSV infection in a plaque reduction assay, as detailed in Example 6.
  • Vero cells after overnight incubation, were exposed to serial dilutions of KY compound, from 0.625-10 ⁇ g/ml, and HSV-l or HSV-2 virus for two hours. After washing to remove drug and extracellular virus, the cells were further incubated for 2 days, then stained and counted for plaque formation. Percent inhibition was determined by dividing plaques produced by total number of plaques in infected, untreated controls. From the concentration effect curve of plaque inhibition (expressed as percent of control) , the concentration of compound required to produce 50% plaque reduction, IC J Q, was determined. The IC ⁇ values for infection by HSV-1 and HSV-2 infection is given in the right-hand columns in Table 3.
  • R-group features can be identified as contributing to low activity (no protection of cells from CPE effect seen at 10-20 ⁇ g/ml: in KY-48, KY-49 and KY-110, a bulky side chain in the methylene bridge; in KY-143, an OH R 3 group; in KY-147 and KY-148, a sulfonamide with a non-polar alkyl group at the R 2 position; in KY-158 and KY-175, a sulfinate ester or sulfonate ester with a non-polar alkyl group at the R 2 position; and in KY-395, a trimethylamine bridge in combination with a methyl ether substituent at the Rj position.
  • the "GC” symbol for KY-193 means that some giant cells were formed, indicating partial inhibitory activity.
  • the optimal radicals at the R 2 position are sulfonic acid or sulfonic acid salts, sulfinic acid and salts thereof, and sulfonamides with polar amine groups, such as NH 2 , NHOH, N-glycosides (KY-352) , and amino acids.
  • the preferred radicals at the R 3 position are H or Br.
  • the optimal bridge linkage groups are substituted and unsubstituted methylenes, where the R group is not a bulky alkyl group, and is preferably a carboxylic acid group.
  • the R 2 groups are sulfonic acid or sulfonic acid salts or sulfonamides with a terminal carboxylic acid. This feature indicates that an R 2 position acid group favors high activity.
  • the R, bridge is methylene or a methylene carrying a carboxylic acid group.
  • Example 7 Briefly, cultured Hela cells were exposed to serially diluted KY compound and virus, allowed to grow for 24 hours, then freeze/thawed 3 times to release virus particles. Vero cells were infected serial dilutions of the viral lysates were assayed for plaque counts as described in Example 6. The drop in viral yield, as a function of drug concentra ⁇ tion, is plotted in Figures 29A and 29B for compounds KY-1 and KY-42 respectively. The dose dependent drop in viral yield was between about 3-5 orders of magnitude, depending on drug and virus. The degree of inhibition of viral yield was generally greater for HSV-1 than for HSV-2. Similar results were observed with several other KY compounds.
  • the highest activities observed for the phenyl- subunit compounds are comparable to the highest activities seen with the naphthyl compounds, e.g., from about 1-3 ⁇ g/ml IC J0 values.
  • Addition of acetyl groups at the R, position produces little change in the activities of partially oxidized compounds, also consistent with the results observed with the naphthalene-subunit compounds, addition of alkyl esters at the R, positions gave activities comparable to the partially oxidized analog.
  • the same rules discussed above generally apply.
  • highest activity is expected when the R 2 group terminates in a negatively charged group, such as a sulfonic acid.
  • the present invention contemplates, for use in treatment of infection by enveloped virus, a calix(n)arene compound which whose R 2 group is a polar substituent which terminates in a sulfonic acid, phosphonic acid, or carboxylic acid group, including esters and amides of these acids which can be converted to the corresponding acid by hydrolytic cleavage in vivo.
  • the present invention contemplates a macrocyclic compound whose R 2 groups are negatively charged substituent which terminates in a sulfonate, sulfinate, phosphonate, or carboxylate group.
  • the compounds may also include esters and amides of the negatively charged groups which can be converted to acid groups by hydrolytic cleavage in vivo.
  • esters and amides of sulfonic acid, phosphonic acid, and carboxylic acid have been shown to undergo hydrolytic cleavage in vivo to the corresponding acids (Svensson, 1988, 1991; Stella; Bundgaard) including esters and amides of lower alkyl groups.
  • Methods for preparing a variety of exemplary compounds of this type are described in Section II above.
  • the calix(n)arene compounds having terminal acid groups may be substituted at other ring and bridge positions, as indicated in Section IIB and IIC above.
  • Inhibition of viral yield was determined, as above, by infecting Hela cells in the presence of wild type or drug- resistant strains of HSV-1 or HSV-2, and serial dilutions of a selected anti-viral compound, and infecting Vero cells with serial dilutions of the Hela cell lysate, as above. Details of the inhibition study are given in Example 8.
  • the IDso concentration (which effects 90% inhibition of viral yield) is given in Table 5.
  • the KOS (HSV-1) and 333 (HSV-2) are wild type viruses; the KOS(PMEA)' and KOS(PFA)' are drug-resistant HSV-1 strains having a DNA polymerase mutation.
  • the 333(DHPG) strain is a drug-resistant HSV-2 strain having a thymidine kinase mutation.
  • DHPG as an inhibitor of drug-resistant strains of HSV-l
  • PMEA as an inhibitor of drug- resistant strains of HSV-2
  • all of the nucleoside analogs were at least about 20 times less active against drug-resistant strains than wildtype strains of either HSV-1 or HSV-2, as measured by drug concentration required to inhibit yield.
  • the aryl macrocyclic compound showed substantially the same specific activity against drug-resistant strains of HSV-1 and HSV-2 as against wildtype strains.
  • Example 9 Twenty-four hours later the cells were examined for percent clum- ping, based on the percent of clumped cells of total cell particles in a given view field. The inhibition of clumping was plotted as a function of drug con ⁇ centration, to determine the dose effective to produce a 50% reduction in the percent clumped cells, measured with respect to control (no drug treatment) . The measured ED ⁇ values are given in Table 6 below. A similar method was employed to determine the ED 50 of RSV inhibition of cytopathic effect (cell clumping) in HEp2 cells, with the results shown in Table 6. Details are given in Example 9.
  • RSV was significantly more sensitive to compound inhibition than the Influenza A/Taiwan virus.
  • Highest-IAV activity was seen with a sulfonamide with polar amine (S0 2 NH 2 ) at the R 2 position, and with selected methylene bridge groups.
  • Relatively high anti-RSV activity was seen with all compounds except KY-47.
  • Representative macrocyclic compounds from Table 1 were tested for inhibition of cytopathic effects in cells infected with one of two HTLV-III strains, HTLV-III B and RF-II strains, as described in Example 12. Briefly, cells chronically infected with HTLV- III B or RF-II HIV strains were incubated in the presence of serial dilutions of the selected KY com ⁇ pound, then further cocultured with indicator cells. The extent of syncytia formation was scored under phase microscopy. The concentration ( ⁇ g/mL) effective to produce complete inhibition of syncytia formation, ED 100 , is shown in Table 7 for the two HIV strains. The "N" means that the compound was not tested for that virus. 10
  • R-group features can be identified as contributing to sub-optimal activity (ED 5 o values > 63 ⁇ g/ml for both strains): in KY-48, a bulky side chain in the methylene bridge; in KY-110, a methyl ketone group in the bridge; in KY-143, an OH R 3 group; in KY-147 and KY-148, a sulfonamide with a non-polar alkyl group at the R 2 position; in KY-158 and KY-175, a sulfonate ester or sulfinate ester having a non-polar alkyl group, at the R 2 position; and in KY-272, a methyl ester at the R, position combined with a acetyl-group bridge.
  • These features are substantially the same as those which gave reduced activity against HSV viral infectivity, i.e., showed no inhibitory effect on CPE at 10-20 ⁇ g/ml.
  • those factors which promote high activity against HSV activity are in general the same as those which give highest activity against HIV infectivity.
  • the preferred radicals at the R 2 position are sulfonic acid or sulfonic acid salts, sulfinic acid and salts thereof, and sulfonamides with polar amine groups, such as NH 2 , NHOH, N-glycosides (KY-352) , and amino acids, with sulfonic acid.
  • sulfonic acid, sulfonate salts, and sulfonamides having a terminal carboxylic acid group.
  • the optimal radicals at the R 3 position is H, with both OH and Br giving reduced activity.
  • the bridge groups are preferably substituted and unsubstituted methylenes, where for a >CHR or >CHR bridge, the R group is not a bulky alkyl group.
  • the R 2 groups are sulfonic acid or sulfonic acid salts or sulfonamides with a terminal carboxylic acid. This feature indicates that an R 2 position acid group favors high activity.
  • the j bridge is methylene or a methylene carrying a carboxylic acid group.
  • R-groups are intended to provide guidance in the selection of R groups at the R 1 -R 4 positions, for optimizing compound efficiency.
  • calix(n)arene compounds from Table 2 were tested for inhibition of cytopathic effects in cells infected with one of two HTLV-III strains, HTLV-III B and RF-II, as described in Example
  • IC 50 values measured for the HXB and RS-11 strains of HIV are given in units of ⁇ g/ml in Table 8 below.
  • Section A examines the specificity of the viral-inhibition method to enveloped viruses.
  • the studies reported in subsection A show that the macrocyclic compounds used in the method act, at least in part, by binding selectively to viral envelope proteins, and that this binding blocks virus attachment to infectable cells, thereby inhibiting virus infectivity. These studies are detailed in U.S. patent application Serial No. 647,720, filed January 29, 1991, now U.S. Patent No. 5,196,452.
  • Subsection B examines the inhibitory effect of the macrocyclic on non-enveloped viruses.
  • Example 14 the ability of a macrocyclic com ⁇ pound to block HSV binding to infectable cells was examined as described in Example 14. Briefly, Vero cells were exposed to radiolabeled HSV-1 or HSV-2 in the absence of KY compound or in the presence of 10 ⁇ g/ml KY-1, and binding of the virus at times up to 4 hours after exposure to the virus was measured.
  • Figure 30 shows a plot of virus (radiolabel) binding to cells over the four-hour incubation period. In the absence of drug, the amount of bound virus increased steadily over two hours, and slightly from 2-4 hours. By contrast, virus binding to cells peaked at about 1/2 hour in the presence of drug, presumably reflecting the time during which the binding events effective to block virus binding to the cells are equilibrating.
  • Example 15 In a second study, the effect of compound when administered prior to, during, or after cell infection by HSV-1 was examined as described in Example 15.
  • cells were exposed to one of a series of increasing KY-2 concentrations, and the extent of infection was measured by number of plaques observed 24 hours after infection.
  • the reduction in plaque formation, expressed as a percent of control, is shown in Figure 31 for cells treated with drug prior to (solid rectangles) , during (closed circles) , and after (open rectangles) .
  • Virus inhibition was seen most significantly when the cells were treated with drug during exposure to virus, indicating that virus inhibition occurs at the period of virus binding to and entry into infectable cells.
  • the major bands of drug binding in HSV-1 have molecular weights, as determined from SDS-PAGE, of 45, 66, and about 130 kilodaltons.
  • the major bands of drug binding in HSV-2 have similar molecular weights.
  • the major bands which show KY binding in Figure 32B correspond in molecular weight, to HSV glycoproteins gD, gB, and gC
  • Vero cells were infected with adenovirus in the presence of KY-1, also at concentrations ranging between 1-100 ⁇ g, and twenty-four hours after virus infection, the cells were examined for cytopathic effect. No reduction in cell clumping was observed at any of the KY-1 drug concentrations.
  • a broad range of macrocyclic com ⁇ pounds are effective inhibitors of cell infection by each of the several enveloped viruses which were studied. Binding studies carried out in particular with respect to HSV viruses indicate that the anti ⁇ viral activity of the compounds is dependent on binding to virus envelope components, which in turn inhibits virus attachment to infectable cells. The apparent inability of the compounds to inhibit infection of non-enveloped viruses is consistent with this mechanism.
  • the invention also includes a composition containing a macrocylcic compound of the type described above in combination with a nucleoside analog anti-viral compound.
  • the nucleoside analog compound is one effective to inhibit viral replication at the level of viral replication or transcription.
  • the nucleoside analog compounds which are useful in combination with a macrocyclic compound are: (1) Pyrophosphate analoges, such as phosphoformic acid (PFA) , phosphonoacetic acid (PAA) , methanediphosphonic acid (MDP) , carbonyldiphosphonic acid (COMDP) , phosphonoglyoxalic acid (COPAA) , and various halogen- and/or methyl-substituted derivatives thereof, which are inhibitors of viral nucleic acid polymerases.
  • PFA phosphoformic acid
  • PAA phosphonoacetic acid
  • MDP methanediphosphonic acid
  • COMDP carbonyldiphosphonic acid
  • COPAA
  • Base-modified analogs such as IUDR, trifluorothymidine, AraA, and azidothymidine (AZT) , didieoxyinosine (DDI) , D4T, dideoxycytidine (DDC) , and ribavirin.
  • Trifluorothymidine, IUDR, and AraA are active mainly against herpes virues (Nicolson, 1984a, 1984b) .
  • Ribavirin is active against several RNA and DNA viruses (Sidwell)
  • AZT is active against HIV (Fischl) , as are other dideoxynucleoside analogs, such as DDI.
  • Sugar-modified analogs such as N-acyl derivatives of 5'-amino-2' ,5'-dideoxy 5'-ioduridine, sulphonamide derivatives of 5'-amino-5'- deoxythymidine, 2'-deoxy-5-ethyluridine, and N-acyl derivatives, 5'-Sulfate and 5'-sulfamate nucleoside analogs, such as nucleocidin, adenosine 5' sulfamate, and ribavarin, which may act primarily at the level of protein synthesis inhibition (Martin) .
  • N-acyl derivatives of 5'-amino-2' ,5'-dideoxy 5'-ioduridine sulphonamide derivatives of 5'-amino-5'- deoxythymidine, 2'-deoxy-5-ethyluridine
  • N-acyl derivatives 5'-Sulfate and 5'-sulfamate nucleoside analog
  • Phosphate analogs including acyclonucleoside phosphonates, such as acyclovir and gangiclovir, and their isosteric phosphonate analogs. These compounds can act as virus-selective substrates for viral thymidine kinases, in the synthesis of nucleoside triphosphate analogs intracellularly (Galbraith) . Subsequently, the nucleoside triphosphate analogs can act as selective substrates for viral DNA polymerase, acting as a chain terminator since the analog does not have the bifunctionality necessary for chain extension (Allen) . These compounds have demonstrated anti ⁇ viral activity against herpes viruses (Collins) , including HSV-l, HSV-2, varicella zoster (VZV) , and cytomegalovirus (CMV) (Smith) .
  • Collins herpes viruses
  • VZV varicella zoster
  • CMV cytomegalovirus
  • phosphonomethyl ethers of nucleosides and their acyclic analogs, such as N-(3-hydroxy-2-phosphonylmethoxypropyl)- (HPMP-) and N-(2-phosphonylmethoxyethyl-) (PME-) derivatives of heterocyclic bases.
  • HPMP- N-(3-hydroxy-2-phosphonylmethoxypropyl)-
  • PME- N-(2-phosphonylmethoxyethyl-) derivatives of heterocyclic bases.
  • HPMP- N-(3-hydroxy-2-phosphonylmethoxypropyl)-
  • PME- N-(2-phosphonylmethoxyethyl-) derivatives of heterocyclic bases.
  • These compounds act specifically against herpes viruses, adenoviruses, cytomegalovirus (DeClercq) , poxviruses, vaccinia viruses, and retroviruses.
  • Example 18 The ability of the two-compound composition to inhibit viral infection by an enveloped virus is demonstrated in the study reported in Example 18, which examines the viral yields after infection of Vero cells with serial dilutions of HSV-1 or HSV-2 particles, as described above.
  • Figure 34A shows the drop in HSV-1 viral yields when infected cells are exposed to increasing concentrations of the macrocyclic compound Y-l alone (solid circles) , increasing concentrations of acyclovir alone (open circles) , increasing concentrations of acyclovir plus 25 ⁇ g/ml Y-l (solid rectangles) , and increasing concentrations of acyclovir plus 50 ⁇ g/ml Y-l (solid ovals) .
  • a maximum decrease in viral yield was slightly less than three logs (orders of magnitude) .
  • the two compounds are formulated, preferably in a jelly form, at a preferred weight ratio of between about 10:1 to 1:1 macrocyclic compound to nucleoside analog, respectively.
  • Figures 34A and 34B show that a significantly higher level of inhibition was observed when the co- administered compounds were at a ratio of about 5:1 macrocyclic compound to nucleoside.
  • the macrocyclic compound in the composition is preferably selected for optimal activity against the target virus, e.g., a herpes virus, respiratory syncytial virus, or retrovirus, as detailed above.
  • the preferred nucleoside analog compound is selected for activity against the target virus (Martin)
  • One advantage of the combined-drug composition is that substantially lower doses of both types of compounds are required for achieving a selected viral inhibition level, reducing drug side effects in a composition that also is characterized by greater anti-viral activity.
  • Section A examines the specificity of the viral-inhibition method to enveloped viruses.
  • the studies reported in subsection A show that the macrocyclic compounds used in the method act, at least in part, by binding selectively to viral envelope proteins, and that this binding blocks virus attachment to infectable cells, thereby inhibiting virus infectivity. These studies are detailed in parent U.S. patent application Serial No. 647,720, filed January 29, 1991.
  • Subsection B examines the inhibitory effect of the macrocyclic on non-enveloped viruses.
  • Example 15 In a second study, the effect of compound when administered prior to, during, or after cell infection by HSV-1 was examined as described in Example 15.
  • cells were exposed to one of a series of increasing KY-2 concentrations, and the extent of infection was measured by number of plaques observed 24 hours after infection.
  • the reduction in plaque formation, expressed as a percent of control, is shown in Figure 31 for cells treated with drug prior to (solid rectangles) , during (closed circles) , and after (open rectangles) .
  • Virus inhibition was seen most significantly when the cells were treated with drug during exposure to virus, indicating that virus inhibition occurs at the period of virus binding to and entry into infectable cells.
  • the major bands of drug binding in HSV-1 have molecular weights, as determined from SDS-PAGE, of 45, 66, and about 130 kilodaltons.
  • the major bands of drug binding in HSV-2 have similar molecular weights.
  • the major bands which show KY binding in Figure 32B correspond in molecular weight, to HSV glycoproteins gD, gB, and gC.
  • Vero cells were infected with adenovirus in the presence of KY-1, also at concentrations ranging between 1-100 ⁇ g, and twenty-four hours after virus infection, the cells were examined for cytopathic effect. No reduction in cell clumping was observed at any of the KY-1 drug concentrations.
  • a broad range of macrocyclic com ⁇ pounds are effective inhibitors of cell infection by each of the several enveloped viruses which were studied. Binding studies carried out in particular with respect to HSV viruses indicate that the anti ⁇ viral activity of the compounds is dependent on binding to virus envelope components, which in turn inhibits virus attachment to infectable cells. The apparent inability of the compounds to inhibit infection of non-enveloped viruses is consistent with this mechanism.
  • Viral Inhibition bv a Composition Con ⁇ taining a Macrocyclic Compound and a Nucleoside Analog Compound
  • the invention also includes a composition containing a macrocylcic compound of the type described above in combination with a nucleoside analog anti-viral compound.
  • the nucleoside analog compound is one effective to inhibit viral replication at the level of viral replication or transcription.
  • nucleoside analog compounds which are useful in combination with a macrocyclic compound, in accordance with the invention are:
  • Pyrophosphate analoges such as phosphoformic acid (PFA) , phosphonoacetic acid (PAA) , methanediphosphonic acid (MDP) , carbonyldiphosphonic acid (COMDP) , phosphonoglyoxalic acid (COPAA) , and various halogen- and/or methyl-substituted derivatives thereof, which are inhibitors of viral nucleic acid polymerases.
  • PFA phosphoformic acid
  • PAA phosphonoacetic acid
  • MDP methanediphosphonic acid
  • COMDP carbonyldiphosphonic acid
  • COPAA phosphonoglyoxalic acid
  • these compounds are known to inhibit herpes virus (Blackburn, Sidwell) and Influenza (Sidwell) infections, and reverse transcriptase activity in retroviruses, such as human HIV.
  • Base-modified analogs such as IUDR, trifluorothymidine, AraA, and azidothymidine (AZT) , didieoxyinosine (DDI) , D4T, dideoxycytidine (DDC) , and ribavirin.
  • Trifluorothymidine, IUDR, and AraA are active mainly against herpes virues (Nicolson, 1984a, 1984b) .
  • Ribavirin is active against several RNA and DNA viruses (Sidwell)
  • AZT is active against HIV (Fischl) , as are other dideoxynucleoside analogs, such as DDI.
  • Sugar-modified analogs such as N-acyl derivatives of 5'-amino-2' ,5'-dideoxy 5'-ioduridine, sulphonamide derivatives of 5'-amino-5'- deoxythymidine, 2'-deoxy-5-ethyluridine, and N-acyl derivatives, 5'-Sulfate and 5'-sulfamate nucleoside analogs, such as nucleocidin, adenosine 5' sulfamate, and ribavarin, which may act primarily at the level of protein synthesis inhibition (Martin) .
  • N-acyl derivatives of 5'-amino-2' ,5'-dideoxy 5'-ioduridine sulphonamide derivatives of 5'-amino-5'- deoxythymidine, 2'-deoxy-5-ethyluridine
  • N-acyl derivatives 5'-Sulfate and 5'-sulfamate nucleoside analog
  • Phosphate analogs including acyclonucleoside phosphonates, such as acyclovir and gangiclovir, and their isosteric phosphonate analogs. These compounds can act as virus-selective substrates for viral thymidine kinases, in the synthesis of nucleoside triphosphate analogs intracellularly (Galbraith) . Subsequently, the nucleoside triphosphate analogs can act as selective substrates for viral DNA polymerase, acting as a chain terminator since the analog does not have the bifunctionality necessary for chain extension (Allen) . These compounds have demonstrated anti ⁇ viral activity against herpes viruses (Collins) , including HSV-1, HSV-2, varicella zoster (VZV) , and cytomegalovirus (CMV) (Smith) .
  • Collins herpes viruses
  • VZV varicella zoster
  • CMV cytomegalovirus
  • phosphonomethyl ethers of nucleosides and their acyclic analogs, such as N-(3-hydroxy-2-phosphonylmethoxypropyl)- (HPMP-) and N-(2-phosphonylmethoxyethyl-) (PME-) derivatives of heterocyclic bases.
  • HPMP- N-(3-hydroxy-2-phosphonylmethoxypropyl)-
  • PME- N-(2-phosphonylmethoxyethyl-) derivatives of heterocyclic bases.
  • HPMP- N-(3-hydroxy-2-phosphonylmethoxypropyl)-
  • PME- N-(2-phosphonylmethoxyethyl-) derivatives of heterocyclic bases.
  • These compounds act specifically against herpes viruses, adenoviruses, cytomegalovirus (DeClercq) , poxviruses, vaccinia viruses, and retroviruses.
  • FIG. 34A shows the drop in HSV-1 viral yields when infected cells are exposed to increasing concentrations of the macrocyclic compound Y-l alone (solid circles) , increasing concentrations of acyclovir alone (open circles) , increasing concentrations of acyclovir plus 25 ⁇ g/ml Y-l (solid rectangles) , and increasing concentrations of acyclovir plus 50 ⁇ g/ml Y-l (solid ovals) .
  • the two compounds are formulated (e.g., in tablet, jelly, ointment, or injectable form) at a preferred weight ratio of between about 10:1 to 1:1 macrocyclic compound and nucleoside analog, respectively.
  • the viral-yield plots in Figures 34A and 34B a significantly higher level of inhibition was observed when the co-administered compounds were at a ratio of about 5:1 macrocyclic compound to nucleoside.
  • the macrocyclic compound in the composition is preferably selected for optimal activity against the target virus, e.g., a herpes virus, respiratory syncytial virus, or retrovirus, as detailed above.
  • the preferred nucleoside analog compound is selected for activity against the target virus (Martin) .
  • One advantage of the combined-drug composition is that substantially lower doses of both types of compounds are required for achieving a selected viral inhibition level, reducing drug side effects in a composition that also is characterized by greater anti-viral activity.
  • calix(n)arene compounds of the type described herein are administered to the site of infection in an individual infected with an enveloped virus, for treatment of the invention.
  • the composition of the invention includes novel includes the calix(n)arene compound contained in a pharmaceutical carrier which is suitable for oral, topical or parenteral administration of the compound.
  • the composition may contain the calix(n)arene compound alone, or in combination with an anti-viral nucleoside analog.
  • the dosage form of the composition is one which is pharmaceutically effective, i.e., effective to inhibit viral infection of host cells.
  • compound doses in the range 1-50 ⁇ g/ml are generally effective in inhibiting viral infection of cells.
  • an effective dose is preferably one which produces a concentration of compound in this range at the site of infection.
  • a composition containing between 1-5% or more calix(n)arene is suitable.
  • composition dose may be substantially lower in one or both compounds, as discussed in the section above.
  • calix(n)arene compound particularly the sulfonic acid compound
  • the calix(n)arene compound may show anti-coagulant activity after oral and intravenous administration.
  • a polycationic compound such as protamine sulfate
  • the protamine administration is timed to correspond to highest blood levels of the calix(n)arene compounds.
  • a dose of protamine equivalent to about l mg per 100 heparin anticoagulant units is administered intravenously simultaneously with IV administration of the calix(n)arene, or 1-2 hours after oral administration of the macrocyclic drug. It is generally recommended that protamine be infused slowly (i.e., not more than a total of 50 mg/10 minutes) . Therefore in the case of simultaneous administration of calix(n)arene compound, the rate of co-infusion of the two compounds would be adjusted such that the protamine sulfate was not introduced to the subject at a rate exceeding 50 mg/10 minutes.
  • composition of the invention can therefore include protamine in an amount effective to reduce the anti-coagulant effect of the macrocyclic compound, when the compound is administered for uptake into the bloodstream.
  • the composition also contains a nucleoside analog drug, and lower amounts of macrocyclic drug, the protamine may be reduced or eliminated, due to the lower amounts of macrocyclic compound.
  • viral e.g., HSV-1, HSV-2, RSV, and HIV
  • the injectable composition contains the calix(n)arene in a suitable IV solution, such as sterile physiological salt solution.
  • a suitable IV solution such as sterile physiological salt solution.
  • the solution may additionally contain nucleoside analog compound and/or protamine.
  • the composition is preferably formulated in an ointment form.
  • a topical composition for treatment of genital herpes lesions is illustrated in the following study, which is detailed in Example 13. Briefly, female guinea pigs were infected intravaginally with HSV-2, then treated topically three times daily beginning 6 hours or 48 hours after inoculation with HSV-2, as described in Example 13. Animal groups included control animals (no treatment following virus inoculation) , placebo (vehicle treatment) , KY-1 in vehicle, or acyclovir. Swabs of vaginal secretion were obtained and assayed for viral activity by a standard CPE assay.
  • the severity of genital lesions was scored on a 0-5+ scale through the period of primary infection (21 days) . Three to four days after HSV-2 inoculation, vesicular lesions appeared on the external genital skin. Lesions progressed to an ulcerative stage by days 7-8 and gradually healed by days 15-21.
  • the effect of topical treatment with the KY-1 preparations on lesion development and severity is shown in Table 10.
  • the group treated with placebo at +6h had a significantly increased lesion score-day AUC (P ⁇ 0.05); however, mean peak lesion scores were not different when compared to the untreated control group.
  • Lesion development as determined by both AUC values and mean peak lesion scores was significantly reduced by treatment with 5% KY-1 when given at 6h after infection compared to the placebo (P ⁇ 0.001). Treatment with 1% KY-1 significantly reduced the AUC at +6h (P ⁇ 0.01) but not mean peak lesion scores.
  • Treatment with placebo resulted in significantly worse infection scores than no treatment in this study.
  • Drug treatment with 2% or 6% Y-l, administered 6 hours post infection resulted in reduced numbers of animals exhibiting lesions, decreased mean lesion scores and decreased peak lesion score, in comparison to placebo treatment.
  • treatment with a 6% formulation of either KY-1 or Y-l or a 2% formulation of KY-1 administered 24 hours post-infection, resulted in reduced numbers of lesion bearing animals and reduced severity of lesions.
  • the topical composition includes a calix(n)arene compound in a ointment or solution form suitable for administering the compound to the eye, e.g., to the corneal surfaces of the eye.
  • composition may also include a nucleoside compound effective against the target viral infection.
  • a nucleoside compound effective against the target viral infection.
  • a topical concentration of 12.5 ⁇ g/50 ⁇ l was the most effective ocular therapy.
  • the epithelial disease scores decreased through day 6 post infection, and rebounded slightly on day 7 post infection.
  • this concentration was effective in reducing the development of HSV-1 disease in the eye and was associated with only mild conjunctival, iris, and stromal disease development parameters.
  • a higher concentration (18.75 ⁇ g/50 ⁇ l) was also effective in reducing the development of corneal epithelial HSV-1- induced disease.
  • this concentration of Y-l appeared to be somewhat toxic to the corneal epithelial surface and to the conjunctiva, iris and stroma. This toxicity was evidenced as an increase in all disease parameters on days 6 and 7 post infection.
