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NZ625636B2 - Novel pyrrole derivatives - Google Patents

Novel pyrrole derivatives Download PDF

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Publication number
NZ625636B2
NZ625636B2 NZ625636A NZ62563612A NZ625636B2 NZ 625636 B2 NZ625636 B2 NZ 625636B2 NZ 625636 A NZ625636 A NZ 625636A NZ 62563612 A NZ62563612 A NZ 62563612A NZ 625636 B2 NZ625636 B2 NZ 625636B2
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NZ
New Zealand
Prior art keywords
methoxyphenyl
pyrrole
bis
mmol
benzyloxy
Prior art date
Application number
NZ625636A
Other versions
NZ625636A (en
Inventor
Peter William Andrew
Mafalda Pires Damaso
Mark William Davies
Fritz Frieder Frickel
Daniel Hamza
Simon Christopher Hirst
Rana Lonnen
Original Assignee
University Of Leicester
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from EP11191986.6A external-priority patent/EP2602248A1/en
Application filed by University Of Leicester filed Critical University Of Leicester
Publication of NZ625636A publication Critical patent/NZ625636A/en
Publication of NZ625636B2 publication Critical patent/NZ625636B2/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/402—1-aryl substituted, e.g. piretanide
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    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/4025—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil not condensed and containing further heterocyclic rings, e.g. cromakalim
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    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
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    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/4439—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. omeprazole
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    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
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    • A61K31/5375—1,4-Oxazines, e.g. morpholine
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Abstract

The disclosure relates to pyrrole derivative compounds of formula (I) which are cytolysin inhibitors, wherein R1, R2, R3, R4a and R4b are as defined in the specification. These compounds are suitable for use in the treatment of bacterial infections caused by bacteria selected from 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) which produce pore-forming toxins, such as cholesterol dependent cytolysins. These compounds are also suitable 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. pp. (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) which produce pore-forming toxins, such as cholesterol dependent cytolysins. These compounds are also suitable 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.

Description

NOVEL PYRROLE DERIVATIVES Field of the invention The invention relates to nds which are cytolysin tors 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 ngly high in the ping world. Nearly 20% of hospitalised patients still die despite otic killing of the pneumococcus, while many survivors of pneumococcal meningitis suffer severe neurological aps, ing 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 s 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 ia resulting from the release of toxic coccal 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 s on the affected cells. In the bacterium, the toxin lysin is cytoplasmic and is mainly ed 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 tutes a ntial unmet medical need that is internationally recognised.
Currently, corticosteroids, principally dexamethasone, are used as an tive 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 t (2002) 360 2 11-218]. 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 lysin 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 icant reduction in the severity of pulmonary inflammation. Other evidence ed 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 9].
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) 65 441 1-4418]. 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 cocci on the cultured ependymal cells is clearly caused by the toxin pneumolysin released from the otic-lysed bacteria, as this damage was abolished in the presence of anti-pneumolysin antibodies [Infect. Immun. (2004) 72 6694-6698]. This finding ts the gy that antibiotic-induced toxaemia is prevented by combination with anti-pneumolysin agents.
Evidence for the significant involvement of pneumolysin in pneumococcal infections and the ntial 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 coccal 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 ar 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 tes the involvement of pneumolysin in pneumococcal keratitis and the therapeutic benefit obtained following its tion. 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 ent in garlic extract, has been usly found to t the ytic activity of pneumolysin in vitro [Toxicon (201 1) 57 540-545]. This compound is a ne inhibitor that irreversibly binds to the ve thiol group of the toxin. Compounds exhibiting such a property are urable as drug candidates because of their potential unspecific binding to other cysteine-containing proteins in the body.
There s a need to provide inhibitors of cytolysins, such as pneumolysin, which are suitable for use in the treatment of bacterial infections.
The present invention provides compounds that specifically inhibit the direct toxic effect of pneumolysin and other cholesterol dependent cytolysins that are pivotal in the virulence of their respective hosts. The compounds of the invention have no structural similarity to Allicin and do not bind covalently to the reactive thiol groups of the toxins.
Certain N-phenyl substituted pyrroles are known, however their use as pharmaceuticals in particular for the treatment of bacterial ions had not been suggested. The compounds diethyl 3,4-dihydroxy(4-methoxyphenyl)-1/-/-pyrrole-2,5-dicarboxylate (CAS 6540523) and diethyl 3,4-dihydroxyphenyl-1/-/-pyrrole-2,5-dicarboxylate (CAS 559323) are commercially available. The nds dimethyl 3,4-dihydroxy(4-bromophenyl)-1/-/-pyrrole- carboxylate (CAS 98), dimethyl 3,4-dihydroxy(4-chlorophenyl)-1/-/-pyrrole- 2,5-dicarboxylate (CAS 10826557), di-te/f-butyl 3,4-dihydroxy(4-nitrophenyl)-1/-/-pyrrole- 2,5-dicarboxylate (CAS 1103322) and dimethyl hydroxy(4-methoxyphenyl)-1/-/- pyrrole-2,5-dicarboxylate (CAS 1010906) are described in Justus s Annalen der Chemie (1961), 639, . The compounds dimethyl 3,4-dihydroxyphenyl-1/-/-pyrrole-2, 5- dicarboxylate (CAS 78038) and dimethyl s(acetyloxy)phenyl-1/-/-pyrrole-2,5- dicarboxylate (CAS 73425) are described in Hoppe-Seyler's hrift fuer Physiologische Chemie (1956), 306, 49-55. The compound l s(acetyloxy)phenyl-1/-/-pyrrole-2,5- dicarboxylate (CAS 559324) is disclosed in Chemische Berichte (1975), 108(2), 569-75.
The compounds of the present invention also prevent stimulation of host-derived toxic effects induced by pneumolysin and 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): wherein: R and R2 are independently selected from -C(0)N R R6, -C(0)OR 7, C N, -C(0)R 7, - C(0)N HC(0)R 7, -NO2, -SO 3R7, -SO2R 7, -SOR 7, -S0 R6, -S0 8, -POR 2 2NR 2NH-C(0)OR R22 and optionally substituted phenyl or heteroaryl; R3 is optionally substituted phenyl; R4a and R4 are independently selected from hydrogen; C C6 alkyl which alkyl group may optionally be substituted by hydroxyl, COOR 2 or CON R R14 ; aryl and -C C3 ryl in which said aryl groups may be optionally substituted; R5 and R6 are independently selected from: (a) hydrogen, (b) Ci-C 6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C 3-C1 0 cycloalkyl, C 5-C1 0 cycloalkenyl, heterocyclyl, -C1-C3 alkyl-C3-C 0 cycloalkyl, -C1-C3 C5-C 0 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 r 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, C1-C6 alkoxy, C1-C6 fluoroalkoxy, C1-C6 alkyl, C1-C6 fluoroalkyl and -C(O)NRaRb, where Ra and Rb are ndently selected from hydrogen and C1-C6 alkyl, and any of the entioned R5 and R6 groups may be optionally substituted by one or more halogen atoms, and (c) aryl, heteroaryl, C1-C3 alkylaryl and -C1-C3 alkylheteroaryl, said aryl and heteroaryl groups being optionally substituted; R7 is selected from: (a) C1-C6 alkyl, C2C6 alkenyl, C2-C6 alkynyl, C3-C10 cycloalkyl, C5-C10 cycloalkenyl, heterocyclyl, -C1-C3 alkyl-C3-C10 cycloalkyl, -C1-C3 alkyl-C5-C10 cycloalkenyl or -C1-C3 alkylheterocyclyl, in which any of the aforementioned R7 groups may be optionally substituted by a group selected from cyano, C1-C6 alkoxy, C1-C6 fluoroalkoxy, C1-C6 alkyl, C1-C6 alkyl and RaRb, where Ra and Rb are independently selected from hydrogen and C1-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 ryl and -C1-C3 alkylheteroaryl, said aryl and heteroaryl groups being optionally substituted; R8 is C1-C6 alkyl; R9 is hydrogen, C1-C6 alkyl; -C(O)R10 or -C(O)OR11; R10 is C1-C6 alkyl; R11 is C1-C6 alkyl; R12 is C1-C6 alkyl; R13 is hydrogen or C1-C6 alkyl; R14 is hydrogen or C1-C6 alkyl; and R21 and R22 are independently selected from C1-C6 alkyl and C1-C6 hydroxyalkyl; or a pharmaceutically acceptable prodrug derivative thereof, or a pharmaceutically acceptable salt or solvate f; provided that the compound is not: a) diethyl hydroxy(4-methoxyphenyl)-1H-pyrrole-2,5-dicarboxylate; b) diethyl 3,4-dihydroxyphenyl-1H-pyrrole-2,5-dicarboxylate; c) yl 3,4-dihydroxy(4-bromophenyl)-1H-pyrrole-2,5-dicarboxylate; d) dimethyl 3,4-dihydroxy(4-chlorophenyl)-1H-pyrrole-2,5-dicarboxylate; e) di-tert-butyl 3,4-dihydroxy(4-nitrophenyl)-1H-pyrrole-2,5-dicarboxylate; f) dimethyl 3,4-dihydroxy(4-methoxyphenyl)-1H-pyrrole-2,5-dicarboxylate; g) dimethyl 3,4-dihydroxyphenyl-1H-pyrrole-2,5-dicarboxylate; h) diethyl s(acetyloxy)phenyl-1H-pyrrole-2,5-dicarboxylate; or i) dimethyl 3,4-bis(acetyloxy)phenyl-1H-pyrrole-2,5-dicarboxylate.
In another aspect, there is provided a compound of a (I) or a pharmaceutically acceptable prodrug tive thereof, or a pharmaceutically acceptable salt or solvate thereof: wherein: R1 and R2 are independently selected from -C(O)NR5R6, -C(O)OR7, CN, -C(O)R7, - C(O)NHC(O)R7, -NO2, -SO3R7, , -SOR7, -SO2NR5R6, -SO2NH-C(O)OR8, -POR21R22 and substituted phenyl or optionally substituted heteroaryl, wherein the aryl is selected from pyrrole, furan, thiophene, oxazole, thiazole, isothiazole, oxadiazole, tetrazole, imidazole, pyrazole, ole, pyridine, pyridazine, pyrimidine, pyrazine, benzothiophene, benzofuran, 1, 2, 3-triazole and 1, 2, 4-triazole; R3 is optionally substituted phenyl; R4a and R4b are independently selected from hydrogen; C1-C6 alkyl which alkyl group may optionally be substituted by hydroxyl, COOR12 or R14; aryl and -C1-C3 alkylaryl in which said aryl groups may be ally substituted; R5 and R6 are independently selected from: (a) hydrogen, (b) C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C10 cycloalkyl, C5-C10 cycloalkenyl, heterocyclyl, -C1-C3 alkyl-C3-C10 cycloalkyl, -C1-C3 alkyl-C5-C10 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, C1-C6 alkoxy, C1-C6 fluoroalkoxy, C1-C6 alkyl, C1-C6 fluoroalkyl and -C(O)NRaRb, where Ra and Rb are independently ed from hydrogen and C1-C6 alkyl, and any of the aforementioned R5 and R6 groups may be optionally substituted by one or more halogen atoms, and (c) aryl, heteroaryl, C1-C3 alkylaryl and -C1-C3 alkylheteroaryl, said aryl and heteroaryl groups being optionally substituted; R7 is selected from: (a) C1-C6 alkyl, C2C6 alkenyl, C2-C6 alkynyl, C3-C10 cycloalkyl, C5-C10 lkenyl, cyclyl, -C1-C3 alkyl-C3-C10 cycloalkyl, -C1-C3 alkyl-C5-C10 cycloalkenyl or -C1- C3 alkylheterocyclyl, in which any of the aforementioned R7 groups may be optionally substituted by a group selected from cyano, C1-C6 alkoxy, C1-C6 fluoroalkoxy, C1-C6 alkyl, C1-C6 fluoroalkyl and -C(O)NRaRb, where Ra and Rb are independently selected - 4A - from en and C1-C6 alkyl, and any of the aforementioned R7 groups may be optionally substituted by one or more n atoms, and (b) aryl, heteroaryl, C1-C3 alkylaryl and -C1-C3 alkylheteroaryl, said aryl and heteroaryl groups being optionally tuted; R8 is C1-C6 alkyl; R9 is hydrogen, C1-C6 alkyl; -C(O)R10 or R11; R10 is C1-C6 alkyl; R11 is C1-C6 alkyl; R12 is C1-C6 alkyl; R13 is hydrogen or C1-C6 alkyl; R14 is hydrogen or C1-C6 alkyl; and R21 and R22 are independently selected from C1-C6 alkyl and C1-C6 hydroxyalkyl; wherein, when the compound of formula (I) is in the form of a prodrug derivative, one or both of R4a and R4b are independently selected from -C(O)R16, -SO2NH2 , -PO(OR19)(OR20), - CHR26-OPO(OR19)(OR20) where R26 is hydrogen or C1-C6 alkyl, and -C(O)NR17R18, wherein R16, R17, R18, R19 and R20 are independently selected from: (a) C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C10 cycloalkyl, C5-C10 cycloalkenyl, cyclyl, -C1-C3 alkyl-C3-C10 cycloalkyl, -C1-C3 alkyl-C5-C10 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 NR25aR25b where R25a is hydrogen, C1-C6 alkyl, - CH2-OPO(OR19)(OR20) or a 5- or 6-membered heterocyclic ring, and R25b is absent or C1-C6 alkyl; and in which any of the aforementioned R16, R17 or R18 groups may be optionally substituted by one or more groups selected from cyano, - OPO(OR19)(OR20), -(O(CH2)z)rOR24, wherein each z, which may be the same or different, represents 2 or 3, r represents an integer ed from 1 to 20, and R24 is hydrogen, C1-C3 alkyl or -PO(OR19)(OR20), C1-C6 alkoxy, C1-C6 fluoroalkoxy, C1-C6 alkyl, C1-C6 fluoroalkyl and -C(O)NRaRb, where Ra and Rb are independently selected from hydrogen and C1-C6 alkyl, and any of the entioned 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; or wherein, when the compound of formula (I) is in the form of a prodrug derivative, one or both of R4a and R4b are independently selected from -SO2NH2, -PO(OH)2, -CH2- PO(OH)2, -PO(OEt)2, -CON-(4-N-piperidinyl-piperidine), -COt-butyl, propyl, N- methyl)piperazine, iperazine, -CON(CH3)2, COCH3, -CO-(CH2)2-OMe, -CO(CH2)2- (O(CH2)2)pOMe where p is 1 to 12, -CO-CMe2-CH2-(O(CH2)3)pOMe where p is 1 to 12, -CO- - 4B - \Interwoven\NRPortbl\DCC\REC\7918383_1.docx-15/06/2015 CMe2-CH2-(O(CH2)2)pO-PO(OH)2 where p is 1 to 12, -CO-CMe2-CH2-(O(CH2)2)pO-PO(OH)2 where p is 1 to 12, -CO-(4-phosphonoxymethylbenzene) and -CO-(4- phosphonoxymethylcyclohexane); wherein when only one of R4a and R4b represents a prodrug derivative group as defined above the other of R4a and R4b is hydrogen; provided that the compound is not: a) diethyl 3,4-dihydroxy(4-methoxyphenyl)-1H-pyrrole-2,5-dicarboxylate; b) diethyl 3,4-dihydroxyphenyl-1H-pyrrole-2,5-dicarboxylate; c) yl 3,4-dihydroxy(4-bromophenyl)-1H-pyrrole-2,5-dicarboxylate; d) dimethyl 3,4-dihydroxy(4-chlorophenyl)-1H-pyrrole-2,5-dicarboxylate; e) di-tert-butyl 3,4-dihydroxy(4-nitrophenyl)-1H-pyrrole-2,5-dicarboxylate; f) dimethyl 3,4-dihydroxy(4-methoxyphenyl)-1H-pyrrole-2,5-dicarboxylate; g) dimethyl 3,4-dihydroxyphenyl-1H-pyrrole-2,5-dicarboxylate; h) diethyl 3,4-bis(acetyloxy)phenyl-1H-pyrrole-2,5-dicarboxylate; or i) dimethyl 3,4-bis(acetyloxy)phenyl-1H-pyrrole-2,5-dicarboxylate.
The compounds of formula (I) including the compounds which are the subject of provisos a) to i) have therapeutic ty. In a further aspect, the present invention provides a compound of formula (I) without os a) to i) for use as a medicament.
Brief description of the figures Figure 1 shows the in vitro inhibition of pneumolysin-induced LDH e by the compound UL1-012 using A549 human lung epithelial cells. - 4C - Figure 2A shows the effect of the nd UL1-012 in inhibiting pneumolysin from damaging the ciliary on of ependymal cells in an ex vivo meningitis efficacy assay.
Figure 2B shows the effect of the compound UL1-012 in inhibiting a bacterial lysate from damaging the ciliary function of ependymal cells in an ex vivo meningitis efficacy assay.
Figure 3 shows the effect of the compound UL2-001 in inhibiting pneumolysin from damaging the ciliary function of ependymal cells in an ex vivo meningitis efficacy assay.
Figure 4 shows the experimental design for an in vivo mouse nia model efficacy assay using the compound UL1-012.
Figure 5 shows the survival of infected control mice and treated groups administered with 4 mg/kg of the compound UL1-012 in an in vivo mouse pneumonia model efficacy assay.
Figure 6 shows the survival of infected control mice and treated groups administered with 16 mg/kg of the compound 2 in an in vivo mouse pneumonia model cy assay.
Figure 7 shows a comparison of the signs of disease between an infected/non-treated control group and an infected/ treated group that received 16 mg/kg of the compound UL1-012 in an in vivo mouse pneumonia model efficacy assay.
Figure 8 shows the experimental design for an in vivo mouse pneumonia model efficacy assay using the compound UL2-001 .
Figure 9 shows the survival of infected control mice and treated groups administered with 8 mg/kg of the compound UL2-001 in an in vivo mouse pneumonia model efficacy assay.
Detailed description of the invention R and R2 may be independently selected from -C(0)NR R6, R 7, CN, -C(0)R 7, - C(0)NHC(0)R 7, -N0 , -SO R7, -SO R7, -SOR7, -S0 NR R6, -S0 )OR 8 and optionally 2 3 2 2 2 substituted phenyl or aryl; for example R and R2 may be independently selected from - C(0)NR R6, -C(0)OR 7, CN, -C(0)R 7, -C(0)NHC(0)R 7, -SO R7, -S0 R7, -SOR7, -S0 NR R6, - 3 2 2 S0 NH-C(0)OR 8 and optionally tuted phenyl or heteroaryl. R and R2 are preferably independently selected from -C(0)NR R6, -C(0)OR 7, CN, -C(0)R 7, -C(0)NHC(0)R 7 and - S0 NH-C(0)OR 8; more ably R and R2 are independently selected from -C(0)NR R6, - C(0)OR 7 and CN; even more preferably R and R2 are independently selected from -C(0)NR R' and -C(0)OR 7.
R is preferably -C(0)NR R6.
In one ment R2 is -C(0)NR R6. In another embodiment R2 is -C(0)OR 7. Thus in one embodiment R is -C(0)NR R6 and R2 is -C(0)NR R6 and in another embodiment R is - C(0)NR R6 and R2 is -C(0)OR 7.
When R and R2 are both -C(0)NR R6 they may be same or different, preferably they are the same.
In an alternative embodiment R and R2 are both -C(0)OR 7 and they may be same or different, preferably they are the same.
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 ed from halo, cyano, hydroxyl, C C6 alkoxy, C C6 yalkoxy, C C6 fluoroalkoxy, C C6 alkyl, C C6 fluoroalkyi, -C(0)NR aR , where Ra and R are independently ed from hydrogen and C C6 alkyl; -O-R 5 n R is -(CH 2)X- P(0)(OR 2 ) (where x is 0 , 1 , 2 , 3 or 4 and R23 is independently selected from hydrogen and C C3 alkyl), -(CH 2)y-S(0) Me (where y is 1 , 2 , 3 or 4), -C C6 alkylheterocyclyl which heterocyclyl group may be optionally substituted e.g. by C 3 alkyl, -C C6 alkylphenyl which phenyl group may be optionally substituted e.g. by C 3 alkoxy, or phenyl or 5- or 6-membered heteroaryl which phenyl or heteroaryl group may optionally be substituted by a group e.g. ed from C - 4 alkyl and halo; or -(0(CH ) ) OR24, where each z , which may be the same or different, 2 p 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 CH .
When R 5 is -C C6 alkylheterocyclyl, particular heterocyclyl groups which may be mentioned include 5- or 6-membered, clic 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. es of heterocyclic rings include morpholine, piperazine, and the like, which may be optionally substituted e.g. by C1-C3 alkyl, such as methyl. r 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 R aR , where Ra and R are independently selected from hydrogen and C - 6 alkyl. In addition, when R3 is substituted phenyl, said phenyl may be provided with a single substituent -O-R 15 wherein R 5 is selected from phenyl and 5- or 6-membered heteroaryl which phenyl or aryl group may optionally be substituted by a group selected from C C alkyl and halo.
Preferred optional substituents for R3 include 1 or more, e.g. 1, 2 or 3 , substituents (e.g. 1 substituent) ndently selected from C C6 alkoxy; -O-R 15 wherein R is -(CH 2)X- P(0)(OR 2 ) , where x is 0 , 1, 2 , 3 or 4 and R23 is independently selected from hydrogen and C C3 alkyl or R 5 is -(CH ) -S(0) Me where y is 1, 2 , 3 or 4 ; and -(0(CH ) ) OR24, where each z , 2 2 2 p 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 tuents for R3 are described in further detail below.
When R3 is tuted 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 ment, 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 ment, R3 is phenyl substituted by three substituents. When R3 is tuted 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, OCH , OCH2CH3, OCH2CH2CH3, CF , OCF3, CON(CH ) , O-phenyl, methyl, ethyl, isopropyl, t- 3 3 3 2 butyl, hydroxyl, -OP(0)(OH) , -(0(CH 2)2)pOMe where p is 1 , 2 , 3 or 4 , holinopropoxy, 3- (4-methylpiperazinyl)propoxy, 3-(diethoxyphosphoryl)propoxy, -(0(CH ) )-P(0)(OH) , 3- 2 3 2 (methylsulfonyl)propoxy, and 4-methoxybenzyloxy. In a further group of nds that may be mentioned R3 may represent phenyl g a para substituent selected from F, CI, I , OCH3, OCH CH , OCH CH CH , CF , OCF , CON(CH ) , O-phenyl, methyl, ethyl, isopropyl and t- 2 3 2 2 3 3 3 3 2 butyl. A particular R3 group which may be mentioned is phenyl bearing a para OCH substituent.
For e, R3 may represent phenyl bearing an ortho substituent which is OCH .
For example, R3 may represent phenyl bearing a meta tuent which is O-phenyl or OCH .
For example, R3 may ent phenyl substituted in the meta position by I and in the para position by OCH , or phenyl substituted in the meta position by OCH and in the para position 3 3 by OCH , or phenyl linked in the meta and para positions by CH . 3 2 For example, R3 may represent phenyl substituted in the ortho position by OCH and in the para position by OCH .
For example, R3 may represent phenyl substituted in the 3 , 4 and 5 positions by OCH , or phenyl substituted in the 3 and 5 positions by F and in the para position by OCH CH . 2 3 Hence a particularly suitable substituent for the phenyl of R3 is OCH , especially in the para position. Further particularly suitable substituents for R3 include -O-R 5 wherein R 5 is as defined above and -(0(CH ) ) OR24, where z , p and R24 are as defined above, especially in the 2 p para on.
When an alkyl group or R4a and/or R4 is substituted by hydroxyl, COOR 2 or CONR R14, es of R4a and/or R4 groups include -CH COOt-butyl, CH CONH and CH CH OH. 2 2 2 2 2 R4a and R4 may be independently selected from hydrogen; C C alkyl which alkyl group may optionally be substituted by hydroxyl, COOR 12 or CONR R14; and -C C alkylaryl in which said aryl groups may be optionally substituted. R4a and R4 are preferably independently selected from hydrogen, C C alkyl, aryl and -C1-C3 alkylaryl in which aryl may be optionally substituted.
For e R4a and R4 are preferably independently selected from hydrogen, C C alkyl and - C1-C3 alkylaryl in which aryl may be optionally substituted. R4a and R4 are more preferably hydrogen or -C1-C3 alkylaryl, e.g. benzyl. Most preferably R4a and R4 are hydrogen.
R5 and R6 are preferably independently selected from hydrogen, C C alkyl e.g. , ethyl, or propyl, aryl e.g. phenyl, or C1-C3 alkylaryl, e.g. benzyl in which said aryl may be optionally tuted, 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. line, dine or piperazine (optionally N substituted with an R9 group).
In one embodiment of the invention one of R5 and R6 is en. Preferably at least one of R5 and R6 is not hydrogen, more preferably both of R5 and R6 are not hydrogen.
Specific- NR R6 groups of interest e NMe , NHethyl, -N-morpholinyl and N-piperidinyl, especially NMe .
R7 is preferably C C alkyl e.g. methyl, ethyl, propyl or butyl, such as /so-propyl or -butyl.
R8 is preferably methyl.
R9 is preferably hydrogen, methyl, COCH or -CO-t-butyl.
R is preferably methyl.
R is preferably methyl.
R 2 is preferably .
R 3 is preferably H or methyl.
R 4 is preferably H or methyl.
In an embodiment R 5 is -(CH ) OR 2 ) or -(CH ) -S(0) Me. 2 x 2 2 2 In an embodiment R 5 group is optionally tuted phenyl, e.g. tituted phenyl.
In an embodiment R 5 is-C C alkylheterocyclyl which heterocyclyl group may be optionally substituted e.g. by C1-C3 alkyl.
R2 are R22 are preferably independently selected from C C alkyl, e.g. methyl.
R23 is preferably hydrogen, methyl or ethyl.
R24 is preferably C1-C3 alkyl, e.g. methyl. x is ably 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) 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 tives 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 R4a and R4 are independently selected from -C(0)R 16 , - S0 NH , -PO(OR )(OR20) , -CHR26-OPO(OR )(OR20) (where R26 is hydrogen or C C alkyl), 2 2 6 and -C(0)NR 7R18, wherein R 6 , R 7 , R 8 , R 9 and R20 are independently selected from: (a) C - 6 alkyl, C -C alkenyl, C -C alkynyl, 2 6 2 6 C3-C10 cycloalkyl, C5-C10 cycloalkenyl, heterocyclyl, -C1 - C 3 alkyl-C3-C 0 cycloalkyl, -C1 - C 3 alkyl-C5-C 0 cycloalkenyl or -C1 - C 3 alkylheterocyclyl, or R 7 and R 8 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 NR2 aR2 where R2 a is hydrogen, C C alkyl, -CH -OPO(OR ) or 6 2 a 5- or 6-membered heterocyclic ring, and R2 is absent or C C alkyl; and in which any of the aforementioned R 6 , R 7 or R 8 groups may be optionally tuted by one or more groups, e.g. one group, selected from cyano, -OPO(OR ) , -(0(CH ) ) OR24 (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, C 1- C 3 alkyl or - PO(OR )(OR20)), C C alkoxy, C C fluoroalkoxy, C C alkyl, C C fluoroalkyi and - 6 6 6 6 C(0)NR aR , where Ra and R are independently selected from hydrogen and C C alkyl, and any of the aforementioned R 6 , R 7 or R 8 groups may be optionally substituted by one or more n atoms; and (b) aryl, heteroaryl, C 1- C 3 alkylaryl and -C1 - C 3 alkylheteroaryl, said aryl and heteroaryl groups being optionally substituted; or R 8 , R 9 and R2 may independently represent hydrogen.
Optional tuents for phenyl, aryl and heteroaryl groups within the definitions of R , R2, R3, R4a, R4 , R5, R6, R7, R 6 , R 7 , R 8 , R 9 and R20 are suitably selected from hydroxyl, halo, cyano, - (CHR26) -OPO(OR )(OR20) wherein q represents 0 or 1 (said group not being substituted by another R 9 or R2 containing group), C C alkoxy or C C alkoxy, e.g. C 1- C 3 alkoxy or 6 6 C 1- C 3 fluoroalkoxy such as methoxy, ethoxy or trifluoromethoxy, C C alkyl or C C fluoroalkyi, 6 6 e.g. C 1- C 3 alkyl or C 1- C 3 fluoroalkyi such as methyl or trifluoromethyl, and -C(0)NR aR , where Ra and R are independently selected from hydrogen and C C alkyl e.g. C 1- C 3 alkyl such as ; and also when two adjacent hydroxyl tuents are present they may optionally be connected by a methylene group to form an acetal. Another possible al substituent is - SF . Said aryl and heteroaryl groups, if substituted, may be substituted by 1 , 2 or 3 , ably 1 or 2 , more preferably 1 substituent.
Optional tuents for the C C alkyl, C -C alkenyl, C -C alkynyl, 6 2 6 2 6 C3-C10 cycloalkyl, C5-C10 cycloalkenyl, heterocyclyl, -C1 - C 3 alkyl-C3-C 0 cycloalkyl, -C1 - C 3 alkyl-C5-C 0 cycloalkenyl, -C C alkylheterocyclyl or heterocyclic ring groups of R5, R6, R7, R 6 , R 7 , R 8 , R 9 and R20 include substituents selected from cyano, -OPO(OR )(OR2 ) (said group not being substituted by another R 9 or R2 ning group), C C alkoxy or C C fluoroalkoxy, e.g. C 1- C 3 alkoxy or 6 6 C 1- C 3 alkoxy such as methoxy, ethoxy or trifluoromethoxy, C C alkyl or C C fluoroalkyi, 6 6 e.g. C 1- C 3 alkyl or C 1- C 3 fluoroalkyi such as methyl or trifluoromethyl, and R aR , where Ra and R are ndently selected from en and C C alkyl e.g. C 1- C 3 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 C I atoms (especially F atoms).
R 6 preferably ents C C 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(OR )(OR20) and -(0(CH ) ) OR24, 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 C alkyl or -PO(OR )(OR20) .
Alternatively, R 6 preferably represents phenyl optionally substituted (and is preferably substituted) by -(CHR26) -OPO(OR )(OR20) n q represents 0 or 1.
R 7 preferably represents C C alkyl e.g. methyl. R 8 preferably represents C C alkyl e.g. 6 6 methyl. Alternatively, R 7 and R 8 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 NR2 a where R2 a is hydrogen, C C alkyl, -CH -OPO(OR )(OR20) or a 5- or 6-membered 6 2 heterocyclic ring.
R 9 is preferably hydrogen, methyl or ethyl, ally hydrogen.
R2 is preferably hydrogen, methyl or ethyl, especially hydrogen.
R2 a is preferably hydrogen or methyl.
R2 is preferably absent.
R26 is preferably hydrogen or methyl, more preferably .
In one embodiment q represents 0 . In another embodiment q represents 1.
In one embodiment one of R4a and R4 represents a prodrug derivative group as defined above.
In r ment both of R4a and R4 represent a g group as defined above. When only one of R4a and R4 represents a prodrug derivative group as defined above the other of R4a and R4 is preferably hydrogen.
In one embodiment both of R4a and R4 are independently selected from -C(0)R 16 , -S0 NH , - 2 2 PO(OR )(OR20) , -CHR26-OPO(OR ) where R26 is hydrogen or C C alkyl, and - C(0)NR 7R18. In a further embodiment one of R4a and R4 is selected from -C(0)R 16 , -S0 NH , 2 2 -PO(OR )(OR20) , -OPO(OR )(OR20) where R26 is hydrogen or C C alkyl, and - C(0)NR 7R18; and the other of R4a and R4 is hydrogen.
One or both of R4a and R4 are ably ndently selected from -C(0)R 16 .
When the prodrug is a carboxylate ester prodrug, e.g. n one or both of R4a and R4 are - C(0)R 16 , the carbon atom nt 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 R4a and R4 are independently selected from -S0 NH , -PO(OH) , -CH -PO(OH) , t) , 2 2 2 2 2 2 -CON-(4-N-piperidinyl-piperidine), -COt-butyl, -COisopropyl, -CON-(N-methyl)piperazine, -CON- piperazine, -CON(CH ) , COCH , -CO-(CH ) -OMe, -CO(CH ) -(0(CH ) ) OMe where p is 1 to 3 2 3 2 2 2 2 2 2 p 12, -CO-CMe -CH -(0(CH ) ) OMe where p is 1 to 12, -CO-CMe -CH -(0(CH ) ) O-PO(OH) 2 2 2 3 p 2 2 2 2 p 2 where p is 1 to 12, -CO-CMe -CH -(0(CH ) ) O-PO(OH) where p is 1 to 12, -CO-(4- 2 2 2 2 p 2 phosphonoxymethylbenzene) and -CO-(4-phosphonoxymethylcyclohexane); wherein when only one of R4a and R4 represents a prodrug derivative group as defined above the other of R4a and R4 is hydrogen. A group of specific examples of prodrug derivatives include compounds of formula (I) wherein R4a and R4 are independently selected from -S0 NH , ) , -CON-(4- 2 2 2 N-piperidinyl-piperidine), -COt-butyl, -COisopropyl, -CON-(N-methyl)piperazine, -CON(CH ) While the red groups for each variable have generally been listed above tely for each variable, preferred compounds of this invention include those in which several or each variable in formula (I) is ed from the preferred, more preferred or particularly listed groups for each le. 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 ably less than 1000, even more preferably less than 800, for example less than 600.
