EP1406631A2 - Dopamine receptor ligands and therapeutic methods based thereon - Google Patents

Dopamine receptor ligands and therapeutic methods based thereon

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Publication number
EP1406631A2
EP1406631A2 EP02744349A EP02744349A EP1406631A2 EP 1406631 A2 EP1406631 A2 EP 1406631A2 EP 02744349 A EP02744349 A EP 02744349A EP 02744349 A EP02744349 A EP 02744349A EP 1406631 A2 EP1406631 A2 EP 1406631A2
Authority
EP
European Patent Office
Prior art keywords
receptor
compound
subject
dopamine
substituted
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP02744349A
Other languages
German (de)
French (fr)
Other versions
EP1406631A4 (en
Inventor
Shaomeng Wang
Judith Varady
Xihan Wu
Ji Min
Ke Ding
Beth Levant
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of Michigan
Original Assignee
University of Michigan
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Filing date
Publication date
Application filed by University of Michigan filed Critical University of Michigan
Publication of EP1406631A2 publication Critical patent/EP1406631A2/en
Publication of EP1406631A4 publication Critical patent/EP1406631A4/en
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/04—Ortho-condensed systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system

Definitions

  • the present invention relates to compounds designed as ligands (e.g., agonists, antagonists, and partial agonists) for dopamine receptors (e.g., D], D 2 , D 3 , D , and D 5 ), and in particular, the D ⁇ D 2 , and D 3 dopamine receptors.
  • the present invention also relates to methods for rationally designing these compounds and to therapeutic methods of using these compounds.
  • the present invention relates to compounds designed as ligands (e.g., agonists, antagonists, and partial agonists) for dopamine receptors (e.g., D ⁇ , D 2 , D 3 , D 4 , and D 5 ), and in particular, the Di, D , and D 3 dopamine receptors.
  • the present invention also relates to methods for rationally designing these compounds and to therapeutic methods of using these compounds.
  • the present invention is not intended, however, to be limited to compositions that modulate (e.g., agonists, antagonists, and partial agonists) dopamine receptors.
  • compositions that modulate e.g., agonists, antagonists, and partial agonists
  • G- protein coupled receptors generally, and to methods for discovering and designing these compounds.
  • Preferred embodiments of the present invention provide compounds that are rationally designed to control dopamine flow in the brain.
  • these compounds are highly selective antagonists of the dopamine family receptors (e.g., D 3 ).
  • rational design of the compounds of the present invention includes identifying a mechanism associated with dopamine flow in the brain. Information relating to the mechanism is then analyzed such that compound structures having possible activity in interfering with such a mechanism are formulated.
  • structures are synthesized based on "building blocks," wherein each building block has a feature potentially capable of interfering with a particular mechanism associated with dopamine flow, particularly, a mechanism involving one ore more members of the D receptors family.
  • Compounds having different building block combinations are then synthesized and their activity in relation to the identified mechanism tested. Such tests are preferably conducted in vitro and/or in vivo. The information obtained through such tests is then incorporated in a new cycle of rational drug design. The design-synthesis-testing cycle is repeated until a candidate compound having the desired properties for a targeted therapy; e.g., dopamine flow control, is obtained. The candidate compound is then preferably clinically tested.
  • a targeted therapy e.g., dopamine flow control
  • One approach for modulating (e.g., controlling) dopamine flow in the brain for the treatment of cocaine addiction is to design cocaine antagonists which can affect dopamine uptake. More specifically, this approach is based on rationally designing compounds that are antagonists of cocaine such that they reduce or block dopamine binding to the D receptors family, and particularly, receptor D 3 . In preferred embodiments, antagonists are designed to reduce or block cocaine binding while leaving other aspects of dopamine transport unaffected. The designed antagonists should provide a basis for therapeutic protocols based on the selective control of dopamine transport and thereby control of synaptic signaling with no or little disruption of the normal flow of dopamine in the brain.
  • the present invention provides a composition comprising one or more of the compounds disclosed herein, or derivatives of such compounds (e.g., derivatives having minor chemical substitutions).
  • the present invention also provides methods of using such compounds. It will be appreciated that the methods described herein find use with the compounds disclosed herein, and their derivatives.
  • the present invention provides a composition comprising a compound of formula (including derivatives of this formula):
  • X, Y, and Z independently represent C, O, N, S;
  • R 7 , R 8 , and R 9 independently represent H, F, Cl, Br, I, OH, CN, NO 2 , OR', CO 2 R', OCOR',
  • R'R CONR'R
  • NR"COR' NR'SO 2 R
  • SO 2 NR'R NR'R
  • R'R independently represents H, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, or heterocyclic, substituted or unsubstituted;
  • a and B independently represent O, S, SO, SO 2 , NR', CR'R" where R' and R" independently represent H, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, or heterocyclic, substituted or unsubstituted;
  • R represents alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, or heterocyclic, substituted or unsubstituted;
  • the present invention provides a composition comprising a compound of formula (including derivatives of this formula):
  • the present invention provides a composition comprising a compound of formula (including derivatives of this formula):
  • X, Y, Z, D and M independently represent C, O, N, S;
  • the present invention provides a composition comprising a compound of formula (including derivatives of this formula):
  • X, Y, Z, D, and M independently represent C, O, N, S;
  • R 7 , R 8 , R 9 , and R 10 independently represent H, F, Cl, Br, I, OH, CN, NO 2 , OR', CO 2 R ⁇ OCOR', CONR'R", NR"COR', NR'SO 2 R", SO 2 NR'R", NR'R" where R' and R" independently represents H, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, or heterocyclic, substituted or unsubstituted;
  • a and B independently represent O, S, SO, SO 2 , NR', CR'R" where R' and R" independently represent H, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, or heterocyclic, substituted or unsubstituted;
  • E represents a linker group that does not contain an amide group and F represents cycloalkyl, cycloalkenyl, or heterocyclic, substituted or unsubstituted;
  • Group E can be any linking chemistry including, but not limited to, carbon chains, repeating polymer units, and the like.
  • Group F is preferably a bulky group, such as a substituted or unsubstituted benzene ring or a multi-ring complex.
  • the present invention also provides methods for identifying agonists and antagonists of dopamine receptors, comprising the step of assessing an interaction between a candidate ligand and an amino acid of a dopamine receptors.
  • the contemplated amino acids of the dopamine receptors include, but are not limited to, Y32, S35, Y36, L39, F345, F346, T368, T369, Y365, P200, S192, S193, S197, VI 11, 1183, F188, V164, S182, and A161 of receptor D 3 , Y37, L39, L41, L44, F198, F389, F390, F411, S192, S193, S196, VI 15, 1194, F189, and S163 of receptor D 2 , and the like.
  • the contemplated amino acids of the dopamine receptors include, but are not limited to, N47, D75, F338, N375, and N379 of receptor D 3 , N52, D80, F382, N418, and N422 of receptor D 2 .
  • the contemplated amino acids of the dopamine receptors include, but are not limited to, N47, D75, V78, V82, V86, F106, DUO, Vl l l, C114, L121, V164, L168, 1183, F188, S192, S193, V195, S196, F197, F338, W342, F345, F346, H349, V350, Y365, T369, Y373, N375, and N379 of receptor D3, N52, D80, V85, V87, V91, F110, D114, V115, C118, L125, 1166, L170, 1184, F189, S193, S194, V196, S197, F198, F382, W386, F389, F390, H393, 1394, Y408, T412, Y416, N418, and N422 of receptor D 2 .
  • the assessing step when identifying agonists and antagonists of dopamine receptors, comprises investigating a computer model for a predicted interaction between the ligand and the amino acid. In still other preferred embodiments, the assessing step comprises binding the ligand to the dopamine receptor and determining binding between the ligand and the amino acid. However, in still further embodiments, determining the binding between the ligand and the amino acid comprises determining the crystal structure of the ligand bound to the receptor.
  • determining the binding between the ligand and the amino acid comprises comparing a binding affinity between the ligand and the receptor with a binding affinity between the ligand and a receptor lacking the amino acid. Some other additional embodiments further contemplate that determining the binding between the ligand and the amino acid comprises measuring the ability of the ligand to displace a molecule bound to the amino acid of the receptor.
  • the present invention provides a method of treating a subject having a disease, addiction, or other pathological condition (e.g., cocaine abuse, depression, anxiety, an eating disorder, alcoholism, chronic pain, obsessive compulsive disorder, schizophrenia, restless legs syndrome (RLS), and Parkinson's disease, and the like) comprising administering to the subject a therapeutic dose of a composition of the present invention.
  • a disease, addiction, or other pathological condition e.g., cocaine abuse, depression, anxiety, an eating disorder, alcoholism, chronic pain, obsessive compulsive disorder, schizophrenia, restless legs syndrome (RLS), and Parkinson's disease, and the like
  • RLS restless legs syndrome
  • Parkinson's disease e.g., Parkinson's disease, and the like
  • the present invention provides methods of treating (e.g., administering an effective therapeutic amount/dose) a subject with a composition comprising a compound of formula (including derivatives of this formula):
  • X, Y, and Z independently represent C, O, N, S;
  • R 7 , R 8 , and R 9 independently represent H, F, Cl, Br, I, OH, CN, NO 2 , OR', CO 2 R', OCOR', CONR'R", NR"COR', NR'SO 2 R", SO 2 NR'R", NR'R" where R' and R" independently represents H, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, or heterocyclic, substituted or unsubstituted; A and B independently represent O, S, SO, SO 2 , NR', CR'R' ' where R' and R' ' independently represent H, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, or heterocyclic, substituted or unsubstituted;
  • the present invention provides methods of treating (e.g. administering an effective therapeutic amount/dose) a subject with a composition comprising a compound of formula (including derivatives of this formula):
  • compositions comprising a compound of formula (including derivatives of this formula):
  • X, Y, Z, D and M independently represent C, O, N, S;
  • R 7 , R 8 , R 9 , R ⁇ o,R ⁇ ,Ri2,Ri3,Ri4, andRi 5 independently represent H, F, Cl, Br, I, OH, CN,
  • R' independently represents H, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, or heterocyclic, substituted or unsubstituted;
  • a and B independently represent O, S, SO, SO 2 , NR', CR'R" where R' and R" independently represent H, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, or heterocyclic, substituted or unsubstituted;
  • R represents H, O, S, SO, SO 2 , NR', CR'R" where R' and R" independently represent
  • alkyl alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, or heterocyclic, substituted or unsubstituted.
  • the present invention provides methods of treating a subject (e.g., administering an effective therapeutic amount/dose) with a composition comprising a compound of formula (including derivatives of this formula):
  • X, Y, Z, D, and M independently represent C, O, N, S;
  • R 7 , R 8 , R 9 , and R, 0 independently represent H, F, Cl, Br, I, OH, CN, NO 2 , OR', CO 2 R', OCOR', CONR'R", NR"COR', NR'SO 2 R", SO 2 NR'R", NR'R" where R' and R" independently represents H, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, or heterocyclic, substituted or unsubstituted;
  • a and B independently represent O, S, SO, SO 2 , NR', CR'R" where R' and R" independently represent H, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, or heterocyclic, substituted or unsubstituted;
  • the present invention has a condition characterized by faulty (e.g., aberrant) regulation and/or function of a G-protein couple receptor (e.g., a dopamine family receptor).
  • a G-protein couple receptor e.g., a dopamine family receptor
  • the subject being treated has one or more conditions characterized by the faulty regulation and/or function of a dopamine receptor (e.g., D l s D 2 , D 3 , D , and /or D 5 ).
  • the subject has one or more conditions characterized by faulty (e.g., aberrant) regulation and/or function of the D 3 receptor.
  • the present invention provides therapeutic methods and compositions for treating cocaine addiction/abuse.
  • cocaine addiction/abuse is considered a disease.
  • Still further embodiments of the present invention provide methods of modulating
  • the present invention provides methods of administering an agonist of the action of a G-protein coupled receptor to a subject. In some other embodiments, the present invention provides methods of administering an antagonist of the action of a G-protein coupled receptor to a subject. It is understood that the subjects contemplated for treating (e.g., receiving the administration) with the therapeutic methods and compositions of the present invention include mammals, and more particularly, include humans. Other embodiments of the present invention provide methods of controlling dopamine flow in a subject in need of such control comprising administering to the subject an effective amount of at least one composition of present invention.
  • Still further embodiments of the present invention provide methods of treating a subject with a D 3 receptor-specific modulator, comprising administering to the subject a compound of formula (I) or formula (II), wherein formula (I) is (including derivatives of this formula):
  • X, Y, Z and M independently represent C, O, N, S;
  • R 7 , R 8 , R 9 , and R 10 independently represent H, F, Cl, Br, I, OH, CN, NO 2 , OR', CO 2 R', OCOR', CONR'R", NR"COR', NR'SO 2 R", SO 2 NR'R", NR'R" where R' and R" independently represents H, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, or heterocyclic, substituted or unsubstituted;
  • a and B independently represent O, S, SO, SO 2 , NR', CR'R" where R' and R" independently represent H, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, or heterocyclic, substituted or unsubstituted;
  • X, Y, and Z independently represent C, O, N, S;
  • R 7 , R 8 , and R 9 independently represent H, F, Cl, Br, I, OH, CN, NO 2 , OR', CO 2 R', OCOR', CONR'R", NR"COR', NR'SO 2 R", SO 2 NR'R", NR'R" where R' and R" independently represents H, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, or heterocyclic, substituted or unsubstituted;
  • a and B independently represent O, S, SO, SO 2 , NR', CR'R" where R' and R" independently represent H, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, or heterocyclic, substituted or unsubstituted;
  • R represents alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, or heterocyclic, substituted or unsubstituted;
  • preferred embodiments of the therapeutic methods of the present invention contemplate administering an effective dose of one or more compounds of the present invention to a subject.
  • a receptor e.g., dopamine receptor
  • modulating e.g., antagonizing and/or agonizing
  • the present invention additionally comprises the coadministration of one or more (e.g., at least one) additional therapeutic compounds that are relevant to the treatment of a particular disease in the subject (e.g., a disease characterized by the aberrant regulation and/or function of a G-protein coupled [e.g., dopamine] receptor) in additional to the administration of a therapeutic compound of the present invention.
  • a particular disease in the subject e.g., a disease characterized by the aberrant regulation and/or function of a G-protein coupled [e.g., dopamine] receptor
  • the present invention provides methods of treatment or prophylaxis of conditions characterized by the faulty (e.g., aberrant ) regulation or function of dopamine receptors.
  • the methods of the present invention are particularly well suited for the treatment of cocaine abuse, depression, anxiety, an eating disorder, alcoholism, chronic pain, obsessive compulsive disorder, schizophrenia, restless legs syndrome (RLS), and Parkinson's disease, and the like.
  • compositions comprising: one or more compositions of the present invention; and instructions for administering the composition(s) to a subject, wherein the subject has a disease characterized by aberrant receptor (e.g., dopamine receptor) function/regulation.
  • aberrant receptor e.g., dopamine receptor
  • the instructions included with these kits meet U.S. Food and Drug Administrations rules, regulations, and suggestions for provision of therapeutic compounds.
  • the present invention provides methods of screening a G-protein coupled receptor (e.g., D ) modulating compound and a test compound comprising: providing: a G-protein coupled receptor modulating compound; a test compound; a first group of cells; and contacting the first group of cells with the G-protein coupled receptor modulating compound and the test compound; and observing the effects of contacting the first group of cells with the G-protein coupled receptor modulating compound and the test compound.
  • a G-protein coupled receptor e.g., D
  • the present invention further provides the additional step of comparing the effects observed in the first cells against a second group of the cells contacted with the G-protein coupled receptor modulating compound alone, or with the test compound alone. Effects that may be observed include, but are not limited to, changes in dopamine metabolism.
  • the present invention further contemplates additional methods for selling test compounds screened/identified by the above methods.
  • test compounds may be offered for sale by a third party in one or more forms (e.g., a kit, including, instructions for administering the test compound to a patient).
  • Fig. 1 shows the modeled 3D structure of dopamine receptor D 3 and its ligand binding site.
  • Fig. 2 shows nine relatively selective D3 partial agonists used to derive a pharmacophore model for 3D-database searching in one embodiment of the present invention.
  • Fig. 3 shows a simple pharmacophore model used in one embodiment of present invention.
  • Fig. 4 shows several compounds contemplated so use in one embodiment of the present invention.
  • Fig. 5 shows one novel approach to identifying compounds useful in the present invention.
  • Fig. 6A shows a superposition of 9 D 3 partial agonists and agonists contemplated in one embodiment of the present invention.
  • Fig. 6B shows a simple pharmacophore model derived from contemplated D 3 ligands.
  • Fig. 7 shows several compounds that are useful in some embodiments of the present invention.
  • Fig. 8 shows the functional activity of compound 4 at Di, D 2 , and D 3 receptors and comparison to standard ligands in one embodiment of the present invention.
  • Fig. 9 shows that compound 4 forms a strong salt bridge between its protonated nitrogen and Dl 10 in D 3 in one embodiment of the present invention.
  • Fig. 10 shows several compounds that are useful in some embodiments of the present invention.
  • Fig. 11 shows one contemplated synthesis scheme for making compounds use in some methods of the present invention.
  • Fig. 12 shows one contemplated synthesis scheme for making compounds use in some methods of the present invention.
  • Fig. 13 shows several compounds that are useful in some embodiments of the present invention.
  • Fig. 14 shows functional assays using transfected cells in one embodiment of the present invention.
  • Fig. 15 shows one contemplated synthesis scheme for making compounds use in some methods of the present invention.
  • Fig. 16 shows one contemplated synthesis scheme for making compounds use in some methods of the present invention.
  • Fig. 17 shows several compounds that are useful in some embodiments of the present invention.
  • Fig. 18A show a predicted binding model for compounds 14 with the D 3 receptor in one embodiment of the present invention.
  • Fig. 18B show a predicted binding model for compounds 32 with the D 3 receptor in one embodiment of the present invention.
  • Fig. 19 shows a schematic representation of compound 14 in the D3 receptor's binding site in one embodiment of the present invention.
  • Fig. 20 shows one contemplated synthesis scheme for making compounds use in some methods of the present invention.
  • Fig. 21 shows one contemplated synthesis scheme for making compounds use in some methods of the present invention.
  • Fig. 22 shows one contemplated binding model of the present invention.
  • Fig. 23 shows one contemplated synthesis scheme for making compounds use in some methods of the present invention.
  • Fig. 24 shows one contemplated synthesis scheme for making compounds use in some methods of the present invention.
  • Fig. 25 shows one contemplated synthesis scheme for making compounds use in some methods of the present invention.
  • Fig. 26 shows a diagrammatic representation of one embodiment of the present invention.
  • the term "lead compound” refers to a chemical compound selected (e.g., rationally selected based on 3D structural analysis) for use directly as therapeutic compound, or for chemical modification (e.g., optimization) to design analog compounds useful in the treatment of a given condition.
  • “lead compounds” are selected for modification to increase binding and specificity to the D 3 receptor.
  • Lead compounds can be known compounds or compounds designed de novo.
  • a "pharmacophore” according to the present invention is a chemical motif, including, but not limited to, a number of binding elements and their three-dimensional geometric arrangement. The elements are presumed to play a role in the activity of compounds to be identified as a lead compound. The pharmacophore is defined by the chemical nature of the binding elements as well as the three-dimensional geometric arrangement of those elements.
  • in vitro refers to an artificial environment and to processes or reactions that occur within an artificial environment.
  • in vitro environments can consist of, but are not limited to, test tubes and cell cultures.
  • in vivo refers to the natural environment (e.g. , an animal or a cell) and to processes or reaction that occur within a natural environment.
  • the term "host cell” refers to any eukaryotic or prokaryotic cell (e.g., mammalian cells, avian cells, amphibian cells, plant cells, fish cells, and insect cells), whether located in vitro or in vivo.
  • eukaryotic or prokaryotic cell e.g., mammalian cells, avian cells, amphibian cells, plant cells, fish cells, and insect cells
  • the term "cell culture” refers to any in vitro culture of cells. Included within this term are continuous cell lines (e.g., with an immortal phenotype), primary cell cultures, finite cell lines (e.g., non-transformed cells), and any other cell population maintained in vitro, including oocytes and embryos.
  • the term "subject” refers to organisms to be treated by the methods of the present invention. Such organisms include, but are not limited to, humans.
  • the term “subject” generally refers to an individual who will receive or who has received treatment (e.g., administration of antagonists and/or agonists of the G-protein coupled receptors).
  • the G-protein coupled receptors comprise dopamine receptors (e.g., Ox, D 2 , D 3 , and D 4 ).
  • the dopamine receptor is the D receptor.
  • diagnosis refers to the to recognition of a disease by its signs and symptoms (e.g., aberrant G-protein coupled receptor regulation and/or function, and more particularly, aberrant dopamine receptor regulation and/or function), or genetic analysis, pathological analysis, histological analysis, and the like.
  • signs and symptoms e.g., aberrant G-protein coupled receptor regulation and/or function, and more particularly, aberrant dopamine receptor regulation and/or function
  • genetic analysis pathological analysis, histological analysis, and the like.
  • antisense is used in reference to RNA sequences that are complementary to a specific RNA sequence (e.g., mRNA). included within this definition are antisense RNA (“asRNA”) molecules involved in gene regulation by bacteria.
  • asRNA antisense RNA
  • Antisense RNA may be produced by any method, including synthesis by splicing the gene(s) of interest in a reverse orientation to a viral promoter that permits the synthesis of a coding strand. For example, once introduced into an embryo, this transcribed strand combines with natural mRNA produced by the embryo to form duplexes. These duplexes then block either the further transcription of the mRNA or its translation. In this manner, mutant phenotypes may be generated.
  • antisense strand is used in reference to a nucleic acid strand that is complementary to the "sense” strand.
  • the designation (-) i.e., "negative” is sometimes used in reference to the antisense strand, with the designation (+) sometimes used in reference to the sense (i.e., "positive") strand.
  • Regions of a nucleic acid sequences that are accessible to antisense molecules can be determined using available computer analysis methods.
  • sample as used herein is used in its broadest sense.
  • a sample suspected of indicating a condition characterized by the aberrant regulation and/or function of a G- protein coupled receptor, and more particularly, the aberrant regulation and/or function of a dopamine receptor may comprise a cell, tissue, or fluids, chromosomes isolated from a cell (e.g., a spread of metaphase chromosomes), genomic DNA (in solution or bound to a solid support such as for Southern blot analysis), RNA (in solution or bound to a solid support such as for Northern blot analysis), cDNA (in solution or bound to a solid support) and the like.
  • a sample suspected of containing a protein may comprise a cell, a portion of a tissue, an extract containing one or more proteins and the like.
  • the term “purified” or “to purify” refers, to the removal of undesired components from a sample.
  • substantially purified refers to molecules, either nucleic or amino acid sequences, that are removed from their natural environment, isolated or separated, and are at least 60% free, preferably 75% free, and most preferably 90% free from other components with which they are naturally associated.
  • an "isolated polynucleotide” is therefore a substantially purified polynucleotide.
  • the term “genome” refers to the genetic material (e.g., chromosomes) of an organism or a host cell.
  • nucleotide sequence of interest refers to any nucleotide sequence (e.g., RNA or DNA), the manipulation of which may be deemed desirable for any reason (e.g., treat disease, confer improved qualities, etc.), by one of ordinary skill in the art.
  • nucleotide sequences include, but are not limited to, coding sequences, or portions thereof, of structural genes (e.g., reporter genes, selection marker genes, oncogenes, drug resistance genes, growth factors, etc.), and non-coding regulatory sequences which do not encode an mRNA or protein product (e.g., promoter sequence, polyadenylation sequence, termination sequence, enhancer sequence, etc.).
  • Nucleic acid sequence and “nucleotide sequence” as used herein refer to an oligonucleotide or polynucleotide, and fragments or portions thereof, and to DNA or RNA of genomic or synthetic origin which may be single- or double-stranded, and represent the sense or antisense strand.
  • nucleic acid molecule encoding refers to the order or sequence of deoxyribonucleotides or ribonucleotides along a strand of deoxyribonucleic acid or ribonucleic acid. The order of these deoxyribonucleotides or ribonucleotides determines the order of amino acids along the polypeptide (protein) chain translated from the mRNA. The DNA or RNA sequence thus codes for the amino acid sequence.
  • gene refers to a nucleic acid (e.g., DNA or RNA) sequence that comprises coding sequences necessary for the production of a polypeptide or precursor
  • polypeptide e.g., proinsulin
  • the polypeptide can be encoded by a full length coding sequence or by any portion of the coding sequence so long as the desired activity or functional properties
  • the term also encompasses the coding region of a structural gene and includes sequences located adjacent to the coding region on both the 5' and 3' ends for a distance of about 1 kb or more on either end such that the gene corresponds to the length of the full-length mRNA.
  • the sequences that are located 5' of the coding region and which are present on the mRNA are referred to as 5' untranslated sequences.
  • the sequences that are located 3' or downstream of the coding region and which are present on the mRNA are referred to as 3 1 untranslated sequences.
  • genomic form or clone of a gene contains the coding region interrupted with non-coding sequences termed "introns” or “intervening regions” or “intervening sequences.”
  • Introns are segments of a gene which are transcribed into nuclear RNA (hnRNA); introns may contain regulatory elements such as enhancers. Introns are removed or “spliced out” from the nuclear or primary transcript; introns therefore are absent in the messenger RNA (mRNA) transcript.
  • mRNA messenger RNA
  • the term "exogenous gene” refers to a gene that is not naturally present in a host organism or cell, or is artificially introduced into a host organism or cell.
  • the term “vector” refers to any genetic element, such as a plasmid, phage, transposon, cosmid, chromosome, virus, virion, etc., which is capable of replication when associated with the proper control elements and which can transfer gene sequences between cells.
  • the term includes cloning and expression vehicles, as well as viral vectors.
  • RNA expression refers to the process of converting genetic information encoded in a gene into RNA (e.g., mRNA, rRNA, tRNA, or snRNA) through “transcription” of the gene (i.e., via the enzymatic action of an RNA polymerase), and for protein encoding genes, into protein through “translation” of mRNA.
  • Gene expression can be regulated at many stages in the process.
  • Up-regulation” or “activation” refers to regulation that increases the production of gene expression products (i.e., RNA or protein), while “down-regulation” or “repression” refers to regulation that decrease production.
  • nucleic acid molecule encoding refers to the order or sequence of deoxyribonucleotides or ribonucleotides along a strand of deoxyribonucleic acid or ribonucleic acid. The order of these deoxyribonucleotides or ribonucleotides determines the order of amino acids along the polypeptide (protein) chain translated from the mRNA. The DNA or RNA sequence thus codes for the amino acid sequence.
  • a partially complementary sequence is one that at least partially inhibits a completely complementary sequence from hybridizing to a target nucleic acid sequence and is referred to using the functional term "substantially homologous.”
  • the inhibition of hybridization of the completely complementary sequence to the target sequence may be examined using a hybridization assay (Southern or Northern blot, solution hybridization and the like) under conditions of low stringency.
  • a substantially homologous sequence or probe i.e., an oligonucleotide which is capable of hybridizing to another oligonucleotide of interest
  • conditions of low stringency are such that non-specific binding is permitted; low stringency conditions require that the binding of two sequences to one another be a specific (i.e., selective) interaction.
  • the absence of non-specific binding may be tested by the use of a second target which lacks even a partial degree of complementarity (e.g., less than about 30% identity); in the absence of non-specific binding the probe will not hybridize to the second non-complementary target.
  • a partial degree of complementarity e.g., less than about 30% identity
  • the probe will not hybridize to the second non-complementary target.
  • stringency is used in reference to the conditions of temperature, ionic strength, and the presence of other compounds such as organic solvents, under which nucleic acid hybridizations are conducted. With “high stringency” conditions, nucleic acid base pairing will occur only between nucleic acid fragments that have a high frequency of complementary base sequences.
  • conditions of "weak” or “low” stringency are often required with nucleic acids that are derived from organisms that are genetically diverse, as the frequency of complementary sequences is usually less. While the present invention can be practiced utilizing low stringency conditions, in a preferred embodiment, medium to high stringency conditions are employed.
  • High stringency conditions when used in reference to nucleic acid hybridization comprise conditions equivalent to binding or hybridization at 42 °C (e.g., overnight) in a solution consisting of 5X SSPE (43.8 g/1 NaCl, 6.9 g/1 NaH 2 PO 4 H 2 O and 1.85 g/1 EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS, 5X Denhardt's reagent [50X Denhardt's contains per 500 ml, 5 g Ficoll (Type 400, Pharamcia), 5 g BSA (Fraction V; Sigma)], 100 ⁇ j/ml denatured salmon sperm DNA and 50% (V/V) of formamide.
  • 5X SSPE 4. g/1 NaCl, 6.9 g/1 NaH 2 PO 4 H 2 O and 1.85 g/1 EDTA, pH adjusted to 7.4 with NaOH
  • 5X Denhardt's reagent 50X Denhardt's contains per 500 ml, 5 g Ficoll (Type
  • hybridized DNA samples are then washed twice in a solution comprising 2 X SSPE, 0.1% SDS at room temperature followed by 0.1X SSPE, 1.0% SDS at 42 °C when a probe of about 500 nucleotides in length is employed.
  • a solution comprising 2 X SSPE, 0.1% SDS at room temperature followed by 0.1X SSPE, 1.0% SDS at 42 °C when a probe of about 500 nucleotides in length is employed.
  • conditions that promote hybridization under conditions of high stringency e.g., increasing the temperature of the hybridization and/or wash steps, the use of formamide in the hybridization solution, etc.).
  • “Medium stringency conditions” when used in reference to nucleic acid hybridization comprise conditions equivalent to binding or hybridization at 42 °C in a solution consisting of 5X SSPE (43.8 g/1 NaCl, 6.9 g/1 NaH 2 PO 4 H 2 O and 1.85 g/1 EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS, 5X Denhardt's reagent [50X Denhardt's contains per 500 ml, 5 g Ficoll (Type 400, Pharamcia), 5 g BSA (Fraction V; Sigma)] 100 uj/ml denatured salmon sperm DNA, and 50% (V/V) formamide.
  • 5X SSPE 43.8 g/1 NaCl, 6.9 g/1 NaH 2 PO 4 H 2 O and 1.85 g/1 EDTA, pH adjusted to 7.4 with NaOH
  • 5X Denhardt's reagent [50X Denhardt's contains per 500 ml, 5 g Ficoll (Type 400, Ph
  • Low stringency conditions comprise conditions equivalent to binding or hybridization at 42 °C in a solution consisting of 5X SSPE (43.8 g/1 NaCl, 6.9 g/1 NaH 2 PO 4 H 2 O and 1.85 g/1 EDTA, pH adjusted to 7.4 with NaOH), 0.1% SDS, 5X Denhardt's reagent [5 OX Denhardt's contains per 500 ml, 5 g Ficoll (Type 400, Pharamcia), 5 g BSA (Fraction V; Sigma)], 100 g/ml denatured salmon sperm DNA, and 50%(V/V) formamide.
  • a solution comprising 5X SSPE, 0.1% SDS at 42 °C when a probe of about 500 nucleotides in length is employed.
  • numerous equivalent conditions may be employed to comprise low stringency conditions; factors such as the length and nature (DNA, RNA, base composition) of the probe and nature of the target (DNA, RNA, base composition, present in solution or immobilized, etc.) and the concentration of the salts and other components (e.g., the presence or absence of formamide, dextran sulfate, polyethylene glycol) are considered and the hybridization solution may be varied to generate conditions of low stringency hybridization different from, but equivalent to, the above listed conditions.
  • substantially homologous refers to any probe that can hybridize (i.e., it is the complement of) the single-stranded nucleic acid sequence under conditions of low stringency as described above.
  • a gene may produce multiple RNA species that are generated by differencial splicing of the primary RNA transcript.
  • cDNAs that are splice variants of the same gene will contain regions of sequence identity or complete homology (representing the presence of the same exon or portion of the same exon on both cDNAs) and regions of complete non-identity (for example, representing the presence of exon "A” on cDNA 1 wherein cDNA 2 contains exon "B” instead).
  • the two cDNAs contain regions of sequence identity they will both hybridize to a probe derived from the entire gene or portions of the gene containing sequences found on both cDNAs; the two splice variants are therefore substantially homologous to such a probe and to each other.
  • the present invention is not limited to the situation where hybridization takes place only between completely homologous sequences. In some embodiments, hybridization takes place with substantially homologous sequences.
  • the term "protein of interest” refers to a protein encoded by a nucleic acid of interest.
  • the term “native” when used in reference to a protein, refers to proteins encoded by partially homologous nucleic acids so that the amino acid sequence of the proteins varies.
  • variant encompasses proteins encoded by homologous genes having both conservative and nonconservative amino acid substitutions that do not result in a change in protein function, as well as proteins encoded by homologous genes having amino acid substitutions that cause decreased (e.g., null mutations) protein function or increased protein function.
  • pathogen refers a biological agent that causes a disease state (e.g., infection, cancer, etc.) in a host.
  • a disease state e.g., infection, cancer, etc.
  • fungi include, but are not limited to, viruses, bacteria, archaea, fungi, protozoans, mycoplasma, and parasitic organisms.
  • organism is used to refer to any species or type of microorganism, including but not limited to, bacteria, archaea, fungi, protozoans, mycoplasma, and parasitic organisms.
  • fungi is used in reference to eukaryotic organisms such as the molds and yeasts, including dimorphic fungi.
  • virus refers to minute infectious agents, which with certain exceptions, are not observable by light microscopy, lack independent metabolism, and are able to replicate only within a living host cell.
  • the individual particles i.e., virions
  • the term "virus” encompasses all types of viruses, including animal, plant, phage, and other viruses.
  • bacteria and "bacterium” refer to all prokaryotic organisms, including those within all of the phyla in the Kingdom Procaryotae. It is intended that the term encompass all microorganisms considered to be bacteria including Mycoplasma, Chlamydia, Actinomyces, Streptomyces, and Rickettsia. All forms of bacteria are included within this definition including cocci, bacilli, spirochetes, spheroplasts, protoplasts, etc. Also included within this term are prokaryotic organisms which are gram negative or gram positive. "Gram negative” and “gram positive” refer to staining patterns with the Gram-staining process which is well known in the art. (See e.g. , Finegold and Martin,
  • Gram positive bacteria are bacteria which retain the primary dye used in the Gram stain, causing the stained cells to appear dark blue to purple under the microscope.
  • Gram negative bacteria do not retain the primary dye used in the Gram stain, but are stained by the counterstain. Thus, gram negative bacteria appear red.
  • antigen binding protein refers to proteins which bind to a specific antigen.
  • Antigen binding proteins include, but are not limited to, immunoglobulins, including polyclonal, monoclonal, chimeric, single chain, and humanized antibodies, Fab fragments, F(ab')2 fragments, and Fab expression libraries.
  • immunoglobulins including polyclonal, monoclonal, chimeric, single chain, and humanized antibodies, Fab fragments, F(ab')2 fragments, and Fab expression libraries.
  • Fab fragments fragments, F(ab')2 fragments, and Fab expression libraries.
  • the peptide is conjugated to an immunogenic carrier (e.g., diphtheria toxoid, bovine serum albumin (BSA), or keyhole limpet hemocyanin [KLH]).
  • an immunogenic carrier e.g., diphtheria toxoid, bovine serum albumin (BSA), or keyhole limpet hemocyanin [KLH]
  • Various adjuvants are used to increase the immunological response, depending on the host species, including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (Bacille Calmette-Guerin) and Corynebacterium parvum.
  • any technique that provides for the production of antibody molecules by continuous cell lines in culture may be used (See e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). These include, but are not limited to, the hybridoma technique originally developed by K ⁇ hler and Milstein (K ⁇ hler and Milstein, Nature, 256:495-497).
  • Antibody fragments that contain the idiotype (antigen binding region) of the antibody molecule can be generated by known techniques.
  • fragments include but are not limited to: the F(ab')2 fragment that can be produced by pepsin digestion of an antibody molecule; the Fab' fragments that can be generated by reducing the disulfide bridges of an F(ab')2 fragment, and the Fab fragments that can be generated by treating an antibody molecule with papain and a reducing agent.
  • Genes encoding antigen binding proteins can be isolated by methods known in the art. In the production of antibodies, screening for the desired antibody can be accomplished by techniques known in the art (e.g., radioimmunoassay, ELISA (enzyme-linked immunosorbant assay), "sandwich” immunoassays, immunoradiometric assays, gel diffusion precipitin reactions, immunodiffusion assays, in situ immunoassays (using colloidal gold, enzyme or radioisotope labels, for example), Western Blots, precipitation reactions, agglutination assays (e.g., gel agglutination assays, hemagglutination assays, etc.), complement fixation assays, immuno fluorescence assays, protein A assays, and immunoelectrophoresis assays, etc.) etc.
  • radioimmunoassay e.g., ELISA (enzyme-linked immunosorbant assay), "sandwich” immunoas
  • the term "instructions for administering said dopamine receptor modulating compound to a subject,” and grammatical equivalents, includes instructions for using the compositions contained in the kit for the treatment of conditions characterized by the aberrant regulation and or function of a dopamine receptor (e.g., D 3 receptor), including, but not limited to, drug [e.g., cocaine] addiction, depression, anxiety, schizophrenia, Tourette's syndrome, eating disorders, alcoholism, chronic pain, obsessive compulsive disorders, restless leg syndrome, Parkinson's Disease, and the like) in a cell or tissue.
  • the instructions further comprise a statement of the recommended or usual dosages of the compositions contained within the kit pursuant to 21 CFR ⁇ 201 et seq.
  • test compound refers to any chemical entity, pharmaceutical, drug, and the like, that can be used to treat or prevent a disease, illness, sickness, or disorder of bodily function, or otherwise alter the physiological or cellular status of a sample (e.g., aberrant regulation and/or function of a G-protein coupled receptor, and more particularly, the aberrant regulation and/or function of a dopamine receptor [e.g., D 3 ]).
  • Test compounds comprise both known and potential therapeutic compounds.
  • a test compound can be determined to be therapeutic by using the screening methods of the present invention.
  • a "known therapeutic compound” refers to a therapeutic compound that has been shown (e.g. , through animal trials or prior experience with administration to humans) to be effective in such treatment or prevention.
  • test compounds are G-protein coupled receptor modulators.
  • the term "third party” refers to any entity engaged in selling, warehousing, distributing, or offering for sale a test compound contemplated for co- administered with a receptor (e.g., G-protein coupled receptor, and more particularly, a dopamine receptor [e.g., D 3 ]) modulating compound for treating conditions characterized by the aberrant regulation and/or function of a G-protein coupled receptor, and more particularly, the aberrant regulation and/or function of a dopamine receptor (e.g., D 3 ).
  • a receptor e.g., G-protein coupled receptor, and more particularly, a dopamine receptor [e.g., D 3 ]
  • a dopamine receptor e.g., D 3
  • modulate refers to the activity of a compound (e.g., a dopamine receptor modulating compound) to affect (e.g., to promote or retard) an aspect of a cellular or a molecular function, including, but not limited to, neurotransmitter cycling, enzyme activity, and the like.
  • a compound e.g., a dopamine receptor modulating compound
  • affect e.g., to promote or retard an aspect of a cellular or a molecular function, including, but not limited to, neurotransmitter cycling, enzyme activity, and the like.
  • agonists of dopamine receptors refers to compounds that promote the activity of one or more dopamine receptors, as compared to the activity of corresponding dopamine receptors not subjected to the agonist compound(s).
  • antagonists of dopamine receptors refers to compounds that retard the activity of one or more dopamine receptors, as compared to the activity of corresponding dopamine receptors not subjected to the antagonist compound(s).
  • Dopamine is a neurotransmitter that plays an essential role in normal brain functions. As a chemical messenger, dopamine is similar to adrenaline. In the brain, dopamine is synthesized in the pre-synaptic neurons and released into the space between the pre-synaptic and post-synaptic neurons. Dopamine affects brain processes that control movement, emotional response, and ability to experience pleasure and pain. Regulation of dopamine plays a crucial role in mental and physical health. Neurons containing the neurotransmitter dopamine are clustered in the midbrain in an area called the substantia nigra.
  • Abnormal dopamine signaling in the brain has been implicated in many pathological conditions, including drug (e.g., cocaine) abuse, depression, anxiety, schizophrenia, Tourette's syndrome, eating disorders, alcoholism, chronic pain, obsessive compulsive disorders, restless leg syndrome, Parkinson's Disease, and the like.
  • drug e.g., cocaine
  • depression anxiety
  • schizophrenia Tourette's syndrome
  • eating disorders anxiety
  • alcoholism chronic pain
  • obsessive compulsive disorders restless leg syndrome
  • Parkinson's Disease and the like.
  • Dopamine molecules bind to and activate the dopamine receptors on the post- synaptic neurons. Dopamine molecules are then transported through the dopamine transporter protein (DAT) back into the pre-synaptic neurons, where they are metabolized by monoamine oxidase (MAO). In conditions such as cocaine abuse, cocaine binds to the dopamine transporter and blocks the normal flow of dopamine molecules. Excess concentrations of dopamine cause over-activation of dopamine receptors. In other conditions, such as Parkinson's Disease, lack of sufficient dopamine receptors in the brain causes insufficient activation of dopamine receptors.
  • DAT dopamine transporter protein
  • MAO monoamine oxidase
  • the D 3 receptor is concentrated almost exclusively in limbic brain regions such as the nucleus accumbens, olfactory tubercle, and islands of Calleja.
  • limbic brain regions such as the nucleus accumbens, olfactory tubercle, and islands of Calleja.
  • B. Landwhermeyer et al Brain Res. Mol. Brain Res., 18:187-192 [1993]; A.M. Murray et al, Proc. Natl. Acad. Sci. USA, 91:11271-11275 [1994]; and M.L. Bouthenet et al, Brain Res., 564:203-219 [1991]).
  • These brain areas are terminal fields of the mesolimbic dopamine projection that are associated with reinforcement and reward mechanisms.
  • the present invention contemplates that the dopamine 3 (D 3 ) subtype receptor is involved in the positive reinforcing effects of cocaine and other psychostimulants. Accordingly, preferred embodiments of the present invention contemplate designing dopamine receptor ligands (e.g., partial agonists) to treat cocaine and other psychostimulant abuse and dependence.
  • dopamine receptor ligands e.g., partial agonists
  • D 3 agonists produced an aversive effect when administered alone (F. Chaperon and M.H. Thiebot, Behav. Pharmacol., 7:105-109 [1996]) and attenuated the cocaine- and amphetamine-conditioned place preference (T.V. Khroyan et al, Psychopharmacology, 139:332-341 [1998]; and T.V. Khroyan et al, Psychopharmacology, 142:383-392 [1999]).
  • D 3 partial agonists have been shown to decrease cocaine self-administration.
  • the dopamine 3 (D 3 ) receptor has been reported to have 52% sequence homology to the D 2 receptor and a similar, but unique pharmacological profile. (P. Sokoloff et al, Nature, 347:146-151[1990]). Data suggests that the D 3 receptor may be involved in the positive reinforcing effects of cocaine and other psychostimulants. (See e.g., S.B. Caine and G.F. Koob, Science, 260:1814-1816 [1993]; G.F. Koob and M. Le Moal, Science, 278:52- 58 [1997]; S.B. Caine et al, J. Pharmacol. Exp. Ther., 291:353-360 [1999]; M.
  • Partial D 3 agonists with unambiguous selectivity for the D 3 receptor were not available prior to the present invention.
  • most existing D 3 ligands also have good activity at the D 2 subtype receptor.
  • highly selective D 3 ligands serve as useful pharmacological tools to facilitate the elucidation of the functional role of the D 3 receptor in cocaine addiction.
