JP2005517630A - Dopamine receptor ligand and therapeutic method based thereon - Google Patents

Abstract

The present invention relates to dopamine receptors (eg D ₁ , D ₂ , D ₃ , D ₄ and D ₅ ), in particular ligands (eg agonists, antagonists and partial agonists) to D ₁ , D ₂ , and D ₃ dopamine receptors. Relates to compounds designed as The invention also relates to methods for the rational design of these compounds and therapeutic methods using these compounds.

Description

This application claims priority to US Provisional Patent Application No. 60 / 297,445 filed on June 13, 2001 and US Provisional Patent Application No. EV092300689US filed on June 7, 2002. . Both of which are hereby incorporated by reference in their entirety. This research is supported by the National Institutes of Health grant number: Assisted by. The US government has certain rights to the invention.

FIELD OF THE INVENTION The present invention relates to dopamine receptors (eg, D ₁ , D ₂ , D ₃ , D ₄ , and D ₅ ), particularly ligands (eg, agonists) to D ₁ , D ₂ , and D ₃ dopamine receptors. , Antagonists and partial agonists). The invention also relates to methods for the rational design of these compounds and therapeutic methods using these compounds.

BACKGROUND OF THE INVENTION The value of drug abuse, including cocaine abuse, is enormous and constantly increasing. In a recent report prepared for the Office of National Drug Control Policy, in 1998 alone, illegal drug abuse in the United States cost approximately 143,400,000,000, and from 1992 to 1998 It was estimated that the overall social consideration of drug abuse in the United States increased during the year at a rate of 5.9 percent. Lost productivity (98,500,000,000) and expenses for the treatment of related medical conditions (12,900,000,000) accounted for two major parts of this enormous burden. In 2000, an estimated 14,000,000 Americans – nearly 6.3% of the US population over the age of 12 were illegal drug users.

  Addiction to some illegal drugs such as heroin can be successfully treated with drug therapy (eg, methadone, naloxone, and naltrexone), but cocaine and crack cocaine (“crack” is a free base because of smoking There is no effective pharmacotherapy available to treat addiction to the cocaine hydrochloride processed to the same name). Thus, cocaine addiction is particularly problematic for both drug addicts and society as a whole. In addition, cocaine is one of the most commonly addicted known substances, and as a result, cocaine addicts often lose their ability to work and their interpersonal abilities.

  A 1998 National Household Survey on Drug Abuse estimated that 1,700,000 Americans were currently cocaine or crack cocaine users. Also noteworthy is the fact that cocaine users appear to be rising in American youth. Among the 1st to 4th graders who graduated from high school, in 1995, 3.6% used cocaine within the past year and 0.7% used cocaine in the previous month. The proportion of high school seniors who have used cocaine at least once increased from 5.9% in 1994 to 9.8% in 1999. In 1999, the number of 10th graders who had tried cocaine at least once rose to 7.7% from 3.3% in 1992. The proportion of eighth graders who have tried cocaine so far increased from a low of 2.3% in 1991 to 4.7% in 1999.

  Despite considerable progress towards understanding the neuropharmacological basis of cocaine addiction, there is no effective pharmacotherapy for treating cocaine addiction. (F. I. Carroll et al., J. Med. Chem., 42: 2721-2731 [1999]). Correspondingly, the clinical demands of cocaine-dependent patients are broad and patients are likely to benefit from medications that act to interrupt any stage of the cocaine-dependent cycle. Thus, cocaine abuse remains a major health problem and social concern.

  Accordingly, there is a need for improved compositions and methods for treating cocaine abuse, and more generally, improved compositions and methods for discovering and designing potent and specific drug therapies.

SUMMARY OF THE INVENTION The present invention relates to dopamine receptors (eg, D ₁ , D ₂ , D ₃ , D ₄ , and D ₅ ), in particular, ligands (eg, agonists) to D ₁ , D ₂ , and D ₃ dopamine receptors. , Antagonists and partial agonists). The invention also relates to methods for the rational design of these compounds and therapeutic methods using these compounds. However, it is not intended that the present invention be limited to compositions that modulate dopamine receptors (eg, agonists, antagonists, and partial agonists). Indeed, some aspects of the invention generally provide methods compositions (eg, agonists, antagonists, and partial agonists) for modulating G protein-coupled receptors, and discover and design these compounds. Directed to a method for.

A preferred embodiment of the invention provides compounds that are rationally designed to control dopamine flow in the brain. In other preferred embodiments, these compounds are highly selective antagonists of dopamine family receptors (eg, D ₃ ). In yet other embodiments, rational design of the compounds of the invention involves identifying mechanisms associated with dopamine flux in the brain. The information about the mechanism is then analyzed so that compound structures with activities that can interfere with such a mechanism are expressed. In some specific embodiments, the structure is synthesized based on “building blocks”, each building block involving a particular mechanism associated with dopamine flux, particularly one or more members of the D receptor family It has the feature that it can potentially interfere with the mechanism to do.

  Compounds with various building block combinations are then synthesized and tested for their activity with respect to the identified mechanism. Such tests are preferably performed in vitro and / or in vivo. The information obtained through such testing is then incorporated into a new cycle of rational drug design. The design-synthesis-test cycle is repeated until a candidate compound is obtained that has the desired properties for controlling the targeted therapy; eg, dopamine flow. The candidate compound is then preferably tested clinically.

One approach to modulating (eg, controlling) dopamine flux in the brain to treat cocaine addiction is to design cocaine antagonists that can affect dopamine uptake. More specifically, this approach is based on rational design D receptor family, is an antagonist of cocaine, such as to reduce dopamine or block particularly bind to receptors D ₃ compounds. In preferred embodiments, the antagonist is designed to reduce or block cocaine binding but not be affected by other aspects of dopamine transport. Engineered antagonists provide the basis for therapeutic protocols based on selective control of dopamine transport, thereby causing no or little disruption of the normal flow of dopamine in the brain Should control synaptic signaling.

  In preferred embodiments, the present invention provides compositions comprising one or more compounds disclosed herein or derivatives of such compounds (eg, derivatives having minor chemical substitutions). The present invention also provides methods of using such compounds. It will be understood that the methods described herein can be used with the compounds disclosed herein and their derivatives.

式中、X、Y、およびZは、独立してC、O、N、Sを表し；
R₇、R₈、およびR₉は、独立してH、F、Cl、Br、I、OH、CN、NO₂、OR’、CO₂R’、OCOR’、CONR'R''、NR''COR’、NR'SO₂R''、SO₂NR'R''、NR'R''を表し、式中R'およびR''は、独立してH、アルキル、シクロアルキル、アルケニル、シクロアルケニル、アルキニル、アリール、もしくは複素環の、置換または非置換のものを表し；
AおよびBは、独立してO、S、SO、SO₂、NR'、CR'R''を表し、式中R'およびR''は、独立してH、アルキル、シクロアルキル、アルケニル、シクロアルケニル、アルキニル、アリール、もしくは複素環の、置換または非置換のものを表し；
R₁、R₂、R₃、R₄、R₅、およびR₆は、独立してH、アルキル、シクロアルキル、アルケニル、シクロアルケニル、アルキニル、アリール、もしくは複素環の、置換または非置換のものを表し；式中、n=0、1、または2；
Rは、アルキル、シクロアルキル、アルケニル、シクロアルケニル、アルキニル、アリール、もしくは複素環の、置換または非置換のものを表し；例には、n=1〜6、R'''がアリールの、置換または非置換のものを表すC_nH_2n+1,（CH₂）_nR'''を含む。 In a preferred embodiment, the present invention provides a composition comprising a compound of the following formula (including derivatives of this formula):

In which X, Y and Z independently represent C, O, N, S;
R ₇ , R ₈ , and R ₉ are independently H, F, Cl, Br, I, OH, CN, NO ₂ , OR ′, CO ₂ R ′, OCOR ′, CONR′R ″, NR ′. Represents' COR ', NR'SO ₂ R'', SO ₂ NR'R'',NR'R'', wherein R' and R '' are independently H, alkyl, cycloalkyl, alkenyl, Represents a substituted or unsubstituted cycloalkenyl, alkynyl, aryl, or heterocyclic ring;
A and B independently represent O, S, SO, SO ₂ NR ′, CR′R ″, wherein R ′ and R ″ are independently H, alkyl, cycloalkyl, alkenyl, Represents a substituted or unsubstituted cycloalkenyl, alkynyl, aryl, or heterocyclic ring;
R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , and R ₆ are independently H, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, or heterocyclic, substituted or unsubstituted Wherein n = 0, 1, or 2;
R represents alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, or heterocyclic substituted or unsubstituted; for example, n = 1-6, R ′ ″ is aryl substituted Or C _n H _{2n + 1} , (CH ₂ ) _n R ′ ″, which represents an unsubstituted one.

In yet another preferred embodiment, the present invention provides a composition comprising a compound of the following formula (including derivatives of this formula):

式中、X、Y、Z、D、およびMは、独立してC、O、N、Sを表し；
R₇、R₈、R₉、R₁₀、R₁₁、R₁₂、R₁₃、R₁₄、およびR₁₅は、独立してH、F、Cl、Br、I、OH、CN、NO₂、OR’、CO₂R’、OCOR’、CONR'R''、NR''COR’、NR'SO₂R''、SO₂NR'R''、NR'R''を表し、式中R'およびR''は、独立してH、アルキル、シクロアルキル、アルケニル、シクロアルケニル、アルキニル、アリール、もしくは複素環の、置換または非置換のものを表し；
AおよびBは、独立してO、S、SO、SO₂、NR’、CR'R''を表し、式中R'およびR''は、独立して、H、アルキル、シクロアルキル、アルケニル、シクロアルケニル、アルキニル、アリール、もしくは複素環の、置換または非置換のものを表し；
R₁、R₂、R₃、R₄、R₅、およびR₆は、独立してH、アルキル、シクロアルキル、アルケニル、シクロアルケニル、アルキニル、アリール、もしくは複素環の、置換または非置換のものを表し；式中、n=0、1、または2；式中、m=1〜20；Rは、H、O、S、SO、SO₂、NR’、CR'R''を表し、式中R'およびR''は、独立してH、アルキル、シクロアルキル、アルケニル、シクロアルケニル、アルキニル、アリール、もしくは複素環の、置換または非置換のものを表す。 In yet another preferred embodiment, the present invention provides a composition comprising a compound of the following formula (including derivatives of this formula):

In which X, Y, Z, D and M independently represent C, O, N, S;
R ₇ , R ₈ , R ₉ , R ₁₀ , R ₁₁ , R ₁₂ , R ₁₃ , R ₁₄ , and R ₁₅ are independently 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 ″ each independently represents a substituted or unsubstituted H, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, or heterocyclic ring;
A and B, O _{independently, S, SO, SO 2,} NR ', CR'R' represent ', in R' and R '' formula, independently, H, alkyl, cycloalkyl, alkenyl Represents substituted, unsubstituted, cycloalkenyl, alkynyl, aryl, or heterocyclic;
R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , and R ₆ are independently H, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, or heterocyclic, substituted or unsubstituted Wherein n = 0, 1, or 2; where m = 1-20; R represents H, O, S, SO, SO ₂ NR ′, CR′R ″, and the formula R ′ and R ″ each independently represents a substituted or unsubstituted H, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, or heterocyclic ring.

式中、X、Y、Z、D、およびMは、独立してC、O、N、Sを表し；
R₇、R₈、R₉、およびR₁₀は、独立してH、F、Cl、Br、I、OH、CN、NO₂、OR’、CO₂R’、OCOR’、CONR'R''、NR''COR’、NR'SO₂R''、SO₂NR'R''、NR'R''を表し、式中R'およびR''は、独立してH、アルキル、シクロアルキル、アルケニル、シクロアルケニル、アルキニル、アリール、もしくは複素環の、置換または非置換のものを表し；
AおよびBは、独立してO、S、SO、SO₂、NR'、CR'R''を表し、式中R'およびR''は、独立してH、アルキル、シクロアルキル、アルケニル、シクロアルケニル、アルキニル、アリール、もしくは複素環の、置換または非置換のものを表し；
R₁、R₂、R₃、R₄、R₅、およびR₆は、独立してH、アルキル、シクロアルキル、アルケニル、シクロアルケニル、アルキニル、アリール、もしくは複素環の、置換または非置換のものを表し；式中、n=0、1、または2；Eは、アミド基を含まないリンカー基を表し、およびFは、シクロアルキル、シクロアルケニル、もしくは複素環の、置換または非置換のものを表し；例には、n=1〜6、R'''がアリールの、置換または非置換のものを表すC_nH_2n+1,（CH₂）_nR'''を含む。基Eは、炭素鎖、繰り返しポリマーユニットなどを含むどのような結鎖化学であることもできるが、これらに限定されない。基Fは、好ましくは置換もしくは非置換のベンゼン環または多環複合体などの大きな基である。 In a further preferred embodiment, the present invention provides a composition comprising a compound of the following formula (including derivatives of this formula):

In which X, Y, Z, D and M independently represent C, O, N, S;
R ₇ , R ₈ , R ₉ , and R ₁₀ are independently 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'', wherein R' and R '' are independently H, alkyl, cycloalkyl Represents substituted, unsubstituted, alkenyl, cycloalkenyl, alkynyl, aryl, or heterocyclic;
A and B independently represent O, S, SO, SO ₂ NR ′, CR′R ″, wherein R ′ and R ″ are independently H, alkyl, cycloalkyl, alkenyl, Represents a substituted or unsubstituted cycloalkenyl, alkynyl, aryl, or heterocyclic ring;
R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , and R ₆ are independently H, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, or heterocyclic, substituted or unsubstituted Wherein n = 0, 1, or 2; E represents a linker group that does not include an amide group, and F represents a substituted or unsubstituted cycloalkyl, cycloalkenyl, or heterocyclic ring Examples; examples include C _n H _{2n + 1} , (CH ₂ ) _n R ′ ″, where n = 1-6, R ′ ″ represents aryl, substituted or unsubstituted. Group E can be any chain chemistry including, but not limited to, carbon chains, repeating polymer units, and the like. The group F is preferably a large group such as a substituted or unsubstituted benzene ring or a polycyclic complex.

The present invention also provides a method for identifying dopamine receptor agonists and antagonists comprising the step of assessing the interaction between a candidate ligand and an amino acid of the dopamine receptor. In some embodiments, the amino acid of the dopamine receptor envisioned, Y32 receptors _{D 3, S35, Y36, L39} , F345, F346, T368, T369, Y365, P200, S192, S193, S197, V111, I183 , F188, V164, S182, and A161, Y37 receptors _{D 2, L39, L41, L44} , F198, F389, F390, F411, S192, S193, S196, V115, I194, F189, and although S163 and the like, It is not limited to these. In some preferred embodiments, the amino acid of the dopamine receptor envisioned, N47 receptors _{D 3, D75, F338, N375} , and N379, N52 receptors _{D 2, D80, F382, N418} , and an N422 However, it is not limited to these. In still other embodiments, the contemplated amino acids of the dopamine receptor are N47, D75, V78, V82, V86, F106, D110, V111, C114, L121, V164, L168, I183, F188 of receptor D3. , S192, S193, V195, S196 , F197, F338, W342, F345, F346, H349, V350, Y365, T369, Y373, N375, and N379, N52 receptors _{D 2, D80, V85, V87} , V91, F110 D114, V115, C118, L125, I166, L170, I184, F189, S193, S194, V196, S197, F198, F382, W386, F389, F390, H393, I394, Y408, T412, Y416, N418, and N422 Including, but not limited to.

  In a preferred embodiment, the evaluating step in identifying agonists and antagonists of dopamine receptors includes examining a computer model for the predicted interaction between the ligand and the amino acid. In still other preferred embodiments, the evaluating step comprises binding a ligand to the dopamine receptor to determine 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.

  In some further aspects of the invention, determining the binding between the ligand and the amino acid comprises binding affinity between the ligand and the receptor lacking the amino acid, and between the ligand and the receptor. It is assumed to include comparing the binding affinity between. Some other further aspects further envision that determining the binding between the ligand and the amino acid comprises measuring the ability of the ligand to displace the molecule bound to the amino acid of the receptor.

式中、X、Y、およびZは、独立してC、O、N、Sを表し；
R₇、R₈、およびR₉は、独立してH、F、Cl、Br、I、OH、CN、NO₂、OR’、CO₂R’、OCOR’、CONR'R''、NR''COR’、NR'SO₂R''、SO₂NR'R''、NR'R''を表し、式中R'およびR''は、独立してH、アルキル、シクロアルキル、アルケニル、シクロアルケニル、アルキニル、アリール、もしくは複素環の、置換または非置換のものを表し；
AおよびBは、独立してO、S、SO、SO₂、NR’、CR'R''を表し、式中R'およびR''は、独立してH、アルキル、シクロアルキル、アルケニル、シクロアルケニル、アルキニル、アリール、もしくは複素環の、置換または非置換のものを表し；
R₁、R₂、R₃、R₄、R₅、およびR₆は、独立してH、アルキル、シクロアルキル、アルケニル、シクロアルケニル、アルキニル、アリール、もしくは複素環の、置換または非置換のものを表し；式中、n=0、1、または2；
Rは、アルキル、シクロアルキル、アルケニル、シクロアルケニル、アルキニル、アリール、もしくは複素環の、置換または非置換のものを表し；例には、n=1〜6、R'''がアリールの、置換または非置換のものを表すC_nH_2n+1,（CH₂）_nR'''を含む。 In one preferred embodiment, the invention relates to a disease, epilepsy, or other pathological condition (eg, cocaine abuse, depression, anxiety, eating disorders, alcoholism, chronic pain, obsessive compulsive disorder, schizophrenia) , Restless leg syndrome (RLS), and Parkinson's disease, etc.), comprising administering to the subject a therapeutic dose of a composition of the invention. For example, in some embodiments, the invention provides a method of treating a subject (eg, administering an effective therapeutic amount / dose) with a composition comprising a compound of the following formula (including derivatives of the formula): provide:

In which X, Y, and Z independently represent C, O, N, S;
R ₇ , R ₈ , and R ₉ are independently H, F, Cl, Br, I, OH, CN, NO ₂ , OR ′, CO ₂ R ′, OCOR ′, CONR′R ″, NR ′. Represents' COR ', NR'SO ₂ R'', SO ₂ NR'R'',NR'R'', wherein R' and R '' are independently H, alkyl, cycloalkyl, alkenyl, Represents a substituted or unsubstituted cycloalkenyl, alkynyl, aryl, or heterocyclic ring;
A and B, O _{independently, S, SO, SO 2,} NR ', CR'R' represent ', in R' and R '' formula, H are independently alkyl, cycloalkyl, alkenyl, Represents a substituted or unsubstituted cycloalkenyl, alkynyl, aryl, or heterocyclic ring;
R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , and R ₆ are independently H, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, or heterocyclic, substituted or unsubstituted Wherein n = 0, 1, or 2;
R represents alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, or heterocyclic substituted or unsubstituted; for example, n = 1-6, R ′ ″ is aryl substituted Or C _n H _{2n + 1} , (CH ₂ ) _n R ′ ″, which represents an unsubstituted one.

In some other embodiments, the invention provides a method of treating a subject (eg, administering an effective therapeutic amount / dose) with a composition comprising a compound of the following formula (including derivatives of the formula): I will provide a:

式中、X、Y、Z、D、およびMは、独立してC、O、N、Sを表し；
R₇、R₈、R₉、R₁₀、R₁₁、R₁₂、R₁₃、R₁₄、およびR₁₅は、独立してH、F、Cl、Br、I、OH、CN、NO₂、OR’、CO₂R’、OCOR’、CONR'R''、NR''COR'、NR'SO₂R''、SO₂NR'R''、NR'R''を表し、式中R'およびR''は、独立してH、アルキル、シクロアルキル、アルケニル、シクロアルケニル、アルキニル、アリール、もしくは複素環の、置換または非置換のものを表し；
AおよびBは、独立してO、S、SO、SO₂、NR’、CR'R''を表し、式中R'およびR''は、独立して、H、アルキル、シクロアルキル、アルケニル、シクロアルケニル、アルキニル、アリール、もしくは複素環の、置換または非置換のものを表し；
R₁、R₂、R₃、R₄、R₅、およびR₆は、独立してH、アルキル、シクロアルキル、アルケニル、シクロアルケニル、アルキニル、アリール、もしくは複素環の、置換または非置換のものを表し；式中、n=0、1、または2；式中、m=1〜20；Rは、H、O、S、SO、SO₂、NR’、CR'R''を表し、式中R'およびR''は、独立してH、アルキル、シクロアルキル、アルケニル、シクロアルケニル、アルキニル、アリール、もしくは複素環の、置換または非置換のものを表す。 In yet another example, a further aspect of the invention treats a subject with a composition comprising a compound of the following formula (including derivatives of the formula) (eg, administering an effective therapeutic amount / dose): ) Provide a method:

In which X, Y, Z, D and M independently represent C, O, N, S;
R ₇ , R ₈ , R ₉ , R ₁₀ , R ₁₁ , R ₁₂ , R ₁₃ , R ₁₄ , and R ₁₅ are independently 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 ″ each independently represents a substituted or unsubstituted H, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, or heterocyclic ring;
A and B, O _{independently, S, SO, SO 2,} NR ', CR'R' represent ', in R' and R '' formula, independently, H, alkyl, cycloalkyl, alkenyl Represents substituted, unsubstituted, cycloalkenyl, alkynyl, aryl, or heterocyclic;
R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , and R ₆ are independently H, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, or heterocyclic, substituted or unsubstituted Wherein n = 0, 1, or 2; where m = 1-20; R represents H, O, S, SO, SO ₂ NR ′, CR′R ″, and the formula R ′ and R ″ each independently represents a substituted or unsubstituted H, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, or heterocyclic ring.

式中、X、Y、Z、D、およびMは、独立してC、O、N、Sを表し；
R₇、R₈、R₉、およびR₁₀は、独立してH、F、Cl、Br、I、OH、CN、NO₂、OR’、CO₂R’、OCOR’、CONR'R''、NR''COR’、NR'SO₂R''、SO₂NR'R''、NR'R''を表し、式中R'およびR''は、独立してH、アルキル、シクロアルキル、アルケニル、シクロアルケニル、アルキニル、アリール、もしくは複素環の、置換または非置換のものを表し；
AおよびBは、独立してO、S、SO、SO₂、NR'、CR'R''を表し、式中R'およびR''は、独立してH、アルキル、シクロアルキル、アルケニル、シクロアルケニル、アルキニル、アリール、もしくは複素環の、置換または非置換のものを表し；
R₁、R₂、R₃、R₄、R₅、およびR₆は、独立してH、アルキル、シクロアルキル、アルケニル、シクロアルケニル、アルキニル、アリール、もしくは複素環の、置換または非置換のものを表し；式中、n=0、1または2；Eは、アミド基を含まないリンカー基を表し、およびFは、シクロアルキル、シクロアルケニル、もしくは複素環の、置換または非置換のものを表し；例には、n=1〜6、R'''がアリールの、置換または非置換のものを表すC_nH_2n+1,（CH₂）_nR'''を含む。 In yet a further aspect, the invention provides a method of treating a subject (eg, administering an effective therapeutic amount / dose) with a composition comprising a compound of the following formula (including derivatives of the formula):

In which X, Y, Z, D and M independently represent C, O, N, S;
R ₇ , R ₈ , R ₉ , and R ₁₀ are independently 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'', wherein R' and R '' are independently H, alkyl, cycloalkyl Represents substituted, unsubstituted, alkenyl, cycloalkenyl, alkynyl, aryl, or heterocyclic;
A and B, O _{independently, S, SO, SO 2,} NR ', CR'R' represent ', in R' and R '' formula, H are independently alkyl, cycloalkyl, alkenyl, Represents a substituted or unsubstituted cycloalkenyl, alkynyl, aryl, or heterocyclic ring;
R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , and R ₆ are independently H, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, or heterocyclic, substituted or unsubstituted In which n = 0, 1 or 2; E represents a linker group that does not contain an amide group, and F represents a substituted or unsubstituted cycloalkyl, cycloalkenyl, or heterocyclic ring Examples include C _n H _{2n + 1} , (CH ₂ ) _n R ′ ″, where n = 1-6, R ′ ″ represents aryl, substituted or unsubstituted.

In some embodiments, the subject treated with the therapeutic composition (eg, formulation) of the present invention is deficient in the regulation and / or function of G protein-coupled receptors (eg, dopamine family receptors). Have symptoms characterized by (for example, abnormal). In a preferred embodiment, the subject to be treated is characterized by a deficiency in the regulation and / or function of dopamine receptors (eg, D ₁ , D ₂ , D ₃ , D ₄ , and / or D ₅ ) 1 Has one or more symptoms. In yet another preferred embodiment, the subject has one or more symptoms characterized by a defective regulation and / or function of D ₃ receptors (e.g., abnormal). In particularly preferred embodiments, the present invention provides therapeutic methods and compositions for treating cocaine addiction / abuse. In some embodiments of the invention, cocaine addiction / abuse is considered a disease.

  A still further aspect of the invention modulates the action of a G protein coupled receptor in a subject (eg, antagonizes and comprises administering to the subject an effective amount of one or more compositions of the invention). (Or agonize) a method. In some embodiments, the present invention provides a method of administering to a subject an agonist of G protein-coupled receptor action. In some other embodiments, the present invention provides a method of administering to a subject an antagonist of G protein coupled receptor action. It is understood that subjects envisioned to be treated (eg, administered) with the therapeutic methods and compositions of the present invention include mammals, and more particularly humans.

  Another aspect of the invention is a method 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 the invention. Provide a method.

