WO2001013958A2 - Conjugues de medicaments et leurs procedes de preparation - Google Patents

Conjugues de medicaments et leurs procedes de preparation Download PDF

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Publication number
WO2001013958A2
WO2001013958A2 PCT/US2000/023593 US0023593W WO0113958A2 WO 2001013958 A2 WO2001013958 A2 WO 2001013958A2 US 0023593 W US0023593 W US 0023593W WO 0113958 A2 WO0113958 A2 WO 0113958A2
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WIPO (PCT)
Prior art keywords
target
vector
pharmacophore
linker
affinity
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PCT/US2000/023593
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English (en)
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WO2001013958A3 (fr
Inventor
Sydney Brenner
Philip Goelet
Joseph Stackhouse
Steven W. Millward
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Sydney Brenner
Philip Goelet
Joseph Stackhouse
Millward Steven W
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Application filed by Sydney Brenner, Philip Goelet, Joseph Stackhouse, Millward Steven W filed Critical Sydney Brenner
Priority to JP2001518093A priority Critical patent/JP2003507439A/ja
Priority to AU70824/00A priority patent/AU7082400A/en
Priority to CA002382202A priority patent/CA2382202A1/fr
Priority to EP00959512A priority patent/EP1212096A2/fr
Publication of WO2001013958A2 publication Critical patent/WO2001013958A2/fr
Publication of WO2001013958A3 publication Critical patent/WO2001013958A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound

Definitions

  • the invention relates to methods for designing drug conjugates, methods of improving the delivery of drugs, methods of improving the therapeutic indices of drugs, methods of decreasing the toxicities of drugs, and drug conjugates and compositions comprising the conjugates.
  • the therapeutic effectiveness, or therapeutic index, of a drug can depend on a variety of factors which include its ability to reach its intended site of action.
  • a drug may provide a therapeutic effect when delivered to a specific site within the body of a patient, but cause unpleasant or harmful side-effects when delivered to other sites.
  • the preferred site of action of a drug is referred to as its target (e.g., a tissue, organ, cell, receptor, enzyme, or endogenous signaling molecule). Because the accurate delivery of a drug to its target may increase its efficacy and reduce undesired side effects, targeted drug delivery is one method by which the safety and efficacy of a drug can be improved.
  • Attempts at targeted drug delivery include the use of different routes of administration (e.g., topical instead of oral delivery). It has further been attempted by attaching pharmacologically active compounds, or "pharmacophores,” to moieties that have an affinity for the organs or tissues to which the pharmacophores are preferably delivered. Examples of targeted pharmacophores, which are sometimes referred to as "drug conjugates,” may be found in U.S. patent nos.: 5,466,681; 5,502,037; 5,723,589; 5,739,287; 5,827,819; and 5,869,465.
  • This invention is directed, in part, to a method of designing compounds which are referred to herein as "vector-linker-pharmacophore” or “VLP” conjugates, and which comprise three components: a “vector,” a “linker,” and a “pharmacophore.”
  • each VLP conjugate comprises at least one pharmacophore, which exerts a pharmacological effect by interacting with a "pharmacophore target" (e.g., a receptor or an enzyme).
  • a "pharmacophore target” e.g., a receptor or an enzyme.
  • Each VLP conjugate further comprises at least one vector, which has an affinity for a "vector target,” which is different from the pharmacophore target but which is either located in close proximity to the pharmacophore target, or can easily travel (e.g., via the bloodstream or by diffusion) to it.
  • the vector(s) and pharmacophore(s) of each conjugate are connected by covalent attachment to at least one linker.
  • the invention encompasses a method of improving the delivery of a pharmacophore to a patient, a method of improving the therapeutic efficacy of a pharmacophore, and a method of decreasing the toxicity of a pharmacophore. Further encompassed by the invention is a method of increasing the concentration of a pharmacophore in or on a cell.
  • the methods disclosed herein are facilitated by the use of computers and automated screening devices.
  • the invention therefore encompasses an electronic device for the design of VLP conjugates.
  • the invention also encompasses methods of managing and manipulating data or information concerning vectors, linkers, and pharmacophores such as approved drugs, drugs in clinical development, and drugs that failed during clinical development. Such methods encompass the use of existing software to search, mix, and match databases of existing data and information.
  • the invention further encompasses the generation of databases or packages of information useful for the design of VLP conjugates.
  • the invention also encompasses VLP conjugates.
  • Preferred VLP conjugates of the invention have molecular weights of less than about 2000, more preferably less than about 1500, and most preferably less than about 1000 daltons.
  • the term “small” when used to describe a molecule means that the molecule has a molecular weight of less than about 2000, preferably less than about 1500, and most preferably less than about 1000 daltons.
  • therapeutic index refers to the ratio of the concentration at which a pharmacophore exerts an undesired effect to the concentration at which it exerts a desired effect. A higher therapeutic index provides a greater margin of safety than a lower therapeutic index, and is therefore desirable.
  • toxicity index of a compound is the concentration at which an undesired effect of the compound is, or is estimated to be, no longer tolerable by a typical patient.
  • the abbreviation "MIC 50 " refers to the minimum concentration of a compound that will kill or inhibit the growth of 50 percent of test cultures of a microbial strain.
  • the abbreviation “IC 50” refers to the concentration of an antimicrobial compound that will displace 50 percent of labeled compound bound to a receptor. The abbreviation also encompasses the concentration of an antimicrobial compound that will inhibit the binding of 50 percent of a labeled compound that binds to the same receptor.
  • the term "patient” means a plant or animal suffering or likely to suffer from a disease or condition. Examples of animals include, but are not limited to, vertebrates such as mammals (e.g., humans), reptiles, birds, and fish.
  • target refers to a biological entity upon which a compound or chemical moiety (e.g., a pharmacophore) acts or to which a compound (e.g., a vector) has an affinity.
  • targets include, but not limited to, organs, tissues, cells, cellular parts (e.g., ribosomes, surface receptors, and DNA), proteins, peptides, polypeptides, and nucleotides.
  • the term "affect,” when used herein to describe the interaction between a compound or chemical moiety and a target means to alter the behavior, physical properties, or structure of the target.
  • pharmacophores typically affect their targets by associating (e.g. , ligand-receptor binding) with them.
  • associating e.g. , ligand-receptor binding
  • affinity for as used herein to describe the characteristics of a molecule or chemical moiety with regard to target, means that the molecule or chemical moiety will associate with the target when the two are contacted.
  • association can be measured by techniques known in the art and expressed, for example, with a rate constant or binding constant.
  • a molecule or chemical moiety that has an affinity for a target will bind to the target (e.g. , by ligand-receptor or enzyme-substrate interaction) with a dissociation constant of less than about 10 "6 , more preferably less than about 10 "8 , and most preferably less than about 10 "10 molar.
  • affinity of a molecule or chemical moiety for a given target may be lowered because of factors present in the target's natural environment such as, but not limited to, diffusion within a cell or across cell membranes, such that in vivo affinity is less than that which would be measured if the molecule or chemical moiety were contacted with a target removed from its natural environment.
  • the term "associated,” when used to describe the relationship between a biological entity (e.g., a receptor, protein, or enzyme) with a disease or condition, means that the inhibition, destruction, modification, production, or accumulation of the biological entity causes or aggravates the disease or condition, or causes or aggravates a symptom thereof.
  • a biological entity e.g., a receptor, protein, or enzyme
  • the phrase "likely to be located near,” as used herein to describe the relationship between a first target and a second target, means that there is a probability of at least about 50, 60, 70, 80, 90, 95, or 99 percent that the first target is located within a distance of less than about 10 "4 , 10 "5 , 10 "6 , or 10 "7 meters from the second target. If a first and/or second target are mobile within a patient or biological system, the phrase means that there is a probability of at least about 50, 60, 70, 80, 90, 95, or 99 percent that the first target will pass within a distance of less than about 10 '4 , 10 "5 , 10 "6 , or 10 "7 meters from the second target.
  • FIG. 1 is a schematic diagram of a VLP conjugate
  • FIG. 2 is a schematic diagram of the mode of action of a VLP conjugate
  • FIG. 3 is a table of fungal pharmacophores
  • FIG. 4 is a table of fungal vectors
  • FIG. 5 is a table of fungal linkers
  • FIG. 6 is a flow chart of the process by which a VLP conjugate may be designed
  • FIG. 7 provides the structures of some fluorescent probes
  • FIG. 8 is a table of antifungal ligands
  • FIG. 9 provides the chemical structure of sordarin
  • FIG. 10 provides the chemical structure of fluconazole
  • FIG. 11 is a representation of a sordarin-linker-fluconazole conjugate
  • FIG. 12 provides a synthetic scheme for the preparation of a sordarin-linker- fluconazole conjugate
  • FIGS. 13a-i provide examples of tables used in the design of VLP conjugates with antibiotic properties
  • FIG. 14 is a diagram of bio tin-penicillin VLP conjugates
  • FIG. 15 provides structures of conjugates of tetracycline and trimethoprim with a mechansim-based inhibitor of ⁇ -galactosidase
  • FIG. 16 provides the chemical structure of a kirromycin-trimethoprim conjugate
  • FIG. 17 provides the chemical structure of a kirromycin-tetracycline conjugate
  • FIGS. 18-20 provide a synthetic scheme for the preparation of biotin-penicillin conjugates
  • FIG. 21 shows the expected inhibition of microbial growth caused by an antibiotic
  • FIG. 22 provide a synthetic scheme for the preparation of modified trimethoprim pharmacophores ;
  • FIG. 23-25 provide a synthetic scheme for the preparation of modified tetracycline pharmacophores ;
  • FIG. 26 provides a synthetic scheme for the preparation of a mechanism-based inhibitor of ⁇ -galactosidase, which can be used as a vector;
  • FIG. 27-38 provide a synthetic scheme for the preparation of kirromycin derivatives useful as vectors
  • the invention encompasses a rational and systematic method that can be used to lower the cost and decrease the time associated with the development of vector-linker- pharmacophore ("VLP") conjugates.
  • VLP conjugates A representation of a VLP conjugate is shown in FIG. 1.
  • the invention also encompasses methods of improving the delivery, increasing the therapeutic efficacy, and decreasing the toxicity of a class of pharmacophores, a pharmacophore, or a drug delivery molecule.
  • VLP conjugates are also encompassed by the invention that can be used to treat and/or prevent diseases and conditions in plants and animals, including vertebrates such as mammals (e.g., humans), reptiles, birds, and fish.
  • the invention is based, at least in part, on the use of a vector that has an affinity for a target that is different from the target of a given pharmacophore, and which can exist in a concentration and/or location sufficient to concentrate the VLP conjugate near the pharmacophore target. It is believed that the use of such vector/target interactions in the targeted delivery of pharmacophores is novel, and has not been previously reported.
  • a first embodiment of the invention encompasses a method of designing a VLP conjugate for use in the treatment or prevention of a disease or condition in a patient, which comprises: selecting a pharmacophore that can affect a first target associated with the disease or condition, and that has a first affinity for the first target; selecting a vector that has a second affinity for a second target likely to be located near the first target; and selecting a linker to which the first pharmacophore and the first vector can both be covalently attached to provide the VLP conjugate; wherein the first target is not the same as the second target, and either the second affinity is greater than the first affinity or the concentration of the second target in the patient is greater than that of the first target.
  • a preferred method of this embodiment further comprises covalently attaching the pharmacophore and vector to the linker to provide a conjugate, and testing the ability of the conjugate to affect the first target.
  • the pharmacophore is selected using at least one criterion selected from the group which includes, but is not limited to: mode of action; target of action (e.g., location in the body, cellular or intracellular location, concentration, and molecules that react with it); molecular weight; solubility; types and/or severities of adverse effects; therapeutic index; chemical stability; presence of chemically reactive (and preferably modifiable) moieties; and structure-activity relationship data.
  • target of action e.g., location in the body, cellular or intracellular location, concentration, and molecules that react with it
  • molecular weight solubility
  • types and/or severities of adverse effects e.g., therapeutic index, chemical stability; presence of chemically reactive (and preferably modifiable) moieties; and structure-activity relationship data.
  • This method can be used to design a plurality of VLP conjugates, which can then be ranked by at least one pharmacological or chemical characteristic.
  • characteristics include, but are not limited to: affinity for the first target (i.e., the pharmacophore target); affinity for the second target (i.e., the vector target); mechanism- directed inhibition of a specific enzyme; modification of a specific enzyme; ability to inhibit or modify enzyme production, DNA or RNA synthesis, or signal transduction; chemical stability (e.g., ability to withstand cleavage under certain conditions); physiological concentration of the first target; physiological concentration of the second target; enzyme kinetic constants of the first and/or second targets; diffusion characteristics (e.g., ability to enter particular types of cells); solubility; estimated systemic concentration when administered to a patient under specific conditions; resistance to metabolic degradation; and estimated systemic clearance and metabolism when administered to a patient under specific conditions.
  • a second embodiment of the invention encompasses a method of designing a VLP conjugate of a pharmacophore having a first target, and a first affinity for the first target, which comprises: selecting a vector that has a second affinity for a second target likely to be located near the first target; and selecting a linker to which the first pharmacophore and the first vector can both be covalently attached to provide the VLP conjugate; wherein the first target is not the same as the second target, and either the second affinity is greater than the first affinity or the concentration of the second target in a patient to whom the VLP conjugate could be administered is greater than that of the first target.
  • a preferred method of this embodiment further comprises covalently attaching the pharmacophore and vector to the linker to provide a conjugate, and testing the ability of the conjugate to affect the first target.
  • a third embodiment of the invention encompasses a method of improving the delivery of a pharmacophore to a first target located in or on a cell, wherein the pharmacophore has a first affinity for the first target, which comprises: selecting a vector that has a second affinity for a second target likely to be located near the first target; selecting a linker; covalently binding the pharmacophore and the vector to the linker to provide a conjugate; testing the ability of the conjugate to concentrate near the first target; and repeating the process with a different vector if the ability of the conjugate to concentrate near the first target is less than the ability of the pharmacophore alone to concentrate near the first target; wherein the first target is not the same as the second target, and either the second affinity is greater than the first affinity or the concentration of the second target in or on the cell is greater than that of the first target.
  • information provided by the testing is used in the selection of a different vector if the process is repeated.
  • a related method encompassed by this embodiment is a method of targeting a pharmacophore having an affinity for a first target in vivo, which comprises: chemically linking the pharmacophore and a vector to a linker to provide a VLP conjugate; and administering the VLP conjugate to a host; wherein the vector can associate with a second target with a dissociation constant of less than about 10 "6 , the second target is different from the first target, and the second target is located within 10 "4 meters of the first target.
  • the second target is present in the host in a concentration of greater than 10 times that of the first target.
  • a fourth embodiment of the invention encompasses a method of improving the therapeutic index of a pharmacophore having a first target, a first affinity for the first target, and a first therapeutic index, which comprises: selecting a vector that has a second affinity for a second target likely to be located near the first target; selecting a linker; covalently binding the pharmacophore and the vector to the linker to provide a conjugate which has a second therapeutic index; testing the conjugate to determine the second therapeutic index; and repeating the process if the second therapeutic index is less than the first therapeutic index; wherein the first target is not the same as the second target, and either the second affinity is greater than the first affinity or the concentration of the second target in a patient to whom the VLP conjugate could be administered is greater than that of the first target.
  • information provided by the testing is used in the selection of a different vector if the process is repeated.
  • the pharmacophore has a poor first affinity and an acceptable toxicity index.
  • Another specific method encompassed by this embodiment is a method of decreasing the toxicity of a pharmacophore having a first affinity for a first target and a first toxicity index, which comprises: selecting a vector that has a second affinity for a second target likely to be located near the first target; selecting a linker; covalently binding the pharmacophore and the vector to the linker to provide a conjugate which has a second toxicity index; testing the conjugate to determine the second toxicity index; and repeating the process if the second toxicity index is greater than the first toxicity index; wherein the first target is not the same as the second target, and either the second affinity is greater than the first affinity or the concentration of the second target in a patient to whom the VLP conjugate could be administered is greater than that of the first target.
  • a fifth embodiment of the invention encompasses a method for treating or preventing a disease which comprises the systemic (e.g., oral or parenteral) or local (e.g., topical) administration of a VLP conjugate to a patient in need of such treatment or prevention, which comprises: a pharmacophore that has a first affinity for a target associated with the disease; a vector that has a second affinity for a second target likely to be located near the first target in the patient; and a linker covalently linking the pharmacophore and the vector; wherein the first target is not the same as the second target, and either the second affinity is greater than the first affinity or the concentration of the second target in the patient is greater than that of the first target.
  • a disease which comprises the systemic (e.g., oral or parenteral) or local (e.g., topical) administration of a VLP conjugate to a patient in need of such treatment or prevention, which comprises: a pharmacophore that has a first affinity for a
  • the disease is fungal or bacterial infection.
