RS20080604A - Penem prodrugs - Google Patents

Abstract

Orally bioavailable prodrugs of sulopenem, e.g., Formula (I) and solvates and hydrates thereof, preparation thereof, formulation thereof, and use to treat and prevent infection in mammals such as humans.

Description

whether

PRODRUGS PENEMA-

FIELD AND BASIS OF THE INVENTION

This invention relates to anti-infective agents, antibiotics, oral antibiotics, and prodrugs, especially sulopenem prodrugs, their preparation and use.

US5013729 describes sulopenem, which is a broad-spectrum antibiotic and is (5R,6S)-6-[(1R)-1-hydroxyethyl]-7-oxo-3-[[(1R,3S)-tetrahydro-1-oxido-3-thienyl]thio]-4-thia-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid. See also J. Org. Chem., 57, 4352-61 (1992).

Other penems and prodrugs are discussed in, for example, US4952577; US5506225; WO1992/003444; and WO2004/067532.

Various preclinical and clinical trials have been performed with sulopenem and some of its prodrugs. Sulopenem itself is not orally bioavailable to a significant extent. US5013729 also discusses prodrugs of sulopenem, including the pivaloyloxymethyl prodrug of sulopenem (sulopenem POM ester). When given orally as a mixture of the two stereoisomers, the POM ester has been shown to be orally bioavailable in humans. See Foulds et al., Antimicrobial Agents and Chemotherapy, pp. 665-671 (Apr. 1991). However, POM ester prodrugs are associated with tissue depletion of camitin, following hydrolysis and release of pivalic acid or trimethylacetic acid. See Brass, Pharmacological Reviews, 54, 589-598 (2002).

DESCRIPTION

This invention expresses the desire for new sulopenem prodrugs that combine one or more properties: high oral exposure or bioavailability, absence of tendency to deplete camitin from tissues, physicochemical properties, such as crystallization ability, melting point, water solubility, and permeability, which favor practical pharmaceutical formulation and use.

In some embodiments, the present invention includes compounds of formula I

in other aspects, the present invention includes compounds of formula II

The invention further includes formulations and administration of the compounds for the treatment or prevention of bacterial infections.

DETAILED DESCRIPTION

COMPOUNDS

The present invention includes prodrug compounds of formula I and II, as shown and described above. All stereoisomers and mixtures thereof, as indicated by the drawings, which permit and include both R and S configurations at the stereocenters are contemplated and included.

The most preferred configuration of the compounds of Formulas I and II is

In particular, the oxothiolanyl group most often has the 1R 3S configuration, as shown below

For example, (2-ethyl-1-oxobutoxy)methyl (5R,6S)-6-[(1R)-1-hydroxyethyl]-7-oxo-3-[[(1R,3S)-tetrahydro-1-oxido-3-thienyl]thio]-4-thia-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylate (herein compound 1) was obtained, shown below:

Another example is provided by (2-ethoxy-2-methyl-1-oxopropoxy)methyl (5R,6S)-6-[(1R)-1-hydroxyethyl]-7-oxo-3-[[(1R,3S)-tetrahydro-1-oxido-3-thienyl]thio]-4-thia-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylate (Compound 2 herein), shown below:

The prodrugs of this invention may be amorphous or may exist in various crystalline forms, or may be polymorphic, including solvates and hydrates. Polymorphs of the prodrugs form part of the present invention and can be obtained by crystallizing the prodrugs of the present invention under various conditions. Polymorphs can also be obtained by heating or melting the prodrug, followed by gradual or rapid cooling. The presence of polymorphs can be determined on a solid sample by NMR spectroscopy, IR spectroscopy, differential scanning calorimetry, X-ray powder diffraction, and other similar techniques. Thus, a representation of a compound is created for its polymorphs, including water or solvent molecules bound to them.

REFUTATION

The prodrugs of the present invention can be obtained, for example, from sulopenem free acid according to known methods, such as the methods given in US3951954; US4234579; US4287181; US4452796; US4342693; US4348264; US4416891; US4457924; and US5013729, which are incorporated herein by reference in their entirety.

USE

The prodrugs of this invention can be used to treat a whole range of in-hospital and out-of-hospital human infections, such as respiratory tract, surgical, central nervous system, gastrointestinal, genitourinary, gynecological, as well as skin and soft tissue, eye, and community-acquired pneumonia infections. The antibacterial activity of these prodrugs can also be usefully applied for preventive purposes. They are most often administered orally. Data on biological activity are given below.

The minimum amount of prodrug that is given is the therapeutically effective amount. The maximum amount of prodrug that is given is the amount that is toxicologically acceptable. In some embodiments, the amount of sulopenem prodrug administered is that amount which will maintain a plasma concentration of sulopenem antibiotic above the MIC90S

(90% minimum inhibitory concentration; eg, about 0.5 ug/mL, or about 1 pg/mL)

of the pathogen that caused the infection for at least about 30% of the interval between doses (eg, at least about 3.5 hours for BID (2x daily) or 2.4 hours for TID (3x daily) dosing). In some embodiments, the blood level is maintained at or above the target level for at least 40% (eg, at least about 4.8 hours for BID or 3.2 hours for TID) of the dosing interval.

Generally, a daily dose of the sulopenem prodrug for adults may be about 500 mgA (milligram equivalents of sulopenem) to about 6 gA, or about 1 gA to about 5 gA. The dosage regimen of the sulopenem prodrug for adults may be about 500 mgA given twice a day at intervals of about 12 hours. This regimen can be carried out for a period of one week to about two weeks. For some infections, it may be necessary or desirable to use doses outside these ranges.

