WO2014195697A1 - Novel pyrrole derivative - Google Patents

Abstract

There is provided inter alia a novel N-phenyl substituted pyrrole derivative and its use in therapy, especially in the treatment of bacterial (e.g. pneumococcal) infections.

Description

NOVEL PYRROLE DERIVATIVE

Field of the invention 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.

Background of the invention

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.

Even after decades of using antibiotics and steroids as adjunctive to antibiotics the mortality and morbidity from pneumococcal diseases remains very high in the developed world and alarmingly high in the developing world. Nearly 20% of hospitalised patients still die despite antibiotic killing of the pneumococcus, while many survivors of pneumococcal meningitis suffer severe neurological handicaps, including cognitive impairment, vision and hearing loss, hence imposing huge distress on patients and their families and a very significant cost to healthcare systems. Today, infection with pneumococcus remains a major global public health problem that is widely recognised by leaders in the field and by health organisations, including the WHO.

One of the leading factors for this consistently high mortality and morbidity that is not addressed by the current standard therapy, is the toxaemia resulting from the release of toxic

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. In the bacterium, 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. Thus, even if treatment with antibiotics is successful in clearing the bacteria from the patients, the subsequent release of the toxin is detrimental and can be fatal or cause long-term handicaps. This toxaemia constitutes a substantial unmet medical need that is internationally recognised. Currently, corticosteroids, principally dexamethasone, are used as an adjunctive to antibiotic therapy for pneumococcal meningitis. However, even when dexamethasone is used, significant mortality and morbidity are seen and the widespread use of dexamathasone is still debated due to its non-specific effect, limited clinical impact and in some cases its detrimental effect in increasing neuronal apoptosis in meningitis [Lancet (2002) 360 211-218]. Therefore, the present state of the art is not adequate for the efficient treatment of invasive pneumococcal diseases. There is considerable evidence substantiating the validity of pneumolysin as a therapeutic target. In the laboratories of the inventors it has been demonstrated that, using a mouse pneumonia model, a mutated strain of S. pneumoniae (PLN-A) that does not produce pneumolysin is no longer lethal, causes substantially less bacteraemia and exhibits a significant reduction in the severity of pulmonary inflammation. Other evidence obtained in a rat meningitis model, has shown that infection with the pneumolysin-negative mutant was markedly less severe than with wild-type pneumococci, with no observed damage to the ciliated epithelium of the brain and no apoptosis of the cells surrounding the epithelium [J. Infect, (2007) 55 394-399]. In pneumococcal meningitis in guinea pigs, wild-type pneumococci induced severe cochlear damage and hearing loss, while infection with PLN-A left the organ of Corti intact [Infect.

Immun. (1997) 65 4411-4418]. An ex vivo model using cultured ciliated brain epithelial cells, enabled recreation of the in vivo situation, where cells lining the brain ventricles are exposed to S. pneumoniae. Both intact and antibiotic-killed wild-type pneumococci induced damage to the epithelial cells in culture and significantly impaired ciliary beating; effects not seen with PLN-A [Infect. Immun. (2000) 68 1557-1562]. This damaging effect of antibiotic-lysed pneumococci on the cultured ependymal cells is clearly caused by the toxin pneumolysin released from the antibiotic-lysed bacteria, as this damage was abolished in the presence of anti-pneumolysin antibodies [Infect. Immun. (2004) 72 6694-6698]. This finding supports the strategy that antibiotic-induced toxaemia is prevented by combination with anti-pneumolysin agents.

Evidence for the significant involvement of pneumolysin in pneumococcal infections and the substantial improvement of the disease prognosis in the absence of pneumolsyin, has led to the conclusion that pneumolysin constitutes a potential therapeutic target to develop new

treatments for pneumococcal diseases. Previous research has shown the ability of cholesterol to inhibit pneumolysin [Biochem. J. (1974) 140 95-98], however, this inhibition is merely due to the fact that cholesterol is a natural cellular receptor of pneumolysin that is required for the pore formation in the target cell membrane. The topical application of cholesterol on the cornea of rabbits demonstrated a positive therapeutic effect in pneumococcal keratitis [Invest. Ophtalmol. Vis. Sci. (2007) 48 2661-2666]. This indicates the involvement of pneumolysin in

pneumococcal keratitis and the therapeutic benefit obtained following its inhibition. However, 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. There remains a need to provide inhibitors of cytolysins, such as pneumolysin, which are suitable for use in the treatment of bacterial infections.

International Patent Application PCT/GB2012/053022, published after the priority date of the present application and herein incorporated by reference in its entirety, discloses 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. Thus 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

According to the invention, there is provided a compound of formula (I):

(I) or a pharmaceutically acceptable solvate thereof. In a further aspect, the present invention provides a compound of formula (I) for use as a medicament.

Brief description of the figures 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.

Detailed description of the invention

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):

(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")

VIVO.

Examples of 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. Examples of isotopes that can be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, and phosphorus, such as ²H, ³H, ¹¹C, ¹⁴C, ¹⁵N, ³²P and ³³P. 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.

Thus according to a further aspect of the invention there is provided a process for the production of the compound of formula (I) which comprises reacting a compound of formula (III):

with a basic sodium salt such as NaHC0₃, Na₂C0₃ or NaOH, particularly NaHC0₃.

The compound of formula (III) may be produced by a process comprising reaction of a compound of formula (IV):

(IV) with a compound of formula (V):

P(NEt₂)(OR)₂

(V) where 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.

