JP2004159664A - Rxr homodimer formation, bridged bicyclic aromatic compound and use thereof in controlled gene expression - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method of screening a substance for an ability to effect the formation of a retinoid X receptor homodimer comprising combining the substance and a solution containing the retinoid X receptor and determining the presence of homodimer formation. <P>SOLUTION: The method of screening a substance for an ability to affect the formation of the retinoid X receptor homodimer, comprises processes for combining the substance and the solution containing the retinoid X receptor, determining the presence of homodimer formation and determining the presence of the homodimer by detecting activation of transcription through the retinoid X receptor homodimer. <P>COPYRIGHT: (C)2004,JPO

Description

  The present invention relates generally to the regulation of gene expression by retinoid receptors, and more particularly, is useful in regulating gene expression by retinoid X receptors and retinoic acid receptors, retinoid X receptors, vitamin D receptors and thyroid receptors. The present invention relates to a novel bridged bicyclic aromatic compound.

The vitamin A metabolite all-trans-retinoic acid (RA) and its natural and synthetic derivatives (retinoids) exert a wide range of biological effects ^1,2 . Clinically, retinoids are important therapeutics in the treatment of skin diseases and cancers ^3-6 . An understanding of how the actions of many retinoids can be mediated at the molecular level is through the cloning and characterization of specific nuclear receptors, retinoic acid receptor (RAR) ^7-12 and retinoid X receptor (RXR) ^13-17 . Notably enhanced. RAR and RXR is a part of the steroid / thyroid hormone receptor superfamily ^{18 and 19.} Both types of receptors are encoded by three different genes (α, β, and γ), from which multiple isoforms can be generated in the case of RAR ^20-22 . Interestingly, RAR is specific in vertebrates, while RXR is well conserved from Drosophila to human ^17,23 . Despite considerable progress in understanding the molecular mechanisms of retinoid receptor action in recent years, the answer to the central question of whether different molecular pathways exist for naturally occurring retinoids remains unanswered. Recent observations ^23,24 of stereoisomers 9-cis -RA of RA binds with high affinity to RXR, the total - suggesting a different retinoid response pathway is the route of the trans -RA. However, RAR effectively requires interaction with coreceptor for DNA binding and functional, yet RXR be said that it is such a co-receptor, it has been almost simultaneously discovered by several laboratories ^{15 , 16, 26-29} . Thus, RARs appear to function effectively only as heterodimeric RAR / RXR complexes or in combination with the corresponding accessory proteins that need to be further identified. Similarly, RXR is for effective DNA binding, RAR, thyroid hormone receptor (TR), or may require vitamin _{D 3} receptor (VDR) was shown ^15,16,26-29. Thus, these DNA binding studies reveal that RXR can function preferentially even if not exclusively as a co-receptor, thereby generating a high degree of diversity and specificity of transcriptional regulation and It plays an important role in mediating the highly versatile effects of different hormones by increasing DNA affinity and specificity for at least three different classes of activating receptors.

  Contrary to these findings, the present invention provides that RXRs form homodimers. The present invention provides that these homodimers bind effectively to specific response elements in the absence of co-receptors, and that their DNA binding specificities differ from those of RXRs containing heterodimers. I do. The present invention describes a novel mechanism for the action of retinoids, whereby ligand-induced homodimers mediate distinct retinoid pathways. In addition, ligands are provided that selectively activate RXR homodimer formation.

  The present invention also provides a new class of retinoids in the form of bridged bicyclic aromatic compounds, as described in detail herein. These novel compounds are effective for modulating and / or inducing selective gene expression by receptors in the retinoic acid family (eg, RAR, RXR, vitamin D receptor (VDR), thyroid hormone receptor (THR)). . Without intending to be bound by theory, the compounds disclosed and claimed herein are effective in regulating gene expression due to their ability to interact with receptor proteins that bind to the aforementioned receptors. It is. The compounds are also useful in regulating gene expression by inducing the formation of RXR homodimers, in addition to RAR-RXR, VDR-RXR, and THR-RXR heterodimers.

  The novel compounds are therefore useful for controlling cellular processes regulated by retinoids (natural ligands for RXR), such as thyroid hormone, vitamin D, and 9-cis-retinoic acid. Therefore, acne, leukemia, psoriasis, and skin aging (all regulated by retinoic acid) are similar to bone mineralization (which can be regulated by vitamin D) and energy levels (which can be regulated by thyroid hormone) In addition, it can be treated with the compounds of the present invention. Unlike prior art synthetic retinoids, the compounds of the present invention are believed to provide substantially reduced side effects and teratogenicity.

  1. In one aspect, the invention provides a method of screening a substance for the ability to affect the formation of a retinoid X receptor homodimer, comprising combining a solution containing the substance and a retinoid X receptor, and determining the presence of homodimer formation. Providing a method comprising:

  2. In a preferred embodiment, there is provided the method of paragraph 1, wherein the presence of the homodimer formation is measured by detecting activation of transcription by the retinoid X receptor homodimer.

  3. In a preferred embodiment, there is provided the method of paragraph 1, wherein the presence of the homodimer formation is measured by co-precipitation.

  4. In a preferred embodiment, the method according to item 1 is provided, wherein the effect is induction of homodimer formation.

  5. In a preferred embodiment, there is provided the method of paragraph 1 above, wherein said effect is inhibition of homodimer formation.

  6. In a preferred embodiment, there is provided the method according to item 1, wherein the retinoid X receptor is retinoid X receptor α.

  7. In a preferred embodiment, the method according to item 4, further comprising a step of determining whether the substance induces a retinoid X receptor heterodimer, and a step of selecting the substance that selectively induces the formation of a retinoid X receptor homodimer. Are provided.

8. In one aspect, the present invention provides a method of screening a substance for its ability to selectively induce the formation of a retinoid X receptor heterodimer over a retinoid X receptor homodimer, comprising the steps of:
a) determining whether the substance induces retinoid X receptor homodimer formation;
b) determining whether the substance induces retinoid X receptor heterodimer formation; and c) selecting a compound that selectively induces formation of retinoid X receptor heterodimer over retinoid X receptor homodimer.

  9. In one aspect, the present invention provides a method of screening a substance for its effect on the ability of a retinoid X receptor homodimer to bind DNA, comprising combining the substance with the homodimer, and reducing the ability of the homodimer to bind DNA. Measuring the effect of a compound of the formula (I).

  10. In one aspect, the present invention provides a method for screening a response element for binding to a retinoid X receptor homodimer, comprising the steps of: combining the response element with the retinoid X receptor homodimer; and detecting the presence of binding. A method is provided.

  11. In a preferred embodiment, there is provided the method of clause 10, wherein the presence of the binding is detected by transcriptional activation of a marker operably linked to the response element.

  12. In one aspect, the present invention provides a purified retinoid X receptor homodimer.

  13. In one aspect, the present invention provides a bicyclic aromatic compound having the following structural formula:

ここで、
Ｒ^１は、低級アルキルおよびアダマンチルからなる群より選択され；
Ｒ^２は、−Ｏ−Ｒ^６または−Ｓ−Ｒ^６であり、ここでＲ^６は低級アルキルであり；または、Ｒ^１がＲ^２に対してオルトであるとき、Ｒ^１およびＲ^２が互いに連結して５または６員のシクロアルキレン環を形成し得、非置換であるかまたは１〜４個の低級アルキル基で置換され、そして任意にＯ、Ｓ、およびＮＲからなる群より選択される１または２個のヘテロ環式原子を含み、ここでＲは水素または低級アルキルであり；
Ｒ^３は、カルボニル、

here,
R ¹ is selected from the group consisting of lower alkyl and adamantyl;
R ² is —O—R ⁶ or —S—R ⁶ , wherein R ⁶ is lower alkyl; or when R ¹ is ortho to R ² , R ¹ and R ² are Can be linked to form a 5- or 6-membered cycloalkylene ring, is unsubstituted or substituted with 1-4 lower alkyl groups, and is optionally selected from the group consisting of O, S, and NR Containing one or two heterocyclic atoms, wherein R is hydrogen or lower alkyl;
R ³ is carbonyl,

からなる群より選択され、ここでＸ^１およびＸ^２は、独立して、Ｏ、Ｓ、およびメチレンからなる群より選択され、ここでＸ^１およびＸ^２の少なくとも１つはＯまたはＳであり、またはＸ^１およびＸ^２の１つがＮＲでありそして他方がメチレンであり、ｍは２または３であり、Ｒ^６、Ｒ^７、Ｒ^８、およびＲ^９は、独立して、水素または低級アルキルであり、ただし、ｎが０、Ｒ^６およびＲ^７であるときは両方とも水素ではなくそしてＲ^８およびＲ^９は両方とも水素ではなく、またはＲ^８およびＲ^９は互いに連結して３〜６個の炭素原子を含むシクロアルキレン環を形成し得、そして^＊は分子の残りの部分へのＲ^３置換基の付加位置を示し；そして
Ｒ^４は、

Wherein X ¹ and X ² are independently selected from the group consisting of O, S, and methylene, wherein at least one of X ¹ and X ² is O or S Or ^one of X ¹ and X ² is NR and the other is methylene, m is 2 or 3, and R ⁶ , R ⁷ , R ⁸ , and R ⁹ are independently hydrogen or lower alkyl. With the proviso that when n is 0, R ⁶ and R ⁷ are not both hydrogen and R ⁸ and R ⁹ are not both hydrogen, or R ⁸ and R ⁹ are linked to each other by 3-6 Can form a cycloalkylene ring containing six carbon atoms, and ^* indicates the position of addition of the R ³ substituent to the rest of the molecule; and R ⁴ is

からなる群より選択され、ここでＲ^１０は水素またはメチルであり、ｌは０または１であり、そして^＊＊は分子の残りの部分へのＲ^４置換基の付加位置を示し、
Ｒ^５は、独立して、低級アルキルおよび低級アルコキシからなる群より選択され；そして
ｎは０、１、２、または３であり、
ただし、ｎが０であるとき、Ｒ^３はカルボニル、^＊Ｃ＝ＣＨ_２、またはＣＨ_２以外であり、
および上記化合物の薬学的に受容可能なエステル、アミド、および塩を提供する。

Wherein R ¹⁰ is hydrogen or methyl, 1 is 0 or 1, and ^** indicates the position of addition of the R ⁴ substituent to the rest of the molecule;
R ⁵ is independently selected from the group consisting of lower alkyl and lower alkoxy; and n is 0, 1, 2, or 3;
Provided that when n is 0, R ³ is other than carbonyl, ^* C = CH ₂ , or CH ₂ ;
And pharmaceutically acceptable esters, amides, and salts of the above compounds.

  14．

からなる群より選択される構造を有する、上記項１３に記載の化合物であって、ここでＲ^１１がＯ、Ｓ、（ＣＨ_３）_２Ｃ、およびＣＨ_２からなる群より選択され、そしてＲ^１２が水素またはメチルである、化合物を提供する。

14. The compound according to above 13, having a structure selected from the group consisting of: wherein R ¹¹ is selected from the group consisting of O, S, (CH ₃ ) ₂ C, and CH ₂ ; ^A compound is provided wherein ¹² is hydrogen or methyl.

  15. In a preferred embodiment, there is provided the compound according to item 14, having the above structural formula (II).

  16. In a preferred embodiment, there is provided the compound according to item 15, wherein n is 0.

17. In a preferred embodiment, R ⁴ is

であり、そしてｌが１である、上記項１６に記載の化合物を提供する。

16. The compound according to item 16, wherein l is 1.

18. In a preferred embodiment, R ⁴ is

である、上記項１７に記載の化合物を提供する。

Item 18. The compound according to Item 17, wherein the compound is

19. In a preferred embodiment, R ⁴ is

である、上記項１７に記載の化合物を提供する。

Item 18. The compound according to Item 17, wherein the compound is

20. In a preferred embodiment, R ³ has the structural formula

を有する、上記項１５に記載の化合物を提供する。

16. The compound according to the above item 15, having the following formula:

21. In a preferred embodiment, R ³ has the structural formula

を有する、上記項１７に記載の化合物を提供する。

18. The compound according to the above item 17, having the following formula:

22. In a preferred embodiment, R ³ has the structural formula

を有する、上記項１５に記載の化合物を提供する。

16. The compound according to the above item 15, having the following formula:

23. In a preferred embodiment, R ³ has the structural formula

を有する、上記項１７に記載の化合物を提供する。

18. The compound according to the above item 17, having the following formula:

24. In a preferred embodiment, R ³ is

からなる群より選択される、上記項２３に記載の化合物を提供する。

24. The compound according to the above item 23, which is selected from the group consisting of:

25. In a preferred embodiment, R ³ has the structural formula

を有する、上記項１５に記載の化合物を提供する。

16. The compound according to the above item 15, having the following formula:

26. In a preferred embodiment, R ³ has the structural formula

を有する、上記項１７に記載の化合物を提供する。

18. The compound according to the above item 17, having the following formula:

27. In a preferred embodiment, R ³ is

からなる群より選択される、上記項２６に記載の化合物を提供する。

27. The compound according to the above item 26, which is selected from the group consisting of:

  28. In a preferred embodiment, the structural formula

を有する、上記項１５に記載の化合物を提供する。

16. The compound according to the above item 15, having the following formula:

  29. In a preferred embodiment, the structural formula

を有する、上記項１５に記載の化合物を提供する。

16. The compound according to the above item 15, having the following formula:

  30. In a preferred embodiment, the structural formula

を有する、上記項１５に記載の化合物を提供する。

16. The compound according to the above item 15, having the following formula:

  31. In a preferred embodiment, the structural formula

を有する、上記項１５に記載の化合物を提供する。

16. The compound according to the above item 15, having the following formula:

  32. In a preferred embodiment, the structural formula

を有する、上記項１５に記載の化合物を提供する。

16. The compound according to the above item 15, having the following formula:

  33. In a preferred embodiment, the structural formula

を有する、上記項１５に記載の化合物であって、ここで、Ｘ^１およびＸ^２が、独立して、ＯおよびＳからなる群より選択される化合物、および上記化合物のアンモニウム塩を提供する。

16. The compound according to item 15, wherein X ¹ and X ² are independently selected from the group consisting of O and S, and an ammonium salt of the compound.

  34. In a preferred embodiment, the structural formula

を有する、上記項１５に記載の化合物であって、ここで、Ｘ^１およびＸ^２が、独立して、ＯおよびＳからなる群より選択される化合物、および上記化合物のアンモニウム塩を提供する。

16. The compound according to item 15, wherein X ¹ and X ² are independently selected from the group consisting of O and S, and an ammonium salt of the compound.

  35. In a preferred embodiment, the structural formula

を有する、上記項１５に記載の化合物を提供する。

16. The compound according to the above item 15, having the following formula:

  36. In a preferred embodiment, the structural formula

を有する、上記項１５に記載の化合物を提供する。

16. The compound according to the above item 15, having the following formula:

  37. In a preferred embodiment, the structural formula

を有し、Ｒ^１がＯまたはＳである、上記項１５に記載の化合物を提供する。

16. The compound according to item 15, wherein R ¹ is O or S.

  38. In a preferred embodiment, the structural formula

を有する、上記項１５に記載の化合物、および上記化合物のアンモニウム塩を提供する。

Item 15. The compound according to item 15, and an ammonium salt of the compound,

  39. In a preferred embodiment, the structural formula

を有する、上記項１５に記載の化合物を提供する。

16. The compound according to the above item 15, having the following formula:

  40. In a preferred embodiment, the structural formula

を有する、上記項１５に記載の化合物を提供する。

16. The compound according to the above item 15, having the following formula:

  41. In a preferred embodiment, the structural formula

を有する、上記項１５に記載の化合物を提供する。

16. The compound according to the above item 15, having the following formula:

  42. In a preferred embodiment, the structural formula

を有する、上記項１５に記載の化合物を提供する。

16. The compound according to the above item 15, having the following formula:

  43. In one aspect, the present invention provides a method for inhibiting the activity of a retinoid X receptor heterodimer, comprising the step of inducing the formation of a retinoid X receptor homodimer, thereby suppressing the formation of the retinoid X receptor heterodimer. And inhibiting the resulting heterodimer activity.

  44. In a preferred embodiment, the method according to the above item 43 is provided, wherein the activity is activation or repression of transcription.

  45. In a preferred embodiment, there is provided the method of paragraph 43 above wherein the retinoid X receptor heterodimer is a thyroid hormone receptor and a retinoid X receptor.

  46. In one aspect, the present invention relates to a method for promoting the transcription of a gene activated by a retinoid X receptor homodimer in a cell, the method comprising: providing the cell with an amount of a synthetic compound capable of promoting the formation of a retinoid X receptor homodimer. Contacting with a method.

  47. In a preferred embodiment, the compound has the structural formula

を有する、上記項４６に記載の方法を提供する。

48. The method according to the above item 46, comprising:

  48. In a preferred embodiment, the compound has the structural formula

を有する、上記項４６に記載の方法を提供する。

48. The method according to the above item 46, comprising:

  49. In one aspect, the present invention provides a method for inhibiting the activity of the retinoid X receptor homodimer, comprising the step of suppressing the formation of a retinoid X receptor homodimer.

  50. In a preferred embodiment, the method according to item 49 is provided, wherein the activity is activation or repression of transcription.

  51. In one aspect, the present invention provides a method for inhibiting the activity of the retinoid X receptor homodimer, comprising the step of suppressing the binding of the retinoid X receptor homodimer to its response element.

  52. In a preferred embodiment, the method according to the above item 51 is provided, wherein the activity is activation or repression of transcription.

  53. In one aspect, the present invention is a method of determining the probability of an increase in a pathology associated with retinoid X receptor homodimer formation, wherein the method comprises determining, in a subject, a decrease in retinoid X receptor homodimer formation as compared to a normal subject. A method is provided that comprises the step of detecting.

  54. In one aspect, the present invention is a method of treating a condition associated with a retinoid X receptor homodimer in a subject, comprising the step of inducing retinoid X receptor homodimer formation in the subject, thereby comprising the step of: Methods are provided that increase the expression of genes that can be activated by homodimers.

