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WO2014195697A1 - Nouveau dérivé de pyrrole - Google Patents

Nouveau dérivé de pyrrole Download PDF

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Publication number
WO2014195697A1
WO2014195697A1 PCT/GB2014/051720 GB2014051720W WO2014195697A1 WO 2014195697 A1 WO2014195697 A1 WO 2014195697A1 GB 2014051720 W GB2014051720 W GB 2014051720W WO 2014195697 A1 WO2014195697 A1 WO 2014195697A1
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WIPO (PCT)
Prior art keywords
compound
treatment
pneumococcal
pneumolysin
pharmaceutical composition
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
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PCT/GB2014/051720
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English (en)
Inventor
Peter William Andrew
Mafalda Pires DAMASO
Mark William DAVIES
Daniel HAMZA
Simon Christopher Hirst
Rana LONNEN
Fritz-Frieder Frickel
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University of Leicester
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University of Leicester
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Publication date
Application filed by University of Leicester filed Critical University of Leicester
Publication of WO2014195697A1 publication Critical patent/WO2014195697A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D207/02—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D207/30—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having two double bonds between ring members or between ring members and non-ring members
    • C07D207/32—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having two double bonds between ring members or between ring members and non-ring members with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to ring carbon atoms
    • C07D207/33—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having two double bonds between ring members or between ring members and non-ring members with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to ring carbon atoms with substituted hydrocarbon radicals, directly attached to ring carbon atoms
    • C07D207/337—Radicals substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/553—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having one nitrogen atom as the only ring hetero atom
    • C07F9/572—Five-membered rings

Definitions

  • the invention relates to a compound which is a prodrug of a cytolysin inhibitor and its use in therapy, including in pharmaceutical combinations, especially in the treatment of bacterial, e.g. pneumococcal, infections.
  • Streptococcus pneumoniae (pneumococcus) is one of the most potent human pathogens, affecting over 10 million people worldwide, of all age groups, in particular young children, the elderly and the immunocompromised. It is a leading causative agent of serious, often fatal diseases, such as pneumonia, bacteraemia and meningitis. It is also responsible of other less serious, but nevertheless debilitating diseases such as otitis media and keratitis.
  • pneumococcal products the most important of which is the pneumococcal toxin pneumolysin.
  • This toxin is a major player in pneumococcal virulence and is the primary direct and indirect cause of toxaemia.
  • Pneumolysin belongs to the family of cholesterol dependent cytolysins (CDCs), which bind to cholesterol containing membranes and generate large pores that have lethal and sub-lethal effects on the affected cells.
  • CDCs cholesterol dependent cytolysins
  • the toxin pneumolysin is cytoplasmic and is mainly released from the pneumococcus after its lysis. Consequently, under the effect of lytic antibiotics, a large bolus of toxin is released, compounding the toxaemia.
  • lytic antibiotics a large bolus of toxin is released, compounding the toxaemia.
  • This toxaemia constitutes a substantial unmet medical need that is internationally recognised.
  • pneumolysin constitutes a potential therapeutic target to develop new
  • pneumococcal keratitis and the therapeutic benefit obtained following its inhibition.
  • cholesterol is not considered as a therapeutic agent for the treatment of pneumococcal diseases and has not been clinically used in patients.
  • Another pneumolysin inhibitor, Allicin, a component in garlic extract has been previously found to inhibit the haemolytic activity of pneumolysin in vitro [Toxicon (2011) 57 540-545].
  • This compound is a cysteine inhibitor that irreversibly binds to the reactive thiol group of the toxin. Compounds exhibiting such a property are unfavourable as drug candidates because of their potential unspecific binding to other cysteine-containing proteins in the body.
  • cytolysins such as pneumolysin
  • N-phenyl substituted pyrrole derivatives as cytolysin inhibitors, that specifically inhibit the direct toxic effect of pneumolysin and other cholesterol dependent cytolysins that are pivotal in the virulence of their respective hosts, including the compound 2,5-bis(dimethylcarbamoyl)-1-(4- methoxyphenyl)-1 H-pyrrole-3,4-diyl bis(4-((phosphonooxy)-methyl)benzoate). These compounds have no structural similarity to Allicin and do not bind covalently to the reactive thiol groups of the toxins.
  • the present invention provides a novel prodrug of a N-phenyl substituted pyrrole cytolysin inhibitor which demonstrates particularly advantageous properties e.g. in terms of solubility and physicochemical properties making it particularly suitable for parenteral delivery.
  • the parent active compound of the present invention also prevents stimulation of host-derived toxic effects induced by pneumolysin and, it may be assumed, other cholesterol dependent cytolysins.
  • the compound may be used as a single agent or as an adjunct to antibiotics, to prevent or attenuate pneumolysin-induced toxicity and its anti-host effects seen during infections caused e.g. by S. pneumoniae. Summary of the invention
  • the present invention provides a compound of formula (I) for use as a medicament.
  • Figure 1 shows the in vitro inhibition of pneumolysin-induced LDH release by the compound UL1-005 using A549 human lung epithelial cells.