Any novel intermediates, such as those defined above, may be of use in the synthesis of compounds of formula (I) and are therefore also included within the scope of the invention.

Thus according to a further aspect of the invention there is provided a compound of formula (IVa):

(IVa) wherein 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.

As indicated above the compound of the invention is useful for treatment of bacterial infections caused by bacteria producing pore-forming toxins, such as cholesterol dependent cytolysins.

In particular the compound of the invention is useful for the treatment of toxaemia associated with bacterial infections.

For such use the compound of the invention will generally be administered in the form of a pharmaceutical composition.

Further, the present invention provides 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.

As mentioned above, such 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.

The 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.

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. For buccal administration typical 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. 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). Liquid compositions may be encapsulated in, for example, gelatin to provide a unit dosage form.

Solid oral dosage forms include tablets, two-piece hard shell capsules and soft elastic gelatin (SEG) capsules.

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.

Pharmaceutical compositions of the invention may optionally include one or more anti-oxidants (e.g. ascorbic acid or metabisulfate and salts thereof).

Particular pharmaceutical compositions according to the invention which may be mentioned include the following:

- A pharmaceutical composition for parenteral, e.g. intravenous, administration. - A pharmaceutical composition for oral administration.

- A pharmaceutical composition for parenteral, e.g. intravenous, or oral administration in unit dose form.

- A pharmaceutical composition for parenteral, e.g. intravenous, administration in solid form for reconstitution with a liquid prior to administration.

- A pharmaceutical composition for parenteral, e.g. intravenous, administration in liquid form e.g. a solution.

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 (SLO) 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.

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. Such 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). For example, 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.

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.

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. Encephalitis, enteritis, meningitis, septicaemia/bacteraemia and pneumonia caused by Listeria monocytogenes where the cholesterol dependent cytolysin, listeriolosin O (LLO), plays an important role in the pathogensis of the above diseases.

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.

Thus the invention provides:

-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) or Bacillus spp. (e.g. Bacillus anthracis);

-The compound of the invention for the treatment of bacterial infection which is caused by Streptococcus pneumonia;

-The compound of the invention for use in the treatment of pneumococcal pneumonia, pneumococcal meningitis, pneumococcal septicaemia/bacteraemia, pneumococcal keratitis or pneumococcal otitis media; and

-The compound of the invention for the treatment of conditions selected from gas gangrene, gastrointestinal anthrax, inhalational anthrax, porcine meningitis, encephalitis, septicaemia/bacteraemia and pneumonia which are caused by bacteria other than

pneumococcus.

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

pharmaceutical compositions should be interpreted to cover compositions suitable for either human or animal use.

Thus, in a further aspect, the present invention provides a compound of formula (I) for use in the treatment of the above mentioned conditions. In a further aspect, the present invention provides a compound of formula (I) for the

manufacture of a medicament for the treatment of the above mentioned conditions.

In a further aspect, 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. The word "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. Thus 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. For example, possible combinations for treatment include combinations with antimicrobial agents, e.g. antibiotic agents, including natural, synthetic and semisynthetic antimicrobial agents. Examples of antibiotic agents include β-lactams including, but not limited to, penicillin, benzylpenicillin, amoxicillin and all generations thereof; β-lactams in combination with β-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

chloramphenicol.

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

neurotransmitters, other 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, β-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. Further antiinflammatory agents include non-steroidal anti-inflammatory drugs (NSAID's). Examples of 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.g. chemokine antagonists, such as CCR3 antagonists, or inhibitors of cytokine synthesis, and 5-lipoxygenase inhibitors. Thus 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.

Another aspect of the invention provides a pharmaceutical composition 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.

Similarly, another aspect of the invention provides a combination product comprising:

(A) a compound of formula (I); and

(B) another therapeutic agent,

wherein each of components (A) and (B) is formulated in admixture with a pharmaceutically- acceptable adjuvant, diluent or carrier.

In this aspect of the invention, the combination product may be either a single (combination) pharmaceutical formulation or a kit-of-parts.

Thus, 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").

It also encompasses a kit of parts comprising components:

(i) a pharmaceutical formulation including a compound of formula (I) in admixture with a pharmaceutically acceptable adjuvant, diluent or carrier; and

(ii) a pharmaceutical formulation including another therapeutic agent, in admixture with a pharmaceutically-acceptable adjuvant, diluent or carrier;

which 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

pharmaceutically acceptable adjuvant, diluent or carrier. Similarly, component (ii) is component

(B) above in admixture with a pharmaceutically acceptable adjuvant, diluent or carrier.

The other therapeutic agent (i.e. component (B) above) may be, for example, any of the agents e.g. antimicrobial or immunomodulatory agents mentioned 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.

Thus a further aspect of the invention provides a compound of formula (I) for use in

combination with one or more further active ingredients, for example one or more of the active ingredients described above.

The compound of formula (I) for use in this aspect of the invention may be used in the treatment or prevention of any of the conditions mentioned above. The invention will now be described by reference to the following examples which are for illustrative purposes and are not to be construed as a limitation of the scope of the present invention.

