2,4-Diaminopyrimidines as dual ligands at the

histamine H1 and H4 receptor –

H1/H4-receptor selectivity

Sebastian G. Hammera, Susanne Goblederb, Franziska Naporrab,

Hans-Joachim Wittmannc, Sigurd Elzb, Markus R. Heinricha and

Andrea Strasserc

a Department of Chemistry and Pharmacy, Pharmaceutical Chemistry, Friedrich-

Alexander-Universität Erlangen-Nürnberg, D-91052 Erlangen, Germany

b Department of Pharmaceutical/Medicinal Chemistry I, Institute of Pharmacy,

University of Regensburg, D-93040 Regensburg, Germany

c Department of Pharmaceutical/Medicinal Chemistry II, Institute of Pharmacy,

University of Regensburg, D-93040 Regensburg, Germany

Corresponding authors:

Pharmacology and Molecular Modelling: Dr. Andrea Strasser, Department of

Pharmaceutical and Medicinal Chemistry II, University of Regensburg,

Universitätsstraße 31, D-93053 Regensburg, Germany

Phone: +49-941-943-4821; Fax: +49-941-943-4820

Email: andrea.strasser@chemie.uni-regensburg.de

Synthesis: Prof. Dr. Markus R. Heinrich, Department of Chemistry and Pharmacy,

Pharmaceutical Chemistry, Friedrich-Alexander-Universität Erlangen-Nürnberg,

Schuhstraße 19, D-91052 Erlangen, Germany

Phone: +49-9131-8524115; Fax: +49-9131-8522585

Email: markus.heinrich@fau.de

1. Chemistry1.1. Synthesis of the lead compound 11.2. Synthesis of the final compounds 6a-e and 7a-c1.2.1. Synthesis of 41.2.2. Synthesis of 51.2.3. General procedure for the synthesis of the mono-aminated precursor for 6a-e1.2.4. General procedure for the synthesis of 6a-e 1.2.5. General procedure for the synthesis of the mono-aminated precursor for 7a-c1.2.6. General procedure for the synthesis of 7a-c

1.3. Synthesis of the final compounds 10a-c, 11a-c, 12a and 12b1.3.1. Synthesis of 91.3.2. General procedure for the synthesis of the mono-aminated precursors for 10a-c1.3.3. General procedure for the synthesis of 10a-c1.3.4. General procedure for the synthesis of the mono-aminated precursors for 11a-c1.3.5. General procedure for the synthesis of 11a-c1.3.6. General Procedure for the synthesis of 12a and 12b1.4. Synthesis of the final compounds 15a-d1.4.1. Synthesis of 131.4.2. Synthesis of 141.4.3. General procedure for the synthesis of the mono-aminated precursors for 15a-c1.4.4. General procedure for the synthesis of 15a-c1.4.5. Synthesis of 15d1.5. NOESY spectra

2. Pharmacology2.1. Materials 2.2. Preparation of compound stock solutions2.3. Cell culture and membrane preparation2.4. Radioligand competition binding assays2.5. [35S]GTPγS binding assay2.6. Data analysis

3. Molecular Modelling3.1. Generation of the hH1R and hH4R homology models3.2. Energetical considerations

4. References

1. ChemistryColumn chromatography was performed on silica gel, 230 - 400 mesh ASTM. TLC plates were visualized with UV. All reagents were purchased from commercial sources and used without additional purification. 1H NMR, 13C NMR and 19F NMR spectra were recorded on Bruker Avance 600 or Avance 360 spectrometers using either CDCl3 or CD3OD as solvent with TMS or C6F6 (for 19F NMR) as reference. Mass spectra were recorded on a Jeol GC mate II spectrometer (EI), on a Bruker MicrOTOF II (ESI-HRMS) or a Bruker maXis 4G (ESI-HRMS). High performance liquid chromatography was performed on a Varian 940-LC liquid chromatography system. The detection was done by a photo diode array-detector (Varian, 254 and 280 nm). For preparative HPLC, the chromatography column “Pursuit XRs C8” (RP-8, 150 × 21.2 mm, 5 μm particle size) was used.

1.1. Synthesis of the lead compound 14-(4-Methylpiperazin-1-yl)-5,6-dihydrobenzo[h]quinazolin-2-amine (1). Under nitrogen at-mosphere, α-tetralone (25.0 mmol, 3.33 mL), dimethyl carbonate (300 mmol, 25.0 mL), sodium hydride (60% in mineral oil, 50.0 mmol, 2.00 g) and dry methanol (0.1 mL) are heated to 80 °C and stirred for 3 hours. After cooling, hydrochloric acid (3 N, 30.0 mL) is added, the mixture is extracted with ethyl acetate and the combined organic phases are dried over sodium sulphate. After removing the solvent, a red-brown liquid is obtained. This liquid (5.00 mmol, 1.02 g), guanidine hydrochloride (10.0 mmol, 0.95 g) and potassium carbonate (20.0 mmol, 2.76 g) are dissolved in DMF (15.0 mL) and stirred for 2 hours at 125 °C. After cooling, the reaction mixture is diluted with water and the pH value is adjusted to about 6 using glacial acetic acid. The resulting light brown precipitate is filtered off and dried in vacuo (3.52 mmol, 750 mg, 70%). The previously obtained brown solid (0.94 mmol, 200 mg) is dissolved in phosphorous oxychloride (7.00 mL) and the solution is stirred for 23 hours at 110 °C. After cooling, the solution is poured onto ice and the pH value is adjusted to 10 to 11 using potassium carbonate. The aqueous phase is extracted with ethyl acetate (3 ×). Subsequently, the combined organic phases are washed with brine, dried over sodium sulphate and the solvent is removed under reduced pressure (0.61 mmol, 142 mg, 65%). The obtained brown solid (0.23 mmol, 53.3 mg) is dissolved in 1-methyl-piperazine (2.00 mL). The reaction mixture is heated to 90 °C and is stirred for 18 hours. Subsequently, the excess of amine is removed via Kugelrohr distillation and the crude product is purified via reversed phase-HPLC (mobile phase: water + 0.1% formic acid (A) and methanol (B); linear gradient 0-100% B in 100-0% A over 13 minutes) to yield 3 (15.8 mg, 23%) as yellowish solid. tR (HPLC) 9.50 min; 1H NMR (600 MHz, CDCl3): δ 2.62-2.65 (m, 5 H), 2.82-2.85 (m, 2 H), 3.01-3.04 (m, 4 H), 3.78-3.82 (m, 4 H), 7.24-7.27 (m, 1 H), 7.40-7.43 (m, 2 H), 8.06 (dd, J = 3.8 Hz, J = 5.2 Hz, 1 H); 13C NMR (151 MHz, CDCl3): δ 23.9 (CH2), 28.2 (CH2), 44.0 (2 × CH2), 45.7 (2 × CH2), 53.1 (CH3), 106.0 (CH), 125.5 (CH), 127.7 (CH), 128.8 (Cq), 131.4 (Cq), 138.2 (CH), 153.9 (Cq), 157.1 (Cq), 165.3 (Cq), 166.1 (Cq); MS (EI) m/z (%): 295 (28) [M+], 226 (19), 225 (100), 212 (40), 211 (11), 169 (20), 71 (10), 43 (14), 42 (12); HRMS (EI) m/z calcd. for C17H21N5 [M+]: 295.1797, found: 295.1798. CAS 1027331-46-9.1

1.2. Synthesis of the final compounds 6a-e and 7a-c1.2.1. Synthesis of 4Methyl 1-oxo-1,2,3,4-tetrahydronaphthalene-2-carboxylate (4). Under nitrogen atmo-sphere, α-tetralone (25.0 mmol, 3.33 mL), dimethyl carbonate (300 mmol, 25.0 mL), sodium hydride (60% in mineral oil, 50.0 mmol, 2.00 g) and dry methanol (0.1 mL) are heated to 80 °C and stirred for 3 hours. After cooling, hydrochloric acid (3 N, 30.0 mL) is added, the mixture is extracted with ethyl acetate and the combined organic phases are dried over sodium sulphate. After removing the solvent, 4 (5.06 g, 100%) is obtained as a red-brown liquid; 1H NMR (600 MHz, CDCl3, 2 tautomers, ratio 1:1): δ 2.32-2.38 (m, 1 H), 2.46-2.54 (m, 1 H), 2.56 (dd, J = 7.5 Hz, J = 8.2 Hz, 2 H), 2.78-2.82 (m, 2 H), 2.96-3.02 (m, 1 H), 3.04-3.09 (m, 1 H), 3.62 (dd, J = 4.7 Hz, J = 10.4 Hz, 1 H), 3.78 (s, 3 H), 3.82 (s, 3 H), 7.17 (d, J = 7.3 Hz, 1 H),7.25 (d, J = 7.7 Hz, 1 H), 7.28 (t, J = 7.5 Hz, J = 7.5 Hz, 1 H), 7.32 (t,

J = 7.5 Hz, J = 7.5 Hz, 2 H), 7.49 (dt, J = 1.4 Hz, J = 7.5 Hz, J = 7.5 Hz, 1 H), 7.80 (dd, J = 1.4 Hz, J = 7.6 Hz, 1 H), 8.04 (dd, J = 1.3 Hz, J = 7.8 Hz, 1 H); 13C NMR (91 MHz, CDCl3, 2 tautomers): δ 20.5 (CH2), 26.4 (CH2), 27.6 (CH2), 27.7 (CH2), 51.6 (CH3), 52.3 (CH3), 54.4 (CH), 96.8 (Cq),124.3 (CH), 126.6 (CH), 126.9 (CH), 127.4 (CH), 127.8 (CH), 128.8 (CH), 129.9 (Cq), 130.5 (CH), 131.7 (Cq), 133.9 (CH), 139.4 (Cq), 143.6 (Cq), 165.1 (Cq), 170.6 (Cq), 173.0 (Cq), 193.1 (Cq). CAS 7442-52-6 (keto form), 95448-04-7 (enol form).2

