CS198112B2 - Process for preparing phthalidestere of 6-/d/-/alpha-aminophemylacetamido/penicilanic acid - Google Patents

Description

The present invention relates to a process for the preparation of 6- [D (-) α-aminophenylacetamido] penicillanic acid phthalide ester and pharmaceutically acceptable acid addition salts thereof.

6- [D (-) α-aminophenylacetamido ]penicillanic acid is very often used for its broad antibiotic spectrum. However, this acid is absorbed incompletely into the bloodstream when administered orally. Some experts believe this is not advantageous, and as a result some have attempted to find 6- [D (-) α-aminophenylacetamido) penicillanic acid derivatives which, after oral administration

4 »administration will result in a higher level of penmillin in the blood than would be achieved by the administration of the original penicidin alone.

It is an object of the present invention to provide a novel 6- [D (-}. Alpha.-aminophenylacetamido] penicillanic acid ester which causes high serum penicillin serum concentrations when administered orally.

According to the invention, the phthalide ester of 6- [D (-) α-aminophenylacetamido] penicillanic acid of formula I (D) / ~ \ ^lu '^ \ - CH-CO-NH-CH CH ^C NH »

WHAT

CH ₃

CH-CO-O-CHO

II

r /;

and its pharmaceutically acceptable acid addition salts are prepared by reacting a compound of formula V,

X ^D / ^S \ <VCN-CO-WW-CW-CW c \ = / ii I XCW,

CO-N

-CU (V) coow where

X is amino, protonated amino, benzyloxycarbonylamino or enamino III

R *

I ^{1 1} r ^ ³ -C him (III) wherein R

R 1 is C 1 -C 6 alkyl

R ³ is alkyl having 1-6 carbon atoms or alkoxy having 1 to 6 carbon atoms, or a tautomeric modification with the compound of formula VIa, NY product is optionally converted into an acid addition salt.

Preferably the hydrochloride is used as the acid addition salt of the compound of the invention, but salts of other inorganic or organic acids may also be used, especially when they form salts with 6- [D (-) - aminophenylacetamido-penicillanic acid itself. The compounds of the present invention also form salts with other penicillanic acids, such as 3- (2‘-chloro-6‘-fluorophenyl) -5-methyl-4-isoxazolylpenicilane.

The reaction is usually carried out in an inert solvent at room temperature.

The reaction may also be carried out with a reactive esterified derivative of the starting compounds within the scope of the substituent delimitation.

The term reactive esterified derivative of the compounds of the above general formula (V) means those derivatives which, when reacted with each other, form an ester bond containing group of the formula under reaction conditions.

-COO ^

II o

where

B is a halogen atom, and when X is other than amino, then under acidic or neutral conditions X is converted to an amino group and a variety of esterification methods are known from the literature using several different combinations of reactive esterified derivatives with carboxyl and hydroxyl groups. The reaction can be carried out, for example, by reacting a compound of formula V,

-A (D)

VCW-CO-NH-Cl-I ^J CH-CO-N-CH ³ COOH, ^X (V) wherein

X is as defined above, with a compound of formula VIa,

g (Via.) where

B has the meaning given above, to form the ester of formula VII, σ (0) /

CH-CO-NH-CH-CHX

where

X has the meaning given above, and in the case that:

X is other than amino, optionally converted thereafter to amino.

Specific methods of esterification known from the literature can be applied to the above process. Although X in V may be a free amino group, it is preferable to carry out the reaction with V in which X is a protected amino group, preferably an enamino group of the above formula III, or a group convertible to an amino group such as an azido group. In such a case, the sodium or potassium salt of the N-protected compound of formula (V) is usually reacted with a compound of formula (VI) wherein B is as defined above.

In the compound of formula (V), X is an amino group which can be converted to a protected amino group, and vice versa can be liberated from it.

Examples of protected amino include protonated amino (X = NHs ⁺ ), which, after acylation, can be converted to the free amino group simply by neutralization, benzyloxycarbonylamino (X = NH-CO 2 -CH 2 -Ph-, where Ph is phenyl) or substituted benzyloxycarbonylamino, which are then converted to the amino group by catalytic hydrogenation and various groups from which the amino group can be obtained after acylation by hydrolysis under mildly acidic conditions. (Alkaline hydrolysis is not commonly used, as the phthalide group undergoes hydrolysis under alkaline conditions.)

