Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
  • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
  • C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
  • C07D311/22—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4
  • C07D311/24—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 4 with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached in position 2
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P33/00—Antiparasitic agents
  • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
  • C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
  • C07D405/12—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings linked by a chain containing hetero atoms as chain links
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D407/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen atoms as the only ring hetero atoms, not provided for by group C07D405/00
  • C07D407/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen atoms as the only ring hetero atoms, not provided for by group C07D405/00 containing two hetero rings
  • C07D407/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen atoms as the only ring hetero atoms, not provided for by group C07D405/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
  • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

• the present invention relates to a new class of anti-infective agents, to their use in medicine, to compositions containing them, to processes for their preparation and to intermediates used in such processes.
• the present invention provides chromone-2-carboxamide compounds for use in the treatment or prevention of infectious diseases including: Malaria; Cryptosporidiosis; tuberculosis (TB); Schistosomiasis, African sleeping sickness (HAT and/or AAT); Chagas disease; and/or Leishmaniasis, and in the treatment or prevention of bacterial infections including: bacterial infections stemming from Streptococcus pneumonia , and/or Enterococcus ; or bacterial infections stemming from the ESKAPE bacterial species.
• Malaria is a devastating disease with over 214 million clinical cases in 2015. In 2015, there have been an estimated 438 000 deaths attributed to malaria (WHO, Malaria Report 2015) mostly amongst children under five in sub-Saharan Africa. Malaria is caused by an infection of the red blood cells by a protozoa parasite. Five species of the protozoa Plasmodium are known to cause infection in humans: Plasmodium falciparum; Plasmodium vivax; Plasmodium ovale; Plasmodium malariae ; and Plasmodium knowlesi .
• Plasmodium falciparum Plasmodium falciparum
• Plasmodium vivax Plasmodium ovale
• Plasmodium malariae Plasmodium malariae
• the most life-threatening form of malaria is attributable to blood cells infected with the Plasmodium falciparum parasite, and can cause kidney or liver failure, coma and death. It is estimated that one child dies every minute from falciparum malarial infections therefore the need for an effective treatment could not be higher.
• agents which are: effective against Plasmodium falciparum infections; effective against Plasmodium falciparum and Plasmodium vivax infections; effective against Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale and Plasmodium knowlesi infections.
• Plasmodium species require two hosts, human and mosquito for completion of its life-cycle.
• the infection is initiated by the inoculation of sporozoites in the saliva of an infected mosquito.
• the sporozoites migrate to the liver and there infect hepatocytes where they differentiate, via the exoerythrocytic intracellular stage, into the merozoite stage which infects red blood cells to initiate cyclical replication in the asexual blood stage.
• the life-cycle is completed by the differentiation of a number of merozoites in the red blood cells into sexual stage gametocytes which are ingested by the mosquito, where they develop through a series of stages in the mid gut to produce sporozoites which migrate to the salivary gland.
• new and effective anti-malarial agents with novel modes of action.
• new anti-malarial agents which: are effective against drug-resistant parasites; are effective against drug-resistant Plasmodium falciparum infections such as for example chloroquine-resistant Plasmodium falciparum infections; which are active against liver stage; which are active against the hypnozoite form; and/or which can be used for single-dose treatment; and/or which can be used for prophylactic treatment.
• Cryptosporidiosis is a diarrhoeal disease caused by the parasite species Cryptosporidium .
• Cryptosporidium Currently, there are 27 recognized species of Cryptosporidium , including 20 species infecting humans with C. parvum or C. hominis being responsible for the majority of human infections, Int. J. For Parasitology 2015, 45, 367-373.
• Cryptosporidiosis was first identified as a cause of human infection in 1976, Gastroenterology, 1976, 70, 592-598.
• Cryptosporidiosis is an opportunistic infection and individuals with underdeveloped immune systems, such as children under 5 years and immunocompromised individuals with HIV co-infection are at a higher risk of infection and mortality. Malnutrition in early childhood is also associated with persistent diarrhoea and Cryptosporidium infection, Lancet Infect. Dis., 2015, 15, 85-94. Nitazoxanide is the only FDA-approved drug for the treatment of Cryptosporidisosis.
• Nitazoxanide was also shown to be ineffective in clinical trials for Cryptosporidium -HIV co-infected patients, whom were not co-treated with HIV antiretroviral therapy, Trans. R. Soc. Trop. Med. Hyg. 1998, 92, 663-66 and BMC Infect. Dis. 2009, 9, 195.
• Leishmaniasis is caused by several leishmania species transmitted to hosts (humans and animals) by the bites of infected female phlebotomine sandflies.
• leishmaniasis There are three main human forms of leishmaniasis, visceral (the most serious form of the disease), cutaneous (the most common), and mucocutaneous (the most disfiguring). Most leishmaniases can be transmitted from animals to humans and the reservoir hosts include many species of mammals. Dogs are important reservoirs of Leishmania infantum ( L. infantum ) which is one of the species responsible for visceral leishmaniasis. Animals can also suffer from visceral, cutaneous and mucocutaneous forms of the disease.
• guyanensis L. v. panamensis, L. v, peruviana which are associated with other forms of the disease and in particular with the causation of cutaneous leishmaniasis; Leishmania v. braziliensis, L. v. guyanensis and L . v. panamensis which can also cause muco-cutaneous leishmaniasis.
• Chagas disease is due to the protozoan parasite Trypanosoma cruzi .
• the main route of transmission to humans and other mammals is by infected faeces of a blood-sucking triatominae bug. However it can also be transmitted from mother to un-born child and through blood transfusion with contaminated blood.
• Chagas disease is endemic throughout Central America and South America where an estimated 7 to 8 million people are infected. Migration of populations from endemic countries has increased the geographic distribution of Chagas disease, with a rising number of Chagas disease cases in the USA, Canada and in many parts of Europe. Approximately there are 13000 deaths each year due to Chagas-induced heart disease caused by Chagas chronic infection.
• HAT Human African Trypanosomiasis
• HAT Human African Trypanosomiasis
• tik-tik Human African Trypanosomiasis
• Trypanosoma brucei gambiense T.b. gambiense
• Trypanosoma brucei rhodesiense T.b. rhodesiense
• Trypanosoma brucei gambiense which is prevalent in west and central Africa, represents approximately 95% of the reported cases of sleeping sickness and causes a chronic infection.
• Trypanosoma brucei rhodesiense which is found in eastern and southern Africa and represents approximately 5% of the reported cases.
• stage 1 presents with non-specific symptoms including fever, rash, and fatigue. Untreated stage 1 HAT results in stage 2 disease or neurological phase, where parasites invade the central nervous system causing severe neurological symptoms and eventually death.
• Stage 1 of the disease is treated with intravenous or intramuscular pentamidine, for T. b. gambiense , or intravenous suramin, for T. b. rhodesiense .
• Stage 2 of the disease is treated with intravenous melarsoprol, or intravenous melarsoprol in combination with oral nifurtimox, or intravenous eflornithine only, or eflornithine in combination with nifurtimox. All four drugs, whether used individually or in these combination therapies have serious adverse effects. As such, new, safer and more efficacious treatments for HAT are urgently needed.
• Animal trypanosomiasis is also known as animal African trypanosomiasis (AAT), and is a disease of vertebrate non-human animals.
• Human African trypanosomiasis (HAT) is commonly known as sleeping sickness.
• Animal trypanosomiasis is caused by various parasite species and sub-species of the Trypanosoma genus, trypanosomes which are pathogenic to animals, including Trypanosoma congolense, Trypanosoma vivax, Trypanosoma brucei, Trypanosoma simiae, Trypanosoma godfreyi, Trypanosoma suis , and Trypanosoma evansi .
• HAT Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense.
• Trypanosomes are protozoan parasites in the family Trypanosomatidae and most trypanosomes are transmitted by tsetse flies with the trypanosomes infecting the blood of the animal. As such, an infected animal can act as a disease reservoir with resultant potential for further disease spread in areas affected by the tsetse fly. In Africa, the disease is most common in areas affected by tsetse flies and is spread by the bite of an infected tsetse or other infected flies. Many different animals can be infected by animal trypanosomiasis, including domestic livestock, such as cattle, goats, pigs, sheep and camels. Wild animals, including elephants and leopards have also been found to have trypanosomiasis. Different parasites affect different ranges of organism.
• T. vivax Trypanosoma vivax
• Spickler “African Animal Trypanosomiasis” http://www.cfsph.iastate.edu/Diseaselnfo/factsheets.php
• T. vivax is also found in South and Central America and in the Caribbean with the associated potential risk of transmission to animals via mechanical vectors in these regions.
• Schistosomiasis is a deadly neglected infectious disease caused by parasitic trematode worms of the genus Schistosoma (mainly Schistosoma haematobium, S. mansoni or S. japonicum ). WHO estimates show that at least 218 million people required preventive treatment for schistosomiasis in 2015 (WHO, http://www.who.int/schistosomiasis/en/) The number of deaths due to schistosomiasis estimated by WHO are approximately 200.000 globally each year. The economic and health effects of Schistosomiasis are considerable due to the disability caused by the disease. In children, schistosomiasis can cause anemia, stunting and reduced ability to learn. Schistosomiasis can also exacerbate the effects of malaria, tuberculosis, HIV and hepatitis, see Lancet, 2014; 383: 2253-64.
• PZQ Praziquantel
• PZQ Whilst PZQ is effective against adult schistosomes worms it has poor activity against immature schistosome larvae, Lancet, 2014; 383: 2253-64. In areas of constant reinfection, repeated treatment 3-6 weeks apart are required to kill resistant juvenile worms and to improve drug treatment. Resistance to PZQ can be induced experimentally, Trans. R. Soc. Trop. Med. Hyg. 2002: 96, 465-69. The threat of emerging resistance to PZQ caused by mass monotherapy is a major concern. These factors create an unmet medical need.
• Tuberculosis is a bacterial infection caused by Mycobacterium tuberculosis and is easily spread from person-to-person when an subject with an active TB infection expels droplets containing the TB bacteria from their lungs via coughing or sneezing. TB is the second leading cause of death due to an infectious agent. It has been reported that in 2013, 9 million people developed TB, which resulted in 1.5 million deaths (WHO 2014).
• MDR-TB multi-drug-resistant form of TB
• Bacteria involved in human disease include gram-negative bacteria, Neisseria gohorrhoease, Klebsielle, Acinetobacter, Pseudomonas aeruginosa, E. coli ., and Yersinia pestis , and gram-positive bacteria Streptococcus, Staphylococcus, Corynebacterium, Listeria (a coccobacillus), Bacilus and Clostridium.
• Gram-negative bacteria cause infections including pneumonia, bloodstream infections, wound or surgical site infections, and meningitis in healthcare settings. Gram-negative bacteria are resistant to multiple drugs and are increasingly resistant to most available antibiotics. Gram-positive bacteria cause infections including Anthrax, septicemia and meningitis.
• bacteria examples include methicillin resistant Staphylococcus aureus (MRSA), penicillin resistant Streptococcus pneumoniae , and vancomycin resistant Enterocossus .
• MRSA methicillin resistant Staphylococcus aureus
• Streptococcus pneumoniae penicillin resistant Streptococcus pneumoniae
• vancomycin resistant Enterocossus vancomycin resistant Enterocossus .