  • Figure 2 shows the effect of the compound UL1-005 in inhibiting pneumolysin from damaging the ciliary function of ependymal cells in an ex vivo meningitis efficacy assay.
  • Figure 3 shows the experimental design for an in vivo mouse pneumonia model efficacy assay using the compound UL5-001.
  • Figure 4 shows the survival of infected control mice and treated groups administered with 36 mg/kg of the compound UL5-001 in an in vivo mouse pneumonia model efficacy assay.
  • the compound of formula (I), herein after referred to as “the compound of the invention”, is a prodrug derivative of the compound of formula (II):
  • the compound of the invention will break down after administration to a subject to form an active compound of formula (II) (sometimes referred to herein as "parent active compound”)
  • solvates of the compound of formula (I) include hydrates.
  • the invention also extends to all polymorphic forms of the compound of formula (I).
  • the invention also extends to isotopically-labelled compounds of formula (I) in which one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number most commonly found in nature.
  • isotopes that can be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, and phosphorus, such as 2 H, 3 H, 11 C, 14 C, 15 N, 32 P and 33 P.
  • Isotopically labelled compounds of formula (I) may be prepared by carrying out the synthetic methods described below and substituting an isotopically labelled reagent or intermediate for a non-isotopically labelled reagent or intermediate.
  • the compound of the invention may be prepared as described in the Examples.
  • a basic sodium salt such as NaHC0 3 , Na 2 C0 3 or NaOH, particularly NaHC0 3 .
  • the compound of formula (III) may be produced by a process comprising reaction of a compound of formula (IV):
  • R represents a protecting group, such as te/f-butyl, in the presence of an oxidising agent followed by deprotection to convert the groups R to H.
  • a suitable oxidising agent is an organic peroxide such as m-CPBA and the deprotection step may be conducted in the presence of TFA or HCI in an organic solvent such as DCM, THF or dioxan.
  • R represents H or a protecting group such as Boc, PMB, TMS, TBDMS, THP or trityl, especially Boc.
  • Protecting groups may be required to protect chemically sensitive groups during the synthesis of the compound of the invention, to ensure that the process is efficient. Thus if desired or necessary, intermediate compounds may be protected by the use of conventional protecting groups. Protecting groups and means for their removal are described in "Protective Groups in Organic Synthesis", by Theodora W. Greene and Peter G.M. Wuts, published by John Wiley & Sons Inc; 4 th Rev Ed., 2006, ISBN-10: 0471697540.
  • the compound of the invention is useful for treatment of bacterial infections caused by bacteria producing pore-forming toxins, such as cholesterol dependent cytolysins.
  • the compound of the invention is useful for the treatment of toxaemia associated with bacterial infections.
  • the compound of the invention will generally be administered in the form of a pharmaceutical composition.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a compound of formula (I) optionally in combination with one or more pharmaceutically acceptable diluents or carriers.
  • Diluents and carriers may include those suitable for parenteral, oral, topical, mucosal and rectal administration.
  • compositions may be prepared e.g. for parenteral, subcutaneous, intramuscular, intravenous, intra-articular or peri-articular administration, particularly in the form of liquid solutions or suspensions; for oral administration, particularly in the form of tablets or capsules; for topical e.g. intravitreal, pulmonary or intranasal administration, particularly in the form of eye drops, powders, nasal drops or aerosols and transdermal administration; for mucosal administration e.g. to buccal, sublingual or vaginal mucosa, and for rectal
  • administration e.g. in the form of a suppository.
  • compositions may conveniently be administered in unit dosage form and may be prepared by any of the methods well-known in the pharmaceutical art, for example as described in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, PA., (1985).
  • Formulations for parenteral administration may contain as excipients sterile water or saline, alkylene glycols such as propylene glycol, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, hydrogenated naphthalenes and the like.
  • Formulations for parenteral administration may be provided in solid form, such as a lyophilised composition, the lyophilised composition may be re-constituted, preferably just before administration.
  • Re- constitution may involve dissolving the lyophilised composition in water or some other pharmaceutically acceptable solvent, for example physiological saline, an aqueous solution of a pharmaceutically acceptable alcohol, e.g. ethanol, propylene glycol, a polyethylene glycol, e.g. polyethylene glycol 300, and the like, or some other sterile injectable.
  • a pharmaceutically acceptable solvent for example physiological saline, an aqueous solution of a pharmaceutically acceptable alcohol, e.g. ethanol, propylene glycol, a polyethylene glycol, e.g. polyethylene glycol 300, and the like, or some other sterile injectable.
  • Formulations for nasal administration may be solid and may contain excipients, for example, lactose or dextran, or may be aqueous or oily solutions for use in the form of nasal drops or metered spray.
  • excipients include sugars, calcium stearate, magnesium stearate, pregelatinated starch, and the like.
  • compositions suitable for oral administration may comprise one or more physiologically compatible carriers and/or excipients and may be in solid or liquid form.
