JP2006514614A - 11β-hydroxysteroid dehydrogenase type 1 and type 2 inhibitors - Google Patents

Abstract

ここで、Ｒ_１およびＲ_２のうちの１つは、式（ａ）の基であり、ここで、Ｒ_４は、Ｈおよびヒドロカルビルから選択され、Ｒ_５は、ヒドロカルビル基であり、Ｌは任意のリンカー基であるか、または、Ｒ_１およびＲ_２は一緒になって、基（式（ａ））で置換された環を形成し、ここで、Ｒ_３は、Ｈもしくは置換基であり、ここで、Ｘは、Ｓ、Ｏ、ＮＲ_６およびＣ（Ｒ_７）（Ｒ_８）から選択され、ここで、Ｒ_６は、Ｈおよびヒドロカルビル基から選択され、ここで、Ｒ_７およびＲ_８の各々は、独立して、Ｈおよびヒドロカルビル基から選択される。Compounds having the formula (I) are provided:
Embedded image

Wherein one of R ₁ and R ₂ is a group of formula (a), wherein R ₄ is selected from H and hydrocarbyl, R ₅ is a hydrocarbyl group, and L is optional Or R ₁ and R ₂ together form a ring substituted with a group (formula (a)), wherein R ₃ is H or a substituent; Wherein X is selected from S, O, NR ₆ and C (R ₇ ) (R ₈ ), wherein R ₆ is selected from H and a hydrocarbyl group, wherein R ₇ and R ₈ Each is independently selected from H and hydrocarbyl groups.

Description

(Field of Invention)
The present invention relates to certain compounds. In particular, the present invention provides compounds that can inhibit 11β-hydroxysteroid dehydrogenase (Up-HSD).

(Introduction)
The role of the glucocorticoid, glucocorticoid, is synthesized in the adrenal cortex from cholesterol. The major glucocorticoid in the human body is cortisol, which is synthesized and secreted in response to adrenocorticotropic hormone (ACTH) from the pituitary gland in a circadian sporadic manner. However, secretion of this hormone can also be stimulated by stress, exercise and infection. Cortisol circulates primarily in association with transcortin (cortisol binding protein) or albumin, and for biological processes, only a small fraction is free (5-10%) [1].

  Cortisol has a wide range of physiological effects, including regulation of carbohydrate, protein and lipid metabolism, regulation of normal growth and development, effects on cognitive function, resistance to stress, and mineralocorticoid activity. Can be mentioned. Cortisol acts in the opposite direction compared to insulin, i.e. stimulates blood gluconeogenesis, inhibits peripheral glucose uptake and increases blood glucose concentration. Glucocorticoids are also essential for regulating the immune response. When circulating at high concentrations, glucocorticoids are immunosuppressive and are used pharmacologically as anti-inflammatory agents.

  Glucocorticoids, like other steroid hormones, are lipophilic and freely penetrate cell membranes. Cortisol primarily binds to intracellular glucocorticoid receptors (GR) and then functions as a transcription factor to induce the expression of glucocorticoid responsive genes and consequently to induce protein synthesis.

(Role of 11β-HSD enzyme)
The conversion of cortisol (F) to its inactive metabolite, cortisone (E), by 11β-HSD was first described in the 1950s, after which biological significance for this conversion has been suggested. [2]. In 1983, Krozowski et al. Showed that the mineralocorticoid receptor (MR) has comparable binding affinity for glucocorticoids and mineralocorticoids [3]. The circulating concentration of cortisol is 100 times higher than that of aldosterone, and it is still clear how MR remains mineral corticoid-specific and not constantly occupied by glucocorticoids during stress or high activity I didn't. Earlier Ulick et al. [4] described a hypertensive condition known as “apparent mineralocorticoid excess” (AME), which interfered with peripheral metabolism of cortisol when aldosterone secretion from the adrenal gland was actually low. Was observed. These findings suggested a protective role for these enzymes. By converting cortisol to cortisone in mineralocorticoid-dependent tissues, the 11β-HSD enzyme protects MR from occupation by mineralocorticoids and allows it to be mineralcorticoid-specific. Aldosterone itself is protected from the enzyme by the presence of an aldehyde group at the C-18 position.

  The congenital deficiency of the 11β-HSD enzyme results in an overall occupation of MR by cortisol and high blood pressure and low potassium symptoms are seen in AME.

  Localization of 11β-HSD indicated that this enzyme and its activity are highly present in MR-dependent tissues (kidney and parotid gland). However, in tissues where MR is not mineralocorticoid-specific and is usually occupied by glucocorticoids, 11β-HSD is not present in these tissues (eg, heart and hippocampus) [5]. This study also showed that inhibition of 11β-HSD resulted in a loss of MR aldosterone specificity in these mineralocorticoid-dependent tissues.

  It has been shown that there are two isoenzymes of 11β-HSD. Both are members of the short-chain alcohol dehydrogenase (SCAD) superfamily that is widely conserved during evolution. The 11β-HSD type 2 functions as a dehydrogenase and converts a secondary alcohol group at the C-11 position of cortisol to a secondary ketone, resulting in cortisone with low metabolic activity. 11β-HSD type 1 is thought to function primarily as a reductase in vivo, which is the opposite of type 2 [6] [see below]. 11β-HSD type 1 and type 2 have only 30% amino acid homology.

（１１β−ＨＳＤ酵素活性）。

(11β-HSD enzyme activity).

  The intracellular activity of cortisol depends on the concentration of glucocorticoids and can be modified and independently controlled without being involved in the overall secretion and synthesis of hormones.

(Role of 11β-HSD type 1)
The orientation of 11β-HSD type 1 in vivo is generally accepted to be the opposite of type 2 dehydrogenation. Homozygous mice with a disrupted type 1 gene in vivo are unable to convert cortisone to cortisol, providing further evidence for the reductive activity of the enzyme [7]. 11β-HSD type 1 is expressed in tissues that are regulated by many important glucocorticoids (liver, pituitary, gonadal, brain, fat and adrenal gland), but the function of the enzyme in many of these tissues is Little is understood [8].

  The concentration of cortisone in the body is higher than that of cortisol, and cortisone also binds little to the binding globulins, making cortisone many times more bioavailable. Although cortisol is secreted by the adrenal cortex, there is increasing evidence that intracellular conversion of E to F can be an important mechanism in regulating the action of glucocorticoids [9].

  11β-HSD type 1 may allow certain tissues to convert cortisone to cortisol, increase local glucocorticoid activity, enhance the fit response, and cause failure of glucocorticoid activity. Offsets type 2 activity [10]. Enhancement of the stress response is particularly important in the brain, and high levels of 11β-HSD type 1 are found around the hippocampus and further elucidate the role of this enzyme. 11β-HSD type 1 also appears to play an important role in hepatocyte maturation [8]. Another emerging role for the 11β-HSD type 1 enzyme is in the detoxification process of many non-steroidal carbonyl compounds, since the reduction of the carbonyl group of many toxic compounds increases solubility and thus increases its exocrine secretion. Is a general method. The 11β-HSD type 1 enzyme has recently been shown to be active in lung tissue [11]. Type 1 activity is not seen until after birth, and therefore the harmful cigarettes of a mother who smokes during pregnancy before her child can metabolically detoxify this compound. Gives action.

(Role of 11β-HSD type 2)
As previously mentioned, 11β-HSD type 2 converts cortisol to cortisone and thus protects MR in many important regulatory tissues of the body. The importance of protecting MR from occupancy by glucocorticoids is seen in patients with AME or licorice intoxication. A deficiency or inability of type 2 enzyme results in hypertension syndrome and studies have shown that patients with hypertension syndrome have an increased urinary excretion ratio of cortisol: cortisone. This suggests a decrease in 11β-HSD type 2 activity with a reported decrease in the half-life of radiolabeled cortisol [12].

(Principle for the development of 11β-HSD inhibitors)
As mentioned above, prior cortisol counters the action of insulin, i.e. stimulates hepatic gluconeogenesis, inhibits peripheral glucose uptake and increases blood glucose levels. This action of cortisol appears to be enhanced in patients suffering from glucose intolerance or diabetic meritis. Inhibition of the enzyme 11β-HDS type 1 increases glucose uptake, inhibits hepatic gluconeogenesis, leading to a decrease in circulating glucose levels. Therefore, the development of potent 11β-HSD type 1 inhibitors may have a therapeutic potential to consider for symptoms associated with elevated blood glucose levels.

  Glucocorticoid excess can result in neuronal dysfunction and can also impair cognitive function. Specific 11β-HSD type 1 inhibitors may be somewhat important by reducing neuronal dysfunction and loss of cognitive function associated with aging and blocking the conversion of cortisone to cortisol.

  Glucocorticoids also have an important role in the regulatory part of the immune response [13]. Glucocorticoids can suppress cytokine production and regulate receptor levels. They are also involved in determining whether T helper (Th) lymphocytes progress to the Th1 or Th2 phenotype. These two different types of Th cells secrete different profiles of cytokines, and Th2 is dominant in the glucocorticoid environment. By inhibiting 11β-HSD type 1, the Th1 cytokine response works favorably. It is also possible to inhibit 11β-HSD type 2, and therefore it may be possible to enhance the anti-inflammatory action of glucocorticoids by inhibiting the inactivation of cortisol.

  Aspects of the invention are defined in the appended claims.

(Summary of the present invention)
In one aspect, the present invention provides a compound of formula I:

を有する化合物を提供し、ここで、
Ｒ_１およびＲ_２のうちの１つは、以下の式：

Wherein a compound having the formula:
One of R ₁ and R ₂ has the following formula:

の基であり、ここで、Ｒ_４は、Ｈおよびヒドロカルビルから選択され、Ｒ５はヒドロカルビル基であり、かつＬは任意のリンカー基であるか、または、
Ｒ_１およびＲ_２が一緒になって、以下の基：

Where R ₄ is selected from H and hydrocarbyl, R 5 is a hydrocarbyl group and L is any linker group, or
R ₁ and R ₂ together are the following groups:

で置換された環を形成し、ここで、Ｒ_３は、Ｈまたは置換基であり、ここで、Ｘは、Ｓ、Ｏ、ＮＲ_６およびＣ（Ｒ_７）（Ｒ_８）から選択され、ここで、Ｒ_６は、Ｈおよびヒドロカルビル基から選択され、ここで、Ｒ_７およびＲ_８の各々は、独立して、Ｈおよびヒドロカルビル基から選択される。

In which R ₃ is H or a substituent, wherein X is selected from S, O, NR ₆ and C (R ₇ ) (R ₈ ), wherein Wherein R ₆ is selected from H and hydrocarbyl groups, wherein each of R ₇ and R ₈ is independently selected from H and hydrocarbyl groups.

In one aspect, the present invention provides:
(I) Formula I:

を有する化合物を含み、
（ｉｉ）必要に応じて薬学的に受容可能なキャリア、希釈剤、賦形剤またはアジュバントと配合された薬学的組成物を提供し、ここで、
Ｒ_１およびＲ_２のうちの１つは、以下の式：

Comprising a compound having
(Ii) providing a pharmaceutical composition optionally combined with a pharmaceutically acceptable carrier, diluent, excipient or adjuvant, wherein
One of R ₁ and R ₂ has the following formula:

の基であり、ここで、Ｒ_４は、Ｈおよびヒドロカルビルから選択され、Ｒ５は、ヒドロカルビル基であり、かつ、Ｌは任意のリンカー基であるか、または、
Ｒ_１およびＲ_２は、一緒になって、以下の基：

Where R ₄ is selected from H and hydrocarbyl, R 5 is a hydrocarbyl group and L is an optional linker group, or
R ₁ and R ₂ together are the following groups:

で置換された環を形成し、ここで、Ｒ_３は、Ｈまたは置換基であり、ここで、Ｘは、Ｓ、Ｏ、ＮＲ_６およびＣ（Ｒ_７）（Ｒ_８）から選択され、ここで、Ｒ_６は、Ｈおよびヒドロカルビル基から選択され、ここで、Ｒ_７およびＲ_８の各々は、独立して、Ｈおよびヒドロカルビル基から選択される。

In which R ₃ is H or a substituent, wherein X is selected from S, O, NR ₆ and C (R ₇ ) (R ₈ ), wherein Wherein R ₆ is selected from H and hydrocarbyl groups, wherein each of R ₇ and R ₈ is independently selected from H and hydrocarbyl groups.

  In one aspect, the present invention provides a compound of formula I:

を有する、医療における使用のための化合物を提供し、ここで、
Ｒ_１およびＲ_２のうちの１つは、以下の式：

Having a compound for use in medicine, wherein:
One of R ₁ and R ₂ has the following formula:

の基であり、ここで、Ｒ_４は、Ｈおよびヒドロカルビルから選択され、Ｒ５は、ヒドロカルビル基であり、かつ、Ｌは任意のリンカー基であるか、または、
Ｒ_１およびＲ_２は一緒になって、以下の基：

Where R ₄ is selected from H and hydrocarbyl, R 5 is a hydrocarbyl group and L is an optional linker group, or
R ₁ and R ₂ together are the following groups:

で置換された環を形成し、ここで、Ｒ_３は、Ｈまたは置換基であり、ここで、Ｘは、Ｓ、Ｏ、ＮＲ_６およびＣ（Ｒ_７）（Ｒ_８）から選択され、ここで、Ｒ_６は、Ｈおよびヒドロカルビル基から選択され、ここで、Ｒ_７およびＲ_８の各々は、独立して、Ｈおよびヒドロカルビル基から選択される。

In which R ₃ is H or a substituent, wherein X is selected from S, O, NR ₆ and C (R ₇ ) (R ₈ ), wherein Wherein R ₆ is selected from H and hydrocarbyl groups, wherein each of R ₇ and R ₈ is independently selected from H and hydrocarbyl groups.

  In one aspect, the invention provides the use of a compound in the manufacture of a medicament for use in the treatment of a condition or disease associated with 11β-HSD, wherein the compound has the formula I:

を有し、ここで、Ｒ_１およびＲ_２のうちの１つは、以下の式：

Where one of R ₁ and R ₂ has the following formula:

の基であり、ここで、Ｒ_４はＨおよびヒドロカルビルから選択され、Ｒ５は、ヒドロカルビル基であり、かつ、Ｌは任意のリンカー基であるか、または、Ｒ_１およびＲ_２は一緒になって、以下の基：

Where R ₄ is selected from H and hydrocarbyl, R 5 is a hydrocarbyl group and L is an optional linker group, or R ₁ and R ₂ are taken together The following groups:

で置換された環を形成し、ここで、Ｒ_３は、Ｈまたは置換基であり、ここで、Ｘは、Ｓ、Ｏ、ＮＲ_６およびＣ（Ｒ_７）（Ｒ_８）から選択され、ここで、Ｒ_６は、Ｈおよびヒドロカルビル基から選択され、ここで、Ｒ_７およびＲ_８の各々は、独立して、Ｈおよびヒドロカルビル基から選択される。

In which R ₃ is H or a substituent, wherein X is selected from S, O, NR ₆ and C (R ₇ ) (R ₈ ), wherein Wherein R ₆ is selected from H and hydrocarbyl groups, wherein each of R ₇ and R ₈ is independently selected from H and hydrocarbyl groups.

(Several advantages)
One important advantage of the present invention is that the compounds of the present invention can function as 11β-HSD inhibitors. This compound can inhibit the interchange of an inactive 11-ketosteroid with its active hydroxy equivalent. Thus, the present invention provides a method by which the conversion of the inactive form to the active form can be controlled, which translates into a useful therapeutic effect that can be obtained as a result of such control. More specifically (but not limited to), the present invention relates to the interchange between cortisone and cortisol in humans.

  Another advantage of the compounds of the present invention is that they can be potent 11β-HSD inhibitors in vivo.

  Some of the compounds of the present invention are also advantageous in that they can be orally active.

  The present invention includes (i) regulation of carbohydrate metabolism, (ii) regulation of protein metabolism, (iii) regulation of lipid metabolism, (iv) regulation of normal growth and / or development, (v) impact on cognitive function, (Vi) may be provided for a medicament for one or more of resistance to stress and mineralocorticoid activity.

  Some of the compounds of the present invention may also be useful for inhibiting liver gluconeogenesis. The present invention may also provide a medicament for reducing the effects of endogenous glucocorticoids in diabetes mellitus, obesity (including afferent obesity), neuronal loss and / or old age cognitive impairment. Accordingly, in a further aspect, the present invention provides the use of an inhibitor of 11β-HSD in the manufacture of a medicament for producing one or more therapeutic effects in a patient to which the medicament is administered, wherein said therapeutic effect Results from inhibition of hepatic gluconeogenesis, increased insulin sensitivity in adipose tissue and muscle, and inhibition or reduction of neuronal loss / cognitive impairment due to glucocorticoid-enhanced neurotoxicity or neurological deficit or injury Selected.

  From an alternative perspective, the present invention relates to liver insulin resistance, adipose tissue insulin resistance, muscle insulin resistance, neuronal loss or dysfunction caused by glucocorticoid-enhanced neurotoxins, as well as the conditions described above. A method for the treatment of a human or animal subject suffering from a condition selected from the group consisting of any combination of the above, wherein the method comprises administering to the patient a pharmaceutically active amount of a compound described in the present invention. Administering a penalty comprising:

  Some of the compounds of the invention may be used to treat cancer (eg, breast cancer), as well as (alternatively) non-malignant conditions (eg, when the drug may need to be administered from an early age) It can be useful for the prevention of autoimmune diseases).

(Detailed Description of the Invention)
In one aspect, the present invention provides compounds of formula I

を有する化合物を提供し、ここで、
Ｒ_１およびＲ_２のうちの１つは、以下の式：

Wherein a compound having the formula:
One of R ₁ and R ₂ has the following formula:

の基であり、ここで、Ｒ_４は、Ｈおよびヒドロカルビルから選択され、Ｒ_５は、ヒドロカルビル基であり、かつ、Ｌは任意のリンカー基であるか、または、
Ｒ_１およびＲ_２は一緒になって、以下の基：

Wherein R ₄ is selected from H and hydrocarbyl, R ₅ is a hydrocarbyl group and L is an optional linker group, or
R ₁ and R ₂ together are the following groups:

で置換された環を形成し、ここで、Ｒ_３は、Ｈまたは置換基であり、ここで、Ｘは、Ｓ、Ｏ、ＮＲ_６およびＣ（Ｒ_７）（Ｒ_８）から選択され、ここで、Ｒ_６は、Ｈおよびヒドロカルビル基から選択され、ここで、Ｒ_７およびＲ_８の各々は、独立して、Ｈおよびヒドロカルビル基から選択される。

In which R ₃ is H or a substituent, wherein X is selected from S, O, NR ₆ and C (R ₇ ) (R ₈ ), wherein Wherein R ₆ is selected from H and hydrocarbyl groups, wherein each of R ₇ and R ₈ is independently selected from H and hydrocarbyl groups.

In one aspect, the present invention provides:
(I) comprising a compound having the formula I as defined above,
(Ii) Provide a pharmaceutical composition, optionally combined with a pharmaceutically acceptable carrier, diluent, excipient or adjuvant.

  In one aspect, the present invention provides a compound having formula I as defined above for use in medicine.

  In one aspect, the invention provides the use of a compound having formula I as defined above in the manufacture of a medicament for use in the treatment of a condition or disease associated with 11β-HSD.

  In one aspect, the present invention provides the use of a compound having formula I as defined above in the manufacture of a medicament for use in the treatment of a condition or disease associated with harmful 11β-HSD levels.

  In one aspect, the present invention provides the use of a compound having formula I as defined above in the manufacture of a medicament for inhibiting 11β-HSD activity.

  In one aspect, the present invention provides the use of a compound having formula I as defined above in the manufacture of a medicament for inhibiting 11β-HSD activity.

  In one aspect, the present invention provides a method, wherein (a) performing an 11β-HSD assay with one or more candidate compounds having formula I as defined above; (b) ) Determining whether the one or more candidate compounds can modulate 11β-HSD activity; and (c) selecting the one or more candidate compounds capable of modulating 11β-HSD activity. .

  In one aspect, the present invention provides a method, wherein (a) performing an 11β-HSD assay with one or more candidate compounds having formula I as defined above; (b) ) Determining whether the one or more candidate compounds can inhibit 11β-HSD activity; and (c) selecting the one or more candidate compounds capable of inhibiting 11β-HSD activity. .

In one aspect, the present invention provides the following:
A compound identified by the above method,
The use of the above compounds in medicine,
A pharmaceutical composition comprising the above compound, optionally combined with a pharmaceutically acceptable carrier, diluent, excipient or adjuvant, for use in the treatment of a condition or disease associated with 11β-HSD Use of the compound in the manufacture of a medicament, and Use of the compound in the manufacture of a medicament for use in the treatment of a condition or disease associated with harmful 11β-HSD levels.

  For ease of reference, these and other aspects of the invention will now be discussed under the appropriate section headings. However, the teachings under each section need not be limited to each particular section.

(Preferred aspects)
In one preferred aspect, the compounds of the invention have the formula II:

を有する。

Have

  In one preferred aspect, L is absent. In this aspect, the present invention provides a compound of formula I:

を有する化合物を提供し、ここで、
Ｒ_１およびＲ_２のうちの１つは、以下の式：

Wherein a compound having the formula:
One of R ₁ and R ₂ has the following formula:

の基であり、ここで、Ｒ_４は、Ｈおよびヒドロカルビルから選択され、Ｒ_５は、ヒドロカルビル基であり、かつ、Ｌは任意のリンカー基であるか、または、
Ｒ_１およびＲ_２は一緒になって、以下の基：

Wherein R ₄ is selected from H and hydrocarbyl, R ₅ is a hydrocarbyl group and L is an optional linker group, or
R ₁ and R ₂ together are the following groups:

で置換された環を形成し、ここで、Ｒ_３は、Ｈまたは置換基であり、ここで、Ｘは、Ｓ、Ｏ、ＮＲ_６およびＣ（Ｒ_７）（Ｒ_８）から選択され、ここで、Ｒ_６は、Ｈおよびヒドロカルビル基から選択され、ここで、Ｒ_７およびＲ_８の各々は、独立して、Ｈおよびヒドロカルビル基から選択される。

In which R ₃ is H or a substituent, wherein X is selected from S, O, NR ₆ and C (R ₇ ) (R ₈ ), wherein Wherein R ₆ is selected from H and hydrocarbyl groups, wherein each of R ₇ and R ₈ is independently selected from H and hydrocarbyl groups.

In one preferred aspect of the compounds of the present invention, R ₁ and R _{2 taken} together are the following groups:

で置換された環を形成する。

To form a ring substituted with.

In one preferred aspect of the compounds of the present invention, R ₁ and R ₂ are taken together to form a carbocyclic ring.

In one preferred aspect of the compounds of the present invention, R ₁ and R ₂ are taken together to form a 6-membered ring.

In one preferred aspect of the compounds of the present invention, R ₁ and R ₂ are taken together to form a 6-membered carbocyclic ring.

In one preferred aspect of the compounds of the present invention, R ₁ and R ₂ are taken together to form an aryl ring.

  Preferred compounds of the invention have the following formula:

のうちの１つを有するものである。

One of them.

In one preferred aspect of the invention, R ₃ is selected from H, hydrocarbyl, —S hydrocarbyl, —S—H, halogen and N (R ₉ ) (R ₁₀ ), each of R ₉ and R _{10 being} Independently selected from H and hydrocarbyl groups.

In one preferred aspect of the invention, R ₃ is H, hydroxy, alkyl (especially a C ₁ -C ₁₀ alkyl group, a C ₁ -C ₆ alkyl group (eg, a C ₁ -C ₃ alkyl group), methyl, ethyl , N-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, n-pentyl and other pentyl isomers, and n-hexyl and other hexyl isomers), alkoxy (especially C ₁ -C ₁₀ It is selected from an alkoxy group, C ₁ -C ₆ alkoxy (eg, C ₁ -C ₃ alkoxy group, methoxy, ethoxy, propoxy, etc.), alkynyl (eg, ethynyl) or halogen (eg, fluoro substituent).

If R ₃ is -S- hydrocarbyl, preferably, _{R 3} is, -S- alkyl, -S- carboxylic acid is selected from S- ether and -S- amides, preferably, _{-S-C. 1 to} Selected from C ₁₀ alkyl, —S—C _1-10 carboxylic acid, —S—C _1-10 ether and —S—C _1-10 amide.

In one preferred aspect of the invention, R ₃ is —CH ₃ .

  Further preferred compounds of the invention are those having one of the following formulas:

化合物が式Ｉａ、式ＶＩＩＩ、式ＩＸ、式Ｘ、式Ｘａ、式ＸＩまたは式ＸＩａを有する場合のような、本発明のさらなる好ましい局面において、Ｒ_３は、Ｏ、ヒドロカルビルおよびＮ（Ｒ_９）から選択され、ここで、Ｒ_９は、Ｈおよびヒドロカルビル基から選択される。より好ましくは、Ｒ_３は、Ｏ、Ｃ_１〜Ｃ_１０アルケニル基（例えば、Ｃ_１〜Ｃ_６アルケニル基およびＣ_１〜Ｃ_３アルケニル基）ＮＨおよびＮ−Ｃ_１〜Ｃ_１０アルキル基（例えば、Ｎ−Ｃ_１〜Ｃ_６アルキル基およびＮ−Ｃ_１〜Ｃ_３アルキル基）から選択される。

In further preferred aspects of the invention, such as when the compound has Formula Ia, Formula VIII, Formula IX, Formula X, Formula Xa, Formula XI or Formula XIa, R ₃ is O, hydrocarbyl and N (R ₉ ) Where R ₉ is selected from H and a hydrocarbyl group. More preferably, R ₃ is O, a C ₁ -C ₁₀ alkenyl group (eg, a C ₁ -C ₆ alkenyl group and a C ₁ -C ₃ alkenyl group) NH and an N—C ₁ -C ₁₀ alkyl group (eg, _N-C 1 ~C ₆ alkyl group and _N-C 1 _{~C 3} is selected from alkyl groups).

In a further preferred aspect of the invention, R ₄ is selected from H and a C ₁ -C ₁₀ alkyl group (eg a C ₁ -C ₆ alkyl group and a C ₁ -C ₃ alkyl group). Preferably R ₄ is H.

In a further preferred aspect of the invention, R ₄ has the following formula:

の基である。

It is the basis of.

  In these aspects,

のような、上に示される基は、以下の式：

The groups shown above, such as, have the following formula:

であり得、ここで、各Ｒ_５は、独立して、ヒドロカルビル基から選択される。各Ｒ_５は、他のＲ_５とは同じであっても異なっていてもよい。１つの局面において、２つのＲ_５基は、同じである。

Where each R ₅ is independently selected from hydrocarbyl groups. Each R ₅ may be the same as or different from the other R ₅ . In one aspect, the two R ₅ groups are the same.

In some preferred aspects of the invention, R ₅ is a cyclic hydrocarbyl group. Preferably R ₅ is a cyclic hydrocarbyl group containing a hydrocarbon ring.

R ₅ can be a substituted ring or an unsubstituted ring. In some preferred aspects of the invention, R ₅ is a substituted ring.

Preferably R ₅ is a carbocyclic ring.

Preferably R ₅ is a six-membered ring.

Preferably R ₅ is a 6-membered carbocyclic ring. More preferably, R ₅ is a substituted 6 membered carbocyclic ring.

In some preferred aspects of the invention, R ₅ is an aryl ring. Preferably R ₅ is a substituted aryl ring.

In one highly preferred aspect, R ₅ has the following formula:

を有する基であり、ここで、Ｒ_１１、Ｒ_１２、Ｒ_１３、Ｒ_１４およびＲ_１５の各々は、独立して、Ｈ、ハロゲンおよびヒドロカルビル基から選択される。

Wherein each of R ₁₁ , R ₁₂ , R ₁₃ , R ₁₄ and R ₁₅ is independently selected from H, halogen and hydrocarbyl groups.

Preferably, each of R ₁₁ , R ₁₂ , R ₁₃ , R ₁₄ and R ₁₅ is independently H, halogen, alkyl (eg, C ₁ -C ₆ alkyl, phenyl, O-alkyl, O-phenyl, Selected from nitrile, haloalkyl (eg CF ₃ , CCl ₃ and CBR ₃ ), carboxyalkyl, —CO ₂ H, CO ₂ alkyl and NH-acetyl groups.

Two or more of R ₁₁ , R ₁₂ , R ₁₃ , R ₁₄ and R ₁₅ can be linked to form a ring. Two or more of R ₁₁ , R ₁₂ , R ₁₃ , R ₁₄ and R ₁₅ may or may not be in close proximity. The ring can be a carbocyclic ring or a heterocyclic ring. The ring can be optionally substituted with any of the R ₁₁ , R ₁₂ , R ₁₃ , R ₁₄ and R ₁₅ substituents listed above. Two or more of R ₁₁ , R ₁₂ , R ₁₃ , R ₁₄ and R ₁₅ are linked to form the following group:

の環を形成し得、ナフチル、キノリル、テトラヒドロキノリルまたはベンゾテトラヒドロピラニルを提供し得、これらの各々は、置換されていても置換されていなくてもよい。

And can provide naphthyl, quinolyl, tetrahydroquinolyl or benzotetrahydropyranyl, each of which can be substituted or unsubstituted.

(Substituent)
The compounds of the present invention may have substituents other than the ring systems shown herein. Furthermore, the ring systems herein are given as general formulas and should thus be interrupted. In the absence of a specifically indicated substituent on a given ring member, the ring member may be substituted with any substituent and H is only one example. A ring system can contain one or more degrees of unsaturation, for example, in some aspects, one or more rings of the ring system are aromatic. The ring system can be carbocyclic or can contain one or more heteroatoms.

  The compounds of the present invention, in particular, the ring system compounds of the present invention may contain substituents other than those shown herein. By way of example, these other substituents can be one or more of the following: one or more halo groups, one or more O groups, one or more hydroxy groups, one or more amino groups, one type The sulfur-containing group (S) may be one or more hydrocarbyl groups (S) (for example, an oxyhydrocarbyl group).

In general terms, the ring system of the compounds of the invention may contain various non-interfering substituents. In particular, the ring system comprises one or more hydroxy, alkyl (especially lower (C ₁ -C ₆ ) alkyl (eg methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, n-pentyl and other pentyl isomers, and n-hexyl and other hexyl isomers)), alkoxy (eg methoxy, ethoxy, propoxy, etc.), alkynyl (eg ethynyl) or halogen (eg fluoro substituents) ).

  For some compounds of the invention, the compound may be substituted with a hydrocarbylsulfanyl group. The term “hydrocarbylsulfanyl” means a group comprising at least one hydrocarbyl group (as defined herein) and sulfur, preferably —S-hydrocarbyl, more preferably —S—. Means hydrocarbon. This sulfur group can be oxygenated as required.

Preferably, the hydrocarbylsulfanyl group is —S—C _1-10 alkyl, more preferably —S—C _1-5 alkyl, more preferably —S—C _1-3 alkyl, and more _{preferably, -S-CH 2 CH 2 CH} 3, a _-S-CH 2 _{CH 3} or -SCH _3.

(Further aspects)
For some applications, preferably the compound has a reversible action.

  For some applications, preferably the compound has an irreversible action.

  In one embodiment, the compounds of the invention are useful for the treatment of breast cancer.

  The compounds of the present invention may be in the form of a salt.

  The present invention also includes novel intermediates useful for preparing the compounds of the present invention. For example, the present invention includes novel alcohol precursors for compounds. As a further example, the present invention includes bis-protected precursors of compounds. Examples of each of these precursors are presented herein. The invention also encompasses processes involving each or both of these precursors for the synthesis of the compounds of the invention.

(Steroid dehydrogenase)
11β steroid dehydrogenase may be referred to as “11β-HSD” or shortly “HD”.

  In some aspects of the invention, the 11β-HSD is preferably of the 11β-HSD type 1.

  In some aspects of the invention, the 11β-HSD is preferably of the 11β-HSD type 2.

(Inhibition of steroid dehydrogenase)
Several disease states associated with HD activity are believed to be due to the conversion of inactive cortisone to active cortisol. In disease states associated with HD activity, it is desirable to inhibit HD activity.

  As used herein, the term “inhibit” includes reducing and / or eliminating and / or masking and / or avoiding the harmful activity of HD.

(HD inhibitor)
In accordance with the invention, the compounds of the invention can act as HD inhibitors.

  Here, as used herein with respect to the compounds of the invention, the term “inhibitor” may inhibit HD activity—eg, reduce and / or eliminate and / or mask and / or eliminate the harmful activity of HD. Or it means a compound that can be avoided. HD inhibitors can function as antagonists.

  The ability of a compound to inhibit steroid dehydrogenase activity can be assessed using an appropriate assay protocol presented in the Examples section.

  The compounds of the present invention may have other advantageous properties in addition to or in place of its ability to inhibit HD activity.

(Hydrocarbyl)
As used herein, the term “hydrocarbyl group” means a group that includes at least C and H and may optionally include one or more other suitable substituents. Examples of such substituents can include halo, alkoxy, nitro, alkyl groups, cyclic groups, and the like. In addition to the possibility of a substituent being a cyclic group, a combination of substituents can form a cyclic group. If the hydrocarbyl group contains one or more C, these carbons need not necessarily be bonded to each other. For example, at least two carbons can be bonded via a suitable element or group. Thus, the hydrocarbyl group can contain heteroatoms. Suitable heteroatoms will be apparent to those skilled in the art and include, for example, sulfur, nitrogen and oxygen. A non-limiting example of a hydrocarbyl group is an acyl group.

  A typical hydrocarbyl group is a hydrocarbon group. Here, the term “hydrocarbon” means any one of an alkyl group, an alkenyl group, an alkynyl group in which the group may be linear, branched or cyclic, or an aryl group. The term “hydrocarbon” also includes those groups that are optionally substituted. Where the hydrocarbon is a molecular structure with substituents, the substitution can be either on the hydrocarbon backbone or on the branched chain; alternatively, the substitution can be on the hydrocarbon backbone and branched chain.

