JPH06505477A - Anticoagulants and treatment methods for high cyclic compounds - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] High cyclic compound anticoagulant and treatment method The present invention is based on the U.S. patent application “High cyclic compound” Antiviral Compounds and Methods°, Serial No. 647.720, and “Herpes Simplex ``Methods for the Treatment of Viral Infections in Japan'', serial number 647.469 (both 1999 This is a continuation-in-part application filed on November 29, 2013.

■、Technical field The present invention relates to highly cyclic anticoagulant compounds and methods for inhibiting blood coagulation.

2. References Andriuof i, c, et al. (1990) Hemostasis 20 (Appendix 1): 154-158゜785-789゜ New York, Chapter 55: 1311-1331° and Technical Principles, C, V. Mo5by Co, St, Louis, pp, 135-143゜3, this publication Ming background Blood clotting or clotting is the result of a complex series of biochemical reactions. Normality of the incident In the normal course, blood flow interruption and related processes related to blood coagulation are caused by damage to blood vessels. prevents excessive blood loss. However, within its circulatory system, pathological conditions, e.g. e.g. in atherothrombotic conditions or including surgical procedures and implantation of medical devices. Inappropriate blood clotting may occur in response to various disorders.

This can lead to vascular occlusion and/or thromboembolism. Here, all of the blood clots or pieces break apart and become trapped as blood clots in other areas of the circulatory system. It becomes like a full circle. Such blood clots are particularly important for pulmonary arterial or cerebrovascular circulatory obstruction. When it causes blockage, it can be fatal in some cases.

Therefore, prevention of blood clots is important in the treatment of patients at risk of developing thromboembolism. It is considered an essential part of the curing method. Diseases and treatments for which treatment with anticoagulants is indicated Medical conditions include heart valve replacement, transplant surgery, chronic hospital bed, surgery, venous thrombosis and Pulmonary thrombosis, arterial thrombosis, stroke, presence of abnormal clotting factors, certain stem cell diseases , and homocy, including stinuria.

In order to understand the various means by which blood clotting can be controlled, it is necessary to leading to fibrin formation and blood clot formation (a basic understanding of the cascade of reactions is essential) be. These reactions and their components have been extensively investigated (Majeru s, Baboir) and is briefly cited as a reference in Figure 14 of this specification. It can be summarized as follows.

Blood coagulation occurs through two different but interconnected routes - the intrinsic route and the extrinsic route. can be stimulated by causal pathways. In both pathways, blood coagulation relies on inactive enzymatically cleaves the protein molecule into an active protease, which in turn is used by the next enzyme in the pathway. results from a series of zymogen activation steps, including activating zymogens. See Figure 14 Then, the link between the intrinsic and extrinsic pathways is the activation of the zymogen factor [X]. Activation to protease, factor IXa.

The intrinsic pathway is so called because it follows the first contact stimulus and is Only the factors that are present are included in the action. In this route, in vitro With factor X[I, prekallikrein, and foreign surfaces, as studied in The interaction of high molecular weight kininogens, such as glass or china clay, is due to factor X1. resulting in conversion to factor X1a (which activates factor IX to factor IXa) Become.

Factor IXa is a protease that converts inactive factor X to active factor Xa. child The conversion of platelets or phospholipids (both named PL in the figure), cofactor V illa, and is accelerated by the presence of calcium. Factor 11 (prothrombin ) to form factor 11aO (thrombin), platelets or phospholipids, factor It is enhanced by the presence of Va and calcium. Factor Va is stimulated platelet can be released by

Thrombin is a protein that cleaves high-molecular-weight fibrinogen into fibrin monomers. It is rotease. These monomers form a gel and attach red blood cells to that gel. However, they coalesce to form blood clots. The strength of the clot is catalyzed by factor X1lla. It is increased by the transglutaminosis reaction of the inner chains of fibrin monomers.

To complete the general pathway shown in FIG. more destroyed (“dissolved”). This active protease, plasmin, is l or more endogenous activities, including plasminogen activator (t-PA) Enzymatic cleavage catalyzed in vivo by sexing factors releases inactive plasmids. Formed from nogen.

Streptokinase, a microbial product, or urokiner isolated from human cells plasminogen can also activate plasminogen, as shown in FIG.

Plasmin non-specifically binds fibrin and other plasma proteins, including some clot-forming factors. Disintegrate differently.

In the extrinsic route, exposing the blood to tissue factor converts factor 1x to factor IXa. It is a stimulus for conversion.

Tissue factor is used in non-circulating cells, e.g. fibroblasts, or in blood in certain pathological conditions. present on the surface of activated monocytes or endothelial cells that can be exposed to It is a riboprotein. As shown in Figure 14, factor V[[a is ,factor! Conversion of X to factor IXa and conversion of factor X to factor Xa are performed. Factor V IE itself has a proteolytic activity of about 1/100 that of factor Vlra; and therefore clot formation can begin. Tissue factor includes factor Vll and factor V[Ia enhances both activities approximately 30.000 times. Formation of factor Xa is also still more The process is accelerated by converting the factor Vll to the factor VI[a.

Generally, there are three types of agents that affect blood blockage: those that interfere with platelet activation and aggregation; agents that interfere with some of the coagulation cascades mentioned above, and agents that promote clot disintegration. are categorized. Aspirin, dipyridazole, and ticlopidine are antiplatelet agents. This is an example. Their use as anti-clotting agents is commonly used in atherothrombotic diseases. limited to prophylaxis against recurrent myocardial infarction, temporary ischemic attack, and or in conjunction with anticoagulants in certain heart valve disorders.

They are commonly used in the treatment of other abnormal clot events, such as arterial thrombosis. are not commonly used and there is no mechanical basis for their use in such ataxias. It is not believed that there is a foundation.

Agents that promote the disintegration of blood clots (fibrin-degrading agents) activate tissue plasminogen. including the factors streptokinase and urokinase.

These substances are used after myocardial infarction to prevent thromboembolism. the current Anticoagulants that can be used only when given intravenously and oral coumarin anticoagulants Limited to active heparin-like compounds IXa, Xa, Xla, XI Ia, kallikrein and thrombin).

This reaction results in inhibition of these factors and therefore inhibits coagulation. do. Heparin is not well absorbed orally and has a relatively short half-life in the bloodstream. One. Side effects of long-term heparin treatment include associated paradoxical arterial thrombosis and, rarely, may include thrombocytopenia with osteoporosis. Overdosing on heparin can cause sulfur It can be antagonized by injection of acid protamine.

Oral anticoagulants, including warfarin and other coumarin derivatives, are thus producing their effect on blood coagulation.

These compounds inhibit the liver's reproduction of vitamins. Vitamins have Child 11 (prothrombin), Vl+, [X, and therefore, depletion of the vitamin results in inhibition of coagulation. Become. As would be expected from their mechanism, the coumarin drugs have a relatively long Raises therapeutic activity.

because their effects depend on the depletion of endogenous stores of active vitamins. It is et al. Treatment with coumarin has been shown to increase or increase the effective dosage level of many pharmaceutical drugs. requires careful management due to nutritional interactions that help reduce coumarin Treatment with derivatives is associated with several serious side effects including bleeding incidents and hereditary malformations. is also relevant.

Numerous analytical tests have been devised to measure the patency of the blood coagulation cascade. It has been. These tests are described in more detail below, but generally involve plasma testing. It will be carried out. For example, the prothrombin time assay (PT) is a test for exogenous blood coagulation. system and therefore detect deficiencies of factors II, V, VU and X. used for PT is a treatment option in patients receiving coumarin anticoagulants. It is also used to monitor medical treatment. Because factors l] and V[[but vitamin This is because it is among the things that depend on K.

The activated partial thromboplastin time assay (APTT) Coagulation factors present in the endogenous system of blood coagulation, excluding Xtl, are measured. So This is commonly used to monitor heparin therapy. Plasma clot time are other measurements related to the intrinsic blood coagulation pathway and are useful for monitoring heparin therapy. It can be used even if you leave it there.

As mentioned above, current anticoagulation regimens include various heparin or coumarin drugs. Including treatment by form. Of those two, heparin drugs are far more tolerable. tolerant and easy to titrate. However, the use of these substances is limited by the current requirement for an intravenous route of administration. these Although prescriptions of substances have been administered enterally, their anticoagulant activity remains It has been observed only after intraduodenal administration (Andriuoli, Cara mazza).

Coumarin drugs, such as warfarin, can be given orally; however However, the utilization of these medicines is limited by their relatively long attack times, as mentioned above. Limited by titration difficulties, interactions with other drugs, and side effects. Therefore, with shorter attack and duration of action for improved oral control of blood coagulation; It is a principal object of the present invention to provide materials and methods for oral anticoagulation therapy. It is a purpose.

4. Summary of the invention The main object of the present invention is to provide compounds and methods effective for the treatment of blood coagulation pathologies in human specimens. It is to provide law.

The present invention provides a method of inhibiting blood coagulation. The method consists of a series of bonded skeleton atoms. Aryl rings bonded one-to-one by ring-added crosslinks forming a continuation chain The method includes administering to the specimen a highly cyclic compound composed of subunits of. this The subunit contains a sulfonic acid on a non-skeletal atom of its aryl subunit ring. has a substitution product induced by .

The cyclic subunit preferably has sulfonic acids at its 3- and 6-ring positions. Naphthalene subunit with a substituent derived from the ring position at the 4-position This compound contains a phenyl subunit with a sulfonic acid-derived substituent. Here, the high ring crosslinking bond is the cyclic bond at the 2-position of one naphthalene or phenyl group. carbon position and the 7-position cyclic carbon group of the adjacent naphthalene group or the 6-position ring of the adjacent phenyl group between the carbon positions. The compound preferably contains from 4 to 8 such subgroups. Contains units. The sulfonic acid-derived substituent is preferably a sulfonic acid-derived substituent. fonic acid, sulfonate, sulfinic acid, sulfinate, sulfone, or It is honamide.

In one major embodiment, the highly cyclic compound comprises carbon atoms at the 3- and 6-ring carbon positions. Substituents derived from sulfonic acid, polar groups at the 1- and 8-position cyclic positions, and the cyclic carbon position at position 2 of one subunit and the cyclic carbon position at position 7 of the adjacent subunit. at least four naphthalenes each having a crosslink between carbon positions Contains subunits of. One preferred compound of this type has the following main structure: [Here, R1 is sulfonic acid, sulfonate, sulfinic acid, sulfinate , an alkyl sulfone, or a polar sulfonamide, such as 502Nl(R(coco Te, NHR is Nf (,, NHQHl is also an amino acid), and R5 is OH, = 0, alkyl or aryl ester, ester, or acid acid or a mixture thereof, and n=4.6 or 8, and R 4 is >CHR' or C>CR" (here, R" is H or a carboxylic acid group) ) has 1.

In another major embodiment, the high cyclic compound is derivatized with a sulfonic acid at the para position. The substituents are placed at the 2nd ring carbon position of one subunit and the adjacent subunit. at least four phenyl subunits with a bridge bond between the ring carbon position 6 and including the unit. One preferred compound of this type has the following main structure: (where n, R+, R2 and R4 are the same as above).

For use as an anticoagulant, this compound can be administered orally or parenterally. can be administered. Such treatment methods rely on the anticoagulant effects of this compound. further comprising administering to the subject a dosage of protamine sufficient to offset the

In another aspect, the present invention provides alkyl sulfones, and 5O2NHR (where NH R is NHi, NHO)I, or an amino acid) at least six regularly arranged groups selected from groups consisting of of highly cyclic biocompatible polymer constructs with sulfonic acid-derived substituents. Including methods of inhibiting blood coagulation by administering to a subject a therapeutically effective dose .

In a preferred embodiment, the polymer has a high cyclic component or an aryl ring substituent. unit, forming a continuous chain of bonded atoms that make up this highly cyclic skeleton. bonded by a cycloaddition bridge and on a non-skeletal atom of the aryl subunit. containing substituents derived from sulfonic acid.

