JP5888667B2 - ADAM action inhibitor screening method, temporary foot retention method, temporary foot retention agent, provisional foot control substance screening method, and ADAM activity inhibitor - Google Patents

Description

  The present invention relates to a screening method for an ADAM action inhibitor, a temporary foot retention method, a temporary foot retention agent, a temporary foot control substance screening method, and an ADAM activity inhibitor.

  There are roughly two types of snake venoms, hemorrhagic snake venom possessed by rattlesnakes and the like, and nervous snake venom possessed by cobras and the like.

  As disclosed in the following Non-Patent Documents 1 to 4, vascular apoptosis-inducing protein (hereinafter referred to as VAP) 1 and 2 were isolated from rattlesnake venom as hemorrhagic snake toxins by the present inventors. It is a protein and induces apoptosis with the formation of intense apoptotic vesicles on vascular endothelial cells. The inventors of the present application have also confirmed that VAP1 and VAP2 are homologs of the ADAM family.

  ADAM is an abbreviation for a-disintegrin and metalloprotease, and is a protein having a disintegrin domain and a metalloprotease domain. Matrix metalloprotease (hereinafter referred to as MMP) is also a protein having a metalloprotease domain and is considered to be involved in wound healing and the like. The metalloprotease site of these proteins has a metal ion at the active center and is activated to cleave the protein. Moreover, the ADAM inhibitor in the following patent documents 1-3 is disclosed.

JP-T-2005-533789 This patent document 1 discloses a compound useful for inhibiting ADAM-10 protein. JP, 2008-184387, A This patent documents 2 discloses an ADAM inhibitor which is an ascorbic acid derivative. JP, 2008-201772, A This patent documents 3 discloses an ADAM inhibitor which is alkyl glucoside.

Masuda, S., Hayashi, H., Araki, S., 1998. Two vascularapoptosis-inducing proteins from snake venom are members of the metalloprotease / disintegrin family. Eur. J. Biochem. 253, 36-41. Masuda, S., Ohta, T., Kaji, K., Fox, JW, Hayashi, H., Araki, S., 2000.cDNA cloning and characterization of vascular apoptosis-inducing protein 1. Biochem. Biophys. Res. Commun. 278, 197-204. Maruyama, J., Hayashi, H., Miao, J., Sawada, H., Araki, S., 2005.Severe cell fragmentation in the endothelial cell apoptosis induced by snakeapoptosis toxin VAP1 is an apoptotic characteristic controlled by caspases. , 1-6. Kikushima, Y., Nakamura, S., Ohshima, Y., Shibuya, T., Miao, JY, Hayashi, H., Nikai, T., Araki, S., 2008. Hemorrhagic activity of thevascular apoptosis-inducing proteins VAP1 and VAP2 from Crotalus atrox. Toxicon 52, 589-593.

  It has been clarified that ADAM is involved in various diseases, and the ADAM inhibitor is used to treat the disease. However, conventional ADAM inhibitors have the following problems.

  In the present application, a signal transduction system including a protease reaction by a metalloprotease site of ADAM in the signal transduction process is referred to as an ADAM signal transduction system. The ADAM signal transmission system also includes a signal transmission system caused by administration / introduction of ADAM. The upstream of ADAM refers to the stage before the protease reaction by ADAM in the ADAM signaling system. Similarly, downstream of ADAM refers to a stage after the protease reaction by ADAM.

  First, the target metalloprotease site of ADAM has very high homology with the metalloprotease site of other proteins such as MMP, and it has been difficult to create a metalloprotease-specific inhibitor of ADAM. On the other hand, a protein having each metalloprotease site is strictly regulated in terms of expression site, amount, time, etc. in the living body, and maintains homeostasis.

  As a direct inhibitor of the metalloprotease site of ADAM, for example, there is a compound having a peptide-like structure containing a metal ion chelate moiety such as hydroxamic acid disclosed in Patent Document 1. The direct inhibitor acts not only on the metalloprotease site of ADAM but also on the metalloprotease site of other proteins such as MMP, so that the function of the other protein is inhibited and the side effect is great. There was a problem.

  As an indirect inhibitor of ADAM, for example, there are ADAM inhibitors described in Patent Document 2 and Patent Document 3. These were evaluated for the TNF-α release inhibitory effect, and inhibit signal transduction upstream of ADAM. When such upstream signal transduction of ADAM is inhibited, there is a problem in that the release of cell growth factor, which is one of the direct actions of ADAM, is suppressed and side effects such as suppression of normal cell growth occur.

  As described above, there has been no disclosure of an inhibitor that works downstream of ADAM to inhibit ADAM action. In addition, it has been thought that the cause of bleeding caused by snake venom is that metalloproteinases degrade vascular proteins.

  As a result of intensive studies to solve the above-mentioned problems, the present inventor has found that bleeding caused by rattlesnake venom is not simply caused by the degradation of proteins constituting blood vessels, but a signal related to VAP, a homolog of ADAM. I found out that it was the result of the transmission. From this, it was speculated that, for example, a plurality of actions resulting from signal transduction by the ADAM signal transduction system can be individually inhibited by controlling the signal transduction system downstream of ADAM. And this inventor discovered the substance which inhibits the signal transduction downstream of ADAM, and also discovered the substance which inhibits ADAM action specifically. That is, the present inventors have found that it is possible to inhibit the action of ADAM without inhibiting the signal transduction upstream of ADAM and leaving the activity of the metalloprotease site of other proteins such as MMP.

  In addition, the present inventor has obtained an interesting finding that provisional limbs are maintained in apoptosis induced by VAP, while conducting research on apoptosis induced by VAP1 and VAP2. Therefore, it has been found that ADAM is also useful for research on pseudopods, particularly screening of pseudopod control substances.

  The present inventor has completed the present invention based on these studies. Accordingly, it is an object of the present invention to provide a method for screening an ADAM action inhibitor, a method for retaining a provisional foot, a provisional foot retention agent, a method for screening a provisional foot control substance, and an ADAM action inhibitor.

  In the present application, the action of ADAM (hereinafter also referred to as ADAM action) first refers to an action that is a result of signal transmission by the ADAM signal transmission system. For example, in the above-mentioned ADAM homologues VAP1 and VAP2, “production of intense apoptotic vesicles” and “maintenance of pseudopods” correspond to the action of ADAM. As will be described later, when VAP1 and VAP2 are administered into the mouse skin, bleeding occurs at the administration site, and “bleeding” also corresponds to the action of ADAM. Secondly, when it is caused by a change in the amount or activity of ADAM, it also includes an effect caused by signal amplification or reduction in the signal transduction system. In addition, since VAP1 and VAP2 are substances derived from snake venom, “production of intense apoptotic vesicles” and “maintenance of pseudopods” are also actions of snake venom.

  In the present invention, inhibition of ADAM action (hereinafter also referred to as ADAM action inhibition) is a concept that includes reducing or suppressing ADAM action in addition to inhibiting one or more ADAM actions. Similarly, the inhibition of the action of snake venom is a concept including reducing or suppressing the action of snake venom in addition to inhibiting the action of one or more snake venoms.

(First invention)
The configuration of the first invention of the present application for solving the above problems is as follows.
The substance in which the progress of the ADAM protease reaction is confirmed from the following steps a) and b) and the inhibition of the ADAM action from the following steps a) and c) is determined as an ADAM action inhibitor. This is a screening method for an ADAM action inhibitor.
a) A step of administering or introducing a substance to be screened into a specimen having ADAM protease activity or having ADAM protease activity.
b) A step of confirming the progress of the ADAM protease reaction in the specimen provided with the ADAM protease activity and the screening target substance by the step a).
c) A step of confirming the inhibition of ADAM action in the specimen provided with the protease activity of ADAM and the substance to be screened by the above step a).

  The first invention only needs to include each step a) to c) at least once. For example, each step is performed in the order of a), b), c) or a), c), b), and the reaction progress of the ADAM protease is confirmed from the steps a) and b), and a) and c). The inhibition of ADAM action may be confirmed from the process. In addition, the experimental system was divided into two, the progress of the ADAM protease reaction was confirmed from steps a) and b), and the inhibition of ADAM action was confirmed from steps a) and c). You may determine an action inhibitory substance.

(Second invention)
The configuration of the second invention of the present application for solving the above problem is as follows.
The specimen in the step a) is a cell, an organ, a tissue, or an individual, and the inhibition of ADAM action in the step c) is inhibition of holding of a temporary foot or inhibition of formation of a temporary foot. This is a screening method for an ADAM action inhibitor.

(Third invention)
The configuration of the third invention of the present application for solving the above problem is as follows.
The specimen in the step a) is an organ, tissue, or individual, and the ADAM action-inhibiting substance according to the first invention, wherein the inhibition of ADAM action in the step c) is inhibition of bleeding.

(Fourth invention)
The configuration of the fourth invention of the present application for solving the above problem is as follows.
The screening method for an ADAM action inhibitor according to any one of the first to third inventions, wherein the method for screening an ADAM action inhibitor comprises confirming ADAM specificity from the step a) and the following step d). Is the method.
d) A step of confirming the remaining protease activity of a protease that is not ADAM in a sample to which a screening target substance has been administered or introduced.

  The 4th invention should just contain each process of a) -d) at least once. For example, after the step a), the steps b) to d) are performed in random order, the progress of the ADAM protease reaction is confirmed from the steps a) and b), and the ADAM action from the steps a) and c) is confirmed. Inhibition may be confirmed, and ADAM specificity may be confirmed from steps a) and d). Moreover, it is the same as that of the above-mentioned 1st invention that an experimental system may be divided into plurality.

(Fifth invention)
The configuration of the fifth invention of the present application for solving the above problem is a method for holding a temporary foot using a snake venom for a specimen capable of forming a temporary foot.

(Sixth invention)
The structure of the sixth invention of the present application for solving the above problem is a temporary foot retainer containing snake venom as an active ingredient.

(Seventh invention)
The configuration of the seventh invention of the present application for solving the above problem is as follows.
A treatment process including the following steps e) and f) in random order is performed, and then a substance for which the control of the temporary foot is confirmed by the confirmation process including the following step g) is determined as a temporary foot control substance. This is a screening method for a pseudopod control substance.
e) A step of administering or introducing a snake venom into a cell, organ, tissue, or individual as a specimen.
f) A step of administering or introducing a substance to be screened into a cell, organ, tissue, or individual that is a specimen.
g) A step of confirming the control of the temporary foot.

  The seventh invention only needs to include the steps e) to g) at least once. In the processing step, the steps e) and f) may be performed simultaneously, or one of them may be performed first, and the other one may be performed later. A specimen is administered or introduced with a snake venom and a substance to be screened.

(Eighth invention)
The configuration of the eighth invention of the present application for solving the above problem is as follows.
Effectively one or more compounds selected from a compound having a structure in which a nitrogen atom constituting one amino group in an alkylpolyamine or a derivative thereof is bonded to the isoquinoline skeleton, with or without a linker, and a salt thereof. It is an ADAM action inhibitor contained as a component.

(9th invention)
The configuration of the ninth invention of the present application for solving the above problems is as follows.
The ADAM action inhibitor according to the eighth invention, wherein the linker is selected from sulfur or phosphorus oxoacids.

(10th invention)
The configuration of the tenth invention of the present application for solving the above-described problems is as follows.
The ADAM action inhibitor according to the eighth or ninth invention, wherein the linker or, if no linker is present, an alkylpolyamine or a derivative thereof is bonded to the carbon atom at the 5-position of the isoquinoline skeleton.