  In some aspects of the invention, one or more hydrocarbyl groups are independently an optionally substituted alkyl group, an optionally substituted haloalkyl group, an aryl group, an alkylaryl group, an alkylaryl. Selected from alkyl and alkene groups.

In some aspects of the invention, the one or more hydrocarbyl groups are independently selected from C ₁ -C ₁₀ alkyl groups (eg, C ₁ -C ₆ alkyl groups and C ₁ -C ₃ alkyl groups). The Representative alkyl groups include C ₁ alkyl, C ₂ alkyl, C ₃ alkyl, C ₄ alkyl, C ₅ alkyl, C ₇ alkyl and C ₈ alkyl.

In some aspects of the present invention, one or more hydrocarbyl groups is _{independently,} C 1 _{-C 10} haloalkyl _group, C 1 -C ₆ haloalkyl _group, C 1 -C ₃ haloalkyl _group, C 1 _{-C 10} bromo alkyl groups _are selected from C 1 -C ₆ bromoalkyl group, and _C 1 -C ₃ bromoalkyl group. Representative haloalkyl groups include C ₁ haloalkyl, C ₂ haloalkyl, C ₃ haloalkyl, C ₄ haloalkyl, C ₅ haloalkyl, C ₇ haloalkyl, C ₈ haloalkyl, C ₁ bromoalkyl, C ₂ bromoalkyl, C ₃ bromoalkyl. , C ₄ bromoalkyl, C ₅ bromoalkyl, C ₇ bromoalkyl and C ₈ bromoalkyl.

In some aspects of the invention, one or more hydrocarbyl groups are independently an aryl group, an alkylaryl group, an alkylarylalkyl group, — (CH ₂ ) _1-10 -aryl, — (CH ₂ ) _{1. ~10 -Ph, (CH 2) 1~10} -Ph-C 1~10 _{alkyl, - (CH 2) 1~5 -Ph} , (CH 2) 1~5 -Ph-C 1~5 alkyl, - ( CH ₂₎ 1~3 _-Ph, is selected from _{(CH 2) 1~3 -Ph-C} 1~3 alkyl, _-CH 2 -Ph and _{-CH 2 -Ph-C (CH 3} ) 3. The aryl group can contain heteroatoms. Thus, one aryl group or one or more aryl groups can be carbocyclic or heterocyclic. Representative heteroatoms include O, N and S, with N being particularly preferred.

In some aspects of the present invention, one or more hydrocarbyl groups is independently, _{- (CH 2) 1 to 10} - _{cycloalkyl, - (CH 2) 1~10 -C} 3~10 cycloalkyl, - (CH ₂ ) _1-7 -C _3-7 cycloalkyl,-(CH ₂ ) _1-5 -C _3-5 cycloalkyl,-(CH ₂ ) _1-3 -C _3-5 cycloalkyl and -CH ₂ Selected from the group consisting of —C ₃ cycloalkyl.

In some aspects of the invention, one or more hydrocarbyl groups are independently alkene groups. Representative alkene _group, C 1 _{-C 10} alkene _group, C i -C _e alkene _group, C 1 -C ₃ alkene group _{(e.g., C 1, C 2, C} 3, C 4, C 5, C ₆ or C ₇ alkene group). In preferred aspects, the alkene group contains 1, 2 or 3 C═C bonds. In a preferred aspect, the alkene group contains one C═C bond. In some preferred aspects, at least one C═C bond is included or only one C═C bond is at the C-terminus of the alkene chain, ie, the bond is distal of the chain relative to the ring system. At the end.

  In some aspects of the invention, the one or more hydrocarbyl groups are independently selected from the group consisting of oxyhydrocarbyl groups.

(Oxyhydrocarbyl)
The term “oxyhydrocarbyl” group, as used herein, means a group that includes at least C, H, and O, and may optionally include one or more other suitable substituents. Examples of such substituents may include halo groups, alkoxy groups, nitro groups, alkyl groups, cyclic groups and the like.

  In addition to the possibility that the substituents are cyclic groups, combinations of substituents can form cyclic groups. If the oxyhydrocarbyl group contains one or more C, these carbons need not necessarily be bonded to each other. For example, at least two carbons can be bonded via a suitable element or group. Thus, an oxyhydrocarbyl group can contain heteroatoms. Suitable heteroatoms will be apparent to those skilled in the art and include, for example, sulfur and nitrogen.

  In one embodiment of the invention, the oxyhydrocarbyl group is an oxygen hydrocarbon group.

  Here, the term “oxygen hydrocarbon” means any one of an alkoxy group, an oxyalkenyl group, an oxyalkynyl group or an oxyaryl group which may be linear, branched or cyclic. The term “oxygen hydrocarbon” also includes those groups that are optionally substituted. When the oxygen hydrocarbon is a branched structure with substituents, the substitution can be either on the hydrocarbon backbone or on the branched chain; alternatively, the substitution is on the hydrocarbon backbone and on the branched chain possible.

Typically, the oxyhydrocarbyl group is of the formula C _1-4 O (eg C _1-3 O).

(Animal assay model for determining osteogenic activity (protocol 1))
(Lack of in vivo estrogen production)
The compounds of the invention can be studied using animal models, particularly in ovariectomized rats. In this model, compounds that are estrogenic stimulate uterine growth.

  The compound (10 mg / Kg / day for 5 days) was orally administered to rats, and another group of animals received vehicle only (propylene glycol). A further group was administered estate, an estrogenic compound, at 10 μg / day subcutaneously for 5 days. At the end of the study, the uterus was removed and weighed, and the results were expressed as uterine weight / total weight × 100.

  Compounds that did not have a significant effect on uterine growth are not estrogenic.

(Reporter)
A variety of reporters can be used in the assay methods (and screens) of the invention, with preferred reporters conveniently providing a detectable marker (eg, by spectroscopy). As an example, the reporter gene can encode an enzyme that catalyzes a reactant that alters light absorption properties.

  Other protocols include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and fluorescence cell analysis separation device (FACS). A two-site monoclonal-based immunoassay using monoclonal antibodies against two non-interfering epitopes can further be used. These and other assays can be found elsewhere in Hampton R et al. (1990, Serial Methods, A Laboratory Manual, APS Press, St Paul MN) and Madox DE et al. (1983, J Exp Med 15: 8: 121 1). be written.

  Examples of reporter molecules include (but are not limited to (β-galactosidase, invertase, green fluorescent protein, luciferase, chloramphenicol, acetyltransferase, (-glucuronidase, exo-glucanase and glucoamylase). Radiolabeled or fluorescently labeled nucleotides can be incorporated into the generator transcript, which is then identified when bound to the oligonucleotide probe.

  As a further example, many companies (eg, Pharmacia Biotech (Piscataway, NJ), Promega (Madison, Wis.) And US Biochemical Corp (Cleveland, OH) provide protocols for commercial kits and assay procedures. Suitable reporter molecules or labels include radionuclides, enzymes, fluorescence, chemiluminescence, or chromogenic factors and substrates, cofactors, inhibitors, magnetic particles, etc. As US patents teaching the use of such labels US-A-3817837; US-A-3850752; US-A-3939350: US-A-3996345; US-A-4277437: US-A-4275149 and US-A-4366241. It is.

(Host cell)
In the context of the present invention, the term “host cell” includes any cell that may contain a target for an agent of the invention.

  Accordingly, a further embodiment of the present invention provides a host cell transformed or transfected with a polynucleotide that is a target of the present invention or expresses a target of the present invention. Preferably, the polynucleotide is carried in a vector for replication and expression of the polynucleotide that is the target or expresses the target. The cells are selected to be compatible with the vector and can be, for example, prokaryotic (eg, bacterial) cells, fungal cells, yeast cells, or plant cells.

  Gram-negative bacteria E. coli is widely used as a host for heterologous gene expression. However, large amounts of heterologous proteins tend to accumulate in the cell. E. Subsequent purification of the desired protein from the E. coli intracellular protein bulk can sometimes be difficult.

  E. In contrast to E. coli, bacteria from the genus Bacillus are very suitable as heterologous hosts because of their ability to secrete proteins into the culture medium. Other bacteria suitable as hosts are those from the genera Streptomyces and Pseudomonas.

  Depending on the nature of the polynucleotide encoding the polypeptide of the invention and / or the desirability for further processing of the expressed protein, eukaryotic hosts (eg, yeast or other fungi) may be preferred. . Usually, yeast cells are preferred over fungal cells because they are easier to manipulate. However, some proteins are either secreted little from yeast cells, or in some cases are not properly processed (eg, hyperglycosylation in yeast). Different fungal host organisms should be selected.

  Examples of suitable expression hosts within the scope of the present invention include fungi (eg Aspergillus species (eg as described in EP-A-0184438 and EP-A-0284603) and Trichoderma species); bacteria ( For example, Bacillus species (eg, as described in EP-A-0134048 and EP-A-0253455); Streptomyces species and Pseudomonas species; and yeast (eg, Kluyveromyces species (eg, EP-A-0096430 and EP-A-0301670))) and Saccharomyces species. By way of example, representative expression hosts are Aspergillus niger, Aspergillus niger var. tubeigenis, Aspergillus niger var. awamori, Aspergillus aculeatis, Aspergillus nidulans, Aspergillus orvzae, Trichoderma reesei, Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, may be selected from Kluyveromyces lactis and Saccharomyces cerevidiae.

  The use of appropriate hosts (eg, yeast host cells, fungal host cells, and plant host cells) can be post-translational as may be required to confer optimal biological activity in the recombinant expression products of the invention. Modifications such as myristoylation, glycosylation, cleavage, lipidation and phosphorylation of tyrosine, serine or threonine may be provided.

(Living)
In the context of the present invention, the term “organism” includes any organism that may comprise a target according to the present invention and / or products obtained from that organism. Examples of organisms can include fungi, yeasts, or plants.

  In the context of the present invention, the term “transgenic organism” includes any organism comprising a target according to the present invention and / or the resulting product.

(Host cell / host organism transformation)
As indicated initially, the host organism can be prokaryotic or eukaryotic. Examples of suitable prokaryotic hosts include E. coli. E. coli and Bacillus subtilis. Teachings about transformation of prokaryotic hosts are well documented in the art, for example, Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd edition, 1989, Cold Spring Harbor Laboratory Press) and Ausubel et al. Molecular Biology (1995), John Wiley & Sons, Inc. checking ...

  If a prokaryotic host is used, the nucleotide sequence may need to be appropriately modified (eg, by removal of introns) prior to transformation.

  In another embodiment, the transgenic organism can be a yeast. In this regard, yeast is also widely used as a vehicle for heterologous gene expression. Saccaromyces cerevisiae species has a long history of industrial use, including use for heterologous gene expression. For the expression of heterologous genes in Saccharomyces cerevisiae, see Goodey et al. (1987, Yeast Biotechnology, D. R. Berry et al., Pages 401-429, Allen and Unwin, London and King et al. (1989, Molecule Y). EF Walton and GT Yarronton, pp. 107-133, Blackie, Glasgow).

  For various reasons, Saccharomyces cerevisiae is well suited for heterologous gene expression. First, it is non-pathogenic to humans and cannot produce certain endotoxins. Second, it has a long history of safe use, centuries after commercial development for various purposes. This resulted in wide public approval. Third, extensive commercial use and research directed at this organism has provided an insight into the genetics and physiology of Saccharomyces cerevisiae and the characterization of large-scale fermentation.

  A review of the principles of heterologous gene expression and gene product secretion in Saccharomyces cerevisiae can be found in E. Hinchcliffe E.M. Kenny (1993, “Yeast as a vehicle for the expression of heterogenes”, Yeasts, Vol. 5, Anthony H. Rose and J. St. Harrison, 2nd edition, Ped. Accade.

  Various types of yeast vectors are available, including integration vectors (vectors that require recombination with the host genome to maintain them), and autonomously replicating plasmid vectors.

  To prepare transgenic Saccaromyces, an expression construct is prepared by inserting a nucleotide sequence into a construct designed to be expressed in yeast. Various types of constructs used for heterologous expression have been developed. In yeast that is fused to a nucleotide sequence, a construct containing promoter activity (usually a promoter of yeast origin such as the GAL1 promoter) is used. Usually, a signal sequence derived from yeast (for example, a sequence encoding a SUC2 signal peptide) is used. Terminator activity in yeast terminates the expression system.

  Various transformation protocols have been developed for yeast transformation. For example, transgenic Saccaromyces according to the present invention are described by Hinnen et al. (1978, Proceedings of the National Academy of Sciences USA 75, 1929); Beggs, JD (1978, Nature, London, 75; (1983, J. Bacteriology 153, 163-168).

  Transformed yeast cells are selected using various selectable markers. Among the markers used for transformation are many auxotrophic markers such as LEU2, HIS4, and TRP1, and many dominant antibiotic resistance markers such as aminoglycoside antibiotic markers (eg, G418) .

  Another host organism is a plant. The basic principle in the construction of genetically modified plants is to insert genetic information into the plant genome and obtain stable retention of the inserted genetic material. Various techniques exist for inserting genetic information, and the two main principles are to introduce genetic information directly and to introduce genetic information using a vector system. General technical reviews are available in Potrykus (Annu. Rev. Plant Physiol. Plant Mol. Biol. (1991) 42: 205-225) and Christou (Agro-Food-Industry Hi-Tech March / April 1994). Can be found in Further teachings regarding plant transformation can be found in EP-A-0449375.

  Accordingly, the present invention also provides a method of transforming a host cell with a nucleotide sequence that is targeted or expresses a target. Host cells transformed with this nucleotide sequence can be cultured under conditions suitable for expression of the encoded protein. Proteins produced by recombinant cells can be presented on the surface of the cells. As desired and as understood by one of skill in the art, expression vectors containing coding sequences can be designed with signal sequences to allow the coding sequences to be secreted through the cell membrane of a particular prokaryotic or eukaryotic organism. Other recombinant constructs can link the coding sequence to a nucleotide sequence that encodes a polypeptide region that facilitates purification of soluble proteins (Kroll DJ (1993) DNA Cell Biol. 12: 441-53).

(Modified / Homologue / Derivative)
In addition to the specific amino acid and nucleotide sequences referred to herein, the present invention also encompasses the use of these variants, homologues, and derivatives. As used herein, the term “homology” may be equated with “identity”.

  In the context herein, homologous sequences are taken to include amino acid sequences that can be at least 75, 85 or 90% identical (preferably at least 95 or 98% identical). While homology can also be viewed as similarity (ie, amino acid residues with similar chemical properties / functions), in the context of the present invention, it is preferred to express homology with respect to sequence identity.

  Homology comparisons can be made visually or with the aid of more commonly available sequence comparison programs. These commercially available computer programs can calculate homology (%) between two or more.

  Homology (%) can be calculated across consecutive sequences. That is, one sequence is aligned with another sequence, and each amino acid in one sequence is directly compared to the corresponding amino acid in the other sequence, one residue at a time. This is referred to as an “ungapped” alignment. Typically, such ungapped alignments are performed only over a relatively small number of residues.

  This is a very simple and consistent method, but, for example, in another sequence of identity pairs, one insertion or deletion causes the next amino acid residue to be aligned, thus potentially Furthermore, it is not considered to cause a large reduction in homology (%) when the entire alignment is performed. Thus, most sequence comparison methods are designed to produce optimal alignments that take into account possible insertions and deletions without unduly degrading the overall homology score. This is achieved by inserting “gaps” in the sequence alignment and attempting to make the best use of local homology.

  However, these more complex methods assign “gap penalties” to each gap that occurs in the alignment, resulting in a sequence alignment that has as few gaps as possible for the same number of identical amino acids (thus Reflects a higher association between the two compared sequences), achieving a higher score than an alignment with many gaps. “Affine gap costs” is typically used to require a relatively high cost for the presence of a gap and the existence of a smaller penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system. A high gap penalty naturally results in an optimized alignment with fewer gaps. Most alignment programs allow gap penalties to be changed. However, it is preferred to use the default values when using such software for sequence comparisons. For example, when using the GCG Wisconsin Bestfit package (see below), the default gap penalty for amino acid sequences is -12 for gaps and -4 for each extension.

  Thus, the calculation of maximum homology (%) requires firstly the generation of an optimal alignment taking into account the gap penalty. A suitable computer program for performing such an alignment is the GCG Wisconsin Bestfit package (University of Wisconsin, USA; Devereux et al., 1984, Nucleic Acids Research 12: 387). Examples of other software that can perform sequence comparisons include the BLAST package (see Ausubel et al., 1999 ibid, chapter 18), FASTA (Atschul et al., 1990, J. Mol. Biol., 403-410). ) And GENEWORKS package software in comparison tools. BLAST and FASTA are available for offline and online research (see Ausubel et al., 1999 ibid, pages 7-58 to 7-60). However, it is preferred to use the GCG Bestfit program.

  Further useful references can be found in FEMS Microbiol Lett 1999 May 15: 174 (2): 247-50 (and the published error is revealed in FEMS Microbiol Lett 1999 Aug 1: 177 (1): 187-8). Found).

  The final homology (%) is measured in terms of identity, but the alignment process itself is typically not dependent on comparison of all-or-nothing pairs. Instead, the measured similarity score matrix is typically used to assign a score to each pair comparison based on chemical similarity or evolutionary distance. An example of such a commonly used matrix is the BLOSUM62 matrix (the default matrix for BLAST package software programs). The GCG Wisconsin program uses either publicly available default values or its own symbol comparison table if provided (see user manual for further details). It is preferred to use the published default values for the GCG package. In the case of other software, it is preferable to use a default matrix such as BLOSUM62.

  Once the software has produced an optimal alignment, homology (%), preferably sequence identity (%), can be calculated. The software typically does this as part of the sequence comparison and produces a real result.

  The sequence also has deletions, insertions or substitutions of amino acid residues, which results in static changes and functionally equivalent substances. Intentional amino acid substitutions can be made based on similarity in residue polarity, charge, solubility, hydrophobicity, hydrophilicity and / or amphiphilicity so long as the secondary binding activity of the substance is retained. . For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups with similar hydrophilicity values, Examples include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine.

  Conservative substitutions can be made, for example, according to the following table. Amino acids in the same block of the second column, and preferably in the same row of the third column, can be substituted for each other.

  (Table 1)

（発現ベクター）
標的として使用されるヌクレオチド配列または標的を発現するヌクレオチド配列は、組換え複製可能ベクターに組み込まれ得る。ベクターを用いて、適合可能な宿主細胞内で、および／または宿主細胞からヌクレオチド配列を複製し、発現し得る。発現は、プロモーター／エンハンサーおよび他の発現調節シグナルを含む制御配列を用いて制御され得る。原核生物プロモーターおよび真核生物細胞中で機能するプロモーターが用いられ得る。組織特異的または刺激特異的なプロモーターが用いられ得る。上記の２つ以上の異なるプロモーター由来の配列エレメントを含むキメラプロモーターもまた用いられ得る。

(Expression vector)
The nucleotide sequence used as the target or the nucleotide sequence expressing the target can be incorporated into a recombinant replicable vector. Vectors can be used to replicate and express nucleotide sequences in and / or from compatible host cells. Expression can be controlled using control sequences including promoters / enhancers and other expression control signals. Prokaryotic promoters and promoters that function in eukaryotic cells can be used. Tissue specific or stimulus specific promoters may be used. Chimeric promoters comprising sequence elements from two or more different promoters as described above can also be used.

  The protein produced by the host recombinant cell by expression of the nucleotide sequence can be secreted or contained within the cell, depending on the sequence and / or vector used. The coding sequence can be designed to include a signal sequence so that the substance coding sequence is secreted through a particular prokaryotic or eukaryotic cell membrane.

(Fusion protein)
The target amino acid sequence can be produced, for example, as a fusion protein to aid in extraction and purification. Examples of fusion protein partners include glutathione-S-transferase (GST), 6xHis, GAL4 (DNA binding and / or transcriptional activation domain) and (-galactosidase. Allows removal of fusion protein sequences. Thus, it may also be convenient to include a proteolytic cleavage site between the fusion protein partner and the protein of interest, preferably the fusion protein does not interfere with the activity of the target.

  A fusion protein may comprise an antigen or antigenic determinant fused to a substance of the invention. In this embodiment, the fusion protein can be a non-naturally occurring fusion protein comprising a substance that can act as an adjuvant in the sense of providing a general purpose stimulus of the immune system. The antigen or antigenic determinant can be attached to either the amino terminus or the carboxy terminus of the substance.

  In another embodiment of the invention, the amino acid sequence can be linked to a heterologous sequence encoding a fusion protein. For example, for screening peptide libraries for agents that can affect the activity of this substance, it may be useful to encode a chimeric substance that expresses a heterologous epitope recognized by a commercially available antibody.

(Treatment)
The compounds of the present invention can be used as therapeutic agents, ie in therapeutic applications.

  The term “treatment” includes curative effects, alleviation effects, and prophylactic effects.

  The treatment can be on humans or animals, preferably female animals.

(Pharmaceutical composition)
In one aspect, the invention provides a pharmaceutical composition comprising a compound according to the invention, optionally a pharmaceutically acceptable carrier, diluent or excipient (these Including a combination of

  The pharmaceutical composition may be for human or veterinary use in human medicine and veterinary medicine, typically any one or more pharmaceutically acceptable diluents, carriers or excipients. Contains agents. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro, 1985). The choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical composition may include any suitable binder, lubricant, suspending agent, coating agent, solubilizer as or in addition to, as a carrier, excipient or diluent.

  Preservatives, stabilizers, dyes and flavoring agents may be provided in the pharmaceutical composition. Examples of preservatives include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. Antioxidants and suspending agents may also be used.

  There may be different composition / formulation requirements dependent on different delivery systems. By way of example, a pharmaceutical composition of the invention can be formulated and delivered using a minipump or by a mucosal route (eg, as a nasal spray or aerosol for inhalation or an ingestible solution) or composition The article is delivered parenterally formulated in an injectable form for delivery (eg, by intravenous, intramuscular or subcutaneous route).

  If the drug is delivered mucosally via the gastrointestinal mucosa, the drug should remain stable during passage through the gastrointestinal tract; for example, the drug is resistant to proteolytic degradation. It should be resistant, stable at acidic pH, and resistant to the bile cleansing effect.

  Where appropriate, the pharmaceutical compositions may be formulated by inhalation in the form of suppositories or pessaries, topically in the form of lotions, solutions, creams, ointments or powders, by the use of skin patches, such as starch or lactose Administered orally in the form of tablets containing the agent, or in capsules or eggs, either alone or mixed with excipients, or in the form of elixirs, solutions or suspensions containing flavoring or coloring agents. Or they can be injected parenterally, eg intravenously, intramuscularly or subcutaneously. For parenteral administration, the composition may best be used in the form of a sterile aqueous solution, which is sufficient salt or simple substance to make the solution isotonic with other substances, such as blood. Sugars can be included. For buccal or sublingual administration, the composition can be administered in the form of a tablet or lozenge that can be formulated in a conventional manner.

(Combination drug)
The compounds of the present invention can be used in combination with one or more other active agents (eg, one or more other pharmaceutically active agents).

  By way of example, the compounds of the present invention may include other 11β-HSD inhibitors and / or other inhibitors (eg, aromatase inhibitors (eg, 4 hydroxyandrostenedione (4-OHA)) and / or steroid sulfatase inhibitors (eg, EMATE). ) And / or steroids (such as naturally occurring sterneurosteroid dehydroepiandrosterone sulfate (DHEAS) and pregnenolone sulfate (PS) and / or other structurally similar organic compounds) Can be used together.

By way of example, the compounds of the present invention may be used in combination with other STS inhibitors and / or other inhibitors, such as aromatase inhibitors. In addition or alternatively, the compounds of the present invention may be used in combination with biological response modifiers. obtain.

  The term biological response modifier ("BRM") includes cytokines, immune regulators, growth factors, hematopoietic regulators, colony stimulating factors, chemotactic factors, hemolytic and thrombolytic factors, cell surface receptors, Examples include ligands, leukocyte adhesion molecules, monoclonal antibodies, prophylactic and therapeutic vaccines, hormones, extracellular matrix components, fibronectin and the like. In some indications, preferably the modulator of biological response is a cytokine. Examples of cytokines include: interleukin (IL) (eg, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL- 9, IL-10, IL-11, IL-12, IL-19); tumor necrosis factor (TNF) (eg, TNF-α); interferon α, interferon β and interferon γ; TGF-β. For some applications, preferably the cytokine is tumor necrosis factor (TNF). In some applications, the TNF can be any type of TNF, such as TNF-α, TNF-β (including derivatives or mixtures thereof). More preferably, the cytokine is TNF-α. Teachings regarding TNF can be found in the art (eg, WO-A-98 / 08870 and WO-A-98 / 13348).

(Administration)
Typically, a physician will determine the actual dosage that will be most appropriate for an individual subject, and this dosage will vary with the age, weight and response of the particular patient. Lower dosages are typical of the average case. There can, of course, be individual instances where higher or lower dosage ranges are merited.

  The compositions of the invention can be administered by direct injection. The composition may be formulated for parenteral administration, mucosal administration, intramuscular administration, intravenous administration, subcutaneous administration, intraocular administration, or transdermal administration. Depending on the need, the drug may be administered at a dose of 0.01-30 mg / kg body weight (eg, a dose of 0.1-10 mg / kg body weight, more preferably 0.1-1 mg / kg body weight). .

  As a further example, the agents of the present invention may be administered according to a regimen of 1 to 4 times per day, preferably once or twice per day. The particular dose level and dosing frequency for any particular patient can vary, and various factors (activity of the particular compound used, metabolic stability and length of action of this compound, age, weight, systemic Health, sex, diet, mode of administration and time, elimination rate, combination of drugs, severity of particular condition, and host being treated).

  Apart from typical delivery modalities (shown above), the term “administration” also refers to lipid-mediated transfection, liposomes, immunoliposomes, lipofectin, cationic facial amphiphiles (CFA) and combinations thereof. Delivery by various techniques. Routes for such delivery mechanisms include, but are not limited to, mucosal routes, nasal routes, buccal routes, parenteral routes, gastrointestinal routes, topical routes, or sublingual routes.

  The term “administered” refers to a mucosal route (eg, as a nasal spray or aerosol for inhalation or as an ingestible solution); a parenteral route (eg, intravenous, intramuscular or Subcutaneous route) delivery, but is not limited to.

  Thus, for pharmaceutical administration, the STS inhibitors of the present invention are usually for parenteral administration in any suitable manner using conventional pharmaceutical formulation techniques as well as pharmaceutical carriers, adjuvants, excipients, diluents and the like. Can be prescribed. The approximate effective dose rate can range from 1-1000 mg / day, such as 10-900 mg / day or even 100-800 mg / day, depending on the individual activity of the compound in question, and this is average For patients with body weight (70 kg). More useful dosage rates for compounds that are preferred and more active range from 200 to 800 mg / day, more preferably from 200 to 500 mg / day, most preferably from 200 to 250 mg / day. These can be provided in a single dosing regimen, with an excess dosing regimen and / or multiple dosing regimes lasting for several days. For oral administration, they can be formulated in tablets, capsules, solutions or suspensions containing 100-500 mg of compound per single dose. Alternatively and preferably, the compound is formulated for parenteral administration in a suitable parenterally administrable carrier and is 200-800 mg, preferably 200-500 mg, more preferably 200-250 mg. Provides a range of single daily dosages. However, such effective daily doses will vary depending on the original activity of the active ingredient, the patient's weight, and such variations are within the judgment of the art and physician.

(Cell cycle)
The compounds of the present invention may be useful in methods of treating cell cycle disorders.

  As discussed in “Molecular Cell Biology” 3rd edition, Rodish et al., Pp. 177-181, different eukaryotic cells can grow and divide at very different rates. For example, yeast cells divide every 120 minutes and the first division of a fertilized egg in sea urchin and insect embryo cells takes only 1530 minutes. Because one large existing cell is subdivided. However, the most growing plant and animal cells take 10-20 hours to double in number, and some replicas are slower and faster. Many adult cells (eg, neurons and striated muscle cells) do not divide a little; other cells such as fibroblasts that assist wound healing proliferate on demand but otherwise Paused.

  Furthermore, all eukaryotic cells that divide must be prepared to provide genetic material equally to the two daughter cells. DNA synthesis in eukaryotes does not always occur during the cell division cycle, but is limited to a portion prior to cell division.

The link between eukaryotic DNA synthesis and cell division has been thoroughly analyzed in the culture of mammalian cells, all of which could proliferate and divide. In contrast to bacteria, eukaryotic cells have been found to spend only a portion of their time in DNA synthesis and complete in time prior to cell division (mitosis). Thus, it has been found that a time gap occurs after DNA synthesis and before cell division; another gap occurs after division and before the next round of DNA synthesis. This analysis shows that the eukaryotic cell cycle consists of M (mitotic) phase, G ₁ phase (first gap), S phase (DNA synthesis) phase, G ₂ phase (second gap), and M Led the conclusion to return. The intermitotic phase (G ₁ , S, and G ₂ ) is collectively known as the interphase.

  Many non-dividing cells in tissues (eg, all resting fibroblasts) stop cycling after mitosis and just before DNA synthesis; such “resting” cells are removed from the cell cycle. (Exit) and is said to be in the G0 state.

If the cell is in one of the three interphases of the cell cycle, identify this cell by using a fluorescence activated cell classifier (FACS) that measures the relative DNA content of the cell Is possible; cells in G ₁ (before DNA synthesis) have a defined amount x of DNA; during S (DNA replication), cells have between x and 2x; and G _{When in 2} (or M), the cell has 2x DNA.

  The stages of mitosis and cytokinesis in animal cells are as follows.

((A) Interphase) interphase G ₂ phase is immediately before the start mitosis. Chromosomal DNA is replicated during S phase and bound to protein, but the chromosome is not yet observed as a well-defined structure. The nucleolus is the only nuclear substructure visible under an optical microscope. In diploid cells, there are two morphological chromosomes of each type prior to DNA replication, and this cell is said to be 2n. In G _2, after DNA replication, the cell is 4n. There are 4 copies of each chromosomal DNA. Since these sister chromosomes are not yet separated from each other, they are called sister chromatids.

  (B) Early early centriole, each with a newly formed daughter centriole, initiates migration of the cell to the counter electrode; the chromosome can be observed as a long thread. The nuclear membrane begins to disaggregate into vesicles.

  (C) Early middle and late Chromosome condensation is complete; each visible chromosome structure consists of two chromatids held together in their centromere. Each chromatid contains one of two newly replicated daughter DNA molecules. The microtubule spindles begin to grow radially from the area adjacent to the central body, and they move closer to their poles. Some spindle fibers reach from pole to pole; most migrate to chromatids and attach at centromeres.

  (D) Metaphase Chromosomes move towards the equator of the cell where they become aligned with the equator plane. This sister chromatid is not yet separated.

  (E) Late stage Two sister chromatids segregate into individual chromosomes. Each contains centromeres bound to spindle fibers to the one pole they have traveled. Thus, one copy of each chromosome is given to each daughter cell. At the same time, the cells extend in the same way as the polar-polar spindle. Cytokinesis begins as the dividing groove begins to form.

  (F) Termination A new membrane is formed around the daughter nucleus; the chromosome is unraveled and becomes less clear, the nucleolus becomes visible again, and the nuclear membrane is around each daughter nucleus Formed. Cytokinesis is almost complete and the spindle disappears as microtubules and other fibers depolymerize. Throughout mitosis, the “daughter” centriole at each pole extends until it is full length. At the end, replication of each of the original centriole is completed and a new daughter centriole is generated during the next interphase.

(G) the interphase at the completion of cytokinesis, the cell enters the G ₁ phase of the cell cycle, and again travels through this period.

  It is understood that cell cycle movement is a very important cellular process. Deviations from normal cell cycle movement can cause many medical disorders. Increased and / or unlimited cell cycle movement can result in cancer. A decrease in cell cycle movement can result in a degenerative state. By using the compounds of the present invention, a means for treating such disorders and conditions may be provided.

  Thus, the compounds of the present invention may be suitable for use in the treatment of cell cycle movement disorders such as cancer (including hormone dependent and hormone independent cancers).

  In addition, the compounds of the present invention may be suitable for the treatment of cancers such as breast cancer, ovarian cancer, endometrial cancer, sarcoma, melanoma, prostate cancer, pancreatic cancer, and other solid tumors.

For some applications, cell cycle movement is inhibited and / or prevented and / or prevented, preferably where cell cycle movement is prevented and / or prevented. In one aspect, the cell cycle movement, in G 2 _{/ M} phase, or may be inhibited, and / or may interfere and / or be blocked. In one aspect, cell cycle movement can be irreversibly inhibited and / or prevented and / or prevented, preferably cell cycle movement is irreversibly prevented, And / or blocked.

  The term “irreversibly prevented and / or inhibited and / or prevented” refers to the effect of the compound upon removal of the compound after application of the compound of the invention ( That is, the prevention and / or inhibition and / or prevention of cell cycle movement) is still observable. More particularly, the term “irreversibly prevented, and / or inhibited and / or prevented” is assayed according to the cell cycle motility assay protocol provided herein. Means that cells treated with the compound of interest show lower proliferation after stage 2 of protocol 1 than control cells. Details of this protocol are shown below.

  Accordingly, the present invention provides the following compounds: Estrogen receptor positive (ER +) and ER negative (ER−) breast cancer in vitro by preventing and / or inhibiting and / or preventing cell cycle movement. Cause cell growth inhibition; and / or cause nitroso-methylurea (NMU) induced breast tumor regression and / or cell cycle motility in cancer cells in intact animals (ie, not ovariectomized) Hinder and / or inhibit and / or prevent; and / or act in vivo by preventing and / or inhibit and / or prevent cell cycle movement and / or act as a cell cycle movement agonist A compound.

(Cell cycle assay (protocol 2))
Procedure Stage 1
MCF-7 breast cancer cells are seeded in multiwell culture plates at a density of 10 ⁵ cells / well. Cells were allowed to attach and were grown to approximately 30% confluence when treated as follows.

