Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/16—Amides, e.g. hydroxamic acids
  • A61K31/165—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide
• • 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
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
  • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
  • A61K31/404—Indoles, e.g. pindolol
  • A61K31/4045—Indole-alkylamines; Amides thereof, e.g. serotonin, melatonin
• • 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
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
  • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
  • A61K31/404—Indoles, e.g. pindolol
  • A61K31/405—Indole-alkanecarboxylic acids; Derivatives thereof, e.g. tryptophan, indomethacin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
  • A61P31/14—Antivirals for RNA viruses
  • A61P31/18—Antivirals for RNA viruses for HIV
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
  • A61P35/04—Antineoplastic agents specific for metastasis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D233/00—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings
  • C07D233/54—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members
  • C07D233/64—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members with substituted hydrocarbon radicals attached to ring carbon atoms, e.g. histidine

Definitions

• the present invention relates to compounds for use in the treatment of AIDS and other viral diseases and EDN+ related infections and compositions containing such compounds.
• the present invention also provides methods for the treatment of such diseases and infections and methods of making such compounds and compositions.
• HIV Acquired Immunodeficiency Syndrome
• a retrovirus designated human hnmunodeficiency virus (HIV) is the etiological agent of AIDS, a complex disease that includes the progressive destruction of the irnmune system and degeneration of the central and peripheral nervous system.
• HIV is one of the deadliest diseases to have struck humans in recent times, and it has reached epidemic proportions. It is estimated that over eighteen million people are infected with HIV worldwide. AIDS has been reported in more than one hundred and twenty-three countries.
• the present invention provides improvements in or relating to compounds for use in the treatment of AIDS and other viral diseases and HIV+ related infections and the like and compositions comprising such compounds.
• the present invention also provides methods for making such compounds and compositions and methods of treating or controlling such diseases or infections. According to one aspect of the present invention therefore there is provided a method of treating, preventing or controlling a viral disorder by admimstering to a patient in need thereof a compound represented by the structure of formula I:
• Ri and R 2 may be the same or different and independently of each other may represent -CH 2 OH, -CH(OH)CH 3 , -CH(OR 4 )CH 3 or a group represented by the formula:
• ILi is a linear or branched - alkyl
• R 5 is H, OH or O 5 (where R $ is a linear or branched C ⁇ -C 4 alkyl)
• A-B is a group represented by the formula: O O O II II II C-NH — — NH— C — — C- CH 2 - > ' O II NH-CH 2 - H 2 C-C — or — CH ⁇ NH-
• m may be an integer of 0 or 1
• n may be an integer of 1-500
• X may be O, -CH 2 O, -CH 2 CH 2 O, -CH(CH 3 )CH 2 O or -CH 2 CH(CH 3 )O.
• n may be an integer of 0 to 500
• X may be -CH 2 O, - CH 2 CH 2 O, -CH(CH 3 )CH 2 O or -CH 2 CH(CH 3 )O
• Z may be -CH 2 CH 2 O, -CH(CH 3 )CH 2 O or -CH 2 CH(CH 3 )O.
• n may be an integer of from 1-200, particularly 1-100.
• X may be O, -CH 2 O, -CH 2 CH 2 O, -CH(CH 3 )CH 2 O or - CH 2 CH(CH 3 )O
• Z may be -CH 2 CH 2 O, -CH(CH 3 )CH 2 O or -CH 2 CH(CH 3 )O
• m may be 0 or 1 and n may be 0-50, but preferably m and n may not both be 0. More preferably, n may be an integer of from 1-50. In some embodiments, n may be an integer of from 5-75.
• n may be 5, 6, 7, 8, 10, 11, 12, 13, 15, 16, 17, 18, 20, 25, 30, 33, 34, 35, 40, 45, 50, 60, 65, 68, 69, 70, or 75.
• n is 7, 12, 17, 34 or 69.
• Ri may be -CH 2 OH, -CH 2 OR 4 , -CH(OH)CH 3 or - CH(OR4)CH 3 .
• Ri may be:
• Ri may be phenyl or:
• R 2 may be -CH 2 OH, -CH ⁇ OR ⁇ -CH(OH)CH 3 or ⁇ H(OR 4 )CH 3 .
• R 2 may be:
• R 5 is H or OH.
• R 2 may be phenyl or
• R 5 and Rs' independently of each other, are H, OH or OR 6 (where Re is a linear or branched Cj-C 4 alkyl).
• m may be an integer of 0 or 1
• n may be an integer of 1-500
• X may be O, -CH 2 O, -CH 2 CH 2 O, -CH(CH 3 )CH 2 O or -CH 2 CH(CH 3 )O.
• m may be 1, n may be an integer of 0 to 500, and X may be -CH 2 O, -CH 2 CH 2 O,
• X may be O, -CH 2 O, -CH 2 CH 2 O, -CH(CH 3 )CH 2 O or - CH 2 CH(CH 3 )O
• Z may be -CH 2 CH 2 O, -CH(CH 3 )CH 2 O or -CH 2 CH(CH 3 )O
• n may be an integer of from 1-200, particularly 1-100.
