1.5. Experimental Part
1.5.3. Synthesis of New Compounds
Safety
Sodium azide is toxic and can generate the extremely hazardous hydrazoic acid (volatile, toxic, explosive) if it gets in contact with acids. Organic azides with a saturated carbon:azide ratio of < 6 (such as azidoethylamine or azidopropylamine) may be heat and shock sensitive and should be handled carefully. Phenylacetylene is suspected to cause cancer.
Chemicals
Dansyl chloride, fluorescein di-sodium salt, 2-bromoethylamine hydrobromide, 3-bromopropylamine hydrobromide, propargylamine and phenyl acetylene were obtained from Fluka Chemicals. Benzyl azide was synthesized from benzyl bromide and sodium azide.
N+ H H
H N3
Cl
2-azidoethylamine hydrochloride:
The hydrobromide (2.0 g, 9.8 mmol) was dissolved in a saturated aqueous solution of NaN3 (2.6 g, 39.4 mmol, 4 eq) and heated to 90 °C for 16 h. CAUTION! as the solution is acidic this causes the formation of volatile and toxic HN3! After cooling the solution to room temperature NaOH was added to make the solution basic (pH > 10).
This produces the free amine which was separated from all inorganic compounds by distillation under reduced pressure (100 mbar, bp: 50 – 60 °C). The distillate was an aqueous solution of the amine which was afterwards acidified with 6 N hydrochloric acid. Lyophilisation of this solution gave the desired compound as a colourless, hygroscopic solid (1.10 g, 92 %).
1H-NMR (300 MHz, MeOD): δ = 3.12 (t, 3J = 5.6 Hz, 2 H), 3.75 (t, 3J = 5.6 Hz, 2 H);
13C-NMR (75.5 MHz, MeOD): δ = 40.1 (–, 1 C), 49.5 (–, 1 C); MS (ESI, H2O/MeCN):
m/z (%) = 87.0 [MH+] (100), 127.9 [MH++MeCN] (17); IR (KBr): ν [cm-1] = 3530, 2988, 2145, 1605, 1498, 1276, 1148; MF: C2H7N4Cl; MW = 122.56 g/mol;
N+ H H
H
N3 Cl
3-azidopropylamine hydrochloride:
The hydrobromide (2.0 g, 9.8 mmol) was dissolved in a saturated aqueous solution of NaN3 (2.6 g, 39.4 mmol, 4 eq) and heated to 90 °C for 16 h. CAUTION! as the solution is acidic this causes the formation of volatile and toxic HN3! After cooling the solution to room temperature NaOH was added to make the solution basic (pH > 10).
This produces the free amine which was separated from all inorganic compounds by distillation under reduced pressure (100 mbar, bp: 50 – 60 °C). The distillate was an aqueous solution of the amine which was afterwards acidified with 6 N hydrochloric acid. Lyophilisation of this solution gave the desired compound as a colourless, hygroscopic solid (1.1 g; 92 %).
1H-NMR (300 MHz, MeOD): δ = 1.97 (tt, 3J = 6.6 Hz, 3J = 7.4 Hz, 2 H), 3.01 – 3.06 (m, 2 H), 3.52 (t, 3J = 6.6 Hz, 2 H); 13C-NMR (75.5 MHz, MeOD): δ = 28.0 (–, 1 C), 38.5 (–, 1 C), 49.6 (–, 1 C); MS (ESI, H2O/MeCN): m/z (%) = 101.0 [MH+] (100), 142.0 [MH++MeCN] (13); IR (KBr): ν [cm-1] = 3532, 2987, 2147, 1604, 1497, 1278, 1149; MF: C3H9N4Cl; MW = 136.58 g/mol;
O
O O
O H
O
O
O O
O H
O
Propargylfluorescein (5) and Methylfluorescein (6):
Fluorescein-disodium-salt (3.8 g, 10 mmol) was suspended in 100 ml DMF and 8.6 ml of a solution of propargyl bromide in toluene (80 %, 11.9 g, 80 mmol) was added.