  • Viral titers were recovered from tear film at days 0, 3, 5, and 7 post-inoculation and from epithelial scrapings performed on day 7 post- infection (sacrifice) . Viral titers were determined by plaque reduction and multiple regression analysis, as described in Example 10. In the tear film study, a marked reduction of viral titer was observed in all animals given topical doses of Y-l, and this reduction appeared dose-dependent, although no difference was seen at the highest doses (12.5 and 18.75 ⁇ g/50 ⁇ l) . A dose-dependent reduction in viral titer was observed in the scrapings taken on day 7. Based upon these studies, dose efficacy/range was generated. The optimal concentration of compound appeared to be 12.5 ⁇ g/50 ⁇ l in this study.
  • D. Oral Composition Studies conducted in support of the present invention have shown that a macrocyclic drug of the type used in the invention is available in the plasma for a period from about 0.5 hrs. after oral administration (e.g., by gavage) , with a peak at about 2-4 hours.
  • the period of effective drug concentration in the bloodstream is roughly between 4 and 18 hours after IV administration.
  • the relatively short distribution volume halflife of the drug, reflecting distribution to extracorporeal body compartments when the compound is administered intravenously, is generally advantageous in the case where drug is one which shows anti-coagulant side effects, since the concentration of compound in the bloodstream can be more closely titrated.
  • a topical composition containing a macrocyclic compound such as described above is administered to an area or areas of the body where sexual contact is likely to occur.
  • Such areas may include the skin surfaces of the anal- genital regions and the mouth, and also mucous membrane tissues of the vagina, rectum, mouth, and throat.
  • the macrocyclic compounds described above are compatible with topical administration on skin and mucous membranes; i.e., that the compounds do not give rise to signs of irritation, such as swelling or redness.
  • the topical composition may include a pharmaceutically acceptable carrier adapted for topical administration.
  • the composition may take the form of a suspension, solution, ointment, lotion, sexual lubricant, cream, foam, aerosol, spray, suppository, implant, inhalant, tablet, capsule, dry powder, syrup, balm or lozenge, for example. Methods for preparing such compositions are well known in the pharmaceutical industry.
  • the composition may include an antiviral nucleoside analog such as discussed in section V above.
  • the composition is a lubricating jelly (sexual lubricant) which includes a lubricating jelly vehicle, and dissolved in the vehicle, a macrocyclic compound of the invention.
  • the jelly vehicle acts in part to minimize abrasion during sexual contact, thereby reducing the likelihood of entry of an enveloped virus into damaged tissue and subsequent entry into the blood stream.
  • the macrocyclic compounds of the invention are effective to bind tightly to enveloped viruses, thereby inhibiting virus attachment to infectable cells.
  • the macrocyclic compound in the jelly can intercept enveloped virus particles before infection can occur.
  • the dosage form of the composition is one which is pharmaceutically effective, i.e., effective to inhibit infection by a sexually transmitted enveloped virus.
  • an effective dose is preferably one which produces a concentration of compound in this range at the site of infection.
  • a composition containing between 1-10% of macrocylic compound is suitable.
  • the amount of compound in the composition may be substantially lower for one or both compounds.
  • Y-l as part of a lubricating jelly formulation, to inhibit cytopathic effects in cells exposed to HSV-1 and HSV-2 was assessed in the study detailed in Example 19. Jelly formulations consisting of a commercial lubricating jelly (K-Y Jelly, Johnson & Johnson) and a selected amounts of Y-l were used. In the study, cells were exposed to ⁇ - HSV-i or HSV-2 virus and, at the same time, to a Y-l jelly composition at final drug concentrations of 5- 40 ⁇ g/ml. Twenty-four hours after inoculation, the cells were inspected for cytopathic effects (i.e., round cell formation) .
  • lubricating jelly formulations containing various amounts of Y-l were tested for inhibition of cytopathic effects in cells infected with one of two HTLV-III strains, HTLV-III B and RF-II (Example 20) . Briefly, cells chronically infected with HTLV-III B or RF-II were incubated in the presence of serial dilutions of the selected Y-l- containing jelly compositions, and were then cocultured with indicator cells. The extent-of syncytia formation was scored under phase microscopy.
  • Y-l formulated in a jelly composition is effective for inhibiting cell infection by the HTLV strains tested.
  • the invention includes a physical-barrier type device, in combination with a macrocyclic of the type described above, for use in inhibiting infection by sexually transmitted enveloped viruses.
  • the device includes a physical barrier-type device (e.g., a contraceptive device) , and coating the device, a lubricating composition composed of a lubricating jelly vehicle containing a macrocyclic compound of the invention.
  • the device is a condom, where the lubricating composition is coated on the outer surface of the condom.
  • the contraceptive can also be a condom in which the macrocycle-containing composition coats the inner surface of the condom, for the purpose of combining with any semen expressed into the condom.
  • the condom may be of the conventional type used by males (i.e., for placement on a male penis) , or may be of an insert-type such as can be worn by a female, where the device includes a condom element that is inserted into the vagina prior to initiation of heterosexual intercourse, for example.
  • the device may also take the form of a cervical cap, diaphragm, or contraceptive sponge which is placed in the vicinity of the cervix. Accordingly, a cervical cap or diaphragm is coated or infused with a jelly or cream, for example, which contains a macrocyclic compound of the invention. Similarly, a contraceptive sponge may be imbued with a solution, cream, or the like, which contains a macrocyclic compound of the invention.
  • the macrocyclic compounds of the invention are compatible with latex contraceptive devices for preventing the transmission of viral infection during sexual intercourse.
  • KY-1 (2mM) was treated with 5 ml chlorosulfonic acid and the mixture was stirred at 50°C for one-half hour. The resultant mixture was added to 20 g of crushed ice to precipitate the product, which was collected by filtration and then washed with ether.
  • the crude product was dissolved in 100 ml of 25% ammonium water solution and allowed to react for 2 hours at room temperature. The mixture was concentrated in vacuo and the remaining oil was dissolved in a small amount of water and filtered.
  • the product was precipitated by adding acetonitrile to the filtrate and collected by filtration and washing with acetonitrile.
  • the compound was characterized as follows:
  • UV (H 2 0) 238.0, 351.5, 520 nm;
  • KY-1 (50mM) was dissolved in 80 ml of NaOH water solution (0.2M NaOH) and heated to 50°C, dimethyl ⁇ sulfate (0.2M) was added slowly for 1 hour. The mixture was continuously stirred for another 2 hours and left at room temperature for 2 days. Saturated NaCl solution (100 ml) was added to the resultant substance and filtered. The precipitate was washed with ethanol, acetonitrile and ether sequentially. The dry substance was dissolved in 100 ml of methanol and filtered. The filtrate was concentrated and ether was added to precipitate the dimethyl ether of chromotropic acid, disodium.
  • Chromotropic acid disodium salt was dissolved in 80 ml of water at a concentration of 50 mM with ⁇ tirring at 50°C until the solution turned to clear, hexamethylenetetramine (50 mM) was then added to above solution with continuous stirring at the same temperature for additional two hours. At this time, the color of this mixture converted to dark blue. The mixture was allowed to stir at room temperature for 2 days. The resultant dark blue solution was filtered and the filtrate was concentrated, evaporated by flask, which was subsequently treated with 200 ml methanol to precipitate the product KY- 346. The yield of KY-346 was 85%.
  • the compound was characterized a ⁇ follows: M.P.>300°C;
  • Y-l (1 g) is heated at 60-70°C with chlorosulfonic acid (20 ml) for 1 hour. After cooling to room temperature, the oily material is poured into ice water, and the precipitate is filtered. After washing the precipitate with cold water, the material is added to 50 ml of solution containing 5.7 g glycine and 2.1 g NaOH, and stirred for 2 hours at room temperature. After removal of all ⁇ olvent from the resultant substance, the residue is dis ⁇ olved in a 200 ml of cold methanol and filtered. The filtrate i ⁇ added with acetonitrile to precipitate the product.
  • trifluoromethanesulfonic anhydride (1.0 ml) i ⁇ added to ice cold dry dichloromethane solution (10 ml) of 2,6, di-tert- butyl-4-methylpyridine (1.25 g) and 4-tert- butylcalix[4]arene (0.65 g) .
  • the mixture is diluted with pentane (10 ml) and filtered.
  • the filtrate is extracted with ice cold IN aqueous NaOH solution, ice cold IN aqueous HCl solution, then saturated aqueous NaCl ⁇ olution, dried over anhydrous sodium sulfate, filtered through a pad of silica gel and concentrated in vacuo.
  • Calix(4)arene XXVI is prepared from 4-tert- butylcalix(4)arene (XXV; Fig. 13) as described (Gutsche, Levine, and Sujeeth, 1985) .
  • Calix(4)arene XXVIII is prepared as described (Yilmaz and Vural) .
  • Known p-acetyl-calix(4)arene (XXVII; 1.3 g) (Yilmaz and Vural, 1991; No et al., 1986) is dissolved in 50 ml of 2 N aqueous NaOH.
  • a solution of iodine (8 g) and potassium iodide (20 g) in 40 ml of water is added and the mixture stirred. The solution i ⁇ warmed on a water bath for 1 h.
  • Derivative XXXVI is prepared by adaptation of the methods of Arduini et al. and Hirao et al.
  • Calix(4)arene (XXVI) is refluxed with Hg(OCOCF 3 ) 2 in CHC1 3 , giving an almost quantitative yield of the tetra-(Hg-OCOCFj) calixarene derivative.
  • metal iodine exchange is carried out by reaction of the calixarene derivative with I 2 in CHCl j , giving p-iodo-calix(4)arene XXXIV as a brown compound in 40% yield.
  • Calix(4)arene derivative XLIV is prepared via p- chloromethyl-calix(4)arene as described (Almi et al.) .
  • Calix(4)arene derivative XLVI i ⁇ prepared from p-2-bromoethyl derivative XLIV (Example P) by modification of the method used to make XXXIX from p- chloromethyl-calix(4)arene (Example 0).
  • UV-active fractions containing product are concentrated under vacuum and the re ⁇ idue i ⁇ recry ⁇ tallized from methanol to give purified XLVII.
  • Calix(4)arene derivative XLIX i ⁇ prepared from bromoethyl derivative XLIV (Example P) by applying sequentially the sulfonation method of Example R to give XLVIII, the hydroly ⁇ is step of Example Q to remove the tosyl group, and the Amberlite IR-120 ⁇ tep of Example R to produce ⁇ ulfonic acid XLIX.
  • Derivative LII (Example T; 0.5 mmol) i ⁇ refluxed in S0C1 2 (5 ml) under nitrogen atmo ⁇ phere. After evolution of S0 2 ha ⁇ ceased, excess S0C1 2 is removed by distillation under high vacuum ( ⁇ 0.1 mm Hg) . To the re ⁇ idue i ⁇ added THF (5 ml) , and distillation is repeated to remove residual S0C1 2 , yielding chloro- derivative LIII.
  • Calix(4)arene XXVI (1.54 mmol) is dis ⁇ olved in THF (100 ml) at 50°C under nitrogen atmo ⁇ phere. After cooling, ⁇ odium hydride (1.20 g, 30 mmol; 60% dispersion in oil) is added and the mixture is stirred until evolution of hydrogen ceases (-1 h) . Propane-l,3-sulfone (2.26 g, 18.5 mmol) i ⁇ then added dropwi ⁇ e and the mixture is stirred at room temerature for 24 h.
  • Remaining NaH is decomposed by addition of methanol, after which the solvent is evaporated under reduced pre ⁇ ure, and the re ⁇ idue i ⁇ di ⁇ olved in hot water (500 ml) . Any insoluble material is removed by centrifugation. The product i ⁇ then precipitated by the ⁇ alting-out method with sodium acetate to give purified LXII (10% yield) . m.p. > 300°C
  • Example 4 Cytotoxicity in Proliferating Cells A panel of human cell lines was used to check the toxicity of the drugs, including: KB (nasopharyngeal carcinoma) , HeLaS 3 (cervical epithelial carcinoma) , PLC (hepatocarcinoma) , HepG 2 (human hepatocarcinoma) HepG 2 T 14 (hepatocarcinoma tran ⁇ fected with HBV) , WI38 (normal human lung fibrobla ⁇ t) , BT549 (brea ⁇ t cancer) , SW480 (brea ⁇ t cancer) , and A549 (lung cancer) .
  • KB nasopharyngeal carcinoma
  • HeLaS 3 cervical epithelial carcinoma
  • PLC hepatocarcinoma
  • HepG 2 human hepatocarcinoma
  • HepG 2 T 14 hepatocarcinoma tran ⁇ fected with HBV
  • WI38 normal human lung fibrobla ⁇ t
  • Example 5 Inhibition of HSV Activity: Cytopathic Effect Vero cell ⁇ were maintained in RPMI-1640 medium supplemented with 5% fetal calf serum, 100 Units of penicillin per ml and 100 ⁇ g of streptomycin per ml at 37°C in a humidified incubator containing 7% C0 2 .
  • the HSV strain ⁇ HSV-1 (Kos-l) and HSV-2 (333) were used. 1 x 10 s Vero cell ⁇ were plated in each well of a
  • the cells In the absence of viral infection, the cells form an even monolayer of fibrobla ⁇ t cell ⁇ . With viral infection, the cells form a suspension of round cell ⁇ , followed by cell clumping, whose appearance is easily distingui ⁇ hable from normal fibrobla ⁇ t cells. If no detectable cytopathic effect was produced, the test was repeated with 10 ⁇ g/ml. A parallel set of cells without virus inoculation were done as a control for cytotoxicity to Vero cells.
  • Table 1 above shows the structure ⁇ of the com ⁇ pound ⁇ which were tested, and Table 3, column 2, the compounds which protected the cells from cytopathic effect (+) .
  • Example 6 Inhibition of HSV Activity: Plague Reduction Vero cells were maintained in RPMI-1640 medium supplemented with 5% fetal calf serum, as in Example 5. 4 x 10 5 Vero cells were plated in a 24-well plate, in 1 ml RPMI-1640 medium containing 5% FCS and 0.1% methyl cellulo ⁇ e (15 cps) .
  • the medium was aspirated and replaced with 100 ⁇ l of the same medium containing 2% FCS, which contained 50 ⁇ l control or drug solution to a final drug concentration of 0.25, 2.5, 5, 10, or 20 ⁇ g/ml and 50 ⁇ l viru ⁇ , containing about lxlO 3 PFU/ml, i.e., 50 PFU/well, of HSV-1 or HSV-2, a ⁇ in Example 5.
  • Example 7 Inhibition of HSV Activity Viral Yield Inhibition 1 x 10 6 HeLa S 3 were plated in 25 T flasks in 5 ml RPMI-1640 + 5% FCS + P/S. 24 hours later, the medium was aspirated and replaced with 6 x 10 6 PFU HSV-1 or HSV-2, and serial dilutions of selected KY compounds, at 10, 5, 2.5, 1.25, and 0.625 ⁇ g/ml drug. After growth at 37°C for 24 hours in 2 ml of RPMI- 1640 containing 2% FCS and P/S, the cells were frozen at -70°C until the time for titration. The cells were freeze/thawed 3 times to release virus from the cells, and serially diluted 10 fold.
  • IX 10 5 Vero cells were plated in each well of 24 well multi-dish in 1 ml RPMI-1640 + 5% FCS + P/S + 0.1% methylcellulose (15 cp ⁇ ) .
  • the 10 fold ⁇ erially diluted virus in 100 ⁇ l was added in duplicate.
  • the viru ⁇ wa ⁇ removed and 0.5 ml methycellulose (4K cps) in RPMI- 1640 and 2% FCS + P/S was added.
  • the cell ⁇ were ⁇ tained in 0.8% crystal violet in 50% ethanol. The plaques formed were counted and the titer was calculated from the fold of dilutions.
  • HSV-l and HSV-2 Activity against Drug-Re ⁇ istant Strains of HSV-l and HSV-2
  • KOS a wild type HSV-1 viru ⁇
  • KOS PMEA
  • KOS PFA
  • DHPG DHPG
  • Inhibition of viral yield was by KY-1, acyclovir (ACV), DHPG, PFA, and FMEA wa ⁇ examined in each of the five HSV strains substantially as de ⁇ cribed in Example 7. Briefly, Hela S 3 were plated in 25 T fla ⁇ k ⁇ in culture, and 24 hour ⁇ later, the medium was aspirated and replaced with 6 x 10 6 PFU of the selected HSV ⁇ train, and serial dilutions of KY-1, ACV, DHGP, PFA, and PMEA. After growth at 37°c for 24 hours in 2 ml of RPMI-1640 containing 2% FCS and penicillin and ⁇ treptomycin (P/S) , the cell ⁇ were frozen at -70°C until the time for titration.
  • the cell ⁇ were freeze/thawed 3 time ⁇ to relea ⁇ e virus from the cells, ⁇ erially diluted 10 fold, and the serial dilutions were added to Vero cell ⁇ in culture. After 2 hour ⁇ incubation at 37°C the viru ⁇ wa ⁇ removed and 0.5 ml methycellulo ⁇ e (4K cps) in RPMI- 1640 and 2% FCS + P/S wa ⁇ added. Two day ⁇ later, the medium was removed. The cells were stained in 0.8% crystal violet in 50% ethanol. The plaques formed were counted and the titer was calculated from the fold of dilutions. From the drug dose response, the concentration of each drug required to effect a 90% inhibition of virus yield, the ICgo concentration was determined. These values are ⁇ hown in Table 5 above.
  • Example 9 Inhibition of RSV Activity Assays to assess the antiviral activity of KY- and Y- compounds in tissue culture were performed in 96-well flat-bottom tissue culture plates (Falcon
  • Example 10 Activity against HSV virus: Topical activity against in vivo ocular cultures of HSV-1
  • New Zealand white rabbits were acclimated for a minimum of two day ⁇ prior to inoculation to allow the animals to accommodate to conditions in the vivarium facility. After the accommodation period, animals received a slit lamp ocular examination to exclude any animals with preexi ⁇ ting anterior segment ocular defects. Animals were bilaterally inoculated topically with an 80 ⁇ l drop of Minimal Essential Medium (MEM; Gibco) containing 10 s pfu/ml McKrae strain HSV-1; eyes were massaged for 30 seconds. Animals were replaced individually in cages.
  • MEM Minimal Essential Medium
  • mice On day 4 post inoculation (PI) , animals were evaluated by ⁇ lit lamp micro ⁇ copy. Corneal epithelial, iris, and conjunctival disease were graded on an increasing scale of severity from 0+ to 4+. After evaluation, animals were divided into 4 groups of 5 animals with matched corneal, stromal and conjunctival involvement. Topical therapy was initiated immediately after animal grouping. Therapy groups included:
  • Group #1 5 rabbit ⁇ , Y-l topical therapy (6.25 ⁇ g/50 ⁇ l) 5x/day for 4 day ⁇ ;
  • Group #2 5 rabbit ⁇ , Y-l topical therapy (12.5 ⁇ g/50 ⁇ l) 5x/day for 4 day ⁇ ;
  • Group #3 5 rabbits, Y-l topical therapy (18.75 ⁇ g/50 ⁇ l) 5x/day for 4 days;
  • Group #4 5 rabbit ⁇ ., placebo therapy ( ⁇ terile water) 5x/day for 5 days.
  • the concentration of Y-l for the ascending do ⁇ e tolerance ⁇ tudy were ba ⁇ ed upon the ED90 concentration ⁇ determined in the viru ⁇ yield or CPE assays.
  • Group l received topical eyedrop therapy containing 6.25 ⁇ g/50 ⁇ l [one-half of the ED90 concentration] ;
  • Group 2 received eyedrop therapy containing 12.5 ⁇ g/50 ⁇ l [the ED90 concentration];
  • Group 3 received eyedrop therapy containing 18.75 ⁇ g/50 ⁇ l [1.5 times the ED90 concentration]. All Y-l doses were formulated to contain these concentrations in a volume of 50 ⁇ l (a ⁇ tandard eye drop) .
  • Topical therapy with 0-19 ⁇ g Y-l in 50 ⁇ l wa ⁇ initiated on day 4 po ⁇ t-inoculation (PI) and continued to day 7 PI. All animal ⁇ received daily ocular ⁇ lit lamp evaluations from day 3 through day 7 PI. The ocular HSV-1 induced disease severity was recorded daily.
  • Eyes of all animal ⁇ were additionally sampled for the presence of infectious HSV-1 on days 0 (pre- inoculation) , 3, 5, and 7 PI. Briefly, tear film was obtained by swabbing the lower and upper conjunctival sacs and retaining the swab in the nasal fornix for 10 seconds. The swabs were eluted individually in Hank's Buffered Saline (HBSS, Gibco Laboratories). Fifty microliter aliquots of the viru ⁇ -HBSS eluate was adsorbed onto confluent HFF cell monolayer ⁇ for 5 minute ⁇ .
  • HBSS Hank's Buffered Saline
  • HSV CPE HSV infection
  • the anti-influenza A activity of KY compounds was evaluated as described in Example 9, except that MDCK cells (kidney cell line) was u ⁇ ed for infection in vitro by influenza viru ⁇ ( ⁇ train A/Taiwan) .
  • Example 12 Inhibition of HIV-Induced Cell Fu ⁇ ion Human CD 4 + indicator cell ⁇ (VB) and chronically infected H, cells were maintained in RPMI-1640 medium supplemented with 5% fetal calf serum, 100 Units of penicillin per ml and 100 ⁇ g of streptomycin per ml at 37°C in a humidified incubator containing 7% C0 2 .
  • the HIV strain ⁇ that were u ⁇ ed were HTLV-III B and RF- II strains obtained from the National Institute ⁇ of Health (Bethesda, MD) .
  • serial dilution ⁇ between 1:2 and 1:2 8 of a selected KY compound, l mg/ml in PBS were made in a 96 well round bottom plate.
  • the diluted KY compound was transferred to a 96 well flat-bottom plate.
  • To each well was added 25 ⁇ g chronically infected H 9 cells (at 2 x 10 6 , cell ⁇ /ml) , or cell ⁇ chronically infected with RF-II ⁇ train HIV, followed by incubation at 37°C for 45 minutes.
  • the MS ⁇ train of HSV-2 wa ⁇ utilized for the experimental animal infection.
  • Female Hartley ⁇ train guinea pigs (Charles River Breeding Laboratories, Springfield, NY) weighing 250-300 g were inoculated intravaginally with 2.0 x 10 s plaque-forming unit ⁇ of HSV-2 one hour after being ⁇ wabbed for removal of vaginal secretions.
  • vaginal secretions were obtained on days l, 3, 5, 7 and 10 after HSV inoculation, placed in a tube containing 2.0 ml of media, vortexed and frozen at -70°C until titrated for HSV-2. When all samples were collected, they were thawed, diluted serially and HSV-2 titers determined u ⁇ ing rabbit kidney cell ⁇ •in a microtiter CPE assay.
  • lesion severity was scored on a 0-5+ scale through the primary infection period (19-21 days) .
  • Lesion ⁇ core-day area ⁇ and virus titer-day areas under the curve, and peak lesion scores and peak virus titer ⁇ between untreated and placebo-treated or placebo- treated and drug-treated animal ⁇ were compared u ⁇ ing the Mann-Whitney U range sum test.
  • a p-value of 0.05 or les ⁇ was considered significant. The results are discussed with reference to Table ⁇ 10 and 11 in Section IV above.
  • Animal ⁇ were ⁇ cored daily for 19 days following inoculation for presence of le ⁇ ion ⁇ and ⁇ everity of le ⁇ ion ⁇ (on a 0-5+ point scale) .
  • Lesion scores were tabulated as area under the curve of daily lesion ⁇ core vs. time (days) and peak lesion score observed. Data are presented in Table 11.
  • a known antiviral agent, acyclovir (ACV) was administered in a 5% formulation to 8 animals as a positive control in the study.
  • Vero cell ⁇ were maintained in RPMI-1640 medium, a ⁇ de ⁇ cribed in Example 5. After overnight incubation, and cell doubling, the medium wa ⁇ a ⁇ pirated and replaced with 100 ⁇ l of medium containing 2% FCS composed of 50 ⁇ l control or drug solution to a final drug concentration of lO ⁇ g/ml and 50 ⁇ l virus, containing about 3 PFU/cell, i.e., 6 x 10 s PFU/well, of H 3 -labeled HSV-1. At time intervals of 5, 30, 60, 120, and 240 minutes, cells were removed from the suspension, washed two times with PBS, and assayed for bound virus (cpm 3 H) . The results are given in Figure 30, where the control virus binding is indicated by solid circles, and the drug-inhibited binding, by open rectangles.
  • Example 15 Effect of Drug/Virus Exposure on HSV Inhibition Vero cell ⁇ were maintained in RPMI-1640 medium, as above. After overnight incubation, and cell doubling, the medium was aspirated and replaced with 100 ⁇ l of medium containing 2% FCS. In one group of well ⁇ , serial dilutions of KY-1 compound, between
  • 0.625 and 10 ⁇ g/ml drug were added in 50 ⁇ l, together with 50 ⁇ l of HSV-1 virus suspension, 5 x 10 6 PFU per well. The cells were incubated for 2 hour ⁇ at 37°C, then washed with PBS and assayed for number of virus plaques, as in Example 6.
  • a second group of cell ⁇ serial dilutions of the drug were added to the cells, prior to the addition of the HSV-1 virus, and the cell ⁇ were incubated for 2 hour ⁇ at 37°C in the presence of the virus. After wa ⁇ hing the cell ⁇ to remove free drug, viru ⁇ suspension was added, 5 x 10 6 PFU per well. The cell ⁇ were incubated for 2 hours at 37°C, then washed with PBS and assayed for number of virus plaques, a ⁇ in Example 6. In a third group of cell ⁇ , 100 ⁇ l viru ⁇ suspension was added to the cells, 5 x 10 6 PFU per well, and the cells were incubated for 2 hours at 37°c, then washed with PBS to remove unbound virus.
  • the numbers of plaques observed in each of the above treatment methods, expressed as percent of untreated control, are plotted in Figure 31.
  • the solid circles indicate co-exposure of the cells to drug and virus; the solid squares, preincubation of the cells with drug before addition of virus; and the open ⁇ quare ⁇ , preincubation of the cell ⁇ with virus before addition of drug.
  • Purified HSV-1 was suspended in RPMI-1640 medium (Gibco Laboratories) containing 2% FCS, penicillin and ⁇ treptomycin. To aliquot ⁇ of the suspensions were added control, KY-1, or KY-217 solution, to a final drug concentration of 10 ⁇ g/ml, and a final virus particle concentration of 6 x 10 6 or 6 x 10 s PFU/ml. The suspensions were incubated for 1 hour at 37°C, then diluted serially at 10 fold dilutions to final drug concentrations of 10, 10°, 10" 1 , 10' 2 , 10 "3 , and 10" 4 ⁇ g/ml drug concentration ⁇ . The serially diluted particles were then added to Vero cells for two hour ⁇ , as in Example 6, and the cells examined for plaques 48 hours later. The number of plaques counted on each of two plates, for each viru ⁇ and drug concentration, are given in Table 9.
  • Example 17 Binding of KY Compound ⁇ to HSV Protein ⁇ A. Binding of KY compound to HSV Proteins HSV-1 and HSV-2 viral suspensions from above, each at a concentration of about 5 x 10 7 CFU/ml, were incubated for 2 hours at 37°C with 5 x 10 s cpm I4 C- labeled KY-1 (50 ⁇ g/ml) . Each viral suspension was divided into two aliquot ⁇ and solubilized with 0.5% sodium dodecyl sulfate (SDS) , with or without 1% mercaptoethanol. The four solubilized samples were fractionated on 8.5% polyacrylamide gel, and the gels developed by autoradiography, according to standard procedures.
  • SDS sodium dodecyl sulfate
  • HSV-1 and HSV-2 virus ⁇ u ⁇ pen ⁇ ion ⁇ were solubilized with SDS and fractionated on SDS-PAGE as above. Each sample was run in triplicate, corresponding to groups D, B, and C in Figure 32B. The two gels in each group were analyzed by We ⁇ tern blotting as follows: The gels in groups D, B, and C were fir ⁇ t reacted with mouse monoclonal antibody specific against HSV glycoprotein gD, gB, and dC, re ⁇ pectively. The antibodies were obtained from Dr. S. Chatterjee from the University of Alabama.
  • the gels were then incubated with alkaline phosphata ⁇ e- labeled goat anti-mouse antibody, to label the glycoprotein in each group.
  • the glycoprotein with bound antibody was identified by reaction with H 2 0 2 in the presence of nitroblue tetrazolium and bromochloroindolephosphate, according to standard methods. The results are shown in Figure 32B.
  • HSV-1 particle ⁇ were obtained from infected HeLa cell ⁇ , a ⁇ described in Example 7. IX 10 s Vero cells were plated in each well of 24 well multi-dish in 1 ml RPMI-1640 + 5% FCS + P/S + 0.1% methylcellulose (15 cp ⁇ ) .
  • the 10 fold ⁇ erially diluted viru ⁇ in 100 ⁇ l wa ⁇ added in duplicate, plus (i) a selected concentration of GL-288 alone (up to 50 ⁇ g/ml) , (ii) a selected concentration of acyclovir alone (up to 50 ⁇ g/ml) ; (iii) a selected concentration of acyclovir (up to 50 ⁇ g/ml) plus 25 ⁇ g/ml GL-288; or (iv) a selected concentration of acyclovir (up to 50 ⁇ g/ml) plus 50 ⁇ g/ml GL-288, to Vero cell ⁇ in culture.