Particular compounds of the invention include the following: J , , -tetraethyl(4-fluorophenyl)-3,4-dihydroxy-1/-/-pyrrole-2,5-dicarboxamide; 1-(4-ethoxyphenyl)-A J , , -tetraethyl-3,4-dihydroxy-1/-/-pyrrole-2,5-dicarboxamide; N2,N2, , -tetraethyl-3,4-dihydroxy(4-methoxyphenyl)-1/-/-pyrrole-2,5-dicarboxamide; (3,4-dihydroxy(4-methoxyphenyl)-1/-/-pyrrole-2,5-diyl)bis(morpholinomethanone); 3,4-dihydroxy(4-methoxyphenyl)-/V 2 L/2 , -tetramethyl-1/-/-pyrrole-2,5-dicarboxamide; (1-(4-fluorophenyl)-3,4-dihydroxy-1/-/-pyrrole-2,5-diyl)bis(morpholinomethanone); 1-(4-fluorophenyl)-3,4-dihydroxy-/V 2 -dimethyl-/V2 nyl-1H-pyrrole-2,5-dicarboxamide; 1-(4-fluorophenyl)-3,4-dihydroxy-/V 2 L/2 , -tetramethyl-1/-/-pyrrole-2,5-dicarboxamide; di-tert-butyl 4,4'-(1-(4-fluorophenyl)-3,4-dihydroxy-1/-/-pyrrole-2,5-dicarbonyl)bis(piperazine carboxylate); 1,1'-(4,4'-(1-(4-fluorophenyl)-3,4-dihydroxy-1/-/-pyrrole-2,5-dicarbonyl)bis(piperazine-4,1- diyl))diethanone; (1-(4-fluorophenyl)-3,4-dihydroxy-1/-/-pyrrole-2,5-diyl)bis(piperidinylmethanone); ethyl 5-(dimethylcarbamoyl)-3,4-dihydroxy(4-methoxyphenyl)-1/-/-pyrrolecarboxylate; 1-(4-fluorophenyl)-3,4-dihydroxy-/V 2 -diisopropyl-/\/2 -dimethyl-1/-/-pyrrole-2,5- dicarboxamide; L/2 -dibenzyl(4-fluorophenyl)-3,4-dihydroxy-/V 2 -dimethyl-1H-pyrrole-2,5-dicarboxamide; di-te/f-butyl 4,4'-(3,4-dihydroxy(4-methoxyphenyl)-1/-/-pyrrole-2,5-dicarbonyl)bis(piperazine carboxylate); (3,4-dihydroxy(4-methoxyphenyl)-1/-/-pyrrole-2,5-diyl)bis(piperazinylmethanone); 1-(4-fluorophenyl)-3,4-dihydroxy-A J J , -tetraisopropyl-1/-/-pyrrole-2,5-dicarboxamide; (3,4-dihydroxy(4-methoxyphenyl)-1/-/-pyrrole-2,5-diyl)bis(piperidinylmethanone); 3,4-dihydroxy(2-methoxyphenyl)-/V 2 L/2 , -tetramethyl-1/-/-pyrrole-2,5-dicarboxamide; (3,4-dihydroxy(2-methoxyphenyl)-1/-/-pyrrole-2,5-diyl)bis(morpholinomethanone); 1-(4-ethoxyphenyl)-3,4-dihydroxy-/V 2 L/2 , methyl-1/-/-pyrrole-2,5-dicarboxamide; (1-(4-ethoxyphenyl)-3,4-dihydroxy-1/-/-pyrrole-2,5-diyl)bis(morpholinomethanone); (3,4-dihydroxy(4-(trifluoromethyl)phenyl)-1/-/-pyrrole-2,5-diyl)bis(morpholinomethanone); -ethyl-3,4-dihydroxy(4-methoxyphenyl)-/\/ , -dimethyl-1/-/-pyrrole-2,5-dicarboxamide; 3,4-dihydroxy(4-methoxyphenyl)-/\/,A/-dimethyl(piperidinecarbonyl)-1/-/-pyrrole carboxamide; 3,4-dihydroxy(4-methoxyphenyl)-/\/,A/-dimethyl(morpholinecarbonyl)-1/-/-pyrrole carboxamide; 3,4-dihydroxy(4-methoxyphenyl)-A J\/-dimethyl(4-methylpiperazine 2-carboxamide; 3,4-d ihydroxy-A/2,L2, -te dicarboxamide; 1-(4-(dimethylcarbamoyl)phenyl)-3,4-dihydroxy-A J oxamide; -butyl (5-(dimethylcarbamoyl)-3,4-dihydroxy(4-rnethoxyphenyl)-1/-/-pyrrol yl)sulfonylcarbamate; -cyano-3,4-dihydroxy(4-methoxyphenyl)-A J\/-dimethyl-1H-pyrrolecarboxarTiide; ethyl 5-(dimethylcarbamoyl)(4-fluorophenyl)-3,4-dihydroxy-1/-/-pyrrolecarboxylate; 1,1'-(4,4'-(3,4-dihydroxy(4-methoxyphenyl)-1/-/-pyrrole-2,5-dicarbonyl)bis(piperazine-4,1- diyl))diethanone; 3,4-dihydroxy(4-methoxyphenyl)-1/-/-pyrrole-2,5-dicarbonitrile; isopropyl 5-(dimethylcarbamoyl)-3,4-dihydroxy(4-methoxyphenyl)-1/-/-pyrrolecarboxylate; -butyl 5-(dimethylcarbamoyl)-3,4-dihydroxy(4-methoxyphenyl)-1/-/-pyrrolecarboxylate; ethyl 3,4-dihydroxy(4-methoxyphenyl)(4-methylpiperazinecarbonyl)-1/-/-pyrrole carboxylate; ethyl utylcarbamoyl)-3,4-dihydroxy(4-methoxyphenyl)-1/-/-pyrrolecarboxylate; ethyl 5-cyano(4-fluorophenyl)-3,4-dihydroxy-1/-/-pyrrolecarboxylate; ethyl 3,4-dihydroxy(4-methoxyphenyl)(morpholinecarbonyl)-1/-/-pyrrolecarboxylate; ethyl 5-(diethylcarbamoyl)-3,4-dihydroxy(4-methoxyphenyl)-1/-/-pyrrolecarboxylate; ethyl 5-(dimethylcarbamoyl)-3,4-dihydroxy(4-iodophenyl)-1/-/-pyrrolecarboxylate; ethyl 5-(dimethylcarbamoyl)-3,4-dihydroxy(3-iodomethoxyphenyl)-1/-/-pyrrole carboxylate; 3,4-bis(benzyloxy)(4-ethoxyphenyl)-A J J J -tetraethyl-1H-pyrrole-2,5-dicarboxarTiide; 3,4-bis(benzyloxy)-A J J J -tetraethyl(4-methoxyphenyl)-1H-pyrrole-2,5-dicarboxamide; 3,4-bis(benzyloxy)(4-methoxyphenyl)-A J J J -tetramethyl-1H-pyrrole-2,5-dicarboxami t-butyl 3,4-bis(benzyloxy)(4-methoxyphenyl)-1/-/-pyrrole-2,5- dicarbonyl)bis(piperazine-l-carboxylate); (3,4-bis(benzyloxy)(4-methoxyphenyl)-1H-pyrrole-2,5-diyl)bis(piperidinylmethanone); 3,4-bis(benzyloxy)(2-methoxyphenyl)-A J J J -tetramethyl-1H-pyrrole-2,5-dicarboxami (3,4-bis(benzyloxy)(2-methoxyphenyl)-1H-pyrrole-2,5-diyl)bis(morpholinornethanone); 3,4-bis(benzyloxy)(4-ethoxyphenyl)-A J J J -tetramethyl-1H-pyrrole-2,5-dicarboxamide; (3,4-bis(benzyloxy)(4-ethoxyphenyl)-1/-/-pyrrole-2,5-diyl)bis(morpholinomethanone); 3,4-bis(benzyloxy)-/V2 N2, , methyl(4-(trifluoromethyl)phenyl)-1/-/-pyrrole-2,5- dicarboxamide; 3,4-bis(benzyloxy)-A/2-ethyl(4-methoxyphenyl)-A J -dimethyl-1H-pyrrole-2,5-dicarboxarTiide; 3,4-bis(benzyloxy)(4-methoxyphenyl)-A J\/-dimethyl(4-rnethylpiperazinecarbonyl)-1/-/- ecarboxamide; 3,4-bis(benzyloxy)(4-methoxyphenyl)-A J\/-dimethyl(piperidinecarbonyl)-1/-/-pyrrole carboxamide; 3,4-bis(benzyloxy)(4-methoxyphenyl)-A J\/-dimethyl(rnorpholinecarbonyl)-1/-/-pyrrole carboxamide; 1,1'-(4,4'-(3,4-bis(benzyloxy)(4-methoxyphenyl)-1/-/-pyrrole-2,5-dicarbonyl)bis(piperazine-4,1- diyl))diethanone; ethyl 3,4-bis(benzyloxy)cyano(4-fluorophenyl)-1/-/-pyrrolecarboxylate; s(benzyloxy)cyano(4-fluorophenyl)-A J\/-dimethyl-1H-pyrrolecarboxamide; ethyl 3,4-bis(benzyloxy)(dimethylcarbamoyl)(4-iodophenyl)-1/-/-pyrrolecarboxylate; 3,4-bis(benzyloxy)(4-methoxyphenyl)-1/-/-pyrrole-2,5-dicarboxamide; 3,4-dihydroxy(4-methoxyphenyl)-1/-/-pyrrole-2,5-dicarboxamide; 3,4-bis(benzyloxy)-/V2, -diethyl(4-methoxyphenyl)-1/-/-pyrrole-2,5-dicarboxamide; 3,4-bis(benzyloxy)(4-fluorophenyl)-1/-/-pyrrole-2,5-dicarboxamide; 1-(4-fluorophenyl)-3,4-dihydroxy-1/-/-pyrrole-2,5-dicarboxamide; 1-(4-ethoxyphenyl)-3,4-dihydroxy-1/-/-pyrrole-2,5-dicarboxamide; thoxyphenyl)-/V2, -diethyl-3,4-dihydroxy-1/-/-pyrrole-2,5-dicarboxamide; ethyl s(benzyloxy)carbamoyl(4-methoxyphenyl)-1/-/-pyrrolecarboxylate; ethyl 5-carbamoyl-3,4-dihydroxy(4-methoxyphenyl)-1/-/-pyrrolecarboxylate; ethyl 3,4-bis(benzyloxy)(2-methoxyphenyl)(4-methoxyphenyl)-1/-/-pyrrolecarboxylate; ethyl 3,4-bis(benzyloxy)(4-methoxyphenyl)(pyridinyl)-1/-/-pyrrolecarboxylate; ethyl 3,4-dihydroxy(4-methoxyphenyl)(pyridinyl)-1/-/-pyrrolecarboxylate; ethyl 3,4-bis(benzyloxy)(4-methoxyphenyl)(pyridinyl)-1/-/-pyrrolecarboxylate; ethyl s(benzyloxy)(4-methoxyphenyl)(pyridinyl)-1/-/-pyrrolecarboxylate; ethyl 5-(4-ethylthiazolyl)-3,4-dihydroxy(4-methoxyphenyl)-1/-/-pyrrolecarboxylate; ethyl 3,4-bis(benzyloxy)(4-methoxyphenyl)(5-methyl-1,3,4-oxadiazolyl)-1/-/-pyrrole carboxylate; ethyl 3,4-bis(benzyloxy)(5-ethyl-1,3,4-oxadiazolyl)(4-methoxyphenyl)-1/-/-pyrrole carboxylate; ethyl 3,4-dihydroxy(4-methoxyphenyl)(2/-/-tetrazolyl)-1/-/-pyrrolecarboxylate; ethyl 3,4-bis(benzyloxy)(4-methoxyphenyl)(2/-/-tetrazolyl)-1/-/-pyrrolecarboxylate; diethyl 3,4-dihydroxy(4-methoxyphenyl)-1/-/-pyrrole-2,5-dicarboxylate (this compound is not novel per se) ; diethyl 3,4-dihydroxyphenyl-1/-/-pyrrole-2,5-dicarboxylate (this compound is not novel per se) ; l 1-(4-fluorophenyl)-3,4-dihydroxy-1/-/-pyrrole-2,5-dicarboxylate; diethyl 3,4-dihydroxy(3-methoxyphenyl)-1/-/-pyrrole-2,5-dicarboxylate; diethyl 3,4-dihydroxy(4-(trifluoromethoxy)phenyl)-1/-/-pyrrole-2,5-dicarboxylate; diethyl 3,4-dihydroxy(4-isopropylphenyl)-1/-/-pyrrole-2,5-dicarboxylate; l 3,4-dihydroxy(4-phenoxyphenyl)-1/-/-pyrrole-2,5-dicarboxylate; diethyl 1-(4-ethoxyphenyl)-3,4-dihydroxy-1/-/-pyrrole-2,5-dicarboxylate; diethyl 1-(4-ethylphenyl)-3,4-dihydroxy-1/-/-pyrrole-2,5-dicarboxylate; diethyl 3,4-dihydroxy(p-tolyl)-1/-/-pyrrole-2,5-dicarboxylate; diethyl 1-(3,4-dimethoxyphenyl)-3,4-dihydroxy-1/-/-pyrrole-2,5-dicarboxylate; diethyl 1-(4-chlorophenyl)-3,4-dihydroxy-1/-/-pyrrole-2,5-dicarboxylate; diethyl 3,4-dihydroxy(4-(trifluoromethyl)phenyl)-1/-/-pyrrole-2,5-dicarboxylate; diethyl 3,4-dihydroxy(3,4,5-trimethoxyphenyl)-1/-/-pyrrole-2,5-dicarboxylate; diethyl -dimethoxyphenyl)-3,4-dihydroxy-1/-/-pyrrole-2,5-dicarboxylate; diethyl 3,4-dihydroxy(4-propoxyphenyl)-1/-/-pyrrole-2,5-dicarboxylate; diethyl 1-(4-ethoxy-3,5-difluorophenyl)-3,4-dihydroxy-1/-/-pyrrole-2,5-dicarboxylate; diethyl 3,4-dihydroxy(3-phenoxyphenyl)-1/-/-pyrrole-2,5-dicarboxylate; diethyl 1-(benzo[d][1,3]dioxolyl)-3,4-dihydroxy-1/-/-pyrrole-2,5-dicarboxylate; diisopropyl 3,4-dihydroxy(4-methoxyphenyl)-1/-/-pyrrole-2,5-dicarboxylate; 2-ethyl 5-isopropyl 3,4-dihydroxy(4-methoxyphenyl)-1/-/-pyrrole-2,5-dicarboxylate; di-tert-butyl 3,4-dihydroxy(4-methoxyphenyl)-1/-/-pyrrole-2,5-dicarboxylate; diethyl hydroxy(2-methoxyphenyl)-1/-/-pyrrole-2,5-dicarboxylate; diethyl 1-(4-cyanophenyl)-3,4-dihydroxy-1/-/-pyrrole-2,5-dicarboxylate; diethyl 1-(4-fluorophenyl)hydroxy(2-hydroxyethoxy)-1/-/-pyrrole-2,5-dicarboxylate; diethyl 3-hydroxy(2-hydroxyethoxy)(4-methoxyphenyl)-1/-/-pyrrole-2,5-dicarboxylate; diethyl 3-(2-(tert-butoxy)oxoethoxy)(4-fluorophenyl)hydroxy-1/-/-pyrrole-2,5- dicarboxylate; diethyl 3-(benzyloxy)(2-(tert-butoxy)oxoethoxy)(4-methoxyphenyl)-1/-/-pyrrole-2,5- dicarboxylate; diethyl 3-(2-aminooxoethoxy)(4-fluorophenyl)hydroxy-1/-/-pyrrole-2,5-dicarboxylate; diethyl 3-(2-(tert-butoxy)oxoethoxy)hydroxy(4-methoxyphenyl)-1/-/-pyrrole-2,5- dicarboxylate; diethyl 3-(2-aminooxoethoxy)hydroxy(4-methoxyphenyl)-1/-/-pyrrole-2,5-dicarboxylate; diethyl 1-(4-(tert-butyl)phenyl)-3,4-dihydroxy-1/-/-pyrrole-2,5-dicarboxylate;and diethyl 3,4-dihydroxy(3-iodomethoxyphenyl)-1 H-pyrrole-2,5-dicarboxylate; or a ceutically acceptable g derivative of any one thereof, or a pharmaceutically acceptable salt or e of any one thereof.
Further particular compounds of the invention include the following: -acetyl-3,4-dihydroxy(4-methoxyphenyl)-/\/,A/-dimethyl-1/-/-pyrrolecarboxamide; ethyl 5-(dimethylcarbamoyl)-3,4-dihydroxy(4-(2-methoxyethoxy)phenyl)-1/-/-pyrrole carboxylate; ethyl ethoxyphosphoryl)oxy)(dimethylcarbamoyl)hydroxy(4-methoxyphenyl)-1/-/- pyrrolecarboxylate; ethyl 1-(4-(2,5,8,1 1-tetraoxatridecanyloxy)phenyl)(dimethylcarbamoyl)-3,4-dihydroxy-1/-/- pyrrolecarboxylate; ethyl 3,4-dihydroxy(4-methoxyphenyl)(methylcarbamoyl)-1/-/-pyrrolecarboxylate; ethyl 5-(dimethylcarbamoyl)-3,4-dihydroxy(4-hydroxyphenyl)-1/-/-pyrrolecarboxylate; ethyl 5-(dimethylcarbamoyl)-3,4-dihydroxy(4-(phosphonooxy)phenyl)-1/-/-pyrrole carboxylate; ethyl 5-(dimethylcarbamoyl)-3,4-dihydroxy(4-(3-morpholinopropoxy)phenyl)-1/-/-pyrrole carboxylate; ethyl 5-(dimethylcarbamoyl)-3,4-dihydroxy(4-(3-(4-methylpiperazinyl)propoxy)phenyl)-1/-/- pyrrolecarboxylate; ethyl 1-(4-(3-(diethoxyphosphoryl)propoxy)phenyl)(dimethylcarbamoyl)-3,4-dihydroxy-1/-/- pyrrolecarboxylate; (3-(4-(2-(dimethylcarbamoyl)(ethoxycarbonyl)-3,4-dihydroxy-1/-/-pyrrol yl)phenoxy)propyl)phosphonic acid; ethyl 5-(dimethylcarbamoyl)-3,4-dihydroxy(4-(3-(methylsulfonyl)propoxy)phenyl)-1/-/-pyrrole- 2-carboxylate; tyl 5-(dimethylcarbamoyl)-3,4-dihydroxy(4-methoxyphenyl)-1/-/-pyrrolecarboxylate; 2,6-dimethylcyclohexyl 5-(dimethylcarbamoyl)-3,4-dihydroxy(4-methoxyphenyl)-1/-/-pyrrole carboxylate; cyclopentyl 5-(dimethylcarbamoyl)-3,4-dihydroxy(4-methoxyphenyl)-1/-/-pyrrole ylate; -(dimethylphosphoryl)-3,4-dihydroxy(4-methoxyphenyl)-/\/,A/-dimethyl-1/-/-pyrrole carboxamide; diethyl 3,4-dihydroxy(4-(2-methoxyethoxy)phenyl)-1/-/-pyrrole-2,5-dicarboxylate; diethyl 3,4-dihydroxy(4-(2-(2-(2-methoxyethoxy)ethoxy)ethoxy)phenyl)-1/-/-pyrrole-2,5- dicarboxylate; diethyl 1-(4-(2,5,8,1 1-tetraoxatridecanyloxy)phenyl)-3,4-dihydroxy-1/-/-pyrrole-2,5- dicarboxylate; diethyl 3,4-dihydroxy(4-(3-morpholinopropoxy)phenyl)-1/-/-pyrrole-2,5-dicarboxylate; and diethyl hydroxy(4-((4-methoxybenzyl)oxy)phenyl)-1/-/-pyrrole-2,5-dicarboxylate; or a pharmaceutically acceptable prodrug derivative of any one thereof, or a pharmaceutically acceptable salt or solvate of any one thereof.
Particular g derivatives of the compounds of the invention include the following: 2-(dimethylcarbamoyl)(ethoxycarbonyl)(4-methoxyphenyl)-1/-/-pyrrole-3,4-diyl diacetate; ethyl 5-(dimethylcarbamoyl)-3,4-bis((dimethylcarbamoyl)oxy)(4-methoxyphenyl)-1/-/-pyrrole- 2-carboxylate; 2-(dimethylcarbamoyl)(ethoxycarbonyl)(4-methoxyphenyl)-1/-/-pyrrole-3,4-diyl bis(4- methylpiperazinecarboxylate); 2-(dimethylcarbamoyl)(ethoxycarbonyl)(4-methoxyphenyl)-1/-/-pyrrole-3,4-diyl bis([1 ,4'- bipiperidine]-1 oxylate); ethyl 5-(dimethylcarbamoyl)((dimethylcarbamoyl)oxy)hydroxy(4-methoxyphenyl)-1/-/- pyrrolecarboxylate; 2-(dimethylcarbamoyl)(ethoxycarbonyl)(4-methoxyphenyl)-1/-/-pyrrole-3,4-diyl bis(2- methylpropanoate); 2-(dimethylcarbamoyl)(ethoxycarbonyl)(4-methoxyphenyl)-1/-/-pyrrole-3,4-diyl bis(2,2- dimethylpropanoate); ethyl 5-(dimethylcarbamoyl)hydroxy(4-methoxyphenyl)(sulfamoyloxy)-1/-/-pyrrole carboxylate; ethyl 5-(dimethylcarbamoyl)hydroxy(4-methoxyphenyl)(phosphonooxy)-1/-/-pyrrole carboxylate; and 2-(dimethylcarbamoyl)(ethoxycarbonyl)hydroxy(4-methoxyphenyl)-1/-/-pyrrolyl [ 1 ,4'- bipiperidine]-1 '-carboxylate; or a ceutically acceptable salt or e of any one thereof.
Further particular prodrug derivatives of the compounds of the invention include the following: ethyl 3,4-bis((diethoxyphosphoryl)oxy)(dimethylcarbamoyl)(4-methoxyphenyl)-1/-/-pyrrole- 2-carboxylate; ethyl 3-((diethoxyphosphoryl)oxy)(dimethylcarbamoyl)hydroxy(4-methoxyphenyl)-1/-/- pyrrolecarboxylate; ethyl ethoxyphosphoryl)oxy)(dimethylcarbamoyl)hydroxy(4-methoxyphenyl)-1/-/- pyrrolecarboxylate; 2-(dimethylcarbamoyl)(ethoxycarbonyl)(4-(2-(2-(2-methoxyethoxy)ethoxy)ethoxy)phenyl)- 1/-/-pyrrole-3,4-diyl bis(2-methylpropanoate); 2-(dimethylcarbamoyl)(ethoxycarbonyl)(4-methoxyphenyl)-1/-/-pyrrole-3,4-diyl bis(3- methoxypropanoate); 2-(dimethylcarbamoyl)(ethoxycarbonyl)(4-methoxyphenyl)-1/-/-pyrrole-3,4-diyl bis(3-(2- methoxyethoxy)propanoate); 2-(dimethylcarbamoyl)(ethoxycarbonyl)(4-methoxyphenyl)-1H-pyrrole-3,4-diyl bis(3-(2- methoxyethoxy)-2,2-dimethylpropanoate); ethyl 5-(dimethylcarbamoyl)hydroxy(4-methoxyphenyl)((phosphonooxy)methoxy)-1/-/- pyrrolecarboxylate; 2-(dimethylcarbamoyl)(ethoxycarbonyl)(4-methoxyphenyl)-1/-/-pyrrole-3,4-diyl bis(3-(2-(2- methoxyethoxy)ethoxy)-2,2-dimethylpropanoate); 2,5-bis(dimethylcarbamoyl)(4-methoxyphenyl)-1/-/-pyrrole-3,4-diyl bis(2-methylpropanoate); ethyl ethylcarbamoyl)hydroxy(4-methoxyphenyl)((phosphonooxy)methoxy)-1/-/- pyrrolecarboxylate; 2-(dimethylcarbamoyl)(ethoxycarbonyl)(4-methoxyphenyl)-1/-/-pyrrole-3,4-diyl bis(2,2- dimethyl(2-(2-(phosphonooxy)ethoxy)ethoxy)propanoate); 2-(dimethylcarbamoyl)(ethoxycarbonyl)(4-methoxyphenyl)-1/-/-pyrrole-3,4-diyl bis(2,2- yl(3-(phosphonooxy)propoxy)propanoate); 2-(dimethylcarbamoyl)(ethoxycarbonyl)(4-methoxyphenyl)-1/-/-pyrrole-3,4-diyl bis(25,25- dimethyl-2,5,8, 11,14 ,17,20,23-octaoxahexacosanoate); 2,5-bis(dimethylcarbamoyl)(4-methoxyphenyl)-1/-/-pyrrole-3,4-diyl bis(25,25-dimethyl- 2,5,8, 11,14 ,17,20,23-octaoxahexacosanoate); 2,5-bis(dimethylcarbamoyl)(4-methoxyphenyl)-1/-/-pyrrole-3,4-diyl bis(2,2-dimethyl(2-(2- (phosphonooxy)ethoxy)ethoxy)propanoate); 2-(dimethylcarbamoyl)(isopropoxycarbonyl)(4-methoxyphenyl)-1/-/-pyrrole-3,4-diyl 2- yl(2-(2-(phosphonooxy)ethoxy)ethoxy)propanoate); 2,5-bis(dimethylcarbamoyl)(4-methoxyphenyl)-1/-/-pyrrole-3,4-diyl bis(piperidine carboxylate); 2,5-bis(dimethylcarbamoyl)(4-methoxyphenyl)-1/-/-pyrrole-3,4-diyl bis(4- ((phosphonooxy)methyl)benzoate); and ( 1 4R,4'R)-2,5-bis(dimethylcarbamoyl)(4-methoxyphenyl)-1/-/-pyrrole-3,4-diyl bis(4- phonooxy)methyl)cyclohexanecarboxylate); or a pharmaceutically acceptable salt or solvate of any one thereof.
Particular compound of the ion and prodrugs which may be mentioned include those designated as compounds UL1-004, UL1-005, UL1-012, UL1-024, UL1-028, UL1-035, UL1- 049, UL1-070, UL1-089, UL1-098, UL1-106, 9, UL1-1 11 , UL1-1 14, UL1-1 15, UL1-1 16, UL1-1 17, UL1-1 18, UL1-120, UL1-121, UL1-122, UL1-124, UL1-126 and UL2-001, in Table 1 below.
Alkyl as used herein refers to straight chain or branched chain alkyl, such as, without limitation, methyl, ethyl, propyl, /so-propyl, butyl, and -butyl. In one embodiment alkyl refers to straight chain alkyl in another ment 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 alkyl groups include -CH F, -CHF and -CF . 2 2 3 Cycloalkyl 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 yclic) but are suitably monocyclic.
Alkoxy as used herein refers to straight or branched chain alkoxy, for e methoxy, ethoxy, y, butoxy. Alkoxy as used herein also extends to embodiments in which the oxygen atom is located within the alkyl chain, for example -CH OCH . In one embodiment the alkoxy is 2 3 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 e, tetrahydrofuran, tetrahydropyran, oxepane, oxocane, thietane, tetrahydrothiophene, tetrahydrothiopyran, thiepane, thiocane, ine, pyrrolidine, piperidine, azepane, azocane, [ 1 ,4]dioxane, oxazolidine, piperazine, and the like a further example is line. Other examples of cyclic 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 ered to be heterocyclic rings.
Aryl as used herein includes C -C mono or bicyclic groups having 1 or 2 rings wherein at least 6 4 one ring is aromatic, ing 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, zine, pyrimidine, ne, hiophene, benzofuran, 1, 2 , 3-triazole and 1, 2 , 4-triazole. In a bicyclic ring system the definition of aryl will be satisfied if at least one ring contains a atom and at least one ring is aromatic. The heteroaryl may be linked to the remainder of the le 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, ium and sodium salts and the like. Salts derived from acids, include those derived from nic 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 ically-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 ion include isotopes of hydrogen, carbon, nitrogen, fluorine, such as 2H , H , C, 4C and 8F. Isotopically labelled compounds of formula (I) may be prepared by carrying out the synthetic methods bed 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 nds illustrated herein (particularly enol-keto tautomers). For example whereas formula (I) illustrates in some embodiments (e.g. when R4a and/or R4 represents H) an enol form, the corresponding keto form is also embraced as part of the invention. The same s to other structures herein which illustrate enol or keto forms of compounds. rly, the disclaimed compounds are disclaimed in all their eric forms.
Compounds of the invention may be prepared by the ing 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 R4a and R4 represent en is shown below in Scheme A : Scheme A In the third step shown in Scheme A , Rx typically represents -C alkyl such as methyl or ethyl.
A method for preparing certain compounds of formula (I) in which R is -C(0)NR R6, R2 is - C(0)OR 7 and R4a and R4 ent hydrogen is shown below in Scheme B : (I) (I) Scheme B In the second step shown in Scheme B, Rx typically represents CrC 6alkyl such as methyl ethyl.
An alternative method for preparing certain compounds of formula (I) in which R is - C(0)NR R6, R2 is -C(0)OR 7and R4a and R4 represent hydrogen is shown below in Scheme C : Scheme C A method for preparing certain nds of formula (I) in which R is -C(0)NHR 6, R2 is C(0)NR R6 and R4a and R4 represent hydrogen is shown below in Scheme D : Scheme D A method for preparing certain compounds of formula (I) in which R is -C(0)NR R6, R2 is C(0)NR R6 and R4a and R4 ent hydrogen is shown below in Scheme E : Scheme E A method for preparing certain compounds of formula (I) in which R is -CN, R2 is C(0)NR R' and R4a and R4 represent hydrogen is shown below in Scheme F : (I) (I) Scheme F A method for preparing certain nds of formula (I) in which R is -C(0)OR 7, R2 is CN and R4a and R4 represent hydrogen is shown below in Scheme G : Scheme G Scheme H A method for preparing certain compounds of formula (I) in which R is -CN, R2 is -CN and R' and R4 represent hydrogen is shown below in Scheme I : Scheme I A method for preparing certain nds of formula (I) in which R and R represent groups other than hydrogen is shown below in Scheme J : (I) (I) Scheme J Scheme J may be adapted to convert one or both hydroxyl groups to OR and/or OR depending on the molar excess of reagent(s) employed. When R4a and R4 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).
A method for preparing certain compounds of formula (I) where R2 is -C(0)R 7 and R4a and R4 represent H is shown below in Scheme K : Scheme K A method for preparing certain compounds of a (I) where R2 is aryl/heteroaryl is shown below in Scheme L : Scheme L Compounds where R2 is -POR2 R22 may be prepared by reaction of a compound of formula (XVII) shown above with a compound of formula POR2 R22CI, followed by deprotection.
A method for preparing certain compounds of a (I) where R is -C(0)OR 7 and R2 is a thiazole containing group and R4a and R4 represent H is shown below in Scheme M : Scheme M A method for preparing certain compounds of formula (I) where R is -C(0)OR 7, R2 is tetrazole and R4a and R4 represent H is shown below in Scheme N : (I) (I) (I) Scheme N A method for preparing n compounds of formula (I) where R is -C(0)OR 7 and R2 is a oxadiazole containing group and R4a and R4 ent H is shown below in Scheme O : (XIX) (XX) (I) Scheme O In the above Schemes A to O the general conditions for ming 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 e the skilled person will be familiar with rd 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 R and/or R2 represents -C(0)NHC(0)R 7 may be ed by on of a compound of formula (I) in which R and/or R2 represents -C(0)NH 2 with a compound of formula R7C(0)L wherein L represents a leaving group, such as halogen.
Protecting groups may be required to t 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 Wiley & Sons Inc; 4th 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 ing to a further aspect of the invention there is ed a compound of formula (II): (II) wherein R and R2 are as defined above for the compounds of formula (I), and R3 is phenyl substituted by 1 or more, e.g. 1, 2 or 3 , substituents (e.g. 1 substituent) ndently selected from halo, cyano, hydroxyl, C C6 alkoxy, C C6 hydroxyalkoxy, C C6 fluoroalkoxy, C C6 alkyl, C - 6 fluoroalkyl, -C(0)NRaR , where Ra and R are independently selected from hydrogen and Ci-Ce alkyl; -O-R 5 n R 5 is -(CH 2 ) 2)x-P(0)(OR 2, where x is 0 , 1, 2 , 3 or 4 and R23 is independently selected from hydrogen and C 3 alkyl, -(CH2)y-S(0) 2Me where y is 1 , 2 , 3 or 4 , -Ci-C6 alkylheterocyclyl which heterocyclyl group may be optionally substituted by C 3 alkyl, - C - 6 alkylphenyl which phenyl group may be optionally substituted by C 3 alkoxy, or phenyl or 5- or 6-membered heteroaryl which phenyl or heteroaryl group may optionally be tuted by a group selected from C C alkyl and halo; or -(0(CH 24, where 2) )pOR each z , which may be the same or different, ents 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 CH 2, or a salt or protected derivative thereof; provided that when R5 or R6 is ally substituted aryl, said aryl is optionally substituted by 1 , 2 or 3 groups selected from hydroxyl, halo, cyano, C C6 alkoxy or C C6 fluoroalkoxy, C C6 alkyl or C C6 fluoroalkyl, and -C(0)NRaR , where Ra and R are independently selected from en and C C6 alkyl; or when two adjacent hydroxyl substituents are present they may optionally be connected by a methylene group to form an acetal; and provided that the compound is not: a) methyl 2-((2-oxo(phenylamino)ethyl)(phenyl)amino)acetate; b) methyl 2-((4-methoxyphenyl)(2-oxo(phenylamino)ethyl)amino)acetate; c) ethyl 2-(phenyl(tosylmethyl)amino)acetate; d) ethyl 2-((cyanomethyl)(3,4-dichlorophenyl)amino)acetate; e) methyl 2-((cyanomethyl)(p-tolyl)amino)acetate; f) ethyl 2-(mesityl(2-oxopropyl)amino)acetate; g) dimethyl 2,2'-((3-bromophenyl)azanediyl)diacetate; or h) dimethyl 2,2'-((4-iodophenyl)azanediyl)diacetate.
The specific compounds disclaimed from the definition of formula (II) above are found in CAS as follows: methyl 2-((2-oxo(phenylamino)ethyl)(phenyl)amino)acetate (CAS 8626995), methyl 2-((4-methoxyphenyl)(2-oxo(phenylamino)ethyl)amino)acetate (CAS 8626998), ethyl 2-(phenyl(tosylmethyl)amino)acetate (CAS 47), ethyl 2-((cyanomethyl)(3,4- dichlorophenyl)amino)acetate (CAS 10038783), methyl anomethyl)(ptolyl )amino)acetate (CAS 1001341) and ethyl 2-(mesityl(2-oxopropyl)amino)acetate (CAS 9357581); or otherwise as s: dimethyl 2,2'-((3-bromophenyl)azanediyl)diacetate and dimethyl 2,2'-((4-iodophenyl)azanediyl)diacetate national Patent Application WO2006/020004).
Compounds of formula (II) which may be mentioned in particular are those in which R is - C(0)NR R6 and R2 is -C(0)NR R6 or wherein R is -C(0)NR R6 and R2 is -C(0)OR 7.
According to a further aspect of the d a compound of formula (II): (II) wherein R is -C(0)NR R6 and R2 is -C(0)NR R6 or wherein R is -C(0)NR R6 and R2 is - C(0)OR 7, and R3, R5 and R6 are as defined above for the compounds of formula (I), or a salt or protected derivative thereof; provided that when R5 or R6 is optionally substituted aryl, said aryl is optionally substituted by 1 , 2 or 3 groups selected from yl, halo, cyano, C C alkoxy or C C fluoroalkoxy, C C 6 6 6 alkyl or C C fluoroalkyi, and R aR , where Ra and R are independently selected from hydrogen and C C alkyl; or when two adjacent hydroxyl substituents are present they may optionally be connected by a ene group to form an acetal; and provided that the compound is not: a) methyl 2-((2-oxo(phenylamino)ethyl)(phenyl)amino)acetate; or b) methyl methoxyphenyl)(2-oxo(phenylamino)ethyl)amino)acetate.
Any preferences or examples of specific groups for R , R2, R3, R5, R6, Ra and R as described above for the compounds of formula (I) also apply to the definitions of R , R2, R3, R5, R6, Ra and R in the compounds of formula (II).
Particular compounds of formula (II) include those ned in the examples.
There is also provided a process for preparing nds of formula (I) in which R4a and R4 represent H which comprises reacting a compound of formula (II) with a compound of formula ROCOCOOR x in which Rx represents C C 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) without provisos a) to i) are referred to below as unds of the invention".
As indicated above the compounds of the invention are useful for treatment of bacterial ions caused by bacteria producing orming toxins, such as cholesterol dependent cytolysins.
In particular the compounds of the invention are useful for the treatment of ia 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 ceutical composition sing a compound of formula (I) without provisos a) to i) optionally in combination with one or more pharmaceutically acceptable diluents or carriers.
Diluents and carriers may include those suitable for parenteral, oral, l, 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 stration 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 e as bed in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, PA., (1985). Formulations for parenteral administration may n as excipients sterile water or saline, alkylene glycols such as propylene glycol, polyalkylene glycols such as hylene glycol, oils of vegetable origin, hydrogenated naphthalenes and the like. Formulations for nasal stration may be solid and may contain ents, 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 ents 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 e. Liquid compositions may contain conventional ves such as suspending agents, for example sorbitol syrup, methyl cellulose, sugar syrup, n, ymethyl-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 ted hydroxytoluene (BHT). Liquid compositions may be encapsulated in, for example, gelatin to provide a unit dosage form.
Solid oral dosage forms include tablets, ece 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. ceutical compositions of the ion may optionally include one or more anti-oxidants (e.g. ascorbic acid or metabisulfate and salts thereof).
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 ed 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 us anthracis and Suilysin (SLY) produced by Streptococcus suis.
The nds of the invention are useful for the treatment of bacterial infections, e.g. pneumococcal infections including the associated toxaemia where the lysin toxin has been demonstrated to play a pivotal role in the es ed. Such diseases include, but are not limited to, pneumococcal pneumonia, coccal meningitis, pneumococcal septicaemia/bacteraemia, pneumococcal tis and pneumococcal otitis media. The nds of the invention are also useful for the treatment of pneumococcal infections associated with other conditions. Such conditions e (without tion) 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), ing 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 es.
The compounds of the invention are useful for the treatment of infections caused by Clostridium perfringens including, but not limited to, gas gangrene, terized 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 sins, examples of which include, but are not limited to: Porcine meningitis, septicaemia/bacteraemia and septic shock caused by Streptococcus suis which es 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 ensis of the above diseases.
The compounds of the invention may well also be useful for the tion 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 es the pore-forming toxin staphylococcal a- hemolysis and peritonitis caused by pathogenic ichia 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 ococcus 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. us 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, coccal meningitis, pneumococcal septicaemia/bacteraemia, pneumococcal keratitis or coccal otitis media; and -A compound of the invention for the treatment of conditions selected from gas gangrene, gastrointestinal anthrax, inhalational anthrax, porcine itis, 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 a (I) without provisos a) to i) for use in the treatment of the above mentioned conditions.
In a further aspect, the present ion provides a compound of formula (I) without provisos a) to i) for the manufacture of a medicament for the treatment of the above mentioned conditions.
