3.5. Synthesis
3.5.1. Substrate preparation
The monohydrochlorides of
L-α,β-diaminopropionic acid (
1a) and
L-α,γ-diaminobutyric acid (
1b) were obtained according to the Rao protocol as white crystals, with the correct melting points, elemental analysis and optical rotation values: α[D]
28,5°C = +25 ± 1º and +24 ± 1º (C=2 in 1M HCl) for
1a and
1b, respectively, corresponding to the literature data [
46].
To a stirred aqueous solution containing 0.1 mol of a basic amino acid and 0.1 mol of NaOH, an excess of acrylonitrile (0.6 mol) and THF (30-40 mL) was added. The mixture was stirred for 48 h at room temperature and then refluxed for 3 h. To a cold reaction mixture, 0.1 mol of conc. HCl was added and left in refrigerator overnight. The crystalline product was separated and washed with several portions of cold MeOH-H2O (1:1). The washings were collected and evaporated to dryness. Then acetone (150 mL) was added to the oily residue and filtered. Two crops of precipitate were combined and recrystallized from hot water or MeOH-H2O (7:3) giving the tris-N-cyanoethylated amino acids 2a-d as white crystals in 49.5-69.2% yield.
The acetone filtrate was evaporated to dryness and purified by flash chromatography, eluting with an 8:2 mixture of ethyl acetate and hexane plus 3% of methanol, yielding the tetrakis-N-cyanoethylated derivatives 3a-d as amber-colored gums (except Nα,Nβ-tetrakis(cyanoethyl)-L-α,β-diaminopropionic acid, which slowly crystallized to give a white solid) in 21.1-28.6% yield. Both the yields and the 2:3 ratio depend on the reaction temperature. The yield of 3 can reach 50% when the reaction is performed in a boiling mixture of water-methanol (1:1) overnight.
N,N,N’-tris(cyanoethyl)-L-α,β-diaminopropionic acid (2a): Yield: 13.0 g (49.5%); mp 178-180 °C (dec.); ESI MS 264 (M+1), 286 (M+23); 1H-NMR (D2O) δ 2.74 (m, 4H, βCH2CN), 2.97 (m, 2H, αCH2CN), 2.99 (dd, J = 3.96 Hz, 1H, βCHa), 3.05 [m, 4H, Nβ-(CH2)2], 3.15 (dd, J = 3.96 Hz, 1H, βCHb), 3.6 (t, J = 6.96 Hz, 2H, NHα-CH2), 3.9 (dd, J = 3.96 Hz, 1H, αCH); 13C-NMR δ 15.2 (αCH2-CN), 15.4 (βCH2-CN), 42.7 (NHα-CH2), 48.8 [Nβ-(CH2)2], 53.2 (βC), 61.1 (αC), 117.6 (αCN), 121.0 (βCN), 170.9 (COOH); Anal. Calcd for C12H17O2N5: C, 54.7; H, 6.5; N, 26.6. Found: C, 54.53; H, 6.42; N, 26.44; α[D]26.2 °C = +24.3 ± 1° (c = 2 in 1M HCl).
N,N,N’-tris(cyanoethyl)-L-α,γ-diaminobutyric acid (2b): Yield: 17.3 g (62.6%); mp 170-172 °C; ESI MS 278 (M+1), 300 (M+23); 1H-NMR (D2O) δ 2.09 (q, J = 6.55 Hz, 2H, βCH2), 2.73 (m, 6H, CH2CN), 2.93 [m, 4H, Nγ-(CH2)2], 3.03 (t, J = 6.85 Hz, 2H, NHα-CH2), 3.46 (t, J = 6.85 Hz, 2H, γCH2), 3.84 (t, J = 6.08 Hz, 1H, αCH); 13C-NMR δ 15.5 (αCH2-CN), 15.5 (γCH2-CN), 26.9 (βC), 42.7 (NHα-CH2), 48.2 [Nγ-(CH2)2], 50.0 (γC), 62.4 (αC), 117.9 (αCN), 121.2 (γCN), 172.9 (COOH); Anal. Calcd for C13H19O2N5: C, 56.30; H, 6.90; N, 25.25. Found: C, 56.05; H, 6.80; N, 25.20; α[D]27.8°C = +24.2 ° 1° (c = 2 in 1M HCl).
N,N,N’-tris(cyanoethyl)-L-ornithine (2c): Yield: 18.4 g (63.3%); mp 192-194 °C; ESI MS 292 (M+1), 314 (M+23); 1H-NMR (D2O) δ 1.51, 1.92 (2m, 4H, γCH2, βCH2), 2.62 (t, J = 7.28 Hz, 2H, αCH2CN), 2.69 (t, J = 6.65 Hz, 4H, δCH2CN), 2.91 [t, J = 6.73 Hz, 4H, Nδ-(CH2)2], 3.02 (t, 2H, NHα-CH2), 3.42 (m, 2H, δCH2), 3.72 (dd, J = 4.95 Hz, 1H, αCH); 13C-NMR δ 15.3 (αCH2-CN), 15.7 (δCH2-CN), 22.0 (γC), 27.6 (βC), 42.3 (NHα-CH2), 48.5 [Nδ-(CH2)2], 52.3 (δC), 62.9 (αC), 117.9 (αCN), 121.1 (δCN), 173.4 (COOH); Anal. Calcd for C14H21O2N5: C, 57.71; H, 7.26; N, 24.03. Found: C, 57.52; H, 7.28; N, 23.85; α[D]27.5°C = +24.5 ± 1° (c = 2 in 1M HCl).
N,N,N’-tris(cyanoethyl)-L-lysine (2d): Yield: 21.1 g (69.2%); mp 214-216 °C; ESI MS 306 (M+1), 328 (M+23); 1H-NMR (DMSO) δ 1.30-1.38 (bm, 4H, γCH2, δCH2), 1.50-1.55 (2m, 2H, βCH2), 2.45 (t, J = 7.05 Hz, 2H, εCH2), 2.55 (m, 6H, CH2CN), 2.73 [t, J = 6.76 Hz, 4H, Nε-(CH2)2], 2.65, 2.81 (2m, 2H, NHα-CH2), 3.15 (t, J = 6.42 Hz, 1H, αCH); 13C-NMR δ 15.5 (εCH2-CN), 17.7 (αCH2-CN), 22.8 (γC), 26.5 (δC), 32.0 (βC), 42.9 (NHα-CH2), 48.4 [Nε-(CH2)2], 52.3 (εC), 60.2 (αC), 119.7 (αCN), 119.9 (εCN), 175.2 (COOH); Anal. Calcd for C15H23O2N5: C, 58.99; H, 7.59; N, 22.9. Found: C, 58.9; H, 7.47; N, 22.89; α[D]22°C = +23 ± 1° (c = 2 in 1M HCl).
