JP2001515907A - Flopyrrolidine derivatives and their use as serine proteases - Google Patents

Abstract

  (57) [Summary] Serine protease enzyme inhibitors that are substituted derivatives of trans-hexahydrofuro [3,2-b] pyrrole-2-one are provided.

Description

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a new class of chemical compounds and their use as serine protease enzyme inhibitors. These compounds are useful as medicaments, and the inventors provide methods for their preparation and formulations containing them.

Serine proteases are one of a class of proteolytic enzymes characterized by having an active site at the serine residue that interacts with the carbonyl carbon of the peptide bond to cleave the peptide bond through an acyl enzyme intermediate. One. By conventional residue numbering based on homology to the serine protease enzyme chymotrypsin, the active site serine is generally Ser-195. Most members of the serine protease family activate the serine hydroxyl group and have histidine and aspartic acid residues at the active site that attack the truncated peptide carbonyl (numbered His-57 and Asp-102 based on chymotrypsin). ).
Asp-10 in a small number of enzymes, especially herpesvirus proteases
The role of 2 is played by an additional histidine residue (His-157 in cytomegalovirus protease). From residue mutation studies, these three residues are essential for activity and usually they are referred to as "catalytic triads".
) ".

[0003] In all enzymes of this family, although the mechanism of hydrolysis of peptide bonds by serine proteases is thought to be similar, their substrate specificities differ significantly. In general, the conventional nomenclature recognizes specificity for peptide bonds having a particular moiety α to a truncated peptide carbonyl that is said to be at the P ₁ position and occupy an S ₁ specific subsite (Schlecter and Ber
ger (1967) Biochem Biophys Res Common 27 157). For example, a preferred substrate for thrombin is a peptide having a basic residue (eg, an arginine, ie, a (CH ₂ ) ₃ NHC (＝ NH) NH ₂ ) moiety is at the P ₁ position, while
Preferred substrates for elastase are peptides containing a valine residue (ie, C
H (CH ₃ ) ₂ is P ₁ position).

In recent years, the X-ray crystal structures of a number of serine protease enzymes have been revealed. In the description of the observation, "triad catalyst" generally are highly conserved in terms of its spatial arrangement in the active site, leading factor differences in substrate specificity Shaped S ₁ specific subsite It is concluded that this is attributable to the characteristics. Serine proteases are widely distributed in the human body, and abnormal or overactive activity of serine proteases implies a variety of diseases and conditions (“protein inhibitors”, Barrett and Saiveson (1986), Elsevier, p56; Drugs Future (1996). ), twenty one(
8), 811-816: Exp. Opin. Ther. Patents (1997) 7 (1) 17-28).

The following are examples of enzymes and related conditions: Neutrophil elastase is found in neutrophil azul granules associated with tissue inflammation, and causes emphysema, chronic bronchitis and adult respiratory distress syndrome (ARDS) And many other inflammatory diseases.

[0006] Members of the blood coagulation cascade (eg, thrombin, factor VIIa, factor Xa, factor XIa, factor XIIa) and members of the fibrinolytic cascade (eg, tissue plasminogen activator and plasmin) are It is a potential target for the treatment of systemic diseases. For example, thrombin is a potential target for treating thrombosis. Tissue plasminogen activator and plasmin are also involved in tumor metastasis.

[0007] Tryptase is present in mast cells, and tryptase inhibitors show efficacy in asthma models. Pancreatic elastase, trypsin and chymotrypsin are involved in gastrointestinal diseases such as pancreatitis.

[0008] Cathepsin G is involved in emphysema. Serine proteases are also widely present, especially in human pathogens such as viruses, which make them attractive targets for the treatment of pathogenic diseases and conditions. For example, herpes viruses encode serine proteases that are critical for viral replication, and thus are targets for the treatment of conditions caused by these viruses.

[0009] Viruses of the herpes family include varicella and herpes zoster (varicella and herpes simplex virus, respectively), herpes simplex and genital herpes (herpes simplex virus), retinitis, pneumonia, and keratitis (human cytomegalovirus, hCMV).
) And Epstein-Barr virus (EBV), human herpes virus 6 (H
HV6), HHV7 and HHV8 and are responsible for a wide range of human infections.

[0010] Hepatitis C virus is also a target for treatment of hepatitis C virus infection and encodes a serine protease involved in liver damage (known as the NS3 serine protease).

[0011] In addition to the above enzymes and conditions, many other serine protease enzymes are believed to be known as suitable targets for drug therapy, and in fact, more serine proteases will be identified in the future. It is.

The inventors have now found a new chemical class of molecules that can inhibit a wide range of serine protease enzymes. They may be effective in treating diseases such as those discussed above.

More specifically, according to the present invention we provide trans-hexahydrofuro [3,2
-B] a serine protease enzyme inhibitor which is a substituted derivative of pyrrol-2-one.

More specifically, the present invention provides a compound of formula I:

Embedded image (Expressed as relative stereochemistry) where R ¹ is the moiety adapted to fit into the S ₁ specificity subsite of the enzyme; R ² is the potency, pharmacokinetics, pharmacodynamics, The present invention relates to a serine protease enzyme inhibitor, which is a compound represented by the following formula, which is a moiety modified to optimize selectivity and physicochemical properties, and physiologically acceptable salts and solvates thereof.

[0015] Without being bound by theory, we believe that the translactone template of Formula I is highly complementary to the active site of serine proteases, and that the lactone carbonyl is similar to peptide carbonyl, the natural substrate of the enzyme. Think about it. It is thought that the attack of serine, the enzyme active site on translactone carbonyl, causes the ring opening of the distorted lactone ring, resulting in the acylation of the enzyme in the serine side chain and the time-dependent (acylation) inhibition.

The advantages of the present invention are, inter alia, (a) trans-hexahydrofuro [3,2-b]
Pyrrole-2-one templates are completely novel and therefore highly desirable especially in medicaments for the treatment of pathogenic diseases in which drug resistance is likely to occur, and (b) such trans-hexahydrofuro [3,2-b] pyrroles The -2-one template is highly functionalizable, making it ideal for specific and selective inhibition of a wide variety of enzymes, and (c) such trans-hexahydrofuro [3,2-b]. The pyrrol-2-one template can be functionalized to have high or low metabolic stability.

With respect to determining the optimal substitution of a trans-hexahydrofuro [3,2-b] pyrrole-2-one derivative, particularly with respect to the selection of the R ¹ and R ² groups for a particular serine protease enzyme, I.e., (a) producing several compounds with sufficient diversity, in particular in the R ¹ and R ² groups, and (b) preparing a sample of the enzyme of interest with a sample of each compound thus produced. Treatment, and (c) measuring the extent to which enzyme inhibition occurs.

[0018] Enzyme inhibition assays are generally well known and may be performed in any case by those skilled in the art.

More specifically: Suitable R ¹ groups are suitably embedded in S ₁ -specific subsites of the target enzyme. The selection of the R ¹ group takes into account the selection of the known substrate specificity of the target enzyme, crystallographic information regarding the geometry of the S ₁ -specific subsite of the target enzyme, and / or empirical determinations based on screening data. (Eg, “proteinase inhibitors” Barrett a
nd Salveson (1986), Elsevier, p9 and p59).

Categorized by their major substrate specificities, there are three major types of serine proteases: elastase-like, trypsin-like and chymotrypsin-like. The differences between these types can be understood in terms of structure (Kraut J (1977) Am. Re
v. Biochem. 46 331-358).

For the inhibition of neutrophil elastase and elastase-like enzymes, the R ¹ group is small and hydrophobic, for example C _2-4 alkyl or C _2-4 alkenyl, especially propyl or isopropyl, especially isopropyl. Is preferred.

For inhibition of chymotrypsin-like enzymes (including chymotrypsin and cathepsin G)
As for R¹The groups are large, hydrophobic, eg, (CH₂)_1-2Ph, (CH ₂ )_0-2Cyclohexyl and t-butyl are preferred.

Ph represents phenyl or substituted phenyl (eg C _1-6 alkyl, phenyl substituted with halogen). Planar aromatic side chains (eg benzyl) are particularly preferred.