  • Tablets and capsules may be prepared with binding agents, for example, syrup, acacia, gelatin, sorbitol, tragacanth, or poly-vinylpyrollidone; fillers, such as lactose, sucrose, corn starch, calcium phosphate, sorbitol, or glycine; lubricants, such as magnesium stearate, talc, polyethylene glycol, or silica; and surfactants, such as sodium lauryl sulfate.
  • binding agents for example, syrup, acacia, gelatin, sorbitol, tragacanth, or poly-vinylpyrollidone
  • fillers such as lactose, sucrose, corn starch, calcium phosphate, sorbitol, or glycine
  • lubricants such as magnesium stearate, talc, polyethylene glycol
  • Liquid compositions may contain conventional additives such as suspending agents, for example sorbitol syrup, methyl cellulose, sugar syrup, gelatin, carboxymethyl-cellulose, or edible fats; emulsifying agents such as lecithin, or acacia; vegetable oils such as almond oil, coconut oil, cod liver oil, or peanut oil; preservatives such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT).
  • suspending agents for example sorbitol syrup, methyl cellulose, sugar syrup, gelatin, carboxymethyl-cellulose, or edible fats
  • emulsifying agents such as lecithin, or acacia
  • vegetable oils such as almond oil, coconut oil, cod liver oil, or peanut oil
  • preservatives such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT).
  • BHA butylated hydroxyanisole
  • BHT butylated hydroxytoluen
  • Solid oral dosage forms include tablets, two-piece hard shell capsules and soft elastic gelatin (SEG) capsules.
  • SEG soft elastic gelatin
  • a dry shell formulation typically comprises of about 40% to 60% concentration of gelatin, about a 20% to 30% concentration of plasticizer (such as glycerin, sorbitol or propylene glycol) and about a 30% to 40% concentration of water. Other materials such as preservatives, dyes, opacifiers and flavours also may be present.
  • the liquid fill material comprises a solid drug that has been dissolved, solubilized or dispersed (with suspending agents such as beeswax, hydrogenated castor oil or polyethylene glycol 4000) or a liquid drug in vehicles or combinations of vehicles such as mineral oil, vegetable oils, triglycerides, glycols, polyols and surface-active agents.
  • compositions of the invention may optionally include one or more anti-oxidants (e.g. ascorbic acid or metabisulfate and salts thereof).
  • anti-oxidants e.g. ascorbic acid or metabisulfate and salts thereof.
  • compositions according to the invention include the following:
  • a pharmaceutical composition for parenteral e.g. intravenous, or oral administration in unit dose form.
  • the compound of the invention is an inhibitor of the cholesterol-dependent cytolysin, pneumolysin, produced by the bacterium Streptococcus pneumoniae. It also inhibits
  • Streptolysin O produced by Group A Streptococci and Perfringolysin O (PFO) produced by Clostridium perfringens. It is also expected to inhibit other members of the closely related cholesterol-dependent cytolysins, examples of which include, but are not limited to, Listeriolysin O (LLO) produced by Listeria monocytogenes, Anthrolysin O (ALO) produced by Bacillus anthracis and Suilysin (SLY) produced by Streptococcus suis.
  • LLO Listeriolysin O
  • ALO Anthrolysin O
  • SLY Suilysin
  • the compound of the invention is useful for the treatment of bacterial infections, e.g.
  • pneumococcal infections including the associated toxaemia where the pneumolysin toxin has been demonstrated to play a pivotal role in the diseases produced.
  • diseases include, but are not limited to, pneumococcal pneumonia, pneumococcal meningitis, pneumococcal septicaemia/bacteraemia, pneumococcal keratitis and pneumococcal otitis media.
  • the compound of the invention is also useful for the treatment of pneumococcal infections associated with other conditions.
  • Such conditions include (without limitation) cystic fibrosis and chronic obstructive pulmonary disease (COPD).
  • COPD chronic obstructive pulmonary disease
  • S pneumoniae has been isolated from patients with COPD and is believed to be an exacerbatory factor in this disease.
  • the compound of the invention is useful for the treatment of infections caused by group A Streptococci (GAS), including but not limited to, invasive group A Streptococcal diseases, where the toxin Streptolysin O (SLO) has been demonstrated to play a crucial role in the pathogenesis of systemic GAS diseases.
  • GAS group A Streptococci
  • SLO toxin Streptolysin O
  • the compound of the invention is useful for the treatment of infections caused by Clostridium perfringens including, but not limited to, gas gangrene, characterized by myonecrosis, septic shock and death, where the toxin Perfringolysin O has been demonstrated to be a major virulence factor in the pathogenesis of this disease.
  • the compound of the invention is useful for the treatment of infections caused by Bacillus anthracis, where the cholesterol dependent cytolysin Anthrolysin O (ALO) plays an essential role in gastrointestinal (Gl) anthrax, and contributes to the pathogenesis of inhalational anthrax.
  • ALO cholesterol dependent cytolysin Anthrolysin O
  • the compound of the invention is useful for the treatment of other diseases caused by Gram positive bacteria, producing cholesterol-dependent cytolysins, examples of which include, but are not limited to: Porcine meningitis, septicaemia/bacteraemia and septic shock caused by Streptococcus suis which produces a cholesterol dependent cytolysin, Suilysin, involved in the pathogenesis of diseases by S. suis.