N,N’,N,N’-tetrakis(cyanoethyl)-L-α,β-diaminopropionic acid (
3a): Yield: 9.05 g (28.6%); mp 126-128 °C; ESI MS 317 (M+1), 339 (M+23);
1H-NMR (DMSO) δ 2.57-2.67 (bm, 9H, CH
2CN, βCH
a), 2.81-2.98 [bm, 9H, N-(CH
2)
2, βCH
b], 3.48 (dd,
J = 6.24 Hz, 1H, αCH), 12.64 (s, 1H, COOH);
13C-NMR δ 15.3, 17.4 (α
CH
2-CN, β
CH
2-CN), 47.1, 48.6 [N
α-(CH
2)
2, N
β-(CH
2)
2], 53.3 (βC), 61.7 (αC), 119.68, 119.72 (αCN, βCN), 172.9 (COOH); Anal. Calcd for C
15H
20O
2N
6: C, 56.95; H, 6.37; N, 26.56. Found: C, 56.99; H, 6.37; N, 26.67; α[D]
29.1°C = -71° (±1°, c=2 in acetone). Monocrystals of
rac-3a prepared in a separate procedure were subjected to X-ray analysis [
31].
N,N’,N,N’-tetrakis(cyanoethyl)-L-α,γ-diaminobutyric acid (3b): Yield: 7.1 g (21.1%); semisolid; ESI MS 353 (M+23); 1H-NMR (DMSO) δ 1.61 (m, 2H, βCH2), 2.47 (t, J=6.95 Hz, 2H, γCH2), 2.58 [m, 8H, CH2CN), 2.75 [m, 8H, N-(CH2)2], 3.67 (t, J = 6.00 Hz, 1H, αCH), 11.55 (s, 1H, COOH); 13C-NMR δ 15.6, 18.0 (αCH2-CN, γCH2-CN), 31.1 (βC), 47.8, 48.4 [Nα-(CH2)2, Nγ-(CH2)2], 51.6 (γC), 65.1 (αC), 119.1, 120.0 (αCN, γCN), 174.6 (COOH); Anal. Calcd for C16H22O2N6: C, 58.16; H, 6.71; N, 25.43. Found: C, 57.95; H, 6.62; N, 25.23; α[D]29.1°C = -64° (±1°, c = 2 in acetone).
N,N’,N,N’-tetrakis(cyanoethyl)-L-ornithine (3c): Yield: 8.2 g (23.6%); semisolid; ESI MS 345 (M+1), 367 (M+23); 1H-NMR (CDCl3) δ 1.40, 1.54, 1.68 (3m, 4H, γCH2, βCH2), 2.50 (m, 2H, δCH2), 2.59 [m, 8H, CH2CN), 2.76, 2.88 [2m, 8H, N-(CH2)2], 3.34 (dd, J = 4.45 Hz, 1H, αCH); 13C-NMR δ 15.5, 17.4 (αCH2-CN, δCH2-CN), 23.5 (γC), 27.1 (βC), 46.9, 48.4 [Nα-(CH2)2, Nδ-(CH2)2], 51.9 (δC), 62.5 (αC), 119.86, 119.91 (αCN, δCN), 174.2 (COOH); Anal. Calcd for C17H24O2N6: C, 59.28; H, 7.02; N, 24.40. Found: C, 59.04; H, 6.96; N, 24.19; α[D]29.1°C = -65.2° (±1°, c = 2 in acetone).
N,N’,N,N’-tetrakis(cyanoethyl)-L-lysine (3d): Yield: 8.5 g (23.8%); semisolid; ESI MS 359 (M+1), 381 (M+23); 1H-NMR (CDCl3) δ 1.52, 1.68, 1.85 (3m, 6H, γCH2, δCH2, βCH2), 2.52 (m, 10H, CH2CN, εCH2), 2.85, 3.04 [2m, 8H, N-(CH2)2], 3.38 (dd, J = 5.34 Hz, 1H, αCH); 13C-NMR δ 16.9, 18.4 (αCH2-CN, εCH2-CN), 24.1 (γC), 26.9 (δC), 29.6 (βC), 47.5, 49.5 [Nα-(CH2)2, Nε-(CH2)2], 52.9 (εC), 63.4 (αC), 118.77, 118.84 (αCN, εCN), 177.2 (COOH); Anal. Calcd for C18H26O2N6: C, 60.3; H, 7.3; N, 23.4. Found: C, 60.08; H, 7.26; N, 23.15; α[D]28.6°C = -63° (±1°, c = 2 in acetone).
3.5.2. Synthesis of N,N-bis(cyanoethyl)-L-α,β-diamino propionic acid (11)
L-α,β-Diaminopropionic acid hydrochloride (14.05 g, 0.1 mol) was suspended in MeOH (100 mL), followed by slow addition of NaOH (8 g) in MeOH (100 mL). The reaction mixture was cooled to 0 °C in an ice-water bath. Then CH2=CH-CN (26.33 mL, 0.4 mol) was added in small portions and vigorous stirring was continued overnight, followed by addition of concentrated HCl (16.9 mL, 0.2 mol). The resulting white precipitate was collected on a filter and recrystallized from a MeOH-H2O mixture (1:1, v/v) to give 16.9 g (66.92%) of 11. Yield: 16.9g (66.9%); mp 180-182 °C (dec.); ESI MS 211 (M+1), 233 (M+23), 265 (M+23+32); 1H-NMR (D2O) δ 3.03, 3.07 (2t, J = 6.74 Hz, 4H, α, βCH2CN), 3.45-3.65 (bm, 6H, N-CH2), 4.06 (dd, J = 5.50 Hz, 1H, αCH); 13C-NMR δ 15.3, 15.8 (α, βCH2-CN), 42.74, 43.78 (α, βNH-CH2-), 45.8 (βC), 56.4 (αC), 117.7, 118.0 (α, βCN), 170.2 (COOH); Anal. Calcd for C9H15O2N4Cl: C, 43.82; H, 6.12; N, 22.71; Cl, 14.37. Found: C, 43.66; H, 6.20; N, 22.55; Cl, 14.41; α[D]24.5°C = +24.2 ± 1° (c = 2 in 1 M HCl).