Trypsin-like enzymes (trypsin, thrombin, tryptase, factor VIIa,
For inhibition of factor Xa, factor XIa, factor XIIa), the R ¹ group is a group such as (CH ₂ ) _2-4 NHC (＝ NH) NH ₂ , (CH ₂ ) _1-2 PhC
(= NH) NH ₂ , (CH ₂ ) _3-5 C (= NH) NH ₂ , CH ₂ (cyclohexyl) NH ₂ , (CH ₂ ) _1-3 (NH) _0-1 Het (where Het is It contains one or more nitrogen atoms, optionally an aromatic ring may also be 5 or 6 membered optionally substituted with an amine), or _{(CH 2)} 3-5 NH _2, in particular _{(CH 2) 4 C (=} NH
) NH ₂ or _{(CH 2) 3 NHC (=} NH) NH 2, especially _{(CH 2)} 4 C
(= NH) NH ₂ are preferred. R ² Efficacy of inhibitors, pharmacokinetics, pharmacodynamics, a modified part as selectivity and physicochemical properties is optimized. Water-soluble and oral activity (if desired)
Other pharmacological properties may be modified to optimize.

In general, R ² varies over a very wide range, so that a person skilled in the art will be able to determine from a suitable test whether a given R ² is suitable for the above purpose. R ² portion increased efficacy when combined with a distal specific subsite such as S _3, S ₄ or S ₅ is often occur ( "proteinase inhibitor", Barrett and
Salveson (1986) Elsevier, p6, 69).

CO, SO ₂ or COO wherein R ² is directly attached to the pyrrolidine nitrogen (especially CO 2
Or SO ₂ ) moieties, for example of the formula R ²⁰ CO
, R ²⁰ SO ₂ or R ²⁰ OCO (especially R ²⁰ CO, R ²⁰ SO ₂ ). R ²⁰ is also a moiety that has been modified to optimize the potency, pharmacokinetics, pharmacodynamics, selectivity, and physicochemical properties of a serine protease inhibitor, for example, alkyl (eg, C _1-8 alkyl) , Alkenyl (eg, C _1-8 alkenyl), aryl, alkylaryl (eg, C _1-8 alkylaryl), or alkenylaryl (eg, C _1-8 alkenylaryl).

As used herein, alkyl includes branched and cyclic alkyl. Alkenyl includes branched and cyclic alkenyl. Aryl is optionally heteroatom, such as O, N and S atoms (e.g.
Monocyclic and bicyclic aromatic rings containing four heteroatoms).

Alkyl, alkenyl, aryl, alkylaryl and alkenylaryl groups may be optionally substituted, for example by amines and halogens, and may be optionally heteroatom (eg, nitrogen or oxygen) or other functionalized. May be inserted.

The amine groups include primary, secondary and tertiary amine groups including cyclic amines. Thus, in accordance with the present invention, we also provide a method of inhibiting a serine protease enzyme, comprising treating with a compound of the present invention. The inventors also provide a method for screening for a serine protease inhibitor, comprising treating a serine protease enzyme with a compound of the present invention and measuring the extent to which inhibition occurs.

The inventors have also prepared (a) several trans-hexahydrofuro [3,2-b] pyrrole-2-one-substituted derivatives; and (b) a sample of each derivative produced (C) measuring the extent to which inhibition of the enzyme has occurred. A method for identifying a serine protease enzyme inhibitor, comprising: The extent to which inhibition occurs can be measured by conventional assay techniques, including but not limited to chromogenic assays, fluorescent assays, HPLC, and scintillation proximity assays.

One particularly advantageous method of drug development involves the use of multiple trans-hexahydrofuro [3,
2-b] Prepare a library containing substituted derivatives of pyrrol-2-one. Such a library is preferably a plurality of Formula I:

Embedded image Where R ¹ is a moiety adapted to fit into the S ₁ -specific subsite of the enzyme; R ² is the inhibitor, pharmacokinetics, pharmacodynamics, selectivity and physicochemical properties of the inhibitor. It comprises a serine protease enzyme inhibitor which is a compound represented by the following formula, which is a moiety modified to be optimal, and a physiologically acceptable salt and solvate thereof.

The library comprises at least 10 (eg, 10, 100, 1000 or more) different compounds. Libraries of compounds of formula I wherein R ¹ represents a small and hydrophobic group, for example C _2-4 alkyl or C _2-4 alkenyl, especially propyl or isopropyl, especially isopropyl, are for example neutrophil elastase It would be particularly useful for screening for elastase-like enzymes.

R ¹ is (CH ₂ ) _2-4 NHC (＝ NH) NH ₂ , (CH ₂ ) _1-2 PhC (
ＮＨNH) NH ₂ , (CH ₂ ) _3-5 C (＝ NH) NH ₂ , CH ₂ (cyclohexyl) NH ₂ , (CH ₂ ) _1-3 (NH) _0-1 Het (where Het is 1 Or a (CH ₂ ) _3-5 NH ₂ , particularly a (CH ₂ ) ₄ C (＝ NH) N, containing a nitrogen atom and representing a 5- or 6-membered aromatic ring optionally substituted with an amine
H ₂ or (CH ₂ ) ₃ NHC (＝ NH) NH ₂ , especially (CH ₂ ) ₄ C (=
Libraries of compounds of formula I representing a basic group such as NH) NH ₂ may be particularly useful for screening inhibitors of trypsin-like enzymes (such as thrombin or tryptase). A compound library of Formula I wherein R ¹ represents a large and hydrophobic group, such as (CH ₂ ) _1-2 Ph, (CH ₂ ) _{0 -2} cyclohexyl or t-butyl, is for example a chymotrypsin or cathepsin G Such a chymotrypsin-like enzyme would be useful for screening for inhibitors.

Library techniques are known to those of skill in the art and are described in Drug Discovery Today (1996) 1 (4).
134-144 and Annual Reports in Combinational Chemistry and Molecular Di
versity 1.Ed. Moos Walter H, Pavia Michael R, Kay Brian K, Ellington And
y D. This library may be a solid phase library or a liquid phase library. It can be a separate or pooled library.

The inventors also provide a method of treating a disease involving serine protease activity, comprising administering to a patient an effective amount of a compound of the invention, and for treating a disease involving serine protease activity. The use of a compound of the invention in the manufacture of a medicament.

As used herein, treatment is intended to extend to prevention as well as treatment of established conditions. A particularly preferred embodiment of the present invention relates to the application of the compounds of the present invention in inhibiting neutrophil elastase, thrombin, and tryptase.

The compounds of the present invention have the formula II:

Embedded image (Represented as relative stereochemistry) wherein R ¹ is a moiety adapted to fit into the S ₁ -specific subsite of the enzyme or a protected derivative thereof, is reacted can be prepared by introducing the desired R ² substituent.

[0038] Conditions for such continuous reactions are known to those skilled in the art. Generally, these reactions consist of alkylation (usually with an alkyl halide), sulfonylation (with a sulfonyl halide), or acylation (reaction with a carboxylic acid, halogenated acid, or anhydride).

Compounds of formula II can be prepared according to Scheme 1 below (the compounds are depicted by relative stereochemistry).

Scheme 1

Embedded image {Where R ^p and R ^q are amino and hydroxyl protecting groups, respectively} Steps (i)-(xi) of Scheme 1 above are discussed below.

Step (i) comprises treating β-alanine hydrochloride to introduce a suitable amino protecting group.

Step (ii) comprises reacting the compound of formula (III) with diethyl fumarate in a suitable solvent such as an aromatic hydrocarbon (eg toluene) or an ether (eg tetrahydrofuran) in the presence of a strong base such as sodium hydride. Treating the compound of formula (I). The reaction is conveniently carried out at an elevated temperature (eg, reflux).

Step (iii) comprises treating the compound of formula (IV) with dimethyl sulfoxide at an elevated temperature (eg, reflux) in the presence of a solution of sodium chloride.

