JPH0680673A - Soluble cephalosporin compound - Google Patents

Abstract

PURPOSE:To provide a new compound useful as an enzyme inhibitor having excellent activity, stability and water-solubility. CONSTITUTION:The compound of formula I {R<1> is CX<1>3 (X<1> is H or halogen); R<2> is 0-Q-Z<1> [Q is group bonding O and Z<1> through a 3-6C chain; Z<1> is CONH-(CH2)1-3-Z<2> or SO2NH-(CH2)1-3-Z<2> ( <2> is COOH or SO2H)]; R<3> is H, OCOR<4> (R<4> is 1-6C alkyl), etc.} and its pharmaceutically permissible salt, e.g. (4-carboxymethylcarbamoyl-benzyl)-7alpha-trifluoroacetamido-3-acetoxym ethyl-3- cephem-4-carboxylate-1,1-dioxide. The compound of formula I is produced e.g. by producing a compound of formula II from 7-amino-cephalosporanic acid, etc., used as starting raw materials, reacting the compound of formula II with a compound of the formula HOQ-Z<3> (Z is COOH or SO3H) and reacting the resultant compound of formula III with an amino acid or an aminosulfonic acid.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a soluble cephalosporin compound, and particularly to serine proteinase inhibitory activity,
Particularly, it relates to a cephalosporin compound having an elastase inhibitory activity and being excellent in stability in plasma and water solubility.

[0002]

2. Description of the Related Art Elastase, which is a type of serine proteinase, is an enzyme that exists in leukocytes and decomposes elastin. This elastase is deeply involved in inflammatory diseases such as pancreatitis, emphysema, and rheumatism, and its treatment with α ₁ -proteinase inhibitor is being studied.
Various low molecular weight compounds have been investigated as elastase inhibitors, but none have been put to practical use because of problems such as toxicity and hydrolysis resistance in vivo.

Incidentally, a sulfone derivative of penicillin or cephalosporin is known as a β-lactamase inhibitor, and attempts have been made to utilize it as an elastase inhibitor (US Pat. No. 4,54).
7, 371, JP-A-63-198688 and JP-A-2-28183).

However, according to the studies by the present inventors, the compounds specifically disclosed in the above-mentioned patent application specifications were not always satisfactory for use as pharmaceuticals. That is, in order to be usable as a medicine, not only is it highly active, but when it is administered to the body, it can be stably present in blood until it reaches a predetermined affected area, and for example, injection In order to use it as a medicine, it must satisfy further requirements such as high water solubility. The compounds specifically disclosed in the above specification also included compounds having a high elastase inhibitory activity in an in vitro test, but having both the stability in blood and the water solubility as described above. Nothing was found.

That is, as a compound similar to the present invention, for example, in US Pat. No. 4,547,371, a compound represented by the formula (I) is used.

[0006]

[Chemical 4]

The above compounds and R ₁ and R ₂ are the same as above, and a compound in which --CH ₂ --R ₃ is --CF ₃ is specifically produced and is listed as a compound showing high elastase inhibitory activity (Table II). Although they show some activity in an in vitro test, they have very poor stability in blood and low water solubility.

A table of Japanese Patent Laid-Open No. 63-198688
In II, similar compounds, which are not the same as the present invention, are listed as examples of specifically prepared compounds together with their activities. Among these compounds are the compounds of formula (I) herein
At

[0009]

[Chemical 5]

Although the above-mentioned substances are described as exhibiting high activity, they also have poor stability in blood and low water solubility.

Further, Japanese Patent Laid-Open No. 2-28183, No. 21
The table on the page also lists similar compounds, although not identical to the present invention, as specific examples of their activity. This compound is represented by formula (I) herein.

[0012]

[Chemical 6]

[0013] Corresponding to the above, the activity is not so high, the stability in blood is poor, and it is not soluble in water at all.

In addition, in these specifications, many examples of substituents that do not exhibit any activity are listed.
It was impossible to speculate at all whether any compound had high activity, high stability in blood, and high water solubility.

