WO2014195705A1 - Novel pyrrole derivatives - Google Patents

Abstract

There are provided inter alia compounds of formula (I), wherein R¹, R², R³, R^4a and R^4b are as defined in the specification and their use in therapy, especially in the treatment of bacterial (e.g. pneumococcal) infections.

Description

NOVEL PYRROLE DERIVATIVES

Field of the invention The invention relates to compounds which are prodrugs of cytolysin inhibitors and their 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 21 1-218]. 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. 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 novel N-phenyl substituted pyrrole cytolysin inhibitors which are expected to demonstrate particularly advantageous properties e.g. in terms of solubility. The compounds of the present invention also are expected to prevent stimulation of host-derived toxic effects induced by pneumolysin and, it may be assumed, other cholesterol dependent cytolysins. Thus these compounds may be used as single agents or as 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) wherein:

R¹ is -C(0)NR⁸R⁹ or -C(0)OR^8a;

R² is -C(0)NR⁸R⁹, -C(0)NR⁵R⁶,-C(0)OR⁷ or -C(0)OR^8a;

R³ is optionally substituted phenyl;

R^4a and R^4b are independently selected from hydrogen; C C₆ alkyl which alkyl group may optionally be substituted by hydroxyl, COOR¹² or CONR¹³R¹⁴; aryl and -C C₃ alkylaryl in which said aryl groups may be optionally substituted;

R⁵ and R⁶ are independently selected from:

(a) hydrogen,

(b) Ci-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-Ci₀ cycloalkyl, C₅-Ci₀ cycloalkenyl,

heterocyclyl, -CrC₃ alkyl-C₃-Ci₀ cycloalkyl, -C C₃ alkyl-C₅-Ci₀ cycloalkenyl or -C C₃ alkylheterocyclyl, or R⁵ and R⁶ together with the N to which they are attached may form a 5- or 6-membered heterocyclic ring optionally containing a further heteroatom selected from O, S and NR⁹, in which any of the aforementioned R⁵ and R⁶ groups may be optionally substituted by a group selected from cyano, C C₆ alkoxy, C C₆ fluoroalkoxy, Ci-C₆ alkyl, C C₆ fluoroalkyl and -C(0)NR^aR^b, where R^a and R^b are independently selected from hydrogen and C C₆ alkyl, and any of the aforementioned R⁵, R⁶ and R⁹ groups may be optionally substituted by one or more halogen atoms, and

(c) aryl, heteroaryl, C C₃ alkylaryl and -C C₃ alkylheteroaryl, said aryl and heteroaryl

groups being optionally substituted;

R⁷ is selected from:

(a) Ci-C₆ alkyl, C₂C₆ alkenyl, C₂-C₆ alkynyl, C₃-Ci₀ cycloalkyl, C₅-Ci₀ cycloalkenyl,

heterocyclyl, -C C₃ alkyl-C₃-Ci₀ cycloalkyl, -C C₃ alkyl-C₅-Ci₀ cycloalkenyl or -C C₃ alkylheterocyclyl, in which any of the aforementioned R⁷ groups may be optionally substituted by a group selected from cyano, C C₆ alkoxy, C C₆ fluoroalkoxy, C C₆ alkyl, C C₆ fluoroalkyl and -C(0)NR^aR^b, where R^a and R^b are independently selected from hydrogen and C C₆ alkyl, and any of the aforementioned R⁷ groups may be optionally substituted by one or more halogen atoms, and

(b) aryl, heteroaryl, C1-C₃ alkylaryl and -C1-C₃ alkylheteroaryl, said aryl and heteroaryl

groups being optionally substituted;

R⁸ is selected from C C₆ alkyl substituted by -NR¹⁰R¹¹ , C₃-C1₀ cycloalkyl substituted by - NR¹⁰R¹¹ or a 5- or 6-membered, optionally substituted, heterocyclic or heteroaromatic ring containing at least one nitrogen atom;

R^8a is selected from C C₆ alkyl substituted by -NR^10aR^11a, C₃-C1₀ cycloalkyl substituted by - NR^10aR^{11 a} or a 5- or 6-membered, optionally substituted, heterocyclic or heteroaromatic ring containing at least one nitrogen atom;

R⁹ is selected from:

(a) hydrogen,

(b) Ci-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C1₀ cycloalkyl, C5-C1₀ cycloalkenyl,

heterocyclyl, -C1-C₃ alkyl-C₃-Ci₀ cycloalkyl, -C1-C₃ alkyl-C₅-Ci₀ cycloalkenyl or -C1-C₃ alkylheterocyclyl, or R⁵ and R⁶ together with the N to which they are attached may form a 5- or 6-membered heterocyclic ring optionally containing a further heteroatom selected from O, S and NR⁹, in which any of the aforementioned R⁵ and R⁶ groups may be optionally substituted by a group selected from cyano, C C₆ alkoxy, C C₆ fluoroalkoxy, Ci-C₆ alkyl, C C₆ fluoroalkyl and -C(0)NR^aR^b, where R^a and R^b are independently selected from hydrogen and C C₆ alkyl, and any of the aforementioned R⁵, R⁶ and R⁹ groups may be optionally substituted by one or more halogen atoms,

(c) aryl, heteroaryl, C1-C3 alkylaryl and -C1-C3 alkylheteroaryl, said aryl and heteroaryl

groups being optionally substituted, and

(d) Ci-C_e alkyl substituted by -NR¹⁰R¹¹ , C₃-C1₀ cycloalkyl substituted by -NR¹⁰R¹¹ or a 5- or 6-membered, optionally substituted, heterocyclic or heteroaromatic ring containing at least one nitrogen atom;

R¹⁰, R^10a, R¹¹ and R^11a are independently selected from hydrogen, C C₆ alkyl, C₂-C₆ alkenyl, C₂- C₆ alkynyl, C₃-C1₀ cycloalkyl, C5-C1₀ cycloalkenyl, heterocyclyl, -C1-C3 alkyl-C₃-Ci₀ cycloalkyl, - C1-C3 alkyl-C₅-Cio cycloalkenyl and -C1-C3 alkylheterocyclyl;

R¹² is Ci-Ce alkyl;

R¹³ is hydrogen or C C₆ alkyl; and

R¹⁴ is hydrogen or C C₆ alkyl;

or a pharmaceutically acceptable prodrug derivative thereof, or a pharmaceutically acceptable salt or solvate thereof.

The compounds of formula (I) have therapeutic activity. In a further aspect, the present invention provides a compound of formula (I) for use as a medicament.

Detailed description of the invention

In one embodiment R¹ and R² are independently -C(0)NR⁸R⁹, and may be the same or different, preferably the same. In another embodiment R¹ is -C(0)NR⁸R⁹ and R² is -C(0)NR⁵R⁶. In a further embodiment R¹ is -C(0)NR⁸R⁹ and R² is -C(0)OR⁷. In a further embodiment R² is - C(0)NR⁵R⁶ and R¹ is -C(0)OR^8a. In a further embodiment R¹ is -C(0)OR⁷ and R² is -C(0)OR^8a. In a further embodiment R¹ and R² are independently -C(0)OR^8a, and may be the same or different, preferably the same. In a further embodiment R¹ is -C(0)NR⁸R⁹ and R² is -C(0)OR^8a. R³ is preferably substituted phenyl.

Suitable optional substituents for R³ include 1 or more, e.g. 1 , 2 or 3, substituents (e.g. 1 substituent) independently selected from halo, cyano, hydroxyl, C C₆ alkoxy, C C₆

hydroxyalkoxy, C C₆ fluoroalkoxy, C C₆ alkyl, C C₆ fluoroalkyi, -C(0)NR^aR^b, where R^a and R^b are independently selected from hydrogen and C C₆ alkyl; -O-R¹⁵ wherein R¹⁵ is -(CH₂)_X-

P(0)(OR²³)₂ (where x is 0, 1 , 2, 3 or 4 and R²³ is independently selected from hydrogen and C C₃ alkyl), -(CH₂)y-S(0)₂Me (where y is 1 , 2, 3 or 4), -C C₆ alkylheterocyclyl which heterocyclyl group may be optionally substituted e.g. by C C₃ alkyl, -C C₆ alkylphenyl which phenyl group may be optionally substituted e.g. by C C₃ alkoxy, or phenyl or 5- or 6-membered heteroaryl which phenyl or heteroaryl group may optionally be substituted by a group e.g. selected from C1-C4 alkyl and halo; or -(0(CH₂)_z)_pOR²⁴, where each z, which may be the same or different, represents 2 or 3, p represents 1 , 2, 3, 4 or 5 and R²⁴ is hydrogen or C1-C3 alkyl; or two adjacent carbon atoms within R³ may be linked by -0-CH₂-0-. When R¹⁵ is -C C₆ alkylheterocyclyl, particular heterocyclyl groups which may be mentioned include 5- or 6-membered, monocyclic non-aromatic ring systems, containing up to two heteroatoms selected from N, O and S. Such rings are suitably linked to -C C₆ alkyl via an N atom. Examples of heterocyclic rings include morpholine, piperazine, and the like, which may be optionally substituted e.g. by C1-C3 alkyl, such as methyl. Further examples of heterocyclic rings include piperidine and pyrrolidine.

A group of suitable optional substituents for R³ which may be mentioned include 1 , 2 or 3 substituents selected from halo, cyano, C C₆ alkoxy, C C₆ fluoroalkoxy, C C₆ alkyl, C C₆ fluoroalkyi and -C(0)NR^aR^b, where R^a and R^b are independently selected from hydrogen and Ci-C₆ alkyl. In addition, when R³ is substituted phenyl, said phenyl may be provided with a single substituent -O-R¹⁵ wherein R¹⁵ is selected from phenyl and 5- or 6-membered heteroaryl which phenyl or heteroaryl group may optionally be substituted by a group selected from C C₄ alkyl and halo. Preferred optional substituents for R³ include 1 or more, e.g. 1 , 2 or 3, substituents (e.g. 1 substituent) independently selected from C C₆ alkoxy; -O-R¹⁵ wherein R¹⁵ is -(CH₂)_X- P(0)(OR²³)₂, where x is 0, 1 , 2, 3 or 4 and R²³ is independently selected from hydrogen and C C₃ alkyl or R¹⁵ is -(CH₂)_y-S(0)₂Me where y is 1 , 2, 3 or 4; and -(0(CH₂)_z)_pOR²⁴, where each z, which may be the same or different, represents 2 or 3, p represents 1 , 2, 3, 4 or 5 and R²⁴ is hydrogen or C1-C3 alkyl.

Suitable optional substituents for R³ are described in further detail below.

When R³ is substituted phenyl, it preferably has a substituent in the meta or para position relative to the pyrrole ring, more preferably it has a substituent in the para position relative to the pyrrole ring. Alternatively, when R³ is substituted phenyl it may have a substituent in the ortho position relative to the pyrrole ring. In one embodiment, R³ is phenyl substituted by a single substituent. In another embodiment, R³ is phenyl substituted by two substituents. When R³ is substituted phenyl having 2 substituents, these may, for example, be in the meta and para positions relative to the pyrrole ring. In another embodiment, R³ is phenyl substituted by three substituents. When R³ is substituted phenyl having 3 substituents, these may, for example, be in the 3, 4 and 5 positions relative to the pyrrole ring.

For example, R³ may represent phenyl bearing a para substituent selected from F, CI, I, cyano, OCH₃, OCH2CH3, OCH2CH2CH3, CF₃, OCF3, CON(CH₃)₂, O-phenyl, methyl, ethyl, isopropyl, t- butyl, hydroxyl, -OP(0)(OH)₂, -(0(CH₂)₂)pOMe where p is 1 , 2, 3 or 4, 3-morpholinopropoxy, 3- (4-methylpiperazin-1-yl)propoxy, 3-(diethoxyphosphoryl)propoxy, -(0(CH₂)₃)-P(0)(OH)₂, 3- (methylsulfonyl)propoxy, and 4-methoxybenzyloxy. In a further group of compounds that may be mentioned R³ may represent phenyl bearing a para substituent selected from F, CI, I, OCH₃, OCH₂CH₃, OCH₂CH₂CH₃, CF₃, OCF₃, CON(CH₃)₂, O-phenyl, methyl, ethyl, isopropyl and t- butyl. A particular R³ group which may be mentioned is phenyl bearing a para OCH₃ substituent.

For example, R³ may represent phenyl bearing an ortho substituent which is OCH₃. For example, R³ may represent phenyl bearing a meta substituent which is O-phenyl or OCH₃.

For example, R³ may represent phenyl substituted in the meta position by I and in the para position by OCH₃, or phenyl substituted in the meta position by OCH₃ and in the para position by OCH₃, or phenyl linked in the meta and para positions by -0-CH₂-0-.

For example, R³ may represent phenyl substituted in the ortho position by OCH₃ and in the para position by OCH₃.