A daily dose of a prodrug of the present invention may typically be administered 1 to 4 times per day, usually in equal doses. In some embodiments, the dosage of the prodrug may be about 500 to about 2500 mg BID or TID; about 800 mg to about 1 g BID; or about 2 g BID or TID in more serious infections. In some embodiments, the dosage may be about 7 to about 25 mg/kg BID; about 17 to about 45 mg/kg BID; or about 17 to about 45 mg/kg TID.

In some embodiments, treatment begins with intravenous administration of sulopenem alone or another antibiotic, followed by oral administration of a prodrug of the invention.

As will be discussed later, the prodrug of Compound 1 was found to maintain human blood levels above 0.5 pg/mL for 3.18 to 4.84 hours after oral administration of 1000 mg (about 730 mg sulopenem equivalents) of the prodrug. In another experiment, the prodrug of Compound 1 was found to produce human blood levels of 1 µg/mL between 4.28 and 5.94 hours after oral administration of 2000 mg (about 1400 mg sulopenem equivalents) of the prodrug.

The prodrug can be used in combination with some other active agents. The use of sulopenem or a prodrug of sulopenem can also be in combination with probenecid or an agent with a similar effect that has an inhibitory effect on renal tubular secretion.

FORMULATION

The present invention encompasses pharmaceutical compositions containing the compound(s) of the prodrugs of the present invention formulated for oral administration with or without one or more excipients and/or with one or more other active ingredients. The prodrug can be in the form of a solvate or a hydrate.

Forms of the present invention for oral administration may be tablets, including chewable tablets, capsules, pills, lozenges, pastilles, powders, syrups, elixirs, solutions and suspensions, and the like. in accordance with standard pharmaceutical practice. The pharmaceutical composition of the present invention may also be administered directly to the gastrointestinal tract of a patient via a nasogastric tube.

In some embodiments, the oral dosage form may contain about 800 to about 2500 mg of the prodrug.

Excipients can be selected based on the intended dosage form. Some examples include polyvinylpyrrolidone, hydroxypropylmethylcellulose, hydroxypropylcellulose, sucrose, gelatin, acacia, tragacanth gum, or corn starch; bulking agents such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate, glycine and starch; disintegrants such as corn starch, potato starch, alginic acid, sodium starch glycolate, croscarmellose sodium and some complex silicates; lubricants such as magnesium stearate, sodium lauryl sulfate and talc; and sweeteners such as sucrose, lactose or saccharin. When the unit dose is in the form of a capsule, it may contain, in addition to the listed substances, a liquid carrier, such as fatty oil. Excipients may also include suspending aids such as xantham gum or hydroxypropylmethylcellulose, glidants such as colloidal silica; diluents or fillers, such as silica; flavors, especially in the case of pediatric oral suspensions and sachets. Stabilizers, such as succinic acid, can also be used. Various other substances may be present, such as tablet coatings, or which modify the physical form of the unit dose. For example, tablets may be coated with shellac, sugar, or both. Modified release dosage forms are also being considered.

The prodrug(s) will be present in the pharmaceutical composition in an amount sufficient to provide the desired amount of therapeutic dose within the described range. The proportional ratio of prodrugs to excipients will naturally depend on factors such as the chemical nature, solubility and stability of the active ingredients, as well as on the given dosage form. Typically, the pharmaceutical compositions of the present invention may contain about 20 to about 95 percent by weight of the prodrug.

BIOLOGICAL ACTIVITY OF SULOPENEM

Sulopenem is active against a wide range of pathogens, including nosocomial pathogens. This includes strong activity against members of the Enterobacteriaceae that express an extended spectrum of β-lactamases that give them resistance to cephalosporins (K. pneumoniae, ESBL+). In addition, many of these isolates are also resistant to fluoroquinolones. Sulopenem is highly active against many clinically relevant anaerobes.

The in vitro activity of sulopenem (source acid (5R,6S)-6-[(1R)-1-hydroxyethyl]-7-oxo-3-[[(1R,3S)-tetrahydro-1-oxido-3-thienyl]thio]-4-thia-1-azabicyclo [3.2.0]hept-2-ene-2-carboxylic acid) was tested against pathogens involved in nosocomial and community-acquired infections, such as summarized in Table 1.

Thus, sulopenem is active against a wide range of pathogens, including nosocomial pathogens that are resistant to cephalosporins and fluoroquinolones. This spectrum supports widespread use in hospitals, where the infectious pathogen has been identified and its susceptibility to sulopenem confirmed. This would include a broad list of respiratory indications and surgical indications where mixed flora are likely to be involved, particularly as part of a multidrug regimen when mixed infections are suspected.

ORAL EFFICACY OF PRODRUGS

The compounds were tuned for oral efficacy in three different in vivo infection models. The bacterial pathogens used to establish each of these infections were selected based on their resistance profiles and ability to cause infection in models relevant to human disease. Isolates 1109 and 6485 Klebsiella pneumoniaes from a recent collection of clinical isolates with extended-spectrum P-lactamase positive (ESBL<+>, from extended-spectrum P-lactamase positive) strains and have increased MICs to ciprofloxacin and ceftazidime, as well as to other β-lactam antibiotics. Both isolates exhibited the ability to cause lethal systemic infection in mice. Streptococcus pneumoniae1095 is a penicillin-tolerant, macrolide-resistant strain that is pathogenic in murine models of systemic and respiratory tract infections. Strain Rd7AH5-3Haemophilus influenzae was derived from laboratory strain Rd; a directed point mutation in PBP3 renders this P-lactamase negative strain penicillin resistant (BLNAR). This strain is able to cause inflammation of the middle ear in the Mongolian guinea pig (Meriones unguiculatus), which is a model of the disease. The results are summarized later in Table 2.