The mixture was stirred at r.t. for 24 h, then a reflux condenser was put on and the suspension was heated to 40 °C for further 24 h. After addition of 150 ml water, a precipitate fell out of solution. The mixture was extracted twice with 300 ml EE each time. In order to redissolve the precipitate completely the mixture had to be heated to approx. 40 °C. The organic layer was washed with 100 ml water, 50 ml brine and dried over Na2SO4. Removal of the solvent at reduced pressure and drying in vacuum gave a red solid which was suspended in 160 ml MeOH. After addition of 25 ml aqueous NaOH (c = 2 mol/L) the solid dissolved slowly. The mixture was stirred at 40 °C for 4 h. The MeOH was evaporated at reduced pressure and 50 ml water was added. The aqueous solution was washed with diethylether (50 ml, twice).
The organic layers were discarded. The aqueous layer was acidified to pH 2 with hydrochloric acid (c = 10 mol/L) to precipitate the product. The suspension was stirred at r.t. for 1 h and filtered. The residue was dissolved in 300 ml EE. The solution was washed with 100 ml water, 50 ml brine and dried over Na2SO4. Removal of the solvent and drying in vacuum gave a red solid which was purified by column chromatography (diethylether:PE = 2:1). This yielded a mixture of compound 5 and 6 in a ratio of 15:65 (determined by 1H-NMR). To separate both compounds the yellow solid is treated with small amounts of diethyl ether several times. Solutions were checked by TLC (eluent: diethylether:PE = 2:1, Rf (5) = 0.38, Rf (6) = 0.35). Pure solutions of 5 were combined. Removal of the solvent and drying in vacuum finally yielded 555 mg of 5 (15 %, 1.5 mmol).
Compound 5:
Mp: 125 °C; 1H-NMR (300 MHz, DMSO-d6): δ = 3.64 (t, 4J = 2.2 Hz, 1 H, CH), 4.89 (d, 4J = 2.2 Hz, 2 H, CH2), 6.57 (s, 1 H), 6.58 (s, 1 H), 6.66 – 6.77 (m, 3 H), 7.00 (d,
4J = 2.5 Hz, 1 H), 7.30 (d, 3J = 7.7 Hz, 1 H), 7.72 (dt, 3J = 7.4 Hz, 4J = 1.1 Hz, 1 H), 7.80 (dt, 3J = 7.5 Hz, 4J = 1.2 Hz, 1 H), 8.01 (d, 3J = 7.1 Hz, 1 H), 10.21 (bs, 1 H, OH); 13C-NMR (75.5 MHz, DMSO-d6): δ = 55.7 (–), 78.7 (+, alkyne-CH), 78.8 (Cquat, alkyne), 82.5 (Cquat), 101.7 (+), 102.1 (+), 109.3 (Cquat), 111.6 (Cquat), 112.3 (+), 112.8 (+), 123.9 (+), 124.6 (+), 125.9 (Cquat), 128.9 (+), 129.0 (+), 130.1 (+), 135.6 (+), 151.6 (Cquat), 151.6 (Cquat), 152.3 (Cquat), 158.7 (Cquat), 159.5 (Cquat), 168.6 (Cquat); MS (ESI, MeOH): m/z (%) = 371.1 [MH+] (100); UV (MeOH) λmax (log ε) = 453 nm (3.370), 478 nm (3.280); MF: C23H14O5; MW = 370.36 g/mol;
Compound 6:
Mp: 135 °C; 1H-NMR (300 MHz, DMSO-d6): δ = 3.83 (s, 3 H, CH3), 6.57 (s, 1 H), 6.58 (s, 1 H), 6.66 – 6.77 (m, 3 H), 6.93 (d, 4J = 2.5 Hz, 1 H), 7.29 (d, 3J = 7.7 Hz, 1 H), 7.71 (dt, 3J = 7.4 Hz, 4J = 1.1 Hz, 1 H), 7.79 (dt, 3J = 7.5 Hz, 4J = 1.2 Hz, 1 H), 8.00 (d, 3J = 7.1 Hz, 1 H), 10.22 (bs, 1 H, OH); 13C-NMR (75.5 MHz, DMSO-d6): δ = 55.5 (+), 82.5 (Cquat), 100.7 (+), 102.1 (+), 109.3 (Cquat), 110.8 (Cquat), 112.3 (+), 112.8 (+), 123.9 (+), 124.6 (+), 125.9 (Cquat), 128.9 (+), 129.0 (+), 130.1 (+), 135.6 (+), 151.6 (Cquat), 151.6 (Cquat), 152.3 (Cquat), 159.5 (Cquat), 160.9 (Cquat), 168.6 (Cquat);
MS (ESI, MeOH): m/z (%) = 347.1 [MH+] (100); UV (MeOH) λmax (log ε) = 453 nm (3.366), 478 nm (3.275); MF: C21H14O5; MW = 346.34 g/mol;
S O O
N H N
N3
5-Dimethylamino-naphtalene-1-sulfonic acid (2-azido-ethyl)-amide (7):
Dansyl chloride (864 mg, 3.2 mmol) was dissolved in 10 ml DCM. To this solution 1.78 ml of NEt3 (1296 mg, 12.8 mmol, 4 eq) and 2-azido-ethylamine hydrochloride (785 mg, 6.4 mmol, 2 eq) were added. The mixture was stirred 1 h at room temperature and diluted with 40 ml DCM as TLC showed almost 100 % conversion.