Forming part of the invention are the following forms: [wherein R2 is sulfur] fonic acid, sulfonate, sulfinic acid, sulfinate, sulfone, or honamide, and R1 is ;0 and -〇　, alkyl or aryl; is a ster, ester, or acid or a mixture thereof, and R4 is >CHR “or >CR” (where R” is H or a carboxylic acid group) and A novel compound composed of phenyl subunits with 1 where n=4.6 or 8 It is also a highly cyclic compound.

These and other objects and features of the invention will be appreciated in the following detailed description of the invention. It will be more fully clear when read in conjunction with the figures.゛ Brief description of the drawing FIG. 1 shows a high-density polymer composed of naphthalene subunits for use in the present invention. The main structure of the cyclic compound is shown; Figures 2A and 2B show the unoxidized (2A) structure of Figure 1. and partially oxidized (2B) form, where n=4 and the subunits Nit shows the form of chromotropic acid.

Figures 3A and 3B show two main types of highly cyclic compounds, such as the compound shown in Figure 2A. Describe the synthesis method: Figures 4A and 4B show mixed phenyl and sulfonated naphthalene subunits. shows a non-oxidized (4A) and a partially oxidized (4B) hypercyclic structure with Figure 5 shows how the sulfonic acid substituent of a highly cyclic chromotropic acid can be converted into a glycyl sulfonamide. and a reaction method for converting it into a sulfonamide group; Figure 6 shows that the sulfonic apparatus residues of highly cyclic chromotropic acids are converted to sulfinic acids or Describe the reaction method for converting these into methyl (aryl) esters.

FIG. 7 shows a sulfonic acid induced position at the para position for use in the present invention. Shows the main structure of a high cyclic compound composed of phenyl groups with substituents; FIG. 8 shows a non-oxidized version of the structure of FIG. buunit is parasulfonic acid.

Figures 9A and 9B illustrate the main synthesis method for the non-oxidized and partially oxidized forms of the compound of Figure 8. Explain: Figure 1O is for substitution of a ring hydroxyl group within the compound of Figure 8 with an acetyl group. shows the reaction mechanism of; Figure 11 shows the glycylation of sulfonic acid substituents within the high ring compound of the phenyl subunit. Figure 12 shows the reaction for conversion to the sulfonamide group; Although it is similar to a compound, it is possible to produce a highly cyclic compound with a carboxylic acid containing a cross-linked bond. The reaction mechanism for the production is shown.

Figure 13 shows that for substitution of hydroxyl groups within the compound of Figure 8 with carboxylic acid groups. shows the reaction mechanism of; Figure 14 shows the cascade of biochemical events that occur during the blood clotting process in mammalian blood. Show the diagram: Figure 15 shows the prototyping in seconds as a function of the concentration of KY-1, Y-1 and Y-49. Figure 16 shows a plot of rhombin time (PT); Activated partial thromboplastin time in seconds as a function of concentration of 49 (APTT): Figure 17 shows a plot of 2.5 mg/kg in mice. Measured at various times after intravenous injection of Y-1 (X) or 5.0 mg/kg (△) shows a plot of APTT performed in parallel, where APTT is expressed as a percentage of the value of the control sample; FIG. 18 shows control AP as a function of intravenous injection dose of Y-1 in rats. Shows a plot of the percentage of TT, where APTT is the untreated Expressed as a percentage of the control value: Figure 2 shows a plot of thrombin time (TT) as a function of concentration.

Figure 20. Atroxin as a function of the concentration of KY-1, Y-1 and Y-49. Shows a plot of time (AT); Figure 21 (A-G) shows collagen (A), collagen plus 24 (B) or is 48 (C) μg/ml Y-49, collagen plus 24 (D) or 4 8 (E) μg/ml Y-1, plus 24 (F) or 48 ( G) Time relationship in platelet aggregation measured in the presence of μg/ml KY-1. shows a trace of the change in absorbance, as a number; Figure 22 (A-B) shows Y-1 (A), KY-1 and henoin activity in plasmin activity. Figure 22C shows a plot of the effect of varying the concentration of Curin (B); This is a graph summarizing the data of 2B (CB=Y-1): Figure 23A shows the clod time measured as PT, TT, APTT and AT. Figure 238 shows a composite plot of the concentration change of KY-1 for PT, TT. , Y-1 concentration change with respect to clod time measured as APTT and AT and FIGS. 24A and 24B show a composite plot of KY-1 (2 4A) or Heno(phosphorus (24B)) of TT and APTT as a function of concentration. Show the plot.

Detailed description of the invention 1.! Respect Unless otherwise specified, the terms defined in this chapter have the following meanings: "Anticoagulant activity" refers to the inhibition of the normal blood coagulation or clotting process. , as measured by one or more standard in vitro clot assays. as an extension of the time required for fibrin or clod formation. shown.

An “aryl ring” subunit includes at least one aromatic ring, i.e. a single or fused ring containing a 5- or 6-membered ring with the 6 pi electrons required for be. Examples are benzene, naphthalene, mixed aromatic and non-aromatic fused ring structures, e.g. For example, tetralin, as well as heterocyclic structures (fused rings, e.g. quinoline, isoquinoline , and indole).

"Highly cyclic compounds composed of aryl ring subunits" form a cyclic chain It is a cyclic compound formed by connecting ring atoms in aryl ring subunits.

"Cycloaddition bridge" refers to a ring atom of one aryl subunit in a highly cyclic compound. and the ring atoms of adjacent aryl subunits; This cycloaddition bridge and the ring atoms (such as (shorter paths) collectively form a "continuous chain of bonded backbone atoms". Explained in Figure 1 In the compound in which the chain is at the 2- and 7-positions of the naphthalene ring, and Formed by a crosslinking bond (R4) with 05 ring atoms in the naphthalene ring between the 7-positions. It will be done. In the compound illustrated in Figure 7, the chain at positions 2 and 6 of the benzene ring is and a crosslinking bond by three ring atoms in the benzene ring between the 2- and 6-positions (R4) formed by.

Similarly, the "non-chain ring atoms" in this high ring are those ring atoms that are outside of the bridge bond. Ru. In the compound illustrated in Figure 1, this non-chain atom is 5 naphthalene from ring positions 3-6. Contains ren ring atoms: In the compound of Figure 7, includes 3 ring atoms from positions 3-5.

“Sulfonic acid-derived substituent” refers to sulfonic acid, sulfonate, sulfonate. Phosphate, Sulfinate, Alkyl and Aryl Sulfone, SotN)IR sulfonamides of the form (where R is H or OH, ether, ester substituents with a radical, a ketone, or an acid component).

It, Synthesis of Highly Cyclic Compounds of Aryl Subunits In this section, the present invention Two general types of aryl hypercyclic compounds useful in antiviral therapeutic methods The synthesis of is described. The first type is as described in subsection eight. , composed of naphthalene subunits with substituents derived from sulfonic acid. It will be done. The second general type is the slug in the para position, as described in subsection B. It consists of a phenyl subunit with a substituent derived from fonic acid. 2 From the synthetic routes given in two sections, mixed subunits, e.g. How can a high ring composed of both phthalene and phenyl be synthesized? will become clear. This method of synthesis generally involves sulfonic acid derivatization. It can also be applied to high cyclic compounds composed of heterocyclic subunits with substituents. .

A. Highly cyclic compounds with substituted naphthalene subunits Figure 1 shows compounds for use in the present invention. The main structural formula of a high-cyclic compound composed of substituted naphthalene subunits for shows. One exemplary compound of this type is shown in Figures 2A and 2B, respectively. It is in the oxidized (I) and partially oxidized (II) forms. This compound is methyl or is a chromotope linked by a methylene (>CHz or >CH) bridge (R4). Tetramer of rope acid (l, 8-dihydroxy, 3,6-disulfonic acid naphthalene) It is. As shown, the methylene bridge and the "internal" ring atoms (currently l, 2.7 , and 8) are continuous chains with bonds R, =OH or 10 groups attached to the l and 8 positions. form. Non-chain atoms (current positions 3-6 of each substituent) are the 3rd and 6th ring atoms. Rz = sulfonic acid substituent on the atom.

The nature of the partially oxidized structure was determined by Hl and C'' NMR analysis and mass spectrometry. It is inferred from the evidence.

For the purpose of the following discussion and to illustrate the synthetic route, the above compounds are commonly used. Only the non-oxidized subunit form of the product is given. This compound is shown in FIG. As shown in B, 1 or more (R1 ketone (・0) groups, as well as rings and linked After the double bonds between the bridging methylene groups are exposed to air under heating and acid conditions, It will be understood that it can be partially oxidized. A similar reaction mechanism , a partially oxidized form of this compound, i.e., the structure shown in Figure 2B, or an additional It will also be understood that it is also generally applicable to similar structures containing R1=0 groups. Dew. However, the R3 modification reaction typically selectively modifies the R3-OH group and and leave the corresponding R1=0 group as is.

As shown below, the present compound is preferably a derivative of chromotropic acid [herein, R, is a polar substituent, such as OH1.0, cotH or ether, thioether -ether, ester, or thioether linked to an alkyl or aryl group , and combinations of these groups (e.g. only the OH group in the partially oxidized structure (substituted by one of the groups listed below).

R2 is a sulfonic acid, a sulfonate, a sulfine as shown in FIG. acids (-3O2H), and sulfinates, sulfones, and sulfonamides. is a substituent derived from sulfonic acid that can be R1 is H or B r or other halogen. In addition, as shown below, chromotopic acid derivative subunits The R4 bridge linked to the nit is preferably >CHR or >CR (partially acid (in which R is H or a small carbon-containing group, e.g. For example, lower alkyl, alkenyl, ketone, or carboxylic acid groups, or aryl This is the form of the group 1. This crosslinking is CH2NR''CH2-[wherein R' is Similarly, it may also be in the form of 1, which is H or a less carbon-containing group, such as a lower alkyl group. Ru.

Alternatively, the bridges in this high ring can be linked to aryl ring structures, such as the dimer shown in Figure 4. or bridges through heterocyclic rings, such as pyrrole or furan. It can be a ring structure, including similar structures formed by bridges.

The number of subunits can vary from 4 to 8, with high cyclics being preferred. However, it contains 4.6 and 8 subunits. In the reaction mechanism described below, The formed macrocycles have different subunit number (n) values, e.g. 6 and 8 subunits. A structure with a predominant n-4 structure (4 subunits) with an additional structure containing the unit mixtures of compounds.

Representative highly cyclic compounds that have been synthesized and tested for antiviral activity are: are identified in Table 1 by the R1, R2, R3, and R4 substituents. left in this table The numbers KY and Y in the hand column refer to the analogous name of the corresponding compound. for example , in which R1 is OH, R2 is SO2NH2, R2 is H, and Compounds where R4 is -CH2- are designated as KY-3.

Although not shown in this table, this compound may contain one or more of the formulas where the R+ group of is =0 and the adjacent bridge contains the carbon linkage of the double bond to the ring. Can exist in partially oxidized states.

Table 1 Figures 3A and 3B show two general schemes for the synthesis of highly cyclic chromotropic acid compounds. We will explain how to create it. The method illustrated in Figure 3A is based on chromotropic acid derivatives (chromotropic acid derivatives) It is a ring formation between an aldehyde (including motolope acid itself) and an aldehyde (RCHO), and a high ring compounds, such as the tetramer shown in FIG. 2 [wherein the chromotropic acid subunit is linked by an R-substituted methylene group, i.e., where R4 is >CHR( > CR). This synthesis mechanism is based on the form of RCHO A variety of different bridge-methylene provides a convenient method for the construction of highly cyclic compounds with R groups.