  • the viru ⁇ was removed and 0.5 ml methycellulose (4K cps) in RPMI- 1640 and 2% FCS + P/S was added. Two day ⁇ later, the medium wa ⁇ removed. The cells were stained in 0.8% crystal violet in 50% ethanol. The plaques formed were counted and the titer wa ⁇ calculated from the fold of dilutions.
  • HSV-2 particles were obtained from infected HeLa cells, a ⁇ de ⁇ cribed in Example 7. Vero cell ⁇ were infected with serial dilutions of the virus particles plus GL-228 alone, acyclovir alone, or acyclovir plu ⁇ GL-228 a ⁇ de ⁇ cribed in Section A. After 2 hour ⁇ incubation at 37°C, the viru ⁇ wa ⁇ removed, and 0.5 ml methycellulo ⁇ e (4K cp ⁇ ) in RPMI-1640 and 2% FCS + P/S wa ⁇ added. Two day ⁇ later, the medium was removed. The cells were ⁇ tained in 0.8% crystal violet in 50% ethanol. The plaques formed were counted and the titer was calculated from the fold of dilutions.
  • Example 19 Inhibition of HSV Activity: Cytopathic Effect Vero cells were maintained in RPMI-1640 medium supplemented with 5% fetal calf serum, 100 Units of penicillin per ml and 100 ⁇ g of streptomycin per ml at 37°C in a humidified incubator containing 7% C0 2 .
  • the HSV strain ⁇ HSV-1 (Ko ⁇ -1) and HSV-2 (333) were u ⁇ ed.
  • a topical formulation of compound Y-l wa ⁇ created by dissolving Y-l in K-Y Jelly (Johnson & Johnson) at 5, 10, 15 and 20% (weight/weight) concentrations. Each of the Y-l formulations was then dissolved and diluted in RPMI-1640 containing 2% FCS.
  • Vero cells were plated in each well of a 96 well microtitre plate in 0.2 ml RPMI-1640 medium containing 5% FCS and 0.1% methyl cellulose (15 cps). After overnight incubation and cell doubling, the medium was aspirated and replaced with 100 - ⁇ l of the same medium containing 2% FCS, and 50 ⁇ l control or drug solution yielding final drug concentrations of 5, 10, 20 and 40 ⁇ g/ml and 50 ⁇ l virus, containing about 3 PFU/cell, i.e., 6 x 10 5 PFU/well, of HSV-1 or HSV-2.
  • the cells were cultured for 24 hours at 37°C, at which time cytopathic effects are clearly visible.
  • the cell ⁇ form an even monolayer of fibrobla ⁇ t cell ⁇ .
  • the cell ⁇ form a suspension of round cells, followed by cell clumping, who ⁇ e appearance is easily distinguishable from normal fibroblast cells.
  • a parallel set of cells without virus inoculation were done as a control for cytotoxicity to Vero cells. No cytotoxicity by K-Y Jelly alone was observed. The results are shown in Table 12, where a "+" indicates full inhibition of cytopathic effects, and a "-" indicate ⁇ no inhbition of cytopathic effect ⁇ .
  • Human CD 4 + indicator cells (VB) and chronically infected H 9 cells were maintained in RPMI-1640 medium supplemented with 5% fetal calf ⁇ erum, 100 units of penicillin per ml and 100 ⁇ g of streptomycin per ml at 37°C in a humidified incubator containing 7% C0 2 .
  • the HIV ⁇ train ⁇ that were u ⁇ ed were HTLV-III B and RF- II strains obtained from the National Institutes of Health (Bethesda, MD) .
  • Topical formulation ⁇ of compound Y-l were prepared by dissolving Y-l in K-Y Jelly (Johnson & Johnson) at 5, 10, 15 and 20% (weight/weight) concentrations. Each Y-l formulation was then dis ⁇ olved in PBS to a final Y-l concentration of 1 mg/ml.

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Abstract

A method for inhibiting cell infection by an enveloped virus, by administering to an infection site, a therapeutically effective amount of a calix(n) arene compound derivatized, at its ring positions meta to the bridge attachments to the ring, with polar substituent having a terminal carboxylate, phosphate, or sulfonate groups, including esters and amides which are cleavable in vivo. The compound may be administered orally, or topically, e.g., for treatment of herpes virus. The invention also includes a method of inhibiting infection by sexually transmitted enveloped viruses, by topically administering to an area of likely sexual contact a composition containing a prophylactically effective amount of a macrocyclic compound such as described above.

Description

INHIBITION AND TREATMENT OF INFECTION BY ENVELOPED VIRUS WITH CALIX.N.ARENE COMPOUNDS
1. Field of the Invention The present invention relates to a method for inhibiting cell infection by enveloped viruses, and more particularly, to a method which employs a defined class of calix(n)arene compounds; and in a related aspect, to a method for inhibiting infection by sexually transmitted enveloped viruses.
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Yilmaz, M., and Vural, U.S. (1991) Synth. React. Inorg. Met. Org. Chem. 21, 1231-1241.
3. Background of the Invention
The challenge in developing an effective therapy and prophylaxis for viral disease is to achieve inhibition of viral processes without producing extreme side effects and preferably without inducing viral resistance. Since viral replication requires use of the cellular apparatus of the host, treating virus infection by inhibiting viral replication can be lethal to the infected host cells as well. Ideally, the virus should be destroyed or inactivated in the host prior to its invasion of host cells. This is normally accomplished, with varying degrees of success, by the host's immune system, but this mechanism requires an earlier immune response, either by a prior infection or by vaccination. Further, many viruses, such as Herpes Simplex viruses (HSV) are able to effectively elude a host's immune systems, and at least one virus, the human immunodeficiency virus (HIV) is known to cripple the host's immune system (Gottlieb) .
Currently, the most widely used anti-viral agents are nucleoside analogs. This class of drugs acts by disrupting viral replication, either by inhibiting enzymes required for nucleic acid processing, or by producing defective viral genomes, such as by premature termination of replication. As an example, acyclovir, a purine analog used in treating a variety of viral diseases, including herpes simplex virus-1 (HSV-1) and herpes simplex viruε-2 (HSV-2) inhibits viral replication at several key points, including inhibition of viral thymidine kinase and DNA polymerase, and DNA strand elongation (Elion) . Ribavirin, another purine analog, is the drug of choice in treating respiratory syncytial viruses (RSV) infection. This compound appears to act by reducing cellular GTP levels, blocking the action of several GTP-dependent viral processes (Smith) . To date, the most common drug treatment of HIV infection is with zidovudine (Azidothy idine; AZT) , a thymidine analog which is particularly effective against human retroviruses. AZT acts with high affinity to block viral RNA-dependent DNA polymerase (reverse transcriptase) , but does also block human DNA- polymerase and causes chain termination (Mitsuya) .
Other nucleic acid analogs include ganciclovir, vidarabine, idoxuridine, trifluridine and foscarnet (an inorganic phosphate analog) . As indicated above, all of these drugs, by blocking viral replication, also have the capacity to disrupt normal host replication and/or DNA transcription processes as well (see, e.g., Martin).
Understanding of the mechanisms of infection and replication of viruses has lead to alternate drug therapies, including attempts to block viral entry into cells, alter protein synthesis at the host ribosomes, complexation of viral DNA/RNA, and immunomodulation. Interferons are glycoproteins which have complex actions including enhancement of certain immune responses as well as direct antiviral action. They are more competent in preventing infec¬ tion, rather than treating established viral infec¬ tion, and their use leads to undesirable problems including acute, serious discomfort, bone marrow suppression, viral resistance, and development of host immune response to the interferon.
Treatment with "anti-sense" polymers of nucleic acids is a method in which the particular viral genome is the select target. The treatment provides a highly discriminating approach which would be expected to have minimal side-effects; its use as a therapeutic is hampered by problems of targeting, introduction into cells, and the quantity of material that would be required to block each strand produced. Agents which bind to and interfere with host riboso al protein synthesis will block viral replica¬ tion. These include the toxin ricin, various plant proteins such as pokeweed anti-viral protein, alpha sarcin, and other low molecular weight compounds. The weakness with the use of these materials is their lack of selectivity. In the treatment of HIV, additional therapy has been developed by specifically targeting the unique retroviral enzyme, reverse tran¬ scriptase. Non-retroviral systems do not produce or use this enzyme, but tha virus cannot replicate without it.
In some instances, an understanding of structural aspects of the mechanisms of replication of viruses has provided additional drug therapies. Certain viruses, including orthomyxoviruses and paramyxovirus, herpes viruses, togaviruses and retroviruses, contain a viral envelope which surrounds the viral capsid and nucleic acid. During cell infection by an enveloped virus, the plasma membrane of the host cell is altered to include some viral-coded proteins and, as the viral nucleoprotein core exits the host cell in which it was assembled, it becomes enveloped with the modified membrane, thus forming the viral envelope. Because this structure is unique to host cells when they are virally irfectious and distinct from normal cells, it can serve as an additional target for therapeutic assault.
4. Summary of the Invention
The present invention includes a method for treating infection by an enveloped virus. The method includes administering to the site of infection a therapeutically effective dose of a calix(n)arene compound which is derivatized, at its ring positions meta to the bridge attachments to the ring, with polar substituents having terminal carboxylate, phosphate, sulfinate, or sulfonate groups. In one general embodiment, the calix(n)arene has the general structure:
Figure imgf000010_0001
where n = 4-10, R, is OH, =0, an alkyl or aryl ether, an ester, an acid, or a mixture thereof; R2 is a polar substitutent with a terminal carboxylate, phosphonate, sulfinate, or sulfonate group, including cleavable esters and amides thereof; and R, is >CHR", > CR", or a mixture thereof, where R" is H or a carboxylate group. R, is preferably OH, or, in a partially oxidized form of the compound, a combination of OH and =0.
In one specific embodiment, the compound has the structure shown above, where n = 4, 6, or 8; R, is OH or =0 or a mixture thereof; R2 is as above; and R4 is >CH2 or >CH, or a mixture thereof.
In a more specific embodiment, R2 has the form: (CH2)mR2', where m= 1-3, and R2' is a sulfonate group of the form S03R or S02NRR', where R and R' are each H or a lower alkyl group. In another embodiment, R2 has the form: (CH2)m-
R2', where m= 1-2, and R2' is a sulfinate group of the form S02R or S(=0)NRR', where R and R' are each H or a lower alkyl group.
In another embodiment, R2 has the form: (CH2) m-R2' , where m= 0-3, and R2' is a carboxylate group of the form C02R or C(0)NRR', where R and R' are each H or a lower alkyl group.
In another embodiment, Rj has the form: (CH2)m-R2', where m= 0-3, and R2' is a phosphonate group of the form PO(OR)2, PO(OH)(OR), PO(OR) (NRR') , or PO(NRR')2, where R and R' are each H or a lower alkyl group. In another aspect, the invention contemplates novel calix(n)arene compounds of the type described above, and having sulfinate, sulfonate, phosphonate, and carboxylate terminal groups.
In one embodiment, the novel compounds include oxidized and partially oxidized calix(n)arenes having the general structure shown above, where R, is OH, =0, an alkyl or aryl ether, an ester, an acid, or a mixture thereof, and at least one of the R, groups in the calix(n)arene compound is =0; R2 is a polar substituent with a terminal carboxylate, phosphonate, sulfinate or sulfonate group; and R4 is >CH2 or ≥CH, or a mixture thereof. In a preferred embodiment, Rx is OH or =0, and at least one of the R, groups in the calix(n)arene compound is =0. In another embodiment, the novel compounds include calix(n)arenes having the general structure shown above, where n = 4-10; R, is OH, =0, an alkyl or aryl ether, an ester, an acid, or a mixture thereof; R2 is a polar substitutent with a terminal carboxylate or sulfinate group, and R4 is >CH2 or ≥CH, or a mixture thereof.
The compound may be administered orally, for treatment, for example of human immunodeficiency virus (HIV) respiratory syncytial virus (RSV) , or herpes simplex viruses HSV-1 or HSV-2.
The compound may be administered by inhalation, for treatment of respiratory syncytial virus, and topically for treatment of HSV-1 or HSV-2. Other modes of administration, e.g., intravenous, are also contemplated.
Also contemplated is a method which employs a combination of a calix(n)arene compound and an antiviral nucleoside analog compound for treating viral infection. The calix(n)arene and nucleoside analog compounds may be formulated in an ointment vehicle, for topical administration, e.g., in treating lesions due to HSV-1 or HSV-2. Alternatively, the compounds may be formulated in liquid or tablet form for oral administration, for treatment of systemic viral infection, or in solution form for systemic administration.
In another aspect, the invention includes a method of inhibiting infection by a sexually- transmitted enveloped virus. In the method, a prophylactically effective amount of a macrocyclic compound is administered topically to an area of likely sexual contact. The macrocyclic compound is composed of aryl ring subunits which are connected by ring-attached bridge linkages which form a continuous chain of connected atoms making up the backbone of the macrocycle, and which contain negatively charged substituents on non-backbone atoms of the aryl subunits. Preferably, the negatively charged substituents include a sulfonate group, a sulfinate group, a carboxylate group, or a phosphonate group. In one embodiment, the compound includes amide or ester forms of the negatively charged substituent, which can be cleaved to a negatively charged form in vivo.
The ring subunits in the macrocyclic compound preferably include naphthalene subunits having 3- and 6-position sulfinate or sulfonate groups; and/or phenyl subunits having 4-position negatively charged substituents, where the bridge linkages are between the 2 ring-carbon position of one naphthalene or phenyl group, and the 7 ring-carbon group of an adjacent naphthalene group or 5 ring-carbon position of an adjacent phenyl group.
In one general embodiment, the macrocyclic compound includes at least four naphthalene subunits, each having polar groups at 1- and 8-position, sulfinate or sulfonate groups at 3- and 6-positions, and bridge linkages between the 2 ring-carbon position of one subunit and the 7 ring-carbon position of an adjacent subunit. One preferred compound of this type has the general structure:
Figure imgf000013_0001
where n = 4, 6, or 8, R, is OH, =0, an alkyl or aryl ether, an ester, an acid, or a mixture thereof, R2 is a sulfonate group or sulfinate group, and R4 is >CHR", ≥CR", or a mixture thereof, where R" is H or a carboxylate group.
In a more specific embodiment, R, is OH, =0 or a mixture thereof, R2 is a sulfonate group, and R4 is >CH2 or ≥CH, or a mixture thereof.
In another general embodiment, the macrocyclic compound includes at least four phenyl subunits having negatively charged substituents at 4- positions, and bridge linkages between a 2 ring- carbon position of one phenyl subunit and a 5 ring- carbon position of an adjacent phenyl subunit. One preferred compound of this type has the general structure:
Figure imgf000014_0001
where n, R,, R2, and R,, are as above.
In a more specific embodiment, R2 has the form: (CH2)mR2', where m= 0-3, and R2' is a sulfonate group. In another specific embodiment, R2 has the form: (CH2) mR2' , where m= 0-3, and R2' is a sulfinate group. In another specific embodiment, R2 has the form: (CH2)mR2', where m= 0-3, and R2' is a carboxylate group.
In yet another specific embodiment, R2 has the form: (CH2)mR2', where m= 0-3, and R2' is a phosphonate group.
Also contemplated is a method which employs a composition containing a macrocyclic compound such as described above and an antiviral nucleoside analog compound.
Compositions in accordance with the invention are administered topically for inhibiting transmission of viral infection between sexual partners. The compositions may take the form of a lubricating jelly, suppository, for example, and include a vehicle in which the macrocyclic compound is carried. In addition, the compositions may be applied to the surfaces of physical barrier-type devices as a prophylactic measure against infection. In another aspect, the invention includes a lubricating jelly composition for use in inhibiting infection by sexually transmitted enveloped viruses. The jelly composition includes a lubricating jelly vehicle, and dissolved in the vehicle, a macrocyclic compound such as described above.
In another aspect, the invention includes a physical-barrier type device in combination with a macrocylic compound of the type described above, for use in inhibiting infection by sexually transmitted enveloped viruses. In one embodiment, the device is coated with a lubricating jelly composition contain¬ ing a macrocyclic compound such as described above. Sexually transmitted enveloped viruses against which the compounds of the invention are prophyl- actically effective include hepatitis delta virus (HDV) , hepatitis B virus (HBV) , hepatitis C virus (HCV) , papillomavirus, herpes simplex viruses 1 and 2 (HSV-1 and HSV-2), HIV-1, HIV-2, HTLV-I and HTLV-II. These and other objects and features of the invention will become more fully apparent when the following detailed description of the invention is read in conjunction with the accompanying drawings.
Brief Description of the Drawings
Figure 1 shows the general structure of a macrocyclic compound composed of naphthalene subunits, for use in the present invention;
Figures 2A and 2B show non-oxidized (2A) and partially oxidized (2B) forms of the Figure-1 structure, where n=4 and the subunit is chromotropic acid;
Figures 3A and 3B illustrate two general methods of synthesis of a macrocyclic compound like the one shown in Figure 2A; Figures 4A and 4B show an unoxidized (4A) and partially oxidized (4B) macrocycle with mixed phenyl and sulfonated naphthalene subunits;
Figure 5 illustrates reaction methods for converting the sulfonic acid substituents of macrocyclic chromotropic acid to glycyl sulfonamide and sulfonamide groups;
Figure 6 illustrates a reaction method for converting sulfonate groups in a macrocycle containing chromotropic acid subunits to a sulfinate salt or sulfinate methyl or aryl ester;
Figure 7 shows the general structure of a macrocyclic compound composed of phenyl groups, for use in the present invention; Figure 8 shows a non-oxidized form of the
Figure-7 structure, where n=4 and the subunit is para-sulfonic acid phenol;
Figures 9A and 9B illustrate general methods of synthesis of non-oxidized and partially oxidized forms of the Figure 8 compound;
Figure 10 shows a reaction scheme for replacing the ring hydroxyl groups in the Figure-8 compound with acetyl groups;
Figure 11 shows a reaction for converting sulfonic acid substituents to glycyl sulfonamide groups in a phεnyl-subunit macrocyclic compound;
Figure 12 shows a reaction scheme for producing a macrocylic compound like that shown in Figure 8 but with carboxylic acid-containing bridge linkages; Figure 13 shows a reaction scheme for replacing hydroxyl groups in the Figure-8 compound with carboxylic acid groups.
Figure 14 shows a reaction scheme for producing a calix(n)arene from a para-tert-butyl-precursor; Figure 15 shows a reaction scheme for preparing a calix(n)arene having para-carboxyl substituents;
Figure 16 shows a reaction scheme for preparing a calix(n)arene having carboxyl substituents linked to the para position by a methylene linker;
Figure 17 shows a reaction scheme for preparing a calix(n)arene like that of Figure 16 but where the carboxyl substituents are linked to the para position by an ethylene linker; Figure 18 shows a reaction scheme for preparing a calix(n)arene having para-phosphonate substituents;
Figure 19 shows a reaction scheme for preparing a calix(n)arene having a phosphonate substituent linked to the para position (C-4) by a methylene group;
Figure 20 shows a reaction scheme for preparing a p-2-bromoethyl-0-tosyl-calix(n)arene as a precursor for preparing other calix(n)arene derivatives;
Figure 21 shows a reaction scheme employing the bromoethyl-calix(n)arene of Figure 20 to prepare a calix(n)arene having a phosphonate group linked to C- 4 by an ethylene group;
Figure 22 shows a reaction scheme for preparing a calix(n)arene derivative having a sulfonate group linked to C-4 by a methylene group;
Figure 23 shows a reaction scheme for preparing a calix(n)arene derivative having a sulfonate group linked to C-4 by an ethylene group;
Figure 24 shows a reaction scheme for preparing a calix(n)arene having a chlorine atom at the methylene bridge (R4 in Fig. 7) for introduction of other substituents at the methylene bridge; Figure 25 shows a reaction scheme for preparing calix(n)arenes like that of Figure 12, starting from the cyclized precursor from Figure 24;
Figure 26 shows a reaction scheme for preparing a calix(n)arene having a carboxymethyl group attached to the bridge methylene (structure LVIII) , and a general approach for preparing a variety of calix(n)arenes having selected R groups at the methylene bridge (structure LX) , using organocuprate reagents;
Figure 27 shows reaction schemes for preparing a number of calix(n)arenes having 3-sulfonylpropyloxy groups attached to calix(n)arenes at various ring positions; Figure 28 shows a reaction scheme for preparing a macrocyclic compound having alternating phenyl and naphthyl rings;
Figures 29A and 29B are plots of HSV viral yields, as a function of drug dose, for the macrocyclic compounds KY-1 (29A) and KY-42 (29B) ;
Figure 30 shows the inhibition of 3H-labeled HSV-1 binding to cells by the compound KY-1;
Figure 31 is a plot of the inhibition in plaque formation of HSV-1 virus when the virus is exposed to the compound KY-1 before (open squares) , (ii) after (closed squares) , and during (closed circles) incubation with Vero cells;
Figure 32A shows SDS-PAGE autoradiogra s of HSV- 1 proteins in the presence (lane A) and absence (lane B) of mercaptoethanol, and of HSV-2 proteins in the presence (lane C) and absence (lane D) of mercaptoethanol, all with bound radiolabeled KY-1, and stained marker proteins (lane E) ; Figure 32B shows SDS-PAGE autoradiograms of radiolabeled KY-1 compound bound to HSV-1 glycoproteins gD (lanes A and B) , gB (lanes C and D) , and gC (lanes E and F) ; Figures 33 (A-D) show plots of effects of topical Y-l on epithelial damage (33A) , conjunctivitis (33B) , iritis (33C) , and stromal disease (33D) subsequent to ocular application of HSV-1 in rabbits; and Figures 34A and 34B show the drop in HSV-1 (34A) and HSV-2 (34B) viral yields when infected cells are exposed to increasing concentrations of Y-l alone (solid circles) , increasing concentrations of acyclovir alone (open circles) , increasing concentrations of acyclovir plus 25 μg/ml Y-l (solid squares) , and increasing concentrations of acyclovir plus 50 μg/ml Y-l (solid ovals) .
Detailed Description of the Invention I. Definitions
The terms defined in this section have the following meanings unless otherwise indicated.
An "enveloped virus" means a virus containing a proteinaceous viral envelope which surrounds the viral capsid. Such enveloped viruses include orthomyxoviruses and paramyxovirus, herpes viruses, togaviruses and retroviruses. During cell infection by an enveloped virus, the plasma membrane of the host cell is altered to include some viral-coded proteins and, as the viral nucleoprotein core exits the host cell in which it was assembled, it becomes enveloped with the modified membrane, thus forming the viral envelop.
A "sexually transmitted enveloped virus" is an enveloped virus which is known or suspected to be transmitted by sexual contact. Specific examples include hepatitis delta virus (HDV) , hepatitis B virus (HBV) , hepatitis C virus (HCV) , papillomavirus, herpes simplex viruses 1 and 2 (HSV-1 and HSV-2) , HIV-1 (also known as HTLV-III) , HIV-2, HTLV-I and HTLV-II.
An "aryl ring" subunit is a single ring or fused ring structure containing at least one aromatic ring, i.e., a 5- or 6-member ring with 6 pi electrons necessary for aromaticity. Examples include benzene, naphthalene, fused ring structures, such as tetralin, and heterocyclic structures, including fused-ring structures, such as quinoline, isoquinoline, and indole. A "macrocyclic compound composed of aryJL ring subunits" is a cyclic compound formed by linking ring atoms in aryl ring subunits to form a cyclic chain.
A "calix(n)arene" or "calixarene compound" is a macrocyclic compound having a skeletal structure of the form:
Figure imgf000020_0001
where n is preferably 4-10, and in a preferred embodiment, is 4, 6 or 8.
A partially oxidized calix(n)arene refers to a compound having the structure shown above, where at least one of the R, groups is =0. In a fully oxidized calix(n)arene, all of the R, groups are =0. An exemplary structure of a partially oxidized calix(n)arene is shown in Figure 9B. In addition, for each R, group that is =0, it is to be understood that the , group attached to the same aryl ring has the form ≥CR; i.e., the group is double-bonded to the aryl ring, as illustrated in Fig. 9B. The "positions of bridge attachments to the ring" in a calixarene compound refer to ring positions 2 and 6 in each ring of the compound. The "non-bridge positions" in a calixarene compound refer to ring positions l, 3, 4, and 5 in each ring of the compound.
The "ring position meta to the bridge attachments" in a calixarene compound refer to ring position 4 in each ring of the compound.
A "polar substituent" refers to a radical R whose octanol/water partition coefficient is less than 1.
A "polar substituent having a terminal carboxylate, phosphonate, sulfonate, or sulfinate refers to R having the form -C02" or R'-C02 " (carboxylate) , -P03" or R'-P03" (phosphonate) , -S03' or R'-S03" (sulfonate) , -S02" or R'-S02" (su finate) , where R' is a linear chain 1-4 atoms in length which is effective to link the associated carboxylate, phosphonate, or sulfonate group to the phenyl ring of calixarene. One preferred R' linear chain is (CH2) m, where m = 1-3.
A "carboxylate" group includes the carboxylic acid group -C02 *, carboxylate salts, and carboxylic acid esters and amides which are cleavable in vivo. A carboxylic acid ester has the general form -C02-R, where R is an unsubstituted lower alkyl or a substituted alkyl group, and a carboxylic acid amide has the general form CONRR'f where NRR' is a secondary or tertiary amine, i.e., R and R' are each H or lower substituted or unsubstituted lower alkyl groups. A carboxylic acid ester or amide is cleavable in vivo if it is hydrolysed by serum esterases or amidases, respectively, to the corresponding carboxylic group.
A "phosphonate" group includes the phosphonic acid group -P03 '2, including phosphonate salts, and phosphonic acid esters and amides which are cleavable in vivo. A phosphonic acid ester has the general form -P03RR' where R and R' are lower alkyl groups, or substituted lower alkyl groups, and a phosphonic acid amide has the general form PO(OR) (NRR') , PO(NRR')2, where R and R' are each H or a lower alkyl group. A phosphonic acid ester or amide is cleavable in vivo if it is hydrolysed by serum phosphatases or phosphoamidases, respectively, to the corresponding sulfonic acid group.
A "sulfonate" group includes the sulfonic acid group -S03', including sulfonate salts, and sulfonic acid esters and amides which are cleavable in vivo. A sulfonic acid ester has the general form -S03R, where R is an unsubstituted lower alkyl or substituted lower alkyl group, and a sulfonic acid amide has the general form S02NRR', where R and R' are each H or a lower alkyl group. A sulfonic acid ester or amide is cleavable in vivo if it is hydrolysed by serum esterases or sulfoamidases, respectively, to the corresponding sulfonic acid group. A "sulfinate" group includes the sulfinic acid group -S03", including sulfinate salts, and sulfinic acid esters and amides which are cleavable in vivo. A sulfinic acid ester has the general form -S02R, where R is an unsubstituted lower alkyl or substituted lower alkyl group, and a sulfinic acid amide has the general form SONRR', where R and R' are each H or a lower alkyl group. A sulfinic acid ester or amide is cleavable in vivo if it is hydrolysed by serum esterases or sulfoamidases, respectively, to the corresponding sulfinic acid group.
The terms "sulfonate", "sulfinate", "phosphonate", and "carboxylate" include the corresponding acid forms and any pharmaceutically acceptable salts thereof.
A "negatively charged substituent" refers to a substituent which is negatively charged under physiological conditions. Exemplary groups which can impart negative charge include sulfonate groups (-S03' ) , sulfinate groups (-S02") , phosphonate groups (-P03 2" ) , and carboxylate groups (-C02') . Preferably, the negatively charged substituent has the form -X-S03", - X-S02', -X-P03", or -X-C02", where X is a linear chain 1-4 atoms in length which is effective to link the associated carboxylate, phosphonate, sulfinate, or sulfonate group to the phenyl ring of a calixarene. One preferred chain is (CH2)m, where m = 0-3.
A "lower alkyl group" is a linear or branched alkyl group containing 1-5 carbon atoms. A "substituted lower alkyl group" is a lower alkyl group having one or more substitutions at its carbon atoms.
II. Preparing Aryl-Subunit Macrocyclic Compounds This section describes the synthesis of two general types of aryl macrocyclic compounds which are useful in the method of the invention. Methods for synthesizing the first type of compounds (naphthylene subunits) are discussed in section A below, and methods for preparing compounds in accordance with the second type (phenyl subunits) are described in sections B and C From the synthetic routes given in sections A-C, it will be apparent how macrocycles composed of mixed subunits, e.g., both naphthalene and phenyl subunits can be prepared. The synthetic methods are also generally applicable to macrocycles composed of heterocyclic subunits, particularly those having sulfonate or sulfinate substituents.