3.5.3. General procedure for the catalytic reduction of the nitriles 2a-d to the tris-N-propylamino derivatives 4a-d and the synthesis of the Z-protected derivatives 4e-h
To a methanolic solution (150 mL) containing a tris-N-cyanoethylated derivative of basic amino acid 2a-d (5 g, ca. 15 mmol) and NaOH (0.64 g, 16 mmol), Raney-Ni suspension (5 g) was added. The resulting mixture was agitated at room temperature for 14-16 h under 90 psi (6 bar) of H2 pressure. The progress of the reaction was monitored by TLC using 1% ninhydrin in ethanol. After completion of the reaction, the catalyst was separated by filtration on Celite and the remaining solution was evaporated to dryness giving the compounds 4a-d as white hygroscopic oils in almost quantitative yields. The crude products were used for the next step without further purification.
N,N,N’-tris(3-aminopropyl)-L-α,β-diaminopropionic acid (4a): Yield: ~100%; ESI MS 298 (M+23), 330 (M+23+32); 1H-NMR (D2O) δ 1.50-1.70 (m, 6H, C-CH2-C), 2.50-2.70 (bm, 14H, N-CH2), 3.15 (dd, J = 5.65 Hz, 1H, αCH), 3.34 (s, 6H, NH2); 13C-NMR δ 28.3 (βCH2-CH2NH2), 28.9 (αCH2-CH2NH2), 38.9 (αCH2NH2), 39.3 (βCH2NH2), 44.9 (NHα-CH2), 49.1 [Nβ-(CH2)2], 62.0 (αC), 181.8 (COO-).
N,N,N’-tris(3-aminopropyl)-L-α,γ-diaminobutyric acid (4b): Yield: ~100%; ESI MS 344 (M+23+32); 1H-NMR (D2O) δ 1.55-1.75 (bm, 8H, C-CH2-C), 2.40-2.80 (bm, 14H, N-CH2), 2.98 (dd, J = 5.33 Hz, 1H, αCH), 3.35 (s, 6H, NH2); 13C-NMR δ 28.8 (γCH2-CH2NH2), 29.0 (αCH2-CH2NH2), 32.0 (βC), 38.9 (αCH2NH2), 39.4 (γCH2NH2), 45.1 (NHα-CH2), 49.1 [Nγ-(CH2)2], 50.9 (γC), 62.67 (αC), 182.3 (COO-).
N,N,N’-tris(3-aminopropyl)-L-ornithine (4c): Yield: ~100%; ESI MS 326 (M+23); 1H-NMR (D2O) δ 1.39 (m, 4H, β,γCH2), 1.53 (m, 6H, C-CH2-C), 2.30-2.60 (bm, 14H, N-CH2), 2.95 (dd, J = 6.02 Hz, 1H, αCH), 3.28 (s, 6H, NH2); 13C-NMR δ 21.5 (αCH2-CH2NH2), 28.1 (δCH2-CH2NH2), 30.7 (γC), 31.6 (βC), 38.4 (αCH2NH2), 38.9 (δCH2NH2), 44.6 (NHα-CH2), 48.6 [Nδ-(CH2)2], 50.4 (δC), 63.4 (αC), 182.3 (COO-).
N,N,N’-tris(3-aminopropyl)-L-lysine (4d): Yield: ~100%; ESI MS 318 (M+1); 1H-NMR (D2O) δ 1.50 (m, 2H, γCH2), 1.81 (m, 2H, δCH2), 2.06 (m, 2H, βCH2), 2.15 (m, 6H, C-CH2-C), 3.10 (t, J = 7.7 Hz, 6H, CH2NH2), 3.20-3.30 (bm, 8H, Nε-CH2, NHα-CH2), 3.34 (s, 6H, NH2), 3.97 (dd, J = 5.09 Hz, 1H, αCH); 13C-NMR δ 21.5 (αCH2-CH2NH2), 21.7 (εCH2-CH2NH2), 22.9(γC), 23.9 (δC), 28.6 (βC), 36.6 (εCH2NH2), 36.7 (αCH2NH2), 43.9 (NHα-CH2), 50.1 [Nε-(CH2)2], 52.7 (εC), 60.7 (αC), 181.5 (COO-).
N,N-bis(3-aminopropyl)-L-α,β-diaminopropionic acid (12): Yield: ~100%; ESI MS 272 (M+NH4++ 2H2O), 273 (M+Na++MeOH), 275 (M+K++H2O); 1H-NMR (D2O) δ 1.60 (m, 4H, C-CH2-C), 2.50-2.75 (bm, 10H, βCH2, N-CH2), 3.19 (t, J = 6.46 Hz, 1H, αCH), 3.35 (s, 6H, NH); 13C=NMR δ 31.9, 32.1 (α, βCH2-CH2NH2), 38.9 (α, βCH2NH2), 45.4, 46.4 (NH-CH2), 51.4 (βCH2), 63.2 (αC), 181.44 (COO-).
3.5.4. General synthesis of the Z-protected derivatives 4e-h
To the crude products from the previous reaction (8.5-9.5 g, ca. 15 mmol, 100%), a dioxane-water mixture (1:1, v/v, 100 mL) was added, followed by addition of benzyloxycarbonyl chloride (12 mL, 85.7 mmol). The pH value of the mixture was maintained between 9 and 10 by adding 1M NaOH (88 mL). The reaction mixture was cooled in an ice-water bath; after addition of substrates, it was allowed to warm to room temperature and was stirred overnight. Then the aqueous phase was washed three times with diethyl ether (50 mL; the ether extracts were discarded), acidified using 10% citric acid, and extracted three times with chloroform (100 mL). The CHCl3 layers were combined, dried over Na2SO4 overnight and concentrated in vacuo. The residue was purified by flash chromatography (CHCl3:MeOH 8:1) to give 4e-h (58.7-69%) as pale colored gums.
