Aryl-piperidine derivatives

Abstract

An aryl-piperidine derivative of formula I, wherein the meaning of R.sub.3, X, Cz, and Cy is that specified in the description, for use as inhibitors of T cells. ##STR00001##

Claims

1. A compound selected from: ##STR00242## ##STR00243## ##STR00244## ##STR00245## ##STR00246## ##STR00247## ##STR00248## ##STR00249## ##STR00250## ##STR00251## ##STR00252## ##STR00253## or a pharmaceutically acceptable salt thereof.

2. A pharmaceutical composition comprising the compound of claim 1, or the compound of formula: ##STR00254## or a compound selected from: ##STR00255## ##STR00256## ##STR00257## or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, adjuvant, or vehicle.

3. The compound of claim 1, wherein the compound is selected from: ##STR00258## ##STR00259## ##STR00260## ##STR00261## ##STR00262## ##STR00263## or a pharmaceutically acceptable salt thereof.

4. The compound of claim 1, wherein the compound is selected from: ##STR00264## ##STR00265## or a pharmaceutically acceptable salt thereof.

5. The compound of claim 1, wherein the compound is selected ##STR00266## ##STR00267## ##STR00268## ##STR00269## or a pharmaceutically acceptable salt thereof.

6. The compound of claim 1, wherein the compound is: ##STR00270## or a pharmaceutically acceptable salt thereof.

7. A compound selected from: ##STR00271## ##STR00272## ##STR00273## ##STR00274## or a pharmaceutically acceptable salt thereof.

8. The compound of claim 7, wherein the compound is: ##STR00275## or a pharmaceutically acceptable salt thereof.

9. The compound of claim 7, wherein the compound is selected from: ##STR00276## ##STR00277## ##STR00278## or a pharmaceutically acceptable salt thereof.

10. The compound of claim 7, wherein the compound is selected from: ##STR00279## or a pharmaceutically acceptable salt thereof.

11. A pharmaceutical composition comprising the compound of claim 7, or the compound of formula: ##STR00280## or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, adjuvant, or vehicle.

12. A compound selected from: ##STR00281## or a pharmaceutically acceptable salt thereof.

13. The compound of claim 12, wherein the compound is: ##STR00282## or a pharmaceutically acceptable salt thereof.

14. The compound of claim 12, wherein the compound is: ##STR00283## or a pharmaceutically acceptable salt thereof.

15. The pharmaceutical composition of claim 2, wherein the compound is: ##STR00284## or a pharmaceutically acceptable salt thereof.

16. A pharmaceutical composition comprising the compound of claim 12, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, adjuvant, or vehicle.

17. A method for treating an autoimmune disease, a lymphoma, or a T-cell leukemia in a patient, comprising administering to the patient the compound of claim 1, or the compound of formula: ##STR00285## or a compound selected from: ##STR00286## ##STR00287## ##STR00288## or a pharmaceutically acceptable salt thereof.

18. A method for treating an autoimmune disease, a lymphoma, or a T-cell leukemia in a patient, comprising administering to the patient the compound of claim 7, or the compound of formula: ##STR00289## or a pharmaceutically acceptable salt thereof.

19. A method for treating an autoimmmune disease, a lymphoma, or a T-cell leukemia in a patient, comprising administering to the patient the compound of claim 12, or the compound of formula: ##STR00290## or a pharmaceutically acceptable salt thereof.

20. The method of claim 17, wherein the autoimmune disease is selected from the group consisting of: rheumatoid arthritis, vitiligo, autoimmune hepatitis, myasthenia gravis, ankylosing spondylitis, inflammatory bowel disease, Crohn's disease, ulcerative colitis, psoriatic arthritis, transplant rejection, psoriasis, type I diabetes, multiple sclerosis, systemic lupus erythematosus, asthma, uveitis and atopic dermatitis.