  • the linker is selected using at least one criterion selected from the group which includes, but is not limited to: chemical stability under physiological conditions (e.g., those typically surrounding the first and second targets); types and number of reactive moieties; metabolic stability; solubility (e.g., hydrophobicity); length; and flexibility.
  • each vector is selected using at least one criterion selected from the group which includes, but is not limited to: pharmacological effects; and lack of affinity for the first target.
  • Preferred vectors bind to their targets with high affinity, but bind to the pharmacophore target with low affinity.
  • a preferred vector binds to its target with a binding constant that is greater than about 10, 100, or 1000 times the binding constant that describes the vector's affinity for the pharmacophore target.
  • the vector and linker are selected using information obtained by screening a plurality of vector- linker conjugates for their affinities for the second target.
  • vector- linker conjugates include radioactively-labeled vector-linker conjugates and vector-linker conjugates attached to probe molecules (e.g., fluorescent probes).
  • Vector-linker conjugates can also be screened prior to the selection of their components using combinatorial chemistry techniques such as those disclosed herein.
  • both can be selected using information obtained by screening a plurality of pharmacophore-linker conjugates for their affinities for the first target.
  • the VLP conjugate has a molecular weight of less than about 2000, more preferably less than about 1500, and most preferably less than about 1000 daltons.
  • the second target i.e., vector target
  • the concentration of the second target in that cell is greater than about 10 "5 , more preferably greater than about 10 ⁇ 4 , and most preferably greater than about 10 "3 molar.
  • a sixth embodiment of the invention encompasses an electronic device for the design of VLP conjugates which comprises: a searchable database containing information about the pharmacological, chemical, and/or physical properties of vectors, linkers, and pharmacophores; an input device configured to receive instructions from a user; an output device configured to display at least one string of text data; memory configured to store the database; and a processor configured to rank data entries in the database (e.g., rank a plurality of pharmacophores, a plurality of linkers, and/or a plurality of vectors), and to identify a pharmacophore, a linker, and a vector that can be bound together to form a VLP conjugate.
  • a searchable database containing information about the pharmacological, chemical, and/or physical properties of vectors, linkers, and pharmacophores
  • an input device configured to receive instructions from a user
  • an output device configured to display at least one string of text data
  • memory configured to store the database
  • a seventh embodiment of the invention encompasses a VLP conjugate that can be used in the treatment or prevention of a disease or condition in a patient, which comprises: a pharmacophore moiety having an affinity for a first target; a linker moiety; and a vector moiety having an affinity for one or more second targets; wherein the pharmacophore and vector moieties are covalently attached to the linker, each second target is likely to be located near the first target in a typical patient suffering or likely to suffer from the disease or condition, and the first target is not the same as any of the second targets.
  • Preferred VLP conjugates have a molecular weight of less than about 2000, more preferably less than about 1500, and most preferably less than about 1000 daltons.
  • the second target is not associated with the disease or condition to be treated, but the first target is so associated.
  • both first and second targets can be associated with a disease or condition if they otherwise meet the criteria described herein.
  • the covalent bonds attaching the vector to the linker and the linker to the pharmacophore do not cleave under the physiological conditions surrounding the first target.
  • VLP conjugates of the invention comprise vector moieties derived from one of the following molecules: sordarin; biotin; and kirromycin.
  • Specific VLP conjugates of the invention comprise pharmacophore moieties derived from one of the following molecules: fluconazole; penicillin; trimethoprim; and tetracycline.
  • the vector is sordarin and the pharmacophore is an antifungal preferably of the conazole class.
  • conjugates of the invention include, but are not limited to: sordarin-linker-fluconazole conjugates; biotin-linker-penicillin conjugates; kirromycin-linker-trimethoprim conjugates; and kirromycin-linker- tetracyline conjugates.
  • methods of this invention are based on a unique systematic and rational approach to the design of VLP conjugates and drug targeting.
  • methods of the invention are not limited to specific types of vectors, linkers, or pharmacophores, and can be used to design VLP conjugates useful in the treatment or prevention of a wide variety of diseases and conditions. Methods of the invention can further be used to develop conjugates of small pharmacophores and small vectors.
  • compositions disclosed herein are based on the ability of some chemical moieties, referred to herein as "vectors,” to interact with target macromolecules in such a way that pharmacophores attached to the vectors and administered to a patient will be concentrated near those macromolecules. As shown in FIG. 2, this can improve the delivery of pharmacophores to their sites of action -or targets- if those sites of action are proximate to the vector targets.
  • a database or “table”.
  • the term “table” can be used to refer to a part of a database.
  • the database can be stored in the memory of a computer, on magnetic media (e.g., floppy disks, hard disks, and tapes), on optical media (e.g., CD-ROMS), or even on paper.
  • the terms “database” and “table” therefore refer to a collection of information which is not limited to a particular method of storage, manipulation, or organization.
  • a preferred database contains data that is divisible into three subsets, each of which is related to pharmacophores, linkers, or vectors.
  • at least one, and more preferably at least two of the three subsets of data contain information for a plurality of compounds or chemical moieties.
  • a database can contain one subset of information concerning physical properties, chemical properties, pharmacological (including pharmacokinetic, metabolic, toxicity, potency, receptor binding, and enzyme inhibitory) properties, and/or physiological properties of one pharmacophore, another subset containing similar information for a plurality of linkers (e.g., at least about 5, 10, 20, 50, 100, or 500), and a third subset containing similar information for a plurality of vectors (e.g., at least about 5, 10, 20, 50, 100, or 500).
  • linkers e.g., at least about 5, 10, 20, 50, 100, or 500
  • a third subset containing similar information for a plurality of vectors
  • Each of these combinations represents the components of one VLP conjugate, the estimated physical, chemical, and/or physiological properties of which are preferably improved with regard to the pharmacophore(s) alone, and which can be used to rank potential VLP conjugates according to a particular preference.
  • the information that is assembled into a database is preferably obtained prior to application of sorting algorithms.
  • the information those tests provide can be used to augment and/or modify the database in such a way that information obtained from it can be used to predict the properties of potential VLP conjugates with more accuracy.
  • databases already in existence can be used in methods of invention.
  • databases include, but are not limited to: the Merck Index ® ; the Merck Manual ® ; the Physicians' Desk Reference ® ; online service databases such as those available from Lexis ® , Westlaw ® , Dialog ® (e.g., Medline ® ), and Chemical Abstracts Service ® ; and governmental registries and databases such as those maintained by or on the behalf of the Food and Drug Administration, the United States Patent and Trademark Office, the Japanese Patent and Trademark Office; the European Patent and Trademark Office; and the United States Pharmacopea.
  • these and other sources of information known to those skilled in the art are preferably used to assemble a modifiable database of the invention.
  • a general database that can be used to design VLP conjugates contains information about a variety of pharmacophores, including their ability to treat certain diseases and their sites and mechanisms of action.
  • the database further contains information about a variety of vectors, including their targets (e.g., cells, parts of cells, or proteins to which they have a particular affinity) and pharmacological effects, as well as information about a variety of linkers, such as their chemical stabilities, solubilities, and lengths.
  • targets e.g., cells, parts of cells, or proteins to which they have a particular affinity
  • linkers such as their chemical stabilities, solubilities, and lengths.
  • a database may contain information about only one pharmacophore, and the method used to analyze it may focus only on ways of more accurately targeting its delivery.
  • a database and method can focus on VLP conjugates of a particular vector, given the concentration and location of its target in a particular group of patients (e.g., humans suffering from a particular disease or condition).
  • a database of the invention comprises information about at least one pharmacophore, at least one vector, and at least one linker. Examples of such information are provided below.
  • a pharmacophore is a molecule or chemical moiety having an affinity for a particular target and thus active against a biological condition or a disease state.
  • Pharmacophores include, but are not limited to, pharmaceutical compounds recognized by the medical and scientific communities as safe and effective (e.g., compounds disclosed in the Physicians' Desk Reference ® ), as well as compounds that have adverse effects that outweigh their therapeutic effects. Examples of such compounds include those that have been rejected as drug candidates during clinical trials and those that have few or no adverse effects but which interact poorly with their targets.
  • Examples of pharmacophores that can be including in a database of the invention include, but are not limited to, antibiotics, anti-tumor agents, angiogenesis inhibitors, antivirals, antifungals, molecules that bind tightly to metabolites, inhibitors or activators that bind to macromolecular receptors, neural receptor agonists or antagonists, transcription factors inhibitors or activators, enzyme inhibitors or activators, inhibitors or activators of binding interactions between or catalytic activities of proteins in cell signaling pathways; protein synthesis inhibitors, ionophores, antigens, and molecules that modify DNA or RNA.
  • Specific pharmacophores include, but are not limited to, penicillin, tetracycline, trimethoprim, and those listed in FIG. 3. Additional pharmacophores are disclosed by U.S. patent nos. 4,873,088; 5,190,969; 5,466,681; 5,795,909, each of which is inco ⁇ orated herein by reference. Potential pharmacophores further include: cytotoxic phenolic compounds, such as those disclosed by U.S. patent no. 5,639,737, which is inco ⁇ orated herein by reference; antibodies, such as those disclosed in U.S. patent no. 5,824,805, which is inco ⁇ orated herein by reference; psychotropic, neurotropic, and neurological agents, such as those disclosed by U.S.
  • bradykinin analogs such as those disclosed by U.S. patent no. 5,863,899, which is inco ⁇ orated herein by reference
  • lysomotropic moieties intracellular polymerizing moieties, protein sorting signals or sequences, conditional membrane binding peptides and bi- or multi-valent receptor cross linking moieties, such as those disclosed by U.S. patent no. 5,869,465, which is inco ⁇ orated herein by reference
  • chimeric fused proteins such as those disclosed by U.S. patent no. 5,871,753, which is inco ⁇ orated herein by reference.
  • information about each pharmacophore includes information about its preferred site(s) of action (i.e., its target(s)). Examples of such information include, but are not limited to, the location, concentration, and structure of the target, other drugs that interact with it, macromolecules that interact with it, and other members its family (e.g., receptors of similar structure or function).
  • pharmacophores can be correlated to disease states.
  • Disease states can be classified as those caused by microorganisms or as those associated with mammalian cells or quality of life states. Classes of microorganisms include prokaryotes, single cell eukaryotes, multicellular eukaryotes, and viruses. Disease conditions of mammalian cells, or quality of life states, can be classified by cell types and locations.
  • a database used in a method of the invention may contain information regarding the organism that causes the disease of interest, or the types and locations of the cells associated with the disease.
  • Pharmacophore information stored in a database can further include: the molecular structure of the pharmacophore; the minimum concentration at which it is typically effective; structure-activity relationship data; the MIC 50 of the pharmacophore; binding and/or kinetic constants for the pharmacophore's association with its target; the mechanism or mode of the pharmacophore's action; its therapeutic effects; and its adverse effects.
  • Structure-activity data can, for example, include the structures of derivatives of the pharmacophore, together with the minimum concentration at which each of those derivatives exhibits a desired or undesired effect (e.g., the therapeutic index of each derivative), and the binding and/or kinetic constant of each for its target.
  • Structural modifications that consisted of addition of long chain substituents to some part of the pharmacophore may also be of interest, as they can provide information relevant to the selection of a linker, as described elsewhere herein.
  • a table of pharmacophore targets can be prepared that corresponds to the pharmacophores chosen to combat a disease state of interest.
  • Pharmacophore targets can be classified as, for example: receptors for, or enzymes that process, known pharmacophores; as undesired or unintended targets of known pharmacophores; or as macromolecules or structures that have a function which, if modified, can cause a pharmacological effect.
  • Information about each target can include: its cellular or extracellular abundance; its cellular or extracellular location; the location of the cell types that express it; the location of the target within a particular organism; and the cellular or physiological function of the target. Additional information for targets that are proteins can include their amino acid sequences and 3-dimensional structures.
  • a database may also include the identities of small and large molecules that associate with each target in its native environment.
  • Pharmacophore targets can thus be characterized in any number of ways, and can be indexed, classified, or arranged by any of those characteristics. Examples of ways by which a pharmacophore target can be classified or identified include, but are not limited to: a. whether it is a target for known pharmacophores or compounds which, if modified, can have a pharmacological effect (e.g., prodrugs); b. pharmacophores with which it interacts; c. its cellular or physiological function(s); d. its cellular or extracellular location; e. the location of the cell types that express it; f. if the target is a protein, its molecular properties, which in turn can be determined or characterized by: i.
  • vectors are compounds or moieties which have an affinity for targets (“vector targets”) located in the vicinity of pharmacophore targets.
  • vector targets are compounds or moieties which have an affinity for targets (“vector targets") located in the vicinity of pharmacophore targets.
  • vector targets Preferably, the interaction between a vector and its target will cause few physiological effects, or will cause effects that augment the therapeutic effects of the pharmacophore.
  • vector targets there are many potential vector targets within, or in the vicinity of, bacteria, fungi, mammalian cells, and virally infected cells.
  • the target of the vector it comprises must either be present at a sufficiently high concentration or associate with the vector with a sufficiently high binding or good kinetic constants such that the increase in the local concentration of the pharmacophore due to the vector/vector target interaction is sufficient to improve the activity of the pharmacophore.
  • Vector targets can be abundant proteins (preferably comprising more than one percent of the mass of a target cell), with binding sites that have very low dissociation constants for the vector. They can also be enzymes that process the vector as a substrate. Alternatively, vector targets can be cellular systems that increase the local concentration of the conjugate by other means. Whatever the vector target, it must be located in close proximity to the cellular or molecular target upon which the pharmacophore preferably acts (i.e., the pharmacophore target).
  • vector targets include, but are not limited to, macromolecular receptors for small molecules, enzymes for which mechanism-based inhibitors are known, polymerases, and synthetases. Proteins that are uniquely found in specific cells (e.g., diseased or infected cells) are also potential vector targets. Vector targets further include specific and abundant protein, DNA, RNA, or polysaccharide products of a given pathogen, such as structural proteins of a pathogen, or abundant enzymes of a pathogen or of a diseased cell or tissue, or protein products of a disease.
  • bacterial ribosomes include, but are not limited to: bacterial ribosomes, ribosomal RNAs, tRNAs, and ribosomal proteins; peptidyl prolyl cis-trans isomerases or rotamases; structural proteins of viruses, such as the nucleoproteins, and coat proteins; abundant proteins of specific eukaryotic cells, such as steroid hormone receptors and the receptors for retinols and vitamin D; and cleavage enzymes.
  • Potential vector targets also include antibiotic resistance elements, including those which have genes carried on plasmids such as the aminoglycoside phosphokinases and acetylases, bacterial beta lactamases, and bacterial transamidases.
  • Vectors are selected to achieve preferential targeting or concentration at vector targets.
  • a suitable vector may bind with high affinity to a protein receptor or be a substrate for an enzyme, for example a polymerase.
  • Such vector targets are associated with pathogens or have a distinct cellular distribution in organisms suffering from certain diseases.
  • Vectors can also be small molecules that bind with high affinity to particular macromolecules. New small molecules with the sole characteristic that they bind with high affinity to some macromolecular target or are the substrate for some enzyme may also be used as vectors. Similarly, small molecules that are known to accumulate in, on, or in the vicinity of cells or intercellular or intracellular structures, by other means may be used as the vectors in VLP conjugates. Specific vectors that can be included in a database of the invention are listed in
  • FIG. 4 Other vectors are disclosed by U.S. patent nos. 5,466,681; 5,639,737; 5,723,859; 5,739,287; 5,824,805; 5,827,819; 5,837,690; 5,863,899; 5,869,465; and 5,871,753, each of which is inco ⁇ orated herein by reference.
  • Methods and conjugates of the invention are premised on the interactions between small molecules and target macromolecules.
  • a typical database of the invention will thus contain information about ligands and their receptors or substrates and their corresponding enzymes. Because many small molecules that interact with macromolecules have been identified on the basis of their pharmacological activity, most initial entries in a database will be pharmacophores and their receptors. However, the data used to provide a database of the invention need not be limited to intermolecular interactions that lead to a pharmacological effect; those that do not can simply be characterized as vectors and their targets.
  • a table of ligands and receptors or substrates and enzymes which can be used as vector information within a database can include information such as, but are not limited to: the identity of the macromolecule to which a small molecule binds or the enzyme for which it is a substrate; the cellular location and concentration of such a macromolecule; the molecular structure of the small molecule; structure activity relationship data regarding the interaction between the small molecule and its target receptor or enzyme; and reaction catalyzed by the enzyme.
  • Vector information can also include the pharmacological effects of each potential vector, its pharmacologically effective concentration, its mode of action, and its side effects. Of particular interest is structure activity data that can be used to determine what effects, if any, the attachment of a linker to a vector will have on its affinity for its target.