• X may be O, -CH 2 O, -CH 2 CH 2 O, -CH(CH 3 )CH 2 O or - CH 2 CH(CH 3 )O
• Z may be -CH 2 CH 2 O, -CH(CH 3 )CH 2 O or -CH 2 CH(CH 3 )O
• m may be 0 or
• n may be 0-50, but preferably m and n may not both be 0. More preferably, n may be an integer of from 1-50. In some embodiments, n may be an integer of from 5-75. For example, n may be 5, 6, 7, 8, 10, 11, 12, 13, 15, 16, 17, 18, 20, 25, 30, 33, 34, 35, 40, 45, 50, 60, 65, 68, 69, 70, or 75. Preferably, n is 7, 12, 17, 34 or 69. In some embodiments, m may be 0. Alternatively, X may be -CH O, and m may be
• the present invention comprehends methods of treating, preventing or controlling a viral disorder by administering to a patient in need thereof a compound represented by the structure of formula III:
• Z may be -CH(CH )CH 2 O, and accordingly the present invention embraces methods of treating, preventing or controlling a viral disorder by administering to a patient in need thereof a compound represented by the structure of formula IV:
• R 5 is H.
• R 5 is OH.
• R 5 ' is H.
• R 5 ' is OH.
• n is an integer of 1-20.
• n is an integer of 10-20.
• n is 17.
• the present invention also includes salts or hydrates of the compounds represented by the structures of formula? I, II, III and IV.
• R is a polyalkylene glycol polymer having p units, where p is an integer from 1-10O.
• the polyalkylene glycol polymer may be polyisopropylene glycol.
• p may be an integer of from 5-75.
• p can be 5, 6, 7, 8, 10, 11, 12, 13, 15, 16, 17, 18, 20, 25, 30, 33, 34, 35, 40, 45, 50, 60, 65, 68, 69, 70 or 75.
• p is 7, 12, 17, 34 or 69.
• the present invention provides methods of treating, preventing or controlling a viral disorder by administering to a patient in need thereof compounds of formulas B, C, D, E or F: Formula B
• compositions for treating, preventing or controlling a viral disorder comprising one or more compounds of formulae I, II, III, IV, A, B, C, D, E or F.
• the present invention provides a pharmaceutical composition for treating, preventing or controlling a viral disorder comprising as an active ingredient one or more compounds of formulae I, II, III, IV, A, B, C, D, E or F, together with one or more pharmaceutically acceptable excipients or adjuvants.
• the pharmaceutical composition of the invention may comprise one or more compounds of formulae I or II.
• a method for the treatment, prevention or control of AIDS and other viral diseases and HIV+ related infections comprises administering one or more compounds of formulae I, II, III, IV, A, B, C, D, E or F and/or a pharmaceutical composition comprising one or more compounds of formulae I, II, III, IV, A, B, C, D, E or F to a patient in need thereof.
• one or more compounds of formulae I or II may be used.
• the present invention comprehends the use of one or more compounds of formulae I, II, III, IV, A, B, C, D, E or F as hereinbefore defined in the manufacture of a medicament for the treatment, prevention or control of AIDS and other viral diseases and HIV+ related infections.
• methods for inducing AICD, inducing apoptosis, inhibiting a chemokine receptor, inhibiting malignant metastasis or inhibiting fibrosis or aberrant fibroblast proliferation which methods each comprise admiriistering one or more compounds of formulae I, II, III, IV, A, B, C, D, E or
• one or more compounds of formulae I or II are used.
• Said compounds of the invention may suitably be administered in a carrier which minimises micellar formation or van der Waals attraction of molecules of the compounds; an example of such a carrier is DMSO.
• the compounds of the present invention may exclude N- cinnamoyl-D,L-phenylalaninol, N-[ 1 -hydroxymethyl-2-( 1 H-indol-3 -yl)-ethyl]-3 -phenyl- propionamide, N-[l-hydroxymethyl-2-phenyl-ethyl]-3-(4-hydroxy-phenyl)-propionamide,
• the S-enantiomeric forms of the compounds of formulae A to F may be particularly advantageous in that embodiments thereof have been found to exhibit useful cell division inhibitory properties whilst at the same time demonstrating little or no toxicity to animal cells at the concentration levels required to achieve such cell inhibition. Accordingly, in a particular aspect of the present invention there is provided a compound of formula A', B', C, D', E' or F' as follows: Formula A'
• R represents a polyalkylene glycol polymer having p units, in which p is an integer from 1- 100.
• R is polyethylene glycol or polypropylene glycol and p is an integer in the range 1 to 20.
• Particularly preferred are compounds where p is 7 or 17.
• Said compounds of formula A', B', C, D E' or F' as hereinbefore defined may be used in methods of treatment of the human or animal body by therapy, for example for the treatment, prevention or control of AIDS and other viral diseases and HIV+ related infections as described above or for the treatment or prophylaxis of immuno-allergical or autoimmune diseases or for the treatment, prevention or control of organ or tissue transplantation rejection in humans or animals as described in copending PCT/IB2003/04993, the contents of which are incorporated herein by reference.
