ANTIBACTERIAL AND ANTIPARASITIC QUINOXALINE-2,3-DIAMINE DERIVATIVES
20220259159 · 2022-08-18
Inventors
Cpc classification
C07D405/12
CHEMISTRY; METALLURGY
A61K45/06
HUMAN NECESSITIES
C07D241/44
CHEMISTRY; METALLURGY
A61K31/498
HUMAN NECESSITIES
International classification
C07D241/44
CHEMISTRY; METALLURGY
A61K45/06
HUMAN NECESSITIES
Abstract
A compound of formula (I), or a pharmaceutically acceptable salt or solvate thereof, for use in the treatment of a patient infected with pathogenic organisms; wherein X and Y are each independently selected from C, N, O and S, provided at least one of X and Y is N, O or S; wherein R.sup.1 and R.sup.2 are each independently selected from an optionally substituted C.sub.1-C.sub.8 alkyl, C.sub.1-C.sub.8 alkenyl, aryl, heteroaryl or alkylaryl group; and wherein Z is selected from H, —CN, —NO.sub.2, —NR.sup.3R.sup.4, —NR.sup.5(CO)R.sup.6; C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 alkoxy, —(CO)WR.sup.12, hydroxy, amino, thiol, chloro, fluoro, CF.sub.3, CHF.sub.2 or CH.sub.2F groups. The compounds may be effective in treating patients/animals infected with parasites selected from Schistosoma, Haemonchus, Eimeria, Echinococcus, Dirofilaria, Fasciola or Plasmodium parasites. Additionally or alternatively, the compounds of formula (I) may be effective in treating patients infected with pathogenic bacteria selected from S. aureus, MRSA and Enterococcus faecalis.
Claims
1. A compound of formula (I), or a pharmaceutically acceptable salt or solvate thereof, for use in the treatment of a patient infected with pathogenic organisms; ##STR00038## wherein X and Y are each independently selected from C, N, O and S, provided at least one of X and Y is N, O or S; wherein R.sup.1 and R.sup.2 are each independently selected from a C.sub.1-C.sub.8 alkyl, a C.sub.1-C.sub.8 alkenyl, an aryl group, a heteroaryl group or an alkylaryl group, optionally substituted with one or more of C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 alkoxy, C.sub.1-C.sub.4 alkylamino, C.sub.1-C.sub.4 alkylthio, hydroxy, amino, thiol, chloro, fluoro, CF.sub.3, CHF.sub.2 or CH.sub.2F groups; or a (CO)OR.sup.13 or (CO)NHR.sup.13 group wherein R.sup.13 is selected from said groups; wherein Z is selected from H, —CN, —NO.sub.2, —NR.sup.3R.sup.4, —NR.sup.5(CO)R.sup.6; C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 alkoxy, —(CO)WR.sup.12, hydroxy, amino, thiol, chloro, fluoro, CF.sub.3, CHF.sub.2 or CH.sub.2F groups; wherein W is a bond, N or O and wherein R.sup.12 is selected from C.sub.1-C.sub.6 alkyl, a C.sub.1-C.sub.6 alkenyl, an aryl or a heteroaryl group, optionally substituted with one or more of C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 alkoxy, C.sub.1-C.sub.4 alkylamino, C.sub.1-C.sub.4 alkylthio, hydroxy, amino, thiol, chloro, fluoro, CF.sub.3, CHF.sub.2 or CH.sub.2F groups; wherein R.sup.3 and R.sup.4 are each independently selected from H, a C.sub.1-C.sub.6 alkyl, optionally forming a ring, a C.sub.1-C.sub.6 alkenyl, an aryl group, or a heteroaryl group, optionally substituted with one or more of C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 alkoxy, C.sub.1-C.sub.4 alkylamino, C.sub.1-C.sub.4 alkylthio, hydroxy, amino, thiol, chloro, fluoro, CF.sub.3, CHF.sub.2 or CH.sub.2F groups; or a (CO)OR.sup.14 or (CO)NHR.sup.14 group wherein R.sup.14 is selected from said groups; wherein R.sup.5 is selected from H, a C.sub.1-C.sub.6 alkyl or a C.sub.1-C.sub.6 alkenyl group, optionally substituted with one or more of C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 alkoxy, C.sub.1-C.sub.4 alkylamino, C.sub.1-C.sub.4 alkylthio, hydroxy, amino, thiol, chloro, fluoro, CF.sub.3, CHF.sub.2 or CH.sub.2F groups; wherein R.sup.6 is selected from a C.sub.1-C.sub.6 alkyl, a C.sub.1-C.sub.6 alkenyl, an aryl or a heteroaryl group, optionally substituted with one or more of C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 alkoxy, C.sub.1-C.sub.4 alkylamino, C.sub.1-C.sub.4 alkylthio, hydroxy, amino, thiol, chloro, fluoro, CF.sub.3, CHF.sub.2 or CH.sub.2F groups; or a (CO)OR.sup.15 or (CO)NHR.sup.15 group wherein R.sup.15 is selected from said groups; wherein R.sup.7 and R.sup.8, when present, are each independently selected from a H or a C.sub.1-C.sub.4 alkyl; wherein R.sup.9, R.sup.10 and R.sup.11 are each independently selected from H, C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 alkoxy, —(CO)WR.sup.12, hydroxy, amino, thiol, chloro, fluoro, CF.sub.3, CHF.sub.2 or CH.sub.2F groups; wherein W is a bond, N or O and wherein R.sup.12 is selected from C.sub.1-C.sub.6 alkyl, a C.sub.1-C.sub.6 alkenyl, an aryl or a heteroaryl group, optionally substituted with one or more of C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 alkoxy, C.sub.1-C.sub.4 alkylamino, C.sub.1-C.sub.4 alkylthio, hydroxy, amino, thiol, chloro, fluoro, CF.sub.3, CHF.sub.2 or CH.sub.2F groups.
2. The compound according to claim 1, wherein the compound has the formula ##STR00039## wherein R.sup.1, R.sup.2, R.sup.9, R.sup.10, R.sup.11 and Z are as defined in claim 1.
3. The compound according to claim 1, wherein the compound has the formula ##STR00040## wherein R.sup.1 and R.sup.2 are each independently selected from a C.sub.1-C.sub.8 alkyl, a C.sub.1-C.sub.8 alkenyl, an aryl group, a heteroaryl group or an alkylaryl group, optionally substituted with one or more of C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 alkoxy, C.sub.1-C.sub.4 alkylamino, C.sub.1-C.sub.4 alkylthio, hydroxy, amino, thiol, chloro, fluoro, CF.sub.3, CHF.sub.2 or CH.sub.2F groups; wherein Z is selected from —NO.sub.2, —NR.sup.3R.sup.4, —NR.sup.5(CO)R.sup.6; C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 alkoxy, —(CO)WR.sup.12, hydroxy, amino, thiol, chloro, fluoro, CF.sub.3, CHF.sub.2 or CH.sub.2F groups; wherein W is a bond, N or O and wherein R.sup.12 is selected from C.sub.1-C.sub.6 alkyl, a C.sub.1-C.sub.6 alkenyl, an aryl or a heteroaryl group, optionally substituted with one or more of C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 alkoxy, C.sub.1-C.sub.4 alkylamino, C.sub.1-C.sub.4 alkylthio, hydroxy, amino, thiol, chloro, fluoro, CF.sub.3, CHF.sub.2 or CH.sub.2F groups; wherein R.sup.3 and R.sup.4 are each independently selected from H, a C.sub.1-C.sub.6 alkyl group, optionally forming a ring, a C.sub.1-C.sub.6 alkenyl, an aryl group, or a heteroaryl group, optionally substituted with one or more of C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 alkoxy, C.sub.1-C.sub.4 alkylamino, C.sub.1-C.sub.4 alkylthio, hydroxy, amino, thiol, chloro, fluoro, CF.sub.3, CHF.sub.2 or CH.sub.2F groups; wherein R.sup.5 is selected from H, a C.sub.1-C.sub.6 alkyl or a C.sub.1-C.sub.6 alkenyl group, optionally substituted with one or more of C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 alkoxy, C.sub.1-C.sub.4 alkylamino, C.sub.1-C.sub.4 alkylthio, hydroxy, amino, thiol, chloro, fluoro, CF.sub.3, CHF.sub.2 or CH.sub.2F groups; wherein R.sup.6 is selected from a C.sub.1-0.sub.6 alkyl, a C.sub.1-C.sub.6 alkenyl, an aryl or a heteroaryl group, optionally substituted with one or more of C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 alkoxy, C.sub.1-C.sub.4 alkylamino, C.sub.1-C.sub.4 alkylthio, hydroxy, amino, thiol, chloro, fluoro, CF.sub.3, CHF.sub.2 or CH.sub.2F groups; wherein R.sup.7 and R.sup.8 are each independently selected from a H or a C.sub.1-C.sub.4 alkyl; wherein R.sup.9, R.sup.10 and R.sup.11 are each independently selected from H, C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 alkoxy, —(CO)WR.sup.12, hydroxy, amino, thiol, chloro, fluoro, CF.sub.3, CHF.sub.2 or CH.sub.2F groups; wherein W is a bond, N or O and wherein R.sup.12 is selected from C.sub.1-C.sub.6 alkyl, a C.sub.1-C.sub.6 alkenyl, an aryl or a heteroaryl group, optionally substituted with one or more of C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 alkoxy, C.sub.1-C.sub.4 alkylamino, C.sub.1-C.sub.4 alkylthio, hydroxy, amino, thiol, chloro, fluoro, CF.sub.3, CHF.sub.2 or CH.sub.2F groups.
4. The compound according to claim 1, wherein R.sup.1 and R.sup.2 are each independently selected from an aryl group or an alkylaryl group, optionally substituted with one or more of C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 alkoxy, OH, NH.sub.2, SH, chloro, fluoro, CF.sub.3, CHF.sub.2 or CH.sub.2F groups.
5. The compound according to claim 1, wherein the groups R.sup.1 and R.sup.2 are the same.
6. The compound according to claim 1, wherein R.sup.7 and R.sup.8 are both H.
7. The compound according to claim 1, wherein Z is —NR.sup.3R.sup.4 or —NR.sup.5(CO)R.sup.6.
8. The compound according to claim 7, wherein Z is —NR.sup.3R.sup.4 and wherein R.sup.3 and R.sup.4 are independently selected from H, a C.sub.1-C.sub.6 alkyl, optionally forming a ring, optionally substituted with one or more of C.sub.1-C.sub.2 alkoxy, F, Cl and CF.sub.3 groups.
9. The compound according to claim 7, wherein Z is NR.sup.5(CO)R.sup.6, wherein R.sup.5 is H or methyl; wherein R.sup.6 is selected from a C.sub.1-C.sub.6 alkyl, or an aryl group, optionally substituted with one or more of C.sub.1-C.sub.2 alkyl, C.sub.1-C.sub.2 alkoxy, F, Cl or CF.sub.3 groups.
10. The compound according to claim 1, wherein the pathogenic organisms which the patient is infected with are parasites selected from schistosome, Fasciola or Plasmodium parasites.
11. The compound according to claim 1, wherein the pathogenic organisms which the patient is infected with are pathogenic bacteria selected from S. aureus, MRSA and Enterococcus faecalis.
