KR20030070011A - Pharmaceutical compositions containing oxapenem-3-carboxylic acids - Google Patents

Abstract

The present invention relates to an oxapene compound which is a zwitter ion of formula Ia or formula Ib or capable of forming a zwitter ion of formula Ia or formula Ib, wherein R comprises a proton-added basic substituent C ₁ -C ₈ branched or straight chain alkyl group. The compounds are used as β-lactamase inhibitors with particularly high bioavailability.

Description

Pharmaceutical composition comprising oxapene-3-carboxylic acid {PHARMACEUTICAL COMPOSITIONS CONTAINING OXAPENEM-3-CARBOXYLIC ACIDS}

β-lactam antibiotics (eg, penicillin, cephalosporin and carbapenem) are known to treat bacterial infections, but their long-term use is associated with increased bacterial resistance.

The main mechanism for this bacteria's resistance is by β-lactamase. There are four kinds of β-lactamases from class A to class D. Clinically, β-lactamases of class A and class C are the most important. Combination treatment with the β-lactam antibiotics and β-lactamase inhibitors has proven successful in offsetting some forms of resistance. Illustrating such known combination products, Tazocim (RTM), a combination of piperacillin antibiotics and tazobactam inhibitors, and Augmentin (RTM), a combination of amoxycillin antibiotics and clavulanic acid inhibitors Can be mentioned. However, tazobactam and clavulanic acid are only effective against β-lactamases of class A, and have the problem of being defenseless antibiotic partners for class B, class D and most importantly class C.

Although β-lactamase inhibitors having activity against the above class C β-lactamases are known, are commercially available as "broad spectrum" inhibitors active against both class A and class C β-lactamases. There is nothing.

EP 0 301394 describes various variants of the oxapene compound which is an antimicrobial agent. EP 0 362622 also describes oxapene compounds, which have very high activity against β-lactamases of class A, class C and class D and have broad spectrum β-lactamase inhibitors. EP 0 548790 describes that the stereochemistry of the chiral side chains on the 6-carbon of the oxapene structure has a significant effect on the in vitro β-lactamase inhibitor activity. Compounds having a (1'S) -1-hydroxyalkyl side chain opposite to thienamicin and having a (1'S) -1-hydroxyalkyl side chain can be used as TEM 1 β-lactamase derived from E. coli as a common class A β-lactamase. Increased in vitro activity. However, none of the above documents have proved to exhibit particularly in vivo activity.

In addition, it is important for β-lactam to obtain sufficiently high blood levels and long biological half-lives to treat bacterial infections. Ceftazidime and Ceftriaxon (biological half-life in human serum in the body show 1.7 to 2.1 hours and 5.8 to 8.7 hours when using classic β-lactam antibiotics, such as cephalosporin antibiotics. Compounds such as) have been used to solve these requirements. In contrast, the biological half-life of nonclassical β-lactams, such as carbapenem imipenem, is shorter (0.9 and 1.0 hours). The half-life of clavulanate is 0.7 to 1.4 hours. The relatively long biological half-life is important for the practical application of antibiotics and β-lactamase inhibitors.

As of present application, it is known that several novel compounds within the specification range described in EP 0 362622 are surprisingly high in bioavailability and have high activity against β-lactamases of class A and class B. In addition, they are known to exhibit unexpectedly high stability in serum, increasing blood levels and half-life.

The present invention relates to an oxapene compound, a pharmaceutical composition comprising the compound and a therapeutic use thereof.

The invention will be described with reference to the following figures.

FIG. 1 shows oxapene analog plasma blood levels after subcutaneous administration in an amount of 50 mg per kg mouse subject.

2 is a schematic representation of the synthesis of a compound (PFOB) embodying the present invention from commercially available compounds.

According to one embodiment of the present invention, there is provided an oxapene compound which is a zwitterion of formula Ia or formula Ib or capable of forming the zwitter ion.

Wherein each R is a C ₁ to C ₈ branched or straight chain alkyl group comprising a protonated basic substituent.

The protonated basic substituent is preferably a protonated nitrogen base. More preferably, the protonated basic substituent is a protonated amine or a protonated aminomethyleneamino substituent.

As the compound of Formula Ia, In the above formula, R is-(CH ₂ ) ₄ NH ₃ ⁺ (hereinafter, referred to as compound E); R is — (CH ₂ ) ₃ NH ₃ ⁺ (hereinafter referred to as compound PFOB); R is — (CH ₂ ) ₂ NH ₃ ⁺ (hereinafter referred to as Compound A); R is (CH ₂ ) ₂ NHCH: NH ₂ ⁺ (hereinafter referred to as Compound B); Or R is -CH ₂ NHCH: NH ₂ ⁺ (hereinafter referred to as compound D).

Note that protonation of nitrogen in R of compounds B and D shown above can occur in either nitrogen. Accordingly, the compounds B and D may also be represented by the formula Ia wherein R is — (CH ₂ ) ₂ NH ₂ ⁺ CH: NH and —CH ₂ NH ₂ ⁺ CH: NH.

In addition, the R a - is (CH _{2) 3} NH ₃ ⁺ A compound of the formula Ib (hereinafter referred to as YOB) is preferred.

Compounds of the present invention that are in the Zwitter ion form (e.g., protons are added to the basic (i.e., amine) group and protons are released from the carboxyl group) and their "non-ionic" forms (basic groups) That is, the equilibrium between the amine) and the carboxyl group is neutral). The "nonionic" form of the composition, and the equilibrium mixtures of the zwitterion and "nonionic" forms are all included within the scope of the present invention. Thus, "oxaphenem compound capable of forming zwitter ions" includes an oxapene compound in the "non-ionic" form, ie CO ₂ ⁻ has been added protons with CO ₂ H, The basic group to which protons are added "or" nitrogen base to which protons are added "is the oxapene compound of formula Ia or formula Ib above, deprotonated. To illustrate the oxapene compound capable of forming the zwitter ion, CO ₂ ^- is protonated with CO ₂ H, and R is-(CH ₂ ) ₄ NH ₂ ,-(CH ₂ ) ₃ NH ₂ ,-( A compound of Formula Ia which is CH ₂ ) ₂ NH ₂ ,-(CH ₂ ) ₂ NHCH: NH, or -CH ₂ NHCH: NH; And compounds of formula Ib wherein CO ₂ ⁻ is deprotonated with CO ₂ H and R is — (CH ₂ ) ₃ NH ₂ .