  55. In a preferred embodiment, the retinoid X receptor homodimer formation has the structural formula

を有する化合物の添加により誘導される、上記項５４に記載の方法を提供する。

55. The method according to the above item 54, wherein the method is derived by adding a compound having

  56. In a preferred embodiment, the retinoid X receptor homodimer formation has the structural formula

を有する化合物の添加により誘導される、上記項５４に記載の方法を提供する。

55. The method according to the above item 54, wherein the method is derived by adding a compound having

  57. In a preferred embodiment, there is provided the method of paragraph 54 wherein the condition is skin-related.

  58. In a preferred embodiment, there is provided the method of paragraph 54 above wherein the skin condition is selected from the group consisting of acne and psoriasis.

  59. In a preferred embodiment, there is provided the method of paragraph 54 above wherein the condition is cancer.

  60. In a preferred embodiment, there is provided the method according to the above item 54, wherein the gene is an apolipoprotein AI gene.

  61. In one aspect, the present invention is a method for selectively activating retinoid X receptor homodimer formation in a cell, comprising the step of adding a homodimer formation specific ligand to the cell, whereby the cell comprises Wherein the retinoid X receptor homodimer formation is selectively activated.

  62. In a preferred embodiment, the compound has the structural formula

を有する、上記項６１に記載の方法を提供する。

61. The method according to the above item 61, comprising:

  63. In a preferred embodiment, the compound has the structural formula

を有する、上記項６１に記載の方法を提供する。

61. The method according to the above item 61, comprising:

  64. In one aspect, the present invention provides a method of promoting retinoid X receptor homodimer formation in a cell, including expression of 9-cis-RA in the cell.

  65. In one aspect, the present invention provides a cell regulated by retinoic acid, vitamin D, or thyroid hormone comprising an effective modulating amount of the compound of item 13 above in combination with a pharmaceutically acceptable carrier. Pharmaceutical compositions for controlling a process are provided.

  66. In one aspect, the present invention provides a cell regulated by retinoic acid, vitamin D, or thyroid hormone, comprising an effective modulating amount of the compound of item 15 above in combination with a pharmaceutically acceptable carrier. Pharmaceutical compositions for controlling a process are provided.

  67. In one aspect, the present invention provides a method for regulating gene expression in a subject by a receptor selected from the group consisting of a retinoic acid receptor, a retinoid X receptor, a vitamin D receptor, and a thyroid receptor. And administering to said subject an effective modulating amount of the compound described in or a pharmaceutical composition thereof.

  68. In one aspect, the present invention provides a method for regulating gene expression in a subject by a receptor selected from the group consisting of a retinoic acid receptor, a retinoid X receptor, a vitamin D receptor, and a thyroid receptor. And administering to said subject an effective modulating amount of the compound described in or a pharmaceutical composition thereof.

  69. In one aspect, the present invention is directed to a method of treating a subject suffering from a disease caused by a dysfunction of a cell differentiation process regulated by a retinoid, thyroid hormone, or vitamin D, comprising a therapeutically effective amount of Item 13. A method comprising the step of administering the compound according to Item 13 or a pharmaceutical composition thereof to the subject.

  70. In one aspect, the present invention is directed to a method of treating a subject suffering from a disease caused by a dysfunction of a cell differentiation process regulated by a retinoid, thyroid hormone, or vitamin D, comprising a therapeutically effective amount of 16. A method comprising administering the compound or the pharmaceutical composition thereof according to the above item 15 to the subject.

(Summary of the Invention)
The present invention provides a method of screening a substance for the ability to affect the formation of a retinoid X receptor homodimer, comprising combining the substance with a solution containing a retinoid X receptor, and determining the presence of homodimer formation. The step of performing Also provided is a method of screening a substance for an effect on the ability of a retinoid X receptor homodimer to bind to DNA, the method comprising combining the substance with a homodimer, and determining the effect of the compound on the ability of the homodimer to bind to DNA. The step of performing It also includes the step of increasing the formation of a retinoid X receptor homodimer, thereby inhibiting the retinoid X receptor from forming a heterodimer, and inhibiting the retinoid X receptor heterodimer activity that inhibits the activity of the resulting heterodimer. A method is provided for doing so. Further, a method for inhibiting the activity of a retinoid X receptor homodimer is provided. Further provided are methods of determining the probability of an increase in a condition associated with retinoid X receptor homodimer formation, and treating such a condition. Further provided is a method of screening a response element for binding to a retinoid X receptor homodimer. Finally, the present invention provides a method for activating retinoid X receptor homodimer formation.

  Furthermore, meeting the above-mentioned need in the art by providing novel compounds useful for modulating gene expression by receptors in the retinoic acid family of receptors, it is a first object of the present invention to provide The purpose is. Another object of the present invention is to provide novel compounds useful for controlling cell differentiation processes regulated by retinoids such as thyroid hormone, vitamin D, and / or 9-cis-retinoic acid. Yet another object of the present invention is to provide novel compounds in the form of bridged bicyclic structures. Yet another object of the present invention is to provide a pharmaceutical composition containing one or more novel compounds. Yet another object of the present invention is to provide a method for regulating gene expression of a receptor selected from the group consisting of retinoic acid receptor, retinoid X receptor, vitamin D receptor, and thyroid receptor. Yet another object of the present invention is to provide a method for controlling a cell differentiation process regulated by retinoids such as thyroid hormone, vitamin D, and / or 9-cis-retinoic acid.

  Further objects, advantages and novel features of the invention will be set forth in part in the description which follows, and in part will be obvious to those skilled in the art from the following description, or may be learned by practice of the invention. Next, in one aspect, the invention relates to novel compounds having structural formula (I):

ここで、
Ｒ^１は低級アルキルおよびアダマンチルから成る群から選択され；
Ｒ^２は−Ｏ−Ｒ^６または−Ｓ−Ｒ^６であり、ここでＲ^６は低級アルキルであり；または
ここでＲ^１はＲ^２に対してオルトであり、Ｒ^１およびＲ^２は共に連結して、５員環または６員環シクロアルキレン環を形成し、このシクロアルキレン環は、炭素数１〜４個の低級アルキル基で置換されないまたは置換され、そして必要に応じてＯ、ＳおよびＮＲからなる群から選択される１個または２個のヘテロ環原子を含み、ここでＲは水素または低級アルキル、好ましくは芳香族環に隣接し；
Ｒ^３はカルボニル、

here,
R ¹ is selected from the group consisting of lower alkyl and adamantyl;
R ² is ^{-O-R 6} or ^{-S-R 6,} where ^{R 6} is lower alkyl; or wherein ^{R 1} is ortho to ^{R 2, R 1} and ^{R 2} are linked together To form a 5- or 6-membered cycloalkylene ring, which cycloalkylene ring is unsubstituted or substituted by a lower alkyl group having 1 to 4 carbon atoms, and optionally O, S and NR Comprising one or two hetero ring atoms selected from the group consisting of: wherein R is adjacent to hydrogen or lower alkyl, preferably an aromatic ring;
R ³ is carbonyl,

からなる群より選択され、
ここで、Ｘ^１およびＸ^２はＯ、Ｓおよびメチレンからなる群から独立して選択され、ここでＸ^１およびＸ^２の少なくとも１つはＯまたはＳであり、またはここでＸ^１およびＸ^２の１つはＮＲであり、および他はメチレンであり、ｍは２または３であり、Ｒ^６、Ｒ^７、Ｒ^８およびＲ^９はそれぞれ独立して水素または低級アルキルであり、ただし、ｎが０のとき、Ｒ^６およびＲ^７は両方とも水素でなくかつＲ^８およびＲ^９は両方とも水素でなく、またはＲ^８およびＲ^９は共に連結して、３〜６個の炭素原子を有するシクロアルキレン環を形成し得、そして＊は分子の残り部分へのＲ^３置換基の結合位置を示す；そして
Ｒ^４は以下からなる群から選択され、

Selected from the group consisting of
Wherein X ¹ and X ² are independently selected from the group consisting of O, S and methylene, wherein at least one of X ¹ and X ² is O or S, or wherein X ¹ and X ² Is NR, and the other is methylene, m is 2 or 3, and R ⁶ , R ⁷ , R ⁸ and R ⁹ are each independently hydrogen or lower alkyl, where n is When 0, R ⁶ and R ⁷ are not both hydrogen and R ⁸ and R ⁹ are not both hydrogen, or R ⁸ and R ⁹ are linked together to form a cyclo having 3-6 carbon atoms. Can form an alkylene ring, and * indicates the point of attachment of the R ³ substituent to the rest of the molecule; and R ⁴ is selected from the group consisting of:

ここで、Ｒ^１０は水素またはメチルであり、ｌは０または１であり、^＊＊は分子の残りへのＲ^４置換基の結合位置表し、
Ｒ^５は低級アルキルおよび低級アルコキシからなる群から独立して選択され；そして
ｎは０、１、２または３であり、
ただし、ｎが０のとき、Ｒ^３はカルボニル、^＊Ｃ＝ＣＨ_２またはＣＨ_２以外である。

Wherein R ¹⁰ is hydrogen or methyl, l is 0 or 1, ^** represents the position of attachment of the R ⁴ substituent to the rest of the molecule,
R ⁵ is independently selected from the group consisting of lower alkyl and lower alkoxy; and n is 0, 1, 2, or 3;
However, when n is 0, R ³ is other than carbonyl, ^* C = CH ₂ or CH ₂ .

  The present invention also includes pharmaceutically acceptable esters, amides and salts of such compounds, as described in detail below.

  In another aspect, the present invention relates to pharmaceutical compositions containing the aforementioned compounds, as well as methods of using said compounds for modulating selective gene expression by receptors belonging to the retinoic acid family of receptors.

Detailed Description of the Invention Definitions and Names:
In disclosing and describing the compounds, compositions, and methods of the present invention, the present invention is not limited to any particular synthetic method, any particular pharmaceutical carrier, or any particular pharmaceutical formulation or regime of administration. It should be understood that this can naturally vary. It is also to be understood that the terminology used herein is used only for describing particular embodiments and is not intended to be limiting.

  As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a bicyclic aromatic compound" includes a mixture of the bicyclic aromatic compounds, and "a pharmaceutical carrier" includes a mixture of two or more such carriers and the like. .

In this specification and the appended claims, references that may be made to a number of terms are defined to have the following meanings:
The term “alkyl,” as used herein, refers to a branched or unbranched saturated hydrocarbon group of 1 to 24 carbon atoms. That is, they are methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, octyl, decyl, tetradecyl, hexadecyl, eicosyl, tetracosyl and the like. Preferred alkyl groups herein contain 1 to 12 carbon atoms. The term "lower alkyl" intends an alkyl group of one to six carbon atoms, preferably one to four carbon atoms. The term "cycloalkyl" intends a cyclic alkyl group of 3 to 8, preferably 5 or 6, carbon atoms.

  The term “alkoxy” as used herein intends an alkyl group attached through a single terminal ether bond; that is, an “alkoxy” group is one in which R is alkyl as defined above— It can be defined as OR. A "lower alkyl" group intends an alkyl group containing one to six, more preferably one to four, carbon atoms.

The term "alkylene" as used herein, includes from 1 to 24 carbon atoms refers to a hydrocarbon chain that is not saturated branched or branched bifunctional and such as methylene (-CH ₂ - ), ethylene _(-CH 2 -CH 2 -), polyethylene _{(-CH 2 -CH 2 -CH 2 -} ), 2 -methylpropylene _{[--CH 2 -CH (CH 3)} -CH 2 -], hexylene [ - including such] - _{(CH 2) 6.} "Lower alkylene" refers to an alkene group of one to six, more preferably one to four, carbon atoms. The term "cycloalkylene," as used herein, refers to a cyclic alkylene group, typically a 5- or 6-membered ring.

  The term "alkene" as used herein intends a mono- or di-unsaturated hydrocarbon group of 2 to 24 carbon atoms. Preferred groups falling into this class contain 2 to 12 carbon atoms. Asymmetric structures such as (AB) C = C (CD) are intended to include both E and Z isomers. This can be assumed in the structural formulas herein where the asymmetric alkene is present or the binding symbol

で明瞭に示され得る。

Can be clearly shown.

  "Optional" or "as needed" means that the subsequently described event or circumstance may or may not occur, and that description includes instances where this event or circumstance occurs and instances where it does not. . For example, the phrase "optionally substituted phenyl" means that the phenyl can be substituted or unsubstituted, and the description includes both unsubstituted phenyl and substituted phenyl.

  By the term "effective amount" of a compound provided herein is meant a nontoxic amount of the compound that is sufficient to provide the desired modulation of gene expression. As can be pointed out below, the exact requirements can vary from subject to subject. It depends on the species, age, general condition of the subject, the severity of the ailment being treated, the particular bicyclic compound used, the manner of administration, and the like. Therefore, it is not possible to state an exact "effective amount" in the specification. However, an appropriate effective amount can be determined by one of ordinary skill in the art using only routine experimentation.

  The term “pharmaceutically acceptable” means not a biologically or otherwise undesirable material. That is, with the selected bicyclic compound without producing any undesired biological effects or interacting in a deleterious manner with any other components contained in the pharmaceutical composition. It can be a material that can be administered to an individual.

  “Inducing,” “modulating,” or “modulating” selective gene expression is intended to mean that the compound can act as an activator or antagonist of gene expression by a particular receptor (ie, the retinoic acid family). .

  The “retinoic acid family” of receptors, also called “retinoid receptors”, is intended to include retinoic acid receptors, retinoid X receptors, vitamin D receptors, and thyroid hormone receptors. The aforementioned three groups of receptors are also broadly referred to herein as "retinoic acid receptors." That is, the term includes, in addition to the retinoid acid receptor itself, retinoid X receptor, vitamin D receptor, and thyroid hormone receptor.

  "Bridged bicyclic aromatic compound" refers to any compound encompassed by the structure of Formula (I). These compounds are also referred to as "retinoids," which term is intended to further include species such as retinoic acid and 9-cis retinoic acid.

  The present invention provides a method of screening a substance for the ability to affect RXR homodimer formation, comprising combining a solution containing the substance and RXR, and measuring the presence of homodimer formation. . The presence of homodimer formation can be determined, for example, by detecting transcriptional activation by RXR homodimer or co-precipitation. The effect can be the induction of homodimer formation, for example, an activity similar to the activity activated by 9-cis-RA, or an activity that selectively activates homodimer formation over heterodimer formation. The term "selectively activate" refers to compounds that activate homodimer formation but do not significantly activate heterodimers. Alternatively, "selectively activate" includes compounds that activate heterodimer formation but do not significantly activate homodimer formation. The term "significant activation" encompasses activation sufficient to cause a deleterious pharmacological effect in the subject. This effect may also be an inhibition of homodimer formation. Examples of inhibition are substances that compete for 9-cis-RA binding to the receptor but do not themselves activate or induce dimerization, or 9-cis-RA to block its activity. Includes substances that bind. Such screening of substances, if present in the subject, can routinely perform the discovery of homodimer formation. The specific assay set presented below can be commonly used for screening by simply replacing 9-cis-RA with the substance of interest. A good starting point for screening such "substances" is the 9-cis-RA activity described herein. Substitutes for 9-cis-RA may be altered to make a 9-cis-RA analog and screened in a manner to measure any increase or decrease in homodimer formation. However, any substance can be screened in this assay to determine any effect on homodimer formation. Such compounds can then be used to promote homodimer formation and gene transcription in cells. Cells as used herein include cells found either in vitro or in vivo. Thus, these compounds can be administered to a human subject to affect RXR homodimer formation and promote transcription of a gene activated by the RXR homodimer. Such compounds are provided in the examples below.

  The data presented herein below utilize RXRα. However, given high homology between RXRα, β, and γ, each protein should form a homodimer and have the activity described for the RXRα homodimer. Relatedly, homodimers can be formed between different RXRs. For example, homodimers can be formed between RXRα and RXRβ, or between RXRβ and RXRγ, or between RXRα and RXRγ. The activity of these homodimers can be confirmed using the methods presented herein.

  The invention also provides a method of screening a substance for its effect on the ability of an RXR homodimer to bind DNA, comprising the step of combining the substance with the above homodimer, and the effect of the compound on the ability of the homodimer to bind DNA. Measuring the effect. For example, compounds that can bind homodimers or bind DNA response elements recognized by RXR homodimers can be screened in this manner.

  The invention further provides a method of inhibiting the activity of an RXR-containing heterodimer, the method increasing the formation of an RXR homodimer, whereby the RXR suppresses the formation of the heterodimer and reduces the resulting heterodimer activity. Including the step of suppressing. The activity can be any activity, but is generally activation or repression of transcription. This activity can be blocked, for example, by utilizing RXR, which would otherwise be available to form a heterodimer, to form a homodimer. As the number of heterodimers decreases, the activity of the heterodimers decreases. In one embodiment, the RXR heterodimer consists of a thyroid hormone receptor and RXR. The activity of the RXR / TR heterodimer was reduced. Other heterodimers can be tested using the standard methods provided herein.

  The invention also provides a method of inhibiting RXR receptor homodimer activity, the method comprising the step of inhibiting RXR homodimer formation. Such inhibition can be obtained, for example, by inhibiting 9-cis-RA, or transcription or activation by 9-cis-RA. This inhibited activity is generally the activation or repression of transcription.

  The invention also provides a method of inhibiting the activity of an RXR homodimer, comprising the step of inhibiting the binding of the RXR homodimer to its response element. For example, the activity of a receptor that competes with the same response element can be enhanced. Generally, the activity that is inhibited is the activation or repression of transcription.

  The invention further provides a method for determining the probability of an increase in a pathology associated with RXR homodimer formation, comprising detecting the modulation of RXR homodimer formation in a subject as compared to a normal subject. I do. This modulation may increase or decrease homodimer formation. Such regulation may result, for example, from a mutated RXR. This reduction can be detected by assaying for homodimers in the sample, or by detecting variants that are known to reduce homodimer formation.

  Relatedly, the present invention provides a method of treating a condition associated with RXR homodimer formation in a subject, comprising modulating homodimer formation in the subject. This regulation may be increased or decreased depending on the condition. Such an increase can be achieved, for example, by utilizing a compound that promotes RXR transcription. This condition can be associated with skin, such as, for example, acne and psoriasis. Further, the condition may be cancer. The exact amount of such compounds will vary from subject to subject, including: species, age, general condition of the subject, the severity of the ailment being treated, the particular compound employed, and the manner of administration thereof. And so on. Therefore, it is impossible to specify an accurate activity promoting amount. However, an appropriate amount, given the teachings herein, can be determined by one of ordinary skill in the art using only routine experimentation.