Step (iv) is carried out at about room temperature in a suitable solvent such as an alcohol (eg, ethanol) in the presence of a lanthanide salt (eg, cerium trichloride) and a odorant such as sodium hydrogen bromide. The compound of formula (V) is stereospecifically reduced using hydride and then at about room temperature in a suitable solvent such as an ether (eg, tetrahydrofuran) and triphenylphosphine and diethylazodicarboxy. Performing a Mitsunobu rearrangement with benzoic acid in the presence of a rate.

Step (v) is carried out at about room temperature in a solvent such as an alcohol (eg, methanol) using a base such as an inorganic base such as a carbonate (eg, potassium carbonate) using a compound of the formula ( Removing the benzoyl group from the compound of VI).

Step (vi) comprises treating the compound of formula (VII) to introduce a suitable hydroxyl protecting group.

[0047]Step (vii)Is R¹Treatment of a compound of formula (VIII) with a reagent capable of introducing a group
To do. R¹Is generally in the presence of a strong base such as lithium hexamethyldisyl azide in a solvent such as THF at a temperature of 78 ° C. to 20 ° C. ¹ Hal (where Hal is a halogen atom such as bromine) represented by the formula (VIII)
It can be introduced by treating the compound.

[0048] Step (viii) comprises removing the hydroxyl protecting group ^{R Q} from compounds of formula (IX).

Step (ix) comprises base-catalyzed hydrolysis of the compound of formula (X).
This hydrolysis may conveniently be carried out using an alkali metal hydroxide such as lithium hydroxide or potassium hydroxide.

Step (x) is carried out in the presence of an organic base (for example, triethylamine) in the presence of an activating agent such as a pyridinium salt (for example, 2-chloro-1-methylpyridinium iodide).
Cyclization of the compound of formula (XI) in the presence of a base such as The reaction may be performed at a temperature between 0 and 100 <0> C in a suitable inert solvent (e.g. DCM). For the pyridinium salt and the reaction, add 4-dimethylaminopyridine in an aromatic hydrocarbon solvent (eg, toluene) and heat the mixture (eg, reflux).
Is completed.

[0051] Step (xi) comprises a removal of the amino protecting group ^{R P.} Compounds of formula (II) wherein R ^P represents benzyloxycarbonyl group may conveniently in a solvent such as ethyl acetate, a palladium catalyst (e.g., palladium on carbon) can be prepared by hydrogenation in the presence of.

In Scheme 1, diastereomeric separation will be required to obtain a compound of the desired stereochemistry. A compound of formula (I) may also be prepared from another compound of formula (I) by one or more conventional chemical transformations.

In the course of routine optimization of experimental conditions, the above synthetic procedure for producing compounds of formula (I) may be modified to include or remove protecting groups, or to provide another protecting group (eg,
Those skilled in the art will appreciate that they may use TW Greene "Inorganic Synthesis" 2nd Ed (1991) J Wiley & Sons).

For example, where R ¹ contains an amidine moiety, it is preferred to introduce a substituted R ¹ (eg, of Scheme 1) as an oxadiazolinone derivative. It may optionally be O- or N-protected in subsequent chemical steps. Hydrogenation of this derivative in the presence of Pd / C produces free amidine.

The present invention includes the compounds of the present invention in racemic forms as well as in forms where one enantiomer predominates or exists exclusively. In general, it is preferred that compounds of formula (I) be supplied in diastereomerically and enantiomerically pure form. With respect to elastase inhibition, preferably of the formula (Ia):

Embedded image Diastereomers having the relative stereochemistry shown by are preferred.

For the inhibition of thrombin and tryptase, diastereoisomers having the relative stereochemistry of formula (Ia) are also preferred. Enantiomerically pure compounds can be prepared by chiral separation or by synthesis based on chiral starting materials.

The present invention also includes physiologically acceptable salts of the compounds of the present invention. Suitable physiologically acceptable salts include inorganic salts, such as alkali metal salts (eg, sodium and potassium salts) and ammonium salts, and organic basic salts. Suitable organic basic salts include trialkylamines (eg, triethylamine), dialkylamines (eg, dicyclohexylamine), optionally substituted benzylamines (eg, phenylbenzylamine or p-
Amine salts such as bromobenzylamine), procaine, ethanolamine, diethanolamine, N-methylglucosamine and tri (hydroxymethyl) methylamine salts and amino acid salts (eg, lysine and arginine salts). Suitable inorganic and organic acid salts include hydrochloric acid, trifluoroacetic acid, and tartrate.

The compounds of the present invention may be formulated for administration by any convenient method, and the present invention also includes within its scope the compounds of the present invention or a physiologically acceptable salt or solvate thereof. It also includes a therapeutic pharmaceutical composition comprising a substance in admixture with one or more physiologically acceptable excipients or carriers. Also according to the present invention there is provided a process for the manufacture of such a pharmaceutical composition comprising admixing components deemed suitable for the present invention.

The compounds of the invention may be formulated for oral, buccal, parenteral, topical or rectal administration, for example.

Tablets and capsules for oral administration include binders such as syrup, acacia,
Gelatin, sorbitol, tragacanth, starch mucilage or polyvinylpyrrolidone; fillers, such as lactose, microcrystalline cellulose, sugar, corn starch, calcium phosphate or sorbitol; lubricants, such as magnesium stearate, stearic acid, talc, polyethylene glycol or silica Conventional excipients may be included such as disintegrating agents, for example potato starch, croscarmellose sodium or sodium starch glycolate; or wetting agents, such as sodium lauryl sulfate. Tablets may be coated according to methods well known to those skilled in the art. Oral liquid preparations may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or provided as a dry preparation prior to use in water or other suitable vehicle. May be done. Such liquid formulations include suspending agents such as sorbitol syrup, methylcellulose,
Glucose / sugar syrup, gelatin, hydroxymethylcellulose, carboxymethylcellulose, aluminum stearate gel or hydrogenated edible fat; emulsifiers such as lecithin, sorbitan monooleate or acacia; non-aqueous vehicles (including edible oils) such as almond oil; Distilled coconut oil, oily esters, polyethylene glycol or ethyl alcohol; or preservatives, such as conventional additives such as ethyl or propyl p-hydroxybenzoate or sorbic acid, may be included. The formulation may also optionally contain buffer salts, flavoring, coloring and / or sweetening agents such as mannitol.

For buccal administration, the compositions formulated in conventional manner may take the form of tablets or lozenges. The compound may also be formulated as a suppository, with conventional suppository bases such as cocoa butter or other glycerides.

The compounds of the present invention may also be formulated for parenteral administration by bolus injection or continuous infusion, or in unit dosage form, eg, in ampules, vials, small doses or pre-filled syringes, or in storage. It may be provided in a multidose container with the agent added. The compositions may take such forms as solutions, suspensions or emulsions in aqueous or non-aqueous vehicles, as well as antioxidants, buffers, antibacterial agents and / or
Alternatively, a compounding agent such as a toxicity modifier may be contained. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, eg, sterile pyrogen-free water, before use. Dry solid formulations may be prepared by aseptically filling sterile powders into individual sterilized containers, or by aseptically filling sterilized solutions into each container and then lyophilizing.

As topical administration herein, we include administration by insufflation and inhalation. Examples of various formulations for topical administration include ointments, creams, lotions,
Powders, vaginal suppositories, sprays, aerosols, capsules, or cartridges for inhalation or insufflation or instillation (eg eye drops or nose drops).

Ointments and creams may, for example, be formulated with an aqueous or oily base with the addition of suitable thickening and / or gelling agents and / or solvents. Such a base may thus include, for example, water and / or an oil such as liquid paraffin, or a vegetable oil such as peanut oil or castor oil, or a solvent such as polyethylene glycol. Thickeners that can be used may include paraffin, aluminum stearate, cetostearyl alcohol, polyethylene glycol, microcrystalline wax and beeswax.

Lotions may be formulated with aqueous or oily bases and are generally formulated with
The above-mentioned emulsifier, stabilizer, dispersant, suspending agent or thickener may be included. External powders may be formed with any suitable powder base, for example, talc, lactose or starch. Drops may be formulated with an aqueous or non-aqueous base also comprising one or more aqueous dispersing agents, solubilizing agents or suspending agents.