An example of a group X that can then be hydrolyzed to an amino group under mildly acidic conditions is the enamino group of formula III,

I (Ac η

O (III) where

R ¹ and R ³ are as defined above, or tautomeric modifications thereof.

An example of a group X which can be converted to an amino group is an azido group. In this case, the final conversion to the amino group can be carried out either by catalytic hydrogenation or by electrolytic reduction. Of course, the choice of reactive derivative depends on the effect of the chemical nature of the α-substituent X. When X represents an acid stable group such as a protonated NH 3+ amino group or an azido group, conversion may occur. To avoid this, mixed anhydride is often used. Particularly suitable for this purpose are mixed anhydrides, such as alkoxy formic anhydrides, which are preferably prepared by reacting an alkali metal salt or a tertiary amine of the formula (V) with a suitable alkyl chloroformate in an anhydrous environment at or below room temperature.

Other reactive N-acylated derivatives of the compound of formula (V) include reactive intermediates formed by in situ reaction with carbodiimide or carbonyldiimidazole.

The compound of this invention is well tolerated and is preferably administered orally, especially in the form of an acid addition salt. It is usually administered in combination with a suitable pharmaceutically acceptable carrier. The compound of the invention may be present in the compositions in an amount of 1 to 95% by weight of the total weight. The composition may be used as a powder for the preparation of a syrup, such as tablets, capsules, pills, or other conventional forms.

The esters of the invention or salts thereof are usually administered in dosage units containing an amount equivalent to 0.025 to 1 g of 6- [D (ω-aminophenylacetamide) -penicillanic acid and in particular in an amount equivalent to 0.1 to 0.7 g of penicillanic acid. Dosage units containing 250 to 500 mg of penicillanic acid are suitable, and the daily dose depends on the patient's condition, but usually a reasonable dose is 1 to 3 g of the ester of the invention (calculated on penicillanic acid).

The compound of the invention, 6- [D (-) - aminophenylacetamido] penicillanic acid phthalide ester, is well absorbable when administered orally to humans or animals. Serum levels of 6- [D (—-) α-aminophenyl lacetamido] penicillanic acid are high.

The following examples illustrate the invention. Example 1

(a) 3-Bromophthalid- [3-bromo-1- (3H) -isobenzofuranone]

10.0 g (0.075 mol) of phthalide were refluxed with N-bromosuccinimide in 200 ml of anhydrous carbon tetrachloride in the presence of a catalytic amount of α-azoisobutyronitrile for 3-4 hours. The end of the reaction was reported as the N-bromosuccinimide disappeared from the bottom of the reaction vessel and the succinimide accumulated on the surface. The succinimide is filtered off and the filtrate is evaporated in vacuo to 15-20 ml. Cooling and concentrating the concentrate gave 13.0 g (81% yield) of crude 3-bromophthalide, m.p. 75-80 ° C, as a white crystalline solid. Recrystallization of the product from cyclohexane gave colorless plates in 95% yield, which melted at 78-80 ° C.

NMR (CCl4):

d = 7.67 (4H, m, aromatics), δ = 7.38 (1H, m, CH-).

and

Br

b) D (-) α-Aminobenzylpeniciline phthalide ester hydrochloride

17.5 g (0.05 mol) of anhydrous D (-) α-aminobenzylpenicillin and 7.10 ml, i.e. 1 equivalent, of triethylamine are mixed with 350 ml of acetone containing 1% water. After half an hour, 5 g of potassium bicarbonate and 10.65 g (0.05 mol) of 3-bromophthalide were added and the mixture was stirred at room temperature for 4 hours. The mixture was filtered and the filtrate evaporated in vacuo to a volume of about 75 mL. Ethyl acetate (500 ml) was added and the resultant solution was washed twice with 100 ml of 2% aqueous sodium bicarbonate solution twice and then twice with 100 ml of water. 150 ml of water were added to the ethyl acetate solution and 1 N hydrochloric acid was added dropwise with vigorous stirring until the aqueous phase reached pH 2.5. The ethyl acetate layer was separated and dried over anhydrous magnesium sulfate. Ether was then added to the clear yellow ethyl acetate filtrate until precipitation of the white amorphous material was complete. 7.8 g (28.8%) of product are obtained.