In addition, the "oxaphenem compound capable of forming zwitter ions" includes mixtures of oxaphenem compounds in the zwitter ion form and the "nonionic" form.

Applicants disclose that (a) specific stereochemistry at positions 5 and 6 of the double ring structure of oxapene; And (b) a combination of specific zwitterionic structures (or the ability to form such zwitterionic structures) has been found to yield compounds with significant bioavailability and broad activity spectrum.

In addition, the compounds of the present invention exhibit very high bioavailability compared to conventional compounds. The compounds of the present invention show very high activity and selectivity against β-lactamases of class A and class C. In particular, compound YOB shows very high activity and selectivity against β-lactamases of class A.

The compounds and β-lactam antibiotics described herein are active against a wide range of Gram-positive (staphylococci, streptococci) and Gram-negative (enterobacteriaceae) bacteria that cause infections of the urinary tract, respiratory tract, wounds and intraperitoneal sepsis. The oxapene compound and the antibiotic may be administered simultaneously (eg, as a mixture), or for example as separate agents. The oxapene and antibiotic may be administered at different times.

As another embodiment of the present invention, the present invention provides a pharmaceutical composition comprising an pharmacologically effective amount of an oxapene compound according to the first embodiment of the present invention. In addition, the pharmaceutical composition preferably further comprises a pharmacologically effective amount of antibiotic. The antibiotic may be a β-lactam antibiotic (eg ceftazidime).

As another embodiment of the invention, the invention provides a method for treating infection comprising administering to a patient in need thereof a pharmacologically effective amount of a zwitter ion represented by the following formula Ia or Ib:

Wherein each R is a C ₁ to C ₈ branched or straight chain alkyl group comprising a protonated basic substituent. The proton-added basic substituent is preferably a proton-added nitrogen base. More preferably, the protonated basic substituent is a protonated amine or a protonated aminomethyleneamino substituent. The method of treatment may further comprise administering a pharmacologically effective amount of antibiotic to the patient. The oxapene compound and the antibiotic may be administered simultaneously or at different times.

The treatment may be by β-lactamase inhibition.

Preferably, the method of treatment is for treating infection of urinary tract, respiratory tract, wound and intraperitoneal sepsis. Said patient may be a human or animal.

More than 40 oxapene analogs were synthesized and tested in vitro. Structure activity relationship (SAR) studies have been conducted to examine different parts of the oxapene molecule related to chemical stability, target binding affinity, antibiotic activity, and β-lactamase inhibition spectrum and degree of inhibition. The function was confirmed. Structural studies did not predict bioavailability.

The present inventors have synthesized and tested several kinds of oxapenem compounds known as PFOB, YOB, A, E, B, D, U and XOB. These structures are shown in Table 1. These PFOBs, YOBs, Es, Bs, Ds, and As can be represented by embodiments of the present invention.

The composite is discussed below.

Table 1: Structure of oxapene compound

A. Pharmacokinetic Experiments

Compositions XOB, YOB, PFOB and YOB were administered subcutaneously (SC), respectively, in an amount of 50 mg per kg of mouse subject, followed by plasma blood levels of 5, 10, 20 and Measurement was made at a time point of 30 minutes. This result is shown in Table 2, and is shown in FIG.

TABLE 2

From the plasma blood levels at all of the above time points, it is clear that the compounds of the invention (Zwitter ion compounds PFOB and YOB) administered subcutaneously show significantly better bioavailability compared to salts XOB and U. It should also be noted that the difference between the PFOB and U and YOB and XOB is merely an amine substituted chain (instead of the methyl group) in the PFOB and YOB compounds. Such amine substituted chains provide a zwitterionic structure. Thus, the compositions are well suited for use in hospitals for intraperitoneal and subcutaneous dosing regimens.

B. In vitro activity on combination with ceftazidime

Analysis by agar dilution was performed according to the NCCLS guidelines (2000) to determine the Minimum Inhibitory Concentration (MIC). In the data below, the lowest MIC shows the strongest activity.

Β-lactamase of class A

Ceftazidime alone (CAZ) and PFOB, YOB, U and XOB mixed with ceftazidime at a 2: 1 ratio (CAZ + PFOB, CAZ + YOB, CAZ + U and CAZ + XOB), respectively. Each was tested for class A β-lactamase. The results are shown in Table 3.

Table 3: Inhibitory activity against β-lactamase of class A

All of the oxafenem compounds in combination with CAZ showed significantly better activity against E. coli TEM-3, TEM-6, TEM-9 and TEM-10 than in the case of CAZ alone.

The composition YOB according to the present invention showed very good activity compared to XOB (structurally and stereochemically close to a composition which is not a zwitter ion). This is especially demonstrated when the MIC values (0.03 and 0.06) for E. coli ATCC 25922 and TEM-1 of YOB are compared to those of XOB (0.25 and 0.25). This superiority can also be seen in the MIC values for the TEM-9 and TEM-10.

Β-lactamase of class C

Ceftazidime alone (CAZ) and PFOB, YOB, U and XOB mixed with ceftazidime in 1: 1 ratio (CAZ + PFOB, CAZ + YOB, CAZ + U and CAZ + XOB) Each was tested for class C β-lactamase. The results are shown in Table 4.1.

Table 4.1: Activating β-lactamase of class C Enterobacteriacaeae Inhibitory activity against

All of the oxaphenem compounds using the combination with CAZ showed significantly better activity against the whole bacteria than when using CAZ alone.

The composition PFOB according to the present invention showed superior activity compared to U (structurally and stereochemically close to a composition which is not a zwitter ion). This is especially true when MIC values (4, 0.03 and 0.03, respectively) for E. cloacae 84-con, C Freundii C2-con and S marcescens S2-con of PFOB are compared to those of U (8, 2 and 0.25). Well proved. In addition, it can be seen that the MIC value of YOB for the S marcescens S2-con is very superior to that of XOB.

Ceftazidime alone (CAZ) and PFOB, YOB, U and XOB mixed with ceftazidime at a 2: 1 ratio (CAZ + PFOB, CAZ + YOB, CAZ + U and CAZ + XOB), respectively. Each was tested for class C β-lactamase. The results are shown in Table 4.2.