For example, R³ may represent phenyl substituted in the 3, 4 and 5 positions by OCH₃, or phenyl substituted in the 3 and 5 positions by F and in the para position by OCH₂CH₃.

Hence a particularly suitable substituent for the phenyl of R³ is OCH₃, especially in the para position. Further particularly suitable substituents for R³ include -O-R¹⁵ wherein R¹⁵ is as defined above and -(0(CH₂)_z)_pOR²⁴, where z, p and R²⁴ are as defined above, especially in the para position.

When an alkyl group or R^4a and/or R^4b is substituted by hydroxyl, COOR¹² or CONR¹³R¹⁴, examples of R^4a and/or R^4b groups include -CH₂COOt-butyl, CH₂CONH₂ and CH₂CH₂OH. R^4a and R^4b may be independently selected from hydrogen; C C₆ alkyl which alkyl group may optionally be substituted by hydroxyl, COOR¹² or CONR¹³R¹⁴; and -C C₃ alkylaryl in which said aryl groups may be optionally substituted. R^4a and R^4b are preferably independently selected from hydrogen, C C₆ alkyl, aryl and -C1-C3 alkylaryl in which aryl may be optionally substituted. For example R^4a and R^4b are preferably independently selected from hydrogen, C C₆ alkyl and - C1-C3 alkylaryl in which aryl may be optionally substituted. R^4a and R^4b are more preferably hydrogen or -C1-C3 alkylaryl, e.g. benzyl. Most preferably R^4a and R^4b are hydrogen. R⁵ and R⁶ are preferably independently selected from hydrogen, C C₆ alkyl e.g. methyl, ethyl, or propyl, aryl e.g. phenyl, or C1-C₃ alkylaryl, e.g. benzyl in which said aryl may be optionally substituted, or R⁵ and R⁶ together with the N to which they are attached may form a 5- or 6- membered heterocyclic ring optionally containing a further heteroatom selected from O, S and NR⁹, e.g. morpholine, piperidine or piperazine (optionally N substituted with an R⁹ group).

In one embodiment of the invention one of R⁵ and R⁶ is hydrogen. Preferably at least one of R⁵ and R⁶ is not hydrogen, more preferably both of R⁵ and R⁶ are not hydrogen. Preferably R⁹ is not hydrogen.

Specific -NR⁵R⁶ groups of interest include NMe₂, NHethyl, -N-morpholinyl and N-piperidinyl, especially NMe₂. R⁷ is preferably C C₆ alkyl e.g. methyl, ethyl, propyl or butyl, such as /^'so-propyl or te/f-butyl. R⁸ is preferably C C₆ alkyl (e.g. (CH₂)₂-3, particularly (CH₂)₂) substituted by -NR¹⁰R¹¹.

When R⁸ is C₃-C1₀ cycloalkyl substituted by -NR¹⁰R¹¹ or a 5- or 6-membered, optionally substituted, heterocyclic or heteroaromatic ring containing at least one nitrogen atom, it is preferably C₅-C₆ cycloalkyl substituted by -NR¹⁰R^{1 1} or a 5- or 6-membered, optionally substituted, heterocyclic or heteroaromatic ring containing at least one nitrogen atom. Suitable 5- or 6-membered heterocyclic rings include rings containing one nitrogen atom and optionally one additional heteroatom selected from N, O and S, such as pyrrolidine, imidazolidine, pyrazolidine, piperidine, piperazine, morpholine and thiomorpholine. Suitable 5- or 6-membered heteroaromatic rings include rings containing one nitrogen atom and optionally one, two or three additional heteroatoms selected from N, O and S, such as pyrazole, imidazole, oxazole, isoxazole, pyridine, pyrimidine, pyrazine, thiazole, triazole, oxadiazole, thiadiazole, tetrazole and pyridazine. Optional substituents for heterocyclic or heteroaromatic rings within the definition of R⁸ are suitably selected from hydroxyl, halo, cyano, C C₆ alkoxy or C C₆ fluoroalkoxy, e.g. C C₃ alkoxy or C1-C3 fluoroalkoxy such as methoxy, ethoxy or trifluoromethoxy, C C₆ alkyl or C C₆ fluoroalkyl, e.g. C1-C₃ alkyl or C1-C₃ fluoroalkyl such as methyl or trifluoromethyl, and - C(0)NR^aR^b, where R^a and R^b are independently selected from hydrogen and C C₆ alkyl e.g. C1-C₃ alkyl such as methyl. Said groups, if substituted, may be substituted by 1 , 2 or 3, preferably 1 or 2, more preferably 1 substituent.

R^8a is preferably C C₆ alkyl (e.g. (CH₂)₂.₃, particularly (CH₂)₂) substituted by -NR^10aR^11a.

When R^8a is C₃-C1₀ cycloalkyl substituted by -NR^10aR^11a or a 5- or 6-membered, optionally substituted, heterocyclic or heteroaromatic ring containing at least one nitrogen atom, it is preferably C₅-C₆ cycloalkyl substituted by -NR^10aR^11a or a 5- or 6-membered, optionally substituted, heterocyclic or heteroaromatic ring containing at least one nitrogen atom. Suitable 5- or 6-membered heterocyclic rings include rings containing one nitrogen atom and optionally one additional heteroatom selected from N, O and S, such as pyrrolidine, imidazolidine, pyrazolidine, piperidine, piperazine, morpholine and thiomorpholine. Suitable 5- or 6-membered heteroaromatic rings include rings containing one nitrogen atom and optionally one, two or three additional heteroatoms selected from N, O and S, such as pyrazole, imidazole, oxazole, isoxazole, pyridine, pyrimidine, pyrazine, thiazole. triazole, oxadiazole, thiadiazole, tetrazole and pyridazine. Optional substituents for heterocyclic or heteroaromatic rings within the definition of R^8a are suitably selected from hydroxyl, halo, cyano, C C₆ alkoxy or C C₆ fluoroalkoxy, e.g. C C₃ alkoxy or C1-C3 fluoroalkoxy such as methoxy, ethoxy or trifluoromethoxy, C C₆ alkyl or C C₆ fluoroalkyl, e.g. C1-C₃ alkyl or C1-C₃ fluoroalkyl such as methyl or trifluoromethyl, and - C(0)NR^aaR^ba, where R^aa and R^bb are independently selected from hydrogen and C C₆ alkyl e.g. C1-C₃ alkyl such as methyl. Said groups, if substituted, may be substituted by 1 , 2 or 3, preferably 1 or 2, more preferably 1 substituent.

R⁹ is preferably selected from hydrogen, C C₆ alkyl e.g. methyl, ethyl, or propyl, aryl e.g.

phenyl, or C1-C₃ alkylaryl, e.g. benzyl in which said aryl may be optionally substituted.

Preferably R⁹ is not hydrogen. For example, R⁹ is C C₆ alkyl e.g. methyl, ethyl, or propyl R¹⁰ and R¹¹ are preferably independently selected hydrogen and C C₆ alkyl. A suitable

NR¹⁰R¹¹ group is NMe₂.

R^10a and R^11a are preferably independently selected hydrogen and C C₆ alkyl. A suitable NR^10aR^{11 a} group is NMe₂.

R¹² is preferably methyl.

R¹³ is preferably H or methyl.

R¹⁴ is preferably H or methyl.

In an embodiment R¹⁵ is -(CH₂)_x-P(0)(OR²³)₂ or -(CH₂)_y-S(0)₂Me.

In an embodiment R¹⁵ group is optionally substituted phenyl, e.g. unsubstituted phenyl.

In an embodiment R¹⁵ is-C C₆ alkylheterocyclyl which heterocyclyl group may be optionally substituted e.g. by C1-C₃ alkyl.

R²¹ are R²² are preferably independently selected from C C₆ alkyl, e.g. methyl.

R²³ is preferably hydrogen, methyl or ethyl.

R²⁴ is preferably C1-C₃ alkyl, e.g. methyl,

x is preferably 0, 1 , 2, 3 or 4.

y is preferably 1 , 2 or 3.

z is preferably 2.

p is preferably 2, 3, 4 or 5.

Prodrug derivatives of compounds of the invention will break down after administration to a subject to form an active compound of formula (I) (sometimes herein after referred to as "parent active compound") in vivo. Prodrug derivatives of compounds of the invention may have some intrinsic biological activity (e.g. as pneumolysin inhibitors) however typically they have little or no such intrinsic activity.

Prodrug derivatives of the compounds of formula (I) include ester prodrug derivatives. Ester prodrug derivatives include carboxylate ester, sulfamate ester, phosphate ester and carbamate ester derivatives, preferably carboxylate ester, sulfamate ester or phosphate ester derivatives, more preferably carboxylate ester or phosphate ester derivatives, even more preferably carboxylate ester derivatives. Examples of ester prodrug derivatives thus include compounds of formula (I) wherein one or both of R^4a and R^4b are independently selected from -C(0)R¹⁶, - S0₂NH₂ , -PO(OR¹⁹)(OR²⁰), -CHR²⁶-OPO(OR¹⁹)(OR²⁰) (where R²⁶ is hydrogen or C C₆ alkyl), and -C(0)NR¹⁷R¹⁸, wherein R¹⁶, R¹⁷, R¹⁸, R¹⁹ and R²⁰ are independently selected from:

(a) C C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C10 cycloalkyl, C5-C10 cycloalkenyl,

heterocyclyl, -C1-C₃ alkyl-C₃-Ci₀ cycloalkyl, -C1-C₃ alkyl-C₅-Ci₀ cycloalkenyl or -C1-C₃ alkylheterocyclyl, or R¹⁷ and R¹⁸ together with the N to which they are attached may form a 5- or 6-membered heterocyclic ring optionally containing a further heteroatom selected from O, S and NR^25aR^25b where R^25a is hydrogen, C C₆ alkyl, -CH₂-OPO(OR¹⁹)(OR²⁰) or a 5- or 6-membered heterocyclic ring, and R^25b is absent or C C₆ alkyl; and in which any of the aforementioned R¹⁶, R¹⁷ or R¹⁸ groups may be optionally substituted by one or more groups, e.g. one group, selected from cyano, -OPO(OR¹⁹)(OR²⁰), -(0(CH₂)_z)_rOR²⁴ (wherein each z, which may be the same or different, represents 2 or 3, r represents an integer selected from 1 to 20, e.g. 7 to 12, and R²⁴ is hydrogen, C1-C₃ alkyl or - PO(OR¹⁹)(OR²⁰)), C C₆ alkoxy, C C₆ fluoroalkoxy, C C₆ alkyl, C C₆ fluoroalkyi and - C(0)NR^aR^b, where R^a and R^b are independently selected from hydrogen and C C₆ alkyl, and any of the aforementioned R¹⁶, R¹⁷ or R¹⁸ groups may be optionally substituted by one or more halogen atoms; and

(b) aryl, heteroaryl, C1-C₃ alkylaryl and -C1-C₃ alkylheteroaryl, said aryl and heteroaryl

groups being optionally substituted;

or R¹⁸, R¹⁹ and R²⁰ may independently represent hydrogen.

Optional substituents for phenyl, aryl and heteroaryl groups within the definitions of R¹ , R², R³, R^4a, R^4b, R⁵, R⁶, R⁷, R¹⁶, R¹⁷, R¹⁸, R¹⁹ and R²⁰ are suitably selected from hydroxyl, halo, cyano, - (CHR²⁶)_q-OPO(OR¹⁹)(OR²⁰) wherein q represents 0 or 1 (said group not being substituted by another R¹⁹ or R²⁰ containing group), C C₆ alkoxy or C C₆ fluoroalkoxy, e.g. C1-C₃ alkoxy or C1-C₃ fluoroalkoxy such as methoxy, ethoxy or trifluoromethoxy, C C₆ alkyl or C C₆ fluoroalkyi, e.g. C1-C₃ alkyl or C1-C₃ fluoroalkyi such as methyl or trifluoromethyl, and -C(0)NR^aR^b, where R^a and R^b are independently selected from hydrogen and C C₆ alkyl e.g. C1-C₃ alkyl such as methyl; and also when two adjacent hydroxyl substituents are present they may optionally be connected by a methylene group to form an acetal. Another possible optional substituent is - SF₅. Said aryl and heteroaryl groups, if substituted, may be substituted by 1 , 2 or 3, preferably 1 or 2, more preferably 1 substituent.