Murine model of acute systemic infection: For this model, CF-1 mice are infected by intraperitoneal injection of a lethal inoculum of either K.pneumoniae1109 or 6485, orS. pneumoniae1095. Four dose groups consisting of eight to ten mice per group were infected and treated with a wide range of dose levels. Mice were given BID therapy at either 30 minutes/four hours post-infection, or one/five hours post-infection. PD50 (the dose at which 50% of infected and treated mice survive) was calculated based on the number of surviving animals on the fourth day after infection.

Murine model of respiratory tract infection: This model was established by intranasal inoculation of a lethal challenge with S . pneumoniae1095, which leads to pneumonia. Four dose groups consisting of eight to ten mice per group were infected and treated with a wide range of dose levels. BID therapy was started eighteen hours after infection and continued for two days. PD50 was calculated based on the number of surviving mice in each dose group on day 10 post-infection.

Guinea pig model of otitis media: To induce otitis media, Mongolian guinea pigs were infected intrabulally by inoculation with BLANAR strain H. influenzae. Four dose groups consisting of five guinea pigs per group were infected and treated to cover a wide range of dose levels. TID therapy was started eighteen hours after infection and continued for two days. On the fourth day after infection, the animals were euthanized, middle ear fluid washes were collected and the number of bacteria found in them was determined. The ED50 was calculated based on bacterial levels, with middle ear fluid lavage samples with less than 100 colony-forming units/mL considered clean.

Data were collected for compounds Compound 1 and 2, for compound A (shown below); and for compound BI, which is a pivaloyl methyl ester (POM-ester) of sulopenem (stereochemistry of (lsolv,3S) oxothiolane) and which is shown below. Compound B2 is a diastereomeric mixture (shown below).

Compound A

Compound BI

Compound B2

CLINICAL PHARMACOKINETICS OF PRODRUG

Clinical pharmacokinetic (PK) data in healthy volunteers for the sulopenem prodrug compounds Compound 1, Compound B2 (data from Foulds et al.), and Compound A are summarized below in Table 3. Compound B2 is a diastereomeric mixture in which the diastereomer is of the (1R,3S) configuration on the oxothiolanyl group of Compound BI (see figure above). There are no clinical data for the prodrug of Compound 2.

For Compound 1 and Compound A, six subjects received increasing doses. Whole blood samples were taken before dosing and plasma obtained 0.5, 1, 2, 3, 4, 6, 8 and 12 hours after dosing. Serum and plasma samples were then quantified for sulopenem concentrations using the prescribed HPLC method. Data for Compound A Tmax are given as medians and ranges.

In total, ten subjects received single doses of Compound B2. See Foulds et al., supra. Blood samples were taken before dosing at 0.08, 0.17, 0.33, 0.5, 1, 1.5, 2, 3, 4, 6 and 8 hours and serum was obtained from them after oral administration of Compound B2 of 500 mg equivalents of the parent sulopenem compound (five subjects) and 1000 mg equivalents of the parent sulopenem compound (five subjects). Foulds et al. also evaluate the PK contributions of the 1R,3S (Compound BI) and 1S,3R diastereomers present in Compound B2.

Sulopenem exposure following oral administration of Compound B2 was expressed as the fraction absorbed relative to intravenous AUCs in the same study (Table 4, Foulds et al.). The absorbed fraction ranged from 38.5 to 33.5% for Compound B2 at doses of 205 to 409 mg sulopenem equivalents. Using the same intravenous data from Table 4 of Foulds et al., an absorbed fraction of 37.1 and 28.0% can be estimated for Compound 1 at doses of 292 and 438 mg sulopenem equivalents, respectively.

Although different dose equivalents have been administered, the tendency for prodrugs is for systemic exposures to produce increases that are less than the increases that would correspond to the doses. The data for compound A at least show that increased lipophilicity of the prodrug does not necessarily translate into improved oral exposure, or oral uptake. Calculations of lipophilicities (ClogP) using the ACD Labs 9.0 program (LogP/DB; www. aclabs. com) gave the following results: Compound 1: -0.29; Compound A: 0.83; Compound BI: -1.0; Compound B2: -1.0. Further evaluation of Compound A revealed its inherent instability in the gastrointestinal tract. Improved gastrointestinal stability, as demonstrated by in vitro use of human intestinal juice on Compound 1, was correlated with a dose-proportional increase in oral uptake.

EVALUATION AND SELECTION OF MEDICINES

Prodrugs are evaluated with the ultimate goal of identifying compounds that exhibit, or are predicted to exhibit, one or more properties: favorable PK, such as high exposure or bioavailability in humans upon oral administration; lack of tendency to deplete carnitine from tissues; and physicochemical properties that favor practical pharmaceutical formulation and use.