21. The method of claim 18, wherein the autoimmune disease is selected from the group consisting of: rheumatoid arthritis, vitiligo, autoimmune hepatitis, myasthenia gravis, ankylosing spondylitis, inflammatory bowel disease, Crohn's disease, ulcerative colitis, psoriatic arthritis, transplant rejection, psoriasis, type I diabetes, multiple sclerosis, systemic lupus erythematosus, asthma, uveitis and atopic dermatitis.

22. The method of claim 19, wherein the autoimmune disease is selected from the group consisting of: rheumatoid arthritis, vitiligo, autoimmune hepatitis, myasthenia gravis, ankylosing spondylitis, inflammatory bowel disease, Crohn's disease, ulcerative colitis, psoriatic arthritis, transplant rejection, psoriasis, type I diabetes, multiple sclerosis, systemic lupus erythematosus, asthma, uveitis and atopic dermatitis.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1 shows the percentage T cell inhibition (% Inh) of compounds IV-3, V-1, IV-2, IV-4, IV-5, IV-6, IV-7, VII-1-01, VII-1-02, I-1, IV-10, V-2, V-3, V-5, V-6 and V-9, at a concentration of 0.1 nm.

(2) FIG. 2 shows the percentage T cell inhibition (% Inh) of compounds IV-1, IV-9 and V-4, at a concentration of 1.0 nM.

(3) FIG. 3 shows a schematic diagram of the Pulse protocol.

EXAMPLES

Example 1. Synthesis of the Compounds of the Invention

(4) General: The starting reagents, solvents and products were acquired from commercial sources. The term “concentration” refers to vacuum evaporation using a Büchi rotary evaporator. When specified, the reaction products were purified by means of silica gel flash chromatography (40-63 microns) using the specified solvent system. The spectroscopic data were measured using a Varian Mercury 400 spectrometer. The melting points were measured using a Büchi 535 instrument. The HPLC-MS was performed using a Gilson instrument equipped with a Gilson 321 piston pump, a Gilson 864 vacuum degasser, a Gilson 189 injection module, a Gilson 1/1000 splitter, a Gilson 307 pump, a Gilson 170 detector, and a ThermoQuest Finnigan AQA detector.

(5) Synthesis of compounds: Subseries-1, Subseries-2, Subseries-3 and Subseries-4

(6) ##STR00240##

(7) Synthesis of Compound 2: EDC.HCl (3.76 g, 0.019 mol) and HOBT (2.0 g, 0.013 mol) were added to a solution of acid 1 (3.0 g, 0.013 mol), N, O-dimethylhydroxylamine hydrochloride (1.59 g, 0.016 mol) and DIPEA (6.8 ml, 0.039 mol) in DMF (15 ml) and stirred for 12 h at room temperature (r.t.). The reaction mixture was then diluted with ice-cold water (150 ml) and extracted with EtOAC (3×50 ml). The organic extracts were combined, washed with sat. NaHCO.sub.3 solution (50 ml, aqueous) followed by water (50 ml) and brine (50 ml) separated and then dried over anhydrous Na.sub.2SO.sub.4 and concentrated to dryness to afford amide 2 (3.0 g, crude) as a pale yellow oil.

(8) Synthesis of Compound 3: To a cooled solution of amide 2 (3.0 g, 0.011 mol) in dry THE (20 ml) under N.sub.2 at −78° C. was added 7.3 ml (0.022 mol) of 3.0 M solution of PhMgBr in THF over 30 min. Reaction temperature was gradually increased to room temperature and stirred at the same temperature for 2 h. After consumption of starting material, the reaction mixture was cooled to 0° C. and quenched by drop wise addition of sat. aq. NH.sub.4Cl (30 ml). The reaction mixture was then diluted with water (100 ml); extracted with EtOAc (3×75 ml) and the organic layers were separated. The combined organic extract was washed with by water (100 ml) and brine solution (50 ml) and dried over anhydrous Na.sub.2SO.sub.4 and concentrated to dryness to get a crude residue which was purified (Silica gel column chromatography) using EtOAC::Hexanes to afford compound 3 (2.1 g) as a pale yellow oil.