In a further aspect, the t invention es 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 a (I) without provisos a) to i) or a pharmaceutical ition thereof.
The word "treatment" is intended to embrace prophylaxis as well as eutic ent.
The compounds of the invention may be used either alone or in combination with further eutically active ingredients. Thus compounds of the invention may be administered in combination, simultaneously, sequentially or separately, with further eutically active ingredients either er 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 ions. For example, possible combinations for treatment include combinations with antimicrobial agents, e.g. antibiotic agents, including natural, synthetic and semisynthetic antimicrobial . Examples of antibiotic agents include b-lactams including, but not limited to, penicillin, benzylpenicillin, amoxicillin and all generations thereof; b-lactams in combination with b-lactamase tors including, but not limited to, clavulanic acid and sulbactam; cephalosporins including, but not limited to, cefuroxime, cefotaxime and ceftriaxone; fluoroquinolones including, but not d to, levofloxacin and moxifloxacin; tetracyclines including, but not limited to, doxycycline; ides 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 ing, but not limited to, rifampicin; and chloramphenicol.
Further combinations e combinations with immunomodulatory , such as anti- inflammatory agents.
Immunomodulatory agents can include for e, 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 , for example, es, 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 , vascular or lymphatic systems or combinations thereof. Examples of nflammatory and immunomodulatory agents include, but are not limited to, interferon derivatives such as betaseron, b-interferon, prostane derivatives such as iloprost and ost, corticosteroids such as prednisolone, methylprednisolone, dexamethasone and fluticasone, COX2 inhibitors, immunsuppressive agents such as porine 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 t receptors of interleukins, other cytokines and T-cell-proteins. Further ant i inflammatory 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 tors.
Thus an aspect of the invention provides a compound of formula (I) without provisos a) to i) in combination with one or more r active ingredients, for example one or more of the active ients described above.
Another aspect of the invention provides a pharmaceutical composition comprising a compound of formula (I) without provisos a) to i) optionally in combination with one or more pharmaceutically acceptable adjuvants, ts 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) without provisos a) to i); and (B) another eutic agent, n each of components (A) and (B) is formulated in admixture with a pharmaceuticallyacceptable adjuvant, diluent or r.
In this aspect of the invention, the combination product may be either a single nation) 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 asses a kit of parts comprising components: (i) a pharmaceutical formulation including a compound of formula (I) without provisos a) to i) in admixture with a pharmaceutically acceptable adjuvant, diluent or carrier; and (ii) a pharmaceutical formulation including another therapeutic agent, in ure with a ceutically-acceptable adjuvant, diluent or carrier; which ents (i) and (ii) are each ed 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, ent (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 modulatory agents mentioned above.
The combination product (either a combined preparation or kit-of-parts) of this aspect of the ion may be used in the treatment or prevention of any of the conditions mentioned above.
The nds of formula (I) without provisos a) to 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 es a compound of formula (I) without provisos a) to 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) without provisos a) to 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 ion.
Examples iations AcOH l acetic acid aq. aqueous Bn benzyl br broad Boc te/f-butoxycarbonyl COPD chronic obstructive pulmonary disease d doublet DCM dichloromethane DIPEA A/,A/-diisopropylethylamine DMAP 4-dimethylaminopyridine DMF A/,A/-dimethylformamide DMSO dimethylsulfoxide EtOAc ethyl acetate h hour(s) HATU A/,A/,yV',yV'-tetramethyl(7-azabenzotriazolyl)uronium PF HPLC high performance liquid chromatography m multiplet MeCN itrile MeOH methanol min minute(s) NMR nuclear magnetic resonance quin. quintet RT room temperature s singlet sat. saturated SAX solid supported strong cation exchange resin sept. septet sext. sextet triplet TFAA trifluoroacetic acid anhydride THF tetrahydrofuran General ures 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 med on pre-packed silica (230-400 mesh, 40-63 mM) cartridges. ical Methods e Phase High Performance Liquid Chromatography: Agilent Zorbax Extend RRHT column 1.8 mhi (4.6x30 mm) flow rate 2.5 mL/min eluting with a H 0-MeCN gradient containing 0.1% v/v formic acid employing UV detection at 215 and 254 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 a (I) were prepared using the ing general methods: Example A : , , , -Tetraethyl-3,4-dihydroxy(4-methoxyphenyl)-1/-/-pyrrole-2,5- dicarboxamide (UL1-003) UL1-003 Step (i): 3,4-Bis(benzyloxy)(4-methoxyphenyl)-1/-/-pyrrole-2,5-dicarboxylic acid (2) A e of diethyl 3,4-bis(benzyloxy)(4-methoxyphenyl)-1/-/-pyrrole-2,5-dicarboxylate (1) (2.8 g , 5.29 mmol), 2M NaOH (aq.) (26.4 mL, 52.9 mmol), in ethanol (12 mL) and THF (20 mL) was stirred at 60°C for 72 h . After cooling to RT, the e was acidified with 6M HCI (aq.) and the ing precipitate was collected by filtration, washed with water (5 mL), and Et 0 (5 mL) to afford 4-bis(benzyloxy)(4-methoxyphenyl)-1/-/-pyrrole-2,5-dicarboxylic acid (2) ( 1 .94 g , 67 %) as an off-white solid: m/z 474 (M+H)+ (ES+) ; 472 (M-H) (ES ) . H NMR (400 MHz, DMSO-d ) d 12.61 (s, 2H), 7.46-7.40 (m, 4H), 7.39-7.29 (m, 6H), 7.16-7.07 (m, 2H), 6.92-6.84 (m, 2H), .07 (s, 4H), 3.78 (s, 3H).
Step (ii): 3,4-Bis(benzyloxy)-/V , , , -tetraethyl(4-methoxyphenyl)-1/-/-pyrrole-2,5- dicarboxamide 6) To a solution of 4-bis(benzyloxy)(4-methoxyphenyl)-1/-/-pyrrole-2,5-dicarboxylic acid (2) (400 mg, 0.85 mmol) and DIPEA (386 m , 2.1 1 mmol) in DMF (4 mL) was added HATU (803 mg, 2.1 1 mmol) followed by diethylamine (219 m , 2.1 1 mmol). The reaction mixture was stirred at RT for 1h and then partitioned between Et 0 (50 mL) and sat. NaOAc (aq.) (30 mL). The organic phase was washed succesively with sat. NaOAc (aq.) (20 mL), sat. NaHC0 (aq.) (20 mL) and brine (2 x 20 mL), dried (MgS0 ) , filtered and concentrated in vacuo to afford the crude product. The residue was purified by silica gel chromatography (12 g , 30% EtOAc/isohexane) to afford 3,4-bis(benzyloxy)-/\/ , , , -tetraethyl(4-methoxyphenyl)-1/-/-pyrrole-2,5- dicarboxamide (UL1-046) (201 mg, 4 1 %) as a yellow solid: m/z 584 (M+H)+ (ES+) . H NMR (400 MHz, DMSO-de) d : 7.47-7.22 (m, 10H), 7.09-6.99 (m, 2H), 6.94-6.76 (m, 2H), 4.97 (s, 4H), 3.74 (s, 3H), .03 (m, 8H), 0.98-0.71 (m, 12H).
Step (iii): , , -Tetraethyl-3,4-^ dicarboxamide (UL1-003) A solution of 3,4-bis(benzyloxy)-A J , , -tetraethyl(4-methoxyphenyl)-1/-/-pyrrole-2,5- dicarboxamide UL1-046 (50 mg, 0.086 mmol) in ol/DCM (4 ml_/4 mL) was passed through a Thales Ή -cube' cartridge (10% Pd/C) at a flow rate of 1 mL/min at 25°C under H2 (full H2 mode). The output was concentrated in vacuo to afford N2,N2, , ethyl-3,4-dihydroxy- 1-(4-methoxyphenyl)-1/-/-pyrrole-2,5-dicarboxamide (UL1-003) (32 mg, 93 %) as a light yellow solid: m/z 404 (M+H)+ (ES+) ; 402 (M-H) (ES ) . H NMR (400 MHz, DMSO-d ) d 8.26 (s, 2H), 6.96-6.88 (m, 2H), 6.87-6.81 (m, 2H), 3.72 (s, 3H), 3.27 (q, J = 7.1 Hz, 8H), 0.96 (t, J = 7.0 Hz, 12H).
Example B : Ethyl 5-(dimethylcarbamoyl)-3,4-dihydroxy(4-methoxyphenyl)-1/-/-pyrrole UL1 -012 Step (i): Diethyl 2,2'-((4-methoxyphenyl)azanediyl)diacetate (4) Ethyl 2-bromoacetate (146 mL, 1.3 mol) was added dropwise to a stirred solution of 4- methoxyaniline (3) (75 g , 0.61 mol) and DIPEA (265 mL, 1.5 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 cs were washed succesively with 2 M HCI (aq.) (2 x 300 mL), water (500 mL), and brine (500 mL), dried (MgS0 ) , ed and solvents removed in vacuo to give diethyl 2,2'- ((4-methoxyphenyl)azanediyl)diacetate (4) (180 g , 100 %) as a purple oil: m/z 296 (M+H)+ (ES+) . H NMR (400 MHz, CDCI ) d 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(4-methoxyphenyl)-1/-/-pyrrole-2,5-dicarboxylate (UL2-001) Diethyl oxalate (83 ml, 0.61 mol) was added dropwise to a stirred solution of l 2,2'-((4- yphenyl)azanediyl)diacetate (4) (180 g , 0.61 mol) in NaOEt (21% by wt in EtOH) (506 ml, 1.3 mol), the mixture was stirred at 100°C for 1h. The reaction was quenched with acetic acid (210 ml, 3.7 mol) and the resulting suspension was poured into iced water ( 1 L), the ing off-white solid collected by vacuum filtration. The crude product was recrystallised from hot EtOH (3.5 L) to give diethyl 3,4-dihydroxy(4-methoxyphenyl)-1/-/-pyrrole-2,5-dicarboxylate (UL2-001) (152 g , 7 1 %) as a white solid: m/z 350 (M+H)+ (ES+) ; 348 (M-H) (ES ) . H NMR (400 MHz, e) d 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)(4-methoxyphenyl)-1/-/-pyrrole-2,5-dicarboxylate (1) Benzyl bromide (42.6 ml, 358 mmol) was added dropwise to a stirred suspension of 3,4- dihydroxy(4-methoxyphenyl)-1/-/-pyrrole-2,5-dicarboxylate (UL2-001) (50 g , 143 mmol) and K C0 (49.5 g , 358 mmol) in DMF ( 1 L), the reaction mixture was stirred at 60°C for 4h. After 2 3 cooling to RT the reaction mixture was poured into ether (500 mL) and washed with brine (3 x 250 mL), dried (MgS0 ) , filtered and concentrated in vacuo to afford a bright yellow solid. The crude product was triturated with isohexane to give diethyl s(benzyloxy)(4- methoxyphenyl)-1/-/-pyrrole-2,5-dicarboxylate (1) (64.8 g , 85 %) as a white solid: m/z 530 (M+H)+ (ES+) . H NMR (400 MHz, DMSO-d ) d : 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)(ethoxycarbonyl)(4-methoxyphenyl)-1/-/- pyrrolecarboxylate (5) To a solution of diethyl 3,4-bis(benzyloxy)(4-methoxyphenyl)-1/-/-pyrrole-2,5-dicarboxylate (1) (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% Et N), then 20%MeOH/EtOAc (+2% Et N)) to 3 3 afford triethylammonium 3,4-bis(benzyloxy)(ethoxycarbonyl)(4-methoxyphenyl)-1/-/- ecarboxylate (5) (39.3 g , 83 %) as a yellow oil: m/z 502 (M+H)+ (ES+) ; 500 (M-H) (ES ) .
H NMR (400 MHz, DMSO-d ) d : 7.51-7.26 (m, 10H), 7.1 1-7.05 (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)(dimethylcarbamoyl)(4-methoxyphenyl)-1/-/-pyrrole carboxylate (6) To a solution of triethylammonium 3,4-bis(benzyloxy)(ethoxycarbonyl)(4-methoxyphenyl)- 1H-pyrrolecarboxylate (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 1M HCI (aq.) (250 ml_). The organic phase was washed succesively with 1M HCI (aq.) (250 ml_), sat. NaHC0 (aq.) (2 x 250 ml_), and brine (2 x 250 ml_), dried (MgS0 ) , ed and 3 4 concentrated in vacuo to afford ethyl 3,4-bis(benzyloxy)(dimethylcarbamoyl)(4- methoxyphenyl)-1/-/-pyrrolecarboxylate (6) (7.62 g , 79 %) as a light yellow oil, that fied on standing: m/z 529 (M+H)+ (ES+) . H NMR (400 MHz, DMSO-d ) d : 7.51-7.21 (m, 10H), 7.14- 7.03 (m, 2H), .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): Ethyl 5-(dimethylcarbamoyl)-3,4-dihydroxy(4-methoxyphenyl)-1/-/-pyrrole carboxylate (UL1-012) Ethyl 3,4-bis(benzyloxy)(dimethylcarbamoyl)(4-methoxyphenyl)-1/-/-pyrrolecarboxylate (6) (1.03 g , 1.94 mmol) was dissolved in EtOH and then treated with 10% Pd/C (37 mg). The reaction mixture was purged with N for 5 min then Hydrogen gas was bubbled through the e with stirring at RT for 1.5h. The mixture was filtered through Celite and concentrated in vacuo. The al yellow solid was triturated with Et 0 to afford ethyl 5-(dimethylcarbamoyl)- 3,4-dihydroxy(4-methoxyphenyl)-1/-/-pyrrolecarboxylate 12) (602 mg, 89 %) as a white solid: m/z 349 (M+H)+ (ES+) , 347 (M-H) (ES ) . 1H NMR (400 MHz, DMSO-d ) d : 8.60 (s, 1H), 8.46 (s, 1H), 7.08-7.01 (m, 2H), 6.90-6.82 (m, 2H), 4.00 (q, J = 7.0 Hz, 2H), 3.76 (s, 3H), 2.83 (br s , 6H), 0.99 (t, J = 7.1 Hz, 6H).
Example C : -Ethyl-3,4-dihydroxy(4-methoxyphenyl)-/\/ , -dimethyl-1/-/-pyrrole-2,5- dicarboxamide (UL1-024) UL1 -055 UL1 -024 Step (i): 3,4-Bis(benzyloxy)-/\/ -ethyl(4-methoxyphenyl)-/\/ , -dimethyl-1/-/-pyrrole-2,5- dicarboxamide (UL1 -055) To a stirred suspension of ethyl 3,4-bis(benzyloxy)(dimethylcarbamoyl)(4-methoxyphenyl)- 1/-/-pyrrolecarboxylate (6) (200 mg, 0.38 mmol) and ethylamine hydrochloride (61 .7 mg, 0.75 mmol) in THF (2 mL) was added isopropylmagnesium chloride (2 M in THF) (757 m I_ , 1.51 mmol) and the reaction stirred at RT for 2h, further portions of ethylamine hydrochloride (120 mg, 1.5 mmol) and isopropylmagnesium chloride (2 M in THF) ( 1 .5 mL, 3.0 mmol) were added and the reaction stirred for 16h at RT. The reaction e was partitioned between sat. NH4C I on (aq.) (5 mL) and EtOAc (20 mL) the aqueous was further acidified with 1 M HCI (5 mL).
The organic layer was separated and washed with brine (25 mL), dried (MgS0 ) , filtered and concentrated in vacuo to afford crude product. The residue was purified by silica gel chromatography (25 g , 30-80% EtOAc in isohexane) to afford 3,4-bis(benzyloxy)-/V -ethyl(4- methoxyphenyl)-/\/ , -dimethyl-1/-/-pyrrole-2,5-dicarboxamide (UL1-055) (192 mg, 95 %) as a yellow oil: m/z 528 (M+H)+ (ES+) . H NMR (400 MHz, CDCI3) d : 7.44-7.30 (m, 10H), 7.20-7.09 (m, 2H), 7.06-6.97 (m, 1H), .80 (m, 2H), 5.20 (s, 2H), 5.07 (s, 2H), 3.79 (s, 3H), 3.25-3.14 (m, 2H), 2.79 (s, 3H), 2.72 (s, 3H), 0.97 (t, J = 7.3 Hz, 3H).
Step (ii) -Ethyl-3,4-dihydroxy(4-methoxyphenyl)-/\/ , -dimethyl-1/-/-pyrrole-2,5- dicarboxamide (UL1-024) A solution of 3,4-bis(benzyloxy)-/\/ -ethyl(4-methoxyphenyl)-/\/ , -dimethyl-1/-/-pyrrole-2,5- dicarboxamide (UL1-055) ( 1 18 mg, 0.22 mmol) in methanol (4.5 mL) was passed through a Thales Ή -cube' cartridge (10% Pd/C) at a flow rate of 1 mL/min at 30°C under H2 (full H2 mode).
The output was trated in vacuo to afford -3,4-dihydroxy(4-methoxyphenyl)- , -dimethyl-1/-/-pyrrole-2,5-dicarboxamide (UL1-024) (66 mg, 82 %) as a brown solid: m/z 348 (M+H)+ (ES+) ; 346 (M-H) (ES ) . H NMR (400 MHz, DMSO-d ) d : 9.44 (s, 1H), 8.42 (s, 1H), 7.10-7.03 (m, 1H), 7.02-6.97 (m, 2H), 6.86-6.77 (m, 2H), 3.75 (s, 3H), 3.14-3.06 (m, 2H), 2.81 (br s , 6H), 0.97 (t, J = 7.2 Hz, 3H).
Example D : isopropyl 5-(dimethylcarbamoyl)-3,4-dihydroxy(4-methoxyphenyl)-1/-/-pyrrole carboxylate (UL1-035) 7 8 UL1 -035 Step (i): Isopropyl 3,4-bis(benzyloxy)(dimethylcarbamoyl)(4-methoxyphenyl)-1/-/-pyrrole carboxylate (8) To a stirred suspension of triethylammonium 3,4-bis(benzyloxy)(dimethylcarbamoyl)(4- yphenyl)-1/-/-pyrrolecarboxylate (7) [prepared as example B-step (iv) using ethyl 3,4- bis(benzyloxy)(dimethylcarbamoyl)- 1-(4-methoxyphenyl)-1H-pyrrolecarboxylate (6) as starting al] (360 mg, 0.60 mmol) and Cs2C0 3 (429 mg, 1.32 mmol) in DMF (3 mL) was added 2-bromopropane (197 m , 2.10 mmol) and the reaction stirred at 40°C for 2h then partitioned between EtOAc (50 ml_) and water (20 ml_). The organic was separated and washed with water (2 x 50 ml_), brine (2 x 50 ml_), dried (MgS0 ) , filtered and concentrated in vacuo to afford a yellow oil. The residue was purified by silica gel chromatography (40 g , 20- 60% EtOAc in ane) to afford isopropyl s(benzyloxy)(dimethylcarbamoyl)(4- yphenyl)-1/-/-pyrrolecarboxylate (8) (108 mg, 33 %) as a yellow oil: H NMR (400 MHz, DMSO-de) d : 7.51-7.43 (m, 2H), 7.43-7.28 (m, 8H), 7.19-7.05 (m, 2 H), 6.96-6.84 (m, 2H), .13 (s, 2H), 4.96 (s, 2H), 4.85 (sept., J = 6.2 Hz, 1H), 3.78 (s, 3H), 2.72 (s, 3H), 2.71 (s, 3H), 1.10 (d, J = 6.2 Hz, 6H).
Step (ii): Isopropyl 5-(dimethylcarbamoyl)-3,4-dihydroxy(4-methoxyphenyl)-1/-/-pyrrole carboxylate (UL1-035) A solution of isopropyl 3,4-bis(benzyloxy)(dimethylcarbamoyl)(4-methoxyphenyl)-1/-/- pyrrolecarboxylate (8) (130 mg, 0.240 mmol) in methanol (5 ml_) was passed through a Thales Ή -cube' dge (10% Pd/C) at a flow rate of 1 mL/min at 30°C under H (full H mode). 2 2 The output was concentrated in vacuo to afford isopropyl 5-(dimethylcarbamoyl)-3,4-dihydroxy- 1-(4-methoxyphenyl)-1/-/-pyrrolecarboxylate (UL1-035) (69 mg, 79 %) as a white solid: m/z 363 (M+H)+ (ES+) ; 361 (M-H) (ES ) . H NMR (400 MHz, DMSO-d ) d : 8.60 (s, 1H), 8.42 (s, 1H), 7.10-6.99 (m, 2H), 6.91-6.82 (m, 2H), 4.83 (sept., J = 6.2 Hz, 1H), 3.77 (s, 3H), 2.83 (br s , 6H), 0.96 (d, J = 6.2 Hz, 6H).
Example E : -Butyl (5-(dimethylcarbamoyl)-3,4-dihydroxy(4-methoxyphenyl)-1/-/-pyrrol yl)sulfonylcarbamate (UL1-030) UL1 -030 Step (i): 3,4-Bis(benzyloxy)(4-methoxyphenyl)-/\/,A/-dimethyl-1/-/-pyrrolecarboxamide (9) A solution of triethylammonium s(benzyloxy)(dimethylcarbamoyl)(4-methoxyphenyl)- 1/-/-pyrrolecarboxylate (7) (3.5 g , 6.70 mmol) in acetic acid (100 ml_) was stirred at RT for 4h and then 110°C for 1h. The les were removed in vacuo and the crude material partitioned between EtOAc (100 ml_) and 1M NaOH (aq.) (20 ml_)/brine (50 ml_), the organic layer was then dried (MgS0 ) , filtered and concentrated in vacuo to afford 3,4-bis(benzyloxy)(4- methoxyphenyl)-/\/,A/-dimethyl-1/-/-pyrrolecarboxamide (9) (3.05 g , 100 %) as a yellow oil: m/z 457 (M+H)+ (ES+) .
Step (ii): -Butyl (3,4-bis(benzyloxy)(dimethylcarbamoyl)(4-methoxyphenyl)-1/-/-pyrrol yl)sulfonylcarbamate (10) To a solution of chlorosulfonyl isocyanate (70.9 m I_ , 0.82 mmol) at -5°C in ous DCM (4 ml_) was added te/f-butanol (solution in 0.5 ml_ anhydrous DCM) (81 m I_ , 0.85 mmol) dropwise over 5 min and the reaction stirred at -5°C for 15 min. After this time, DIPEA (296 m I_ , 1.70 mmol) was added se. After 15 min a solution of 3,4-bis(benzyloxy)(4-methoxyphenyl)- A/,A/-dimethyl-1/-/-pyrrolecarboxamide (9) (310 mg, 0.68 mmol) in DCM ( 1 ml_) was added and the reaction was allowed to warm to RT and stirred for 16h. The reaction was partitioned between water (10 ml_) and DCM (20 ml_) and the organic washed with 1 M HCI (aq.) (2 x 20 ml_), and brine (10 ml_), dried (MgS0 ) , filtered and solvent removed in vacuo to afford crude product. The residue was purified by silica gel chromatography (40 g , 0-4% MeOH in DCM) to afford -butyl is(benzyloxy)(dimethylcarbamoyl)(4-methoxyphenyl)-1/-/-pyrrol fonylcarbamate (10) (166 mg, 35 %) as a colourless oil: m/z 636 (M+H)+ (ES+) ; 634 (M-H) (ES-).
Step (iii): -Butyl (5-(dimethylcarbamoyl)-3,4-dihydroxy(4-methoxyphenyl)-1/-/-pyrrol yl)sulfonylcarbamate (UL1-030) -Butyl (3,4-bis(benzyloxy)(dimethylcarbamoyl)(4-methoxyphenyl)-1/-/-pyrrolyl) sulfonyl carbamate (10) (166 mg, 0.26 mmol) in methanol (2.5 ml_) was passed through a Thales Ή -cube' cartridge (10% Pd/C) at a flow rate of 1 mL/min at 30°C under H (full H mode). 2 2 The output was concentrated in vacuo to afford -butyl (5-(dimethylcarbamoyl)-3,4-dihydroxy- 1-(4-methoxyphenyl)-1/-/-pyrrolyl)sulfonylcarbamate (UL1-030) (65 mg, 52 %) as a yellow solid: m/z 456 (M+H)+ (ES+) ; 454 (M-H) (ES ) . H NMR (400 MHz, DMSO-d ) d : 11.04 (s, 1H), 8.75 (s, 1H), 8.35 (s, 1H), 7.07-7.00 (m, 2H), 6.92-6.86 (m, 2H), 3.77 (s, 3H), 2.80 (br s , 6H), 1.35 (s, 9H).
Example F : 5-Cyano-3,4-dihydroxy(4-methoxyphenyl)-/\/,A/-dimethyl-1/-/-pyrrole carboxamide (UL1-031) UL1 -031 Step (i): 3,4-Bis(benzyloxy)(4-methoxyphenyl)- , -dimethyl-1/-/-pyrrole-2,5-dicarboxamide ( 1 1) 3,4-Bis(benzyloxy)(4-methoxyphenyl)-A J -dimethyl-1H-pyrrole-2,5-dicarboxamide ( 1 1) was prepared using the same procedure as Example B step (iv) except using triethylammonium 3,4- bis(benzyloxy)(dimethylcarbamoyl)(4-methoxyphenyl)-1/-/-pyrrolecarboxylate (7) (570 mg, 0.76 mmol) and ammonium chloride (405 mg, 7.58 mmol) to afford 3,4-bis(benzyloxy)(4- methoxyphenyl)-/V2 -dimethyl-1/-/-pyrrole-2,5-dicarboxamide ( 1 1) as yellow oil: m/z 500 (M+H)+ (ES+) .
Step (ii): 3,4-Bis(benzyloxy)cyano(4-methoxyphenyl)-/\/,A/-dimethyl-1/-/-pyrrole carboxamide (12) Trifluoroacetic anhydride (27.0 m I_ , 0.19 mmol) was added dropwise to a stirred solution of 3,4- bis(benzyloxy)(4-methoxyphenyl)-/V 2 -dimethyl-1/-/-pyrrole-2,5-dicarboxamide ( 1 1) (88 mg, 0.18 mmol) and triethylamine (74.3 m I_ , 0.53 mmol) in DCM (4 mL) at 0°C. The reaction mixture was d for 1h before and trifluoroacetic anhydride (27.0 m I_ , 0.19 mmol) was added and the mixture allowed to warm to RT. The reaction was partitioned between sat. NaHC0 (aq.) (5 mL) and DCM (5 mL) the organic layer was ted and volatiles removed in vacuo to afford 4- bis(benzyloxy)cyano(4-methoxyphenyl)-/\/,A/-dimethyl-1/-/-pyrrolecarboxamide (12) (85 mg, 100 %), as a yellow oil: m/z 482 (M+H)+ (ES+) .
Step (iii): 5-Cyano-3,4-dihydroxy(4-methoxyphenyl)-/\/,A/-dimethyl-1/-/-pyrrolecarboxamide 4-Bis(benzyloxy)cyano(4-methoxyphenyl)-/\/,A/-dimethyl-1/-/-pyrrolecarboxamide (12) (85 mg, 0.17 mmol) in methanol (2 mL) was passed through a Thales Ή -cube' cartridge (10% Pd/C) at a flow rate of 1.5 mL/min at 25°C under H (full H mode). The output was concentrated 2 2 in vacuo to afford 5-cyano-3,4-dihydroxy(4-methoxyphenyl)-/\/,A/-dimethyl-1/-/-pyrrole amide (UL1-031) ( 1 1 mg, 2 1 %) as a yellow solid: m/z 302 (M+H)+ (ES+) ; 300 (M-H) (ES ) . H NMR (400 MHz, DMSO-d ) d : 9.80 (s, 1H), 8.80 (s, 1H), 7.21-7.1 1 (m, 2H), 7.03-6.93 (m, 2H), 3.79 (s, 3H), 2.88 (br s , 6H).
Example G : 3,4-Dihydroxy(4-methoxyphenyl)-1/-/-pyrrole-2,5-dicarbonitrile (UL1-034) 13 UL1 -034 Step (i): 3,4-Bis(benzyloxy)(4-methoxyphenyl)-1/-/-pyrrole-2,5-dicarbonitrile (14) To a d solution of 3,4-bis(benzyloxy)(4-methoxyphenyl)-1/-/-pyrrole-2,5-dicarboxamide (13) [prepared using the same procedure as Example A step (ii) using ammonium chloride] (267 mg, 0.57 mmol) and triethylamine (710 m I_ , 5.10 mmol) in DCM (8 ml_) at 0°C was added trifluoroacetic ide (240 m I_ , 1.70 mmol) and the reaction allowed to warm to RT over 30 min. The reaction mixture was partitioned between DCM (50 ml_) and 1 M HCI (aq.) (30 ml_), the organic was washed with 1 M HCI (aq.) (20 ml_), sat. NaHC0 (aq.) (30 ml_), and brine (20 ml_), dried (MgS0 ) , filtered and volatiles removed in vacuo to afford a yellow solid. The crude residue was purified by silica gel tography (40 g , 0-50% EtOAc in isohexane) to afford 3,4-bis(benzyloxy)(4-methoxyphenyl)-1/-/-pyrrole-2,5-dicarbonitrile (14) (240 mg, 97 %) as a pale yellow solid: H NMR (400 MHz, DMSO-d ) d : 7.47-7.27 (m, 10H), 7.30-7.20 (m, 2H), 7.05- 6.91 (m, 2H), 5.26 (s, 4H), 3.79 (s, 3H).
Step (ii): 3,4-Dihydroxy(4-methoxyphenyl)-1/-/-pyrrole-2,5-dicarbonitrile (UL1-034) 3,4-Bis(benzyloxy)(4-methoxyphenyl)-1/-/-pyrrole-2,5-dicarbonitrile (14) (70 mg, 0.16 mmol) in THF (5 ml_) was passed through a Thales Ή -cube' dge (10% Pd/C) at a flow rate of 1.5 mL/min at 25°C under H (full H mode). The output was concentrated in vacuo to afford the 2 2 crude product. The compound was purified by preparative HPLC (C-18 column, 21.2 mm i.d. x 100 mm, 5 micron particle size, gradient 5-95 % MeCN in 0.1 % aqueous formic acid over 16 min) to afford 3,4-dihydroxy(4-methoxyphenyl)-1/-/-pyrrole-2,5-dicarbonitrile (UL1-034) (6 mg, 14%) as a yellow powder: m/z 254 (M-H) (ES ) . H NMR (400 MHz, DMSO-d ) d : 7.44-7.36 (m, 2H), 7.14-7.05 (m, 2H).
Example H : Ethyl 5-cyano(4-fluorophenyl)-3,4-dihydroxy-1/-/-pyrrolecarboxylate (UL1-039) UL1 -060 UL1-039 Step (i): Ethyl 3,4-bis(benzyloxy)cyano(4-fluorophenyl)-1/-/-pyrrolecarboxylate (UL1- 060) To a stirred solution of ethyl 3,4-bis(benzyloxy)carbamoyl(4-fluorophenyl)-1/-/-pyrrole carboxylate (15) ( 1 .1 g , 2.25 mmol) [prepared using the same procedure as Example B step (v) using triethylammonium 3,4-bis(benzyloxy)(dimethylcarbamoyl)- 1-(4-fluorophenyl)- 1H- pyrrolecarboxylate and um chloride] and triethylamine ( 1 .26 mL, 9.0 mmol) in DCM (60 mL) at 0°C was added trifluoroacetic anhydride (0.48 mL 3.38 mmol). The reaction mixture was allowed to warm to RT and partitioned n 1M HCI (aq.) (25 mL) and DCM (50 mL) the c layer was washed with 1M HCI (aq.), and sat. NaHC0 (aq.) (25 mL), dried (MgS0 ) , 3 4 filtered and the volatiles removed in vacuo to afford the crude product. The crude residue was purified by silica gel chromatography (40 g , 0-20% EtOAc in isohexane) to afford ethyl 3,4- nzyloxy)cyano(4-fluorophenyl)-1/-/-pyrrolecarboxylate (UL1-060) ( 1 .01 g , 94 %) as yellow solid: m/z 471 (M+H)+ (ES+) . H NMR (400 MHz, DMSO-d ) d : 7.57-7.27 (m, 14H), .27 (s, 2H), 5.1 1 (s, 2H), 4.05 (q, J = 7.1 Hz, 2H), 1.00 (t, J = 7.1 Hz, 3H).
Step (ii): Ethyl 5-cyano(4-fluorophenyl)-3,4-dihydroxy-1/-/-pyrrolecarboxylate (UL1-039) Ethyl 3,4-bis(benzyloxy)cyano(4-fluorophenyl)-1/-/-pyrrolecarboxylate (UL1-060) (102 mg, 0.22 mmol) in THF (5 mL) was passed through a Thales Ή -cube' dge (10% Pd/C) at a flow rate of 1.5 mL/min at 20°C under H (full H mode). The output was concentrated in vacuo 2 2 to afford the crude product. The crude residue was purified by silica gel chromatography (4 g , 0- % EtOAc in isohexane) to afford ethyl 5-cyano(4-fluorophenyl)-3,4-dihydroxy-1/-/-pyrrole carboxylate (UL1-039) (18 mg, 29 % yield) as a white solid: m/z 291 (M+H)+ (ES+) ; 289 (M-H) (ES ) . H NMR (400 MHz, DMSO-d ) d : 10.14 (s, 1H), 8.99 (s, 1H), 7.44-7.36 (m, 2H), 7.35-7.26 (m, 2H), 4.05 (q, J = 7.1 Hz, 2H), 1.01 (t, J = 7.1 Hz, 3H).
Example I : -Butyl 5-(dimethylcarbamoyl)-3,4-dihydroxy(4-methoxyphenyl)-1/-/-pyrrole carboxylate (UL1-036) 16 UL1 -036 To a solution of -butyl 2-((2-(dimethylarnino)oxoethyl)(4-methoxyphenyl)arnino)acetate (16) (1.98 g , 6.14 mmol) and di-te/f-butyl e ( 1 .37 g , 6.76 mmol) in anhydrous utanol (6 mL) was added potassium te/f-butoxide (20 wt. % in THF) (10.77 ml, 15.35 mmol) and the reaction was stirred at 100°C for 1.5 h , di-te/f-butyl oxalate (300 mg, 1.5 mmol), ium tert- butoxide (20 wt. % in THF) (4 mL, 5.71 mmol), THF (10 mL), and te/f-butanol (4 mL) were added and the mixture stirred for a further 1h. The e was allowed to cool to RT and acetic acid (4 ml, 69.9 mmol) added, the mixture was poured on to ice-cold water (50 mL) and the resulting precipitate was collected by vacuum filtration and recrystallised from hot ethanol to afford -butyl 5-(dimethylcarbamoyl)-3,4-dihydroxy(4-methoxyphenyl)-1/-/-pyrrole carboxylate (UL1-036) (890 mg, 37 %) as a white powder: m/z 377 (M+H)+ (ES+) ; 375 (M-H) (ES ) . H NMR (400 MHz, DMSO-d ) d : 8.62 (s, 1H), 8.45 (s, 1H), 7.12-6.99 (m, 2H), 6.94-6.81 (m, 2H), 3.76 (s, 3H), 2.83 (br s , 6H), 1.17 (s, 9H).
Example J : 2-(Dimethylcarbamoyl)(ethoxycarbonyl)(4-methoxyphenyl)-1/-/-pyrrole-3,4-diyl diacetate (UL1-044) UL1-012 UL1-044 To a suspension of ethyl 5-(dimethylcarbamoyl)-3,4-dihydroxy(4-methoxyphenyl)-1/-/-pyrrole- 2-carboxylate (UL1-012) (120 mg, 0.35 mmol) in acetic anhydride ( 1 mL, 10.6 mmol) was added NaOAc (90 mg, 1.06 mmol), the suspension was d at 100°C for 2h. The reaction mixture was partitioned between DCM (25 mL) and iced water (25 mL), dried (MgS0 ) , ed and volatiles removed in vacuo to afford a colourless oil. Trituration of the colourless oil from Et 0 gave 2-(dimethylcarbamoyl)(ethoxycarbonyl)(4-methoxyphenyl)-1/-/-pyrrole-3,4-diyl diacetate (UL1-044) (80 mg, 52 %) as a white solid: m/z 433 (M+H)+ (ES+) . H NMR (400 MHz, DMSO-de) d : 7.27-7.1 1 (2H, m), 7.00-6.88 (2H, m), 4.00 (q, J = 7.1 Hz, 2H), 3.79 (s, 3H), 2.79 (s, 3H), 2.71 (s, 3H), 2.26 (s, 6H) 1.05 (t, J = 7.1 Hz, 3H).