(11th invention)
The structure of the eleventh invention of the present application for solving the above problems is as follows.
The ADAM action inhibitor according to any one of the eighth to tenth aspects, wherein the compound that is an active ingredient of the ADAM action inhibitor is a compound having a structure represented by the following general formula (I).
[General Formula (I)]
[In the general formula (I), X represents SO ₂ or PO ₂ H, and an isoquinoline and a nitrogen atom are bonded to the sulfur atom or phosphorus atom;
R ₁ represents the following (A) or (B):
(A) Hydrogen, halogen, hydroxyl group, thiol group, aldehyde group, carboxyl group, cyano group or nitro group.
(B) a hydrocarbon group, an alkoxy group, a thioalkoxy group, an allyloxy group, an allylthio group, an ether group, a thioether group, an ester group or a thioester group, and these groups are linear or branched having 1 to 5 carbon atoms Saturated or unsaturated having and may be halogenated, hydroxylated, thiolated, aldehyded, oxolated, carboxylated, cyanated or nitrated.
R ₂ represents a linear or branched saturated or unsaturated hydrocarbon group having 1 to 5 carbon atoms;
R ₃ represents a substituent represented by the following general formula (II), general formula (III) or general formula (IV).
[General Formula (II)]
(In general formula (II), R ₄ and R ₅ each represent one of the following (c) to (f), and R ₄ and R ₅ may be the same or different.
(C) Hydrogen, halogen, hydroxyl group, thiol group, aldehyde group, thioaldehyde group, carboxyl group, cyano group, azide group, nitro group, pyridyl group, pyrrolidinyl group, thienyl group or furanyl group.
(D) an amino group, an amidino group, a guanidino group, a ureido group, a hydrazino group, a diazenyl group or a hydroxyamino group, and these groups are first saturated or straight-chained or branched or having 1 to 5 carbon atoms It may have an unsaturated hydrocarbon group, and secondly, including the hydrocarbon group described in the first, halogenated, hydroxylated, thiolated, aldehyded, oxoated, carboxylated, cyanated or It may be nitrated.
(E) an aryl group, a naphthyl group or an anthracenyl group with or without a substituent;
(F) A linear, branched or cyclic C1-C20 saturated or unsaturated hydrocarbon group having or not having a substituent, alkoxy group, thioalkoxy group, allyloxy group, allylthio group, ether group, thioether Group, ester group or thioester group.
The “substituent” referred to in (e) and (f) is halogen, hydroxyl group, thiol group, aldehyde group, thioaldehyde group, carboxyl group, cyano group, azide group, nitro group, pyridyl group, pyrrolidinyl group, thienyl group. A furanyl group, a phenyl group, a halogenated phenyl group, a benzyl group, a halogenated benzyl group, a benzoyl group or a halogenated benzoyl group,
Among them, in the “substituent” referred to in (e), in addition to the above options, a hydrocarbon group, an alkoxy group, a thioalkoxy group, an allyloxy group, an allylthio group, an ether group, a thioether group, an ester group, a thioester group These groups have 1 to 5 carbon atoms, are saturated or unsaturated, linear or branched, and are halogenated, hydroxylated, thiolated, aldehyded, oxoated, carboxylated May be cyanated or nitrated. )
[General Formula (III)]
(In General Formula (III), R ₆ and R ₇ are groups that R ₆ and R ₇ in R ₁ and R ₅ in General Formula (II) can form a bond with each other at one or more positions, respectively. Show).
[General Formula (IV)]
(In the general formula (IV), R ₈ represents any one of the following (g) to (g).
(G) Hydrogen, halogen, hydroxyl group, thiol group, pyridyl group, pyrrolidinyl group, thienyl group, or furanyl group.
(H) an aryl group, a naphthyl group, or an anthracenyl group with or without a substituent;
(I) A linear, branched or cyclic C1-C20 saturated or unsaturated hydrocarbon group having or not having a substituent, alkoxy group, thioalkoxy group, allyloxy group, allylthio group, ether group, thioether Group, ester group or thioester group.
The “substituents” referred to in (T) and (L) are halogen, hydroxyl group, thiol group, aldehyde group, thioaldehyde group, carboxyl group, cyano group, azide group, nitro group, pyridyl group, pyrrolidinyl group, thienyl group. A furanyl group, a phenyl group, a halogenated phenyl group, a benzyl group, a halogenated benzyl group, a benzoyl group or a halogenated benzoyl group,
Among them, in the “substituent” referred to in (h), in addition to the above options, further, a hydrocarbon group, an alkoxy group, a thioalkoxy group, an allyloxy group, an allylthio group, an ether group, a thioether group, an ester group, and a thioester group These groups have 1 to 5 carbon atoms, are saturated or unsaturated, linear or branched, and are halogenated, hydroxylated, thiolated, aldehyded, oxoated, carboxylated May be cyanated or nitrated. )]

(Twelfth invention)
The configuration of the twelfth invention of the present application for solving the above-described problems is as follows.
One nitrogen atom of a nitrogen-containing 6-membered ring structure or a derivative thereof containing two nitrogen atoms in the 1-position and 4-position relative to each other with or without a linker with respect to the isoquinoline skeleton It is an ADAM action inhibitor which contains as an active ingredient 1 or more types chosen from the compound which has the structure which bonded | bonded, and its salt.

(13th invention)
The configuration of the thirteenth invention of the present application for solving the above problems is as follows.
The ADAM action inhibitor according to the twelfth aspect, wherein the linker is selected from sulfur or phosphorus oxoacids.

(14th invention)
The configuration of the fourteenth invention of the present application for solving the above problem is as follows.
The ADAM according to the twelfth or thirteenth invention, wherein the linker, or when no linker is present, the nitrogen-containing six-membered ring structure or derivative thereof is bonded to the carbon atom at the 5-position of the isoquinoline skeleton. It is an action inhibitor.

(15th invention)
The configuration of the fifteenth aspect of the present invention for solving the above problem is as follows.
The ADAM action inhibitor according to any one of the twelfth to fourteenth inventions, wherein the compound that is an active ingredient of the ADAM action inhibitor is a compound having a structure represented by the following general formula (V).
[General formula (V)]
[In the general formula (V), X represents SO ₂ or PO ₂ H, and an isoquinoline and a nitrogen atom are bonded to the sulfur atom or phosphorus atom;
R ₁₁ represents a substituent represented by the following general formula (VI), general formula (VII) or general formula (VIII).
[General formula (VI)]
(In the general formula _{(VI), R} 21 _{~R 29} may be either a single bond, a double bond listed _below, if R 21 _{to R 29} are bonded by a single _{bond, R} 21 to R ₂₉ represents one of the following (i) to (ha), and R _{21 to} R ₂₉ may be the same as or different from each other.
(Ii) Hydrogen, halogen, hydroxyl group, thiol group, aldehyde group, thioaldehyde group, carboxyl group, cyano group, azide group, or nitro group.
(B) an amino group, an amidino group, a guanidino group, a ureido group, a hydrazino group, a diazenyl group or a hydroxyamino group, and these groups are primarily saturated or straight-chain or branched having 1 to 5 carbon atoms or It may have an unsaturated hydrocarbon group, and secondly, including the hydrocarbon group described in the first, halogenated, hydroxylated, thiolated, aldehyded, oxoated, carboxylated, cyanated or It may be nitrated.
(Ha) a linear, branched or cyclic C1-C10 saturated or unsaturated hydrocarbon group having or not having a substituent, alkoxy group, thioalkoxy group, allyloxy group, allylthio group, ether group, thioether Group, ester group or thioester group.
When R ₂₁ and R ₂₂ , R ₂₃ and R ₂₄ , R ₂₆ and R ₂₇ , R ₂₈ and R ₂₉ are bonded by a double bond, R ₂₁ and R ₂₂ , R ₂₃ and R ₂₄ , R ₂₆ and R ₂₇ , R ₂₈ and R ₂₉ each represent one of the following (ni) to (e), and R ₂₁ and R ₂₂ , R ₂₃ and R ₂₄ , R ₂₆ and R ₂₇ , R ₂₈ and R ₂₉ are respectively They may be the same or different.
(Ii) an oxo group or a thioxy group.
(E) A hydroxyimino group or a hydrazono group with or without a substituent.
Note that the “substituent” in (ha) and (e) is a halogen, a hydroxyl group, a thiol group, an aldehyde group, a thioaldehyde group, or a carboxyl group. )
[General formula (VII)]
(In the general formula _{(VII), R} 31 _{~R 37} is either a single bond may be a double bond listed _below, if R 31 _{to R 37} are bonded by a single _{bond, R} 31 to R ₃₇ represents any one of (i) to (ha) in the general formula (VI), and R _{31 to} R ₃₇ may be the same as or different from each other.
When R ₃₄ and R ₃₅ , R ₃₆ and R ₃₇ are bonded by a double bond, R ₃₄ and R ₃₅ , R ₃₆ and R ₃₇ are respectively represented by (ni) to (v) in the general formula (VI). R ₃₄ and R ₃₅ , R ₃₆ and R ₃₇ may be the same or different.
The “substituent” is the same as in the general formula (VI). )
[General formula (VIII)]
(In the general formula (VIII), R _{41 to} R ₄₅ each represent any one of (i) to (ha) in the general formula (VI), and R _{41 to} R ₄₅ may be the same as each other. , May be different.
The “substituent” is the same as in the general formula (VI). )]

(16th invention)
The configuration of the sixteenth invention of the present application for solving the above problem is as follows.
The substance selected using the ADAM action inhibitor screening method according to any one of the first to fourth inventions is a compound corresponding to the following general formula (IX) or general formula (X), and these It is an ADAM action inhibitor containing at least one selected from a compound and a salt thereof as an active ingredient.
[General Formula (IX)]
(In General Formula (IX), X represents SO ₂ or PO ₂ H, and an isoquinoline and a nitrogen atom are bonded to the sulfur atom or phosphorus atom; R ₅₁ and R ₅₂ are each hydrogen or an optional substituent; is there.)
[General Formula (X)]
(In the general formula (X), X represents SO ₂ or PO ₂ H, and an isoquinoline and a nitrogen atom are bonded to the sulfur atom or phosphorus atom; R _{61 to} R ₆₉ are each hydrogen or an arbitrary substituent. is there.)

(First invention)
According to the first invention, a screening method for a substance that acts downstream of ADAM and inhibits ADAM action is provided.

(Second invention)
According to the second invention, there is provided a screening method for an ADAM action inhibitor using as an index the activity of inhibiting the retention of pseudopods or the inhibition of formation of pseudopods. VAP, a homologue of ADAM, causes apoptosis of vascular endothelial cells, and a temporary foot is formed and maintained in the apoptosis. Therefore, since the formation of the temporary foot and the retention of the temporary foot are ADAM action, the substance that inhibits the retention of the temporary foot and the formation of the temporary foot is a substance that inhibits the ADAM action.

(Third invention)
According to the third invention, there is provided a screening method for an ADAM action inhibitor using as an index the inhibition activity of bleeding, particularly the activity of inhibiting bleeding caused by acting on blood vessels rather than the blood coagulation system. VAM, an ADAM homolog, does not degrade the intercellular matrix collagen, but induces apoptosis of vascular endothelial cells. Furthermore, it has been conventionally thought that proteases bleed due to degradation of vascular proteins. However, due to a new finding by the present inventor that bleeding is inhibited even if the protease reaction of VAP progresses, downstream of ADAM, It has become clear that the signal transmission system is involved in bleeding, particularly bleeding that is caused by blood vessels. Therefore, since bleeding caused by blood vessels has an ADAM action, a substance that works downstream of ADAM and inhibits bleeding inhibits the ADAM action.