Examples

Abbreviations

AcOH glacial acetic acid

aq. aqueous

Bn benzyl

br broad

Boc te/f-butoxycarbonyl

m-CPBA mefa-chloroperoxybenzoic acid

COPD chronic obstructive pulmonary disease

d doublet

DCM dichloromethane

DIPEA A/,A/-diisopropylethylamine

DMAP 4-dimethylaminopyridine

DMF A/,A/-dimethylformamide

DMSO dimethylsulfoxide

EDC 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide

EtOAc ethyl acetate

h hour(s)

HATU A/,A/, V', V'-tetramethyl-0-(7-azabenzotriazol-1-yl)uronium PF₆ HPLC high performance liquid chromatography

m multiplet

MeCN acetonitrile

MeOH methanol

min minute(s)

NMR nuclear magnetic resonance

PBS phosphate buffered saline

PMB paramethoxybenzyl

quin. quintet

RT room temperature

s singlet

sat. saturated SAX solid supported strong cation exchange

sept, septet

sext. sextet

triplet

TBDMS tert-butyldimethylsilyl

TFAA trifluoroacetic acid anhydride

THF tetrahydrofuran

THP tetrahydropyranyl

TMS trimethylsilyl

UV ultra violet

General Procedures

All starting materials and solvents were obtained from commercial sources or prepared according to literature conditions.

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 μΜ) cartridges.

PBS solutions for solubility and stability studies were prepared by dissolving 1 Oxoid™ 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 Methods

Analytical HPLC was carried out using an Agilent Zorbax Extend C18, Rapid Resolution HT 1.8 μηι 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. Alternatively, a Waters Xselect CSH C18 3.5 μηι eluting with a 5-95% gradient of 0.1 % formic acid in MeCN in 0.1 % aqueous formic acid. 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 μηι 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. Alternatively, a Waters Xselect CSH C18 3.5 μηι eluting with a 5-95% gradient of 0.1 % formic acid in MeCN in 0.1 % aqueous formic acid. 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.

¹ H NMR Spectroscopy:

NMR spectra were recorded using a Bruker Avance III 400 MHz instrument, using either residual non-deuterated solvent or tetra-methylsilane as reference.

Chemical Synthesis: The compound of formula (I) and comparator compounds (UL1-005 and UL1-124) were prepared using the following general methods:

Example A: 3,4-Dihydroxy-1-(4-methoxyphenyl)-/V²,/\/²,/\/⁵,/\/⁵-tetramethyl-1 /-/-pyrrole-2,5- dicarboxamide (UL1 -005)

3 4

UL1-005

Step (i): Diethyl 2,2'-((4-methoxyphenyl)azanediyl)diacetate (1 ) Ethyl 2-bromoacetate (146 mL, 1.30 mol) was added dropwise to a stirred solution of 4- methoxyaniline (75.0 g, 0.610 mol) and DIPEA (265 mL, 1.50 mol) in MeCN (300 mL). The 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 (MgS0₄), 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⁺). ¹ H NMR (400 MHz, CDCI₃) δ 6.82-6.78 (m, 2H), 6.64-6.59 (m, 2H), 4.19 (q, J = 7.1 Hz, 4H), 4.10 (s, 4H), 3.74 (s, 3H), 1.27 (t, J =7.1 Hz, 6H).

Step (ii): Diethyl 3,4-dihydroxy-1-(4-methoxyphenyl)-1 /-/-pyrrole-2,5-dicarboxylate (2)

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. The crude product was recrystallised from hot EtOH (3.50 L) to give diethyl 3,4-dihydroxy-1-(4-methoxyphenyl)-1 /-/-pyrrole-2,5- dicarboxylate (2) (152 g, 71 %) as a white solid: m/z 350 (M+H)⁺ (ES⁺); 348 (M-H)^" (ES^"). ¹ H NMR (400 MHz, DMSO-d₆) δ 8.64 (s, 2H), 7.13-7.01 (m, 2H), 6.92-6.81 (m, 2H), 3.99 (q, J = 7.1 Hz, 4H), 3.78 (s, 3H), 0.99 (t, J =7.1 Hz, 6H).

Step (iii): Diethyl 3,4-bis(benzyloxy)-1-(4-methoxyphenyl)-1 /-/-pyrrole-2,5-dicarboxylate (3)

Benzyl bromide (42.6 mL, 358 mmol) was added dropwise to a stirred suspension of 3,4- dihydroxy-1-(4-methoxyphenyl)-1 /-/-pyrrole-2,5-dicarboxylate (2) (50.0 g, 143 mmol) and K₂C0₃ (49.5 g, 358 mmol) in DMF (1 L), the reaction mixture was stirred at 60°C for 4h. After cooling to RT the reaction mixture was poured into ether (500 mL) and washed with brine (3 x 250 mL), dried (MgS0₄), filtered and concentrated in vacuo to afford a bright yellow solid. The crude product was triturated with isohexane to give diethyl 3,4-bis(benzyloxy)-1-(4-methoxyphenyl)- 1 H-pyrrole-2,5-dicarboxylate (3) (64.8 g, 85 %) as a white solid: m/z 530 (M+H)⁺ (ES⁺). ¹ H NMR (400 MHz, DMSO-de) δ 7.48-7.29 (m, 10H), 7.17-7.09 (m, 2H), 6.95-6.87 (m, 2H), 5.09 (s, 4H), 3.99 (q, J = 7.1 Hz, 4H), 3.80 (s, 3H), 0.99 (t, J =7.1 Hz, 6H).