Evaluation of Compound A, among others, led to the conclusion that gastrointestinal stability can be predicted to play a significant role in oral bioavailability. New prodrug compounds were evaluated and classified, as described later, with respect to stability in the presence of porcine pancreatic lipase (PPE) and stability in human intestinal juice (HIJ). And the efficiency of conversion to sulopenem in human liver homogenates was considered a significant parameter related to the oral bioavailability of the prodrug. Table 4 summarizes the endpoints for Liver S9, PPE and HIJ. Prodrugs were tested according to the following general procedures.

Conversion efficiency of Liver S9

Prodrugs were evaluated for stability and conversion efficiency in human liver homogenates (fraction S9). Liver S9 was freshly prepared for each analysis from pieces of liver stored at -70 °C. About 5 g of frozen liver tissue was completely homogenized in 15 mL of ice-cold 100 mM potassium phosphate buffer (pH 7.4). The homogenate was then centrifuged at 9,000 g for 20 min at 5 °C to isolate the supernatant fraction S9. Each incubation was performed in a 1:10 dilution of S9 supernatant in 100 mM potassium phosphate buffer (pH 7.4). Reactions (1 mL) were initiated by addition of substrate (final 50 µM) at 37°C. Aliquots (75 pL) were taken at 0, 0.5, 1, 2, 3, 5, 10, and 20 minutes and the reaction was stopped with 150 pL of 80/20 acetonitrile/100 mM ammonium acetate pH 4.5 with internal standard (ampicillin, 5 pg/mL). The samples were centrifuged at 3000 g for 10 minutes and the supernatants transferred to injection vials. First-order degradation of the prodrug was checked by LC/MS/MS as described later. Conversion to sulopenem was expressed as a percentage of molar equivalents (50 pM) in the fortified sample. Compounds that achieved conversion efficiencies of around 75% or more generally went on to further screening.

Stability

In these experiments, the contents of one ku-zyme® HP (USP pancrelipase preparation consisting of: lipase, 8000 USP units, protease 30,000 USP units, and amylase, 30,000 USP units; Schwarz Pharma Inc., Milwaukee, WI) capsule was mixed with 50 mL of 100 mM potassium phosphate pH 7.4 and mixed until homogenous. Each incubation (1 mL) was performed at 37°C and started with the addition of substrate (final 50 pM). Aliquots (100 µL) were taken at 0, 0.5, 1, 2, 3, 5, 10 and 20 minutes after addition of substrate and the reaction was stopped with 200 µL each of 80/20 acetonitrile/100 mM ammonium acetate pH 4.5 with internal standard (ampicillin, 5 pg/mL). The samples were centrifuged at 3000 g for 10 minutes and the supernatants transferred to injection vials. First-order degradation of the prodrug was checked by LC/MS/MS as described later. Conversion to sulopenem was expressed as a percentage of molar equivalents (50 pM) in the fortified sample. Compounds that achieved stable half-lives of about 10 minutes or more went for further screening.

In Table 4, each value represents the mean of two determinations. Where multiple determinations were made for a given compound, data are expressed as mean values with standard deviation. All compounds were tested using the first lot (Lot 1) of co-enzymes.

Compounds 1, A, BI and B2 were also evaluated using a second lot of co-enzyme (Lot 2), for which data are given in parentheses.

In HIJ experiments, HIJ from 4 individuals (1 mL of each) were combined with 1 mL of 600 mM potassium phosphate buffer pH 7.4. Aliquots of 300 pL x 6 buffered human intestinal juice were incubated at 37°C after substrate amplification at concentrations of 300, 100, 30, 10, 3, and 1 pM. Two prodrug compounds can be tested at the same time. Samples of 35 pL were taken at 0, 0.5, 1, 2, 10, and 20 minutes and the reaction was stopped with 70 pL of 80/20 acetonitrile/100 mM ammonium acetate pH 4.5 with an internal standard (ampicillin, 5 pg/mL). The samples were centrifuged at 3000 g for 10 minutes and the supernatants transferred to injection vials. First-order prodrug degradation was checked by LC/MS/MS as described later. The percentage of prodrug that remains as a function of time, for each concentration, was fitted to a first-order decay function to determine the substrate loss rate constant kdep A linear log plot of k^cp as a function of concentration can be fitted by the following equation

The value of lc^ at the infinitesimal concentration of the substrate (where k<iep~ kdep[S]=o) represents the maximum rate of consumption or the characteristic clearance of the system, and kmućkaseje the concentration at which half of the maximum rate (V^) of the system is reached. Expressed in Michaelis-Menten terms, the intrinsic clearance Cljntrepresents the ratio of Vmax/Km, where [S] is significantly less than Km. The substrate units for these tests are Km given in pM and the intrinsic clearance (Cli„t) in mL/min. Generally, compounds with an intrinsic clearance of less than 0.1 mL/min or with a Km that is three times less than its water solubility (to saturate the enzyme action) went to further verification.

Solubility

Equilibrium solubility was determined in 25 mM phosphate buffer (pH 5) at room temperature. Vials containing excess prodrug in phosphate buffer were rotated for about 48 h. After an equilibration period, the samples were pooled, filtered through a 0.45<p>m Gelman Acrodisc Nylon syringe filter, and analyzed for drug concentration using HPLC. HPLC conditions were: column: Cl8, SymmetrvShield RP, Waters, 4.6x150 mm, 3.5 micron; mobile phase A: acetonitrile; mobile phase B: 0.1% TFA in water; flow rate: 1 mL/min; duration of chromatography (Run Time): 30 min; inj. ox: 20 pL; detection: 210 nm; dissolving solution: acetonitrile/water (50:50 v:v). The results are shown in Table 4.