[0015]

As described above, when a cephalosporin derivative is used as an elastase inhibitor, a compound that satisfies the high activity, high stability and high water solubility required for a practical drug is actually found. Has not been issued,
Thus, there has been a demand for the development of compounds that simultaneously satisfy the above three requirements.

[0016]

Means for Solving the Problems As a result of earnest studies, the present inventors have found that in a cephalosporin-based compound, the substituents at the 3-position, 4-position and 7-position are combined and constituted by certain limited specific ones. As a result, they have found that it is possible to obtain a compound that can simultaneously satisfy the above-mentioned required properties, and have reached the present invention.

[0017]

That is, the present invention has the following general formula (I).

[0018]

[Chemical 7]

"However, in the formula R¹Is-CX¹ ₃Represents,
Where X¹Represents H or a halogen atom; R²Is-O
-Q-Z¹Represents a group represented by, where Q is R²To
Let ’s say –O– and –Z¹And carbon atoms with 3 to 6 carbon atoms
Is a group that¹Is CONH- (CH₂)_1-3-Z²
Or SO₂NH- (CH₂)_1-3-Z²And Z²Is C
OOH or SO₃H; R³Is H, OH, OCOR
^Four, OR^Four, OCONH ₂,

[0020]

[Chemical 8]

Represents X, where X²Is -S- or -NH
Represents-and X³Is H, CH₃, NH ₂Or S-CH₂
Represents COOH, X^FourIs H, COOH or N (C
H₃)₂Represents; R_FourIs C_1-6Represents alkyl. ]
Cephalosporin compounds represented by
It is a physiologically acceptable salt.

In the above formula (I), R ¹ is --CX ¹ ₃
X ¹ is H or a halogen atom, preferably Cl
Or F, particularly preferably F. This substituent is the part that contributes most to the elastase inhibitory activity of the compound, and also the part that contributes to the selectivity for eliminating the inhibitory effect on other enzymes.

The most preferred R ¹ in terms of activity and selectivity is —CF ₃ .

R ² represents a group represented by --O--Q--Z ¹ , and Q represents a group which connects --O-- and --Z with a carbon atom length of 3 to 6. The carbon atom length of 3 to 6 means that the carbon atom passes through at least 3 to 6 from -O- to -Z ¹ , and Q itself may be linear or cyclic. I don't care. In particular

[0025]

[Chemical 9]

Hydrocarbon groups which may contain heteroatoms such as

Z¹Is CONH- (CH₂)_1-3-Z²or
Is SO₂NH- (CH₂)_1-3-Z ²Represents

Z ² represents COOH or SO ₃ H. R
^As a concrete example of ²

[0029]

[Chemical 10]

And the like.

In the compound of the present invention, the group R ² contributes most to stability in blood and also contributes to improvement of activity and water solubility.

From this point of view, preferable R ² is

[0033]

[Chemical 11]

And particularly preferably

[0035]

[Chemical 12]

And the like.

R ³ may be a side chain generally used in cephalosporin antibiotics. In particular,

[0038]

[Chemical 13]

And the like.

Preferred R ³ is

[0041]

[Chemical 14]

And the like.

The cephalosporin compound of the present invention has excellent properties in terms of elastase inhibitory activity, blood stability and water solubility, but representative compounds having such excellent properties will be exemplified. The following may be mentioned.

[0044]

[Chemical 15]

[0045]

[Chemical 16]

[0046]

[Chemical 17]

[0047]

[Chemical 18]

The compounds of the present invention show a high elastase inhibitory activity with an IC ₅₀ of 0.03 to 10 μM, preferably 0.03 to 1.0 μM. Here, the elastase inhibitory activity means that the inhibitor is dissolved in dimethyl sulfoxide (finally 6.7% concentration), and human leukocyte elastase (manufactured by Calbiochem, finally in Tris buffer (50 mM, pH 8.0) is dissolved. 0.02 μM concentration), and a dimethylformamide solution of methoxysuccinyl-alanyl-alanyl-prolyl-valine-p-nitroanilide (manufactured by Sigma, finally 0.2 mM concentration) is added, and the mixture is added at 25 ° C. for 2 minutes. This is a value measured by measuring the absorbance of p-nitroaniline released later, obtaining the concentration of the inhibitor that shows half the absorbance in the absence of the inhibitor, and setting this as the IC ₅₀ .