  The present invention also provides a purified RXR homodimer. The term "purified" means the absence of at least some cellular components associated with the RXR homodimer in its natural environment.

  The present invention provides a method of screening for a response element for binding to an RXR homodimer, the method comprising combining the response element with the RXR homodimer and detecting the presence of binding. The presence of a bond can be determined by a number of standard methods. In one method, binding is detected by transcriptional activation of a marker operably linked to a response element. The term "operably linked" means that the marker can be transcribed in the presence of a transcription activator.

This study comes from studies of the effects of the natural vitamin A derivative 9-cis-RA on retinoid receptor DNA binding and transcriptional activation. In contrast to all-trans-RA, the 9-cis analog dramatically enhances RXRa binding to some RXR-specific RARE, but not to the native TRE or ERE, at a concentration of ^10-9 to ^10-8 M. Facilitate. This effect is specific to RXR since 9-cis-RA does not induce RXRα, β or γ binding to the response element (FIGS. 1, 3). It is presumed that 9-cis-RA induces homodimer formation, although judging from the migration pattern in the gel shift assay, larger complexes including RXR trimers or tetramers may occur (especially in the case of CRBPII-RARE). Such trimer or tetramer formation can be tested using the methods provided herein.

RXRa homodimers exhibit different response element specificities than heterodimers. The rCRBPI response element does not interact with the RXR homodimer, whereas only the CRBPII response element (a previously identified natural RARE including full repeats) is the potent 9-cis-RA-induced RXRa homodimer. It was a binder. It has previously been shown that this response element is well activated by RXRα ^24,30 . This response element is also effectively bound by RXRα-RARa heterodimers (FIG. 3) ^27,29, but the heterodimers appear to have repressor function ³⁰ .

  Although CV-1 cells, like all other mammalian tissue culture cells tested, embrace endogenous retinoid receptors that can partially obscure the effects, the results obtained in transcriptional activation studies are: Good agreement with results obtained in DNA binding studies. Nevertheless, response elements that bind strongly to 9-cis-RA-RXRa homodimers also respond strongly to co-transfected RXRa in the presence of 9-cis-RA. On the other hand, response elements that do not bind well to RXRa homodimers could not be activated by RXRa alone, such as rCRBPI-RARE or MHC-TRE.

It is generally believed that dimerization-homodimerization or heterodimerization of nuclear hormone receptors is important for high affinity interactions of receptors with their cognate response elements. RXR is present mainly as a monomer in solution ^16, the effective DNA binding activity high concentration to indicate or RAR, the presence of TR or VDR requires ^{13,15,16,26-30.} Observation of increased cooperative RXR DNA binding activity in the presence of 9-cis-RA indicates that 9-cis-RA induced the formation of RXR homodimers with increased affinity for DNA. Thus, binding of 9-cis-RA to RXR can induce a conformational change, which results in homodimerization. Although 9-cis-RA and RA can bind to RAR ^24,25 , it is interesting that they do not induce RAR homodimer formation.

RXRa homodimer formation can occur in solution in the absence of DNA. Thus, when 9-cis-RA becomes available to cells, the equilibrium between monomeric and dimeric receptors changes, and an additional species, the RXR homodimer, can be formed, providing a novel response pathway . The concept of ligand-induced homodimer binding as observed by in vitro gel shift assays has been previously observed for nuclear receptors except for the mutated estrogen receptor (ER-val-400) ^44,45 .

For related TRs, ligand effects have been reported for homodimer binding ^46,47 , but ligand (T3) reduced homodimer response element interactions. Because the carboxy-terminal halves of TR and RAR encode both ligand and dimerization functions ^36,46,48 , the strong effect of ligand on dimerization as observed here is quite surprising. It should not be. However, the specificity of this effect is quite dramatic, as it only affects homodimer formation and not heterodimer formation. The overall picture is that the carboxy-terminal region of the receptor (which also contains the transcriptional activation region) ⁴⁹ through its intermixed domains ⁴⁹ is an activity of the individual receptors multiple that may involve interaction with other regulatory proteins ⁵⁰ Resulting from allowing

  The data described in this application demonstrated a central role for RXR with two functions. It allows to act as co-receptors for three classes of hormone receptors, RAR, TR and VDR, through heterodimerization. Two functions of RXR-homodimerization and heterodimerization-illustrate two distinct transcriptional regulatory controls. It can be expected to affect distinct physiological processes. Thus, 9-cis-RA may have therapeutic properties that are distinct from those of all-trans-RA.

  The novel compounds provided herein are those defined by structural formula (I) above. Preferred compounds falling into this general structure include:

ここで、Ｒ^１１は、Ｏ、Ｓ、（ＣＨ_３）_２ＣおよびＣＨ_２からなる群から選択され、そしてＲ^１２は水素またはメチルである。この群に入る特に好ましい化合物は構造式（ＩＩ）に示されるような化合物である。

Wherein R ¹¹ is selected from the group consisting of O, S, (CH ₃ ) ₂ C and CH ₂ , and R ¹² is hydrogen or methyl. Particularly preferred compounds falling into this group are those as shown in structural formula (II).

Preferred R ³ substituents are those in which R ³ has the following general structure:

以下よりなる群から選択される：

Selected from the group consisting of:

。
好ましいＲ^３置換体は、Ｒ^３が以下の一般構造の場合であり、

.
Preferred R ³ substituents are when R ³ has the following general structure:

以下よりなる群から選択される：

Selected from the group consisting of:

。
式（ＩＩ）に包含される好ましい構造の例は以下を包含する：

.
Examples of preferred structures encompassed by formula (II) include:

式（ＩＩ）により定義される化合物のクラスに入る特定の詳細な好ましい化合物の例は以下を包含する：

Examples of certain detailed preferred compounds falling into the class of compounds defined by formula (II) include:

本発明はまた、カルボン酸部分を含む式（Ｉ）の化合物の薬学的に受容可能な無毒性エステル、アミド、および塩の誘導体を包含する。

The present invention also includes pharmaceutically acceptable non-toxic ester, amide, and salt derivatives of the compounds of formula (I) that contain a carboxylic acid moiety.

  Pharmaceutically acceptable salts are prepared by treating the free acid with an appropriate amount of a pharmaceutically acceptable base. Representative pharmaceutically acceptable bases are ammonium hydroxide, sodium hydroxide, potassium hydroxide, lithium hydroxide, calcium hydroxide, magnesium hydroxide, ferrous hydroxide, zinc hydroxide, copper hydroxide, Aluminum hydroxide, ferric hydroxide, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, lysine, arginine, histidine and the like. The reaction is carried out in water at a temperature of about 0 ° C. to about 100 ° C., preferably at room temperature, in combination with water alone or an inert water-miscible organic solvent. The molar ratio of the compound of structural formula (I) to the base used is selected to provide the desired ratio for any particular salt. For example, for the preparation of the ammonium salt of the free acid starting material (a particularly preferred embodiment herein), the starting material is treated with about 1 equivalent of a pharmaceutically acceptable base to give the neutral salt. Can be When the calcium salt is prepared, about 1/2 molar equivalent of base is used to obtain the neutral salt, whereas for ammonium salt, about 1/3 molar equivalent of base is used.

Ester derivatives are typically prepared as precursors to the acid form of the compound (illustrated in the examples below) and can therefore be used as prodrugs. Generally, these derivatives are lower alkyl esters such as acetate, propionate and the like. Amide _{derivatives, - (CO) NH 2,} - (CO) NHR, and - (CO) NR ₂ (where R is lower alkyl) is prepared by reaction of a carboxylic acid-containing compound with ammonia or a substituted amine (Illustrated in Example VII below).

Synthesis method:
The compounds of the present invention can be readily synthesized using techniques generally known to synthetic organic chemists. Experimental methods suitable for the preparation and derivatization of bridged bicyclic aromatic compounds are described, for example, in the above-cited Maignnan et al. Patent, the disclosure of which is incorporated herein by reference. ing. Methods for making certain and preferred compounds of the present invention are described in detail in Examples III-XVII below.

Usefulness and administration:
The compounds of the present invention (including their pharmaceutically acceptable esters, amides, or salts) as defined by structural formula (I) induce and / or modulate selective gene expression by receptors in the retinoic acid family. And controlling cell differentiation processes regulated by retinoids, vitamin D, and / or thyroid hormone. Thus, as described above, the compounds of the present invention are useful for acne, leukemia, psoriasis, skin aging, bone calcification, and energy levels, as well as retinoic acid, vitamin D, thyroid hormone, and 9-cis. -Useful for treating other indications related to cellular processes regulated by retinoic acid.

The compounds of the present invention may be conveniently formulated in a pharmaceutical composition composed of one or more compounds together with a pharmaceutically acceptable carrier. See, for example, Remington's Pharmaceutical Sciences , latest edition, E.C. W. Martin (Mack Publ. Co., Easton PA) discloses representative carriers and conventional methods of preparing pharmaceutical compositions that can be used in connection with preparing formulations of the compounds of the present invention. checking ...

  The compounds can be administered orally, parenterally (eg, intravenously), intramuscularly, intraperitoneally, topically, transdermally, and the like, but typically oral or topical administration is preferred. Of course, the amount of active compound administered will be dependent on the subject being treated, on the subject's weight, the manner of administration and the judgment of the prescribing physician. However, in general, dosages will approximate those typical for retinoic acid administration, and will preferably range from about 2 μg / kg / day to 2 mg / kg / day.

  Depending on the given mode of administration, the pharmaceutical composition may be in solid, semi-solid or liquid dosage form, such as tablets, suppositories, pills, capsules, powders, solutions, suspensions Agents, lotions, creams, gels and the like, preferably in unit dosage forms appropriate for the precise unit dosage. The composition comprises a selected effective amount of the drug in combination with a pharmaceutically acceptable carrier, as described above, and further comprises other medical agents, medicaments, carriers, adjuvants, diluents, and the like. May be included.

For solid compositions, conventional non-toxic solid carriers include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium carbonate, and the like. Liquid pharmaceutically administrable compositions can be provided, for example, by shaping the active compound as described herein and any pharmaceutical adjuvants, such as, for example, water, saline, aqueous dextrose, glycerol, ethanol and the like. It may be prepared by dissolving, dispersing, etc. in the agent, thereby forming a solution or suspension. If desired, the pharmaceutical composition to be administered can also include a wetting or emulsifying agent, a pH buffering agent and the like (eg, sodium acetate, sorbitan monolaurate, sodium triethanolamine acetate, triethanolamine oleate, etc.). It may contain small amounts of non-toxic adjuvants. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art; see, for example, Remington's Pharmaceutical Sciences , referenced above.

  For oral administration, the fine powder or granules may contain diluents, dispersants, and / or surfactants and may be in water or in a syrup, in a dry state in capsules or sachets, or in suspension. It may be present in a non-aqueous solution or suspension which may contain the agent, in a tablet which may contain a binder or lubricant, or in a suspension in water or syrup. If desired or necessary, flavoring, preserving, suspending, thickening, or emulsifying agents may be included. Tablets and granules are the preferred oral dosage forms, and they can be coated.

  Parenteral administration, if used, is generally characterized by injection. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsifiers. Recently reviewed approaches to parenteral administration include the use of sustained release or sustained release systems such that a constant level of dosage is maintained. See, for example, U.S. Patent No. 3,710,795. This is incorporated herein by reference.

(Experiment)
The following examples are submitted to provide those of ordinary skill in the art with a complete disclosure and description of how to make and evaluate the methods, compositions and compounds recited herein. The examples are not intended to limit the scope of the inventors' invention. Efforts have been made to ensure accuracy with respect to numbers (eg, amounts, temperature, etc.) but some errors and deviations must be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in ° C., and pressure is at or near atmospheric.

  The positions of the groups and substituents in the description employ the following numbering system throughout the examples:

実施例Ｉ
ホモダイマー形成の同定
９−シス−ＲＡは、ＴＲＥｐａｌ上でＲＸＲホモダイマー結合を誘導する
ＲＸＲを高濃度で用いる場合、ＲＸＲがＲＡ応答エレメント（ＲＡＲＥ）に結合することを示されているが^{２８，３０，３１}、さらに最近の研究により、ＲＸＲは溶液中でモノマーとして主に存在すること^１０、そして効果的なＤＮＡ相互作用に、ＲＡＲまたはＴＲまたはＶＤＲとのヘテロダイマー形成が必要であること^{１５，１６，２６−２９}が明らかになった。種々の応答エレメントへのヘテロダイマーの結合は、リガンド非依存性であることが見出された^３２。新たに発見された天然のＲＡ異性体９−シス−ＲＡは、ＲＡＲまたはＴＲのいずれも含んでいないことが知られているＤｒｏｓｏｐｈｉｌａ Ｓｃｈｎｅｉｄｅｒ細胞中のＲＸＲの効果的なアクティベーターであることが報告された^{１７，２４}。９−シス−ＲＡが、ＲＸＲに対する確かに真のリガンドである場合、このリガンドが、ＲＸＲ応答エレメント相互作用を調節すると予想される。従って、コレセプター（ＴＲおよびＲＡＲ）の非存在下および存在下で、パリンドローム性ＴＲＥ（ＴＲＥｐａｌ）、ＲＸＲ応答エレメント^１４へのＲＸＲαの結合に及ぼす９−シス−ＲＡの影響を研究した。

Example I
Identification of homodimer formation 9-cis-RA induces RXR homodimer binding on TREpal When RXR is used at high concentrations, it has been shown that RXR binds to the RA response element (RARE) ^{28,30, 31} , and more recent studies show that RXR is predominantly present as a monomer in solution ¹⁰ and that effective DNA interaction requires heterodimer formation with RAR or TR or VDR ^{15,16, 26-29} became apparent. Heterodimer binding to various response elements has been found to be ligand independent ³² . The newly discovered natural RA isomer 9-cis-RA was reported to be an effective activator of RXR in Drosophila Schneider cells known not to contain either RAR or TR ^{17, 24} . If 9-cis-RA is indeed the true ligand for RXR, this ligand is expected to modulate RXR response element interactions. Therefore, the effect of 9-cis-RA on the binding of RXRα to the palindromic TRE (TREpal), RXR response element ¹⁴ , in the absence and presence of coreceptors (TR and RAR) was studied.

Cloning of RXRα, RARβ, or TRα receptor cDNA into pBluescript (Stratagene, San Diego, Calif.) Has been previously described ²⁶ . Flag-RXRα encodes a double-stranded oligonucleotide containing an ATG codon and a flag (Arg-Tyr-Lys-Asp-Asp-Asp-Asp-Lys) [SEQ ID NO: 1] corresponding to the N-terminus of RXRα. It was constructed as described above ³³ by ligation with DNA sequence. The fusion product was then cloned into pBluescript. The synthesis of the receptor protein using an in vitro transcription / translation system, and the gel retardation assay ³⁴ using the synthesized receptor protein and double-stranded TREpal is as described ³³ . 9-cis-RA (mp 184-187 ° C.) was prepared from 9-cis-retinal by _two successive MnO ₂ oxidations ^{35 in} the presence of HOAc-MeOH to give the methyl ester (69%), followed by Hydrolyzed (80%) in 0.5N KOH in 25% aqueous MeOH and crystallized (MeOH); HPLC (Novapak _C18 , 32% MeOH, 27% MeCH, 16% I- PrOH, 24) _{% H 2 O, 1% HOAc} , 1.9mL / min, 260 _nM) t R 15.4 min (100%). To analyze the effect of the hormone, the receptor protein was incubated with the appropriate concentration of the hormone for 30 minutes at room temperature before performing the DNA binding assay. One microliter of antiserum was incubated with the receptor protein for an additional 30 minutes at room temperature before performing the DNA binding assay using an anti-flag antibody (Immunex, Seattle, WA).

In the absence of ligand, RXR alone did not effectively bind to TREpal and required TR or RAR for response element interaction, but RXR binding was dramatically reduced in the presence of 9-cis-RA. (Figure 1a) and did not require TR or RAR. The RXR-specific band observed in the presence of 9-cis-RA was as prominent as the band obtained with the heterodimer TR-RXR and the RAR / RXR complex. The RXR complex migrated to a position very similar to that of the RAR / RXR heterodimeric TREpal complex, slower than the TR-RXR complex. Thus, these data indicate that 9-cis-RA induces RXR homodimer formation. Because the RAR-RXR complex migrated at the same position as the 9-cis-RA-induced RXR homodimer complex, it could not be determined in this experiment if both complexes were formed. Notably, the total - also if a trans -RA was added at a concentration of 10 -6 ^M, but the RXRα homodimer binding was induced to some extent, T ₃ did not induce. The effect of RA could be observed using several freshly prepared RA solutions, however, this homodimer formation in the presence of RA indicates that the homodimer formation in the all-trans-RA solution used herein was 9-cis-. It is not clear at this time whether it is due to RA impurities or a direct effect of all-trans-RA (see below). Interestingly, 9-cis -RA, unlike its effect on RXR, but the RAR has been reported ²⁴ and capable of binding, 9-cis -RA is, induce RAR homodimer binding to TREpal Was not found to do so.

To further provide evidence that the complex observed in the presence of 9-cis-RA was indeed an RXR complex, DNA binding experiments were performed with Flag-RXR. This derivative retains an eight amino acid amino terminal epitope specifically recognized by anti-flag (αF) antibodies ³³ . As shown in FIG. 1b, αF shifted the 9-cis-RA-induced complex, but not the non-specific antibody. This demonstrated that the complex observed by TREpal in the presence of 9-cis-RA did indeed contain the RXR protein. To determine how 9-cis-RA-induced homodimer formation depends on the concentration of RXR protein, increasing concentrations of translated RXR protein in vitro were measured in the presence of 9-cis-RA or Mixed with labeled TREpal in the absence (FIG. 2a). When these mixtures were analyzed by a gel retardation assay, a strong cooperative effect was found on the DNA binding of the homodimers, which was positively dependent on the RXRa protein concentration (Fig. 2a, b). When high concentrations of RXR were used, slight binding of RXR could be observed, but at all receptor concentrations used, 9-cis-RA was required for effective complex formation. At all receptor concentrations used, the concentration of 9-cis-RA required for homodimer complex formation was further determined. The concentration of 9-cis-RA required for homodimer complex formation was further determined. Significant effects were already observed at 10 ⁻⁹ M, but optimal binding was seen at 10 ⁻⁸ M (FIG. 2c). Thus, effective RXR homodimer DNA interactions depend on RXR protein concentration and can occur at low levels of 9-cis-RA.

