Phthalide penicillin ester intermediates

(a) 3-Bromophthalide [3-Bromo-1-(3H)-Isobenzofuranone]

Phthalide (10.0 g; 0.075 moles) and N-bromosuccinimide were refluxed in dry carbon tetrachloride (200 ml.) in the presence of a catalytic amount of α-azo-iso-butyronitrile for 3-4 hours. The end of the reaction was indicated by the disappearance of N-bromosuccinimide from the bottom of the reaction vessel and the accumulation of succinimide at the top. The succinimide was removed by filtration and the filtrate concentrated in vacuo to 15-20 ml. Cooling of this concentrate followed by filtration gave 13.0 g. (81% yield) of crude 3-bromophthalide, m.p. 75-80°, as a white crystalline solid. The product was recrystallised from cyclohexane as colourless plates, m.p. 78-80° with a 95% recovery.

N.m.r. (CCl 4 ) Br δ = 7.67 (4H.m. aromatic), δ = 7.38 (1H.s. CH--)

(b) D(-)α-(aminobenzylpenicillinphthalide ester, hydrochloride

Anhydrous D(-)α-aminobenzylpenicillin (17.5; 0.05 mole) and triethylamine (7.10 ml; 1 equiv.) were mixed with acetone containing 1% of water (2350 ml). After 1/2 hour potassium bicarbonate (5 g) and 3-bromophthalide (10.65 g; 0.05 mole) were added and the mixture stirred at room temperature for 4 hours. After filtration, the filtrate was concentrated in vacuo to about 75 ml, ethyl acetate (500 ml was added and the resulting solution washed with a 2% aqueous solution of sodium bicarbonate (2 × 100 ml) followed by water (2 × 100 ml). Water (150 ml) was added to the ethyl acetate solution, and, with vigorous stirring, 1N hydrochloric acid was added drop by drop until the pH of the aqueous phase was 2.5. The ethyl acetate layer was separated and dried over anhydrous magnesium sulphate. Ether was then added to the clear yellow ethyl acetate filtrate until no further precipitation of a white amorphous solid occurred. The product was collected (7.8 g; 28.8%). Further material (0.8 g; 3.0%), was obtained from the aqueous layer as follows. To the aqueous layer, n-butanol (750 ml) was added and the resulting mixture was evaporated in vacuo until all the water was removed. The resulting butanolic solution was poured into ether (2000 ml) whereby an amorphous precipitate separated. Combined yields were 31.8%.

The I.R. spectrum (KBr) contains inter alia strong bonds at: 1178 cm -1 1682 cm -1 1500 cm -1 1285 cm -1 1149 cm -1 978 cm -1 752 cm -1 697 cm -1

n.m.r. ((CD 3 ) 2 SO/D 2 O):, δ = 7.88 (4H.m. phthalide aromatics); δ = 7.60 (1H.s. --CO.OCH--); δ = 7.48 (5/6H.m. aromatic); δ = 5.50 (2H.m. β-lactams); δ = 5.16 (1H.s. α-proton); δ = 4.54 (1H.s. C 3 proton); δ = 1.45 (6H.d. gem-dimethyls). The purity as assessed by hydroxylamine and cysteine assays was 92.4% and 86.5% respectively.

C 24 h 24 o 6 n 3 scl requires: C, 55.65; N, 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.

Phthalide 6-[D(-)α-aminophenylacetamido] penicillanate, Hydrochloride

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Method 1

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##STR11##

##STR12##

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A fine suspension of potassium salt of enamine protected ampicillin I (25.18 g; 0.05 M) and 3-bromophthalide (10.65 g; 0.05 M) were reacted in a 1:2 mixture of acetone/ethyl acetate. (1500 ml) for 24 hours. After filtration the organic layer was washed twice with 250 ml. portions of IN sodium bicarbonate and brine, dried over anhydrous magnesium sulphate and concentrated in vacuo. Addition of ether crystallised the phthalide enamine protected α-aminophenylacetamido penicillanate (II) in 85% yield.

n.m.r. [(CD 3 ) 2 SO]; δ = 7.86 (4H.m. phthalide aromatics); δ = 7.60 (1H.s. CO.O.CH); δ = 7.35 (5H.S. aromatics) δ = 5.30 - 5.65 (3H.m. β-lactams and α-proton); δ = 4.53 (1H.s. C--3 proton) δ = 4.50 (1H.s. H) δ = 3.56 (3H.s. O CH 3 ) δ = 1.78 (3H.s. CH 3 ) δ = 1.50 (6H.m. gem di CH 3 ). C 28 H 29 N 3 O 8 S requires: C, 59.26; H, 5.11; N, 7.40 S, 5.68. Found: C, 58.83; H, 5.00; 1N, 6.89 S, 5.34.