• ESKAPE group of bacterial species Enterococcus faecium , which also includes Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa , and Enterobacter species.
• Lysyl t-RNA Synthetase (LysRS or KRS1)
• aaRS Aminoacyl-tRNA synthetases
• MRSA methicillin-resistant Staphylococcus aureus
• cladosporin is poorly bioavailable which is a key requirement for any active therapeutic agent, not only for a potential anti-malarial agent.
• the present invention provides a novel class of class of anti-infective agents which are chromone-2-carboxamide compounds and Plasmodium falciparum 3D7 inhibitors having potential as treatment of infectious diseases and especially Malaria, Cryptosporidiosis, tuberculosis (TB), Schistosomiasis, African sleeping sickness (HAT), African animal trypanosomiasis (AAT), Chagas disease and Leishmaniasis.
• novel class of chromone-2-carboxamide compounds according to the present invention have potential for the treatment of Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae and Plasmodium knowlesi infections.
• novel class of anti-infective agents according to the present invention have potential for the treatment of: Plasmodium falciparum infections; Plasmodium falciparum and Plasmodium vivax infections; Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae and Plasmodium knowlesi infections; drug-resistant Plasmodium falciparum infections, such as for example Chloroquine-resistant Plasmodium falciparum infections; which are active against liver stage schizont forms; activity against liver stage dormant forms of P. vivax ; which are active against the hypnozoite form; and/or which can be used for single-dose treatment; and/or which can be used for prophylactic treatment.
• novel class of chromone-2-carboxamide compounds according to the present invention have potential for the treatment of Cryptosporidium infections.
• novel class of anti-infective agents according to the present invention have potential for the treatment of: Cryptosporidium -only infected subjects; immunocompromised subjects infected with Cryptosporidium such as Cryptosporidium -HIV co-infected subjects.
• novel class of chromone-2-carboxamide compounds according to the present invention have potential for the treatment of new anti-infective agents which are effective against Leishmania infections.
• novel class of anti-infective agents according to the present invention have potential for the treatment of: Leishmania infantum infections; subjects infected with: of: Leishmania donovani, Leishmania infantum and/or L chagasi , which are all causes of visceral leishmaniasis; L. mexicana, L. amazonensis, L. venezuelensis, L. tropica, L. major, L. aethiopica, Leishmania viannia braziliensis, L . v. guyanensis, L . v.
• novel class of chromone-2-carboxamide compounds according to the present invention have potential for the treatment of Chagas disease.
• novel class of class of anti-infective agents according to the present invention have potential for the treatment of Trypanosoma cruzi . infections.
• novel class of chromone-2-carboxamide compounds according to the present invention have potential for the treatment of Schistosomiasis.
• novel class of class of anti-infective agents according to the present invention have potential for the treatment of: Schistosoma haematobium, Schistosoma mansoni and Schistosoma japonicum infections.
• novel class of chromone-2-carboxamide compounds according to the present invention have potential for the treatment of African sleeping sickness (HAT).
• novel class of class of anti-infective agents according to the present invention have potential for the treatment of: Trypanosoma brucei gambiense ( T.b. gambiense ) and/or Trypanosoma brucei rhodesiense ( T.b. rhodesiense ) infections.
• novel class of chromone-2-carboxamide compounds according to the present invention have potential for the treatment of tuberculosis (TB).
• novel class of class of anti-infective agents according to the present invention have potential for the treatment of TB via: reducing the duration of treatment required; provision of effective treatment of Mycobacterium tuberculosis infections; provision of effective treatment of TB, MDR, and/or XDR-TB; that are effective for use as a single drug therapy; are effective for use in combination with one or more existing drug therapies.
• the present invention provides a novel class of class of anti-infective agents which are chromone-2-carboxamide compounds having potential as new antibiotics for use in the treatment or prevention of Gram negative and/or Gram positive bacterial infections and in particular for use in the effective treatment of bacterial infections stemming from: one or more of Streptococcus pneumonia ; and/or Enterococcus ; or one or more of the ESKAPE group of bacterial species, Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa , and/or Enterobacter.
• Desirable properties of compounds of formula (I) according to the present invention include: potency against Pf-KRS1; potency against Plasmodium falciparum 3D7; potency against Mtb; potency against Cryptosporidium parvum ; potency against Leishmani donovani; Plasmodium falciparum 3D7; Plasmodium falciparum 3D7; Plasmodium falciparum 3D7; worms of the genus Schistosoma , such as Schistosoma haematobium, Schistosoma mansoni, Schistosoma japonicum ; low toxicity in MRC-5 or HepG2 cells; both desirable Plasmodium falciparum (Pf) 3D7 potency and low toxicity in MRC-5 or HepG2; desirable Plasmodium falciparum and Plasmodium vivax (Pv) activity against clinical isolates; desirable transmission blocking activity; gametocyte inhibitory potential; activity against dormant liver stage forms;
• the present invention additionally provides preferred compounds of formula I, and in particular the compounds of formulae C-I, C-II, C-III, A-I, A-II, S-I, S-II, and S-III as defined hereinafter.
• moieties are described as being “each independently” selected from a list of atoms or groups, this means that the moieties may be the same or different. The identity of each moiety is therefore independent of the identities of the one or more other moieties.
• alkyl groups having two or more carbon atoms may be unsaturated or saturated, and are preferably saturated; alkyl groups having three or more carbon atoms, may be straight chain or branched chain.
• a C 3 alkyl substituent can be in the form of normal-propyl (n-propyl), or iso-propyl (i-propyl).
• the chromone scaffold or a cyclic or heterocyclic R 7 group is optionally substituted by an alkyl group said alkyl substitutent group(s) may not be further substituted by further (unsubstituted) alkyl groups.
• optionally substituted as used herein indicates that the particular group or groups may have one or more non-hydrogen substituents.
• the total number of such substituents which may be present is equal to the number of H atoms present on the unsubstituted form of the particular group.
• pharmaceutically acceptable includes reference to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings or animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. This term includes acceptability for both human and veterinary purposes.
• the present invention provides compounds of formula (I):
• the compounds of formula C-I are compounds of formula I; wherein n is 0, 1, 2 or 3; wherein X is a bond, an —O-link, or an —S-link; wherein R 1 to R 6 and R x are as defined in accordance with formula (I) hereinbefore; wherein when R p is CH 3 n is 0 or 1; or a veterinarily or pharmaceutically acceptable, salt, hydrate, solvate, isomer, prodrug or polymorph thereof.
• R 1 to R 6 are all H, —(CH p ) n —X—R x is not —(CH 2 )-cyclohexyl.
• the compounds of formula C-II are compounds of formula I wherein R 1 , R 5 and R 6 are all H; wherein n is 1; X is a bond; R p is H; wherein R 2 is H, OH, F, Cl, Br, —CH 3 , —CH 2 CH 3 , —OCH 3 , or —ON; wherein R 3 is H, F, or Cl; wherein R 4 is H, F, OH, —CH 2 OH, —C(O)OH, —NH 2 , —NHSO 2 CH 3 , or a 1H-tetrazol-5-yl group; wherein R x is a C-linked 6-membered saturated cycloalkyl group, which is optionally substituted with one or more groups independently selected from: OH, or F; or a veterinarily or pharmaceutically acceptable, salt, hydrate, solvate, isomer, prodrug or polymorph thereof.
• R 2 to R 4 are all H, —(CH)
• the compounds of formula C-III are compounds of formula I wherein R 1 is H; wherein n is 1 or 2; wherein X is a bond; wherein R x is a cyclohexyl, cyclopentyl, cyclobutyl, tetrahydropyranyl, or a benzoxazole group, wherein each of said R x groups may be optionally substituted by one or more groups independently selected from: OH, CH 3 , or F; wherein R p is H or CH 3 and wherein when R p is H, n is 1 or 2, and wherein when R p is CH 3 , n is 1; wherein R 1 (not shown) is H; wherein R 2 is H, OH, F, Cl, Br, —CH 3 , —CH 2 CH 3 , —OCH 3 , or —CN; wherein R 3 is H, OH, F, Cl, or —O(CH 2 ) 2 NH 2 ; wherein R 4 is H,
• the compounds of formula A-I are compounds of formula I wherein R 1 , R 3 , R 5 and R 6 are all H; wherein n, R 2 and R 4 are as defined in accordance with formula I hereinbefore, and wherein R x is as defined in accordance with formula (I) hereinbefore, or a veterinarily or pharmaceutically acceptable, salt, hydrate, solvate, isomer, prodrug or polymorph thereof.
• R 2 and R 4 are H and n is 1, R x is not cyclohexyl.
• the compounds of formula A-II are compounds of formula I wherein R 1 , R 2 , R 3 , R 5 and R 6 are all H; wherein n, R 2 and R 4 are as defined in accordance with formula I hereinbefore, and wherein R x is as defined in accordance with formula (I) hereinbefore, or a veterinarily or pharmaceutically acceptable, salt, hydrate, solvate, isomer, prodrug or polymorph thereof.
• R 2 is H and n is 1, R x is not cyclohexyl.
• the compounds of formula S-I are compounds of formula I wherein R 4 is an —N(R 10 )SO 2 R 10 group; wherein R 1 , R 2 , R 3 , R 5 , R 6 , R 7 and R 10 are as defined in accordance with formula (I) hereinbefore, or a veterinarily or pharmaceutically acceptable, salt, hydrate, solvate, isomer, prodrug or polymorph thereof.
• the compounds of formula S-II are compounds of formula I wherein R 1 , R 3 , R 5 and R 6 are all H and R 4 is an —N(R 10 )SO 2 R 10 group; wherein R 2 , R 7 and R 10 are as defined in accordance with formula (I) hereinbefore, or a veterinarily or pharmaceutically acceptable, salt, hydrate, solvate, isomer, prodrug or polymorph thereof.
• the compounds of formula S-III are compounds of formula I wherein R 1 , R 3 , R 5 and R 6 are all H, R 4 is an —N(R 10 )SO 2 R 10 group, and R 7 is a cyclohexylmethyl group; wherein said cyclohexylmethyl (R 7 ) group may be optionally substituted by one or more R o groups independently selected from: OH, CH 3 , or F; wherein R 2 and R 10 are as defined in accordance with formula (I) hereinbefore, or a veterinarily or pharmaceutically acceptable, salt, hydrate, solvate, isomer, prodrug or polymorph thereof.
• Particularly preferred compounds according to the present invention are compounds of formula (I)
• R 1 is H; wherein R 2 is OH, Cl, F or CH 3 , preferably OH, Cl or F; wherein R 3 is H, Cl or F, preferably H; wherein R 5 is H or CH 3 , preferably H; wherein R 6 is H or CH 3 , preferably H; wherein n is 1 or 2; wherein R p is H or CH 3 , preferably H; wherein R x is cyclohexyl optionally substituted at C-1 by OH or CH 2 OH OH and/or at C-4 with one or more F groups, or wherein R x is a cyclopentyl, tetrahydropyranyl, norbornanyl, spiro[3.3]heptanyl, dihydrobenzo[b][1,4]dioxinylyl, or a dihydrobenzo[b][1,3]dioxayl group wherein said groups are optionally substituted at the C-1 position by OH, or CH 2 CO 2 H
• Preferred halogen substituents for use here are F, Cl and/or Br, more preferably F and Cl, most especially F.