Control-untreated compound of interest (COI) 20 μM
Cells are grown for 6 days in growth medium containing COI (medium / COI change every 3 days). At the end of this period, cell numbers were counted using a Coulter cell counter.

Stage 2
After 6 days of cell treatment with COI, cells are replated at a density of 10 ⁴ cells / well. Does not add any further processing. Cells are allowed to grow for an additional 6 days in the presence of growth medium. At the end of this period, the number of cells is counted again.

(cancer)
As indicated, the compounds of the invention may be useful in the treatment of cell cycle motility disorders. A specific cell cycle movement disorder is cancer.

  Cancer remains the leading cause of mortality in most Western countries. Cancer therapies developed to date have included blocking the action or synthesis of hormones to inhibit the growth of hormone-dependent tumors. However, more aggressive chemotherapy is currently used for the treatment of hormone-independent tumors.

  Thus, the development of medicaments for anti-cancer treatment of hormone-dependent and / or hormone-independent tumors, which lack some or all of the side effects associated with chemotherapy, represents a major therapeutic advance.

  We believe that the compounds of the invention provide a means for the treatment of cancer, and in particular breast cancer.

  Additionally or alternatively, the compounds of the present invention may be useful in blocking the growth of cancer (including leukemia) and solid tumors (eg, breast, endometrial, prostate, ovarian and pancreatic tumors).

(Other treatments)
It is also understood that the compounds / compositions of the present invention may have other important medical meanings.

For example, the compounds or compositions of the invention may be useful in the treatment of disorders listed in WO-A-99 / 52890 (ie):
Additionally (or) the compounds or compositions of the invention may be useful in the treatment of disorders listed in WO-A-98 / 05635. For ease of reference, some of that list are listed here: cancer, inflammation or inflammatory disease, dermatological disorders, fever, cardiovascular effects, bleeding, coagulation and acute phase reaction, cachexia, anorexia , Acute infection, HIV infection, shock condition, host versus graft reaction, autoimmune disease, reperfusion injury, meningitis, migraine, and aspirin-dependent antithrombosis; tumor growth, invasion and spread, angiogenesis , Metastasis, malignant, ascites and malignant pleural effusion; cerebral ischemia, ischemic heart disease, osteoarthritis, rheumatoid arthritis, osteoporosis, asthma, multiple sclerosis, neurodegeneration, Alzheimer's disease, atherosclerosis , Seizures, vasculitis, Crohn's disease and ulcerative colitis; periodontitis, gingivitis; psoriasis, atopic dermatitis, chronic ulcer, epidermolysis bullosa; corneal ulcer, retinopathy and surgical wound healing; rhinitis, allergic conjunctivitis ,eczema, Nafirakishi; restenosis, congestive heart failure, endometriosis, atherosclerosis or intimal sclerosis.

  Additionally (or alternatively), the compounds or compositions of the invention may be useful for the treatment of disorders listed in WO-A-98 / 07859. For ease of reference, some of that list are listed here: cytokines and cell proliferation / differentiation activity; immunosuppressive or immunostimulatory activity (eg, immunodeficiency (including infection with human immunodeficiency virus); lymph To treat regulation of sphere proliferation; to treat cancer and many autoimmune diseases and to prevent transplant rejection or tumor immunity induction); regulation of hematopoiesis, eg treatment of bone marrow or lymphocyte disease; bone, cartilage Promotes growth of tendons, ligaments and nerve tissue (eg for wound healing) treatment of burns, ulcers and periodontal diseases and neurodegeneration; inhibition of follicle stimulating hormone or active compounds (modulation of fertilization); chemotaxis Activity / chemokinesis activity (eg to mobilize cell types specific to the site of injury or infection); hemostatic and thrombolytic activity (eg hemophilia and Anti-inflammatory activity (eg to treat septic shock or Crohn's disease); as an antibacterial agent; eg as a metabolic or behavioral modulator; as an analgesic; to treat certain deficient injuries For example in the treatment of psoriasis (in human or veterinary medicine).

  Additionally (or alternatively) the compositions of the present invention may be useful in the treatment of disorders listed in WO-A-98 / 09985. For ease of reference, some of the listings are listed here: macrophage inhibitory activity and / or T cell inhibitory activity, thereby anti-inflammatory activity; anti-immune activity; ie cellular immunity and / or body fluids Inhibitory effects on innate immune responses (including responses not related to inflammation); inhibits macrophage and T cell ability to immobilize extracellular matrix components and fibronectin, and upregulated fas receptor expression in T cells; Inhibit: unwanted immune responses and inflammation (including arthritis including chronic arthritis, inflammation associated with hypersensitivity, allergic reactions, asthma, systemic lupus erythematosus, collagen disease and other autoimmune diseases), atherosclerosis , Arteriosclerosis, atherosclerotic heart disease, reperfusion wound , Cardiac arrest, myocardial infarction, vascular inflammatory disorder, respiratory depression syndrome or other cardiopulmonary disease, inflammation associated with peptic ulcer, ulcerative colitis and other gastrointestinal tract diseases, liver fibrosis, cirrhosis or other liver Diseases, thyroiditis or other secretory gland diseases, glomerulonephritis or other renal and urological diseases, otitis or other ENT diseases, dermatitis or other skin diseases, periodontal diseases or other dental diseases Testicularitis or epididymis, infertility, testicular trauma or other immune-related testicular disease, placental dysfunction, placental failure, habitual abortion, eclampsia, pre-eclampsia and other immune and / or inflammation-related gynecological diseases, Posterior uveitis, middle uveitis, anterior uveitis, conjunctivitis, chorioretinitis, uveoretinitis, optic neuritis, intraocular inflammation, eg retinitis or cystoid macular edema, sympathetic ophthalmitis , Scleritis, pigmented net Inflammation, immune and inflammatory components of degenerative fundus disease, inflammatory components of ocular trauma, ocular inflammation caused by infection, proliferative vitreoretinopathy, acute ischemic optic neuropathy, excessive scar formation, eg glaucoma Immune and / or inflammatory response to intraocular implants after filtration surgery, and inflammation associated with other immune and inflammation related eye diseases, autoimmune diseases or conditions or disorders, where the central nervous system (CNS) or any In other organs, suppression of immunity and / or inflammation is beneficial), Parkinson's disease, complications and / or side effects from treatment of Parkinson's disease, AIDS-related dementia complex HIV-related brain disorders, Dovic's disease, Sydenham chorea, Alzheimer's disease and other degenerative diseases, conditions or disorders of the CNS, inflammatory component of seizures, post-polio syndrome (p ost-polio syndrome), immune and inflammatory elements of psychiatric disorders, myelitis, encephalitis, subacute sclerosing panencephalitis, encephalomyelitis, acute neuropathy, subacute neuropathy, chronic neuropathy, Guyan- Valley syndrome, Sydenham chorea, myasthenia gravis, pseudobrain tumor, Down syndrome, Huntington's disease, muscular atrophy lateral sclerosis, inflammatory component of CNS compression or CNS trauma or CNS infection, muscular atrophy and inflammatory component of dystrophy, And immune and inflammation related diseases, conditions or disorders of the central and peripheral nervous system, post-traumatic inflammation, septic shock, infectious diseases, inflammatory complications or side effects of surgery, bone marrow transplantation or other transplant complications and / or Or side effects, inflammatory and / or immune complications of gene therapy and side effects (e.g. viruses Monocyte or leukocyte proliferative disease (e.g., due to infection by the body) or AIDS related inflammation, suppression or inhibition of humoral and / or cellular immune responses, reducing the amount of monocytes or lymphocytes (e.g. Leukemia) treatment or mitigation, prevention of graft rejection in the case of transplantation of natural or artificial cells, tissues, and organs (eg, cornea, bone marrow, organ, lens, pacemaker, natural or artificial skin tissue) and / or For treatment.

  As mentioned above, in one aspect, the invention provides the use of a compound as described herein in the manufacture of a medicament for use in the treatment of a condition or disease associated with 11β-HSD. I will provide a.

  Conditions and diseases associated with 11β-HSD are described in Walker, E .; A, Stewart, P.A. M.M. Reviews in Trends in Endocrinology and Metabolism, 2003, 14 (7), 334-339.

In a preferred aspect, the condition or disease is selected from a list consisting of:
Metabolic disorders (eg diabetes and obesity)
・ Cardiovascular disorders (eg hypertension)
Glaucoma / inflammatory disorders (eg arthritis or asthma)
・ Immune disorders / bone disorders (eg osteoporosis)
・ Cancer ・ Intrauterine growth delay ・ Overtension mineralocorticoid excess syndrome (AME)
・ Polycystic ovary syndrome (PCOS)
• Hairy hair, acne, rare or amenorrhea, adrenocortical adenoma and carcinoma, Cushing syndrome, pituitary tumor, invasive carcinoma, breast cancer; and • endometrial cancer.

(Summary)
In summary, the present invention provides compounds for use as steroid dehydrogenase inhibitors and pharmaceutical compositions for the same use.

  The invention will now be described by way of example only.

(Materials and methods)
(material)
(enzyme)
Rat liver and rat kidney were obtained from normal Wistar rats (Harlan Olac, Bicester, Oxon, UK). Both kidney and liver were homogenized with Ultra-Turrax on ice in PBS-sucrose buffer (1 g / 10 ml). After homogenizing the liver and kidney, the homogenate was centrifuged at 4000 rpm for 5 minutes. The resulting supernatant was removed and stored in a glass vial at −20 ° C. The amount of protein per μl of rat liver and kidney cortisol was determined using the Bradford method [14].

(apparatus)
Incubator: mechanical shaking water bath, SW 20, Germany.
Evaporator, Techne Driveblock DB 3A, UK
TLC aluminum sheet 20 × 20 cm silica gel 60 F ₂₅₄ , Merck, Germany.
Scintillation vial: 20 ml polypropylene vial with cap, SARSTED, Germany.
Scintillation counter: Beckman LS 6000 SC, Beckman Instruments Inc. , USA.

(solution)
Assay medium: PBS-sucrose buffer containing 0.25 M sucrose, Dulbecco phosphate buffered saline, 1 tablet / 100 ml, pH 7.4 BDH Laboratory supplements, UK.
Scintillation fluid: Ecoscint A (National Diagnostics, USA).
Radioactive compound solution: [1,2,6,7- ³ H] -cortisol (Sp. Ac. 84 Ci / mmol) NEN Germany, [4- ¹⁴ C] -cortisol (Sp. Ac. 53 mCi / mmol) NEN Germany .
CrO ₃ and acetic acid (Sigma Chemical Co., UK).
Extraction fluid: diethyl ether, Fischer Chemicals, UK.
Bradford reagent solution: Coomassie Brilliant Blue G-250, 100 mg in 95% ethanol, diluted to 1 liter, 100 ml phosphoric acid (85% w / v).

(Compound)
Inhibitor: The compound was synthesized according to the following synthetic route Cofactors: NADPH and NADP, Sigma Chemical Co. , UK.

(Method)
(Synthesis of radiolabeled cortisone)
To synthesize the corresponding labeled cortisone ( ³ H-E and ¹⁴ C-E), labeled cortisol (F) ( ³ H-F and ¹⁴ C-F) was oxidized to the C-11 position with CrO _3. did.

For this reaction, F was oxidized in 0.25% CrO ₃ (w / v) dissolved in 50% acetic acid / distilled water (v / v) solution. Labeled F was added to 1 ml of CrO ₃ solution, mixed vigorously and placed in an incubator at 37 ° C. for 20 minutes. The aqueous reaction mixture was extracted with 4 ml of diethyl ether, then the diethyl ether was evaporated and the residue was transferred to a TLC plate, which was developed in the following system: chloroform: methanol 9: 1 (v / v ). Unlabeled cortisone (E) was also developed on the TLC plate to locate the labeled steroid. After positioning the labeled steroid spot, this region was cut out of the TLC plate and extracted with 0.5 ml of methanol.

(Amount of protein per μl of rat liver and rat kidney)
It was necessary to determine the amount of protein in rat liver and rat kidney. The experiment was performed according to the Bradford method [14]. The following method was used: First, a BSA (protein) solution was prepared (1 mg / ml). A protein solution containing 10 to 100 gag proteins was pipetted into a tube and the volume was adjusted with distilled water. 5 ml of protein reagent was then added to the tube and mixed vigorously. Absorbance was measured against a reagent blank in a 3 ml cuvette 15 minutes and 1 hour before at 595 nm. The weight of the protein was plotted against the corresponding absorbance to obtain a standard curve used to determine the protein concentration in the cytoplasm of rat liver and rat kidney.

(Assessment of assay-enzyme concentration and time dependence of 11β-HSD activity)
Prior to performing the 11β-HSD assay to examine E to F and F to E conversions and the effects of different inhibitors on these conversions, the amount of rat liver homogenate and rat kidney homogenate and incubation It is necessary to determine the time.

The 11β-HSD type 1 is an enzyme responsible for the conversion of E to F, and this type of enzyme is present in the rat liver. The substrate solution used in this assay contained 70,000 cpm / ml ³ H-E and 0.5 μM unlabeled E and cofactor NADPH (9 mg / 10 ml substrate solution) in PBS-sucrose. 1 ml of substrate solution and different amounts of rat liver homogenate were added to all tubes.

The amount of rat liver homogenate required for the assay is determined by mechanically shaking the tube at 37 ° C. in a water bath for 30, 60, 90 and 120 minutes with 25, 50, 100 and 150 μl of substrate solution. Determined by incubating. After incubation, add 50 μL of recovery solution containing 8,000 cpm / 50 μL of 14C-F and 50 μg / 50 μL of unlabeled F to visualize the spots on the TLC plate, and reduce the loss that occurs in the next two steps Corrected. F was then extracted from the aqueous phase with 4 ml of ether (2 × 30 second cycle, vigorously mixed). The aqueous phase was then frozen using dry ice, the organic layer was decanted, poured into a small tube and evaporated. Then 6 drops of ether was added to the small tube to redissolve the residue and transferred to an aluminum thin layer chromatography plate (TLC plate). The TLC plate was developed in a TLC tank under standard conditions. The solvent system used was chloroform: methanol 9: 1 (v / v). The spot of F on the TLC plate was visualized under UV light and cut out from the TLC plate (R _f = 0.45). The spot from the TLC plate was then poured into scintillation vials and 0.5 ml of methanol was added to all vials to elute the radioactivity from the TLC plate for 5 minutes. 10 ml of Ecoscint was added to the scintillation vial and they were poured into a scintillation counter to count the amount of product formed.

The same procedure was used for the 11β-HSD type 2 assay (F to E conversion) to determine the amount of rat kidney to be used and the incubation time. Except for this time, the substrate solution contains ³ H-F and unlabeled F, the collection contains ¹⁴ C-E and unlabeled E, and cortisone is 0.65 on the TLC plate. R _f value of

(Assay Procedure-11 β-HSD Inhibitor)
In these assays, the effect of different inhibitors on 11β-HSD activity in both reduced (type 1) and oxidized (type 2) directions was evaluated. In the reduced direction, E is the substrate, F is the product, and vice versa for the participatory type. The method described here is for the direction of the oxidation type.

The substrate solution contained 50,000 cpm / ml ³ H-F and 0.5 μM F in PBS-sucrose. 1 ml of substrate solution was added to each tube, and inhibitors were also added to each tube at a concentration of 10 μM, except for the “control” and “blank” tubes. 150 μL was added to all tubes except the blank, which corrected for the amount of ³ H-F formed simultaneously. The tube was incubated for 60 minutes at 37 ° C. in a mechanically shaking water bath. The amount of kidney, liver homogenate and the incubation time used were obtained from enzyme-dependent and time-dependent assays. After incubation, 50 psi of collection containing 500 cpm / 50 μL of ¹⁴ C-E and 50 μg / 50 μL of unlabeled E was added to correct for the loss that occurs in the next step (to visualize the spot on the TLC plate For). The aqueous mixture was then extracted with 4 ml ether (2 × 30 sec cycle, vigorous mixing). After freezing the aqueous phase, the ether (upper) layer was decanted into a small tube and evaporated at 45 ° C. until completely dry. The residue was then redissolved in 6 drops of ether and transferred to a TLC plate. The TLC plate was developed with a chloroform: methanol (9: 1 v / v) solvent system, and the TLC plate was developed for about 90 minutes until the tip of the solvent moved about 18 cm. The position of product E was visualized under UV light, cut out from the TLC plate and placed in a scintillation vial. Radioactivity was eluted with 0.5 ml methanol over 5 minutes. 0.5 ml PBS-sucrose and 10 ml Ecoscint were then added, mixed vigorously and then counted in a scintillation counter. Two total active vials were prepared before counting the samples. These included 0.5 ml substrate solution, 50 μL harvest, 0.5 ml methanol and 10 ml Ecscint. These two all active vials were required to determine the amount of ¹⁴ C-E and ³ H-F initially added and perform the calculations.

The same method was used for the reduced direction (E to F). Only the substrate solution containing ³ H-E and unlabeled E and the recovery containing ¹⁴ C-F and unlabeled F differ from the method used in the oxidized direction.

  After all inhibitors were tested at 10 μM, dose-dependent experiments were performed on the most potent 11β-HSD type 1 and type 2 inhibitors. Four different concentrations, 1, 5, 10 and 20 μM were used to see the% inhibition. The methods for both rat liver, type 1 (reduced) and rat kidney, type 2 (oxidized) remain the same throughout all experiments.

(result)
(Amount of protein per μL of rat liver and rat kidney)
Initial experiments were performed to determine the amount of protein in rat liver cytoplasm and rat kidney cytoplasm to be added to each tube. Graph 1 in FIG. 1 shows a standard curve from which the amount of protein used in both experiments was calculated. In the rat liver experiment, the amount of protein added to each tube was 75 μg (per 25 μl). In rat kidney experiments, the protein added to each tube was 135.6 μg (per 150 μL).

(Enzyme concentration and time dependence of 11β-HSD activity)
In this experiment, the amount of rat liver homogenate and rat kidney homogenate added to each tube and the incubation time were determined. Graph 2 in FIG. 2 shows the enzyme concentration and time-dependent course of the rat liver experiment (E to F, 11β-HSD type 1 activity). Graph 3 in FIG. 3 shows the enzyme concentration and time-dependent course from F to E (11β-HSD type 2 activity). After drawing the graph, the optimal amount of rat liver cytoplasm and rat kidney cytoplasm, as well as the incubation time for both, were selected. One important rule in selecting both varieties is to select the amount of rat liver and rat kidney and the incubation time on the linear part of the graph. This is done to avoid fluctuations in enzyme activity. The amount of rat liver cytoplasm selected was 25 μL with an incubation time of 90 minutes, and the amount of rat kidney cytoplasm selected was 150 μL with an incubation time of 60 minutes.

(11β-HSD inhibitor)
In this experiment, the effect of different inhibitors on E to F and F to E conversions was determined. The reason why inhibition in both directions was tested was to compare the inhibitor and which type of 11β-HSD was more inhibitory. Compounds were screened for the ability to inhibit 11β-HSD type 1 (E to F) and type 2 (F to E). All inhibitors were first tested at a 10 μM concentration. The% inhibition was calculated as the% reduction in the ratio of labeled 3H-F and 2H-F of the product formed when compared to the control activity (tube without inhibitor). All calculated results are averages (n = 2).

  (Table 2: Inhibitory effect)

（ヒト１１β−ヒドロキシステロイドデヒドロゲナーゼ１型を用いる生物学的アッセイの開発）
（１１−ヒドロキシステロイドデヒドロゲナーゼ１型コルチゾールラジオイムノアッセイのための標準的な操作手順）
（１１β−ＨＳＤ１コルチゾールＲＩＡ）
試薬：コルチゾン、コルチゾール（ヒドロコルチゾン）、ＮＡＤＰＨ、グルコース−６−リン酸、グリシレチン酸（ＧＡ）、デキストランでコーティングした炭（Ｃ６１９７）およびＤＭＳＯをＳｉｇｍａ Ａｌｄｒｉｃｈから入手し、カルベノキソロンをＩＣＮ Ｂｉｏｍｅｄｉｃａｌｓから入手し、製品２１５４９３００１，^３Ｈ−コルチゾンをＡｍｅｒｉｃａｎ Ｒａｄｉｏｌａｂｅｌｌｅｄ Ｃｏｍｐｏｎｄｓ Ｉｎｃから入手し、製品ＡＲＴ−７４３，^３Ｈ−コルチゾールをＮＥＮから入手し、製品ＮＥＴ ３９６，^１４Ｃ−コルチゾールをＮＥＮから入手し、製品ＮＥＣ １６３，ヒト肝臓ミクロソームをＸｅｎｏＴｅｃｈから入手し、製品Ｈ０６１０／ロット０２１００７８，ラット肝臓ミクロソームをＸｅｎｏＴｅｃｈから入手し、ＳＰＡビーズをＡｍｅｒｓｈａｍから入手し、製品ＲＰＮＱ００１７，イムノアッセイキットをＡｓｓａｙ Ｄｅｓｉｇｎｓから入手し、製品９００−０７１，Ｉｍｍｕｎｏｌｏｇｉｃａｌｓ Ｄｉｒｅｃｔ抗コルチゾール抗体は製品ＯＢＴ ０６４６であり、Ｓｉｇｍａ抗コルチゾール抗体はＰｒｏｄｕｃｔ Ｃ８４０９であり、そして、Ｉｍｍｕｎｏｔｅｃｈ抗体を、Ｂｅｃｋｍａｎ，製品ＩＭＢＵＬＫ３ ６Ｄ６から入手した。

(Development of biological assay using human 11β-hydroxysteroid dehydrogenase type 1)
Standard operating procedure for 11-hydroxysteroid dehydrogenase type 1 cortisol radioimmunoassay
(11β-HSD1 cortisol RIA)
Reagents: Cortisone, cortisol (hydrocortisone), NADPH, glucose-6-phosphate, glycyrrhetinic acid (GA), dextran-coated charcoal (C6197) and DMSO are obtained from Sigma Aldrich and carbenoxolone is obtained from ICN Biomedicals 215493001, ³ H-cortisone is obtained from American Radiolabeled Components Inc, product ART-743, ³ H-cortisol is obtained from NEN, product NET 396, ¹⁴ C-cortisol is obtained from NEN, product NEC 163, human liver Microsomes were obtained from XenoTech, product H0610 / Lot 01000078, rat liver microsomes were obtained from XenoTech SPA beads are obtained from Amersham, product RPNQ0017, immunoassay kit is obtained from Assay Designs, product 900-071, Immunologicals Direct anti-cortisol antibody is product OBT 0646, Sigma anti-cortisol antibody is Product C840, Immunotech antibody was obtained from Beckman, product IMBULK3 6D6.

(Buffer solution)
Buffer 1 [15] from Barf: 30 mM Tris-HCL containing 1 mM EDTA, pH 7.2
Buffer 2: Sterix protocol PBS containing 0.25 M sucrose (pH 7.4)
Buffer 3 from Sigma RIA protocol: 50 mM Tris-HCl, pH 8 containing 0.1 M NaCl and 0.1% gelatin.
Stop solution from Barf [15]: 1 mM glycyrrhetinic acid in 100% DMSO.

Enzyme assays were performed in the presence of 10 μM NADPH, 1 mM glucose-6-phosphate and cortisone concentrations specified for each experiment:
Enzyme assay buffer:
30 mM Tris-HCl containing 1 mM EDTA, pH 7.2
Antibody binding buffer:
50 mM Tris-HCl, pH 8, containing 0.1 M NaCl and 0.1% gelatin.

Compound preparation:
A 10 mM stock solution in 100% DMSO is prepared at 100 times the required assay concentration. Dilute 1:25 in assay buffer. As a control, neat DMSO is diluted 1:25 in assay buffer.

Substrate preparation:
Prepare a cortisone solution in ethanol at 600 times the required assay concentration (175 μM). Dilute 1:50 in assay buffer.
NADPH is prepared as a 1.8 mg / ml solution in assay buffer.
G-6-P is prepared as a 3.65 mg / ml solution in assay buffer.
These three solutions are mixed 1: 1: 1 to make enough solution for the addition of 25 μl to each sample. Cortisone titrated to 0.5 mCi per 25 μl is added and the solution is mixed well.

Preparation of microsomes:
Dilute a 20 mg / ml solution of stock 1: 100 with assay buffer.

Antibody preparation:
The stock antibody solution is diluted to 17 μg / ml in antibody binding buffer.
Preparation of dextran-coated charcoal: Make a 20 mg / ml solution in antibody binding buffer and cool on ice.

Enzyme assay:
Add the following to a u-bottom polypropylene 96 well plate:
25 μl compound dilution or diluted DMSO for control, NSB and blank
Blank contains 10 μl of 1 mM GA (enzyme stop solution) in DMSO
25 μl substrate mix for all samples 50 μl of diluted microsome plate for all samples Incubate for 30 minutes at 37 ° C. with shaking Add all wells except NSB to which 10 μl enzyme stop solution is added to all wells except blank Add 100 μl antibody solution to the wells, add antibody binding buffer to these wells, incubate at 37 ° C. for 1 hour, add 50 μl / well charcoal solution to cool plate on ice for 15 minutes, mix with 8-channel pipette Do (suction 4-5 times)
Centrifuge the plate on ice for 15 minutes at 4 ° C. and 2000 × g. Transfer 100 μl of supernatant to Optiplate and add 25 μl of substrate mixture to two empty wells to incubate counting efficiency. Microscint-40 is added to all wells and counted on the Topcount.

(Radioimmunoassay)
The 11β-HSD1 enzyme assay was performed according to the standard operating procedure described above in either u-bottom polypropylene 96 well plates or 1.5 ml Eppendorf tubes for each experiment. After stopping the enzyme reaction, unless otherwise indicated, 100 μl of antibody prepared in buffer 3 was added to the test sample and 100 μl of buffer was added to the NSB sample. Samples were incubated at 37 ° C. for 1 hour and chilled on ice for 15 minutes. Charcoal (50 μl / sample) coated with dextran adjusted to the prescribed concentration in buffer 3 was added and the sample mixed (vortexed for tubes and aspirated 5 times for 8-well pipettes for 96-well plates) Cooled for another 10 minutes. The sample was centrifuged at 2000 × g for 15 minutes at 4 ° C. to precipitate charcoal. An aliquot (100 mu l) of the supernatant was transferred to an Optiplate and counted on a Topcount in 150-200 [mu] l Microscint 40. In some experiments, aliquots of the supernatant were transferred to scintillation vials and counted on a 5 ml Ultima Gold field and in a Ticlab LSC.

(Development of 11β-HSD1 assay)
(11β-HSD1 TLC format assay)
(Separation of cortisone and cortisol)
Prior to performing the enzyme assay, the solvent system reported in the literature for the separation of cortisone from cortisol was examined [16, 17]. A 10 mg / ml solution of cortisone and cortisol was prepared in methanol and aliquots were spotted on silica gel TLC plates. The plate was run with CH ₂ Cl ₂ : IMS 92: 8 v / v (2). The plates were then air dried and sprayed with 0.1% rhodamine in methanol to visualize the spots. The following table describes the separations obtained.

  (Table 3: Separation of cortisone from cortisol by TLC)

この分離が、酵素アッセイにおいて使用するために適切であると考えた。

This separation was considered suitable for use in enzyme assays.

The literature details the extraction of cortisol from several aqueous solutions [16, 17]. [ ⁴ C] -labeled cortisol was obtained from NEN to select the method of use. Stocks were prepared by adding chilled cortisol (1 μg) as a carrier in phosphate buffered saline (PBS) containing 4000 DPM in 50 μl. The final ethanol concentration was 0.4%. An aliquot of this solution was added to a glass tube (100 μl) and the following extractions were performed: 2. 1 ml CH ₂ Cl ₂ , vortexed and passed through phase separation filter paper (Whatman, IPS), 2.1 ml ethyl acetate, vortexed and passed through phase separation filter paper _3. Vortex 1 ml CH ₂ Cl ₂ and 200 μl 0.05% CaCl ₂ , centrifuge (500 g for 5 min) and remove the upper aqueous phase. Vortex 1 ml ethyl acetate and 200 μl 0.05% CaCl ₂ , centrifuge (500 g for 5 min) and collect upper organic layer. The organic phase was dried and the residue was taken up in 100 μl IMS. This aliquot was spotted on a TLC plate and the plate was developed as before. After visualization with rhodamine B, the spots were scraped into scintillation vials and counted in a liquid scintillation counter (Packard TriCarb) in 5 ml of Ultima Gold scintillant. The extraction efficiency is calculated and shown in FIG.

From these results it is clear that 90% of cortisol was lost by phase separation filtration. Ethyl acetate appears to extract cortisol more efficiently than CH ₂ Cl ₂ , probably because the organic phase is easier to recover. Ethyl acetate appears to be a suitable extraction method.

(11β-HSD1 activity of human and rat liver microsomes)
11β-HSD1 activity in rat and human liver microsomes was evaluated to determine the minimum microsomal protein concentration required to measure enzyme activity. Experiments were performed according to the Bradford method [14]. These assays were performed in buffer 2 and the concentration of cortisone used was 2 μM containing 0.5 μCi [ ³ H] -cortisone per incubation. Microsomes were tested in glass tubes at concentrations ranging from 50 μg to 400 g, with a final incubation volume of 100 μl per incubation. Samples were incubated for 1 hour in a 37 ° C. shaking water bath and 1 ml of ethyl acetate was added to stop the assay. To recover the collected substance, ^[14 C] of 50 [mu] l - added cortisol in the sample, followed by addition of 0.05% CaCl ₂ in 200 [mu] l. Samples were mixed vigorously and centrifuged as above. The upper organic phase was removed and dried, and the residue was spotted on a TLC plate with 100 μl methanol and 50 μl aliquot. This was developed as described above. Samples were counted on a TriCarb liquid scintillation counter using a double labeling program. Recovery efficiency was determined from DPM obtained in 50 μl of [ ¹⁴ C] -cortisol solution, which was counted with the sample. The results are shown in FIG.

  11β-HSD1 activity in rat and human microsomes was similar, with 0.7 μmol / mg / min and 0.5 μmol / mg / min for rat and human microsomes, respectively. Activity in human microsomes appears to be independent of microsomal protein concentration, which may suggest that the extracted protein concentration range is too high.

  Lower human microsomal protein concentrations were assessed; 3.7 μg to 100 μg per sample. The time course of activity was also determined over a period of 0-60 minutes at 37 ° C. The extraction conditions were as described above. The results from these experiments are shown in FIGS.

  The results shown in FIGS. 6 and 7 show that enzyme activity is linear at incubation times at all microsomal protein concentrations tested up to 30 minutes and enzyme activity is linear at microsomal protein concentrations below 30 μg per sample. Indicates that there is.

The effect of substrate concentration on activity was tested. [ ³ H] -cortisone concentration was kept constant at 0.5 μCi / sample, and unlabeled cortisone was varied from 44 nM to 2 μM. The assay was performed at 37 ° C. with 10 μg microsomal protein per sample, 30 min incubation time. The results are shown in FIG. A double reciprocal plot of these data (Lineweaver-Burke) gives the apparent Km for 660 μM cortisone, FIG.

  Standard compounds glycyrrhetinic acid and carbenoxolone were tested in this assay system as part of the evaluation process. The assay was performed as described in Barf [15] using 175 nM cortisone substrate, 10 μg microsomal protein, and incubation at 37 ° C. for 30 minutes. The data in FIGS. 8 and 9 above suggest that this substrate concentration is not saturated under these assay conditions. Glycyretic acid and carbenoxolone were tested at concentrations from 0.012 μM to 3 μM and the DMSO concentration was 1% in all samples. The results are shown in FIGS.

Glycyretic acid and carbenoxolone yield IC ₅₀ values of 40 nM and 119 nM, respectively. The IC ₅₀ reported for carbenoxolone by Barf et al. Using the SPA format and recombinant 11β-HSD is 330 nM [15], which is approximately 3 times less potent. The difference in capacity between the two assay systems is probably due to different assay conditions (SPA compared to the tic endpoint and also the enzyme source: liver enzyme naive to the recombinant enzyme).

  While the above assay conditions support good enzyme activity, these should also apply to the 96-well format.

(Development of high-throughput 11β-HSD1 assay)
The supply of antibodies used by Barf [15] in the scintillation approximation assay (SPA) has proven to be a problem. A sample batch of antibody (Immunotech) was tested for suitability and the second order was replaced in large quantities. A robust 96-well plate assay using the radioimmunoassay (RIA) format was developed using available Immunotech antibodies and is described below.

(Immunoassay format)
The Assay Designs enzyme immunoassay system was evaluated as a powerful assay format. The basis of this assay is the competition for antibody binding between sample cortisol (produced by 11β-HSD1) and labeled cortisol binding. The anti-cortisol detection antibody provided in the kit is a mouse monoclonal and has been reported to cross-react with cortisone by less than 0.1%. This kit is designed for the analysis of cortisol levels in saliva, urine, serum and plasma, and also in tissue culture media, not to determine enzyme activity.

  The 11β-HSD1 enzyme assay conditions described by Barf [15] were used; human liver microsomes (25 μg-200 μg protein concentration) in buffer 1, cortisone at a concentration of 44 nM-700 nM, at 37 ° C. for 60 minutes. incubation. The effect of 0.9% Tween 80 was also investigated. This is because this reagent has been reported to improve the activity of enzymes involved in steroid metabolism. The results are shown in FIG.

  FIG. 12 (A) shows the effect of the protein. Data were taken from the 700 μM cortisone group tested in the presence of Tween 80.

  FIG. 12 (B) shows the effect of cortisone. Data were taken from a group of 25 μg microsomal proteins tested in the presence of Tween 80.

  FIG. 12C shows the effect of Tween 80. Data were taken from a group of 25 μg microsomal proteins tested in the presence of 700 μM cortisone.

  This assay detected cortisol in the standard curve (313 μg / ml-10,000 μg / ml) as expected, but the signal obtained from the enzyme assay sample decreased with increasing microsomal protein concentration, This suggests that microsomal proteins can affect immunoassays (FIG. 12 (A)). The addition of exogenous cortisone has no effect on the level of cortisol detected in the enzyme assay sample, suggesting that the antibody does not cross-react with cortisone (FIG. 12 (B)). Inclusion of a surfactant in the enzyme assay buffer had little effect, FIG. 12 (C).