Single spot on biochromatogram at R f = 0.95.

The enamine protecting group was removed from the product (II) by dissolving 10 g. in aqueous acetone (250 ml water to 250 ml acetone) and vigorously stirring this solution at pH 2.5 for 1 hour. The acetone was removed in vacuo and the ester (III), which was salted out of the aqueous phase as a sticky yellow gum, was dissolved in ethyl acetate (200 ml) and washed twice with 200 ml portion, of 1N sodium bicarbonate and brine and dried over anhydrous magnesium sulphate. Careful addition of dry ester (ca.50 ml) to the dry ethyl acetate layer yielded the ampicillin phthalide ester as hydrochloride salt as a fine white amorphous solid in 80% yield.

n.m.r. [(CD 3 ) 2 SO/D 2 O); δ = 7.88 (4H.m. phthalide aromatics); δ = 7.60 (1H.s. CO.O CH--); δ = 7.48 (5/6H.m. aromatic) δm = 5.50 (2H.m. β-lactams); δ = 51.6 (1H.s. α-proton) δ = 4.54 (1H.s. C 3 -proton) δ = 1.45 (6H.d. gem-dimethyls). Purity as assessed by hydroxylamine assay = 110.3%.

Single spot on biochromatogram at R f = 0.85

C 24 H 24 N 3 O 6 SCl requires: C, 55.65; 1H, 4.67; N, 8.11; S, 6.19. Found C, 54.60; H, 4.70; N, 7.92; S, 6.40.

METHOD 2

A mixture of acetone (250) ml, sodium D(+)N-methoxycarbonylpropen-2-yl-α-aminophenylacetate (30.51g), ethyl chloroformate (10.9ml) and N-methyl morpholine (4-6 drops) were stirred together for 10-15 minutes at -20° to -30° C.

To this solution was added, all at once, a solution of 6-APA (25.4 g) dissolved in water (50ml) with the aid of triethylamine (11.9 g) and then diluted with acetone (150 ml) and cooled to -20° C.

The reaction mixture was stirred for 45 mins. without further cooling and a solution of 3-bromophthalide (25 g) in acetone (100 ml) added all at once, after which stirring was continued for a further 5 hours, the temperature rising meanwhile to ambient (23° C.).

Acetone was next removed in vacuo, after first clarifying the mixture by filtration and to the residue was added ethyl acetate (375 ml.) and 2% sodium bicarbonate solution (200ml.). After stirring for a short while the phases were separated and the organic layer washed again with 2% sodium bicarbonate solution (200 ml).

To the ethyl acetate solution thus obtained was added water (375 ml) and 2N/HCl (60 ml) and this mixture stirred at ambient temperature (23° C) for 45 mins. Petrol (600 ml) was then added and after a short period of stirring the phases were allowed to separate. The organic layer was discarded and the aqueous layer was filtered with a little decolourising charcoal.

Sufficient sodium chloride to saturate the filtrate was next added and after a few minutes stirring the precipitated oil was extracted with methylene dichloride (1 × 400 ml, 1 × 100 ml). These extracts were combined, dried with anhydrous magnesium sulphate, filtered and evaporated under reduced pressure to approximately 100 ml. Ether (500ml) was then added quickly, with stirring to the residue and the resulting precipitate stirred for about 30 mins. at ambient temperature. The product was filtered at the pump, washed with ether (2 × 50 ml) and dried for 3 hours in a forced-air oven at 35° - 40° C. The product was identical with an authenic sample of phthalide 6-[D(-)α-aminophenylacetamido) penicillanate.

Phthalide 6-aminopenicillanate.

METHOD 1

A mixture of 6-aminopenicillanic acid (10.8 g 0.05 M) and triethylamine (6.9 ml; 0.05 M) was stirred in dry acetone (20 ml) for 1/2 hour at ambient temperatures. The mixture was cooled to 0° C and a solution of 3-bromophthalide (10.65 g. 0.05 M) in dry acetone (20 ml) added in one portion and the resulting yellow mixture stirred at ambient temperatures for 5 hours. The reaction mixture was diluted with dry diethyl ether (150 ml) and filtered. The clear yellow filtrate was washed with 1N sodium bicarbonate (100 ml) and a saturated brine solution (100 ml). A solution of p-toluenesulphonic acid monohydrate (9.5 g- 1 equiv.) in dry acetone (150 ml) was added to the clear dry yellow filtrate and immediately phthalide epi 6-aminopenicillanate as the p-toluenesulphonate salt crystallised out of solution.

