Piperazine derivatives for treating disorders

Abstract

Anti-angiogenic treatments, treatments of hyperpermeability disorders, treatments of neuropathic and neurodegenerative disorders, pain treatments, methods of reducing the risk of pre-eclampsia and compounds for use in such methods are described.

Claims

1. A method of treating pain, comprising administering to a subject in need thereof a compound of Formula (I) ##STR00110## or a pharmaceutically acceptable salt thereof; wherein: n=1, 2, 3 or 0; R.sub.1=a 4- to 8-membered carbocyclic group, which may have one or more substituent; a 4- to 8-membered heterocyclic group comprising one oxygen atom, which may have one or more substituent; a 4- to 8-membered heterocyclic group comprising one nitrogen atom, which may have one or more substituent, a 4- to 8-membered heterocyclic group comprising one nitrogen atom and one oxygen atom which may have one or more substituent; a 4- to 8-membered heterocyclic group comprising two nitrogen atoms which may have one or more substituent; a 4- to 8-membered heterocyclic group comprising three nitrogen atoms which may have one or more substituent, or a condensed aromatic heterocyclic group, which may have one or more substituent; XCH, NH or N; YCH, NH or N; ZO, S, N or NH; and R.sub.2H, a C.sub.1-6 alkyl group; a phenyl group; a 4- to 8-membered heterocyclic group; or a condensed aromatic heterocyclic group, each of which may have one or more substituent.

2. A method according to claim 1, wherein n=1, 2, 3 or 0; R.sub.1=a 4- to 8-membered carbocyclic group, which may have one or more substituent; a 4- to 8-membered heterocyclic group having one nitrogen atom, which may have one or more substituent, a 4- to 8-membered heterocyclic group comprising one nitrogen atom and one oxygen atom which may have one or more substituent, or a 4- to 8-membered heterocyclic group comprising two nitrogen atoms which may have one or more substituent; XCH or N; YCH or N; ZO, S or NH; and R.sub.2=a phenyl group; a 4- to 8-membered heterocyclic group or a condensed aromatic heterocyclic group.

3. A method according to claim 1, wherein R.sub.1 represents a 4- to 8- membered heterocyclic group comprising one nitrogen atom, which may have one or more substituent.

4. A method according to claim 1, wherein R.sub.1 represents a 6-membered heteroaromatic group comprising one nitrogen atom, which may have one or more substituent.

5. A method according to claim 1, wherein R.sub.1 represents a 2-, 3- or 4-pyridyl group, each of which may have one or more substituent.

6. A method according to claim 1, wherein R.sub.1 represents a pyrimidinyl group, which may have one or more substituent.

7. A method according to claim 1, wherein R.sub.1 represents a 4- to 8-membered carbocyclic group, which may have one or more substituent.

8. A method according to claim 1, wherein R.sub.1 represents a phenyl group, which may have one or more substituent.

9. A method according to claim 1, wherein R.sub.2 represents a nitrogen- or oxygen-containing 4- to 8-membered heteroaromatic ring, which may have one or more substituent.

10. A method according to claim 1, wherein R.sub.2 represents a nitrogen-containing 4- to 8-membered heteroaromatic ring, which may have one or more substituent.

11. A method according to claim 1, wherein R.sub.2 represents a 2- or 3- or 4-pyridyl group, each of which may have one or more substituent.

12. A method according to claim 1, wherein R.sub.2 represents an oxygen-containing 4- to 8- membered heterocyclic ring, which may have one or more substituent.

13. A method according to claim 1, wherein R.sub.2 represents a 4- to 8-membered carbocyclic group, which may have one or more substituent.

14. A method according to claim 1, wherein R.sub.2 represents a phenyl group, which may have one or more substituent.

15. A method according to claim 1, wherein R.sub.2 represents H, or C.sub.1-6 alkyl which may have one or more substituent.

16. A method according to claim 1, wherein R.sub.2 represents a methyl group, which may have one or more substituent.

17. A method according to claim 1, wherein R.sub.2 represents a tetrahydropyranyl group, which may have one or more substituent.

18. A method according to claim 1, wherein: XYCH and ZO; XYCH and ZS; XYN and ZO; or XN, YCH, and ZO.

19. A method according to claim 1, wherein n=1 or 2.

20. A method according to claim 1, wherein the pain comprises non-inflammatory neuropathic or nociceptive pain.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) Embodiments of the present invention will now be described, purely by way of example, and with reference to the accompanying drawings, in which:

(2) FIG. 1 shows the activity of compounds 12 to 14 (termed SPHINX31, SPHINX32 and SPHINX33 respectively) of Formula (I) against SRPK1;

(3) FIG. 2 shows the effects of SPHINX31 relative to reference compounds SPHINX and SPHINX7 on SRSF1 phosphorylation in SRPK1 de-repressed (DDS) cells;

(4) FIG. 3 shows the dose response curve for SPHINX31 relative to reference compounds SPHINX and SPHINX7 in DDS cells;

(5) FIGS. 4 shows the results from the laser-induced mouse CNV model, where (A) and (B) show the effects of SPHINX31 in a laser-induced mouse CNV model relative to reference compounds SRPIN340 and SPHINX7; (C) shows the dose response curve for SPHINX31 in the laser-induced mouse CNV model; and (D) shows images of fluorescein angiography showing representative lesion sizes in the laser-induced mouse CNV model after treatment with SPHINX31 or reference compound SPHINX7;

(6) FIG. 5 shows the hERG inhibition profile of SPHINX31;

(7) FIG. 6 shows the TREEspot results of a kinome screen of SPHINX31 against all known kinases using the DiscoverX KINOMEscan binding affinity assay;

(8) FIG. 7 shows the equivalent binding affinity calculated using differential scanning fluorimetry;

(9) FIG. 8 shows that HCl salts of compounds of the invention salts dose-dependently inhibit SRPK1 activity in vitro and as efficiently as unconjugated SPHINX compounds;

(10) FIG. 9 shows that SPHINX31 also inhibits SRSF1 phosphorylation in PC-3 cells (at 1 M SPHINX31);

(11) FIGS. 10a shows that SPHINX31 blocks nuclear localisation of SRSF1 in PC-3 cancer cells;

(12) FIG. 10b shows that SPHINX31 blocks nuclear localisation of SRSF1 in MDA-MB-231 cancer cells;

(13) FIG. 11 shows that SPHINX compounds dose dependently increase expression of anti-angiogenic VEGF.sub.165b in Denys Drash (DDS) podocytes and normal podocytes;

(14) FIG. 12 shows that SPHINX compounds increase expression of anti-angiogenic VEGF.sub.165b in MCF7 breast cancer cells (left hand chart) and MDA-MB-231 breast cancer cells (right hand chart);

(15) FIG. 13 shows that SPHINX compounds dose dependently increase expression of anti-angiogenic VEGF.sub.165b RPE cells;

(16) FIG. 14 shows the results of testing compounds of the invention, where (A) shows a permeability chamber used to test whether compounds of the invention can penetrate through the sclera. Pazaponib was used as a control; (B) shows the concentration of compounds in the lower chamber (vitreous) after 0, 4 or 24 hours in rabbit eye tissue; and (C) shows retinal concentration after 4 hrs as a percentage of applied concentration in rabbit eye tissue;

(17) FIGS. 14D-G show data for the compounds, where (D) shows concentration of compounds in sclera tissue after 24 hrs as a percentage of applied concentration in porcine eye tissue; (B) shows concentration of compounds in RPE/choroid tissue after 24 hrs as a percentage of applied concentration in porcine eye tissue; (F) shows concentration of compounds in retinal tissue after 24 hrs as a percentage of applied concentration in porcine eye tissue; and (G) shows concentration of compounds in the lower chamber (vitreous) after 24 hrs as a percentage of applied concentration in porcine eye tissue;

(18) FIG. 15 shows (A) substantial accumulation of compound in the retina of a mouse 24 hours after addition of SPHINX31; and (B) accumulation of SPHINX31 in other tissues;

(19) FIG. 16 shows greater retinal penetration for SPHINX31 relative to pazopanib in in vivo rabbit studies;

(20) FIG. 17 shows that SPHINX31 binds to melanin substantially less than pazopanib does;

(21) FIG. 18 shows the stability of compounds in human plasma relative to propantheline bromide;

(22) FIG. 19 shows that neither SPHINX31 nor its metabolite SPHINX46 induce genotoxicity in an Ames test;

(23) FIGS. 20A-D show Ganzfeld ERG recordings in mice taken 24 h after treatment with control or SPHINX31 at 2 g/mL by topical eye drop administration, following stimulation with increasing intensities of (A) green light; (B) UV light; (C) green light; (D) UV light;

(24) FIGS. 20E-F show ERG recordings in mice after treatment with control or SPHINX31 at 2 g/ml by topical eye drop administration, following stimulation with increasing intensities of (E) green light; (F) UV light;

(25) FIGS. 20G-J show ERG recordings in mice after treatment with control or SPHINX31 at 2 g/ml by topical eye drop administration, following stimulation with increasing intensities of (G) green light; (H) UV light; and (I) extraction efficiency in the retina; and (J) extraction efficiency in the choroid;

(26) FIG. 21 shows Ganzfeld ERG recordings in mice taken 24 h after treatment with control or SPHINX31 at 2 g/ml by topical eye drop administration, where the graphs show ERG amplitude for (A) A-wave; and (B) B-wave following stimulation with green light, UV light, or white light.