Optional substituents for the C C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C10 cycloalkyl, C5-C10 cycloalkenyl, heterocyclyl, -C1-C₃ alkyl-C₃-Ci₀ cycloalkyl, -C1-C₃ alkyl-C₅-Ci₀ cycloalkenyl, -C C₃ alkylheterocyclyl or heterocyclic ring groups of R⁵, R⁶, R⁷, R¹⁶, R¹⁷, R¹⁸, R¹⁹ and R²⁰ include substituents selected from cyano, -OPO(OR¹⁹)(OR²⁰) (said group not being substituted by another R¹⁹ or R²⁰ containing group), C C₆ alkoxy or C C₆ fluoroalkoxy, e.g. C1-C₃ alkoxy or C1-C₃ fluoroalkoxy such as methoxy, ethoxy or trifluoromethoxy, C C₆ alkyl or C C₆ fluoroalkyi, e.g. C1-C₃ alkyl or C1-C₃ fluoroalkyi such as methyl or trifluoromethyl, and -C(0)NR^aR^b, where R^a and R^b are independently selected from hydrogen and C C₆ alkyl e.g. C1-C₃ alkyl such as methyl. Optional substituents for the groups R⁵, R⁶ and R⁷ also include one or more (e.g. 1 , 2, or 3) halogen atoms e.g. F or CI atoms (especially F atoms). R¹⁶ preferably represents C C₆ alkyl or C3-C10 cycloalkyl in which either of the aforementioned groups may be optionally substituted (and is preferably substituted) by a group selected from - OPO(OR¹⁹)(OR²⁰) and -(0(CH₂)_z)_rOR²⁴, where each z, which may be the same or different, represents 2 or 3, r represents an integer selected from 1 to 20, e.g. 7 to 12, and R²⁴ is hydrogen, C C₃ alkyl or -PO(OR¹⁹)(OR²⁰). Alternatively, R¹⁶ preferably represents phenyl optionally substituted (and is preferably substituted) by -(CHR²⁶)_q-OPO(OR¹⁹)(OR²⁰) wherein q represents 0 or 1.

R¹⁷ preferably represents C C₆ alkyl e.g. methyl. R¹⁸ preferably represents C C₆ alkyl e.g. methyl. Alternatively, R¹⁷ and R¹⁸ together with the N to which they are attached may form a 5- or 6-membered heterocyclic ring optionally containing a further heteroatom selected from O, S and NR^25a where R^25a is hydrogen, C C₆ alkyl, -CH₂-OPO(OR¹⁹)(OR²⁰) or a 5- or 6-membered heterocyclic ring.

R¹⁹ is preferably hydrogen, methyl or ethyl, especially hydrogen.

R²⁰ is preferably hydrogen, methyl or ethyl, especially hydrogen.

R^25a is preferably hydrogen or methyl.

R^25b is preferably absent.

R²⁶ is preferably hydrogen or methyl, more preferably methyl.

In one embodiment q represents 0. In another embodiment q represents 1.

In one embodiment one of R^4a and R^4b represents a prodrug derivative group as defined above. In another embodiment both of R^4a and R^4b represent a prodrug group as defined above. When only one of R^4a and R^4b represents a prodrug derivative group as defined above the other of R^4a and R^4b is preferably hydrogen.

In one embodiment both of R^4a and R^4b are independently selected from -C(0)R¹⁶, -S0₂NH₂ , - PO(OR¹⁹)(OR²⁰), -CHR²⁶-OPO(OR¹⁹)(OR²⁰) where R²⁶ is hydrogen or C C₆ alkyl, and -

C(0)NR¹⁷R¹⁸. In a further embodiment one of R^4a and R^4b is selected from -C(0)R¹⁶, -S0₂NH₂ , -PO(OR¹⁹)(OR²⁰), -CHR²⁶-OPO(OR¹⁹)(OR²⁰) where R²⁶ is hydrogen or C C₆ alkyl, and - C(0)NR¹⁷R¹⁸; and the other of R^4a and R^4b is hydrogen. One or both of R^4a and R^4b are preferably independently selected from -C(0)R¹⁶.

When the prodrug is a carboxylate ester prodrug, e.g. wherein one or both of R^4a and R^4b are - C(0)R¹⁶, the carbon atom adjacent to the C(O) moiety is preferably a tertiary or quaternary carbon atom.

Specific examples of prodrug derivatives include compounds of formula (I) wherein one or both of R^4a and R^4b are independently selected from -S0₂NH₂, -PO(OH)₂, -CH₂-PO(OH)₂, -PO(OEt)₂, -CON-(4-N-piperidinyl-piperidine), -COt-butyl, -COisopropyl, -CON-(N-methyl)piperazine, -CON- piperazine, -CON(CH₃)₂, COCH₃, -CO-(CH₂)₂-OMe, -CO(CH₂)₂-(0(CH₂)₂)_pOMe where p is 1 to 12, -CO-CMe₂-CH₂-(0(CH₂)₃)_POMe where p is 1 to 12, -CO-CMe₂-CH₂-(0(CH₂)₂)_pO-PO(OH)₂ where p is 1 to 12, -CO-CMe₂-CH₂-(0(CH₂)₂)_pO-PO(OH)₂ where p is 1 to 12, -CO-(4- phosphonoxymethylbenzene) and -CO-(4-phosphonoxymethylcyclohexane); wherein when only one of R^4a and R^4b represents a prodrug derivative group as defined above the other of R^4a and R^4b is hydrogen. A group of specific examples of prodrug derivatives include compounds of formula (I) wherein R^4a and R^4b are independently selected from -S0₂NH₂, -PO(OH)₂, -CON-(4- N-piperidinyl-piperidine), -COt-butyl, -COisopropyl, -CON-(N-methyl)piperazine, -CON(CH₃)₂

While the preferred groups for each variable have generally been listed above separately for each variable, preferred compounds of this invention include those in which several or each variable in formula (I) is selected from the preferred, more preferred or particularly listed groups for each variable. Therefore, this invention is intended to include all combinations of preferred, more preferred and particularly listed groups. The molecular weight of the compounds of the invention is preferably less than 2000, more preferably less than 1000, even more preferably less than 800, for example less than 600.

Particular compounds of the invention include the following:

A/²-(2-(dimethylamino)ethyl)-3,4-dihydroxy-1-(4-methoxyphenyl)-A/²J\/⁵,A/⁵-trimethyl-1 /-/-pyrrole- 2,5-dicarboxamide, or a pharmaceutically acceptable prodrug derivative thereof, or a

pharmaceutically acceptable salt or solvate thereof.

Particular prodrug derivatives of the compounds of the invention include the following:

2-((2-(dimethylamino)ethyl)(methyl)carbamoyl)-5-(dimethylcarbamoyl)-1-(4-methoxyphenyl)-1 /-/- pyrrole-3,4-diyl bis(2-methylpropanoate), or a pharmaceutically acceptable salt or solvate thereof.

Alkyl as used herein refers to straight chain or branched chain alkyl, such as, without limitation, methyl, ethyl, propyl, /^'so-propyl, butyl, and te/f-butyl. In one embodiment alkyl refers to straight chain alkyl in another embodiment alkyl refers to branched chain alkyl. Alkenyl and alkynyl should be interpreted accordingly.

FluoroalkyI groups are as described above for alkyl, but may have one or more hydrogen atoms replaced by fluoro. Examples of fluoroalkyl groups include -CH₂F, -CHF₂ and -CF₃.

CycloalkyI as used herein refers to a cyclic alkyl group, containing 3-10 carbon atoms, optionally branched, for example cyclobutyl, cyclopentyl, cyclohexyl or cycloheptyl. A branched example is 2-methylcyclopentyl. Cycloalkenyl refers to a cyclic alkenyl group containing typically 5-10 carbon atoms, for example cyclopentyl, cyclohexenyl or cycloheptenyl. CycloalkyI and cycloalkenyl groups may for example be monocyclic or bicyclic (including spirocyclic) but are suitably monocyclic.

Alkoxy as used herein refers to straight or branched chain alkoxy, for example methoxy, ethoxy, propoxy, butoxy. Alkoxy as used herein also extends to embodiments in which the oxygen atom is located within the alkyl chain, for example -CH₂OCH₃. In one embodiment the alkoxy is linked through oxygen to the remainder of the molecule. In one embodiment the disclosure relates to straight chain alkoxy.

Halo includes fluoro, chloro, bromo or iodo, in particular fluoro, chloro or bromo, especially fluoro or chloro. Heterocyclyl as used herein includes 4- to 10-membered mono or bicyclic non-aromatic ring systems, e.g. 4- to 7-membered monocyclic saturated rings, containing up to three heteroatoms selected from N, O and S. Examples of heterocyclic rings include oxetane, tetrahydrofuran, tetrahydropyran, oxepane, oxocane, thietane, tetrahydrothiophene, tetrahydrothiopyran, thiepane, thiocane, azetidine, pyrrolidine, piperidine, azepane, azocane, [1 ,4]dioxane, oxazolidine, piperazine, and the like a further example is morpholine. Other examples of heterocyclic rings include the oxidised forms of the sulfur-containing rings. Thus,

tetrahydrothiophene-1 -oxide, tetrahydrothiophene-1 , 1 -dioxide, tetrahydrothiopyran-1 -oxide and tetrahydrothiopyran-1 , 1 -dioxide are also considered to be heterocyclic rings.

Aryl as used herein includes C₆-Ci₄ mono or bicyclic groups having 1 or 2 rings wherein at least one ring is aromatic, including phenyl, naphthyl, 5,6,7,8-tetrahydronaphthyl and the like, such as phenyl and napthyl particularly phenyl. Heteroaryl as used herein includes 5- to 10-membered aromatic mono or bicyclic ring systems comprising one or more, (for example 1 , 2, 3 or 4) heteroatoms independently selected from O, N and S. Examples of heteroaryl groups include pyrrole, furan, thiophene, oxazole, thiazole, isothiazole, oxadiazole, tetrazole, imidazole, pyrazole, isoxazole, pyridine, pyridazine, pyrimidine, pyrazine, benzothiophene, benzofuran, 1 , 2, 3-triazole and 1 , 2, 4-triazole. In a bicyclic ring system the definition of heteroaryl will be satisfied if at least one ring contains a heteroatom and at least one ring is aromatic. The heteroaryl may be linked to the remainder of the molecule through a carbocyclic ring or a ring comprising a heteroatom.

Examples of salts of the compounds of formula (I) include all pharmaceutically acceptable salts prepared from pharmaceutically acceptable non-toxic bases or acids. Salts derived from bases include, for example, potassium and sodium salts and the like. Salts derived from acids, include those derived from inorganic and organic acids such as, for example, hydrochloric,

methanesulfonic, sulfuric and p-toluenesulfonic acid and the like. Examples of solvates include hydrates.

The compounds described herein may include one or more chiral centers, and the disclosure extends to include racemates, enantiomers and stereoisomers resulting therefrom. In one embodiment one enantiomeric form is present in a substantially purified form that is

substantially free of the corresponding enantiomeric form.

The invention also extends to all polymorphic forms of the compounds 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, fluorine, such as ²H, ³H, ¹¹C, ¹⁴C and ¹⁸F. 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 invention extends to all tautomeric forms of the compounds illustrated herein (particularly enol-keto tautomers). For example whereas formula (I) illustrates in some embodiments (e.g. when R^4a and/or R^4b represents H) an enol form, the corresponding keto form is also embraced as part of the invention. The same applies to other structures herein which illustrate enol or keto forms of compounds. Similarly, the disclaimed compounds are disclaimed in all their tautomeric forms.

Compounds of the invention may be prepared by the following methods or by methods analogous thereto or by using conventional methods known to a skilled person:

A general method for preparing compounds of formula (I) in which R and R represent hydrogen is shown below in Scheme A:

R³ Base R³ _Base R³ NaOEt/EtOH _r3

(I)

Scheme A

In the third step shown in Scheme A, R^x typically represents CrC₆ alkyl such as methyl or ethyl.

A method for preparing certain compounds of formula (I) in which R¹ is -C(0)NR⁸R⁹, R² is - C(0)OR⁷ and R^4a and R^4b represent hydrogen is shown below in Scheme B:

Scheme B

In the second step shown in Scheme B, R^x typically represents CrC₆ alkyl such as methyl or ethyl.

An alternative method for preparing certain compounds of formula (I) in which R¹ is - C(0)NR⁸R⁹, R² is -C(0)OR⁷and R^4a and R^4b represent hydrogen is shown below in Scheme C:

Scheme C A method for preparing certain compounds of formula (I) in which R¹ is -C(0)NHR⁸, R² is - C(0)NR⁵R⁶ (or -C(0)NR⁸R⁹) and R^4a and R^4b represent hydrogen is shown below in Scheme D:

(I) (I) (I)

Scheme D

A method for preparing certain compounds of formula (I) in which R¹ and R² are -C(0)NR⁸R^! and R^4a and R^4b represent hydrogen is shown below in Scheme E:

(I)

Scheme E

A method for preparing certain compounds of formula (I) in which R^4a and R^4b represent groups other than hydrogen is shown below in Scheme F:

(I) (I)

Scheme F Where X are independently leaving groups such as halogen, an ester (-OCOR', giving a mixed anhydride), or hydrogen, when used in combination with a suitable coupling agent, such as: 1- ethyl-3-(3-dimethylaminopropyl)carbodiimide) (EDC), Λ/,Λ/'-diisopropylcarbodiimide (DIC) or 1 , 1'- carbonyldiimidazole (CDI). Suitably X is halogen. Scheme F may be adapted to convert one or both hydroxyl groups to OR^4a and/or OR^4b depending on the molar excess of reagent(s) employed. When R^4a and R^4b are different, it may be necessary to employ a protection strategy to incorporate one and then the other group. This process is also suitable for preparing prodrug derivatives of compounds of formula (I). In the above Schemes A to F the general conditions for performing the reactions specified will be well known to a skilled person.