Table 4.1: Activating β-lactamase of class C Enterobacteriacaeae Inhibitory activity against (ratio 2: 1)

In an embodiment of the present invention, the composition PFOB once again showed superior activity compared to U (structurally and stereochemically close to a composition that is not a zwitter ion). This is especially true for the MIC values (4, 4 and 2, respectively) for E. cloacae Hennessy, E. cloacae 84-con, C Freundii C2-con and S marcescens S2-con of PFOB. This proves well compared to). In addition, it can be seen that the MIC value of YOB for the C. freundii C2-con is superior to that of XOB.

summary

The compositions embodying the present invention have an excellent combination of "broad spectrum" activity and good bioavailability for both class A and class C β-lactamases.

Stereochemistry of substituents at the activity of the compound and C-6 (XOB and YOB have the (1'S) -1-hydroxyethyl side chain, while U and PFOB have the (1'R) -1-hydroxyethyl side chain) The dependency or relationship between the two is not simply foreseen. Therefore, excellent activity of PFOB and YOB could not be predicted from them.

C.1 Chemical Stability of XOB and YOB

Oxaphenem hydrolysis half-life at a concentration of 10 ⁻⁴ M was measured using UV spectroscopy at 265 nm with 37 ° C., physiological phosphate buffer pH 7.4.

XOB YOB Hydrolysis half-life 6.5 hours 7.0 hours

C.2 Stability of XOB and YOB in Serum

Serum stability was measured microbiologically using agar diffusion method using Escherichia coli TEM 1 (penicillin resistance). The oxapene was incubated with sterile bovine serum (OXOID) at 37 ° C. at a concentration of 0.033%. At intervals of 0 hours, 1.5 hours, 3 hours, 4.5 hours and 6 hours, the sample samples (30 mL) were contaminated on an industrial filter disc (OXOID) containing piperacillin (75 mg) and the residual amount of active β -Blood that contaminated various amounts (10 mg, 5 mg, 2.5 mg, 1.25 mg, 0 mg) of the β-lactamase inhibitor was observed by monitoring the reduced inhibition diameter (27-12 mm) of the lactamase inhibitor (oxapenem). Calculations were made in contrast to the reduced inhibition diameter (24-0 mm) obtained using a peracillin (75 mg) disc.

XOB YOB Serum half-life at 37 ° C 1.0 hours 2.4 hours

As such, the zwitter ion compound YOB is more stable than XOB in serum, but the chemical stability of the two compounds is actually equivalent. This surprising increase in serum stability of Zwitter ion oxapemene YOB (compared structurally and stereochemically to compositions other than Zwitter ions compared to XOB) is the most effective and important. This stability provides high blood levels and long biological half-lives, demonstrating excellent ability to treat humans and animals.

D. Synthesis of Compounds PFOB, A, E and YOB

D.1 (5R, 6R, 1'R) -3- (4-amino-1, 1-dimethylbutyl) -6- (1'-hydroxyethyl) -7-oxo-4-oxa-1-aza Synthesis of Bicyclo [3.2.0] hept-2-ene-2-carboxylic Acid [PFOB]

The following synthesis is shown schematically in FIG. 2 in the accompanying drawings.

Acetonitrile (16.7 kg) and (3R, 4R) -4- (acetoxy) -3-[(1R) -1-[[(1,1-dimethyl) in a 35 L Pfaudler vessel (GLMS) Ethyl) dimethylsilyl] oxy] ethyl] -2-azetidinone (3.34 kg, 11.6 mol) was charged. The header flask was charged with 21% sodium methyl mercaptan (5.8 L, 17.4 mol, 1.5 equiv). To this batch was added over 15 hours at 15-20 ° C. (cooling temperature required for exothermic control). This batch was then stirred for 1 hour before TLC analysis indicated completion. The lower aqueous phase was drained off and the product (acetonitrile) phase was washed with 6.7 L of 20% brine and then dried on a rotary evaporator (20 L) to remove brine. The crude product obtained was then refluxed while cooling to 0 ° C. to crystallize from hexane (13.3 L). The crystal product was filtered off and washed with hexane (1 L). This was dried in vacuo to obtain Compound III of FIG. 2 in a white needle type at a melting point of 93 ° C. (2.49 kg, 78%).

A 20 L tube (glass) was charged with THF (8.25 L) followed by the above compound (III) (1.65 kg, 6.0 mol). The batch was cooled to −40 ° C. and charged with 2.5 M butyl lithium for over 1 hour at a temperature below −25 ° C. (typically −45 ° C. to −35 ° C.). This was to be -25 ° C. A second tube was charged with THF (4.1 L) and para-nitrobenzyl iodoacetate (1.92 kg, 6.02 mol), which was cooled to -10 ° C. The compound (III) solution was then transferred to the second solution for over 30 minutes using a cannula under vacuum while maintaining the temperature below 0 ° C. (typically below −8 ° C.). The result was stirred (less than 2 hours at -5 ° C), then the batch was cooled to -10 ° C and added to a 50 L tube containing 20% brine (16 L). The lower aqueous phase was back extracted with dichloromethane (13 L). Then, the two organic phases were combined and the solvent was dried to give crude compound (IV) of FIG. 2 (approximately 3 kg). THF was charged (approximately 2 L) to store the product in about 40% solution.

THF solution (3.54 L) containing about 40% Compound IV (1292 g, 2.76 mol) was removed with KF <0.1% and redissolved in 7.5 L of fresh THF (KF <0.05%). To this was added 5-azido-2,2-dimethylpentanoic acid chloride (1.23 kg, 6.5 mol, 2.35 equiv) at less than -50 ° C. A 20% (1.04 M) lithium bis (trimethylsilyl) amide solution was added dropwise (6000 mL, 6.24 mol, 2.25 equiv) below -65 ° C. The mixture became very dark in color and was stirred at -70 ° C for 1 hour. Stirring was then stopped and toluene (12.5 L) and 10% HCl (12.5 L) were charged. The organic phase was then washed successively using KHCO ₃ (12.5 L), water (12.5 L) and saturated brine (6 L). The organic phase was concentrated and the solvent was evaporated to yield a dark colored concentrated solution (approximately 30% product).