Nα-benzyloxycarbonyl-N,N,N’-tris(benzyloxycarbonyl-3-aminopropyl)-L-α,β-diaminopropionic acid (4e): Yield: 9.05g (58.7%); yellow gum; ESI MS 826 (esterification in MeOH in the spectrometer!); 1H-NMR (DMSO, 350K) δ 1.60-1.80 (m, 6H, C-CH2-C), 2.80-3.30 (bm, 12H, N-CH2), 3.60 (m, 2H, βCH2), 4.30 (m, 1H, αCH), 5.04 (s, 8H, CH2Ar), 7.29 (m, 20H, Ar-H); 13C-NMR δ 28.5, 29.5 (C-CH2-C), 39.79, 39.81 (CH2NH), 43.7 (Nα-CH2), 49.6 [Nβ-(CH2)2], 51.8 (βC), 59.1 (αC), 65.4, 66.8 (CH2-Ar), 127.7, 127.8, 128.3, 137.0 (CAr), 155.1, 156.4 (O-CO-N-), 172.4 (COOH); α[D]26.6°C = -10.30 ± 0.5° (c=2 in acetone).
Nα-benzyloxycarbonyl-N,N,N’-tris(benzyloxycarbonyl-3-aminopropyl)-L-α,γ-diaminobutyric acid (4f): Yield: 10.25 g (69%); colorless gum; ESI MS 839 (M’ + 1, esterification in MeOH in the spectrometer!), 916 (M + C7H7+); 1H-NMR (DMSO, 350K) δ 1.49-1.66 (bm, 8H, C-CH2-C), 3.0-3.6 (bm, 15H, N-CH2) 4.15 (m, 1H, αCH), 5.02 (s, 8H, CH2-Ar), 7.33 (bs, 20H, Ar-H); 13C-NMR δ 25.6, 26.1 (C-CH2-C), 28.5 (βC), 38.5 (CH2NH), 45.0 (NHα-CH2), 50.7 [Nγ-(CH2)2], 51.0 (γC), 60.1 (αC) 64.8, 66.0 (CH2-Ar), 126.8, 127.0, 127.8, 136.9 (CAr), 155.6 (O-CO-N-), 172.3 (COOH); α[D]26.3°C = -10.1 ± 0.5° (c = 2 in acetone).
Nα-benzyloxycarbonyl-N,N,N’-tris(benzyloxycarbonyl-3-aminopropyl)-L-ornithine (4g):Yield: 8.16 g (56.6%); pale yellow gum; ESI MS 840 (M+1), 854 (M’ + 1, esterification in MeOH in the spectrometer!); 1H-NMR (DMSO, 350K) δ 1.48, 2.31 (2m, 10H, C-CH2-C, β, γCH2), 2.90-3.80 (bm, 14H, N-CH2), 4.24 (m, 1H, αCH), 5.02, 5.18 (s, 8H, CH2Ar), 7.31 (m, 20H, Ar-H); 13C-NMR δ 23.2 (γC), 26.8 (C-CH2-C), 29.4 (βC), 38.7 (CH2NH), 45.1 (Nα-CH2), 50.8 [Nδ-(CH2)2], 52.8 (δC), 59.8 (αC), 65.1, 66.3 (CH2-Ar), 127.8, 128.3, 137.3 (CAr), 155.4, 156.0 (O-CO-N-), 171.5 (COOH); α[D]26.5°C = -10.12 ± 0.5° (c = 2 in acetone).
Nα-benzyloxycarbonyl-N,N,N’-tris(benzyloxycarbonyl-3-aminopropyl)-L-lysine (4h): Yield: 8.50 g (62.27%); yellow gum; ESI MS 854 (M+1), 944 (M + C7H7+), 1034 (M-1+2C7H7+); 1H-NMR (DMSO, 350K) δ 1.15-2.0 (bm, 12H, β, γ, δCH2, C-CH2-C), 2.50, 3.10 (2 bm, 14H, NCH2), 4.10 (m, 1H, αCH), 4.97, 5.03 (bs, 8H, CH2Ar), 7.29 (bm, 20H, Ar-H); 13C-NMR δ 22.6 (γC), 28.6 (δC), 28.8 (C-CH2-C), 29.8 (βC), 37.6, 38.5 (CH2NH), 44.5 (Nα-CH2), 50.6 [Nε-(CH2)2], 52.2 (εC), 58.4 (αC), 66.3, 66.9 (CH2-Ar), 127.9, 128.3, 136.7, 136.8 (CAr), 156.6, 157.1 (O-CO-N-), 172.7 (COOH); α[D]23.6°C = -10.33 ± 0.5° (c = 2 in acetone).
N,Nβ-bis(benzyloxycarbonyl)-Nα,Nβ-bis(benzyloxycarbonyl-3-aminopropyl)-L-α,β-diaminopropionic acid (13): Yield: 11.1g (61.83%); amber-colored gum; ESI MS 777 (M+23, CHCl3); 1H-NMR (DMSO, 350K) δ 1.60-1.70 (m, 4H, C-CH2-C), 2.96 (m, 4H, CH2NH), 3.20, 3.30 (2 m, 4H, N-CH2), 3.70 (m, 2H, βCH2), 4.25 (m, 1H, αCH), 5.04 (s, 8H, CH2Ar), 7.29 (m, 20H, Ar-H); 13C-NMR δ 28.7 (C-CH2-C), 38.8 (CH2NH), 45.8 (NH-CH2), 47.1 (βC), 59.2 (αC), 64.9, 66.0 (CH2-Ar), 128.1, 128.7, 137.79, 137.82 (CAr), 155.7, 156.5 (O-CO-N-), 171.39 (COOH); α[D]24.5°C = -52.50 ± 1° (c = 2 in acetone).
3.5.5. General procedure for the reduction of the nitriles to the corresponding tetrakis-N-propylamino derivatives 5a-d
To a derivative 3a-d (5 g, ca. 15 mmol) dissolved in dry THF (250 mL), 10 M solution of BH3 × SMe2 (Aldrich, 15.8 mL) were added. The reaction mixture was heated to reflux and stirred vigorously overnight. After cooling to room temperature, 1M HCl in MeOH (94.8 mL) and additional MeOH (100 mL) were added. The mixture was evaporated in vacuo, the oily residue was treated three times with 50 mL of MeOH and evaporated to dryness, to give 8.5-9 g (ca. 100%) of a white, very hygroscopic foam. The elemental analysis shows a mixture of penta- and hexahydrochlorides of 5a-d.