式中、X、Y、Z、およびMは、独立してC、O、N、Sを表し；
R₇、R₈、R₉、およびR₁₀は、独立してH、F、Cl、Br、I、OH、CN、NO₂、OR’、CO₂R’、OCOR’、CONR'R''、NR''COR’、NR'SO₂R''、SO₂NR'R''、NR'R''を表し、式中R'およびR''は、独立してH、アルキル、シクロアルキル、アルケニル、シクロアルケニル、アルキニル、アリール、もしくは複素環の、置換または非置換のものを表し；
AおよびBは、独立してO、S、SO、SO₂、NR’、CR'R''を表し、式中R'およびR''は、独立してH、アルキル、シクロアルキル、アルケニル、シクロアルケニル、アルキニル、アリール、もしくは複素環の、置換または非置換のものを表し；
R₁、R₂、R₃、R₄、R₅、およびR₆は、独立してH、アルキル、シクロアルキル、アルケニル、シクロアルケニル、アルキニル、アリール、もしくは複素環の、置換または非置換のものを表し；式中n=0、1、または2；
Rは、アルキル、シクロアルキル、アルケニル、シクロアルケニル、アルキニル、アリール、もしくは複素環の、置換または非置換のものを表し；例には、n=1〜6、R'''がアリールの、置換または非置換のものを表すC_nH_2n+1,（CH₂）_nR'''を含み；ならびに、式（II）は、以下の通りである：

式中、X、Y、およびZは、独立してC、O、N、Sを表し；
R₇、R₈、およびR₉は、独立してH、F、Cl、Br、I、OH、CN、NO₂、OR’、CO₂R’、OCOR’、CONR'R''、NR''COR’、NR'SO₂R''、SO₂NR'R''、NR'R''を表し、式中R'およびR''は、独立してH、アルキル、シクロアルキル、アルケニル、シクロアルケニル、アルキニル、アリール、もしくは複素環の、置換または非置換のものを表し；
AおよびBは、独立してO、S、SO、SO₂、NR’、CR'R''を表し、式中R'およびR''は、独立してH、アルキル、シクロアルキル、アルケニル、シクロアルケニル、アルキニル、アリール、もしくは複素環の、置換または非置換のものを表し；
R₁、R₂、R₃、R₄、R₅、およびR₆は、独立してH、アルキル、シクロアルキル、アルケニル、シクロアルケニル、アルキニル、アリール、もしくは複素環の、置換または非置換のものを表し；式中、n=0、1、または2；
Rは、アルキル、シクロアルキル、アルケニル、シクロアルケニル、アルキニル、アリール、もしくは複素環の、置換または非置換のものを表し；例には、n=1〜6、R'''がアリールの、置換または非置換のものを表すC_nH_2n+1,（CH₂）_nR'''を含み、被験者は、D₃およびD₂もしくはD₁受容体に特異的に結合するドーパミン受容体修飾因子に以前に十分に応答しなかったことによって特徴づけられるか、または十分に応答しない可能性があると考えられる方法を提供する。 A still further aspect of the present invention is a method of treating a subject with specific modifiers to D ₃ receptors, comprising administering a compound of formula (I) or formula (II) to the subject, wherein ( I) (including derivatives of this formula) is as follows:

Wherein X, Y, Z, and M independently represent C, O, N, S;
R ₇ , R ₈ , R ₉ , and R ₁₀ are independently 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'', wherein R' and R '' are independently H, alkyl, cycloalkyl Represents substituted, unsubstituted, alkenyl, cycloalkenyl, alkynyl, aryl, or heterocyclic;
A and B independently represent O, S, SO, SO ₂ NR ′, CR′R ″, wherein R ′ and R ″ are independently H, alkyl, cycloalkyl, alkenyl, Represents a substituted or unsubstituted cycloalkenyl, alkynyl, aryl, or heterocyclic ring;
R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , and R ₆ are independently H, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, or heterocyclic, substituted or unsubstituted Wherein n = 0, 1, or 2;
R represents alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, or heterocyclic substituted or unsubstituted; for example, n = 1-6, R ′ ″ is aryl substituted Or C _n H _{2n + 1} , (CH ₂ ) _n R ′ ″, which represents an unsubstituted one; and formula (II) is as follows:

In which X, Y and Z independently represent C, O, N, S;
R ₇ , R ₈ , and R ₉ are independently H, F, Cl, Br, I, OH, CN, NO ₂ , OR ′, CO ₂ R ′, OCOR ′, CONR′R ″, NR ′. Represents' COR ', NR'SO ₂ R'', SO ₂ NR'R'',NR'R'', wherein R' and R '' are independently H, alkyl, cycloalkyl, alkenyl, Represents a substituted or unsubstituted cycloalkenyl, alkynyl, aryl, or heterocyclic ring;
A and B independently represent O, S, SO, SO ₂ NR ′, CR′R ″, wherein R ′ and R ″ are independently H, alkyl, cycloalkyl, alkenyl, Represents a substituted or unsubstituted cycloalkenyl, alkynyl, aryl, or heterocyclic ring;
R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , and R ₆ are independently H, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, or heterocyclic, substituted or unsubstituted Wherein n = 0, 1, or 2;
R represents alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, or heterocyclic substituted or unsubstituted; for example, n = 1-6, R ′ ″ is aryl substituted Or a non-substituted C _n H _{2n + 1} , (CH ₂ ) _n R ′ ″, where the subject is a dopamine receptor modulator that specifically binds to D ₃ and D ₂ or D ₁ receptors Provides a method that may be characterized by having not previously responded sufficiently or may not respond sufficiently.

  The therapeutic methods of the invention (eg, treating a subject having symptoms characterized by abnormal function and / or regulation of a receptor [eg, dopamine receptor], modulating the action of a G protein-coupled receptor [ Preferred embodiments of (eg antagonizing and / or agonizing), controlling dopamine flow in a subject, etc.) envisage administering to the subject an effective dose of one or more compounds of the invention. It is understood.

  In still further embodiments, the invention is characterized by abnormalities in the modulation and / or function of certain diseases (eg, G protein-coupled [eg, dopamine] receptors, in addition to administration of therapeutic compounds of the invention). Further co-administration of one or more (eg, at least one) additional therapeutic compound (s) associated with treatment of the (

  In other preferred embodiments, the present invention provides methods for the treatment or prevention of symptoms characterized by defective modulation or function (eg, abnormalities) of dopamine receptors.

  The method of the present invention is particularly useful for the treatment of cocaine abuse, depression, anxiety, eating disorders, alcoholism, chronic pain, obsessive compulsive disorder, schizophrenia, restless leg syndrome (RLS), and Parkinson's disease. Very suitable for.

  A further aspect of the invention provides a pharmaceutical composition comprising: one or more compositions of the invention; and characterized by abnormalities in function / regulation of a receptor (eg dopamine receptor) Instructions for administering the composition to a subject having the disease. In preferred embodiments, the instructions included in these kits meet US Food and Drug Administration rules, regulations, and suggestions for the supply of therapeutic compounds.

In yet another embodiment, the invention provides a method for screening a compound and test compound that modulates a G protein-coupled receptor (eg, D ₃ ), wherein the compound modulates a G protein-coupled receptor; a test compound; Providing a first cell group; and contacting the first cell group with a compound and test compound that modulates a G protein-coupled receptor; and a compound and test compound that modulates a G protein-coupled receptor; And a method comprising the step of observing the effect of contacting the first cell group.

  In some of these embodiments, the present invention provides an effect observed in the first cell against a second group of cells contacted only with a compound that modulates a G protein-coupled receptor or only with a test compound. Further steps for comparison are further provided.

  Effects that would be observed include, but are not limited to, changes in dopamine metabolism. In yet other embodiments, the present invention further envisions additional methods for selling test compounds screened / identified by the above methods. In some of these embodiments, the test compound may be sold by a third party in one or more forms (eg, a kit that includes instructions for administering the test compound to a patient).

  Other advantages, benefits, and preferred embodiments of the present invention will be apparent to those skilled in the art.

  The following figures form part of the specification and are included to further demonstrate certain aspects and embodiments of the present invention. However, it is not intended that the present invention be limited to the embodiments specifically detailed in these figures.

Definitions To facilitate understanding of the invention, a number of terms and expressions are defined below.

As used herein, a “lead compound” is an analog useful for the treatment of a given condition, either for direct use as a therapeutic compound or with chemical modification (eg, optimization). A chemical selected to design a body compound (for example, a rational selection based on 3D structural analysis). For example, as used herein, a “lead compound” is selected to modify to increase binding and specificity for the D ₃ receptor. The lead compound can be a known compound or a newly designed compound.

  A “pharmacophore” according to the present invention is a chemical motif that includes, but is not limited to, many binding elements and their three-dimensional geometry. Elements are presumed to play a role in the activity of compounds identified as lead compounds. A pharmacophore is defined by the chemical nature of the binding elements, as well as the three-dimensional geometry of these elements.

  As used herein, the term “in vitro” refers to an artificial environment and processes or reactions that occur within the 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 (eg, animal or cell) and processes or reactions that occur within the natural environment.

  As used herein, the term “host cell” refers to any eukaryotic or prokaryotic cell (eg, mammalian cell, avian cell, amphibian cell, whether or not located in vitro or in vivo, Plant cells, fish cells, and insect cells).

  As used herein, the term “cell culture” refers to any cell culture in vitro. Continuous cell lines (eg, those having an immortal phenotype), primary cell cultures, finite cell lines (eg, non-transformed cells), and any other maintained in vitro, including oocytes and embryos Are also included within the scope of this term.

As used herein, the term “subject” refers to an organism to be treated by the methods of the invention. Such organisms include, but are not limited to, humans. In the present invention, the term “subject” generally refers to an individual who will receive or will receive treatment (eg, administration of an antagonist and / or agonist of a G protein coupled receptor). In a preferred embodiment, the G protein coupled receptor comprises a dopamine receptor (eg, D ₁ , D ₂ , D ₃ , and D ₄ ). In a particularly preferred embodiment, the dopamine receptor is a D ₃ receptor.

  As used herein, the term “diagnosed” refers to signs and symptoms of disease (eg, modulation of G protein-coupled receptors and / or abnormal functioning, in particular modulation of dopamine receptors and / or Abnormality of function), or recognition of disease by genetic analysis, pathological analysis, histological analysis or the like.

  As used herein, the term “antisense” refers to an RNA sequence that is complementary to a specific RNA sequence (eg, mRNA). Antisense RNA (“asRNA”) molecules involved in bacterial gene regulation are included within this definition. Antisense RNA may be produced by any method, including synthesis by splicing the gene of interest in the opposite direction to a viral promoter that allows synthesis of the coding strand. For example, once introduced into an embryo, this transcribed strand combines with the natural mRNA produced by the embryo to form a duplex. These duplexes then block further transcription of the mRNA or its translation. In this way, a mutant phenotype may be created. The term “antisense strand” is used in reference to a nucleic acid strand that is complementary to the “sense” strand. The symbol (−) (ie “negative”) is sometimes used for the antisense strand, and the symbol (+) is sometimes used for the sense (ie “positive”) strand. The region of the nucleic acid sequence accessible to the antisense molecule can be determined using available computer analysis methods.

  As used herein, the term “sample” is used in the broadest sense. Samples that are considered to be indicative of symptoms characterized by abnormalities in the regulation and / or function of G protein-coupled receptors, and more particularly abnormalities in the regulation and / or function of dopamine receptors, include cells, tissues, or body fluids, Chromosomes isolated from cells (eg, spread metaphase chromosomes), genomic DNA (in solution or bound to a solid support, such as for Southern blot analysis), RNA (for Northern blot analysis) And the like (in solution or bound to a solid support), cDNA (in solution or bound to a solid support), and the like. A sample suspected of containing a protein may include cells, some tissues, extracts containing one or more proteins, and the like.

  As used herein, the term “purified” or “purify” refers to the removal of unwanted components from a sample. As used herein, the term “substantially purified” is a molecule of either nucleic acid sequence or amino acid sequence that has been removed, isolated, or from their natural environment, or Molecules that have been separated and from which at least 60%, preferably 75%, and most preferably 90% of other naturally associated components have been removed. For example, an “isolated polynucleotide” is therefore a substantially purified polynucleotide.

  As used herein, the term “genome” refers to the genetic material (eg, chromosome) of an organism or host cell.

  The term “nucleotide sequence of interest” is used by any person of ordinary skill in the art to indicate that treatment would be desirable for any reason (eg, treating a disease, providing improved quality, etc.). Refers to a nucleotide sequence (eg, RNA or DNA). Such nucleotide sequences include coding sequences for structural genes (eg, reporter genes, selectable marker genes, oncogenes, drug resistance genes, growth factors, etc.) or portions thereof, and non-coding regulatory sequences that do not encode mRNA or protein products. (For example, but not limited to, promoter sequence, polyadenylation sequence, termination sequence, enhancer sequence, etc.).

  As used herein, “nucleic acid sequence” and “nucleotide sequence” refers to an oligonucleotide or polynucleotide, and fragments or portions thereof, as well as a single strand or double strand, and a sense strand. Or genomic or synthetic DNA or RNA that may represent the antisense strand. As used herein, the term "nucleic acid molecule encoding", "DNA sequence encoding", "DNA encoding", "RNA sequence encoding", or "RNA encoding" Refers to the order or sequence of deoxyribonucleotides or ribonucleotides along the chain 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. Thus, a DNA or RNA sequence encodes an amino acid sequence.

  The term “gene” refers to a nucleic acid (eg, DNA or RNA) sequence that comprises coding sequences necessary for the production of a polypeptide or precursor (eg, proinsulin). A polypeptide can be encoded by a full-length coding sequence or by any part of the coding sequence, as long as the desired activity or functional properties of the full-length or fragment (eg, enzymatic activity, ligand binding, signaling, etc.) are retained. Can be done. The term also encompasses the coding region of a structural gene, and in the coding region over a distance of about 1 kb or more at both ends of both the 5 ′ and 3 ′ ends so that the gene matches the length of the full-length mRNA. Includes adjacently located sequences. A sequence located 5 ′ of the coding region and present in the mRNA is referred to as a 5 ′ untranslated sequence. A sequence located 3 'or downstream of the coding region and present in the mRNA is referred to as a 3' untranslated sequence. The term “gene” encompasses both cDNA and genomic forms of a gene. A genomic form or clone of a gene includes a coding region interrupted with a non-coding sequence termed “intron” or “intervening region” or “intervening sequence”. Introns are segments of a gene that are transcribed into nuclear RNA (hnRNA); introns may contain regulatory elements such as enhancers. Introns are removed or “spliced out” from the nucleus or primary transcript; therefore, introns are not present in messenger RNA (mRNA) transcripts. The mRNA functions during translation and specifies the amino acid sequence or order of the nascent polypeptide.

  As used herein, the term “exogenous gene” refers to a gene that is not naturally present in the host organism or host cell, or has been artificially introduced into the host organism or host cell.

  As used herein, the term “vector” refers to a plasmid, phage, transposon that is capable of replicating and transferring a gene sequence between cells when combined with an appropriate regulatory element. , Any genetic element such as cosmid, chromosome, virus, virion, etc. The term thus includes cloning and expression media, and viral vectors.

  As used herein, the term “gene expression” refers to the genetic information encoded in a gene via RNA “transcription” (ie, through the enzymatic action of RNA polymerase) , MRNA, rRNA, tRNA, or snRNA), and for genes encoding proteins, refers to the process of converting to protein via mRNA “translation”. Gene expression can be regulated at many stages in the process. “Up-regulation” or “activation” refers to a regulation that increases the production of a gene expression product (ie, RNA or protein), while “down-regulation” or “suppression” refers to a regulation that reduces production. Molecules (eg, transcription factors) involved in up-regulation or down-regulation are often referred to as “activators” and “repressors”, respectively.

  As used herein, the terms “nucleic acid molecule encoding”, “DNA sequence encoding”, “DNA encoding”, “RNA sequence encoding” and “RNA encoding” are: Deoxyribonucleic acid or the order or sequence of deoxyribonucleotides or ribonucleotides along a ribonucleic acid chain. The order of these deoxyribonucleotides or ribonucleotides determines the order of amino acids along the polypeptide (protein) chain translated from the mRNA. Thus, a DNA or RNA sequence encodes an amino acid sequence.

  The terms “homology” and “percent identity” when used in reference to nucleic acids refer to a degree of complementarity. There can be partial homology (ie, partial identity) or complete homology (ie, 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 uses the functional term “substantially homologous”. To do. Inhibition of hybridization of sequences completely complementary to the target sequence may be investigated using hybridization assays (Southern or Northern blots, solution hybridization, etc.) under conditions of low stringency. A substantially homologous sequence or probe (ie, an oligonucleotide that can hybridize to another oligonucleotide of interest) binds a completely homologous sequence to the target sequence under conditions of low stringency. Will compete and inhibit (ie, hybridization). Low stringency requirements to allow non-specific binding; low stringency that requires the binding of two sequences to each other to be a specific (ie, selective) interaction It is a condition of jenshi. The absence of non-specific binding may be tested by use of a second target that lacks even a partial degree of complementarity (eg, less than about 30% identity); In the absence, the probe will not hybridize to the second non-complementary target.

  As used herein, the term “stringency” is used in reference to conditions for the presence of other compounds, such as temperature, ionic strength, and organic solvents, under which nucleic acid hybridization occurs. The Under the condition of “high stringency”, base pairing of nucleic acids is considered to occur only between nucleic acid fragments having a high frequency of nucleotide sequence complementarity. Thus, nucleic acid derived from genetically diverse organisms usually requires less stringent conditions and often requires conditions of “weak” or “low” stringency. Although the present invention can be practiced using low stringency conditions, in preferred embodiments, medium to high stringency conditions are used.

When used for nucleic acid hybridization, “high stringency conditions” are 5X SSPE (43.8 g / l NaCl, 6.9 g / l NaH ₂ PO ₄ H ₂ O, and 1.85 g / l EDTA, pH is NaOH. 7.4), 0.5% SDS, 5X Denhart reagent [50X Denhart with 5g Ficoll (type 400, Pharmcia), 5g BSA (Fraction V; Sigma) per 500ml], 100μg / ml denatured salmon semen DNA and Includes conditions equivalent to binding or hybridization at 42 ° C. (eg overnight) in a solution consisting of 50% (V / V) formamide. Then, when using a probe of about 500 nucleotides in length, the hybridized DNA sample was washed in a solution containing 2X SSPE and 0.1% SDS at room temperature, followed by 0.1X SSPE, 1.0% SDS at 42 ° C. Washed twice. Those skilled in the art are aware of conditions that promote hybridization under conditions of high stringency (eg, increasing the temperature in the hybridization and / or washing step, using formamide in the hybridization solution, etc. ).

When used for nucleic acid hybridization, the “intermediate stringency conditions” are 5X SSPE (43.8 g / l NaCl, 6.9 g / l NaH ₂ PO ₄ H ₂ when using a probe of approximately 500 nucleotides in length. O, and 1.85 g / l EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS, 5X Denhart reagent (contains 5 g Ficoll (type 400, Pharamcia), 5 g BSA (Fraction V; Sigma) per 500 ml) 50X Denhart], 100 μg / ml denatured salmon semen DNA, and binding or hybridization at 42 ° C in a solution consisting of 50% (V / V) formamide, followed by 1.0X SSPE at room temperature, 1.0% SDS Includes conditions equivalent to washing in, followed by 2X SSPE at 42 ° C, 0.1% SDS.

“Low stringency conditions” are 5X SSPE (43.8 g / l NaCl, 6.9 g / l NaH ₂ PO ₄ H ₂ O, and 1.85 g / l EDTA, pH when using a probe of approximately 500 nucleotides in length. Adjusted to 7.4 with NaOH), 0.1% SDS, 5X Denhart reagent [50X Denhart containing 5g Ficoll (type 400, Pharamcia), 5g BSA (Fraction V; Sigma) per 500ml], 100g / ml denatured salmon semen Includes conditions equivalent to binding or hybridization at 42 ° C. in a solution consisting of DNA and 50% (V / V) formamide, followed by washing at 42 ° C. in a solution containing 5 × SSPE, 0.1% SDS. Furthermore, it is well known to those skilled in the art that a number of equivalent conditions may be used for low stringency; probe length and nature (DNA, RNA, base composition), and target nature (DNA, Factors such as RNA, base composition, other present or fixed in solution, as well as salt concentration and other components (eg presence or absence of formamide, dextran sulfate, polyethylene glycol) By varying, low stringency hybridization conditions can be made which are different but equivalent to the above conditions.

  The term “substantially homologous” when used in reference to a single stranded nucleic acid sequence is capable of hybridizing to a single stranded nucleic acid sequence under conditions of low stringency (ie, complementary). ) Any probe. A gene can produce many RNA species that are generated by differential splicing of the primary RNA transcript. CDNAs that are splice variants of the same gene are regions of sequence identity or complete homology (representing the presence of the same exon or part of the same exon in both cDNAs) and non-identity (eg, exon “ Although it represents the presence of “A”, cDNA2 would instead contain a region containing exon “B”). Since the two cDNAs contain regions of sequence identity, they are thought to hybridize both to probes derived from all or part of the gene containing sequences found in both cDNAs; thus, two splice variants Are substantially homologous to such probes and to each. The present invention is not limited only to the case where hybridization occurs between completely homologous sequences. In some embodiments, hybridization occurs in a substantially homologous sequence.

  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), as used for a protein, is encoded by a partially homologous nucleic acid such that the amino acid sequence of the protein changes. Refers to the protein that is produced. As used herein, the term “variant” refers to proteins encoded by homologous genes having both conservative and non-conservative amino acid substitutions that do not alter protein function, and proteins Includes proteins encoded by homologous genes that have amino acid substitutions that result in reduced function (eg, non-expressed mutations) or increased protein function.

  As used herein, the term “pathogen” refers to a biological agent that causes a disease state (eg, infection, cancer, etc.) in a host. “Pathogens” include, but are not limited to, viruses, bacteria, archaea, fungi, protozoa, mycoplasma, and parasites.

  As used herein, the term “organism” refers to any species and type of microorganism, including but not limited to bacteria, archaea, fungi, protozoa, mycoplasma, and parasites. As used herein, the term “fungi” is used in reference to eukaryotes such as molds and yeasts including dimorphic fungi.

  As used herein, the term “virus”, with some exceptions, is not observable by light microscopy, lacks independent metabolism, and replicates only in biological host cells. A microscopic infectious factor that is possible. Individual particles (ie, virions) consist of nucleic acid and protein shells or coatings; some virions also have lipids, including membranes. The term “virus” encompasses all types of viruses, including animals, plants, phages, and other viruses.

  The terms “bacteria” and “bacteria” refer to all prokaryotes, including everything within the gates of the prokaryotic world. The term is intended to encompass all microorganisms regarded as bacteria, including mycoplasma, chlamydia, actinomycetes, streptomyces, and rickettsia. All forms of bacteria are included within this definition, including cocci, bacilli, spirochetes, spheroplasts, protoplasts, and others. Gram negative or gram positive prokaryotes are also included within the scope of the term. “Gram negative” and “Gram positive” refer to staining patterns by the Gram staining process well known to those skilled in the art (eg, Finegold and Martin, Diagnostic Microbiology, 6th Ed., CV Mosby St. Louis, pp 13-15 [1982 ]). “Gram-positive bacteria” are bacteria that retain the primary dye used in Gram staining, yielding stained cells that appear dark blue to purple under the microscope, and “Gram-negative bacteria” are used in Gram staining Does not retain primary dye, but is stained by counterstaining. Thus, Gram negative bacteria appear red.

  As used herein, the term “antigen binding protein” refers to a protein that binds to a specific antigen. “Antigen-binding protein” includes immunoglobulins, Fab fragments, F (ab ′) 2 fragments, and Fab expression libraries, including polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, and humanized antibodies. However, it is not limited to these. Various procedures known to those skilled in the art are used to produce polyclonal antibodies. For antibody production, various host animals, including but not limited to rabbits, mice, rats, sheep, goats, etc., can be immunized by injection with a peptide corresponding to the desired epitope. it can. In preferred embodiments, the peptide is conjugated to an immunogenic carrier (eg, diphtheria toxoid, bovine serum albumin (BSA), or keyhole limpet hemocyanin [KLH]). Depending on the host species, various adjuvants are used to increase the immune response and include Freund (complete and incomplete), mineral gels such as aluminum hydroxide, surfactants such as lysolecithin, pluronic polyols, polyanions, Includes but is not limited to peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG (Bacille Calmette-Guerin) and Corynebacterium parvum Not.

  For preparation of monoclonal antibodies, any technique provided to produce antibody molecules by cell lines passaged in culture may be used (eg, Harlow and Lane, Antibodies: A Laboratory Manual, (See Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). These include hybridoma technology originally developed by Kohler and Milstein (Kohler and Milstein, Nature, 256: 495-497 [1975]), and trioma technology, human B cell hybridoma technology (eg, Kozbor et al., Immunol. Today). , 4:72 [1983]), and EBV-hybridoma technology for producing human monoclonal antibodies (Cole et al., In Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77- 96 [1985]), but not limited to.

  In accordance with the present invention, the techniques described for producing single chain antibodies (US Pat. No. 4,946,778; incorporated herein by reference) can be used to produce specific single chain antibodies as desired. Can be adapted to. A further embodiment of the present invention utilizes a technique known to those skilled in the art for the construction of Fab expression libraries (Huse et al., Science, 246: 1275-1281 [1989]) to produce monoclonal Fabs with the desired specificity. Allows for quick and easy identification of fragments.

  Antibody fragments that contain the idiotype (antigen binding region) of the antibody molecule can be generated by known techniques. For example, such fragments can be produced by pepsin digestion of antibody molecules; F (ab ′) 2 fragments; Fab ′ fragments that can be generated by reducing disulfide bridges of F (ab ′) 2 fragments And Fab fragments that can be generated by treating antibody molecules with papain and a reducing agent.

  The gene encoding the antigen binding protein can be isolated by methods known to those skilled in the art. In producing antibodies, screening for the desired antibody can be accomplished by techniques known to those skilled in the art (eg, radioimmunoassay, ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassay, immunoradioassay, gel diffusion precipitation). Elementary reactions, immunodiffusion assays, in situ immunoassays (eg, using colloidal gold, enzyme, or radioisotope labels), Western blots, precipitation reactions, agglutination assays (eg, gel agglutination assays, Hemagglutination assays, etc.), complement binding assays, immunofluorescence assays, protein A assays, and immunoelectrophoresis assays, etc.).

As used herein, the term and grammatical equivalents of “instructions for administering a compound that modulates the dopamine receptor to a subject” include drugs [eg, cocaine in cells or tissues. ] Dopamine acceptance including (but not limited to) addiction, depression, anxiety, schizophrenia, Tourette syndrome, eating disorders, alcoholism, chronic pain, obsessive compulsive disorder, restless leg syndrome, Parkinson's disease, etc. body (e.g., D ₃ receptors) instructions for using the compositions contained in the kit for the treatment of conditions characterized by abnormal regulation and / or functions. In some embodiments, the instructions further include a description of recommended or normal doses of the composition included in the kit in accordance with 21 Federal Regulations Article 201 et seq. Further information regarding the labels and instructions necessary for application to the present methods and compositions is available on the US FDA Internet web page.

The term “test compound” is capable of treating or preventing a disease, illness, illness, or disorder of physical function, or otherwise the physiological state or cellular state of a sample (eg, G protein-coupled receptor). Any chemical, pharmaceutical, drug that can be used to alter abnormal regulation and / or function of the body, and more particularly, abnormal regulation and / or function of a dopamine receptor [eg, D ₃ ] And so on. Test compounds include known and potential therapeutic compounds. A test compound can be determined to be therapeutic by using the screening methods of the invention. A “known therapeutic compound” refers to a therapeutic compound that has been shown to be effective in such treatment or prevention (eg, through animal studies or prior experience with administration to humans). In a preferred embodiment, the “test compound” is a G protein-coupled receptor modulator.