Step (iv): 3,4-Bis(benzyloxy)-1-(4-methoxyphenyl)-1 /-/-pyrrole-2,5-dicarboxylic acid (4)

A mixture of diethyl 3,4-bis(benzyloxy)-1-(4-methoxyphenyl)-1 /-/-pyrrole-2,5-dicarboxylate (3) (2.80 g, 5.29 mmol), 2M NaOH (aq.) (26.4 mL, 52.9 mmol), in ethanol (12 mL) and THF (20 mL) was stirred at 60°C for 72h. After cooling to RT, the mixture was acidified with 6M HCI_(aq.) and the resulting precipitate was collected by filtration, washed with water (5 mL), and Et₂0 (5.00 mL) to afford 4-bis(benzyloxy)-1-(4-methoxyphenyl)-1 /-/-pyrrole-2,5-dicarboxylic acid (4) (1.94 g, 67 %) as an off-white solid: m/z 474 (M+H)⁺ (ES⁺); 472 (M-H)^" (ES^"). ¹ H NMR (400 MHz, DMSO- cf_e) δ 12.61 (s, 2H), 7.46-7.40 (m, 4H), 7.39-7.29 (m, 6H), 7.16-7.07 (m, 2H), 6.92-6.84 (m, 2H), 5.07 (s, 4H), 3.78 (s, 3H). Step (v): 3,4-Bis(benzyloxy)-A/²,A/²,A/^A/⁵-tetramethyl-1-(4-methoxyphenyl)-1 H-pyrrole-^ dicarboxamide (5)

To a solution of 3,4-bis(benzyloxy)-1-(4-methoxyphenyl)-1 H-pyrrole-2,5-dicarboxylic acid (4) 5 (1 10 mg, 0.232 mmol), and dimethylamine hydrochloride (56.8 mg, 0.697 mmol) in DMF (2 mL) at 0°C was added DIPEA (243 μί, 1.39 mmol) and then immediately HATU (265 mg, 0.697 mmol) and the mixture stirred for 30 min. The reaction mixture was quenched with water and then partitioned between saturated aqueous ammonium chloride (20 mL) and ether (30 mL). The ether layer was taken and washed with further saturated ammonium chloride_(aq.) (15 mL) 10 saturated aqueous sodium bicarbonate (2 x 15 mL), brine (15 mL) and then dried (MgS0₄), filtered and concentrated in vacuo to afford 3,4-bis(benzyloxy)-1-(4-methoxyphenyl)- A/²,A/²,A/⁵,A/⁵-tetramethyl-1 H-pyrrole-2,5-dicarboxamide (5) (124 mg, 100 %). m/z 528.3 (M+H)⁺ (ES⁺). ¹ H NMR (400 MHz, CDCI₃) δ 7.37-7.25 (m, 10H), 7.11-7.06 (m, 2H), 6.82-6.77 (m, 2H), 5.06 (s, 4H), 3.76 (s, 3H), 2.79 (s, 6H), 2.63 (s, 6H).

15

Step (vi): 3,4-Dihydroxy-1-(4-methoxyphenyl)-/V² N², A/⁵,A/⁵-tetramethyl-1 H-pyrrole-2,5- dicarboxamide (UL1 -005)

3,4-Bis(benzyloxy)-1-(4-methoxyphenyl)-/V² Λ/² A/⁵,A/⁵-tetramethyl-1 H-pyrrole-2,5-dicarboxamide 20 (5) (5.00 g, 9.48 mmol) was dissolved in methanol (150 mL) and the solution was hydrogenated in the H-Cube (10% Pd/C, 70x4 mm, Full hydrogen, 40°C, 1 mL/min) then concentrated under vacuum. The resulting residue was recrystallised from isopropanol (100 mL) and dried in a desiccator to afford 3,4-dihydroxy-1-(4-methoxyphenyl)-/V² N², A/⁵,A/⁵-tetramethyl-1 /-/-pyrrole-2,5- dicarboxamide (UL1 -005) (2.70 g, 78 %) as a white crystalline solid: m/z 348.1 (M+H)⁺ (ES⁺); 25 346.0 (M-H)- (ES^"). ¹ H NMR (400 MHz, DMSO-d₆) δ 8.38 (s, 2H), 6.94-6.89 (m, 2H), 6.86-6.81 (m, 2H), 3.73 (s, 3H), 2.88 (s, 12H).

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)

UL5-001

Step (i): Methyl 4-(((di-tert-butoxyphosphoryl)oxy)methyl)benzoate (6) To a stirred solution of 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. The mixture was allowed to warm to RT and stirred for 16h. The reaction mixture was quenched with saturated sodium bisulphite solution (100 mL) and extracted with ethyl acetate (2 x 200 mL). The combined organic layers were washed with saturated sodium bicarbonate_(aq.) (500 mL), dried (MgS0₄), filtered and concentrated in vacuo. The crude product was purified by silica gel chromatography (330 g, 0-100% ethyl acetate in hexane) to afford methyl 4-(((di-tert- butoxyphosphoryl)oxy)methyl)benzoate (6) (9.32 g, 86 %) as a white solid: m/z 381.0 (M+Na)⁺ (ES⁺). ¹ H NMR (400 MHz, DMSO-d₆) δ 7.99-7.97 (m, 1 H), 7.54-7.51 (m, 1 H), 5.01 (d, J = 8.3 Hz, 2H), 3.85 (s, 3H), 1.44-1.37 (m, 18H).