  • the partial agonists specific for the D receptor provide therapeutic compounds for the treatment of cocaine abuse.
  • the compositions and methods of the present invention provide therapeutic treatments for other additions to other psychostimulants characterized by interference with normal dopamine metabolism.
  • compositions and methods of the present invention provide therapeutic treatments for other diseases and conditions characterized by aberrant regulation and/or function of dopamine signaling and/or dopamine receptor activity (e.g., depression, anxiety, schizophrenia, Tourette's syndrome, eating disorders, alcoholism, chronic pain, obsessive compulsive disorders, and Parkinson's Disease, and the like).
  • Additional embodiments of the present invention provide compositions and methods for the treatment of conditions characterized by aberrant regulation and/or function of G-protein couple receptors (GPCRs), and methods for designing these compounds.
  • GPCRs G-protein couple receptors
  • bacteriorhodopsin is not a GPCR and has very low sequence homology with the dopamine receptors and rhodopsin, the topological arrangement of the transmembrane helices differ between the X-ray structure of rhodopsin and bacteriorhodopsin (K. Palczewski et al, Science, 289:739-745 [2000]), and hence the accuracy of the bacteriorhodopsin based dopamine models is limited.
  • the structure of rhodopsin has been used as the template structure for modeling, the low- resolution of existing rhodopsin structures limits their accuracy as well.
  • ligands designed to partially, or fully, modulate e.g., agonism and/or antagonism
  • the present invention also provides compounds designed to block other members of the D family of receptors and other members of the GPCR superfamily. Still other embodiments provide therapeutic compositions and methods for the treatment of conditions characterized by faulty regulation of dopamine signaling.
  • the present invention relates to compounds designed as ligands (e.g., agonists, antagonists, and partial agonists) for dopamine receptors (e.g., Ox, D 2 , D 3 , D4, and D 5 ), and in particular, the Di, D 2 , and D dopamine receptors.
  • ligands e.g., agonists, antagonists, and partial agonists
  • dopamine receptors e.g., Ox, D 2 , D 3 , D4, and D 5
  • the present invention also relates to methods for rationally designing these compounds and to therapeutic methods of using these compounds.
  • compositions and methods of the present invention are described in more detail in the following sections: I. Dopamine receptors; II. Homology modeling of dopamine receptors Dj, D 2 , and D 3 ; III. Computational docking studies of the interactions between D3 ligands and the receptors; IV. General design strategy for identifying novel lead compounds; V. Identification of specific novel lead compounds; VI. Further structure-based design and chemical modifications of lead compounds; VII. Biological testing and characterization of lead compounds and analogues thereof; VIII. Therapeutic agents combined or co-administered with the present compositions; and IX. Pharmaceutical formulations, administration routes, and dosing considerations.
  • compositions and methods directed to providing modulators (e.g., agonists, and/or antagonists, both partial and complete) of the dopamine receptors, and more particularly D 3 receptors.
  • modulators e.g., agonists, and/or antagonists, both partial and complete
  • the present invention is not intended to be limited, however, to providing modulators of dopamine receptors. Indeed, the methods and compositions of the present invention are useful in identifying and designing compositions (e.g., ligands) that modulate an number of receptor family subtypes (e.g., G-protein coupled receptors).
  • Both the Di-like and D 2 -like receptor subtypes are G-protein coupled receptors, but different G-proteins and effectors are involved in their signaling pathways.
  • reserachs have shown that are at least five dopamine receptors (DrD ) and they may be divided into two subfamilies whose properties resemble the original Di and D 2 receptors defined through pharmacological and biochemical techniques.
  • Each known dopamine receptor contains seven transmembrane stretches of hydrophobic amino acids.
  • each dopamine receptor conforms to the general structural model for G-protein coupled receptors (D. Donnelly et al, Receptors Channels, 2(1 ):61-78 [1994]) by having an extracellular amino terminus and seven membrane spanning-helices linked by intracellular and extracellular protein loops.
  • the D ⁇ like receptors have short third intracellular loops and long carboxyl terminal tails, whereas the D 2 -like receptors have long third intracellular loops and short carboxyl terminal tails.
  • the third intracellular loop in dopamine receptors is thought to be important for the interaction of receptor and G-protein and for the D 2 -like receptors, variants of these subtypes exist based on this loop.
  • the D 2 and D 3 receptors with the long forms having an insertion (29 amino acids for D 2 long) in this loop.
  • B. Giros et al Nature, 342(6252):923-926 [1989]
  • polymorphic variants of the D 2 receptor have been described with single amino acid changes in this loop.
  • the D 2 -like receptor variants may have differential abilities to couple to or activate G-proteins (A. Cravchik et al, J. Biol. Chem., 271(42):26013-26017 [1996]; J. Guiramand et al, J. Biol. Chem., 270(13):7354-7358 [1995]; and S.W. Castro and P.G. Strange, FEBS Letts., 315(3):223-226 [1993]) and may also exhibit slightly different pharmacological properties (S.W. Castro and P.G. Strange (1993) J. Neurochem., 60(l):372-375 [1993]; and
  • Dopamine receptor subtypes O and D 2 have different pharmacological profiles, localization, and mechanisms of action.
  • both the Di and D 5 receptors show similar pharmacological properties (e.g., high affinity for benzazepine ligands).
  • the thioxanthines show high affinity for D like receptors, but are not selective for Di-like over D 2 -like receptors.
  • the Di-like receptors also show moderate affinities for typical dopamine agonists such as apomorphine, and selective agonists such as dihydrexidine.
  • ligands selective for either the D] or D 5 dopamine receptors were not known.
  • Di receptors are found at high levels in the typical dopamine rich regions of brain such as the neostriatum, substantia nigra, nucleus accumbens and olfactory tubercle, whereas the distribution of the D 5 receptors is much more restricted.
  • the D 5 subtype is found at lower levels then the Di subtype dopamine receptors. Both Di and D 5 receptors are able to stimulate adenylyl cyclase, with the D 5 receptor showing some constitutive activity for this response.
  • Di and D 5 receptors Inverse agonist activity at the Di and D 5 receptors is seen in recombinant systems with some compounds, such as butaclamol, that were previously considered to be antagonists.
  • some compounds such as butaclamol
  • the Di receptor mediates important actions of dopamine to control movement, cognitive function and cardiovascular function.
  • D 5 receptors a G-protein coupled receptor
  • GABAA receptors ion channel linked receptors
  • D 2 -like receptors exhibit pharmacological properties similar to those of the original pharmacologically defined D 2 receptor (e.g., they all show high affinities for drugs such as the butyrophenones, and the substituted benzamides). These classes of drugs are contemplated to provide selective antagonists for the D 2 -like receptors. As indicated above, the D 2 -like receptors also show high affinities for phenothiazines and thioxanthines. Each D 2 -like receptor has its own pharmacological signature, such that there are some differences in affinities of drugs for the individual D 2 -like receptors.
  • raclopride shows a high affinity for the D 2 and D 3 receptors but a lower affinity for the D 4 receptor.
  • Clozapine shows a slight selectivity for the D 4 receptor.
  • Aminotetralins show some selectivity as D 2 -like autoreceptor antagonists, but theses compounds also show selectivity for D 3 receptors as agonists.
  • Most D 2 -like receptor antagonists show a higher affinity for the D 2 receptor compared to the D 3 and D 4 receptors.
  • the D 2 -like subtypes show moderate affinities for typical dopamine agonists, while the D 3 receptors show slightly higher affinities.
  • There are compounds available that are selective agonists for D 2 -like receptors e.g., quinpirole
  • the D 2 receptor is the predominant D 2 -like receptor subtype in the brain and is found at high levels in typical dopamine rich brain areas. D 3 and D 4 receptors are found at much lower levels and in a more restricted distribution pattern. The D 3 and D 4 receptors are found predominantly in limbic areas of the brain.
  • the D 2 -like receptor subtypes have each been shown to inhibit adenylyl cyclase when expressed in recombinant cells (See e.g., CL. Chio et al, Mol. Pharmacol., 45(l):51-60 [1994]; B. Gardner B et al, Br. J. Pharmacol., 118(6): 1544- 1550 [1996]; L. Tang et al, J. Pharmacol. Exp.
  • the D 2 -like receptors will also stimulate mitogenesis (CL. Chio et al, Mol. Pharmacol., 45(l):51-60 [1994]; and B.C. Swarzenski et al, Proc. Natl. Acad. Sci. U.S.A., 91(2):649-653 [1994]) and extracellular acidification (Chio, infra.) in recombinant systems.
  • D 3 and D 4 receptors The localization of the D 3 and D 4 receptors in limbic areas of brain suggests that they have a role in cognitive, emotional, and behavioral functions. The distribution of the D 3 and D 4 receptors in limbic brain regions has made them particularly attractive targets for the design of potential selective antipsychotic drugs.
  • the D2-like receptors show high affinities for most of the drugs used to treat schizophrenia (antipsychotics) and Parkinson's disease (e.g. bromocriptine).
  • the X-ray structure of rhodopsin was recently determined at 2.8 A resolution. (K. Palczewski et al, Science, 289:739-745 [2000]).
  • the Di, D 2 , and D 3 receptors belong to the rhodopsin family within the GPCR super-family.
  • the Di, D 2 , and D 3 receptors and rhodopsin have a high sequence homology (identity/similarity) of 47%.
  • the high resolution X-ray structure of rhodopsin is used to accurately model the 3D structures of dopamine receptors.
  • homology modeling in addition to the accuracy of the template protein structure, the sequence alignment between the modeled protein (D 1 ⁇ D 2 , and D 3 ) and the template protein (rhodopsin) is important for the accuracy of the modeled structure.
  • a previous sequence analysis of 493 members of the rhodopsin family of GPCR proteins provides an unambiguous sequence alignment in the TM region, that includes the binding site, between Di, D 2 , and D 3 receptors and rhodopsin. (J.M. Baldwin et al, J. Mol. Biol., 272:144-164 [1997]). Therefore, in preferred embodiments of the present invention, homology modeling methods provide accurate 3D structural modeling of Dj, D 2 , and D 3 receptors, especially, of the ligand binding site.
  • the lipid membrane/water environment affects the 3D structures of transmembrane proteins. Accordingly, in some embodiments, to further improve the accuracy of modeled structures, extensive MD simulations including an explicit lipid-water environment in the structural refinement were performed. In some of these embodiments, a highly efficient, parallel version of the CHARMM program (version 2.7) (B.R. Brooks et al, J. Comp. Chem., 4:187-217 [1983]) running on a supercomputer is used in the MD simulations as the modeling environment includes approximately 16,000 atoms, comprising, the Di, D 2 , and D 3 proteins, lipids, and water molecules.
  • ns MD simulations for the Di and D 3 receptor structures have been completed, and 2.5 ns simulations have been completed for the D 2 receptor structure.
  • substantially longer MD simulations are used (e.g., 5 ns, 7 ns, 10 ns, and longer).
  • the present invention is not intended to be limited to an particular length of modeling or simulation. Those skilled in the art will appreciate the potential effects of various modeling and simulation times (e.g., longer MD simulation times allow identification of additional low energy conformational clusters with respect to the binding site conformations if previous MD simulations are not sufficiently long enough).
  • the binding site in D 2 has been experimentally determined through experiments using methanethiosulfonate (MTS) reagents. (See, J.A.
  • the D 2 receptor contains three common Ser residues in TM5, (See e.g., NJ. Pollock et al, J. Biol. Chem., 267:17780-17786 [1992]; B.L. Wiens et al, Mol. Pharmacol., 54:435-444 [1998]; A. Mansour et al, Eur. J. Pharmacol., 227:205-214 [1992]; C Coley et al, J. Neurochem., 74:358-366 [2000]; N. Sartania and P.G. Strange, J.
  • the present invention contemplates that the major structural differences between D 3 and ) ⁇ provides a rational basis for designing ligands that selectively bind to the various dopamine receptors, and in other embodiments, the basis for designing ligands that selectively bind to other members of the G-protein receptor family.
  • Table 2 shows some of the sequence differences in Di and D 3 binding sites (residues for Di are shown in parentheses) in regions accessible by solvent molecules or ligands.
  • TM4 F162 (K167), V164(T169), P167(S172), L168(D173), L169(G174), F170(N175) TM5 F188(Y194), V189(A195), Y191(S197), V195(I201), L199(I205) TM6 V334(277), T348(L291), H349(N292), V350(C293), T353(P296) TM7 L364(T312), Y365(F313), S366(D314), A367(V315), T368(F316), T369(V317), Y373(W321)
  • D 2 and D 3 receptors There are also significant structural differences between the D 2 and D 3 receptors, although, less profound than found in Di and D receptors. Careful structural analysis using high resolution models of D 2 and D 3 was used to identify four regions (i.e., Regions A, B, C, and D) of major sequence and structural differences between D 2 and D 3 receptors. For corresponding residues that are different between the dopamine receptor subtypes, there is some uncertainties with the coordinates of these side chain atoms. To solve this problem, the present invention uses current homology modeling methods employing a rotormer library to provide the most probable conformations of side chain for non-identical residues.
  • the structural differences characterized in these four regions provides a rational basis for designing ligands that selectively bind to a particular receptor of interest (e.g., D 3 receptor).
  • a particular receptor of interest e.g., D 3 receptor.
  • D 3 residues N47, D75, F338, N375, N379 (D 2 residues N52, D80, F382, N418, N422, respectively) in TM1 are part of a large hydrophobic pocket deep in the transmembrane region.
  • the present invention provides ligands that selectively bind either D 3 or D 2 receptors that have a flexible linker tethered to a bulky tail designed to reach the large hydrophobic pocket in Region A.
  • chimeric D 2 /D 3 receptor studies showed that TM6 and TM7 contribute to the selectivity of PD 129807 which is a selective D 3 partial agonist.
  • Chimeric D 2 /D 3 receptor studies also showed that the TM6 and TM7 regions are responsible for the selectivity of several ligands between D 2 and D 3 .
  • Still other studies show that mutation of F198, F389, F390 to alanine in the D 2 receptor abolishes binding of N-0437 which is a ligand that shows a binding preference for D 2 versus D 3 .
  • mutation of T369V significantly increases the binding affinities of two D 3 selective ligands, but not that of non- selective ligands. This suggests that this region is important for ligand selectivity.
  • a hydrophobic pocket in Region D is formed by TM3, TM4, TM5 and E-II loop residues.
  • the orientation and position of VI 11, 1183, F188 in D 3 is significantly different from that of the corresponding D 2 residues, VI 15, 1194, F189.
  • D 3 is an He in D 2 is near the three TM5 serine residues.
  • the difference found in the preferred kink angle of TM5 also contributes to the structural differences of this pocket.
  • Region D of the D 3 and D 2 receptors contain non-conservative sequence differences, for example, in the position adjacent to the disulphide bridge connecting TM3 and the E-II loop residue SI 82 in the D 3 receptor corresponds to residue 1183 in the D 2 receptor. Of note, this position is in the proximity of 1183, F188 in D 3 or 1184, F189 in D 2 .
  • A163 corresponds to T165 in D 2 .
  • a water exposed D 2 residue, S163 is available to interact with ligands in this region and it is replaced by alanine in D 3 (A161).
  • Preferred embodiments of the present invention contemplate designing dopamine receptor subtype specific ligands based on the structural differences in these four specific regions (Regions A, B, C, and D). In particularly preferred embodiments, the present invention contemplates the structure-based design of ligands that specifically bind to Region B of the D receptor.
  • compositions (and methods) of the present invention were designed using accurate modeling of the various dopamine receptor and their transmembrane (TM) regions.
  • the sequences of the dopamine receptors contain about 93- 97% of the set of the conserved amino acids shared by the 493 proteins in the rhodopsin family. (See, J.M. Baldwin et al, J. Mol. Biol., 272:144-164 [1997]).
  • the sequence alignment between Dj, D 2 , and D 3 and rhodopsin used in the homology modeling of the present invention was derived from 500 GPCR members and Di, D 2 , D , and rhodopsin belong to the rhodopsin sub-family within the GPCRs.
  • Dopamine receptors Di, D 2 , and D 3 and rhodopsin share about 47% residue identity/similarity in 7 different TM regions.
  • the sequence identity between rhodopsin and the D 3 receptor is 28%, near the 30% threshold considered sufficient to achieve highly accurate homology modeled structures.
  • the high resolution X-ray structure of rhodopsin is used to provide accurate modeling of the dopamine receptors Di, D 2 , and D 3 receptor structures.
  • the coordinates of the side chain atoms in the rhodopsin structure were copied as the coordinates of the side chain atoms of the corresponding dopamine residues.
  • the residue in rhodopsin was mutated to the corresponding dopamine residue type.
  • the sidechain rotamer library within the homology modeling module was used to model the most likely amino acid sidechain orientation.
  • Table 3 shows the sequence alignment of the transmembrane (TM) regions (TM1- TM7, plus a short TM8) between rhodopsin (RHOA) and D b D 2 and D 3 receptors.
  • Bold letters indicate the conserved amino acids within the 493 members of the rhodopsin family of GPCR proteins that are identical between rhodopsin and all three dopamine receptors at the same time. In preferred embodiments, these conserved amino acids combined with further sequence identities between rhodopsin and the dopamine receptors provide an unambiguous sequence alignment for each transmembrane helix.
  • Underlined letters in Table 3 are additional identical, or similar amino acid residues among rhodopsin, Di, D 2 , and D receptors.
  • the 7 TM helices form a large crevice at the extracellular side of the TM region including the ligand binding pocket in Di, D 2 , and D 3 receptors.
  • several extracellular loops E-I, E-II, E-III
  • the short intracellular loop C-I was also included because its sidechains interacts with the short helical segment connected to the intracellular end of helix 7 in the crystal structure of rhodopsin.
  • Other loops (C-II, C-III) and the N- and C-terminal ends are remote from the ligand binding site.
  • This disulfide bond is contemplated to limit the conformational flexibility of this short loop in dopamine receptors. Therefore, in preferred embodiments the loop structure was generated using modeling tools of the INSIGHTII program package (INSIGHT II, Molecular Simulations Inc., San Diego, CA) to satisfy the spatial requirements for the formation of the disulfide bond between the cysteine residue at the end of TM3 and the cysteine in the E-II loop.
  • INSIGHT II INSIGHT II, Molecular Simulations Inc., San Diego, CA
  • two conserved proline kinks found in TM5 and TM6, are important for ligand binding due to their influence on ligand binding site structure.
  • TM7 Another conserved proline kink is found in the intracellular half of TM7, which is believed to be essential for receptor functions.
  • the absolute conservation of these three important prolines allows the accurate modeling of the proline kinks in TM5, TM6 and TM7 in the modeled structures of D 1 ⁇ D 2 , and D 3 .
  • Two other prolines in rhodopsin, one found in TM1 (Pro53) and the second proline found in the extracellular side of the TM7 (Pro291), are replaced by other residues in Di, D 2 , and D 3 .
  • TM1 is too remote from ligand binding site to not be important for ligand binding.
  • Pro291 found in the extracellular side of the TM7 is close to the extracellular end of this helix, and thus has minimal effect on the ligand binding site conformation.
  • a normal hydrogen bonding pattern of the ⁇ -helix at these two positions was obtained by applying weak NOE restraints between the hydrogen bonding backbone atoms during the initial MD simulation.
  • the accurate modeling of the most important helices in Di, D 2 , and D 3 using the X-ray structure of rhodopsin provides the present invention with high quality structures of the ligand binding sites for Di, D 2 , and D 3 .
  • the lipid membrane/water environment has been shown to significantly affect the 3D structures of TM proteins. (See, R.G. Efremov et al, Biophys. J., 76:2460-2471 [1999]).
  • dopamine receptor modeling efforts avoided modeling the lipid membrane/water environment due to limited computer power.
  • the explicit lipid membrane/water environment was included in additional structural modeling experiments to further refine the models accuracy.
  • the choice of lipid molecule for modeling the membrane is l-palmitoyl-2- oleoyl-sn-glycero-3-phosphatidylcholine (POPC).
  • POPC l-palmitoyl-2- oleoyl-sn-glycero-3-phosphatidylcholine
  • a bilayer model consisting of 200 POPC molecules was used in modeling experiments as specified in W.L.
  • the dopamine receptor structure (e.g., D 3 ) was embedded into the bilayer model according to the position of the predicted membrane boundaries obtained from sequence analysis of 493 GPCR proteins. (See, J.M. Baldwin et al, J. Mol. Biol., 272:144-164 [1997]). First, a number of lipid molecules located at the center of the lipid bilayer were removed to create a hole with a diameter approximately same as that of the receptor structures. Next, the size of the protein structure was scaled down by 50% before placing it into the center of the hole.
  • the reduced scale protein structure was then gradually scaled back to its original size in 5% incremental steps through a MD simulation.
  • the lipid molecules were allowed to adjust themselves to accommodate the protein structure while the protein structure was kept rigid, it is contemplated that this procedure avoids the creation of large voids between the protein structure and the lipid, and the sudden disruption of the structures of both the protein and the bilayer molecules caused by severe van der Walls repulsion that would occur if the protein was simply placed into the bilayer hole without this process.
  • Intracellular and extracellular regions of the receptor structures are then exposed to water.
  • the water environment in these experiments is accurately modeled by using the TIP3P explicit water model as described in B.R. Brooks et al. (B.R.
  • a parallel version of the CHARMM program (version 2.7) (A.D. MacKerell Jr., et al, J. Phys. Chem. B., 102:3586-3616 [1998]) and its latest CHARMM force field (W.D. Cornell et al, J. Am. Chem. Soc, 117:5179-5197 [1995]) are employed for all the energy minimization and MD simulations.
  • the present invention further contemplates running parallel simulations using the latest version of the AMBER force field and program to confirm that the conformational clusters obtained from the MD simulations are not influenced by the CHARMM force field used.
  • AMBER and CHARMM are two most popular MD simulation programs. The force fields for the AMBER and CHARMM programs have been extensively tested for protein simulations.
  • the present invention contemplates running these models and simulations on a 512 CPU Cray T3E supercomputer (e.g., at the Pittsburgh Supercomputer Center), the 32 CPU Origin 2000 computer system at the National Institutes of Health (NTH), and or the Linux 48-CPU cluster at the University of Michigan Cancer Research Center.
  • a 512 CPU Cray T3E supercomputer e.g., at the Pittsburgh Supercomputer Center
  • the 32 CPU Origin 2000 computer system at the National Institutes of Health (NTH)
  • NTH National Institutes of Health
  • Linux 48-CPU cluster at the University of Michigan Cancer Research Center.
  • pre-equilibration of the dopamine receptor structures is achieved via energy minimization and molecular dynamics simulations.
  • the Adopted-Basis Newton Raphson (ABNR) method is used for energy minimization. Simulations were performed using all atom representations in the latest version of the CHARMM force field (W.D. Cornell et al, J. Am. Chem. Soc, 117:5179- 5197 [1995]), except for the hydrocarbon tails of the POPC lipid molecules, to the united CHARMM atom model was applied.
  • the Leapfrog Verlet fl/gorithm was used.
  • the stochastic boundary method was applied if their hydrocarbon tail atoms were further than 35 A away from the origin; the friction coefficient was set to 200.
  • the phosphorus atoms in the lipid headgroups were fixed.
  • the dielectric constant was set to 1, and the time step to 1 fs.
  • the temperature was kept constant at 300 K with coupling decay time of 1.0 ps. Long range electrostatic forces were treated with the force switch method in the range of 12 to 14 A; Van der Waals forces were cut at 14 A.
  • the nonbond list was generated up to 15 A and updated heuristically.
  • the frequency of checking atoms entering the Langevin region was set to 20 steps. Before the final production MD run, the system was pre-equilibrated in three steps of nested energy minimization cycles and extensive MD simulation runs. To extensively refine the dopamine structures and to sample the binding site conformations, lengthy production MD simulations (10 ns or longer) are performed. Trajectory files of final production runs are saved every ps for analysis.
  • the binding site of the dopamine receptors has certain flexibility, especially with respect to the helix bends of TM5 and TM6 and the side chain conformations of the residues that form the binding pocket. Furthermore, different ligands may preferentially bind to distinctively different conformations of the binding crevice.
  • some embodiments of the present invention use more lengthy (e.g., 5, 10, 50, 100 ns, or more) MD simulations for each receptor (e.g., D 1 ⁇ D 2 , D 3 , D 4 , and/or D 5 ) to sample the conformations of the binding crevice. Since many conformations have been generated, use of conformational cluster analysis is necessary to identify the major conformations obtained in the simulations. Conformational cluster analysis was performed using the CHARMM scripts using two criteria. The first criterion was the helical bend angles of TM5 and TM6. The second criterion was the dihedral angles of those residues that form the binding pocket.
  • the receptor structures used in the docking studies of the present invention were obtained from lengthy MD simulations of the 3D structures of Di, D 2 , and D . Cluster analysis was performed to identify the major (most populated) conformational clusters. In preferred embodiments, for each dopamine receptor subtype (e.g., D , D 2 , and D 3 ), each of the major conformational clusters are used in subsequent docking studies. Although an understanding of the mechanism is not necessary to practice the present invention and the present invention is not so limited, it is contemplated that one advantage in using more than one structure for each receptor is that the conformational flexibility of the receptor is better taken into account, in addition to taking into account ligand conformational flexibility.
  • the receptors are treated using the united-atom approximation in the AMBER force field.
  • ACD Available Chemicals Directory
  • Polar hydrogen atoms were added to the receptors, and Kollman united-atom partial charges were assigned.
  • all water molecules were removed, and atomic solvation parameters and fragmental volumes were assigned to the receptor atoms using AddSoll feature in the AUTODOCK program.
  • all identified lead compound D 3 ligands and their selective analogs are studied using the modeling and simulation methods of the present invention.
  • all of the designed lead compound analogs are subjected using extensive docking simulations and modeling.
  • all possible chiral configurations are considered for each ligand.
  • the initial ligand 3D structures are built using the Sybyl (Tripos Associates, Inc., St. Louis, MO) molecular modeling package.
  • the AUTODOCK (version 3.0) program is used for conducting docking studies. (See e.g., G.M. Morris et al, J. Computer- Aided Molecular Design, 10:293-304 [1996]; G.M.
  • the AUTODOCK program has been extensively used to predict accurate protein-inhibitor, peptide-antibody, and protein-protein complexes.
  • the AUTODOCK program takes the ligand's conformational flexibility into account during docking simulations.
  • the Lamarckian genetic algorithm (LGA) method is highly efficient for prediction of the correct ligand binding models.
  • the Lamarckian genetic algorithm (LGA) method in the AUTODOCK program is further used in the docking studies of the present invention. (D.S. Goodsell et al, J. Mol. Recognition, 9:1-5 [1996]).
  • the most recent version of the AUTODOCK program also incorporates a new empirical binding free energy function.
  • the rotatable bonds in the ligand were defined using an AUTODOCK utility, AUTOTORS. In preferred docking simulations, all rotatable bonds of the ligands were not restricted. The position and the orientation of the ligands were also not restricted.
  • the center of the grid maps was defined using the coordinate of the sulfur atom of the Cysl 18 in D 2 , or Cysl 14 in D 3 , or of the oxygen atom of Serl07 in Di, which was determined to be part of the binding site.
  • 10 docking simulations were normally performed.
  • the step sizes used in docking simulations were 0.2 A for translations and 5° for orientations and torsions.
  • the clustering tolerance for the root-mean-square deviation for the ligand was set as 1.0 A.
  • the present invention contemplates using 2 (or more) different docking simulation programs/routines provides cross-validation and improved accuracy. Accordingly, preferred embodiments of the present invention use 2 (or more) docking simulation programs and/or routines.
  • the present invention contemplates using multiple protein (e.g., dopaime receptors and/or other g-protein coupled receptors) conformations obtained from MD simulations for computational docking studies.
  • the receptors e.g., dopamine receptors Di, D 2 , and/or D 3
  • the receptors may assume multiple low energy conformations with respect to their binding sites, thus, different ligands may bind to different low energy conformations of the receptors.
  • some preferred embodiments of the present invention use multiple conformation for a target protein in the design of selective ligands (e.g., D 3 selective ligands).
  • the present invention provides docking studies of 7-OH-DPAT and its analogs to the D 3 receptor structure.
  • (+)-7-OH-DPAT is a partial agonist that has shown some selectivity for dopamine receptor subtype D 3 over subtype D 2 .
  • extensive computational docking studies were performed using the AUTODOCK program package. (G.M. Morris et al, J. Computer-Aided Molecular Design, 10:293-304 [1996]; G.M. Morris et al, J. Comp. Chem., 19:1639-1662 [1998]; D.S. Goodsell et al, J. Mol.
  • the D and D 3 receptors bind protonated amine containing ligands with high affinity, although not limited to any particular mechanism, this binding is believed to occur through the formation of a salt bridge with an Asp at the extracellular end of TM3 (common in other members of the rhodopsin family of GPCRs as well). Mutation of this aspartate in the D 2 (Dl 14) receptor to either Gly or Asn abolishes the binding of both agonists and antagonists. (See, A. Mansour et al, Eur. J. Pharmacol., 227:205-214 [1992]).
  • (+)-7-OH-DPAT is predicted to form hydrogen bonds with SI 92 and SI 96.
  • an S192A mutant D 3 receptor binds the (+)-7-OH-DPAT ligand with 16-fold reduced affinity compared to the wild type receptor.
  • the corresponding residue in D 2 , S193 was also shown to contribute to the binding of this ligand at the D 2 receptor. (See, C Coley et al, J. Neurochem., 74:358-366 [2000]).
  • Cysl 14 is in close contact with one of the propyl groups of the (+)-7-OH-DPAT ligand, thus forming a "propyl" pocket. Indeed, a Cysl 14Ser mutation selectively decreases the affinity of propyl containing (+)-7-OH-DPAT ligands out of a set of 14 compounds tested. (See, G.L. Alberts et al, Br. J. Pharmacol.,
  • Preferred embodiments of the present invention used a novel structural based approach for identifying potential lead compounds as selective ligands of dopamine receptors.
  • these novel structural based design approaches are used to identify and to further design (e.g., optimize) potent and selective modulators (e.g., partial agonists) of dopamine receptor D 3 .
  • potent and selective modulators of dopamine receptor D 3 are useful therapeutic compounds in the treatment of conditions characterized by the faulty regulation of D 3 receptors in a subject (e.g., depression, anxiety, schizophrenia, Tourette's syndrome, eating disorders, alcoholism, chronic pain, obsessive compulsive disorders, and Parkinson's Disease, and the like).
  • the present invention further provides a novel and powerful approach, as shown in Fig. 5, for identifying and designing (e.g., optimizing) lead G-protein receptor binding ligands (e.g., dopamine receptors, including, but not limited to, Di, D 2 , D 3 , D 4 , and D 5 ).
  • a first steps provides identification of potential lead compounds
  • a pharmacophore model was developed based upon the 3D structures of known D 3 ligands and their structure-activity relationships (SAR). The pharmacophore model that was used to search the National Cancer Institute's (NCI) 3D- structure database and/or to search the Available Chemicals Directory (ACD) (See, S. Wang et al, J. Med. Chem., 37:4479-4489 [1994]) database.
  • NCI National Cancer Institute's
  • ACD Available Chemicals Directory
  • the current version of the NCI 3D "open” database consists of 245,000 structures, whose structures and chemical samples can be accessed by researchers. (See, J.H. Voigt et al, J. Chem. Inf. Comput. Sci., 41:702-712 [2001]).
  • the latest version of the ACD database (v99.2) contains 275,000 modeled 3D structures. (Available Chemicals Directory, MDL Information Systems, Inc., Morristown, NJ)
  • the present invention provides a Web-based 3D-pharmacophore docking program package. It was found that the Web-based 3D-pharmacophore docking program of the present invention provides similar results as the Chem-X program. (See, I.D. Kuntz, Science, 257:1078-1082 [1992]). In some embodiments, one or both of the NCI and ACD databases, converted into a 3D-database format, are searched by the present Web- based program as part of identifying potential receptor binding lead compounds.
  • a second structure-based screening step provides a subsequent tool to further define the interactions of the hit compounds (e.g., dopamine receptor ligands), identified in the first step, with the receptor of interest (e.g., D 3 receptor).
  • the hit compounds e.g., dopamine receptor ligands
  • the receptor of interest e.g., D 3 receptor
  • the second structure-based screening step uses a "smaller" database containing only the "hits" identified through the 3D-pharmacophore searching of first step.
  • the present invention is not intended to be limited, however, to selecting potential target receptor ligands based on this methodology.
  • structure-based database searching is used dock each of the hit compounds grouped into the a database into the binding pocket of the receptor of interest (e.g., D receptor binding-pocket).
  • the present invention contemplates ranking the hit compounds according to their scores as calculated by the
  • the scoring function used in the CERIUS 2 program calculates the negative of the potential interaction energy between ligand and receptor.
  • the present invention contemplates, that in limited cases the energy based DOCK program scoring function may not provide a good correlation with the binding affinity of a particular candidate ligand to its target receptor. Accordingly, preferred embodiments of the present invention contemplate overcoming this potential issue by using a novel consensus scoring function developed as part of the present invention.
  • the consensus scoring function consists of three different empirical binding affinity prediction methods, CHEMSCORE, LBSCORE, and LUDISCORE, (See, J. Compu -Aided Mol. Des., 11 :425-445 [1997]; J. Mol.
  • the present invention contemplates, that in limited cases the energy based CERIUS program scoring function may not provide a good correlation with the binding affinity of a particular candidate ligand to its target receptor. Accordingly, preferred embodiments of the present invention contemplate testing the docking and scoring protocol used for structure based database searching as follows. A set of 25 known D 3 ligands were 'mixed' with database compounds.
  • this two-stage computerized screening approach enables the present invention to identify most promising potential ligands based hot only upon their 3D- pharmacophor binding elements but also based upon their interactions with 3D structure of the target receptor of interest (D 3 receptor).
  • this novel approach provides a powerful tool for discovering promising dopamine receptor, and other G-protein receptor, modulators (e.g. , agonists and/or antagonists).
  • modulators e.g. , agonists and/or antagonists.
  • the following example further describes preferred embodiments of the two step structure-based screening approach to identifying potential selective receptor binding ligands (e.g., dopamine receptor [including, but not limited to: Di; D 2 ; D 3 ; D 4 ; and/or D 5 ] agonists and/or antagonists).
  • dopamine receptor including, but not limited to: Di; D 2 ; D 3 ; D 4 ; and/or D 5 ] agonists and/or antagonists.
  • a pharmacophore model is defined as common and essential structural elements for activity. Previous studies have identified a number of potent and relatively selective D 3 partial agonists and full agonists.
  • a representative sub-set of 9 relatively selective partial and full agonists (See, Fig. 2) were chosen for the development of the pharmacophore model(s). Structural analysis of these compounds shows that these compounds contain a common aromatic ring and a Sp3 nitrogen attached to a propyl group. Except for pramipexole, the nitrogen is attached to two additional Sp3 carbons. The distance between the aromatic ring center and the basic Sp3 nitrogen within these compounds was established to be between 4.1 and 6.1 A through conformational analysis using the QUANTA program. (QUANTA, Molecular Simulations Inc., San Diego, CA).
  • the CHEM-X program (Chemical Design Ltd., Oxford England) was used to screen the NCI 3D-database of 245,000 "open" test compounds against the pharmacophore model shown in Figs. 6A and 6B.
  • Fig. 6A shows a superposition of 9 D3 partial agonists and agonists.
  • Fig. 6B shows a simple pharmacophore model derived from contemplated D ligands.
  • compounds were selected that had one or more of the chemical groups required by the pharmacophore model, e.g., an aromatic ring, and/or a tertiary nitrogen attached to three carbon atoms.
  • the CHEM-X program then investigated whether the compound has a conformation that meets the 3D geometric parameters specified in the pharmacophore model. If the CHEM-X program found that the compound indeed had at least one conformation that met the 3D geometric requirements specified in the pharmacophore model, the compound was considered to be "hit.” In preferred embodiments, using this structural-based system, up to 3,000,000 conformations can be examined for hit compound. Using this approach, a total of 6,237 compounds from the NCI 3D-database were identified as hits, that satisfied both the chemical and geometrical requirements specified in the pharmacophore model.
  • the structures of compounds identified as being hits through 3D-pharmacophore searching are extracted from the either the NCI or the ACD databases in SDF format.
  • hydrogen atoms and Gasteiger-Marsili atomic charges are added using the SPL macro in the SYBYL program. (SYBYL, Tripos Associates, Inc., St. Louis, MO).
  • the final coordinates, in mol2 format, for all hit compounds are stored in a single database file.
  • the protonation for each ionizable group such as amine, amidine, and acids, in the hit compounds are assigned according to their protonation state at a physiological pH of 7.4.
  • the 6,237 hit compounds identified from the pharmacophore searching satisfied the basic and necessary chemical and geometrical requirements specified in the pharmacophore model. However, it is expected that many of these 6,237 hit compounds will not show appreciable activity binding to the D 3 receptor for or more reasons (e.g., steric hindrance).
  • a particular ligand may have the essential binding elements specified in the pharmacophore model, but be unable to bind to the receptor of interest (e.g., D 3 ) because the ligand is too big to fit into the receptor's binding site, or some of the ligand's functional groups may interfere with the receptor's residues, thus preventing the ligand from effectively interacting with the target receptor (e.g., D 3 receptor).
  • top scoring hit compounds contemplated as being potential ligands for the receptor of interest are visually examined for their structural novelty and diversity by comparing with known ligands of the receptor of interest (e.g., D 3 receptor ligands).
  • hit compounds that pass the visual inspection stage are tested in binding and functional assays.
  • the present invention contemplates using the most populated conformational cluster for a particular target receptor (e.g., D 3 receptor) in the docking studies obtained from extensive MD simulations in one or more structure-based databases using the CERIUS 2 program.
  • a particular target receptor e.g., D 3 receptor
  • highly potent ligands of a particular target receptor may bind to a conformation of target receptor that is somewhat (or even significantly) different from the most populated conformational cluster. Therefore some potent ligands may not able to dock into the receptor of interest and have a top docking score.
  • the present invention contemplates overcoming this potential limitation by using several populated low energy conformations obtained from lengthy MD simulations for structure-based database searching. It is further contemplated that using multiple receptor conformations for structure-based database searching provides for the discovery of additional novel lead compounds that otherwise might be missed.
  • the present invention contemplates the discovery and design of novel and potent ligands selective for g-protein receptor, and in particular for dopamine receptors, including, but not limited to, Di, D 2 , D 3 , D 4 , and D 5 .
  • accurate modeled structures of the dopamine receptors facilitates the discovery of novel and potent lead compounds as modulators (e.g., partial agonists and/or antagonists) of the D 3 receptor.
  • modulators e.g., partial agonists and/or antagonists
  • the present invention provides novel and powerful approaches for identifying and designing G-protein receptor ligands by combining ligand-based pharmacophore and structure-based database searches.
  • analysis of the modeled structures of target receptors provides further elucidation of the structural bases of ligand binding and selectivity at target receptors (e.g., dopamine receptors).
  • target receptors e.g., dopamine receptors
  • Detailed structural analyses of the Di, D 2 , and D receptors was used to identified four specific regions (Regions A, B, C, and D) that have significant structural differences between D 3 and D 2 receptors in their binding sites. Other detailed structural analyses were used to identify profound differences between the D 3 and Di receptors.
  • differences in the dopamine receptor structural Regions A, B, C, and D are used for rationally designing highly selective dopamine receptor ligands.
  • the present invention contemplates that the lead compounds described herein, as well as the additional lead compounds those skilled in the art will identify in view of the modeling and structure-based ligand identification and design methods disclosed herein, may be further optimized and refined to improve one or more characteristics (e.g., receptor affinity, receptor selectivity, etc.) using the disclosed chemical modification methods.
  • the present invention is not limited, however, to providing highly selective ligands for the D 3 receptor.
  • the present invention provides methods for rationally designing and chemically modifying a number of receptor ligands.
  • the specific examples of lead compound identification, testing, and chemical modifications presented below are intended to provide the reader with non-limiting representative examples of several of various lead compounds (analogs and derivatives) contemplated by the present invention.
  • the initial binding affinity of a first group of 30 potential dopamine receptor ligand test compounds was performed using cell lines transfected with human dopamine receptors. (See, B. Levant, Current Protocols in Pharmacology [J. Ferkany and S.J. Enna, Eds.], John Wiley & Sons, New York, 1.6.1-1.6.16 [1998]).
  • a potent D 2 and D 3 ligand [3H]YM-09151-2 were used as a radioligands for the D 2 and D 3 receptor binding assays.
  • a potent and selective D ⁇ receptor ligand, [3H]SCH 23,390 was used as a radioligand for Oi receptor binding assays.
  • the 30 test compounds were first measured for their ability to compete with [3H]YM-09151-2 binding to the D 3 receptor using CHO cells transfected with human D 3 (hD ) receptors. If sufficient binding was observed for a test compound at 10 ⁇ M, then its IC 50 value was obtained.
  • test compounds were screened in functional assays to elucidate their agonist (partial agonist) or antagonist activity on dopamine receptors (e.g., D 3 receptors).
  • N.D. Not determined due to low affinity. At least two experiments were performed for each compound.
  • the functional studies show compounds 1, 2, 3, 6, and 7 are antagonists, and that compounds 4, 5, and 8 are partial agonists of the D 3 receptor.
  • tests show that compound 4 is a potent D 3 partial agonist with an EC 50 of 9.2 nM with maximum stimulation of 51 % as compared to a full D 3 agonist quinpirole for stimulation of mitogenesis in transfected CHO-D 3 cells.
  • Fig. 8 shows the functional activity of compound 4 (CTDP-31793) at Di, D 2 , and D 3 receptors and comparison to standard ligands.
  • the agonist activity at Dj receptor was measured for stimulation of c AMP accumulation in C6D ⁇ cells.
  • the antagonist activity at Di was measured for inhibition of 10 nM dihydrexidine stimulation of c AMP accumulation in C6D1 cells.
  • the agonist activity at D receptor was measured for stimulation of mitogenesis using CHOp-hD 2 cells.
  • the antagonist activity at D 2 receptor was measured for inhibition of 30 nM quinpirole stimulation of mitogenesis in CHOp-hD cells.
  • the agonist activity at D 3 receptor was measured for stimulation mitogenesis using CHOp-hD cells.
  • the antagonist activity at D 3 receptor was measured for inhibition of 30 nM quinpirole stimulation of mitogenesis in CHOp-hD 3 cells.
  • compound 4 functions as an antagonist at the D 2 receptor with a Ki value of 35.1 nM.
  • compound 4 functions as a weak D ! antagonist with a Ki value of 657 nM (Fig. 8).
  • Compound 5 has a very similar profile in its functional activity but appears to be more selective than compound 4.
  • compound 5 functions as a partial agonist of D 3 , with an EC 50 of 22.2 nM and a 31% of maximum stimulation of mitogenesis as compared to quinpirole, as a D 2 antagonist with an IC 50 value of 378 nM, and as a weak Di antagonist with an IC 50 value of 1722 nM.
  • Compound 4 has a selectivity of 4-fold between D 3 /D 2 and 70-fold between D 3 /D f
  • compound 5 has a selectivity of 17-fold between D 3 /D 2
  • 78-fold between D 3 /D ⁇ in more meaningful functional experiments in measuring selectivity of the compounds.
  • compound 8 functions as a partial agonist of the D 3 receptor with an EC 50 of 146 nM and 53% of maximum stimulation.
  • the present invention contemplates that functional studies show that compounds 4 and 5 are potent D partial agonists, but that compounds 4 and 5 function as antagonists of Di and D 2 receptors.
  • Compounds 4 and 5, and in particular compound 5 display certain selectivity for D versus Di and D 2 in functional assays.