  • the compound of the invention may well also be useful for the inhibition of other bacterial pore- forming toxins, such as the RTX family of toxins, which are essential in the virulence of their host.
  • examples include, but are not limited to, pneumonia and septicaemia/bacteraemia caused by Staphylococcus aureus, which produces the pore-forming toxin staphylococcal a-hemolysis and peritonitis caused by pathogenic Escherichia coli which produces the pore forming toxin a- hemolysin.
  • the compound of the invention for use in the treatment of bacterial infections caused by bacteria producing pore-forming toxins, wherein the bacterial infection is caused by
  • Streptococcus spp. e.g. Streptococcus pneumoniae, Group A Streptococci or Streptococcus suis
  • Clostridium spp. e.g. Clostridium perfringens
  • Listeria spp. e.g. Listeria monocytogenes
  • Bacillus spp. e.g. Bacillus anthracis
  • the compound of the invention for use in the treatment of pneumococcal pneumonia, pneumococcal meningitis, pneumococcal septicaemia/bacteraemia, pneumococcal keratitis or pneumococcal otitis media;
  • the compound of the invention may be used to treat either humans or animals, such as domestic animals or livestock, e.g. pigs, cows, sheep, horses etc, and references to
  • compositions should be interpreted to cover compositions suitable for either human or animal use.
  • the present invention provides a compound of formula (I) for use in the treatment of the above mentioned conditions.
  • the present invention provides a compound of formula (I) for the
  • the present invention provides a method of treatment of the above mentioned conditions which comprises administering to a subject in need thereof an effective amount of a compound of formula (I) or a pharmaceutical composition thereof.
  • treatment is intended to embrace prophylaxis as well as therapeutic treatment.
  • the compound of the invention may be used either alone or in combination with further therapeutically active ingredients.
  • compound of the invention may be administered in combination, simultaneously, sequentially or separately, with further therapeutically active ingredients either together in the same formulation or in separate formulations and either via the same route or via a different route of administration.
  • the compound of the invention may thus be administered in combination with one or more other active ingredients suitable for treating the above mentioned conditions.
  • possible combinations for treatment include combinations with antimicrobial agents, e.g. antibiotic agents, including natural, synthetic and semisynthetic antimicrobial agents.
  • antibiotic agents include ⁇ -lactams including, but not limited to, penicillin, benzylpenicillin, amoxicillin and all generations thereof; ⁇ -lactams in combination with ⁇ -lactamase inhibitors including, but not limited to, clavulanic acid and sulbactam; cephalosporins including, but not limited to, cefuroxime, cefotaxime and ceftriaxone; fluoroquinolones including, but not limited to, levofloxacin and moxifloxacin; tetracyclines including, but not limited to, doxycycline; macrolides including, but not limited to, erythromycin and clarithromycin; lipopeptide antibiotics including, but not limited to, daptomycin;
  • aminoglycosides including, but not limited to, kanamycin and gentamicin; glycopeptide antibiotics, including but not limited to, vancomycin; lincosamides including, but not limited to, clindamycin and lincomycin; rifamycins including, but not limited to, rifampicin; and
  • Further combinations include combinations with immunomodulatory agents, such as antiinflammatory agents.
  • Immunomodulatory agents can include for example, agents which act on the immune system, directly or indirectly, by stimulating or suppressing a cellular activity of a cell in the immune system, for example, T-cells, B-cells, macrophages, or antigen presenting cells, or by acting upon components outside the immune system which, in turn, stimulate, suppress, or modulate the immune system, for example, hormones, receptor agonists or antagonists and
  • immunomodulatory agents can include immunosuppressants or immunostimulants.
  • Anti-inflammatory agents include, for example, agents which treat inflammatory responses, tissue reaction to injury, agents which treat the immune, vascular or lymphatic systems or combinations thereof. Examples of anti-inflammatory and
  • immunomodulatory agents include, but are not limited to, interferon derivatives such as betaseron, ⁇ -interferon, prostane derivatives such as iloprost and cicaprost, corticosteroids such as prednisolone, methylprednisolone, dexamethasone and fluticasone, COX2 inhibitors, immunsuppressive agents such as cyclosporine A, FK-506, methoxsalene, thalidomide, sulfasalazine, azathioprine and methotrexate, lipoxygenase inhibitors, leukotriene antagonists, peptide derivatives such as ACTH and analogs, soluble TNF (tumor necrosis factor) -receptors, TNF-antibodies, soluble receptors of interleukines, other cytokines and T-cell-proteins, antibodies against receptors of interleukins, other cytokines and T-cell-proteins.
  • NSAID's non-steroidal anti-inflammatory drugs
  • NSAID's include sodium cromoglycate, nedocromil sodium, phosphodiesterase (PDE) inhibitors e.g. theophylline, PDE4 inhibitors or mixed PDE3/PDE4 inhibitors, leukotriene antagonists, inhibitors of leukotriene synthesis such as montelukast, iNOS inhibitors, tryptase and elastase inhibitors, beta-2 integrin antagonists and adenosine receptor agonists or antagonists such as adenosine 2a agonists, cytokine antagonists e.