Step (ii): 4-(((Di-tert-butoxyphosphoryl)oxy)methyl)benzoic acid (7)

5 To a stirred solution of methyl 4-(((di-tert-butoxyphosphoryl)oxy)methyl)benzoate (6) (1.63 g, 4.55 mmol) in THF (20 mL) was added sodium hydroxide (364 mg, 9.10 mmol) in water (4 mL) followed by ethanol (4 mL). The reaction mixture was then stirred at RT for 16h. The organic solvents were then removed in vacuo and the resulting aqueous solution was acidifid to ca. pH 6 by dropwise addition of 1 M phosphoric acid. The solution was was then extracted with DCM 10 (2 x 10.0 mL) and the combined organic layers were dried (MgS0₄), filtered and concentrated in vacuo. The resulting residue was triturated with ethyl acetate (10 mL) and isolated by filtration to afford 4-(((di-tert-butoxyphosphoryl)oxy)methyl)benzoic acid (7) (1.01 g, 61 %) as a white solid: m/z 367.0 (M+Na)⁺ (ES⁺); 343.0 (M-H)^" (ES^"). ¹ H NMR (400 MHz, DMSO-d₆) δ 12.99 (s, 1 H), 7.98-7.94 (m, 2H), 7.51-7.47 (m, 2H), 5.00 (d, J = 8.2 Hz, 2H), 1.41 (s, 18H).

15

Step (iii): 2,5-Bis(dimethylcarbamoyl)-1-(4-methoxyphenyl)-1 /-/-pyrrole-3,4-diyl bis(4-(((di-te/f- butoxyphosphoryl)oxy)methyl)benzoate) (8)

EDC (1.99 mL, 1 1.3 mmol) was added to a solution of 3,4-dihydroxy-1-(4-methoxyphenyl)- 20 A/²,A/²,A/⁵,A/⁵-tetramethyl-1 H-pyrrole-2,5-dicarboxamide (UL1 -005) (1.30 g, 3.75 mmol), 4-(((di- te/f-butoxyphosphoryl)oxy)methyl)benzoic acid (7) (3.36 g, 9.75 mmol) and DMAP (183 mg, 1.50 mmol) in THF (60 mL) and the reaction mixture was allowed to stir at RT for 16h. The mixture was poured into brine (100 mL) and extracted with EtOAc (2 x 200 mL), the combined organics were dried (MgS0₄), filtered and concentrated in vacuo. The crude residue was 25 purified by silica gel chromatography (120 g, EtOAc) to afford 2,5-bis(dimethylcarbamoyl)-1-(4- methoxyphenyl)-1 /-/-pyrrole-3,4-diyl bis(4-(((di-tert-butoxyphosphoryl)oxy)methyl)benzoate) (8) (2.19 g, 59 %) as a pale yellow solid: m/z 1001 (M+H)⁺ (ES⁺). ¹ H NMR (400 MHz, DMSO-d₆) δ 8.00 (d, J =8.3 Hz, 4H), 7.53 (d, J = 8.3 Hz 4H), 7.21-7.18 (m, 2H), 7.01-6.99 (m, 2H), 5.00 (d, J = 8.0 Hz, 4H), 3.80 (s, 3H), 2.89 (s, 6H), 2.74 (s, 6H), 1.38 (s, 36H).

30

Step (iv) 2,5-Bis(dimethylcarbamoyl)-1-(4-methoxyphenyl)-1 /-/-pyrrole-3,4-diyl bis(4- ((phosphonooxy)methyl)benzoate) (UL1 -124)

TFA (5.00 mL, 65.3 mmol) was added dropwise to a solution of 2,5-bis(dimethylcarbamoyl)-1- 35 (4-methoxyphenyl)-1 /-/-pyrrole-3,4-diyl bis(4-(((di-fe/f-butoxyphosphoryl)oxy)methyl)benzoate) (8) (2.19 g, 2.19 mmol) in DCM (100 mL). The mixture was left to stand for 1 h, and then concentrated in vacuo to afford 2,5-bis(dimethylcarbamoyl)-1-(4-methoxyphenyl)-1 /-/-pyrrole- 3,4-diyl bis(4-((phosphonooxy)methyl)benzoate) (UL1 -124) (1.56 g, 91 %) as a white solid: m/z 776 (M+H)⁺ (ES⁺); 774 (M-H)^" (ES^"). ¹ H NMR (400 MHz, DMSO-d₆) δ 8.00-7.98 (m, 4H), 7.54- 40 7.52 (m, 4H), 7.20-7.18 (m, 2H), 7.00-6.98 (m, 2H), 4.98-4.96 (d, 4H), 3.79 (s, 3H), 2.89 (s, 6H), 2.74 (s, 6H).

Step (v): 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- 45 001 ) To 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). The mixture was allowed to stand for 10 min and then the acetonitrile was removed in vacuo. The resulting aqueous solution was freeze- dried to afford 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(hydrogenphosphate) (UL5- 001) (1.49 g, 98 %) as a white solid: m/z 776.0 (M-2Na+3H)⁺ (ES⁺); 773.8 (M-2Na+H)^" (ES^"). ¹ H NMR (400 MHz, D₂0) δ 8.05 (d, J = 8.4 Hz, 4H), 7.52 (d, J = 8.4 Hz, 4H), 7.30-7.25 (m, 2H), 7.10-7.05 (m, 2H), 4.97 (d, J = 7.2 Hz, 4H), 3.86 (s, 3H), 3.05 (s, 6H), 2.84 (s, 6H).

