Tertiary amines for use in the treatment of cardiac disorders

Abstract

The present invention relates to tertiary amines of formula (I) for use in therapy, particularly for use in treating cardiovascular disorders. The compounds have been found to regulate phospholamban phosphorylation by interfering with the A-kinase anchor protein 18delta (AKAP18) binding to the PKA substrate phospholamban. The compounds share a tri(alkylaryl/alkylheteroaryl) amine structure.

Claims

1. A compound of formula (I) or a pharmaceutically acceptable salt thereof: ##STR00252## wherein L.sup.1, L.sup.2 and L.sup.3 independently denote C.sub.1-C.sub.4-alkylene optionally substituted with one phenyl; Ar.sup.1 denotes phenyl substituted with one or more R; Ar.sup.2 denotes phenyl substituted with one or two R; Ar.sup.3 denotes phenyl; R denotes NR.sup.2R.sup.3; and R.sup.2 and R.sup.3 independently denote H or C.sub.1-C.sub.4-alkyl.

2. The compound of claim 1, wherein L.sup.1, L.sup.2 and L.sup.3 independently denote C.sub.1-C.sub.4-alkylene.

3. The compound of claim 1, wherein L.sup.1, L.sup.2 and L.sup.3 denote CH.sub.2.

4. The compound of claim 1, wherein Ar.sup.1 denotes phenyl substituted with one R.

5. The compound of claim 1, wherein Ar.sup.2 denotes phenyl substituted with one R.

6. The compound of claim 1, wherein R.sup.2 and R.sup.3 independently denote C.sub.1-C.sub.4-alkyl.

7. The compound of claim 1, wherein R.sup.2 and R.sup.3 denote methyl.

8. The compound of claim 1, wherein L.sup.1, L.sup.2 and L.sup.3 denote CH.sub.2, Ar.sup.1 denotes phenyl substituted with one R, Ar.sup.2 denotes phenyl substituted with one R; and R.sup.2 and R.sup.3 independently denote C.sub.1-C.sub.4-alkyl.

9. The compound of claim 1 having the following structure: ##STR00253## ##STR00254## ##STR00255##

10. The compound of claim 1 having the following structure: ##STR00256##

11. The compound of claim 1 having the following structure: ##STR00257##

Description

(1) The following Examples are given by way of illustration only in which the Figures referred to are as follows:

(2) FIG. 1: Set-up of GST-AKAP18: PLB-biotin interaction assay by AlphaScreen (A) and cross-titration of the preparations of GST-AKAP18 and biotinylated PLB (B)

(3) FIG. 2: Concentration--response curves of compound 2b in the AKAP18-PLB AlphaScreen assay. Compound concentration (x-axis) is logarithmic.

(4) FIG. 3: Concentration-response curves of compound 2g in the AKAP18-PLB AlphaScreen assay. Compound concentration (x-axis) is logarithmic.

(5) FIG. 4: Concentration-response curves of compound 3b in the AKAP18-PLB AlphaScreen assay. Compound concentration (x-axis) is logarithmic.

(6) FIG. 5: Concentration-response curves of compound 3c in the AKAP18-PLB AlphaScreen assay. Compound concentration (x-axis) is logarithmic.

(7) FIG. 6: Concentration-response curves of compound 3d in the AKAP18-PLB AlphaScreen assay. Compound concentration (x-axis) is logarithmic.

(8) FIG. 7: Disruption of the AKAP18-PLB complex influences PLB-Ser.sup.16 phosphorylation. Rat neonatal cardiomyocytes treated with compounds 24 hours prior stimulation with isoproterenol (100 nM, 5 minutes). The histogram shows levels of phosphorylated Ser.sup.16-PLB quantified by densiometery relative to actin levels.

(9) FIG. 8: Percent viability after 24 hours incubation with synthesized compounds.

(10) FIG. 9: Disruption of the AKAP18-PLB complex influences PLB-Ser.sup.16 phosphorylation. Rat neonatal cardiomyocytes treated with compound 2g (A) or 3d (C) 24 hours prior stimulation with isoproterenol (100 nM, 5 minutes). Rat adult cardiomyocytes treated with compound 2g (B) 1 hour prior to stimulation with isoproterenol (10 nM, 5 minutes). The histograms show levels of phosphorylated Ser.sup.16-PLB quantified by densitometry on Western blots (relative to actin levels).

(11) FIG. 10: Compound 2g regulates SERCA2-activity. A novel patch clamp based technique was performed to directly assess SERCA-activity independently of receptor stimulation in intact cardiomyocytes. By dialyzing cardiomyocytes with a fixed level of cAMP with and without presence of compound 2g, preliminary data indicates ability of compound 2g to regulate SERCA2-activity in intact adult cardiomyocytes.

EXAMPLES

(12) Binding Assay

(13) Stable and optimal assay conditions were determined by cross-titrating GST-AKAP18 and biotinylated PLB using 10 g/ml glutathione acceptor beads and 10 g/ml streptavidin donor beads in an AlphaScreen assay. The excitation wavelength was 680 nm, with the emission wavelength being 520-560 nm. Signal intensity in each well was registered and the optimal concentration to use was chosen to be the concentration prior to the peak of the signal. The set up of the assay and cross-titration results are shown in FIGS. 1A and 1B.

(14) Concentrations to give a reliable signal were determined to be between 2 and 16 nM for AKAP18-GST and 4 and 20 nM PLB-biotin for relevant preparations of protein, for example 4 nM and 20 nM, respectively.

Example 1, Screening

(15) A compound library consisting of 79,000 different small molecular compounds was screened with the above assay. The following compounds of formulae (l) were identified as having relatively low EC.sub.50 values (2.0 M-137 M).

(16) TABLE-US-00001 TABLE 1 Group of compounds of formulae (I) Structure EC.sub.50 41.6 M 137 M 10.6 M 18 M 21.1 M 0 36.4 M 39.5 M 60.5 M 94.3 M 98.3 M 25.6 M 35.4 M 29.6 M 3.8 M 215.9 M 0 7.5 M 9.3 M 3.6 M 2.9 M 22.2 M 2.5 M 5.2 M 2.0 M

SYNTHETIC EXAMPLES

Synthesis of Intermediate 1

Preparation of N-(4-fluorobenzyl)-1-(pyridin-4-yl)methanamine

(17) ##STR00178##

(18) To a round bottom flask with a magnetic stirrer bar 4-picolylamine (0.43 g, 4 mmol) and 4-fluorobenzaldehyde (0.63 g, 5 mmol) were added together with 10 m methanol, NaCNBH.sub.3 (0.32 g, 5 mmol) and a few granules of 4 molecular sieve. The solution was stirred overnight at room temperature and the progress of the reaction was monitored by thin layer chromatography (TLC). After filtration the solution was concentrated in vacuo and the resulting liquid was diluted with 30 ml dichloromethane and extracted with 330 ml 0.05 M HCl. The combined aqueous layer was made basic with NaOH until the pH 10-11 and the solution was extracted with 2100 ml dichloromethane. The organic layers were combined and dried over anhydrous MgSO.sub.4, filtered and evaporated to give N-(4-fluorobenzyl)-1-(pyridin-4-yl)methanamine as a pale yellow oil. Yield: 0.66 g (76%). .sup.1H NMR (300 MHz, CDCl.sub.3): 8.54 (2H, d), 7.30 (4H, m), 7.01 (2H, m), 3.81 (2H, s), 3.77 (2H, s).

Synthesis of Intermediate 2

Preparation of N-(4-(tert-butyl)benzyl)-1-(pyridin-3-yl)methanamine

(19) ##STR00179##

(20) To a round bottom flask with a magnetic stirrer bar 3-picolylamine (0.43 g, 4 mmol) and 4-tert-butylbenzaldehyde (0.81 g, 5 mmol) were added together with 10 ml methanol, NaCNBH.sub.3 (0.32 g, 5 mmol) and a few granules of 4 molecular sieve. The solution was stirred for 2-12 hours at room temperature and the progress of the reaction was monitored by thin layer chromatography (TLC). After filtration the solution was concentrated in vacuo and the resulting liquid was diluted with 30 ml dichloromethane and extracted with 330 ml 0.05 M HCl. The combined aqueous layer was made basic with NaOH until the pH 10-11 and the solution was extracted with 2100 ml dichloromethane. The organic layers were combined and dried over anhydrous MgSO.sub.4, filtered and evaporated to give N-(4-(tert-butyl)benzyl)-1-(pyridin-3-yl)methanamine as a pale yellow oil. Yield: 0.61 g (60%). .sup.1H NMR (300 MHz, CDCl.sub.3): 8.59 (1H, d), 8.53 (1H, d), 7.78 (1H, d), 7.39 (2H, d), 7.29 (3H, m), 3.86 (2H, s), 3.81 (2H, s), 1.33 (9H, s). .sup.13C NMR (75 MHz, CDCl.sub.3): 150.5, 150, 148.9, 136.2, 135.9, 134.8, 128.2, 125.6, 123.6, 52.5, 50.1, 34.6, 31.5.

Synthesis of Intermediate 3

Preparation of 2-(((pyridin-4-ylmethyl)amino)methyl)benzene-1,4-diol)

(21) ##STR00180##

(22) To a round bottom flask with a magnetic stirrer bar 4-picolylamine (0.43 g, 4 mmol) and 2,5-dihydroxybenzaldehyde (0.69 g, 5 mmol) were added together with 10 ml methanol, NaCNBH.sub.3 (0.32 g, 5 mmol) and a few granules of 4 molecular sieve. The solution was stirred for 2-12 hours at room temperature and the progress of the reaction was monitored by thin layer chromatography (TLC). After filtration the solution was concentrated in vacuo and the resulting liquid was diluted with 50 ml dichloromethane and extracted with 350 ml 0.05 M HCl. The combined aqueous layer was made basic with NaOH until the pH was 10 and the solution was extracted with 2150 ml dichloromethane. The organic layers were combined and dried over anhydrous MgSO.sub.4, filtered and evaporated to give 2-(((pyridin-4-ylmethyl)amino)methyl)-benzene-1,4-diol as a highly viscous brown oil. Yield: 0.26 g (28%). .sup.1H NMR (300 MHz, DMSO): 8.51 (2H, m), 7.34 (2H, d), 6.51 (2H, m). .sup.13C NMR (75 MHz, DMSO): 149.3, 149.2, 148.8, 148.5, 125, 122.7, 115.2, 113.7, 78.8, 50.4, 48.7.