  • chemokine antagonists such as CCR3 antagonists, or inhibitors of cytokine synthesis, and 5-lipoxygenase inhibitors.
  • CCR3 antagonists CCR3 antagonists
  • 5-lipoxygenase inhibitors CCR3 antagonists
  • an aspect of the invention provides a compound of formula (I) in combination with one or more further active ingredients, for example one or more of the active ingredients described above.
  • compositions comprising a compound of formula (I) optionally in combination with one or more pharmaceutically acceptable adjuvants, diluents or carriers and comprising one or more other therapeutically active ingredients.
  • each of components (A) and (B) is formulated in admixture with a pharmaceutically- acceptable adjuvant, diluent or carrier.
  • the combination product may be either a single (combination) pharmaceutical formulation or a kit-of-parts.
  • this aspect of the invention encompasses a pharmaceutical formulation including a compound of the present invention and another therapeutic agent, in admixture with a pharmaceutically acceptable adjuvant, diluent or carrier (which formulation is hereinafter referred to as a "combined preparation").
  • kit of parts comprising components:
  • a pharmaceutical formulation including a compound of formula (I) in admixture with a pharmaceutically acceptable adjuvant, diluent or carrier;
  • a pharmaceutical formulation including another therapeutic agent, in admixture with a pharmaceutically-acceptable adjuvant, diluent or carrier;
  • components (i) and (ii) are each provided in a form that is suitable for administration in conjunction with the other.
  • Component (i) of the kit of parts is thus component (A) above in admixture with a
  • component (ii) is component
  • the other therapeutic agent i.e. component (B) above
  • the combination product (either a combined preparation or kit-of-parts) of this aspect of the invention may be used in the treatment or prevention of any of the conditions mentioned above.
  • the compound of formula (I) may also be provided for use, e.g. with instructions for use, in combination with one or more further active ingredients.
  • Hydrogenations were performed either on a Thales H-cube flow reactor or with a suspension of the catalyst under a balloon of hydrogen. Column chromatography was performed on pre-packed silica (230-400 mesh, 40-63 ⁇ ) cartridges.
  • PBS solutions for solubility and stability studies were prepared by dissolving 1 OxoidTM tablet (obtained from Thermo Scientific) in deionised water (100 ml_).
  • Stability studies were carried out by dissolving 1-2 mg of compound in DMSO (1 ml_) followed by addition of 0.4 ml_ of the resulting solution to stirred PBS solution (9.6 ml_) at 37.5°C. A sample (ca. 0.5 ml_) was immediately taken for HPLC analysis. Further samples were then taken for analysis at various timepoints thereafter. Half-lives were determined from the decrease in concentration of compound with respect to time.
  • Analytical HPLC was carried out using an Agilent Zorbax Extend C18, Rapid Resolution HT 1.8 ⁇ column eluting with a 5-95% gradient of either 0.1 % formic acid in MeCN in 0.1 % aqueous formic acid or a 5-95% gradient of MeCN in 50 mM aqueous ammonium acetate.
  • UV spectra of the eluted peaks were measured using either a diode array or variable wavelength detector on an Agilent 1100 system.
  • Analytical LCMS was carried out using an Agilent Zorbax Extend C18, Rapid Resolution HT 1.8 ⁇ column eluting with a 5-95% gradient of either 0.1 % formic acid in MeCN in 0.1 % aqueous formic acid or a 5-95% gradient of MeCN in 50 mM aqueous ammonium acetate.
  • UV and mass spectra of the eluted peaks were measured using a variable wavelength detector on either an Agilent 1100 with or an Agilent Infinity 1260 LC with 6120 quadrupole mass spectrometer with positive and negative ion electrospray.
  • NMR spectra were recorded using a Bruker Avance III 400 MHz instrument, using either residual non-deuterated solvent or tetra-methylsilane as reference.
  • Example A 3,4-Dihydroxy-1-(4-methoxyphenyl)-/V 2 ,/ ⁇ / 2 ,/ ⁇ / 5 ,/ ⁇ / 5 -tetramethyl-1 /-/-pyrrole-2,5- dicarboxamide (UL1 -005)
  • reaction mixture was stirred at 60°C for 16h and then partitioned between 2 M HCI (aq.) (500 mL), and EtOAc (300 mL), the aqueous phase was extracted with EtOAc (300 mL) and the combined organics were washed successively with 2M HCI (aq.) (2 x 300 mL), water (500 mL), and brine (500 mL), dried (MgS0 4 ), filtered and solvents removed in vacuo to give diethyl 2,2'-((4- methoxyphenyl)azanediyl)diacetate (1) (180 g, 100 %) as a purple oil: m/z 296 (M+H) + (ES + ).
  • Diethyl oxalate (83.0 mL, 0.610 mol) was added dropwise to a stirred solution of diethyl 2,2'-((4- methoxyphenyl)azanediyl)diacetate (1) (180 g, 0.610 mol) in NaOEt (21 % by wt in EtOH) (506 mL, 1.30 mol), the mixture was stirred at 100°C for 1 h. The reaction was quenched with acetic acid (210 mL, 3.70 mol) and the resulting suspension was poured into iced water (1 L), the resulting off-white solid collected by vacuum filtration.