12. A compound of formula (Ill), or a pharmaceutically acceptable salt or solvate thereof; ##STR00041## wherein R.sup.1 and R.sup.2 are independently selected from a C.sub.1-C.sub.8 alkyl, a C.sub.1-C.sub.8 alkenyl, an aryl group or an alkylaryl group, optionally substituted with one or more of C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 alkoxy, C.sub.1-C.sub.4 alkylamino, C.sub.1-C.sub.4 alkylthio, hydroxy, amino, thiol, fluoro, chloro, CF.sub.3, CHF.sub.2 or CH.sub.2F groups; wherein Z is selected from —NR.sup.3R.sup.4 or —NR.sup.5(CO)R.sup.6; wherein R.sup.3 and R.sup.4 are each independently selected from H, a C.sub.1-C.sub.6 alkyl, optionally forming a ring, a C.sub.1-C.sub.6 alkenyl, an aryl group, or a heteroaryl group, optionally substituted with one or more of C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 alkoxy, C.sub.1-C.sub.4 alkylamino, C.sub.1-C.sub.4 alkylthio, hydroxy, amino, thiol, chloro, fluoro, CF.sub.3, CHF.sub.2 or CH.sub.2F groups; wherein R.sup.5 is selected from H, a C.sub.1-C.sub.6 alkyl or a C.sub.1-C.sub.6 alkenyl group, optionally substituted with one or more of C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 alkoxy, C.sub.1-C.sub.4 alkylamino, C.sub.1-C.sub.4 alkylthio, hydroxy, amino, thiol, chloro, fluoro, CF.sub.3, CHF.sub.2 or CH.sub.2F groups; wherein R.sup.6 is selected from a C.sub.1-C.sub.6 alkyl, a C.sub.1-C.sub.6 alkenyl, an aryl or a heteroaryl group, optionally substituted with one or more of C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 alkoxy, C.sub.1-C.sub.4 alkylamino, C.sub.1-C.sub.4 alkylthio, hydroxy, amino, thiol, chloro, fluoro, CF.sub.3, CHF.sub.2 or CH.sub.2F groups; wherein R.sup.7 and R.sup.8 are each independently selected from a H or a C.sub.1-C.sub.4 alkyl; wherein R.sup.9, R.sup.10 and R.sup.11 are each independently selected from H, C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 alkoxy, —(CO)WR.sup.12, hydroxy, amino, thiol, chloro, fluoro, CF.sub.3, CHF.sub.2 or CH.sub.2F groups; wherein W is a bond, N or O and wherein R.sup.12 is selected from C.sub.1-C.sub.6 alkyl, a C.sub.1-C.sub.6 alkenyl, an aryl or a heteroaryl group, optionally substituted with one or more of C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 alkoxy, C.sub.1-C.sub.4 alkylamino, C.sub.1-C.sub.4 alkylthio, hydroxy, amino, thiol, chloro, fluoro, CF.sub.3, CHF.sub.2 or CH.sub.2F groups.
13. The compound according to claim 12, wherein R.sup.1 and R.sup.2 are independently selected from an aryl group or an alkylaryl group, optionally substituted with one or more of C.sub.1-C.sub.4 alkoxy, chloro, fluoro, CF.sub.3, CHF.sub.2 or CH.sub.2F groups.
14. The compound according to claim 12, for use as a medicament.
15. The compound according to claim 12, for use in the treatment of a patient infected with pathogenic organisms.
16. A method for disinfecting a surface, the method comprising the step of applying a compound according to claim 1 to a surface.
Description
GENERAL EXPERIMENTAL DETAILS
Reagents, Solvents and Other General Information
[0103] All solvents and reagents commercially available were used as supplied from Sigma-Aldrich, Fluorochem or other commercial sources without further purification or treatment. The only exception was compound 2,3-Dichloro-6-nitroquinoxaline (product code 49489—Fluorochem, compound 2, MW=244.03) that contained also compound 3 (6-nitroquinoxaline-2,3-diol, MW=207.15). From NMR analysis, this Fluorochem product resulted a mixture of compounds 2 and 3 in a ratio 3:1.
##STR00013##
[0104] This mixture was purified by dissolving 500 mg in dichloromethane (DCM, 200 ml) and washing four times with sodium hydroxide (NaOH 1N, 20 ml for each wash) until no traces of compound 3 were detected using Thin Layer Chromatography (TLC). The organic phase was washed once with brine (15 ml), dried over anhydrous Na.sub.2SO.sub.4, filtered and reduced to dryness to give the pure compound 2 (350 mg). The basic solution (derived from the extraction) was acidified with 6N hydrochloric acid until pH 5. The aqueous solution was then extracted with ethyl acetate (EtOAc, 15 ml ) for four times. The combined organic phase was washed with brine, dried over anhydrous Na.sub.2SO.sub.4, filtered and reduced to dryness to give the pure compound 3 (125 mg). The structures of these compounds were confirmed by NMR and the spectra agreed with the data reported in public spectra databases.
[0105] The following solvents were purchased as anhydrous: N, N-dimethylsulfoxide (DMSO) and acetonitrile (CH.sub.3CN). Other solvent used in this chapter were: dichloromethane (DCM), ethyl acetate (EtOAc) and n-hexane.
[0106] All glassware was dried in the oven at 130° C. for 2 hours and allowed to cool down to room temperature before use. Reactions under MWI conditions (Microwave Irradiation) were performed in a CEM Discover microwave system in closed vessel mode.
Thin Layer Chromatography
[0107] All reactions were monitored by Thin Layer Chromatography (TLC) on commercially available Merck Kieselgel 60 F254 plates (105554, Merck). The TLC were eluting with n-hexane/EtOAc in different proportions depending on the specific compound being synthesised. Separated components were visualized using ultra violet light (245 and 366 nm).
FLASH Column Chromatography (CC)
[0108] Flash column chromatography was performed on Interchim PuriFlash 430 using high performance silica gel particle size 50 micron cartridges. Samples were applied onto silica gel as a concentrated solution in DCM (replaced by EtOAc if the sample was not soluble in DCM).
[0109] The sample was purified with n-hexane/EtOAc in different proportions depending on the specific compound being synthesised. The fractions containing the desired product were analysed by TLC then combined together and the solvent removed under vacuum.
NMR Spectroscopy p .sup.1H and .sup.13C were recorded on a Brucker Avance 500 spectrometer (500 and 126 MHz respectively) at 25° C. Spectra were calibrated to the residual signal of the deuterated solvent used (Deuterated DMSO—DMSO-d6—was used for all the compounds shown in this chapter). MestReNova (v6.0.2-5475) NMR processing software was used for the assignment of peaks and calculation of coupling constants. Chemical shifts δ were given in parts per million (ppm) and rounded to two decimal places. The following abbreviations were used in the NMR signals assignment: s for singlet, br s for broad singlet, d for doublet, t for triplet, q for quartet, m for multiplet. Coupling constants (J) were measured in Hertz (Hz) and rounded to one decimal place.
High Performance Liquid Chromatography (HPLC)
[0110] Ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) analysis was used for purity/mass determination of the tested compounds using a Waters UPLC system with both Diode Array detection and Electrospray (ESI).sup.+ MS detection. The stationary phase was a Waters Acquity UPLC BEH C18 1.7 um 2.1×50 mm column. The mobile phase was H.sub.2O containing 0.1% Formic acid (A) and CH.sub.3CN containing 0.1% Formic acid (B). Column temperature was 40° C., the sample diluent was acetonitrile and the sample concentration was 1 μg/mL. Injection volume of sample was 2 μL under a flow rate of 0.5 mL/min. The method used included a run time of 3 min, compromising an isocratic elution at 90% A (0.1 min), followed by a 1.4 min gradient from 10% to 100% of B, followed by 1.4 min isocratic elution at 100% of B, followed by 0.1 min gradient to initial conditions. Galaxie Chromatography Data System was used for analysis of data.
[0111] All compounds tested in the biological assays were >95% pure. Purity of intermediates was >90%, unless otherwise stated.
Preparation of Compounds
[0112] Standard Procedure 1a: Preparation of N-(CH.sub.2).sub.n aromatic analogues of compound 1
[0113] To a stirring solution of mixture of purified 2,3-dichloroquinoxaline (compound 2,1 equivalent—1 eq.) in anhydrous DMSO under a nitrogen atmosphere, the appropriate aniline or phenyl-alkyl amine (5 eq.) was added. The reaction mixture was left stirring at 130° C. for 30 min. The reaction was monitored by TLC with n-hexane/EtOAc (7:3). After completion of the reaction, the mixture was diluted with EtOAc and poured into ice water. The aqueous phase was extracted with EtOAc three times, the combined organic phase was then washed with 6N hydrochloric acid (4 times) and once with brine solution. The organic phase was then dried over anhydrous Na.sub.2SO.sub.4, filtered and reduced to dryness to give a crude product. The crude product was purified by flash chromatography (Biotage Isolera One) eluting with n-hexane/EtOAc in different proportions depending on the specific analogue being synthesised. This procedure provided compounds of general formula (VII):
##STR00014##
wherein n=0, 1 or 2;
Standard Procedure 1b: Preparation of N-acyl derivatives of compound 1
[0114] To a solution of compound 15 (1 eq.) dissolved in anhydrous DCM, anhydrous pyridine (3.6 eq.) was added under nitrogen. The appropriate acyl derivate (1.1 eq.) was added dropwise to the above solution cooled to 0° C. in an ice-bath under nitrogen. The resulting mixture was stirred at room temperature for 1 hour. After completion, the reaction mixture was diluted with DCM and quenched with saturated aqueous of sodium bicarbonate (NaHCO.sub.3). The organic phase was washed first with saturated aqueous NaHCO.sub.3 (three times) and brine (once). The organic layer was then dried over anhydrous Na.sub.2SO.sub.4, filtered and reduced to dryness to give a crude product.
[0115] The crude product was purified by flash chromatography (Biotage Isolera One) eluting with n-hexane/EtOAc in different proportions depending on the specific analogue being synthesised. This procedure provided compounds of general formula (VIII):
##STR00015##
Experimental Section and Spectra Data
[0116] Synthesis of N.sup.2, N.sup.3-bis(3,4-dichlorophenyl)-6-nitroquinoxaline-2,3-diamine (compound 1)
##STR00016##
Chemical Formula: C.SUB.20.H.SUB.11.Cl.SUB.4.N.SUB.5.O.SUB.2., Molecular Weight: 495.14
[0117] Prepared according to Standard Procedure 1a, compound 1 was prepared from 2,3-dichloroquinoxaline 2 (0.50 g, 2.00 mmol) and 3,4-dichloroaniline (1.62 g, 10 mmol) in DMSO (25 ml) for 30 min at 130° C. After completion of the reaction, the mixture was diluted with EtOAc (20 ml) and poured into ice water. The aqueous phase was extracted with EtOAc (3×10 ml), the combined organic phase was then washed with 6N hydrochloric acid (4×10 ml) and once with brine (15 ml). The combined organic phase was dried over anhydrous Na.sub.2SO.sub.4, filtered and evaporated. The crude product was purified by automated flash column chromatography eluting with n-hexane:EtOAc 100:0 v/v increasing to 70:30 v/v in 10 column volumes (CV). The final product 1 was obtained in 36% yield as a red powder. .sup.1H NMR (500 MHz, DMSO-d6) δ 9.52 (s, 2H, 2×NH), 8.34-8.18 (m, 3H, 3×ArH), 8.11 (d, J=8.9 Hz, 1H, ArH), 7.87 (m, 2H, 2×ArH), 7.65 (dd, J=8.8, 6.9 Hz, 3H, 3×ArH). .sup.13C NMR (126 MHz, DMSO) δ 139.65 (ArC), 130.95 (ArC), 130.57 (3×ArCH), 124.98 (ArC), 124.50 (ArC), 122.26 (ArCH), 121.66 (ArCH), 121.13 (ArCH), 120.82 (ArCH), 120.61 (ArCH), 119.87 (ArCH). UPLC-MS: t.sub.R 2.526 min. MS (ESI).sup.+ m/z calculated 496.14 [M+H].sup.+, observed 494.00-496.08-498.02 [M+H].sup.+. Note: Data agrees with previously published papers (Jonathan, 2002; Kher et al., 2015).
Synthesis of N.sup.2, N.sup.3-bis(4-chlorophenyI)-6-nitroquinoxaline-2,3-diamine (compound 4)
##STR00017##
Chemical Formula: C.SUB.20.H.SUB.13.Cl.SUB.2.N.SUB.5.O.SUB.2., Molecular Weight: 426.26
[0118] Prepared according to Standard Procedure 1a, compound 4 was derived from 2,3-dichloroquinoxaline 2 (0.17 g, 0.70 mmol) and 4-chloroaniline (0.45 g, 3.52 mmol) in DMSO (9 ml) for 30 min at 130° C. The work-up of the reaction was performed similarly to compound 1. The crude product was purified by automated flash column chromatography eluting with n-hexane:EtOAc 100:0 v/v increasing to 70:30 v/v in 10 CV. The final product 4 was obtained at 41% yield as a brown powder. .sup.1H NMR (500 MHz, DMSO-d6) δ 9.57 (s, 1H, NH), 9.41 (s, 1H, NH), 8.34-8.27 (m, 1H, ArH), 8.10 (dd, J=7.9, 4.2 Hz, 1H, ArH), 7.95 (ddd, J=13.9, 8.7, 1.9 Hz, 4H, ArH), 7.64 (dd, J=9.4, 4.1 Hz, 1 H, ArH), 7.48 (ddd, J=8.9, 4.0, 1.4 Hz, 4H, ArH). .sup.13C NMR (126 MHz, DMSO) δ 144.39 (ArC), 143.41(ArC), 142.70 (ArC), 141.40 (ArC), 138.87 (ArC), 138.54 (ArC), 135.59 (ArC), 129.06 (4×ArCH), 127.89 (ArCCI), 127.36 (ArCCI), 126.50 (ArCH), 123.49 (2×ArCH), 122.96 (2×ArCH), 121.12 (ArCH), 119.84 (ArCH). UPLC-MS: t.sub.R 2.763 min. MS (ESI).sup.+ *m/z calculated 427.26 [M+H].sup.+, observed 426.18-428.19 [M+H].sup.+.