For example, high cyclic compounds with >CH2 bridges, such as KY-1 compounds (where m is constructed For this purpose, chromotropic acid (Ire) is reacted with formaldehyde. typical Reaction conditions are provided in Example IA for the synthesis of KY-1. Similarly, Y-4 8 is synthesized by ring formation with glyoxylic acid (Example 1c). KY-85 is in the presence of benzaldehyde, KY-97 in the presence of acrolein; and KY-110 is formed in the presence of pyruvaldehyde. small alkyl, A variety of other RCHO aldehydes with alkenyl, acid, and other hydrocarbon R groups are preferred. It will be well understood that this would be appropriate. Furthermore, the R bridging group is further removed after ring formation. can be qualified. For example, KY-193 is produced by bromination of Y-97. are synthesized.

In the method explained in Figure 3B, the ring formation of the chromotropic acid derivative (III) is , carried out by reaction with hexamethylenetetramine to form -CH2N(CH,)CH , -forms a three-atom chain bridge of the type. Ring-forming reaction for the synthesis of KY-346 The response is given in Example IJ. After the ring formation reaction, -CI(.

N(CH3)CI, - The bridge can be modified according to known synthetic methods. N-substituted bridge of -C8, N(R')CH, - [wherein R' is is one of the carbon-containing groups]. Some of the crosslinks in the partially oxidized structure are , =CHN(R')CH2 will have one form.

As mentioned above, in the compound of FIG. 4A (m also represents a cyclic bridge, in this case a phenyl bridge) It is a typical highly cyclic naphthalene. This compound, as detailed in Example 3, Combination of 1,2-benzenedimetatool and talomotorovic acid in acetic acid in the presence of hydrochloric acid Formed by reaction. Similar methods can be used with other cyclic bridges, e.g. used to link chromotropic acid subunits by role, thiophene, etc. can be used. Figures 4A and 4B show the unoxidized (vl) and partially oxidized (vl) of the present compound. ■ Shows the accounting of 11D.

For the synthesis of high cyclic compounds with selected Rt, Rt, and R1 substituents, 2 Two general approaches are available.

In one approach, chromotropic acid derivatives are used to form a ring-formed product. , including the selected R1, R2, and R8 substituents, or directly on the selected substituents. In order to be able to include substituents that can be modified immediately, Ru. This approach also includes SO□NH2R2 substituents, as detailed in Example IB. This is explained by the synthesis of KY-3. Here, the ring-formed chromotropic acid ( VII[) is first reacted with chlorosulfonic acid as described in Example IB, ゛It forms its corresponding R 1 =SO2C1 derivative (IX, Figure 5). next, This high cyclic compound reacts with aqueous ammonia to obtain the desired R2=S02 NHZ induction. Form a conductor (X, Figure 5).

A similar strategy was used to combine the final subunit with glycine (XI, Figure 5) at basic pH. used for the synthesis of KY-357 (R2=S02NHCH,CO,H) by reaction. was used.

Figure 6 shows that the ring-formed sulfonyl group of chromotropic acid is sulfinyl (Xl+ ) and alkyl sulfone or methyl sulfinyl ester (can be converted to Explain. The first step in this reaction is to convert its corresponding salt, as outlined earlier. formation of a sulfonyl derivative (IX). This compound is then treated with sodium sulfite. dicarbonate to form its corresponding sulfinyl salt (X[D. Reaction with dimethyl sulfate in the presence of IV, KY-158, n=4).

Similarly, highly cyclic compounds with various R1 substituents are capable of producing chromotropic acids after ring formation. It can be synthesized by modification. In the synthesis of KY-151, for example, (R , =OCHj) ring-formed chromotropic acids, as described in Example IF: Under basic conditions, the chromotropic acid methane was reacted with dimethyl sulfate to form a ring. form a ether. Similarly, KY-307(R, =O(CH2), co In the synthesis of tH), the ring-formed chromotropic acid is By the initiation reaction between motolope acid and 6-bromohexanoic acid under basic reaction conditions, It is first converted to a diether of hexanoic acid. − Further examples include compounds such as KY-272 and KY-294 [where R1 has the form of 0COR], by ring formation of chromotropic acid. The high ring compound formed was as detailed in Example 1 [for the synthesis of KY-270]. Under basic conditions, sea urchins are reacted with acid chlorides in the form of RCOCI.

A second general approach is that the selected substituents are The conductor is formed on the Natsutareno ring of the subunit, and then the subunit is The knit forms rings to form the desired high ring. KY-175 (R, For the synthesis of =SO,CH3), the chromotropic acid is converted into its corresponding R, = So3 React with thionyl chloride as described above to produce the C1 substituent be done. Furthermore, reaction with Na0CH1 and ring formation leads to the desired R2 substituent. . Reaction details are given in Example IH. Among other examples of this approach are KY- 123 (Example IG) and KY-147 (Example IE).

A synthetic method for forming highly cyclic compounds with selected substituents was developed by Chromotro Both prior derivatization of the subacid and subsequent derivatization of the subunit after ring formation. It is recognized that it can include.

For example, in the formation of KY-397 (R, = OCH,, R2 = S02 NHz) In this case, the chromotropic acid subunit reacts first at the R+ position and then reacts as described above. Sea urchins form methyl ether derivatives. After ring formation with formaldehyde, the compound The product is further derivatized at the R2 position, also as described above, to place the SO2Na group in place. Convert to the desired SO, NH2 substituent.

The above-mentioned KYY compounds can also be prepared by reaction in acids according to well-known methods. is rapidly converted to various sulfonic acids or sulfonate salts in the presence of suitable salts. It will be done. Thus, for example, some of the compounds shown in Table 1 contain ammonium Formed by cation exchange of protons in the presence of salts, e.g. ammonium chloride It is an ammonium salt. Furthermore, the highly cyclic compound can be combined with various metal cations (e.g. For example, Ca, Ba, Pt, Cu5Bi, Ge, Zn, La, Nd5 Exposure to (Ni, Hf, or Pd cations) causes metal salts and their metals to oxidize. Both metal chelates of highly cyclic compounds are chelated in the internal polar pocket of the compound. can be made.

The physical properties of some of the high cyclic compounds synthesized according to the present invention are illustrated in Examples IA, IB , by absorption, and by mass spectrometry, and by nuclear magnetism, as detailed in IC and IJ. It has been studied by gas resonance spectroscopy (NMR).

These compounds are tetrameric high cyclic compounds, such as those shown in Figure 2. , or mixtures with predominantly tetrameric forms.

B. Highly cyclic compounds with substituted phenolic subunits. The general structural formula of a high-cyclic compound composed of substituted phenolic subunits is show. One exemplary compound of this type is shown in Figure 8, which is a methylene bridge a tetramer of para-sulfonic acid subunits of phenol (XV) linked by It is. As mentioned, the methylene bridge and the “internal” ring atoms (currently 2.1 and and 6) form a continuous chain with R, =OH group bonded to the first ring position.

The non-chain atoms (current positions 3-5 on each substituent) have R,=sp on their 4 ring atoms. Has a sulfonic acid substituent.

Figure 9 illustrates a general method for forming this type of highly cyclic compound. this The high-cycle precursor shown on the left (XVI) is a tert-butyl calix )(n) is a class of compounds commonly known as arenes, where n is Phenol subunits (with t-butyl substituents at the para position) in high-cyclic compounds ), and the crosslinking bond is a methylene group. 4.6th and 8th place T-butyl calix arene with subunits is commercially available. .

In the sulfonation reaction shown in Figure 9A, the t-block with the selected number of subunits is Calix allene is a 4-position t-butyl group replaced by a sulfonic acid group. typically at 75-85°C for about 4 to 5 hours to substantially complete the conversion. Treated with concentrated sulfuric acid. Details of this sulfonation reaction are given in Example 2A. this The method uses a high cyclic compound with n=4 as shown in Figure 8, and 6 and 8 phenol subs. It has been used to produce related high cyclic compounds with units.

A similar method is used with a partially oxidized l-OH group as shown in 9B. Used for the synthesis of sulfonated calix arenes. here, The starting material for t-butyl calix allene is preferably at a temperature of about 100°C. is treated with concentrated sulfuric acid at between 150-170°C. This reaction is a subunit Effective against sulfonation of the ring and partial oxidation of internal OH groups. It is fruitful. As shown in Figure 9B, partial oxidation allows cross-linking to contribute to delocalized electrons. A conjugated high ring structure (XVI[I) can be derived. This conjugated structure is colored , and the evolution of the colored product can be used to monitor the progress of the oxidation reaction. child Details of the reaction are given in Example 2B.

The desired high cyclics can be produced under suitable crosslinking conditions, e.g. Para-sulfonic acid phenol ( or their precursors) can also be formed directly by the reaction of , will be recognized. This is due to the production of high cyclic products with carboxylic acid-containing crosslinking groups. This is explained by the reaction shown in FIG. In this method, phenol Para-sulfonic acid was reacted with glyoxylic acid under the same conditions as described in Example 2C. The reaction results in the formation of a cyclic structure represented by (XXII).

The hypercyclic compound formed as described above has a selected R3 group, a modified sulfonate. In order to accomplish the addition of Nyl groups and/or R2 groups, in Section IIA above. Modifications can be made according to the general procedures outlined. of R1 and R2 substituents The range is substantially the same as previously discussed. Figures 10.11 and 13 are Various reaction methods for modifying the R1 group of the high ring formed in the following will be described. figure In 10, the sulfonated structure shown in Figure 8 was treated with acetic anhydride and Forms a chill R3 group. Details of this reaction are given in Example 2C.

One would expect this structure to be hydrolyzed in the presence of serum esterases. Differences in the activity of its ester compound and its free OH compound would be expected to occur after administration of (m) in Example 2G. A similar reaction mechanism for forming sulfonic acid esters is described.

FIG. 11 shows sulfonamides, such as glycylsulfonamide (X A general method for forming XI) is described. The reaction described with respect to Figure 5 and Similarly, a sulfonated phenyl high ring compound (XV(1) is a chlorosulfonate Treatment with fonic acid forms its corresponding sulfonyl chloride analog (XX). Selection Further reaction with a selected amine, in this case glycine, provides the desired sulfonate. give amide. The details of the reaction are as follows for the synthesis of the R,=S02NH2 compound. in Example 2D for the synthesis of the glycylsulfonamide compound, and in Example 2E for the synthesis of the glycylsulfonamide compound. Given.

Figure 13 shows the general rule for the net substitution of R2.OH by the R, = carbon atom moiety. represents a synthetic method. In Example 2H, this reaction is carried out with the substrate (R+=OH, R2= tert-butyl, R4"CH3, n"4) is the intermediate (R,=CN, R2=te The process for providing rt-butyl, R4=CI (2, n=4) will be described in detail.

Next, further modifications are made to the product (R+・C02H, R3=SO, H, R4・CH2 , n=4). The modification of the substituent at the R position is a partially oxidized high ring It will be appreciated that Dew. That is, a reaction specific to the OH group of the ring leaves the -0 group intact and therefore , • provides a mixed R1 group containing 0 groups.

The R4 bridge linking to the chromotropic acid derivative subunit is linked to a group containing atoms, e.g. for example lower alkyl, alkenyl, ketone, or carboxylic acid groups, or as mentioned above. The form of an aryl group or the form of -CH2NR'CH2-! ! ! (here, Ro is likewise H or a group containing fewer carbons, such as a lower alkyl group); There is].

Alternatively, the crosslinks in the hypercyclics may be similar to the dimeric hypercyclics shown in Figure 4. It can be a ring structure including a ring.

Of course, as mentioned above, the number of subunits can vary from 4 (e.g., the structure of FIG. 4) to 8. High cyclics can vary up to 4,6 and 8 sars as preferred. including the unit. In the reaction mechanism described below, the formed macrocycle is Mixtures of compounds with different subunit number (n) values, e.g. 5-8 subunits. Predominant n4 structure (4 subunits) in addition to additional structures including knits including.