  The assay conditions were changed to determine if it was feasible to use an immunoassay system to detect 11β-HSD1 activity; 2 μM cortisone substrate in 24 μg microsomal protein and buffer 2 per sample. Enzyme activity was also measured in the sample after addition of a steroid displacement reagent (kit component that releases cortisol from the cortisol-binding protein) if present in the sample. The assay detected cortisol within a standard curve (313 μg / ml to 10,000 μg / ml). FIG. 13 shows the absorbance at 406 nm obtained for the different groups.

  The minimum and maximum concentrations of the cortisol standard are shown in FIG. 13 to be 313 μg / ml and 10,000 μg / ml, and NSB absorbance indicates the dynamic range obtained in this assay.

  The absorbance (“enzyme”) obtained in the presence of the reaction mixture taken from the sample incubated with the microsomal protein is higher than the steep altitude (“no enzyme”) obtained in the presence of the reaction mixture without the microsomal protein. Low, showing increased cortisol levels.

  In the presence of a kit steroid displacement reagent (“DR”), these two reaction mixtures show the same pattern, but the signal is suppressed.

  Glycyretic acid (GA) in the presence of the highest concentration of cortisol standard does not affect the kit's ability to measure cortisol concentration.

  The signal background ratio of 2.5 for these assays is quite low, but these data indicate that the antibody can bind to the cortisol: AP conjugate and that it can be replaced by cortisol. . Experiments were conducted to investigate the effect of increasing microsomal protein concentration in an attempt to improve the resulting signal to noise ratio.

  Microsomal proteins were tested from 100 μg / incubation to 5 μg / incubation using 2 μM cortisone in buffer 2. All other conditions were the same as described above. The results are shown in FIG.

  Decreasing the microsomal protein from 10 μg / incubation to 5 μg / incubation results in a corresponding decrease in enzyme activity. Increasing the microsomal protein to more than 10 μg / incubation results in a loss of signal that can be attributed to microsomal color. Therefore, the dynamic range of this assay cannot be improved by increasing the microsomal protein concentration.

(Development of RIA using Immunotech antibody)
The 11β-HSD1 assay was performed using 10 μg / well of human liver microsomal protein. Immunotech antibody was used in RIA at a concentration of 6.25 μg / well to 25 μg / well, and the results are shown in FIG.

  The Immunotech antibody worked well in this assay and gave a good signal background ratio at all concentrations tested. A signal-to-noise ratio of 12.5 and 6.1 μg antibody per well are similar, suggesting that antibody concentration may be reduced.

  Antibody titers at concentrations from 0.67 μg / well to 6.7 μg / well were tested. The Up-HSD1 assay was performed with 20 μg / well of human microsomal protein and produced an optimal signal background ratio. Each antibody concentration was tested against a “no enzyme” blank (buffer composed of microsomes), a “GA blank” (10 μl stop solution added before microsomes) and a control group. The results are shown in FIGS.

  The saturation curve shows that there is no difference in the detection of the enzyme activity 1.68 μg / well. The signal background ratio at this antibody concentration is good (6 times). Consequently, the antibody will be used at 1.7 μg / well in future assays.

RIA detection is used to test the linearity of enzyme activity with human liver microsomal protein concentrations. The Up-HSD1 assay was performed with the microsomal protein concentration changed from 1 μg / well to 40 μg / well. Up-HSD1 activity is linear with protein up to 20 μg / well (FIG. 18) and results obtained with a conventional enzyme assay (FIG. 7)
It was confirmed.

  The optimal concentration of human microsomal protein for use in this assay appears to be 10 μg / well.

  The effect of including Tween 80 in the enzyme assay buffer was also investigated. This assay was performed under the same conditions except that the enzyme assay buffer (Buffer 2) contained 0.05% Tween 80 in parallel with the above assay. Microsomal proteins were tested at 4 concentrations. Tween 80 was found to increase the blank CPM and reduce the signal-to-noise ratio of the assay. The respective data from the groups tested with 10 μg / well of microsomal protein are shown in FIG. Similar results were obtained at all microsomal protein concentrations tested, and as a result, Tween will not be used in future studies.

  Both enzyme assay and RIA stage are performed in the same buffer, and both phases are performed either in enzyme assay buffer (buffer 2) or buffer 3 (RIA buffer). Simplify the protocol. The microsomal protein concentration used was 10 μg / well and the cortisone concentration was 175 μM. When both the enzyme assay and RIA are performed in buffer 3, the data appears to improve slightly, FIG.

  The linearity of enzyme activity with incubation time was examined. Enzyme assays were performed with 10 μg / well microsomal protein and 175 nM cortisone and stopped at various time points. The results are shown in FIG.

With 10 μg / well microsomal protein and 175 nM substrate, the reaction is linear up to 30 minutes. These results indicate that the substrate concentration of 175 nM is too low. The apparent Km observed in the conventional 11β-HSD1 assay was 660 nM (FIGS. 8 and 9), but these assays were endpoint measurements, so the initial rate was 30 min. It is not clear whether the incubation time was measured in the low substrate group. However, published Km values for cortisone in the human liver microsome 11β-HSD1 assay are in the μmol range [18, 19]. The 175 nM substrate is well below the apparent Km, but there is no possibility of significantly increasing the concentration for two reasons:
(I) If the compound does not compete with cortisone, the measured inhibition falls when the substrate increases above the concentration used in ref. 1 (ii) Increasing the substrate concentration increases the specific activity of the label Decrease the sensitivity of the assay. This can be overcome by adding higher concentrations of [ ³ H] -cortisone, but this protocol uses 0.5 μCi / well and the cost impact when higher levels of radioactivity are used. Exists.

Substrate saturation was tested. Enzymatic assays were performed in Buffer 3 containing 10 μg / well of microsomal protein and [cold cortisone] as indicated, entirely as described in the Methods section. [ ³ H] -cortisone was overall 0.5 μCi / sample. The reaction was stopped after 30 minutes by adding 10 μl of stop solution. RIA was performed exactly as shown in the method section. The results are shown in FIGS.

  The apparent Km (700 nM) determined from the Lineweaver-Burke plot of these data shown in FIG. 23 is very similar to that determined in the tic-form 11β-HSD1 assay (FIG. 9, apparent Km about 660 nM). To do. These data suggest that at 10 μg of microsomal protein, the enzyme is not saturated even at 175 nM cortisone over a 30 minute incubation period.

  Bringing the reaction within the linear range by reducing the microsomal protein concentration or incubation time partially overcomes the problem. However, either of these modulations reduces the sensitivity of the assay and reduces the apparent intensity of the inhibitor. Consequently, the first experiment was performed with 175 nM cortisone.

(Evaluation of 11β-HSD1 assay)
Prior to testing the compounds, the resistance of the enzyme assay to DMSO was determined. Inclusion of 1% DMSO in the enzyme assay does not affect all or blank values, but slightly increases enzyme activity and signal to noise ratio (Table 4). This experiment was reported over a range of 0.3-10% DMSO concentrations, FIG.

(Table 4: Control and blank CPMs obtained in the IC ₅₀ assay for glycyrrhetinic acid show the effect of 1% DMSO and the signal to noise ratio obtained)

０．３％および１％のＤＭＳＯの存在下でのミクロソーム酵素活性にはわずかな上昇が見られる。１％を上回るＤＭＳＯ濃度において、酵素活性における線形の減少が存在する。ＤＭＳＯは、濃度に依存して、おそらくは、ミクロソーム膜上の効果に起因して、ミクロソーム酵素活性を増加も減少もし得る。これらのデータに基づいて、化合物は、１％ ＤＭＳＯの存在下でスクリーニングすることを意図する。

There is a slight increase in microsomal enzyme activity in the presence of 0.3% and 1% DMSO. There is a linear decrease in enzyme activity at DMSO concentrations above 1%. DMSO can increase or decrease microsomal enzyme activity, depending on concentration, possibly due to effects on the microsomal membrane. Based on these data, compounds are intended to be screened in the presence of 1% DMSO.

IC ₅₀ values were generated for the standard inhibitor glycyrrhetinic acid, and compounds were tested at concentrations between 0.012 μM and 3 μM at a final DMSO concentration of 1%, FIG.

Glycyretic acid produces a concentration-related inhibition of the enzyme with an IC ₅₀ of 41 nM, with a good fit value (R ₂ = 0.962) and Hillslope. This is similar to the 40 nM value generated using the tic format assay (see FIG. 10). An IC ₅₀ value of 30 nM has been reported for glycyrrhetinic acid inhibition of 11β-HSD1 in human liver microsomes using dehydro-dexamethasone as a substrate [19]. However, these values are lower than those reported by Barf et al. [15].

  (Table 5: Inhibition data)

（スルホンアミド合成）
（方法Ａ）
ピリジン（３当量）中に溶解したアミン（１当量）に、対応するスルホニルクロリド（１．２当量）を添加し、反応混合物を、Ｎ_２下、室温にて一晩撹拌した。得られた混合物をＨＣｌ水溶液中に注ぎ、有機層を酢酸エチルで抽出し、乾燥させ（ＭｇＳＯ_４）、濾過して、減圧下で濃縮して、結晶固体としてもしくは粘性シロップとして所望のスルホンアミドを得た。粗製化合物を次いで、溶離液としてＥｔＯＡｃ／ヘキサン（３：２）またはＣＨ_２Ｃｌ_２／ＥｔＯＡｃ（４：１）を用いるフラッシュクロマトグラフィーにより精製して、結晶固体を得た。

(Sulfonamide synthesis)
(Method A)
Pyridine amine dissolved in (3 eq) (1 eq), was added the corresponding sulfonyl chloride (1.2 eq), the reaction mixture, N ₂ was stirred under at room temperature overnight. The resulting mixture is poured into aqueous HCl and the organic layer is extracted with ethyl acetate, dried (MgSO ₄ ), filtered and concentrated under reduced pressure to give the desired sulfonamide as a crystalline solid or as a viscous syrup. Obtained. The crude compound was then purified by flash chromatography using EtOAc / hexane (3: 2) or CH ₂ Cl ₂ / EtOAc (4: 1) as eluent to give a crystalline solid.

(Method B)
To the amine (1 eq) dissolved in Et ₃ N (5 eq) was added the corresponding sulfonyl chloride (1.2 eq) and the reaction mixture was stirred overnight at room temperature under N ₂ . The resulting mixture was poured into water and the organic layer was extracted with ethyl acetate, dried (MgSO ₄ ), filtered and concentrated under reduced pressure to give the desired sulfonamide as a crystalline solid or as a viscous syrup. . The crude compound was then purified by flash chromatography using EtOAc / hexane (3: 2) or CH ₂ Cl ₂ / EtOAc (4: 1) as eluent to give a crystalline solid.

Note: Insoluble amine and sulfonyl chloride were dissolved in a minimum amount of CH ₂ Cl ₂ , THF or DMF.

(Method C)
To a solution of arylsulfonyl chloride (1.1 eq) in DCM was added pyridine (2.2 eq) and a catalytic amount of DMAP. The solution was stirred at room temperature for 10 minutes under nitrogen. Amine (1 eq) was then added and the reaction mixture was stirred at room temperature for 4-16 hours under nitrogen. The resulting mixture was partitioned between DCM and 5% sodium bicarbonate. The organic layer was washed with brine, dried over MgSO ₄ and concentrated to give a solid or viscous syrup. The crude compound was then purified by flash chromatography to give the desired arylsulfonamide as a crystalline solid.

DGS03020A (STX412)
Synthesized by Method A. Off-white crystalline DGS03020A (186 mg; 55%). mp 189-190 <0>C; TLC _Rf : 0.68 EtOAc / hexane (3: 2);

ＤＧＳ０３０２２Ａ（ＳＴＸ４１３）
方法Ａにより合成した。オフホワイトの結晶ＤＧＳ０３０２２Ａ（２３３ｍｇ；７２％）。ｍｐ １７８℃；ＴＬＣ Ｒ_ｆ：０．７１ ＥｔＯＡｃ／ヘキサン（３：２）；

DGS03022A (STX413)
Synthesized by Method A. Off-white crystalline DGS03022A (233 mg; 72%). mp 178 ° C .; TLC R _f : 0.71 EtOAc / hexane (3: 2);

ＤＧＳ０３０２４Ａ（ＳＴＸ４２１）
方法Ａにより合成した。白色結晶ＤＧＳ０３０２４Ａ（２４０ｍｇ；７６％）。ｍｐ １３３〜１３４℃；ＴＬＣ Ｒ_ｆ：０．７ ＥｔＯＡｃ／ヘキサン（３：２）；

DGS03024A (STX421)
Synthesized by Method A. White crystalline DGS03024A (240 mg; 76%). mp 133-134 ° C .; TLC R _f : 0.7 EtOAc / hexane (3: 2);

ＤＧＳ０３０３４Ａ（ＳＴＸ４２４）
方法Ａにより合成した。白色結晶ＤＧＳ０３０３４Ａ（２６２ｍｇ；８６％）。ｍｐ １５２℃；ＴＬＣ Ｒ_ｆ：０．４８ ＥｔＯＡｃ／ヘキサン（３：２）；

DGS03034A (STX424)
Synthesized by Method A. White crystalline DGS03034A (262 mg; 86%). mp 152 ° C .; TLC R _f : 0.48 EtOAc / hexane (3: 2);

ＤＧＳ０３０３６Ａ（ＳＴＸ４２５）
方法Ａにより合成した。白色結晶ＤＧＳ０３０３６Ａ（１３６ｍｇ；４２％）。ｍｐ ２９５〜２９６℃；ＴＬＣ Ｒ_ｆ：０．５６ ＥｔＯＡｃ／ヘキサン（３：２）；

DGS03036A (STX425)
Synthesized by Method A. White crystalline DGS03036A (136 mg; 42%). mp 295-296 [deg.] C; TLC _Rf : 0.56 EtOAc / hexane (3: 2);

ＤＧＳ０３０５８Ａ（ＳＴＸ５１９）
方法Ａにより合成した。白色結晶ＤＧＳ０３０５８Ａ（１９９ｍｇ；５７％）。ｍｐ １７２℃；ＴＬＣ Ｒ_ｆ：０．５６ ＥｔＯＡｃ／ヘキサン（３：２）；

DGS03058A (STX519)
Synthesized by Method A. White crystalline DGS03058A (199 mg; 57%). mp 172 ° C .; TLC R _f : 0.56 EtOAc / hexane (3: 2);

ＤＧＳ０３０６２Ｂ（ＳＴＸ４６９）
無水ＤＭＦ（５ｍｌ）およびＮａＨ（７ｍｇ，０．１６ｍｍｏｌ，１．１当量）中のＤＧＳ０３０２２Ａ（５０ｍｇ，０．１４ｍｍｏｌ，１当量）の撹拌溶液に、ＭｅＩ（３ｍｌ，０．２１ｍｍｏｌ，１．５当量）を添加し、混合物を１時間撹拌した。得られた混合物を水中に注ぎ、有機層を酢酸エチルで抽出し、乾燥させ（ＭｇＳＯ_４）、濾過して、減圧下で濃縮して、黄色の懸濁液を得た。粗製化合物（７０ｍｇ）を溶離液としてＥｔＯＡｃ／ヘキサン（３：２）を用いるフラッシュクロマトグラフィーにより精製して、白色結晶ＤＧＳ０３０６２Ａ（３６ｍｇ；６９％）を得た。ｍｐ ９７〜９８℃；ＴＬＣ Ｒ_ｆ：０．６１ ＥｔＯＡｃ／ヘキサン（３：２）；

DGS03062B (STX469)
To a stirred solution of DGS03022A (50 mg, 0.14 mmol, 1 eq) in anhydrous DMF (5 ml) and NaH (7 mg, 0.16 mmol, 1.1 eq), MeI (3 ml, 0.21 mmol, 1.5 eq) Was added and the mixture was stirred for 1 hour. The resulting mixture was poured into water and the organic layer was extracted with ethyl acetate, dried (MgSO ₄ ), filtered and concentrated under reduced pressure to give a yellow suspension. The crude compound (70 mg) was purified by flash chromatography using EtOAc / hexane (3: 2) as eluent to give white crystalline DGS03062A (36 mg; 69%). mp 97-98 <0>C; TLC _Rf : 0.61 EtOAc / hexane (3: 2);

ＤＧＳ０３０７２Ａ（ＳＴＸ４７０）
無水ＤＭＦ（５ｍｌ）およびＮａＨ（１０ｍｇ，０．１６ｍｍｏｌ，１．１当量）中のＤＧＳ０３０２２Ａ（５０ｍｇ，０．１４ｍｍｏｌ，１当量）の撹拌溶液に、Ｅｔｌ（２３ｍｇ，０．２１ｍｍｏｌ，１．５当量）を添加し、混合物を１時間撹拌した。得られた混合物を水中に注ぎ、有機層を酢酸エチルで抽出し、乾燥させ（ＭｇＳＯ_４）、濾過して、減圧下で濃縮して、黄色の懸濁液を得た。粗製化合物（７０ｍｇ）を溶離液としてＥｔＯＡｃ／ヘキサン（３：２）を用いるフラッシュクロマトグラフィーにより精製して、ＤＧＳ０３０７２Ａ（１６ｍｇ；３０％）の淡黄色の粘性シロップを得た。ＴＬＣ Ｒ_ｆ：０．７１ ＥｔＯＡｃ／ヘキサン（３：２）；

DGS03072A (STX470)
To a stirred solution of DGS03022A (50 mg, 0.14 mmol, 1 eq) in anhydrous DMF (5 ml) and NaH (10 mg, 0.16 mmol, 1.1 eq), Etl (23 mg, 0.21 mmol, 1.5 eq) Was added and the mixture was stirred for 1 hour. The resulting mixture was poured into water and the organic layer was extracted with ethyl acetate, dried (MgSO ₄ ), filtered and concentrated under reduced pressure to give a yellow suspension. The crude compound (70 mg) was purified by flash chromatography using EtOAc / hexane (3: 2) as eluent to give DGS03072A (16 mg; 30%) as a pale yellow viscous syrup. TLC _Rf : 0.71 EtOAc / hexane (3: 2);

ＤＧＳ０３０８２Ａ（ＳＴＸ５２１）
方法Ａにより合成した。白色結晶ＤＧＳ０３０８２Ａ（２３０ｍｇ；６７％）。ｍｐ ８５〜８６℃；ＴＬＣ Ｒ_ｆ：０．６４ ＥｔＯＡｃ／ヘキサン（３：２）；

DGS03082A (STX521)
Synthesized by Method A. White crystalline DGS03082A (230 mg; 67%). mp 85-86 ° C; TLC _Rf : 0.64 EtOAc / hexane (3: 2);

ＤＧＳ０３０８４Ａ（ＳＴＸ５２２）
方法Ａにより合成した。．黄色結晶ＤＧＳ０３０８４Ａ（４６ｍｇ；１０％）。ｍｐ ２５３〜２５４℃；ＴＬＣ Ｒ_ｆ：０． ７４ ＥｔＯＡｃ／ヘキサン（３：２）；

DGS03084A (STX522)
Synthesized by Method A. . Yellow crystalline DGS03084A (46 mg; 10%). mp 253-254 [deg.] C; TLC _Rf : 0. 74 EtOAc / hexane (3: 2);

ＤＧＳ０３０８６Ａ（ＳＴＸ５２３）
方法Ａにより合成した。淡黄色結晶ＤＧＳ０３０８６Ａ（１０１ｍｇ；５７％）。ｍｐ ２１９℃；ＴＬＣ Ｒ_ｆ：０．７１ＥｔＯＡｃ／ヘキサン（３：２）；

DGS03086A (STX523)
Synthesized by Method A. Pale yellow crystalline DGS03086A (101 mg; 57%). mp 219 ° C .; TLC R _f : 0.71 EtOAc / hexane (3: 2);

ＤＧＳ０３０６４
２，４−ジクロロ安息香酸（１０ｇ，０．０５２３ ｍｏｌ，１当量）を過剰量のクロロ硫酸（１０．５ｍＬ，０．１５７１ｍｏｌ，３当量）と共に、Ｎ_２下で、１８時間、１１５℃まで加熱した。得られた混合物を冷却し、その後、氷−水中にそそ板。得られた白色沈殿物を濾過して除き、十分な量の水で洗浄し、減圧下で一晩乾燥させた。粗製ＤＧＳ０３０６４（１１．５ｇ，７６％）を、さらに精製することなくその後の反応に用いた。ｍｐ １７３〜１７４℃；ＴＬＣ Ｒ_ｆ；０．４８（４：１，ＣＨ_２Ｃｌ_２／ＥｔＯＡｃ）；^１Ｈ ＮＭＲ（ＣＤＣｌ_３）δ ８．２８（１Ｈ，ｓ，Ａｒ−Ｈ），７．６５（１Ｈ，ｓ，Ａｒ−Ｈ）；ＭＳ ｍ／ｚ（ＦＡＢ＋）２８６．９［１００，（Ｍ＋Ｈ）＋］；ＨＲＭＳ ｍ／ｚ（ＦＡＢ＋）２８７．８７９８，Ｃ_７Ｈ_３ ^３５Ｃｌ_３Ｏ_４Ｓは２８７．８８１８，２９１．８７５５を必要とし，Ｃ_７Ｈ_３ ^３７Ｃｌ_３Ｏ_４Ｓは２９１．８７５９を必要とする。

DGS03064
2,4-Dichlorobenzoic acid (10 g, 0.0523 mol, 1 eq) was heated to 115 ° C. under N ₂ with excess chlorosulfuric acid (10.5 mL, 0.1571 mol, 3 eq) for 18 hours. did. Cool the resulting mixture and then pour into ice-water. The resulting white precipitate was filtered off, washed with a sufficient amount of water and dried overnight under reduced pressure. Crude DGS03064 (11.5 g, 76%) was used in subsequent reactions without further purification. mp 173-174 ° C .; TLC R _f ; 0.48 (4: 1, CH ₂ Cl ₂ / EtOAc); ¹ H NMR (CDCl ₃ ) δ 8.28 (1H, s, Ar—H), 7.65 (1H, s, Ar-H ); MS m / z (FAB +) 286.9 [100, (M + H) +]; HRMS m / z (FAB +) 287.8798, C 7 H 3 35 Cl 3 O 4 S Requires 287.8818, 291.8755, and C ₇ H ₃ ³⁷ Cl ₃ O ₄ S requires 291.8759.

DGS03088A (STX524)
Synthesized by Method B. Two compounds were isolated-DGS03088A and DGS03088A. White crystalline DGS03088A (48 mg; 13%). mp 153-155 <0>C; TLC _Rf : 0.79 EtOAc / hexane (3: 2);

ＤＧＳ０３０８８−１（ＳＴＸ５７５）
白色結晶ＤＧＳ０３０８８−１（３１ｍｇ；１２％）。ｍｐ １４７〜１４８℃；ＴＬＣ Ｒ_ｆ：０．４５ ＥｔＯＡｃ／ヘキサン（３：２）；

DGS03088-1 (STX575)
White crystalline DGS03088-1 (31 mg; 12%). mp 147-148 <0>C; TLC _Rf : 0.45 EtOAc / hexane (3: 2);

ＤＧＳ０３１００Ａ（ＳＴＸ５５２）
方法Ｂにより合成した。白色結晶ＤＧＳ０３１００Ａ（２２４ｍｇ；６９％）。ｍｐ ２２２〜２２３℃；ＴＬＣ Ｒ_ｆ：０．５６ ＣＨ_２Ｃｌ_２／ＥｔＯＡｃ（４：１）；

DGS03100A (STX552)
Synthesized by Method B. White crystalline DGS03100A (224 mg; 69%). mp 222-223 ° C .; TLC R _f : 0.56 CH ₂ Cl ₂ / EtOAc (4: 1);

ＤＧＳ０３１０２Ａ（ＳＴＸ５５３）
方法Ｂにより合成した。淡黄色結晶ＤＧＳ０３１０２Ａ（１７０ｍｇ；５２％）。ｍｐ ８９〜９０℃；ＴＬＣ Ｒ_ｆ：０．５５ ＣＨ_２Ｃｌ_２／ＥｔＯＡｃ（４：１）；

DGS03102A (STX553)
Synthesized by Method B. Pale yellow crystalline DGS03102A (170 mg; 52%). mp 89-90 ° C .; TLC R _f : 0.55 CH ₂ Cl ₂ / EtOAc (4: 1);

ＤＧＳ０３１０４Ａ（ＳＴＸ５５４）
方法Ｂにより合成した。黄色結晶 ＤＧＳ０３１０４Ａ（２３０ｍｇ；６３％）。ｍｐ ８５〜８６℃；ＴＬＣ Ｒ_ｆ：０．６５ ＣＨ_２Ｃｌ_２／ＥｔＯＡｃ（４：１）；

DGS03104A (STX554)
Synthesized by Method B. Yellow crystals DGS03104A (230 mg; 63%). _{mp 85~86 ℃; TLC R f:} 0.65 CH 2 Cl 2 / EtOAc (4: 1);

ＤＧＳ０３１１６Ａ（ＳＴＸ５８０）
方法Ｂにより合成した。淡黄色結晶ＤＧＳ０３１１６Ａ（１５１ｍｇ；４４％）。ｍｐ １５３℃；ＴＬＣ Ｒ_ｆ：０．５５ ＣＨ_２Ｃｌ_２／ＥｔＯＡｃ（４：１）；

DGS03116A (STX580)
Synthesized by Method B. Pale yellow crystalline DGS03116A (151 mg; 44%). mp 153 ° C .; TLC R _f : 0.55 CH ₂ Cl ₂ / EtOAc (4: 1);

ＤＧＳ０３１１８Ａ（ＳＴＸ５８１）
方法Ｂにより合成した。白色結晶ＤＧＳ０３１１８Ａ（４１６ｍｇ；４２％）。ｍｐ ８８〜８９℃；ＴＬＣ Ｒ_ｆ：０．４９ ＣＨ_２Ｃｌ_２／ＥｔＯＡｃ（４：１）；

DGS03118A (STX581)
Synthesized by Method B. White crystalline DGS03118A (416 mg; 42%). mp 88-89 ° C .; TLC R _f : 0.49 CH ₂ Cl ₂ / EtOAc (4: 1);

ＤＧＳ０３１２０Ａ（ＳＴＸ５８２）
方法Ｂにより合成した。淡黄色結晶ＤＧＳ０３１２０Ａ（１８５ｍｇ；５５％）。ｍｐ ９１〜９２℃；ＴＬＣ Ｒ_ｆ：０．５１ ＣＨ_２Ｃｌ_２／ＥｔＯＡｃ（４：１）；

DGS03120A (STX582)
Synthesized by Method B. Pale yellow crystalline DGS03120A (185 mg; 55%). mp 91-92 ° C .; TLC R _f : 0.51 CH ₂ Cl ₂ / EtOAc (4: 1);

ＤＧＳ０３１２２Ａ（ＳＴＸ７３１）
方法Ｂにより合成した。２つの化合物を単離した−ＤＧＳ０３１２２ＡおよびＤＧＳ０３１２２Ｂ．黄色結晶ＤＧＳ０３１２２Ａ（６７ｍｇ；２２％）。ｍｐ ２７２〜２７３℃；ＴＬＣ Ｒ_ｆ：０．５９ ＣＨ_２Ｃｌ_２／ＥｔＯＡｃ（４：１）；

DGS03122A (STX731)
Synthesized by Method B. Two compounds were isolated-DGS03122A and DGS03122B. Yellow crystalline DGS03122A (67 mg; 22%). _{mp 272~273 ℃; TLC R f:} 0.59 CH 2 Cl 2 / EtOAc (4: 1);

ＤＧＳ０３１２２Ｂ（ＳＴＸ５８３）
黄色結晶ＤＧＳ０３１２２Ｂ（４７ｍｇ；１６％）。ｍｐ ２０４−２０６ Ｃ；ＴＬＣ Ｒ_ｆ：０．４８ ＣＨ_２Ｃｌ_２／ＥｔＯＡｃ（４：１）；

DGS03122B (STX583)
Yellow crystalline DGS03122B (47 mg; 16%). _{mp 204-206 C; TLC R f:} 0.48 CH 2 Cl 2 / EtOAc (4: 1);

ＤＧＳ０３１２４Ａ（ＳＴＸ５８４）
方法Ｂにより合成した。淡黄色結晶ＤＧＳ０３１２４Ａ（１２５ｍｇ；５５％）。ｍｐ １８８〜１８９℃；ＴＬＣ Ｒ_ｆ：０．３７ ＣＨ_２Ｃｌ_２／ＥｔＯＡｃ（４：１）；

DGS03124A (STX584)
Synthesized by Method B. Pale yellow crystalline DGS03124A (125 mg; 55%). mp 188-189 ° C .; TLC R _f : 0.37 CH ₂ Cl ₂ / EtOAc (4: 1);

ＤＧＳ０３１２６Ａ（ＳＴＸ５８５）
方法Ｂにより合成した。淡黄色結晶ＤＧＳ０３１２６Ａ（１４５ｍｇ；４０％）。ｍｐ ８４〜８６℃；ＴＬＣ Ｒ_ｆ：０．７１ ＣＨ_２Ｃｌ_２／ＥｔＯＡｃ（４：１）；

DGS03126A (STX585)
Synthesized by Method B. Pale yellow crystalline DGS03126A (145 mg; 40%). mp 84-86 ° C .; TLC R _f : 0.71 CH ₂ Cl ₂ / EtOAc (4: 1);

ＤＧＳ０３１３０Ａ（ＳＴＸ７３０）
方法Ｂにより合成した。２つの化合物を単離した−ＤＧＳ０３１３０ＡおよびＤＧＳ０３１３０Ｂ。方法Ｂにより合成した。淡黄色結晶ＤＧＳ０３１３０Ａ（１０５ｍｇ；３３％）。ｍｐ １２５〜１２６℃；ＴＬＣ Ｒ_ｆ：０．５５ ＣＨ_２Ｃｌ_２／ＥｔＯＡｃ（４：１）；

DGS03130A (STX730)
Synthesized by Method B. Two compounds were isolated-DGS03130A and DGS03130B. Synthesized by Method B. Pale yellow crystalline DGS03130A (105 mg; 33%). mp 125-126 ° C .; TLC R _f : 0.55 CH ₂ Cl ₂ / EtOAc (4: 1);

ＤＧＳ０３１３０Ｂ（ＳＴＸ７０１）
淡黄色結晶ＤＧＳ０３１３０Ａ（４５ｍｇ；１４％）。ｍｐ１６９ Ｃ；ＴＬＣ Ｒ_ｆ：０．４２ ＣＨ_２Ｃｌ_２／ＥｔＯＡｃ（４：１）；

DGS03130B (STX701)
Pale yellow crystalline DGS03130A (45 mg; 14%). mp 169 C; TLC R _f : 0.42 CH ₂ Cl ₂ / EtOAc (4: 1);

ＤＧＳ０３１３４Ａ（ＳＴＸ７０３）
方法Ｂにより合成した。淡黄色結晶ＤＧＳ０３１３４Ａ（９１ｍｇ；２３％）。ｍｐ ２０６〜２０７℃；ＴＬＣ Ｒ_ｆ：０．８１ ＣＨ_２Ｃｌ_２／ＥｔＯＡｃ（４：１）；

DGS03134A (STX703)
Synthesized by Method B. Pale yellow crystalline DGS03134A (91 mg; 23%). _{mp 206~207 ℃; TLC R f:} 0.81 CH 2 Cl 2 / EtOAc (4: 1);

ＤＧＳ０３１３６Ａ（ＳＴＸ７０４）
方法Ｂにより合成した。淡黄色結晶ＤＧＳ０３１３６Ａ（２２５ｍｇ；７１％）。ｍｐ ５４〜５５℃；ＴＬＣ Ｒ_ｆ：０．５０ ＣＨ_２Ｃｌ_２／ＥｔＯＡｃ（４：１）；

DGS03136A (STX704)
Synthesized by Method B. Pale yellow crystalline DGS03136A (225 mg; 71%). _{mp 54~55 ℃; TLC R f:} 0.50 CH 2 Cl 2 / EtOAc (4: 1);

ＤＧＳ０３１３８Ｂ（ＳＴＸ７０５）
方法Ｂにより合成した。淡黄色結晶ＤＧＳ０３１３８Ｂ（２４ｍｇ；７％）。ｍｐ ２４８℃；ＴＬＣ Ｒ_ｆ：０．５２ ＣＨ_２Ｃｌ_２／ＥｔＯＡｃ（４：１）

DGS03138B (STX705)
Synthesized by Method B. Pale yellow crystalline DGS03138B (24 mg; 7%). _{mp 248 ℃; TLC R f:} 0.52 CH 2 Cl 2 / EtOAc (4: 1)

ＤＧＳ０３１４０Ａ（ＳＴＸ７１１）
方法Ｂにより合成した。褐色結晶ＤＧＳ０３１４０Ａ（８５ｍｇ；２６％）。ｍｐ ７３〜７５℃；ＴＬＣ Ｒ_ｆ：０．５９ ＣＨ_２Ｃｌ_２／ＥｔＯＡｃ（４：１）

DGS03140A (STX711)
Synthesized by Method B. Brown crystals DGS03140A (85 mg; 26%). _{mp 73~75 ℃; TLC R f:} 0.59 CH 2 Cl 2 / EtOAc (4: 1)

ＤＧＳ０３１４２Ａ（ＳＴＸ７０６）
方法Ｂにより合成した。淡黄色結晶ＤＧＳ０３１４２Ａ（７９ｍｇ；２４％）。ｍｐ ８９〜９１℃；ＴＬＣ Ｒ_ｆ：０．６５ ＣＨ_２Ｃｌ_２／ＥｔＯＡｃ（４：１）；

DGS03142A (STX706)
Synthesized by Method B. Pale yellow crystalline DGS03142A (79 mg; 24%). mp 89-91 ° C .; TLC R _f : 0.65 CH ₂ Cl ₂ / EtOAc (4: 1);