(Fourth invention)
According to the fourth invention, a method for screening an ADAM action inhibitor having ADAM specificity is provided. Conventionally, it has been difficult to create a metalloprotease-specific inhibitor of ADAM. However, a substance that acts downstream of ADAM to inhibit ADAM action and has ADAM specificity is a metalloprotease-specific inhibitor of ADAM. It can be expected to have an approximate effect. In other words, the remaining protease activity of a protease that is not ADAM means fewer side effects.

(5th invention, 6th invention)
According to the fifth invention, a useful temporary foot holding method is provided. The sixth invention provides a useful temporary foot retainer.
The inventor of the present application found that, in the apoptosis caused by snake venom, in addition to the formation of a temporary foot, the temporary foot was retained, and paid particular attention to this point. Usually, the temporary foot dynamically changes such as formation / disappearance. The creation of a static state of the temporary foot provides a way of researching the structure of the temporary foot, studying the effect of maintaining the temporary foot, and developing a control agent for the temporary foot.
In addition to snake venom, ADAM is used to form and hold a temporary foot. Therefore, even when ADAM is used, the same effect as that obtained when snake venom is used can be obtained.

(Seventh invention)
According to the seventh aspect of the invention, a method for screening a useful provisional foot control substance is provided. While the pseudopods dynamically change such as formation and disappearance, the snake venom holds the pseudopods, that is, can create a static state of the pseudopods. Thereby, first, it becomes possible to keep the screening system of the control substance stable for a long time. Further, the change from the static state can be recognized as a temporary foot control effect of the screening target substance.
In addition to snake venom, ADAM is used to form and hold a temporary foot. Therefore, even when ADAM is used, the same effect as that obtained when snake venom is used can be obtained.

(8th invention to 11th invention, 12th invention to 15th invention and 16th invention)
A useful ADAM action inhibitor is provided by the eighth invention to the eleventh invention, the twelfth invention to the fifteenth invention, and the sixteenth invention. These ADAM action inhibitors inhibit ADAM action without inhibiting ADAM protease reaction. That is, it can be considered that it acts on signal transmission downstream of ADAM. This eliminates the problem of side effects in conventional ADAM inhibitors.
Furthermore, when the protease activity of other proteins such as MMP remains, a highly specific ADAM inhibitor is provided.
In addition, since there were few effective ADAM inhibitors in the past, it was conventionally treated in diseases involving ADAM action such as a snake bite bleeding inhibitor that was suggested but not found to be involved in ADAM. A therapeutic for a disease for which no drug was present is provided.

It is a bar graph which shows the bleeding amount about Chemical | chemical_formation 1-5 and chemical | medical_expression 11-13 with the relative ratio with respect to control (a control display is abbreviate | omitted). It is a bar graph which shows the bleeding amount about Chemical formula 14 and Chemical formula 15 by relative ratio to control. It is a bar graph which shows the bleeding amount about Chemical formula 11 and Chemical formula 16-Chemical formula 19 by the relative ratio with respect to control. It is a photograph which shows the administration result of Chemical formula 4 and Chemical formula 3. It is a photograph which shows the administration result of Chemical formula 11 and Chemical formula 16-Chemical formula 19. The result of having investigated the influence which Chemical formula 4 has on the reaction progress of VAP is shown. The process of apoptosis by survival factor removal and retention of temporary limbs by snake venom ADAM is shown over time. 6 is a photograph showing the influence of Chemical Formula 6. VAP1 indicates that it does not degrade collagen. Figure 2 shows a speculative model for bleeding caused by snake venom ADAM. The influence of an ADAM action inhibitor on the action of collagenase is shown. The formation and maintenance of a protrusion-like temporary foot due to snake venom and the suppression of temporary foot formation due to chemical formula 2 are shown.

  Next, modes for carrying out the present invention will be described including the best mode.

[Screening method of ADAM action inhibitor]
In the screening method for an ADAM action inhibitor of the present invention, the progress of the ADAM protease reaction is confirmed from the following steps a) and b), and inhibition of the ADAM action is confirmed from the following steps a) and c). The determined substance is determined as an ADAM action inhibitor.
a) A step of administering or introducing a substance to be screened into a specimen having ADAM protease activity or having ADAM protease activity.
b) A step of confirming the progress of the ADAM protease reaction in the specimen provided with the ADAM protease activity and the screening target substance by the step a).
c) A step of confirming the inhibition of ADAM action in the specimen provided with the protease activity of ADAM and the substance to be screened by the above step a).

  In the screening method for an ADAM action inhibitor of the present invention, the specimen is not particularly limited. In addition to cells, organs, tissues, or individuals, in vitro systems that have constructed or reproduced ADAM signal transduction systems can also be used as specimens. Among these, cells, organs, tissues, and individuals can be exemplified as preferred specimens, and cells and individuals can be exemplified as further preferred specimens.

  Although a cell is not specifically limited, A vascular endothelial cell, a white blood cell, etc. can be illustrated.

  Although an organ is not specifically limited, A blood vessel, a brain, a liver, a lung, etc. can be illustrated.

  The tissue is not particularly limited, and examples thereof include epithelial tissue, connective tissue, and muscle tissue.

Although an individual is not specifically limited, A mouse | mouth, a rat, a human etc. can be illustrated.
When the temporary foot is a temporary foot with a podosome as described later, the specimen is preferably a leukocyte, macrophage, vascular endothelial cell, or osteoclast, and more preferably a vascular endothelial cell or macrophage. .

  The above-described specimen is required to have ADAM protease activity or ADAM protease activity. In other words, the sample needs to have ADAM protease activity, but the order in which the sample is administered or introduced into the sample and the sample has ADAM protease activity is not particularly limited.

  There is no particular limitation on the method by which the specimen has ADAM protease activity. ADAM itself may be added to, mixed with, or administered to the specimen, or ADAM may be introduced into the specimen using gene expression. When the specimen itself has ADAM protease activity and has ADAM action, it is not necessary to manipulate the specimen.

  ADAM is not particularly limited, but snake venom ADAM, human ADAM, mouse ADAM can be exemplified, snake venom ADAM, human ADAM can be preferably exemplified, and snake venom ADAM is particularly preferably hemorrhagic snake venom ADAM. It can be illustrated. Snake venom ADAM is a concept that includes hemorrhagic snake toxins such as VAP1 and VAP2.

  The ADAM action is not particularly limited, and examples thereof include hemorrhage, vascular retraction, provisional foot formation, provisional foot retention, and formation of severe apoptotic vesicles.

  The inhibition of ADAM action is not particularly limited, and examples thereof include bleeding inhibition, blood vessel strengthening, provisional foot formation inhibition, provisional foot retention inhibition, and apoptosis suppression. Bleeding inhibition, provisional foot holding inhibition, and provisional foot formation inhibition can be preferably exemplified, and bleeding inhibition and provisional foot retention inhibition can be particularly preferably exemplified.

  A temporary foot refers to a site formed by a cytoplasmic protrusion. Usually, the temporary foot dynamically changes such as formation / disappearance. The temporary foot may be a temporary foot with a podosome. Podosomes are known to release proteases for invasion.

  Inhibition of formation of a temporary foot is a concept that includes the number of temporary feet formed and the suppression of extension / expansion of temporary feet, in addition to not forming temporary feet.

  Holding the temporary foot is a concept including maintaining the protrusion of the formed temporary foot and prolonging the time during which the temporary foot is formed. In apoptosis caused by removal of a survival factor, a temporary foot is formed when apoptosis begins, whereas the temporary foot disappears 1 hour after the formation. On the other hand, in snake venom, once formed temporary foot is maintained not only for 1 hour but also for more than 6 hours. In other words, holding the temporary foot includes extending the time during which the temporary foot is formed.

  Inhibition of the retention of the temporary foot means that the formed temporary foot maintains the protrusion and that the time during which the temporary foot is formed is prolonged. That is, as a result of the holding inhibition, the temporary foot again shows a dynamic change. From the viewpoint of screening for an ADAM action inhibitor, it is preferable that the temporary foot disappears as a result of the retention of the temporary foot being inhibited.

  In the method for screening for an ADAM action inhibitor, it is sufficient that the temporary foot of the screening system is maintained for at least 1 hour. For example, it can be realized by using snake venom ADAM, VAP1 or VAP2. If the temporary foot disappears within one hour in the above example under the influence of the screening target substance before the retention time elapses, it can be said that the temporary foot retention is inhibited. On the other hand, regarding the temporary foot holding method and the temporary foot holding agent described later, the holding time is preferably 1 hour or longer, and more preferably 3 hours or longer, 6 hours or longer, 24 hours or longer, etc. preferable. The holding time of the temporary foot can be adjusted by a substantial ADAM dose.

  Examples of the method for confirming the inhibition of the formation and retention of the temporary foot include time-lapse observation using a microscope. For example, when VAP is administered to cultured cells, the behavior of the cell may be observed, and when VAP is administered to an individual mouse to have ADAM protease activity, the administration site of VAP is excised. By observing the excision site using a microscope, it is possible to confirm the inhibition of provisional foot formation or inhibition of provisional foot retention.

  When the inhibition of the ADAM action is inhibition of bleeding, the inhibition of bleeding includes not only the prevention of bleeding, but also the concept of suppressing bleeding such as suppressing the amount of bleeding. In this case, the specimen is particularly preferably an individual including a mouse. For the determination of bleeding inhibition, for example, the method described in Examples described later is preferable. When bleeding inhibitory activity is used as an index, it is preferable to separate the screening target substance administration group and the control group. Moreover, it is preferable that the substance to be screened and the protease activity of ADAM are provided almost simultaneously.

  That is, the ADAM action inhibitory substance is a concept including a hemorrhage inhibitory substance, a blood vessel strengthening substance, a pseudopodia formation inhibitor, a pseudopodia retention inhibitor, an apoptotic vesicle formation inhibitor, and the like.

  The substance to be screened is not particularly limited. Preferable examples include compounds, proteins, nucleic acids, antibodies, antigens, carbohydrate lipids, etc. More preferable examples include compounds, proteins, nucleic acids, and antibodies, and particularly preferable examples include compounds and proteins. can do.

  The method for administering / introducing these screening substances is not particularly limited. In administration, addition, mixing, oral administration, transdermal administration, vascular administration, subcutaneous injection, intraperitoneal administration, intradermal injection, intramuscular injection can be exemplified, and in introduction, a specimen is provided with a gene, or A screening target substance can be expressed using a gene provided in advance or can be produced by metabolism or the like.

  The method for confirming the progress of the ADAM protease reaction is not particularly limited. Preferably, a method for detecting the result of the protease reaction, a method for confirming a decrease in the substrate of the protease reaction, and the like can be exemplified.

The screening method for an ADAM action inhibitor according to the present invention may further include confirming ADAM specificity from the step a) and the following step d).
d) A step of confirming the remaining protease activity of a protease that is not ADAM in a sample to which a screening target substance has been administered or introduced.

  Remaining protease activity of proteases other than ADAM is a concept that includes a decrease in the protease activity, but does not include loss of activity.

  The protease other than ADAM is not particularly limited, but MMP, collagenase, aggrecanase, elastase and the like can be preferably exemplified. Moreover, it is preferable that a sample is provided with any one or more of these enzymes.