The spray compositions are prepared with suitable propellants, for example dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, 1,1,1,2,3,3.
, 3-heptafluoropropane, 1,1,1,2-tetrafluoroethane, carbon dioxide or other suitable gas, for example as an aqueous solution or suspension,
Alternatively, they may be formulated as an aerosol delivered from a pressurized pack. Capsules and cartridges for inhalation or insufflation, for example, of gelatin, may be formulated containing a powder mix of the compound of the invention and a suitable powder base such as lactose or starch.

The compounds of the present invention may also be used in purification and diagnostic applications for serine protease enzymes. For example, if the compound of the present invention is immobilized, a serine protease capable of binding to the compound can be isolated. By labeling the compound of the present invention, a serine protease capable of binding to the compound can be identified.

Abbreviations BOC t-butyloxycarbonyl CBZ benzyloxycarbonyl (BOC) ₂ O di-t-butyldicarbonate THF tetrahydrofuran LHMDS lithium bis (trimethylsilyl) amide DMPU 1,3-dimethyl-3,4,5,6- Tetrahydro2 (1H) pyridinone DMAP 4-dimethylaminopyridine DMF dimethylformamide EDC 1- (3-N, N-dimethyl-aminopropyl) -3-ethylcarbodiimide DEAD diethylazodicarboxylate DCM dichloromethane TMEDA tetramethylethylenediamine DMSO dimethylsulfoxide

The present invention will be described by the following examples. Assay Example The above enzyme activity is generally determined at 15 minutes. Enzyme kinetics can be determined by determining enzyme activity at other times (eg, 30 minutes). Assay Example 1 In Vitro Assay for Inhibition of Human Neutrophil Elastase Assay Content: 50 mM Tris / HCl (pH 8.6) 150 mM NaCl 11.8 nM Purified human neutrophil elastase The appropriate concentration of compound to be tested is dimethyl sulfoxide. The 10 mM stock solution was diluted with water. The above values are the final concentrations after addition of the substrate solution (see below). The mixture was incubated at 30 ° C. for 15 minutes, at which point the remaining elastase activity was reduced to 0.6 mM MeO-succinyl-Ala-Ala-Pro.
-After addition of Val-p-nitroanilide, measure for 10 minutes on a BioTek 340i plate reader. The rate of increase in absorbance at 405 nm is proportional to elastase activity. Enzyme activity is plotted against inhibitor concentration and the IC ₅₀ is determined using curve fitting software.

Assay Example 2 In Vitro Assay for Inhibition of Human Thrombin The compound of the present invention was prepared using Np-tosyl-Gly-Pro-Lys as a chromogenic substrate.
In a chromogenic assay using p-nitroanilide, it can be tested for its thrombin inhibitory activity as determined in vitro by its ability to inhibit human α-thrombin. All diluents were 50 mM HEPES, 150 mM NaCl, 5m
Made of M CaCl ₂ , 0.1% PEG and made with a buffer of pH 7.4. Conveniently, the substrate (final concentration 100 μM) is added to thrombin (final concentration 1 nM)
The reaction was monitored at 405 nm for 10 minutes using a Biotek EL340 plate reader and the assay was performed at room temperature. To obtain IC ₅₀ values, the data was analyzed in Kineticalc (registered trademark) using a four-parameter curve fitting procedure to obtain _{IC 50} values. To determine the IC ₅₀ at 15 minutes, the compounds were preincubated with thrombin for that hour before the chromogenic substrate was added.

Assay Example 3 In Vitro pNA Assay of Viral Serine Protease Inhibitor The hCMV serine protease used was an internal cleavage site (Ala142 / Al
a143) is a mutant form of the 30K protease lacking the active enzyme (hCMV
This was cloned into E. coli to produce (δAla protease). After pre-incubating the enzyme with the test inhibitor compound for 15 minutes, the IC ₅₀ data of the test compound is determined. Test compound is DMSO
, Followed by dilution and further concentration in the concentration range (100 μM-0.195 μM) to 0
. 5 μM CMV δAla protease, 100 mM HEPES pH7.
5, 0.2 mM EDTA, 10 mM NaCl, 1 mM DTT, and 30 mM
% To the reaction containing glycerol. 4 mM oligopeptide substrate (Arg-G
Before adding lu-Ser-Tyr-Val-Lys-Ala-pNA), the reaction mixture was pre-incubated at 32 ° C. for 15 minutes, then BIO-TEK Bi.
o Analyze at 32 ° C. on Kinetics Reader EL340i. A plate reader monitors the production of pNA and calculates the reaction rate over 30 minutes. The rate is plotted against the inhibitor concentration and an IC ₅₀ value is determined.

Assay Example 4 In Vitro Assay for Inhibition of Human Mast Cell Tryptase The compound of the present invention was prepared using Np-tosyl-Gly-Pro-Lys as a chromogenic substrate.
In a chromogenic assay using -p-nitroanilide, it can be tested for its tryptase inhibitory activity as determined in vitro by its ability to inhibit human lung mast cell tryptase. Compounds were diluted in a 10 mM stock solution of dimethyl sulfoxide. All diluents were 10 mM Tris-HCl, 120 mM NaCl, pH7.
4 was prepared. Conveniently, the substrate (final concentration 400 μM) is added to tryptase (final concentration 0.11 μg.ml ⁻¹ ) and the appropriate concentration of compound,
The reaction was monitored at 405 nm for 30 minutes using a Molecular Devices Thermomax microplate reader and the assays were performed at room temperature. To obtain IC ₅₀ values, the data was analyzed using the curve fitting software. To determine the IC ₅₀ of 30 min time point, and the compound which time tryptase before adding the chromogenic substrate was pre-incubated.

Assay Example 5 In Vitro Assay for Inhibition of Trypsin The compound of the present invention was prepared using N-benzoyl-Ile-Glu-Gly as a chromogenic substrate.
In a chromogenic assay using -Arg-p-nitroanilide, it can be tested for its trypsin inhibitory activity as determined in vitro by its ability to inhibit bovine trypsin. Compounds were diluted in a 10 mM stock solution of dimethyl sulfoxide. All dilutions were made with a buffer consisting of 50 mM Tris-HCl, 15 mM CaCl ₂ , pH 8.4. For convenience, the substrate (final concentration 160 μM) is converted to trypsin (
Final concentration 0.25 ng. ml ^-1 ) and the appropriate concentration of compound incubated for 15 minutes, and the reaction was monitored at 405 nm for 10 minutes using a BioTek EL340 plate reader and the assay was performed at 37 ° C. Kineticcalc using a four parameter curve fitting procedure to obtain IC ₅₀ values (
(Registered trademark).

Assay Example 6 In Vitro Assay for Inhibition of Factor Xa The compound of the present invention was used as a chromogenic substrate for N-α-benzyloxycarbonyl-D-
In a chromogenic assay using Arg-Gly-Arg-p-nitroanilide,
They can be tested for their factor Xa inhibitory activity as determined in vitro by their ability to inhibit human factor Xa. Compounds were diluted in a 10 mM stock solution of dimethyl sulfoxide. All diluents were 50 mM Tris-HCl, 150 mM NaCl, 5m
It was made with a buffer consisting of M CaCl ₂ , pH 7.4. Conveniently, the substrate (final concentration 200 μM) is added to factor Xa (final concentration 0.02 U.ml ⁻¹ ) and the appropriate concentration of compound incubated for 15 minutes and reacted at 405 nm for 10 minutes using a BioTek EL340 plate reader. Was monitored and the assay was performed at 37 ° C. To obtain IC ₅₀ values, the data was analyzed in Kineticalc (registered trademark) using a four-parameter curve fitting procedure.