  • Vectors and their targets can thus be characterized in any number of ways, including, but not limited to: a. the identity of the primary target (e.g., protein receptor, enzyme, or another macromolecule) with which the vector interacts; b. the position of the target (e.g. , in relation to a particular pharmacophore target); c. molecular properties such as, but not limited to, structure and molecular weight; d. the pharmacological effects of each vector or its metabolites; e. the binding constant of each vector for its target (e.g., kinetic constants with regard to a target enzyme); and fi structure-activity relationship data.
  • the primary target e.g., protein receptor, enzyme, or another macromolecule
  • the position of the target e.g. , in relation to a particular pharmacophore target
  • molecular properties such as, but not limited to, structure and molecular weight
  • d. the pharmacological effects of each vector or its metabolites e. the binding constant of each
  • a table of vector targets associated with each target microorganism or cell type can be classified, for example, according to their abundance and/or subcellular or extracellular location.
  • a typical table of a database of the invention comprises at least one of the following pieces of information for each target: a. its cellular or extracellular abundance; b. its cellular or extracellular location; c. in the case of multicellular organisms, the location within the organism; d. its cellular or physiological function; e. its molecular properties, which can be characterized or determined using information such as, but not limited to: i. its amino acid sequence; ii. its 3-dimensional structure; iii. small molecules that bind to it in its native environment, and their binding constants; and iv. large molecules (e.g., macromolecules) that bind to it in its native environment; and f. the identities of other small molecules that bind to it.
  • a typical table of a database of the invention comprises at least one of the following pieces of information for each target: a. reaction(s) catalyzed by the enzyme; b. its cellular or extracellular abundance and location; c. in the case of multicellular organisms, its location within the organism; d. the cellular or physiological function of the enzyme; e. its molecular properties, which can be characterized or determined using information such as, but not limited to: i. its amino acid sequence; ii. its 3-dimensional structure; iii. the small molecules that are substrates for, or inhibitors of, the enzyme in its native environment, and their binding or kinetic constants; and iv. large molecules (e.g., macromolecules) that bind or interact with it in its native environment; and f. other small molecules that are known to interact with the enzyme.
  • a table can be formed that contains information about vectors known to bind to the vector targets of interest.
  • the pharmacological effects -if any- exerted by the vectors when bound to their targets are further included. Consequently, the vector information stored in a database can include data such as, but not limited to: the identity of each vector target (e.g., receptor or enzyme); its function; its structure; its molecular weight; its binding or kinetic constants with regard to its target; structure activity relationship data; and its pharmacological effects.
  • a preferred database of the invention contains a table of vectors that encompasses at least one piece of the following information: a. small molecule(s) (i.e., vectors) known to interact with vector targets.
  • each vector i. if the vector is a molecule that binds to a receptor protein, it should be noted whether the receptor is activated or inhibited by the action of the vector; and ii. if the vector is a molecule that interacts with an enzyme, it should be noted whether it is a substrate for the enzyme, an inhibitor for the enzyme, or as a mechanism based inhibitor of the enzyme; c. the molecular properties of the vector, such as: i. molecular structure; ii. molecular weight; iii. binding constant of the vector for its receptor; and/or iv. kinetic or inhibition constants for the vector target if this is an enzyme; d. available structure activity relationship data such as, but not limited to, binding constants or inhibition constants of each structure; and e. the pharmacological effects of a vector.
  • a linker is a chemical moiety that connects a pharmacophore to a vector.
  • a typical database of the invention contains information about at least one linker that can be used to design a VLP conjugate having desired pharmacological, chemical and/or physical properties.
  • a linker can be chosen on the basis of its length, and thus the spacing allowed between the vector and pharmacophore in the conjugate; the solubility characteristics that it will impart to the conjugate; the degree to which it will affect the ability of the conjugate to gain access to the interior of a cell or compartments therein; the degree to which it will affect systemic clearance of the conjugate; the degree to which it will affect distribution of the conjugate; the degree to which it will affect the metabolism of the conjugate; conformational flexibility or rigidity; and commercial availability or ease of synthesis.
  • Typical linkers of the invention can be of a length of from about 10 to greater than about 50, 75, 100, 150, or 200 angstroms. Linkers can be also be charged, polar, or non- polar, depending on, for example, the desired solubility properties of the VLP conjugate. Linkers can also be prepared which contain hydrophobic chains, or ring structures that limit the conformation or freedom of movement of the conjugates that contain them. Preferably, linkers are selected such that they will remain intact under the conditions with which the conjugates that contain them will interact. Preferred linkers also will not diminish the activity of the pharmacophore or the simultaneous interaction of the pharmacophore with its target and the vector its target. Preferred linkers also permit conjugates to gain access to the interior or interior compartments of a cell.
  • linkers include, but are not limited to: polyethylene glycol based linkers; polyethylene enimine based linkers; linear alkane based linkers; and combinations thereof. They can include, but are not limited to, those having chains joining the end groups that are, or are combinations of, linear alkanes, linear alkenes, alkynes, di- and multi-substituted phenyl rings, di- and multi-substituted napthalene rings, di- and multi-substituted cyclohexyl rings, multi- (e.g., tri- and di-) substituted decalin rings, fused aryl or alkyl rings of varying conformational rigidity, linear alkylamines, linear alkyl alcohols, polyalkylamines, polyalkylethers, and polyalkylthioethers.
  • each linker can be the same or different.
  • end groups include, but are not limited to, amines, imines, amine oxides, hydrazines, alcohols, thiols, azo compounds, ethers, thioethers, sulfoxides, sulfones, sulfonamides, sulfonyl esters, phosphate esters, phosphines, methylenes, methines, carboxyl amides, carboxyl esters, and imidates.
  • Specific linkers are shown in FIG. 5. Other examples are disclosed by U.S. patent nos.
  • a database of the invention thus contains a table of linker data containing at least one piece of the following information for each linker: a. reactive end groups; b. chain structure and repeating units in chain; c. multiplicity of reactive end groups; d. chain length; e. chain charge and hydrophobicity; and f metabolism and chemical stability under in vivo conditions.
  • VLP conjugates It is possible to design VLP conjugates rapidly and efficiently once a database has been formed as described herein.
  • pharmacophores are identified that may be therapeutically effective in its treatment. Based on the location of the pharmacophore target, one or more candidate vector targets are identified that are typically located within close proximity to it. Vectors are then identified that bind to those targets, and are ranked using relationships such as those provided herein.
  • Combinations of vectors, linkers, and pharmacophores are then selected and ranked according to relationships known in the art or described herein. For example, quantitative relationships can be used to estimate the solution or systemic concentration at which the VLP conjugate can exhibit activity similar to that of the unconjugated pharmacophore. Such relationships may take into consideration: the local concentration of a vector target; the dissociation constant of a particular vector from that target, or, if the target is an enzyme, the rate at which it processes the vector; and the minimum effective concentration needed by the pharmacophore for it to exhibit activity. From this information, only those vectors that are estimated to concentrate a particular conjugate to a level sufficient for its particular pharmacophore to exhibit activity at its target are selected.
  • Suitable linkers are then chosen that should not interfere with solubility or access of the conjugate to its site of action.
  • Other design issues can then be addressed, such as systemic distribution, stability to metabolic degradation, rate of systemic clearance, ease of synthesis, and commercial availability of starting materials.
  • a flow chart of a design method of the invention is shown in FIG. 6.
  • the table of pharmacophores within a database is preferably sorted by at least one criterion.
  • compounds or moieties suitable for use as pharmacophores in a conjugate can be selected from the database as follows: a. Pharmacophores with the lowest molecular weight are ranked above those with higher molecular weight; b. Pharmacophores, which when modified by the attachment of a long-chain substituent still affect their targets, are ranked above those which do not; and c. Those pharmacophores which exhibit activity at low concentrations are ranked above those which require higher concentrations.
  • the vectors in the database are also preferably sorted or ranked to identify those which are most likely to assist in the targeted delivery of the pharmacophore(s) identified according to Section 4.1.2.1 above.
  • Vectors are typically ranked according to their suitability in delivering a given pharmacophore to its target such that the concentration of pharmacophore necessary for it to provide a therapeutic or prophylactic effect is decreased. This can be done by using quantitative relationships disclosed herein and information such as, but not limited to: the concentration of a vector target; and the dissociation constant of the vector from its target, or the kinetic constants of the vector for its target if that target is an enzyme. This ranking can further be made with regard to the minimum concentration at which the pharmacophore can exhibit its desired activity.
  • Suitable vector candidates can be selected, organized, or ranked as follows: a. Vectors can be ranked according to the concentration of their targets in the cellular compartment being targeted. Penicillins and cephalosporins, for example, can be ranked according to the concentration of penicillin binding proteins in the pathogen targeted. Target concentrations should be modified by the number of binding sites available on the target; b. Vectors that bind to receptors can be ranked according to their dissociation constants for their targets. Vectors with low dissociation constants are ranked above those with higher dissociation constants; c. Vectors that are substrates for enzymes can be ranked according to their kinetic constants for their targets. Vectors favorable kinetic constants are ranked above those with less favorable constants; d.
  • Vectors which when modified by the attachment of a long-chain substituent still bind to or affect their targets, are ranked above those which do not; e.
  • Vectors can also be ranked in a quantitative or semi-quantitative manner using the quantitative relationships described below in Example 3 and knowledge of the dissociation constant of the pharmacophore from its receptor or the MIC 50 of the pharmacophore. A threshold value for these numbers can be used, or the values can be taken from a representative set of pharmacophores that are likely to be used with the vector (because the vector target and pharmacophore target are adjacent to one another). This information is reported in the literature; f. Vectors with the lowest molecular weight should be ranked above those with higher molecular weight; and g. Vectors with undesirable pharmacological effects of their own should be ranked lower than those having no effect or beneficial effects.
  • potential vectors can be bound to probes (e.g., fluorescent moieties) that are representative of pharmacophores, but which can be used to test the affinity of a vector conjugate for the vector's target.
  • probes e.g., fluorescent moieties
  • probes are known in the art, and include those shown in FIG. 7.
  • vectors and pharmacophores are matched to form pairs using quantitative relationships disclosed herein.
  • vectors and pharmacophores can be scored according to how well they may function in an actual conjugate. They are then paired such that the vector target of each conjugate is concentrated in the proximity of the pharmacophore target. Other criteria may also be used to determine optimal pharmacophore/vector pairs. For example, the binding or kinetic constants of each vector with its target can be taken into account.
  • the vector/vector target interaction is known to yield pharmacological effects, those effects must be compatible with those of the pharmacophore.
  • linkage is accomplished by covalently coupling a functional group of the pharmacophore to a functional group bound to one end of a linker, and coupling a functional group on the other end of the linker to a functional group of the vector.
  • Linkers are consequently selected from the database with reference to their functional groups. Linkers are further selected based on their length, flexibility, chemical stability, and solubility.
  • linkers can be chosen to be as small as possible if their influence on the properties of the conjugate are a concern. They can also be chosen on the basis of charge, polarity, and hydrophobicity. Several different linkers can be selected for each vector-pharmacophore pair. Further, although pharmacophores and vectors are typically joined by a single linker, they may also be joined to one another by more than one linker, thereby forming a ring containing both the pharmacophore and vector.
  • VLP conjugates that comprise two or more pharmacophores attached to one vector, two or more vectors attached to one pharmacophore, or clusters or rings of pharmacophores and vectors.
  • Multiple linkers further allow the formation of compounds of the structure X-VLP-Y, wherein VLP is a conjugate of the invention and X and Y are the same or different, and are moieties (e.g., a non-cleavable or cleavable small or large molecule such as polyethylene glycol) or substrates (e.g., a surface or insoluble polymer) useful in the delivery of the VLP to a patient.
  • moieties e.g., a non-cleavable or cleavable small or large molecule such as polyethylene glycol
  • substrates e.g., a surface or insoluble polymer
  • the pharmacophore, vector, and linker are selected to maintain the small size of the assembled conjugate, which is preferably less than about 2000 daltons, more preferably less than about 1500 daltons, and most preferably less than about 1000 daltons.
  • the small size of the conjugates can facilitate their entry into target cells or organisms by diffusion or by the same mechanisms as are used by the pharmacophore alone.
  • smaller conjugates may be absorbed (e.g., by the intestine or following oral administration) at a greater rate than larger conjugates.
  • Combinations of candidate vectors and pharmacophores are preferably selected as follows: a. Vectors and pharmacophores can be paired according to proximity of their receptors (i.e., those associated with a particular pathogen or in a particular cellular compartment). The site of action and probably the mechanism of action, of the pharmacophore can be identified directly from the tables or the literature, or by experiment or conjecture based on characteristics of known analogues. This information can be used to search the database for vector targets with the same cellular or extracellular location as the site of action of the pharmacophore.
  • the concentration of a vector target should be at least ten times greater than the MIC 50 of the unconjugated pharmacophore or the dissociation constant of the pharmacophore from the pharmacophore target. Preferably, the concentration is also at least time times greater than the concentration of the pharmacophore target, b.
  • Structure activity relationships for both vectors and pharmacophores can be examined. Structure activity relationship data can include the structures of derivatives of pharmacologically active molecules, together with the concentrations at which they exhibit activity. Structures that contain moderate- to long-chain substituents attached to some part of them, but which still exhibit activity or affinity can be identified when present, since the substituents may provide information relevant to the attachment of linkers, c. Linkers of a charge and polarity similar to those of the vector and pharmacophore are preferably chosen. Linkers are further selected which have end groups that can be covalently bound to the attachment sites on the pharmacophore and vector.
  • Methods of the invention need not be used to design a single VLP conjugate. Indeed, preferred methods allow the identification of groups of potential conjugates which can subsequently be tested.
  • designs can include different possible linkers, different linker attachment sites on the pharmacophores, different vectors, different linker attachment sites on the vectors, and variations in the length and composition of the linker chain. Since the linker can be selected to make beneficial contributions to the solubility, resistance to degradation, systemic clearance, and tissue penetration of the conjugate, it can be expected that many of the candidates can have adequate solubility, and that for some, the VLP conjugate can have the same access to the pharmacophore and vector targets as the pharmacophores and vectors do alone. Linker chain length can be varied to ensure that it does not interfere with the pharmacophore/pharrnacophore target and vector/vector target interactions.
  • Data preferably used during the design include structure- activity relationships and linkage chemistry. Such data can be obtained from the literature or by experiment. Data can also be estimated from published 3-dimensional structures of vector and pharmacophore targets. Additional information may be gathered by constructing test conjugates with reporter groups in place of the vector or the pharmacophore. These can be used for preliminary screening of vector-linker and pharmacophore-linker combinations to determine which are suitable for use together in conjugates. Structure-activity relationship data allows the determination of the locations on the vector and pharmacophore to which linkers can be attached without interfering with their interactions with their targets.
  • the part of a pharmacophore that interacts with its target cannot be modified by inco ⁇ oration of large functional groups, since such groups can diminish the activity of the pharmacophore.
  • those portions of a pharmacophore that do not interact with its target may support the attachment of linkers.
  • this information has been reported in the literature in connection with attempts to identify structural modifications that improve the interactions of a small molecule with a macromolecular receptor or enzyme.
  • numbers of structural variants of the small molecule are often prepared, and the interaction of each of these with the macromolecular receptor or enzyme is determined. Frequently, such variants include those with short to moderately long chain substituents, which can serve as models for linkers.
  • structure activity data is not available. But if, for example, the three dimensional structure of a target has been reported, it might be possible to estimate where on a particular pharmacophore or vector a linker could be attached without substantially affecting its activity or affinity. Alternatively, routine experimentation can be conducted using techniques known in the art.
  • labels and reporter groups can be used to select the best combinations of vectors, linkers, and pharmacophores.
  • labels and reporter groups can be used to determine optimal vector-linker conjugates which have a desired affinity for a specific vector target, and which can be attached to a large number of pharmacophores with targets proximate to that vector target.
  • Candidate pharmacophores and vectors can be conjugated to different linkers and tested to determine which combinations do not diminish desired interactions.
  • test conjugates can be prepared which consist of a vector, a linker, and a model pharmacophore or a reporter group.
  • assays can be performed wherein a labeled, but unconjugated, vector is allowed to interact with its target in the presence of varying concentrations of a test conjugate.
  • the vector can be labeled with a radioactive isotope that will not alter its structure or perturb its interaction with the vector target.
  • concentration at which the test conjugate inhibits the interaction between the labeled vector and the target can then be determined.
  • Assays can be performed on isolated preparations of vector target. These can be direct measurements of inhibition of vector binding to receptors or of enzymatic activity that processes the vector. For vectors that bind to receptors, the degree of inhibition of vector binding by the test conjugate can be assayed by measuring diminution of binding of the labeled vector to its target caused by the presence of varying concentrations of the test conjugate. For vectors that are substrates for enzymes, the inhibition constant of the test conjugate can be determined by measuring the diminution in the rate of turnover of labeled vector caused by the presence of varying concentrations of the test conjugate. Assays can also be performed in vivo on whole cells or tissue preparations to determine if the test conjugate can gain access to the vector target.