9-cis-RA induces homodimer interaction with specific response elements RXR-containing heterodimers have highly specific interactions with various natural response elements, and TR-RXR heterodimers It only binds strongly to TRE, not RARE. However, the opposite is true for RAR-RXR heterodimers ^15,32 . A number of natural and synthetic response elements were used to determine the sequence required for DNA binding of the 9-cis-RA induced RXR homodimer (FIG. 3a).

A gel retardation assay using an in vitro synthesized receptor protein is described in FIG. The following oligonucleotides and their complementary strands were used as probes in FIGS. ApoAI-RARE, a direct repeat response element ³¹ with a 2 bp spacer,

［配列番号：２］；ＣＲＢＰＩＩ−ＲＡＲＥ、１ｂｐスペーサーを有する直接反復ＲＸＲ特異的応答エレメント^３０、

[SEQ ID NO: 2]; a direct repeat RXR-specific response element ³⁰ with CRBPII-RARE, 1 bp spacer,

［配列番号：３］；βＲＡＲＥ、ＲＡＲβプロモーターに存在するＲＡ応答エレメントの直接反復^{３６，３７}、

[SEQ ID NO: 3]; βRARE, direct repeat ³⁶ , ^{37 of} RA response element present in RARβ promoter;

［配列番号：４］；ＣＲＢＰＩ−ＲＡＲＥ、ラットＣＲＢＰＩプロモーターに存在する直接反復ＲＡ特異的応答エレメント^３８、

[SEQ ID NO: 4]; CRBPI-RARE, a direct repeat RA-specific response element ³⁸ present in the rat CRBPI promoter,

［配列番号：５］；ＭＨＣ−ＴＲＥ、ラットα−ミオシンＨ鎖遺伝子に存在する、直接反復Ｔ_３特異的応答エレメント^４０、

[SEQ ID NO: 5]; MHC-TRE, present in rat α- myosin H chain gene, direct repeat _{T 3} specific response elements ^40,

［配列番号：６］；ＭＥ−ＴＲＥ、ラットリンゴ酸酵素遺伝子に存在する直接反復Ｔ_３特異的応答エレメント^４１、

[SEQ ID NO: 6]; ME-TRE, direct repeat _{T 3} specific response elements ⁴¹ present in the rat malic enzyme gene,

［配列番号：７］；ＤＲ−４、４ｂｐのスペーサーを有する理想化した直接反復Ｔ_３特異的応答エレメント^３９、

[SEQ ID NO: 7]; idealized direct repeat with a spacer DR-4,4bp _{T 3} specific response elements ^39,

［配列番号：８］；ＤＲ−５、５ｂｐスペーサーを有する理想化した直接反復ＲＡ特異的応答エレメント^３９、

[SEQ ID NO: 8]; an idealized direct repeat RA-specific response element ³⁹ with DR-5, 5 bp spacer

［配列番号：９］；ＥＲＥ、完全なパリンドローム性のＥＲ応答エレメント^４２、

[SEQ ID NO: 9]; ERE, complete palindromic ER response element ⁴² ,

［配列番号：１０］。ＴＲＥｐａｌ配列［配列番号：１１］を、比較のために示す。

[SEQ ID NO: 10]. The TREpal sequence [SEQ ID NO: 11] is shown for comparison.

ApoAI-RARE, a direct repeat response element containing a 2 bp spacer, has been suggested ³¹ to be RXR-specific, but was obtained as a strong RXR complex in the presence of 9-cis-RA and RA (10 ^-6 M) was obtained as a lower degree complex. The RXR-RAR heterodimer also effectively bound this response element. Since the heterodimer complex migrated at the same position as the RXR homodimer, the effect of the 9-cis-RA RXR-RAR heterodimer cannot be clearly determined. RAR homodimer binding was not induced by 9-cis-RA (FIG. 3b). When studying another RXR response element ³⁰ , 9-cis-RA, which induces RXR binding to CRBPII-RARE, we observed a complex that migrated more slowly (in the absence of ligand) than the heterodimer, In the presence of 9-cis-RA and RAR, both homodimeric and heterodimeric binding showed a decrease in their strength (FIG. 3); similar effects were observed at high RA concentrations or in 9-cis -Seen when both RA and RA are present. 9-cis -RA also induced RXR of BetaRARE, the interaction of (RAR response elements ^{36 and 37} from the human RARβ promoter containing spacer 5 bp) (FIG. 4a). However, the RXR homodimer band is significantly weaker than the RAR-RXR heterodimer band at the protein concentration used. Interestingly, other native RARE ³⁸ from the rat CRBPI promoter (similar to ApoAI-RARE containing a 2 bp spacer) did not show any binding of RXR in the presence of 9-cis-RA ( Figure 4a). This indicates that the actual sequence of the repeat core motif is important for RXR homodimer binding. Similarly, DR-5-RARE, a complete repetitive element ³⁹ from β-RARE, did not show any interaction with RXR in the presence of 9-cis-RA, but when RARE was present, RXR Strongly interact with (FIG. 4a).

To RXR homodimers bind to determine whether specific for a particular RARE, rat α- myosin H chain promoter (MHC-TRE) derived of _{T 3} response elements ^40, rat malic enzyme (ME-TRE) _{T 3} response element ⁴¹ from, and the full repeat DR-4 derived of _{T 3} response elements ³⁹ also were performed gel shift experiments using. In all three cases, no specific binding of RXR could be observed in the presence or absence of 9-cis-RA, but all three response elements were effective with the TR / RXR heterodimer. And these elements are consistent with the notion that they are not induced by retinoids. Similarly, intact palindromic ERE ⁴² also did not interact with the RXR homodimer (FIG. 4c).
RXR, which results in 9-cis-RA-induced homodimer formation in the absence of DNA, was reported to be present primarily as a monomer in solution ¹⁶ . An important question is whether 9-cis-RA-induced RXR homodimers (similar to those containing heterodimers) can form in solution in the absence of DNA. To solve this problem, the advantage of the Flag-RXR derivative, that is, the ability to specifically precipitate with the anti-flag antibody, but not the RXR wild type, was used.

Flag-RXRα was cloned in reading frame into the expression vector pGex 2T (Pharmacia) and expressed in bacteria using the procedures provided by the manufacturer. The protein was partially purified on a pre-packed Glutathione Sepharose 4B column (Pharmacia) and tested for its function by gel retardation assay using anti-flag antibody and Western blotting. The co-immunoprecipitation assay was performed essentially as described ²⁶ . Briefly, 10 μl of ³⁵ S-labeled synthetic RXRα protein in vitro was combined with 5 μl (approximately 0.1 μg) of a Flag-RXRα fusion protein expressed in partially purified bacteria or a similarly prepared glutathione transferase control protein; Incubated for 30 minutes at room temperature in 100 μl buffer (containing 10 ⁻⁷ M 9-cis-RA, 50 mM KCl, and 10% glycerol). When assayed in the presence of a chemical crosslinker or oligonucleotide, add 2 μl of 100 mM dithiobissuccinimidyl propionate (DSP) dissolved in DMSO, or 10 ng of oligonucleotide and incubate for 15 minutes at room temperature Continued. The reaction mixture was then incubated with 1 μl of anti-flag antibody or non-specific pre-immune serum for 2 hours on ice. The immune complexes were precipitated by adding 50 μl of protein-A-Sepharose slurry and mixing continuously for 1 hour in a cold room. The immune complex was thoroughly washed with cold NET-N buffer containing ^10-7 M9-cis-RA (20 mM Tris, pH 8.0, 100 mM NaCl, 1 mM DTT, 0.5% NP-40), and SDS Boiled in sample buffer and analyzed by SDS-polyacrylamide gel electrophoresis. Gels were fixed, dried and visualized by autoradiography.

A flag -RXR, when mixed with labeled ^{35 S-RXR} protein in vitro, labeled RXR has been obtained by precipitation in the presence of anti-Flag antibody, it could not be precipitated in the presence of non-specific serum ( (Fig. 5). In the presence of ApoAI-RARE, the coprecipitation efficiency increased slightly, but not in the presence of MHC-TRE. In addition, incubation with a crosslinker (DSP) further enhanced co-precipitation of labeled RXR. In all cases, specific co-precipitation was only observed in the presence of 9-cis-RA. Thus, these data strongly support the hypothesis that 9-cis-RA-induced RXR homodimer formation occurs in solution and does not require RXR-DNA interactions.
Response element-specific transcriptional activation by 9-cis-RA and RXRα 9-cis-RA / RXRα homodimer response element interaction is associated with transcriptional activation by such response elements by 9-cis-RA / RXR complex To study whether this could be done, a series of transient transfection assays were performed on CV-1 cells. Here, the receptor expression vector was co-transfected with a CAT reporter construct having various upstream response elements of the tk promoter. CV-1 cells were transiently transfected using a calcium phosphate precipitation procedure modified as described ⁴³ above. CAT activity was normalized for transfection efficiency by measuring enzyme activity from the co-transfected β-galactosidase expression plasmid (pCH110, Pharmacia). Transfected cells were grown in the absence or presence of ^10-7 M 9-cis-RA or all-trans-RA.

Using the TREpal-containing reporter, strong activation by RXRa in the presence of 9-cis-RA and little activation was observed when RA was added. Activation could be further enhanced by co-transfection of RARα. However, in this case, RA also served as an effective activator, although not as efficiently as 9-cis-RA. Overall, in the presence of RARα and RXRα, activation by 9-cis-RA is about twice as strong as that seen with RXRα alone, and when both receptors are present, binding is 9-cis-RA. Consistent with the DNA binding data observed by both heterodimers and homodimers in the presence of RA (FIG. 6a). When βRARE was tested (FIG. 6b), this response element was observed to be highly activated by endogenous CV-1 cell receptors, consistent with previous observations ^37,42 , but at lower concentrations. No further activation by the co-transfected receptor could be observed. However, 9-cis -RA is Interestingly, in ^{10 -7} M, was a strong activator from RA. Thus, these data indicate that CV-1 cells contain endogenous retinoid receptor activity, which is particularly active for βRARE and responsive to 9-cis-RA.

The ApoAI element-containing reporter was also very effectively activated by RXRa in the presence of 9-cis-RA (Figure 6c), but did not induce RA above the levels obtained in the absence of RXRa. Was. Like TREpal, maximal activation was seen when both receptors RXRα and RARα were co-transfected. Under these conditions, RA also led to strong activation. In contrast, the CRBPI element (where no DNA binding by RXR was observed in the presence of 9-cis-RA) was also activated by RXR and 9-cis-RA in transient transfection studies. Although not (FIG. 6d), RARα alone led to significant activation, which was largely 9-cis-RA dependent. The heterodimer RARaRXRa allowed maximal activation in the presence of 9-cis-RA. Although not unexpected, induction by RXRa and 9-cis-RA was not observed for MHC-TRE, ME-TRE, or ERE. These in vivo analyzes correlated significantly with the results obtained in in vitro DNA binding studies. There, strong activation by RXRa in the presence of 9-cis-RA is only observed for response elements that interact strongly with 9-cis-RA-induced RXRa homodimers.
9 Shisurechirein acid in the presence of a thyroid hormone to inhibit activation by TR / RXR heterodimer _{(T 3),} genes report by CAT activated by thyroid hormone receptor / RXR heterodimers, 9- It can be inhibited by adding cis-RA. This type of inhibition is most easily measured using a transfection assay.

Example II
Identification and Selection of Compounds that Induce RXR Activity The TREpal-tk-reporter gene was used in a transient transfection assay essentially as described ⁵¹ to evaluate compounds for induction of RXR activity. Briefly, CV-1 cells or Hep G2 cells were grown in DME medium supplemented with 10% fetal calf serum. Cells were plated at 1.0 × 10 ⁵ per well in 24-well plates for 16-24 hours prior to transfection. Generally, 100 ng of reporter plasmid, 150 ng of β-galactosidase expression vector (pCH110, Pharmacia), and various amounts of receptor expression vector were mixed with carrier DNA (pBluescript) to give 1,000 ng total DNA per well. . Chloramphenicol acetyltransferase (CAT) activity was normalized for transfection efficiency by the corresponding β-galactosidase activity as previously described ⁵² . As shown in Example I, TREpal represents a response element that is activated by both RAR / RXR heterodimers and RXR homodimers. When the RXR expression vector is co-transfected with TREpal-tk-reporter gene into CV-1 cells, all-trans-RA does not effectively activate the reporter, but 9-cis-RA does. Evaluation of a series of retinoids indicated that some exhibited activity with RXR. The pharmacophoric elements of these constructs were then combined and further modified to produce a subset of retinoids whose activation profile for RXR was similar to that of 9-cis-RA. The induction curves for some retinoid activities with RXR are shown in FIG. 7a. Interestingly, none of the active compounds showed activity at 10 ⁻⁸ M, but at 10 ⁻⁷ M, all showed activity similar to 9-cis-RA. Next, a reporter gene having RARE ³⁰ of cytoplasmic retinol binding protein II (CRBPII) was used. It is a response element that is activated only by RXR homodimers but not by RAR / RXR heterodimers. The induction profile obtained with this response element resembled the TREpal responder (FIG. 7b). Thus, the synthetic retinoids SR11203, SR11217, SR11234, SR11235, SR11236, and SR11237 appeared to be effective activators of RXRα. The structure of the compound is as follows:

この一連のレチノイドについての活性ランキングは、ＴＲＥｐａｌおよびＣＲＢＰＩＩリポーター遺伝子の両者について同じであった。ケタールＳＲ１１２３７が最も活性であり、続いてイソプロピリデニルレチノイドＳＲ１１２１７、ヘミチオケタールＳＲ１１２３５およびチオケタールＳＲ１１２３４であった。ジチアンＳＲ１１２０３およびジオキサンＳＲ１１２３６は最も低い活性を有した。構造分析により、これらのレチノイドは、９−シス−ＲＡの親油性の頭部およびカルボキシル末端の空間的配向と類似の空間的配向を有し、しかも、活性がテトラヒドロナフタレンおよびフェニル環系に結合している置換基（ＣＲＲ^１）の長さおよび体積に関係し得ることが示された。

The activity ranking for this series of retinoids was the same for both TREpal and CRBPII reporter genes. Ketal SR11237 was the most active, followed by isopropylidenyl retinoid SR11217, hemithioketal SR11235 and thioketal SR11234. Dithiane SR11203 and dioxane SR11236 had the lowest activities. Structural analysis shows that these retinoids have a spatial orientation similar to that of the lipophilic head and carboxyl terminus of 9-cis-RA, while having activity bound to the tetrahydronaphthalene and phenyl ring systems. It has been shown that this can be related to the length and volume of the substituent (CRR ¹ ).

Example I showed that 9-cis-RA specifically activates RXRα by inducing RXRα homodimer formation, so that a gel retardation assay was used to retinoid-induced RXR homodimer binding to TREpal. Studied. Briefly, gel retardation assays were performed essentially as described ³³ above. Receptor protein translated in vitro (1-5 ml depending on translation efficiency) was mixed with a ³² P-labeled oligonucleotide in a 20 ml reaction mixture (10 mM Hepes buffer, pH 7.9, 50 mM KCl, 1 mM DTT, 2.5 mM Incubated in MgCl, 10% glycerol, and 1 mg poly (dI-dC) for 20 minutes at 25 ° C. The reaction mixture was then loaded on a 5% non-denaturing polyacrylamide gel containing 0.5 × TBE (1 × TBE = 0.089 M tris borate, 0.089 M boric acid, and 0.002 M EDTA).

  In the absence of 9-cis-RA, RXR did not bind to this response element. Retinoids SR11217 and SR11237 induced RXR homodimer binding to the response element in a concentration dependent manner. Retinoid 11203, which acted as a weak activator in the transient transfection assay, also induced only weak RXR binding. SR11231, which did not activate RXR homodimers, also failed to induce RXR homodimer binding. Similar results were obtained with CRBPII-RARE and ApoAI-RARE. Accordingly, the class of synthetic retinoids that activate RXRα is defined herein by homodimer formation and binding to DNA.

The key question was whether these RXR activating compounds like 9-cis-RA could also activate RAR / RXR heterodimers or whether they could be truly RXR selective . To analyze this problem, four different reporter constructs were used that retained any of the following: i) the rat cytoplasmic retinol binding protein I (CRBPI) gene RARE ³⁸ , which was the only RAR / RXR heterodimer coupled is activated; ii) RAR [beta] 2 gene promoter ^{RARE 36, 37,} this is the most efficient binding by heterodimers are activated also to some degree by RXR homodimers; iii) CRBPII-RARE, It is activated only by the RXR homodimer, and RAR suppresses RXR activity ³⁰ ; iv) the apolipoprotein AI (apoAI) gene RARE ³¹ , which is due to the RAR / RXR heterodimer and to the RXR homodimer. Bound and activated. Four different reporter constructs, RARα, RARβ, were cotransfected together with or RXR [alpha and RARα, RXRα ^51. Retinoids were analyzed at a concentration of 5 × 10 ⁻⁷ (almost complete induction was obtained at the indicated dose (FIG. 7)).

CV-1 cells were co-transfected with 100 ng of the reporter plasmids a) CRBPI-tk-CAT, b) βRARE-tk-CAT, c) CRBPII-tk-CAT, and d) apoAI-tk-CAT. Retinoids were applied at 5 × 10 ⁻⁷ M. The results of a representative experiment are shown.