[Method of holding temporary foot, temporary foot holding agent]
ADAM is used as the temporary foot holding method and temporary foot holding agent of the present invention. ADAM itself may be added to, mixed with, or administered to the specimen, or ADAM may be introduced into the specimen using gene expression. ADAM is not particularly limited, but snake venom ADAM, human ADAM, and mouse ADAM can be exemplified, and snake venom ADAM and human ADAM can be preferably exemplified. As human ADAM, ADAM28, ADAM12, and ADAM9 are preferable, and ADAM28 is more preferable.
The temporary foot holding method and temporary foot holding agent of the present invention may form and hold temporary feet with podosomes. Podosomes are known to release proteases for invasion. There are also provided ways to study the structure of the pseudopods with podosomes, the effect of maintaining the pseudopods, and the development of a control agent for the pseudopods.

  Although snake venom is not particularly limited, hemorrhagic snake venom is preferred. As the hemorrhagic snake venom, rattlesnake venom, hub venom, viper venom and the like can be preferably exemplified, and rattlesnake venom can be particularly preferably exemplified. Furthermore, the snake venom is a concept including VAP1 and VAP2 which are hemorrhagic snake toxins.

  The snake venom may be obtained from a snake that is an individual such as a rattlesnake, or a commercially available snake venom may be purchased. Further, VAP1 and VAP2 can be obtained from the rattlesnake venom (purchased by Sigma, Wako Pure Chemicals, etc.) by the method described in Non-Patent Document 4 described above. Human ADAM is available from Enzo Life Sciences, R & D Systems, and the like.

  The temporary foot and the holding of the temporary foot are the same as the contents described in the screening method for the ADAM action inhibitor.

  The specimen capable of forming a temporary foot is not particularly limited, but is preferably a specimen having cells, and examples thereof include cells, organs, tissues, and individuals. These are the same as described in the above-mentioned screening method for ADAM action inhibitor. In the case of a method for holding a temporary foot with podosome, the specimen is preferably a leukocyte, macrophage, vascular endothelial cell, or osteoclast, and more preferably a vascular endothelial cell or macrophage.

[Screening method for temporary foot control substances]
The screening method for a provisional foot control substance according to the present invention includes a processing step including the following steps e) and f) in random order, and then confirms the control of the temporary foot by a confirmation step including the following step g): The determined substance is determined as a temporary foot control substance.
e) A step of administering or introducing ADAM to a cell, organ, tissue, or individual as a specimen.
f) A step of administering or introducing a substance to be screened into a cell, organ, tissue, or individual that is a specimen.
g) A step of confirming the control of the temporary foot.

The provisional foot, cell, organ, tissue, individual, ADAM, screening target substance, administration, and introduction are the same as those described in the above-mentioned screening method for ADAM action inhibitor. About the snake venom, it is the same as that of the content as described in the holding | maintenance method of a said temporary foot, and a temporary foot holding agent. The specimen is preferably a cell or an individual, and a cell is particularly preferred. The cell is required to be able to form a temporary foot, but its ability is potentially sufficient.
In addition, the temporary foot is preferably a temporary foot with a podosome. It is preferable that the specimen can form a temporary foot with a podosome. Leukocytes, macrophages, vascular endothelial cells, and osteoclasts are preferable, and vascular endothelial cells and macrophages are more preferable. When human ADAM is used, ADAM28, ADAM12 and ADAM9 are preferable, and ADAM28 and ADAM12 are more preferable. Human ADAM is available from Enzo Life Sciences, R & D Systems, and the like.

  The control of the temporary foot refers to controlling the dynamic change of the temporary foot that dynamically changes such as formation / disappearance. Preferable examples include inhibition of formation of a temporary foot, inhibition of retention of a temporary foot, stretching / deformation or disappearance of the temporary foot, a change in adhesion of the temporary foot, and the like.

  In other words, a provisional foot control substance is a substance that controls a dynamic change of the temporary foot. For example, a provisional foot formation inhibitor, a temporary foot retention inhibitor, a temporary foot extension material, a temporary foot deforming material, a temporary foot disappearance material, a temporary foot adhesion change material, etc. it can.

  Examples of the method for confirming the control of the temporary foot include time-lapse observation using a microscope as described above, detection of temporary foot-holding cells using antibodies (including staining and flow cytometry), enzymatic detection, and the like. Can also be confirmed. Actin staining and phosphatidylinositol diphosphate (PIP2) antibody staining can be exemplified.

[ADAM inhibitor]
The ADAM action inhibitor is not particularly limited, and examples thereof include a bleeding inhibitor, a blood vessel strengthening agent, a provisional foot formation inhibitor, and a provisional foot retention inhibitor. A bleeding inhibitor, a temporary foot formation inhibitor, Any of the foot retention inhibitors can be preferably exemplified. As a bleeding inhibitor, an antihemorrhagic snake poison bite agent can be preferably exemplified.

  The salt is not particularly limited, but a pharmaceutically acceptable salt is preferable. Pharmaceutically acceptable salts include alkali metal salts (eg, lithium salts, sodium salts, potassium salts), alkaline earth metal salts (eg, magnesium salts, calcium salts), ammonium salts, choline salts, organic amine salts, Examples thereof include iron salts, zinc salts, copper salts, manganese salts, and aluminum salts. Of these, alkali metal salts, alkaline earth metal salts, ammonium salts, choline salts, and organic amine salts are preferred. Furthermore, sodium salt, potassium salt, calcium salt, ammonium salt, choline salt, and organic amine salt are particularly preferable.

  The ADAM inhibitor of the present invention includes a compound having a structure in which a nitrogen atom constituting one amino group in an alkylpolyamine or a derivative thereof is bonded to an isoquinoline skeleton with or without a linker interposed therebetween, and It contains at least one selected from salts as an active ingredient.

  Alkyl polyamines and derivatives thereof have two or more amino groups. That is, “poly” here means “two or more”. The amino group is a concept including an amino group having a substituent.

  The isoquinoline skeleton has at least isoquinoline, and an alkylpolyamine, an alkylpolyamine derivative, or a linker to which any of them is bonded to the isoquinoline in the skeleton.

  The 5-position of isoquinoline is a site to which X is bonded in the general formula (I) described later.

The linker is not particularly limited but is preferably selected from sulfur or phosphorus oxoacids. The oxo acid of sulfur (S) or phosphorus (P) is not particularly limited, and includes sulfuric acid, sulfinic acid, sulfenic acid, phosphoric acid, phosphorous acid and the like. When these become linkers, a sulfur atom (S), a phosphorus atom (P), or an oxygen atom (O) is bonded to an alkylpolyamine or a derivative thereof, and an isoquinoline skeleton. For example, if the sulfenic acid linker, -SO 2 _- sulfur atom next sulfenic acid linking alkyl polyamine or a derivative thereof and the isoquinoline skeleton.

Furthermore, it is preferable that the compound which is an active ingredient of an ADAM action inhibitor is a compound having a structure represented by the following general formula (I).
[General Formula (I)]
[In the general formula (I), X represents SO ₂ or PO ₂ H, and an isoquinoline and a nitrogen atom are bonded to the sulfur atom or phosphorus atom;
R ₁ represents the following (A) or (B):
(A) Hydrogen, halogen, hydroxyl group, thiol group, aldehyde group, carboxyl group, cyano group or nitro group.
(B) a hydrocarbon group, an alkoxy group, a thioalkoxy group, an allyloxy group, an allylthio group, an ether group, a thioether group, an ester group or a thioester group, and these groups are linear or branched having 1 to 5 carbon atoms Saturated or unsaturated having and may be halogenated, hydroxylated, thiolated, aldehyded, oxolated, carboxylated, cyanated or nitrated.
R ₂ represents a linear or branched saturated or unsaturated hydrocarbon group having 1 to 5 carbon atoms;
R ₃ represents a substituent represented by the following general formula (II), general formula (III) or general formula (IV).
[General Formula (II)]
(In general formula (II), R ₄ and R ₅ each represent one of the following (c) to (f), and R ₄ and R ₅ may be the same or different.
(C) Hydrogen, halogen, hydroxyl group, thiol group, aldehyde group, thioaldehyde group, carboxyl group, cyano group, azide group, nitro group, pyridyl group, pyrrolidinyl group, thienyl group or furanyl group.
(D) an amino group, an amidino group, a guanidino group, a ureido group, a hydrazino group, a diazenyl group or a hydroxyamino group, and these groups are first saturated or straight-chained or branched or having 1 to 5 carbon atoms It may have an unsaturated hydrocarbon group, and secondly, including the hydrocarbon group described in the first, halogenated, hydroxylated, thiolated, aldehyded, oxoated, carboxylated, cyanated or It may be nitrated.
(E) an aryl group, a naphthyl group or an anthracenyl group with or without a substituent;
(F) A linear, branched or cyclic C1-C20 saturated or unsaturated hydrocarbon group having or not having a substituent, alkoxy group, thioalkoxy group, allyloxy group, allylthio group, ether group, thioether Group, ester group or thioester group.
The “substituent” referred to in (e) and (f) is halogen, hydroxyl group, thiol group, aldehyde group, thioaldehyde group, carboxyl group, cyano group, azide group, nitro group, pyridyl group, pyrrolidinyl group, thienyl group. A furanyl group, a phenyl group, a halogenated phenyl group, a benzyl group, a halogenated benzyl group, a benzoyl group or a halogenated benzoyl group,
Among them, in the “substituent” referred to in (e), in addition to the above options, a hydrocarbon group, an alkoxy group, a thioalkoxy group, an allyloxy group, an allylthio group, an ether group, a thioether group, an ester group, a thioester group These groups have 1 to 5 carbon atoms, are saturated or unsaturated, linear or branched, and are halogenated, hydroxylated, thiolated, aldehyded, oxoated, carboxylated May be cyanated or nitrated. )

  In the above (f), “having or not having a substituent ... saturated or unsaturated” relates to the subsequent “hydrocarbon group,... Or thioester group”.

[General Formula (III)]
(In General Formula (III), R ₆ and R ₇ are groups that R ₆ and R ₇ in R ₁ and R ₅ in General Formula (II) can form a bond with each other at one or more positions, respectively. Show).
[General Formula (IV)]
(In the general formula (IV), R ₈ represents any one of the following (g) to (g).
(G) Hydrogen, halogen, hydroxyl group, thiol group, pyridyl group, pyrrolidinyl group, thienyl group, or furanyl group.
(H) an aryl group, a naphthyl group, or an anthracenyl group with or without a substituent;
(I) A linear, branched or cyclic C1-C20 saturated or unsaturated hydrocarbon group having or not having a substituent, alkoxy group, thioalkoxy group, allyloxy group, allylthio group, ether group, thioether Group, ester group or thioester group.
The “substituents” referred to in (T) and (L) are halogen, hydroxyl group, thiol group, aldehyde group, thioaldehyde group, carboxyl group, cyano group, azide group, nitro group, pyridyl group, pyrrolidinyl group, thienyl group. A furanyl group, a phenyl group, a halogenated phenyl group, a benzyl group, a halogenated benzyl group, a benzoyl group or a halogenated benzoyl group,
Among them, in the “substituent” referred to in (h), in addition to the above options, further, a hydrocarbon group, an alkoxy group, a thioalkoxy group, an allyloxy group, an allylthio group, an ether group, a thioether group, an ester group, and a thioester group These groups have 1 to 5 carbon atoms, are saturated or unsaturated, linear or branched, and are halogenated, hydroxylated, thiolated, aldehyded, oxoated, carboxylated May be cyanated or nitrated. )]

  In the above (i), “with or without a substituent... Saturated or unsaturated” relates to the subsequent “hydrocarbon group,... Or thioester group”.

In the general formula (III), the phrase “R ₆ and R ₇ can form a bond with each other at one or more sites” means that a bond can be formed by dehydrogenation, dehydration, or the like. A bond may be formed using an enzyme or the like, or a bond may be formed through a chemical reaction.