Synthesis of N.sup.2, N.sup.3-bis(3-chlorophenyl)-6-nitroquinoxaline-2,3-diamine (compound 5)
##STR00018##
Chemical Formula: C.SUB.20.H.SUB.13.Cl.SUB.2.N.SUB.5.O.SUB.2., Molecular Weight: 426.26
[0119] Prepared according to Standard Procedure 1a, compound 5 was synthesised from 2,3-dichloroquinoxaline 2 (0.09 g, 0.38 mmol) and 3-chloroaniline (0.24 g, 1.90 mmol) in DMSO (5 ml) for 30 min at 130° C. The work-up of the reaction was performed similarly to compound 1. The crude product was purified by automated flash column chromatography eluting with n-hexane:EtOAc 100:0 v/v increasing to 70:30 v/v in 14 CV. The final product 5 was obtained at 40% yield as a yellow powder. .sup.1H NMR (500 MHz, DMSO-d6) δ 9.57 (s, 1H, NH), 9.42 (s, 1H, NH), 8.31 (d, J=2.7 Hz, 1H, ArH), 8.17-8.03 (m, 3H, 3 xArH), 7.86 (dd, J=23.1, 8.2 Hz, 2H, 2×ArH), 7.68 (d, J=8.8 Hz, 1H, ArH), 7.46 (td, J=8.1, 2.5 Hz, 2H, 2×ArH), 7.20 (dd, J=11.9, 8.5 Hz, 2H, 2×ArH). .sup.13C NMR (126 MHz, DMSO) δ 144.04 (ArC), 142.92 (ArC), 142.20 (ArC), 140.96 (ArC), 140.82 (ArC), 140.69 (ArC),135.04 (ArCCI), 132.98 (ArCCI), 130.41 (3×ArCH), 126.22 (ArCH), 123.31 (ArCH), 122.88 (ArCH), 120.74 (ArCH), 120.64 (ArCH), 120.09 (ArCH), 119.64 (ArCH), 119.13 (ArCH). UPLC-MS: t.sub.R 2.785 min. MS (ESI).sup.+ m/z calculated 427.26 [M+H].sup.+, observed 426.20-428.18 [M+H].sup.+.
Synthesis of N.sup.2, N.sup.3-bis(4-fluorophenyl)-6-nitroquinoxaline-2,3-diamine (compound 6)
##STR00019##
Chemical Formula: C.SUB.20.H.SUB.13.F.SUB.2.N.SUB.6.O.SUB.2., Molecular Weight: 393.35
[0120] Compound 6 was prepared according to Standard Procedure 1a from 2,3-dichloroquinoxaline 2 (0.10 g, 0.41 mmol) and 4-fluoroaniline (0.23 g, 2.05 mmol) in DMSO (5 ml) for 30 min at 130° C. The work-up of the reaction was performed similarly to compound 1. The crude product was purified by automated flash column chromatography eluting with n-hexane:EtOAc 100:0 v/v increasing to 70:30 v/v in 10 CV. The final product 6 was obtained at 10% yield as an orange powder. .sup.1H NMR (500 MHz, DMSO-d6) δ 9.56 (s, 1H, NH), 9.39 (s, 1H, NH), 8.29 (d, J=2.6 Hz, 1H, ArH), 8.19 (dd, J=9.1, 5.0 Hz, 1H, ArH), 8.10 (d, J=8.9 Hz, 1H, ArH), 7.94 (dd, J=16.4, 10.6 Hz, 3H, 3×ArH), 7.67-7.60 (m, 1 H, ArH), 7.29 (td, J=8.6, 4.3 Hz, 4H, 4×ArH). .sup.13C NMR (126 MHz, DMSO) δ 176.35 (2×ArC), 143.88 (ArC), 143.35 (ArC), 142.61 (ArC), 141.32 (ArC), 135.84 (ArC), 135.83 (ArC), 135.37 (ArC), 126.11 (ArCH), 123.86 (ArCH), 123.79 (ArCH), 123.26 (ArCH), 123.20 (ArCH), 120.69 (ArCH), 119.43 (ArCH), 115.71 (ArCH), 115.67 (ArCH), 115.53 (ArCH), 115.49 (ArCH). UPLC-MS: t.sub.R 2.557 min. MS (ESI).sup.+ m/z calculated 394.35 [M+H].sup.+, observed 394.24 [M+H].sup.+.
Synthesis of N.sup.2, N.sup.3-bis(4-fluoro-3-(trifluoromethyl)phenyI)-6-nitroquinoxaline-2,3-diamine (compound 7)
##STR00020##
Chemical Formula: C.SUB.22.H.SUB.11.F.SUB.8.N.SUB.6.O.SUB.2., Molecular Weight: 529.35
[0121] According to Standard Procedure 1a, compound 7 was prepared from 2,3-dichloroquinoxaline 2 (0.12 g, 0.49 mmol) and 4-fluoro-3-(trifluoromethyl) aniline (0.44 g, 2.46 mmol) in DMSO (6 ml) for 30 min at 130° C. The work-up of the reaction was performed similarly to compound 1. The crude product was purified by automated flash column chromatography eluting with n-hexane:EtOAc 100:0 v/v increasing to 70:30 v/v in 10 CV. The final product 7 was obtained at 60% yield as a red powder. .sup.1H NMR (500 MHz, DMSO-d6) δ 9.61 (s, 2H, 2×NH), 8.32 (br s, 2H, 2×ArH), 8.23 (d, J=13.8 Hz, 3H, 3×ArH), 8.10 (br s, 1H, ArH), 7.57 (ddd, J=17.4, 11.5, 7.0 Hz, 3H, 3×ArH). .sup.13C NMR (126 MHz, DMSO) δ 174.16 (ArC), 144.17 (ArC), 142.96 (ArC), 142.25 (ArC), 140.72 (ArC), 136.27 (ArC), 135.96 (ArC), 135.04 (ArC), 127.22 (ArCH), 126.74 (ArCH), 126.26 (ArCH), 124.73 (ArC), 123.71 (ArC), 120.74 (ArCH), 119.81 (ArCH), 119.18 (ArCH), 118.62 (ArCH), 117.80 (ArCH), 117.63 (ArCH). UPLC-MS: t.sub.R 2.801 min. MS (ESI).sup.+ m/z calculated 530.35 [M+H].sup.+, observed 530.08 [M+H].sup.+.
Synthesis of N.sup.2, N.sup.3-bis(3-fluoro-5-(trifluoromethyl)phenyl)-6-nitroquinoxaline-2,3-diamine (compound 8)
##STR00021##
Chemical Formula: C.SUB.22.H.SUB.11.F.SUB.8.N.SUB.6.O.SUB.2., Molecular Weight: 529.35
[0122] Prepared according to Standard Procedure 1a, compound 8 was synthesised from 2,3-dichloroquinoxaline 2 (0.09 g, 0.36 mmol) and 3-fluoro-5-(trifluoromethyl) aniline (0.32 g, 1.8 mmol) in DMSO (5 ml) for 30 min at 130° C. The work-up of the reaction was performed similarly to compound 1. The crude product was purified by automated flash column chromatography eluting with n-hexane:EtOAc 100:0 v/v increasing to 70:30 v/v in 15 CV. The final product 8 was obtained at 10% yield as an orange powder. .sup.1H NMR (500 MHz, DMSO-d6) δ 9.71 (br s, 2H, 2×NH), 8.34-8.18 (m, 3H, 3×ArH), 8.10 (d, J=8.9 Hz, 1H, ArH), 7.92 (d, J=31.7 Hz, 2H, 2×ArH), 7.65 (d, J=9.0 Hz, 1H, ArH), 7.33 (dd, J=24.4, 8.3 Hz, 2H, 2×ArH). .sup.13C NMR (126 MHz, DMSO) δ 163.17 (ArC), 161.24 (ArC), 147.21 (ArC), 144.53 (ArC), 142.61 (ArC), 141.94 (ArC), 140.41 (ArC), 134.88 (ArC), 131.00 (ArC), 126.63 (ArCH), 124.55 (ArC), 122.38 (ArC), 121.11 (ArCH), 120.20 (ArCH), 113.19 (ArCH), 112.71 (ArCH), 111.02 (ArCH), 110.82 (ArCH), 110.58 (ArCH), 110.36 (ArCH). UPLC-MS: t.sub.R 2.882 min. MS (ESI).sup.+ m/z calculated 530.35 [M+H].sup.+, observed 529.98 [M].sup.+.
Synthesis of N.sup.2, N.sup.3-bis(2-fluoro-5-(trifluoromethyl)phenyl)-6-nitroquinoxaline-2,3-diamine (compound 9)
##STR00022##
Chemical Formula: C.SUB.22.H.SUB.11.F.SUB.8.N.SUB.5.O.SUB.2., Molecular Weight: 529.35
[0123] According to Standard Procedure 1a, compound 9 was prepared from 2,3-dichloroquinoxaline 2 (0.16 g, 0.66 mmol) and 2-fluoro-5-(trifluoromethyl) aniline (0.59 g, 3.3 mmol) in DMSO (5 ml) for 30 min at 130° C. The work-up of the reaction was performed similarly to compound 1. The crude product was purified by automated flash column chromatography eluting with n-hexane:EtOAc 100:0 v/v increasing to 70:30 v/v in 12 CV. The final product 9 was obtained at 10% yield as a yellow powder. .sup.1H NMR (500 MHz, DMSO-d6) δ 9.63 (br s, 2H, 2×NH), 7.67-7.48 (m, 4H, 4×ArH), 7.26 (dd, J=11.6, 8.3 Hz, 1H, ArH), 7.12 (dd, J=20.4, 10.5 Hz, 1H, ArH), 6.93-6.86 (m, 1H, ArH), 6.74-6.67 (m, 1H, ArH), 6.63 (dd, J=8.2, 1.8 Hz, 1H, ArH). .sup.13C NMR (126 MHz, DMSO) δ 157.91 (ArC), 153.77 (ArC), 143.90 (ArC), 135.84 (ArC), 132.90 (ArCH), 131.10 (ArCH), 128.72 (ArCH), 125.29 (ArC), 124.53 (ArCH), 122.03 (ArCH), 118.78 (ArC), 115.49 (ArCH), 115.33 (ArCH), 115.19 (ArCH), 115.03 (ArCH), 114.93 (ArC), 111.95 (ArC). UPLC-MS: t.sub.R 2.378 min. MS (ESI).sup.+m/z calculated 530.35 [M+H].sup.+, observed 530.05 [M+H].sup.+.
Synthesis of 6-nitro-N.sup.2, N.sup.3-bis(3-(trifluoromethyl)phenyl)quinoxaline-2,3-diamine (compound 10)
##STR00023##
Chemical Formula: C.SUB.22.H.SUB.13.F.SUB.6.N.SUB.6.O.SUB.2., Molecular Weight: 493.37
[0124] Compound 10 was prepared according to Standard Procedure 1a from 2,3-dichloroquinoxaline 2 (0.15 g, 0.61 mmol) and 3-(trifluoromethyl) aniline (0.49 g, 3.1 mmol) in DMSO (8 ml) for 30 min at 130° C. The work-up of the reaction was performed similarly to compound 1. The crude product was purified by automated flash column chromatography eluting with n-hexane:EtOAc 100:0 v/v increasing to 70:30 v/v in 12 CV. The final product 10 was obtained at 30% yield as a yellow powder. .sup.1H NMR (500 MHz, DMSO-d6) δ 9.74 (s, 1H, NH), 9.60 (s, 1H, NH), 8.36-8.21 (m, 5H, 5×ArH), 8.15 (dd, J=9.0, 2.7 Hz, 1H, ArH), 7.73-7.62 (m, 3H, 3×ArH), 7.49 (dd, J=12.8, 7.9 Hz, 2H, 2×ArH). .sup.13C NMR (126 MHz, DMSO) δ 144.16 (ArC), 142.99 (ArC), 142.27 (ArC), 140.77 (ArC), 140.29 (ArC), 140.02 (ArC), 135.04 (ArC), 130.01 (ArCH), 129.97 (ArCH), 129.53 (ArC), 126.26 (ArCH), 125.32 (ArC), 124.64 (ArCH), 124.18(ArCH), 120.76 (ArCH), 119.86 (ArCH), 119.79 (ArCH), 119.42 (ArCH), 117.28 (ArCH), 116.75 (ArCH). UPLC-MS: t.sub.R 2.344 min. MS (ESI).sup.+ m/z calculated 494.37 [M+H].sup.+, observed 494.15 [M+H].sup.+.