Compounds of formula (I) may be converted to different compounds of formula (I) by the above methods and/or by conventional methods.

For example the skilled person will be familiar with standard procedures for converting carboxylic acids to esters, amides, carbamates and ureas and for converting amines to amides and sulphonamides. Thus compounds of formula (I) in which R¹ and/or R² represents -C(0)NHC(0)R⁷ may be prepared by reaction of a compound of formula (I) in which R¹ and/or R² represents -C(0)NH₂ with a compound of formula R⁷C(0)L wherein L represents a leaving group, such as halogen.

Protecting groups may be required to protect chemically sensitive groups during one or more of the reactions described above, 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 Wley & Sons Inc; 4^th Rev Ed., 2006, ISBN-10: 0471697540.

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, for example, compounds of formula (XII) and analogous compounds in which Bn (benzyl) is replaced by another hydroxyl protecting group are claimed as an aspect of the invention.

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

wherein R¹ R² and R³ are as defined above for the compounds of formula (I), or a salt or protected derivative thereof. Any preferences or examples of specific groups as described above for the compounds of formula (I) also apply to the definitions of specific groups in the compounds of formula (II).

There is also provided a process for preparing compounds of formula (I) in which R^4a and R^4b represent H which comprises reacting a compound of formula (II) with a compound of formula ROCOCOOR^x in which R^x represents C C₆ alkyl. This process is typically performed in a polar protic solvent such as ethanol in the presence of a strong base such as sodium ethoxide.

Compounds of formula (I) are referred to below as "compounds of the invention".

As indicated above the compounds of the invention are useful for treatment of bacterial infections caused by bacteria producing pore-forming toxins, such as cholesterol dependent cytolysins. In particular the compounds of the invention are useful for the treatment of toxaemia associated with bacterial infections.

For such use the compounds 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 compounds of the invention are inhibitors of the cholesterol-dependent cytolysin, pneumolysin, produced by the bacterium Streptococcus pneumoniae. They also inhibit Streptolysin O (SLO) produced by Group A Streptococci and Perfringolysin O (PFO) produced by Clostridium perfringens. They are 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 compounds of the invention are 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 compounds of the invention are 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 compounds of the invention are 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 compounds of the invention are 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 compounds of the invention are 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 compounds of the invention are 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 compounds 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:

-A 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);

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

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

-A 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 compounds 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 compounds of the invention may be used either alone or in combination with further therapeutically active ingredients. Thus compounds 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 compounds 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 compounds 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

COPD chronic obstructive pulmonary disease

d doublet

DCM dichloromethane

DIPEA Ay.Ay-diisopropylethylamine

DMAP 4-dimethylaminopyridine

DMF Ay.Ay-dimethylformamide

DMSO dimethylsulfoxide

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

EtOAc ethyl acetate

h hour(s)

HATU Ay,Ay,Ay',Ay-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

quin. quintet

RT room temperature

s singlet

sat. saturated

SAX solid supported strong cation exchange resin

sept. septet

sext. sextet

t triplet

TFAA trifluoroacetic acid anhydride

THF tetrahydrofuran

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_). Solubility studies were carried out by charging a vial with 5-10 mg of compound followed by the addition of PBS solution 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. 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 1 100 with or an Agilent Infinity 1260 LC with 6120 quadrupole mass spectrometer with positive and negative ion electrospray.

Preparative HPLC was carried out using an Agilent Prep-C18 5 μηι Preparative Cartridge using either a gradient of 0.1 % formic acid in MeCN in 0.1 % aqueous formic acid or a gradient of MeCN in 10 mM Ammonium Bicarbonate, Alternatively, a Waters Xselect CSH C18 5 μηι column using a gradient 0.1 % MeCN in 0.1 % aqueous formic acid. Fractions were collected following detection by UV at 254nm.

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 compounds of formula (I) are prepared using the following general methods:

Example A: N -(2-(Dimethylamino)ethyl)-3,4-dihydroxy-1-(4-methoxyphenyl)-/\/ ,N ,N -trimethyl 1 /-/-pyrrole-2,5-dicarboxamide (UL9-001)

1

step ( vi)

6 UL9-001 The above route was used to prepare UL9-001 as its hydrochloride salt, as follows:

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): Triethylammonium 3,4-bis(benzyloxy)-5-(ethoxycarbonyl)-1-(4-methoxyphenyl)-1 /-/- pyrrole-2-carboxylate (4)

To a solution of diethyl 3,4-bis(benzyloxy)-1-(4-methoxyphenyl)-1 /-/-pyrrole-2,5-dicarboxylate (3) (39.6 g, 74.8 mmol) in THF/EtOH (300/50 mL) was added NaOH (3.07 g, 77 mmol) as a solution in water (20 mL). The reaction was stirred at 50°C for 16h. Triethylamine was added (30 mL, 215 mmol) and the volatiles were removed in vacuo. The residue was purified by silica gel chromatography (50% isohexane:DCM (+2% Et₃N), then 20%MeOH/EtOAc (+2% Et₃N)) to afford triethylammonium 3,4-bis(benzyloxy)-5-(ethoxycarbonyl)-1-(4-methoxyphenyl)-1 /-/- pyrrole-2-carboxylate (5) (39.3 g, 83 %) as a yellow oil: m/z 502 (M+H)⁺ (ES⁺); 500 (M-H)^" (ES^"). ¹ H NMR (400 MHz, DMSO-d₆) δ: 7.51-7.26 (m, 10H), 7.11-7.05 (m, 2H), 6.92-6.83 (m, 2H), 5.09 (s, 2H), 5.06 (s, 2H), 3.95 (q, J = 7.1 Hz, 2H), 3.79 (s, 3H), 2.85-2.62 (m, 6H), 1.08-0.92 (m, 12H).

Step (v): Ethyl 3,4-bis(benzyloxy)-5-(dimethylcarbamoyl)-1-(4-methoxyphenyl)-1 /-/-pyrrole-2- carboxylate (5)

To a solution of triethylammonium 3,4-bis(benzyloxy)-5-(ethoxycarbonyl)-1-(4-methoxyphenyl)- 1 H-pyrrole-2-carboxylate (5) (10.84 g, 17.99 mmol) in DMF (150 mL), at 0°C was added HATU (10.26 g, 27.0 mmol), dimethylamine hydrochloride (2.93 g, 36.0 mmol) and DIPEA (18.8 mL, 108 mmol). The reaction mixture was stirred at RT for 16h and partitioned between EtOAc (500 mL) and 1 M HCI (aq.) (250 mL). The organic phase was washed succesively with 1 M HCI (aq.) (250 mL), sat. NaHC0₃ (aq.) (2 x 250 mL), and brine (2 x 250 mL), dried (MgS0₄), filtered and concentrated in vacuo to afford ethyl 3,4-bis(benzyloxy)-5-(dimethylcarbamoyl)-1-(4- methoxyphenyl)-1 /-/-pyrrole-2-carboxylate (6) (7.62 g, 79 %) as a light yellow oil, that solidified on standing: m/z 529 (M+H)⁺ (ES⁺). ¹ H NMR (400 MHz, DMSO-d₆) δ: 7.51-7.21 (m, 10H), 7.14- 7.03 (m, 2H), 6.94-6.84 (m, 2H), 5.12 (s, 2H), 4.96 (s, 2H), 4.00 (q, J = 7.1 Hz, 2H), 3.77 (s, 3H), 2.70 (s, 6H), 1.00 (t, J = 7.1 Hz, 6H).

Step (vi): 3,4-bis(benzyloxy)-/V²-(2-(dimethylamino)ethyl)-1-(4-methoxyphenyl)-/\/²,/\/⁵,/\/⁵- trimethyl-1 /-/-pyrrole-2,5-dicarboxamide (6)

To a stirred solution of ethyl 3,4-bis(benzyloxy)-5-(dimethylcarbamoyl)-1-(4-methoxyphenyl)-1 H- pyrrole-2-carboxylate (1.0 g, 1.892 mmol) and A/\A/\A/²-trimethylethane-1 ,2-diamine (0.49 mL, 3.78 mmol) in THF (20 mL) at 0 °C was added isopropylmagnesium chloride (2.36 mL, 4.73 mmol). The reaction mixture was allowed to warm to RT and stirred for 1 h. The reaction mixture was quenched with ammonium chloride (aq.) (10 mL). The solution was then diluted with water (60 mL) and extracted with ethyl acetate (2 x 60 mL). The combined organic layers were washed with brine (80 mL), dried over magnesium sulphate, filtered and concentrated in vacuo to give a pale yellow oil. The residue was purified by silica gel chromatography (0-7% metha- nol(1 % NH₃) in DCM) to afford 3,4-bis(benzyloxy)-/V²-(2-(dimethylamino)ethyl)-1-(4- methoxyphenyl)-/V²,/\/⁵,/\/⁵-trimethyl-1 /-/-pyrrole-2,5-dicarboxamide (6) (0.75 g, 67%) as a cream solid, m/z 585 (M+H)⁺ (ES⁺).

Step (vii): /\/²-(2-(Dimethylamino)ethyl)-3,4-dihydroxy-1-(4-methoxyphenyl)-/\/², A/⁵,A/⁵-trimethyl- 1 /-/-pyrrole-2,5-dicarboxamide (UL9-001) hydrochloride

A solution of 3,4-bis(benzyloxy)-/V²-(2-(dimethylamino)ethyl)-1-(4-methoxyphenyl)-/\/²,/\/⁵,/\/⁵- trimethyl-1 /-/-pyrrole-2,5-dicarboxamide (6) (220 mg, 0.376 mmol) in methanol (20 mL) was passed through a Thales Ή-cube' cartridge (10% Pd/C, 30x4 mm) at a flow rate of 1 mL/min at 40°C under H₂ (full H₂ mode). The output was collected in a flask containing 4 M HCI in dioxane (941 μί, 3.76 mmol). The resulting solution was concentrated in vacuo then was re-dissolved in water (10 mL). The solution was freeze-dried to afford A/²-(2-(dimethylamino)ethyl)-3,4- dihydroxy-1 -(4-methoxyphenyl)-/\/²,/\/⁵,/\/⁵-trimethyl-1 H-pyrrole-2,5-dicarboxamide (UL9-001 ) hydrochloride (0.13 g, 76 %) as a yellow solid, m/z 405.3 (M+H)⁺ (ES⁺); 403.1 (M-H)^" (ES^"). ¹ H NMR (400 MHz, DMSO-d₆, 60 °C) δ 10.37 (br s, 1 H), 7.01 -6.97 (m, 2H), 6.87-6.83 (m, 2H), 3.77 (s, 3H), 3.66 (t, J = 7.0 Hz, 2H), 3.20-3.14 (m, 2H), 3.03 (s, 3H), 2.89 (s, 6H), 2.75-2.74 (m, 6H).

Example B: 2-((2-(Dimethylamino)ethyl)(methyl)carbarnoyl)-5-(dirnethylcarbarnoyl)-1 -(4- methoxyphenyl)-1 /-/-pyrrole-3,4-diyl bis(2-methylpropanoate) (UL9-002)

This compound (e.g. as the formate salt) may be prepared from A/²-(2-(Dimethylamino)ethyl)- 3,4-dihydroxy-1 -(4-methoxyphenyl)-/V²,/\/⁵,/\/⁵-trimethyl-1 /-/-pyrrole-2,5-dicarboxamide (UL9-001 ) e.g. as the hydrochloride which is synthesised by methods described in the description above.