  The above-mentioned “saturated” and “unsaturated” represent a hydrocarbon moiety. For example, “unsaturated alkoxy group” means having an unsaturated bond at an alkyl site in an alkoxy group.

In the general formula (I), X is preferably SO ₂ .

In the general formula (I), R ₁ is preferably hydrogen, halogen, hydroxyl group, aldehyde group or carboxyl group, more preferably hydrogen, hydroxyl group or aldehyde group, particularly preferably hydrogen.

R ₂ is preferably a linear or branched saturated or unsaturated hydrocarbon group having 1 to 3 carbon atoms, particularly preferably a saturated hydrocarbon group having 2 carbon atoms (—CH ₂ CH ₂ —).

R ₃ is preferably a substituent represented by the above general formula (II) or (III), and particularly preferably a substituent represented by the general formula (II).

In the above general formula (II), R ₄ and R ₅ are preferably the groups (c), (d) or (f) shown below.
(C) Hydrogen, hydroxyl group, aldehyde group or carboxyl group.
(D) Amidino group, guanidino group or ureido group.
(F) A linear or branched saturated or unsaturated hydrocarbon group having 1 to 10 carbon atoms, which may or may not have a substituent.
In addition, the substituent referred to in (f) is a phenyl group, a halogenated phenyl group, a benzyl group, a halogenated benzyl group, a benzoyl group or a halogenated benzoyl group.

In the general formula (II), R ₄ is more preferably hydrogen, a hydroxyl group, an aldehyde group or a carboxyl group, and R ₅ is a group (ha), (d) or (f) shown below.
(C) Hydrogen.
(D) Amidino group, guanidino group or ureido group.
(F) A saturated or unsaturated hydrocarbon group having 1 to 5 carbon atoms, preferably 1 to 3 carbon atoms, which may or may not have a substituent.
In addition, the substituent referred to in (f) is a phenyl group, a halogenated phenyl group, a benzyl group, a halogenated benzyl group, a benzoyl group or a halogenated benzoyl group.

In the above general formula (IV), R ₈ is hydrogen, halogen, hydroxyl group, thiol group, pyridyl group, pyrrolidinyl group, thienyl group, or furanyl group, aryl group with or without substituent, naphthyl group, Alternatively, an anthracenyl group is preferable, and hydrogen, halogen, hydroxyl group, thiol group, pyridyl group, pyrrolidinyl group, thienyl group, or furanyl group is particularly preferable. The preferred substituent is halogen, hydroxyl group, thiol group, aldehyde group, thioaldehyde group, carboxyl group, pyridyl group, pyrrolidinyl group, thienyl group, furanyl group, phenyl group, halogenated phenyl group, benzyl group, halogenated benzyl group. Benzoyl group or halogenated benzoyl group.

  The above “having or not having a substituent” relates to the subsequent “aryl group, naphthyl group, or anthracenyl group”.

  More specifically, it is preferable that the compound which is an active ingredient of an ADAM action inhibitor is the following Chemical Formula 1 to Chemical Formula 5 used in Examples described later. The compounds of Chemical Formulas 1 to 5 can be obtained by purchasing commercial products as appropriate.

  Moreover, the ADAM action inhibitor of the present invention is a nitrogen-containing 6-membered ring containing two nitrogen atoms in a 1-position and 4-position relationship with respect to the isoquinoline skeleton, with or without a linker. One or more selected from a compound having a structure in which one of the nitrogen atoms of the structure or its derivative is bonded and a salt thereof may be contained as an active ingredient.

  The description regarding the isoquinoline skeleton, linker, salt and ADAM action inhibitor is the same as described above. However, the portion corresponding to “alkyl polyamine” is “nitrogen-containing 6-membered ring structure”.

  The nitrogen-containing 6-membered ring structure contains two nitrogen atoms in a 1-position and 4-position relationship with each other. That is, when one nitrogen atom is positioned at the 1st position, the other nitrogen atom is positioned at the 4th position (in the case of a benzene ring, it is in the para position).

  The 5-position of isoquinoline is a site to which X is bonded in the general formula (V) described later.

Furthermore, it is also preferable that the compound which is an active ingredient of an ADAM action inhibitor is a compound having a structure represented by the following general formula (V).
[General formula (V)]
[In the general formula (V), X represents SO ₂ or PO ₂ H, and an isoquinoline and a nitrogen atom are bonded to the sulfur atom or phosphorus atom;
R ₁₁ represents a substituent represented by the following general formula (VI), general formula (VII) or general formula (VIII).
[General formula (VI)]
(In the general formula _{(VI), R} 21 _{~R 29} may be either a single bond, a double bond listed _below, if R 21 _{to R 29} are bonded by a single _{bond, R} 21 to R ₂₉ represents one of the following (i) to (ha), and R _{21 to} R ₂₉ may be the same as or different from each other.
(Ii) Hydrogen, halogen, hydroxyl group, thiol group, aldehyde group, thioaldehyde group, carboxyl group, cyano group, azide group, or nitro group.
(B) an amino group, an amidino group, a guanidino group, a ureido group, a hydrazino group, a diazenyl group or a hydroxyamino group, and these groups are primarily saturated or straight-chain or branched having 1 to 5 carbon atoms or It may have an unsaturated hydrocarbon group, and secondly, including the hydrocarbon group described in the first, halogenated, hydroxylated, thiolated, aldehyded, oxoated, carboxylated, cyanated or It may be nitrated.
(Ha) a linear, branched or cyclic C1-C10 saturated or unsaturated hydrocarbon group having or not having a substituent, alkoxy group, thioalkoxy group, allyloxy group, allylthio group, ether group, thioether Group, ester group or thioester group.
In the above (ha), “having or not having a substituent ... saturated or unsaturated” relates to the subsequent “hydrocarbon group,... Or thioester group”.
When R ₂₁ and R ₂₂ , R ₂₃ and R ₂₄ , R ₂₆ and R ₂₇ , R ₂₈ and R ₂₉ are bonded by a double bond, R ₂₁ and R ₂₂ , R ₂₃ and R ₂₄ , R ₂₆ and R ₂₇ , R ₂₈ and R ₂₉ each represent one of the following (ni) to (e), and R ₂₁ and R ₂₂ , R ₂₃ and R ₂₄ , R ₂₆ and R ₂₇ , R ₂₈ and R ₂₉ are respectively They may be the same or different.
(Ii) an oxo group or a thioxy group.
(E) A hydroxyimino group or a hydrazono group with or without a substituent.
Note that the “substituent” in (ha) and (e) is a halogen, a hydroxyl group, a thiol group, an aldehyde group, a thioaldehyde group, or a carboxyl group. )
[General formula (VII)]
(In the general formula _{(VII), R} 31 _{~R 37} is either a single bond may be a double bond listed _below, if R 31 _{to R 37} are bonded by a single _{bond, R} 31 to R ₃₇ represents any one of (i) to (ha) in the general formula (VI), and R _{31 to} R ₃₇ may be the same as or different from each other.
When R ₃₄ and R ₃₅ , R ₃₆ and R ₃₇ are bonded by a double bond, R ₃₄ and R ₃₅ , R ₃₆ and R ₃₇ are respectively represented by (ni) to (v) in the general formula (VI). R ₃₄ and R ₃₅ , R ₃₆ and R ₃₇ may be the same or different.
The “substituent” is the same as in the general formula (VI). )
[General formula (VIII)]
(In the general formula (VIII), R _{41 to} R ₄₅ each represent any one of (i) to (ha) in the general formula (VI), and R _{41 to} R ₄₅ may be the same as each other. , May be different.
The “substituent” is the same as in the general formula (VI). )]

Further, for example, in the general formula (VI), R _{21 to} R ₂₉ may all be a single bond, or may form a double bond at a site where a double bond can be formed. These combinations are not particularly limited. The same applies to the general formula (VII).

In the general formula (V), X is preferably SO ₂ .

In the above general formula (VI), when R _{21 to} R ₂₉ are bonded by a single bond, preferably R _{21 to} R ₂₉ are groups of (ii) or (ha) shown below, respectively.
(I) Hydrogen, halogen, hydroxyl group, thiol group, aldehyde group, thioaldehyde group or carboxyl group.
(Ha) a linear or branched C1-C5 saturated or unsaturated hydrocarbon group having or not having a substituent, alkoxy group, thioalkoxy group, allyloxy group, allylthio group, ether group, thioether Group, ester group or thioester group.
In addition, the substituent referred to in () is a halogen, a hydroxyl group, an aldehyde group, or a carboxyl group.

_{If R} 21 _{to R 29} are bonded by a single bond in the general formula (VI) described above, more preferably a group of each _R 21 _{to R 29,} the following (ii) or ().
(Ii) Hydrogen, hydroxyl group, aldehyde group or carboxyl group.
(Ha) A linear or branched, saturated or unsaturated hydrocarbon group having 1 to 3 carbon atoms, which may or may not have a substituent.
In addition, the substituent referred to in () is a halogen, a hydroxyl group, an aldehyde group, or a carboxyl group.

  These preferable and more preferable substituents are the same in general formula (VII) and general formula (VIII).

In the general formula (VI), when R ₂₁ and R ₂₂ , R ₂₃ and R ₂₄ , R ₂₆ and R ₂₇ , and R ₂₈ and R ₂₉ are bonded by a double bond, R ₃₄ in general formula (VII) And R ₃₅ , R ₃₆ and R ₃₇ are preferably bonded to each other by a double bond.

  More specifically, it is preferable that the compound which is an active ingredient of an ADAM action inhibitor is the following chemical formula 6 used in Examples described later. Chemical formula 6 can be obtained by purchasing a commercial product. For example, H-7 (product number: I6891) manufactured by Sigma may be used.

  Further, in the ADAM action inhibitor of the present invention, the substance selected using the above-mentioned screening method for ADAM action inhibitor is a compound corresponding to the following general formula (IX) or general formula (X), and these One or more selected from a compound and a salt thereof may be contained as an active ingredient.

[General Formula (IX)]
(In General Formula (IX), X represents SO ₂ or PO ₂ H, and an isoquinoline and a nitrogen atom are bonded to the sulfur atom or phosphorus atom; R ₅₁ and R ₅₂ are each hydrogen or an optional substituent; is there.)

  The above chemical formulas 1 to 5 have bleeding inhibitory activity, which is an inhibitory activity of ADAM action, and formation and retention inhibitory activity of pseudopods, and a common structure represented by the general formula (IX) can be seen. On the other hand, the compounds represented by the following chemical formulas 11 to 19 do not have the structure represented by the general formula (IX) and do not have ADAM action inhibitory activity. That is, it is presumed that the compound having a common structure represented by the general formula (IX) has ADAM action inhibitory activity.

In the general formula (IX), X is SO ₂ are preferred.