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 )

R = f-Bu, 8

T

UL5-001

The following compounds in Table 1 were prepared using the methods provided above: Table 1

* compounds are prodrug derivatives of UL1-005

Biological Testing

There is provided below a summary of the biological assays performed with all the compound of the invention, and further assays performed with the compounds UL1-005 and UL1-024 as comparators.

A. PRIMARY IN VITRO ASSAY: INHIBITION OF THE HAEMOLYTIC ACTIVITY OF

PNEUMOLYSIN Rationale

The basis of this assay is that when pneumolysin is added to red blood cells, it induces their lysis and leads to the release of haemoglobin. In the presence of an inhibitory compound, pneumolysin-induced lysis is abolished, the red blood cells pellet at the bottom of the microtitre plate well and the supernatant is clear. However, if the compound is not inhibitory, the red blood cells are lysed and haemoglobin is released into the supernatant.

Experimental procedure

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 IC₅₀ for each test compound. The IC₅₀ 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 A₅₉₅ values, followed by fitting a Hill Slope to the data.

Results

This assay is principally relevant for the determination of the inhibitory activity of the parent active compound UL1-005. Generally, in the case of the parent prodrug, 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. However, in our primary in vitro assay, blood is a component of the assay and is used to assess the inhibition of haemolysis induced by pneumolysin. Therefore, we observe some inhibitory activity in the presence of the prodrug UL5-001 , due a certain degree of enzymatic cleavage of the prodrug moiety, occurring during the 40 min incubation in blood, which leads to the release of the parent active compound UL1-005. In summary, 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.

IC₅₀ values of examples shown in Table 1 are as follows: Parent active compound UL1-005: IC₅₀ 0.2 μΜ; prodrug UL1-124: IC₅₀ 2.6 μΜ and prodrug UL5-001 : IC₅₀ 12.3 μΜ.

B. SECONDARY IN VITRO ASSAY: INHIBITION OF PNEUMOLYSIN-INDUCED LACTATE DEHYDROGENASE RELEASE

Rationale

Pneumolysin induces the release of lactate dehydrogenase (LDH) from human monocytes and lung epithelial cells: a phenomenon that is indicative of plasma membrane damage or rupture [Infect. Immun. (2002) 70 1017-1022]. The LDH assay was applied to demonstrate the ability of the disclosed compounds to inhibit the cytotoxic effect of pneumolysin on human lung epithelial cells in culture. The use of 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. Experimental procedure

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% C0₂, 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% C0₂, for 30 min. The following controls were included on the plate (1) Negative controls, called low control (PBS only) to measure the natural release of LDH from the cells in culture, (2) positive controls (1 % (v/v) Triton-X in PBS) to measure the maximum release of LDH from the cells (3) Pneumolysin solution only to measure pneumolysin-induced LDH release, (4) 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. Incubation in a light-proof chamber at RT for 5-10 min was followed by the addition of 1 N HCI to all wells. Absorbance at 490 nm and 655 nm was then measured. The percentage of LDH release induced by pneumolysin in the presence and absence of test compounds was plotted against the Log of the concentration of the compound and the IC₅₀ was determined, as described above in the inhibition of haemolysis assay, Section A.

Results

UL1-005, the parent active compound of the prodrug UL5-001 , was tested in the LDH assay in triplicate over a range of concentrations from 62.5 μΜ to 0.49 μΜ. The results obtained are shown in Figure 1.

In Figure 1 : (1) The horizontal dotted line at 100%, PLY control (-), indicates the maximum release of LDH from the cells under the effect of pneumolysin, as opposed to the horizontal solid line at 0% (low control), which corresponds to the supernatant of cells exposed to the assay buffer alone that shows the natural LDH release under the assay conditions. (2) The grey solid line (^) shows that the LDH release from cells exposed to pneumolysin was significantly reduced in the presence of UL1-005, in a dose response manner, when compared to the PLY control. This demonstrates that UL1-005 prevents pneumolysin from damaging the human lung epithelial cells in culture, with an IC₅₀ < 0.49 μΜ. (3) The solid black line (- X -) shows that UL1- 005 does not exhibit cytotoxicity at the concentrations tested, up to approximately 150 times the therapeutic IC₅₀ value. Conclusion

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 IC₅₀ value. C. EX VIVO ASSAY: INHIBITION OF THE EFFECT OF PNEUMOLYSIN ON THE CILIARY FUNCTION OF CULTURED EPENDYMAL CELLS

Rationale

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. This layer acts as a selective brain barrier to and from the cerebrospinal fluid and plays a role in controlling the CSF volume. To study if the inhibitors prevent the damage caused by pneumolysin on the ependymal layer, 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.

The use of the ex vivo model of meningitis constitutes a powerful means to predict the ability of 5 a compound to prevent pneumolysin from causing damage in vivo.

Experimental procedure

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

10 37°C in 5% (v/v) C0₂ for 2h before use. The growth medium was minimum essential medium (MEM) with added penicillin (100 lU/mL), streptomycin (100 μg/mL), fungizone (2.5 μg/mL), BSA (5 μg/mL), insulin (5 μg/ml), transferrin (10 μg/mL) and selenium (5 μg/mL). Neo-natal (0-1 day old) rats were killed by cervical dislocation, and their brains were removed. The cerebellum was removed along with edge regions of the left and right cortical hemispheres and the frontal

15 cortex. 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 μΙ/well), each containing 2.5 ml_ of growth medium. The cells then were incubated at 37°C in 5% (v/v) C0₂. 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.