1.2.2. Synthesis of 55,6-Dihydrobenzo[h]quinazoline-2,4(1H,3H)-dione (precursor to 5). 4 (20.0 mmol, 4.08 g) and urea (500 mmol, 30.0 g) are heated to 190 °C and the mixture is stirred for 45 minutes. After cooling, but before congelation of the molten mass, water (100 mL) is added and the mixture is stirred for further 15 minutes. A precipitate is formed, filtered of and dried in vacuo. The product (4.27 g, 100%) is obtained as a light brown solid; 1H NMR (360 MHz, DMSO-d6): δ 2.44-2.49 (m, 2 H), 2.77-2.82 (m, 2 H), 7.31-7.44 (m, 3 H), 7.87-7.90 (m, 1 H); 13C NMR (91 MHz, DMSO-d6): δ 18.5 (CH2), 27.1 (CH2), 106.2 (CH), 124.0(Cq), 126.8(Cq), 128.3 (CH), 130.7 (CH), 139.0 (CH), 143.9(Cq), 151.4(Cq), 159.8(Cq), 164.0 (Cq). CAS 236093-45-1.3

2,4-Dichloro-5,6-dihydrobenzo[h]quinazoline (5). 5,6-Dihydrobenzo[h]quinazoline-2,4(1H,3H)-dione (0.93 mmol, 200 mg) is dissolved in phosphorous oxychloride (15.0 mL) and 5 drops of DMF are added. The reaction mixture is heated to 110 °C and stirred for 46 hours. After cooling, the solution is poured onto ice and the pH value is adjusted to 10 to 11 using potassium carbonate. The aqueous phase is extracted with ethyl acetate (3 ×). Subsequently, the combined organic phases are dried over sodium sulphate and the solvent is removed under reduced pressure. 5 (59.1 mg, 26%) is obtained as a yellow solid; 1H NMR (360 MHz, CDCl3): δ 3.00-3.10 (m, 4 H), 7.27 (d, J = 7.6 Hz, 1 H), 7.39 (dt, J = 1.4 Hz, J = 7.6 Hz, 1 H), 7.46 (dt, J = 1.5 Hz, J = 7.5 Hz, 1 H), 8.31 (dd, J = 1.3 Hz, J = 7.9 Hz, 1 H); 13C NMR (91 MHz, CDCl3): δ 23.3 (CH2), 26.6 (CH2), 125.4 (CH), 126.7 (Cq), 127.6 (Cq), 128.2 (CH), 130.1 (Cq), 132.5 (CH), 139.1 (CH), 158.3 (Cq), 160.7 (Cq), 163.5(Cq). CAS 1186410-71-8.4

1.2.3. General procedure for the synthesis of the mono-aminated precursor for 6a-eThe respective dichloropyrimidine derivative 5 (200 mg) and 1.5 equivalents of 1-methylpiperazine are dissolved in DMF (2.00 mL). The reaction mixture is heated to 40 °C and stirred for 18 hours. Subsequently, the solvent is removed under reduced pressure and the crude product is purified via column chromatography (SiO2, ethyl acetate / methanol = 3:1).2-Chloro-4-(4-methylpiperazin-1-yl)-5,6-dihydrobenzo[h]quinazoline (precursor to 6a-e). Dark yellow solid (34%); Rf 0.3 (ethyl acetate / methanol = 3:1) [UV]; 1H NMR (600 MHz, CDCl3): δ 2.37 (s, 3 H), 2.56-2.59 (m, 4 H), 2.75 (dd, J = 5.8 Hz, J = 8.4 Hz, 2 H), 2.86 (dd, J = 6.2 Hz, J = 8.2 Hz, 2 H), 3.48-3.51 (m, 4 H), 7.21-7.23 (m, 1 H), 7.33-7.38 (m, 2 H), 8.17 (dd, J = 1.9 Hz, J = 7.2 Hz, 1 H); 13C NMR (151 MHz, CDCl3): δ 24.3 (CH2), 27.8 (CH2), 45.9 (2 × CH2), 47.8 (CH3), 54.6 (2 × CH2), 113.9 (CH), 125.9 (CH), 127.2 (CH), 127.3 (Cq), 130.5 (CH), 132.7 (Cq), 137.9 (Cq), 158.2 (Cq), 161.5 (Cq), 166.1 (Cq).

1.2.4. General procedure for the synthesis of 6a-eThe respective chloropyrimidine derivative (between 30 mg and 60 mg) is dissolved in the respective amine (2.00 mL). The reaction mixture is heated to 90 °C and is stirred for 18 hours. Subsequently, the amine is removed via Kugelrohr distillation and the crude product is purified either via column chromatography (SiO2, EtOAc / MeOH = 3:1) or reversed phase-HPLC (mobile phase: water + 0.1% formic acid (A) and methanol (B); linear gradient 10-100% B in 90-0% A over 15 minutes).

N-Benzyl-4-(4-methylpiperazin-1-yl)-5,6-dihydrobenzo[h]quinazolin-2-amine (6a). Yellowish solid (24%, purification via HPLC); tR (HPLC) 9.75 min; 1H NMR (600 MHz, CDCl3): δ 2.47 (s, 3 H), 2.61 (dd, J = 6.3 Hz, J = 8.1 Hz, 2 H), 2.73-2.83 (m, 6 H), 3.62-3.66 (m, 4 H), 4.62 (s, 2 H), 7.22-7.26 (m, 2 H), 7.32 (t, J = 7.7 Hz, 2 H), 7.36-7.40 (m, 4 H), 8.09-8.11 (m, 1 H); 13C NMR (91 MHz, CDCl3): δ 23.8 (CH2), 28.4 (CH2), 44.2 (2 × CH2), 45.4 (CH3), 46.1 (CH2), 53.0 (2 × CH2), 125.5 (CH), 127.0 (CH), 127.2 (2 × CH), 127.4 (Cq), 127.5 (Cq), 128.5 (2 × CH), 130.8 (CH), 138.4 (CH), 139.4 (Cq), 165.0 (Cq), 4 signals missing (CH, Cq, Cq, Cq); MS (EI) m/z (%): 386 (12) [MH+], 385 (43) [M+], 316 (25), 315 (100), 303 (12), 302 (59), 91 (38), 85 (13), 83 (19), 71 (10), 57 (14), 43 (12); HRMS (EI) calcd. for C24H27N5 [M+]: 385.2266, found: 385.2267.N-(4-Methylbenzyl)-4-(4-methylpiperazin-1-yl)-5,6-dihydrobenzo[h]quinazolin-2-amine (6b). Yellowish solid (17%, purification via HPLC); tR (HPLC) 10.55 min; 1H NMR (600 MHz, CDCl3): δ 2.32 (s, 3 H), 2.47 (s, 3 H), 2.59-2.62 (m, 2 H), 2.73-2.82 (m, 6 H), 3.60-3.65 (m, 4 H), 4.58 (s, 2 H), 7.13 (d, J = 7.8 Hz, 2 H), 7.21-7.23 (m, 1 H), 7.25-7.27 (m, 2 H), 7.36-7.39 (m, 2 H), 8.10 (d, J = 7.4 Hz, 1 H); 13C NMR (91 MHz, CDCl3): δ 21.0 (CH3), 23.8 (CH2), 28.4 (CH2), 44.2 (2 × CH2), 45.1 (CH3), 46.1 (CH2), 53.1 (2 × CH2), 125.5 (CH), 127.3 (2 × CH), 127.4 (CH), 127.5 (CH), 129.2 (2 × CH), 136.6 (Cq), 138.4 (Cq), 165.0 (Cq), 6 signals missing (CH, Cq, Cq, Cq, Cq, Cq); MS (EI) m/z (%): 399 (4) [M+], 329 (15), 270 (15), 235 (16), 105 (17), 44 (100), 43 (12); HRMS (EI) calcd. for C25H29N5 [M+]: 399.2423, found: 399.2423.N-(4-Fluorobenzyl)-4-(4-methylpiperazin-1-yl)-5,6-dihydrobenzo[h]quinazolin-2-amine (6c). Yellowish solid (21%, purification via HPLC); tR (HPLC) 11.10 min; 1H NMR (600 MHz, CDCl3): δ 2.47 (s, 3 H), 2.62 (dd, J = 6.2 Hz, J = 8.2 Hz, 2 H), 2.72-2.78 (m, 4 H), 2.80-2.83 (m, 2 H), 3.56-3.60 (m, 4 H), 4.59 (s, 2 H), 7.01 (t, J = 8.7 Hz, 2 H), 7.21-7.24 (m, 1 H), 7.34 (dd, J = 5.5 Hz, J = 8.6 Hz, 2 H), 7.36-7.38 (m, 2 H), 8.11 (dd, J = 2.7 Hz, J = 6.3 Hz, 1 H); 13C NMR (91 MHz, CDCl3): δ 23.8 (CH2), 28.5 (CH2), 44.8 (2 × CH2), 45.3 (CH3), 47.1 (CH2), 54.1 (2 × CH2), 115.2 (d, JCF = 21.4 Hz, 2 × CH), 125.3 (CH), 127.2 (Cq), 127.3 (CH), 129.0 (d, JCF = 8.0 Hz, 2 × CH), 138.4 (Cq), 161.9 (d, JCF = 244.7 Hz, Cq), 165.4 (Cq), 6 signals missing (CH, CH, Cq, Cq, Cq, Cq); 19F NMR (339 MHz, CDCl3): δ -119.0; MS (EI) m/z (%): 403 (23) [M+], 347 (10), 334 (19), 333 (63), 332 (15), 331 (30), 320 (20), 319 (12), 318 (20), 308 (14), 252 (46), 250 (25), 235 (19), 233 (11), 208 (11), 124 (16), 123 (13), 110 (10), 109 (92), 85 (16), 83 (27), 57 (10), 44 (100), 43 (11); HRMS (EI) calcd. for C24H26FN5 [M+]: 403.2172, found: 403.2172.N-(4-Methoxybenzyl)-4-(4-methylpiperazin-1-yl)-5,6-dihydrobenzo[h]quinazolin-2-amine (6d). Red-brown solid (31%, purification via column chromatography); Rf 0.2 (EtOAc / MeOH = 3:1) [UV]; 1H NMR (600 MHz, CDCl3): δ 2.36 (s, 3 H), 2.53-2.57 (m, 4 H), 2.66 (dd, J = 6.1 Hz, J = 8.2 Hz, 2 H), 2.80-2.83 (m, 2 H), 3.38-3.42 (m, 4 H), 3.79 (s, 3 H), 4.59 (d, J = 5.9 Hz, 2 H), 6.86 (d, J = 8.8 Hz, 2 H), 7.18-7.21 (m, 1 H), 7.30-7.34 (m, 4 H), 8.15 (d, J = 8.5 Hz, 1 H); 13C NMR (91 MHz, CDCl3): δ 23.8 (CH2), 28.6 (CH2), 45.1 (2 × CH2), 45.9 (CH3), 47.7 (CH2), 54.7 (2 × CH2), 55.3 (CH3), 113.9 (2 × CH), 125.3 (CH), 127.0 (CH), 127.2 (CH), 128.5 (Cq), 128.9 (2 × CH), 138.5 (Cq), 158.7 (Cq), 165.8 (Cq), 5 signals missing (CH, Cq, Cq, Cq, Cq); MS (EI) m/z (%): 415 (19) [M+], 346 (17), 345 (72), 332 (43), 237 (21), 121 (100), 43 (19); HRMS (EI) calcd. for C25H29N5O [M+]: 415.2372, found: 415.2370.4-(4-Methylpiperazin-1-yl)-N-phenethyl-5,6-dihydrobenzo[h]quinazolin-2-amine (6e). Yellowish solid (18%, purification via column chromatography); Rf 0.2 (EtOAc / MeOH = 3:1) [UV]; 1H NMR (600 MHz, CDCl3): δ 2.36 (s, 3 H), 2.54-2.57 (m, 4 H), 2.67 (dd, J = 6.0 Hz, J = 8.3 Hz, 2 H), 2.80-2.83 (m, 2 H), 2.94-2.97 (m, 2 H), 3.36-3.41 (m, 4 H), 3.71 (dd, J = 6.4 Hz, J = 14.3 Hz, 2 H), 7.18-7.24 (m, 3 H), 7.27-7.28 (m, 1 H), 7.30-7.33 (m, 4 H), 8.14 (d, J = 7.3 Hz, 1 H); 13C NMR (91 MHz, CDCl3): δ 23.8 (CH2), 28.6 (CH2), 36.3 (CH2), 43.1 (2 × CH2), 46.1 (CH3), 48.0 (CH2), 54.9 (2 × CH2), 125.2 (CH), 126.2 (CH), 126.9 (CH), 127.2 (CH), 127.7 (Cq), 128.5 (2 × CH), 128.7 (Cq), 128.9 (2 × CH), 129.5 (CH), 138.5 (Cq), 139.8 (Cq), 166.0 (Cq), 2 signals missing (Cq, Cq); MS (EI) m/z (%): 399 (19) [M+], 330 (24), 329 (100), 317 (12), 316 (39), 105 (42), 43 (14), 28 (15); HRMS (EI) calcd. for C25H29N5 [M+]: 399.2423, found: 399.2423.