  • Combinations of a vector and reporter group with various linkers can also be tested to determine the identity of linkers that do not interfere with the vector's interaction with its target.
  • Assays can directly measure the accumulation of the test conjugate in a cell or tissue sample at various concentrations.
  • Reporter groups which are not themselves pharmacophores, can be chosen to span the range of molecular characteristics of potential pharmacophores; e.g., size, charge, polarity, and hydrophobicity. This can assure that the test conjugates accurately model the behavior of real conjugates.
  • a combinatorial approach to identifying good vector-linker combinations may also be used.
  • a combinatorial synthesis can be used to produce a library of test conjugates in which the structure of the linker is varied, with the test conjugates being attached through their non-vector end to the pixels of a charged-coupled device.
  • the target macromolecule can be labeled with a fluorescent probe, and its binding to individual pixels can be determined. This procedure can be used to identify vector-linker-probe combinations in which the linker does not interfere with the vector/vector target interaction.
  • suitable vector-linker and pharmacophore-linker combinations can be determined as follows: a.
  • a reporter group or model pharmacophore can be selected by the experimenter. The group can be similar to a proposed pharmacophore, if this had been chosen.
  • Each proposed linker can be joined at the pharmacophore end to the model pharmacophore or reporter group, and at the vector end to the vector to produce a test conjugate.
  • iii. Inhibition of interaction between labeled vector and the vector target by the test conjugate can be measured.
  • interaction between the test conjugate and the vector target can be measured directly through use of the reporter group.
  • a reporter group or a model vector can be selected by the experimenter.
  • the group can be similar to a proposed vector.
  • Each proposed linker can be joined at the vector end to the reporter group or model vector, and at the pharmacophore end to the pharmacophore.
  • iii. Inhibition of interaction between labeled pharmacophore and the pharmacophore target by the test conjugate can be measured, iv. Alternately, interaction between the test conjugate and the pharmacophore target can be measured directly through use of the reporter group.
  • Combinations i. The results of steps a and b can be examined for a number of test conjugates; and ii. vector-linker or pharmacophore-linker pairs which exhibit normal or enhanced performance are selected.
  • VLP conjugates designed by the approach described above can be prepared by known techniques and screened for desired characteristics. Such screening can provide information such as whether a conjugate is adequately soluble and whether it can indeed concentrate a pharmacophore at its site of action. Further testing of successful candidates can address systemic distribution, resistance to metabolic degradation, and systemic clearance. All such information can then be entered into a database of the invention, thereby facilitating the more accurate predication of suitable VLP conjugates. Conjugates are preferably tested using methods well known in the art, and those described above in Section 4.1.2.4.
  • VLP CONJUGATES VLP conjugates designed by methods of the invention can be used to treat or prevent innumerable diseases and conditions caused by, for example, viruses, bacteria, mycoplasmas, fungi, protists, parasites, or prions.
  • the target of the pharmacophore of a conjugate is typically contained in, or associated with, such causes.
  • viruses include, but are not limited to, HBV (Hepatitis B Virus), HIV (Human Immunodeficiency Virus), HCV (Hepatitis C Virus), and influenza.
  • bacteria include, but are not limited to, Tuberculosis, Streptococci, Chlamydia, Borrelia, Haemophilus, Neisseria, Heliobacter, Shingella, Pasteurella, Coxiella, Mycobacteria, Salmonella, Fusobecteria, Camphlobacteria, and Staphylococci.
  • fungi include, but are not limited to, Candida; Cryptococci; Histoplasma; Sporothrix; Trichophyton; Microsporum; and Epidermon.
  • prions include, but are not limited to, those causing or associated with, Kreuzfeldt Jacob Disease, scrapie, and Alzheimers disease.
  • Specific disease that can be treated or prevented by the conjugates of the invention include, but are not limited to, cancer, heart disease, neurodegenerative disease, HIV, and diabetes.
  • Quality of life states that can be improved by the conjugates of the invention include, but are not limited to, memory loss, balding, obesity, impotence, and aging.
  • Conjugates of the invention include conjugates of pharmacophores selected from the group that includes, but is not limited to: antibiotics; antibacterials; antimycoplasmals; antivirals; antifungals; antiprotozoals; molecules active against single celled eukaryotes; molecules active against parasites; molecules that bind tightly to metabolites; inhibitors or activators of binding to macromolecular receptors, including antagonists or agonists of neural receptors and inhibitors or activators of transcription factors; enzyme inhibitors or activators; inhibitors or activators of binding interactions between macromolecules; inhibitors or activators of binding interactions between or catalytic activities of the proteins in cell signaling pathways; nucleic acid polymerase inhibitors; protein synthesis inhibitors; protease inhibitors or activators; kinase and phosphatase inhibitors or activators; glycosylation inhibitors; dihydrofolate reductase inhibitors; ionophores
  • Conjugates of the invention include conjugates of vectors selected from the group that includes, but is not limited to those that interact with: polymerases; transcriptases; ribosomes; proteins involved with protein folding or other chaperones; structural proteins of viruses; abundant proteins of specific eukaryotic cells; antibiotic resistance elements; and enzymes, such as bacterial transamidases, that function in the formation the bacterial cell wall.
  • Conjugates of the invention also include conjugates of vectors that are mechanism directed inhibitors that result in a covalent modification of the target enzyme with covalent binding of the vector to it or that result in covalent binding of the vector to structures in the vicinity of the pharmacophore target.
  • the linkers used to connect the pharmacophore and vector moieties of a VLP conjugate of the invention include those molecules which comprise a polyethylene glycol, polyethylene enimine, or linear alkane moiety; molecules which comprise an end- group selected from the group consisting of: amines, imines, amine oxides, hydrazines, azo compounds, ethers, thioethers, sulfoxides, sulfonamides, sulfonyl esters, phosphate esters, phosphines, methylenes, methines, carboxyl amides, carboxyl esters, and imidates; and molecules having chains joining the end groups that are, or are combinations of, linear alkanes, linear alkenes, alkynes, di- and multi- substituted phenyl rings, di- and multi- substituted napthalene rings, di- and multi-substituted cyclohexyl rings, di- and multi- substituted decalin rings,
  • EXAMPLE 1 QUANTITATIVE ANALYSIS
  • Various relationships known to those skilled in the art can be used to rank and select the pharmacophores, vectors, and linkers contained in a database of the invention. Examples of such relationships are provided in FIG. 40. 5.2.
  • EXAMPLE 2 DESIGN OF AN ANTIFUNGAL VLP CONJUGATE
  • VLP conjugate useful in the treatment of fungal infections.
  • the vector in this conjugate is sordarin, and the pharmacophore is fluconazole.
  • Step 1 Building a database i. Choose the Disease Condition
  • VLP conjugate that is active against fungi, which are the disease organisms in this case.
  • Step 2 Making Selections from the Database i. Vector and Vector Target
  • Choice of the vector target Fungi are chosen as the demonstration microorganisms.
  • the fungal protein Elongation Factor 2 (EF2) is chosen as the vector target.
  • Elongation Factor 2 functions as part of the ribosomal protein synthesis machinery. It is present at around the same high copy number as the ribosome within the cell. The protein is present in the cytosol of the cell.
  • all eukaryotes contain proteins with the function of EF2
  • experimental work with the sordarin family of antibiotics demonstrated that these small molecules interact specifically with the fungal protein. See, e.g., B. Tse, J. M. Balkovec, C. M. Blazey, M-J. Hsu, J. Neilsen, and D. Schmatz, Bioorganic and Medicinal Chemistry Letters 8:2269-2272 (1998).
  • Choice of the vector A modified version of sordarin is chosen as the test vector.
  • Sordarin reportedly interacts specifically with EF2. As such, its site of interaction is the fungal cytosol. It exhibits antifungal activity at concentrations between micromolar and nanomolar. Binding of sordarin to EF2 inhibits fungal protein synthesis. Sufficient structure activity relationship data exist to indicate that a long aliphatic chain can be attached to the sordarin system without disrupting its antifungal effect, and this implies that binding to EF2 will not be disrupted. Sordarin is a weakly polar molecule, and probably gains access to its site of action by diffusion through the fungal cell wall. The molecule is not expected to be highly water soluble. The molecular weight of sordaricin (sordarin with its sugar moiety removed) is 332 daltons.
  • Sordarin derivatization Methodology in the literature describes the removal of the sugar moiety from sordarin and the attachment of a long aliphatic chain. Molecules that are so modified retain antifungal activity. The experimental plan for modification of sordarin, to produce thiopropyl and aminopropyl sordarin, is described in the experimental section in step 4. These structural modifications will allow facile attachment of linker molecules with a high probability that the attached linkers will not interfere with the molecule's interaction with fungal EF2.
  • Linking derivatized sordarin to probe molecules and model pharmacophores Candidate linkers are obtained from commercial sources. FIG. 5 shows a table of representative linkers. Candidate probes are obtained from commercial sources. FIG.
  • Linkers are chosen that should not limit the aqueous solubility of the VLP conjugate or its ability to diffuse into the fungal cytosol.
  • Probe molecules model pharmacophores
  • the experimental plan for construction of model VLP conjugates using sordarin, linkers, and a probe molecule is described below in step 4.
  • Assays of activity of model VLP conjugates of sordarin and probe molecules are conducted as described below in step 4.
  • Fluconazole an azole antifungal, was chosen as the test pharmacophore.
  • FIG. 10 shows the structure of fluconazole.
  • Fluconazole is a competitive inhibitor of the fungal enzyme lanosterol 14C demethylase. Fluconazole reportedly binds to the enzyme lanosterol 14C demethylase at concentrations in the submicromolar range. Inhibition of this enzyme disrupts the synthesis of ergosterol, a steroid that is essential for the formation of fungal cell membrane. Inhibition also causes a harmful buildup of intermediates that lie on the fungal synthetic pathway to ergosterol.
  • Fungal lanosterol 14C demethylase the pharmacophore target, is located on the endoplasmic reticulum membrane, presumably on the cytosolic side of the membrane. Sufficient structure activity relationship data exist to indicate that considerable structural modifications can be made to the azole system without disrupting its antifungal effect, or binding to lanosterol 14C demethylase.
  • Fluconazole is a moderately polar molecule, and probably gains access to its site of action by diffusion through the fungal cell wall. The molecule is significantly water soluble. The molecular weight of fluconazole is 306 daltons.
  • Fluconazole synthesis and derivatization Methodology in the literature demonstrates the synthesis of fluconazole and similar azole antifungals. It also indicates that azole antifungals can have considerable structural elaboration, presumably including addition of a linker chain, while retaining antifungal activity.
  • FIG. 5 shows a table of representative linkers.
  • FIG. 7 shows a diagram of representative fluorescent probes (model pharmacophores), which are small fluorescent molecules. Probes may also be small molecules that inco ⁇ orate a radioisotope. These are obtained from commercial sources. The experimental plan for attachment of linkers and probe molecules to fluconazole is described in step 4.
  • step 4 Assays to determine antifungal activity of the combination of the fluconazole vector, with a linker and a probe molecule are described in step 4. Assays to determine the concentrating effect of the combination of the fluconazole vector, with a linker and a probe molecule are also described in step 4.
  • FIG. 5 shows a table of representative linkers. The experimental plan for the synthesis of conjugates of sordarin with fluconazole is described in step 4.
  • VLP conjugates consisting of the combination of the sordarin vector, a linker, and the fluconazole pharmacophore, are conducted as described step 4. The structure of one of these is shown in FIG. 11.
  • Step 4 Building and Testing the VLP Conjugate
  • FIG. 12 details the construction of the sordarin-linker- fluconazole conjugates, the components of which have been selected above in steps 1-3 of this example. Two different lengths of linker are proposed.
  • the "compound/scheme” notation used herein refers to the steps of the synthesis as shown in FIG. 12, which also details construction of test (or surrogate) conjugates with a dansyl group as a flourescent probe attached to either the sordarin vector or fluconazole pharmacophore. Again, two different lengths of linker are proposed.
  • the experiments and assay methods following the construction scheme are proposed to test the sordarin-linker-fluconazole (VLP) construct and the surrogate conjugates.
  • Sordarin (compound 1. scheme 1): This material is prepared by fermentation of Sordaria araneosa as reported in D. Hauser and H. P. Sigg, Helvetica Chimica Acta 54:1178-1190 (1971).
  • Sordaricin paramethoxybenzyl ester (compound 2. scheme 1): This material is prepared by the method reported by B. Tse, J. M. Balkovec, C. M. Blazey, M-J. Hsu, J. Neilsen, and D. Schmatz, Bioorganic and Medicinal Chemistry Letters 8:2269-2272 (1998).
  • Iodopropyl sordarin paramethoxybenzyl ester (compound 3. scheme 1): This compound is prepared by a method analogous to the alkylation procedure of Tse, et. al. Thus, sordaricin paramethoxybenzyl ester (compound 2, scheme 1) (904 mg, 2 mmol) is dissolved in dimethylformamide (30 ml) and diiodopropane (5.9 g, 20 mmol) and sodium hydride (240 mg of a 60% dispersion, 6 mmol) is added. The mixture is stirred overnight at room temperature. Ethyl ether (100 ml) is added and the solution extracted with water (3x, 100 ml).
  • mercaptopropylsordarin paramethoxybenzyl ester (compound 4, scheme 1) (0.53 g, 1 mmol) is dissolved in methanol (90 ml) and a hydrogenation catalyst (for example, palladium hydroxide on charcoal) is added. The mixture is stirred under hydrogen (using a hydrogen balloon). Progress of the reaction is followed by thin layer chromatography on silica gel. When the hydrogenation is complete, the reaction mixture is filtered, the solvent removed on the rotary evaporator and the product dried under vacuum. It may be purified by chromatography on silica gel or used directly.
  • a hydrogenation catalyst for example, palladium hydroxide on charcoal
  • mercaptopropylsordarin paramethoxybenzyl ester (0.53 g, 1 mmol) is dissolved in water-acetone (100 ml) and hydrochloric acid (12 N, 10 ml) is added. The reaction mixture is stirred at room temperature or is heated, and the progress of the reaction followed by thin layer chromatography. When the reaction is complete, the product is dissolved in methylene chloride (50 ml) then extracted with water (3x, 100 ml). It is purified by chromatography on silica gel with chloroform-ethyl acetate-pentane as the eluent.
  • Aminopropylsordarin paramethoxybenzyl ester (compound 1. scheme 2).
  • Iodopropyl sordarin paramethoxybenzyl ester compound 3. scheme 1: (1.24 g, 2 mmol) is dissolved in ether and a solution of ammonia dissolved in ether (about 0.34 g, 20 mmol of ammonia) is added. The progress of the reaction is followed by thin layer chromatography. When the reaction is complete, The reaction mixture is extracted with water (3x, 100 ml). The solvent is removed on the rotary evaporator and the residue dried under vacuum.
  • the crude product may be used directly in the next step or may be dissolved in a minimal volume of ethyl acetate-pentane and chromatographed on silica gel with chloroform-ethyl acetate-pentane as the eluent. The solvent is then removed on the rotary evaporator and the product dried under vacuum.
  • Aminopropylsordarin (compound 2, scheme 2): The paramethoxybenzyl ester is removed from aminopropylsordarin paramethoxybenzyl ester (compound 1, scheme 2) by the methods used for mercaptopropylsordarin (compound 5, scheme 1) above.
  • 5-(4-Methoxybenzylthio)-l-imido valeric acid ethyl ester hvdrochloride (compound 2. scheme 3): This compound is prepared by an adaptation of the method of Reynaud and Moreau. Thus 5-(4-methoxybenzylthio)valeronitrile (compound 1, scheme 3) (23.5 g. 100 mmol) is dissolved in toluene (125 ml) and methanol (23 ml) is added. The solution is cooled in an ice bath and hydrogen chloride gas is added at atmospheric pressure over 3 hours. The reaction mixture is stirred under hydrogen chloride overnight at room temperature.
  • reaction mixture is stirred at room temperature and progress of the reaction is followed by thin layer chromatography.
  • the reaction mixture is filtered, then the filtrate washed with water (3x, 100 ml).
  • the organic solution is dried over magnesium sulfate, and the solvent removed on the rotary evaporator.
  • the residue is dried under vacuum and the product is purified by chromatography on silica gel with pentane-ethyl acetate-chloroform as the eluent.
  • 5 -Amino val eronitrile This compound is prepared by dissolving 5-bromovaleronitrile (16.2 g, 100 mmol) in chloroform (200 ml). The solution is cooled in an ice bath and a solution of anhydrous ammonia (17.0 g, 1.0 mol) dissolved in chloroform (200 ml) is slowly added. When the addition is complete, the reaction is stirred and allowed to warm to room temperature. Progress of the reaction is followed by thin layer chromatography. When the reaction is complete, the solvent is removed on the rotary evaporator and the residue dried under vacuum. It is resuspended in ethyl ether, and suction filtered. The solvent is removed on the rotary evaporator and the residue dried under high vacuum. The crude material may be used without further purification, or purified by chromatography on silica gel.