Example K : Ethyl 5-(dimethylcarbamoyl)-3,4-dihydroxy(4-methoxyphenyl)-1/-/-pyrrole carboxylate (UL1-012) Step (i): Ethyl 2-((4-methoxyphenyl)amino)acetate (17) To a solution of sodium iodide (122 g , 0.81 mol) and sodium acetate trihydrate (221 g , 1.62 mol) in water (200 mL) was added 4-methoxyaniline (3) (100 g , 0.81 mol) followed by ethyl 2- bromoacetate (90 mL, 812 mmol) the reaction was d at 90°C for 3h. The reaction mixture was cooled to RT and partitioned with EtOAc ( 1 L), the c phase was separated, washed with water (400 mL), 1 M HCI (aq.) (3 x 500 mL), and the organic phase ded. The combined acidic extracts were cooled to 0°C and solid NaOH was added to pH 14 and the aqueous phase was extracted with EtOAc (3 x 400 mL). The combined organics were washed with brine (250 mL), dried (MgS0 ) , filtered and solvents removed in vacuo to give ethyl 2-((4- methoxyphenyl)amino)acetate (17) (128 g , 75 %) as a purple oil that crystallised on standing: m/z 210 (M+H)+ (ES+) . H NMR (400 MHz, DMSO-d ) d : 6.75-6.69 (m, 2H), 6.55-6.46 (m, 2H), 3.99 (t, J = 6.5 Hz, 1H), 4.10 (q, J = 7.1 Hz, 2H), 3.82 (d, J = 6.5 Hz, 2H), 3.63 (s, 3H), 1.19 (t, J =7.1 Hz, 3H).
Step (ii): 2-((4-Methoxyphenyl)amino)acetic acid (18) 2M NaOH (aq.) (149 mL, 0.30 mol) was added dropwise to a stirred solution of ethyl 2-((4- methoxyphenyl)amino)acetate (17) (59.2 g , 0.28 mol) in EtOH/THF (300 ml_/80 mL). The reaction was stirred at 40°C for 3h. The mixture was diluted with water (100 mL) and phosphoric acid (85 % wt in water) (35.9 g , 0.31 mol) was added to pH 3 . The resulting brown precipitate was collected by tion and washed with water (100 mL) and dried under vacuum to give 2- thoxyphenyl)amino)acetic acid (18) (45.5 g , 84 %) as a brown solid: m/z 182 (M+H)+ (ES+) ; 180 (M-H) (ES ) . H NMR (400 MHz, DMSO-d ) d : 6.78-6.64 (m, 2H), 6.55-6.44 (m, 2H), 3.74 (s, 2H), 3.64 (s, 3H).
Step (iii): 2-((4-Methoxyphenyl)amino)-A/,A/-dimethylacetamide (19) To a stirred suspension of 2-((4-methoxyphenyl)amino)acetic acid (18) (45 g , 0.25 mol) and dimethylamine hydrochloride (40.5 g , 0.5 mol) in acetonitrile ( 1 L) at 0°C, was added DIPEA (174 ml, 0.99 mol) followed by HATU (99 g , 0.26 mol). The reaction mixture was stirred at RT for 1 h , and then ioned between EtOAc ( 1 L) and 5% NaH P0 (aq.) (250 mL), the c 2 4 layer was washed with sat. NaHC0 (aq.) (3 x 300 mL), and 3M HCI (aq.) (4 x 250 mL). The combined acidic extracts were cooled to 0°C and solid NaOH was added to pH 14 and the aqueous phase was extracted with EtOAc (3 x 400 mL), the combined organics were washed with brine (500 mL) dried (MgS0 ) , filtered and solvents removed in vacuo to give 2-((4- methoxyphenyl)amino)-A/,A/-dimethylacetamide (19) (37. 1g, 7 1 %) as a brown solid: m/z 209 (M+H)+ (ES+) . H NMR (400 MHz, DMSO-d ) d : 6.74-6.70 (m, 2H), 6.65-6.59 (m, 2H), 5.10 (br s , 1H), 3.81 (s, 2H), 3.64 (s, 3H), 3.01 (s, 3H), 2.87 (s, 3H).
Step (iv): Ethyl 2-((2-(dimethylamino)oxoethyl)(4-methoxyphenyl)amino)acetate (20) To a stirred solution of 2-(4-methoxyphenylamino)-A/,A/-dimethylacetamide (19) (10.7 g , 5 1 .2 mmol) in MeCN (80 mL) and DIPEA (13.4 ml, 77 mmol) was added ethyl cetate (6.84 ml, 6 1.4 mmol) the reaction was stirred at 60°C for 16h. The volatiles were removed in vacuo, and the crude mixture was ved in EtOAc (150 mL), washed with 1M HCI (aq.) (150 mL), brine (150 mL), dried (MgS0 ) , filtered and solvents removed in vacuo to afford a purple oil. The residue was purified by silica gel chromatography (120 g , 60-100% EtOAc in isohexane) to afford ethyl 2-((2-(dimethylamino)oxoethyl)(4-methoxyphenyl)amino)acetate (X) (12.9 g , 85 %) as a purple oil: m/z 295.4 (M+H)+ (ES+) . H NMR (400 MHz, DMSO-d ) d : 6.81-6.71 (m, 2H), 6.60-6.47 (m, 2H), 4.24-3.96 (m, 6H), 3.66 (s, 3H), 2.99 (br s , 3H), 2.83 (br s , 3H), 1.19 (t, J = 7.1 Hz, 3H).
Step (v): Ethyl 5-(dimethylcarbamoyl)-3,4-dihydroxy(4-methoxyphenyl)-1/-/-pyrrole carboxylate (UL1-012) Diethyl oxalate (2.54 ml, 18.6 mmol) was added dropwise to a stirred solution of ethyl 2-((2- (dimethylamino)oxoethyl)(4-methoxyphenyl)amino)acetate (20) (5.21 g , 17.7 mmol) in NaOEt (21% by wt in EtOH) (27.8 ml, 74.3 mol), the mixture was stirred at 85°C for 1.5h. The reaction was quenched with acetic acid (10.1 ml, 177 mmol) and the resulting suspension was poured into iced water (200 mL), and extracted with EtOAc (3 x 100 mL), combined organics were washed with brine (200 mL), dried (MgS0 ) , filtered and ts removed in vacuo to afford a brown oil. The e was purified by silica gel tography (80 g , 0-10% MeOH (+1% NH ) in DCM) to afford ethyl 5-(dimethylcarbamoyl)-3,4-dihydroxy(4-methoxyphenyl)-1/-/- pyrrolecarboxylate (UL1-012) (3.64 g , 53 %) as a white solid: m/z 349 (M+H)+ (ES+) , 347 (M- H) (ES ) . H NMR (400 MHz, DMSO-d ) d : 8.60 (s, 1H), 8.46 (s, 1H), 7.08-7.01 (m, 2H), 6.90- 6.82 (m, 2H), 4.00 (q, J = 7.0 Hz, 2H), 3.76 (s, 3H), 2.83 (br s , 6H), 0.99 (t, J = 7.1 Hz, 6H).
Example L : Diisopropyl hydroxy(4-methoxyphenyl)-1/-/-pyrrole-2,5-dicarboxylate (UL2- 020) and 2-ethyl 5-isopropyl 3,4-dihydroxy(4-methoxyphenyl)-1/-/-pyrrole-2,5-dicarboxylate (UL2-021) 2 1 UL2-020 UL2-021 Lithium isopropoxide (2M in THF) (10.4 mL, 20.8 mmol) was added to a mixture of diisopropyl 2,2'-((4-methoxyphenyl)azanediyl)diacetate (21) (2.69 g , 8.32 mmol) [prepared using the same procedure as Example B step (i) using isopropyl 2-bromoacetate] and diethyl oxalate (1.13 mL, 8.32 mmol), the e was d at 65°C for 16h. A further n of lithium isopropoxide (2M in THF) (5.2 ml, 10.4 mmol) was added and the reaction stirred at 65°C for 3h. The reaction was quenched with acetic acid (5 mL) and the volatiles removed in vacuo, the crude product was took up in EtOAc (100 mL) and partitioned with water (100 mL), the aqueous was washed with EtOAc (2 x 100 mL) combined organics were washed with brine (150 mL), dried (MgS0 ) , filtered and solvents d in vacuo to afford a brown oil. The crude product was recrystallised from hot ' PrOH (50 mL), to provide a white solid. The solid was purified by silica gel chromatography (12 g , 0-50% EtOAc in isohexane) to afford diisopropyl 3,4-dihydroxy(4- methoxyphenyl)-1/-/-pyrrole-2,5-dicarboxylate (UL2-020) (21 mg, 1 %) as a white solid: m/z 378 (M+H)+ (ES+) . H NMR (400 MHz, DMSO-d ) d : 8.62 (s, 2H), 7.12-7.01 (m, 2H), 6.93-6.84 (m, 2H), 4.80 (sept., J = 6.3 Hz, 2H), 3.78 (s, 3H), 0.95 (d, J =6.2 Hz, 12H).
Impure ons were combined and purified by preparative HPLC (C-18, 5 mhi , 2 1.2x50 mm column, 5-95% MeCN in Water 0.1% Formic Acid) to afford 2-ethyl 5-isopropyl 3,4-dihydroxy (4-methoxyphenyl)-1/-/-pyrrole-2,5-dicarboxylate (UL2-021) (22 mg, 1%) as a white solid: m/z 364 (M+H)+ (ES+) . H NMR (400 MHz, DMSO-d ) d : 8.66 (s, 1H), 8.62 (s, 1H), 7.09-7.03 (m, 2H), .85 (m, 2H), 4.80 (sept., J = 6.2 Hz, 1H), 3.98 (q, J = 7.1 Hz, 2H), 3.77 (s, 3H), 1.00 (t, J = 7.1 Hz, 3H), 0.93 (d, J =6.2 Hz, 6H).
Example M : l 3-(2-(benzyloxy)ethoxy)(4-fluorophenyl)hydroxy-1/-/-pyrrole-2,5- dicarboxylate (UL2-025) UL2-003 22 UL2-025 Step (i) Diethyl 3-(2-(benzyloxy)ethoxy)(4-fluorophenyl)hydroxy-1/-/-pyrrole-2,5- dicarboxylate (22) Diisopropyl azodicarboxylate (317 m I_ , 1.63 mmol) was added to a solution of diethyl 1-(4- fluorophenyl)-3,4-dihydroxy-1/-/-pyrrole-2,5-dicarboxylate (UL2-003) (500 mg, 1.48 mmol), 2- (benzyloxy)ethanol (232 m I_ , 1.63 mmol) and triphenylphosphine (428 mg, 1.63 mmol) in DCM (5 ml), at 0°C. The reaction mixture was allowed to warm to RT and stirred for 1h. The reaction was washed with water (5 ml_) and the phases ted using a phase separation dge, the organic was concentrated in vacuo to afford a yellow oil. The residue was purified by silica gel chromatography (80 g , 0-25% EtOAc in isohexane) to afford diethyl 3-(2-(benzyloxy)ethoxy)- luorophenyl)hydroxy-1/-/-pyrrole-2,5-dicarboxylate (22) (122 mg, 17 %) as a yellow oil: m/z 472 (M+H)+ (ES+) ; 470 (M-H) (ES ) . H NMR (400 MHz, DMSO-d ) d : 8.70 (s, 1H), 7.38- 7.26 (m, 5H), 7.24-7.12 (m, 4H), 4.54 (s, 2H), 4.27-4.16 (m, 2H), 4.09-3.89 (m, 4H), 3.78-3.71 (m, 2H), .94 (m, 6H).
Step (ii) Diethyl 1-(4-fluorophenyl)hydroxy(2-hydroxyethoxy)-1/-/-pyrrole-2,5-dicarboxylate (UL2-025) A on of diethyl 3-(2-(benzyloxy)ethoxy)(4-fluorophenyl)hydroxy-1/-/-pyrrole-2,5- dicarboxylate (22) (122 mg, 0.26 mmol) in MeOH (120 ml) was passed through a Thales Ή - cube' cartridge (10% Pd/C) at a flow rate of 1 mL/min at 30°C under H (full H mode). The 2 2 output was concentrated in vacuo to afford diethyl 3-(2-(benzyloxy)ethoxy)(4-fluorophenyl) hydroxy-1/-/-pyrrole-2,5-dicarboxylate (UL2-025) (122 mg, 85 %) as a pale yellow oil: m/z 382 (M+H)+ (ES+) , 380 (M-H) (ES ) . H NMR (400 MHz, DMSO-d ) d : 8.94 (br s , 1H), 7.29-7.22 (m, 2H), 7.21-7.14 (m, 2H), 5.01 (br s , 1H), 4.08-3.97 (m, 6H), 3.73-3.65 (m, 2H), 1.02 (t, J = 7.1 Hz, 3H), 0.99 (t, J = 7.1 Hz, 3H).
Example N : Diethyl 3-(2-aminooxoethoxy)hydroxy(4-methoxyphenyl)-1/-/-pyrrole-2,5- dicarboxylate (UL2-031) Step (i): Diethyl 3-(benzyloxy)hydroxy(4-methoxyphenyl)-1/-/-pyrrole-2,5-dicarboxylate (23) Benzyl e (2.13 ml, 17.9 mmol) was added dropwise to a suspension of DIPEA (3.12 mL, 17.9 mmol) and diethyl 3,4-dihydroxy(4-methoxyphenyl)-1/-/-pyrrole-2,5-dicarboxylate (UL2- 001) (5 g , 14.3 mmol) in EtOH (200 mL). The reaction mixture was d at 50°C for 18h.
Additional benzyl e (0.85 mL, 7.20 mmol) and DIPEA ( 1 .25 mL, 7.20 mmol) were added and the reaction stirred at 60°C for 24h, after which further portions of benzyl e (0.85 ml, 7.20 mmol) and DIPEA ( 1 .25 mL, 7.20 mmol) were added and the reaction mixture stirred at 60°C for 24h. The reaction mixture was allowed to cool to RT, and partitioned between EtOAc (200 mL) and sat. NaHC0 (aq.) (200 mL), the organic layer was separated and washed with sat. NaHC0 (aq.) (2 x 200 mL) dried (MgS0 ) , filtered and solvents removed in vacuo to give 3 4 an orange oil. The residue was purified by silica gel chromatography (80 g , 0-100% EtOAc in isohexane) to afford a yellow solid. The product was recrystallised from EtOH (100 mL) to afford diethyl 3-(benzyloxy)hydroxy(4-methoxyphenyl)-1/-/-pyrrole-2,5-dicarboxylate (23) (2.89 g , 37 %) as a pale yellow solid: m/z 440 (M+H)+ (ES+) . H NMR (400 MHz, DMSO-d ) d : 8.82 (s, 1H), 7.52-7.30 (m, 5H), 7.16-7.03 (m, 2H), 6.94-6.83 (m, 2H), 5.09 (s, 2H), 4.10-3.91 (m, 4H), 3.79 (s, 3H), 1.02-0.92 (m, 6H).
Step (ii): Diethyl 3-(benzyloxy)(2-(fe/f-butoxy)oxoethoxy)(4-methoxyphenyl)-1/-/-pyrrole- 2,5-dicarboxylate (UL2-028) -Butyl 2-bromoacetate (0.55 ml, 3.41 mmol) was added dropwise to a stirred on of diethyl 3-(benzyloxy)hydroxy(4-methoxyphenyl)-1/-/-pyrrole-2,5-dicarboxylate (23) ( 1 .0 g , 2.28 mmol) and K C0 (0.63 g , 4.55 mmol) in DMF (20 mL). The reaction mixture was stirred at 2 3 RT for 18h, and partitioned between EtOAc (200 mL) and sat. NaHC0 (aq.) (200 mL), the organic layer was separated and washed with sat. NaHC0 (aq.) (2 x 200 mL), dried (MgS0 ) , 3 4 filtered and solvents removed in vacuo to give a yellow oil. The residue was purified by silica gel chromatography (120 g , 0-20% EtOAc in isohexane) to afford diethyl 3-(benzyloxy)(2-(te/fbutoxy oethoxy)(4-methoxyphenyl)-1/-/-pyrrole-2,5-dicarboxylate (UL2-028) (912 mg, 72 %) as a pale yellow oil: m/z 554 (M+H)+ (ES+) . H NMR (400 MHz, DMSO-d ) d : 7.49-7.44 (m, 2H), 7.43-7.31 (m, 3H), 7.15-7.08 (m, 2H), 6.94-6.88 (m, 2H), 5.12 (s, 2H), 4.59 (s, 2H), 4.02- 3.94 (m, 4H), 3.79 (s, 3H), 1.40 (s, 9H), 1.02 (t, J = 7.1 Hz, 3H), 0.97 (t, J = 7.1 Hz, 3H).
Step (iii): 2-((4-(benzyloxy)-2,5-Bis(ethoxycarbonyl)(4-methoxyphenyl)-1/-/-pyrrol yl)oxy)acetic acid (24) Diethyl 3-(benzyloxy)(2-(fe/f-butoxy)oxoethoxy)(4-methoxyphenyl)-1/-/-pyrrole-2,5- oxylate (UL2-028) (250 mg, 0.45 mmol) was dissolved in 4 M HCI in dioxane (2.5 mL, .0 mmol), and the on mixture was stirred at RT for 2h, volatiles were removed in vacuo and the product was oped with toluene (2 x 5 mL) to afford 2-((4-(benzyloxy)-2,5- bis(ethoxycarbonyl)(4-methoxyphenyl)-1/-/-pyrrolyl)oxy)acetic acid (24) (216 mg, 87 %) as a yellow oil: m/z 498 (M+H)+ (ES+) ; 496 (M-H) (ES ) .
Step (iv): l 3-(2-aminooxoethoxy)(benzyloxy)(4-methoxyphenyl)-1/-/-pyrrole-2,5- dicarboxylate (25) HATU (153 mg, 0.40 mmol) was added to a stirred solution 2-((4-(benzyloxy)-2,5- bis(ethoxycarbonyl)(4-methoxyphenyl)-1/-/-pyrrolyl)oxy)acetic acid (24) (100 mg, 0.20 mmol), DIPEA (176 m , 1.00 mmol), and NH C I (53.8 mg, 1.00 mmol) in THF (2 mL) at O°C. The reaction mixture was stirred at 0°C for 20 min and then at RT for 2h les were removed in vacuo, dissolved in EtOAc (30 mL) and washed with 1 M HCI (3 x 30 mL), sat. NaHC0 (aq.) (3 x 30 mL) and brine (30 mL), dried (MgS0 ) , ed and solvents removed in vacuo to give a yellow oil. The product was purified by silica gel chromatography (12 g , 0-100% EtOAc in isohexane) to afford diethyl 3-(2-aminooxoethoxy)(benzyloxy)(4-methoxyphenyl)-1/-/- pyrrole-2,5-dicarboxylate (25) (66 mg, 66 %) as a white solid: m/z 497 (M+H)+ (ES+) . H NMR (400 MHz, DMSO-de) d : 7.58 (br s , 1H), 7.51 (br s , 1H), 7.49-7.44 (m, 2H), 7.43-7.33 (m, 3H), .09 (m, 2H), 6.95-6.86 (m, 2H), 5.10 (s, 2H), 4.46 (s, 2H), 4.00 (q, J = 7.1 Hz, 2H), 3.98 (q, J = 7.1 Hz, 2H), 3.79 (s, 3H), 0.97 (t, J = 7.1 Hz, 3H), 0.96 (t, J = 7.1 Hz, 3H).
Step (v): Diethyl 3-(2-aminooxoethoxy)hydroxy(4-methoxyphenyl)-1/-/-pyrrole-2,5- dicarboxylate (UL2-031).
Diethyl 3-(2-aminooxoethoxy)(benzyloxy)(4-methoxyphenyl)-1/-/-pyrrole-2,5- dicarboxylate (25) (66 mg, 0.133 mmol) in MeOH (66 mL) was passed through a Thales Ή - cube' cartridge (10% Pd/C) at a flow rate of 1 mL/min at 20°C under H (full H mode). The 2 2 output was concentrated in vacuo to afford a yellow oil. The residue was purified by silica gel chromatography (4 g , 0-100% EtOAc in isohexane) to afford diethyl 3-(2-aminooxoethoxy) hydroxy(4-methoxyphenyl)-1/-/-pyrrole-2,5-dicarboxylate (UL2-031) (25 mg, 46 %) as a white solid: m/z 407 (M+H)+ (ES+) ; 405 (M-H) (ES ) . H NMR (400 MHz, DMSO-d ) d : 9.61 (s, 1H), .63 (m, 2H), 7.10-7.06 (m, 2H), 6.93-6.84 (m, 2H), 4.49 (s, 2H), 4.03-3.93 (m, 4H), 3.79 (s, 3H), 1.05-0.94 (m, 6H).
Example O : Ethyl 5-(dimethylcarbamoyl)-3,4-bis((dimethylcarbamoyl)oxy)(4-methoxyphenyl)- 1/-/-pyrrolecarboxylate (UL1-063) Dimethylcarbamic chloride (160 m I_ , 1.74 mmol) was added dropwise to a stirred sion of ethyl 5-(dimethylcarbamoyl)-3,4-dihydroxy(4-methoxyphenyl)-1/-/-pyrrolecarboxylate (UL1- 012) (202 mg, 0.58 mmol) and K C0 (240 mg, 1.74 mmol) in MeCN (4 mL). The resulting 2 3 mixture was stirred at 80°C for 16h then partitioned between sat. NaHC0 (aq.) (10 mL) and Et 0 (10 mL) the aqueous layer was extracted with Et 0 (2 x 10 mL), the combined organics 2 2 were washed with 10% NaOH (aq.) (2 x 10 mL), water (2 x 10 mL) and brine (2 x 10 mL), dried (MgS0 ) , filtered and solvents removed in vacuo to give an orange oil. Trituration of the oil with isohexane provided ethyl 5-(dimethylcarbamoyl)-3,4-bis((dimethylcarbamoyl)oxy)(4- methoxyphenyl)-1/-/-pyrrolecarboxylate (UL1-063) (40 mg, 14 %) as a cream solid: m/z 491 (M+H)+ (ES+) . H NMR (400 MHz, DMSO-d ) d : 7.21-7.1 1 (m, 2H), .88 (m, 2H), 4.00 (q, J = 7.1 Hz, 2H), 3.79 (s, 3H), 3.01 (s, 3H), 2.96 (s, 3H), 2.90 (s, 3H), 2.88 (s, 3H), 2.80 (s, 3H), 2.70 (s, 3H), 1.05 (t, J = 7.1 Hz, 3H).
Example P : Ethyl 5-(dimethylcarbamoyl)((dimethylcarbamoyl)oxy)hydroxy(4- methoxyphenyl)-1 /-/-pyrrolecarboxylate (UL1 -066) Dimethylcarbamic chloride (163 \ , 1.77 mmol) was added dropwise to a stirred suspension of ethyl 5-(dimethylcarbamoyl)-3,4-dihydroxy(4-methoxyphenyl)-1/-/-pyrrolecarboxylate (UL1- 012) (494 mg, 1.42 mmol) and K C0 (255 mg, 1.85 mmol) in MeCN (8 ml_). The resulting 2 3 mixture was stirred at RT for 24h then partitioned between water (30 ml_) and DCM (20 ml_) the s layer was ted with DCM (2 x 20 ml_), the combined organics were dried (MgS0 ) , filtered and solvents d in vacuo to give an orange oil. The residue was purified by silica gel chromatography (12 g , 0-10% MeOH (+1% NH ) in DCM) to afford ethyl 5- (dimethylcarbamoyl)((dimethylcarbamoyl)oxy)hydroxy(4-methoxyphenyl)-1/-/-pyrrole ylate (UL1-066) (35 mg, 6 %) as a white solid: m/z 420 (M+H)+ (ES+) . H NMR (400 MHz, DMSO-de) d : 8.85 (s, 1H), 7.16-7.06 (m, 2H), 6.95-6.86 (m, 2H), 4.00 (q, J = 7.1 Hz, 2H), 3.77 (s, 3H), 2.98 (s, 3H), 2.88 (s, 3H), 2.78 (s, 3H), 2.69 (s, 3H), 1.00 (t, J = 7.1 Hz, 3H).
Example Q : Ethyl 5-(dimethylcarbamoyl)hydroxy(4-methoxyphenyl)(sulfamoyloxy)-1/-/- pyrrolecarboxylate (UL1-068) UL1 -01 2 UL1 -068 Formic acid (165 m I_ , 4.31 mmol) was added dropwise to neat sulfurisocyanatidic de (374 m I_ , 4.31 mmol) at 0°C with stirring. The reaction mixture d to warm to RT and stirred for 2h, after which a solution of ethyl 5-(dimethylcarbamoyl)-3,4-dihydroxy(4-methoxyphenyl)-1/-/- pyrrolecarboxylate (UL1-012) (0.5 g , 1.44 mmol) in NMP (3 ml_) was added dropwise. The reaction mixture was stirred at 0°C for 30 min and allowed to warm to RT and stirred for a further 2h. The reaction mixture was partitioned with brine (30 ml_) and EtOAc (25 ml_), the aqueous layer was ted with EtOAc (2 x 25 ml_) the combined organics were dried (MgS0 ) , filtered and solvents removed in vacuo to give a yellow oil. The compound was purified by preparative HPLC (C-18 column, 21.2 mm i.d. x 100 mm, 5 micron particle size, gradient 5-95 % MeCN in 0.1 % aqueous formic acid over 16 min) to afford ethyl 5- (dimethylcarbamoyl)hydroxy(4-methoxyphenyl)(sulfamoyloxy)-1/-/-pyrrolecarboxylate (UL1-068) (76 mg, 12 %) as a white solid: m/z 428 (M+H)+ (ES+) ; 426 (M-H) (ES ) . H NMR (400 MHz, DMSO-de) d : 8.87 (s, 1H), 7.92 (s, 2H), 7.16-7.08 (m, 2H), 6.96-6.88 (m, 2H), 4.03 (q, J = 7.1 Hz, 2H), 3.79 (s, 3H), 2.75 (s, 3H), 2.67 (s, 3H), 1.02 (t, J = 7.1 Hz, 3H).
Example R : Ethyl 5-(dimethylcarbamoyl)hydroxy(4-methoxyphenyl)(phosphonooxy)-1/-/- pyrrolecarboxylate (UL1-070) Step (i): Ethyl 4-((bis(benzyloxy)phosphoryl)oxy)(dimethylcarbamoyl)hydroxy(4- methoxyphenyl)-1 rrolecarboxylate (26) momethane (286 mg, 0.86 mmol) and A/-ethyl-A/-isopropylpropanamine (376 m I_ , 2.15 mmol) were added successively to a solution of A/,A/-dimethylpyridinamine (8.8 mg, 0.07 mmol) and ethyl 5-(dimethylcarbamoyl)-3,4-dihydroxy(4-methoxyphenyl)-1/-/-pyrrole carboxylate (UL1-012) (250 mg, 0.72 mmol) in MeCN (3 mL) at -10°C. The reaction mixture was allowed to stir for 30 min and bis(phenoxymethyl)phosphine oxide (166 m I_ , 0.75 mmol) was added, the reaction was allowed to slowly warm to 0°C and stirred for 1h. The reaction was quenched with 5 % NaH P0 (aq.) (20 mL) and extracted with EtOAc (3 x 25 mL). The 2 4 combined organics were washed with brine (50 mL), dried (MgS0 ) , filtered and solvents removed in vacuo. The crude residue was purified by silica gel chromatography (40 g , 0-4 % MeOH in DCM) to afford an orange oil. The compound was purified by ative HPLC (C-18 column, 21.2 mm i.d. x 100 mm, 5 micron particle size, gradient 5-95 % MeCN in 0.1 % aqueous formic acid over 16 min) to afford ethyl 4-((bis(benzyloxy)phosphoryl)oxy) (dimethylcarbamoyl)hydroxy(4-methoxyphenyl)-1/-/-pyrrolecarboxylate (26) (228 mg, 5 1 %) as a pale yellow solid: m/z 609 (M+H)+ (ES+) ; 607 (M-H) (ES ) .
Step (ii): Ethyl 5-(dimethylcarbamoyl)hydroxy(4-methoxyphenyl)(phosphonooxy)-1/-/- pyrrolecarboxylate (UL1-070) A solution of ethyl 4-((bis(benzyloxy)phosphoryl)oxy)(dimethylcarbamoyl)hydroxy(4- methoxyphenyl)-1/-/-pyrrolecarboxylate (26) (225 mg, 0.37 mmol) in MeOH (10 mL) was passed through a Thales Ή -cube' cartridge (10 % Pd/C) at a flow rate of 1 mL/min at 30°C under H (full H mode). The output was concentrated in vacuo to afford ethyl 5- 2 2 (dimethylcarbamoyl)hydroxy(4-methoxyphenyl)(phosphonooxy)-1/-/-pyrrole ylate (UL1-070) (151 mg, 94 %) as a pale yellow solid: m/z 429 (M+H)+ (ES+) ; 427 (M-H) (ES ) . H NMR (400 MHz, DMSO-d ) d : 7.1 1-7.03 (m, 2H), 6.93-6.85 (m, 2H), 3.99 (q, J = 7.1 Hz, 2H), 3.77 (s, 3H), 2.82 (s, 3H), 2.71 (s, 3H), 1.02 (t, J = 7.1 Hz, 3H). e S : Ethyl 3,4-dihydroxy(4-methoxyphenyl)(pyridinyl)-1/-/-pyrrolecarboxylate (UL1-083) UL1 -083 Step (i): Ethyl 3,4-bis(benzyloxy)iodo(4-methoxyphenyl)-1/-/-pyrrolecarboxylate (28) /V-lodosuccinimide (1.77g, 7.57 mmol) was added to a on of ethyl 3,4-bis(benzyloxy)(4- methoxyphenyl)-1/-/-pyrrolecarboxylate (27) [prepared using the same procedure as Example E step (i) using (5) as ng al] (3.0 g , 6.56 mmol) in DMF (60 ml_) and stirred at 0°C for min. The mixture was partitioned between sat. Na S 0 (aq.) (100 ml_) and Et 0 (250 ml_), 2 2 3 2 the organic layer was separated and washed with sat. Na S 0 (aq.) (100 ml_), brine (100 ml_) 2 2 3 dried (MgS0 ) , filtered and solvents removed in vacuo. The crude residue was purified by silica gel tography (120 g , 0-40 % Et 0 in isohexane) to afford ethyl 3,4-bis(benzyloxy) iodo(4-methoxyphenyl)-1/-/-pyrrolecarboxylate (28) (2.92 g , 76 %) as a white solid: m/z 584 (M+H)+ (ES+) . H NMR (400 MHz, DMSO-d ) d : 7.51-7.29 (m, 10 H), 7.14-7.05 (m, 2H), 7.03-6.92 (m, 2H), 5.12 (s, 2H), 5.00 (s, 2H), 3.97 (q, J = 7.1 Hz, 2H) 3.82 (s, 3H), 0.99 (t, J = 7.1 Hz, 3H).
Step (ii): Ethyl 3,4-bis(benzyloxy)(4-methoxyphenyl)(pyridinyl)-1/-/-pyrrolecarboxylate (UL1-082) To a solution of ethyl 3,4-bis(benzyloxy)iodo(4-methoxyphenyl)-1/-/-pyrrolecarboxylate (28) (300 mg, 0.51 mmol) in DMF (5 ml_) was added 2-(tributylstannyl)pyridine (280 m I_ , 0.87 mmol), copper(l) iodide (19.6 mg, 0.10 mmol) and dichlorobis(triphenylphosphine)palladium(ll) (36.1 mg, 0.05 mmol). The mixture was heated in a microwave at 140°C for 30 min, AcOH (2 ml_) was added and the crude product was loaded onto a column of SCX (5 g). The column was washed with MeOH and then the product was eluted with 1 % NH in MeOH. The resultant mixture was concentrated in vacuo and the residue was purified by silica gel chromatography (12 g , 20-100 % EtOAc in isohexane) to afford ethyl 3,4-bis(benzyloxy)(4-methoxyphenyl) (pyridinyl)-1/-/-pyrrolecarboxylate (UL1-082) (231 mg, 83 %) as a pale yellow oil: m/z 535 (M+H)+ (ES+) . H NMR (400 MHz, DMSO-d ) d : H NMR (400 MHz, DMSO-d ) d : 8.47-8.40 (m, 1H), 7.71-7.63 (m, 1H), 7.54-7.47 (m, 2H), 7.45-7.16 (m, 10H), 7.12-7.03 (m, 2H), 6.83-6.76 (m, 2H), 5.18 (s, 2H), 4.96 (s, 2H), 4.02 (q, J = 7.1 Hz, 2H), 3.72 (s, 3H), 1.03 (t, J = 7.1 Hz, 3H).
Step (iii): Ethyl 3,4-dihydroxy(4-methoxyphenyl)(pyridinyl)-1/-/-pyrrolecarboxylate (UL1-083) A solution of ethyl 3,4-bis(benzyloxy)(4-methoxyphenyl)(pyridinyl)-1/-/-pyrrole carboxylate (UL1-082) (146 mg, 0.273 mmol) in MeOH/THF (9:1 ; 20 mL) was passed through a Thales Ή -cube' cartridge (10 % Pd/C) at a flow rate of 1 mL/min at 30°C under H (full H 2 2 mode). The output was concentrated in vacuo and the resulting oil was purified by silica gel chromatography (12 g , 0-2 % MeOH in DCM) to afford ethyl 3,4-dihydroxy(4-methoxyphenyl)- -(pyridinyl)-1/-/-pyrrolecarboxylate (UL1-083) (86 mg, 89 %) as a pale orange solid: m/z 355 (M+H)+ (ES+) . H NMR (400 MHz, DMSO-d ) d : 10.84 (s, 1H), 8.62 (s, 1H), 8.56-8.47 (m, 1H), .55 (m, 1H),7.26-7.12 (m, 3H), 7.01-6.89 (m, 2H), .49 (m, 1H), 4.01 (q, J = 7.1 Hz, 2H), 3.80 (s, 3H), 1.02 (t, J = 7.1 Hz, 3H).
Example T : Ethyl 5-(4-ethylthiazolyl)-3,4-dihydroxy(4-methoxyphenyl)-1/-/-pyrrole carboxylate (UL1-086) 29 30 UL1 -086 Step (i): Ethyl 3,4-bis(benzyloxy)(4-ethylthiazolyl)(4-methoxyphenyl)-1/-/-pyrrole carboxylate (30) A solution of 1-bromobutanone (24 m I_ , 0.21 mmol) and ethyl 3,4-bis(benzyloxy) carbamothioyl(4-methoxyphenyl)-1/-/-pyrrolecarboxylate (29) [prepared under standard conditions from 9 and on reagent] (100 mg, 0.19 mmol) in EtOH(4 mL) was heated at 80°C for 45 min. The les were removed in vacuo and the product was purified by silica gel chromatography (12 g cartridge, 0-25 % EtOAc in isohexane) to afford ethyl 3,4- bis(benzyloxy)(4-ethylthiazolyl)(4-methoxyphenyl)-1/-/-pyrrolecarboxylate (30) (82 mg, 75 %) as a yellow oil: m/z 569 (M+H)+ (ES+).
Step (ii) Ethyl 5-(4-ethylthiazolyl)-3,4-dihydroxy(4-methoxyphenyl)-1/-/-pyrrole carboxylate (UL1 -086) A solution of ethyl 3,4-bis(benzyloxy)(4-ethylthiazolyl)(4-methoxyphenyl)-1/-/-pyrrole carboxylate (30) (70 mg, 0.12 mmol) in MeOH/THF (3:1 ; 5 mL) was passed through a Thales Ή -cube' cartridge (10 Pd/C) at a flow rate of 1 mL/min at 40°C under H (full H mode). The 2 2 output was concentrated in vacuo and the resulting oil was purified by silica gel tography (12 g , 0-20 % EtOAc in ane) to afford ethyl 5-(4-ethylthiazolyl)-3,4-dihydroxy(4- methoxyphenyl)-1/-/-pyrrolecarboxylate (UL1-086) (30 mg, 63 %) as an off-white solid: m/z 389 (M+H)+ (ES+) . H NMR (400 MHz, DMSO-d ) d : H NMR (400 MHz, DMSO-d6) d : 9.82 (s, 1H), 8.68 (s, 1H), 7.28-7.19 (m, 2H), 7.06-7.03 (m, 1H), 7.03-6.97 (m, 2H), 4.00 (q, J = 7.1 Hz, 2H), 3.82 (s, 3H), 2.67 (q, J = 7.5 Hz, 2H), 1.16 (t, J = 7.5 Hz, 3H), 0.99 (t, J = 7.1 Hz, 3H).