(27) FIG. 22a shows the effects of SPHINX compounds on RPE cells expressing genes with alternative splicing, which are not linked to SRPK1;

(28) FIG. 22b shows the effects of SPHINX compounds on RPE expressing genes with alternative splicing, which are linked to SRPK1; and

(29) FIG. 23 shows the effect of SPHINX compounds on MKNK2, an RPE expressed gene with SRPK1 dependent alternative splicing, where (A) shows the alternative splicing of the MKNK2 gene; and (B) and (C) show the effect of SPHINX compounds on this alternative splicing.

METHODS

(30) Synthetic Protocol

(31) The general synthetic protocol for compounds is shown in Scheme 1 below, with an exemplary synthesis for compound 12, also referred to herein as SPHINX31, shown in Scheme 2 below. These compounds can be synthesized in a variety of ways, but this is the shortest and most efficient. Variations of this protocol to synthesize other compounds described herein are within the wherewithal of the skilled person.

(32) ##STR00003##

(33) ##STR00004##

SPHINX31 Experimental

t-Butyl 4-(pyridin-2-ylmethyl)piperazine-1-carboxylate (3)

(34) ##STR00005##

(35) 2-(Chloromethyl)pyridine hydrochloride (2) (1.97 g, 10.58 mmol) was added as a solid in one portion to a suspension of 1-Bocpiperazine (1) (2.24 g, 13.67 mmol) and potassium carbonate (4.98 g, 36.02 mmol) in anhydrous DMF (12 mL) at room temperature. The suspension was stirred at room temperature for 16 hours then poured onto saturated aqueous sodium bicarbonate solution. The mixture was extracted with ethyl acetate (3). The organic extracts were combined and washed with water and brine, then dried (Na.sub.2SO.sub.4). The solvent was removed under reduced pressure and the crude product purified by flash chromatography on deactivated silica gel, eluting with 60% ethyl acetate/n-hexane, to afford the product (3) as a colourless gum (2.98 g, 98%), with all analytical material matching that reported in the literature (E. Carceller, M. Merlos, M. Giral, C. Almansa, J. Bartroli, J. Garcia-Rafanell J. Forn; J. Med. Chem., 1993, 36, 2984-2997). .sup.1H NMR (300 MHz; CDCl.sub.3) 1.44 (s, 9H), 2.42-2.45 (m, 4H), 3.43-3.46 (m, 4H), 3.65 (s, 2H), 7.14-7.18 (m, 1H), 7.38 (d, J=7.8 Hz, 1H), 7.61-7.67 (m, 1H), 8.55-8.57 (m, 1H).

1-(Pyridin-2-ylmethyl)piperazine (4)

(36) ##STR00006##

(37) Trifluoroacetic acid (21.5 mL, 280.96 mmol) was added dropwise to a solution of Boc-piperazine (3) (2.98 g, 10.76 mmol) in dichloromethane (21.5 mL) at 0 C. (ice). The solution was stirred at 0 C. for 10 min then the cold bath was removed and the solution was stirred at room temperature for 4 hours. The solution was neutralised to pH 9 using saturated aqueous sodium bicarbonate solution. The dichloromethane layer was removed and the remaining aqueous solution was extracted with dichloromethane (2). The organic extracts were combined and washed with saturated aqueous sodium bicarbonate solution, water and brine, then dried (Na.sub.2SO.sub.4). The solvent was removed under reduced pressure to afford the product (4) as a light yellow oil (1.91 g, 99%), which was of sufficient purity to use in the next step, with all analytical data matching that reported in the literature (E. Carceller, M. Merlos, M. Giral, C. Almansa, J. Bartroli, J. Garcia- Rafanell, J. Forn J. Med. Chem., 1993, 36, 2984-2997). .sup.1H NMR (300 MHz; CDCl.sub.3) 1.95 (s, 1H), 2.44-2.47 (m, 4H), 2.88-2.91 (m, 4H), 3.62 (s, 2H), 7.13 (dd, 7.6 and 1.2 Hz, 1H), 7.38 (d, J=7.8 Hz, 1H), 7.59-7.65 (m, 1H), 8.52-8.55 (m, 1H).

1-(2-Nitro-4-(trifluoromethyl)phenyl)-4-(pyridin-2-ylmethyl)piperazine (5)

(38) ##STR00007##

(39) A solution of piperazine (4) (7.01 g, 39.56 mmol), 4-chloro-3-nitrobenzotrifluoride (6) (6.1 mL, 41.51 mmol) and solid sodium bicarbonate (8.31 g, 98.91 mmo) in anhydrous THF (39.5 mL) was heated reflux for 16 hours. The solution was allowed to cool to room temperature and the reaction solution was filtered through a short pad of Celite, eluting with ethyl acetate. The solvent was removed under reduced pressure to afford the product (5) as an orange gum (10.72 g, 74%), which was of sufficient purity to use in the next step. On occasion when the crude product was impure it could be purified by flash chromatography on deactivated silica gel, eluting with 2% methanol/ethyl acetate to afford the product. .sup.1H NMR (500 MHz CDCl.sub.3) 2.67-2.69 (m, 4H), 320-3.22 (m, 4H), 3.73 (s, 2H), 7.15 (d, J=8.8 Hz, 1H), 7.17-7.20 (m, 1H), 7.39 (d, J=7.8 Hz, 1H), 7.63-7.68 (m, 2H), 8.03 (br s, 1H), 8.59 (d, J=4.8 Hz, 1H); .sup.13C NMR (75 MHz; CDCl.sub.3) 50.9, 52.9, 64.5, 120.6, 122.1 (q, J.sub.C-F=34.7 Hz), 122.4, 123.4 (q, J.sub.C-F=270.8 Hz), 123.5, 124.3 (q, J.sub.C-F=3.9 Hz), 130.2 (q, J.sub.C-F=3.5 Hz), 136.6, 140.6, 148.1, 149.6, 158.0; IR (NaCl, neat) 1625 cm.sup.1; HRMS (ESI-MS): m/z calcd for C.sub.17H.sub.17F.sub.3N.sub.4O.sub.2Na [M+Na].sup.+ 389.1201, found 389.1185.

2-(4-(Pyridin-2-ylmethyl)piperazin1-yl)-5-(trifluoromethyl)aniline (7)

(40) ##STR00008##

(41) Hydrazine hydrate (35.5 mL, 731.84 mmol) was added dropwise to a solution of piperazine (5) (10.72 g, 29.25 mmol), iron(III) chloride hexahydrate (1.59 g, 5.87 mmol) and charcoal (1.17 g) in methanol (290 mL) at room temperature. The solution was heated at reflux for 2 hours. The solution was allowed to cool to room temperature then filtered through a short pad of Celite, eluting with ethyl acetate. The solvent was removed under reduced pressure. The residue was diluted with water and extracted with ethyl acetate (3). The organic extracts were combined and dried (Na.sub.2SO.sub.4). The solvent was removed under reduced pressure to afford the product 7 as a white solid (9.35 g, 95%), which was of sufficient purity to use in the next step. Mp 126-1.27 C.; .sup.1H NMR (300 MHz; CDCl.sub.3) 2.67-2.69 (m, 4H), 2.97-3.00 (m, 4H), 3.74 (s, 2H), 4.07 (br s, 2H), 6.92-7.04 (m, 3H), 7.16-7.20 (m, 1H), 7.44 (d, J=7.8 Hz, 1H), 7.64-7.70 (m, 1H), 8.57-8.60 (m, 1H); .sup.13C NMR (75 MHz; CDCl.sub.3) 50.7, 54.0, 64.8, 111.6 (q, J=3.9 Hz), 115.5 (q, J=4.1 Hz), 119.7, 122.3, 123.4, 124.6 (q, J.sub.C-F=271.2 Hz), 126.5 (q, J.sub.C-F=32.2 Hz), 136.6, 141.7, 142.2, 149.5, 158.5; IR (NaCl, neat) 3187, 3283 cm.sup.1; HRMS (ESI-MS): m/z calcd for C.sub.17H.sub.20F.sub.3N.sub.4 [M+Na].sup.+ 337.1594.