25 kg of flash silica, consisting of about 50 L toluene, was charged with the material redissolved in 20% hexane in toluene (20 L) (obtained in three batches of the reaction). It was eluted under 0.5 bar pressure and the material was added to the column and circulated until (fraction 1) was obtained. A small front was separated from this fraction and discarded. This eluted the following fractions:

Fractions 2-4 toluene (25 L each)

6% ethyl acetate in fractions 5 and 6 toluene (25 L each)

8% ethyl acetate in fractions 7 and 8 toluene (25 L each)

10% ethyl acetate in fractions 9 and 10 toluene (25 L each)

The fraction containing the product was removed to give 13.5% of a solution of compound V (3.51 kg, 68%) in FIG. 2 with a volume of 25 L.

The tetrahydrofuran solution of compound V was removed on a rotary evaporator to give an oil (containing 4.4 kg, 3.2 kg of compound V, including 5.15 moles, some THF). The oil was redissolved in THF (10.75 L) to form a final solution (KF 0.0326%). The solution was charged to a 100 L tube and further charged with acetic acid (2.98 L), tetra-n-butyl-ammonium fluoride (5.36 kg, 17.13 mol) and THF (21.5 L). Some foaming was involved during the filling process. This batch was heated to reflux (65 ° C.) and refluxed for 16 hours. Sampling through TLC analysis revealed only trace amounts of starting material. Toluene (32 L) was added and the contents of the tube were cooled to 20 ° C. and then quenched with 1 M potassium bicarbonate solution for more than 15 minutes while bubbling.

The organic phase was washed with 1 M potassium bicarbonate solution (2 × 27 L), 10% brine (4 × 11 L) and 20% brine solution (3 L). This wash regimen was confirmed through the removal of acetic acid.

The toluene solution was removed to approximately 5 L volume and stored in the freezer while the second batch was prepared, yielding 4.2 kg of additional crude product.

The crude product was purified using dry flash column chromatography under nitrogen pressure (0.5 bar).

A slurry of flash silica (Chrogel Silica I 254, 25 kg) in toluene (50 L) was charged and placed in a 30 cm diameter column, and a bed having a depth of 80 cm was formed. Toluene was eluted until the silica was partially dried. The crude product (8.5 kg) obtained above was dissolved in toluene (20 L), filled with silica, and dropped onto the column under nitrogen pressure. This product was then eluted with the following solvent mixture.

100% Toluene 50 ℓ

Toluene: ethyl acetate (98: 2) 75 l

Toluene: ethyl acetate (95: 5) 25 l

Toluene: ethyl acetate (80:20) 100 l

Toluene: ethyl acetate (70:30) 50 l

Toluene: ethyl acetate (60:40) 50 l

Toluene: ethyl acetate (60:40)

+ 0.5% isopropanol 50 l

The following fractions were collected in size.

Fraction number Fraction size

1-725 ℓ

8-125 ℓ

13-2025 ℓ

Vacuum concentration was performed on a 20 L rotary evaporator to give a pale red-orange oil as Compound VI of FIG. 2 (single spot by TLC, 4.004 kg, 7.88 mol, 76.5%).

Dichloromethane (1.02 L) and methyl disulfide (398 g, 4.23 mol) were charged to a 2 L flask. The radical scavenger (3-tert-butyl-4-hydroxy-5-methylphenylsulfide, 1.85 g, 0.005 mol) was then charged to cool the batch to -35 ° C. . The solution was sparged with chlorine gas (296 g, 8.35 moles) for more than 2 hours to give an orange methylsulphenyl chloride solution.

To a 20 L flask containing dichloromethane (11.4 L) and compound VI (4.97 kg 38.2% solution in dichloromethane, 1.9 kg activity, 3.74 moles), the solution was added for 25 minutes at -25 ° C to -15 ° C. . The batch was then stirred at -25 ° C for 20 minutes. If TLC suggests that the reaction is complete (without starting material), the batch is placed in a 50 L flask containing a solution of sodium hydrogen sulphite (1.196 kg) and sodium bicarbonate (0.975 kg) in water (23 L). Was cooled rapidly. The obtained phases were separated and the aqueous phase was back extracted with dichloromethane (1.5 kg, 2 L). The combined organic phases were washed with saturated brine solution (6 L), dried over magnesium sulfate (1 kg) and concentrated in vacuo to oil in a 20 L rotary evaporator to give tetrahydrofuran (2.71 kg) as compound VII in FIG. Red crude oil dissolved in) was obtained (1.74 kg, 3.51 mol, 93.7% crude yield) and stored at -30 ° C for use in the next step.

Compound VIII (1.028 kg) was concentrated in vacuo to afford crude oil, dissolved in tetrahydrofuran (9.5 kg, 10.6 L, KF value = 0.02%) and charged into a 20 L flask. The flask was then cooled to below −50 ° C. and triethylamine (848 g, 1.168 L) was added over 5 minutes. The batch was stirred at −50 ° C. for 1 hour, heated to 20 ° C. for over 2 hours and then at 20-25 ° C. for an additional 2 hours. Completion of the reaction was confirmed by TLC (in the absence of starting material). Toluene (17.7 L) was charged to a 50 L flask, to which the reaction mixture was added and washed with toluene (3.55 L). After placing and separating the mixture, the organic phase was washed with 10% brine solution (3 × 12 L), then saturated brine solution (3 L), dried over sodium sulfate (1 kg) and concentrated in vacuo. 2 L of a compound VII solution of FIG. 2 consisting of 4.5 kg of toluene were obtained.

A slurry of silica gel 60 (1500 g) in diethyl ether: n-pentane (1.5: 1) was formed and charged into a jacketed column (inner diameter 80 mm) and circulated glycol at -15 ° C. To cool. Crude product VII (22.5-25.0% w / w, 450 g solution, 101.3-112.5 g active crude mixture) was charged to the silica and precooled to -20 ° C diethylether: n-pentane (1.5: 1 Eluted with). The product was eluted with 25 L of mixed solvent and 2 L of diethyl ether with 1 L fractions collected and concentrated in vacuo at -20 ° C to give the transcompound VII of Figure 2 (typically 46 g) to -20 ° C. Stored at.

Demineralized water (750 ml) in 10 l flask, 10% palladium on charcol (Johnson Matthey, type 87L, 60% water, 55 g damp solid, 2.2 g palladium, 0.0207 mol, 0.0211 Equivalent) and ethyl acetate (1750 mL) were charged. The flask was purged with nitrogen for 15 minutes and then hydrogen and the batch were cooled to below 0 ° C. This was followed by vigorous stirring (450-500 rpm).