Example U : Ethyl 3,4-bis(benzyloxy)(4-methoxyphenyl)(5-methyl-1,3,4-oxadiazolyl)-1/-/- 0 pyrrolecarboxylate (UL1-087) 3 1 UL1-087 Step (i): Ethyl 5-(2-acetylhydrazinecarbonyl)-3,4-bis(benzyloxy)(4-methoxyphenyl)-1/-/- pyrrolecarboxylate (31) HATU (739 mg, 1.94 mmol) was added to a solution of triethylammonium 3,4-bis(benzyloxy) (ethoxycarbonyl)(4-methoxyphenyl)-1/-/-pyrrolecarboxylate (5) (325 mg, 0.65 mmol), DIPEA (566 m I_ , 3.24 mmol) and acetohydrazide (53 mg, 0.71 mmol) in DMF (5 ml_) at 0°C. The 0 mixture was stirred for 30 min and allowed to warm to RT. The reaction was partitioned between water (10 ml_) and Et 0 (30 ml_) and NH OAc (aq.) (20 ml_) was added, the c layer was 2 4 ted and washed with sat. NaHC0 (aq.) (20 ml_), brine (20 ml_), dried (MgS0 ) , filtered and solvents removed in vacuo. The product was ed by silica gel chromatography (40 g , 0- 80 % EtOAc in isohexane) to afford ethyl 5-(2-acetylhydrazinecarbonyl)-3,4-bis(benzyloxy)(4- methoxyphenyl)-1/-/-pyrrolecarboxylate (31) (196 mg, 100 %) as a colourless oil: m/z 558 (M+H)+ (ES+) .
Step (ii): Ethyl 3,4-bis(benzyloxy)(4-methoxyphenyl)(5-methyl-1,3,4-oxadiazolyl)-1/-/- pyrrolecarboxylate (UL1-087) A solution of ethyl 3,4-bis(benzyloxy)(4-methoxyphenyl)(2-acetylhydrazinecarbonyl)-1/-/- pyrrolecarboxylate (31) (155 mg, 0.56 mmol) and s reagent (132 mg, 0.56 mmol) were dissolved in THF (2 ml_). The mixture was heated in a microwave at 100°C for 30 min and the volatiles removed in vacuo. The crude product was purified by silica gel chromatography (12 g , 0-30 % EtOAc in isohexane) to afford ethyl 3,4-bis(benzyloxy)(4-methoxyphenyl)(5-methyl- 1,3,4-oxadiazolyl)-1/-/-pyrrolecarboxylate (UL1-087) ( 1 15 mg, 77 %) as a colourless oil: m/z 540 (M+H)+ (ES+) . H NMR (400 MHz, DMSO-d ) d : 7.51-7.46 (m, 2H), 7.43-7.31 (m, 8H), 7.18-7.12 (m, 2H), 6.93-6.87 (m, 2H), 5.17 (s, 2H), 5.07 (s, 2H), 4.02 (q, J = 7.1 Hz, 2H), 3.78 (s, 3H), 2.35 (s, 3H), 1.01 (t, J = 7.1 Hz, 3H).
Example V : Ethyl 3,4-dihydroxy(4-methoxyphenyl)(2H-tetrazolyl)-1/-/-pyrrole carboxylate (UL1-089) 32 UL1 -090 UL1 -089 Step (i): Ethyl 3,4-bis(benzyloxy)(4-methoxyphenyl)(2H-tetrazolyl)-1/-/-pyrrole carboxylate (UL1 -090) A on of ethyl 3,4-bis(benzyloxy)cyano(4-methoxyphenyl)-1/-/-pyrrolecarboxylate (32) [prepared using the same procedure as Example H step(i) using UL1-079 as starting al] (400 mg, 0.83 mmol), NH C I (222 mg, 4.14 mmol) and sodium azide (269 mg, 4.14 mmol) were dissolved in DMF (4 mL). The reaction mixture was heated in a microwave at 120°C for 2h, partitioned between EtOAc (15 mL) and water (15 mL). The organic layer was separated and washed with water (3 x 15 mL), dried (MgS0 ) , filtered and solvents removed in vacuo. The t was ed by silica gel chromatography (40 g , 0-40 % MeOH in DCM/2.5 %Et N) to afford the triethylammonium salt of (UL1-090). The salt was dissolved in DCM (10 mL) and the organic layer was washed with 1M HCI (aq.) (10 mL), brine (10 mL), sat. NaHC0 (aq.) (10 mL), brine (10 mL), dried (MgS0 ) , filtered and solvents removed in vacuo to afford ethyl 3,4- bis(benzyloxy)(4-methoxyphenyl)(2H-tetrazolyl)-1/-/-pyrrolecarboxylate (UL1-090) (150 mg, 34 %) as a white solid: m/z 526 (M+H)+ (ES+) ; 524 (M-H) (ES ) . H NMR (400 MHz, DMSO-de) d : 7.51-7.45 (m, 2H), 7.42-7.25 (m, 8H), .03 (m, 2H), 6.83-6.76 (m, 2H), 5.15 (s, 2H), 4.95 (s, 2H), 4.00 (q, J = 7.1 Hz, 2H), 3.74 (s, 3H), 1.00 (t, J = 7.1 Hz, 3H).
Step (ii): Ethyl 3,4-dihydroxy(4-methoxyphenyl)(2H-tetrazolyl)-1/-/-pyrrolecarboxylate (UL1-089) A solution of ethyl 3,4-bis(benzyloxy)(4-methoxyphenyl)(2H-tetrazolyl)-1/-/-pyrrole carboxylate (UL1-090) (85 mg, 0.16 mmol) in MeOH (6 mL) was passed through a Thales Ή - cube' cartridge (10 % Pd/C) at a flow rate of 1 mL/min at 40°C under H (full H mode). The 2 2 output was concentrated in vacuo. The crude material was dissolved in DCM (5 mL) and washed with 1M HCI (aq.), brine (5 mL), sat. NaHC0 (aq.) (5 mL), the basic aqueous layer was acidified with 1M HCI (aq.) (5 mL) and washed with DCM (2 x 5 mL), the combined organics were dried (MgS0 ) , filtered and solvents d in vacuo to afford ethyl 3,4-dihydroxy(4- methoxyphenyl)(2H-tetrazolyl)-1/-/-pyrrolecarboxylate (UL1-089) (45 mg, 79 %) as a white solid: m/z 346 (M+H)+ (ES+) ; 344 (M-H) (ES ) . H NMR (400 MHz, DMSO-d ) d : 9.30 (br s , 1H), 8.67 (s, 1H), 7.10-7.05 (m, 2H), 6.87-6.81 (m, 2H), 4.02 (q, J = 7.1 Hz, 2H), 3.76 (s, 0.99 (t, J = 7.1 Hz, 3H).
Example W : 5-Acetyl-3,4-dihydroxy(4-methoxyphenyl)-N,N-dimethyl-1 H-pyrrole carboxamide (UL1-091) UL1 -091 Step (i): 3,4-Bis(benzyloxy)-A/ -methoxy(4-methoxyphenyl)-A J J -trimethyl-1/-/-pyrrole-2,5- dicarboxamide (33) (COCI) (560 m I_ , 6.39 mmol) was added dropwise to a stirred solution of 3,4-bis(benzyloxy) hylcarbamoyl)(4-methoxyphenyl)-1/-/-pyrrolecarboxylic acid (7) ( 1 .6 g , 3.20 mmol) in DCM (20 mL) at 0°C, followed by 2 drops of DMF. The reaction mixture was d at RT for 5h, concentrated in vacuo and the resulting residue was dissolved in DCM (15 mL). N,O- dimethylhydroxylamine was added (215 mg, 3.52 mmol) followed by pyridine (620 m , 7.67 mmol), and the reaction allowed to stir at RT for 18h. The mixture was diluted with DCM (25 mL) and water (50 mL), the organic layer was separated and washed with water (2 x 25 mL), brine (25 mL), dried (MgS0 ) , filtered and concentrated in vacuo. The product was purified by silica gel chromatography (40 g , 0-80 % EtOAc in isohexane) to afford 3,4-bis(benzyloxy)-/V 2- methoxy(4-methoxyphenyl)-/V 2, , -trimethyl-1/-/-pyrrole-2,5-dicarboxamide (33) (505 mg, 22 %) as a colourless oil. m/z 544 (M+H)+ (ES+) .
Step (ii): 5-Acetyl-3,4-bis(benzyloxy)(4-methoxyphenyl)-/\/,A/-dimethyl-1/-/-pyrrole carboxamide (34) Methylmagnesium bromide (3 M in Et 0 ) (294 m , 0.88 mmol) was added se to a stirred on of 3,4-bis(benzyloxy)-/V -methoxy(4-methoxyphenyl)-/\/ 2 , -trimethyl-1/-/-pyrrole- 2,5-dicarboxamide (33) (320 mg, 0.59 mmol) in THF ( 1 mL) at 0°C. The reaction e was allowed to warm to RT and d for 20h. The reaction mixture was cooled to -10°C and 1 M HCI (aq.) (10 ml_) was added. The aqueous layer was extracted with DCM (3 x 10 ml_). The combined organic phases were washed with water (20 ml_), brine (20 ml_), dried (MgS0 ) , ed and concentrated in vacuo to give a yellow oil.
The product was purified by silica gel chromatography (40 g , 0-100 % EtOAc in ane) to afford 5-acetyl-3,4-bis(benzyloxy)(4-methoxyphenyl)-/\/,A/-dimethyl-1/-/-pyrrolecarboxamide (34) ( 1 10 mg, 38 %) as a yellow oil: m/z 499 (M+H)+ (ES+) . H NMR (400 MHz, DMSO-d ) d : 7.46-7.33 (m, 10H), 7.05-7.03 (m, 2H), 6.88-6.86 (m, 2H), 5.25 (s, 2H), 5.01 (s, 2H), 3.77 (s, 3H), 2.71 (s, 6H), 2.26 (s, 3H).
Step (iii): yl-3,4-dihydroxy(4-methoxyphenyl)-/\/,A/-dimethyl-1/-/-pyrrolecarboxamide (UL1-091) A solution of 5-acetyl-3,4-bis(benzyloxy)(4-methoxyphenyl)-/\/,A/-dimethyl-1/-/-pyrrole amide (34) (100 mg, 0.20 mmol) in MeOH (5 ml_) was passed through a Thales Ή -cube' cartridge (10 % Pd/C) at a flow rate of 1 mL/min at 20°C under H (full H mode). The output 2 2 was concentrated in vacuo. The crude compound was purified by preparative HPLC (C-18 column, 21.2 mm i.d. x 100 mm, 5 micron particle size, gradient 5-95 % MeCN in 0.1 % aq. formic acid over 16 min) to afford 5-acetyl-3,4-dihydroxy(4-methoxyphenyl)-/\/,A/-dimethyl-1/-/- pyrrolecarboxamide (UL1-091) (45 mg, 70 %) as a pale yellow solid: m/z 319 (M+H)+ (ES+) , 317 (M-H) (ES ) . H NMR (400 MHz, DMSO-d ) d : 9.77 (s, 1H), 8.57 (s, 1H), 7.09-7.07 (m, 2H), 6.88-6.86 (m, 2H), 3.77 (s, 3H), 2.81 (br s , 6H), 2.05 (s, 3H).
Example X : Ethyl 3,4-bis((diethoxyphosphoryl)oxy)(dimethylcarbamoyl)(4-methoxyphenyl)- 1/-/-pyrrolecarboxylate (UL1 -092) UL1-012 UL1-092 Perbromomethane (207 mg, 0.62 mmol) and DIPEA (131 m I_ , 0.75 mmol) were added successively to a solution of DMAP (3.05 mg, 0.03 mmol) and ethyl ethylcarbamoyl)-3,4- dihydroxy(4-methoxyphenyl)-1/-/-pyrrolecarboxylate 12) (87 mg, 0.25 mmol) in MeCN (3 ml_) at -10°C. The reaction mixture was allowed to stir for 30 min and diethyl phosphite (166 m I_ , 0.75 mmol) added, the reaction was allowed to warm slowly to RT and stirred for 3h.
The reaction was quenched with 5 % NaH P0 (aq.) (20 ml_) and extracted with EtOAc (3 x 25 ml_). The combined organics were washed with brine (50 ml_), dried (MgS0 ) , filtered and concentrated in vacuo. The crude residue was purified by silica gel chromatography (40 g , 0-4 % MeOH in DCM) to afford an orange oil. The compound was further purified by preparative HPLC (C-18 column, 2 1.2 mm i.d. x 100 mm, 5 micron particle size, gradient 5-95 % MeCN in 0.1 % aq. formic acid over 16 min) to afford ethyl 3,4-bis((diethoxyphosphoryl)oxy) (dimethylcarbamoyl)(4-methoxyphenyl)-1/-/-pyrrolecarboxylate (UL1-092) (35 mg, 22 %) as a yellow oil: m/z 621 (M+H)+ (ES+) . H NMR (400 MHz, DMSO-d ) d : 7.18-7.15 (m, 2H), 6.97- 6.94 (m, 2H), 4.22-4.02 (m, 10H), 3.79 (s, 3H), 2.75 (s, 3H), 2.71 (s, 3H), 1.31-1.24 (m, 12H),1 .07 (t, J = 7.2 Hz, 3H).
Example Y: Ethyl 5-(dimethylcarbamoyl)-3,4-dihydroxy(4-hydroxyphenyl)-1/-/-pyrrole carboxylate (UL1-100) 36 0 Step (i): Ethyl 3,4-bis(benzyloxy)(dimethylcarbamoyl)(4-hydroxyphenyl)-1/-/-pyrrole carboxylate (36) A on of ethyl s(benzyloxy)(dimethylcarbamoyl)(4-((4- methoxybenzyl)oxy)phenyl)-1/-/-pyrrolecarboxylate (35) [prepared using the same procedure as Example B except methoxybenzyl)oxy)aniline used in step (i)] (46 mg, 0.07 mmol) in AcOH (5 mL) was stirred at 105°C for 18h. The volatiles were removed in vacuo and the residue was purified by silica gel chromatography (4 g , 0-10 % MeOH in DCM) to afford ethyl 3,4- bis(benzyloxy)(dimethylcarbamoyl)(4-hydroxyphenyl)-1/-/-pyrrolecarboxylate (36) (35 mg, 9 1 %) as a yellow solid: m/z 515 (M+H)+ (ES+) . H NMR (400 MHz, DMSO-d ) d : 9.63 (1H, s), 7.47-7.42 (m, 2H), 7.41-7.29 (m, 8H), 6.99-6.91 (m, 2H), 6.73-6.66 (m, 2H), 5.10 (s, 2H), 4.95 (s, 2H), 3.99 (q, J = 7.1 Hz, 2H), 2.69 (s, 3H), 2.67 (s, 3H),1 .00 (t, J = 7.1 Hz, 3H).
Step (ii): Ethyl 5-(dimethylcarbamoyl)-3,4-dihydroxy(4-hydroxyphenyl)-1/-/-pyrrole carboxylate (UL1 -100) A solution of ethyl 3,4-bis(benzyloxy)(dimethylcarbamoyl)(4-hydroxyphenyl)-1/-/-pyrrole carboxylate (36) (30 mg, 0.06 mmol) in MeOH (2 mL) was passed through a Thales Ή -cube' cartridge (10 % Pd/C) at a flow rate of 1 mL/min at 25°C under H (full H mode). The output 2 2 was concentrated in vacuo, and a solid was ted by filtration after trituration with isohexane to afford ethyl 5-(dimethylcarbamoyl)-3,4-dihydroxy(4-hydroxyphenyl)-1/-/-pyrrole carboxylate (UL1-100) ( 1 1 mg, 56 %) as a yellow solid: m/z 335 (M+H)+ (ES+) , 333 (M-H) (ES ) .
H NMR (400 MHz, DMSO-d ) d : 9.50 (s, 1H), 8.54 (s, 1H), 8.42 (s, 1H), 6.94-6.86 (m, 2H), 6.69-6.62 (m, 2H), 3.99 (q, J = 7.0 Hz, 2H), 2.81 (br s , 6H), 0.98 (t J = 7.0 Hz, 3H).
Example Z : Ethyl 5-(dimethylcarbamoyl)-3,4-dihydroxy(4-(3-morpholinopropoxy)phenyl)-1/-/- pyrrolecarboxylate (UL1-102) 36 37 UL1 -1 02 Step (i): Ethyl 3,4-bis(benzyloxy)(dimethylcarbamoyl)(4-(3-morpholinopropoxy)phenyl)-1/-/- pyrrolecarboxylate (37) A solution of ethyl 3,4-bis(benzyloxy)(dimethylcarbamoyl)(4-hydroxyphenyl)-1/-/-pyrrole carboxylate (36) (45 mg, 0.09 mmol), 4-(3-chloropropyl)morpholine hydrochloride (19 mg, 0.10 mmol) and K C0 (25 mg, 0.18 mmol) in DMF ( 1 mL) was stirred at 60°C for 18h. The on 2 3 mixture was partitioned between EtOAc (10 mL) and water (5 mL), the organic layer was separated and washed with brine (5 mL), dried (MgS0 ) , filtered and concentrated in vacuo.
The residue was dissolved in MeOH (2 mL) and loaded onto a column of SCX (5 g). The column was washed with MeOH and then the t was eluted with 1 % NH in MeOH, removal of the solvents in vacuo afforded ethyl 3,4-bis(benzyloxy)(dimethylcarbamoyl)(4- (3-morpholinopropoxy)phenyl)-1/-/-pyrrolecarboxylate (37) (32 mg, 57 %) as a yellow oil: m/z 642 (M+H)+ (ES+) . H NMR (400 MHz, DMSO-d ) d : 7.47-7.42 (m, 2H), 7.41-7.30 (m, 8H), 7.10- 7.05 (m, 2H), 6.99-6.91 (m, 2H), 5.1 1 (s, 2H), 4.96 (s, 2H), 4.04-3.95 (m, 4H), 3.59-3.52 (m, 5H), 2.72-2.67 (m, 6H), 2.44-2.38 (m, 3H), 2.37-2.32 (m, 5H), 1.92-1 .81 (m, 2H), 1.00 (t, J = 7.0 Hz, 3H).
Step (ii): Ethyl 5-(dimethylcarbamoyl)-3,4-dihydroxy(4-(3-morpholinopropoxy)phenyl)-1/-/- pyrrolecarboxylate (UL1-102) A on of ethyl 3,4-bis(benzyloxy)(dimethylcarbamoyl)(4-(3- morpholinopropoxy)phenyl)-1/-/-pyrrolecarboxylate (37) (105 mg, 0.16 mmol) in MeOH (4 mL) was passed through a Thales Ή -cube' cartridge (10 % Pd/C) at a flow rate of 1 mL/min at 25°C under H (full H mode). The output was concentrated in vacuo, and a solid was collected by 2 2 filtration after trituration with isohexane to afford ethyl 5-(dimethylcarbamoyl)-3,4-dihydroxy(4- (3-morpholinopropoxy)phenyl)-1/-/-pyrrolecarboxylate (UL1-102) (20 mg, 25 %) as a yellow solid: m/z 462 (M+H)+ (ES+) , 460 (M-H) (ES ) . H NMR (400 MHz, DMSO-d ) d : 8.58 (s, 1H), 8.44 (s, 1H), 7.05-6.99 (m, 2H), 6.86-6.82 (m, 2H), 4.03-3.95 (m, 4H), .53 (m, 4H), 2.82 (br s , 6H), .34 (m, 6H), 1.93-1 .81 (m, 2H), 0.98 (t, J = 7.0 Hz, 3H).
Example A 1: 2-(Dimethylcarbamoyl)(ethoxycarbonyl)(4-methoxyphenyl)-1/-/-pyrrole-3,4- diyl bis(3-methoxypropanoate) (UL1-104) UL1 -012 UL1 -104 Isobutyl chloroformate (410 m I_ , 3.16 mmol) was added dropwise to a d solution of 3- methoxypropanoic acid (297 m I_ , 3.16 mmol) and 4-methylmorpholine (790 m I_ , 7.18 mmol) in DCM (20 mL) at -15°C and the reaction mixture was allowed to stir for 20 min. A solution of ethyl 5-(dimethylcarbamoyl)-3,4-dihydroxy(4-methoxyphenyl)-1/-/-pyrrolecarboxylate (UL1- 012) (0.5 g , 1.44 mmol) in DCM (20 mL) was added, and the reaction allowed to warm to RT and stirred for 18h. The solvents were removed in vacuo, and the mixture was partitioned between DCM (30 mL) and 1 M HCI (aq.) (10 mL), the organic layer was separated and washed with sat. NaHC0 (aq.) (10 mL), dried (MgS0 ) , filtered and concentrated in vacuo. The crude 3 4 residue was purified by silica gel chromatography (40 g , 50-70 % EtOAc in isohexane) to afford ethylcarbamoyl)(ethoxycarbonyl)(4-methoxyphenyl)-1/-/-pyrrole-3,4-diyl bis(3- methoxypropanoate) (UL1-104) (300 mg, 39 %) as a yellow oil: m/z 521 (M+H)+ (ES+) . H NMR (400 MHz, DMSO-de) d : .15 (m, 2H), 6.98-6.89 (m, 2H), 4.00 (q, J = 7.1 Hz, 2H), 3.79 (s, 3H), 3.65-3.59 (m, 4H), 3.27 (s, 3H), 3.25 (s, 3H), 2.82 (s, 3H), 2.78 (t, J = 6.0 Hz, 4H), 2.71 (s, 3H), 1.05 (t, J = 7.1 Hz, 3H).
Example B 1 : 2-(Dimethylcarbamoyl)(ethoxycarbonyl)(4-methoxyphenyl)-1/-/-pyrrole-3,4- diyl bis(3-(2-methoxyethoxy)-2,2-dimethylpropanoate) (UL1-108) UL1 -108 Step (i): Methyl 3-(2-methoxyethoxy)-2,2-dimethylpropanoate (39) Sodium hydride (60 % wt in oil) (3.14 g , 78 mmol) was added to a stirred solution of methyl 3- hydroxy-2,2-dimethylpropanoate (5 mL, 39.2 mmol) in DMF (5.6 mL) at 0°C, after 5 min 1- bromomethoxyethane (7.4 mL, 78 mmol) was added dropwise, and the reaction mixture was allowed to stir for 3h. The reaction was quenched with sat. NH C I (aq.) (30 mL) and the aqueous layer was extracted with DCM (2 x 50 mL), the combined organic layers were dried (MgS0 ) , ed and concentrated in vacuo. The crude oil was dissolved in EtOAc (125 mL) and washed with water (3 x 40 mL), dried (MgS0 ) , filtered and concentrated in vacuo. The material was purified by vacuum distillation (68-69°C, 4.4 mbar) to afford methyl 3-(2- yethoxy)-2,2-dimethylpropanoate (39) ( 1 .83 g , 25 %) as a colourless oil: H NMR (400 MHz, CDCIs) d : 3.68 (s, 3H), 3.60-3.58 (m, 2H), 3.52-3.50 (m, 2H), 3.49 (s, 2H), 3.37 (s, 3H), 1.19 (s, 6H).
Step (ii): 3-(2-Methoxyethoxy)-2,2-dimethylpropanoic acid (40) A suspension of methyl 3-(2-methoxyethoxy)-2,2-dimethylpropanoate (39) ( 1 .83 g , 9.62 mmol) and KOH (2.16 g , 38.5 mmol) in water (20 mL) was stirred at RT for 3 days. The aqueous layer was washed with DCM (3 x 20 mL) and acidified to pH 1-2 with 6 M HCI (aq.) and extracted with DCM (3 x 20 mL). The combined organics were dried (MgS0 ) , filtered and concentrated in vacuo to afford 3-(2-methoxyethoxy)-2,2-dimethylpropanoic acid (40) ( 1 .62 g , 96 %) as colourless oil: H NMR (400 MHz, CDCI ) d : 3.67-3.65 (m, 2H), 3.56-3.54 (m, 2H), 3.51 (s, 2H), 3.38 (s, 3H), 1.23 (s, 6H).
Step (iii): 2-(Dimethylcarbamoyl)(ethoxycarbonyl)(4-methoxyphenyl)-1/-/-pyrrole-3,4-diyl bis(3-(2-methoxyethoxy)-2,2-dimethylpropanoate) (UL1 -108) DMF (2.2 m , 0.03 mmol) was added to a d solution of 3-(2-methoxyethoxy)-2,2- dimethylpropanoic acid (40) (500 mg, 2.84 mmol) followed by a solution of (COCI) (0.25 mL, 2.85 mmol) in DCM ( 1 1.4 mL), the reaction e was allowed to stir for 1h. To this mixture was added ethyl 5-(dimethylcarbamoyl)-3,4-dihydroxy(4-methoxyphenyl)-1/-/-pyrrole carboxylate (UL1-012) (450 mg, 1.29 mmol), followed by Et N (900 m I_ , 6.45 mmol) and the reaction allowed to stir for 45 min. The reaction was then filtered, and the filtrate trated in vacuo. The crude residue was purified by silica gel chromatography (40 g , 0-3 % MeOH in DCM) to afford 2-(dimethylcarbamoyl)(ethoxycarbonyl)(4-methoxyphenyl)-1/-/-pyrrole-3,4- diyl bis(3-(2-methoxyethoxy)-2,2-dimethylpropanoate) (UL1-108) (447 mg, 52 %) as a light yellow oil: m/z 665 (M+H)+. H NMR (400 MHz, DMSO-d ) d 7.23-7.15 (m, 2H), .89 (m, 2H), 4.02 (q, J = 7.2 Hz, 2H), 3.79 (s, 3H), 3.58-3.39 (m, 12 H), 3.24 (s, 3H), 3.23 (s, 3H), 2.81 (s, 3H), 2.70 (s, 3H), 1.25 (s, 6H), 1.19 (s, 6H), 1.05 (t, J = 7.2 Hz, 3H).
Example C 1: Ethyl 5-(dimethylcarbamoyl)hydroxy(4-methoxyphenyl) ((phosphonooxy)methoxy)-1 /-/-pyrrolecarboxylate (UL1 -109) UL1 -109 Step (i): Ethyl 3-(benzyloxy)(dimethylcarbamoyl)hydroxy(4-methoxyphenyl)-1/-/-pyrrole- 2-carboxylate (41) Benzyl bromide (3.94 ml_, 33.2 mmol) was added to a stirred suspension of ethyl 5- (dimethylcarbamoyl)-3,4-dihydroxy(4-methoxyphenyl)-1/-/-pyrrolecarboxylate (UL1-012) (10.5 g , 30.1 mmol), potassium iodide (5.50 g , 33.2 mmol) and K C0 (4.58 g , 33.2 mmol) in 2 3 DMF (100 ml_). The reaction was allowed to stir at 80°C for 24h. The reaction e was poured into water (200 ml_), washed with Et 0 (2 x 200 ml_), and the combined organic layers were washed with 1M HCI (aq.) (400 ml_), brine (2 x 400 ml_), dried (MgS0 ) , filtered and concentrated in vacuo. The crude product was ed by silica gel chromatography (330 g , 0- 50 % EtOAc in toluene) to afford ethyl 3-(benzyloxy)(dimethylcarbamoyl)hydroxy(4- methoxyphenyl)-1/-/-pyrrolecarboxylate (41) (4.36 g , 33 %) as a pale yellow solid: m/z 439 (M+H)+ (ES+) . H NMR (400 MHz, DMSO-d ) d : 8.82 (s, 1H), 7.53-7.47 (m, 2H), 7.42-7.29 (m, 3H), 7.08-7.01 (m, 2H), 6.90-6.84 (m, 2H), 5.09 (s, 2H), 3.96 (q, J = 7.0 Hz, 2H), 3.76 (s, 3H), 2.82 (br s , 6H), 0.97 (t, J = 7.0 Hz, 3H).
Step (ii): Ethyl zyloxy)(((bis(benzyloxy)phosphoryl)oxy)methoxy) (dimethylcarbamoyl)(4-methoxyphenyl)-1/-/-pyrrolecarboxylate (42) A on of dibenzyl (chloromethyl) phosphate (0.23 g , 0.70 mmol) in DMF ( 1 mL) was added se to a stirred suspension of ethyl 3-(benzyloxy)(dimethylcarbamoyl)hydroxy(4- methoxyphenyl)-1/-/-pyrrolecarboxylate (41) (0.26 g , 0.58 mmol) and K C0 (80 mg, 0.58 2 3 mmol) in DMF (4 mL) at 0°C, the reaction was stirred at for 1h, then allowed to warm to RT over 16 h . A solution of yl (chloromethyl) phosphate (0.23 g , 0.70 mmol) in DMF ( 1 mL) and K C0 (80 mg, 0.58 mmol) were added and the reaction was stirred for a further 24h. A further 2 3 solution of dibenzyl (chloromethyl) phosphate (0.1 1 g , 0.35 mmol) in DMF ( 1 mL) and K C0 (40 2 3 mg, 0.29 mmol) were added and the reaction was stirred for a further 4h. The reaction mixture was poured into water (20 mL) and extracted with Et 0 (2 x 20 mL). The combined organic layers were washed with brine (3 x 100 mL), dried (MgS0 ) , filtered and concentrated in vacuo.
The crude product was purified by silica gel chromatography (12 g , 0-50 % EtOAc in toluene) to afford ethyl 3-(benzyloxy)(((bis(benzyloxy)phosphoryl)oxy)methoxy)(dimethylcarbamoyl) (4-methoxyphenyl)-1/-/-pyrrolecarboxylate (42) (0.10 g , 23 %) as a colourless gum: m/z 729 (M+H)+ (ES+) . H NMR (400 MHz, DMSO-d ) d : 7.36-7.30 (m, 13H), .05 (m, 2H), 6.88-6.92 (m, 2H), 5.49 (d, J= 11.1 Hz, 2H), 5.1 1 (s, 2H), 5.06-4.96 (m, 2H), .05 (m, 4H), 4.00 (q, J = 7.1 Hz, 2H), 3.77 (s, 3H), 2.72 (s, 3H), 2.65 (s, 3H), 0.99 (t, J = 7.0 Hz, 3H).
Step (iii): Ethyl ethylcarbamoyl)hydroxy(4-methoxyphenyl) ((phosphonooxy)methoxy)-1 /-/-pyrrolecarboxylate (UL1 -109) A solution of ethyl 3-(benzyloxy)(((bis(benzyloxy)phosphoryl)oxy)methoxy) (dimethylcarbamoyl)(4-methoxyphenyl)-1/-/-pyrrolecarboxylate (42) (2.17 g , 2.99 mmol) in MeOH (20 mL) was passed through a Thales Ή -cube' cartridge (10 % Pd/C) at a flow rate of 1 mL/min at 40°C under H (full H mode). The output was concentrated in vacuo, the residue was 2 2 taken up in water (50 mL) and washed with EtOAc (50 mL), the organic layer was washed with water (2 x 50 mL) and the combined aqueous layers were freeze-dried to afford ethyl 5- (dimethylcarbamoyl)hydroxy(4-methoxyphenyl)((phosphonooxy)methoxy)-1/-/-pyrrole carboxylate (UL1-109) (720 mg, 52 %) as a white solid: m/z 459 (M+H)+ (ES+) . H NMR (400 MHz, DMSO-de) d : 7.14-7.05 (m, 2H), .85 (m, 2H), 5.35-5.23 (br m , 2H), 3.99 (q, J = 7.2 Hz, 2H), 3.77 (s, 3H), 2.73 (s, 3H), 2.70 (s, 3H), 1.02 (t, J = 7.2 Hz, 3H).
Example D1: Ethyl 5-(dimethylcarbamoyl)-3,4-dihydroxy(4-(3- (methylsulfonyl)propoxy)phenyl)-1 /-/-pyrrolecarboxylate (UL1 -1 10) UL1 - 0 Step (i): Ethyl 3,4-bis(benzyloxy)(dimethylcarbamoyl)(4-(3-(methylthio)propoxy)phenyl)- 1/-/-pyrrolecarboxylate (43) To a stirred solution of ethyl 3,4-bis(benzyloxy)(dimethylcarbamoyl)(4-hydroxyphenyl)-1/-/- pyrrolecarboxylate (36) (350 mg, 0.68 mmol) in THF (2 mL) at 0°C was added 3- lthio)propan-l-ol (84 m I_ , 0.82 mmol), nylphosphine (214 mg, 0.82 mmol), and DIAD (159 m I_ , 0.82 mmol). After 2h further portions of 3-(methylthio)propanol (84 m I_ , 0.82 mmol), triphenylphosphine (214 mg, 0.82 mmol) and DIAD (159 m I_ , 0.82 mmol) were added, and the reaction mixture was allowed to stir at RT for 4 days. The reaction mixture was quenched with water (10 mL) and extracted with EtOAc (2 x 20 mL), the combined organics were washed with brine (30 mL), dried (MgS0 ) filtered and concentrated in vacuo. The crude product was purified by silica gel chromatography (12 g , 0-100 % EtOAc in ane) to afford ethyl 3,4- bis(benzyloxy)(dimethylcarbamoyl)(4-(3-(methylthio)propoxy)phenyl)-1/-/-pyrrole carboxylate (43) (389 mg, 85 %) as a clear yellow oil: m/z 603 (M+H)+ (ES+) .
Step (ii): Ethyl 3,4-bis(benzyloxy)(dimethylcarbamoyl)(4-(3- (methylsulfonyl)propoxy)phenyl)-1 rrolecarboxylate (44) To a stirred solution of ethyl 3,4-bis(benzyloxy)(dimethylcarbamoyl)(4-(3- (methylthio)propoxy)phenyl)-1/-/-pyrrolecarboxylate (43) (389 mg, 0.65 mmol) in DCM (8 mL) at 0°C was added m-CPBA (445 mg, 2.58 mmol) the mixture was allowed to warm up to RT and stirred for 1h, the reaction was quenched with sat. Na C0 (aq.) (20 mL) and extracted with 2 3 DCM (3 x 10 mL) the combined cs were washed with brine (40 mL), passed through a phase separator, and the volatiles removed in vacuo. The crude product was purified by silica gel chromatography (12 g , 0-2 % MeOH(1 % NH ) in DCM) to afford ethyl 3,4-bis(benzyloxy) (dimethylcarbamoyl)(4-(3-(methylsulfonyl)propoxy)phenyl)-1/-/-pyrrolecarboxylate (44) (96 mg, 23 %) as a brown oil: m/z 635 (M+H)+ (ES+) .
Step (iii): Ethyl 5-(dimethylcarbamoyl)-3,4-dihydroxy(4-(3-(methylsulfonyl)propoxy)phenyl)- 1/-/-pyrrolecarboxylate (UL1 -1 10) A solution of ethyl 3,4-bis(benzyloxy)(dimethylcarbamoyl)(4-(3- (methylsulfonyl)propoxy)phenyl)-1/-/-pyrrolecarboxylate (44) (95 mg, 0.15 mmol) in MeOH (4 mL) was passed through a Thales Ή -cube' cartridge (10 % Pd/C) at a flow rate of 1 mL/min at 40°C under H (full H mode). The output was trated in vacuo, and 2 2 the compound was purified by preparative HPLC (C-18 column, 21.2 mm i.d. x 100 mm, 5 micron particle size, gradient 15-40 % MeCN in 0.1 % aq. formic acid over 16 min) to afford ethyl 5-(dimethylcarbamoyl)-3,4-dihydroxy(4-(3-(methylsulfonyl)propoxy)phenyl)-1/-/-pyrrole- 2-carboxylate (UL1-110) (40 mg, 58 %) as a yellow solid: m/z 455 (M+H)+ (ES+) ; 453 (M-H) (ES ) . H NMR (400 MHz, DMSO-d ) d : 8.58 (s, 1H), 8.45 (s, 1H), 7.07-7.02 (m, 2H), 6.89-6.84 (m, 2H), 4.08 (t, J = 7.2 Hz, 2H), 3.99 (q, J = 7.1 Hz, 2H), .25 (m, 2H), 3.02 (s, 3H), 2.92- 2.71 (br s , 6H), 2.21-2.07 (m, 2H), 0.99 (t, J = 7.1 Hz, 3H).