(9) Synthesis of Compound 4: To a cooled solution of compound 3 (2.1 g, 0.007 mol) in CH.sub.2Cl.sub.2(15 ml) was added 2.5 ml of 20% HCl in dioxane drop wise and stirred at room temperature for 4 h under N.sub.2 atmosphere. The reaction mixture was evaporated and the residue was washed with n-Pentane (50 ml) to afford a white solid which was dried under vacuum to get compound 4 (1.35 g, 90% yield).

(10) Synthesis of V-1: To a cooled solution of compound 4 (450 mg, 1.9 mmol) and DIPEA (0.69 ml, 3.9 mmol) in acetonitrile (10 ml) at 0° C. was added benzoyl chloride (0.23 ml, 1.9 mmol) and stirred at room temperature for 3 h. The reaction mixture was diluted with EtOAC (100 ml) and washed with sat. aq. NaHCO.sub.3 solution (25 ml) followed by water (30 ml). The organic layers were combined, dried over anhydrous Na.sub.2SO.sub.4 and concentrated to a crude compound which was purified (prep HPLC) to afford compound V-1 (151 mg, 25% yield) as a pale yellow oil.

(11) Remaining compounds from subseries 1 can be synthesize using above experimental procedure.

(12) TABLE-US-00006 R R.sub.1 Compound Name H 4-Methyl IV-1 H 4-fluoro IV-2 H H IV-3 2-pyridyl H IV-4 3 -pyridyl H IV-5 4-pyridyl H IV-6 2-pyrimidyl H IV-7 4-pyrimidyl H IV-8 5-pyrimidyl H IV-9 4-methoxy H IV-10 4-Chloro H IV-11 4-Methyl H IV-12 4-Cyano H IV-13 H 4-methoxy IV-14 H 4-Chloro IV-15 H 4-Cyano IV-16 2-methoxy H IV-17 2-Chloro H IV-18 3-methoxy H IV-19 3-Chloro H IV-20 3,4-Dimethoxy H IV-21 3,4-Dichloro H IV-22 H 2-methoxy IV-23 H 2-Chloro IV-24 H 3-methoxy IV-25 H 3-Chloro IV-26 H 3,4-Dimethoxy IV-27 H 3,4-Dichloro IV-28 4-methoxy 4-methoxy IV-29 4-Chloro 4-Chloro IV-30 4-methoxy 4-Chloro IV-31 4-Chloro 4-methoxy IV-32 2-Thiazole H IV-33 1-Methyl-3-pyrazole H IV-34

(13) Synthesis of Compound IV-1: To a cooled solution of compound 4 (200 mg, 0.8 mmol) and DIPEA (0.18 ml, 1.0 mmol) in acetonitrile (10 ml) at 0° C. was added 0.16 mg of 4-Methyl Benzyl bromide (0.8 mmol) and stirred at room temperature for 3 h. The reaction mixture was diluted with EtOAC (100 ml) and washed with sat. NaHCO.sub.3 solution (25 ml) followed by water (30 ml). The organic layer was separated and dried over anhydrous Na.sub.2SO.sub.4 and concentrated to yield a crude compound which was purified (Silica gel column chromatography) using EtOAC:Hexane to afford compound IV-1 (114 mg, 43% yield) as pale yellow oil.

(14) Remaining compounds from subseries 2 can be synthesize using above experimental procedure.