An additional 0.8 g, i.e. 3.0%, of the product is recovered from the aqueous layer as follows:

750 ml of n-butanol are added to the aqueous layer and the resulting mixture is evaporated in vacuo until all the water has been removed. The resulting butanolic solution was poured into 2000 ml of ether, whereupon the precipitated amorphous substance was separated. The overall yield was 31.8%.

The IR spectrum (KBr) contains, among other things, strong bands:

1778 cm ¹ ,

1682 cm ^-1 ,

1500 cm ^-1 ,

1285 cm ^-1 ,

1149 cm ^-1 ,

978 cm ¹ ,

752 cm ^-1 , *

697 cm ^1st

NMR [(CD 3) 2 CO / D 2 O]: δ = 7.88 (4H, m, phthalide aromatics), δ = 7.60 (1H, s, —CO — O — CH—}, δ '= 7.48 ( 5 / 6H, m, aromatics), δ = 5.50 (2H, m, β-lactams),

Δ = 5.16 (1H, s, α-proton), δ = 4.54 (1H, s, C3-proton), δ = 1.45 (6H, d, dimethyl).

The purity determined by hydroxylamine and cysteine tests was 92.4 and 86.5% respectively.

Analysis for

C24H21O6N3SCI:

calculated

C 55.65, H 4.67, N 8.11, S 6.19,

Cl 6.84%, found

C 54.49, H 4.67, N 7.83, S 6.20,

Cl, 5.18%.

Example 2

Phthalid-6- [D (-) α-aminophenyla cetamido] penlcilanate hydrochloride

25.18 g (0.05 mol) of an enamine-protected ampicillin potassium salt of formula Ib is reacted with 10.65 g (0.05 mol) of bromophthalide in 1500 ml of a 1: 2 mixture of acetone and ethyl acetate for 24 hours. After filtration, the organic layer was washed twice with 250 ml of 1 N sodium bicarbonate solution and brine, dried over anhydrous magnesium sulfate, and evaporated in vacuo. After addition of ether, the enamine-protected phthalide-aminophenylacetamidopenicillanate of formula II crystallizes in 85% yield.

NMR [(CD3) 2 SO]:

? = 7.86 (4H, m, phthalide aromatics), δ = 7.60 (1H, s, —CO — O — CH), (5-7.35 (5H, s, aromatics), δ = 5, 30-5.65 (3H, m, (Hactams and α-proton), δ = 4.53 (1H, s, C3 proton), δ = 4.50 (1H, s, —H), = 3.56 (3H, s, O-CH ₃ ), = 1.78 (3H, s, CH ₃ -), = 1.50 (6H, m, dimethyl).

Analysis for

C28H29N3O8S:

calculated

C 59.26, H 5.11, N 7.40, S 5.68%, found

C 58.83, H 5.01, N 6.89, S 5.34%.

Single spot on biochromatogramu at _Rf = 0.95.

From 10 g of the product of formula IIb containing an enamine protecting group in aqueous acetone (250 ml of water and 250 ml of acetone), the protecting group is removed by stirring the solution at pH 2.5 for 1 hour. The acetone is evaporated in vacuo and the ester of formula IIIb is salted out of the aqueous phase as a sticky yellow gum which is dissolved in 200 ml of ethyl acetate and washed twice with 200 ml of 1 N sodium bicarbonate solution and brine and dried over anhydrous magnesium sulfate. The addition of approximately 50 ml of anhydrous ester to the anhydrous ethyl acetate layer yields ampicillin phthalide ester as the salt, i.e. the hydrochloride, as a fine white amorphous substance in 80% yield.

NMR [(CD ₃₎ 2 SO / D20]:

δ = 7.88 (4H, m, phthalide aromatics), = 7.60 (1H, s, —CO — O — CH—), = 7.48 (5 / 6H, m, aromatic), = 5.50 (2H, m, d-lactams j = 5.16 (1H, s, or-proton),

S = 4.54 (1H, s, C3-proton), d = 1.45 (8H, d, dimethyl).

The purity determined by the hydroxylamine assay is 110.3%.