  RXR-specific retinoids act significantly differently than 9-cis-RA (or RA) in that they only activate RXR homodimers but do not activate RAR / RXR heterodimers. Like 9-cis-RA, both SR11217 and SR11237 are strong activators of CRBPII-RARE (ie, RARE that is significantly activated only by RXR homodimers). However, compared to 9-cis-RA, they did not induce CRBPI-RARE, which is activated only by RAR / RXR heterodimers. Thus, SR11217 and SR11237 act very similar to CRBPII-RARE to 9-cis-RA, but they show no response for CRBPI-RARE, where 9-cis-RA is optimally Activator. βRARE was slightly activated by SR11217 and SR11237, consistent with the relatively low affinity of the RXR homodimer for this response element. apoAI-RARE was most effectively activated by the RAR / RXR heterodimer in the presence of 9-cis-RA. In addition to the activity found in CV-1 cells, marked and RXR-specific activation by retinoids SR11217 and SR11237 was also observed in various other cell lines, including Hep G2 cells. Here, a particularly high response was observed. RARα and RARβ, when only co-transfected, were not significantly activated by any of the synthetic retinoids for any of the response elements tested. Similar negative results were obtained for RARγ. RARα and β are hypothesized to form heterodimers with endogenous RXR-like proteins in CV-1 cells. Therefore, these heterodimers are also non-responsive to synthetic retinoids.

  These data indicate that a new class of retinoids has been identified. These retinoids specifically induce RXR homodimer formation and specifically activate RXR homodimers for response elements but do not activate RAR / RXR heterodimers. These retinoids allow specific activation of the RXR-selective response pathway but do not induce an RAR-dependent response pathway. These retinoids provide a more limited physiological response than currently used RA isomers or other retinoids.

Example III

（ａ．）メチル４−［（５，６，７，８−テトラヒドロ−５，５，８，８−テトラメチル−２−ナフタレニル）カルボニル］ベンゾエート（３）：
１．５ｍＬの１，２−ジクロロメタン中の塩化アルミニウム（１．１３ｇ、８．５ｍｍｏｌ）の懸濁液に、０℃で、アルゴン雰囲気下にて、６ｍＬの１，２−ジクロロエタン中の１，２，３，４−テトラヒドロ−１，１，４，４−テトラメチルナフタレン１（１．４５ｇ、７．７ｍｍｏｌ）（Ｋａｇｅｃｈｉｋａ，Ｈ．ら、Ｊ．Ｍｅｄ．Ｃｈｅｍ．３１：２１８２（１９８８））および４−カルボメトキシベンゾイルクロライド２（１．５６ｇ、７．９ｍｍｏｌ）の溶液を添加した（４−カルボメトキシベンゾイルクロライド２は、Ａｌｄｒｉｃｈから容易に入手可能なモノメチルテレフタレートから、１工程（ＳＯＣｌ_２、ＤＭＦ）により得た）。得られた溶液を室温まで昇温し、その後１６時間撹拌した。反応混合物を、氷水上に注ぎ、４０％酢酸エチル／ヘキサンで抽出した。合わせた有機層を、飽和ＮａＨＣＯ_３水溶液および塩水で洗浄した。溶液を、無水ＭｇＳＯ_４で乾燥し、濾過し、そして濃縮して橙色の固体（４．５ｇ）を得た。フラッシュクロマトグラフィー（５０％ジクロロメタン／ヘキサン）により、所望の生成物３を、淡黄色の固体（２．０７ｇ）として得た。ジクロロメタン／ヘキサンからの再結晶により、所望の生成物３を、白色の結晶性固体として得た（１．９６ｇ、５０％）：融点１４６〜１４８℃；Ｒ_ｆ ０．１４（５０％ＣＨ_２Ｃｌ_２／ヘキサン）。生成物の構造もまた、ＩＲ、^１Ｈ ＮＭＲおよび質量分析を用いて確認した。

(A.) Methyl 4-[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl) carbonyl] benzoate ( 3 ):
To a suspension of aluminum chloride (1.13 g, 8.5 mmol) in 1.5 mL of 1,2-dichloromethane at 0 ° C. under an atmosphere of argon was added 1,2-dichloroethane in 6 mL of 1,2-dichloroethane. , 3,4-tetrahydro-1,1,4,4-tetramethylnaphthalene 1 (1.45 g, 7.7 mmol) (Kagechika, H. et al., J. Med . Chem . 31: 2182 (1988)) and 4 A solution of -carbomethoxybenzoyl chloride 2 (1.56 g, 7.9 mmol) was added (4-carbomethoxybenzoyl chloride 2 was obtained from monomethyl terephthalate readily available from Aldrich by one step (SOCl ₂ , DMF) Obtained). The resulting solution was warmed to room temperature and then stirred for 16 hours. The reaction mixture was poured over ice water and extracted with 40% ethyl acetate / hexane. The combined organic layers were washed with saturated aqueous NaHCO ₃ and brine. The solution was dried over anhydrous MgSO _4, filtered, and concentrated to give an orange solid (4.5 g). Flash chromatography (50% dichloromethane / hexane) provided the desired product 3 as a pale yellow solid (2.07 g). Recrystallization from dichloromethane / hexane provided the desired product 3 as a white crystalline solid (1.96 g, 50%): mp 146-148 ° C .; R _f 0.14 (50% CH ₂ Cl) ₂ / hexane). The structure of the product was also confirmed using IR, ¹ H NMR and mass spectroscopy.

(B.) [2- (5,6,7,8-tetrahydro-5,5,8,8-tetramethylnaphthalen-2-yl) -2- (4-carbomethoxyphenyl)]-1,3- Zhitian ( 4 ):
To a solution of keto-ester 3 (97 mg, 0.277 mmol) in 3 mL of chloroform at 0 ° C. under an argon atmosphere, a solution of 1,3-propanediol (33 μL, 36 mg, 0.332 mmol) followed by Boron trifluoride etherate (17 μL, 0.140 mmol) was added. The resulting mixture was stirred at 0 ° C. for 1 hour, then warmed to room temperature overnight. The reaction mixture was quenched by pouring into saturated aqueous Na ₂ CO ₃ and extracted with dichloromethane. The combined organic layers were dried over anhydrous MgSO _4, filtered, and concentrated to give a yellow solid (0.108 g). Recrystallization from ethyl acetate / hexane gave the desired dithiane 4 as a white crystalline solid (0.087 g, 71%): mp 195-197 ° C .; R _f 0.32 (50% CH ₂ Cl _{2). 2} / hexane). The structure of the product was also confirmed using IR, ¹ H NMR and mass spectroscopy.

(C.) [2- (5,6,7,8-tetrahydro-5,5,8,8-tetramethylnaphthalen-2-yl) -2- (4-carboxyphenyl)]-1,3-dithiane ( 5 ):
To a suspension of ester 4 (85 mg, 0.193 mmol) in a 75% aqueous methanol solution (2 mL), add 0.024 g of potassium hydroxide and allow the mixture at 50 ° C. for 2 hours until the material dissolves Stirred. The solution was cooled to room temperature, acidified with 1N aqueous hydrochloric acid, and then extracted with 80% ethyl acetate / hexane. The combined organic layers were dried over anhydrous MgSO _4, filtered, and concentrated to afford a white solid. Recrystallization from benzene / hexane gave the desired acid 5 as a white powder (0.076 g, 92%): mp 229-231 ° C. The structure of the product was also confirmed using IR, ¹ H NMR and mass spectroscopy.

Example IV

（ａ．）［２−（５，６，７，８−テトラヒドロ−５，５，８，８−テトラメチル−２−ナフタレニル）−２−（４−カルボメトキシフェニル）］−１，３−ジチオラン（６）：
ジクロロメタン（２ｍＬ）中のケト−エステル３（８０ｍｇ、０．２２８ｍｍｏｌ）の溶液に、０℃で、アルゴン雰囲気下にて、ジクロロメタン（０．５ｍＬ）中のエタンジチオール（２６ｍｇ、０．２７ｍｍｏｌ）、続いて三フッ化ホウ素エーテル錯体（０．０４ｍＬ、０．３ｍｍｏｌ）を添加した。得られた混合物を、０℃で１時間撹拌し、次いで一晩室温まで温めた。反応混合物を、飽和Ｎａ_２ＣＯ_３水溶液中に注ぐことによりクエンチし、そして４０％酢酸エチル／ヘキサンで抽出した。合わせた有機層を、無水ＭｇＳＯ_４で乾燥し、濾過し、そして濃縮して固体を得た。フラッシュクロマトグラフィー（３０；４０％ＣＨ_２Ｃｌ_２／ヘキサン）により、所望のジチアン６を白色固体として得た（０．０８８ｇ、９０％）：融点１０５〜１０７℃；Ｒ_ｆ ０．３３（５０％ＣＨ_２Ｃｌ_２／ヘキサン）。生成物の構造もまた、ＩＲ、^１Ｈ ＮＭＲおよび質量分析を用いて確認した。

(A.) [2- (5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl) -2- (4-carbomethoxyphenyl)]-1,3-dithiolane ( 6 ):
To a solution of keto-ester 3 (80 mg, 0.228 mmol) in dichloromethane (2 mL) at 0 ° C. under an atmosphere of argon, ethanedithiol (26 mg, 0.27 mmol) in dichloromethane (0.5 mL) followed by Then boron trifluoride etherate (0.04 mL, 0.3 mmol) was added. The resulting mixture was stirred at 0 ° C. for 1 hour, then warmed to room temperature overnight. The reaction mixture was quenched by pouring into saturated aqueous Na ₂ CO ₃ and extracted with 40% ethyl acetate / hexane. The combined organic layers were dried over anhydrous MgSO _4, filtered, and concentrated to give a solid. Flash chromatography (30; 40% CH ₂ Cl ₂ / hexane) provided the desired dithiane 6 as a white solid (0.088 g, 90%): mp 105-107 ° C .; R _f 0.33 (50% CH ₂ Cl ₂ / hexane). The structure of the product was also confirmed using IR, ¹ H NMR and mass spectroscopy.

(B.) [2- (5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl) -2- (4-carboxyphenyl)]-1,3-dithiolane ( 7 ):
To a suspension of ester 6 (85 mg, 0.199 mmol) in a 75% aqueous methanol solution (3 mL), add one drop of potassium hydroxide (0.11 g) and dissolve the mixture at 70 ° C. And stirred for 1 hour. The solution was cooled to room temperature, acidified with 1N aqueous hydrochloric acid, and then extracted with 80% ethyl acetate / hexane. The combined organic layers were dried over anhydrous MgSO _4, filtered, and concentrated to afford a white solid. Recrystallization from dichloromethane-hexane gave the desired acid 7 as a white powder (0.064 g, 79%): mp 218-221 ° C. The structure of the product was also confirmed using IR, ¹ H NMR and mass spectroscopy.

Example V

（ａ．）［２−（５，６，７，８−テトラヒドロ−５，５，８，８−テトラメチル−２−ナフタレニル）−２−（４−カルボメトキシフェニル）］−１，３−ジオキソラン（８）：
１ｍＬのベンゼン中のケト−エステル３（８０ｍｇ、０．２２８）の溶液に、エチレングリコール（１ｍＬ）、１，２−ビス（トリメチルシリルオキシ）エタン（２ｍＬ）、および触媒量のｐ−ＴｓＯＨを添加した。反応混合物を４時間還流加熱し、次いで室温まで冷却した。溶液を飽和ＮａＨＣＯ_３水溶液中に注ぎ、そして４０％酢酸エチル／ヘキサンで抽出した。合わせた有機層を、無水ＭｇＳＯ_４で乾燥し、濾過し、そして濃縮して固体を得た。フラッシュクロマトグラフィー（５０％ＣＨ_２Ｃｌ_２／ヘキサン）により、所望のケタール８を白色固体として得た（０．０８２ｇ、９１％）：融点１４５〜１４７℃；Ｒ_ｆ ０．１６（５０％ＣＨ_２Ｃｌ_２／ヘキサン）。生成物の構造もまた、ＩＲ、^１Ｈ ＮＭＲおよび質量分析を用いて確認した。

(A.) [2- (5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl) -2- (4-carbomethoxyphenyl)]-1,3-dioxolane ( 8 ):
To a solution of keto-ester 3 (80 mg, 0.228) in 1 mL of benzene was added ethylene glycol (1 mL), 1,2-bis (trimethylsilyloxy) ethane (2 mL), and a catalytic amount of p- TsOH. . The reaction mixture was heated at reflux for 4 hours and then cooled to room temperature. The solution was poured into saturated aqueous NaHCO ₃ and extracted with 40% ethyl acetate / hexane. The combined organic layers were dried over anhydrous MgSO _4, filtered, and concentrated to give a solid. Flash chromatography (50% CH ₂ Cl ₂ / hexane) provided the desired ketal 8 as a white solid (0.082 g, 91%): mp 145-147 ° C .; R _f 0.16 (50% CH ₂ Cl ₂ / hexane). The structure of the product was also confirmed using IR, ¹ H NMR and mass spectroscopy.

(B.) [2- (5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl) -2- (4-carboxyphenyl)]-1,3-dioxolanammonium Salt ( 10 ):
To a suspension of ester 8 (50 mg, 0.127 mmol) in a 75% aqueous methanol solution (2 mL), add one drop of potassium hydroxide (0.1 g) and dissolve the reaction mixture at 70 ° C. The mixture was stirred for 1 hour. The solution was cooled to room temperature, acidified with 1N aqueous hydrochloric acid, and then extracted with 80% ethyl acetate / hexane. The combined organic layers were dried over anhydrous MgSO _4, filtered and to give a white solid 9 and concentrated. This acid 9 was dissolved in dichloromethane (4 mL) under an argon atmosphere and the ammonia gas was condensed into the solution, which was stirred for 5 minutes at -33 <0> C. The solution was warmed to room temperature for 20 minutes, the ammonia was evaporated and concentrated to give the ammonium salt 10 as a white powder (47 mg, 93%): mp 259-261 ° C. The structure of the product was also confirmed using IR, ¹ H NMR and mass spectroscopy.

Example VI

（ａ．）［２−（５，６，７，８−テトラヒドロ−５，５，８，８−テトラメチル−２−ナフタレニル）−２−（４−カルボメトキシフェニル）］−１，３−オキサチオラン（１１）：
２ｍＬのベンゼン中のケト−エステル３（８８ｍｇ、０．２５１）の溶液に、２−メルカプトエタノール（１ｍＬ）、および触媒量のｐ−ＴｓＯＨを添加した。反応混合物を一晩還流加熱し、次いで室温まで冷却した。溶液を、飽和ＮａＨＣＯ_３水溶液中に注ぎ、そして４０％酢酸エチル／ヘキサンで抽出した。合わせた有機層を、無水ＭｇＳＯ_４で乾燥し、濾過し、そして濃縮して固体を得た。フラッシュクロマトグラフィー（５０％ＣＨ_２Ｃｌ_２／ヘキサン）により、所望のケタール１１を白色固体として得た（０．０９ｇ、８７％）：融点１２２〜１２４℃；Ｒ_ｆ ０．２４（５０％ＣＨ_２Ｃｌ_２／ヘキサン）。生成物の構造もまた、ＩＲ、^１Ｈ ＮＭＲおよび質量分析を用いて確認した。

(A.) [2- (5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl) -2- (4-carbomethoxyphenyl)]-1,3-oxathiolane ( 11 ):
To a solution of keto-ester 3 (88 mg, 0.251) in 2 mL of benzene was added 2-mercaptoethanol (1 mL) and a catalytic amount of p- TsOH. The reaction mixture was heated at reflux overnight and then cooled to room temperature. The solution was poured into saturated aqueous NaHCO ₃ and extracted with 40% ethyl acetate / hexane. The combined organic layers were dried over anhydrous MgSO _4, filtered, and concentrated to give a solid. Flash chromatography (50% CH ₂ Cl ₂ / hexane) provided the desired ketal 11 as a white solid (0.09 g, 87%): mp 122-124 ° C .; R _f 0.24 (50% CH ₂ Cl ₂ / hexane). The structure of the product was also confirmed using IR, ¹ H NMR and mass spectroscopy.

(B.) [2- (5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl) -2- (4-carboxyphenyl)]-1,3-oxathiolane ( 12 ):
To a suspension of ester 11 (64 mg, 0.156 mmol) in a 75% aqueous methanol solution (3 mL), add 1 potassium hydroxide (0.12 g) and dissolve the reaction mixture at 75 ° C. The mixture was stirred for 1 hour. The solution was cooled to room temperature, acidified with 1N aqueous hydrochloric acid, and then extracted with 80% ethyl acetate / hexane. The combined organic layers were dried over anhydrous MgSO _4, filtered, and concentrated to afford a white solid. Recrystallization from dichloromethane-hexane gave the desired acid 12 as a white powder (0.06 g, 97%): mp 216-217.5 ° C. The structure of the product was also confirmed using IR, ¹ H NMR and mass spectroscopy.

Example VII

（ａ．）［２−（５，６，７，８−テトラヒドロ−５，５，８，８−テトラメチル−２−ナフタレニル）−２−（４−カルボメトキシフェニル）］−１，３−ジオキサン（１３）：
５ｍＬのベンゼン中のケト−エステル３（１５０ｍｇ、０．４２８ｍｍｏｌ）の溶液に、１，３−プロパンジオール（１．５ｍＬ）、および触媒量のｐ−ＴｓＯＨを添加した。反応混合物を、一晩還流加熱し、次いで室温まで冷却した。溶液を飽和ＮａＨＣＯ_３水溶液中に注ぎ、そして４０％酢酸エチル／ヘキサンで抽出した。合わせた有機層を、無水ＭｇＳＯ_４で乾燥し、濾過し、そして濃縮して固体を得た。フラッシュクロマトグラフィー（５０％ＣＨ_２Ｃｌ_２／ヘキサン）により、所望のケタール１３を白色固体として得た（０．１６４ｇ、９４％）：融点１５７〜１５９℃；Ｒ_ｆ ０．２４（５％酢酸エチル／ヘキサン）。生成物の構造もまた、ＩＲ、^１Ｈ ＮＭＲおよび質量分析を用いて確認した。

(A.) [2- (5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl) -2- (4-carbomethoxyphenyl)]-1,3-dioxane ( 13 ):
To a solution of keto-ester 3 (150 mg, 0.428 mmol) in 5 mL of benzene was added 1,3-propanediol (1.5 mL) and a catalytic amount of p- TsOH. The reaction mixture was heated at reflux overnight and then cooled to room temperature. The solution was poured into saturated aqueous NaHCO ₃ and extracted with 40% ethyl acetate / hexane. The combined organic layers were dried over anhydrous MgSO _4, filtered, and concentrated to give a solid. Flash chromatography (50% _{CH 2} Cl 2 / hexane), the desired ketal 13 as a white solid (0.164 g, 94%): mp _{157~159 ℃; R f 0.24 (5} % ethyl acetate / Hexane). The structure of the product was also confirmed using IR, ¹ H NMR and mass spectroscopy.