• Example 54 N-[(4,4-difluoro-1-hydroxy-cyclohexyl)methyl]-6-fluoro-4-oxo-chromene-2-carboxamide and pharmaceutically and veterinarily acceptable, acid salts, hydrates, solvates, isomers, pro-drugs or polymorphs thereof.
• Example 70 N-(cyclohexylmethyl)-6-fluoro-8-hydroxy-4-oxo-4H-chromene-2-carboxamide and pharmaceutically and veterinarily acceptable, acid salts, hydrates, solvates, isomers, pro-drugs or polymorphs thereof.
• Example 74 6-fluoro-8-hydroxy-N-((1-hydroxycyclohexyl)methyl)-4-oxo-4H-chromene-2-carboxamide and pharmaceutically and veterinarily acceptable, acid salts, hydrates, solvates, isomers, pro-drugs or polymorphs thereof.
• Example 77 Fluoro-8-hydroxy-N-((1-hydroxycyclohexyl)methyl)-4-oxo-4H-chromene-2-carboxamide and pharmaceutically and veterinarily acceptable, acid salts, hydrates, solvates, isomers, pro-drugs or polymorphs thereof.
• Example 79 N-((4,4-difluorocyclohexyl)methyl)-7-fluoro-8-hydroxy-4-oxo-4H-chromene-carboxamide and pharmaceutically and veterinarily acceptable, acid salts, hydrates, solvates, isomers, pro-drugs or polymorphs thereof.
• Example 81 8-fluoro-6-hydroxy-N-((1-hydroxycyclohexyl)methyl)-4-oxo-4H-chromene-2-carboxamide and pharmaceutically and veterinarily acceptable, acid salts, hydrates, solvates, isomers, pro-drugs or polymorphs thereof.
• Example 54 N-[(4,4-difluoro-1-hydroxy-cyclohexyl)methyl]-6-fluoro-4-oxo-chromene-2-carboxamide;
• Example 77 Fluoro-8-hydroxy-N-((1-hydroxycyclohexyl)methyl)-4-oxo-4H-chromene-2-carboxamide;
• Example 78 N-(cyclohexylmethyl)-7-fluoro-8-hydroxy-4-oxo-4H-chromene-2-carboxamide, Example 74.
• Example 100 N-(cyclohexylmethyl)-6-fluoro-8-(methanesulfonamido)-4-oxo-chromene-2-carboxamide;
• Example 99 8-amino-N-(cyclohexylmethyl)-6-fluoro-4-oxo-chromene-2-carboxamide;
• Example 97 8-amino-6-fluoro-N-[(1-fluorocyclohexyl)methyl]-4-oxo-4H-chromene-2-carboxamide;
• Example 96 8-amino-6-fluoro-N-[(1-hydroxycyclohexyl)methyl]-4-oxo-4H-chromene-2-carboxamide;
• Example 93 8-amino-N-[(4,4-difluoro-1-hydroxy-cyclohexyl)methyl]-6-fluoro-4-oxo-chromene-2-carboxamide;
• Example 92 8-amino
• compositions of certain compounds of the formula (I) may be readily prepared in a conventional manner by mixing together solutions of a compound of the formula (I) and the desired acid, as appropriate. For example, a solution of the free base is treated with the appropriate acid, either neat or in a suitable solvent, and the resulting salt isolated either by filtration or by evaporation under reduced pressure of the reaction solvent.
• suitable salts see “Handbook of Pharmaceutical Salts: Properties Selection, and Use” by Stahl and Wermuth (Wiley-VCH, Weinheim, Germany, 2002).
• Suitable acid addition salts for use herein include: fumarate, acetate, adipate, aspartate, benzoate, besylate, bicarbonate/carbonate, bisulphate/sulphate, borate, camsylate, citrate, cyclamate, edisylate, esylate, formate, fumarate, gluceptate, gluconate, glucuronate, hexafluorophosphate, hibenzate, hydrochloride/chloride, hydrobromide/bromide, hydroiodide/iodide, isethionate, lactate, malate, maleate, malonate, mesylate, methylsulphate, naphthylate, 2-napsylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate/hydrogen phosphate/dihydrogen phosphate, pyroglutamate, saccharate, stearate, succinate
• the compounds of the invention may exist in a continuum of solid states ranging from fully amorphous to fully crystalline.
• the compounds of the invention may also exist in unsolvated and solvated forms.
• solvate as used herein describes a molecular complex comprising the compound of the invention and one or more pharmaceutically acceptable solvent molecules, for example, ethanol.
• hydrate is employed when said solvent is water.
• multi-component complexes other than salts and solvates
• Complexes of this type include clathrates (drug-host inclusion complexes) and co-crystals.
• references to compounds of formula (I) include references to salts, solvates, and multi-component complexes.
• the compounds of the invention include compounds of formula (I) as hereinbefore defined, and polymorphs and crystal habits thereof.
• Isomers of compounds of formula (I) as used herein, and included in the present invention include optical, geometric and tautomeric isomers. Stereoisomers such as enantiomers and diastereomers, all geometric isomers and tautomeric forms of the compounds of formula (I), including compounds exhibiting more than one type of isomerism, and mixtures of one or more thereof are included in the present invention. Also included are acid addition salts wherein the counterion is optically active, for example, d-lactate or l-lysine, or racemic, for example, dl-tartrate or dl-arginine. Geometric isomers may be separated by conventional techniques well known to those skilled in the art, for example, by chromatography and fractional crystallisation. Stereoisomers may be separated by conventional techniques known to those skilled in the art—see, for example, “Stereochemistry of Organic Compounds” by E L Eliel (Wiley, New York, 1994).
• prodrugs of the present compounds are also within the scope of the invention.
• certain derivatives of compounds of formula (I) which may have little or no pharmacological activity themselves, can, when administered into or onto the body, be converted into compounds of formula (I) having the desired activity, for example, by hydrolytic cleavage.
• Such derivatives are referred to as ‘prodrugs’.
• Further information on the use of prodrugs may be found in Pro - drugs as Novel Delivery Systems , Vol. 14, ACS Symposium Series (T Higuchi and W Stella) and Bioreversible Carriers in Drug Design , Pergamon Press, 1987 (Ed. E B Roche, American Pharmaceutical Association).
• Prodrugs in accordance with the invention can, for example, be produced by replacing appropriate functionalities present in the compounds of formula (I) with certain moieties known to those skilled in the art as ‘pro-moieties’ as described, for example, in Design of Prodrugs by H Bundgaard (Elsevier, 1985). Finally, certain compounds of formula (I) may themselves act as prodrugs of other compounds of formula (I).
• metabolites of compounds of formula I that is, compounds formed in vivo upon administration of the drug.
• An example of a metabolite in accordance with the invention is a phenol derivative of a compound of formula I (-Ph->-PhOH).
• the present invention includes all pharmaceutically acceptable isotopically-labelled compounds of formula (I) wherein one or more atoms are replaced by atoms having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number usually found in nature.
• Isotopically-labelled compounds of formula (I) can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described in the accompanying Examples and Preparations using an appropriate isotopically-labelled reagent in place of the non-labelled reagent previously employed.
• suitable reagents for effecting the chemical transformations from a 2-hydroxyacetophenone starting material of general formula (II) in steps a to c to furnish intermediate chromone acid compounds of general formula (III) are as follows: a. EtONa, diethyl oxalate, EtOH, 80° C. then HCl 37%, 90° C.; b. AcOH, HCl 37%, 90° C.; bb. AcOH, HBr, 90° C.; c. LiOH, water.
• suitable reagents for effecting the chemical transformations from an intermediate chromone acid compound of general formula (III) via an amidation reaction to provide using any one of steps d, e, f, g, h or i, to furnish chromone compounds of general formula (I) having the desired “R 7 ” group in the final product are as follows: d. PyBOP, DIPEA, DCM, R 7 NH 2 ; e. C(O)Cl 2 , DCM, a drop of DMF, R 7 NH 2 ; f. COMU, DIPEA, ACN, R 7 NH 2 ; g. CDMT, NMO, DCM, R 7 NH 2 ; h.
• Scheme 1A illustrates the same overall process as Scheme 1, with the further illustration of the acid-catalysed cyclization of a non-isolated intermediate to provide a chromone ester intermediate.
• R 5 can be any of the R 5 substituent groups indicated in the definition of formula (I) hereinbefore.
• R 6 is H and again R 6 can be any of the R 6 substituent groups indicated in the definition of formula (I) hereinbefore.
• the present invention provides a process for the preparation of chromone-2-carboxamide compounds of general formula (I) comprising amination of a chromone acid intermediate of general formula (III), and optionally wherein said process additionally provides for the preparation of the chromone acid intermediate via hydrolysis of a chromone ester intermediate of general formula (II).
• n is 0, 1, 2 or 3; wherein X is a bond, an —O-link, or an —S-link; wherein R 1 to R 6 and R x are as defined in accordance with formula (I) hereinbefore; wherein when R p is CH 3 n is 0 or 1.
• the present invention provides a general process for the preparation of chromone-2-carboxamide compounds wherein wherein n is 0, 1, 2 or 3; wherein X is a bond, an —O-link, or an —S-link; wherein R 1 to R 6 and R x are as defined in accordance with formula (I) hereinbefore; wherein when R p is CH 3 n is 0 or 1 of general formula (C-I) comprising amination of a chromone acid intermediate of general formula (III), and optionally wherein said process additionally provides for the preparation of the chromone acid intermediate via hydrolysis of a chromone ester intermediate of general formula (II).
• suitable reagents for effecting the chemical transformations for the conversion of the protected intermediate-oxyamine compounds of general formula (IV) into further secondary amine intermediate compounds of general formula (V) wherein R 4 is a de-protected salt form of the R 4 group (from the corresponding N—BOC protected compounds of general formula (IV)) is as follows: c. HCl 4M in dioxane.
• suitable reagents for effecting the chemical transformations for the conversion of the amine-salt intermediate compounds of general formula (V) into compounds of general formula (I) wherein R 4 is an —O(CH 2 ) n NR 11 R 12 group is as follows: d. formaldehyde 37%, formic acid, reflux, 1 h to 8 h.
• the present invention provides a process for the preparation of compounds of general formula (I) via the process as illustrated in Scheme 2.
• Scheme 3 illustrates an alternative route for the preparation of chromone-2-carboxamide compounds of general formula (I) starting from phenol or substituted phenol groups.
• suitable reagents for effecting the chemical transformations from the starting phenolic material in steps a to to furnish a mixture of intermediate ester compounds of general formulae (VIA) and (VIB) are as follows: a. dimethyl but-2-ynedioate, Et 3 N, DCM, 25° C., 1 h.
• suitable reagents for effecting the chemical transformations from the mixture of intermediate ester compounds of general formulae (VIA) and (VIB) to provide a mixture of further intermediate olefin di-acid compounds of general formulae (VIIA) and (VIIB) are as follows: b. NaOH or LiOH, THF/H 2 O, 40° C., 3 h.