  • reaction mixture is washed with water (3x, 100 ml) and the organic layer dried over magnesium sulfate.
  • the solvent is removed on the rotary evaporator and the residue dried under vacuum.
  • the crude material is purified by chromatography on silica gel.
  • reaction mixture is cooled to room temperature.
  • Water (180 ml) is added and the mixture extracted twice with chloroform (2x, 60 ml).
  • the combined chloroform extracts are washed with water (2x, 100 ml), dried over magnesium sulfate, filtered, and the solvent removed on the rotary evaporator.
  • the product is purified by chromatography on silica gel with pentane-ethyl acetate-chloroform as the eluent.
  • the solvent is removed on the rotary evaporator and the residue dried under vacuum.
  • the product is purified by chromatography on silica gel with pentane-ethyl acetate-chloroform as the eluent.
  • the combined chloroform extracts are washed with water (2x, 100 ml), dried over magnesium sulfate, filtered, and the solvent removed on the rotary evaporator.
  • the product is purified by chromatography on silica gel with pentane-ethyl acetate-chloroform as the eluent.
  • reaction mixture is filtered, the filtrate washed with ethanol, and the solvent removed from the combined ethanol washings on the rotary evaporator.
  • the residue is purified by chromatography on silica gel with pentane ethyl acetate-chloroform as the eluent.
  • scheme 7 A, scheme 7B1 This compound is prepared from mercaptopropylsordarin (compound 5, scheme 1) and 2-(2,4-difluorophenyl)-l- ( 1 H- 1 ,2,4-triazol- 1 -yl)-3 -(3 -(4-aminobutyl)- 1 H- 1 ,2,4-triazol- 1 -yl)propan-2-ol (compound 4, scheme 6).
  • succinimidyl 6-[6-(((iodoacetyl)amino)hexanoyl)amino]hexanoate (SIAXX, 0.51 g, 1.0 mmol) or succinimidyl 6-((iodoacetyl)amino)hexanoate (SIAX, 0.39 g, 1.0 mmol) is dissolved in dry methylene chloride (20 ml) and mercaptopropylsordarin (compound 5, scheme 1) (0.37 g, 1.0 mmol) is added. The reaction mixture is stirred at room temperature and its progress is followed by thin layer chromatography.
  • succinimidyl 6-[6-(((iodoacetyl)amino)hexanoyl)amino]hexanoate (SIAXX, 0.51 g, 1.0 mmol) or succinimidyl 6-((iodoacetyl)amino)hexanoate (SIAX, 0.39 g, 1.0 mmol) is dissolved in dry methylene chloride (10 ml) and mercaptopropylsordarin (compound 5, scheme 1) (0.37 g, 1.0 mmol) is added. The reaction mixture is stirred at room temperature and its progress is followed by thin layer chromatography.
  • dansyl chloride (2.69 g, 10 mmol) is dissolved in dry methylene chloride (100 ml) and 1,4-diaminobutane (8.8 g, 100 mmol) is added. The reaction mixture is stirred at room temperature and its progress is followed by thin layer chromatography. When the reaction is complete, the reaction mixture is washed with aqueous sodium hydroxide (0.5 N), then water. It is dried over sodium sulfate, then filtered. The solvent is removed on the rotary evaporator, and the residue dried under vacuum. The crude material is used without further purification.
  • succinimidyl 6-[6-(((iodoacetyl)amino)hexanoyl)amino]hexanoate (SIAXX, 0.51 g, 1.0 mmol) or succinimidyl 6-((iodoacetyl)amino)hexanoate (SIAX, 0.39 g, 1.0 mmol) is dissolved in dry methylene chloride (20 ml) and 2-(2,4-difluorophenyl)-l-(lH-l,2,4-triazol- l-yl)-3-(3-(4-thiobutyl)-lH-l,2,4-triazol-l-yl)propan-2-ol (compound 2, scheme 6) (0.39 g, 1.0 mmol) is added.
  • the reaction mixture is sti ⁇ ed at room temperature and its progress is followed by thin layer chromatography.
  • the reaction product of dansyl chloride and 1,4-diaminobutane is dissolved in methylene chloride (10 ml) and one tenth of this material (1.0 mmol) is added.
  • the reaction is again sti ⁇ ed at room temperature and its progress followed by thin layer chromatography.
  • the solvent is removed on the rotary evaporator and the residue dried under vacuum. The residue is purified by high performance liquid chromatography.
  • VLP conjugates which contain vector moieties that bind to targets not usually present in high concentrations in bacteria.
  • bacteria can be modified by placing the genes for the vector targets on plasmids such that the target proteins are expressed in the bacteria at a high concentration. This approach therefore demonstrates that VLP conjugates can be designed which will target diseased cells to the exclusion of non-diseased cells. Construction of Antibacterial Vector/Pharmacophore Tables
  • Ligand Tables Information on bacterial protein ligands is gathered from the literature. This information preferably includes: molecular weight, receptor(s), mode of inhibition, association constant (Ka), IC50, binding sites, and MIC 50 . This information is organized into a tabular format. An example is given in FIGS. 13a-d.
  • Ligand Receptor Tables Information on ligands that bind to bacterial proteins is gathered from the literature. This information preferably includes: species, receptor concentration, molecular weight (daltons), cellular compartment, solubility, natural substrate, function, and three-dimensional structure. An example is given in FIG. 13e.
  • ligands are separated into vectors and pharmacophores.
  • a ligand For a ligand to be considered a good vector, it must have (1) a high receptor concentration in the cellular compartment being targeted and (2) a high association constant for its receptor.
  • a concentrating factor is calculated for each ligand. The concentrating factor is determined by first multiplying the number of receptor molecules in the cell by the number of ligand binding sites on the receptor to give the number of ligand binding sites per cell.
  • Ligands with concentrating factors above 50 are designated vectors.
  • Ligands with concentrating factors below 50 are designated pharmacophores. An example of this process is given in FIG. 13f.
  • the ligands are then ranked according to concentrating factor.
  • Ligands with high concentrating factors will efficiently concentrate a wider range of pharmacophores than ligands with low concentrating factors.
  • a ligand with a low concentrating factor can serve as a vector for a pharmacophore with a relatively high association constant or low MIC 50 .
  • all ligands with concentrating factors above 50 are designated as vectors, and all ligands with concentrating factors below 50 as designated as pharmacophores.
  • An example vector table is given in FIG. 13g.
  • An example pharmacophore table is given in FIG. 13h.
  • This table gives the possible vector/pharmacophore pairs that satisfy the inequalities given above.
  • a similar set of computations can be performed for other promising cytosolic vectors.
  • SAR data is collected for each pharmacophore, with special attention to pharmacophores whose activity is unaffected by the attachment of a long chain.
  • promising V/P pairs are ranked according to the combined molecular weights of the vector and pharmacophore. Those with the lowest combined molecular weight are then investigated as potential VLP conjugates.
  • VLP conjugates are shown in FIG. 14-17, and discussed in detail below. These conjugates are designed by first selecting a disease state (bacterial infection), and then pharmacophores that are reportedly active against it.
  • the selected anti-bacterials include: penicillin, tetracycline and trimethoprim.
  • penicillin tetracycline
  • trimethoprim For each pharmacophore, sufficient structure activity data was present to allow a good estimate of one or more locations on the pharmacophore molecule to which a linker can be attached without significant disruption to the activity. These sites of action are in the bacterial periplasmic space for penicillin and in the bacterial cytosol for tetracycline and trimethoprim.
  • model bacterial vector targets two proteins are chosen as model bacterial vector targets, one that can be expressed in the periplasm and one that can be intracellularly expressed, since interaction with these targets can thus result in accumulation of the respective antibiotics at their sites of action.
  • bacteria contain unique targets, the genes for these model target proteins can be introduced on plasmids, and the level of expression of the proteins can be controlled.
  • vector targets with high levels of expression are desirable, and in these systems, expression can be adjusted to high levels.
  • the vectors for these targets are then chosen.
  • the tightly binding molecule biotin can be used as a vector.
  • beta galactosidase a substrate, which is a galactose based mechanism based inhibitor, can be used as a vector.
  • Biotin binds to avidin with a dissociation constant of around 10 "14 molar. Sufficient data exists to indicate that the mechanism based beta galactosidase inhibitor chosen can be processed at a rate resulting in a change in the bound inhibitor concentration of at least 10 "9 molar per second.
  • the pharmacophores are then paired with their respective vectors.
  • the minimum concentration at which the antibiotic can exert an effect is known.
  • the expected concentration of the vector target, and the dissociation constant of the vector from its target or the expected rate of enzyme processing of its substrate it is possible to estimate the solution concentration of the VLP conjugate at which the antibiotic pharmacophore can be concentrated to an effective level at its site of action. This is substantially lower than that required by the unconjugated pharmacophore molecule.
  • Only selection of the linkers remains. These are selected to have polarity and solubility similar to that of the vectors and pharmacophores.
  • Linker lengths of 15 to 20 bonds are selected. Linker length may need to be optimized, however, to allow the interaction of the VLP conjugate with both the vector target and pharmacophore target. This is achieved by routine methods known to those skilled in the art.
  • 6-(5-oxopentyl)aminopenicillanic acid (compound 1 FIG. 18): (+) 6-Aminopenicillanic acid (1.08 g, 5 mmol) is dissolved in water-methanol (50 miililiters), with the pH adjusted to 8-9 with aqueous sodium bicarbonate. An excess of freshly distilled glutardialdehyde (10.0 g, 100 mmol), will then be added. The mixture is stirred for 1 hour at room temperature, then the solvent is removed on a rotary evaporator, and the residue dried under vacuum, washed with methylene chloride, and redried under vacuum. This material is used in the next step without further purification.
  • 6-(5-biocvtinhydrazidopentyl " )aminopenicillanic (compound 2 FIG. 18): The material is prepared by the general methods in Greg T. Hermanson, Bioconjugate Techniques (Academic Press, New York: 1996) ("Hermanson”).
  • the crude 6-(5- oxopentyl)aminopenicillanic acid (compound 1 FIG. 18), from the above reaction, is dissolved in water, and the pH is adjusted to 8-9 with aqueous sodium bicarbonate.
  • biocytin-LC-hydrazide (1.93 g, 5 mmol). The mixture is stirred for 1 hour at room temperature, then cooled in an ice bath.
  • This material is purified by high performance liquid chromatography on silica gel with ethyl acetate- methylene chloride-triethyl amine as the eluant or by reverse phase high performance liquid chromatography with water-acetonitrile as the eluant, or by ion exchange chromatography using a sulfate or carboxylate ion exchange resin.
  • 6-(5-(6-(biotinyl)-aminocaproyl)-hvdrazidopentyl)-aminopenicillanic compound 3 FIG. 18:
  • the material is prepared by the general methods in Hermanson.
  • the crude 6- (5-oxopentyl)aminopenicillanic acid compound 1 FIG. 18
  • the pH is adjusted to 8-9 with aqueous sodium bicarbonate.
  • biotin-LC -hydrazide (1.86 g, 5 mmol). The mixture is stirred for 1 hour at room temperature, then cooled to around 0 degrees in an ice bath.
  • This material is purified by high performance liquid chromatography on silica gel with ethyl acetate-methylene chloride-triethyl amine as the eluant or by reverse phase high performance liquid chromatography with water- acetonitrile as the eluant, or by ion exchange chromatography using a sulfate or carboxylate ion exchange resin.
  • 6-(6-(biotinyl)-aminocaproyl)aminopenicillanic acid (6(LC- biotin)aminopenicillanic acid (compound 4 FIG. 19): The material is prepared by the general methods in Hermanson. (+) 6-Aminopenicillanic acid (1.08 g, 5 mmol) is dissolved in water-methanol (50 milliliters), with the pH adjusted to 8-9 with aqueous sodium bicarbonate. NHS-LC-biotin (2.28 g, 5 mmol) dissolved in methanol is added and the mixture is stirred for one hour or until thin layer chromatography shows the reaction to be complete. The pH is maintained at around 8 by additions of sodium bicarbonate solution. The solvent is removed under vacuum.
  • the product is purified by reverse phase high performance liquid chromatography with a linear gradient of water-acetonitrile as the eluant, or by high performance liquid chromatography on silica gel with methylene chloride-ethyl acetate-tri ethyl amine as the eluant, or by high performance ion exchange liquid chromatography with a sulfate or carboxylate ion exchange resin.
  • reaction is stirred at room temperature for 1 hour, then N-iodoacetyl-N-biotinylhexylenediamine (iodoacetyl-LC- biotin, 2.55 g, 5 mmol), dissolved in methanol, is added and the reaction is sti ⁇ ed for an additional hour or until thin layer chromatography shows the reaction to be complete.
  • iodoacetyl-LC- biotin 2.55 g, 5 mmol
  • the solvent is then removed under vacuum, and the product purified by high performance liquid chromatography on silica gel with chloroform-ethyl acetate-triethyl amine as the eluant or reverse phase high performance liquid chromatography on silica gel with a linear gradient of water-acetonitrile as the eluant, or by high performance ion exchange liquid chromatography with a sulfate or carboxylate ion exchange resin.
  • Biotin-penicillin conjugates may be tested for antibacterial activity against strains of E. coli K 12 containing cloned chicken avidin, that will either be expressed and secreted into the periplasmic space or is delivered to the outer cell membrane as a fusion protein with the transmembrane portion of the aspartate receptor.
  • avidin is the vector receptor
  • biotin is the vector
  • penicillin is the antibiotic pharmacophore.
  • the gene for avidin is carried by a plasmid and expression of the protein to a high concentration is induced by materials added to the growth medium. Testing of inhibition of bacterial growth is performed with a series of increasing concentrations of the biotin-penicillin conjugate, alone in the growth medium.
  • a series of increasing concentrations of an unconjugated penicillin derivative, ampicillin is also be tested.
  • a control experiment is also be performed, in which a sufficiently high concentration of free biotin is added to the medium to saturate all avidin expressed by the bacteria, in the presence of the biotin penicillin conjugates.
  • the degree of bacterial growth is measured in the presence of a series of increasing concentrations of the biotin- penicillin conjugate, and a parallel measurement is performed with a series of increasing concentrations of the unconjugated penicillin derivative, ampicillin.
  • the conjugates is tested against a strain of E. coli K 12 that does not express cloned chicken avidin.
  • biotin-penicillin conjugates is pre-treated with beta lactamase, then tested as above. Again, in both cases, a parallel experiment is performed with ampicillin. In each case, bacterial growth in the presence of increasing concentrations of the conjugate or of unconjugated antibiotic is measured, and plotted as the curves seen in the accompanying graph (FIG. 21).
  • unconjugated penicillins such as ampicillin
  • the biotin-penicillin conjugates should inhibit bacterial growth at a significantly lower concentration range (10 to 100 fold lower).
  • the biotin-penicillin conjugates should inhibit bacterial growth at around the same concentration range as the unconjugated penicillins.
  • beta lactamase neither the biotin-penicillin nor the unconjugated penicillins should show any inhibition of growth.
  • 2,4-diamino-5-(3,5- dimethoxy-4-hydroxybenzyl)pyrimidine (compound 2 FIG. 22, 3.5 g, 12.7 mmol) is dissolved under nitrogen in dry dimethylsulfoxide (50 ml) by warming to 50 degrees. After cooling the solution, sodium methoxide (0.69 g, 12.7 mmol) is added under nitrogen and when this has been dissolved, 1,3-dichloropropane (1.44 g, 12.7 mmol) is added, under nitrogen. The reaction mixture is allowed to stand at room temperature under nitrogen for 4 hours, then neutralized with acetic acid. The solvent is removed under vacuum and the residue washed with aqueous sodium hydroxide solution, then water. The product is dried under vacuum, and optionally purified by high performance liquid chromatography. Melting point should be 163-164 degrees.
  • 9-Bromoacetamido-5-hvdroxy-6-deoxytetracvc ⁇ ine (compound 5 FIG. 24): This compound is prepared by the method of Barden, et. al. Thus 9-amino-5-hydroxy-6- deoxytetracycline (compound 4 FIG. 24, 3.43 g, 7.47mmol) is mixed in N- methylpyrrolidin-2-one (40 ml) with sodium bicarbonate (2.0g, 23.8 mmol) at room temperature. Bromoacetyl bromide (0.75 ml, 8.62 mmol) is added and the solution sti ⁇ ed for 30 minutes.
  • the material is flash chromatographed on silica gel with chloroform-mefhanol as the eluant and the solvent removed under vacuum.
  • the material is then dissolved in water or methanol, and 2- iminothiolane (Traut's reagent, 0.30 g, 2.2 mmol) is added.
  • the solution is stirred at room temperature for 1 hour and the solvent removed under vacuum.
  • the crude material is washed with acetone then ethyl ether and dried under vacuum.