(15) TABLE-US-00007 R R.sub.2 Compound Name H H V-1 4-methoxy H V-2 H 4-methoxy V-3 4-Chloro H V-4 H 4-Chloro V-5 H 4-Methyl V-6 4-Methyl H V-7 4-Cyano H V-8 4-Methyl 4-Cyano V-9 H 4-fluoro V-10 2-methoxy H V-11 2-Chloro H V-12 3-methoxy H V-13 3-Chloro H V-14 3,4-Dimethoxy H V-15 3,4-Dichloro H V-16 H 2-methoxy V-17 H 2-Chloro V-18 H 3-methoxy V-19 H 3-Chloro V-20 H 3,4-Dimethoxy V-21 H 3,4-Dichloro V-22 4-methoxy 4-methoxy V-23 4-Chloro 4-Chloro V-24 4-methoxy 4-Chloro V-25 4-Chloro 4-methoxy V-26 2-pyridyl H V-27 3-pyridyl H V-28 4-pyridyl H V-29 2-pyrimidyl H V-30 4-pyrimidyl H V-31 5-pyrimidyl H V-32 2-Thiazole H V-33 1-Methyl-3-pyrazole H V-34

(16) Synthesis of VI-1-01 and VI-1-02: Sodium borohydride (0.08 g, 2.15 mmol) was added to a cooled solution of compound IV-3 (400 mg, 1.4 mmol) in MeOH (20 ml) at 0-5° C., and stirred at room temperature for 2 h. After this time, the reaction mixture was concentrated and quenched with aq. NH.sub.4Cl solution, extracted with EtOAC (2×100 ml) and washed with water (50 ml) followed by brine (25 ml). The organic layers were collected and dried over anhydrous Na.sub.2SO.sub.4 and concentrated. The crude compound VI-1 (400 mg) was purified by prep HIPLC to afford VI-1-01 (60 mg) and VI-1-02 (40 mg).

(17) Remaining compounds from subseries 3 can be synthesize using above experimental procedure.

(18) TABLE-US-00008 R R.sub.1 Compound Name H H VI-1-01 and VI-1-02 3 -methoxy H VI-2-01 and VI-2-02

(19) Synthesis of VII-1-01 and VII-1-02: Sodium borohydride (0.1 g, 2.5 mmol) was added to a cooled solution of compound V-1 (300 mg, 1.0 mmol) in MeOH (20 ml) at 0-5° C., and stirred at room temperature for 2 h. After this time, the reaction mixture was concentrated and quenched with aq. N.sub.4Cl solution, extracted with EtOAC (2×100 ml) and washed with water (50 ml) followed by brine (25 ml). The organic layers were collected and dried over anhydrous Na.sub.2SO.sub.4 and concentrated. The crude compound VII-1 (250 mg) was purified by prep HPLC to afford VII-1-01 (60 mg) and VII-1-02 (60 mg).

(20) Remaining compounds from subseries 4 can be synthesize using above experimental procedure.

(21) Subseries-4 Compounds

(22) TABLE-US-00009 R R.sub.2 Compound Name H H VII-1-01 and VII-1-02 4-methoxy H VII-2-01 and VII-2-02 4-Chloro H VII-3-01 and VII-3-02 H 4-Chloro VII-4-01 and VII-4-02 4-Methyl H VII-5-01 and VII-5-02 4-Cyano H VII-6-01 and VII-6-02 2-methoxy H VII-7-01 and VII-7-02 2-Chloro H VII-8-01 and VII-8-02 3-methoxy H VII-9-01 and VII-9-02 3-Chloro H VII-10-01 and VII-10-02 3,4-Dichloro H VII-11-01 and VII-11-02 H 2-Chloro VII-12-01 and VII-12-02 H 3-Chloro VII-13-01 and VII-13-02 H 3,4-Dichloro VII-14-01 and VII-14-02 4-methoxy 4-methoxy VII-15-01 and VII-15-02 4-Chloro 4-Chloro VII-16-01 and VII-16-02 1-Methyl-3-pyrazole H VII-17-01 and VII-17-02
Synthetic Route for Subseries-5 (Compound I-1)

(23) ##STR00241##

(24) Synthesis of Compound 5: EDC.HCl (376 mg, 1.9 mmol) and HOBT (200 mg, 1.3 mmol) were added to a solution of acid 1 (300 mg, 1.3 mmol), N,O-dimethylhydroxylamine hydrochloride (159 mg, 1.6 mmol) and DIPEA (0.45 ml, 2.6 mmol) in DMF (15 ml) and the contents stirred for 12 h at room temperature.