• suitable reagents for effecting the chemical transformations to convert the mixture of intermediate olefin di-acid compounds of general formulae (VIIA) and (VIIB) into chromone-2-carboxylic acid intermediate compounds of general formulae (VIII) are as follows: c. H 2 SO 4 , acetyl chloride, 50° C., 1 h.
• suitable reagents for effecting the chemical transformations to convert the chromone-2-carboxylic acid intermediate compounds of general formulae (VIII) into chromone-2-carboxamide compounds of general formula (I) via reaction with a suitable amine substrate for the desired “R 7 ” group in the final product using either one of steps d, or e are as follows: d. HATU, DIPEA, R 7 NH 2 , 60° C., 15 h; e. C(O)Cl 2 , DCM, drop of DMF, R 7 NH 2 .
• Scheme 3A illustrates the same overall process as Scheme 3, where intermediates labelled A and B are consolidated into a single intermediate structure and also includes suitable reagents for each transformational step.
• chormone-2-carboxamides were synthesised from either phenol or commercially available substituted phenols.
• Treatment of the corresponding phenol with dimethylacetylenedicarboxylate (DMAD, dimethyl but-2-yenedioate) followed by ester hydrolysis gave a mixture of olefin diacids.
• DMAD dimethylacetylenedicarboxylate
• ester hydrolysis methods are provided hereinafter and are disclosed in Lynch et al. J. Med. Chem. 2006, 49: 6569. Ring closure was achieved by heating this mixture with sulfuric acid and acetyl chloride to obtain chomone-2-carboxylic acids.
• the present invention provides a process for the preparation of chromone-2-carboxamide compounds of general formula (I) from phenol or a substituted phenol via the process as illustrated in Scheme 3, or Scheme 3A, wherein said substituted phenol has one, two, three or four substitutent groups, and wherein those substituent groups are R 1P , R 2P , R 3P , and R 4P groups wherein R 1 , R 2P , R 3P , and R 4P correspond to R 1 , R 2 , R 3 and R 4 as defined hereinbefore, both in respect of their functionality and their positions on the phenyl-ring relative to the final chromone-2-carboxamide compounds of formula (I).
• the acid intermediate compounds of general formula (VIII) are firstly converted to their corresponding esters (step 1, transformation a) i.e.
• step 1 the intermediates of general formula (IX) having a 8-methoxy substituent, and thereafter are converted to the corresponding 8-hydroxy containing ester intermediates of general formula (X) (step 1, transformation b) prior to reaction with the desired amine having an R 7 group to provide the final 8-hydroxy-4-oxo-4H-chromene-2-carboxamides compounds of general formula (I) in final step, transformation c.
• 6-bromo-8-nitro intermediate compounds of general formula (XI) are firstly converted to their corresponding amines (step 1, transformation a) i.e. compounds of general formula (I) having an R 4 /8-amino substituent, which can thereafter be converted to the corresponding R 4 /8-sulfonamino compounds of general formula (I) (step 2, transformation b).
• 8-bromo-8 intermediate compounds of general formula (XII) are firstly converted to their corresponding cyano analogues (step 1, transformation a) i.e. compounds of general formula (I) having an R 4 /8-cyano substituent, which can thereafter be converted to the corresponding R 4 /8-HET compounds of general formula (I) (step 2, transformation b).
• Scheme 7 illustrates a synthetic approach for the interconversion of 8-substituted chromone-2-carboxamide compounds of general formula (I) into further 8-substituted chromone-2-carboxamide compounds of general formula (I).
• 8-bromo-compounds of general formula (I) are firstly converted to their corresponding methylesters, (step 1, transformation a) i.e. compounds of general formula (I) having an R 4 /8-alkyl-ester substituent, which can thereafter be converted to the corresponding R 4 /8-carboxylates (step 2, transformation b), and then R 4 /8-carbamoyl compounds of general formula (I) (step 3, transformation c).
• Scheme 8 illustrates a synthetic approach for the preparation of 3-substituted chromone-2-carboxamide compounds of general formula (I).
• 3-substituted compounds of general formula (I) are prepared via the reaction of a suitable acid-intermediate (which contains the desired substituent group in the 3-position relative to the final compound) of general formula (XIV) and a suitable amine-intermediate of general formula (XV).
• the acid intermediate can be prepared from commercially available starting materials via preparation of an ester-substituted chromone (step 1, transformation a) which is converted to the corresponding acid-substituted chromone (step 2, transformation b), and then combined with the amine-intermediate to furnish 3-substituted chromone-2-carboxamide compounds of general formula (I) (step 3, transformation c).
• Scheme 9 illustrates a synthetic approach for the preparation of 3, 6-di-substituted chromone-2-carboxamide compounds of general formula (I).
• 3, 6-di-substituted compounds of general formula (I) are prepared via the reaction of a suitable acid-intermediate (which contains the desired substituent group in the 3-position relative to the final compound) of general formula (XVI) and a suitable amine-intermediate of general formula (XV).
• the acid intermediate can be prepared from commercially available starting materials in 2-steps as indicated by step 1, transformation a, and step 2, transformation b.
• the acid intermediate is combined with the amine-intermediate to furnish 3, 6-di-substituted chromone-2-carboxamide compounds of general formula (I) (step 3, transformation c).
• compounds of general formula (I) are prepared via the reaction of a suitable ester-intermediate of general formula (XVIII) and an amine having the desired R 6 and R 7 groups.
• the ester intermediate can be prepared from commercially available starting materials in 3-steps as indicated by step 1, transformation a, and step 2, transformation b and c and step 3, transformation d.
• the ester intermediate is combined with the amine-intermediate to furnish 8-amino-4-oxo-chromene-2-carboxamides compounds of general formula (I) (step 4, transformation e).
• the present invention provides a process for the preparation of 8-amino-4-oxo-chromene-2-carboxamides compounds of general formula (I) comprising amidation of the ester-intermediate of general formula (XVIII) in accordance with the process illustrated in Scheme 10.
• conventional methods of heating and cooling may be employed, for example temperature-regulated oil-baths or temperature-regulated hot-blocks, and ice/salt baths or dry ice/acetone baths respectively.
• Conventional methods of isolation for example extraction from or into aqueous or non-aqueous solvents may be used.
• Conventional methods of drying organic solvents, solutions, or extracts such as shaking with anhydrous magnesium sulfate, or anhydrous sodium sulfate, or passing through a hydrophobic frit, may be employed.
• Conventional methods of purification for example crystallisation and chromatography, for example silica chromatography or reverse-phase chromatography, may be used as required.
• Crystallisation may be performed using conventional solvents such as ethyl acetate, methanol, ethanol, or butanol, or aqueous mixtures thereof. It will be appreciated that specific reaction times temperatures may typically be determined by reaction-monitoring techniques, for example thin-layer chromatography and LC-MS.
• references to treatment as used herein includes prophylaxis as well as palliative treatment via the alleviation of established symptoms of a condition i.e. prevention or control.
• Treating” or “treatment” of a state, disorder or condition includes: (1) preventing or delaying the appearance of clinical symptoms of the state, disorder or condition developing in a human that may be afflicted with or predisposed to the state, disorder or condition but does not yet experience or display clinical or subclinical symptoms of the state, disorder or condition, (2) inhibiting the state, disorder or condition, i.e., arresting, reducing or delaying the development of the disease or a relapse thereof (in case of maintenance treatment) or at least one clinical or subclinical symptom thereof, or (3) relieving or attenuating the disease, i. e., causing regression of the state, disorder or condition or at least one of its clinical or subclinical symptoms.
• references herein to treatment refer to the treatment of established conditions.
• the compounds of general formula (I) and pharmaceutically acceptable salts thereof may, depending on the condition, also be useful in the prevention (prophylaxis) of certain diseases.
• treat means: (1) to ameliorate the disease or one or more of the biological manifestations of the disease (2) to interfere with (a) one or more points in the biological cascade that leads to or is responsible for the disease or (b) one or more of the biological manifestations of the disease, (3) to alleviate one or more of the symptoms or effects associated with the disease, (4) to slow the progression of the disease or one or more of the biological manifestations of the disease, and/or (5) to diminish the likelihood of severity of a disease or biological manifestations of the disease.
• prevention means the prophylactic administration of a drug to diminish the likelihood of the onset of or to delay the onset of a disease or biological manifestation thereof.
• prevention is not an absolute term. In medicine, “prevention” is understood to refer to the prophylactic administration of a drug to substantially diminish the likelihood or severity of a disorder or biological manifestation thereof, or to delay the onset of such disorder or biological manifestation thereof.
• the treatment or prevention of a disease there is provided the treatment or prevention of a disease. In another embodiment, there is provided the treatment of a disease. In a further embodiment, there is provided the prevention of a disease.
• a compound of general formula (I), or a pharmaceutically acceptable salt thereof for use in therapy.
• a compound of general formula (I), or a pharmaceutically acceptable salt thereof for use as a medicament in therapy, which therapy is human or veterinary.
• treatment includes references to curative, palliative and prophylactic treatment.
• composition comprising a compound of formula (I), or a pharmaceutically acceptable, salt, solvate, hydrate, isomer, prodrug or polymorph thereof, together with one or more pharmaceutically acceptable, carrier, diluent or excipient;
• compound of formula (I) or a pharmaceutically acceptable, salt, solvate, hydrate, isomer, prodrug or polymorph thereof for the preparation of a pharmaceutical formulation for the treatment of one or more infectious diseases, and particularly for use in the prophylactic treatment of one or more infectious diseases independently selected from:
• compound of formula (I), or a pharmaceutically acceptable, salt, solvate, hydrate, isomer, prodrug or polymorph thereof for the preparation of a pharmaceutical formulation for the treatment of one or more bacterial infections, and particularly for use in the prophylactic treatment of one or more bacterial infections independently selected from: treatment of one or more Gram positive and/or Gram negative bacterial infections wherein the bacterial infections are independently selected from: bacterial infections stemming from: one or more of Streptococcus pneumonia ; and/or Enterococcus ; or one or more of the ESKAPE group of bacterial species, Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa , and/or Enterobacter.;
• diseases or conditions independently selected from: drug-resistant malaria; a trypanosomal infection; visceral leishmaniasis; cutaneous leishmaniasis; Cryptosporidiosis; human African trypanosomiasis (HAT); African animal trypanosomiasis; Schistosomiasis;
• a veterinary composition comprising a compound of formula (I), or an acceptable salt, solvate, hydrate, isomer, prodrug or polymorph thereof, together with one or more acceptable carrier, diluent or excipient;
• Prophylactic treatment of malaria includes the treatment of a subject with a prophylaxis-effective amount of compound of formula (I) wherein said prophylaxis-effective amount is an amount of compound that is effective in inhibiting, decreasing the likelihood of the disease by malarial parasites, or preventing malarial infection or preventing the delayed onset of the disease by malarial parasites, when administered before infection, i.e. before, during and/or slightly after the exposure period to malarial parasites
• Treatment of malaria as defined herein includes: treatment of Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae and/or Plasmodium knowlesi infections; treatment of Plasmodium falciparum infections; treatment of Plasmodium falciparum and Plasmodium vivax infections; treatment of Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae and Plasmodium knowlesi infections; treatment of the latent forms of vivax malaria.
• a further aspect of the invention provides a method for the prophylaxis or treatment of trypanosomal infection comprising the administration of a compound of formula (I) to a subject suffering from or likely to be exposed to said T. cruzi infection.
• a related aspect of the invention provides the use of a compound of formula (I) in the treatment or prophylaxis of trypanosomal infection.
• a further related aspect provides the use of the compounds of formula (I) for the treatment or prophylaxis of T. cruzi infection.
• a further aspect of the invention provides a method for the treatment of trypanosomal infection comprising the administration of a compound of formula (I) to a subject suffering from or likely to be exposed to said trypanosomal infection.
• a related aspect of the invention provides the use of a compound of formula I in the treatment of trypanosomal infection.
• a further related aspect provides the use of the compounds of formula (I) for the treatment of T. cruzi infection.
• Other related aspects provide, a compound of formula (I) for use in the treatment of trypanosomal infection, and a compound of formula (I) for use in the treatment of a T. cruzi infection.
• the trypanosomal infection is a T. cruzi infection.
• the method or use of the invention relates to treatment of an ongoing infection in human subjects.
• the anti-infective agents of formula (I) according to the present invention are believed to be suitable for treatment those infectious diseases in which the pathogen is present in organs such as the liver, spleen or kidney, and in particular to muscles such as heart.
• the invention provides a kit comprising an effective amount of one or more compounds of the formulae herein in unit dosage form, together with instructions for administering the compound to a subject suffering from or susceptible to a trypanosomal infections, such as Chagas disease.
• a compound of formula (I), or a pharmaceutically acceptable salt thereof for use in the treatment or prevention of cutaneous leishmaniasis.
• a compound of formula (I), or a pharmaceutically acceptable salt thereof for use as a medicament for the treatment or prevention of leishmaniasis, particularly visceral leishmaniasis.
• a method of treatment or prevention of leishmaniasis which method comprises administering to a human subject in need thereof, a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof.
• a method of treatment or prevention of cutaneous leishmaniasis which method comprises administering to a mammal in need thereof, a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof.
• the above uses, and/or methods provide compounds of formula (I), or a pharmaceutically acceptable salt thereof effective against agents which are effective against Leishmania , and particularly for agents which are suitable for use in the treatment or prevention of Leishmania infantum.
• a method of treatment or prevention of schistosomiasis comprises administering to a human subject in need thereof, a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof.
• the above uses, and/or methods provide compounds of formula (I), or a pharmaceutically acceptable salt thereof effective against agents which are effective against worms of the genus Schistosoma , and particularly effective against Schistosoma haematobium, Schistosoma mansoni and/or Schistosoma japonicum.
• a method of treatment or prevention of Cryptosporidiosis comprises administering to a human subject in need thereof, a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof.
• a method of treatment or prevention of Cryptosporidiosis comprises administering to a mammal in need thereof, a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof.
• the above uses, and/or methods provide compounds of formula (I), or a pharmaceutically acceptable salt thereof effective against Cryptosporidium , and particularly compounds of formula (I) suitable for use in the treatment or prevention of; Cryptosporidium -only infected subjects; immunocompromised subjects infected with Cryptosporidium such as Cryptosporidium -HIV co-infected subjects.
• a method of treatment or prevention of HAT comprises administering to a human subject in need thereof, a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof.
• a method of treatment or prevention of HAT comprises administering to a mammal in need thereof, a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof.
• the above uses, and/or methods provide compounds of formula (I), or a pharmaceutically acceptable salt thereof effective against Trypanosoma brucei gambiense ( T.b. gambiense ) and/or Trypanosoma brucei rhodesiense ( T.b. rhodesiense ) infections.
• a method of treatment or prevention of TB comprises administering to a human subject in need thereof, a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof.
• a method of treatment or prevention of TB comprises administering to a mammal in need thereof, a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof.
• a method of treatment or prevention of bacterial infections comprises administering to a human subject in need thereof, a therapeutically effective amount of a compound of formula (I), or a pharmaceutically acceptable salt thereof.
• a method of treatment or prevention of one or more bacterial infections comprises administering to a mammal in need thereof, a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof.
• the above uses, and/or methods provide anti-bacterial compounds of formula (I), or a pharmaceutically acceptable salt thereof suitable for use in the treatment or prevention of, or which provide effective treatment of bacterial infections stemming from: Staphylococcus aureus; Streptococcus pneumonia; Enterococcus ; and/or Mycobacterium tuberculosis.
• Artemisinin and derivatives thereof such as for example Artesunate; Quinine and related agents; Chloroquine; OZ439; NITD609; ferroquine; napthoquine; pipe
• the suitability of a potential combination of two, or more, antimalarial drugs can be assessed on the basis of their in vitro drug interactions wherein the interactions of the two selected antimalarial drugs are investigated in vitro using standard dose-response assays over a range of individualised concentrations.
• the selection of suitable conditions and concentrations for carrying out such investigations would be within the remit of the skilled practitioner.
• the present invention provides a pharmaceutical composition
• a pharmaceutical composition comprising: a compound of formula (I) or a pharmaceutically acceptable, salt, solvate, hydrate, isomer, prodrug, or polymorph thereof; one or more additional antimalarial agents; and one or more pharmaceutically acceptable, carriers, diluents or excipients.
• Suitable combinations herein include a compound of the present invention and one or more additional therapeutic agents selected from: artesunate; mefloquine; OZ439, piperaquine and mixtures thereof.
• the composition comprising a compound of formula (I) as detailed hereinbefore may be administered to an individual prior to, simultaneously, separately or sequentially with other therapeutic regiments or co-agents useful in the treatment of malaria.
• the different actives may be formulated for the same or different delivery, for example one active formulated for immediate and another for sustained release.
• the active agents may be formulated for the same or different routes of administration, for example in a dual-therapy one active may be formulated for oral administration and another for parenteral administration.
• compounds of formula (I) should be assessed for their biopharmaceutical properties, such as for example, solubility, solution stability (across a range of pHs), likely dose level and permeability. Initial biopharmaceutical testing for potential as anti-malarial treatment has provided positive results.
• Compounds of the invention intended for pharmaceutical use may be administered as crystalline or amorphous products. They may be obtained, for example, as solid plugs, powders, or films by methods such as precipitation, crystallization, freeze-drying, spray drying, or evaporative drying. Microwave or radio frequency drying may be used for this purpose.
• excipient is used herein to describe any ingredient other than the compound(s) of the invention. The choice of excipient will to a large extent depend on factors such as the particular mode of administration, the effect of the excipient on solubility and stability, and the nature of the dosage form.
• Pharmaceutically acceptable excipients include one or more of: lubricants, binding agents, diluents, surface-active agents, anti-oxidants, colorants, flavouring agents, preservatives, flavour enhancers, preservatives, salivary stimulating agents, cooling agents, co-solvents (including oils), emollients, bulking agents, anti-foaming agents, surfactants and taste-masking agents.
• compositions suitable for the delivery of compounds of the present invention and methods for their preparation will be readily apparent to those skilled in the art. Such compositions and methods for their preparation may be found, for example, in Remington's Pharmaceutical Sciences, 19th Edition (Mack Publishing Company, 1995).
• Formulations suitable for oral administration include solids, semi-solids or liquids such as tablets; soft or hard capsules; bolus; powders; lozenges (including liquid-filled); chews; multi and nano-particulates; gels; solid solutions; fast-dispersing dosage forms; fast-dissolving dosage forms; fast-disintegrating dosage forms; films; ovules; sprays; buccal/mucoadhesive patches; and liquid formulations.
• Liquid formulations include suspensions, solutions, elixirs and syrups.
• Oral administration may involve swallowing, so that the compound enters the gastrointestinal tract, and/or buccal, lingual or sublingual administration by which the compound enters the blood stream directly from the mouth.
• Liquid formulations may be employed as fillers in soft or hard capsules and typically comprise a carrier, for example, water, ethanol, polyethylene glycol, propylene glycol, methylcellulose, or a suitable oil, and one or more emulsifying agents and/or suspending agents. Liquid formulations may also be prepared by the reconstitution of a solid, for example, from a sachet.
• a carrier for example, water, ethanol, polyethylene glycol, propylene glycol, methylcellulose, or a suitable oil
• emulsifying agents and/or suspending agents may also be prepared by the reconstitution of a solid, for example, from a sachet.
• Formulations for oral administration may be formulated to be immediate and/or modified release.
• Modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted and programmed release.
• the formulation of tablets is discussed in “Pharmaceutical Dosage Forms: Tablets, Vol. 1”, by H. Lieberman and L. Lachman, Marcel Dekker, N. Y., N.Y., 1980 (ISBN 0-8247-6918-X).
• the present invention provides a pharmaceutical composition formulated for oral delivery comprising a compound of formula (I) or a pharmaceutically acceptable, salt, solvate or hydrate thereof, according to any preceding claim, together with one or more pharmaceutically acceptable excipients.
• the present invention further provides said pharmaceutical composition formulated for oral delivery as an immediate release, or as a modified release tablet formulation.
• the compounds of the invention may also be administered parenterally, or by injection directly into the blood stream, into muscle, or into an internal organ.
• Suitable means for parenteral administration include intravenous, intraarterial, intraperitoneal, intrathecal, intraventricular, intraurethral, intrasternal, intracranial, intramuscular, intrasynovial and subcutaneous.
• Suitable devices for parenteral administration include needle (including microneedle) injectors, needle-free injectors and infusion techniques.
• the present invention provides a pharmaceutical composition formulated for parenteral delivery comprising a compound of formula (I) or a pharmaceutically acceptable, salt, solvate or hydrate thereof, according to any preceding claim, together with one or more pharmaceutically acceptable excipients.
• the present invention further provides said pharmaceutical composition formulated for parenteral delivery as an immediate release, or as a modified release tablet formulation suitable for intramuscular or intravenous administration.
• the compounds of the invention may also be administered topically, (intra)dermally, or transdermally to the skin or mucosa.
• Typical formulations for this purpose include gels, hydrogels, lotions, solutions, creams, ointments, dusting powders, dressings, foams, films, skin patches, wafers, implants, sponges, fibres, bandages and microemulsions. Liposomes may also be used.
• the compounds of the invention may be administered rectally or vaginally, for example, in the form of a suppository, pessary, or enema.
• Cocoa butter is a traditional suppository base, but various alternatives may be used as appropriate.
• compositions containing compounds of the invention may be formulated to be immediate and/or modified release.
• Modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted and programmed release.
• a physician will determine the actual dosage which will be most suitable for an individual subject.
• the specific dose level and frequency of dosage for any particular individual may be varied and will depend upon a variety of factors including the condition being treated, the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the individual undergoing therapy.
• a suitable dose will be in the range of from about 0.001 to about 50 mg/kg of body weight per day, in a further embodiment, of from about 0.001 to about 5 mg/kg of body weight per day; in a further embodiment of from about 0.001 to about 0.5 mg/kg of body weight per day and in yet a further embodiment of from about 0.001 to about 0.1 mg/kg of body weight per day.
• the ranges can be of from about 0.001 to about 750 mg/kg of body weight per day, in the range of 0.5 to 60 mg/kg/day, and in the range of 1 to 20 mg/kg/day.
• the desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example as one, two, three, four or more doses per day. If the compounds are administered transdermally or in extended release form the compounds could be dosed once a day or less.