Further, the compound of the present invention exhibits excellent properties such that the stability in blood is represented by the half-life (t _1/2 ) in plasma of 7 minutes or longer, preferably 10 minutes or longer. Here, the half-life in plasma is 0.01 to 0.1 mg / ml of a test compound dissolved in plasma of a hamster, pH 7.0,
It is a value expressed by incubating at 7 ° C., measuring the decrease by the internal standard method using high performance liquid chromatography, and determining the time at which the concentration of the test compound is ½ of the initial concentration. In order to recognize the above, it is desired that the time is 10 minutes or more depending on the activity.

The compounds of the present invention are also excellent in water solubility. When the compound of the present invention is used as an elastase inhibitor, it is required to be water-soluble regardless of whether it is applied through the respiratory tract or intravenously. Especially for intravenous injection, high water solubility, for example, 10 mg / ml or more is required.

The compound of the present invention has a water solubility of 5 mg / ml or more, more preferably 20 mg / ml or more, and particularly preferably 100 mg / ml or more.

The water solubility is the solubility of the present compound in distilled water adjusted to pH 7, and is the value measured at 25 ° C. In the case of dissolution, when the compound is a free carboxylic acid, it is dispersed in distilled water, and NaHCO ₃ , Na ₂ C
O _3, NaOH or dissolved by neutralization to pH7 was added these 1~2N solution, the 1~2N alkaline aqueous solution after dissolved in distilled water in the case of such Na salt soluble the compound as it Alternatively, it is preferable to adjust the pH to 7 using 1-2N hydrochloric acid.

Further, in general, enzyme inhibitors are required to have, in addition to the above properties, selectivity, that is, a property of acting only on the target enzyme and not on other enzymes.

The compounds of the present invention have a selectivity of 1000 or more when human thrombin and human trypsin are selected as other enzymes and their action specificity is expressed.

Here, the selectivity is the value of the ratio of the IC ₅₀ of elastase to the IC ₅₀ of thrombin and trypsin. The IC ₅₀ of thrombin and trypsin can be measured by the same method as that for elastase except that the enzyme concentration is 0.01 μM and N-carbobenzoxy-glycyl-prolyl-arginine-p-nitroanilide is used as the substrate.

The compound of the present invention described above can be produced by using a known raw material in combination with a method known in the technical field for producing a cephalosporin derivative.

For example, 7ACA (7-amino-cephalosporanic acid), 7ADCA (7-amino-deacetoxycephalosporanic acid), GCLE (7-phenylacetamido-3-chloromethyl-3-cephem-4-carboxylic acid-p) -Methoxybenzyl ester), 3BMY (7-
Phenylacetamide-3-bromomethyl-3-cephem-4-carboxylic acid) etc.
It is produced from the compound of I), (III) or (V).

The compounds of formulas (II), (III) and (V) can be obtained by known methods, for example, as follows.

(Compounds of Formulas (II) and (III)) Compounds of Formulas (II) and (III) in which R ³ is other than a sulfide derivative are obtained by starting from 7ACA, 7ADCA or other corresponding compound (VI). To be

[0060]

[Chemical 19]

[0061] Compound (VI) in R ¹ COOH (R ¹ C
O) ₂ O (R ¹ is as described above) to react with the compound (VI
I) get

[0062]

[Chemical 20]

Compound (VII) in tetrahydrofuran
-With chloroperbenzoic acid or in aqueous solution 2 KHSO ₅
・ Oxidized with KHSO ₄ · K ₂ SO ₄ (“Oxone” manufactured by Aldrich) and treated with alkali if necessary to obtain formula (III)
The compound of

[0064]

[Chemical 21]

Compound (III) is converted to an acid chloride with, for example, PCl ₅ and pyridine, and this is reacted with protected or unprotected HOQ-Z ³ and, if necessary, deprotected to obtain compound (II).

[0066]

[Chemical formula 22]

(Compound of Formula (V)) Compound (V) is obtained from GCLE or 3BMY as follows.