(25) The reaction mixture was diluted with ice-cold water (150 ml) and extracted with EtOAC (3×50 ml). The organic extracts were combined, washed with sat. NaHCO.sub.3 solution (50 ml) followed by water (50 ml) and brine (50 ml), dried over anhydrous Na.sub.2SO.sub.4 and concentrated to dryness to afford amide 5 (340 mg, crude) as a pale yellow oil.

(26) Synthesis of Compound 6: To a cooled solution of compound 5 (340 mg, 1.1 mmol) in CH.sub.2Cl.sub.2(10 ml) was added drop wise 20% HCl in dioxane (1 ml) and stirred at room temperature for 4 h under N.sub.2 atmosphere. The reaction mixture was the evaporated under reduced pressure and the residue obtained was washed with n-pentane (50 ml). The resultant oily substance was dried under vacuum to afford crude compound 6 (200 mg, 90% yield).

(27) Synthesis of Compound I-1: Benzoyl chloride (0.137 mg, 0.9 mmol) was added to a cooled solution of compound 6 (200 mg, 0.9 mmol) and DIPEA (0.35 ml, 2.0 mmol) in acetonitrile (10 ml) at 0° C., and stirred at room temperature for 3 h. The reaction mixture was then diluted with EtOAC (100 ml), washed with sat. NaHCO.sub.3 solution (25 ml) followed by water (30 ml). The organic layer was dried over anhydrous Na.sub.2SO.sub.4 and concentrated to get the crude compound which was purified (Silica gel column chromatography) using EtOAC: Hexane to afford I-1 (157 mg, 51% yield) as off-white solid.

Example 2. Activity

(28) The effect of compounds IV-3, V-1, IV-1, IV-2, IV-4, IV-5, IV-6, IV-7, IV-9, VII-1-01, VII-1-02, I-1, IV-10, V-2, V-4, V-3, V-5, V-6 and V-9, on the capacity of the TCR to induce T cells proliferation was evaluated on primary T cells obtained from the blood of healthy human donors (PBMCs; peripheral blood mononuclear cells). The volunteers' PBMCs were isolated by means of density gradient centrifugation of venous blood using Ficoll-Paque Plus. The purified cells (NWT; Nylon Wood T cells) were labelled with carboxyfluorescein succinimidyl ester (CFSE) in order to analyse the cell division capacity. The labelled cells were pre-incubated for 1 hour in the absence or in the presence of the compounds at a concentration of 0.1 and 1.0 nM. Subsequently, the TCR/CD3 complex was stimulated using immobilised OKT3 (10 μg/ml) on flat-bottom P96 plates from Costar. The cells were cultured in triplicate at a density of 0.2×10.sup.5 in 200 μl of full medium, and the proliferation was evaluated by means of flow cytometry after 5-7 days, by quantifying the fluorescence of the CFSE. As the cells divide, they dilute the amount of labelling incorporated into the daughter cells, which gives an idea of the degree of cell proliferation. The inhibitory capacity of the compounds tested at a concentration of 0.1 and 1.0 nM, is shown in FIGS. 1 and 2, respectively.

Example 3. CYP Inhibition

(29) The effect of compounds IV-6, IV-7, and V-4 on Cytochromes P450 (CYP) inhibition was evaluated.