Synthesis of Intermediate 4

Preparation of N,N-dimethyl-4-(((pyridin-4-ylmethyl)amino)methyl)aniline

(23) ##STR00181##

(24) To a round bottom flask with a magnetic stirrer bar 4-picolylamine (0.43 g, 4 mmol) and 4-(dimethylamino)benzaldehyde (1.34 g, 9 mmol) were added together with 10 ml methanol, NaCNBH.sub.3 (0.57 g, 9 mmol) and a few granules of 4 molecular sieve. The solution was stirred for 2-12 hours at room temperature and the progress of the reaction was monitored by thin layer chromatography (TLC). After filtration the solution was concentrated in vacuo and the resulting liquid was diluted with 50 ml dichloromethane and extracted with 350 ml 0.05 M HCl. The combined aqueous layer was made basic with NaOH until the pH was 10 and the solution was extracted with 2150 ml dichloromethane. The organic layers were combined and dried over anhydrous MgSO.sub.4, filtered and evaporated to give N,N-dimethyl-4-(((pyridin-4-ylmethyl)amino)methyl)aniline as a dark brown oil. Yield: 0.85 g (88%), .sup.1H NMR (300 MHz, CDCl.sub.3): 8.56 (2H, d), 7.25 (4H, m), 6.74 (2, d), 3.82 (2H, s), 3.73 (2H, s), 2.96 (6H, s). .sup.13C NMR (75 MHz, CDCl.sub.3): 150.1, 149.9, 149.7, 129.2, 128.7, 127.7, 123.2, 112.8, 52.8, 51.7, 40.8.

Synthesis of Intermediate 5

Preparation of methyl 4-((benzylamino)methyl)benzoate

(25) ##STR00182##

(26) To a round bottom flask with a magnetic stirrer bar benzylamine (0.215 g, 2 mmol) and methyl 3-formylbenzoate (0.41 g, 2.5 mmol) were added together with 10 ml methanol, NaCNBH.sub.3 (0.16 g, 2.5 mmol) and a few granules of 4 molecular sieve. The solution was stirred for 2-12 hours at room temperature and the progress of the reaction was monitored by thin layer chromatography (TLC). After filtration the solution was concentrated in vacuo and the resulting liquid was diluted with 50 ml dichloromethane and extracted with 350 ml 0.05 M HCl. The combined aqueous layer was made basic with NaOH until the pH 10-11 and the solution was extracted with 2150 ml dichloromethane. The organic layers were combined and dried over anhydrous MgSO.sub.4, filtered and evaporated to give methyl 4-((benzylamino)methyl)benzoate as a light brown oil. Yield: 0.37 g (36%). .sup.1H NMR (300 MHz. CDCl.sub.3): 8.00 (2H, m), 7.59 (1H, d), 7.36 (6H, m), 3.94 (3H, s), 3.87 (2H, s), 3.83 (2H, s) .sup.13C NMR (75 MHz, CDCl.sub.3): 167.3, 140.9, 140.3, 132.9, 130.4, 129.4, 128.6, 128.4, 128.3, 127.2, 53.4, 52.9, 52.2.

Synthesis of Intermediate 6

Preparation of 4-((benzylamino)methyl)-N,N-dimethylaniline

(27) ##STR00183##

(28) To a round bottom flask with a magnetic stirrer bar benzylamine (1.07 g, 10 mmol) and 4-(dimethylamino)benzaldehyde (1.79 g, 12 mmol) were added together with 10 ml methanol, NaCNBH.sub.3 (0.76 g, 12 mmol) and a few granules of 4 molecular sieve. The solution was stirred for 3 hours at room temperature and the progress of the reaction was monitored by thin layer chromatography (TLC). After filtration the mixture was concentrated in vacuo and 300 mg of the crude product was purified by flash chromatography using ethyl acetate/CH.sub.2Cl.sub.2 (1:9) giving 4-((benzylamino)methyl)-N,N-dimethylaniline as a yellow/brown oil. .sup.1H NMR (300 MHz, CDCl.sub.3): 7.34 (5H, m), 7.19 (2H, d), 6.68 (2H, d), 3.81 (2H, s), 3.75 (2H, s), 2.92 (6H, s).

Synthesis of Intermediate 7

Preparation of N-(4-methoxybenzyl)-1-(pyridin-4-yl)methanamine

(29) ##STR00184##

(30) To a round bottom flask with a magnetic stirrer bar, 4-picolylamine (0.43 g, 4 mmol) and 4-methoxybenzaldehyde (1.22 g, 9 mmol) were added with 10 ml methanol, NaCNBH.sub.3 (0.57 g, 9 mmol) and a few granules of 4 molecular sieve. The solution was stirred for 2-12 hours at room temperature and the progress of the reaction was monitored by thin layer chromatography (TLC). After filtration the solution was concentrated in vacuo and to the resulting liquid was added 50 ml dichloromethane and extracted with 350 ml 0.05 M HCl. The combined aqueous layer was made basic with NaOH until the pH 10-11 and the solution was extracted with 2150 ml dichloromethane. The organic layers were combined and dried over anhydrous MgSO.sub.4, filtered and evaporated to give N-(4-methoxybenzyl)-1-(pyridin-4-yl)methanamine as a brown oil. Yield: 0.36 g (38%) .sup.1H NMR (300 MHz, CDCl.sub.3): 8.56 (2H, d), 7.28 (4H, m), 6.90 (2H, m), 3.82 (5H, s), 3.76 (2H, s), .sup.13C NMR (75 MHz, CDCl.sub.3): 158.9, 149.9, 149.6, 132, 129.4, 128.7, 123.1, 114, 65, 55.4, 52.7, 51.8.

Synthesis of Intermediate 8

Preparation of N-benzyl-2,2-diphenylethanamine

(31) ##STR00185##

(32) To a round bottom flask with a magnetic stirrer bar benzylamine (0.86 g, 8 mmol) and diphenylacetaldehyde (1.96 g, 10 mmol) were added together with 10 ml methanol, NaCNBH.sub.3 (0.64 g, 10 mmol) and a few granules of 4 molecular sieve. The solution was stirred for 2-12 hours at room temperature and the progress of the reaction was monitored by thin layer chromatography (TLC). After filtration the mixture was concentrated in vacuo and purified by flash chromatography using ethyl acetate/CH.sub.2Cl.sub.2 (1:9) giving N-benzyl-2,2-diphenylethanamine as a viscous yellow oil. Yield: 0.45 g (20%), .sup.1H NMR (300 MHz, CDCl.sub.3): 7.19 (15H, m), 4.16 (1H, m), 3.74 (2H, s), 3.18 (2H, m), .sup.13C NMR (75 MHz, CDCl.sub.3): 142.5, 139.7, 128.3, 128.1, 128, 127.8, 126.7, 126.3, 125.8, 53.5, 50.9.

Example 2

Preparation of N,N-bis(4-fluorobenzyl)-1-(pyridin-4-yl)methanamine

(33) ##STR00186##

(34) To a round bottom flask with a magnetic stirrer bar 4-picolylamine (0.43 g, 4 mmol) and 4-fluorobenzaldehyde (1.12 g, 9 mmol) were added together with 10 ml methanol, NaCNBH.sub.3 (0.57 g, 9 mmol) and a few granules of 4 molecular sieve. The solution was stirred for 4 hours at room temperature and the progress of the reaction was monitored by thin layer chromatography (TLC). After filtration the mixture was concentrated in vacuo and the resulting liquid was diluted with 50 ml dichloromethane and extracted with 350 ml 0.05 M HCl. The combined aqueous layer was made basic with NaOH until the pH was 10-11 and the solution was extracted with 2150 ml dichloromethane. The organic layers were combined and dried over anhydrous MgSO.sub.4, filtered and evaporated to give N,N-bis(4-fluorobenzyl)-1-(pyridin-4-yl)methanamine as a yellow oil. Yield: 0.49 g (38%) .sup.1H NMR (300 MHz, CDCl.sub.3): 8.53 (2H, d), 7.17 (10H, m), 3.76 (4H, d), 2.34 (2H, s), .sup.13C NMR (75 MHz, CDCl.sub.3): 163.4, 160.1, 149.4, 148.9, 135.1, 135, 129.4, 129.3, 128.3, 122.7, 115.1, 114.8, 64.1, 52.1, 51.4.

Example 3

Preparation of 2,2-(((pyridin-4-ylmethyl)azanediyl)bis(methylene))bis(benzene-1,4-diol)

(35) ##STR00187##

(36) To a round bottom flask with a magnetic stirrer bar 4-picolylamine (0.43 g, 4 mmol) and 2,5-dihydroxybenzaldehyde (1.24 g, 9 mmol) were added together with 10 ml methanol, NaCNBH.sub.3 (0.57 g, 9 mmol) and a few granules of 4 molecular sieve. The solution was stirred for 4 hours at room temperature and the progress of the reaction was monitored by thin layer chromatography (TLC). After filtration the mixture was concentrated in vacuo and the resulting liquid was diluted with 50 ml dichloromethane and extracted with 350 ml 0.05 M HCl. The combined aqueous layer was made basic with NaOH until the pH was 10-11 and the solution was extracted with 2150 ml dichloromethane. The organic layers were combined and dried over anhydrous MgSO.sub.4, filtered and evaporated to give 2,2-(((pyridin-4-ylmethyl)azanediyl)bis (methylene))bis(benzene-1,4-diol) as highly viscous dark brown oil. Yield: 0.88 g (62%), .sup.1H NMR (300 MHz, DMSO): 8.5 (5H, m), 7.35 (4H, m), 6.55 (8H, m), 3.69 (3H, d), 3.16 (4H, s), .sup.13C NMR (75 MHz, DMSO): 150.6, 150.4, 150, 149.3, 126.1, 124.8, 124.7, 123.9, 116.6, 116.5, 116.3, 114.8, 60, 51.5, 49.8, 49.5.