In the general formula (IX), R ₅₁ and R ₅₂ are each hydrogen or an arbitrary substituent. Preferably, R ₅₁ and R ₅₂ each represent one of the following (C) to (F), and R ₅₁ and R ₅₂ may be the same or different.
(C) Hydrogen, halogen, hydroxyl group, thiol group, aldehyde group, thioaldehyde group, carboxyl group, cyano group, azide group, nitro group, pyridyl group, pyrrolidinyl group, thienyl group or furanyl group.
(D) an amino group, an amidino group, a guanidino group, a ureido group, a hydrazino group, a diazenyl group or a hydroxyamino group, and these groups are first saturated or straight-chained or branched or having 1 to 5 carbon atoms It may have an unsaturated hydrocarbon group, and secondly, including the hydrocarbon group described in the first, halogenated, hydroxylated, thiolated, aldehyded, oxoated, carboxylated, cyanated or It may be nitrated.
(E) an aryl group, a naphthyl group or an anthracenyl group with or without a substituent;
(F) A linear, branched or cyclic C1-C20 saturated or unsaturated hydrocarbon group having or not having a substituent, alkoxy group, thioalkoxy group, allyloxy group, allylthio group, ether group, thioether Group, ester group or thioester group.
The “substituent” referred to in (e) and (f) is halogen, hydroxyl group, thiol group, aldehyde group, thioaldehyde group, carboxyl group, cyano group, azide group, nitro group, pyridyl group, pyrrolidinyl group, thienyl group. A furanyl group, a phenyl group, a halogenated phenyl group, a benzyl group, a halogenated benzyl group, a benzoyl group or a halogenated benzoyl group,
Among them, in the “substituent” referred to in (e), in addition to the above options, a hydrocarbon group, an alkoxy group, a thioalkoxy group, an allyloxy group, an allylthio group, an ether group, a thioether group, an ester group, a thioester group These groups have 1 to 5 carbon atoms, are saturated or unsaturated, linear or branched, and are halogenated, hydroxylated, thiolated, aldehyded, oxoated, carboxylated May be cyanated or nitrated. )

  In the above (f), “having or not having a substituent ... saturated or unsaturated” relates to the subsequent “hydrocarbon group,... Or thioester group”.

  The above-mentioned “saturated” and “unsaturated” represent a hydrocarbon moiety. For example, “unsaturated alkoxy group” means having an unsaturated bond at an alkyl site in an alkoxy group.

In the above general formula (IX), R ₅₁ and R ₅₂ are preferably the groups (c), (d) or (f) shown below.
(C) Hydrogen, hydroxyl group, aldehyde group or carboxyl group.
(D) Amidino group, guanidino group or ureido group.
(F) A linear or branched saturated or unsaturated hydrocarbon group having 1 to 10 carbon atoms, which may or may not have a substituent.
In addition, the substituent referred to in (f) is a phenyl group, a halogenated phenyl group, a benzyl group, a halogenated benzyl group, a benzoyl group or a halogenated benzoyl group.

In the general formula (IX), R ₅₁ is more preferably hydrogen, a hydroxyl group, an aldehyde group or a carboxyl group, and R ₅₂ is a group (ha), (d) or (f) shown below.
(C) Hydrogen.
(D) Amidino group, guanidino group or ureido group.
(F) A saturated or unsaturated hydrocarbon group having 1 to 5 carbon atoms, preferably 1 to 3 carbon atoms, which may or may not have a substituent.
In addition, the substituent referred to in (f) is a phenyl group, a halogenated phenyl group, a benzyl group, a halogenated benzyl group, a benzoyl group or a halogenated benzoyl group.

  More specifically, it is preferable that the compound which is an active ingredient is the said chemical formula 1-chemical formula 5.

[General Formula (X)]
(In the general formula (X), X represents SO ₂ or PO ₂ H, and an isoquinoline and a nitrogen atom are bonded to the sulfur atom or phosphorus atom; R _{61 to} R ₆₉ are each hydrogen or an arbitrary substituent. is there.)

  Comparing the above chemical formula 6 with chemical formula 12 described later, the chemical formula 6 is a 6-membered ring, whereas the chemical formula 12 is a 7-membered ring. In addition, Chemical Formula 6 has a bleeding inhibitory activity that is an inhibitory activity of ADAM action, and a pseudopodia formation and retention inhibitory activity. On the other hand, Chemical Formula 12 does not have ADAM action inhibitory activity. Therefore, it is estimated that the compound having the structure represented by the general formula (X) has ADAM action inhibitory activity.

In general formula (X), X is preferably SO ₂ .

R _{61 to} R ₆₉ in the general formula (X) are each hydrogen or an arbitrary substituent. Preferably, R _{61 to} R ₆₉ may be a single bond or a double bond listed below. When R _{61 to} R ₆₉ are bonded by a single bond, R _{61 to} R ₆₉ are respectively Any one of (i) to (ha) is shown, and R _{61 to} R ₆₉ may be the same or different from each other.
(Ii) Hydrogen, halogen, hydroxyl group, thiol group, aldehyde group, thioaldehyde group, carboxyl group, cyano group, azide group, or nitro group.
(B) an amino group, an amidino group, a guanidino group, a ureido group, a hydrazino group, a diazenyl group or a hydroxyamino group, and these groups are primarily saturated or straight-chain or branched having 1 to 5 carbon atoms or It may have an unsaturated hydrocarbon group, and secondly, including the hydrocarbon group described in the first, halogenated, hydroxylated, thiolated, aldehyded, oxoated, carboxylated, cyanated or It may be nitrated.
(Ha) a linear, branched or cyclic C1-C10 saturated or unsaturated hydrocarbon group having or not having a substituent, alkoxy group, thioalkoxy group, allyloxy group, allylthio group, ether group, thioether Group, ester group or thioester group.

  In the above (ha), “having or not having a substituent ... saturated or unsaturated” relates to the subsequent “hydrocarbon group,... Or thioester group”.

When R ₆₁ and R ₆₂ , R ₆₃ and R ₆₄ , R ₆₆ and R ₆₇ , R ₆₈ and R ₆₉ are bonded by a double bond, R ₆₁ and R ₆₂ , R ₆₃ and R ₆₄ , R ₆₆ and R ₆₇ , R ₆₈ and R ₆₉ each represent one of the following (ni) to (ho), R ₆₁ and R ₆₂ , R ₆₃ and R ₆₄ , R ₆₆ and R ₆₇ , R ₆₈ and R ₆₉ are respectively They may be the same or different.
(Ii) an oxo group or a thioxy group.
(E) A hydroxyimino group or a hydrazono group with or without a substituent.
Note that the “substituent” in (ha) and (e) is a halogen, a hydroxyl group, a thiol group, an aldehyde group, a thioaldehyde group, or a carboxyl group. )

In the above general formula (X), when R _{61 to} R ₆₉ are bonded by a single bond, preferably each of R _{61 to} R ₆₉ is any of the groups shown below.
(I) Hydrogen, halogen, hydroxyl group, thiol group, aldehyde group, thioaldehyde group or carboxyl group.
(Ha) a linear or branched C1-C5 saturated or unsaturated hydrocarbon group having or not having a substituent, alkoxy group, thioalkoxy group, allyloxy group, allylthio group, ether group, thioether Group, ester group or thioester group.
In addition, the substituent referred to in () is a halogen, a hydroxyl group, an aldehyde group, or a carboxyl group.

In the above general formula (X), when R _{61 to} R ₆₉ are bonded by a single bond, R _{61 to} R ₆₉ are each preferably any one of the groups shown below.
(Ii) Hydrogen, hydroxyl group, aldehyde group or carboxyl group.
(Ha) A linear or branched, saturated or unsaturated hydrocarbon group having 1 to 3 carbon atoms, which may or may not have a substituent.
In addition, the substituent referred to in () is a halogen, a hydroxyl group, an aldehyde group, or a carboxyl group.

In the general formula (X), when R ₆₁ and R ₆₂ , R ₆₃ and R ₆₄ , R ₆₆ and R ₆₇ , and R ₆₈ and R ₆₉ are bonded by a double bond, an oxo group is preferable.

  More specifically, it is preferable that the compound which is an active ingredient is the above-mentioned chemical formula 6.

  The description regarding the salt and the ADAM action inhibitor is the same as described above.

[Others]
A compound that is an active ingredient of an ADAM action inhibitor can be chemically synthesized by appropriately combining known methods. Moreover, the salt of the said compound can be obtained by a well-known method. Commercially available products can be used as appropriate for the compounds and salts thereof described in Chemical Formulas 1 to 6 above.

  The above active ingredients are mixed with various known carriers such as liquids and solids, and if necessary, stabilizers, colorants, flavoring agents, flavoring agents, excipients, solvents, surfactants, emulsifiers, suspensions. Additives, dispersants, preservatives, solubilizers, tonicity agents, buffers, soothing agents, moisturizers, binders, coating agents, lubricants, disintegrants, etc., liquids, oral preparations, tablets , Powders, suppositories, external preparations, ointments, patches, eye drops, injections, powders, granules, dragees, capsules, pills, suspensions, ampoules, injection solutions, etc. Is possible. Moreover, it can also be set as a pharmaceutical by adding a pharmaceutically acceptable agent to the said active ingredient.

The dosage of an ADAM action inhibitor varies depending on the subject of administration, age, sex, body weight, dosage form or mode of administration, but if the subject of administration is a mammal, particularly a human adult, one or more days per week, per day of administration It is preferable to administer 1 to 100 mg / kg in terms of active ingredient.
The same applies to the case where the ADAM action inhibitor is a bleeding inhibitor, a blood vessel strengthening agent, a provisional foot formation inhibitor, or a temporary foot retention inhibitor.

  In addition, “effective component conversion” in an ADAM inhibitor is, for example, when the active ingredient contained in the agent is a compound, it is the mass of the active ingredient, and when it is a salt, it is calculated by converting to the mass of the compound. It means to do.

  A commercially available product can be used as appropriate for ADAM, which is an active ingredient of a temporary foot retainer. The snake venom may be obtained from a snake that is an individual such as a rattlesnake, or a commercially available snake venom may be purchased. Further, VAP1 and VAP2 can be obtained from the rattlesnake venom (purchased by Sigma, Wako Pure Chemicals, etc.) by the method described in Non-Patent Document 4 described above.

  The above active ingredients are mixed with various known carriers such as liquids and solids, and if necessary, stabilizers, colorants, flavoring agents, flavoring agents, excipients, solvents, surfactants, emulsifiers, suspensions. Additives, dispersants, preservatives, solubilizers, tonicity agents, buffers, soothing agents, moisturizers, binders, coating agents, lubricants, disintegrants, etc., liquids, oral preparations, tablets , Powders, suppositories, external preparations, ointments, patches, eye drops, injections, powders, granules, dragees, capsules, pills, suspensions, ampoules, injection solutions, etc. Is possible. Moreover, it can also be set as a pharmaceutical by adding a pharmaceutically acceptable agent to the said active ingredient.

  The dosage of the temporary foot retainer varies depending on the subject of administration, age, sex, weight, dosage form or mode of administration, but when the subject of administration is a mammal, particularly a human adult, one day or more per week, per day of administration It is preferable to administer 0.1-1 mg / kg by mass of the active ingredient.

  The administration frequency of each of the above-mentioned agents can be administered once or divided into several times. Administration schedules include daily administration, intermittent administration, or a combination thereof. In these administrations, it is necessary to use the judgment of a doctor.

  When the administration target of each of the above-mentioned agents is a cell, organ, tissue, etc., the dose is not particularly limited, but an ADAM action inhibitor is preferably administered in an amount of 1 to 100 μg / ml in terms of active ingredient for the administration target. In the case of a temporary foot retainer, it is preferably administered in an amount of 0.1 to 10 μg / ml in terms of the effective amount.