1.2.5. General procedure for the synthesis of the mono-aminated precursors for 7a-cThe respective dichloropyrimidine derivative 5 (between 60 mg and 120 mg) and 1.5 equivalents of the respective amine are dissolved in DMF (2.00 mL). The reaction mixture is heated to 40 °C and stirred for 18 hours. Subsequently, the solvent is removed under reduced pressure and the crude product is purified via column chromatography (SiO2, dichloromethane / ethyl acetate = 20:1).N-Benzyl-2-chloro-5,6-dihydrobenzo[h]quinazolin-4-amine (precursor to 7a). Yellowish solid (47%); Rf 0.4 (dichloromethane) [UV]; 1H NMR (600 MHz, CDCl3): δ 2.56-2.59 (m, 2 H), 2.94-2.98 (m, 2 H), 4.74 (d, J = 5.3 Hz, 2 H), 7.19-7.22 (m, 1 H), 7.32-7.36 (m, 3 H), 7.37-7.39 (m, 4 H), 8.24 (dd, J = 2.7 Hz, J = 6.5 Hz, 1 H); 13C NMR (151 MHz, CDCl3): δ 20.3 (CH2), 26.8 (CH2), 45.6 (CH2), 108.5 (Cq), 125.8 (CH), 127.2 (CH), 127.6 (CH), 127.8 (CH), 128.2 (2 × CH), 128.9 (2 × CH), 130.5 (CH), 132.0 (Cq), 137.4 (Cq), 138.1 (Cq), 158.1 (Cq), 159.0 (Cq), 161.4 (Cq).2-Chloro-N-phenethyl-5,6-dihydrobenzo[h]quinazolin-4-amine (precursor to 7b). Yellowish solid (48%); Rf 0.4 (dichloromethane) [UV]; 1H NMR (600 MHz, CDCl3): δ 2.44 (dd, J = 7.1 Hz, J = 8.3 Hz, 2 H), 2.91-2.94 (m, 2 H), 2.96 (t, J = 6.8 Hz, 2 H), 3.82 (dt, J = 5.7 Hz, J = 6.8 Hz, 2 H), 7.18-7.20 (m, 1 H), 7.22-7.24 (m, 2 H), 7.32-7.36 (m, 5 H), 8.23 (dd, J = 2.3 Hz, J = 6.8 Hz, 1 H); 13C NMR (151 MHz, CDCl3): δ 20.1 (CH2), 26.8 (CH2), 35.3 (CH2), 42.4 (CH2), 108.5 (Cq), 125.7 (CH), 126.7 (CH), 127.2 (CH), 127.6 (CH), 128.7 (2 × CH), 128.8 (2 × CH), 130.4 (CH), 132.0 (Cq), 137.3 (Cq), 138.7 (Cq), 157.8 (Cq), 159.0 (Cq), 161.5 (Cq).

(4'-Chloro-6-methoxy-[1,1'-biphenyl]-3-yl)methanamine (precursor to 7c) A solution of 4-chlorophenyldiazonium tetrafluoroborate (2.00 mmol, 452 mg in 6.00 mL of water / acetonitrile = 2:1) is added dropwise to a degassed solution of 4-methoxyaniline (10.0 mmol, 1.23 g) and titanium(III)-chloride(1 M in 3 N hydrochloric acid, 4.00 mmol, 4.00 mL) in water (6.00 mL) over a period of 15 minutes. The reaction mixture is stirred for another 10 minutes and a solution of sodium hydroxide (2.00 g) and sodium sulfite (2.00 g) in water (20.0 mL) is added. Subsequently, the aqueous solution is extracted with ethyl acetate (3 × 100 mL) and the combined organic phases are washed with brine, dried over sodium sulphate and the solvent is removed under reduced pressure. Residual 4-methoxyaniline is removed by Kugelrohr distillation at 120 °C and the product is used without further purification. 1H NMR (360 MHz, CDCl3): δ 3.79 (s, 3H), 3.85 (s, 2 H), 6.94 (d, J = 8.4 Hz, 1 H), 7.23 (d, J = 2.3 Hz, 1 H), 7.27 (dd, J = 2.3 Hz, J = 8.4 Hz, 1 H), 7.34 - 7.37 (m, 2 H), 7.45-7.48 (m, 2 H).2-Chloro-N-((4'-chloro-6-methoxy-[1,1'-biphenyl]-3-yl)methyl)-5,6-dihydrobenzo[h]quinazolin-4-amine (precursor to 7c). Yellowish solid (27%); Rf 0.1 (hexane / dichloromethane = 2:1) [UV]; 1H NMR (600 MHz, CDCl3): δ 2.56 (dd, J = 7.0 Hz, J = 8.3 Hz, 2 H), 2.94 (t, J = 7.6 Hz, 2 H), 3.81 (s, 3 H), 4.69 (d, J = 5.3 Hz, 2 H), 6.98 (d, J = 2.2 Hz, 1 H), 7.08 (dd, J = 2.5 Hz, J = 8.5 Hz, 1 H), 7.17-7.20 (m, 1 H), 7.31-7.37 (m, 5 H), 6.46 (d, J = 8.7 Hz, 2 H), 8.23 (dd, J = 2.2 Hz, J = 7.0 Hz, 1 H).

1.2.6. General procedure for the synthesis of 7a-cThe respective chloropyrimidine derivative (between 30 mg and 60 mg) is dissolved in the respective amine (2.00 mL). The reaction mixture is heated to 90 °C and is stirred for 18 hours. Subsequently, the amine is removed via Kugelrohr distillation and the crude product is purified either via column chromatography (SiO2, EtOAc / MeOH = 3:1) or reversed phase-HPLC (mobile phase: water + 0.1% formic acid (A) and methanol (B); linear gradient 10-100% B in 90-0% A over 15 minutes).N-Benzyl-2-(4-methylpiperazin-1-yl)-5,6-dihydrobenzo[h]quinazolin-4-amine (7a). Yellowish solid (33%, purification via column chromatography); Rf 0.1 (EtOAc) [UV]; 1H NMR (600 MHz, CDCl3): δ 2.41 (s, 3 H), 2.51-2.60 (m, 6 H), 2.92 (t, J = 7.6 Hz, 2 H), 3.92-4.00 (m, 4 H), 4.70 (d, J = 5.4 Hz, 2 H), 7.16-7.19 (m, 1 H), 7.27-7.38 (m, 7 H), 8.22-8.25 (m, 1 H); 13C NMR (91 MHz, CDCl3): δ 20.2 (CH2), 27.7 (CH2), 43.5 (2 × CH2), 45.2 (CH3), 45.9 (CH2), 54.9