Example 4

Preparation of N,N-bis(4-fluorobenzyl)-1-(thiazol-2-yl)methanamine

(37) ##STR00188##

(38) To a round bottom flask with a magnetic stirrer bar 2-(aminomethyl)thiazole (0.46 g, 4 mmol) and 4-fluorobenzaldehyde (1.12 g, 9 mmol) were added together with 10 ml methanol, NaCNBH.sub.3 (0.57 g, 9 mmol) and a few granules of 4 molecular sieve. The solution was stirred overnight at room temperature and the progress of the reaction was monitored by thin layer chromatography (TLC). After filtration the mixture was concentrated in vacuo and the resulting liquid was diluted with 50 ml dichloromethane and extracted with 350 ml 0.05 M HCl. The combined aqueous layer was made basic with NaOH until the pH was 10-11 and the solution was extracted with 2150 ml dichloromethane. The organic layers were combined and dried over anhydrous MgSO.sub.4, filtered and evaporated to give N,N-bis(4-fluorobenzyl)-1-(thiazol-2-yl)methanamine as a dark brown oil. Yield: 0.91 g (69%), .sup.1H NMR (300 MHz, CDCl.sub.3): 7.73 (1H, d), 7.32 (5H, m), 7.04 (4H, m), 4.67 (2H, s), 4.14 (2H, s), 3.86 (2H, s), .sup.13C NMR (75 MHz, CDCl.sub.3): 171.7, 164, 163.8, 160.8, 160.6, 142.7, 135.5, 135.4, 129.9, 129.8, 128.8, 119, 115.6, 115.5, 115.3, 115.2, 64.6, 52.5, 50.2.

Example 5

Preparation of N,N-bis(4-methoxybenzyl)-1-(thiazol-2-yl)methanamine

(39) ##STR00189##

(40) To a round bottom flask with a magnetic stirrer bar 2-(aminomethyl)thiazole (0.46 g, 4 mmol) and 4-methoxybenzaldehyde (1.22 g, 9 mmol) were added together with 10 ml methanol, NaCNBH.sub.3 (0.57 g, 9 mmol) and a few granules of 4 molecular sieve. The solution was stirred overnight at room temperature and the progress of the reaction was monitored by thin layer chromatography (TLC). After filtration the mixture was concentrated in vacuo and the resulting liquid was diluted with 50 ml dichloromethane and extracted with 350 ml 0.05 M HCl. The combined aqueous layer was made basic with NaOH until the pH was 10-11 and the solution was extracted with 2150 ml dichloromethane. The organic layers were combined and dried over anhydrous MgSO.sub.4, filtered and evaporated to give N,N-bis(4-methoxybenzyl)-1-(thiazol-2-yl)methanamine as a dark brown oil. Yield: 1 g (70%), .sup.1H NMR (300 MHz, CDCl.sub.3): 7.73 (1H, d), 7.29 (5H, m), 6.9 (4H, m), 4.62 (2H, s), 4.13 (2H, s), 3.81 (8H, s), .sup.13C NMR (75 MHz, CDCl.sub.3): 172, 159.2, 158.9, 142.6, 133.4, 131.8, 129.5, 128.7, 118.9, 114, 65, 55.4, 52.7, 50.1.

Example 6

Preparation of N-benzyl-N-4-methoxybenzyl)-1-(4-methoxyphenyl)methanamine

(41) ##STR00190##

(42) To a round bottom flask with a magnetic stirrer bar benzylamine (0.43 g, 4 mmol) and 4-methoxybenzaldehyde (1.22 g, 9 mmol) were added together with 10 ml methanol, NaCNBH.sub.3 (0.57 g, 9 mmol) and a few granules of 4 molecular sieve. The solution was stirred for 6 hours at room temperature and the progress of the reaction was monitored by thin layer chromatography (TLC). After filtration the mixture was concentrated in vacuo and the resulting liquid was diluted with 50 ml dichloromethane and extracted with 350 ml 0.05 M HCl. The combined aqueous layer was made basic with NaOH until the pH was 10-11 and the solution was extracted with 2150 ml dichloromethane. The organic layers were combined and dried over anhydrous MgSO.sub.4, filtered and evaporated to give N-benzyl-N-(4-methoxybenzyl)-1-(4-methoxyphenyl)methanamine as a pale yellow oil. Yield: 0.29 g (20%) NMR (300 MHz, CDCl.sub.3): 7.35 (9H, m), 6.91 (4H, m), 3.8 (12H, m), .sup.13C NMR (75 MHz, CDCl.sub.3): 159.2, 140.9, 133, 129.9, 129.2, 128.9, 128.7, 127.5, 114.5, 114.3, 65.6, 55.8, 53.6, 53.1.

Example 7

Preparation of dimethyl 4,4-((benzylazanediyl)bis(methylene))dibenzoate

(43) ##STR00191##

(44) To a round bottom flask with a magnetic stirrer bar benzylamine (0.107 g, 1 mmol) and methyl 3-formylbenzoate (0.49 g, 3 mmol) were added together with 10 ml methanol, NaCNBH.sub.3 (0.19 g, 3 mmol) and a few granules of 4 molecular sieve. The solution was stirred for 6 hours at room temperature and the progress of the reaction was monitored by thin layer chromatography (TLC). After filtration the mixture was concentrated in vacuo and the resulting liquid was diluted with 20 ml dichloromethane and extracted with 320 ml 0.05 M HCl. The combined aqueous layer was made basic with NaOH until the pH was 10-11 and the solution was extracted with 360 ml dichloromethane. The organic layers were combined and dried over anhydrous MgSO.sub.4, filtered and evaporated to give dimethyl 4,4-((benzylazanediyl)bis (methylene))dibenzoate as a pale yellow oil. Yield: 0.14 g (18%), .sup.13C NMR (75 MHz, CDCl.sub.3): 169.3, 167.1, 141.5, 140.7, 140, 132.9, 131.5, 130.5, 130.4, 129.4, 128.9, 128.7, 128.6, 128.4, 128.3, 128, 127.2, 64.8, 53.3, 52.8, 52.3, 52.4.

Example 8

Preparation of 4-((benzyl(4-(dimethylamino)benzyl)amino)methyl)-N,N-dimethylaniline

(45) ##STR00192##

(46) To a round bottom flask with a magnetic stirrer bar benzylamine (0.43 g, 4 mmol) and 4-(dimethylamino)benzaldehyde (1.6 g, 10.7 mmol) were added together with 10 ml methanol, NaCNBH.sub.3 (0.57 g, 9 mmol) and a few granules of 4 molecular sieve. The solution was stirred overnight at room temperature and the progress of the reaction was monitored by thin layer chromatography (TLC). After filtration the mixture was concentrated in vacuo and 100 mg of the crude product was purified by flash chromatography using ethyl acetate/CH.sub.2Cl.sub.2 (1:9) giving 4-((benzyl(4-(dimethylamino)benzyl)amino)methyl)-N,N-dimethylaniline as a highly viscous brown oil. .sup.13C NMR (75 MHz, CDCl.sub.3): 150.3, 140.9, 130.2, 129.3, 128.6, 128.3, 127.1, 113.1, 57.9, 57.5, 41.4.

Example 9

Preparation of 4-(4-(dimethylamino)benzyl)(1-phenylethyl)amino)methyl)-N,N-dimethylaniline

(47) ##STR00193##

(48) To a round bottom flask with a magnetic stirrer bar 1-phenylethylamine (0.48 g, 4 mmol) and 4-(dimethylamino)benzaldehyde (1.34 g, 9 mmol) were weighed in and added 10 ml methanol, NaCNBH.sub.3 (0.57 g, 9 mmol) and a few granules of molecular sieve 4 . The solution was stirred for 6 hours at room temperature and the progress of the reaction was monitored by thin layer chromatography (TLC). After filtration the mixture was concentrated in vacuo and 100 mg of the crude product was purified by flash chromatography using ethyl acetate/CH.sub.2Cl.sub.2 (1:9) giving 4-(((4-(dimethylamino)benzyl)(1-phenylethyl)amino)methyl)-N,N-dimethylaniline as a highly viscous brown oil. .sup.1H NMR (300 MHz, CDCl.sub.3): 7.28 (10H, m), 3.72 (2H, d), 3.84 (1H, q), 2.94 (6H, s), 2.19 (12H, s), 1.38 (3H, d), .sup.13C NMR (75 MHz, CDCl.sub.3): 150, 146.1, 145.5, 129.3, 129.2, 128.6, 128.4, 127.1, 126.9, 125.4, 112.9, 57.4, 51.1, 40.9, 25.5, 24.4.

Example 10

Preparation of N-(4-(tert-butyl)benzyl)-N-(4-fluorobenzyl)-1-(pyridin-4-yl)methanamine

(49) ##STR00194##

(50) To a round bottom flask with a magnetic stirrer bar Intermediate 1 (0.66 g, 3 mmol) and 4-tert-butylbenzaldehyde (1.29 g, 8 mmol) were added together with 10 ml methanol, NaCNBH.sub.3 (0.50 g, 8 mmol) and a few granules of 4 molecular sieve. The solution was stirred for 8 hours at room temperature and the progress of the reaction was monitored by thin layer chromatography (TLC). After filtration the mixture was concentrated in vacuo and the resulting liquid was diluted with 30 ml dichloromethane and extracted with 330 ml 0.05 M HCl. The combined aqueous layer was made basic with NaOH until the pH was 10-11 and the solution was extracted with 2100 ml dichloromethane. The organic layers were combined and dried over anhydrous MgSO.sub.4, filtered and evaporated to give N-(4-(tert-butyl)benzyl)-N-(4-fluorobenzyl)-1-(pyridin-4-yl)methanamine as a yellow oil. Yield: 0.19 g (18%), .sup.1H NMR (300 MHz, CDCl.sub.3): 8.57 (2H, d), 7.2 (10H, m), 3.81 (4H, d), 3.55 (2H, m), 1.3 (9H, m), .sup.13C NMR (75 MHz, CDCl.sub.3): 149.6, 149, 135.2, 129.4, 129.3, 126.6, 125.2, 125, 123.3, 122.7, 115.1, 114.9, 52.1, 51.5, 31.1, 29.4.

