Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K9/00—Peptides having up to 20 amino acids, containing saccharide radicals and having a fully defined sequence; Derivatives thereof
  • C07K9/006—Peptides having up to 20 amino acids, containing saccharide radicals and having a fully defined sequence; Derivatives thereof the peptide sequence being part of a ring structure
  • C07K9/008—Peptides having up to 20 amino acids, containing saccharide radicals and having a fully defined sequence; Derivatives thereof the peptide sequence being part of a ring structure directly attached to a hetero atom of the saccharide radical, e.g. actaplanin, avoparcin, ristomycin, vancomycin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• the multibinding agents of the invention comprise from 2-10 ligands covalently connected by a linker or linkers, wherein each of said ligands in their monovalent (i.e. unlinked) state have the ability to bind to a cell surface or a precursor used in the synthesis of the bacterial cell wall and thereby interfere with the synthesis of the precursor and the cell wall.
• the manner in which the ligands are linked is such that the multibinding agents so constructed demonstrate an increased biological and/or therapeutic effect as compared to the same number of unlinked ligands available for binding to the ligand binding site.
• the invention also relates to pharmaceutical compositions comprising a pharmaceutically acceptable excipient and an effective amount of a compound of the invention, and to methods of using such compounds and pharmaceutical compositions containing them as antibacterial agents. Still further, the invention also relates to methods of preparing such compounds.
• a bacterial cell wall consists of linear polysaccharide chains that are cross-linked by short peptides. This arrangement confers mechanical support to the cell wall, and prevents the bacteria from bursting due to the high internal osmotic pressure.
• Cross linking takes place after lipid-linked disaccharide-pentapeptide constructs (lipid intermediate II) are incorporated into linear polysaccharide chains by a transglycolase enzyme.
• the cross-linking reaction is the last step of the synthesis of the cell wall, and is catalyzed by an enzyme known as peptidoglycan transpeptidase.
• One method by which antibacterial agents exert their antibacterial activity is by inhibiting the transglycosylase enzyme, thus interfering with the penultimate step of the synthesis of the bacteria cell wall.
• a glycopeptide for example vancomycin
• vancomycin inhibits the bacterial transglycosylase that is responsible for adding lipid intermediate II subunits to growing peptidoglycan chains. This step preceeds the cross-linking transpeptidation step which is inhibited by beta-lactams antibiotics. It is likely that vancomycin also inhibits transpeptidation which involves the D-alanyl-D-alanine termini; however, as this step occurs subsequent to transglycosylation, inhibition of transpeptidation is not be directly observed.
• Antibacterial agents have proved to be important weapons in the fight against pathogenic bacteria.
• an increasing problem with respect to the effectiveness of antibacterial agents relates to the emergence of strains of entrococci that are highly resistant to such agents; for example, vancomycin-resistant entrococci (VRE), which are also multi-drug resistant.
• VRE vancomycin-resistant entrococci
• a preferred ligand is vancomycin.
• vancomycin bivalent compounds which demonstrate greatly enhanced biological effect when compared to vancomycin monomer, or vancomycin monomer to which is attached the linking structure. They are also highly effective when tested against VRE strains.
• the surprising activity of the compounds of the invention arises from their ability to bind in a multivalent manner with their target and thus lower the energetic costs of binding (i.e. the phenomena of energetically coupled binding), which is produced by the optimum positioning of two or more molecules of a ligand in relationship to its binding site, i.e., a multivalent interaction. That is to say, the compounds act as multibinding agents, in which ligands that are covalently attached by a linker or linkers simultaneously (or contemporaneously) bind to multiple binding sites on another component, such as an enzyme substrate.
• Vancomycin derivatives are disclosed in Patent Applications EP 0 802 199, EP 0 801 075, EP 0 667 353, WO 97/28812, WO 97/38702, and in JACS U8, pp 13107-
• the present invention addresses the above needs by providing novel multibinding agents. Accordingly, in one aspect, the present invention relates to novel multibinding agents; wherein a multibinding agent comprises 2-10 ligands, which may be the same or different, covalently connected by a linker or linkers, which may be the same or different, each of said ligands comprising a ligand domain capable of binding to a transglycosylase enzyme substrate.
• the preferred multibinding agents are represented by Formula I: (L) p (X) q
• L is a ligand that may be the same or different at each occurrence;
• X is a linker that may be the same or different at each occurrence;
• p is an integer of 2-10; and
• q is an integer of 1-20; or a salt thereof; wherein each of said ligands comprises a ligand domain capable of binding to a transglycosylase enzyme substrate, thereby modulating the activity of the enzyme substrate.
• q is less than p. More preferably p is 2 and q is 1.
• L at each occurrence represents optionally substituted vancomycin; and X represents a linker between any hydroxyl group, carboxyl group or amino group of the first vancomycin to any hydroxyl group, carboxyl group or amino group of the second vancomycin.
• the invention in a second aspect, relates to a method of treatment of mammals having a disease state that is treatable by an antibacterial agent, comprising administering a therapeutically effective .amount of a novel multibinding agent thereto; wherein a multibinding agent comprises 2-10 ligands, which may be the same or different, covalently connected by a linker or linkers, which may be the same or different, each of said ligands comprising a ligand domain capable of binding to a transglycosylase enzyme substrate.
• the preferred multibinding agent is a compound of Formula I, or a mixture of compounds of Formula I.
• the invention in a third aspect, relates to a method of treatment of mammals having a bacterial disease characterized by resistance to vancomycin, comprising administering a therapeutically effective amount of a multibinding agent as defined above, preferably a compound of Formula I, thereto, or a mixture of multibinding agents.
• a multibinding agent as defined above, preferably a compound of Formula I, thereto, or a mixture of multibinding agents.
• the invention relates to a pharmaceutical composition
• a pharmaceutical composition comprising a therapeutically effective amount of one or more multibinding agents, or a pharmaceutically acceptable salt thereof, said multibinding agent comprising 2-10 ligands, which may be the same or different, covalently connected by a linker or linkers, which may be the same or different, each of said ligands comprising a ligand domain capable of binding to a transglycosylase enzyme substrate, admixed with at least one pharmaceutically acceptable excipient, wherein the multibinding agent is preferably a compound of Formula I
• the invention relates to processes for preparing the multibinding agents of the invention. Definitions
• alkyl refers to a monoradical branched or unbranched saturated hydrocarbon chain, preferably having from 1 to 40 carbon atoms, preferably 1-10 carbon atoms, more preferably 1-6 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n- butyl, secondary butyl, tert-butyl, n-hexyl, n-octyl, n-decyl, n-dodecyl, 2-ethyldodecyl, tetradecyl, and the like, unless otherwise indicated.
• substituted alkyl refers to an alkyl group as defined above having from 1 to 5 substituents selected from the group consisting of alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-ary
• alkylene refers to a diradical of a branched or unbranched saturated hydrocarbon chain, preferably having from 1 to 40 carbon atoms, preferably 1-10 carbon atoms, more preferably 1-6 carbon atoms. This term is exemplified by groups such as methylene
• substituted alkylene refers to:
• an alkylene group as defined above having from 1 to 5 substituents selected from the group consisting of alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino (including, for example, N- glucosaminecarbonyl, benzoylamino, biphenylcarbonylamino, and the like), acyloxy, amino, aminoacyl, aminoacyloxy, oxyacylamino, azido, cyano, halogen, hydroxyl, keto, thioketo, carboxyl, carboxylalkyl, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, thioaryloxy, heteroaryl, heteroaryloxy, thioheteroaryloxy, heterocyclic, heterocyclooxy, thioheterocyclooxy, nitro, and
• substituted alkylene groups include those where 2 substituents on the alkylene group are fused to form one or more cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, heterocyclic or heteroaryl groups fused to the alkylene group.
• alkylene group as defined above that is interrupted by 1-20 atoms or substituents independently chosen from oxygen, sulfur .and NR a -, wherein R a is chosen from hydrogen, optionally substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl and heterocyclic; or
• alkylene group as defined above that has both from 1 to 5 substituents as defined above and is also interrupted by 1-20 atoms as defined above.
• substituted alkylenes are chloromethylene (-CH(Cl)-), aminoethylene (-CH(NH 2 )CH ), l-(dodecanoylamino)propylene (-CH[NHC(O)-(CH 2 ) admir-CH 3 ] CH 2 -), l-(4-phenylbenzoylamino)pentylene (-CH[NHC(O)-Z] (CH 2 ) 4 ) ,2-carboxypropylene isomers (-CH 2 CH(CO 2 H)CH 2 -), ethoxyethyl (-CH 2 CH 2 O-CH 2 CH 2 -), -), ethylmethylaminoethyl (-CH 2 CH 2 N(CH 3 ) CH 2 CH 2 -), l-ethoxy-2-(2-ethoxy-
• alkaryl or "aralkyf'refers to the groups -alkylene-aryl and -substituted alkylene-aryl in which alkylene and aryl are as defined herein. Such alkaryl groups are exemplified by benzyl, phenethyl and the like.
• alkoxy refers to the groups alkyl-O-, alkenyl-O-, cycloalkyl-O-, cycloalkenyl-O-, and alkynyl-O-, where alkyl, alkenyl, cycloalkyl, cycloalkenyl, and alkynyl are as defined herein.
• Preferred alkoxy groups are alkyl-O- and include, by way of example, methoxy, ethoxy, n-propoxy, iso-propoxy, n-butoxy, tert-butoxy, sec- butoxy, n-pentoxy, n-hexoxy, 1 ,2-dimethylbutoxy, and the like
• substituted alkoxy refers to the groups substituted alkyl-O-, substituted alkenyl-O-, substituted cycloalkyl-O-, substituted cycloalkenyl-O-, and substituted alkynyl-O- where substituted alkyl, substituted alkenyl, substituted cycloalkyl, substituted cycloalkenyl and substituted alkynyl are as defined herein. 7
• alkylalkoxy refers to the groups -alkylene-O-alkyl, alkylene-O-substituted alkyl, substituted alkylene-O-alkyl and substituted alkylene-O- substituted alkyl wherein alkyl, substituted alkyl, alkylene and substituted alkylene are as defined herein.
• alkyl, substituted alkyl, alkylene and substituted alkylene are as defined herein. Examples of such groups are methylenemethoxy (-CH 2 OCH 3 ), ethylenemethoxy (-CH 2 CH 2 OCH 3 ), n-propylene-iso-propoxy (-
• alkylthioalkoxy refers to the group -alkylene-S-alkyl, alkylene-S-substituted alkyl, substituted alkylene-S-alkyl and substituted alkylene-S- substituted alkyl wherein alkyl, substituted alkyl, alkylene and substituted alkylene are as defined herein.