As used herein, the term “third party” refers to an abnormal regulation and / or function of a G protein-coupled receptor, more specifically an abnormal condition of a dopamine receptor [eg, D ₃ ]. Co-administration with a compound that modulates a receptor (eg, a G protein-coupled receptor, more particularly a dopamine receptor [eg, D ₃ ]) to treat a condition characterized by modulation and / or function Refers to any entity involved in sales, storage, distribution, or provision for selling test compounds.

  As used herein, the term “modulation” affects (eg, promotes) the functional aspects of a cell or molecule, including but not limited to neurotransmitter cycling, enzyme activity, and the like. Or delayed), refers to the activity of a compound (eg, a compound that modulates dopamine receptors).

  As used herein, the term “dopamine receptor agonist” refers to one or more dopamine receptors as compared to the activity of the corresponding dopamine receptor that has not been subjected to the agonist compound (s). A compound that promotes body activity.

  As used herein, the term “antagonist of a dopamine receptor” refers to one or more dopamine receptors compared to the activity of the corresponding dopamine receptor that has not been subjected to the antagonist compound (s). A compound that delays body activity.

General Description of the Invention Dopamine (DA) is a neurotransmitter that plays an essential role in normal brain function. As a chemical messenger, dopamine is similar to adrenaline. In the brain, dopamine is synthesized in presynaptic neurons and released into the space between presynaptic and postsynaptic neurons.

  Dopamine affects brain processes that control exercise, emotional responses, and the ability to experience pleasure and pain. Dopamine regulation plays a critical role in mental and physical health. Neurons containing the neurotransmitter dopamine form clusters in a region called the substantia nigra of the midbrain. Abnormal dopamine signaling in the brain includes drug (eg, cocaine) abuse, depression, anxiety, schizophrenia, Tourette syndrome, eating disorders, alcoholism, chronic pain, obsessive compulsive disorder, restless leg syndrome, Parkinson It is related to many medical conditions including diseases.

  Dopamine molecules bind to and activate dopamine receptors in postsynaptic neurons. Dopamine molecules are then transported back to presynaptic neurons via dopamine transporter protein (DAT), where they are metabolized by monoamine oxidase (MAO). In symptoms such as cocaine abuse, cocaine binds to the dopamine transporter and blocks the normal flow of dopamine molecules. Excess concentrations of dopamine cause excessive 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.

D ₃ receptors, almost exclusively, nucleus accumbens, are concentrated olfactory tubercle, and limbic brain regions, such as Kaeha island. (For example, B. Landwhermeyer et al., Brain Res. Mol. Brain Res., 18: 187-192 [1993]; AM Murray et al., Proc. Natl. Acad. Sci. USA, 91: 11271-11275 [1994]; ML Bouthenet et al., Brain Res., 564: 203-219 [1991]). These brain regions are the terminal areas of mesolimbic dopamine projections associated with reinforcement and reward mechanisms. (See, eg, HC Fibiger and AG Phillips, Ann. NY Acad. Sci., 537: 206-215 [1988]; and WJ McBride et al., Behav. Brain Res., 101: 129-152 [1999]) .

  An understanding of this mechanism is not necessary to practice the invention, and the invention is not so limited, but cocaine strongly inhibits reuptake of norepinephrine (NE) and serotonin (5-HT). It is thought that its ability to act as a strengthening factor stems from inhibiting dopamine reuptake into dopaminergic neurons. (Eg, MC Ritz et al., Science, 237-1219 [1987]; MJ Kuhar et al., Trends Neurosci., 14: 299-302 [1991]; FI Carroll et al., J. Med. Chem., 35: 969 [1992] And FI Carroll et al., J. Med. Chem., 42: 2721-2731 [1999]). Cocaine exerts this inhibitory action through specific interaction with a dopamine transporter (DAT) protein (cocaine receptor) located at the dopamine nerve ending. The resulting increase in synaptic dopamine availability in the mesolimbic system of the brain that mediates reward is the essence of the dopamine hypothesis on the action of cocaine. Thus, pharmacological and neurobiological evidence indicates that the effects associated with cocaine abuse are mediated by the dopaminergic system.

The present invention contemplates that dopamine 3 (D ₃ ) subtype receptors are involved in the positive enhancing effects of cocaine and other psychostimulants. Accordingly, preferred embodiments of the present invention envision designing dopamine receptor ligands (eg, partial agonists) to treat the abuse and dependence of cocaine and other psychostimulants. Compound 7-OH-DPAT and pramipexole (pramipexole) and several other D ₃ parts / full agonists, such as PD128907, in mitogenesis functional assays in vitro, and these potency in D ₃ receptors Reduce cocaine self-administration with a highly correlated rank order of action. (Eg, SB Caine and GF Koob, Science, 260: 1814-1816 [1993]; GF Koob and M. Le Moal, Science, 278: 52-58 [1997]; SB Caine et al., J. Pharmacol. Exp. Ther , 291: 353-360 [1999]; M. Pilla et al., Nature, 400: 371-375 [1999]; GF Koob, Ann. NY Acad. Sci., 654: 171-191 [1992]; GF Koob, J. Exp. Anal. Behav., 61: 213-221 [1994]; and LH Parsons et al., J. Neurochem., 67: 1078-1089 [1996]). Similarly, D ₃ sites of stimulation, decrease in self-stimulation in 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 reduced food-enhanced response rates in a fixed rate operant paradigm (DJ Sanger et al., Behav. Pharmacol., 7: 477). -482 [1996]). In studies evaluating place preference, D ₃ agonists produce aversive effects when administered alone (F. Chaperon and MH Thiebot, Behav. Pharmacol., 7: 105-109 [1996]), cocaine and amphetamines. Conditioned place preference (TV Khroyan et al., Psychopharmacology, 139: 332-341 [1998]; and TV Khroyan et al., Psychopharmacology, 142: 383-392 [1999]).

Several D ₃ partial agonists have been shown to reduce cocaine self-administration. (SB Caine et al, Neuroreport, 8: 2373-2377 [1997 ]) The efficacy of these compounds on reducing self-administration of cocaine, as an agonist of the D ₃ receptors in mitogenesis functional assay in vitro of Correlates with efficacy. (For example, SB Caine and GF Koob, Science, 260: 1814-1816 [1993]; SB Caine et al., J. Pharmacol. Exp. Ther., 291: 353-360 [1999]; and LH Parsons et al., J. Neurochem. ., 67: 1078-1089 [1996]). Since experimental animals cannot be trained to self-administer D ₃ partial agonists, partial D ₃ agonists do not appear to be prone to abuse. (MA Nader and RH Mach, Psychopharmacology [Berl], 125: 13-22 [1996]; and L. Pulvirenti et al., J. Pharmacol. Exp. Ther., 286: 1231-1238 [1998]). Recent studies relatively selective partial D ₃ agonists BP897 has been shown to inhibit behavior in response to cues associated with drug in rats look cocaine. (M. Pilla et al., Nature, 400: 371-375 [1999]).

The density of D ₃ receptors by cocaine use has been reported to vary. D density of ₃ sites, caudate nucleus of dying human cocaine dose excess, nucleus accumbens, and was increased 1-3 fold in the substantia nigra. (JK Staley and DC Mash, J. Neurosci., 16: 6100-6106 [1996]). Similarly, in the nucleus accumbens of deaths cocaine was seen 6-fold increase in D ₃ receptor mRNA. (DM Segal and CT Moraes, Brain Res. Mol. Brain Res., 45: 335-339 [1997]). Furthermore, gene polymorphism studies, homozygous for BalI D ₃ receptor polymorphisms and possibly D ₄ receptor polymorphism is associated with other traits that can contribute to cocaine dependence and drug addiction It has been suggested. (RP Ebstein et al., Am. J. Med. Gen., 74: 65-72 [1997]; and DE Comings et al., Mol. Psychiatry, 4: 484-487 [1999]).

The dopamine 3 (D ₃ ) receptor is reported to have 52% sequence homology with and similar to the D ₂ receptor, but with a unique pharmacological profile. (P. Sokoloff et al., Nature, 347: 146-151 [1990]). Data suggest that D ₃ receptors are likely to be involved in the positive reinforcing effect of cocaine and other psychostimulants. (See, eg, SB Caine and GF Koob, Science, 260: 1814-1816 [1993]; GF Koob and M. Le Moal, Science, 278: 52-58 [1997]; SB Caine et al., J. Pharmacol. Exp. Ther. , 291: 353-360 [1999]; M. Pilla et al., Nature, 400: 371-375 [1999]; GF Koob, Ann. NY Acad. Sci., 654: 171-191 [1992]; GF Koob, J. Exp. Anal. Behav., 61: 213-221 [1994]; LH Parsons et al., J. Neurochem., 67: 1078-1089 [1996]; RS Sinnott et al., Drug Alcohol Depend., 54: 97-110 [1999]; see JK Staley and DC Mash, J. Neurosci., 16: 6100-6106 [1996]; and LH Parsons et al., J. Neurochem., 67: 1078-1089 [1996]) .

  Previous attempts to develop cocaine antagonists as a treatment for the treatment of cocaine abuse have focused on the modification of cocaine itself; this provides a wealth of information on the structure-activity relationship (SAR) of cocaine analogs. lead. Despite active research efforts in this area, few compounds with significant cocaine antagonist activity have been reported. Accordingly, preferred embodiments of the invention provide novel compounds that antagonize the addictive effects of all or part of cocaine and other psychostimulants and methods for designing these compounds.

Partial D ₃ agonists with clear selectivity for the D ₃ receptor, such as those described in some preferred embodiments herein, were not available prior to the present invention. For example, existing D ₃ ligand Most have also excellent activity in D ₂ subtype receptors.

In some embodiments, highly selective D ₃ ligand serves as a pharmacological tool for facilitating understanding of the functional role of D ₃ receptors in cocaine addiction. Furthermore, in other embodiments, specific partial agonist against D ₃ receptors provide a therapeutic compound for the treatment of cocaine abuse. In still some other embodiments, the compositions and methods of the present invention provide therapeutic treatment for other epilepsy versus other psychostimulants characterized by interfering with normal dopamine metabolism. In yet other embodiments, the compositions and methods of the present invention may be used to treat other diseases and conditions characterized by aberrant regulation and / or function of dopamine signaling and / or dopamine receptor activity (eg, depression, anxiety). , Schizophrenia, Tourette syndrome, eating disorders, alcoholism, chronic pain, obsessive compulsive disorder, and Parkinson's disease). Further aspects of the invention provide compositions and methods for treating conditions characterized by abnormal regulation and / or function of G protein-coupled receptors (GPCRs), and methods for designing these compounds. To do.

One factor that had prevented the development of highly selective D ₃ ligands prior to the present invention was the lack of accurate 3D structural information, mainly due to the lack of dopamine ligand binding and Sometimes the structural basis of selectivity was poorly understood. Since dopamine receptors are membrane-bound proteins, measuring these 3D structures experimentally is a difficult task. In the past, computational homology modeling approaches using either the low-resolution structure of rhodopsin or the high-resolution structure of bacteriorhodopsin have been used to construct 3D models of dopamine receptors. (For example, EJ Homan et al., Bioorg. Med. Chem., 7: 1805-1820 [1999]; MM 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 JE Leysen, Receptors Channels, 1: 89-97. [1993]; see CD Livingstone et al., Biochem. J., 287: 277-282 [1992]; and SG 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 dopamine receptors and rhodopsin, and the topological arrangement of the transmembrane helix differs between the X-ray structures of rhodopsin and bacteriorhodopsin (K. Palczewski Science, 289: 739-745 [2000]), therefore, the accuracy of dopamine models based on bacteriorhodopsin is limited. The structure of rhodopsin was also used as a template structure for modeling, but their accuracy is limited by the low resolution of existing rhodopsin structures. Furthermore, due to the limited computational power, prior to the present invention, structural improvements were made in these models without considering the proper lipid / water environment required for accurate modeling. . (RG Efremov et al., Biophys. J., 76: 2460-2471 [1999]).

In the present invention previously, yet another difficulty with determining the specific features of dopamine receptor subtypes, especially in vivo, the ligand can be reliably discriminate between D ₂ and D ₃ subtypes The availability was limited. In contrast, preferred embodiments of the present invention include ligands designed to selectively bind to various dopamine receptor (eg, D ₁ , D ₂ , D ₃ , D ₄ , and D ₅ ) subtypes. provide. In another preferred aspect, a high D ₃ receptors selective partially or fully adjusted (e.g., agonism and / or antagonism) so designed ligand. The present invention also provides compounds designed to block other members of the D family receptors and other members of the GPCR superfamily. Yet another aspect provides therapeutic compositions and methods for the treatment of conditions characterized by deficiencies in dopamine signaling regulation.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to dopamine receptors (eg, D ₁ , D ₂ , D ₃ , D ₄ , and D ₅ ), and in particular, ligands for D ₁ , D ₂ , and D ₃ dopamine receptors. (Eg, agonists, antagonists, and partial agonists). The invention also relates to methods for the rational design of these compounds and therapeutic methods using these compounds.

Exemplary compositions and methods of the invention are described in further detail in the following sections: I. Dopamine receptors; II. Homology modeling of dopamine receptors D ₁ , D ₂ , and D ₃ ; III.D3 Computational docking studies of ligand-receptor interactions; IV. General design strategies for identifying new lead compounds; V. Identification of specific new lead compounds; VI. Further structure of lead compounds VII. Biological testing and characterization of lead compounds and analogs thereof; VIII. Therapeutic agents combined or co-administered with the composition; and IX. Pharmaceutical formulations, administration Route and dose studies.

In the following a number of examples, mention may primarily dopamine receptors, in particular modulators of D ₃ receptors (e.g., agonists and / or antagonists, both partial and complete ones) directed to providing a composition It is considered an object and method. However, it is not intended that the present invention be limited to providing modulators of dopamine receptors. Indeed, the methods and compositions of the invention are useful for identifying and designing compositions (eg, ligands) that modulate many receptor family subtypes (eg, G protein coupled receptors).

I. Dopamine receptor
In the 1950s, scientists began to recognize that dopamine is an important neurotransmitter based on the heterogeneous distribution of dopamine in the brain. A study by Cools and Van Rossum suggested that the brain might have multiple types of receptors for dopamine based on anatomical, electrophysiological, and pharmacological studies. (AR Cools and JM Van Rossum, Psychopharmacology, 45 (3): 243-245 [1976]). Biochemical analysis of dopamine receptors based on the use of second messenger assays (eg, stimulation of cAMP production) and ligand binding assays have different biochemical and pharmacological properties and different physiological functions Other researchers have suggested that there are two classes of dopamine receptors, D ₁ and D ₂ , that mediate the expression of (eg JW Kebabian and DB Calne, Nature, 277 (5692): 93-96 [1979] See). Table 1 lists some properties of each of the D ₁ and D ₂ classes of dopamine receptors (see PG Strange, Neurochem. Int., 22 (3): 223-236 [1993]) .

While D ₁ -like and D ₂ like receptor subtypes are G-protein coupled receptors Both various G proteins and effectors involved in these signaling pathways. The application of gene cloning techniques, researchers, there are at least five dopamine receptors (D ₁ to D _5), these are the original D ₁ and which is defined through the pharmacological and biochemical techniques It has been shown to be divided into two subfamilies that are similar in nature to the D ₂ receptor (DR Sibley and FJ Monsma Jr., Trends Pharmacol. Sci., 13 (2): 61-69 [1992] O. Civelli et al., Ann. Rev. Pharmacol. Toxicol., 33: 281-307 [1993]; and KR Jarvie and MG Caron, Adv. Neurol., 60: 325-333 [1993]). Two subfamilies are often referred to as D _1-like (D _1, D ₅₎ and D ₂ like _{(D 2, D 3, D} 4). Table 1, a number of important properties of ₁ -like and D ₂ like dopamine receptors D are summarized. For example, additional D ₁ like receptor, Xenopus, chicken, and so have been cloned from Drosophila, may exist other subtypes of dopamine receptors.

Analysis of the amino acid sequence of the dopamine receptor, have been shown to very significant homology between subtypes are present, the highest homology among members of the subfamily (e.g., subfamily ₁ -like or D ₂ like D) Sex was seen.

Each known dopamine receptor contains a seven transmembrane sequence of hydrophobic amino acids. Thus, each dopamine receptor has a general structural structure of G protein-coupled receptors by having an extracellular amino terminus and a seven-transmembrane helix connected by intracellular and extracellular protein loops. It matches the model (D. Donnelly et al., Receptors Channels, 2 (1): 61-78 [1994]). The D ₁ -like receptor has a short third intracellular loop and a long carboxyl terminal tail, while the D ₂ -like receptor has a long third intracellular loop and a short carboxyl terminal tail. This provides a structural basis for dividing the receptor into two subfamilies, but may have functional significance with respect to the specificity of the receptor / G protein interaction.

Present third intracellular loop of the dopamine receptors, believed to be important for receptor interactions with the G protein, for D ₂ like receptors, these subtypes variant based on the loop To do. For example, D ₂ and D ₃ receptors short variant and long variants exist, long form, it has an insertion into the loop (length of 29 amino acids for D _2). (B. Giros et al., Nature, 342 (6252): 923-926 [1989]). For example, polymorphic variants of the D ₂ receptor have been described as changing a single amino acid in this loop (A. Cravchik et al., J. Biol. Chem., 271 (42): 26013- 26017 [1996]). In humans, D ₄ receptor has an insertion of polymorphism in this loop. (HH Van Tol et al., Nature, 358 (6382): 149-152 [1992]).

D ₂ like receptors variants Shirezu be have the unique ability or activate, bind to G protein (A. Cravchik et, J Biol Chem, 271 (42 ):... 26013-26017 [1996]; J. Guiramand et al., J. Biol. Chem., 270 (13): 7354-7358 [1995]; and SW Castro and PG Strange, FEBS Letts., 315 (3): 223-226 [1993] ) And may also exhibit slightly different pharmacological properties (SW Castro and PG Strange (1993) J. Neurochem., 60 (1): 372-375 [1993]; and A. Malmberg et al., Mol. Pharmacol., 43 (5): 749-754 [1993]). However, variants of D ₄ receptor, the binding of the ligand, or binding to G protein, it does not show any differences. (MA Kazmi et al., Biochemistry, 39 (13): 3734-3744 [2000]). The individual properties of the different subtypes were explored by expressing the receptor in recombinant cells and by examining the subtype localization at the mRNA and protein level.

Dopamine receptor subtypes D ₁ and D ₂ have different pharmacological profiles, localization, and mechanism of action. Respect D _1-like dopamine receptors, D ₁ and D ₅ receptors Both exhibit similar pharmacological properties (e.g., high affinity for benzazepine ligand). Thioxanthine, while indicating a high affinity for D ₁ like receptor, not selective for D ₁ like receptor over the D ₂ like receptors. D ₁ -like receptors also show moderate affinity for typical dopamine agonists such as apomorphine and selective agonists such as dihydrexidine. Prior to the present invention, no ligand selective for either D ₁ or D ₅ dopamine receptors was known.

D ₁ receptors, neostriatum, substantia nigra, nucleus accumbens, and is found in high levels in the typical dopamine-rich brain regions such as the olfactory tubercle, the distribution of D ₅ receptors is more limited ing. D ₅ subtypes are found in lower levels than D ₁ subtype dopamine receptor. Both D ₁ and D ₅ receptor can stimulate adenylyl cyclase, D ₅ receptors show some constitutive activity for this reaction.

Inverse agonist activity at the D ₁ and D ₅ receptors is found in some compounds, such as butaclamol, previously thought to be antagonists in the recombinant system. Understanding the mechanisms for carrying out the present invention is not required, the invention is not so limited, D ₁ receptor mediates important effects of dopamine, motor, cognitive, and cardiovascular function Is assumed to be controlled. Furthermore, the interaction between the D ₅ receptor (G-protein coupled receptors) GABAA receptor (ion channel-linked receptor) has been described to exhibit a functional role for the D ₅ receptors.

D ₂ -like receptors (eg, D ₂ , D ₃ , and D ₄ ) exhibit pharmacological properties similar to those of the original pharmacologically defined D ₂ receptor (eg, they are all High affinity for drugs such as butyrophenone and substituted benzamides). Classification of these drugs, it is contemplated to provide selective antagonists to D ₂ like receptors. As described above, D ₂ like receptors also shows high affinity for phenothiazine and thioxanthine. Each D ₂ like receptors have a unique pharmacological characteristics, there are some differences in the affinity of the drug to an individual D ₂ like receptors. For example, raclopride (raclopride) may exhibit high affinity for D ₂ and D ₃ receptors indicate a low affinity for D ₄ receptors. Clozapine shows a slight selectivity for the D ₄ receptor. Aminotetralin, while indicating some selectivity as D ₂ like autoreceptor antagonists, these compounds also shows selectivity for D ₃ receptor as an agonist. D ₂ like receptor antagonists Most, compared to D ₃ and D ₄ receptors, indicating a higher affinity for D ₂ receptors. D ₂ like subtypes show moderate affinity for typical dopamine agonist, D ₃ receptors indicate a slightly higher affinity. D ₂ like receptor selective agonist is available compounds against body (e.g., quinpirole) but is present, prior to the present invention, was not highly selective agonist for each D ₂ like receptors .

D ₂ receptors are the major D ₂ like receptor subtypes in the brain, a typical dopamine is found in high levels in rich brain regions. D ₃ and D ₄ receptors, at lower levels, and are found in more restricted distribution pattern. D ₃ and D ₄ receptors are primarily found in the limbic area of the brain. .. D ₂ like receptor subtype, when expressed in recombinant cells, it has been shown respectively to inhibit adenylyl cyclase (e.g., CL Chio et al, Mol Pharmacol, 45 (1) : 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 (1) :.. 495-502 [1994]; and DA Hall and PG Strange, Biochem Pharmacol , 58 (2): 285-289 see [1999]), to indicate a signal passing through the D ₃ receptor, and more It has proven difficult and is generally lower than that of the other two subtypes. D ₂ like receptors also provides a recombinant system, mitogenesis (CL Chio et al, Mol Pharmacol, 45 (1) :.... 51-60 [1994]; and BC Swarzenski et, Proc Natl Acad. Sci. USA, 91 (2): 649-653 [1994]) and will stimulate extracellular acidification (Chio, below). For D ₂ receptors, in arachidonic acid release and MAP kinases (P. Sokoloff et al., Biochem. Pharmacol., 43 (4): 659-666 [1992]; and GI Welsh et al., J. Neurochem., 70 (5 ): 2139-2146 [1998]), and the D ₂ and D ₃ receptors showed an effect in the Ca ²⁺ channel. (GR Seabrook et al., Br. J. Pharmacol., 111 (2): 391-393 [1994]). The relevance of these effects on responses in vivo is completely unknown.

D ₂ receptors mediate the effects of dopamine, exercise is important for controlling the prolactin secretion from certain aspects and anterior pituitary, the behavior in the brain. Localization of D ₃ and D ₄ receptors in the limbic area of the brain suggests these recognition, to have a role in the function of emotional, and behavioral. Distribution of D ₃ and D ₄ receptors in limbic brain regions, they are particularly attractive targets for designing potent selective antipsychotic. D ₂ like receptors, a high affinity for most drugs used to treat schizophrenia (antipsychotic) and Parkinson's disease (e.g., bromocriptine).

II. Homology modeling of dopamine receptors D ₁ , D ₂ , and D ₃ The X-ray structure of rhodopsin was recently determined with a resolution of 2.8 Å. (K. Palczewski et al., Science, 289: 739-745 [2000]). As mentioned above, the D ₁ , D ₂ , and D ₃ receptors belong to the rhodopsin family within the GPCR superfamily. Within the seven transmembrane (TM) helices that form the ligand binding site, the D ₁ , D ₂ , and D ₃ receptors and rhodopsin have 47% high sequence homology (identity / similarity) is important. Thus, in a preferred embodiment of the invention, the high resolution X-ray structure of rhodopsin is used to accurately model the 3D structure of the dopamine receptor. In 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) Important to accuracy. The previous sequence analysis of 493 members of the rhodopsin family of GPCR protein, distinct sequences Alan placement of the TM region containing a binding site is provided between the D _1, D _2, and D ₃ receptors and rhodopsin (JM Baldwin et al., J. Mol. Biol., 272: 144-164 [1997]). Thus, in a preferred embodiment of the present invention, the homology modeling method provides accurate 3D structural modeling of the D ₁ , D ₂ , and D ₃ receptors, particularly the ligand binding site.

The lipid membrane / water environment affects the 3D structure of transmembrane proteins. Thus, in some embodiments, large-scale MD simulations involving a well-defined lipid-water environment in structural refinement were performed to further improve the accuracy of the modeled structure. In some of these embodiments, a parallel version of the highly effective CHARMM program (version 2.7) running on a supercomputer (BR Brooks et al., J. Comp. Chem., 4: 187-217 [1983]) It is used in MD simulations as a modeling environment containing approximately 16,000 atoms including D ₁ , D ₂ , and D ₃ proteins, lipids, and water molecules. The 3 ns MD simulation was completed for the D ₁ and D ₃ receptor structures and the 2.5 ns simulation was completed for the D ₂ receptor structure. In other embodiments, substantially longer MD simulations are used (eg, 5 ns, 7 ns, 10 ns, and longer). However, the present invention is not intended to be limited to a particular length of modeling or simulation. Those skilled in the art will be able to identify lower energy conformation clusters with respect to binding site conformation when various modeling and simulation times (for example, with longer MD simulation times, the previous MD simulation is not long enough) Will recognize the potential effects of

Binding site of D ₂ was determined experimentally through experiments using methane thiosulfonate (MTS) reagent. (JA Javitch et al., Neuron, 14: 825-831 [1995]; JA Javitch et al., Biochemistry, 34: 16433-16439 [1995]; JA Javitch et al., Mol. Pharmacol., 49: 692-698 [1996]; D Fu et al., Biochemistry, 35: 11278-11285 [1996]; JA Javitch et al., Biochemistry, 37: 998-1006 [1998]; JA Javitch et al., Biochemistry, 38: 7961-7968 [1999]; and JA Javitch et al., Biochemistry, 39: 12190-12199 [2000]).