Example 11

Preparation of 2-(((4-fluorobenzyl)(pyridin-4-ylmethyl)amino)methyl)benzene-1,4-diol

(51) ##STR00195##

(52) To a round bottom flask with a magnetic stirrer bar Intermediate 3 (0.23 g, 1 mmol) and 4-fluorobenzaldehyde (0.37 g, 3 mmol) were added together with 10 ml methanol, NaCNBH.sub.3 (0.19 g, 3 mmol) and a few granules of 4 molecular sieve. The solution was stirred for 8 hours at room temperature and the progress of the reaction was monitored by thin layer chromatography (TLC). After filtration the mixture was concentrated in vacuo and the resulting liquid was diluted with 30 ml dichloromethane and extracted with 330 ml 0.05 M HCl. The combined aqueous layer was made basic with NaOH until the pH was 10-11 and the solution was extracted with 2100 ml dichloromethane. The organic layers were combined and dried over anhydrous MgSO.sub.4, filtered and evaporated to give 2-(((4-fluorobenzyl)(pyridin-4-ylmethyl)amino)methyl)benzene-1,4-diol as a brown oil. Yield: 0.39 g (100%), .sup.1H NMR (300 MHz, DMSO): 8.51 (2H, d), 7.36 (4H, m), 7.14 (2H, m), 6.80 (1H, s), 6.60 (1H, s), 6.48 (1H, s), 3.32 (6H, s), .sup.13C NMR (75 MHz, DMSO): 149.9, 149.6, 148.2, 148, 138.7, 134.6, 130.5, 130.4, 128.4, 128.3, 124.4, 123.5, 115.7, 115.6, 115.2, 114.9, 114.6, 114.3, 62.1, 56.5, 56, 52.1.

Example 12

Preparation of 4-((benzyl(2,2-diphenylethyl)amino)methyl)-N,N-dimethylaniline

(53) ##STR00196##

(54) To a round bottom flask with a magnetic stirrer bar Intermediate 8 (0.24 g, 0.83 mmol) and 4-(dimethylamino)benzaldehyde (0.18 g, 1.2 mmol) were added together with 10 ml methanol, NaCNBH.sub.3 (0.10 g, 1.59 mmol) and a few granules of 4 molecular sieve. The solution was stirred for 6 hours at room temperature and the progress of the reaction was monitored by thin layer chromatography (TLC). After filtration the mixture was concentrated in vacuo and the crude product was purified by flash chromatography using ethyl acetate/CH.sub.2Cl.sub.2 (1:9) giving 4-((benzyl(2,2-diphenylethyl)amino)methyl)-N,N-dimethylaniline as a yellow oil. .sup.1H NMR (300 MHz, CDCl.sub.3): 7.15 (15H, m), 6.91 (2H, d), 6.57 (2H, d), 4.17 (1H, t), 3.47 (4H, d), 2.96 (2H, d). 2.84 (6H, s).

Example 13

Preparation of 4-((benzyl((2,3-dihydrobenzofuran-6-yl)methyl)amino)methyl)-N,N dimethylaniline

(55) ##STR00197##

(56) To a round bottom flask with a magnetic stirrer bar Intermediate 6 (0.4 g, 1.4 mmol) and 2,3-dihydrobenzofuran-5-carboxaldehyde (0.37 g, 2.5 mmol) were added together with 10 ml methanol, NaCNBH.sub.3 (0.16 g, 2.5 mmol) and a few granules of 4 molecular sieve. The solution was stirred for 8 hours at room temperature and the progress of the reaction was monitored by thin layer chromatography (TLC). After filtration the mixture was concentrated in vacuo and 100 mg of the crude product was purified by flash chromatography using 5-10% ethyl acetate in dichloromethane giving 4-((benzyl((2,3-dihydrobenzofuran-6-yl)methyl)amino)methyl)-N,N-dimethylaniline as a viscous yellow oil. .sup.1H NMR (300 MHz, CDCl.sub.3): 7.22 (9H, m), 7.02 (1H, d), 6.64 (2H, d), 4.47 (2H, t), 3.45 (2H, s), 3.38 (4H, s), 3.12 (2H, t), 2.85 (6H, s), .sup.13C NMR (75 MHz, CDCl.sub.3): 159.4, 150.1, 140.5, 132.2, 130, 129.1, 128.9, 128.5, 127.9, 127.1, 127, 125.8, 113, 109, 71.6, 57.9, 57.7, 57.4, 51.2, 30.2.

Example 14

Preparation of methyl 4-((benzyl(4-fluorobenzyl)amino)methyl)benzoate

(57) ##STR00198##

(58) First Step:

(59) To a round bottom flask with a magnetic stirrer bar benzylamine (2.14 g, 10 mmol) and 4-fluorobenzaldehyde (1.55 g, 12.5 alma) were added together with 10 ml methanol, NaCNBH.sub.3 (0.79 g, 12.5 mmol) and a few granules of 4 molecular sieve. The solution was stirred for 4 hours at room temperature and the progress of the reaction was monitored by thin layer chromatography (TLC). After filtration the solution was concentrated in vacuo and the resulting liquid was diluted with 50 ml dichloromethane and extracted with 350 ml 0.05 M HCl. The combined aqueous layer was made basic with NaOH until the pH was 10-11 and the solution was extracted with 2150 ml dichloromethane. The organic layers were combined and dried over anhydrous MgSO.sub.4, filtered and evaporated to give N-benzyl-1-(4-fluorophenyl)methanamine.

(60) Second Step:

(61) N-benzyl-1-(4-fluorophenyl)methanamine (0.61 g, 2.7 mmol) was added to 20 ml acetone in a round bottom flask. To the solution methyl 4-(bromomethyl)benzoate (1.15 g, 5 mmol) and K.sub.2CO.sub.3 (1.38 g, 10 mmol) were added and the reaction mixture was stirred for 4 hours at 55 C. at reflux. The progress of the reaction was monitored by thin layer chromatography (TLC), and when the reaction was complete the mixture was filtered and concentrated in vacuo. The crude product was purified by flash chromatography using 5-10% ethyl acetate in dichloromethane giving methyl 4-((benzyl(4-fluorobenzyl)amino)methyl)benzoate as a pale yellow oil. .sup.1H NMR (300 MHz, CDCl.sub.3): 7.92 (2H, d), 7.38 (2H, d), 7.24 (7H, m), 6.92 (2H, t), 3.83 (3H, s), 3.5 (2H, s), 3.46 (2H, s), 3.43 (2H, s).

Example 15

Preparation of 4-((benzyl(4-fluorobenzyl)amino)methyl)benzoic acid

(62) ##STR00199##

(63) The product from Example 13 was diluted in 0.1 M NaOH (15 ml) and added to 10 ml methanol. The reaction mixture was stirred for 8 hours at 60 C. at reflux, and methanol was evaporated off on rotary evaporator. The remaining aqueous phase was acidified with HCl until pH 5 and extracted with 320 ml dichloromethane. The combined organic phase was dried over anhydrous MgSO.sub.4 and concentrated in vacuo to give 4-((benzyl(4-fluorobenzyl)amino)methyl)benzoic acid as a pale yellow oil. .sup.1H NMR (300 MHz, CDCl.sub.3): 7.98 (2H, d), 7.41 (2H, d), 7.24 (7H, m), 6.92 (2H, t), 3.52 (2H, s), 3.46 (2H, s), 3.44 (2H, s).

Example 16

Preparation of 1-(2-(2H-tetrazol-5-yl)-[1,1-biphenyl]-4-yl)-N-benzyl-N-(4-fluorobenzyl)methanamine

(64) ##STR00200## ##STR00201##

(65) First Step:

(66) To a round bottom flask with a magnetic stirrer bar N-benzyl-1-(4-fluorophenyl)methanamine (0.4 g, 1.97 mmol) and 5-(4-bromo-[1,1-biphenyl]-2-yl)-2-trityl-2H-tetrazole (1.4 g, 2.5 mmol) were added together with 10 ml acetone and K.sub.2CO.sub.3 (0.69 g, 5 mmol). The reaction mixture was stirred for 4 hours at 55 C. at reflux. The progress of the reaction was monitored by thin layer chromatography (TLC). After filtration the solution was concentrated in vacuo and the crude product was purified by flash chromatography using 5-10% ethyl acetate in dichloromethane giving 0.2 g of the tertiary amine.

(67) Second Step:

(68) The tertiary amine from the first step of the reaction (0.2 g, 0.29 mmol) was added 10 ml of a solution of trifluoro acetic acid/dichloromethane/methanol (2:1:1) and stirred for 3 hours at room temperature. The crude product was purified by flash chromatography using 5-10% ethyl acetate in dichloromethane giving 1-(2-(2H-tetrazol-5-yl)-[1,1-biphenyl]-4-yl)-N-benzyl-N-(4-fluorobenzyl) methanamine as a pale brown oil. .sup.1H NMR (300 MHz, CDCl.sub.3): 10.77 (1H, s), 8.08 (2H, d), 7.42 (10H, m), 7.03 (2H, t), 3.57 (6H, m), .sup.13C NMR (75 MHz, CDCl.sub.3): 164, 160.7, 146.3, 139.4, 135.2, 130.6, 130.5, 129.1, 129, 128.7, 127.5, 115.6, 115.4, 58.4, 58, 57.7.