Single spot on biochromatogramu at _Rf = 0.85.

Analysis for C24H21N3O6SCI:

calculated:

C 55.65, H 4.67, N 8.11, S 6.19%, found:

C 54.60, H 4.70, N 7.92, S 6.40%.

Example 3

Phthalid-6-aminopenicilane Method 1

A mixture of 10.8 g (0.05 mol) of 6-aminopenicillanic acid and 6.9 ml (0.05 mol) of triethylamine is stirred in 20 ml of anhydrous acetone at room temperature for 1/2 hour. The mixture was cooled to 0 ° C and a solution of 10.65 g (0.05 mol) of 3-bromophthalide in 20 mL of anhydrous acetone was added in one portion. The resulting yellow mixture was stirred at room temperature for 5 hours. The reaction mixture was diluted with 150 mL of anhydrous diethyl ether and filtered. The clear yellow filtrate was washed with 100 mL of 1 N sodium bicarbonate and 100 mL of saturated brine. To the clear, dried yellow filtrate was added 9.5 g, i.e., 1 equivalent of p-toluenesulfonic acid monohydrate in 150 mL of anhydrous acetone. The epi-6-aminopenicillanate crystallizes out of solution in the form of a salt with p-toluenesulfonic acid.

6-a (trans jisoiner:

NMR [(CD3) 2SO]:

L <5 = 7.84 (4H, s, phthalide aromatics),

S = 7.58 (1H, S, -CO-O-CH),

S = 7.30 (4H, q, sulfonate aromatics),

S = 5.35 (1H, d, C-proton, J = 2Hz), λ = 4.89 (1H, s, C-proton), d = 4.70 (1H, d, C6-proton, J = 2Hz),

S = 2.30 (3H, s, CH), ~ 1.48 (6H, d, dimethyl).

IR (KBr): thick bands at

1780 cm ^-1 , 1210 cm ^-1 . 1170 cm ^-1 , 1010 cm ' ¹ , 970 cm ' ¹ , 682 cm ¹ , 574 cm ' ¹ ,

The remaining oil is recovered from the mother liquor. A small amount (about 5%) of appropriately pure material corresponding to the natural cis-isomer is obtained, in the form of p-toluenesulfonate, by repeated fractional crystallization of the 6 - ((trans) -isomer from acetone / ether 3: 1).

6 -, (1 (cis isomer:

NMR [(CD3) 2SO]:

d = 7.84 (4H, s, phthalide aromatics), d = 7.58 (1H, s, —CO — O — CH), d = 7.30 (4H, q, sulfate aromatics), d = 5, 50 (1H, d, C5-proton, J = 4Hz), d = 5.14 (1H, d, C6-proton, J = 4Hz), d = 4.68 (1H, s, C3-proton), d = 2.27 d (3H, s, CH 3), d = 1.53 (5H, d, dimethyl).

IR (KBr): thick bands at

1780 cm-i,

1210 cm ^-1 ,

1170 cm ^-1 ,

1010 cm ^-1 ,

970 cm ^-1 ,

682 cm ^-1 ,

574 cm ^-1 .

Method 2

A solution of 9.8 g (0.02 mol) of 6-tritylaminopenicillanic acid in 100 ml of anhydrous acetone was cooled to 0 ° C and 2.9 mg (0.02 mol) of triethylamine was added followed by 4.1 g (0, 02 mol) of 3-bromophthalide in 20 ml of anhydrous acetone.

The reaction mixture was stirred at 0 ° C for 2 hours and then at room temperature for 1 hour. The precipitated triethylammonium bromide was removed by filtration. The filtrate is evaporated, diluted with 150 ml of ethyl acetate, washed twice with 150 ml of cold 2% sodium bicarbonate solution and twice with 100 ml of ice-water. The ethyl acetate layer was dried over anhydrous magnesium sulfate. The solvent was removed in vacuo to give phthalide-6-tritylaminoperricilanoate as a white amorphous solid.

NMR [(CD3) 2SO]:

d = 7.4 (20H, low inflex broad singlet at d = 7.80 (aromatic protons and —CO — O — CH—), d = 4.41 (2H, m, β-lactam protons): d = 4.15 (1H, broad singlet, C3-protons), d = 1.38 (6H, d, dimethyl).