• a method for making a compound of formula A', D' or E' as defined above which comprises: (i) providing a compound of formula V: V wherein the chiral centre indicated by * is the S-enantiomer, said compound of formula V comprising at least one hydroxy phenyl group; (ii) reacting said compound of formula V with a protecting agent adapted to protect the phenylic hydroxyl group(s) on said compound; (iii) forming an alkali metal salt of the alkyl hydroxyl group; (iv) reacting said alkali metal salt with a polyalkylene glycol comprising a leaving group; and (v) thereafter deprotecting said phenylic hydroxyl group(s) to obtain said compound of formula A', D' or E'.
• said compound of formula V comprises a phenyl group and an hydroxy phenyl group, such as a 4-hydroxy phenyl group.
• Said polyalkylene glycol may be polyethylene glycol or polypropylene glycol.
• a preferred protecting agent in step (ii) is di-tert-butyl dicarbonate, but any other protecting group known to those skilled in the art for use in peptide synthesis may be used.
• the alkali metal salt formed in step (iii) may be the potassium or sodium salt which may be obtained, for example, by reacting the (protected) compound of formula V with sodium or potassium ethoxide.
• a preferred leaving group in step (iv) is mesyl (methane sulfonyl), but other suitable leaving groups are known to those skilled in the art.
• Said compound of formula V may be formed by coupling a compound of formula X:
• N-hydroxybenzotriazole (HOBt) and dicyclohexylcarbodiimide (DCC) may be employed as coupling agents, but other suitable coupling agents are known and available to those skilled in the art of peptide synthesis.
• Said compound of formula X may be selected from 3-(4-hydroxyphenyl)-propionic acid and hydrocinnamic acid.
• Said compound of formula Y may be selected from L- tyrosinol hydrochloride and 3-(4-hydroxyphenyl)-propionic acid.
• the method for making a compound of formula A', D' or E' in accordance with the present invention gives surprisingly high yields as compared with other possible methods such, for example, as those described in copending PCT/IB2003/004993.
• the yield of said compound of formula A', D' or E' may be at least 20% wt. or 30% wt., with the majority of any other product(s) or residue being composed substantially of unreacted polyalkylene oxide.
• yields of 40% wt. or more may be obtained.
• the yield of the desired compound may be greater for lower values of p.
• a particularly preferred value of p is 7. Another preferred value is 17.
• a composition comprising at least 20% wt. of a compound of formula A', D' or
• composition may further comprise unreacted polyalkylene glycol as a side product.
• said composition may comprise more than about 30 % wt. or 40% wt. of said compound, preferably more than 50% wt., and more preferably more than 75% wt., e.g. about 80% wt. or about 85% wt.
• all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below by way of example only.
• an "alkyl” group refers to a saturated aliphatic hydrocarbon, including straight-chain, branched-chain and cyclic alkyl groups.
• the alkyl group may have 1-4 carbons.
• the alkyl group may be a methyl group.
• the alkyl group may be an ethyl group.
• the alkyl group may be a propyl group.
• the alkyl group may be a butyl group.
• the alkyl group may be unsubstituted or substituted by one or more groups selected from halogen, hydroxy, alkoxy carbonyl, amido, alkylamido, dialkylamido, nitro, amino, alkylamino, dialkylamino, carboxyl, thio and tbioalkyl.
• Synthetic methodologies for obtaining the compounds of the present invention are disclosed in detail in the Examples section below. However, it should be apparent to those skilled in the art that the compounds of the present invention may be prepaired by any feasible synthetic method and that, except where stated otherwise, the syntheses set forth in the Experimental Details Section are in no way limiting. Compounds of the invention may be further modified as allowed by the rules of chemistry.
• Such modifications include the addition of various substituents (e.g., hydroxylation, carboxylation, methylation, etc.), generation of enantiomers, creation of acid- or base-addition salts or the like.
• Other modifications include adding polyalkylene glycol polymers.
• the compounds of the invention may he synthesised as polyalkylene glycol (PAG) conjugates.
• PAG polyalkylene glycol
• Typical polymers used for conjugation include poly(ethylene glycol) (PEG) - also known as or poly(ethylene oxide)
• PEG polyalkylene glycol
• the above polymer, alpha-, omega-dihydroxylpoly(ethylene glycol), can also be represented as HO-PEG-OH, where it is understood that the -PEG-symbol represents the following structural unit: -CH 2 CH 2 O-(CH 2 CH 2 O) q -CH 2 CH 2 - where q typically ranges from about 4 to about 10,000.
• PEG is commonly used as methoxy-PEG-OH, or mPEG, in which one terminus is the relatively inert methoxy group, while the other terminus is a hydroxyl group that is subject to ready chemical modification.
• PAGs are polymers which typically have the properties of solubility in water and in many organic solvents, lack of toxicity and lack of immunogenicity.
• One use of PAGs is to attach covalently the polymer to insoluble molecules to make the resulting PAG-molecule "conjugate" soluble. For example, it has been shown that the water-insoluble drug paclitaxel, when coupled to PEG, becomes water-soluble. Greenwald, et al, J. Org. Chetn., 60:331-336 (1995).
• Polyalkylated compounds of the invention may typically contain between 1 and 500 monomeric units.
• Other PAG compounds of the invention may contain between 1 and 200 monomeric units.
• Still other PAG compounds of the invention may contain between 1 and 100 monomeric units.
• the polymer may contain 1, 10, 20, 30, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 monomeric units.