Representative highly cyclic compounds that have been synthesized and tested for antiviral activity include: Identified by R,, R2 and R2 substituents in Table 2 below. In the left hand column of this table The numbers KY and Y refer to their corresponding similar names, as shown in Table 1. R1 place bridging methylene carbons, both saturated and unsaturated, that are partially oxidized at Compounds that can carry groups are shown in Table 1.

Table 2 Compound RIR-3R4rL Y-10HSo, -C Toshi-8 KY-226010HSo, -CH,/-CH-8Y-490HSo3 -% -4 KY-225010HSo, -C)t, /-CH-4Y''-770HSo, - CM26 Y-4807OHSo, -C)L, /-CH-6KY-268010HSo, -cK2/-CH-3KY-2690/Co2CM,, So, -C)L, /-CH-4KY-2710/Co, CH, SO3-C)L, /-CH-3Y- 7807OHSo, Pigeon-C--8 Y-400010H5o2OCR3-C) I, -8 The above compound shown in Table 2, and their combination with R-groups according to well-known methods, as described above. various sulfonic acids or sulfonic acids by reaction in acids or in the presence of suitable salts. It can be easily converted to the sulfonate salt.

+j [, anticoagulant properties of high cyclic compounds as shown in one or more standard blood coagulation assays of a compound for use in Describes the ability to inhibit blood coagulation. Anticoagulant activity, or to some extent The assay used in the evaluation of the mechanism of anticoagulant activity, until it is limited activated partial thromboplastin time (APTT) assay, prothrombin time (PT) assay, thrombin (TT) time assay, fib Rinogen assay, levutylase (atroxin time, AT) assay, and plasma clot Contains a calcium re-deposition time assay. Such a test and its implementation Specific methods are generally known to those skilled in the art and are described by Brown (198 8).

The blood used for test compounds in such assays can be from a variety of vertebrate sources. human sources are particularly preferred, although they can be from mammalian sources. child In performing assays such as obtained using a clean venipuncture procedure to prevent contamination of the sample. Ru. Blood samples used in screening compounds useful in the methods of the invention Many standard blood collection samples contain calcium-binding or chelating agents. can be collected anywhere in the tube.

Plastic tubing is preferred; however, as a calcium binder Glass-walled VACUTA containing sodium enoate [NERTM tube is Suitable for carrying out most experiments in support of the invention.

Freshly aspirated samples should be kept on ice for up to 2-4 hours prior to further processing. and checked for the presence of clods or hemolytic phenomena: The tube is disposed of. Plasma was collected using the centrifugation procedure described in Example 4A. obtained from that sample. Plasma samples showing evidence of hemolysis are discarded.

This is because hemolysis is known to shorten clod formation time. . Ideally, plasma samples are stored on ice and tested within 8 hours of blood collection. be struck. Alternatively, samples may be collected for testing within 1 week of blood collection. Can be frozen at °C. Collection of blood samples for experiments supporting the present invention The general method used for assembly and processing is shown in Example 4A.

Tests for anticoagulant activity can be performed in vitro. where the compound is added to the isolated plasma or blood sample and The effect on clod time is measured. Anticoagulant activity in whole animals It can also be measured by administering a compound. Such in vivo , the evaluation of the effectiveness of a compound depends on how the drug is absorbed, distributed, and throughout the animal. indicates the extent to which it is biotransformed and provides a measure of bioavailability.

Plasma clot or recalcification time measures the integrity of the endogenous blood coagulation system. Set. Deficiency or inhibition of any of the factors of the endogenous blood coagulation system can lead to This results in an extended agglomeration time. Anticoagulants, both heparin and coumarin but prolongs the clotting time of plasma. In this test, as described in Example 4D, At 37 °C, the plasma was mixed with calcium chloride, mixed, and clot-formed. observed.

Effect of compounds on clotting time in vitro Various highly cyclic compounds Effect on clotting time of plasma in vitro as described in Example 5. Therefore, three concentrations were tested. The results of the study are shown in Table 3. phenyl Highly cyclic compounds KY225 and KY-47 have the highest anticoagulant properties in this assay. It showed solid activity. A concentration of 12.5ig of each of these compounds is , produced anticoagulant activity equivalent to 7.54 and 5.68 μg of heparin.

Phenyl derivatives Y-48, Y-77, Y-78, Y-100 and Y-1 and Naphthyl derivatives Y-20, KY-42, KY-1, KY-357 and KYY- 19 has the same ability as Ton in this test, and based on mass, Heparin The activity was approximately 1/10 to 1/20 of that of the active protein.

° Cannot be measured in this assay due to high anti-blood coagulation activity.

5. The activity of anti-blood coagulants has not been detected.

ゞ Values are expressed as microgram equivalents of heparin; i.e. 25 μg of KY-3 produces an anticoagulant activity equivalent to 1.5 μg of heparin.

Effect of Y-1 on clod formation in vivo Regarding clod formation time by oral administration The effects of Y-1 were studied in mice as described in Example 13. Table 4 is The results of the study are presented, where 500 or 600 mg/kg + 7 of y-i, respectively. ) 2 (D doses were administered at 30 minute intervals to female 5w1ss-W by gastric gavage) ebster mice.

Blood samples were collected 2.5 hours after the first dose. Blood plasma is blood plasma were analyzed for clot formation time. Compared to control (PBS treated) animals, Y-1 treated animals of animals showed increased clod time at both doses tested.

For comparison purposes, Y-1 (12 or 20 μg/ml) was added to blood from control animals. Plasma samples were also added directly, and the plasma clot times obtained indicated that the compound was within the time range reported after oral administration.

The reversal of the effect of Y-1 on plasma clot time by protamine is also shown in Table 4. vinegar. This will be described in more detail in Part 4 below.

Table 4 IN, no fasting, F, 24-hour fasting before testing’ Direct injection of Y-1 into control plasma addition at 3.2 minutes (12 μg/ml Y-1) and 4.1 minutes (20 ag/ml Y-1). The soil clod formation time of Y-1> was given.

2. Prothrombin time (PT) test Prothrombin time assesses the competency of the extrinsic blood coagulation pathway and factors If, Measure the presence of V, VI [and X. This assay is less than approximately 80 mg/dL It also serves as an indicative analysis of fibrinogen levels.

Therefore, prothrombin time regulates the production of factors II, Vll, IX and X. Inhibiting coumarin can be used in the evaluation of anticoagulant therapy. Blood service The presence of a relatively high concentration of heparin in the sample also distorts the measured prothrombin time. Extend.

The method used in the PT measurements can be shown in Example 4B. Simply put , this assay uses tissue factor, e.g. thromboplastin-calcium reagent (Dad e■ Thromboplastin OC, Becton Dickinson ) to the plasma sample. Soil clod formation that can be observed with the naked eye from the time of addition The duration of time is observed as PT.

Highly cyclic compounds KY-1, Y-1 and Y-49 were prepared in human blood as described in Example 6. Tested in PT assay using liquid. Change in test compound (0-25 Samples of human plasma containing thromboplastin (0 μg/ml, final concentration) - The clotting time that occurs upon mixing with calcium reagents was tested.

Among several highly cyclic compounds tested in this assay, KY-1 has the highest Figure 15) showed activity, and at concentrations as low as 30 μg/ml, protron showed an increase in bin time. Y-] showed intermediate activity. Y-49 is 90 It was inactive at concentrations as high as 0 μg/ml (not shown in graph). (but shown in the raw data).

b. In vivo administration of high cyclic compounds The high cyclic compounds of the present invention It was given intravenously to rats at a dose of mg/kg (2 rats/dose). Ru. Next (5 hours post-dose), venous blood samples were collected for PTSAPTT and fibrin. Tested for linogen. Table 5 shows the results from saline-treated controls. Shows the percentage change in observed PT as compared to blood plasma PT . A moderate increase in PT was observed for some of the compounds tested. It was. Most prominently KY-225 and KY-226 are partially oxidized hypercyclic compounds with 4 and 8 phenyl subunits, respectively.

Table 5 Intravenous administration of highly cyclic compounds in PT, APTT and fibrinogen effect IN, no effect ! (3 and 131%) 8 (21 and 142%) In another series of studies, Y-1 was administered at 300 and 450 mg/kgc7) (p.

0) dose was administered to rats. A blood sample is taken and in Example 14 PT measurements were taken at times from 0.5 to 24 hours post-dose, as described.

The results of these experiments are shown in Table 6. Here, a significant increase in PT (18% ) was observed in the mouth for 4 hours after administration of 450 mg/kg Y-1 by gastric gavage. It was. The reproducibility of this effect indicates that additional compound was administered to some of the animals at 23 hours. administered and tested by PT test at 24 hours. also, A significant (19%) prolongation of PT was observed. APTT extensions are also listed below. was observed at both doses of Y-1.

Table 6 Time course of the effects of oral Y-1 on plasma PT and APTT 1, 300 mg/ kg p, 0゜ Il, 450 mg/kg p, o.

reproduction 1 0 hour control animals given saline PT and APTT at 2 seconds, 4 standard error of the mean for animals pretreated with 450 mg/kg p,o. received an additional oral dose of 225 mg/kg at 23 hours.

3. Activated Partial Thromboplastin Time (APTT) Assay This APTT The assay is used as a measure of the integrity of the endogenous blood coagulation pathway. it is , the presence of all blood clotting factors in the intrinsic pathway except platelets and factor XIII. , and that heparin is associated with some of the factors of the intrinsic pathway (Xla, [Xa , Xa, thrombin) to monitor treatment with heparin. commonly used in

The detailed method used in performing this assay can be shown in Example 4C.

Briefly, plasma samples contain activated thromboplastin, e.g. Ac tin■ activated cephaloblastin reagent (Becton Dicki nson). The tube containing this mixture is made of calcium chloride. Place in a 37°C water bath for 3 minutes prior to addition. This sample then observed for the formation of a fibrin network.

Platelet-depleted plasma (human) is KY-1, Y-!, as described in Example 7.　 , and was used to test the effects of Y-49. Figure 16 shows these experiments. shows the effect of Now, to create a concentration-effect plot, for each compound Varying concentrations of were tested. Among the three compounds tested, KY-1 Y-1 produced the highest activity in the assay, while Y-1 showed less activity. Y- 49 was inactive at the highest concentration tested.

In another experiment, compound KY-1 was administered to human blood samples at a concentration of 50 μg/ml. and one set of standard clinical tests where one of them is APTT. Tested in Tuto. As shown in Table 8 in Part 9 of this section, the availability of APTT is A significant extension was observed.

b. Effect of in vivo administration of highly cyclic compounds in APTT when rats were quiescent. Results from an experiment in which polysulfonated hypercyclic compounds were given intravenously. are shown in Table 5 above. Percentage change in APTTs compared to untreated animals Determined by comparison. With some exceptions, the processes described in the methods of the invention The compound showed a dose-dependent prolongation of APTT. In particular, partially oxidized The phenyl derivatives KY-225 and KY-226 performed best in this assay. also showed high activity, while Y-48 and Y-42 followed in descending order of activity. there was.

Compounds Y-49, Y-20 and KY-3 showed slight activity.

Whole animal treatment with polysulfonated hypercyclic compounds has been shown to prolong APTT. This observation was further investigated using rats as test animals. It was done. In this case, as illustrated in FIG. 17, compound Y-1 is administered intravenously to ．． Animals were given doses of 5 and 5.0 mg/kg, and blood samples were , were aspirated from four different animals at various times after administration. APTT is AP Induces rapid lengthening of TT to approximately 300% or higher than normal However, the anticoagulant effect remained until 4 to 6 hours after administration of 2.5 mg/kg. , remained approximately 20% above normal at 12 hours after administration of 5.0 mg/kg. day data for each set of animals to normalize the values obtained in different experiments. It is expressed as a percentage of the control value.