  • Compounds 4 and 5 have a functional profile identical to that of BP-897, which has been shown to have great therapeutic potential for the treatment of cocaine addiction. (See e.g., M. Pilla et al, Nature, 400:371-375 [1999]). Therefore, in preferred embodiments, compounds 4, 5, and 8 are promising lead compounds for further optimization to design potent and selective D 3 partial agonists.
  • Still further embodiments provide computational docking studies of lead compound 4 with the D 3 receptor. Since compound 4 is a flexible ligand it is beneficial to use a docking program, that considers ligand flexibility in the docking process. For this reason, the AUTODOCK program (See, D.S. Goodsell and A.J. Olson, Proteins: Str. Func Genet., 8:195-202 [1990]; G.M. Morris et al, J. Computer-Aided Molecular Design, 10:293-304 [1996]; G.M. Morris et al, J. Comp. Chem., 19:1639-1662 [1998]; D.S. Goodsell et al, J. Mol. Recognition, 9:1-5 [1996]; and G.M.
  • the present invention contemplates that other racemate lead compounds, analogs, and derivatives are also amenable to docking studies using (R)- and (S)-configurations of compound to determine whether one configuration binds to the target receptor more favorably.
  • Out of the 10 docking simulations 4 docking simulations with lowest predicted binding free-energy converged to a single binding model. (See, Fig. 9).
  • Fig. 9 shows that compound 4 forms a strong salt bridge between its protonated nitrogen and Dl 10 in D 3 , similar to that shown in (R)-(+)-7-OH-DPAT. (See, Fig. 3).
  • the tricyclic ring in compound 4 is in close contact with a number of hydrophobic residues in D 3 , including VI 11, V189, F346, F197, F345, W342, and Cl 14.
  • three serine residues, S192, SI 93, and SI 96 of D 3 are in close proximity with docked compound 4, thus, in some embodiments the present invention provides, derivatives of compound 4 designed to form additional hydrogen bonds with the D 3 receptor.
  • D 3 receptor model structure suggests that fairly large hydrophobic groups may be accommodated in the pocket of Region A.
  • atom groups In order to reach region A located deep in the transmembrane region, such atom groups must be attached at the end of a flexible linker.
  • the linker is contemplated to be a hydrocarbon chain substituent of the protonated nitrogen group, which is expected to interact with a common Asp in TM3 (Dl 10 in D 3 ).
  • TM3 Asp in TM3
  • Still further embodiments of the present invention take advantage of the significant differences between D 3 , D 2 , in the region A pocket for the design of ligands with high potency and/or selectivity for particular dopamine receptor subtypes of interest.
  • the present invention provides additional ligand compounds optimized for enhanced potency and/or selectivity when binding to additional receptors types of interest (e.g., G-protein coupled receptors) through rationally directed ligand chemical modifications.
  • chemical modifications of lead compounds are guided by structure-based design. For example, based upon the binding model shown in Fig. 9, new analogs of compound 4 were designed. In one embodiments, compounds 8, 9, and 10 were designed to investigate the optimal length of linker. (See, Fig. 10). In another embodiment, compounds 11 and 12 were designed to investigate the effects of the terminal phenyl group in compounds 4, 8, 9 and 10. (See, Fig. 10). Based on docking studies, the 4-F-phenyl group of compound 4 binds to a large hydrophobic pocket in the D 3 receptor.
  • the intermediate, compound 19 was alkylated with the commercially available compounds 20, 21, 22, 24, or 25, respectively, in acetonitrile using cesium carbonate as the base, to yield compounds 8, 4, and 20, 11, and 12, respectively. (See, L.A. van Vliet et al, J. Med. Chem., 43:2871 [2000]).
  • the intermediate compound 19 was first converted to its sodium salt with sodium hydride in DMF, and then reacted with commercially available compound 23 in DMF at room temperature to afford the final product compound 9.
  • rat brain binding assays are used to determine the binding affinities of the new analogues of lead compound 4. (See, B. Levant, Current Protocols in Pharmacology [J. Ferkany and S.J. Enna, Eds.], John Wiley & Sons, New York, 1.6.1-1.6.16 [1998]). The results of analogs tested in rat brain binding affinity assays are summarized in Table 6.
  • the data are average of 3-5 replicates.
  • compound 4 is more potent than compound 8 (e.g., about 7-12 fold more potent at D 2 , and 6-8 fold more potent at D 3 ) indicating that the binding affinities obtained from these two different assays are consistent in terms of relative potencies between analogues, although absolute values may differ.
  • This is consistent with previous observations that binding affinities of a ligand to a particular dopamine receptor depends upon the particular expression system or tissue, radioligand, or in vitro assay conditions used. (See, B. Levant, Pharmacol. Rev., 49:231-252 [1997]).
  • Compounds 11 and 12 were designed to investigate the importance of the interactions of the terminal 4-F-phenylkentone group with D 3 for binding affinity.
  • compound 11 is as potent as compound 4, and compound 12 is 3-times more potent than compound 4 at binding the D 3 receptor.
  • This finding is consistent with the predicted binding model for compound 4 (See, Fig. 9) that the 4-F-phenyl group is in close contact with a number of hydrophilic/charged residues in D 3 and may have some unfavorable interactions with the receptor.
  • compound 12 has a 9-fold selectivity for receptor D 3 versus receptor D 2 and is 11 -fold more selective than lead compound 4 for receptor D versus receptor D 2 .
  • the present invention provides compound 29 (CTDP- 31819) as a partial agonist of receptor D 3 .
  • Compound 29 (Fig. 13) was found to have potent affinity at D 3 and to have a selectivity of 39-fold between receptors D 3 and D 2 , and 10-fold selectivity between receptors D 3 and Di in binding assays using rat brain. (See, Table 7). Table 7 shows the binding affinities of lead compound 29 and several new analogues thereof in rat brain binding assays.
  • Compound 31, with an additional methoxyl group in the meta -position of the phenyl ring, is a potent D3 ligand (K, value 46 nM) and a good selectivity for receptor D 3 .
  • the selectivity for compound 31 is 15-fold between receptors D 3 and D 2 , and 175-fold between receptors D and Di.
  • the binding model for compound 14 shows that its naphthenyl ring reaches a portion of the D binding region that has significant differences between the D and D 2 receptors.
  • compound 32 with a naphthenyl ring, was contemplated designed and synthesized.
  • compound 32 provides a highly selective D 3 ligand.
  • chemical modifications of the lead compounds are guided by structure-based design strategies to yielded new analogs with improved binding affinity and selectivity for a target receptor (e.g., D 3 ) receptor versus other D family receptors (e g ., affinity and selectivity for D 3 receptor versus the Di and D 2 receptors).
  • a target receptor e.g., D 3
  • other D family receptors e.g ., affinity and selectivity for D 3 receptor versus the Di and D 2 receptors.
  • some of the lead compounds, and analogues/derivatives thereof have specific binding properties to Di or D 2 or other member of the D family of receptors.
  • the chemical modifications contemplated in preferred embodiments of the present invention are based, in part, on consideration of four different aspects: 1) analysis of the important structural differences among D ls D 2 , and D 3 receptors ,and especially between D 2 and D 3 receptors, that are contemplated to contribute to ligand selectivity; 2) discovery of novel and potent D 3 partial agonists as highly promising lead compounds; 3) effective optimization of lead compounds designed to improve the potency and the selectivity of candidate compounds at the D 3 receptor; and 4) characterization of novel ligands for their potency, selectivity and functional activity.
  • Some embodiments of the present invention for example, preformed extensive docking studies on lead compounds 4 and 29 and new more potent/ selective analogues of (compounds 14 and 32) thereof. Since compounds 14 and 32 are potent and selective D 3 ligands (Fig. 17), these two compounds are used as new lead compounds for further design and optimization.
  • the binding models for compounds 14 and 32 predicted using the CERTUS2 docking program is shown in Figs. 19A and 19B.
  • D 3 receptor residues that interact with compounds 14 and 32 are structurally identical or very similar in the D 3 and D 2 receptors, such as, Dl 10, Cl 14, F106, V86, H349 and V350. Therefore, it is contemplated that the interactions between ligands and these common D 3 and D 2 residues contributes to ligand affinity but not to selectivity.
  • structural analyses of the dopamine receptor structures shows that there are considerable structural differences between the D 3 and D 2 receptors, mainly located in four regions. (See, Table 2 and Figs. 18A and 18B).
  • Fig. 18A and 18B show the predicted binding models for compounds 14 (Fig. 18A) and 32 (Fig. 18B) with the D 3 receptor.
  • Residues within 5 A distance from the ligand are depicted and some specific interactions between the ligands and D 3 receptor are shown in dotted lines.
  • Four regions (Regions A, B, C, and D) that contribute to ligand selectivity, especially between D 3 and D 2 are shown in orange circles. Therefore, preferred embodiments of the present invention provide, new analogues with binding groups (elements) that specifically target these four regions are contemplated to provide greater selectivity.
  • One particular embodiment of the present invention provides, four new groups (Groups I, II, II, and IV) of analogues designed based upon the binding models of compounds 14 and 32 and the D 3 receptor. (See, Fig. 19). Fig.
  • the present invention provides analogues (Group I analogues) of compounds 14 and 32 with modifications in the tricyclic ring to better bind to dopamine receptor Region C In this region, among the dopamine receptors, there are 3 common serine residues.
  • lead compound analogues with hydrogen bonding donor/acceptor groups as substituent on the phenyl ring in different positions are provided to explore the structural differences between the 3 common dopamine receptor serine residues in Region C.
  • a general scheme for synthesis of the compounds shown in Group I (a) is illustrated in Scheme V. (See, Fig. 20). The steps of Scheme V are similar to the synthetic methods shown in Scheme I, the 2-substituted aminomethylquinoline, compound 42, was prepared from the starting material, compound 40, by bromination with NBS to yield 2-bromomethylquinoline, compound 41, followed by reaction with ethanolamine in ethanol.
  • the substituted quinaldines can be prepared directly by condensation of the corresponding substituted aniline with crotonaldehyde using the well-known Skraup method. (See, G. Jones, Quinolines Part I, 100-117 [1977]).
  • the present invention provides Scheme VII an alternative method for the synthesis of proposed new analogues in Group 1(a).
  • compounds 46 and 47 are commercially available, or they can be easily obtained using a known method. (See e.g., B. Scholl et al, Helv. Chim. Acta., 69:184-194 [1986]).
  • Compound 47 is epoxidized to obtain compound 48.
  • Condensation of compound 48 with phthalimide yields intermediate compound 49, which can be oxidized to ketone (compound 50).
  • Reduction of nitro group of compound 50 to an amine group produces compound 51. Through reductive amination of compound 51, compound 52 is obtained.
  • the present invention provides a synthesis routine for the analogues in Group 1(b) by using methods similar to those shown in Scheme III and IV. (See also, U.S. 3,931,188 [incorporated herein by reference in its entirety]; L.A. van Vliet et al, J. Med. Chem., 43:2871 [2000]; and K.Y. Avenell et al, Med. Chem. Lett., 8:2859- 2864 [1998]).
  • an alternative method for the synthesis of the analogues in Group 1(b) is as provided in Scheme VIII.
  • the present invention provides analogues of lead compounds 14 and 32 having modifications in the tail portion designed to target D3 receptor Region A (Group II analogues).
  • the ligands provide a general chemical structure having a flexible 5-6 bond linker (9-10 A) and a bulky hydrophobic tail.
  • new analogues of compounds 14 and 32 having a 4-carbon and amide group as a linker and a bulky naphthyl ring in their tail portions show much improved selectivity between D 3 and D 2 receptors as compared to the original lead compounds 4 and 29.
  • the amide group in the linker portion may play a role in ligand potency and selectivity by forming a hydrogen bond with residue N375, thus positioning the naphthyl group in proper orientation to interact with residues in Region A. More specifically, the naphthyl ring in compounds 14 and 32 is predicted interact with residues N379, F338, N47, L121 of the D 3 receptor in a region of structural differences between D 2 /D 3 as discussed in detail. Thus, although an understanding of the mechanism is not necessary to practice the present invention and the present invention is not so limited, it is contemplated that residue N47 appears plays a major role for the selectivity of compounds 14 and 32.
  • residues F338, N375 in the D 3 receptor corresponding to F382, N418 in D 2 are also believed to contribute to the ability of compounds 14 and 32 to distinguish among the dopamine receptor subtypes.
  • Fig. 22 shows the distance requirements of ligands to reach Region.
  • Some preferred embodiments of the present invention provide additional modifications of the naphthyl ring of lead compounds 14 and 32 to explore the structural differences in dopamine receptor Region A to achieve even greater selectivity.
  • small hydrophobic groups e.g., CH 3 , F, Cl, Br, I, and the like
  • polar substituents OH, OCH 3 , NH 2 , CN, and the like
  • the synthesis of analogues in Group 11(a) is accomplished by using methods similar to those shown in Schemes I, II, V, VI, and VII.
  • Yet other embodiments of the present invention provide analogues of lead compounds 14 and 32 having (Group III analogues) modifications in the linker designed to target dopamine receptor Region B.
  • Group III analogues modifications in the linker designed to target dopamine receptor Region B.
  • significant structural differences are found among the dopamine receptors in Region B, for instance, difference are found in the orientations of residues F345 and F346 in TM6 of receptors D 3 and D 2 .
  • the orientation and position of residues Y365 and T369 in TM7 are also significantly different, in part due to the different helical packing between TM6 and TM7.
  • some preferred embodiments of the present invention provide lead compound analogues that are optimized to extend ligand interactions with a particular dopamine receptor subtype (e.g., Dj, D 2 , D , D 4 , and/or D 5 ) in Region B of the receptor, by modifying the lead compound to provide one or more small hydrophobic groups that are designed to specifically interact with this region.
  • a particular dopamine receptor subtype e.g., Dj, D 2 , D , D 4 , and/or D 5
  • the present invention provides analogues of lead compounds 14 and 32 having modifications in the tricyclic ring that are designed to target dopamine receptor region D (Group IV analogues).
  • dopamine receptor Region D has a well-defined hydrophobic pocket in proximity to 3 serine residues. Further structural analysis shows that there are profound structural differences in this region among the dopamine receptor subtypes. These structural differences are believed to be primarily due to differences in the residues that make Region D. For example, several D 3 receptor residues in this region, A161, A163, S182, correspond to residues S163, T165, and 1183, respectively, in the D 2 receptor.
  • Preferred embodiments of the present invention provide lead compound analogues 14 and 32 have an additional substituted phenyl group linked to the tricyclic ring in through a sulphonylamide linker.
  • the sulphonylamide linker forms hydrogen bonds with the serine residues in TM5, while the substituted phenyl group interacts with the hydrophobic pocket in Region D that is formed, in the D 3 receptor, by residues 1183, Phel88, V164, L168, and VI 11.
  • the analogues in Group IV(a) and IV(b) are obtained by treating compounds 70 or 73 with different substituted benzenesulfonyl chlorides as shown in Scheme X. (See, Fig. 24). It is of note that compound 70 is obtained using synthetic methods shown in Schemes V and VII and that compound 73 is obtained using the methods outlined in Scheme VIII.
  • the present invention further provides methods for estimating docking of the new analogues to D 3 , D 2 , and Di and for the estimation of their binding affinities.
  • computational docking studies of the new analogues in with Dj, D 2 , and D 3 are performed using the methods outlined above.
  • the complex ligand-receptor structures are further refined through extensive MD simulation for 2 ns, or longer, in an explicit water and lipid environment using the CHARMM program. Based upon the predicted models, binding affinities are predicted.
  • DPAT DPAT to the D 3 receptor is stereo-specific
  • all of the compounds synthesized in Schemes I, II, and V are racemic since they all contain one chiral center.
  • Prefe ⁇ ed embodiments separate (+) and (-) stereoisomers using known stereoisomer separation methods. (See, L.A. van Vliet et al, J. Med. Chem., 43:2871 [2000]; and H. Wikstrom et al, J. Med. Chem., 28:215 [1985]).
  • Scheme XI Fig.
  • the novel compounds and ligands identified and designed using the methods of the present invention are subjected to a step-wise screening process for affinity, efficacy, and selectivity at the dopamine receptors as outlined in Fig. 26.
  • the present invention contemplates that, initial studies be used to focus on determining receptor affinity and selectivity in in vitro radioligand binding assays using the rat brain model and cloned human receptors. (See, Fig. 26, steps 1, 2, 3, and 4).
  • the present invention further contemplates that, additional studies be used to determine the efficacy and D 2 /D 3 selectivity in two different in vitro functional assays. (See, Fig. 26, step 5).
  • the present invention provides the following non-limiting examples, to further describe several of the biological tests and assays contemplated for characterizing the novel compounds and ligands identified and designed using the methods of the present invention.
  • the determination of affinity and selectivity of novel compounds at their target receptors e.g., dopamine receptors
  • their selectivity for the these receptors is initially determined in radioligand binding assays.
  • 3 series of mutually complementary radioligand binding studies are performed in a step-wise manner.
  • the invention contemplates that this complementary approach compensates for the particular limitations of any one of the individual assays, thus providing a more accurate determination of the affinity and selectivity of novel compounds at their target receptors than is provided using a single assay approach.
  • the invention further contemplates that this complementary approach provides a thorough and definitive assessment of the in vitro pharmacological profile of the compounds with respect to their target receptors (e.g., dopamine receptors).
  • potential therapeutic compounds designed using the methods of the present invention, are first subjected to screens in the rat brain D 3 receptor model describe above. If a test compound exhibits sufficient affinity and selectivity for the D 3 receptor, then the affinity and selectivity for the D 3 receptor is further determined additional in rat brain assays and in cloned human dopamine receptors assays. Finally, compounds that exhibit selectivity for the D receptor in both the rat brain membranes and in cloned human receptors are further evaluated using an autoradiographic methods to simultaneously determine D 2 , and D 3 receptor affinities.
  • affinity of test compounds at the D3 receptor is measured using [ 3 H]PD 128907 binding in membranes prepared from rat ventral striatum (e.g., nucleus accumbens and olfactory tubercle).
  • Rat ventral striatum tissues are used for D 3 receptor assays because these tissues express the highest density of D 3 receptors in the rat CNS. (See, M.L. Bouthenet et al, Brain Res., 564:203-219 [1991]).
  • the present invention contemplates that the [3H]PD128907 agonist exhibits greater that 300-fold selectivity for the D 3 receptor over the D 2 receptor in rat brain.
  • the affinity of test compounds for Di and D 2 receptors is determined by using antagonists (e.g., radioligands [ H]SCH 23390 and [ H]spiperone, respectively) and membranes from rat caudate-putamen (striatum) that expresses high densities of Di and D 2 receptors and low densities of other dopamine receptors according to the methods in Levant et al. (B. Levant, CNS Neuro transmitters and Neuromodulators, 3 [T.W. Stone, Ed.], CRC Press, Boca Raton, FL, 77-88 [1996]).
  • the [ 3 H]SCH 23390 and [ 3 H]spiperone antagonists are highly selective for the Di-like and D 2 receptors, respectively. It was found that [ 3 H]spiperone exhibited an 80- fold higher affinity for D receptors than D 3 receptors. With the low density of D receptors expressed in rat striatum, labeling of the D 3 receptor by [ 3 H]spiperone is negligible. Likewise, rat striatum expresses very low densities of the D 5 receptor. Thus, the present invention contemplates that, although [ 3 H]SCH 23390 has similar affinity for Di and D 5 receptors, the binding assays in these studies are representative of the Di receptor.
  • [ 3 H]SCH 23390 and [ 3 H]spiperone binding assays are performed using in vitro assay conditions that favor agonist binding at dopamine receptors (e.g., inclusion of Mg 2+ , and the exclusion of NaCl [See e.g., D. Grigoriadis and P. Seeman, J. Neurochem., 44:1925-1935 (1985)]).
  • the present invention contemplates that assays performed under these in vitro conditions enhance the ability to detect high-affinity binding of agonists in assays using antagonist radioligands.
  • assays are performed as previously described in Wang et al. (See, S. Wang et al, J. Med.
  • the K; value for each test compound at each dopamine receptor subtype is determined.
  • Table 8 describes the determination of binding affinity at Di, D 2 , and D 3 receptors in rat brain.
  • the primary advantage of performing the initial evaluation of test compounds in rat brain is that this approach allows the study of receptors expressed in their native tissue. Hence, the source tissue will contain all components required for normal physiological function. With the use of thoroughly characterized radioligands and brain areas with known densities of dopamine receptor subtypes, it is possible to determine the affinities of novel test compounds at Di, D 2 , and D 3 receptors in brain tissue. This approach has the additional advantage that rat brains are readily available and are relatively inexpensive compared to cloned receptors. Table 9, provided below, shows the affinity (Kj or K ⁇ j values) of reference ligands used in contemplated binding assays in rat brain.
  • a second phase of biological test compound evaluation involves evaluation of the affinity and selectivity of test compounds for the D 3 receptor using cloned human receptors (hDi, hD 2s h 0 r t> hD 3 , and hD 4 ) expressed in transfected cell lines.
  • test compounds progress to the second phase of evaluation if they exhibit selectivity for the D 3 receptor in rat brain.
  • the affinity of the test compounds is determined in hDi, hD 2s hort > hD 3 , and hD 4 receptors expressed in CHO cells (NEN Life Science and Sigma- Aldrich) using [ 3 H]SCH 23390 for hDi binding and [ 3 H]spiperone for the hD 2s h 0 rt, hD 3 , and hD 44 receptors. Assays are performed as previously described in Wang et al. (See, S. Wang et al, J. Med. Chem., 39:2047-2054 [1996]). In yet other prefe ⁇ ed embodiments, the K s value for each test compound at each dopamine receptor subtype is determined.
  • the present invention contemplates that the use of cloned human receptors has the advantage of enabling the study of human, rather than rat receptors, thus increasing the relevance to human therapeutics. Because the host cells do not normally express dopamine receptors, this approach also enables the study of each receptor subtype in isolation, obviating the dependence on selective pharmacological tools. Although different radioligands are used for hDi and the hD 2 -like receptor studies, the in vitro assay conditions otherwise remain the same. Hence, it is further contemplated that this approach compensates for the potential limitations of the use of rat brain membranes for determination of dopamine receptor affinity and selectivity.
  • the present invention provides methods for the simultaneous autoradiographic determination of D 2 /D 3 -selectivity in rat brain.
  • the quantitative autoradiographic method for the simultaneous determination of D 2 and D receptor affinity in rat brain using the D 2 -D 3 ligand [ Hjquinpirole according to Levant and De Souza, and Levant and Flietstra. (See, B. Levant and E.B. De Souza, Synapse, 14:90-95 [1993]).
  • This method is based on the observation that dopamine receptors in the molecular layer of the vestibulocerebellum, which appear to be co-localized exclusively with dopamine D 3 receptor mRNA, exhibit a lack of guanyl nucleotide regulation and a pharmacological profile consistent with the D 3 site. As such, this brain area can serve as a discrete source of D 3 receptors in brain tissue. In contrast, the caudate- putamen, which expresses substantially greater amounts of D 2 receptor mRNA and exhibits relatively little D 3 receptor binding, proves useful as a prototypical dopamine D 2 tissue.
  • One contemplated advantage of this approach is that it measures D and D 3 receptor affinity in brain tissues using a radiolabeled agonist while avoiding the potentially confounding variable of using separate assays to measure D 2 and D 3 receptor affinity. This is an important advantage because the D 2 /D 3 selectivities of dopaminergic compounds have been shown to vary depending on the in vitro assay system used.
  • this approach provides a confirmatory assessment of D 2 /D 3 receptor selectivity in addition to those obtained using receptors in heterologous expression systems or using different in vitro assays for each receptor in brain.
  • the present invention contemplates that D 2 /D 3 selectivities determined by this method are generally more consistent with those determined in functional assays than radioligand binding assays using human receptors expressed in transfected cells.
  • Additional embodiments of the present invention provide methods for the determination of potency, efficacy, and selectivity of novel compounds in functional assays.
  • functional assays are used in prefe ⁇ ed embodiments to determine the potency, efficacy, and selectivity of novel ligands at the D receptor.
  • These assays enable the identification of agonists and antagonists as well as partial and inverse agonists.
  • Two functional assays are contemplated to provide cross validation of experimental results (e.g., mitogenesis assays and [ S]GTPgS binding assays), however, the present invention is not intended to be limited to using the two specifically mentioned functional assays. Indeed, any relevant functional assay that is compatible with the compositions and methods of the present invention are suitable for use herein.
  • test compounds that exhibit higher affinity for the D receptor than other dopamine receptors in the radioligand binding studies are further evaluated in functional assays.
  • one type of functional assay suitable for use with the compositions and methods of the present invention are mitogenesis assays.
  • Agonist- induced mitogenesis in CHO cells expressing hD 2 or hD 3 receptors has been developed as a su ⁇ ogate marker of dopamine receptor activation. (See e.g., CL. Chio et al, Mol. Pharmacol., 45:51-60 [1994]; and F. Sautel et al, Neuroreport, 6:329-332 [1995]).
  • test compound efficacy and potency are used to determine test compound efficacy and potency.
  • test compounds lacking efficacy are evaluated for antagonist activity and potency as determined by their ability to block agonist-induced mitogenesis. Assays are performed as previously described in Milne et al. to determine the Emax and ED 50 values for each test compound. (See, G.W. A. Milne et al, J. Chem. Inf. Comput. Sci., 34:1219-1224 [1994]). IC 50 values are determined for test compounds found to block agonist-induced mitogenesis.
  • Another type of functional assay suitable for use with the compositions and methods of the present invention are [ S]GTPgS binding assays. These assays study receptor activation of G-proteins in membranes by assaying agonist- stimulated binding of [ 35 S]GTPgS, a non-hydrolyzable analog of GTP, in the presence of excess GDP. This physiologically relevant endpoint has been used to assess G-protein activation by D 2 and D 3 receptors in brain and transfected cells. (See, S.L. Gilliland et al, Eur. J. Pharmacol., 392:125-128 [2000]; S.L. Gilliland and R.H.
  • the dose-response effects of test compounds on [ 35 S]GTPgS binding in membranes prepared for CHO cells expressing hD 2 or hD 3 receptors is used to determine efficacy and potency.
  • Compounds lacking efficacy are evaluated for antagonist activity and potency as determined by their ability to block agonist-induced [35S]GTPgS binding.
  • [ 35 S]GTPgS binding assays are also performed in CHO cell membranes containing hD sh0rt , hD 3 , or hD 44 receptors as previously described to determine Emax and ED 50 values for each compound. IC 50 values are determined for test compounds found to block agonist-induced [ 35 S]GTPgS binding.
  • a wide range of therapeutic agents find use with the present invention. Any therapeutic agent that can be co-administered with the disclosed compounds, or associated with the disclosed compounds is suitable for us in the present invention.
  • Some embodiments of the present invention provide administering to a subject an effective amount of a dopamine receptor modulator (e.g., antagonists or agonists) and one or more compounds indicated for the therapeutic treatment of cocaine abuse, depression, anxiety, an eating disorder, alcoholism, chronic pain, obsessive compulsive disorder, schizophrenia, restless legs syndrome (RLS), and Parkinson's disease, and the like.
  • the subject is a mammal (e.g., human).
  • Additional embodiments of the present invention provide therapeutic compositions and methods directed to modulating (e.g. , agonizing or antagonizing) G-protein coupled receptors.
  • the therapeutic compositions and methods of the present invention are directed to modulating (e.g., agonizing or antagonizing) faulty dopamine receptor functions (e.g., reuptake of dopamine by the D3 receptor in limbic brain regions, including, but not limited to, the nucleus accumbens, olfactory tubercle, and the islands of Calleja).
  • the present invention also provides the opportunity to monitor therapeutic success following administration of the compounds and therapeutic methods of the present invention to a subject.
  • these measurements are taken by observing the amelioration of a condition characterized, at least in part, by faulty dopamine (e.g., D 3 ) receptor function.
  • patient observations are made in a clinical setting (e.g., a hospital, doctor's office, or clinic).
  • one or more physiological samples are taken from a patient at various time points (e.g., prior to, during, or after) administration of the therapeutic compositions and methods of the present invention to determine the effectiveness of administration.
  • patient administration data is used to alter or modify the administration (e.g., dosing levels, frequency of administration, periodicity of admimstration, administration method, etc.) of the therapeutic compositions and methods of the present invention.
  • compositions of the present invention may be delivered via any suitable method, including, but not limited to, injection intravenously, subcutaneously, intratumorally, intraperitoneally, or topically (e.g., to mucosal surfaces).
  • compositions which comprise at least one receptor (e.g., dopamine, G-protein couple receptors, and the like) modulation composition administered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water.
  • the pharmaceutical compositions of the present invention may contain one agent (e.g., a D 3 receptor modulating ligand).
  • the pharmaceutical compositions may contain a mixture of at least two agents (e.g., one or more D 3 receptor modulating ligand, and one or more additional therapeutic compositions).
  • the pharmaceutical compositions of the present invention contain at least two agents (e.g., one or more D 3 receptor modulating ligand, and one or more additional therapeutic compositions) that are administered to a patient under one or more of the following conditions: at different periodicities, different durations, different concentrations, different administration routes, etc.
  • agents e.g., one or more D 3 receptor modulating ligand, and one or more additional therapeutic compositions
  • compositions and methods of the present invention find use in treating diseases or altering physiological states characterized by faulty (abe ⁇ ant) regulation and/or function of dopamine receptors and other receptor, (e.g., Di, D 2 , D , D 4 , and D 5 ).
  • abe ⁇ ant receptor e.g., D 3 receptor
  • the abe ⁇ ant receptor function is caused, or is a result of, an organic disease or state or condition.
  • the present invention contemplates administering a dopamine receptor ligand and, in some embodiments, one or more traditional therapeutic compositions directed to treating a condition associated with faulty receptor regulation and/or function (e.g., compositions directed to treating substance abuse, depression, Parkinson's disease, schizophrenia, and the like) in accordance with acceptable pharmaceutical delivery methods and preparation techniques.
  • one or more D 3 receptor modulating ligands, and one or more additional therapeutic compositions can be administered to a subject intravenously in a pharmaceutically acceptable carrier such as physiological saline.
  • Standard methods for intracellular delivery of pharmaceutical agents can be used (e.g., delivery via liposome).
  • the formulations of the present invention are useful for parenteral administration, such as intravenous, subcutaneous, intramuscular, and intraperitoneal.
  • parenteral administration such as intravenous, subcutaneous, intramuscular, and intraperitoneal.
  • Therapeutic co-administration of some contemplated therapeutic agent agents can also be accomplished using gene therapy techniques. Gene therapy techniques are now widely known in the art.
  • dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and interaction with other drugs being concurrently administered.
  • one or more D receptor modulating ligands are administered to a patient alone, or in combination with one or more substance abuse treatment agents, or in pharmaceutical compositions where it is mixed with excipient(s) or other pharmaceutically acceptable carriers.
  • the pharmaceutically acceptable carrier is pharmaceutically inert.
  • these pharmaceutical compositions may be formulated and administered systemically or locally. Techniques for formulation and administration may be found in the latest edition of "Remington's Pharmaceutical Sciences” (Mack Publishing Co, Easton Pa.). Suitable routes may, for example, include oral or transmucosal administration; as well as parenteral delivery, including intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, or intranasal administration.
  • the pharmaceutical compositions of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiologically buffered saline.
  • the pharmaceutical compositions of the present invention can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral or nasal ingestion by a patient to be treated.
  • the therapeutic compounds are administered orally to a patient orally.
  • compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended purpose.
  • an effective amount of D 3 receptor modulating ligands may be that amount that induces reuptake of dopamine in a patient's cell or tissue having depressed (or elevated) elevated levels of dopamine uptake as compared to normal nonpathological examples of the particular cells or tissues. Determination of effective amounts is well within the capability of those skilled in the art, especially in light of the disclosure provided herein.
  • prefe ⁇ ed pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries that facilitate processing of the active compounds into preparations which can be used pharmaceutically.
  • the preparations formulated for oral admimstration may be in the form of tablets, dragees, capsules, or solutions.
  • the pharmaceutical compositions of the present invention may be manufactured in a manner that is itself known (e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes).
  • Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form.
  • suspensions of the active compounds may be prepared as appropriate oily injection suspensions.
  • Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or Iiposomes.
  • Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
  • the suspension may also contain suitable stabilizers or agents that increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
  • compositions for oral use can be obtained by combining the active compounds (e.g., receptor ligands) with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
  • suitable excipients are carbohydrate or protein fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato, etc; cellulose such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; and gums including arabic and tragacanth; and proteins such as gelatin and collagen.
  • disintegrating or solubilizing agents may be added, such as the cross-linked polyvinyl py ⁇ olidone, agar, alginic acid or a salt thereof such as sodium alginate.
  • Ingestible formulations of the present compositions may further include any material approved by the United States Department of Agriculture for inclusion in foodstuffs and substances that are generally recognized as safe (GRAS), such as, food additives, flavorings, colorings, vitamins, minerals, and phytonutrients.
  • GRAS United States Department of Agriculture
  • phytonutrients refers to organic compounds isolated from plants that have a biological effect, and includes, but is not limited to, compounds of the following classes: isoflavonoids, oligomeric proanthcyanidins, indol-3-carbinol, sulforaphone, fibrous ligands, plant phytosterols, ferulic acid, anthocyanocides, triterpenes, omega 3/6 fatty acids, polyacetylene, quinones, terpenes, cathechins, gallates, and quercitin.
  • Dragee cores are provided with suitable coatings such as concentrated sugar solutions, which may also contain gum arabic, talc, polyvinylpy ⁇ olidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments may be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound, (i.e., dosage).
  • Pharmaceutical preparations that can be used orally include push- fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating such as glycerol or sorbitol.
  • the push-fit capsules can contain the active ingredients mixed with a filler or binders such as lactose or starches, lubricants such as talc or magnesium stearate, and, optionally, stabilizers.
  • a filler or binders such as lactose or starches
  • lubricants such as talc or magnesium stearate
  • stabilizers optionally, stabilizers.
  • the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycol with or without stabilizers.
  • compositions comprising a compound of the invention formulated in a pharmaceutical acceptable carrier may be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.
  • conditions indicated on the label may include treatment of conditions related to substance abuse (e.g., cocaine abuse), depression, Parkinson's disease, and schizophrenia, and the like.
  • the pharmaceutical compositions may be provided as a salt and can be formed with many acids, including but not limited to hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents that are the co ⁇ esponding free base forms.
  • the prefe ⁇ ed preparation may be a lyophilized powder in 1 mM-50 mM histidine, 0.1%-2% sucrose, 2%-7% mannitol at a pH range of 4.5 to 5.5 that is combined with buffer prior to use.
  • the therapeutically effective dose can be estimated initially from cell culture assays. Then, preferably, dosage can be formulated in animal models (particularly murine or rat models) to achieve a desirable circulating concentration range that induces normal function/regulation of receptors (e.g., reuptake of dopamine by D 3 receptors).
  • a therapeutically effective dose refers to that amount of receptor modulating ligand (and in some embodiments, one or more other therapeutic agents) that ameliorate symptoms of the disease state (e.g., depressed reuptake of dopamine).
  • Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index, and it can be expressed as the ratio LD50/ED50.
  • a range of dosage for example, mammalian use (e.g., humans, Equus caballus, Felis catus, and Canis familiaris, etc.).
  • the dosage of such compounds lies preferably, however the present invention is not limited to this range, within a range of circulating concentrations that include the ED50 with little or no toxicity.
  • the dosage varies within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration. The exact dosage is chosen by the individual physician in view of the patient to be treated. Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect.
  • Additional factors which may be taken into account include the severity of the disease state; age, weight, and gender of the patient; diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy.
  • Long acting pharmaceutical compositions might be administered every 3 to 4 days, every week, or once every two weeks depending on half- life and clearance rate of the particular formulation.
  • Other pharmaceutical compositions may be administered daily or several times a day.
  • Normal dosage amounts may vary from 0.1 to 100,000 micrograms, up to a total dose of about 1 g, depending upon the route of admimstration.
  • Guidance as to particular dosages and methods of delivery is provided in the literature. Administration of some agents to a patient's bone marrow may necessitate delivery in a mariner different from intravenous injections.
  • Prefe ⁇ ed embodiments of the present invention provide pharmaceutical compositions and methods for administering an effective amount of receptor modulating ligands to a patient to modulate (e.g., increase or decrease the activity of) faulty receptor function/regulation.
  • diseases suspected of being characterized by having faulty receptor (e.g., D 3 receptor) function/regulation suitable for treatment by the present invention are selected by obtaining a sample of interest (e.g., cells, tissues, fluids, etc.) suspected of having abe ⁇ ant levels of native (e.g., endogenous) receptor ligands (e.g., dopamine), measuring the levels of native receptor ligands in the sample using one or more well established immunohistochemical techniques (e.g., ELISA and Western blots, etc.), and comparing the levels of native receptor ligands in the sample with levels of co ⁇ esponding receptor ligands in relevant reference nonpathological samples.
  • a sample of interest e.g., cells, tissues, fluids, etc.
  • native receptor ligands e.g., endogenous receptor ligands
  • immunohistochemical techniques e.g., ELISA and Western blots, etc.
  • diseases suspected of being characterized by having abe ⁇ ant levels of one or more native receptor ligands are selected by comparing levels of one or more markers (e.g., polynucleotides, polypeptides, lipids, etc.) in a sample (e.g., cells, tissues, fluids, etc.) that directly or indirectly indicate elevated aberrant levels of the receptor ligand of interest as compared to levels of these markers relevant nonpathological samples.
  • markers e.g., polynucleotides, polypeptides, lipids, etc.
  • the present invention is not intended to be limited to the administration routes chosen for delivering agents to a subject. Indeed, a number of suitable administration routes are contemplated the selection of which is within the skill of those in the art.
  • therapeutic compositions are administered to a patient at a dosage range of about 1 to 200 mg/day, from about 5 to 50 mg/day, and most preferably from about 10 to 40 mg/day.
  • the therapeutic compounds are administered to a patient (e.g., orally) in a tolerable daily dose (e.g., 30 to 40 mg/day) shown to have some biologic activity (e.g., alterations in Rb and Cyclin Di levels).
  • standard immunohistochemical techniques are employed on samples obtained from patients following, or during, treatments with the methods and compositions of the present invention to determine changes in the patient's disease state.
  • cocaine abuse and/or addiction is considered to be a pathological disease state.
  • D 3 dopamine receptor Therapeutic potential of the D3 receptor in schizophrenia.
  • Schizophrenia is a neurodevelopmental disorder involving abnormal dopaminergic function.
  • the importance and therapeutic potential of the D 3 dopamine receptor in schizophrenia is supported by several lines of evidence.
  • D 2 and D 3 dopamine receptors are the main targets for nearly all cu ⁇ ently known agents with antipsychotic activity (recent reviews: Strange, 2001; Schwartz et al, 2000; Emilien et al, 1999).
  • the D 3 subtype is primarily localized to forebrain limbic areas, known of being involved in schizophrenia.
  • antipsychotic drugs administered to schizophrenic patients appear to act as down regulators of the D receptor in the ventral striatum region, which is supported by the over-expression of the D 3 (and not D 2 ) receptors in case of drug- free schizophrenic patients compared to schizophrenic patients taking anti-psychotic agents (Gurevich et al, 1997). This would also account for the behavioral sensitization to psychostimulants observed in schizophrenia.
  • D3 receptor Therapeutic implication of the D3 receptor in Parkinson 's disease.
  • Long-term levodopa therapy for Parkinson's disease leads to severe side effects, dyskinesias, fluctuations in motor performance, hallucinations.
  • An induction of D receptor gene expression was shown to accompany levodopa administration in conditions in which it leads to behavioral sensitization. This involvement has therapeutic relevance in the prevention of side effects and also in the treatment of Parkinson's disease (Bordet et al, 1997; S. Perachon et al, 366:293-300 [1999]).
  • Combined administration of levodopa and dopamine agonist is considered an effective way of reducing and delaying the side effects of chronic levodopa therapy.
  • RLS Restless legs syndrome
  • Levodopa and ergoline derivatives are effective treating this condition but their usefulness is limited by major side effects. Also, no complete relief of the RLS symptoms may be achieved using these drugs.
  • a D receptor agonist, pramipexole was found highly effective in the treatment of RLS, which also supports the involvement and therapeutic potential of the D 3 subtype receptor in RLS (Montplaisir, et al, 1999).
  • D3 receptor Therapeutic implications of the D3 receptor in the treatment of depression. Dopamine function was found reduced in patients with severe depression (Shah et al, 1997). Also, antidepressent agents that have dopaminergic effects are generally effective for treating depression (Brown and Gershon, 1993). Specifically, chronic antidepressants effect D2/D3 receptor function in mesolimbic terminal regions (Emilien et al, 1999).
  • Reagent and condition i. a) 1.1 eq. ethanolamine, benzene, under N 2 , reflux, remove H O, overnight; b) 3.5 eq. NaBH 4 , absolute ethanol, under N , reflux, overnight, yield 87- 90%.
  • Table 10 shows the structure and activity of several D ligands.
  • Example 17 3-(p-Fluorobenzoylbutyl)-8-methoxy-2,3,4,4a,5,6-hexahydro-l/y-pyrazino[l,2- ]quinoline (Compound 7c) Alkylation of compound 5b with l-(4-fluorophenyl)-5-chloro-l-oxopentane as described in method A (Example 9) gave 269mg of compound 7c as a colorless oil, yield 68%.
  • [ H]PD 128907 binding assays for D 3 receptors dopamine receptors are performed as previously described in Levant. (See, B. Levant, Cu ⁇ ent Protocols in Pharmacology (J Ferkany and SJ Enna, Eds) John Wiley & Sons, New York, 1.6.1-1.6.16 [1998]).
  • Rat ventral striatum (nucleus accumbens and olfactory tubercles) is prepared in assay buffer (50 mM Tris, 1 mM EDTA; pH 7.4 at 23 °C) to yield a final concentration of 10 mg original wet weight (o.w.w.)/ml.
  • Membranes are incubated with
  • [ H] spiperone binding assays are performed as previously
  • the assay buffer is 50 mM Tris-HCI, 5 mM KCl, 2 mM MgCl2, and 2 mM CaCl2, pH 7.4 at 23°C; the concentration of [ 3 H]spiperone (24 Ci/mmol; Amersham) is 200 pM; and the incubation time was 2 h at 23 °C Nonspecific binding is defined in the presence of 1 ⁇ M (+)-butaclamol.
  • [ ⁇ H] SCH 23390 binding assays for Drlike dopamine receptors are performed as previously described in Levant (See, B. Levant, Cu ⁇ ent Protocols in Pharmacology (J Ferkany and SJ Enna, Eds) John Wiley & Sons, New York,
  • sagittal brain sections (20 ⁇ m, lateral 1.00 - 1.40 mm) are cut on a cryostat, thaw-mounted onto chrome alum/gelatin-coated slides, and processed as previously described in Levant. (B. Levant, and E.B. De Souza, Synapse, 14:90-95 [1993]).
  • Sections are incubated for 2 hr at 23 °C with 10 nM [ H]quinpirole (50.6 Ci/mmol; NEN Life Science, Boston, MA), in the presence or absence of 5 concentrations of competing drug (IO “9 to l ⁇ "5 M or l ⁇ "8 to IO “4 M), in assay buffer (50 mM Tris-HCI, 5 mM KCl, 2 mM MgCl2, and 2 mM CaCl2, pH 7.4 at 23 °C). Duplicate sections from each animal are used for each data point. Autoradiograms are generated using H-Hyperfilm (Amersham,
  • mitogenensis assays are performed as previously described in
  • CHO cells expressing hD 2s or hD 3 are seeded into 96-well plates at density of 5,000 cells/well and grown at 37° C in MEM with 10% fetal calf serum for 48 hr. Wells are washed with serum-free MEM and incubated with various concentrations of drug (10 "10 to IO "4 M) or vehicle and cultured for 16 hr. Antagonism of agonist-stimulated mitogenesis is determined in the presence 10 nM quinpirole. [ 3 H]Thymidine (1 ⁇ Ci/well; 25 Ci/mmol; Amersham) are then be added to each well. After 2 hr incubation, cells are trypsinized and harvested by rapid vacuum filtration.