Example 17

Synthesis of dimethyl 4,4-((benzylazanediyl)bis(methylene))dibenzoate

(69) ##STR00202##

(70) To a suspension of benzylamine (1.07 g, 10 mmol) and potassium carbonate (2.76 g, 20 mmol) in acetone (20 ml) was added methyl 4-(bromomethyl)benzoate (4.58 g, 20 mmol) and the reaction mixture was heated to reflux overnight, cooled to room temperature and evaporated in vacuo. The residue was separated with flash chromatography (dichloromethane) to give dimethyl 4,4-((benzylazanediyl)bis(methylene))dibenzoate as a brown viscous oil. Yield: 1.4 g (35%); TLC: R.sub.f: 0.85 (10% methanol in dichloromethane); .sup.1H NMR (400 MHz, CDCl.sub.3): 7.91 (4H, d), 7.38 (4H, d), 7.23 (5H, m), 3.81 (6H, s), 3.51 (4H, s), 3.46 (2H, s); .sup.13C NMR (400 MHz, CDCl.sub.3): 167.0, 144.9, 138.8, 129.7, 129.0, 128.8, 128.6, 128.3, 127.2, 58.2, 57.8, 52.0 (.sup.13C NMR had some overlapping signals).

Example 18

Synthesis of 4,4-((benzylazanediyl)bis(methylene)dibenzoic acid

(71) ##STR00203##

(72) Compound 17 (0.4 g, 1 mmol) was added to a suspension of potassium hydroxide (0.17 g, 3 mmol) in 2 ml of 50:50 water/methanol. After 15 minutes 1M HCl was added until the mixture became acidic and then washed with water. The residue was subsequently suspended in toluene and dried in vacuo. Removal of toluene (as an azeotropic mixture with water) yielded 4,4-((benzylazanediyl)bis(methylene)dibenzoic acid as light brown crystals. Yield: 0.21 g (56%); TLC: R.sub.f: 0.17 (10% methanol in dichloromethane); .sup.1H NMR (400 MHz, DMSO): 12.83 (2H, s), 7.93 (4H, d), 7.52 (4H, d), 7.38 (5H, m), 3.58 (4H, s), 3.52 (2H, s); .sup.13C NMR (400 MHz, DMSO): 167.1, 144.2, 138.6, 129.6, 129.4, 128.5, 128.3, 127.0, 57.2, 56.8 (.sup.13C NMR had some overlapping signals).

Example 19

Synthesis of N-benzyl-N-(4-(tert-butyl)benzyl)-1-(4-(tert-butyl)phenyl)methanamine

(73) ##STR00204##

(74) Benzylamine (0.536 g, 5 mmol), sodium hydrogen carbonate (0.92 g, 11 mmol) and sodium dodecyl sulfate (approx. 20 mg) were added to water (20 ml) in a 50 ml round-bottom flask, along with a stirring magnet. The mixture was heated at 80 C. for 5 min. 4-tert-Butylbenzyl bromide (2.5 g, 11 mmol) was added to the mixture and subsequently heated to 80 C. for 1 hour. The reaction mixture was cooled to room temperature and the residue filtered and dried, before being recrystallized from a 1:1 mixture of hexane and ethyl acetate to yield N-benzyl-N-(4-(tert-butyl)benzyl)-1-(4-(tert-butyl)phenyl)methanamine as a white crystalline powder. Yield: 1.55 g (78%); TLC: R.sub.f: 0.88 (10% methanol in dichloromethane); .sup.1H NMR (400 MHz, CDCl.sub.3): 7.38 (13H, m), 3.60 (6H, d), 1.34 (18H, s); .sup.13C NMR (400 MHz, CDCl.sub.3): 127.7, 127.3, 126.6, 125.9, 124.7, 124.5, 124.3, 124.1 (.sup.13C NMR had some overlapping signals).

Example 20

Synthesis of N-benzyl-N-(4-nitrobenzyl)-1-(4-nitrophenyl)methanamine

(75) ##STR00205##

(76) Benzylamine (0.536 g, 5 mmol), sodium hydrogen carbonate (0.92 g, 11 mmol) and sodium dodecyl sulfate (approx. 20 mg) were added to water (20 ml) in a 50 ml round-bottom flask, along with a stirring magnet. The mixture was heated at 80 C. for 5 min. 4-nitrobenzylbromide (2.37 g, 11 mmol) was added to the mixture and subsequently heated to 80 C. for 1 hour. The reaction mixture was cooled to room temperature and the residue filtered and dried, before being recrystallized from a 1:1 mixture of hexane and ethyl acetate to yield N-benzyl-N-(4-nitrobenzyl)-1-(4-nitrophenyl)methanamine as a white crystalline powder. Yield: 0.94 g (50%); TLC: R.sub.f=0.86 (10% methanol in dichloromethane); .sup.1H NMR (400 MHz, CDCl.sub.3): 8.21 (4H, d), 7.58 (4H, d), 7.32 (5H, m), 3.69 (4H, s), 3.60 (2H, s); .sup.13C NMR (400 MHZ, CDCl.sub.3): 147.3, 146.8, 138.0, 129.2, 128.7, 128.6, 127.6, 123.7, 58.5, 57.6.

Example 21

Synthesis of tris(4-nitrobenzyl)amine

(77) ##STR00206##

(78) A mixture of 4-nitrobenzyl bromide (6.48 g, 30 mmol) and aqueous ammonia solution (30-33%, 2 ml) in methanol (15 ml) was heated in a sealed tube at 100 C. for 4 hours. Then it was cooled to room temperature and poured into water (80 ml). The resulting mixture was extracted three times with dichloromethane and the combined organic layers were dried over sodium sulfate. Removal of the solvents yielded a yellow solid, which was recrystallized in ethyl acetate to give tris(4-nitrobenzyl)amine as a light yellow crystal. Yield: 0.63 g (15%); TLC: R.sub.f: 0.87 (10% methanol in dichloromethane); .sup.1H NMR (400 MHz, CDCl.sub.3): 8.25 (6H, d), 7.56 (6H, d), 3.70 (6H, s); .sup.13C NMR (400 MHz, CDCl.sub.3): 147.5, 145.8, 129.2, 123.9, 57.8.

Example 22

Synthesis of 4-(((4-aminobenzyl)(benzyl)amino)methyl)aniline

(79) ##STR00207##

(80) N-benzyl-N-(4-nitrobenzyl)-1-(4-nitrophenyl)methanamine (compound 20) (2.19 g, 5.81 mmol), calcium chloride (0.782 g, 5.32 mmol) and acid-washed zinc (15 g) were suspended in 125 ml of ethanol. The mixture was refluxed for 2 hours, cooled to room temperature, filtered and dried in vacuo, before being recrystallized from a 1:1 mixture of hexane and diethyl ether to yield 4-(((4-aminobenzyl)(benzyl)amino)methyl)aniline as a light yellow crystalline powder. Yield: 0.37 g (20%); TLC: R.sub.f: 0.51 (10% methanol in dichloromethane); .sup.1H NMR (400 MHz, DMSO): 7.33 (5H, m), 7.21 (4H, d), 6.97 (4H, d), 4.90 (4H, s), 3.40 (2H, s), 3.27 (4H, s); .sup.13C NMR (400 MHz, DMSO): 147.3, 139.9, 129.2, 128.3, 128.1, 126.7, 125.4, 113.7, 56.4, 48.6.

Example 23

Synthesis of 4-((benzyl(4-(diethylamino)benzyl)amino)methyl)-N,N-diethylaniline

(81) ##STR00208##

(82) A suspension of 4-((benzyl(4-(diethylamino)benzyl)amino)methyl)-N,N-diethylaniline (compound 22) (0.6 g, 1.9 mmol) and potassium carbonate (2.76 g, 20 mmol) in acetone (50 ml) was added ethyl iodide (3.92 g, 20 mmol) and the reaction mixture was heated to reflux overnight, cooled to room temperature and evaporated in vacuo. The residue was separated with flash chromatography (10% methanol in dichloromethane) to give 4-((benzyl(4-(diethylamino)benzyl)amino)methyl)-N,N-diethylaniline as a viscous brown oil. Yield: 0.05 g (6.1%-NB .sup.1H NMR indicated that the substance was not completely dry); TLC: R.sub.f: 0.82 (10% methanol in dichloromethane); .sup.1H NMR (300 MHz, CDCl.sub.3): 7.40 (9H, m), 6.74 (4H, m), 3.66 (2H, s), 3.58 (4H, s), 3.42 (8H, q), 1.25 (12H, t).

Example 24

Synthesis of 4-((bis(4-aminobenzyl)amino)methyl)aniline

(83) ##STR00209##

(84) A mixture of tris(4-nitrobenzyl)amine (compound 21) (848 mg, 2 mmol), 10% PdC (42.4 mg) and aqueous HCl (37%, 4 ml) in methanol (30 ml) was hydrogenated at room temperature and atmospheric hydrogen pressure for 3 hours. After the catalyst was filtered off and methanol was evaporated, the residue was dissolved in ethanol and toluene. Removal of these solvents (as azeotropic mixtures with water) yielded a light yellow solid which was washed with diethyl ether to give 4-((bis(4-ammoniobenzyl)ammonio)methyl)benzenaminium as light yellow crystals. Yield: 0.56 g (84% NB the NMR indicated that the substance was not completely dried); TLC: R.sub.f: 0.16 (10 methanol in dichloromethane); .sup.1H NMR (300 MHz, D.sub.2O): 7.39 (6H, d), 7.33 (6H, d), 4.33 (6H, s).

Example 25

Synthesis of 1,1-(((benzylazanediyl)bis(methylene))bis(4,1-phenylene))bis(3-(p-tolyl)urea)

(85) ##STR00210##

(86) 4-(((4-Aminobenzyl)(benzyl)amino)methyl)aniline (compound 22) (0.1 g, 0.315 mmol) was dissolved in dry dichloromethane (4 ml) and p-tolyl isocyanate (0.084 g, 0.63 mmol) was slowly added at 0 C. After stirring at room temperature for 18 hours, the solvent was removed and diethyl ether (3 ml) was added. The white solid was filtered off and dried to yield 1,1-(((benzylazanediyl)bis(methylene))bis(4,1-phenylene))bis(3-(p-tolyl)urea) as a white powder. Yield: 0.147 g (80%); TLC: R.sub.f: 0.54 (10% methanol in dichloromethane); .sup.1H NMR (400 MHz, DMSO): 8.54 (4H, d), 7.36 (17H, m), 7.07 (4H, d), 3.45 (6H, d), 2.24 (6H, s); .sup.13C NMR (400 MHz, DMSO): 152.5, 138.6, 137.1, 132.2, 130.5, 129.1, 128.9, 128.4, 128.2, 118.2, 118.1, 56.7, 56.3, 20.3 (.sup.13C NMR had some overlapping signals).