Figure 18 shows the APT of Y-1 by plotting the 30 minute values of different experiments. T dose-response relationship is illustrated, where Y-1 at 2.5.5 and 25 mg/kg was administered intravenously to rats. High doses of Y-1 are associated with significant effect, and the linearity of its dose-response curve indicates that the blood coagulation effect of Y-] We demonstrate a high degree of predictability for

The effect of oral administration of Y-1 was also tested in the above assay for PT.

As shown earlier in Table 6, the prolongation of APTT was increased by 300 and 450 mg/kg Y -1 observed. At a dose of 450 mg/kg, this effect occurs approximately 4 hours after administration. Although the peak occurred shortly after administration, it was still clear even 16 hours after administration. These training The present study shows that the highly cyclic compounds of the present invention are active when administered orally and that they We are confident that the effects of this will last for a relatively long time.

4. Reversal of blood coagulation effect of high cyclic compounds by protamine sulfate High cyclic compounds of the present invention Blood and plasma treated with the compound are used to measure blood clotting activity induced by the compound. In order to treated with protamine as indicated. Murine plasma is described in Example 13. and as shown in Table 4 above, the soil that occurs after oral administration of Compound Y-1 Tested for clumping time. In these studies, protamine sulfate was was added to plasma samples from treated animals at a concentration of 10.4 μg/ml. . The addition of protamine sulfate to the sample was induced by the clotting time Y-1. This resulted in a reversal of the extension.

The effect of protamine sulfate on reversing the effects of Y-1 was demonstrated when animals received 25 mg/kg of Y-1. Using the procedure described in Example 14, except that 1 was given intravenously, further tested in the kit. After 26 to 28, protamine sulfate was added as well. was administered intravenously at a dose of 25 mg/kg. blood sample first drug were collected 30 minutes after injection. The results of these studies suggest that PT and APTT The result was an increase of 161% and 831% of the PBS control value, respectively. Ta. Protamine sulfate treatment resulted in an overall reversal of the effects of Y-1 administration on PT; and resulted in an overall reversal of most of the effects in APTT.

Table 7 1 Value expressed as standard error above the mean 4. Fibrinogen assay Fibrinogen is a fibril that naturally polymerizes to initiate clot formation. is a polymeric precursor from monomers. As explained in Figure 14, this blood In the coagulation cascade, fibrinogen plays a role in the proteolytic action of thrombin. is converted into fibrin. The fibrinogen content of the blood is associated with many disorders will be affected by Fibrinogen deficiency reduces clot formation .

The presence of a relatively high concentration of heparin in the sample indicates that exogenous and added heparin For inhibition of added thrombin, use a thrombin time assay (see next section). with artificially low values of fibrinogen content, as determined by The result can be as follows.

The fibrinogen content of the blood is determined by excess thrombin, as described in Example 4H. was measured by adding chloride to a diluted plasma sample and recording the clot time. It can be done. Intravenous administration of various highly cyclic compounds was withdrawn from rats previously given The fibrinogen content of the collected plasma samples is shown in Table 5 above. The higher one At doses of KY225, KY226 and, to a lesser extent, Y-48 It is clear that this resulted in a decrease in fibrinogen content in the blood. be.

In addition, KY-1 analyzes blood plasma in a standard set of clinical tests. In a study, it was added directly to human blood at a concentration of 75 μg/ml prior to A significant decrease in fibrinogen content was observed (shown in Table 8 below). ).

5. Thrombin time Thrombin time is the rate of fibrinogen in the blood, catalyzed by the enzyme thrombin. Another measure of the conversion of fibrin to fibrin. extended thrombin Time lowers fibrinogen levels, heparin, and other thrombin inhibitors. inhibitors can be caused by a number of factors, including e.g. fibrin degradation products. can be done.

This assay uses a reserve amount of purified thrombin for platelet deficiency as described in Example 4G. added to the plasma sample and record the amount of time required for clot formation in the plasma It is done by doing.

Compounds KY-1, Y-1 and Y-49 in TT as described in Example 8 The results of the study where the effectiveness was tested are shown in Figure 19. Here, the plasma sample The presence of KY-1 in the sample significantly increases thrombin time, whereas Y-49 , indicating no activity at the concentrations tested.

Levutilase, which converts brinogen to fibrin, is not affected by heparin. do not have. Therefore, it is important to note that it is important to understand that fibrils in the blood of patients undergoing treatment with heparin. Available in tests for brinogen content. Fibrinolytic agents, e.g. For example, blood from patients receiving streptokinase may be Figure 2 shows Xin and thrombin times. General procedure for performing a labutilase assay is described in Example 4F.

Figure 20 shows KY-1 in human plasma atroxin time in vitro; The effect of increasing the concentration of Y-1 and Y48 is shown. Atroxin time by KY-1 Only the extension of concentration (25 μg/ml) than required to significantly prolong the incubation time. Occurs at higher concentrations. These results are similar to those observed with heparin. Here, higher concentrations can be shown to prolong levutylase time. and, as reported in Part 4 above, the content of fibrinogen The effect of KY-1 on heparin in the presence of thrombin during this assay may be indirectly We show that this can be explained, at least in part, by a ton-like effect.

7. Aggregation of platelets Compounds that interfere with platelet aggregation, such as aspirin, can cause prolonged bleeding times. This results in The integrity of the platelet population in a blood sample was determined as described in Example 4H. Sea urchins, platelets increase in response to platelet aggregation promoting factors such as ADP or collagen. It can be determined by the characteristic change in absorbance of an enriched plasma sample. Example 1 Regarding collagen-activated platelet aggregation performed as described in 6. In a study, KY at concentrations of 24 μg/mt and 48 tt g/ml -1 and Y-49 have measurable effects on collagen-induced assembly. No fruit was produced (Fig. 21, B, C, F, G). 24μg/ml and 48μg/ml Y-1 in ml significantly inhibits collagen-induced platelet aggregation (Figure 2L D, E).

8. Plasmin activity Compounds KY-1, Y-1 and Y-49 were prepared using plasmin as described in Example 4I. tested for its pigmentation effects. Figures 22A and 22B show that these studies Show the results. Both KY-1 and Y-1 play a role in the pigment-forming activity of plasmin. , showed a concentration-dependent effect. Enzyme activity measured by change in absorbance per minute In terms of gender, it is 2o, 40. and the inhibitory effect of Y-1 at 80 Bg/ml compared to the control 15%, 28%, and 31% of plasmin activity, respectively (Figure 22A). : The inhibitory effect of KY-1 at 9 and 19Bg/ml was 35% of the control activity, and 52%. At higher doses of KY-1 and C8, the inhibition remains KY-1 (64 μg/ml) caused 35% inhibition; and Y-1 (233 μg/ml) caused 34% inhibition. Convert to TT Heparin at equivalent anticoagulant doses calculated had no inhibitory effect. (Figure 22B).

9. Effect of KY-1 in human plasma in clinical assay Human blood samples were aspirated. , and sampled in an accredited clinical testing laboratory for a set of standard clinical assays. Prior to transferring the pull, KY-1 was added to the final concentration (75 μg/ml). It was done. The results of these tests are shown in Table 8. All standards at the concentrations used Standard assays recorded abnormal blood coagulation parameters.

Table 8 Effect of KY-1 (75 μg/ml) on blood coagulation of human plasma: ratio of clinical assays comparison Naf in analyzing the effects of these classes of compounds on blood coagulation. KY-1 and Y-1 serve as representative of thyl and phenyl macrocyclic compounds. both The latter compound overall extended the clod time, with almost equivalent ability (Table 3). As mentioned above and summarized below, these Compounds can have slightly different predominant mechanisms of action.

KY-1 significantly inhibited PT, APTT, TT, and AT in vitro. It was extended (Figure 23). However, concentration-effect studies (0,6-180Bg/ ml), the prolongation of AT by KY-1 is similar to the heparin-like effect at 150 μg/ml. It was clearly shown that this only occurs at concentrations near 1. This is all like heparin Further experiments were conducted to determine whether the effect was due to , heparin and KY-1 at concentrations that gave similar prolongations in thrombin time. tested (Fig. 24 A-B). 19Bg/ml KY gave a TT of 38 seconds -1 indicates a pronounced APTT prolongation up to >300 seconds, while a TT of 49 seconds Heparin at 0.41 II g/ml gave an APTT of 81 seconds. child These data suggest that the blood coagulation effect of KY-1 is entirely due to the heparin-like effect. This suggests that it is not something that exists.

y-i also extended PTSAPTT, TT and AT. Contrary to KY-1 , the effect of Y-1 on AT was minimal, showing prolongation even at 400 μg/ml. There was no (Figure 20). Concentration-effect studies were performed on PT and PTT at 20 Bg/ml. 2 x base), but there was no significant extension in TT at this concentration. No significant extension was shown. Prolongation of TT was only seen at ≥75 μg/ml (Fig. 1 9). These data indicate that the blood coagulation effect of Y-1 in the range of #20 μg/ml is , suggesting that it is unlikely to be due to heparin-like effects. P Simultaneous lengthening of T and APTT allows Y-1 to be directed towards a common pathway. It suggests that it can be done. Potential targets include factor X, factor ■, prothrombin (Factor II) or phospholipids. Furthermore, Y-1 showed antiplatelet activity .

Y-1 and KY-1 (high cyclic phenyl derivative and high cyclic naphthyl derivative, respectively) The profile of the different activities of the compounds (chosen as representatives of suggests that it can be preferred in different indications requiring treatment by . For example, in these cases, antiplatelet and anticoagulant treatments may be In certain forms of coexisting valvular heart disease, compounds exhibiting Y-1 activity may be indicated. shown.

Both Y-1 and KY-1 are effective in vivo when administered parenterally or orally. It was shown to be active in The peak effect after oral administration is dose dependent. and 450 mg/kg of Y-1 as assessed by APTT. observed between approximately 0.5 and 4 hours after oral administration (Table 6). Evaluated by APTT As shown, the apparent amount of this compound after intravenous administration of 2.5 and 5 mg/kg The half-life of the fabric is less than ## and the reduction half-life at about 3-4 hours in APTT. The decline is defined as being above control levels for at least 4 hours in a dose-related manner. remained (Figure 17).

Studies using Y-1, mentioned earlier, show that this compound has a heparin-like mechanism of action. It appears that the effect of protamine sulfate can be antagonized by protamine sulfate. proved. This finding is a relative convenience against accidental overdosing with this compound. suggests an approved antidote.

Il, treatment method In the methods of treatment of the invention, aryl rings of the type described in Section 11 The compound is administered into the bloodstream of a patient at risk of developing thromboembolism. Said As such, the compounds of the present invention are useful in blood cells where they produce anticoagulant effects. It appears to have a direct effect on a factor or groups of factors present in the coagulation cascade. be.

The main routes of drug delivery are intravenous and oral, but the preferred route is oral. Ru. Other methods of drug administration are effective at delivering drugs into the bloodstream. Methods of insufflation, eg intra-arterial, subcutaneous or nasal, are also contemplated.

The dose administered was determined to be effective in prolonging blood clotting time within the patient. A determined pharmaceutically effective dose. As seen above, 10-10 Concentrations of compounds in the range of 0 μg/ml have been shown to increase plasma K. cerevisiae in vitro. Inhibition of blood coagulation as assessed by mucous redeposition time, APTT or PT. generally effective against Therefore, for most indications, the effective dosage is This will produce a concentration of the compound within the above range in the liquid.

One consideration in treatment regimens with anticoagulants is that they may produce life-threatening bleeding. In the perspective of its inherent potential, it equates with an antidote to its treatment; That is, the sudden onset of the action of the compound in the event of excess of that compound. This is the mode of disconnection. In the treatment method of the present invention, an effective antidote, sulfate protein, is used. Rotamine has been identified. In an overdose incident, the amount of the administered compound An amount of protamine sulfate that is approximately equal to or less than the amount of The amount of protamine sulfate will be effective in antagonizing the It is expected that this will depend on the time after administration.