Example E1: 2,6-Dimethylcyclohexyl 5-(dimethylcarbamoyl)-3,4-dihydroxy(4-methoxyphenyl)- 1/-/-pyrrolecarboxylate (UL1 -1 13) UL1 -1 13 Step (i): 2,6-Dimethylcyclohexyl 3,4-bis(benzyloxy)(dimethylcarbamoyl)(4-methoxyphenyl)- 1/-/-pyrrolecarboxylate (45) Sodium hydride (60 wt % in oil) (78 mg, 1.95 mmol) was added to a stirred solution of 2,6- ylcyclohexanol (250 mg, 1.95 mmol) in DMF ( 1 .95 mL). The mixture was heated at 60°C for 10 min and then ethyl 3,4-bis(benzyloxy)(dimethylcarbamoyl)(4-methoxyphenyl)-1/-/- ecarboxylate (6) (206 mg, 0.39 mmol) was added in one portion. The reaction was allowed to stir at 70°C for 16h. The cooled reaction mixture was filtered through Celite, and the filtrate diluted with EtOAc (20 mL) and washed with water (3 x 10 mL), the organic layer was dried (MgS0 ) , filtered and concentrated in vacuo. The crude product was purified by silica gel chromatography (12 g , 0.5 % MeOH ( 1 % NH ) in DCM) to afford 2,6-dimethylcyclohexyl 3,4- bis(benzyloxy)(dimethylcarbamoyl)(4-methoxyphenyl)-1/-/-pyrrolecarboxylate (45) (73 mg, 20 %) as a brown oil. m/z 6 11 (M+H)+ (ES+) Step (ii): 2,6-Dimethylcyclohexyl 5-(dimethylcarbamoyl)-3,4-dihydroxy(4-methoxyphenyl)-1/-/- pyrrolecarboxylate (UL1 -1 13) A solution of 2,6-dimethylcyclohexyl 3,4-bis(benzyloxy)(dimethylcarbamoyl)(4- methoxyphenyl)-1/-/-pyrrolecarboxylate (45) (73 mg, 0.12 mmol) in MeOH (6 ml_) was passed through a Thales Ή -cube' cartridge (10 % Pd/C) at a flow rate of 1 mL/min at 26°C under H (full H mode). The output was concentrated in vacuo and the crude product was purified by preparative HPLC (Waters, Acidic (0.1 % formic acid), Waters X-Select Prep-C18, 5 mhi , 19x50 mm column, 5-95 % MeCN in Water) to afford 2,6-dimethylcyclohexyl 5-(dimethylcarbamoyl)- 3,4-dihydroxy-1 -(4-methoxyphenyl)-1 /-/-pyrrolecarboxylate (UL1 -1 13) (15 mg, 29 %) as a yellow solid: m/z 431 (M+H)+ (ES+) ; 429 (M-H) (ES ) . H NMR (400 MHz, DMSO-de) d : 8.67-8.40 (m, 2H), .05 (m, 2H), 6.91-6.85 (m, 2H), 5.05 (br s , 1H), 3.76-3.75 (m, 3H), 2.82 (br s , 6H), .94 (m, 6H), 0.74-0.57 (m, 6H), 0.57-0.40 (m, 2H).
Example F1: 2-(Dimethylcarbamoyl)(ethoxycarbonyl)(4-methoxyphenyl)-1/-/-pyrrole-3,4- diyl bis(3-(2-(2-((bis(benzyloxy)phosphoryl)oxy)ethoxy)ethoxy)-2,2-dimethylpropanoate) (UL1 117) 46 48 step (i) step (ii) Step (i): 2-(2-(Benzyloxy)ethoxy)ethyl methanesulfonate (47) MsCI (15.8 mL. 204 mmol) was added to a solution of 2-(2-(benzyloxy)ethoxy)ethanol (46) (25 g , 127 mmol) and Et N (36 mL, 255 mmol) in DCM (180 mL) at 0°C, the reaction mixture was allowed to warm up to RT and stirred for 16h. The reaction mixture was diluted with DCM (50 mL) and washed with water (2 x 50 mL), 1M HCI (aq.) (2 x 50 mL), the organic layer was dried (MgS0 ) , filtered and concentrated in vacuo to afford 2-(2-(benzyloxy)ethoxy)ethyl methanesulfonate (47) (35 g , 100 %) as an orange oil: m/z 275 (M+H)+ (ES+) . H NMR (400 MHz, CDCIs) d : 7.37-7.27 (m, 5H), 4.55 (s, 2H), 4.40-4.37 (m, 2H), .76 (m, 2H), 3.71-3.68 (m, 2H), 3.64-3.62 (m, 2H), 3.02 (s, 3H).
Step (ii): -Butyl 2-(benzyloxy)ethoxy)ethoxy)-2,2-dimethylpropanoate (48) To a solution of -butyl 3-hydroxy-2,2-dimethylpropanoate (353 mg, 2.03 mmol) and 2-(2- (benzyloxy)ethoxy)ethyl methanesulfonate (47) (500 mg, 1.82 mmol) in DMF (6 mL) at 0°C was added NaH (60 wt % in oil) (109 mg, 2.73 mmol), and the on mixture was allowed to stir at RT for 30 min. The reaction mixture was quenched with sat. NH C I (aq.) ( 1 mL) diluted with water (20 mL) and washed with EtOAc (4 x 20 mL). The combined organic layers were dried (MgS0 ) , filtered and trated in vacuo. The crude product was purified by silica gel chromatography (12 g , 0-40 % Et 0 in isohexane) to afford te -butyl 3-(2-(2- (benzyloxy)ethoxy)ethoxy)-2,2-dimethylpropanoate (48) (619 mg, 96 %) as a colourless oil: m/z 375 + (ES+) . H NMR (400 MHz, CDCI ) d : 7.32-7.25 (m, 5H), 4.54 (s, 2H), 3.66-3.56 (m, 8H), 3.41 (s, 2H), 1.40 (s, 8H), 1.10 (s, 6H).
Step (iii): -Butyl 3-(2-(2-hydroxyethoxy)ethoxy)-2,2-dimethylpropanoate (49) A suspension of -butyl 3-(2-(2-(benzyloxy)ethoxy)ethoxy)-2,2-dimethylpropanoate (48) (13 g , 37 mmol) and 10 % Pd/C (1.3g) in EtOH (92 mL) were placed under 5 bar of H pressure ted system), and stirred for 16h. The mixture was filtered h celite and the filtrate concentrated in vacuo to afford -butyl 3-(2-(2-hydroxyethoxy)ethoxy)-2,2-dimethylpropanoate (49) (8.2 g , 85 %) as a less oil: m/z 285 (M+Na)+ (ES+) . H NMR (400 MHz, CDCI ) d : 3.73-3.69 (m, 2H), 3.66-3.58 (m, 6H), 3.44 (s, 2H), 2.37 (t, J = 6.2 Hz, 1H), 1.43 (s, 9H), 1.14 (s, 6H).
Step (iv): -Butyl 3-(2-(2-((bis(benzyloxy)phosphoryl)oxy)ethoxy)ethoxy)-2,2- dimethylpropanoate (50) A mixture of te -butyl 3-(2-(2-hydroxyethoxy)ethoxy)-2,2-dimethylpropanoate (49) (6.87 g , 26.2 mmol), 5-methyl-1/-/-tetrazole (4.40 g , 52.4 mmol), and dibenzyl diethylphosphoramidite (12.47 g , 39.3 mmol) in THF (66 mL) was allowed to stir at RT for 1.5h. The solution was cooled to 0°C and m-CPBA (10.85 g , 47.1 mmol) was slowly added, the mixture was allowed to stir at RT for 16h. The reaction was diluted with DCM (200 mL) and washed with sat. NaHC0 (aq.) (4 x 100 mL), the organic layer was dried (MgS0 ) , filtered and concentrated in vacuo. The crude product was purified by silica gel chromatography (220 g , 0-40 % EtOAc in isohexane) to afford -butyl 3-(2-(2-((bis(benzyloxy)phosphoryl)oxy)ethoxy)ethoxy)-2,2-dimethylpropanoate (50) (12.2 g , 89 %) as a colourless oil: m/z 545 (M+Na)+ (ES+) . H NMR (400 MHz, CDCI ) d : 7.35- 7.31 (m, 10H), 5.09-5.00 (m, 4H), .09 (m, 2H), 3.65-3.63 (m, 2H), 3.59-3.52 (m, 4H), 3.39 (s, 2H), 1.41 (s, 9H), 1.1 1 (s, 6H).
Step (v): 3-(2-(2-((Bis(benzyloxy)phosphoryl)oxy)ethoxy)ethoxy)-2,2-dimethylpropanoic acid (51) TFA (6.63 ml_, 86 mmol) was added dropwise to a stirred solution of -butyl 3-(2-(2- ((bis(benzyloxy)phosphoryl)oxy)ethoxy)ethoxy)-2,2-dimethylpropanoate (50) (4.5 g , 8.61 mmol) in DCM (43 ml_) and the reaction mixture was d to stir at RT for 16h then concentrated in vacuo. The crude product was dissolved in EtOAc (150 ml_) and washed with 1M HCI (aq.) (50 ml_), water (2 x 50 ml_), and brine (50 ml_) the organic layer was dried (MgS0 ) , filtered and concentrated in vacuo to afford 3-(2-(2-((bis(benzyloxy)phosphoryl)oxy)ethoxy)ethoxy)-2,2- ylpropanoic acid (51) (3.98 g , 99 %) as a colourless oil: m/z 467 (M+H)+ (ES+) ; 465 (M- H) (ES ) . H NMR (400 MHz, CDCI ) d : 7.36-7.31 (m, 10H), 5.12-5.03 (m, 4H), 4.14-4.09 (m, 2H), 3.69-3.64 (m, 2H), 3.60 (s, 4H), 3.47 (s, 2H), 1.20 (s, 6H).
Step (vi): 2-(Dimethylcarbamoyl)(ethoxycarbonyl)(4-methoxyphenyl)-1/-/-pyrrole-3,4-diyl bis(3-(2-(2-((bis(benzyloxy)phosphoryl)oxy)ethoxy)ethoxy)-2,2-dimethylpropanoate) (52) DMF (0.2 m I_ , 2.14 mhioI) was added to a d solution of 3-(2-(2- benzyloxy)phosphoryl)oxy)ethoxy)ethoxy)-2,2-dimethylpropanoic acid (51) (100 mg, 0.21 mmol), followed by a solution of (COCI) (19 m I_ , 0.22 mmol) in DCM (860 m I_), the reaction mixture was allowed to stir for 45 min. To this mixture was added ethyl 5-(dimethylcarbamoyl)- hydroxy(4-methoxyphenyl)-1/-/-pyrrolecarboxylate (UL1-012) (30 mg, 0.09 mmol), the reaction mixture was cooled to 0°C and Et N (60 m I_ , 0.43 mmol) was added, the resulting mixture was d to stir for 45 min. The volatiles were concentrated in vacuo, the crude was suspended in Et 0 (5 ml_) ed, concentrated in vacuo and the residue purified by silica gel chromatography (4 g , 0-2 % MeOH in DCM) to afford a yellow oil. The compound was further purified by preparative HPLC (C-18 column, 19 mm i.d. x 50 mm, 5 micron particle size, gradient 5-95 % MeCN in 0.1 % aq. formic acid over 16 min) to afford 2-(dimethylcarbamoyl) (ethoxycarbonyl)(4-methoxyphenyl)-1/-/-pyrrole-3,4-diyl bis(3-(2-(2- ((bis(benzyloxy)phosphoryl)oxy)ethoxy)ethoxy)-2,2-dimethylpropanoate) (52) ( 11 mg, 10 %) as a colourless oil: H NMR (400 MHz, CDCI ) d 7.40-7.30 (m, 20H), 7.25-710 (m, 2H), 3.87-6.85 (m, 2H), 5.09-4.99 (m, 8H), 4.14-4.05 (m, 6H), 3.81 (s, 3H), 3.65-3.48 (m, 16H), 2.80 (s, 3H), 2.75 (s, 3H), 1.32 (s, 6H), 1.23 (s, 6H). 1.06 (t, J =7.2 Hz, 3H).
Step (vii): 2-(Dimethylcarbamoyl)(ethoxycarbonyl)(4-methoxyphenyl)-1/-/-pyrrole-3,4-diyl bis(3-(2-(2-((bis(benzyloxy)phosphoryl)oxy)ethoxy)ethoxy)-2,2-dimethylpropanoate) (UL1 -1 1 ) A solution of methyl 2-(dimethylcarbamoyl)(ethoxycarbonyl)(4-methoxyphenyl)-1/-/-pyrrole- 3,4-diyl bis(3-(2-(2-((bis(benzyloxy)phosphoryl)oxy)ethoxy)ethoxy)-2,2-dimethylpropanoate (52) (10 mg, 8.03 mhioI) in MeOH (4 ml_) was passed through a Thales Ή -cube' cartridge (10 % Pd/C) at a flow rate of 1 mL/min at 25°C under H (10 bar). The output was concentrated in vacuo to afford 2-(dimethylcarbamoyl)(ethoxycarbonyl)(4-methoxyphenyl)-1/-/-pyrrole-3,4- diyl bis(3-(2-(2-((bis(benzyloxy)phosphoryl)oxy)ethoxy)ethoxy)-2,2-dimethylpropanoate) (UL1 - 117) (5 mg, 70 %) as a white solid: m/z 885 (M+H)+ (ES+) ; 883 (M-H) (ES ) . H NMR (CDCI ) d : 7.25-7.15 (m, 2H), 6.91-6.86 (m, 2H), 4.20-4.02 (m, 6H), 3.82 (s, 3H), 3.75-3.50 (m, 16H), 2.83 (s, 3H), 2.77 (s, 3H), 2.69 (br s , 4H), 1.35 (s, 6H), 1.28 (s, 6H), 1.08 (t, J = 7.1 Hz, 3H).
Example G1: 2-(Dimethylcarbamoyl)(ethoxycarbonyl)(4-methoxyphenyl)-1/-/-pyrrole-3,4- diyl bis(2,2-dimethyl(3-(phosphonooxy)propoxy)propanoate) (UL1-1 18) UL1-1 18 Step (i): -Butyl 3-(3-((bis(benzyloxy)phosphoryl)oxy)propoxy)-2,2-dimethylpropanoate (54) DIAD ( 1 .36 mL, 6.97 mmol) was added dropwise to a stirred solution of PPh (76 mg, 0.30 mmol), dibenzyl phosphite ( 1 .94 g , 6.97 mmol) and -butyl 3-(3-hydroxypropoxy)-2,2- dimethylpropanoate (53) ( 1 .08 g , 4.65 mmol) [prepared using the same procedure as Example F 1 steps (ii)-(iii) using 3-(benzyloxy)propyl 4-methylbenzenesulfonate in step (77)] in THF (100 mL) at 0°C. The reaction was allowed to warm to RT and stirred for 16h, and the les were removed in vacuo. The crude t was purified by silica gel chromatography (120 g , 0-60 % EtOAc in isohexane) to afford -butyl 3-(3-((bis(benzyloxy)phosphoryl)oxy)propoxy)-2,2- ylpropanoate (54)(1 .76 g , 54 %) as a clear colourless oil: m/z 515 (M+Na)+ (ES+) . H NMR (400 MHz, DMSO-d ) d : 7.40-7.35 (m, 10H), 5.07-4.96 (m, 4H), 4.04-3.92 (m, 2H), 3.39 (t, J = 6.2 Hz, 2H), 3.28 (s, 2H), 1.77 (quin, J = 6.2 Hz, 2H), 1.35 (s, 9H), 1.02 (s, 6H).
Step (ii): 3-(3-((Bis(benzyloxy)phosphoryl)oxy)propoxy)-2,2-dimethylpropanoic acid (55) To a stirred solution of -butyl 3-(3-((bis(benzyloxy)phosphoryl)oxy)propoxy)-2,2- dimethylpropanoate (54) (1.76 g , 3.57 mmol) in DCM (30 mL) was added TFA (1.38 mL, 17.9 mmol), and the reaction was allowed to stir at RT for 4h. The volatiles were d in vacuo and the crude product was purified by silica gel chromatography (40 g , 0-30 % EtOAc in isohexane (+1 % AcOH)) to afford 3-(3-((bis(benzyloxy)phosphoryl)oxy)propoxy)-2,2- dimethylpropanoic acid (55) (1.03 g , 58 %) as a colourless oil: m/z 437 (M+H)+ (ES+) ; 435 (M- H) (ES ) . H NMR (400 MHz, DMSO-d ) d : 7.49-7.26 (m, 10H), 5.08-4.95 (m, 4H), .96 (m, 2H), 3.42-3.35 (m, 2H), 3.31 (s, 2H), 1.77 (quin, J = 6.2 Hz, 2H), 1.05 (s, 6H).
Step (iii): 2-(Dimethylcarbamoyl)(ethoxycarbonyl)(4-methoxyphenyl)-1/-/-pyrrole-3,4-diyl bis(3-(3-((bis(benzyloxy)phosphoryl)oxy)propoxy)-2,2-dimethylpropanoate) (56) 2 (28 m I_ , 0.32 mmol) was added dropwise to a stirred solution of 3-(3- ((bis(benzyloxy)phosphoryl)oxy)propoxy)-2,2-dimethylpropanoic acid (55) (0.14 g , 0.32 mmol) at 0°C, followed by a solution DMF (0.25 m I_ , 3.2 mhioI) in DCM (0.1 ml_), the reaction was d to warm to RT and left to stir for 2.5h. The reaction was cooled to 0°C and a further n of (COCI)2 (28 m I_ , 0.32 mmol) and a solution DMF (0.25 m I_ , 3.2 mhioI) in DCM (0.1 ml_) was added and the reaction allowed to warm to RT over 4h. The reaction was cooled to 0°C and ethyl ethylcarbamoyl)-3,4-dihydroxy(4-methoxyphenyl)-1/-/-pyrrolecarboxylate (UL1- 012) (55 mg, 0.16 mmol) was added, followed by Et3N (133 m I_ , 0.96 mmol). The reaction was d to warm to RT and d for 16h. The volatiles were concentrated in vacuo, the crude was suspended in EtOAc (5 ml_) filtered, concentrated in vacuo and the crude product was purified by preparative HPLC (C-18 column, 19 mm i.d. x 50 mm, 5 micron particle size, gradient 60-90 % MeCN in water + 0.1 % aq. formic acid over 16 min) to afford 2- (dimethylcarbamoyl)(ethoxycarbonyl)(4-methoxyphenyl)-1/-/-pyrrole-3,4-diyl bis(3-(3- ((bis(benzyloxy)phosphoryl)oxy)propoxy)-2,2-dimethylpropanoate) (56) (42 mg, 11 %) as a pale yellow gum: m/z: no ionisation observed. H NMR (400 MHz, DMSO-d ) d : 7.37-7.32 (m, 2H), 7.18-7.1 1 (m, 2H), 6.95-6.89 (m, 2H), 5.06-4.95 (m, 8H), 4.05-3.95 (m, 6H) 3.78 (s, 3H), 3 . 45- 3.36 (m, 8H), 2.76 (s, 3H), 2.66 (s, 3H), 1.80-1.75 (m, 4H), 1.23 (s, 6H), 1.13 (s, 6H), 0.98 (t, J = 7.1 Hz, 3H).
Step (iv): 2-(Dimethylcarbamoyl)(ethoxycarbonyl)(4-methoxyphenyl)-1/-/-pyrrole-3,4-diyl bis(2,2-dimethyl(3-(phosphonooxy)propoxy)propanoate) (UL1 -118) A solution of 2-(dimethylcarbamoyl)(ethoxycarbonyl)(4-methoxyphenyl)-1/-/-pyrrole-3,4-diyl bis(3-(3-((bis(benzyloxy)phosphoryl)oxy)propoxy)-2,2-dimethylpropanoate) (56) (37 mg, 0.03 mmol) in MeOH (4 ml_) was passed through a Thales Ή -cube' cartridge (10 % Pd/C) at a flow rate of 1 mL/min at 40°C under H2 (full H2 mode). The output was concentrated in vacuo to afford 2-(dimethylcarbamoyl)(ethoxycarbonyl)(4-methoxyphenyl)-1/-/-pyrrole-3,4-diyl bis(2,2-dimethyl(3-(phosphonooxy)propoxy)propanoate) (UL1-118) (25 mg, 95 %) as a white gum: m/z 825 (M+H)+ (ES+) ; 823 (M-H) (ES ) . H NMR (400 MHz, DMSO-d ) d : 7.24-7.16 (m, 2H), 6.98-6.91 (m, 2H), 4.01 (q, J = 7.1 Hz, 2H), 3.90-3.81 (m, 4H), 3.79 (s, 3H), 3.51-3.43 (m, 8H), 2.82 (s, 3H), 2.70 (s, 3H), 1.86-1 .73 (m, 4H), 1.25 (s, 6H), 1.19 (s, 6H), 1.01 (t, J = 7.1 Hz, 3H).
Example H1: 2-(Dimethylcarbamoyl)(ethoxycarbonyl)(4-methoxyphenyl)-1/-/-pyrrole-3,4- diyl bis(25,25-dimethyl-2,5,8, 11,14 ,17,20,23-octaoxahexacosanoate) (UL1 -119) UL1 -119 Step (i): r -Butyl 25,25-dimethyl-2,5,8,1 1,14,17,20,23-octaoxahexacosanoate (58) Sodium hydride (60 % wt in oil) (400 mg, 10 mmol) was added to a d solution of -butyl 3-hydroxy-2,2-dimethylpropanoate (57) (1.74 g , 10 mmol) in DMF (20 mL) at 0°C, after 30 min a solution of 2,5,8,1 1,14,17,20-heptaoxadocosanyl methanesulfonate (2.79 g , 6.67 mmol) in DMF (10 mL) was added dropwise, and the reaction mixture was allowed to warm to RT and d for 16h. The reaction was quenched with water (100 mL) and the aqueous layer was extracted with DCM (2 x 100 mL), the combined organic layers were washed with brine (3 x 100 mL), dried (MgS0 ) , filtered and concentrated in vacuo. The crude product was purified by silica gel chromatography (120 g , 0-10 % MeOH in EtOAc) to afford te -butyl 25,25-dimethyl- 2,5,8,1 1,14,17,20,23-octaoxahexacosanoate (58) ( 1 .59 g , 48 %) as a colourless oil: H NMR (400 MHz, CDCI ) d : 3.65-3.51 (m, 32H), 3.40 (s, 2H), 3.36 (s, 3H), 1.41 (s, 9H), 1.1 1 (s, 6H).
Step (ii): 25,25-Dimethyl-2,5,8,1 1,14,17,20,23-octaoxahexacosanoic acid (59) TFA (1.5 mL, 19.3 mmol) was added dropwise to a d solution of -butyl 25,25-dimethyl- 2,5,8,1 1,1 4,1 7,20,23-octaoxahexacosanoate (58) (0.48 g , 0.97 mmol) in DCM (5 mL), and the reaction was stirred at RT for 4h. The volatiles were d in vacuo to afford 25,25- dimethyl-2,5,8,1 7,20,23-octaoxahexacosanoic acid (59) (0.49 g , 95 %) as a colourless oil: H NMR (400 MHz, CDCI ) d : 3.68-3.60 (m, 26H), .52 (m, 2H), 3.47 (s, 2H), 3.36 (s, 3H), 1.18 (s, 6H).
Step (iii): 2-(Dimethylcarbamoyl)(ethoxycarbonyl)(4-methoxyphenyl)-1/-/-pyrrole-3,4-diyl bis(25,25-dimethyl-2,5,8, 11,14 ,17,20,23-octaoxahexacosanoate) (UL1 -119) Thionyl chloride (43 mg, 0.36 mmol) was added to a stirred solution of 25,25-dimethyl- 2,5,8,1 1,14,17,20,23-octaoxahexacosanoic acid (59) (140 mg, 0.32 mmol) in DCM (2 mL) and the reaction was stirred at RT for 2h. The volatiles were removed in vacuo, the residue dissolved in DCM (2 mL) and added to a stirred solution of ethyl 5-(dimethylcarbamoyl)-3,4- dihydroxy(4-methoxyphenyl)-1/-/-pyrrolecarboxylate (UL1-012) (50 mg, 0.14 mmol) and DIPEA (46 mg, 0.36 mmol) in DCM (3 mL) at 0°C. The reaction mixture was d to warm to RT and stirred for 16h. The volatiles were concentrated in vacuo and the resulting residue was suspended in EtOAc (10 mL), filtered and the te concentrated in vacuo. The crude residue was purified by silica gel chromatography (12 g , 0-3 % MeOH in DCM) to give a crude oil. The product was further purified by preparative HPLC (Waters, Acidic (0.1 % Formic acid), Waters X-Select Prep-C18, 5 m h , 19x50 mm column, 25-70 % MeCN in water) to afford 2- (dimethylcarbamoyl)(ethoxycarbonyl)(4-methoxyphenyl)-1/-/-pyrrole-3,4-diyl bis(25,25- dimethyl-2,5,8,1 1,14,17,20,23-octaoxahexacosanoate) (UL1-1 19) (52 mg, 30 %) as a colourless oil: m/z 1193 (M+H)+ (ES+) . H NMR (400 MHz, DMSO-d ) d : 7.21-7.16 (m, 2H), 6.96- 6.92 (m, 2H), 4.02 (q, J = 7.1 Hz, 2H), 3.79 (s, 3H), 3.58-3.46 (m, 56H), 3.43-3.40 (m, 4H), 3.23 (s, 6H), 2.82 (s, 3H), 2.70 (s, 3H), 1.25 (s, 6H), 1.19 (s, 6H), 1.02 (t, J = 7.1 Hz, 3H).
Example 11 : Ethyl 5-(dimethylcarbamoyl)hydroxy(4-methoxyphenyl) ((phosphonooxy)methoxy)-1 /-/-pyrrolecarboxylate (UL1 -1 15) UL1 -115 Step (i): Ethyl 4-(benzyloxy)(dimethylcarbamoyl)hydroxy(4-methoxyphenyl)-1/-/-pyrrole- 2-carboxylate (60) HBr (33 % in AcOH, 970 m , 5.34 mmol) was added to a stirred on of ethyl 3,4- bis(benzyloxy)(dimethylcarbamoyl)(4-methoxyphenyl)-1 H-pyrrolecarboxylate (6) (2.82 g , 5.34 mmol) in AcOH (40 mL) and the reaction was d to stir for 16h at RT. The reaction mixture was diluted with DCM (100 mL), washed with water (100 mL), brine (2 x 100 mL), the organic layer was dried (MgS0 ) , filtered and concentrated in vacuo. The residue was redissolved in AcOH (40 ml_) and HBr (33 % in AcOH, 970 m I_ , 5.34 mmol) was added dropwise and the reaction was allowed to stir at RT for 40h. The reaction mixture was diluted with DCM (100 ml_), washed with water (100 ml_) and brine (2 x 100 ml_). The organic layer was dried (MgS0 4) , filtered and concentrated in vacuo. The crude product was purified by silica gel tography (40 g , 0-30 % EtOAc in toluene) to afford ethyl zyloxy) (dimethylcarbamoyl)hydroxy(4-methoxyphenyl)-1/-/-pyrrolecarboxylate (60) (1.48 g , 57 %) as a colourless oil: m/z 439 (M+H)+ (ES+) ; 437 (M-H) (ES ) . H NMR (400 MHz, DMSO-d ) d : 8.75 (s, 1H), 7.42-7.28 (m, 5H), 7.12-7.05 (m, 2H), 6.91-6.84 (m, 2H), 5.01 (s, 2H), 4.02 (q, J = 7.1 Hz, 2H), 3.76 (s, 3H), 2.68 (s, 3H), 2.66 (s, 3H), 1.0 (t, J = 7.1 Hz, 3H).
Step (ii): Ethyl 4-(benzyloxy)(((bis(benzyloxy)phosphoryl)oxy)methoxy) (dimethylcarbamoyl)(4-methoxyphenyl)-1/-/-pyrrolecarboxylate (61) A solution of dibenzyl (chloromethyl) phosphate (227 mg, 0.70 mmol) in DMF ( 1 ml_) was added dropwise to a stirred suspension of ethyl 4-(benzyloxy)(dimethylcarbamoyl)hydroxy(4- methoxyphenyl)-1/-/-pyrrolecarboxylate (60) (203 mg, 0.46 mmol) and K2C0 3 (128 mg, 0.93 mmol) in DMF (4 ml_) at 0°C, the reaction was stirred for 1h, then allowed to warm to RT over 16h. The reaction mixture was poured into water (50 ml_), extracted with Et20 (2 x 50 ml_). The combined organic layers were washed with brine (3 x 100 ml_), dried (MgS0 ) , filtered and concentrated in vacuo. The crude t was purified by silica gel chromatography (40 g , 0-30 % EtOAc in toluene) to afford ethyl 4-(benzyloxy)(((bis(benzyloxy)phosphoryl)oxy)methoxy)- -(dimethylcarbamoyl)(4-methoxyphenyl)-1/-/-pyrrolecarboxylate (61) (178 mg, 5 1 %) as a clear colourless oil: m/z 729 (M+H)+ (ES+) . H NMR (400 MHz, DMSO-d ) d : 7.38-7.27 (m, 15H), .03 (m, 2H), 6.92-6.86 (m, 2H), 5.49 (d, J= 11.1 Hz, 2H), 5.1 1 (s, 2H), 5.07-4.95 (m, 6H), 4.00 (q, J = 7.1 Hz, 2H), 3.78 (s, 3H), 2.69 (s, 3H), 2.68 (s, 3H), 0.99 (t, J = 7.0 Hz, 3H).
Step (iii): Ethyl 5-(dimethylcarbamoyl)hydroxy(4-methoxyphenyl) ((phosphonooxy)methoxy)-1 /-/-pyrrolecarboxylate (UL1 -1 15) A solution of ethyl 4-(benzyloxy)(((bis(benzyloxy)phosphoryl)oxy)methoxy) (dimethylcarbamoyl)(4-methoxyphenyl)-1/-/-pyrrolecarboxylate (61) (102 mg, 0.14 mmol) in MeOH (5 ml_) was passed h a Thales Ή -cube' cartridge (10 % Pd/C) at a flow rate of 1 mL/min at 45°C under H2 (full H2 mode). The output was concentrated in vacuo to afford ethyl 5- (dimethylcarbamoyl)hydroxy(4-methoxyphenyl)((phosphonooxy)methoxy)-1/-/-pyrrole ylate (UL1 -1 15) (61 mg, 93 %) as a white solid: m/z 459 (M+H)+ (ES+) ; 457 (M-H) (ES ) , H NMR (400 MHz, DMSO-d ) d : 7.1 1-7.05 (m, 2H), 6.91-6.84 (m, 2H), 5.45 (d, J = 16 Hz, 2H), 3.98 (q, J = 7.1 Hz, 2H), 3.77 (s, 3H), 2.98 (br s , 3H) 2.74 (br s , 3H), 1.01 (t, J = 7.0 Hz, 3H).
Example J1: 2,5-Bis(dimethylcarbamoyl)(4-methoxyphenyl)-1/-/-pyrrole-3,4-diyl bis(2,2- dimethyl(2-(2-(phosphonooxy)ethoxy)ethoxy)propanoate) (UL1-121) Step (i): 2,5-Bis(dimethylcarbamoyl)(4-methoxyphenyl)-1/-/-pyrrole-3,4-diyl bis(3-(2-(2- ((bis(benzyloxy)phosphoryl)oxy)ethoxy)ethoxy)-2,2-dimethylpropanoate) (62) DIC (160 mg, 1.27 mmol) was added to a solution of 3,4-dihydroxy(4-methoxyphenyl)- , , , methyl-1H-pyrrole-2,5-dicarboxamide 05) (200 mg, 0.58 mmol), 3-(2-(2- ((bis(benzyloxy)phosphoryl)oxy)ethoxy)ethoxy)-2,2-dimethylpropanoic acid (51) (591 mg, 1.27 mmol), and DMAP (28 mg, 0.23 mmol) in EtOAc (4 mL) and the reaction mixture was allowed to stir at RT for 96h. The mixture was diluted with EtOAc (150 mL) washed with 1M HCI (aq.) (50 mL), water (2 x 50 mL), and brine (50 mL), dried (MgS0 ) , filtered and concentrated in vacuo.
The crude e was purified by preparative HPLC (Waters, Acidic (0.1 % Formic acid), Waters X-Select Prep-C18, 5 m h , 19x50 mm column, 50-95 % MeCN in water) to afford 2,5- bis(dimethylcarbamoyl)(4-methoxyphenyl)-1/-/-pyrrole-3,4-diyl bis(3-(2-(2- ((bis(benzyloxy)phosphoryl)oxy)ethoxy)ethoxy)-2,2-dimethylpropanoate) (62) (228 mg, 32 %) as a yellow solid: m/z not observed. H NMR (400 MHz, DMSO-d ) d : .31 (m, 20H), 7.09- 7.07 (m, 2H), 6.95-6.93 (m, 2H), 5.02 (d, J = 7.6 Hz, 8H), 4.08-4.04 (m, 4H), 3.77 (s, 3H), 3.58- 3.56 (m, 4H), 3.56-3.47 (m, 8H), 3.44 (s, 4H), 2.72 (s, 6H), 1.13 (s, 12H).
Step (ii) 2,5-Bis(dimethylcarbamoyl)(4-methoxyphenyl)-1/-/-pyrrole-3,4-diyl bis(2,2-dimethyl (2-(2-(phosphonooxy)ethoxy)ethoxy)propanoate) (UL1-121) A solution of 2,5-bis(dimethylcarbamoyl)(4-methoxyphenyl)-1/-/-pyrrole-3,4-diyl bis(3-(2-(2- ((bis(benzyloxy)phosphoryl)oxy)ethoxy)ethoxy)-2,2-dimethylpropanoate) (62) (278 mg, 0.22 mmol) in MeOH ( 1 12 mL) was passed through a Thales Ή -cube' cartridge (10 % Pd/C) at a flow rate of 1 mL/min at 45°C under H (10 bar). The output was concentrated in vacuo to afford 2,5- bis(dimethylcarbamoyl)(4-methoxyphenyl)-1/-/-pyrrole-3,4-diyl bis(2,2-dimethyl(2-(2- (phosphonooxy)ethoxy)ethoxy)propanoate) (UL1-121) (175 mg, 88 %) as a light yellow oil: m/z 884 (M+H)+ (ES+) ; 882 (M-H) (ES ) . H NMR (400 MHz, DMSO-d ) d : 7.1 1-7.09 (m, 2H), 6.97- 6.95 (m, 2H), 3.91-3.87 (m, 4H), 3.78 (s, 3H), 3.58-3.50 (m, 16H), 2.89 (s, 6H), 2.76 (s, 6H), 1.19 (s, 12H). e K 1 : 2,5-Bis(dimethylcarbamoyl)(4-methoxyphenyl)-1/-/-pyrrole-3,4-diyl bis(piperidinecarboxylate) (UL1-123) UL1-005 R=Boc, 63 Step (i): 0 ,0 -(2,5-Bis(dimethylcarbamoyl)(4-methoxyphenyl)-1/-/-pyrrole-3,4-diyl) 1-di- - butyl bis(piperidine-1,4-dicarboxylate) (63) DIC (633 m I_ , 4.10 mmol) was added to a solution of 3,4-dihydroxy(4-methoxyphenyl)- , , , -tetramethyl-1H-pyrrole-2,5-dicarboxamide (UL1-005) (284 mg, 0.82 mmol), 1-( e - carbonyl)piperidinecarboxylic acid (525 mg, 2.29 mmol) and DMAP (40 mg, 0.33 mmol) in EtOAc (5 mL) the reaction mixture was allowed to stir at RT for 2h. The reaction mixture was washed with 1M HCI (aq.) (20 mL), dried (MgS0 ) , filtered and concentrated in vacuo. The crude residue was purified by silica gel chromatography (40 g , 0-3 % MeOH in DCM) to afford 0 ' ,0 bis(dimethylcarbamoyl)(4-methoxyphenyl)-1/-/-pyrrole-3,4-diyl) 1- di-te/f-butyl bis(piperidine-1,4-dicarboxylate) (63) (565 mg, 76 %) as a colourless oil: 792 (M+Na)+ (ES+) . H NMR (400 MHz, DMSO-d ) d : 7.12-7.08 (m, 2H), 6.98-6.93 (m, 2H), 3.88- 3.81 (br m , 4H), 3.77 (s, 3H), 2.96-2.73 (m, 18H), 1.89-1 .81 (br m , 4 H), 1.50-1.37 (m, 22H).