  • [ 3 H]Thymidine incorporation in each sample is calculated as fmol incorporated/mg protein. Data are present as percent stimulation over basal.
  • the EC 50 , IC 50 , and E ma ⁇ values are calculated by nonlinear regression analysis using a four-parameter model (Sigma Plot, SPSS, Chicago, IL).
  • [ S]GTP-S binding assays are performed in CHO cell membranes containing hD 2s , hD 3 , or hD 4 . 4 receptors as previously described in Wang et al. (See, S. Wang et al, J. Med. Chem., 39:2047-2054 [1996]; and S. Wang et al, J. Med. Chem., 43:351-360 [2000]).
  • 1 HNMR spectra were recorded at 300MHz and 13 CNMR spectra were recorded at 75MHz both on Varian Mercury 300 and Bruker DPX 300 spectrometer. Chemical shifts are given in ⁇ units (ppm) and are relative to the solvent. Coupling constants are given in hertz (Hz). Elemental analyses were performed by the Micro-Analysis, Inc., (Wilmington, DE) and are within 0.4% of the theoretical values, except where noted. All the reagents and chemicals were purchased from Aldrich Chemical Co., Fisher Scientific, or Lancaster Synthesis, Inc., and used without further purification. All the reactions were run under nitrogen unless otherwise indicated.
  • the 8-methoxyl quinaldine is obtained very easily by methylation of commercially available 8-hydroixyl quinaldine.
  • To create 5-methoxyl quinaldine first the amine 163 is treated with acyl acetate ethyl ester to get the coupling product (compound 164).
  • Compound 64 is cyclized by heating to 250°C to yield compound 165, and the hydroxyl group in compound 165 is converted to a chloride, then the chloride is removed by hydrogenation to yield the 5-methoxyl quinaldine.
  • 7-methoxyl quinaldine is obtained by the same method.
  • 5-methoxyl, or 7-methoxyl, or 8-methoxyl quinaldine is oxidized by SeO 2 to the aldehyde (compound 174).
  • the aldehyde (compound 174) is treated with 2-amino ethanol and NaBH 4 to yield compound 178.
  • hydrogenation and cyclization yield the important intermediate compound 177.
  • D 3 ligands are obtained by treating compound 177 with different alkyl halides.
  • Nine D 3 ligands are obtained.
  • the synthetic method is different.
  • Compound 183 is obtained by treating compound 177c with 4-heptanone and NaBH 4 .
  • the tail of compounds 186 or 182 is synthesized by using compound 187 as the starting material.
  • the hydroxyl group is selectively protected with TBS group, and the amine is treated with 2-naphthoyl chloride to yield the amide (compound 189).
  • the TBS group is removed, and the alcohol (compound 190) is treated with CBr and Ph P in THF.
  • Compound 192 comes from the reaction between compound 190 and the solvent (THF).
  • Compound 192 is treated the MsCl to get the compound 191.
  • Reagent and condition i. a) 1.1 eq. ethanolamine, benzene, under N 2 , reflux, remove H 2 O, overnight; b) 3.5 eq. NaBH 4 , absolute ethanol, under N 2 , reflux, overnight, yield 87-90%o.
  • Reagent and condition v. 2 eq. hydrazine, EtOH, reflux, 2h, yield 87-94%. vi. 1.2 eq. 2- naphthoyl chloride , 3 eq. triethylamines, 0°C, 4h, yield 91-95%. vii. 1.2 eq. 4- biphenylcarbonyl chloride , 3 eq. triethylamines, 0°C, 4h, yield 72-75%.
  • Reagent and condition viii. . 4 eq. BBr 3 , CH 2 C1 2 , under N 2 , 0°C, 4h, yield 52%.

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Abstract

The present invention relates to compounds designed as ligands (e.g., agonists, antagonists, and partial agonists) for dopamine receptors (e.g., D1, D2, D3, D4, and D5), and in particular, the D1, D2, and D3 dopamine receptors. The present invention also relates to methods for rationally designing these compounds and to therapeutic methods of using these compounds.

Description

DOPAMINE RECEPTOR LIGANDS AND THERAPEUTIC METHODS BASED THEREON
This application claims priority to U.S. Provisional Patent Application Serial Number 60/297,445, filed June 13, 2001, and to a U.S. Provisional Patent Application, filed June 7, 2002, Express Mail No. : EV092300689US, both of which are herein incorporated by reference in their entirety. This work was supported by National Institutes of Health grant No.: . The government has certain rights in this invention.
FIELD OF THE INVENTION The present invention relates to compounds designed as ligands (e.g., agonists, antagonists, and partial agonists) for dopamine receptors (e.g., D], D2, D3, D , and D5), and in particular, the D^ D2, and D3 dopamine receptors. The present invention also relates to methods for rationally designing these compounds and to therapeutic methods of using these compounds.
BACKGROUND OF THE INVENTION
The cost of drug abuse, including cocaine abuse, is enormous and ever increasing. A recent report prepared for the Office of National Drug Control Policy estimated that in 1998 alone illicit drug abuse in the U.S. cost society approximately 143.4 billion, and that between 1992 and 1998 the overall societal cost of drug abuse in the U.S. increased at a rate of 5.9 percent annually. Lost productivity (98.5 billion), and expenditures for treating drug related medical conditions (12.9 billion) accounted for the two largest portions of the this huge burden. In the year 2000, an estimated 14.0 million Americans were illicit drug users- -nearly 6.3% of U.S. population 12 years old and older. While addiction to some illicit drugs, such as heroin, can be successfully treated with pharmacotherapies (e.g. , methadone, naloxone, and naltrexone), there are no effective pharmacotherapies available for treating addiction to cocaine and crack cocaine ("crack" is the street name given to cocaine hydrochloride that has been processed to free base for smoking). Thus, cocaine addiction is particularly problematic for both the drug addict and for society as a whole. Moreover, cocaine is one of the most addictive substances known, such that, cocaine addicts often lose their ability to function at work and in their interpersonal relationships.
A 1998 National Household Survey on Drug Abuse estimated that 1.7 million Americans are current cocaine or crack cocaine users. Even more alarming is the fact that use of cocaine use appears to be on the rise with America's youth. Of college students 1 to 4 years beyond high school, in 1995, 3.6 % had used cocaine within the past year, and 0.7% had used cocaine in the past month. The proportion of high school seniors who used cocaine at least once increased from a low of 5.9 % in 1994, to 9.8 % in 1999. Also in 1999, 7.7 % of lOth-graders had tried cocaine at least once, up from 3.3% in 1992. The percent of 8th-graders who have ever tried cocaine increased from a low of 2.3% in 1991 to 4.7% in 1999.
Despite the considerable progress toward an understanding of the neuropharmacological bases of cocaine addiction, there remains no effective pharmacotherapy for the treating cocaine addiction. (F.I. Carroll et al, J. Med. Chem., 42:2721-2731 [1999]). Correspondingly, the clinical needs of cocaine-dependent patients are broad, and patients would likely benefit from pharmacotherapies acting to interrupt any stage of the cycle of cocaine dependence. Thus, cocaine abuse remains a major health problem and social concern. Accordingly, what is needed are improved compositions and methods of treating cocaine abuse, and more generally, improved compositions and methods of discovering and designing potent and specific pharmacotherapy agents.
SUMMARY OF THE INVENTION The present invention relates to compounds designed as ligands (e.g., agonists, antagonists, and partial agonists) for dopamine receptors (e.g., D\, D2, D3, D4, and D5), and in particular, the Di, D , and D3 dopamine receptors. The present invention also relates to methods for rationally designing these compounds and to therapeutic methods of using these compounds. The present invention is not intended, however, to be limited to compositions that modulate (e.g., agonists, antagonists, and partial agonists) dopamine receptors. Indeed, some embodiments of the present invention are directed to providing methods compositions that modulate (e.g., agonists, antagonists, and partial agonists) G- protein coupled receptors generally, and to methods for discovering and designing these compounds. Preferred embodiments of the present invention provide compounds that are rationally designed to control dopamine flow in the brain. In other preferred embodiments, these compounds are highly selective antagonists of the dopamine family receptors (e.g., D3). In still other embodiments, rational design of the compounds of the present invention includes identifying a mechanism associated with dopamine flow in the brain. Information relating to the mechanism is then analyzed such that compound structures having possible activity in interfering with such a mechanism are formulated. In some particular embodiments, structures are synthesized based on "building blocks," wherein each building block has a feature potentially capable of interfering with a particular mechanism associated with dopamine flow, particularly, a mechanism involving one ore more members of the D receptors family.
Compounds having different building block combinations are then synthesized and their activity in relation to the identified mechanism tested. Such tests are preferably conducted in vitro and/or in vivo. The information obtained through such tests is then incorporated in a new cycle of rational drug design. The design-synthesis-testing cycle is repeated until a candidate compound having the desired properties for a targeted therapy; e.g., dopamine flow control, is obtained. The candidate compound is then preferably clinically tested.
One approach for modulating (e.g., controlling) dopamine flow in the brain for the treatment of cocaine addiction is to design cocaine antagonists which can affect dopamine uptake. More specifically, this approach is based on rationally designing compounds that are antagonists of cocaine such that they reduce or block dopamine binding to the D receptors family, and particularly, receptor D3. In preferred embodiments, antagonists are designed to reduce or block cocaine binding while leaving other aspects of dopamine transport unaffected. The designed antagonists should provide a basis for therapeutic protocols based on the selective control of dopamine transport and thereby control of synaptic signaling with no or little disruption of the normal flow of dopamine in the brain.
In preferred embodiments, the present invention provides a composition comprising one or more of the compounds disclosed herein, or derivatives of such compounds (e.g., derivatives having minor chemical substitutions). The present invention also provides methods of using such compounds. It will be appreciated that the methods described herein find use with the compounds disclosed herein, and their derivatives. In preferred embodiments, the present invention provides a composition comprising a compound of formula (including derivatives of this formula):
wherein,
X, Y, and Z independently represent C, O, N, S;
R7, R8 , and R9 independently represent H, F, Cl, Br, I, OH, CN, NO2, OR', CO2R', OCOR',
CONR'R", NR"COR', NR'SO2R", SO2NR'R", NR'R" where R' and R" independently represents H, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, or heterocyclic, substituted or unsubstituted;
A and B independently represent O, S, SO, SO2, NR', CR'R" where R' and R" independently represent H, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, or heterocyclic, substituted or unsubstituted; Ri , R2) R3ι Rj, R5, and R5 independently represent H, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, or heterocyclic, substituted or unsubstituted; where n= 0, 1, or 2;
R represents alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, or heterocyclic, substituted or unsubstituted; The examples include CnH2n+ι, (CH2)nR'" where n=l-6, R'" represents aryl, substituted or unsubstituted. In still other preferred embodiments, the present invention provides a composition comprising a compound of formula (including derivatives of this formula):
In yet other preferred embodiments, the present invention provides a composition comprising a compound of formula (including derivatives of this formula):
wherein,
X, Y, Z, D and M independently represent C, O, N, S;
R7, R8 , R9 , R10>Rιι,Ri2,Ri3,Ri4, andR15 independently represent H, F, Cl, Br, I, OH, CN, NO2, OR', CO2R', OCOR', CONR'R", NR"COR', NR'SO2R", SO2NR'R", NR'R" where R' and R" independently represents H, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, or heterocyclic, substituted or unsubstituted; A and B independently represent O, S, SO, SO2, NR\ CR'R" where R' and R" independently represent H, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, or heterocyclic, substituted or unsubstituted; Ri, R2>R3;R ,R5) and Rό independently represent H, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, or heterocyclic, substituted or unsubstituted; where n= 0, 1, or 2; where m= 1- 20; R represents H, O, S, SO, SO2, NR\ CR'R" where R' and R" independently represent H, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, or heterocyclic, substituted or unsubstituted.
In additional preferred embodiments, the present invention provides a composition comprising a compound of formula (including derivatives of this formula):
wherein,
X, Y, Z, D, and M independently represent C, O, N, S;
R7, R8 , R9 , and R10 independently represent H, F, Cl, Br, I, OH, CN, NO2, OR', CO2R\ OCOR', CONR'R", NR"COR', NR'SO2R", SO2NR'R", NR'R" where R' and R" independently represents H, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, or heterocyclic, substituted or unsubstituted;
A and B independently represent O, S, SO, SO2, NR', CR'R" where R' and R" independently represent H, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, or heterocyclic, substituted or unsubstituted; Ri, R2) R3, f , R5, and Re independently represent H, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, or heterocyclic, substituted or unsubstituted; where n= 0, 1, or 2; E represents a linker group that does not contain an amide group and F represents cycloalkyl, cycloalkenyl, or heterocyclic, substituted or unsubstituted; The examples include CnH2n+1, (CH2)nR'" where n=l-6, R'" represents aryl, substituted or unsubstituted. Group E can be any linking chemistry including, but not limited to, carbon chains, repeating polymer units, and the like. Group F is preferably a bulky group, such as a substituted or unsubstituted benzene ring or a multi-ring complex.
The present invention also provides methods for identifying agonists and antagonists of dopamine receptors, comprising the step of assessing an interaction between a candidate ligand and an amino acid of a dopamine receptors. In some embodiments, the contemplated amino acids of the dopamine receptors, include, but are not limited to, Y32, S35, Y36, L39, F345, F346, T368, T369, Y365, P200, S192, S193, S197, VI 11, 1183, F188, V164, S182, and A161 of receptor D3, Y37, L39, L41, L44, F198, F389, F390, F411, S192, S193, S196, VI 15, 1194, F189, and S163 of receptor D2, and the like. In some preferred embodiments, the contemplated amino acids of the dopamine receptors, include, but are not limited to, N47, D75, F338, N375, and N379 of receptor D3, N52, D80, F382, N418, and N422 of receptor D2. In still some other embodiments, the contemplated amino acids of the dopamine receptors, include, but are not limited to, N47, D75, V78, V82, V86, F106, DUO, Vl l l, C114, L121, V164, L168, 1183, F188, S192, S193, V195, S196, F197, F338, W342, F345, F346, H349, V350, Y365, T369, Y373, N375, and N379 of receptor D3, N52, D80, V85, V87, V91, F110, D114, V115, C118, L125, 1166, L170, 1184, F189, S193, S194, V196, S197, F198, F382, W386, F389, F390, H393, 1394, Y408, T412, Y416, N418, and N422 of receptor D2.
In preferred embodiments, the assessing step, when identifying agonists and antagonists of dopamine receptors, comprises investigating a computer model for a predicted interaction between the ligand and the amino acid. In still other preferred embodiments, the assessing step comprises binding the ligand to the dopamine receptor and determining binding between the ligand and the amino acid. However, in still further embodiments, determining the binding between the ligand and the amino acid comprises determining the crystal structure of the ligand bound to the receptor.
Some additional embodiments of the present invention contemplate, that determining the binding between the ligand and the amino acid comprises comparing a binding affinity between the ligand and the receptor with a binding affinity between the ligand and a receptor lacking the amino acid. Some other additional embodiments further contemplate that determining the binding between the ligand and the amino acid comprises measuring the ability of the ligand to displace a molecule bound to the amino acid of the receptor.
In one preferred embodiment, the present invention provides a method of treating a subject having a disease, addiction, or other pathological condition (e.g., cocaine abuse, depression, anxiety, an eating disorder, alcoholism, chronic pain, obsessive compulsive disorder, schizophrenia, restless legs syndrome (RLS), and Parkinson's disease, and the like) comprising administering to the subject a therapeutic dose of a composition of the present invention. For example, in some embodiments, the present invention provides methods of treating (e.g., administering an effective therapeutic amount/dose) a subject with a composition comprising a compound of formula (including derivatives of this formula):
wherein, X, Y, and Z independently represent C, O, N, S;
R7, R8 , and R9 independently represent H, F, Cl, Br, I, OH, CN, NO2, OR', CO2R', OCOR', CONR'R", NR"COR', NR'SO2R", SO2NR'R", NR'R" where R' and R" independently represents H, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, or heterocyclic, substituted or unsubstituted; A and B independently represent O, S, SO, SO2, NR', CR'R' ' where R' and R' ' independently represent H, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, or heterocyclic, substituted or unsubstituted;
Ri, R2ι R3j R , R5; and Rό independently represent H, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, or heterocyclic, substituted or unsubstituted; where n= 0, 1, or 2; R represents alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, or heterocyclic, substituted or unsubstituted; The examples include CnH2n+ι, (CH2)nR'" where n=l-6, R'" represents aryl, substituted or unsubstituted.
In some other embodiments, the present invention provides methods of treating (e.g. administering an effective therapeutic amount/dose) a subject with a composition comprising a compound of formula (including derivatives of this formula):
In yet another example, further embodiments of the present invention provide methods of treating a subject (e.g., administering an effective therapeutic amount/dose) with a composition comprising a compound of formula (including derivatives of this formula):
wherein,
X, Y, Z, D and M independently represent C, O, N, S;
R7, R8 , R9 , Rιo,Rιι,Ri2,Ri3,Ri4, andRi5 independently represent H, F, Cl, Br, I, OH, CN,
NO2, OR', CO2R\ OCOR', CONR'R", NR"COR', NR'SO2R", SO2NR'R", NR'R" where R' and R" independently represents H, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, or heterocyclic, substituted or unsubstituted;
A and B independently represent O, S, SO, SO2, NR', CR'R" where R' and R" independently represent H, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, or heterocyclic, substituted or unsubstituted;
Ri, R2, R3) R4, R5j and Rό independently represent H, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, or heterocyclic, substituted or unsubstituted; where n= 0, 1, or 2; where m= 1-
20; R represents H, O, S, SO, SO2, NR', CR'R" where R' and R" independently represent
H, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, or heterocyclic, substituted or unsubstituted.
In still further embodiments, the present invention provides methods of treating a subject (e.g., administering an effective therapeutic amount/dose) with a composition comprising a compound of formula (including derivatives of this formula):
wherein,
X, Y, Z, D, and M independently represent C, O, N, S;
R7, R8 , R9 , and R,0 independently represent H, F, Cl, Br, I, OH, CN, NO2, OR', CO2R', OCOR', CONR'R", NR"COR', NR'SO2R", SO2NR'R", NR'R" where R' and R" independently represents H, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, or heterocyclic, substituted or unsubstituted;
A and B independently represent O, S, SO, SO2, NR', CR'R" where R' and R" independently represent H, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, or heterocyclic, substituted or unsubstituted; Rl5 R2, R3, R4, R5, and Re independently represent H, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, or heterocyclic, substituted or unsubstituted; where n= 0, 1, or 2; E represents a linker group that does not contain an amide group and F represents cycloalkyl, cycloalkenyl, or heterocyclic, substituted or unsubstituted; the examples include CnH2n+ι, (CH2)nR'" where n=l-6, R'" represents aryl, substituted or unsubstituted. In some embodiments, the subject being treated with the therapeutic compositions
(e.g., formulations) of the present invention has a condition characterized by faulty (e.g., aberrant) regulation and/or function of a G-protein couple receptor (e.g., a dopamine family receptor). In preferred embodiments, the subject being treated has one or more conditions characterized by the faulty regulation and/or function of a dopamine receptor (e.g., Dl s D2, D3, D , and /or D5). In still other preferred embodiments, the subject has one or more conditions characterized by faulty (e.g., aberrant) regulation and/or function of the D3 receptor. In particularly preferred embodiments, the present invention provides therapeutic methods and compositions for treating cocaine addiction/abuse. In some embodiments of the present invention, cocaine addiction/abuse is considered a disease. Still further embodiments of the present invention provide methods of modulating
(e.g., antagonizing and/or agonizing) the action of a G-protein coupled receptor in a subject comprising administering to the subject an effective amount of a one or more compositions of the present invention. In some embodiments, the present invention provides methods of administering an agonist of the action of a G-protein coupled receptor to a subject. In some other embodiments, the present invention provides methods of administering an antagonist of the action of a G-protein coupled receptor to a subject. It is understood that the subjects contemplated for treating (e.g., receiving the administration) with the therapeutic methods and compositions of the present invention include mammals, and more particularly, include humans. Other embodiments of the present invention provide methods of controlling dopamine flow in a subject in need of such control comprising administering to the subject an effective amount of at least one composition of present invention.
Still further embodiments of the present invention provide methods of treating a subject with a D3 receptor-specific modulator, comprising administering to the subject a compound of formula (I) or formula (II), wherein formula (I) is (including derivatives of this formula):
wherein,
X, Y, Z and M independently represent C, O, N, S;
R7, R8 , R9 , and R10 independently represent H, F, Cl, Br, I, OH, CN, NO2, OR', CO2R', OCOR', CONR'R", NR"COR', NR'SO2R", SO2NR'R", NR'R" where R' and R" independently represents H, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, or heterocyclic, substituted or unsubstituted;
A and B independently represent O, S, SO, SO2, NR', CR'R" where R' and R" independently represent H, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, or heterocyclic, substituted or unsubstituted;
Ri, R2!R3)R4>R5> and Rό independently represent H, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, or heterocyclic, substituted or unsubstituted; where n= 0, 1, or 2; R represents alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, or heterocyclic, substituted or unsubstituted; the examples include CnH2n+1, (CH2)nR'" where n=l-6, R'" represents aryl, substituted or unsubstituted; and formula (II) is:
wherein,
X, Y, and Z independently represent C, O, N, S;
R7, R8 , and R9 independently represent H, F, Cl, Br, I, OH, CN, NO2, OR', CO2R', OCOR', CONR'R", NR"COR', NR'SO2R", SO2NR'R", NR'R" where R' and R" independently represents H, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, or heterocyclic, substituted or unsubstituted;
A and B independently represent O, S, SO, SO2, NR', CR'R" where R' and R" independently represent H, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, or heterocyclic, substituted or unsubstituted;
Ri, R2; R3, RΛ, R5, and s independently represent H, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, or heterocyclic, substituted or unsubstituted; where n= 0, 1, or 2;
R represents alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, or heterocyclic, substituted or unsubstituted; The examples include CnH2n+ι, (CH2)nR'" where n=l-6, R'" represents aryl, substituted or unsubstituted, wherein the subject is characterized as having previously responded poorly or is believed likely to respond poorly to a dopamine receptor modulator that binds specifically to D3 and D2 or D] receptors.
It is understood that preferred embodiments of the therapeutic methods of the present invention (e.g., treating a subject having a condition characterized by aberrant function and/or regulation of a receptor [e.g., dopamine receptor], modulating [e.g., antagonizing and/or agonizing] the action of a G-protein coupled receptor, and controlling dopamine flow in a subject, and the like) contemplate administering an effective dose of one or more compounds of the present invention to a subject. In still further embodiments, the present invention additionally comprises the coadministration of one or more (e.g., at least one) additional therapeutic compounds that are relevant to the treatment of a particular disease in the subject (e.g., a disease characterized by the aberrant regulation and/or function of a G-protein coupled [e.g., dopamine] receptor) in additional to the administration of a therapeutic compound of the present invention.
In other preferred embodiments, the present invention provides methods of treatment or prophylaxis of conditions characterized by the faulty (e.g., aberrant ) regulation or function of dopamine receptors.
The methods of the present invention are particularly well suited for the treatment of cocaine abuse, depression, anxiety, an eating disorder, alcoholism, chronic pain, obsessive compulsive disorder, schizophrenia, restless legs syndrome (RLS), and Parkinson's disease, and the like.
Further embodiments of the present invention provide pharmaceutical compositions comprising: one or more compositions of the present invention; and instructions for administering the composition(s) to a subject, wherein the subject has a disease characterized by aberrant receptor (e.g., dopamine receptor) function/regulation. In preferred embodiments, the instructions included with these kits meet U.S. Food and Drug Administrations rules, regulations, and suggestions for provision of therapeutic compounds. In yet another embodiment, the present invention provides methods of screening a G-protein coupled receptor (e.g., D ) modulating compound and a test compound comprising: providing: a G-protein coupled receptor modulating compound; a test compound; a first group of cells; and contacting the first group of cells with the G-protein coupled receptor modulating compound and the test compound; and observing the effects of contacting the first group of cells with the G-protein coupled receptor modulating compound and the test compound.
In some of these embodiments, the present invention further provides the additional step of comparing the effects observed in the first cells against a second group of the cells contacted with the G-protein coupled receptor modulating compound alone, or with the test compound alone. Effects that may be observed include, but are not limited to, changes in dopamine metabolism. In still other embodiments, the present invention further contemplates additional methods for selling test compounds screened/identified by the above methods. In some of these embodiments, test compounds may be offered for sale by a third party in one or more forms (e.g., a kit, including, instructions for administering the test compound to a patient).
Other advantages, benefits, and preferable embodiments of the present invention will be apparent to those skilled in the art.
DESCRIPTION OF THE FIGURES
The following figures form part of the specification and are included to further demonstrate certain aspects and embodiments of the present invention. The present invention is not intended to be limited however to the embodiments specifically recited in these figures. Fig. 1 shows the modeled 3D structure of dopamine receptor D3 and its ligand binding site.
Fig. 2 shows nine relatively selective D3 partial agonists used to derive a pharmacophore model for 3D-database searching in one embodiment of the present invention. Fig. 3 shows a simple pharmacophore model used in one embodiment of present invention.
Fig. 4 shows several compounds contemplated so use in one embodiment of the present invention.
Fig. 5 shows one novel approach to identifying compounds useful in the present invention.
Fig. 6A shows a superposition of 9 D3 partial agonists and agonists contemplated in one embodiment of the present invention.
Fig. 6B shows a simple pharmacophore model derived from contemplated D3 ligands. Fig. 7 shows several compounds that are useful in some embodiments of the present invention.
Fig. 8 shows the functional activity of compound 4 at Di, D2, and D3 receptors and comparison to standard ligands in one embodiment of the present invention. Fig. 9 shows that compound 4 forms a strong salt bridge between its protonated nitrogen and Dl 10 in D3 in one embodiment of the present invention.
Fig. 10 shows several compounds that are useful in some embodiments of the present invention. Fig. 11 shows one contemplated synthesis scheme for making compounds use in some methods of the present invention.
Fig. 12 shows one contemplated synthesis scheme for making compounds use in some methods of the present invention.
Fig. 13 shows several compounds that are useful in some embodiments of the present invention.
Fig. 14 shows functional assays using transfected cells in one embodiment of the present invention.
Fig. 15 shows one contemplated synthesis scheme for making compounds use in some methods of the present invention. Fig. 16 shows one contemplated synthesis scheme for making compounds use in some methods of the present invention.
Fig. 17 shows several compounds that are useful in some embodiments of the present invention.
Fig. 18A show a predicted binding model for compounds 14 with the D3 receptor in one embodiment of the present invention.
Fig. 18B show a predicted binding model for compounds 32 with the D3 receptor in one embodiment of the present invention.
Fig. 19 shows a schematic representation of compound 14 in the D3 receptor's binding site in one embodiment of the present invention. Fig. 20 shows one contemplated synthesis scheme for making compounds use in some methods of the present invention.
Fig. 21 shows one contemplated synthesis scheme for making compounds use in some methods of the present invention.
Fig. 22 shows one contemplated binding model of the present invention. Fig. 23 shows one contemplated synthesis scheme for making compounds use in some methods of the present invention.
Fig. 24 shows one contemplated synthesis scheme for making compounds use in some methods of the present invention. Fig. 25 shows one contemplated synthesis scheme for making compounds use in some methods of the present invention.
Fig. 26 shows a diagrammatic representation of one embodiment of the present invention.
DEFINITIONS
To facilitate an understanding of the present invention, a number of terms and phrases are defined below.
As used herein, the term "lead compound" refers to a chemical compound selected (e.g., rationally selected based on 3D structural analysis) for use directly as therapeutic compound, or for chemical modification (e.g., optimization) to design analog compounds useful in the treatment of a given condition. For example, as used herein, "lead compounds" are selected for modification to increase binding and specificity to the D3 receptor. Lead compounds can be known compounds or compounds designed de novo. A "pharmacophore" according to the present invention, is a chemical motif, including, but not limited to, a number of binding elements and their three-dimensional geometric arrangement. The elements are presumed to play a role in the activity of compounds to be identified as a lead compound. The pharmacophore is defined by the chemical nature of the binding elements as well as the three-dimensional geometric arrangement of those elements.
As used herein the term, "in vitro" refers to an artificial environment and to processes or reactions that occur within an artificial environment. In vitro environments can consist of, but are not limited to, test tubes and cell cultures. The term "in vivo" refers to the natural environment (e.g. , an animal or a cell) and to processes or reaction that occur within a natural environment.
As used herein, the term "host cell" refers to any eukaryotic or prokaryotic cell (e.g., mammalian cells, avian cells, amphibian cells, plant cells, fish cells, and insect cells), whether located in vitro or in vivo.
As used herein, the term "cell culture" refers to any in vitro culture of cells. Included within this term are continuous cell lines (e.g., with an immortal phenotype), primary cell cultures, finite cell lines (e.g., non-transformed cells), and any other cell population maintained in vitro, including oocytes and embryos. As used herein, the term "subject" refers to organisms to be treated by the methods of the present invention. Such organisms include, but are not limited to, humans. In the context of the invention, the term "subject" generally refers to an individual who will receive or who has received treatment (e.g., administration of antagonists and/or agonists of the G-protein coupled receptors). In preferred embodiments, the G-protein coupled receptors comprise dopamine receptors (e.g., Ox, D2, D3, and D4). In particularly preferred embodiments, the dopamine receptor is the D receptor.
The term "diagnosed," as used herein, refers to the to recognition of a disease by its signs and symptoms (e.g., aberrant G-protein coupled receptor regulation and/or function, and more particularly, aberrant dopamine receptor regulation and/or function), or genetic analysis, pathological analysis, histological analysis, and the like.
As used herein, the term "antisense" is used in reference to RNA sequences that are complementary to a specific RNA sequence (e.g., mRNA). included within this definition are antisense RNA ("asRNA") molecules involved in gene regulation by bacteria. Antisense RNA may be produced by any method, including synthesis by splicing the gene(s) of interest in a reverse orientation to a viral promoter that permits the synthesis of a coding strand. For example, once introduced into an embryo, this transcribed strand combines with natural mRNA produced by the embryo to form duplexes. These duplexes then block either the further transcription of the mRNA or its translation. In this manner, mutant phenotypes may be generated. The term "antisense strand" is used in reference to a nucleic acid strand that is complementary to the "sense" strand. The designation (-) (i.e., "negative") is sometimes used in reference to the antisense strand, with the designation (+) sometimes used in reference to the sense (i.e., "positive") strand. Regions of a nucleic acid sequences that are accessible to antisense molecules can be determined using available computer analysis methods.
The term "sample" as used herein is used in its broadest sense. A sample suspected of indicating a condition characterized by the aberrant regulation and/or function of a G- protein coupled receptor, and more particularly, the aberrant regulation and/or function of a dopamine receptor, may comprise a cell, tissue, or fluids, chromosomes isolated from a cell (e.g., a spread of metaphase chromosomes), genomic DNA (in solution or bound to a solid support such as for Southern blot analysis), RNA (in solution or bound to a solid support such as for Northern blot analysis), cDNA (in solution or bound to a solid support) and the like. A sample suspected of containing a protein may comprise a cell, a portion of a tissue, an extract containing one or more proteins and the like.
As used herein, the term "purified" or "to purify" refers, to the removal of undesired components from a sample. As used herein, the term "substantially purified" refers to molecules, either nucleic or amino acid sequences, that are removed from their natural environment, isolated or separated, and are at least 60% free, preferably 75% free, and most preferably 90% free from other components with which they are naturally associated. For example, an "isolated polynucleotide" is therefore a substantially purified polynucleotide.
As used herein, the term "genome" refers to the genetic material (e.g., chromosomes) of an organism or a host cell.
The term "nucleotide sequence of interest" refers to any nucleotide sequence (e.g., RNA or DNA), the manipulation of which may be deemed desirable for any reason (e.g., treat disease, confer improved qualities, etc.), by one of ordinary skill in the art. Such nucleotide sequences include, but are not limited to, coding sequences, or portions thereof, of structural genes (e.g., reporter genes, selection marker genes, oncogenes, drug resistance genes, growth factors, etc.), and non-coding regulatory sequences which do not encode an mRNA or protein product (e.g., promoter sequence, polyadenylation sequence, termination sequence, enhancer sequence, etc.).
"Nucleic acid sequence" and "nucleotide sequence" as used herein refer to an oligonucleotide or polynucleotide, and fragments or portions thereof, and to DNA or RNA of genomic or synthetic origin which may be single- or double-stranded, and represent the sense or antisense strand. As used herein, the terms "nucleic acid molecule encoding," "DNA sequence encoding," "DNA encoding," "RNA sequence encoding," and "RNA encoding" refer to the order or sequence of deoxyribonucleotides or ribonucleotides along a strand of deoxyribonucleic acid or ribonucleic acid. The order of these deoxyribonucleotides or ribonucleotides determines the order of amino acids along the polypeptide (protein) chain translated from the mRNA. The DNA or RNA sequence thus codes for the amino acid sequence.
The term "gene" refers to a nucleic acid (e.g., DNA or RNA) sequence that comprises coding sequences necessary for the production of a polypeptide or precursor
(e.g., proinsulin). The polypeptide can be encoded by a full length coding sequence or by any portion of the coding sequence so long as the desired activity or functional properties
(e.g., enzymatic activity, ligand binding, signal transduction, etc.) of the full-length or fragment are retained. The term also encompasses the coding region of a structural gene and includes sequences located adjacent to the coding region on both the 5' and 3' ends for a distance of about 1 kb or more on either end such that the gene corresponds to the length of the full-length mRNA. The sequences that are located 5' of the coding region and which are present on the mRNA are referred to as 5' untranslated sequences. The sequences that are located 3' or downstream of the coding region and which are present on the mRNA are referred to as 31 untranslated sequences. The term "gene" encompasses both cDNA and genomic forms of a gene. A genomic form or clone of a gene contains the coding region interrupted with non-coding sequences termed "introns" or "intervening regions" or "intervening sequences." Introns are segments of a gene which are transcribed into nuclear RNA (hnRNA); introns may contain regulatory elements such as enhancers. Introns are removed or "spliced out" from the nuclear or primary transcript; introns therefore are absent in the messenger RNA (mRNA) transcript. The mRNA functions during translation to specify the sequence or order of amino acids in a nascent polypeptide.
As used herein, the term "exogenous gene" refers to a gene that is not naturally present in a host organism or cell, or is artificially introduced into a host organism or cell. As used herein, the term "vector" refers to any genetic element, such as a plasmid, phage, transposon, cosmid, chromosome, virus, virion, etc., which is capable of replication when associated with the proper control elements and which can transfer gene sequences between cells. Thus, the term includes cloning and expression vehicles, as well as viral vectors.
As used herein, the term "gene expression" refers to the process of converting genetic information encoded in a gene into RNA (e.g., mRNA, rRNA, tRNA, or snRNA) through "transcription" of the gene (i.e., via the enzymatic action of an RNA polymerase), and for protein encoding genes, into protein through "translation" of mRNA. Gene expression can be regulated at many stages in the process. "Up-regulation" or "activation" refers to regulation that increases the production of gene expression products (i.e., RNA or protein), while "down-regulation" or "repression" refers to regulation that decrease production. Molecules (e.g., transcription factors) that are involved in up-regulation or down-regulation are often called "activators" and "repressors," respectively. As used herein, the terms "nucleic acid molecule encoding," "DNA sequence encoding," "DNA encoding," "RNA sequence encoding," and "RNA encoding" refer to the order or sequence of deoxyribonucleotides or ribonucleotides along a strand of deoxyribonucleic acid or ribonucleic acid. The order of these deoxyribonucleotides or ribonucleotides determines the order of amino acids along the polypeptide (protein) chain translated from the mRNA. The DNA or RNA sequence thus codes for the amino acid sequence.
The terms "homology" and "percent identity" when used in relation to nucleic acids refers to a degree of complementarity. There may be partial homology (i.e., partial identity) or complete homology (i.e., complete identity). A partially complementary sequence is one that at least partially inhibits a completely complementary sequence from hybridizing to a target nucleic acid sequence and is referred to using the functional term "substantially homologous." The inhibition of hybridization of the completely complementary sequence to the target sequence may be examined using a hybridization assay (Southern or Northern blot, solution hybridization and the like) under conditions of low stringency. A substantially homologous sequence or probe (i.e., an oligonucleotide which is capable of hybridizing to another oligonucleotide of interest) will compete for and inhibit the binding (i.e., the hybridization) of a completely homologous sequence to a target sequence under conditions of low stringency. This is not to say that conditions of low stringency are such that non-specific binding is permitted; low stringency conditions require that the binding of two sequences to one another be a specific (i.e., selective) interaction. The absence of non-specific binding may be tested by the use of a second target which lacks even a partial degree of complementarity (e.g., less than about 30% identity); in the absence of non-specific binding the probe will not hybridize to the second non-complementary target. As used herein the term "stringency" is used in reference to the conditions of temperature, ionic strength, and the presence of other compounds such as organic solvents, under which nucleic acid hybridizations are conducted. With "high stringency" conditions, nucleic acid base pairing will occur only between nucleic acid fragments that have a high frequency of complementary base sequences. Thus, conditions of "weak" or "low" stringency are often required with nucleic acids that are derived from organisms that are genetically diverse, as the frequency of complementary sequences is usually less. While the present invention can be practiced utilizing low stringency conditions, in a preferred embodiment, medium to high stringency conditions are employed.
"High stringency conditions" when used in reference to nucleic acid hybridization comprise conditions equivalent to binding or hybridization at 42 °C (e.g., overnight) in a solution consisting of 5X SSPE (43.8 g/1 NaCl, 6.9 g/1 NaH2PO4 H2O and 1.85 g/1 EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS, 5X Denhardt's reagent [50X Denhardt's contains per 500 ml, 5 g Ficoll (Type 400, Pharamcia), 5 g BSA (Fraction V; Sigma)], 100 μj/ml denatured salmon sperm DNA and 50% (V/V) of formamide. The hybridized DNA samples are then washed twice in a solution comprising 2 X SSPE, 0.1% SDS at room temperature followed by 0.1X SSPE, 1.0% SDS at 42 °C when a probe of about 500 nucleotides in length is employed. The art knows conditions that promote hybridization under conditions of high stringency (e.g., increasing the temperature of the hybridization and/or wash steps, the use of formamide in the hybridization solution, etc.).
"Medium stringency conditions" when used in reference to nucleic acid hybridization comprise conditions equivalent to binding or hybridization at 42 °C in a solution consisting of 5X SSPE (43.8 g/1 NaCl, 6.9 g/1 NaH2PO4 H2O and 1.85 g/1 EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS, 5X Denhardt's reagent [50X Denhardt's contains per 500 ml, 5 g Ficoll (Type 400, Pharamcia), 5 g BSA (Fraction V; Sigma)] 100 uj/ml denatured salmon sperm DNA, and 50% (V/V) formamide. followed by washing in a solution comprising 1.0X SSPE, 1.0 % SDS at room temperature followed by 2X SSPE, 0.1% SDS at 42 °C when a probe of about 500 nucleotides in length is employed. "Low stringency conditions" comprise conditions equivalent to binding or hybridization at 42 °C in a solution consisting of 5X SSPE (43.8 g/1 NaCl, 6.9 g/1 NaH2PO4 H2O and 1.85 g/1 EDTA, pH adjusted to 7.4 with NaOH), 0.1% SDS, 5X Denhardt's reagent [5 OX Denhardt's contains per 500 ml, 5 g Ficoll (Type 400, Pharamcia), 5 g BSA (Fraction V; Sigma)], 100 g/ml denatured salmon sperm DNA, and 50%(V/V) formamide. followed by washing in a solution comprising 5X SSPE, 0.1% SDS at 42 °C when a probe of about 500 nucleotides in length is employed. In addition, the art knows well that numerous equivalent conditions may be employed to comprise low stringency conditions; factors such as the length and nature (DNA, RNA, base composition) of the probe and nature of the target (DNA, RNA, base composition, present in solution or immobilized, etc.) and the concentration of the salts and other components (e.g., the presence or absence of formamide, dextran sulfate, polyethylene glycol) are considered and the hybridization solution may be varied to generate conditions of low stringency hybridization different from, but equivalent to, the above listed conditions.
When used in reference to a single-stranded nucleic acid sequence, the term "substantially homologous" refers to any probe that can hybridize (i.e., it is the complement of) the single-stranded nucleic acid sequence under conditions of low stringency as described above. A gene may produce multiple RNA species that are generated by differencial splicing of the primary RNA transcript. cDNAs that are splice variants of the same gene will contain regions of sequence identity or complete homology (representing the presence of the same exon or portion of the same exon on both cDNAs) and regions of complete non-identity (for example, representing the presence of exon "A" on cDNA 1 wherein cDNA 2 contains exon "B" instead). Because the two cDNAs contain regions of sequence identity they will both hybridize to a probe derived from the entire gene or portions of the gene containing sequences found on both cDNAs; the two splice variants are therefore substantially homologous to such a probe and to each other. The present invention is not limited to the situation where hybridization takes place only between completely homologous sequences. In some embodiments, hybridization takes place with substantially homologous sequences. As used herein, the term "protein of interest" refers to a protein encoded by a nucleic acid of interest.
As used herein, the term "native" (or wild type) when used in reference to a protein, refers to proteins encoded by partially homologous nucleic acids so that the amino acid sequence of the proteins varies. As used herein, the term "variant" encompasses proteins encoded by homologous genes having both conservative and nonconservative amino acid substitutions that do not result in a change in protein function, as well as proteins encoded by homologous genes having amino acid substitutions that cause decreased (e.g., null mutations) protein function or increased protein function.
As used herein, the term "pathogen" refers a biological agent that causes a disease state (e.g., infection, cancer, etc.) in a host. "Pathogen" include, but are not limited to, viruses, bacteria, archaea, fungi, protozoans, mycoplasma, and parasitic organisms.
As used herein, the term "organism" is used to refer to any species or type of microorganism, including but not limited to, bacteria, archaea, fungi, protozoans, mycoplasma, and parasitic organisms. As used herein, the term "fungi" is used in reference to eukaryotic organisms such as the molds and yeasts, including dimorphic fungi.
As used herein, the term "virus" refers to minute infectious agents, which with certain exceptions, are not observable by light microscopy, lack independent metabolism, and are able to replicate only within a living host cell. The individual particles (i.e., virions) consist of nucleic acid and a protein shell or coat; some virions also have a lipid containing membrane. The term "virus" encompasses all types of viruses, including animal, plant, phage, and other viruses.
The terms "bacteria" and "bacterium" refer to all prokaryotic organisms, including those within all of the phyla in the Kingdom Procaryotae. It is intended that the term encompass all microorganisms considered to be bacteria including Mycoplasma, Chlamydia, Actinomyces, Streptomyces, and Rickettsia. All forms of bacteria are included within this definition including cocci, bacilli, spirochetes, spheroplasts, protoplasts, etc. Also included within this term are prokaryotic organisms which are gram negative or gram positive. "Gram negative" and "gram positive" refer to staining patterns with the Gram-staining process which is well known in the art. (See e.g. , Finegold and Martin,
Diagnostic Microbiology, 6th Ed., CV Mosby St. Louis, pp 13-15 [1982]). "Gram positive bacteria" are bacteria which retain the primary dye used in the Gram stain, causing the stained cells to appear dark blue to purple under the microscope. "Gram negative bacteria" do not retain the primary dye used in the Gram stain, but are stained by the counterstain. Thus, gram negative bacteria appear red.