  • the product may be purified by high performance liquid chromatography using a Hamilton PRP-1 column and a linear gradient of water-acetonitrile with added 0.1% trifluoroacetic acid.
  • N-Hydroxymethylmaleimide (compound 8 FIG. 25): This material is prepared by the method in Martell, M.J., et al, J. Med. Chem. 10:359-363 (1967) (referencing P. O. Tawney, P.O., et al, J.O.C 26:15 (1961).
  • aqueous formaldehyde (0.81 ml, 11 mmol)
  • aqueous sodium hydroxide 0.3 ml to adjust the pH to ca. 7.5.
  • the solid should dissolve and the solution is allowed to stand at room temperature for 3 hours.
  • the solvent is then removed under vacuum to leave an oil which can be crystallized.
  • the product may be further purified by sublimation.
  • the precipitate is dissolved in water (18 ml) and the pH adjusted to 5.0 with 1 N sodium hydroxide. The resulting precipitate is filtered, washed with two portions of water (1 ml each) and dried under vacuum.
  • the product may be purified by reverse phase high performance liquid chromatography using a linear gradient of water- acetonitrile.
  • MECHANISM BASED INHIBITOR MBP AS A VECTOR l-Bromo-2.3.4.6-tetraacetyl-beta-D-galactose (compound 1 FIG. 26): This material is prepared by a method analogous to that of Halazy, S., et al, Bioorganic Chem.
  • FIG. 26 This material is prepared by the method of Janda, K.D., et al, Science 275:945- 948 (1997) according to the method of Halazy, S., et al, Bioorganic Chem. 18:330-344 (1990).
  • a solution of 5-nitrosalicylaldehyde (7.0 g, 42 mmol) in dichloromethane is vigorously stirred at room temperature with 5% sodium hydroxide (70 ml) and tetramethylammonium bromide (2.26 g, 7 mmol).
  • This material is prepared by the method of Janda et.al. according to the method of S. Halazy et. al.
  • l-(2-difluoromethyl-4-aminophenoxy)-2,3,4,6-tetraacetyl- ⁇ -D- galactose (compound 4 FIG. 26, 0.86 g, 1.75 mmol) is dissolved at 20 degrees in methanol (15 ml) to which sodium methoxide (13 mg) had been added.
  • the reaction mixture is allowed to stand for 2 hours, then neutralized with IN hydrochloric acid (0.2 ml) and filtered.
  • the solvent is removed from the filtrate under vacuum. This should yield the product (around 0.54 g, 95%).
  • the material may be further purified by reverse phase chromatography with a linear gradient of water-acetonitrile as the eluant.
  • l-(2-Difluoromethyl-4-(6-(6-(((iodoacetyl)amino)-hexanoyl)amino)hexanoyl)- amidophenoxy)- ⁇ -D-galactose compound 6 FIG. 26
  • l-(2-Difluoromethyl-4- aminophenoxy)- ⁇ -D-galactose compound 5 FIG. 26, 0.32 g, 1 mmol
  • methanol-water or it is dissolved in 100 mm sodium bicarbonate solution, and methanol is added.
  • Succinimidyl 6-(6-(((iodoacetyl)amino)-hexanoyl)amino)hexanoate (0.39 g, 1 mmol) is then added, dissolved in methanol or ethanol.
  • the solution is sti ⁇ ed at room temperature, and the progress of the reaction monitored by thin layer chromatography.
  • the solvent is removed under vacuum, and the residue dried under vacuum.
  • the material may be purified by reverse phase chromatography with a linear gradient of water- acetonitrile as the eluant.
  • Succinimidyl 6-(6-(((iodoacetyl)amino)- hexanoyl)amino)hexanoate (0.39 g, lmmol) is then added.
  • the solution is sti ⁇ ed at room temperature, and the progress of the reaction is monitored by thin layer chromatography.
  • the solvent is removed under vacuum, and the residue dried under vacuum.
  • the material is purified by chromatography on silica gel with petroleum ether-chloroform as the eluant.
  • EXAMPLE 9 SYNTHESIS OF MBI/BGM CONJUGATES Trimethprim-beta galactosidase MBI and tetracycline-beta galactosidase MBI conjugates can be used as pharmacophores, to which can be attached beta galactosidase mechanism (BGM) based inhibitors as a vector to form VLP conjugates of the invention.
  • BGM beta galactosidase mechanism
  • the solvent is removed under vacuum with minimal heating, and the residue is dried under vacuum.
  • the product is purified by reverse phase high performance liquid chromatography with a linear gradient of water-acetonitrile as the eluant, or high performance ion exchange liquid chromatography using a sulfate or carboxylate ion exchange resin.
  • the crude starting materials may also be used in the coupling reaction.
  • the solvent is removed under vacuum with minimal heating, and the residue is dried under vacuum.
  • the product may be purified by high performance liquid chromatography on silica gel, reverse phase high performance liquid chromatography with a linear gradient of water- acetonitrile as the eluant, or high performance ion exchange liquid chromatography using a sulfate or carboxylate ion exchange resin.
  • the product may also be deacylated, as below, before purification.
  • the crude starting materials may also be used in the coupling reaction.
  • the material from the above coupling reaction is dissolved in a small volume methanol or methanol-water to which sodium methoxide (around 0.15 equivalents) had been added.
  • the reaction mixture is allowed to stand for 2 hours, and the pH checked periodically, and adjusted to remain basic, as necessary, by the addition of further sodium methoxide.
  • the reaction is then neutralized with IN hydrochloric acid and filtered.
  • the solvent is removed from the filtrate under vacuum to yield the crude product, which can be purified by reverse phase high performance liquid chromatography with a linear gradient of water-acetonitrile as the eluant, or high performance ion exchange liquid chromatography using a sulfate or carboxylate ion exchange resin.
  • EXAMPLE 10 PROPERTIES OF MBI/BGM CONJUGATES Conjugates of a mechanism based inhibitor (or MBI) of beta-galactosidase and tetracycline or trimethoprim may be tested for antibacterial activity against strains of E. coli K 12 containing cloned beta galactosidase, which is expressed in soluble form in the bacterial cytosol.
  • the beta galactosidase is the vector receptor
  • the MBI galactose analog is the vector
  • tetracycline or trimethoprim is the antibiotic pharmacophore.
  • the gene for beta galactosidase is carried by a plasmid and expression of the enzyme to a high concentration is induced by materials added to the growth medium. Testing of inhibition of bacterial growth is performed with a series of increasing concentrations of the MBI galactose analog-antibiotic conjugate, alone in the growth medium. In parallel, a series of concentrations of the unconjugated antibiotic tetracycline or triemthoprim is also be tested. A control experiment is also performed, in which a sufficiently high concentration of lactose, or of a galactose analog that is not conjugated to an antibiotic, is added to the medium in order to saturate all beta galactosidase expressed by the bacteria, in the presence of the vector-antibiotic conjugates.
  • the degree of bacterial growth is measured in the presence of a series of increasing concentrations of the MBI galactose analog-antibiotic conjugate, and in parallel, a series of increasing concentrations of the unconjugated antibiotic tetracycline or trimethoprim is tested. Additionally, the conjugates is tested against a strain of E. coli K 12 that does not express any beta galactosidase, and in parallel, the unconjugated antibiotic tetracycline or trimethoprim is also be tested. In each case, bacterial growth in the presence of increasing concentrations of the conjugate, or of the unconjugated antibiotic, is measured, and plotted as curves. Calculated curves are shown in FIG. 21. For the E.
  • unconjugated antibiotics should inhibit bacterial growth at the expected concentration ranges (around 1-10 ug/ml for tetracycline and 0.01-0.1 ug/ml for trimethoprim).
  • the MBI galactose analog- antibiotic conjugates should inhibit bacterial growth at a significantly lower concentration range (10 to 100 fold lower).
  • the MBI galactose analog-antibiotic conjugates should inhibit bacterial growth at around the same concentration range as the respective unconjugated antibiotics.
  • EXAMPLE 11 MEASURING ANTIBIOTIC ACTIVITY
  • the antibiotic activity of each VLP conjugate is performed upon several representative, non-pathogenic gram positive and gram negative strains of bacteria using the following methods. For compounds expected to have enhanced antibiotic activity against actual pathogens, these measurements can constitute a preliminary screening procedure. Bacteria, stored as frozen cultures, is streaked onto LB agar plates, containing any necessary antibiotics, and is grown at 37 degrees in an incubator. On the day before a measurement, individual colonies is picked and used to inoculate LB medium (5 or 25 ml) containing any necessary antibiotics or supplemental nutrients, in culture tubes or Erlenmeyer flasks. These are shaken overnight at 37 degrees to produce saturated cultures.
  • LB medium 5 or 25 ml
  • Serial dilutions of the antibiotic or test substance are made in water or the appropriate organic solvent.
  • aliquots of these dilutions are added to culture tubes containing LB media (5 or 10 ml) containing any necessary additional nutrients or activators or inhibitors of antibiotic or test substance activity, as required by the experiment.
  • the tubes are inoculated with aliquots (50 to 100 ul) of the bacterial culture, and shaken in an incubator, usually at 37 degrees. At intervals of several hours, for a period of up to several days, they are scored visually for bacterial growth. Additionally, aliquots may be withdrawn and the absorbance at 550 nm determined, for a more precise spectrophotometric determination of the cell concentration.
  • Serial dilutions of the antibiotic or test substance are made in water or the appropriate organic solvent.
  • Molten LB agar is prepared, containing any necessary additional nutrients or activators or inhibitors of antibiotic or test substance activity, as required by the experiment. It is cooled below 60 degrees and aliquoted into pre-warmed culture tubes.
  • To each tube is added an aliquot of one of the set of serial dilutions of the antibiotic or test substance. As soon as the aliquot has been added to a tube, the contents of the tube is mixed rapidly with a vortex mixer, and poured into a petri dish. This is covered, let stand to allow the agar to harden, and dried briefly in an incubator.
  • Aliquots of the bacterial culture (50 to 100 ul), prepared as above, are spread onto the medium in each petrie dish, and these are covered and placed in an incubator. After 24 hours or more, the number of bacterial colonies growing on each plate are counted. To determine the degree of cell killing, as opposed to growth stasis, aliquots of a growing or saturated bacterial culture are treated with concentrations of antibiotic or test substance sufficient to halt growth. After an incubation period, the cells from each aliquot of culture are collected by centrifugation, washed twice in fresh LB medium (without antibiotic), and resuspended in fresh LB medium (without antibiotic, 5 or 10 ml). Aliquots of these suspensions are then spread onto fresh LB plates, that also have no added antibiotic.
  • the experimental data is inte ⁇ reted as follows.
  • the growth of E.coli should be inhibited by the unconjugated antibiotic at the concentration expected for each antibiotic.
  • the antibiotics and bacteria thus should behave normally.
  • the growth of E.coli should be inhibited by significantly lower concentrations of the conjugates because the vector should cause the conjugate molecule, and thus the pharmacophore, to accumulate in the bacteria to a concentration that can inhibit growth or kill the bacteria, when the concentration of conjugate in the medium was sufficient for significant amounts of the vector to become bound to the vector receptor. This is the effect expected for vector reagents.
  • the conjugate When there is no vector receptor in the bacteria or when the vector receptor is saturated by a molecule which is not the conjugate, the conjugate cannot accumulate in the bacteria to a concentration higher than it can have in the su ⁇ ounding medium because it cannot bind to the vector receptor. Under these conditions, the concentration at which the conjugate exerts an effect can be about the same as the concentration at which the pharmacophore alone is effective. Finally, when the antibiotic portion (pharmacophore) of the conjugate molecule is converted to an inactive form, the conjugate molecule is expected to show no inhibition of bacterial growth, demonstrating that the vector and linker portions of the conjugate are devoid of antibiotic activity.
  • vector reagents are expected to exhibit the pharmacological activity of the unconjugated pharmacophore, but at a concentration much lower than that needed by the unconjugated pharmacophore.
  • EXAMPLE 12 ELONGATION FACTOR BINDING MOLECULE.
  • the bacterial elongation factor EF Tu which is present at the high concentration of around 10 "4 molar in the bacterial cytoplasm, is selected as the vector target.
  • the vector selected is the antibiotic kirromycin, which binds to this elongation factor with a dissociation constant of around 10 "6 molar.
  • Sufficient structure activity relationship data is present for ki ⁇ omycin to allow the design of modified molecules that should bind to the EF Tu elongation factor about as well as ki ⁇ omycin itself. Kirromycin reportedly gains access to the cytosol of gram positive bacteria by diffusion.
  • the kirromycin -EF Tu vector- vector receptor pair can be a candidate for use in a VLP conjugate that can enhance the concentration of many pharmacophores in the bacterial cytosol of gram positive bacteria.
  • potential pharmacophores could be the antibiotics trimethoprim and tetracycline. Conjugates of ki ⁇ omycin with these antibiotics may therefore be useful in the treatment of bacterial infections.
  • 1-N-Desmethylgoldinamine is prepared as its formate salt by the method of Tavecchia, P., et al, Journal of Antibiotics 49:1249-57 (1996).
  • ki ⁇ omycin (20g, 25.12 mmol) dissolved in dioxane (200 ml) and formic acid (99%, 50 ml) is stirred at 350 for 22 hours.
  • the mixture is concentrated to half its volume, then ethyl ether (250 ml) is added.
  • the solvent is decanted and saved and the gummy residue sti ⁇ ed in ethyl ether/ethyl acetate (1 :1 v/v).
  • the solid material obtained is washed with ethyl ether and dried at room temperature to give 1-N- desmethylgoldinamine as the formic acid salt (22 mmol, 87%).
  • the goldinonic acid is recovered by a modification of the procedure of Maehr, H, et. al, Canadian Journal of Chemistry 58:501-26 (1980).
  • the decanted solvent and the collected washings are combined, and the volatile materials removed on the rotary evaporator.
  • the residue is dried under vacuum. It is then washed with hexane-ethyl ether.
  • the residue is dissolved in a minimal volume of methanol and purified by reverse phase chromatography on silanized silica gel or by reverse phase high performance liquid chromatography with a linear gradient of chloroform-methanol or chloroform-acetonitrile as the eluant.
  • the goldinonic acid recovered by this procedure may be used without further purification.
  • the modified goldinonic acid or goldinonic acid analog (1 mmol), with its hydroxyl groups protected as silyl ethers, is dissolved in dry chloroform (20 ml) and EDC (l-(3-(dimethylamino)propyl)-3-ethylcarbodiimide) (155) (155 mg, 1 mmol) or DCC (dicyclohexylcarbodiimide) (206) (206 mg, 1 mmol) is added.
  • EDC l-(3-(dimethylamino)propyl)-3-ethylcarbodiimide)
  • DCC dicyclohexylcarbodiimide
  • the chloroform solution is dried over sodium sulfate, and the solvent is removed on the rotary evaporator. The residue is dried under vacuum.
  • the crude product can be used directly in the next step or purified by chromatography, although the silyl ether protecting group is likely to have been partially hydrolyzed during the workup, leading to inhomogenous material, all of which should be usable product.
  • reaction mixture is sti ⁇ ed at room temperature, until thin layer chromatography indicates that all of the starting material has reacted.
  • the reaction mixture is washed with water, and dried over sodium sulfate.
  • the solvent is removed on the rotary evaporator.
  • the crude material is fully resilylated using the procedure for tris- O-triisopropylsilyl-goldinonic acid (compound 1 FIG. 29).
  • An aqueous workup should be sufficient to hydrolyze any silyl carboxyate ester or silyl thioether formed.
  • the resulting material is purified by high performance liquid chromatography on silica gel.
  • FIG. 29 (785) (785 mg, 1 mmol) is dissolved in methylene chloride (20 ml) in a 100 ml round bottom flask equipped with a gas inlet, outlet, thermometer, and magnetic sti ⁇ ing bar and placed in a fume hood.
  • the solution is cooled in to around -30 degrees by partial immersion of the flask in a dry ice- acetone bath and a stream of oxygen containing ozone gas (from an ozone generator) is bubbled into it with stirring.
  • the temperature is allowed to fall to around -60 degrees.
  • the progress of the reaction mixture is monitored by thin layer chromatography.
  • reaction mixture When all of the starting material has reacted, the reaction mixture is purged with a stream of dry nitrogen to evaporate excess ozone. Then dimethylsulfide (62) (0.124 g, 2 mmol) in methanol (5 ml) is added at dry ice temperatures, and the reaction mixture is allowed to warm to -10 degrees. It is sti ⁇ ed at this temperature for an hour, then placed in an ice bath and sti ⁇ ed for an hour, then sti ⁇ ed at room temperature for an hour. The solvent is removed on the rotary evaporator, and the residue dissolved in methylene chloride. The methylene chloride solution is washed with water, and dried over sodium sulfate.