(2 × CH2), 100.3 (Cq), 125.1 (CH), 126.7 (CH), 127.3 (CH), 127.4 (CH), 127.8 (2 × CH), 128.6 (2 × CH), 129.3 (CH), 134.0 (Cq), 138.0 (Cq), 139.7 (Cq), 156.3 (Cq), 160.4 (Cq), 160.6 (Cq); MS (EI)m/z (%): 386 (25) [MH+], 385 (84) [M+], 383 (13), 329 (15), 328 (12), 327 (10), 317 (11), 316 (97), 315 (100), 314 (19), 313 (64), 303 (24), 302 (27), 301 (14), 223 (10), 91 (96), 43 (11); HRMS (EI) calcd. for C24H27N5 [M+]: 385.2266, found: 385.2267.2-(4-Methylpiperazin-1-yl)-N-phenethyl-5,6-dihydrobenzo[h]quinazolin-4-amine (7b). Yellowish solid (32%, purification via column chromatography); Rf 0.1 (EtOAc) [UV]; 1H NMR (600 MHz, CDCl3): δ 2.38-2.52 (m, 5 H), 2.56 (t, J = 5.0 Hz, 4 H), 2.88 (t, J = 7.5 Hz, 2 H), 2.94 (t, J = 7.1 Hz, 2 H), 3.74 (dt, J = 5.9 Hz, J = 7.2 Hz, 2 H), 3.93-3.98 (m, 4 H), 4.54 (t, J = 5.7 Hz, 1 H), 7.15-7.17 (m, 1 H), 7.20-7.25 (m, 3 H), 7.26-7.33 (m, 4 H), 8.24 (dd, J = 1.6 Hz, J = 7.4 Hz, 1 H); 13C NMR (91 MHz, CDCl3): δ 20.0 (CH2), 27.7 (CH2), 35.8 (CH2), 42.4 (2 × CH2), 43.7 (CH3), 46.1 (CH2), 55.0 (2 × CH2), 100.4 (CH), 125.0 (CH), 126.4 (CH), 126.7 (CH), 127.4 (CH), 128.6 (2 × CH), 128.8 (2 × CH), 129.2 (Cq), 134.1 (Cq), 137.9 (Cq), 139.5 (Cq), 156.0 (Cq), 160.5 (Cq), 160.6 (Cq); MS (EI) m/z (%): 400 (21) [MH+], 399 (75) [M+], 398 (15), 397 (19), 343 (31), 342 (20), 331 (19), 330 (100), 329 (100), 328 (32), 327 (100), 317 (36), 316 (47), 315 (12), 252 (30), 251 (12), 250 (18), 237 (33), 235 (12), 225 (14), 223 (10), 212 (17), 211 (11), 208 (14), 181 (10), 154 (12), 153 (22), 105 (53), 57 (14), 44 (11), 43 (16); HRMS (EI) calcd. for C25H29N5 [M+]: 399.2423, found: 399.2424.N-((4'-Chloro-6-methoxy-[1,1'-biphenyl]-3-yl)methyl)-2-(4-methylpiperazin-1-yl)-5,6-dihydrobenzo[h]quinazolin-4-amine (7c). Yellowish solid (37%, purification via column chromatography); Rf 0.3 (EtOAc / MeOH = 10:1) [UV]; 1H NMR (600 MHz, CDCl3): δ 2.50-2.54 (m, 5 H), 2.70-2.75 (m, 4 H), 2.91 (t, J = 7.5 Hz, 2 H), 3.81 (s, 3 H), 4.02-4.12 (m, 4 H), 4.65 (d, J = 5.4 Hz, 2 H), 4.85 (t, J = 5.3 Hz, 1 H), 6.95 (d, J = 8.4 Hz, 1 H), 7.16-7.19 (m, 1 H), 7.28-7.34 (m, 4 H), 7.37 (d, J = 8.7 Hz, 2 H), 7.44 (d, J = 8.7 Hz, 2 H), 8.21 (dd, J = 2.2 Hz, J = 7.0 Hz, 1 H); 13C NMR (151 MHz, CDCl3): δ 20.2 (CH2), 27.6 (CH2), 42.6 (CH2), 44.7 (CH3), 54.2 (2 × CH2), 55.7 (CH3), 111.5 (CH), 125.0 (CH), 126.7 (CH), 127.5 (CH), 128.2 (2 × CH), 128.4 (CH), 129.4 (CH), 129.5 (Cq), 130.4 (Cq), 130.7 (2 × CH), 131.9 (Cq), 133.1 (Cq), 133.7 (Cq), 136.6 (Cq), 137.9 (CH), 155.7 (Cq), 156.3 (Cq), 160.5 (Cq), 2 signals missing (Cq, Cq); MS (EI) m/z (%): 528 (10) [37Cl-MH+], 527 (32) [37Cl-M+], 526 (34) [35Cl-MH+], 525 (91) [35Cl-M+], 468 (10), 458 (25), 457 (98), 456 (80), 455 (100), 453 (20), 444 (14), 443 (27), 442 (29), 233 (30), 232 (14), 231 (88), 181 (12); HRMS (EI) calcd. for C31H32ClN5O [M+]: 525.2295, found: 525.2295.

1.3. Synthesis of the final compounds 10a-c, 11a-c, 12a and 12b1.3.1. Synthesis of 92,4-Dichloro-6-phenylpyrimidine (9). Phenylboronic acid (5.00 mmol, 610 mg) and 2,4,6-trichloropyrimidine (6.00 mmol, 0.70 mL) are dissolved in a sodium carbonate solution (1 M in water, 10.0 mmol, 10.0 mL), and palladium(II) acetate (0.10 mmol, 22.5 mg) and triphenylphosphine (0.20 mmol, 52.5 mg) are added. The reaction mixture is heated to 60 °C and stirred for 3 hours. After cooling, the solution is extracted with diethyl ether (3 × 30 mL). The combined organic phases are washed with brine and dried over sodium sulphate. Subsequently, the solvent is removed under reduced pressure and the crude product is purified via column chromatography (SiO2, NEt3 (1 %), hexane / dichloromethane = 2:1) to yield 9 (478 mg, 42%) as white solid; Rf 0.3 (hexane / dichloromethane = 2:1) [UV]; 1H NMR (360 MHz, CDCl3): δ 7.50-7.60 (m, 3 H), 7.67 (s, 1 H), 8.07 (dd, J = 1.5 Hz, J = 8.3 Hz, 2 H); 13C NMR (151 MHz, CDCl3): δ 115.3 (CH), 127.6 (2 × CH), 129.2 (2 × CH), 132.4 (CH), 134.1 (Cq), 161.0 (Cq), 162.9 (Cq), 168.2(Cq). CAS 26032-72-5.5

1.3.2. General procedure for the synthesis of the mono-aminated precursor for 10a-cThe respective dichloropyrimidine derivative 9 (75 mg) and 1.5 equivalents of 1-methylpiperazine are dissolved in DMF (2.00 mL). The reaction mixture is heated to 40 °C and stirred for 18 hours. Subsequently, the solvent is removed under reduced pressure and the crude product is purified via column chromatography (SiO2, ethyl acetate / methanol = 3:1).

2-Chloro-4-(4-methylpiperazin-1-yl)-6-phenylpyrimidine (precursor to 10a-c). Brownish solid (42%); Rf 0.5 (ethyl acetate / methanol = 3:1) [UV]; 1H NMR (360 MHz, CDCl3): δ 2.36 (s, 3 H), 2.49-2.53 (m, 4 H), 3.72-3.76 (m, 4 H), 6.74 (s, 1 H), 7.41-7.46 (m, 3 H), 7.91-7.95 (m, 2 H); 13C NMR (151 MHz, CDCl3): δ 44.1 (2 × CH2), 46.0 (CH3), 54.4 (2 × CH2), 96.7 (CH), 127.1 (2 × CH), 128.7 (2 × CH), 130.6 (CH), 136.8 (Cq), 161.0 (Cq), 163.6 (Cq), 165.7 (Cq).