A. Treatment by intravenous administration・ Studies on the pharmacokinetics and efficacy of compounds of the invention administered intravenously have been previously It is described in. Briefly, aryl highly cyclic compounds are Producing an anticoagulant effect for 4 hours or longer in a dose-related manner Show off.

B. Treatment by oral administration Earlier studies supporting the present invention showed that after oral (gavage) administration, the high cyclic form of the present invention The compound produces significant anticoagulant activity for 4 or longer periods of time and is effective. shows that the duration of is dependent on its dose.

The following examples describe methods for synthesizing tetrameric high ring compounds according to the present invention, as well as enveloping. Their use in the inhibition of blood coagulation by membrane viruses is described. child The examples are intended to be illustrative and not to limit the scope of the invention.

material All reagents were from Aldrich Chemical Co, or other commercial sources. Obtained from.

A, KY-1(R+”OH,R*”SosNa,R2=H,R4= >CHt) 41mM aqueous solution (50ml) of disodium chromotrope , 15 ml of 37% formaldehyde was added to give a final molar ratio of 5:1 formaldehyde. Dehyde: gave chromotropic acid. This mixture was kept in a fixed flask for one week at room temperature. The reaction was carried out while stirring inside the container. The resulting dark red solution (70 ml) was purified under vacuum. Filter and the filtrate, after concentration, is precipitated by adding 200 ml of acetonitrile. I set it. The precipitate was collected by filtration and removed as dry under vacuum. K.Y. The yield of -1 was 95%. This compound was characterized as follows: Point (M, P,) >300°C: HPLC in CH, CN/MeOH/H, 0/TFA: 14° 48" single block Load peak: (IR/KBR) = 3425 (OH), 1638 (Ar), 1181.1044 (So 3) cm-': UV (H, 0): 238. 0.358.5 nm Molecular weight: 1505 by mass spectrometry Ql+1):HIN MR (CD20D), chemical shift on δ scale: 5.20 (CHt), 8 ．． 01 (ArH) ppm; CI3N9IR(0,0), chemical shift on δ scale・27.19.120. 18.121゜69, 122.06. 122.67, 133.30. 1 42.97. 154.42 and 181 ppm.

Analysis: (CzJ + oO + sS 4 Na4) 2 X 6 H20 or ( C2J ++0 +sSa Nai) t X 5 Ht O detection value (Fou nd): C33,17, H2,54, Na 11.93 Calculated value (Calcul ated): C32,75, H2,23, Na 11.41: C33,16 , H2゜13, Na 11.56゜ B, KY-3 (Rr = OH, R*・So 2NH,, R, = H, R, ・-C H2-)KY-2 (2mM) was treated with 5ml of chlorosulfonic acid and mixed. The mixture was stirred at 50°C for 1.5 hours. Crushed 20g of the resulting mixture. added to ice to precipitate the crude chloride product, which is collected by filtration and then evaporated. Washed with water.

Dissolve the crude chloride product in 100 ml of 25% ammonium aqueous solution and incubate for 2 hours at room temperature. I responded. Concentrate the mixture in vacuo and dissolve the remaining oil in a small amount of water. and filtered. This product was precipitated by adding acetonitrile to the filtrate. , and collected by filtration and washed with acetonitrile. This compound is It was characterized as follows: Melting point (M, P,) >300°C; Mass spectrometry: 1452 (M-7NH2): CH, CN/MeOH/H, 0/T HPLC in FA: 11’46” single peak; ([, R/KBR) = 3430 (OH), 3187.1686 (NH2), 1637 (Ar ), 1211.1110, +044 (SOs) cm”’: LIV ( H20): 246 nm: H' NMR (D, 0), chemistry on δ scale Shift: 5.15 (CH2), 7.5-8゜2 (Ar) ppm: Analysis (C44H4002J +2Na4) -16820 detection value (Found ): C22, 62, H3, 93, Na 8.82. S 17.17. Na 5.44 + Calculated value: C28,51, H3,89, Na　9.07. S 17.28. Na 4゜97゜ C, KY-42(R,=OH,R,=So,Na,R,=H,R,=>C Chromotropic acid disodium salt (10 mM) in 50 ml of water (HCOOH) Glyoxylic acid (in 5 ml water) 0. OmM) and 10ml of 37% hydrogen chloride. Mix together at room temperature. Boil this mixture for 8 hours and the color of the solution will be dark red. It changed color. The resulting solution was added to 50ml of water and filtered. filtrate was concentrated and ethanol was added to precipitate the KY-42 product. The yield is , 87%. This compound was characterized as follows: Melting point (M, P,): >300°C: Mass spectrometry: 1623 (M-3H,0 ); HPLC in CHs CN/MeOH/H, 0/TFA: IO’36” Single peak: ([R/KBR) = 3452 (OH), 1801.1 719 (Co), 1638 (Ar), 1206.11050 (So) cm-'; UV (H20): 238.0.351.5.520 nm+H' NMR ( D20), chemical shift near on δ scale, 10 (CHCo2H), 8° Oo (ArH) ppm; C” NMR (D20), chemical shift on δ scale: l16.04.118. 90.120°94, 121.27.122.30.124.30.124.6 8. +26.60. +28.37.136.48゜136.71.140. 50.143.93.144.26.145.75.152.0+, 154. 33.156°01, 156.67: Analysis: (C41H4040S I Nai) 4-4820 detection value (Fou nd): C32,74,H2,50; Calculated: C 32,58,H2,71;D,KY-123(RI=OH,R2=S02Na , R,=H,R4=>CH2) KY-1 (2mM) was added to 5ml of chlorosulfonic acid. and the mixture was stirred for 1.5 hours at 50°C. The resulting mixture The liquid was added to 50 g of crushed ice to precipitate the product, which was collected by filtration. , and then washed with ether. Add the crude sulfonyl chloride product to 4ml of sulfite in water. treated with sodium chloride (20mM). The reaction mixture was divided into small portions over 2 days. Maintain slightly alkalinity by adding 50% NaOH in several portions. It was. After solvent removal, ethanol was added to precipitate the product and 50% HSOa was added. acidify by addition and then precipitate the sodium sulfate by addition of ethanol Ta. Mix the ethanol phase with ether (l·2.V/V) to precipitate the desired product. I let it happen. Product yield was 39%.

E, KY-147 (R, = OH, R2 = = S02 NHCH,, R, = H, R, =>CHri N-Methyl chromotropic acid chloride with chromotropic acid (disodium salt) It was formed by reacting with thionyl chloride in the presence of DRIF. this reaction was carried out for 4 hours at 80°C with stirring. Remove solvent and excess thionyl chloride. After removal in vacuo, ether is added to precipitate the chromotropic acid chloride and then was collected by filtration and washed with ether. Add 20ml of the crude product to methyl Added to amine and stirred for 2 hours. Remove all solvent from the resulting material. After evaporation, the residue was dissolved in 200 ml of cold methanol and filtered. to the filtrate Add acetonitrile to produce the product chromotropic acid methylsulfonamide. precipitated. The yield was 56%.

Chromotropic acid methylsulfonamide (2mM) in 3ml water at room temperature for 1 week. The mixture was reacted with 37% formaldehyde (1 ml). Add acetonitrile , the product was precipitated, collected by filtration, and washed with acetonitrile. yield was 85%.

F, KY-151 (RI=OCHi, R2=Sow Na, R2=H , R, =>CHt), Y-1 (50mM) was added to 80ml of Na0)1 aqueous solution ( Dissolve in dimethyl sulfate (0.2M NaOH), heat to 50°C, and dimethyl sulfate (0.2M ) was slowly added over 1 hour. Stir the mixture continuously for 2 hours, then The mixture was left at room temperature for 2 days.

Saturated NaCl solution (100 ml) was added to the resulting material and filtered. . The precipitate was washed sequentially with ethanol, acetonitrile and ether. dry matter was dissolved in 100 ml of methanol and filtered. Concentrate the filtrate and was added to precipitate the methyl ether of KT-1.

G, KY-123 (R, = OH, R2...SO□CH,, R!, H, R4= > KY-1 from CHt Example IA was first treated with thionyl chloride and chromotrope sulfonyl chloride was produced. This compound in the presence of sodium dicarbonate The corresponding ring-formed chromotropic acids were reduced with excess sodium sulfite. A sulfonic acid sodium salt (R2-.SO2Na) was produced. This sulfonic acid was treated with dimethyl sulfate in the presence of NaHCO2 and as described in Example IA. I made it up. Product yield was approximately 21%.

H, KY-175 (R, = 0)1. R,=SO,CH,,R,=)1. R4 ・>C)I 1) Chromotropic acid is first treated with thionyl chloride, and then chromotropic acid is treated with thionyl chloride. Trope acid sulfonyl chloride was produced. This compound is then treated with the sodium salt treated with sodium methoxide in the presence of sodium methoxide. This product as described in Example IA and formed a highly cyclic compound. Product yield was approximately 29%.

1, KY-270 (Rl = OCOCH2, R2 = = S03 Na, R, =H, R4=>CH KY-1 from Example IA (0.1 g of N Dissolved in 3 ml of water containing aOH. To this, 1 g of acetyl chloride (13 mmo 1) was added and the reaction was allowed to proceed overnight with stirring at room temperature. 25m1 of ethanol was added to precipitate the product. Dissolve the crude product in methanol and and filtered.

The filtrate was precipitated giving a yield of 87%.

J, KY-346 (R, = OH, R, = SO, Na, R, = H, R4 = -CH2-N(CH,)CH2-) Disodium salt of chromotropic acid was added to a concentration of 50mM in 80ml of water. Stir at 50°C until the solution becomes clear, then add hexamethylene. Tetramine (50mM) was added to the above solution and stirred continuously at the same temperature for an additional 2 hours. I added it while doing so. At this time, the color of this liquid mixture changed to dark blue. This mixture The combined solution was stirred at room temperature for 2 days. Filter the resulting dark blue solution and strain the filtrate. was concentrated, evaporated by flask and then treated with 200 ml of methanol to give the product KY-346 was precipitated. The yield of KY-346 was 85%. This compound The object was characterized as follows: Melting point (81.P,) >300°C: CH=HPLC in CN/MeOH/H20/TFA: 13’07” Thin Gulbeek; (IR/KBR) = 3425 (OH), +626 (Ar ), 1197.1052 (SO3) cm-': UV (H20). 232 ．． 0.377.5 nm analysis: (CI3H++Os Nst Nat) a X 12 H20 detection value (Found): C33, 17, H3, 13, N 2.75 Calculated value: C33, 98, H3, 59, N 2.96° molecular weight/1668° by gel filtration A, Y-49 (R, = OH, R, = SO, + (, R4''-CH2-, n = 4) 4-tert-butyl calix (4) arene (10 g) at 0 ．． treated with 200 ml concentrated H2Na4 at room temperature for 5 hours, and then for a further 4 hours 7 Placed in an oil bath at 5-85°C. This occurs when no water-insoluble substances are detected. The reaction was completed. Drop the resulting oil onto 500g of crushed ice, The solution was then filtered under reduced pressure. After removing water from the filtrate, acetonitrile (5 00 ml) was added to the residue and left for 4 hours to precipitate the crude product, which was then was collected by filtration and washed with acetonitrile, ethyl acetate and ether. Ta. The yield was 8 g (yield 73%). methanol-ether or methano Recrystallization of the crude compound using alcohol-acetonitrile system gave the pure product. .

Single crystal compounds were also discovered during this recrystallization process.

A similar method is used for Y47(R+ = OH, Rs = SOa H, R4 = -CH 2-1n=6) and Y-1(R+=OH,R,=SO,H,R,=-CH , -, n''8).