It is not necessary to understand the mechanism for practicing the present invention, and the present invention is not so limited, but the D ₂ receptor contains three common Ser residues in TM5 (eg, NJ Pollock et al., J Biol. Chem., 267: 17780-17786 [1992]; BL 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 PG Strange, J. Neurochem., 72: 2621-2624 [1999]; BA 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 (see A. Mansour et al., Below) are assumed to be important residues for ligand binding. Cysteine residues of the D ₂ and D ₃ is important for ligand binding. (GL Alberts et al., Br. J. Pharmacol., 125: 705-710 [1998]). In the modeled structure, all of these residues are exposed to water and are part of the ligand binding site formed primarily by TM3, TM5, TM6, and TM7. Modeled 3D structure and its ligand binding site of D ₃ is shown in (FIG. 1). Further analysis was performed to investigate the structural differences between D ₁ , D ₂ , and D ₃ .

Structural difference between D ₁ and D ₃ receptors have been found for these ligand binding sites is very serious. Differences in major residues when considering residues exposed to water in the transmembrane region of TM2 to TM7 at the binding site are shown below (Table 2). TM3 residues and structural differences affect ligand selectivity.

In D ₁ / D ₃ using a chimeric receptor further experiments, the extracellular portion of TM3 of D ₃ is substituted with the corresponding residues of D _1, that the binding affinity of a number of D ₃ ligands significantly reduced It has been shown. Furthermore, a single mutation from C114 of D ₃ to the corresponding residues Ser of D _1, binding affinity, 37-fold and about quinpirole (-) - about 3-PPP reduced to 57 times. (GL Alberts et al., Br. J. Pharmacol., 125: 705-710 [1998]). In some embodiments, the Y-373 to replace more by larger Trp (W321) of D _1. The present invention provides a major difference in the structure, a reasonable basis for designing ligands that selectively bind to various dopamine receptors, and other aspects between D ₃ and D ₁ is Suppose that we provide a basis for designing ligands that selectively bind to other members of the G protein receptor family. Table 2 shows some sequence differences in D ₁ and D ₃ binding site region that can contact solvent molecules or ligands (residues D ₁ are shown in parentheses).

D ₂ receptors and D ₃ receptors differences significant structural also between the presence, but not critical than that seen between the D ₁ receptor and D ₃ receptors. Using careful structural analysis using high resolution models of D ₂ and D _3, 4 a region of the main sequence between D ₂ receptors and D ₃ receptors (i.e., the region A, B, C, And D) and structural differences were identified. For corresponding residues that differ between dopamine receptor subtypes, there is some uncertainty about the position of these side chain atoms. To solve this problem, the present invention uses the latest homology modeling method using a rotormer library to provide the most accurate conformation in the side chains of non-identical residues To do.

In a preferred embodiment, structural differences which characterized by these four regions is reasonable to design specific receptor of interest (e.g., D ₃ receptor) ligands that selectively bind to the base I will provide a.

In the region A, residues of TM1 of _{D 3 N47, D75, F338,} N375, N379 ( respectively, residues of _{D 2 N52, D80, F382,} N418, N422) , the transmembrane regions deep large hydrophobic pocket It was found to be a part. In a preferred embodiment, the present invention has a flexible linker that is chained to a large tail which is designed to reach a large hydrophobic pocket region A, either selectively D ₃ or D ₂ receptors A ligand that binds to is provided. These findings are based in part on experimental data obtained in the studies referred to below. For example, the SAR analysis classification of several proposed lead compound, a long group linker with a large tail section is shown to selectively bind to D ₃ receptors.

In the region B, residues F345 and F346 of TM6 of D ₃ was found to have an orientation different side chains compared to the corresponding residues F389 and F390 of D _2. Also, the structural differences of TM6 were found to affect the interhelical packing between TM6 and TM7. Residues T368 of D ₃ is exposed to a solvent, the corresponding D ₂ residues F411 plays a role in helix interactions between TM6-TM7. Furthermore, residues T369, Y365, F345, and F346 are important structural differences of TM6 and TM7 of D ₂ and D _3. These residues are assumed to interact directly with their respective ligands. These findings are based in part on experimental data obtained in the studies referred to below. For example, studies of chimeric D ₂ / D ₃ receptor, TM6 and TM7 has been shown to contribute to the selectivity of the PD one hundred twenty-nine thousand eight hundred and seven a selective D ₃ partial agonist. Moreover, studies of chimeric D ₂ / D ₃ receptor, TM6 and TM7 region was shown to be responsible for the selectivity of some ligands between D ₂ and D _3. According to yet another study, D ₂ receptors F198, F389, mutation to F390 of alanine, be eliminated binding of N-0437 is a ligand showing binding priority to D ₂ with respect to D ₃ Indicated. In still other research, mutations T369V is, but significantly increases the binding affinity of the two D ₃ selective ligand, it has been shown not to increase those ligands not selective. This suggests that this region is important for ligand selectivity.

In the region C, D ₃ and the comparison of the most conformations clusters in D ₂ (from MD simulations), preferably kink (kink) angle of Pro200 (TM5), D ₃ in (16.3 °) D ₂ ( 24.0 °). This kink, serine residue three important TM5 (each, S193 of D _2, S194, and S197, and, respectively, of the D ₃ S192, S193, and S196) between D ₃ and D ₂ comprising Move the position of the residue at the extracellular end of TM5. These findings are based in part on experimental data obtained in the studies referred to below. Studies show that the nature of this conserved Pro previous residue in TM5 affects the binding / selectivity of some ligands (eg, in D ₁ , D ₂ , and D ₃ Related residues are Ile, Val, and Leu, respectively). Further studies, such as having a different effect on the binding affinity of the D ₂ and D ₃ receptors for the same ligand, mutations in serine corresponding TM5 is shown, thus a preferred embodiment of the present invention, by mutation by utilizing the difference of these receptors, it is designed to selectively bind to either D ₂ or D ₃ receptor. In yet further studies, (R) - (+) - 7-OH-DPAT show that interact with S192 of D ₃ receptors. The selectivity of this compound decreased 5-fold when the hydroxyl substituent moved to the 5-position.

The hydrophobic pocket in region D is formed by residues of TM3, TM4, TM5, and E-II loops. V111 in D _3, I183, orientation and position of the F188 is the residue of the corresponding D _2, V115, I194, very different from those of F189. TM4 residue V164 of D ₃ is Ile in D ₂ is close to three serine residues in TM5. Also, the difference seen in the preferred kink angle of TM5 contributes to this pocket structural difference. These findings are based in part on experimental data obtained in the studies referred to below. Studies region D of the D ₃ and D ₂ receptors, for example the TM3, the disulfide bridge linking the residues S182 of E-II loop of D ₃ receptors corresponding to residue I183 in D ₂ receptors It is shown to contain non-conservative sequence differences in adjacent positions. This position, it is important to close the D ₃ of I183, F188 or D ₂ of I184, F189,. A163 corresponds to T165 of D _2. Residues S163 of D ₂ which is exposed to water, is utilized in order to interact with the ligand in this region, which is in the D ₃ is replaced by an alanine (A161).

Preferred embodiments of the invention envisage designing ligands specific for dopamine receptor subtypes based on structural differences in these four specific regions (regions A, B, C, and D). To do. In a particularly preferred embodiment, the present invention contemplates the design based on the structure of the ligand that specifically binds to a region B of the D ₃ receptor.

  Preferred embodiments of the compositions (and methods) of the invention were designed using accurate modeling of various dopamine receptors and their transmembrane (TM) regions.

The sequence of the dopamine receptor comprises about 93-97% of the conserved amino acid set shared by the 493 proteins of the rhodopsin family. (See JM Baldwin et al., J. Mol. Biol., 272: 144-164 [1997]). The sequence alignment between D ₁ , D ₂ , and D ₃ , and rhodopsin used in the homology modeling of the present invention, shows 500 GPCR members and D ₁ , D ₂ , D ₃ , and the rhodopsin subfamily within the GPCR. It was derived from rhodopsin.

The dopamine receptors D ₁ , D ₂ , and D ₃ , and rhodopsin share about 47% residue identity / similarity in seven different TM regions. Sequence identity between rhodopsin and D ₃ receptors are 28%, close to 30% of the threshold value is considered to be sufficient to achieve a highly accurate homology model structure. There is no gap or insertion in the sequence alignment between D ₁ , D ₂ , and D ₃ that form the binding site for the ligand, and the rhodopsin helix region. Thus, in a preferred embodiment, the high resolution X-ray structure of rhodopsin is used to provide an accurate modeling of the structure of the dopamine receptors D ₁ , D ₂ , and D ₃ receptors. As shown in Table 3 below, rhodopsin and D ₁ , D ₁ in the transmembrane region, due to the conserved amino acid residues of the previously obtained rhodopsin family and also the sequence identity between rhodopsin and dopamine receptors. _2, and D ₃ distinct sequence alignment is possible between the receptor. (See JM Baldwin et al., J. Mol. Biol., 272: 144-164 [1997]). Use alignment and X-ray of the rhodopsin (K. Palczewski et al., Science, 289: 739-745 [2000 ]), D 1, D 2, and the initial coordinates of the transmembrane region of D ₃ receptors, InsightII Created using the package Homology Module. (AM van Rhee and KA Jacobson, Drug Dev. Res., 37: 1-38 [1996]). Since there are no insertions or deletions in the sequence alignment for the TM region, the coordinates of the rhodopsin backbone atoms were copied as the backbone atom coordinates of the aligned residues of the dopamine receptor. If the residues aligned between rhodopsin and the dopamine receptor are identical, the side chain atom coordinates in the rhodopsin structure were copied as the side chain atom coordinates of the corresponding dopamine residue. If the aligned residues were not the same, the rhodopsin residue was mutated to the corresponding dopamine residue type. The side chain rotamer library within the homology modeling module was used to model the most likely amino acid side chain orientation.

Table 3 shows the sequence alignment of rhodopsin transmembrane between (RHOA) and D _1, D _2, and D ₃ receptors (TM) region (TM1～TM7, plus short TM8). Bold letters indicate amino acids that are conserved within 493 members of the rhodopsin family of GPCR proteins, and are identical between rhodopsin and all three dopamine receptors at the same time. In preferred embodiments, together with additional sequence identity between rhodopsin and the dopamine receptor, these conserved amino acids provide a clear sequence alignment for each transmembrane helix. The underlined letters in Table 3 are additional amino acid residues that are the same or similar between the rhodopsin, D ₁ , D ₂ , and D ₃ receptors.

Seven TM helices (TM1~TM7) is, D _1, D _2, and to form a large gap on the extracellular side of the TM region containing a ligand-binding pocket of D ₃ receptors. In a preferred embodiment, some extracellular loops (EI, E-II, E-III) are close to the ligand binding pocket and may have some effect on the structure of the ligand binding pocket. included. A short intracellular loop CI was also included because its side chain interacted with a short helix moiety linked to the intracellular end of helix 7 in the rhodopsin crystal structure. The other loops (C-II, C-III) and the N and C termini are far from the ligand binding site. Therefore, they are not expected to affect ligand binding (DH van Leeuwen et al., Mol. Pharmacol., 48: 344-351 [1995]; and H. Heller et al., J. Phys. Chem., 97: 8343-8360 [1993]) and therefore not included in the preferred model. Residues before and after the omitted loop were covered with acetyl and N-methyl groups. The extracellular loop region (EI, E-II, and E-III) and one intracellular loop CI were modeled based on sequence homology between rhodopsin and dopamine receptors. For some of these loop regions, fairly high sequence homology was seen between the rhodopsin, D ₁ , D ₂ , and D ₃ receptors. Amino acid identity between rhodopsin and D ₃ receptors, 33% for EI, respectively, 25% for E-III, 33% for CI loop. TM3 end conserved cysteines (Table 3) (D ₁ of Cys96, D ₂ of Cys107, and D ₃ of Cys103) and very short cysteine residues E-II loop (D ₁ Cys186, D ₂ of Cys182, and there is a conserved disulphide bond is formed between the D Cys181 of _3). This disulfide bond is supposed to limit the conformational flexibility of this short loop of the dopamine receptor. Thus, in a preferred embodiment, the loop structure is such that the INSIGHTII program package (INSIGHT II, INSIGHT II, Molecular Simulations Inc., San Diego, CA).

No understanding of the mechanism is necessary to practice the invention, and the invention is not so limited, but the two conserved proline kinks found in TM5 and TM6 affect the ligand binding site structure. It is assumed that it is important for ligand binding. In addition, another conserved proline kink is found in the intracellular portion of TM7, which is thought to be essential for receptor function. (See H. Heller et al., J. Phys. Chem., 97: 8343-8360 [1993]). Complete conservation of these three important prolines allows for the accurate modeling of proline kinks in TM5, TM6, and TM7 of the modeled D ₁ , D ₂ , and D ₃ structures. Two other proline rhodopsin, one is seen in TM1 (Pro53), a second proline was seen in cells outside of TM7 (Pro291), the D _1, D _2, and D _3, the other residues Is replaced by Also, an understanding of the mechanism is not required to practice the present invention, and the present invention is not so limited, but it is assumed that TM1 is not critical for ligand binding because it is so distant from the ligand binding site. . Pro291 found on the extracellular side of TM7 is near the extracellular end of this helix and therefore has little effect on the conformation of the ligand binding site. The standard hydrogen bond pattern of the α-helix at these two sites was obtained by applying a weak NOE restriction between the hydrogen bond backbone atoms during the initial MD simulation. Accurate modeling of the most important helices in D ₁ , D ₂ , and D ₃ using rhodopsin's X-ray structure allows the present invention to structure high-quality ligand binding sites for D ₁ , D ₂ , and D ₃ I will provide a.

The lipid membrane / water environment has been shown to greatly affect the 3D structure of TM proteins. (See RG Efremov et al., Biophys. J., 76: 2460-2471 [1999]). Prior to the present invention, dopamine receptor modeling work avoided modeling lipid membrane / water environments due to limited computer capabilities. In the present invention, a clear lipid membrane / water environment was included in further structural modeling experiments to further improve model accuracy. In a preferred embodiment, the lipid molecule selected to model the membrane is 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC). A bilayer model consisting of 200 POPC molecules was used in modeling experiments as specified in WL Jorgensen et al., J. Chem. Phys., 79: 926-935 (1983). In a preferred embodiment, dopamine receptor structures (eg, D ₃ ) were incorporated into the bilayer model according to the predicted membrane boundary location obtained from sequence analysis of 493 GPCR proteins. (See JM Baldwin et al., J. Mol. Biol., 272: 144-164 [1997]). First, many lipid molecules located in the center of the lipid bilayer were removed to create a hole with approximately the same diameter as that of the receptor structure. The size of the protein structure was then reduced to 50% before placing it in the center of the hole. The reduced size protein structure was then stepped back to its original size in 5% increments via MD simulation. Lipid molecules can adjust themselves to accommodate protein structures during MD simulations, but protein structures are preserved strictly. This procedure creates large voids between the protein structure and lipids, and the proteins and twos created by the strict van der Waals repulsion that would occur if the protein was simply placed in a bilayer hole without this process. It is supposed to prevent sudden degradation of the structure of the stratified molecule.

  The intracellular and extracellular regions of the receptor structure are then exposed to water. In a preferred embodiment, the water environment in these experiments is a clear water model of TIP3P as described in BR Brooks et al. (BR Brooks et al., J. Comp. Chem., 4: 187-217 [1983]). Is accurately modeled. Approximately 2,000-2,200 water molecules were used to cover the protein residues and charged lipid heads exposed to water with a minimum of 20 cm water layer. The total size of the system is about 16,000 atoms as a whole.

  In some other embodiments, extensive structural improvements in the modeled structure are made by including distinct lipids and water molecules. A parallel version of the CHARMM program (version 2.7) (AD MacKerell Jr. et al., J. Phys. Chem. B., 102: 3586-3616 [1998]) and its latest CHARMM force field (WD Cornell et al., J. Am Chem. Soc., 117: 5179-5197 [1995]) is used for all energy minimization and MD simulations. The present invention performs parallel simulation using the latest version of AMBER force field and program to confirm that the cluster of conformations obtained from MD simulation is not affected by the CHARMM force field used. Assume further. AMBER and CHARMM are the two most popular MD simulation programs. The AMBER and CHARMM program force fields have been extensively tested for protein simulation.

  The present invention is a 512 CPU Clay T3E supercomputer (eg, Pittsburgh Supercomputer Center), a 32 CPU Origin 2000 computer system from the National Institutes of Health (NIH), and / or a Linux 48-CPU cluster from the University of Michigan Cancer Research Center, Assume that these models and simulations operate.

  In yet other embodiments, pre-equilibration of dopamine receptor structure 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. The simulation was performed using the presentation of all atoms in the latest version of the CHARMM force field, except that the hydrocarbon tail of the POPC lipid molecule was applied to the integrated CHARMM atom model (WD Cornell et al., J. Am. Chem. Soc., 117: 5179-5197 [1995]). In a preferred embodiment, the Leapfrog Verlet algorithm was used. In yet other embodiments, these hydrocarbon tail atoms are more than 35 mm away from the origin to reduce artifacts caused by the lipid molecular layer at the edge where there is no "external" adjacency. Applied a stochastic boundary method; the coefficient of friction was set to 200. The phosphorus atom in the lipid head was fixed. The dielectric constant was set to 1 and the time step was set to 1 fs. The temperature was held constant at 300 K with a bond decay time of 1.0 ps. Long-distance electrostatic forces were processed at 12-14 法 with the force switch method; van der Waals forces were cut at 14 Å. The unbonded list was made up to 15 cm and heuristically updated. The frequency of checking atoms entering the Langevin region was set to 20 steps. Prior to running the final production MD, the system is pre-equilibrated in three steps of a nested energy minimization cycle to run a large MD simulation. To significantly improve dopamine structure and to sample binding site conformation, very long production MD simulations (at least 10 ns) are performed. The trajectory file of the final production run is saved for each ps for analysis.

The dopamine receptor binding site has certain flexibility, particularly with respect to the bending of the TM5 and TM6 helices, and the side chain conformation of the residues that form the binding pocket. In addition, different ligands will preferentially bind to the characteristic different conformations of the binding gap. For these reasons, in some embodiments of the present invention, the respective receptors (eg, D ₁ , D ₂ , D ₃ , D ₄ , and Use very long (eg, 5, 10, 50, 100 ns, or longer) MD simulations for (or D ₅ ). Since many conformations are created, it is necessary to use conformational cluster analysis to identify the major conformations obtained in the simulation. Conformational cluster analysis was performed using a CHARMM script that uses two criteria. The first criterion was the flexion angle of the TM5 and TM6 helices. The second criterion was the dihedral angle of these residues forming the binding pocket. Based on the 2000 receptor conformations preserved during the 2.0 ns MD simulation of the D ₃ receptor, the four major conformational clusters are 30%, 16%, 13%, and 10% Obtained by distribution. Since the first cluster occupies almost twice as much as the next largest, the central conformation of this cluster is preferred and good for D ₃ receptors in its natural environment This is considered to be a typical structure. The purpose of the database search based on the structure, the representative D ₃ conformationally considered acceptor structure.

Receptor structure used in the docking studies of computational docking studies present invention the interaction between III.D ₃ ligand and receptor, D _1, D _2, and a very long 3D structure D ₃ Obtained from MD simulation. Cluster analysis was performed to identify the major (most populated) conformational clusters. In a preferred embodiment, for each dopamine receptor subtype (eg, D ₁ , D ₂ , and D ₃ ), each major conformational cluster is used for subsequent docking studies. An understanding of the mechanism is not required to practice the invention, and the invention is not so limited, but one advantage of using multiple structures for each receptor is that the conformation of the receptor It is envisaged to better take into account the flexibility of the ligand, and also to take into account the flexibility of the ligand conformation. In a preferred embodiment, the receptor is processed using the fused atom approximation in the AMBER force field. (See Available Chemicals Directory [ACD]: Database of chemicals and their suppliers, MDL Information Systems, Inc., Morristown, NJ). A polar hydrogen atom was added to the acceptor and assigned a partial charge for the Kollman fusion atom. In some embodiments, all water molecules were removed and atom solvation parameters and fragment volumes were assigned to acceptor atoms using the AddSoll function of the AUTODOCK program.

In a preferred embodiment, all of the lead compound D ₃ ligands and their selective analogs identified is studied using modeling and simulation method of the present invention. In a further preferred embodiment, all of the designed lead compound analogs are provided using extensive docking simulation and modeling. In still further preferred embodiments, all possible chiral coordinations are considered for each ligand. In some embodiments, the initial ligand 3D structure is generated using the Sybyl (Tripos Associates, Inc., St. Louis, MO) molecular modeling package.

  In yet another embodiment, the AUTODOCK (version 3.0) program is used to perform docking studies. (See, eg, GM Morris et al., J. Computer-Aided Molecular Design, 10: 293-304 [1996]; GM Morris et al., J. Comp. Chem., 19: 1639-1662 [1998]; DS Goodsell et al., J. Mol. Recognition, 9: 1-5 [1996]; and GM Morris et al., J. Comp. Chem., 19: 1639-1662 [1998]). The AUTODOCK program is widely used to accurately predict protein-inhibitors, peptide-antibodies, and protein-protein complexes. The AUTODOCK program takes into account the conformational flexibility of the ligand during docking simulation. Among the three search methods available in the AUTODOCK program, the Lamarckian genetic algorithm (LGA) method has been found to be very efficient in predicting accurate ligand binding models. . Thus, in some embodiments, the Lamarckian genetic algorithm (LGA) method in the AUTODOCK program is further used in the docking study of the present invention. (D. S. Goodsell et al., J. Mol. Recognition, 9: 1-5 [1996]). The latest version of the AUTODOCK program also incorporates a new empirical coupled free energy function.

In some embodiments, rotatable binding in the ligand was defined using the AUTODOCK utility (AUTOTORS). In the preferred docking simulation, all rotatable binding of the ligand was not limited. Also, the position and orientation of the ligand was not limited. The center of the lattice map was defined using the coordinates of the sulfur atom of D ₂ Cys118 or D ₃ Cys114 or the oxygen atom of D ₁ Ser107, which was determined to be part of the binding site. For each docking study, typically 10 docking simulations were performed. The step size used for the docking simulation was 0.2 mm for translation and 5 ° for orientation and twist. In the analysis of the docked structure, the clustering tolerance for the root mean square deviation of the ligand was set to 1.0 Å.

In a further embodiment, a docking module in the CERIUS ² (Accelrys, Inc., San Diego, CA) program is used to perform docking simulations. The present invention contemplates the use of two (or more) different docking simulation programs / routines that provide cross validation and improved accuracy. Accordingly, preferred embodiments of the present invention use two (or more) docking simulation programs and / or routines.

One limitation of modern computational docking methods, including the AUTODOCK program we used in this project, is that due to the limitations of computational power, the flexibility of protein conformation is hardly considered during docking. is there. To overcome this limitation, the present invention provides a conformation of a number of proteins (eg, dopamine receptors and / or other g-protein coupled receptors) derived from MD simulations for computational docking studies. Suppose you use a motion. The present invention may assume a number of low energy conformations for these binding sites at receptors (eg, dopamine receptors D ₁ , D ₂ , and / or D ₃ ), and thus different ligands It is further envisioned that it may bind to receptors of different low energy conformations.

It has been found that using a large number of conformations of a particular protein (eg, dopamine receptor) greatly improves the accuracy of computational docking studies. Thus, some preferred embodiments of the present invention, selective ligands (e.g., D ₃ selective ligand) using a number of conformation design leave the target protein.

  Another preferred embodiment of the present invention uses large scale MD simulations for the expected complex structure obtained to correct the ligand design for optimal performance (eg selectivity). Solves the potential problem of receptor conformational change (inductive adaptation) that can occur when binding its ligand.

In one aspect, the present invention is, D ₃ receptor structure to provide a docking studies of 7-OH-DPAT and its analogs. (+) - 7-OH- DPAT is a partial agonist showing some selectivity for subtype D ₂ or more dopamine receptor subtypes D _3. D ₃ binding to the receptor (+) - 7-OH- DPAT the foundation of the structure in order to survey, the large-scale computational docking study was performed using the AUTODOCK program package. (GM Morris et al., J. Computer-Aided Molecular Design, 10: 293-304 [1996]; GM Morris et al., J. Comp. Chem., 19: 1639-1662 [1998]; DS Goodsell et al., J. Mol. Recognition, 9: 1-5 [1996] and GM Morris et al., J. Comp. Chem., 19: 1639-1662 [1998]). The most populated D ₃ receptor conformation obtained from cluster analysis was used as a receptor structure in the docking studies. Of the 10 AUTODOCK simulations performed, the four lowest energy complexes converged to a single binding model for the ligand, as shown in FIG. (+) Since there is a wealth of experimental information about the ligand (+)-7-OH-DPAT (Figure 4) obtained via mutagenesis analysis and extensive structure-activity relationship (SAR) information (+) binding model for D ₃ predicted for -7-OH-DPAT ligand was easily confirmed.

(+) - 7-OH- DPAT and D ₃ predicted receptor with the receptor - ligand interactions include the following (but not limited to) is supported by experimental evidence . To a 1, D ₂ and D ₃ receptors, which binds the ligand containing the protonated amine with high affinity, is not limited to any particular mechanism, this binding, extracellular TM3 It is thought to occur through the formation of a salt bridge with the terminal Asp (also common to other members of the rhodopsin family GPCR). This mutation of aspartate to Gly or Asn at the D ₂ (D114) receptor abolishes both agonist and antagonist binding. (See A. Mansour et al., Eur. J. Pharmacol., 227: 205-214 [1992]). Second, the hydroxyl group of (+)-7-OH-DPAT is expected to form hydrogen bonds with S192 and S196. Indeed, S192A mutant D ₃ receptor, as compared to the wild type receptor, with 16-fold reduced affinity (+) - coupling the 7-OH-DPAT ligand. (See N. Sartania and PG Strange, J. Neurochem., 72: 2621-2624 [1999]). Moreover, the corresponding residue S193 of D ₂ have been shown to contribute to the binding of the ligand in D ₂ receptors. (See C. Coley et al., J. Neurochem., 74: 358-366 [2000]). Third, Cys114 forms a “propyl” pocket by intimate contact with one of the propyl groups of the (+)-7-OH-DPAT ligand. In fact, the Cys114Ser mutation selectively reduces the affinity of (+) 7-OH-DPAT ligand containing propyl out of 14 compounds tested. (See GL Alberts et al., Br. J. Pharmacol., 125: 705-710 [1998]; and GL Alberts et al., Mol. Pharmacol., 54: 379-388 [1998]). Fourth, one of the (+)-7-OH-DPAT propyl groups binds to a highly hydrophobic pocket, while the other is in a mixed hydrophobic / hydrophilic environment. A prediction based on this model showed that a 10-fold decrease in binding affinity was observed when both propyl groups were removed, but removing only one propyl did not appear to change the binding capacity of the ligand It is consistent with being. (See DA Norman and LH Naylor, Biochem. Soc. Trans., 22: 143S [1994]). For the 5-OH-DPAT (Figure 4) analog of (+)-7-OH-DPAT, the change due to removal of both propyl groups is even more dramatic. It is removed one of a propyl group, but no effect on ligand binding in D _3, by removing both propyl groups, decrease the affinity of the ligand to the 635-fold. (See DA Norman and LH Naylor, below). One D ₃ hydrophobic pocket, rather large, very makes it possible to tolerate large group, the other pocket is limited in size, they have a hydrophobic / hydrophilic mixed environment That is important. Taken together, the docking results predicted by ligand design method disclosed in the present invention, the existing experimental mutation of data and (+) - SAR data for binding of 7-OH-DPAT ligand and D ₃ receptors There was an excellent agreement with

This excellent agreement serves as a confirmation of the modeled D ₃ structure envisioned by the present invention, and this provides insights into the structural basis of D ₃ binding to potentially strong D ₃ ligands. provide.