Example 26

Synthesis of 1,1-(((benzylazanediyl)bis(methylene))bis(4,1-phenylene))bis(3-(4-fluorophenyl)urea)

(87) ##STR00211##

(88) 4-(((4-Aminobenzyl)(benzyl)amino)methyl)aniline (compound 22) (0.1 g, 0.315 mmol) was dissolved in dry dichloromethane (4 ml) and 4-fluorophenyl isocyanate (0.084 g, 0.63 mmol) was slowly added at 0 C. After stirring at room temperature for 18 hours, the solvent was removed and diethyl ether (3 ml) was added. The white solid was filtered off and dried to yield 1,1-(((benzylazanediyl)bis(methylene))bis(4,1-phenylene))bis(3-(4-fluorophenyl)urea) as a white powder. Yield 0.14 g (75%); TLC: R.sub.f: 0.59 (10% methanol in dichloromethane); .sup.1H NMR (400 MHz, DMSO): 8.66 (4H, d), 7.27 (21H, m), 3.48 (2H, s), 3.42 (4H, s); .sup.13C NMR (300 MHz, DMSO): 153.5, 139.3, 136.94, 136.90, 133.2, 129.8, 129.3, 129.1, 120.8, 120.6, 119.1, 116.3, 116.0, 80.3, 80.1 (.sup.13C NMR had some overlapping signals).

Example 27

Synthesis of 1,1-(((benzylazanediyl)bis(methylene))bis(4,1-phenylene))bis(3-phenylurea)

(89) ##STR00212##

(90) 4-(((4-Aminobenzyl)(benzyl)amino)methyl)aniline (compound 22) (0.1 g, 0.315 mmol) was dissolved in dry dichloromethane (4 ml) and phenyl isocyanate (0.075 g, 0.63 mmol) was slowly added at 0 C. After stirring at room temperature for 18 hours, the solvent was removed and diethyl ether (3 ml) was added. The white solid was filtered off and dried to yield 1,1-(((benzylazanediyl)bis(methylene))bis(4,1-phenylene))bis(3-phenylurea) as a white powder. Yield: 0.08 g (46%); TLC; R.sub.f: 0.76 (10% methanol in dichloromethane); .sup.1H NMR (400 MHz, DMSO): 8.61 (4H, s), 7.22 (23H, m), 3.49 (4H, s), 3.43 (2H, s); .sup.13C NMR (400 MHz, DMSO): 152.5, 139.7, 139.3, 138.5, 132.4, 128.9, 128.7, 128.4, 128.2, 126.8, 121.7, 118.1, 56.7, 56.3.

Example 28

Synthesis of 1,1,1-((nitrilotris(methylene))tris(benzene-4,1-diyl))tris(3-(p-tolyl)urea)

(91) ##STR00213##

(92) 4-((Bis(4-ammoniobenzyl)ammonio)methyl)benzenaminium (compound 24) (100 mg, 0.297 mmol) was dissolved in water and stirred at room temperature, before potassium carbonate was added until the pH became 11-12. The mixture was extracted three times with dichloromethane and subsequently dried in vacuo. The residue was dissolved in dry dichloromethane (4 ml) and p-tolyl isocyanate (0.136 g, 0.9 mmol) was slowly added at 0 C. After stirring at room temperature for 18 hours, the solvent was removed and diethylether (3 ml) was added. The white solid was filtered off to give 1,1,1-((nitrilotris(methylene))tris(benzene-4,1-diyl))tris(3-(p-tolyl)urea) as a white powder. Yield: 0.07 g (32%); TLC: R.sub.f: 0.76 (10% methanol in dichloromethane); .sup.1H NMR (300 MHz, DMSO): 8.57 (6H, d), 7.25 (24H, m), 3.41 (6H, s), 2.24 (9H, s).

Example 29

Synthesis of 4-((benzyl(4-(dimethylamino)benzyl)amino)methyl)-N,N-dimethylaniline

(93) ##STR00214##

(94) A solution of benzylamine (1.29 g, 12 mmol) and 4-(dimethylamino)benzaldehyde (3.75 g, 25 mmol) in methanol (50 ml) was stirred for 30 minutes before sodium cyanoborohydride (1.6 g, 25 mmol) and 4 molecular sieves were added. The mixture was stirred overnight, filtered and thereafter dried in vacuo. The residue was separated with flash chromatography (5-10% ethyl acetate in dichloromethane) to give 4-((benzyl(4-(dimethylamino)benzyl)amino)methyl)-N,N-dimethylaniline as a brown viscous oil. Yield: 1.6 g (35%); TLC: R.sub.f: 0.83 (10% methanol in dichloromethane); .sup.1H NMR (400 MHz, CDCl.sub.3): 7.32 (2H, m), 7.18 (7H, m), 6.63 (4H, d), 3.45 (2H, s), 3.38 (4H, s), 2.84 (12H, 5); .sup.13C NMR (400 MHz, CDCl.sub.3): 149.7, 140.3, 129.7, 128.7, 128.0, 127.7, 126.5, 112.7, 57.4, 57.0, 40.8.

Example 30

Synthesis of N-benzyl-N-(4-(methoxycarbonyl)benzyl)-1-(4(methoxycarbonyl)phenyl)methanaminium chloride

(95) ##STR00215##

(96) Dimethyl 4,4-((benzylazanediyl)bis(methylene))dibenzoate (compound 17) (0.2 g, 0.5 mmol) was added to stirring diethyl ether in a round bottom flask and 2.0 M hydrochloride solution in diethyl ether (0.3 mL, 0.6 mmol) was slowly added. After 5 minutes, the precipitate was filtered off and dried under vacuum to yield N-benzyl-N-(4-(methoxycarbonyl)benzyl)-1-(4-(methoxycarbonyl)phenyl)methanaminium chloride as a light brown powder.

Example 31

Synthesis of N-benzyl-N-(4-(methoxycarbonyl)benzyl)-1-(4-(methoxycarbonyl)phenyl)methanaminium methanesulfonate

(97) ##STR00216##

(98) Dimethyl 4,4-((benzylazanediyl)bis(methylene))dibenzoate (compound 17) (0.2 g, 0.5 mmol) was added to stirring diethyl ether in a round bottom flask and methane sulfonic acid (0.05 g, 0.52 mmol) was slowly added. After 5 minutes, the precipitate was filtered off and dried under vacuum to yield N-benzyl-N-(4-(methoxycarbonyl)benzyl)-1-(4-(methoxycarbonyl)phenyl)methanaminium methanesulfonate as a brown viscous substance.

Example 32

Synthesis of N-benzyl-N-(4-carboxybenzyl)-1-(4-carboxyphenyl)methanaminium chloride

(99) ##STR00217##

(100) 4,4-((Benzylazanediyl)bis(methylene)dibenzoic acid (compound 18) (0.2 g, 0.53 mmol) was added to stirring diethyl ether in a round bottom flask and 2.0 M hydrochloride solution in diethyl ether (0.3 mL, 0.6 mmol) was slowly added. After 5 minutes, the precipitate was filtered off and dried under vacuum to yield N-benzyl-N-(4-carboxybenzyl)-1-(4-carboxyphenyl)methanaminium chloride as a light grey crystal-like powder.

Example 33

Synthesis of N-benzyl-N-(4-carboxybenzyl)-1-(4-carboxyphenyl)methanaminium methanesulfonate

(101) ##STR00218##

(102) 4,4-((Benzylazanediyl)bis(methylene)dibenzoic acid (compound 18) (0.2 g, 0.53 mmol) was added to stirring diethyl ether in a round bottom flask and methane sulfonic acid (0.06 g, 0.62 mmol) was slowly added. After 5 minutes, the precipitate was filtered off and dried under vacuum to yield N-benzyl-N-(4-carboxybenzyl)-1-(4-carboxyphenyl)methanaminium methanesulfonate chloride as a red viscous substance.

Example 34

Synthesis of N-benzyl-N-(4-(tert-butyl)benzyl)-1-(4-(tert-butyl)phenyl)methanaminium chloride

(103) ##STR00219##

(104) N-Benzyl-N-(4-(tert-butyl)benzyl)-1-(4-(tert-butyl)phenyl)methanamine (compound 19) (0.2 g, 0.46 mmol) was added to stirring diethyl ether in a round bottom flask and 2.0 M hydrochloride solution in diethyl ether (0.25 mL, 0.5 mmol) was slowly added. After 5 minutes, the precipitate was filtered off and dried under vacuum to yield of N-benzyl-N-(4-(tert-butyl)benzyl)-1-(4-(tert-butyl)phenyl)methanaminium chloride as a white crystal-like powder.

Example 35

Synthesis of N-benzyl-N-(4-(tert-butyl)benzyl)-1-(4-(tert-butyl)phenyl)methanaminium methanesulfonate

(105) ##STR00220##

(106) N-Benzyl-N-(4-(tert-butyl)benzyl)-1-(4-(tert-butyl)phenyl)methanamine (compound 19) (0.2 g, 0.46 mmol) was added to stirring diethyl ether in a round bottom flask and methane sulfonic acid (0.05 g, 0.52 mmol) was slowly added. After 5 minutes, the precipitate was filtered off and dried under vacuum to yield N-benzyl-N-(4-(tert-butyl)benzyl)-1-(4-(tert-butyl)phenyl)methanaminium methanesulfonate as a viscous substance.