6-α(trans)isomer:

n.m.r.[ (CD 3 ) 2 SO] :L δ = 7.84 (4H.s. phthalide aromatics); δ = 7.58 (1H.s. CO.O.CH); δ = 7.30 (4H.q. sulphonate aromatics); δ = 5.35 (1H.d. C -- 5 proton J = 2Hz); δ = 4.89 (1H.s. C -- 3 proton); δ = 4.70 (1H.d. C -- 6 proton- J = 2Hz); δ = 2.30 (3H.s. CH 3 ); δ = 1.48 (6H.d. gem-di CH 3 ).

i.r. (KBr disc) strong bands at 1780 cm -1 , 1210 cm -1 ; 1170 cm -1 ; 1010 cm -1 ; 970 cm -1 ; 682 cm -1 ; 574 cm -1 .

A residual oil was obtained from the mother liquor from which a small amount (ca. 5%) of a reasonably pure sample of the natural cis isomer of phthalide 6-aminopenicillanate as its p-toluenesulphonate salt was obtained by repeated fractional crystallisation of the 6-α(trans) isomer from an acetone:ether (3:1) solvent mixture.

6-β(cis)isomer:

n.m.r. [(CD 3 ) 2 SO]; δ = 7.84 (4H.s. phthalide aromatics) δ = 7.58 (1H.s. CO O CH); δ = 7.30 (4H.q. sulphate aromatics); δ = 5.50 (1H.d. C -- 5 proton; J = 4Hz); δ = 5.14 (1H.d. C -- 6 proton) J = 4Hz) δ = 4.68 (1H.s. C -- 3 proton); δ = 2.27d(3H.s. CH 3 ); δ = 1.53 (6H.d. gem-dimethyls).

i.r. (KBr disc) strong bands at 1780 cm -1 ; 1210 cm -1 ; 1170 cm -1 ; 1010 cm -1 970 cm -1 682 cm -1 574 cm -1 .

METHOD 2

A solution of 6-tritylaminopenicillanic acid (9.8 g; 0.02M) in dry acetone (100 ml) was cooled to 0° C, triethylamine (2.9 mg, 0.02 M) was added, followed by 3-bromophthalide (4.1 g; 0.02M) in dry acetone (20 ml.), and the reaction mixture kept at 0° C with stirring for 2 hours and finally at ambient temperatures for 1 hour. The triethylammonium bromide which precipitated was removed by filtration, the evaporated filtrate was dissolved in ethyl acetate (150 ml) and after two washings with cold 2% aqueous sodium bicarbonate (2 × 150 ml) and with ice-water (2 × 100 ml) the ethyl acetate layer was dried over anhydrous magnesium sulphate to yield, on removal of solvent in vacuo, phthalide 6-tritylaminopenicillanate as a white amorphous solid.

n.m.r. [(CD 3 ) 2 SO]; δ = 7.4 (20 H. broad singlet with smaller shoulder at δ = 7.80 aromatic protons and CO O CH--) S = 4.41 (2H.m. β-lactam protons); δ = 4.15 (1H broad singlet C -- 3 proton); δ = 1.38 (6H.d. gem dimethyls).

i.r. (KBrdisc) strong bands at 1745 cm -1 ; 980 cm -1 750 cm -1 708 cm -1 .

Phthalide 6-tritylaminopenicillanate (5.9 g; 0.01 m) in acetone (200 ml containing 0.2% H 2 O) was treated with p-toluenesulphonic acid monohydrate (1.9 g; 0.01 M). After standing at room temperature for 2 hours, water (0.25 ml) was added, and the precipitation of phthalide 6-aminopenicillanate p-toluenesulphonate was achieved by the slow addition of petroleum ether, b.p. 40-60° (250 ml). Filtration and consecutive washings with petroleum ether left the crude p-toluenesulphonate salt. The sample was recrystallised from acetone-diethyl ether with an 85% recovery.

n.m.r. [(CD 3 ) 2 SO]; δ = 7.84 (4H.s. phthalide aromatics); δ = 7.58 (1H.s. --CO.O CH--); δ = 7.30 (44.9 q. sulphonate aromatics)- β = 5.50 (1H.d. β-lactams; J = 4Hz) δ = 5.14 (1H.d. β -lactam, J = 4Hz); δ = 4.68 (1H.s. C -- 3 proton); δ = 2.27 (3H.s. CH 3 ); δ = 1.53 (6H.d. gem dimethyls).