Assay Example 7 In Vitro Assay for Inhibition of Factor XIa The compound of the present invention was prepared using L-pyroglutamyl-Pro-Arg-p as a chromogenic substrate.
-In a chromogenic assay using nitroanilide, they can be tested for their factor XIa inhibitory activity as determined in vitro by their ability to inhibit human factor XIa. Compounds were diluted in a 10 mM stock solution of dimethyl sulfoxide. All diluents are 8
. 1 mM NaH ₂ PO ₄ , 147 mM KH ₂ PO ₄ , 2.7 mM KCl,
It was prepared with a buffer consisting of 137 mM NaCl, pH 7.2. Conveniently, substrate (final concentration 400 μM) is added to factor XIa (final concentration 0.25 μg.ml ⁻¹ ) and the appropriate concentration of compound incubated for 15 minutes and incubated at 405 nm for 10 minutes using a BioTek EL340 plate reader. And monitor 2
The assay was performed at 5 ° C. To obtain IC ₅₀ values, the data was analyzed in Kineticalc (registered trademark) using a four-parameter curve fitting procedure.

Assay Example 8 In Vitro Assay for Inhibition of Factor XIIa The compounds of the present invention have the ability to inhibit human factor XIIa in a chromogenic assay using HD-Pro-Phe-Arg-p-nitroanilide as a chromogenic substrate. Can be tested for their factor XIIa inhibitory activity as determined in vitro. Compounds were diluted in a 10 mM stock solution of dimethyl sulfoxide. All diluents are 2
It was prepared with a buffer consisting of 8 mM sodium barbital, 125 mM NaCl, 1 mM EDTA, pH 7.35. For convenience, the substrate (final concentration 200 μM
) Was added to factor XIIa (final concentration 1.25 μg.ml ^-1 ) and the appropriate concentration of compound incubated for 15 minutes, the reaction was monitored at 405 nm for 10 minutes using a BioTek EL340 plate reader, and the assay was performed at 25 ° C. went.

Assay Example 9 In Vitro Assay for Inhibition of tPa The compound of the present invention was prepared using MeSO ₂ -D-CHT-Gly-Arg as a chromogenic substrate.
-Can be tested for their tissue plasminogen activator inhibitory activity as determined in vitro by the ability to inhibit human tissue plasminogen activator in a chromogenic assay using p-nitroanilide. Compounds were diluted in a 10 mM stock solution of dimethyl sulfoxide. All dilutions were made with a buffer consisting of 50 mM Tris-HCl, 150 mM NaCl, pH 8.4. Conveniently, the substrate (750 μM final concentration) is added to tissue plasminogen activator (1.0 μg.ml ⁻¹ final concentration) and the appropriate concentration of the compound incubated for 15 minutes and read at 405 nm using a BioTek EL340 plate reader. The reaction was monitored for 10 minutes and the assay was performed at 30 ° C. To obtain IC ₅₀ values, the data was analyzed in Kineticalc (registered trademark) using a four-parameter curve fitting procedure.

Assay Example 10 In Vitro Assay for Inhibition of Plasmin The compounds of the present invention were tested for their ability to inhibit human plasmin in a chromogenic assay using HD-Val-Leu-Lys-p-nitroanilide as a chromogenic substrate. They can be tested for their plasmin inhibitory activity required in vitro.
Compounds were diluted in a 10 mM stock solution of dimethyl sulfoxide. All diluents are 50m
M Tris-HCl, 150 mM NaCl, 5 mM CaCl ₂ , pH 7.4
And a buffer solution consisting of: For convenience, substrate (final concentration 363 μM) is added to plasmin (final concentration 0.02 U.ml ⁻¹ ) and the appropriate concentration of compound incubated for 15 minutes, and the reaction is performed at 405 nm for 10 minutes using a BioTek EL340 plate reader. Monitored and performed the assay at 37 ° C. To obtain IC ₅₀ values, Kineticalc using 4 parameter curve fitting procedure
(Registered trademark).

Assay Example 11 In Vitro Assay for Inhibition of Factor VIIa The compounds of the present invention have the ability to inhibit human Factor VIIa in a chromogenic assay using HD-Ile-Pro-Arg-p-nitroanilide as a chromogenic substrate. Can be tested for their factor VIIa inhibitory activity as determined in vitro. Compounds were diluted in a 10 mM stock solution of dimethyl sulfoxide. All diluents are 2
0 mM Tris-HCl, 150 mM NaCl, 5 mM CaCl ₂ , 0.1
% Bovine serum albumin, pH 7.5. Conveniently, substrate (final concentration 400 μM) is added to factor VIIa (final concentration 10 nM in the presence of optimal concentration of soluble recombinant tissue factor) and the appropriate concentration of compounds incubated for 15 minutes, using a BioTek EL340 plate reader. The reaction was monitored at 405 nm for 30 minutes and the assay was performed at 37 ° C. To obtain IC ₅₀ values, the data was analyzed in Kineticalc (registered trademark) using a four-parameter curve fitting procedure.

Assay Example 12 In Vitro Assay for Inhibition of Chymotrypsin The compound of the present invention was prepared using MeO-succi-Arg-Pro-Tyr as a chromogenic substrate.
-Can be tested for their chymotrypsin inhibitory activity as determined in vitro by its ability to inhibit human pancreatic chymotrypsin in a chromogenic assay using pNA hydrochloride. Compounds were diluted in a 10 mM stock solution of dimethyl sulfoxide. All diluents were 50 mM Tris-HCl, 150 mM NaCl, 25 mM CaC
It was made with a buffer consisting of l ₂ , pH 8.4. For convenience, the substrate (final concentration 178)
μM) was added to chymotrypsin (final concentration 0.2 μg / ml) and the appropriate concentration of compound, the reaction was monitored at 405 nm for 10 minutes using a BioTek EL340 plate reader, and the assay was performed at 30 ° C. To obtain IC ₅₀ values, the data was analyzed in Kineticalc (registered trademark) using a four-parameter curve fitting procedure. Compounds were pre-incubated with chymotrypsin for that time before adding the chromogenic substrate to determine the IC ₅₀ at 15 minutes.

Assay Example 13 In Vitro Assay for Inhibition of Cathepsin G The compound of the present invention was prepared using N-succinyl-Ala-Ala-Pro as a chromogenic substrate.
In a chromogenic assay using -Phe-p-nitroanilide, it can be tested for its cathepsin G inhibitory activity as determined in vitro by its ability to inhibit human neutrophil cathepsin G. Compounds were diluted in a 10 mM stock solution of dimethyl sulfoxide. All dilutions were 100 mM HEPES, 300 mM NaCl, pH 7
. 2 was prepared. For convenience, enzymes (final 1.25 μg / mL),
The buffer and the appropriate concentration of compound were incubated at 30 ° C. for 15 minutes. Substrate (final 7.25 mM) was added and the reaction was monitored at 405 nm, 30 ° C. for 30 minutes using a BioTek EL340 plate reader. Ac using 4-parameter curve fitting procedure (XLFIT®) to obtain IC ₅₀ values
The data was analyzed with Microsoft Excel (registered trademark) of the activityBase (registered trademark).

Assay Example 14 In Vitro Assay for Plasma Stability The stability of the compounds of the present invention against exposure to cryptic esterification (and other) activities was evaluated in rat plasma and blood. Conveniently, compounds were mixed with fresh rat plasma or rat blood, then incubated at 37 ° C., and after further mixing, extracted several times by precipitation with acetonitrile. Compounds at each time point were quantified using reverse phase high performance liquid chromatography. Compound half-life was calculated from the time-course data by log linear regression analysis. Compounds were considered unstable if the half-life was less than 10 minutes.

Compound Example Intermediate Intermediate 1 3- (benzyloxycarbonylamino) propionic acid ethyl ester β-alanine hydrochloride (181 g) in 1,4-dioxane (200 ml) at 20 ° C.
)), A suspension of sodium bicarbonate (218 g) in water (600 ml) was added. The mixture was cooled to 0-5 <0> C and a solution of benzyl chloroformate (239 g) in 1,4-dioxane (200 ml) was added over 40 minutes. The mixture was stirred at 0-5 <0> C for 5 hours, filtered and concentrated. Then it was diluted with ethyl acetate (800 ml), water (2 × 400 ml) and brine (400 ml).
), Dried (MgSO ₄ ) and concentrated in vacuo to give the title compound as a colorless oil (300 g). Analysis: C, 62.3; H, 6.6 ; N, 5.5; C 13 H 17 NO 4 theoretical value: C, 62.4; H, 6.8 ; N, 5.7%.