(Acquire VAP1)
In accordance with the purification method of VAP1 of Masuda et al. Described in Non-Patent Document 2, a crude poison of rattlesnake (C. atox) (manufactured by SIGMA: product name Snake venom, Crotalus atrox (V7000)) was added to 25 mM NaCl buffer (25 mM). It is dissolved in NaCl, 10 mM Tris-HCl (pH 7.0), 1 mM CaCl ₂ ), purified using CM-Sephadex C25 (SIGMA-ALDRICH) column (1.5 cm × 9 cm), and NaCl concentration gradient (25 mM) -500 mM, 150 ml). Each of the obtained fractions was subjected to SDS-PAGE (non-reducing conditions) with SDS-PAGE (10% polyacrylamide gel (separation gel 9.73% Acrylamide (Sigma A8887), 0,27% Bis-acrylamide (Sigma M7279), 10% ammonium persulfate 0.04 ml and TEMED 0.005 ml were added to 10 ml of a solution of 1% SDS, 0.375 M Tris-HCl (pH 8.8) for gelation), gel size 90 mm wide x 80 mm long x 1 mm thick Electrophoresis at a constant current of 20 mA in an electrophoresis buffer (0.025 M Tris, 0.192 M glycine, 0.1% SDS) The molecular weight marker is Kit for Molecular Weights 30,000-200,000 for SDS-PAGE (MWDSDS200 manufactured by Sigma). The use.) Is performed, and fractions band of detection has been confirmed to 110kDa by silver staining and elution fractions of VAP1. Further, using hydroxyapatite (Seikagaku Corporation) column (1.5 cm × 8.5 cm), an equal amount of 25 mM sodium phosphate buffer (pH 7.0) was mixed with the elution fraction and added to the column. Elution was performed with a sodium phosphate concentration gradient (0.25 mM-500 mM, 150 ml). Each obtained fraction was subjected to SDS-PAGE under the same non-reducing conditions as described above, and a single band of VAP1 was confirmed at 110 kDa by silver staining. The fraction containing the 110 kDa band was used as a purified VAP1 preparation.

(Acquire VAP2)
Each fraction obtained with CM-Sephadex C25 column at the first stage of VAP1 purification was subjected to SDS-PAGE under non-reducing conditions, and a fraction in which a 60 kDa band was detected by silver staining was used as a VAP2 elution fraction. . Further purification was performed using Phenyl Sepharose CL-4B column (0.8 cm × 3 cm) in accordance with the Masuda et al. VAP2 purification method described in Non-Patent Document 1. An equal amount of 1M ammonium sulfate buffer (1M ammonium sulfate, 10 mM Tris-HCl (pH 7.0), 1 mM CaCl ₂ ) was mixed with the VAP2 elution fraction and added to the column. Elution was carried out using an ammonium sulfate concentration gradient (500 mM-0 mM). Each obtained fraction was subjected to SDS-PAGE under non-reducing conditions similar to the acquisition of VAP1, and a VAP2 band was confirmed at 60 kDa by silver staining. The fraction containing the 60 kDa band was used as a purified VAP2 standard.

(Homology of VAP1, VAP2 and human ADAM)
The amino acid sequences of VAP1 and VAP2 have been specified by the present inventors, and are described in the following documents.
VAP1: Non-patent document 2, VAP2: Masuda
S, Maeda H, Miao JY, Hayashi H, Araki S. cDNA
Cloning and Some Additional Peptide Characterization of a Single-Chain Vascular
Apoptosis-Inducing Protein, VAP2. Endothelium. 2007; 14 (2): 89-96.

  VAP1, VAP2 and human ADAM28 have amino acid sequence agreements of 41% and 42%, respectively (from BLAST homology search). This figure shows that the human ADAM closest to human ADAM28 is human ADAM7 and human ADAM12 in this order, and the amino acid sequence matches are 41% and 36%, respectively. VAP1 and VAP2 are strong homologs of human ADAM. It can be said. Concerning the three-dimensional structure, the three-dimensional structure deviation of the protease domain is 0.987Å (angstrom) with a three-dimensional structure deviation RMSD (Root Mean Square Deviation) with VAP1 as compared to human ADAM33 whose three-dimensional structure is known. Shows a high structure match. Therefore, it is very strongly suggested that VAP1 and VAP2 are homologs of the ADAM family.

(Search for ADAM inhibitory substances)
The bleeding caused by VAP was regarded as ADAM action, and the inhibitory activity of ADAM action was measured for the above chemical formulas 1 to 6 and the following chemical formulas 11 to 19. In the following Examples, these compounds were used by purchasing commercially available products as appropriate.

  The bleeding activity caused by VAP was measured as follows. As animals, adult ICR mice (after 2 months of age) were used. The skin on the back of mice is suitable for viewing bleeding and is often used to see the activity of snake venom. The bleeding activity of VAP was measured by intradermal injection into the skin on the back of mice.

After fixing the tail of the mouse and calming the mouse, the hair on the back was carefully cut with scissors to make the hair cutting site 4 × 4 cm in size. After the hair cutting site was sterilized with ethanol, an injection solution was injected using a syringe (1 ml of Terumo syringe, Terumo injection needle 27G × 3/4 ″). The injection solution was 10 μl of 1 mg / ml purified VAP1 standard solution and 10 mM compound solution (in addition, phosphate buffered saline (PBS ⁻ ) or dimethyl sulfoxide (DMSO) was used as a control, and VAP1 alone was administered. A mixture of 10 μl) was used. Four hours after the injection, it was sacrificed with diethyl ether, and the skin with the bleeding spots was peeled off from the back and observed.

  Each compound was tested in triplicate, and the mean value of the blood loss was 0.36 for chemical 1, 0.32 for chemical 2, 0.21 for chemical 3, 0.16 for chemical 4 5 is 0.40, Chemical 6 is 0.38, Chemical 11 is 1.48, Chemical 12 is 1.38, Chemical 13 is 1.33, Chemical 14 is 1.20, Chemical 15 is 1.48, Chemical 16 The chemical compound was 1.26, the chemical compound 17 was 1.30, the chemical compound 18 was 1.13, and the chemical compound 19 was 1.36.

  FIG. 1 is a bar graph showing the amount of bleeding for Chemical Formulas 1 to 5 and Chemical Formulas 11 to 13 as a relative ratio to the control, where 1 is the amount of bleeding in the control administered with VAP1 alone. FIG. 2 is a bar graph showing the amount of bleeding for Chemical 14 and Chemical 15 as a relative ratio to the control. FIG. 3 is a bar graph showing the amount of bleeding for Chemical Formula 11 and Chemical Formula 16 to Chemical Formula 19 as a relative ratio to the control. Each bar also shows the standard deviation. Note that illustration of the chemical formula 6 is omitted.

  Next, hemorrhage activity using VAP2 was measured for Chemical 3 and Chemical 4 by the same procedure as in the injection experiment described above. The injection solution was mixed with the purified VAP2 standard instead of the purified VAP1 standard. FIG. 4 is a photograph showing the administration results of Chemical 4 (upper) and Chemical 3 (lower). The left side is the result of administration (control) of VAP2 alone, and the right side is the result of administration of a mixture of VAP2 and Chemical Formula 4 or Chemical Formula 3. Chemical formula 3 and chemical formula 4 showed bleeding inhibitory activity.

  FIG. 5 is a photograph showing the administration results of Chemical Formula 11 and Chemical Formula 16 to Chemical Formula 19. As in FIG. 4, the left side is the result of administration (control) of VAP1 alone, and the right side is the result of administration of a mixture of VAP1 and each compound. Chemical Formula 11 and Chemical Formula 16 to Chemical Formula 19 did not show bleeding inhibitory activity.

From the experimental results, the chemical compounds 1 to 6 were found to have bleeding inhibitory activity. As described above, since VAP1 and VAP2 are homologs of ADAM, Chemical Formulas 1 to 6 are substances showing ADAM action inhibitory activity. In Formulas 1 to 6, a common structure in which a nitrogen atom is bonded to isoquinoline through a linker, sulfenic acid (—SO ₂ —), can be seen.

(Confirmation downstream of ADAM)
Whether the compound directly inhibited the proteolytic activity of VAP was examined by the following method. Cleavage was judged by whether fibronectin, which is one of the degradation substrates of VAP1, is cleaved. DMSO was used for the purpose of dissolving chemical formula 4 in the solution.

(1) As a control, fibronectin (also expressed as “fib” in FIG. 6) or VAP1 alone, (2) 10 μl of 1 mg / ml purified VAP1 preparation 50 mM phosphate buffer solution and 200 μg of fibronectin (experiment) The results are shown in the third column from the left in FIG. 6), (3) a mixture of 10 μl of 1 mg / ml purified VAP1 standard 50 mM phosphate buffer solution and 10 μl DMSO mixed with 200 μg of fibronectin (experimental results are shown in FIG. 6). (Shown in the fourth column from the middle left), (4) A mixture of 10 μl of 1 mg / ml purified VAP1 preparation 50 mM phosphate buffer solution and 10 μl of 5.2 mg / ml DMSO solution of “Chemical Formula 4” and 200 μg of fibronectin were mixed. (The experimental results are shown in the rightmost column in FIG. 6, and the description of DMSO is omitted in the figure). Each was incubated at 37 ° C. for 24 hours in 100 μl of acid buffered saline (PBS ⁻ ) (pH 7.0)). These mixed solutions were collected and subjected to SDS-PAGE, and then subjected to silver staining.

  The experimental results are shown in FIG. As is clear from the results of (2) to (4) above, it was confirmed that the reaction of VAP1 proceeded even when chemical formula 4 was present. Similar results were obtained for other compounds of Chemical Formulas 1 to 6.

  Moreover, it was speculated that the same phenomenon can be seen in VAP2 from the homology between VAP1 and VAP2 and the above-mentioned experimental results of searching for an ADAM action inhibitor.

  That is, it was shown that Chemical Formulas 1 to 6 act downstream of ADAM in the ADAM signaling system.

(VAP and temporary foot)
Using VAP1 which is a snake venom ADAM and vascular endothelial cells, the formation and maintenance of pseudopodia caused by VAP was examined.

The cells were prepared by the method of Jaffe et al. (Jaffe,
EA, Nachman, RL, Becher,
CG, and Minick, RC (1973) Culture of human
endothelial cells derived from umbilical veins. J. Clin. Invest. 52,
2745-2756.) Human umbilical vascular endothelial cells (HUVEC) were used. The culture solution was MCDB105 culture solution (manufactured by Sigma), 10% (v / v) of fetal bovine serum at the final concentration after mixing, and 70 ng / ml of FGF (the method of Lobb and Fett (Lobb, Fett
RR, and Fett, JW (1984) Purification of two
distinct growth factors bovine neural tissue by heparin affinity
chromatography. Biochemistry 23,
6295-6299) extracted from bovine brain) and heparin added at 100 μg / ml. As culture conditions, HUVECs were plated on a plastic dish coated with 0.1% gelatin and cultured with the culture medium at 37 ° C., 5% CO ₂ , 95% air.

  The removal of the survival factor was performed by replacing the culture solution with a culture solution without addition of serum / FGF. On the other hand, the toxin added in the apoptosis caused by snake venom was the above-described crude poison of rattlesnake (C. atox), and added at a final concentration of 1 μg / ml. Immediately after exchanging the culture solution, a rough poison of the diamondback rattlesnake was added, and the cells were photographed by time-lapse video (using a phase contrast microscope).

The experimental results are shown in FIG. A to C show the process of apoptosis by removal of survival factor over time. The cells at 0 hours (A) form a pseudopod when apoptosis begins about 24 hours after removal of the survival factor (small bumps indicate the formation of a pseudopod compared to the cell body) (B), One hour after formation, the temporary foot disappears and becomes spherical (C), and the shape remains as it is thereafter.
In addition, it has been found from recent findings that blebbing (vesicles) similar to that seen in apoptosis is one of the forms of pseudopodia. For example, it is described in: T. T. et al. Charras, A Short History of Blebbing. Journal of Microscopy, 231: 466-478 (2008).