ＤＧＳ０３１４４Ａ（ＳＴＸ７０７）
方法Ｂにより合成した。淡黄色結晶ＤＧＳ０３１４４Ａ（７９ｍｇ；２４％）。ｍｐ ８９〜９１℃；ＴＬＣ Ｒ_ｆ：０．６９ ＣＨ_２Ｃｌ_２／ＥｔＯＡｃ（４：１）；

DGS03144A (STX707)
Synthesized by Method B. Pale yellow crystalline DGS03144A (79 mg; 24%). mp 89-91 ° C .; TLC R _f : 0.69 CH ₂ Cl ₂ / EtOAc (4: 1);

ＤＧＳ０３１４６Ａ（ＳＴＸ７０８）
方法Ｂにより合成した。淡黄色結晶ＤＧＳ０３１４６Ａ（１８１ｍｇ；５１％）。ｍｐ １７５℃；ＴＬＣ Ｒ_ｆ：０．５７ ＣＨ_２Ｃｌ_２／ＥｔＯＡｃ（４：１）；

DGS03146A (STX708)
Synthesized by Method B. Pale yellow crystalline DGS03146A (181 mg; 51%). _{mp 175 ℃; TLC R f:} 0.57 CH 2 Cl 2 / EtOAc (4: 1);

ＤＧＳ０３１４８Ａ（ＳＴＸ７０９）
方法Ｂにより合成した。オフホワイトの結晶ＤＧＳ０３１４８Ａ（１０２ｍｇ；３１％）。ｍｐ ２１４〜２１５℃；ＴＬＣ Ｒ_ｆ：０．６２ ＣＨ_２Ｃｌ_２／ＥｔＯＡｃ（４：１）；

DGS03148A (STX709)
Synthesized by Method B. Off-white crystalline DGS03148A (102 mg; 31%). mp 214-215 ° C .; TLC R _f : 0.62 CH ₂ Cl ₂ / EtOAc (4: 1);

ＤＧＳ０３１５０Ａ（ＳＴＸ７１０）
方法Ｂにより合成した。淡黄色結晶ＤＧＳ０３１５０Ａ（１０１ｍｇ；３０％）。ｍｐ ２００〜２０１℃；ＴＬＣ Ｒ_ｆ：０．５０ ＣＨ_２Ｃｌ_２／ＥｔＯＡｃ（４：１）；

DGS03150A (STX710)
Synthesized by Method B. Pale yellow crystalline DGS03150A (101 mg; 30%). _{mp 200~201 ℃; TLC R f:} 0.50 CH 2 Cl 2 / EtOAc (4: 1);

ＤＧＳ０３１５２Ａ（ＳＴＸ７１２）
方法Ｂにより合成した。淡黄色結晶ＤＧＳ０３１５２Ａ（１２０ｍｇ；３３％）。ｍｐ １８１〜１８２℃；ＴＬＣ Ｒ_ｆ：０．６５ ＣＨ_２Ｃｌ_２／ＥｔＯＡｃ（４：１）；

DGS03152A (STX712)
Synthesized by Method B. Pale yellow crystalline DGS03152A (120 mg; 33%). mp 181 ° -182 ° C .; TLC R _f : 0.65 CH ₂ Cl ₂ / EtOAc (4: 1);

ＤＧＳ０３１５８Ａ（ＳＴＸ７１３）
方法Ｂにより合成した。黄色結晶ＤＧＳ０３１５８Ａ（１５８ｍｇ；４０％）。ｍｐ ３３４〜３３５℃；ＴＬＣ Ｒ_ｆ：０．４７ ＣＨ_２Ｃｌ_２／ＥｔＯＡｃ（４：１）；

DGS03158A (STX713)
Synthesized by Method B. Yellow crystalline DGS03158A (158 mg; 40%). _{mp 334~335 ℃; TLC R f:} 0.47 CH 2 Cl 2 / EtOAc (4: 1);

（ベンゾチアゾールアリールスルホンアミド誘導体の合成）

(Synthesis of benzothiazole arylsulfonamide derivatives)

（Ｎ−ベンゾチアゾールベンゼンスルホンアミド誘導体の調製のための一般方法）
ＤＣＭ（５〜１０ｍＬ）中のアリールスルホニルクロリド（１．１当量）の溶液に、ピリジン（２．２当量）および触媒量のＤＭＡＰを添加した。溶液を窒素下、室温にて１０分間撹拌した。次いで、アミン（１当量）を添加し、反応混合物を窒素下、室温にて４〜１６時間撹拌した。得られた混合物をＤＣＭと５％炭酸水素ナトリウムとの間で分配した。有機層をブラインで洗浄し、ＭｇＳＯ_４で乾燥させ、濃縮して、黄色の残留物を得た。粗製化合物を次いで、フラッシュクロマトグラフィーで精製して、結晶固体として所望のベンゼンスルホンアミドを得た（収率４０〜９０％）。

(General method for the preparation of N-benzothiazolebenzenesulfonamide derivatives)
To a solution of arylsulfonyl chloride (1.1 eq) in DCM (5-10 mL) was added pyridine (2.2 eq) and a catalytic amount of DMAP. The solution was stirred at room temperature for 10 minutes under nitrogen. Amine (1 eq) was then added and the reaction mixture was stirred at room temperature for 4-16 hours under nitrogen. The resulting mixture was partitioned between DCM and 5% sodium bicarbonate. The organic layer was washed with brine, dried over MgSO ₄ and concentrated to give a yellow residue. The crude compound was then purified by flash chromatography to give the desired benzenesulfonamide as a crystalline solid (yield 40-90%).

(Synthesis of 2-alkylsulfanyl-benzothiazol-6-yl-amine)
To a solution of 6-amino-2-mercaptobenzothiazole (273 mg, 1.5 mmol) in anhydrous THF (10 mL) was added NaH (60% dispersion, 1.5 mmol) followed by alkyl halide (1. 5 mmol) was added. The mixture was stirred at room temperature for 24 hours and partitioned between ethyl acetate and 5% sodium bicarbonate. The organic layer was washed with brine, dried over sodium sulfate and concentrated under vacuum to a yellow solid, which was purified by recrystallization or flash chromatography (yield 60-90%).

The following amines were synthesized as described above:
(2-ethylsulfanylbenzothiazol-6-ylamine)
Yellow crystalline solid. mp 77-78 ° C (77 ° C in the literature). Single spot of TLC R _f 0.78 (8% methanol / DCM);

（２−（２−メトキシエチルスルファニル）−ベンゾチアゾール−６−イルアミン）
黄色の粘性シロップ。ＴＬＣ Ｒ_ｆ ０．６５の単一スポット（３０％ 酢酸エチル／ＤＣＭ）；

(2- (2-methoxyethylsulfanyl) -benzothiazol-6-ylamine)
Yellow viscous syrup. TLC _Rf 0.65 single spot (30% ethyl acetate / DCM);

（６−アミノベンゾチアゾール−２−イルメルカプト）−酢酸エチルエステル
オフホワイトの固体。ｍｐ ８７〜８９℃（文献では９２℃，［２０］）；ＴＬＣ Ｒ_ｆ ０．７２の単一スポット（８％ メタノール／ＤＣＭ）；

(6-Aminobenzothiazol-2-ylmercapto) -acetic acid ethyl ester Off-white solid. mp 87-89 ° C (92 ° C in literature, [20]); TLC R _f 0.72 single spot (8% methanol / DCM);

以下の化合物を、Ｎ−ベンゾチアゾールベンゼンスルホンアミドについての一般法を用いて合成した：
３−クロロ−Ｎ−（２−エチルスルファニルベンゾチアゾール−６−イル）−２−メチルベンゼンスルホンアミド（ＳＴＸ７５１，ＸＤＳ０１１４１）
オフホワイトの固体（２２０ｍｇ；５５％）。ＴＬＣ Ｒ_ｆ：０．８３の単一スポット（１７％ ＥｔＯＡｃ／ＤＣＭ）；ＨＰＬＣ純度 ９６％（ｔ_Ｒ メタノール中１．９分）；

The following compounds were synthesized using the general method for N-benzothiazolebenzenesulfonamide:
3-chloro-N- (2-ethylsulfanylbenzothiazol-6-yl) -2-methylbenzenesulfonamide (STX751, XDS01141)
Off-white solid (220 mg; 55%). TLC R _f : 0.83 single spot (17% EtOAc / DCM); HPLC purity 96% (1.9 min in t _R methanol);

［６−（３−クロロ−２−メチルベンゼンスルホニルアミノ）−ベンゾチアゾール−２−イルスルファニル］−酢酸エチルエステル（ＳＴＸ７５２，ＸＤＳ０１１４２）
白色結晶固体（２１０ｍｇ；４６％）。ＴＬＣ Ｒ_ｆ：０．６９の単一スポット（１７％ ＥｔＯＡｃ／ＤＣＭ）；ＨＰＬＣ純度 ９９％（ｔ_Ｒ １０％ 水−メタノール中２．９分）９９％（ｔ_Ｒ １０％ 水−メタノール中２．３分）；

[6- (3-Chloro-2-methylbenzenesulfonylamino) -benzothiazol-2-ylsulfanyl] -acetic acid ethyl ester (STX752, XDS01142)
White crystalline solid (210 mg; 46%). TLC R _f : 0.69 single spot (17% EtOAc / DCM); HPLC purity 99% (2.9% in t _R 10% water-methanol) 99% (t _R 10% in water-methanol 2. 3 minutes);

（３−クロロ−Ｎ−［２−（２−メトキシエチルスルファニル）−ベンゾチアゾール−６−イル］−２−メチルベンゼンスルホンアミド（ＳＴＸ７５４，ＸＤＳ０１１４４））
オフホワイトの固体（１５０ｍｇ；７７％）。ＴＬＣ Ｒ_ｆ ０．６０の単一スポット（１７％ ＥｔＯＡｃ／ＤＣＭ）；ＨＰＬＣ純度９４％（ｔ_Ｒ １０％ 水−メタノール中３．１分）；

(3-Chloro-N- [2- (2-methoxyethylsulfanyl) -benzothiazol-6-yl] -2-methylbenzenesulfonamide (STX754, XDS01144))
Off-white solid (150 mg; 77%). TLC R _f 0.60 single spot (17% EtOAc / DCM); HPLC purity 94% (t _R 10% water-3.1 min in methanol);

（２−［６−（３−クロロ−２−メチルベンゼンスルホニルアミノ）−ベンゾチアゾール−２−イルスルファニル］−Ｎ，Ｎ−ジエチルアセトアミド（ＳＴＸ７５５，ＸＤＳ０１１４５）および２−［６−（３−クロロ−２−メチルベンゼンスルホニルアミノ）−ベンゾチアゾール−２−イル］−Ｎ，Ｎ−ジエチルアセトアミド（ＳＴＸ７６３，ＸＤＳ０１１４５Ｂ））
ＤＭＣ（５ｍｌ）中のＡｌＣｌ_３（５０ｍｇ）の懸濁液に、ジエチルアミン（０．４ｍｌ）を添加した。溶液を窒素下、室温にて１０分間撹拌した。［６−（３−クロロ−２−メチルベンゼンスルホニルアミノ）−ベンゾチアゾール−２−イルスルファニル］−酢酸エチルエステル（ＳＴＸ７５２，１００ｍｇ）を添加し、混合物を室温にて３０分間撹拌し続けた。反応を水でクエンチし、ＤＣＭと５％ ＮａＨＣＯ_３との間で分配した。有機層を水で洗浄し、ＭｇＳＯ_４で乾燥させ、真空下でエバポレートして、黄色の残留物を生じ、これを、溶離溶媒として２０〜３０％ 酢酸エチル−ＤＣＭを用いるフラッシュカラムクロマトグラフィーにより精製した。ＳＴＸ７５５（５０ｍｇ，４７％）を白色固体として得た。ＴＬＣ Ｒ_ｆ ０．６０の単一スポット（２５％ ＥｔＯＡｃ／ＤＣＭ）；ＨＰＬＣ純度 ８９％（ｔ_Ｒ １０％ 水−メタノール中２．７分）；

(2- [6- (3-Chloro-2-methylbenzenesulfonylamino) -benzothiazol-2-ylsulfanyl] -N, N-diethylacetamide (STX755, XDS01145) and 2- [6- (3-chloro- 2-methylbenzenesulfonylamino) -benzothiazol-2-yl] -N, N-diethylacetamide (STX763, XDS01145B))
To a suspension of AlCl ₃ (50 mg) in DMC (5 ml) was added diethylamine (0.4 ml). The solution was stirred at room temperature for 10 minutes under nitrogen. [6- (3-Chloro-2-methylbenzenesulfonylamino) -benzothiazol-2-ylsulfanyl] -acetic acid ethyl ester (STX752,100 mg) was added and the mixture continued to stir at room temperature for 30 minutes. The reaction was quenched with water and partitioned between DCM and 5% NaHCO ₃ . The organic layer is washed with water, dried over MgSO ₄ and evaporated under vacuum to give a yellow residue which is purified by flash column chromatography using 20-30% ethyl acetate-DCM as the eluting solvent. did. STX755 (50 mg, 47%) was obtained as a white solid. TLC R _f 0.60 single spot (25% EtOAc / DCM); HPLC purity 89% (t _R 10% water-2.7 min in methanol);

ＳＴＸ７６３（２５ｍｇ，２５％）を白色固体として得た。ＴＬＣ Ｒ_ｆ ０．３９の単一スポット（２５％ ＥｔＯＡｃ／ＤＣＭ）；ＨＰＬＣ純度 ９８％（ｔ_Ｒ １０％ 水−ＣＨ_３ＣＮ中６．９分）；

STX763 (25 mg, 25%) was obtained as a white solid. Single spot (25% EtOAc / DCM) of TLC R _f 0.39; HPLC purity _{98% (t} R _10% water _-CH 3 CN in 6.9 min);

３−クロロ−Ｎ−ベンゾチアゾール−６−イル−２−メチルベンゼンスルホンアミド（ＳＴＸ７５０，ＸＤＳ０１１３９）
淡ピンクの針状物質（２６０ｍｇ；７７％）。ＴＬＣ Ｒ_ｆ ０．４６の単一スポット（１７％ ＥｔＯＡｃ／ＤＣＭ）；ＨＰＬＣ純度９９％（ｔ_Ｒ １０％ 水−メタノール中２．５分）；

3-Chloro-N-benzothiazol-6-yl-2-methylbenzenesulfonamide (STX750, XDS01139)
Pale pink needles (260 mg; 77%). TLC R _f 0.46 single spot (17% EtOAc / DCM); HPLC purity 99% (t _R 10% water-2.5 min in methanol);

３−クロロ−Ｎ−（２−メチルベンゾチアゾール−６−イル）−２−メチルベンゼンスルホンアミド（ＳＴＸ８８６，ＸＤＳ０１１８７Ｂ）
オフホワイトの固体。ＴＬＣ Ｒ_ｆ ０．６５の単一スポット（１０％ メタノール／ＤＣＭ）；ＨＰＬＣ純度＞９９％（ｔ_Ｒ １０％ 水−メタノール中２．４分）；

3-Chloro-N- (2-methylbenzothiazol-6-yl) -2-methylbenzenesulfonamide (STX886, XDS01187B)
Off-white solid. TLC R _f 0.65 single spot (10% methanol / DCM); HPLC purity> 99% (t _R 10% water-2.4 min in methanol);

Ｎ−（２−メチルベンゾチアゾール−６−イル）−Ｎ−（３−クロロ−２−メチルフェニルスルホニル）−３−クロロ−２−メチルベンゼンスルホンアミド（ＳＴＸ８８７，ＸＤＳ０１１８７Ａ）
オフホワイトの粉末。ＴＬＣ Ｒ_ｆ ０．８９の単一スポット（１０％ メタノール／ＤＣＭ）；ＨＰＬＣ純度９１％（ｔ_Ｒ １０％ 水−メタノール中３．１分）；

N- (2-methylbenzothiazol-6-yl) -N- (3-chloro-2-methylphenylsulfonyl) -3-chloro-2-methylbenzenesulfonamide (STX887, XDS01187A)
Off-white powder. TLC R _f 0.89 single spot (10% methanol / DCM); HPLC purity 91% (t _R 10% water-3.1 min in methanol);

２，５−ジクロロ−Ｎ−（２−メチルベンゾチアゾール−６−イル）−ベンゼンスルホンアミド（ＳＴＸ８８８，ＸＤＳ０１１８８Ｂ）
白色結晶固体。ＴＬＣ Ｒ_ｆ ０．６８の単一スポット（１０％ メタノール／ＤＣＭ）；ＨＰＬＣ純度＞９９％（ｔ_Ｒ １０％ 水−メタノール中２．３分）；

2,5-Dichloro-N- (2-methylbenzothiazol-6-yl) -benzenesulfonamide (STX888, XDS01188B)
White crystalline solid. TLC R _f 0.68 single spot (10% methanol / DCM); HPLC purity> 99% (t _R 10% water-2.3 min in methanol);

Ｎ−（２−メチルベンゾチアゾール−６−イル）−Ｎ−（２，５−ジクロロフェニルスルホニル）−２，５−ジクロロ−ベンゼンスルホンアミド（ＳＴＸ８８９，ＸＤＳ０１１８８Ａ）
黄色固体。ＴＬＣ Ｒ_ｆ ０．７２の単一スポット（１０％ メタノール／ＤＣＭ）；ＨＰＬＣ純度 ９４％（ｔ_Ｒ １０％ 水−メタノール中２．９分）；

N- (2-methylbenzothiazol-6-yl) -N- (2,5-dichlorophenylsulfonyl) -2,5-dichloro-benzenesulfonamide (STX889, XDS01188A)
Yellow solid. TLC R _f 0.72 single spot (10% methanol / DCM); HPLC purity 94% (t _R 10% water-2.9 min in methanol);

Ｎ−（２−メチルベンゾチアゾール−６−イル）−４−プロピルベンゼンスルホンアミド（ＳＴＸ８９０，ＸＤＳ０１１８９）
オフホワイトの固体。ＴＬＣ Ｒ_ｆ ０．７２の単一スポット（１０％ メタノール／ＤＣＭ）；ＨＰＬＣ純度 ９９％（ｔ_Ｒ １０％ 水−メタノール中２．３分）；

N- (2-methylbenzothiazol-6-yl) -4-propylbenzenesulfonamide (STX890, XDS01189)
Off-white solid. TLC R _f 0.72 single spot (10% methanol / DCM); HPLC purity 99% (t _R 10% water-2.3 min in methanol);

３−クロロ−２−メチル−Ｎ−（２−オキソ−２，３−ジヒドロ−ベンゾチアゾール−６−イル）−ベンゼンスルホンアミド（ＳＴＸ７５３，ＸＤＳ０１１４３）
白色結晶固体（１６０ｍｇ；４５％）。ＴＬＣ Ｒ_ｆ ０．４２の単一スポット（１７％ ＥｔＯＡｃ／ＤＣＭ）；ＨＰＬＣ純度 ９８％（ｔ_Ｒ １０％ 水−メタノール中２．３分）；

3-Chloro-2-methyl-N- (2-oxo-2,3-dihydro-benzothiazol-6-yl) -benzenesulfonamide (STX753, XDS01143)
White crystalline solid (160 mg; 45%). TLC R _f 0.42 single spot (17% EtOAc / DCM); HPLC purity 98% (t _R 10% water-2.3 min in methanol);

３−クロロ−Ｎ−メチル−Ｎ−（３−メチル−２−オキソ−２，３−ジヒドロ−ベンゾチアゾール−６−イル）−２−メチルベンゼンスルホンアミド（ＳＴＸ８３１，ＸＤＳ０１１６３）
アセトン（３ｍＬ）中のＳＴＸ７５３（６６ｍｇ、０．１９ｍｍｏｌ）の溶液に、炭酸カリウム（６６ｍｇ）を添加し、その後、メチルヨージド（６６ｍｇ）を添加した。混合物を室温にて２時間撹拌し、ＤＣＭ中に抽出し、ブラインで洗浄した。硫酸ナトリウムで乾燥させた後、溶媒を真空下で除去して、油状の残留物を得、これを、フラッシュクロマトグラフィーにより精製した。オフホワイトの固体（５９ｍｇ、８０％）をＥｔＯＨ。ＴＬＣ Ｒ_ｆ ０．３７の単一スポット（１００％ ＤＣＭ）；ＨＰＬＣ純度 ９９％（ｔ_Ｒ １０％ 水−メタノール中２．０分）；^１Ｈ ＮＭＲ（４００ＭＨｚ，

3-Chloro-N-methyl-N- (3-methyl-2-oxo-2,3-dihydro-benzothiazol-6-yl) -2-methylbenzenesulfonamide (STX831, XDS01163)
To a solution of STX753 (66 mg, 0.19 mmol) in acetone (3 mL) was added potassium carbonate (66 mg) followed by methyl iodide (66 mg). The mixture was stirred at room temperature for 2 hours, extracted into DCM and washed with brine. After drying over sodium sulfate, the solvent was removed in vacuo to give an oily residue that was purified by flash chromatography. An off-white solid (59 mg, 80%) in EtOH. TLC R _f 0.37 single spot (100% DCM); HPLC purity 99% (2.0% in t _R 10% water-methanol); ¹ H NMR (400 MHz,

［（３−クロロ−２−メチルベンゼンスルホニル）−（３−エトキシカルボニルメチル−２−オキソ−２，３−ジヒドロ−ベンゾチアゾール−６−イル）−アミノ］−酢酸エチルエステル（ＳＴＸ７６４，ＸＤＳ０１１４９）
アセトン（３ｍＬ）中のＳＴＸ７５３（２０ｍｇ，０．０５６ｍｍｏｌ）の溶液に、炭酸カリウム（２０ｍｇ）を添加し、その後、メチル ２−ブロモ酢酸エチル（５０μｌ）を添加した。混合物を室温にて４時間撹拌し、ＥｔＯＡｃ中に抽出し、ブラインで洗浄した。硫酸ナトリウムで乾燥させた後、溶媒を真空下で除去して、油状の残留物を生じ、これを、フラッシュクロマトグラフィーにより精製した。白色結晶固体（２０ｍｇ，６８％）を得た。ＴＬＣ Ｒ_ｆ ０．５１の単一スポット（３０％ 酢酸エチル／ヘキサン）；ＨＰＬＣ純度 ９８％（ｔ_Ｒ １０％ 水−メタノール中２．６分）；

[(3-Chloro-2-methylbenzenesulfonyl)-(3-ethoxycarbonylmethyl-2-oxo-2,3-dihydro-benzothiazol-6-yl) -amino] -acetic acid ethyl ester (STX764, XDS01149)
To a solution of STX753 (20 mg, 0.056 mmol) in acetone (3 mL) was added potassium carbonate (20 mg) followed by methyl 2-bromoethyl acetate (50 μl). The mixture was stirred at room temperature for 4 hours, extracted into EtOAc and washed with brine. After drying with sodium sulfate, the solvent was removed in vacuo to yield an oily residue that was purified by flash chromatography. White crystalline solid (20 mg, 68%) was obtained. Single spot of TLC R _f 0.51 (30% ethyl acetate / hexane); HPLC purity 98% (t _R 10% water-2.6 min in methanol);

（２−クロロベンゾチアゾール−６−イル−アミンおよび２−クロロ−ベンゾチアゾール−５−イル−アミンの合成）
濃Ｈ_２ＳＯ_４（６０ｍＬ）中の２−クロロベンゾチアゾール（１２．０ｇ，７０．７ｍｍｏｌ）の溶液に、ＨＮＯ_３（６９％溶液，６ｍＬ）を、０℃にて２０分間にわたって滴下した。混合物を５℃にて３時間撹拌し、氷−水（１５０ｍＬ）中に注いだ。沈殿物を回収し、５％炭酸水素ナトリウムおよび水で洗浄して、真空下で乾燥させた。^１Ｈ ＮＭＲ分析は、混合物が、７８％の６−ニトロ−２−クロロベンゾチアゾールおよび８％の５−ニトロ−２ クロロベンゾチアゾールを含んだことを示した。エタノールから再結晶させて、白色固体として６−ニトロ−２−クロロベンゾチアゾールを得た（１１ｇ，７２％）。３．５ｇの固体を還流中のエタノール−酢酸（１５０：１５ｍＬ）中に溶解させ、鉄粉末を一度に添加した。混合物を１．５時間還流させ、濾過した。濾液を真空下で半分の容量まで濃縮して、１０％ ＮａＯＨを用いてｐＨ ７．５まで中和し、酢酸エチルで抽出した。有機相をブラインで洗浄し、硫酸マグネシウムで乾燥させ、エバポレートして、残留物を得た。これを、エタノールから再結晶させた。淡紫結晶（２．５ｇ，８３％）を得た。Ｍｐ １６０〜１６４℃；ＴＬＣ Ｒ_ｆ ０．２７の単一スポット（３０％ ＥｔＯＡｃ／ヘキサン）；^１Ｈ ＮＭＲ（２７０ＭＨｚ，ＤＭＳＯ−ｄ６）δ ７．５８（１Ｈ，ｄ，Ｊ＝９．０Ｈｚ，４−Ｈ），７．０３（１Ｈ，ｄ，Ｊ＝２．０Ｈｚ，７−Ｈ），６．７７（１Ｈ，ｄｄ，Ｊ＝９．０，２．０Ｈｚ，５−Ｈ），５．５５（２Ｈ，ｓ，ＮＨ２）。ニトロ化生成物の再結晶からの母液をエバポレートして、上記のように鉄粉末による還元に供した。粗製生成物をフラッシュクロマグラフィー（酢酸エチル−ＤＣＭ勾配溶出）により精製して、黄色固体として２−クロロ−ベンゾチアゾール−５−イル−アミンを生じた。Ｍｐ １４６〜１４９℃；ＴＬＣ Ｒ_ｆ ０．５２の単一スポット（１０％ ＥｔＯＡｃ／ＤＣＭ）；^１Ｈ ＮＭＲ（２７０ＭＨｚ，ＤＭＳＯ−ｄ６）δ ７．６３（１Ｈ，ｄ，Ｊ＝８．６Ｈｚ，７Ｈ），７．０５（１Ｈ，ｄ，Ｊ＝２．３Ｈｚ，４Ｈ），６．７８（１Ｈ，ｄｄ，Ｊ＝８．６，２．３Ｈｚ，６Ｈ），５．４０（２Ｈ，ｓ，ＮＨ_２）。

(Synthesis of 2-chlorobenzothiazol-6-yl-amine and 2-chloro-benzothiazol-5-yl-amine)
To a solution of 2-chlorobenzothiazole (12.0 g, 70.7 mmol) in concentrated H ₂ SO ₄ (60 mL) was added HNO ₃ (69% solution, 6 mL) dropwise at 0 ° C. over 20 minutes. The mixture was stirred at 5 ° C. for 3 hours and poured into ice-water (150 mL). The precipitate was collected, washed with 5% sodium bicarbonate and water and dried under vacuum. ¹ H NMR analysis indicated that the mixture contained 78% 6-nitro-2-chlorobenzothiazole and 8% 5-nitro-2 chlorobenzothiazole. Recrystallization from ethanol gave 6-nitro-2-chlorobenzothiazole as a white solid (11 g, 72%). 3.5 g of solid was dissolved in refluxing ethanol-acetic acid (150: 15 mL) and iron powder was added in one portion. The mixture was refluxed for 1.5 hours and filtered. The filtrate was concentrated to half volume under vacuum, neutralized to pH 7.5 with 10% NaOH and extracted with ethyl acetate. The organic phase was washed with brine, dried over magnesium sulfate and evaporated to give a residue. This was recrystallized from ethanol. Pale purple crystals (2.5 g, 83%) were obtained. Mp 160-164 ° C .; TLC R _f 0.27 single spot (30% EtOAc / hexane); ¹ H NMR (270 MHz, DMSO-d6) δ 7.58 (1H, d, J = 9.0 Hz, 4 -H), 7.03 (1H, d, J = 2.0 Hz, 7-H), 6.77 (1H, dd, J = 9.0, 2.0 Hz, 5-H), 5.55 ( 2H, s, NH2). The mother liquor from recrystallization of the nitrated product was evaporated and subjected to reduction with iron powder as described above. The crude product was purified by flash chromatography (ethyl acetate-DCM gradient elution) to give 2-chloro-benzothiazol-5-yl-amine as a yellow solid. Mp 146-149 ° C .; TLC R _f 0.52 single spot (10% EtOAc / DCM); ¹ H NMR (270 MHz, DMSO-d6) δ 7.63 (1H, d, J = 8.6 Hz, 7H) ), 7.05 (1H, d, J = 2.3 Hz, 4H), 6.78 (1H, dd, J = 8.6, 2.3 Hz, 6H), 5.40 (2H, s, NH ₂ ).

The following compounds were synthesized by the general method for N-benzothiazolebenzenesulfonamide:
(N- (2-chlorobenzothiazol-6-yl) -N- (3-chloro-2-methylphenylsulfonyl) -3-chloro-2-methylbenzenesulfonamide (STX767, XDS01151A))
White crystalline solid. TLC R _f 0.78 single spot (33% EtOAc / DCM); HPLC purity 95% (t _R 10% water-6.4 min in methanol);

（３−クロロ−Ｎ−（２−クロロベンゾチアゾール−６−イル）−２−メチルベンゼンスルホンアミド（ＳＴＸ７６８，ＸＤＳ０１１５１ Ｂ））
オフホワイトの結晶固体。ＴＬＣ Ｒ_ｆ ０．６８の単一スポット（３３％ ＥｔＯＡｃ／ＤＣＭ）；ＨＰＬＣ純度 ９９％（ｔ_Ｒ メタノール中１．７分）；

(3-Chloro-N- (2-chlorobenzothiazol-6-yl) -2-methylbenzenesulfonamide (STX768, XDS01151 B))
Off-white crystalline solid. TLC R _f 0.68 single spot (33% EtOAc / DCM); HPLC purity 99% (t _R 1.7 min in methanol);

（３−クロロ−Ｎ−（２−クロロベンゾチアゾール−５−イル）−２−メチルベンゼンスルホンアミド（ＳＴＸ８３４，ＸＤＳ０１１６８））
白色結晶固体。ＴＬＣ Ｒ_ｆ ０．５２の単一スポット（３０％ ＥｔＯＡｃ／ヘキサン）；ＨＰＬＣ純度 ９９％（ｔ_Ｒ メタノール中１．７）；

(3-Chloro-N- (2-chlorobenzothiazol-5-yl) -2-methylbenzenesulfonamide (STX834, XDS01168))
White crystalline solid. Single spot (30% EtOAc / hexanes) of TLC R _f 0.52; HPLC purity 99% _{(t R} in methanol 1.7);

（３−クロロ−２−メチル−Ｎ−（２−メチルアミノベンゾチアゾール−６−イル）−ベンゼンスルホンアミド（ＳＴＸ８３３，ＸＤＳ０１１６７））
ＣＨ_３ＮＨ−ＴＨＦ（２Ｍ，３ｍＬ）中の３−クロロ−Ｎ−（２−クロロベンゾチアゾール−６−イル）−２−メチルベンゼンスルホンアミド（ＳＴＸ７６８，１５０ｍｇ，０．４０ｍｍｏｌ）の溶液を、密封されたチューブ内、８２℃にて２４時間撹拌し、酢酸エチルで抽出した。有機相をブラインで洗浄し、硫酸ナトリウムで乾燥させ、真空下で濃縮して、残留物を生じ、これを、フラッシュクロマトグラフィー（酢酸エチル／ＤＣＭ勾配溶出）により精製した。白色結晶（１００ｍｇ，６８％）を得た。ＴＬＣ Ｒ_ｆ ０．２７の単一スポット（３０％ ＥｔＯＡｃ／ＤＣＭ）；ＨＰＬＣ純度 ９９％（ｔ_Ｒ ４％ 水−メタノール中１．８分）；

(3-Chloro-2-methyl-N- (2-methylaminobenzothiazol-6-yl) -benzenesulfonamide (STX833, XDS01167))
_{CH 3 NH-THF (2M,} 3mL) solution of 3-chloro-N-(2-chloro-6-yl) -2-methyl-benzenesulfonamide (STX768,150mg, 0.40mmol) A solution of sealing The tube was stirred at 82 ° C. for 24 hours and extracted with ethyl acetate. The organic phase was washed with brine, dried over sodium sulfate and concentrated in vacuo to give a residue that was purified by flash chromatography (ethyl acetate / DCM gradient elution). White crystals (100 mg, 68%) were obtained. TLC R _f 0.27 single spot (30% EtOAc / DCM); HPLC purity 99% (t _R 4% water-1.8 min in methanol);

３−クロロ−２−メチル−Ｎ−（２−メチルアミノベンゾチアゾール−５−イル）−ベンゼンスルホンアミド（ＳＴＸ８３５，ＸＤＳ０１１７６）
３−クロロ−Ｎ−（２−クロロベンゾチアゾール−５−イル）−２−メチルベンゼンスルホンアミド（ＳＴＸ８３４，８０ｍｇ，０．２１ｍｍｏｌ）を出発物質として用いて、ＳＴＸ８３３について記載したように、化合物を調製した。白色結晶（６０ｍｇ，７８％）を得た。ＴＬＣ Ｒ_ｆ ０．２５の単一スポット（３０％ ＥｔＯＡｃ／ＤＣＭ）；ＨＰＬＣ純度 ９９％（ｔ_Ｒ １０％ 水−メタノール中２．３分）；

3-Chloro-2-methyl-N- (2-methylaminobenzothiazol-5-yl) -benzenesulfonamide (STX835, XDS01176)
The compound was prepared as described for STX833 using 3-chloro-N- (2-chlorobenzothiazol-5-yl) -2-methylbenzenesulfonamide (STX834, 80 mg, 0.21 mmol) as starting material. did. White crystals (60 mg, 78%) were obtained. TLC R _f 0.25 single spot (30% EtOAc / DCM); HPLC purity 99% (t _R 10% water-2.3 min in methanol);