• Preferred alkylthioalkoxy groups are alkylene-S-alkyl and include, by way of example, methylenethiomethoxy (-CH 2 SCH 3 ), ethylenethiomethoxy (- CH 2 CH 2 SCH 3 ), n-propylene-iso-thiopropoxy (-CH 2 CH 2 CH 2 SCH(CH 3 ) 2 ), methylene-t- thiobutoxy (-CH 2 SC(CH 3 ) 3 ) and the like.
• Alkenyl refers to a monoradical of a branched or unbranched unsaturated hydrocarbon preferably having from 2 to 40 carbon atoms, preferably 2-10 carbon atoms, more preferably 2-6 carbon atoms, and preferably having 1-6 double bonds. This term is further exemplified by such radicals as vinyl, prop-2-enyl, pent-3-enyl, hex-5-enyl, 5- ethyldodec-3,6-dienyl, and the like.
• substituted alkenyl refers to an alkenyl group as defined above having from 1 to 5 substituents selected from the group consisting of alkoxy, substituted alkoxy, acyl, acylamino, acyloxy, amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, keto, thioketo, carboxyl, carboxylalkyl, thiol, thioalkoxy, substituted thioalkoxy, aryl, heteroaryl, heterocyclic, aryloxy, thioaryloxy, heteroaryloxy, thioheteroaryloxy, heterocyclooxy, thioheterocyclooxy, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO 2 -alkyl, -SO 2 -substitute
• R a and R b may be the same or different and are chosen from hydrogen, optionally substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl and heterocyclic.
• Alkenylene refers to a diradical of an unsaturated hydrocarbon, preferably having from 2 to 40 carbon atoms, preferably 2-10 carbon atoms, more preferably 2-6 carbon atoms, and preferably having 1-6 double bonds. This term is further exemplified 8
• substituted alkenylene refers to an alkenylene group as defined above having from 1 to 5 substituents, selected from the group consisting of alkoxy, substituted alkoxy, acyl, acylamino, acyloxy, amino, aminoacyl, aminoacyloxy, oxyacylamino, azido, cyano, halogen, hydroxyl, keto, thioketo, carboxyl, carboxylalkyl, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, thioaryloxy, heteroaryl, heteroaryloxy, thioheteroaryloxy, heterocyclic, heterocyclooxy, thioheterocyclooxy, nitro, and NR a R , wherein R a and R may be the same or different and are chosen from hydrogen, optionally substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkyn
• substituted alkenylene groups include those where 2 substituents on the alkenylene group are fused to form one or more cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, heterocyclic or heteroaryl groups fused to the alkenylene group.
• Alkynyl refers to a monoradical of an unsaturated hydrocarbon, preferably having from 2 to 40 carbon atoms, preferably 2-10 carbon atoms, more preferably 2-6 carbon atoms, and preferably having 1-6 triple bonds.
• This term is further exemplified by such radicals as acetylenyl, prop-2-ynyl, pent-3-ynyl, hex-5-ynyl, 5-ethyldodec-3,6- diynyl, and the like.
• substituted alkynyl refers to an alkynyl group as defined above having from 1 to 5 substituents, selected from the group consisting of alkoxy, substituted alkoxy, acyl, acylamino, acyloxy, amino, aminoacyl, aminoacyloxy, oxyacylamino, azido, cyano, halogen, hydroxyl, keto, thioketo, carboxyl, carboxylalkyl, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, thioaryloxy, heteroaryl, heteroaryloxy, thioheteroaryloxy, heterocyclic, heterocyclooxy, thioheterocycloxy, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO 2 -alkyl, -SO 2 -substit
• acyl refers to the groups -CHO, alkyl-C(O)-, substituted alkyl-C(O)-, cycloalkyl-C(O)-, substituted cycloalkyl-C(O)-, cycloalkenyl-C(O)-, substituted cycloalkenyl-C(O)-, aryl-C(O)-, heteroaryl-C(O)- and heterocyclic-C(O)- where alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, heteroaryl and heterocyclic are as defined herein.
• acylamino refers to the group -C(O)NRR where each R is independently hydrogen, alkyl, substituted alkyl, aryl, heteroaryl, heterocyclic or where both R groups are joined to form a heterocyclic group (e.g., morpholino) wherein alkyl, substituted alkyl, aryl, heteroaryl and heterocyclic are as defined herein.
• aminoacyl refers to the group -NRC(O)R where each R is independently hydrogen, alkyl, substituted alkyl, aryl, heteroaryl, or heterocyclic wherein alkyl, substituted alkyl, aryl, heteroaryl and heterocyclic are as defined herein.
• aminoacyloxy refers to the group -NRC(O)OR where each R is independently hydrogen, alkyl, substituted alkyl, aryl, heteroaryl, or heterocyclic wherein alkyl, substituted alkyl, aryl, heteroaryl and heterocyclic are as defined herein.
• acyloxy refers to the groups alkyl-C(O)O-, substituted alkyl-C(O)O-, cycloalkyl-C(O)O-, substituted cycloalkyl-C(O)O-, aryl-C(O)O-, heteroaryl-C(O)O-, and heterocyclic-C(O)O- wherein alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, heteroaryl, and heterocyclic are as defined herein.
• alkylamino refers to the group -NHR a , where R a is alkyl as defined above.
• alkylaminoalkyl rfers to the group -R b -NHR a , where R a is alkyl as defined above, and R is alkylene as defined above.
• alky laminoalkyl are n- decylaminoethyl, 3-(dimethylamino)propyl, and the like.
• aryl refers to an unsaturated aromatic carbocyclic group of from 6 to 20 carbon atoms having a single ring (e.g., phenyl) or multiple condensed (fused) rings (e.g., naphthyl or anthryl). Unless otherwise constrained by the definition for the aryl substituent, such aryl groups can optionally be substituted with from 1 to 5 substituents selected from the 10
• acyloxy hydroxy, thiol, acyl, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl, substituted alkoxy, substituted alkenyl, substituted alkynyl, substituted cycloalkyl, substituted cycloalkenyl, amino, aminoacyl, acylamino, alkaryl, aryl, aryloxy, azido, carboxyl, carboxylalkyl, cyano, halo, nitro, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, -SO-alkyl, -SO- substituted alkyl, -SO-aryl, -SO-heteroaryloxy, -SO 2 , -
• aryloxy refers to the group aryl-O- wherein the aryl group is as defined above including optionally substituted aryl groups as also defined above.
• arylene refers to a diradical derived from aryl or substituted aryl as defined above, and is exemplified by 1,2-phenylene, 1,3-phenylene, 1 ,4-phenylene, 1,2- naphthylene and the like.
• carboxyalkyl refers to the group “-C(O)Oalkyl” where alkyl is as defined above.
• cycloalkyl refers to cyclic alkyl groups of from 3 to 20 carbon atoms having a single cyclic ring or multiple condensed rings.
• Such cycloalkyl groups include, by way of example, single ring structures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, and the like, or multiple ring structures such as adamantanyl, and the like.
• substituted cycloalkyl refers to cycloalkyl groups having from 1 to 5 substituents selected from the group consisting of alkoxy, substituted alkoxy, cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO- substituted alkyl, -
• alkyl chosen from hydrogen, optionally substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl and heterocyclic.
• cycloalkenyl refers to cyclic alkenyl groups of from 4 to 20 carbon atoms having a single cyclic ring or fused rings and at least one point of internal unsaturation.
• suitable cycloalkenyl groups include, for instance, cyclobut- 2-enyl, cyclopent-3-enyl, cyclooct-3-enyl and the like.
• substituted cycloalkenyl refers to cycloalkenyl groups having from 1 to 5 substituents selected from the group consisting of alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl,
• halo or “halogen” refers to fluoro, chloro, bromo and iodo.
• Haloalkyl refers to alkyl as defined above substituted by 1-4 halo groups as defined above, which may be the same or different, such as 3-fiuorododecyl, 12,12,12- trifluorododecyl, 2-bromooctyl, -3-bromo-6-chloroheptyl, and the like.
• heteroaryl refers to an aromatic group of from 1 to 15 carbon atoms and 1 to 4 heteroatoms selected from oxygen, nitrogen and sulfur within at least one ring (if there is more than one ring). Unless otherwise constrained by the definition for the heteroaryl substituent, such heteroaryl groups can be optionally substituted with 1 to 5 substituents selected from the group consisting of acyloxy, hydroxy, thiol, acyl, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl, substituted alkoxy, substituted alkenyl, substituted alkynyl, substituted cycloalkyl, substituted cycloalkenyl, amino, aminoacyl, acylamino, alkaryl, aryl, aryloxy, azido, carboxyl, carboxylalkyl, cyano, halo, nitro, heteroaryl, heteroaryloxy, heterocyclic
• thioalkoxy substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, -SO-alkyl, -SO- substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO 2 -alkyl, -SO 2 -substituted alkyl, -SO 2 -aryl, -SO 2 -heteroaryl, trihalomethyl, mono-and di-alkylamino, and NR a R , wherein R a and R may be the same or different and are chosen from hydrogen, optionally substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl and heterocyclic. Preferred heteroaryls nclude pyridyl, pyrrolyl and furyl.
• heteroaryloxy refers to the group heteroaryl-O-.
• heteroarylene refers to the diradical group derived from heteroaryl or substituted heteroaryl as defined above, and is exemplified by the groups 2,6-pyridylene, 2,4-pyridiylene, 1,2-quinolinylene, 1,8-quinolinylene, 1 ,4-benzofuranylene, 2,5- pyridinylene, 1,3-morpholinylene, 2,5-indolenyl, and the like.
• heterocycle or “heterocyclic” refers to a monoradical saturated or unsaturated group having a single ring or multiple condensed rings, from 1 to 40 carbon atoms and from 1 to 10 hetero atoms, preferably 1 to 4 heteroatoms, selected from nitrogen, sulfur, phosphorus, and/or oxygen within the ring.
• heterocyclic groups can be optionally substituted with 1 to 5, and preferably 1 to 3 substituents, selected from the group consisting of alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, aminoacyl, aminoacyloxy, oxyaminoacyl, cyano, halogen, hydroxyl, keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl
• nitrogen heterocycles and heteroaryls include, but are not limited to, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, 13
• naphthylpyridine quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, phenazine, isoxazole, phenoxazine, phenothiazine, imidazolidine, imidazoline, piperidine, piperazine, indoline, morpholino, piperidinyl, tetrahydrofuranyl, and the like as well as N-alkoxy-nitrogen containing heterocycles.