Step (ii): 4,4'-(((2,5-Bis(dimethylcarbamoyl)(4-methoxyphenyl)-1/-/-pyrrole-3,4- diyl)bis(oxy))bis(carbonyl))bis(piperidinium) 2,2,2-trifluoroacetate (UL1-123) TFA ( 1 .12 mL, 14.7 mmol) was added dropwise to a solution of 0 ,0 -(2,5- bis(dimethylcarbamoyl)(4-methoxyphenyl)-1/-/-pyrrole-3,4-diyl) e/f-butyl bis(piperidine- 1,4-dicarboxylate) (565 mg, 0.73 mmol) in DCM (20 mL) and the mixture was allowed to stir at RT for 2h. The reaction was concentrated in vacuo to afford 4,4'-(((2,5-bis(dimethylcarbamoyl)- 1-(4-methoxyphenyl)-1/-/-pyrrole-3,4-diyl)bis(oxy))bis(carbonyl))bis(piperidinium) 2,2,2- trifluoroacetate (UL1-123) (360 mg, 58 %) as a white solid: m/z 570 (M+H)+ (ES+) . H NMR (400 MHz, DMSO-de) d : 8.71-8.61 (br m , 2H), 8.49-8.36 (br m , 2H), 7.13-7.08 (m, 2H), 7.00-6.95 (m, 2H), 3.78 (s, 3H), .25 (br m , 4H), 3.05-2.92 (m, 6H), 2.79 (br s , 6H), 2.75 (br s , 6H), 2.08- 2.00 (br m , 4H), 1.82-1 .69 (m, 4H). e L 1 : 2,5-Bis(dimethylcarbamoyl)(4-methoxyphenyl)-1/-/-pyrrole-3,4-diyl bis(4- ((phosphonooxy)methyl)benzoate) (UL1-124) Step (i): 2,5-Bis(dimethylcarbamoyl)(4-methoxyphenyl)-1/-/-pyrrole-3,4-diyl (((di-te/fbutoxyphosphoryl ethyl)benzoate) (64) DIC (633 m I, 4.10 mmol) was added to a solution of 3,4-dihydroxy(4-methoxyphenyl)- , , , -tetramethyl-1H-pyrrole-2,5-dicarboxamide (UL1-005) (252 mg, 0.73 mmol), 4-(((dite /f-butoxyphosphoryl)oxy)methyl)benzoic acid (500 mg, 1.45 mmol) and DMAP (71 .0 mg, 0.58 mmol) in EtOAc (8 mL) the reaction mixture was allowed to stir at RT for 16h. The mixture was quenched with water (20 mL) and extracted with EtOAc (3 x 20 mL), the combined organics were washed with brine (100 mL), dried (MgS0 4) , filtered and concentrated in vacuo. The crude residue was purified by silica gel chromatography (12 g , 0-5 % MeOH in EtOAc) to afford 2,5- bis(dimethylcarbamoyl)(4-methoxyphenyl)-1/-/-pyrrole-3,4-diyl bis(4-(((di-tertbutoxyphosphoryl )oxy)methyl)benzoate) (64) (91 mg, 6 %) as a yellow solid: m/z 1001 (M+H)+ (ES+) . H NMR (400 MHz, DMSO-d ) d : 8.01-7.99 (m, 4H), 7.55-7.52 (m, 4H), 7.21-7.18 (m, 2H), 7.01-6.99 (m, 2H), 5.00 (s, 4H), 3.80 (s, 3H), 2.89 (s, 6H), 2.74 (s, 6H), 1.38 (s, 36H).
Step (ii) 2,5-Bis(dimethylcarbamoyl)(4-methoxyphenyl)-1/-/-pyrrole-3,4-diyl bis(4- phonooxy)methyl)benzoate) (UL1-124) TFA (16 mL, 208 mmol) was added dropwise to a solution of 2,5-bis(dimethylcarbamoyl)(4- methoxyphenyl)-1 /-/-pyrrole-3,4-diyl bis(4-(((di-fe/f-butoxyphosphoryl)oxy)methyl)benzoate) (64) (88 mg, 0.09 mmol) in DCM (64 mL) the mixture was left to stand for 72h, and then concentrated in vacuo to afford 2,5-bis(dimethylcarbamoyl)(4-methoxyphenyl)-1/-/-pyrrole- yl bis(4-((phosphonooxy)methyl)benzoate) (UL1-124) as a yellow oil: m/z 776 (M+H)+ (ES+) ; 774 (M-H) (ES ) . H NMR (400 MHz, DMSO-d ) d : 8.00-7.98 (m, 4H), 7.54-7.52 (m, 4H), .18 (m, 2H), 7.00-6.98 (m, 2H), 4.98-4.96 (d, 4H), 3.79 (s, 3H), 2.89 (s, 6H), 2.74 (s, 6H).
Example M1: 5-(Dimethylphosphoryl)-3,4-dihydroxy(4-methoxyphenyl)-/V,/\/-dimethyl-1/-/- pyrrolecarboxamide (UL1-125) Step (i): 3,4-Bis(benzyloxy)(dimethylphosphoi7l)(4-methoxyphenyl)-A/,A/-dimeth pyrrolecarboxamide (66) Isopropylmagnesium chloride lithium chloride x ( 1 .3 M in THF, 315 m I_ , 0.410 mmol) was added dropwise to a stirred solution of 3,4-bis(benzyloxy)iodo(4-methoxyphenyl)-/V,/\/- dimethyl-1/-/-pyrrolecarboxamide (65) [prepared using the same procedure as Example S step (i) using (9) as starting material] (21 1 mg, 0.36 mmol) in THF (1.2 mL) at 0°C, the reaction mixture was allowed to stir for 15 min and isopropylmagnesium de lithium chloride complex ( 1 .3 M in THF, 100 m I_ , 0.13 mmol) was added, and the reaction mixture was allowed to stir for a further 15 min. A solution of dimethylphosphinic chloride (61 mg, 0.54 mmol) in THF (0.6 mL) was added and the reaction allowed to warm to RT and stirred for a further 16h. The reaction mixture was quenched with 5 % AcOH in MeOH (5 mL) and the volatiles removed in vacuo. The residue was purified by silica gel chromatography (12 g , 0-5 % MeOH in DCM) to afford a crude residue. The crude residue was r purified by preparative HPLC s, Acidic (0.1 % Formic acid), Waters X-Select Prep-C18, 5 mhi , 19x50 mm column, 30-60 % MeCN in water) to afford s(benzyloxy)(dimethylphosphoryl)(4-methoxyphenyl)-/V,/\/- dimethyl-1/-/-pyrrolecarboxamide (66) (29 mg, 15 %) as a colourless gum: m/z 533 (M+H)+ (ES+) . H NMR (400 MHz, CDCI ) d : 7.44-7.28 (m, 12H), .87 (m, 2H), 5.22 (s, 2H), 5.05 (s, 2H), 3.82 (s, 3H), 2.76 (s, 3H), 2.63 (s, 3H), 1.41 (br d , J =13 Hz, 6H).
Step (ii): 5-(Dimethylphosphoryl)-3,4-dihydroxy(4-methoxyphenyl)-/V,/\/-dimethyl-1/-/-pyrrole carboxamide (UL1-125) A solution of 3,4-bis(benzyloxy)(dimethylphosphoryl)(4-methoxyphenyl)-/V,/\/-dimethyl-1/-/- pyrrolecarboxamide (66) (29 mg, 0.054 mmol) in MeOH (5 mL) was passed through a Thales Ή cartridge (10 % Pd/C) at a flow rate of 1 mL/min at 25°C under H (full H mode). The 2 2 output was concentrated in vacuo to afford 5-(dimethylphosphoryl)-3,4-dihydroxy(4- methoxyphenyl)-A/,A/-dimethyl-1H-pyrrolecarboxamide (UL1-125) (13 mg, 67 %) as a pale yellow solid: m/z 353 (M+H)+ (ES+) , 351 (M-H) (ES ) . H NMR (400 MHz, DMSO-d ) d : 9.35 (s, 1H), 8.55 (s, 1H), 7.23-7.14 (m, 2H), 6.96-6.88 (m, 2H), 3.77 (s, 3H), 2.86 (br s , 6H), 1.33 (s, 3H), 1.30 (s, 3H).
Example N1: ( 1 R,1'R,4R,4'R)-2,5-Bis(dimethylcarbamoyl)(4-methoxyphenyl)-1/-/-pyrrole-3,4- diyl bis(4-((phosphonooxy)methyl)cyclohexanecarboxylate) (UL1-126) Step (i): ( 1 R,4R)-Methyl 4-(((bis(benzyloxy)phosphoryl)oxy)methyl)cyclohexanecarboxylate (67) -methyl-1/-/-tetrazole (4.4 g , 52.3 mmol) was added to a stirred solution of ( 1 R,4R)-methyl 4- (hydroxymethyl)cyclohexanecarboxylate (4.5 g , 26.1 mmol) and dibenzyl diethylphosphoramidite ( 1 1.73 mL, 39.2 mmol) in THF (60 mL), after 2h the reaction mixture was cooled to 0°C and a solution of m-CPBA (10.5 g , 47 mmol) in THF (30 mL) was added dropwise. The reaction was allowed to warm to RT and stirred for 16h. The volatiles were removed in vacuo and the reaction was diluted with Et20 (200 mL) and washed with sat.
NaHC0 3 (aq.) (5 x 50 mL), brine (30 mL), dried (MgS0 4) filtered and the concentrated in vacuo.
The crude residue was purified by silica gel chromatography (80 g , 0-50 % Et20 in isohexane) to afford ( 1 R,4R)-methyl 4-(((bis(benzyloxy)phosphoryl)oxy)methyl)cyclohexanecarboxylate (67) (6.35 g , 53 %) as a less oil: m/z 433 (M+H)+ (ES+) . H NMR (400 MHz, DMSO-d ) d : 7.42- 7.32 (m, 10H), 5.02 (dd, J = 1.0, 5.4 Hz, 4H), 3.74 (t, J = 6.5 Hz, 2H), 3.58 (s, 3H), 2.20 (tt, J = 12.2, 3.5 Hz, 1H), 1.90-1 .83 (m, 2H), 1.70-1 .62 (m, 2H), 1.26 (ddd, J = 3.4, 13.1 , 16.6 Hz, 2H), 0.93 (ddd, J = 3.4, 13.1, 16.6 Hz, 2H).
Step (ii): ( 1 4-(((Bis(benzyloxy)phosphoryl)oxy)methyl)cyclohexanecarboxylic acid (68) NaOH (290 mg, 7.17 mmol) was added to a stirred solution of ( 1 R,4R)-methyl 4- (((bis(benzyloxy)phosphoryl)oxy)methyl)cyclohexanecarboxylate (67) (3.1 g , 7.17 mmol) in MeOH (50 mL) and water (50 mL) and the reaction was allowed to stir for 24h. The volatiles were removed in vacuo diluted with 1 M HCI (aq.) (20 mL) and extracted with DCM (2 x 100 mL), the ed organic layers were dried (MgS0 ) , filtered and trated in vacuo to afford ( 1 R,4R)(((bis(benzyloxy)phosphoryl)oxy)methyl)cyclohexanecarboxylic acid (68) (2.74 g , 88 %) as a white solid: m/z 419 (M+H) + (ES+) ; 417 (M-H) (ES ) . H NMR (400 MHz, DMSO- c e) d : 12.03 (s, 1H), 7.42-7.30 (m, 10H), 5.02 (dd, J = 8.4, 1.0 Hz, 4H), 3.74 (t, J = 6.5 Hz, 2H), 2.12-2.03 (tt, J = 12.3, 3.5 Hz, 1H), 1.91-1.81 (m, 2H), 1.71-1 .61 (m, 2H), 1.54-1.41 (m, 1H), 1.24 (ddd, J = 3.3, 13.1 , 16.3 Hz, 2H), 0.92 (ddd, J = 3.3, 13.1, 16.3 Hz, 2H).
Step (iii): ( 1 R,TR,4R,4'R)-2,5-Bis(dimethylcarb^^ bis(4-(((bis(benzyloxy)phosphoryl)oxy)methyl)cyclohexanecarboxylate) (69) PS-DCC (2.3 mmol/g, 1.25 g , 17.6 mmol) was added to a solution of 3,4-dihydroxy(4- methoxyphenyl)-A/ , , , -tetramethyl-1/-/-pyrrole-2,5-dicarboxarTiide (UL1-005) (200 mg, 0.58 mmol), DMAP (0.028 g , 0.230 mmol) and (1R,4R) (((bis(benzyloxy)phosphoryl)oxy)methyl)cyclohexanecarboxylic acid (68) (578 mg, 1.38 mmol) in THF (20 mL) and the reaction mixture was shaken at RT for 96h.
The reaction e was diluted with EtOAc (50 mL) and washed with 1 M HCI (aq.) (50 mL), sat. NaHC0 3 (aq.) (50 mL), brine (50 mL), dried (MgS0 4) , ed and concentrated in vacuo.
The product was purified by silica gel chromatography (12 g , 0-3 % MeOH in DCM) to afford ( 1 R,1'R,4R,4'R)-2,5-bis(dimethylcarbamoyl)(4-methoxyphenyl)-1/-/-pyrrole-3,4-diyl bis(4- (((bis(benzyloxy)phosphoryl)oxy)methyl)cyclohexanecarboxylate) (69) (325 mg, 49 %) as a yellow oil: m/z not ed. H NMR (400 MHz, DMSO-d ) d : 7.41-7.32 (m, 20H), 7.1 1-7.07 (m, 2H), 6.97-6.92 (m, 2H), 5.07-4.98 (m, 8H), 3.77 (s, 3H), 3.75 (d, J = 6.2 Hz, 4H), 2.83 (br s , 6H), 2.75 (br s , 6H), 2.44 (tt, J = 3.5, 12.2 Hz, 2H), 1.96-1 .87 (m, 4H), 1.75-1 .65 (m, 4H), 1.59-1.46 (m, 2H), 1.33 (ddd, J = 3.4, 13.1 , 16.2 Hz, 4H), 1.01 (ddd, J = 3.4, 13.1, 16.2 Hz, 4H).
Step (iv): ( 1 R,1'R,4R,4'R)-2,5-Bis(dimethylcarbamoyl)(4-methoxyphenyl)-1/-/-pyrrole-3,4-diyl bis(4-((phosphonooxy)methyl)cyclohexanecarboxylate) (UL1 -126) A solution of ( 1 R,TR,4R,4'R)-2,5-bis(dimethylcarbamoyl)(4-methoxyphenyl)-1/-/-pyrrole-3,4- diyl bis(4-(((bis(benzyloxy)phosphoryl)oxy)methyl)cyclohexanecarboxylate) (69) (323 mg, 0.28 mmol) in MeOH (8 mL) was passed through a Thales Ή -cube' cartridge (10 % Pd/C) at a flow rate of 1 mL/min at 50°C under H2 (full H2 mode). The output was concentrated in vacuo, and a solid was collected by filtration after trituration with EtOAc (5 mL) to afford ( 1 4R,4'R)-2,5- bis(dimethylcarbamoyl)(4-methoxyphenyl)-1/-/-pyrrole-3,4-diyl bis(4- ((phosphonooxy)methyl)cyclohexanecarboxylate) (UL1-126) (121 mg, 43 %) as a pale yellow solid: m/z 788 (M+H) + (ES+) ; 786 (M-H) (ES ) . H NMR (400 MHz, DMSO-d ) d : 10.90 (br s , 2H), 7.12-7.06 (m, 2H), 6.97-6.92 (m, 2H), 3.77 (s, 3H), 3.63 (t, J = 6.4 Hz, 4H), 2.83 (br s , 6H), 2.75 (br s , 6H), 2.50-2.45 (m, 2H), 2.00-1 .93 (m, 4H), 1.84-1 .76 (m, 4H), 1.63-1 .51 (m, 2H), 1.38 (ddd, 3.1 , 12.7, 16.0 Hz, 4H), 1.05 (ddd, 3.1, 12.7, 16.0 Hz, 4H). above: s outlined the general prepared using 1 were in Table following Examples 1 The Table 65522.26 I 85 new As Lin F. .A__.
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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 ce 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. r, if the compound is not inhibitory, the red blood cells are lysed and haemoglobin is released into the supernatant.
Experimental ure Test compound solutions (typically at 5 mM in DMSO) were diluted 1:1 in 100% DMSO. The compounds were then two-fold serially diluted in 100% DMSO across 11 wells of 96-well ottomed microtitre plate. PBS was then added to all the wells to achieve a 1:10 (v/v) dilution of the compound in PBS. Pneumolysin was then added at a concentration equal to its LD100.
Plates were then incubated at 37°C for 30-40 min. After the incubation period, an equal volume of 4% (v/v) sheep erythrocyte suspension was added to each well and the plates incubated again at 37°C, for at least 30 min. Controls with only erythrocytes in PBS (control for no lysis) or erythrocytes plus pneumolysin (control for lysis) were prepared following the same ure. Following the incubation with the erythrocytes, the Absorbance at 595 nm of each well was measured and the data used to ine the IC50 for each test compound. The IC50 values were determined using non-linear sion curve fitting. For that, the Log of the concentrations of the test nd was plotted against the percentage inhibition, estimated from the A595 values, followed by fitting a Hill Slope to the data.
Results IC50 values for compounds of the invention generated in this assay are shown in Table 1 as follows: +++ = IC50 < 10 mM; ++ = IC50 10 to <20 mM; + = IC50 20 to <60 mM.
Specific IC50 values for representative compounds of the invention are: 4: ICso 0.17 mM; UL1-012: IC50 0.15 mM; UL1-024: IC50 0.182 mM ; UL1-028: IC50 0.068 mM ; 9: IC50 0.479 mM ; 1: IC50 0.3 mM; UL1-005: IC50 0.15 mM; UL1-035: IC50 0.15 mM ; UL1-089: IC50 0.4 mM ; UL1-106: IC50 0.17 mM; UL1-1 16: IC50 0.18 mM.
B. PRIMARY IN VITRO ASSAY: INHIBITION OF THE HAEMOLYTIC ACTIVITY OF OTHER CHOLESTEROL DEPENDENT CYTOLYSINS Compounds UL1-012 and UL2-001 were tested for their ability to inhibit the haemolytic ty 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 . Inhibition of haemolysis of these toxins was obtained with IC as ted in the table below (Table 2).
Table 2 +++ = ICso < 10 mM; ++ = IC50 10 to < 20 mM; + = IC50 20 to < 60 mM C. SECONDARY IN VITRO ASSAY: INHIBITION OF LYSIN-INDUCED LACTATE OGENASE RELEASE Rationale Pneumolysin induces the release of lactate dehydrogenase (LDH) from human monocytes and lung epithelial cells: a phenomenon that is indicative of plasma membrane damage or rupture [Infect. Immun. (2002) 70 1017-1022]. The LDH assay was applied to demonstrate the ability of the disclosed compounds to inhibit the cytotoxic effect of pneumolysin on human lung epithelial cells in culture. The use of this assay can provide two main pieces of information on (1) Activity, to demonstrate the inhibition of LDH release from cells exposed to pneumolysin in the presence of inhibitory nds versus the LDH release from cells exposed to pneumolysin alone, (2) Compound toxicity, the assay format was designed so it allows, in the control wells, the testing of the LDH release from cells exposed to the nd only.
Experimental procedure Human lung epithelial cells (A549) were seeded in flat-bottomed 96-well tissue culture plates and grown in RPMI 1640 medium supplemented with ine, at 37°C, 5% C0 , for 24h.
Before use, the cells were washed with PBS. Test compound dilutions were incubated with pneumolysin as described in Section A , then transferred to wells containing the human lung lial cells and the plates were incubated at 37°C, 5% C0 , for 30 min. The following ls were included on the plate (1) Negative controls, called low control (PBS only) to measure the natural release of LDH from the cells in culture, (2) positive controls (1% (v/v) Triton-X in PBS) to measure the m release of LDH from the cells (3) Pneumolysin solution only to measure pneumolysin-induced LDH release, (4) Test compound solution to assess the toxicity of the compound alone. After incubation, the supernatant was transferred to the wells of round-bottomed 96-well microtitre plates containing a double volume of lactate dehydrogenase assay e (TOX7, Sigma) prepared ing to manufacturer's instructions. Incubation in a light-proof chamber at RT for 5-10 min was followed by the addition of 1N HCI to all wells. Absorbance at 490 nm and 655 nm was then measured. The percentage of LDH release induced by pneumolysin in the presence and absence of test compounds was plotted against the Log of the concentration of the nd and the IC was determined, as described above in the inhibition of haemolysis assay, Section A .
Results UL1-012 was tested in the LDH assay in triplicate over a range of concentration from 62.5 mM to 0.49 mM . The results obtained are shown in Figure 1 .
In Figure 1 : (1) The horizontal dotted line at 100%, PLY l (o), indicates the maximum release of LDH from the cells under the effect of pneumolysin, as opposed to the horizontal solid line at 0% (low control), which corresponds to the supernatant of cells exposed to the assay buffer alone that shows the natural LDH release under the assay conditions. (2) The grey solid line (Q ) shows that the LDH release from cells exposed to lysin was significantly reduced in the presence of UL1-012, in a dose response manner, when compared to the PLY control. This demonstrates that UL1-012 prevents pneumolysin from ng the human lung epithelial cells in culture, with an IC50 < 0.4 mM. (3) The dotted black line (x) shows that UL1- 012 does not exhibit cytotoxicity at the concentrations tested, up to approximately 150 times the therapeutic IC50 value.
Conclusion UL1-012 inhibits the damaging ty of pneumolysin on human lung epithelial cells in e.
UL1-012 did not exhibit cytotoxic effects on the human lung epithelial cells at 150 times the therapeutic IC50 value.
D. EX VIVO ASSAY: INHIBITION OF THE EFFECT OF PNEUMOLYSIN ON THE CILIARY FUNCTION OF CULTURED EPENDYMAL CELLS The mal ed cells line the cerebral cles of the brain and the central canal of the spinal cord and are covered with cilia responsible for the ation 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. Long standing research in this field in the laboratory of the inventors resulted in the development of a rat ex vivo model of meningitis that was proven to predict the in vivo situation during meningitis. This model is based on culturing and differentiating ciliated mal 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 y of a compound to prevent pneumolysin from causing damage in vivo.
Experimental procedure Ependymal cell cultures were prepared by the method previously described [Microb. Pathog. (1999) 27 303-309]. Tissue e trays were coated with bovine fibronectin and incubated at 37°C in 5% (v/v) C0 2 for 2h before use. The growth medium was minimum essential medium (MEM) with added penicillin (100 lU/ml), streptomycin (100 Mg/ml), fungizone (2.5 Mg/ml), BSA (5 mg/ml), insulin (5 mg/ml), errin (10 mg/ml) and selenium (5 mg/ml). Neo-natal (0-1 day old) rats were killed by cervical dislocation, and their brains were removed. The cerebellum was d along with edge regions of the left and right cortical hemispheres and the frontal cortex. The ing brain areas were ically dissociated in 4 ml of growth medium. The dissociated tissue from one or two brains was added to the wells of the tissue culture trays (500 m I/well), each containing 2.5 ml of growth medium. The cells then were ted at 37°C in 5% (v/v) C0 . The medium was replaced after three days and thereafter the ependymal cells were fed every two days with 2 ml of fresh growth medium supplemented with thrombin.
After approximately two weeks, the cells were fully ciliated and ready for experiments. For experiments, the growth medium replaced with 1 ml of medium MEM containing 25 mM HEPES, pH 7.4. The tissue culture trays were placed inside a thermostatically controlled incubation chamber surrounding the stage of an inverted light cope. The cell cultures were allowed to equilibrate until the temperature of the assay medium was 37°C. At this point, either recombinant purified pneumolysin or S.pneumoniae cell lysate containing native pneumolysin - obtained ing the lytic effect of the otic Penicillin - with and without test compound, pre-incubated in 1 ml of medium MEM at 37°C for 40 min, were added to the wells containing the ciliated cells. To the control cells, 1 ml of MEM medium was added. Beating cilia were recorded before and after exposure over 30 min, with a digital high-speed video camera at a rate of 500 frames/s. The recorded video sequences were played back at reduced frame rates and the y beat frequency (CBF) was determined by the following equation: 500frames/s , , CBF (Hz) = - — x 5 (conversion per beat cyclej. (frames elapsed for 5 ciliary beat cycles) Results The ter measured was the ciliary beating frequency (CBF). lysin or bacterial lysate, added to ciliated cells in culture induces a severe or total loss of ciliary beating. UL1-012 and UL2-001 inhibited this damaging effect induced by pneumolysin on the y function of ependymal cells in culture (Figure 2 .A and Figure 3 , respectively). Furthermore, UL1-012 was also shown to inhibit the detrimental effect caused by a bacterial lysate, using the same ex vivo model (Figure 2.B).
In Figure 2.A: Each time point represents the normalised mean ± SD of ciliary beating frequency (CBF) measurements of four individual cilia from each well, in three ndent experiments. (1) Control 1, assay medium only: the symbol (-|-) ents measurements of the CBF in the assay medium which was used as a reference for the normal cilia beating. No damaging effect on the CBF was seen throughout the recording. (2) Control 2 , pneumolysin only: The symbol (¨) represents ements of the CBF in the wells where pneumolysin was added. A progressive drop in the CBF to 27% of the al frequency was observed. (3) Treatment with UL1-012: Symbols (¾; ; * ) represent the measurements of the CBF in the presence of pneumolysin and UL1-012 (1.56 mM - 25 mM) . No significant loss of the CBF was seen, showing that UL1-012 inhibits pneumolysin-induced damage on the ciliary beating frequency of the brain ependymal cells. There was no statistical difference between the CBF of Control 1 (medium only) and the CBF in the presence of the treatment (¾; ;»; *), indicating that the inhibition of the ng effect of lysin by UL1-012 was achieved to an extent comparable to the control medium alone.
In Figure 2.B: Each time point represents the normalised mean ± SD of ciliary beating frequency (CBF) measurements of four individual cilia from each well, in three independent experiments. (1) Control 1, assay medium only: the symbol (-|-) represents ements of the CBF in the assay medium which was used as a reference for the normal cilia beating. No damaging effect on the CBF was seen throughout the recording. (2) Control 2 , bacterial lysate: the symbol (*) ents measurements of the CBF in the wells where the bacterial lysate obtained from penicillin-lysed S. pneumoniae induced a significant reduction in the CBF within the first five minutes of the recording, to 16% of the original frequency. (3) Treatment with 2: the symbol ( ) represents the measurements of the CBF in the presence of treatment UL1-012 and bacterial lysate obtained from penicillin-lysed S. pneumoniae. UL1-012 significantly prevented the drop of the CBF, indicating that UL1-012 inhibits pneumolysin released from penicillin-lysed bacteria, to a similar extent of the control 1 (-|-); y cilia in the presence of the assay medium.
In Figure 3 : Each time point represents the normalised mean ± SD of ciliary beating frequency (CBF) ements of four individual cilia from each well, in three independent experiments. (1) Control 1, assay medium only: the symbol (-|-) represents ements of the CBF in the assay medium which was used as a reference for the normal cilia beating. No damaging effect on the CBF was seen throughout the recording (2) Control 2 , pneumolysin only: The symbol (¨) represents measurements of the CBF in the wells where pneumolysin was added. A rapid drop in the CBF, with complete inhibition by 1 minute after the addition of pneumolysin was observed (3) ent with UL2-001 : Symbol (*) represent the measurements of the CBF in the presence of lysin and UL2-001 (2.00 mM) . No icant loss of the CBF was seen, g that UL2-001 inhibits pneumolysin-induced damage on the ciliary beating frequency of the brain ependymal cells. There was no statistical difference between the CBF of Control 1 m only) and the CBF in the presence of the treatment, indicating that the inhibition of the damaging effect of pneumolysin by UL2-001 was achieved to an extent comparable to the control medium alone.
Conclusion 2 and UL2-001 inhibit the damaging effect that pneumolysin induces on the brain epen dymal ciliated cells in culture which predicts its y to prevent pneumolysin from causing damage in vivo. In addition, UL1-012 demonstrated the ability to also inhibit the native pneumo lysin, released by pneumococcus following antibiotic-mediated lysis. It is remarkable that the inhibition of pneumolysin only, amongst all other bacterial products present in S.pneumoniae bacterial lysate, was sufficient to h the damaging effect of the whole bacterial lysate, which highlights again the substantial ement of pneumolysin in the damaging effect of antibiotic lysed bacteria.
These findings support the use of this novel-approach as an adjunctive therapy in patients.
E . IN VIVO EFFICACY ASSAY USING A MOUSE PNEUMONIA MODEL This model has been long developed and 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. niae mutant deficient in pneumolysin (PLN-A), ted (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 mation and less bacteraemia (infiltration of the bacteria from the lungs to the circulation). Therefore, this in vivo disease model constitutes a powerful tool to study the disease progression of mice infected with wild-type S.pneumoniae and treated with pneumolysin inhibitors. To assess the severity of the disease, the parameters that are followed are the survival and the disease score.
Experimental Procedures: Infection, Treatment and e Signs Scoring Outbred MF1 female mice, 8 weeks old or more and weighing 25-30 g were used. The animals were maintained under controlled conditions of temperature, humidity and day . They had free access to tap water and ed food. The in vivo experiments were performed using two control groups: Control 1 (infected and not treated), Control 2 (not infected and treated) and one Treatment group (infected and treated). Mice in control group 1 and in the treatment group were infected intranasally with Streptococcus pneumoniae strain D39 (procedure bed below).
After completing the infections, the viable count of the given dose was determined (as described below). Subsequently, every six hours, animals in the ent group and in Control group 2 , received the test compound intravenously or intranasally as appropriate while excipient alone was administered to Control group 1. The progress of the signs of e (Table 3) was assessed every 6h based on the scheme of Morton and Griffiths [Veterinary Record. (1985) 1 1 1. 431-436].
Animals were killed if they became 2+ lethargic and the time was recorded. After imately 72h the experiment was ended. The survival rates of control and test groups were compared with a log-rank test, while the signs of disease were ed with the Mann-Whitney test.
Table 3 Scoring scheme of the disease signs Sign Description Score Healthy appearance.
Normal 0 Highly active.
Slight (1+) or pronounced (2+) 1+/2+ Hunched ; 2 convex curvature of the upper spine. 1+/2+ Starey coat Slight (1+) or pronounced (2+) 3· 4 (Piloerection) piloerection of the coat.
Pronounced hunching and piloerection accompanied by a 1+/2+ Lethargic 5; 6 considerable (1+) or severe (2+) reduction of activity.
Complete inertia. nd Drastic ion of breathing rate. N/A Drop of the body temperature.
N/A - Not applicable The administration of S. pne on/ae/Treatment and the determination of the bacterial viable counts ned in the above procedure are detailed as follows: - Intranasal administration Mice were lightly anaesthetised with 2.5% (v/v) isoflurane over 1.6-1 .8 L 0 /min. The confirmation of effective anaesthesia was made by observation of no pedal reflex. A mouse was held by the scruff of the neck in a vertical position with its nose upward. The dose was then administered in sterile PBS, given drop by drop into the nostrils, allowing the animal to inhale it in between drops. Once the dose was given, the mouse was ed to its cage, placed on its back to recover from the s of hetic. - enous administration Mice were placed in a cage inside an incubator at 37°C, for 20 min, to dilate their veins. After incubation, the cage was brought outside and the mice were kept warm under an infra-red (IR) lamp. A mouse was then placed inside a restrainer, leaving the tail of the animal exposed. The tail was disinfected with 10% (v/v) ol in water. The dose was then gently administered intravenously using a 0.5 ml_ insulin syringe inserted carefully into one of the tail lateral veins.
- Determination of viable counts Viable counting was performed by the method of Miles and Misra [J. Hyg. (1938) 38 732-749). m I of the sample were serially diluted in 180 m I PBS in round-bottomed 96-wells microtitre plates, up to a dilution of 106. Blood agar plates were divided into six sectors and 60 m I of each dilution plated onto an individual sector. The plates were incubated in C0 gas jars overnight at 37°C. The following day, colonies were counted in the sector where 30-300 colonies were visible. The concentration of colony forming units (CFU) per millilitre was determined by using the following equation: Number of colonies in sector _ ., . \ CFU per ml = x Dilution x 1000 (conversion lactor ). 60 mΐ Results of the in vivo cy assay obtained with Example UL1-012 0 - Experimental design The experimental design is shown in Figure 4 . The treatment with UL1-012 was administered intravenously every 6h. Based on the IC100 tration seen in the primary assay, the amount of compound required to neutralise pneumolysin released in vivo during the most severe phase of infection, was calculated. A dose of 4 mg/kg prepared in 2% (v/v) DMSO in PBS was given.
This proved effective. In subsequent PD experiments, 16 mg/kg prepared in 4% (v/v) DMSO in PBS was also used. The doses were prepared freshly and administered enously to the Survival Results 0 4 mg/kg of body weight of UL1-012 was administered intravenously every 6h to mice infected with S. niae and the outcome compared against a control group of infected mice, which had not ed the compound (only the excipient). The p-value was calculated by means of the log-rank (Mantel-Cox) test (n = 10/group). The survival curves of the control (solid line) and ent (dotted line) groups obtained with this experiment are presented in Figure 5 . Up to approximately 40h after ion, there was a significant improvement in the survival of the treatment group over the control group (p<0.05). 4 mg/kg of UL1-012 produced a good e with a icant increase of the survival at critical time points where we usually see a rapid disease ssion of untreated animals (p<0.05). 16 mg/kg of body weight of UL1-012 was used. Six hours post infection with wild-type S. 0 pneumoniae, animals received intravenously UL1-012 (16 mg/kg) and every 6h thereafter. The p-value was calculated by means of the nk (Mantel-Cox) test (n = 10/group). This was performed to test if protection could be enhanced but also to test if a higher dose is as tolerated.
As shown in Figure 6 , a significant increase in the survival was seen with 50% of mice surviving at the endpoint of the ment at 72h post infection (p<0.05).
Disease score results During the course of the experiment, the signs of the disease were assessed at least every 6h and disease scores were noted for each mouse. At various time points of the experiment, infected/non-treated mice exhibited pronounced signs of the e, reflected by their hunched 0 spine, piloerect coat and reduction in their activity. On the other hand, a significantly higher number of infected mice that received the ent with UL1-012 had a healthy appearance (spine not hunched, coat not standing) and were highly , exploring the cage surroundings.
Disease scoring with 16 mg/kg of UL1-012 recorded at multiple time points during the course of the experiment is shown in Figure 7 . Each symbol (■ ;·) represents an individual mouse in the control and treatment group, respectively; horizontal lines represent medians; p-values were calculated by using a Mann-Whitney test. A delay in the onset of the signs of the disease for the group that was treated with UL1-012 was obtained. The difference in the disease scoring between the two groups was statistically significant (p<0.05) at 30 h , 36 h , 60 h and 66 h post infection. The results indicate a delay in the onset of the disease and a significant attenuation of the signs of the disease.
Conclusion (1) In vivo protection was obtained with UL1-012 at both dosing regimen tested. (2) Higher dose of UL1-012 ed an enhanced protection showing that there is a dose dependent response. (3) Protection is seen even in the e of an antibiotic, which is a remarkable outcome, suggesting that the neutralisation of pneumolysin alone, without the killing of the bacterium, is providing tion to the animals. This is consistent with the disease profile obtained with S. pneumoniae deficient in pneumolysin ). (4) Even at the highest concentration of UL1- 012, no visible adverse effects were seen in the control group of mice receiving the compound alone.
Results of the in vivo cy assay obtained with e UL2-001 Experimental set up The in vivo efficacy of Example UL2-001 was tested using a pneumonia model. The experimental design is shown in Figure 8 . The ent with 1 was administered intranasally every 6h. Based on the IC100 concentration seen in the primary assay, the amount of compound required to lise pneumolysin released in vivo during the most severe phase of infection, was calculated. A dose of 0.8 mg/kg prepared in 2% (v/v) DMSO in PBS was given.
This proved effective. The doses were prepared freshly and administered intranasally to the animals.
Survival Results 0.8 mg/kg of body weight of UL2-001 was administered asally every 6h to mice infected intranasally with S. pneumoniae and the outcome compared against a control group of infected mice, which had not received the compound (only the excipient). The p-value was calculated by means of the nk (Mantel-Cox) test (n = 5/group). The survival curves of the control (solid line) and treatment (dotted line) groups obtained with this experiment are presented in Figure 9 . 36 hours post-infection infection, all mice in the control group had died. At this time-point, the percentage of survival of the treatment group had dropped to 60%, and then remained constant until the endpoint of the experiment at 60 hours nfection. This experimental e has shown a significant improvement in the survival of the treatment group over the control group (p<0.05).