Synthesis of 6-nitro-N.sup.2, N.sup.3-bis(4-(trifluoromethyl)phenyl)quinoxaline-2,3-diamine (compound 11)
##STR00024##
Chemical Formula: C.SUB.22.H.SUB.13.F.SUB.6.N.SUB.6.O.SUB.2., Molecular Weight: 493.37
[0125] Prepared according to Standard Procedure 1a, compound 11 was synthesised from 2,3-dichloroquinoxaline 2 (0.20 g, 0.85 mmol) and 4-(trifluoromethyl) aniline (0.69 g, 4.25 mmol) in DMSO (10 ml) for 30 min at 130° C. The work-up of the reaction was performed similarly to compound 1. The crude product was purified by automated flash column chromatography eluting with n-hexane:EtOAc 100:0 v/v increasing to 80:20 v/v in 12 CV. The final product 11 was obtained at 50% yield as a yellow powder. .sup.1H NMR (500 MHz, DMSO-d6) δ 9.74 (s, 1H, NH), 9.61 (s, 1 H, NH), 8.34 (s, 1 H, ArH), 8.18-8.07 (m, 5H, 5 x ArH), 7.77 (d, J=8.3 Hz, 4H, 4×ArH), 7.68 (d, J=8.9 Hz, 1H, ArH). .sup.13C NMR (126 MHz, DMSO) δ 144.22 (ArC), 143.16 (ArC), 142.87 (ArC), 142.79 (ArC), 142.07 (ArC), 140.67 (ArC), 135.03 (ArC), 125.90 (5×ArCH), 123.06 (ArCF.sub.3), 122.81 (ArCF.sub.3), 120.94 (3×ArCH), 120.42 (2×ArCH), 119.78 (ArCH). UPLC-MS: t.sub.R 2.357 min. MS (ESI).sup.+ m/z calculated 494.371_[M+H].sup.+, observed 494.13 [M+H].sup.+.
Synthesis of N.sup.2, N.sup.3-bis(4-methoxy-3-(tritluoromethyl)phenyI)-6-nitroquinoxaline-2,3-diamine (compound 12)
##STR00025##
Chemical Formula: C.SUB.24.H.SUB.17.F.SUB.6.N.SUB.5.O.SUB.4., Molecular Weight: 553.42
[0126] According to Standard Procedure 1a, compound 12 was prepared from 2,3-dichloroquinoxaline 2 (0.16 g, 0.61 mmol) and 4-methoxy-3-(trifluoromethyl) aniline (0.58 g, 3.05 mmol) in DMSO (8 ml) for 30 min at 130° C. The work-up of the reaction was performed similarly to compound 1. The crude product was purified by automated flash column chromatography eluting with n-hexane:EtOAc 100:0 v/v increasing to 50:50 v/v in 10 CV. The final product 12 was obtained at 88% yield as an orange powder. .sup.1H NMR (500 MHz, DMSO-d6) δ 9.50 (s, 1H, NH), 9.34 (s, 1H, NH), 8.28-8.03 (m, 6H, 6×ArH), 7.55 (d, J=8.8 Hz, 1 H, ArH), 7.35 (d, J=9.1 Hz, 2H, 2×ArH), 3.92 (s, 3H, CH.sub.3), 3.91 (s, 3H, CH.sub.3). .sup.13C NMR (126 MHz, DMSO) δ 153.16 (ArC-OCH.sub.3), 152.86 (ArC-OCH.sub.3), 143.68 (ArC), 142.99 (ArC), 142.31 (ArC), 141.01 (ArC), 135.07 (ArC), 132.16 (ArC), 131.83 (ArC), 126.92 (ArCH), 126.53 (ArCH), 125.80 (ArCH), 124.70 (ArCF.sub.3), 122.57 (ArCF.sub.3), 120.35 (ArCH), 119.96 (ArCH), 119.44 (ArCH), 119.29 (ArCH), 116.73 (ArC), 116.50 (ArC), 113.37 (ArCH), 113.36 (ArCH), 56.28 (2×CH.sub.3). UPLC-MS: t.sub.R 2.273 min. MS (ESI).sup.+ m/z calculated 554.42 [M+H].sup.+, observed 554.13 [M+H].sup.+.
Synthesis of N.sup.2, N.sup.3-dibenzyl-6-nitroquinoxaline-2,3-diamine (compound 13)
##STR00026##
Chemical Formula: C.SUB.22.H.SUB.19.N.SUB.5.O.SUB.2., Molecular Weight: 385.43
[0127] Prepared according to Standard Procedure 1a, compound 13 was synthesised from 2,3-dichloroquinoxaline 2 (0.13 g, 0.51 mmol) and phenylmethanamine (0.27 g, 2.5 mmol) in DMSO (6 ml) for 30 min at 130° C. The work-up of the reaction was performed similarly to compound 1. The crude product was purified by automated flash column chromatography eluting with n-hexane:EtOAc 100:0 v/v increasing to 70:30 v/v in 10 CV. The final product 13 was obtained at 65% yield as an orange powder. .sup.1H NMR (500 MHz, DMSO-d6) δ 8.16 (d, J=2.6 Hz, 1H, ArH), 8.09 (t, J=5.2 Hz, 1H, NH), 7.97 (dd, J=8.9, 2.7 Hz, 1H, ArH), 7.85 (t, J=5.2 Hz, 1H, NH), 7.51 (d, J=8.9 Hz, 1H, ArH), 7.46-7.39 (m, 4H, ArH), 7.36 (t, J=7.6 Hz, 4H, ArH), 7.30-7.25 (m, 2H, ArH), 4.76 (d, J=5.2 Hz, 2H, CH.sub.2), 4.73 (d, J=5.1 Hz, 2H, CH.sub.2). .sup.13C NMR (126 MHz, DMSO) δ 145.08 (ArC), 144.39 (ArC), 142.59 (ArC), 141.92 (ArC), 138.68 (ArC), 138.42 (ArC), 135.58 (ArC), 128.43 (2×ArCH), 128.40 (2×ArCH), 128.13 (2×ArCH), 128.12 (2×ArCH), 127.20 (ArCH), 127.13 (ArCH), 125.04 (ArCH), 119.77 (ArCH), 118.01 (ArCH), 44.50 (2×CH.sub.2). UPLC-MS: t.sub.R 2.186 min. MS (ESI).sup.+ m/z calculated 386.43 [M+H].sup.+, observed 386.32 [M+H].sup.+.
Synthesis of 6-nitro- N.sup.2, N.sup.3-diphenethylquinoxaline-2,3-diamine (compound 15)
##STR00027##
Chemical Formula: C.SUB.24.H.SUB.23.N.SUB.5.O.SUB.2., Molecular Weight: 413.48
[0128] According to Standard Procedure 1a, compound 14 was prepared from 2,3-dichloroquinoxaline 2 (0.12 g, 0.49 mmol) and 2-phenylethan-1-amine (0.30 g, 2.5 mmol) in DMSO (6 ml) for 30 min at 130° C. The work-up of the reaction was performed similarly to compound 1. The crude product was purified by automated flash column chromatography eluting with n-hexane:EtOAc 100:0 v/v increasing to 70:30 v/v in 10 CV. The final product 14 was obtained at 75% yield as an orange powder. .sup.1H NMR (500 MHz, DMSO-d6) δ 8.18 (d, J=2.7 Hz, 1H, ArH), 7.97 (dd, J=8.9, 2.7 Hz, 1H, ArH), 7.67 (t, J=5.2 Hz, 1H, NH), 7.52 (d, J=8.9 Hz, 1H, ArH), 7.43 (t, J=5.1 Hz, 1H, NH), 7.34-7.26 (m, 8H, 8×ArH), 7.25-7.18 (m, 2H, 2×ArH), 3.73 (td, J=12.6, 6.6 Hz, 4H, 2×CH.sub.2-N), 2.97 (dd, J=8.1, 6.6 Hz, 4H, 2×H.sub.2). .sup.13C NMR (126 MHz, DMSO) δ 145.18 (ArC), 144.48 (ArC), 142.48 (ArC), 141.96 (ArC), 139.58 (ArC), 139.46 (ArC), 135.58 (ArC), 128.74(2×ArCH), 128.73 (2×ArCH), 128.39 (2×ArCH), 128.38 (2×ArCH), 126.20 (ArCH), 126.16 (ArCH), 125.04 (ArCH), 119.83 (ArCH), 117.87 (ArCH), 42.61 (CH.sub.2), 42.57 (CH.sub.2), 34.20 (CH.sub.2), 34.15 (CH.sub.2). UPLC-MS: t.sub.R 2.271 min. MS (ESI).sup.+ m/z calculated 414.48 [M+H].sup.+, observed 414.35 [M+H].sup.+.
Synthesis of N.sup.2, N.sup.3-bis(3,4-dichlorophenyl)quinoxaline-2,3,6-triamine (compound 15)
##STR00028##
Chemical Formula: C.SUB.20.H.SUB.13.Cl.SUB.4.N.SUB.5., Molecular Weight: 465.16
[0129] A mixture of compound 1 (0.24 g, 0.48 mmol) and 10% Pd/C (0.25 g) was stirred under hydrogen atmosphere at room temperature in EtOAc (24 ml) for 8 hours. After completion of the reaction, it was filtered through Celite. The filtrate was dried over anhydrous Na.sub.2SO.sub.4, filtered and reduced to dryness to give a crude product. The crude was purified by automated flash column chromatography eluting with n-hexane:EtOAc 100:0 v/v increasing to 50:50 v/v in 15 CV. The final product 15 was obtained at 50% yield as a yellow powder. .sup.1H NMR (500 MHz, DMSO-d6) δ 9.06 (s, 1H, NH), 8.88 (s, 1H, NH), 8.36 (d, J=2.5 Hz, 1H, ArH), 8.14 (d, J=2.6 Hz, 1H, ArH), 7.78 (dd, J=8.9, 2.6 Hz, 1H, ArH), 7.70 (dd, J=8.9, 2.6 Hz, 1 H, ArH), 7.61 (d, J=8.8 Hz, 1H, ArH), 7.56 (d, J=8.8 Hz, 1H, ArH), 7.34 (d, J=8.7 Hz, 1H, ArH), 6.83 (dd, J=8.8, 2.5 Hz, 1H, ArH), 6.72 (d, J=2.4 Hz, 1H, ArH), 5.42 (s, 2H, NH.sub.2). .sup.13C NMR (126 MHz, DMSO) δ 147.98 (ArC), 141.44 (ArC), 140.98(ArC) , 140.83 (ArC), 137.91(ArC), 137.21 (ArC), 130.76 (ArC), 130.37 (2×ArCH), 128.38 (ArC), 126.31 (ArCH), 122.97 (ArC), 122.12 (ArC), 120.63 (ArCH), 119.83 (ArCH), 119.74 (ArCH), 118.98 (ArCH), 116.86 (ArCH), 105.85 (ArCH). UPLC-MS: t.sub.R 2.66 min. MS (ESI).sup.+ m/z calculated 466.16 [M+H].sup.+, observed 464.07-466.04-468.05 [M+H].sup.+.
Synthesis of N.sup.2, N.sup.3-bis(3,4-dichlorophenyl)-6-(pyrrolidin-1-yl)quinoxaline-2,3-diamine (compound 16)
##STR00029##
Chemical Formula: C.SUB.24.H.SUB.19.Cl.SUB.4.N.SUB.5., Molecular Weight: 519.25.