  • Example B Sodium ((((2,5-bis(dimethylcarbamoyl)-1-(4-methoxyphenyl)-1 H-pyrrole-3,4- diyl)bis(oxy))bis(carbonyl))bis(4, 1-phenylene))bis(methylene) bis(hydrogen phosphate) (UL5- 30 001)
  • methyl 4-(hydroxymethyl)benzoate (5.00 g, 30.1 mmol) and di-tert-butyl diethylphosphoramidite (12.56 mL, 45.1 mmol) in THF (150 mL) was added 5-methyl-1 H- tetrazole (2.53 g, 30.1 mmol) and the reaction was stirred at RT. After 4h, the reaction mixture was cooled to -78°C, and 3-chlorobenzoperoxoic acid (12.1 g, 54.2 mmol) was added.
  • a suspension of 2,5-bis(dimethylcarbamoyl)-1-(4-methoxyphenyl)-1 H-pyrrole-3,4-diyl bis(4- ((phosphonooxy)methyl)benzoate) (UL1 -124) (1.44 g, 1.85 mmol) in acetonitrile (50 mL) was added 0.1 M NaHC0 3(aq.) (37.0 mL, 3.70 mmol).
  • Example C Alternative potential synthesis of 3,4-dihydroxy-1-(4-methoxyphenyl)-/ ⁇ / 2 ,A/ 2 ,A/ 5 ,A/ 5 - tetramethyl-1 /-/-pyrrole-2,5-dicarboxamide (UL1 -005)
  • Example D Alternative potential synthesis of sodium ((((2,5-bis(dimethylcarbamoyl)-1-(4- methoxyphenyl)-1 H-pyrrole-3,4-diyl)bis(oxy))bis(carbonyl))bis(4, 1-phenylene))bis(methylene) bis(hydrogen phosphate) (UL5-001 )
  • Test compound solutions (typically at 5 mM in DMSO) were diluted 1 : 1 in 100% DMSO. The compounds were then two-fold serially diluted in 100% DMSO across 1 1 wells of 96-well round- bottomed microtitre plate. PBS was then added to all the wells to achieve a 1 :10 dilution of the compound in PBS. Pneumolysin was then added at a concentration equal to its LD100. Plates were then incubated at 37°C for 30-40 min. After the incubation period, an equal volume of 4% (v/v) sheep erythrocyte suspension was added to each well and the plates incubated again at 37°C, for at least 30 min.
  • Controls with only erythrocytes in PBS (control for no lysis) or erythrocytes plus pneumolysin (control for lysis) were prepared following the same procedure. Following the incubation with the erythrocytes, the Absorbance at 595 nm of each well was measured and the data used to determine the IC 50 for each test compound. The IC 50 values were determined using non-linear regression curve fitting. For that, the Log of the
  • concentrations of the test compound was plotted against the percentage inhibition, estimated from the A 595 values, followed by fitting a Hill Slope to the data.
  • This assay is principally relevant for the determination of the inhibitory activity of the parent active compound UL1-005.
  • the inhibitory activity is expected to be absent in vitro, as the prodrug requires the presence of plasma enzymes to hydrolyse the prodrug moiety and allow the formation of the parent active compound.
  • blood is a component of the assay and is used to assess the inhibition of haemolysis induced by pneumolysin.
  • this assay demonstrates the in vitro activity of the parent active compound UL1-005 and indicates that the prodrug converts to the parent active compound in the presence of blood. This conversion to the parent active compound is further demonstrated in Section F.
  • IC 50 values of examples shown in Table 1 are as follows: Parent active compound UL1-005: IC 50 0.2 ⁇ ; prodrug UL1-124: IC 50 2.6 ⁇ and prodrug UL5-001 : IC 50 12.3 ⁇ .
  • LDH lactate dehydrogenase
  • This assay can provide two main pieces of information on (1) Activity, to demonstrate the inhibition of LDH release from cells exposed to pneumolysin in the presence of inhibitory compounds versus the LDH release from cells exposed to pneumolysin alone, (2) Compound toxicity, the assay format was designed so it allows, in the control wells, the testing of the LDH release from cells exposed to the compound only.
  • Human lung epithelial cells (A549) were seeded in flat-bottomed 96-well tissue culture plates and grown in RPMI 1640 medium supplemented with Glutamine, at 37°C, 5% C0 2 , for 24h. Before use, the cells were washed with PBS. Test compound dilutions were incubated with pneumolysin as described in Section A, then transferred to wells containing the human lung epithelial cells and the plates were incubated at 37°C, 5% C0 2 , for 30 min.
  • Negative controls called low control (PBS only) to measure the natural release of LDH from the cells in culture
  • positive controls (1 % (v/v) Triton-X in PBS) to measure the maximum release of LDH from the cells
  • Pneumolysin solution only to measure pneumolysin-induced LDH release
  • Test compound solution to assess the toxicity of the compound alone. After incubation, the supernatant was transferred to the wells of round-bottomed 96-well microtitre plates containing a double volume of lactate dehydrogenase assay mixture (TOX7, Sigma) prepared according to manufacturer's instructions.