Methyl 5-bromofuran-2-carboxylate (8)

(42) ##STR00009##

(43) Concentrated sulfuric acid (0.56 mL, 10.51 mmol) was added dropwise to a solution of carboxylic acid (9) (20.0 g, 0.105 mol) in methanol (1050 mL) at room temperature. The solution was heated at reflux for 17 hours. The solution was allowed to cool to room temperature and the methanol was removed under reduced pressure. The residue was diluted with water and the pH of the solution was adjusted to pH 9 using solid sodium bicarbonate. The mixture was extracted with ethyl acetate (3). The organic extracts were combined and washed with water and brine, then dried (Na.sub.2SO.sub.4). The solvent was removed under reduced pressure to afford the product 8 as a white solid (19.47 g, 91%), which was of sufficient purity to use in the next step, with all analytical data matching that reported in the literature (Y. Zhu, H. Yan, L. Lu, D. Liu, G. Rong, J. Mao J. Org. Chem., 2013, 78, 9898-9905). Mp 67-68 C.; .sup.1H NMR (300 MHz; CDCl.sub.3) 3.90 (s, 3H), 6.46 (d, J=3.5 Hz, 1H), 7.13 (d, J=3.5 Hz, 1H).

Methyl 5-(pyridin-4-yl)furan-2-carboxylate (10)

(44) ##STR00010##

(45) A flask was charged with ester 8 (2.17 g, 10.58 mmol), 4-pyridinylboronic acid (II) (1.00 g, 8.14 mmol), PdCl.sub.2(PPh.sub.3).sub.2 (0.29 g, 0.41 mmol), 2 M aqueous sodium carbonate solution (10.2 mL, 22.4 mmol) and 1,2-dimethoxyethane (81 mL). The flask was freeze-pump-thawed (3), backfilled with argon and heated at reflux for 17 hours. The solution was cooled to room temperature and the DME was removed under reduced pressure. The pH of the residue was adjusted to pH 1 using 2 M aqueous hydrochloric acid solution. The solution was extracted with dichloromethane (3). The dichloromethane extracts were discarded. The remaining aqueous solution was neutralised to pH 9 using solid sodium bicarbonate and extracted with ethyl acetate (3). The respective organic extracts were combined and washed with water and brine, then dried (Na.sub.2SO.sub.4). The solvent was removed under reduced pressure to afford the product 10 as a white solid (1.41 g, 85%), which was of sufficient purity to use in the next step, with all analytical data matching that reported in the literature (H. Y. Fu, H. Doucet, Eur. J. Org. Chem., 2011, 7163-7173). Mp 95-97 C.; .sup.1H NMR (400 MHz; CDCl.sub.3) 3.94 (s, 3H), 6.95 (d, J3.6 Hz, 1H), 7.27 (d, J=3.5 Hz, 1H), 7.62-7.64 (m, 2H), 8.66-8.68 (m, 2H).

N-(2-(4-(Pyridin-2-ylmethyl)piperazin-1-yl)-5-trifluoromethyl)phenyl)-5-(pyridin-4-yl)furan-2-carboxamide (SPHINX31) (12)

(46) ##STR00011##

(47) A 2M solution of trimethylaluminium in toluene (0.84 mL, 1.68 mmol) was added dropwise to a solution of aniline 7 (0.189 g, 0.56 mmol) in dichloromethane (1.1 mL) at room temperature. The solution was stirred at room temperature for 1 hour after which, a solution of ester 10 (0.114 g, 0.56 mmol) in dichloromethane (0.6 mL) was added dropwise at room temperature. The reaction solution was stirred at room temperature for an additional 16 hours. To quench the reaction saturated aqueous Rochelle's salt solution was added dropwise at room temperature and the solution allowed to stir at room temperature for a further 15 minutes. The mixture was diluted with saturated aqueous sodium bicarbonate solution and extracted with dichloromethane (3). The organic extracts were combined and washed with water and brine, then dried (Na.sub.2SO.sub.4). The solvent was removed under reduced pressure to afford the product as a white solid (0.19 g, 67%), which was of sufficient purity to use in the next step. On occasion when the crude product was impure it could be purified by flash chromatography on deactivated silica gel, eluting with 5% methanol/ethyl acetate to afford the product. Mp 157-159 C.; .sup.1H NMR (300 MHz, CDCl.sub.3) 2.85 (br, s, 4H), 3.04 (br s, 4H), 3.78 (s, 2H), 7.06 (d, J=3.7 Hz, 1H), 7.20 (m, 1H), 7.31-7.41 (m, 4H), 7.65-7.72 (m, 3H), 8.60 (d, J=4.5 Hz, 1H), 8.80-8.87 (m, 3H), 9.65 (br s, 1H); .sup.C NMR (100 MHz, CDCl.sub.3) 52.2, 54.5, 65.0, 111.3, 116.7 (q, J.sub.C-F=4.5 Hz), 117.7, 118.5, 121.1, 121.3 (q, J.sub.C-F=4.5 Hz), 122.5, 123.5, 124.1 (q, J.sub.C-F=272 Hz), 128.0 (q, J.sub.C-F=34 Hz), 133.5, 136.7, 148.6, 149.7, 150.8, 153.2, 155.7, 157.9; HRMS (ESI): Calcd for C.sub.27H.sub.24F.sub.3N.sub.5O.sub.2 (MH+) 508.19603, found 508.19315; IR: (neat) 1669, 3332 cm.sup.1

(48) Analytical data For all compounds is presented in Table 4.

(49) In Vitro Kinase Assay

(50) Candidate compounds were screened by the Kinase-Glo assay (Promega; Koresawa and Okabe, 2004), the results of which are shown in Table 1 and Table 2. A reaction buffer containing 9.6 mM MOPS pH7 and 0.2 nM EDTA Ph8 was added to 10 M SRSF1 RS peptide (NH.sub.2-RSPSSYGRSRSRSRSRSRSRSRSNSRSRSYOH (SEQ ID NO: 1)) and 0.1 g of purified SRPK1 kinase. Candidate compounds were serially diluted from 10 M-0.5 nM and added to the reaction mixture, wells with omitted SRPK1 kinase and omitted compounds were also added as controls. All wells contained one percent DMSO. One micromolar ATP was added, wells minus ATP were used as background controls. The plate was then incubated at 30 C. for 10 minutes. An equal volume of Kinase-Glo (Promega, 25 l) was added to each well and the plate read for luminescence using an Fluostar Optima (BMG Labtech).

(51) Inhibition of SRSF1 Phosphorylation.

(52) Denys Drash podocytes, also referred to as DDS cells (DDS=Denys Drash Syndrome), with a WTI mutant that fails to repress SRPK1 expression, were treated with increasing concentrations of SPHINX31, or reference compounds SPHINX7 or SPHINX.

(53) Both whole cell lysate (nuclear and cytoplasmic) protein extraction and nuclear protein extracts were used. The extracts were then immunoblotted using either mouse anti-SRPK1 (anti-SRPK1;BD 611072; 1:1000), rabbit anti-panVEGF (Santa Cruz A20 sc-152; 1:500), mouse anti-VEGF.sub.xxxb (MAB3045; R&D; 1:500), goat anti-SRSF1 (SC10255; 1:500), mouse anti-SRSF1 (AK96) (Santa Cruz SC-33562) or rabbit anti-GAPDH (Sigma G9545, 1:2000). For immunoprecipitation phospho-SRSF1 studies, cell lysates were incubated with mouse anti-SRSF1 (Santa Cruz SC-33562) or anti-Pan-phospho-SR antibody (Santa Cruz, SC-13509) and Protein G Dynabeads (Invitrogen). To detect phosphorylated SRSF1, the eluent was immunoblotted with either anti-SRSF1 or the anti-Pan-phospho-SR antibody (1:500).

(54) PC3 prostate cancer cells were treated with 10 nM EGF in the presence of either DMSO (Vehicle), compound 12 (SPHINX31) or reference compounds at 10 M for 1 hr. Cells were lysed, and subjected to immunoblotting as described above.