UL9-001

UL9-002 Step (i): 2-((2-(Dimethylamino)ethyl)(methyl)carbamoyl)-5-(dimethylcarbamoyl)-1 -(4- methoxyphenyl)-1 /-/-pyrrole-3,4-diyl bis(2-methylpropanoate) formate (UL9-002)

To a suspension of 2-fe/f-Butylimino-2-diethylamino-1 ,3-dimethylperhydro-1 ,3,2- diazaphosphorine (polymer-bound_:2.2 mmol/g) (310 mg, 0.680 mmol) and N²-(2- (dimethylamino)ethyl)-3,4-dihydroxy-1 -(4-methoxyphenyl)-/\/², A/⁵,A/⁵-trimethyl-1 /-/-pyrrole-2,5- dicarboxamide hydrochloride (UL9-001 ) (100 mg, 0.227 mmol) in DCM (5 ml_) at 0°C was added isobutyryl chloride (180 μΙ_, 1.70 mmol) and the mixture allowed to warm to ambient temperature and shaken for 30 mins. After this time it was filtered, solvents removed under reduced pressure and the resulting pale yellow oil purified by preparative HPLC (Gilson, Acidic (0.1 % Formic acid), Acidic, Waters X-Select Prep-C18, 5 μηι, 19x50 mm column, 15-40%

MeCN in Water) to afford 2-((2-(dimethylamino)ethyl)(methyl)carbamoyl)-5-(dimethylcarbamoyl)- 1 -(4-methoxyphenyl)-1 /-/-pyrrole-3,4-diyl bis(2-methylpropanoate) formate (UL9-002) (0.03 g, 21 %) as a pale yellow gum/solid, m/z 545 (M+H)⁺ (ES). ¹ H NM R (400 MHz, DMSO-d₆) δ 8.14 (s, 1 H), 7.19-7.1 1 (m, 2H), 6.97-6.95 (m, 2H), 3.78 (s, 3H), 3.44-3.39 (m, 2H), 2.88-2.72 (m, 1 1 H), 2.39-2.26 (m, 6H), 2.17-2.09 (m, 2H), 1 .18-1 .16 (m, 12 H).

Example C: 3,4-Dihydroxy-1 -(4-methoxyphenyl)-/V²,/\/⁵-dimethyl-/\/²,/\/⁵-bis(1 -methylpiperidin-4- yl)-1 /-/-pyrrole-2,5-dicarboxamide (UL9-003) This compound as its dihydrochloride salt may be prepared by the following method:

UL9-003

Step (i): Ethyl 3,4-bis(benzyloxy)-1-(4-methoxyphenyl)-5-(methyl(1-methylpiperidin-4- yl)carbamoyl)-1 /-/-pyrrole-2-carboxylate (7) and 3,4-bis(benzyloxy)-1-(4-methoxyphenyl)-/V²,/\/⁵- dimethyl-/V²,/\/⁵-bis(1-methylpiperidin-4-yl)-1 /-/-pyrrole-2,5-dicarboxamide (8)

To a stirred solution of diethyl 3,4-bis(benzyloxy)-1-(4-methoxyphenyl)-1 H-pyrrole-2,5- dicarboxylate (0.5 g, 0.944 mmol) and A/,1-dimethylpiperidin-4-amine (0.484 g, 3.78 mmol) in THF (10 mL) at 0 °C was added isopropylmagnesium chloride (1.89 mL, 3.78 mmol). The reaction mixture was allowed to warm to RT and stirred for 2 h. The reaction mixture was quenched with ammonium chloride (aq.) (10 mL). The solution was then diluted with water (60 mL) and extracted with ethyl acetate (2 x 60 mL). The combined organic layers were washed with brine (40 mL), dried over magnesium sulphate, filtered and concentrated in vacuo to give a pale orange oil. The residue was purified by silica gel chromatography (0-10% methanol(1 % NH₃) in DCM) to afford ethyl 3,4-bis(benzyloxy)-1-(4-methoxyphenyl)-5-(methyl(1- methylpiperidin-4-yl)carbamoyl)-1 /-/-pyrrole-2-carboxylate (7) (0.23 g, 39%) as the first eluting product, as an off-white solid, m/z 612 (M+H)⁺ (ES⁺). And 3,4-bis(benzyloxy)-1-(4- methoxyphenyl)-/V²,/\/⁵-dimethyl-/\/²,/\/⁵-bis(1-methylpiperidin-4-yl)-1 /-/-pyrrole-2,5-dicarboxamide (8) (0.06 g, 9 %) as the second eluting product, as a cream solid, m/z 347 ((M+2)/2)⁺ (ES⁺).

Step (ii): 3,4-Dihydroxy-1-(4-methoxyphenyl)-/V²,/\/⁵-dimethyl-/\/²,/\/⁵-bis(1-methylpiperidin-4-yl)- 1 /-/-pyrrole-2,5-dicarboxamide dihydrochloride (UL9-003)

A solution of 3,4-bis(benzyloxy)-1-(4-methoxyphenyl)-/V²,/\/⁵-dimethyl-/\/²,/\/⁵-bis(1- methylpiperidin-4-yl)-1 /-/-pyrrole-2,5-dicarboxamide (8) (60 mg, 0.086 mmol) in methanol (6 mL) was passed through a Thales Ή-cube' cartridge (10% Pd/C, 30x4 mm) at a flow rate of 1 mL/min at 40°C under H₂ (full H₂ mode). The output was collected in a flask containing 4 M HCI in dioxane (216 μί, 0.865 mmol). The resulting solution was concentrated in vacuo then was re- dissolved in water (5 mL). The solution was freeze-dried to afford 3,4-dihydroxy-1-(4- methoxyphenyl)-/V²,/\/⁵-dimethyl-/\/²,/\/⁵-bis(1-methylpiperidin-4-yl)-1 /-/-pyrrole-2,5-dicarboxamide dihydrochloride (UL9-003) (0.026 g, 48%) as a yellow solid, m/z 514 (M+H)⁺ (ES⁺); 512 (M-H)^" (ES^"). ¹ H NMR (400 MHz, DMSO-d₆, 60 °C) δ 10.21 (br s, 2H), 8.42 (br s, 2H), 6.97-6.93 (m, 2H), 6.87-6.83 (m, 2H), 4.25-4.19 (m, 2H), 3.76 (s, 3H), 3.42 (d, J = 10.4 Hz, 4H), 3.05 (q, J = 10.4 Hz, 4H), 2.81 (s, 6H), 2.72 (d, J = 2.9 Hz, 6H), 2.09 (dq, J = 13.2, 3.5 Hz, 4H), 1.65 (d, J = 13.2 Hz, 4H).

Example D: Ethyl 3,4-dihydroxy-1-(4-methoxyphenyl)-5-(methyl(1-methylpiperidin-4- yl)carbamoyl)-1 /-/-pyrrole-2-carboxylate (UL9-004)

This compound as its hydrochloride salt may be prepared by the following method:

Step (i): ethyl 3,4-dihydroxy-1-(4-methoxyphenyl)-5-(methyl(1-methylpiperidin-4-yl)carbamoyl)- 1 /-/-pyrrole-2-carboxylate hydrochloride (UL9-004) A solution of ethyl 3,4-bis(benzyloxy)-1-(4-methoxyphenyl)-5-(methyl(1-methylpiperidin-4- yl)carbamoyl)-1 /-/-pyrrole-2-carboxylate (7) (230 mg, 0.376 mmol) in methanol (20 mL) was passed through a Thales Ή-cube' cartridge (10% Pd/C, 30x4 mm) at a flow rate of 1 mL/min at 40°C under H₂ (full H₂ mode). The output was collected in a flask containing 4 M HCI in dioxane (940 μί, 3.76 mmol). The resulting solution was concentrated in vacuo then was re-dissolved in water (10 mL). The solution was freeze-dried to afford ethyl 3,4-dihydroxy-1-(4-methoxyphenyl)- 5-(methyl(1-methylpiperidin-4-yl)carbamoyl)-1 /-/-pyrrole-2-carboxylate hydrochloride (UL9-004) (0.16 g, 87%) as an off-white solid, m/z 432 (M+H)⁺ (ES⁺); 430 (M-H)^" (ES^"). ¹ H NMR (400 MHz, DMSO-de, 60 °C) δ 10.61 (br s, 1 H), 8.45 (s, 1 H), 8.38 (s, 1 H), 7.10-7.06 (m, 2H), 6.89-6.85 (m, 2H), 4.11-4.00 (br s, 1 H), 4.03 (q, J = 7.2 Hz, 2H), 3.78 (s, 3H), 3.37 (d, J = 10.4 Hz, 2H), 3.07- 3.00 (m, 2H), 2.73 (s, 3H), 2.70 (s, 3H), 2.02 (dq, J = 13.2, 3.9 Hz, 2H), 1.51 (d, J = 13.2 Hz, 2H), 1.02 (t, J = 7.2 Hz, 3H).

Example E: A/²,A/⁵-bis(3-(dimethylamino)propyl)-3,4-dihydroxy-1-(4-methoxyphenyl)-/\/²,/\/⁵- dimethyl-1 /-/-pyrrole-2,5-dicarboxamide (UL9-005)

This compound as its dihydrochloride salt may be prepared by the following method:

UL9-005

Step (i): 3,4-bis(benzyloxy)-/V²,/\/⁵-bis(3-(dimethylamino)propyl)-1-(4-methoxyphenyl)-/\/²,/\/⁵- dimethyl-1 /-/-pyrrole-2,5-dicarboxamide (9) To a stirred solution of diethyl 3,4-bis(benzyloxy)-1-(4-methoxyphenyl)-1 H-pyrrole-2,5- dicarboxylate (3) (0.5 g, 0.944 mmol) and ^A/^-trimethylpropane-I .S-diamine (0.55 mL, 3.78 mmol) in THF (10 mL) at 0 °C was added isopropylmagnesium chloride (1.89 mL, 3.78 mmol). The reaction mixture was allowed to warm to RT and stirred for 1 h. The reaction mixture was quenched with ammonium chloride (aq.) (10 mL). The solution was then diluted with water (20 mL) and extracted with ethyl acetate (40 mL). The organic layer was washed with saturated sodium bicarbonate solution (20 mL) and brine (20 mL), dried over magnesium sulphate, filtered and concentrated in vacuo to give a pale yellow solid. The residue was purified by silica gel chromatography (0-10% methanol(1 % NH₃) in DCM) to afford 3,4-bis(benzyloxy)-/V²,/V⁵-bis(3- (dimethylamino)propyl)-1-(4-methoxyphenyl)-/\/²,/\/⁵-dimethyl-1 /-/-pyrrole-2,5-dicarboxamide (9) (0.37 g, 57%) as a white solid, m/z 670.5 (M+H)⁺ (ES⁺). Step (ii): 3,4-Dihydroxy-1-(4-methoxyphenyl)-A/²,A/⁵-dimethyl-A/²,A/⁵-bis(1-methy

1 /-/-pyrrole-2,5-dicarboxamide dihydrochloride (UL9-005)

3,4-bis(benzyloxy)-A/²,A/⁵-bis(3-(dimethylamino)propylH

pyrrole-2,5-dicarboxamide (9) (360 mg, 0.537 mmol) in methanol (20 mL) was passed through a Thales Ή-cube' cartridge (10% Pd/C, 30x4 mm) at a flow rate of 1 mL/min at 40°C under H₂ (full H₂ mode). The output was collected in a flask containing 4 M HCI in dioxane (941 μί, 3.76 mmol). The resulting solution was concentrated in vacuo then was re-dissolved in water (15 mL). The solution was freeze-dried to afford 3,4-dihydroxy-1-(4-methoxyphenyl)-A/²,A/⁵-dimethyl- A/²,/\/⁵-bis(1-methylpiperidin-4-yl)-1 /-/-pyrrole-2,5-dicarboxamide dihydrochloride (UL9-005) (0.28 g, 88%) as a yellow solid, m/z 490 (M+H)⁺ (ES⁺); 488 (M-H)^" (ES^"). ¹ H NMR (400 MHz, DMSO- d₆, 60 °C) δ 10.44 (br s, 2H), 7.06-7.02 (m, 2H), 6.92-6.88 (m, 2H), 3.77 (s, 3H), 3.38 (t, J = 6.9 Hz, 4H), 2.99 (s, 6H), 2.90-2.85 (m, 4H), 2.67 (d, J = 4.8 Hz, 12H), 1.93-1.85 (m, 4H). Example F: 3,4-Dihydroxy-1-(4-methoxyphenyl)-/V²,/\/⁵-dimethyl-/\/²,/\/⁵-di(pyridin-3-yl)-1 /-/- pyrrole-2,5-dicarboxamide (UL9-006)

This compound as its dihydrochloride salt may be prepared by the following method:

UL9-006

Step (i): Ethyl 3,4-bis(benzyloxy)-1-(4-methoxyphenyl)-5-(methyl(pyridin-3-yl)carbamoyl)-1 /-/- pyrrole-2-carboxylate (10) and 3,4-bis(benzyloxy)-1-(4-methoxyphenyl)-/V²,/\/⁵-dimethyl-/\/²,/\/⁵- di(pyridin-3-yl)-1 H-pyrrole-2,5-dicarboxamide (11)

To a stirred solution of diethyl 3,4-bis(benzyloxy)-1-(4-methoxyphenyl)-1 H-pyrrole-2,5- dicarboxylate (0.5 g, 0.944 mmol) and A/-methylpyridin-3-amine (0.39 ml_, 3.78 mmol) in THF (10 ml_) at 0 °C was added isopropylmagnesium chloride (1.89 ml_, 3.78 mmol). The reaction mixture was allowed to warm to RT and stirred for 4 h. The reaction mixture was quenched with ammonium chloride (aq.) (10 ml_). The solution was then diluted with water (20 ml_) and extracted with ethyl acetate (40 ml_). The organic layer was washed with saturated sodium bicarbonate solution (20 ml_) and brine (40 ml_), dried over magnesium sulphate, filtered and concentrated in vacuo to give a pale orange oil. The residue was purified by silica gel chromatography (0-10% methanol(1 % NH₃) in DCM) to afford ethyl 3,4-bis(benzyloxy)-1-(4- methoxyphenyl)-5-(methyl(pyridin-3-yl)carbamoyl)-1 /-/-pyrrole-2-carboxylate (10) (0.34 g, 57%) as the first eluting product, as a pale yellow solid, m/z 592 (M+H)⁺ (ES⁺). And 3,4- bis(benzyloxy)-1-(4-methoxyphenyl)-/\/²,/\/⁵-dimethyl-/\/²,/\/⁵-di(pyridin-3-yl)-1 H-pyrrole-2,5- dicarboxamide (11) (0.19 g, 29%) as the second eluting product, as an off-white solid, m/z 654 (M+H)⁺ (ES⁺).