[0068]

[Chemical formula 23]

However, GCLE: n = 0, X = Cl, R ⁴ =
Deprotection of t-butyl, 3-BMY: n = 1, X = Br, R ⁴ = methoxybenzyl GCLE or 3-BMY with phosphorus pentachloride by a known method gives compound (VIII).

[0070]

[Chemical formula 24]

Compound (VIII) and acid chloride R ¹ COC
Compound (IX) is obtained from 1.

[0072]

[Chemical 25]

The compound (IX) is oxidized with, for example, m-chloroperbenzoic acid or oxone to obtain the compound (X).

[0074]

[Chemical formula 26]

Compound (XI) is obtained by deprotecting compound (X) with trifluoroacetic acid or the like.

[0076]

[Chemical 27]

The compound (XI) or a reactive derivative thereof such as an acid chloride or an active ester derivative thereof is reacted with a protected or unprotected compound of formula (IV) HOQ-Z ¹ and, if necessary, deprotected, And, if necessary, the compound (V) can be obtained by treating with triethylamine.

[0078]

[Chemical 28]

The target compound can be obtained by further reacting these compounds.

For example a compound of formula (II)

[0081]

[Chemical 29]

[However, R ¹ , R ³ and Q are as described above. Z ³
Is --COOH, --SO ₃ H or its reactive derivative]
It is obtained by reacting with protected or unprotected amino acid or aminosulfonic acid, and if necessary, deprotecting.

Also a compound of formula (III)

[0084]

[Chemical 30]

[However, R ¹ and R ³ are as described above. ] And protected or unprotected compound of formula (IV) HOQ-Z ¹ ... (IV) [wherein Q and Z ¹ are as described above]. ] Is reacted with, and if necessary, deprotected to obtain.

When R ³ is a sulfide derivative, a compound of formula (V)

[0087]

[Chemical 31]

[However, R ¹ and R ² are as described above. X is a halogen atom. ] And a mercapto derivative are made to react.

Here, there may exist R ¹ CONH at the 7-position at the α-position and at the β-position, but they can be isomerized to obtain a pure isomer. Thereby, for example, only one isomer can be obtained as shown in the following reaction formula.

[0090]

[Chemical 32]

The compound of the present invention or a salt thereof can be used as an active ingredient of a pharmaceutical preparation.

As a method of administration, it is used in the form of injection or aerosol.

The injection is used by dissolving it in physiological saline, sugar solution of budo, etc., but it is prepared by adding a preservative, a stabilizer and the like as necessary.

The aerosol administration agent may be used as it is or as a nebulizer liquid agent together with a surfactant such as fatty acid.
Alternatively, it is used as an aerosol liquid agent together with a propellant such as alkane having 5 or less carbon atoms.

Usually, this compound is 0.1 to 10 per day for an adult.
The dose of 00 mg is set, but it may be appropriately increased or decreased depending on the age, sex, weight of the patient, indication and its degree, and administration method.

The present invention will be described in detail below with reference to examples.

[0097]

Example 1 1.34-g of (4-carboxybenzyl) -7α-trifluoroacetamido-3-acetoxymethyl-3-cephem-4-carboxylate-1,1-dioxide was dissolved in 50 ml of tetrahydrofuran and cooled with ice. Lower 5.2 g of phosphorus pentachloride and 2.0 ml of pyridine are added.
Then glycine-t-butyl ester hydrochloride 0.42 g
And 0.4 ml of pyridine are added and reacted for 2 hours.

Ethyl acetate was added and a 5% KHSO ₄ aqueous solution was added.
After washing with 4% NaHCO ₃ aqueous solution and saturated saline,
Dry over SO ₄ and evaporate the solvent.

50 ml of trichloroacetic acid was added to this, and the mixture was cooled to 0 ° C.
Stir for 2 hours.