(55) Laser Lesion Induction Protocol

(56) Six to eight week-old C57/B6 mice (B & K Laboratories) and adult Norway-Brown rats (Harlan Laboratories) were anaesthetized with an intraperitoneal injection of a mixture of 50 mg/kg ketamine and 0.5 mg/kg medetomidine. The pupils were dilated with 2.5% phenylephrine hydrochloride and 1% tropicamide. Four photocoagulation lesions were delivered with a krypton red laser (Mice: 250 mW, 0.01 s, 75 m, Rats: 200 mW, 0.01 s, 75 m, IRIS Medical 810 nm Oculight Six laser) between the retinal vessels in a peripapillary distribution at a distance of 1-2 disc-diameters in each eye. Only laser lesions with a subretinal bubble at the time of treatment were included in the study. Immediately following laser photocoagulation the animals either received intravitreal injections in both eyes (day 0 and day 7), or given topical eye drops twice daily of reference compounds SRPIN340, SPHINX7 or SPHINX31 in one eye and control vehicle in the other eye. Animals were culled on either day 4 or day 14 and eyes were either unfixed for retinal dissection and protein extraction, or fixed and enucleated and choroids stained for isolectin-B4 and examined, or imaged by fluorescein angiography.

(57) During topical administration tests compounds were made up into a gel based drug delivery vehicle to aid duration of drug exposure to the eye (Doukas et al., 2008), 0.05% DMSO was used to dissolve the compound before it was added to control vehicle.

(58) hERG Inhibition

(59) Compounds were tested for inhibition of the human ether a go-go related gene (hERG) K.sup.+ channel using IonWorks patch clamp electrophysiology. 8-Point concentration-response curves were generated using 3-fold serial dilutions (Essen Biosciences).

(60) Differential Scanning fluorimetry was performed as described in Federov et al (2011).

(61) Isoform specific ELISA was performed as described in Varey et al (2008) and Carter et al (2014).

(62) Scleral permeability was measured using a modified Ussing chamber assembly in isotonic assay buffer (pH 7.4). Rabbit or porcine excised eye tissues were mounted in the chambers such that the episcleral side faced the donor chamber and the retinal side faced the receiver chamber. The chambers were filled with equal volumes of assay buffer, with (donor side) or without (receiver side) 1 g/ml compound. After 4 or 24 hours tissue was removed from the chamber and the receiver side (vitreous) sampled. Tissue was dissected into sclera, choroid/RPE, and retina and homogenised. A tracer (SPHINX7) was added, and tissue extracted by acetonitrile extraction as described in Gammons et al (2013), Compounds were then analysed by mass spectrometry as described in Gammons et al (2013).

(63) Rabbit Pharmacokinetic Study

(64) Rabbits were treated thrice daily for six days, with 50 g SPHINX31 in one eye and 50 g pazopanib as 200 l eye drops. Rabbits were killed 12 hours after the last eye drop, blood and liver taken, and the retina dissected from the choroid/sclera, incisions male, and laid out flat, and photographs taken. Both eye compartments were dissected into 17 different areas. All samples were weighted. Compound was extracted by reverse phase extraction from the retina and choroid/sclera samples and the liver and plasma as above, and amount determined by mass spectrometry in different areas of the eye, in the blood and in the liver. Amounts per gram of tissue were calculated for SPHINX31 and pazopanib for each sample, and averaged.

(65) Mouse Electroretinography Toxicity Test.

(66) Mice were treated for six days with 2 g per eye SPHINX31 as eye drops, and ERG carried out using a Micron IV Ganzfield ERG system as recommended by the manufacturers instructions.

(67) Melanin Binding Assay

(68) 10 g/ml SPHINX31 or pazopanib were incubated in 10 g/ml melanin for 1hr at 37 C. Solutions were then spun at 15 kg for 15 mins and supernatant collected and compounds extracted in acetonitrile. They were then subjected to mass spectometry for quantitation.

Results

(69) Identification of Novel SRPK1 Inhibitors

(70) To identify novel SRPK1 inhibitors, a range of inhibitors were screened in an in vitro kinase assay (Promega; Koresawa and Okabe, 2004). The previously identified SRPK inhibitors SPHINX and SPHINX7 were used as positive controls for the identification of novel candidates. Kinase assays showed that compounds 12 to 14 in Table 1 (termed SPHINX31-33 respectively) had a 10-20 fold increase in potency compared with the previously reported compounds, resulting in IC.sub.50 values of 3.2-17 nM (FIG. 1).

(71) A structure activity relationship study to identify the mechanism and potential for new compounds was undertaken, with new compounds generated with structures shown in Table 2. Additional activity was reached with these compounds down to sub nM potencies for compound 61. A kinome screen of SPHINX31 against all known kinases using substrate DiscoverX binding affinity assay demonstrated that the only other kineses that showed binding were the closely related Clk1 and Clk4, which showed 27% and 14% binding at 1 M (FIG. 6). Using differential scanning fluorimetry, we determined that SPHINX31 had a binding affinity 44 greater for SRPK1 (Tm 12.8 C.) than SRPK2 ((Tm 6.7 C.) or Clk1 (Tm 6.7 C.) and 88 fold greater than Clk4 (Tm 5.7 C.). Binding activity to Clk2, Clk3, PIM1, PIM2, DYRK1, DYRK2, PRPF4B and SRPK3 was negligible (Tm<3 C.) (FIG. 7). The salt forms of the compounds (SPHINX31, 32 and 33) were also potent inhibitors, and were more freely soluble in water (FIG. 8).

(72) To determine whether these compounds could inhibit SRPK1 activity in cells, Denys Drash podocytes with constitutively active SRPK1 (caused by a mutation in the SRPK1 repressor, WT1) were treated with increasing concentration of compound 12, termed SPHINX31. FIG. 2 shows that increasing amounts of SPHINX31 increase inhibition of SRSF1 phosphorylation, and FIG. 3 shows that SRSF1 phosphorylation was dose dependently inhibited by treatment with SPHINX31.

(73) This was repeated in prostate cancer cells (PC3), previously shown to be sensitive to SRPK1 inhibition and again SRSF1 phosphorylation was inhibited (FIG. 9). The effect on SRSF1 localisation (known to be a result of SRPK1 phosphorylation) was also measured by immunofluorescence in PC3 prostate cancer cells and MDA-MB231 (FIGS. 10a (PC3 cells) and 10b MDA-MB231)), SPHINX31 treatment inhibited cellular localisation in both cell types.

(74) The effect on downstream splicing activity was also investigated, with the data showing that the compounds dose dependently switched splicing from VEGF-A.sub.165a to VEGF-A.sub.165b in Denys Drash podocytes and normal podocytes (FIG. 11). Using, isoform specific ELISA, this was also shown to be the case in breast cancer cells (MCF7 and MDA-MB231) cells (FIG. 12). In RPE cells (FIG. 13a) which have been shown to be the primary source of VEGF in angiogenic eye disease), SPHINX31 showed a dose dependent increase in VEGF.sub.165b with an EC.sub.50 of 20 nM (FIG. 13).

(75) To determine whether SPHINX31 could get across the sclera of a larger animal, rabbit sclera was clamped between two chambers and SPHINX31 or pazopanib (a VEGFR2 TKI) were added to the sclera with saline added to the bottom chamber and compounds added to the top chamber. After 0, 4 or 24 hours, the fluid from the bottom chamber (vitreous) and the retinal tissue was isolated and compounds purified by acetonitrile extraction and HPLC. FIG. 14 shows that SPHINX31 could be detected at significant concentrations in both retina and vitreous at 4 hrs, whereas pazopanib could not. At 24 hours both were detected in the vitreous, but more SPHINX31 than pazopanib. We also determined whether SPHINX31 could cross porcine eyes. While pazopanib accumulated in the sclera and in the RPE/Choroid layer, it did not penetrate the retina. In contrast, SPHINX31 crossed into the retina and vitreous.

(76) We also investigated accumulation of compounds of Formula (I) in various tissues in mice following treatment with 10 l eye drop of 5 g/ml SPHINX31. Mice were killed after 30 min, 1 hr, 4 hrs, 8 hrs or 24 hrs. Eyes were removed, and eye tissues dissected. Samples were subjected to extraction with control tracer chemical added to correct for extraction efficiency and subjected to mass spectroscopy for determination of quantity of compound per mg of tissue. FIG. 15A shows accumulation of SPHINX31 in different tissues of the eye. FIG. 15B shows accumulation of SPHINX31 in other tissues. These results show substantial accumulation of compound in the retina 24 hours after addition of SPHINX31.

(77) To determine if SPHINX31 could access the retina of an animal with a large eye a rabbit was exposed to 150 g per day SPHINX31 or pazopanib (FIG. 16). After 6 days the animal was killed and the eyes harvested. Individual sections of sclera and retina from the rear half of the eye were then assayed for pazopanib or SPHINX31, Retinal penetration was seen for both pazopanib and SPHINX31, but the concentrations for SPHINX31 were 10 the IC.sub.50 for the compound whereas for pazopanib the concentration was similar to the IC.sub.50.