N,N’,N,N’-tetrakis(3-aminopropyl)-L-α,β-diaminopropanol (5a): Yield: 8.9g (~100%); ESI MS 319 (M+1); 1H-NMR (D2O) δ 1.90-2.10 (m, 8H, C-CH2-C), 3.00-3.70 (3m, 20H, N-CH2, CH2OH), 3.90 (2m, 1H, αCH); 13C-NMR δ 26.7, 29.3 (αC-CH2-C, βC-CH2-C), 37.6, 38.0 (CH2NH2), 48.7, 57.8 [Nα-(CH2)2, Nβ-(CH2)2], 58.34 (βC), 59.2 (αC), 61.7 (CH2OH); α[D]26.5 °C = +15.6° (±1°, c = 2 in 1M HCl).
N,N’,N,N’-tetrakis(3-aminopropyl)-L-α,γ-diaminobutanol (5b): Yield: 8.8g (~100%); ESI MS 353 (M+23); 1H-NMR (D2O) δ 1.60-2.2 (bm, 10H, βCH2, C-CH2C), 2.80-3.60 (bm, 20H, N-CH2, CH2OH), 3.87 (m, 1H, αCH); 13C-NMR δ 21.7, 22.9 (C-CH2-C), 28.8 (βC), 36.5, 36.6 (CH2NH2), 49.0, 50.1 [Nα-(CH2)2, Nγ-(CH2)2], 52.1 (γC), 57.35 (αC), 61.9 (CH2OH); α[D]29.1°C = +12.7° (±1°, c=2 in 1M HCl).
N,N’,N,N’-tetrakis(3-aminopropyl)-L-α,δ-diaminopentanol (5c): Yield: 8.7g (~100%); ESI MS 347 (M+1); 1H-NMR (CDCl3) δ 1.55, 1.80, 2.10 (3m, 12H, β, γCH2, C-CH2-C), 2.90-3.40 (bm, 20H, N-CH2, CH2OH), 3.95 (2m, 1H, αCH); 13C-NMR δ 21.8 (C-CH2-C), 22.9 (γC), 27.9 (βC), 36.6, 36.7 (CH2NH2), 50.0 [α, δN-(CH2)2], 52.9 (δC), 57.8 (αC), 61.6 (CH2OH); α[D]29.1°C = +12.3° (±1°, c=2 in 1M HCl).
N,N’,N,N’-tetrakis(3-aminopropyl)-L-α,ε-diaminohexanol (5d): Yield: 8.9 g (~100%); ESI MS 361 (M+1), 383 (M+23); 1H-NMR (CDCl3) δ 1.50-2.30 (3m, 14H, β, γ, δCH2, C-CH2-C), 2.90-3.40 (bm, 20H, N-CH2, CH2OH), 3.95 (m, 1H, αCH); 13C-NMR δ 22.0 (C-CH2-C), 23.3 (γC), 23.9 (δC), 26.3 (βC), 36.9, 37.3 (CH2NH2), 49.2, 50.21 [α, ε N-(CH2)2], 53.2 (εC), 58.9 (αC), 61.8 (CH2OH); α[D]28.6°C = +13.2 ± 1°, (c = 2 in 1M HCl).
3.5.6. Preparation of dendrimeric compounds 7e-7h
To benzylamine (3.21 g, 3.3 mL = 30 mmol) dissolved in dry THF (15 mL), 4g (2.55 g, 3 mmol), HOBt (0.46 g, 3 mmol) and DCC (0.63 g, 3 mmol) were added, the mixture was stirred for 24 h and the solvent was evaporated in vacuo. The residue was dissolved in CHCl3 (50 mL) and washed consecutively with 10% Na2CO3, H2O, 1% citric acid and saturated NaCl solution, then dried over Na2SO4 and evaporated to dryness: yield 2.07 g (74.3%) of the amide 6g as a light yellow gum.
N,N,N’-tris(benzyloxycarbonyl-3-aminopropyl)-L-diaminoalanine benzylamide (6e): C51H60O9N6, Yield: 1,92g (65.3%); yellow gum; ESI MS 901 (M+H+), 923 (M+Na+); 1H-NMR (DMSO, 350K) δ 1.66-1.85 (m, 6H, C-CH2-C), 2.79-3.38 (bm, 12H, N-CH2), 3.63 (m, 2H, βCH2), 4.23 (s, 2H, Ar-CH2-NH), 4.3 (m, 1H, αCH), 5.05, 5.09 (2s, 8H, CH2-Ar), 7.2-7.3 (bm, 25H, Ar-H); 13C-NMR δ 28.6, 29.1 (C-CH2-C), 40.1 (CH2NH), 42.3 (Ar-CH2-NH), 43.7 (Nα-CH2), 49.5 [Nβ-(CH2)2], 51.1 (βC), 59.6 (αC), 65.1, 66.3 (CH2-Ar), 127.7, 127.8, 128.2 (CHAr), 136.6, 136.9 (CAr), 155.0, 156.2 (O-CO-N-), 173.1 (CONH).
N,N,N’-tris(benzyloxycarbonyl-3-aminopropyl)-L-diaminobutyric benzylamide (6f): Yield 2.15 g (70.1%); pale yellow gum; ESI MS 915 (M+H+), 937 (M+Na+); 1H-NMR (DMSO, 350K) δ 1.45-1.7 (3m, 8H, βCH2, C-CH2-C), 2.9-3.5 (bm, 14H, N-CH2), 4.2 (s, 2H, Ar-CH2-NH), 4.28 (m, 1H, αCH), 5.07, 5.11 (2s, 8H, CH2-Ar), 7.2-7.3 (bm, 25H, Ar-H). 13C-NMR δ 25.5, 26.4 (C-CH2-C), 29.1 (βC), 38.5 (CH2NH), 42.4 (Ar-CH2-NH), 44.5 (NHα-CH2), 50.3 [Nγ-(CH2)2], 51.1 (γC), 59.8 (αC), 65.0, 66.1 (CH2-Ar), 126.9, 127.0, 127.8, 128.0 (CHAr), 136.8, 137.0 (CAr), 155.3, 156.2 (O-CO-N-), 173.0 (CONH).