IV. General Design Strategy for Identifying New Lead Compounds A preferred embodiment of the present invention is a novel structure-based approach to identify potential lead compounds as selective ligands for dopamine receptors. use. In a particularly preferred embodiment, the design approach based on the structure of these novel, potent and selective modulators of dopamine receptor D ₃ (e.g., partial agonist) in order to identify and further design (e.g., optimum Used). In yet another preferred embodiment, potent and selective modulators of dopamine receptors D ₃ are symptoms (e.g. depression characterized by a defect in regulation of D ₃ receptors in a subject, anxiety, schizophrenia, Tourette Syndrome, eating disorders, alcoholism, chronic pain, obsessive compulsive disorder, and Parkinson's disease).

The present invention relates to a lead G protein receptor binding ligand (eg, but not limited to D ₁ , D ₂ , D ₃ , D ₄ , and D ₅ ) as shown in FIG. It further provides a new and powerful approach for identifying and designing (eg, optimizing)). In a preferred embodiment, the first step is to search a 500,000 library of highly structurally diverse synthetic and natural organic compounds to identify initial receptor binding candidate ligands (“hits”). using the 3D- database search technique based on pharmacophore, provides for the identification of potential lead compound (e.g., D ₃ agonists and / or antagonists). For example, in one embodiment, pharmacophore model of a known D ₃ ligand 3D structure and their structures - have been developed based on activity relationship (SAR). This pharmacophore model can be used to search the National Cancer Institute (NCI) 3D structure database and / or the Available Chemicals Directory (ACD) (S. Wang et al., J. Med. Chem., 37: 4479-4489). (See [1994]) Used to search the database. The latest version of NCI 3D's “public” database consists of 245,000 structures, allowing researchers access to these structures and chemical samples. (See JH 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. The web-based 3D-pharmacophore docking program of the present invention has been found to provide 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 to 3D-database format are searched by the web-based program as part of identifying potential receptor binding lead compounds.

In yet another preferred embodiment, the screening step based on the second structure, receptor of interest (e.g., D ₃ receptors) and hit compounds identified in the first step (e.g., dopamine receptor ligands The following means are provided to further clarify the interaction.

In a preferred embodiment, the screening step based on the second structure uses a “smaller” database that contains only “hits” identified through the 3D-pharmacophore search of the first step. However, it is not intended that the present invention be limited to selecting potential target receptor ligands based on this methodology. In yet another preferred embodiment, the Ligand Fit module of program CERIUS ² version 4.5 (Accelrys, Inc., San Diego, Calif.) Is used to perform structure-based database searches. In some embodiments, a database search based on the structure is used that each hit compounds classified in the database, to dock the binding pocket of the receptor of interest (e.g., D ₃ receptor binding pocket) . In still some other embodiments, the present invention contemplates ranking hit compounds according to these scores calculated by the CERIUS ² program. In some embodiments of the invention, the scoring function used in the CERIUS ² program calculates the negative number of potential interaction energies between the ligand and the receptor. Although the present invention is limited, the scoring function of the energy-based DOCK program will not provide a good correlation with the binding affinity of a particular candidate ligand for its target receptor Is assumed. Accordingly, the preferred embodiment of the present invention contemplates overcoming this potential problem by using a novel consensus scoring function developed as part of the present invention. In a preferred embodiment, the consensus scoring function comprises three different empirical binding affinity prediction methods, CHEMSCORE, LBSCORE, and LUDISCORE (J. Comput.-Aided Mol. Des., 11: 425-445 [1997]; Mol. Model., 4,379-394 [1998]; and J. Comput.-Aided Mol. Des., 12,309-323 [1998]) and used more than 200 protein-ligand complexes. Obtained. In some aspects, it is further envisaged to use a consensus scoring function to improve the “hit” rate.

While the present invention is limited, the scoring function of the energy-based CERIUS ² program will not provide a good correlation with the binding affinity of a particular candidate ligand for its target receptor Assume that. Accordingly, a preferred aspect of the present invention envisions testing a docking and scoring protocol used for structure-based database searching as follows. A set of 25 known D ₃ ligands was "mixed" with the database compounds. After docking these compounds into a representative D ₃ receptor conformation and scoring based on these Cerius ² dock cores, 24 of the 25 known D ₃ ligands It was found to be among the top 25% in the rank order of the highest to worst scores of compounds. Therefore, applicable Index and docking protocols, it is possible to select ligands with a good D ₃ binding affinities among the top ranked King compound. Thus, compounds selected for further biological analysis (eg, in vivo and / or in vitro assays) have scores comparable to known D ₃ ligands, ie, they are the top 25 ranked database compounds. Selected from%.

The computerized screening approaches the two-stage, not only based on their 3D pharmacophore binding element, also based on the 3D structure and their interaction target receptor of interest (D ₃ receptors) It is envisioned that the present invention will allow identification of the most promising potential ligands.

  As demonstrated herein, this novel approach provides a powerful means for discovering promising dopamine receptors and other G protein receptors, modifiers (eg, agonists and / or antagonists).

The following examples are potential selective receptor binding ligand (e.g. dopamine receptor _{[D 1; D 2; D} 3; D 4; including and / or D ₅ is not limited to] agonists and / or antagonists A preferred embodiment of a two-step structure-based screening approach for identifying) is further described.

In a preferred embodiment of the invention, the pharmacophore model is defined as a common and essential structural element for activity. Previous studies, many potent and relatively selective D ₃ partial agonists and full agonists was identified. In one embodiment, nine relatively selective moieties and a representative subset of full agonists (see FIG. 2) were selected for development of the pharmacophore model. Structural analysis of these compounds shows that these compounds contain a Sp3 nitrogen attached to a common aromatic ring and propyl group. With the exception of pramipexole, nitrogen is attached to two additional Sp3 carbons. The distance between the aromatic ring center and the basic Sp3 nitrogen in these compounds is between 4.1 and 6.1 mm using a QUANTA program (QUANTA, Molecular Simulations Inc., San Diego, CA). Confirmed through home analysis. A simple pharmacophore model used for structure-based receptor ligand identification and modification (eg, optimization of selectivity) was developed from these analyzes (see FIG. 3). Chemical elements and geometric parameters identified in the pharmacophore model, represents several important D ₃ receptor binding element, in the pharmacophore model alone, high affinity for D ₃ receptors and It is important not to provide enough binding elements to identify those that select or design selectivity.

In one embodiment, the CHEM-X program (Chemical Design Ltd., Oxford England) was used to screen 245,000 “public” test compounds in the NCI 3D-database against the pharmacophore model shown in FIGS. 6A and 6B. )It was used. 6A shows the superposition of nine D ₃ partial agonists and agonists. 6B shows a simple pharmacophore model derived from the D ₃ ligands contemplated. During the exploration process, compounds were selected that had one or more chemical groups required for the pharmacophore model, such as an aromatic ring and / or a tertiary nitrogen attached to three carbon atoms. If the chemical structural requirements were met, the CHEM-X program investigated whether the compound had a conformation that met the 3D geometric parameters specified in the pharmacophore model. A compound is considered a “hit” if the CHEM-X program finds that the compound actually has at least one conformation that meets the 3D geometrical requirements specified in the pharmacophore model. In a preferred embodiment, up to 3,000,000 conformations can be examined for "hit" compounds using a system based on this structure. Using this approach, a total of 6,237 compounds from the NCI 3D-database were identified as hits that met both the chemical and geometric requirements specified in the pharmacophore model.

  In a preferred embodiment, the structure of a compound identified as a hit via a 3D-pharmacophore search is extracted in SDF format from either the NCI or ACD database. In yet another preferred embodiment, each extracted structure is assigned a hydrogen atom and a Gasteiger-Marsili charge using the SPL macro of the SYBYL program. (SYBYL, Tripos Associates, Inc., St. Louis, MO). In a further preferred embodiment, the final coordinates are stored in a single database file for all hit compounds in mol2 format. The protonation of ionizable groups such as amines, amidines, and acids in hit compounds is assigned according to their physiological protonation state at pH 7.4.

The 6,237 hit compounds identified from the pharmacophore search met the basic and essential chemical and geometrical requirements identified in the pharmacophore model. However, many of these hit compounds of 6,237, the more reasons (e.g., steric hindrance) for, it is expected that would show no obvious activity binding to the D ₃ receptor. For example, a specific ligand (hit compound) may have the essential binding elements identified in the pharmacophore model, but the ligand is too large to fit the receptor binding site, or some ligand functional groups interferes with the residues of the receptor ligand is a target receptor (e.g., D ₃ receptor) because might prevent effectively interact with, interest (e.g., D ₃₎ It is envisaged that it may not be able to bind to its receptor.

Comparison of In a further preferred embodiment, the hit compounds of the upper scoring which is assumed to be potential ligands for the receptor of interest, the known ligand of the receptor of interest (e.g., D ₃ receptor ligands) To visually examine their structural novelty and diversity. In a preferred embodiment, hit compounds that pass the visual inspection stage are tested in binding and functional assays.

The present invention is most populated for a particular target receptor in the docking studies obtained from a large MD simulations of the database based on one or more structures using CERIUS ² program (e.g., D ₃ receptors) Assume that you use a cluster of conformations. However, in some instances, very strong ligands of a particular target receptor bind to a target receptor conformation that is slightly different (or even very different) from the most populated conformation cluster. It may be. Thus, some strong ligands may not be able to dock to the receptor of interest and may not have a high docking score. In some embodiments, the present invention overcomes this potential limitation by using several populated low energy conformations derived from very long MD simulations for structure-based database searches. Assume that. It is further envisioned that by using multiple receptor conformations for structure-based database searching, discovery of additional new lead compounds that might otherwise be missed.

V. Identification of Specific Novel Lead Compounds As noted above, the present invention specifically includes D ₁ , D ₂ , D ₃ , D ₄ , and D ₅ for (but not including) g protein receptors. We envisage the discovery and design of new and potent ligands selective for (but not limited to) dopamine receptors.

In a preferred embodiment, the structure of accurately modeled dopamine receptors facilitates the discovery of novel and potent lead compounds as modulators of D ₃ receptors (e.g., partial agonists and / or antagonists). As described above, the present invention provides a novel and powerful approach for identifying and designing G protein receptor ligands by combining ligand-based pharmacophores and structure-based database searches.

In a further embodiment, analysis of the modeled structure of the target receptor (eg, dopamine receptor) and computational docking studies of known relatively selective D ₃ ligands result in the target receptor (eg, dopamine receptor). Provides further elucidation of the structural basis of ligand binding and selectivity in Using a detailed structural analysis of the D ₁ , D ₂ , and D ₃ receptors, four identifications with significant structural differences between their binding sites on the D ₂ and D ₃ receptors Regions (regions A, B, C, and D) were identified. Using an analysis of the other detailed structures were identified significant differences between D ₃ and D ₁ receptors. In a preferred embodiment, as described below, the differences in structural regions A, B, C, and D of the dopamine receptor are used to rationally design highly selective dopamine receptor ligands.

The present invention is directed to lead compounds described herein, as well as additional lead compounds that would be identified by one of ordinary skill in the art in light of the modeling and structure-based ligand identification and design methods described herein. May be further optimized and refined using the disclosed chemical modification methods to improve one or more characteristics (eg, receptor affinity, receptor selectivity, etc.) Is assumed. Indeed, the design and chemical modification methods based on the structure of the present invention, high affinity (Ki = 6 nM or higher), and great selectivity for D ₃ with respect to D ₁ and D ₂ (> 100 times) Derivatives and analogs of lead compounds having However, the present invention provides a highly selective ligand with respect to D ₃ receptors, but is not limited thereto. Indeed, the present invention provides a method for rationally designing and chemically modifying many receptor ligands.

  The following specific examples of lead compound identification, assay, and chemical modification provide the reader with some non-limiting representative examples of the various lead compounds (analogs and derivatives) envisioned by the present invention. Contemplate that.

In one embodiment, the initial binding affinity of the 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 SJ Enna, Eds.], John Wiley & Sons, New York, 1.6.1-1.6.16 [1998]). The potent D ₂ and D ₃ ligand [ ³ H] YM-09151-2 was used as the radioligand for the D ₂ and D ₃ receptor binding assays. Similarly, [ ³ H] SCH23,390, a potent and selective D ₁ receptor ligand, was used as a radioligand for the D ₁ receptor binding assay. The ability of 30 test compounds to compete with [ ³ H] YM-09151-2 for binding to the D ₃ receptor using CHO cells first transfected with the human D ₃ (hD ₃ ) receptor Was measured. When sufficient binding was observed for 10 μM test compound, its IC ₅₀ value was obtained. For compounds with IC ₅₀ values less than 10 μM, LHD1 cells transfected with human D ₁ receptor (hD ₁ ) are used to determine their D ₁ binding capacity and human D ₂ receptor (hD ₂ ) These D ₂ binding capacities were determined using CHO cells transfected with. Ki values were calculated using the Cheng-Prusoff equation, which assumes classical competitive inhibition.

  The results are summarized in Table 4. Table 4 shows the binding affinity of the eight best test compounds measured using cell lines transfected with human dopamine receptor.

Of the 30 test compounds evaluated, 8 compounds (named compounds 1-8, respectively) had Ki values in the nanomolar range (eg, about 11 nM to about 465 nM). One compound has a Ki value of 11 nM, a fairly potent D ₃ ligands. 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, 10-fold selectivity between D ₁ and D ₃ receptors, and 23-fold selectivity between D ₂ and D ₃ receptors, respectively. . The results show that the method of the present invention is to identify novel and potent dopamine receptor ligand is effective in particular for identifying the D ₃ receptor ligands.

To characterize the new D ₃ ligands, additional experiments were performed in a functional assay. Many studies have shown that the binding affinity of a ligand for dopamine receptors can vary considerably, at least in part, depending on the expression system or tissue, the radioligand, and the in vitro assay conditions used. . (See B. Levant, Pharmacol. Rev., 49: 231-252 [1997]). In one embodiment, the dopamine receptor (e.g., D ₃ receptor) to elucidate these agonists (partial agonist) or an antagonist activity against were screened test compound in a functional assay. Eight rather powerful D ₃ ligands shown in Table 4, Chio et al. And Sautel et al (CL Chio et al, Mol Pharmacol, 45:.. 51-60 [1994]; and F. Sautel et al, Neuroreport, 6: 329 -332 [1995]) were tested for their in vitro functional activity against the human hD ₁ , hD ₂ , and hD ₃ receptors. The results of these functional assays are summarized in Table 5.

N.D. 低い親和性のために測定できず。それぞれの化合物について少なくとも2回の実験を行った。

ND Cannot be measured due to low affinity. At least two experiments were performed for each compound.

In this embodiment, the functional studies show that the compounds 1, 2, 3, 6, and 7 is an antagonist of D ₃ receptors, and compounds 4, 5, and 8 are partial agonists. (See Figure 7). In fact, the study showed that compound 4 gave a 9.2 nM EC ₅₀ of 51% maximal stimulation for mitogenic stimulation in transfected CHO-D ₃ cells compared to the full D ₃ agonist quinpirole. it is shown a potent D ₃ partial agonist having. (Figure 8). FIG. 8 shows the functional activity of compound 4 (CTDP-31793) at the D ₁ , D ₂ , and D ₃ receptors and a comparison with standard ligands. Agonist activity at the D ₁ receptor was measured for stimulation of cAMP accumulation in C6D ₁ cells. Antagonist activity at D ₁ measured the inhibition of cAMP accumulation in 10 nM C6D ₁ cells by dihydroxy Jin (dihydrexidine) stimulation. Agonist activity of D ₂ receptors was measured stimulation of mitogenesis using Chop-hD ₂ cells. D ₂ receptor antagonist activity measured the inhibition of mitogenesis in CHOp-hD ₂ cells by 30 nM quinpirole stimulation. Agonist activity of D ₃ receptors, was determined stimulation of mitogenesis using Chop-hD ₃ cells. Antagonist activity of the D ₃ receptor, was measured the inhibition of mitogenesis in Chop-hD ₃ cells with 30nM quinpirole stimulation. In contrast to quinpirole, Compound 4 acts as an antagonist having a Ki value of 35.1nM in D ₂ receptors.

In addition, compound 4 functions as a weak D ₁ antagonist with a Ki value of 657 nM (FIG. 8). Compound 5 appears to be more selective, although it has a profile that is very similar in function activity to compound 4. However, Compound 5 is a partial agonist of D ₃ with an EC ₅₀ of 22.2 nM and 31% mitogenic maximal stimulation compared to quinpirole, as a D ₂ antagonist with an IC ₅₀ value of 378 nM, and 1722 nM It functions as a weak D ₁ antagonist with an IC ₅₀ value of.

To determine the selectivity of the compound, in a more meaningful functional experiments, Compound 4, 4 times between the D ₃ / D _2, has a 70-fold selectivity between D ₃ / D _1, Compound 5 The selectivity is 17 times between D ₃ / D ₂ and 78 times between D ₃ / D ₁ . Like the compounds 4 and 5, compound 8 serves as a partial agonist of D ₃ receptors with maximum stimulation of EC ₅₀ and 53% of 146 nm.

Subsequently, the present invention contemplates functional studies showing that compounds 4 and 5 are potent D ₃ partial agonists, but compounds 4 and 5 function as antagonists of the D ₁ and D ₂ receptors. Compounds 4 and 5, in particular compound 5 in D ₃ with respect to D ₁ and D ₂ in a functional assay showing a certain selectivity. Compounds 4 and 5 have the same functional profile as that of BP-897, which has been shown to have strong therapeutic potential for the treatment of cocaine addiction. (See, eg, M. Pilla et al., Nature, 400: 371-375 [1999]). Accordingly, in a preferred embodiment, it compounds 4, 5, and 8 is a promising lead compound for further optimization to design potent and selective D ₃ partial agonist.

A still further aspect provides a computational docking studies of D ₃ receptors and lead compound 4. Since compound 4 is a flexible ligand, it is beneficial to use a docking program that considers the flexibility of the ligand in the docking process. For this reason, the AUTODOCK program (DS Goodsell and AJ Olson, Proteins: Str. Func. Genet., 8: 195-202 [1990]; GM Morris et al., J. Computer-Aided Molecular Design, 10: 293-304 [1996]; GM Morris et al., J. Comp. Chem., 19: 1639-1662 [1998]; DS Goodsell et al., J. Mol. Recognition, 9: 1-5 [1996]; Comp. Chem., 19: 1639-1662 [1998]) was used. The most populated conformation clusters of D ₃ receptors obtained from MD simulations of 2 ns, was used as the receptor structures for docking studies. Since compound 4 has a chiral center and the chemical sample used for the receptor binding and functional assays was racemic, both (R)-and (S) -configurations of compound 4 Was used in docking studies. In one embodiment, 10 docking simulations were performed for each structure. Results docking studies of compound 4 (R) - indicates that interacts with preferably D ₃ receptors than that of the arrangement - arrangement (S). The present invention uses other racemic lead compounds, analogs, and derivatives to determine whether one configuration binds to the target receptor more advantageously, (R)-and (S)- It is also envisioned for use in docking studies that use configuration. Of the 10 docking simulations, 4 docking simulations with the lowest predicted binding free energy converged to a single binding model (see FIG. 9).

9, compound 4, (R) - indicates that a strong salt bridge between the 7-OH-DPAT as with, D110 nitrogen and D _3, which is the protonated - (+). (See Figure 3). Tricyclic ring of compound 4, V111, V189, F346, F197 , F345, W342, and in intimate contact with a number of hydrophobic residues of D ₃ including C114. Importantly, three serine residues of D _3, S192, S193, and S196 are the compounds 4 docked are fairly close, thus in some embodiments, the present invention is further a D ₃ receptor Derivatives of compound 4 designed to form hydrogen bonds are provided. It is envisioned that incorporation of additional donor / acceptor into the derivative of compound 4 will improve the potency and / or selectivity of the ligand. Similarly, further embodiments are based on the somewhat different positions of these three common serines in D ₁ , D ₂ , D ₄ , and D ₅ based on other dopamine receptor subtypes (eg, D ₁ , D ₂ , envisage designing lead compound derivatives with increased potency and / or selectivity for D ₄ , and / or D ₅ ). Docking studies also show that the 4-F phenyl group of compound 4 interacts with hydrophobic residues W370, Y373. In yet a further docking studies, D ₃ hydrophobic pocket named area A shows that can be accessed from the extracellular space of the receptor. D ₃ receptors model structure suggests that would quite large hydrophobic group is contained in the pocket regions A. In order to reach region A located deep in the transmembrane region, such a group of atoms must be attached to the end of the mobile linker. The linker is assumed to be a hydrocarbon chain substituent protonated nitrogen groups, which is thought to interact with a common Asp of TM3 (D110 in D _3). Our structural analysis shows that the distance between the protonated nitrogen and the center of the large group housed in region A will be about 14 cm. A further aspect of the present invention provides a significant difference between D ₃ and D ₂ in the region A pocket for the design of ligands with high potency and / or selectivity for the particular dopamine receptor subtype of interest. Use. In still further embodiments, the present invention provides potency and / or selectivity in binding to additional receptor types of interest (eg, G protein coupled receptors) via rational directed ligand chemical modifications. Further ligand compounds optimized to enhance are provided.

In a further aspect of the invention, chemical modification of the lead compound is guided by a structure based design. For example, a novel analog of Compound 4 was designed based on the binding model shown in FIG. In one embodiment, compounds 8, 9, and 10 were designed to investigate optimal length linkers. (See Figure 10). In another embodiment, compounds 11 and 12 were designed to investigate the effect of the terminal phenyl group in compounds 4, 8, 9, and 10. (See Figure 10). Based on the docking studies, 4-F- phenyl group of compound 4 is bound to a large hydrophobic pocket of D ₃ receptors. In a further docking studies, the hydrophobic pocket of D _3, indicating that the potential additional space available a larger hydrophobic group such as a biphenyl ring or a naphthyl ring of the ligand binding partner in order to receive there. As previously mentioned, there is a significant difference between the ends of this large hydrophobic pocket at dopamine receptors D ₃ , D ₁ , and D ₂ . Thus, by exploring this large hydrophobic pocket difference between dopamine receptors, compounds 13 and 14 were designed to investigate whether a larger hydrophobic group on the ligand would improve selectivity. (See Figure 10).

In one embodiment of the invention, compounds 4, 8, 9, 10, 11, and 12 were synthesized using the method shown in Scheme I (FIG. 11). Scheme I, Reagents: 20, 3-Chloro-4′-fluoropriofenone; 21, 4-fluorobutyrophenone; 22, 1- (4-flurophenyl-5-chloro-1-oxopentane; 23, 2- Chloro-4'-fluoroacetophenone; 24, iodopropane; and 25, iodobutane, in particular, condensation of ethanolamine with commercially available 2-quinoline-carboxaldehyde (compound 15) gave imine compound 16, which was Reduction to amine compound 17 with ₄ (eg CAR Baxter and HC Richards, J. Med. Chem., 15: 351 [1972]; and VA Rao et al., J. Med. Chem., 13: 516 [1970] Reduction of compound 17 to the corresponding 1,2,3,4-tetrahydroquinoline (compound 18) was achieved in an acetic acid solution of NaBH ₃ CN (GW Gribble and PW Heald, Synthesis, 650 [1975]) cyclization of compound 18 was achieved with a solution of P ₂ O ₅ in xylene. Produced an important intermediate compound 19 (2,3,4,4a, 5,6-hexahydro-1H-pyrazino [1,2-a] quinoline) (CAR Baxter and HC Richards, J. Med. Chem., 15: 351 [1972]) intermediate (compound 19) is alkylated with commercially available compound 20, 21, 22, 24, or 25, respectively, in acetonitrile using cesium carbonate as the base. Yielded compounds 8, 4, and 20, 11, and 12, respectively (see LA van Vliet et al., J. Med. Chem., 43: 2871 [2000]). 19 was first converted to its sodium salt with a DMF solution of sodium hydride and then reacted with a commercial DMF solution of Compound 23 at room temperature to give the final product, Compound 9.

The synthesis of compounds 13 and 14 is provided in Scheme II (Figure 12). In Scheme II: i. 1.2 eq; 26, 2 eq Cs ₂ CO ₃ , CH ₃ CN, N ₂ , reflux, overnight, yield 75-88%; ii. 2 eq hydrazine, EtOH, reflux for 2 h, Yield 82-87%. For compounds 13 and 14: iii. 1.2 eq 2-naphthoyl chloride, 3 eq triethylamine, 0 ° C., 4 h, yield 83-87%. In particular, reaction of phthalimide protected 1-bromoalkylamine compound 26 with intermediate compound 19 (Scheme I) yielded compound 27. Deprotection of compound 26 with hydrazine gave compound 28. Amidation of compound 28 using 2-naphthoyl chloride as the amide drug provides target compounds 13 and 14, respectively. (See JM Robarge et al., J. Med. Chem., 44: 3175-3186 [2001]). Additional lead compound modifications and additional analog designs are described below.

  In yet another embodiment, a rat brain binding assay is used to determine the binding affinity of the novel analog 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 the rat brain binding affinity assay are summarized in Table 6.

データは、3〜5回の繰り返しの平均値である。

Data are average values of 3-5 repeats.