Melting point

Melting points (Ttsu) determined on a MEL-TEMP 3.0 capillary melting point apparatus and are uncorrected.

Quantitative determination of prodrugs

Stopped samples from these in vitro experiments were quantified by LC/MS/MS. Separation was achieved on a Phenomonex Primesphere C18-HC column (5 pm, 30 x 2.0 mm) using a binary gradient of solvent A (95% water/ 5% acetonitrile /0.1% acetic acid) and solvent B (5% water/ 95% acetonitrile / 0.1% acetic acid). The injected volume was 20 pL. The column was equilibrated and the gradient started as 100% A at a flow rate of 1000 pL/min. The gradient was switched to 100% B within 0.4 min and then back to 100% A in 0.9 min. Ampicillin (5 pg/mL) was used as an internal standard. The effluent was analyzed by a mass spectrometer detector (Sciex API 3000) with a turbo ion atomizer, in the positive ion mode, at a cluster breaking potential of 10V, at a temperature of 400 °C and a collision energy of 25V. All prodrugs, , sulopenem, and ampicillin, were determined via MRM transitions of the protonated initial mass to the major ion fragment by collision-induced dissociation spectra. The typical dynamic range of the assay was 10.0 to 10,000 ng/mL.

Depletion of wood

Malecarboxylic acids, such as pivalic acid, which are completely substituted on the alpha carbon atom with the carboxylate, are insufficiently catabolized via p-oxidation. As a result, carnitine is acylated and acyl carnitine accumulates in tissue and the bloodstream, depleting free carnitine concentrations. As such, acids that are fully substituted on the alpha carbon atom have the potential to deplete carnitine reserves in the body. See earlier Brass. This has been demonstrated in short-term therapies with prodrugs containing pivalic acid, when carnitine depletion occurs due to reduced fatty acid oxidation and reduced ketogenesis. See Abrahamsson et al. Biochem. Med. Metab. Biol., 52, 18-21 (1994). Therefore, a prodrug side chain that is rapidly and safely eliminated and does not deplete the body's carnitine reserves would be desirable. Metabolic conversion of certain small carboxylic acids into their glucuronic conjugates is an efficient route of elimination from the body. Valproic acid, for example, has been shown to be largely eliminated by glucuronidation (See Zaccara et al. Clin. Pharmaool., 15, 367-389 (1988)), while pivalic acid in humans is excreted almost entirely as its acylcarnitine conjugate. See Totsuka et al. Antimicrob. Agents and Chemother., 36, 757-761 (1992). It is estimated that subtle structural changes can be translated into significant differences in the metabolic distribution of these carboxylic acids.

Metabolic conversion of certain small carboxylic acids into their glucuronic conjugates is an efficient way of their elimination from the body. Valproic acid, for example, has been shown to be largely eliminated by glucuronidation (See Zaccara et al. Clin. Pharmaool., 15, 367-389 (1988)), while pivalic acid in humans is excreted almost entirely as its acylcarnitine conjugate.

Of interest was the comparison between Compound 1 and Compound BI regarding the tendency, or lack of tendency, of the side chains to deplete plasma carnitine after prodrug metabolism. This was assessed in vivo using an acute carnitine depletion model in Sprague-Dawley rats. To understand the potential impact in vivo, radioactive pivalic acid (side chain of Compound BI) and 2-ethylbutyric acid (side chain of Compound 1) were administered orally at a dose of 200 mg/kg BID for 4 days to two separate groups of animals. Pivalic acid was <14>C labeled at the carbon atom of the carbonyl group (position 1) and had a specific activity of 0.482 pCi/mg. 2-Ethylbuterene was <14>C labeled on the carbon atom adjacent to the carboxyl group (position 2) and had a specific activity of 0.503 pCi/mg. Doses were administered in 100 mM sodium phosphate pH 6.6, with a dose volume of 10 mL/kg. Blood samples were taken at intervals of 24 hours after the start of this study, plasma was obtained from them and tests were performed to determine carnitine levels using LC/MS/MS. A control consisting of oral administration of an equal volume of compound-free buffer was performed as a baseline for comparison. As shown in Figure 1, animals given 200 mg/kg BID pivalic acid showed lower plasma carnitine levels compared to controls. In contrast, animals given the same dose of 2-ethylbutyric acid for 4 days showed statistically insignificant changes in plasma carnitine, indicating that this compound does not cause carnitine depletion.

A second study using a single dose of 200 mg/kg of each of the radioactive compounds (pivalic acid and 2-ethylbuterene) was performed in rats to determine the systemic dose exposure after oral administration. The oral route of administration was chosen because the majority of prodrug hydrolysis is expected to take place in the gut, before entering the systemic circulation. Plasma samples were taken before dosing, and at 0.25, 0.5, 1, 4, 8, and 24 hours after dosing. In the samples, the radioactivity was quantitatively determined in a scintillation counter, and the beats were translated into pg equivalents/mL. As shown in Table 5 and on S1.2, from the moment of absorption, the radioactivity attached to 2-ethylbutyric acid is removed 4.5 times faster than pivalic acid, which reflects the efficient metabolic processing and excretion of the given compound.

Therefore, oral administration of the prodrug of Compound 1 would not be expected to lead to carnitine depletion, whereas administration of Compound BI would.