  • Osmium tetroxide olefin cleavage product of tris-O-triisopropylsilyl goldinonic acid (1 FIG. 30): Purified tris-O-triisopropylsilylgoldinonic acid (compound 1 FIG. 29) (785) (785 mg, 1 mmol) is dissolved in dioxane (30 ml). To this is added osmium tetroxide (26 mg, 0.1 mmol). The solution is stirred in the dark for 15 minutes, to allow the osmate ester to form.
  • the solution is then diluted with 10 ml of deionized water, and to this, over several hours, in the dark, is added a solution of sodium periodate (504 mg, 2.3 mmol) dissolved in 15 ml of deionized water.
  • the addition is performed in the dark and the progress of the reaction mixture is monitored by thin layer chromatography.
  • chloroform 40 ml
  • the salt filtrate is washed with methylene chloride.
  • the combined filtrate and washings is washed with a solution of sodium sulfide (10%) in saturated aqueous sodium chloride, until the washings become free from precipitate or color. Then they is washed with saturated aqueous sodium chloride, and dried over sodium sulfate.
  • the solvent is removed on the rotary evaporator, and the residue dried under vacuum.
  • the crude material is used without further purification in the next step.
  • Reductive amination of olefin cleavage product of tris-O-triisopropylsilyl goldinonic acid (compound 2 FIG. 30):
  • the aldehyde from the oxidative olefin cleavage of tris-O-triisopropylsilylgoldinonic acid (compound 1 FIG. 30) (747) (3.73g, 5 mmol) is dissolved in methanol, and concentrated ammonia solution is added to 5% of the total volume.
  • the reaction mixture is placed in a fume hood and allowed to stand for one hour.
  • Aqueous hydrochloric acid (IN) is added until the pH of an aliquot, when added to a 10 fold excess of water, is around 9 (measured by pH paper).
  • the product may be purified by chromatography on silica gel or used directly in the next step.
  • Addition product of (gamma-thiobutyrolactone to the amine adduct of the olefin cleavage product of tris-O-triisopropylsilyl goldinonic acid (compound 3 FIG. 30), (compound 4 FIG. 30): The amination product of the aldehyde from the olefin cleavage of tris-O-triisopropylsilylgoldinonic acid (compound 2 FIG.
  • N-Triisopropylsilyl-4-piperidone (compound 1 FIG. 31): Anhydrous 4-piperidone (99) (9.9 g, 100 mmol) is distilled from the hydrate under vacuum (0.1mm Hg), using a short path still, and collected in a tared receiver cooled in a dry ice bath. • The flask is then weighed. To its contents is added, for 9.9 g of distillate, a chilled solution of triethyl amine (101) (10.1 g, 100 mmol) and triisopropylsilyl chloride (193.5) (19.35 g, 100 mmol) in chloroform (200 ml). The mixture is sti ⁇ ed in the receiving flask and allowed to warm to room temperature. The solvent is removed on the rotary evaporator and the product is purified by flash chromatography on silica gel.
  • 4-hydroxypiperidine (compound 2 FIG. 31) (159) (7.95g, 50 mmol) is dissolved in dry chloroform (200 ml) and dry triethylamine (101) (5.05g, 50 mmol) is added. Then (gamma-thiobutyrolactone (102) (5.1 g, 50 mmol) is added. The progress of the reaction mixture is monitored by thin layer chromatography. When the starting material has reacted, water is added. The pH is adjusted to 3 (measured by pH paper) with hydrochloric acid (IN) and the chloroform solution is washed with this, then water. It is then extracted into sodium bicarbonate solution (0.1 N) and washed with ether.
  • N-(4-Thiobutyryl)-4-aceto-4-hydroxypiperidine (compound 3 FIG. 31) is converted to the silyl ether by the same procedure used for tris-O-triisopropylsilylgoldinonic acid (compound 1 FIG. 29).
  • An aqueous workup should be sufficient to hydrolyze any silyl carboxyate ester or silyl thioether formed.
  • the resulting material is purified by chromatography on silica gel.
  • This material is prepared from 4-dimethylaceto-4-hydroxypiperidine (compound 5 FIG. 31) by the procedure for N-(4-thiobutyryl)-4-aceto-4-hydroxypiperidine (compound 3 FIG. 31) above.
  • N-(4-Thiobutyryl)-4-dimethylaceto-4-triisopropylsiloxypiperidine (compound 7 FIG. 31): This material is prepared from N-(4-thiobutyryl)-4-dimethylaceto-4- hydroxypiperidine (compound 6 FIG. 31) by the procedure N-(4-thiobutyryl)-4-aceto-4- triisopropylsiloxypiperidine (compound 4 FIG. 31) above.
  • 1,3-dithiolane (5.3 g, 0.05 mol) is dissolved in dry tetrahydrofuran (50 ml). This is also be cooled in a dry ice bath, under dry nitrogen. To this is added butyl lithium solution (0.053 mole butyl lithium in hexane) by means of a dry syringe. The solution is allowed to warm to around -40 degrees and sti ⁇ ed for 2 hours while keeping it at this temperature. It is then added by syringe to the 2-(triisopropylsilyloxy)acetic acid methyl ester.
  • the reaction mixture is sti ⁇ ed for 2 hours at -78 degrees, then allowed to warm to room temperature and sti ⁇ ed for an additional 1/2 hour.
  • the reaction mixture is again cooled to around -40 degrees, and a second portion of butyl lithium solution (0.053 mol) is added.
  • the reaction mixture is sti ⁇ ed at this temperature for 2 hours, and then cooled to -78 degrees.
  • a second portion of 2-(triisopropylsilyloxy)acetic acid methyl ester (12.35 g, 0.05 mol) is added.
  • the reaction mixture is stirred for 2 hours at -78 degrees, then allowed to warm to room temperature, and stirred for an additional half hour.
  • the pH is adjusted to 9-10 (measured by pH paper) and the solvent is removed on the rotary evaporator. The residue is dissolved in chloroform, and washed with water saturated with sodium chloride. The solvent is again removed on the rotary evaporator and the crude material purified chromatography on silica gel or by crystallization of the hydrochloride salt. A similar reduction may be performed using lithium tris-sec-butylborohydride as the reducing agent.
  • Benzyl bromide (171) (5.64 g, 33 mmol) is then added slowly, with rapid stirring.
  • the reaction mixture is sti ⁇ ed at room temperature, and monitored by thin layer chromatography. After an hour, additional benzyl bromide (0.56 g, 3.3 mmol) and sodium hydride (79 mg, 3.3 mol) or sodamide (129 mg, 3.3 mol), is added, and stirring is continued. The process is continued until no further change is apparent.
  • the reaction mixture is neutralized by the addition of hydrochloric acid (IN) and the solvent is removed on the rotary evaporator.
  • the product is dissolved in chloroform and washed with aqueous sodium hydroxide (0.1 N), then water.
  • the solution is dried over sodium sulfate, and the solvent removed on the rotary evaporator.
  • the residue is purified by chromatography on silica gel or by crystallization of the hydrochloride salt.
  • N-Benzyl-bis-3.5-O-benzyl-4-piperidone (compound 8 FIG. 32): N-Benzyl-bis- 3,5-O-benzyl-4,4-(S,S-cylo-l,2-dithioethyl)piperidine (compound 7 FIG. 32) (477) (4.8 g, 10 mmol) is dissolved in a minimum volume of tetrahydrofuran. This is dropped over a period of 15 minutes into a rapidly sti ⁇ ed mixture of red mercuric oxide (224) (4.48 g, 20 mmol) and boron trifluoride etherate (20 mmol) in 15% aqueous tetrahydrofuran (50 ml) under nitrogen.
  • red mercuric oxide (224) (4.48 g, 20 mmol)
  • boron trifluoride etherate (20 mmol
  • N-Benzyl-bis-3,5-O-benzyl-4-aceto-4-hvdroxypiperidine (compound 9 FIG. 32): N-Benzyl-bis-3,5-O-benzyl-4-piperidone (compound 8 FIG. 32)(401) (4.01 g, 10 mmol) is dissolved in dry dioxane or tetrahydrofuran, chilled in an ice bath. To this is added a mixture of tert-butylacetate (116) (1.28 g, 11 mmol) and fresh potassium tert-butoxide (112) (1.23 g, 11 mmol) in the same solvent, with rapid stirring. Stirring is continued and the solution is allowed to warm to room temperature.
  • the resulting material is dissolved in aqueous-methanolic hydrochloric acid (I N) and warmed until thin layer chromatography indicates that the tert-butyl ester has been hydrolyzed, then all solvents is removed on the rotary evaporator, and the residue is dried under vacuum.
  • This material is dissolved in chloroform, extracted into aqueous sodium hydroxide (0.1N), and washed with toluene. Then the aqueous solution is neutralized with hydrochloric acid (IN), and the product extracted into chloroform. It is dried over sodium sulfate and the solvent is removed on the rotary evaporator. The residue is dried under vacuum.
  • the crude material may be purified by chromatography on silica gel, by crystallization of the hydrochloride salt, or it may be used directly.
  • 4-Aceto-3A5-trihvdroxypiperidine (compound 10 FIG. 32): Sodium (23) (1.4 g, 60 mmol) is placed in a dry, 3-neck 300 ml round bottom flask, under dry nitrogen and equipped with a magnetic sti ⁇ ing bar, in a fume hood. The flask is placed in a dry ice bath and anhydrous ammonia (around 50 ml) is condensed into it.
  • the solution is sti ⁇ ed at room temperature for an additional hour, then any remaining sodium is destroyed by the slow addition of ethanol.
  • water is cautiously added, and the aqueous solution is extracted with toluene. It is then neutralized by the addition of aqueous hydrochloric acid (0.1 N), saturated with sodium chloride, and extracted into chloroform.
  • aqueous hydrochloric acid 0.1 N
  • saturated sodium chloride saturated with sodium chloride
  • extracted into chloroform The chloroform solution is washed with saturated sodium chloride solution, then dried over sodium sulfate.
  • the solvent is removed on the rotary evaporator and the residue dried under vacuum.
  • the product may be purified by chromatography on silica gel, reverse phase chromatography on silanized silica gel, or crystallization of the hydrochloride salt.
  • N-(4-Thiobutyryl)-4-aceto-3.4,5-trihydroxypiperidine compound 11 FIG. 32: 4- Aceto-3,4,5-trihydroxypiperidine (compound 10 FIG. 32) (191) (1.91g, 10 mmol) is dissolved in dry chloroform (200 ml) and dry triethylamine (101) (l.Olg, 10 mmol) is added. Then (gamma-thiobutyrolactone (102) (1.02 g, 10 mmol) is added. The progress of the reaction mixture is monitored by thin layer chromatography. When the starting material has reacted, saturated sodium chloride solution is added.
  • N-Benzyl-bis-3.5-O-benzyl-4-dimethylaceto-4-hvdroxypiperidine (compound 13 FIG. 32): This material is prepared from N-benzyl-bis-3,5-O-benzyl-4-piperidone (compound 8 FIG. 32) and tert-butylvalerate by the procedure for N-benzyl-bis-3,5-O- benzyl-4-aceto-4-hydroxypiperidine (compound 9 FIG. 32) above.
  • FIG. 32 This material is prepared from 4-dimethylaceto-3,4,5-trihydroxypiperidine (compound 14 FIG. 32) and (gamma-thiobutyrolactone by the procedure for N-(4- thiobutyryl)-4-aceto-3,4,5-trihydroxypiperidine (compound 11 FIG. 32) above.
  • N-(4-Thiobutyryl)-4-dimethylaceto-3.4.5-tris-triisopropylsiloxypiperidine (compound 16 FIG. 32): This material is prepared from N-(4-thiobutyryl)-4- dimethylaceto-3,4,5-trihydroxypiperidine (compound 15 FIG. 32) and triisopropylsilyl chloride by the procedure for N-(4-thiobutyryl)-4-aceto-3,4,5-tris- triisopropylsiloxypiperidine (compound 12 FIG. 32) above.
  • the residue is washed with dry ethyl ether and dry ethyl acetate, to remove the unreacted carbonyldiimadazole, then it is dried under vacuum.
  • the residue is dissolved in dry chloroform and the aniline derivative (1 mmol) is added.
  • the reaction mixture is sti ⁇ ed for an additional hour. It is then extracted with water and dried over sodium sulfate.
  • the solvent is removed under vacuum to yield a crude residue that is washed with ethyl ether and ethyl acetate. This is purified by reverse phase high performance liquid chromatography on silica gel with a linear gradient of water-acetonitrile or acetone as the eluant.
  • 2-Aminoresorcinol (compound 1 FIG. 34): 2-Nitrororesorcinol (155) (31 g, 0.2 mol) is dissolved in methanol and placed in a hydrogenation bottle. To this is added 10% Pd on charcoal (1 gm). The bottle is sealed, evacuated, and hydrogen gas is applied at a pressure of 50 psi. The reaction mixture is shaken at room temperature until no further hydrogen uptake is apparent. The reaction mixture is removed, filtered, and the solvent removed by evaporation to yield a solid. The crude material is used without further purification.
  • 2-Acetylaminoresorcinol (compound 2 FIG. 34): The crude 2-aminoresorcinol (compound 1 FIG. 34) (125) (12.5 g, 0.1 mol) is dissolved in chloroform (200 ml), and triethyl amine (101) (10.1 g, 0.1 mmol) is added. To this is added acetic anhydride (102) (10.2 g, 0.1 mol). The reaction mixture is sti ⁇ ed for 3 hours, then the solvent is removed on the rotary evaporator. The residue is dissolved in chloroform and washed with hydrochloric acid (0.1 N) once, then with water. The chloroform solution is dried over sodium sulfate.
  • the product is then extracted into aqueous hydrochloric acid (0.1 N).
  • the aqueous layer is washed with chloroform then the pH is adjusted to 9-10 (measured by pH paper) by the addition of aqueous sodium hydroxide (0.1 N) and the product extracted into chloroform.
  • the solution is dried over sodium sulfate, the solvent is removed on the rotary evaporator, and the residue dried under vacuum.
  • the product is purified by chromatography on silica gel or by crystallization of the hydrochloride salt. 3-(3.5-dihvdroxy-4-acetylamino)-thiobenzyl-l-thiopropane (compound 4 FIG.
  • 2-Thiobenzylethylamine (compound 1 FIG. 35): 2-Thioethylamine (76) (15.2 g, 0.2 mol) is dissolved in chloroform (200 ml) in a round bottom flask. Into the flask is placed finely ground, anhydrous sodium carbonate (106) (21.2 g, 0.2 mol). The suspension is rapidly sti ⁇ ed and a solution of benzyl bromide (171) (34.2 g, 0.2 mol) in chloroform (200 ml) is added dropwise. The reaction mixture is assayed by thin layer chromatography and stining is continued until all of the benzyl bromide has reacted.
  • N-methyl-2-thiobenzylethylamine (compound 2 FIG. 35): 2- Thiobenzylethylamine (compound 1 FIG. 35) (167) (33.4 g, 0.2 mol) and freshly distilled formaldehyde (30) (9 g, 0.20 mol) is mixed. A minimum volume of absolute ethanol may be added to assist dissolution.
  • the crude material is dissolved in chloroform, filtered again, washed with sodium hydroxide solution (0.1 N), extracted into hydrochloric acid solution (0.1 N), then washed with chloroform.
  • the aqueous solution is again made basic with sodium hydroxide, and the product is extracted into chloroform.
  • the crude material may be purified by flash chromatography on silica gel or by fractional crystallization of the hydrochloride salt
  • FIG. 35 Sodium (23) (2.3 g, 100 mmol) is placed in a dry, 3-neck 500 ml round bottom flask, equipped with a magnetic stirring bar, under dry nitrogen, in a fume hood. The flask is placed in a dry ice bath and anhydrous ammonia (around 100 ml) is condensed into it. Into this solution is slowly dripped a solution of N-methyl-N-(2-thiobenzylethyl)-3,5- dihydroxy-4-acetylaminobenzylamine (compound 3 FIG. 35) (344) (17.2 g, 50 mmol) dissolved in tetrahydrofuran and tert-butanol.
  • reaction mixture is rapidly sti ⁇ ed for an hour at dry ice temperatures.
  • the progress of the reaction mixture is followed by quenching aliquots of the reaction mixture in water, neutralizing them with hydrochloric acid (0.1 N), saturating them with sodium chloride, extracting them into chloroform, then analyzing material by thin layer chromatography.
  • hydrochloric acid 0.1 N
  • the reaction mixture is allowed to warm to room temperature with continued rapid stining.
  • the solution is stirred at room temperature for an additional hour, then any remaining sodium is destroyed by the slow addition of ethanol.
  • hydrogen evolution has ceased, methanol, then water is cautiously added with rapid stirring.
  • the pH is adjusted to 9 (measured by pH paper) by the addition of hydrochloric acid (IN).
  • the reaction mixture is extracted into chloroform, then washed with water.