A. Macrocyclic Compounds with Substituted Naphthalene Subunits
Figure 1 shows the general structural formula of a macrocyclic compound composed of substituted naphthalene subunits, for use in the present^ invention. One exemplary compound of this type is shown in non-oxidized (I) and partially oxidized (II) form in Figures 2A and 2B, respectively. The compound is a tetramer of chromotropic acid (1,8- dihydroxy-3,6-disulfonic acid naphthalene) subunits linked by methylene or methine (>CH2 or ≥CH) bridges (R4) . As seen, the methylene/methine bridges and the "interior" ring atoms (ring positions 1, 2, 7, and 8) form a continuous chain having R, = OH or =0 polar groups attached at the 1 and 8 positions. The non- chain atoms (ring positions 3-6 on each substituent) have R2 = sulfonate or sulfinate substituents on the 3 and 6 ring atoms. The nature of the partially oxidized structure was deduced from H1 and C13 NMR studies, and from mass spectroscopy evidence.
For the discussions below (sections A-C) , and for illustrating synthetic routes, usually only the non-oxidized subunit form of the compound is given. It will be understood that the compound may be partially or fully oxidized, after exposure to air under heat and acidic conditions, i.e., contain one or more R- ketone (=0) groups, and a double bond between the ring and the associated bridge group (RJ , as indicated in Figure 2B. It will also be understood that the same reaction mechanisms will apply generally to the partially oxidized form of the compound, i.e., the structure shown in Figure 2B, or similar structures containing additional R! =0 groups, except that R, modification reactions will typically selectively modify an R, -OH group, and leave the corresponding R, =0 group intact.
As will be seen below, the compound preferably includes the chromotropic acid derivatives in which R, is a polar substituent, such as OH, =0, C02H or an ether, thioether, ester, or thioester-linked alkyl or aryl group, and combinations of these group, e.g., where the OH groups in the partially oxidized structure are substituted by one of the above groups. R2, as noted, is a sulfonate or sulfinate group. R3 is H or an uncharged or negatively charged substituent, subject to the activity constraints discussed below. Preferably, R3 is H.
Also as will be seen below, the R4 bridge linking the chromotropic acid derivative subunits is preferably of the form >CHR or ≥CR (indicating unsaturated bridges in the partially oxidized form) , where R is H or a small carbon-containing group, such as lower alkyl, alkenyl, ketone, or carboxylic acid group, or aryl group. The bridge may also be of the form -CH2NR'CH2-, where R' is similarly H or a small carbon-containing group, such as a lower alkyl group. Alternatively, the bridges in the macrocycle may be ring structures, including aryl ring structures, such as in the dimeric macrocycle shown in Figure 4, or analogous structures formed by bridging through heterocyclic rings, such as pyran or furan rings.
The number of subunits may vary from 4 (e.g.. Fig. 2A structure) to 8, with macrocycles containing 4, 6 and 8 subunits being preferred. In the reaction schemes described below, the macrocycle formed may include mixtures of compounds with different subunit numbers (n) values, e.g., a dominant n=4 structure (4 subunits) with additional structures containing 6 and 8 subunits.
Representative macrocyclic compounds which have been synthesized and tested for anti-viral activity are identified by their R,, R2, R3, and R4 substituents in Table 1 below. The KY and Y number in the left and column in the table refers to the analog designation of the corresponding compound. For example, the compound in which R, is OH, R2 is S02NH2, R3 is H, and R4 is -CH2- is designated KY-3. Although not shown in the table, the compounds may exist in a partially oxidized state in which one of more R, groups are =0, and adjacent bridges contain a double-bond carbon linkage to the ring.
Figure imgf000026_0001
Figure imgf000027_0001
Figures 3A and 3B illustrate two preferred synthetic methods for preparing macrocyclic chromotropic acid compounds. The method illustrated in Figure 3A involves cyclization of a chromotropic acid derivative (including chromotropic acid itself) with an aldehyde (RCHO) to form a macrocyclic com¬ pound, such as the tetramer shown in Figure 2, in which the chromotropic acid subunits are linked by R- substituted methylene groups, i.e., in which R4 is
>CHR (including ≥CR) . i'his synthetic scheme provides a convenient method for constructing macrocyclic com¬ pounds having a variety of different bridge-methylene R groups, by carrying out the cyclization reaction in the presence of an aldehyde of the form RCHO. For example, to construct a macrocyclic compound with a -CH2- bridge, such as the KY-1 compound (IV), chromotropic acid (III) is reacted with formaldehyde. Typical reaction conditions are given in Example 1A for the synthesis of KY-1. Similarly, KY-42 is prepared by cyclization with glyoxylic acid (Example IC) ; KY-48, in the presence of glyceraldehyde; KY-85, in the presence of benzaldehyde; KY-97,, in the presence of acrolein; and KY-110, in the presence of pyruvic aldehyde. It will be appreciated that a variety of other RCHO aldehydes having small alkyl, alkenyl, acid and other hydrocarbon R groups would be suitable. Further, the R bridge group may be further modified after the cyclization reaction. For example, KY-193 may be prepared by brominatien of the KY-97 compound.
In the method illustrated in Figure 3B, cyclization of the chromotropic acid derivatives (III) is carried out by reaction with hexamethylenetetramine, to form a 3-atom chain bridge of the type -CH2N(CH3)CH2- (V). The cyclization reaction for the synthesis of KY-346 is given in Example 1J. The -CH2N(CH3)CH2- bridge may be modified, after the cyclization reaction, to form a variety of N-substituted bridges of the -CH2N(R')CH2-, where R' is one of a variety of small carbon- containing groups, according to known synthetic methods. Some of the bridges in the partially oxidized structure will have the form =CHN(R')CH2-. As noted above, the Figure-4A compound (VT) is representative of macrocyclic naphthalene having a cyclic bridge, in this case a phenyl bridge. The compound is formed by reacting chromotropic acid, in the presence of hydrochloric acid with 1,2- benzenedimethanol in acetic acid, as detailed in Example 3. Similar methods can be employed to linked chromotropic acid subunits by other cyclic bridges, such as furan, pyran, pyrrole, and the like. Figures 4A and 4B show the non-oxidized (VI) and partially oxidized (VIII) forms of one compound) .
For synthesis of macrocyclic compounds with selected R,, R2, and substituents, two general approaches are available. In one approach, the chromotropic acid derivative is modified after cyclization so that the cyclized product will either contain the selected Rt, R2, and R4 substituent, or contain a substituent which can be readily modified to the selected substituent. This approach is illustrated by the synthesis of KY-3, which has an S02NH2 R2 substituent, as detailed in Example IB. Here cyclized chromotropic acid (VIII) is reacted first with chlorosulfonic acid, to form the corresponding R2 = S02C1 derivative (IX, Figure 5) . The macrocyclic compound is then reacted with ammonia water to form the desired R2 = S02NH2 derivative (X, Figure 5) .
A similar strategy was employed for the synthesis of KY-357 (R2 = S02NHCH2C02H) by final subunit reaction with glycine (XI, Figure 5), at basic pH.
Figure 6 (in combination with Fig. 5) illustrates the conversion of the sulfonate groups of cyclized chromotropic acid to sulfinate salts (XII) and sulfinate methyl esters (XIV) . The first stage of the reaction involves formation of the corresponding sulfonyl chloride derivative (IX) , as outlined above. This compound is then treated with sodium sulfite, to form the corresponding sulfinate salt (XII) . Reaction with dimethyl sulfate in the presence of sodium bicarbonate produces the corresponding sulfinate methyl ester (XIV) .
Similarly, macrocyclic compounds with a variety of Rj substituents may be prepared by modification of chromotropic acid after cyclization. In synthesizing KY-151, for example, (R, = OCH3) cyclized chromotropic acid is reacted with dimethylsulfate under basic conditions, as detailed in Example IF, to form the dimethylether of cyclized chromotropic acid.
Similarly, in preparing KY-307 (R, = 0(CH2)SC02H) , cyclized chromotropic acid is first converted to the diether of hexanoic acid by initial reaction of cyclized chromotropic acid with 6-bromohexanoic acid under basic reaction conditions.
As a further example, in preparing compounds such as KY-272 and KY-294, in which R, has the form OCOR, the macrocyclic compound formed by cyclization of chromotropic acid is reacted with an acid chloride of the form RCOC1, under basic conditions, as detailed in Example 1J for the synthesis of KY-285.
In a second general approach, the selected substituent is formed on the subunit naphthalene rings by derivatization of the naphthalene subunit, with subsequent subunit cyclization to form the desired macrocycle. For the synthesis of KY-175 (R2 = SO3CB3) , chromotropic acid is reacted with sulfonylchloride, as above, to produce the corresponding R2 = S02C1 substituents. Further reaction with NaOCH3 leads to the desired R2 substituent. Reaction details are given in Example IH.
It will be appreciated that the synthetic method for forming selected-substituent macrocyclic com- pounds may include both prior derivatization of chromotropic acid and subsequent derivatization of the subunits after cyclization. For example, in forming KY-397 (R, = OCH3, R2 = S02NH2) , chromotropic acid subunits are first reacted at the R, positions, to form the dimethyl ether derivative as described above. After cyclization with formaldehyde, the com¬ pound is further derivatized at the R2 position, also as described above, to convert the S03 group to the desired S02NH2 substituent.
The KY compounds described above can be converted readily to a variety of sulfinate and sulfonate acids and salts. Salt and acid forms can be interchanged by passage over standard cation- exchange resins, to displace one cationic counter ion for another, according to well known methods. Thus, for example, several of the KY compounds shown in Table 1 are ammonium salts formed by cation exchange of protons in the presence of an ammonium salt, such as ammonium chloride. In addition, exposure of the macrocyclic compound to a variety of metal cations, such as the cations of Ca, Ba, Pt, Cu, Bi, Ge, Zn, La, Nd, Ni, Hf, or Pb, may produce both a metal salt and a metal chelate of the macrocyclic compound in which the metal is chelated at interior polar pocket in the compound.
The physical properties of several macrocyclic compounds prepared in accordance with the invention have been studied by absorption and mass spectrometry and by nuclear resonance spectroscopy (NMR) , as detailed in Examples 1A, IB, IC, and 1J. These com¬ pounds include tetrameric macrocyclic compounds, such as indicated in Figure 2, or mixtures with predominantly tetrameric forms. B. Ca1i (n.arene Compounds
Figure 7 shows the general structural formula of a calix(n)arene compound of the type that can be used in the method of the present invention. One exemplary compound of this type is shown in Figure 8, which is a tetramer of phenol parasulfonic acid subunits linked by methylene bridges (XV) . As seen, the methylene bridges and the "interior" ring atoms (ring positions 2, 1, and 6) form a continuous chain having R, = OH groups attached at the 1 ring positions. The non-chain atoms (ring positions 3-5 on each substituent) have R2 = sulfonic acid substituents on the 4 ring atoms.
Figure 9A illustrates a general method for forming calix(n)arene compounds. The precursor shown at the left (XVI) is a tert-butyl calix(n)arene, where n is the number of phenolic subunits (with para-position t-butyl substituents) in the macrocycle, and the bridge connections are methylene groups, t-butyl calixarenes with 4, 6, and 8, subunits are commercially available, and larger and uneven-numbered subunit calix(n)arenes can be prepared by standard purifying methods.
In the sulfonation reaction shown in Figure 9A, a t-butyl calixarene with a selected subunit number is treated with concentrated sulfuric acid, typically for about 4 to 5 hours at 75-85°C to effect substantially complete displacement of the 4-position t-butyl group by a sulfonic acid group. Details of the sulfonation reaction are given in Example 2A. The method has been used to produce the n=4 macrocycle compound shown in Figure 8, and related macrocycles with 6 and 8 phenol subunits. A similar method is used for preparing a sulfonated calixarene with partially oxidized 1- position OH groups, as shown at 9B. Here the t-butyl calixarene starting material is treated with cone. sulfuric acid at a temperature above 100°C, preferably between 150-170°C The reaction is effective to sulfonate the subunit rings and to partially oxidize the interior OH groups. As indicated in Figure 9B, partial oxidation can lead to a conjugated calix(n)arene structure (XVIII) in which the bridge contributes delocalized electrons. This conjugated structure is colored, and the development of a colored product can be used to monitor the course of the oxidation reaction. Details of the reaction are given in Example 2B.
It will be appreciated that the desired macrocycle can also be formed directly by reacting parasulfonic acid phenol (or precursors thereof) under suitable bridging conditions, such as described above for producing naphthalene-subunit macrocycles. This is illustrated by the reaction shown in Figure 12, for production of a macrocyle having carboxylic acid-containing bridge groups. In this method, phenol parasulfonic acid is reacted with glyoxylic acid, under conditions similar to those described in Example IC, to form the cyclized structure shown (XXII) . A synthesis of XXII is described in Example 2F.
The calix(n)arene compounds formed as above can be modified, according to general procedures outlined in Section IIA above, to achieve selected Rj groups, modified sulfonyl groups, and/or addition of R2 groups. The range of R, and R2 substitutents iε substantially the same as that discussed in Section IIA above. Figures 10, 11, and 13 illustrate various reaction methods for modifying the Rj or R4 group of an already formed macrocycle. In Figure 10, the sulfonated structure shown in Figure 8 is treated with acetic anhydride, to form an O-acetyl Rj group. Details of the reaction are given in Example 2C
Example 2G describes a similar reaction scheme for forming a toluene sulfonic acid ester at the R} position. Figure 11 illustrates a general method for forming sulfonamides, such as glycylsulfonamide (XXI) of the Figure 8 compound. Analogous to the reactions described with respect to Figure 5, the sulfonated phenyl calix(n)arene compound (XVII) is treated with chlorosulfonic acid, to form the corresponding sulfonyl chloride analog (XX) . Further reaction with a selected amine, in this case glycine, gives the desired sulfonamide. Reaction details are given in Example 2D for the synthesis of the R2 = S02NH2 compound and in Example 2E, for the synthesis of the glycyl sulfonamide compound.
Figure 13 depicts a general non-exclusive synthetic method for a net substitution of R, = OH by R, = carbon moieties. In Example 2H, the reactions detail a process from which a substrate (R, = OH, R2 = tert-butyl, » CH2, n = 4) affords an intermediate (R, = CN, R2 - tert-butyl, R< = CH2, n = 4) . Further modification then provides the product (R, - C02H, R2 - S03H, R„ = CH2, n «= 4) . It will be appreciated that substituent modifications at the R, site can be selectively carried out at OH sites in a partially oxidized macrocycle, such as the structure shown at Figure 9B. That is, reactions which are specific for ring OH groups will leave the =0 group intact, thus providing a mixed R, group containing =0 groups.
The R3 is generally H, but may be an uncharged or negatively charged substituent, similar to the R3 group described in Section IIA above.
The Rή bridge linking the chromotropic acid derivative subunits is preferably of the form >CHR or > CR, where R is H or a small carbon-containing group, such as lower alkyl, alkenyl, ketone, or carboxylic acid group, or aryl group, as noted above, or of the form -CH2NR'CH2-, where R' is similarly H or a small carbon-containing group, such as a lower alkyl group. Alternatively, the bridges in the macrocycle may be ring structures, including aryl ring structures, analogous to the dimeric macrocycle shown in Figure 4.
Also as above, the number of subunits may vary from four (e.g., Figure 8 structure) to 8, with macrocycles containing 4, 6 and 8 subunits being preferred. In the reaction schemes described below, the macrocycle formed may include mixtures of compounds with different subunit numbers (n) values, e.g., a dominant n=4 structure (4 subunits) plus additional structures containing 5-8 subunits. Representative calix(n)arene compounds which have been synthesized and tested for anti-viral activity are identified by their Rj, R2, and R4 substituents in Table 2 below. The KY and Y number in the lefthand column in the table refers to the analog designation of the corresponding compound, as in Table 1. Compounds which are partially oxidized at the R, position, and which may have both saturated and unsaturated bridge methylene carbon groups are indicated in column 2 of the table.
Figure imgf000036_0001
The compounds shown in Table 2, and R-group " combinations thereof, described above can be converted readily to a variety of sulfonic acid or sulfonate salts, by reaction in acid or in the presence of a suitable salt, according to well known methods, as described above.
C Macrocyclic Compounds with Sulfonate.
Sulfinate. Phosphonate. and Carboxylate Groups One general class of compounds which are useful in inhibiting infection by enveloped viruses, in accordance with the present invention, are calix(n)arene compounds in which the ring position meta to the bridge attachments, i.e., 4-position carrying substituent Rj in Fig 7, is substituted with a negatively charged substitutent having a terminal sulfonate, sulfinate, phosphonate, or carboxylate group.
Methods for preparing calix(n)arene compounds in which a sulfonate group is carried at the ring 4 position are given in Examples 2A, 2B, and 2C, including compounds with different substitutions at the ring 1 position. Compounds having sulfonamide group, including a group which terminates with an end terminal carboxyl group are given in Examples 2D and 2E, respectively.
Fig. 14 illustrates a method for converting a t- butyl calix(n)arene (XXV) to the unsubstituted compound (XXVI) , which can be used as a starting material for some of the syntheses described below. Details of the reaction are given in Example 2J.
Fig. 15 shows the conversion of a calix(n)arene (XXVII) carrying a p-acetyl group to the corresponding calix(n)arene with a p-carboxyl group (XXVIII) . Details are given in Example 2K.
To form the p-carboxyethyl compound shown at XXXI in Fig. 16, compound XXVI produced above is converted to the corresponding (dimethylamino)methyl compound (XXIX) by reaction with dimethylamine and formaldehyde. This compound is then taken to the corresponding cyanomethyl compound (XXX) , which when heated in acid, is converted to the desired carboxymethyl compound (XXXI) . Details are given in Example 2L.
Fig. 17 illustrates the synthesis of a carboxyethyl calix(n)arene (XXXIII). Here the intermediate (XXIX) from above (Fig. 16) is treated sequentially with Mel and the sodium salt of diethylmalonate to give the diethylmalonylmethyl compound (XXXII) . Heating in acid gives the desired compound XXXIII. Details are given in Example 2M. Fig. 18 illustrates the synthesis of a p- phosphonate calix(n)arene (XXXVI). In this synthesis, compound XXVI from above is iodinated and then reacted with diethylphosphite to give the diethylphosphonate compound (XXXV) . Refluxing in acid gives the desired compound XXXVI. Details are given in Example 2N. The synthesis of a phosphonomethyl calix(n)arene (XXXIX) is shown in Fig. 19. As seen, compound XXVI from above is chloromethylated (compound XXXVII) , and further reaction with triethylphosphite gives a diethylphosphonyl esrer compound (XXXVIII) . Heating in acid gives the desired phosphonomethyl compound. Details are given in Example 20. Note that the chloromethyl intermediate is also useful in synthesis of the sulfonylmethyl calix(n)arene analog. The synthesis of a p-2-bromoethyl compound useful in the synthesis of a phosphonoethyl or sulfonylethyl calix(n)arene is outlined in Fig. 20, with details given in Example 2P. With reference to the figure, compound XXVI from above is allylated at the phenol hydroxyl (compound XL) , and heated to give the rearrangement product XLI. Tosylation serves to protect the phenyl hydroxyl position (compound XLII) , allowing conversion to the p-hydroxyethyl derivative (XLIII) . Further reaction with triphenylphosphine dibromide gives the desired p-bromoethyl XLIV compound.
The p-bromoethyl calix(n)arene (XLIV) is used in the synthesis of the p-phosphonoethyl compound (XLVI) , by a reaction sequence which is analogous to that shown in Fig. 19, described above. Details of the reaction scheme shown in Fig. 21 are given in Example 2Q.
The intermediate p-chloromethyl calix(n)arene (compound XXXVII) used above can also be used in the synthesis of a p-sulfonylmethyl calix(n)arene
(XLVII) , as shown in Fig. 22, with details provided in Example 2R.
Similarly, the p-2-bromoethyl intermediate (XLIV) described above can be used in the synthesis of the p-sulfonylethyl calix(n)arene (XLIX) as shown in Fig. 23, with details given in Example 2S.
It will be appreciated that when desired, sulfonate groups can be converted to sulfinate groups by the methodology illustrated in Fig. 6 and exemplified in Example ID.
The foregoing synthetic methods can be used to produce calix(n)arene compound having at the position meta to the bridge positions (the C4 ring positions) , negatively charged substituents which terminate with sulfonate, sulfinate, phosphonate, and carboxylate groups. The syntheses outlined show both direct acid-group attachment to the rings, or attachment through alkyl linkages, such as methyl and ethyl linkages. It will be appreciated from the discussion above how acid groups linked to the rings through longer alkyl groups can be prepared. Also, as detailed above, the acid groups can be converted to the corresponding salts. It will also be appreciated how a variety of esters and amides of the terminal acid groups in the macrocyclic compounds of the invention (e.g., calixarenes) can be prepared. Such derivative can be used as "pro-drugs", where an ester or amide is converted in vivo to the corresponding negatively charged acid group by enzyme-catalyzed hydrolysis (e.g., by an esterase) .
Generally, the acid esters of carboxylic acid and sulfonic acid can be prepared by standard esterification reactions in which the acid is converted to, for example, an acid chloride, then reacted with an alcohol, such as an alkyl alcohol. The amides of carboxylic and sulfonic acid can similarly be formed by reaction of the acid chloride with an amine, such as an alkyl amine. Preferred esters include aryl and lower alkyl carbonate esters, such as n-butyl alkyl group. Preferred amides include amides of lower alkyl groups.
The conversion of a phosphonic acid calix(n)arene to a corresponding ester or amide likewise follows conventional phosphonate esterification or amidation reaction methods. One method for generating a diethylphosphonyl ester has been described above with reference to Fig. 21. In addition to the polar substituents at the C4 position in the calix(n)arene rings, the present invention contemplates, for use in the method of the invention, calix(n)arene compounds which are substituted at other ring positions and at the bridge positions in the macrocycle. For example, the C3 and/or C5 ring positions may be substituted with halogens such as F of Cl. Also as described above with respect to several of the naphthyl-ring macrocycles, substitutions at the "inner" ring positions (the Cl ring positions in calix(n)arene) are compatible with anti-viral activity. Also as described above, substitutions at the bridge positions in naphthyl-ring macrocycles are compatible with activity. Figs. 24 and 25 illustrate one method of attaching carboxyl groups to the bridge methylene in calix(n)arene. In this method, the hydroxyl group of p-t-butyl calix(n)arene (XXV) is acetylated (L) , and the product is oxidized at the bridge methylene, to give the bridge ketone (LI) . Reduction with sodium borohydride, and subsequent reaction with thionyl chloride yield the compound LIII which is chlorinated at the methylene bridge. Details are given in Examples 2T and 2U. With reference to Fig. 25, the compound LIII is cyanylated, then treated with acid to form the carboxylic acid group at the bridge methylene. The resulting compound LV can be de-t-butylated by treatment with aluminum chloride to give the bridge- carboxylated calix(n)arene shown at LVI. Alternatively, compound LV may be sulfonated, at the C4 ring position, by treatment with sulfuric acid, as above. It will be appreciated that similar methods, but involving initial protection of the bridge carboxyl and/or ring hydroxyl groups, can be used to form corresponding p-phosphonic acid or p-carboxylic acid calix(n)arenes.
Using compound LIII from above, a variety of bridge substitutions can be produced, by the method outlined in Fig. 26, using a suitable cuprate reagent as illustrated at the left in the figure. The reaction at the right in the figure shows how a calix(n)arene LIII can be converted to a compound having a carboxymethyl group attached to the bridge methylene group. Details of this reaction sequence are given in Examples 2W and 2X. The final reaction product (LVIII) can be p-sulfonated or derivatized with other acid groups at the para position as above. Fig. 27 shows a variety of derivatization reactions involving calix(n)arenes and propane-1,3- sulfone. The reactions are effective to add alkylsulfonate groups at ring hydroxyl positions, as shown. This method provides an alternative approach for producing calix(n)arenes with ring-attached sulfonic acid groups. Exemplary reaction conditions are given in Examples 2Y and 2Z.
Finally, Fig. 28 shows the preparation of a mixed macrocyle containing alternating phenyl and naphthyl groups. The reaction method is described in Example 3B.
III. Inhibition of Virus Infectivitv This section examines the ability of compositions containing a macrocyclic compound to inhibit infection by a variety of enveloped viruses. The enveloped viruses which were examined are the herpes viruses, herpes simplex virus-1 (HSV-1) and herpes simplex virus-2 (HSV-2) , which are double- stranded DNA viruses (Roizman) ; human immuno¬ deficiency virus (HIV) , an RNA retrovirus (Popovic; Barre-Simoussi) ; and influenza A and B and respiratory syncytial viruses (RSV) , all RNA viruses (Chanock) .
For comparative purposes, selected non-enveloped viruses, including adenovirus, a double-stranded DNA virus (Rowe; Hilleman) , and rhinovirus, a single- strand RNA virus (Dick) , were examined. Typically, inhibition of virus infectivity was measured by the extent of inhibition of cytopathic effects detectable in infected cultured cells. Inhibition of HSV-1 and HSV-2 infectivity in cultured cells was also shown by inhibition of virus binding to infectable cells, and inhibition of viral plaque formation in infected cells, as described below.
In addition, a large number of representative aryl macrocyclic compounds (including those shown in Tables 1 and 2) were examined for toxicity in cell culture, using a panel of human cell lines, as detailed in Example 4. Briefly, the selected KY- or Y- compound was added to cell cultures at a final concentration of 5, 10, 25, 50, or 100 μg/ml. Three days later the cells were washed to remove drug, and stained with a vital stain, to determine the percentage of dead cells in each culture. The IC50 drug concentration, i.e., value concentration of drug which produced 50% cell death, was 50μg/ml for KY- 143, KY-151, and KY-163, and lOOμg/ml or greater for all of the other KY compounds tested. For KY-1, which has a molecular weight of 1404 daltons, a drug concentration of 100 μg/ml is equivalent to about 66 μM.
A. Inhibition of HSV Infectivity: Naphthalene-
Subunit Compounds
Several compositions containing one of the compounds in Tables 1 and 2 were tested for inhibi¬ tion of cytopathic effects (CPE's) in cultured, HSV- infected cells. In the method reported in Example 5, Vero cells were infected with HSV-1 or HSV-2 and allowed to grow in culture until cytopathic effects were clearly visible. In the absence of infection, the cells form an even monolayer of fibroblast-like cells. With HSV infection, a cytopathic effect characterized by round cells in suspension is clearly evident after 24 hours, followed by clumping and lysis of infected cells after 24-72 hours.
In the drug inhibition study reported in Example 5, cells were exposed to HSV-1 or HSV-2 virus and, at the same time, to a selected aryl macrocyclic compound, at a final drug concentration of 10 μg/ml. Twenty-four hours later the cells were examined for cytopathic effect. If a clear cytopathic effect was not observed with lOμg/ml of the drug, the study was repeated at a drug concentration of 20 μg/ml for some compounds.
Table 3 below lists 50 naphthalene-subunit macrocycles which were tested in this assay. A "+" symbol in the second column indicates that the compound was effective in inhibiting cytopathic effects at 10 or 20 μg/ml. A "-" symbol indicates that CPE was observed at 10 or 20 μg/ml.
Figure imgf000044_0001
Figure imgf000045_0001
1 N, no inhibition of CPE observed at highest concentration tested, or insufficient inhibition observed to predict IC^,.
The compounds used in Table 3 were further tested for activity against HSV infection in a plaque reduction assay, as detailed in Example 6. Here Vero cells, after overnight incubation, were exposed to serial dilutions of KY compound, from 0.625-10 μg/ml, and HSV-l or HSV-2 virus for two hours. After washing to remove drug and extracellular virus, the cells were further incubated for 2 days, then stained and counted for plaque formation. Percent inhibition was determined by dividing plaques produced by total number of plaques in infected, untreated controls. From the concentration effect curve of plaque inhibition (expressed as percent of control) , the concentration of compound required to produce 50% plaque reduction, ICJQ, was determined. The IC^ values for infection by HSV-1 and HSV-2 infection is given in the right-hand columns in Table 3.
With reference to the compound structures given in Table 1, the following R-group features can be identified as contributing to low activity (no protection of cells from CPE effect seen at 10-20 μg/ml: in KY-48, KY-49 and KY-110, a bulky side chain in the methylene bridge; in KY-143, an OH R3 group; in KY-147 and KY-148, a sulfonamide with a non-polar alkyl group at the R2 position; in KY-158 and KY-175, a sulfinate ester or sulfonate ester with a non-polar alkyl group at the R2 position; and in KY-395, a trimethylamine bridge in combination with a methyl ether substituent at the Rj position. The "GC" symbol for KY-193 means that some giant cells were formed, indicating partial inhibitory activity. Looking now at the compounds which give complete inhibition of CPE at 10-20 μg/ml, the following R- group structures can be identified as preferred radicals: The R, position contains OH, including combinations of OH and =0 groups; alkyl and aryl esters, including combination of such esters and =0; and alkyl ethers, including combinations of such ethers and =0. The optimal radicals at the R2 position are sulfonic acid or sulfonic acid salts, sulfinic acid and salts thereof, and sulfonamides with polar amine groups, such as NH2, NHOH, N-glycosides (KY-352) , and amino acids. The preferred radicals at the R3 position are H or Br.
The optimal bridge linkage groups are substituted and unsubstituted methylenes, where the R group is not a bulky alkyl group, and is preferably a carboxylic acid group.
As a further guide to R-group selection, compounds having an EDX value of < lμg/ml for at least one of the two HSV tests have one of the following R-group characteristics: Compounds whose R, groups are lower-alky1 ethers or esters, or contain a terminal carboxylic acid group are typically most active, especially in combination with =0 groups at other R, sites in the compound.