• heterocyclics includes “crown compounds” which refers to a specific class of heterocyclic compounds having one or more repeating units of the formula [- (CH 2 -) m Y-] where m is equal to or greater than 2, and Y at each separate occurrence can be O, N, S or P.
• Examples of crown compounds include [-(CH 2 ) 3 -NH-] 3 , [-((CH 2 ) 2 -O) 4 - ((CH 2 ) 2 -NH) 2 ] and the like. Typically such crown compounds can have from 4 to 10 heteroatoms and 8 to 40 carbon atoms.
• heterocyclooxy refers to the group heterocyclic-O-.
• thioheterocyclooxy refers to the group heterocyclic-S-.
• heterocyclene refers to the diradical group derived from a heterocycle as defined herein, and is exemplified by the groups 2,6-morpholino, 2,5-morpholino and the like.
• oxyacylamino refers to the group -OC(O)NRR where each R is independently hydrogen, alkyl, substituted alkyl, aryl, heteroaryl, or heterocyclic wherein alkyl, substituted alkyl, aryl, heteroaryl and heterocyclic are as defined herein.
• thiol refers to the group -SH.
• thioalkoxy refers to the group -S-alkyl.
• substituted thioalkoxy refers to the group -S-substituted alkyl.
• thioaryloxy refers to the group aryl-S- wherein the aryl group is as defined above including optionally substituted aryl groups also defined above.
• thioheteroaryloxy refers to the group heteroaryl-S- wherein the heteroaryl group is as defined above including optionally substituted aryl groups as also defined above.
• the compounds of this invention include all stereochemical isomers arising from the substitution of these compounds.
• Alkyl optionally interrupted by 1-5 atoms chosen from O, S, or N refers to alkyl as defined above in which the carbon chain is interrupted by O, S, or NR a , where R a is hydrogen, alkyl, aryl, or heteroaryl, all of which may be optionally substituted.
• ethers, sulfides, and amines for example 1-methoxydecyl, 1- pentyloxynonane, 1 -(2-isopropoxyethoxy)-4-methylnonane, 1 -(2-ethoxyethoxy)dodecyl, 2-(t-butoxy)heptyl, 1-pentylsulfanylnonane, nonylpentylamine, and the like.
• Heteroarylalkyl refers to heteroaryl as defined above linked to alkyl as defined above, for example pyrid-2-ylmethyl, 8-quinolinylpropyl, and the like.
• Ligand denotes an antibiotic compound that is a binding partner for a transglycosylase enzyme substrate and is bound thereto by complementarity.
• the specific region or regions of the ligand that is (are) recognized by the enzyme substrate is designated as the "ligand domain".
• a ligand may be either capable of binding to a transglycosylase enzyme substrate by itself, or may require
• non-lig.and components for binding e.g. Ca , Mg , or a water molecule is required for the binding of a ligand domain.
• ligands are not intended to be limited to compounds known to be useful as antibiotics (for example, known drugs).
• ligand can equally apply to a molecule that is not normally recognized as having useful antibacterial properties, in that ligands that exhibit minimally useful properties as monomers can be highly active as multibinding agents, due to the biological benefit (increased biological effect) conferred by multivalency.
• the primary requirement for a ligand as defined herein is that it has a ligand domain as defined above.
• a preferred class of ligands are heptapeptides, known as the glycopeptide antibiotics, characterized by a multi-ring peptide core, and at least one sugar attached at 15
• glycopeptide class of ligands included in this definition may be found in "Glycopeptides Classification, Occurrence, and Discovery", by Raymond C. Rao and Louise W. Crandall, ("Drugs and the Pharmaceutical Sciences”Volume 63, edited by Ramakrishnan Nagarajan, published by Marcal Dekker, Inc.), which is hereby incorporated by reference.
• Another preferred class of ligands is the general class of glycopeptides disclosed above on which the sugar moiety is absent, i.e. the aglycone series of heptapeptides.
• aglycone series of heptapeptides For example, removal of the disaccharide moiety appended to the phenol on vancomycin (as shown below as Formula II) by mild hydrolysis gives vancomycin aglycone.
• a further preferred class of ligands are glycopeptides that have been further appended with additional saccharide residues, especially aminoglycosides, in a manner similar to vancosamine.
• “Vancomycin” refers to the antibacterial compound whose structure is reproduced below as Formula II.
• Optional or “optionally” means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances in which it does not.
• optionally substituted glycopeptide with respect to a compound of Formula I refers to a ligand as defined above in which those positions that are not linked to X may or may not be substituted by various groups as defined below.
• the term also includes those instances in which one amino acid of the basic core structure is replaced by another amino acid, for example as described in "Preparation and conformational analysis of vancomycin hexapeptide and aglucovancomycin hexapeptide", by Booth, Paul M.; Williams, Dudley 17
• Optionally substituted vancomycin with respect to the multibinding agents of the invention refers to vancomycin in which the hydroxy group at any position, the [R] position, the carboxyl groups at the [C] position, or the amine groups at the [V] or [N] position that are not attached to the linker X may or may not be substituted by various groups.
• Such groups include: R a , where R a at each occurrence is chosen from alkyl, alkyl optionally interrupted by 1-5 atoms chosen from O, S, or NR D -, where R D is alkyl, aryl, or heteroaryl, all of which are optionally substituted, haloalkyl, alkenyl, alkynyl, alkylamino, alkylaminoalkyl, cycloalkyl, alkanoyl, aryl, heteroaryl, heterocyclic, additional saccharide residues, especially aminoglycosides, all of which are optionally substituted as defined above; and: NR c Rd, in which R c and Rd are independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, alkanoyl, aryl, heteroaryl, arylalkyl, or heteroarylalkyl, or R c and Rd when taken together with the nitrogen to which they are attached represent a hetero
• An example of a preferred [C] substitution is dimethylaminopropylamino and glucosamino; an example of a preferred [V] substitution is alkyl, for example n-decyl, or alkylaminoalkyl, for example n- decylaminoethyl.
• Optionally substituted vancomycin aglycone with respect to the multibinding agents of the invention refers to vancomycin aglycone in which the hydroxy group at any position, particularly the hydroxy group at the [O] position, the [R] position, the carboxyl groups at the [C] position, or the amine group at the [N] position, that are not attached to the linker X may or may not be substituted by various groups -R a as defined above.
• Transglycosylase enzyme substrate denotes the molecular target of the transpeptidase enzyme.
• the substrate binds to the enzyme and eventually results in synthesis of the bacterial cell wall.
• the action of this enzyme is inhibited by a ligand domain that binds to the enzyme substrate.
• a ligand such as vancomycin binds to this 18
• Multibinding agent or “multibinding compound” as used herein refers to a compound that is capable of multivalency as defined below, and which has 2-10 ligands, which may be the same or different, connected by one or more covalent linker or linkers, which may be the same or different, preferably from 1-20.
• a multibinding agent provides a biological and/or therapeutic effect greater than the aggregate of the unlinked ligands equivalent thereto. That is to say, an improved biological and/or therapeutic effect of the multibinding agent is obtained as measured against that achieved by the same number of unlinked ligands available for binding to the ligand binding site of the transglycosylase enzyme substrate.
• Examples of increased biological and/or therapeutic effect with respect to the target include, for example, increased specificity, increased affinity, increased selectivity, increased potency, increased efficacy, increased therapeutic index, a change in the duration of action, decreased toxicity, decreased side effects, improved bioavailability, improved pharmacokinetics, improved activity spectrum, improved ability to kill bacteria, and the like.
• the multibinding compounds of the invention will exhibit one or more of the foregoing effects.
• “Potency” as used herein refers to the minimum concentration at which a ligand is able to achieve a desirable biological or therapeutic effect.
• the potency of a ligand is typically proportional to its affinity for its ligand binding site. In some cases, the potency may be non-linearly correlated with its affinity.
• the dose- response curve of each is determined under identical test conditions (e.g., in an in vitro or in vivo assay or in an appropriate animal model, such as a human patient. The finding that the multibinding agent produces an equivalent biological or therapeutic effect at a lower concentration than the aggregate unlinked ligand (e.g., on a per weight, per mole, or per ligand basis) is indicative of enhanced potency.
• Univalency refers to a single binding interaction between the ligand domain of one ligand as defined herein with the ligand recognition site of a transglycosylase enzyme substrate as defined herein. It should be noted that a compound having multiple copies of a ligand (or ligands) exhibits univalency when only one ligand 19
• Multivalency refers to the concurrent binding of 2 to 10 linked ligands (which may be the same or different) .and two or more corresponding ligand binding sites.
• two ligands connected by a linker that bind concurrently to two ligand binding sites would be considered as a bivalent compound; similarly, three ligands thus connected provide a trivalent compound.
• Selectivity in general is a measure of the binding preferences of a ligand for different receptors and/or different ligands for the same receptor.
• the selectivity of a ligand with respect to its target receptor relative to another receptor is given by the ratio of the respective values of K d (i.e., the dissociation constants for each ligand-receptor complex), or in cases where a biological effect is observed below the K d , selectivity is given by the ratio of the respective EC 50 s (i.e. the concentrations that produce 50% of the maximum response for the ligand interacting with the two distinct 20
• ligand recognition site or "ligand binding site” as used herein denotes the site on a transglycosylase enzyme substrate that recognizes a ligand domain and provides a binding partner.
• the ligand binding site may be defined by monomeric or multimeric structures.
• inert organic solvent or “inert solvent” mean a solvent inert under the conditions of the reaction being described in conjunction therewith [including, for example, benzene, toluene, acetonitrile, tetrahydrofuran (“THF”), dimethylformamide (“DMF”), chloroform (“CHCI3”), methylene chloride (or dichloromethane or "CH2CI2), diethyl ether, ethyl acetate, acetone, methylethyl ketone, methanol, ethanol, propanol, isopropanol, tert-butanol, dioxane, pyridine, and the like].
• the solvents used in the reactions of the present invention are inert solvents.
• “Pharmaceutically acceptable salt” means those salts which retain the biological effectiveness and properties of the compounds of Formula I, and which are not biologically or otherwise undesirable.
• the compounds of Formula I are capable of forming both acid and base salts by virtue of the presence of amino and carboxyl groups respectively.
• Pharmaceutically acceptable base addition salts may be prepared from inorganic and organic bases. Salts derived from inorganic bases include, but are not limited to, the sodium, potassium, lithium, ammonium, calcium, and magnesium salts.