３−クロロ−Ｎ−（２−ジエチルアミノベンゾチアゾール−５−イル）−２−メチルベンゼンスルホンアミド（ＳＴＸ８３６，ＸＤＳ０１１７７）
３−クロロ−Ｎ−（２−クロロベンゾチアゾール−５−イル）−２−メチルベンゼンスルホンアミド（ＳＴＸ８３４，７０ｍｇ，０．１８ｍｍｏｌ）およびジエチルアミン−ＴＨＦ（３ｍＬ）を出発物質として用いて、ＳＴＸ８３３について記載した通りに化合物を調製した。白色結晶（５０ｍｇ，６８％）を得た。ＴＬＣ Ｒ_ｆ ０．６０の単一スポット（３０％ ＥｔＯＡｃ／ＤＣＭ）；ＨＰＬＣ純度 ９７％（ｔ_Ｒ １０％ 水−メタノール中３．０分）；

3-Chloro-N- (2-diethylaminobenzothiazol-5-yl) -2-methylbenzenesulfonamide (STX836, XDS01177)
Described for STX833 using 3-chloro-N- (2-chlorobenzothiazol-5-yl) -2-methylbenzenesulfonamide (STX834, 70 mg, 0.18 mmol) and diethylamine-THF (3 mL) as starting materials. Compounds were prepared as described. White crystals (50 mg, 68%) were obtained. TLC R _f 0.60 single spot (30% EtOAc / DCM); HPLC purity 97% (t _R 10% water-3.0 min in methanol);

３−クロロ−Ｎ−（２−ジエチルアミノベンゾチアゾール−６−イル）−２−メチルベンゼンスルホンアミド（ＳＴＸ８７８，ＸＤＳ０１１６４）
３−クロロ−Ｎ−（２−クロロベンゾチアゾール−６−イル）−２−メチルベンゼンスルホンアミド（ＳＴＸ７６８，２４０ｍｇ，０．６４ｍｍｏｌ）およびジエチルアミン−ＩＰＡ（３ｍＬ）を出発物質として用いて、ＳＴＸ８３３について記載した通りに化合物を調製した。フホワイトの結晶固体（１２８ｍｇ，４９％）を得た。ＴＬＣ Ｒ_ｆ ０．３３の単一スポット（３０％ ＥｔＯＡｃ／ヘキサン）；ＨＰＬＣ純度 ９６％（ｔ_Ｒ ４％ 水−メタノール中２．２分）；

3-Chloro-N- (2-diethylaminobenzothiazol-6-yl) -2-methylbenzenesulfonamide (STX878, XDS01164)
Described for STX833 using 3-chloro-N- (2-chlorobenzothiazol-6-yl) -2-methylbenzenesulfonamide (STX768, 240 mg, 0.64 mmol) and diethylamine-IPA (3 mL) as starting materials. Compounds were prepared as described. A white crystalline solid (128 mg, 49%) was obtained. TLC R _f 0.33 single spot (30% EtOAc / hexane); HPLC purity 96% (t _R 4% water-2.2 min in methanol);

（２，６−ジメチルベンゾチアゾール−７−イルアミンの合成）
濃Ｈ_２ＳＯ_４（４ｍＬ）中の２，６−ジメチルベンゾチアゾール（３５０ｍｇ，２．１５ｍｍｏｌ）の溶液に、０℃にてＨＮＯ_３（６９％，０．３ｍｍｏｌ）を添加した。０℃にて０．５時間撹拌した後、混合物を氷−水に注いだ。沈殿を回収し、５％ 炭酸水素ナトリウムおよび水で洗浄し、エタノールから再結晶させて、黄色固体（１６０ｍｇ）として７−ニトロ−２，６−ジメチルベンゾチアゾールを生じた。生成物（１５０ｍｇ）をエタノール−ＴＨＦ（１０：２ｍＬ）中、大気圧下で５％ Ｐｄ／Ｃで水素化して、黄色固体として２，６−ジメチル−ベンゾチアゾール−７−イルアミン（１２０ｍｇ）を得た。ＴＬＣ Ｒ_ｆ ０．５５の単一スポット（１０％ ＥｔＯＡｃ／ＤＣＭ）；^１Ｈ ＮＭＲ（２７０ＭＨｚ，ＤＭＳＯ）：δ ７．０７（２Ｈ，ｓ，ＡｒＨ），５．２３（２Ｈ，ｓ，ＮＨ_２），２．７３（３Ｈ，ｓ，ＣＨ_３），２．２０（３Ｈ，ｓ，ＣＨ_３）。

(Synthesis of 2,6-dimethylbenzothiazol-7-ylamine)
To a solution of 2,6-dimethylbenzothiazole (350 mg, 2.15 mmol) in concentrated H ₂ SO ₄ (4 mL) was added HNO ₃ (69%, 0.3 mmol) at 0 ° C. After stirring at 0 ° C. for 0.5 hour, the mixture was poured into ice-water. The precipitate was collected, washed with 5% sodium bicarbonate and water and recrystallized from ethanol to give 7-nitro-2,6-dimethylbenzothiazole as a yellow solid (160 mg). The product (150 mg) was hydrogenated in ethanol-THF (10: 2 mL) with 5% Pd / C at atmospheric pressure to give 2,6-dimethyl-benzothiazol-7-ylamine (120 mg) as a yellow solid. It was. TLC R _f 0.55 single spot (10% EtOAc / DCM); ¹ H NMR (270 MHz, DMSO): δ 7.07 (2H, s, ArH), 5.23 (2H, s, NH ₂ ) _{, 2.73 (3H, s, CH} 3), 2.20 (3H, s, CH 3).

(Synthesis of 6-methoxy-2-methylbenzothiazol-7-ylamine)
Compounds were prepared starting from 6-methoxy-2-methylbenzothiazole as described above. A yellow solid was obtained. mp 117-119 ° C. (121-122 ° C. in literature); TLC R _f 0.55 single spot (40% EtOAc / DCM);

（２，５−ジメチルベンゾチアゾール−４−イルアミンおよび２，５−ジメチルベンゾチアゾール−６−イルアミンの合成）
濃Ｈ_２ＳＯ_４（１２ｍＬ）中の２，５−ジメチルベンゾチアゾール（１．６３ｇ，１０ｍｍｏｌ）の溶液に、−５℃にてＨＮＯ_３（６９％，１ｍｍｏｌ）を添加した。−５〜０℃にて２時間撹拌した後、混合物を氷−水（１５０ｍＬ）に注いだ。沈殿を回収し、５％炭酸水素ナトリウム、水および７０％エタノールで洗浄した。生成物（１．９８ｇ）は、ＮＭＲにより判断すると、４−ニトロ−２，５−ジメチルベンゾチアゾールおよび６−ニトロ−２，５−ジメチルベンゾチアゾールの１：１の比の混合物であった。生成物（９９８ｍｇ）を、エタノール−ＴＨＦ（５０：２０ｍＬ）中、大気圧下で、５％ Ｐｄ／Ｃ（６００ｍｇ）で水素化し、黄色固体（８８０ｍｇ）を得た。フラッシュクロマトグラフィー（ＥｔＯＡｃ／ＤＣＭ勾配溶出）により分離して、２，５−ジメチルベンゾチアゾール−４−イルアミンを黄色結晶として得た（４００ｍｇ）。ＴＬＣ Ｒ_ｆ ０．６０の単一スポット（１５％ ＥｔＯＡｃ／ＤＣＭ）；

(Synthesis of 2,5-dimethylbenzothiazol-4-ylamine and 2,5-dimethylbenzothiazol-6-ylamine)
To a solution of 2,5-dimethylbenzothiazole (1.63 g, 10 mmol) in concentrated H ₂ SO ₄ (12 mL) was added HNO ₃ (69%, 1 mmol) at −5 ° C. After stirring at −5 to 0 ° C. for 2 hours, the mixture was poured into ice-water (150 mL). The precipitate was collected and washed with 5% sodium bicarbonate, water and 70% ethanol. The product (1.98 g) was a 1: 1 ratio mixture of 4-nitro-2,5-dimethylbenzothiazole and 6-nitro-2,5-dimethylbenzothiazole as judged by NMR. The product (998 mg) was hydrogenated with 5% Pd / C (600 mg) in ethanol-THF (50:20 mL) at atmospheric pressure to give a yellow solid (880 mg). Separation by flash chromatography (EtOAc / DCM gradient elution) gave 2,5-dimethylbenzothiazol-4-ylamine as yellow crystals (400 mg). TLC R _f 0.60 single spot (15% EtOAc / DCM);

２，５−ジメチルベンゾチアゾール−６−イルアミンを、黄色固体として得た（３２０ｍｇ）。ＴＬＣ Ｒ_ｆ ０．５５の単一のスポット（１５％ ＥｔＯＡｃ／ＤＣＭ）；

2,5-Dimethylbenzothiazol-6-ylamine was obtained as a yellow solid (320 mg). Single spot of TLC R _f 0.55 (15% EtOAc / DCM);

（４−クロロ−２−メチルベンゾチアゾール−５−イルアミンおよび４，６−ジクロロ−２−メチルベンゾチアゾール−５−イルアミンの合成）
イソプロパノール（１２ｍＬ）中の５−アミノ−２−メチルベンゾチアゾール（８１８ｍｇ，４．９９ｍｍｏｌ）の溶液に、Ｎ−クロロスクシンイミド（７３２ｍｇ，５．４８ｍｍｏｌ）を添加した。混合物を６０℃にて１５分間撹拌し、ＤＣＭと５％炭酸水素ナトリウムとの間で分配した。有機相をブラインで洗浄し、硫酸ナトリウムで乾燥させ、真空下で濃縮して、残留物を生じ、これを、フラッシュクロマトグラフィー（ＥｔＯＡｃ／ＤＣＭ勾配溶出）で精製した。４−クロロ−２−メチルベンゾチアゾール−５−イルアミンをオフホワイトの結晶固体（５１０ｍｇ，５１％）として得た。ｍｐ １２１〜１２２℃（文献では１２４℃）；ＴＬＣ Ｒ_ｆ ０．５１の単一スポット（２０％ ＥｔＯＡｃ／ＤＣＭ）；^１Ｈ ＮＭＲ（２７０ＭＨｚ，ＤＭＳＯ）：δ ７．６１（１Ｈ，ｄ，Ｊ＝８．６Ｈｚ，ＡｒＨ），６．９１（１Ｈ，ｄ，Ｊ＝８．６Ｈｚ，ＡｒＨ），５．４９（２Ｈ，ｓ，ＮＨ２），２．７５（３Ｈ，ｓ，ＣＨ_３）；ＡＰＣＩ−ＭＳ １９９（ＭＨ）^＋。
４，６−ジクロロ−２−メチルベンゾチアゾール−５−イルアミンを、黄色固体として得た（６０ｍｇ，５％）。ＴＬＣ Ｒ_ｆ ０．５７の単一スポット（２０％ ＥｔＯＡｃ／ＤＣＭ）；^１Ｈ ＮＭＲ（２７０ＭＨｚ，ＤＭＳＯ）：δ ７．８９（１Ｈ，ｓ，ＡｒＨ），５．２６（２Ｈ，ｓ，ＮＨ２），２．７７（３Ｈ，ｓ，ＣＨ_３）；ＡＰＣＩ−ＭＳ ２３３（ＭＨ）^＋。

(Synthesis of 4-chloro-2-methylbenzothiazol-5-ylamine and 4,6-dichloro-2-methylbenzothiazol-5-ylamine)
To a solution of 5-amino-2-methylbenzothiazole (818 mg, 4.99 mmol) in isopropanol (12 mL) was added N-chlorosuccinimide (732 mg, 5.48 mmol). The mixture was stirred at 60 ° C. for 15 minutes and partitioned between DCM and 5% sodium bicarbonate. The organic phase was washed with brine, dried over sodium sulfate and concentrated in vacuo to give a residue that was purified by flash chromatography (EtOAc / DCM gradient elution). 4-Chloro-2-methylbenzothiazol-5-ylamine was obtained as an off-white crystalline solid (510 mg, 51%). mp 121-122 ° C. (124 ° C. in literature); TLC R _f 0.51 single spot (20% EtOAc / DCM); ¹ H NMR (270 MHz, DMSO): δ 7.61 (1H, d, J = 8.6Hz, ArH), 6.91 (1H , d, J = 8.6Hz, ArH), 5.49 (2H, s, NH2), 2.75 (3H, s, CH 3); APCI-MS 199 (MH) ⁺ .
4,6-Dichloro-2-methylbenzothiazol-5-ylamine was obtained as a yellow solid (60 mg, 5%). TLC R _f 0.57 single spot (20% EtOAc / DCM); ¹ H NMR (270 MHz, DMSO): δ 7.89 (1H, s, ArH), 5.26 (2H, s, NH 2), _{2.77 (3H, s, CH 3} ); APCI-MS 233 (MH) +.

  The following compounds were synthesized by the general method for N-benzothiazolebenzenesulfonamide.

3-chloro-N- (6-methoxy-2-methylbenzothiazol-7-yl) -2-methylbenzenesulfonamide (STX989, XDS02038)
White crystalline solid. TLC R _f 0.71 single spot (30% EtOAc / DCM); HPLC purity 99% (t _R 10% water-2.3 min in methanol);

３−クロロ−Ｎ−（２，６−ジメトキシ−ベンゾチアゾール−７−イル）−２−メチル−ベンゼンスルホンアミド（ＳＴＸ１０２１，ＸＤＳ０２０６９）
オフホワイトの結晶固体。ＴＬＣ Ｒ_ｆ ０．４９の単一スポット（１０％ ＥｔＯＡｃ／ＤＣＭ）；ＨＰＬＣ純度 ９８％（ｔ_Ｒ ２０％ 水−メタノール中２．０分）；

3-Chloro-N- (2,6-dimethoxy-benzothiazol-7-yl) -2-methyl-benzenesulfonamide (STX1021, XDS02069)
Off-white crystalline solid. TLC R _f 0.49 single spot (10% EtOAc / DCM); HPLC purity 98% (t _R 20% water-2.0 min in methanol);

３−クロロ−Ｎ−（２，５−ジメトキシ−ベンゾチアゾール−４−イル）−２−メチル−ベンゼンスルホンアミド（ＳＴＸ９９６，ＸＤＳ０２０４７）
白色結晶固体。ＴＬＣ Ｒ_ｆ ０．７６の単一スポット（１０％ ＥｔＯＡｃ／ＤＣＭ）；ＨＰＬＣ純度＞９９％（ｔ_Ｒ １０％ 水−メタノール中２．９分）；

3-Chloro-N- (2,5-dimethoxy-benzothiazol-4-yl) -2-methyl-benzenesulfonamide (STX996, XDS02047)
White crystalline solid. TLC R _f 0.76 single spot (10% EtOAc / DCM); HPLC purity> 99% (t _R 10% water-2.9 min in methanol);

Ｎ−（２，５−ジメトキシベンゾチアゾール−６−イル）−Ｎ（−３−クロロ−２−メチルフェニルスルホニル）−３−クロロ−２−メチルベンゼンスルホンアミド（ＳＴＸ９９７，ＸＤＳ０２０４８Ａ）
オフホワイトのシロップ。ＴＬＣ Ｒ_ｆ ０．７８の単一スポット（１０％ ＥｔＯＡｃ／ＤＣＭ）；ＨＰＬＣ純度 ８５％（ｔ_Ｒ １０％ 水−メタノール中４．２分）；

N- (2,5-dimethoxybenzothiazol-6-yl) -N (-3-chloro-2-methylphenylsulfonyl) -3-chloro-2-methylbenzenesulfonamide (STX997, XDS02048A)
Off-white syrup. TLC R _f 0.78 single spot (10% EtOAc / DCM); HPLC purity 85% (t _R 10% water-4.2 min in methanol);

３−クロロ−Ｎ−（２，５−ジメチルベンゾチアゾール−６−イル）−２−メチルベンゼンスルホンアミド（ＳＴＸ９９８，ＸＤＳ０２０４８Ｂ）
白色結晶固体。ＴＬＣ Ｒ_ｆ ０．３９の単一スポット（１０％ ＥｔＯＡｃ／ＤＣＭ）；ＨＰＬＣ純度 ９６％（ｔ_Ｒ １０％ 水−メタノール中２．１分）；

3-Chloro-N- (2,5-dimethylbenzothiazol-6-yl) -2-methylbenzenesulfonamide (STX998, XDS02048B)
White crystalline solid. TLC R _f 0.39 single spot (10% EtOAc / DCM); HPLC purity 96% (t _R 10% water-2.1 min in methanol);

２，５−ジクロロ−Ｎ−（２，５−ジメチルベンゾチアゾール−６−イル）−ベンゼンスルホンアミド（ＳＴＸ９９９，ＸＤＳ０２０４９）
白色結晶固体。ＴＬＣ Ｒ_ｆ ０．４３の単一スポット（１０％ ＥｔＯＡｃ／ＤＣＭ）；ＨＰＬＣ純度 ９８％（ｔ_Ｒ １０％ 水−メタノール中２．０分）；

2,5-Dichloro-N- (2,5-dimethylbenzothiazol-6-yl) -benzenesulfonamide (STX999, XDS02049)
White crystalline solid. Single spot of TLC R _f 0.43 (10% EtOAc / DCM); HPLC purity 98% (2.0% in t _R 10% water-methanol);

Ｎ−（４−クロロ−２−メチル−ベンゾチアゾール−５−イル）−Ｎ−（３−クロロ−２−メチルフェニルスルホニル）−３−クロロ−２−メチル−ベンゼンスルホンアミド（ＳＴＸ９９１，ＸＤＳ０２０４２Ａ）
白色粉末。ＴＬＣ Ｒ_ｆ ０．７５の単一スポット（８％ ＥｔＯＡｃ／ＤＣＭ）；ＨＰＬＣ純度 ＞９９％（ｔ_Ｒ １０％ 水−メタノール中４．４分）；

N- (4-Chloro-2-methyl-benzothiazol-5-yl) -N- (3-chloro-2-methylphenylsulfonyl) -3-chloro-2-methyl-benzenesulfonamide (STX991, XDS02042A)
White powder. TLC R _f 0.75 single spot (8% EtOAc / DCM); HPLC purity> 99% (t _R 10% water-4.4 min in methanol);

３−クロロ−Ｎ−（４−クロロ−２−メチル−ベンゾチアゾール−５−イル）−２−メチルベンゼンスルホンアミド（ＳＴＸ９９２，ＸＤＳ０２０４２Ｂ）
白色結晶固体。ＴＬＣ Ｒ_ｆ ０．６９の単一スポット（８％ ＥｔＯＡｃ／ＤＣＭ）；ＨＰＬＣ純度 ９９％（ｔ_Ｒ １０％ 水−メタノール中２．５分）；

3-Chloro-N- (4-chloro-2-methyl-benzothiazol-5-yl) -2-methylbenzenesulfonamide (STX992, XDS02042B)
White crystalline solid. TLC R _f 0.69 single spot (8% EtOAc / DCM); HPLC purity 99% (t _R 10% water-2.5 min in methanol);

２，５−ジクロロ−Ｎ−（４−クロロ−２−メチルベンゾチアゾール−５−イル）−ベンゼンスルホンアミド（ＳＴＸ９９３，ＸＤＳ０２０４３Ｂ）
白色結晶固体。ＴＬＣ Ｒ_ｆ ０．７１の単一スポット（８％ ＥｔＯＡｃ／ＤＣＭ）；ＨＰＬＣ純度 ９９％（ｔ_Ｒ １０％ 水−メタノール中５．０分）；

2,5-Dichloro-N- (4-chloro-2-methylbenzothiazol-5-yl) -benzenesulfonamide (STX993, XDS02043B)
White crystalline solid. TLC R _f 0.71 single spot (8% EtOAc / DCM); HPLC purity 99% (t _R 10% water-5.0 min in methanol);

Ｎ−（４−クロロ−２−メチルベンゾチアゾール−５−イル）−４−プロピルベンゼンスルホンアミド（ＳＴＸ９９４，ＸＤＳ０２０４４Ｂ）
白色結晶固体。ＴＬＣ Ｒ_ｆ ０．７０の単一スポット（８％ ＥｔＯＡｃ／ＤＣＭ）；ＨＰＬＣ純度 ９９％（ｔ_Ｒ １０％ 水−メタノール中２．７分）；

N- (4-Chloro-2-methylbenzothiazol-5-yl) -4-propylbenzenesulfonamide (STX994, XDS02044B)
White crystalline solid. TLC R _f 0.70 single spot (8% EtOAc / DCM); HPLC purity 99% (t _R 10% water-2.7 min in methanol);

Ｎ−（４−クロロ−２−メチルベンゾチアゾール−５−イル）−Ｎ−（４−プロピルフェニルスルホニル）−４− プロピルベンゼンスルホンアミド（ＳＴＸ９９５，ＸＤＳ０２０４４Ａ）
白色粉末。ＴＬＣ Ｒ_ｆ ０．７０の単一スポット（８％ ＥｔＯＡｃ／ＤＣＭ）；ＨＰＬＣ純度 ９９％（ｔ_Ｒ １０％ 水−メタノール中３．８分）；

N- (4-chloro-2-methylbenzothiazol-5-yl) -N- (4-propylphenylsulfonyl) -4-propylbenzenesulfonamide (STX995, XDS02044A)
White powder. TLC R _f 0.70 single spot (8% EtOAc / DCM); HPLC purity 99% (t _R 10% water-3.8 min in methanol);

（３−クロロ−２−メチル−Ｎ−（２−メチル−ベンゾチアゾール−５−イルメチル）−ベンゼンスルホンアミド（ＳＴＸ１０２９，ＸＤＳ０２０７０Ａ）および３−クロロ−２−メチル−Ｎ，Ｎ−ビス−（２−メチル−ベンゾチアゾール−５−イルメチル）−ベンゼンスルホンアミド（ＳＴＸ１０３０，ＸＤＳ０２０７０Ｂ）の合成）
ＣＨ_３ＣＮ中の３−クロロ−２−メチルベンゼンスルホンアミド（１０３ｍｇ，０．５ｍｍｏｌ）の溶液に、炭酸水素カリウム（１００ｍｇ）を添加し、その後、５−ブロモメチル−２−メチルベンゾチアゾール（１２１ｍｇ，０．５ｍｍｏｌ）を添加した。混合物をＮ_２下、６時間還流させ、酢酸エチルと水との間で分配した。有機相をブラインで洗浄し、硫酸ナトリウムで乾燥させ、真空下で濃縮させて、黄色残留物を生じ、これを、フラッシュクロマトグラフィー（酢酸エチル／ＤＣＭ，勾配溶出）で分離した。ＳＴＸ１０２９を白色固体として得た。ＴＬＣ Ｒ_ｆ ０．５５の単一スポット（１０％ＥｔＯＡｃ／ＤＣＭ）；ＨＰＬＣ純度 ＞９９％（ｔ_Ｒ １０％ 水−メタノール中２．０分）；

(3-Chloro-2-methyl-N- (2-methyl-benzothiazol-5-ylmethyl) -benzenesulfonamide (STX1029, XDS02070A) and 3-chloro-2-methyl-N, N-bis- (2- Methyl-benzothiazol-5-ylmethyl) -benzenesulfonamide (synthesis of STX1030, XDS02070B)
To a solution of 3-chloro-2-methylbenzenesulfonamide (103 mg, 0.5 mmol) in CH ₃ CN was added potassium bicarbonate (100 mg) followed by 5-bromomethyl-2-methylbenzothiazole (121 mg, 0.5 mmol) was added. The mixture was refluxed under N ₂ for 6 hours and partitioned between ethyl acetate and water. The organic phase was washed with brine, dried over sodium sulfate and concentrated under vacuum to give a yellow residue which was separated by flash chromatography (ethyl acetate / DCM, gradient elution). STX1029 was obtained as a white solid. TLC R _f 0.55 single spot (10% EtOAc / DCM); HPLC purity> 99% (t _R 10% water-2.0 min in methanol);

ＳＴＸ１０３０を白色固体として得た。ＴＬＣ Ｒ_ｆ ０．５０の単一スポット（１０％ ＥｔＯＡｃ／ＤＣＭ）；ＨＰＬＣ純度 ９９％（ｔ_Ｒ ２０％水−メタノール中６．１分）；^１Ｈ ＮＭＲ（２７０ＭＨｚ，ＤＭＳＯ−ｄ６）δ ７．８７（３Ｈ，ｄ，Ｊ＝８．１Ｈｚ，ＡｒＨ），７．７５（１Ｈ，ｄ，Ｊ＝８．０Ｈｚ，ＡｒＨ），７．５９（２Ｈ，ブロード，２＝１．１Ｈｚ，ＡｒＨ），７．３８（１Ｈ，ｔ，Ｊ＝８．０Ｈｚ，ＡｒＨ），７．１１（２Ｈ，ｄｄ，Ｊ＝８．１，１．１Ｈｚ，ＡｒＨ），４．５６（４Ｈ，ｓ，２×ＮＣＨ_２），２．７８（６Ｈ，ｓ，２×ＣＨ_３），２．５８（３Ｈ，ｓ，ＣＨ_３）；ＡＰＣＩ−ＭＳ ５２８（ＭＨ）^＋；ＦＡＢ−ＨＲＭＳ Ｃ_２５Ｈ_２３ＣｌＮ_３Ｏ_２Ｓ_３（ＭＨ＋）についての計算値５２８．０６４１，実測値５２８．０６３０。

STX1030 was obtained as a white solid. 6. TLC R _f 0.50 single spot (10% EtOAc / DCM); HPLC purity 99% (t _R 20% water-6.1 min in methanol); ¹ H NMR (270 MHz, DMSO-d6) δ 87 (3H, d, J = 8.1 Hz, ArH), 7.75 (1H, d, J = 8.0 Hz, ArH), 7.59 (2H, broad, 2 = 1.1 Hz, ArH), 7 .38 (1H, t, J = 8.0 Hz, ArH), 7.11 (2H, dd, J = 8.1, 1.1 Hz, ArH), 4.56 (4H, s, 2 × NCH ₂ ) , 2.78 (6H, s, 2 × CH 3), 2.58 (3H, s, CH 3); APCI-MS 528 (MH) +; FAB-HRMS C 25 H 23 ClN 3 O 2 S 3 ( Calculated value 528.0641, measured value 528.06 for (MH +) 30.

  (Synthesis of N-indole or N-indoline arylsulfonamide derivative)

（Ｎ−インドールまたはＮ−インドリンアリールスルホンアミド誘導体（ＳＴＸ８３２，ＳＴＸ９８１〜９８２，ＳＴＸ９８４，ＳＴＸ９８６−９８７，ＳＴＸ１０１８〜１０２０）の合成のための一般方法：）
ＤＣＭ中のアリールスルホニルクロリド（１．１当量）の溶液に、ピリジン（２．２当量）および触媒量のＤＭＡＰを添加し、その後、対応するアミン（１当量）を添加した。反応混合物を窒素下、室温にて４〜６時間撹拌し、ＴＬＣが反応の完了を示した後、次いで、酢酸エチルと５％炭酸水素ナトリウムとの間で分配した。有機層をブラインで洗浄し、硫酸ナトリウムで乾燥させ、真空下で濃縮して、固体もしくは粘性シロップとして粗生成物を得た。化合物を次いで、フラッシュクロマトグラフィー（メタノール−ＤＣＭ勾配溶出）により精製して、結晶固体として所望のアリールスルホンアミドを得た。収率は５０〜８５％の範囲である。

(General method for the synthesis of N-indole or N-indoline arylsulfonamide derivatives (STX832, STX981 to 982, STX984, STX986-987, STX1018 to 1020) :)
To a solution of arylsulfonyl chloride (1.1 eq) in DCM was added pyridine (2.2 eq) and a catalytic amount of DMAP followed by the corresponding amine (1 eq). The reaction mixture was stirred at room temperature for 4-6 hours under nitrogen and then partitioned between ethyl acetate and 5% sodium bicarbonate after TLC showed the reaction was complete. The organic layer was washed with brine, dried over sodium sulfate and concentrated under vacuum to give the crude product as a solid or viscous syrup. The compound was then purified by flash chromatography (methanol-DCM gradient elution) to give the desired arylsulfonamide as a crystalline solid. Yields range from 50 to 85%.

3-Chloro-2-methyl-N- (2-methyl-1H-indol-5-yl) -benzenesulfonamide (STX832, XDS01165)
White crystalline solid. TLC R _f 0.68 single spot (30% ethyl acetate / hexane); HPLC purity> 99% (t _R 4% water-1.8 min in methanol);

３−クロロ−２−メチル−Ｎ−（１Ｈ−インドール−５−イル）−ベンゼンスルホンアミド（ＳＴＸ９８１，ＸＤＳ０２０１９）
白色結晶固体。ＴＬＣ Ｒ_ｆ ０．７２の単一スポット（６％ メタノール／ＤＣＭ）；ＨＰＬＣ純度 ９８％（ｔ_Ｒ １０％ 水−メタノール中２．１分）；

3-Chloro-2-methyl-N- (1H-indol-5-yl) -benzenesulfonamide (STX981, XDS02019)
White crystalline solid. TLC R _f 0.72 single spot (6% methanol / DCM); HPLC purity 98% (t _R 10% water-2.1 min in methanol);

３−クロロ−２−メチル−Ｎ−（１Ｈ−インドール−６−イル）−ベンゼンスルホンアミド（ＳＴＸ９８２，ＸＤＳ０２０２０）
白色結晶固体。ＴＬＣ Ｒ_ｆ ０．８８の単一スポット（１０％ メタノール／ＤＣＭ）；ＨＰＬＣ純度 ９８％（ｔ_Ｒ ２０％水−メタノール中２．５分）；

3-Chloro-2-methyl-N- (1H-indol-6-yl) -benzenesulfonamide (STX982, XDS02020)
White crystalline solid. TLC R _f 0.88 single spot (10% methanol / DCM); HPLC purity 98% (t _R 20% water-2.5 min in methanol);

５−（３−クロロ−２−メチルベンゼンスルホニルアミノ）−１Ｈ−インドール−２−カルボン酸エチルエステル（ＳＴＸ９８６，ＸＤＳ０２０３０）
白色結晶固体。ＴＬＣ Ｒ_ｆ ０．８２の単一スポット（８％ メタノール／ＤＣＭ）；ＨＰＬＣ純度＞９９％（ｔ_Ｒ １０％ 水−メタノール中２．３分）；

5- (3-Chloro-2-methylbenzenesulfonylamino) -1H-indole-2-carboxylic acid ethyl ester (STX986, XDS02030)
White crystalline solid. TLC R _f 0.82 single spot (8% methanol / DCM); HPLC purity> 99% (t _R 10% water-2.3 min in methanol);

３−クロロ−２−メチル−Ｎ−（２，３−ジメチル−１Ｈ−インドール−５−イル）−ベンゼンスルホンアミド（ＳＴＸ１０１８，ＸＤＳ０２０６１）
白色結晶固体。ＴＬＣ Ｒ_ｆ ０．８３の単一スポット（３０％ 酢酸エチル／ヘキサン）；ＨＰＬＣ純度 ９７％（ｔ_Ｒ ２０％水−メタノール中２．９分）；

3-Chloro-2-methyl-N- (2,3-dimethyl-1H-indol-5-yl) -benzenesulfonamide (STX1018, XDS02061)
White crystalline solid. TLC R _f 0.83 single spot (30% ethyl acetate / hexane); HPLC purity 97% (t _R 20% water-2.9 min in methanol);

２，５−ジクロロ−Ｎ−（２，３−ジメチル−１Ｈ−インドール−５−イル）−ベンゼンスルホンアミド（ＳＴＸ１０１９，ＸＤＳ０２０６２）
白色非結晶性 ｐｏｗｄｅｒ．ＴＬＣ Ｒ_ｆ ０．８２の単一スポット（１０％ 酢酸エチル／ヘキサン）；ＨＰＬＣ純度 ９８％（ｔ_Ｒ ２０％水−メタノール中３．０分）；

2,5-Dichloro-N- (2,3-dimethyl-1H-indol-5-yl) -benzenesulfonamide (STX1019, XDS02062)
White amorphous powder. TLC R _f 0.82 single spot (10% ethyl acetate / hexane); HPLC purity 98% (t _R 20% water-3.0 min in methanol);

４−ｎ−プロピル−Ｎ−（２，３−ジメチル−１Ｈ−インドール−５−イル）−ベンゼンスルホンアミド（ＳＴＸ１０２０，ＸＤＳ０２０６３）
オフホワイトの結晶固体。ＴＬＣ Ｒ_ｆ ０．８２の単一スポット（１０％ 酢酸エチル／ヘキサン）；ＨＰＬＣ純度 ９７％（ｔ_Ｒ ２０％水−メタノール中２．９分）；

4-n-propyl-N- (2,3-dimethyl-1H-indol-5-yl) -benzenesulfonamide (STX1020, XDS02063)
Off-white crystalline solid. TLC R _f 0.82 single spot (10% ethyl acetate / hexane); HPLC purity 97% (t _R 20% water-2.9 min in methanol);

３−クロロ−２−メチル−Ｎ−（１−アセチル−２，３−ジヒドロ−１Ｈ−インドール−５−イル）−ベンゼンスルホンアミド（ＳＴＸ９８４，ＸＤＳ０２０２５）
白色結晶固体。ＴＬＣ Ｒ_ｆ ０．５８の単一スポット（５％ メタノール／ＤＣＭ）；ＨＰＬＣ純度 ９５％（ｔ_Ｒ ２０％水−メタノール中２．２分）；

3-Chloro-2-methyl-N- (1-acetyl-2,3-dihydro-1H-indol-5-yl) -benzenesulfonamide (STX984, XDS02025)
White crystalline solid. TLC R _f 0.58 single spot (5% methanol / DCM); HPLC purity 95% (t _R 20% water-2.2 min in methanol);

５−（３−クロロ−２−メチル−ベンゼンスルホニルアミノ）−１−エチル−２，３−ジヒドロ−１Ｈ−インドリウムクロリド（ＳＴＸ９８７，ＸＤＳ０２０３１）
ＳＴＸ９８７の遊離塩基を上記のように合成した。紫色の非結晶性粉末を得た；ＴＬＣ Ｒ_ｆ ０．７９の単一スポット（８％ メタノール／ＤＣＭ）；

5- (3-Chloro-2-methyl-benzenesulfonylamino) -1-ethyl-2,3-dihydro-1H-indolium chloride (STX987, XDS02031)
The free base of STX987 was synthesized as described above. A purple amorphous powder was obtained; a single spot with TLC R _f 0.79 (8% methanol / DCM);

遊離塩基をＨＣｌ−エーテル溶液で処理し、淡いピンクの結晶固体としてＳＴＸ９８７を得た。ＨＰＬＣ純度 ９３ ％（ｔ_Ｒ ２０％水−メタノール中３．６分）；

The free base was treated with HCl-ether solution to give STX987 as a pale pink crystalline solid. HPLC purity 93% _(t R 20% water - 3.6 minutes in methanol);

（１−アセチル−５−アミノインドリン）
エタノール−ＴＨＦ（１００ｍＬ：３０ｍＬ）中の１−アセチル−５−ニトロインドリン（１．０ｇ，４．８５ｍｍｏｌ）の溶液を、大気圧下、５％ Ｐｄ／Ｃ（６００ｍｇ）で２時間水素化し、Ｃｅｌｉｔｅを通して濾過し、真空下で濃縮し、白色固体を生じ、これをエタノールから再結晶させた。白色結晶固体（５８０ｍｇ，６８％）を得た。Ｍｐ １８５〜１８６．５℃（文献では１８４〜１８５℃，［２１］）；

(1-acetyl-5-aminoindoline)
A solution of 1-acetyl-5-nitroindoline (1.0 g, 4.85 mmol) in ethanol-THF (100 mL: 30 mL) was hydrogenated with 5% Pd / C (600 mg) at atmospheric pressure for 2 hours, and Celite. Filtered through and concentrated in vacuo to yield a white solid which was recrystallized from ethanol. White crystalline solid (580 mg, 68%) was obtained. Mp 185-186.5 ° C (184-185 ° C in literature, [21]);

（１−エチル−５−アミノインドリン）
無水ＴＨＦ（１０ｍＬ）中の１−アセチル−５−アミノインドリン（１３０ｍｇ，０．７４ｍｍｏｌ）の懸濁液に、ＬｉＡｌＨ_４（４２ｍｇ，１．１１ｍｍｏｌ）を添加した。混合物を室温にて６時間撹拌し、飽和ＮＨ_４Ｃｌでクエンチし、酢酸エチルで抽出した。有機相をブラインで洗浄し、硫酸ナトリウムで乾燥させ、真空下で濃縮して、紫色の残留物（８０ｍｇ，６７％）を生じ、これを、さらに精製することなく用いた。

(1-ethyl-5-aminoindoline)
To a suspension of 1-acetyl-5-aminoindoline (130 mg, 0.74 mmol) in anhydrous THF (10 mL) was added LiAlH ₄ (42 mg, 1.11 mmol). The mixture was stirred at room temperature for 6 hours, quenched with saturated NH ₄ Cl and extracted with ethyl acetate. The organic phase was washed with brine, dried over sodium sulfate and concentrated in vacuo to give a purple residue (80 mg, 67%) that was used without further purification.