[0130] Compound 16 was prepared adding dibromobutane (0.03 g, 0.14 mmol, 2.2 eq.) to a mixture of compound 15 (0.03 g, 0.06 mmol, 1 eq.), anhydrous K.sub.2CO.sub.3 (0.01 g, 0.07 mmol, 1.1 eq.) in CH.sub.3C (2 ml). The reaction mixture was irradiated at 150° C. for 15 min. After complete reaction, the mixture was filtered with EtOAc (20 ml). The results organic phase was washed with saturated aqueous NaHCO.sub.3 (3×5 ml) and brine (10 ml). The organic layer then dried over anhydrous Na.sub.2SO.sub.4, filtered and reduced to dryness to give a crude product. The crude was purified by automated flash column chromatography eluting with n-hexane:EtOAc 100:0 v/v increasing to 80:20 v/v in 10 CV. The final product 16 was obtained at 28% yield as a brown powder. .sup.1H NMR (500 MHz, DMSO-d6) δ 9.12 (s, 1H, NH), 8.97 (s, 1H, NH), 8.27 (d, J=2.6 Hz, 1H, ArH), 8.19 (d, J=2.5 Hz, 1H, ArH), 7.94 (dd, J=8.9, 2.5 Hz, 1H, ArH), 7.78 (dd, J=8.9, 2.5 Hz, 1H, ArH), 7.67 (d, J=8.8 Hz, 1H, ArH), 7.62 (d, J=8.8 Hz, 1H, ArH), 7.51 (d, J=8.9 Hz, 1 H, ArH), 6.93 (dd, J=9.0, 2.6 Hz, 1H, ArH), 6.61 (d, J=2.6 Hz, 1H, ArH), 3.35 (t, J=6.6 Hz, 4H, 2×CH.sub.2), 2.04-1.96 (m, 4H, 2×CH.sub.2). .sup.13C NMR (126 MHz, DMSO) δ 145.75 (ArC), 141.34 (ArC), 141.20 (ArC), 140.63 (ArC), 138.11 (ArC), 136.89 (ArC), 131.13 (ArC), 131.04 (ArC), 130.72 (ArCH), 130.58 (ArCH), 128.61(ArC), 126.51 (ArCH), 124.09 (ArC), 122.91 (ArC), 121.69 (ArCH), 120.62 (ArCH), 120.43 (ArCH), 119.49 (ArCH), 114.87 (ArCH), 105.12 (ArCH), 48.97 (2×CH.sub.2), 25.01 (2×CH.sub.2). UPLC-MS: t.sub.R 2.707 min. MS (ESI).sup.+ m/z calculated 520.25 [M+H].sup.+, observed 515.95-518.05-591.13 [M+H].sup.+.
Synthesis of N-(2,3-bis((3,4-dichlorophenyl)amino)quinoxalin-6-yl)acetamide (compound 17)
##STR00030##
Chemical Formula: C.SUB.22.H.SUB.15.Cl.SUB.4.N.SUB.5.O, Molecular Weight: 507.20
[0131] According to Standard Procedure 1b, compound 17 was prepared adding dropwise acetyl chloride (0.01 g, 0.13 mmol) to a solution of anhydrous DCM (4 ml) containing compound 15 (0.06 g, 0.12 mmol) and anhydrous pyridine (0.03 g, 0.43 mmol) under a nitrogen atmosphere. The resulting mixture was stirred at room temperature for 1 hour. After completion, the reaction was diluted with DCM (10 ml) and quenched with saturated aqueous NaHCO.sub.3 (15 ml). The organic phase was washed with saturated aqueous NaHCO.sub.3 (3×10ml) and brine (15 ml) respectively. The organic layer then dried over anhydrous Na.sub.2SO.sub.4, filtered and reduced to dryness to give a crude product. The crude product was purified by automated flash column chromatography eluting with n-hexane:EtOAc 100:0 v/v increasing to 50:50 v/v in 10 CV. The final product 17 was obtained at 52% yield as a pale-yellow powder. .sup.1H NMR (500 MHz, DMSO-d6) δ 10.11 (s, 1H, NH—C═O), 9.23 (s, 1H, NH), 9.15 (s, 1H, NH), 8.31 (d, J =2.5 Hz, 1H, ArH), 8.24 (d, J=2.6 Hz, 1 H, ArH), 8.00 (d, J=2.1 Hz, 1 H, ArH), 7.81 (ddd, J=9.3, 6.9, 2.5 Hz, 2H, 2×ArH), 7.63 (dd, J=18.7, 8.8 Hz, 2H, 2×ArH), 7.58-7.50 (m, 2H, 2×ArH), 2.09 (s, 3H, CH.sub.3). .sup.13C NMR (126 MHz, DMSO) δ 168.38 (C═O), 141.15 (ArC), 140.64 (ArC), 140.43 (ArC), 139.82 (ArC), 137.46 (ArC), 136.30 (ArC), 132.18 (ArC), 130.79 (2×ArC), 130.47 (ArCH), 130.43 (ArCH), 125.78 (ArCH), 123.52 (ArC), 123.20 (ArC), 121.04 (ArCH), 120.78 (ArCH), 120.05 (ArCH), 119.81 (ArCH), 118.78 (ArCH), 113.96 (ArCH), 24.12 (CH.sub.3). UPLC-MS: t.sub.R 2.195 min. MS (ESI).sup.+ m/z calculated 508.20 [M+H].sup.+, observed 506.10-508.02-510.04 [M+H].sup.+.
Synthesis of N-(2,3-bis((3,4-dichlorophenyl)amino)quinoxalin-6-yl)hexanamide (compound 18)
##STR00031##
Chemical Formula: C.SUB.26.H.SUB.23.Cl.SUB.4.N.SUB.5.O, Molecular Weight: 563.30
[0132] Compound 18 was synthesised according to Standard Procedure 1b by addition dropwise of hexanoyl chloride (0.02 g, 0.12 mmol) to a solution of anhydrous DCM (5 ml) containing compound 15 (0.05 g, 0.11 mmol) and anhydrous pyridine (0.03 g, 0.39 mmol) under a nitrogen atmosphere. The resulting mixture was stirred at room temperature for 1 hour. The work-up of the reaction was performed similarly to compound 17. The crude product was purified by automated flash column chromatography eluting with n-hexane:EtOAc 100:0 v/v increasing to 70:30 v/v in 10 CV. The final product 18 was obtained at 66% yield as a brown powder. .sup.1H NMR (500 MHz, DMSO-d6) δ 10.04 (s, 1H, NH—C═O), 9.24 (s, 1H, NH), 9.17 (s, 1H, NH), 8.33 (d, J=2.6 Hz, 1H, ArH), 8.25 (d, J=2.5 Hz, 1H, ArH), 8.05 (d, J=2.2 Hz, 1H, ArH), 7.82 (ddd, J=13.8, 8.9, 2.5 Hz, 2H, 2×ArH), 7.63 (dd, J=18.7, 8.8 Hz, 2H, 2×ArH), 7.59-7.51 (m, 2H, 2×ArH), 2.34 (t, J=7.4 Hz, 2H, CH.sub.2), 1.62 (p, J=7.4 Hz, 2H, CH.sub.2), 1.36-1.28 (m, 4H, 2×CH.sub.2), 0.91-0.86 (m, 3H, CH.sub.3). .sup.13C NMR (126 MHz, DMSO) δ 171.39 (C═O), 141.06 (ArC), 140.64 (ArC), 140.44 (ArC), 139.73 (ArC), 137.48 (ArC), 136.28 (ArC), 132.13 (ArC), 130.80 (ArC), 130.43 (ArCH), 130.39 (ArCH), 125.73 (ArCH), 123.50 (ArC), 123.20 (ArC), 120.98 (ArCH), 120.76 (ArCH), 119.99 (ArCH), 119.78 (ArCH), 118.84 (ArCH), 114.01 (ArCH), 36.49 (CH.sub.2), 30.95 (CH.sub.2), 24.84 (CH.sub.2), 21.96 (CH.sub.2), 13.90 (CH.sub.3). UPLC-MS: t.sub.R 2.429 min. MS (ESI).sup.+ m/z calculated 564.30 [M+H].sup.+, observed 562.04-564.08-566.01 [M+H].sup.+.
Synthesis of N-(2,3-bis((3,4-dichlorophenyl)amino)quinoxalin-6-yl)furan-2-carboxamide (compound 19)
##STR00032##
Chemical Formula: C.SUB.26.H.SUB.16.C.SUB.14.N.SUB.6.O.SUB.2., Molecular Weight: 559.23
[0133] According to Standard Procedure 1b, compound 19 was prepared adding dropwise furan-2-carbonyl chloride (0. 02 g, 0.14 mmol) to a solution of anhydrous DCM (4 ml) containing compound 15 (0.06 g, 0.13 mmol) and anhydrous pyridine (0.04 g, 0.47 mmol) under a nitrogen atmosphere. The resulting mixture was stirred at room temperature for 1 hour. The work-up of the reaction was performed similarly to compound 17. The crude product was purified by automated flash column chromatography eluting with n-hexane:EtOAc 100:0 v/v increasing to 70:30 v/v in 10 CV. The final product 19 was obtained at 55% yield as a yellow powder. .sup.1H NMR (500 MHz, DMSO-d6) δ 10.35 (s, 1H, NH—C═O), 9.26 (s, 1H, NH), 9.20 (s, 1H, NH), 8.35 (d, J=2.5 Hz, 1 H, ArH), 8.26 (d, J=2.5 Hz, 1 H, ArH), 8.14 (d, J =2.3 Hz, 1 H, ArH), 7.96 (dd, J=1.8, 0.8 Hz, 1H, ArH), 7.83 (dt, J=8.9, 2.0 Hz, 3H, 3×ArH), 7.69-7.55 (m, 3H, 3×ArH), 7.39 (dd, J=3.5, 0.9 Hz, 1H, ArH), 6.72 (dd, J=3.5, 1.7 Hz, 1H, ArH). .sup.13C NMR (126 MHz, DMSO) δ 156.61 (C═O), 147.88 (ArC), 146.23 (ArCH), 141.57 (ArC), 140.97 (ArC), 140.80 (ArC), 140.45
[0134] (ArC), 137.07 (ArC), 136.55 (ArC), 133.03 (ArC), 131.20 (ArC), 130.85(ArCH), 130.83 (ArCH), 126.07 (ArCH), 123.95 (ArC), 123.70 (ArC), 121.45 (ArCH), 121.26(ArCH), 120.47 (ArCH), 120.28 (ArCH), 120.18(ArCH), 115.79(ArCH), 115.20 (ArCH), 112.59 (ArCH). UPLC-MS: t.sub.R 2.297 min. MS (ESI).sup.+ m/z calculated 560.23 [M+H].sup.+, observed 557.99-559.93-561.85 [M+H].sup.+.
[0135] Compounds 20 and 21 having the structures shown below were commercially obtained from SPECS.
##STR00033##
[0136] Comparative compounds “Comp. 1” (prepared by similar methods to those described above), “Comp. 2” (obtained from Sigma-Aldrich) and “Comp. 3” (obtained from Sigma-Aldrich) were included in the experiments described below as comparative examples. The results produced by these compounds are shown in Table 1 (first two rows). Compound “Comp. 1” has the following structure.
##STR00034##
[0137] Compound “Comp. 2” is praziquantel, the known treatment for Schistosomiasis in humans, having the following structure:
##STR00035##
[0138] Compound “Comp. 3” is triclabendazole (Tbz) which is a known anti-parasitic with the following structure:
##STR00036##
Biological Screening
72 Hours Schistosomula Drug Screen
[0139] S. mansoni (Puerto Rican Strain, Naval Medical Research Institute—NMRI) cercariae were collected from infected Biomphalaria glabrata (NMRI) snails after exposure to 2 hours of light at 26° C. and then mechanically transformed into schistosomula as described in D. G. Colley, S. K. Wikel, Schistosoma mansoni: simplified method for the production of schistosomules., Exp. Parasitol. 35 (1974) 44-51. Newly transformed schistosomula were prepared for 72 hour high throughput screening (HTS) in 384-well black-sided microtiter plates (Perkin Elmer, Mass., USA) as described in M. Nur-e-alam, M. Yousaf, S. Ahmed, E. S. Al-sheddi, I. Parveen, D. M. Fazakerley, A. Bari, H.A. Ghabbour, M.D. Threadgill, K. C. L. Whatley, K. F. Ho, A. J. Al-rehaily, Neoclerodane Diterpenoids from Reehal Fatima, Teucrium yemense, J. Nat. Prod. 80 (2017) 1900-1908, with a final DMSO concentration of 0.625%. The effect of compounds on 72 hour cultured schistosomula was deduced by analysing the effect on both motility and phenotype of treated schistosomula using the image analysis model described by Paveley, R., Mansour, N., Hallyburton, I., Bleiche, L., Alex, E., Mikic, I., Guidi, A., H Gilbert, I., L Hopkins, A., D Bickle, Q., in “Whole Organism High -Content Screening by Label-Free, Image-Based Bayesian Classification for Parasitic Diseases”, PLoS Negl Trop Dis 6(7): e1762 (2012). The data obtained in this manner is shown in Table 1 in the columns labelled “Phenotype”, “Motility” and “Schistosomula average”.