From the results of Table 6, among the respective compounds 4, 8, 9, and 10, compound 4 having 3 carbon linker is clear that the most potent compounds of the D ₃ receptor, therefore, this aspect in three carbon linker are optimal analog of the compound 4 which bind to dopamine receptors D _3. Compounds 9 and 8 with either a 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 a compound at the binding D ₃ receptor. About 2 times less effective than 4. Of note, the binding affinity obtained for both compounds 4 and 8 using the rat striatum binding assay is greater than that obtained using the CHO transfected cells shown above. . However, in both assays, compound 4 is more potent than compound 8 (eg, about 7-12 times stronger in D _{2 and} 6-8 times stronger in D ₃ ), so these two different assays The binding affinities obtained from the method indicate that the absolute values may be different, but are consistent with respect to the relative potency between the analogs. This is consistent with previous observations that the binding affinity of a ligand for a particular dopamine receptor depends on the particular expression system or tissue, the radioligand, or the 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 D ₃ and terminal 4-F-phenyl ketone group interactions in binding affinity. As can be seen in Table 6, compound 11 is a potent in the same manner as Compound 4, Compound 12, the D ₃ receptor binding is 3 times more potent than compound 4. That this finding, 4-F- phenyl group, in intimate contact with a number of hydrophilic / charged residues D _3, it may have some interactions unfavorable and receptor Consistent with the binding model predicted for compound 4 (see FIG. 9). Further, compound 12 has a 9-fold selectivity for receptors D ₃ for the receptor D _2, which is 11 times more selective than the lead compound 4 receptor D ₃ for the receptor D ₂ .

In a preferred embodiment, the present invention provides compound 29 as a partial agonist of the receptor D ₃ a (CTDP-31819). Compound 29 (FIG. 13) has a strong affinity at D _3, in binding assays using rat brain, 39-fold selectivity between the receptor D ₃ and D _2, receptors D ₃ And D ₁ has a 10-fold selectivity. (See Table 7). Table 7 shows the binding affinity of lead compound 29 and some new analogs thereof in the rat brain binding assay.

Functional assays using transfected cells further show that compound 29 is a potent D ₃ partial agonist and a weaker D ₁ and D ₂ antagonist. (See Figure 14). In D ₃ receptor, compound 29, as compared to quinpirole of full agonist, 45% of the maximum stimulation has an EC ₅₀ value of 7.1 nM. At the D ₁ and D ₂ receptors, compound 29 has IC ₅₀ values of 384 nM and 355 nM, respectively. Accordingly, the present invention is that the selectivity of the compound 29 shown in the above functional assays is a 54-fold between 50 times and receptor D ₃ and D ₁ between the receptor D ₃ and D ₂ Consider. Accordingly, the invention provides compounds 29, even before optimizing shown to provide a highly selective D ₃ ligands.

Since compound 29 is a potent D ₃ partial agonist and shows good selectivity for D ₃ over D ₁ and D ₂ , chemical modifications were made to further improve compound potency and selectivity. Computational docking studies showed that compound 29 has a similar binding model as lead compound 4. (See Figure 9). The present invention is described in Robarge et al. And US 3,931,188. (See JM Robarge et al., J. Med. Chem., 44: 3175-3186 [2001]; US 3,931,188, which is incorporated herein in its entirety) Many analogs and chemical derivatives of compound 29 are envisioned using the methods shown in Scheme III (FIG. 15) and Scheme IV (FIG. 16) according to the methods described in (see FIG. 13). Scheme III, Reagents and Conditions: i. 1.2 eq MeMgBr, EtOH, 0 ° C., then 0.5 hour reflux; ii. P-toluenesulfonic acid, toluene, reflux, —H ₂ O, 4 hours, 2 steps yield 72 ˜82%; iii. CH ₂ O, RNH ₃ Cl, AcOH, H ₂ O, 6 hours, yield 18-28%. Scheme IV, Reagents and Conditions: i. 36, (BOC) ₂ O = 7: 1, Dioxane, rt, overnight; ii. 1.2 eq trifluoroacetic anhydride, Et ₃ N, CH ₂ Cl ₂ , rt, 1 Overnight, 2 steps yield 88%; iii. AcCl, MeOH, rt, quantitative; iv. 35, CH ₂ O, AcOH, H ₂ O, 6 hours, yield 38%; v. A) K ₂ CO ₃ , MeOH, rt, overnight; b) 1.2 eq 2-naphthoyl chloride, Et ₃ N, CH ₂ Cl ₂ , 0 ° C., rt, 1 hour, 83% yield over 2 steps. The binding affinity of analogs of compound 29 was tested in a rat brain assay and the results are shown in Table 7 above. Compound 30 has a butyl group and has potent activity at the receptor D ₃ (K _i = 25 nM), but only moderate D ₃ receptor selectivity. Compound 31 has an additional methoxyl group in the meta position of the phenyl ring, is a strong D ₃ ligand (K _i value = 46 nM) and has good selectivity for the receptor D ₃ . The selectivity of Compound 31 is a 175 times between 15 times and receptor D ₃ and D ₁ between the receptor D ₃ and D _2. Binding model for Compound 14, the naphthenyl ring indicates that reaching a portion of the D ₃ binding domain have a significant difference between D ₃ and D ₂ receptors. Thus, in one embodiment of the invention, it is envisioned that compound 32 having a naphthenyl ring is designed and synthesized. Compound 32 is a very potent D ₃ ligand (K _i = 6.1 nM), 133-fold and 163-fold between D ₃ and D ₂ receptors and between D ₃ and D ₁ receptors, respectively. Has great selectivity. Accordingly, in a preferred embodiment, even before optimization, compound 32 provides a highly selective D ₃ ligands.

VI. Further structure-based design and chemical modification of lead compounds In a preferred embodiment, chemical modification of the lead compound is induced by a structure-based design strategy to target receptors (e.g., other D family receptors) , D ₃ ) novel analogs with improved binding affinity and selectivity for the receptor (eg, affinity and selectivity for D ₃ receptor versus D ₁ and D ₂ receptors) are obtained. In some embodiments of the invention, some of the lead compounds and analogs / derivatives thereof have specific binding properties for D ₁ or D ₂ receptors or for other members of D family receptors. The envisaged chemical modifications are, in preferred embodiments of the invention, in part due to consideration of four different aspects, 1) of the D ₁ , D ₂ , and D ₃ receptors that are supposed to contribute to ligand selectivity. between, in particular the analysis of differences in important structures between D ₂ and D ₃ receptors; 2) very discovery of novel and potent D ₃ partial agonist as promising lead compounds; 3) D ₃ receptor Efficient optimization of designed lead compounds to improve the potency and selectivity of candidate compounds in the body; and 4) based on characterization of novel ligands for their potency, selectivity, and functional activity .

In some embodiments of the present invention, extensive docking studies of lead compounds 4 and 29, and new, more potent / selective analogs thereof (compounds 14 and 32), for example, were performed. Compounds 14 and 32 are the potent and selective D ₃ ligand (Figure 17), the use of these two compounds as a novel lead compound for further design and optimization. The binding models for compounds 14 and 32 predicted using the CERIUS2 docking program are shown in FIGS. 19A and 19B.

Many of the compounds 14 and 32 that interact with D ₃ receptor residues, D110, C114, F106, V86 , H349, and V350, etc., in the D ₃ and D ₂ receptors, structurally identical or very It is similar. Thus, the interaction between the ligand and these common D ₃ and D ₂ residues, contributes to ligand affinity, does not contribute is assumed selectivity. However, as described above, the structural analysis of the dopamine receptors structure, between D ₃ and D ₂ receptors, there is a difference in the substantial structure located mainly into four regions. (See Table 2 and FIGS. 18A and 18B). 18A and 18B show the predicted binding model of D ₃ of the acceptor compound 14 (FIG. 18A) and 32 (Figure 18B). It represents a residue within a distance of 5Å from the ligand, are shown some specific interaction between the ligand and the D ₃ receptor by a dotted line. In particular D ₃ and four regions that contribute to the selectivity of the ligand between D ₂ (areas A, B, C, and D) are shown by a circle of orange. Accordingly, preferred embodiments of the present invention provide novel analogs having binding groups (elements) that specifically target these four regions that are supposed to provide greater selectivity. One particular aspect of the present invention provides compounds 14 and 32 and D ₃ 4 single new class of analogues was designed based on binding model with the receptor (group I, II, II, and IV) a . (See Figure 19). FIG. 19 shows a schematic diagram of compound 14 at the D ₃ receptor binding site (notation: residue number / helix number of D ₃ ; EII is extracellular loop 2; black ellipse: primarily responsible for potency Red ellipse: residue predicted to affect ligand selectivity most significantly; blue ellipse: predicted to affect selectivity but less than indicated by red ellipse Residue; point gland shows the expected receptor-ligand interaction).

In one embodiment, the present invention provides analogs of compounds 14 and 32 (Group I analogs) that have modifications in the tricyclic ring to better bind to region C of the dopamine receptor. In this region, there are three common serine residues between dopamine receptors. However, due to the different kink angles in TM5, the positions of the three serine residues in D ₃ (S192, S193, and S196) are altered compared to those of dopamine receptors D ₂ and D ₁ . In a preferred embodiment, compounds are designed to explore differences in the orientation and position of the three common serine residues. For example, the selectivity of compound 31 with one additional methoxyl group is improved compared to that of compound 30. (Table 7). The selectivity of compound 30 between the receptor D ₃ and D ₁ is a 63-fold, selectivity of compounds 31 between these receptors is 175-fold. Thus, in a preferred embodiment of the present invention, a lead compound analog having a hydrogen bond donor / acceptor group as a substituent on a phenyl ring at a different site is a structure between serine residues of three common dopamine receptors in region C. Provided to explore the above differences (see Figure 19).

Synthesis of novel analogues in group I (a). A general scheme for the synthesis of compounds shown in Group I (a) is illustrated in Scheme V (see FIG. 20). The process of Scheme V is the same as the synthetic method shown in Scheme I. The compound 42 of 2-substituted aminomethylquinoline was prepared from the starting compound 40 by bromination with NBS to give 2-bromomethylquinoline. Is obtained, followed by reaction with an ethanolamine solution in ethanol. Reduction of compound 42 with an acetic acid solution of NaBH ₃ CN yields 2-substituted aminomethyl-1,2,3,4-tetrahydroquinoline (compound 43). An important intermediate (compound 44) was synthesized by cyclizing compound 43 with a solution of P ₂ O ₅ in xylene. Finally, compound 44 was alkylated using the synthetic method outlined in Scheme II to give the desired derivative of compound 45.

  Most of the starting materials used in Scheme V are commercially available. If starting materials are not available, they can be easily synthesized. A method for preparing starting materials for many analogs is shown in Scheme VI. (Figure 21). In general, substituted quinaldines can be prepared directly by condensing the corresponding substituted aniline with crotonaldehyde using the well-known Skraup method. (See G. Jones, Quinolines Part I, 100-117 [1977]).

  In yet another embodiment, the present invention provides Scheme VII, which is an alternative way to synthesize the novel analogs provided in Group I (a). Briefly, compounds 46 and 47 are commercially available, or they can be readily obtained using known methods. (See, eg, B. Scholl et al., Helv. Chim. Acta., 69: 184-194 [1986]). Compound 47 is epoxidized to give compound 48. By condensation of phthalimide and compound 48, intermediate compound 49 can be obtained, which can be oxidized to a ketone (compound 50). Reduction of the nitro group of compound 50 to an amine group produces compound 51. Compound 52 is obtained via reductive amination of compound 51. Removal of the phalamido group of compound 52 yields diamine compound 53, which is then converted to compound 54. Reaction of chloroacetic acid chloride with compound 54 produces compound 55. Treatment of compound 55 with a base provides the tricyclic lactam compound 56. Removal of the protecting group following reduction of the tricyclic lactam compound 56 produces the key intermediate compound 57. (See E. W. Baxter and A. Reitz, Bioor. & Med. Chem. Lett., 7: 763-768 [1997]). Intermediate compound 57 can be directly alkylated with various alkyl halides to provide compound 58 of the desired tricyclic derivative.

  In another embodiment, the present invention provides synthetic routines for group I (b) analogs by using methods similar to those shown in Schemes III and IV. (US 3,931,188 [incorporated herein in its entirety by reference]; LA van Vliet et al., J. Med. Chem., 43: 2871 [2000]; and KY Avenell et al., Med. Chem. Lett., 8: (See also 2859-2864 [1998]). In another embodiment, an alternative scheme for the synthesis of group I (b) analogs is also provided in Scheme VIII. In Scheme VIII, compound 60 is formed by reaction of n-butyllithium and acetonitrile with 1-tetralones (compound 59) at −78 ° C. followed by reduction with lithium aluminum and dehydration. Ring closure with formaldehyde under acidic conditions yields the key intermediate hexahydrobenz [f] isoquinoline (Compound 61). The intermediate (compound 61) is combined with various RX (X = Cl or Br) to produce the final target compound 62.

In still some other embodiments, the present invention provides analogs of the lead compound 14 and 32 having a designed tails modified to target D ₃ receptor regions A (Group II analogs). In a preferred embodiment, based on the binding model predicted for compounds 4 and 29, in order to target the dopamine receptor D ₃ regions A, Ligands, mobility of 5-6 binding linker (9 to 10) and a large Provide a general chemical structure with a hydrophobic tail. For example, in some embodiments, novel analogs of compounds 14 and 32 that have a 4 carbon and amide group as a linker and a large naphthyl ring at their tail are compared to the original lead compounds 4 and 29 shows a very improved selectivity between ₃ and D ₂ receptors. In some embodiments, the amide group in the linker moiety plays a role in the potency and selectivity of the ligand by forming a hydrogen bond with residue N375, thus placing the naphthyl group in the appropriate orientation. It was found that it may interact with the residues in region A. More particularly, the naphthyl ring of compounds 14 and 32, as discussed in detail, in the area of structural differences between D ₂ / D _3, residues D ₃ receptors N379, F338, N47, L121 Is expected to interact with.

Thus, no mechanism understanding is required to practice the invention, and the invention is not so limited, but residue N47 appears to play a major role in the selectivity of compounds 14 and 32. It is assumed that In addition, D ₃ receptor residues F338, N375 corresponding to F382, N418 of D ₂ respectively are also compounds 14 and 32 are thought to contribute to the ability to identify between dopamine receptor subtypes. FIG. 22 is the distance required for the ligand to reach the region.

Some preferred embodiments of the present invention further modify the naphthyl ring of lead compounds 14 and 32 to explore structural differences in region A of the dopamine receptor to achieve even greater selectivity. I will provide a. In some of these embodiments, small hydrophobic groups (eg, CH ₃ , F, Cl, Br, I, etc.) or polar substituents (OH, OCH ₃ , NH ₂ , CN, etc.) are introduced into the naphthyl ring Is done.

  In some preferred embodiments, the synthesis of Group II (a) analogs is accomplished by using methods similar to those shown in Schemes I, II, V, VI, and VII.

Furthermore, other embodiments of the present invention provide analogs (group III analogs) of lead compounds 14 and 32 having modifications in the linker designed to target region B of the dopamine receptor. In one embodiment of the invention, based on the binding model predicted for compounds 14 and 32, significant structural differences are seen between region B of the dopamine receptor, such as receptors D ₃ and D _2. There are differences in the orientation of residues F345 and F346 of TM6. Also, the orientation and position of residues Y365 and T369 of TM7 are significantly different due in part to the different helix packing between TM6 and TM7. Accordingly, some preferred embodiments of the present invention are similar to lead compounds to provide one or more small hydrophobic groups designed to specifically interact with region B of the receptor. Leads optimized to extend ligand interactions with specific dopamine receptor subtypes (eg, D ₁ , D ₂ , D ₃ , D ₄ , and / or D ₅ ) by modifying the body A compound is provided.

  One method for the synthesis of Group III (a) and (b) analogs is illustrated in Scheme IX (Figure 23). Compound 64 is obtained from commercially available compound 63. The methyl ester (compound 64) is selectively reduced to give the aldehyde (compound 65). (See, for example, L. I. Zakharkin, Tetrahydron Lett., 2087 [1963]). Treatment of the aldehyde (Compound 65) with a Grignard reagent followed by Swern oxidation gives the key intermediate ketone (Compound 67). Compound 67 is treated with an amine (compound 44, 57, or 61) to give target compound 68 or 69, respectively.

In some further embodiments, the present invention provides analogs of lead compounds 14 and 32 (group IV analogs) having modifications in a tricyclic ring designed to target region D of the dopamine receptor To do. For example, based on structural analysis, dopamine receptor region D was found to have a distinct hydrophobic pocket adjacent to three serine residues. Further structural analysis shows that there are significant structural differences in this region between dopamine receptor subtypes. These structural differences are considered to be mainly due to differences in residues constituting the region D. For example, some D ₃ receptor residues A161, A163, S182 of this region, respectively D ₂ receptor residues S163, T165, and corresponds to I183.

Preferred embodiments of the present invention provide lead compound analogs 14 and 32 having additional substituted phenyl groups linked to the tricyclic ring via a sulfonylamide linker. An understanding of the mechanism is not required to practice the invention, and the invention is not so limited, although the sulfonylamide linker forms a hydrogen bond with the serine residue of TM5, but the substituted phenyl group is D ₃ residues in receptor I183, Phel88, V164, L168, and is assumed to interact with the hydrophobic pocket region D formed by V111.

  In certain embodiments, analogs of groups IV (a) and IV (b) are obtained by treating compound 70 or 73 with various substituted benzenesulfonyl chlorides as shown in Scheme X (see FIG. 24). See). Importantly, compound 70 is obtained using the synthetic methods shown in Schemes V and VII, and compound 73 is obtained using the method outlined in Scheme VIII.

The present invention further provides methods for assessing the docking of novel analogs to D ₃ , D ₂ , and D _{1 and} for assessing their binding affinity. In a preferred embodiment, computational docking studies of D ₁ , D ₂ , and D ₃ and novel analogs are performed using the methods outlined above. The ligand-receptor complex structure is further refined through 2 ns or longer extensive MD simulations in a well-defined water and lipid environment using the CHARMM program. Based on the predicted model, the binding affinity is predicted.

Because the binding of many possible ligands (eg, 7-OH-DPAT and 5-OH-DPAT) to the D ₃ receptor is stereospecific, other aspects of the invention provide for stereoisomeric resolution. Providing a method for In particular, all of the compounds synthesized in Schemes I, II, and V are racemic because they all contain one chiral center. In a preferred embodiment, the (+) and (−) stereoisomers are resolved using known stereoisomer separation methods. (See LA 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), the key intermediate compound 41 used in Scheme V is coupled with (S)-(+)-α-methoxyphenylacetic chloride, which produces two diastereomers. Resolution is achieved by forming a mixture of amide compound 75 (a) and compound 75 (b). Column chromatographic separation is used to obtain pure diastereomers 75 (a) and 75 (b). Cleavage of the amide group of compounds 75 (a) and 75 (b) with potassium tert-butoxide and water in THF gives the pure stereoisomeric compounds (-)-41 and (+)-41, respectively. .

VII. Biological Testing and Characterization of Lead Compounds and Analogues In some embodiments, novel compounds and ligands identified and designed using the methods of the invention are as outlined in FIG. In addition, they are subjected to a step-by-step screening process for affinity, efficacy, and selectivity at dopamine receptors. In a preferred embodiment, the present invention is used in an in vitro radioligand binding assay using rat brain models and cloned human receptors, with a focus on determining receptor affinity and selectivity. Assume the first study. (See FIG. 26, steps 1, 2, 3, and 4). The present invention further envisions further studies used to determine 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 some of the biological tests and assays envisioned for characterizing novel compounds and ligands identified and designed using the methods of the invention. Limited examples are provided.

  In some embodiments, the determination of the affinity and selectivity of novel compounds at their target receptors (eg, dopamine receptors) and their selectivity for these receptors is first determined by a radioligand binding assay. To decide. To this end, in one embodiment, three series of mutually complementary radioligand binding studies are performed in a stepwise manner. The present invention is that this complementary approach compensates for the specific limitations of any one of the individual assays, and therefore their targets for novel compounds than those provided using a single assay approach. Assume that it provides a more accurate determination of affinity and selectivity at the receptor. The present invention further envisions that this complementary approach provides a thorough and definitive assessment of the in vitro pharmacological profile of compounds for these target receptors (eg, dopamine receptors).

In some embodiments, potential therapeutic compounds designed using the method of the present invention (test compound) is first subjected to a screen in the rat brain D ₃ receptor model. If the test compound exhibits sufficient affinity and selectivity for D ₃ receptors, the affinity and selectivity for D ₃ receptors, in a further rat brain assays and cloned human dopamine receptor assay Determine further. Finally, a compound that exhibits selectivity for D ₃ receptors in both rat brain membranes and cloned human receptors, using autoradiography methods to determine D ₂ and D ₃ receptor affinity simultaneously And further evaluate.

For example, in some embodiments, the affinity of a test compound at the D ₃ receptor uses [ ³ H] PD128907 binding in membranes prepared from rat ventral striatum (eg, nucleus accumbens and olfactory nodules). To measure. Rat ventral striatum tissue is used in the D ₃ receptor assay because these tissues express the highest density of D ₃ receptors in the rat CNS. (See ML Bouthenet et al., Brain Res., 564: 203-219 [1991]). The present invention envisions that [ ³ H] PD128907 agonists exhibit a better 300-fold selectivity for D ₃ receptors over D ₂ receptors in rat brain. The affinity of test compounds for the D ₁ and D ₂ receptors is described by Levant et al. (B. Levant, CNS Neurotransmitters and Neuromodulators, 3 [TW Stone, Ed.], CRC Press, Boca Raton, FL, 77-88 [1996]. Rat tails expressing antagonists (eg, radioligand [ ³ H] SCH23390 and [ ³ H] spiperone, respectively) and high density D ₁ and D ₂ receptors and low density other dopamine receptors Determined by using membranes from the nuclear shell (striatum). [ ³ H] SCH23390 and [ ³ H] spiperone antagonists are highly selective for D ₁ -like and D ₂ receptors, respectively. [ ³ H] spiperone was shown to have an 80-fold higher affinity for the D ₂ receptor than the D ₃ receptor. Due to the low density of D ₃ receptors expressed in rat striatum, labeling of D ₃ receptors with [ ³ H] spiperone is negligible. Similarly, the rat striatum express D ₅ receptor a very low density. Thus, the present invention shows that [ ³ H] SCH23390 has similar affinity for D ₁ and D ₅ receptors, but the binding assays in these studies are representative examples of D ₁ receptors. Assume that. In preferred embodiments, [ ³ H] SCH23390 and [ ³ H] spiperone binding assays are performed using in vitro assay conditions that favor the binding of agonists at dopamine receptors (eg, Mg ²⁺ content, And elimination of NaCl [see, for example, 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 of agonists to detect high affinity binding in assays using antagonist radioligands. In a preferred embodiment, the assay is performed as previously described in Wang et al. (See S. Wang et al., J. Med. Chem., 43: 351-360 [2000]). In yet another preferred embodiment, the Ki value of each test compound at each dopamine receptor subtype is determined. Table 8 describes the determination of binding affinity at the D ₁ , D ₂ , and D ₃ receptors in the rat brain.

In a preferred embodiment, the main advantage of conducting an initial assessment of the test compound in the rat brain is that this approach allows the study of receptors expressed in these natural tissues. Therefore, the source tissue is believed to contain all components necessary for normal physiology. Determining the affinity of new test compounds at D ₁ , D ₂ , and D ₃ receptors in brain tissue using brain regions with a fully characterized radioligand and dopamine receptor subtype density known Is possible. This approach has the additional advantage that the rat brain is readily available and relatively inexpensive compared to the cloned receptor. Table 9 provided below shows the affinity (K _i or K _d value) of the reference ligand used in the binding assay in the rat brain envisaged.

In a preferred embodiment, the second stage of evaluation of the biological test compound is D using cloned human receptors (hD ₁ , hD _2short , hD ₃ , and hD _4.4 ) expressed in the transfected cell line. Includes assessment of test compound affinity and selectivity for ₃ receptors. In a particularly preferred embodiment, the test compound, if they exhibit a selectivity for D ₃ receptors in rat brain, the process proceeds to the second stage of evaluation. In yet another preferred embodiment, the affinity of the test _{compound, hD 1, hD 2short, hD} 3, and in CHO cells expressing hD _4.4 (NEN Life Science and Sigma-Aldrich), the hD ₁ binding ^[3 H ] Determined _using SCH23390, and [ ³ H] spiperone for hD _2short , hD ₃ , and hD _4.4 receptors. The assay is performed as previously described in Wang et al. (See S. Wang et al., J. Med. Chem., 39: 2047-2054 [1996]). In yet another preferred embodiment, the Ki value of each test compound at each dopamine receptor subtype is determined.

The present invention envisions that the use of cloned human receptors has the advantage of allowing human studies rather than rat receptors, thus increasing their relevance to human therapy. Since host cells do not normally express dopamine receptors, this approach also allows individual studies of each receptor subtype and eliminates reliance on selective pharmacological means. A variety of radioligands are used for hD ₁ and hD _2- like receptor studies, but in vitro assay conditions remain the same. It is therefore envisioned that this approach compensates for the potential limitations of using rat brain membranes for measuring dopamine receptor affinity and selectivity.

In a further aspect, the present invention provides a method for the simultaneous autoradiography determination of D ₂ / D ₃ selectivity in the rat brain. In some of these methods, quantitative autoradiographic methods for the simultaneous determination of D ₂ and D ₃ receptor affinity in rat brain are Levant and De Souza and Levant and Flietstra (B. Levant and EB De Souza , Synapse, 14: 90-95 [1993]), using D ₂ -D ₃ ligand [ ³ H] quinpirole. The method, the molecular layer of the vestibular cerebellum (which dopamine D ₃ appears to localize only with receptor mRNA) dopamine receptors in the, indicating a lack of guanyl nucleotide regulatory, and consistent with D ₃ sites medicine Based on the observation of showing a physical profile. Thus, the brain region, can donate a discrete source of D ₃ receptors in the brain tissue. In contrast, express large amounts of D ₂ receptor mRNA by substantial and relatively little of D ₃ only shows receptor binding caudate putamen, it ensures useful as prototype of dopamine D ₂ tissue To do. Thus, by labeling these brain regions with non-selective radioligands such as [ ³ H] quinpirole, in tissues under uniform in vitro assay conditions, and in the same brain section, the D ₂ / D ₃ receptor The selectivity of the test compound for can be evaluated. In a preferred embodiment, studies are performed as described above to determine the IC ₅₀ value of the test compound at the D ₂ and D ₃ receptors and the D ₂ / D ₃ selectivity. (See B. Levant and EB De Souza, Synapse, 14: 90-95 [1993]).