As used herein, the term "antigen binding protein" refers to proteins which bind to a specific antigen. "Antigen binding proteins" include, but are not limited to, immunoglobulins, including polyclonal, monoclonal, chimeric, single chain, and humanized antibodies, Fab fragments, F(ab')2 fragments, and Fab expression libraries. Various procedures known in the art are used for the production of polyclonal antibodies. For the production of antibody, various host animals can be immunized by injection with the peptide corresponding to the desired epitope including but not limited to rabbits, mice, rats, sheep, goats, etc. In a preferred embodiment, the peptide is conjugated to an immunogenic carrier (e.g., diphtheria toxoid, bovine serum albumin (BSA), or keyhole limpet hemocyanin [KLH]). Various adjuvants are used to increase the immunological response, depending on the host species, including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (Bacille Calmette-Guerin) and Corynebacterium parvum.
For preparation of monoclonal antibodies, any technique that provides for the production of antibody molecules by continuous cell lines in culture may be used (See e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). These include, but are not limited to, the hybridoma technique originally developed by Kδhler and Milstein (Kδhler and Milstein, Nature, 256:495-497
[1975]), as well as the trioma technique, the human B-cell hybridoma technique (See e.g.,
Kozbor et al, Immunol. Today, 4:72 [1983]), and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al, in Monoclonal Antibodies and Cancer Therapy,
Alan R. Liss, Inc., pp. 77-96 [1985]). According to the invention, techniques described for the production of single chain antibodies (U.S. 4,946,778; herein incorporated by reference) can be adapted to produce specific single chain antibodies as desired. An additional embodiment of the invention utilizes the techniques known in the art for the construction of Fab expression libraries (Huse et al, Science, 246:1275-1281 [1989]) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity.
Antibody fragments that contain the idiotype (antigen binding region) of the antibody molecule can be generated by known techniques. For example, such fragments include but are not limited to: the F(ab')2 fragment that can be produced by pepsin digestion of an antibody molecule; the Fab' fragments that can be generated by reducing the disulfide bridges of an F(ab')2 fragment, and the Fab fragments that can be generated by treating an antibody molecule with papain and a reducing agent.
Genes encoding antigen binding proteins can be isolated by methods known in the art. In the production of antibodies, screening for the desired antibody can be accomplished by techniques known in the art (e.g., radioimmunoassay, ELISA (enzyme-linked immunosorbant assay), "sandwich" immunoassays, immunoradiometric assays, gel diffusion precipitin reactions, immunodiffusion assays, in situ immunoassays (using colloidal gold, enzyme or radioisotope labels, for example), Western Blots, precipitation reactions, agglutination assays (e.g., gel agglutination assays, hemagglutination assays, etc.), complement fixation assays, immuno fluorescence assays, protein A assays, and immunoelectrophoresis assays, etc.) etc.
As used herein, the term "instructions for administering said dopamine receptor modulating compound to a subject," and grammatical equivalents, includes instructions for using the compositions contained in the kit for the treatment of conditions characterized by the aberrant regulation and or function of a dopamine receptor (e.g., D3 receptor), including, but not limited to, drug [e.g., cocaine] addiction, depression, anxiety, schizophrenia, Tourette's syndrome, eating disorders, alcoholism, chronic pain, obsessive compulsive disorders, restless leg syndrome, Parkinson's Disease, and the like) in a cell or tissue. In some embodiments, the instructions further comprise a statement of the recommended or usual dosages of the compositions contained within the kit pursuant to 21 CFR §201 et seq. Additional information concerning labeling and instruction requirements applicable to the methods and compositions of the present are available at the Internet web page of the U.S. FDA. The term "test compound" refers to any chemical entity, pharmaceutical, drug, and the like, that can be used to treat or prevent a disease, illness, sickness, or disorder of bodily function, or otherwise alter the physiological or cellular status of a sample (e.g., aberrant regulation and/or function of a G-protein coupled receptor, and more particularly, the aberrant regulation and/or function of a dopamine receptor [e.g., D3]). Test compounds comprise both known and potential therapeutic compounds. A test compound can be determined to be therapeutic by using the screening methods of the present invention. A "known therapeutic compound" refers to a therapeutic compound that has been shown (e.g. , through animal trials or prior experience with administration to humans) to be effective in such treatment or prevention. In preferred embodiments, "test compounds" are G-protein coupled receptor modulators.
As used herein, the term "third party" refers to any entity engaged in selling, warehousing, distributing, or offering for sale a test compound contemplated for co- administered with a receptor (e.g., G-protein coupled receptor, and more particularly, a dopamine receptor [e.g., D3]) modulating compound for treating conditions characterized by the aberrant regulation and/or function of a G-protein coupled receptor, and more particularly, the aberrant regulation and/or function of a dopamine receptor (e.g., D3).
As used herein, the term "modulate" refers to the activity of a compound (e.g., a dopamine receptor modulating compound) to affect (e.g., to promote or retard) an aspect of a cellular or a molecular function, including, but not limited to, neurotransmitter cycling, enzyme activity, and the like.
As used herein, the term "agonists of dopamine receptors," refers to compounds that promote the activity of one or more dopamine receptors, as compared to the activity of corresponding dopamine receptors not subjected to the agonist compound(s). As used herein, the term "antagonists of dopamine receptors," refers to compounds that retard the activity of one or more dopamine receptors, as compared to the activity of corresponding dopamine receptors not subjected to the antagonist compound(s).
GENERAL DESCRIPTION OF THE INVENTION Dopamine (DA) is a neurotransmitter that plays an essential role in normal brain functions. As a chemical messenger, dopamine is similar to adrenaline. In the brain, dopamine is synthesized in the pre-synaptic neurons and released into the space between the pre-synaptic and post-synaptic neurons. Dopamine affects brain processes that control movement, emotional response, and ability to experience pleasure and pain. Regulation of dopamine plays a crucial role in mental and physical health. Neurons containing the neurotransmitter dopamine are clustered in the midbrain in an area called the substantia nigra. Abnormal dopamine signaling in the brain has been implicated in many pathological conditions, including drug (e.g., cocaine) abuse, depression, anxiety, schizophrenia, Tourette's syndrome, eating disorders, alcoholism, chronic pain, obsessive compulsive disorders, restless leg syndrome, Parkinson's Disease, and the like.
Dopamine molecules bind to and activate the dopamine receptors on the post- synaptic neurons. Dopamine molecules are then transported through the dopamine transporter protein (DAT) back into the pre-synaptic neurons, where they are metabolized by monoamine oxidase (MAO). In conditions such as cocaine abuse, cocaine binds to the dopamine transporter and blocks the normal flow of dopamine molecules. Excess concentrations of dopamine cause over-activation of dopamine receptors. In other conditions, such as Parkinson's Disease, lack of sufficient dopamine receptors in the brain causes insufficient activation of dopamine receptors.
The D3 receptor is concentrated almost exclusively in limbic brain regions such as the nucleus accumbens, olfactory tubercle, and islands of Calleja. (See e.g., B. Landwhermeyer et al, Brain Res. Mol. Brain Res., 18:187-192 [1993]; A.M. Murray et al, Proc. Natl. Acad. Sci. USA, 91:11271-11275 [1994]; and M.L. Bouthenet et al, Brain Res., 564:203-219 [1991]). These brain areas are terminal fields of the mesolimbic dopamine projection that are associated with reinforcement and reward mechanisms. (See e.g., H.C. Fibiger and A.G. Phillips, Ann. N.Y. Acad. Sci., 537:206-215 [1988]; and W. J. McBride et al, Behav. Brain Res., 101:129-152 [1999]). Although an understanding of the mechanism is not necessary to practice the present invention and the present invention is not so limited, it is contemplated that while cocaine potently inhibits the reuptake of both norepinephrine (NE) and serotonin (5-HT), its ability to act as a reinforcer stems from inhibiting the reuptake of dopamine into dopaminergic neurons. (See e.g., M.C. Ritz et al, Science, 237-1219 [1987]; M.J. Kuhar et al, Trends Neurosci., 14:299-302 [1991]; F.I. Carroll et al, J. Med. Chem., 35:969 [1992]; and F.I. Carroll et al, J. Med. Chem., 42:2721-2731 [1999]). Cocaine exerts this inhibitory effect through specific interactions with dopamine transporter (DAT) proteins (cocaine receptor) located on dopamine nerve terminals. The resulting increase in the synaptic availability of dopamine in the reward mediating brain mesolimbic system is the essence of the dopamine hypothesis for cocaine action. Thus, pharmacological and neurobiologic evidence indicates that the abuse-related effects of cocaine are mediated by dopaminergic systems.
The present invention contemplates that the dopamine 3 (D3) subtype receptor is involved in the positive reinforcing effects of cocaine and other psychostimulants. Accordingly, preferred embodiments of the present invention contemplate designing dopamine receptor ligands (e.g., partial agonists) to treat cocaine and other psychostimulant abuse and dependence. The compound 7-OH-DPAT and several other D3 partial/full agonists, such as pramipexole and PD 128907, decrease self-administration of cocaine with a rank order of potency that is highly correlated with their potency at the D3 receptor in in vitro mitogenesis functional assays. (See e.g., S.B. Caine and G.F. Koob, Science,
260:1814-1816 [1993]; G.F. Koob and M. Le Moal, Science, 278:52-58 [1997]; S.B. Caine et al, J. Pharmacol. Exp. Ther., 291 :353-360 [1999]; M. Pilla et al, Nature, 400:371-375 [1999]; G.F. Koob, Ann. N. Y. Acad. Sci., 654:171-191 [1992]; S.B. Caine and G.F.
Koob, J. Exp. Anal. Behav., 61 :213-221 [1994]; and L.H. Parsons et al, J. Neurochem., 67: 1078-1089 [1996]). Likewise, stimulation of D3 sites is implicated in decreasing self- stimulation of the ventral tegmental area (R. Depoortere et al, Psychopharmacology, 124:231-240 [1996]), blocking the reinforcing effects of cocaine and d-amphetamine (T. Kling-Petersen et al, Pharmacol. Biochem. Behav., 49:345-351 [1994]), and decreasing the rate of food-reinforced responding in a fixed-ratio operant paradigm (DJ. Sanger et al, Behav. Pharmacol., 7:477-482 [1996]). In studies assessing conditioned place preference, D3 agonists produced an aversive effect when administered alone (F. Chaperon and M.H. Thiebot, Behav. Pharmacol., 7:105-109 [1996]) and attenuated the cocaine- and amphetamine-conditioned place preference (T.V. Khroyan et al, Psychopharmacology, 139:332-341 [1998]; and T.V. Khroyan et al, Psychopharmacology, 142:383-392 [1999]). Several D3 partial agonists have been shown to decrease cocaine self-administration.
(S.B. Caine et al, Neuroreport, 8:2373-2377 [1997]) The potency of these compounds in decreasing self-administration of cocaine is correlated with their potency as agonists of the D3 receptor in in vitro mitogenesis functional assays. (See e.g., S.B. Caine and G.F. Koob, Science, 260:1814-1816 [1993]; S.B. Caine et al, J. Pharmacol. Exp. Ther., 291:353-360 [1999]; and L.H. Parsons et al, J. Neurochem., 67:1078-1089 [1996]). Partial D3 agonists appear to lack the abuse liability themselves because experimental animals could not be trained to self-administer D3 partial agonists. (M.A. Nader and R.H. Mach,
Psychopharmacology [Berl], 125:13-22 [1996]; and L. Pulvirenti et al, J. Pharmacol. Exp.
Ther., 286:1231-1238 [1998]). A recent study showed that a relatively selective partial D3 agonist BP 897 inhibited cocaine-Seeking behavior in response to drug-associated cues in rats. (M. Pilla et al, Nature, 400:371-375 [1999]).
Alterations in the density of the D3 receptor have been reported with cocaine use. The density of D3 sites was increased from l-to-3-fold in the caudate, nucleus accumbens, and substantia nigra of persons dying from a cocaine over-dose. (J.K. Staley and D.C. Mash, J. Neurosci., 16:6100-6106 [1996]). Likewise, a 6-fold increase in D3 receptor mRNA was found in the nucleus accumbens of cocaine fatalities. (D.M. Segal and CT. Moraes, Brain Res. Mol. Brain Res., 45:335-339 [1997]). In addition, studies of gene polymorphisms suggest that the homozygosity for the Ball D3 receptor polymorphism, and perhaps also D receptor polymorphisms, is associated with cocaine dependence and other traits that may contribute to drug addiction. (R.P. Ebstein et al, Am. J. Med. Gen., 74:65- 72 [1997]; and D.E. Comings et al, Mol. Psychiatry, 4:484-487 [1999]).
The dopamine 3 (D3) receptor has been reported to have 52% sequence homology to the D2 receptor and a similar, but unique pharmacological profile. (P. Sokoloff et al, Nature, 347:146-151[1990]). Data suggests that the D3 receptor may be involved in the positive reinforcing effects of cocaine and other psychostimulants. (See e.g., S.B. Caine and G.F. Koob, Science, 260:1814-1816 [1993]; G.F. Koob and M. Le Moal, Science, 278:52- 58 [1997]; S.B. Caine et al, J. Pharmacol. Exp. Ther., 291:353-360 [1999]; M. Pilla et al, Nature, 400:371-375 [1999]; G.F. Koob, Ann. N. Y. Acad. Sci., 654:171-191 [1992]; S.B. Caine and G.F. Koob, J. Exp. Anal. Behav., 61 :213-221 [1994]; L.H. Parsons et al, J. Neurochem., 67:1078-1089 [1996]; R.S. Sinnott et al, Drug Alcohol Depend., 54:97-110 [1999]; J.K. Staley and D.C. Mash, J. Neurosci., 16:6100-6106 [1996]; and L.H. Parsons et al, J. Neurochem., 67:1078-1089 [1996]).
Prior efforts to develop cocaine antagonists as therapies for the treatment of cocaine abuse have focused on modifications of cocaine itself; this has led to a wealth of information about the structure-activity relationships (SARs) of cocaine analogs. Despite intense research efforts in this area, very few compounds with significant cocaine antagonist activity have been reported. Accordingly, preferred embodiments of the present invention provide novel compounds and method of designing these compounds, that antagonize all or some of addictive effects of cocaine and other psychostimulants.
Partial D3 agonists with unambiguous selectivity for the D3 receptor, such as those described herein in some preferred embodiments, were not available prior to the present invention. For instance, most existing D3 ligands also have good activity at the D2 subtype receptor. In some embodiments, highly selective D3 ligands serve as useful pharmacological tools to facilitate the elucidation of the functional role of the D3 receptor in cocaine addiction. Furthermore, in other embodiments, the partial agonists specific for the D receptor provide therapeutic compounds for the treatment of cocaine abuse. In still some other embodiments, the compositions and methods of the present invention provide therapeutic treatments for other additions to other psychostimulants characterized by interference with normal dopamine metabolism. In yet other embodiments, the compositions and methods of the present invention provide therapeutic treatments for other diseases and conditions characterized by aberrant regulation and/or function of dopamine signaling and/or dopamine receptor activity (e.g., depression, anxiety, schizophrenia, Tourette's syndrome, eating disorders, alcoholism, chronic pain, obsessive compulsive disorders, and Parkinson's Disease, and the like). Additional embodiments of the present invention provide compositions and methods for the treatment of conditions characterized by aberrant regulation and/or function of G-protein couple receptors (GPCRs), and methods for designing these compounds.
One factor that has impeded the development of highly selective D3 ligands prior to the present invention, is that the structural basis of dopamine ligand binding and selectivity was not well understood, primarily due to the lack of accurate 3D structural information of dopamine receptors. Since dopamine receptors are membrane bound proteins, experimental determination of their 3D structures is a difficult task. In the past, computational homology modeling approaches were used to construct 3D models of dopamine receptors using either low-resolution structures of rhodopsin or high-resolution structures of bacteriorhodopsin. (See e.g., E.J. Homan et al, Bioorg. Med. Chem., 7:1805-1820 [1999]; M.M. Teeter et al, J. Med. Chem., 37:2874-2888 [1994]; A. Malmberg et al, Mol. Pharmacol., 46:299-312 [1994]; S. Trumpp-Kallmeyer et al, J. Med. Chem., 35:3448-3462 [1992]; H. Moereels and J.E. Leysen, Receptors Channels, 1:89-97 [1993]; CD. Livingstone et al, Biochem. J., 287:277-282 [1992]; and S.G. Dahl et al, Proc. Natl. Acad. Sci. USA, 88:8111-8115 [1991]). However, bacteriorhodopsin is not a GPCR and has very low sequence homology with the dopamine receptors and rhodopsin, the topological arrangement of the transmembrane helices differ between the X-ray structure of rhodopsin and bacteriorhodopsin (K. Palczewski et al, Science, 289:739-745 [2000]), and hence the accuracy of the bacteriorhodopsin based dopamine models is limited. Although the structure of rhodopsin has been used as the template structure for modeling, the low- resolution of existing rhodopsin structures limits their accuracy as well. Furthermore, due to limited computing power, prior to the present invention structural refinements in these models were performed without the inclusion of proper lipid/water environment that is required for accurate modeling. (R.G. Efremov et al, Biophys. J., 76:2460-2471 [1999]). Prior to the present invention, yet another difficulty in determining the specific functions of the dopamine receptor subtypes was the limited availability of ligands that could reliably discriminate between the D2 and D3 subtypes, especially in vivo. In contrast, preferred embodiments of the present invention provide ligands designed to selectively bind to the various dopamine receptor (e.g., O , D2, D3, D4, and D5) subtypes. Other preferred embodiments, provide ligands designed to partially, or fully, modulate (e.g., agonism and/or antagonism) the D3 receptor with high selectivity. The present invention also provides compounds designed to block other members of the D family of receptors and other members of the GPCR superfamily. Still other embodiments provide therapeutic compositions and methods for the treatment of conditions characterized by faulty regulation of dopamine signaling.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to compounds designed as ligands (e.g., agonists, antagonists, and partial agonists) for dopamine receptors (e.g., Ox, D2, D3, D4, and D5), and in particular, the Di, D2, and D dopamine receptors. The present invention also relates to methods for rationally designing these compounds and to therapeutic methods of using these compounds.
Exemplary compositions and methods of the present invention are described in more detail in the following sections: I. Dopamine receptors; II. Homology modeling of dopamine receptors Dj, D2, and D3; III. Computational docking studies of the interactions between D3 ligands and the receptors; IV. General design strategy for identifying novel lead compounds; V. Identification of specific novel lead compounds; VI. Further structure-based design and chemical modifications of lead compounds; VII. Biological testing and characterization of lead compounds and analogues thereof; VIII. Therapeutic agents combined or co-administered with the present compositions; and IX. Pharmaceutical formulations, administration routes, and dosing considerations.
In the numerous examples that follow, reference is principally taken to compositions and methods directed to providing modulators (e.g., agonists, and/or antagonists, both partial and complete) of the dopamine receptors, and more particularly D3 receptors. The present invention is not intended to be limited, however, to providing modulators of dopamine receptors. Indeed, the methods and compositions of the present invention are useful in identifying and designing compositions (e.g., ligands) that modulate an number of receptor family subtypes (e.g., G-protein coupled receptors).
I. Dopamine receptors
In the 1950s scientists began to recognize that dopamine was an important neurotransmitter based on the non-uniform distribution of dopamine in the brain. Research by Cools and Van Rossum suggested, based on anatomical, electrophysiological, and pharmacological studies, that there might be more than one kind of receptor for dopamine in the brain. (A.R. Cools and J.M. Van Rossum, Psychopharmacology, 45(3):243-245
[1976]). Biochemical analysis of dopamine receptors based on using second messenger assays (e.g., stimulation of cAMP production), and ligand binding assays, led other researchers to propose that there were two classes of dopamine receptor, Di and D , with different biochemical and pharmacological properties and mediating different physiological functions. (See e.g., J.W. Kebabian and D.B. Caine, Nature, 277(5692):93-96 [1979]). Table 1 lists some of the respective properties of the Di and D2 classes of dopamine receptors. (See, P.G. Strange, Neurochem. Int., 22(3):223-236 [1993]).
Table 1
Both the Di-like and D2-like receptor subtypes are G-protein coupled receptors, but different G-proteins and effectors are involved in their signaling pathways. Through application of gene cloning techniques, reserachs have shown that are at least five dopamine receptors (DrD ) and they may be divided into two subfamilies whose properties resemble the original Di and D2 receptors defined through pharmacological and biochemical techniques. (See, D.R. Sibley and F.J. Monsma Jr., Trends Pharmacol. Sci., 13(2):61-69
[1992]; O. Civelli et al, Ann. Rev. Pharmacol. Toxicol., 33:281-307 [1993]; and K.R. Jarvie and M.G. Caron, Adv. Neurol., 60:325-333 [1993]). The two subfamilies are often termed D^like (D1 ? D5) and D2-like (D2, D3, D4). Table 1 summarizes some of the key properties of the Di-like and D2 like dopamine receptors. There may be other subtypes of dopamine receptors, for example, as additional Dplike receptors have been cloned from Xenopus, chicken, and drosophila.
Analysis of the amino acid sequences of the dopamine receptors has shown that significant homologies exist among subtypes, with the greatest homologies being found between members of a subfamily (e.g., Di-like or D2-like subfamilies).
Each known dopamine receptor contains seven transmembrane stretches of hydrophobic amino acids. Thus, each dopamine receptor conforms to the general structural model for G-protein coupled receptors (D. Donnelly et al, Receptors Channels, 2(1 ):61-78 [1994]) by having an extracellular amino terminus and seven membrane spanning-helices linked by intracellular and extracellular protein loops. The D^like receptors have short third intracellular loops and long carboxyl terminal tails, whereas the D2-like receptors have long third intracellular loops and short carboxyl terminal tails. This provides a structural basis for the division of the receptors into two subfamilies but is also likely to have a functional significance possibly related to the specificity of receptor/G-protein interaction. The third intracellular loop in dopamine receptors is thought to be important for the interaction of receptor and G-protein and for the D2-like receptors, variants of these subtypes exist based on this loop. For example, there are short and long variants of the D2 and D3 receptors with the long forms having an insertion (29 amino acids for D2 long) in this loop. (B. Giros et al, Nature, 342(6252):923-926 [1989]). For example, polymorphic variants of the D2 receptor have been described with single amino acid changes in this loop. (A. Cravchik et al, J. Biol. Chem., 271(42):26013-26017 [1996]). In the humans, the D4 receptor has polymorphic insertions in this loop. (H.H. Van Tol et al, Nature, 358(6382):149-152 [1992]).
The D2-like receptor variants may have differential abilities to couple to or activate G-proteins (A. Cravchik et al, J. Biol. Chem., 271(42):26013-26017 [1996]; J. Guiramand et al, J. Biol. Chem., 270(13):7354-7358 [1995]; and S.W. Castro and P.G. Strange, FEBS Letts., 315(3):223-226 [1993]) and may also exhibit slightly different pharmacological properties (S.W. Castro and P.G. Strange (1993) J. Neurochem., 60(l):372-375 [1993]; and
A. Malmberg et al, Mol. Pharmacol., 43(5):749-754 [1993]). However, variants of the D4 receptor have not been found to exhibit any differences in the binding of ligands or in coupling to G proteins. (M.A. Kazmi et al, Biochemistry, 39(13):3734-3744 [2000]). The individual properties of the different subtypes have been probed by expressing the receptors in recombinant cells and by examining the localization of the subtypes at the mRNA and protein level.
Dopamine receptor subtypes O and D2 have different pharmacological profiles, localization, and mechanisms of action. In regard to the Drlike dopamine receptors, both the Di and D5 receptors show similar pharmacological properties (e.g., high affinity for benzazepine ligands). The thioxanthines show high affinity for D like receptors, but are not selective for Di-like over D2-like receptors. The Di-like receptors also show moderate affinities for typical dopamine agonists such as apomorphine, and selective agonists such as dihydrexidine. Prior to the present invention, ligands selective for either the D] or D5 dopamine receptors were not known.
Di receptors are found at high levels in the typical dopamine rich regions of brain such as the neostriatum, substantia nigra, nucleus accumbens and olfactory tubercle, whereas the distribution of the D5 receptors is much more restricted. The D5 subtype is found at lower levels then the Di subtype dopamine receptors. Both Di and D5 receptors are able to stimulate adenylyl cyclase, with the D5 receptor showing some constitutive activity for this response.
Inverse agonist activity at the Di and D5 receptors is seen in recombinant systems with some compounds, such as butaclamol, that were previously considered to be antagonists. Although an understanding of the mechanism is not necessary to practice the present invention and the present invention is not so limited, it is contemplated that the Di receptor mediates important actions of dopamine to control movement, cognitive function and cardiovascular function. Moreover, interaction between D5 receptors (a G-protein coupled receptor) and GABAA receptors (ion channel linked receptors) have been described that point towards a functional role for the D5 receptor.
D2-like receptors (e.g., D2, D , and D4) exhibit pharmacological properties similar to those of the original pharmacologically defined D2 receptor (e.g., they all show high affinities for drugs such as the butyrophenones, and the substituted benzamides). These classes of drugs are contemplated to provide selective antagonists for the D2-like receptors. As indicated above, the D2-like receptors also show high affinities for phenothiazines and thioxanthines. Each D2-like receptor has its own pharmacological signature, such that there are some differences in affinities of drugs for the individual D2-like receptors. For example, raclopride shows a high affinity for the D2 and D3 receptors but a lower affinity for the D4 receptor. Clozapine shows a slight selectivity for the D4 receptor. Aminotetralins show some selectivity as D2-like autoreceptor antagonists, but theses compounds also show selectivity for D3 receptors as agonists. Most D2-like receptor antagonists show a higher affinity for the D2 receptor compared to the D3 and D4 receptors. The D2-like subtypes show moderate affinities for typical dopamine agonists, while the D3 receptors show slightly higher affinities. There are compounds available that are selective agonists for D2-like receptors (e.g., quinpirole), however, prior to the present invention, there were no highly selective agonists for individual D2-like receptors.
The D2 receptor is the predominant D2-like receptor subtype in the brain and is found at high levels in typical dopamine rich brain areas. D3 and D4 receptors are found at much lower levels and in a more restricted distribution pattern. The D3 and D4 receptors are found predominantly in limbic areas of the brain. The D2-like receptor subtypes have each been shown to inhibit adenylyl cyclase when expressed in recombinant cells (See e.g., CL. Chio et al, Mol. Pharmacol., 45(l):51-60 [1994]; B. Gardner B et al, Br. J. Pharmacol., 118(6): 1544- 1550 [1996]; L. Tang et al, J. Pharmacol. Exp. Ther., 268(l):495-502 [1994]; and D.A. Hall and P.G. Strange, Biochem. Pharmacol., 58(2):285-289 [1999]) although, the signal via the D3 receptor has proven more difficult to demonstrate and is generally lower than for the other two subtypes. The D2-like receptors will also stimulate mitogenesis (CL. Chio et al, Mol. Pharmacol., 45(l):51-60 [1994]; and B.C. Swarzenski et al, Proc. Natl. Acad. Sci. U.S.A., 91(2):649-653 [1994]) and extracellular acidification (Chio, infra.) in recombinant systems. Effects have been shown on arachidonic acid release and MAP kinase for the D2 receptor (P. Sokoloff et al, Biochem. Pharmacol., 43(4):659-666 [1992]; and G.I. Welsh et al, J. Neurochem., 70(5):2139-2146 [1998]) and on Ca2+ channels for the D2 and D3 receptors. (G.R. Seabrook et al, Br. J. Pharmacol., 111(2):391-393 [1994]). The relationship of these effects to the in vivo responses is entirely unclear. The D2 receptor is important for mediating the effects of dopamine to control movement, certain aspects of behavior in the brain and prolactin secretion from the anterior pituitary gland. The localization of the D3 and D4 receptors in limbic areas of brain suggests that they have a role in cognitive, emotional, and behavioral functions. The distribution of the D3 and D4 receptors in limbic brain regions has made them particularly attractive targets for the design of potential selective antipsychotic drugs. The D2-like receptors show high affinities for most of the drugs used to treat schizophrenia (antipsychotics) and Parkinson's disease (e.g. bromocriptine). II. Homology modeling of dopamine receptors Di, D2, and D3
The X-ray structure of rhodopsin was recently determined at 2.8 A resolution. (K. Palczewski et al, Science, 289:739-745 [2000]). As mentioned above, the Di, D2, and D3 receptors belong to the rhodopsin family within the GPCR super-family. Of note, within the seven transmembrane (TM) helices, which form the ligand binding site, the Di, D2, and D3 receptors and rhodopsin have a high sequence homology (identity/similarity) of 47%. Thus, in preferred embodiments of the present invention, the high resolution X-ray structure of rhodopsin is used to accurately model the 3D structures of dopamine receptors. In homology modeling, in addition to the accuracy of the template protein structure, the sequence alignment between the modeled protein (D1} D2, and D3) and the template protein (rhodopsin) is important for the accuracy of the modeled structure. A previous sequence analysis of 493 members of the rhodopsin family of GPCR proteins provides an unambiguous sequence alignment in the TM region, that includes the binding site, between Di, D2, and D3 receptors and rhodopsin. (J.M. Baldwin et al, J. Mol. Biol., 272:144-164 [1997]). Therefore, in preferred embodiments of the present invention, homology modeling methods provide accurate 3D structural modeling of Dj, D2, and D3 receptors, especially, of the ligand binding site.
The lipid membrane/water environment affects the 3D structures of transmembrane proteins. Accordingly, in some embodiments, to further improve the accuracy of modeled structures, extensive MD simulations including an explicit lipid-water environment in the structural refinement were performed. In some of these embodiments, a highly efficient, parallel version of the CHARMM program (version 2.7) (B.R. Brooks et al, J. Comp. Chem., 4:187-217 [1983]) running on a supercomputer is used in the MD simulations as the modeling environment includes approximately 16,000 atoms, comprising, the Di, D2, and D3 proteins, lipids, and water molecules. Three ns MD simulations for the Di and D3 receptor structures have been completed, and 2.5 ns simulations have been completed for the D2 receptor structure. In other embodiments, substantially longer MD simulations are used (e.g., 5 ns, 7 ns, 10 ns, and longer). The present invention, however, is not intended to be limited to an particular length of modeling or simulation. Those skilled in the art will appreciate the potential effects of various modeling and simulation times (e.g., longer MD simulation times allow identification of additional low energy conformational clusters with respect to the binding site conformations if previous MD simulations are not sufficiently long enough). The binding site in D2 has been experimentally determined through experiments using methanethiosulfonate (MTS) reagents. (See, J.A. Javitch et al, Neuron, 14:825-831 [1995]; J.A. Javitch et al, Biochemistry, 34:16433-16439 [1995]; J.A. Javitch et al, Mol. Pharmacol., 49:692-698 [1996]; D. Fu et al, Biochemistry, 35:11278-11285 [1996]; J.A. Javitch et al, Biochemistry, 37:998-1006 [1998]; J.A. Javitch et al, Biochemistry, 38:7961- 7968 [1999]; and J.A. Javitch et al, Biochemistry, 39:12190-12199 [2000]).
Although an understanding of the mechanism is not necessary to practice the present invention and the present invention is not so limited, it is contemplated that the D2 receptor contains three common Ser residues in TM5, (See e.g., NJ. Pollock et al, J. Biol. Chem., 267:17780-17786 [1992]; B.L. Wiens et al, Mol. Pharmacol., 54:435-444 [1998]; A. Mansour et al, Eur. J. Pharmacol., 227:205-214 [1992]; C Coley et al, J. Neurochem., 74:358-366 [2000]; N. Sartania and P.G. Strange, J. Neurochem., 72:2621-2624 [1999]; B.A. Cox et al, J. Neurochem., 59:627-635 [1992]; and R. Woodward et al, J. Neurochem., 66:394-402 [1996]) and a common Asp residue at the extracellular end of TM3 (A. Mansour et al. infra) are important residues in ligand binding. A cysteine residue in D2 and D3 is important for ligand binding. (G.L. Alberts et al, Br. J. Pharmacol., 125:705-710 [1998]). In the modeled structures, all these residues are water exposed and are part of the ligand binding site, primarily formed by TM3, TM5, TM6 and TM7. The modeled 3D structure of D3 and its ligand binding site are shown in (Fig. 1). Further analyses were performed to investigate the structural differences between the Di, D2, and D3.
It was found that the structural differences between Di and D3 receptors, with respect to their ligand binding sites, are very profound. The major residue differences when considering the water-exposed residues in transmembrane regions TM2 to TM7 of the binding site are shown below in (Table 2). Residue and structural differences in TM3 impact ligand selectivity.
Further experiments using Dι/D3 chimeric receptors showed that replacing the extracellular portion of TM3 in D3 with corresponding residues in Dj significantly reduces the binding affinity of many D3 ligands. Furthermore, a single mutation of Cl 14 in D3 to Ser, the corresponding residue in Di, reduces the binding affinity by 37-fold for quinpirole and 57-fold for (-)-3-PPP. (G.L. Alberts et al, Br. J. Pharmacol., 125:705-710 [1998]). In some embodiments, Y-373 is replaced by a bulkier Trp (W321) in Di. The present invention contemplates that the major structural differences between D3 and )χ provides a rational basis for designing ligands that selectively bind to the various dopamine receptors, and in other embodiments, the basis for designing ligands that selectively bind to other members of the G-protein receptor family. Table 2 shows some of the sequence differences in Di and D3 binding sites (residues for Di are shown in parentheses) in regions accessible by solvent molecules or ligands.
Table 2
TM3 D104(N97), V105(I98), F106(W99), L109(F102), VI 11(1104), Cl 14(S107)
TM4 F162(K167), V164(T169), P167(S172), L168(D173), L169(G174), F170(N175) TM5 F188(Y194), V189(A195), Y191(S197), V195(I201), L199(I205) TM6 V334(277), T348(L291), H349(N292), V350(C293), T353(P296) TM7 L364(T312), Y365(F313), S366(D314), A367(V315), T368(F316), T369(V317), Y373(W321)
There are also significant structural differences between the D2 and D3 receptors, although, less profound than found in Di and D receptors. Careful structural analysis using high resolution models of D2 and D3 was used to identify four regions (i.e., Regions A, B, C, and D) of major sequence and structural differences between D2 and D3 receptors. For corresponding residues that are different between the dopamine receptor subtypes, there is some uncertainties with the coordinates of these side chain atoms. To solve this problem, the present invention uses current homology modeling methods employing a rotormer library to provide the most probable conformations of side chain for non-identical residues.
In preferred embodiments, the structural differences characterized in these four regions provides a rational basis for designing ligands that selectively bind to a particular receptor of interest (e.g., D3 receptor). In Region A, it was found that D3 residues N47, D75, F338, N375, N379 (D2 residues N52, D80, F382, N418, N422, respectively) in TM1 are part of a large hydrophobic pocket deep in the transmembrane region. In preferred embodiments, the present invention provides ligands that selectively bind either D3 or D2 receptors that have a flexible linker tethered to a bulky tail designed to reach the large hydrophobic pocket in Region A. These findings are based in part on experimental data collected in the studies mentioned below.
For instance, S AR analysis of several proposed classes of lead compounds shows that a long group linker with a bulky tail section selectively bind to D3 receptors.
In Region B, it was found that residues F345 and F346 in TM6 of D3 have different sidechain orientations compared to D2 corresponding residues F389 and F390. It was also found that structural differences in TM6 affect the interhelical packing between TM6 and TM7. D3 residue T368 is exposed to solvent, the corresponding D2 residue F411, plays a role in the interhelical interactions between TM6-TM7. Residues T369, Y365, F345, and F346 are also important in structural differences in D2 and D3 TM6 and TM7. It is contemplated that these residues directly interact with their respective ligands. These findings are based in part on experimental data collected in the studies mentioned below. For example, chimeric D2/D3 receptor studies showed that TM6 and TM7 contribute to the selectivity of PD 129807 which is a selective D3 partial agonist. Chimeric D2/D3 receptor studies also showed that the TM6 and TM7 regions are responsible for the selectivity of several ligands between D2 and D3. Still other studies show that mutation of F198, F389, F390 to alanine in the D2 receptor abolishes binding of N-0437 which is a ligand that shows a binding preference for D2 versus D3. Yet other studies show that mutation of T369V significantly increases the binding affinities of two D3 selective ligands, but not that of non- selective ligands. This suggests that this region is important for ligand selectivity.
In Region C, a comparison of the most populated conformational clusters (from MD simulations) of D and D shows that the preferred kink angle at Pro200 (TM5) is smaller in D3 (16.3°) than D2 (24.0°). It is contemplated that this kink shifts the position of residues at the extracellular end of TM5 between D3 and D , including, three important TM5 serine residues (SI 93, SI 94, and SI 97, respectively, in D2, and SI 92, SI 93, and SI 96, respectively, in D3). These findings are based in part on experimental data collected in the studies mentioned below. Studies show that the nature of the residue preceding this conserved Pro in TM5 affects the binding/selectivity of some ligands (e.g., in Di, D2, and D3 the relevant residues are He, Val, and Leu, respectively). Further studies show that mutations in corresponding TM5 serines to have a distinct effect on the binding affinity at the D2 and D3 receptors for the same ligand, thus, preferred embodiments of the present invention are designed to selectively bind to either the D2 or D3 receptors by taking advantage of the differences in these receptors due to mutations. Still further studies show that (i?)-(+)-7-OH-DPAT interacts with SI 92 of the D3 receptor. The selectivity of this compound decreased 5-fold if the hydroxyl substituent of is moved to position 5.
A hydrophobic pocket in Region D is formed by TM3, TM4, TM5 and E-II loop residues. The orientation and position of VI 11, 1183, F188 in D3 is significantly different from that of the corresponding D2 residues, VI 15, 1194, F189. The TM4 residue V164 in
D3 is an He in D2 is near the three TM5 serine residues. The difference found in the preferred kink angle of TM5 also contributes to the structural differences of this pocket.
These findings are based in part on experimental data collected in the studies mentioned below. Studies show that Region D of the D3 and D2 receptors contain non-conservative sequence differences, for example, in the position adjacent to the disulphide bridge connecting TM3 and the E-II loop residue SI 82 in the D3 receptor corresponds to residue 1183 in the D2 receptor. Of note, this position is in the proximity of 1183, F188 in D3 or 1184, F189 in D2. A163 corresponds to T165 in D2. A water exposed D2 residue, S163, is available to interact with ligands in this region and it is replaced by alanine in D3 (A161). Preferred embodiments of the present invention contemplate designing dopamine receptor subtype specific ligands based on the structural differences in these four specific regions (Regions A, B, C, and D). In particularly preferred embodiments, the present invention contemplates the structure-based design of ligands that specifically bind to Region B of the D receptor.
Preferred embodiments of the compositions (and methods) of the present invention were designed using accurate modeling of the various dopamine receptor and their transmembrane (TM) regions. The sequences of the dopamine receptors contain about 93- 97% of the set of the conserved amino acids shared by the 493 proteins in the rhodopsin family. (See, J.M. Baldwin et al, J. Mol. Biol., 272:144-164 [1997]). The sequence alignment between Dj, D2, and D3 and rhodopsin used in the homology modeling of the present invention was derived from 500 GPCR members and Di, D2, D , and rhodopsin belong to the rhodopsin sub-family within the GPCRs.
Dopamine receptors Di, D2, and D3 and rhodopsin share about 47% residue identity/similarity in 7 different TM regions. The sequence identity between rhodopsin and the D3 receptor is 28%, near the 30% threshold considered sufficient to achieve highly accurate homology modeled structures. There is no gap or insertion in the sequence alignment between Di, D2, and D3, and rhodopsin in the helix regions, which form the binding sites for ligands. Accordingly, in preferred embodiments, the high resolution X-ray structure of rhodopsin is used to provide accurate modeling of the dopamine receptors Di, D2, and D3 receptor structures. As shown below in Table 3, the conserved amino acid residues of the rhodopsin family obtained previously and further sequence identities between the rhodopsin and dopamine receptors allow unambiguous sequence alignment between rhodopsin and Di, D2, and D3 receptors in the transmembrane regions. (See, J.M. Baldwin et al, J. Mol. Biol., 272:144-164 [1997]). Using this alignment and the X-ray of rhodopsin, (K. Palczewski et al, Science, 289:739-745 [2000]) initial coordinates of the transmembrane regions of the Di, D2, and D receptors were generated using the Homology Module in the Insightll package. (A.M. van Rhee and K.A. Jacobson, Drug Dev. Res., 37:1-38 [1996]). Since there is no insertion or deletion in the sequence alignment for the TM regions, the coordinates of the backbone atoms in rhodopsin were copied as the coordinates of the backbone atoms for aligned residues in dopamine receptors. If the aligned residues between rhodopsin and the dopamine receptor are identical, the coordinates of the side chain atoms in the rhodopsin structure were copied as the coordinates of the side chain atoms of the corresponding dopamine residues. In cases where the aligned residues are not the same, the residue in rhodopsin was mutated to the corresponding dopamine residue type. The sidechain rotamer library within the homology modeling module was used to model the most likely amino acid sidechain orientation.
Table 3 shows the sequence alignment of the transmembrane (TM) regions (TM1- TM7, plus a short TM8) between rhodopsin (RHOA) and Db D2 and D3 receptors. Bold letters indicate the conserved amino acids within the 493 members of the rhodopsin family of GPCR proteins that are identical between rhodopsin and all three dopamine receptors at the same time. In preferred embodiments, these conserved amino acids combined with further sequence identities between rhodopsin and the dopamine receptors provide an unambiguous sequence alignment for each transmembrane helix. Underlined letters in Table 3 are additional identical, or similar amino acid residues among rhodopsin, Di, D2, and D receptors.
Table 3
The 7 TM helices (TM1-TM7) form a large crevice at the extracellular side of the TM region including the ligand binding pocket in Di, D2, and D3 receptors. In preferred embodiments, several extracellular loops (E-I, E-II, E-III) were included in the models because they are close to the ligand binding pocket and may have some affects on the structure of the ligand binding pocket. The short intracellular loop C-I was also included because its sidechains interacts with the short helical segment connected to the intracellular end of helix 7 in the crystal structure of rhodopsin. Other loops (C-II, C-III) and the N- and C-terminal ends are remote from the ligand binding site. Thus, they are not contemplated to affect ligand binding (D.H. van Leeuwen et al, Mol. Pharmacol., 48:344-351 [1995]; and H. Heller et al, J. Phys. Chem., 97:8343-8360 [1993]) and were thus not included in preferred models. Residues before and after omitted loops were capped with acetyl and N- methyl groups. The extracellular loop regions (E-I, E-II, and E-III) and one intracellular loop C-I were modeled based on sequence homology between rhodopsin and dopamine receptors. For several of these loop regions, fairly high sequence homology was found between rhodopsin, Di, D2, and D3 receptors. Between rhodopsin and D receptors, the amino acid identities are 33% for E-I, 25% for E-III, 33% for C-I loops, respectively. There is a conserved disulfide bond formed between a conserved cysteine at the end of the TM3 (Table 3), Cys96 in D1 } Cys 107 in D2, and Cys 103 in D3) and a cysteine residue (Cys 186 in Di, Cysl82 in D2, and Cysl81 in D3) in the very short E-II loop. This disulfide bond is contemplated to limit the conformational flexibility of this short loop in dopamine receptors. Therefore, in preferred embodiments the loop structure was generated using modeling tools of the INSIGHTII program package (INSIGHT II, Molecular Simulations Inc., San Diego, CA) to satisfy the spatial requirements for the formation of the disulfide bond between the cysteine residue at the end of TM3 and the cysteine in the E-II loop. Although an understanding of the mechanism is not necessary to practice the present invention and the present invention is not so limited, it is contemplates that two conserved proline kinks found in TM5 and TM6, are important for ligand binding due to their influence on ligand binding site structure. In addition, another conserved proline kink is found in the intracellular half of TM7, which is believed to be essential for receptor functions. (See, H. Heller et al, J. Phys. Chem., 97:8343-8360 [1993]). The absolute conservation of these three important prolines allows the accurate modeling of the proline kinks in TM5, TM6 and TM7 in the modeled structures of D1} D2, and D3. Two other prolines in rhodopsin, one found in TM1 (Pro53) and the second proline found in the extracellular side of the TM7 (Pro291), are replaced by other residues in Di, D2, and D3. Again, although an understanding of the mechanism is not necessary to practice the present invention and the present invention is not so limited, it is contemplated that TM1 is too remote from ligand binding site to not be important for ligand binding. Pro291 found in the extracellular side of the TM7 is close to the extracellular end of this helix, and thus has minimal effect on the ligand binding site conformation. A normal hydrogen bonding pattern of the α -helix at these two positions, was obtained by applying weak NOE restraints between the hydrogen bonding backbone atoms during the initial MD simulation. The accurate modeling of the most important helices in Di, D2, and D3 using the X-ray structure of rhodopsin provides the present invention with high quality structures of the ligand binding sites for Di, D2, and D3.