(78) We have previously shown that SRPK1 inhibition by SRPIN340 (IC.sub.50 1 M), or SPHINX (IC.sub.50 0.44 M) was anti-angiogenic in mouse models of choroidal neovascularisation, as eye drops with a maximum effect at 10 g/ml, as these compounds are relatively lipophilic and have high penetrance into the eye. We therefore tested the effect of SPHINX31 as an eyedrop in this same model. SPHINX31 exerted a dose dependent inhibition of choroidal neovascularisation, with greater efficacy at 2 g/ml, and an IC.sub.50 of 0.24 g/ml (FIG. 4).

(79) Concerns have been raised that compounds in the eye may be sequestered by melanin. We therefore measured the melanin binding of the compounds, and determined that SPHINX31 was substantially less bound than pazopanib to melanin. (FIG. 17). We also tested the half life of these compounds when exposed to human liver microsomes. This showed that the half lives of the compounds were as shown in Table 3 below.

(80) TABLE-US-00001 TABLE 3 Compound Number Half life (mm) 12 161.50 20 17.00 21 62.50 Verapamil 11.75

(81) However, the compounds were stable in plasma (FIG. 18) indicating that they would most likely be taken to the liver and broken down there.

(82) To test whether compounds of Formula (I) are safe to administer to patients we began a basic safety test of these compounds in vitro. A dose dependence of cytotoxicity to SPHINX7, SPHINX and SPHINX31 revealed that SPHINX31 had no effect, whereas reference compound SPHINX7 was toxic at doses greater than 10 M.

(83) We also tested whether these compounds could inhibit the human ether a go-go related gene (hERG) potassium channel using patch clamp electrophysiology. Novel drug candidates are typically screened for an ability to inhibit the hERG potassium channel, due to the established association between pharmacological blockade of hERG channels and drug-induced long QT syndrome and torsades de poinses arrhythmia (Hancox et al., 2008; Gintant, 2008). SPHINX did not inhibit hERG, as has previously been described (Gammons et al., 2013).

(84) Plasma level of all compounds of Formula (I) tested during topical local application to the eye were extremely low (below detection level of 1 pM) and consequently substantial hERG channel block in the heart is unlikely to occur during in vivo use of these compounds as eye drops. The finding that known compounds SRPIN340 and SPHINX do not inhibit hERG suggests that it is possible to have significant pharmacological actions against SRPK1 without substantial hERG activity, (Gammons et al, 2013). We therefore tested SPHINX31, which inhibited hERG with an IC.sub.50 of 0.3 M, 100-fold higher than its IC.sub.50 value against SRPK1 (3.2 nM) (FIG. 5).

(85) We also tested SPHINX31 and its metabolite (termed SPHINX46, in FIG. 19) in an Ames test of genotoxicity, and both compounds induced no genotoxicity (FIG. 19).

(86) To determine whether there was any indication of a toxic effect on nerve function normal mice were dosed with 2 g/ml SPHINX31 and electroretinography determined on a Phoenix Ganzfeld ERG system. Scotopic ERG recordings were taken in dark adapted animals following stimulation with increasing intensities of green (FIGS. 20A, 20C, 20E, 20G) light (to activate M-cones and rods) or UV (FIGS. 20B, 20D, 20F, 20H) light (to activate S-cones and rods). The average ERG amplitude over time following stimulation with green light (FIG. 20A) and UV light (FIG. 20B) at 3.756 cd.s.m.sup.2. ERG amplitude at different intensities of green (FIGS. 20C, 20E) or UV (FIGS. 20D, 20F) are shown for the A wave (FIGS. 20C, 20D) and B wave (FIGS. 20E, 20F). The ratio of A-wave: B-wave was unaffected by SPHINX31 treatment (FIG. 20G, FIG. 20H). After ERG recordings, eyes were enucleated, dissected, homogenised and spiked with SPHINX7 to measure extraction efficiency, then analysed by mass spectrometry (FIG. 20I, FIG. 20J). SPHINX31 was detected in the retina (0.165% of applied eye drop dose when normalised to extraction efficiency) and choroid (0.0175% of applied dose). SPHINX31 levels in control eyes are shown for comparison as background levels. Photopic ERG recordings were taken to isolate cone responses from rod responses following 10 minutes light adaptation and with continuous background stimulation with white light at an intensity of 30 cd.m.s.sup.2. ERG amplitude for A-wave (A) and B-wave (B) at 120 and 1920 cd.s.m.sup.2 green light (to activate M-cones and rods), UV light (to activate S-cones and rods) or white light. No effect was seen on scotopic or photopic activity, indicating that no visual functional toxicity effects were seen (FIG. 20,21).

(87) Off target splicing effects were also screened by examining alternative splicing of a number of genes expressed in RPE cells and no changes in generic splicing were seen (FIG. 22), although VEGFR splicing was altered slightly. The known SRPK1 target MKNK2 was altered in these cell lines (FIG. 23).

(88) The data presented in this study shows novel small molecular weight compound inhibitors for reducing pro-angiogenic VEGF mediated CNV associated with AMD. Furthermore we have shown that the compounds of the present invention penetrate into the back of the eye in large animal models, are effective at reducing CNV following topical administration in mice, for reducing tumour cell growth and are safe on tests undertaken so far.

(89) TABLE-US-00002 TABLE 1 IC.sub.50 data for compounds of Formula (I) tested in the SRPK1 inhibition assay Compound Name Structure IC.sub.50 (nM) Reference Compound SPHINX 440 Reference Compound SPHINX7 54.7 12 SPHINX31 3.18 13 SPHINX32 17.19 14 SPHINX33 7.18

(90) Additional compounds tested are presented in Table 2 below:

(91) TABLE-US-00003 TABLE 2 IC.sub.50 data for compounds of Formula (I) tested in the SRPK1 inhibition assay Compound n R.sub.1 R.sub.2 X Y Z IC.sub.50 (nM) 15 1 CH CH O 16.6 16 1 0 CH CH O 38.2 17 1 CH CH O 110.7 18 1 CH CH O 13.9 19 1 CH CH O 14.1 20 1 CH CH O 4.4 21 1 0 CH CH O 4.6 22 1 CH CH O 14.8 23 1 CH CH O 4008.7 24 1 CH CH O 3507.5 25 1 CH CH O 13.5 26 1 0 N N O 10000 27 1 N CH O 10000 28 1 N N O 10000 29 1 N N O 10000 30 1 CH CH O 335.7 31 1 0 CH CH O 1.9 32 1 CH CH O 1547.0 33 1 CH N O 4027.2 34 1 O N N 5308.8 35 1 N O N 10000 36 1 0 CH CH O 170.0 37 1 CH CH O 323.0 38 1 CH CH O 5.0 39 1 CH CH O 2.6 40 1 H H CH CH S 4688 41 1 H NH CH N 493 42 1 CH CH O 164 43 1 0 CH CH O 183 44 1 NH N N 8413 45 1 CH CH O 119