One possible advantage of this approach is to measure D ₂ and D ₃ receptor affinity while using radiolabeled agonists to measure D ₂ and D ₃ receptor affinity in brain tissue It is to avoid potential disruptive changes due to the use of separate assays. This is an important advantage since the D ₂ / D ₃ selectivity of dopaminergic compounds has been shown to vary according to the in vitro assay system used. Accordingly, in a preferred embodiment, this approach, in addition to those using receptors in heterologous expression systems, or obtained using a variety of in vitro assays of the respective receptors in the brain, D ₂ / D ₃ Provides a definitive assessment of receptor selectivity. In addition, the present invention, D ₂ / D ₃ selectivity as determined by this method, than radioligand binding assay using human receptor expressed on the transfected cells, in general, functional assays Assume that it is more consistent with what is determined.

A further aspect of the invention provides a method for measuring the potency, efficacy, and selectivity of novel compounds in functional assays. Unlike radioligand binding assay to evaluate only the affinity of the compounds for a given receptor, functional assays of novel ligands at D ₃ receptors efficacy, to determine the efficacy, and selectivity Used in a preferred embodiment. These assays allow the identification of agonists and antagonists, as well as partial agonists and inverse agonists. Two functional assays are envisioned to provide cross-validation of experimental results (eg, a mitogenic assay and a [ ³⁵ S] GTPgS binding assay), but the present invention It is not intended to be limited to using the specific mentioned functional assays. Indeed, any relevant functional assay that is compatible with the compositions and methods of the present invention is suitable for use herein.

In some embodiments of the present invention, only the test compound showing a higher affinity for D ₃ receptor over other dopamine receptors in the radioligand binding studies it is further evaluated in a functional assay.

In a preferred embodiment, one type of functional assay suitable for use in the compositions and methods of the invention is a mitogenic assay. hD ₂ or hD ₃ mitogenesis induced by agonists in CHO cells expressing the receptor has been developed as a surrogate marker for dopamine receptor activation. (See, eg, 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 effect of novel compounds on mitogenesis in CHO cells, assessed based on [ ³ H] thymidine incorporation, is used to determine the efficacy and potency of test compounds. In yet other embodiments, test compounds lacking efficacy are evaluated for antagonist activity and potency as determined by their ability to block agonist-induced mitogenesis. The assay is performed as previously described in Milne et al. To determine E _max and ED ₅₀ values for each test compound. (See GWA 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.

In other preferred embodiments, another type of functional assay suitable for use in the compositions and methods of the invention is a [ ³⁵ S] GTPgS binding assay. These assays determine the activity of G protein receptors in the membrane by assaying agonist-stimulated [ ³⁵ S] GTPgS, a hydrolysis-resistant analog of GTP, in the presence of excess GDP. Studying chemistry. The physiologically relevant indicators are used to assess the activation of G proteins by D ₂ and D ₃ receptors in the brain and transfected cells. (SL Gilliland et al., Eur. J. Pharmacol., 392: 125-128 [2000]; SL Gilliland and RH Alper, Naunyn-Schmiedeberg's Arch Pharmacol., 361: 498-504 [2000]; and JF Pregenzer et al., Neurosci. Lett., 226: 91-94 [1997]). In some embodiments, the dose response effect of a test compound 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. The Compounds lacking efficacy are evaluated for antagonist activity and potency, as determined by their ability to block agonist-induced [ ³⁵ S] GTPgS binding. In some other embodiments, the [ ³⁵ S] GTPgS binding assay is performed using hD _2short , hD ₃ , or hD _4.4 receptor as previously described to determine E _max and ED ₅₀ values for each compound. Also on CHO cell membranes containing the body. IC ₅₀ values are determined for test compounds found to block agonist-induced [ ³⁵ S] GTPgS binding.

  One possible advantage of these functional approaches is that the use of studies on human dopamine receptors increases the relevance of experimental data obtained for human therapy.

VIII. Therapeutic Agents Combined or Coadministered with the Composition A wide range of therapeutic agents are useful for use with the present invention. Any therapeutic agent that can be co-administered with or disclosed with the disclosed compounds is suitable for us in the present invention.

  Some aspects of the invention include dopamine receptor modulators (eg, antagonists or agonists) and cocaine abuse, depression, anxiety, eating disorders, alcoholism, chronic pain, obsessive compulsive disorder, schizophrenia, It is provided that an effective amount of one or more compounds indicated for therapeutic treatment such as restless leg syndrome (RLS) and Parkinson's disease is administered to a subject. In some embodiments, the subject is a mammal (eg, a human).

Further aspects of the invention provide therapeutic compositions and methods directed to modulating (eg, agonizing or antagonizing) G protein coupled receptors. In some other further embodiments, the therapeutic compositions and methods of the invention are directed to modulating (eg, agonizing or antagonizing) defective dopamine receptor function (eg, nucleus accumbens, olfactory tubercle, and Kaeha island (but not limited to) reuptake of dopamine by D ₃ receptors in limbic brain regions including).

The present invention also provides an opportunity to monitor the success of treatment following administration of a compound of the present invention to a subject and methods of treatment. In some embodiments, these measurements are made at least in part by observing the recovery of symptoms characterized by a defect in dopamine (eg, D ₃ ) receptor function. In some embodiments, patient observation is made in a clinical environment (eg, a hospital, clinic, or clinic). In other embodiments, to determine the efficacy of administration, one or more from the patient at various times (eg, before, during, or after) administration and methods of the therapeutic compositions of the invention Collect multiple physiological samples. In still further embodiments, patient administration data is used to alter or modify the administration and method (eg, dosage level, frequency of administration, administration cycle, administration method, etc.) of the therapeutic compositions of the invention. Used for.

  The compositions of the invention may be delivered via any suitable method including intravenous, subcutaneous, intratumoral, intraperitoneal, or local (eg, mucosal surface) injection. However, it is not limited to these.

IX. Review of Pharmaceutical Formulations, Routes of Administration, and Dose The present invention is in any sterile, biocompatible pharmaceutical carrier, including but not limited to saline, buffered saline, dextrose and water. A pharmaceutical composition is provided that comprises a composition that modulates at least one receptor (eg, dopamine, G protein coupled receptor, etc.) administered in In some embodiments, the pharmaceutical compositions of the present invention, one agent (e.g., ligand for adjusting the D ₃ receptor) may be contained. In other embodiments, the pharmaceutical composition comprises at least two agents (e.g., one or more D ₃ receptor modulators ligand and the one or more additional therapeutic compositions) may comprise a mixture of . In a still further embodiment, the pharmaceutical compositions of the present invention, at least two agents are administered to the patient in one or more of the conditions below (e.g., one or more D ₃ receptor modulators ligand and 1 One or more additional therapeutic compositions): different cycles, different periods, different concentrations, different routes of administration, etc.

The compositions and methods of the present invention provide for the treatment of disease or defective (abnormal) modulation of dopamine receptors and other receptors (eg, D ₁ , D ₂ , D ₃ , D ₄ , and D ₅ ). And / or useful in the modification of physiological conditions characterized by function. In some aspects the functionality of the abnormal receptor (e.g., D ₃ receptors) is psychostimulant drugs (eg, cocaine) is caused to the patient's body by. In other embodiments, the abnormal receptor function occurs by or as a result of causing an organic disease or condition or symptom.

The present invention relates to dopamine receptor ligands and, in some embodiments, one or more conventional therapeutic compositions directed to treating symptoms associated with the modulation and / or function of defective receptors. It is envisioned that a composition (eg, a composition directed to treating substance abuse, depression, Parkinson's disease, schizophrenia, etc.) will be administered according to acceptable pharmaceutical delivery methods and preparation techniques. For example, one or more D ₃ receptor modulators ligand and the one or more additional therapeutic compositions, in a pharmaceutically acceptable carrier such as physiological saline, can be administered intravenously to a subject . Standard methods for intracellular delivery of pharmaceuticals can be used (eg, delivery via liposomes). Such methods are well known to those skilled 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 several contemplated therapeutic agents (eg, therapeutic polypeptides) can also be achieved using gene therapy techniques. Gene therapy techniques are now widely known in the art.

  As is well known in the medical field, the dose for any single patient is determined by the patient's size, body surface area, age, the particular compound being administered, sex, time and route of administration, overall health, As well as interactions with other drugs administered in parallel.

In some embodiments of the present invention therefore, a ligand for adjusting one or more of D ₃ receptors, to a patient alone, or in combination with one or more substances abuse treatment agent, or which excipients or It is administered in a pharmaceutical composition mixed with other pharmaceutically acceptable carriers. In one embodiment of the invention, the pharmaceutically acceptable carrier is pharmaceutically inert.

  Depending on the condition being treated, these pharmaceutical compositions may be formulated or administered systemically or locally. Techniques for formulation and administration will be found in the latest edition of “Remington's Pharmaceutical Sciences” (Mack Publishing Co, Easton Pa.). Suitable routes include, for example, oral or transmucosal administration, as well as parenteral including intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, or intranasal administration Delivery may be included.

  For injection, the pharmaceutical compositions of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiologically buffered saline. For tissue or cell administration, penetrants appropriate to the particular barrier to be permeated are used in the formulation. Such penetrants are known to those skilled in the art.

  In other embodiments, the pharmaceutical compositions of the invention can be formulated using pharmaceutically acceptable carriers well known in the art at dosages suitable for oral administration. Such carriers allow the pharmaceutical composition to be formulated as tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions, etc. for oral or nasal ingestion by the patient being treated. To do. In preferred embodiments, the therapeutic compound is orally administered to the patient orally.

Pharmaceutical compositions suitable for use in the present invention include compositions wherein the active ingredients are included in an amount effective to achieve the intended purpose. For example, an effective amount of a ligand for adjusting the D ₃ receptor, as compared to Example not a normal pathological specific cell or tissue, is suppressed (or rise to) have elevated levels of dopamine uptake It may be an amount that induces reuptake of dopamine in the patient's cells or tissues. Effective amount measurements are well within the ability of those skilled in the art, especially in light of the disclosure provided herein.

  In addition to the active ingredient, preferred pharmaceutical compositions include suitable pharmaceutically acceptable carriers containing excipients and additives that facilitate the processing of the active compound in a pharmaceutically acceptable preparation. May be included. Formulations formulated for oral administration may be in the form of tablets, dragees, capsules, or solutions.

  The pharmaceutical compositions of the present invention may be manufactured in a manner known per se (for example by conventional mixing, dissolving, granulating, dragee making, grinding, emulsifying, encapsulating, embedding or lyophilizing processes). Good.

  Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. In addition, 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 liposomes. 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 contain suitable stabilizers or agents that increase the solubility of the compound and allow for the preparation of highly concentrated solutions.

  Pharmaceutical formulations for oral use combine solid excipients and active compounds (eg, receptor ligands), grind the resulting mixture selectively, process the mixture of granules, and if necessary It can be obtained after adding suitable additives to obtain tablets or sugar-coated nuclei. Suitable excipients include sugars including lactose, sucrose, mannitol or sorbitol; starches from corn, wheat, rice, potato, etc .; celluloses such as methylcellulose, hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; and Arabic Gum including gum and tragacanth; and carbohydrate or protein fillers such as proteins such as gelatin and collagen. If desired, disintegrating or solubilizing agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof such as sodium alginate.

  The ingestible formulations of this composition are US agricultural for food ingredients and generally recognized as safe substances (GRAS), such as food additives, flavors, colorings, vitamins, minerals, and phytonutrients. It may further include any material approved by the Ministry. As used herein, the term “phytonutrient” refers to an organic compound isolated from a plant with biological effects, including but not limited to the following classes of compounds: isoflavonoids, oligomers Proanthocyanidins, indole-3-carbinol, sulforaphone, fibrous ligand, plant phytosterol, ferulic acid, anthocyanoside, triterpene, omega-3 / 6 fatty acid, polyacetylene, quinone, terpene, catechin, Gallate and quercitin.

  In addition, sugar-coated nuclei are made of suitable coatings such as gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and / or concentrated sugar solutions that will contain titanium dioxide, lacquer solutions, As well as a suitable organic solvent or solvent mixture. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification of the product or to characterize the quantity of active compound (ie, dosage).

  Pharmaceutical formulations that can be used orally include push-fit capsules made of gelatin, soft sealed capsules made of gelatin, and a coating such as glycerol or sorbitol. Push-fit capsules can also contain active ingredients mixed with fillers or binders such as lactose or starch, 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 glycols in the presence or absence of stabilizers.

Compositions comprising a compound of the invention formulated in a pharmaceutically acceptable carrier may be prepared by placing in a suitable container and labeling for the treatment of the indicated condition. The ligand to adjust the D ₃ receptor, the condition indicated on the label, substance abuse (eg, cocaine abuse), depression, Parkinson's disease, and it may include treatment of conditions related to such schizophrenia. The pharmaceutical composition may be provided as a salt and can be formed with many acids including, but not limited to, hydrochloric acid, sulfuric acid, acetic acid, lactic acid, tartaric acid, malic acid, succinic acid, and the like. Salts tend to be soluble in aqueous or other protic solvents in the corresponding free base form. In other cases, the preferred formulation is a powder lyophilized in a pH range of 4.5 to 5.5, 1 mM to 50 mM histidine, 0.1% to 2% sucrose, 2% to 7% mannitol. May be combined with buffer before use.

For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. Then, the dose preferably, in normal function / regulation of the receptor (e.g., re-uptake of dopamine by D ₃ receptor) to achieve a desirable circulating concentration range that induces an animal model (in particular, a mouse or rat model) Can be prescribed. A therapeutically effective dose is a receptor-modulating ligand (and, in some embodiments, one or more other therapeutic agents) that restores symptoms of the disease state (eg, suppression of dopamine reuptake). ). The toxicity and therapeutic efficacy of such compounds determine, for example, LD ₅₀ (dose that is lethal to 50% of the population) and ED ₅₀ (dose that is therapeutically effective in 50% of the population). Can be determined by standard pharmaceutical procedures in cell cultures or laboratory animals. The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD ₅₀ / ED ₅₀ . Compounds that exhibit large therapeutic indices are preferred. Data from cell culture assays and further animal studies, for example in mammals (eg, humans, horses (Equus caballus), cats (Felis catus), dogs (Canis familiaris), etc.) Can be used. The dosage of such compounds is not limited to this range, but is preferably within a circulating concentration range that includes ED ₅₀ with little or no toxicity. The dose will vary within this range depending upon the dosage form used, patient sensitivity, and route of administration.

  The exact dose is selected from the perspective of the patient being treated by the individual physician. Dosage and administration are adjusted to provide a sufficient level of the active moiety or to maintain the desired effect. Additional factors that may be considered include: severity of disease state; patient age, weight, and sex; diet, time and frequency of administration, drug combination, response sensitivity, and tolerance / response to therapy Including. Long-acting pharmaceutical compositions may be administered every 3-4 days, every week, or once every two weeks, depending on the half-life and clearance rate of the particular formulation. Other pharmaceutical compositions may be administered daily or several times daily.

  The normal dose may vary from 0.1 to 100,000 micrograms to a total dose of about 1 g, depending on the route of administration. Guidance on specific doses and methods for delivery is provided in the literature. Administration of some drugs into the patient's bone marrow will require delivery by means other than intravenous injection.

  Preferred embodiments of the invention are pharmaceuticals for administering to a patient an effective amount of a ligand that modulates the receptor to modulate the function / modulation of the defective receptor (eg, increase or decrease activity). Compositions and methods are provided.

In some embodiments, suitable for treatment according to the present invention, receptor (e.g., D ₃ receptors) diseases suspected of being characterized by a defect in the function / regulation, abnormal levels natural (e.g., intrinsic Obtain a sample (eg, cell, tissue, fluid, etc.) of interest suspected of having a (sex) receptor ligand (eg, dopamine) and one or more well-established immunohistochemical techniques (E.g., ELISA and Western blot, etc.) to measure the natural receptor ligand level in a sample and the natural receptor ligand level in the sample to the corresponding non-pathological reference sample Selected by comparison with the ligand level. In other embodiments, a disease suspected of being characterized by an abnormal level of one or more natural receptor ligands (eg, dopamine) directly or indirectly with an abnormally high level of the receptor ligand of interest. The level of one or more markers (eg, polynucleotides, polypeptides, lipids, etc.) in a sample (eg, cell, tissue, fluid, etc.) Selected by comparing to the level of the marker.

  It is not intended that the present invention be limited to the route of administration selected to deliver the drug to the subject. Indeed, many suitable routes of administration are envisioned and the selection is within the skill of the artisan.

In some preferred embodiments, the therapeutic composition is administered to the patient in a dosage range of about 1-200 mg / day, about 5-50 mg / day, and most preferably about 10-40 mg / day. In a particularly preferred embodiment, the therapeutic compound is an acceptable daily dose (eg, 30-40 mg / day) that has been shown to have some biological activity (eg, changes in Rb and cyclin D ₁ levels). To the patient (eg, orally).

  In some embodiments, standard immunohistochemical techniques are used on samples obtained from patients after or during treatment with the methods and compositions of the invention to determine changes in the patient's disease state Is used. As used herein, cocaine abuse and / or sputum is considered a disease state.

Potential treatment of D ₃ receptors in schizophrenia. Schizophrenia is a neurogenic disease involving abnormal dopaminergic function. Potential importance and treatment of D ₃ dopamine receptors in schizophrenia is supported by the backing of some evidence. First, D ₂ and D ₃ dopamine receptor is a major target for almost all current known drugs having antipsychotic activity (recent review: Strange, 2001; Schwartz et al., 2000; Emilien et al., 1999). Moreover, D ₃ subtype is predominantly localized in the mesolimbic area before known to be involved in schizophrenia. In addition, antipsychotics administered to people with schizophrenia appear to act as downregulators of D ₃ receptors in the ventral striatum region, compared to schizophrenic patients taking antipsychotics And is supported by overexpression of D ₃ (not D ₂ ) receptors in drug-free schizophrenic patients (Gurevich et al., 1997). This will also explain the behavioral sensitization to mental stimulation observed in schizophrenia. Pharmacological discussion, support that is another factor contributing to the symptoms of schizophrenia might be unbalanced relative abundance between D ₁ and D ₃ receptors (Schwartz et al. 1998). However, overexpression of D ₃ receptors in schizophrenia, and in order to confirm the role of the symptomatology of the disease, further studies are needed.

Typical antipsychotic drugs tend to have severe extrapyramidal side effects, unlike second generation atypical drugs. D ₃ receptors, since it is mainly restricted to brain regions associated with edges, this is an attractive target for the development of side effects without antipsychotic therapy extrapyramidal or neuroendocrine It is generally considered to be. For example, in the case of the recently discovered D ₃ antagonist SB-277011-A, which shows significant selectivity for D ₃ over D ₂ in the rat brain, this D ₃ ligand can be used to treat schizophrenia. Evidence has shown that beneficial effects may be achieved without the side effects characteristic of non-selective dopamine receptor antagonists (Reavill et al., 2000). In the case of neuroleptics used to treat schizophrenia, the antipsychotic effects of this class of drugs are related to blocking D ₃ rather than D ₂ receptors, but the extrapyramidal side effects are is supported by the evidence that these effects on D ₂ receptors expressed in the striatum (Schwartz et al., 2000; Sigmundson, 1994; Missale et al., 1998; Joyce and Meador-Woodruff , 1997). On the other hand, as shown in the case of haloperidol and clozapine (Strange, 2001), the extrapyramidal side effects of antipsychotics can reduce D ₂ receptor occupancy in the striatum by administering low doses. It is also discussed that it may be removed by decreasing.

Therapeutic association of D ₃ receptors in Parkinson's disease. Long-term levodopa treatment for Parkinson's disease causes serious side effects, movement disorders, movement swings, and hallucinations. Levodopa administration in conditions that cause sensitization action has been shown to involve the induction of D ₃ receptor gene expression. This association has therapeutic relevance in the prevention of side effects and in the treatment of Parkinson's disease (Bordet et al., 1997; S. Perachon et al., 366: 293-300 [1999]. The combined administration of levodopa and dopamine agonists It is considered an effective way to reduce and delay the side effects of chronic levodopa therapy, and two recently developed drugs for Parkinson's disease, pramipexole, and ropinirole, Although primarily targeting the dopamine D ₃ receptor, another antiparkinsonian drug, pergolide, is potent against all three D ₁ , D ₂ , and D ₃ subtypes.

Therapeutic of D ₃ receptors in restless leg syndrome related. Restless Leg Syndrome (RLS) is characterized by abnormal sensations in the legs associated with urges for movement and movement unrest. Levodopa and ergoline derivatives effectively treat this condition, but their usefulness is limited by major side effects. Also, complete relief of RLS symptoms using these agents cannot be achieved. Recently, the D ₃ receptor agonist pramipexole has been found to be very effective in the treatment of RLS, which also supports the involvement of D ₃ subtype receptors in RLS and its therapeutic potential. (Montplaisir et al., 1999).

Therapeutic association of D ₃ receptors in the treatment of depression. In patients with severe depression, a decrease in dopamine function was found (Shah et al., 1997). In addition, antidepressants with dopaminergic effects are generally effective for treating depression (Brown and Gershon, 1993). In particular, chronic antidepressants are effective for D ₂ / D ₃ receptor function in the terminal region of the mesolimbic system (Emilien et al., 1999).

Examples The following examples are provided to illustrate and further illustrate certain preferred embodiments of the invention and are not to be construed as limiting its scope.

Example 1
Preparation of Various Lead Compounds This example describes the synthesis of various lead compounds and their key analogs. The important intermediate compound 5 was synthesized using known modified methods. (CAR Baxter et al., J. Med. Chem., 15 (4): 351-356 [1972]; VA Rao et al., J. Med. Chem., 13 (3): 516-522 [1970]; and GW Gribble and PW Heald, Synthesis, 650-651 [1975]). Condensation of ethanolamine and 2-quinoline-carboxaldehyde compound 2 (compound 2b was obtained in dioxane as shown in G. Lunn and EB Sansone, J. Org. Chem., 51: 513-517 [1986]. Prepared by oxidation of 6-methoxyquinaldine with SeO ₂ ) to give a Schiff base, which was then reduced with NaBH ₄ to a 2-substituted aminomethylquinoline (compound 3). (See CAR Baxter et al., J. Med. Chem., 15 (4): 351-356 [1972]; and VA Rao et al., J. Med. Chem., 13 (3): 516-522 [1970]. Wanna) By reducing Compound 3 with a nickel aluminum alloy in an aqueous KOH solution, 2-substituted aminomethyl-1,2,3,4-tetrahydro-quinoline (Compound 4) was easily obtained in good yield. (See N. Rabjohn, Org. React., 24: 261-415 [1976]). Important intermediate compound 5 was prepared by refluxing compound 4 with P ₂ O ₅ in xylene. (See CAR Baxter et al., J. Med. Chem., 15 (4): 351-356 [1972]; and VA Rao et al., J. Med. Chem., 13 (3): 516-522 [1970]. Wanna) Alkylation of compound 5 with various alkyl halides in the presence of an acetonitrile solution of Cs ₂ CO ₃ gave lead compounds and analogs 6a-e and compounds 7a-e. (See LA Vliet, J. Med. Chem., 43: 2871-2882 [2000]). Demethylation of the compound 7a~e by a CH ₂ Cl ₂ solution of the BBr _3, to give the analog compounds 8a~e having free hydroxy groups to the phenyl ring. (See EH Vickery et al., J. Org. Chem., 44: 4444-4446 [1979]).

Reagents and conditions: ia) 1.1 eq ethanolamine, benzene, reflux under N ₂ , H ₂ O removal, overnight; b) 3.5 eq NaBH ₄ , absolute ethanol, under N ₂ , reflux, overnight, yield 87- 90%. ii. Approximately 5 g nickel-aluminum alloy / 1 g substrate, 1M KOH, methanol, rt, overnight, 85-89% yield. iii.3 equivalent P ₂ O _5, xylene, N ₂ under reflux, overnight, 82 to 86% yield. iv.1.2 eq R ₂ Cl / NaI or 1.2 eq R ₂ I, 2 eq Cs ₂ CO ₃ , CH ₃ CN, under N ₂ , reflux, overnight, 61-76% yield. v. 4 eq BBr ₃ , CH ₂ Cl ₂ , N ₂ , 0 ° C., 4 h, yield 71-75%.

Table 10 shows the structure and activity of some D ₃ ligands.

Example 2
6-Methoxyquinoline-2-carboxaldehyde (Compound 2b)
5.5 g SeO ₂ (50 mmol) was added to 8.7 g 6-methoxyquinaldine (50 mmol) in 100 ml dioxane and 5 ml water and the resulting mixture was refluxed for 3 hours, cooled and filtered. The filtrate was concentrated and the residue was purified by chromatography on silica gel eluting with hexane: EtOAc = 6: 1 to give 8.5 g of compound 2b as colorless crystals in 91% yield.

Example 3
2- (β-Hydroxyethylaminomethyl) -quinoline (compound 3a)
To 85 ml benzene solution of 7.85 g 2-quinolinecarboxaldehyde (compound 2a) (50 mmol), add 3.3 ml ethanolamine (3.36 g, 55 mmol) and mix the resulting mixture until no water is separated. Boiled. Benzene was removed under reduced pressure and the crude Schiff base was dissolved in absolute ethanol. The resulting mixture was treated with 6.62 g NaBH ₄ (0.175 mol) at 0 ° C. and refluxed overnight. After removal of ethanol, the crude product was dissolved in water and extracted with 3 × 50 ml CHCl ₃ . The organic layer was dried over Na ₂ SO ₄ , concentrated and the residue was purified by chromatography on silica gel eluting with 10% MeOH in CHCl ₃ to afford 9.1 g of compound 3a as a colorless solid in 90% yield. Got in. The sample was converted to its hydrochloride salt.

Example 4
2- (β-Hydroxyethylaminomethyl) -6-methoxyquinoline (Compound 3b)
10.1 g of compound 3b was prepared in 87% yield from 9.35 g of compound 2b (50 mmol) as described above for the preparation of compound 3a (Example 3).

Example 5
2- (β-Hydroxyethylaminomethyl) -1,2,3,4-tetrahydroquinoline (compound 4a)
8.1 g of compound 3a (40 mmol) was taken up in 200 ml of methanol and 200 ml of 1M KOH was added. Nickel-aluminum alloy (40 g) was added in 5 portions over 1 hour and the resulting mixture was stirred at rt overnight. The mixture was filtered and concentrated, extracted with 3 × 100 ml of CHCl ₃ and the extract was dried over Na ₂ SO ₄ . After removal of the solvent, the residue was purified by chromatography on silica gel eluting with 20% MeOH in CHCl ₃ to give 7.3 g of compound 4a as a colorless solid in 89% yield.