Step (ii): 3,4-Dihydroxy-1-(4-methoxyphenyl)-A/²,^

2,5-dicarboxamide dihydrochloride (UL9-006)

3,4-bis(benzyloxy)-1-(4-methoxyphenyl)-A/²,A/⁵-dimethyl-A/²,A/⁵-di(pyridin-3-yl)-1 H

dicarboxamide (11) (190 mg, 0.291 mmol) in methanol (20 mL) was passed through a Thales Ή-cube' cartridge (10% Pd/C, 30x4 mm) at a flow rate of 1 mL/min at 40°C under H₂ (full H₂ mode). The output was collected in a flask containing 4 M HCI in dioxane (727 μΙ_, 2.91 mmol). The resulting solution was concentrated in vacuo then was re-dissolved in water (10 mL). The solution was freeze-dried to afford 3,4-dihydroxy-1-(4-methoxyphenyl)-/V²,/\/⁵-dimethyl-/\/²,/\/⁵- di(pyridin-3-yl)-1 /-/-pyrrole-2,5-dicarboxamide dihydrochloride (UL9-006) (0.13 g, 83%) as an orange sticky solid, m/z 474 (M+H)⁺ (ES⁺); 472 (M-H)^" (ES^"). ¹ H NMR (400 MHz, DMSO-d₆, 60 °C) δ 8.51 (d, J = 4.9 Hz, 1 H), 8.39 (br s, 1 H), 8.02 (d, J = 2.7 Hz, 1 H), 8.00-7.98 (m, 1 H), 7.77- 7.75 (m, 2H), 7.74-7.70 (m, 2H), 7.67-7.63 (m, 4H), 6.98-6.94 (m, 2H), 6.92-6.88 (m, 2H), 3.82 (s, 3H), 3.27 (s, 6H).

Example G: Ethyl 3,4-dihydroxy-1-(4-methoxyphenyl)-5-(methyl(pyridin-3-yl)carbamoyl)-1 /-/- pyrrole-2-carboxylate (UL9-007)

This compound as its hydrochloride salt may be prepared by the following method:

UL9-007

Step (i): ethyl 3,4-dihydroxy-1-(4-methoxyphenyl)-5-(methyl(pyridin-3-yl)carbamoyl)-1 /-/-pyrrole- 2-carboxylate hydrochloride (UL9-007)

A solution of ethyl 3,4-bis(benzyloxy)-1-(4-methoxyphenyl)-5-(methyl(pyridin-3-yl)carbamoyl)- 1 /-/-pyrrole-2-carboxylate (10) (340 mg, 0.575 mmol) in methanol (20 mL) was passed through a Thales Ή-cube' cartridge (10% Pd/C, 30x4 mm) at a flow rate of 1 mL/min at 40°C under H₂ (full H₂ mode). The output was collected in a flask containing 4 M HCI in dioxane (1437 μί, 5.75 mmol). The resulting solution was concentrated in vacuo then was re-dissolved in water (15 mL). The solution was freeze-dried to afford ethyl 3,4-dihydroxy-1-(4-methoxyphenyl)-5- (methyl(pyridin-3-yl)carbamoyl)-1 /-/-pyrrole-2-carboxylate hydrochloride (UL9-007) (0.22 g, 82%) as a pale yellow solid, m/z 412 (M+H)⁺ (ES⁺). ¹ H NMR (400 MHz, DMSO-d₆, 60 °C) δ 8.40 (dd, J = 4.7, 1.4 Hz, 1 H), 8.25 (d, J = 1.4 Hz, 1 H), 7.50 (ddd, J = 8.2, 2.5, 1.4 Hz, 1 H), 7.39 (dd, J = 8.2, 4.7 Hz, 1 H), 7.04-7.00 (m, 2H), 6.93-6.89 (m, 2H), 3.99 (q, J = 7.1 Hz, 2H), 3.81 (s, 3H), 3.22 (s, 3H), 0.98 (t, J = 7.1 Hz, 3H). Example H: A/²,A/⁵-bis(2-(dimethylamino)ethyl)-3,4-dihydroxy-1-(4-methoxypheny dimethyl-1 /-/-pyrrole-2,5-dicarboxamide (UL9-008) This compound as its dihydrochloride salt may be prepared by the following method:

UL9-008 Step (i): 3,4-bis(benzyloxy)-/V²,/\/⁵-bis(2-(dimethylamino)ethyl)-1-(4-methoxyphenyl)-/\/²,/\/⁵- dimethyl-1 /-/-pyrrole-2,5-dicarboxamide (12)

To a stirred solution of diethyl 3,4-bis(benzyloxy)-1-(4-methoxyphenyl)-1 H-pyrrole-2,5- dicarboxylate (3) (0.5 g, 0.944 mmol) and A/\A/\A/²-trimethylethane-1 ,2-diamine (0.49 mL, 3.78 mmol) in THF (10 mL) at 0 °C was added isopropylmagnesium chloride (1.89 mL, 3.78 mmol). The reaction mixture was allowed to warm to RT and stirred for 1 h. The reaction mixture was quenched with ammonium chloride (aq.) (10 mL). The solution was then diluted with water (20 mL) and extracted with ethyl acetate (40 mL). The organic layer was washed with saturated sodium bicarbonate solution (20 mL) and brine (20 mL), dried over magnesium sulphate, filtered and concentrated in vacuo to give a pale yellow solid. The residue was triturated with EtOAc (5 mL) and isolated by filtration to afford 3,4-bis(benzyloxy)-/V²,/\/⁵-bis(2-(dimethylamino)ethyl)-1-(4- methoxyphenyl)-/V²,/\/⁵-dimethyl-1 /-/-pyrrole-2,5-dicarboxamide (12) (0.28 g, 46%) as a white solid, m/z 642 (M+H)⁺ (ES⁺). Step (ii): A/²,/\/⁵-bis(2-(dimethylamino)ethyl)-3,4-dihydroxy-1-(4-methoxyphenyl)-/\/²,/\/⁵-dimethyl- 1 /-/-pyrrole-2,5-dicarboxamide dihydrochloride (UL9-008) 3,4-bis(benzyloxy)-A/²,A/⁵-bis(2-(dimethylamino)ethyl)-1-(4-methoxyphen

pyrrole-2,5-dicarboxamide (12) (50 mg, 0.078 mmol) in methanol (5 mL) was passed through a Thales Ή-cube' cartridge (10% Pd/C, 30x4 mm) at a flow rate of 1 mL/min at 40°C under H₂ (full H₂ mode). The output was collected in a flask containing 4 M HCI in dioxane (195 μί, 0.779 mmol). The resulting solution was concentrated in vacuo then was re-dissolved in water (10 mL). The solution was freeze-dried to afford A/²,/\/⁵-bis(2-(dimethylamino)ethyl)-3,4-dihydroxy-1- (4-methoxyphenyl)-A/²,A/⁵-dimethyl-1 /-/-pyrrole-2,5-dicarboxamide dihydrochloride (UL9-008) (0.04 g, 98%) as a pale yellow solid, m/z 462 (M+H)⁺ (ES⁺); 460 (M-H)^" (ES^"). ¹ H NMR (400 MHz, DMSO-cfe, 60 °C) δ 10.50 (br s, 2H), 7.05-7.01 (m, 2H), 6.87-6.83 (m, 2H), 3.77 (s, 3H), 3.67 (t, J = 6.9 Hz, 4H), 3.20-3.31 (m, 4H), 3.03 (s, 6H), 2.74 (s, 12H).

The following Examples in Table 1 were prepared using the methods above.

Table 1

- xamp e

Biological Testing

There is provided below a summary of the primary in vitro assay performed with the examples presented in Table 1 and a description of further assays which may be performed with the compounds of the invention.

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) are diluted 1 : 1 in 100% DMSO. The compounds are then two-fold serially diluted in 100% DMSO across 1 1 wells of 96-well round- bottomed microtitre plate. PBS is then added to all the wells to achieve a 1 : 10 (v/v) dilution of the compound in PBS. Pneumolysin is then added at a concentration equal to its LD100. Plates are then incubated at 37°C for 30-40 min. After the incubation period, an equal volume of 4% (v/v) sheep erythrocyte suspension is 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) are prepared following the same procedure. Following the incubation with the erythrocytes, the Absorbance at 595 nm of each well is measured and the data used to determine the IC₅₀ for each test compound. The IC₅₀ values are determined using non-linear regression curve fitting. For that, the Log of the concentrations of the test compound is 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 compounds UL9-001 , UL9-003, UL9-004, UL9-005, UL9-006, UL9-007 and UL9-008. Generally, in the case of prodrugs, 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 inhibitory activity in the presence of the example UL9-002, due to the enzymatic cleavage of the prodrug moieties, occurring during the 40 min incubation in blood, which leads to the release of the parent active compound. In summary, this assay demonstrates the in vitro inhibitory activity of all the examples of this invention. IC₅₀ values obtained following the above experimental procedure are presented in Table 2.

Table 2

B. PRIMARY IN VITRO ASSAY: INHIBITION OF THE HAEMOLYTIC ACTIVITY OF OTHER CHOLESTEROL DEPENDENT CYTOLYSINS

Compounds may be tested for their ability to inhibit the haemolytic activity of Streptolysin O (SLO), Perfringolysin O (PFO), Listeriolysin O (LLO), Anthrolysin O (ALO) and Suilysin (SLY) using the assay protocol outlined in the above Section A.

C. 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 may be 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 is 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) are 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 are washed with PBS. Test compound dilutions are incubated with pneumolysin as described in Section A, then transferred to wells containing the human lung epithelial cells and the plates are incubated at 37°C, 5% C0₂, for 30 min. The following controls are 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 is 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 is followed by the addition of 1 N HCI to all wells. Absorbance at 490 nm and 655 nm is then measured. The percentage of LDH release induced by pneumolysin in the presence and absence of test compounds is plotted against the Log of the concentration of the compound and the IC₅₀ is determined, as described above in the inhibition of haemolysis assay, Section A. D. 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 may be 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 a compound to prevent pneumolysin from causing damage in vivo. Experimental procedure

Ependymal cell cultures are prepared by the method previously described [Microb. Pathog. (1999) 27 303-309]. Tissue culture trays are coated with bovine fibronectin and incubated at 37°C in 5% (v/v) C0₂ for 2h before use. The growth medium is 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 are killed by cervical dislocation, and their brains are removed. The cerebellum is removed along with edge regions of the left and right cortical hemispheres and the frontal cortex. The remaining brain areas are mechanically dissociated in 4 mL of growth medium. The dissociated tissue from one or two brains is added to the wells of the tissue culture trays (500 μΙ/well), each containing 2.5 mL of growth medium. The cells then are incubated at 37°C in 5% (v/v) C0₂. The medium is replaced after three days and thereafter the ependymal cells are fed every two days with 2 mL of fresh growth medium supplemented with thrombin.

After approximately two weeks, the cells are fully ciliated and ready for experiments. To perform the experiments, the growth medium is replaced with 1 mL of medium MEM containing 25 mM HEPES, pH 7.4. The tissue culture trays are placed inside a thermostatically controlled incubation chamber surrounding the stage of an inverted light microscope. The cell cultures are 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, is added to the wells containing the ciliated cells. To the control cells, 1 mL of MEM medium is added. Beating cilia are recorded before and after exposure over 30 min, with a digital high-speed video camera at a rate of 500 frames/s. The recorded video sequences are played back at reduced frame rates and the ciliary beat frequency (CBF) is 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)

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. neumoniae and treated with pneumolysin inhibitors. Survival is 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 are used. The animals are maintained under controlled conditions of temperature, humidity and day length. They have free access to tap water and pelleted food. The in vivo experiments are 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 are infected intranasally with Streptococcus pneumoniae strain D39 (procedure described below). After completing the infections, the viable count of the given dose is determined (as described below). Subsequently, every six hours, animals in the treatment group and in the control group 2, receive the test compound intravenously, while excipient alone is administered to control group 1. The progress of the signs of disease (Table 3) is assessed every 6h based on the scheme of Morton and Griffiths [Veterinary Record. (1985) 111 , 431-436]. Animals are killed if they became 2+ lethargic and the time is recorded. The survival rates of control and test groups are 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 procedures which may be 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 are lightly anaesthetised with 2.5% (v/v) isoflurane over 1.6-1.8 L 0₂/min. The confirmation of effective anaesthesia is made by observation of no pedal reflex. A mouse is held by the scruff of the neck in a vertical position with its nose upward. The infectious dose is then administered in sterile PBS, given drop by drop into the nostrils, allowing the animal to inhale it in between drops. Once the dose is given, the mouse is returned to its cage, placed on its back to recover from the effects of anaesthetic.