N,N,N’-tris(benzyloxycarbonyl-3-aminopropyl)-L-ornithine benzylamide (6g): Yield: 74.3%; C53H64O9N6, pale yellow gum; ESI MS 929 (M+1), 951 (M+23), 967 (M+39), 1,019 (M + 91 = C7H7+); 1H-NMR (DMSO, 350K) δ 1.40 - 2.30 (2m, 10H, C-CH2-C, β, γCH2,), 2.85-3.70 (bm, 14H, N-CH2), 4.22 (s, 2H, Ar-CH2-NH), 4.31 (m, 1H, αCH), 5.05, 5.20 (s, 8H, CH2Ar), 7.29 (m, 25H, Ar-H); 13C- NMR δ 23.2 (γC), 26.3 (C-CH2-C), 29.6 (βC), 38.6 (CH2NH), 42.2 (Ar-CH2-NH), 44.2 (Nα-CH2), 50.6 [Nδ-(CH2)2], 52.6 (δC), 59.7 (αC), 65.0, 66.3 (OCH2-Ar), 127.6, 127.7, 128.3, 137.3 (CAr), 155.2, 156.1 (O-CO-N-), 171.3 (CONH).
N,N,N’-tris(benzyloxycarbonyl-3-aminopropyl)-L-lysine benzylamide (6h): Yield: 78.6%; C54H66O9N6, yellow gum; ESI MS 943 (M+1), 965 (M+23), 981 (M+39) 1,033 (M+91 = C7H7+); 1H-NMR (DMSO, 350K) δ 1.2-2.1 (bm, 12H, β, γ, δCH2, C-CH2-C), 2.52, 3.12 (2 bm, 14H, NCH2), 4.19 (m, 1H, αCH), 4.26 (s, 2H, Ar-CH2-NH), 5.03, 5.1 (bs, 8H, CH2Ar), 7.15-7.3 (bm, 25H, Ar-H); 13C-NMR δ 22.7(γC), 28.4 (δC), 28.5 (C-CH2-C), 29.4 (βC), 37.4, 38.1 (CH2NH), 42.1 (Ar-CH2-NH), 43.9 (Nα-CH2), 49.9 [Nε-(CH2)2], 52.1 (εC), 58.5 (αC), 66.4, 66.8 (OCH2-Ar), 127.9, 128.4, 136.8, 136.9 (CAr), 156.6, 156.9 (O-CO-N-), 171.9 (CONH).
A) 6g (1.5 g, 1.6 mmol) dissolved in MeOH (25 mL) was stirred for 24 h with 10% Pd/C (150 mg) under an atmospheric pressure of H2. Then the catalyst was separated on Celite® and washed with MeOH. The collected methanol fractions were evaporated to dryness yielding 0.59 g (93.1%) of deprotected 6g as a white waxy solid. To the solution of deprotected 6g in DMF (25 mL), (2-Cl-Z)Lys(Boc) (2.04 g, 4.95 mmol), HOSu (0.57 g, 4.95 mmol) and DCC (1.02 g, 4.95 mmol) were added. The mixture was stirred for 24 h (until disappearance of free amino groups in the ninhydrin test), filtered and evaporated in vacuo. The residue was dissolved in CHCl3 (50 mL) and washed with 10% Na2CO3, H2O, 1% citric acid, dried over Na2SO4 overnight and evaporated to dryness. The residue was purified by flash chromatography (CHCl3-MeOH 8:1) to give 1.7 g (71.3%) of Boc-protected 7g as a dark yellow resin.
B) Boc-protected 7g (0.5 g, 0.32mmol) was dissolved in CH2Cl2 (5 mL) and trifluoroacetic acid (TFA, 5 mL) was added. The reaction mixture was stirred at room temperature for 3 h. Then the reaction mixture was evaporated in vacuo, the residue was dissolved in ethyl acetate (5 mL) and evaporated (3 times) and then in diethyl ether (5 mL) and evaporated (twice) – to remove all remaining trifluoroacetic acid. Trifluoroacetate ions were replaced by chlorides by dissolving the oily residue in HCl-saturated ethyl acetate and evaporation in vacuo (four times) to give 470 mg (99%) of 7g hexahydrochloride as a yellow glassy gum.
N,N,N’-tris[(Nα-2-chlorobenzyloxycarbonyl)-L-lysil-3-aminopropyl]-L-diaminoalanine benzylamide pentahydrochloride (7e): C61H87O10N12Cl3·5HCl; Yield 0.99g (97.5%); ESI MS 627 (M+2H+)2+ - base peak, 647 (M+H++Na++H2O)2+, 1,253 (M+H+); 1H-NMR (DMSO, 350K) δ1.1 – 2.1 (4bm, 24H, C-CH2-C, β, γ,δCH2 G-1 Lys), 2.6-3.5 (3 bm, 20H, αNH-CH2, βN-(CH2)2, CH2-NH core, εCH2 G-1 Lys), 3.53 (m, 1H, αCH core), 4.18 (m, 3H, αCH G-1 Lys), 4.30 (Ar-CH2-NH), 5.11, 5.18 (2bs, 6H, Ar-CH2O- from 2-Cl-Z), 7.2-7.4 (bm; 17H, Ar-H); 13C-NMR δ 22.2 (γC G-1 Lys), 25.1 (C-CH2-C core), 29.7 (δC G-1 Lys), 31.0 (βC G-1 Lys), 38.5 (εC G-1 Lys), 40.1 (CH2NH core), 43.4 (ArCH2-NH), 44.6 (Nα-CH2 core), 50.9 [Nβ-(CH2)2 core], 52.7 (βC core), 55.4 (αC G-1 Lys), 59.5 (αC core Lys), 62.1, 62.3 (Ar-CH2-O), 126.5, 126.6, 127.4, 127.8, 128.7, 129.0, 129.1, 129.2 (CHAr), 132.0 (CAr-Cl), 133.7 (CAr-CH2-NH), 134.1 (CAr-CH2-O), 155.6, 156.7 (O-CO-NH-), 171.3 (CONH G-1 Lys), 172.2 (CONH core); Anal. Calcd for C61H87O10N12Cl3·5HCl, (12 days/P2O5): C, 51.0; H, 6.45; N, 11.70; Cl, 19.73. Found: C, 50.67; H, 6.51; N, 11.43; Cl, 19.5.