• Salts derived from organic bases include, but are not limited to, salts of primary, secondary and tertiary amines, substituted amines including naturally-occurring substituted amines, and cyclic amines, including isopropylamine, trimethyl amine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol, tromethamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, N-alkylglucamines, theobromine, purines, piperazine, piperidine, and N-ethylpiperidine.
• carboxylic acid derivatives would be useful in the practice of this invention, for example carboxylic acid amides, including carboxamides, lower alkyl carboxamides, di(lower alkyl) carboxamides, and the like.
• Pharmaceutically acceptable acid addition salts may be prepared from inorganic and organic acids. Salts derived from inorganic acids include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like. Salts derived from organic acids 21
• the compounds of the invention include racemic ligands as well as the individual stereoisomers of the ligands, including enantiomers and non- racemic mixtures thereof.
• treatment covers any treatment of a condition or disease in an animal, particularly a mammal, more particularly a human, and includes: (i) preventing the disease or condition from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (ii) inhibiting the disease or condition, i.e. arresting its development; (iii) relieving the disease or condition, i.e. causing regression of the condition; or. (iv) relieving the conditions caused by the disease, i.e. symptoms of the disease.
• disease state that is alleviated by treatment with an antibacterial agent is intended to cover all disease states which are acknowledged in the art to be usefully treated with an antibacterial agent in general, and those disease states which have been found to be usefully treated by the specific antibacterials of our invention, including the compounds of Formula I.
• disease states include, but are not limited to, treatment of a mammal afflicted with pathogenic bacteria, in particular staphylococci (methicillin sensitive and resistant), streptococci (penicillin sensitive and resistant), enterococci (vancomycin sensitive and resistant), and Clostridium difficile
• therapeutically effective amount refers to that amount which is sufficient to effect treatment, as defined above, when administered to a mammal in need of such treatment.
• protecting group refers to any group which when bound to one or more hydroxyl, thiol, amino or carboxyl groups of the compounds prevents reactions from occurring at these groups and which protecting group can be 22
• removable blocking group employed is not critical and preferred removable hydroxyl blocking groups include conventional substituents such as allyl, benzyl, acetyl, chloroacetyl, thiobenzyl, benzylidine, phenacyl, t-butyl-diphenylsilyl and any other group that can be introduced chemically onto a hydroxyl functionality and later selectively removed either by chemical or enzymatic methods in mild conditions compatible with the nature of the product.
• Protecting groups are disclosed in more detail in T.W. Greene and P.G.M.
• removable amino blocking groups include conventional substituents such as t-butyoxycarbonyl (t-BOC), benzyloxycarbonyl (CBZ), fluorenylmethoxycarbonyl (FMOC), allyloxycarbonyl (ALOC) and the like, which can be removed by conventional conditions compatible with the nature of the product.
• Preferred carboxyl protecting groups include esters such as methyl, ethyl, propyl, t-butyl etc. which can be removed by mild conditions compatible with the nature of the product.
• Linker or “linkers” as used herein, identified where appropriate by the symbol X, refers to a group or groups that covalently link(s) from 2-10 ligands (as defined herein) in a manner that provides a multibinding agent capable of multivalency.
• the linker is a ligand domain orienting entity that permits attachment of multiple copies of ligands (which may be the same or different) thereto. The extent to which multivalent binding is realized depends upon the efficiency with which the linker that joins the ligands permits the ligand domains to be presented to the ligand recognition sites (on enzymes and enzyme substrates).
• linker spatially constrains these interactions to occur within dimensions defined by the linker.
• structural features of the linker valency, geometry, orienting capabilities, size, flexibility, chemical composition
• linker does not include solid inert supports such as beads, resins, glass particles, rods, fibres, and the like, but it should be understood that the multibinding compounds of the invention can be attached 23
• a solid support if desired to provide, for example, a material useful for separation and purification processes (e.g. affinity chromatography).
• the ligands are covalently attached to the linker or linkers using conventional chemical techniques, for example reaction between a carboxylic acid and an amine to form an amide, an amine and a sulfonyl halide to form a sulfonamide, an alcohol or phenol with an alkyl or aryl halide to form an ether, and the like.
• the linker (or linkers) is attached to the ligand at a position such that the ligand domain is permitted to orient itself appropriately in order to bind to the ligand binding site.
• the term linker embraces everything that is not considered to be part of the ligand.
• the relative orientation in which the ligand domains are displayed derives from the particular point or points of attachment of the ligands to the linker, and on the framework geometry.
• the determination of where acceptable substitutions can be made on a ligand is typically based on prior knowledge of structure-activity relationships of the ligand and/or congeners and/or structural information about ligand-receptor complexes (e.g., from X-ray crystallography, NMR). Such positions and the synthetic methods for covalent attachment are well known in the art. Suitable linkers are discussed below.
• the multibinding agent is a bivalent compound, in which two ligands are covalently linked.
• a glycopeptide bivalent compound may be constructed by linking any hydroxyl group, carboxyl group, "resorcinol” group [R], or amino group of a first glycopeptide ligand to any hydroxyl group, carboxyl group, "resorcinol” group [R], or amino group of a second glycopeptide.
• hydroxyl group includes a hydroxyl group obtained by reducing the carboxyl group at the [C] position to a hydroxymethyl group, as well as the hydroxyl groups on the sugars and on the peptide core structure.”
• Increased biological effect includes, but is not limited to, increased affinity, increased selectivity, increased potency, increased efficacy, increased duration of action, decreased toxicity, and the like.
• the invention relates to multivalent compounds as defined above, comprising ligands that are binding partners for a transglycosylase enzyme substrate.
• the preferred ligands are optionally substituted glycopeptides, or their corresponding aglycones.
• glycopeptides are depicted in a simplified form as a shaded box that shows only the carboxyl terminus, labeled [C], the sugar amine terminus (for example, vancosamine), labeled [V], and the "non-sugar” amino terminus, labeled [N], as follows:
• R is hydrogen (as in N-desmethylvancomycin) or methyl (as in vancomycin).
• one general class of multivalent compounds that fall within the scope of the definition of Formula I include compounds in which ligands, including optionally substituted ligands, are connected by one or more linkers X at the [C-C], [V-V], [N-N], [C-V], [C-N], and [V-N] termini.
• aglycone derivatives of glycopeptides are depicted as a shaded triangle that shows only the carboxyl terminus, labeled [C], the aglycone hydroxy terminus, labeled [O], and the "non-sugar” amino terminus, labeled [N], as follows:
• R is hydrogen (as in N-desmethylvancomycin aglycone) or methyl (as in vancomycin aglycone).
• glycopeptides linked via their [C], [V], or [N] termini to aglycone derivatives of glycopeptides linked via their [C], [O], or [N] termini.
• a third class of compounds falling within the scope of the invention are those in which the glycopeptides, or aglycone derivatives thereof, are linked via the [R] position.
• Reaction schemes that exemplify this linking strategy depict the ligands in a simplified form as above, i.e. as a shaded box in which the carboxyl terminus is labeled [C], the vancosamine amino terminus is labeled [V], and the "non-sugar” amino terminus is labeled [N], with the addition of the [R] position as a resorcinol derivative, as below:
• R is hydrogen or methyl
• glycopeptides are generally depicted in a simplified form as a shaded box that shows only the glycopeptide carboxyl terminus, labeled [C], the sugar amine (vancosamine in vancomycin) terminus, labeled [V], and the "non-sugar” amino terminus, labeled [N].
• the preferred compounds of the invention are those represented by compounds of Formula I, (L) p X q , in which p is 2 and q is 1.
• the following nomenclature system will be used for naming the compounds of the invention and substituted ligands used in their preparation, using vancomycin as an example (Formula II), but it should be understood that all compounds of the invention may be named following these principles.
• glycopeptides are within the scope of the definition of "ligand", and the point of substitution will be indicated as follows.
• vancomycin substituted at the [C] terminus that has the formula:
• R represents two glycopeptides (vancomycin in this example) linked by a 1,8-diaminooctane group at the [C] positions of the glycopeptide. It will be named as:
• [C-C]-octane- 1 ,8-diamino-bis(vancomycin) which indicates that two unsubstituted vancomycin molecules are linked by -NH-(CH 2 ) 8 .NH-, each NH groups being attached to the carboxy termini of each vancomycin.
• the linkage at the [C] terminus of the glycopeptides is an amide, the name indicates the linker only, i.e. it is named as a diamine, not a diamide.
• [V-V]-heptan-l,7-dioyl-bis-(vancomycin) indicates that two unsubstituted vancomycin molecules are linked by -C(O)-(CH 2 ) 5 -C(O)-, both C(O) groups being attached to the vancosamine (V) termini of each vancomycin, i.e. a 27
• [N-N] indicates linking of two vancomycin molecules by the methylamino termini
• [R-R] indicates linking of two vancomycin molecules by the "resorcinol” termini
• [O-O] indicates that two aglycone molecules are linked by the 28
• both C(O) termini being attached to the vancosamine (V) termini of each vancomycin.
• both vancomycins are not identically substituted, for example a compound in which the first ligand is [C]-dimethylamino-vancomycin, and a second ligand is [C]-butylamino vancomycin), linked by
• L are both vancomycin linked via their carbon [C] termini by: -NH-(CH 2 ) 2 NHC(O)(CH 2 ) 3 C(O)NH(CH 2 ) 2 -NH- is named as :
• a compound of Formula I wherein p is 2 and the first ligand L is vancomycin linked via its carbon [C] terminus and the second ligand L is vancomycin linked via its methylamino [N] terminus by:
• the multibinding agents of the invention including the compounds of Formula I, and their pharmaceutically acceptable salts, are useful in medical treatments and exhibit biological activity, in particular antibacterial activity, which can be demonstrated in the tests described in the Examples. Such tests are well known to those skilled in the art, and are referenced and described in the fourth edition of "Antibiotics in Laboratory Medicine", by Victor Lorian, M.D., published by Williams and Wilkins, which is hereby inco ⁇ orated by reference.
• Those compounds of the invention including the compounds of Formula I, and their pharmaceutically acceptable salts that are active when given orally can be formulated as liquids for example syrups, suspensions or emulsions, tablets, capsules and lozenges.
• a liquid composition will generally consist of a suspension or solution of the compound or pharmaceutically acceptable salt in a suitable liquid carrier(s), for example ethanol, glycerine, sorbitol, non-aqueous solvent such as polyethylene glycol, oils or water, with a suspending agent, preservative, surfactant, wetting agent, flavoring or coloring agent.