(30) For each isozyme, microsomes-buffer-substrate mixture (MBS mix) is prepared by premixing appropriate volumes of buffer, microsomes and substrate. MBS mixture (179 μL) is transferred to a 96-well reaction plate. An aliquot (1 μL) is spiked from corresponding wells of Test Item stock solution plate to reaction plate. The reaction plate is pre-incubated for 5 minutes at 37° C. Reaction is initiated by the addition of 20 μL of NADPH solution. Each experiment is performed in duplicate. Reaction plate is incubated for predetermined time at 37° C. and quenched using either 200 μL of acetonitrile (for CYP2C9, CYP2D6, CYP2C19 and CYP3A4) or 200 μL of a mixture of 70:30 1% formic acid:acetonitrile (for CYP1A2). The protein concentration, incubation time, substrate concentrations and metabolite monitored for each CYP is given in the table below:

(31) TABLE-US-00010 Experimental conditions Isozyme Protein (mg/mL) Metabolite CYP1A2 0.15 Acetaminophen CYP2C9 0.15 4-hydroxy diclofenac CYP2C19 0.25 4′-hydroxy mephenytoin CYP2D6 0.15 Dextrorphan CYP3A4 0.10 1-hydroxy midazolam 0.15 6-β-hydroxy testosterone

(32) Certain CYP inhibition data is shown in Table 5, below. Class A: IC50≤5 uM; Class B: 5 uM<IC50≤20 uM; Class C: 20 uM<IC50≤50 uM; Class D: IC50 >50 uM.

(33) TABLE-US-00011 TABLE 5 CYP Inhibition Compound CYP IC50 uM # CYP1A2 CYP2C9 CYP2C19 CYP2D6 CYP3A4.sup.a CYP3A4.sup.b IV-6 C B A A A B IV-7 D D D B D D V-4 A B A A D C

Example 4. Microsomal Stability

(34) The microsomal stability of compounds IV-6, IV-7, and V-4 was evaluated.

(35) Intrinsic clearance studies are performed individually with mouse, rat and human liver microsomes at 0.5 mg/mL protein concentration. Briefly, for protein concentration 0.5 mg/mL assay, liver microsomal protein (12.5 uL), NAPDH (50 uL) and phosphate buffer (435 uL) are co-incubated (pre-incubation) in a 96-well deep well plate in an orbital incubator (10 min, 37° C.). Reactions are initiated by the addition of 2.5 uL of test item working stock solution (100 uM). Aliquots (50 uL) are withdrawn from the reaction tube at 0, 5, 10, 20 and 30 minutes and the reaction is immediately terminated by transferring to a 96 deep well plate containing 50 uL of acetonitrile. Verapamil is used as a positive control in mouse, rat and human liver microsomes. To the quenched samples internal standard is added and vortex mixed followed by centrifugation at 4000 rpm for 10 minutes and an aliquot of supernatant is taken for LC-MS/MS analysis. Samples are analyzed by a suitable fit-for-purpose multiple reaction monitoring method developed on LC-MS/MS using and API 4000 mass spectrometer to estimate the area ratio (analyte peak area/internal standard peak area).

(36) Certain microsomal stability data is shown in Table 6, below. Class A: T1/2≥30 minutes; Class B: 10 minutes ≤TI/2<30 minutes; Class C: TI/2<10 minutes.

(37) TABLE-US-00012 TABLE 6 Microsomal Stability Microsomal stability (T1/2 min) Compound # Mouse Rat Human IV-6 C C B IV-7 A B A V-4 C C B

Example 5. hERG Inhibition

(38) The effect of compounds IV-6, IV-7, and V-4 on hERG inhibition was evaluated.

(39) HEK cells stably transfected with the hERG clone are maintained at 37±2° C. in a 5% CO2 incubator. The cells are initially revived and grown in DMEM/F12+GlutaMAX-I medium supplemented with 9% fetal bovine serum (FBS) and antibiotics (Penicillin 100 IU/mL Streptomycin 100 μg/mL) (complete medium). Furthermore, the cells are continuously maintained and passaged in complete media in addition to the appropriate concentration of selection antibiotic (Geneticin (G418) 200 μg/mL) (Selection media). The cells are sub cultured every 2-3 days and medium is changed with complete medium at least a day before the experiment.

(40) The cells are harvested on the day of experiment, dislodged and suspended in sterile filtered external solution (NaCl 140 mM, KCl 4 mM, MgCl2 1 mM, CaCl2 2 mM, D-Glucose monohydrate 5 mM, Hepes/NaOH 10 mM, pH 7.4, Osmolarity:298+/−5 mOsmol).