20

After approximately two weeks, the cells were fully ciliated and ready for experiments. To perform the 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

25 microscope. The cell cultures were allowed to equilibrate until the temperature of the assay medium was 37°C. At this point, recombinant purified pneumolysin, with and without test compound, pre-incubated in 1 ml of medium MEM at 37°C for 40 min, was added to the wells containing the ciliated cells. To the control cells, 1 ml_ of MEM medium was added. Beating cilia were recorded before and after exposure over 30 min, with a digital high-speed video camera at

30 a rate of 500 frames/s. The recorded video sequences were played back at reduced frame rates and the ciliary beat frequency (CBF) was determined by the following equation:

„_πτ. _{TT x} 500frames/s _ , , \

CBF (Hz) = -; — x 5 (conversion per beat cyclej.

(frames elapsed for 5 ciliary beat cycles)

35

Results

The parameter measured was the ciliary beating frequency (CBF). 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).

In 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. (1) 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. (2) 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. (3) Treatment with UL1-005: The symbol (▼) represents the measurements of the CBF in the presence of pneumolysin and UL1-005 (1.56 μΜ). No significant loss of the CBF was seen, showing that UL1-005 inhibits pneumolysin-induced damage on the ciliary beating frequency of the brain ependymal cells. There was no statistical difference between the CBF of Control 1 (medium only) and the CBF in the presence of the treatment (▼), indicating that the inhibition of the damaging effect of pneumolysin by UL1-005 was achieved to an extent comparable to the control medium alone.

Conclusion

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.

These findings support the use of this novel-approach as an adjunctive therapy in patients.

D. SOLUBILITY TESTING FOR THE DETERMINATION OF A SUITABLE FORMULATION FOR INTRAVENOUS ADMINISTRATION OF PRODRUGS OF THE INVENTION

Rationale

Parenteral delivery is one preferred routes of administration of the compound of the invention. Therefore, the aqueous solubility and pH of resulting solutions are important parameters for the pharmaceutical utility of the compound of the invention. 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. Experimental procedure

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.

Results

The formulations obtained with prodrugs UL1-124 and UL5-001 are shown in Table 2. UL5-001 proved to be readily soluble in aqueous formulations that are compatible with safe intravenous dosing at the desired concentrations. Thus UL5-001 demonstrates superior properties to ULI- 124 which showed low solubility and resulted in a formulation having a pH making it

incompatible for intravenous dosing. Table 2 Properties of the formulations of compounds of the invention

E. IN VIVO EFFICACY ASSAY USING A MOUSE PNEUMONIA MODEL

Rationale

This model has been well established in the laboratory of the inventors and has become adapted by other research groups working in this field. Using this model, pneumolysin was shown to be essential for the pathogenesis of S. pneumoniae and for its survival in vivo. With this disease model, mice infected with a strain of S. pneumoniae mutant deficient in

pneumolysin (PLN-A), exhibited (1) a significant increase in the survival, (2) significant delay and attenuation of the signs of the disease and (3) substantial decrease in the pulmonary inflammation and less bacteraemia (infiltration of the bacteria from the lungs to the circulation). Therefore, this in vivo disease model constitutes a powerful tool to study the disease progression of mice infected with wild-type S. pneumoniae and treated with pneumolysin inhibitors. Survival was used as an endpoint parameter for the study.

Experimental Procedures: Infection, Treatment and Disease Signs Scoring

Outbred MF1 female mice, 8 weeks old or more and weighing 25-30 g were used. The animals were maintained under controlled conditions of temperature, humidity and day length. They had free access to tap water and pelleted food. The in vivo experiments were performed using two control groups: Control 1 (infected and not treated), Control 2 (not infected and treated) and one Treatment group (infected and treated). Mice in control group 1 and in the treatment group were infected intranasally with Streptococcus pneumoniae strain D39 (procedure described below). After completing the infections, the viable count of the given dose was determined (as described below). Subsequently, every six hours, animals 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 (Table 3) 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

Sign Description

Healthy appearance.

Normal

Highly active.

Slight (1+) or pronounced (2+)

1+/2+ Hunched

convex curvature of the upper spine.

1+/2+ Starey coat Slight (1+) orpronounced (2+)

(Piloerection) piloerection of the coat.

Pronounced hunching and

piloerection accompanied by a

1+/2+ Lethargic

considerable (1+) or severe (2+)

reduction of activity.

The experimental design is shown in Figure 3. The procedures used for infection with S. pneumoniae, the delivery of the treatment and for the determination of the bacterial viable counts, mentioned above, are detailed as follows: - Intranasal instillation of infection

Mice were lightly anaesthetised with 2.5% (v/v) isoflurane over 1.6-1.8 L 0₂/min. The

confirmation of effective anaesthesia was made by observation of no pedal reflex. A mouse was held by the scruff of the neck in a vertical position with its nose upward. The infectious dose was then administered in sterile PBS, given drop by drop into the nostrils, allowing the animal to inhale it in between drops. Once the dose was given, the mouse was returned to its cage, placed on its back to recover from the effects of anaesthetic.

- Intravenous administration of treatment

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.

- Determination of viable count of the infectious dose

Viable counting was performed by the method of Miles and Misra [J. Hyg. (1938) 38 732-749). 20 μΙ_ of the sample were serially diluted in 180 μΙ_ PBS in round-bottomed 96-wells microtitre plates, up to a dilution of 10⁶. Blood agar plates were divided into six sectors and 60 μΙ_ of each dilution plated onto an individual sector. The plates were incubated in C0₂ 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

CFU per ml x Dilution x lOOO (conversion factor).