（５−Ｈ−インドール−３−カルボン酸メチルエステル，ＳＴＸ１０５０（ＫＲＢ０１１３２）の合成）

(Synthesis of 5-H-indole-3-carboxylic acid methyl ester, STX1050 (KRB01132))

５−アミノ−１Ｈ−インドール−３−カルボン酸メチルエステル（ＫＲＢ０１１３１）：
メタノール（４０ｍＬ）中の５−ニトロ−１Ｈ−インドール−３−カルボン酸メチルエステル（２０６ｍｇ，０．９４０ｍｍｏｌ）の溶液に、炭素上の５％パラジウム（４０ｍｇ）を添加し、混合物を１ａｔｍのＨ_２下、５時間撹拌した。混合物をＣｅｌｉｔｅを通してろ過紙、濾液をエバポレートして、褐色の固体を得、これを、さらに精製することなく用いた（１７３ｍｇ，９７％），Ｒ_ｆ ０．６４の単一スポット（酢酸エチル）。

5-Amino-1H-indole-3-carboxylic acid methyl ester (KRB01131):
To a solution of 5-nitro-1H-indole-3-carboxylic acid methyl ester (206 mg, 0.940 mmol) in methanol (40 mL) was added 5% palladium on carbon (40 mg) and the mixture was added 1 atm of H _2. The mixture was stirred for 5 hours. The mixture was filtered through Celite and the filtrate was evaporated to give a brown solid which was used without further purification (173 mg, 97%), single spot of R _f 0.64 (ethyl acetate).

ジクロロメタン（４ｍＬ）中の３−クロロ−２−メチルベンゼンスルホニルクロリド（１２４ｍｇ，０．５５２ｍｍｏｌ）の溶液に、ピリジン（１００μＬ，１．３ｍｍｏｌ）を添加し、混合物をＮ_２下で５分間撹拌し、その後、５−アミノ−１Ｈ−インドール−カルボン酸メチルエステル（１００ｍｇ，０．５２６ｍｍｏｌ）を添加した。得られた混合物を室温にて１．５時間撹拌し、次いで、飽和ＮａＨＣＯ_３溶液（１５ｍＬ）を添加し、混合物を、酢酸エチル（２０ｍＬ）中に抽出した。有機相をブラインで洗浄し、乾燥させ（Ｎａ_２ＳＯ_４）、濾過して、エバポレートさせ、残留物を生じた。これを、フラッシュクロマトグラフィーを用いて精製して、白色固体（１２９ｍｇ，６５％）を得た。Ｒ_ｆ ０．８４の単一スポット（酢酸エチル）。ｍｐ ２１６．８〜２１９．３℃［２２］，ＨＰＬＣ純度 ９９＋％（ｔ_Ｒ １０％ 水−アセトニトリル中２．０７分）。

To a solution of 3-chloro-2-methylbenzenesulfonyl chloride (124 mg, 0.552 mmol) in dichloromethane (4 mL) was added pyridine (100 μL, 1.3 mmol) and the mixture was stirred under N ₂ for 5 min. Subsequently, 5-amino-1H-indole-carboxylic acid methyl ester (100 mg, 0.526 mmol) was added. The resulting mixture was stirred at room temperature for 1.5 hours, then saturated NaHCO ₃ solution (15 mL) was added and the mixture was extracted into ethyl acetate (20 mL). The organic phase was washed with brine, dried (Na ₂ SO ₄ ), filtered and evaporated to give a residue. This was purified using flash chromatography to give a white solid (129 mg, 65%). Single spot with R _f 0.84 (ethyl acetate). mp 216.8~219.3 ℃ [22], HPLC purity 99 _{+% (t} R _10% water - 2.07 minutes in acetonitrile).

（ベンズイミダゾールアリールスルホンアミド誘導体の合成）

(Synthesis of benzimidazole arylsulfonamide derivatives)

（１−アルキル−５−アミノ−２−メチルベンズイミダゾールおよび１−アルキル−６−アミノ−２メチルベンズイミダゾールの調製）
アセトン（５０ｍＬ）中の５−ニトロベンズイミダゾール（１．０ｇ，５．６ｍｍｏｌ）の溶液に、炭酸カリウム（１．０ｇ）を添加し、その後、アルキルハロゲン化物（１．２〜１．５当量）を添加した。混合物を窒素下、室温にて撹拌し、ＴＬＣが反応の完了を示した後、次いで、酢酸エチルと水との間で分配した。有機相をブラインで洗浄し、硫酸ナトリウムで乾燥させ、真空下で濃縮して、１−アルキル−５−ニトロ−２−メチルベンズイミダゾールおよび１−アルキル−６−ニトロ−２−メチルベンズイミダゾールの混合物を生じ、これを、エタノール−ＴＨＦ（１００ｍＬ，２：１）中に溶解させ、大気圧下で、８時間にわたって５％ Ｐｄ−Ｃで水素化した。Ｃｅｌｉｔｅを通して濾過した後、濾液をエバポレートして、黄色固体を生じ、これを、フラッシュクロマトグラフィー（メタノール−ＤＣＭ勾配溶出）により分離した。１−アルキル−５−アミノベンズイミダゾールおよび１−アルキル−６−アミノベンズイミダゾールを黄色固体または粘性シロップとして得た。

(Preparation of 1-alkyl-5-amino-2-methylbenzimidazole and 1-alkyl-6-amino-2methylbenzimidazole)
To a solution of 5-nitrobenzimidazole (1.0 g, 5.6 mmol) in acetone (50 mL) was added potassium carbonate (1.0 g) followed by alkyl halide (1.2-1.5 eq). Was added. The mixture was stirred at room temperature under nitrogen and then partitioned between ethyl acetate and water after TLC showed the reaction was complete. The organic phase is washed with brine, dried over sodium sulfate, concentrated in vacuo, and a mixture of 1-alkyl-5-nitro-2-methylbenzimidazole and 1-alkyl-6-nitro-2-methylbenzimidazole This was dissolved in ethanol-THF (100 mL, 2: 1) and hydrogenated with 5% Pd-C at atmospheric pressure for 8 hours. After filtration through Celite, the filtrate was evaporated to yield a yellow solid that was separated by flash chromatography (methanol-DCM gradient elution). 1-alkyl-5-aminobenzimidazole and 1-alkyl-6-aminobenzimidazole were obtained as yellow solids or viscous syrups.

5-amino-1,2-dimethylbenzimidazole (XDS01191B, XDS02082B):
Yellow solid, mp 126-127 ° C (128 ° C in literature, [23]). Single spot of TLC R _f 0.30 (5% methanol / DCM);

６−アミノ−１，２−ジメチルベンズイミダゾール（ＸＤＳ０１１９１Ａ，ＸＤＳ０２０８２Ａ）：
黄色固体。ＴＬＣ Ｒ_ｆ ０．３３の単一スポット（５％ メタノール／ＤＣＭ）；

6-amino-1,2-dimethylbenzimidazole (XDS01191A, XDS02082A):
Yellow solid. TLC R _f 0.33 single spot (5% methanol / DCM);

５−アミノ−１−エチル−２−メチルベンズイミダゾール（ＸＤＳ０２０７９Ｂ）：
黄色シロップ。ＴＬＣ Ｒ_ｆ ０．２７の単一スポット（５％ メタノール／ＤＣＭ）；

5-Amino-1-ethyl-2-methylbenzimidazole (XDS02079B):
Yellow syrup. Single spot of TLC R _f 0.27 (5% methanol / DCM);

６−アミノ−１−エチル−２−メチルベンズイミダゾール（ＸＤＳ０２０７９Ａ）：
黄色固体。ＴＬＣ Ｒ_ｆ ０．３０の単一スポット（５％ メタノール／ＤＣＭ）；

6-amino-1-ethyl-2-methylbenzimidazole (XDS02079A):
Yellow solid. Single spot of TLC R _f 0.30 (5% methanol / DCM);

５−アミノ−１−ｉ−ブチル−２−メチルベンズイミダゾール（ＸＤＳ０２０９３Ｂ）：
黄色シロップ。ＴＬＣ Ｒ_ｆ ０．４２の単一スポット（１０％ メタノール／ＤＣＭ）；

5-Amino-1-i-butyl-2-methylbenzimidazole (XDS02093B):
Yellow syrup. TLC R _f 0.42 single spot (10% methanol / DCM);

６−アミノ−１−ｉ−ブチル−２−メチルベンズイミダゾール（ＸＤＳ０２０９３Ａ）：
黄色固体。ＴＬＣ Ｒ_ｆ ０．４５の単一スポット（１０％ メタノール／ＤＣＭ）；

6-amino-1-i-butyl-2-methylbenzimidazole (XDS02093A):
Yellow solid. Single spot of TLC R _f 0.45 (10% methanol / DCM);

５−アミノベンゾイミダゾール−１−イル）−酢酸エチルエステル（ＸＤＳ０２０１２Ｂ）
黄色固体。 ＴＬＣ Ｒ_ｆ ０．３６の単一スポット（５％メタノール／ＤＣＭ）；

5-Aminobenzimidazol-1-yl) -acetic acid ethyl ester (XDS02012B)
Yellow solid. TLC R _f 0.36 single spot (5% methanol / DCM);

６−アミノベンゾイミダゾール−１−イル）−酢酸エチルエステル（ＸＤＳ０２０１２Ａ）
黄色固体。 ＴＬＣ Ｒ_ｆ ０．４０の単一スポット（５％ メタノール／ＤＣＭ）；

6-aminobenzimidazol-1-yl) -acetic acid ethyl ester (XDS02012A)
Yellow solid. TLC R _f 0.40 single spot (5% methanol / DCM);

５−アミノ−１−ベンジル−２−メチルベンズイミダゾール（ＸＤＳ０２０８６Ｂ）：
黄色シロップ。ＴＬＣ Ｒ_ｆ ０．２７の単一スポット（５％ メタノール／ＤＣＭ）；

5-Amino-1-benzyl-2-methylbenzimidazole (XDS02086B):
Yellow syrup. Single spot of TLC R _f 0.27 (5% methanol / DCM);

６−アミノ−１−ベンジル−２−メチルベンズイミダゾール（ＸＤＳ０２０８６Ａ）：
黄色固体。 ＴＬＣ Ｒ_ｆ ０．３０の単一スポット（５％ メタノール／ＤＣＭ）；

6-amino-1-benzyl-2-methylbenzimidazole (XDS02086A):
Yellow solid. Single spot of TLC R _f 0.30 (5% methanol / DCM);

（５−アミノ−４−クロロ−１，２−ジメチルベンズイミダゾール（ＸＤＳ０２０９６Ａ）の調製）
ＩＰＡ（１５ｍＬ）中の５−アミノ−１，２−ジメチルベンズイミダゾール（６００ｍｇ，３．７３ｍｍｏｌ）の溶液に、Ｎ−クロロスクシンイミド（５４８ｍｇ，４．１０ｍｍｏｌ）を添加した。混合物を室温にて２０分間撹拌し、ＤＣＭ（８０ｍＬ）で希釈して、５％炭酸水素ナトリウムおよびブラインで洗浄した。暗褐色の溶液を、硫酸ナトリウムで乾燥させ、真空下で濃縮して、褐色の残留物を生じ、これを、フラッシュクロマトグラフィー（メタノール−ＤＣＭ勾配溶出）に供した。黄色固体（２２０ｍｇ，３３％）を得た。ＴＬＣ の単一スポットＲ_ｆ ０．６９（１０％ メタノール／ＤＣＭ）；

(Preparation of 5-amino-4-chloro-1,2-dimethylbenzimidazole (XDS02096A))
To a solution of 5-amino-1,2-dimethylbenzimidazole (600 mg, 3.73 mmol) in IPA (15 mL) was added N-chlorosuccinimide (548 mg, 4.10 mmol). The mixture was stirred at room temperature for 20 minutes, diluted with DCM (80 mL) and washed with 5% sodium bicarbonate and brine. The dark brown solution was dried over sodium sulfate and concentrated in vacuo to give a brown residue that was subjected to flash chromatography (methanol-DCM gradient elution). A yellow solid (220 mg, 33%) was obtained. TLC single spot R _f 0.69 (10% methanol / DCM);

（Ｎ−ベンズイミダゾールアリールスルホンアミド誘導体の合成のための一般法）
ＤＣＭ中のアリールスルホニルクロリド（１．１当量）の溶液に、ピリジン（２．２当量）および触媒量のＤＭＡＰを添加し、その後、対応するアミン（１当量）を添加した。反応混合物を窒素下、室温にて、４〜１６時間撹拌し、ＴＬＣが反応の完了を示した後、次いで、酢酸と５％炭酸水素ナトリウムとの間で分配した。有機層をブラインで洗浄し、硫酸ナトリウムで乾燥させ、真空下で濃縮して、固体または粘性シロップとして粗生成物を生じた。次いで、化合物をフラッシュクロマトグラフィー（メタノール−ＤＣＭ勾配溶出）により精製して、結晶固体として所望のアリールスルホンアミドを生じた。収率は、５０〜８０％の範囲である。

(General method for the synthesis of N-benzimidazole arylsulfonamide derivatives)
To a solution of arylsulfonyl chloride (1.1 eq) in DCM was added pyridine (2.2 eq) and a catalytic amount of DMAP followed by the corresponding amine (1 eq). The reaction mixture was stirred at room temperature under nitrogen for 4-16 hours and then partitioned between acetic acid and 5% sodium bicarbonate after TLC showed the reaction was complete. The organic layer was washed with brine, dried over sodium sulfate and concentrated under vacuum to give the crude product as a solid or viscous syrup. The compound was then purified by flash chromatography (methanol-DCM gradient elution) to yield the desired arylsulfonamide as a crystalline solid. The yield is in the range of 50-80%.

3-Chloro-N-2-dimethyl-1H-benzimidazol-6-yl) -2-methylbenzenesulfonamide (STX975, XDS02001)
White crystalline solid. Mp265-266 C; single spot of TLC R _f 0.43 (5% methanol / DCM); HPLC purity> 99% (t _R 10% water-2.min in methanol);

３−クロロ−Ｎ−（１，２−ジメチル−１Ｈ−ベンゾイミダゾール−５−イル）−２−メチルベンゼンスルホンアミド（ＳＴＸ９７６，ＸＤＳ０２００３）
白色結晶固体。Ｍｐ ２８３−２８３℃。９９％（ｔ_Ｒ １０％ 水−メタノール中２．０分）；

3-Chloro-N- (1,2-dimethyl-1H-benzimidazol-5-yl) -2-methylbenzenesulfonamide (STX976, XDS02003)
White crystalline solid. Mp 283-283 ° C. 99% (t _R 10% water-2.0 min in methanol);

３−クロロ−Ｎ−（４−クロロ−１，２−ジメチル−１Ｈ−ベンゾイミダゾール−５−イル）−２−メチルベンゼンスルホンアミド（ＳＴＸ１１２１，ＸＤＳ０２１０２Ｂ）
オフホワイトの結晶固体。ＴＬＣ Ｒ_ｆ ０．５０の単一スポット（１０％ メタノール／ＤＣＭ）；ＨＰＬＣ純度 ９５％（ｔ_Ｒ ２０％水−メタノール中２．１分）；

3-chloro-N- (4-chloro-1,2-dimethyl-1H-benzimidazol-5-yl) -2-methylbenzenesulfonamide (STX1121, XDS02102B)
Off-white crystalline solid. TLC R _f 0.50 single spot (10% methanol / DCM); HPLC purity 95% (t _R 20% water-2.1 min in methanol);

Ｎ−（１，２−ジメチル−１Ｈ−ベンゾイミダゾール−５−イル）−４−プロピルベンゼンスルホンアミド（ＳＴＸ１１１２，ＸＤＳ０２０８８）
白色結晶固体。ＴＬＣ Ｒ_ｆ ０．３８の単一スポット（５％ メタノール／ＤＣＭ）；ＨＰＬＣ純度＞９９％（ｔ_Ｒ ２０％水−メタノール中２．１分）；

N- (1,2-dimethyl-1H-benzimidazol-5-yl) -4-propylbenzenesulfonamide (STX1112, XDS02088)
White crystalline solid. TLC R _f 0.38 single spot (5% methanol / DCM); HPLC purity> 99% (t _R 20% water-2.1 min in methanol);

２，５−ジクロロ−Ｎ−（１，２−ジメチル−１Ｈ−ベンゾイミダゾール−５−イル）−ベンゼンスルホンアミド（ＳＴＸ１１１３，ＸＤＳ０２０８９）
白色結晶固体。ＴＬＣ Ｒ_ｆ ０．６７の単一スポット（１０％ メタノール／ＤＣＭ）；ＨＰＬＣ純度 ９９％（ｔ_Ｒ ２０％水−メタノール中２．０分）；

2,5-dichloro-N- (1,2-dimethyl-1H-benzimidazol-5-yl) -benzenesulfonamide (STX1113, XDS02089)
White crystalline solid. TLC R _f 0.67 single spot (10% methanol / DCM); HPLC purity 99% (t _R 20% water-2.0 min in methanol);

２，４−ジクロロ−Ｎ−（１，２−ジメチル−１Ｈ−ベンゾイミダゾール−５−イル）−ベンゼンスルホンアミド（ＳＴＸ１１１４，ＸＤＳ０２０９０）
オフホワイトの結晶固体。ＴＬＣ Ｒ_ｆ ０．５９の単一スポット（１０％メタノール／ＤＣＭ）；ＨＰＬＣ純度＞９９％（ｔ_Ｒ ２０％水−メタノール中２．０分）

2,4-Dichloro-N- (1,2-dimethyl-1H-benzimidazol-5-yl) -benzenesulfonamide (STX1114, XDS02090)
Off-white crystalline solid. TLC R _f 0.59 single spot (10% methanol / DCM); HPLC purity> 99% (t _R 20% water-2.0 min in methanol)

４−ブロモ−Ｎ−（１，２−ジメチル−１Ｈ−ベンゾイミダゾール−５−イル）−２−メチルベンゼンスルホンアミド（ＳＴＸ１１１５，ＸＤＳ０２０９１）
白色結晶固体。ＴＬＣ Ｒ_ｆ ０．６７の単一スポット（１０％ メタノール／ＤＣＭ）；ＨＰＬＣ純度＞９９％（ｔ_Ｒ ２０％水−メタノール中２．１分）；

4-Bromo-N- (1,2-dimethyl-1H-benzimidazol-5-yl) -2-methylbenzenesulfonamide (STX1115, XDS02091)
White crystalline solid. TLC R _f 0.67 single spot (10% methanol / DCM); HPLC purity> 99% (t _R 20% water-2.1 min in methanol);

Ｎ−（１，２−ジメチル−１Ｈ−ベンゾイミダゾール−５−イル）−４−フェニルベンゼンスルホンアミド（ＳＴＸ１１１６，ＸＤＳ０２０９２）
白色結晶固体。ＴＬＣ Ｒ_ｆ ０．７２の単一スポット（５％ メタノール／ＤＣＭ）；ＨＰＬＣ純度＞９９％（ｔ_Ｒ ２０％水−メタノール中２．１分）；

N- (1,2-dimethyl-1H-benzimidazol-5-yl) -4-phenylbenzenesulfonamide (STX1116, XDS02092)
White crystalline solid. TLC R _f 0.72 single spot (5% methanol / DCM); HPLC purity> 99% (t _R 20% water-2.1 min in methanol);

３−クロロ−Ｎ−（１−エチル−２−メチル−Ｉ Ｈ−ベンゾイミダゾール−６−イル）−２−メチルベンゼンスルホンアミド（ＳＴＸ１１１０，ＸＤＳ０２０８４）
オフホワイトの固体。ＴＬＣ Ｒ_ｆ ０．４５の単一スポット（８％ メタノール／ＤＣＭ）；ＨＰＬＣ純度＞９９％（ｔ_Ｒ ２０％水−メタノール中２．２分）；

3-Chloro-N- (1-ethyl-2-methyl-I H-benzimidazol-6-yl) -2-methylbenzenesulfonamide (STX1110, XDS02084)
Off-white solid. TLC R _f 0.45 single spot (8% methanol / DCM); HPLC purity> 99% (t _R 20% water-2.2 min in methanol);

３−クロロ−Ｎ−（１−エチル−２−メチル−１Ｈ−ベンゾイミダゾール−５−イル）−２−メチルベンゼンスルホンアミド（ＳＴＸ１１１１，ＸＤＳ０２０８５）
オフホワイトの固体。ＴＬＣ Ｒ_ｆ ０．４２の単一スポット（８％ メタノール／ＤＣＭ）；ＨＰＬＣ純度＞９９％（ｔ_Ｒ ２０％水−メタノール中２．２分）；

3-chloro-N- (1-ethyl-2-methyl-1H-benzimidazol-5-yl) -2-methylbenzenesulfonamide (STX1111, XDS02085)
Off-white solid. TLC R _f 0.42 single spot (8% methanol / DCM); HPLC purity> 99% (t _R 20% water-2.2 min in methanol);

３−クロロ−Ｎ−（１−イソブチル−２−メチル−１Ｈ−ベンゾイミダゾール−６−イル）−２−メチルベンゼンスルホンアミド（ＳＴＸ１１１９，ＸＤＳ０２１００）
オフホワイトの固体。ＴＬＣ Ｒ_ｆ ０．５７の単一スポット（８％ メタノール／ＤＣＭ）；ＨＰＬＣ純度 ９９％（ｔ_Ｒ ２０％水−メタノール中２．２分）；

3-chloro-N- (1-isobutyl-2-methyl-1H-benzimidazol-6-yl) -2-methylbenzenesulfonamide (STX1119, XDS02100)
Off-white solid. TLC R _f 0.57 single spot (8% methanol / DCM); HPLC purity 99% (t _R 20% water-2.2 min in methanol);

３−クロロ−Ｎ−（１−イソブチル−２−メチル−１Ｈ−ベンゾイミダゾール−５−イル）−２−メチルベンゼンスルホンアミド（ＳＴＸ１１２０，ＸＤＳ０２１０１）
オフホワイトの固体。ＴＬＣ Ｒ_ｆ ０．５２の単一スポット（８％ メタノール／ＤＣＭ）；ＨＰＬＣ純度 ９９％（ｔ_Ｒ ２０％水−メタノール中２．３分）；

3-chloro-N- (1-isobutyl-2-methyl-1H-benzimidazol-5-yl) -2-methylbenzenesulfonamide (STX1120, XDS02101)
Off-white solid. TLC R _f 0.52 single spot (8% methanol / DCM); HPLC purity 99% (t _R 20% water-2.3 min in methanol);

［６−（３−クロロ−２−メチルベンゼンスルホニルアミノ）−２−メチルベンゾイミダゾール−１−イル］−酢酸エチルエステル（ＳＴＸ９７７，ＸＤＳ０２０１５）
オフホワイトの固体。ＴＬＣ Ｒ_ｆ ０．４６の単一スポット（６％ メタノール／ＤＣＭ）；ＨＰＬＣ純度＞９９％（ｔ_Ｒ １０％ 水−メタノール中２．０分）；

[6- (3-Chloro-2-methylbenzenesulfonylamino) -2-methylbenzimidazol-1-yl] -acetic acid ethyl ester (STX977, XDS02015)
Off-white solid. TLC R _f 0.46 single spot (6% methanol / DCM); HPLC purity> 99% (t _R 10% water-2.0 min in methanol);

［５−（３−クロロ−２−メチルベンゼンスルホニルアミノ）−２−メチルベンゾイミダゾール−１−イル］−酢酸エチルエステル（ＳＴＸ９７８，ＸＤＳ０２０１７）
オフホワイトの固体。ＴＬＣ Ｒ_ｆ ０．４０の単一スポット（６％ メタノール／ＤＣＭ）；ＨＰＬＣ純度 ９９％（ｔ_Ｒ １０％ 水−メタノール中２．０分）；

[5- (3-Chloro-2-methylbenzenesulfonylamino) -2-methylbenzimidazol-1-yl] -acetic acid ethyl ester (STX978, XDS02017)
Off-white solid. TLC R _f 0.40 single spot (6% methanol / DCM); HPLC purity 99% (t _R 10% water-2.0 min in methanol);

３−クロロ−Ｎ− ベンジル−２−メチル−１Ｈ−ベンゾイミダゾール−６−イル）−２−メチルベンゼンスルホンアミド（ＳＴＸ１１１７，ＸＤＳ０２０９８）
オフホワイトの固体。ＴＬＣ Ｒ_ｆ ０．７０の単一スポット（１０％ メタノール／ＤＣＭ）；ＨＰＬＣ純度 ９９％（ｔ_Ｒ ２０％水−メタノール中２．２分）；

3-chloro-N-benzyl-2-methyl-1H-benzimidazol-6-yl) -2-methylbenzenesulfonamide (STX1117, XDS02098)
Off-white solid. TLC R _f 0.70 single spot (10% methanol / DCM); HPLC purity 99% (t _R 20% water-2.2 min in methanol);

３−クロロ−Ｎ−（１−ベンジル−２−メチル−１Ｈ−ベンゾイミダゾール−５−イル）−２−メチルベンゼンスルホンアミド（ＳＴＸ１１１８，ＸＤＳ０２０９９）
オフホワイトの固体。ＴＬＣ Ｒ_ｆ ０．６５の単一スポット（１０％ メタノール／ＤＣＭ）；ＨＰＬＣ純度 ９９％（ｔ_Ｒ １０％ 水−メタノール中２．２分）；

3-Chloro-N- (1-benzyl-2-methyl-1H-benzimidazol-5-yl) -2-methylbenzenesulfonamide (STX1118, XDS02099)
Off-white solid. TLC R _f 0.65 single spot (10% methanol / DCM); HPLC purity 99% (t _R 10% water-2.2 min in methanol);

３−クロロ−Ｎ−（２−トリフルオロメチル−１Ｈ−ベンゾイミダゾール−５−イル）−２−メチルベンゼンスルホンアミド（ＳＴＸ８７９，ＸＤＳ０１１７３）
白色結晶固体。ＴＬＣ Ｒ_ｆ ０．５８の単一スポット（２０％ 酢酸エチル／ＤＣＭ）；ＨＰＬＣ純度 ９９％（ｔ_Ｒ ２０％水−メタノール中２．４分）；

3-Chloro-N- (2-trifluoromethyl-1H-benzimidazol-5-yl) -2-methylbenzenesulfonamide (STX879, XDS01173)
White crystalline solid. TLC R _f 0.58 single spot (20% ethyl acetate / DCM); HPLC purity 99% (t _R 20% water-2.4 min in methanol);

（３−クロロ−Ｎ−（２−メチル−１Ｈ−ベンゾイミダゾール−５−イル）−２−メチルベンゼンスルホンアミド（ＳＴＸ９８５，ＸＤＳ０２０２６）の調製）
上記の条件下での３−クロロ−２−メチルベンゼンスルホニルクロリド（２当量）の２−メチルベンズイミダゾール（１当量）とのカップリング反応により、３−クロロ−Ｎ−［１−（３−クロロ−２−メチルベンゼンスルホニル）−２−メチル−１Ｈ−ベンゾイミダゾール−５−イル］−２−メチル−ベンゼンスルホンアミドおよび３−クロロ−Ｎ−［１−（３−クロロ−２−メチルベンゼンスルホニル）−２−メチル−１Ｈ−ベンゾイミダゾール−６−イル］−２−メチル−ベンゼンスルホンアミドの１：１比（^１ＨＮＭＲにより判断）の混合物を生じた。

(Preparation of 3-chloro-N- (2-methyl-1H-benzimidazol-5-yl) -2-methylbenzenesulfonamide (STX985, XDS02026))
A coupling reaction of 3-chloro-2-methylbenzenesulfonyl chloride (2 eq) with 2-methylbenzimidazole (1 eq) under the above conditions yields 3-chloro-N- [1- (3-chloro -2-methylbenzenesulfonyl) -2-methyl-1H-benzimidazol-5-yl] -2-methyl-benzenesulfonamide and 3-chloro-N- [1- (3-chloro-2-methylbenzenesulfonyl) A mixture of 2-methyl-1H-benzimidazol-6-yl] -2-methyl-benzenesulfonamide in a 1: 1 ratio (determined by ¹ HNMR) was produced.