Trans Compound (VII) (45 g, 0.098 mol) was dissolved in ethyl acetate (300 mL) at -30 ° C and a final solution was obtained at a temperature of -5 ° C. While maintaining the temperature of the reaction and header below 0 ° C., it was added to the batch.

The batch was hydrogenated for up to 90 minutes while maintaining a constant hydrogen flow. The reaction was deemed complete when no starting material was present via HPLC analysis of the organic layer.

The catalyst was removed as quickly as possible by filtration through Celite 521 (50 g, premineralized with demineralized water and ethyl acetate) while maintaining a temperature below 0 ° C. The remaining catalyst was washed at 0 ° C. with pre-cooled ethyl acetate (100 mL) and demineralized water (2 × 100 mL). After settling, the aqueous phase was separated and left to stand at 0 ° C. and washed with n-pentane: toluene (3: 1) (225 mL) cooled to −20 ° C.

The aqueous phase comprising the product was filtered by passing 1.6 mm glass fiber paper (GF / A grade) and polyethersulfone membrane (pore size 0.2 mm, PES grade) with a glass fiber prefilter cartridge (GFP grade), A light pale yellow solution was obtained. This was frozen in a 200 ml aliquot on a 500 ml flask wall at -78 ° C. After freeze drying for 72 hours, (5R, 6R, 1'R) -3- (4-amino-1, 1-dimethylbutyl) -6- (1'-hydroxyethyl) -7-oxo-4- Oxa-1-azabicyclo [3.2.0] hept-2-ene-2-carboxylic acid (typically -50% corrected yield) was separated into bulky off-white to pale yellow solids. .

D.2 Synthesis of A, E

Compounds A and E were prepared by applying the above synthesis method of PFOB.

D.3 (5R, 6R, 1'S) -3- (4-amino-1, 1-dimethylbutyl) -6- (1'-hydroxyethyl) -7-oxo-4-oxa-1-azabicyclo [3.2.0] hept-2-ene-2-carboxylic acid [YOB]

In a 500 ml Schlenk flask equipped with a magnetic stirrer and a reflux condenser connected to a nitrogen-filled balloon, p-nitrobenzyl (3S, 4R)-(3- (in dry tetrahydrofuran) 1 '(R) -tert-butyldimethylsiloxyethyl) -4-methylthio-2-oxoazetidinyl) acetate (14.13 g, 30.15 mmol) [Compound IV in PFOB synthesis], acetic acid (17.3 mL, 302 mmol) and tetrabutylammonium fluoride (23.2 g, 88.6 mmol) were refluxed for 12 hours. Complete conversion via Tlc on silica gel was implied. The mixture was diluted with toluene (2500 mL) and then washed with 500 mL of 10% KHCO ₃ , 10% NaCl (2x) and saturated NaCl. The organic phase was dried over magnesium sulfate, filtered and the filtrate was evaporated in vacuo to give an amorphous residue (12 g). Purification by column chromatography on silica gel (440 g, 40-63 mm) with toluene-ethylacetate (2: 1, 3: 2 and 1: 1) using 440 mL fractions was performed. Crystalline product (6.38 g, 60%), p-nitrobenzyl (3S, 4R)-(3- (1 '(R) -hydroxyethyl) -4-, from 10 to 16 fractions at melting point 77-79 ° C. Methylthio-2-oxoazetidinyl) acetate was obtained.

In a 500 ml Schlenk flask equipped with a magnetic stirrer, rubber septum and a nitrogen filled instrument, p-nitrobenzyl (3S, 4R)-(3- (1 '(R) -hydroxyethyl) -4 -Methylthio-2-oxazetidinyl) acetate (6.35 g, 17.93 mmol), triphenylphosphine (9.4 g, 35.86 mmol) and dry tetrahydrofuran (134 mL) were added. Dry formic acid (2.03 mL, 53.79 mmol) was added to the mixture, and diisopropyl azodicarboxylate (7.03 mL, 35.86 mmol) was slowly added to the stirred solution at −10 ° C. for at least 30 minutes. After 10 minutes a precipitate formed. The suspension is stirred for an additional 30 minutes at 0 ° C. and for 90 minutes at room temperature where the precipitate is redissolved. This gave a dark yellow solution. The reaction mixture was diluted with toluene (1500 mL) and then washed with 1200 mL of water, 10% KHCO ₃ and 10% NaCl. The organic layer was dried over magnesium sulfate, filtered and the solvent was removed by vacuum drying to give an oil. The oil was purified by silica gel chromatography (205 g, 63-200 mm) using 200 ml of a fraction of toluene-ethylacetate (9: 1). The fractions were examined by silica gel tlc (chloroform-ethyl acetate 4: 1). P-nitrobenzyl (3S, 4R)-(3- (1 '(S) -formyloxyethyl) -4-methylthio-2-oxoazeti as impure semicrystalline solid (10.01 g) from fractions 6-15 Dinyne) acetate was collected (6.85% = 100%).

In a 500 ml round bottom flask with a magnetic stirrer, crude p-nitrobenzyl (3S, 4R)-(3- (1 '(S) -formyloxyethyl) -4-methylthio-2-oxoazetidinyl Acetate (10.01 g, prepared from 17.93 mmol of the precursor) and methanol (180 mL) were added. 1N HCl (17.9 mL, 17.9 mmol) was added to the mixture and the solution was stirred overnight at room temperature. The complete reaction was confirmed by Tlc on silica gel. The reaction mixture was diluted with toluene (1600 mL) and then the solution was washed with cold water (1100 mL), 10% KHCO ₃ (500 mL) and 10% NaCl (500 mL). The organic layer was dried over magnesium sulfate and filtered, and the resulting solution was evaporated in vacuo to give an oil. It was purified by silica gel chromatography column using 200 ml fractions and toluene-ethylacetate (2: 1 and 1: 1). P-nitrobenzyl (3S, 4R)-(3- (1 '(S) -hydroxyethyl) -4-methylthio-2-oxoazeti as amorphous solid (3.77 g, 59%) from fractions 9-19 Diyl) acetate was obtained.