• Some compounds of the invention may contain polymers which include between 5 and 75 or between 1 and 50 monomeric units.
• the polymer may contain 2, 3, 5, 6, 7, 8, 10, 11, 12, 13, 15, 16, 17, 18, 20, 25, 30, 33, 34, 35, 40, 45, 50, 60, 65, 68, 69, 70, or 75 monomeric units.
• m or n is 7, 12, 17, 34 or 69.
• the present invention comprehends all sucht compounds, including cis- and tr ⁇ r ⁇ -isomers, R- and S-enantiomers, diastereomers, (D)-isomers, (L)-isomers, racemic mixtures thereof and other mixtures thereof as falling within- the scope of the invention.
• Additional asymmetric carbon atoms may be present in a substituent such as an alkyl group. All such isomers, as well as mixtures thereof, are included in this invention.
• a carbon atom which contains four different substituents is referred to as a chiral centre.
• a chiral centre can occur in two different isomeric forms.
• a solution of one stereoisomer of a given compound will rotate plane polarized light to the left and is called the levorotatory isomer [designated (-)]; the other stereoisomer for the compound will rotate plane polarized light to the same extent but to the right and is called dextrorotatory isomer [designated (+)].
• the R S system was invented to avoid ambiguities when a compound contains two or more chiral centres.
• the system is designed to rank the four different substituent atoms around an asymmetric carbon atom in order of decreasing atomic number or in order of decreasing valance density when the smallest or lowest-rank group is pointing directly away from the viewer.
• the different rankings are well known in the art and are described on page 99 ofLehninger.
• the configuration around the chiral centre is referred to as R; if the decreasing rank order is counter-clockwise, the configuration is referred to as S.
• R the configuration around the chiral centre
• S the configuration around the chiral centre
• Each chiral centre is named accordingly using this system. If, for instance, a particular enantiomer of a compound of the present invention is desired, for example the S enantiomer, then it may be prepared by asymmetric synthesis or by derivation with a chiral auxiliary where the resulting diastereomeric mixture is separated and the auxiliary group cleaved to provide the pure desired enantiomers.
• compositions and pharmaceutical compositions of the present invention may comprise one or more of the compounds of the present invention either in a pure form or a partially pure form.
• the methods of the present invention comprise using one or more compounds, wherein the compounds are in a pure form or a partially pure form.
• a composition of the invention may comprise at least one of the compounds of the present invention, i.e.
• a composition of the invention may comprise a mixture of at least two of the compounds represented by the structures of formulae I, II, III, IV, A, B, C, D, E, F, A', B'.C, D', E' and F'.
• a composition of the invention may comprises a mixture of at least five of the compounds represented by the structures of formulae I, II, III, IN A, B, C, D, E, F, A', B',C, D ⁇ E' or F'.
• a composition of the invention may comprise a mixture of at least ten of the compounds represented by the structures of formulae I, II, III, IV, A, B, C, D, E, F, A', B',C, D', E' and F'. It has been surprisingly found that one or more compounds represented by the structures of formulae I, II, III, IN A, B, C, D, E, F, A', B',C, D', E' and F' are effective against AIDS and other viral diseases and against HIV+ related infections.
• the present invention provides a method for the treatment, prevention or control of AIDS and other viral diseases and HIV+ related infections in human as well as in veterinary applications.
• said method may comprise a ⁇ ininistering to a subject one or more compounds represented by the structures of formulae I, II, III, IN, A, B, C, D, E, F, A', B',C, D', E' and F'.
• the method may comprise administering to a subject a pharmaceutical composition comprising one or more compounds represented by the structures of formulae I, II, III, IV, A, B, C, D, E, F, A', B',C', D', E' and F'. It has also been found that compounds of the invention should be useful for treating AIDS and HIV+ related infections. In experiments described in the Examples below, compounds of the invention have shown activity in vitro.
• chemokine receptor CXCR4 In binding experiments which involved the chemokine receptor CXCR4, compounds of the invention showed activity by preventing the function of this receptor, which is the most important receptor for the entrance of the HIV-1 T tropic into its target cell. Treatment of other conditions in which chemokine receptor inhibition is important or desirable are also contemplated. For example, control and/or prevention of malignant metastasis is highly important and desirable in cancer treatment. Since the chemokine receptor CXCR4 (and to an extent CXCR3) is involved in cell migration and is possibly the most prominent and important receptor in the movement of the malignant cells, compounds of the invention, which prevent the function of this receptor, may contribute to control/prevent the movement of such cells.
• Compounds of the invention are also intended for inducing apoptosis and/or inducing AICD.
• the inventors have found that compounds of the invention exert an inhibitory effect under certain conditions against apoptosis, as described in PCT/IB03/04993 cited above. According to PCT/IB03/04993, however, the compounds are administered in a more hydrophilic carrier, e.g., a water-containing carrier.
• the compounds of the invention when dissolved in even a small amount of a carrier which minimizes micellar formation or van der Waals attraction of molecules of the compounds, like DMSO, appear to enhance the development of an immune response, as evidenced by enhanced lymphocyte proliferation and apoptotic effect which are characteristic of activation-induced cell death (AICD.)