Other features

In order to obtain suitable formulations and applicability for a pharmaceutical product, it is desirable that in some forms the compound is solid at room temperature and readily forms crystalline solids, as well as being sufficiently stable against degradation.

Discussion

Compound 1 was found to have a suitable combination of properties. In addition to being crystalline, and adequately soluble in water, Compound 1 was completely converted to sulopenem in liver fraction S9 experiments, showed a relatively long PPE half-life, and relatively low intrinsic clearance and saturation of intestinal enzymes in human intestinal juice. Based on these data, Compound 1 was predicted to exhibit favorable clinical PK, which was confirmed by the clinical data described above.

Moreover, as seen from its structure and work with carnitine described above, Compound 1 is not responsible for carnitine. Thus, Compound 1 combines at least good oral bioavailability, no reduced responsibility for carnitine, and favorable physical properties. In contrast, other prodrugs, particularly those with alkyl side chains, are not predicted to have these properties. For example, several Compounds C through AA have a tertiary alpha carbon atom of the carbonyl group of the starting group ester (eg, Compound C). They are predicted to have a potential responsibility for carnitine. Other compounds tested had relatively low PPE stability and/or low S9 conversion, predicting lower GI stability and oral bioavailability. There are other compounds that have not been tested due to difficulties in obtaining samples that can be easily tested. See Table 4.

A prodrug of Compound 2 has also been shown to have favorable properties, including its predicted GI stability and bioavailability, as well as physical properties. See Table 4.

EXAMPLES OF COMPOUNDS

This invention will be further illustrated by the following non-limiting examples. The crystalline sulopenem used in this illustration was obtained according to Primer US5013729.

Example 1: (2-Ethyl-1-oxobutoxy)methyl (5R,6S)-6-[(1R)-1-hydroxyethyl]-7-oxo-3-[[(1R,3S)-tetrahydro-1-oxido-3-thienyl]thio]-4-thia-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylate (Compound 1)

The title compound was obtained according to the following scheme and description:

1st step: 2-ethyl butyric acid (1500 g) is added to a solution of thionyl chloride (1800 g) in dichloromethane (0.75 L) over 1 hour. The mixture was heated to reflux and checked by GC (gas chromatography). After approximately 2 hours, the reaction mixture was concentrated by distillation at atmospheric pressure. It was then cooled to 22 °C, dichloromethane (0.75 L) was added and the mixture was re-concentrated by distillation at atmospheric pressure. Due to the extreme corrosiveness of the reagents used, all released gases were passed through a wet caustic cleaner.

Step 2: Meanwhile, a mixture of zinc chloride (18 g) and paraformaldehyde (480 g) was prepared. Semi-crude, pure acid chloride was added to this mixture over 1 hour at room temperature with mechanical stirring. After a short induction period, a significant exothermic reaction occurred. The temperature of the reaction mixture increased from room temperature (25 °C) to 50 °C in 5 minutes. The addition rate was slowed to control heat generation and the reaction was maintained at 50 °C. After the addition was complete, the reaction mixture was allowed to cool and stirred at room temperature for an additional 18 hours.

n-Heptane (4 L) and 10% aqueous sodium bicarbonate solution (9 L) were then added and the phases were separated. The aqueous phase was extracted with n-heptane (3.4 L). The combined organic phases were filtered and distilled under vacuum to give the crude product. The product was purified by vacuum distillation (10-20 mm Hg) to give 587 g of 2-ethylbutyric acid chloromethyl ester.

Step 3: 2-ethylbutyric acid chloromethyl ester (700 g) was dissolved in acetone (3 L). To this solution was added sodium iodide (1.0 kg). The resulting reaction mixture was heated at reflux until the reaction was complete (2 h as checked by GC). The solution was then cooled to room temperature, when tert-butyl methyl ether (7 L) and 5% aqueous sodium thiosulfate (4 L) were added. The phases were separated and the organic phase was washed with aqueous sodium thiosulfate (4 L), low pyroxene water (4 L) and 10% sodium chloride solution (4 L). The organic layer was dried over magnesium sulfate (350 g), filtered and the resulting cake washed with tert-butyl methyl ether (2 x 0.7 L). The filtrate was evaporated to a small volume {ca. 2 L) and gave the iodomethyl ester of 2-ethylbutyric acid as a solution in tert-butyl methyl ether.

Step 4: A solution of 2-ethylbutyric acid iodomethyl ester in semi-crude tert-butyl methyl ether from step 3 was added to a slurry of sulopenem (750 g) in acetone (5.9 L). N,N-diisopropylethylamine (DIEA) (319 g) in acetone (0.5 L) was added and the mixture was stirred at room temperature until the reaction was complete. Low pyrogen water (6.5 L) and heptane (3.75 L) were added and the phases were separated. The aqueous layer was first extracted with heptane (5 L) and then with ethyl acetate (2x 6 L). The ethyl acetate extracts were combined and washed with 5% aqueous sodium thiosulfate (6 L), low pyrogen water (6 L), and 10% aqueous sodium chloride (6 L). The organic extract was treated with activated carbon (75 g) and magnesium sulfate (150 g) and then filtered. The resulting cake was washed with ethyl acetate (2 x 1 L) and the filtrate evaporated to dryness to give the crude product (0.8 kg). Ethyl acetate (2.4 L) was added and the solution heated (45°C) to achieve dissolution. This hot solution was then filtered and tert-butyl methyl ether (4.7 L) was added. The resulting slurry was granulated for 10 minutes at 40 °C to 50 °C and then slowly cooled to less than 10 °C. The resulting solid was then collected, washed with a mixture of ethyl acetate and tert-butyl methyl ether, 1:2 (4 x 0.5 L) and dried under vacuum to constant weight at a temperature of up to 50°C to give 0.57 kg of the desired product (60% yield).