• a liquid formulation can be prepared from a reconstitutable powder.
• a powder containing active compound, suspending agent, sucrose and a sweetener can be reconstituted with water to form a suspension; and a syrup can be prepared from a powder containing active ingredient, sucrose and a sweetener.
• a composition in the form of a tablet can be prepared using any suitable pharmaceutical carrier(s) routinely used for preparing solid compositions. Examples of such carriers include magnesium stearate, starch, lactose, sucrose, microcrystalline cellulose and 31
• binders for example polyvinylpyrrolidone.
• the tablet can also be provided with a color film coating, or color included as part of the carrier(s).
• active compound can be formulated in a controlled release dosage form as a tablet comprising a hydrophilic or hydrophobic matrix.
• a composition in the form of a capsule can be prepared using routine encapsulation procedures, for example by inco ⁇ oration of active compound and excipients into a hard gelatin capsule.
• a semi-solid matrix of active compound and high molecular weight polyethylene glycol can be prepared and filled into a hard gelatin capsule; or a solution of active compound in polyethylene glycol or a suspension in edible oil, for example liquid paraffin or fractionated coconut oil can be prepared and filled into a soft gelatin capsule.
• the compounds of the invention including the compounds of Formula I, and their pharmaceutically acceptable salts that are active when given parenterally can be formulated for intramuscular, intrathecal, or intravenous administration.
• a typical composition for intra-muscular or intrathecal administration will consist of a suspension or solution of active ingredient in an oil, for example arachis oil or sesame oil.
• a typical composition for intravenous or intrathecal administration will consist of a sterile isotonic aqueous solution containing, for example active ingredient and dextrose or sodium chloride, or a mixture of dextrose and sodium chloride.
• lactated Ringer's injection lactated Ringer's injection
• lactated Ringer's plus dextrose injection Normosol-M and dextrose
• Isolyte E acylated Ringer's injection
• a co-solvent for example polyethylene glycol
• a chelating agent for example ethylenediamine tetracetic acid
• an anti-oxidant for example, sodium metabisulphite
• the solution can be freeze dried and then reconstituted with a suitable solvent just prior to administration.
• the compounds of the invention including the compounds of Formula I, and their pharmaceutically acceptable salts which are active on rectal administration can be formulated as suppositories.
• a typical suppository formulation will generally consist of active ingredient with a binding and/or lubricating agent such as a gelatin or cocoa butter or other low melting vegetable or synthetic wax or fat.
• compositions include, for example, a backing, active compound reservoir, a control membrane, liner and contact adhesive.
• the typical daily dose of a compound of Formula I varies according to individual needs, the condition to be treated and with the route of administration. Suitable doses are in the general range of from 0.01-100 mg/kg/day, preferably 0.1-50 mg/kg/day. For an average 70 kg human, this would amount to 0.7mg to 7g per day, or preferably 7mg to 3.5g per day.
• linker when covalently attached to multiple copies of the ligands, provides a biocompatible, substantially non-immunogenic multibinding agent.
• the biological effects of the multibinding agent are highly sensitive to the valency, geometry, composition, size, flexibility or rigidity, the presence or absence of anionic or cationic charge, and similar considerations (including hydrophilicity and hydrophobicity as discussed below) with respect to the linker. Accordingly, the linker is preferably chosen to maximize the desired biological effect.
• the linker may be biologically "neutral", i.e. not itself contribute any biological activity to the compound of Formula I, or it may be chosen to enhance the biological effect of the molecule.
• the linker may be chosen from any organic molecule that orients two or more ligands to the receptors (enzymes or enzyme substrates), and permits multivalency.
• the linker can be considered as a "framework" on which the ligands are arranged in order to bring about the desired ligand-orienting result, and thus produce a multibinding agent.
• different orientations can be achieved by including in the framework groups containing monocyclic or polycyclic groups, including aryl and heteroaryl groups, or structures inco ⁇ orating one or more carbon-carbon multiple bonds (i.e., alkenes and alkynes).
• Other groups can also include oligomers and polymers which 33
• ring is branched- or straight-chain species.
• rigidity is imparted by the presence of cyclic groups (e.g., aryl, heteroaryl, cycloalkyl, heterocycles, etc.).
• the ring is a six-or ten membered ring.
• the ring is an aromatic group such as, for example, phenyl or naphthyl.
• frameworks can be designed to provide preferred orientations of the ligands.
• Such frameworks may be represented by using an array of dots (as shown below) wherein each dot may potentially be an atom, such as C, O, N, S, P, H, F, Cl, Br, and F, or the dot may alternatively indicate the absence of an atom at that position.
• the framework is illustrated as a two dimensional array in the following diagram, although clearly the framework is a three dimensional array in practice:
• Each dot is either an atom, chosen from carbon, hydrogen, oxygen, nitrogen, sulfur, phosphorus, or halogen, or the dot represents a point in space (i.e. an absence of an atom). Only certain atoms on the grid have the ability to act as an attachment point for the ligands, namely C, O, N, S, and P.
• Atoms can be connected to each other via bonds (single, double, or triple with acceptable resonance and tautomeric forms), with regard to the usual constraints of chemical bonding.
• Ligands may be attached to the framework via single, double, or 34
• ligand groups (2 to 10) can be attached to the framework such that the minimal, shortest path distance between adjacent ligand groups does not exceed 100 atoms or 40 angstroms.
• the intersection of the framework (linker) and the ligand group, and indeed, the framework (linker) itself can have many different bonding patterns. Examples of acceptable patterns of three contiguous atom arrangements within the linker and at the linker-ligand interface are shown in the following diagram.
• a bivalent compound can comprise ligands (represented as L) 36
• core structures other than those shown here can be used for determining the optimal framework display orientation of the ligands.
• the process may require the use of multiple copies of the same central core structure or combinations of different types of display cores.
• linkers include aliphatic moieties, aromatic moieties, steroidal moieties, peptides, and the like. Specific examples are peptides or polyamides, hydrocarbons, aromatic groups, ethers, lipids, cationic or anionic groups, or a combination thereof, and many specific examples of linkers are shown below. However, it should be understood that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention.
• properties of the linker can be modified by the addition or insertion of ancillary groups into the linker, for example, to change solubility of the multibinding agent (in water, fats, lipids, biological fluids, etc.), hydrophobicity, hydrophilicity, linker flexibility, antigenicity, molecular size, molecular weight, in vivo half-life, in vivo distribution, biocompatability, immunogenicity, stability, and the like.
• the introduction of one or more poly or preferably oligo(ethylene glycol) (PEG) groups onto the linker enhances hydrophilicity and water solubility of the multibinding agent, increases both molecular weight and molecular size and, depending on the nature of the unPEGylated linker, may increase the in vivo retention time. Further, PEG may decrease antigenicity and potentially enhances the overall rigidity of the linker.
• ancillary groups that enhance the water solubility/hydrophilicity of the linker are useful in practicing the present invention.
• ancillary groups such as, for example, poly(ethylene glycol), alcohols, polyols (e.g., glycerin, glycerol propoxylate, saccharides, including mono-, oligo- and polysaccharides, etc.), carboxylates, polycarboxylates (e.g., polyglutamic acid, polyacrylic acid, etc.), amines, polyamines (e.g., polylysine, poly(ethyleneimine), etc) to enhance the water solubility and or hydrophilicity of the compounds of Formula I.
• the ancillary group used to improve water solubility/hydrophilicity will be a polyether.
• the ancillary group will be a poly(ethylene glycol).
• lipophilic ancillary groups within the structure of the linker to enhance the lipophilicity and/or hydrophobicity of the compounds of Formula I is within the scope of the present invention.
• Lipophilic groups of use in practicing the instant invention include, but are not limited to, aryl and heteroaryl groups.
• the aromatic groups may be either unsubstituted or substituted with other groups, but are at least substituted with a group which allows their covalent attachment to the linker.
• Other lipophilic groups of use in practicing the instant invention include fatty acid derivatives which do not form bilayers in aqueous medium until higher concentrations are reached.
• lipid refers to any fatty acid derivative that is capable of forming a bilayer or micelle such that a hydrophobic portion of the lipid material orients toward the bilayer while a hydrophilic portion orients toward the aqueous phase. Hydrophilic characteristics derive from the presence of phosphato, carboxylic, sulfato, amino, sulfhydryl, nitro, and other like groups.
• Hydrophobicity could be conferred by the inclusion of groups that include, but are not limited to, long chain saturated and unsaturated aliphatic hydrocarbon groups of up to 20 carbon atoms and such groups substituted by one or more aryl, heteroaryl, cycloalkyl and/or heterocyclic group(s).
• Preferred lipids are phosphoglycerides and sphingolipids, representative examples of which include phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoyl phosphatidylcholine, lysophosphatidylcholine, lysophosphatidyl-ethanolamine, dipalmitoylphosphatidylcholine, 39
• dioleoylphosphatidylcholine distearoyl-phosphatidylcholine or dilinoleoylphosphatidylcholine could be used.
• Other compounds lacking phosphorus, such as sphingolipid and glycosphingolipid families are also within the group designated as lipid.
• the amphipathic lipids described above may be mixed with other lipids including triglycerides and sterols.
• the flexibility of the linker can be reduced by the inclusion of ancillary groups which are bulky and/or rigid.
• ancillary groups which are bulky and/or rigid.
• the presence of bulky or rigid groups can hinder free rotation about bonds in the linker or bonds between the linker and the ancillary group(s) or bonds between the linker and the functional groups.
• Rigid groups can include, for example, those groups whose conformational lability is restrained by the presence of rings and/or multiple bonds, for example, aryl, heteroaryl, cycloalkyl, and/or heterocyclic.
• Other groups which can impart rigidity include polymeric groups such as oligo- or polyproline chains.
• Rigidity can also be imparted electrostatically.
• the ancillary groups are either negatively or positively charged, the similarly charged ancillary groups will force the presenter linker into a configuration affording the maximum distance between each of the like charges.
• the energetic cost of bringing the like-charged groups closer to each other will tend to hold the linker in a configuration that maintains the separation between the like- charged ancillary groups.
• ancillary groups bearing opposite charges will tend to be attracted to their oppositely charged counte ⁇ arts and potentially may enter into both inter- and intramolecular ionic bonds. This non-covalent bonding mechanism will tend to hold the linker into a conformation which allows bonding between the oppositely charged groups.
• Rigidity may also be imparted by internal hydrogen bonding, or by hydrophobic collapse.