(41) The NPC®-1 chip of the Port-a-Patch® is filled with 5 μL of internal solution (KCl 50 mM, NaCl 10 mM, KFl 60 mM, EGTA 20 mM, Hepes/KOH 10 mM, pH 7.2, Osmolarity: 285+/−5 mOsmol) and it is screwed onto the chip holder. The Faraday cage is then fixed on the chip holder, such that the external electrode is placed near to the chip aperture. 5 μL of external solution is added to the center of the aperture so that the external electrode is also in contact with the solution.

(42) The experiment is be initiated. Once the set threshold of resistance attained (i.e., 2-3.5 MOhm), 5 μL of cell suspension is added in the middle of the external solution droplet after the Suction Control unit has generated the suction pulse. The suction automatically attracts a cell to the aperture, resulting in an increase in the chip resistance. When the cell is captured and the set threshold for the resistance is reached, the software recognizes this increase in resistance and proceeds to the next step of the sealing procedure. 20 μL of seal enhancer solution (NaCl 80 mM, KCl 4 mM, MgCl2 10 mM, CaCl.sub.2) 35 mM, Hepes (Na+salt)/HCl pH 7.4, Osmolarity: 298+/−5 mOsmol) is added 2-3 times. When the threshold resistance is reached, Patch Control automatically moves to the step of improving the seal. When the desirable resistance (Rpip) is reached, whole cell mode is attained and maintained.

(43) Finally, hERG protocol is selected to run the experiment. The seal enhancer solution is replaced with 20 μL external solution after 2-4 rinses and then the test item is added (from lower to higher concentration) and checked for current inhibition. All the experiments are performed at ambient temperature (22-25° C.).

(44) A schematic diagram (FIG. 3) of the Pulse protocol: Cells are stimulated every 10 seconds using the following protocol. The holding potential, sweep intervals and the depolarization/repolarization potentials are furnished in the table below.

(45) TABLE-US-00013 Protocol Voltage (mili-volts) Duration (mili-seconds) Holding potential −80 51 Depolarization +40 500 Repolarization −40 500 Holding potential −80 200

(46) Once the stable current is attained (at least 2-5 minutes) vehicle control is added and the current measured till the stable current is achieved. 3-5 consecutive data points from the last stable current phase are considered for further analysis. The test concentrations are analyzed from the lower to the higher concentrations (1, 3, 10 and 30 μM) in duplicates till stable current is achieved along with positive control. The system performs on-line analysis for several parameters which include functions that acquire data from traces and functions that perform calculations on the results of other functions.

(47) Certain hERG inhibition data is shown in Table 7, below.

(48) TABLE-US-00014 TABLE 7 hERG Inhibition Concentration Percent Inhibition Compound # (μM) Trial I Trial II Mean (%) Effect of IV-6 on IKr current IV-6 1 0.00 4.74 2.37 3 0.00 0.00 0.00 10 7.16 13.14 10.15 30 39.88 31.05 35.47 Propafenone 10 80.53 N/A 80.53 IC50 = 44.57 μM Effect of IV-7 on IKr current IV-7 1 0.00 0.00 0.00 3 1.46 0.00 0.73 10 3.44 0.54 1.99 30 17.25 25.87 21.56 Propafenone 10 70.20 70.83 70.52 IC 50 = 73.23 μM Effect of V-4 on IKr current V-4 1 16.19 10.20 13.20 3 20.54 17.01 18.78 10 26.38 19.75 23.07 30 39.46 38.69 39.08 Propafenone 10 68.16 69.79 68.98 IC 50 = 76.03 μM

(49) While we have described a number of embodiments of this invention, it is apparent that our basic examples may be altered to provide other embodiments that utilize the compounds and methods of this invention. Therefore, it will be appreciated that the scope of this invention is to be defined by the appended claims rather than by the specific embodiments that have been represented by way of example