Juveniles Drug Screen
[0140] Juvenile 3 wk old worms were obtained by the perfusion of outbred mice (TO or MF-1) percutaneously infected three weeks earlier with 4,000 S. mansoni cercaria. Juvenile worm screens (15-20 worms/well) were performed in a 96 well plate format (flat bottom) containing a final volume of 200 μL of media (DMEM (Gibco, Paisley, UK) supplemented with 10% v/v Hepes (Sigma-Aldrich, Gillingham, UK), 10% v/v Foetal Bovine Serum (Gibco, Paisley, UK), 0.7% v/v 200 mM L-Glutamine (Gibco, Paisley, UK) and 1X v/v penicillin-streptomycin (Fisher Scientific, UK)) as previously described in Padalino G, Ferla S, Brancale A, Chalmers I W, Hoffmann K F. Int J Parasitol Drugs Drug Resist. 2018 Dec;8(3):559-570. The data obtained in this manner is shown in Table 1 in the columns labelled “Juveniles”.
Adult Worm Drug Screen
[0141] Mice (MF-1 mice, Harlan, UK), infected 7 weeks previously with 180 cercaria, were euthanised with an intraperitoneal injection of phenobarbital/heparin solution. Adult worms were recovered by perfusion of the portal system and subjected to drug screening as previously described in Padalino G, Ferla S, Brancale A, Chalmers I W, Hoffmann K F. Int J Parasitol Drugs Drug Resist. 2018 Dec;8(3):559-570; in Whiteland H L, Chakroborty A, Forde-Thomas J E, Crusco A, Cookson A, Hollinshead J, Fenn C A, Bartholomew B, Holdsworth P A, Fisher M, Nash R J, Hoffmann K F. Int J Parasitol Drugs Drug Resist. 2018 Dec;8(3):465-474; and in Crusco A, Bordoni C, Chakroborty A, Whatley K C L, Whiteland H, Westwell A D, Hoffmann K F. Eur J Med Chem. 2018 May 25;152:87-100. The data obtained in this manner is shown in Table 1 in the columns labelled “Adult worm”.
[0142] Regarding the quantification of drug treatment on adult worms (e.g. decreased activity, paralysis or sluggishness), two different approaches were used, as described below. Where egg deposition was noticed, eggs were removed and counted using a Sedgewick rafter slide.
WHO Scoring Function
[0143] Adult worms were scored using the standardized microscopic WHO-TDR scoring system previously described by Ramirez, B., Bickle, Q., Yousif, F., Fakorede, F., Mouries, M.-A., Nwaka, S. in Schistosomes: Challenges in compound screening, Expert Opin Drug Discov. 2007 Oct;2(s1): S53-61. This scoring system (0-4) identifies changes in worm movement and is defined according to the following scoring matrix: 0=no movement, 1=no movement with visible gut peristalsis, 2=minimal movement (occasional movement from anterior or posterior end of the worm), 3=slow continuous movement and 4=normal movement.
WormassayGP2
[0144] A digital image processing-based system was also used to assess parasite motility after drug treatment and is a modification of methods previously described by Marcellino C., Gut, J., Lim, K., Singh R., McKerrow, L. and Sakanari, J. WormAssay: a novel computer application for whole-plate motion-based screening of macroscopic parasites. PLoS Negl Trop Dis. 2012 Jan:6(1):e1494. This allowed for objective verification of the WHO method described above. The data obtained with this quantification method is shown in Table 1 in the columns labelled “Adult worm”.
Schistosoma mansoni RNAi
[0145] Following the perfusion of 7-week infected mice, adult worms were recovered and RNAi performed as previously described in Geyer K K, Munshi S E, Whiteland H L, Fernandez-Fuentes N, Phillips D W, Hoffmann K F. PLoS Pathog. 2018 Jun 28;14(6):e1007107 and Padalino G, Ferla S, Brancale A, Chalmers I W, Hoffmann K F. Int J Parasitol Drugs Drug Resist. 2018 Dec;8(3):559-570. Briefly, smp 138030 and non-specific Luciferase siRNA duplexes were purchased from Sigma (siSmp_138030=CGUUUGGUCCCAUCGGACA[dT][dT] and UGUCCGAUGGGACCAAACG[dT][dT]; siLuc=CUUACGCUGAGUACUUCGA[dT][dT] and UCGAAGUACUCAGCGUAAG[dT][dT]). Mixed sex adult worm pairs (for knockdown assessment by quantitative reverse transcription PCR, qRT-PCR) were cultured at 37° C. in DMEM supplemented with 10% fetal calf serum, 2 mM L-glutamine, 10,000 Units/ml penicillin and 10,000 μg/ml streptomycin in an atmosphere of 5% CO.sub.2 with a 70% media exchange performed every 48 hr. Quantitative reverse transcription PCR (qRT-PCR) of smp_138030 abundance was performed at 48 hr and adult worm motility [39] as well as egg counts were quantified at 168 hr. Levels of H3K4me detected in schistosome histone extracts were assessed at 72 hr.
Quantitative Reverse Transcription PCR (qRT-PCR)
[0146] Following RNAi with siSmp 138030 and siLuc, mixed-sex adult worms were incubated for a total of 48 hr before processing them for RNA isolation. Briefly, worms were homogenised using a TissueLyser LT (Qiagen, UK) in TRIzol Reagent (Invitrogen, UK) before isolation of total RNA using the Direct-zol RNA Kit (Epigentek, UK). cDNA was then generated using the SensiFAST cDNA synthesis kit (Bioline), qRT-PCR performed and data analysed as previously described in Padalino G, Ferla S, Brancale A, Chalmers I W, Hoffmann K F. Int J Parasitol Drugs Drug Resist. 2018 Dec;8(3):559-570. qRT-PCR primers for amplifying smp_138030 (Forward primer FW: 5′-GTCTACCGGGTGTTCGACG-3′, Reverse 5′-TCCAAATCCCGTGCAGC-3′) and internal standard alpha tubulin (SmAT1; Forward primer 5′-CGAAGCTTGGGCGCGTCTAGAT -3′, Reverse 5′-CTAATACTCTTCACCTTCCCCT -3′) were purchased from Sigma.
Histone Extraction and Detection of H3K4 Methylation
[0147] Drug or RNAi treated adult worms were separated by sex and transferred to fresh microcentrifuge tubes (with Snap Cap, 2 ml Capacity, Round Bottom). Total histone extraction from these worms was performed using EpiQuik™ Total Histone Extraction Kit (OP-0006, Epigentek) following the manufacturer's instructions.
[0148] The global histone H3-K4 methylation was measured using the EpiQuik™ Global Histone H3-K4 Methylation Assay Kit (P-3017-96) following the manufacturer's instructions.
Newly Excysted Juveniles (NEJs) Drug Screen
[0149] F. hepatica (Italian strain) metacercariae were obtained from Ridgeway Research, Gloucestershire, UK and stored at 4° C. Newly excysted juveniles (NEJs) from metacercariae were produced based on an optimisation introduced to the excystment method published by Dixon et al. and Wilson et al (Dixon, K. E., The physiology of excystment of the metacercaria of Fasciola hepática L. Parasitology, 1966, 56, 431-456.; Wilson, L. R., Good, R. T., Panaccio, M., Wijffels, G. L., Sandeman, R. M., Spithill, T. W, Fasciola hepatica: Characterization and Cloning of the Major Cathepsin B Protease Secreted by Newly Excysted Juvenile Liver Fluke. Experimental Parasitology 1998, 88, 85-94).
[0150] After excystment, NEJs were distributed into a 24 well tissue culture plate at a density of 25 parasites per well containing 1 mL of pre-warmed NEJs culture media. Compounds were added to respective wells and NEJ/compound co-cultures were incubated at 37° C. in an atmosphere containing 5% CO.sub.2 for 72 hours at a final concentration of 10 μM. The effect of the drug on the parasite phenotype and motility was independently scored using the scoring matrix as described previously in Edwards, J., Brown, M., Peak, E., Bartholomew, B., Nash, R. J., Hoffmann, K. F., The diterpenoid 7-keto-sempervirol, derived from Lycium chinense, displays anthelmintic activity against both Schistosoma mansoni and Fasciola hepatica, PLoS neglected tropical diseases 2015, 9, e0003604-e0003604.
MTT Assay on HepG2 Cells
[0151] Human Caucasian Hepatocyte Carcinoma (HepG2) cells (85011430, Sigma Aldrich) were used to assess the overt toxicity of these anti-infective compounds.
[0152] Briefly, HepG2 cells were treated with compounds for 24 hrs as previously described in Crusco A, Bordoni C, Chakroborty A, Whatley K C L, Whiteland H, Westwell A D, Hoffmann K F., Eur J Med Chem. 2018 May 25;152:87-100. Each compound was used to dose the cells at different concentration points: 200, 100, 75, 50, 20, 10 and 1 μM, each of them in triplicate. Each compound solution was prepared in HepG2 culture media at double the concentration required for the cytotoxicity assay (200-2 μM for 100-1 μM final concentration). 50 μl of the compound solution was added to the corresponding well of the plate according to a predefined layout. Media and a DMSO controls (in triplicate as well) were prepared by addition of 50 μl of fresh media without compound and DMSO (1.25 % v/v) respectively. A negative control (1% v/v Triton X-100; X100, Sigma-Aldrich) was also included in each screen. After addition of compounds, each plate was then incubated for a further 20 hrs before application of MTT reagent (prepared as described in Nur-E-Alam M, Yousaf M, Ahmed S, Al-Sheddi E S, Parveen I, Fazakerley D M, Bari A, Ghabbour H A, Threadgill M D, Whatley K C L, Hoffmann K F, Al-Rehaily A J, J Nat Prod. 2017 Jun 23;80(6):1900-1908 for the assessment of compound cytotoxicity using the MTT assay.
[0153] Following incubation with test compounds for 20 hrs, 10 μl (10% final volume) of 12 mM MTT reagent was added to each well. The plate was then further incubated for 4 hrs. After incubation, each plate was blotted dry under the laminar flow to remove the MTT treated culture media. A 100 μl DMSO: isopropanol (1:1) solution was then added to each well and the plate was then subsequently incubated for 10 minutes at 37° C. Following incubation, each plate was shaken again and then absorbance measured at 570 nm. The absorbance reading was corrected based on the average value of the wells used as blank (no cells). Dose response curves to calculate CC.sub.50 values of the compounds tested were generated in GraphPad Prism 7.02 based on the average absorbance of the three replicates for each concentration.
[0154] The data obtained in this manner is shown in Table 1 in the columns labelled “HepG2”. This data was also used to calculate a selectivity index of activity against schistosomula and adult worms over activity in the HepG2 assay (an indication of toxicity). The selectivity indexes for schistosomula and adult worms are shown in the final two columns in Table 1.
In vitro Determination of Antibacterial Activity
[0155] Bacteria strains of Escherichia coli—E.coli—ATCC 25922; Staphylococcus aureus—S. aureus—ATCC 29213; Mycobacterium smegmatis—M. smegmatis mc.sup.215—ATCC 700084, MRSA (USA300, ATCC 33591, EM RSA), Pseudomonas aeruginosa—ATCC 27853, S. epidermidis—NTCT11077, B. cereus strains were used for testing all the compouds, as previously described in “Untargeted metabolomics reveals a new mode of action of pretomanid (PA-824)”, Baptista, Rafael, Fazakerley, David M., Beckmann, Manfred, Baillie, Les and Mur, Luis A. J. 2018. Scientific Reports 8 (1), 2018. Bacterial strains of Enterococcus faecalis—ATCC 29212, NCTC 12201, ATCC 51299; Streptococcus pneumoniae—ATCC 49619, 18778, 21394, 21395; Haemophilus influenza—ATCC 49766, 18391, 17428; Staphylococcus aureus—NCTC 12493, ATCC BAA-077, ATCC 700698, 11051; used for testing the compounds, as previously described in “Clinical laboratory testing and in vitro diagnostics test systems—Susceptibility testing of infectious agents and evaluation of performance of antimicrobial susceptibility devices—Part 1: Reference methods for testing the in vitro activity of antimicrobial agentes against bacteria involved in infectious diseases, ISO/FDIS 20776-1:2006(E). All the procedures were carried out in a biosafety containment level 2.
Anti-Schistosomal in vivo Efficacy Screen
[0156] An anti-schistosomal efficacy trial of the compounds described herein was conducted on a murine model of Schistosomiasis using the following procedure. Schistosoma mansoni cercaria were prepared from infected Biomphalaria glabrata snails by inducing synchronous shedding upon exposure to light for 40 minutes (in artificial pond water). Mice were infected percutaneously by exposing tails to 120 S. mansoni cercariae per mouse for a duration of 45 minutes [Smithers S, Terry R. The infection of laboratory hosts with cercariae of Schistosoma mansoni and the recovery of the adult worms. Parasitology. 1965;55(4):695-700]. A maximum of 6 mice were housed in a cage for the duration of the 7-week infection.