• Bulky groups can include, for example, large atoms and/or ions (e.g., iodine, sulfur, metal ions, etc.) groups containing large atoms, polycyclic groups, including aromatic groups, non-aromatic groups and structures inco ⁇ orating one or more carbon-carbon multiple bonds (i.e., alkenes and alkynes). Bulky groups can also include oligomers and polymers which are branched- or straight-chain species. Species that are branched are 40 expected to increase the rigidity of the structure more per unit molecular weight gain than are straight-chain species.
• ions e.g., iodine, sulfur, metal ions, etc.
• Bulky groups can also include oligomers and polymers which are branched- or straight-chain species. Species that are branched are 40 expected to increase the rigidity of the structure more per unit molecular weight gain than are straight-chain species.
• Eliminating or reducing antigenicity of the compounds of Formula I by judicious choice of ancillary group(s) is within the scope of the present invention.
• the antigenicity of a compound of Formula I may be reduced or eliminated by the use of groups such as, for example, poly(ethylene glycol).
• the multibinding agents of the invention comprise 2-10 ligands attached to a linker that connects the ligands in such a manner that they are presented to the enzyme multivalent receptors for multivalent interactions with the appropriate receptors (ligand binding site).
• the linker spatially constrains these interactions to occur within dimensions defined by the linker, thus increasing the biological effect of the multibinding agent as compared to the same number of individual units of the ligand.
• the multivalent compounds of the invention are represented by the empirical formula (L) p (X) q .
• L the empirical formula
• X the ligands can be linked together in order to achieve the objective of multivalency, and a more detailed explanation is given below.
• the linker can be considered as a framework, and it should be understood that the ligands can be attached to this framework at any intermediate point on the framework, and/or on the termini of the framework.
• the linker is a linear chain
• a bivalent compound can be constructed by attaching two ligands at the two ends of the linear chain, or alternatively attaching two ligands at some intermediate atom along the chain.
• the simplest (and preferred) multibinding agent is a bivalent compound, which can be represented as L-X-L, where L is a ligand and is the same or different, and X is the linker.
• the linker X can be linear or cyclic, or a combination of both linear and cyclic constructs, and that the two ligands may be located at the termini of the linker or may be attached at some intermediate attachment point.
• a trivalent compound which can also be represented in a linear fashion, i.e. as a sequence of repeated units L-X-L-X-L, in which L is a ligand and is the same or 41
• X or a compound comprising three ligands attached to a central core, and thus represented as (L) 3 X, where the linker X could include, for example, an aryl or cycloalkyl group.
• a tetravalent compound could be represented as L-X-L-X-L-X-L, or L-X-L-X-L
• L i.e. a branched construct analogous to the isomers of butane (n-butyl, sec-butyl, tert- butyl). Alternatively, it could be represented as an aryl or cycloalkyl derivative as above with four ligands attached to the core linker.
• the same principles apply to the higher multibinding agents, e.g. pentavalent to decavalent compounds.
• the preferred linker length will vary depending upon the distance between adjacent ligand recognition sites, and the geometry, flexibility and composition of the linker.
• the length of the linker will preferably be in the range of about 2-100
• Angstroms more preferably about 2-50 Angstroms, and even more preferably about 5-20 Angstroms.
• m is an integer of 0-20;
• X' at each separate occurrence is -0-, -S-, -S(O)-, -S(0)2-, -NR- (where R is as defined below),
• Z at each separate occurrence is alkylene, cycloalkylene, alkenylene, alkynylene, arylene, heteroarylene, or a covalent bond;
• n 0, 1 or 2;
• R, R' and R" at each separate occurrence are chosen from hydrogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl, and heterocyclo.
• linker moiety can be optionally substituted at any atom in the chain by alkyl, cycloalkyl, alkenyl, alkynyl, alkoxy, halo, nitro, aryl, heteroaryl, or heterocyclo. 43
• the preferred compounds of the invention are those in which p is 2 and q is 1, and for the sake of simplicity the following description is directed toward the preparation of such compounds. However, it should be understood that the same synthetic principles can be applied to all compounds within the scope of the invention.
• shaded circle in the following Figures and Reaction Schemes represents, for the sake of simplicity, the "core" of a linker. That is to say, a shaded circle showing two attached amino grouips (formula (2)) represents any diamino compound that can be used in a coupling reaction, for example 1,6- diaminohexane, l,2-bis-(aminomethyl)phenyl, l,4-bis-(aminomethyl)cyclohexane, and the like.
• a complete list of commercially available diamines are available on the ACD catalogue.
• FIGURE 1 A first figure.
• Figure 2 shows reaction of an amine, for example the [V] or [N] terminus, with a carboxylic acid or dicarboxylic acid to form an amide or diamide.
• the amine can also be reacted to produce a protected intermediate with a second amino group or carboxy functional group, which can be reacted further with a dicarboxylic acid or diamine in order to produce a compound of the invention.
• A— i ' ⁇ PG A— N ⁇ OH H H H where A represents a ligand or substituted ligand to which an amino group is attached, PG is a protecting group, and the shaded circle represents a linker "core”.
• an amine for example the [V] or [N] termini
• a reductive alkylation reaction with an aldehyde to form an alkyl substituent, or reacted with a dialdehyde under reductive alkylation conditions to give a compound of the invention.
• Reaction with appropriately protected aldehydes or carboxylic acids gives an intermediates with an amino or carboxy functional groups (Reactions L and M), the products of which can be used to react further with a carboxylic acid or amine respectively to form an amide.
• A represents a ligand or substituted ligand to which an amino group acid is attached
• PG is a protecting group
• the shaded circle represents a linker "core”.
• Figure 4 similarly shows the derivatization of a hydroxy group, for example the aglycone hydroxy group designated as [O].
• A represents a ligand or substituted ligand to which a hydroxy group is attached
• PG is a protecting group
• the shaded circle represents a linker "core”.
• Figure 5 shows the preparation of unsymmetrical compounds of the invention, by reaction of those products described above having amino and carboxyl functional groups
• diamines of Formula (19) may be prepared as shown below in Reaction Scheme 1.
• PG is a protecting group, preferably t-butyl carbamate, and the shaded circle represents a linker core
• step 1 about two molar equivalents of an omega-amino carbamic acid ester [formula (3)] reacted with about one molar equivalent of a dicarboxylic acid halide, preferably chloride, of formula (17).
• the reaction is conducted in the presence of a hindered base, preferably diisopropylethylamine, in an inert solvent, preferably methylene chloride, at a temperature of about 0-5°C.
• a hindered base preferably diisopropylethylamine
• an inert solvent preferably methylene chloride
• step 2 the carbamate protecting group is hydrolyzed under acid conditions.
• a preferred acid is trifluoroacetic acid.
• the reaction is conducted in an inert solvent, preferably methylene chloride, at about room temperature.
• an inert solvent preferably methylene chloride
• linking groups used as coupling reagents in the figures above are either commercially available or are prepared by methods well known in the art. Several examples of such a preparation are shown below in Reaction Scheme 2-3.
• Reaction Scheme 2 shows the preparation of an aminoaldehyde from the corresponding aminoalcohol.
• PG is a protecting group, preferably 9-fluorenylmethoxycarbonyl.
• Illustrated in Reaction Scheme 2 is a method for preparing a protected- aminoaldehyde (7) from the corresponding aminoalcohol (20).
• the aminoalcohol is protected by conventional technique, for example, by treatment with 9-fluorenylmethyl chloroformate in the presence of base, to yield F-moc protected aminoalcohol (21).
• R is hydrogen, alkyl, aryl, heteroaryl, arylalkyl, or
• R is alkyl, arylalkyl, or heteroarylalkyl, ,
• Reaction Scheme 4 shows one method by which a [C-C] bivalent compound can be made. Also produced is a monomer of formula (22).
• a glycopeptide for example vancomycin
• a diamine of general formula (2) for example, a diamine of formula (19)
• a hindered base is employed, preferably diisopropylethylamine, in the presence of benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) and 1-hydroxybenzotriazole.
• the reaction is conducted in an inert polar solvent, for example N,N-dimethylformamide (DMF) or dimethyl sulfoxide (DMSO), or preferably a mixture of both, at about room temperature.
• DMF N,N-dimethylformamide
• DMSO dimethyl sulfoxide
• the compound of Formula I is isolated and purified by conventional means, preferably purified by reverse-phase HPLC. Also isolated is the monoadduct, a compound of formula (22).
• an intermediate of formula (22) may be prepared as shown below in Reaction Scheme 5, and used to prepare a [C-C] compound of Formula I.
• R is hydrogen or methyl
• PG represents a protecting group, preferably 9- fluorenylmethoxycarbonyl.
• step 1 a glycopeptide, for example vancomycin, is reacted with about 1.1 molar equivalents of a carbamic ester terminated by an alkylamino group [formula (3)].
• the ester moiety is chosen for ease of removal under mild conditions in subsequent reactions, and is preferably 9-fluorenylmethyl.
• Conventional amide coupling conditions are employed, preferably using PyBOP and 1- hydroxybenzotriazole.
• the reaction is conducted in the presence of a hindered base, preferably diisopropylethylamine, in an inert polar solvent, preferably DMF or DMSO, preferably a mixture of both, at about room temperature.
• a hindered base preferably diisopropylethylamine
• an inert polar solvent preferably DMF or DMSO
• step 2 the compound of formula (21) is reacted with a mild base to remove the protecting ester group, which also affords decarboxylation.
• the base is preferably piperidine, and the reaction is conducted in an inert polar solvent, preferably dimethylformamide, at about room temperature for about 10 minutes to one hour.
• the compound of formula (22) is isolated and purified by conventional means, preferably using reverse-phase HPLC.
• the intermediate of formula (22) may then be converted into a [C-C] glycopeptide bivalent compound.
• step 3 the compound of formula (22) is reacted with a dicarboxylic acid.
• a dicarboxylic acid In general, about 3 molar equivalents of the compound of formula (22) is reacted with about 1 molar equivalent of the dicarboxylic acid of formula (6), under conventional amide coupling conditions.
• a hindered base is employed, preferably diisopropylethylamine, in the presence of PyBOP and 1- 53
• reaction is conducted in an inert polar solvent, preferably
• [C-C] compounds of the invention can also be prepared by bis reductive alkylation of
• Reaction Scheme 5 shows unsubstituted glycopeptides as the ligand for reaction, the same reaction is possible starting with a glycopeptide substituted (or protected) at the [V] position. The [V] amino group can then be further modified in later steps if desired.