The lipid membrane/water environment has been shown to significantly affect the 3D structures of TM proteins. (See, R.G. Efremov et al, Biophys. J., 76:2460-2471 [1999]). Prior to the present invention, dopamine receptor modeling efforts avoided modeling the lipid membrane/water environment due to limited computer power. In the present invention, the explicit lipid membrane/water environment was included in additional structural modeling experiments to further refine the models accuracy. In preferred embodiments, the choice of lipid molecule for modeling the membrane is l-palmitoyl-2- oleoyl-sn-glycero-3-phosphatidylcholine (POPC). A bilayer model consisting of 200 POPC molecules was used in modeling experiments as specified in W.L. Jorgensen et al, J. Chem. Phys., 79:926-935 (1983). In a preferred embodiment, the dopamine receptor structure (e.g., D3) was embedded into the bilayer model according to the position of the predicted membrane boundaries obtained from sequence analysis of 493 GPCR proteins. (See, J.M. Baldwin et al, J. Mol. Biol., 272:144-164 [1997]). First, a number of lipid molecules located at the center of the lipid bilayer were removed to create a hole with a diameter approximately same as that of the receptor structures. Next, the size of the protein structure was scaled down by 50% before placing it into the center of the hole. The reduced scale protein structure was then gradually scaled back to its original size in 5% incremental steps through a MD simulation. During the MD simulation, the lipid molecules were allowed to adjust themselves to accommodate the protein structure while the protein structure was kept rigid, it is contemplated that this procedure avoids the creation of large voids between the protein structure and the lipid, and the sudden disruption of the structures of both the protein and the bilayer molecules caused by severe van der Walls repulsion that would occur if the protein was simply placed into the bilayer hole without this process. Intracellular and extracellular regions of the receptor structures are then exposed to water. In preferred embodiments, the water environment in these experiments is accurately modeled by using the TIP3P explicit water model as described in B.R. Brooks et al. (B.R. Brooks et al, J. Comp. Chem., 4:187-217 [1983]). Approximately 2,000-2,200 water molecules were used to cover the water exposed protein residues and the charged lipid head groups with a minimal 20 A water layer. The size of the total systems is approximately 16,000 atoms in total.
In some other embodiments, extensive structural refinements on the modeled structures are performed by including the explicit lipid and water molecules. A parallel version of the CHARMM program (version 2.7) (A.D. MacKerell Jr., et al, J. Phys. Chem. B., 102:3586-3616 [1998]) and its latest CHARMM force field (W.D. Cornell et al, J. Am. Chem. Soc, 117:5179-5197 [1995]) are employed for all the energy minimization and MD simulations. The present invention further contemplates running parallel simulations using the latest version of the AMBER force field and program to confirm that the conformational clusters obtained from the MD simulations are not influenced by the CHARMM force field used. AMBER and CHARMM are two most popular MD simulation programs. The force fields for the AMBER and CHARMM programs have been extensively tested for protein simulations.
The present invention contemplates running these models and simulations on a 512 CPU Cray T3E supercomputer (e.g., at the Pittsburgh Supercomputer Center), the 32 CPU Origin 2000 computer system at the National Institutes of Health (NTH), and or the Linux 48-CPU cluster at the University of Michigan Cancer Research Center.
In still other embodiments, pre-equilibration of the dopamine receptor structures is achieved via energy minimization and molecular dynamics simulations. In some of these embodiments, the Adopted-Basis Newton Raphson (ABNR) method is used for energy minimization. Simulations were performed using all atom representations in the latest version of the CHARMM force field (W.D. Cornell et al, J. Am. Chem. Soc, 117:5179- 5197 [1995]), except for the hydrocarbon tails of the POPC lipid molecules, to the united CHARMM atom model was applied. In preferred embodiments, the Leapfrog Verlet fl/gorithm was used. In still other embodiments, in order to reduce artifacts caused by a layer of lipid molecules at the edges lacking their 'outer' neighbors, the stochastic boundary method was applied if their hydrocarbon tail atoms were further than 35 A away from the origin; the friction coefficient was set to 200. The phosphorus atoms in the lipid headgroups were fixed. The dielectric constant was set to 1, and the time step to 1 fs. The temperature was kept constant at 300 K with coupling decay time of 1.0 ps. Long range electrostatic forces were treated with the force switch method in the range of 12 to 14 A; Van der Waals forces were cut at 14 A. The nonbond list was generated up to 15 A and updated heuristically. The frequency of checking atoms entering the Langevin region was set to 20 steps. Before the final production MD run, the system was pre-equilibrated in three steps of nested energy minimization cycles and extensive MD simulation runs. To extensively refine the dopamine structures and to sample the binding site conformations, lengthy production MD simulations (10 ns or longer) are performed. Trajectory files of final production runs are saved every ps for analysis. The binding site of the dopamine receptors has certain flexibility, especially with respect to the helix bends of TM5 and TM6 and the side chain conformations of the residues that form the binding pocket. Furthermore, different ligands may preferentially bind to distinctively different conformations of the binding crevice. For these reasons, some embodiments of the present invention use more lengthy (e.g., 5, 10, 50, 100 ns, or more) MD simulations for each receptor (e.g., D1} D2, D3, D4, and/or D5) to sample the conformations of the binding crevice. Since many conformations have been generated, use of conformational cluster analysis is necessary to identify the major conformations obtained in the simulations. Conformational cluster analysis was performed using the CHARMM scripts using two criteria. The first criterion was the helical bend angles of TM5 and TM6. The second criterion was the dihedral angles of those residues that form the binding pocket. Based on 2000 receptor conformations saved during 2.0 ns MD simulation of the D3 receptor four major conformational clusters were obtained with distributions 30% 16%, 13% and 10%. Since the first cluster is nearly twice as more populated than the next largest one, the center conformation of this cluster may be considered a favored and good representative structure for the D3 receptor in its native environment. For the purpose of structure based database searching this representative D3 conformation was considered as receptor structure.
III. Computational docking studies of the interactions between D3 ligands and the receptors
The receptor structures used in the docking studies of the present invention were obtained from lengthy MD simulations of the 3D structures of Di, D2, and D . Cluster analysis was performed to identify the major (most populated) conformational clusters. In preferred embodiments, for each dopamine receptor subtype (e.g., D , D2, and D3), each of the major conformational clusters are used in subsequent docking studies. Although an understanding of the mechanism is not necessary to practice the present invention and the present invention is not so limited, it is contemplated that one advantage in using more than one structure for each receptor is that the conformational flexibility of the receptor is better taken into account, in addition to taking into account ligand conformational flexibility. In preferred embodiments, the receptors are treated using the united-atom approximation in the AMBER force field. (See, Available Chemicals Directory [ACD]: Database of chemicals and their suppliers, MDL Information Systems, Inc., Morristown, NJ). Polar hydrogen atoms were added to the receptors, and Kollman united-atom partial charges were assigned. In some embodiments, all water molecules were removed, and atomic solvation parameters and fragmental volumes were assigned to the receptor atoms using AddSoll feature in the AUTODOCK program.
In preferred embodiments, all identified lead compound D3 ligands and their selective analogs are studied using the modeling and simulation methods of the present invention. In further preferred embodiments, all of the designed lead compound analogs are subjected using extensive docking simulations and modeling. In still further preferred embodiments, all possible chiral configurations are considered for each ligand. In some embodiments, the initial ligand 3D structures are built using the Sybyl (Tripos Associates, Inc., St. Louis, MO) molecular modeling package. In yet further embodiments, the AUTODOCK (version 3.0) program is used for conducting docking studies. (See e.g., G.M. Morris et al, J. Computer- Aided Molecular Design, 10:293-304 [1996]; G.M. Morris et al, J. Comp. Chem., 19:1639-1662 [1998]; D.S. Goodsell et al, J. Mol. Recognition, 9:1-5 [1996]; and G.M. Morris et al, J. Comp. Chem., 19: 1639-1662 [1998]). The AUTODOCK program has been extensively used to predict accurate protein-inhibitor, peptide-antibody, and protein-protein complexes. The AUTODOCK program takes the ligand's conformational flexibility into account during docking simulations. Among the 3 searching methods available in the AUTODOCK program, it was found that the Lamarckian genetic algorithm (LGA) method is highly efficient for prediction of the correct ligand binding models. Accordingly, in some of embodiments, the Lamarckian genetic algorithm (LGA) method in the AUTODOCK program is further used in the docking studies of the present invention. (D.S. Goodsell et al, J. Mol. Recognition, 9:1-5 [1996]). The most recent version of the AUTODOCK program also incorporates a new empirical binding free energy function. In some embodiments, the rotatable bonds in the ligand were defined using an AUTODOCK utility, AUTOTORS. In preferred docking simulations, all rotatable bonds of the ligands were not restricted. The position and the orientation of the ligands were also not restricted. The center of the grid maps was defined using the coordinate of the sulfur atom of the Cysl 18 in D2, or Cysl 14 in D3, or of the oxygen atom of Serl07 in Di, which was determined to be part of the binding site. For each docking study, 10 docking simulations were normally performed. The step sizes used in docking simulations were 0.2 A for translations and 5° for orientations and torsions. I n the analysis of the docked structures, the clustering tolerance for the root-mean-square deviation for the ligand was set as 1.0 A. In additional embodiments, the docking module within the CERTUS (Accelrys, Inc.,
San Diego, CA) program is used for performing docking simulations. The present invention contemplates using 2 (or more) different docking simulation programs/routines provides cross-validation and improved accuracy. Accordingly, preferred embodiments of the present invention use 2 (or more) docking simulation programs and/or routines. One limitation with current computational docking methods, including the
AUTODOCK program we used in this project, is that little conformational flexibility of the protein is taken into account during docking, due to limitations in computing power. To overcome this limitation, the present invention contemplates using multiple protein (e.g., dopaime receptors and/or other g-protein coupled receptors) conformations obtained from MD simulations for computational docking studies. The present invention further contemplates that the receptors (e.g., dopamine receptors Di, D2, and/or D3) may assume multiple low energy conformations with respect to their binding sites, thus, different ligands may bind to different low energy conformations of the receptors.
It was found that using multiple conformations of a particular protein (e.g., a dopamine receptor) significantly improves the accuracy of computational docking studies. Accordingly, some preferred embodiments of the present invention use multiple conformation for a target protein in the design of selective ligands (e.g., D3 selective ligands).
Other preferred embodiments of the present invention overcome the potential problems of conformational changes in a receptor (induced fit) that can occur when binding its ligand by using extensive MD simulations for the predicted the complex structures obtained to compensate ligand design for optimal performance (e.g., selectivity).
In one embodiment, the present invention provides docking studies of 7-OH-DPAT and its analogs to the D3 receptor structure. (+)-7-OH-DPAT is a partial agonist that has shown some selectivity for dopamine receptor subtype D3 over subtype D2. To investigate the structural basis of (+)-7-OH-DPAT binding to the D3 receptor, extensive computational docking studies were performed using the AUTODOCK program package. (G.M. Morris et al, J. Computer-Aided Molecular Design, 10:293-304 [1996]; G.M. Morris et al, J. Comp. Chem., 19:1639-1662 [1998]; D.S. Goodsell et al, J. Mol. Recognition, 9:1-5 [1996] and G.M. Morris et al, J. Comp. Chem., 19:1639-1662 [1998]). The most populated D3 receptor conformation obtained from the cluster analysis was used as the receptor structure in the docking studies. Out of 10 AUTODOCK simulations performed, the top 4 lowest energy complexes converged to a single binding model for the ligand, as shown in Fig. 3. Since there is rich experimental information for the (+)-7-OH-DPAT (Fig. 4) ligand obtained through mutagenesis analysis and extensive structure-activity relationship (SAR) information, the predicted binding model for the (+)-7-OH-DPAT ligand to D3 was readily validated.
The predicted receptor-ligand interactions between (+)-7-OH-DPAT and the D3 receptor are supported by experimental evidence, including, but not limited to, the following. First, the D and D3 receptors bind protonated amine containing ligands with high affinity, although not limited to any particular mechanism, this binding is believed to occur through the formation of a salt bridge with an Asp at the extracellular end of TM3 (common in other members of the rhodopsin family of GPCRs as well). Mutation of this aspartate in the D2 (Dl 14) receptor to either Gly or Asn abolishes the binding of both agonists and antagonists. (See, A. Mansour et al, Eur. J. Pharmacol., 227:205-214 [1992]). Second, the hydroxyl group of (+)-7-OH-DPAT is predicted to form hydrogen bonds with SI 92 and SI 96. Indeed, an S192A mutant D3 receptor binds the (+)-7-OH-DPAT ligand with 16-fold reduced affinity compared to the wild type receptor. (See, N. Sartania and P.G. Strange, J. Neurochem., 72:2621-2624 [1999]). The corresponding residue in D2, S193, was also shown to contribute to the binding of this ligand at the D2 receptor. (See, C Coley et al, J. Neurochem., 74:358-366 [2000]). Third, Cysl 14 is in close contact with one of the propyl groups of the (+)-7-OH-DPAT ligand, thus forming a "propyl" pocket. Indeed, a Cysl 14Ser mutation selectively decreases the affinity of propyl containing (+)-7-OH-DPAT ligands out of a set of 14 compounds tested. (See, G.L. Alberts et al, Br. J. Pharmacol.,
125:705-710 [1998]; and G.L. Alberts et al, Mol. Pharmacol., 54:379-388 [1998]). Fourth, one of the propyl groups of (+)-7-OH-DPAT binds in a highly hydrophobic pocket, while the other is in a mixed hydrophobic/hydrophilic environment. This model based prediction is consistent with the observed 10-fold reduction of the binding affinity if both propyl groups are removed, while deleting only one propyl does not appear to change the ligand's binding potency. (See, D.A. Norman and L.H. Naylor, Biochem. Soc. Trans., 22:143S [1994]). For the 5-OH-DPAT (Fig. 4) analog of (+)-7-OH-DPAT, the change upon removing both propyl groups is even more dramatic. Removing one of the propyl groups has no effect on the ligand binding at D3, but removing both propyl groups decreases the affinity of the ligand by 635-fold. (See, D.A. Norman and L.H. Naylor, infra.). It is of note, that one D3 hydrophobic pocket is fairly large such that much larger groups can be tolerated, while the other pocket is more limited in size and has a mixed hydrophobic/hydrophilic environment. Taken together, an excellent agreement was found between the docking results predicted by the methods of ligand design disclosed in the present invention and existing experimental mutational data and SAR data for (+)-7-OH- DPAT ligand and D3 receptor binding.
This excellent agreement serves as a validation of the modeled D3 structure contemplated by the present invention and it also provides insights into the structural basis of the binding of D3 to potential potent D ligands.
IV. General design strategy for identifying novel lead compounds
Preferred embodiments of the present invention used a novel structural based approach for identifying potential lead compounds as selective ligands of dopamine receptors. In particularly preferred embodiments, these novel structural based design approaches are used to identify and to further design (e.g., optimize) potent and selective modulators (e.g., partial agonists) of dopamine receptor D3. In still other preferred embodiments, potent and selective modulators of dopamine receptor D3 are useful therapeutic compounds in the treatment of conditions characterized by the faulty regulation of D3 receptors in a subject (e.g., depression, anxiety, schizophrenia, Tourette's syndrome, eating disorders, alcoholism, chronic pain, obsessive compulsive disorders, and Parkinson's Disease, and the like).
The present invention further provides a novel and powerful approach, as shown in Fig. 5, for identifying and designing (e.g., optimizing) lead G-protein receptor binding ligands (e.g., dopamine receptors, including, but not limited to, Di, D2, D3, D4, and D5). In a preferred embodiment, a first steps provides identification of potential lead compounds
(e.g., D3 agonists and/or antagonists) using a ligand pharmacophore-based 3D-database search technique to search a library of 500,000 highly structurally diverse synthetic and natural organic compounds to identify initial candidate receptor binding ligands ("hits"). For example, in one embodiment, a pharmacophore model was developed based upon the 3D structures of known D3 ligands and their structure-activity relationships (SAR). The pharmacophore model that was used to search the National Cancer Institute's (NCI) 3D- structure database and/or to search the Available Chemicals Directory (ACD) (See, S. Wang et al, J. Med. Chem., 37:4479-4489 [1994]) database. The current version of the NCI 3D "open" database consists of 245,000 structures, whose structures and chemical samples can be accessed by researchers. (See, J.H. Voigt et al, J. Chem. Inf. Comput. Sci., 41:702-712 [2001]). The latest version of the ACD database (v99.2) contains 275,000 modeled 3D structures. (Available Chemicals Directory, MDL Information Systems, Inc., Morristown, NJ)
In one embodiment, the present invention provides a Web-based 3D-pharmacophore docking program package. It was found that the Web-based 3D-pharmacophore docking program of the present invention provides similar results as the Chem-X program. (See, I.D. Kuntz, Science, 257:1078-1082 [1992]). In some embodiments, one or both of the NCI and ACD databases, converted into a 3D-database format, are searched by the present Web- based program as part of identifying potential receptor binding lead compounds.
In still other preferred embodiments, a second structure-based screening step provides a subsequent tool to further define the interactions of the hit compounds (e.g., dopamine receptor ligands), identified in the first step, with the receptor of interest (e.g., D3 receptor).
In preferred embodiments, the second structure-based screening step, uses a "smaller" database containing only the "hits" identified through the 3D-pharmacophore searching of first step. The present invention is not intended to be limited, however, to selecting potential target receptor ligands based on this methodology. In still other preferred embodiments, the Ligand Fit module of the program CERIUS2 version 4.5
(Accelrys, Inc., San Diego, CA) is used to perform structure-based database searching. In some embodiments, structure-based database searching is used dock each of the hit compounds grouped into the a database into the binding pocket of the receptor of interest (e.g., D receptor binding-pocket). In yet some other embodiments, the present invention contemplates ranking the hit compounds according to their scores as calculated by the
CERIUS program. In some embodiments of the present invention, the scoring function used in the CERIUS2 program calculates the negative of the potential interaction energy between ligand and receptor. The present invention contemplates, that in limited cases the energy based DOCK program scoring function may not provide a good correlation with the binding affinity of a particular candidate ligand to its target receptor. Accordingly, preferred embodiments of the present invention contemplate overcoming this potential issue by using a novel consensus scoring function developed as part of the present invention. In preferred embodiments, the consensus scoring function consists of three different empirical binding affinity prediction methods, CHEMSCORE, LBSCORE, and LUDISCORE, (See, J. Compu -Aided Mol. Des., 11 :425-445 [1997]; J. Mol. Model, 4, 379-394 [1998]; and J. Comput. -Aided Mol. Des., 12, 309-323 [1998]) and is obtained using more than 200 protein- ligand complexes. It is further contemplated, that in some embodiments use of the consensus scoring function improves the "hits" rate. The present invention contemplates, that in limited cases the energy based CERIUS program scoring function may not provide a good correlation with the binding affinity of a particular candidate ligand to its target receptor. Accordingly, preferred embodiments of the present invention contemplate testing the docking and scoring protocol used for structure based database searching as follows. A set of 25 known D3 ligands were 'mixed' with database compounds. After docking these compounds into the representative D receptor conformation and scoring based on their Cerius2 dock score, it is found that 24 out of the 25 known D3 ligands are among the top 25% of all compounds in a rank order from best to worst scores. Thus, the ranking and docking protocol applied is able to select ligands with good D3 binding affinities among top ranking compounds. Therefore, compounds selected for further biological analyses (e.g., in vivo and/or in vitro assays) have comparable scores to the known D3 ligands, i.e., they are selected from the top 25% of the ranked database compounds.
It is contemplated that this two-stage computerized screening approach enables the present invention to identify most promising potential ligands based hot only upon their 3D- pharmacophor binding elements but also based upon their interactions with 3D structure of the target receptor of interest (D3 receptor).
As shown herein, this novel approach provides a powerful tool for discovering promising dopamine receptor, and other G-protein receptor, modulators (e.g. , agonists and/or antagonists). The following example further describes preferred embodiments of the two step structure-based screening approach to identifying potential selective receptor binding ligands (e.g., dopamine receptor [including, but not limited to: Di; D2; D3; D4; and/or D5] agonists and/or antagonists). In a preferred embodiments of the present invention, a pharmacophore model is defined as common and essential structural elements for activity. Previous studies have identified a number of potent and relatively selective D3 partial agonists and full agonists. In one embodiment, a representative sub-set of 9 relatively selective partial and full agonists (See, Fig. 2) were chosen for the development of the pharmacophore model(s). Structural analysis of these compounds shows that these compounds contain a common aromatic ring and a Sp3 nitrogen attached to a propyl group. Except for pramipexole, the nitrogen is attached to two additional Sp3 carbons. The distance between the aromatic ring center and the basic Sp3 nitrogen within these compounds was established to be between 4.1 and 6.1 A through conformational analysis using the QUANTA program. (QUANTA, Molecular Simulations Inc., San Diego, CA). A simple pharmacophore model, used in the structure- based receptor ligand identification and modification (e.g., selectivity optimization) was developed from these analyses. (See, Fig. 3). It is of note that the chemical elements and the geometrical parameters specified in the pharmacophore model represent several of the important D3 receptor binding elements, however, the pharmacophore model alone does not provide sufficient binding elements for identifying selecting or designing high affinity and selectivity for the D3 receptor.
In one embodiment, the CHEM-X program (Chemical Design Ltd., Oxford England) was used to screen the NCI 3D-database of 245,000 "open" test compounds against the pharmacophore model shown in Figs. 6A and 6B. Fig. 6A shows a superposition of 9 D3 partial agonists and agonists. Fig. 6B shows a simple pharmacophore model derived from contemplated D ligands. During the search process, compounds were selected that had one or more of the chemical groups required by the pharmacophore model, e.g., an aromatic ring, and/or a tertiary nitrogen attached to three carbon atoms. If the chemical structural requirements were met, the CHEM-X program then investigated whether the compound has a conformation that meets the 3D geometric parameters specified in the pharmacophore model. If the CHEM-X program found that the compound indeed had at least one conformation that met the 3D geometric requirements specified in the pharmacophore model, the compound was considered to be "hit." In preferred embodiments, using this structural-based system, up to 3,000,000 conformations can be examined for hit compound. Using this approach, a total of 6,237 compounds from the NCI 3D-database were identified as hits, that satisfied both the chemical and geometrical requirements specified in the pharmacophore model. In preferred embodiments, the structures of compounds identified as being hits through 3D-pharmacophore searching are extracted from the either the NCI or the ACD databases in SDF format. In still other preferred embodiments, for each of structure extracted, hydrogen atoms and Gasteiger-Marsili atomic charges are added using the SPL macro in the SYBYL program. (SYBYL, Tripos Associates, Inc., St. Louis, MO). In additional preferred embodiments, the final coordinates, in mol2 format, for all hit compounds are stored in a single database file. The protonation for each ionizable group such as amine, amidine, and acids, in the hit compounds are assigned according to their protonation state at a physiological pH of 7.4. The 6,237 hit compounds identified from the pharmacophore searching satisfied the basic and necessary chemical and geometrical requirements specified in the pharmacophore model. However, it is expected that many of these 6,237 hit compounds will not show appreciable activity binding to the D3 receptor for or more reasons (e.g., steric hindrance). For example, it is contemplated that a particular ligand (hit compound) may have the essential binding elements specified in the pharmacophore model, but be unable to bind to the receptor of interest (e.g., D3) because the ligand is too big to fit into the receptor's binding site, or some of the ligand's functional groups may interfere with the receptor's residues, thus preventing the ligand from effectively interacting with the target receptor (e.g., D3 receptor). In additional preferred embodiments, top scoring hit compounds contemplated as being potential ligands for the receptor of interest, are visually examined for their structural novelty and diversity by comparing with known ligands of the receptor of interest (e.g., D3 receptor ligands). In preferred embodiments, hit compounds that pass the visual inspection stage are tested in binding and functional assays. The present invention contemplates using the most populated conformational cluster for a particular target receptor (e.g., D3 receptor) in the docking studies obtained from extensive MD simulations in one or more structure-based databases using the CERIUS2 program. However, in some instances, highly potent ligands of a particular target receptor may bind to a conformation of target receptor that is somewhat (or even significantly) different from the most populated conformational cluster. Therefore some potent ligands may not able to dock into the receptor of interest and have a top docking score. In some embodiments, the present invention contemplates overcoming this potential limitation by using several populated low energy conformations obtained from lengthy MD simulations for structure-based database searching. It is further contemplated that using multiple receptor conformations for structure-based database searching provides for the discovery of additional novel lead compounds that otherwise might be missed.
V. Identification of specific novel lead compounds As described above, the present invention contemplates the discovery and design of novel and potent ligands selective for g-protein receptor, and in particular for dopamine receptors, including, but not limited to, Di, D2, D3, D4, and D5.
In preferred embodiments, accurate modeled structures of the dopamine receptors facilitates the discovery of novel and potent lead compounds as modulators (e.g., partial agonists and/or antagonists) of the D3 receptor. As described above, the present invention provides novel and powerful approaches for identifying and designing G-protein receptor ligands by combining ligand-based pharmacophore and structure-based database searches.
In additional embodiments, analysis of the modeled structures of target receptors (e.g., dopamine receptors) and computational docking studies of known relatively selective D3 ligands provides further elucidation of the structural bases of ligand binding and selectivity at target receptors (e.g., dopamine receptors). Detailed structural analyses of the Di, D2, and D receptors was used to identified four specific regions (Regions A, B, C, and D) that have significant structural differences between D3 and D2 receptors in their binding sites. Other detailed structural analyses were used to identify profound differences between the D3 and Di receptors. In preferred embodiments, as described below, differences in the dopamine receptor structural Regions A, B, C, and D are used for rationally designing highly selective dopamine receptor ligands.
The present invention contemplates that the lead compounds described herein, as well as the additional lead compounds those skilled in the art will identify in view of the modeling and structure-based ligand identification and design methods disclosed herein, may be further optimized and refined to improve one or more characteristics (e.g., receptor affinity, receptor selectivity, etc.) using the disclosed chemical modification methods. Indeed, the structure-based design and chemical modification methods of the present invention, provide derivatives and analogs of the lead compounds with high affinity (Ki = 6 nM, and greater) and impressive selectivity (>100- fold, and greater) for D3 versus Di and D2. The present invention is not limited, however, to providing highly selective ligands for the D3 receptor. Indeed, the present invention provides methods for rationally designing and chemically modifying a number of receptor ligands. The specific examples of lead compound identification, testing, and chemical modifications presented below are intended to provide the reader with non-limiting representative examples of several of various lead compounds (analogs and derivatives) contemplated by the present invention. In one embodiment, the initial binding affinity of a first group of 30 potential dopamine receptor ligand test compounds was performed using cell lines transfected with human dopamine receptors. (See, B. Levant, Current Protocols in Pharmacology [J. Ferkany and S.J. Enna, Eds.], John Wiley & Sons, New York, 1.6.1-1.6.16 [1998]). A potent D2 and D3 ligand [3H]YM-09151-2 were used as a radioligands for the D2 and D3 receptor binding assays. Likewise, a potent and selective D\ receptor ligand, [3H]SCH 23,390, was used as a radioligand for Oi receptor binding assays. The 30 test compounds were first measured for their ability to compete with [3H]YM-09151-2 binding to the D3 receptor using CHO cells transfected with human D3 (hD ) receptors. If sufficient binding was observed for a test compound at 10 μM, then its IC50 value was obtained. For compounds with IC50 values less than 10 μM, their Di binding potency was determined using LHD1 cells transfected with human Di receptor (hDi) and their D2 binding was determined using CHO cells transfected with human D2 (hD2) receptor. Ki values were calculated according to the Cheng-Prusoff equation assuming classical competitive inhibition. The results are summarized in Table 4. Table 4 shows the binding affinity of the 8 best test compounds as measured using cell lines transfected with human dopamine receptors.
Table 4
Out of the 30 test compounds evaluated, 8 compounds (named compounds 1-8, respectively) had Ki values in the nanomolar range (e.g., about 11 nM to about 465 nM). One compound had a Ki value of 11 nM, a quite potent D3 ligand. Test compounds 2, 3, and 4 had Ki values of 43, 63, and 84 nM, respectively. Test Compound 7 had a Ki value of 442 nM and showed 10-fold selectivity between Di and D3 receptors, and more than 23 -fold selectivity between D2 and D3 receptors, respectively. The results show that the methods of the present invention are effective for identifying novel and potent dopamine receptor ligands, and especially for identifying D3 receptor ligands.
Further experiments were performed to characterize novel D3 ligands in functional assays. Many studies have shown that binding affinities of a ligand to dopamine receptors can vary considerably, depending at least in part on the expression system or tissue, the radioligand, and the in vitro assay conditions used. (See e.g., B. Levant, Pharmacol. Rev., 49:231-252 [1997]). In one embodiment, test compounds were screened in functional assays to elucidate their agonist (partial agonist) or antagonist activity on dopamine receptors (e.g., D3 receptors). The 8 fairly potent D ligands shown in Table 4 were tested for their in vitro functional activity on human hD], hD2 and hD3 receptors according to the methods described in Chio et al. and Sautel et al. (See, CL. Chio et al, Mol. Pharmacol., 45:51-60 [1994]; and F. Sautel et al, Neuroreport, 6:329-332 [1995]). The results of these functional assays are summarized in Table 5. Table 5
N.D. Not determined due to low affinity. At least two experiments were performed for each compound. In this embodiment, the functional studies show compounds 1, 2, 3, 6, and 7 are antagonists, and that compounds 4, 5, and 8 are partial agonists of the D3 receptor. (See, Fig. 7). Indeed, tests show that compound 4 is a potent D3 partial agonist with an EC50 of 9.2 nM with maximum stimulation of 51 % as compared to a full D3 agonist quinpirole for stimulation of mitogenesis in transfected CHO-D3 cells. (Fig. 8). Fig. 8 shows the functional activity of compound 4 (CTDP-31793) at Di, D2, and D3 receptors and comparison to standard ligands. The agonist activity at Dj receptor was measured for stimulation of c AMP accumulation in C6Dι cells. The antagonist activity at Di was measured for inhibition of 10 nM dihydrexidine stimulation of c AMP accumulation in C6D1 cells. The agonist activity at D receptor was measured for stimulation of mitogenesis using CHOp-hD2 cells. The antagonist activity at D2 receptor was measured for inhibition of 30 nM quinpirole stimulation of mitogenesis in CHOp-hD cells. The agonist activity at D3 receptor was measured for stimulation mitogenesis using CHOp-hD cells. The antagonist activity at D3 receptor was measured for inhibition of 30 nM quinpirole stimulation of mitogenesis in CHOp-hD3 cells. In contrast to quinpirole, compound 4 functions as an antagonist at the D2 receptor with a Ki value of 35.1 nM.
Furthermore, compound 4 functions as a weak D! antagonist with a Ki value of 657 nM (Fig. 8). Compound 5 has a very similar profile in its functional activity but appears to be more selective than compound 4. However, compound 5 functions as a partial agonist of D3, with an EC50 of 22.2 nM and a 31% of maximum stimulation of mitogenesis as compared to quinpirole, as a D2 antagonist with an IC50 value of 378 nM, and as a weak Di antagonist with an IC50 value of 1722 nM.
Compound 4 has a selectivity of 4-fold between D3/D2 and 70-fold between D3/Df, while compound 5 has a selectivity of 17-fold between D3/D2, and 78-fold between D3/Dι in more meaningful functional experiments in measuring selectivity of the compounds. Similar to compounds 4 and 5, compound 8 functions as a partial agonist of the D3 receptor with an EC50 of 146 nM and 53% of maximum stimulation.
Consequently, the present invention contemplates that functional studies show that compounds 4 and 5 are potent D partial agonists, but that compounds 4 and 5 function as antagonists of Di and D2 receptors. Compounds 4 and 5, and in particular compound 5, display certain selectivity for D versus Di and D2 in functional assays. Compounds 4 and 5 have a functional profile identical to that of BP-897, which has been shown to have great therapeutic potential for the treatment of cocaine addiction. (See e.g., M. Pilla et al, Nature, 400:371-375 [1999]). Therefore, in preferred embodiments, compounds 4, 5, and 8 are promising lead compounds for further optimization to design potent and selective D3 partial agonists.
Still further embodiments provide computational docking studies of lead compound 4 with the D3 receptor. Since compound 4 is a flexible ligand it is beneficial to use a docking program, that considers ligand flexibility in the docking process. For this reason, the AUTODOCK program (See, D.S. Goodsell and A.J. Olson, Proteins: Str. Func Genet., 8:195-202 [1990]; G.M. Morris et al, J. Computer-Aided Molecular Design, 10:293-304 [1996]; G.M. Morris et al, J. Comp. Chem., 19:1639-1662 [1998]; D.S. Goodsell et al, J. Mol. Recognition, 9:1-5 [1996]; and G.M. Morris et al, J. Comp. Chem., 19:1639-1662 [1998]) was used. The most populated conformational cluster of the D3 receptor obtained from a 2ns MD simulation was used as the receptor structure for docking studies. Since compound 4 has a chiral center and the chemical sample used in receptor binding and functional assays was a racemate, both the (R)- and (S)-configurations of compound 4 were used in the docking studies. In certain embodiments, 10 docking simulations were performed for each structure. Docking study results show that the (Reconfiguration of compound 4 interacts more favorably with the D3 receptor than does the (S)-configuration. The present invention contemplates that other racemate lead compounds, analogs, and derivatives are also amenable to docking studies using (R)- and (S)-configurations of compound to determine whether one configuration binds to the target receptor more favorably. Out of the 10 docking simulations, 4 docking simulations with lowest predicted binding free-energy converged to a single binding model. (See, Fig. 9).
Fig. 9 shows that compound 4 forms a strong salt bridge between its protonated nitrogen and Dl 10 in D3, similar to that shown in (R)-(+)-7-OH-DPAT. (See, Fig. 3). The tricyclic ring in compound 4 is in close contact with a number of hydrophobic residues in D3, including VI 11, V189, F346, F197, F345, W342, and Cl 14. Of note, three serine residues, S192, SI 93, and SI 96 of D3 are in close proximity with docked compound 4, thus, in some embodiments the present invention provides, derivatives of compound 4 designed to form additional hydrogen bonds with the D3 receptor. It is contemplated that installing additional donors/acceptors to compound 4 derivatives improves the potency and/or selectivity of the ligand. Similarly, additional embodiments contemplate designing lead compound derivatives with increased potency and/or selectivity for the other dopamine receptor subtypes (e.g., Di, D , D4, and/or D5) based on the somewhat different locations for these three common serines in Di, D2, D4, and D5. Docking studies also show that the 4-F- phenyl group of compound 4 interacts with the hydrophobic residues W370, Y373. Still further docking studies show that the D3 hydrophobic pocket named region A is accessible from the extracellular crevice of the receptor. D3 receptor model structure suggests that fairly large hydrophobic groups may be accommodated in the pocket of Region A. In order to reach region A located deep in the transmembrane region, such atom groups must be attached at the end of a flexible linker. The linker is contemplated to be a hydrocarbon chain substituent of the protonated nitrogen group, which is expected to interact with a common Asp in TM3 (Dl 10 in D3). Our structural analysis shows that the distance between the protonated nitrogen and the center of a bulky group accommodated in Region A might be around 14 A. Still further embodiments of the present invention, take advantage of the significant differences between D3, D2, in the region A pocket for the design of ligands with high potency and/or selectivity for particular dopamine receptor subtypes of interest. In still further embodiments, the present invention provides additional ligand compounds optimized for enhanced potency and/or selectivity when binding to additional receptors types of interest (e.g., G-protein coupled receptors) through rationally directed ligand chemical modifications.
In further embodiments of the present invention, chemical modifications of lead compounds are guided by structure-based design. For example, based upon the binding model shown in Fig. 9, new analogs of compound 4 were designed. In one embodiments, compounds 8, 9, and 10 were designed to investigate the optimal length of linker. (See, Fig. 10). In another embodiment, compounds 11 and 12 were designed to investigate the effects of the terminal phenyl group in compounds 4, 8, 9 and 10. (See, Fig. 10). Based on docking studies, the 4-F-phenyl group of compound 4 binds to a large hydrophobic pocket in the D3 receptor. Further docking studies shows that there is additional room in the hydrophobic pocket of D3 available for accepting larger hydrophobic group such as a biphenyl ring or a naphthyl ring on potential ligand binding partners. As mentioned previously, there is a significant difference between the end of this large hydrophobic pocket in dopamine receptors D , Dj, and D2. Compounds 13 and 14 were thus designed to investigate whether larger hydrophobic groups on the ligands would improve selectivity by probing the differences between dopamine receptors in this large hydrophobic pocket. (See,
Fig. 10).
In one embodiment of the present invention, compounds 4, 8, 9, 10, 11, and 12 were synthesized using the methods shown in shown Scheme I (Fig. 11). Scheme I, reagents: 20,
3-chloro-4'-fluoropriophenone; 21, 4-fluorobutyrophenone; 22, l-(4-flurophenyl)-5-chloro- 1-oxopentane; 23, 2-chloro-4'-fluoroacetophenone; 24, iodopropane; and 25, iodobutane. In particular, condensation of commercially available 2-quinoline-carbox-aldehyde (compound 15) with ethanolamine gave imine compound 16, which was then reduced to amine compound 17 with NaBH4. (See e.g., C.A.R. Baxter and H.C. Richards, J. Med. Chem., 15:351 [1972]; and V.A. Rao et al, J. Med. Chem., 13:516 [1970]). The reduction of compound 17 to the corresponding 1,2,3,4-tetrahydroquinoline (compound 18) was achieved with NaBH3CN in acetic acid. (See, G.W. Gribble and P.W. Heald, Synthesis, 650 [1975]). Cyclization of compound 18 was accomplished with P2O5 in xylene affording the important intermediate compound 19 (2,3,4,4a,5,6-hexahydro-lH-pyrazino[l,2-a]quinoline) (See, C.A.R. Baxter and H.C. Richards, J. Med. Chem., 15:351 [1972]). The intermediate, compound 19, was alkylated with the commercially available compounds 20, 21, 22, 24, or 25, respectively, in acetonitrile using cesium carbonate as the base, to yield compounds 8, 4, and 20, 11, and 12, respectively. (See, L.A. van Vliet et al, J. Med. Chem., 43:2871 [2000]). For compound 9, the intermediate compound 19 was first converted to its sodium salt with sodium hydride in DMF, and then reacted with commercially available compound 23 in DMF at room temperature to afford the final product compound 9.
The synthesis of compounds 13 and 14 is provided in Scheme II (Fig. 12). In Scheme II: i. 1.2 eq.; 26, 2 eq. Cs2CO3, CH3CN, under N2, reflux, overnight, yield 75-88%; ii. 2 eq. hydrazine, EtOH, reflux 2 hr, yield 82-87%. For compounds 13 and 14: iii. 1.2 eq. 2-naphthoyl chloride, 3 eq. triethylamines, 0°C, 4 hr, yield 83-87%. In particular, the reaction of the intermediate compound 19 (Scheme I) with phthalimido protected 1- bromoalkylamines, compound 26, generated compound 27. Deprotection of compound 26 with hydrazine yielded compound 28. Amidation of compound 28 using 2-naphthoyl chloride as amide agents yields target compounds 13 and 14, respectively. (See, J.M. Robarge et al, J. Med. Chem., 44:3175-3186 [2001]). Further lead compound modifications and design of additional analogs is described below.
In still other embodiments, rat brain binding assays are used to determine the binding affinities of the new analogues of lead compound 4. (See, B. Levant, Current Protocols in Pharmacology [J. Ferkany and S.J. Enna, Eds.], John Wiley & Sons, New York, 1.6.1-1.6.16 [1998]). The results of analogs tested in rat brain binding affinity assays are summarized in Table 6.
The data are average of 3-5 replicates.
From the results Table 6, it is clear that compound 4 with a 3 -carbon linker is the most potent compound at the D receptor among compounds 4, 8, 9, and 10, respectively, thus in this embodiment a 3-carbon linker is optimal for compound 4 analog binding to dopamine receptor D3. Compounds 9 and 8 with either 1- or 2-carbon linker are substantially less potent than compound 4 with a 3-carbon linker, and compound 10 with a 4-carbon linker is about 2-fold less potent than compound 4 at binding the D3 receptor. Of note, the binding affinities obtained using the rat striatum binding assay for both compounds 4 and 8 are larger than the values obtained using the CHO transfected cells shown above. However, in both assays, compound 4 is more potent than compound 8 (e.g., about 7-12 fold more potent at D2, and 6-8 fold more potent at D3) indicating that the binding affinities obtained from these two different assays are consistent in terms of relative potencies between analogues, although absolute values may differ. This is consistent with previous observations that binding affinities of a ligand to a particular dopamine receptor depends upon the particular expression system or tissue, radioligand, or in vitro assay conditions used. (See, B. Levant, Pharmacol. Rev., 49:231-252 [1997]). Compounds 11 and 12 were designed to investigate the importance of the interactions of the terminal 4-F-phenylkentone group with D3 for binding affinity. As can be seen in Table 6, compound 11 is as potent as compound 4, and compound 12 is 3-times more potent than compound 4 at binding the D3 receptor. This finding is consistent with the predicted binding model for compound 4 (See, Fig. 9) that the 4-F-phenyl group is in close contact with a number of hydrophilic/charged residues in D3 and may have some unfavorable interactions with the receptor. Furthermore, compound 12 has a 9-fold selectivity for receptor D3 versus receptor D2 and is 11 -fold more selective than lead compound 4 for receptor D versus receptor D2.
In a preferred embodiments, the present invention provides compound 29 (CTDP- 31819) as a partial agonist of receptor D3. Compound 29 (Fig. 13) was found to have potent affinity at D3 and to have a selectivity of 39-fold between receptors D3 and D2, and 10-fold selectivity between receptors D3 and Di in binding assays using rat brain. (See, Table 7). Table 7 shows the binding affinities of lead compound 29 and several new analogues thereof in rat brain binding assays.
Functional assays using transfected cells further show that compound 29 is a potent D3 partial agonist, and a weaker Di and D2 antagonist. (See, Fig. 14). At the D3 receptor, compound 29 has an EC50 value of 7.1 nM with a maximum stimulation of 45% as compared to a full agonist quinpirole. At the Di and D2 receptors, compound 29 has IC50 values of 384 nM and 355 nM, respectively. Therefore, the present invention contemplates that the selectivity of compound 29 shown in the above functional assays is 50-fold between receptors D3 and D2, and 54-fold between receptors D3 and Di. Thus, the present invention shows that compound 29, even before optimization, provides a highly selective D3 ligand.