In a 100 ml Schlenk flask equipped with a magnetic stirrer and an instrument filled with nitrogen, p-nitrobenzyl (3S, 4R)-(3- (1 '(S) -hydroxyethyl) -4-methylthio-2-oxoa Zetidinyl) acetate (3.72 g, 10.50 mmol) and dry (ethanol-free) methylene chloride (23 mL) were added. A solution of p-nitrobenzyl chloroformate (3.08 g, 14.28 mmol) in methylene chloride was added slowly at -18 ° C. The stirred solid N, N-dimethylaminopyridine (1.74 g, 14.28 mmol) was added in small amounts over 20 minutes. After stirring at −18 ° C. for 4 hours, the complete reaction was examined by silica gel tlc. The reaction mixture was diluted with methylene chloride (230 mL) and the resulting solution was then washed with a ratio of 1N HCl, saturated NaHCO ₃ and 10% NaCl (120 mL). The HCl phase was reextracted with methylene chloride (40 mL) and the extraction solution was washed with NaHCO ₃ . The combined extraction solution was dried over magnesium sulfate and filtered to remove the solvent in vacuo to afford crude product (5.24 g). Silica gel chromatography (170 g, 63-200 mm) using 170 ml of fraction of toluene-ethylacetate (6: 1) was carried out to give p-nitrobenzyl (3S, 3) as an amorphous solid (4.80 g, 86%). 4R)-(3- (1 '(S) -p-nitrobenzyloxcarbonyloxyethyl) -4-methylthio-2-oxoazetidinyl) acetate was obtained.

In a 250 ml Schlenk flask equipped with a magnetic stirrer, a rubber diaphragm and a nitrogen filled instrument, p-nitrobenzyl (3S, 4R)-(3- (1 '(S) -p-nitrobenzyloxcarbonyloxyethyl) 4-Methylthio-2-oxoazetidinyl) acetate (1.00 g, 1.87 mmol) and dry tetrahydrofuran (46 mL) were added. To this mixture was added 5-azido-2,2-dimethyl-pentanoyl chloride (0.37 g, 1.96 mmol) at -78 ° C, followed by lithium bis-trimethylsilylamide in tetrahydrofuran (3.74) at -78 ° C. ML, 3.74 mmol) solution 1M was added. The orange solution formed initially turned pale yellow. After a further 15 minutes of stirring, the reaction was terminated via tlc on silica gel. The reaction mixture was diluted with toluene (240 mL) and washed with 2N HCl and 2x saturated NaCl in a ratio of 1: 2 (220 mL). The organic phase was dried over magnesium sulphate and filtered, the filtrate was evaporated in vacuo and then dried briefly in high vacuum to give an amorphous crude product (1.38 g). Purification was carried out by silica gel column chromatography (92 g, 63-200 mm) using 90 ml of a fraction of toluene-ethyl acetate (9: 1), followed by double distillation with methylene chloride, and high vacuum. Dried over, p-nitrobenzyl 7-azido-4,4-dimethyl-2-[(3S, 4R) -4-methylthio-3-[(S) as a pale yellow amorphous solid (1.01 g, 79%). -1-p-nitrobenzyloxcarbonyloxyethyl] -2-oxo-1-azetidinyl] -3-oxo-heptanoate was obtained.

In a 250 ml Schlenk flask equipped with a magnetic stirrer, a rubber septum and a nitrogen filled instrument, p-nitrobenzyl 7-azido-4,4-dimethyl-2-[(3S, 4R) -4-methylthio-3 -[(S) -1-p-nitrobenzyloxcarbonyloxyethyl] -2-oxo-1-azetidinyl] -3-oxoheptanoate (0.97 g, 1.41 mmol) and dry methylene without ethanol Chloride (56 mL) was added. The mixture was infused with dry chlorine gas through the surface using a syringe at -60 ° C through the inlet. After 10 minutes, the reaction was completed by tlc on silica gel at -50 ° C. The cold solution was poured into an aqueous solution containing NaHSO ₃ (2.82 g, anhydride) and sodium carbonate (2.23 g, anhydride). The mixture was stirred for 4 minutes, the organic phase was collected and the aqueous phase was extracted with a small amount of methylene chloride. The organic solution was then washed twice with 10% NaCl (37 mL) to combine the organic solutions and dried over magnesium sulfate. After evaporation in vacuo, an amorphous solid (0.95 g) was obtained. Purification by rapid silica gel column chromatography (2.3 g, 63-200 mm) using 5 ml fractions of toluene-ethyl acetate 19: 1 and 4: 1. The solvent was evaporated in vacuo and the residue was dried in high vacuum to yield p-nitrobenzyl 7-azido-2-[(3S, 4R)-as a light yellow amorphous solid (930 mg, 97%) from fractions 2-14. Obtain 4-chloro-3-[(S) -1-p-nitrobenzyloxcarbonyloxyethyl] -2-oxo-1-azetidinyl] -4,4-dimethyl-3-oxo-heptanoate It was.

In a 100 ml Schlenk flask equipped with a magnetic stirrer, a rubber septum and a nitrogen filled instrument, p-nitrobenzyl 7-azido-[(3S, 4R) -4-chloro-3-[(S) -1-p -Nitrobenzyloxcarbonyloxyethyl] -2-oxo-1-azetidinyl] -4,4-dimethyl-3-oxo-heptanoate (910 mg, 1.35 mmol) and dry tetrahydrofuran (27 mL) ) Was added. To the mixture was slowly added 0.92 M of a solution of potassium tert-butoxide (1.54 mL, 1.42 mmol) in dry tert-butanol at -30 ° C. The reaction mixture was stirred at -30 ° C for 150 minutes. Reaction completion was confirmed via tlc on silica gel. The dark yellow solution was diluted with ethyl acetate (150 mL), left for 5 minutes and washed with 10% NaCl (130 mL), 10% NaCl (65 mL) and saturated NaCl (65 mL). The organic layer was collected and dried over magnesium sulfate. After filtration and solvent evaporation in vacuo, it was dried in high vacuum for 90 minutes to give a yellow oil (860 mg). The purification process was carried out by intermediate pressure column chromatography at low temperature (-13 ° C.) on silica gel (60 g, 5-20 mm) using toluene-butyl acetate (19: 1), each of which collected 30 ml of fractions. Was performed. The fractions were left at -20 ° C. After evaporation of the solvent in high vacuum, from the fractions 11 to 13, pure trans p-nitrobenzyl (5R, 6R) -3- [4-azido-1,1-dimethylbutyl] -6- as a colorless amorphous solid [(S) -1-p-nitrobenzyloxycarbonyloxyethyl] -7-oxo-4-oxa-1-azabicyclo [3.2.0] hept-2-ene-carboxylate (306 mg, 35 %) Was obtained. From fractions 14-21 similarly a cis - trans isomer mixture (277 mg, 32%) was obtained.