1.3.3. General procedure for the synthesis of 10a-cThe respective chloropyrimidine derivative (between 30 mg and 60 mg) is dissolved in the respective amine (2.00 mL). The reaction mixture is heated to 90 °C and is stirred for 18 hours. Subsequently, the amine is removed via Kugelrohr distillation and the crude product is purified either via column chromatography (SiO2, EtOAc / MeOH = 3:1) or reversed phase-HPLC (mobile phase: water + 0.1% formic acid (A) and methanol (B); linear gradient 10-100% B in 90-0% A over 15 minutes).N-Benzyl-4-(4-methylpiperazin-1-yl)-6-phenylpyrimidin-2-amine (10a). Yellowish solid (79%, purification via column chromatography); Rf 0.5 (EtOAc / MeOH = 1:1) [UV]; 1H NMR (600 MHz, CDCl3): δ 2.34 (s, 3 H), 2.46-2.49 (m, 4 H), 3.68-3.71 (m, 4 H), 4.67 (d, J = 5.8 Hz, 2 H), 6.29 (s, 1 H), 7.24 (t, J = 7.3 Hz, 1 H), 7.32 (t, J = 7.6 Hz, 2 H), 7.38-7.40 (m, 2 H), 7.42-7.44 (m, 3 H), 7.90-7.93 (m, 2 H); 13C NMR (91 MHz, CDCl3): δ 44.0 (2 × CH2), 45.5 (CH3), 46.0 (CH2), 54.6 (2 × CH2), 89.8 (CH), 126.9 (2 × CH), 127.0 (CH), 127.5 (2 × CH), 127.9 (Cq), 128.2 (Cq), 128.4 (2 × CH), 128.5 (Cq), 128.6 (2 × CH), 129.9 (CH), 2 signals missing (Cq, Cq); MS (EI)m/z (%): 359 (27) [M+], 290 (22), 289 (100), 276 (25), 91 (60), 43 (15); HRMS (EI) cald. for C22H25N5 [M+]: 359.2110, found: 359.2110.N-(4-Methoxybenzyl)-4-(4-methylpiperazin-1-yl)-6-phenylpyrimidin-2-amine (10b). Yellowish solid (70%, purification via column chromatography); Rf 0.3 (EtOAc / MeOH = 3:1) [UV]; 1H NMR (600 MHz, CDCl3): δ 2.35 (s, 3 H), 2.47-2.50 (m, 4 H), 3.69-3.72 (m, 4 H), 3.79 (s, 3 H), 4.60 (d, J = 5.8 Hz, 2 H), 6.30 (s, 1 H), 6.86 (d, J = 8.8 Hz, 2 H), 7.31 (d, J = 8.7 Hz, 2 H), 7.42-7.45 (m, 3 H), 7.91-7.94 (m, 2 H); 13C NMR (151 MHz, CDCl3): δ 44.0 (2 × CH2), 45.0 (CH2), 46.1 (CH3), 54.7 (2 × CH2), 55.3 (CH3), 89.8 (CH), 113.8 (2 × CH), 126.9 (2 × CH), 128.6 (2 × CH), 128.9 (2 × CH), 130.0 (Cq), 158.7 (Cq), 5 signals missing (Cq, Cq, Cq, Cq, CH); MS (EI) m/z (%): 390 (20) [MH+], 389 (72) [M+], 320 (37), 319 (89), 307 (13), 306 (52), 305 (12), 211 (65), 194 (14), 136 (11), 128 (13), 122 (12), 121 (100), 70 (13), 43 (18), 42 (12); HRMS (EI) calcd. for C23H27N5O [M+]: 389.2216, found: 389.2216.4-(4-Methylpiperazin-1-yl)-N-phenethyl-6-phenylpyrimidin-2-amine (10c). Yellowish solid (88%, purification via column chromatography); Rf 0.4 (EtOAc / MeOH = 3:1) [UV]; 1H NMR (600 MHz, CDCl3): δ 2.35 (s, 3 H), 2.49-2.52 (m, 4 H), 2.93-2.97 (m, 2 H), 3.67-3.75 (m, 6 H), 6.28 (s, 1 H), 7.22 (t, J = 7.3 Hz, 1 H), 7.25-7.27 (m, 2 H), 7.29-7.32 (m, 2 H), 7.42-7.46 (m, 3 H), 7.90-7.94 (m, 2 H); 13C NMR (151 MHz, CDCl3): δ 36.1 (CH2), 42.9 (CH2), 44.0 (2 × CH2), 46.0 (CH3), 54.6 (2 × CH2), 89.6 (CH), 126.2 (CH), 126.9 (2 × CH), 128.5 (2 × CH), 128.6 (2 × CH), 128.8 (2 × CH), 139.6 (Cq), 5 signals missing (CH, Cq, Cq, Cq, Cq); MS (EI) m/z (%): 373 (27) [M+], 304 (25), 303 (100), 290 (24), 282 (15), 211 (10), 199 (14), 128 (11), 105 (18), 42 (11); HRMS (EI) calcd. for C23H27N5 [M+]: 373.2266, found: 373.2267.

1.3.4. General procedure for the synthesis of the mono-aminated precursors for 11a-cThe respective dichloropyrimidine derivative 9 (between 60 mg and 120 mg) and 1.5 equivalents of the respective amine are dissolved in DMF (2.00 mL). The reaction mixture is heated to 40 °C and stirred for 18 hours. Subsequently, the solvent is removed under reduced pressure and the crude product is purified via column chromatography (SiO2, dichloromethane / ethyl acetate = 20:1).

N-Benzyl-2-chloro-6-phenylpyrimidin-4-amine (precursor to 11a). Yellowish solid (39%); Rf 0.5 (dichloromethane / ethyl acetate = 20:1) [UV]; 1H NMR (600 MHz, CDCl3): δ 4.62 (br s, 2 H), 6.61 (s, 1 H), 7.30-7.45 (m, 5 H), 7.42-7.47 (m, 3 H), 7.91 (dd, J = 1.7 Hz, J = 7.9 Hz, 2 H).2-Chloro-N-(4-methoxybenzyl)-6-phenylpyrimidin-4-amine (precursor to 11b). Yellowish solid (26%); Rf 0.5 (dichloromethane / ethyl acetate = 20:1) [UV]; 1H NMR (360 MHz, CDCl3): δ 3.81 (s, 3 H), 4.53 (d, J = 5.1 Hz, 2 H), 6.60 (s, 1 H), 6.90 (d, J = 8.7 Hz, 2 H), 7.27 (d, J = 9.0 Hz, 2 H), 7.40-7.47 (m, 3 H), 7.90-7.94 (m, 2 H).2-Chloro-N-phenethyl-6-phenylpyrimidin-4-amine (precursor to 11c). Yellowish solid (72%); Rf 0.5 (dichloromethane / ethyl acetate = 20:1) [UV]; 1H NMR (600 MHz, CDCl3): δ 2.95 (t, J = 6.9 Hz, 2 H), 3.68 (br s, 2 H), 6.54 (s, 1 H), 7.22-7.26 (m, 3 H), 7.33 (t, J = 7.4 Hz, 2 H), 7.42-7.48 (m, 3 H), 7.93 (dd, J = 1.9 Hz, J = 7.6 Hz, 2 H); 13C NMR (151 MHz, CDCl3): δ 30.9 (CH2), 35.3 (CH2), 126.8 (CH), 127.0 (2 × CH), 128.7 (2 × CH), 128.7 (2 × CH), 128.8 (2 × CH), 130.7 (CH), 136.3 (Cq), 164.3 (Cq), 4 signals missing (Cq, Cq, Cq, CH).

1.3.5. General procedure for the synthesis of 11a-cThe respective chloropyrimidine derivative (between 30 mg and 60 mg) is dissolved in the respective amine (2.00 mL). The reaction mixture is heated to 90 °C and is stirred for 18 hours. Subsequently, the amine is removed via Kugelrohr distillation and the crude product is purified either via column chromatography (SiO2, EtOAc / MeOH = 3:1) or reversed phase-HPLC (mobile phase: water + 0.1% formic acid (A) and methanol (B); linear gradient 10-100% B in 90-0% A over 15 minutes).N-Benzyl-2-(4-methylpiperazin-1-yl)-6-phenylpyrimidin-4-amine (11a). Yellowish solid (89%, purification via column chromatography); Rf 0.6 (EtOAc / MeOH = 1:1) [UV]; 1H NMR (600 MHz, CDCl3): δ 2.34 (s, 3 H), 2.47-2.49 (m, 4 H), 3.89-3.93 (m, 4 H), 4.59 (d, J = 5.7 Hz, 2 H), 6.13 (s, 1 H), 7.26-7.29 (m, 1 H), 7.32-7.37 (m, 4 H), 7.39-7.43 (m, 3 H), 7.93-7.95 (m, 2 H); 13C NMR(91 MHz, CDCl3): δ 43.7 (2 × CH2), 45.4 (CH3), 46.2 (CH2), 55.1 (2 × CH2), 126.8 (2 × CH), 127.3 (CH), 127.5 (2 × CH), 128.4 (2 × CH), 128.6 (2 × CH), 129.6 (CH), 138.8 (Cq), 161.9 (Cq), 163.9 (Cq), 3 signals missing (CH, Cq, Cq); MS (EI) m/z (%): 360 (11) [MH+], 359 (42) [M+], 302 (12), 290 (48), 289 (100), 277 (28), 276 (10), 128 (14), 91 (87), 43 (22), 42 (14); HRMS (EI) calcd. for C22H25N5 [M+]: 359.2110, found: 359.2108.N-(4-Methoxybenzyl)-2-(4-methylpiperazin-1-yl)-6-phenylpyrimidin-4-amine (11b). Yellowish solid (88%, purification via column chromatography); Rf 0.3 (EtOAc / MeOH = 3:1) [UV]; 1H NMR (600 MHz, CDCl3): δ 2.37 (s, 3 H), 2.53 (t, J = 4.9 Hz, 4 H), 3.80 (s, 3 H), 3.92-3.96 (m, 4 H), 4.51 (d, J = 5.4 Hz, 2 H), 6.13 (s, 1 H), 6.87 (d, J = 8.7 Hz, 2 H), 7.28 (d, J = 8.7 Hz, 2 H), 7.39-7.43 (m, 3 H), 7.94 (dd, J = 2.2 Hz, J = 7.5 Hz, 2 H); 13C NMR (151 MHz, CDCl3): δ 43.5 (2 × CH2), 44.9 (CH2), 46.0 (CH3), 54.9 (2 × CH2), 55.3 (CH3), 114.0 (2 × CH), 126.8 (2 × CH), 128.4 (2 × CH), 128.9 (2 × CH), 129.6 (CH), 138.7 (Cq), 158.9 (Cq), 161.9 (Cq), 163.8 (Cq), 3 signals missing (CH, Cq, Cq); MS (EI) m/z (%): 389 (28) [M+], 320 (23), 319 (100), 307 (13), 121 (78); HRMS (EI) calcd. for C23H27N5O [M+]: 389.2216, found: 389.2214.2-(4-Methylpiperazin-1-yl)-N-phenethyl-6-phenylpyrimidin-4-amine (11c). Yellowish solid (77%, purification via column chromatography); Rf 0.4 (EtOAc / MeOH = 3:1) [UV]; 1H NMR (600 MHz, CDCl3): δ 2.36 (s, 3 H), 2.49-2.52 (m, 4 H), 2.93 (t, J = 7.1 Hz, 2 H), 3.64 (dd, J = 6.6 Hz, J = 13.0 Hz, 2 H), 3.90-3.95 (m, 4 H), 6.08 (s, 1 H), 7.22-7.25 (m, 3 H), 7.32 (t, J = 7.4 Hz, 2 H), 7.39-7.43 (m, 3 H), 7.96 (dd, J = 1.9 Hz, J = 7.8 Hz, 2 H); 13C NMR (151 MHz, CDCl3): δ 35.8 (CH2), 42.5 (CH2), 43.6 (2 × CH2), 46.2 (CH3), 55.1 (2 × CH2), 126.5 (CH), 126.8 (2 × CH), 128.3 (2 × CH), 128.6 (2 × CH), 128.8 (2 × CH), 129.5 (CH), 138.8 (Cq), 139.1 (Cq), 161.9 (Cq), 163.8 (Cq), 2 signals missing (Cq, CH); MS (EI) m/z (%): 373 (10) [M+], 304 (23), 303 (100), 291 (15), 105 (11); HRMS (EI) calcd. for C23H27N5 [M+]: 373.2266, found: 373.2268.