(B.) [2- (5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl) -2- (4-carboxyphenyl)]-1,3-dioxan ammonium Salt ( 15 ):
To a suspension of ester 13 (0.1 g, 0.245 mmol) in a 75% aqueous methanol solution (3 mL) was added one drop of potassium hydroxide (0.12 g). The reaction mixture was stirred at 80 ° C. for 30 minutes until the material dissolved. The solution was cooled to room temperature, acidified with 1N aqueous hydrochloric acid, and then extracted with 80% ethyl acetate / hexane. The combined organic layers were dried over anhydrous MgSO _4, filtered and to give a white solid 14 and concentrated. Acid 14 was dissolved in dichloromethane (4 mL) under an argon atmosphere. Ammonia gas was condensed into the flask and the mixture was stirred for 5 minutes at -33C. The solution was warmed to room temperature for 20 minutes, the ammonia was evaporated and concentrated to give the ammonium salt 15 as a white powder (0.238 g, 97%): mp 228-230 ° C. The structure of the product was also confirmed using IR, ¹ H NMR and mass spectroscopy.

Example VIII

（ａ．）メチル４−［１−（５，６，７，８−テトラヒドロ−５，５，８，８−テトラメチル−２−ナフタレニル）−１−エテニル］ベンゾエート（１６）：
５ｍＬのベンゼン中のメチルトリフェニルホスホニウムブロマイド（０．７８ｇ、２．１８ｍｍｏｌ）の懸濁液に、室温でアルゴン雰囲気下にて、ヘキサメチルジシラジドカリウムの０．５Ｍトルエン溶液（４．４ｍＬ、２．２ｍｍｏｌ）を添加し、そして黄色の溶液を５分間撹拌した。７ｍＬのベンゼン中のケトエステル３（０．５１ｇ、１．４５５ｍｍｏｌ）の溶液を添加し、そして橙色の溶液を、１時間室温で撹拌した。反応混合物を、飽和ＮａＨＣＯ_３水溶液中に注ぎ、そして４０％酢酸エチル／ヘキサンで抽出した。合わせた有機層を、無水ＭｇＳＯ_４で乾燥し、シリカゲルのプラグを通して濾過し、そして濃縮して固体を得た。フラッシュクロマトグラフィー（３０％ジクロロメタン／ヘキサン）により、所望の生成物１６を白色固体として得た（０．４０５ｇ、８０％）：融点１１７〜１１８℃；Ｒ_ｆ ０．２（２５％ＣＨ_２Ｃｌ_２／ヘキサン）。生成物の構造もまた、ＩＲ、^１Ｈ ＮＭＲおよび質量分析を用いて確認した。

(A.) Methyl 4- [1- (5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl) -1-ethenyl] benzoate ( 16 ):
To a suspension of methyltriphenylphosphonium bromide (0.78 g, 2.18 mmol) in 5 mL of benzene at room temperature under an argon atmosphere was added a 0.5 M solution of potassium hexamethyldisilazide in toluene (4.4 mL, 2.2 mmol) was added and the yellow solution was stirred for 5 minutes. A solution of ketoester 3 (0.51 g, 1.455 mmol) in 7 mL of benzene was added and the orange solution was stirred for 1 hour at room temperature. The reaction mixture was poured into saturated aqueous NaHCO ₃ and extracted with 40% ethyl acetate / hexane. The combined organic layers were dried over anhydrous MgSO _4, filtered through a plug of silica gel, and concentrated to give a solid. Flash chromatography (30% dichloromethane / hexane), the desired product 16 was obtained as a white solid (0.405 g, 80%): mp _{117~118 ℃; R f 0.2 (25} % CH 2 Cl 2 / Hexane). The structure of the product was also confirmed using IR, ¹ H NMR and mass spectroscopy.

(B.) [1- (5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl) -1- (4-carbomethoxyphenyl)] cyclopropane ( 17 ):
To a solution of ethene 16 (0.130 g, 0.373 mmol) in 10 mL of benzene at room temperature under an argon atmosphere was added a 1 M solution of diethylzinc in hexane (5.6 mL, 5.6 mmol) and the reaction mixture Was heated to 60 ° C. Diiodomethane (0.48 mL, 6.0 mmol) in 2 mL of benzene was added dropwise for 5 minutes. The reaction mixture was cooled to room temperature and oxygen was passed in for 3 hours. The cloudy solution was diluted with 40% ethyl acetate / hexane and washed with aqueous hydrochloric acid, water, and saturated aqueous NaHCO ₃ . The organic layer was dried over anhydrous MgSO _4, filtered, and concentrated to give a solid. Flash chromatography (30%; 40% CH ₂ Cl ₂ / hexane) provided the desired product 17 as a white solid (0.08 g, 59%): mp 100-102 ° C .; R _f 0.36 ( 50% _CH 2 Cl ₂ / hexane). The structure of the product was also confirmed using IR, ¹ H NMR and mass spectroscopy.

(C.) [1- (5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl) -1- (4-carboxyphenyl)] cyclopropane ( 18) :
To a suspension of ester 17 (60 mg, 0.166 mmol) in a 75% aqueous methanol solution (2 mL) was added 1 potassium hydroxide (0.12 g) and the reaction mixture was dissolved at 70 ° C. The mixture was stirred for 1 hour. The solution was cooled to room temperature, acidified with 1N aqueous hydrochloric acid, and then extracted with 80% ethyl acetate / hexane. The combined organic layers were dried over anhydrous MgSO _4, filtered, and concentrated to afford a white solid. Recrystallization from dichloromethane-hexane gave the desired acid 18 as a white powder (0.055 g, 95%): mp 333-335 ° C. The structure of the product was also confirmed using IR, ¹ H NMR and mass spectroscopy.

Example IX

（ａ．）メチル４−［１−（５，６，７，８−テトラヒドロ−５，５，８，８−テトラメチル−２−ナフタレニル）−２−メチル−１−プロペニル］ベンゾエート（１９）：
３ｍＬのベンゼン中のイソプロピルトリフェニルホスホニウムヨージド（０．３５ｇ、０．８０７ｍｍｏｌ）の懸濁液に、室温でアルゴン雰囲気下にて、トルエン（１．８ｍＬ、０．８９ｍｍｏｌ）中のヘキサメチルジシラジドカリウムの０．５Ｍ溶液を添加し、そして赤色の溶液を、５分間撹拌した。３ｍＬのベンゼン中のケト−エステル３（０．１６９ｇ、０．４８１ｍｍｏｌ）を添加し、そして、約４ｍＬのベンゼンが蒸発する間、赤色の溶液を１１０℃まで加熱した。１時間後、反応混合物を、４０％の酢酸エチル／ヘキサンで希釈し、そして飽和ＮａＨＣＯ_３水溶液および塩水で洗浄した。有機層を無水ＭｇＳＯ_４で乾燥し、シリカゲルのプラグを介して濾過し、そして濃縮して固体を得た。フラッシュクロマトグラフィー（４０％ジクロロメタン／ヘキサン）により、所望の生成物１９を白色粉末として得た（０．１２８ｇ、７１％）：Ｒ_ｆ ０．４４（５０％ＣＨ_２Ｃｌ_２／ヘキサン）。生成物の構造もまた、ＩＲ、^１Ｈ ＮＭＲおよび質量分析を用いて確認した。

(A.) Methyl 4- [1- (5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl) -2-methyl-1-propenyl] benzoate ( 19 ):
To a suspension of isopropyltriphenylphosphonium iodide (0.35 g, 0.807 mmol) in 3 mL of benzene was added hexamethyldisila in toluene (1.8 mL, 0.89 mmol) at room temperature under an argon atmosphere. A 0.5 M solution of potassium zide was added and the red solution was stirred for 5 minutes. Keto-ester 3 (0.169 g, 0.481 mmol) in 3 mL of benzene was added and the red solution was heated to 110 ° C. while about 4 mL of benzene was evaporated. After 1 hour, the reaction mixture was diluted with 40% ethyl acetate / hexane and washed with saturated aqueous NaHCO ₃ and brine. The organic layer was dried over anhydrous MgSO _4, filtered through a plug of silica gel, and concentrated to give a solid. Flash chromatography (40% dichloromethane / hexane) to give the desired product 19 as a white powder _{(0.128g, 71%): R} f 0.44 (50% CH 2 Cl 2 / hexane). The structure of the product was also confirmed using IR, ¹ H NMR and mass spectroscopy.

(B.) 4- [1- (5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl) -2-methyl-1-propenyl] benzoic acid ( 20 ):
To a suspension of ester 19 (0.115 g, 0.304 mmol) in a 75% aqueous methanol solution (3 mL) was added one drop of potassium hydroxide (0.12 g). The mixture was stirred at 75 ° C. for 1 hour until the material dissolved. The solution was cooled to room temperature, acidified using 1N aqueous hydrochloric acid, and then extracted with 80% ethyl acetate / hexane. The combined organic layers were dried over anhydrous MgSO _4, filtered, and concentrated to give the desired acid 20 as a white powder (0.11g, 99%): mp 204 - 206 ° C.. The structure of the product was also confirmed using IR, ¹ H NMR and mass spectroscopy.

Example X

（ａ．）メチル４−［（４，４−ジメチル−３，４−ジヒドロ−２Ｈ−１−ベンゾピラン−６−イル）カルボニル］ベンゾエート（２２）（Ｍａｉｇｎａｎ，Ｊ．ら、ＢＥ １，０００，１９５）：
１ｍＬの１，２−ジクロロエタン中の塩化アルミニウム（１．６ｇ、１２ｍｍｏｌ）の懸濁液に、室温でアルゴン雰囲気下にて９ｍＬの１，２−ジクロロエタン中の４，４−ジメチル−３，４−ジヒドロ−２Ｈ−１−ベンゾピラン２１（１．５ｇ、９．２５ｍｍｏｌ）（Ｄａｗｓｏｎ，Ｍ．Ｉ．ら、Ｊ．Ｍｅｄ．Ｃｈｅｍ．２７：１５１６−１５３１（１９８４））および４−カルボメトキシベンゾイルクロライド２（１．７９ｇ、９ｍｍｏｌ）を添加した。反応混合物を一晩撹拌し、氷水上に注ぎ、４０％酢酸エチル／ヘキサンで抽出した。合わせた有機層を飽和ＮａＨＣＯ_３水溶液および塩水で洗浄した。溶液を無水ＭｇＳＯ_４で乾燥し、濾過し、そして濃縮して黄色の固体（３．２４ｇ）を得た。フラッシュクロマトグラフィー（８０％ジクロロメタン／ヘキサン）により、所望の生成物２２を白色粉末として得た（１．４２ｇ、４９％）：融点１２９〜１３１℃；Ｒ_ｆ ０．２６（ＣＨ_２Ｃｌ_２）。生成物の構造もまた、ＩＲ、^１Ｈ ＮＭＲおよび質量分析を用いて確認した。

(. A) Methyl 4 -. [(4,4-dimethyl-3,4-dihydro -2 H-1-benzopyran-6-yl) carbonyl] benzoate (22) (Maignan, J et al., BE 1,000, 195):
To a suspension of aluminum chloride (1.6 g, 12 mmol) in 1 mL of 1,2-dichloroethane was added at room temperature under an argon atmosphere, 9 mL of 4,4-dimethyl-3,4-in 1,2-dichloroethane. dihydro -2 H-1-benzopyran 21 (1.5g, 9.25mmol) (Dawson , M.I , et al, J.Med.Chem.27:. 1516-1531 (1984) ) and 4-carbomethoxy benzoyl chloride 2 (1.79 g, 9 mmol) was added. The reaction mixture was stirred overnight, poured onto ice water and extracted with 40% ethyl acetate / hexane. The combined organic layers were washed with saturated aqueous NaHCO ₃ and brine. The solution was dried over anhydrous MgSO ₄ , filtered, and concentrated to give a yellow solid (3.24 g). Flash chromatography (80% dichloromethane / hexane) to give the desired product 22 as a white powder (1.42 g, 49%): mp _{129~131 ℃; R f 0.26 (CH} 2 Cl 2). The structure of the product was also confirmed using IR, ¹ H NMR and mass spectroscopy.

(. B) [2- (4,4- dimethyl-3,4-dihydro -2 H-1-benzopyran-6-yl) -2- (4-carbomethoxy phenyl)] - 1,3-dithiane (23 ):
To a solution of keto-ester 22 (0.152 g, 0.469 mmol) in dichloromethane (3 mL) at 0 ° C. under an argon atmosphere was added 1,3-propanedithiol (0.061 g, 0.563 mmol); Subsequently, boron trifluoride etherate (0.07 mL, 0.57 mmol) was added. The resulting mixture was stirred at 0 ° C. for 1 hour and at room temperature overnight. The reaction mixture was quenched by pouring into saturated aqueous Na ₂ CO ₃ and then extracted with 40% ethyl acetate / hexane. The combined organic layers were dried over anhydrous MgSO _4, filtered, and concentrated to give an oil. Flash chromatography (80% dichloromethane / hexane) provided the desired dithiane 23 as a white solid (0.175 g, 90%): mp 103-105 ° C .; R _f 0.2 (50% CH ₂ Cl ₂ / Hexane). The structure of the product was also confirmed using IR, ¹ H NMR and mass spectroscopy.

(. C) [2- (4,4- dimethyl-3,4-dihydro -2 H-1-benzopyran-6-yl) -2- (4-carboxyphenyl)] - 1,3-dithiane (24) :
To a suspension of dithiane 23 (0.145 g, 0.349 mmol) in a 75% aqueous methanol solution (4 mL) was added one drop of sodium hydroxide (0.106 g). The reaction mixture was stirred at 70 ° C. for 1 hour until the material dissolved. The solution was cooled to room temperature, acidified using 1N aqueous hydrochloric acid, and then extracted with 80% ethyl acetate / hexane. The combined organic layers were dried over anhydrous MgSO _4, filtered, and concentrated to afford a white solid. Recrystallization from dichloromethane-hexane gave the desired acid 24 as a white powder (0.127 g, 90%): mp 204-205 ° C. The structure of the product was also confirmed using IR, ¹ H NMR and mass spectroscopy.

Example XI

（ａ．）メチル４−［（４，４−ジメチル−３，４−ジヒドロ−２Ｈ−１−ベンゾチオピラン−６−イル）カルボニル］ベンゾエート（２６）：
１ｍＬの１，２−ジクロロエタン中の塩化アルミニウム（１．６５ｇ、９．２５ｍｍｏｌ）の懸濁液に、室温でアルゴン雰囲気下で、９ｍＬの１，２−ジクロロエタン中の４，４−ジメチル−３，４−ジヒドロ−２Ｈ−１−ベンゾチオピラン２５（１．６５ｇ、９．２５ｍｍｏｌ）（Ｗａｕｇｈ，Ｋ．Ｍ．ら、Ｊ．Ｍｅｄ．Ｃｈｅｍ．２８：１１６−１２４（１９８５））および４−カルボメトキシベンゾイルクロライド２（１．８ｇ、９．０６ｍｍｏｌ）の溶液を添加した。反応混合物を、一晩撹拌し、氷水上に注ぎ、そして４０％酢酸エチル／ヘキサンで抽出した。合わせた有機層を飽和ＮａＨＣＯ_３水溶液および塩水で洗浄した。溶液を無水ＭｇＳＯ_４で乾燥し、濾過し、そして濃縮して緑色の固体（２．１ｇ）を得た。フラッシュクロマトグラフィー（８０％ジクロロメタン／ヘキサン）により、所望の生成物２６を白色粉末として得た（１．２３ｇ、４０％）：融点１１８〜１２０℃；Ｒ_ｆ ０．３５（ＣＨ_２Ｃｌ_２）。生成物の構造もまた、ＩＲ、^１Ｈ ＮＭＲおよび質量分析を用いて確認した。

(. A) Methyl 4 - [(4,4-dimethyl-3,4-dihydro -2 H-1-benzothiopyran-6-yl) carbonyl] benzoate (26):
To a suspension of aluminum chloride (1.65 g, 9.25 mmol) in 1 mL of 1,2-dichloroethane at room temperature under an argon atmosphere was added 4,4-dimethyl-3,9 in 1,2-dichloroethane. 4-dihydro -2 H-1-benzothiopyran 25 (1.65g, 9.25mmol) (Waugh , K.M , et al, J.Med.Chem.28:. 116-124 (1985) ) and 4-carbomethoxy benzoyl A solution of chloride 2 (1.8 g, 9.06 mmol) was added. The reaction mixture was stirred overnight, poured onto ice water and extracted with 40% ethyl acetate / hexane. The combined organic layers were washed with saturated aqueous NaHCO ₃ and brine. The solution was dried over anhydrous MgSO _4, filtered, and concentrated to give a green solid (2.1 g). Flash chromatography (80% dichloromethane / hexane) to give the desired product 26 as a white powder (1.23 g, 40%): mp _{118~120 ℃; R f 0.35 (CH} 2 Cl 2). The structure of the product was also confirmed using IR, ¹ H NMR and mass spectroscopy.

(. B) [2- (4,4- dimethyl-3,4-dihydro -2 H-1-benzothiopyran-6-yl) -2- (4-carbomethoxy phenyl)] - 1,3-dithiane (27 ):
To a solution of keto-ester 26 (0.12 g, 0.353 mmol) in dichloromethane (3 mL) at 0 ° C. under an argon atmosphere was added 1,3-propanedithiol (0.06 mL, 0.53 mmol); Subsequently, boron trifluoride etherate (0.07 mL, 0.57 mmol) was added. The resulting mixture was stirred at 0 ° C. for 1 hour and warmed at room temperature overnight. The reaction mixture was quenched by pouring into saturated aqueous Na ₂ CO ₃ and extracted with 40% ethyl acetate / hexane. The combined organic layers were dried over anhydrous MgSO _4, filtered, and concentrated to give an oil. Flash chromatography (80% _{CH 2} Cl 2 / hexane), the desired dithiane 27 as a white solid (0.145 g, 96%): mp _{164~166 ℃; R f 0.27 (50} % CH 2 Cl ₂ / hexane). The structure of the product was also confirmed using IR, ¹ H NMR and mass spectroscopy.