The solution was washed once with 50 ml of saturated aqueous solution of NaHCO3, dried over Na2SO4, filtered and concentrated at reduced pressure. The crude product was purified by column chromatography over a short silica gel column, eluting with EE:PE = 7:3. This gave the dansyl derivative 7 as light yellow oil, which crystallized after drying in vacuum (972 mg, 95 %). Rf (EE:PE = 1:3) = 0.30.
1H-NMR (300 MHz, CDCl3): δ = 2.88 (s, 6 H, 2 CH3), 3.05 (dt, 3J = 5.8 Hz, 3J = 6.3 Hz, 2 H, CH2), 3.28 (t, 3J = 5.8 Hz, 2 H, CH2), 5.43 (t, 3J = 6.3 Hz, 1 H, NH), 7.18 (dd, 3J = 7.7 Hz, 4J = 1.0 Hz, 1 H), 7.48 – 7.58 (m, 2 H), 8.24 (dd, 3J = 7.3 Hz, 4J = 1.3 Hz, 1 H), 8.29 (d, 3J = 8.8 Hz, 1 H), 8.55 (dt, 3J = 8.5 Hz, 4J = 1.1 Hz, 1 H);
122.1 (+), 127.6 (+), 128.4 (Cquat), 128.5 (+), 128.8 (Cquat), 129.7 (+), 133.5 (Cquat), 150.9 (Cquat); MS (ESI, DCM/MeOH): m/z (%) = 320.2 [MH+] (100), 639.4 [2M+H+] (20); EA (C14H17N5SO2) calc.: C 52.65 H 5.37 N 21.93, found: C 52.71 H 5.44 N 21.79; UV (MeOH) λmax (log ε) = 336 nm (3.684); MF: C14H17N5SO2; MW = 319.38 g/mol;
O O O
OH O
N N N
S O O
N H N
(5-Dimethylamino-naphtalene-1-sulfonic acid [2-(4-methyl-[1,2,3]triazol-1-yl)-ethyl]-amidyl)-fluorescein (8):
The azide 7 (51 mg, 0.16 mmol) and the alkyne 5 (59 mg, 0.16 mmol) were dissolved in 4 ml EtOH and a solution of 63 mg sodium ascorbate (0.32 mmol, 2 eq) in 1 ml water was added. Finally a solution of 40 mg CuSO4 x 5 H2O (0.16 mmol, 1 eq) in 0.5 ml water was added and the mixture was stirred at r.t. for 3 h. TLC after that time showed complete conversion. The mixture was diluted with 20 ml water and extracted with EE (50 ml, twice). The organic phases were combined, dried over Na2SO4 and concentrated at reduced pressure. The resulting orange solid was purified by column chromatography (DCM:MeOH = 98:2 to 95:5). This gave 8 as yellow solid in a yield of 101 mg (92 %, 0.15 mmol). Rf (DCM:MeOH = 97:3) = 0.2.