(92) TABLE-US-00004 TABLE 4 Analytical data for synthesized compounds Compound 12 Mp: 157-159 C. .sup.1H NMR (300 MHz, CDCl.sub.3) 2.85 (br s, 4H), 3.04 (br s, 4H), 3.78 (s, 2H), 7.06 (d, J = 3.7 Hz, 1H), 7.20 (m, 1H), 7.31-7.41 (m, 4H), 7.65-7.72 (m, 3H), 8.60 (d, J = 4.5 Hz, 1H), 8.80-8.87 (m, 3H), 9.65 (br s, 1H) HRMS (ESI-MS): m/z calcd for C.sub.27H.sub.24F.sub.3N.sub.5O.sub.2 (M.sup.+ + H) 508.19603, found 508.19315 Compound 13 Mp: 166-168 C. .sup.1H NMR (400 MHz, CDCl.sub.3) 2.79 (br s, 4H), 3.04 (br s, 4H), 3.73 (s, 2H), 7.06 (d, J = 3.7 Hz, 1H), 7.23 (m, 1H), 7.31-7.41 (m, 4H), 7.62-7.69 (m, 3H), 8.54 (d, J = 4.5 Hz, 1H), 8.67-8.76 (m, 3H), 9.65 (br s, 1H) HRMS (ESI-MS): m/z calcd for C.sub.27H.sub.24F.sub.3N.sub.5O.sub.2 (M.sup.+ + H) 508.19603, found 508.19315 Compound 14 Mp: 188-190 C. .sup.1H NMR (400 MHz, CDCl.sub.3) 2.77 (br s, 4H), 3,04 (m, 4H), 3.61 (s, 2H), 7.07 (d, J = 3.7 Hz, 1H), 7.29 (m, 2H), 7.32-7.42 (m, 3H), 7.72 (m, 2H), 8.56 (m, 2H), 8.78 (m, 2H), 8.86 (d, J = 1.8 Hz, 1H), 9.63 (br s, 1H) HRMS (ESI-MS): m/z calcd for C.sub.27H.sub.24F.sub.3N.sub.5O.sub.2 (M.sup.+ + H) 508.19603, found 508.19423 Compound 15 .sup.1H NMR (300 MHz, CDCl.sub.3) 2.75 (br s, 4H), 2.97 (t, J = 4.6 Hz, 4H), 3.77 (s, 2H), 6.56 (dd, J = 3.5, 1.8 Hz, 1H), 7.15-7.23 (m, 3H), 7.28-7.32 (m, 1H), 7.44 (d, J = 7.8 Hz, 1H), 7.54 (dd, J = 1.8, 0.8 Hz, 1H), 7.63-7.69 (m, 1H), 8.55-8.57 (m, 1H), 8.78 (d, J = 1.8 Hz, 1H), 9.39 (br s, 1H). HRMS (ESI-MS): m/z calcd for C.sub.22H.sub.21F.sub.3N.sub.4O.sub.2 (M.sup.+ + H) 431.1695, found 431.1644 0 Compound 16 .sup.1H NMR (400 MHz, CDCl.sub.3) 2.43 (s, 3H), 2.78 (br s, 4H), 3.00 (t, J = 4.6 Hz, 4H), 3.77 (s, 2H), 6.18 (d, J = 3.4 Hz, 1H), 7.13 (d, J = 3.4 Hz, 1H), 7.19 (dd, J = 7.2, 5,0 Hz, 1H), 7.23 (d, J = 8.3 Hz, 1H), 7.30 (d, J = 8.3, 1.7 Hz, 1H), 7.46 (d, J = 7.8 Hz, 1H), 7.66-7.70 (m, 1H), 8.57-8.58 (m, 1H), 8.79 (d, J = 1.7 Hz, 1H), 9.42 (s, 1H) HRMS (ESI-MS): m/z calcd for C.sub.23H.sub.23F.sub.3N.sub.4O.sub.2 (M.sup.+ + H) 467.1671, found 467.1616 Compound 17 .sup.1H NMR (400 MHz, CDCl.sub.3) 2.84 (br s, 4H), 3.04 (t, J = 4.5 Hz, 4H), 3.77 (s, 2H), 6.83 (d, J = 3.6 Hz, 1H), 7.18 (ddd, J = 7.6, 4.9, 1.1 Hz, 1H), 7.30 (d, J = 8.3 Hz, 1H), 7.34-7.46 (m, 4H), 7.53-7.57 (m, 2H), 7.64-7.68 (m, 1H), 7.86-7.88 (m, 2H), 8.58-8.60 (m, 1H), 8.89 (d, J = 1.8 Hz, 1H), 9.63 (s, 1H) HRMS (ESI-MS): m/z calcd for C.sub.28H.sub.25F.sub.3N.sub.4O.sub.2 (M.sup.+ + H) 507.2008, found 507.1943 Compound 18 .sup.1H NMR (400 MHz, d6-DMSO) 2.78 (br s, 4H), 2.98 (br s, 4H), 3.71 (s, 2H), 6.63 (br s, 1H), 7.08 (d, J = 3.6 Hz, 1H), 7.24 (dd, J = 6.9, 5.5 Hz, 1H), 7.39 (d, J = 3.6 Hz, 1H), 7.42-7.51 (m, 5H), 7.59 (d, J = 8.5 Hz, 1H), 7.72-7.76 (m, 2H), 8.20 (s, 1H), 8.48 (br d, J = 4.5 Hz, 1H), 8.70 (s, 1H), 9.61 (s, 1H), 11.42 (s, 1H) HRMS (ESI-MS): m/z calcd for C.sub.30H.sub.27F.sub.3N.sub.5O.sub.2 (M.sup.+ + H) 546.2117, found 546.2073 . Compound 19 Mp: 96-99 C. .sup.1H NMR (300 MHz, CDCl.sub.3) 2.72 (br s, 4H), 2.98 (t, J = 4.8 Hz, 4H), 3.66 (s, 2H), 6.60 (dd, J = 3.5, 1.8 Hz, 1H), 7.22-7.40 (m, 8H), 7.54 (dd, J = 1.9, 0.8 Hz, 1H), 8.81 (d, J = 1.8 Hz), 9.43 (br s, 1H) HRMS (ESI-MS): m/z calcd for C.sub.23H.sub.22F.sub.3N.sub.3O.sub.2 (M.sup.+ + H) 430.17369, found 430.16879 Compound 20 Mp: 124-128 C. .sup.1H NMR (300 MHz, CDCl.sub.3) 2.44 (s, 3H), 2.72 (br s, 4H), 2.98 (t, J = 4.8 Hz, 4H), 3.63 (s, 2H), 6.20 (dd, J = 0.8, 3.5 Hz, 1H), 7.14 (d, J = 3.2 Hz, 1H), 7.21-7.26 (m, 1H), 7.28-7.38 (m, 7H), 8.81 (d, J = 1.8 Hz, 1H), 9.42 (br s, 1H) HRMS (ESI-MS): m/z calcd for C.sub.24H.sub.24F.sub.3N.sub.3O.sub.2 (M.sup.+ + H) 444.18934, found 444.18488 Compound 21 Mp: 62-64 C. .sup.1H NMR (300 MHz, CDCl.sub.3) 2.76 (br s, 4H), 3.01 (t, J = 4.8 Hz, 4H), 3.63 (s, 2H), 6.94 (d, J = 3.6 Hz, 1H), 7.27-7.40 (m, 8H), 7.47 (ddd, J = 8.2, 4.9, 0.8 Hz, 1H), 8.13 (dt, J = 8.0, 1.9 Hz, 1H), 8.70 (dd, J = 4.9, 1.6 Hz, 1H), 8.86 (d, J = 1.7 Hz, 1H), 9.20 (dd, J = 2.3, 0.7 Hz, 1H), 9.67 (br s, 1H) HRMS (ESI-MS): m/z calcd for C.sub.28H.sub.25F.sub.3N.sub.4O.sub.2 (M.sup.+ + H) 507.20024, found 507.2031 Compound 22 Mp: 178-180 C. .sup.1H NMR (300 MHz, CDCl.sub.3) 2.77 (br s, 4H), 3.02 (t, J = 4.7 Hz, 4H), 3.62 (s, 2H), 7.07 (d, J = 3.6 Hz, 1H), 7.31-7.42 (m, 8H), 7.74 (dd, J = 4.4, 1.7 Hz, 2H), 8.79 (dd, J = 4.4, 1.8 Hz, 2H), 8.87 (d, J = 1.6 Hz, 1H), 9.67 (br s, 1H) HRMS (ESI-MS): m/z calcd for C.sub.28H.sub.25F.sub.3N.sub.4O.sub.2 (M.sup.+ + H) 507.20024, found 507.19342 Compound 23 Mp: 150-154 C. .sup.1H NMR (300 MHz, CDCl.sub.3) 2.78 (br s, 4H), 3.01 (t, J = 4.6 Hz, 4H), 3.60 (s, 2H), 6.85 (d, J = 3.7 Hz, 1H), 7.27-7.39 (m, 8H), 7.43-7.49 (m, 1H), 7.51-7.58 (m, 2H), 7.90 (dd, J = 8.6, 1.6 Hz), 8.89 (d, J = 1.6 Hz), 9.65 (br s, 1H) HRMS (ESI-MS): m/z calcd for C.sub.29H.sub.26F.sub.3N.sub.3O.sub.2 (M.sup.