Step 5: The semi-crude product (0.55 kg) was dissolved in ethyl acetate (1.65 L) at room temperature. The temperature was then adjusted to about 50 °C to achieve dissolution. This hot solution was filtered to remove insoluble impurities and then tert-butyl methyl ether (3.6 L) was added. The resulting solution was slowly cooled to less than 5 °C to initiate crystallization. The solid product was collected and washed with a mixture of ethyl acetate and tert-butyl methyl ether, 1:2 (4 x 150 mL) and dried under vacuum to constant weight at a temperature of up to 50 °C to give 0.48 kg of the desired product (86% yield). It was determined that the crystalline product was not solvated.

1H NMR (DMSO-d6,400 MHz): 5.71 (m, 3H), 5.19 (d, 1H, J = 4.56 Hz), 3.92 (m, 2H), 3.81 (m, 1H), 3.70 (m, 1H), 2.96 (m, 1H), 2.80 (m, 1H), 2.65 (m, 2H), 2.36 (m, 1H), 2.19 (m, 1H), 1.45 (m, 4H), 1.10 (d, 3H, J = 6.22 Hz), 0.78 (t, 6H).

Tt: 105 °C; Mass. Spec.: (M+H)<+>478.

MW: 477.92 g/mol; Molek. form.: C!9H27N 07S3,

Solubility in water (pH 5 phosphate buffer, 25 °C): 1209 pg/mL.

Crystals of Compound 1, obtained as in Step 5, were subjected to powder X-ray diffraction. The samples were analyzed on a Siemens D500 Automated Powder Diffractometer, equipped with a graphite monochromator and a Cu (k=\.54 A) X-ray source operating at 50 kV, 40 mA. A two-theta calibration was performed using NBS mica standards. Sample preparation was performed using a zero background sample plate. The diffraction pattern of these crystals of Compound 1 is shown in SI. 3 and tabulated on SI.5. Example 2: (2-Ethoxy-2-methyl-1-oxopropoxy)methyl (5R,6S)-6-[(1R)-1-hydroxyethyl]-7-oxo-3-[I(1R,3S)-tetrahydro-1-oxido-3-thienyl]thio]-4-thia4-azabicyclo[3.2.0]hept-2-ene-2-carboxylate (Compound 2)

The title compound was obtained according to the following scheme and description:

In steps 1-4, 2-hydroxy isobutyric acid was protected with benzyl bromide, alkylated with ethyl iodide, deprotected, and esterified to give 2-ethoxy-isobutyric acid chloromethyl ester.

In step 5, in a suitable reaction vessel, sodium iodide (23.9 g, 159.45 mmol, 1.6 eq.) was dissolved in acetone (96 mL). Then, 2-ethoxy-isobutyric acid chloromethyl ester (18 g, 99.65 mmol, 1 eq.) was added in an additional amount of acetone (18 mL), and the resulting reaction mixture was heated at reflux, under a nitrogen atmosphere, for about 2 h. The reaction was checked by GC. After the conversion was complete, the reaction mixture was allowed to cool to room temperature with stirring. The reaction mixture was then partitioned between heptane (120 mL) and 10% aqueous sodium thiosulfate (105 mL). The contents of the reaction vessel were stirred for at least 5 minutes and then the phases were allowed to separate. The light organic phase was saved and the heavier aqueous phase was discarded. The organic compounds were washed with another portion of 10% aqueous sodium thiosulfate (105 mL), and, after separation, the heavier phase was again discarded. The organic layer was then washed with 10% aqueous sodium thiosulfate (105 mL). The heavier aqueous phase was discarded, and the organic phase was concentrated under reduced pressure (< 35 °C). This gave 16.26 g of 2-ethoxy-isobutyric acid chloromethyl ester, which was used in subsequent chemical reactions without further purification (check: ~60%).

In step 6, sulopenem (13.92 g, 39.83 mmol, 1 eq.) and acetone (110 mL) were added to a suitable reaction vessel under nitrogen atmosphere. 2-Ethoxy-isobutyric acid iodomethyl ester (16.26 g, 59.9 mmol, 1.5 equiv) was then added at 100% potency in acetone (14 mL) and the suspension was stirred for at least 10 min. °C (exothermic reaction). The reaction mixture was then stirred overnight at room temperature. The reaction mixture was then partitioned between heptane (80 mL), and the contents of the reaction vessel were separated for at least 5 minutes. The heavier phase was washed with additional heptane (80 mL). Discarded.The contents of the reaction vessel was then concentrated under reduced pressure to approximately 50%, with internal temperatures maintained below 35 °C. Ethyl acetate (120 mL) was added and the contents of the reaction vessel were stirred for at least 5 minutes. The phases were allowed to separate and the light organic phase was saved. The heavy aqueous phase was re-extracted with additional ethyl acetate (2 x 120 mL). The combined organic phases were washed with 10% aqueous sodium thiosulfate (120 mL), water (120 mL), and 10% aqueous sodium chloride (120 mL). The organic phase was treated with activated carbon (2.9 g), celite (2.9 g) and magnesium sulfate (MgSO 4 ) (8.2 g) at room temperature and stirred for at least lh. After removal of the solids by filtration, the solution was concentrated under reduced pressure while maintaining the internal temperature below 45 °C (ethyl acetate, b.p. 76.5-77.5 °C).