[0157] At 6 wks post infection (Day 42 post infection), mice were dosed with anthelmintic chemicals using autoclaved gavage needles for oral delivery. The gavage needle was dipped into a concentrated solution of sucrose (approx. 1 g/ml) before dosing as a refinement measure to reduce stress-related reactions.
[0158] For oral dosing, the compounds (dry powder) were weighed and prepared in an appropriate excipient (e.g. 10% DMSO, 20% Kolliphor EL and 70% sterile water). Compound formulations were prepared fresh daily and any leftover discarded.
[0159] For each selected compound, a defined number of animals (in the range of 3-6) were dosed orally (e.g. dose level 100 mg/kg, dose volume 10 ml/kg). Each mouse was dosed once per day on two non-consecutive days (Day 42 and 44 post infection). Each mouse of the control treatment was dosed with only the vehicle (10% DMSO, 20% Kolliphor EL and 70% sterile water) at a dose volume of 10 ml/kg following the same dosing interval.
[0160] At the end of the treatment (on day 48 post-infection), mice were perfused after receiving an intra-peritoneal administration of non-recoverable dose of sodium pentobarbital (100 μl/10 g body weight) containing 100 U/ml heparin solution (in 1X PBS). Adult S. mansoni were obtained from each mouse by hepatic portal vein perfusion with pre-warmed (37° C.) perfusion media (DMEM supplemented with 0.1% 100 U/ml heparin solution in 1X PBS). Worms were collected in a sedimentation flask placed directly below the perfused animal. Following that, worms were washed by sedimentation with perfusion media first and then pre-warmed adult worm media (DMEM (Gibco, Paisley, UK) supplemented with 10% v/v FCS (Gibco, Paisley, UK), 1% v/v L-glutamine (Gibco, Paisley, UK) and an antibiotic mixture (150 Units/ml penicillin and 150 μg/ml streptomycin; Gibco, UK)) [as described in Crusco A, Whiteland H, Baptista R, Forde-Thomas J E, Beckmann M, Mur L A J, et al. Antischistosomal Properties of Sclareol and Its Heck-Coupled Derivatives: Design, Synthesis, Biological Evaluation, and Untargeted Metabolomics. ACS Infectious Diseases. 2019;5(7):1188-99; Whatley KCL, Padalino G, Whiteland H, Geyer KK, Hulme B J, Chalmers I W, et al. The repositioning of epigenetic probes/inhibitors identifies new anti-schistosomal lead compounds and chemotherapeutic targets. PLOS Neglected Tropical Diseases. 2019;13(11):e0007693; and Whiteland H, Chakroborty A, Forde-Thomas J, Crusco A, Cookson A, Hollinshead J, et al. An Abeis procera—Derived tetracyclic triterpene containing a steroid-like nucleus core and a lactone side chain attenuates in vitro survival of both Fasciola hepatica and Schistosoma mansoni. International Journal for Parasitology: Drugs and Drug Resistance. 2018;8]. After incubation in a humidified environment containing 5% CO.sub.2 at 37° C. for at least 1 h, the quantification of worm burden per each mouse of each treatment was performed.
[0161] Following perfusion, livers were removed and weighed before being placed into a 50 mL falcon tube to perform an overnight digestion by 4% KOH treatment (at 37° C.). Subsequent schistosome egg count (expressed as number of eggs per organ weight) was performed for each sample to assess any differences between the drug treated cage and the control cage (e.g. vehicle).
Results
[0162] The data obtained by the above methods for compounds 1 and 4-21 were converted into dose response curves to calculate EC.sub.50 values of these compounds using GraphPad Prism 7.02, based on the average score for each concentration. Table 1 shows the anti-schistosomal screening data of compounds 1 and 4-21. Table 2 shows the activity of compounds 1, 4, 5, 7, 9-16, 18, 19 and Comp. 3 (Tbz) on newly excysted juveniles (NEJs) of F. hepatica against a DMSO and RPMI media control, wherein statistical evaluation of all treatment conditions was performed (compared to the DMSO control) using a Kruskal-Wallis Anova (followed by Dunn's post-hoc analysis test). Table 3 shows the antibacterial screening data for compounds 2, 5, 7, 10-14 and 16-19. Table 4 shows additional antibacterial screening data for compounds 2, 5, 7, 11, 12 and 19.
[0163] Table 1 demonstrates that the example compounds show a similar or improved activity against Schistosomula, juveniles and adult worms of S. mansoni than the comparative compounds
[0164] “Comp. 1” and “Comp. 2”, and that many of these compounds show a greater selectivity index over HepG2. The compounds of the present invention may therefore provide safe and effective treatments for infections of S. mansoni and also, by way of inhibition of homologous enzymes, treatments for Fasciola hepatica, Plasmodium falciparum and Staphylococcus aureus infections.
[0165] The data in Table 2 demonstrates that the example compounds show activity against F. hepatica NEJs, which is similar to or an improvement over compound Comp. 3 (Tbz).
[0166] Tables 3 and 4 show that the many of the example compounds of the present invention are also effective in combatting Staphylococcus aureus and MRSA and therefore could provide effective treatments for infections of these organisms. Also, this data demonstrates that the example compounds may be effective in disinfecting surfaces, for example on surgical devices or implants, comprising pathogenic bacteria, such as Staphylococcus aureus and MRSA.
TABLE-US-00001 TABLE 1 EC.sub.50 EC.sub.50 (μM) (μM) EC.sub.50 CC.sub.50 Selectivity Selectivity Schistosomula Adult (μM) (μM) Index Index Compound Phenotype Motility average worm Juveniles HepG2 Schistosomula Adult Comp. 1 2.21 1.22 1.71 0.14 ND 9.89 6 73 Comp. 2 1.11 0.84 0.98 0.0107 ND >100 — — 1 0.42 0.54 0.48 0.07 0.0771 7.40 15 107 4 0.40 0.53 0.47 ND ND 13.05 28 ND 5 1.22 1.30 1.26 0.012 ND 6.76 5 568 6 2.14 1.40 1.77 0.18 ND 22.04 12 121 7 0.54 0.54 0.54 0.011 ND 7.75 14 705 8 0.37 0.43 0.40 0.0065 ND 10.07 25 1549 9 0.48 0.47 0.48 0.0036 ND 106.07 223 29464 10 0.38 0.57 0.48 0.0070 ND 5.85 12 836 11 0.42 0.53 0.48 0.0067 ND 15.55 33 2320 12 0.57 0.65 0.61 0.0085 ND 5.64 9 664 13 3.19 2.64 2.92 ND ND 11.67 4 ND 14 2.20 1.47 1.84 0.20 ND 4.40 2 23 15 3.51 3.25 3.38 ND ND 25.11 7 ND 16 >10 >10 ND ND ND ND ND ND 17 2.60 2.09 2.35 0.70 ND 8.60 4 12 18 1.02 1.80 1.41 0.32 ND 64.75 46 203 19 2.54 1.33 1.94 ND ND 14.84 8 ND 20 4.31 4.51 4.41 ND ND 13.75 3 ND 21 1.62 1.23 1.42 ND ND 15.58 11 ND
TABLE-US-00002 TABLE 2 Phenotype (P) Motility (M) Signif- Signif- Com- icance icance pound/ (compared (compared control to to name Mean* SD DMSO)** Mean* SD DMSO)** DMSO 1.48 0.96 — 1.36 0.64 — RPMI 1.44 0.77 ns 1.36 0.64 ns (media) Comp. 3 4.72 0.46 **** 4.72 0.46 **** (Tbz) 1 5.00 0.82 **** 4.69 0.48 **** 4 5.13 0.85 **** 4.71 0.46 **** 5 4.70 0.98 **** 4.20 0.62 **** 7 5.00 0.69 **** 4.56 0.51 **** 9 4.63 0.96 **** 3.68 0.48 *** 10 2.76 0.97 ns 2.71 0.77 ns 11 4.28 0.83 **** 4.17 0.51 **** 12 4.70 0.86 **** 4.45 0.51 **** 13 3.14 0.35 ns 3.14 0.35 ns 14 4.27 1.28 **** 4.00 0.93 **** 15 5.09 0.61 **** 4.86 0.35 **** 16 2.95 1.22 ns 2.84 0.96 ns 18 4.09 0.43 **** 4.05 0.21 **** 19 3.40 0.87 * 3.36 0.76 ** *Phenotype and motility scores recorded at 72 h are presented in the form of a mean score and standard deviation. **Statistical evaluation of all treatment conditions was performed (compared to the DMSO control) using a Kruskal-Wallis Anova (followed by Dunn's post-hoc analysis test).
TABLE-US-00003 TABLE 3 All data points in Table 3 below are MIC90 values in ug/ml. *M. smegmatis Pseudomonas E. coli S. aureus mc.sup.2 15 3 MRSA strains aeruginosa S. Com- ATCC ATCC ATCC USA ATCC EM ATCC epidermidis pound 25922 29213 700084 300 33591 RSA 27853 NTCT11077 B. cereus 2 62.5 <3.91 1.95 15.63 15.63 15.63 125 <62.50 <62.50 5 >125 <3.91 >125 3.91 3.91 3.91 >125 <62.5 125 7 62.5 1.95 62.5 <3.91 <3.91 7.81 >125 <62.5 125 10 >125 7.81 >125 >125 >125 >125 >125 >125 >125 11 >125 0.98 31.25 <3.91 <3.91 3.91 >125 <62.5 <62.5 12 >125 62.5 >125 15.63 31.25 31.25 >125 <62.5 <62.5 13 >125 >125 >125 ND ND ND ND ND ND 14 >125 >125 31.25 ND ND ND ND ND ND 16 >125 15.63 >125 62.5 31.25 62.5 >125 >125 >125 17 >125 62.5 >125 ND ND ND >125 <62.5 <62.5 18 >125 62.5 >125 ND ND ND >125 >125 125 19 >125 31.25 >125 62.5 15.63 62.5 >125 125 <62.5
TABLE-US-00004 TABLE 4 All data points in Table 4 below are MIC values in mg/l Compound (MIC values in mg/l) Organism ID Reference strain Resistances Mechanism 2 5 7 11 12 19 Enterococcus faecalis ATCC 29212 — — 32 0.25 1 0.06 0.5 4 Streptococcus pneumoniae ATCC 49619 — — 64 4 8 2 8 8 Haemophilus influenzae ATCC 49766 — — 32 4 >64 16 64 64 Staphylococcus aureus NCTC 12493 Flucloxacillin mecA 16 0.06 0.5 0.125 1 1 Staphylococcus aureus ATCC BAA-977 ERY/CLIND MLSB 32 0.125 0.5 0.125 1 8 Staphylococcus aureus ATCC 700698 Vancomycin hVISA 32 0.125 0.5 0.125 1 8 Staphylococcus aureus 11051 Tetracycline — 16 0.125 0.5 0.125 2 8 Enterococcus faecalis NCTC 12201 Vancomycin vanA 8 0.06 0.5 0.06 0.5 0.25 Enterococcus faecalis ATCC 51299 Vancomycin vanB 32 0.25 0.5 0.25 1 2 Streptococcus pneumoniae 18778 ERY, TET — 64 8 32 4 16 8 Streptococcus pneumoniae 21394 Penicillin PBP 64 4 32 4 8 8 Streptococcus pneumoniae 21395 ERY, CLIND MLSB 64 4 8 2 8 8 Haemophilus influenzae 18391 AMP/COA PBP/β-lactamase 32 8 >64 16 64 64 Haemophilus influenzae 17428 Ampicillin PBP 32 8 >64 16 >128 64
Homology Modelling and Compound Docking
[0167] Homology modelling of Smp_138030 (a protein lysine methyltransferase) and docking of compound 1 was performed as previously described Padalino G, Ferla S, Brancale A, Chalmers I W, Hoffmann K F. Int J Parasitol Drugs Drug Resist. 2018 Dec;8(3):559-570.
Effect of Drug Treatment on the Parasite Epitope
[0168]
RNAi-mediated Knockdown of smp_138030 Affects Worm Movement and the in vitro Production of Schistosome Eggs
[0169]
[0170] Panels D and E (
Activity Against Fasciola hepatica Newly Excysted Juveniles (NEJs)
[0171]
Identification of Putative Smp_138030 Homologs in Other Infectious Agents.
[0172] The protein sequence of the putative target of these compounds (Smp_138030) was used as queries for protein BLAST (BLASTp) searches against the predicted protein database derived from genomes of the different species hosted in NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi) and Wormbase-Parasite (VVormBase ParaSite—a comprehensive resource for helminth genomics. Howe K L, Bolt B J, Shafie M, Kersey P and Berriman, M. Mol Biochem Parasitol. 2017 Jul;215:2-10) using default settings except for S. aureus where a lower threshold value and BLOSUM45 as scoring matrix was used.