Intermediate 2 5-ethoxycarbonylmethyl-4-oxopyrrolidine-1,3-dicarboxylic acid 1-benzyl ester 3-ethyl ester Sodium hydride (80% suspension in oil) in anhydrous toluene (900 ml) at 20 ° C. To a stirred suspension of the suspension (22 g) was added intermediate 1 (16 ml) in anhydrous toluene (100 ml).
9g) The solution was added over 1 hour. After stirring the mixture for 10 minutes, a solution of diethyl fumarate (127 g) in anhydrous toluene (100 ml) was added over 30 minutes. The reaction was then stirred at reflux for 3.5 hours. The reaction mixture was cooled to 20 ° C. and acidified to pH 5.0 with hydrochloric acid (2N; about 300 ml). The mixture was extracted with ethyl acetate (3x600ml). Combined extract with water (1000 ml
) And brine (1000 ml) and dried (MgSO ₄ ). The solvent was removed in vacuo to give the title compound as a brown oil (252g).
. T. l. c. Silica, ether, Rf 0.41

[0085] Intermediate 3 2-ethoxycarbonyl-3-oxo-1-carboxylic acid benzyl glycol ester dimethyl sulfoxide (2L) and brine (545 ml) of intermediate 2 (
252 g) The emulsion was stirred vigorously under reflux for 5 hours. The reaction mixture was cooled to 20 ° C. and diluted with water (2 L). The mixture was extracted with ethyl acetate (3 × 1 L) and the combined extracts were washed with water (2 L) and brine (2 L) and dried (Na ₂ SO ₄ ). The solvent was evaporated and the residue was purified by flash column chromatography using ether: hexane (1: 1) to 100% ether (gradient elution) to give the title compound as a colorless oil (138 g). T. l. c. Silica, ether, Rf 0.53

Intermediate 4 trans-3-benzyloxy-2-ethoxycarbonylmethylpyrrolidine-1 -carboxylic acid benzyl ester Intermediate 3 (20 g) and cerium chloride heptahydrate (24 g) in ethanol (200 ml) at 20 ° C. ) (2.5 g) was added over 15 minutes and the mixture was stirred for 2.5 hours. The reaction was quenched by the dropwise addition of a saturated aqueous solution of ammonium chloride (250 ml) and brine (250 ml) and then extracted with ethyl acetate (3 × 500 ml). The combined extracts were dried _(Na 2 _SO 4), and evaporated. The remaining oil was dissolved in anhydrous tetrahydrofuran (600 ml) and benzoic acid (12 g) and triphenylphosphine (26 g) were added. A solution of diethyl azodicarboxylate (17 g) in anhydrous tetrahydrofuran (25 ml) was added dropwise at 20 ° C. over 0.5 hours. The solution was stirred at 20 ° C. for 20 hours, then concentrated in vacuo. The residue was treated with sodium bicarbonate solution (
1 L) and ethyl acetate (3 × 500 ml). Dry the combined extracts (
Na ₂ SO ₄ ), concentrated in vacuo and purified by flash column chromatography on silica (Merck 9385) using ether: hexane (1: 3) as eluent to afford the title compound as a yellow oil ( 12.6g)
(Once the product starts to be recovered, the eluent is replaced with ether: hexane (1: 2)
Up). T. l. c. Silica, ether: hexane (1: 1), Rf 0.32

[0087] To a stirred solution of Intermediate 5 trans-3-hydroxy-2-ethoxycarbonylmethyl-l- mosquito carboxylic acid benzyl ester 20 ° C. intermediates in absolute ethanol (50ml) 4 (4.0g), carbonate Potassium (4.0 g) was added and the suspension was stirred at 20 ° C. for 2 days. Then hydrochloric acid (2N, 30 ml) was added and the mixture was extracted with ethyl acetate (3 × 40 ml).
). The combined extracts were dried (MgSO ₄ ) and concentrated in vacuo to a yellow oil. Silica (Me) using ether: hexane (10: 1) as an eluent.
Purification by flash chromatography on rck 9385) provided the title compound as a yellow oil (1.6 g). Analytical value: C, 62.35; H, 6.9; N, 4.5; C ₁₆ H ₂₁ NO ₅ Theoretical value: C, 62.5; H, 6.9; N, 4.6%.

[0088] Intermediate 6 trans-3- (butyldimethylsiloxy) -2-ethoxycarbonyl-piperidine Roridin-1 Intermediate 5 in carboxylic acid benzyl ester 20 ° C. in anhydrous dimethylformamide (120 ml) (10.5 g
To a stirred solution of), imidazole (4.6 g) and t-butylchlorodimethylsilane (8.8 g) were added. The resulting solution was stirred at 20 ° C. for 16 hours. Water (3
00 ml) and the mixture was extracted with ethyl acetate (3 × 40 ml). The combined extracts were dried (MgSO ₄ ) and concentrated in vacuo to a yellow oil. Silica (Merck 9385) using ether: hexane (1: 5) as an eluent.
Purification by flash chromatography under 1) provided the title compound as a colorless oil (13.8 g). T. l. c. Silica, ether: hexane (1: 3), Rf 0.30

[0089] Intermediate 7 trans-3- (t-butyldimethylsilyloxy) -2- (1 Etokishikaru Bonirubuto-3-enyl) pyrrolidine-1-carboxylic acid benzyl ester -78 ° C. in anhydrous tetrahydrofuran (140 ml) solution of Intermediate 6 (10.4 g
) In a stirred solution of lithium hexamethyldisilazide (1M in hexane, 37 ml)
) Was added dropwise over 20 minutes. The solution was stirred at -78 C for 2 hours. Allyl bromide (3.8 g) was added over 5 minutes, and the solution was added at 78 ° C. for 1 hour and 2
Stirred at 0 ° C. for 3 hours. A saturated aqueous solution of ammonium chloride (100 ml) was added and the mixture was extracted with ethyl acetate (3 × 40 ml). The combined extracts are dried (Mg
SO ₄ ), concentrated in vacuo to give a yellow oil which was purified by flash chromatography on silica (Merck 9385) using ether: hexane (1: 5) as eluent to give a pale yellow oil. The title compound was obtained (6.9 g). Analysis: C, 65.05; H, 8.6 ; N, 3.3; C 25 H 41 NO 5 Si theoretical: C, 64.8; H, 8.9 ; N, 3.0%.

Intermediate 8 Trans-2- (1-ethoxycarbonylbut-3-enyl) -3-hydroxypyrrolidine -1-carboxylic acid benzyl ester To a solution of Intermediate 7 (280 g) in anhydrous tetrahydrofuran was added tetrabutyl fluoride. Ammonium (1M in tetrahydrofuran, 750 ml) was added over 10 minutes. The mixture was stirred at 20 ° C. under nitrogen for 18 hours. The solvent was removed in vacuo and the residue was purified by flash column chromatography on silica (Merck 9385) using ether: hexane (1: 1) to 100% ether (gradient elution) to give the title compound as a colorless oil. (157.5g)
. T. l. c. Silica, ether, Rf 0.71 / 0.67 (diastereomer)

[0091] Intermediate 9 trans-2- (1-Karubokishibuto-3-enyl) -3- intermediate in a mixture of hydroxy pyrrolidinone emission-1-carboxylic acid benzyl ester in tetrahydrofuran (700 ml) and water (80 ml) 8 (45.0 g) To the solution was added a suspension of lithium hydroxide (16.0 g) in water (50 ml). This suspension was stirred at 50 ° C. for 18 hours. The reaction was cooled.
Water (500 ml) was added and the mixture was extracted with diethyl ether (3x150m
l). The aqueous layer was acidified to pH 4 with dilute hydrochloric acid (2N; 100 ml) and extracted with ethyl acetate (3 × 400 ml). The combined extracts were washed with brine (500 ml), dried (MgSO _4), and evaporated to give the title compound as a pale yellow oil (34g). T. l. c. Silica, ether, Rf0.32