N,N,N’-tris[(Nα-2-chlorobenzyloxycarbonyl)-L-lysil-3-aminopropyl]-L-diaminobutyro benzylamide pentahydrochloride (7f): C62H89O10N12Cl3·5HCl; Yield 1.0g (98.3%); hygroscopic yellow gum; ESI MS 634 (M+2H+)2+- base peak, 654 (M+H++Na++H2O)2+, 1,267 (M+H+); 1H-NMR (DMSO, 350K) δ 1.1 – 2.1 (bm, 26H, C-CH2-C, βCH2 core and β, γ,δCH2 G-1 Lys), 2.5-3.55 (2 bm, 20H, αNH-CH2, γ N-(CH2)2, CH2-NH core, εCH2 G-1 Lys), 3.68 (m, 1H, αCH core), 4.15 (m, 3H, αCH G-1 Lys), 4.28 (Ar-CH2-NH), 5.09, 5.15 (2bs, 6H, Ar-CH2O- grup 2-Cl-Z), 7.2-7.4 (bm; 17H, Ar-H); 13C-NMR δ 22.1 (γC G-1 Lys), 25.3 (C-CH2-C core), 29.1 (βC core), 29.6 (δC G-1 Lys), 31.1 (βC core Lys), 38.2 (εC G-1 Lys), 39.9 (CH2NH core), 43.2 (ArCH2-NH), 44.9 (Nα-CH2 core), 50.8 [Nγ-(CH2)2 core], 53.1 (γC core), 55.3 (αC G-1 Lys), 59.7 (αC core), 62.2, 62.4 (Ar-CH2-O), 126.5, 126.7, 127.3, 127.8, 128.7, 129.0, 129.1, 129.2 (CHAr), 131.9 (CAr-Cl), 133.8 (CAr-CH2-NH), 134.0 (CAr-CH2-O), 155.7, 156.8 (O-CO-NH-), 171.4 (CONH G-1 Lys), 172.2 (CONH core); Anal. Calcd for C62H89O10N12Cl3·5HCl, (12 days/P2O5): C, 51.3; H, 6.52; N, 11.58; Cl, 19.54. Found: C, 50.96; H, 6.61; N, 11.31; Cl, 19.42.
N,N,N’-tris[(Nα-2-chlorobenzyloxycarbonyl)-L-lysil-3-aminopropyl]-L-ornithine benzylamide penta- hydrochloride (7g): Yield: 99%; C63H91O10N12Cl3·5HCl, hygroscopic, glass-like gum; ESI MS 641 (M+2)2+, 1,281 (M+1), 1,299 (M+18+1); 1H-NMR (DMSO, 350K) δ 1.3 – 2.2 (bm, 28H, core β, γCH2, C-CH2-C, G1 β, γ,δCH2), 2.4-3.3 (bm, 20H, core αNH-CH2, δN-(CH2)2, CH2-NH, G1 εCH2), 3.60 (m, 1H, core αCH), 4.19 (m, 3H, G1 αCH), 4.38 (Ar-CH2-NH), 5.17 (m, 6H, G1 Ar-CH2O-), 7.2-7.4 (bm; 17H, Ar-H); 13C-NMR δ 22.4 (G1 γC), 23.1 (core γC), 27.6 (core C-CH2-C), 29.3 (G1 δC), 29.5 (core βC), 32.3(G1 βC), 36.3 (core CH2NH), 39.8, 40.0 (G1 εC), 43.2 (ArCH2-NH), 44.1 (core Nα-CH2), 49.7 [core Nδ-(CH2)2], 51.6 (core δC), 55.0, 55.2 (G1 αC), 58.8 (core αC), 66.5, 66.8 (Ar-CH2-O), 126.67, 126.7, 126.8, 127.6, 128.5, 128.9, 129.0, 129.3 (CHAr), 132.8 (G1 CAr-Cl), 133.9 (CAr-CH2-NH), 134.4 (G1 CAr-CH2-O), 155.8, 156.0 (G1 O-CO-N-), 174.0 (core CONH), 175.2 (G1 CONH); Anal. Calcd for C63H91O10N12Cl3·5HCl: C, 51.65; H, 6.26; N, 11.47; Cl, 19.35. Found: C, 51.43; H, 6.45; N, 10.31; Cl, 19.05.
N,N,N’-tris[(Nα-2-chlorobenzyloxycarbonyl)-L-lysil-3-aminopropyl]-L-lysine benzylamide penta- hydrochloride (7h): Yield: 99%; C64H93O10N12Cl3·5HCl, hygroscopic, glassy solid; ESI MS 648 (M+2)2+, 659 (M+1+23)2+ 1,295 (M+1), 1,313 (M+18+1); 1H-NMR (DMSO, 350K) δ 1.1–2.1 (bm, 30H, C-CH2-C, core and G1 β, γ,δCH2), 2.6-3.5 (2 bm, 20H, core αNH-CH2, εN-(CH2)2, CH2-NH, G1 εCH2), 3.65 (m, 1H, core αCH), 4.05 (m, 3H, G1 αCH), 4.33 (Ar-CH2-NH), 5.12 (m, 6H, G1 Ar-CH2O-), 7.2-7.4 (bm; 17H, Ar-H); 13C-NMR δ 21.9 (G1 γC), 22.1 (core γC), 24.5 (core δC) 25.6 (core C-CH2-C), 28.8 (core βC), 29.8 (G1 δC), 31.0 (G1 βC), 38.2 (G1 εC), 39.7 (core CH2NH), 43.1 (ArCH2-NH), 45.3 (core Nα-CH2), 51.4 [core Nε-(CH2)2], 53.6 (core εC), 55.6 (G1 αC), 59.9 (core αC), 62.4, 62.5 (Ar-CH2-O), 126.5, 126.7, 127.1, 127.8, 128.7, 129.0, 129.1, 129.2 (CHAr), 131.8 (G1 CAr-Cl), 133.9 (CAr-CH2-NH), 134.0 (G1 CAr-CH2-O), 156.7, 157.0 (G1 O-CO-N-), 171.5 (G1 CONH), 172.0 (core CONH); Anal. Calcd for C64H93O10N12Cl3*5HCl: C, 51.9; H, 6.67; N, 11.36; Cl, 19.17. Found: C, 51.63; H, 6.8; N, 11.19; Cl, 18.93.
3.5.7. Preparation of the dendrimeric compounds 9a-d
5c (450 mg, 0.72 mmol) was suspended in DMF (15 mL) with addition of N(Et)3 (2.40 mL, 17.28 mmol) and stirred at 40 °C until all of 5c was dissolved. Then (Boc)-L-Phe-2,4,5-trichlorophenyl ester 1.76 g (3.96 mmol, 5.5 eq) was added and the reaction mixture was stirred at room temperature for 7 days until the complete disappearance of free amino groups in the ninhydrin test. The solution was evaporated in vacuo, the residue was dissolved in CHCl3 (50 mL) and washed with 20% K2CO3 and saturated NaCl solution, dried over Na2SO4 overnight, filtered and evaporated to dryness. The residue was purified by flash chromatography (CHCl3-MeOH, 8:1) to give 520 mg of 9c (54%) as a white foam.