The R2 groups are sulfonic acid or sulfonic acid salts or sulfonamides with a terminal carboxylic acid. This feature indicates that an R2 position acid group favors high activity. The R, bridge is methylene or a methylene carrying a carboxylic acid group.
The ability of selected naphthalene-subunit compounds to inhibit HSV-1 and HSV-2 viral yields at selected drug concentrations up to 10 μg/ml was assessed in the viral inhibition assay described in
Example 7. Briefly, cultured Hela cells were exposed to serially diluted KY compound and virus, allowed to grow for 24 hours, then freeze/thawed 3 times to release virus particles. Vero cells were infected serial dilutions of the viral lysates were assayed for plaque counts as described in Example 6. The drop in viral yield, as a function of drug concentra¬ tion, is plotted in Figures 29A and 29B for compounds KY-1 and KY-42 respectively. The dose dependent drop in viral yield was between about 3-5 orders of magnitude, depending on drug and virus. The degree of inhibition of viral yield was generally greater for HSV-1 than for HSV-2. Similar results were observed with several other KY compounds.
B. Inhibition of HSV Activity: Calixfn.arene Compounds
A similar study of anti-HSV activities was carried out with several of the calix(n)arene compounds listed in Table 2 above, with the results shown in Table 4. As seen from column 2 of the table, all of the compounds which were tested gave inhibition of CPE at 10-20 μg/ml. Inhibition activity of the tested compounds against HSV-I and HSV-2 in the plaque reduction assay is shown in the two righthand columns in the table (given as IC50 in μg/ml) .
Figure imgf000048_0001
The highest activities observed for the phenyl- subunit compounds are comparable to the highest activities seen with the naphthyl compounds, e.g., from about 1-3 μg/ml ICJ0 values.
The most active compounds, Y-226 (n=8) , Y-48 (n=6) , and Y-225 (n=4) all have partially oxidized Rj OH/=0 groups, and each partially oxidized compound is substantially more active than its corresponding non- oxidized analog.
The partially oxidized n=3 compound, KY-268, is somewhat less active than its n=4, 6, and 8 analogs. Among the non-oxidized compounds, the n=8 compound, Y-l, is somewhat more active than the corresponding n=4 and n=6 compounds. Addition of acetyl groups at the R, position produces little change in the activities of partially oxidized compounds, also consistent with the results observed with the naphthalene-subunit compounds, addition of alkyl esters at the R, positions gave activities comparable to the partially oxidized analog.
As a general guide to optimizing compound activity in the phenyl-subunit compounds, the same rules discussed above generally apply. Thus, for example, highest activity is expected when the R2 group terminates in a negatively charged group, such as a sulfonic acid. More generally, the present invention contemplates, for use in treatment of infection by enveloped virus, a calix(n)arene compound which whose R2 group is a polar substituent which terminates in a sulfonic acid, phosphonic acid, or carboxylic acid group, including esters and amides of these acids which can be converted to the corresponding acid by hydrolytic cleavage in vivo. For use in inhibiting infection by a sexually transmitted enveloped virus, the present invention contemplates a macrocyclic compound whose R2 groups are negatively charged substituent which terminates in a sulfonate, sulfinate, phosphonate, or carboxylate group.
The compounds may also include esters and amides of the negatively charged groups which can be converted to acid groups by hydrolytic cleavage in vivo. A variety of esters and amides of sulfonic acid, phosphonic acid, and carboxylic acid have been shown to undergo hydrolytic cleavage in vivo to the corresponding acids (Svensson, 1988, 1991; Stella; Bundgaard) including esters and amides of lower alkyl groups. Methods for preparing a variety of exemplary compounds of this type are described in Section II above.
The calix(n)arene compounds having terminal acid groups (or groups cleavable to terminal acid groups) may be substituted at other ring and bridge positions, as indicated in Section IIB and IIC above. In one preferred embodiment, the Rt substituent is an OH group, or, where the compound is partially oxidized, a combination of OH and =0 groups.
C. Comparison of Anti-HSV Compounds The inhibitory effect of KY-1 against drug- resistant strains of HSV-1 and HSV-2 was compared with several anti-viral agents which have been used in treating HSV infection. The compounds tested were the nucleoside analogs acyclovir (ACV) , ganciclovir (DHPG) , phosphonofor ate (PFA) , and phosphomethoxyethyladenine (PMEA) . Inhibition of viral yield was determined, as above, by infecting Hela cells in the presence of wild type or drug- resistant strains of HSV-1 or HSV-2, and serial dilutions of a selected anti-viral compound, and infecting Vero cells with serial dilutions of the Hela cell lysate, as above. Details of the inhibition study are given in Example 8.
The IDso concentration (which effects 90% inhibition of viral yield) is given in Table 5. The KOS (HSV-1) and 333 (HSV-2) are wild type viruses; the KOS(PMEA)' and KOS(PFA)' are drug-resistant HSV-1 strains having a DNA polymerase mutation. The 333(DHPG) strain is a drug-resistant HSV-2 strain having a thymidine kinase mutation. With the exception of DHPG as an inhibitor of drug-resistant strains of HSV-l, and PMEA as an inhibitor of drug- resistant strains of HSV-2, all of the nucleoside analogs were at least about 20 times less active against drug-resistant strains than wildtype strains of either HSV-1 or HSV-2, as measured by drug concentration required to inhibit yield. By contrast, the aryl macrocyclic compound showed substantially the same specific activity against drug-resistant strains of HSV-1 and HSV-2 as against wildtype strains.
Figure imgf000051_0001
The data demonstrate that aryl macrocyclic compounds are effective against drug-resistant HSV strains at drug concentrations comparable to those which are effective against wild type virus strains. By contrast, and with the exception of DHPG as an inhibitor of HSV-1 strains, both drug-resistant strains showed a significant resistance to ACV, DHPG, PFA, and PMEA, as evidenced by the severalfold greater ID^ drug concentrations required for virus inhibition.
D. Inhibition of RSV and Influenza A Virus Infectivity
Representative macrocyclic compounds from Table
1 were tested for inhibition of cytopathic effects in cultured MDCK or HEp2 cells after infection by Influenza A virus (A/Taiwan strain) or RSV virus. In the method of inhibiting virus infectivity by influenza A, MDCK cells were infected with the virus, and the cells were allowed to grow in culture until cytopathic effects were clearly visible. In the absence of infection, the cells form an even monolayer of fibroblast-like cells. With virus infection, a cytopathic effect characterized by cell clumping is observed. For each compound tested, drug concentrations of 0.1, 1, 10, 25, and 100 μg/ml were added to cultured cells at the time of virus infec¬ tion, as detailed in Examples 9 and 11. Twenty-four hours later the cells were examined for percent clum- ping, based on the percent of clumped cells of total cell particles in a given view field. The inhibition of clumping was plotted as a function of drug con¬ centration, to determine the dose effective to produce a 50% reduction in the percent clumped cells, measured with respect to control (no drug treatment) . The measured ED∞ values are given in Table 6 below. A similar method was employed to determine the ED50 of RSV inhibition of cytopathic effect (cell clumping) in HEp2 cells, with the results shown in Table 6. Details are given in Example 9.
Figure imgf000052_0001
Figure imgf000053_0001
In general, RSV was significantly more sensitive to compound inhibition than the Influenza A/Taiwan virus. Highest-IAV activity was seen with a sulfonamide with polar amine (S02NH2) at the R2 position, and with selected methylene bridge groups. Relatively high anti-RSV activity was seen with all compounds except KY-47.
E. Inhibition of HIV Infectivity: Naphthalene- Subunit Compounds
Representative macrocyclic compounds from Table 1 were tested for inhibition of cytopathic effects in cells infected with one of two HTLV-III strains, HTLV-IIIB and RF-II strains, as described in Example 12. Briefly, cells chronically infected with HTLV- IIIB or RF-II HIV strains were incubated in the presence of serial dilutions of the selected KY com¬ pound, then further cocultured with indicator cells. The extent of syncytia formation was scored under phase microscopy. The concentration (μg/mL) effective to produce complete inhibition of syncytia formation, ED100, is shown in Table 7 for the two HIV strains. The "N" means that the compound was not tested for that virus. 10
15
20
25
30
35
40
45
Figure imgf000054_0001
Figure imgf000055_0001
As seen from these results there is a general correlation between ώnti-viral activity against the two strains; that is, compounds which are most active against the HTLV-IIIB strains are also most active against the RF-11 strain.
With reference to the compound structures given in Table 1, the following R-group features can be identified as contributing to sub-optimal activity (ED5o values > 63 μg/ml for both strains): in KY-48, a bulky side chain in the methylene bridge; in KY-110, a methyl ketone group in the bridge; in KY-143, an OH R3 group; in KY-147 and KY-148, a sulfonamide with a non-polar alkyl group at the R2 position; in KY-158 and KY-175, a sulfonate ester or sulfinate ester having a non-polar alkyl group, at the R2 position; and in KY-272, a methyl ester at the R, position combined with a acetyl-group bridge. These features are substantially the same as those which gave reduced activity against HSV viral infectivity, i.e., showed no inhibitory effect on CPE at 10-20 μg/ml.
Similarly, those factors which promote high activity against HSV activity are in general the same as those which give highest activity against HIV infectivity. These factors include: the groups at the R, position are OH, including combinations of OH and =0 groups (partial oxidation) ; alkyl and aryl esters, including combination of such esters and =0; and alkyl ethers, including combinations of such ethers and =0.
The preferred radicals at the R2 position are sulfonic acid or sulfonic acid salts, sulfinic acid and salts thereof, and sulfonamides with polar amine groups, such as NH2, NHOH, N-glycosides (KY-352) , and amino acids, with sulfonic acid. In particular, high activity was seen with sulfonic acid, sulfonate salts, and sulfonamides having a terminal carboxylic acid group.
The optimal radicals at the R3 position is H, with both OH and Br giving reduced activity.
The bridge groups are preferably substituted and unsubstituted methylenes, where for a >CHR or >CHR bridge, the R group is not a bulky alkyl group. As a further guide to R-group selection, compounds having an ED^ value of < lμg/ml for at least one of the two HSV tests have one of the following R-group characteristics: Compounds whose Rj groups are lower-alky1 ethers or esters, or contain a terminal carboxylic acid group are typically most active, especially in combination with =0 groups at other R, sites in the compound. The R2 groups are sulfonic acid or sulfonic acid salts or sulfonamides with a terminal carboxylic acid. This feature indicates that an R2 position acid group favors high activity. The j bridge is methylene or a methylene carrying a carboxylic acid group.
These preferred R-groups are intended to provide guidance in the selection of R groups at the R1-R4 positions, for optimizing compound efficiency.
F. Inhibition of HIV Infectivity: Calixfn.arene Compounds
Representative calix(n)arene compounds from Table 2 were tested for inhibition of cytopathic effects in cells infected with one of two HTLV-III strains, HTLV-IIIB and RF-II, as described in Example
12, and in the subsection above. The IC50 values measured for the HXB and RS-11 strains of HIV are given in units of μg/ml in Table 8 below.
Figure imgf000057_0001
Interestingly, the Y-l compound and KY-226 (the corresponding partially oxidized analog) have comparable activities against the HXB strain, in contrast to the significantly higher activity of KY- 226 seen against HSV viruses. All of the other compounds tested have partially oxidized R, =0 groups, and all compounds give comparable activity. IV. Specificity Toward Enveloped Viruses
This section examines the specificity of the viral-inhibition method to enveloped viruses. The studies reported in subsection A show that the macrocyclic compounds used in the method act, at least in part, by binding selectively to viral envelope proteins, and that this binding blocks virus attachment to infectable cells, thereby inhibiting virus infectivity. These studies are detailed in U.S. patent application Serial No. 647,720, filed January 29, 1991, now U.S. Patent No. 5,196,452. Subsection B examines the inhibitory effect of the macrocyclic on non-enveloped viruses.
A. Mechanism of Viral-Infection Inhibition
In one study, the ability of a macrocyclic com¬ pound to block HSV binding to infectable cells was examined as described in Example 14. Briefly, Vero cells were exposed to radiolabeled HSV-1 or HSV-2 in the absence of KY compound or in the presence of 10 μg/ml KY-1, and binding of the virus at times up to 4 hours after exposure to the virus was measured. Figure 30 shows a plot of virus (radiolabel) binding to cells over the four-hour incubation period. In the absence of drug, the amount of bound virus increased steadily over two hours, and slightly from 2-4 hours. By contrast, virus binding to cells peaked at about 1/2 hour in the presence of drug, presumably reflecting the time during which the binding events effective to block virus binding to the cells are equilibrating.
In a second study, the effect of compound when administered prior to, during, or after cell infection by HSV-1 was examined as described in Example 15. In these studies, cells were exposed to one of a series of increasing KY-2 concentrations, and the extent of infection was measured by number of plaques observed 24 hours after infection. The reduction in plaque formation, expressed as a percent of control, is shown in Figure 31 for cells treated with drug prior to (solid rectangles) , during (closed circles) , and after (open rectangles) . Virus inhibition was seen most significantly when the cells were treated with drug during exposure to virus, indicating that virus inhibition occurs at the period of virus binding to and entry into infectable cells.
In a third study, purified HSV-1 virus suspensions were incubated with KY-1 or the sodium salt thereof, or a control solution for 1 hour, then serially diluted to drug concentrations between 101 to 10"* μg/ml as described in Example 16. Addition of the serially diluted virus suspensions gave the plaque counts, measured in duplicate, shown in Table 9. The "X" symbol in the table indicates plaques too numerous to count. The results of the study demonstrate that inhibition of HSV infection by KY compounds is due, at least in part, to binding of drug to HSV particles. Further, complete virus inhibition was seen at drug final drug concentration of 10"2 to 10^ μg/ml (which are much lower than those needed to inhibit HSV in Vero cell culture) . It can be concluded that the drug-binding/inactivation of the virus is effectively irreversible, i.e., not reversed by high dilution effects.
Figure imgf000060_0001
In a fourth study, the binding of radiolabeled KY-l compound to HSV-1 and HSV-2 viral proteins was examined (Example 17) . After compound binding, virus proteins were fractionated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) , and the gel patterns developed by autoradiography. In Figure 32A, lanes A and B in the figure are auto- radiographs of HSV-1 proteins in the presence (lane B) and absence (lane B) of mercaptoethanol, and lanes C and D, analogous patterns for HSV-2 proteins. The lane at the right contains the molecular weight mar¬ kers, as indicated. The major bands of drug binding in HSV-1 have molecular weights, as determined from SDS-PAGE, of 45, 66, and about 130 kilodaltons. The major bands of drug binding in HSV-2 have similar molecular weights. The major bands which show KY binding in Figure 32B correspond in molecular weight, to HSV glycoproteins gD, gB, and gC
B. Effect on Non-Enveloped Viruses The ability of KY compounds to inhibit cell infection by a rhinovirus and adenoviruses 5 and 7 which are non-enveloped viruses, was similarly studied. Vero cells (10s) were infected with a rhinovirus in the presence of KY-1, at concentrations ranging between 1-100 μg. Twenty-four hours after virus infection, the cells were examined for cyto¬ pathic effect, evidencing viral infection. No reduc- tion in cell clumping was observed at any of the KY drug concentrations tested.
Vero cells were infected with adenovirus in the presence of KY-1, also at concentrations ranging between 1-100 μg, and twenty-four hours after virus infection, the cells were examined for cytopathic effect. No reduction in cell clumping was observed at any of the KY-1 drug concentrations.
In summary, a broad range of macrocyclic com¬ pounds are effective inhibitors of cell infection by each of the several enveloped viruses which were studied. Binding studies carried out in particular with respect to HSV viruses indicate that the anti¬ viral activity of the compounds is dependent on binding to virus envelope components, which in turn inhibits virus attachment to infectable cells. The apparent inability of the compounds to inhibit infection of non-enveloped viruses is consistent with this mechanism.
V. Viral Inhibition by Combination of Macrocyclic Compound and an Antiviral Nucleoside Analog
The invention also includes a composition containing a macrocylcic compound of the type described above in combination with a nucleoside analog anti-viral compound. The nucleoside analog compound is one effective to inhibit viral replication at the level of viral replication or transcription. Among the nucleoside analog compounds which are useful in combination with a macrocyclic compound, in accordance with the invention are: (1) Pyrophosphate analoges, such as phosphoformic acid (PFA) , phosphonoacetic acid (PAA) , methanediphosphonic acid (MDP) , carbonyldiphosphonic acid (COMDP) , phosphonoglyoxalic acid (COPAA) , and various halogen- and/or methyl-substituted derivatives thereof, which are inhibitors of viral nucleic acid polymerases. In particular, these compounds are known to inhibit herpes virus (Blackburn, Sidwell) and Influenza (Sidwell) infections, and reverse transcriptase activity in retroviruses, such as human HIV.
(2) Base-modified analogs, such as IUDR, trifluorothymidine, AraA, and azidothymidine (AZT) , didieoxyinosine (DDI) , D4T, dideoxycytidine (DDC) , and ribavirin. Trifluorothymidine, IUDR, and AraA are active mainly against herpes virues (Nicolson, 1984a, 1984b) . Ribavirin is active against several RNA and DNA viruses (Sidwell) , and AZT is active against HIV (Fischl) , as are other dideoxynucleoside analogs, such as DDI.
(3) Sugar-modified analogs, such as N-acyl derivatives of 5'-amino-2' ,5'-dideoxy 5'-ioduridine, sulphonamide derivatives of 5'-amino-5'- deoxythymidine, 2'-deoxy-5-ethyluridine, and N-acyl derivatives, 5'-Sulfate and 5'-sulfamate nucleoside analogs, such as nucleocidin, adenosine 5' sulfamate, and ribavarin, which may act primarily at the level of protein synthesis inhibition (Martin) .
(4) Phosphate analogs, including acyclonucleoside phosphonates, such as acyclovir and gangiclovir, and their isosteric phosphonate analogs. These compounds can act as virus-selective substrates for viral thymidine kinases, in the synthesis of nucleoside triphosphate analogs intracellularly (Galbraith) . Subsequently, the nucleoside triphosphate analogs can act as selective substrates for viral DNA polymerase, acting as a chain terminator since the analog does not have the bifunctionality necessary for chain extension (Allen) . These compounds have demonstrated anti¬ viral activity against herpes viruses (Collins) , including HSV-l, HSV-2, varicella zoster (VZV) , and cytomegalovirus (CMV) (Smith) .
Also included in this class are phosphonomethyl ethers of nucleosides, and their acyclic analogs, such as N-(3-hydroxy-2-phosphonylmethoxypropyl)- (HPMP-) and N-(2-phosphonylmethoxyethyl-) (PME-) derivatives of heterocyclic bases. These compounds act specifically against herpes viruses, adenoviruses, cytomegalovirus (DeClercq) , poxviruses, vaccinia viruses, and retroviruses.
The ability of the two-compound composition to inhibit viral infection by an enveloped virus is demonstrated in the study reported in Example 18, which examines the viral yields after infection of Vero cells with serial dilutions of HSV-1 or HSV-2 particles, as described above.
Figure 34A shows the drop in HSV-1 viral yields when infected cells are exposed to increasing concentrations of the macrocyclic compound Y-l alone (solid circles) , increasing concentrations of acyclovir alone (open circles) , increasing concentrations of acyclovir plus 25 μg/ml Y-l (solid rectangles) , and increasing concentrations of acyclovir plus 50 μg/ml Y-l (solid ovals) . With either drug alone, a maximum decrease in viral yield was slightly less than three logs (orders of magnitude) .
The effect of combined compounds was tested at two Y-l concentrations. At the lower Y-l concentration of 25 μg/ml, the two compounds together gave over seven logs inhibition in viral yield, i.e., more than tenfold greater than the sum of the inhibition produced by the two drugs alone. At the higher Y-l concentration, the combined inhibitory effect of the two compounds was several orders of magnitude greater than than the sum of effect of macrocyclic compound and acyclovir alone. Similar results were observed for inhibition of HSV-2 viral yields with combined compound administration, as seen in Figure 34B.
The two compounds are formulated, preferably in a jelly form, at a preferred weight ratio of between about 10:1 to 1:1 macrocyclic compound to nucleoside analog, respectively. The viral-yield plots in
Figures 34A and 34B show that a significantly higher level of inhibition was observed when the co- administered compounds were at a ratio of about 5:1 macrocyclic compound to nucleoside. The macrocyclic compound in the composition is preferably selected for optimal activity against the target virus, e.g., a herpes virus, respiratory syncytial virus, or retrovirus, as detailed above. Similarly, the preferred nucleoside analog compound is selected for activity against the target virus (Martin)
One advantage of the combined-drug composition is that substantially lower doses of both types of compounds are required for achieving a selected viral inhibition level, reducing drug side effects in a composition that also is characterized by greater anti-viral activity.
IV. Specificity Toward Enveloped Viruses
This section examines the specificity of the viral-inhibition method to enveloped viruses. The studies reported in subsection A show that the macrocyclic compounds used in the method act, at least in part, by binding selectively to viral envelope proteins, and that this binding blocks virus attachment to infectable cells, thereby inhibiting virus infectivity. These studies are detailed in parent U.S. patent application Serial No. 647,720, filed January 29, 1991. Subsection B examines the inhibitory effect of the macrocyclic on non-enveloped viruses.
A. Mechanism of Viral-Infection Inhibition In one study, the ability of a macrocyclic com¬ pound to block HSV binding to infectable cells was examined as described in Example 14. Briefly, Vero cells were exposed to radiolabeled HSV-1 or HSV-2 in the absence of KY compound or in the presence of 10 μg/ml KY-1, and binding of the virus at times up to 4 hours after exposure to the virus was measured. Figure 30 shows a plot of virus (radiolabel) binding to cells over the four-hour incubation period. In the absence of drug, the amount of bound virus increased steadily over two hours, and slightly from 2-4 hours. By contrast, virus binding to cells peaked at about 1/2 hour in the presence of drug, presumably reflecting the time during which the binding events effective to block virus binding to the cells are equilibrating.
In a second study, the effect of compound when administered prior to, during, or after cell infection by HSV-1 was examined as described in Example 15. In these studies, cells were exposed to one of a series of increasing KY-2 concentrations, and the extent of infection was measured by number of plaques observed 24 hours after infection. The reduction in plaque formation, expressed as a percent of control, is shown in Figure 31 for cells treated with drug prior to (solid rectangles) , during (closed circles) , and after (open rectangles) . Virus inhibition was seen most significantly when the cells were treated with drug during exposure to virus, indicating that virus inhibition occurs at the period of virus binding to and entry into infectable cells. In a third study, purified HSV-1 virus suspensions were incubated with KY-1 or the sodium salt thereof, or a control solution for 1 hour, then serially diluted to drug concentrations between 101 to 10" μg/ml as described in Example 16. Addition of the serially diluted virus suspensions gave the plaque counts, measured in duplicate, shown "in Table 9. The "X" symbol in the table indicates plaques too numerous to count. The results of the study demonstrate that inhibition of HSV infection by KY compounds is due, at least in part, to binding of drug to HSV particles. Further, complete virus inhibition was seen at drug final drug concentration of 10"2 to 10^ μg/ml (which are much lower than those needed to inhibit HSV in Vero cell culture) . It can be concluded that the drug-binding/inactivation of the virus is effectively irreversible, i.e., not reversed by high dilution effects.
Figure imgf000067_0001
In a fourth study, the binding of radiolabeled KY-1 compound to HSV-1 and HSV-2 viral proteins was examined (Example 17) . After compound binding, virus proteins were fractionated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) , and the gel patterns developed by autoradiography. In Figure 32A, lanes A and B in the figure are auto- radiographs of HSV-1 proteins in the presence (lane B) and absence (lane B) of mercaptoethanol, and lanes C and D, analogous patterns for HSV-2 proteins. The lane at the right contains the molecular weight mar¬ kers, as indicated. The major bands of drug binding in HSV-1 have molecular weights, as determined from SDS-PAGE, of 45, 66, and about 130 kilodaltons. The major bands of drug binding in HSV-2 have similar molecular weights. The major bands which show KY binding in Figure 32B correspond in molecular weight, to HSV glycoproteins gD, gB, and gC.
B. Effect on Non-Enveloped Viruses The ability of KY compounds to inhibit cell infection by a rhinovirus and adenoviruses 5 and 7 which are non-enveloped viruses, was similarly studied. Vero cells (10s) were infected with a rhinovirus in the presence of KY-1, at concentrations ranging between 1-100 μg. Twenty-four hours after virus infection, the cells were examined for cyto¬ pathic effect, evidencing viral infection. No reduc- tion in cell clumping was observed at any of the KY drug concentrations tested.
Vero cells were infected with adenovirus in the presence of KY-1, also at concentrations ranging between 1-100 μg, and twenty-four hours after virus infection, the cells were examined for cytopathic effect. No reduction in cell clumping was observed at any of the KY-1 drug concentrations.
In summary, a broad range of macrocyclic com¬ pounds are effective inhibitors of cell infection by each of the several enveloped viruses which were studied. Binding studies carried out in particular with respect to HSV viruses indicate that the anti¬ viral activity of the compounds is dependent on binding to virus envelope components, which in turn inhibits virus attachment to infectable cells. The apparent inability of the compounds to inhibit infection of non-enveloped viruses is consistent with this mechanism.
V. Viral Inhibition bv a Composition Con¬ taining a Macrocyclic Compound and a Nucleoside Analog Compound
The invention also includes a composition containing a macrocylcic compound of the type described above in combination with a nucleoside analog anti-viral compound. The nucleoside analog compound is one effective to inhibit viral replication at the level of viral replication or transcription. Among the nucleoside analog compounds which are useful in combination with a macrocyclic compound, in accordance with the invention are:
(1) Pyrophosphate analoges, such as phosphoformic acid (PFA) , phosphonoacetic acid (PAA) , methanediphosphonic acid (MDP) , carbonyldiphosphonic acid (COMDP) , phosphonoglyoxalic acid (COPAA) , and various halogen- and/or methyl-substituted derivatives thereof, which are inhibitors of viral nucleic acid polymerases. In particular, these compounds are known to inhibit herpes virus (Blackburn, Sidwell) and Influenza (Sidwell) infections, and reverse transcriptase activity in retroviruses, such as human HIV.
(2) Base-modified analogs, such as IUDR, trifluorothymidine, AraA, and azidothymidine (AZT) , didieoxyinosine (DDI) , D4T, dideoxycytidine (DDC) , and ribavirin. Trifluorothymidine, IUDR, and AraA are active mainly against herpes virues (Nicolson, 1984a, 1984b) . Ribavirin is active against several RNA and DNA viruses (Sidwell) , and AZT is active against HIV (Fischl) , as are other dideoxynucleoside analogs, such as DDI.
(3) Sugar-modified analogs, such as N-acyl derivatives of 5'-amino-2' ,5'-dideoxy 5'-ioduridine, sulphonamide derivatives of 5'-amino-5'- deoxythymidine, 2'-deoxy-5-ethyluridine, and N-acyl derivatives, 5'-Sulfate and 5'-sulfamate nucleoside analogs, such as nucleocidin, adenosine 5' sulfamate, and ribavarin, which may act primarily at the level of protein synthesis inhibition (Martin) .
(4) Phosphate analogs, including acyclonucleoside phosphonates, such as acyclovir and gangiclovir, and their isosteric phosphonate analogs. These compounds can act as virus-selective substrates for viral thymidine kinases, in the synthesis of nucleoside triphosphate analogs intracellularly (Galbraith) . Subsequently, the nucleoside triphosphate analogs can act as selective substrates for viral DNA polymerase, acting as a chain terminator since the analog does not have the bifunctionality necessary for chain extension (Allen) . These compounds have demonstrated anti¬ viral activity against herpes viruses (Collins) , including HSV-1, HSV-2, varicella zoster (VZV) , and cytomegalovirus (CMV) (Smith) .
Also included in this class are phosphonomethyl ethers of nucleosides, and their acyclic analogs, such as N-(3-hydroxy-2-phosphonylmethoxypropyl)- (HPMP-) and N-(2-phosphonylmethoxyethyl-) (PME-) derivatives of heterocyclic bases. These compounds act specifically against herpes viruses, adenoviruses, cytomegalovirus (DeClercq) , poxviruses, vaccinia viruses, and retroviruses.
The ability of the two-compound composition to inhibit viral infection in enveloped virus is demonstrated in the study reported in Example 18, which examines the viral yields after infection of Vero cells with serial dilutions of HSV-1 or HSV-2 particles, as described above. Figure 34A shows the drop in HSV-1 viral yields when infected cells are exposed to increasing concentrations of the macrocyclic compound Y-l alone (solid circles) , increasing concentrations of acyclovir alone (open circles) , increasing concentrations of acyclovir plus 25 μg/ml Y-l (solid rectangles) , and increasing concentrations of acyclovir plus 50 μg/ml Y-l (solid ovals) . With either drug alone, a maximum decrease in viral yield was slightly less than three logs (orders of magnitude) . The effect of combined compounds was tested at two Y-l concentrations. At the lower Y-l concentration of 25 μg/ml, the two compounds together gave over seven logs inhibition in viral yield, i.e., more than tenfold greater than the sum of the inhibition produced by the two drugs alone. At the higher Y-l concentration, the combined inhibitory effect of the two compounds was several orders of magnitude greater than than the sum of effect of macrocyclic compound and acyclovir alone. Similar results were observed for inhibition of HSV-2 viral yields with combined compound treatment, as seen in Figure 34B.