60 μΐ

Results of the in vivo efficacy assay obtained with Example UL5-001

Survival Results

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 p-value was calculated by means of the log-rank (Mantel- Cox) test (n = 10/group). 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).

Conclusion

(1 ) In vivo protection was obtained with UL5-001 at the dosing regimen tested. (2) Protection is seen even in the absence of an antibiotic, which is a remarkable outcome, suggesting that the neutralisation of pneumolysin alone, without the killing of the bacterium, is providing protection to the animals. This is consistent with the disease profile obtained with S. pneumoniae deficient in pneumolysin (PLN-A). (3) No visible adverse effects were seen in the control group of mice receiving the prodrug UL5-001 alone.

F. CONVERSION OF PRODRUG DERIVATIVES TO ACTIVE INHIBITORS IN MOUSE, RAT OR HUMAN PLASMA

Rationale

To demonstrate that the prodrug derivative is converted to the parent active compound in the presence of plasma enzymes, 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.

Experimental procedure

Prodrug derivative were assessed in the mouse, rat or human plasma stability assay at a concentration of 10 μΜ. Test compounds were diluted in DMSO to a final stock concentration of 10 mM. For the purpose of the assay, the stocks prepared were further diluted in DMSO to a concentration of 400 μΜ and 5 μΙ_ were added to 195 μΙ_ 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 μΙ_ of acetonitrile containing 0.55 μΜ metoprolol and 1 % (v/v) formic acid. The plate was then centrifuged at 3000 rpm, for 45 min, at 4°C. 80 μΙ_ of supernatant were transferred into a conical bottom 96 well glass coated plate. 40 μΙ_ 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.

Results

The quantification of the prodrug compound remaining and the parent active compound appearing was performed as follows:

(1 ) The parent active compound was quantified using a 6 point calibration curve prepared in deactivated mouse, rat or human plasma. (2) 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 μΜ) at time 0 min.

The conversion of the prodrugs UL5-001 and UL1-124 to the parent active compound UL1-005 is shown in Table 4.

Table 4

Conclusion

The results presented in Table 4 clearly indicate the therapeutic benefits of the prodrug of the invention, which is demonstrated by their conversion in plasma into the parent active

compound. Besides the demonstration of its therapeutic benefits, the physicochemical properties of UL5-001 are favourable for the preparation of formulations and is particularly suitable for parenteral delivery thus demonstrating clear advantages over UL1-124.

Throughout the specification and the claims which follow, unless the context requires otherwise, the word 'comprise', and variations such as 'comprises' and 'comprising', will be understood to imply the inclusion of a stated integer, step, group of integers or group of steps but not to the exclusion of any other integer, step, group of integers or group of steps. All patents and patent applications referred to herein are incorporated by reference in their entirety.

Claims

or a pharmaceutically acceptable solvate thereof.

2. A compound of formula (I) which is not a solvate.

3. A pharmaceutical composition comprising a compound according to claim 1 or 2, optionally in combination with one or more pharmaceutically acceptable diluents or carriers.

4. A pharmaceutical composition according to claim 3, for parenteral, e.g. intravenous, administration.

5. A pharmaceutical composition according to claim 3, for oral administration.

6. A pharmaceutical composition according to any one of claims 3 to 5 in unit dose form.

7. A pharmaceutical composition according to claim 4 or 6, in solid form for reconstitution with a liquid prior to administration.

8. A pharmaceutical composition according to claim 4 or 6, in liquid form e.g. a solution.

9. A pharmaceutical composition according to any one of claims 3 to 8 comprising one or more other therapeutically active ingredients.

10. A compound according to claim 1 or 2 for use as a medicament.

1 1. A compound according to any one of claims 1 , 2 or 10 for use in combination with one or more other therapeutically active ingredients.

12. A compound according to any one of claims 1 , 2, 10 or 11 for use in the treatment of bacterial infections caused by bacteria producing pore-forming toxins, such as cholesterol dependent cytolysins.

13. A compound for use according to claim 12 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) or Bacillus spp. (e.g. Bacillus anthracis).

14. A compound for use according to claim 13 for the treatment of bacterial infection which is caused by Streptococcus pneumoniae.

15. A compound for use according to claim 14 for the treatment of pneumococcal pneumonia, pneumococcal meningitis, pneumococcal septicaemia/bacteraemia, pneumococcal keratitis or pneumococcal otitis media.

16. A compound for use according to claim 13 for the treatment of conditions selected from gas gangrene, gastrointestinal anthrax, inhalational anthrax, porcine meningitis, encephalitis, septicaemia/bacteraemia and pneumonia which are caused by bacteria other than

pneumococcus.

17. A compound for use according to any one of claims 10 to 16 wherein the compound is administered in combination with one or more other therapeutically active ingredients (e.g. one or more antimicrobial or immunomodulatory agents).

18. A method of treatment of bacterial infections caused by bacteria producing pore-forming toxins, such as cholesterol dependent cytolysins which comprises administering to a subject in need thereof an effective amount of a compound according to any one of claims 1 , 2, 10 or 1 1.

19. A process for preparing a compound of formula (I) as defined in claim 1 or 2, which comprises reacting a compound of formula (III):

(III) with a basic sodium salt such as NaHC0₃.

20. A compound of formula (IVa):

wherein R represents H or a protecting group such as Boc.