Intermediate 10 rel- (3R, 3aR, 6aS) -3-allyl-2-oxohexahydrofuro [3,2-b] pyrrole-4-carboxylic acid benzyl ester anhydrous dichloromethane at 20 ° C. (600 ml) and Triethylamine (3.6
To a solution of Intermediate 9 (10 g) in g) was added 2,4,6-trichlorobenzoyl chloride (26 g). The mixture was stirred at 20 ° C. for 3 hours. It was then diluted with toluene (1600 ml) and added dropwise to a stirred solution of 4-dimethylaminopyridine (17.22 g) in anhydrous toluene (800 ml) over 4 hours under reflux. The mixture was stirred under reflux for another hour. The reaction was cooled and the solvent was removed in vacuo. Water (300 ml) and dilute hydrochloric acid (2N, 300 ml) were added and the mixture was extracted with ethyl acetate (3 × 500 ml). Dry the combined extracts (MgSO ₄ )
Concentrate and use silica (Merc) with ether: hexane (1: 1) as eluent
k9385) by flash column chromatography twice.
The title compound was obtained as an off-white solid (9.08g). Analysis: C, 68.5; H, 6.6 ; N, 4.65; C 17 H 19 NO 4 theoretical value: C, 67.8; H, 6.4 ; N, 4.7%.

Intermediate 11 rel- (3R, 3aR, 6aS) -3-propylhexahydrofuro [3,2- b] pyrrole-2-one 5% palladium / carbon catalyst was placed under a hydrogen atmosphere for 1/2 hour. Ethyl acetate (50m
1) stirred. To the catalyst suspension was added intermediate 10 (150 ml) in ethyl acetate (150 ml).
5.0 g) was added and the mixture was stirred under a hydrogen atmosphere for 2 hours. The catalyst was removed by filtration and the filtrate was evaporated to give the title compound as a white solid (2.7g).
. T. l. c. Silica, ethyl acetate: methanol (19: 1), Rf 0.21 Analysis: C, 63.85; H, 8.8; N, 7.9; C ₁₇ H ₁₉ NO ₄ Theoretical: C, 63.9; H, 8.9; N, 8.3 %.

[0094] Compound Example 1 rel- (3R, 3aR, 6aS ) -4- (2- oxo-3-propyl-hexa arsenide Dorofuro [3,2-b] pyrrol-4-sulfonyl) benzoic acid Intermediate 11 (0. 05g), 4- (chlorosulfonyl) benzoic acid (0.076
g) and triethylamine (0.076 g) in dichloromethane (5 ml)
Mix at 0 ° C. The mixture was warmed to room temperature. After 3 days, the reaction was diluted with dichloromethane (10 ml) and hydrochloric acid (1 M 10 ml). Then organics were washed with brine, dried (MgSO _4). The solvent was removed in vacuo and the residue was purified by flash chromatography on silica (Merck 9385) using ethyl acetate: hexane (1: 1) as eluent to give the title compound as a white solid (0.006 g). ). Proton NMR (CH ₃ OD) δ 1.01 (3H, t), 1.35 (2H, m), 1.6 (2H, m), 1.75-1
.92 (2H, m), 2.07 (1H, m), 2.3 (1H, m), 2.92-3.03 (2H, m), 3.65-3.81 (2H
, M), 4.09 (1H, ddd), 7.93 and 8.25 (4H, AA'BB ') analysis: C, 54.5; H, 5.5 ; N, 3.6; C 16 H 19 NO 6 S theory: C, 54.4 ; H, 5.4; N, 4.0%.

Compound Example 2 rel- (3R, 3aR, 6aS)-[3- (2-oxo-3-propylhexahydrofuro [3,2-b] pyrrole-4-sulfonylmethyl) benzoic acid benzyl ester dichloromethane (2 A mixture of intermediate 11 (0.025 g), triethylamine (0.017 g) and benzyl 3-chlorosulfonylmethylbenzoate (0.052 g) in 0.5 ml) was stirred at room temperature for 2.5 hours. The mixture is concentrated in vacuo, then silica (4: 1) with ether: hexane (4: 1) as eluent.
Merck 9385) gave the title compound as a white solid (0.040 g) as determined by flash column chromatography. Proton NMR (CDCl ₃ ) δ 0.87 (3H, t), 1.0-1.8 (4H, m), 2.01 (1H, m), 2.
24-2.45 (2H, m), 3.17 (1H, dd), 3.52 (1H, m), 3.7-3.84 (2H, m), 4.3 (2H,
AB), 5.37 (2H, s), 7.35-7.47 (5H, m), 7.51 (1H, t), 7.62 (1H, ddd), 8.0
. 3 (1H, t), 8.12 (1H, ddd) analysis: C, 62.4; H, 5.9 ; N, 2.9; C 24 H 29 NO 8 S theory: C, 62.7; H, 6.4 ; N, 3.0 %.

Compound Example 3 Ethyl acetate of rel- (3R, 3aR, 6aS) -3- (2-oxo-propylhexahydrofuro [3,2-b] pyrrole-4-sulfonylmethyl) benzoate (25 ml) A mixture of Example 2 (0.034 g) and 5% palladium on carbon catalyst (0.030 g) was stirred vigorously under a hydrogen atmosphere at room temperature for 2 hours. The catalyst was removed by filtration, and the filtrate was concentrated in vacuo. The residue was redissolved in ethyl acetate and the solution was filtered and evaporated to give the title compound (0.032g). Analytical value: C, 56.3; H, 6.0; N, 3.6; C ₁₇ H ₂₁ NO ₆ S Theoretical value: C, 55.6; H, 5.8; N, 3.8%. Proton NMR (d ₅ -DMSO) δ 0.82 ( 3H, t), 1.14 (2H, m), 1.38 (2H, m), 1.53 (2H
, m), 1.67 (2H, m), 2.07 (2H, m), 2.34 (2H, m), 2.83 (1H, m), 3.32 (1H, dd),
3.8 (1H, m), 3.94 (1H, t), 4.08 (1H, ddd), 4.67 (2H, AB), 7.56 (1H, t), 7.68 (
1H, dt), 7.75 (1H, dt), 8.05 (1H, t).

Compound Example 4 rel- (3R, 3aR, 6aS) -4- (1H-indole-2-carbonyl ) -3-propyl-hexahydro-furo [3,2-b] pyrrole-2-one To a stirred solution of intermediate 11 (100 mg) and triethylamine (0.2 ml) in dichloromethane (10 ml) was added 1H-indole-2-carbonyl chloride. The suspension was stirred for 1.5 hours. Water (20 ml) was added and the mixture was extracted with dichloromethane (3 × 20 ml). The combined extracts were dried (MgSO ₄ ) and concentrated in vacuo to give a foam (304 mg). This material was combined with silica (Merck) using ether: hexane (3: 1) as eluent.
Purification by flash chromatography on 9385) afforded the title compound as a pale yellow foam (118 mg). T. l. c. Silica, ether: hexane (3: 1); Rf 0.53 NMR (CDCI ₃ ) δ9.23 (1H, brs), 7.69 (1H, brd), 7.41 (1H, brd), 7.32 (1H, dt)
, 7.16 (1H, dt), 6.91 (1H, d), 4.41-4.18 (2H, m), 4.16-4.0 (1H, m), 3.59 (1H,
brt), 2.97 (1H, m), 2.55 (1H, m), 2.35-2.10 (3H, m), 1.75 (1H, m), 1.40 (1H,
m), 1.03 (3H, t). * Ref.WH Parsons et al 3 Med Chem (1989) 32, 1681

The results obtained by testing the example compounds in the Example Biological Data Assay are shown below.