• a carrier which minimizes micellar formation or van der Waals attraction of molecules of the compounds like DMSO
• the effect of the DMSO or other agent which would interfere with micellar formation
• the compounds to behave differently i.e., proliferation followed by apoptosis
• Methods of inhibiting fibrosis or aberrant fibroblast proliferation are also within the scope of the invention. Preventing fibrosis or aberrant fibroblast proliferation is important in treating or preventing liver cirrhosis, for example, and compounds of the invention may exert an inhibitory effect on fibroblast proliferation as shown in the Examples. As such, the compounds of the invention will have usefulness in this regard.
• Methods of administration are well known to a person skilled in the art. Methods of administration include, but are not limited to, parenterally, transdermally, intramuscularly, intravenously, intradermally, intranasally, subcutaneously, intraperitoneally or intraventricularly or rectally. Methods and means of administration are known to those skilled in the art from, for example, U.S.
• the present invention provides a pharmaceutical composition comprising as an active ingredient one or more compounds of the present invention, together with one or more pharmaceutically acceptable excipients.
• pharmaceutical composition means a therapeutically effective amount of one or more compounds of the present invention together with suitable excipients and/or carriers useful for the treatment of immuno-allergical diseases, autoimmune diseases and organ or tissue transplantation rejection.
• a "therapeutically effective amount” as used herein refers to that amount that provides a therapeutic effect for a given condition and administration regimen.
• Such compositions can be administered by any one of the methods listed hereinabove.
• a further aspect of the invention comprehends a compound of the invention in combination with other compounds of the invention.
• a compound of the invention may also be administered in combination with an anti-inflammatory agent, an immunosuppressant, an antiviral agent or the like.
• the compounds of the invention may be adirunistered in combination with a chemotherapeutic agent such as an alkylating agent, anti-metabolite, mitotic inhibitor or cytotoxic antibiotic as described above.
• chemotherapeutic agent such as an alkylating agent, anti-metabolite, mitotic inhibitor or cytotoxic antibiotic as described above.
• Combination therapy includes the adniinistration of a compound of the invention and at least a second agent as part of a specific treatment regimen intended to provide the beneficial effect from the co-action of these therapeutic agents.
• the beneficial effect of the combination includes, but is not limited to, pharmacokinetic or pharmacodynamic co-action resulting from the combination of therapeutic agents.
• Administration of these therapeutic agents in combination is typically carried out over a defined time period (usually minutes, hours, days or weeks depending upon the combination selected).
• “Combination therapy” may, but generally is not, intended to encompass the administration of two or more of these therapeutic agents as part of separate monotherapy regimens that incidentally and arbitrarily result in the combinations of the present invention.
• “Combination therapy” is intended to embrace admimstration of these therapeutic agents in a sequential manner; that is wherein each therapeutic agent is administered at a different time, as well as administration of these therapeutic agents, or at least two of the therapeutic agents, in a substantially simultaneous manner.
• Substantially simultaneous administration can be accomplished, for example, by administering to the subject a single capsule having a fixed ratio of each therapeutic agent or in multiple, single capsules for each of the therapeutic agents.
• Sequential or substantially simultaneous administration of each therapeutic agent can be effected by any appropriate route including, but not limited to, oral routes, intravenous routes, intramuscular routes and direct absorption through mucous membrane tissues.
• the therapeutic agents can be administered by the same route or by different routes.
• a first therapeutic agent of the combination selected may be administered by intravenous injection while the other therapeutic agents of the combination may be administered orally.
• all therapeutic agents may be administered orally or all therapeutic agents may be administered by intravenous injection.
• the sequence in which the therapeutic agents are administered is not narrowly critical.
• Combination therapy also can embrace the administration of the therapeutic agents as described above in further combination with other biologically active ingredients and non-drug therapies (e.g., surgery or radiation treatment.)
• the combination therapy further comprises a non-drug treatment
• the non-drug treatment may be conducted at any suitable time so long as a beneficial effect from the co-action of the comt ination of the therapeutic agents and non-drug treatment is achieved.
• the beneficial effect is still achieved when the non-drug treatment is tem rally removed from the administration of the therapeutic agents, perhaps by days or e ⁇ ven weeks.
• the compounds of the invention and the other pharmacologically active -agent may be adniinistered to a patient simultaneously, sequentially or in combination.
• the compound of th--e invention and the other pharmacologically active agent may be in the same pharmaceuticatlly acceptable carrier and therefore administered simultaneously. They may be in separate pharmaceutical carriers such as conventional oral dosage forms which are taken simultaneously.
• the term “combination” further refers to the case where the compounds are provided in separate dosage forms and are administered sequentially.
• the compositions and combination therapies of the invention may be administered in combination with a variety of pharmaceutical excipients, including stabilising agents, carriers and/or encapsulation formulations as described herein.
• compositions of the present invention are formulated as oral or parenteral dosage forms, such as uncoated tablets, coated tablets, pills, capsules, powders, granulates, dispersions or suspensions.
• the com-positions of the present invention are formulated for intravenous administration.
• the compounds of the present invention are formulated in ointment, cream or gel form for transdermal administration.
• the compounds of the present invention are formulated as an aerosol or spray for nasal application.
• the compositions of the present invention are formulated in a liquid dosage form.