Example 6
2- (β-Hydroxyethylaminomethyl) -6-methoxy-1,2,3,4-tetrahydroquinoline (compound 4b)
8.0 g of compound 4b was prepared in 85% yield from 9.3 g of compound 3b (40 mmol) as described above for the preparation of compound 4a (Example 5). The sample was converted to its hydrochloride salt.

Example 7
2,3,4,4a, 5,6-Hexahydro-1H-pyrazino [1,2-a] quinoline (Compound 5a)
A mixture of 7.21 g of compound 4a (35 mmol) and 14.9 g of P ₂ O ₅ (1005 mmol) in 100 ml of xylene was refluxed overnight, cooled and concentrated. The residue was hydrolyzed with 5N NaOH, extracted with 3 × 100 ml of CHCl ₃ and the extract was dried over K ₂ CO ₃ . After removal of the solvent, the residue was purified by chromatography on silica gel eluting with 10% MeOH in CHCl ₃ to give 5.4 g of compound 5a as a colorless oil in 82% yield. The sample was converted to its hydrochloride salt.

Example 8
8-methoxy-2,3,4,4a, 5,6-hexahydro-1H-pyrazino [1,2-a] quinoline (compound 5b)
6.56 g of compound 5b was prepared in 86% yield from 8.26 g of compound 4b (35 mmol) as described above for the preparation of compound 5a (Example 7).

Example 9
General procedure for alkylation of compounds 5a and 5b (Method A)
In one embodiment, 10 ml anhydrous of compound 5 (1 mmol), appropriate alkyl chloride (1.2 mmol), and anhydrous NaI (1.2 mmol), or appropriate alkyl iodide (1.2 mmol), anhydrous Cs ₂ CO ₃ (2 mmol) The mixture of CH ₃ CN solution was refluxed overnight. The reaction mixture was cooled, filtered and concentrated. The residue was purified by chromatography on silica gel eluting with hexane: EtOAc = 1: 1 to give the required product.

Example 10
3- (p-Fluorobenzoylethyl) -2,3,4,4a, 5,6-hexahydro-1H-pyrazino [1,2-a] quinoline (Compound 6a)
Alkylation of compound 5a with 3-chloro-4′-fluoropropiophenone as described in Method A (Example 9) gave 206 mg of compound 6a as a colorless oil in 61% yield. The sample was converted to its hydrochloride salt.

Example 11
3- (p-Fluorobenzoylpropyl) -2,3,4,4a, 5,6-hexahydro-1H-pyrazino [1,2-a] quinoline (Compound 6b)
Alkylation of compound 5a with 4-chloro-4'-fluorobutyrophenone as described in Method A (Example 9) gave 225 mg of compound 6b as a colorless oil in 64% yield. The sample was converted to its hydrochloride salt.

Example 12
3- (p-Fluorobenzoylbutyl) -2,3,4,4a, 5,6-hexahydro-1H-pyrazino [1,2-a] quinoline (Compound 6c)
Alkylation of compound 5a with 1- (4-fluorophenyl) -5-chloro-1-oxopentane as described in Method A (Example 9) yielded 252 mg of compound 6c as a colorless oil in 69% yield. Obtained in%. The sample was converted to its hydrochloride salt.

Example 13
3-propyl-2,3,4,4a, 5,6-hexahydro-1H-pyrazino [1,2-a] quinoline (compound 6d)
Alkylation of compound 5a with 1-iodopropane as described in Method A (Example 9) gave 165 mg of compound 6d as a colorless oil in 72% yield. The sample was converted to its hydrochloride salt.

Example 14
3-Butyl-2,3,4,4a, 5,6-hexahydro-1H-pyrazino [1,2-a] quinoline (Compound 6e)
Alkylation of compound 5a with 1-iodobutane as described in Method A (Example 9) gave 180 mg of compound 6e as a colorless oil in 74% yield. The sample was converted to its hydrochloride salt.

Example 15
3- (p-Fluorobenzoylethyl) -8-methoxy-2,3,4,4a, 5,6-hexahydro-1H-pyrazino [1,2-a] quinoline (Compound 7a)
Alkylation of compound 5b with 3-chloro-4′-fluoropropiophenone as described in Method A (Example 9) gave 239 mg of compound 7a as a colorless oil in 65% yield.

Example 16
3- (p-Fluorobenzoylpropyl) -8-methoxy-2,3,4,4a, 5,6-hexahydro-1H-pyrazino [1,2-a] quinoline (compound 7b)
Alkylation of compound 5b with 4-chloro-4'-fluorobutyrophenone as described in Method A (Example 9) gave 252 mg of compound 7b as a colorless oil in 66% yield.

Example 17
3- (p-Fluorobenzoylbutyl) -8-methoxy-2,3,4,4a, 5,6-hexahydro-1H-pyrazino [1,2-a] quinoline (compound 7c)
Alkylation of compound 5b with 1- (4-fluorophenyl) -5-chloro-1-oxopentane as described in Method A (Example 9) yielded 269 mg of compound 7c as a colorless oil. Obtained at 68%.

Example 18
8-methoxy-3-propyl-2,3,4,4a, 5,6-hexahydro-1H-pyrazino [1,2-a] quinoline (compound 7d)
Alkylation of compound 5b with 1-iodopropane as described in Method A (Example 9) gave 190 mg of compound 7d as a colorless oil in 73% yield.

Example 19
8-methoxy-3-butyl-2,3,4,4a, 5,6-hexahydro-1H-pyrazino [1,2-a] quinoline (compound 7e)
Alkylation of compound 5b with 1-iodobutane as described in Method A (Example 9) gave 208 mg of compound 7e as a colorless oil in 76% yield.

Example 20
[ ³ H] PD 128907 Binding Assay In some embodiments, the [ ³ H] PD 128907 binding assay for the D ₃ receptor dopamine receptor was previously described by Levant (B. Levant, Current Protocols in Pharmacology). (See 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 nodule) was prepared in assay buffer (50 mM Tris, 1 mM EDTA; pH 7.4, 23 ° C.) to a final concentration of 10 mg original wet weight (oww ) / Ml. Membranes are incubated with [ ³ H] PD 128907 0.3 nM; 116 Ci / mmol; (Amersham, Arlington Heights, IL) and various concentrations of competing compounds (10 ⁻¹⁰ to 10 ⁻⁴ M). Nonspecific binding is defined by 1 μM spiperone. Incubate the assay tubes at 23 ° C. for 3 hours. The reaction is terminated by rapid vacuum filtration. Data is analyzed using a non-linear least squares curve fitting program LIGAND.

Example 21
[ ³ H] Spiperone Binding Assay In some embodiments, the [ ³ H] spiperone binding assay was previously described for [ ³ H] PD128907 with the exception of Levant (B. Levant, Current Protocols in Pharmacology (J Ferkany and SJ Enna, Eds) John Wiley & Sons, New York, 1.6.1-1.6.16 [1998]). D ₂ dopamine receptor binding in the brain is assayed using membranes prepared from rat caudate putamen and the final membrane homogenate concentration is 1.5 mg oww / ml. hD _2s, for assays in hD _3, and hD _4.4 receptors determined for each receptor expression cell line membrane concentration in a preliminary study. Assay buffer is 50 mM Tris-HCl, 5 mM KCl, 2 mM MgCl ₂ , and 2 mM CaCl ₂ , pH 7.4, 23 ° C .; [ ³ H] spiperone concentration (24 Ci / mmol; Amersham) Is 200 pM; and the incubation time is 2 hours at 23 ° C. Non-specific binding is defined in the presence of 1 μM (+)-butaclamol.

Example 22
[ ³ H] SCH 23390 Binding Assay In some embodiments, the [ ³ H] SCH 23390 binding assay for D ₁ -like dopamine receptors has been previously described by Levant (B. Levant, Current Protocols in Pharmacology (see J Ferkany and SJ Enna, Eds) John Wiley & Sons, New York, 1.6.1-1.6.16 [1998]), and [ ³ H] PD spiperone binding Perform as described for the assay. The concentration of [ ³ H] SCH 23390 (73 Ci / mmol; Amersham) is 300 pM, and the incubation time is 30 minutes at 37 ° C.

Example 23
Receptor autoradiography with [ ³ H] quinpirole In some embodiments, a sagittal brain section (20 μm, lateral 1.00-1.40 mm) is cut with a cryostat, melt mounted onto a chrome alum / gelatin coated slide, Process as previously described in Levant (B. Levant, and EB De Souza, Synapse, 14: 90-95 [1993]). Sections were incubated at 23 ° C. for 2 hours with 10 nM [ ³ H] quinpirole (50.6 Ci / mmol; NEN Life Science, Boston, Mass.) At 5 concentrations of competitor (10 ⁻⁹ to 10 ⁻⁵ M or 10 ⁻⁸ Incubate in assay buffer (50 mM Tris-HCl, 5 mM KCl, 2 mM MgCl ₂ , 2 mM CaCl ₂ , pH 7.4, 23 ° C.) in the presence or absence of ˜10 ⁻⁴ M) To do. Duplicate sections from each animal are used for each data point. Autoradiograms are made using ³ H-Hyperfilm (Amersham, Arlington Heights, IL) and 20 μm [ ³ H] methyl methacrylate standard (Amersham). Autoradiographic images are quantified with NIH "Image" version 1.6.

Example 24
Mitosis Induction Assay In some embodiments, the mitogenesis assay was previously described in Chio et al. (See CL Chio, Mol. Pharmacol., 45: 51-60 [1994]). Do as follows. CHO cells expressing hD _2s or hD ₃ are seeded in 96-well plates at a density of 5,000 cells / well and cultured for 48 hours at 37 ° C. in MEM containing 10% fetal calf serum. The wells are washed with serum-free MEM, incubated with various concentrations of drugs (10 ⁻¹⁰ to 10 ⁻⁴ M) or solvent and incubated for 16 hours. Agonist-stimulated mitogenic antagonism is determined in the presence of 10 nM quinpirole. [ ³ H] thymidine (1 μCi / well; 25 Ci / mmol; Amersham) is then added to each well. After a 2 hour incubation, cells are trypsinized and harvested by rapid vacuum filtration. [ ³ H] thymidine incorporation in each sample is calculated as fmol incorporation / mg protein. Data are presented as percent stimulation relative to the base. EC ₅₀ , IC ₅₀ , and E _max values are calculated by non-linear regression analysis using a 4-parameter model (Sigma Plot, SPSS, Chicago, IL).

Example 25
[ ³⁵ S] GTP-S binding assay In some embodiments, the [ ³⁵ S] GTP-S binding assay has been previously described in Wang et al. (S. Wang et al., J. Med. Chem., 39: 2047-2054 [1996]; and S. Wang et al., J. Med. Chem., 43: 351-360 [2000]), including hD _2S , hD ₃ , or hD _4.4 receptors. Perform on CHO cell membrane.

Example 26
¹ HNMR spectra In some embodiments, ¹ HNMR spectra were recorded at 300 MHz and ¹³ CNMR spectra were recorded at 75 MHz, both on Varian Mercury 300 and Bruker DPX 300 spectrometers. The chemical shift is given in δ units (ppm) and is related to the solvent. The coupling constant is given in hertz (Hz). Element analysis is performed by Micro-Analysis, Inc., (Wilmington, DE) and is within 0.4% of the theoretical value except as noted. All reagents and chemicals were purchased from Aldrich Chemical Co., Fisher Scientific, or Lancaster Synthesis, Inc. and used without further purification. All reactions were performed under nitrogen unless otherwise stated.

Example 27
Additional compounds This example describes additional compounds envisioned for use in the present invention.

Synthesis of 5-methoxyl, 7-methoxyl, or 8-methoxylquinaldine
8-Methoxylquinaldine is obtained very easily by methylation of commercially available 8-hydroxylquinaldine. To make 5-methoxylucinaldine, amine 163 is first treated with acylacetic acid ethyl ester to give the coupling product (compound 164). Compound 64 is cyclized by heating to 250 ° C. to give compound 165, the hydroxyl group of compound 165 is converted to chloride, and the chloride is then removed by hydrogenation to give 5-methoxylucinaldine. 7-methoxylquinardine is obtained by the same method.

Synthesis of key intermediate compound 177
Methoxyl 5-, or 7-methoxyl, or 8-methoxy quinic Le Jin, is oxidized to the aldehyde (compound 174) by SeO _2. The aldehyde (compound 174) is treated with 2-aminoethanol and NaBH ₄ to give compound 178. The important intermediate compound 177 is then obtained by hydrogenation and cyclization.

Synthesis of D ₃ Ligand Along with the key intermediates at hand, potential D ₃ ligands are obtained by treating compound 177 with various alkyl halides. Nine D ₃ ligands is obtained. Compound 183 has a different synthesis method. Compound 183 is obtained by treating compound 177c with ₄ -heptanone and NaBH ₄ .

Synthesis of the tail of compound 186 or 182 The tail of compound 186 or 182 is synthesized by using compound 187 as the starting material. The amine is treated with 2-naphthoyl chloride with the hydroxyl group selectively protected with a TBS group to give the amide (Compound 189). Next, the TBS group is removed and the alcohol (compound 190) is treated with a THF solution of CBr ₄ and Ph ₃ P. Compound 192 is derived from the reaction between compound 190 and solvent (THF). Compound 192 is treated with MsCl to give compound 191.

Example 28
Additional compounds This example describes additional compounds envisioned for use in the present invention.

Reagents and conditions: ia) 1.1 eq ethanolamine, benzene, reflux under N ₂ , H ₂ O removal, overnight; b) 3.5 eq NaBH ₄ , absolute ethanol, under N ₂ , reflux, overnight, yield 87- 90%. ii. Approximately 5 g nickel-aluminum alloy / 1 g substrate, 1M KOH, methanol, rt, overnight, 85-89% yield. iii.3 equivalent P ₂ O _5, xylene, N ₂ under reflux, overnight, 82 to 86% yield. iv.1.2 eq R ₂ Cl / NaI or 1.2 eq R ₂ I, 2 eq Cs ₂ CO ₃ , CH ₃ CN, under N ₂ , reflux, overnight, 61-76% yield.

試薬および条件：v.2当量ヒドラジン、EtOH、還流、2時間、収率87〜94%。vi.1.2当量2-ナフトイルクロリド、3当量トリエチルアミン、0℃、4時間、収率91〜95%。vii.1.2当量4-ビフェニルカルボニルクロリド、3当量トリエチルアミン、0℃、4時間、収率72〜75%。

Reagents and conditions: v. 2 equivalents hydrazine, EtOH, reflux, 2 hours, yield 87-94%. vi.1.2 equivalent 2-naphthoyl chloride, 3 equivalent triethylamine, 0 ° C., 4 hours, yield 91-95%. vii.1.2 eq 4-biphenylcarbonyl chloride, 3 eq triethylamine, 0 ° C., 4 h, yield 72-75%.

試薬および条件：viii.4当量BBr₃、CH₂Cl₂、N₂下、0℃、4時間、収率52%。ix.1.2当量p-トルエンスルホニルクロリド、3当量トリエチルアミン、0℃、4時間、収率84%。

Reagents and conditions: viii.4 equivalents BBr ₃ , CH ₂ Cl ₂ , N ₂ at 0 ° C., 4 hours, 52% yield. ix.1.2 eq p-toluenesulfonyl chloride, 3 eq triethylamine, 0 ° C., 4 h, yield 84%.

  All publications and patents mentioned in the above specification are herein incorporated by reference. It will be apparent to those skilled in the art that various modifications and variations of the described method and system of the present invention do not depart from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it is to be understood that the claimed invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the described methods for carrying out the invention which are obvious to those skilled in the relevant fields are intended to be within the scope of the following claims.

Figure ₃ shows a model 3D structure of dopamine receptor D3 and its ligand binding site. Shows nine relatively selective D ₃ partial agonist used to induce pharmacophore model for the 3D- database search in one embodiment of the present invention. Figure 2 shows a simple pharmacophore model used in one embodiment of the present invention. Several compounds envisioned for use in one embodiment of the invention are shown. One novel approach for identifying compounds useful in the present invention is shown. 6A shows the superposition of agonists contemplated in the nine aspect of D ₃ partial agonist and the present invention. 6B shows a simple pharmacophore model derived from the D ₃ ligands contemplated. Several compounds are shown that are useful in some embodiments of the present invention. ₂ shows the functional activity of compound 4 at the D ₁ , D ₂ , and D ₃ receptors and a comparison of standard ligands in one embodiment of the invention. In one aspect of the present invention, it indicates that the compound 4 to form a strong salt bridge between D110 of nitrogen and D ₃ that its protonated. Several compounds are shown that are useful in some embodiments of the present invention. 1 shows one possible synthetic scheme for making compounds for use in some methods of the invention. 1 shows one possible synthetic scheme for making compounds for use in some methods of the invention. Several compounds are shown that are useful in some embodiments of the present invention. Figure 2 shows a functional assay using transfected cells in one embodiment of the invention. 1 shows one possible synthetic scheme for making compounds for use in some methods of the invention. 1 shows one possible synthetic scheme for making compounds for use in some methods of the invention. Several compounds are shown that are useful in some embodiments of the present invention. Figure 18A shows binding model predicted for compound 14 and D ₃ receptors in one aspect of the present invention. Figure 18B shows binding model predicted for Compound 32 and D ₃ receptors in one aspect of the present invention. It is a schematic view of a compound 14 in the binding site of D ₃ receptors in one aspect of the present invention. 1 shows one possible synthetic scheme for making compounds for use in some methods of the invention. 1 shows one possible synthetic scheme for making compounds for use in some methods of the invention. 1 shows one possible combined model of the present invention. 1 shows one possible synthetic scheme for making compounds for use in some methods of the invention. 1 shows one possible synthetic scheme for making compounds for use in some methods of the invention. 1 shows one possible synthetic scheme for making compounds for use in some methods of the invention. 1 shows a diagram of one embodiment of the present invention.

Claims (23)

A composition comprising a compound of the following formula:

In which X, Y, and Z independently represent C, O, N, S;
R ₇ , R ₈ , and R ₉ are independently H, F, Cl, Br, I, OH, CN, NO ₂ , OR ′, CO ₂ R ′, OCOR ′, CONR′R ″, NR ′. Represents' COR ', NR'SO ₂ R'', SO ₂ NR'R'',NR'R'', wherein R' and R '' are independently H, alkyl, cycloalkyl, alkenyl, Represents a substituted or unsubstituted cycloalkenyl, alkynyl, aryl, or heterocyclic ring;
A and B, O _{independently, S, SO, SO 2,} NR ', CR'R' represent ', in R' and R '' formula, independently, H, alkyl, cycloalkyl, alkenyl Represents substituted, unsubstituted, cycloalkenyl, alkynyl, aryl, or heterocyclic;
R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , and R ₆ are independently H, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, or heterocyclic, substituted or unsubstituted Wherein n = 0, 1, or 2;
R represents alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, or heterocyclic substituted or unsubstituted; for example, n = 1-6, R ′ ″ is aryl substituted Or C _n H _{2n + 1} , (CH ₂ ) _n R ′ ″, which represents an unsubstituted one. A composition comprising a compound of the following formula:

A composition comprising a compound of the following formula:

In which X, Y, Z, D and M independently represent C, O, N, S;
R ₇ , R ₈ , R ₉ , R ₁₀ , R ₁₁ , R ₁₂ , R ₁₃ , R ₁₄ , and R ₁₅ are independently 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 ″ each independently represents a substituted or unsubstituted H, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, or heterocyclic ring;
A and B, O _{independently, S, SO, SO 2,} NR ', CR'R' represent ', in R' and R '' formula, H are independently alkyl, cycloalkyl, alkenyl, Represents a substituted or unsubstituted cycloalkenyl, alkynyl, aryl, or heterocyclic ring;
R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , and R ₆ are independently H, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, or heterocyclic, substituted or unsubstituted Wherein n = 0, 1, or 2; where m = 1-20; R represents H, O, S, SO, SO ₂ , NR ′, CR′R ″ In which R ′ and R ″ independently represent substituted or unsubstituted H, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, or a heterocyclic ring. A composition comprising a compound of the following formula:

In which X, Y, Z, D and M independently represent C, O, N, S;
R ₇ , R ₈ , R ₉ , and R ₁₀ are independently 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'', wherein R' and R '' are independently H, alkyl, cycloalkyl Represents substituted, unsubstituted, alkenyl, cycloalkenyl, alkynyl, aryl, or heterocyclic;
A and B independently represent O, S, SO, SO ₂ NR ′, CR′R ″, wherein R ′ and R ″ are independently H, alkyl, cycloalkyl, alkenyl, Represents a substituted or unsubstituted cycloalkenyl, alkynyl, aryl, or heterocyclic ring;
R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , and R ₆ are independently H, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, or heterocyclic, substituted or unsubstituted Wherein n = 0, 1, or 2; E represents a linker group that does not include an amide group, and F represents a substituted or unsubstituted cycloalkyl, cycloalkenyl, or heterocyclic ring Examples include: C _n H _{2n + 1} , (CH ₂ ) _n R ′ ″, where n = 1-6, R ′ ″ represents aryl, substituted or unsubstituted.   A method for identifying agonists and antagonists of dopamine receptors, comprising candidate ligands and receptors D3 from N47, D75, F338, N375, and N379, receptor D2 from N52, D80, F382, N418, and N422 A method comprising evaluating an interaction between an amino acid of a dopamine receptor selected from the group consisting of:   6. The method of claim 5, wherein the step of assessing comprises examining a computer model for a predicted interaction between the ligand and the amino acid.   6. The method of claim 5, wherein the step of assessing comprises binding the ligand to a dopamine receptor and determining the binding between the ligand and the amino acid.   8. The method of claim 7, wherein the determining step comprises determining the crystal structure of the ligand bound to the receptor.   8. The method of claim 7, wherein the determining comprises comparing the binding affinity between a ligand and a receptor lacking the amino acid and the binding affinity between the ligand and the receptor.   8. The method of claim 7, wherein the determining step comprises measuring the ability of the ligand to displace a molecule bound to a receptor amino acid.   Of a subject having symptoms selected from the group consisting of cocaine abuse, depression, anxiety, eating disorders, alcoholism, chronic pain, obsessive compulsive disorder, schizophrenia, restless leg syndrome (RLS), and Parkinson's disease A method of treatment comprising administering to a subject a therapeutic dose of the composition of claim 1.   Of subjects having symptoms selected from the group consisting of cocaine abuse, depression, anxiety, eating disorders, alcoholism, chronic pain, obsessive compulsive disorder, schizophrenia, restless leg syndrome (RLS), and Parkinson's disease A method of treatment comprising administering to a subject a therapeutic dose of the composition of claim 2.   Of a subject having symptoms selected from the group consisting of cocaine abuse, depression, anxiety, eating disorders, alcoholism, chronic pain, obsessive compulsive disorder, schizophrenia, restless leg syndrome (RLS), and Parkinson's disease A method of treatment comprising administering to a subject a therapeutic dose of the composition of claim 3.   Of a subject having symptoms selected from the group consisting of cocaine abuse, depression, anxiety, eating disorders, alcoholism, chronic pain, obsessive compulsive disorder, schizophrenia, restless leg syndrome (RLS), and Parkinson's disease A method of treatment comprising administering to a subject a therapeutic dose of the composition of claim 4.   2. A method for modulating the action of a G protein-coupled receptor in a subject comprising the step of administering to the subject an effective amount of the composition of claim 1.   3. A method for modulating the action of a G protein-coupled receptor in a subject comprising the step of administering to the subject an effective amount of the composition of claim 2.   4. A method for modulating the action of a G protein-coupled receptor in a subject comprising the step of administering to the subject an effective amount of the composition of claim 3.   5. A method for modulating the action of a G protein-coupled receptor in a subject comprising the step of administering to the subject an effective amount of the composition of claim 4.   A method for controlling dopamine flow in a subject in need of such control, comprising the step of administering to the subject an effective amount of the composition of claim 1.   A method for controlling dopamine flow in a subject in need of such control, comprising the step of administering to the subject an effective amount of the composition of claim 2.   A method for controlling dopamine flow in a subject in need of such control, comprising the step of administering to the subject an effective amount of the composition of claim 3.   A method for controlling dopamine flow in a subject in need of such control, comprising the step of administering to the subject an effective amount of the composition of claim 4. A method of treating a subject with a modulator specific for the D3 receptor, comprising the step of administering to the subject a compound of formula (I) or formula (II), wherein formula (I) is as follows: :

In which X, Y, Z, and M independently represent C, O, N, S;
R ₇ , R ₈ , R ₉ , and R ₁₀ are independently 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' represent ', wherein, R' and R '', H are independently alkyl, cycloalkyl Represents an alkyl, alkenyl, cycloalkenyl, alkynyl, aryl, or heterocyclic ring, substituted or unsubstituted;
A and B independently represent O, S, SO, SO ₂ NR ′, CR′R ″, wherein R ′ and R ″ are independently H, alkyl, cycloalkyl, alkenyl, Represents a substituted or unsubstituted cycloalkenyl, alkynyl, aryl, or heterocyclic ring;
R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , and R ₆ are independently H, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, or heterocyclic, substituted or unsubstituted Wherein n = 0, 1, or 2;
R represents alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, or heterocyclic substituted or unsubstituted; for example, n = 1-6, R ′ ″ is aryl substituted Or C _n H _{2n + 1} , (CH ₂ ) _n R ′ ″, which represents an unsubstituted one; and formula (II) is as follows:

In which X, Y and Z independently represent C, O, N, S;
R ₇ , R ₈ , and R ₉ are independently H, F, Cl, Br, I, OH, CN, NO ₂ , OR ′, CO ₂ R ′, OCOR ′, CONR′R ″, NR ′. Represents' COR ', NR'SO ₂ R'', SO ₂ NR'R'',NR'R'', wherein R' and R '' are independently H, alkyl, cycloalkyl, alkenyl, Represents a substituted or unsubstituted cycloalkenyl, alkynyl, aryl, or heterocyclic ring;
A and B independently represent O, S, SO, SO ₂ NR ′, CR′R ″, wherein R ′ and R ″ are independently H, alkyl, cycloalkyl, alkenyl, Represents a substituted or unsubstituted cycloalkenyl, alkynyl, aryl, or heterocyclic ring;
R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , and R ₆ are independently H, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, or heterocyclic, substituted or unsubstituted Wherein n = 0, 1, or 2;
R represents alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, or heterocyclic substituted or unsubstituted; for example, n = 1-6, R ′ ″ is aryl substituted Or C _n H _{2n + 1} , (CH ₂ ) _n R ′ ″, which represents an unsubstituted one, wherein the subject has previously been associated with a dopamine receptor modulator that specifically binds to D3 and D2 or D1 receptors A method that is characterized by not responding sufficiently to it or may not respond sufficiently.

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