[Table 1]

[Procedure amendment]

[Submission date] February 15, 2001 (2001.1.25)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Claims

[Correction method] Change

[Correction contents]

[Claims]

Embedded image (Expressed as relative stereochemistry) wherein R ¹ is the moiety modified to fit into the S ₁ -specific subsite of the enzyme; R ² is the potency, pharmacokinetics, pharmacodynamics, A serine protease enzyme inhibitor, which is a compound represented by the following formula, which is a moiety modified to optimize selectivity and physicochemical properties, and physiologically acceptable salts and solvates thereof.

Embedded image (Displayed as relative stereochemistry) {wherein, R ¹ is a moiety modified so as to fit into the S ₁ -specific subsite of the enzyme}.

Embedded image (Expressed as relative stereochemistry) wherein R ¹ is the moiety modified to fit into the S ₁ -specific subsite of the enzyme; R ² is the potency, pharmacokinetics, pharmacodynamics, A library comprising a compound represented by the following general formula (I), which is a moiety modified to optimize selectivity and physicochemical properties, and a pharmaceutically acceptable salt and solvate thereof.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 31/12 A61P 31/12 31/22 31/22 43/00 111 43/00 111 C07B 61/00 C07B 61/00 A // C07M 7:00 C07M 7:00 (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE) , LS, MW, SD, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG , BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GE, GH, GM, HR, HU, ID, IL, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG , SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZW (71) Applicant Glaxo Wellcome House, Berkeley Avenue Greenfield, Middlesex UB6N0 in (72) Inventors Michael, Dennis, Doll, Hertfordshire, England, Stevenage, Gunnels, Wood, Road, Glaxo, C In Lucham, PLC (72) Inventor Harry, Finch In Hertfordshire, England, Stevenage, Gunnels, Wood, Road, Glaxo, Welcome, plc (72) Inventor Michael, Mentis, Han Hertfordshire, England (72) Inventor Henry, Anderson, Kelly Hertfordshire, England, Stevenage, Gunnels, Wood, Road, Glaxo, Welcome, PLC (72) 72) Inventors Simon, John, Fauset, McDonald, Hertfordshire, England, Stevenage, Gunnels, Wood, Road, Glaxo, Welcome, Plc. (72) Inventors Nail, Anthony, Peg in Hertfordshire, England, Stevenage, Gunnels, Wood, Road, Glaxo, Welcome, Plc (72) Inventor Nigel, Graham, Ramsden In Hertfordshire, England, Stevenage, Gunnels, Wood, Road, Glaxo, Welcome, plc (72) Inventor Nigel, Steven, Watson Hertfordshire, England, Stevenage, Gunnels, Wood, Road, Glaxo, Welcome, plc F term (reference) 4C086 AA01 AA02 AA03 CB22 MA01 MA04 NA14 ZA54 ZA59 ZC20 4H006 AA01 AA03 AB20 AB24 AB25

Claims (27)

[Claims] 1. A serine protease enzyme inhibitor which is a substituted derivative of trans-hexahydrofuro [3,2-b] pyrrole-2-one. 2. Formula I: ## STR1 ## (Expressed as relative stereochemistry) wherein R ¹ is a moiety modified to fit into the S ₁ -specific subsite of the enzyme; R ² is the potency, pharmacokinetics, pharmacodynamics, A serine protease enzyme inhibitor, which is a compound represented by the following formula, which is a moiety modified to optimize selectivity and physicochemical properties, and physiologically acceptable salts and solvates thereof. 3. Use of a substituted derivative of trans-hexahydrofuro [3,2-b] pyrrole-2-one as a serine protease enzyme inhibitor. 4. The inhibitor according to claim 1, which is used as a medicament in the treatment of a disease associated with serine protease enzyme activity. 5. The inhibitor according to claim 2, wherein R ¹ represents C _2-4 alkyl or C _2-4 alkenyl and is used as an inhibitor of an elastase-like enzyme such as human neutrophil elastase. 6. The inhibitor according to claim 5, wherein R ¹ represents propyl or isopropyl. 7. R ¹ is (CH ₂ ) _2-4 NHC (＝ NH) NH ₂ , (CH ₂ ) _1-2 PhC (
ＮＨNH) NH ₂ , (CH ₂ ) _3-5 C (＝ NH) NH ₂ , CH ₂ (cyclohexyl) NH ₂ , (CH ₂ ) _1-3 (NH) _0-1 Het (where Het is 1 includes more nitrogen atoms, inhibition of optionally represents an aromatic ring may also be 5 or 6 membered optionally substituted with an amine) or (CH ₂₎ represents a _3-5 NH _2, trypsin-like enzymes, such as thrombin or tryptase The inhibitor according to claim 2, which is used as an agent. 8. The inhibitor according to claim 2, wherein R ¹ represents benzyl and is used as an inhibitor of a chymotrypsin-like enzyme such as cathepsin G. 9. A compound of formula II: (Expressed as relative stereochemistry) {wherein, R ¹ is a moiety modified to be compatible with the S ₁ -specific subsite of the enzyme}. 10. R ¹ is (CH ₂ ) _2-4 NHC (＝ NH) NH ₂ , (CH ₂ ) _1-2 PhC (
ＮＨNH) NH ₂ , (CH ₂ ) _3-5 C (＝ NH) NH ₂ , CH ₂ (cyclohexyl) NH ₂ , (CH ₂ ) _1-3 (NH) _0-1 Het (where Het is 1 includes more nitrogen atoms, optionally an aromatic ring may also be 5 or 6 membered optionally substituted with an amine) or (CH _{2) 3-5} represents a NH _2, the compound of claim 9. 11. The compound according to claim 9, wherein R ¹ represents C _2-4 alkyl or C _2-4 alkenyl. 12. A method for treating chronic bronchitis or ARDS, comprising administering an effective amount of the neutrophil elastase inhibitor according to claim 5 or 6 to a patient. 13. A method for treating a vascular disease, particularly thrombosis, comprising administering to a patient an effective amount of the thrombin inhibitor according to claim 7. 14. A method for treating asthma, comprising administering to a patient an effective amount of the tryptase inhibitor according to claim 7. 15. A library comprising a plurality of substituted derivatives of trans-hexahydrofuro [3,2-b] pyrrole-2-one. 16. A compound of formula I: (Expressed as relative stereochemistry) wherein R ¹ is a moiety modified to fit the S ₁ specific subsite of the enzyme; R ² is the potency, pharmacokinetics, pharmacodynamics, A library comprising a compound represented by the following general formula (I), which is a moiety modified to optimize selectivity and physicochemical properties, and a pharmaceutically acceptable salt and solvate thereof. 17. The library according to claim 15, which comprises at least 10 different compounds. 18. The library according to claim 16, wherein R ¹ represents C _2-4 alkyl or C _2-4 alkenyl. 19. R ¹ is _{(CH 2) 2-4 NHC (=} NH) NH 2, (CH 2) 1-2 PhC (
ＮＨNH) NH ₂ , (CH ₂ ) _3-5 C (＝ NH) NH ₂ , CH ₂ (cyclohexyl) NH ₂ , (CH ₂ ) _1-3 (NH) _0-1 Het (where Het is 1 more it includes a nitrogen atom, optionally an aromatic ring may also be 5 or 6 membered optionally substituted with an amine) or (CH _{2) 3-5} represents a NH _2, claim 16 or 17 library described. 20. The library according to any one of claims 15 to 19, which is a solid phase library. 21. The library according to any one of claims 15 to 19, which is a liquid phase library. 22. The library according to claim 20, which is a separate library. 23. The library of claim 20, which is a pooled library. 24. Use of the library according to any one of claims 15 to 18 for screening for an inhibitor of neutrophil elastase. 25. The method for screening a thrombin or tryptase inhibitor according to claim 1.
Use of the library according to any one of 5, 16, 17 or 19. 26. A method for screening a serine protease enzyme inhibitor, comprising treating a serine protease enzyme with the inhibitor according to claim 1 and measuring the degree of inhibition. . 27. (a) preparing several kinds of trans-hexahydrofuro [3,2-b] pyrrole-2-one-substituted derivatives; (C) measuring the degree to which inhibition of the enzyme has occurred. A method for identifying a serine protease enzyme inhibitor, comprising: