Patent Grant
11046700
This invention relates to compounds of formula (1a), (1b), (1c) and (1d) and pharmaceutically acceptable salts or solvates thereof which have activity as inhibitors of Schistosoma growth. The invention also relates to pharmaceutical compositions comprising such compounds, salts or solvates and to the use of such compounds as medicaments, in particular in the treatment or prevention of schistosomiasis, also known as bilharzia.
Schistosomiasis is one of the major neglected diseases affecting over 200 million people across sub-Saharan Africa, the Middle East and South America. It is a parasitic disease caused by flatworms of the genus Schistosoma, such as S. mansoni, S. haemotobium and S. japonicum. Infections are due to the larval stage of the worm, which then develop through a juvenile stage to adult worms.
Two drugs, praziquantel and oxamniquine, are approved for the treatment of schistosomiasis. Oxamniquine has a narrow spectrum of activity (only S. mansoni). Praziquantel is used worldwide against all three worm species but is known to lack efficacy against juvenile worms. Arthemether also shows activity against schistosomes in humans when tested in repeated doses but as it is used extensively in artemesin-based combination therapies for malaria treatment, it is not considered a viable compound for schistosomiasis treatment and control as its use against helminth infections might generate drug-resistant malaria parasites (Keiser J, Utzinger J. Curr Opin Infect Dis. 2007 December; 20(6):605-12).
The current status of research into novel anti-schistomes was the subject of an edition of Future Medicinal Chemistry (2015 Volume 7, Issue 6). There are many examples of repurposing of existing drug molecules and applications of known human drug mechanisms to discover new treatments for schistosomiasis. The former includes Abdulla, M. H., et al. (2009) “Drug discovery for schistosomiasis; hit and lead compounds identified in a library of known drugs by medium-throughput phenotypic screening.” PLoS Negl Trap Dis 3(7): e478; Dissous, C. and C. G. Grevelding (2011) “Piggy-backing the concept of cancer drugs for schistosomiasis treatment; a tangible perspective?” Trends Parasitol 27(2): 59-66; and Neves B. J. “The antidepressant drug paroxetine as a new lead candidate in schistosome drug discovery” Medicinal Chemistry Communications, 2016, 7, 1176.
Applications of known human drug targets in the field include Kuntz, A. N., et al. (2007). “Thioredoxin glutathione reductase from Schistosoma mansoni; an essential parasite enzyme and a key drug target.” PLoS Med 4(6): e206; Long, T., et al. (2010). “Schistosoma mansoni Polo-like kinase 1; A mitotic kinase with key functions in parasite reproduction.” Int J Parasitol 40(9):1075-1086; Rojo-Arreola, L., et al. (2014). “Chemical and genetic validation of the statin drug target to treat the helminth disease, schistosomiasis PLoS One 9(1): e87594; Jacques, S. A., et al. (2015). “Discovery of Potent Inhibitors of Schistosoma mansoni NAD(+) Catabolizing Enzyme.” Journal of Medicinal Chemistry 2015 58(8): 3582-3592; Mader, P., et al. (2016). “Biarylalkyl Carboxylic Acid Derivatives as Novel Antischistosomal Agents.” ChemMedChem 2016, 11, 1-11; Heimburg, T., et al. (2016). “Structure-Based Design and Synthesis of Novel Inhibitors Targeting HDAC8 from Schistosoma mansoni for the Treatment of Schistosomiasis.” J Chem Inf Model 2014 54(10): 3005-3019 and Journal of Medicinal Chemistry 2016 59(6): 2423-2435.
Large-scale testing on intact Schistosoma was limited by the available technology Ramirez B, B. Q et al (2007) “Schistosomes; challenges in compound screening.” Expert Opinion on Drug Discovery 2: S53-361; Sayed, A. A., et al. (2008) “Identification of oxadiazoles as new drug leads for the control of schistosomiasis.” Nat Med 14(4): 407-412. We have recently described a novel method for high throughput screening using larval stage Schistosoma and subsequently used this methodology to identify a set of hit molecules. Paveley, R. A., et al. (2012) “Whole organism high-content screening by label-free, image-based Bayesian classification for parasitic diseases.” PLoS Negl Trap Dis 6(7): e1762 and Mansour, N. R., et al. (2016) “High Throughput Screening Identifies Novel Lead Compounds with Activity against Larval, Juvenile and Adult Schistosoma mansoni PLoS Negl Trap Dis 10(4): e0004659. The latter paper disclosed an imidazopyrazine derivative (LSHTM-1945) with relatively weak activity of 4.9-6.7 μM against the larval, juvenile and adult stages of S. mansoni.
WO2014078813A1 discloses the preparation of imidazopyrazines for treating parasitic diseases, predominantly malaria, leishmaniasis and trypanosomiasis. WO2012080232A1 discloses the preparation of substituted imidazopyrazines as Mps-1 and TKK inhibitors useful in the treatment of hyperproliferative disorders. WO2007096764A2 discloses the preparation of bicyclic heteroaryl derivatives as cannabinoid receptor modulators. In addition, Kayagil, I. and S. Demirayak (2011). “Synthesis of some 2,3,6,8-tetraarylimidazo[1,2-a]pyrazine derivatives by using either reflux or microwave irradiation method, and investigation their anticancer activities.” Turk. J. Chem. 35(1): 13-24. WO2016133935 discloses a series of pyrazolo[1,5-c]pyrimidines as kinase inhibitors for the treatment of a variety of cancers.
There remains a need in the art for further compounds active as anti-schistosomes with good pharmacokinetic properties, combined with sufficient activity against all three main infective species of worm and against both juvenile and adult worms.
In a first embodiment, the invention provides a compound of formula (1a), (1b), (1c) or (1d) or a pharmaceutically acceptable salt or solvate thereof,
wherein:
R1 is C1-C4 alkyl optionally substituted with up to five F atoms, C3-C6 cycloalkyl optionally substituted with one methyl group, or C4-C7 cycloalkylmethyl;
R2 is H, F, Cl or OMe;
R3 is H, OH, OMe, OPO(OH)OH or OCH2OPO(OH)OH;
R4 is H, OH, OMe, OPO(OH)OH or OCH2OPO(OH)OH;
provided that R3 and R4 cannot both be H;
or R3 and R4 combine, together with the phenyl ring to which they are attached, to form an indazole group as shown below
R5 is H, F, Cl or OMe;
R6 is H, F, Cl or OMe;
X is N or C—R7
R7 (where present) is H or F;
R8 is SF5, Br, C1-C3 alkyl optionally substituted with up to seven F atoms, C3-C4 cycloalkyl, OCH2C≡CH or OC1-C3 alkyl optionally substituted with up to seven F atoms;
R9 is H; and
R10 is H, F or Me.
In a second embodiment, the present invention provides a compound of formula (1a), (1b), (1c) or (1d) as defined above, or a pharmaceutically acceptable salt or solvate thereof, for use in therapy.
In a third embodiment, the present invention provides a compound of formula (1a), (1b), (1c) or (1d), as defined above, or a pharmaceutically acceptable salt or solvate thereof, for use in the treatment of schistosomiasis.
In a fourth embodiment, the present invention provides the use of a compound of formula 1a), (1b), (1c) or (1d) as defined above, or a pharmaceutically acceptable salt or solvate thereof, for the manufacture of a medicament for the treatment of schistosomiasis.
In a fifth embodiment, the present invention provides a method for treating schistosomiasis comprising administering a therapeutically effective amount of a compound of formula (1a), (1b), (1c) or (1d) as defined above, or a pharmaceutically acceptable salt or solvate thereof, to a patient in need thereof.
In a sixth embodiment, the present invention provides a pharmaceutical composition comprising (i) a therapeutically effective amount of a compound of formula (1a), (1b), (1c) or (1d) as defined above, or a pharmaceutically acceptable salt or solvate thereof; and (ii) a pharmaceutically acceptable excipient.
The following definitions apply to the terms as used throughout this specification, unless otherwise limited in specific instances.
As used herein, the term “alkyl” means both straight and branched chain saturated hydrocarbon groups. Examples of alkyl groups include methyl, ethyl, n-propyl, iso-propyl, n-butyl, t-butyl, i-butyl, and sec-butyl groups.
As used herein, the term “cycloalkyl” means a cyclic saturated hydrocarbon group. Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.
As used herein, the term “cycloalkylmethyl” means a cyclic saturated hydrocarbon group linked to the rest of the molecule via a methylene bridge. Examples of cycloalkylmethyl groups include cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl and cyclohexylmethyl.
As used herein, the term “halogen” or “halo” means fluorine, chlorine, bromine or iodine. Fluorine, chlorine and bromine are particularly preferred.
“Pharmaceutically acceptable salt” means a salt such as those described in standard texts on salt formation, see for example: P. Stahl, et al. Handbook of Pharmaceutical Salts: Properties, Selection and Use (VCHA/Wiley-VCH, 2002), or S. M. Berge, et at, “Pharmaceutical Salts” (1977) Journal of Pharmaceutical Sciences, 66, 1-19. Suitable salts according to the invention include those formed with organic or inorganic acids or bases. In particular, suitable salts formed with acids according to the invention include those formed with mineral acids, strong organic carboxylic acids, such as alkanecarboxylic acids of 1 to 4 carbon atoms which are unsubstituted or substituted, for example, by halogen, such as saturated or unsaturated dicarboxylic acids, such as hydroxycarboxylic acids, such as amino acids, or with organic sulfonic acids, such as C1-C4 alkyl- or aryl-sulfonic acids which are unsubstituted or substituted, for example by halogen. Pharmaceutically acceptable acid addition salts include those formed from hydrochloric, hydrobromic, sulphuric, nitric, citric, tartaric, acetic, phosphoric, lactic, pyruvic, acetic, trifluoroacetic, succinic, perchloric, fumaric, maleic, glycolic, lactic, salicylic, oxaloacetic, methanesulfonic, ethanesulfonic, p-toluenesulfonic, formic, benzoic, malonic, naphthalene-2-sulfonic, benzenesulfonic, isethionic, ascorbic, malic, phthalic, aspartic, and glutamic acids, lysine and arginine. Other acids, which may or may not in themselves be pharmaceutically acceptable, may be useful as intermediates in obtaining the compounds of the invention and their pharmaceutical acceptable acid addition salts.
Pharmaceutically acceptable base salts include ammonium salts, alkali metal salts, for example those of potassium and sodium, alkaline earth metal salts, for example those of calcium and magnesium, and salts with organic bases, for example dicyclohexylamine, N-methyl-D-glucomine, morpholine, thiomorpholine, piperidine, pyrrolidine, a mono-, di- or tri-lower alkylamine, for example ethyl-, tert-butyl-, diethyl-, diisopropyl-, triethyl-, tributyl- or dimethyl-propylamine, or a mono-, di- or trihydroxy lower alkylamine, for example mono-, di- or triethanolamine. Corresponding internal salts may furthermore be formed.
“Pharmaceutically acceptable solvate” means a molecular complex comprising the compound of the invention and one or more pharmaceutically acceptable solvent molecules, for example, water or ethanol. Those skilled in the art of organic chemistry will appreciate that many organic compounds can form complexes with solvents in which they are reacted or from which they are precipitated or crystallized. These complexes are known as “solvates”. For example, a complex with water is known as a “hydrate”. Solvates, such as hydrates, exist when the drug substance incorporates solvent, such as water, in the crystal lattice in either stoichiometric or non-stoichiometric amounts. Drug substances are routinely screened for the existence of hydrates since these may be encountered at any stage of the drug manufacturing process or upon storage of the drug substance or dosage form. Solvates are described in S. Byrn et al. Pharmaceutical Research, 1995. 12(7): p. 954-954, and Water-Insoluble Drug Formulation, 2nd ed. R. Liu, CRC Press, page 553, which are incorporated herein by reference.
“Therapy”, “treatment” and “treating” include both preventative and curative treatment of a condition, disease or disorder. It also includes slowing, interrupting, controlling or stopping the progression of a condition, disease or disorder. It also includes preventing, curing, slowing, interrupting, controlling or stopping the symptoms of a condition, disease or disorder.
The invention provides a compound of formula (1a), (1b), (1c) or (1d) as defined above, or a pharmaceutically acceptable salt or solvate thereof
The invention also provides a compound of formula (1a′) or (1b′) or a pharmaceutically acceptable salt or solvate thereof,
wherein:
R1 is C1-C4 alkyl optionally substituted with up to five F atoms, C3-C6 cycloalkyl optionally substituted with one methyl group, or C4-C7 cycloalkylmethyl;
R2 is H, F, Cl or OMe;
R3 is H, OH, OMe, OPO(OH)OH or OCH2OPO(OH)OH;
R4 is H, OH, OMe, OPO(OH)OH or OCH2OPO(OH)OH;
provided that R3 and R4 cannot both be H;
or R3 and R4 combine, together with the phenyl ring to which they are attached, to form an indazole group as shown below
R5 is H, F, Cl or OMe;
R6 is H, F, Cl or OMe;
R7 is H or F;
R8 is SF5 or C1-C3 alkyl substituted with three to seven F atoms;
R9 is H; and
R10 is H, F or Me.
B0. Core Structures
In some embodiments the compound of the invention is of formula (1a), as defined above.
In other embodiments the compound of the invention is of formula (1b), as defined above.
In other embodiments the compound of the invention is of formula (1c), as defined above.
In other embodiments the compound of the invention is of formula (1d), as defined above.
In other embodiments the compound of the invention is of formula (1a′), as defined above.
In other embodiments the compound of the invention is of formula (1b′), as defined above.
B1. Substituent R1
R1 is selected from the group consisting of C1-C4 alkyl optionally substituted with up to five F atoms, C3-C6 cycloalkyl optionally substituted with one methyl group, and C4-C7 cycloalkylmethyl.
R1 is preferably selected from the group consisting of methyl, ethyl, n-propyl, isopropyl, cyclopropyl optionally substituted with one methyl group, cyclobutyl, cyclopropylmethyl, CHF2, CF3 and CH2CF3.
R1 is more preferably selected from the group consisting of ethyl, isopropyl, cyclopropyl, cyclobutyl, CF3 and CH2CF3.
B2. Substituent R2
R2 is selected from the group consisting of H, F, Cl and OMe.
R2 is preferably selected from the group consisting of H, F and Cl.
R2 is more preferably F or Cl.
R2 is most preferably F
B3. Substituent R3
R3 is selected from the group consisting of H, OH, OMe, OPO(OH)OH and OCH2OPO(OH)OH.
R3 is preferably selected from the group consisting of H, OH, OMe and OPO(OH)OH.
R3 is more preferably H.
B4. Substituent R4
R4 is selected from the group consisting of H, OH, OMe, OPO(OH)OH and OCH2OPO(OH)OH.
R4 is preferably selected from the group consisting of H, OH, OMe and OPO(OH)OH.
R4 is more preferably OH.
Alternatively, and preferably, substituents R3 and R4 may combine, together with the phenyl ring to which they are attached, to form an indazole group as shown below
wherein:
R9 is H; and
R10 is selected from the group consisting of H, F and Me.
In a preferred embodiment of the indazole, R10 is selected from the group consisting of H and Me
In a more preferred embodiment of the indazole, substituents R9 and R10 are both H.
B5. Substituent R5
R5 is selected from the group consisting of H, F, Cl and OMe.
R5 is preferably selected from the group consisting of H and OMe.
R5 is more preferably H.
B6. Substituent R6
R6 is selected from the group consisting of H, F, Cl and OMe.
R6 is preferably selected from the group consisting of H and F.
B7. Substituent X
X is N or C—R7, where R7 is selected from the group consisting of H and F.
In one embodiment, X is C—R7.
B8. Substituent R8
R8 is selected from the group consisting of SF5, Br, C1-C3 alkyl optionally substituted with up to to seven F atoms; C3-C4 cycloalkyl; OCH2C≡CH and OC1-C3 alkyl optionally substituted with up to to seven F atoms.
Alternatively, R8 is selected from the group consisting of SF5 and C1-C3 alkyl substituted with three to seven F atoms.
R8 is preferably selected from the group consisting of cyclopropyl, cyclobutyl, isopropyl, CH2CF3, OCF3, OiPr, CF3, CF2CF3 and SF5.
Alternatively, R8 is preferably selected from the group consisting of CF3, CF2CF3 and SF5.
B9. Combinations of Substituents R2 to R6
Preferred combinations of substituents R2 to-R6 include:
B10. Combinations of Substituents X and R8
Preferred combinations of substituents X and R8 include:
Alternative preferred combinations of substituents X and R8 include:
B11. Specific Embodiments of Compounds of Formula (1a), (1b), (1c) and (1d)
Various embodiments of substituents R1, R2, R3, R4, R5, R6, X, R7, R8, R9 and R10 have been discussed in B1 to B10 above. These “substituent” embodiments can be combined with any of the “core structure” embodiments, discussed in BO above, to form further embodiments of compounds of formula (1a), (1a′), (1b), (1b′), (1c) and (1d). All embodiments of compounds of formula (1a), (1a′), (1b), (1b′), (1c) and (1d) formed by combining the “substituent” embodiments and “core structure” embodiments, discussed above, are within the scope of the present invention, and some further preferred embodiments of the compounds of formula (1a), (1a′), (1b), (1b′), (1c) and (1d) are provided below.
In a preferred aspect of the first embodiment, the invention provides a compound of formula (1a), (1a′), (1b), (1b′), (1c) or (1d) or a pharmaceutically acceptable salt or solvate thereof,
wherein:
R1 is methyl, ethyl, n-propyl, isopropyl, cyclopropyl optionally substituted with one methyl group, cyclobutyl, cyclopropylmethyl, CHF2, CF3 or CH2CF3;
R2 is H, F or OMe; or R2 is H, F or Cl;
R3 is H, OH, OMe or OPO(OH)OH;
R4 is H, OH, OMe or OPO(OH)OH;
provided that R3 and R4 cannot both be H;
or R3 and R4 combine, together with the phenyl ring to which they are attached, to form an indazole group as shown below;
R5 is H or OMe;
R6 is H or F;
X is N or C—R7 where R7 is H or F; or X is C—R7 where R7 is H or F;
R8 is CF3, CF2CF3, CH2CF3, CH(CH3)2, OCF3, OCH(CH3)2, cyclopropyl, cycolbutyl or SF5; or
R8 is CF3, CF2CF3 or SF5;
R9 is H; and
R10 is H.
In a more preferred aspect of the first embodiment, the invention provides a compound of formula (1a), (1a′), (1b), (1b′), or (1c) or a pharmaceutically acceptable salt or solvate thereof, wherein:
R1 is ethyl, isopropyl, cyclopropyl, cyclobutyl, CF3 or CH2CF3; or R1 is ethyl, isopropyl, cyclopropyl, CF3 or CH2CF3;
R2 is F;
R3 is H;
R4 is OH;
or R3 and R4 combine, together with the phenyl ring to which they are attached, to form an indazole group as shown below;
R5 is H;
R6 is H or F;
X is C—R7 where R7 is H or F;
R8 is CF3, CF2CF3 CH(CH3)2 or SF5; or R8 is CF3, CF2CF3 or SF5;
R9 is H; and
R10 is H.
In an even more preferred aspect of the first embodiment, the invention provides a compound of formula (1a), (1a′), (1 b), (1 b′), (1c) or (1 d) or a pharmaceutically acceptable salt or solvate thereof,
wherein:
R1 is ethyl, isopropyl, cyclopropyl, CF3 or CH2CF3;
R2 to R6 are selected to provide the structure:
and
R7 and R8 are selected to provide the structure
or R7 and R8 are selected to provide the structure:
The following compounds represent specific embodiments of the invention:
The compounds of the invention intended for pharmaceutical use may be administered alone or in combination with one or more other compounds of the invention or in combination with one or more other drugs (or as any combination thereof). Generally, they will be administered as a formulation in association with one or more pharmaceutically acceptable excipients. Accordingly, the present invention is also directed to a pharmaceutical composition comprising (i) a therapeutically effective amount of a compound of formula (1a), (1b), (1c) or (1d) as defined above, or a pharmaceutically acceptable salt or solvate thereof; and (ii) a pharmaceutically acceptable excipient.
Pharmaceutical 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).
This invention is also directed to compounds of formula (1a), (1b), (1c) and (1d) as defined above, or a pharmaceutically acceptable salt or solvate thereof, for use in therapy, in particular for the treatment of schistosomiasis.
This invention is also directed to the use of compounds of formula (1a), (1b), (1c) and (1d), as defined above, or a pharmaceutically acceptable salt or solvate thereof, in the manufacture of a medicament for treatment of schistosomiasis.
This invention is also directed to a method for treating schistosomiasis comprising administering a therapeutically effective amount of a compound of formula (1a), (1b), (1c) or (1d) as defined above, or a pharmaceutically acceptable salt or solvate thereof, to a patient in need thereof.
The amount of active ingredient which is required to achieve a therapeutic effect will, of course, vary with the particular compound, the route of administration, the subject under treatment, including the type, species, age, weight, sex, and medical condition of the subject and the renal and hepatic function of the subject, and the particular disorder or disease being treated, as well as its severity. An ordinarily skilled physician, veterinarian or clinician can readily determine and prescribe the effective amount of the drug required to prevent, counter or arrest the progress of the condition.
The methods used for the synthesis of the compounds of the invention are illustrated by the schemes below. The starting materials and reagents used in preparing these compounds are available from commercial suppliers or can be prepared by methods obvious to those skilled in the art.
General Method 1A
The invention also provides a process for the preparation of Schistosoma growth inhibitors where R1-R6 and X are defined according to the invention and R8 is CF3, CF2CF3 or CH(CH3)2. The process involves reacting an appropriate 3-chloro or 3-bromo-imidazo[1,2-a]pyrazine with an appropriate arylboronic acid or aryl pinacol borane under Suzuki conditions in the presence of a palladium catalyst.
General Method 1B
The invention also provides a process for the preparation of Schistosoma growth inhibitors where R1-R6 and X are defined according to the invention and R8 is CF3 or CF2CF3. The process involves reacting an appropriate 3-bromo-pyrazolo[1,5-c]pyrimidine with an appropriate arylboronic acid or aryl pinacol borane under Suzuki conditions in the presence of a palladium catalyst.
General Method 2
The invention also provides a process for the preparation of Schistosoma growth inhibitors where R1-R6 and X are defined according to the invention and R8 is CF2CF3 or SF5. The process involves reacting an appropriate 6-bromo-imidazo[1,2-a]pyrazine with an appropriate arylboronic acid or aryl pinacol borane under Suzuki conditions in the presence of a palladium catalyst.
General Method 3A
The invention also provides a process for the preparation of Schistosoma growth inhibitors where R1-R6 and X are defined according to the invention and R8 is CF3, CF2CF3, CH(CH3)2, CH2CF3, OCF3, OCH(CH3)2, OCH2C≡CH, cPr, cBu or SF5. The process involves reacting an appropriate 3-bromo or 3-chloro-imidazo[1,2-a]pyrazine with an appropriate THP-protected indazole pinacol borane under Suzuki conditions in the presence of a palladium catalyst. The THP group can be removed under acidic conditions e.g. with TFA or HCl in an alcoholic solvent.
General Method 3B
The invention also provides a process for the preparation of Schistosoma growth inhibitors where R1-R6 and X are defined according to the invention and R8 is CF3 or CF2CF3. The process involves reacting an appropriate 3-bromo-pyrazolo[1,5-c]pyrimidine with an appropriate THP-protected indazole pinacol borane under Suzuki conditions in the presence of a palladium catalyst. The THP group can be removed under acidic conditions e.g. with TFA or HCl in an alcoholic solvent.
General Method 3C
The invention also provides a process for the preparation of Schistosoma growth inhibitors where R1-R8 and X are defined according to the invention. The process involves reacting an appropriate 3-bromo-pyrazolo[1,5-c]pyridine with an appropriate THP-protected indazole pinacol borane under Suzuki conditions in the presence of a palladium catalyst. The THP group can be removed under acidic conditions e.g. with TFA or HCl in an alcoholic solvent.
General Method 3D
The invention also provides a process for the preparation of Schistosoma growth inhibitors where R1-R8 and X are defined according to the invention. The process involves reacting an appropriate 3-iodo-imidazo[1,2-a]pyrazine with an appropriate THP-protected indazole pinacol borane under Suzuki conditions in the presence of a palladium catalyst. The THP group can be removed under acidic conditions e.g. with TFA or HCl in an alcoholic solvent.
General Method 4
The invention also provides a process for the preparation of Schistosoma growth inhibitors where R1, R2 and R5-R8 and X are defined according to the invention, and R3 or R4 are OPO(OH)OH. The process involves reacting an appropriate hydroxyphenyl imidazo[1,2-a]pyrazine with phosphorus oxychloride in pyridine, followed by treatment of the crude product with acetone/water.
General Method 5
The invention also provides a process for the preparation of Schistosoma growth inhibitors where R1, R2, R6, R8 and X are defined according to the invention. The process involves coupling of an appropriate chloropyrimidine and arylboronic acid to give an intermediate, which is reduced with Raney Ni. The resulting diamine is reacted with an alkylaldehyde to provide an intermediate that is aromatised to a purine derivative and finally deprotected under acidic conditions.
General Experimental Details
LC-MS
Compounds requiring purification under basic conditions were purified on an LC-MS system equipped with a YMC Actus Triart C18 5 μm (20×250 mm) column or Gemini NX 5 μm C18 (100×30 mm) columns, using a gradient elution of acetonitrile in water containing 20 mM Ammonium bicarbonate (10-45% over 30 min then 95% acetonitrile for 2 minutes).
UPLC
Method A: Formic Acid/Ammonium acetate (3 min runtime-UPLC) (3 min) Column—Restek Ultra AQ C18 (30×2.1 mm, 3u), (mobile phase: 98% [0.05% modifier in water] and 2% [CH3CN] held for 0.75 min, then to 90% [0.05% Modifier in water] and 10% [CH3CN] in 1.0 min, further to 2% [0.05% Modifier in water] and 98% [CH3CN] in 2.0 min, held this mobile phase composition up to 2.25 min and finally back to initial condition in 3.0 min).
Flow=1.5 ml/min
Method E: (General-5 min)
Column—ZorbaxC18 (50×4.6 mm, 5u, 130A), (mobile phase: from 90% [10 mM NH4OAc in water] and 10% [CH3CN] to 70% [10 mM NH4OAc in water] and 30% [CH3CN] in 1.5 min, further to 10% [10 mM NH4OAc in water] and 90% [CH3CN] in 3.0 min, held this mobile phase composition up to 4.0 min and finally back to initial condition in 5.0 min). Flow=1.2 ml/min.
NMR
1H NMR and 13C spectra were recorded on 400 MHz and 101 MHz respectively instruments at room temperature unless specified otherwise were referenced to residual solvent signals. Data are presented as follows: chemical shift in ppm, integration, multiplicity (br=broad, app=apparent, s=singlet, d=doublet, t=triplet, q=quartet, p=pentet, m=multiplet) and coupling constants in Hz.
Preparation of Starting Materials
All of the starting materials for making the intermediates and example compounds were obtained from commercial sources or using literature methods with the exception of the following compounds.
To a stirred solution of 5-bromo-4-fluoro-1-(oxan-2-yl)-1H-indazole (800 mg, 2.7 mmol) in dioxane (20 mL) were added bis(pinacolato)diboron (1.36 g, 5.3 mmol) and potassium acetate (787 mg, 8.0 mmol) in sealed tube and the resulting mixture was stirred and degased by using argon gas for 5 min. After that Pd(dppf)Cl2.DCM (218 mg, 0.27 mmol) was added and the resulting mixture was refluxed at 110° C. for 6 h. After completion, reaction mixture was evaporated under reduced pressure. The residue was suspended into water and extracted with ethyl acetate. Combined organic layer was dried over anhydrous Na2SO4 and evaporated under reduced pressure. The crude compound was purified by column chromatography using silica gel eluted with 0-10% EtOAc in hexane to give the title compound as a yellow color oil (900 mg, 97%). UPLC rt 1.9 min MH+ 347. 1H NMR (400 MHz, CDCl3) δ 8.09 (s, 1H), 7.67 (dd, 1H), 7.31 (d, 1H), 5.69 (dd, 1H), 4.01 (d, 1H), 3.73 (t, 1H), 2.53 (m, 1H), 2.12 (m, 1H), 2.05 (m, 1H), 1.75 (m, 2H), 1.68 (m, 1H), 1.40 (s, 12H).
To a stirred solution of 4-Bromo-1-fluoro-2-pentafluoroethyl-benzene (1 g, 3.4 mmol) in 1,4-dioxane (25 ml) was added bispinacolatodiborane (1.7 g, 6.8 mmol) followed by potassium acetate (1 g, 10.2 mmol). The reaction mixture was deoxygenated with argon, then to the reaction mixture was added [1,1′-Bis(diphenylphosphino)ferrocene]palladium(II) chloride, complex with dichloromethane (0.28 g, 0.34 mmol) and the reaction mixture was allowed to stir at 90° C. for 16 hours under nitrogen. After complete consumption of the starting material (monitored by TLC) the reaction mixture was filtered throw a celite bed to remove the catalyst, the mother liquor was evaporated under reduced pressure, the residue was diluted with ethyl acetate, washed successively with water and brine, the organic layer dried over sodium sulphate and evaporated under reduced pressure to get the product, which was used without further purification.
To a stirred solution of 3-fluoro-2-methylaniline (15.0 g, 120 mmol) in ACN (300.0 mL) was added N-Bromosuccinimide (23 g, 132 mmol) portion wise at 10° C. The reaction mixture was stirred at ambient temperature for 3 h, and was evaporated under reduced pressure. The reaction mixture was diluted with saturated Na2S2O3 (100.0 mL) at 10° C. and extracted with EtOAc (2×100 mL). Combined organic layer was washed with brine, dried over anhydrous Na2SO4 and evaporated under reduced pressure to get desired crude, which was purified by column chromatography(100-200 mesh silica gel, eluent: 15% ethyl acetate in hexane) of the title compound (15 g, 61%) as a brown solid. LCMS rt 3.27 min MH+204. 1H NMR (400 MHz, DMSO-d6) δ 7.09 (t, J=8.2, 1H), 6.40 (d, J=8.52, 1H), 1.98 (s, 3H).
To a stirred solution of 4-Bromo-3-fluoro-2-methyl-phenylamine (15.0 g, 73.5 mmol) in acetic acid (200 mL) was added sodium nitrite (10 g, 147 mmol) portion wise at 10° C. and reaction mixture was stirred at rt for 16 h. Upon completion, aqueous NaOH (50%) was added to the reaction mixture at −10° C. dropwise with vigorous stirring until pH was 7-8. The mixture was then extracted with ethyl acetate. The organic layer was dried over sodium sulphate and concentrated under reduced pressure to get the crude compound which was purified by column chromatography on silica gel (0-40% EtOAc in Hexane) to afford the title compound (10 g, 63%) as a off white solid. LCMS rt 3.03 min MH+214. 1H NMR (400 MHz, CDCl3) δ 10.44 (brs, 1H), 8.14 (s, 1H), 7.49-7.45 (m, 1H), 7.19 (d, J=8.76, 1H).
To a stirred solution of 5-Bromo-4-fluoro-1H-indazole (10.0 g, 46 mmol) in dichloromethane (300.0 mL) was 3,4-dihydropyran (11.7 g, 139 mmol) followed by PTSA (800 mg, 4.6 mmol) and the reaction mixture was stirred at ambient temperature for 12 h under nitrogen. After completion, the reaction mixture was diluted with DCM, washed successively with saturated NaHCO3 solution and brine, combined organic layer was dried over anhydrous Na2SO4 and evaporated under reduced pressure to get the crude compound, which was purified by column chromatography (100-200 mesh silica gel, eluent: 5% ethyl acetate in hexane) to get the title compound (8 g, 57%) as a off white solid.
LCMS rt 3.83 min MH+299. 1H NMR (400 MHz, CDCl3) δ 8.05 (s, 1H), 7.46-7.43 (m, 1H), 7.28 (d, J=8.84, 1H), 5.69-5.67 (m, 1H), 3.99-3.86 (m, 1H), 3.74-3.70 (m, 1H), 2.53-2.45 (m, 1H), 2.13-2.09 (m, 2H), 1.86-1.71 (m, 4H).
To a stirred solution of 5-Bromo-4-fluoro-1-(tetrahydro-pyran-2-yl)-1H-indazole (8 g, 26.7 mmol) in 1,4-dioxane (200 ml) was added bispinacolatodiborane (13.6 g, 53.5 mmol) followed by potassium acetate (7.8 g, 80.3 mmol). The reaction mixture was deoxygenated with argon, then to the reaction mixture was added [1,1′-Bis(diphenylphosphino)ferrocene]-palladium(II) chloride, complex with dichloromethane (2.2 g, 2.67 mmol) and the reaction mixture was allowed to stir at 90° C. for 16 hours under nitrogen. The solvent was evaporated under reduced pressure, the residue was diluted with ethyl acetate, filtered over celite bed. Filtrate was then washed successively with water and brine. The organic layer was dried over sodium sulphate and evaporated under reduced pressure to give the crude product, which was purified by column chromatography(100-200 mesh silica gel, eluent: 5% ethyl acetate in hexane) to get the title compound (6 g, 64%) as white solid. 1H NMR (400 MHz, CDCl3) δ 8.08 (s, 1H), 7.68-7.65 (m, 1H), 7.31 (d, J=8.56, 1H), 5.70-5.67 (m, 1H), 4.02-3.99 (m, 1H), 3.76-3.70 (m, 1H), 2.54-2.52 (m, 1H), 2.16-2.03 (m, 2H), 1.77-1.65 (m, 4H), 1.36 (s, 12H).
2-amino-5-bromopyrazine (10 g, 57 mmol) and 1-bromo-3-methyl-butan-2-one (20 mL) were dissolved in acetonitrile (50 mL) and heated at 100° C. in a sealed tube for 3 days. The reaction was quenched with sodium bicarbonate solution and filtered and extracted with ethyl acetate, organic layer was dried over sodium sulphate and concentrated to give a brown liquid that was purified by column chromatography (100-200 mesh silica gel, eluent; 50% ethyl acetate in DCM) to give the title compound as a brown semi solid (3.5 g, 25%). UPLC rt 2.7 min MH+ 242. 1H NMR (400 MHz, DMSO-d6) δ 8.79 (s, 1H), 8.18 (s, 1H), 7.45 (s, 1H), 3.15 (h, 1H), 1.36 (d, 6H).
A mixture of 6-bromo-2-(propan-2-yl)imidazo[1,2-a]pyrazine (2.0 g, 8.3 mmol) and 3-(trifluoromethyl)phenylboronic acid (1.9 g, 10 mmol) was dissolved in dioxane:H2O (3:1, 20 mL) and treated with K3PO4 (5.3 g, 25 mmol). The mixture was degassed for 20-30 min, treated with Pd-dppf-DCM complex (2.0 g, 2.5 mmol) and heated at 90° C. for 16 h, After completion, the reaction was quenched with water and extracted with ethyl acetate. The organic layer was dried over sodium sulphate and concentrated to get crude compound, which was purified by column chromatography (100-200 mesh silica gel, eluent: 10% ethyl acetate in DCM) to give the title compound (2.3 g, 90%). UPLC rt 3.4 min MH+ 306. 1H NMR (400 MHz, DMSO-d6) δ 9.12 (s, 1H), 8.43 (s, 1H), 8.19 (s, 1H), 8.10 (d, 1H), 7.65 (d, 1H), 7.59 (t, 1H), 7.54 (s, 1H), 3.21 (h, 1H), 1.40 (d, 6H).
2-(propan-2-yl)-6-[3-(trifluoromethyl)phenyl]imidazo[1,2-a]pyrazine (2.3 g, 7.5 mmol) was dissolved in DCE (25 mL) and treated with N-Bromosuccinimide (1.6 g, 9.1 mmol) and the mixture was heated at 85-90° C. for 8 h, The reaction mixture was concentrated and the crude was purified by combiflash chromatography to get the title compound as a brown solid (1.5 g, 52%) UPLC rt 4.0 min MH+ 386. 1H NMR (400 MHz, DMSO-d6) δ 9.07 (s, 1H), 8.38 (s, 1H), 8.25 (s, 1H), 8.15 (d, 1H), 7.68 (d, 1H), 7.62 (t, 1H), 3.30 (h, 1H), 1.40 (d, 6H).
To a stirred solution of 2-amino-5-bromo-pyrazine (2 g, 11.5 mmol) in isopropanol (60 ml) was added 3-Bromo-1,1,1-trifluoroacetone (3 g, 3 mmol) and the mixture was allowed to stir at 90° C. for 72 h. After complete consumption of the starting material (monitored by both TLC and LCMS) the solvent was evaporated under reduced pressure, the residue was diluted with ethyl acetate basified with saturated bicarbonate solution, filtered overcelite bed. Aqueous part was discarded and organic part was washed successively with saturated sodium bicarbonate solution and brine, the organic layer dried over sodium sulphate, evaporated under reduced pressure to get crude compound, which was purified by column chromatography (100-200 mesh silica gel, eluent:20% ethyl acetate in DCM) to give the title compound (1 g, 32.7%) as brown solid. LCMS rt 2.87 min MH+ 266. 1H NMR (400 MHz, DMSO-d6) δ 9.13 (s, 1H) 8.99 (s, 1H), 8.64 (s, 1H).
To a stirred solution of 6-Bromo-2-trifluoromethyl-imidazo[1,2-a]pyrazine (1 g, 3.75 mmol) in dry DMF (25 ml) was added N-Chlorosuccinimide (753 mg, 5.63 mmol) and the reaction mixture was allowed stir at 90° C. 24 h. After complete consumption of the starting material (monitored by both TLC and LCMS) the reaction mixture was diluted with ethyl acetate, washed successively with water and brine. The organic part was dried over sodium sulphate and evaporated under reduced pressure to get crude compound which was purified by column chromatography (100-200 mesh silica gel, eluent: 10% ethyl acetate in DCM) to give the title compound (900 mg, 79.7%) of as light brown solid.
LCMS rt 3.16 min MH+ 301. 1H NMR (400 MHz, DMSO-d6) δ 9.17 (s, 1H) 8.94 (s, 1H).
A mixture of 6-Bromo-3-chloro-2-trifluoromethyl-imidazo[1,2-a]pyrazine (800 mg, 2.67 mmol) and 2-(4-Fluoro-3-pentafluoroethyl-phenyl)-4,4,5,5-tetramethyl-[1,3,2]dioxaborolane) (1 g, 2.93 mmol) was dissolved in 1,4-dioxane:H2O (4:1, 15 mL) and treated with K3PO4 (1.7 g, 8 mmol). The mixture was degassed for 20-30 min, treated with Bis(diphenylphosphino)ferrocene]palladium(II) chloride, complex with dichloromethane (217 mg, 0.27 mmol) and heated at 90° C. for 16 h. After completion, the reaction was quenched with water and extracted with ethyl acetate. The organic layer was dried over sodium sulphate and concentrated to get crude compound, which was purified by column chromatography (100-200 mesh silica gel, eluent: 10% ethyl acetate in DCM) to give the title compound (700 mg, 60.5%). LCMS rt 4 min MH+ 434. 1H NMR (400 MHz, DMSO-d6) δ 9.40 (s, 1H), 9.26 (s, 1H), 8.63 (br. s, 1H), 8.53 (d, J=5.08 Hz, 1H), 7.75-7.70 (m, 1H).
Prepared using a similar method to Intermediate 1 or 37 from the appropriate halo-ketone and boronic acid or boronate.
Prepared by a modification of the route to the above intermediates using the scheme below.
Prepared from the appropriate 3-iodo imidazo[1,2-a]pyrazine using the method below
2-amino-5-bromopyrazine (10 g, 57 mmol) and 1-bromo-2,2-dimethoxy-propane (15 g) were dissolved in IPA (30 mL) and heated at 100° C. in a sealed tube for 3 days. The reaction was quenched with sodium bicarbonate solution and filtered and extracted with ethyl acetate; the organic layer was dried over sodium sulphate and concentrated to give a brown solid that was used for the next step without further purification.
To a well stirred solution of 6-bromo-2-methyl imidazo[1,2-a]pyrazine (3 g, 14.15 mmol) in DMF (15 ml) was added N-Iodoosuccinimide (3.82 g, 16.98 mmol) and the reaction mixture was allowed to stir at 80° C. for 12 h under nitrogen. After complete consumption of the SM (Monitored by LCMS) the reaction mixture was diluted with ethyl acetate, washed successively with water and brine, dried over sodium sulphate and evaporated under reduced pressure to get the crude compound, which was purified by column chromatography (100-200 mesh silica gel, eluent; 20% ethyl acetate in hexane) to give the title compound as a yellowish solid [1.5 g, 31% (After two steps)]. LCMS rt 2.89 min MH+ 338. 1H NMR (400 MHz, DMSO-d6) δ 8.74 (s, 1H), 8.49 (s, 1H), 2.44 (s, 3H).
A mixture of 6-bromo-3-iodo-2-methyl-imidazo[1,2-a]pyrazine (120 mg, 0.355 mmol) and 2,6-difluoro-4-hydroxyphenylboronic acid (61.8 mg, 0.355 mmol) was dissolved in THF:H2O (3:1, 4 mL) and treated with KF (62 mg, 1.065 mmol). The mixture was degassed for 20-30 min, treated with bis(tri-tert-butylphosphine)palladium(0) (18 mg, 0.036 mmol) and heated at 120° C. for 1 h under microwave irradiation (200 watt), After completion, the reaction was quenched with water and extracted with ethyl acetate. The organic layer was dried over sodium sulphate and concentrated to get crude compound, which was used in the preparation of Example 31 without further purification.
To the stirred solution of ethyl 4-bromo-3-(propan-2-yl)-1H-pyrazole-5-carboxylate (400 mg, 1.5 mmol) in THF (4 mL) was added 2.5 M LAH (Lithium Aluminium Hydride) solution (2.5M in THF, 87 μL, 2.3 mmol) in THF at 0° C. and stirred for 3 h. The reaction was then quenched by saturated solution of Na2SO4 and filtered with celite and evaporated under reduced pressure to give the title compound (290 mg, 86%). UPLC rt 3.1 min MH+ 261. 1H NMR (400 MHz, DMSO-d6) δ 12.79 (s, 1H), 4.95 (br.s, 1H), 4.37 (s, 2H), 2.94 (m, 1H), 1.21 (d, 6H).
To ice cold [4-bromo-3-(propan-2-yl)-1H-pyrazol-5-yl]methanol (280 mg, 1.28 mmol) was added SOBr2 (7 mL) and the mixture was heated at 40° C. for 2 h. Volatiles were removed under vacuum and the residue triturated with hexane to afford the title compound as a pale yellow solid HBr salt (300 mg, 83%) UPLC rt 2.5 min MH+ 283. 1H NMR (400 MHz, DMSO-d6) δ 4.53 (s, 2H), 2.98 (m, 1H), 1.22 (d, 6H).
To a solution of 4-bromo-5-(bromomethyl)-3-(propan-2-yl)-1H-pyrazole (100 mg, 0.36 mmol) in DCM (2 mL) at −10° C. was added 1-(3-trifluoromethylphenyl)-1-tosyl methyl isocyanide (120 mg, 0.36 mmol) and benzyl triethylammonium chloride (16 mg, 0.07 mmol) and then added drop wise a solution of of 30%-NaOH in water (2 mL). The resultant reaction mixture was maintained at −10° C. for 3 h. The mixture was extracted into DCM, the organic layer was dried and concentrated under vacuum to afford crude product. The crude was chromatographed on silica gel column using 3%-EtOAc in hexane to afford title compound as a colourless solid (30 mg, 22%). UPLC rt 2.7 min MH+ 384. 1H NMR (400 MHz, DMSO-d6) δ 9.59 (s, 1H), 8.57 (s, 1H), 8.54 (d, 1H), 8.27 (s, 1H), 7.82 (d, 1H), 7.75 (t, 1H), 3.25 (h, 1H), 1.36 (d, 6H).
Prepared from the appropriate pyrazole derivative and tosyl isocyanide. 1-(4-fluoro-3-trifluoromethylphenyl)-1-tosyl methyl isocyanide was prepared from (4-fluoro-3-trifluoromethylphenyl) methyl isocyanide and tosyl fluoride. 1-(4-fluoro-3-pentafluoroethylphenyl)-1-tosyl methyl isocyanide was prepared from (4-fluoro-3-pentafluoroethylphenyl) methyl isocyanide and tosyl fluoride.
TFA (160 ml) was added drop wise to tert-butyl [(mesitylsulfonyl)oxy]carbamate 1 (40 g, 126.98 mmol) at 0° C. and the reaction mixture was allowed to stir at this temperature for 1 h. The reaction mixture was poured slowly into ice water. The precipitate formed was filtered and washed thoroughly with water to remove trace amount of TFA to afford 2-[(aminooxy)sulfonyl]-1,3,5-trimethylbenzene. The solid was dissolved in DCM (200 mL) and the solution was used in the next step immediately without further analysis.
To a solution of Pyridin-4-yl-carbamic acid tert-butyl ester (2.30 g, 11.06 mmol) in DCM (30 mL) was added drop wise a solution of compound 2-[(aminooxy)sulfonyl]-1,3,5-trimethylbenzene in DCM (30 mL) at 0-5° C. and continued stirring at this temperature for 2 h. The reaction mixture was concentrated under reduced pressure to afford the target compound (2 g, 50% in two step) as brown gum. It was used in the next step without further purification. 1H NMR (400 MHz, DMSO-d6) δ 10.93 (1H, br. s), 8.54 (2H, d, J=6.9 Hz), 7.85 (2H, d, J=6.8 Hz s), 6.73 (2H, s), 2.16 (3H, s), 1.51 (9H, s) [possibly SO3H proton was not seen in the NMR].
Ethyl isobutyrylacetate (5 g, 31 mmol) was added to a round-bottom flask and dissolved in toluene (150 ml). The solution was cooled with an ice bath to 5-10° C. (internal temperature) followed by addition of a saturated aqueous solution of LiOH (50 mL, 240 mmol) in one portion. The resulting biphasic mixture was vigorously stirred at 5-10° C. for 5 minutes followed by the addition of triflic anhydride (13 ml, 79 mmol) dropwise at a rate to maintain the internal temperature between 5-15° C. Upon completion of the reaction (as judged by TLC, typically <10 min), the biphasic solution was diluted with water and the layers were separated. The aqueous layer was extracted with EtOAc. The combined organic layers were washed with water, brine and dried over sodium sulphate. The organic layer was filtered and concentrated under reduced pressure to get the title compound (6.2 g, 67%) as colourless liquid.
1H NMR (400 MHz, DMSO-d6) 6.09 (1H, s), 4.19-4.13 (2H, m), 2.57-2.53 (1H, m), 1.24-1.14 (9H, m)
To a stirred solution of Ethyl ethyl 4-methyl-2-[(trifluoromethane)sulfonyloxy]pent-2-enoate (6.1 g, 21 mmol) in dry THF (40 ml) was added triethyl amine (4 ml, 29 mmol) and the reaction mixture was allowed to stir at 80° C. for 16 h under nitrogen. The reaction mixture was then cooled to RT and evaporated under reduced pressure to afford the title compound (2.5 g, 84.2%) as yellow oil.
1H NMR (400 MHz, DMSO-d6) 4.16-4.10 (1H, m), 3.09-3.06 (2H, m), d 1.24-1.14 (9H, m).
To a stirred solution of tert-butyl N-(1-amino{4}-pyridin-4-yl)carbamate. 2,4,6-trimethylbenzene-1-sulfonic acid (4.5 g, 11 mmol) in dry DMF (12 ml) was added ethyl-4-methylpent-2-ynoate (1.5 g, 11 mmol) followed by potassium carbonate (3 g, 22 mmol) and the reaction mixture was allowed to stir at room temperature for 16 h under nitrogen. After complete consumption of the SM (Monitored by LCMS) the reaction mixture was diluted with ethyl acetate, washed successively with water and brine, dried over sodium sulphate and evaporated under reduced pressure to get the crude compound, which was purified by column chromatography (100-200 mesh silica gel, eluent; 20% ethyl acetate in hexane) to give the title compound as a brown solid (1.6 g, 44%). LCMS rt 4.20 min MH+ 348. 1H NMR (400 MHz, DMSO-d6) δ 9.93 (s, 1H). 8.61 (d, 1H, J=7.44 Hz), 8.29 (s, 1H), 7.08 (d, 1H, J=7.44 Hz) 4.28-4.24 (m, 2H), 3.68-3.65 (m, 1H), 1.38-1.15 (m, 18H).
To a stirred solution of ethyl-5-{[(tert-butoxy)carbonyl]amino}-2-(propan-2-yl)pyrazolo[1,5-a]pyridine-3-carboxylate (1.7 g, 4.8 mmol) in dry DCM (12 ml) was added trifluoroacetic acid (3.6 ml) at 0° C. and the reaction mixture was allowed to stir at room temperature for 1 h under nitrogen. After complete consumption of the SM (Monitored by LCMS) the volatiles were evaporated under reduced pressure to get the crude compound, which was purified by column chromatography (100-200 mesh silica gel, eluent; 30% ethyl acetate in hexane) to give the title compound as a off white solid (0.9 g, 75%). LCMS rt 3.34 min MH+ 248. 1H NMR (400 MHz, DMSO-d6) δ 8.30 (d, 1H, J=7.32 Hz), 6.90 (s, 1H), 6.41 (d, 1H, J=7.44 Hz), 6.18 (brs, 2H), 4.23-4.18 (m, 2H), 3.63-3.56 (m, 1H), 1.32-1.18 (m, 9H).
To a stirred solution of ethyl-5-amino-2-(propan-2-yl)pyrazolo[1,5-a]pyridine-3-carboxylate (800 mg, 3.23 mmol) in dry acetonitrile (20 ml) at 0° C. were added tert-Butyl nitrite (0.8 ml, 6.47 mmol) followed by potassium iodide (1.1 g, 6.47 mmol) and the reaction mixture was allowed to stir at 70° C. for 16 h under nitrogen. After complete consumption of the SM (Monitored by LCMS) the reaction mixture was diluted with ethyl acetate, washed successively with water and brine, dried over sodium sulphate and evaporated under reduced pressure to get the crude compound, which was purified by column chromatography over silica gel (100-200 mesh silica gel, eluent; 10% ethyl acetate in hexane) to give the title compound as a white solid (550 mg, 47.41%). LCMS rt 2.78 min MH+ 359 (non-polar method). 1H NMR (400 MHz, DMSO-d6) δ 0.8.59 (d, 1H, J=7.28 Hz), 8.39 (s, 1H), 7.33 (d, 1H, J=5.08 Hz) 4.31-4.30 (m, 2H), 3.72-3.69 (m, 1H), 1.35-1.29 (m, 9H).
Ethyl-5-iodo-2-(propan-2-yl)pyrazolo[1,5-a]pyridine-3-carboxylate (0.25 g, 0.69 mmol) and 4-fluoro-3-trifluoromethylphenylboronic acid (0.21 g, 1.03 mmol) were dissolved in dioxane/water (4:1, 5 mL) and treated with K3PO4 (0.44 g, 2.07 mmol). The solution was degassed 20-30 min before addition of PdCl2(dppf). DCM catalyst (57 mg, 0.07 mmol), The reaction mixture was heated to 90° C. for 16 h, cooled to room temperature, and partitioned between ethyl acetate and water. The organic layer was dried over sodium sulphate and concentrated under reduced pressure. The crude product was purified by column chromatography (100-200 mesh silica gel, by elution with 10% ethyl acetate in hexane). Fractions containing the desired product were combined and evaporated under reduced pressure to give the title compound (240 mg, 85%) UPLC rt 1.94 min MH+ 395 (3 min run) 1H NMR (400 MHz, DMSO-d6) δ 8.91 (d, J=7.36 Hz, 1H), 8.26 (s, 1H) 8.20-8.19 (m, 1H), 8.13 (d, J=6.64 Hz, 1H), 7.70 (t, J=10.16 Hz, 1H), 7.52 (dd, J=7.2 Hz, 1.02 Hz, 1H), 4.36-4.30 (m, 2H), 3.77-3.74 (m, 1H), 1.38-1.32 (m, 9H).
Ethyl-5-[4-fluoro-3-(trifluoromethyl)phenyl]-2-(propan-2-yl)pyrazolo[1,5-a]pyridine-3-carboxylate (0.25 g, 0.63 mmol) was taken in cone HCl (60 ml) and the reaction mass was heated to reflux for 16 h. After complete consumption of the SM (Monitored by LCMS) the reaction mixture was cooled to 0° C., to it was added 2N NaOH dropwise until pH8. It was then extracted with ethyl acetate, washed successively with water and brine, dried over sodium sulphate and evaporated under reduced pressure to get the crude compound, which was purified by column chromatography over silica gel (100-200 mesh silica gel, eluent; 10% ethyl acetate in hexane) to give the title compound as a white solid (90 mg, 44%). UPLC rt 1.87 min (3 min run) MH+ 323.
1H NMR (400 MHz, DMSO-d6) δ 0.8.66 (d, J=7.04 Hz, 1H), 8.16-8.10 (m, 2H) 8.01 (s, 1H), 7.65 (t, J=9.64 Hz, 1H), 7.21 (d, J=7.08 Hz, 1H) 6.49 (s, 1H), 3.12-3.09 (m, 1H), 1.34-1.30 (m, 6H).
To a stirred solution of 5-[4-fluoro-3-(trifluoromethyl)phenyl]-2-(propan-2-yl)pyrazolo[1,5-a]pyridine (86 mg, 0.26 mmol) in dry acetonitrile (5 ml) at 0° C. were added N-Bromosuccinimide (57 mg, 0.32 mmol) and the reaction mixture was allowed to stir at 70° C. for 1 h under nitrogen. After complete consumption of the SM (Monitored by LCMS) the reaction mixture was diluted with ethyl acetate, washed successively with water and brine, dried over sodium sulphate and evaporated under reduced pressure to get the 89 mg of the title compound as brown solid which was used for the next step without further purification.
9-[4-(benzyloxy)-2,6-difluorophenyl]-8-(propan-2-yl)-2-[3-(trifluoromethyl)phenyl]-9H-purine was prepared by an analogous route to Example 89 step 6 product starting from 2,4,6-trifluoronitrobenzene according the following scheme
3-bromo-2-(propan-2-yl)-6-[3-(trifluoromethyl)phenyl]imidazo[1,2-a]pyrazine (1.5 g, 3.9 mmol) and 2-methoxy-5-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-phenol (1.46 g, 5.9 mmol) were dissolved in dioxane/water (4:1, 30 mL) and treated with K3PO4 (2.48 g, 11.7 mmol. The solution was degassed 20-30 min before addition of Pd-118 catalyst (0.25 g, 0.39 mmol), The reaction mixture was heated to 90° C. for 16 h, cooled to room temperature, and partitioned between ethyl acetate and water. The organic layer was dried over sodium sulphate and concentrated under reduced pressure. The crude product was purified by column chromatography (100-200 mesh silica gel, by elution with 20% ethyl acetate in DCM). Fractions containing the desired product were combined and evaporated under reduced pressure. The product was crystallised from methanol to give the title compound (600 mg, 36%) UPLC rt 1.9 min MH+ 428.
1H NMR (400 MHz, DMSO-d6) δ 9.37 (s, 1H), 9.15 (s, 1H), 8.66 (s, 1H), 8.36 (s, 1H), 8.27 (d, 1H), 7.75 (d, 1H), 7.70 (d, 1H), 7.16 (d, 1H), 7.03 (d, 1H), 6.96 (s, 1H), 3.87 (s, 3H), 3.17 (h, 1H), 1.28 (d, 6H).
3-bromo-2-(propan-2-yl)-6-[4-fluoro-3-(trifluoromethyl)phenyl]imidazo[1,2-a]pyrazine (500 mg, 1.24 mmol) and 4-fluoro-1-(oxan-2-yl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indazole (861 mg, 2.49 mmol) were dissolved in dioxane/water (4:1.15 mL) and treated with K3PO4 (791 mg, 3.73 mmol. The solution was degassed with argon for 5 min before addition of tetrakis(triphenylphosphine palladium (0) catalyst (143 mg, 0.12 mmol). The reaction vessel was sealed and the mixture was heated to 100° C. for 3 h, cooled to room temperature, filtered over a celite bed to remove the solids, then partitioned between ethyl acetate and water. The organic layer was dried over sodium sulphate and concentrated under reduced pressure. The crude product was purified by column chromatography (100-200 mesh silica gel, by elution with 20% ethyl acetate in DCM). Fractions containing the desired product were combined and evaporated under reduced pressure then triturated with ether and pentane to give the title compound 4-fluoro-5-{6-[4-fluoro-3-(trifluoromethyl)phenyl]-2-(propan-2-yl)imidazo[1,2-a]pyrazin-3-yl}-1-(oxan-2-yl)-1H-indazole (600 mg, 89%) UPLC rt 4.27 min MH+ 542.
4-fluoro-5-{6-[4-fluoro-3-(trifluoromethyl)phenyl]-2-(propan-2-yl)imidazo[1,2-a]pyrazin-3-yl}-1-(oxan-2-yl)-1H-indazole (600 mg, 1.1 mmol) was dissolved in a 30% solution of TFA in DCM (4 mL) and was stirred at room temperature for 2 h. The solvents were removed under reduced pressure then the crude reaction mixture was diluted with dicholoromethane, washed successively with sodium bicarbonate solution, water and brine, the organic layer was dried over sodium sulphate and concentrated under reduced pressure to get the crude product which was purified by reverse-phase prep-HPLC to give the title compound (125 mg, 25%). UPLC rt 3.5 min MH+ 458.
1H NMR (400 MHz, DMSO-d6) δ 13.85 (s, 1H), 9.20 (s, 1H), 8.76 (s, 1H), 8.41 (d, 2H), 8.35 (s, 1H), 7.61 (t, 1H), 7.54 (t, 1H), 7.50 (t, 1H), 3.05 (h, 1H), 1.27 (dd 6H).
A solution of 3-fluoro-4-[2-(propan-2-yl)-6-[3-(trifluoromethyl)phenyl]imidazo[1,2-a]pyrazin-3-yl]phenol (Example 9 600 mg, 1.45 mmol) in DCM (10 mL) was cooled to 0° C. and treated with pyridine (4 mL) followed by dropwise addition of phosphorous oxychloride (4 mL) solution in DCM (10 mL). The reaction mixture was stirred for 5 h at RT. Two further charges of pyridine and POCl3 were added at 0° C. to drive the reaction to completion over 3 days. The reaction was quenched by dropwise addition of (1:1) acetone:water) (100 ml). Volatile solvents were then removed under reduced pressure. The resulting oil was dissolved in DMF and purified by reveres-phase prep-HPLC to give the title compound (104 mg, 15%). UPLC rt 2.6 min MH+ 496.
1H NMR (400 MHz, DMSO-d6) δ 9.17 (s, 1H), 8.61 (s, 1H), 8.39 (s, 1H), 8.30 (d, 1H), 7.72 (d, 1H), 7.67 (t, 1H), 7.44 (t, 1H), 7.32 (d, 1H), 7.16 (d, 1H), 3.17 (h, 1H), 1.25 (d, 6H).
A mixture 4-{6-bromo-2-methylimidazo[1,2-a]pyrazin-3-yl}-3,5-difluorophenol (40 mg, 0.118 mmol) and 3-(pentafluorosulfanyl)benzeneboronic acid, pinacol ester (58 mg, 0.176 mmol) was dissolved in dioxane/water (4:1, 5 mL) and treated with K3PO4 (49.8 mg, 0.235 mmol). The solution was degassed 20-30 min before addition of [1,1′-bis(diphenylphosphino)ferrocene]palladium(II) chloride, complex with dichloromethane (19 mg, 0.024 mmol). The reaction mixture was heated to 90° C. for 16 h. The solvents were removed under reduced pressure then the crude reaction mixture was diluted with dicholoromethane, washed successively with water and brine, the organic layer was dried over sodium sulphate and concentrated under reduced pressure to get the crude product which was purified by reverse-phase prep-HPLC to give the title compound (4 mg, 7%).
UPLC rt 3.02 min MH+ 464. 1H NMR (400 MHz, DMSO-d6) δ 9.12 (s, 1H), 9.20 (s, 1H), 8.33 (s, 1H), 7.98 (m, 2H), 7.77 (d, 1H), 7.56 (t, 1H), 6.68 (d, 1H), 6.41 (m, 1H), 2.47. (s, 3H)
A mixture of 3-Chloro-6-(4-fluoro-3-pentafluoroethyl-phenyl)-2-trifluoromethyl-imidazo[1,2-a]pyrazine (620 mg, 1.43 mmol) and 4-Fluoro-1-(tetrahydro-pyran-2-yl)-5-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-1H-indazole (1 g, 2.9 mmol) was dissolved in 1,4-dioxane:H2O (4:1, 15 mL) and treated with K3PO4 (0.91 g, 4.3 mmol). The mixture was degassed for 20-30 min, treated with palladium-tetrakis(triphenylphosphine) (331 mg, 0.28 mmol) and heated at 90° C. for 16 h. After completion, the reaction was quenched with water and extracted with ethyl acetate. The organic layer was dried over sodium sulphate and concentrated to get crude compound, which was purified by column chromatography (100-200 mesh silica gel, eluent: 10% ethyl acetate in DCM) to give the title compound (480 mg, 54.3%). LCMS rt 4.33 min MH+ 618. 1H NMR (400 MHz, DMSO-d6) δ 9.47 (s, 1H), 8.92-8.86 (m, 1H), 8.44-8.41 (m, 3H), 7.87-7.84 (m, 1H), 7.66-7.59 (m, 2H), 6.02 (d, J=8.84 Hz, 1H), 3.96-3.80 (m, 1H), 2.08-1.62 (m, 5H).
To a stirred solution of 6-(4-Fluoro-3-pentafluoroethyl-phenyl)-3-[4-fluoro-1-(tetrahydro-pyran-2-yl)-1H-indazol-5-yl]-2-trifluoromethyl-imidazo[1,2-a]pyrazine (480 mg, 0.78 mmol) was added 20 ml of 30% TFA in DCM at 0° C. and the reaction mixture was allowed to stir at room temperature for 1 h under nitrogen. After complete consumption of the starting material (monitored by both TLC and LCMS) the volatiles were evaporated under reduced pressure, the crude compound was diluted with dichloromethane, washed successively with sodium bicarbonate solution and brine, the organic layer dried over sodium sulphate, evaporated under reduced pressure to get crude, which was purified by reverse phase prep-HPLC to get 150 mg of the title compound (150 mg, 36.2%). UPLC rt 1.96 min (3 min run) MH+ 534. 1H NMR (400 MHz, DMSO-d6) δ 13.73 (s, 1H), 9.47 (s, 1H) 8.44-8.38 (m, 3H), 7.64-7.53 (m, 3H).
Synthetic Scheme:
To a stirred solution of 3-Fluoro-4-nitro-phenol (3.8 g, 24.2 mmol) in DMF (30 mL) was added sodium hydride (1.74 g, 72.6 mmol) portion wise at 0° C. under argon, the reaction mass was stirred at the same temperature for 30 min, after that was added benzyl bromide (3.18 ml, 26.6 mmol) to the reaction mass dropwise at 0° C. and the reaction mixture was allowed to stir for another 4 h at room temperature. After complete conversion of the starting material, the reaction mixture was pored into crushed ice to precipitate a solid which was collected by filtration to get 5.5 g (91%) of the title compound as a yellow solid. LCMS rt 3.52 min MH− 245. 1H NMR (400 MHz, CDCl3) δ 8.10-7.97 (m, 1H), 7.46-7.32 (m, 5H), 6.82-6.78 (m, 1H), 6.64-6.56 (m, 1H), 5.19-5.08 (m, 2H).
To a stirred solution of 4-Benzyloxy-2-fluoro-1-nitro-benzene (2) (5.5 g, 22.26 mmol) in ethanol (150 mL) and water (30 mL) was added iron powder (4.98 g, 89.07 mmol) followed by ammonium chloride (9.44 g, 178.2 mmol) and the reaction mixture was allowed to stir at 70° C. for 4 h. After complete consumption of the SM the reaction mass was filtered through celite bed, the mother liquors were evaporated, the crude reaction mass was then diluted with ethyl acetate, washed successively with water and brine, dried over sodium sulphate, evaporated under reduced pressure to give crude product, which was purified by column chromatography (100-200 mesh silica gel, eluent:30% ethyl acetate in hexane) to get 1.4 g (28.9%) of the title compound as a brown liquid. LCMS rt 3.32 min MH+ 218. 1H NMR (400 MHz, CDCl3) δ 7.38-7.29 (m, 5H), 6.72-6.-6.59 (m, 3H), 5.03 (s, 2H), 3.43 (br.s, 2H).
To a stirred solution of 4-Benzyloxy-2-fluoro-phenylamine (3) (1.4 g, 6.45 mmol) in THF(25 mL) was added 2,4-Dichloro-5-nitro-pyrimidine (1.5 g, 7.74 mmol) at 0° C. under argon and the reaction mixture was allowed to stir at room temperature for 4 h. After complete consumption of the SM, the reaction mass was poured into crushed ice to give precipitate, which was collected by filtration to get 2.4 g (99.2%) the title compound as a red solid. LCMS rt 3.65 min MH+ 375. 1H NMR (400 MHz, CDCl3) δ 10.10 (brs, 1H), 9.16 (s, 1H), 7.93-7.89 (m, 1H), 7.43-7.34 (m, 6H), 6.85-6.-6.82 (m, 2H), 5.07 (s, 2H).
N-[4-(benzyloxy)-2-fluorophenyl]-2-chloro-5-nitropyrimidin-4-amine (4) (2.4 g, 6.41 mmol) and 3-trifluoromethylphenylboronic acid (1.81 g, 9.62 mmol) were dissolved in dioxane/water (4:1.40 mL) and treated with Cs2CO3 (4.17 g, 12.83 mmol). The solution was degassed 20-30 min before addition of PdCl2(dppf). DCM catalyst (524 mg, 0.64 mmol), The reaction mixture was heated to 90° C. for 16 h, cooled to room temperature, and partitioned between ethyl acetate and water. The organic layer was dried over sodium sulphate and concentrated under reduced pressure. The crude product was purified by column chromatography (100-200 mesh silica gel, by elution with 20% ethyl acetate in hexane). Fractions containing the desired product were combined and evaporated under reduced pressure to give the title compound as orange solid (2.5 g, 80.4%) LCMS rt 4.3 min MH+ 483
1H NMR (400 MHz, DMSO-d6) δ 9.98 (s, 1H), 9.4 (s, 1H), 8.64 (s, 1H), 8.52-8.51 (m, 1H) 7.89-7.57 (m, 3H), 7.47-7.37 (m, 5H), 6.9-6.71 (m, 3H), 5.11 (s, 2H).
To a stirred solution of 4 N-[4-(benzyloxy)-2-fluorophenyl]-5-nitro-2-[3-(trifluoromethyl)phenyl]pyrimidin-4-amine (5) (220 mg, 0.455 mmol) in THF(10 mL) was added Raney nickel (100 mg). The reaction vessel was filled with hydrogen and the reaction mixture was allowed to stir at 1 atm hydrogen pressure for 1 h. After complete consumption of the SM (monitored by TLC) the reaction mass was filtered through a Celite bed, the mother liquor was evaporated to get 200 mg (96.8%) of the title compound as crude which was used for the next step without further purification. LCMS rt 3.75 min MH+ 353.
To a stirred solution of 4-N-[4-(benzyloxy)-2-fluorophenyl]-2-[3-(trifluoromethyl)phenyl]pyrimidine-4,5-diamine (6) (200 mg, 0.441 mmol) in acetic acid (20 mL) was added isobutyraldehyde (44 μL, 0.485 mmol) followed by copper acetate (80 mg, 0.441 mmol) and the reaction mixture was allowed to stir at 100° C. for 2 h under nitrogen. After complete consumption of the SM (monitored by TLC) the reaction mixture was diluted with DCM, washed successively with sodium bicarbonate and brine, dried over sodium sulphate, evaporated under reduced pressure to get the crude title compound, which was used for the next step without further purification. LCMS rt 4.50 min MH+ 507.
To a stirred solution of 9-[4-(benzyloxy)-2-fluorophenyl]-8-(propan-2-yl)-2-[3-(trifluoromethyl)phenyl]-9H-purine (7) (150 mg, 0.29 mmol) in 1,2-Dichloroethane (7 mL) was added trifluoroacetic acid (4.5 mL) and the reaction mixture was allowed to stir at 90° C. for 16 h. After complete consumption of the starting material (monitored by both TLC and LCMS) the volatiles were evaporated under reduced pressure, the crude compound was diluted with dichloromethane, washed successively with sodium bicarbonate solution and brine, the organic layer dried over sodium sulphate, evaporated under reduced pressure to get crude, which was purified by reverse phase prep-HPLC to get 50 mg of the title compound (40.5%) as off white solid. UPLC rt 3.23 min MH+ 417. 1H NMR (400 MHz, DMSO-d6) δ 10.61 (s, 1H), 9.21 (s, 1H) 8.58-8.54 (m, 2H), 7.85-7.57 (m, 3H), 6.96-6.87 (m, 2H), 3.04-3.01 (m, 1H), 1.31-1.22 (m, 6H).
The following compounds were made by analogous methods:
In Vitro Testing
The routine biological assays using adult or juvenile worms and cytotoxicity assays using MRC-5 cells have been disclosed previously in Mansour, N. R., et al. (2016). “High Throughput Screening Identifies Novel Lead Compounds with Activity against Larval, Juvenile and Schistosoma mansoni PLoS Negl Trap Dis 10(4): e0004659. Cytotoxicity assays using the HepG2 cell line were carried out as described in Molecular Diversity (2015) 19, 251-261.
(a) S. mansoni adult worms
(b) S. mansoni juvenile worms
(c) S. haemotobium adult worms
(d) Cytotoxicity against human MRC-5 cell line
(e) Cytotoxicity against human HepG2 cell lines
The compounds of Examples 1-90 exhibit one or more of the following compared to the compound disclosed in PLoS Negl Trap Dis 10(4): e0004659
haemotobium
Preferred compounds of the invention are those which by inspection, appear to kill or significantly damage the worms at lower concentrations within the IC50 range shown above. For example, preferred are compounds with IC50 values against both adult and juvenile worms below 0.5 μM; More preferred compounds are those with IC50 values against both adult and juvenile worms below 0.1 μM;
In Vivo Testing
Infection of Mice and Worm Recovery
Methods for the subcutaneous infection of mice and subsequent worm recovery were as described in Mansour et al (2016) except that infection was with 150 cercariae and the perfusion medium was citrate saline (0.9% sodium chloride, 1.5% tri-sodium citrate). Perfusion was carried out 8 days (adult infections) or 15 days (juvenile infections) after treatment. Perfuseate was collected into 30 mL universal tubes. RBC (Red Blood Cells) were removed by allowing the perfuseate to settle for 10 min, removal of most of the supernatant and washing once as above with 10 mL perfusion medium. A drop of dilute aqueous saponin solution was added to lyse any remaining RBC and the worm suspension poured into a grid-marked small petri dish. The tube was rinsed out into the petri dish and examined for any remaining worms. Worms were counted using a dissecting microscope. The mouse livers removed after perfusion were squashed between two thick glass plates and examined visually and any remaining worms added to those counted as above.
Drug Treatment
For testing efficacy against the juvenile worms, treatment was on day 25 post infection and for testing against the adult worms on day 42 post infection. Drugs were suspended in 7% Tween-80/3% Ethanol/double distilled water and drug dispersal was facilitated by vortexing and using a sonicating water bath (Formulation F1). Alternatively, drugs were suspended in 10% DMSO, 90% double distilled water containing 50 mM Na2HPO4 with 0.5% Tween-80 by first dissolving or suspending the drug in DMSO then adding the DMSO solution/suspension to the aqueous solution (Formulation F2). In a further set of conditions, drugs were first dissolved in DMSO, then diluted with corn oil to give a 5% DMSO+drug solution/suspension (Formulation F3)
The drug solution/suspensions were given by oral gavage at the rate of 10 ml/kg. Positive controls (artemether for juvenile worms and praziquantel for adult worms) were used in each experiment. Oral artemether at 400 mg/kg single dose is equally or more effective in mice against juvenile compared with adult S. mansoni (Am J Trap Med Hyg. 2010 January; 82(1):112-4. Activity of artemether and mefloquine against juvenile and adult Schistosoma mansoni in athymic and immunocompetent NMRI mice. Keiser J1, Vargas M, Doenhoff M J) and so is a useful positive control for drug testing against juvenile stages in the murine screen.
Preferred compounds are those which by inspection show a statistically significant (P value <0.05) reduction in worm numbers of at least 50% when administered orally in the mouse model of infection as described above. For example, compounds 2, 17, 29, 50, 63 and 67.
More preferred compounds are those which show a statistically significant (P value <0.05) reduction in worm numbers of at least 50% when administered orally in the mouse model of infection as described above in a single dose of 25 mg/kg or less. For example, compounds 2, 29, 50 and 63.
| Number | Date | Country | Kind |
|---|---|---|---|
| 1700692 | Jan 2017 | GB | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/GB2018/050101 | 1/15/2018 | WO | 00 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2018/130853 | 7/19/2018 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 8748435 | Du et al. | Jun 2014 | B2 |
| 20120252778 | Miltz et al. | Oct 2012 | A1 |
| Number | Date | Country |
|---|---|---|
| 2011092293 | Aug 2011 | WO |
| 2012080232 | Jun 2012 | WO |
| WO 18130853 | Jul 2018 | WO |
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| Vippagunta, et al. Advanced Drug Delivery Reviews, 48, 2001, 18. |
| International Preliminary Report on Patentability for corresponding PCT application No. PCT/GB2018/050101 dated Jul. 16, 2019. |
| Number | Date | Country | |
|---|---|---|---|
| 20200123158 A1 | Apr 2020 | US |