In a 100 ml flask equipped with a magnetic stirrer, rubber diaphragm and two limbs connected to a hydrogenation unit, ethyl acetate (13 ml) and palladium catalyst on carbon (10%, 300 mg) in water (13 ml) at 0 ° Hydrogenated 20 minutes in advance. This was followed by p-nitrobenzyl (5R, 6R) -3- [4-azido-1,1-dimethylbutyl] -6-[(S) -1-p-nitrobenzyloxy in ethyl acetate (10 mL). While carbonyloxyethyl] -7-oxo-4-oxa-1-azabicyclo [3.2.0] hept-2-ene-2-carboxylate (280 mg, 0.44 mmol) was stirred at 0 ° C, Add using syringe. After stirring for 50 min at 0 ° C., hydrogen aspiration was terminated (90 mL of hydrogen was consumed). The catalyst was filtered off, washed with a small amount of ethyl acetate and water and the cooled biphasic mixture was separated. The aqueous phase was washed twice with a small amount (3 mL) of cold ethyl acetate and water and the ethyl acetate phase was reextracted twice with 3 mL of water. The remaining ethyl acetate was removed from the combined aqueous phase in vacuo and dried in high vacuum at 0 ° C. to reduce the volume to 13 ml in high vacuum. The solution was lyophilized at −25 ° C. for 3 days to give (5R, 6R) -3- [4-amino-1,1-dimethylbutyl] -6 as a colorless bulky powder (98 mg, 75%). -[(S) -1-hydroxyethyl] -7-oxo-4-oxa-1-azabicyclo [3.2.0] hept2-ene-2-carboxylic acid (YOB) was obtained.

E. Pharmaceutical Preparation

The formulations can be prepared as follows.

Example 1 (Method A)

(5R, 6R, 1'R) -3- (4-amino-1,1-dimethylbutyl) -6- (1'-hydroxyethyl) -7-oxo-4-oxa-1-azabi in lactose Very stable formulation of cyclo [3.2.0] hept-2-ene-2-carboxylic acid (PFOB).

3.79 g (8.25 mmol) p-nitrobenzyl (5R, 6R, 1'R) 3- (4-azido-1-dimethylbutyl) -6- (1'-hydroxyethyl)-in 30 mL ethyl acetate A solution of 7-oxo-4-oxa-1-azabicyclo [3.2.0] hept-2-ene-2-carboxylate was injected into a 120 ml ethyl acetate and 60 ml water by syringe through a rubber septum at 0 ° C. To the pre-hydrogenated mixture of 3.1 g of palladium on charcoal was added. After reacting for 70 minutes at 0 ° C., 840 ml of hydrogen was injected (theoretical amount was 740 ml). The reaction mixture was filtered through a 10 cm diameter G5 glass filter, the residue washed with 30 ml of cold water and 30 moles of cold ethyl acetate, and the ethyl acetate layer was separated from the mixed filtrates. The aqueous layer was washed with 50 ml cold ethyl acetate and 50 ml cold toluene at 0 ° C. and the resulting aqueous colloidal solution was compressed through a membrane filter using a syringe onto a separate layer. The aqueous layer was evacuated in high vacuum to remove the remaining organic solvent. To an aqueous solution (89.8 mL) was added a cold solution containing 7.20 g lactose monohydrate in 180 mL water and 3 mL of the resulting solution was filled in a glass ampoule. The contents of the ampoule were frozen in a dry ice-acetone bath and freeze dried for 4 hours in a freeze dryer at −25 ° C., 0.01 bar to remove moisture. The resulting white powder was dried overnight in a desiccator with phosphorus pentoxide at room temperature, 0.001 mbar to give 98.3 mg of white powder in each ampoule. In water, UV spectroscopy at 262 nm was used to reveal the title compound and 80.1 mg lactose in each ampoule with 18.2 mg content. The ampoule was filled with dry nitrogen and sealed, or stored using a desiccant, respectively.

In addition, the pharmaceutical composition may be prepared as follows:

Example 2

(5R, 6R, 1'R) -3- (4-amino-1,1-dimethylbutyl) -6- (1'-hydroxyethyl) -7-oxo-4-oxa-1-azabicyclo [ 3.2.0] hept-2-ene-2-carboxylic acid (PFOB)

Following the procedure of section C1, freeze drying at −25 ° C. (without lactose) and overnight drying at 0.001 bar, phosphorus pentoxide at room temperature, afforded the pure title compound as a white powder. The compounds thus obtained are suitable for administration as pharmaceutical compositions prepared according to known methods or according to the methods discussed below.

Example 3

Preparation of the formulation

The novel oxapenem compounds described above can be used in pharmaceutical compositions. The oxapene compound may be prepared in a pharmaceutical composition / pharmaceutical according to conventional methods as described in EP 0 301394. Other methods of making a medicament include the following:

Lactose Monohydrate and (5R, 6R, 1'R) -3- (4-Amino-1,1-dimethylbutyl) -6- (1'-hydroxyethyl) -7-oxo-4-oxa-1- Azabicyclo [3.2.0] hept-2-ene-2-carboxylic acid (PFOB, Example 1) (4: 1) 300 mg co-freeze dried 120 mg cefaclor and 5 mg magnesium stearate And 425 mg of the mixture is added to gelatin number 3 capsules to prepare a unit dosage form. As such, when a co-freeze-dried product of high content of oxapene-3-carboxylic acid is used, other formulations may be prepared and filled in No. 3 capsules, and if more than 425 mg of ingredients need to be mixed together, more Large capsules can also be prepared in compressed tablets and pills. The following examples illustrate the preparation of pharmaceutical formulations.