混合物（２００ｍｇ）をＴＨＦ（１５ｍＬ）中に溶解させ、Ｎヒドロキシベンゾトリアゾール（２００ｍｇ）を添加した。室温にて４８時間撹拌した後、混合物を酢酸エチルと５％炭酸水素ナトリウムとの間で分配した。有機相をブラインで洗浄し、硫酸ナトリウムで乾燥させ、真空下で濃縮して、黄色の残留物を生じ、これをフラッシュクロマトグラフィー（メタノール／ＤＣＭ勾配溶出）で精製した。オフホワイトの非結晶性粉末を得た。ＴＬＣ Ｒ_ｆ ０．３８の単一スポット（１０％ メタノール／ＤＣＭ）；ＨＰＬＣ純度 ９９％（ｔ_Ｒ １０％ 水−メタノール中２．０分）；

The mixture (200 mg) was dissolved in THF (15 mL) and N hydroxybenzotriazole (200 mg) was added. After stirring at room temperature for 48 hours, the mixture was partitioned between ethyl acetate and 5% sodium bicarbonate. The organic phase was washed with brine, dried over sodium sulfate and concentrated in vacuo to give a yellow residue that was purified by flash chromatography (methanol / DCM gradient elution). An off-white amorphous powder was obtained. TLC R _f 0.38 single spot (10% methanol / DCM); HPLC purity 99% (t _R 10% water-2.0 min in methanol);

（Ｎ−ベンズイミダゾールアリールスルホンアミド誘導体の合成）

(Synthesis of N-benzimidazole arylsulfonamide derivative)

（Ｎ^１−フェニル−ベンゼン−１，２，４−トリアミン：）
エタノール−ＴＨＦ（１５０：５０ｍＬ）中の２，４−ジニトロフェニルアミン（１．５ｇ，５．８ｍｍｏｌ）の溶液に、ヒドラジン水和物（２ｍＬ，６５ｍｍｏｌ）およびＲａｎｅｙニッケル（２．０ｇ）を添加した。反応混合物を室温にて２０分間撹拌し、Ｃｅｌｉｔｅを通して濾過した。溶媒をエバポレートして、黒色の残留物を生じ、これをフラッシュクロマトグラフィー（メタノール−ＤＣＭ勾配溶出）により精製した。黒色結晶固体（１．０ｇ，８７％）を得た。Ｍｐ １２８〜１２９℃；ＴＬＣ Ｒ_ｆ ０．４６の単一スポット（８％メタノール／ＤＣＭ）；

^{(N 1} - phenyl - benzene-1,2,4-triamine :)
To a solution of 2,4-dinitrophenylamine (1.5 g, 5.8 mmol) in ethanol-THF (150: 50 mL) was added hydrazine hydrate (2 mL, 65 mmol) and Raney nickel (2.0 g). . The reaction mixture was stirred at room temperature for 20 minutes and filtered through Celite. The solvent was evaporated to give a black residue that was purified by flash chromatography (methanol-DCM gradient elution). A black crystalline solid (1.0 g, 87%) was obtained. Mp 128-129 ° C; TLC R _f 0.46 single spot (8% methanol / DCM);

Ｎ−（２−メチル−１−フェニル−１Ｈ−ベンズイミダゾール−５−イル）−アセトアミド：
Ｎ’−フェニル−ベンゼン−１，２，４−トリアミン（８００ｍｇ，４ｍｍｏｌ）を、酢酸（１０ｍＭＬ）中に溶解させ、無水酢酸（１．０ｍＬ）をこの溶液に添加した。混合物を８０℃にて６時間撹拌し、室温まで冷却して、５％炭酸ナトリウムで中和し、次いで、酢酸エチルで抽出した。有機相をブラインで洗浄し、硫酸マグネシウムで乾燥させ、濃縮して残留物を生じ、これをエタノールから結晶化した。褐色の結晶固体（０．８５ｇ，８０％）を得た。Ｍｐ ２３１〜２３２℃；ＴＬＣ Ｒ_ｆ ０．３９の単一スポット（１０％ メタノール／ＤＣＭ）；

N- (2-Methyl-1-phenyl-1H-benzimidazol-5-yl) -acetamide:
N′-phenyl-benzene-1,2,4-triamine (800 mg, 4 mmol) was dissolved in acetic acid (10 mM L) and acetic anhydride (1.0 mL) was added to this solution. The mixture was stirred at 80 ° C. for 6 hours, cooled to room temperature, neutralized with 5% sodium carbonate, and then extracted with ethyl acetate. The organic phase was washed with brine, dried over magnesium sulfate and concentrated to give a residue that was crystallized from ethanol. A brown crystalline solid (0.85 g, 80%) was obtained. Mp 231-232 ° C; TLC R _f 0.39 single spot (10% methanol / DCM);

（２−メチル−１−フェニル−１Ｈ−ベンゾイミダゾール−５−イルアミン：）
６Ｎ ＨＣｌ（５ｍＬ）中のＮ−（２−メチル−１−フェニル−１Ｈ−ベンズイミダゾール−５−イル）−アセトアミド（８００ｍｇ，３ｍｍｏｌ）の溶液を、７５℃にて３時間撹拌し、炭酸ナトリウムでｐＨ ７まで中和し、次いで、酢酸エチルで抽出した。有機相をブラインで洗浄し、硫酸マグネシウムで乾燥させ、濃縮して、暗褐色の固体（６００ｍｇ，９０％）を生じた。ｍｐ １４５〜１４６℃；ＴＬＣ Ｒ_ｆ ０．４７の単一スポット（１０％ メタノール／ＤＣＭ）；

(2-Methyl-1-phenyl-1H-benzimidazol-5-ylamine :)
A solution of N- (2-methyl-1-phenyl-1H-benzimidazol-5-yl) -acetamide (800 mg, 3 mmol) in 6N HCl (5 mL) was stirred at 75 ° C. for 3 hours and washed with sodium carbonate. Neutralized to pH 7, then extracted with ethyl acetate. The organic phase was washed with brine, dried over magnesium sulfate and concentrated to give a dark brown solid (600 mg, 90%). mp 145-146 ° C; TLC R _f 0.47 single spot (10% methanol / DCM);

３−クロロ−２−メチル−Ｎ−（２−メチル−１−フェニル−１Ｈ−ベンゾイミダゾール−５−イル）−ベンゼンスルホンアミド（ＳＴＸ１１４０，ＸＤＳ０２１１０）
化合物を、ベンゼンスルホンアミド形成の一般方法により調製した。淡いピンクの結晶固体を得た。Ｍｐ ２５４〜２５６℃；ＴＬＣ Ｒ_ｆ ０．６２の単一スポット（８％ メタノール／ＤＣＭ）；ＨＰＬＣ純度 ＞９９％（ｔ_Ｒ ２０％水−メタノール中２．６分）；

3-Chloro-2-methyl-N- (2-methyl-1-phenyl-1H-benzimidazol-5-yl) -benzenesulfonamide (STX1140, XDS02110)
The compound was prepared by the general method of benzenesulfonamide formation. A pale pink crystalline solid was obtained. Mp 254-256 ° C .; TLC R _f 0.62 single spot (8% methanol / DCM); HPLC purity> 99% (t _R 20% water-2.6 min in methanol);

３−クロロ−Ｎ−［１−（２−ヒドロキシエチル）−２−メチル−１Ｈ−ベンゾイミダゾール−６−イル］−２−メチル−ベンゼンスルホンアミド（ＳＴＸ１１４１，ＸＤＳ０２１１５）
無水ＴＨＦ（１０ｍＬ）中の［６−（３−クロロ−２−メチル−ベンゼンスルホニルアミノ）−２−メチル−ベンゾイミダゾール−１−イル］−酢酸エチルエステル（１００ｍｇ，０．２３７ｍｍｏｌ）の溶液に、０℃にてＬｉＡｌＨ_４（５４ｍｇ，１．４２ｍｍｏｌ）を添加した。混合物を０℃にて０．５時間撹拌させ、飽和塩化アンモニウム溶液でクエンチし、６Ｎ ＨＣｌで中和し、酢酸エチルで抽出した。有機相をブラインで洗浄し、硫酸ナトリウムで乾燥させ、真空下で濃縮して、淡いピンクの結晶固体（８２ｍｇ，９１％）を生じた。ｍｐ ２１３〜２１４．５℃；ＴＬＣ Ｒ_ｆ ０．３９の単一スポット（１２％ メタノール／ＤＣＭ）；ＨＰＬＣ純度＞９９％（ｔ_Ｒ ２０％水−メタノール中２．０分）；

3-chloro-N- [1- (2-hydroxyethyl) -2-methyl-1H-benzimidazol-6-yl] -2-methyl-benzenesulfonamide (STX1141, XDS02115)
To a solution of [6- (3-chloro-2-methyl-benzenesulfonylamino) -2-methyl-benzimidazol-1-yl] -acetic acid ethyl ester (100 mg, 0.237 mmol) in anhydrous THF (10 mL), LiAlH ₄ (54 mg, 1.42 mmol) was added at 0 ° C. The mixture was allowed to stir at 0 ° C. for 0.5 h, quenched with saturated ammonium chloride solution, neutralized with 6N HCl, and extracted with ethyl acetate. The organic phase was washed with brine, dried over sodium sulfate and concentrated under vacuum to give a pale pink crystalline solid (82 mg, 91%). mp 213-214.5 ° C; TLC R _f 0.39 single spot (12% methanol / DCM); HPLC purity> 99% (t _R 20% water-2.0 min in methanol);

３−クロロ−Ｎ−［１−（２−ヒドロキシ−エチル）−２−メチル−１Ｈ−ベンゾイミダゾール−５−イル］−２−メチル−ベンゼンスルホンアミド（ＳＴＸ１１４２，ＸＤＳ０２１１６）
上記のように、［５−（３−クロロ−２−メチル−ベンゼンスルホニルアミノ）−２−メチル−ベンゾイミダゾール−１−イル］−酢酸エチルエステル（３５ｍｇ，０．０８３ｍｍｏｌ）から化合物を調製した。白色結晶固体（２２ｍｇ，８９％）を得た。Ｍｐ ２４５〜２４７℃；ＴＬＣ Ｒ_ｆ ０．３８の単一スポット（１２％ メタノール／ＤＣＭ）；ＨＰＬＣ純度＞９９％（ｔ_Ｒ ２０％水−メタノール中２．０分）；

3-Chloro-N- [1- (2-hydroxy-ethyl) -2-methyl-1H-benzimidazol-5-yl] -2-methyl-benzenesulfonamide (STX1142, XDS02116)
The compound was prepared from [5- (3-chloro-2-methyl-benzenesulfonylamino) -2-methyl-benzimidazol-1-yl] -acetic acid ethyl ester (35 mg, 0.083 mmol) as described above. White crystalline solid (22 mg, 89%) was obtained. Mp 245-247 ° C; TLC R _f 0.38 single spot (12% methanol / DCM); HPLC purity> 99% (t _R 20% water-2.0 min in methanol);

（ベンズオキサゾール誘導体の合成）

(Synthesis of benzoxazole derivatives)

（３−クロロ−２−メチル−Ｎ−（２−メチル−ベンゾオキサゾール−６−イル）−ベンゼンスルホンアミド，ＳＴＸ８３９（ＫＲＢ０１００９）の合成）

(Synthesis of 3-chloro-2-methyl-N- (2-methyl-benzoxazol-6-yl) -benzenesulfonamide, STX839 (KRB01009))

ジクロロメタン（３ｍＬ）中の３−クロロ−２−メチルベンゼンスルホニルクロリド（１６３ｍｇ，０．７２３ｍｍｏｌ）の溶液に、ピリジン（１４０μＬ，１．７２ｍｍｏｌ）を添加し、混合物をＮ_２下で５分間撹拌し、その後、６−アミノ−２−メチルベンゾオキサゾール（１０２ｍｇ，０．６８８ｍｍｏｌ）を添加した。得られた混合物を室温にて１時間撹拌し、次いで、飽和ＮａＨＣＯ_３溶液を添加し（８ｍＬ）、混合物を酢酸エチル（１５ｍＬ）中に抽出した。有機相をブラインで洗浄し、乾燥させ（Ｎａ_２ＳＯ_４）、濾過して、エバポレートし、残留物を生じ、これを、フラッシュクロマトグラフィーを用いて精製して、白色固体（１５１ｍｇ，６５％）を得た。Ｒ_ｆ ０．５０の単一スポット（６０：４０ ヘキサン：酢酸エチル）。ｍｐ １２７．１〜１２７．５℃，ＨＰＬＣ純度 ９７％（ｔ_Ｒ １０％水−アセトニトリル中２．０５分）。

To a solution of 3-chloro-2-methylbenzenesulfonyl chloride (163 mg, 0.723 mmol) in dichloromethane (3 mL) was added pyridine (140 μL, 1.72 mmol) and the mixture was stirred under N ₂ for 5 min. Then 6-amino-2-methylbenzoxazole (102 mg, 0.688 mmol) was added. The resulting mixture was stirred at room temperature for 1 hour, then saturated NaHCO ₃ solution was added (8 mL) and the mixture was extracted into ethyl acetate (15 mL). The organic phase was washed with brine, dried (Na ₂ SO ₄ ), filtered and evaporated to give a residue that was purified using flash chromatography to give a white solid (151 mg, 65%) Got. Single spot with R _f 0.50 (60:40 hexane: ethyl acetate). mp 127.1-127.5 ° C., HPLC purity 97% (t _R 10% water-acetonitrile in 2.05 min).

（Ｎ−（２−メチル−ベンゾオキサゾール−６−イル）−４−プロピル−ベンゼンスルホンアミド，ＳＴＸ８４０（ＫＲＢ０１０１０）の合成）

(Synthesis of N- (2-methyl-benzoxazol-6-yl) -4-propyl-benzenesulfonamide, STX840 (KRB01010))

ジクロロメタン（３ｍＬ）中の４ｎ−プロピルベンゼンスルホニルクロリド（１６３ｍｇ，０．７４４ｍｍｏｌ）の溶液に、ピリジン（１４０μＬ，１．７２ｍｍｏｌ）を添加し、混合物をＮ_２下で５分間撹拌し、その後、６−アミノ−２−メチルベンゾオキサゾール（１０５ｍｇ，０．７０９ｍｍｏｌ）を添加した。得られた混合物を室温にて１時間撹拌し、次いで、飽和ＮａＨＣＯ_３溶液を添加し（８ｍＬ）、混合物を酢酸エチル（１５ｍＬ）中に抽出した。有機相をブラインで洗浄し、乾燥させ（Ｎａ_２ＳＯ_４）、濾過し、エバポレートして、残留物を生じ、これを、フラッシュクロマトグラフィーを用いて精製し、淡いピンクの固体（１６４ｍｇ，７０％）を得た。Ｒ_ｆ ０．４９の単一スポット（６０：４０ ヘキサン：酢酸エチル）。ｍｐ １０１．７〜１０２．３℃，ＨＰＬＣ 純度９９％（ｔ_Ｒ １０％水−アセトニトリル中２．０２分）。

To a solution of 4n-propylbenzenesulfonyl chloride (163 mg, 0.744 mmol) in dichloromethane (3 mL) was added pyridine (140 μL, 1.72 mmol) and the mixture was stirred under N ₂ for 5 minutes, then 6- Amino-2-methylbenzoxazole (105 mg, 0.709 mmol) was added. The resulting mixture was stirred at room temperature for 1 hour, then saturated NaHCO ₃ solution was added (8 mL) and the mixture was extracted into ethyl acetate (15 mL). The organic phase was washed with brine, dried (Na ₂ SO ₄ ), filtered and evaporated to give a residue that was purified using flash chromatography to give a pale pink solid (164 mg, 70% ) Single spot with R _f 0.49 (60:40 hexane: ethyl acetate). mp 101.7-102.3 ° C., HPLC purity _{99% (t} R _10% water - 2.02 minutes in acetonitrile).

（２，５−ジクロロ−（２−メチル−ベンゾオキサゾール−６−イル）−ベンゼンスルホンアミド，ＳＴＸ８４１（ＫＲＢ０１０１１）の合成）

(Synthesis of 2,5-dichloro- (2-methyl-benzoxazol-6-yl) -benzenesulfonamide, STX841 (KRB01011))

ジクロロメタン（３ｍＬ）中の２，５−ジクロロベンゼンスルホニルクロリド（１７４ｍｇ，０．７０９ｍｍｏｌ）の溶液に、ピリジン（１４０μＬ，１．７２ｍｍｏｌ）を添加し、混合物をＮ_２下で５分間撹拌し、その後、６−アミノ−２−メチルベンゾオキサゾール（１００ｍｇ，０．６７５ｍｍｏｌ）を添加した。得られた混合物を室温にて１時間撹拌し、次いで、飽和ＮａＨＣＯ_３溶液を添加し（８ｍＬ）、混合物を酢酸エチル（１５ｍＬ）中に抽出した。有機相をブラインで洗浄し、乾燥させ（Ｎａ_２ＳＯ_４）、濾過し、エバポレートして、残留物を生じ、これを、フラッシュクロマトグラフィーを用いて精製し、白色固体（１５４ｍｇ，６４％）を得た。Ｒ_ｆ ０．５０の単一スポット（６０：４０ ヘキサン：酢酸エチル）。ｍｐ １６７．０〜１６７．３℃，ＨＰＬＣ純度 ９７％（ｔ_Ｒ １０％水−アセトニトリル中１．９７分）。

To a solution of 2,5-dichlorobenzenesulfonyl chloride (174 mg, 0.709 mmol) in dichloromethane (3 mL) was added pyridine (140 μL, 1.72 mmol) and the mixture was stirred under N ₂ for 5 minutes, then 6-Amino-2-methylbenzoxazole (100 mg, 0.675 mmol) was added. The resulting mixture was stirred at room temperature for 1 hour, then saturated NaHCO ₃ solution was added (8 mL) and the mixture was extracted into ethyl acetate (15 mL). The organic phase was washed with brine, dried (Na ₂ SO ₄ ), filtered and evaporated to give a residue that was purified using flash chromatography to give a white solid (154 mg, 64%). Obtained. Single spot with R _f 0.50 (60:40 hexane: ethyl acetate). mp from 167.0 to 167.3 ° C., HPLC purity _{97% (t} R _10% water - 1.97 minutes in acetonitrile).

（３−クロロ−２−メチル−Ｎ−（２−メチル−ベンゾオキサゾール−５−イル）−ベンゼンスルホンアミド，ＳＴＸ８４２（ＫＲＢ０１０１４）の合成）

(Synthesis of 3-chloro-2-methyl-N- (2-methyl-benzoxazol-5-yl) -benzenesulfonamide, STX842 (KRB01014))

ジクロロメタン（２ｍＬ）中の３−クロロ−２−メチルベンゼンスルホニルクロリド（９６ｍｇ，０．４３ｍｍｏｌ）の溶液に、ピリジン（８０μＬ，１．０ｍｍｏｌ）を添加し、混合物をＮ_２下で５分間撹拌し、その後、５−アミノ−２−メチルベンゾオキサゾール（６０ｍｇ，０．４０ｍｍｏｌ）を添加した。得られた混合物を室温にて１時間撹拌し、次いで、飽和ＮａＨＣＯ_３溶液を添加し（８ｍＬ）、混合物を酢酸エチル（１５ｍＬ）中に抽出した。有機相をブラインで洗浄し、乾燥させ（Ｎａ_２ＳＯ_４）、濾過し、エバポレートして、残留物を生じ、これを、フラッシュクロマトグラフィーを用いて精製し、白色固体（８９ｍｇ，６４％）を得た。Ｒ_ｆ ０．５２の単一スポット（１：１ ヘキサン：酢酸エチル）。ｍｐ １８０．２〜１８０．５℃，ＨＰＬＣ純度 ９９％（ｔ_Ｒ １０％水−アセトニトリル中２．３２分）。

To a solution of 3-chloro-2-methylbenzenesulfonyl chloride (96 mg, 0.43 mmol) in dichloromethane (2 mL) was added pyridine (80 μL, 1.0 mmol) and the mixture was stirred under N ₂ for 5 min. Then 5-amino-2-methylbenzoxazole (60 mg, 0.40 mmol) was added. The resulting mixture was stirred at room temperature for 1 hour, then saturated NaHCO ₃ solution was added (8 mL) and the mixture was extracted into ethyl acetate (15 mL). The organic phase was washed with brine, dried (Na ₂ SO ₄ ), filtered and evaporated to give a residue that was purified using flash chromatography to give a white solid (89 mg, 64%). Obtained. Single spot with R _f 0.52 (1: 1 hexane: ethyl acetate). mp from 180.2 to 180.5 ° C., HPLC purity _{99% (t} R _10% water - 2.32 minutes in acetonitrile).

（Ｎ−（２−メチル−ベンゾオキサゾール−５−イル）−４−プロピル−ベンゼンスルホンアミド，ＳＴＸ８４３（ＫＲＢ０１０１５）の合成）

(Synthesis of N- (2-methyl-benzoxazol-5-yl) -4-propyl-benzenesulfonamide, STX843 (KRB01015))

ジクロロメタン（２ｍＬ）中の４ｎ−プロピルベンゼンスルホニルクロリド（９３ｍｇ，０．４３ｍｍｏｌ）の溶液に、ピリジン（８０μＬ，１．０ｍｍｏｌ）を添加し、混合物をＮ_２下で５分間撹拌し、その後、５−アミノ−２−メチルベンゾオキサゾール（６０ｍｇ，０．４０ｍｍｏｌ）を添加した。得られた混合物を室温にて１時間撹拌し、次いで、飽和ＮａＨＣＯ_３溶液を添加し（８ｍＬ）、混合物を酢酸エチル（１５ｍＬ）中に抽出した。有機相をブラインで洗浄し、乾燥させ（Ｎａ_２ＳＯ_４）、濾過し、エバポレートして、残留物を生じ、これを、フラッシュクロマトグラフィーを用いて精製し、淡いピンクのオイル（１１２ｍｇ，８５％）を得た。Ｒ_ｆ ０．５３の単一スポット（１：１ ヘキサン：酢酸エチル）。ＨＰＬＣ純度 ９９＋％（ｔ_Ｒ １０％水−アセトニトリル中２．３８分）

To a solution of 4n-propylbenzenesulfonyl chloride (93 mg, 0.43 mmol) in dichloromethane (2 mL) is added pyridine (80 μL, 1.0 mmol) and the mixture is stirred under N ₂ for 5 minutes, then 5- Amino-2-methylbenzoxazole (60 mg, 0.40 mmol) was added. The resulting mixture was stirred at room temperature for 1 hour, then saturated NaHCO ₃ solution was added (8 mL) and the mixture was extracted into ethyl acetate (15 mL). The organic phase was washed with brine, dried (Na ₂ SO ₄ ), filtered and evaporated to give a residue that was purified using flash chromatography to give a pale pink oil (112 mg, 85% ) Single spot with R _f 0.53 (1: 1 hexane: ethyl acetate). HPLC purity 99 _{+% (t} R _10% water - 2.38 minutes in acetonitrile)

（２，５−ジクロロ−Ｎ−（２−メチル−ベンゾオキサゾール−５−イル）−ベンゼンスルホンアミド，ＳＴＸ８４６（ＫＲＢ０１０１６）の合成）

(Synthesis of 2,5-dichloro-N- (2-methyl-benzoxazol-5-yl) -benzenesulfonamide, STX846 (KRB01016))

ジクロロメタン（１．５ｍＬ）中の２，５−ジクロロベンゼンスルホニルクロリド（５２ｍｇ，０．２１ｍｍｏｌ）の溶液に、ピリジン（４０μＬ，０．５ｍｍｏｌ）を添加し、混合物をＮ_２下で５分間撹拌し、その後、５−アミノ−２−メチルベンゾオキサゾール（３０ｍｇ，０．２０ｍｍｏｌ）を添加した。得られた混合物を室温にて１時間撹拌し、次いで、飽和ＮａＨＣＯ_３溶液を添加し（８ｍＬ）、混合物を酢酸エチル（１５ｍＬ）中に抽出した。有機相をブラインで洗浄し、乾燥させ（Ｎａ_２ＳＯ_４）、濾過し、エバポレートして、残留物を生じ、これを、フラッシュクロマトグラフィーを用いて精製し、淡いピンクの固体（４５ｍｇ，６３％）を得た。Ｒ_ｆ ０．５３の単一スポット（１：１ ヘキサン：酢酸エチル）。ｍｐ １９３．５〜１９３．９℃，ＨＰＬＣ純度 ９８％（ｔ_Ｒ １０％水−アセトニトリル中２．２７分）。

To a solution of 2,5-dichlorobenzenesulfonyl chloride (52 mg, 0.21 mmol) in dichloromethane (1.5 mL), pyridine (40 μL, 0.5 mmol) is added and the mixture is stirred under N ₂ for 5 min. Then 5-amino-2-methylbenzoxazole (30 mg, 0.20 mmol) was added. The resulting mixture was stirred at room temperature for 1 hour, then saturated NaHCO ₃ solution was added (8 mL) and the mixture was extracted into ethyl acetate (15 mL). The organic phase was washed with brine, dried (Na ₂ SO ₄ ), filtered and evaporated to give a residue that was purified using flash chromatography to give a pale pink solid (45 mg, 63% ) Single spot with R _f 0.53 (1: 1 hexane: ethyl acetate). mp 193.5 to 193.9 ° C., HPLC purity _{98% (t} R _10% water - 2.27 minutes in acetonitrile).

上記の明細書中で言及した全ての刊行物は、本明細書中に参考として援用される。記載される本発明の方法および系の種々の改変および変更は、本発明の範囲および精神から逸脱することなく、当業者に明らかである。本発明は、特定の好ましい実施形態と組み合せて記載されているが、特許請求される本発明は、このような特定の実施形態に不当に限定されるべきでないことが理解されるべきである。実際、化学または関連分野の当業者に明らかである、本発明の実施するための記載される方法の種々の改変が、添付の特許請求の範囲内であることが意図される。

All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in conjunction with certain preferred embodiments, it is to be understood that the claimed invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the described methods for carrying out the invention which are obvious to those skilled in chemistry or related fields are intended to be within the scope of the following claims.

  (References)

The invention will now be described in further detail, by way of example only, with reference to the accompanying drawings.
FIG. 1 is graph 1 showing the amount of protein per liter of rat liver and rat kidney. FIG. 2 is a graph 2 showing the enzyme concentration and time-dependent course of 11β-HSD type 1 activity in rat liver. FIG. 3 is a graph 3 showing a time-dependent course of 11β-HSD type 2 activity in rat kidney. FIG. 4 is a graph showing the extraction efficiency obtained by the four extraction methods. FIG. 5 is a graph showing a comparison of 11β-HSD1 activity in rat and human liver microsomes. FIG. 6 is a series of graphs showing the effect of incubation time on human microsomal 11β-HSD1 activity. FIG. 7 is a series of graphs showing the effect of microsomal protein concentration on human microsomal 11β-HSD1 activity. FIG. 8 is a graph showing a substrate (cortisone) saturation curve for human liver microsomes Up HSD1. FIG. 9 is a Lineweaver-Burke plot of substrate saturation data for human liver microsome 11HSD1. FIG. 10 is a graph showing IC ₅₀ determination for glycyrrhetinic acid. FIG. 11 is a graph showing IC ₅₀ determinations for carbenoxolone. FIG. 12A is a graph showing 11β-HSD1 activity measured by immunoassay. FIG. 12A shows the effect of the protein. FIG. 12B is a graph showing 11β-HSD1 activity measured by immunoassay. FIG. 12B shows the effect of cortisone. FIG. 12C is a graph showing 11β-HSD1 activity measured by immunoassay. FIG. 12C shows the effect of Tween-80. FIG. 13 is a graph showing the evaluation of the Assay Designs Cortisol immunoassay. FIG. 14 is a graph showing the effect of increasing microsomal protein on the measurement of 11β-HSD1 activity detected by Assay Designs immunoassay. FIG. 15 is a graph showing the detection of Up HSD1 activity by RIA using an Immunotech anti-cortisol antibody. FIG. 16 is a graph showing the effect of Immunotech antibody concentration on the signal-to-noise ratio (microsome group compared to GA blank group). FIG. 17 is a graph showing an Immunotech antibody saturation curve for detection of 11β HSD1 activity by RIA. FIG. 18 is a graph showing the linearity of human liver microsomal Up HSD1 activity by RIA. FIG. 19 is a graph showing the effect of Tween 80 on the detection of human liver microsomal Up HSD1 activity by RIA. FIG. 20 is a graph showing the effect of the buffer system on the detection of human liver microsomal Up HSD1 activity by RIA. FIG. 21 is a graph showing linearity with the incubation time of human liver microsomal Up HSD1 activity detected by RIA. FIG. 22 is a graph showing a substrate saturation curve for human liver microsome 11β-HSD1 activity detected by RIA. FIG. 23 is a Lineweaver-Burke plot of substrate saturation data for human liver microsomal 11β-HSD1 activity detected by RIA. FIG. 24 is a graph showing DMSO resistance of human liver microsome 11β-HSD1 activity. FIG. 25 is an IC ₅₀ curve for inhibition of human liver microsomal 11β-HSD1 activity by glycyrrhetinic acid.

Claims (51)

Formula I:

A compound having the following formula:
One of R ₁ and R ₂ has the following formula:

Where R ₄ is selected from H and hydrocarbyl, R ₅ is a hydrocarbyl group, and L is an optional linker group, or R ₁ and R ₂ are taken together The following groups:

In which R ₃ is H or a substituent, wherein X is selected from S, O, NR ₆ and C (R ₇ ) (R ₈ ), wherein Wherein R ₆ is selected from H and hydrocarbyl groups, wherein each of R ₇ and R ₈ is independently selected from H and hydrocarbyl groups.
Compound. Formula II below:

The compound of claim 1 having 3. A compound according to claim 1 or 2, wherein L is absent. R ₁ and R ₂ together are the following groups:

4. A compound according to claim 1, 2 or 3 which forms a ring substituted with. R ₁ and R ₂ are taken together to form a carbocyclic ring A compound according to any one of claims 1 to 4. 6. A compound according to any one of claims 1 to 5, wherein R < ₁ > and R < ₂ > together form a 6-membered ring. R ₁ and R ₂ are taken together to form an aryl ring, a compound according to any one of claims 1-6. Formula III:

The compound of any one of Claims 1-7 which has these. Formula IV:

The compound of any one of Claims 1-8 which has these. Formula V:

10. The compound according to any one of claims 1 to 9, wherein Formula VI:

11. The compound according to any one of claims 1 to 10, which has Formula VII:

11. The compound according to any one of claims 1 to 10, which has R ₃ is selected from H, hydrocarbyl, —S-hydrocarbyl, —S—H—, halogen and N (R ₉ ) (R ₁₀ ), wherein each of R ₉ and R ₁₀ is independently H 13. A compound according to any one of claims 1 to 12, which is selected from and a hydrocarbyl group. R ₃ is, H, and _are selected from _C 1 _{-C 10} alkyl groups such as C 1 -C ₆ alkyl groups and _C 1 -C ₃ alkyl group, according to any one of claims 1 to 13 Compound. R ₃ is -CH _3, A compound according to any one of claims 1 to 14. Formula VIII:

The compound of any one of Claims 1-7 which has these. Formula IX:

The compound of any one of Claims 1-7 which has these. Formula X:

The compound of any one of Claims 1-7 which has these. Formula XI:

The compound of any one of Claims 1-7 which has these. R ₃ is, O, is selected from hydrocarbyl and N _{(R 9),} wherein, _{R 9} is selected from H and hydrocarbyl groups, the compounds according to any one of claims 16 to 19. R ₃ is O, a C ₁ -C ₁₀ alkenyl group, such as a C ₁ -C ₆ alkenyl group and a C ₁ -C ₃ alkenyl group, NH and an N—C ₁ -C ₁₀ alkyl group, such as N—C ₁ -C ₆ are selected from alkyl groups and _N-C 1 -C ₃ alkyl group, a compound according to any one of claims 1 to 20. R ₄ is H, and is selected from _C 1 _{-C 10} alkyl groups such as _C 1 -C ₆ alkyl groups and _C 1 -C ₃ alkyl group, A compound according to any one of claims 1 to 21 . R ₄ is H, A compound according to any one of claims 1 to 22. R ₄ represents the following formula:

The compound according to any one of claims 1 to 21, which is a group of: R ₅ is a ring substituted compound according to any one of claims 1 to 24. R ₅ is a carbocyclic ring A compound according to any one of claims 1 to 25. R ₅ is a six-membered ring, A compound according to any one of claims 1 to 26. R ₅ is an aryl ring, a compound according to any one of claims 1 to 27. R ₅ represents the following formula:

A group having the wherein _each of _{R 11, R 12, R 13} , R 14 and _{R 15} are, independently, H, are selected from halogen and hydrocarbyl groups, claim 1 to 28 The compound according to item 1. Each of R ₁₁ , R ₁₂ , R ₁₃ , R ₁₄ and R ₁₅ is independently H, halogen, alkyl, phenyl, O-alkyl, O-phenyl, nitrile, haloalkyl, carboxyalkyl, —CO ₂ H, It is selected from CO ₂ alkyl and NH- acetyl group, the compound according to claim 29. 31. A pharmaceutical composition comprising a compound according to any one of claims 1 to 30, optionally formulated with a pharmaceutically acceptable carrier, diluent, excipient or adjuvant. 31. A compound according to any one of claims 1 to 30 for use in medicine. 31. Use of a compound according to any one of claims 1-30 in the manufacture of a medicament for use in the treatment of a condition or disease associated with 11 [beta] -HSD. Said condition or disease is metabolic disorders such as diabetes and obesity; cardiovascular disorders such as hypertension; glaucoma; inflammatory disorders such as arthritis or asthma; immune disorders; bone disorders such as osteoporosis; Intrauterine growth retardation; overt mineralocorticoid syndrome (AME); polycystic ovary syndrome (PCOS); hirsutism; acne; rare or amenorrhea; adrenal cortical adenoma and carcinoma; Cushing syndrome; 34. Use according to claim 33, selected from the group consisting of invasive carcinoma; breast cancer; and endometrial cancer. 31. Use of a compound according to any one of claims 1 to 30 in the manufacture of a medicament for use in the treatment of a condition or disease associated with harmful 11 [beta] -HSD levels. 31. Use of a compound according to any one of claims 1 to 30 in the manufacture of a medicament for modulating 11 [beta] -HSD activity. 31. Use of a compound according to any one of claims 1 to 30 in the manufacture of a medicament for inhibiting 11 [beta] -HSD activity. Less than:
(A) performing an 11β-HSD assay using one or more candidate compounds having a formula as defined in any one of claims 1-30;
(B) determining whether the one or more candidate compounds can modulate 11β-HSD activity; and (c) selecting the one or more candidate compounds capable of modulating 11β-HSD activity. Including the method. Less than:
(A) performing an 11β-HSD assay using one or more candidate compounds having a formula as defined in any one of claims 1-30;
(B) determining whether the one or more candidate compounds can inhibit 11β-HSD activity; and (c) selecting the one or more candidate compounds capable of inhibiting 11β-HSD activity. Including the method. 40. A compound identified by the method of claim 38 or claim 39. 41. A compound according to claim 40 for use in medicine. 41. A pharmaceutical composition comprising a compound according to claim 40, optionally formulated with a pharmaceutically acceptable carrier, diluent, excipient or adjuvant. 41. Use of a compound according to claim 40 in the manufacture of a medicament for use in the treatment of a condition or disease associated with 11 [beta] -HSD. Said condition or disease is metabolic disorders such as diabetes and obesity; cardiovascular disorders such as hypertension; glaucoma; inflammatory disorders such as arthritis or asthma; immune disorders; bone disorders such as osteoporosis; Intrauterine growth retardation; overt mineralocorticoid syndrome (AME); polycystic ovary syndrome (PCOS); hirsutism; acne; rare or amenorrhea; adrenal cortical adenoma and carcinoma; Cushing syndrome; 44. Use according to claim 43, selected from the group consisting of invasive carcinoma; breast cancer as well as endometrial cancer. 41. Use of a compound according to claim 40 in the manufacture of a medicament for use in the treatment of a condition or disease associated with harmful 11 [beta] -HSD levels. The invention according to any one of claims 33 to 45, wherein 11β-HSD is 11β-HSD type 1. The invention according to any one of claims 33 to 45, wherein 11β-HSD is 11β-HSD type 2. A compound substantially as described herein with reference to any of the Examples. A composition substantially as described herein with reference to any of the Examples. A method substantially as described herein with reference to any of the Examples. Use substantially as described herein with reference to any of the Examples.

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