• suitable liquid dosage forms include solutions or suspensions in water, pharmaceutically acceptable fats and oils, alcohols or other organic solvents, including esters, emulsions, syrups or elixirs, solutions and/or suspensions.
• Suitable excipients and carriers can be solid or liquid and the type is generally chosen based on the type of administration being used. Liposomes may also be used to deliver the composition.
• suitable solid carriers include lactose, sucrose, gelatin and agar.
• Oral dosage forms may contain suitable binders, lubricants, diluents, disintegrating agents, colouring agents, flavouring agents, flow-inducing agents, and melting agents.
• Liquid dosage forms may contain, for example, suitable solvents, preservatives, emulsifying agents, suspending agents, diluents, sweeteners, thickeners, and melting agents
• suitable solvents for example, suitable solvents, preservatives, emulsifying agents, suspending agents, diluents, sweeteners, thickeners, and melting agents
• Parenteral and intravenous forms should also include minerals and other materials to make them compatible with the type of injection or delivery system chosen. This invention is further illustrated in the Examples section which follows. This section is set forth to aid in an understanding of the invention but is not intended to, and should not be construed to, limit in any way the invention as set forth in the claims that follow thereafter.
• EXAMPLE 2 Synthesis of Polyalkylene Glvcol Compounds Polyalkylene glycol compounds were generally synthesised by preparation of the appropriate alcohol compound (e.g., one of the compounds described in Example 1, or a hydroxylated derivative thereof) and then conjugation of the alcohol with a polyalkylene glycol (PAG) polymer (e.g., polyethylene glycol (PEG) or polypropylene glycol (PPG)) of the desired length.
• PAG polyalkylene glycol
• PEG polyethylene glycol
• PPG polypropylene glycol
• This compound can also be represented as Formula A, where R is a polypropylene glycol polymer and n is the total number of polypropylene monomers in the polymer:
• THF and acetonitrile were dried over KOH pellets for at least 48 biours prior to use.
• methanesulfonyl chloride (mesyl chloride) and pyridine were distilled prior to use.
• Proton NMR tests were made on Bruker's Avance 500 and Avance 200 instruments.
• Mass-spectral analyses of small molecular weight molecules were made on Bruker's Esquire 3000 pl " s mass- spectrometer and of the PPG-containing molecules on Bruker's MALDI-TOjF (reflex IV) mass-spectrometer. Using a chromatotron is recommended for a better controlled chromatographic separations.
• the reaction mixture was allowed to stir for additional 1 hour at low temperature and then for another 2 hours at room temperature.
• the white precipitate formed was filtered out and the filtrate was evaporated to dryness.
• the residue was dissolved in 10 mL ethyl acetate and the organic phase washed twice with 1M HCl, then twice with a saturated solution of sodium bicarbonate solution and then once with water.
• the organic phase was dried over anhydrous magnesium sulfate, paper filtered and evaporated to about a quarter of its original volume. The remaining solution was allowed to cool and the crystalline precipitate formed was recovered by vacuum filtration to yield 0.24g of 1 (67%).
• PPGiooo analogue of AV74S (AV78S): spot at R f 0.43 (eluant: ethyl acetate).
• EXAMPLE 5 Effect of AV Compounds on inhibiting CXCR4 and CXCR3
• VCAM-1 ligands
• active 2 ⁇ g/ml
• heat denatured SDF-1 or Mig and HSA 2mg/ml
• washed and quenched as above.
• a polystyrene plate with coated adhesive substrates was assembled as the lower wall in a parallel plate flow chamber (260 ⁇ m gap) mounted on the stage of an inverted phase contrast microscope (Diaphot 300, Nikon) and extensively washed with binding medium. All experiments were conducted at 37 C.
• Treated (1 hr incubation with 0.1, 1, 10 ⁇ g/ml of AV 61, 63, 75, 77) and untreated T cells were diluted with binding medium and perfused into the chamber at 10 6 cells/ml by an automated syringe pump (Harvard Apparatus, Natick, MA). All experiments were recorded on videotape by a long integration camera LIS-700 CCD (Applitech, Holon Israel) and a SVHS time lapse video recorder (AG-6730 Panasonic). The human T cells were allowed to accumulate for 1 min on the substrate and then the flow rate was increased to 22 dyn/cm 2 . All recorded images of cells interacting with the adhesive substrates were analyzed and quantified by computer tracking individual cells.
• EXAMPLE 6 Effect of AV Compounds on AICD T cells were isolated from buffy coats (BC) of consenting normal human donors (Hadassah Hospital Blood Bank). The BC preparations were diluted 1:4 with phosphate- buffered saline (PBS) that contained 10 U/mL heparin. Peripheral blood mononuclear cells were separated by Ficoll Paque density centrifugation. Monocytes and B cells were depleted by plastic adherence and passage through nylon wool columns, respectively.
• PBS phosphate- buffered saline
• Small T lymphocytes were harvested from the pellet of a discontinuous Percoll gradient. The cells were found to be >80%> CD3+ by FACS analysis. Cells were cultured in the presence of various concentrations of compound and/or phytohemaglutinin (PHA) (1 ⁇ g/ml) T cell mitogen. Proliferation was measured by culturing 1x10 s cells in each well of a 96-well flat-bottomed microtiter plates. 48 hrs and 7 days following addition of compound, 1 ⁇ Ci 3 [H] thymidine was added to each well and the cultures were incubated for an additional 24 hrs. Samples were harvested and incorporated radioactivity was measured.