+ + H) 506.20499, found 506.19922 Compound 24 Mp: 228-230 C. .sup.1H NMR (300 MHz, CDCl.sub.3) 2.80 (br s, 4H), 3.02 (t, J = 4.5 Hz, 4H), 3.60 (s, 2H), 6.71 (t, J = 2.1 Hz), 6.78 (d, J = 3.6 Hz), 7.19-7.40 (m, 9H), 7.54 (d, J = 8.4 Hz, 1H), 7.72 (dd, J = 8.6, 0.8 Hz, 1H), 8.21 (s, 1H), 8.38 (br s, 1H), 8.89 (d, J = 1.5 Hz, 1H), 9.62 (br s, 1H) HRMS (ESI-MS): m/z calcd for C.sub.31H.sub.27F.sub.3N.sub.4O.sub.2 (M.sup.+ + H) 545.21589, found 545.21100 Compound 25 .sup.1H NMR (400 MHz, CDCl.sub.3) 2.81 (br s, 4H), 3.02 (t, J = 4.5 Hz, 4H), 3.77 (s, 2H), 6.91 (d, J = 3.7 Hz, 1H), 7.16 (ddd, J = 7.6, 4.8, 1.0 Hz, 1H), 7.29 (d, J = 8.3 Hz, 1H), 7.34-7.36 (m, 2H), 7.41 (d, J = 8.0 Hz, 1H), 7.49 (dd, J = 8.1, 4.8 Hz, 1H), 7.61-7.67 (m, 1H), 8.10-8.13 (m, 1H), 8.56-8.58 (m, 1H), 8.66 (dd, J = 4.9, 1.6 Hz, 1H), 8.84 (d, J = 1.7 Hz, 1H), 9.14 (d, J = 2.2 Hz, 1H), 9.64 (s, 1H) HRMS (ESI-MS): m/z calcd for C.sub.27H.sub.24F.sub.3N.sub.4O.sub.2 (M.sup.+ + H) 508.1960, found 508.1902 0 Compound 26 .sup.1H NMR (400 MHz; CDCl.sub.3) 2.84 (br s, 4H), 3.03 (m, 4H), 3.80 (s, 2H), 7.18 (ddd, J = 7.4, 4.9, 0.9 Hz, 1H), 7.30 (d, J = 8.3 Hz, 1H), 7.41-7.44 (m, 2H), 7.54-7.64 (m, 3H), 7.67 (m, 1H), 8.18-8.21 (m, 2H), 8.60-8.61 (m, 1H), 8.75 (d, J = 1.8 Hz, 1H), 10.2 (br s, 1H) HRMS (ESI-MS): m/z calcd for C.sub.26H.sub.24F.sub.3N.sub.4O.sub.2 (M.sup.+ + H) 509.1913, found 509.1875 Compound 27 .sup.1H NMR (300 MHz; CDCl.sub.3) 2.44 (d, J = 1.1 Hz, 3H), 2.79 (br s, 4H), 2.99 (t, J = 4.8 Hz, 4H), 3.79 (s, 2H), 6.93 (q, J = 1.1 Hz, 1H), 7.18 (ddd, J = 7.6, 5.0, 1.1 Hz, 1H), 7.23 (d, J = 8.3 Hz, 1H), 7.35 (dd, J = 8.3, 2.1 Hz, 1H), 7.45 (d, J = 7.8 Hz, 1H), 7.67 (td, J = 7.7, 1.8 Hz, 1H), 8.56-8.59 (m, 1H), 8.73 (d, J = 1.8 Hz, 1H), 9.94 (br s, 1H) HRMS (ESI-MS): m/z calcd for C.sub.22H.sub.22F.sub.3N.sub.5O.sub.2Na (M.sup.+ + Na) 468.1623, found 468.1610 Compound 28 .sup.1H NMR (300 MHz; CDCl.sub.3) 2.67 (s, 3H), 2.79 (br s, 4H), 2.99 (t, J = 4.6 Hz, 4H), 3.76 (s, 2H), 7.16 (dd, J = 7.1, 5.3 Hz, 1H), 7.28 (d, J = 8.1 Hz, 1H), 7.38-7.41 (m, 2H), 7.54-7.64 (m, 3H), 7.65 (td, J = 7.7, 1.8 Hz, 1H), 8.18-8.21 (m, 2H), 8.57-8.59 (m, 1H), 8.68 (br s, 1H), 10.04 (br s, 1H) HRMS (ESI-MS): m/z calcd for C.sub.21H.sub.21F.sub.3N.sub.6O.sub.2Na (M.sup.+ + Na) 469.1576, found 469.1527 Compound 29 .sup.1H NMR (300 MHz; CDCl.sub.3) 2.83 (br s, 4H), 3.03 (t, J = 4.5 Hz, 4H), 3.79 (s, 2H), 7.18 (dd, J = 6.9, 5.1 Hz, 1H), 7.31 (d, J = 8.3 Hz, 1H), 7.41-7.45 (m, 2H), 7.67 (td, J = 7.6, 1.9 Hz, 1H), 8.02-8.04 (m, 2H), 8.59-8.60 (m, 1H), 8.73 (d, J = 1.4 Hz, 1H), 8.87-8.89 (m, 2H), 10.21 (br s, 1H) HRMS (ESI-MS): m/z calcd for C.sub.25H.sub.23F.sub.3N.sub.7O.sub.2 (M.sup.+ + H) 510.1865, found 510.1860 Compound 30 Mp: 165-167 C. .sup.1H NMR (300 MHz, CDCl.sub.3) 2.74 (br s, 4H), 3.00 (t, J = 4.4 Hz, 4H), 3.54 (s, 2H), 3.78 (s, 3H), 6.85 (d, J = 8.4 Hz, 2H), 7.05 (d, J = 3.6 Hz, 1H), 7.23 (d, J = 8.4 Hz, 2H), 7.30 (d, J = 8.1 Hz, 1H), 7.34-7.40 (m, 2H), 7.73 (d, J = 5.2 Hz, 2H), 8.78 (d, J = 5.2 Hz, 2H), 8.85 (s, 1H), 9.66 (br s, 1H) HRMS (ESI-MS): m/z calcd for C.sub.29H.sub.27F.sub.3N.sub.4O.sub.3 (M.sup.+ + H) 537.21080, found 537.21030 Compound 31 .sup.1H NMR (400 MHz, CDCl.sub.3) 2.43 (s, 3H), 2.56 (s, 3H), 2.79 (br s, 4H), 3.00 (t, J = 4.4 Hz, 4H), 3.68 (s, 2H), 6.19 (d, J = 3.0 Hz, 1H), 7.13 (d, J = 3.2 Hz, 1H), 7.21 (d, J = 5.0 Hz, 1H), 7.24 (d, J = 8.1 Hz, 1H), 7.32 (d, J = 8.1 Hz, 1H), 8.50 (d, J = 5.0 Hz, 1H), 8.80 (s, 1H), 9.37 (s, 1H) HRMS (ESI-MS): m/z calcd for C.sub.23H.sub.24F.sub.3N.sub.5O.sub.2S (M.sup.+ + Na) 514.1501, found 514.1456 Compound 32 Mp: 188-190 C. .sup.1H NMR (400 MHz, CDCl3) 2.76 (br s, 4H), 3.01 (t, J = 4.8 Hz, 4H), 3.59 (s, 2H), 3.80 (s, 3H), 6.81 (ddd, J = 8.3, 2.8, 0.8 Hz, 1H), 6.89-6.92 (m, 2H), 7.06 (d, J = 3.7 Hz, 1H), 7.22 (d, J = 8.0 Hz, 1H), 7.32 (d, J = 8.0 Hz, 1H), 7.37-7.40 (m, 2H), 7.73 (dd, J = 4.4, 1.6 Hz, 2H), 8.78 (dd, J = 4.4, 1.6 Hz, 2H), 8.86 (d, J = 1.8 Hz, 1H), 9.66 (br s, 1H) ) HRMS (ESI-MS): m/z calcd for C.sub.29H.sub.27F.sub.3N.sub.4O.sub.3 (M.sup.+ + H) 537.21080, found 537.20560 Compound 33 .sup.1H NMR (300 MHz; CDCl.sub.3) 2.84 (br s, 4H), 3.04 (t, J = 4.3, 4H), 3.79 (s, 2H), 7.19 (dd, J = 6.7, 5.0 Hz, 1H), 7.31 (d, J = 8.3 Hz, 1H), 7.38 (dd, J = 8.5, 1.5 Hz, 1H), 7.42 (d, J = 7.8 Hz, 1H), 7.60-7.63 (m, 3H), 7.67 (td, J = 7.6, 1.8 Hz, 1H), 7.92 (s, 1H), 8.20-8.24 (m, 2H), 8.58-8.60 (m, 1H), 8.83 (d, J = 1.3 Hz, 1H), 9.57 (br s, 1H) HRMS (ESI-MS): m/z calcd for C.sub.27H.sub.24F.sub.3N.sub.5O.sub.2Na (M.sup.