  DF shows the process of the change of the temporary foot by the above-mentioned Nishidia Rattlesnake coarse poisoning with time. The cells (D) at 0 hours form pseudopods about 24 hours after the removal of the survival factor and the addition of the rough rattlesnake poison (E), but at least 6 hours after the formation of the pseudopods. Even if it passes, the temporary foot is not lost and is maintained (F), and the form is maintained thereafter.

Similar experimental results were obtained for VAP1 and VAP2.
That is, it was shown that the provisional foot can be formed and the temporary foot can be held by a simple method using snake venom.

(Snake venom ADAM and ADAM action inhibitor)
Using VAP1 which is a snake venom ADAM and Chemical 6 which is an ADAM action inhibitor, the effect of Chemical 6 on the formation and retention of pseudopods was examined.

Human umbilical cord vascular endothelial cells (HUVEC) and culture solution prepared by the same method as described above were used. As for the culture conditions, HUVECs were plated on 0.1% gelatin-coated plastic dishes and cultured with the culture medium at 37 ° C., 5% CO ₂ , 95% air.

  The culture solution was replaced with a culture solution without addition of serum / FGF, and as a control, VAP1, which was purified immediately after the replacement of the culture solution, was added to a final concentration of 10 μg / ml, and the culture was continued for 24 hours. On the other hand, the chemical group 6 was added so that the final concentrations of VAP1 were 10 μg / ml and chemical 6 was 30 μM, and the culture was continued for 24 hours.

The experimental results are shown in FIG. FIG. 8A shows the results of the control and shows cells 24 hours after the addition of VAP1. Bumps that are smaller than the cell body indicate the formation of pseudopods, and it can be seen that they are retained after 24 hours. FIG. 8B shows cells 24 hours after administration of VAP1 and The cell body has a smooth spherical shape, and no temporary foot is seen.
Moreover, although illustration is abbreviate | omitted, when VAP1 was added and the cell was made to form a temporary foot, and chemical | medical agent 6 was administered after that, the temporary foot disappeared. Similar results were obtained for other compounds of Chemical Formulas 1 to 6.

  That is, the temporary foot retention by the snake venom ADAM was inhibited by Chemical Formulas 1 to 6. Moreover, formation of a temporary foot by VAP was inhibited by Chemical Formula 1 to Chemical Formula 6. That is, Chemical Formula 1 to Chemical Formula 6 showed ADAM action inhibitory activity. In addition, Chemical Formula 1 to Chemical Formula 6 also showed provisional foot control activity.

(Effects of snake venom ADAM on blood vessels)
The following paper shows that snake venom ADAM acts on blood vessels.
Araki
S, Masuda S, Maeda H, Ying MJ, Hayashi H. Involvement of specific integrins in apoptosis induced by vascular
apoptosis-inducing protein 1. Toxicon.
2002 May; 40 (5): 535-42.

The upper part of FIG. 9 (decomposition of the collagen solution) will be described. 50 μg of collagen was incubated at 37 ° C. for 24 hours with 0.2 μg of purified preparation VAP1 or 2 μg of rattlesnake venom. In the reaction solution, 10 μg / ml of VAP1 or 100 μg / ml of rattlesnake venom was added to 10 mM Tris-HCl (pH 7.0), 1 mM CaCl ₂ , 2.5 mg / ml α-gelatin (Sigma). Each sample was fractionated by polyacrylamide SDS electrophoresis, stained with Coomassie brilliant blue, and detected. The left side of FIG. 9 (lane 1) is the one in which only collagen is incubated, the center of FIG. 9 (lane 2) is the one in which collagen and VAP1 are incubated, and the right side in FIG. 9 (lane 3) is the one in which collagen and rattlesnake venom are incubated.

  The lower part of FIG. 9 (decomposition of the culture dish gelatin coat) will be described. Collagen-coated culture dishes were incubated with VAP1 or rattlesnake venom for 5 days, and then examined for whether the ability of collagen to adhere was impaired by planting human umbilical cord vascular endothelial cells. The collagen-coated culture dish was incubated for 5 days in a culture solution (A), a rattlesnake toxin (10 μg / ml) -added culture solution (B), or a VAP1 (1 μg / ml) -added culture solution (C). The state 6 hours after planting the cells was photographed with an optical microscope.

As shown in FIG. 9, VAP1 is shown not to degrade collagen. That is, VAP1 does not degrade the extracellular matrix of blood vessels.
The snake venom ADAM has been shown to cause apoptosis of vascular endothelial cells while not degrading the extracellular matrix of blood vessels. Therefore, a bleeding model as shown in FIG. 10 can be estimated. The above-described chemical formulas 1 to 6 are estimated to correspond to the “signal transmission inhibitor” in FIG.

(MMP collagenase and ADAM inhibitor)
The influence of an ADAM action inhibitor on collagenase bleeding was measured as follows. The animals used ICR mouse adults (after 2 months of age), and the collagenase bleeding activity was measured by intradermal injection into the back skin of the mice. After fixing the tail of the mouse and calming the mouse, the hair on the back was carefully cut with scissors to make the hair cutting site 4 × 4 cm in size. After the hair cutting site was sterilized with ethanol, an injection solution was injected using a syringe (1 ml of Terumo syringe, Terumo injection needle 27G × 3/4 ″). The injection solution was 0.8 mg / ml collagenase (manufactured by Sigma, product name Collagenase from Clostridium histolyticum (C0130)) and 10 mM “Chemical Formula 4” solution (in addition, the solvent dimethyl sulfoxide was used as a control substance. 10 μl) was used. Four hours after the injection, it was sacrificed with diethyl ether, and the skin with the bleeding spots was peeled off from the back and observed.

  The experimental results are shown in FIG. The left side is the result of administration (control) of collagenase alone, and the right side is the result of administration of a mixture of collagenase and chemical formula 4. Similar results were obtained for other compounds of Chemical Formulas 1 to 6.

  That is, it was shown that an ADAM action inhibitor cannot inhibit bleeding which is an action of collagenase which is MMP. This indicates that the ADAM action inhibitor does not inhibit the action of other proteases. In other words, an ADAM action inhibitor that acts downstream of ADAM has few side effects. Further, since bleeding which is an action of collagenase cannot be inhibited, it is reasonably estimated that the protease activity of collagenase remains even when an ADAM action inhibitor is added.

(VAP and temporary foot-2-)
Using VAP2 which is snake venom and snake venom ADAM, and chemical formula 2, the effect of chemical formula 2 on the formation and maintenance of pseudopods was confirmed.

Human umbilical cord vascular endothelial cells (HUVEC) and culture medium prepared by the same method as “(VAP and temporary foot)” described above were used. As the culture conditions, they were spread on a plastic dish on which a slide glass coated with 0.1% gelatin of HUVEC was placed, and cultured with the culture solution at 37 ° C., 5% CO ₂ , 95% air.

  The culture solution was replaced with a serum / FGF-free culture solution. Immediately after the replacement of the culture solution, the rough rattlesnake used in the above-mentioned “(Acquisition of VAP1)” and “(Acquisition of VAP2)” was purified or purified. A sample, VAP2, was added to a final concentration of 10 μg / ml, and the culture was continued for 4 hours. In addition, in the chemical administration group, the above-mentioned snake venom or VAP2 was added at a final concentration of 10 μg / ml, and chemical 2 was added at 10 μM, and the culture was continued for 4 hours. The control was continued for 4 hours as it was without adding the rough venom of the rattlesnake, VAP2, and Chemical 2 after the culture medium was changed.

After culturing for 4 hours, the cells were fixed with 4% paraformaldehyde, stained with 100-fold diluted AlexaFluor 488 phalloidin (Molecular Probe) for 1 hour, then washed and observed for fluorescence. The fluorescence observation was performed using a fluorescence microscope BZ-9000 (Keyence Corporation), and observed and photographed with an excitation wavelength of 470 nm, an absorption wavelength of 535 nm, and an objective lens of 40 times.
In the above example of “(VAP and provisional foot)”, apoptosis was started about 24 hours after the removal of the survival factor / poisonous rattlesnake snake venom / VAP was added. Since fixation with paraformaldehyde was performed at 4 hours after removal of survival factor / poisonous rattlesnake venom and VAP, apoptosis did not start.

  FIG. 12 shows the result of an experiment using a rough poison of the diamondback rattlesnake. FIG. 12A shows the experimental results of the control for 4 hours after exchanging the culture solution. The fibrous actin protein skeleton is seen, and the formation of a prominent pseudopod is not seen.

  FIG. 12B shows cells 4 hours after the addition of a rough poison of the diamondback rattlesnake. Since actin which is a skeletal protein is stained, a site exhibiting strong fluorescence means accumulation of actin. A long and slender protrusion-like temporary foot accompanied by accumulation of actin is formed at the tip of the cell. Many pseudopods have granular or ring-shaped podosomes. FIG. 12B is a temporary foot 4 hours after the addition of the snake venom, which was observed 30 minutes after the addition of the snake venom and continued for about 24 hours. After about 24 hours, the temporary foot was crushed and became as shown in FIGS. 7E and 8A.

  FIG. 12C shows the cells 4 hours after the addition of the snake venom and compound 2. Compared with 12B, there were few protrusion-like temporary feet, actin accumulation was weakened, and formation of temporary feet was suppressed.

  Similar results were obtained with snake venom ADAM VAP2. As described above, VAP2 is a homologue of human ADAM. Therefore, it is presumed that it is possible to effectively form a temporary foot and hold the temporary foot by administration / introduction of snake venom ADAM or human ADAM.

  INDUSTRIAL APPLICABILITY According to the present invention, a useful ADAM action inhibitor screening method, provisional foot retention method, provisional foot retention agent, provisional foot control substance screening method, and ADAM action inhibitor are provided.

Claims (7)

The substance in which the progress of the ADAM protease reaction is confirmed from the following steps a) and b) and the inhibition of the ADAM action from the following steps a) and c) is determined as an ADAM action inhibitor. A screening method for an ADAM action inhibitory substance characterized by the above.
a) A step of administering or introducing a substance to be screened into a specimen (excluding a human individual) having ADAM protease activity or having ADAM protease activity.
b) A step of confirming the progress of ADAM protease reaction in the specimen provided with the ADAM protease activity and the substance to be screened by the above step a).
c) A step of confirming the inhibition of the provisional foot or the formation of the temporary foot in the specimen having the ADAM protease activity and the screening target substance by the step a). 2. The screening method for an ADAM action inhibitor according to claim 1, wherein the specimen in the step a) is a cell, an organ, a tissue, or an individual. The method for screening an ADAM action inhibitor according to claim 1 or 2, wherein the ADAM is human ADAM or snake venom ADAM. 4. The ADAM action according to any one of claims 1 to 3, wherein the ADAM action inhibitor screening method comprises confirming ADAM specificity from the step a) and the following step d). Inhibitor screening method.
d) A step of confirming the remaining protease activity of a protease other than ADAM in a sample to which a screening target substance has been administered or introduced. A method for holding a temporary foot, comprising using ADAM for a specimen capable of forming a temporary foot (excluding a human individual) . A temporary foot retainer comprising ADAM as an active ingredient. A processing step including the following steps e) and f) in random order is performed, and then a substance for which provisional foot control is confirmed by a confirmation step including the following step g) is determined as a provisional foot control substance. A screening method for a control substance of a temporary foot characterized by the following.
e) A step of administering or introducing ADAM to cells, organs, tissues, or individuals that are specimens (excluding human individuals).
f) A step of administering or introducing a substance to be screened into a cell, organ, tissue, or individual that is a specimen (excluding a human individual).
g) A step of confirming the control of the temporary foot.