- Intravenous administration of treatment

Mice are placed inside an incubator at 37°C, for 10 min, to dilate their veins. Each mouse is then individually placed inside a restrainer, leaving the tail of the animal exposed. The tail is disinfected with antimicrobial wipes. The treatment with the drug is administered intravenously every 6h using a 0.5 ml insulin syringe inserted carefully into one of the tail lateral veins. Doses are prepared freshly and administered intravenously to the animals.

- Determination of viable count of the infectious dose

Viable counting is performed by the method of Miles and Misra [J. Hyg. (1938) 38 732-749). 20 μΙ_ of the sample are serially diluted in 180 μΙ_ PBS in round-bottomed 96-wells microtitre plates, up to a dilution of 10⁶. Blood agar plates are divided into six sectors and 60 μΙ_ of each dilution plated onto an individual sector. The plates are incubated in C0₂ gas jars overnight at 37°C. The following day, colonies are counted in the sector where 30-300 colonies are visible. The concentration of colony forming units (CFU) per millilitre is determined by using the following equation:

_{™T T} , Number of colonies in sector _ ., . „ _^ \

CFU per ml = x Dilution x 1000 conversion factor .

60 μΐ

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

Rationale

To demonstrate that the prodrug derivatives are converted to the parent active compound in the presence of plasma enzymes, a prodrug derivative can be incubated with mouse or human plasma at 37°C at 5 time points over a 2h period. The samples are then analysed by LC-MS/MS to obtain the amount of parent active compound appearing and prodrug derivative remaining over time. The mouse plasma assay system is considered to be a good model for human behaviour. Nevertheless data obtained in a human plasma assay system may be obtained in some cases.

Experimental procedure

Prodrug derivatives are assessed in the mouse or human plasma stability assay at a

concentration of 10 μΜ. Test compounds are diluted in DMSO to a final stock concentration of 10 mM. For the purpose of the assay, the stocks prepared are further diluted in DMSO to a concentration of 400 μΜ and 5μΙ were added to 195 μΙ of mouse or human plasma (pH 7.4) and then incubated at 37°C. The final concentration of DMSO in the plate is 2.5% (v/v). Reactions were terminated at 0, 15, 30, 60 and 120 min after incubation by adding 400 μί of acetonitrile containing 0.55 μΜ metoprolol and 1 % (v/v) formic acid. The plate is then centrifuged at 3000 rpm, for 45 min, at 4°C. 80 μΙ of supernatant is transferred into a conical bottom 96 well glass coated plate. 40 μί of water is added prior to analysis for prodrug derivative and active species by LC-MS/MS. Results

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

(1 ) The parent active compound is quantified using a 6 point calibration curve prepared in deactivated mouse or human plasma. (2) The percentage of prodrug compound remaining at each time point relative to 0 min sample is calculated from LC-MS/MS peak area ratios (compound peak area/internal standard peak area). This percentage is 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.

G. SOLUBILITY AND CHEMICAL STABILITY TESTING FOR THE DETERMINATION OF A SUITABLE FORMULATION FOR INTRAVENOUS ADMINISTRATION

Rationale

Parenteral delivery is one preferred route of administration of compounds of the invention. Therefore, solubility and chemical stability in aqueous buffers is desirable in order to achieve a readily soluble formulation, with enhanced chemical stability that could be reconstituted at the bed side, at a high concentrations, in safe saline solutions, compatible with intravenous administration.

Experimental procedure

- Solubility testing

Solubility studies may be performed by charging a vial with 5-10 mg of compound followed by the addition of PBS solution to achieve a concentration of 100 mg/ml. If solubility is not observed, the solution may be diluted to concentrations of 50 mg/ml, 25 mg/ml and 4 mg/ml consecutively until complete solubility is observed ("method 1").

Alternatively, solubility studies may be performed by charging a vial with 5-10 mg of compound followed by the addition of PBS solution to achieve a concentration of 200 mg/ml. If solubility is not observed, the solution may be diluted to concentrations of 100 mg/ml, 50 mg/ml, 25 mg/ml and 4 mg/ml consecutively until complete solubility is observed ("method 2").

- Chemical stability assessment

Stability studies may be performed 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 (9.6 ml) at 37.5°C. A sample (~ 0.5 ml) is immediately taken for HPLC analysis. Further samples are then taken for analysis at various time-points thereafter. Half-lives are determined from the decrease in concentration of compound with respect to time. Results

The formulations obtained with the examples of this invention are shown in Table 4. Higher solubility was achieved with UL9-002, UL9-003, UL9-005, UL9-006 and UL9-008. UL9-002 and UL9-003 are selected for their pharmaceutical utility by offering safe formulations that are readily soluble at high concentrations and at a pH compatible with intravenous administration. Table 4 Properties of the formulations of compounds of the invention

*Phosphate Buffered Saline (pH 7.2). Method 2 was employed. 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.

The application of which this description and claims forms part may be used as a basis for priority in respect of any subsequent application. The claims of such subsequent application may be directed to any feature or combination of features described herein. They may take the form of product, composition, process, or use claims and may include, by way of example and without limitation, the claims.

Claims

Claims

1. A compound of formula (I)

(I) wherein:

R¹ is -C(0)NR⁸R⁹ or -C(0)OR^8a;

R² is -C(0)NR⁸R⁹, -C(0)NR⁵R⁶,-C(0)OR⁷ or -C(0)OR^8a;

R³ is optionally substituted phenyl;

R^4a and R^4b are independently selected from hydrogen; C C₆ alkyl which alkyl group may optionally be substituted by hydroxyl, COOR¹² or CONR¹³R¹⁴; aryl and -C C₃ alkylaryl in which said aryl groups may be optionally substituted;

R⁵ and R⁶ are independently selected from:

(a) hydrogen,

(b) Ci-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-Ci₀ cycloalkyl, C₅-Ci₀ cycloalkenyl, heterocyclyl, -CrC₃ alkyl-C₃-Ci₀ cycloalkyl, -C C₃ alkyl-C₅-Ci₀ cycloalkenyl or -C C₃ alkylheterocyclyl, or R⁵ and R⁶ together with the N to which they are attached may form a

5- or 6-membered heterocyclic ring optionally containing a further heteroatom selected from O, S and NR⁹, in which any of the aforementioned R⁵ and R⁶ groups may be optionally substituted by a group selected from cyano, C C₆ alkoxy, C C₆ fluoroalkoxy, Ci-C₆ alkyl, C C₆ fluoroalkyl and -C(0)NR^aR^b, where R^a and R^b are independently selected from hydrogen and C C₆ alkyl, and any of the aforementioned R⁵, R⁶ and R⁹ groups may be optionally substituted by one or more halogen atoms, and

(c) aryl, heteroaryl, C C₃ alkylaryl and -C C₃ alkylheteroaryl, said aryl and heteroaryl groups being optionally substituted;

R⁷ is selected from:

(a) Ci-C₆ alkyl, C₂C₆ alkenyl, C₂-C₆ alkynyl, C₃-Ci₀ cycloalkyl, C₅-Ci₀ cycloalkenyl, heterocyclyl, -C C₃ alkyl-C₃-Ci₀ cycloalkyl, -C C₃ alkyl-C₅-Ci₀ cycloalkenyl or -C C₃ alkylheterocyclyl, in which any of the aforementioned R⁷ groups may be optionally substituted by a group selected from cyano, C C₆ alkoxy, C C₆ fluoroalkoxy, C C₆ alkyl, C C₆ fluoroalkyl and -C(0)NR^aR^b, where R^a and R^b are independently selected from hydrogen and C C₆ alkyl, and any of the aforementioned R⁷ groups may be optionally substituted by one or more halogen atoms, and

(b) aryl, heteroaryl, C C₃ alkylaryl and -C C₃ alkylheteroaryl, said aryl and heteroaryl groups being optionally substituted;

R⁸ is selected from C C₆ alkyl substituted by -NR¹⁰R¹¹, C₃-Ci₀ cycloalkyl substituted by - NR¹⁰R¹¹ or a 5- or 6-membered, optionally substituted, heterocyclic or heteroaromatic ring containing at least one nitrogen atom; R^8a is selected from C C₆ alkyl substituted by -NR^10aR^11a, C3-C10 cycloalkyl substituted by -

NR^10aR^{11 a} or a 5- or 6-membered, optionally substituted, heterocyclic or heteroaromatic ring containing at least one nitrogen atom;

R⁹ is selected from:

(a) hydrogen,

(b) Ci-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-Ci₀ cycloalkyl, C₅-Ci₀ cycloalkenyl, heterocyclyl, -CrC₃ alkyl-C₃-Ci₀ cycloalkyl, -C C₃ alkyl-C₅-Ci₀ cycloalkenyl or -C C₃ alkylheterocyclyl, or R⁵ and R⁶ together with the N to which they are attached may form a 5- or 6-membered heterocyclic ring optionally containing a further heteroatom selected from O, S and NR⁹, in which any of the aforementioned R⁵ and R⁶ groups may be optionally substituted by a group selected from cyano, C C₆ alkoxy, C C₆ fluoroalkoxy, Ci-C₆ alkyl, C C₆ fluoroalkyi and -C(0)NR^aR^b, where R^a and R^b are independently selected from hydrogen and C C₆ alkyl, and any of the aforementioned R⁵, R⁶ and R⁹ groups may be optionally substituted by one or more halogen atoms,

(c) aryl, heteroaryl, C C₃ alkylaryl and -C C₃ alkylheteroaryl, said aryl and heteroaryl groups being optionally substituted, and

(d) Ci-C_e alkyl substituted by -N R¹⁰R^{1 1} , C₃-Ci₀ cycloalkyl substituted by -NR¹⁰R¹¹ or a 5- or 6-membered, optionally substituted, heterocyclic or heteroaromatic ring containing at least one nitrogen atom;

R¹⁰, R^10a, R¹¹ and R^11a are independently selected from hydrogen, C C₆ alkyl, C₂-C₆ alkenyl, C₂-

C₆ alkynyl, C₃-Ci₀ cycloalkyl, C₅-Ci₀ cycloalkenyl, heterocyclyl, -C C₃ alkyl-C₃-Ci₀ cycloalkyl, -

C1-C3 alkyl-C₅-Ci o cycloalkenyl and -C1-C3 alkylheterocyclyl;

R¹² is C C₆ alkyl;

R¹³ is hydrogen or C C₆ alkyl; and

R¹⁴ is hydrogen or C C₆ alkyl;

or a pharmaceutically acceptable prodrug derivative thereof, or a pharmaceutically acceptable salt or solvate thereof.

2. A compound according to claim 1 wherein R¹ and R² are independently -C(0)NR⁸R⁹.

3. A compound according to claim 1 wherein R¹ is -C(0)NR⁸R⁹ and R² is -C(0)NR⁵R⁶.

4. A compound according to claim 1 wherein R¹ is -C(0)NR⁸R⁹ and R² is -C(0)OR⁷.

5. A compound according to any one of claims 1 to 4 wherein R³ is substituted phenyl.

6. A compound according to claim 5 wherein R³ is phenyl substituted by one or more substituents independently selected from halo, cyano, hydroxyl, C C₆ alkoxy, C C₆

hydroxyalkoxy, C C₆ fluoroalkoxy, C C₆ alkyl, C C₆ fluoroalkyi, -C(0)NR^aR^b, where R^a and R^b are independently selected from hydrogen and C C₆ alkyl; -O-R¹⁵ wherein R¹⁵ is -(CH₂)_X-

P(0)(OR²³)₂, where x is 0, 1 , 2, 3 or 4 and R²³ is independently selected from hydrogen and C C₃ alkyl, -(CH₂)_y-S(0)₂Me where y is 1 , 2, 3 or 4, -C C₆ alkylheterocyclyl which heterocyclyl group may be optionally substituted by C C₃ alkyl, -C C₆ alkylphenyl which phenyl group may be optionally substituted by C C₃ alkoxy, or phenyl or 5- or 6-membered heteroaryl which phenyl or heteroaryl group may optionally be substituted by a group selected from C C₄ alkyl and halo; or -(0(CH₂)_z)_pOR²⁴, where each z, which may be the same or different, represents 2 or 3, p represents 1 , 2, 3, 4 or 5 and R²⁴ is hydrogen or C C₃ alkyl; or two adjacent carbon atoms within R³ may be linked by -0-CH₂-0-.