+ + Na) 530.1780, found 530.1705 Compound 34 .sup.1H NMR (300 MHz; CDCl.sub.3) 2.87 (br s, 4H), 3.06 (t, J = 4.7, 4H), 3.82 (s, 2H), 7.19 (ddd, J = 7.5, 5.0, 1.0 Hz, 1H), 7.32 (d, J = 8.4 Hz, 1H), 7.42-7.46 (m, 2H), 7.57-7.62 (m. 3H), 7.68 (td, J = 7.7, 1.8 Hz, 1H), 8.18-8.21 (m, 2H), 8.58-8.60 (m, 1H), 8.75 (d, J = 1.8 Hz, 1H), 10.34 (br s, 1H) HRMS (ESI-MS): m/z calcd for C.sub.26H.sub.23F.sub.3N.sub.6O.sub.2Na (M.sup.+ + Na) 531.1732, found 531.1677 Compound 35 .sup.1H NMR (300 MHz; CDCl.sub.3) 2.85 (br s, 4H), 3.03 (t, J = 4.7, 4H), 3.80 (s, 2H), 7.18 (ddd, J = 7.6, 4.9, 1.2 Hz, 1H), 7.28 (d, J = 8.3 Hz, 1H), 7.39 (d, J = 8.4, 2.0 Hz, 1H), 7.45 (d, J = 7.8 Hz, 1H), 7.59-7.71 (m, 4H), 8.23-8.27 (m, 2H), 8.56-8.59 (m, 1H), 8.82 (d, J = 1.8 Hz, 1H), 10.17 (br s, 1H) HRMS (ESI-MS): m/z calcd for C.sub.25H.sub.23F.sub.3N.sub.6O.sub.2Na (M.sup.+ + Na) 531.1732, found 531.1652 00 Compound 36 .sup.1H NMR (400 MHz, DMSO) 2.73 (br s, 4H) 2.99 (br s, 4H), 3.74 (s, 2H),. 3.96 (s, 3H), 7.27 (m, 1H), 7.38 (s, 1H), 7.46-7.52 (m, 6H), 7.78 (m, 1H), 8.34 (d, J = 5.2 Hz, 1H), 8.51 (d, J = 3.6 Hz, 1H), 8.60 (s, 1H), 9.74 (s, 1H). MS (ESI-MS): m/z calcd for C.sub.28H.sub.26F.sub.3N.sub.5O.sub.3 [M+].sup.+ 538.20, found 538.32. 01 Compound 37 .sup.1H NMR (400 MHz, DMSO) 2.74 (br s, 4H), 2.99 (br s, 4H), 3.75 (s, 2H), 3.98 (s, 3H), 7.05 (d, J = 8.8 Hz, 1H), 7.21 (d, J = 3.6 Hz, 1H), 7.27 (m, 1H), 7.43-7.54 (m, 4H), 7.78 (m, 1H), 8.26 (dd, J = 8.8, 2.4 Hz, 1H), 8.49 (d, J = 4.4 Hz, 1H), 8.61 (s, 1H), 8.87 (d, J = 2.4 Hz, 1H), 9.72 (s, 1H), MS (ESI-MS): m/z calcd for C.sub.28H.sub.26F.sub.3N.sub.5O.sub.3 [MH].sup.+ 538.20, found 538.30. 02 Compound 38 .sup.1H NMR (400 MHz, DMSO) 9.62 (s, 1H), 8.61 (s, 1H), 8.50 (d, J = 3.6 Hz, 1H), 7.88 (d, J = 8.4 Hz, 2H), 7.78 (m, 1H), 7.44- 7.58 (m, 6H), 7.25-7.30 (m, 2H), 5.42 (t, J = 5.2 Hz, 1H), 4.65 (d, J = 4.4 Hz, 2H), 3.70 (s, 2H), 3.01 (br s, 4H), 2.75 (br s, 4H). MS (ESI-MS): m/z calcd for C.sub.29H.sub.27F.sub.3N.sub.4O.sub.3 [MH].sup.+ 537.20, found 537.42. 03 Compound 39 .sup.1H NMR (400 MHz, DMSO) 9.50 (s, 1H), 8.63 (s, 1H), 8.52 (d, J = 4.0 Hz, 1H), 7.80 (m, 1H), 7.49 (m, 3H), 7.29 (m, 1H), 7.25 (d, J = 3.6 Hz, 1H), 6.47 (d, J = 3.2 Hz, 1H), 4.00 (d, J = 9.6 Hz, 2H), 3.69 (s, 2H), 3.53 (t, J = 10.8 Hz, 2H), 3.10 (m, 1H), 2.96 (br s, 4H), 2.71 (br s, 4H), 2.00 (d, J = 11.2 Hz, 2H), 1.67-1.83 (m, 2H). MS (ESI-MS): m/z calcd for C.sub.27H.sub.29F.sub.3N.sub.4O.sub.3 [MH].sup.+ 515.22, found 515.42 04 Compound 40 .sup.1H NMR (300 MHz; CDCl.sub.3) 2.41 (s, 3H), 2.66 (br s, 4H), 2.97 (t, J = 4.7 Hz, 4H), 7.17 (dd, J = 5.0, 3.8 Hz, 1H), 7.27 (d, J = 8.3 Hz, 1H), 7.33 (dd, J = 8.3, 1.8 Hz, 1H), 7.57 (dd, J = 5.0, 1.1 Hz, 1H), 7.67 (dd, J = 3.8, 1.1 Hz, 1H), 8.81 (d, J = 1.8 Hz, 1H), 9.18 (s, 1H) HRMS (ESI-MS): m/z calcd for C.sub.17H.sub.19F.sub.3N.sub.3OS (M.sup.+ + H) 370.1201, found 370.1192 05 Compound 41 .sup.1H NMR (300 MHz; d6-DMSO) 2.34 (s, 3H), 2.69 (br s, 4H), 2.95 (m, 4H), 7.26-7.31 (m, 1H), 7.38-7.44 (m, 4H), 7.96-8.00 (m, 3H), 8.72 (br s, 1H), 10.33 (br s, 1H) HRMS (ESI-MS): m/z calcd for C.sub.22H.sub.23F.sub.3N.sub.5O (M.sup.+ + H) 430.1855, found 430.1832 06 Compound 42 .sup.1H NMR (300 MHz; CDCl.sub.3) 2.82 (br s, 4H), 2.97 (m, 4H), 3.82 (s, 2H), 7.06 (d, J = 3.7 Hz, 1H), 7.08-7.13 (m, 2H), 7.17- 7.23 (m, 1H), 7.30 (d, J = 8.2 Hz, 1H), 7.35-7.40 (m, 3H), 7.72 (d, J = 8.0 Hz, 1H), 7.75-7.77 (m, 2H), 8.30 (s, 1H), 8.78-8.80 (m, 2H), 8.86 (d, J = 1.8 Hz, 1H), 9.68 (s, 1H). HRMS (ESI-MS): m/z calcd for C.sub.30H.sub.27F.sub.3N.sub.5O.sub.2 (M.sup.+ + H) 546.2117, found 546.2101 07 Compound 43 .sup.1H NMR (400 MHz, DMSO) 2.71 (br s, 4H), 2.97 (br s, 4H), 3.81 (s, 2H), 6.78-6.77 (dd, J = 3.6, 1.6, Hz, 1H), 7.35 (d, J = 3.2 Hz, 1H), 7.50-7.45 (m, 2H), 8.01 (d, J = 1.2 Hz, 1H), 8.51 (s, 1H), 8.58 (s, 1H), 8.63 (s, 1H), 8.75 (s, 1H), 9.44 (s, 1H),. MS (ESI-MS): m/z calcd for C.sub.21H.sub.20F.sub.3N.sub.5O.sub.2 [MH].sup.+ 431.16, found 432.37. 08 Compound 44 .sup.1H NMR (400 MHz, DMSO) 2.68 (br s, 4H). 2.97 (br s, 4H), 3.71 (s, 2H), 7.30-7.27 (m, 1H), 7.49-7.51 (m, 3H), 7.80 (td, J = 7.6, 2.0 Hz, 1H), 8.52 (d, J = 4.4 Hz, 1H), 8.70 (s, 1H), 8.74 (s, 1H), 10.07 (s, 1H), 14.90 (s, 1H), MS (ESI-MS): m/z calcd for C.sub.20H.sub.20F.sub.3N.sub.5O [MH].sup.+ 432.17, found 432.37. 09 Compound 45 .sup.1H NMR (400 MHz, DMSO) 2.69 (br s, 4H), 2.93 (br s, 4H), 3.17 (m, 1H), 4.54 (d, J = 4.8 Hz, 2H), 5.62-5.70 (m, 1H), 6.57 (d, J = 3.2 Hz, 1H), 7.24-7.31 (m, 2H), 7.45-7.50 (m, 3H), 7.78- 7.79 (m, 1H), 8.50 (s, 2H), 9.47 (s, 1H). MS (ESI-MS): Calcd for C.sub.23H.sub.23F.sub.3N.sub.4O.sub.3 (MH+) 461.17, found 461.36.

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