Trichloroacetic acid was distilled off under reduced pressure, the residue was dissolved in 50 ml of dichloromethane and triethylamine was added to 0.3
5 ml was added and the mixture was stirred at room temperature for 2 hours, and the solvent was distilled off under reduced pressure. The residue was subjected to high performance liquid chromatography (column: Inertocyl (registered trademark) PREP-ODS; solvent: 50%).
Precipitate with (methanol-water) and freeze-dry to obtain (4-carboxymethylcarbamoyl-benzyl) -7α-trifluoroacetamido-3-acetoxymethyl-3-cephem-4-carboxylate-1,1-dioxide. obtain. (Compound 1) NMR (acetone-d ₆ ) δ (ppm): 4.0-4.3 (m, 4H), 4.64, 5.10 (ABq, J = 13.1 Hz, 1H), 5. 2 to 5.5 (m, 4H), 6.57 (t, J = 5.9Hz, 1H), 7.47 (d, J = 8.0Hz, 2H), 7.83 (d, J = 8) 0.0Hz, 2H), 9.52 (d, J = 7.7Hz, 1H). IR (cm ^-1 ): 1795, 1725, 1640, 155
0, 1330, 1225, 1165, 1120, 104
5

[0101]

Example 2 5.3 g of (4-carboxybenzyl) -7α-trifluoroacetamido-3-acetoxymethyl-3-cephem-4-carboxylate-1,1-dioxide and 1.2 g of N-hydroxysuccinimide Dissolve in 20 ml of N, N'-dimethylformamide. 2.3 g of dicyclohexylcarbodiimide was added under ice cooling to 2
Stir for hours. The precipitated dicyclohexylurea is filtered off and the solvent is distilled off from the filtrate under reduced pressure. Dissolve in 40 ml of dimethyl cellosolve, 20 ml of a solution of 0.90 g of β-alanine in dimethyl cellosolve, then 0.17 of NaHCO _3.
20 g aqueous solution of g was added and the reaction was carried out at room temperature for 1.5 hours.
The reaction mixture is extracted with ethyl acetate, washed with 5% KHSO ₄ aqueous solution and saturated saline, dried over MgSO ₄ , and the solvent is distilled off. This was purified by high performance liquid chromatography in the same manner as in Example 1 to give [4- (2-carboxyethylcarbamoyl) -benzyl] -7α-trifluoroacetamido-3-acetoxymethyl-3-cephem-4-
Obtain the carboxylate-1,1-dioxide. (Compound 2) IR (cm ^-1 ): 1800, 1730, 1645, 155
5, 1330, 1220, 1170.

[0102]

Example 3 [4- (3-Carboxy-n-] was carried out in the same manner as in Example 2 except that 1.03 g of 4-aminobutanoic acid was used.
Butylcarbamoyl) -benzyl] -7α-trifluoroacetamido-3-acetoxymethyl-3-cephem-4-carboxylate-1,1-dioxide is obtained. (Compound 3) IR (cm ^-1 ): 1798, 1728, 1645, 155
5,1335,1230,1165.

[0103]

Example 4 (4-Carboxymethylcarbamoyl-benzyl) -7α-trifluoroacetamido-3-chloromethyl-3-cephem-4-carboxylate-1,
1.5 g of 1-dioxide is dissolved in 100 ml of acetone. 3-amino-5-mercapto-1,2,
Add 306 mg of 4-triazole and dissolve.

222 mg NaHCO ₃ , 2.19 g KI
Is added to 50 ml of water, and the mixture is reacted for 2 hours at room temperature. Acidify with N-HCl and distill off the acetone under reduced pressure. Ethyl acetate is added to separate the layers, the mixture is washed with saturated saline, and then dried with sodium sulfate. After filtering out the sodium sulfate, the solvent was distilled off, and the residue was fractionated by high performance liquid chromatography (column; Inertocyl (registered trademark) PREP-ODS, solvent; 50% methanol-water) (4-carboxymethylcarbamoyl-benzyl). ) -7α-Trifluoroacetamide-3
-[(3-Amino-1,2,4-trizol-5-yl) -thiomethyl] -3-cephem-4-carboxylate-1,1-dioxide is obtained. (Compound 4) NMR (DMSO-d ₆ ) δ (ppm); 3.87, 4.23 (ABq, J = 14.2 Hz, 1H), 3.93 (d, J = 5.9 Hz, 2H), 4.37, 4.56 (ABq, J = 17.8 Hz, 1H), 5.26 (dd, J = 7.7 Hz, 2.2 Hz, 1H), 5.39 (s, 2H), 5.64. (S, 1H), 7.55 (d, J = 8.1Hz, 2H), 7.89 (d, J = 8.1Hz, 2H), 8.85 (t, J = 5.9Hz, 1H) , 10.7 (d, J = 7.7 Hz, 1H). IR (cm ^-1 ): 1800, 1725, 1690, 164
5,1550,1330,1195.