1H-NMR (300 MHz, acetone-d6): δ = 2.87 (s, 6 H, 2 CH3), 3.44 – 3.50 (m, 2 H, CH2), 4.80 (t, 3J = 5.9 Hz, 2 H, CH2), 5.13 (s, 2 H, CH2), 6.62 – 6.81 (m, 5 H), 6.99 – 7.01 (m, 1H), 7.17 (t, 3J = 6.2 Hz, 1 H, NH), 7.23 – 7.28 (m, 2 H), 7.50 – 7.62 (m, 2 H), 7.68 – 7.80 (m, 2 H), 7.84 (s, 1 H, triazole), 7.97 – 8.01 (m, 1 H), 8.21 (dd, 3J = 7.4 Hz, 4J = 1.1 Hz, 1 H), 8.32 (d, 3J = 8.8 Hz, 1 H), 8.57 (d, 3J = 8.5 Hz, 1 H), 9.15 (bs, 1 H, OH); Due to low solubility of 8 in all organic solvents and overlapping of too many signals 13C-NMR spectrum is not evaluable; MS (ESI, DCM/MeOH): m/z (%) = 690.2 [MH+] (100); UV (MeOH) λmax (log ε) = 336 nm (3.688), 453 nm (3.375), 478 nm (3.285); MF: C37H31N5SO7; MW = 689.74 g/mol;
S O O
N
N N3
Ph
5-Dimethylamino-naphtalene-1-sulfonic acid (2-azido-ethyl)-benzyl-amide (9):
The dansyl derivative 7 (200 mg, 0.63 mmol) was dissolved in 3 ml dry DMF. The solution was cooled to 0 °C in an ice-bath. After addition of NaH (60 % suspension in paraffine, 28 mg, 0.69 mmol, 1.1 eq) 150 µl of benzyl bromide (214 mg, 1.25 mmol, 2 eq) was added dropwise. The ice-bath was removed and the solution was stirred for 6 h while warming to room temperature. TLC after that time showed complete conversion. Water (20 ml) is added and the resulting mixture is extracted with EE (30 ml, twice). The organic layer is dried over Na2SO4 and solvent is removed at reduced pressure. This gave a brown oil which was purified by column chromatography (EE:PE = 8:2). Drying in vacuum yielded compound 9 as greenish yellow oil (254 mg, 0.62 mmol, 99 %). Rf (EE:PE = 1:3) = 0.37.
1H-NMR (300 MHz, CDCl3): δ = 2.90 (s, 6 H, 2 CH3), 3.20 (t, 3J = 6.6 Hz, 2 H, N-CH2), 3.38 (t, 3J = 6.6 Hz, 2 H, CH2), 4.53 (s, 2 H, Bn-CH2), 7.17 – 7.28 (m, 6 H, arom. CH), 7.50 – 7.63 (m, 2 H), 8.26 (dd, 3J = 7.3 Hz, 4J = 1.2 Hz, 1 H), 8.38 (d, 3J = 8.8 Hz, 1 H), 8.58 (dt, 3J = 8.5 Hz, 4J = 1.1 Hz, 1 H); 13C-NMR (75.5 MHz, CDCl3): δ = 45.5 (+), 45.6 (–, N-CH2), 49.8 (–), 52.3 (–, Bn), 115.4 (+, 1 C), 119.3 (+, 1 C), 123.2 (+, 1 C), 128.1 (+, 1 C), 128.4 (+, 1 C), 128.6 (+, 2 C), 128.8 (+, 2 C), 130.1 (+, 1 C), 130.1 (Cquat, 1 C), 130.1 (Cquat, 1 C), 130.8 (+, 1 C), 134.6 (Cquat, 1 C), 135.7 (Cquat, 1 C), 151.9 (Cquat); MS (ESI, DCM/MeOH): m/z (%) = 410.1 [MH+] (100), 819.5 [2M+H+] (5); EA (C21H23N5SO2) calc.: C 61.59 H 5.66 N 17.10, found: C 61.72 H 5.74 N 17.00; UV (MeOH) λmax (log ε) = 337 nm (3.694); MF: C21H23N5SO2; MW = 409.51 g/mol;
S O O
N
N N3
O
5-Dimethylamino-naphtalene-1-sulfonic acid acetyl-(2-azido-ethyl)-amide (10):
The sulfonamide 7 (281 mg, 0.88 mmol) was dissolved in 12 ml dry DCM. After addition of 818 µl NEt3 (594 mg, 3.52 mmol, 4 eq), 333 µl acetanhydride (359 mg, 3.52 mmol, 4 eq) and 11 mg DMAP (0.09 mmol, 10 mol%) the solution was stirred at room temeprature for 2 h. TLC after that time showed complete conversion. The solution was diluted with 200 ml DCM and washed with 100 ml sat. aqueous NaHCO3
and 100 ml brine. The organic phase was dried over Na2SO4 and concentrated under reduced pressure. The resulting brown oil was purified by column chromatography (PE:EE = 8:2) to yield 314 mg of 10 as a yellow oil (0.87 mmol, 99 %). Rf (EE:PE = 1:3) = 0.40.