  • TOX7 lactate dehydrogenase assay mixture
  • UL1-005 inhibits the damaging activity of pneumolysin on human lung epithelial cells in culture. UL1-005 did not exhibit cytotoxic effects on the human lung epithelial cells at 150 times the therapeutic IC 50 value.
  • the ependymal ciliated cells line the cerebral ventricles of the brain and the central canal of the spinal cord and are covered with cilia responsible for the circulation of the cerebrospinal fluid (CSF) around the central nervous system.
  • CSF cerebrospinal fluid
  • This layer acts as a selective brain barrier to and from the cerebrospinal fluid and plays a role in controlling the CSF volume.
  • a rat ex vivo model of meningitis was used. This model is based on culturing and differentiating ciliated ependymal cells from neonate rat brains, which recreate the in vivo situation, where cells lining the brain ventricles, are exposed to S. pneumoniae and its toxic products.
  • Ependymal cell cultures were prepared by the method previously described [Microb. Pathog. (1999) 27 303-309]. Tissue culture trays were coated with bovine fibronectin and incubated at
  • the growth medium was minimum essential medium (MEM) with added penicillin (100 lU/mL), streptomycin (100 ⁇ g/mL), fungizone (2.5 ⁇ g/mL), BSA (5 ⁇ g/mL), insulin (5 ⁇ g/ml), transferrin (10 ⁇ g/mL) and selenium (5 ⁇ g/mL).
  • MEM minimum essential medium
  • penicillin 100 lU/mL
  • streptomycin 100 ⁇ g/mL
  • fungizone 2.5 ⁇ g/mL
  • BSA 5 ⁇ g/mL
  • insulin 5 ⁇ g/ml
  • transferrin 10 ⁇ g/mL
  • selenium 5 ⁇ g/mL
  • the remaining brain areas were mechanically dissociated in 4 ml_ of growth medium.
  • the dissociated tissue from one or two brains was added to the wells of the tissue culture trays (500 ⁇ /well), each containing 2.5 ml_ of growth medium. The cells then were incubated at 37°C in 5% (v/v) C0 2 . The medium was replaced after three days and thereafter the ependymal cells were fed every two days with 2 ml_ of fresh growth medium supplemented with thrombin.
  • the cells were fully ciliated and ready for experiments.
  • the growth medium was replaced with 1 ml_ of medium MEM containing 25 mM HEPES, pH 7.4.
  • the tissue culture trays were placed inside a
  • thermostatically controlled incubation chamber surrounding the stage of an inverted light
  • CBF ciliary beat frequency
  • the parameter measured was the ciliary beating frequency (CBF).
  • CBF ciliary beating frequency
  • Pneumolysin added to ciliated cells in culture induces a severe or total loss of ciliary beating.
  • UL1-005 the parent 40 active compound of prodrug UL5-001 , inhibited this damaging effect induced by pneumolysin on the ciliary function of ependymal cells in culture ( Figure 2).
  • Each time point represents the normalised mean ⁇ SD of ciliary beating frequency (CBF) measurements of ten individual cilia from each well, in three independent experiments.
  • Control 1 assay medium only: the symbol (-I-) represents measurements of the CBF in the 45 assay medium which was used as a reference for the normal cilia beating. No damaging effect on the CBF was seen throughout the recording.
  • Control 2 pneumolysin only: The symbol ( ⁇ ) represents measurements of the CBF in the wells where pneumolysin was added. A substantial drop in the CBF to 0% of the original frequency was observed within 5 min. of exposure to the cytotoxin.
  • UL1-005 inhibits the damaging effect that pneumolysin induces on ependymal ciliated cells in culture. This predicts that when the prodrug UL5-001 is converted in vivo to the parent active compound UL1-005, the latter will prevent pneumolysin from causing damage in vivo.
  • Prodrug UL5-001 shows improved solubility over the parent active compound UL1-005 and the prodrug UL1-124.
  • UL5-001 provides a readily soluble formulation that could be reconstituted at the bed side in safe saline solutions, at high concentrations and at a pH compatible with intravenous administration.
  • Solubility studies were carried out by charging a vial with 5-10 mg of compound followed by the addition of PBS solution or 0.9% saline to achieve a concentration of 100 mg/mL. If solubility was not observed the solution was diluted to concentrations of 50 mg/mL, 25 mg/mL and 4 mg/mL consecutively until complete solubility was observed.
  • mice in the treatment group and in the control group 2 received the test compound intravenously, while excipient alone was administered to control group 1.
  • the progress of the signs of disease was assessed every 6h based on the scheme of Morton and Griffiths [Veterinary Record. (1985) 111 , 431-436]. Animals were killed if they became 2+ lethargic and the time was recorded. The survival rates of control and test groups were compared with a log-rank test. Table 3 Scoring scheme of the disease signs
  • mice were lightly anaesthetised with 2.5% (v/v) isoflurane over 1.6-1.8 L 0 2 /min.