Conclusion (1) In vivo protection was obtained with UL2-001 following intranasal administration. (2) Protection is seen even in the absence of an otic, suggesting that the neutralisation of pneumolysin alone, without the killing of the bacterium, is providing protection to the animals.
This is tent with the disease profile obtained with S. niae deficient in pneumolysin (PLN-A).
F. CONVERSION OF PRODRUG DERIVATIVES TO ACTIVE INHIBITORS IN MOUSE AND HUMAN PLASMA Rationale To demonstrate that the g derivatives are converted to the active ingredient in the ce of plasma enzymes, a prodrug derivative was incubated with mouse and human plasma at 37°C at 5 time points over a 2h period. The s were then analysed by LC- MS/MS to obtain the amount of active nd 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 was obtained in some cases.
Experimental procedure Prodrug derivatives were assessed in the mouse and human plasma ity assay at a concentration of 10 mM. Test compounds were diluted in DMSO to a final stock concentration of mM. For the purpose of the assay, the stocks prepared were further diluted in DMSO to a concentration of 400 mM and 5 were added to 195 m I of mouse or human plasma (pH 7.4) and then incubated at 37°C. The final concentration of DMSO in the plate was 2.5% (v/v). Reactions were terminated at 0 , 15, 30, 60 and 120 min after incubation by adding 400 m I of itrile containing 0.55 mM metoprolol and 1% (v/v) formic acid. The plate was then centrifuged at 3000 rpm, for 45 min, at 4°C. 80 m I of supernatant were transferred into a conical bottom 96 well glass coated plate. 40 m I of water were added prior to analysis for g derivative and active species by LC-MS/MS. This assay was performed by a contract ch organisation, Cyprotex Discovery d, UK, at the request of the inventors at Leicester.
Results The quantification of the parent nd (prodrug derivative) remaining and the active ingredient appearing was performed as follows: (1) The active compound was quantified using a 6 point calibration curve prepared in deactivated mouse and human plasma. (2) The percentage of parent compound ing at each time point relative to 0 min sample was calculated from LC-MS/MS peak area ratios (compound peak area/internal standard peak area). This percentage was then used to determine the concentration of the parent compound at each time point in reference to the starting concentration (10 mM) at time 0 min.
A y of the conversion of the prodrug derivatives to active inhibitors is shown Table 4 .
Conclusion The s presented in Table 4 clearly indicate the therapeutic benefits of the prodrugs of the invention, which is demonstrated by their conversion in plasma into the pharmacologically active ingredient. The rate of conversion of the prodrug derivatives to the active ingredients is variable amongst the prodrug derivatives. This offers a range of different therapeutic strategies ranging from immediate to slow release, in order to achieve the desired therapeutic benefits.
Table 4Table 4 Prodrug 122336? 110M) 110M) 110M) 110M) 110M) ID t0 min. t15 min. t30 min. t60 min. t120 min. ”” -——— 0 UL1-044 11 11 13 L1-012) UL 1 -063 ACtiVC 3.10 7.00 5.00 2.20 UL 1 -064 133833) ACtiVC 0.02 0.11 0.24 0.37 L1-012) — 4.21 3.72 2.57 1.14 UL 1 -065 13383;?) ACtiVC 0.05 0.07 0.12 0.19 L1-012) — UL 1 -066 999999999999??? L1-069)L1T044)Active ACtiVC 0.23 0.23 0.21 0.20 ”” -——— 0 1213:” .40 10.60 10.10 10.80 L1-012) — Pmdmg 3.02 1.00 0.136 0 L1-068) Active.
UL1-069 Active 0.44 0.42 0.40 040 (L L 1 -0 12) Prodrug (Mouse 6.40 4.73 2.43 0.93 plasma) (UL 1 -070) Active (Mouse 3.22 5.85 7.72 8.71 plasma) UL1-070 (UL 1 -0 12) Prodrug ) 10 8.02 8.28 7.70 7.48 (UL1-070) Active (Human) 0.25 0.45 0.78 1.29 (UL 1 -0 12) Prodrug (UL 1 -07 1) 9.50 8.72 7.39 UL 1 -071 Active (UL1-012) 0.47 0.87 1.65 2.56 UL1-092 Prodrug 5.12 4.67 4.59 3.70 (UL1-092) Active 0 0.01 0.01 0.01 0.012 (LL1-012) Prodrug 9'22 8'78 8'49 UL1—094 (LU-094) -n Active 0.059 0.01 0.01 0.03 0.05 (LL1-012) Prodrug (Mouse) 10 0 Active (Vlouse) 10.40 11.00 11.40 11.40 UL1-098 (LL1-095) Prodrug (Human) 10 5.42 3.45 1.76 0.65 (LL1-098) Active (Human) 4.45 6.84 8.93 10 Prodrug 0 UL1-105 (LU-105) Active Prodrug (Vlouse) 10 6.87 4.84 2.45 0.73 (LL1-109) Active (VIouse) 3.47 6.01 8.40 9.92 UL1-109 (LL1-012) (Human) 10 6.22 5.37 3.39 1.72 (LL1-109) Active (Human) 1.96 3.31 5.81 8.71 (LL1-012) Prodrug (Vlouse) 10 4.74 2.47 1.16 0.47 (LL1-111) Active (Vlouse) 0.22 6.31 8.55 10.45 13.8 (LLI'OH) UL1-111 Prodrug (Human) 10 2.84 0.80 0.00 (LL1-111) Active (Human) <LOQ 6.98 9.01 9.23 9.23 (LL1-012) Prodrug (Mouse) 10 0 (LL1-114) Active (Vlouse) 14.14 14.16 13.14 10.85 (LLI'OOS) UL1-114 Prodrug (Human) 10 9.53 9.48 8.22 7.22 Active (Human) 0 1.72 3.11 4.96 7.62 (LL1-005) *lf the s is not specified in the table it is referring to mouse.
A set of clauses defining certain aspects of the ion is as follows: 1. A compound of formula (I): wherein: R and R2 are independently selected from -C(0)NR R6, -C(0)OR 7, CN, -C(0)R 7, - C(0)NHC(0)R 7, -NO 7, -SO 7, -S0 R6, -SOR 7, -S0 8 and 2, -SO 3R 2R 2NR 2NH-C(0)OR optionally substituted phenyl or heteroaryl; R3 is optionally substituted phenyl; R4a and R4 are independently ed from hydrogen, C C6 alkyi which alkyi group may optionally be substituted by hydroxyl, COOR 2 or CONR R14, 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-C 6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C 3-C1 0 cycloalkyl, C 5-C1 0 cycloalkenyl, heterocyclyl, -C1-C 3 alkyl-C3-C 0 cycloalkyl, -C1-C 3 alkyl-C5-C 0 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 cyclic 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-C 6 alkyl, C C6 fluoroalkyl and -C(0)NR aR , where Ra and R are independently selected from hydrogen and C C6 alkyl, and any of the aforementioned R5 and R6 groups may be optionally substituted by one or more halogen atoms, and (c) aryl, heteroaryl, C 1-C3 alkylaryl and -C1 -C3 alkylheteroaryl, said aryl and heteroaryl groups being optionally tuted; R7 is selected from: (a) Ci-C 6 alkyl, C2-C6 l, C2-C6 alkynyl, C3-C1 0 cycloalkyl, C5-C1 0 cycloalkenyl, heterocyclyl, -C1-C3 alkyl-C3-C 0 cycloalkyl, -C1-C3 alkyl-C5-C 0 cycloalkenyl or -C1-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 , where Ra and R 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 Ci-C alkyl; R9 is hydrogen, C C6 alkyl; -C(0)R 10 or -C (0)OR 11; R 0 is Ci-Ce alkyl; R is Ci-Ce alkyl; R 2 is Ci-C alkyl; R 3 is hydrogen or C C6 alkyl; and R 4 is hydrogen or C C6 alkyl; or a pharmaceutically acceptable prodrug tive thereof, or a pharmaceutically able salt or solvate thereof; ed that the compound is not: a) diethyl hydroxy(4-methoxyphenyl)-1/-/-pyrrole-2,5-dicarboxylate; b) diethyl 3,4-dihydroxyphenyl-1/-/-pyrrole-2,5-dicarboxylate; c) dimethyl 3,4-dihydroxy(4-bromophenyl)-1/-/-pyrrole-2,5-dicarboxylate; d) yl 3,4-dihydroxy(4-chlorophenyl)-1/-/-pyrrole-2,5-dicarboxylate; e) di- r -butyl 3,4-dihydroxy(4-nitrophenyl)-1/-/-pyrrole-2,5-dicarboxylate; f) dimethyl 3,4-dihydroxy(4-methoxyphenyl)-1/-/-pyrrole-2,5-dicarboxylate; g) yl 3,4-dihydroxyphenyl-1/-/-pyrrole-2,5-dicarboxylate; h) l 3,4-bis(acetyloxy)phenyl-1/-/-pyrrole-2,5-dicarboxylate; or i) dimethyl 3,4-bis(acetyloxy)phenyl-1/-/-pyrrole-2,5-dicarboxylate. 2 . A compound according to clause 1 wherein R and R2 are independently selected from - C(0)NR R6, -C(0)OR 7 and CN. 3 . A compound according to clause 1 wherein (i) R is -C(0)NR R6 and R2 is -C(0)NR R6 or (ii) R is -C(0)NR R6 and R2 is -C(0)OR 7. 4 . A compound ing to clause 1 n R is -C(0)OR 7 and R2 is -C(0)OR 7.
. A compound according to any one of the preceding clauses wherein R3 is substituted phenyl. 6 . A compound ing to clause 5 wherein R3 is phenyl substituted by 1 , 2 or 3 substituents selected from halo, cyano, C C alkoxy, C C fluoroalkoxy, C C alkyl, C C 6 6 6 6 fluoroalkyi and -C(0)NR aR , where Ra and R are independently selected from hydrogen and C C alkyl. 7 . A compound according to clause 5 or 6 wherein R3 is phenyl having a substituent in the meta or para positions relative to the pyrrole ring. 8 . A compound according to any one of the preceding clauses wherein R4a and R4 are hydrogen or -CrC alkylaryl. 9 . A compound according to clause 8 wherein R4a and R4 are hydrogen.
. A compound according to any one of the preceding clauses n R5 and R6 are independently selected from hydrogen, C C alkyl, aryl or C alkylaryl in which said aryl may 6 3 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 ning a further heteroatom ed from O, S and NR9. 11. A compound according to clause 10 wherein R5 and R6 are not both en. 12. A compound according to any one of the preceding clauses wherein R7 is C C alkyl. 13. A compound according to clause 1 which is selected from: N2,N2, , -tetraethyl(4-fluorophenyl)-3,4-dihydroxy-1/-/-pyrrole-2,5-dicarboxamide; thoxyphenyl)-/V2 L/2 , -tetraethyl-3,4-dihydroxy-1/-/-pyrrole-2,5-dicarboxamide; N2,N2, , ethyl-3,4-dihydroxy(4-methoxyphenyl)-1/-/-pyrrole-2,5-dicarboxamide; (3,4-dihydroxy(4-methoxyphenyl)-1/-/-pyrrole-2,5-diyl)bis(morpholinomethanone); 3,4-dihydroxy(4-methoxyphenyl)-/V 2 L/2 , -tetramethyl-1/-/-pyrrole-2,5-dicarboxamide; fluorophenyl)-3,4-dihydroxy-1/-/-pyrrole-2,5-diyl)bis(morpholinomethanone); 1-(4-fluorophenyl)-3,4-dihydroxy-/V 2 -dimethyl-/V2 -diphenyl-1H-pyrrole-2,5-dicarboxamide; 1-(4-fluorophenyl)-3,4-dihydroxy-/V 2 L/2 , -tetramethyl-1/-/-pyrrole-2,5-dicarboxamide; di-tert-butyl 4,4'-(1-(4-fluorophenyl)-3,4-dihydroxy-1/-/-pyrrole-2,5-dicarbonyl)bis(piperazine carboxylate); 1,1'-(4,4'-(1-(4-fluorophenyl)-3,4-dihydroxy-1/-/-pyrrole-2,5-dicarbonyl)bis(piperazine-4,1- diyl))diethanone; (1-(4-fluorophenyl)-3,4-dihydroxy-1/-/-pyrrole-2,5-diyl)bis(piperidinylmethanone); ethyl 5-(dimethylcarbamoyl)-3,4-dihydroxy(4-methoxyphenyl)-1/-/-pyrrolecarboxylate; 1-(4-fluorophenyl)-3,4-dihydroxy-A J -diisopropyl-A/ , hyl-1/-/-pyrrole-2,5- dicarboxamide; J -dibenzyl(4-fluorophenyl)-3,4-dihydroxy-A J -dimethyl-1H-pyrrole-2,5-dicarboxam di-te/f-butyl 4,4'-(3,4-dihydroxy(4-methoxyphenyl)-1/-/-pyrrole-2,5-dicarbonyl)bis(piperazine carboxylate); (3,4-dihydroxy(4-methoxyphenyl)-1/-/-pyrrole-2,5-diyl)bis(piperazinylmethanone); 1-(4-fluorophenyl)-3,4-dihydroxy-A J J J -tetraisopropyl-1H-pyrrole-2,5-dicarboxamide (3,4-dihydroxy(4-methoxyphenyl)-1/-/-pyrrole-2,5-diyl)bis(piperidinylmethanone); 3,4-dihydroxy(2-methoxyphenyl)-A J J (3,4-dihydroxy(2-methoxyphenyl)-1/-/-pyrrole-2,5-diyl)bis(morpholinomethanone); 1-(4-ethoxyphenyl)-3,4-dihydroxy-A J J J -tetramethyl-1H-pyrrole-2,5-dicarboxami ethoxyphenyl)-3,4-dihydroxy-1/-/-pyrrole-2,5-diyl)bis(morpholinomethanone); (3,4-dihydroxy(4-(trifluoromethyl)phenyl)-1H-pyrrole-2,5-diyl)bis(morpholinomethanone -ethyl-3,4-dihydroxy(4-methoxyphenyl)-A J hyl-1H-pyrrole-2,5-dicarboxamide; 3,4-dihydroxy(4-methoxyphenyl)-A/,A/-dimethyl(piperidinecarbonyl)-1/-/-pyrrole amide; 3,4-dihydroxy(4-methoxyphenyl)-A/,A/-dimethyl(morpholinecarbonyl)-1/-/-pyrrole carboxamide; 3,4-dihydroxy(4-methoxyphenyl)-A J\/-dimethyl(4-m^ 2-carboxamide; 3,4-dihydroxy-/V2 N2, , -tetramethyl(4-(trifluoromethyl)phenyl)-1/-/-pyrrole-2,5- oxamide; 1-(4-(dimethylcarbamoyl)phenyl)-3,4-dihydroxy-A J dicarboxamide; -butyl (5-(dimethylcarbamoyl)-3,4-dihydroxy(4-methoxyphenyl)-1/-/-pyrrol yl)sulfonylcarbamate; -cyano-3,4-dihydroxy(4-methoxyphenyl)-A/,A/-dimethyl-1/-/-pyrrolecarboxamide; ethyl 5-(dimethylcarbamoyl)(4-fluorophenyl)-3,4-dihydroxy-1/-/-pyrrolecarboxylate; 1,1'-(4,4'-(3,4-dihydroxy(4-methoxyphenyl)-1/-/-pyrrole-2,5-dicarbonyl)bis(piperazine-4,1- diyl))diethanone; 3,4-dihydroxy(4-methoxyphenyl)-1/-/-pyrrole-2,5-dicarbonitrile; isopropyl 5-(dimethylcarbamoyl)-3,4-dihydroxy(4-methoxyphenyl)-1/-/-pyrrolecarboxylate; -butyl 5-(dimethylcarbamoyl)-3,4-dihydroxy(4-methoxyphenyl)-1/-/-pyrrolecarboxylate; ethyl 3,4-dihydroxy(4-methoxyphenyl)(4-methylpiperazinecarbonyl)-1/-/-pyrrole carboxylate; ethyl 5-(dibutylcarbamoyl)-3,4-dihydroxy(4-methoxyphenyl)-1/-/-pyrrolecarboxylate; ethyl 5-cyano(4-fluorophenyl)-3,4-dihydroxy-1/-/-pyrrolecarboxylate; ethyl 3,4-dihydroxy(4-methoxyphenyl)(morpholinecarbonyl)-1/-/-pyrrolecarboxylate; ethyl 5-(diethylcarbamoyl)-3,4-dihydroxy(4-methoxyphenyl)-1/-/-pyrrolecarboxylate; ethyl 5-(dimethylcarbamoyl)-3,4-dihydroxy(4-iodophenyl)-1/-/-pyrrolecarboxylate; ethyl 5-(dimethylcarbamoyl)-3,4-dihydroxy(3-iodomethoxyphenyl)-1/-/-pyrrole carboxylate; 3,4-bis(benzyloxy)(4-ethoxyphenyl)-^ 3,4-bis(benzyloxy)-A J J J -tetraethyl(4-methoxyphenyl)-1H-pyrrole-2,5-dicarboxam 3,4-bis(benzyloxy)(4-methoxyphenyl)-A J J di-tert-butyl 4,4'-(3,4-bis(benzyloxy)(4-methoxyphenyl)-1/-/-pyrrole-2,5- dicarbonyl)bis(piperazine-l-carboxylate); (3,4-bis(benzyloxy)(4-methoxyphenyl)-1/-/-pyrrole-2,5-diyl)bis(piperidinylmethanone); 3,4-bis(benzyloxy)(2-methoxyphenyl)-A J J J -tetramethyl-1H-pyrrole-2,5-dicarboxam (3,4-bis(benzyloxy)(2-methoxyphenyl)-1/-/-pyrrole-2,5-diyl)bis(morpholinomethanone); 3,4-bis(benzyloxy)(4-ethoxyphenyl)-A J J J -tetramethyl-1H-pyrrole-2,5-dicarboxami (3,4-bis(benzyloxy)(4-ethoxyphenyl)-1/-/-pyrrole-2,5-diyl)bis(morpholinomethanone); 3,4-bis(benzyloxy)-/V2 N2, , -tetramethyl(4-(trifluoromethyl)phenyl)-1/-/-pyrrole-2,5- dicarboxamide; 3,4-bis(benzyloxy)-A/2-ethyl(4-methoxyphenyl)^ s(benzyloxy)(4-methoxyphenyl)-A methyl(4-methylpiperazinecarbonyl)-1/-/- pyrrolecarboxamide; 3,4-bis(benzyloxy)(4-methoxyphenyl)-A J\/-dimethyl(piperidinecarbonyl)-1/-/-pyrrole carboxamide; s(benzyloxy)(4-methoxyphenyl)-A J\/-dimethyl(morpholinecarbonyl)-1/-/-pyrrole carboxamide; 1J'-(4,4'-(3,4-bis(benzyloxy)(4-methoxyphenyl)-1/-/-pyrrole-2,5-dicarbonyl)bis(piperazine-4,1- diyl))diethanone; ethyl 3,4-bis(benzyloxy)cyano(4-fluorophenyl)-1/-/-pyrrolecarboxylate; 3,4-bis(benzyloxy)cyano(4-fluorophenyl)-/\/,A/-dimethyl-1/-/-pyrrolecarboxamide; ethyl 3,4-bis(benzyloxy)(dimethylcarbamoyl)(4-iodophenyl)-1/-/-pyrrolecarboxylate; 3,4-bis(benzyloxy)(4-methoxyphenyl)-1/-/-pyrrole-2,5-dicarboxamide; 3,4-dihydroxy(4-methoxyphenyl)-1/-/-pyrrole-2,5-dicarboxamide; s(benzyloxy)-/V2, yl(4-methoxyphenyl)-1/-/-pyrrole-2,5-dicarboxamide; 3,4-bis(benzyloxy)(4-fluorophenyl)-1/-/-pyrrole-2,5-dicarboxamide; 1-(4-fluorophenyl)-3,4-dihydroxy-1/-/-pyrrole-2,5-dicarboxamide; 1-(4-ethoxyphenyl)-3,4-dihydroxy-1/-/-pyrrole-2,5-dicarboxamide; 1-(4-ethoxyphenyl)-/V2, -diethyl-3,4-dihydroxy-1/-/-pyrrole-2,5-dicarboxamide; ethyl 3,4-bis(benzyloxy)carbamoyl(4-methoxyphenyl)-1/-/-pyrrolecarboxylate; ethyl 5-carbamoyl-3,4-dihydroxy(4-methoxyphenyl)-1/-/-pyrrolecarboxylate; ethyl 3,4-bis(benzyloxy)(2-methoxyphenyl)(4-methoxyphenyl)-1/-/-pyrrolecarboxylate; ethyl 3,4-bis(benzyloxy)(4-methoxyphenyl)(pyridinyl)-1/-/-pyrrolecarboxylate; ethyl 3,4-dihydroxy(4-methoxyphenyl)(pyridinyl)-1/-/-pyrrolecarboxylate; ethyl 3,4-bis(benzyloxy)(4-methoxyphenyl)(pyridinyl)-1/-/-pyrrolecarboxylate; ethyl 3,4-bis(benzyloxy)(4-methoxyphenyl)(pyridinyl)-1/-/-pyrrolecarboxylate; ethyl 5-(4-ethylthiazolyl)-3,4-dihydroxy(4-methoxyphenyl)-1/-/-pyrrolecarboxylate; ethyl 3,4-bis(benzyloxy)(4-methoxyphenyl)(5-methyl-1,3,4-oxadiazolyl)-1/-/-pyrrole carboxylate; ethyl 3,4-bis(benzyloxy)(5-ethyl-1,3,4-oxadiazolyl)(4-methoxyphenyl)-1/-/-pyrrole carboxylate; ethyl 3,4-dihydroxy(4-methoxyphenyl)(2/-/-tetrazolyl)-1/-/-pyrrolecarboxylate; ethyl 3,4-bis(benzyloxy)(4-methoxyphenyl)(2/-/-tetrazolyl)-1/-/-pyrrolecarboxylate; diethyl 1-(4-fluorophenyl)-3,4-dihydroxy-1/-/-pyrrole-2,5-dicarboxylate; diethyl 3,4-dihydroxy(3-methoxyphenyl)-1/-/-pyrrole-2,5-dicarboxylate; diethyl 3,4-dihydroxy(4-(trifluoromethoxy)phenyl)-1/-/-pyrrole-2,5-dicarboxylate; diethyl 3,4-dihydroxy(4-isopropylphenyl)-1/-/-pyrrole-2,5-dicarboxylate; diethyl 3,4-dihydroxy(4-phenoxyphenyl)-1/-/-pyrrole-2,5-dicarboxylate; diethyl 1-(4-ethoxyphenyl)-3,4-dihydroxy-1/-/-pyrrole-2,5-dicarboxylate; diethyl 1-(4-ethylphenyl)-3,4-dihydroxy-1/-/-pyrrole-2,5-dicarboxylate; diethyl 3,4-dihydroxy(p-tolyl)-1/-/-pyrrole-2,5-dicarboxylate; diethyl 1-(3,4-dimethoxyphenyl)-3,4-dihydroxy-1/-/-pyrrole-2,5-dicarboxylate; diethyl 1-(4-chlorophenyl)-3,4-dihydroxy-1/-/-pyrrole-2,5-dicarboxylate; diethyl 3,4-dihydroxy(4-(trifluoromethyl)phenyl)-1/-/-pyrrole-2,5-dicarboxylate; diethyl 3,4-dihydroxy(3,4,5-trimethoxyphenyl)-1/-/-pyrrole-2,5-dicarboxylate; diethyl -dimethoxyphenyl)-3,4-dihydroxy-1/-/-pyrrole-2,5-dicarboxylate; diethyl 3,4-dihydroxy(4-propoxyphenyl)-1/-/-pyrrole-2,5-dicarboxylate; diethyl 1-(4-ethoxy-3,5-difluorophenyl)-3,4-dihydroxy-1/-/-pyrrole-2,5-dicarboxylate; diethyl 3,4-dihydroxy(3-phenoxyphenyl)-1/-/-pyrrole-2,5-dicarboxylate; diethyl 1-(benzo[d][1,3]dioxolyl)-3,4-dihydroxy-1/-/-pyrrole-2,5-dicarboxylate; diisopropyl 3,4-dihydroxy(4-methoxyphenyl)-1/-/-pyrrole-2,5-dicarboxylate; 2-ethyl 5-isopropyl 3,4-dihydroxy(4-methoxyphenyl)-1/-/-pyrrole-2,5-dicarboxylate; di-tert-butyl 3,4-dihydroxy(4-methoxyphenyl)-1/-/-pyrrole-2,5-dicarboxylate; diethyl 3,4-dihydroxy(2-methoxyphenyl)-1/-/-pyrrole-2,5-dicarboxylate; diethyl 1-(4-cyanophenyl)-3,4-dihydroxy-1/-/-pyrrole-2,5-dicarboxylate; diethyl 1-(4-fluorophenyl)hydroxy(2-hydroxyethoxy)-1/-/-pyrrole-2,5-dicarboxylate; diethyl oxy(2-hydroxyethoxy)(4-methoxyphenyl)-1/-/-pyrrole-2,5-dicarboxylate; diethyl 3-(2-(tert-butoxy)oxoethoxy)(4-fluorophenyl)hydroxy-1/-/-pyrrole-2,5- dicarboxylate; diethyl 3-(benzyloxy)(2-(tert-butoxy)oxoethoxy)(4-methoxyphenyl)-1/-/-pyrrole-2,5- dicarboxylate; diethyl 3-(2-aminooxoethoxy)(4-fluorophenyl)hydroxy-1/-/-pyrrole-2,5-dicarboxylate; l 3-(2-(tert-butoxy)oxoethoxy)hydroxy(4-methoxyphenyl)-1/-/-pyrrole-2,5- dicarboxylate; diethyl 3-(2-aminooxoethoxy)hydroxy(4-methoxyphenyl)-1/-/-pyrrole-2,5-dicarboxylate; diethyl 1-(4-(tert-butyl)phenyl)-3,4-dihydroxy-1/-/-pyrrole-2,5-dicarboxylate; and diethyl 3,4-dihydroxy(3-iodomethoxyphenyl)-1/-/-pyrrole-2,5-dicarboxylate; or a pharmaceutically acceptable prodrug derivative of any one thereof, or a pharmaceutically acceptable salt or e of any one thereof. 14. A compound ing to any one of clauses 1 to 13 in the form of a prodrug derivative.
. A compound according to clause 14 n the prodrug derivative is selected from carboxylate ester, sulfamate ester, phosphate ester and ate ester derivatives. 16. A nd according to clause 14 wherein one or both of R4a and R4 are independently selected from -C (0)R 16 , -S0 7R18, n 2NH2 , -PO(OR )(OR20) and -C (0)NR R 6 , R 7 , R 8 , R 9 and R20 are independently selected from (a) C - 6 alkyl, C 2-C 6 alkenyl, C 2-C 6 alkynyl, C3-C10 cycloalkyl, C 5-C 0 cycloalkenyl, heterocyclyl, -CrC 3 C3-C 0 cycloalkyl, -CrC 3 alkyl-C5-C 0 cycloalkenyl or -C C 3 alkylheterocyclyl, in which any of the aforementioned R 6 , R 7 or R 8 groups may be optionally substituted by a group selected from cyano, C C6 , C C6 alkoxy, Ci-C 6 alkyl, C C6 fluoroalkyl and -C(0)N RaR , where Ra and R are independently selected from hydrogen and C C6 alkyl, and any of the aforementioned R 6 , R 7 or R 8 groups groups may be optionally substituted by one or more halogen atoms, and (b) aryl, heteroaryl, C1-C3 alkylaryl and -C1-C 3 alkylheteroaryl, said aryl and heteroaryl groups being optionally substituted; or R 8 , R 9 and R2 may independently represent en. 17. A nd according to clause 14 selected from: 2-(dimethylcarbamoyl)(ethoxycarbonyl)(4-methoxyphenyl)-1/-/-pyrrole-3,4-diyl ate; ethyl 5-(dimethylcarbamoyl)-3,4-bis((dimethylcarbamoyl)oxy)(4-methoxyphenyl)-1/-/-pyrrole- 2-carboxylate; 2-(dimethylcarbamoyl)(ethoxycarbonyl)(4-methoxyphenyl)-1/-/-pyrrole-3,4-diyl bis(4- methylpiperazinecarboxylate); 2-(dimethylcarbamoyl)(ethoxycarbonyl)(4-methoxyphenyl)-1/-/-pyrrole-3,4-diyl bis([1 ,4'- bipiperidine]-1 '-carboxylate); ethyl 5-(dimethylcarbamoyl)((dimethylcarbamoyl)oxy)hydroxy(4-methoxyphenyl)-1/-/- pyrrolecarboxylate; 2-(dimethylcarbamoyl)(ethoxycarbonyl)(4-methoxyphenyl)-1/-/-pyrrole-3,4-diyl bis(2- methylpropanoate); 2-(dimethylcarbamoyl)(ethoxycarbonyl)(4-methoxyphenyl)-1/-/-pyrrole-3,4-diyl bis(2,2- dimethylpropanoate); ethyl ethylcarbamoyl)hydroxy(4-methoxyphenyl)(sulfamoyloxy)-1/-/-pyrrole carboxylate; ethyl 5-(dimethylcarbamoyl)hydroxy(4-methoxyphenyl)(phosphonooxy)-1/-/-pyrrole carboxylate; and 2-(dimethylcarbamoyl)(ethoxycarbonyl)hydroxy(4-methoxyphenyl)-1/-/-pyrrolyl [ 1 ,4'- bipiperidine]-1 '-carboxylate; or a pharmaceutically acceptable salt or solvate of any one thereof. 18. A compound according to clause 1 which is ethyl 5-(dimethylcarbamoyl)-3,4-dihydroxy- 1-(4-methoxyphenyl)-1/-/-pyrrolecarboxylate, or a pharmaceutically acceptable prodrug derivative, or a pharmaceutically acceptable salt or solvate thereof. 19. A pharmaceutical composition comprising a compound ing to any one of clauses 1 to 18, without provisos a) to i), optionally in ation with one or more ceutically acceptable diluents or carriers.
. A pharmaceutical composition according to clause 19 comprising one or more other therapeutically active ingredients. 2 1. A compound according to any one of clauses 1 to 18 without proviso a) to i) for use as a medicament. 22. A compound according to clause 2 1 for use as a ment which is diethyl 3,4- oxy(4-methoxyphenyl)-1/-/-pyrrole-2,5-dicarboxylate or a pharmaceutically acceptable prodrug derivative thereof, or a pharmaceutically acceptable salt or solvate thereof. 23. A compound according to any one of clauses 1 to 18, 2 1 and 22 without provisos a) to i) for use in the ent of bacterial infections caused by bacteria producing pore-forming toxins, such as cholesterol dependent cytolysins. 24. A nd for use according to clause 23 wherein the bacterial infection is caused by ococcus 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 for use according to clause 24 for the treatment of bacterial infection which is caused by Streptococcus niae. 26. A compound for use ing to clause 25 for the treatment of pneumococcal pneumonia, pneumococcal meningitis, pneumococcal septicaemia/bacteraemia, pneumococcal keratitis or pneumococcal otitis media. 27. A compound for use ing to clause 24 for the treatment of conditions selected from gas gangrene, gastrointestinal x, inhalational anthrax, porcine meningitis, encephalitis, septicaemia/bacteraemia and pneumonia which are caused by bacteria other than pneumococcus. 28. A compound for use according to any one of clauses 2 1 to 27 wherein the compound is administered in combination with one or more other eutically active ingredients (e.g. one or more antibiotic agents). 29. A method of ent of bacterial infections caused by bacteria producing pore-forming toxins, such as cholesterol dependent cytolysins which comprises administering to a subject in need thereof an effective amount of a nd according to any one of clauses 1 to 18, 2 1 and 22 without provisos a) to i). 30. A compound of formula (II): (II) wherein R , R2 and R3 are as d in clause 1 for the compounds of formula (I), or a salt or 40 protected derivative thereof; provided that when R5 or R6 is optionally substituted aryl it is optionally substituted by 1 , 2 or 3 groups selected from hydroxyl, halo, cyano, C C alkoxy or C C fluoroalkoxy, C C alkyl or 6 6 6 Ci-C fluoroalkyl, and -C(0)NR aR , where Ra and R are independently selected from hydrogen and C1-C6 alkyl; or when two adjacent hydroxyl substituents are present they may optionally be connected by a methylene group to form an acetal; and provided that the compound is not: a) methyl 2-((2-oxo(phenylamino)ethyl)(phenyl)amino)acetate; b) methyl methoxyphenyl)(2-oxo(phenylamino)ethyl)amino)acetate; c) ethyl 2-(phenyl(tosylmethyl)amino)acetate; d) ethyl 2-((cyanomethyl)(3,4-dichlorophenyl)amino)acetate; e) methyl 2-((cyanomethyl)(p-tolyl)amino)acetate; or f) ethyl 2-(mesityl(2-oxopropyl)amino)acetate. 31. A compound of formula (II) ing to clause 30 wherein R1 is -C(O)NR5R6 and R2 is -C(O)NR5R6 or wherein R1 is -C(O)NR5R6 and R2 is -C(O)OR7. 32. A process for preparing compounds of formula (I) as defined in any one of clauses 1 to 18 in which R4a and R4b represent H which comprises reacting a compound of formula (II) as defined in clause 30 with a compound of formula RxOCOCOORx in which Rx represents C1-C6 alkyl. hout the specification and the claims which follow, unless the context requires otherwise, the word ‘comprise’, and ions 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 nce in their entirety.
The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or ation derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this ication relates.
The application of which this ption and claims forms part may be used as a basis for priority in t 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 t limitation, the claims.
THE

Claims (1)

CLAIMS NG THE INVENTION ARE AS FOLLOWS:
1. A compound of formula (I) or a pharmaceutically acceptable prodrug derivative thereof, or a pharmaceutically acceptable salt or solvate thereof: wherein: R1 and R2 are independently selected from -C(O)NR5R6, -C(O)OR7, CN, -C(O)R7, - C(O)NHC(O)R7, -NO2, -SO3R7, -SO2R7, -SOR7, -SO2NR5R6, -SO2NH-C(O)OR8, -POR21R22 and substituted phenyl or optionally tuted heteroaryl, wherein the heteroaryl is selected from pyrrole, furan, thiophene, oxazole, thiazole, isothiazole, oxadiazole, tetrazole, imidazole, pyrazole, ole, ne, pyridazine, pyrimidine, pyrazine, benzothiophene, benzofuran, 1, 2, 3-triazole and 1, 2, 4-triazole; R3 is optionally substituted phenyl; R4a and R4b are independently selected from hydrogen; C1-C6 alkyl which alkyl group may optionally be substituted by hydroxyl, COOR12 or CONR13R14; aryl and -C1-C3 ryl in which said aryl groups may be optionally substituted; R5 and R6 are independently selected from: (a) hydrogen, (b) C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C10 cycloalkyl, C5-C10 cycloalkenyl, heterocyclyl, -C1-C3 alkyl-C3-C10 cycloalkyl, -C1-C3 alkyl-C5-C10 cycloalkenyl or -C1- C3 alkylheterocyclyl, or R5 and R6 er 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 ed from cyano, C1-C6 alkoxy, C1-C6 fluoroalkoxy, C1-C6 alkyl, C1-C6 fluoroalkyl and -C(O)NRaRb, where Ra and Rb are ndently selected from en and C1-C6 alkyl, and any of the aforementioned R5 and R6 groups may be ally substituted by one or more halogen atoms, and (c) aryl, heteroaryl, C1-C3 alkylaryl and -C1-C3 alkylheteroaryl, said aryl and heteroaryl groups being optionally substituted; R7 is selected from: (a) C1-C6 alkyl, C2C6 alkenyl, C2-C6 alkynyl, C3-C10 cycloalkyl, C5-C10 cycloalkenyl, heterocyclyl, -C1-C3 alkyl-C3-C10 cycloalkyl, -C1-C3 alkyl-C5-C10 cycloalkenyl or -C1- C3 alkylheterocyclyl, in which any of the aforementioned R7 groups may be optionally substituted by a group selected from cyano, C1-C6 alkoxy, C1-C6 fluoroalkoxy, C1-C6 H:\rec\Interwoven\NRPortbl\DCC\REC\7918383_1.docx-
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