The two compounds are formulated (e.g., in tablet, jelly, ointment, or injectable form) at a preferred weight ratio of between about 10:1 to 1:1 macrocyclic compound and nucleoside analog, respectively. The viral-yield plots in Figures 34A and 34B a significantly higher level of inhibition was observed when the co-administered compounds were at a ratio of about 5:1 macrocyclic compound to nucleoside. The macrocyclic compound in the composition is preferably selected for optimal activity against the target virus, e.g., a herpes virus, respiratory syncytial virus, or retrovirus, as detailed above. Similarly, the preferred nucleoside analog compound is selected for activity against the target virus (Martin) .
One advantage of the combined-drug composition is that substantially lower doses of both types of compounds are required for achieving a selected viral inhibition level, reducing drug side effects in a composition that also is characterized by greater anti-viral activity. VI. Use of the Composition in Treatment of Viral Infection
In accordance with the invention, calix(n)arene compounds of the type described herein are administered to the site of infection in an individual infected with an enveloped virus, for treatment of the invention. The composition of the invention includes novel includes the calix(n)arene compound contained in a pharmaceutical carrier which is suitable for oral, topical or parenteral administration of the compound. The composition may contain the calix(n)arene compound alone, or in combination with an anti-viral nucleoside analog.
The dosage form of the composition is one which is pharmaceutically effective, i.e., effective to inhibit viral infection of host cells. As seen above, compound doses in the range 1-50 μg/ml are generally effective in inhibiting viral infection of cells. Thus, for many applications, an effective dose is preferably one which produces a concentration of compound in this range at the site of infection. For topical administration, a composition containing between 1-5% or more calix(n)arene is suitable.
In a composition containing both calix(n)arene and nucleoside analog compounds, the composition dose may be substantially lower in one or both compounds, as discussed in the section above.
One of the considerations in the administering the composition, particularly when the drug is administered parenterally or orally, is systemic side effects. Studies conducted in support of the present invention indicate that the calix(n)arene compound, particularly the sulfonic acid compound, may show anti-coagulant activity after oral and intravenous administration. One conclusion from these studies is that the anticoagulant effect of calix(n)arene in the bloodstream can be effectively blocked by administering a polycationic compound, such as protamine sulfate, by intravenous administration. The protamine administration is timed to correspond to highest blood levels of the calix(n)arene compounds. In a typical method, a dose of protamine equivalent to about l mg per 100 heparin anticoagulant units is administered intravenously simultaneously with IV administration of the calix(n)arene, or 1-2 hours after oral administration of the macrocyclic drug. It is generally recommended that protamine be infused slowly (i.e., not more than a total of 50 mg/10 minutes) . Therefore in the case of simultaneous administration of calix(n)arene compound, the rate of co-infusion of the two compounds would be adjusted such that the protamine sulfate was not introduced to the subject at a rate exceeding 50 mg/10 minutes. The composition of the invention can therefore include protamine in an amount effective to reduce the anti-coagulant effect of the macrocyclic compound, when the compound is administered for uptake into the bloodstream. Where the composition also contains a nucleoside analog drug, and lower amounts of macrocyclic drug, the protamine may be reduced or eliminated, due to the lower amounts of macrocyclic compound.
A. Iniectable Composition
Studies on the pharmacokinetics and efficacy of intravenously administered composition has been studied. Briefly, it was shown that a macrocyclic compound of the type used in the method, when administered intravenously, (a) is cleared relatively slowly from the bloodstream (t1/2= approx. 5-8 hours) , (b) is present predominantly in free form, and (c) retains activity in the bloodstream for inhibiting viral (e.g., HSV-1, HSV-2, RSV, and HIV) infection.
The injectable composition contains the calix(n)arene in a suitable IV solution, such as sterile physiological salt solution. The solution may additionally contain nucleoside analog compound and/or protamine.
B* Topical Composition: Treatment of Genital Herpes Lesions
For inhibiting viral infection of skin and mucosal membrane, the composition is preferably formulated in an ointment form. The use of a topical composition for treatment of genital herpes lesions is illustrated in the following study, which is detailed in Example 13. Briefly, female guinea pigs were infected intravaginally with HSV-2, then treated topically three times daily beginning 6 hours or 48 hours after inoculation with HSV-2, as described in Example 13. Animal groups included control animals (no treatment following virus inoculation) , placebo (vehicle treatment) , KY-1 in vehicle, or acyclovir. Swabs of vaginal secretion were obtained and assayed for viral activity by a standard CPE assay. The severity of genital lesions was scored on a 0-5+ scale through the period of primary infection (21 days) . Three to four days after HSV-2 inoculation, vesicular lesions appeared on the external genital skin. Lesions progressed to an ulcerative stage by days 7-8 and gradually healed by days 15-21. The effect of topical treatment with the KY-1 preparations on lesion development and severity is shown in Table 10. The group treated with placebo at +6h had a significantly increased lesion score-day AUC (P < 0.05); however, mean peak lesion scores were not different when compared to the untreated control group. Lesion development as determined by both AUC values and mean peak lesion scores was significantly reduced by treatment with 5% KY-1 when given at 6h after infection compared to the placebo (P < 0.001). Treatment with 1% KY-1 significantly reduced the AUC at +6h (P <0.01) but not mean peak lesion scores.
Figure imgf000075_0001
No sign of any skin irritation from any of the formulations was observed. Throughout the treatment period, the genital skin remained normal in appearance; no redness or swelling was observed. The guinea pigs also remained normal and healthy in appearance throughout the entire study.
In another study using the guinea pig genital model described above, animals were infected with HSV-2, then treated with KY-1 or Y-l topically at concentrations of 2% or 5% drug. Treatment of animals was initiated 6 or 24 hours post infection, as described in Example 13. Animals were treated and scored daily for severity of infection for 19 days. The effects of topical treatment with KY-1 and Y-l on infection are tabulated in Table 11 and compared to treatment with 5% acyclovir (ACV) .
Treatment with placebo (vehicle only) resulted in significantly worse infection scores than no treatment in this study. Drug treatment with 2% or 6% Y-l, administered 6 hours post infection, resulted in reduced numbers of animals exhibiting lesions, decreased mean lesion scores and decreased peak lesion score, in comparison to placebo treatment. Likewise, treatment with a 6% formulation of either KY-1 or Y-l or a 2% formulation of KY-1, administered 24 hours post-infection, resulted in reduced numbers of lesion bearing animals and reduced severity of lesions.
Figure imgf000076_0001
C Topical Composition: Treatment of Eve Infections
In another embodiment, the topical composition includes a calix(n)arene compound in a ointment or solution form suitable for administering the compound to the eye, e.g., to the corneal surfaces of the eye.
The composition may also include a nucleoside compound effective against the target viral infection. In one treatment method, described in Example
10, graded topical doses of compound Y-l were administered to the corneal regions of rabbits previously infected with HSV-1. Clinical slit lamp biomicroscopy was used to assess disease severity as measured by average epithelial disease involvement (Figure 33A) , conjunctivitis rating (Figure 33B) , iritis rating (Figure 33C) , and stromal disease (Figure 33D) .
By day 7 post infection, the severity of the epithelial disease had peaked, as observed in placebo treated animals (Figure 33A) . Conjunctivitis, iritis and stromal disease parameters also progressed throughout the study (Figures 33B-D) .
In all four assessments of ocular infection, drug treatment resulted in less severe infection as compared to placebo treatment. All concentrations of Y-l were effective in reducing the development of HSV-1 induced ocular disease. Therapy with all concentrations of Y-l were statistically different from each other.
A topical concentration of 12.5 μg/50 μl was the most effective ocular therapy. The epithelial disease scores decreased through day 6 post infection, and rebounded slightly on day 7 post infection. Compared to the other two Y-l therapies, this concentration was effective in reducing the development of HSV-1 disease in the eye and was associated with only mild conjunctival, iris, and stromal disease development parameters. A higher concentration (18.75 μg/50 μl) was also effective in reducing the development of corneal epithelial HSV-1- induced disease. However, this concentration of Y-l appeared to be somewhat toxic to the corneal epithelial surface and to the conjunctiva, iris and stroma. This toxicity was evidenced as an increase in all disease parameters on days 6 and 7 post infection.
Viral titers were recovered from tear film at days 0, 3, 5, and 7 post-inoculation and from epithelial scrapings performed on day 7 post- infection (sacrifice) . Viral titers were determined by plaque reduction and multiple regression analysis, as described in Example 10. In the tear film study, a marked reduction of viral titer was observed in all animals given topical doses of Y-l, and this reduction appeared dose-dependent, although no difference was seen at the highest doses (12.5 and 18.75 μg/50 μl) . A dose-dependent reduction in viral titer was observed in the scrapings taken on day 7. Based upon these studies, dose efficacy/range was generated. The optimal concentration of compound appeared to be 12.5 μg/50 μl in this study.
D. Oral Composition Studies conducted in support of the present invention have shown that a macrocyclic drug of the type used in the invention is available in the plasma for a period from about 0.5 hrs. after oral administration (e.g., by gavage) , with a peak at about 2-4 hours. The period of effective drug concentration in the bloodstream is roughly between 4 and 18 hours after IV administration. The relatively short distribution volume halflife of the drug, reflecting distribution to extracorporeal body compartments when the compound is administered intravenously, is generally advantageous in the case where drug is one which shows anti-coagulant side effects, since the concentration of compound in the bloodstream can be more closely titrated.
VII. Topical Composition for Inhibiting
Infection by Sexually Transmitted Enveloped Viruses
In a method of the invention, a topical composition containing a macrocyclic compound such as described above is administered to an area or areas of the body where sexual contact is likely to occur. Such areas may include the skin surfaces of the anal- genital regions and the mouth, and also mucous membrane tissues of the vagina, rectum, mouth, and throat. Studies carried out in support of the invention indicate that the macrocyclic compounds described above are compatible with topical administration on skin and mucous membranes; i.e., that the compounds do not give rise to signs of irritation, such as swelling or redness.
The topical composition may include a pharmaceutically acceptable carrier adapted for topical administration. Thus, the composition may take the form of a suspension, solution, ointment, lotion, sexual lubricant, cream, foam, aerosol, spray, suppository, implant, inhalant, tablet, capsule, dry powder, syrup, balm or lozenge, for example. Methods for preparing such compositions are well known in the pharmaceutical industry. In addition to the macrocyclic compound, the composition may include an antiviral nucleoside analog such as discussed in section V above.
In one preferred embodiment, the composition is a lubricating jelly (sexual lubricant) which includes a lubricating jelly vehicle, and dissolved in the vehicle, a macrocyclic compound of the invention. The jelly vehicle acts in part to minimize abrasion during sexual contact, thereby reducing the likelihood of entry of an enveloped virus into damaged tissue and subsequent entry into the blood stream. As discussed in section IVA, the macrocyclic compounds of the invention are effective to bind tightly to enveloped viruses, thereby inhibiting virus attachment to infectable cells. Thus, the macrocyclic compound in the jelly can intercept enveloped virus particles before infection can occur. The dosage form of the composition is one which is pharmaceutically effective, i.e., effective to inhibit infection by a sexually transmitted enveloped virus. As seen above, compound doses in the range 1- 7b μg/ml are generally effective in inhibiting viral infection of cells. Thus, for many applications, an effective dose is preferably one which produces a concentration of compound in this range at the site of infection. For topical administration, a composition containing between 1-10% of macrocylic compound is suitable. In a composition that contains an antiviral nucleoside analog compound (e.g., section V) in addition to a macrocyclic compound of the invention, the amount of compound in the composition may be substantially lower for one or both compounds. A. Topical Composition: Prevention of Genital Herpes Infection
The ability of Y-l, as part of a lubricating jelly formulation, to inhibit cytopathic effects in cells exposed to HSV-1 and HSV-2 was assessed in the study detailed in Example 19. Jelly formulations consisting of a commercial lubricating jelly (K-Y Jelly, Johnson & Johnson) and a selected amounts of Y-l were used. In the study, cells were exposed to ι - HSV-i or HSV-2 virus and, at the same time, to a Y-l jelly composition at final drug concentrations of 5- 40 μg/ml. Twenty-four hours after inoculation, the cells were inspected for cytopathic effects (i.e., round cell formation) .
The results are shown in Table 12. A "+" symbol in the indicates that the concentration of Y-l was effective in inhibiting cytopathic effects (CPE's) . A "-" symbol indicates that CPE was observed.
0
25
30
35
Figure imgf000081_0001
The results indicate that the various Y-l- containing jelly compositions were effective to significantly inhibited cell infection at drug concentrations of about 20 μg/ml or greater.
B. Inhibition of HIV Infection In another study, lubricating jelly formulations containing various amounts of Y-l were tested for inhibition of cytopathic effects in cells infected with one of two HTLV-III strains, HTLV-IIIB and RF-II (Example 20) . Briefly, cells chronically infected with HTLV-IIIB or RF-II were incubated in the presence of serial dilutions of the selected Y-l- containing jelly compositions, and were then cocultured with indicator cells. The extent-of syncytia formation was scored under phase microscopy.
The results, shown in Table 13, were scored as follows: "-" represents absence of cell fusion (or complete blockage of fusion) , and 4+ represents a level of cell fusion indistinguishable from untreated controls.
Figure imgf000082_0001
As can be seen, exposure of the cells to Y-l concentrations greater than about 63 ug/ml was associated with inhibition of HIV-induced cell fusion. The results indicate that Y-l formulated in a jelly composition is effective for inhibiting cell infection by the HTLV strains tested.
VIII.Contraceptive Device
In another aspect, the invention includes a physical-barrier type device, in combination with a macrocyclic of the type described above, for use in inhibiting infection by sexually transmitted enveloped viruses. In one embodiment, the device includes a physical barrier-type device (e.g., a contraceptive device) , and coating the device, a lubricating composition composed of a lubricating jelly vehicle containing a macrocyclic compound of the invention.
In one embodiment, the device is a condom, where the lubricating composition is coated on the outer surface of the condom. The contraceptive can also be a condom in which the macrocycle-containing composition coats the inner surface of the condom, for the purpose of combining with any semen expressed into the condom. The condom may be of the conventional type used by males (i.e., for placement on a male penis) , or may be of an insert-type such as can be worn by a female, where the device includes a condom element that is inserted into the vagina prior to initiation of heterosexual intercourse, for example.
The device may also take the form of a cervical cap, diaphragm, or contraceptive sponge which is placed in the vicinity of the cervix. Accordingly, a cervical cap or diaphragm is coated or infused with a jelly or cream, for example, which contains a macrocyclic compound of the invention. Similarly, a contraceptive sponge may be imbued with a solution, cream, or the like, which contains a macrocyclic compound of the invention.
In a study to assess the compatibility of the macrocyclic compounds of the invention with latex condoms, a 20% w/w mixture of Y-l in K-Y jelly was applied to the interior of several brands of latex condoms. A 50 ml conical centrifuge tube was inserted into each condom to simulate an erect penis, and the open end of each condom was sealed with dialysis clips. The sealed condoms were then placed in double-distilled water at 37°C for 24 hours. After dialysis, the water was analyzed by HPLC for the presence of Y-l. At the sensitivity of the assay (5 ug/ml) no Y-l was detected for any of the condoms, showing that the macrocyclic compound did not seep through the latex condoms. Thus, the macrocyclic compounds of the invention are compatible with latex contraceptive devices for preventing the transmission of viral infection during sexual intercourse.
The following examples illustrate methods of preparing macrocyclic compounds in accordance with the invention, and the use in inhibiting infection. The examples are intended to illustrate but not limit the scope of the invention.
Materials
All chemical reagents were obtained from Aldrich Chemical Co., or from other commercial sources. Example 1 Preparation of Naphthalene Macrocyclic Compounds
A. KY-1 fR^OH. R7=SO?Na. R7=H. R^ >O
To a 41 mM aqueous solution (50 ml) of disodium chromotropic acid, 15 ml of 37% formaldehyde was added, giving a final molar ratio of 5:1 formaldehyde:chromotropic acid. The mixture was reacted with stirring in a stoppered flask at room temperature for 1 week. The resulting dark red solution (70 ml) was filtered under vacuum, and the filtrate, after being concentrated was precipitated by adding 200 ml of acetonitrile. The precipitated product was collected by filtration and taken to dryness under vacuum. The yield of KY-1 was 95%. The compound was characterized as follows: Melting point (M.P.)> 300°C; HPLC in CH3CN/MeOH/H20/TFA: 14'48" single broad peak;
(IR/KBr) = 3425 (OH) , 1638 (Ar) , 1181, 1044 (S03) cm1; UV (H20) : 238.0, 358.5 nm
Mol Weight: 1505 (M+l) by mass spectroscopy;
H'NMR(CD3OD) , chemical shifts on the γ scale: 5.20
(CH2, 8.01 (ArH) ppm;
C13 NMR (D20) , chemical shifts on the γ scale: 27. 19 , 120. 18 , 121.69 , 122-06 , 122-67 , 133-30, 142.97 ,
154.42 and 181 ppm. Analysis : (C22H10O16S4Na4) 2 x 6 H20 or (C^H.jO^Na^ x 5 H20
Found: C 33 . 17 , H 2.54 , Na 11.93
Calculated: C 32.75 , H 2.23 , Na 11.41; C 33 .16 , H 2. 13 , Na 11.56.
B. KY-3 .R^OH. R,=SQ7NH.,. R,=H. R= -CH?-.
KY-1 (2mM) was treated with 5 ml chlorosulfonic acid and the mixture was stirred at 50°C for one-half hour. The resultant mixture was added to 20 g of crushed ice to precipitate the product, which was collected by filtration and then washed with ether.
The crude product was dissolved in 100 ml of 25% ammonium water solution and allowed to react for 2 hours at room temperature. The mixture was concentrated in vacuo and the remaining oil was dissolved in a small amount of water and filtered.
The product was precipitated by adding acetonitrile to the filtrate and collected by filtration and washing with acetonitrile. The compound was characterized as follows:
Melting point (M.P.)> 300°C;
Mass spec: 1452 (M-7NH2) ;. HPLC in CH3CN/MeOH/H20/TFA: 11'46" single peak;
(IR/KBr) = 3430 (OH), 3187, 1686 (NH2) , 1637 (Ar) ,
1211, 1110, 1044 (S03) cm1;
UV (H20) : 246 nm;
H1 NMR(D20) , chemical shifts on the y scale: 5.15 (CH2) , 7.5-8.2 (ArH) ppm;
Analysis: (C44H40O26S,0Nj2Na4) -16H20
Found: C 28.62, H 3.93, N 8.82, S 17.17, Na 5.44;
Calculated: C 28.51, H 3.89, N 9.07, S 17.28, Na
4.97;
C KY-42 ιR,=OH. R,=SO.Na. R.=H. ,= >CHCOOH. Chromotropic acid, disodium (lOmM) in 50 ml water was mixed with glyoxylic acid (10.0 mM, in 5 ml water) and 10 ml of 37% hydrogen chloride at room temperature. The mixture was boiled for 8 hours and the color of the solution turned to dark red. The resultant solution was added to 50 ml of water and filtered. The filtrate was concentrated and ethanol was added to precipitate the product of KY-42. The yield was 87%. The compound was characterized as follows:
Melting point (M.P.)> 300°C;
Mass spec: 1623 (M-3H20) . HPLC in CH3CN/Me0H/H20/TFA: 10'36" single peak;
(IR/KBr) = 3452 (OH), 1801, 1719 (Co), 1638 (Ar) ,
1206, 1050 (S03) cm1:
UV (H20) : 238.0, 351.5, 520 nm;
Hl NMR(D20) , chemical shifts on the γ scale: 7.10 (CHC02H) 8.00 (ArH) ppm;
C13 NMR (D20) , chemical shifts on the 7 scale: 116.04,
118.90, 120.94, 121.27, 122.30, 124.30, 124.68,
126.60, 128.37, 136.48, 136.71, 140.50, 143.93,
144.26, 145.75, 152.01, 154.33, 156.01, 156.67; Analysis: (C4gH4O40SgNa8)4-4H2O
Found: C 32.74, H 2.50;
Calculated: C 32.58, H 2.71;
D. KY-123 fRι=OH. R7=SO?Na. R.=H. R^= >CH?. KY-1 (2mM) was treated with 5 ml chlorosulfonic acid and the mixture was stirred at 50°C for one-half hour. The resultant mixture was added to 50 g of crushed ice to precipitate the product which was collected by filtration and then washed with ether. The crude sulfonyl chloride product was treated with sodium sulfite (20 mM) in 4 ml water. The reaction mixture was kept slightly alkaline by addition at intervals of small portions of 50% NaOH for 2 days. After solvent removal, ethanol was added to preci- pitate the product, which was acidified by addition of 50% H2S04, followed by addition of ethanol to precipitate sodium sulfate. The ethanol phase was mixed with ether (1:2, v/v) to precipitate the desired product. Product yield was 39%. E. KY-147 tR^OH . R,=SQ7NHCH R,=H. R^ >CH?. N-methyl chromotropic acid chloride was formed by reacting chromotropic acid (disodium salt) with sulphonylchloride in the presence of DMF. The reaction was carried out with εtirring at 80°C for 4 hours. After removal of solvent and excess of thi- onylchloride in vacuo, ether was added to precipitate the chromotropic acid chloride which was subsequently collected by filtration and washed with ether. The crude product was added to 20 ml of methylamine and stirred for 2 hours. After removal of all solvent from the resultant substance, the residue was dissolved in a 200 ml of cold methanol and filtered. The filtrate was added with acetonitrile to precipitate the product chromotropic acid methyl sulfonamide. Yield 56%.
The chromotropic acid methyl sulfonamide (2mM) in 3 ml water was reacted with 37% formaldehyde (1ml) at room temperature for one week. Acetonitrile was added to precipitate the product which was collected by filtration and washed by acetonitrile. Yield was 85%.
F. KY-151 fR,=OCH.. R?=S0.Na. R.=H. R,= >CH7. KY-1 (50mM) was dissolved in 80 ml of NaOH water solution (0.2M NaOH) and heated to 50°C, dimethyl¬ sulfate (0.2M) was added slowly for 1 hour. The mixture was continuously stirred for another 2 hours and left at room temperature for 2 days. Saturated NaCl solution (100 ml) was added to the resultant substance and filtered. The precipitate was washed with ethanol, acetonitrile and ether sequentially. The dry substance was dissolved in 100 ml of methanol and filtered. The filtrate was concentrated and ether was added to precipitate the dimethyl ether of chromotropic acid, disodium.
G. KY-158 (R-=OU . R7=SO,CH R,=H. R^ >CHT. KY-1 from Example 1A was first treated with thionyl chloride to produce chromotropic acid sulfonyl chloride. This compound was reduced by excess sodium sulfite in the presence of sodium bicarbonate to produce the corresponding sodium sulfonate salt of cyclized chromotropic acid (R2 =
S02Na) . The sulfonate salt was treated with dimethyl sulfate in the presence of NaHC03, and worked up as as described in Example 1A. Product yield was about 21%.
H. KY-175 fR^OH. R?=SO,CH7. R7=H. R,= >CH7. Chromotropic acid was first treated with thionyl chloride to produce chromotropic acid sulfonyl chloride. This compound was then treated with sodium methoxide in methanol in the presence of sodium salt. The product was worked up as described in Example 1A to form the macrocyclic compound. Product yield was about 29%.
I. KY-285 .RT=OCOCH.. R,=SO.Na. R.=H. ,= >CH->.
KY-1 from Example 1A (0.66 mmole) was dissolved in 3 ml water containing 0.1 g NaOH. To this was added 1 g acetyl chloride (13 mmole) and the reaction was allowed to proceed at room temperature overnight with stirring. After solvent removal, 25 ml ethanol was added to precipitate the product. The crude product was dissolved in methanol and filtered. The filtrate was allowed to precipitate, giving a 87% yield.
Figure imgf000090_0001
Chromotropic acid disodium salt, was dissolved in 80 ml of water at a concentration of 50 mM with εtirring at 50°C until the solution turned to clear, hexamethylenetetramine (50 mM) was then added to above solution with continuous stirring at the same temperature for additional two hours. At this time, the color of this mixture converted to dark blue. The mixture was allowed to stir at room temperature for 2 days. The resultant dark blue solution was filtered and the filtrate was concentrated, evaporated by flask, which was subsequently treated with 200 ml methanol to precipitate the product KY- 346. The yield of KY-346 was 85%. The compound was characterized aε follows: M.P.>300°C;
HPLC in CH3CN/MeOH/H20/TFA: 13'07" single peak; (IR/KBr) - 3425 (OH), 1626 (Ar) , 1197, 1052 (S03) cm"1'1 UV (H20) : 232.0, 377.5 nm
Analysis : (Cl}H„OtNS2Na2)4 x 12 H20 Found: C 33.17 , H 3. 13 , N 2.75 Calculated: C 33.98 , H 3 .59 , N 2.96. Molecular weight: 1668 by gel filtration.
Example 2 Preparation of Calix tn)arene Compounds A. Y-49 .R,=0H. R?=SO.H. R,=-CH?-. n=4. 4-tert-butylcalix(4)arene (10 g) was treated with 200 ml of concentrated H2S04 at room temperature for 0.5 hour and then at 75-85°C oil bath for another 4 hours. The reaction was completed when no water- insoluble material was detected. The resultant oil was dropped into 500 g of crushed ice and the solution was filtered by reduced pressure. After the water removed away from the filtrate, acetonitrile (500 ml) was added to the residual and allowed to stand for 4 hours to precipitate the crude product which was then collected by filtration and washed with acetonitrile, ethyl acetate and ether. Yield 8 g (73%) . The pure product was furnished by recrystallization of the crude compound with methanol-ether or methanol-acetonitrile system. The single crystal compound was also found in the recryεtallization process.
Similar methodε were used in the syntheεiε of Y- 77 (R,=0H, R3=S03H, R^ -CH2-, n=6) and Y-l (Rι=OH, R3=S03H, R4=-CH2-, n=8) .
B. KY-225 .R,= -OH. =0.. R?=SQ3H. R.= >CH7. >
CH. n=4.
4-tert-Butylcalix(4)arene (1 g) waε treated with
10 ml of 95-98% εulfuric acid at room temperature for
0.5 hourε then at 160°C for 5 minuteε. After the resultant mixture was cool, the mixture was poured slowly into 100 ml of cruεhed ice and filtrated. The solution was evaporated and the residual was added with 300 ml acetonitrile to produce great amount of precipitate which waε collected by filtration and waεhed with acetonitrile. The crude product was dissolved in 20 ml methanol and the product was precipitated by addition of diethyl ether. Yield was 84%.
Similar methods were used in the synthesis of Y- 48 (R,= -OH or =0, R3=S02H, R,= -CH2-, n=6) and Y-226 (R,= -OH or =0, R2=S03H, R,= -CH2-, n=8) . C. O-Acetylate Of Y-l . R,= -OCOCH. . R7=SQ7H . R^ >CH7. n=8 .
Y-l (0.75 g) was stirred in dry acetic anhydride
(30 ml) overnight. The reaction was continued until the material was disεolved in the εolvent. After cooling to room temperature, the εuεpenεion was filtered. The solid was washed twice with acetonitrile and dried in vacuo. The material was washed and recrystallized. 13CNMR (D20, δ) : 173.9, 151.6, 144.1, 135.6,
130.1, 34.2, and 22.4.
D. Y-78 (R.= -OH. R7=SQ7NH7. R^ >CH7. n=8. Under nitrogen, Y-l (1 g) is heated at 60-70βC with chlorosulfonic acid (20 ml) for 1 hour. After cooling to room temperature, the oily material iε poured into ice water, and the precipitate is filtered. After waεhing the precipitate with cold water, the material is added to 50 ml of solution containing 5.7 g glycine and 2.1 g NaOH, and stirred for 2 hours at room temperature. The crude product was dissolved in 100 ml of 25% ammonium water solution and allowed to react for 2 hours at room temperature. The mixture is concentrated in vacuo and the remaining oil is disεolved in a small amount of water and filtered. The product is precipitated by adding acetonitrile to the filtrate and collected by filtration and washing with acetonitrile.
E. Glvcyl sulfonamide of Y-l (R-= -OH.
Figure imgf000092_0001
Under nitrogen, Y-l (1 g) is heated at 60-70°C with chlorosulfonic acid (20 ml) for 1 hour. After cooling to room temperature, the oily material is poured into ice water, and the precipitate is filtered. After washing the precipitate with cold water, the material is added to 50 ml of solution containing 5.7 g glycine and 2.1 g NaOH, and stirred for 2 hours at room temperature. After removal of all εolvent from the resultant substance, the residue is disεolved in a 200 ml of cold methanol and filtered. The filtrate iε added with acetonitrile to precipitate the product.
F. Acetyl-Bridged Y-49 (R.= -OH. R7=SO^H. R^ -
CHC07H-. n=4.
4.3 g of p-hydroxybenzeneεulfonic acid waε treated with g gram of glyoxylic acid in 30 ml 18% cone. HCl for 2 hourε at 100°C After the reaction product waε dried under reduced preεεure, 50 ml of methanol waε added and insoluble impurities were removed by filtration. The product was precipitated from the filtrate by addition of ether then collected by filtration and dried in vacuo.