1.3.6. General procedure for the synthesis of 12a and 12b2,4-Dichloro-6-phenylpyrimidine (9) (between 80 mg and 82 mg) and 1.5 equivalents of the respective benzyl amine are dissolved in DMF (2.00 mL). The reaction mixture is heated to 90 °C and stirred for 18 hours. Subsequently, the solvent is removed under reduced pressure and the crude product is purified via column chromatography (SiO2, dichloromethane / ethyl acetate = 20:1).N4-(4-Methoxybenzyl)-N2,N2-dimethyl-6-phenylpyrimidin-2,4-diamine (12a). Yellowish solid (25%); Rf 0.7 (CH2Cl2 / EtOAc = 20:1) [UV]; 1H NMR (360 MHz, CDCl3): δ 3.22 (s, 6 H), 3.78 (s, 3 H), 4.51 (d, J = 5.7 Hz, 2 H), 6.08 (s, 1 H), 6.86 (d, J = 8.8 Hz, 2 H), 7.28 (d, J = 8.8 Hz, 2 H), 7.37-7.43 (m, 3 H), 7.95-7.99 (m, 2 H); 13C NMR (91 MHz, CDCl3): δ 36.9 (2 × CH3), 44.8 (CH2), 55.3 (CH3), 114.0 (2 × CH), 126.8 (2 × CH), 128.3 (2 × CH), 128.8 (2 × CH), 129.5 (CH), 139.0 (Cq), 159.9 (Cq), 5 signals missing (Cq, Cq, Cq, Cq, CH); MS (EI) m/z (%): 335 (23) [MH+], 334 (100) [M+], 333 (13), 319 (33), 291 (17), 136 (29), 128 (15), 121 (71); HRMS (EI) calcd. for C20H22N4O [M+]: 334.1794, found: 334.1790.N2,N2-Dimethyl-N4-phenethyl-6-phenylpyrimidin-2,4-diamine (12b). Yellowish solid (29%); Rf 0.7 (CH2Cl2 / EtOAc = 20:1) [UV]; 1H NMR (360 MHz, CDCl3): δ 2.94 (t, J = 7.1 Hz, 2 H), 3.23 (s, 6 H), 3.65 (dd, J = 6.9 Hz, J = 13.3 Hz, 2 H), 6.05 (s, 1 H), 7.21-7.26 (m, 3 H), 7.29-7.35 (m, 2 H), 7.38-7.44 (m, 3 H), 7.96-8.00 (m, 2 H); 13C NMR (91 MHz, CDCl3): δ 35.8 (CH2), 36.9 (2 × CH3), 42.6 (CH2), 126.4 (CH), 126.8 (2 × CH), 128.3 (2 × CH), 128.6 (2 × CH), 128.8 (2 × CH), 129.5 (CH), 139.0 (Cq), 139.2 (Cq), 4 signals missing (Cq, Cq, Cq, CH); HRMS (ESI) calcd. for C20H23N4 [MH+]: 319.1917, found: 319.1922.

1.4. Synthesis of the final compounds 15a-d1.4.1. Synthesis of 13Methyl 2-oxo-1,2,3,4-tetrahydronaphthalene-1-carboxylate (13). Under nitrogen atmosphere, β-tetralone (15.0 mmol, 1.98 mL), dimethyl carbonate (180 mmol, 15.0 mL), sodium hydride (60% in mineral oil, 50.0 mmol, 1.20 g) and dry methanol (0.1 mL) are heated to 80 °C and stirred for 3 hours. After cooling, hydrochloric acid (3 N, 15.0 mL) is added, the mixture is extracted with ethyl acetate and the combined organic phases are dried over sodium sulphate. After removing the solvent, 13 (3.05 g, 100%) is obtained as a red-brown liquid; 1H NMR (600 MHz, CDCl3, enole form): δ 2.52-2.55 (m, 2 H), 2.80-2.83 (m, 2 H), 3.91 (s, 3 H), 7.06 (dt, J = 1.2 Hz, J = 7.4 Hz, J = 7.4 Hz, 1 H), 7.12 (ddd, J = 0.6 Hz, J = 1.5 Hz, J = 7.4 Hz, 1 H), 7.19 (ddd, J = 1.6 Hz, J = 7.4 Hz, J = 8.0 Hz, 1 H), 7.67 (dd, J = 0.8 Hz, J = 8.0 Hz, 1 H), 13.30 (s, 1 H); 13C NMR (91 MHz, CDCl3, enole form):δ 27.7 (CH2), 29.5 (CH2), 51.7 (CH3), 99.9 (Cq), 125.0 (CH), 125.8 (CH), 126.4 (CH), 127.1 (CH), 131.3 (Cq), 133.1 (Cq), 172.4 (Cq), 178.4 (Cq). CAS 31202-23-0 (keto form), 99511-70-3 (enol form).6

1.4.2. Synthesis of 145,6-Dihydrobenzo[f]quinazoline-1,3(2H,4H)-dione (precursor to 14). 13 (10.0 mmol, 2.04 g) and urea (250 mmol, 15.0 g) are heated to 190 °C and the mixture is stirred for 45 minutes. After cooling, but before congelation of the molten mass, water (50 mL) is added and the mixture is stirred for further 15 minutes. A precipitate is formed, filtered of and dried in vacuo. The product (1.14 g, 53%) is obtained as a light brown solid; 1H NMR (600 MHz, DMSO-d6): δ 2.60-2.63 (m, 2 H), 2.78-2.81 (m, 2 H), 7.02-7.06 (m, 1 H), 7.10 (dt, J = 1.3 Hz, J = 7.4 Hz, 1 H), 7.15-7.20 (m, 1 H), 8.36 (dd, J = 1.0 Hz, J = 7.9 Hz, 1 H); 13C NMR (91 MHz, DMSO-d6): δ 25.6 (CH2), 26.3 (CH2), 103.9 (CH), 124.9 (Cq), 126.2 (Cq), 127.1 (CH), 130.2 (CH), 133.2 (CH), 150.2 (Cq), 153.0 (Cq), 155.5 (Cq), 164.4(Cq); MS (EI) m/z (%): 214 (100) [M+], 171 (60), 143 (14), 116 (52), 115 (41), 103 (11), 70 (11), 43 (13); HRMS (EI) calcd. for C12H10N2O2 [M+]: 214.0742, found: 214.0743. CAS 33987-17-6.3

1,3-Dichloro-5,6-dihydrobenzo[f]quinazoline (14). 5,6-Dihydrobenzo[f]quinazoline-1,3(2H,4H)-dione (1.86 mmol, 400 mg) is dissolved in phosphorous oxychloride (25.0 mL) and 10 drops of DMF are added. The reaction mixture is heated to 110 °C and stirred for 46 hours. After cooling, the solution is poured onto ice and the pH value is adjusted to 10 to 11 using potassium carbonate. The aqueous phase is extracted with ethyl acetate (3 ×). Subsequently, the combined organic phases are dried over sodium sulphate and the solvent is removed under reduced pressure. After purification via column chromatography (SiO2, dichloromethane), 14 (180 mg, 39%) is obtained as a yellow solid; Rf 0.6 (CH2Cl2) [UV]; 1H NMR (360 MHz, CDCl3): δ 2.90-2.96 (m, 2 H), 3.00-3.06 (m, 2 H), 7.30-7.38 (m, 3 H), 8.25 (dd, J = 3.8 Hz, J = 5.4 Hz, 1 H); 13C NMR (91 MHz, CDCl3): δ 27.7 (CH2), 32.2 (CH2), 125.8 (Cq), 126.8 (CH), 127.8 (CH), 127.9 (Cq), 128.1 (CH), 129.7 (CH), 138.2 (Cq), 156.7 (Cq), 157.4 (Cq), 172.8 (Cq); MS (EI) m/z (%): 253 (12) [37Cl-MH+], 252 (76) [37Cl-M+], 251 (34) [35Cl-MH+], 250 (100) [35Cl-M+], 249 (19), 215 (17), 213 (17), 179 (22), 154 (16), 153 (16), 152 (17), 127 (21), 126 (12), 107 (13), 83 (15), 77 (11); HRMS (EI) calcd. for C12H8Cl2N2: 250.0065, found: 250.0065.