• R is hydrogen or methyl
• PG represents a protecting group, preferably 9- flfluuoorreennyyllmmeetthhooxxyyccaarrbboonnyyll,, aanndd RR iiss aallkkyyll., aryl, heteroaryl, arylalkyl, or heteroarylalkyl, all of which may be optionally substituted.
• step 1 the amino sugar moiety of the glycopeptide, for example the vancosamine portion of vancomycin, is reacted with a protected amino-aldehyde of formula (7) to form a Schiff s base.
• the ester moiety is chosen for ease of removal under mild conditions in subsequent reactions, and is preferably 9-fluorenylmethoxycarbonyl (Fmoc).
• the reaction is conducted in an inert polar solvent, preferably N,N-dimethylformamide, in the presence of a hindered base, preferably diisopropylethylamine, at about 0-50°C, preferably about 25°C, for 55
• the Schiff s base is further reacted with a mild reducing agent.
• a protic solvent is added, preferably methanol, followed by the reducing agent, preferably sodium cyanoborohydride, and then trifluoroacetic acid .
• the reaction is conducted at about 0-50°C, preferably about 25°C, for about 1 hour.
• the compound of formula (25) is isolated and purified by conventional means, preferably purified by reverse-phase HPLC. It should be noted that other intermediates can be used in place of the compound of formula (7), for example a protected acid (to give an amide), or an alkyl or aryl group can be appended directly on the [V] amine.
• the intermediate of formula (25) may then be converted into a [C-C] vancomycin bivalent compound in which the [V] position is also substituted.
• a hindered base is used, preferably diisopropylethylamine, in the presence of
• step 3 the compound of formula (26) is deprotected conventionally as shown above, for example in Reaction Scheme 5, step 2, to form a compound of formula (27).
• step 4 the compound of formula (27) is reacted with a compound of formula R 5 -CO 2 H or R 5 -COCl, in which R 5 is alkyl, aryl, 56
• conventional amide coupling conditions are employed.
• a hindered base is used, preferably diisopropylethylamine, in the presence of PyBOP and 1- hydroxybenzotriazole.
• the reaction is conducted in an inert polar solvent, preferably DMF, at about room temperature for about 1-3 hours.
• an inert polar solvent preferably DMF
• step 4 can be any reaction that gives a substituted derivative of the amine group; for example, reductive alkylation, alkylation, and the like.
• R is hydrogen or methyl
• R is hydrogen or methyl
• PG represents a protecting group, preferably 9- fluorenylmethoxycarbonyl
• R is, alkyl, aminoalkyl, alkylaminoalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl, or a quarternary salt derivative.
• step 1 the carboxyl function of the compound of formula (25) is reacted with an amine of formula (1).
• an amine of formula (1) In general, about 1 molar equivalent of the compound of formula (25) is reacted with about 1.5 molar equivalents of (1) under conventional amide coupling conditions.
• a hindered base is employed, preferably diisopropylethylamine, in the presence of PyBOP and 1- hydroxybenzotriazole.
• the reaction is conducted in an inert polar solvent, preferably 58
• the compound of formula (31) is isolated and purified by conventional means, preferably by reverse-phase HPLC.
• step 2 the compound of formula (31) is deprotected conventionally as shown above, for example in Reaction Scheme 5, step 2, to form a compound of formula (32).
• the intermediate of formula (32) is then converted into a [V-V] glycopeptide bivalent compound in which the [C] position is also substituted, as shown below.
• step 3 the compound of formula (32) is reacted with a dicarboxylic acid (6).
• a dicarboxylic acid (6) In general, about 3 molar equivalents of the compound of formula (32) is reacted with about 1 molar equivalent of the dicarboxylic acid of formula (6), under conventional amide coupling conditions.
• a hindered base is employed, preferably diisopropylethylamine, in the presence of PyBOP and 1 -hydroxybenzotriazole.
• the reaction is conducted in an inert polar solvent, preferably DMF, at about room temperature for about 1-3 hours.
• the compound of formula (25) can be reacted directly with a "preactivated" dicarboxylic acid of formula (6), as described below.
• R is hydrogen or methyl
• PG represents a protecting group, preferably 9- fluorenylmethyl
• R R are independently alkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl, all of which may be optionally substituted.
• the compound of formula (32) is reacted with a monoester of a dicarboxylic acid (8); preferably the monoester is one easily hydrolyzed under mild conditions, for example a 9-fluorenylmethyl ester.
• a monoester of a dicarboxylic acid (8) preferably the monoester is one easily hydrolyzed under mild conditions, for example a 9-fluorenylmethyl ester.
• about one molar equivalent of (32) is reacted with one molar equivalent of (8).
• the reaction is carried out under conventional amide coupling conditions.
• a hindered base is employed, preferably diisopropylethylamine, in the presence of PyBOP and 1 -hydroxybenzotriazole.
• the reaction is conducted in an inert polar solvent, preferably DMF, at about room temperature for about 1-3 hours, preferably about 80 minutes.
• an inert polar solvent preferably DMF
• the desired product (35) is isolated by conventional means, and reacted with a mild base, preferably piperidine, to remove the protecting group.
• the reaction is conducted in an inert polar solvent, preferably DMF, at 60
• the compound of formula (36) is isolated and purified by conventional means, preferably purified by reverse-phase HPLC.
• the intermediate of formula (36) is then converted into a [V-V] bivalent compound wherein the [C] -position is substituted on both vancomycin subunits by reaction with a compound of formula (32') [not necessarily the same as starting material (32)].
• step 3 the compound of formula (36) is reacted with a compound of formula (32').
• a hindered base is employed, preferably diisopropylethylamine, in the presence of PyBOP and 1 -hydroxybenzotriazole.
• the reaction is conducted in an inert polar solvent, preferably DMF, at about room temperature for about 30 minutes to 3 hours, preferably about 1 hour.
• the compound of Formula I is isolated and purified by conventional means, preferably purified by reverse-phase HPLC.
• the two glycopeptides can be the same or different (or the same but differently substituted), and the linking groups, although always represented by a shaded circle, can be the same or different at each occurrence.
• R is hydrogen or methyl
• PG represents a protecting group, preferably 9- fluorenylmethoxycarbonyl
• R is, alkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl, or a quarternary salt derivative.
• step 1 a glycopeptide, for example vancomycin, is reacted with a protected aminoaldehyde under conditions sufficient to direct the aldehyde to the [N]-position.
• the Schiff s base thus formed is reduced in the same manner as shown in Reaction Scheme 6, step 1, to form a compound of formula (41). 62
• step 2 the compound of formula (41) is reacted with an amine (1) in a coupling reaction in the same manner as shown above, for example in Reaction Scheme 8, step 1, to form an amide of formula (42).
• step 3 the compound of formula (42) is deprotected conventionally as shown above, for example in Reaction Scheme 5, step 2, to form a compound of formula (43).
• step 4 the compound of formula (43) is reacted with a dicarboxylic acid in the same manner as shown above, for example in Reaction Scheme 8, step 3, to give a compound of Formula I which is isolated and purified by conventional means, preferably by reverse-phase HPLC.
• the invention also encompasses aglycone derivatives of glycopeptides, for example vancomycin lacking the amino sugar moiety.
• aglycone derivatives of glycopeptides for example vancomycin lacking the amino sugar moiety.
• Such a compound has a hydroxy group as a point of attachment in place of the [V] amino group of the amino sugar.
• agly cones are represented as described above, i.e. they are depicted as a shaded triangle that shows only the carboxyl terminus, the aglycone hydroxy terminus, and the amino terminus.
• Reaction Scheme 10 shows the preparation of an aglycone bivalent compound from a glycopeptide.
• the glycopeptide is initially substituted at the [C] position with an amide, followed by hydrolysis of the aminosugar, and finally coupling of the phenol thus produced.
• R is hydrogen or methyl, and R is alkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl, all of which may be optionally substituted
• step 1 a glycopeptide, for example vancomycin, is is reacted with an amine (1) in a coupling reaction in the same manner as shown above, for example in Reaction Scheme 8, step 1, to form an amide of formula (46), which is isolated and purified by conventional means.
• a glycopeptide for example vancomycin
• step 2 the glycopeptide of formula (46) is hydrolysed with a strong acid, preferably at about 50°C, to produce an aglycone of formula (47), which is isolated and purified by conventional means.
• step 3 two equivalents of the compound of formula (47) are reacted with a dihalo compound of formula (10) to give a compound of Formula I [O-O].
• the reaction is carried out in a polar solvent, preferably DMF, in the 64
• the compound of Formula I is isolated and purified by conventional means.
• the compound of formula (47) may be first reacted with a protected haloamine, for example tert-butyl N-(2- bromoethyl)carbamate, to give an aminoether of formula (51), which is then deprotected and reacted further with a dicarboxylic acid to give a compound of Formula I.
• a protected haloamine for example tert-butyl N-(2- bromoethyl)carbamate
• R is hydrogen or methyl
• PG represents a protecting group, preferably BOC, aanndd wwhheerree RR iiss aallkkyyll,, aryl, heteroaryl, arylalkyl, heteroarylalkyl, all of which may be optionally substituted
• step 1 .an aglycone of formula (47) is reacted with a protected haloamine, for example tert-butyl N-(2-bromoethyl)carbamate, under conventional coupling conditions, preferably using potassium carbonate as a base in an inert polar solvent, preferably DMF or DMSO, at about room temperature.
• a protected haloamine for example tert-butyl N-(2-bromoethyl)carbamate
• an inert polar solvent preferably DMF or DMSO
• step 2 the protecting group (carbamate) is hydrolyzed under acidic conditions.
• a preferred acid is trifluoroacetic acid, and the reaction is conducted in an inert solvent, at about room temperature.
• the compound of formula (52) is isolated and purified by conventional means.
• step 3 two equivalents of the compound of formula (52) are reacted with a dicarboxylic acid compound of formula (6) to give a compound of Formula I [O-O].
• the reaction is carried out under conventional amide coupling conditions as detailed above, for example, as in Reaction Scheme 8, step 3, to give a compound of Formula I
• the compound of formula (47) may be first reacted with a haloester, for example t-butylbromoacetate, to give a carboxy- substituted ether, which is deprotected and then reacted further with a diamine to give a compound of Formula I.
• a haloester for example t-butylbromoacetate
• R is hydrogen or methyl
• PG represents a protecting group
• R is alkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl, all of which may be optionally substituted
• step 1 an aglycone of formula (47) is reacted with a haloester under conventional coupling conditions, preferably using potassium carbonate as a base in an inert polar solvent, preferably DMF or DMSO, at about room temperature.
• an inert polar solvent preferably DMF or DMSO
• step 2 the protecting group (carbamate) is hydrolyzed under acidic conditions.