[0173] Table 5 shows the identification of homologous Smp_130830 proteins in Fasciola hepatica, S. japonicum, S. haematobium, P. berghei, Plasmodium falciparum, Eimeria tenella, Dirofilaria immitis, Haemonchus contortus, Echinococcus multilocularis and Staphylococcus aureus. The data in Table 5 shows a close similarity of the Smp_130830 protein sequence of Schistosoma mansoni with the protein lysine methyltransferase enzymes listed therein of F. hepatica, E. tenella, D. immitis, H. contortus, E. multilocularis and Plasmodium sp, and a reasonably close similarity with the prmA protein sequence of Staphylococcus aureus. This data suggests that the compounds of the present invention which are considered to be inhibitors of the Smp_138030 enzyme may also be effective in combatting F. hepatica, S. japonicum, S. haematobium, P. berghei, Plasmodium falciparum, E. tenella, D. immitis, H. contortus, E. multilocularis Plasmodium sp and S. aureus due to inhibition activity of the homologous proteins identified in Table 5.
TABLE-US-00005 TABLE 5 BLAST Closest homologue P E value *Plasmodium F. hepatica D915_13540 1.90E−176 S. japonicum Sjp_0076680 1.50E−156 S. haematobium MS3_03745 0.00E+00 P. berghei PBANKA_112850 5.00E−25 P. falciparum 3D7 PF3D7_0629700 3.00E−25 P. falciparum 7G8 Pf7G8_060034700 3.00E−25 P. falciparum CD01 PfCD01_060035100 3.00E−25 P. falciparum Dd2 PfDd2_060034400 3.00E−25 P. falciparum GA01 PfGA01_060034700 3.00E−25 P. falciparum GB4 PfGB4_060034200 3.00E−25 P. falciparum GN01 PfGN01_060035400 3.00E−25 P. falciparum HB3 PfHB3_060033900 3.00E−25 P. falciparum IT PfIT_060033500 3.00E−25 P. falciparum KE01 PfKE01_060035800 3.00E−25 P. falciparum KH01 PfKH01_060036700 3.00E−25 P. falciparum KH02 PfKH02_060036200 3.00E−25 P. falciparum ML01 PfML01_060033500 3.00E−25 P. falciparum SD01 PfSD01_060033600 3.00E−25 P. falciparum SN01 PfSN01_060034500 3.00E−25 P. falciparum TG01 PfTG01_060035000 3.00E−25 *S. aureus prmA 5.60E−03 *Eimeria tenella CDJ39082 5.00E−30 (Houghton Strain, PRJEB4863) Dirofilaria immitis nDi.2.2.2.t05252 1.90E−39 (PRJEB1797) Haemonchus HCON_00084150 1.10E−33 contortus (PRJEB506) Echinococcus EmuJ_000993000 3.20E−135 multilocularis (PRJEB122) Toxoplasma gondii KFG45293 3.00E−34 (PRJNA80767) *NCBI BlastP **WormBase Parasite BlastP
ADME Data
[0174] Table 6 shows ADME data for compounds 4, 9, 11, 12 and 18 compared to the known control compounds listed. This data was obtained using the methods described by Cyprotex (https://www.cyprotex.com/admepk), the CRO that conducted these assays.
[0175] This data shows that most of the tested compounds bind strongly to plasma protein constituents. Furthermore, the metabolic stability (in mouse liver microsomes) of most of the tested compounds is classified as medium (between 9 and 48 μL/min/mg protein). Finally, most of the compounds are partially soluble (between 1 μM and 100μM)
TABLE-US-00006 TABLE 6 Semi thermo- Plasma Metabolic stability** dynamic solubility protein CL.sub.int Mean binding (μL/min/mg t.sub.1/2 solubility fu (± SD) protein) SE (min) (μM) SD Control 0.0727 ± 457 4.21 4.21 35.4 4.74 0.00131 (Diazepam) (Nicardipine) (Warfarin) 58.3 4.24 4.24 >100 NA (Diphenhydramine) (Warfarin) Com- 4 ND 9.18 1.11 151 12.7 2.85 pound 9 ND 16 2.66 86.9 6.95 1.59 11 ND 0.134 4.63 10300 32.8 6.51 12 0.0134* 10.8 3.93 128 4.09 1.02 18 ND 37.4 1.4 37.1 15.5 3.38 ND—could not be determined *Compound detected in one out of the three replicates NA—not applicable SE—Standard error SD—Standard deviation CL.sub.int—Intrinsic clearance **Species = Mouse, SubstrateConc = 1 μM fu—fraction of drug unbound in plasma
Mouse Pharmacokinetics
[0176] Compounds 1, 7, 12, 4, 9, 11 and 18 were formulated as follows: 0.25 mg/mL in 10% DMSO, 20% Cremophor EL, 70% water (clear solution). Non-compartmental mouse pharmacokinetics (Male C57/BL6 Mouse, Fasted; 3 mice per group) were determined after oral administration of a nominal dose of 2.5 mg/kg of these formulations in cassettes (5 compounds/group) according to the protocols employed by WuXi (https://www.wuxiapptec.com; the CRO that conducted these studies).
[0177] The results are shown in Table 7 below.
TABLE-US-00007 TABLE 7 Compound PK parameters 1 7 12 4 9 11 18 C.sub.max (ng/mL) 22.57 21.25 20.10 90.43 79.27 68.83 16.45 T.sub.max (h) 4.00 4.00 4.00 3.33 4.00 4.00 4.00 T.sub.1/2 (h) ND ND ND ND ND 3.74 ND T.sub.last (h) 8.00 ND ND 8.00 8.00 ND ND AUC.sub.0-last (ng .Math. h/mL) 98.93 89.60 77.80 436.33 337.33 419.00 68.20 AUC.sub.0-inf (ng .Math. h/mL) ND ND ND ND ND 444.50 ND MRT.sub.0-last (h) 4.89 4.75 4.76 4.25 4.22 6.50 3.86 MRT.sub.0-inf (h) ND ND ND ND ND 7.84 ND AUC.sub.Extra (%) ND ND ND ND ND 1.82 ND AUMC.sub.Extra (%) ND ND ND ND ND 6.77 ND
[0178] The results in Table 7 show a similar plasma-profile for these compounds as the maximum blood concentration of each compound was observed about 3-4 hours (T.sub.max) post-dosing (e.g. a rapid onset of each compound in the blood stream). Most interestingly, mice dosed with a single dose of compounds 4, 9 and 11 presented a higher Cmax and AUC.sub.0-last compared to the other compounds (1, 7 and 12). Collectively all the compounds are effective for a few hours after dosing as the observed T.sub.last (when detectable) is 8 hours post-dosing.
Anti-Schistosomal in vivo Efficacy Screen
[0179]
[0180] These results indicate that compound 4 partially reduces worm and egg burdens in an experimental murine model of schistosomiasis.
[0181] In summary, the present invention provides compounds of formula (I), or pharmaceutically acceptable salts or solvates thereof, for use in the treatment of a patient infected with pathogenic organisms;
##STR00037##
[0182] wherein X and Y are each independently selected from C, N, O and S, provided at least one of X and Y is N, O or S; wherein R.sup.1 and R.sup.2 are each independently selected from an optionally substituted C.sub.1-C.sub.8 alkyl, C.sub.1-C.sub.8 alkenyl, aryl, heteroaryl or alkylaryl group; and wherein Z is selected from H, —CN, —NO.sub.2, —NR.sup.3R.sup.4, —NR.sup.5(CO)R.sup.6; C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 alkoxy, —(CO)WR.sup.12, hydroxy, amino, thiol, chloro, fluoro, CF.sub.3, CHF.sub.2 or CH.sub.2F groups. The compounds may be effective in treating patients infected with parasites selected from schistosome, Fasciola or Plasmodium parasites. Additionally or alternatively, the compounds of formula (I) may be effective in treating patients infected with pathogenic bacteria selected from S. aureus and MRSA.
[0183] The following definitions apply to the terms as used throughout this specification, unless otherwise limited in specific instances.
[0184] 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. Furthermore, the term alkyl encompasses cyclic hydrocarbon groups, for example cyclopropyl, cylcobutyl, cyclopentyl, cyclohexyl groups etc.
[0185] As used herein, the term “aryl” means a carbocyclic aromatic system containing one, two or three rings wherein such rings may be attached together in a pendent manner or may be fused. The term “aryl” embraces aromatic radicals such as benzyl, phenyl, naphthyl, anthracenyl, phenanthryl, indanyl, indenyl, annulenyl, azulenyl, tetrahydronaphthyl, and biphenyl.
[0186] As used herein, the term “heteroaryl” as used herein refers to 3 to 7 membered, preferably 5 to 7 membered, unsaturated heteromonocyclic rings, or fused polycyclic rings in which at least one of the fused rings is unsaturated, wherein at least one atom is selected from the group consisting of O, S, and N. The term also embraces fused polycyclic groups wherein heterocyclic radicals are fused with aryl radicals, wherein heteroaryl radicals are fused with other heteroaryl radicals, or wherein heteroaryl radicals are fused with cycloalkyl radicals. Examples of heteroaryl groups include pyrrolyl, pyrrolinyl, imidazolyl, pyrazolyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazolyl, pyranyl, furyl, thienyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, thiadiazolyl, isothiazolyl, indolyl, isoindolyl, indolizinyl, benzimidazolyl, quinolyl, isoquinolyl, quinoxalinyl, quinazolinyl, indazolyl, benzotriazolyl, benzodioxolyl, benzopyranyl, benzoxazolyl, benzoxadiazolyl, benzothiazolyl, benzothiadiazolyl, benzofuryl, benzothienyl, chromonyl, coumarinyl, benzopyranyl, tetrahydroquinolinyl, tetrazolopyridazinyl, tetrahydroisoquinolinyl, thienopyridinyl, furopyridinyl, pyrrolopyridinyl and the like. Exemplary tricyclic heterocyclic groups include carbazolyl, benzidolyl, phenanthrolinyl, dibenzofuranyl, acridinyl, phenanthridinyl, xanthenyl and the like.
[0187] As used herein, the term “cycloalkyl” means a cyclic saturated hydrocarbon group. Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.
[0188] As used herein, the term “halogen” or “halo” means fluorine, chlorine, bromine or iodine. Fluorine and chlorine are particularly preferred.
[0189] “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/VVNey-VCH, 2002), or S. M. Berge, et al., “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 C.sub.1-C.sub.4 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.
[0190] 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.
[0191] “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.
[0192] “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.
[0193] Although a few preferred embodiments have been shown and described, it will be appreciated by those skilled in the art that various changes and modifications might be made without departing from the scope of the invention, as defined in the appended claims.
[0194] Throughout this specification, the term “comprising” or “comprises” means including the component(s) specified but not to the exclusion of the presence of other components. The term “consisting essentially of” or “consists essentially of” means including the components specified but excluding other components except for materials present as impurities, unavoidable materials present as a result of processes used to provide the components, and components added for a purpose other than achieving the technical effect of the invention. Typically, when referring to compositions, a composition consisting essentially of a set of components will comprise less than 5% by weight, typically less than 3% by weight, more typically less than 1% by weight of non-specified components.
[0195] The term “consisting of” or “consists of” means including the components specified but excluding addition of other components.
[0196] Whenever appropriate, depending upon the context, the use of the term “comprises” or “comprising” may also be taken to encompass or include the meaning “consists essentially of” 0or “consisting essentially of”, and may also be taken to include the meaning “consists of” or “consisting of”.
[0197] For the avoidance of doubt, wherein amounts of components in a composition are described in wt %, this means the weight percentage of the specified component in relation to the whole composition referred to.
[0198] The optional features set out herein may be used either individually or in combination with each other where appropriate and particularly in the combinations as set out in the accompanying claims. The optional features for each aspect or exemplary embodiment of the invention as set out herein are also to be read as applicable to any other aspect or exemplary embodiments of the invention, where appropriate. In other words, the skilled person reading this specification should consider the optional features for each exemplary embodiment of the invention as interchangeable and combinable between different exemplary embodiments.
[0199] Attention is directed to all papers and documents which are filed concurrently with or previous to this specification in connection with this application and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference.
[0200] All of the features disclosed in this specification (including any accompanying claims, and drawings), and/or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive.
[0201] Each feature disclosed in this specification (including any accompanying claims, and drawings) may be replaced by alternative features serving the same, equivalent or similar purpose, unless expressly stated otherwise. Thus, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features.
[0202] The invention is not restricted to the details of the foregoing embodiment(s). The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.