B, KY-225 (Rl = -OH, = 0. R2 = SO, H, R, = >CH. ＞　CH.

n=4) 4-tert-butyl calix (calixX4) arene (1 g) was prepared at room temperature. 0°C for 5 hours, then treated with 10ml of 95-98% sulfuric acid for 5 minutes at 160°C. Ta. After cooling the resulting mixture, pour the mixture onto 100ml of crushed ice. Pour slowly and filter. The solution was evaporated and the residue was poured into a 3° Oml solution. added to setonitrile to form a significant amount of precipitate, which was recovered by filtration. The mixture was washed with acetonitrile. Dissolve the crude product in 20ml methanol and and the product was precipitated by addition of diethyl ether. The yield was 84%. It was.

A similar method can be used for Y-48(R, = -OH or =0. R2 = SO□H, R 4 = -CHt -, n□6) and Y-226 (R, = -OH, R2 = SO, It was used in the synthesis of H,R, = -CH2-, n.8).

0-acetyley of C, Y-1) (R, = -0COCHs, R2 = S02 Na, R, => C82, n・8) Under nitrogen, Y-1 (0.4 g) was dissolved in NaOAc (305 mg) and acetic anhydride (20 m The mixture was refluxed for 3 days in the stirred mixture of 1). After cooling to room temperature, filter the suspension. passed. The solid was washed three times with ether (25ml) and dried in vacuo. . The resulting solid was mixed with methanol (50 ml) and activated carbon (15 ml). sonicate in a mixture of 0 mg), filter, and dissolve the black precipitate in MeOH (10 ml) twice. The filtrate was concentrated in vacuo. The resulting residue was diluted with MeOH /acetonitrile mixture. After filtration and freeze drying, the product (240 m g) was obtained.

"CNMR (D 20.6'): 173.9.151.6, 144.1.13 5.6.130.1.34.2° and 22.4° D, Y-78(R+　　　　-OH, R2=　So　2　NH2, R4=　>CH 2, n”8) Under nitrogen, Y-1 (Ig) was mixed with oo sulfonic acid (20 ml) for 1 hour. +1: Heat at 60-70"C. Cool to room temperature, pour the oil into ice water and set aside. and filter the precipitate. After washing the precipitate with cold water, the crude product was poured into 100 ml of 25% ammonia. Dissolved in an aqueous sodium solution and allowed to react at room temperature for 2 hours. Concentrate the mixture in vacuum and dissolve the residual oil in a small amount of water and filter.

The product is precipitated by adding acetonitrile to the filtrate and by filtration. Collect and wash with acetonitrile.

E, Y-1 (7) Glycyl sulfonamide (R, = -OH, R2 = SO! NHCH.

COz　H, R4=　>CH1, n=8) Under nitrogen, y-t(xg) was chloroformed for 1 hour. Heat at 60-70″C with sulfonic acid (20ml). Cool to room temperature. , pour the oil onto ice water and filter the precipitate. After washing the precipitate with cold water, the crude product was dissolved in 50 ml of a solution containing 5.7 g of glycine and 2.1 g of NaOH, Then, stir at room temperature for 2 hours. After removing all solvent from the resulting material, Dissolve the residue in 200 ml of cold methanol and filter. Dilute the filtrate with acetonate Add to the rill to precipitate the product.

F, Y-49 crosslinked with acetyl (R+ = -OH, R2 = SOs H, R 4=-CHCO2H, n=4) 4.3 g of p-hydroxybenzenesulfonic acid was heated at 100°C for 2 hours at 30 m 1 g of glyoxylic acid in 18% concentration of HCl. reaction product After drying under reduced pressure, 50 ml of methanol is added and undissolved impurities are removed. Removed by filtration. The product is precipitated from the filtrate by addition of ether and then filtered. Collected by filtration and dried in vacuo.

G, Y-49 toluenesulfonyl ester (R, = -3O3C@H,C Hs, R2=SOs H, R4=>CHCO2H, n=4) Under nitrogen, Luenesulfonyl chloride (1.9g) in dry sodium carbonate (1.06g) , added to dry dimethylformamide (10 ml) and Y-49 (0.75 g). Ru. - After refluxing overnight, the resulting mixture is cooled to room temperature and filtered.

Dilute the filtrate with ether to precipitate the crude product.

Recrystallization from an acetonitrile/ether solution gives the product.

Carboxylic acid derivative of H, Y-49 (RI = -Co□H, R2 = SO, H, R4 =>CHCO2H, n=4) Trifluoromethanesulfonic anhydride (1.0 ml) was heated at 2.6°C under nitrogen. tert-butyl-4-methylpyridine (1,25 g) and 4-tert-butyl Calix (4) water-cooled dry diluted solution containing arene (0,65g) Add to lolomethane solution (10ml). After stirring overnight at room temperature, pour the mixture into a pen. Dilute with chlorine (10 ml) and filter. The filtrate was cooled in aqueous NaOH solution. and extracted with a water-cooled IN aqueous HC1 solution, then with a saturated aqueous NaCl solution, and anhydrous sulfuric acid. Dry over sodium, filter through a pad of silica gel, and concentrate in vacuo. Shrink. The residue was dissolved in dry diisopropylethylamine (10 ml) and trimethylsilane. Lilcyanide (0.5ml) and palladium tetrakis-triphenylphosphine (20 mg). Reflux under nitrogen overnight and cool to room temperature. After cooling, ether (50 ml) was added and the resulting suspension was filtered. Ta. Concentrate the filtrate in vacuo and chromatograph on silica gel (hexane/ ethyl acetate eluent), the cyan intermediate was mixed with concentrated sulfuric acid (10 ml). Heated to 80°C for 3 hours, diluted with water (10ml) and refluxed - overnight. room temperature After cooling to , add the resulting mixture to activated carbon (0.5 g) and ice (50 g). do. After filtration, the resulting filtrate was concentrated in vacuo to a volume of 15 ml and The resulting solid was filtered. Dissolve this solid in a minimum amount of methanol, and precipitated by addition of ether. Reversed phase C18 chromatography (meth Final purification by ethanol/water eluent) gives the product.

[, Y-1 methyl ester CR, = 08 Ie, R2 = S03 Na, R4 =>CHx, n=8) Iodomethane (0.58ml), Y-1 (447mg), Na0)1 (in water) 6N, 1.53ml) and dimethyl sulfoxide (9ml) heated (50° C) was added to the mixed solution in 20 hours. The resulting mixture is being stirred Added dropwise to absolute ethanol (100ml). Centrifuge the resulting suspension Separation (9,00 Orpm, 20 minutes) and then remove the supernatant. Twice, conclusion The fruit solids were dissolved in water (6 ml) and the resulting solution was dissolved as described above. Treated with ethanol as in, centrifuged, and removed supernatant. residual solids was freeze-dried to yield the product (420 mg).

” CNMR (D, o, δ): 161.2.140.9.137.6.1 29.5.63.6. and 33゜5゜ Disodium chromotrope (10g) in 55ml water was dissolved in 30ml 137% H Treated with 22ml of CI. To this solution was added 1,2-benzene in 55 ml of acetic anhydride. Zendimetatool (5g) was added and the reaction was refluxed for 6 hours.

After filtering the resulting mixture, add acetonitrile (500 ml) and The crude product was precipitated and collected by filtration. The crude compound was transferred to LH-20 Further purification was achieved by column chromatography on gin and elution with ethanol. Ta.

A. Blood sample preparation Venous blood samples were taken using a clean venipuncture procedure. sample into a collection tube (VAC [JTA [NER”)] containing sodium citrate. 9 parts of blood were added to 1 part of 3.8%. Prior to centrifugation So, the blood sample is checked for the presence of clods and tested to see if it contains clods. I also disposed of the tube.

Centrifuge the sample at 1500 x g for 15 minutes in a refrigerated clinical centrifuge. separated. Remove the plasma from the sample using a damp plastic pipette. and stored at 4°C in a moisture-proof plastic stoppered tube. . Samples that showed evidence of hemolysis were discarded. Next, a plasma sample is drawn Place on ice for testing within 8 hours of blood collection, or alternatively, It was frozen at -20°C for testing within a week. All in vivo after drug administration Specimens must be stored within 8 hours without freezing for PTSAPTT and fibrinogen. For assay, it was collected into a VACUTA [NER” blood coagulation tube.

B. Prothrombin time test (PT) Plasma samples collected as described in Example 4A were preheated to 37°C in a water bath. and then add 0.2 ml of thromboplastin-calcium reagent ( Dade Thromboplastin OC, Becton Dickin son), reconstitute and store according to manufacturer's instructions. The sample was preheated, stirred, and kept in a 37°C bath. Each tube is hand Measure the time individually while mixing gently using the tilting method of the tube. It was. The time to macroscopic clot formation was obtained for each sample.

All samples were measured in triplicate and the dish washing time (PT waiting time) was averaged. Ta.

Example of C0 activated partial thromboplastin time (APTT) plasma sample 4A and stored in a water bath until testing. O, 1m 1 of the plasma sample was placed in a 13 x 100 mm polyethylene tube at 0.1 mm. 1 ml partial thromboplastin (Actin [F] activated cephaloblast) Rustin reagent: Mix with Becton (Dickinson) and follow the manufacturer's instructions. Therefore, it was reconstructed and handled. Preheat the tube containing this mixture to 0.2 ml. (37°) for 3 min prior to the addition of 0,025λ1 calcium chloride. Placed in a 7° warm bath. The tube was then rotated for a total of 20 seconds at 37° for 5 Tilt the sample gently at intervals of seconds, then remove it from the bath, and ensure that the sample is Periodic tilting was continued until formation of a bilin network was observed. The previous sample is Each was analyzed in duplicate.

Blood was collected in 8% sodium citrate and used to obtain platelet-poor plasma. centrifuged at 1500 x g for at least 20 minutes.

A 50 microliter portion of the plasma was mixed with a 50 microliter saline solution. Mixed and the mixture was placed in a test tube at room temperature. Preheating 25 microliters (37°) 1% (Ig/100ml) calcium chloride into the test tube. Add with rapid mixing. The mixture was then tilted at 1 minute intervals and the clot formed. I observed the growth.

E. Fibrinogen assay Blood was collected as described in Example IA. Thrombin reagent (Data-Pi Toro) Bin reagent, Baxter Healthcare Carp, &Iam i, FL; reconstitution was performed according to the manufacturer's instructions) into the diluted plasma sample. Dishwashing time was measured after addition. This time is used to calculate the known amount of fibrinogen. That diluted sample compared to a standard curve of dish washing time for samples containing The fibrinogen concentration in the sample was determined.

F. Lebutylase Assay (Atroxin Time) Blood samples are described in Example 4A. To obtain platelet-deficient plasma, centrifugation (1500 x g, 15 minutes). Plasma samples (0°2 ml) were incubated at 37°C for 5 min. Ta. ATROX [N■ (Sigma Chemical) preheated to 37° Co., St. Louis, 810) in 0.1 ml portions, respectively. was added to each sample tube while mixing to start the reaction. Time until clot formation The time was recorded as the atroxin time.

G, thrombin time test (TT) Concentrated stock human α-thrombin (4270 u/ml) was added to a buffer at pH 7.35. Prepared by diluting in rubitool-buffered saline and concentrated to 10X. A working stock solution was prepared. Final concentration of thrombin to be used in standard assays A wash time of 20 seconds ± 0.5 seconds was used in control platelet-depleted plasma samples. Test serial dilutions of concentrated stock human a-thrombin to have the ability to make determined by the strike. As described in Example 4A, there was a shortage of platelets. Plasma was prepared and prewarmed to 37° in 0.18 ml portions. test compound or saline to the plasma sample to make a final volume of 0.2 ml. Ta. Add 10 μl of preserved purified human a-thrombin concentrated 10 times to each sample. The reaction was started by adding the solution to the solution. A clod forms within that loop. Using a wire loop, load each sample twice per second until Incubate at 37° with gentle sweeping motions. continued. Time to clot formation was recorded for pre-samples.

