JPH0665253A - Soluble cephalosporin derivative - Google Patents

Abstract

PURPOSE:To obtain a new compound useful as an enzymic inhibitor, especially an elastase inhibitor. CONSTITUTION:The objective compound of formula I [R<1> is CX<1>3 (X<1> is H or halogen); R<2> is O-Q-Z<1> (Q is 3-6C connecting group; Z<1> is COOH, SO3H or OH, with the proviso that Q is formula II when Z<1> is OH or its salt); R<3> is H, OCOCH3, OH, OCH3, OCONH2, etc.], e.g. (4-carboxybenzyl)-7 a- trifluoroacetoamido-3-acetoxymethyl-3-cephem-4-carboxylate-1,1-dioxide. This compound of formula I is produced by starting from a compound of formula III and passing the compound of formula III through a compound of formula IV, etc.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a soluble cephalosporin derivative, and more particularly to a cephalosporin derivative having serine proteinase inhibitory activity, especially elastase inhibitory activity, and excellent in plasma stability 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).

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

Compounds similar to the present invention, for example, in US Pat. No. 4,547,371, are represented by the formula (I) herein.

[0006]

[Chemical 2]

A compound (referred to as compound M1) and R ¹
And R ² are the same as above, and a compound in which —CH ₂ —R ³ is —CF ₃ (referred to as compound M2) has been specifically produced and is listed as 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 3]

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 the formula (I) of the present specification,

[0012]

[Chemical 4]

[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.

[0014]

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,
In addition, in these specifications, many examples of substituents that do not exhibit any activity are listed, and as a result, any compound has high activity, and in addition, has high stability in blood, and I couldn't know if it was also water-soluble. Thus, there has been a demand for the development of compounds that simultaneously satisfy the above three requirements.

[0015]

Means for Solving the Problems As a result of earnest studies, the inventors of the present invention have found that by combining the substituents at the 3-position, 4-position and 7-position of a cephalosporin derivative with certain limited specific ones. The inventors have found that the above required characteristics can be satisfied at the same time, and have reached the present invention.

[0016]

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

[0017]

[Chemical 5]

A soluble cephalosporin derivative represented by and a pharmaceutically acceptable salt thereof.

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 , wherein Q represents a group which connects --O-- and --Z ¹ with a carbon atom length of 3 to 6. The carbon atom length of 3 to 6 means
From -O- to -Z ¹ 3 to 6 carbon atoms at the shortest
This means that the Q itself may be linear or cyclic. In particular

[0022]

[Chemical 6]

Hydrocarbon groups which may contain heteroatoms such as

Z ¹ is --COOH, --SO ₃ H or --OH
Represents However, when Z ¹ is --OH, Q is

[0025]

[Chemical 7]

It is necessary that —OH be bonded to the phenylene group of Q.

As a concrete example of R ² ,

[0028]

[Chemical 8]

And the like.

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

From this point of view, preferable R ² is

[0032]

[Chemical 9]

It is

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

Specifically,

[0036]

[Chemical 10]

And the like, preferably --H, --OCO
CH ₃ may be mentioned.

The compounds of the present invention have been described above in such a manner that each substituent independently contributes individually. However, as can be easily understood by those skilled in the art, only these substituents control specific characteristics of the compound. It goes without saying that the property of the compound is derived from the structure of the entire compound.

The cephalosporin derivative of the present invention has excellent properties in terms of elastase inhibitory activity, blood stability and water solubility. Typical compounds having such excellent properties are shown below. Something like.

[0040]

[Chemical 11]

[0041]

[Chemical 12]

The compounds of the present invention have an elastase inhibitory activity with an IC ₅₀ of 0.03 to 10 μM, preferably 0.
It shows a high activity of 03 to 1.0 μM. Here, the elastase inhibitory activity means that the inhibitor is dissolved in dimethylsulfoxide (finally 6.7% concentration), and then Tris buffer (50%
In addition to human leukocyte elastase (manufactured by Calbiochem, finally 0.02 μM concentration) in mM, pH 8.0, methoxysuccinyl-alanyl-alanyl-prolyl-valine-p-nitroanilide (manufactured by Sigma, final (0.2 mM concentration) in dimethylformamide, and the absorbance of p-nitroaniline released after 2 minutes at 25 ° C is measured to obtain an inhibitor concentration showing an absorbance half that in the absence of the inhibitor. It is a value measured by setting this as IC ₅₀ .