• a preferred acid is trifluoroacetic acid, and the reaction is conducted in an inert solvent, at about room temperature.
• step 3 two equivalents of the compound of formula (57) are reacted with a diamino compound of formula (2) to give a compoimd of Formula I [O-O].
• the reaction is carried out under conventional amide coupling conditions as detailed above.
• a hindered base is employed, preferably diisopropylethylamine, in the presence of PyBOP and 1 -hydroxybenzotriazole.
• the reaction is conducted in an inert polar solvent, preferably DMF, at about room temperature for about 30 minutes to 5 hours, preferably about 2 hours.
• the compound of Formula I is isolated and purified by conventional means, preferably purified by reverse-phase HPLC.
• glycopeptides A new type of chemical modification of glycopeptides is described in a recent publication from The Institute of New Antibiotics, Russian Academy of Medical
• reaction disclosed therein utilizes the Mannich reaction to derivatize the glycopeptide at the position between the two meta hydroxy groups on the "resorcinol" group (identified above in Structure II as the [R] position). This type of reaction can be used to prepare compounds of the invention, as shown below in Reaction Scheme 14.
• a glycopeptide for example vancomycin
• a glycopeptide is linked at the [R] position.
• about two molar equivalents of a glycopeptide, for example vancomycin are reacted with about two molar equivalents of formalin and one molar equivalent of diamine (2).
• the reaction is conducted in an inert polar solvent, preferably aqueous acetonitrile, for about 18 hours.
• an inert polar solvent preferably aqueous acetonitrile
• Secondary amines can be used in place of the primary amines of the linker of formula (2) if desired, and the [C] position can be substituted, for example by NHR 1 , as shown previously if preferred.
• the Mannich reaction can be used to introduce an amino sidechain or an acid sidechain at the [R] position (for example, as shown for the compound of formula (82)), which can then be further reacted with an appropriate linker (a diacid,dialdehyde, dihalo compound) as shown above to provide a compound of Formula I.
• an appropriate linker a diacid,dialdehyde, dihalo compound
• R is hydrogen or methyl
• PG represents a protecting group, preferably 9- fluorenylmethoxycarbonyl
• R 9 is, alkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl, or a quarternary salt derivative.
• the compounds of [C-V] Formula I are prepared by the procedures of Reaction Scheme 15 using reactions described previously.
• the [C] carboxy group of the compound of formula (25) is preferably "preactivated” before reaction with the compound of formula (60), because (60) also contains a free carboxy group.
• R is hydrogen or methyl
• PG represents a protecting group, preferably 9- fluorenylmethoxycarbonyl
• R is, alkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl, or a quarternary salt derivative.
• the compound of formula (32) is reacted with .an N-protected aminoacid (7) in the same manner as shown above, for example in Reaction Scheme 9, step 1 , to form a protected amide, which is then deprotected 71
• step 2 to form a compound of formula (66).
• the compound of formula (66) is then converted into a [C-V] bivalent compound of Formula I by reaction with a glycopeptide, for example vancomycin.
• step 3 the compound of formula (66) is reacted with a glycopeptide in a typical coupling reaction, as shown above, to give a compound of Formula I [C-V].
• Reaction Scheme 17 shows the preparation of [C-V] compounds of Formula I using techniques described above, i.e. reductive alkylation to provide a protected amino "handle", followed by coupling of the [C]-position to a [V] amino substituted compound, and deprotection.
• R is hydrogen or methyl
• PG represents a protecting group, preferably 9-
• R and R are independently alkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl, or a quarternary salt derivative.
• R is hydrogen or methyl
• PG represents a protecting group, for example 9- fluorenylmethyl
• R is alkyl, alkylamino, alkylaminoalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl, or a quarternary salt derivative.
• step 1 the compound of formula (22) is reacted with an acid in the same manner as shown above, for example in Reaction Scheme 9, step 1, to form a protected amide, which is then deprotected conventionally, for example as in Reaction Scheme 9, step 2, to form a compound of formula (72).
• step 2 the compound of formula (22) is reacted with an acid in the same manner as shown above, for example in Reaction Scheme 9, step 1, to form a protected amide, which is then deprotected conventionally, for example as in Reaction Scheme 9, step 2, to form a compound of formula (72).
• the compound of formula (72) is then converted into a [C-V] bivalent compound of Formula I by reaction with a compound of formula (32), prepared as shown previously, as shown in Reaction Scheme 6.
• step 3 the compound of formula (72) is reacted with a compound of formula (32) in a typical coupling reaction as shown above, to give a compound of Formula I [C-V].
• the compound of [C-V] Formula I thus prepared is substituted at the second [C] terminus (by R'NH).
• the free acid i.e., the unsubstiututed [C] terminus
• R is hydrogen or methyl
• PG represents a protecting group, preferably 9- fluorenylmethoxycarbonyl
• R , R are independently chosen from hydroger aryl, heteroaryl, arylalkyl, heteroarylalkyl, and a quarternary salt derivative.
• step 1 a glycopeptide, for example vancomycin, is reacted with an aldehyde of formula R CHO, using excess base in order to to direct the alkylation to the [V] position.
• the Schiff s base thus formed is reduced in the same manner as shown in Reaction Scheme 6, step 1, to form a compound of formula (76).
• step 2 the compound of formula (76) is reacted with a diamine (2) to form an amide of formula (77).
• the compound of formula (77) is then converted into a [C-V] bivalent compound of Formula I (in which the [C] terminus of one glycopeptide and the [V] terminus of a 76
• step 3 the compound of formula (77) is reacted with a compound of formula (36) in a typical coupling reaction as shown above to give a compound of Formula I [C-V] in which the [C] terminus of one glycopeptide and the [V] terminus of a second glycopeptide are additionally substituted.
• R is hydrogen or methyl, and R is alkyl, aryl, heteroaryl, arylalkyl, or heteroarylalkyl, all of which may be optionally substituted.
• one molar equivalent of a compound of Formula (32) and one molar equivalnent of a compound of Formula (22) are reacted with one molar equivalent of a bis-imidate of formula (38) under conventional coupling conditions.
• a hindered base is employed, preferably diisopropylethylamine.
• the reaction is conducted in an inert polar solvent, for example N,N-dimethylformamide (DMF), for about one hour.
• DMF N,N-dimethylformamide
• R is hydrogen or methyl
• R 1 is alkyl, aryl, heteroaryl, arylalkyl, or heteroarylalkyl, all of which may be optionally substituted.
• R is hydrogen or methyl
• R 1 , R 1 is alkyl, aryl, heteroaryl, arylalkyl, or heteroarylalkyl, all of which may be optionally substituted.
• the compound of formula (81) is reacted with a diamine and formaldehyde, using typical reaction conditions for the Mannich reaction, to give a compound of formula (82).
• This amino derivative is then coupled with a glycopeptide, in a typical coupling reaction as shown above, to give a compound of Formula I [C-R].
• Isolation and purification of the compounds and intermediates described herein can be effected, if desired, by any suitable separation or purification procedure such as, for example, filtration, extraction, crystallization, column chromatography, thin-layer 80
• amide (96) (3.0g, 12mmol, l.Oeq) was dissolved in anhydrous tetrahydrofuran (25mL) and cooled in an ice bath. A solution of lithium aluminum hydride (IN, 25mL, 25mmol, 2.0eq) was added carefully. The resulting solution was refluxed under nitrogen overnight, then cooled in an ice bath. 50mL more tetrahydrofuran was added followed by slow addition of sodium sulfate decahydrate until effervescence ceased. The mixture was allowed to warm to room temperature, filtered, then concentrated under vacuum. 2-(n-decylamino)ethanol, a compound of formula (97) (2.3 g, 1 lmmol, 93%) was obtained, and used without further purification.
• Fluorenylmethyl chloroformate (2.6g, 1 Ommol, l.Oeq) in methylene chloride (15mL) was added, the mixture stirred for 30 minutes then washed with 3N hydrochloric acid (50mL) twice and saturated sodium bicarbonate (50mL). The organics were dried over magnesium sulfate, and the solvents removed under reduced pressure.
• vancomycin hydrochloride (3.6g, 2.3mmol) was dissolved in 36mL of dimethylsulfoxide. To this solution was added pentanedioic acid-bis-(2- aminoethyl)amide, a compound of formula (2) (l.Og, 3.4mmol suspended in 27mL N,N- dimethylformamide) followed by N,N-diisopropylethylamine (2.4mL, 13.8mmol). The resulting suspension was stirred at room temperature for several hours until it was mostly homogenous.
• vancomycin hydrochloride (7.3g, 4.7mmol) was dissolved in 75mL of dimethylsulfoxide. To this solution was added N,N-diisopropylethylamine (4.1mL, 23.5mmol) followed by 9-fluorenylmethyl N-(2-aminoethyl)carbamate hydrochloride (1.8g, 5.6mmol). To the resulting solution at room temperature was added rapidly dropwise a solution of PyBOP (2.7g, 5.2mmol) and 1 -hydroxybenzotriazole (630mg, 4.7mmol) in 75mL l,3-dimethyl-3,4,5,6-tetrahydro-2(lH)-pyrimidinone.
• the precipitate was purified by reverse- phase HPLC (50 minutes 10-70% acetonitrile in water containing 0.1 % trifluoroacetic acid, elutes at 24 minutes) giving [V]-[2-Fmoc-aminoethyl] [C-C] -pentane- 1,5 -dioic acid bis-[(2-aminoethyl)amide]-bis-(vancomycin), a compound of formula (26), as its trifluoroacetic acid salt.
• ⁇ indicates that the subsitutent is an aglycone
• V-[Fmoc-2-aminoethyl] vancomycin (the compound of formula (25) prepared above) (300mg, 150 ⁇ mol) was dissolved in NN-dimethylformamide. NN- diisopropylethylamine (65 ⁇ L, 370 ⁇ mol) was added followed by 1 -hydroxybenzotriazole (2Omg, 150 ⁇ mol), 3-(dimethylamino)propylamine (30 ⁇ L, 240 ⁇ mol) and PyBOP (90mg, 170 ⁇ umol). The solution was kept at room temperature for 30 minutes, then dripped into acetonitrile to give a white precipitate.
• the compound of formula (36) (10.8mg, 5.3 ⁇ mol) and the compound of formula (32') (lO.Omg, 5.3 ⁇ mol) and NN-diisopropylethylamine (6.5 ⁇ L, 37 ⁇ mol), prepared as above, were dissolved in 200 ⁇ L of NN-dimethylformamide.

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