Since compound 29 is a potent D3 partial agonist and displays a good selectivity for D3 versus Di and D2, chemical modifications were performed to further improve the compound's potency and selectivity. Computational docking studies showed that compound 29 has a similar binding model as lead compound 4. (See, Fig. 9). The present invention contemplates a number of compound 29 analogs and chemical derivatives (See, Fig. 13) using the methods shown in Scheme III (Fig. 15) and Scheme IV (Fig. 16) according to the methods described in Robarge et al. and U.S. 3,931,188. (See, J.M. Robarge et al, J. Med. Chem., 44:3175-3186 [2001]; U.S. 3,931,188, incorporated herein in its entirety). Scheme III, reagents and conditions: i. 1.2 eq. MeMgBr, EtOH, 0°C then reflux for 0.5 hr; ii. P-toluenesulofonic acid, toluene, reflux, -H2O, 4 hr, yield 72-82% in two steps; iii. CH2O, RNH3C1, AcOH, H2O, 6 hr, yield 18-28%. Scheme IV, reagents and conditions: i. 36, (BOC)2O=7:l, dioxane, it, overnight,; ii. 1.2 eq. trifluoroacetic anhydride, Et3N, CH2C12, it, overnight, yield 88% in two steps; iii. AcCl, MeOH, it, quantative; iv. 35, CH2O, AcOH, H2O, 6 hr, yield 38%; v. a) K2CO3, MeOH, rt, overnight; b) 1.2 eq. 2- naphthoyl chloride, Et3N, CH2C12, 0°C, rt, 1 hr, yield 83% in two steps. The binding affinities of the compound 29 analogs were tested in the rat brain assay, the results are shown above in Table 7. Compound 30, with a butyl group, has a potent activity at receptor D3 (K, = 25 nM) but, only moderate D3 receptor selectivity. Compound 31, with an additional methoxyl group in the meta -position of the phenyl ring, is a potent D3 ligand (K, value = 46 nM) and a good selectivity for receptor D3. The selectivity for compound 31 is 15-fold between receptors D3 and D2, and 175-fold between receptors D and Di. The binding model for compound 14 shows that its naphthenyl ring reaches a portion of the D binding region that has significant differences between the D and D2 receptors. Accordingly, in one embodiment of the present invention, compound 32, with a naphthenyl ring, was contemplated designed and synthesized. Compound 32 is a very potent D3 ligand (K,= 6.1 nM) and has an impressive selectivity of 133- and 163-fold between D3 and D2 receptors, and between D and Di receptors. Thus, in preferred embodiments, even before optimization, compound 32 provides a highly selective D3 ligand.
VI. Further structure-based design and chemical modifications of lead compounds
In preferred embodiments, chemical modifications of the lead compounds are guided by structure-based design strategies to yielded new analogs with improved binding affinity and selectivity for a target receptor (e.g., D3) receptor versus other D family receptors (e g., affinity and selectivity for D3 receptor versus the Di and D2 receptors). In some embodiments of the present invention, some of the lead compounds, and analogues/derivatives thereof, have specific binding properties to Di or D2 or other member of the D family of receptors. The chemical modifications contemplated in preferred embodiments of the present invention are based, in part, on consideration of four different aspects: 1) analysis of the important structural differences among Dls D2, and D3 receptors ,and especially between D2 and D3 receptors, that are contemplated to contribute to ligand selectivity; 2) discovery of novel and potent D3 partial agonists as highly promising lead compounds; 3) effective optimization of lead compounds designed to improve the potency and the selectivity of candidate compounds at the D3 receptor; and 4) characterization of novel ligands for their potency, selectivity and functional activity.
Some embodiments of the present invention, for example, preformed extensive docking studies on lead compounds 4 and 29 and new more potent/ selective analogues of (compounds 14 and 32) thereof. Since compounds 14 and 32 are potent and selective D3 ligands (Fig. 17), these two compounds are used as new lead compounds for further design and optimization. The binding models for compounds 14 and 32 predicted using the CERTUS2 docking program is shown in Figs. 19A and 19B.
Many of the D3 receptor residues that interact with compounds 14 and 32 are structurally identical or very similar in the D3 and D2 receptors, such as, Dl 10, Cl 14, F106, V86, H349 and V350. Therefore, it is contemplated that the interactions between ligands and these common D3 and D2 residues contributes to ligand affinity but not to selectivity. However, as mentioned above, structural analyses of the dopamine receptor structures shows that there are considerable structural differences between the D3 and D2 receptors, mainly located in four regions. (See, Table 2 and Figs. 18A and 18B). Fig. 18A and 18B show the predicted binding models for compounds 14 (Fig. 18A) and 32 (Fig. 18B) with the D3 receptor. Residues within 5 A distance from the ligand are depicted and some specific interactions between the ligands and D3 receptor are shown in dotted lines. Four regions (Regions A, B, C, and D) that contribute to ligand selectivity, especially between D3 and D2 are shown in orange circles. Therefore, preferred embodiments of the present invention provide, new analogues with binding groups (elements) that specifically target these four regions are contemplated to provide greater selectivity. One particular embodiment of the present invention provides, four new groups (Groups I, II, II, and IV) of analogues designed based upon the binding models of compounds 14 and 32 and the D3 receptor. (See, Fig. 19). Fig. 19 shows a schematic representation of compound 14 in the D3 receptor's binding site (Notation: D3 residue number/helix number; EII is extracellular loop 2; black ellipses: residues primarily responsible for potency; red ellipses: residues predicted to affect ligand selectivity most significantly; blue ellipses: residues predicted to affect the selectivity, but less than those residues shown in red ellipses; dotted lines show predicted receptor-ligand interactions). In one embodiment, the present invention provides analogues (Group I analogues) of compounds 14 and 32 with modifications in the tricyclic ring to better bind to dopamine receptor Region C In this region, among the dopamine receptors, there are 3 common serine residues. However, due to the different kink angles in TM5 the position of these three serine residues in D3 (S 192, S 193 , and S 196) are shifted as compared to that in dopamine receptors D2 and Di. In preferred embodiments, compounds are designed to explore the orientation and positional differences of the 3 common serine residues. For example, the selectivity of compound 31, with one additional methoxyl group, is improved as compared to that of compound 30. (Table 7). While the selectivity of compound 30 between receptors D3 and Di is 63-fold, the selectivity of compound 31 between these receptors is 175-fold. Accordingly, in preferred embodiments of the present invention, lead compound analogues with hydrogen bonding donor/acceptor groups as substituent on the phenyl ring in different positions are provided to explore the structural differences between the 3 common dopamine receptor serine residues in Region C. (See, Fig. 19). Synthesis of new analogues in Group I (a). A general scheme for synthesis of the compounds shown in Group I (a) is illustrated in Scheme V. (See, Fig. 20). The steps of Scheme V are similar to the synthetic methods shown in Scheme I, the 2-substituted aminomethylquinoline, compound 42, was prepared from the starting material, compound 40, by bromination with NBS to yield 2-bromomethylquinoline, compound 41, followed by reaction with ethanolamine in ethanol. Reduction of compound 42 with NaBH3CN in acetic acid yields 2-substituted aminomethyl-l,2,3,4-tetrahydroquinoline (compound 43). The important intermediate (compound 44) was synthesized by cyclization of compound 43 with P2O5 in xylene. Finally, compound 44 was alkylated using the synthetic methods outlined in Scheme II to yield the desired derivative, compound 45. Most of the starting materials used in Scheme V are commercially available. In cases whether the starting materials are not available, they can be readily synthesized. The methods for preparing the starting materials for many analogs are shown in Scheme VI. (Fig. 21). Generally, the substituted quinaldines can be prepared directly by condensation of the corresponding substituted aniline with crotonaldehyde using the well-known Skraup method. (See, G. Jones, Quinolines Part I, 100-117 [1977]).
In still another embodiment, the present invention provides Scheme VII an alternative method for the synthesis of proposed new analogues in Group 1(a). Briefly, compounds 46 and 47 are commercially available, or they can be easily obtained using a known method. (See e.g., B. Scholl et al, Helv. Chim. Acta., 69:184-194 [1986]). Compound 47 is epoxidized to obtain compound 48. Condensation of compound 48 with phthalimide yields intermediate compound 49, which can be oxidized to ketone (compound 50). Reduction of nitro group of compound 50 to an amine group produces compound 51. Through reductive amination of compound 51, compound 52 is obtained. Removal of the phalamido group of compound 52 generates diamine compound 53, which is subsequently converted into compound 54. Reaction of compound 54 with chloroacetyl chloride produces compound 55. Treatment of compound 55 with base yields the tricyclic lactam compound 56. Reduction of the tricyclic lactam compound 56, followed by removal of the protected group, produces the important intermediate compound 57. (See, E.W. Baxter and A. Reitz, Bioor. & Med. Chem. Lett., 7:763-768 [1997]). The intermediate compound 57 can be directly alkylated with different alkyl halides to yield the desired tricycle derivatives compound 58.
In another embodiment, the present invention provides a synthesis routine for the analogues in Group 1(b) by using methods similar to those shown in Scheme III and IV. (See also, U.S. 3,931,188 [incorporated herein by reference in its entirety]; L.A. van Vliet et al, J. Med. Chem., 43:2871 [2000]; and K.Y. Avenell et al, Med. Chem. Lett., 8:2859- 2864 [1998]). In yet another embodiment, an alternative method for the synthesis of the analogues in Group 1(b) is as provided in Scheme VIII. In Scheme VIII, reaction of 1- tetralones (compound 59) with n-butyl lithium and acetonitrile at -78°C, followed by reduction with lithium aluminum and dehydration produces compound 60. Ring closure by formaldehyde in acid condition yields the important intermediate hexahydrobenz[ ]isoquinolines (compound 61). The intermediates (compound 61) are coupled with different RX (X=C1, or Br) to produce the final target compound 62.
In still some other embodiments, the present invention provides analogues of lead compounds 14 and 32 having modifications in the tail portion designed to target D3 receptor Region A (Group II analogues). In preferred embodiments, based upon the predicted binding models for compounds 4 and 29, in order to target dopamine receptor D3 Region A, the ligands provide a general chemical structure having a flexible 5-6 bond linker (9-10 A) and a bulky hydrophobic tail. For example, in some embodiments, new analogues of compounds 14 and 32 having a 4-carbon and amide group as a linker and a bulky naphthyl ring in their tail portions show much improved selectivity between D3 and D2 receptors as compared to the original lead compounds 4 and 29. In some embodiments, it was found that the amide group in the linker portion may play a role in ligand potency and selectivity by forming a hydrogen bond with residue N375, thus positioning the naphthyl group in proper orientation to interact with residues in Region A. More specifically, the naphthyl ring in compounds 14 and 32 is predicted interact with residues N379, F338, N47, L121 of the D3 receptor in a region of structural differences between D2/D3 as discussed in detail. Thus, although an understanding of the mechanism is not necessary to practice the present invention and the present invention is not so limited, it is contemplated that residue N47 appears plays a major role for the selectivity of compounds 14 and 32. In addition, residues F338, N375 in the D3 receptor corresponding to F382, N418 in D2, respectively are also believed to contribute to the ability of compounds 14 and 32 to distinguish among the dopamine receptor subtypes. Fig. 22 shows the distance requirements of ligands to reach Region.
Some preferred embodiments of the present invention provide additional modifications of the naphthyl ring of lead compounds 14 and 32 to explore the structural differences in dopamine receptor Region A to achieve even greater selectivity. In some of these embodiments, small hydrophobic groups (e.g., CH3, F, Cl, Br, I, and the like) or polar substituents (OH, OCH3, NH2, CN, and the like) are installed on the naphthyl ring. In some preferred embodiments, the synthesis of analogues in Group 11(a) is accomplished by using methods similar to those shown in Schemes I, II, V, VI, and VII. Yet other embodiments of the present invention provide analogues of lead compounds 14 and 32 having (Group III analogues) modifications in the linker designed to target dopamine receptor Region B. In one embodiment of the present invention, based on the predicted binding models for compounds 14 and 32, significant structural differences are found among the dopamine receptors in Region B, for instance, difference are found in the orientations of residues F345 and F346 in TM6 of receptors D3 and D2. The orientation and position of residues Y365 and T369 in TM7 are also significantly different, in part due to the different helical packing between TM6 and TM7. Accordingly, some preferred embodiments of the present invention provide lead compound analogues that are optimized to extend ligand interactions with a particular dopamine receptor subtype (e.g., Dj, D2, D , D4, and/or D5) in Region B of the receptor, by modifying the lead compound to provide one or more small hydrophobic groups that are designed to specifically interact with this region.
One method for synthesis of Group IΙI(a) and (b) analogues is illustrated in Scheme
IX. (Fig. 23). Compound 64 is obtained from commercial available compound 63. The methyl ester (compound 64) is selectively reduced to obtain the aldehyde (compound 65).
(See e.g., L.I. Zakharkin, Tetrahydron Lett., 2087 [1963]). Treatment of the aldehyde (compound 65) with a Grignard reagent, followed by Swern Oxidization, yields the important intermediate ketone (compound 67). Compound 67 is treated with an amine (compounds 44, 57, or 61) to obtain the target compounds 68 or 69, respectively.
In some further embodiments, the present invention provides analogues of lead compounds 14 and 32 having modifications in the tricyclic ring that are designed to target dopamine receptor region D (Group IV analogues). For example, based on structural analyses, it was found that dopamine receptor Region D has a well-defined hydrophobic pocket in proximity to 3 serine residues. Further structural analysis shows that there are profound structural differences in this region among the dopamine receptor subtypes. These structural differences are believed to be primarily due to differences in the residues that make Region D. For example, several D3 receptor residues in this region, A161, A163, S182, correspond to residues S163, T165, and 1183, respectively, in the D2 receptor.
Preferred embodiments of the present invention provide lead compound analogues 14 and 32 have an additional substituted phenyl group linked to the tricyclic ring in through a sulphonylamide linker. Although an understanding of the mechanism is not necessary to practice the present invention and the present invention is not so limited, it is contemplated that the sulphonylamide linker forms hydrogen bonds with the serine residues in TM5, while the substituted phenyl group interacts with the hydrophobic pocket in Region D that is formed, in the D3 receptor, by residues 1183, Phel88, V164, L168, and VI 11. In certain embodiments, the analogues in Group IV(a) and IV(b) are obtained by treating compounds 70 or 73 with different substituted benzenesulfonyl chlorides as shown in Scheme X. (See, Fig. 24). It is of note that compound 70 is obtained using synthetic methods shown in Schemes V and VII and that compound 73 is obtained using the methods outlined in Scheme VIII. The present invention further provides methods for estimating docking of the new analogues to D3, D2, and Di and for the estimation of their binding affinities. In preferred embodiments, computational docking studies of the new analogues in with Dj, D2, and D3 are performed using the methods outlined above. The complex ligand-receptor structures are further refined through extensive MD simulation for 2 ns, or longer, in an explicit water and lipid environment using the CHARMM program. Based upon the predicted models, binding affinities are predicted.
Other embodiments of the present invention provide methods for the resolution of stereoisomers, as the binding of many contemplated ligands (e.g., 7-OH-DPAT and 5-OH-
DPAT) to the D3 receptor is stereo-specific Notably, all of the compounds synthesized in Schemes I, II, and V are racemic since they all contain one chiral center. Prefeπed embodiments separate (+) and (-) stereoisomers using known stereoisomer separation methods. (See, L.A. van Vliet et al, J. Med. Chem., 43:2871 [2000]; and H. Wikstrom et al, J. Med. Chem., 28:215 [1985]). As shown in Scheme XI (Fig. 25), important intermediate compound 41 used in Scheme V is resolved by coupling with (S)-(+)-α - methoxyphenylacetic acid chloride, which forms a mixture of two diastereomeric amides, compound 75(a) and compound 75(b). Column chromatographic separation is used to give pure diastereomers 75 (a) and 75(b). Cleavage of the amide group in compounds 75(a) and 75(b) by potassium tert-butoxide and water in THF yields pure stereoisomer compounds (-)- 41 and (+)-41 , respectively.
VII. Biological testing and characterization of lead compounds and analogues thereof
In some embodiments, the novel compounds and ligands identified and designed using the methods of the present invention are subjected to a step-wise screening process for affinity, efficacy, and selectivity at the dopamine receptors as outlined in Fig. 26. In prefeπed embodiments the present invention contemplates that, initial studies be used to focus on determining receptor affinity and selectivity in in vitro radioligand binding assays using the rat brain model and cloned human receptors. (See, Fig. 26, steps 1, 2, 3, and 4). The present invention further contemplates that, additional studies be used to determine the efficacy and D2/D3 selectivity in two different in vitro functional assays. (See, Fig. 26, step 5).
The present invention provides the following non-limiting examples, to further describe several of the biological tests and assays contemplated for characterizing the novel compounds and ligands identified and designed using the methods of the present invention. In some embodiments, the determination of affinity and selectivity of novel compounds at their target receptors (e.g., dopamine receptors) and their selectivity for the these receptors is initially determined in radioligand binding assays. To this end, in certain embodiments, 3 series of mutually complementary radioligand binding studies are performed in a step-wise manner. The invention contemplates that this complementary approach compensates for the particular limitations of any one of the individual assays, thus providing a more accurate determination of the affinity and selectivity of novel compounds at their target receptors than is provided using a single assay approach. The invention further contemplates that this complementary approach provides a thorough and definitive assessment of the in vitro pharmacological profile of the compounds with respect to their target receptors (e.g., dopamine receptors).
In certain embodiments, potential therapeutic compounds (test compounds) designed using the methods of the present invention, are first subjected to screens in the rat brain D3 receptor model describe above. If a test compound exhibits sufficient affinity and selectivity for the D3 receptor, then the affinity and selectivity for the D3 receptor is further determined additional in rat brain assays and in cloned human dopamine receptors assays. Finally, compounds that exhibit selectivity for the D receptor in both the rat brain membranes and in cloned human receptors are further evaluated using an autoradiographic methods to simultaneously determine D2, and D3 receptor affinities.
For example, in some embodiments, affinity of test compounds at the D3 receptor is measured using [3H]PD 128907 binding in membranes prepared from rat ventral striatum (e.g., nucleus accumbens and olfactory tubercle). Rat ventral striatum tissues are used for D3 receptor assays because these tissues express the highest density of D3 receptors in the rat CNS. (See, M.L. Bouthenet et al, Brain Res., 564:203-219 [1991]).
The present invention contemplates that the [3H]PD128907 agonist exhibits greater that 300-fold selectivity for the D3 receptor over the D2 receptor in rat brain. The affinity of test compounds for Di and D2 receptors is determined by using antagonists (e.g., radioligands [ H]SCH 23390 and [ H]spiperone, respectively) and membranes from rat caudate-putamen (striatum) that expresses high densities of Di and D2 receptors and low densities of other dopamine receptors according to the methods in Levant et al. (B. Levant, CNS Neuro transmitters and Neuromodulators, 3 [T.W. Stone, Ed.], CRC Press, Boca Raton, FL, 77-88 [1996]). The [3H]SCH 23390 and [3H]spiperone antagonists are highly selective for the Di-like and D2 receptors, respectively. It was found that [3H]spiperone exhibited an 80- fold higher affinity for D receptors than D3 receptors. With the low density of D receptors expressed in rat striatum, labeling of the D3 receptor by [3H]spiperone is negligible. Likewise, rat striatum expresses very low densities of the D5 receptor. Thus, the present invention contemplates that, although [3H]SCH 23390 has similar affinity for Di and D5 receptors, the binding assays in these studies are representative of the Di receptor. In prefeπed embodiments, [3H]SCH 23390 and [3H]spiperone binding assays are performed using in vitro assay conditions that favor agonist binding at dopamine receptors (e.g., inclusion of Mg2+, and the exclusion of NaCl [See e.g., D. Grigoriadis and P. Seeman, J. Neurochem., 44:1925-1935 (1985)]). The present invention contemplates that assays performed under these in vitro conditions enhance the ability to detect high-affinity binding of agonists in assays using antagonist radioligands. In prefeπed embodiments, assays are performed as previously described in Wang et al. (See, S. Wang et al, J. Med. Chem., 43:351-360 [2000]). In still other prefeπed embodiments, the K; value for each test compound at each dopamine receptor subtype is determined. Table 8 describes the determination of binding affinity at Di, D2, and D3 receptors in rat brain.
Table 8
In prefeπed embodiments, the primary advantage of performing the initial evaluation of test compounds in rat brain is that this approach allows the study of receptors expressed in their native tissue. Hence, the source tissue will contain all components required for normal physiological function. With the use of thoroughly characterized radioligands and brain areas with known densities of dopamine receptor subtypes, it is possible to determine the affinities of novel test compounds at Di, D2, and D3 receptors in brain tissue. This approach has the additional advantage that rat brains are readily available and are relatively inexpensive compared to cloned receptors. Table 9, provided below, shows the affinity (Kj or K<j values) of reference ligands used in contemplated binding assays in rat brain.
Table 9
In prefeπed embodiments, a second phase of biological test compound evaluation involves evaluation of the affinity and selectivity of test compounds for the D3 receptor using cloned human receptors (hDi, hD2sh0rt> hD3, and hD 4) expressed in transfected cell lines. In particularly prefeπed embodiments, test compounds progress to the second phase of evaluation if they exhibit selectivity for the D3 receptor in rat brain. In still other prefeπed embodiments, the affinity of the test compounds is determined in hDi, hD2short> hD3, and hD 4 receptors expressed in CHO cells (NEN Life Science and Sigma- Aldrich) using [3H]SCH 23390 for hDi binding and [3H]spiperone for the hD2sh0rt, hD3, and hD44 receptors. Assays are performed as previously described in Wang et al. (See, S. Wang et al, J. Med. Chem., 39:2047-2054 [1996]). In yet other prefeπed embodiments, the Ks value for each test compound at each dopamine receptor subtype is determined.
The present invention contemplates that the use of cloned human receptors has the advantage of enabling the study of human, rather than rat receptors, thus increasing the relevance to human therapeutics. Because the host cells do not normally express dopamine receptors, this approach also enables the study of each receptor subtype in isolation, obviating the dependence on selective pharmacological tools. Although different radioligands are used for hDi and the hD2-like receptor studies, the in vitro assay conditions otherwise remain the same. Hence, it is further contemplated that this approach compensates for the potential limitations of the use of rat brain membranes for determination of dopamine receptor affinity and selectivity.
In additional embodiments, the present invention provides methods for the simultaneous autoradiographic determination of D2/D3-selectivity in rat brain. In some of these methods, the quantitative autoradiographic method for the simultaneous determination of D2 and D receptor affinity in rat brain using the D2-D3 ligand [ Hjquinpirole according to Levant and De Souza, and Levant and Flietstra. (See, B. Levant and E.B. De Souza, Synapse, 14:90-95 [1993]). This method is based on the observation that dopamine receptors in the molecular layer of the vestibulocerebellum, which appear to be co-localized exclusively with dopamine D3 receptor mRNA, exhibit a lack of guanyl nucleotide regulation and a pharmacological profile consistent with the D3 site. As such, this brain area can serve as a discrete source of D3 receptors in brain tissue. In contrast, the caudate- putamen, which expresses substantially greater amounts of D2 receptor mRNA and exhibits relatively little D3 receptor binding, proves useful as a prototypical dopamine D2 tissue. Thus, by labeling these brain areas with a non-selective radioligand, such as [ H]quinpirole, it is possible to evaluate the selectivity of a test compound for the D2/D3 receptors in brain tissues under uniform in vitro assay conditions and in the same brain section. In prefeπed embodiments, studies are performed as previously described to determine a test compound's IC50 values at the D2 and D receptors, and thus D2/D3 selectivity. ((See, B. Levant and E.B. De Souza, Synapse, 14:90-95 [1993])). One contemplated advantage of this approach is that it measures D and D3 receptor affinity in brain tissues using a radiolabeled agonist while avoiding the potentially confounding variable of using separate assays to measure D2 and D3 receptor affinity. This is an important advantage because the D2/D3 selectivities of dopaminergic compounds have been shown to vary depending on the in vitro assay system used. Thus, in prefeπed embodiments, this approach provides a confirmatory assessment of D2/D3 receptor selectivity in addition to those obtained using receptors in heterologous expression systems or using different in vitro assays for each receptor in brain. Additionally, the present invention contemplates that D2/D3 selectivities determined by this method are generally more consistent with those determined in functional assays than radioligand binding assays using human receptors expressed in transfected cells.
Additional embodiments of the present invention provide methods for the determination of potency, efficacy, and selectivity of novel compounds in functional assays. Unlike radioligand binding assays, which assess only the affinity of a compound for a given receptor, functional assays are used in prefeπed embodiments to determine the potency, efficacy, and selectivity of novel ligands at the D receptor. These assays enable the identification of agonists and antagonists as well as partial and inverse agonists. Two functional assays are contemplated to provide cross validation of experimental results (e.g., mitogenesis assays and [ S]GTPgS binding assays), however, the present invention is not intended to be limited to using the two specifically mentioned functional assays. Indeed, any relevant functional assay that is compatible with the compositions and methods of the present invention are suitable for use herein.
In some embodiments of the present invention, only those test compounds that exhibit higher affinity for the D receptor than other dopamine receptors in the radioligand binding studies are further evaluated in functional assays.
In prefeπed embodiments, one type of functional assay suitable for use with the compositions and methods of the present invention are mitogenesis assays. Agonist- induced mitogenesis in CHO cells expressing hD2 or hD3 receptors has been developed as a suπogate marker of dopamine receptor activation. (See e.g., CL. Chio et al, Mol. Pharmacol., 45:51-60 [1994]; and F. Sautel et al, Neuroreport, 6:329-332 [1995]). In certain embodiments, the dose-response effects of novel compounds on CHO cell mitogenesis, as assessed on the basis of [ H]thymidine incorporation, is used to determine test compound efficacy and potency. In still other embodiments, test compounds lacking efficacy are evaluated for antagonist activity and potency as determined by their ability to block agonist-induced mitogenesis. Assays are performed as previously described in Milne et al. to determine the Emax and ED50 values for each test compound. (See, G.W. A. Milne et al, J. Chem. Inf. Comput. Sci., 34:1219-1224 [1994]). IC50 values are determined for test compounds found to block agonist-induced mitogenesis. In other prefeπed embodiment, another type of functional assay suitable for use with the compositions and methods of the present invention are [ S]GTPgS binding assays. These assays study receptor activation of G-proteins in membranes by assaying agonist- stimulated binding of [35S]GTPgS, a non-hydrolyzable analog of GTP, in the presence of excess GDP. This physiologically relevant endpoint has been used to assess G-protein activation by D2 and D3 receptors in brain and transfected cells. (See, S.L. Gilliland et al, Eur. J. Pharmacol., 392:125-128 [2000]; S.L. Gilliland and R.H. Alper, Naunyn- Schmiedeberg's Arch Pharmacol., 361:498-504 [2000]; and J.F. Pregenzer et al, Neurosci. Lett., 226:91-94 [1997]). In some embodiments, the dose-response effects of test compounds on [35S]GTPgS binding in membranes prepared for CHO cells expressing hD2 or hD3 receptors, is used to determine efficacy and potency. Compounds lacking efficacy are evaluated for antagonist activity and potency as determined by their ability to block agonist-induced [35S]GTPgS binding. In some other embodiments, [35S]GTPgS binding assays are also performed in CHO cell membranes containing hD sh0rt, hD3, or hD44 receptors as previously described to determine Emax and ED50 values for each compound. IC50 values are determined for test compounds found to block agonist-induced [35S]GTPgS binding.
One contemplated advantage of these functional approaches is that the use of studies on human dopamine receptors increases the relevance of the experimental data obtained to human therapeutics.
VIII. Therapeutic agents combined or co-administered with the present compositions
A wide range of therapeutic agents find use with the present invention. Any therapeutic agent that can be co-administered with the disclosed compounds, or associated with the disclosed compounds is suitable for us in the present invention. Some embodiments of the present invention provide administering to a subject an effective amount of a dopamine receptor modulator (e.g., antagonists or agonists) and one or more compounds indicated for the therapeutic treatment of cocaine abuse, depression, anxiety, an eating disorder, alcoholism, chronic pain, obsessive compulsive disorder, schizophrenia, restless legs syndrome (RLS), and Parkinson's disease, and the like. In some embodiments, the subject is a mammal (e.g., human).
Additional embodiments of the present invention provide therapeutic compositions and methods directed to modulating (e.g. , agonizing or antagonizing) G-protein coupled receptors. In some other additional embodiments, the therapeutic compositions and methods of the present invention are directed to modulating (e.g., agonizing or antagonizing) faulty dopamine receptor functions (e.g., reuptake of dopamine by the D3 receptor in limbic brain regions, including, but not limited to, the nucleus accumbens, olfactory tubercle, and the islands of Calleja). The present invention also provides the opportunity to monitor therapeutic success following administration of the compounds and therapeutic methods of the present invention to a subject. In some embodiments, these measurements are taken by observing the amelioration of a condition characterized, at least in part, by faulty dopamine (e.g., D3) receptor function. In some embodiments, patient observations are made in a clinical setting (e.g., a hospital, doctor's office, or clinic). In other embodiments, one or more physiological samples are taken from a patient at various time points (e.g., prior to, during, or after) administration of the therapeutic compositions and methods of the present invention to determine the effectiveness of administration. In still further embodiments, patient administration data is used to alter or modify the administration (e.g., dosing levels, frequency of administration, periodicity of admimstration, administration method, etc.) of the therapeutic compositions and methods of the present invention.
The compositions of the present invention may be delivered via any suitable method, including, but not limited to, injection intravenously, subcutaneously, intratumorally, intraperitoneally, or topically (e.g., to mucosal surfaces).
IX. Pharmaceutical formulations, administration routes, and dosing considerations The present invention provides pharmaceutical compositions which comprise at least one receptor (e.g., dopamine, G-protein couple receptors, and the like) modulation composition administered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water. In some embodiments, the pharmaceutical compositions of the present invention may contain one agent (e.g., a D3 receptor modulating ligand). In other embodiments, the pharmaceutical compositions may contain a mixture of at least two agents (e.g., one or more D3 receptor modulating ligand, and one or more additional therapeutic compositions). In still further embodiments, the pharmaceutical compositions of the present invention contain at least two agents (e.g., one or more D3 receptor modulating ligand, and one or more additional therapeutic compositions) that are administered to a patient under one or more of the following conditions: at different periodicities, different durations, different concentrations, different administration routes, etc.
The compositions and methods of the present invention find use in treating diseases or altering physiological states characterized by faulty (abeπant) regulation and/or function of dopamine receptors and other receptor, (e.g., Di, D2, D , D4, and D5). In some of embodiments, abeπant receptor (e.g., D3 receptor) function is induced by a psychostimulant drug (e.g., cocaine) I the patient's system. In other embodiments, the abeπant receptor function is caused, or is a result of, an organic disease or state or condition.
The present invention contemplates administering a dopamine receptor ligand and, in some embodiments, one or more traditional therapeutic compositions directed to treating a condition associated with faulty receptor regulation and/or function (e.g., compositions directed to treating substance abuse, depression, Parkinson's disease, schizophrenia, and the like) in accordance with acceptable pharmaceutical delivery methods and preparation techniques. For example, one or more D3 receptor modulating ligands, and one or more additional therapeutic compositions can be administered to a subject intravenously in a pharmaceutically acceptable carrier such as physiological saline. Standard methods for intracellular delivery of pharmaceutical agents can be used (e.g., delivery via liposome).
, Such methods are well known to those of ordinary skill in the art.
In some embodiments, the formulations of the present invention are useful for parenteral administration, such as intravenous, subcutaneous, intramuscular, and intraperitoneal. Therapeutic co-administration of some contemplated therapeutic agent agents (e.g., therapeutic polypeptides) can also be accomplished using gene therapy techniques. Gene therapy techniques are now widely known in the art.
As is well known in the medical arts, dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and interaction with other drugs being concurrently administered.
Accordingly, in some embodiments of the present invention, one or more D receptor modulating ligands are administered to a patient alone, or in combination with one or more substance abuse treatment agents, or in pharmaceutical compositions where it is mixed with excipient(s) or other pharmaceutically acceptable carriers. In one embodiment of the present invention, the pharmaceutically acceptable carrier is pharmaceutically inert.
Depending on the condition being treated, these pharmaceutical compositions may be formulated and administered systemically or locally. Techniques for formulation and administration may be found in the latest edition of "Remington's Pharmaceutical Sciences" (Mack Publishing Co, Easton Pa.). Suitable routes may, for example, include oral or transmucosal administration; as well as parenteral delivery, including intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, or intranasal administration. For injection, the pharmaceutical compositions of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiologically buffered saline. For tissue or cellular administration, penetrants appropriate to the particular barrier to be permeated are used in the formulation. Such penetrants are generally known in the art. In other embodiments, the pharmaceutical compositions of the present invention can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral or nasal ingestion by a patient to be treated. In prefeπed embodiments, the therapeutic compounds are administered orally to a patient orally.
Pharmaceutical compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended purpose. For example, an effective amount of D3 receptor modulating ligands may be that amount that induces reuptake of dopamine in a patient's cell or tissue having depressed (or elevated) elevated levels of dopamine uptake as compared to normal nonpathological examples of the particular cells or tissues. Determination of effective amounts is well within the capability of those skilled in the art, especially in light of the disclosure provided herein.
In addition to the active ingredients, prefeπed pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries that facilitate processing of the active compounds into preparations which can be used pharmaceutically. The preparations formulated for oral admimstration may be in the form of tablets, dragees, capsules, or solutions. The pharmaceutical compositions of the present invention may be manufactured in a manner that is itself known (e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes). Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or Iiposomes. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
Pharmaceutical preparations for oral use can be obtained by combining the active compounds (e.g., receptor ligands) with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are carbohydrate or protein fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato, etc; cellulose such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; and gums including arabic and tragacanth; and proteins such as gelatin and collagen. If desired, disintegrating or solubilizing agents may be added, such as the cross-linked polyvinyl pyπolidone, agar, alginic acid or a salt thereof such as sodium alginate.
Ingestible formulations of the present compositions may further include any material approved by the United States Department of Agriculture for inclusion in foodstuffs and substances that are generally recognized as safe (GRAS), such as, food additives, flavorings, colorings, vitamins, minerals, and phytonutrients. The term phytonutrients as used herein, refers to organic compounds isolated from plants that have a biological effect, and includes, but is not limited to, compounds of the following classes: isoflavonoids, oligomeric proanthcyanidins, indol-3-carbinol, sulforaphone, fibrous ligands, plant phytosterols, ferulic acid, anthocyanocides, triterpenes, omega 3/6 fatty acids, polyacetylene, quinones, terpenes, cathechins, gallates, and quercitin.
Dragee cores are provided with suitable coatings such as concentrated sugar solutions, which may also contain gum arabic, talc, polyvinylpyπolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound, (i.e., dosage). Pharmaceutical preparations that can be used orally include push- fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients mixed with a filler or binders such as lactose or starches, lubricants such as talc or magnesium stearate, and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycol with or without stabilizers.
Compositions comprising a compound of the invention formulated in a pharmaceutical acceptable carrier may be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition. For D3 receptor modulating ligands, conditions indicated on the label may include treatment of conditions related to substance abuse (e.g., cocaine abuse), depression, Parkinson's disease, and schizophrenia, and the like. The pharmaceutical compositions may be provided as a salt and can be formed with many acids, including but not limited to hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents that are the coπesponding free base forms. In other cases, the prefeπed preparation may be a lyophilized powder in 1 mM-50 mM histidine, 0.1%-2% sucrose, 2%-7% mannitol at a pH range of 4.5 to 5.5 that is combined with buffer prior to use.
For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. Then, preferably, dosage can be formulated in animal models (particularly murine or rat models) to achieve a desirable circulating concentration range that induces normal function/regulation of receptors (e.g., reuptake of dopamine by D3 receptors). A therapeutically effective dose refers to that amount of receptor modulating ligand (and in some embodiments, one or more other therapeutic agents) that ameliorate symptoms of the disease state (e.g., depressed reuptake of dopamine). Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, and it can be expressed as the ratio LD50/ED50.
Compounds that exhibit large therapeutic indices are prefeπed. The data obtained from cell culture assays and additional animal studies can be used in formulating a range of dosage, for example, mammalian use (e.g., humans, Equus caballus, Felis catus, and Canis familiaris, etc.). The dosage of such compounds lies preferably, however the present invention is not limited to this range, within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage varies within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration. The exact dosage is chosen by the individual physician in view of the patient to be treated. Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Additional factors which may be taken into account include the severity of the disease state; age, weight, and gender of the patient; diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. Long acting pharmaceutical compositions might be administered every 3 to 4 days, every week, or once every two weeks depending on half- life and clearance rate of the particular formulation. Other pharmaceutical compositions may be administered daily or several times a day.
Normal dosage amounts may vary from 0.1 to 100,000 micrograms, up to a total dose of about 1 g, depending upon the route of admimstration. Guidance as to particular dosages and methods of delivery is provided in the literature. Administration of some agents to a patient's bone marrow may necessitate delivery in a mariner different from intravenous injections.
Prefeπed embodiments of the present invention provide pharmaceutical compositions and methods for administering an effective amount of receptor modulating ligands to a patient to modulate (e.g., increase or decrease the activity of) faulty receptor function/regulation.
In some embodiments, diseases suspected of being characterized by having faulty receptor (e.g., D3 receptor) function/regulation suitable for treatment by the present invention are selected by obtaining a sample of interest (e.g., cells, tissues, fluids, etc.) suspected of having abeπant levels of native (e.g., endogenous) receptor ligands (e.g., dopamine), measuring the levels of native receptor ligands in the sample using one or more well established immunohistochemical techniques (e.g., ELISA and Western blots, etc.), and comparing the levels of native receptor ligands in the sample with levels of coπesponding receptor ligands in relevant reference nonpathological samples. In other embodiments, diseases suspected of being characterized by having abeπant levels of one or more native receptor ligands (e.g., dopamine) are selected by comparing levels of one or more markers (e.g., polynucleotides, polypeptides, lipids, etc.) in a sample (e.g., cells, tissues, fluids, etc.) that directly or indirectly indicate elevated aberrant levels of the receptor ligand of interest as compared to levels of these markers relevant nonpathological samples.
The present invention is not intended to be limited to the administration routes chosen for delivering agents to a subject. Indeed, a number of suitable administration routes are contemplated the selection of which is within the skill of those in the art.
In some prefeπed embodiments, therapeutic compositions are administered to a patient at a dosage range of about 1 to 200 mg/day, from about 5 to 50 mg/day, and most preferably from about 10 to 40 mg/day. In particularly prefeπed embodiments, the therapeutic compounds are administered to a patient (e.g., orally) in a tolerable daily dose (e.g., 30 to 40 mg/day) shown to have some biologic activity (e.g., alterations in Rb and Cyclin Di levels).
In some embodiments, standard immunohistochemical techniques are employed on samples obtained from patients following, or during, treatments with the methods and compositions of the present invention to determine changes in the patient's disease state. As used herein, cocaine abuse and/or addiction, is considered to be a pathological disease state.
Therapeutic potential of the D3 receptor in schizophrenia. Schizophrenia is a neurodevelopmental disorder involving abnormal dopaminergic function. The importance and therapeutic potential of the D3 dopamine receptor in schizophrenia is supported by several lines of evidence. First, D2 and D3 dopamine receptors are the main targets for nearly all cuπently known agents with antipsychotic activity (recent reviews: Strange, 2001; Schwartz et al, 2000; Emilien et al, 1999). Also, the D3 subtype is primarily localized to forebrain limbic areas, known of being involved in schizophrenia. Furthermore, antipsychotic drugs administered to schizophrenic patients appear to act as down regulators of the D receptor in the ventral striatum region, which is supported by the over-expression of the D3 (and not D2) receptors in case of drug- free schizophrenic patients compared to schizophrenic patients taking anti-psychotic agents (Gurevich et al, 1997). This would also account for the behavioral sensitization to psychostimulants observed in schizophrenia.
Pharmacological arguments also support that another factor contributing to schizophrenia symptoms might be an imbalance between the relative abundance of the Di and D3 receptors (Schwartz, et al, 1998). However, further studies are needed to confirm the over- expression of the D3 receptor in schizophrenia as well as the role it plays in the symptomatology of this disorder. Typical antipsychotic drugs tend to have severe extrapyramidal side effects, unlike second generation atypical drugs. Since the D3 receptor is mainly restricted to limbic associated brain regions, it is generally considered an attractive target for the development of antipsychotic treatments that lack extrapyramidal or neuroendocrine side effects. For example, in case of a recently discovered D3 antagonist showing significant selectivity for D3 over D2 in rat brain, SB-277011-A, evidence is presented that a beneficial effect may be achieved by this D3 ligand in the treatment of schizophrenia without the side effects characteristic of non-selective dopamine receptor antagonists (Reavill et al, 2000). In case of neuroleptics used in the treatment of schizophrenia, evidence supports that the antipsychotic effects of this class of drugs are associated with blocking the D3 rather than the D2 receptor, while extrapyramidal side effects are attributed to their effects on the D receptor expressed in the striatum (Schwartz, et al, 2000; Sigmundson, 1994; Missale et al, 1998; Joyce and Meador- Woodruff, 1997). On the other hand, it is also argued that extrapyramidal side effects of antipsychotics may be eliminated by reducing the occupancy of striatal D2 receptors through administering lower doses, as this was shown in case of haloperidol and clozapine (Strange, 2001).
Therapeutic implication of the D3 receptor in Parkinson 's disease. Long-term levodopa therapy for Parkinson's disease leads to severe side effects, dyskinesias, fluctuations in motor performance, hallucinations. An induction of D receptor gene expression was shown to accompany levodopa administration in conditions in which it leads to behavioral sensitization. This involvement has therapeutic relevance in the prevention of side effects and also in the treatment of Parkinson's disease (Bordet et al, 1997; S. Perachon et al, 366:293-300 [1999]). Combined administration of levodopa and dopamine agonist is considered an effective way of reducing and delaying the side effects of chronic levodopa therapy. Furthermore, two recently developed drugs for Parkinson's disease, pramipexole and ropinirole have the dopamine D3 receptor as their primary target, while another anti-parkinsonian drug, pergolide is potent at all three, Di, D , D3 subtypes.
Therapeutic implications of the D3 receptor in restless legs syndrome. Restless legs syndrome (RLS)is characterized by leg paresthesis associated with an urge to move and motor restlessness. Levodopa and ergoline derivatives are effective treating this condition but their usefulness is limited by major side effects. Also, no complete relief of the RLS symptoms may be achieved using these drugs. Recently, a D receptor agonist, pramipexole was found highly effective in the treatment of RLS, which also supports the involvement and therapeutic potential of the D3 subtype receptor in RLS (Montplaisir, et al, 1999).
Therapeutic implications of the D3 receptor in the treatment of depression. Dopamine function was found reduced in patients with severe depression (Shah et al, 1997). Also, antidepressent agents that have dopaminergic effects are generally effective for treating depression (Brown and Gershon, 1993). Specifically, chronic antidepressants effect D2/D3 receptor function in mesolimbic terminal regions (Emilien et al, 1999).
EXAMPLES
The following examples are provided to demonstrate and further illustrate certain prefeπed embodiments of the present invention and are not to be construed as limiting the scope thereof.