The compound of the present invention has a stability in blood represented by a half-life (t _1/2 ) in plasma of 10 minutes or more,
It preferably exhibits excellent properties for 15 minutes or more. Here, the half-life in plasma means that the test compound is dissolved in plasma of a hamster at 0.01 to 0.1 mg / ml to give a pH of 7.
It is a value expressed by incubating at 0 ° C. and 37 ° C., measuring the decrease of the test compound by an internal standard method using high performance liquid chromatography, and determining the time at which the test compound becomes ½ of the initial concentration. , In vivo, it is desired to be 10 minutes or more in order to recognize 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 compounds of the present invention have a water solubility of 5 mg / ml or more, more preferably 20 mg / ml or more, and particularly preferably 100 mg / ml or more.

Here, the water solubility is the solubility in distilled water at pH 7, which 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 dissolved by adding NaHCO ₃ , Na ₂ CO ₃ , NaOH or a 1 to 2N aqueous solution of these to neutralize to pH 7, When the compound is a soluble Na salt or the like as it is, it is preferably dissolved in distilled water and then adjusted to pH 7 by using the 1 to 2N alkaline aqueous solution or 1 to 2N hydrochloric acid.

Further, in general, the enzyme inhibitor is required to have selectivity in addition to the above properties, that is, a property that it acts only on the target enzyme and does not act 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.

The selectivity is 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 elastase except that the enzyme concentration is 0.01 μM and N-carbobenzoxy-glycyl-propyl-arginine-p-nitroanilide is used as the substrate.

The compound of the present invention described above can be produced by the following procedure.

The overall method for making the compounds of the present invention is:
Various methods in which the methods known in the technical field for producing a cephalosporin derivative are combined are easily conceivable, and the simplest method is shown below by a reaction formula.

[0052]

[Chemical 13]

Here, there may be an R ¹ CONH at the 7-position attached to the α-position and an R ¹ CONH at the β-position, but any one after oxidation of the 1-position sulfide group in order to obtain a pure isomer. It can be isomerized at some stage. Thereby, for example, only one isomer can be obtained as shown in the following reaction formula.

[0054]

[Chemical 14]

A) Compound 1 → Compound 2 Compound 1 and (R ¹ CO) ₂ O at a molar ratio of 1: 1.3 in trifluoroacetic acid at a concentration of 10 to 30 wt% at 0 ° C. to room temperature for 30 minutes Stir for 2 hours. After completion of the reaction, the solvent is evaporated under reduced pressure and the obtained residue is subjected to the next reaction.

(B) Compound 2 → Compound 3 To the product obtained in a), water is added so as to be about 10 wt%, and the pH is adjusted to _{5 to 6} with NaHCO ₃ and dissolved. An equimolar amount of 2KHSO ₅ · KHSO ₄ · K ₂ SO
₄ ("Oxone" manufactured by Aldrich) with 2N-NaOH
Add pH while adjusting to pH 5-6. After stirring at room temperature for 5 hours, an equal volume of methanol was added, the resulting precipitate was separated by filtration, and the same amount of ethyl acetate as methanol was added to the filtrate to give 10
After being washed twice with an aqueous KHSO ₄ solution, it is dried over MgSO ₄ and the solvent is distilled off under reduced pressure. This was dissolved in ethyl ether and reprecipitated with hexane to give compound 3
To get

C) Compound 3 → Compound 4 The product obtained in b) is dissolved in tetrahydrofuran,
Cool on ice. Under ice cooling, PCl ₅ was added to the above product in an equimolar amount, and an equimolar amount of pyridine was added as a deoxidizing agent. Next, R ² —H and equimolar amounts of R ² —H and pyridine are added, and the reaction is continued for 30 minutes with stirring. Ethyl acetate is added, and the mixture is washed twice with a 10% KHSO ₄ aqueous solution, washed with saturated saline, and then dried over MgSO ₄ . Compound 4 is obtained by distilling off the solvent. A compound in which the acetoxymethyl group of compound 4 is a methyl group, a methoxy group or the like can be obtained in the same manner as above.