1.4.3. General procedure for the synthesis of the mono-aminated precursors for 15a-cThe respective dichloropyrimidine derivative 14 (between 60 mg and 120 mg) and 1.5 equivalents of the respective amine are dissolved in DMF (2.00 mL). The reaction mixture is heated to 40 °C and stirred for 18 hours. Subsequently, the solvent is removed under reduced pressure and the crude product is purified via column chromatography (SiO2, dichloromethane / ethyl acetate = 20:1).N-Benzyl-1-chloro-5,6-dihydrobenzo[f]quinazolin-3-amine (precursor to 15a). Yellowish solid (44%); Rf 0.4 (dichloromethane / ethyl acetate = 20:1) [UV]; 1H NMR (360 MHz, CDCl3): δ 2.78-2.88 (m, 4 H), 4.69 (d, J = 5.9 Hz, 2 H), 7.18-7.24 (m, 2 H), 7.26-7.39 (m, 6 H), 8.13 (d, J = 7.7 Hz, 1 H); 13C NMR (91 MHz, CDCl3): δ 28.2 (CH2), 32.6 (CH2), 45.6 (CH2), 116.5 (Cq), 126.3 (CH), 127.0 (CH), 127.2 (CH), 127.4 (Cq), 127.5 (CH), 127.6 (2 × CH), 128.6 (2 × CH), 130.1 (Cq), 137.1 (CH), 138.6 (Cq), 159.3 (Cq), 2 signals missing (Cq, Cq).1-Chloro-N-(4-methoxybenzyl)-5,6-dihydrobenzo[f]quinazolin-3-amine (precursor to 15b). Yellowish solid (55%); Rf 0.4 (dichloromethane / ethyl acetate = 20:1) [UV]; 1H NMR (360 MHz, CDCl3): δ 2.75-2.85 (m, 4 H), 3.79 (s, 3 H), 4.66 (d, J = 5.3 Hz, 2 H), 5.96-6.02 (m, 1 Hz), 6.88 (d, J = 8.7 Hz, 2 H), 7.17-7.32 (m, 5 H), 7.64-7.68 (m, 1 H); 13C NMR (91 MHz, CDCl3): δ (ppm) = 28.1 (CH2), 31.1 (CH2), 45.1 (CH2), 55.2 (CH3), 114.0 (Cq), 114.2 (2 × CH), 123.1 (Cq), 127.1 (CH), 127.7 (CH), 128.7 (CH), 129.4 (2 × CH), 129.9 (Cq), 138.1 (CH), 157.9 (Cq), 159.1 (Cq), 159.2 (Cq), 167.0 (Cq), 1 signal missing (Cq).1-Chloro-N-phenethyl-5,6-dihydrobenzo[f]quinazolin-3-amine (precursor to 15c). Yellowish solid (63%); Rf 0.4 (dichloromethane / ethyl acetate = 20:1) [UV]; 1H NMR (600 MHz, CDCl3): δ 2.74-2.81 (m, 4 H), 2.98 (t, J = 6.8 Hz, 2 H), 3.82 (dt, J = 5.5 Hz, J = 6.8 Hz, 2 H), 5.75 (t, J = 4.6 Hz, 1 H), 7.12 (dt, J = 1.4 Hz, J = 7.6 Hz, 1 H), 7.19 (dt, J = 1.2 Hz, J = 7.5 Hz, 1 H), 7.25-7.28 (m, 5 H), 7.34 (t, J = 7.4 Hz, 2 H); 13C NMR (91 MHz, CDCl3): δ 28.2 (CH2), 31.1 (CH2), 35.1 (CH2), 42.5 (CH2), 110.1 (Cq), 123.0 (CH), 126.7 (CH), 126.9 (CH), 127.6 (CH), 128.6 (Cq), 128.7 (2 × CH), 128.8 (2 × CH), 129.8 (Cq), 138.0 (CH), 138.7 (Cq), 157.9 (Cq), 159.3 (Cq), 166.8 (Cq).

1.4.4. General procedure for the synthesis of 15a-cThe respective chloropyrimidine derivative (between 30 mg and 60 mg) is dissolved in the respective amine (2.00 mL). The reaction mixture is heated to 90 °C and is stirred for 18 hours. Subsequently, the amine is removed via Kugelrohr distillation and the crude product is purified either via column chromatography (SiO2, EtOAc / MeOH = 3:1) or reversed phase-HPLC (mobile phase: water + 0.1% formic acid (A) and methanol (B); linear gradient 10-100% B in 90-0% A over 15 minutes).

N-Benzyl-1-(4-methylpiperazin-1-yl)-5,6-dihydrobenzo[f]quinazolin-3-amine (15a). Yellowish solid (46%, purification via column chromatography); Rf 0.2 (EtOAc / MeOH = 3:1) [UV]; 1H NMR (600 MHz, CDCl3): δ 2.29 (s, 3 H), 2.37-2.44 (m, 4 H), 2.70 (dd, J = 6.2 Hz, J = 8.1 Hz, 2 H), 2.77-2.80 (m, 2 H), 3.32-3.50 (m, 4 H), 4.62 (d, J = 6.0 Hz, 2 H), 7.08 (dt, J = 1.3 Hz, J = 7.4 Hz, 1 H), 7.17-7.22 (m, 2 H), 7.24 (t, J = 7.3 Hz, 1 H), 7.32 (t, J = 7.6 Hz, 2 H), 7.36-7.38 (m, 2 H), 7.69 (d, J = 7.7 Hz, 1 H); 13C NMR (91 MHz, CDCl3): δ 28.2 (CH2), 31.3 (CH2), 45.5 (2 × CH2), 45.9 (CH3), 47.8 (CH2), 54.4 (2 × CH2), 105.0 (Cq), 124.2 (CH), 125.5 (CH), 126.3 (CH), 127.0 (CH), 127.3 (Cq), 127.4 (2 × CH), 128.4 (2 × CH), 132.8 (Cq), 135.2 (Cq), 139.7 (CH), 162.7 (Cq), 2 signals missing (Cq, Cq); HRMS (ESI) calcd. for C24H28N5

[MH+]: 386.2339, found: 386.2346.N-(4-Methoxybenzyl)-1-(4-methylpiperazin-1-yl)-5,6-dihydrobenzo[f]quinazolin-3-amine (15b). Yellowish solid (85%, purification via column chromatography); Rf 0.2 (EtOAc / MeOH = 3:1) [UV]; 1H NMR (600 MHz, CDCl3): δ 2.35 (s, 3 H), 2.47 (t, J = 5.1 Hz, 4 H), 2.68 (dd, J = 5.7 Hz, J = 8.6 Hz, 2 H), 2.78 (dd, J = 5.6 Hz, J = 8.4 Hz, 2 H), 3.80 (s, 3 H), 3.86-3.89 (m, 4 H), 4.63 (d, J = 5.4 Hz, 2 H), 5.69 (t, J = 5.4 Hz, 1 H), 6.87 (d, J = 8.8 Hz, 2 H), 7.07 (dt, J = 1.2 Hz, J = 7.4 Hz, 1 H), 7.18 (dt, J = 1.4 Hz, J = 7.6 Hz, 1 H), 7.22 (dd, J = 0.9 Hz, J = 7.4 Hz, 1 H), 7.29 (d, J = 8.8 Hz, 2 H), 7.60 (d, J = 7.6 Hz, 1 H); 13C NMR (151 MHz, CDCl3): δ 28.8 (CH2), 32.1 (CH2), 43.6 (2 × CH2), 44.7 (CH3), 46.2 (CH2), 55.0 (2 × CH2), 55.3 (CH3), 102.3 (Cq), 114.0 (2 × CH), 122.7 (CH), 125.2 (Cq), 126.7 (CH), 128.2 (CH), 129.0 (2 × CH), 131.6 (CH), 132.4 (Cq), 137.2 (Cq), 158.6 (Cq), 158.8 (Cq), 159.5 (Cq), 166.3 (Cq); HRMS (ESI) calcd. for C25H30N5O [MH+]: 416.2445, found: 416.2444.

1-(4-Methylpiperazin-1-yl)-N-phenethyl-5,6-dihydrobenzo[f]quinazolin-3-amine (15c). Yellowish solid (79%, purification via column chromatography); Rf 0.2 (EtOAc / MeOH = 3:1) [UV]; 1H NMR (600 MHz, CDCl3): δ 2.37 (s, 3 H), 2.51 (t, J = 5.1 Hz, 4 H), 2.66 (dd, J = 5.7 Hz, J = 8.6 Hz, 2 H), 2.76 (dd, J = 5.6 Hz, J = 8.4 Hz, 2 H), 2.96 (t, J = 7.1 Hz, 2 H), 3.75 (dt, J = 5.6 Hz, J = 7.1 Hz, 2 H), 3.88-3.92 (m, 4 H), 5.47 (t, J = 5.4 Hz, 1 H), 7.06 (dt, J = 1.3 Hz, J = 7.4 Hz, 1 H), 7.10 (dt, J = 1.5 Hz, J = 7.6 Hz, 1 H), 7.20 (dd, J = 1.0 Hz, J = 7.3 Hz, 1 H), 7.23-7.26 (m, 3 H), 7.30-7.34 (m, 3 H); 13C NMR (91 MHz, CDCl3): δ 28.8 (CH2), 32.1 (CH2), 35.6 (CH2), 42.4 (2 × CH2), 43.6 (CH3), 46.1 (CH2), 55.0 (2 × CH2), 102.3 (Cq), 122.6 (CH), 125.1 (Cq), 126.4 (CH), 126.6 (CH), 128.1 (Cq), 128.6 (2 × CH), 128.8 (2 × CH), 132.3 (Cq), 137.1 (CH), 139.5 (CH), 158.7 (Cq), 159.6 (Cq), 162.7 (Cq); HRMS (ESI) calcd. for C25H30N5 [MH+]: 400.2496, found: 400.2496.

1.4.5. Synthesis of 15d1,3-Bis(4-methylpiperazin-1-yl)-5,6-dihydrobenzo[f]quinazoline (15d). 1,3-Dichloro-5,6-dihydrobenzo[f]quinazoline (14) (0.23 mmol, 58.9 mg) and 1.5 equivalents of 1-methylpiperazine (0.35 mmol, 0.03 mL) are dissolved in DMF (2.00 mL). The reaction mixture is heated to 40 °C and stirred for 18 hours. Subsequently, the solvent is removed under reduced pressure and the crude product is purified via column chromatography (SiO2, ethyl acetate / methanol = 1:1) to yield 15d (32.6 mg, 38%) as yellowish solid. Rf 0.3 (EtOAc / MeOH = 1:1) [UV]; 1H NMR (600 MHz, CDCl3): δ 2.33 (s, 3 H), 2.36 (s, 3 H), 2.47-2.50 (m, 4 H), 2.70 (dd, J = 6.1 Hz, J = 8.2 Hz, 2 H), 2.76-2.80 (m, 2 H), 3.84-3.87 (m, 4 H), 7.06 (dt, J = 1.3 Hz, J = 7.4 Hz, 1 H), 7.16-7.21 (m, 2 H), 7.79 (d, J = 7.5 Hz, 1 H); 13C NMR (151 MHz, CDCl3): δ 28.5 (CH2), 32.5 (CH2), 43.6 (2 × CH2), 46.1 (CH3), 46.2 (2 × CH2), 47.9 (CH3), 54.6 (2 × CH2), 55.0 (2 × CH2), 104.8 (Cq), 124.3 (CH), 125.2 (CH), 126.1 (CH), 127.2 (CH), 133.4 (Cq), 135.5 (Cq), 159.2 (Cq), 162.7 (Cq), 169.1 (Cq); MS (EI) m/z (%): 379 (14) [MH+], 378 (48) [M+], 309 (23), 308 (100), 251 (26), 70 (10), 43 (18), 42 (14); HRMS (EI) calcd. for C22H30N6 [M+]: 378.2532, found: 378.2534.