  • mice were placed inside an incubator at 37°C, for 10 min, to dilate their veins. Each mouse was then individually placed inside a restrainer, leaving the tail of the animal exposed. The tail was disinfected with antimicrobial wipes. The treatment with the prodrug UL5-001 was administered intravenously every 6h using a 0.5 ml insulin syringe inserted carefully into one of the tail lateral veins. A dose of 36 mg/kg prepared in PBS was given. Doses were prepared freshly and administered intravenously to the animals.
  • Viable counting was performed by the method of Miles and Misra [J. Hyg. (1938) 38 732-749). 20 ⁇ _ of the sample were serially diluted in 180 ⁇ _ PBS in round-bottomed 96-wells microtitre plates, up to a dilution of 10 6 . Blood agar plates were divided into six sectors and 60 ⁇ _ of each dilution plated onto an individual sector. The plates were incubated in C0 2 gas jars overnight at 37°C. The following day, colonies were counted in the sector where 30-300 colonies were visible. The concentration of colony forming units (CFU) per millilitre was determined by using the following equation: Number of colonies in sec tor
  • UL5-001 was administered intravenously every 6h to mice infected with S. pneumoniae and the survival was compared against a control group of infected mice, which had not received the compound (only the excipient).
  • the survival curves of the control (solid line) and treatment (dotted line) groups obtained with this experiment are presented in Figure 4. A significant increase in the survival of the treatment group was seen, with 40% of mice surviving at the endpoint of the experiment at 54h post-infection (p ⁇ 0.05).
  • the prodrug derivative was incubated with mouse, rat or human plasma at 37°C at 5 time points over a 2h period. The samples were then analysed by LC- MS/MS to obtain the amount of parent active compound appearing and prodrug derivative remaining over time.
  • Prodrug derivative were assessed in the mouse, rat or human plasma stability assay at a concentration of 10 ⁇ .
  • Test compounds were diluted in DMSO to a final stock concentration of 10 mM.
  • the stocks prepared were further diluted in DMSO to a concentration of 400 ⁇ and 5 ⁇ _ were added to 195 ⁇ _ of mouse, rat or human plasma (pH 7.4) and then incubated at 37°C.
  • the final concentration of DMSO in the plate was 2.5% (v/v). Reactions were terminated at 0, 15, 30, 60 and 120 min after incubation by adding 400 ⁇ _ of acetonitrile containing 0.55 ⁇ metoprolol and 1 % (v/v) formic acid.
  • the plate was then centrifuged at 3000 rpm, for 45 min, at 4°C. 80 ⁇ _ of supernatant were transferred into a conical bottom 96 well glass coated plate. 40 ⁇ _ of water were added prior to analysis for prodrug derivative and active species by LC-MS/MS. This assay was performed by a contract research organisation, Cyprotex Discovery Limited, UK, at the request of the inventors at Leicester.
  • the parent active compound was quantified using a 6 point calibration curve prepared in deactivated mouse, rat or human plasma.
  • the percentage of prodrug compound remaining at each time point relative to 0 min sample was calculated from LC-MS/MS peak area ratios (compound peak area/internal standard peak area). This percentage was then used to determine the concentration of the prodrug compound at each time point in reference to the starting concentration (10 ⁇ ) at time 0 min.

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Abstract

L'invention concerne, entre autres, de nouveaux dérivés de pyrrole à substitution N-phényle et leur utilisation en thérapie, en particulier dans le traitement d'infections bactériennes (par exemple à pneumocoques).
PCT/GB2014/051720 2013-06-04 2014-06-04 Nouveau dérivé de pyrrole Ceased WO2014195697A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US12384777B2 (en) 2019-04-24 2025-08-12 Tay Therapeutics Limited Compounds comprising N-methyl-2-pyridone, and pharmaceutically acceptable salts

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WO2003057212A1 (fr) * 2001-12-26 2003-07-17 Genelabs Technologies, Inc. Derives de polyamide possedant une activite antibacterienne, antifongique ou antitumorale
WO2013033240A1 (fr) * 2011-08-29 2013-03-07 Ptc Therapeutics, Inc. Composés antibactériens et procédés destinés à leur utilisation
EP2602248A1 (fr) * 2011-12-05 2013-06-12 University Of Leicester Nouveaux composés pyrroliques

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Publication number Priority date Publication date Assignee Title
WO2003057212A1 (fr) * 2001-12-26 2003-07-17 Genelabs Technologies, Inc. Derives de polyamide possedant une activite antibacterienne, antifongique ou antitumorale
WO2013033240A1 (fr) * 2011-08-29 2013-03-07 Ptc Therapeutics, Inc. Composés antibactériens et procédés destinés à leur utilisation
EP2602248A1 (fr) * 2011-12-05 2013-06-12 University Of Leicester Nouveaux composés pyrroliques

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US12384777B2 (en) 2019-04-24 2025-08-12 Tay Therapeutics Limited Compounds comprising N-methyl-2-pyridone, and pharmaceutically acceptable salts

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