(. C) [2- (4,4- dimethyl-3,4-dihydro -2 H-1-benzothiopyran-6-yl) -2- (4-carboxyphenyl)] - 1,3-dithiane (28) :
To a suspension of dithian 27 (0.1 g, 0.232 mmol) in a 75% aqueous methanol solution (4 mL) was added one drop of potassium hydroxide (0.12 g). The reaction mixture was stirred at 75 ° C. for 1 hour until the material dissolved. The solution was cooled to room temperature, acidified using 1N aqueous hydrochloric acid, and then extracted with 80% ethyl acetate / hexane. The combined organic layers were dried over anhydrous MgSO _4, filtered, and concentrated to afford a white solid. Recrystallization from dichloromethane / hexane provided the desired acid 28 as a white powder (0.089 g, 92%): mp 211-212.5 ° C. The structure of the product was also confirmed using IR, ¹ H NMR and mass spectroscopy.

Example XII

（ａ．）メチル［４−（５，６，７，８−テトラヒドロ−３，５，５，８，８−ペンタメチル−２−ナフタレニル）カルボニル］ベンゾエート（３０）：
３０ｍＬの１，２−ジクロロエタン中の塩化アルミニウム（１．１０ｇ、８．２５ｍｍｏｌ）の懸濁液に、室温でアルゴン雰囲気下で、１５ｍＬの１，２−ジクロロエタン中の１，２，３，４−テトラヒドロ−１，１，４，４，６−ペンタメチルナフタレン２９（１．５２ｇ、７．５ｍｍｏｌ）（Ｋａｇｅｃｈｉｋａ，Ｈ．ら、Ｊ．Ｍｅｄ．Ｃｈｅｍ．３１：２１８２（１９８８））および４−カルボメトキシベンゾイルクロライド２（１．５７ｇ、７．８７ｍｍｏｌ）の溶液を添加した。反応混合物を一晩撹拌し、氷水上に注ぎ、そして４０％酢酸エチル／ヘキサンで抽出した。合わせた有機層を飽和ＮａＨＣＯ_３水溶液および塩水で洗浄した。溶液を無水ＭｇＳＯ_４で乾燥し、濾過し、そして濃縮して褐色の固体（２．５６ｇ）を得た。フラッシュクロマトグラフィー（６０％ジクロロメタン／ヘキサン）により、所望の生成物３０を白色の結晶性固体として得た（１．７３３ｇ、６４％）：融点１４６〜１４９℃；Ｒ_ｆ ０．１１（５０％ＣＨ_２Ｃｌ_２／ヘキサン）。生成物の構造もまた、ＩＲ、^１Ｈ ＮＭＲおよび質量分析を用いて確認した。

(A.) Methyl [4- (5,6,7,8-tetrahydro-3,5,5,8,8-pentamethyl-2-naphthalenyl) carbonyl] benzoate ( 30 ):
To a suspension of aluminum chloride (1.10 g, 8.25 mmol) in 30 mL of 1,2-dichloroethane at room temperature under argon atmosphere was added 1,2,3,4-in 15 mL of 1,2-dichloroethane. Tetrahydro-1,1,4,4,6-pentamethylnaphthalene 29 (1.52 g, 7.5 mmol) (Kagechika, H. et al., J. Med . Chem . 31: 2182 (1988)) and 4-carbomethoxy A solution of benzoyl chloride 2 (1.57 g, 7.87 mmol) was added. The reaction mixture was stirred overnight, poured onto ice water and extracted with 40% ethyl acetate / hexane. The combined organic layers were washed with saturated aqueous NaHCO ₃ and brine. The solution was dried over anhydrous MgSO _4, filtered, and concentrated to give brown solid (2.56 g). Flash chromatography (60% dichloromethane / hexane) provided the desired product 30 as a white crystalline solid (1.733 g, 64%): mp 146-149 ° C .; R _f 0.11 (50% CH ₂ Cl ₂ / hexane). The structure of the product was also confirmed using IR, ¹ H NMR and mass spectroscopy.

(B.) [4- (5,6,7,8-tetrahydro-3,5,5,8,8-pentamethyl-2-naphthalenyl) carbonyl] benzoic acid ( 31 ):
To a suspension of ester 30 (0.120 g, 0.329 mmol) in a 75% aqueous methanol solution (2 mL) was added potassium hydroxide (0.055 g). The reaction mixture was stirred at 60 ° C. for 1 hour until the material dissolved. The solution was cooled to room temperature, acidified using 1N aqueous hydrochloric acid, and then extracted with 80% ethyl acetate / hexane. The combined organic layers were dried over anhydrous MgSO _4, filtered, and concentrated to afford a white solid (0.109 g). Recrystallization from benzene / hexane gave 31 as a white crystalline solid (0.102 g, 89%): mp 209-212 ° C. The structure of the product was also confirmed using IR, ¹ H NMR and mass spectroscopy.

Example XIII

（ａ．）メチル４−［１−（５，６，７，８−テトラヒドロ−３，５，５，８，８−ペンタメチル−２−ナフタレニル）−１−エテニル］ベンゾエート（３２）：
１ｍＬのベンゼン中のメチルトリフェニルホスホニウムブロマイド（０．１９６ｇ、０．５５ｍｍｏｌ）の懸濁液に、室温でアルゴン雰囲気下にて、トルエン（１．２ｍＬ、０．６ｍｍｏｌ）中のヘキサメチルジシラジドカリウムの０．５Ｍ溶液を添加し、そしてこの黄色溶液を５分間撹拌した。１．５ｍＬのベンゼン中のケト−エステル３０（０．１ｇ、０．２７４ｍｍｏｌ）の溶液を添加し、そして橙色の溶液を室温で３時間撹拌した。反応混合物を、４０％酢酸エチル／ヘキサンを用いてシリカゲルのプラグを介して濾過した。濾液を濃縮して、固体を得た。フラッシュクロマトグラフィー（３０％；４０％ジクロロメタン／ヘキサン）により、所望の生成物３２を白色の結晶性固体として得た（０．０７７ｇ、７８％）：融点１６７〜１６８℃；Ｒ_ｆ ０．４（５０％ジクロロメタン／ヘキサン）。生成物の構造もまた、ＩＲ、^１Ｈ ＮＭＲおよび質量分析を用いて確認した。

(A.) Methyl 4- [1- (5,6,7,8-tetrahydro-3,5,5,8,8-pentamethyl-2-naphthalenyl) -1-ethenyl] benzoate ( 32 ):
To a suspension of methyltriphenylphosphonium bromide (0.196 g, 0.55 mmol) in 1 mL of benzene was added hexamethyldisilazide in toluene (1.2 mL, 0.6 mmol) at room temperature under an argon atmosphere. A 0.5 M solution of potassium was added and the yellow solution was stirred for 5 minutes. A solution of keto-ester 30 (0.1 g, 0.274 mmol) in 1.5 mL of benzene was added and the orange solution was stirred at room temperature for 3 hours. The reaction mixture was filtered through a plug of silica gel with 40% ethyl acetate / hexane. The filtrate was concentrated to give a solid. Flash chromatography (30%; 40% dichloromethane / hexane) provided the desired product 32 as a white crystalline solid (0.077 g, 78%): mp 167-168 ° C; _Rf 0.4 ( 50% dichloromethane / hexane). The structure of the product was also confirmed using IR, ¹ H NMR and mass spectroscopy.

(B.) [4- [1- (5,6,7,8-Tetrahydro-3,5,5,8,8-pentamethyl-2-naphthalenyl) -1-ethenyl] benzoic acid ( 33 ):
To a suspension of ester 32 (0.058 g, 0.16 mmol) in a 75% aqueous methanol solution (2 mL) was added one drop of potassium hydroxide (0.1 g). The mixture was stirred at 70 ° C. for 1 hour until the material dissolved. The solution was cooled to room temperature, acidified using 1N aqueous hydrochloric acid, and then extracted with 80% ethyl acetate / hexane. The combined organic layers were dried over anhydrous MgSO _4, filtered, and concentrated to afford a white solid. Recrystallization from dichloromethane / hexane provided the desired acid 33 as a white crystalline solid (42 mg, 91%): mp 230-231 ° C. The structure of the product was also confirmed using IR, ¹ H NMR and mass spectroscopy.

Example XIV

（ａ．）５−カルボエトキシチオフェン−２−カルボン酸（３５）：
１０ｍＬのＴＨＦ中のジイソプロピルアミン（３．６ｍＬ、２５．７５ｍｍｏｌ）の溶液に、−７８℃でアルゴン雰囲気下にて、ヘキサン（１６．１ｍＬ、２５．７５ｍｍｏｌ）中のｎ−ブチルリチウムの１．６Ｍ溶液を添加した。混合物を１５分間撹拌し、そして５ｍＬのＴＨＦ中の２−チオフェンカルボン酸３４（１．５ｇ、１１．７０５ｍｍｏｌ）（２−チオフェンカルボン酸３４はＡｌｄｒｉｃｈより容易に入手可能である）の溶液を徐々に添加した。混合物を１５分間撹拌し、そしてエチルクロロホルメート（２．７ｍＬ、２８．３３ｍｍｏｌ）を添加した。混合物を−７８℃で３０分間撹拌し、そして次の３０分間で０℃まで昇温した。反応混合物を飽和ＮａＨＣＯ_３水溶液中に注ぎ、そして８０％酢酸エチル／ヘキサンで洗浄した。水層を酢酸で酸性にし、８０％酢酸エチル／ヘキサンで抽出した。合わせた有機層を無水ＭｇＳＯ_４で乾燥し、濾過し、そして濃縮して黄色の固体を得た。フラッシュクロマトグラフィーにより、所望の酸３５（１．７６ｇ、７５％）を白色固体として得た：融点は３００℃を超える。生成物の構造もまた、ＩＲ、^１Ｈ ＮＭＲおよび質量分析を用いて確認した。

(A.) 5-Carboethoxythiophene-2-carboxylic acid ( 35 ):
To a solution of diisopropylamine (3.6 mL, 25.75 mmol) in 10 mL of THF at −78 ° C. under an argon atmosphere, 1.6 M of n -butyllithium in hexane (16.1 mL, 25.75 mmol). The solution was added. The mixture was stirred for 15 minutes and a solution of 2-thiophene carboxylic acid 34 (1.5 g, 11.705 mmol) (2-thiophene carboxylic acid 34 is readily available from Aldrich) in 5 mL of THF was slowly added. Was added. The mixture was stirred for 15 minutes and ethyl chloroformate (2.7 mL, 28.33 mmol) was added. The mixture was stirred at -78 ° C for 30 minutes and warmed to 0 ° C over the next 30 minutes. The reaction mixture was poured into saturated aqueous NaHCO ₃ and washed with 80% ethyl acetate / hexane. The aqueous layer was acidified with acetic acid and extracted with 80% ethyl acetate / hexane. The combined organic layers were dried over anhydrous MgSO _4, filtered, and concentrated to give a yellow solid. Flash chromatography provided the desired acid 35 (1.76 g, 75%) as a white solid: mp> 300 ° C. The structure of the product was also confirmed using IR, ¹ H NMR and mass spectroscopy.

(B.) 5-carboethoxythiophene-2-carbonyl chloride ( 36 ):
To a suspension of the acid 35 (0.64 g, 3.2 mmol) in dichloromethane (20 mL) at room temperature under an argon atmosphere was added (COCl) ₂ in dichloromethane (2.4 mL, 4.8 mmol). A 0 M solution and 2 drops of DMF were added and stirred for 1 hour. Excess (COCl) ₂ and dichloromethane were removed under reduced pressure and the viscous light yellow product was dried overnight to give the desired benzoyl chloride 36 as a yellow solid. The structure of the product was also confirmed using IR and ¹ H NMR spectroscopy.

(C.) Ethyl 5-[(5,6,7,8-tetrahydro-3,5,5,8,8-pentamethyl-2-naphthalenyl) carbonyl] thiophen-2-carboxylate ( 37 ):
To a suspension of aluminum chloride (0.5 g, 3.75 mmol) in 1 mL of 1,2-dichloroethane at room temperature under an argon atmosphere was added 3.5 mL of 1,2,3 of 1,2-dichloroethane. A solution of 4-tetrahydro-1,1,4,4,6-pentamethylnaphthalene 29 (0.712 g, 3.52 mmol) and 36 (0.7 g, 3.2 mmol) was added dropwise. The reaction mixture was stirred overnight, then poured onto ice water and extracted with 40% ethyl acetate / hexane. The combined organic layers were washed with saturated aqueous NaHCO ₃ and brine. The solution was dried over anhydrous MgSO _4, filtered and concentrated to give a yellow solid (1.5 g). Flash chromatography (50% dichloromethane / hexane) provided a light yellow solid. Recrystallization from dichloromethane / hexane provided 37 as a white crystalline solid (0.62 g, 50%): mp 111-112 ° C .; R _f 0.2 (50% CH ₂ Cl ₂ / hexane). The structure of the product was also confirmed using IR, ¹ H NMR and mass spectroscopy.

(D.) 5-[(5,6,7,8-tetrahydro-3,5,5,8,8-pentamethyl-2-naphthalenyl) carbonyl] thiophen-2-carboxylic acid ( 38 ):
To a suspension of ester 37 (50 mg, 0.13 mmol) in a 75% aqueous methanol solution (3 mL) was added one drop of potassium hydroxide (0.1 g). The reaction mixture was stirred at 70 ° C. for 1.5 hours until the material dissolved. The solution was cooled to room temperature, acidified using 1N aqueous hydrochloric acid, and then extracted with 80% ethyl acetate / hexane. The combined organic layers were dried over anhydrous MgSO _4, filtered, and concentrated to afford a white solid (46 mg). Recrystallization from methanol provided the desired acid 38 as a white crystalline solid (42 g, 91%): mp 214-215 ° C. The structure of the product was also confirmed using IR, ¹ H NMR and mass spectroscopy.

Example XV

（ａ．）２−アセチル−３，５，５，８，８−ペンタメチル−５，６，７，８−テトラヒドロナフタレン（３９）：
１ｍＬの１，２−ジクロロエタン中の塩化アルミニウム（０．９６ｇ、７．１７ｍｍｏｌ）の懸濁液に、室温でアルゴン雰囲気下にて、９ｍＬの１，２−ジクロロエタン中の１，２，３，４−テトラヒドロ−１，１，４，４，６−ペンタメチルナフタレン２９（１．２ｇ、５．９３ｍｍｏｌ）およびアセチルクロライド（０．５１ｇ、６．５２ｍｍｏｌ）の溶液を添加した。反応混合物を１時間撹拌し、次いで、氷水上に注ぎ、そして４０％酢酸エチル／ヘキサンで抽出した。合わせた有機層を、飽和ＮａＨＣＯ_３水溶液および塩水で洗浄した。溶液を、無水ＭｇＳＯ_４で乾燥し、シリカゲルのプラグを介して濾過し、そして濃縮して白色固体３９を得た（１．４５ｇ、９９％）：融点５４〜５７℃；Ｒ_ｆ ０．６２（１０％酢酸エチル／ヘキサン）。生成物の構造もまた、ＩＲ、^１Ｈ ＮＭＲおよび質量分析を用いて確認した。

(A.) 2-acetyl-3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydronaphthalene ( 39 ):
To a suspension of aluminum chloride (0.96 g, 7.17 mmol) in 1 mL of 1,2-dichloroethane was added 1,2,3,4 in 9 mL of 1,2-dichloroethane at room temperature under an argon atmosphere. -A solution of tetrahydro-1,1,4,4,6-pentamethylnaphthalene 29 (1.2 g, 5.93 mmol) and acetyl chloride (0.51 g, 6.52 mmol) was added. The reaction mixture was stirred for 1 hour, then poured onto ice water and extracted with 40% ethyl acetate / hexane. The combined organic layers were washed with saturated aqueous NaHCO ₃ and brine. The solution was dried over anhydrous MgSO ₄ , filtered through a plug of silica gel and concentrated to give a white solid 39 (1.45 g, 99%): mp 54-57 ° C .; R _f 0.62 ( 10% ethyl acetate / hexane). The structure of the product was also confirmed using IR, ¹ H NMR and mass spectroscopy.

(B.) Ethyl ( E ) -4-hydroxy-3-methyl-6-oxo-6- (3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthalenyl) -2-hexenoate ( 41 ):
To a solution of diisopropylamine (0.5 mL, 3.52 mmol) in 7 mL of THF at −78 ° C. under an argon atmosphere, 1.6 M of n -butyllithium in hexane (2.2 mL, 3.52 mmol). The solution was added. The mixture was stirred for 25 minutes and a solution of ketone 39 (0.78 g, 3.2 mmol) in 4 mL of THF was added slowly. The mixture was stirred for 20 minutes and ethyl ( E ) -3-formyl-2-butenoate 40 (0.45 g, 3.2 mmol) (ethyl ( E ) -3-formyl-2-butenoate 40 in 3 mL of THF was added. (Available from Fluka) was slowly added. The mixture was stirred at −78 ° C. for 35 minutes, poured into saturated aqueous NH ₄ Cl and extracted with 40% ethyl acetate / hexane. The combined organic layers were dried over anhydrous MgSO _4, filtered and concentrated to give a light yellow solid. Flash chromatography provided the desired alcohol 41 as a white crystalline solid (0.98 g, 80%): mp 126-128 ° C; _Rf 0.13 (10% ethyl acetate / hexane). The structure of the product was also confirmed using IR, ¹ H NMR and mass spectroscopy.