• the compound is conveniently administered in unit dosage form; for example containing 0.1 to 50 mg, conveniently 0.1 to 10 mg, most conveniently 0.1 to 5 mg of active ingredient per unit dosage form.
• the compound can be conveniently administered in unit dosage form; for example containing 10 to 1500 mg, 20 to 1000 mg, or 50 to 700 mg of active ingredient per unit dosage form.
• These dosages are based on an average human subject having a weight of about 65 kg to 70 kg. The physician will readily be able to determine doses for subjects whose weight falls outside this range, such as infants and the elderly.
• the present invention provides a pharmaceutical composition formulated as a single-dose tablet suitable for oral delivery comprising a compound of formula (I) or a pharmaceutically acceptable, salt, solvate or hydrate thereof, together with one or more pharmaceutically acceptable excipients.
• the present invention further provides said pharmaceutical composition formulated for oral delivery as an immediate release, or as a modified release single-dose tablet formulation.
• the present invention further provides a pharmaceutical composition formulated as a single-dose tablet formulated for oral delivery as an immediate release, or as a modified release single-dose tablet formulation comprising from about 0.1 to about 3000 mg, preferably from about 0.5 to about 1500 mg, more preferably from about 1 to about 750 mg, from about 1 to about 750 mg, and especially from about 5 to about 250 mg of a compound of formula (I) or a pharmaceutically acceptable, salt, solvate or hydrate thereof, together with one or more pharmaceutically acceptable excipients.
• a pharmaceutical composition formulated as a single-dose tablet formulated for oral delivery as an immediate release, or as a modified release single-dose tablet formulation comprising from about 0.1 to about 3000 mg, preferably from about 0.5 to about 1500 mg, more preferably from about 1 to about 750 mg, from about 1 to about 750 mg, and especially from about 5 to about 250 mg of a compound of formula (I) or a pharmaceutically acceptable, salt, solvate or hydrate thereof, together with one or more pharmaceutically acceptable
• a single-dose treatment is highly desirable to increase effective treatment levels; increase compliance rates; as well as to reduce treatment costs.
• the present invention further provides a pharmaceutical composition formulated as a single-dose tablet formulated for oral delivery as an immediate release, or as a modified release single-dose tablet formulation comprising from 0.1 to 3000 mg, preferably from about 0.5 to about 1500 mg, more preferably from about 1 to about 750 mg and especially from about 5 to about 250 mg of a compound of formula (I) or a pharmaceutically acceptable, salt, solvate or hydrate thereof, together with one or more pharmaceutically acceptable excipients.
• a pharmaceutical composition formulated as a single-dose tablet formulated for oral delivery as an immediate release, or as a modified release single-dose tablet formulation comprising from 0.1 to 3000 mg, preferably from about 0.5 to about 1500 mg, more preferably from about 1 to about 750 mg and especially from about 5 to about 250 mg of a compound of formula (I) or a pharmaceutically acceptable, salt, solvate or hydrate thereof, together with one or more pharmaceutically acceptable excipients.
• the dose could be provided by more than one tablet, such as 2 ⁇ 1500 mg, or 3 ⁇ 1000 mg, rather than a single-dose 3000 mg tablet where the tablets may be taken either one after the other, or together according to suitability.
• compositions may conveniently be combined in the form of a kit suitable for coadministration of the compositions.
• the kit of the invention comprises two or more separate pharmaceutical compositions, at least one of which contains a compound of formula (I) in accordance with the invention, and means for separately retaining said compositions, such as a container, divided bottle, or divided foil packet.
• a container, divided bottle, or divided foil packet An example of such a kit is the familiar blister pack used for the packaging of tablets, capsules and the like.
• references herein to treatment refer to the treatment of established conditions.
• the compounds of general formula (I) and pharmaceutically acceptable salts thereof may, depending on the condition, also be useful in the prevention (prophylaxis) of certain diseases.
• treat means: (1) to ameliorate the disease or one or more of the biological manifestations of the disease (2) to interfere with (a) one or more points in the biological cascade that leads to or is responsible for the disease or (b) one or more of the biological manifestations of the disease, (3) to alleviate one or more of the symptoms or effects associated with the disease, (4) to slow the progression of the disease or one or more of the biological manifestations of the disease, and/or (5) to diminish the likelihood of severity of a disease or biological manifestations of the disease.
• prevention means the prophylactic administration of a drug to diminish the likelihood of the onset of or to delay the onset of a disease or biological manifestation thereof.
• prevention is not an absolute term. In medicine, “prevention” is understood to refer to the prophylactic administration of a drug to substantially diminish the likelihood or severity of a disorder or biological manifestation thereof, or to delay the onset of such disorder or biological manifestation thereof.
• the treatment or prevention of a disease there is provided the treatment or prevention of a disease. In another embodiment, there is provided the treatment of a disease. In a further embodiment, there is provided the prevention of a disease.
• a compound of general formula (I), or a pharmaceutically acceptable salt thereof for use in therapy.
• a compound of general formula (I), or a pharmaceutically acceptable salt thereof for use as a medicament in therapy, which therapy is human or veterinary.
• treatment includes references to curative, palliative and prophylactic treatment.
• Compounds of the present invention are useful in the treatment of malaria.
• Compounds according to the present invention have potential for the treatment of Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae and Plasmodium knowlesi infections.
• the novel class of class of quinolone-4-carboxamide compounds according to the present invention have potential for the treatment of Plasmodium falciparum infections; Plasmodium falciparum and Plasmodium vivax infections; Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale and Plasmodium knowlesi infections.
• novel class of class of chromone compounds of general formula (I) have potential for the treatment of malaria attributable to infection from the life-threatening form of malaria attributable to Plasmodium falciparum.
• Malaria is caused by an infection of the red blood cells with a tiny organism or parasite called protozoa. Infection of the five species of the malaria protozoa, Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae and Plasmodium knowlesi occurs through the injection of protozoa into the blood stream, is effected by a single source, the bite of the female Anopheles mosquito.
• Plasmodium species requires two hosts, human and mosquito for completion of its life-cycle.
• the infection is initiated by the inoculation of sporozoites in the saliva of an infected mosquito.
• the sporozoites migrate to the liver and there infect hepatocytes where they differentiate, via the exoerythrocytic intracellular stage, into the merozoite stage which infects red blood cells to initiate cyclical replication in the asexual blood stage.
• the life-cycle is completed by the differentiation of a number of merozoites in the red blood cells into sexual stage gametocytes which are ingested by the mosquito, where they develop through a series of stages in the mid gut to produce sporozoites which migrate to the salivary gland.
• the present invention provides chromone compounds of general formula (I) for use as antimalarial medicaments.
• the gene encoding a truncated form of Plasmodium falciparum lysyl-tRNA-synthetase (Uniprot code Q81DJ8), residues 80-583, was codon optimized and synthesized by Genscript, with additional NdeI and XhoI restriction sites added at the termini of the gene.
• the gene was subsequently digested out the provided pUC57 vector and ligated into a modified pET15B vector encoding an N-terminal Hexa-His tagged Maltose Binding Protein (MBP) tag with an additional Tobacco Etch Virus (TEV) cleavage site between the protein and the tag to allow increased soluble expression, ease of affinity capture and subsequent cleavage during purification. All plasmids were sent for sequencing to confirm identity at the DNA sequencing and services (Dundee University).
• the plasmid translation for PfKRS from a modified pET15B vector (Novagen) is provided hereinafter.
• the gene was synthesised by Genscript (USA) and cloned using FastDigest enzymes (Fermentas).
• the plasmid was transformed into BL21 (DE3) cells (Stratagene) using the heat shock methods prior to plating onto LB agar plates supplemented with 50 ug ⁇ 1 ml ⁇ 1 ampicillin and incubated overnight at 37° C. Cell scrapings were taken and used to inoculate Autoinduction media (Studier, 2005) supplemented with 50 ug ⁇ 1 ml ⁇ 1 ampicillin. Cultures were grown at 37° C. 200 rpm shaking for four hours prior to being grown for 18 hours overnight at 21° C. to allow expression of the protein.
• Cultures were pelleted at 3,500 g, 4° C. for 30 minutes prior to being frozen at ⁇ 20° C. until required. Cells were defrosted and resuspended in buffer A (25 mM TRIS, 500 mM NaCl, 20 mM imidazole, pH 8.5) supplemented with 10 ug ⁇ 1 ml ⁇ 1 DNAse (Sigma) and protease inhibitor tablets (Roche) prior to being lysed at 30 KPSI on a Constant Cell Disruption System (Constant Systems, UK). Cell debris was removed by centrifugation at 37,500 g 4° C. for 30 minutes prior to the supernatant being filtered to 0.25 um.
• buffer A 25 mM TRIS, 500 mM NaCl, 20 mM imidazole, pH 8.5
• 10 ug ⁇ 1 ml ⁇ 1 DNAse Sigma
• protease inhibitor tablets Roche
• Samples for crystallography were then subject to a further round of IMAC purification to remove the His-MBP tag and any uncleaved protein. Both samples were then subjected to gel filtration using a calibrated Superdex 200 26/60 column (GE healthcare) equilibrated with buffer C. Cleaved Pf KRS eluted as a dimer, whilst the tagged version eluted as a trimer. Samples were subjected to ESI-TOF analysis by the University of Dundee Proteomics Facility to further confirm their identity. Typically the final samples were in excess of 95% pure by SDS-PAGE analysis.
• KRS1 Plasmodium falciparum Lysine tRNA Synthetase
• Lysine tRNA Synthetase is an enzyme which catalyses the esterification of L-Lysine to the appropriate transfer ribonucleic acid (tRNA), to form an aminoacyl-tRNA. Once the tRNA is charged, a ribosome transfers the amino acid from the tRNA onto a growing peptide, according to the genetic code.
• the substrates of this reaction are L-lysine, and adenosine triphosphate (ATP), the overall reaction is: amino acid+tRNA+ATP ⁇ aminoacyl-tRNA+AMP+PP i
• reaction AMP is adenosine monophosphate and PP i is inorganic pyrophosphate.
• the reaction progression in a biochemical assay can be tracked by the depletion of one of the substrates, in the case of this invention, ATP.
• ATP adenosine triphosphate
• Kinase Glo® Assay which is a luminescent kinase assay platform suitable for monitoring kinase activities using up to 10 ⁇ m ATP which is commercially available from the Promega Corporation of Madison, Wis., USA.
• the Kinase-Glo® Assay was performed in a single well of a multiwell plate by adding a volume of Kinase-Glo® Reagent equal to the volume of a completed kinase reaction and measuring luminescence.
• the resulting luminescent signal is correlated with the amount of ATP present and is inversely correlated with the amount of kinase activity.
• a reduction of the luminescent signal is relative to the reduction in ATP as the reaction progresses.
• a compound which inhibits the action of KRS will show no drop in the luminescent signal from assay controls.
• Test compounds were solubilized in DMSO at a top concentration of 10 mM and serially diluted 1 in 3 to achieve a range of final assay concentrations of 50 ⁇ M to 2.5 nM in 10 ⁇ l reaction volume.