C 23 H 24 N 2 S 2 O 8 requires: 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:

The potassium salt of benzylpenicillin (20.0 g;10.054 mole) was dissolved in dry dimethylformamide (50 ml) and cooled to 0° C. To this stirred solution 3-bromophthalide (11.5 g; 0.054 mole) in dry dimethylformamide (20 ml) was added in one portion. The reaction mixture was allowed to warm to room temperature and then stirred for a further 2 hours. The mixture was then poured into ice cold water (600 ml) and stirred vigorously. The white solid precipitate which separated was collected and washed well with water. After drying, the material was recrystallised from hot isopropyl alcohol to give a white crystalline product (10.5 g. 41.9%). m.p. 167°-169°. The I.R. spectrum (Nujol) contains inter alia strong bands at: 1770 cm -1 ; 1678 cm -1 ; 1524 cm -1 970 cm -1 .

n.m.r. [(CD 3 ) 2 SO/D 2 O] contains peaks at: 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. β-lactams); δ = 4.55 (1H.s. C 3 proton); δ = 3.56 (2H.s. PhCH 2 CO); δ = 1.53 (6H.d. gem-dimethyls).

The purity as assessed by hydroxylamine assay was 109.2%.

Found C, 61.55; H, 4.90; N, 5.87; S, 6.72; C 24 H 22 N 2 SO 6 requires: C, 61.80; H, 4.72; N, 6.02; S, 6.86.

Benzylpenicillin phthalide ester (11.6 g; 0.025M) was dissolved in dry methylene dichloride (250 ml) and chilled to -25°. N-methyl morpholine (5.60 ml; 0.025M) was added followed by a solution of phosphorus pentachloride (6.0 g) in methylene dichloride (150 ml.) over 5 minutes. A pale yellow colour developed and after stirring for 1/2 hour the temperature rose to 0°. The reaction mixture was recooled to -25° and N-methylmorpholine (5.60 ml) and dry methanol was added to give a slow steady rise in temperature to ca -10°.

After stirring at -5° to 0° for a further 2 hours water (400 ml) was added with vigorous stirring whilst the pH of the mixture was 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 siliconised paper.

A solution of p-toluenesulphonic acid monohydrate (4.75 g 0.025M) in acetone (100 ml) was added with stirring to the organic layer and ether added until the solution appeared cloudy. On standing overnight at 0°, 7.0 g of white crystalline 6-aminopenicillanic acid phthalide ester, p-toluenesulphonate was obtained and a further crop of 2.5 g obtained by concentration of the filtrate. Total yield = 9.5 g 73.4%.

n.m.r. [(CD 3 ) 2 SO] δ = 7.84 (4H.s. phthalide aromatics); δ = 7.58 (1H.s. --CO.O CH) δ = 7.30 (4H.s. sulphonate aromatics) δ = 5.50 (1H.d. β-lactam J = 4Hz) δ = 5.14 (1H.d. β lactam. J = 4Hz) δ = 4.68 (1H.s. C 3 proton) δ = 2.27 (3H.s. CH 3 --) δ = 1.53 (6H.d. gem di-CH 3 ).

C 23 H 24 N 2 S 2 O 8 requires: C, 53.08; N, 4.61; N, 5.39; S, 12.31. Found: C, 52.50; H, 4.62; N, 4.98; S, 12.34.

Ampicillin phthalide ester (I) via coupling of phthalide 6-aminopenicillanate with an enamine protected α-aminophenylacetic acid mixed anhydride. ##STR13##

Phthalide 6-aminopenicillanate p-toluenesulphonate (10.4 g) was suspended in ethyl acetate (60 ml) and stirred vigorously with 1N-sodium bicarbonate (135 ml) for 20 min. at ambient temperatures. The organic layer was separated, washed with water (100 ml) containing 2% sodium bicarbonate (5 ml) and dried over anhydrous magnesium sulphate, filtered and kept at -15° C.

A mixed anhydride of sodium D(-)N-(1-methoxycarbonylpropen-2-yl)-α-aminophenylacetate (5.4 g) in ethyl acetate (30 ml) was prepared by the addition of ethyl chloroformate (2 ml) and pyridine (2 drops) at -15° and stirring the reaction mixture for 10 min. at -15° to -20° C. To this mixed anhydride solution was added the ethyl acetate solution of phthalide 6-aminopenicillanate and the mixed stirred at -15° for 15 mins. and then for a further 45 min. without further cooling.