  • the product is extracted into hydrochloric acid (0.1 N), washed with chloroform, then the pH of the aqueous solution is adjusted to 9-10 (checked with pH paper) with sodium hydroxide solution (0.1N) and the product is extracted into chloroform.
  • the chloroform solution is dried over sodium sulfate, and the solvent removed on the rotary evaporator. The residue is dried under vacuum. It may be purified by chromatography on silica gel or by crystallization of the hydrochloride salt.
  • N-Methyl-N-(2-thioethyl)-3.5-dihydroxy-4-aminobenzylamine (compound 5 FIG. 35): N-Methyl-N-(2-thioethyl)-3,5-dihydroxy-4-acetylaminobenzylamine (compound 4 FIG. 32) (254) (6.35 g, 25 mmol) is dissolved in methanol (100 ml) and hydrazine (32) (3.2 g, 100 mmol) is added. The reaction mixture is allowed to stir overnight, then the solvent is removed on the rotary evaporator. The residue is dissolved in chloroform, washed with water, then dried over sodium sulfate. The solvent is removed on the rotary evaporator and the residue chromatographed on silica gel with hexane-chloroform- triethylamine as the eluant.
  • N-Methyltaurine (compound 6 FIG. 35): In a 500 ml, 3 neck round bottom flask, taurine (125) (25.0 g, 0.2 mol) is dissolved in a minimum volume of methanol.
  • Triethylamine (101) (1.0 g, 0.01 mmol) and freshly distilled formaldehyde (30) (6.6 g, 0.22 mol) is added.
  • the mixture is sti ⁇ ed in a fume hood at room temperature for an hour, then sodium cyanoborohydride (63) (8.2 g, 0.13 mmol) in methanol (100 ml) is carefully added using an addition funnel.
  • the reaction mixture is stined for an additional hour.
  • the excess cyanoborohydride is destroyed by acidification with hydrochloric acid (0.1 N) in the fume hood, and the reaction mixture briefly degassed under an aspirator vacuum in the fume hood.
  • the solvent is then removed on the rotary evaporator.
  • the aqueous layer is saturated with sodium chloride, and the reaction mixture is washed with saturated sodium chloride, then extracted into aqueous hydrochloric acid (0.1 N), and washed with ethyl acetate.
  • the aqueous layer is made neutral (to pH 7 measured by pH paper) by the addition of aqueous sodium hydroxide (0.1 N). It is saturated with sodium chloride, and the product extracted into chloroform. The solvent is removed on the rotary evaporator and the residue dried under vacuum.
  • the crude product may be purified by chromatography on silica gel, reverse phase chromatography on silanized silica gel, or by fractional crystallization.
  • FIG. 35 N-Methyl-N-(2-sulfonoethyl)-3,5-dihydroxy-4-acetylaminobenzylamine (compound 7 FIG. 32) (346) (8.65 g, 25 mmol) is dissolved in anhydrous diglyme (100 ml) under nitrogen in a dry, 3 neck round bottom flask. The flask is cooled in an ice bath and the solution rapidly sti ⁇ ed. Lithium aluminum hydride (38) (0.95 g, 25 mmol) is added in small portions with rapid stirring. Sti ing is continued and the solution is allowed to warm to room temperature. Progress of the reaction mixture is monitored by thin layer chromatography.
  • the excess lithium aluminum hydride is destroyed by cautious addition of methanol, then water.
  • the pH is adjusted to around 9 (measured by pH paper) and chloroform and sufficient water to form two phases is added.
  • the aqueous layer is extracted with chloroform.
  • the chloroform extract is washed with water and dried over sodium sulfate.
  • the solvent is removed on the rotary evaporator and the residue dried under vacuum.
  • the crude product may be purified by chromatography on silica gel or by fractional crystallization of the hydrochloride salt.
  • N-methyl-N-(2-thiobenzylethyl)-4-acetylaminobenzylamine (compound 8 FIG. 35): This material is prepared from N-methyl-2-thiobenzylethylamine (compound 2 FIG. 35) and acetylaniline by the method used to prepare N-methyl-N-(2-thiobenzylethyl)-3,5- dihydroxy-4-acetylaminobenzylamine (compound 3 FIG. 35).
  • N-methyl-N-(2-thioethyl)-4-aminobenzylamine (compound 10 FIG. 35): N- Methyl-N-(2-thiobenzylethyl)-4-acetylaminobenzylamine (compound 8 FIG. 35) (312) (15.6 g, 50 mmol) is dissolved in methanol (100 ml) and hydrazine (32) (3.2 g, 100 mmol) is added. The reaction mixture is allowed to stir overnight, then the solvent is removed on the rotary evaporator. The residue is dissolved in chloroform, washed with water, then dried over sodium sulfate. The solvent is removed on the rotary evaporator and the residue thoroughly dried under vacuum.
  • the benzyl group of the crude material is then cleaved by the method used to prepare N-methyl-N-(2-thioethyl)-3,5-dihydroxy-4- acetylaminobenzylamine (compound 4 FIG. 35) above.
  • N-Methyl-N-(2-sulfonoethyl)-4-acetylaminobenzylamine (compound 11 FIG. 35): This material is prepared from N-methyltaurine (compound 6 FIG. 35) and acetylaniline by the method used to prepare N-methyl-N-(2-sulfonoethyl)-3,5-dihydroxy-4- acetylaminobenzylamine (compound 7 FIG. 35) above.
  • N-Methyl-N-(2-thioethyl)-4-acetylaminobenzylamine (compound 9 FIG. 35):
  • This material is prepared from N-methyl-N-(2-sulfonoethyl)-4-acetylaminobenzylamine (compound 11 FIG. 35) by the reaction used with N-methyl-N-(2-sulfonoethyl)-3,5- dihydroxy-4-acetylaminobenzylamine (compound 7 FIG. 35) above.
  • Hydrazone of ki ⁇ omycin with 3-nitro-4-hvdrazidophenylthioethanol (compound 1 FIG. 36): Hydrazones of kr ⁇ omycin are prepared by the method of Chinali, G, et al, Bollettino Societa Italiana Biologia Sperimentale 57:1706-12 (1981). Thus, ki ⁇ omycin sodium salt (830) (0.83 g, 1 mmol) is dissolved in a minimal volume of absolute ethanol. 3-Nitro-4-hydrazidophenylthioethanol (compound 5 FIG. 37) (0.22 g, 1 mmol), dissolved in the same solvent, is then added.
  • the reaction mixture is sti ⁇ ed at room temperature overnight, then the solvent removed under vacuum. The residue is washed with ethyl ether and ethyl acetate, then dried under vacuum.
  • the product may purified by reverse phase high performance liquid chromatography with a linear gradient of water-acetonitrile or acetone as the eluant, or it may be crystallized.
  • 3-nitro-4-aminophenylethanol (compound 1 FIG. 37): 4-Aminophenylethanol (151) (30.2 g, 0.2 mol) is dissolved in dichlorobenzene or nitrobenzene (200 ml) and concentrated nitric acid (63) (0.44 mol) is dripped in slowly, with rapid stirring. The reaction mixture is cooled in an ice bath, as necessary. The reaction mixture is allowed to stir, then ice water is added. The water layer is separated and saved and the reaction mixture is extracted twice with water. The combined aqueous extracts is made basic with sodium hydroxide (IN) and the product is extracted into chloroform. This is washed twice with water and dried over sodium sulfate. The solvent is removed on the rotary evaporator, and the residue dried under vacuum. The product may be purified by chromatography on silica gel or crystallization of the hydrochloride salt.
  • 3-nitro-4-acetylhydrazidophenylthioethanol compound 4 FIG. 37: 3-Nitro-4- acetylhydrazidophenylethanol (compound 3 FIG. 34) (255) (25.5 g, 0.1 mol) and dry triethylamine (101) (10.1 g, 0.1 mol) is dissolved in chloroform (200 ml). The solution is rapidly sti ⁇ ed and methyl sulfonyl chloride (114.5) (11.45 g, 0.1 mol) in dry chloroform (50 ml) is added by means of a dropping funnel.
  • the reaction mixture is sti ⁇ ed for one hour, then finely powdered solid sodium sulfide (78) (39 g, 0.5 mol), a solution of sodium sulfide (39 g, 0.5 mol) in a minimal volume of water, or tetrabutylammonium sulfide (242) (24.2 g, 0.1 mol) is added.
  • the reaction mixture is sti ⁇ ed overnight, then washed with water.
  • the chloroform solution is dried over sodium sulfate, then the solvent is removed on the rotary evaporator. The residue is dried under vacuum.
  • the crude product may be purified by flash chromatography or used directly in the next step.
  • kinomycin sodium salt (2.2g, 2.65 mmol) is dissolved in dimethylformamide (25 ml) and cooled in an ice bath.
  • the sulfonyl chloride (3 mmol) is added and the reaction mixture is sti ⁇ ed vigorously for 30 minutes. After this, the reaction mixture is quenched by adding it to a well sti ⁇ ed mixture of saturated sodium bicarbonate (100 ml) and chloroform (25 ml).
  • the amino derivative of tetracycline (compound 7 FIG. 24) (0.59 g, lmmol) is dissolved in methanol, methanol-chloroform, or dimethylethylene glycol and succinimidyl 6-[(iodoacetyl)-amino]hexanoate (0.40 g, 0.1 mmol) is added.
  • the reaction mixture is sti ⁇ ed at room temperature, and the progress of the reaction mixture is monitored by thin layer chromatography.
  • the hydrazone of ki ⁇ omycin and 3-nitro-4-hydrazidophenylthioethanol (compound 1 FIG. 36) (0.97 g, lmmol) is added and the reaction stirred further at room temperature.
  • the residue is purified from excess N,N'-hexamethylene-bis(iodoaetamide) by chromatography.
  • To this material is added the hydrazone of krrromycin and 3-nitro-4-hydrazidophenylthioethanol (compound 1 FIG. 36) (0.97 g, lmmol) which is dissolved in methanol- dimethylformamide.
  • the reaction mixture is sti ⁇ ed at room temperature, and the progress of the reaction mixture is monitored by thin layer chromatography. When the reaction mixture is complete, the solvent is removed under vacuum with minimal heating, and the residue is dried under vacuum.
  • the product is purified by reverse phase high performance liquid chromatography with a linear gradient of water-acetonitrile as the eluant, or it may be crystallized.
  • the crude ki ⁇ omycin hydrazone may also be used in the coupling reaction.
  • Serial dilutions of conjugate are made in water or the appropriate organic solvent. Aliquots of these are mixed with portions of freshly grown yeast or fungal cultures (5-10 ml) that have been placed in culture tubes. The tubes are shaken in an incubator. At intervals of 10 minutes to several hours, aliquots of the cultures are withdrawn. The cellular material is collected by centrifugation, washed with media, then recentrifuged. The resulting pellet is analyzed for the presence of conjugate molecule. As a control, the above procedure is performed with an excess of unconjugated vector molecule that is added to the culture medium at the same time as the conjugate of the vector with the probe. Another control uses serial dilutions of the probe molecule alone or in combination with the linker.
  • the washed cellular pellet is suspended in water or culture medium.
  • the suspension is illuminated by ultraviolet light and the degree of fluorescence estimated by visual inspection. Aliquots of the suspension may also be analyzed in a fluorimeter, for a more precise spectrophotometric determination of the degree of fluorescence.
  • the cellular pellet is suspended in distilled water, and a portion of the material aliquoted into scintillation fluid and dispersed. Radioactivity ( 14 C, 3 H, or 35 S) in the resulting suspension is determined by scintillation counting.
  • the cells may also be lysed, and fractionated, and the presence of the probe determined in various cellular fractions.
  • Fungi or yeast taken from stored, frozen cultures, are grown in culture tubes or shake flasks at 25 °C. Aliquots of around 10 5 colony forming units per milliliter is then used as inocula for the assays. Cultures are incubated at 25 °C, for up to 48 hours with yeasts and 120 hours with filamentous fungi, to obtain an adequate cell density.
  • the degree of accumulation of fluorescent or radiolabeled model conjugates of a vector, a linker, and a probe molecule can be determined at a series of concentrations of the conjugate. In parallel, a series of concentrations of the conjugate plus excess free vector is also tested, and the results graphed.

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Abstract

La présente invention concerne des conjugués de médicaments et leurs procédés de préparation. Un des modes de réalisation de cette invention concerne un procédé de préparation des conjugués VLP (vecteur-liant-pharmacophore) pouvant être généralement appliqué à une grande variété de vecteurs, liants, et pharmacophores. En outre, cette invention concerne un procédé d'amélioration de l'administration d'un pharmacophore à un patient, ainsi qu'un procédé d'amélioration de l'efficacité thérapeutique d'un pharmacophore et un procédé de réduction de la toxicité d'un pharmacophore. Par ailleurs, cette invention concerne un procédé d'augmentation de la concentration d'un pharmacophore dans une cellule.
PCT/US2000/023593 1999-08-26 2000-08-28 Conjugues de medicaments et leurs procedes de preparation WO2001013958A2 (fr)

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JP2001518093A JP2003507439A (ja) 1999-08-26 2000-08-28 薬物コンジュゲートおよびその設計方法
AU70824/00A AU7082400A (en) 1999-08-26 2000-08-28 Drug conjugates and methods of designing the same
CA002382202A CA2382202A1 (fr) 1999-08-26 2000-08-28 Conjugues de medicaments et leurs procedes de preparation
EP00959512A EP1212096A2 (fr) 1999-08-26 2000-08-28 Conjugues de medicaments et leurs procedes de preparation

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Cited By (8)

* Cited by examiner, † Cited by third party
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WO2008034122A2 (fr) * 2006-09-15 2008-03-20 Enzon Pharmaceuticals, Inc. Segments de liaison biodégradables à base d'ester encombré pour distribution d'oligonuclotides
JP2008266330A (ja) * 2002-03-08 2008-11-06 Paratek Pharmaceuticals Inc アミノメチル置換されたテトラサイクリン化合物
US8110559B2 (en) 2006-09-15 2012-02-07 Enzon Pharmaceuticals, Inc. Hindered ester-based biodegradable linkers for oligonucleotide delivery
US8268318B2 (en) 2006-09-15 2012-09-18 Enzon Pharmaceuticals, Inc. Polyalkylene oxides having hindered ester-based biodegradable linkers
CN102993116A (zh) * 2012-12-04 2013-03-27 常州大学 一种苯并噁嗪类激动剂的制备方法
CN110963946A (zh) * 2019-12-12 2020-04-07 万华化学集团股份有限公司 一种甲基牛磺酸钠的制备方法
CN111549071A (zh) * 2020-05-15 2020-08-18 乾元康安(苏州)生物科技有限公司 生产蛋白药物偶联物的药物毒性耐受生产细胞株的构建方法及其应用
WO2022191459A1 (fr) * 2021-03-08 2022-09-15 비엔제이바이오파마 주식회사 Procédé de conception de médicament et dispositif mettant en œuvre un tel procédé

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Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008266330A (ja) * 2002-03-08 2008-11-06 Paratek Pharmaceuticals Inc アミノメチル置換されたテトラサイクリン化合物
WO2008034122A2 (fr) * 2006-09-15 2008-03-20 Enzon Pharmaceuticals, Inc. Segments de liaison biodégradables à base d'ester encombré pour distribution d'oligonuclotides
WO2008034122A3 (fr) * 2006-09-15 2008-10-30 Enzon Pharmaceuticals Inc Segments de liaison biodégradables à base d'ester encombré pour distribution d'oligonuclotides
US8110559B2 (en) 2006-09-15 2012-02-07 Enzon Pharmaceuticals, Inc. Hindered ester-based biodegradable linkers for oligonucleotide delivery
US8268318B2 (en) 2006-09-15 2012-09-18 Enzon Pharmaceuticals, Inc. Polyalkylene oxides having hindered ester-based biodegradable linkers
AU2007296054B2 (en) * 2006-09-15 2013-03-28 Enzon Pharmaceuticals, Inc. Hindered ester-based biodegradable linkers for oligonucleotide delivery
CN102993116A (zh) * 2012-12-04 2013-03-27 常州大学 一种苯并噁嗪类激动剂的制备方法
CN110963946A (zh) * 2019-12-12 2020-04-07 万华化学集团股份有限公司 一种甲基牛磺酸钠的制备方法
CN110963946B (zh) * 2019-12-12 2022-03-11 万华化学集团股份有限公司 一种甲基牛磺酸钠的制备方法
CN111549071A (zh) * 2020-05-15 2020-08-18 乾元康安(苏州)生物科技有限公司 生产蛋白药物偶联物的药物毒性耐受生产细胞株的构建方法及其应用
WO2022191459A1 (fr) * 2021-03-08 2022-09-15 비엔제이바이오파마 주식회사 Procédé de conception de médicament et dispositif mettant en œuvre un tel procédé

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EP1212096A2 (fr) 2002-06-12
CA2382202A1 (fr) 2001-03-01

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