G. Toluene Sulfonyl Eεter of Y-49 (R)= - S0.C.H.CH.. R7=S0.H. R,= >CHCO?H. n=4.
Under nitrogen iε added tolueneεulfonyl chloride
(1.9 g) to a suspension of dry sodium carbonate (1.06 g) , dry dimethylformamide (10 ml) and Y-49 (0.75 g) . After an overnight reflux, the resulting mixture iε cooled to room temperature and filtered. The filtrate iε diluted with ether to precipitate out the crude product. Recryεtallization from acetonitrile/ether solvent provided the product.
H. Carboxylic Acid Derivative of Y-49 (R*= - CQ7H. R7=S0.H. R*= >CHCQ7H. n=4.
Under nitrogen, trifluoromethanesulfonic anhydride (1.0 ml) iε added to ice cold dry dichloromethane solution (10 ml) of 2,6, di-tert- butyl-4-methylpyridine (1.25 g) and 4-tert- butylcalix[4]arene (0.65 g) . After overnight stirring at room temperature, the mixture is diluted with pentane (10 ml) and filtered. The filtrate is extracted with ice cold IN aqueous NaOH solution, ice cold IN aqueous HCl solution, then saturated aqueous NaCl εolution, dried over anhydrous sodium sulfate, filtered through a pad of silica gel and concentrated in vacuo. The residue is dissolved in a mixture of dry diisapropylethylamine (10 ml) , tri ethylsilyl cyanide (0.5 ml) and palladium tetrakis- triphenylphoεphine (20 mg) . After an overnight reflux under nitrogen and then cooling to room temperature, ether (50 ml) was added and the- resulting suspension was filtered. After concentration of the filtrate in vacuo and silica gel chromatography (hexane/ethyl acetate eluent) , the cyano intermediate is heated at 80°C with concentrated sulfuric acid (10 ml) for 3 hours, diluted with water (10 ml) and refluxed overnight. After cooling to room temperature, the resulting mix is added to charcoal (0.5 g) and ice (50 g) . After filtration, the resulting filtrate is concentrated in vacuo to ca 15 ml in volume and the resulting solid was filtered. The solid is disεolved in a minimal amount of methanol and precipitated out by adding ether. Final purification by reverse phase C18 chromatography (methanol/water eluent) provide the product.
I- Methyl Ether of Y-l f t =OMe. R,=SQNa. R1 >CH7. n ~- 8.
A mixture of Y-l (447 mg) , NaOH (6 N in water, 1.53 ml), and dimethylsulfate (9 ml) was heated at 60°C for 20 hourε. The reεulting mix was added dropwise into stirring abεolute ethanol (100 ml) . The reεulting suspension was centrifuged (9000 rpm, 20 min) and then the supernatant waε removed. Twice, the reεulting εolid waε dissolved in water (6 ml) , and the reεulting solution was treated as above with ethanol, centrifuged, and εupernatant removed. The remaining εolid waε lyophilized to yield the product (420 mg) . I3CNMR (D20, δ) : 161.2, 140.9, 137.6, 129.5,
63.6, and 33.5.
J. XXVI. fR,= -OH. R?= H. R.=R.= H. <= -CH-,-. n=4. Calix(4)arene XXVI is prepared from 4-tert- butylcalix(4)arene (XXV; Fig. 13) as described (Gutsche, Levine, and Sujeeth, 1985) . A hot εolution of 5.0 g (6.75 mmol) of XXV in 250 ml of toluene iε placed in a 500 ml three-necked round-bottom flask fitted with a mechanical stirrer and a gas inlet tube. The solution is cooled to 50-55°C, treated with 5.0 g (37 mmol) of anhydrous A1C13, and εtirred for 2 h at 50-55°C in an inert atmosphere. The mixture is cooled in an ice bath and stirred with 125 ml of 1 N HCl for 30 min, and the organic phase is separated and waεhed, dried, and evaporated to leave a yellow reεidue. This iε triturated with 500 ml of ether, and the insoluble material is recrystallized from CHC13-CH30H to yield 1.9 g (66%) of XXVI as off- white microcrystalε. m.p. 313-318βC
K.
Figure imgf000095_0001
Calix(4)arene XXVIII is prepared as described (Yilmaz and Vural) . Known p-acetyl-calix(4)arene (XXVII; 1.3 g) (Yilmaz and Vural, 1991; No et al., 1986) is dissolved in 50 ml of 2 N aqueous NaOH. A solution of iodine (8 g) and potassium iodide (20 g) in 40 ml of water is added and the mixture stirred. The solution iε warmed on a water bath for 1 h.
Iodoform iε removed by filtration, and NaHS03 (20 g) iε added to the filtrate. Concentrated HCl iε then added to the filtrate to produce a pale yellow precipitate which iε then filtered off, waεhed with water, and dried. The crude product is disεolved in 10% aq. NaHS03 and treated with charcoal. After filtration, the εolution iε acidified with 1 N HCl. The precipitated product iε collected by filtration, waεhed with distilled water until free of Cl", and dried in a vacuum desiccator, yielding 1.04 g (79%) of XXVIII. m.p. 320°C (dec).
L. XXXI. (R,= -OH. R7= -CH OOH. R=R,= H. Kf ■ CH?-. n=4. Calix(4)arene derivative XXXI is prepared via p-
(dimethylamino)methyl-calix(4)arene (XXIX) as described (Gutsche and Nam, 1989) .
To a solution of 15.9 g (39.5 mmol) of calix(4)arene (XXVI) in 360 ml of THF are added 45 ml of acetic acid, 22.5 g (0.2 mol) of 40% aqueous dimethylamine, and 16.2 g (0.2 mol) of 37% aqueous formaldehyde. The reaction mixture is stirred for 24 h at room temperature, the solvents are removed under vacuum, and the residue is dissolved in 250 ml of water. The aqueous solution is extracted twice with 200 ml of ether and neutralized with 10% K2C03 solution, and the precipitate that forms is removed by suction filtration. The product is dried under vacuum and then recrystallized from chloroform to give 19.1 g (78%) of p-(dimethylamino)methyl- calix(4)arene XXIX as white needles.
To a solution containing 16.3 g of p- (dimethylamino)methyl-calix(4)arene in 220 ml of DMSO is slowly added 9.57 ml (0.15 mol) of methyl iodide. After the reaction mixture is εtirred for 30 min at room temperature, 15 g (0.3 mol) of NaCN is added, and the mixture is heated for 2 h at 80°C under a nitrogen atmosphere. The solution is then cooled, treated with 1 liter of ice water, acidified with 2 N HCl, filtered, and air-dried. The crude product is recrystallized from CH3CN to yield 12.8 g (88%) of p- cyanomethyl-calix(4)arene XXX as a pale yellow solid. p-Cyanomethyl-calix(4)arene (0.5 mmol) is then added to a solution of DMSO (25 ml) and cone, aqueous HCl(5 ml) and refluxed overnight. After dilution with water (100 ml) at room temperature, the precipitate is collected by filtration and recrystalized from methanol to provide purified XXXI.
M. XXXIII . ( R,= -OH . R-»= -CH7CH,COOH . R.=R.= H . R,= -CH2- , n=4 )
Calix(4)arene derivative XXXIII iε prepared aε described (Gutsche and Nam, 1989) . To a solution of 3.26 g (5 mmol) of
(dimethylamino)methyl-calix(4)arene (XXIX; Example L) in 80 ml of DMSO, 1.90 ml (30 mmol) of methyl iodide is added. After the mixture is stirred for 30 min, sodium diethyl malonate, prepared from 1.20 g of Na, 7.28 g of diethyl malonate, and 28 ml of EtOH, is added, and the reaction mixture is heated for 2 h at 80°C in an atmosphere of nitrogen. The solution is then cooled, poured onto 200 ml of ice-water, acidified with 2 N HCl, and worked up in the usual fashion to give 5.50 g (99%) of p- (diethylmalonyl)methyl-calix(4)arene XXXII as crude product. Hydrolysis and decarboxylation is effected by dissolving the crude product in 100 ml of DMSO and 30 ml of cone. HCl and heating at 120°C for 10 h in an atmosphere of nitrogen. The mixture is then cooled, poured onto 500 ml of ice-water, stirred for 10 min, and filtered. The precipitate is recrystallized from acetone-ethyl aςetate to give 2.42 g (69%) of XXXIII aε colorless crystals, m.p. 224.
N. XXXVI (R*= -OH. R7= -PQ7H. R,=Rf= H. R1= -CH7-
. n=4) Derivative XXXVI is prepared by adaptation of the methods of Arduini et al. and Hirao et al.
Calix(4)arene (XXVI) is refluxed with Hg(OCOCF3)2 in CHC13 , giving an almost quantitative yield of the tetra-(Hg-OCOCFj) calixarene derivative. Following evaporation of the CHC13. metal iodine exchange is carried out by reaction of the calixarene derivative with I2 in CHClj, giving p-iodo-calix(4)arene XXXIV as a brown compound in 40% yield.
A concentrated toluene solution of HPO(OEt)2 (10 mmol), triethylamine (10 mmol), Pd(PPh3)4 (0.3 mmol) and p-iodo-calix(4)arene (1.0 mmol) is stirred at 100°C under nitrogen atmosphere for 3 days. After dilution with ether (50 ml) at room temperature, the reaction mixture is filtered and then concentrated under high vacuum (100°C, at < 0.1 mm Hg) . The resultant concentrate is purified by silica chromatography to obtain purified phosphonate diester (XXXV), which is then refluxed overnight in 6 N HCl (5 ml) to produce the phosphonic acid product. After removal of solvent (100°C, at < 0.1 mm Hg) , the solid is recrystallized from methanol to yield purified XXXVI.
O. XXXIX. (R-= -Oh. R,= -CH7PO.H. R7=R.= H. ,= 5 -CH,-. n=4.
Calix(4)arene derivative XLIV is prepared via p- chloromethyl-calix(4)arene as described (Almi et al.) .
To a εolution of 1.0 g (2.4 mmol) of
I . calix(4)arene (XXVI) and 14.4 g (81 mmol) of chloromethyl-n-octyl ether in 100 ml of CHC13 cooled at -10°C, iε added 4.7 ml (40.3 mmol) of SnCl4 dropwiεe over about 15 min. The cooling bath iε then removed, and the reaction mixture iε kept at room
15 temperature until all of the calixarene εtarting material haε reacted (after -50 min) , aε judged by thin layer chromatography (hexane:ethyl acetate = 4:3). Water is then added slowly and the two phases are allowed to separate. The organic layer is washed 0 twice with distilled water and is then dried over
Na2S04. Following removal of the Na2S04, the εolvent iε evaporated to give a reεidue that iε then washed with n-hexane and filtered, giving 1.23 g (80%) of product, p-chloromethyl-calix(4)arene XXXVII.
25 Derivative XXXVII (1 g, 1.6 mmol) is refluxed for 6 h in 20 ml of triethyl phosphite. Excess triethyl phosphite iε then removed by distillation, and the resultant solid residue (phoεphonate dieεter XXXVIII) iε dried under vacuum for 8 h. A solution
30 of 20% HCl (60 ml) iε added and the reεultant reaction mixture iε refluxed for 20 h. The solvent is then removed by evaporation, and the reεultant precipitate iε filtered, waεhed first with methanol and then with CHC13, and dried under vacuum to give 1.11 g (80%) of XXXIX as a white solid, m.p. 360°C,
P. XLIV. (R.= -QTs. R,= -CH7CH7Br. R7=R5= H. R^= -CH,-. n=4. p-2-Bromoethyl-calix(4)arene derivative XLIV is prepared as described (Gutsche, Levine, and Sujeeth,
1985; Gutsche, Dhawan, et al., 1983).
(a) To a solution of calix(4)arene (XXVI; 2.14 g) in 100 ml of THF and 10 ml of DMF is added 2.0 g of NaH followed by 28 g of allyl bromide. The mixture is refluxed 1 h, after which the THF is removed by evaporation, and the reεidue is partitioned between water and CHC13. The CHC13 extract is waεhed with water, dried, and evaporated, and the reεidue is recrystallized from 95% ethanol to give 2.18 g (74 %) of 0-allyl-calix(4)arene XL as colorless needleε.
(b) A solution of 1.66 g (2.84 mmol) of the O- allyl calix(4)arene in 25 ml of N,N-diethylaniline iε heated at reflux for 2 h in an inert atmosphere. The solution is cooled, poured into 250 ml of ice-water, stirred with 250 ml of concentrated HCl, and filtered to yield a crude product, which is then crystallized from isopropanol to afford 1.22 g (74%) of p-allyl- calix(4)arene XLI as off-white needles, m.p. 245- 248°C
(c) A εolution of 2.09 g (3.57 mmol) of p- allyl-calix(4)arene in 100 ml of dry THF iε treated with 1.0 g (42 mmol) of NaH followed by 4.0 g (21 mmol) of p-toluenesulfonyl chloride, and the mixture iε heated at reflux for 1.5 h. The εolvent iε removed by evaporation to leave a light brown oil, which iε dissolved in 100 mL of CHC13, cooled in an ice bath, and treated with 100 ml of ice-water. The organic phase iε dried and evaporated, and the residue is recryεtallized from isopropanol to yield 3.41 g (79.5%) of tosylated p-allyl-calix(4)arene (XLII) . (d) A solution of 3.50 g of toεylated p-allyl- calix(4)arene in 60 ml of CH2C12 and 40 mL of CH3OH is cooled in a dry ice-acetone bath and treated with ozone until it retains a blue color (10-15 min) . Nitrogen iε bubbled through the εolution until the blue color diεappearε, and 2 g of NaBH4 iε added.
The solution is stirred at room temperature for 3-4 h, poured into ice cold, dilute HCl εolution, and worked up in conventiaonal fashion to yield a crude product as a white resin. Recrystallization from 3:5 acetone-hexane produces 1.51 g (43%) of microcrystalline p-2-hydroxyethyl-calix(4)arene phenol-oxygen-toεylate (XLIII) .
(e) A εolution of triphenylphosphine dibromide, prepared from 6.5 g (25 mπol) of triphenylphoεphine and Br2 (Schaefer et al., 1973), in 150 ml of dry acetonitrile is treated with a solution of the product from step (d) , prepared from 3.25 g of the product of step (c) , in 50 ml of acetonitrile. The mixture is stirred for 2 h at room temperature and filtered, and the εolvent iε removed by evaporation to leave a εticky orange oil. This is εtirred with 250 ml of 95% ethanol for 8 h, and 3.10 g (78%) of p- 2-bromoethyl-0-toεyl-calix(4)arene XLIV iε collected aε a white powder by filtration.
Q. XLVI .R,= -OH. R,= -CH7CH7PQ.H. R,=R.= H. R,= -CH7-. n=4.
Calix(4)arene derivative XLVI iε prepared from p-2-bromoethyl derivative XLIV (Example P) by modification of the method used to make XXXIX from p- chloromethyl-calix(4)arene (Example 0).
Purified bromide XLIV (1 mmol) is refluxed in P(OEt)3 (10 ml) overnight under nitrogen atmosphere. After removal of excess phoεphite at high vacuum (100°C,0.1 mm Hg) , the residue (diethyl phoεphite XLV) is added to a mixture of DMSO (5 mL) and 6 N NaOH (1 ml) under nitrogen and heated at 100°C overnight, thus removing the toεylate groups. After removal of DMSO under high vacuum (100°C, < 0.1 mm Hg) , the reεidue is diluted with hot water (25 ml) and acidified with cone. HCl to give upon cooling a precipitate which is then collected by filtration. Recrystallization of the εolid from methanol provideε purified XLVI.
R. XLVII. (R-~- -OH. R,*= -CH,SOH. R7=R«= H. R,= -CH7-. n=4.
To a εolution of p-chloromethyl-calix(4)arene (XXVII, Example O; 2.5 mmol) in 95% ethanol (10 ml) iε added at room temperature an aqueous solution of
Na2S03 (2 M, 11 mmol) . After refluxing overnight, the solvent iε removed by distillation until a precipitate forms. The precipitate iε collected by filtration, waεhed with cold, saturated aqueous NaCl, and then suspended in a minimum of water and passed through a column of Amberlite IR-120 resin in water.
The UV-active fractions containing product are concentrated under vacuum and the reεidue iε recryεtallized from methanol to give purified XLVII.
S. XLIX. (R,= -OH. R7= -CH7CH7SO.H. R.=R.= H. R^= -CH7-. n=4.
Calix(4)arene derivative XLIX iε prepared from bromoethyl derivative XLIV (Example P) by applying sequentially the sulfonation method of Example R to give XLVIII, the hydrolyεis step of Example Q to remove the tosyl group, and the Amberlite IR-120 εtep of Example R to produce εulfonic acid XLIX.
T. LII. (R-= -QAC. R,= t-butyl. R.=R^= H. R.,= - CHfOH.-. n=4.
Calixarene LII iε made via known calixarene LI
(Gormer et al; 1990: R,= -OAc, R2= t-butyl, R3=R5= H, R<= -C(=0)-, n=4) as follows.
A solution of 1.5 g (2.3 mmol) of p-tert-butyl- calix(4)arene (XXV) iε refluxed in a εolution of acetic anhydride (37 ml) and cone, εulfuric acid (0.1 ml) . The reaction mixture is then added to 300 ml of ice water, producing an oil that slowly crystallizeε. The crystalline εolid iε collected, waεhed several times with water, and dried with petroleum ether, giving the 0-acetyl-p-tert-butyl-calix(4)arene (L) as white crystals. To a three-necked round-bottom flask equipped with a condenser, stirrer, and addition funnel, are added 1.2 g (1.5 mmol) of O-acetyl-p-tert-butyl- calix(4)arene in 70 ml of acetic anhydride. To this iε added dropwiεe a εolution of 3.5 g chromium(IV)oxide in a mixture of acetic anhydride
(15 ml) and acetic acid (5 ml) at 20°C with εtirring, and the reaction iε εtirred at 140°C for 8 h. After cooling, the reaction mixture iε added to 600 ml of ice-water and allowed to stand for 12 h. The reεultant yellow precipitate iε collected and waεhed with water. Recryεtallization from methanol yieldε the purified keto derivative LI (O-acetyl-p-tert- butyl-calix(4)arene, R^ -C(=0)-) (Gormer et al., 1990) . m.p. 305°C To the keto derivative (1 mmol) from the previous step, dissolved in abεolute ethanol (10 mL) , is added NaBH4 (8 mmol) in small portions at room temperature under nitrogen atmoεphere. After reduction of the keto-gro p iε complete, aε judged from the disappearance of the carbonyl band at 1670 cm1 observed by infrared εpectroεcopy, acetic acid (1 ml) is added dropwise and the resulting mixture is stirred for 1 h. The solvent iε removed under high vacuum (< 0.1 mm Hg) , and the reεultant εolid iε refluxed in methanol (5 ml) for 20 min. After removal of εolvent, the reεidue iε purified by εilica chromatrography, yielding purified hydroxymethylene- bridged, O-acetyl-p-tert-butyl product LII.
U. LIII. (R-= -OH. R7=R7=R<= H. R^ -CHfCl.-. n=4.
Derivative LII (Example T; 0.5 mmol) iε refluxed in S0C12 (5 ml) under nitrogen atmoεphere. After evolution of S02 haε ceased, excess S0C12 is removed by distillation under high vacuum (< 0.1 mm Hg) . To the reεidue iε added THF (5 ml) , and distillation is repeated to remove residual S0C12, yielding chloro- derivative LIII.
V. LVI. (R-= -OH. R7=R.=R«= H. R,= -CHfCO, -. n=4.
(a) A reaction mixture containing chloro- derivative LIII (1 mmol) and NaCN (1.1 mmol) in DMSO (10 ml) iε heated at 80°C under nitrogen for 6 h. The mixture is then poured in ice-water (50 ml) , acidified with 3 N HCl, and the resultant precipitate iε collected by filtration. The filtrate iε added to a mixture of DMSO (25 ml) and cone, aqueous HCl (5 ml) and refluxed overnight. After dilution with water (100 ml) at room temperature, the reεultant precipitate iε collected by filtration and recryεtallized from CHCl3/methanol to afford LV. (b) To remove the p-tert-butyl groupε, the product from the previous step iε added in εmall portionε under nitrogen atmoεphere to a hot (60°C) toluene suspension (50 ml) of A1C13 (10 mmol) . After εtirring overnight, the mixture is cooled to 0°C, and 1 N HCl (100 ml) is added dropwiεe. After the addition, the organic phaεe iε εeparated and concentrated in vacuo. Recryεtallization from CHCl3/methanol affords purified LVI.
W.
Figure imgf000105_0001
Chloro-derivative LIII (Example U) is disεolved in a minimum amount of THF, and the mixture iε added dropwiεe to a εtirred, cold (-78°C) εolution of THF containing (CH2CH=CH2)2CuLi (0.2 M, 3 mmole). The suspension is then allowed to warm to room temperature. After overnight stirring, the suspension is extracted with a 3:1 mixture (5 ml) of saturated NH4C1 and saturated NH3 solutionε. The organic phase is dried over anhydrouε Na2S04, filtered, and concentrated in vacuo. The reεidue iε purified by εilica chromatography to provide the O- acetyl-p-tert-butyl allyl derivative LVII.
If desired, removal of the acetyl and t-butyl groups iε achieved aε in part b of the following Example.
X- LVIII. (R*= -OH. R,=R.=R<= H. R,= -CH.CH,C0,H1-. n^4.
(a) Calixarene LVII (1 mmol) in CH2C12 (10 ml) is ozonized at -78°C until the reaction mixture turns blue. Formic acid (2 ml) and then hydrogen peroxide (1 ml) are added, and the reεultant mixture iε allowed to warm to room temperature while being purged with nitrogen. Tha mixture is then refluxed overnight, after which the solvent is revoved under vacuum, yielding LVIIa.
(b) Removal of the acetyl groups is achieved by refluxing the O-acetylated product (0.2 mmol) from the previous step in a mixture of methanol (4 ml) and 6 N NaOH (1 ml) overnight. After removal of εolvent under vacuum, the reεidue iε diluted with water (10 ml), acidified to pH 2. The reεultant precipitate iε collected by filtration and recryεtallized from CHC13/hexane provideε. De-t- butylation iε then effected according to step-b of Example V, yielding purified LVIII.
Y. LXII. (R.~= -OfCH7..SO.Na. R7= H. lower alkyl. R.=R.= H. R,= -CH,-. n=4. Cali (4)arene LXII is prepared aε described
(Shinkai et al., 1989).
Calix(4)arene XXVI (1.54 mmol) is disεolved in THF (100 ml) at 50°C under nitrogen atmoεphere. After cooling, εodium hydride (1.20 g, 30 mmol; 60% dispersion in oil) is added and the mixture is stirred until evolution of hydrogen ceases (-1 h) . Propane-l,3-sulfone (2.26 g, 18.5 mmol) iε then added dropwiεe and the mixture is stirred at room temerature for 24 h. Remaining NaH is decomposed by addition of methanol, after which the solvent is evaporated under reduced preεεure, and the reεidue iε diεεolved in hot water (500 ml) . Any insoluble material is removed by centrifugation. The product iε then precipitated by the εalting-out method with sodium acetate to give purified LXII (10% yield) . m.p. > 300°C
Z• LXIV. (R.<= -OfCH7.,SO.Na. R,= SO.Na. R7=R.= H. R-= -CH7-. n=4.
To a mixture of DMSO (10 ml) , p-sulfonyl- calix(4)arene (XV, Figure 8; 1 mmol), and 6 N NaOH (1 ml) iε added propane-l,3-sulfone (9 mmol), and the resultant reaction mixture is heated at 60°C overnight. After removal of solvents under vacuum (< 0.1 mm Hg) , the solid residue is diluted with a minimum amount of water and then added dropwise to 100 ml of ethanol with stirring. The resultant precipitate is collected by filtration, and the steps of dilution in a minimum of water and dropwise addition to 100 ml of ethanol are repeated once. The precipitate is collected by filtration and recrystallized from methanol/CH3CN provides purified LXIV.
Example 3 Preparation of Aryl-Bridged Macrocyclic Compound A. VII. Mixed naphthyl/Phenyl Macrocycle Chromotropic acid, disodium (10 g) .in 55 ml of water was treated with 22 ml of 30 ml 37% HCl. To this solution, 1,2-benzenedimethanol (5 g) in 55 ml of acetic acid was added and this reaction was carried at reflex for 6 hours. After filtration of the resultant mixture, acetonitrile (500 ml) was added to precipitate the crude product and collected it by filtration. The crude compound waε further purified by column chroπatographic purification on LH-20 reεin and elution with ethanol. B. LXXV. Napthyl-Phenyl Macrocycle (n = 2 napthyl + 2 phenyl)
Mixed macrocycle LXXV is prepared using the strategy outlined by de Mendoza et al. Chromotropic acid (III, Figure 3A; 10 mmol) and
2,5-dihydroxymethyl-3-tert-butyl-phenol (LXXII; 1 mmol) iε heated at 100°C overnight in the presence of cone. HCl (5 ml) . After removal of solvent under high vacuum (100°C, < 0.1 mm Hg) , the residue is dissolved in a minimum amount of water and eluted through a column of Sephadex LH-20 in water. The isolated product (LXXIII; 0.6 mmol), which contains two chromotropic acid units and one phenol unit, is again heated at 100°C overnight in the presence of cone. HCl (2 ml) and 2,5-dihydroxymethyl-3-tert- butyl-phenol (LXXII; 0.6 mmol). The product (LXXIV; 0.05 mmol), isolated using a Sephadex LH-20 column, iε dried under vacuum and then heated at 80°C for 6 h in cone, εulfuric acid (1 ml) under nitrogen atmoεphere. After dilution with cold water (5 ml) and treatment with charcoal (100 mg) , the reεulting mixture iε filtered, and moεt of the water in the filtrate iε removed in vacuo (< 0.1 mm Hg) . The residue is dissolved in hot, saturated aqueous NaCl. Upon cooling to 0°C, a precipitate formε. The precipitate iε filtered, dissolved in a minimum of water, and eluted through a column of Amberlite IR- 120 in water. The fractions containing pure product are combined and lyophilized, yielding purified LXXV (0.03 mmol) .
Example 4 Cytotoxicity in Proliferating Cells A panel of human cell lines was used to check the toxicity of the drugs, including: KB (nasopharyngeal carcinoma) , HeLaS3 (cervical epithelial carcinoma) , PLC (hepatocarcinoma) , HepG2 (human hepatocarcinoma) HepG2T14 (hepatocarcinoma tranεfected with HBV) , WI38 (normal human lung fibroblaεt) , BT549 (breaεt cancer) , SW480 (breaεt cancer) , and A549 (lung cancer) .
5 x 104 cells were plated in each well of a 24 well multi-dish in 1 ml of RPMI-1640 containing 5% FCS and P/S. On the second day after plating, one of the fifty test compounds given in Table 3 waε added to the cellε, at concentrationε between 1-100 μg/ml. Three days later, the medium was removed and the cellε were stained with Commasεie Blue in 40% methanol and 7% acetic acid. The results are dis- cussed in Section II above.
Example 5 Inhibition of HSV Activity: Cytopathic Effect Vero cellε were maintained in RPMI-1640 medium supplemented with 5% fetal calf serum, 100 Units of penicillin per ml and 100 μg of streptomycin per ml at 37°C in a humidified incubator containing 7% C02. The HSV strainε HSV-1 (Kos-l) and HSV-2 (333) were used. 1 x 10s Vero cellε were plated in each well of a
96 well microtitre plate in 0.2 ml RPMI-1640 medium containing 5% FCS and 0.1% methyl celluloεe (15 cpε). After overnight incubation, and cell doubling, the medium was aεpirated and replaced with 100 μl of the εame medium containing 2% FCS, and 50μl control or drug εolution to a final drug concentration of lOμg/ml and 50 μl viruε, containing about 3 PFU/cell, i.e., 6 x 105 PFU/well, of HSV-1 or HSV-2. The cellε were cultured for 24 hours at 37°C, at which time cytopathic effects are clearly visible. In the absence of viral infection, the cells form an even monolayer of fibroblaεt cellε. With viral infection, the cells form a suspension of round cellε, followed by cell clumping, whose appearance is easily distinguiεhable from normal fibroblaεt cells. If no detectable cytopathic effect was produced, the test was repeated with 10 μg/ml. A parallel set of cells without virus inoculation were done as a control for cytotoxicity to Vero cells.
Table 1 above shows the structureε of the com¬ poundε which were tested, and Table 3, column 2, the compounds which protected the cells from cytopathic effect (+) .
Example 6 Inhibition of HSV Activity: Plague Reduction Vero cells were maintained in RPMI-1640 medium supplemented with 5% fetal calf serum, as in Example 5. 4 x 105 Vero cells were plated in a 24-well plate, in 1 ml RPMI-1640 medium containing 5% FCS and 0.1% methyl celluloεe (15 cps) . After overnight incubation, and cell doubling, the medium was aspirated and replaced with 100 μl of the same medium containing 2% FCS, which contained 50 μl control or drug solution to a final drug concentration of 0.25, 2.5, 5, 10, or 20 μg/ml and 50 μl viruε, containing about lxlO3 PFU/ml, i.e., 50 PFU/well, of HSV-1 or HSV-2, aε in Example 5.