7. A compound according to claim 6 wherein R³ is phenyl substituted by 1 , 2 or 3 substituents independently selected from C C₆ alkoxy; -O-R¹⁵ wherein R¹⁵ is -(CH₂)_X-

P(0)(OR²³)₂, where x is 0, 1 , 2, 3 or 4 and R²³ is independently selected from hydrogen and C C₃ alkyl or R¹⁵ is -(CH₂)_y-S(0)₂Me where y is 1 , 2, 3 or 4; and -(0(CH₂)_z)_pOR²⁴, where each z, which may be the same or different, represents 2 or 3, p represents 1 , 2, 3, 4 or 5 and R²⁴ is hydrogen or C C₃ alkyl.

8. A compound according to claim 6 wherein R³ is phenyl substituted by 1 , 2 or 3 substituents selected from halo, cyano, C C₆ alkoxy, C C₆ fluoroalkoxy, C C₆ alkyl, C C₆ fluoroalkyl and -C(0)NR^aR^b, where R^a and R^b are independently selected from hydrogen and C C₆ alkyl.

9. A compound according to any one of claims 2 to 8 wherein R³ is phenyl having a substituent in the meta or para positions relative to the pyrrole ring, for example, R³ is 4- methoxyphenyl.

10. A compound according to any one of the preceding claims wherein R^4a and R^4b are hydrogen or -CrC₃ alkylaryl.

1 1. A compound according to claim 10 wherein R^4a and R^4b are hydrogen.

12. A compound according to any one of the preceding claims wherein R⁵ and R⁶ are independently selected from hydrogen, C C₆ alkyl, aryl or C C₃ alkylaryl in which said aryl may be optionally substituted, or R⁵ and R⁶ together with the N to which they are attached form a 5- or 6-membered heterocyclic ring optionally containing a further heteroatom selected from O, S and NR⁹.

13. A compound according to claim 12 wherein R⁵ and R⁶ are not both hydrogen.

14. A compound according to any one of the preceding claims wherein R⁷ is C C₆ alkyl.

15. A compound according to any one of the preceding claims wherein R⁸ is C C₆ alkyl substituted by -NR¹⁰R¹¹.

16. A compound according to any one of the preceding claims wherein R⁹ is selected from hydrogen, C C₆ alkyl, aryl e.g. phenyl, or C C₃ alkylaryl.

17. A compound according to any one of the preceding claims wherein R¹⁰ and R¹¹ are independently selected hydrogen and C C₆ alkyl.

18. A compound according to claim 1 which is A/²-(2-(dimethylamino)ethyl)-3,4-dihydroxy-1- (4-methoxyphenyl)-/V² A/⁵,A/⁵-trimethyl-1 /-/-pyrrole-2,5-dicarboxamide, or a pharmaceutically acceptable prodrug derivative thereof, or a pharmaceutically acceptable salt or solvate thereof.

19. A compound according to claim 1 which is selected from:

3.4- Dihydroxy-1-(4-methoxyphenyl)-A/²,A/⁵-dimethyl-A/²,A/⁵-bis(1-methylpiperidin-4-yl)-1 H-pyrrole-

2.5- dicarboxamide,

Ethyl 3,4-dihydroxy-1-(4-methoxyphenyl)-5-(methyl(1-methylpiperidin-4-yl)carbamoyl)-1 /-/- 5 pyrrole-2-carboxylate,

A/²,/\/⁵-bis(3-(dimethylamino)propyl)-3,4-dihydroxy-1-(4-methoxyphenyl)-/\/²,/\/⁵-dimethyl-1 /-/- pyrrole-2,5-dicarboxamide,

3,4-Dihydroxy-1-(4-methoxyphenyl)-/V²,/\/⁵-dimethyl-/\/²,/\/⁵-di(pyridin-3-yl)-1 /-/-pyrrole-2,5- dicarboxamide,

10 Ethyl 3,4-dihydroxy-1-(4-methoxyphenyl)-5-(methyl(pyridin-3-yl)carbamoyl)-1 /-/-pyrrole-2- carboxylate,

A/²,/\/⁵-bis(2-(dimethylamino)ethyl)-3,4-dihydroxy-1-(4-methoxyphenyl)-/\/²,/\/⁵-dimethyl-1 /-/- pyrrole-2,5-dicarboxamide,

and pharmaceutically acceptable prodrug derivatives thereof, and pharmaceutically acceptable 15 salts and solvates thereof.

20. A compound according to any one of claims 1 to 19 in the form of a prodrug derivative.

21. A compound according to claim 20 wherein the prodrug derivative is selected from 20 carboxylate ester, sulfamate ester, phosphate ester and carbamate ester derivatives.

22. A compound according to claim 21 wherein the prodrug derivative is a carboxylate ester derivative.

25 23. A compound according to claim 21 or 22 wherein one or both of R^4a and R^4b are

independently selected from -C(0)R¹⁶, -S0₂NH₂ , -PO(OR¹⁹)(OR²⁰), -CHR²⁶-OPO(OR¹⁹)(OR²⁰) where R²⁶ is hydrogen or C C₆ alkyl, and -C(0)NR¹⁷R¹⁸, wherein R¹⁶, R¹⁷, R¹⁸, R¹⁹ and R²⁰ are independently selected from:

(a) C C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-Ci₀ cycloalkyl, C₅-Ci₀ cycloalkenyl,

30 heterocyclyl, -CrC₃ alkyl-C₃-Ci₀ cycloalkyl, -CrC₃ alkyl-C₅-Ci₀ cycloalkenyl or -C C₃ alkylheterocyclyl, or R¹⁷ and R¹⁸ together with the N to which they are attached may form a 5- or 6-membered heterocyclic ring optionally containing a further heteroatom selected from O, S and NR^25aR^25b where R^25a is hydrogen, C C₆ alkyl, -CH₂-OPO(OR¹⁹)(OR²⁰) or a 5- or 6-membered heterocyclic ring, and R^25b is absent or C C₆ alkyl; and in which any

35 of the aforementioned R¹⁶, R¹⁷ or R¹⁸ groups may be optionally substituted by one or more groups selected from cyano, -OPO(OR¹⁹)(OR²⁰), -(0(CH₂)_z)_rOR²⁴, wherein each z, which may be the same or different, represents 2 or 3, r represents an integer selected from 1 to 20, and R²⁴ is hydrogen, C C₃ alkyl or -PO(OR¹⁹)(OR²⁰), C C₆ alkoxy, C C₆ fluoroalkoxy, C C₆ alkyl, C C₆ fluoroalkyl and -C(0)NR^aR^b, where R^a and R^b are

40 independently selected from hydrogen and C C₆ alkyl, and any of the aforementioned

R¹⁶, R¹⁷ or R¹⁸ groups may be optionally substituted by one or more halogen atoms; and

(b) aryl, heteroaryl, C C₃ alkylaryl and -C C₃ alkylheteroaryl, said aryl and heteroaryl

groups being optionally substituted;

or R¹⁸, R¹⁹ and R²⁰ may independently represent hydrogen.

24. A compound according to claim 21 or 22 wherein one or both of R and R are independently selected from -C(0)R¹⁶, -S0₂NH₂ , -PO(OR¹⁹)(OR²⁰) and -C(0)NR¹⁷R¹⁸, wherein R¹⁶, R¹⁷, R¹⁸, R¹⁹ and R²⁰ are independently selected from

(a) C C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-Ci₀ cycloalkyl, C₅-Ci₀ cycloalkenyl,

5 heterocyclyl, -CrC₃ alkyl-C₃-Ci₀ cycloalkyl, -CrC₃ alkyl-C₅-Ci₀ cycloalkenyl or -C C₃ alkylheterocyclyl, in which any of the aforementioned R¹⁶, R¹⁷ or R¹⁸ groups may be optionally substituted by a group selected from cyano, C C₆ alkoxy, C C₆ fluoroalkoxy, Ci-C₆ alkyl, C C₆ fluoroalkyl and -C(0)NR^aR^b, where R^a and R^b are independently selected from hydrogen and C C₆ alkyl, and any of the aforementioned R¹⁶, R¹⁷ or R¹⁸ 10 groups groups may be optionally substituted by one or more halogen atoms, and

(b) aryl, heteroaryl, C C₃ alkylaryl and -C C₃ alkylheteroaryl, said aryl and heteroaryl groups being optionally substituted;

or R¹⁸, R¹⁹ and R²⁰ may independently represent hydrogen.

15 25. A compound according to any one of claims 21 to 24 wherein both of R^4a and R^4b are independently selected from -C(0)R¹⁶, -S0₂NH₂ , -PO(OR¹⁹)(OR²⁰), -CHR²⁶-OPO(OR¹⁹)(OR²⁰) where R²⁶ is hydrogen or C C₆ alkyl, and -C(0)NR¹⁷R¹⁸.

26. A compound according to any one of claims 21 to 24 wherein one of R^4a and R^4b is 20 selected from -C(0)R¹⁶, -S0₂NH₂ , -PO(OR¹⁹)(OR²⁰), -CHR²⁶-OPO(OR¹⁹)(OR²⁰) where R²⁶ is hydrogen or C C₆ alkyl, and -C(0)NR¹⁷R¹⁸; and the other of R^4a and R^4b is hydrogen.

27. A compound according to any one of claims 21 to 26 wherein one or both of R^4a and R^4b are independently selected from -C(0)R¹⁶.

25

28. A compound according to claim 27 wherein R¹⁶ is C C₆ alkyl or C₃-Ci₀ cycloalkyl in which either of the aforementioned groups may be optionally substituted by a group selected from -OPO(OR¹⁹)(OR²⁰) and -(0(CH₂)_z)_rOR²⁴, where each z, which may be the same or different, represents 2 or 3, r represents an integer selected from 1 to 20, and R²⁴ is hydrogen,

30 Ci-C₃ alkyl or -PO(OR¹⁹)(OR²⁰) or R¹⁶ is phenyl optionally substituted by -(CHR²⁶)_q- OPO(OR¹⁹)(OR²⁰) wherein q represents 0 or 1.

29. A compound according to claim 20 which is 2-((2-(dimethylamino)ethyl)- (methyl)carbamoyl)-5-(dimethylcarbamoyl)-1-(4-methoxyphenyl)-1 /-/-pyrrole-3,4-diyl bis(2-

35 methylpropanoate), or a pharmaceutically acceptable salt or solvate thereof.

30. A pharmaceutical composition comprising a compound according to any one of claims 1 to 29, optionally in combination with one or more pharmaceutically acceptable diluents or carriers.

40

31. A pharmaceutical composition according to claim 30 comprising one or more other therapeutically active ingredients.

32. A compound according to any one of claims 1 to 29 for use as a medicament.

45

33. A compound according to any one of claims 1 to 29 or 32 for use in combination with one or more other therapeutically active ingredients.

34. A compound according to any one of claims 1 to 29, 32 or 33 for use in the treatment of 5 bacterial infections caused by bacteria producing pore-forming toxins, such as cholesterol

dependent cytolysins.

35. A compound for use according to claim 34 wherein the bacterial infection is caused by Streptococcus spp. (e.g. Streptococcus pneumoniae, Group A Streptococci or Streptococcus

10 suis), Clostridium spp. (e.g. Clostridium perfringens), Listeria spp. (e.g. Listeria monocytogenes) or Bacillus spp. (e.g. Bacillus anthracis).

36. A compound for use according to claim 35 for the treatment of bacterial infection which is caused by Streptococcus pneumoniae.

15

37. A compound for use according to claim 36 for the treatment of pneumococcal pneumonia, pneumococcal meningitis, pneumococcal septicaemia/bacteraemia, pneumococcal keratitis or pneumococcal otitis media.

20 38. A compound for use according to claim 35 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.

25 39. A compound for use according to any one of claims 32 to 38 wherein the compound is administered in combination with one or more other therapeutically active ingredients (e.g. one or more antimicrobial or immunomodulatory agents).

40. A method of treatment of bacterial infections caused by bacteria producing pore-forming 30 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 to 29, 32 or 33.

41. A compound of formula (II):

35

(II) wherein R¹ , R² and R³ are as defined in claim 1 for the compounds of formula (I), or a salt or protected derivative thereof.

40

42. A process for preparing compounds of formula (I) as defined in any one of claims 1 to 29 in which R^4a and R^4b represent H which comprises reacting a compound of formula (II): R³

(II) wherein R¹ , R² and R³ are as defined in claim 1 for the compounds of formula (I), or a salt or protected derivative thereof, with a compound of formula ROCOCOOR^x in which R^x represents C C₆ alkyl.