• PHA phytohemaglutinin
• FIGS. 3C and 4B Data from the PPG-34 experiment is shown in FIGS. 3C and 4B. Data from the AV 131 experiment is shown in FIGS. 3B, 3D, 4C and 7A. Data from the AV 77 experiment is shown in FIG. 5 A and 6C.
• compounds of the invention surprisingly show the enhanced lymphocyte proliferation and apoptotic effect which is characteristic of activation-induced cell death (AICD.)
• EXAMPLE 7 Effect of AV Compounds on inhibiting fibrosis The effect of AV compounds on fibroblast proliferation was investigated.
• a thymidine (TdR) incorporation experiment was conducted to determine the effect of combined AV molecules on human foreskin fibroblast cells (HFF).
• Control Cell culture medium Inhibitor: > 50% decrease of cpm relative to control (drug Doxorubicin)
• HFF HFF are cultured in DMEM medium +1 % Na-Pyruvate, 1 % Pen/Srep, 1 % L-Glu,
• TdR - 1 50 (20 ml in 1 ml) (NET-027 Thymidine [methyl-3H] from NEN 6.7 Ci/mmol Batch 3106446) Cells were harvested by adding denaturated agent and scintillation liquid and then counted for 1 min/sample.
• EXAMPLE 8 AV compound selectivity index Anti-HIV-1 results obtained with AN 61 show activity against HIV-1 (III B strain) with an EC50 and CC50 of as low as 15.6 ⁇ g/ml and 125 ⁇ g/ml, of pure substance, resulting in a selectivity index of greater than 8, and a % PR of as high as 100%, as was determined by an MTT-assay. The following experimental procedures were employed.
• MT-4 cells were grown in RPMI 1640 medium (Life Technologies, Merelbeke, Belgium), supplemented with 10 % (v/v) heat-inactivated fetal calf scrum (FCS), 2 mM L-glutamine, 0.1 %> sodium bicarbonate and 20 ⁇ g /ml gentamicin (Life Technologies, Merelbeke, Belgium).
• FCS heat-inactivated fetal calf scrum
• FCS heat-inactivated fetal calf scrum
• 2 mM L-glutamine 2 mM L-glutamine
• 0.1 %> sodium bicarbonate 20 ⁇ g /ml gentamicin
• the cells were maintained at 37 C in a humidified atmosphere of 5 %> CO 2 in air. Every 3-4 days, cells were seeded at 3 x 10 5 cells/ml.
• Stocks of HIV-1 III B strain were obtained from the culture supernatant of 4 x 10 5 MT-4 cells/ml infected with HIV at 400 CCID 50 immediately after complete cytopathic effect (CPE) has appeared.
• the virus titre of the supernatant was determined in MT-4 cells using the Reed and Muench end-point dilution method.
• the virus stocks were aliquoted and stored at -70 ° C until used.
• Flat-bottom, 96-well plastic microtiter trays ( ⁇ unc, Roskilde, Denmark) were filled with 100ml of complete medium using a Titertek Multidrop dispenser (IC ⁇ Biomedicals -
• the MT-4 cells were resuspended at 6 x 10 5 cells/ml in a flask connected with an autoclavable dispensing cassette of a Titertek Multidrop dispenser. Under slight magnetic stirring, 50 ⁇ l volumes were then transferred to the microtiter tray wells. The outer row wells were filled with 200 ⁇ l of medium. The cell cultures were incubated at 37 C in a humidified atmosphere of 5% CO 2 in air. The cells remained in contact with the test compounds during the whole incubation period. Five days after infection, the viability of mock and HIV-infected cells was examined spectrophotometrically by the MTT method as described hereinbelow.
• the MTT assay is based on the reduction of the yellow coloured 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma Chemical Co., St. Louis, MO) by mitochondrial dehydrogenase of metabolically active cells to a blue formazan which can be measured spectrophotometrically.
• MTT 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
• mitochondrial dehydrogenase of metabolically active cells to a blue formazan which can be measured spectrophotometrically.
• 20 ⁇ l of a solution of MTT (7.5 mg/ml) in phosphate- buffered saline was added using the Titertek Multidrop.
• the trays were further incubated at 37 C in a 5% CO 2 incubator for 1 hr.
• a fixed volume of medium (150 ⁇ l) was then removed from each cup using the Biomek 2000 robot without disturbing the MT-4 cell clusters containing the formazan crystals. Solubilization of the formazan crystals was achieved by adding lOO ⁇ l of 10% (v/v) Triton X-100 in acidified isopropanol (2ml concentrated HCl per 500ml solvent) using the Biomek 2000 robot. Complete dissolution of the formazan crystals could be obtained after the trays had been placed on a plate shaker for 10 min (ICN Biomedicals Flow Laboratories).