Example 36

Synthesis of N-benzyl-N-(4-nitrobenzyl)-1-(4-nitrophenyl)methanaminium chloride)

(107) ##STR00221##

(108) N-Benzyl-N-(4-nitrobenzyl)-1-(4-nitrophenyl)methanamine (compound 20) (0.2 g, 0.53 mmol) was added to stirring diethyl ether in a round bottom flask and 2.0 M hydrochloride solution in diethyl ether (0.3 mL, 0.6 mmol) was slowly added. After 5 minutes, the precipitate was filtered off and dried under vacuum to N-benzyl-N-(4-nitrobenzyl)-1-(4-nitrophenyl)methanaminium chloride as a light yellow crystal-like powder.

Example 37

Synthesis of N-benzyl-N-(4-nitrobenzyl)-1-(4-nitrophenyl)methanaminium methanesulfonate

(109) ##STR00222##

(110) N-benzyl-N-(4-nitrobenzyl)-1-(4-nitrophenyl)methanamine (compound 20) (0.2 g, 0.53 mmol) was added to stirring diethyl ether in a round bottom flask and methane sulfonic acid (0.06 g, 0.62 mmol) was slowly added. After 5 minutes, the precipitate was filtered off and dried under vacuum to yield N-benzyl-N-(4-(dimethylamino)benzyl)-1-(4-(dimethylamino)phenyl)methanaminium methanesulfonate as a light yellow viscous substance.

Example 38

Synthesis of N,N-bis(4-aminobenzyl)-1-phenylmethanaminium chloride

(111) ##STR00223##

(112) 4-(((4-Aminobenzyl)(benzyl)amino)methyl)aniline (compound 22) (0.2 g, 0.63 mmol) was added to stirring diethyl ether in a round bottom flask and 2.0 M hydrochloride solution in diethyl ether (0.35 mL, 0.7 mmol) was slowly added. After 5 minutes, the precipitate was filtered off and dried under vacuum to yield N,N-bis(4-aminobenzyl)-1-phenylmethanaminium chloride as a light yellow crystal-like powder.

Example 39

Synthesis of N,N-bis(4-aminobenzyl)-1-phenylmethanaminium methanesulfonate

(113) ##STR00224##

(114) 4-(((4-Aminobenzyl)(benzyl)amino)methylaniline (compound 22) (0.2 g, 0.63 mmol) was added to stirring diethyl ether in a round bottom flask and methane sulfonic acid (0.07 g, 0.73 mmol) was slowly added. Alter 5 minutes, the precipitate was filtered off and dried under vacuum to yield N,N-bis(4-aminobenzyl)-1-phenylmethanaminium methanesulfonate as a light yellow viscous substance.

Example 40

Synthesis of N-benzyl-N-(4-(diethylamino)benzyl)-1-(4-(diethylamino)phenyl)methanaminium chloride

(115) ##STR00225##

(116) 4-((Benzyl(4-(diethylamino)benzyl)amino)methyl)-N,N-diethylaniline (compound 23) (0.2 g, 0.47 mmol) was added to stirring diethyl ether in a round bottom flask and 2.0 M hydrochloride solution in diethyl ether (0.25 mL, 0.5 mmol) was slowly added. After 5 minutes, the precipitate was filtered off and dried under vacuum to yield N-benzyl-N-(4-(diethylamino)benzyl)-1-(4-(diethylamino)phenyl)methanaminium chloride as a light yellow crystal-like powder.

Example 41

Synthesis of N-benzyl-N-(4-(diethylamino)benzyl)-1-(4-(diethylamino)phenyl)methanaminium methanesulfonate

(117) ##STR00226##

(118) 4-((Benzyl(4-(diethylamino)benzyl)amino)methyl)-N,N-diethylaniline (compound 23) (0.2 g, 0.47 mmol) was added to stirring diethyl ether in a round bottom flask and methane sulfonic acid (0.25 mL, 0.5 mmol) was slowly added. After 5 minutes, the precipitate was filtered off and dried under vacuum to yield N-benzyl-N-(4-(diethylamino)benzyl)-1-(4-(diethylamino)phenyl)methanaminium methanesulfonate as a light yellow viscous substance.

Example 42

Synthesis of N-benzyl-N-(4-(3-(p-tolyl)ureido)benzyl)-1-(4-(3-(p-tolyl)ureido)phenyl)methanaminium chloride

(119) ##STR00227##

(120) 1,1-(((Benzylazanediyl)bis(methylene))bis(4,1-phenylene))bis(3-(p-tolyl)urea) (compound 25) (0.2 g, 0.34 mmol) was added to stirring diethyl ether in a round bottom flask and 2.0 M hydrochloride solution in diethyl ether (0.2 mL, 0.4 mmol) was slowly added. After 5 minutes, the precipitate was filtered off and dried under vacuum to yield N-benzyl-N-(4-(3-(p-tolyl)ureido)benzyl)-1-(4-(3-(p-tolyl)ureido)phenyl)methanaminium chloride as a yellow crystal-like powder.

Example 43

Synthesis of N-benzyl-N-(4-(3-(p-tolyl)ureido)benzyl)-1-(4-(3-(p-tolyl)ureido)phenyl)methanaminium methanesulfonate

(121) ##STR00228##

(122) 1,1-(((Benzylazanediyl)bis(methylene))bis(4,1-phenylene))bis(3-(p-tolyl)urea) (compound 25) (0.2 g, 0.34 mmol) was added to stirring diethyl ether in a round bottom flask and methane sulfonic acid (0.04 g, 0.42 mmol) was slowly added. After 5 minutes, the precipitate was filtered off and dried under vacuum to yield N-benzyl-N-(4-(3-(p-tolyl)ureido)benzyl)-1-(4-(3-(p-tolyl)ureido)phenyl)methanaminium methanesulfonate as a yellow powder.

Example 44

Synthesis of N-benzyl-N-(4-(3-(4-fluorophenyl)ureido)benzyl)-1-(4-(3-(4-fluorophenyl)ureido)phenyl)methanaminium chloride

(123) ##STR00229##

(124) 1,1-(((Benzylazanediyl)bis(methylene))bis(4,1-phenylene))bis(3-(4-fluorophenyl)urea) (compound 26) (0.2 g, 0.34 mmol) was added to stirring diethyl ether in a round bottom flask and 2.0 M hydrochloride solution in diethyl ether (0.2 mL, 0.4 mmol) was slowly added. After 5 minutes, the precipitate was filtered off and dried under vacuum to yield N-benzyl-N-(4-(3-(4-fluorophenyl)ureido)benzyl)-1-(4-(3-(4-fluorophenyl)ureido)phenyl)methanaminium chloride as a yellow powder.

Example 45

Synthesis of N-benzyl-N-(4-(3-(4-fluorophenyl)ureido)benzyl)-1-(4-(3-(4-fluorophenyl)ureido)phenyl)methanaminium methanesulfonate

(125) ##STR00230##

(126) 1,1-(((Benzylazanediyl)bis(methylene))bis(4,1-phenylene))bis(3-(4-fluorophenyl)urea) (compound 26) (0.2 g, 0.34 mmol) was added to stirring diethyl ether in a round bottom flask and methane sulfonic acid (0.04 g, 0.42 mmol) was slowly added. After 5 minutes, the precipitate was filtered off and dried under vacuum to yield N-benzyl-N-(4-(3-(4-fluorophenyl)ureido)benzyl)-1-(4-(3-(4-fluorophenyl)ureido)phenyl)methanaminium methanesulfonate as a yellow substance.

Example 46

Synthesis of N-benzyl-N-(4-(3-phenylureido)benzyl)-1-(4-(3-phenylureido)phenyl)methanaminium chloride

(127) ##STR00231##

(128) 1,1-(((Benzylazanediyl)bis(methylene))bis(4,1-phenylene))bis(3-phenylurea) (compound 27) (0.2 g, 0.36 mmol) was added to stirring diethyl ether in a round bottom flask and 2.0 M hydrochloride solution in diethyl ether (0.2 mL, 0.4 mmol) was slowly added. After 5 minutes, the precipitate was filtered off and dried under vacuum to yield N-benzyl-N-(4-(3-phenylureido)benzyl)-1-(4-(3-phenylureido)phenyl)methanaminium chloride as a yellow powder.

Example 47

Synthesis of N-benzyl-N-(4-(3-phenylureido)benzyl)-1-(4-(3-phenylureido)phenyl)methanaminium methanesulfonate

(129) ##STR00232##

(130) 1,1-(((Benzylazanediyl)bis(methylene))bis(4,1-phenylene))bis(3-phenylurea) (compound 27) (0.2 g, 0.36 mmol) was added to stirring diethyl ether in a round bottom flask and methane sulfonic acid (0.04 g, 0.42 mmol) was slowly added. After 5 minutes, the precipitate was filtered off and dried under vacuum to yield N-benzyl-N-(4-(3-phenylureido)benzyl)-1-(4-(3-phenylureido)phenyl)methanaminium methanesulfonate as a viscous yellow substance.

Example 48

Synthesis of N-benzyl-N-(4-(dimethylamino)benzyl)-1-(4-(dimethylamino)phenyl)methanaminium chloride

(131) ##STR00233##

(132) 4-(Benzyl(4-(dimethylamino)benzyl)amino)methyl)-N,N-dimethylaniline (compound 29) (0.2 g, 0.54 mmol) was added to stirring diethyl ether in a round bottom flask and 2.0 M hydrochloride solution in diethyl ether (0.3 mL, 0.6 mmol) was slowly added. After 5 minutes, the precipitate was filtered off and dried under vacuum to yield N-benzyl-N-(4-(dimethylamino)benzyl)-1-(4-(dimethylamino)phenyl)methanaminium chloride as a light yellow crystal-like powder.

Example 49

Synthesis of N-benzyl-N-(4-(dimethylamino)benzyl)-1-(4-(dimethylamino)phenyl)methanaminium methanesulfonate

(133) ##STR00234##

(134) 4-((Benzyl(4-(dimethylamino)benzyl)amino)methyl)-N,N-dimethylaniline (compound 29) (0.2 g, 0.54 mmol) was added to stirring diethyl ether in a round bottom flask and methane sulfonic acid (0.06 g, 0.62 mmol) was slowly added. After 5 minutes, the precipitate was filtered off and dried under vacuum to yield N-benzyl-N-(4-(dimethylamino)benzyl)-1-(4-(dimethylamino)phenyl)methanaminium methanesulfonate as a light brown viscous substance.