• KRS was diluted to a working concentration of 75 nM in assay buffer (25 mM HEPES, 25 mM KOH, 10 mM MgCl 2 .6H 2 O, 50 mM KCl), and the following ‘Master Mix’ prepared in assay buffer: 0.1 mg/ml BSA, 250 ⁇ M DTT, 200 ⁇ M Spermine, 0.05% NP-40, ATP 3 ⁇ M for Pf KRS1 (4 ⁇ M for Hs KRS1), 0.4 mg/ml tRNA from Yeast, 60 ⁇ M L-Lysine, 0.5 U/ml Pyrophosphatase.
• assay buffer 25 mM HEPES, 25 mM KOH, 10 mM MgCl 2 .6H 2 O, 50 mM KCl
• Master Mix prepared in assay buffer: 0.1 mg/ml BSA, 250 ⁇ M DTT, 200 ⁇ M Spermine, 0.05% NP-40, ATP 3 ⁇ M for
• Hep G2 Human Caucasian hepatocyte carcinoma, HPACC cat. no. 85011430
• Hep G2 in-vitro cytotoxicity can be assessed using the assay procedure as described in “Use of a human-derived liver cell line for the detection of cytoprotective, antigenotoxic and cogenotoxic agents”, Volker Mersch-Sundermann, Siegfried Knasmüller, Xin-jiang Wu, Firouz Darroudi, Fekadu Kassie. J. Tox 198 (2004) 329-340) the contents of which are incorporated herein by reference.
• the gene encoding a truncated form of Mycobacterium tuberculosis lysyl-tRNA-synthetase (Uniprot code I6YCJO) was codon optimized and synthesized by Genscript, with additional NdeI and XhoI restriction sites added at the termini of the gene.
• the gene was subsequently digested out the provided pUC57 vector and ligated into a modified pET15B vector encoding an N-terminal Hexa-His tag with an additional Tobacco Etch Virus (TEV) cleavage site between the protein and the tag to allow increased soluble expression, ease of affinity capture and subsequent cleavage during purification if required. All plasmids were sent for sequencing to confirm identity at the DNA sequencing and services (Dundee University).
• the plasmid translation for MtbKRS 1, from a modified pET15B vector (Novagen) is provided hereinafter.
• the gene was synthesised by Genscript (USA) and cloned using FastDigest enzymes (Fermentas).
• the plasmid was transformed into BL21 (DE3) cells (Stratagene) using the heat shock methods prior to plating onto LB agar plates supplemented with 50 ug ⁇ 1 ml ⁇ 1 ampicillin and incubated overnight at 37° C. Cell scrapings were taken and used to inoculate Autoinduction media (Studier, 2005) supplemented with 50 ug ⁇ 1 ml ⁇ 1 ampicillin. Cultures were grown at 20° C. 200 rpm shaking for forty eight hours to allow expression of the protein.
• Cultures were pelleted at 3,500 g, 4° C. for 30 minutes prior to being frozen at ⁇ 20° C. until required. Cells were defrosted and resuspended in buffer A (100 mM HEPES, 150 mM NaCl, 20 mM imidazole, pH 7.5) supplemented with 10 ug ⁇ 1 ml ⁇ 1 DNAse (Sigma) and protease inhibitor tablets (Pierce) prior to being lysed at 30 KPSI on a Constant Cell Disruption System (Constant Systems, UK). Cell debris was removed by centrifugation at 40,000 g 4° C. for 30 minutes prior to the supernatant being filtered to GFA prefilter.
• buffer A 100 mM HEPES, 150 mM NaCl, 20 mM imidazole, pH 7.5
• 10 ug ⁇ 1 ml ⁇ 1 DNAse Sigma
• protease inhibitor tablets Pieris
• samples were then subjected to gel filtration using a calibrated Superdex 200 50/60 column (GE healthcare) equilibrated with buffer C. Mtb KRS1 eluted as a dimer. Samples were subjected to ESI-TOF analysis by the University of Dundee Proteomics Facility to further confirm their identity. Typically the final samples were in excess of 80% pure by SDS-PAGE analysis.
• KRS1 Mycobacterium tuberculosis Lysine tRNA Synthetase
• Mycobacterium tuberculosis lysyl-tRNA synthetase (or lysyl-tRNA ligase), briefly MtKRS, is a dimeric multistep enzyme catalysing the reaction of L-lysine and ATP in the first step and in the second step the transfer of lysine onto the respective tRNA. In the first step it releases pyrophosphate and in the second step AMP as by-products.
• the main product is lysyl-tRNA which is an essential building block for protein synthesis.
• Step 1 Lysine+ATP->Lysyl-AMP+PP i
• MtKRS enzyme activity is tested in a BioMol Green based endpoint assay.
• a pyrophosphatase enzyme is continuously driving the first step of the reaction by hydrolysing the released inorganic pyrophosphate (PP i ) creating free phosphate (P i ).
• This class of enzyme is a natural driver of pyrophosphate releasing enzymes in cells.
• free inorganic phosphate (P i ) is finally detected via a chemical reaction using Biomol Green (ENZO®).
• BioMol Green consists of the dye Malachite Green, ammonium molybdate and a strong acid (HCl). Free phosphate builds a complex with molybdate and Malachite Green and approximately 13 protons, which are also incorporated and removed from solution. The reaction mix changes colour from orange to green—this is detected by a change in absorbance at 650 nm.
• This assay detects the first step of the MtKRS reaction. And it is not necessary to supply the reaction mixture with tRNA which reduces costs substantially.
• the final assay mixture contained 250 nM MtKRS, 30 mM Tris-HCl pH8, 40 mM MgCl 2 , 140 mM NaCl, 30 mM KCl, 0.01% Brij-35, 1 mM DTT, 3 ⁇ M ATP, 12 ⁇ M L-lysine, 0.5 U/mL yeast pyrophosphatase (Sigma).
• the assay mixture was plated on 384-well plates (Greiner 781101) with a reaction volume of 50 uL per well. After 4 hours of incubation at room temperature the reaction was stopped by addition of an equal amount of 50 uL BioMol Green (ENZO®).
• the stopped reaction mix was incubated for 20 minutes and absorbance read at 650 nm on BMG PHERAstar® FSX plate reader which is a multi-mode reader for use in high-throughput screening available from BMG Labtech if Cary, N.C., USA.
• Test compounds were solubilized in DMSO at a top concentration of 10 mM and serially diluted 1 in 3 to achieve a range of final assay concentrations of 50 ⁇ M to 2.5 nM in 10 ⁇ l reaction volume.
• Isoniazid (Isonicotinyl hydrazine, INHA) was used as a positive control; eight twofold dilutions of isoniazid starting at 160 ⁇ g mL ⁇ 1 were prepared, and this control curve (5 ⁇ L) was added to 95 ⁇ L Middlebrook 7H9 medium (row 11, lines A-H). Neat DMSO (5 ⁇ L) was added to row 12 (growth and blank controls). The inoculum was standardized to ⁇ 1 ⁇ 10 7 CFU mL ⁇ 1 and diluted 1:100 in Middlebrook 7H9 broth (Middlebrook ADC enrichment, a dehydrated culture medium which supports growth of mycobacterial species, available from Becton-Dickinson, cat.
• the cryptosporidiosis NOD SCID gamma mouse model were run as follows: Male NOD SCID gamma mice (NOD.Cg-Prkdc scid II2rg tm1Wjl /SzJ, Jackson Labs) were infected approximately 2 weeks post weaning with 10 ⁇ circumflex over ( ) ⁇ 5 C. parvum Iowa strain oocysts by oral gavage. Four mice were used per experimental group. This establishes a chronic, asymptomatic infection of the small intestine, cecum, and biliary tree. Fecal parasite shedding is monitored by quantitative PCR to amplify C. parvum DNA.
• the intramacrophage Leishmania assay was performed as described in de Rycker et al (Antimicrob Agents Chemother. 2013 July; 57(7):2913-22. doi: 10.1128/AAC.02398-12). Briefly, 1 ⁇ l of compound was pre-dispensed into 384 well sterile intermediary plates. For single point screening, amphotericin B was added to all wells of column 24 as a positive control (final concentration 2 ⁇ M) and DMSO to column 23. For potency determinations, ten-point, one in three dilution curves were created with the highest concentration being 50 ⁇ M and on each plate a control curve of amphotericin B was included.
• Controls were as follows: columns 11 and 12: DMSO, columns 23 and 24: amphotericin B (final concentration 2 ⁇ M).
• THP-1 media 100 ⁇ l of THP-1 media was added and plates were shaken for >5 min to ensure complete mixing.
• THP-1 cells (8,000 per well, 50 ⁇ l) were plated into black clear-bottom 384 well plates (Corning) in presence of 10 nM PMA. After 20 min at room temperature, the plates were incubated at 37° C. under 5% CO2 in a humidified incubator for 75 h.
• the cells were then washed with 450 ⁇ l sterile phosphate buffered saline (PBS) supplemented with 1 mM CaCl 2 , 0.5 mM MgCl 2 , 0.1% (w/v) bovine serum albumin (PBS-A) and amastigotes were added to all wells at a multiplicity of infection of 5 (40,000 amastigotes per well). After 40 min at room temperature, plates were returned to the incubator. Amastigotes were incubated in the presence of macrophages for 16 h.
• PBS sterile phosphate buffered saline
• PBS-A bovine serum albumin
• pIC50 activity data is presented as an pIC50 value this is not the same as an IC50 value.
• pIC 50 and IC 50 are terms of the art and it will be readily appreciated by the skilled person that compounds which have an IC 50 value of less than about 10 ⁇ M will have a corresponding pIC 50 value of greater than about 5.
• an IC 50 value of 1 ⁇ M is equivalent to a pIC50 value of 6, and an IC 50 value of 100 nM is equivalent to a pIC50 value of 7.
• Pf-KRS1 Plasmodium falciparum lysyl t-RNA synthetase
• Mtb KRS Mycobacterium tuberculosis lysyl t-RNA synthetase
• COMU (1-cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate
• Mass-directed preparative HPLC separations were performed using a Waters HPLC (2545 binary gradient pumps, 515 HPLC make up pump, 2767 sample manager) connected to a Waters 2998 photodiode array and a Waters 3100 mass detector.
• Preparative HPLC separations were performed with a Gilson HPLC (321 pumps, 819 injection module, 215 liquid handler/injector) connected to a Gilson 155 UV/vis detector.
• HPLC chromatographic separations were conducted using Waters XBridge C18 columns, 19 ⁇ 100 mm, 5 um particle size; using 0.1% ammonia in water (solvent A) and acetonitrile (solvent B) as mobile phase.
• LC-MS analysis and chromatographic separation were conducted with a Brucker MicrOTOf mass spectrometer or an Agilent Technologies 1200 series HPLC connected to an Agilent Technologies 6130 quadrupole LC/MS, where both instruments were connected to an Agilent diode array detector.
• the column used was a Waters XBridge column (50 mm ⁇ 2.1 mm, 3.5 ⁇ m particle size,) and the compounds were eluted with a gradient of 5 to 95% acetonitrile/water+0.1% Ammonia.
• the compound of preparation 7 is also commercially available from Apollo, Scientific, Cheshire, UK and has CAS number 23866-72-0.
• the compound of preparation 15 is also commercially available from Fluorochem Limited, Hatfield, UK and has CAS number 5006-45-1).