Water (75 ml) was added, followed by 2N-hydrochloric acid (10 ml) and the reaction mixture stirred vigorously for 25 mins. Petroleum ether, b.p. 60-80° (250 ml) was added slowly with stirring. The aqueous layer was separated and saturated with sodium chloride and the oil which separated was extracted into ethyl acetate (2 × 100 ml) and dried over anhydrous magnesium sulphate.

After filtration, the solution was concentrated in vacuo to ca 1/4 volume, dry ether (ca. 250 ml) was added slowly and the ampicillin phthalide which precipitated as a white amorphous hydrochloride salt (4.0 g; 40%) was collected and washed well with ether. Hydroxylamine assay = 76.1% Iodometric assay = 77.5% chlorine content = 7.07% (theoretical = 6.85%).

6-Aminopenicillanic acid (18.5 g- 0.085 mole) and sodium bicarbonate (21g; 0.25 mole) were dissolved in 200 ml of water and 100 ml of acetone. To this solution, chilled in ice, was added α-azidophenylacetyl chloride (16.6 g; 0.085 mole) diluted with 10 ml of dry acetone. The temperature was held at 0° to 5° C and the reaction mixture stirred for 2.5 hours.

The pH of the mixture was adjusted to 7.5 by adding a saturated sodium bicarbonate solution. After being washed twice with diethyl ether, the reaction solution was acidified to pH 2 with dilute HCl, and extracted with ether. The ether solution containing the free penicillin was washed twice with water and then extracted with 50 ml of N potassium bicarbonate solution. After freeze drying the potassium salt of α-azidobenzyl penicillin was obtained as a white powder (2944 g; 84% yield).

The potassium salt of α-azidobenzyl penicillin (21.53 g; 0.05 mole) was dispersed in methylene dichloride (250 ml) and acetone (100 ml) and the mixture chilled to -5° C. To the stirred suspension ethyl chloroformate (5.13 g- 0.048 mole) was added dropwise followed by a catalytic quantity of pyridine. The mixture was stirred at -5° C for 30 minutes.

O-Phthalaldehydic acid (6.5 g; 0.05 mole) was then added to the reaction mixture and after a few minutes, the cooling bath was removed and the temperature was allowed to rise to ambient temperature. The reaction was continued with constant stirring for a further 3 hours. The reaction mixture was then filtered and the filtrate concentrated to low volume by evaporation in vacuo. Lyophilisation gives the crude phthalide ester of α-azidobenzylpenicillin.

Catalytic hydrogenation of the crude phthalide ester of α-azidobenzylpenicillin gave the phthalide ester of α-aminobenzylpenicillin which, after purification by chromatography was found to be identical with an authentic sample prepared by the method of Example 2.

The rates of hydrolysis of the phthalide ester of 6-[D(-)α-aminophenylacetamido penicillanic acid HCl were determined by incubating the ester at the equivalent of 5μg/ml 6-[D(-)α-aminophenylacetamido] penicillanic acid in M/20 pH 7.0 potassium phosphate buffer, 90% human blood and 90% squirrel monkey blood. The ester was also tested for hydrolysis in squirrel monkey small intestine homogenate at the equivalent of 100 μg/ml 6-[D(-)α-aminophenylacetamido] penicillanic acid, the reaction mixtures being diluted to the equivalent of 5.0 μg/ml of the parent penicillanic acid before assay. The tissue homogenate was prepared by homogenising washed squirrel monkey small intestine in four times its weight of M/20 potassium phosphate buffer. This stock preparation was diluted a further 1:10 for the reaction mixture.

All reaction mixtures were incubated at 37° C. After incubation, the ester was separated from the reaction mixture by electrophoresis. 5 μl. aliquots of the reaction mixtures, and 6-[D(-) α-aminophenylacetamido] penicillanic acid standards prepared in the appropriate medium, were applied to starch-agar gel plates buffered at pH 5.5. A potential of 15 volts/cm was applied across the plate for 20 minutes. At this pH the parent penicillanic acid remains near the origin and any ester present migrates towards the cathode. The amount of parent penicillanic acid present in the reaction mixtures was assayed by overlayering the gel plates with nutrient agar (Oxoid blood base agar) seeded with Sarcina lutea NCTC 8340, and incubating for 16 hours at 30° C. Zones of inhibition, resulting from ampicillin in test samples and standards, were measured, and the amount of parent penicillanic acid formed in the reactions calculated.