Step 7: The resulting crude Compound 2 (23 g) in ethyl acetate (100 mL) was heated almost to reflux to completely dissolve the solids, then tert-butyl methyl ether (100 mL) was gradually added, maintaining the internal temperature at 60 °C until reflux. The resulting mixture was slowly stirred at 60 °C until reflux for 5 min and then granulated for at least lh 5-15 °C. The white to off-white product was filtered, washed with methyl t-butyl ether (MTBE) (28 mL) and dried under vacuum at room temperature for at least 18 h. This gave the product (Compound 2) as a white solid (12.57 g, 63.9% yield).

Crystals of this product were subjected to powder X-ray diffraction. The samples were analyzed on a Siemens D500 Automated Powder Diffractometer, equipped with a graphite monochromator and Cu (X=1.54 A). The X-ray source was operated at 50 kV, 40 mA. A two-theta calibration was performed using NBS mica standards. Sample preparation was performed using a zero background sample plate. The diffraction pattern is shown in S1.4 and tabulated in S1.6.

<[>H NMR: (d6-DMSO, 400 MHz): 5.83 (d, IH, J = 5.81 Hz) 5.73 (m, 2H), 5.20 (m, IH), 3.92 (m, 2H), 3.81 (m, IH), 3.70 (m, IH), 3.28 (q, 2H, J = 7.05 Hz), 2.96 (m, IH), 2.80 (m, IH), 2.65 (m, 2H), 2.36 (m, IH), 1.29 (S, 6H), 1.10 (d, 3H, J = 6.63 Hz), 1.00 (t, 3H, J = 6.63 Hz),

Mp: 111-113 °C,

MW: 493.62 g/mol; Molek. formula: Cj^NOgSs,

Solubility in water (pH 5 phosphate buffer, 25 °C): 2900 pg/mL,

Claims (20)

1. Compound of formula: 2. The compound of claim 1, which is (2-ethyl-1-oxobutoxy)methyl (5R,6S)-6-[(1R)-1-hydroxyethyl]-7-oxo-3-[[(1R,3S)-tetrahydro-1-oxido-3-thienyl]thio]-4-thia-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylate 3. The compound of claim 1, which is (2-ethyl-1-oxobutoxy)methyl (5R,6S)-6-[(1R)-1-hydroxyethyl]-7-oxo-3-[[(1R,3S)-tetrahydro-1-oxido-3-thienyl]thio]-4-thia-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylate in crystalline form and has a transmission diffraction pattern The X-ray through the powder is essentially the same as that presented in S1.3 and 5. 4. Compound of formula: 5. The compound of claim 4, which is 2-ethoxy-2-methyl-1-oxopropoxy)methyl (5R,6S)-6-[(1R)-1-hydroxyethyl]-7-oxo-3-[[(1R,3S)-tetrahydro-1-oxido-3-thienyl]thio-4-thia-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylate. 6. The compound of claim 4, which is 2-ethoxy-2-methyl-1-oxopropoxy)methyl (5R,6S)-6-[(1R)-1-hydroxyethyl]-7-oxo-3-[[(1R,3S)-terrahydro-1-oxido-3-thienyl]thio]-4-thia-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylate and has powder X-ray diffraction pattern essentially the same as that presented in S1.4 and 6. 7. A pharmaceutical composition containing the compound of any of claims 1 to 6 formulated for oral administration, with or without one or more excipients and/or one or more other active ingredients. 8. A pharmaceutical composition comprising the compound of any one of claims 1 to 6 and probenecid formulated for oral administration, with or without one or more excipients and/or one or more other active ingredients. 9. A pharmaceutical composition containing about 800 mg to about 2.5 g of the compound of any one of claims 2, 3, 5 or 6, formulated for oral administration, with or without one or more excipients and/or one or more other active ingredients 10. A method for treating bacterial infections comprising orally administering a therapeutically effective amount of the compound of any one of claims 1 to 6 to a person in need thereof. 11. A method for treating bacterial infections comprising orally administering a therapeutically effective amount of the compound of any one of claims 1 to 6 and probenecid to a person in need thereof 12. Use of the compound from any one of claims 1 to 6 for the preparation of a medicament for oral administration in the treatment of bacterial infections in humans in need thereof. 13. A method for treating bacterial infections comprising orally administering a therapeutically effective amount of the compound of any claim 2, 3, 5 or 6 to a person in need thereof. 14. The method of claim 13, wherein the compound is administered orally in an amount of about 500 to about 2500 mg BID or TID. 15. The method of claim 13, wherein the compound is administered orally in an amount of about 800 to about 1000 mg BID. 16. The method of claim 13, wherein the compound is administered orally in an amount of about 2000 mg BID. 17. The method of claim 13, wherein the compound is administered orally in an amount of about 2000 mg TID. 18. The method of claim 13, wherein the compound is administered orally in an amount of about 7 to about 25 mg/kg BID. 19. The method of claim 13, wherein the compound is administered orally in an amount of about 17 to about 45 mg/kg BID. 20. The method of claim 13, wherein the compound is administered orally in an amount of about 17 to about 45 mg/kg TID.