1.5. NOESY spectra

NOESY spectrum of the precursor compound to 6a-e

NN

Cl

NN

abc

2

4

ab

c

The spectrum above shows that the primary substitution of 5 occurs in 4-position on the pyrimidine moiety. A correlation between the protons in positions a, b and c was observed.

NOESY spectrum of 7b

It is shown that the primary substitution of 5 also occurs in 4-position on the pyrimidine moiety when using primary amines instead of 1-methylpiperazine (secondary amine). This is supported by the correlation between the protons in positions a and c, and the proton in position b.

NOESY spectrum of the precursor compound to 10a-c

NN

Cl

NNa

b

2

4

ab

Similar to compound 5, the primary substitution of 9 also occurs in 4-position on the pyrimidine moiety. Correlations between the protons of methylene group a and the aromatic proton in position b were observed.

NOESY spectrum of 11c

It is shown that the primary substitution of 9 also occurs in 4-position on the pyrimidine moiety when using primary amines instead of 1-methylpiperazine (secondary amine). This is supported by the correlation between the protons in positions a and b.

NOESY spectrum of compound 12b

The NOESY spectrum of compound 12b shows that the substitution with phenethylamine occurs in 4-position on the pyrimidine moiety. Correlations between the protons of methylene group a and the aromatic proton in position b were observed.

NOESY spectrum of the precursor compound to 15a

N

N

aCl

HN

b24

b

a

The primary substitution of 14 occurs in 2-position on the pyrimidine moiety. A substitution in 4-position would otherwise lead to a correlation of the signals of the benzylic protons a and the aromatic proton in position b.

2. Pharmacological experiments

2.1. Materials

[3H]Mepyramine (30.0 Ci/mmol) was from PerkinElmer Life Sciences (Boston, MA, USA). [3H]UR-DE257 (33.0 Ci/mmol) was synthesized as described by S. Biselli.7 [3H]Nα-Methylhistamine (80 Ci/mmol) was from American Radiolabeled Chemicals (St. Louis, MO, USA, [3H]Histamine (14.2 Ci/mmol) was from PerkinElmer Life Sciences (Boston, MA, USA) and [35S]GTPγS (1000 Ci/mmol) was from Hartmann Analytic (Braunschweig, Germany). Thioperamide was synthesized as described.8 Histamine was from Tocris (Avonmouth, Bristol, UK). The Rotiszint ecoplus from Roth (Karlsruhe, Germany) was used as liquid scintillation cocktail.

2.2. Preparation of compound stock solutions

All compounds were dissolved in 50% (v/v) DMSO and 50% (v/v) double-distilled-water with appropriate equivalents of aqueous HCl. The final DMSO concentration was adjusted to 5% (v/v) in all assays. DMSO concentrations of 5% (v/v) are well tolerated within the radioligand competition binding assays used within the present study.9,10 Ligand concentrations were used in the range from 0.01 nM up to 1 mM, where appropriate.

2.3. Cell culture and membrane preparation

The construction of the baculoviruses used for infection of Sf9 cells was described earlier.11-13

Cell culture, membrane preparation and determination of protein concentration were performed as described previously.9,11

2.4. Radioligand competition binding assays

The radioligand competition binding assays at the four human histamine receptor subtypes hH1R, hH2R, hH3R and hH4R were performed as described previously.9,11-13 Briefly, the reaction mixtures, containing binding buffer (12.5 mM MgCl2, 1 mM EDTA, 75 mM Tris-HCl, pH 7.4), ligand (different concentrations within a concentration range from 1 nM to 1 mM as appropriate), radioligand (hH1R: 5 nM [3H]mepyramine, hH2R: 20 nM [3H]UR-DE257, hH3R: 3 nM [3H]Nα-Methylhistamine, hH4R: 10 nM [3H]histamine) and Sf9 membranes expressing the appropriate histamine receptor, were incubated with shaking at 250 rpm for 90 min at room temperature. The non-specific binding was determined in presence of 10 µM diphenhydramine (hH1R), famotidine (hH2R) and thioperamide (hH3R and hH4R).14 The bound radioligand was determined by filtration through GF/C filters and liquid scintillation counting.

2.5. [35S]GTPγS binding assayThe [35S]GTPγS binding assay at hH4R was performed as described previously.15 Briefly, the reaction mixtures, containing 1 µM GDP, 100 mM NaCl, 0.05% (w/v) bovine serum albumin, 0.2 nM [35S]GTPγS and ligand (different concentrations within a concentration range from 0.1 nM to 100 µM as appropriate) and Sf9 membranes expressing the hH4R, Gαi2 and Gβ1γ2, were incubated under shaking at 200 rpm at room temperature for 120 min. The non-specific binding was determined in presence of 10 µM unlabelled GTPγS. Before counting, all samples were filtered through GF/C filters and washed three times with cold binding buffer (4 °C), dried and impregnated with meltable scintillation wax.

2.6. Data analysis

The Ki values were calculated according to Cheng and Prusoff16 with KD values (hH1R: [3H]mepyramine, 4.49 ± 0.35 nM; hH2R: [3H]UR-DE257, 31 ± 5 nM; hH3R: 0.62 ± 0.21 nM; hH4R: [3H]histamine, 9.7 ± 1.7 nM) determined previously under the same conditions.14 The resulting Ki values are expressed logarithmically as pKi and were analyzed by nonlinear regression and best-fit to sigmoidal dose-response curves using GraphPad Prism 5.01 (GraphPad Software Inc., San Diego, CA, USA). All data are the means of three independent experiments ± SEM, performed in duplicates each.

3. Molecular Modelling

3.1. Generation of the hH1R and hH4R homology models

The crystal structure of hH1R, with the antagonist doxepine bound (3RZE),17 was used as a template for modelling the inactive conformation of hH1R and hH4R, as already described.18-20

Briefly, the artificially cocrystallized lysozyme was deleted. Afterwards, SYBYL 7.0 (Tripos Inc.) was used to complete the N-Terminus and the E2-loop. To close the gap between the intracellular parts of TM V and TM VI the amino acids of the I3-loop (hH1R: Leu231 to Val404, hH4R: Gly215 to His292) were replaced by eight alanines. Internal water molecules were included into the model of hH1R and hH4R. The compounds 1, 6a and 10a were docked manually into the binding pocket of hH1R and hH4R, using SYBYL 7.0 (Tripos Inc.). Subsequently, the resulting ligand-receptor-complexes were minimized and embedded in a POPC lipid bilayer, whose force field parameters are available at (http://moose.bio.ucalgary.ca/index.php?page=Structures_and_Topologies). Ligand parameterization was obtained from the PRODRG server (http://davapc1.bioch.dundee.ac.uk/prodrg/) and the partial charges were adopted based on the Gasteiger-Hückel partial charges, calculated by SYBYL 7.0. Intracellular and extracellular water, as well as an appropriate number of sodium and chlorine ions were included using the commands genbox and genion of the software package GROMACS 4.0.2 (http://www.gromacs.org). All simulation boxes contained a number of 115 POPC molecules and about 11000 to 12000 water molecules. The ffG53a6 force field21 was used for the hH1R and hH4R. After minimization of the simulation boxes, the MD simulations (5 ns equilibration phase, 10 ns productive phase) were performed with GROMACS 4.0.2.

3.2. Energetical considerations

The equation ∆RGo=−2.303RT pK i provides a good chance to check the quality of the

results gained by MD simulations. The quantity ∆RGo represents the change in standard

Gibbs energy for the transfer of the ligand from the aqueous phase into the binding pocket of the receptor, pKi denotes the Briggs logarithm of the corresponding association constant. From a thermodynamic point of view ∆RG

o is identical to the work which is to be done or released during the association process and may be approximated from the potential energies E(LR) of the ligand-receptor-complex, E(R) of the ligand-free receptor and E(L) of the ligand in the aqueous phase, provided by the MD simulations according to:

∆RGo≅∆ E=E (LR )−E (R )−E (L )

Provided that the sum of the self-energy terms of the ligand and the receptor equals the self-energy term of the ligand-receptor-complex, the right hand side of the above equation may be expressed as the sum of the short range Coulomb- and Lennard-Jones-interaction terms:

∆ E=CSR (LR )+LJSR (LR )+CSR (LW osbp )+ LJSR (LW osbp )−CSR (LW )−LJ SR (LW )+CSR (Glu5.46 ,Trp6.48, Tyr6.51 ,Gln7.42∈RL )+LJ SR (Glu5.46 ,Trp6.48 ,Tyr6.51 ,Gln7.42∈RL)−CSR (Glu5.46 ,Trp6.48 , Tyr6.51,Gln7.42∈R )−LJ SR(Glu5.46 , Trp6.48 , Tyr6.51 ,Gln7.42∈R)

The short range terms for the Coulomb- and Lennard-Jones-interactions are used to avoid artificial effects caused by structural changes of the receptor far away from the binding pocket.

4. References

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Buschauer, A.; Elz, S. J. Pharmacol. Exp. Ther. 2003, 305, 1104.[12] Schnell, D.; Strasser, A.; Seifert, R. Biochem. Pharmacol. 2010, 80, 1437.[13] Schneider, E. H.; Strasser, A.; Thurmond, R. L.; Seifert, R. J. Pharmacol. Exp. Ther.

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