• the absorbances were read in an eight- channel computer controlled Titertek Microplate reader and stacker (Multiskan MCC, ICN Biomedicals - Flow Laboratories) at two wavelengths (540 and 690nm). The absorbance measured at 690nm was automatically subtracted from the absorbance at 540nm, to eliminate the effects of non-specific absorption. Blanking was carried out directly on the microtiter trays with the first column wells which contained all reagents except MT-4 cells, virus and compounds. All data represent the average values for a minimum of three wells. The 50% cytotoxic concentration (CC 50 ) was defined as the concentration of compound that reduced the absorbance (OD 54 o) of the mock-infected control sample by 50%. The percent protection achieved by the compounds in HIV-infected cells was calculated by the following formula:
• AV 61 has activity against HIV-1 (III B strain) with an EC 50 and CC 50 of as low as 15.6 ⁇ g/ml and 125 ⁇ g/ml, of pure substance, resulting in a selectivity index of greater than 8, and a % PR of as high as 100%, as was determined by anMTT-assay.
• FIG. 10 shows the results of an experiment to investigate the inhibitory effect of AV 61 S on PHA-activated PBMCs.
• Human PBMCs were prepared from a blood bank and plated out at a concentration of 10 5 cells per well in a standard 96-well plate.
• PHA was added to each well at a concentration of 10 ⁇ g/ml together with AV 61 S or PPG-7 (both in PBS) at concentrations of 50 ⁇ g/ml, 1 ⁇ g/ml, 100 ng/ml, 1 ng/ml and 100 pg/ml respectively for AV 61 S and 50 ⁇ g ml, 1 ⁇ g/ml and 10 ng/ml for PPG-7.
• the cells were then incubated for 7 days.
• [3 H] -thymidine ( ⁇ ET-027 Thymidine [methyl-3H] fromNEN 6.7 Ci/mmol Batch 3106446) was added to each well 48 hours after plating at a concentration of ImCi/well; a second dose was added to each well 18 hours before the end of the experiment. The cells were then harvested and counted for 1 min. /sample. The controls were medium alone and CsA (1 mg in 1 ml ethanol). As can be seen in FIG. 10, 60% inhibition relative to the controls was shown at a concentration of AV 61 S of 50 ⁇ g/ml.
• EXAMPLE 10 Effect of AV 61S. AV 61 R. AV 74S and AV 74 R on PHA-activated PBMCs
• Example 9 was repeated using separately compounds AV 6 IS, AV 61R and AV 74S and AV 74R.
• the test compound was diluted in DMSO to a final concentration of 0.25%>.
• a single dose of [3H]-thymidine (NET-027 Thymidine [methyl- 3H] from NEN 6.7 Ci/mmol Batch 3106446) was added to each well 18 hours before the end of the experiment.
• FIGS. 11A and 1 IB show the results for AV 61S and AV 61R and FIGS.
• FIG. 12A and 12B show the corresponding results for AV 74S and AV 74R.
• AV 61S shows an inhibitory effect of about 97% relative to the controls at a concentration of 100 ⁇ g/ml and about 49% at 50 ⁇ g/ml.
• AV 61R shows a significantly reduced activity, namely 74%> inhibition at 100 ⁇ g/ml and 42% at 50 ⁇ g/ml.
• the results for AV 74S and AV 74R show an even more marked difference between the S and R enantiomers of the compound.
• FIG. 12 A at 100 ⁇ g/ml AV 74S shows 100%) inhibition relation to the controls and 81 > at 50 ⁇ g/ml.
• AV 74R shows no inhibition of activated PBMCs. at 100 ⁇ g/ml or less.
• EXAMPLE 11 Toxicity of AV Compounds against PBMC Cells bv Alamar Blue Reagent It has been established in Examples 9 and 10 above that the molecules AV 61 S and AV 74S have an inhibitory effect on PBMCs (AV 74R had no effect whereas AV 61R had only a marginal effect) when tested in the concentrations of 100 ⁇ g/ml and 50 ⁇ g/ml (6 IS) and lOOug/ml; 50 ⁇ g/ml and 25 ⁇ g/ml (74S).
• a preliminary experiment with Alamar Blue showed that the inhibitory effect was due to an anti-proliferative activity of the molecules rather than to a "killing" effect.
• Human PBMC cells were obtained from a blood bank and plated out on a standard 96- well plate at a concentration of 10 5 cells/well.
• PHA (10 ⁇ g/ml) and AV 61 S (or AV 74S) at different dilutions in DMSO of 100 ⁇ g/ml, 50 ⁇ g/ml, 25 ⁇ g/ml, 10 ⁇ g/ml and 1 ⁇ g/ml were added simultaneously to each well.
• the cells were incubated for 24 hours. After 24 hours, Alamar Blue reagent was added up to 10% of the total well volume.
• the controls were medium only and CsA (1 mg/ 1 ml EtOH).
• Alamar Blue dyeing confirmed the non-toxic effect of the molecules, except for AV 74S at 100 ⁇ g/ml which has the same activity and toxicity pattern as Cyclosporine (CsA). All other concentrations with AV 61S and AV 74S showed an anti-proliferative effect.
• EXAMPLE 13 Toxicity of AV Compounds against PBMC Cells bv Trvpan Blue
• Reagent Examples 11 and 12 were repeated using, in each case, Trypan Blue reagent instead of Alamar Blue. The results, which confirm those obtained using Alamar Blue, are shown in FIGS. 15A and 15B respectively.

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