(C.) Ethyl (2 E, 4 E) -6- oxo-6- (3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthalenyl) -2,4 -Hexadienoate ( 42 ):
To a solution of 41 (0.33 g, 0.85 mmol) in 8 mL of THF at 0 ° C. was added triethylamine (0.5 mL, 4 mmol) and methylsulfonyl chloride (0.106 g, 0.93 mmol) in 2 mL of THF. The solution was added slowly. The mixture was stirred at 0 ° C. for 1 hour and warmed to room temperature for 30 minutes. The mixture was filtered through a plug of silica gel with 10% ethyl acetate / hexane. The filtrate was concentrated to give 42 as a light yellow solid. Recrystallization from dichloromethane / hexane gave the desired acid 42 as a yellow powder (0.3 g, 95%): mp 101-102 ° C .; R _f 0.42 (10% ethyl acetate / hexane). The structure of the product was also confirmed using IR, ¹ H NMR and mass spectroscopy.

(D.) [2- (5,6,7,8- tetrahydro-3,5,5,8,8-pentamethyl-2-naphthalenyl) -2- (4-carboethoxy -2 E, 4 E -3 -Methoxybutadienyl)]-1,3-dioxolane ( 43 ):
To a solution of keto-ester 42 (0.12 g, 0.33 mmol) in 2 mL of benzene was added ethylene glycol (0.8 mL), 1,2-bis (trimethylsilyloxy) ethane (2 mL) and a catalytic amount of p- TsOH. Was added. The reaction mixture was heated at reflux for 2 days, then cooled to room temperature. The solution was poured into saturated aqueous NaHCO ₃ and extracted with 40% ethyl acetate / hexane. The combined organic layers were dried over anhydrous MgSO _4, filtered, and concentrated to give a solid. Flash chromatography (5% ethyl acetate / hexane) provided the desired ketal 43 as a colorless oil (0.109 _g , 80%): R _f 0.43 (10% ethyl acetate / hexane). The structure of the product was also confirmed using IR, ¹ H NMR and mass spectroscopy.

(E.) [2- (5,6,7,8- tetrahydro-3,5,5,8,8-pentamethyl-2-naphthalenyl) -2- (4-carboxy -2 E, 4 E -3- Methylbutadienyl)]-1,3-dioxolane ( 44 ):
To a solution of ester 43 (30 mg, 0.073 mmol) in a 75% aqueous methanol solution (2 mL) was added one drop of potassium hydroxide (0.1 g). The reaction mixture was stirred at 60 ° C. for 0.5 hours until the material dissolved. The solution was cooled to room temperature, acidified using 1N aqueous hydrochloric acid, and then extracted with 80% ethyl acetate / hexane. The combined organic layers were dried over anhydrous MgSO _4, filtered, and concentrated to afford a white solid. Recrystallization from dichloromethane / hexane gave the desired acid 44 as a white crystalline solid (27 mg, 96%): mp 189-190 ° C. The structure of the product was also confirmed using IR, ¹ H NMR and mass spectroscopy.

Example XVI

（Ｅ）および（Ｚ）−４−［１−（５，６，７，８−テトラヒドロ−５，５，８，８−テトラメチル−２−ナフタレニル）プロペン−１−イル］安息香酸（４６および４７）：
１ｍＬのＴＨＦ中のエチルトリフェニルホスホニウムブロマイド（１６０ｍｇ、０．４３ｍｍｏｌ）の懸濁液に、０℃でアルゴン雰囲気下にて、トルエン（０．９５ｍＬ、０．４７ｍｍｏｌ）中のヘキサメチルジシラジドカリウムの０．５Ｍ溶液を添加し、そして反応混合物を、５分間撹拌した。０．８ｍＬのＴＨＦ中のケト−エステル３の溶液（１００ｍｇ、０．２８ｍｍｏｌ）を添加し、そして橙色の溶液を室温で３時間撹拌した。反応混合物を２０％酢酸エチル／ヘキサンで希釈し、そして２０％酢酸エチル／ヘキサンを用いてシリカのプラグを介して濾過した。溶液を濃縮して黄色のゴム状物を得た。クロマトグラフィー（３８％ジクロロメタン／ヘキサン）により、４５の混合物を淡黄色のゴム状物として得た（５１ｍｇ、５０％）：Ｒ_ｆ ０．４３、０．４７（４０％ＣＨ_２Ｃｌ_２／ヘキサン）。分取ＨＰＬＣ（Ｗａｔｅｒｓ Ｒａｄｉａｌｐａｋ Ｎｏｖａｐａｋシリカ、８ｍｍ×１０ｃｍ、２％エーテル／ヘキサン、１．０ｍＬ／分、２６０ｎｍ）により、無色のガム状物４５ａ（２０ｍｇ、ｔ_ｒ＝９．８分）および白色の固体４５ｂ（２５ｍｇ、ｔ_ｒ＝１０．８分）を得た。

( E ) and ( Z ) -4- [1- (5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl) propen-1-yl] benzoic acid ( 46 and 47 ):
To a suspension of ethyltriphenylphosphonium bromide (160 mg, 0.43 mmol) in 1 mL of THF was added potassium hexamethyldisilazide in toluene (0.95 mL, 0.47 mmol) at 0 ° C. under an argon atmosphere. Was added and the reaction mixture was stirred for 5 minutes. A solution of keto-ester 3 (0.8 mg, 0.28 mmol) in 0.8 mL of THF was added and the orange solution was stirred at room temperature for 3 hours. The reaction mixture was diluted with 20% ethyl acetate / hexane and filtered through a plug of silica with 20% ethyl acetate / hexane. The solution was concentrated to give a yellow gum. Chromatography (38% dichloromethane / hexane) gave a mixture of 45 as a pale yellow gum (51 mg, 50%): R _f 0.43, 0.47 (40% CH ₂ Cl ₂ / hexane). . Preparative HPLC (Waters Radialpak Novapak silica, 8 mm × 10 cm, 2% ether / hexane, 1.0 mL / min, 260 nm), the colorless gum 45a _(20mg, t r = 9.8 min) and a white solid 45b (25 mg, _tr = 10.8 min) was obtained.

To a suspension of the ester 45a (20 mg) in 0.5 mL of ethanol was added a 40% aqueous solution of potassium hydroxide (0.2 g) and the reaction mixture was stirred at 70 ° C. for 2 hours until the material was dissolved. And stirred under an argon atmosphere. The solution was then concentrated under an argon atmosphere. The residue was cooled to room temperature, acidified to pH 2-3 with 1N aqueous sulfuric acid, and then filtered. The precipitate was washed repeatedly with water (6 × 1 mL) and dried to give a white powder (20 mg). Recrystallization from ethyl acetate / hexane provided acid 46 as a white powder (16 mg, 16% overall yield): mp 212 ° C. The structure of the product was also confirmed using IR, ¹ H NMR and mass spectroscopy. The regiochemistry of 46 was confirmed by ¹ H nOe NMR spectroscopy.

Ester 45b was hydrolyzed as above to give 25 mg of a pale yellow solid. Recrystallization from ethyl acetate / hexane provided acid 47 as a pale yellow powder (21 mg, 20% overall yield): mp 229 ° C. The structure of the product was also confirmed using IR, ¹ H NMR and mass spectroscopy. The regiochemistry of 47 was confirmed by ¹ HnOe NMR spectroscopy.

Example XVII

（ａ．）４−［１−（５，６，７，８−テトラヒドロ−５，５，８，８−テトラメチル−２−ナフタレニル）−１−エテニル］安息香酸（４８）：
７５％水性メタノール溶液（２ｍＬ）中のエステル１６（９４ｍｇ、０．２７ｍｍｏｌ）の懸濁液に、水酸化カリウム（０．０４５ｇ）を添加し、そして反応混合物を、この物質が溶解するまで１４時間、５０℃で撹拌した。溶液を室温まで冷却し、２Ｎの塩酸水溶液を用いて酸性にし、次いで、エーテルで抽出した。合わせた有機層を、水および塩水で洗浄した。次いで、有機層を無水ＭｇＳＯ_４で乾燥し、濾過し、そして濃縮して白色固体を得た。ベンゼン−ヘキサンからの再結晶により、所望の酸４８を白色の結晶性固体として得た（０．０７４ｇ、８２％）：融点２０１〜２０４℃。生成物の構造もまた、ＩＲ、^１Ｈ ＮＭＲおよび質量分析を用いて確認した。

(A.) 4- [1- (5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl) -1-ethenyl] benzoic acid ( 48 ):
To a suspension of ester 16 (94 mg, 0.27 mmol) in 75% aqueous methanol solution (2 mL) was added potassium hydroxide (0.045 g) and the reaction mixture was allowed to dissolve for 14 hours until the material was dissolved. And stirred at 50 ° C. The solution was cooled to room temperature, acidified using 2N aqueous hydrochloric acid, and then extracted with ether. The combined organic layers were washed with water and brine. The organic layer was then dried over anhydrous MgSO _4, filtered, and concentrated to afford a white solid. Recrystallization from benzene-hexane gave the desired acid 48 as a white crystalline solid (0.074 g, 82%): mp 201-204 ° C. The structure of the product was also confirmed using IR, ¹ H NMR and mass spectroscopy.

(B.) 4- [1- (5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl) -1-ethyl] benzoic acid ( 49 ):
Olefin 48 (0.0105 g, 0.0314 mmol) was hydrogenated with 5% palladium on charcoal at room temperature and atmospheric pressure. After uptake of one equivalent (0.7 mL) of hydrogen, the catalyst was removed by filtration through a small pad of celite. The solvent was removed under vacuum to give the crude acid as a white solid (0.019g). Recrystallization from benzene-hexane gave the desired acid 49 as a white crystalline solid (0.0078 g, 74%): mp 186-188 ° C. The structure of the product was also confirmed using IR, ¹ H NMR and mass spectroscopy.

  The above examples are intended to illustrate rather than limit the invention. While they are representative that can be used, other procedures known to those skilled in the art can be used instead.

  Throughout this application, various publications are incorporated by number. Thereby, their publications are fully quoted. The disclosures in these publications are incorporated by reference in their entirety in this application in order to more fully describe the state of the art to which this invention pertains.

FIG. 1 shows that 9-cis-retinoic acid induces RXR homodimer binding for TREpal.

(A) In vitro synthesized RXRα was converted to the indicated hormones (10 ⁻⁷ M 9-cis-RA; 10 ⁻⁶ M RA; ^10-6 M) in the presence or absence of in vitro synthesized TRα or RARβ. ^-6 M T ₃₎ with (+) or without (- either in) and incubated for 30 minutes at room temperature. After this preincubation, the reaction mixture was analyzed by gel retardation assay using ³² P-labelled TREpal as a probe. Lane 1 represents non-specific binding of unprogrammed reticulocyte lysate. Open triangles indicate non-specific complexes observed in unprogrammed reticulocyte lysate. Closed triangles indicate specific TRα-RXRα heterodimer binding. Arrows indicate specific RXRα homodimer binding. The RXRα / RARβ heterodimer moves to the same position as the RXRα homodimer. The effect of 9-cis-RA on RARβ binding is shown for comparison.

(B) Flag-RXRα (F-RXR) (which can be recognized by a specific anti-flag monoclonal antibody to determine that the DNA-binding complex induced by 9-cis-RA contains RXRα protein , An RXRa derivative containing an eight amino acid epitope (flag) at the amino terminus). In vitro synthesized F-RXRα was (+) or absent (−) with 10 ⁻⁷ M 9-cis-RA in the presence of either a specific anti-flag antibody (αF) or a non-specific pre-immune serum (NI). For 30 minutes at room temperature. Next, the effect of the anti-flag antibody on F-RXRα binding in the presence of 9-cis-RA was analyzed by a gel retardation assay using ³² P-labeled TREpal as a probe. Lane 1 shows non-specific binding of unprogrammed reticulocyte lysate (open triangles). Arrows indicate specific F-RXRα homodimer and RAR-RXR heterodimer binding. Diamonds indicate F-RXR homodimers shifted up with the anti-flag antibody.
FIG. 2 shows the characterization of 9-cis-RA induced RXR homodimers for TPEpal.

(A) Cooperative binding of RXRa homodimer induced by 9-cis-RA. The formation of RXR-DNA complex at different receptor concentrations in the presence or absence of ^10-7 M 9-cis-RA was analyzed by a gel retardation assay using labeled TPEpal as a probe. Open triangles indicate non-specific binding of unprogrammed reticulocyte lysate. The arrow indicates the complex to which RXR specifically binds.

(B) Quantification of RXR binding complex at different receptor concentrations in the presence of 9-cis-RA. Gel slices containing the RXR binding complex in the presence of 9-cis-RA as shown in FIG. 2 (a) were excised and counted in a scintillation counter and plotted.

(C) 9-cis-RA concentration-dependent binding of RXR homodimer for TREpal. Equal amounts of in vitro synthesized RXR protein were incubated with the indicated concentration of 9-cis-RA. The reaction mixture was then analyzed by a gel retardation assay using labeled TPEpal as a probe. Open triangles indicate non-specific binding of unprogrammed reticulocyte lysate. The arrow indicates the complex to which RXR specifically binds. FIG. 3 shows that 9-cis-RA induces RXR homodimer binding for RXR-specific response elements.

(A) Nuclear receptor binding element used in this study. These oligonucleotides were synthesized with appropriate restriction sites at both ends as indicated in lower case. Arrows indicate sequences closely related to the AGG / TTCA motif.
(B) The effect of 9-cis-RA on RXR homodimer binding of ApoAI-RARE or CRBPII-RARE was analyzed essentially as described in FIG. 1a. Lane 1 represents non-specific binding of unprogrammed reticulocyte lysate, indicated by open triangles. Closed triangles indicate the RAR / RXR heterodimer complex. The arrow indicates a complex to which RXR specifically binds. FIG. 4 shows the response element specific binding of the RXR homodimer. RA-specific response element (a), _{T 3} specific response element (b), or the effect of 9-cis -RA about RXR binding on estrogen specific response element (c), as described in Figure 1a Analyzed by gel retardation assay. As a comparison, the binding of RXR / RAR heterodimer (a), RXR / TR heterodimer (b) or estrogen (c) is shown. Open triangles indicate non-specific binding of unprogrammed reticulocyte lysate. Closed triangles indicate RAR / RXR heterodimer complex (a), TR / RXR heterodimer complex (b) or ER complex (c). FIG. 4 shows the response element specific binding of the RXR homodimer. RA-specific response element (a), _{T 3} specific response element (b), or the effect of 9-cis -RA about RXR binding on estrogen specific response element (c), as described in Figure 1a Analyzed by gel retardation assay. As a comparison, the binding of RXR / RAR heterodimer (a), RXR / TR heterodimer (b) or estrogen (c) is shown. Open triangles indicate non-specific binding of unprogrammed reticulocyte lysate. Closed triangles indicate RAR / RXR heterodimer complex (a), TR / RXR heterodimer complex (b) or ER complex (c). FIG. 4 shows the response element specific binding of the RXR homodimer. RA-specific response element (a), _{T 3} specific response element (b), or the effect of 9-cis -RA about RXR binding on estrogen specific response element (c), as described in Figure 1a Analyzed by gel retardation assay. As a comparison, the binding of RXR / RAR heterodimer (a), RXR / TR heterodimer (b) or estrogen (c) is shown. Open triangles indicate non-specific binding of unprogrammed reticulocyte lysate. Closed triangles indicate RAR / RXR heterodimer complex (a), TR / RXR heterodimer complex (b) or ER complex (c). FIG. 5 shows that RXR homodimerization occurs in solution. ^{The 35} S-labeled in vitro synthesized RXRα protein was purified by partially purified bacterial expression Flag-RXR (F-RXR), either in the presence or absence of a response element or chemical crosslinker DSP as indicated. +) Or similarly prepared glutathione transferase control protein (-). After incubation, either anti-flag antibody (F) or non-specific pre-immune serum (NI) was added. ^10-7 M 9-cis-RA was maintained during the working process. The immune complexes were washed in the presence of ^10-7 M 9-cis-RA, boiled in SDS sample buffer, and separated on 10% SDS-PAGE. ³⁵ S-labeled in vitro synthesized RXRa protein is shown in the right panel. Figure 6 shows the transcriptional activation of RXR and RARa: RXR heterodimer with 9-cis-RA for the natural response element. 100 ng of reporter plasmid (a) TREpal-tk-CAT (b) βRARE-tk-CAT (c) ApoAI-RARE-tk-CAT and (d) CRBPI-RARE-tk-CAT and 5 ng empty pECE expression CV-1 cells were co-transfected with the vector, pECE-RXRα, pECE RARα or a combination of both as indicated. Transfected cells were treated with no hormone (open bars), ^10-7 MRA (shaded bars), or ^10-7 M 9-cis-RA (dark hatched bars). The results of a representative experiment performed twice are shown. Figure 6 shows the transcriptional activation of RXR and RARa: RXR heterodimer with 9-cis-RA for the natural response element. 100 ng of reporter plasmid (a) TREpal-tk-CAT (b) βRARE-tk-CAT (c) ApoAI-RARE-tk-CAT and (d) CRBPI-RARE-tk-CAT and 5 ng empty pECE expression CV-1 cells were co-transfected with the vector, pECE-RXRα, pECE RARα or a combination of both as indicated. Transfected cells were treated with no hormone (open bars), ^10-7 MRA (shaded bars), or ^10-7 M 9-cis-RA (dark hatched bars). The results of a representative experiment performed twice are shown. FIG. 7 shows reporter constructs (a) TREpal-tk-CAT (10) or (b) CRBPII-tk-CAT (10) RXRα-dependent transactivation by 9-cis-RA or retinoids SR11203, SR11217, Indicated by SR11234, SR11235, SR11236, and SR11237. The results of a representative experiment performed four times are shown. Induction profiles did not change significantly in four independent experiments. CAT activity was normalized for transfection and recovery efficiency by measuring enzyme activity from the co-transfected β-galactosidase expression plasmid (pCH110, Pharmacia).

Claims (2)

Purified retinoid X receptor homodimer. A method of screening a substance for its ability to affect the formation of a retinoid X receptor homodimer, comprising combining the substance with a solution containing a retinoid X receptor, and measuring the presence of homodimer formation, wherein The presence of said homodimer formation is measured by detecting activation of transcription by said retinoid X receptor homodimer.

Images