• the compound of preparation 16 is also commercially available from Fluorochem Limited, Hatfield, UK and has CAS number 5527-91-3).
• the compound of preparation 17 is also commercially available from Fluorochem Limited, Hatfield, UK and has CAS number 4940-39-0.
• 4-oxo-chromene-2-carboxylic acid or alternatively a corresponding substituted 4-oxo-chromene-2-carboxylic acid, (2.7 mmol) was dissolved in DMF (4 mL) and di-isopropyl ethyl amine (DIPEA) (346 mg, 2.7 mmol) were added. The reaction mixture was then cooled to 0° C. with an ice water bath and thereafter a solution of PyBOP (1.4 g, 2.7 mmol) in dry DCM (4 mL) was added. The resulting mixture was stirred at 0° C.
• DIPEA di-isopropyl ethyl amine
• Product was purified by preparative HPLC (Gilson) using a 5-50% method and 0.1% HCO 2 H in water and acetonitrile as eluents. The product was then further purified using a mass directed autopreparative (MDA, Waters) using a 5-40% method and 0.1% HCO 2 H in water and acetonitrile as eluents. Purified product obtained in a 3% yield.
• the 1-hydroxy analogue, N-[(1-hydroxycyclopentyl)methyl]-4-oxo-chromene-2-carboxamide, Example 23A was prepared from the appropriate starting materials using analagous chemistry.
• Product was purified by column chromatography using a 12 g silica cartridge (Redisep®) and heptane (A) and ethyl acetate (B) as solvents. The following purification gradient was used: 3 min hold A, 18 min ramp to 100% B, 3 min hold 100% B. Purified product obtained in a 45% yield.
• Product was purified by column chromatography using a 12 g silica cartridge (Redisep®) and heptane (A) and ethyl acetate (B) as solvents. The following purification gradient was used: 3 min hold A, 18 min ramp to 70% B, 3 min hold 70% B. Purified product obtained in a 32% yield.
• Product was purified by column chromatography using a 12 g silica cartridge (Redisep®) and heptane (A) and ethyl acetate (B) as solvents. The following purification gradient was used: 3 min hold A, 18 min ramp to 100% B, 3 min hold 100% B. Purified product obtained in a 29% yield.
• Product was purified by column chromatography using a 12 g silica cartridge (Redisep®) and heptane (A) and ethyl acetate (B) as solvents. The following purification gradient was used: 3 min hold A, 18 min ramp to 100% B, 3 min hold 100% B. Purified product obtained in a 31% yield.
• Product was purified by column chromatography using a 12 g silica cartridge (Redisep®) and heptane (A) and ethyl acetate (B) as solvents. The product was precipitated from a mixture of methanol and DMSO. Purified product obtained in a 10% yield.
• Product was purified by column chromatography using a 12 g silica cartridge (Redisep®) and heptane (A) and ethyl acetate (B) as solvents. The following purification gradient was used: 3 min hold A, 18 min ramp to 100% B, 3 min hold 100% B. Purified product obtained in a 21% yield.
• Product was purified by column chromatography using a 12 g silica cartridge (Redisep®) and heptane (A) and ethyl acetate (B) as solvents. The following purification gradient was used: 3 min hold A, 18 min ramp to 100% B, 3 min hold 100% B. Purified product obtained in a 70% yield.
• Product was purified by column chromatography using a 12 g silica cartridge (Redisep®) and heptane (A) and ethyl acetate (B) as solvents. The following purification gradient was used: 3 min hold A, 18 min ramp to 100% B, 3 min hold 100% B. Purified product obtained in a 54% yield.
• Product was purified by column chromatography using a 12 g silica cartridge (Redisep®) and heptane (A) and ethyl acetate (B) as solvents. The following purification gradient was used: 3 min hold A, 18 min ramp to 100% B, 3 min hold 100% B. Purified product obtained in a 71% yield.
• tert-butyl N-[1-[[(6-fluoro-4-oxo-chromene-2-carbonyl)amino]methyl]cyclohexyl]carbamate (277 mg, 0.63 mmol) was dissolved in a 4 M solution of hydrogen chloride in Dioxane (3.14 mL, 12.58 mmol) at room temperature to give a clear solution. The reaction mixture was stirred at room temperature for 4 h and a white precipitate was formed. Solvents were removed under reduced pressured.
• Example 40A 6-Fluoro-N-((1-(2-hydroxyethyl)cyclohexyl)methyl)-4-oxo-4H-chromene-2-carboxamide
• Product was purified by column chromatography using a 12 g silica cartridge (Redisep® Normal-phase disposable silica flash column commercially available from Teledyne ISCO, Lincoln, Nebr., USA) using heptane (solvent A) and ethyl acetate (solvent B) as solvents. The following purification gradient was used: 3 min hold A, 11 min ramp to 50% B, 10 min hold 50% B. Samples containing product were pooled together to obtain crude product as a yellow solid (260 mg). The product was further purified by preparative HPLC (Gilson) using a 5-50% method and using 0.1% formic acid in water and acetonitrile as eluents. 21% yield.
• Redisep® Normal-phase disposable silica flash column commercially available from Teledyne ISCO, Lincoln, Nebr., USA
• 6-Carbamoyl-4-oxo-chromene-2-carboxylic acid (208 mg, 0.8920 mmol) was suspended in MeCN (2 mL) and di-isopropylethyl amine (115 mg, 0.9 mmol) was added. The reaction mixture was then cooled to 0° C. with an ice water bath and a solution of PyBOP (464 mg, 0.9 mmol) in DCM (2 mL) was added. The mixture was stirred at 0° C. for 30 minutes then cyclohexylmethanamine (101 mg, 0.9 mmol) was added and the reaction was stirred at room temperature for 4 h.
• 4-oxo-chromene-2-carboxylic acid or alternatively a corresponding substituted 4-oxo-chromene-2-carboxylic acid (0.62 mmol) was suspended in dry DCM (2 mL) and oxalyl chloride in DCM (99 mg, 1.2 eq., 0.78 mmol) and a drop of DMF were added. After 10 min. a solution of the selected amine having the desired —R 7 functionality (1.1 eq., 0.68 mmol) and Et 3 N (94 mg, 1.5 eq., 0.93 mmol) in DCM (1 mL) were added to the reaction mixture and the reaction stirred under N 2 at rt for 2 h.
• reaction mixture was diluted with water and extracted with DCM to give a crude material that was purified by flash column chromatography using EtOAc in heptane as eluent, using Redisep® column with the appropriate solvent conditions specified for each example. The desired fractions were concentrated to dryness to give the desired product.
• Example 43A 6-cyano-N-(cyclohexylmethyl)-4-oxo-chromene-2-carboxamide.
• the product was further purified by preparative HPLC (Gilson) eluting with 0.1% HCO 2 H in water and acetonitrile and a 5-50% method. The fractions containing product were pooled together to obtain N-[(4,4-difluoro-1-hydroxy-cyclohexyl)methyl]-6-hydroxy-4-oxo-chromene-2-carboxamide (27 mg, 15% yield) as a white solid.
• Example 57C-1 N-((2,3-dihydrobenzo[b][1,3]dioxa-5-yl)methyl)-4-oxo-4H-chromene-2-carboxamide was prepared from the appropriate starting materials using analagous chemistry.
• the product was purified by preparative HPLC [Instrument: GX-E; Column: Phenomenex Synergi C18 250 ⁇ 21.2 mm, particle size: 4 ⁇ m; Mobile phase: 25-55% acetonitrile in H 2 O (add 0.1% NH 3 .H 2 O, v/v) to afford 6-fluoro-N-(((1S,2S)-2-hydroxycyclohexyl)methyl)-4-oxo-4H-chromene-2-carboxamide (80 mg, 27% yield) as a yellow solid.
• the product was purified by Prep-HPLC (TFA) [Instrument: GX-E; Column: Phenomenex Synergi C18 250 ⁇ 21.2 mm, particle size: 4 ⁇ m; Mobile phase: 25-55% acetonitrile in H 2 O (add 0.05% TFA, v/v)] to afford 6-fluoro-N-(((1S,2R)-2-hydroxycyclohexyl)methyl)-4-oxo-4H-chromene-2-carboxamide (70 mg, 24% yield) as a white solid.
• TFA Prep-HPLC
• Example 57K Ethyl 2-(1-((6-fluoro-4-oxo-4H-chromene-2-carboxamido)methyl)cyclohexyl)acetate
• Example 60 Intermediate Compound, tert-butyl 4-[2-[2-(cyclohexylmethylcarbamoyl)-4-oxo-chromen-8-yl]oxyethyl]piperazine-1-carboxylate
• reaction mixture was diluted with water (5 mL) and extracted with DCM (3 ⁇ 5 mL). The combined organic layers were dried over Na 2 SO 4 , filtered and concentrated. The residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate 10/1 to 1/1) to obtain N-(cyclohexylmethyl)-8-methoxy-4-oxo-4H-chromene-2-carboxamide (268 mg, 35% yield) as a light yellow solid.
• N-(cyclohexylmethyl)-7-fluoro-6-methoxy-4-oxo-4H-chromene-2-carboxamide 200 mg, 0.6 mmol
• BBr 3 751 mg, 3. mmol
• the product was purified by preparative HPLC to give N-(cyclohexylmethyl)-7-fluoro-6-hydroxy-4-oxo-4H-chromene-2-carboxamide (44 mg, 23% yield) as a white solid.
• 6-Chloro-8-methoxy-4-oxo-4H-chromene-2-carboxylic acid 0.5 g, 2 mmol was dissolved in MeOH (10 mL) and SOCl 2 (0.47 g, 4.0 mmol, 0.285 mL) was added at 0° C. The reaction mixture was stirred at 65° C. for 1 hour and then filtered to afford methyl 6-chloro-8-methoxy-4-oxo-4H-chromene-2-carboxylate (300 mg) as a dark brown solid.
• Methyl 6-chloro-8-methoxy-4-oxo-4H-chromene-2-carboxylate 300 mg, 1.12 mmol was dissolved in DCM (10 mL) and BBr 3 (1.68 g, 6.70 mmol, 0.65 mL) was added at ⁇ 78° C. under nitrogen. The reaction mixture was stirred at 25° C. for 12 hours and then poured into ice-water (50 mL). The aqueous phase was extracted with DCM (3 ⁇ 50 mL).
• Methyl 6-chloro-8-hydroxy-4-oxo-4H-chromene-2-carboxylate 100 mg, 0.39 mmol
• 1-(aminomethyl)cyclohexanol 76 mg, 0.59 mmol
• 4-dimethylaminopyridine DMAP
• Et 3 N 120 mg, 1.18 mmol, 0.16 mL
• LiAIH 4 (1M in THF) 49 mg, 1.29 mmol was diluted with anhydrous ether (6.15 mL) and cooled in an ice bath before the dropwise addition of 2-trimethylsilyloxyspiro[3.3]heptane-2-carbonitrile (180 mg, 0.86 mmol) in ether (6.15 mL). After complete addition, the reaction was allowed to warm to room temperature for 2 h. The reaction was then cooled in an ice bath and ice added until no effervescence was observed. Approx. 2 ml of 1M NaOH was added and the mixture stirred at room temperature for approximately 10 mins before being filtered through a celite cartridge.

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