IR (KBr): broad band at

1745 cm ^-1 ,

980 cm ^-1 ,

750 cm ^-1 ,

708 cm ^-1 .

5.9 g (0.01 mol) of phthalid-6-tritylaminopenicillanate in 200 ml of acetone containing 0.2% water are treated with 1.9 g (0.01 mol) of mo198112 p-toluenesulfonic acid footdrate. The mixture was allowed to stand at room temperature for 2 hours and 0.25 ml of water was added. The precipitation of phthalid-6-aminopenicillanate p-toluenesulfonate is achieved by the slow addition of 250 ml of petroleum ether, b.p. 40-60 ° C. The p-toluenesulfonic acid salt was removed by filtration and washed with petroleum ether. The sample was recrystallized from acetone and diethyl ether in 85% yield.

NMR [(CD3) 2SO]:

S = 7.84 (4H, s, phthalide aromatics), δ = 7.58 (1H, s, —CO — O — CH—), δ = 7.30 (44.9, q, sulfonate aromatics), δ = 5.50 (1H, d, β-lactams, J = 4Hz), δ = 5.14 (1H, d, β-lactam, J = 4Hz), δ = 4.68 (1H, s, C3 -protone), δ = 2.27 (3H, s, CH 3), δ = 1.53 (6H, d, dimethyl).

Analysis for C23H24N2S2O8:

calculated:

C 53.08, H 4.61, N '5.39, S 12.31%, found:

C 52.32, H 4.60, N 4.94, S 12.27%. Method 3

Benzylpenicillin phthalide ester

20.0 g (10.054 mol) of benzylpenicillin potassium are dissolved in 50 ml of anhydrous methylformamide and cooled to 0 ° C, and then 11.5 grams (0.054 mol) of 3-bromophthalide in 20 ml of anhydrous dimethylformamide are added in one portion. The reaction mixture was allowed to warm to room temperature and then stirred for an additional 2 hours. The mixture was then poured into 600 ml of ice-cold water and stirred vigorously. The solid white precipitate was collected, collected and washed well with water. After drying, it is recrystallized from hot isopropyl alcohol to give 10.5. g, i.e. 41.9% of white crystalline product, m.p. 167-169 ° C.

The IR spectrum (nujol) contains, among other things, strong bands at

1770 cm-i,

1678 cm ^-1 ,

1524 cm ^-1 ,

970 cm ^-1 .

NMR [(CD3) 2SO / D2O] shows the following peaks:

δ = 7.88 (4H, m, phthalide aromatics), δ = 7.61 (1H, s, -CO-O-CH-), δ = 7.28 (5H, s, aromatics), δ = 5, 55 (2H, m, [beta] -lactams), [delta] = 4.55 (1H, s, C3-proton), <5 = 3.56 (2H, s, PhCH2CO),

S = 1.53 (6H, d, dimethyl).

The purity determined by the hydroxylamine assay is 109.2%.

Analysis for O21H22N2SO6: calculated:

C 61.80, H 4.72, N 6.02, S 6.86%, found:

C 61.55, H 4.90, N 5.87, S 6.72%.

Benzylpenilcillin phthalide ester (11.6 g, 0.025 mol) was dissolved in methylene dichloride (250 ml) and frozen at -25 ° C. 5.60 ml (0.025 mol) of N-methylmorpholine and then 6.0 g of phosphorus pentachloride in 150 ml of methylene dichloride are added to the solution over 5 minutes. The solution turns pale yellow. The temperature of the solution rose to 0 ° C after stirring for 1/2 hour. The reaction mixture was recooled to -25 ° C and 5.60 mL of N-methylmorpholine and anhydrous methanol were added; the temperature slowly rises to approximately -10 ° C.

After stirring for another 2 hours at -5 to 0 ° C, 400 ml of water are added with vigorous stirring and the pH of the mixture is adjusted from 1.2 to 6.0 with dilute sodium hydroxide solution.

The organic phase was separated, washed with water and saturated brine, and filtered through silicone paper.