After 2 hrε. at 37° absorption the virus and the drugs were removed and the cellε were waεhed with PBS and 0.5 ml of 1% methylcelluloεe (4K cpε) in RPMI- 1640 + 2% FCS + penicillin/εtreptomycin (P/S) waε added. Two days later, the media were removed. The cells were εtained with 0.8% cryεtal violet in 50% ethanol. The plaques formed were counted and the percentage of inhibition was calculated by dividing by the plaques formed in control. EDS0 values, indicating the concentration of drug needed to produce 50% inhibition of viral plaques, were calcu¬ lated assuming a linear dose response for viral plaque inhibition. The calculated IC50 values are given in Tables 3 and 4 above.
Example 7 Inhibition of HSV Activity: Viral Yield Inhibition 1 x 106 HeLa S3 were plated in 25 T flasks in 5 ml RPMI-1640 + 5% FCS + P/S. 24 hours later, the medium was aspirated and replaced with 6 x 106 PFU HSV-1 or HSV-2, and serial dilutions of selected KY compounds, at 10, 5, 2.5, 1.25, and 0.625 μg/ml drug. After growth at 37°C for 24 hours in 2 ml of RPMI- 1640 containing 2% FCS and P/S, the cells were frozen at -70°C until the time for titration. The cells were freeze/thawed 3 times to release virus from the cells, and serially diluted 10 fold.
IX 105 Vero cells were plated in each well of 24 well multi-dish in 1 ml RPMI-1640 + 5% FCS + P/S + 0.1% methylcellulose (15 cpε) . On the εecond day, after removal of the medium, the 10 fold εerially diluted virus in 100 μl was added in duplicate. After 2 hours incubation at 37°C, the viruε waε removed and 0.5 ml methycellulose (4K cps) in RPMI- 1640 and 2% FCS + P/S was added. Two days later, the medium waε removed. The cellε were εtained in 0.8% crystal violet in 50% ethanol. The plaques formed were counted and the titer was calculated from the fold of dilutions.
The reduction in virus yield, as a function of KY compound concentration, is seen in Figures 29A and 29B for KY-1 and KY-2.
Example 8
Activity Against Drug-Reεistant Strains of HSV-l and HSV-2 The following strains of HSV-1 and HSV-2 virus were used: KOS, a wild type HSV-1 viruε; KOS (PMEA) and KOS (PFA) , both drug-reεiεtant HSV-1 viruses having a DNA polymerase mutation; 333, a wild type HSV-2 HSV-2 virus, and 333 (DHPG) , a drug-resiεtant HSV-2 virus having a thymidine kinase mutation.
Inhibition of viral yield was by KY-1, acyclovir (ACV), DHPG, PFA, and FMEA waε examined in each of the five HSV strains substantially as deεcribed in Example 7. Briefly, Hela S3 were plated in 25 T flaεkε in culture, and 24 hourε later, the medium was aspirated and replaced with 6 x 106 PFU of the selected HSV εtrain, and serial dilutions of KY-1, ACV, DHGP, PFA, and PMEA. After growth at 37°c for 24 hours in 2 ml of RPMI-1640 containing 2% FCS and penicillin and εtreptomycin (P/S) , the cellε were frozen at -70°C until the time for titration. The cellε were freeze/thawed 3 timeε to releaεe virus from the cells, εerially diluted 10 fold, and the serial dilutions were added to Vero cellε in culture. After 2 hourε incubation at 37°C the viruε waε removed and 0.5 ml methycelluloεe (4K cps) in RPMI- 1640 and 2% FCS + P/S waε added. Two dayε later, the medium was removed. The cells were stained in 0.8% crystal violet in 50% ethanol. The plaques formed were counted and the titer was calculated from the fold of dilutions. From the drug dose response, the concentration of each drug required to effect a 90% inhibition of virus yield, the ICgo concentration was determined. These values are εhown in Table 5 above.
Example 9 Inhibition of RSV Activity Assays to assess the antiviral activity of KY- and Y- compounds in tissue culture were performed in 96-well flat-bottom tissue culture plates (Falcon
307) , using conditions similar to those used in the cytotoxicity assays described above. In these aεεayε, compound was tested in quadruplicate by serially diluting the compound in 2% FCS-MEM using serial two-fold dilutions (0.05 ml/well). A 0.05 ml volume of the appropriate virus containing approximately 100 median tissue culture infectious doses (TCID50) was then added to all wells except those set aside aε antiviral and tissue control wells. Next, approximately 3 x 103 HEp2 cells (0.1 ml) were added to each well. Control wells containing antiviral and no virus (antiviral control) , containing viruε but no antiviral (viruε control) , or containing medium without viruε or antiviral (tissue control) , were included in each test. The challenge virus was then back titrated. All assay plates were incubated at 35°C for 5 to 7 days in a 5% C02 incubator. When virus control wellε exhibited 70% to 100% CPE including εyncytia, all wellε were observed. The median efficacious concentration (IC50) was calculated after determining the final concentration of antiviral in the last wells in each set of quadruplicate rows exhibiting <50% CPE compared to the CPE in virus control wells. The EDj0 values calculated for each of the compounds tested are shown in Table 5.
Example 10 Activity against HSV virus: Topical activity against in vivo ocular cultures of HSV-1
New Zealand white rabbits were acclimated for a minimum of two dayε prior to inoculation to allow the animals to accommodate to conditions in the vivarium facility. After the accommodation period, animals received a slit lamp ocular examination to exclude any animals with preexiεting anterior segment ocular defects. Animals were bilaterally inoculated topically with an 80 μl drop of Minimal Essential Medium (MEM; Gibco) containing 10s pfu/ml McKrae strain HSV-1; eyes were massaged for 30 seconds. Animals were replaced individually in cages.
On day 4 post inoculation (PI) , animals were evaluated by εlit lamp microεcopy. Corneal epithelial, iris, and conjunctival disease were graded on an increasing scale of severity from 0+ to 4+. After evaluation, animals were divided into 4 groups of 5 animals with matched corneal, stromal and conjunctival involvement. Topical therapy was initiated immediately after animal grouping. Therapy groups included:
Group #1: 5 rabbitε, Y-l topical therapy (6.25 μg/50 μl) 5x/day for 4 dayε;
Group #2: 5 rabbitε, Y-l topical therapy (12.5 μg/50 μl) 5x/day for 4 dayε; Group #3: 5 rabbits, Y-l topical therapy (18.75 μg/50 μl) 5x/day for 4 days;
Group #4: 5 rabbitε., placebo therapy (εterile water) 5x/day for 5 days.
The concentration of Y-l for the ascending doεe tolerance εtudy were baεed upon the ED90 concentrationε determined in the viruε yield or CPE assays. Group l received topical eyedrop therapy containing 6.25 μg/50 μl [one-half of the ED90 concentration] ; Group 2 received eyedrop therapy containing 12.5 μg/50 μl [the ED90 concentration]; Group 3 received eyedrop therapy containing 18.75 μg/50 μl [1.5 times the ED90 concentration]. All Y-l doses were formulated to contain these concentrations in a volume of 50 μl (a εtandard eye drop) . Topical therapy with 0-19 μg Y-l in 50 μl waε initiated on day 4 poεt-inoculation (PI) and continued to day 7 PI. All animalε received daily ocular εlit lamp evaluations from day 3 through day 7 PI. The ocular HSV-1 induced disease severity was recorded daily.
Eyes of all animalε were additionally sampled for the presence of infectious HSV-1 on days 0 (pre- inoculation) , 3, 5, and 7 PI. Briefly, tear film was obtained by swabbing the lower and upper conjunctival sacs and retaining the swab in the nasal fornix for 10 seconds. The swabs were eluted individually in Hank's Buffered Saline (HBSS, Gibco Laboratories). Fifty microliter aliquots of the viruε-HBSS eluate was adsorbed onto confluent HFF cell monolayerε for 5 minuteε. Monolayerε were hydrated with Minimal Essential Medium (MEM; Gibco Laboratories) , incubated at 37°C and observed daily for two weeks to detect cytopathology consistent with HSV infection (HSV CPE) . Cultures not exhibiting HSV CPE were blind passaged to confirm negativity.
On day 7 PI (sacrifice) , the corneal epithelium was εcraped from the eyes and HSV was recovered on HFF cell monolayers. Corneal epithelial co-cultures were evaluated daily by inverted light microscopy. Cultures not exhibiting HSV CPE were blind paεεaged to confirm negativity.
Clinical efficacy of the three Y-l concentrations used in single-agent therapies were compared to placebo therapy. Virus recovery during and after topical therapy with the Y-l formulations were compared to each other and to placebo therapy, as illustrated in Figures 33(A-D).
Example 11 Inhibition of Influenza A Activity
The anti-influenza A activity of KY compounds was evaluated as described in Example 9, except that MDCK cells (kidney cell line) was uεed for infection in vitro by influenza viruε (εtrain A/Taiwan) .
Example 12 Inhibition of HIV-Induced Cell Fuεion Human CD4 + indicator cellε (VB) and chronically infected H, cells were maintained in RPMI-1640 medium supplemented with 5% fetal calf serum, 100 Units of penicillin per ml and 100 μg of streptomycin per ml at 37°C in a humidified incubator containing 7% C02. The HIV strainε that were uεed were HTLV-IIIB and RF- II strains obtained from the National Instituteε of Health (Bethesda, MD) .
For the fusion assay, serial dilutionε between 1:2 and 1:28 of a selected KY compound, l mg/ml in PBS were made in a 96 well round bottom plate. The diluted KY compound was transferred to a 96 well flat-bottom plate. To each well was added 25 μg chronically infected H9 cells (at 2 x 106, cellε/ml) , or cellε chronically infected with RF-II εtrain HIV, followed by incubation at 37°C for 45 minutes. To each well was then added 25 μl VB cellε (about 5 x 104 cellε) , and the cellε and virus isolates were cocultured for 18 hours in a humid 5% C02 atmosphere. The extent of syncytia formation waε scored under phaεe microεcopy, and the concentration which completely inhibited εyncytia formation (ED100) waε recorded. The results are given in Table 7.
Example 13 Effect of Topical Adminiεtration on
Genital HSV Infection
A. Viruε and Viral Inoculation
The MS εtrain of HSV-2 waε utilized for the experimental animal infection. Female Hartley εtrain guinea pigs (Charles River Breeding Laboratories, Kingston, NY) weighing 250-300 g were inoculated intravaginally with 2.0 x 10s plaque-forming unitε of HSV-2 one hour after being εwabbed for removal of vaginal secretions.
B. Treatment of Guinea Pigs
In the firεt εtudy, groups of 10 guinea pigs were treated topically (0.1 ml intravaginally + 0.1 ml on external genital skin) three times daily (approximately every eight hours) for seven days beginning 6h or 48h after inoculation with HSV-2. Groups of three uninfected animals were treated in a similar manner to assesε any εkin irritation.
In a εecond εtudy, groups of 8-10 animals were treated three times daily with topical formulationε of 2% or 6% KY-1 or Y-l, with treatment beginning either 6 or 24 hours following viral inoculation, as indicated in Table 8B. Formulations of KY-1 or Y-l were prepared by dissolving the compound in a 1.5% methyl cellulose solution such that final concentration of compound was 2% or 5% (wt/wt) . Control animals were given either no treatment (N=10 animals) or treatment with placebo (1.5% methylcellulose solution) .
C. Sample Collection and Virus Assays
To determine the effect of treatment on vaginal viral replication, swabs of vaginal secretions were obtained on days l, 3, 5, 7 and 10 after HSV inoculation, placed in a tube containing 2.0 ml of media, vortexed and frozen at -70°C until titrated for HSV-2. When all samples were collected, they were thawed, diluted serially and HSV-2 titers determined uεing rabbit kidney cellε •in a microtiter CPE assay.
D. Evaluation of Efficacy
To determine the effect of therapy on the development and spread of external genital lesions, lesion severity was scored on a 0-5+ scale through the primary infection period (19-21 days) . Lesion εcore-day areaε and virus titer-day areas under the curve, and peak lesion scores and peak virus titerε between untreated and placebo-treated or placebo- treated and drug-treated animalε were compared uεing the Mann-Whitney U range sum test. A p-value of 0.05 or lesε was considered significant. The results are discussed with reference to Tableε 10 and 11 in Section IV above. Animalε were εcored daily for 19 days following inoculation for presence of leεionε and εeverity of leεionε (on a 0-5+ point scale) . Lesion scores were tabulated as area under the curve of daily lesion εcore vs. time (days) and peak lesion score observed. Data are presented in Table 11. A known antiviral agent, acyclovir (ACV) was administered in a 5% formulation to 8 animals as a positive control in the study.
Example 14
Inhibition of HSV-1 Binding to Vero Cells Vero cellε were maintained in RPMI-1640 medium, aε deεcribed in Example 5. After overnight incubation, and cell doubling, the medium waε aεpirated and replaced with 100 μl of medium containing 2% FCS composed of 50μl control or drug solution to a final drug concentration of lOμg/ml and 50 μl virus, containing about 3 PFU/cell, i.e., 6 x 10s PFU/well, of H3-labeled HSV-1. At time intervals of 5, 30, 60, 120, and 240 minutes, cells were removed from the suspension, washed two times with PBS, and assayed for bound virus (cpm 3H) . The results are given in Figure 30, where the control virus binding is indicated by solid circles, and the drug-inhibited binding, by open rectangles.
Example 15 Effect of Drug/Virus Exposure on HSV Inhibition Vero cellε were maintained in RPMI-1640 medium, as above. After overnight incubation, and cell doubling, the medium was aspirated and replaced with 100 μl of medium containing 2% FCS. In one group of wellε, serial dilutions of KY-1 compound, between
0.625 and 10 μg/ml drug were added in 50 μl, together with 50μl of HSV-1 virus suspension, 5 x 106 PFU per well. The cells were incubated for 2 hourε at 37°C, then washed with PBS and assayed for number of virus plaques, as in Example 6.
In a second group of cellε, serial dilutions of the drug were added to the cells, prior to the addition of the HSV-1 virus, and the cellε were incubated for 2 hourε at 37°C in the presence of the virus. After waεhing the cellε to remove free drug, viruε suspension was added, 5 x 106 PFU per well. The cellε were incubated for 2 hours at 37°C, then washed with PBS and assayed for number of virus plaques, aε in Example 6. In a third group of cellε, 100 μl viruε suspension was added to the cells, 5 x 106 PFU per well, and the cells were incubated for 2 hours at 37°c, then washed with PBS to remove unbound virus. Serial dilutions of KY-1 compound, between 0.625 and 10 μg/ml drug were added to the cells in 100 μl. The cells were incubated for 2 hours at 37°C in the preεence of the drug, then waεhed with PBS and assayed for number of virus plagues, aε above.
The numbers of plaques observed in each of the above treatment methods, expressed as percent of untreated control, are plotted in Figure 31. The solid circles indicate co-exposure of the cells to drug and virus; the solid squares, preincubation of the cells with drug before addition of virus; and the open εquareε, preincubation of the cellε with virus before addition of drug.
Example 16 Inactivation of HSV-1 bv KY compounds
Purified HSV-1 was suspended in RPMI-1640 medium (Gibco Laboratories) containing 2% FCS, penicillin and εtreptomycin. To aliquotε of the suspensions were added control, KY-1, or KY-217 solution, to a final drug concentration of 10 μg/ml, and a final virus particle concentration of 6 x 106 or 6 x 10s PFU/ml. The suspensions were incubated for 1 hour at 37°C, then diluted serially at 10 fold dilutions to final drug concentrations of 10, 10°, 10"1, 10'2, 10"3, and 10"4 μg/ml drug concentrationε. The serially diluted particles were then added to Vero cells for two hourε, as in Example 6, and the cells examined for plaques 48 hours later. The number of plaques counted on each of two plates, for each viruε and drug concentration, are given in Table 9.
Example 17 Binding of KY Compoundε to HSV Proteinε A. Binding of KY compound to HSV Proteins HSV-1 and HSV-2 viral suspensions from above, each at a concentration of about 5 x 107 CFU/ml, were incubated for 2 hours at 37°C with 5 x 10s cpm I4C- labeled KY-1 (50μg/ml) . Each viral suspension was divided into two aliquotε and solubilized with 0.5% sodium dodecyl sulfate (SDS) , with or without 1% mercaptoethanol. The four solubilized samples were fractionated on 8.5% polyacrylamide gel, and the gels developed by autoradiography, according to standard procedures. The autoradiographs of the four samples are seen in Figure 32A, where the lanes are HSV-1, with (lane A) and without (lane B) mercaptoethanol, and HSV-2, with (lane D) and without (lane E) mercaptoethanol, with the marker proteins in lane C
B. Identification of Binding Proteins HSV-1 and HSV-2 virus εuεpenεionε were solubilized with SDS and fractionated on SDS-PAGE as above. Each sample was run in triplicate, corresponding to groups D, B, and C in Figure 32B. The two gels in each group were analyzed by Weεtern blotting as follows: The gels in groups D, B, and C were firεt reacted with mouse monoclonal antibody specific against HSV glycoprotein gD, gB, and dC, reεpectively. The antibodies were obtained from Dr. S. Chatterjee from the University of Alabama. The gels were then incubated with alkaline phosphataεe- labeled goat anti-mouse antibody, to label the glycoprotein in each group. The glycoprotein with bound antibody was identified by reaction with H202 in the presence of nitroblue tetrazolium and bromochloroindolephosphate, according to standard methods. The results are shown in Figure 32B.
Example 18
Inhibition of HSV By Combined Drug Exposure
A. Inhibition of HSV-1
HSV-1 particleε were obtained from infected HeLa cellε, aε described in Example 7. IX 10s Vero cells were plated in each well of 24 well multi-dish in 1 ml RPMI-1640 + 5% FCS + P/S + 0.1% methylcellulose (15 cpε) . On the εecond day, after removal of the medium, the 10 fold εerially diluted viruε in 100 μl waε added in duplicate, plus (i) a selected concentration of GL-288 alone (up to 50 μg/ml) , (ii) a selected concentration of acyclovir alone (up to 50 μg/ml) ; (iii) a selected concentration of acyclovir (up to 50 μg/ml) plus 25 μg/ml GL-288; or (iv) a selected concentration of acyclovir (up to 50 μg/ml) plus 50 μg/ml GL-288, to Vero cellε in culture.
After 2 hourε incubation at 37°C, the viruε was removed and 0.5 ml methycellulose (4K cps) in RPMI- 1640 and 2% FCS + P/S was added. Two dayε later, the medium waε removed. The cells were stained in 0.8% crystal violet in 50% ethanol. The plaques formed were counted and the titer waε calculated from the fold of dilutions.
The reduction in virus yield, as a function of compound concentration, is seen in Figure 34A.
B. Inhibition of HSV-2
HSV-2 particles were obtained from infected HeLa cells, aε deεcribed in Example 7. Vero cellε were infected with serial dilutions of the virus particles plus GL-228 alone, acyclovir alone, or acyclovir pluε GL-228 aε deεcribed in Section A. After 2 hourε incubation at 37°C, the viruε waε removed, and 0.5 ml methycelluloεe (4K cpε) in RPMI-1640 and 2% FCS + P/S waε added. Two dayε later, the medium was removed. The cells were εtained in 0.8% crystal violet in 50% ethanol. The plaques formed were counted and the titer was calculated from the fold of dilutions.
The reduction in virus yield, aε a function of compound concentration, iε seen in Figure 34B.
Example 19 Inhibition of HSV Activity: Cytopathic Effect Vero cells were maintained in RPMI-1640 medium supplemented with 5% fetal calf serum, 100 Units of penicillin per ml and 100 μg of streptomycin per ml at 37°C in a humidified incubator containing 7% C02. The HSV strainε HSV-1 (Koε-1) and HSV-2 (333) were uεed. A topical formulation of compound Y-l waε created by dissolving Y-l in K-Y Jelly (Johnson & Johnson) at 5, 10, 15 and 20% (weight/weight) concentrations. Each of the Y-l formulations was then dissolved and diluted in RPMI-1640 containing 2% FCS.
1 x 10s Vero cells were plated in each well of a 96 well microtitre plate in 0.2 ml RPMI-1640 medium containing 5% FCS and 0.1% methyl cellulose (15 cps). After overnight incubation and cell doubling, the medium was aspirated and replaced with 100 -μl of the same medium containing 2% FCS, and 50 μl control or drug solution yielding final drug concentrations of 5, 10, 20 and 40 μg/ml and 50 μl virus, containing about 3 PFU/cell, i.e., 6 x 105 PFU/well, of HSV-1 or HSV-2.
The cells were cultured for 24 hours at 37°C, at which time cytopathic effects are clearly visible. In the abεence of viral infection, the cellε form an even monolayer of fibroblaεt cellε. With viral infection, the cellε form a suspension of round cells, followed by cell clumping, whoεe appearance is easily distinguishable from normal fibroblast cells. A parallel set of cells without virus inoculation were done as a control for cytotoxicity to Vero cells. No cytotoxicity by K-Y Jelly alone was observed. The results are shown in Table 12, where a "+" indicates full inhibition of cytopathic effects, and a "-" indicateε no inhbition of cytopathic effectε. Example 20
Inhibition of HIV-Induced Cell Fusion with Y-l Formulated in K-Y Jelly
Human CD4 + indicator cells (VB) and chronically infected H9 cells were maintained in RPMI-1640 medium supplemented with 5% fetal calf εerum, 100 units of penicillin per ml and 100 μg of streptomycin per ml at 37°C in a humidified incubator containing 7% C02.
The HIV εtrainε that were uεed were HTLV-IIIB and RF- II strains obtained from the National Institutes of Health (Bethesda, MD) .
Topical formulationε of compound Y-l were prepared by dissolving Y-l in K-Y Jelly (Johnson & Johnson) at 5, 10, 15 and 20% (weight/weight) concentrations. Each Y-l formulation was then disεolved in PBS to a final Y-l concentration of 1 mg/ml.
For the fusion assay, serial dilutions of each Y-l formulation were transferred to a 96 well flat- bottom plate. To each well was added 25 μl chronically infected Hg cellε (at 2 x 106, cellε/ml) , oi cellε chronically infected with RF-II εtrain HIV, followed by incubation at 37°C for 45 minuteε. To each well waε then added 25 μl VB cellε .(about 5 x 104 cellε) , and the cellε and virus isolates were co- cultured for 18 hours in a humid 5% C02 atmoεphere. The extent of syncytia formation was scored under phase microεcopy. Complete inhibition of εyncytia formation waε recorded aε "4+". The abεence of εyncytia formation waε recorded aε "-". The results are given in Table 13.
Although the invention has been described with reference to preferred compounds and method of viruε inhibition employing the compounds, it will be appreciated that various modification and changes may be made without departing from the invention.

Claims

IT IS CLAIMED:
1. A method of inhibiting cell infection by an enveloped virus comprising administering to the εite of infection a therapeutically effective dose of a calix(n)arene compound which is derivatized, at its ring positions meta to the bridge attachments to the ring, with polar substituents having a terminal carboxylate, phosphonate, sulfinate or sulfonate group.
2. The method of claim 1, wherein the number of subunits in the compound (n) is 4-10.
3. The method of claim 1, wherein the calix(n)arene compound is partially oxidized.
4. The method of claim 1, wherein the calix(n)arene compound has the general structure:
wherein n
Figure imgf000127_0001
R, is OH or =0 or a mixture thereof; R2 is a polar substitutent with a terminal carboxylate, phosphonate, sulfinate or sulfonate group; and
R4 iε >CH2 or >CH, or a mixture thereof.
5. The method of claim 4, wherein R2 iε (CH2)mR2', where m= 1-3, and R2' iε a εulfonate group.
6. The method of claim 5, wherein the εulfonate group is S03R or S02NRR', and R and R' are lower alkyl groups.
7. The method of claim 4, wherein R2 is
(CH2)mR2', where m= 1-3, and R2' iε a εulfinate group.
8. The method of claim 7, wherein the εulfinate group iε S02R or S(=0)NRR', and R and R' are lower alkyl groups.
9. The method of claim 4, wherein R2 is (C 2) m-R2 r , where m= 0-3, and R2' iε a carboxylate group.
10. The method of claim 9, wherein the carboxylate group iε C02R or C(0)NRR', where R and R' are lower alkyl groupε.
11. The method of claim 4, wherein R2 iε
(CH2)m-R2', where m= 0-3, and R2' iε a phoεphonate group.
12. The method of claim 11, wherein the phosphonate group is P0(0R)2, PO(OH) (OR) ,
PO(OR) (NRR') , PO(NRR')2, where R and R' are each H or a lower alkyl group.
13. The method of claim 1, for use in treatment of HIV, RSV, HSV-1 or HSV-2, wherein the compound is adminiεtered orally.
14. The method of claim 1, for use in treatment of HIV, RSV, HSV-l or HSV-2, wherein the compound is adminiεtered intravenouεly.
15. The method of claim 1, for uεe in oral or parenteral adminiεtration, which further includes administering to the εubject, protamine εulfate in an amount effective to inhibit anti-coagulant effects of the calix(n)arene compound.
16. The method of claim 1, which further includes administering to the subject, an antiviral nucleoside analog compound.
17. A method of inhibiting infection by a sexually tranεmitted enveloped virus, comprising topically administering to an area of likely sexual contact, a composition containing a prophylactically effective amount of a macrocyclic compound composed of aryl ring subunits which are connected by ring-attached bridge linkages which form a continuous chain of connected atoms making up the backbone of the macrocycle, and which contain negatively charged substituents on non-backbone atoms of the aryl εubunits in a lubricating jelly.
18. The method of claim 17, wherein the composition iε uεed in conjunction with a physical- barrier type device.
19. The method of claim 17, wherein the compound has the form:
Figure imgf000130_0001
where n = 4, 6, or 8; R, is OH or =0, an alkyl or aryl ether, an ester, an acid, or a mixture thereof;
R2 is a polar substitutent having a terminal, negatively charged substituent selected from the group conεiεting of a εulfonate group, a εulfinate group, a carboxylate group, and a phoεphonate group; and
R4 iε >CH2 or CH, or a mixture thereof.
20. The method of claim 19, wherein at leaεt one of the R2 groupε is =0.
21. The method of claim 17, wherein the enveloped virus is εelected from the group consisting of hepatitis delta virus, hepatitis B virus, hepatitis C virus, papilloma virus, HSV-1, HSV-2, HIV-1, HIV-2, HTLV-I, and HTLV-II.
22. The method of claim 17, where said virus is selected from the group conεiεting of hepatitiε delta viruε, hepatitiε B viruε, and hepatitiε C virus.
23. The method of claim 17, where said virus is selected from the group consiεting of HSV-1 and HSV- 2.
24. The method of claim 17, where said viruε iε selected from the group consisting of HIV-1, HIV-2, HTLV-I and HTLV-II.
25. A calix(n)arene compound having the form
Figure imgf000131_0001
wherein n = 4-10;
R! is OH or =0, where at least one of the Rj groups in the calix(n)arene compound is =0;
R2 is a polar substituent with a terminal carboxylate, phoεphonate, εulfinate or εulfonate group; and
R4 is >CH2 or >CH, or a mixture thereof.
26. The compound of claim 25, wherein R2 is (CH2)mR2', where m= 1-3, and R2' is S03R or S02NR'R" , where R, R', and R' ' are each H or a lower alkyl group.
27. The compound of claim 25, wherein R2 is (CH2)m-R2', where m= 0-3, and R2' is C02R' or C(0)NR'R", where R, R' , and R" are each H or a lower alkyl group.
28. The compound of claim 25, wherein R2 is (CH2)m-R2', where m= 0-3, and R2' is PO(OR)2 or PO(OH) (OR), where R is H or a lower alkyl group.
29. A calix(n)arene compound having the form
Figure imgf000132_0001
wherein n = 4-10; R, is OH; R2 is a polar subεtituent with a terminal carboxylate or sulfinate group, and R4 is >CH2 or >CH, or a mixture thereof.
30. A pharmaceutical composition for inhibiting cell infection by an enveloped viruε comprising administering to the site of infection a therapeutically effective dose of a calix(n)arene compound which iε derivatized, at itε ring positions meta to the bridge attachments to the ring, with polar subεtituentε having a terminal carboxylate, phosphonate, εulfinate or εulfonate group.
31. A pharmaceutical composition, for inhibiting infection by a sexually transmitted enveloped virus, comprising a calix(n)arene compound which is derivatized, at its ring poεitionε meta to the bridge attachments to the ring, with polar substituents having a terminal carboxylate, phosphonate, sulfinate or sulfonate group.
32. The compoεition of claim 31, wherein the enveloped virus is selected from the group consiεting of hepatitiε delta viruε, hepatitiε B viruε. hepatitis C virus, papilloma viruε, HSV-1, HSV-2, HIV-1, HIV-2, HTLV-I, and HTLV-II.
33. A physical barrier type device for use in inhibiting infection by a sexually tranεmitted enveloped viruε, compriεing a phyεical barrier-type device in combination with a composition composed of a vehicle and, disεolved in the vehicle, a macrocyclic compound compoεed of aryl ring εubunitε which are connected by ring-attached bridge linkageε which form a continuous chain of connected atoms making up the backbone of the macrocycle, and which contain negatively charged substituents on non-backbone atoms of the aryl εubunitε.
34. The device of claim 33, wherein the device iε a condom, diaphragm, or cervical sponge.
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