D) Compound 4 → Compound 5 The product obtained in c) is dissolved in acetonitrile, R ³ H is added in the presence of 3 times mole of boron trifluoride ether complex, and the mixture is heated at 50 ° C. for 24 hours. React under stirring. After cooling, ethyl acetate is added, and the mixture is washed with 10% KHSO ₄ water, water and saturated saline and dried over MgSO ₄ . The solvent is distilled off to obtain compound 5. Here, R ³ H is a mercapto compound.

(E) Compound 5 → Compound 6 The product obtained in d) is dissolved in CH ₂ Cl ₂ or tetrahydrofuran under ice cooling, an equimolar amount of Et ₃ N is added, and the mixture is allowed to stand at the same temperature for 2 hours. . Add ethyl acetate 10
% KHSO ₄ water, washed with water, saturated saline, MgSO ₄
To dry. After distilling off the solvent, an equal volume to 10 times the amount of methanol, ethanol or isopropanol is added and dissolved, and the mixture is left standing in a refrigerator and the precipitated solvate is collected by filtration. This solvate is dispersed in water and then filtered or freeze-dried to obtain Compound 6.

At each stage of these reactions, purification by means such as column chromatography separation by silk gel, recrystallization, separation by high performance liquid chromatography and the like may be used.

There is also another method for obtaining the compound 5. Compound 4
It can also be obtained by reacting a halomethyl group such as a chloromethyl group or a bromomethyl group in place of the acetoxymethyl group at the 3-position, and a compound 4 ′ substituted with —CH ₂ X with HR ³ .

[0062]

[Chemical 15] F) Compound 4 ′ → Compound 5 Compound 4 ′ is dissolved in a 20-50% acetone aqueous solution, and H
-R ³ equimolar and NaHCO ₃ equimolar are added and reacted at room temperature. After 30 minutes to several hours, acidify with N-HCl, distill off acetone under reduced pressure, add ethyl acetate, wash with water and saturated saline, and dry with MgSO ₄ . The solvent is distilled off to obtain compound 5.

If KI and NaI are added during this reaction, the reaction proceeds rapidly.

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

[0065]

Example 1 7β-Trifluoroacetamide-3-acetoxymethyl-3-cephem-4-carboxylic acid-1,
1-Dioxide 4.0 g tetrahydrofuran 50 ml
2.1 g of phosphorus pentachloride and 0.8 of pyridine under ice cooling
Add 1 ml. Then, 1.5 g of 4-hydroxymethylbenzoic acid is added, and 0.81 ml of pyridine is further added. After stirring at the same temperature for 30 minutes, ethyl acetate is added for extraction, the mixture is washed with 10% KHSO ₄ aqueous solution and saturated saline, dried over MgSO ₄ , and the solvent is distilled off. The residue was redissolved in tetrahydrofuran and triethylamine 1.
Add 4 ml under ice cooling and react for another 3 hours.

Ethyl acetate is added, and the mixture is washed with a 5% KHSO ₄ aqueous solution and a saturated saline solution, dried over MgSO ₄ , and the solvent is distilled off. 20 ml of ethanol was added to the residue and the mixture was stirred at room temperature for 3
After stirring for 0 minutes, the crystals are filtered off, washed with a small amount of ethanol and dried. 50 ml of water was added to this and dispersed, and then freeze-dried to give (4-carboxybenzyl) -7α-trifluoroacetamido-3-acetoxymethyl-3-cephem-4-carboxylate-1,1-dioxide 3.8 g. Is obtained (Compound I). NMR (DMSO-d ₆ ) δ (ppm): 3.74, 4.26 (A
Bq, J = 18Hz, 1H), 4.68, 7.60 (ABq, J = 13.5Hz, 1H), 5.3-5.5
(m, 4H), 7.51 (d, J = 8.3Hz, 2H), 8.03 (d, J = 8.3Hz, 2H), 10.
2 (d, J = 8.1Hz, 1H). IR (cm ^-1 ): 1820, 1800, 1730, 1680, 1610, 1550,
1320, 1210, 1165, 1120, 1090, 1065, 1020, 975