4.75 g (0.025 mol) of p-toluenesulfonic acid monohydrate in 100 ml of acetone are added to the organic layer with stirring. Ether was then added until the solution became clear. Standing overnight. at 0 DEG C., 7.0 g of 6-aminopenicillanic acid phthalide ester phthalide ester white crystalline p-toluenesulfonate was obtained from the evaporated filtrate for a further 2.5 g. The overall yield is 9.5 g, i.e. 73.4%.

NMR [(CD 3) 2 SO):

δ = 7.84 (4H, s, phthalide aromatics), δ = 7.58 (1H, s, —CO — O — CH—), δ = 7.30 (4H, s, sulphate aromatics), δ = 5 50 (1H, d, β-lactam, J = 4Hz), <5 = 5.14 (1H, d, (3-lactam, J = 4Hz), δ = 4.68 (1H, s, Cs- proton), δ = 2.27 (3H, s, CH 3 -), δ = 1.53 (6H, d, dimethyl).

Analysis for C23H24N2S2O8: calculated:

C 53.08, H 4.61, N 5.39, S 12.31%, found:

C 52.50, H 4.62, N 4.98, S 12.34%. Example 4

Determine the rate of hydrolysis of 6- [D (-) - aminophenylacetamidephenillanic acid phthalide ester hydrochloride by incubating the ester, at the equivalent of 5 (Ug / ml 6- [D (-) - aminophenylacetamidopenicillanic acid) at 1/20

1 mole of buffer (phosphate, potassium) at pH 7.0 in 90% of human blood and in 90% of squirrel's blood. The ester is also found to be hydrolyzed with the homogenized small intestine of the squirrel skue at the equivalent of 100 µg / ml of 6- [D (ω-aminophenylacetamide) -penicillanic acid, and the reaction mixture is diluted to equivalent to 5.0 µg / ml of penicillanic acid prior to assay. is obtained by homogenizing the washed small intestine of squirrel skunk with four times its weight in 1/20 molar potassium phosphate buffer, which is diluted 1:10 for the reaction mixture.

All reaction mixtures were incubated at 37 ° C. After incubation, the ester is separated from the reaction mixture by electrophoresis. 5 µl aliquot of reaction mixture and 6- [D (-) α-aminophenylResults

The medium for the hydrolysis of acetamidojpenicillanic acid, prepared in a standard manner in a suitable medium, is applied to starch gel gel plates maintained at a pH of 5.5 with a buffer. The plates were applied transversely to a voltage of 15 volts per cm for 20 minutes. At this pH the penicillanic acid remains near the original site and the ester present migrates towards the cathode. The amount of penicillanic acid present in the reaction mixtures is determined by overlaying the gel plates with nutrient agar (oxidized blood agar) inoculated with Sarcina Lutea NCTC 8340, and incubating for 16 hours at 30 ° C.

The ampicillin-induced suppression band in the test and standard samples is measured and the amount of penicillanic acid formed in the reaction is calculated.

% hydrolysis at 37 ° C to 6- [D (-) α-aminophenylacetamido] penicillanic acid 3 min. 8 min 15 min. 25 min.

Acid (pH 2.0) 0

Aqueous Buffer 10 (pH 7.0)

Human Blood 50 (pH 7.0)

Squirrel blood 78 (PH 7,0j

The small intestine of the skunk 80 squirrel

0 0 0 15 Dec 20 May 25 62 80 84 84 90 100 ALIGN! 84 92 100 ALIGN!

Claims (1)

Process for preparing 6- [D (-) α-aminophenylacetamido] penicillanic acid phthalide ester of formula I Z - / CH - CO - NH - CH - CH CH _a CH, NH, CO-N CHCO. (I) b-Cl-1c and its acid addition salts thereof, characterized in that a compound of formula V is reacted, IDI / S _{/ C} H ₃ CP-CO-NP-CP-CP (λ ,,, i i i i CH, X CO-N-CH ³ (V) COOH where X is amino, protonated amino, benzyloxycarbonylamino or enamino III, R ¹ is alkyl having 1 to 6 carbon atoms and R ³ is alkyl having 1-6 carbon atoms or alkoxy having 1 to 6 carbon atoms, or a tautomeric modification with the compound of formula VIa H 1 NR ³ -C ^(III) (III) wherein B represents a halogen atom, and when X is other than amino, it is then converted under acidic or neutral conditions X to amino and the resulting product is optionally converted to an acid addition salt.