Table 5

Lactose monohydrate and (5R, 6R, 1'R) -3-

(4-amino-1,1-dimethylbutyl) -6- (1'-hydroxyethyl)-

7-oxo-4-oxa-1-azabicyclo [3.2.0] hept-2-

Co-freeze dried product of -2-carboxylic acid (4: 1) 625 mg

Cefaclor250 mg

Corn Starch V.S.P. 200 mg

Dicalcium phosphate 60 mg

Magnesium Stearate 60mg

The co-lyophilized and other active ingredients (Cefaclor) are mixed with dicalcium phosphate and about half of corn starch. The mixture is then milled and passed through a coarse sieve. It was dried at 45 ° C. and passed again through a sieve (No. 16 screen) having a mesh width of 1.0 mm. The remaining corn starch and magnesium sulfate are added and the mixture is compressed to formulate into tablets weighing 1195 mg each, 1.27 cm (0.5 inch) in diameter.

Parenteral solution

ampoule

Lactose monohydrate and (5R, 6R, 1'R) -3-

(4-amino-1,1-dimethylbutyl) -6- (1'-hydroxyethyl)-

7-oxo-4-oxa-1-azabicyclo [3.2.0] hept-2-ene-

Co-Lyophilized of Carboxylic Acid (4: 1) 1250 mg

Ceftazidime 500 mg

Sterile water (using a syringe immediately before use

5 ml)

Opthalmic solution

Lactose monohydrate and (5R, 6R, 1'R) -3-

(4-amino-1,1-dimethylbutyl) -6- (1'-hydroxyethyl)-

7-oxo-4-oxa-1-azabicyclo [3.2.0] hept-2-ene-

Co-freeze dried product of 2-carboxylic acid (4: 1) 625 mg

Ceftazidim 250 mg

Hydroxypropylmethylcellulose 15 mg

Sterile water (using a syringe immediately before use

2 ml)

Optic solution

Lactose monohydrate and (5R, 6R, 1'R) -3-

(4-amino-1,1-dimethylbutyl) -6- (1'-hydroxyethyl)-

7-oxo-4-oxa-1-azabicyclo [3.2.0] hept-2-ene-

Co-Lyophilized 2-Carboxylic Acid (4: 1) 250 mg

Ceftazidime100 mg

Benzalkonium chloride 0.1 mg

Sterile water (using a syringe immediately before use

2 ml)

Topical creams or ointments

Lactose monohydrate and (5R, 6R, 1'R) -3-

(4-amino-1,1-dimethylbutyl) -6- (1'-hydroxyethyl)-

7-oxo-4-oxa-1-azabicyclo [3.2.0] hept-2-ene-

Co-Lyophilized 2-Carboxylic Acid (4: 1) 250 mg

Ceftazidime100 mg

Polyethylene Glycol 4000 V.S.P.800 mg

Polyethylene Glycol 400 V.S.P.200 mg

YOB, A, B, D and E can be used as pharmaceutically active reagents in an example similar to the above in place of PFOB in the above formulation.

Note that although the co-freeze drying process improves stability, it is not necessary to co-freeze dry the active oxapene (PFOB, YOB, etc.) together with the carrier (ie lactose) before mixing with other reactants. do.

The active ingredients in the above preparations may be used alone or in other biologically active ingredients such as lincomycin, penicillin, streptomycin, novobiocin, gentamycin, neomycin, colistin and klanamycin, or probenidide. It can be mixed with other therapeutic agents such as.

The specification and examples illustrate the invention in detail and are not intended to limit the invention, and other embodiments may be suggested to those skilled in the art within the scope of the invention.

Claims (14)

Oxapenem compounds that are zwitter ions of formula Ia or Ib or capable of forming zwitter ions of formula Ia or Ib: Wherein each R is a C ₁ -C ₈ branched or straight chain alkyl group comprising a protonated basic substituent. The method of claim 1, An oxapene compound, wherein the protonated basic substituent is a protonated nitrogen base. The method according to claim 1 or 2, An oxapene compound, wherein the protonated basic substituent is a protonated amine or a protonated aminomethyleneamino group. The method according to claim 1, 2 or 3, R is-(CH ₂ ) ₄ NH ₃ ⁺ ,-(CH ₂ ) ₃ NH ₃ ⁺ ,-(CH ₂ ) ₂ NH ₃ ⁺ ,-(CH ₂ ) ₂ NHCH: NH ₂ ⁺ , or R is -CH ₂ NHCH: NH ₂ ⁺ expression oxa penem compound having the Ia. The method according to any one of claims 1 to 4, An oxapene compound having formula Ia wherein R is-(CH ₂ ) ₃ NH ₃ ⁺ . The method according to claim 1, 2 or 3, An oxapene compound having formula Ib wherein R is-(CH ₂ ) ₃ NH ₃ ⁺ . A pharmaceutical composition comprising a pharmacologically effective amount of an oxapenem compound according to any one of claims 1 to 6. (Beta) -lactamase inhibitor containing the oxaphenem compound as described in any one of Claims 1-6. The method of claim 7, wherein Pharmaceutical composition wherein the composition further comprises a pharmacologically effective amount of antibiotic. A method of treating infection comprising administering to a patient in need of a pharmacologically effective amount of a zwitterion of Formula Ia or Formula Ib: Wherein each R is a C ₁ -C ₈ branched or straight chain alkyl group comprising a protonated basic substituent. The method of claim 10, Wherein the Zwitter ion is R (-CH ₂ ) ₄ NH ₃ ⁺ ,-(CH ₂ ) ₃ NH ₃ ⁺ ,-(CH ₂ ) ₂ NH ₃ ⁺ ,-(CH ₂ ) ₂ NHCH: NH ₂ ⁺ or -CH ₂ NHCH: NH ₂ ⁺ ; Or wherein in formula Ib R is — (CH ₂ ) ₃ NH ₃ ⁺ . The method according to claim 10 or 11, wherein The method further comprises administering to the patient a pharmacologically effective amount of an antiseptic agent. Use of zwitterion of formula Ia or formula Ib in the manufacture of a medicament for the treatment of infection: Wherein each R is a C ₁ -C ₈ branched or straight chain alkyl group comprising a protonated basic substituent. The method of claim 13, Wherein the Zwitter ion is R (-CH ₂ ) ₄ NH ₃ ⁺ ,-(CH ₂ ) ₃ NH ₃ ⁺ ,-(CH ₂ ) ₂ NH ₃ ⁺ ,-(CH ₂ ) ₂ NHCH: NH ₂ ⁺ or -CH ₂ NHCH: NH ₂ ⁺ ; Or R in formula Ib is-(CH ₂ ) ₃ NH ₃ ⁺ .