[0105]

Examples 5 to 8 Using the corresponding cephalosporin derivative and mercapto derivative, the same reactions and treatments as in Example 4 were carried out to obtain the respective compounds 5 to 8. The IR spectrum is shown in the following Table 1 (sometimes referred to as Table 1).

[0106]

[Table 1]

[0107]

Comparative Compound 1 Benzyl-7α-trifluoroacetamido-3-acetoxymethyl-3-cephem-4-, according to the examples of US Pat. No. 4,547,371.
Obtain the carboxylate-1,1-dioxide.

[0108]

[Comparative compound 2] 7β-trifluoroacetamide-3
-Acetoxymethyl-3-cephem-4-carboxylic acid 1,800 mg of 1,1-dioxide was dissolved in N, N'-dimethylformamide, and phosphorus pentachloride (420 mg) and pyridine (162 µl) were added under ice cooling. Then, 300 mg of 4-carbamoyl-benzyl alcohol is added, the temperature is raised to room temperature, and the reaction is continued at the same temperature for 1 hour. The mixture is ice-cooled again, 556 μl of triethylamine is added, and the mixture is stirred for 3 hours. After adding N-hydrochloric acid to acidify, concentrate under reduced pressure, collect by high-performance liquid chromatography, and freeze-dry (4-carbamoylbenzyl) -7α-trifluoroacetamido-3-acetoxymethyl-3-cephem- 4-carboxylate-1,
1-Obtain Dioxide. IR (cm ^-1 ): 1790, 1730, 1330, 122
5,1170,1120,1050,1030,98
0. Table 2 below (also sometimes referred to as Table 2) shows values of activity, stability and water solubility of the compounds of the present invention and comparative compounds.

The compounds of the present invention each had a selectivity of 1000 or more.

[0110]

[Table 2]

[0111]

[Table 3]

Continuation of front page (51) Int.Cl. ⁵ Identification code Office reference number FI Technical display area C07D 501/28 8829-4C // A61K 31/545 ABE 9360-4C ABG 9360-4C ACD 9360-4C ACJ 9360- 4C AED 9360-4C

Claims (4)

[Claims] 1. The following general formula (I):"However, R in the formula¹Is-CX¹ ₃Where X is¹Is
Represents H or a halogen atom; R²Is -O-Q-Z¹so
Represents a group represented by where Q is R²At-O-
And-Z¹Is a group connecting between and with a carbon atom length of 3 to 6
R, Z¹Is CONH- (CH₂)_1-3-Z²Or SO₂
NH- (CH₂)_1-3-Z²And Z²Is COOH
Is SO₃H; R³Is H, OH, OCOR^Four, OR
^Four, OCONH₂ , [Chemical formula 2]Where X is²Represents -S- or -NH-,
X³Is H, CH₃, NH ₂Or S-CH₂Show COOH
I, X^FourIs H, COOH or N (CH₃)₂Represents
Do; R^FourIs C_1-6Represents alkyl. ] Soluble
Cephalosporin compounds and their pharmaceutically acceptable
Ru salt. 2. R ³ is “Here, X ² represents —S— or —NH—, and X ³ represents N.
Represents H ₂ or S—CH ₂ COOH, and X ⁴ represents COOH
Or N (CH ₃ ) ₂ . ] The soluble cephalosporin compound and its pharmaceutically acceptable salt according to claim 1. 3. The soluble cephalosporin compound according to claim 1, which has a stability in hamster plasma of 7 minutes or more and a solubility in distilled water of 2 mg / ml or more, and a pharmaceutically acceptable compound thereof. Salt. 4. The soluble cephalosporin compound and the pharmaceutically acceptable salt thereof according to claim 1 or 2, which has a solubility in distilled water of 10 mg / ml or more.