1H-NMR (300 MHz, CDCl3): δ = 2.32 (s, 3 H, CH3), 2.86 (s, 6 H, 2 CH3), 3.56 (t, 3J = 6.6 Hz, 2 H, CH2), 4.09 (t, 3J = 6.6 Hz, 2 H, N-CH2), 7.18 (dd, 3J = 7.7 Hz, 4J = 1.0 Hz, 1H), 7.51 – 7.60 (m, 2 H), 7.99 (d, 3J = 8.5 Hz, 1 H), 8.18 (dd, 3J = 7.4 Hz,
4J = 1.4 Hz, 1 H), 8.60 (dt, 3J = 8.5 Hz, 4J = 1.1 Hz, 1 H); 13C-NMR (75.5 MHz, CDCl3): δ = 25.3 (+), 45.2 (–), 45.4 (+), 49.8 (–), 115.7 (+), 117.8 (+), 123.1 (+), 129.2 (+), 129.5 (Cquat), 129.8 (+), 130.0 (Cquat), 132.0 (+), 134.2 (Cquat), 152.3 (Cquat); MS (ESI, DCM/MeOH): m/z (%) = 362.1 [MH+] (100); EA (C16H19N5SO3) calc.: C 53.17 H 5.30 N 19.38, found: C 53.31 H 5.41 N 19.18; UV (MeOH) λmax (log ε) = 350 nm (3.577); MF: C16H19N5SO3; MW = 361.42 g/mol;
N N N
S O O
H N N S
H O
O
N N
Me2N NMe2
5-Dimethylamino-naphtalene-1-sulfonic acid [2-(4-{[4-(4-dimethylamino-phenylazo)-benzenesolfonylamino]-methyl}-[1,2,3]triazol-1-yl)-ethyl]-amide (12):
In a 500 mL round-bottomed flask compounds 7 (192 mg, 0.60 mmol) and 11 (206 mg, 0.60 mmol) were dissolved in 60 mL DMSO. Water (30 mL) was added, followed by aqueous sodium ascorbate (0.2 mL of 1 M stock solution) and aqueous copper sulfate (0.6 mL of 100 mM stock solution). The reaction was followed by TLC, eluting with 1:1 PE:EE. After stirring overnight, the reaction was found to be complete, and 150 mL water was added to precipitate the product. After standing open to air for several hours, the orange solid was filtered and washed with water.
The product was dried to give 314 mg of triazole 12 (0.48 mmol, 79 %). The orange solid was recrystallized from acetonitrile to give red crystals.
1H-NMR (300 MHz, DMSO-d6): δ = 2.79 (s, 6 H, 2 CH3), 3.06 (s, 6 H, 2 CH3), 3.22 (t,
3J = 6.0 Hz, 2 H, CH2), 4.00 (s, 2 H, CH2), 4.32 (t, 3J = 6.0 Hz, 2 H, CH2), 6.83 (d, 3J = 9.3 Hz, 2 H), 7.21 (d, 3J = 7.4 Hz, 1 H), 7.54 – 7.62 (m, 2 H), 7.76 (s, 1 H), 7.83 (d, 3J
= 9.1 Hz, 2 H), 7.88 – 7.95 (m, 4 H), 8.08 (dd, 3J = 7.3 Hz, 4J = 1.0 Hz, 1 H), 8.21 – 8.24 (m, 3 H), 8.44 (d, 3J = 8.5 Hz, 1 H); 13C-NMR (75.5 MHz, DMSO-d6): δ = 37.9 (–), 39.7 (+), 42.25 (–), 44.9 (+), 48.9 (–), 111.5 (+), 115.0 (+), 118.85 (+), 122.0 (+), 123.4 (+), 123.51 (+), 125.3 (+), 127.8 (+), 128.3 (+), 128.8 (Cquat), 128.9 (Cquat), 129.5 (+), 135.2 (Cquat), 140.0 (Cquat), 142.5 (Cquat), 142.9 (Cquat), 151.2 (Cquat), 153.0 (Cquat), 154.4 (Cquat); MS (ESI, DCM/MeOH): m/z (%) = 662.3 [MH+] (100); EA (C31H35N9S2O4) calc.: C 56.26 H 5.33 N 19.05, found: C 56.34 H 5.38 N 19.19; UV (MeCN) λmax (log ε) = 337 nm (3.679), 443 nm (4.478); MF: C31H35N9S2O4; MW = 661.80 g/mol;