Example 1 Preparation of various lead compounds
This example describes the synthesis of various lead compounds and their key analogs. The key intermediate compound 5 was synthesized using modified known methods. (See, C.A.R. Baxter et al, J. Med. Chem., 15(4):351-356 [1972]; V.A. Rao et al, J. Med. Chem., 13(3):516-522 [1970]; and G.W. Gribble and P.W. Heald, Synthesis, 650- 651 [1975]). Condensation of 2-quinoline-carboxaldehyde compound 2 (compound 2b was prepared by oxidation of 6-methoxyquinaldine with SeO2 in dioxane as indicated in G. Lunn and E.B. Sansone, J. Org. Chem., 51:513-517 [1986]) with ethanolamine gave the Schiff base, which was then reduced to 2-substituted aminomethylquinoline (compound 3) with NaBH4. (See, C.A.R. Baxter et al, J. Med. Chem., 15(4):351-356 [1972]; and V.A. Rao et al, J. Med. Chem., 13(3):516-522 [1970]). Reduction of compound 3 with nickel- aluminum alloy in aqueous KOH readily gave 2-substituted aminomethyl-1,2,3,4- tetrahydro-quinoline (compound 4) in good yield. (See, N. Rabjohn, Org. React., 24:261- 415 [1976]). The key intermediate compound 5 was prepared by reflux of compound 4 with
P2O5 in xylenes. (See, C.A.R. Baxter et al, J. Med. Chem., 15(4):351-356 [1972]; and V.A.
Rao et al, J. Med. Chem., 13(3):516-522 [1970]). Alkylation of compound 5 with different alkyl halide in the presence of Cs2CO3 in aceonitrile afforded the lead compound and its analogs compound 6a-e and compounds 7a-e. (See, L.A. Vliet, J. Med. Chem., 43:2871- 2882 [2000]). Demethylation of compounds 7a-e with BBr3 in CH2C12 gave analogs compound 8a-e with free hydroxy group on the phenyl ring. (See, E.H. Vickery et al, J. Org. Chem., 44:4444-4446 [1979]).
Reagent and condition: i. a) 1.1 eq. ethanolamine, benzene, under N2, reflux, remove H O, overnight; b) 3.5 eq. NaBH4, absolute ethanol, under N , reflux, overnight, yield 87- 90%. ii. Ca. 5g nickel-aluminum alloy/lg substrate, IM KOH, methanol, rt, overnight, yield 85-89%. iii. 3 eq. P2O5, xylenes, under N2, reflux, overnight, yield 82-86%. iv. 1.2 eq. R2C1/ Nal or 1.2 eq. R2I, 2 eq. Cs2CO3, CH3CN, under N2, reflux, overnight, yield 61- 76%. v. 4 eq. BBr3, CH2C12, under N2, 0°C, 4h, yield 71-75%.
Table 10 shows the structure and activity of several D ligands.
Table 10
7a OMe (CH2)2COC6H4F-/>
7b OMe (CH2)3COC6H4F-p
7c OMe (CH2)4COC6H4F-/>
7d OMe (CH2)2CH3
7e OMe (CH2)3CH3
Example 2 6-Methoxyquinoline-2-carboxaldehyde (Compound 2b)
To a solution of 8.7g of 6-methoxyquinaldine (50mmol) in 100ml of dioxane and 5ml of water was added 5.5g of SeO2 (50mmol), and the resulting mixture was refluxed for 3h, cooled, filtered. The filtrate was concentrated, and the residue was purified by chromatography on silica gel eluting with hexanes:EtOAc=6:l to give 8.5g of compound 2b as colorless crystals, yield 91%. 1HNMR (CDC13, 300MHz) δ 3.92 (s, 3H), 7.07 (d, J= 2.7Hz, IH), 7.41 (dd, Jj= 9.3Hz, J2= 2.7Hz, IH), 7.93 (d, J- 8.4Hz, IH), 8.07 (d, J= 9.3Hz, IH), 8.11 (d, J= 8.4Hz, IH), 10.13 (s, IH). 13CNMR (CDC13, 75MHz) δ 55.7, 104.9, 117.7, 123.5, 131.4, 131.7, 135.5, 143.8, 150.3, 159.7, 193.1, 193.3.
Example 3 2-(β-Hydroxyethylaminomethyl)-quinoline (Compound 3a)
To a solution of 7.85g of 2-quinolinecarboxaldehyde (compound 2a) (50mmol) in 50ml of benzene was added 3.3ml of ethanolamine (3.36g, 55mmol), and the resulting mixture was azeotroped till no more water seperated. Benzene was removed under reduced pressure and the crude Schiff base was dissolved in absolute ethanol. The resulting mixture was treated with 6.62g of NaBH4 (0.175mol) at 0 C, and was refluxed overnight. After removal of ethanol, the crude was dissolved in water and extracted with 3x50ml of CHC13. The organic layer was dried over Na2SO4 and concentrated, the residue was purified by chromatography on silica gel eluting with 10%MeOH in CHC13 to give 9.1g of compound 3a as a colorless solid, yield 90%. A sample was converted to its hydrochloride salt. 1HNMR (CD3OD, 300MHZ) δ 3.25 (t, J= 6.0Hz, 2H), 3.83 (t, J= 6.0Hz, 2H), 4.53 (s, 2H), 7.43 (d, J= 8.4Hz, IH), 7.52 (t, J= 8.4Hz, IH), 7.69 (t, J= 8.4Hz, IH), 7.85 (d, J= 8.4Hz, IH), 8.01 (d, J= 8.4Hz, IH), 8.27 (d, J= 8.4Hz, IH). 13CNMR (CD3OD, 75MHz) δ 44.8, 45.8, 52.0, 114.7, 122.2, 130.0, 123.0, 123.7, 125.3, 132.8, 142.3, 147.0.
Example 4 2-(β-Hydroxyethylaminomethyl)-6-methoxyquinoline (Compound 3b)
10. lg of compound 3b was prepared from 9.35g of compound 2b (50mmol) as described above for preparation of compound 3a (Example 3), yield 87%. 'HNMR (CDC1 , 300MHz) δ 2.95 (t, J= 6.0Hz, 2H), 3.70 (t, J= 6.0Hz, 2H), 3.90 (s, 3H), 4.13 (s, 2H), 7.04 (d, J= 2.7Hz, IH), 7.33 (dd, J/= 8.7Hz, J2= 2.7Hz, 2H), 7.91 (d, J= 8.7Hz, IH), 7.99 (d, J= 8.7Hz, IH). 13CNMR (CDC13, 75MHz) δ 51.1, 54.4, 54.9, 60.2, 104.6, 120.2, 121.5, 127.5, 129.3, 134.8, 142.8, 156.7, 156.8.
Example 5
2-(β-Hydroxyethylaminomethyl)-l,2,3,4-tetrahydroquinoline (Compound 4a)
8.1g of compound 3a (40mmol) was taken up in 200ml of methanol and 200ml of IM KOH was added. Nickel-aluminum alloy (40g) was added in 5 portions over 1 hr, and the resulting mixture was stiπed overnight at rt. The mixture was filtered, concentrated, extracted with 3χ 100ml CHC13, and the extract was dried over Na2SO4. After removal of solvent, the residue was purified by chromatography on silica gel eluting with 20%MeOH in CHC13 to give 7.3g of compound 4a as a colorless solid, yield 89%. 1HNMR (CDC13, 300MHz) δ 1.60-1.70 (m, IH), 1.85-1.92 (m, IH), 2.20-2.60 (bs, 2H), 2.60-2.90 (m, 6H), 3.31-3.38 (m, IH), 3.69 (t, J= 6.0Hz, 2H), 4.20-4.70 (bs, IH), 6.50 (d, J= 7.5Hz, IH), 6.59 (t, J= 7.5Hz, IH), 6.95 (t, J= 7.5Hz, 2H). 13CNMR (CDC13, 75MHz) δ 26.2, 26.4, 50.9, 51.3, 55.0, 61.0, 114.1, 116.9, 121.1, 126.7, 129.1, 144.1.
Example 6 2-(β-Hydroxyethylaminomethyl)-6-methoxy-l,2,3,4-tetrahydroquinoline (Compound 4b)
8.0g of compound 4b was prepared from 9.3g of compound 3b (40mmol) as described above for preparation of compound 4a (Example 5), yield 85%. A sample was converted to its hydrochloride salt. 1HNMR (DMSO-rf6, 300MHz) δ 1.85-2.0 (m, IH), 2.10- 2.28 (m, IH), 2.70-2.90 (m, 2H), 3.0-3.2 (m, 2H), 3.2-3.6 (m, 2H), 3.6-3.8 (m, 2H), 3.72 (s, 3H), 3.8-3.9 (m, IH), 6.75-6.9 (m, 2H), 7.0-7.10 (m, IH). 13CNMR (OMSO-d6, 75MHz) δ 24.2, 25.0, 49.4, 50.4, 50.6, 56.3, 57.1, 112.1, 114.2, 115.2, 123.2, 139.7, 151.0. Example 7 2,3,4,4a,5,6-Hexahydro-l/y-pyrazino[l,2-α]quinoline (Compound 5a)
A mixture of 7.21g of compound 4a (35mrnol) and 14.9g of P2O5 (105mmol) in 100ml of xylene was refluxed overnight, cooled, and concentrated. The residue was hydrolyzed with 5N NaOH, extracted with 3x 100ml CHC13, and the extract was dried over K2CO . After removal of solvent, the residue was purified by chromatography on silica gel eluting with 10%MeOH in CHC13 to give 5.4g of compound 5a as a colorless oil, yield 82%. A sample was converted to its hydrochloride salt. 1HNMR (CD3OD, 300MHz) δ 1.69-1.1.82 (m, IH), 1.97-2.10 (m, IH), 2.65-2.80 (m, IH), 2.80-2.90 (m, IH), 2.90-3.20 (m, 2H), 3.20-3.40 (m, 2H), 3.50-3.60 (m, 2H), 4.12 (d, J= 7.5Hz, IH), 6.80 (t, J= 7.2Hz, IH), 6.96 (d, J= 7.5Hz, IH), 7.03 (d, J= 7.2Hz, IH), 7.13 (t, J- 7.5Hz, IH). 13CNMR (CD3OD, 75MHz) δ 26.0, 26.3, 43.6, 44.8, 50.2, 53.5, 114.2, 120.4, 125.6, 127.5, 129.6, 144.3.
Example 8
8-Methoxy-2,3,4,4a,5,6-hexahydro-lH-pyrazino[l,2-α]quinoline (Compound 5b)
6.56g of compound 5b was prepared from 8.26g of compound 4b (35mmol) as described above for preparation of compound 5a (Example 7), yield 86%. 'HNMR (CDC13, 300MHz) δ 1.62-1.78 (m, IH), 1.78-1.90 (m, IH), 2.4-2.6 (bs, IH), 2.60-2.72 (m, 3H), 2.74-2.92 (m, 2H), 2.93-3.02 (m, 2H), 3.08-3.15 (m, IH), 3.60-3.68 (m, IH), 3.71 (s, 3H), 6.57 (d, J= 2.7Hz, IH), 6.64 (dd, J,= 9.3Hz, J2= 2.7Hz, IH), 6.72 (d, J= 9.3Hz, IH). 13CNMR (CDC13, 75MHz) δ 27.1, 27.2, 46.0, 48.2, 52.4, 55.6, 56.7, 112.1, 113.4, 114.8, 126.0, 140.7, 152.0.
Example 9
General procedure for alkylation of Compounds 5a and 5b (Method A)
In one embodiment, a mixture of compound 5 (lmmol), the appropriate alkyl chloride (1.2mmol) and anhydrous Nal (1.2mmol) or appropriate alkyl iodide (1.2mmol), anhydrous Cs CO (2mmol) in 10ml of anhydrous CH3CN was refluxed overnight. The reaction mixture was cooled, filtered, and concentrated. The residue was purified by chromatography on silica gel eluting with hexanes:EtOAc=l :1 to give the required products. Example 10 3-(p-Fluorobenzoylethyl)-2,3,4,4a,5,6-hexahydro-l T-pyrazino[l,2-α]quinoline
(Compound 6a)
Alkylation of compound 5a with 3-chloro-4'-fluoropropiophenone as described in method A (Example 9) gave 206mg of compound 6a as a colorless oil, yield 61%. A sample was converted its hydrochloride salt. !HNMR (CD3OD, 300MHZ) δ 1.65-1.80 (m, IH), 1.94-2.02 (m. IH), 2.62-2.72 (m, IH), 2.74-2.90 (m, IH), 2.90-3.10 (m, 2H), 3.15-3.35 (m, 3H), 3.53-3.76 (m, 5H), 4.10 (d, J= 7.2 Hz, IH), 6.71 (t, J- 7.2Hz, IH), 6.93 (d, J= 7.2Hz, IH), 7.03 (d, J= 7.2Hz, IH), 7.08 (t, J= 7.2Hz, IH), 7.42 (t, J= 5.7Hz, 2H), 8.12 (d, J= 5.7Hz, IH), 8.15 (d, J= 5.7Hz, IH). 13CNMR (CD3OD, 75MHz) δ 26.4, 33.6, 44.6, 48.7, 51.7, 52.0, 53.0, 56.0, 114.0, 116.6, 116.9, 119.5, 124.9, 127.8, 130.0, 131.9, 132.0, 133.5, 145.3, 196.0. Anal. (C2ιH23FN2O2HCl), C, H, N.
Example 11 3-(/>-Fluorobenzoylpropyl)-2,3,4,4a,5,6-hexahydro-l.H-pyrazino[l,2- ]quinoline
(Compound 6b)
Alkylation of compound 5a with 4-chloro-4'-fluorbutyrophenone as described in method A (Example 9) gave 225mg of compound 6b as a colorless oil, yield 64%. A sample was converted to its hydrochloride salt. 1HNMR (CD3OD, 300MHz) δ 1.71-1.85 (m, IH), 2.02-2.15 (m, IH), 2.16-2.28 (m, 2H), 2.70-2.80 (m, IH), 2.80-3.02 (m, 2H), 3.06- 3.40 (m, 7H), 3.69 (d, J= 11.4Hz, IH), 3.79 (d, J= 10.2Hz, IH), 4.18 (d, J= 11.4Hz, IH), 6.77 (t, J=7.2Hz, IH), 6.94 (d, J= 7.2Hz, IH), 7.03 (d, J= 7.2Hz, IH), 7.12 (t, J= 7.2Hz, IH), 7.20-7.40 (m, 2H), 8.08-8.16 (m, 2H). 13CNMR (CD3OD, 75MHz) δ 19.2, 27.0, 27.1, 36.1, 45.8, 53.2, 54.6, 57.5, 57.6, 114.6, 116.6, 116.9, 120.7, 125.9, 128.3, 130.4, 132.1, 132.2, 134.2, 145.5, 199.6. Anal. (C22H25FN2O2HCl), C, H, N.
Example 12 3-(p-Fluorobenzoylbutyl)-2,3,4,4a,5,6-hexahydro-l -r-pyrazino[l,2-α]quinoline
(Compound 6c) Alkylation of compound 5a with 1 -(4-fluorophenyl)-5-chloro- 1 -oxopentane as described in method A (Example 9) gave 252mg of compound 6c as a colorless oil, yield 69%>. A sample was converted to its hydrochloride salt. 1HNMR (CD3OD, 300MHz) δ 1.70-2.0 (m, 5H), 2.0-2.10 (m, IH), 2.70-2.80 (m, IH), 2.8-2.98 (m, 2H), 3.05-3.38 (m, 7H), 3.63 (d, J= 12.0Hz, IH), 3.73 (d, J= 7.5Hz, IH), 4.17 (d, J= 12.0Hz, IH), 6.75 (t, J= 7.2Hz, IH), 6.92 (d, J= 7.2Hz, IH), 7.01 (d, J= 7.2Hz, IH), 7.10 (t, J= 7.2Hz, IH), 7.25 (t, J= 8.7Hz, 2H), 8.09 (d, J- 8.7Hz, IH), 8.12 (d, J= 8.7Hz, IH). 13CNMR (CD3OD, 75MHz) δ 21.9, 24.3, 27.0, 27.2, 38.5, 45.7, 53.1, 54.6, 57.4, 58.1, 114.6, 116.6, 116.8, 120.7, 125.9, 128.3, 130.4, 132.1, 132.2, 134.4, 134.5, 145.5, 200.9. Anal. (C23H27FN2O2HCl), C, H, N.
Example 13 3-Propyl-2,3,4,4a,5,6-hexahydro-l//-pyrazino[l,2-α]quinoline (Compound 6d)
Alkylation of compound 5a with 1-iodopropane as described in method A (Example 9) gave 165mg of compound 6d as a colorless oil, yield 72%. A sample was converted to its hydrochloride salt. 1HNMR (CD3OD, 300MHz) δ 1.05 (t, J= 7.2Hz, 3H>, 1.70-1.92 (m, 3H), 2.0-2.09 (m, IH), 2.71-2.80 (m, IH), 2.81-2.96 (m, 2H), 3.05-3.24 (m, 4H), 3.29-3.39 (m, IH), 3.60 (d, J= 12.0Hz, IH), 3.71 (d, J= 9.0Hz, IH), 4.15 (d, J= 12.0Hz, IH), 6.77 (t, J= 7.2Hz, IH), 6.93 (d, J- 7.2Hz, IH), 7.02 (d, J= 7.2Hz, IH), 7.11 (t, J= 7.2Hz, IH). 13CNMR (CD3OD, 75MHz) δ 11.2, 18.3, 27.0, 27.1, 45.7, 53.0, 54.5, 57.3, 59.7, 114.6, 120.7, 126.0, 128.3, 130.4, 145.4. Anal. (Cι5H22N22HCl), C, H, N.
Example 14 3-Butyl-2,3,4,4a,5,6-hexahydro-lH-pyrazino[l,2-α]quinoline (Compound 6e) Alkylation of compound 5 a with 1-iodobutane as described in method A (Example
9) gave 180mg of compound 6e as a colorless oil, yield 74%. A sample was converted to its hydrochloride salt. 1HNMR (CD3OD, 300MHz) δ 1.03 (t, J= 7.2Hz, 3H), 1.40-1.54 (m, 2H), 1.80-1.92 (m, 3H), 2.06-2.14 (m, IH), 276-2.85 (m, IH), 2.86-3.0 (m, IH), 3.08 (t, J= 8.7Hz, IH), 3.18-3.36 (m, 4H), 3.54 (t, J= 10.5Hz, IH), 3.71 (d, J= 12.0Hz, IH), 3.88 (d, J= 9.6Hz, IH), 4.28 (d, J= 12.0Hz, IH), 6.89 (t, J= 7.2Hz, IH), 7.07 (d, J= 7.2Hz, IH), 7.10 (d, J= 7.2Hz, IH), 7.17 (t, J= 7.2Hz, IH). 13CNMR (CD3OD, 75MHz) δ 13.9, 20.9, 26.7, 26.8, 26.9, 46.6, 52.5, 55.6, 56.7, 58.1, 116.0, 122.6, 127.3, 128.4, 130.8, 143.6. Anal. (C,6H24N22HC1), C, H, N. Example 15 3-(p-Fluorobenzoylethyl)-8-methoxy-2,3,4,4a,5,6-hexahydro-lJΪ-pyrazino[l,2- ]quinoline (Compound 7a)
Alkylation of compound 5b with 3-chloro-4'-fluoropropiophenone as described in method A (Example 9) gave 239mg of compound 7a as a colorless oil, yield 65%. 'HNMR (CDC13, 300MHz) δ 1.64-1.80 (m, IH), 1.80-1.82 (m, IH), 2.06 (t, J= 10.8Hz, IH), 2.35 (t, J= 10.8Hz, IH), 2.60-2.70 (m,lH), 2.70-2.92 (m, 6H), 3.01 (d, J= 10.8Hz, IH), 3.20 (t, J= 7.2Hz, 2H), 3.66 (d, J= 9.6Hz, IH), 3.72 (s, 3H), 6.57 (d, J= 2.7Hz, IH), 6.65 (dd, J,= 9.0Hz, J2= 2.7Hz, IH), 6.72 (d, J= 9.0Hz, IH), 7.12 (t, J= 8.7Hz, 2H), 7.98 (t, J= 8.7Hz, 2H). 13CNMR (CDC13, 75MHz) δ 27.2, 27.3, 36.1, 47.2, 53.0, 53.4, 55.3, 55.6, 59.7, 112.1, 113.5, 114.8, 115.5, 115.8, 125.8, 130.5, 130.6, 140.2, 152.1, 163.9, 167.3, 197.0. Anal. (C22H25FN2O22HCl), C, H, N.
Example 16 3-(p-Fluorobenzoylpropyl)-8-methoxy-2,3,4,4a,5,6-hexahydro-l ϊf-pyrazino [1 ,2- α]quinoline (Compound 7b)
Alkylation of compound 5b with 4-chloro-4'-fluorobutyrophenone as described in method A (Example 9) gave 252mg of compound 7b as a colorless oil, yield 66%. 1HNMR (CDC13, 300MHz) δ 1.62-1.77 (m, IH), 1.78-1.92 (m, IH), 1.94-2.06 (m, 3H), 2.20-2.30 (m, IH), 2.42-2.58 (m, 2H), 2.60-2.72 (m, IH), 2.80-2.96 (m, 5H), 3.01 (t, J= 6.9Hz, 2H), 3.64 (d, J= 12.0Hz, IH), 3.72 (s, 3H), 6.56 (d, J= 2.7Hz, IH), 6.64 (dd, J,= 8.7Hz, J2= 2.7Hz, IH), 6.71 (d, J= 8.7Hz, IH), 7.11 (t, J= 8.7Hz, 2H), 7.96 (d, J= 8.7Hz, IH), 7.99 (d, J= 8.7Hz, IH). ,3CNMR (CDC13, 75MHz) δ 20.1, 26.5, 26.7, 35.5, 46.2, 52.5, 54.2, 55.1, 56.9, 58.3, 111.8, 113.2, 114.3, 115.0, 115.3, 125.4, 130.0, 130.2, 139.3, 151.8, 163.4, 166.8, 197.3. Anal. (C23H27FN2O22HCl), C, H, N.
Example 17 3-(p-Fluorobenzoylbutyl)-8-methoxy-2,3,4,4a,5,6-hexahydro-l/y-pyrazino[l,2- ]quinoline (Compound 7c) Alkylation of compound 5b with l-(4-fluorophenyl)-5-chloro-l-oxopentane as described in method A (Example 9) gave 269mg of compound 7c as a colorless oil, yield 68%. 1HNMR (CDC13, 300MHZ) δ 1.56-1.66 (m, IH), 1.70-1.82 (m, 5H), 1.90 (t, J= 10.8Hz, IH), 2.16-2.26 (m, IH), 2.40 (t, J= 7.2Hz, IH), 2.60-2.72 (m, IH), 2.74-2.92 (m, 6H), 2.96 (t, J= 7.2Hz, 2H), 3.64 (d, J= 12.0Hz, IH), 3.70 (s, 3H), 6.56 (d, J= 2.7Hz, IH), 6.64 (J;= 9.0Hz, J2= 3.0Hz, IH), 6.71 (d, J= 3.0Hz, IH), 7.09 (t, J= 8.7Hz, 2H), 7.95 (d, J= 8.7Hz, IH), 7.97 (d, J= 8.7Hz, IH). 13CNMR (CDC13, 75MHz) δ 22.2, 26.3, 27.1, 27.3, 38.2, 47.2, 53.3, 55.3, 55.5, 58.1, 59.7, 112.0, 113.4, 114.7, 115.3, 115.6, 125.7, 130.3, 130.5, 140.3, 151.9, 163.6, 167.0, 198.1. Anal. (C24H29FN2O22HCl), C, H, N.
Example 18 8-Methoxy-3-propyl-2,3,4,4a,5,6-hexahydro-lβ-pyrazino[l,2- ]quinoline (Compound
7d) Alkylation of compound 5b with 1-iodopropane as described in method A (Example 9) gave 190mg of compound 7d as a colorless oil, yield 73%. 'HNMR (CDC13, 300MHz) δ 0.91 (t, J= 7.5Hz, 3H), 1.44-1.60 (m, 2H), 1.62-1.79 (m, IH), 1.80-1.86 (m, IH), 1.90 (t, J= 7.5Hz, IH), 2.14-2.28 (m, IH), 2.32 (t, J= 7.8Hz, 2H), 2.60-2.70 (m, IH), 2.70-2.92 (m, 4H), 2.96 (t, J= 10.2Hz, IH), 3.64 (d, J= 12.0Hz, IH), 3.71 (s, 3H), 6.57 (d, J= 2.7Hz, IH), 6.66 (dd, Jι= 9.3Hz, J2= 2.7Hz, IH), 6.72 (d, J= 9.3Hz, IH). 13CNMR (CDC13, 75MHz) δ 12.4, 20.4, 27.6, 27.8, 47.7, 53.8, 55.8, 56.0, 60.2, 61.0, 112.4, 113.9, 115.2, 126.2, 140.9, 152.4. Anal. (Cι6H24N2O2HCl), C, H, N.
Example 19 8-Methoxy-3-butyl-2,3,4,4a,5,6-hexahydro-l//-pyrazino [1 ,2- ] quinoline (Compound 7e)
Alkylation of compound 5b with 1-iodobutane as described in method A (Example 9) gave 208mg of compound 7e as a colorless oil, yield 76%. 'HNMR (CDC13, 300MHz) δ 0.92 (t, J= 7.5Hz, 3H), 1.20-1.40 (m, 2H), 1.40-1.56 (m, 2H), 1.62-1.84 (m, 2H), 1.89 (t, J= 10.5Hz, IH), 2.10-2.22 (m, IH), 2.34 (t, J= 7.8Hz, 2H), 2.60-3.0 (m, 6H), 3.63 (d, J= 12.0Hz, IH), 3.71 (s, 3H), 6.57 (d, J= 2.7Hz, IH), 6.64 (dd, J,= 8.7Hz, J2= 2.7Hz, IH), 6.72 (d, J= 8.7Hz, IH). 13CNMR (CDC13, 75MHz) δ 13.7, 20.4, 26.8, 27.0, 28.5, 46.8, 53.0, 54.9, 55.1, 58.0, 59.4, 111.6, 113.0, 114.3, 125.3, 140.0, 151.5. Anal. (Cι7H26N2O2HCl), C, H, N. Example 20 [3H]PD 128907 binding assays
3
In some embodiments, [ H]PD 128907 binding assays for D3 receptors dopamine receptors are performed as previously described in Levant. (See, B. Levant, Cuπent Protocols in Pharmacology (J Ferkany and SJ Enna, Eds) John Wiley & Sons, New York, 1.6.1-1.6.16 [1998]). Rat ventral striatum (nucleus accumbens and olfactory tubercles) is prepared in assay buffer (50 mM Tris, 1 mM EDTA; pH 7.4 at 23 °C) to yield a final concentration of 10 mg original wet weight (o.w.w.)/ml. Membranes are incubated with
[ H]PD 128907 0.3 nM; 116 Ci/mmol; (Amersham, Arlington Heights, IL) and various concentrations of competing compounds (10"10 to IO"4 M). Nonspecific binding is defined by 1 μM spiperone. Assay tubes are incubated at 23 °C for 3 hr. The reaction is terminated by rapid vacuum filtration. Data is analyzed using the non-linear least-squares curve-fitting program LIGAND.
Examples 21
3
[ H] Spiperone binding assays
3
In some embodiments, [ H] spiperone binding assays are performed as previously
3 described in Levant for [ H]PD 128907, except for the following. (See, B. Levant, Cuπent Protocols in Pharmacology (J Ferkany and SJ Enna, Eds) John Wiley & Sons, New York, 1.6.1-1.6.16 [1998]). For D2 dopamine receptors binding in brain, assays are performed using membranes prepared from rat caudate-putamen and the final membrane homogenate concentration is 1.5 mg o.w.w./ml. For assays in hD2s, hD3, and hD4 receptors, the membrane concentration is determined for each receptor-expressing cell line in preliminary studies. The assay buffer is 50 mM Tris-HCI, 5 mM KCl, 2 mM MgCl2, and 2 mM CaCl2, pH 7.4 at 23°C; the concentration of [3H]spiperone (24 Ci/mmol; Amersham) is 200 pM; and the incubation time was 2 h at 23 °C Nonspecific binding is defined in the presence of 1 μM (+)-butaclamol. Example 22
[3H]SCH 23390 binding assays
In some embodiments, [^H] SCH 23390 binding assays for Drlike dopamine receptors are performed as previously described in Levant (See, B. Levant, Cuπent Protocols in Pharmacology (J Ferkany and SJ Enna, Eds) John Wiley & Sons, New York,
3
1.6.1-1.6.16 [1998]) and as described for [ H]PD spiperone binding assays, except for the following. The concentration of [3H]SCH 23390 (73 Ci/mmol; Amersham) is 300 pM, and the incubation time is 30 min at 37 °C.
Example 23
Receptor autoradiography with [3 H] quinpirole
In some embodiments, sagittal brain sections (20 μm, lateral 1.00 - 1.40 mm) are cut on a cryostat, thaw-mounted onto chrome alum/gelatin-coated slides, and processed as previously described in Levant. (B. Levant, and E.B. De Souza, Synapse, 14:90-95 [1993]). Sections are incubated for 2 hr at 23 °C with 10 nM [ H]quinpirole (50.6 Ci/mmol; NEN Life Science, Boston, MA), in the presence or absence of 5 concentrations of competing drug (IO"9 to lθ"5 M or lθ"8 to IO"4 M), in assay buffer (50 mM Tris-HCI, 5 mM KCl, 2 mM MgCl2, and 2 mM CaCl2, pH 7.4 at 23 °C). Duplicate sections from each animal are used for each data point. Autoradiograms are generated using H-Hyperfilm (Amersham,
3 Arlington Heights, IL) and 20 μm [ H]methylmethacrylate standards (Amersham).
Autoradiographic images are quantified NTH "Image" version 1.6.
Example 24 Mitogenensis Assays In some embodiments, mitogenesis assays are performed as previously described in
Chio et al. (See, CL. Chio, Mol. Pharmacol., 45:51-60 [1994]). CHO cells expressing hD2s or hD3 are seeded into 96-well plates at density of 5,000 cells/well and grown at 37° C in MEM with 10% fetal calf serum for 48 hr. Wells are washed with serum-free MEM and incubated with various concentrations of drug (10"10 to IO"4 M) or vehicle and cultured for 16 hr. Antagonism of agonist-stimulated mitogenesis is determined in the presence 10 nM quinpirole. [3H]Thymidine (1 μCi/well; 25 Ci/mmol; Amersham) are then be added to each well. After 2 hr incubation, cells are trypsinized and harvested by rapid vacuum filtration.
[3H]Thymidine incorporation in each sample is calculated as fmol incorporated/mg protein. Data are present as percent stimulation over basal. The EC50, IC50, and Emaχ values are calculated by nonlinear regression analysis using a four-parameter model (Sigma Plot, SPSS, Chicago, IL).
Example 25
[35S]GTP-S Binding Assays
35
In some embodiments, [ S]GTP-S binding assays are performed in CHO cell membranes containing hD2s, hD3, or hD4.4 receptors as previously described in Wang et al. (See, S. Wang et al, J. Med. Chem., 39:2047-2054 [1996]; and S. Wang et al, J. Med. Chem., 43:351-360 [2000]).
Example 26 'HNMR spectra
In some embodiments, 1HNMR spectra were recorded at 300MHz and 13CNMR spectra were recorded at 75MHz both on Varian Mercury 300 and Bruker DPX 300 spectrometer. Chemical shifts are given in δ units (ppm) and are relative to the solvent. Coupling constants are given in hertz (Hz). Elemental analyses were performed by the Micro-Analysis, Inc., (Wilmington, DE) and are within 0.4% of the theoretical values, except where noted. All the reagents and chemicals were purchased from Aldrich Chemical Co., Fisher Scientific, or Lancaster Synthesis, Inc., and used without further purification. All the reactions were run under nitrogen unless otherwise indicated.
Example 27 Additional compounds
This Example describes additional compounds contemplated for use in the present invention.
Synthesis 5-methoxyl, or 7-methoxyl, or 8-methoxyl quinaldine
The 8-methoxyl quinaldine is obtained very easily by methylation of commercially available 8-hydroixyl quinaldine. To create 5-methoxyl quinaldine, first the amine 163 is treated with acyl acetate ethyl ester to get the coupling product (compound 164). Compound 64 is cyclized by heating to 250°C to yield compound 165, and the hydroxyl group in compound 165 is converted to a chloride, then the chloride is removed by hydrogenation to yield the 5-methoxyl quinaldine. 7-methoxyl quinaldine is obtained by the same method.
161 162
Synthesis of the important intermediate compound 177
5-methoxyl, or 7-methoxyl, or 8-methoxyl quinaldine is oxidized by SeO2 to the aldehyde (compound 174). The aldehyde (compound 174) is treated with 2-amino ethanol and NaBH4 to yield compound 178. Next, hydrogenation and cyclization yield the important intermediate compound 177.
a) 5-methoxyl 177 b)7-methoxyl a) 5-methoxyl c)-8-methoxyl b)7-methoxyl c)-8-methoxyl Synthesis of the D3 ligands
With the important intermediate in hand, potential D3 ligands are obtained by treating compound 177 with different alkyl halides. Nine D3 ligands are obtained. As to the compound 183, the synthetic method is different. Compound 183 is obtained by treating compound 177c with 4-heptanone and NaBH4.
a) 5-methoxyl b)7-methoxyl c)-8-methoxyl
Synthesis of the tail of the compounds 186 or 182
The tail of compounds 186 or 182 is synthesized by using compound 187 as the starting material. The hydroxyl group is selectively protected with TBS group, and the amine is treated with 2-naphthoyl chloride to yield the amide (compound 189). Next the TBS group is removed, and the alcohol (compound 190) is treated with CBr and Ph P in THF. Compound 192 comes from the reaction between compound 190 and the solvent (THF). Compound 192 is treated the MsCl to get the compound 191.
Example 28 Additional compounds
This Example describes additional compounds contemplated for use in the present invention.
Table 11
No. R R2
6a H (CH2)2Ph 6b H (CH2)3Ph 6c H (CH2)4Ph d H CH2Ph-Ph 6e H (CH2)2Ph-Ph 6f H (CH2)3Ph-Ph 6g H (CH2)4Ph-Ph
6h H σ 6i H
Reagent and condition: i. a) 1.1 eq. ethanolamine, benzene, under N2, reflux, remove H2O, overnight; b) 3.5 eq. NaBH4, absolute ethanol, under N2, reflux, overnight, yield 87-90%o. ii. Ca. 5g nickel-aluminum alloy/lg substrate, IM KOH, methanol, rt, overnight, yield 85- 89%. iii. 3 eq. P2O5, xylenes, under N2, reflux, overnight, yield 82-86%. iv. 1.2 eq. R2C1/ Nal or 1.2 eq. R2I, 2 eq. Cs2CO3, CH3CN, under N2, reflux, overnight, yield 61- 76%.
10a γ=H, n=2 11a Y=H, n=2
10b Y=H, n=3 11b Y=H, n=3
10c Y=H, n=4 11c Y=H, n=4
10d Y=OCH3, n=4
Reagent and condition: v. 2 eq. hydrazine, EtOH, reflux, 2h, yield 87-94%. vi. 1.2 eq. 2- naphthoyl chloride , 3 eq. triethylamines, 0°C, 4h, yield 91-95%. vii. 1.2 eq. 4- biphenylcarbonyl chloride , 3 eq. triethylamines, 0°C, 4h, yield 72-75%.
10d Y=OCH3, n=4
11d Y=OCH3, n=4
12d Y=OCH3, n=4
Reagent and condition: viii. . 4 eq. BBr3, CH2C12, under N2, 0°C, 4h, yield 52%. ix. 1.2 eq. >-toluenesulfonyl chloride, 3 eq. triethylamines, 0°C, 4h, yield 84%.
All publications and patents mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific prefeπed embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the relevant fields are intended to be within the scope of the following claims.

Claims

We claim:
A composition comprising a compound of formula:
wherein,
X, Y, and Z independently represent C, O, N, S;
R7, R8 , and R9 independently represent H, F, Cl, Br, I, OH, CN, NO2, OR', CO2R', OCOR',
CONR'R", NR"COR', NR'SO2R", SO2NR'R", NR'R" where R' and R" independently represents H, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, or heterocyclic, substituted or unsubstituted;
A and B independently represent O, S, SO, SO2, NR', CR'R" where R' and R" independently represent H, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, or heterocyclic, substituted or unsubstituted;
Ri, R2,R3;R4jR5, and R^ independently represent H, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, or heterocyclic, substituted or unsubstituted; where n= 0, 1, or 2;
R represents alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, or heterocyclic, substituted or unsubstituted; the examples include CnH n+ι, (CH2)nR'" where n=l-6, R'" represents aryl, substituted or unsubstituted. A composition comprising a compound of formula:
3. A composition comprising a compound of formula:
wherein,
X, Y, Z, D and M independently represent C, O, N, S; R7, R8 , R9 , Rio, Ri !, R]2, Rι3> Rι , and Rι5 independently represent H, F, Cl, Br, I, OH, CN, NO2, OR', CO2R', OCOR', CONR'R", NR"COR', NR'SO2R", SO2NR'R", NR'R" where R' and R' ' independently represents H, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, or heterocyclic, substituted or unsubstituted; A and B independently represent O, S, SO, SO2, NR', CR'R" where R' and R" independently represent H, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, or heterocyclic, substituted or unsubstituted;
Ri, R2, R3> R4, R5> and Re independently represent H, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, or heterocyclic, substituted or unsubstituted; where n= 0, 1, or 2; where m= 1- 20; R represents H, O, S, SO, SO2, NR', CR'R" where R' and R" independently represent H, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, or heterocyclic, substituted or unsubstituted.
4. A composition comprising a compound of formula:
wherein,
X, Y, Z, D, and M independently represent C, O, N, S;
R7, R8 , R9 , and Rι0 independently represent H, F, Cl, Br, I, OH, CN, NO2, OR', CO2R',
OCOR', CONR'R", NR"COR', NR'SO2R", SO2NR'R", NR'R" where R' and R" independently represents H, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, or heterocyclic, substituted or unsubstituted;
A and B independently represent O, S, SO, SO2, NR', CR'R" where R' and R" independently represent H, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, or heterocyclic, substituted or unsubstituted; Ri, R2, R3, t, R5, and Rό independently represent H, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, or heterocyclic, substituted or unsubstituted; where n= 0, 1, or 2; E represents a linker group that does not contain an amide group and F represents cycloalkyl, cycloalkenyl, or heterocyclic, substituted or unsubstituted; the examples include CnH π+ι, (CH2)nR' ' ' where n=l -6, R' ' ' represents aryl, substituted or unsubstituted.
5. A method for identifying agonists and antagonists of dopamine receptors, comprising the step of assessing an interaction between a candidate ligand and an amino acid of a dopamine receptors selected from the group consisting of N47, D75, F338, N375, and N379 of receptor D3, N52, D80, F382, N418, and N422 of receptor D2.
6. The method of Claim 5, wherein said assessing comprises investigating a computer model for a predicted interaction between said ligand and said amino acid.
7. The method of Claim 5, wherein said assessing comprises binding said ligand to said dopamine receptor and determining binding between said ligand and said amino acid.
8. The method of Claim 7, wherein said determining comprises determining the crystal structure of said ligand bound to said receptor.
9. The method of Claim 7, wherein said determining comprises comparing a binding affinity between said ligand and said receptor with a binding affinity between said ligand and a receptor lacking said amino acid.
10. The method of Claim 7, wherein said determining comprises measuring the ability of said ligand to displace a molecule bound to said amino acid of said receptor.
11. A method of treatment for a subject having a condition selected from the group consisting of cocaine abuse, depression, anxiety, an eating disorder, alcoholism, chronic pain, obsessive compulsive disorder, schizophrenia, restless legs syndrome (RLS), and Parkinson's disease, wherein the method comprises administering to the subject a therapeutic dose of the composition of Claim 1.
12. A method of treatment for a subject having a condition selected from the group consisting of cocaine abuse, depression, anxiety, an eating disorder, alcoholism, chronic pain, obsessive compulsive disorder, schizophrenia, restless legs syndrome (RLS), and Parkinson's disease, wherein the method comprises administering to the subject a therapeutic dose of the composition of Claim 2.
13. A method of treatment for a subject having a condition selected from the group consisting of cocaine abuse, depression, anxiety, an eating disorder, alcoholism, chronic pain, obsessive compulsive disorder, schizophrenia, restless legs syndrome (RLS), and Parkinson's disease, wherein the method comprises administering to the subject a therapeutic dose of the composition of Claim 3.
14. A method of treatment for a subject having a condition selected from the group consisting of cocaine abuse, depression, anxiety, an eating disorder, alcoholism, chronic pain, obsessive compulsive disorder, schizophrenia, restless legs syndrome (RLS), and Parkinson's disease, wherein the method comprises administering to the subject a therapeutic dose of the composition of Claim 4.
15. A method of modulating the action of a G-protein coupled receptor in a subject comprising administering to the subject an effective amount of the composition of Claim 1.
16. A method of modulating the action of a G-protein coupled receptor in a subject comprising administering to the subject an effective amount of the composition of Claim 2.
17. A method of modulating the action of a G-protein coupled receptor in a subject comprising administering to the subject an effective amount of the composition of Claim 3.
18. A method of modulating the action of a G-protein coupled receptor in a subject comprising administering to the subject an effective amount of the composition of Claim 4.
19. A method of control of dopamine flow in a subject in need of such control comprising administering to said subject an effective amount of the composition of Claim 1.
20. A method of control of dopamine flow in a subject in need of such control comprising administering to said subject an effective amount of the composition of Claim 2.
21. A method of control of dopamine flow in a subject in need of such control comprising administering to said subject an effective amount of the composition of Claim 3.
22. A method of control of dopamine flow in a subject in need of such control comprising administering to said subject an effective amount of the composition of Claim 4.
23. A method of treating a subject with a D3 receptor-specific modulator, comprising administering to said subject a compound of formula (I) or formula (II), wherein formula (I) is:
wherein, X, Y, Z and M independently represent C, O, N, S;
R7, R8 , R9 , and R10 independently represent H, F, Cl, Br, I, OH, CN, NO2, OR', CO2R', OCOR', CONR'R", NR"COR', NR'SO2R", SO2NR'R", NR'R" where R' and R" independently represents H, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, or heterocyclic, substituted or unsubstituted;
A and B independently represent O, S, SO, SO2, NR', CR'R" where R' and R" independently represent H, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, or heterocyclic, substituted or unsubstituted;
Ri, R , R3, R4, R5> and Rό independently represent H, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, or heterocyclic, substituted or unsubstituted; where n= 0, 1, or 2;
R represents alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, or heterocyclic, substituted or unsubstituted; the examples include CnH2n+1, (CH2)nR'" where n=l-6, R'" represents aryl, substituted or unsubstituted; and formula (II) is:
wherein,
X, Y, and Z independently represent C, O, N, S;
R7, R8 , and R9 independently represent H, F, Cl, Br, I, OH, CN, NO2, OR', CO2R', OCOR', CONR'R", NR"COR', NR'SO2R", SO2NR'R", NR'R" where R' and R" independently represents H, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, or heterocyclic, substituted or unsubstituted;
A and B independently represent O, S, SO, SO2, NR', CR'R" where R' and R" independently represent H, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, or heterocyclic, substituted or unsubstituted; Ri, R2, R3, R-t, R5, and Re independently represent H, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, or heterocyclic, substituted or unsubstituted; where n= 0, 1, or 2; R represents alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, or heterocyclic, substituted or unsubstituted; the examples include CnH2n-n, (CH2)nR"' where n=l-6, R'" represents aryl, substituted or unsubstituted, wherein said subject is characterized as having previously responded poorly or is believed likely to respond poorly to a dopamine receptor modulator that binds specifically to D3 and D2 or Dl receptors.
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