Aggregation of H1 platelets Aspirate the blood sample with a plastic syringe and add a sufficient amount of sodium citrate. Transfer to a plastic test tube containing thorium and add 0.011 A final concentration of sodium citrate of M was created. First, the sample is 15 Centrifuge at 0 x g for 5 min at room temperature and remove red blood cell-free supernatant (PRP). The remaining blood was then centrifuged at 1500 x g for 15 minutes to remove the PPP supernatant. Platelet-rich plasma (PPP) and platelet-poor plasma by obtaining (PPP) samples were prepared from each sample. Until you test PPP and PPP were packed into tightly capped plastic tubes. of platelets Perform a count and check that the level of platelets is 200,000 per μl of PPPI. PPP was confirmed by measuring between 30o and ooo. need If available, reduce platelets to this level by diluting the PPP sample with PPP. diluted with

Transfer 0.5 ml of PPP sample as a control to the cuvette of the platelet aggregation needle. Ta. Transfer some of the 0.45 ml samples of PRP at room temperature to another cuvette. did. Place the PPP into the platelet aggregation needle according to the platelet aggregometer manufacturer's instructions. The PPP sample by placing and incubating at 37° Obtained a baseline display of. The PRP sample was then washed with 0.05 ml of saline. Place in the platelet aggregation needle and for 2 minutes prior to addition of test reagent containing water. Equilibrium was maintained.

A collection of percentage values was obtained for each sample.

■、Plasmin test Plasmin was tested at the Stafford University Blood Bank using standard clinical procedures. Pigmentation assay was performed.

An intravenous blood sample was collected from the rat tail blood vessel using sodium citrate (8%). and plasma was prepared using the procedure described in Example 1.

Plasma recalcification was carried out as described in Example ID in a total volume of 0.125 ml. provided. 50 μl of plasma dissolved in 50 μl of saline or saline. The compound obtained was added. Plasma recalcification was performed using 25 μl of 1% (wt/v ol) Started with addition of calcium chloride. The effects of these studies are shown in Table 3 , where each compound had a final concentration of 12, 5, 25, and 50 gg/ml. Tested at degrees. The clotting time value is the equivalent weight of heparin, It is expressed as a sum. I LISP unit 6.4 μg heparin.

Example 6 KY-1, Y-1, and Y-49 were added to human blood, similar to that described in Example 4B. It was tested in a PT assay using platelet-deficient plasma prepared from . This test In the test, plasma samples (0.1 ml) were mixed with varying amounts of the test compound (0-25 0 gg/ml) in 10 μl of saline.

The resulting mixed portion was poured into a 0.2 ml preheated (37°) trowel. Boplastin-calcium reagent (Dade Thromboplastin O C. Baxter Healthcare Corp., Hayward. CA). Each tube is Measure the time for individual clod formation while gently mixing using the tilting method. established. Record the time to clod formation as PT for each sample. did.

Y-1, Y-1, and Y-49 were added to human blood similar to that described in Example 4C. was tested in the APTT assay using platelet-deficient plasma prepared from . plasma Samples were collected as described in Example 4A. Plasma sample (0.1 ml) was Premixed with 0-364 μg of test compound in 0 μl of saline. This mixture The combined sample was added to 0.2 ml of 0.025λI calcium chloride. 0,1 preheated to 37° on a fibrometer (f ibrometer) ml APTT reagent (Automated APTT■, Organon T eknikaCorp, Durham, NC). each service The fibrin network formation time for the samples was measured.

Example 8 Compounds were tested for their effects on thrombin time (TT).

Platelet-deficient plasma was injected with 20 μl of test compound (o-t 90 μg/ml maximum). (final concentration) in 0.18 ml portions and preheated to 37°. 10 μ1 ( 42u/ml) purified human a-) thrombin (untreated human platelet-deficient blood) by adding an amount calculated to give a TT of 20 seconds in the plasma. The reaction started. Time to clod formation was recorded for all samples.

A sample (0.2 ml) of human platelet-deficient plasma was treated with the test compound (0- 37 with a 10 μI portion of saline containing (900 gg/ml final concentration) Incubated for 5 minutes at 50°C. ATROX [N■ (Sig ma Chemical co., St. Louis, 810), respectively. to each sample tube in 0.1 ml aliquots to start the reaction. . The time to clod formation was recorded as atroxin time.

KY-1 and Y-1 were tested for their effects on plasmin activity.

20.3. KY-1 was tested at concentrations of 40.6, and 81.1 μg/ml; Y-1 was then tested at final concentrations of 9°4 and 18.8 μg/ml. comparison and Heparin was tested at a final concentration of 0.41 μg/ml. The results are shown in Figure 22. Shown in A-C.

Example 12 Various concentrations of highly cyclic compounds were added to phosphate-buffered saline (pH 7.4). ) and administered to rats intravenously at 2.51.5, or 25 mg/kg. was administered. At various times post-dose, each rat was treated as described in Example 4A. Blood samples were taken from Example 4B, plasma was prepared, and Example 4B, respectively. PT as described in 4C and 4E.

APTT and fibrinogen content were determined.

Two doses of compound or physiological saline (PBS) were administered 30 minutes apart by gastric gavage. The dose (500 or 1625 mg/kg) was applied to female 5w1ss-Webst erv mice (27-28 g each). blood sample, 8% citrate by mouth, via retroorbital venipuncture, 2.5 hours after first dose. collected in. Plasma samples were obtained and processed as described in Example 4A and Example 4C. Plasma was analyzed for clotting time as described in . For comparison, Y-1( 12 or 20 gg/ml) in plasma samples from saline-treated control animals. sample and the sample was tested for clotting time as described in Example 5. I tested it. The results of this test are shown in Table 4.

Y-1 was given to rats at a dose of 450 mg/kg by gastric gavage.

Arterial blood samples were taken at 0.5.4.8.16 and 24 hours orally, tip downwards. While pulling toward the aorta, pull it out through the cannula inserted into the left carotid artery. did. Plasma samples were prepared as described in Example 4A and Examples 4B and 4. Tested for PT and APTT as described in C. 23 days after the first dose An additional dose of 225 mg/kg was administered to a subgroup of animals at Plasma from those animals was also tested at 24 hours. The results are shown in Table 6.

2.5 mg/kg or 5 mg/kg of Y-1 into the lateral tail blood vessels, Male Sprague-Dawley rats (2/dose) were administered intravenously.

At various times after injection, the cannula was inserted into the carotid artery inferiorly toward the aorta. Blood was drawn from rats (47 hour cycles) through a Processed and plasma obtained as described in Example 4A. For each plasma sample APTT test was performed.

A general platelet aggregation assay procedure was used as described in Example 4I. citric acid For platelet aggregation using modified platelet-rich plasma, To achieve this goal, the collagen dosage was titrated.

24μg/m! and KY-1 and Y-49 at a concentration of 48 tt g/ml, It had no effect on collagen-induced assembly. 24μg/m Y-1 at 1 had a slight inhibitory effect, while at 48 μg/ml there was no significant showed inhibition.

X1 work X work V xveeeeee xx engineering Fig, 13 Reagent (μff/ml) Reagent (μg/ml) Time after Y-1 administration Fig, 19 Reagent (μq/ml) Fig, 21E Time (minutes) Time (minutes) ICY-1 (μg/ml) Heparin (μcr/ml) Submission of translation of written amendment (Article 184-8 of the Patent Law) July 23, 1993

Claims (17)

[Claims] 1. A pharmaceutical composition effective in inhibiting blood coagulation in a human specimen, the composition comprising: , a cycloaddition crosslink that forms a continuous chain of bonded atoms that make up the backbone of a high ring. A highly cyclic compound composed of aryl ring subunits connected by sulfonic acid-induced substitutions on non-skeletal atoms of the aryl subunit of containing the peptide group), as well as a pharmaceutically acceptable carrier (wherein the peptide is A pharmaceutical composition comprising: 2. The substituent derived from the above sulfonic acid is a sulfonic acid, a sulfonic acid salt, Composed of sulfinic acids, sulfinates, sulfones, and sulfonamides 2. The composition of claim 1 selected from the group. 3. The above aryl ring subunit has (a) polar groups at the 1- and 8-positions, and Naphthalene subunits with sulfonic acid-derived substituents in the 3- and 6-positions (b) a polar group at the 1-position and a group derived from a sulfonic acid at the 4-position. and (c) a mixture of (a) and (b). selected from the group consisting of The 2nd ring carbon position of the lene or phenyl group and the 7th ring carbon position of the adjacent naphthalene group or the 6th ring carbon position of an adjacent phenyl group. Composition. 4. The above high cyclic compound has the following structure: ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ [wherein R2 is sulfonic acid, sulfonate, sulfinic acid, sulfinate, sulfone or sulfonamide, and R1 is OH, =O, alkyl or is an aryl ether, ester, or acid, or a mixture thereof; R 4 is >CHR'' or >CR'' (where R'' is H or carbon and n=4, 6, or 8]. thing. 5. 5. The composition according to claim 4, wherein R2 is a sulfonic acid or a sulfonic acid salt. 6. R2 is alkyl sulfone or SO2 NHR (where NHR is NH 2, NHOH or an amino acid). 7. The above compound has the following structure: ▲Contains mathematical formulas, chemical formulas, tables, etc.▼ [wherein R2 is sulfonic acid, sulfonate, sulfinic acid, sulfinate, sulfone or sulfonamide, and R1 is OH, =O, alkyl or is an aryl ether, ester, or acid, or a mixture thereof; R 4 is >CHR'' or >CR'' (where R'' is H or carbon and n=4, 6, or 8]. thing. 8. R1 is -OCOCH2, -SO3C6H4CH3, -COOH, and OM The composition according to claim 7, wherein the composition is selected from the group consisting of e. 9. R2 is alkyl sulfone or SO2 NHR (where NHR is NH 8. The composition according to claim 7, which is NHOH or an amino acid. 10. 8. The composition of claim 7, wherein some of the R1 groups are =0. 11. The composition has a maximum concentration of between about 10-100 μg/ml of the compound after oral administration. 2. The composition of claim 1, wherein the composition is formulated to produce large blood concentrations. 12. A pharmaceutical composition effective in inhibiting blood coagulation in human specimens. , alkyl sulfone, and SO2 NHR (where NHR is NH2, NH A group consisting of sulfonamides in the form of OH or amino acids derived from at least six regularly arranged sulfonic acids selected from A medicament comprising a highly cyclic biocompatible polymer composed of substituents the composition, as well as a pharmaceutically acceptable carrier on which the peptide is carried; ). 13. The polymer is a continuous chain of bonded atoms making up the backbone of the high ring. It is composed of aryl ring subunits connected by cycloaddition bridges to form a highly cyclic compound (with sulfonate on the non-skeletal atom of its aryl subunit) 13. The composition of claim 12, wherein the composition comprises a substituent derived from a phosphoric acid. 14. The above-mentioned high cyclic compound has at least six phenyl ring subunits (each containing one sulfonic acid-derived substituent as defined above) The composition according to item 13. 15. Structure below: ▲Contains mathematical formulas, chemical formulas, tables, etc.▼ [wherein R2 is sulfonic acid, sulfonate, sulfinic acid, sulfinate, sulfone or sulfonamide, R1 is =0, and -0H, alkyl or aryl ether, ester, or acid, or mixtures thereof. and R4 is >CHR'' or >CR'' (where R'' is H or carbon carboxylic acid group) and n=4, 6, or 8] thing. 16. R1 is -OCOCH2, -SO3C6H4CH3, -COOH, and O 16. The composition of claim 15, wherein the composition is selected from the group consisting of Me. 17. R2 is alkyl sulfone or SO2 NHR (where NHR is N 16. The composition according to claim 15, which is H2, NHOH or an amino acid.