Example 50 Aqueous Solubility of Free Bases and Various Salts

(135) The aqueous solubility of various amine compounds and their corresponding salts were determined by dissolution of various amounts of the respective compounds at room temperature.

(136) TABLE-US-00002 Solubility Solubility free base Solubility Mesylate Compound (mg/ml) HCl salt salt 17 <0.36 <0.36 <0.36 18 <0.05 <0.05 <0.05 19 <0.05 <0.05 <0.05 20 <3 <3 <3 22 <0.05 4.5 >22 23 <0.05 15 >34 25 <0.05 <0.05 <0.05 26 <1.5 <1.5 <1.5 27 <0.5 <0.5 <0.5 29 <0.16 6 94

Example 51 Aqueous Solubility of Tribenzylamine as Free Bases and Various Salts

(137) Tribenzylamine and the corresponding salts were dissolved in water and the solubility was quantified (area under curve (AUC)) by HPLC

(138) TABLE-US-00003 Solubility of tribenzylamine and its corresponding HCl and mesylate salts, tested on HPLC. Free Mesylate base HCl salt salt Compound (mAu*s) (mAu*s) (mAu*s) AUC 0 406 1975

Examples 52-54, Pharmacological Testing of Newly Synthesized Compounds

Example 52, Testing of Derivatized and Newly Synthesized Tertiary Amines in the AKAP18-PLB AlphaScreen Assay

(139) The compounds of formula (I) and (I) were made in two groups that differed in their substituents on the aromatic rings. Tertiary amines where two of three substituents were similar were defined in one group (Series 2, 2a-2h), while those containing three unique substituents were in the other (Series 3, 3a-3g).

(140) Two compounds in the group 2a-2h were found to have particularly low EC.sub.50 values. Compound 2b contained four hydrogen-bond donors and two hydrogen-bond acceptors, increasing the possibility of binding to the protein complex through hydrogen binding. In compound 2g two nitrogens were introduced in substituents coupled to the aromatic rings, leaving the molecule with three hydrogen-binding domains. FIGS. 2 and 3 show the concentration-response curves of compounds 2b and 2g in the AKAP18 assay.

(141) In the last group of derivatized compounds, the central nitrogen had three different substituents, and by using fragments of the compounds giving a low EC.sub.50 value further highly active compounds where obtained. Compound 3b was based on the structure of 2b, having one substituent with hydroxyl groups, and the other with fluorine in para-position. The EC.sub.50 value of 3b was approximately the same as for 2b, as shown in FIG. 4.

(142) Two compounds were synthesized based on 2g, keeping one of the two dimethylamino substituents. In 3c the other part of the molecule was a diphenyl group, a hydrophobic moiety like the one to be found in some of the original compounds. This resulted in an EC.sub.50 value of 0.327 M and was the most potent compound described. Compound 3d also contained the dimethylamino group, this time in combination with a benzofuran. This compound had an EC.sub.50 of 2.79 M. The concentration response curves of compounds 3c and 3d are shown in FIGS. 5 and 6.

(143) In order to find out whether an acidic moiety was favorable, a compound containing both a basic moiety and a carboxylic acid was synthesised. The synthesis started with making the methylether. After completion of the reaction, a small amount of compound 3e was isolated to before hydrolysis to provide compound 3f. In 3f a carboxylic acid was introduced on the benzene ring, giving the molecule both basic- and acidic properties, like the original group of analogues had. However, compound 3e (EC.sub.50 34.84 M) and compound 3f (36.55 M) were found to have slightly higher EC.sub.50 values.

(144) In the last compound synthesized another acidic group was introduced, this time a tetrazole. The tetrazole is a part of the pharmacophore of a group of existing drugs, angiotensin-II blockers, commonly used in the treatment of hypertension.

(145) ##STR00235##

(146) The rationale for introducing this group was that it had the acidic properties similar to the originally identified compound, and earlier testing had shown that substituents containing nitrogen seemed to be preferable in terms of low EC.sub.50. In this case, four nitrogens were introduced to the molecule, making molecule able to act both as hydrogen binding acceptor and donor. In addition to this an extra benzene ring was added, giving the molecule a larger hydrophobic site. However, compound 3g was found to have the highest EC.sub.50 value of this screen with 47.07 M.

Example 53, Cell-Based Testing of Derivatized Compounds in Cardiomyocytes

(147) Based on data from studies in cardiomyocytes assay using a peptide that blocks AKAP18-PLB interaction, it was expected that effective compounds would also cause disruption of the AKAP18-PLB complex and decrease PLB-Ser.sup.16 phosphorylation (Lygren et al. in EMBO Reports, volume 8, issue 11, page 1061-1067 (2007). A small selection of hits from the derivatized compounds were tested in this cell-based assay, performed as described in Lygren et al.

(148) Briefly, compounds were added to primary cultures of rat neonatal cardiomyocytes were added compounds about 24 hour prior stimulation with isoproterenol, and amount of phosphorylated Ser.sup.16-PLB was analyzed by western blotting using a pSer.sup.16-PLB phosphospecific antibody. Actin was used as a loading control.

(149) Four synthesized compounds were tested, as shown below:

(150) ##STR00236##

(151) Compound 2g displayed a concentration dependent inhibition of PLB-phosphorylation. Even though compound 3c had the lowest EC.sub.50 value of the synthesized compounds in vitro, the effect on PLB phosphorylation in cardiomyocytes was somewhat weaker than that of 2g. Compounds 3d and 3f were tested in the same experiment, but did not appear to have an effect on the phosphorylation of PLB in this assay.

(152) The results are shown in FIG. 7. Rat neonatal cardiomyocytes treated with compounds 24 hours prior stimulation with isoproterenol (100 nM, 5 minutes). The histogram shows levels of phosphorylated Ser.sup.16-PLB quantified by densiometery relative to actin levels.

Example 54, Cell Viability Assay on Derivatized Compounds

(153) Compound toxicity was measured in a luminescence assay that gives a signal proportional to the amount of metabolically active cells in the sample; the assay was performed as described by the supplier. All synthesized compounds were tested in the viability assay, and viability was registered at 24 hours and 72 hours. The results after 24 hours incubation with the synthesised compounds is shown in FIG. 8.

Examples 52-54 Summary of Test Results on Derivatized Compounds

(154) TABLE-US-00004 TABLE 2 Overview of effect and viability of synthesized tertiary amines with two different substituents. EC.sub.50 average is given with standard deviation (n = 3) or half range (n = 2). = no activity, + = possible effect, ++ = effect, n.d = not done Effect on PLB EC.sub.50 phosphorylation Viability in M in (100 M) Compound Structure (n) cardiomyocytes 24 hours 2a n.d 0.85 2b 12.33 3.08 (2) 0.10 2c n.d 1.11 2d 0 n.d 1.15 2e n.d 1.01 2f n.d 0.92 2g 1.7 0.91 (3) ++ 0.56 2h n.d 0.91

(155) TABLE-US-00005 TABLE 3 Overview of effect and viability of synthesized tertiary amines with three different substituents. EC.sub.50 average is given with standard deviation (n = 3) or half range (n = 2). = no activity, + = possible effect, ++ = effect, n.d = not done Effect on PLB phosphorylation Viability EC.sub.50 in (100 M) Compound Structure (n) cardiomyocytes 24 hours 3a n.d 0.79 3b 5.43 1.11 (2) n.d 0.03 3c 0.44 0.18 (3) + 0.74 3d 2.45 0.73 (3) 0.58 3e 34.84 (1) n.d 0.57 3f 0 40.56 7.4 (3) + 0.98 3g 47.07 (1) n.d 0.80

Example 55

(156) Following on Example 53, two compounds, 2g and 3d (FIGS. 9A,B and 9C, respectively), were tested in more detail for their ability to interfere with phosphorylation of phospholamban after stimulation of neonatal (FIGS. 9A and 9C) or adult (FIG. 9B) primary cultures of cardiomyocytes. As can be seen from the Figure, compound 2g inhibited phospholamban phosphorylation with a half-maximal effect of approximately 40 and 100 M in neonatal and adult cardiomyocytes, respectively whereas compound 3d had a half-maximal effect of approximately 100 M in neonatal cells.

Example 56

(157) Adult rat cardiomyocytes were isolated and perfused with external solution (NaCl 125, CsCl 20, D-glucose 5, MgCl.sub.2 1, CaCl.sub.2 1.8, Hepes 10, 4-aminopyridine 5, probenecid 2, pH 7.4 by NaCH). Cell dialysis was achieved by 1-2 M patch pipettes, filled with (in mM): CsCl 115, TEACl 20, Hepes 10, MgATP 5, Na2-Phosphocreatinine 5, EGTA 0.04, cAMP 0.005, adjusted to pH=7.2 with CsOH. Compound 2g (mesylate salt dissolved in water) was included at a concentration of 10 uM. Whole-cell Ca fluorescence measured from voltage-clamped cells was performed with Cairn Research Optoscan Monochromator (Excitation 488 nm, emission 515 nm long pass) (Cain Research Ltd., Faverham, UK). By applying 100 ms square voltage step from 45 to 0 mV at 0.125 Hz by an Axociamp 2B amplifier (Axon Instruments, Foster City, Calif., USA), basal Ca transients were recorded, and tau values were obtained by monoexponential fitting of the Ca.sup.2+ extrusion phase from regular transients () and caffeine transients (caff) after rapid applications of 10 mM caffeine. SERCA2 rate constant were calculated as: k_SERCA=1/1/caff.

(158) As can been seen from the figure, incubation with compound 2g in the presence of cAMP increased SERCA2 activity leading to a faster calcium reuptake in sarcoplasmic reticulum and relaxation of the heart to allow filling. This indicates an ability of this class of compounds to regulation SERCA2 activity, positively or negatively, possibly depending on concentration or other aspects of the structure activity relationship.