[0067]

Example 2 7β-Trifluoroacetamide-3-acetoxymethyl-3-cephem-4-carboxylic acid-1,
800 mg of 1-dioxide is dissolved in N, N′-dimethylformamide, and 420 mg of phosphorus pentachloride and 162 μl of pyridine are added under ice cooling. Then, 420 mg of 4-hydroxymethylbenzenesulfonic acid sodium salt and then 556 μl of triethylamine are added and the temperature is raised to room temperature to react for 1 hour. The mixture is ice-cooled again, 556 μl of triethylamine is added, and the mixture is stirred for 3 hours. 2N-hydrochloric acid (2 ml) was added and the mixture was concentrated under reduced pressure. This was subjected to high performance liquid chromatography (column: Inertocyl (registered trademark) PREP-ODS, solvent: 50).
% Methanol-water) and freeze-dried to give (4-sulfobenzyl) -7α-trifluoroacetamide-3.
-Acetoxymethyl-3-cephem-4-carboxylate-1,1-dioxide is obtained (compound II). IR (cm ^-1 ): 1795, 1720, 1645, 1550, 1425, 1260,
1030, 1005.

[0068]

Example 3 7β-Trifluoroacetamide-3-acetoxymethyl-3-cephem-4-carboxylic acid-1,
400 mg of 1-dioxide is dissolved in 5 ml of N, N'-dimethylformamide, 188 mg of sodium p-hydroxymethylphenylacetic acid salt is added under ice cooling, and the temperature is raised to room temperature, followed by reaction for 1 hour. Triethylamine 250μ
1 was added, the mixture was stirred for 3 hours, the solvent was evaporated under reduced pressure, and the residue was separated by high performance liquid chromatography (4-carboxymethylbenzyl) -7α-trifluoroacetamide-3-acetoxymethyl-3-cephem- Obtaining 4-carboxylate-1,1-dioxide (Compound II
I). IR (cm ^-1 ): 1795, 1725, 1710, 1590, 1335, 1235,
1165, 1120.

[0069]

[Examples 4 to 7] Instead of the hydroxymethylphenylacetic acid sodium salt of Example 3, the corresponding sodium salts were used, and thereafter the same compounds as in Example 3 were obtained to obtain the compounds of the following Table 1 (Compounds IV to VII). .

[0070]

[Table 1]

[0071]

[Examples 8 and 9] (4-Carboxybenzyl) -7β-
1.03 g of trifluoroacetamido-3-chloromethyl-3-cephem-4-carboxylate-1,1-dioxide is dissolved in 50 ml of acetone and 200 mg of 2-mercaptoimidazole is added. NaHCO ₃ 680mg
And a mixture of 3.3 g of KI dissolved in 40 ml of water are added, and the mixture is reacted at room temperature for 3 hours. After acidifying with N-HCl, acetone is distilled off under reduced pressure, and the mixture is extracted with ethyl acetate. MgS
It was dried with O ₄ , the solvent was distilled off, and this was separated by high performance liquid chromatography and lyophilized to give (4-carboxybenzyl) -7α-trifluoroacetamide-3-
[[Imidazol-2-yl) -thiomethyl] -3-cephem-4-carboxylate-1,1-dioxide is obtained (compound VIII). IR (cm ^-1 ): 1800, 1710, 1650, 1235, 1190, 1170,
1135, 1110, 1015. In the same manner except that 220 mg of thiophenol was used (4-
Carboxybenzyl) -7α-trifluoroacetamide-3- (phenylthiomethyl) -3-cephem-4-
Obtain carboxylate-1,1-dioxide (compound IX). IR (cm ^-1 ): 1790, 1710, 1610, 1540, 1315, 1220,
1150, 1105, 1010.

[0072]

Example 10 7β-Trifluoroacetamide-3-
Methyl-3-cephem-4-carboxylic acid-1,1-dioxide (4-sulfobenzyl) -7α-trifluoroacetamido-3-methyl-3-cephem was prepared in the same manner as in Example 2 except that 720 mg was used. -4-carboxylate-1,1-dioxide is obtained (compound X). IR (cm ^-1 ): 1790, 1725, 1330, 1220, 1160, 1125,
1035, 1005.

[0073]

Example 11 The procedure of Example 1 was repeated except that 3.5 g of 7β-acetamido-3-acetoxymethyl-3-cephem-4-carboxylic acid-1,1-dioxide was used (4
-Carboxybenzyl) -7α-acetamido-3-acetoxymethyl-3-cephem-4-carboxylate-1,1-dioxide is obtained (compound XI). IR (cm ^-1 ): 1795, 1740, 1330, 1220, 1160, 1120. Comparative Compound I Benzyl-7α-trifluoroacetamide-3-acetoxymethyl-3--according to the example of US Pat. No. 4,547,371. Cephem-4-carboxylate-1,1
-Get the Geocide.

Comparative Compound II (4-carbamoylbenzyl) -7α-trifluoroacetamido-3-acetoxymethyl-3-cephem-4-carboxy was prepared in the same manner as in Example 2 except that 300 mg of 4-carbamoyl-benzyl alcohol was used. rate-
Get 1,1-Dioxide. IR (cm ^-1 ): 1790, 1730, 1330, 1225, 1170, 1120,
1050, 1030, 980. Comparative compound III Example 3 except that 98 mg of sodium hydroxyacetate was used.
In the same manner as in (Carboxymethylbenzyl) -7α-trifluoroacetamido-3-acetoxymethyl-3-
Obtain cephem-4-carboxylate-1,1-dioxide. IR (cm ^-1 ): 1810, 1740, 1725, 1625, 1545, 1330,
1300, 1230, 1180, 1050. Comparative compound IV 7β-trifluoroacetamido-3-acetoxymethyl-3-cephem-4- [2- (S) -carboxypyrrolidinecarboxamide] -1,1-dioxide 51
Dissolve 2 mg in 5 ml of tetrahydrofuran, add 280 μl of triethylamine and react at room temperature for 2 hours, then distill under reduced pressure and collect the residue by high performance liquid chromatography to obtain 7α-trifluoroacetamide-3-acetoxymethyl-3- Cephem-4- [2- (S) -carboxypyrrolidinecarboxamide] -1,1-dioxide is obtained. IR (cm ^-1 ): 1790, 1725, 1630, 1550, 1330, 1230,
1170, 1130, 1090, 1050. Table 2 below shows the activity of the compounds I to XI produced in Examples 1 to 11.

[0075]

[Table 2]

Table 3 below shows the activity of comparative compounds I to IV.

[0077]

[Table 3]

Table 4 below shows stability t in plasma.
The values of _1/2 (min) and water solubility (mg / ml) are shown.

[0079]

[Table 4]

Compounds I to XI each have a selectivity of 1000 or more for human trypsin and human thrombin.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI Technical indication C07D 501/36 101 9284-4C // A61K 31/545 ABE 9360-4C ABG 9360-4C ABJ 9360- 4C ACD 9360-4C ADZ 9360-4C AED 9360-4C

Claims (3)

[Claims] 1. The following general formula (I): A soluble cephalosporin derivative represented by: 2. The soluble cephalosporin derivative according to claim 1, which has a stability in hamster plasma of 10 minutes or more and a solubility in distilled water of 2 mg / ml or more. 3. The soluble cephalosporin derivative according to claim 2, which has a solubility in distilled water of 10 mg / ml or more.