N,N',N,N’-tetrakis(Boc-L-phenylalanyl-3-aminopropyl)-L-α,δ-diaminopropanol (9a): Yield 2.76g (66%); C71H106O13N10; white foam; ESI MS 654 (M+2H+)2+, 1,307 (M+H+); 1H-NMR (DMSO, 350K) δ1.2-1.3 (m, 36H, Boc C-CH3), 1.47 (m, 8H, C-CH2-C), 2.33 (m, 4H, Nβ-CH2), 2.39 – 2.43 (m, 6H, βCH2, Nα-CH2), 2.83, 2.93 (dd, J 5.3, 8.5 Hz, 8H, Ar-CH2), 3.18 (m, 8H, CH2-NH), 3.42 (m, 2H, CH2OH), 3.86 (m, 1H, αCH core), 4.23 (m, 4H, αCH Phe), 7.2 (m; 20H, Ar-H). 13C-NMR δ 21.2 (C-CH2-C), 28.2 (Boc C-CH3), 36.8, 36.9 (α, βCH2NH core), 50.0 [α, βN-(CH2)2], 52.8 (βC), 58.4 (αC), 62.0 (CH2OH), 77.7 (Boc C-CH3), 125.2, 125.6, 127.6, 127.8, 128.7, 128.8 (CHAr), 137.6, 138.5 (CAr), 154.4, 154.8 (O-CO-NH), 172.2 (CONH Phe).
N,N',N,N’-tetrakis(Boc-L-phenylalanyl-3-aminopropyl)-L-α,δ-diaminobutanol (9b): Yield 3.9 g (71%); C72H108O13N10; ESI MS 662 (M+2H+)2+, 1,321 (M+H+); 1H-NMR (DMSO, 350K) δ1.2-1.3 (m, 36H, Boc C-CH3), 1.46 (m, 10H, βCH2, C-CH2-C), 2.35 (m, 6H, Nγ-CH2), 2.38 (m, 4H, Nα-CH2), 2.82, 2.94 (dd, J 5.3, 8.5 Hz, 8H, Ar-CH2), 3.15 (m, 8H, CH2-NH), 3.39 (m, 2H, CH2OH), 3.96 (m, 1H, αCH core), 4.22 (m, 4H, αCH Phe), 7.2 (m; 20H, Ar-H); 13C-NMR δ 21.6 (C-CH2-C), 27.8 – 28.4 (βC, Boc C-CH3), 36.6, 36.7 (α, γCH2NH core), 50.1 [α, γN-(CH2)2], 52.6 (γC), 57.7 (αC), 61.7 (CH2OH), 77.8 (Boc C-CH3), 125.2, 125.7, 127.6, 127.7, 128.6, 128.8 (CHAr), 137.7, 138.6 (CAr), 154.3, 154.5 (O-CO-NH), 171.3 (CONH Phe).
N,N',N,N’-tetrakis(Boc-L-phenylalanyl-3-aminopropyl)-L-α,δ-diaminopentanol (9c): Yield: 54%; C73H110O13N10, white foam; ESI MS 1,335 (M+1); 1H-NMR (350K, DMSO) δ 1.25-1.35 (m, 36H, C-CH3, Boc), 1.48 (m, 12H, core β, γCH2, C-CH2-C), 2.34 (m, 6H, core Nδ-CH2), 2.40 (m, 4H, core Nα-CH2), 2.80, 2.97 (dd, J = 5.26, 8.55 Hz, 8H, G1 Ar-CH2), 3.10 (m, 8H, core CH2-NH), 3.41 (m, 2H, core CH2OH), 3.98 (m, 1H, core αCH), 4.20 (m, 4H, G1 αCH), 7.19 (m; 20H, G1 Ar-H). 13C-NMR δ 26.5 (γC, δ core C-CH2-C), 28.5 (βC, αC-CH2-C), 36.7, 36.8 (α, δ CH2NH), 37.7 (G1 Ar-CH2), 47.5, 50.9 [core α, δN-(CH2)2], 53.6 (core δC), 55.5, 55.7 (core and G1 αC), 61.1 (core CH2OH), 77.7 [O-C-(CH3)3], 125.2, 125.6, 127.5, 127.2, 128.7, 128.9 (G1 CHAr), 137.6, 138.6 (G1 CAr), 154.3, 154.5 (G1 O-CO-NH), 170.7 (G1 CO-NH).
N,N',N,N’-tetrakis(Boc-L-phenylalanyl-3-aminopropyl)-L-α,ε-diaminohexanol (9d): Yield: 73.5%; C74H112O13N10, white foam; ESI MS 675 (M+2)2+, 1,349 (M+1)+; 1H-NMR (350K, DMSO) δ 1.24-1.36 (m, 36H, C-CH3, Boc), 1.43 (m, 14H, core β, γ, δCH2, C-CH2-C), 2.31 (m, 6H, core Nε-CH2), 2.42 (m, 4H, core Nα-CH2), 2.79, 2.95 (dd, J = 5.27, 8.54 Hz, 8H, G1 Ar-CH2), 3.15 (m, 8H, core CH2-NH), 3.43 (m, 2H, core CH2OH), 3.99 (m, 1H, core αCH), 4.22 (m, 4H, G1 αCH), 7.20 (m; 20H, G1 Ar-H).13C-NMR δ 24.1 (core γC), 26.6 (core δC, ε C-CH2-C), 28.5 (core βC, αC-CH2-C), 36.7, 36.8 (core α, ε CH2NH), 37.7 (G1 Ar-CH2-CH), 47.8, 51.0 [core α, εN-(CH2)2], 53.8 (core εC), 54.9, 55.1 (core and G1 αC), 61.1 (core CH2OH), 77.8 [O-C-(CH3)3], 125.2, 125.5, 127.2, 128.7, 129.0 (G1 CHAr), 137.7, 138.7 (G1 CAr), 154.7, 154.7 (G1 O-CO-NH), 170.6 (G1 CO-NH).
3.5.8. Preparation of the dendrimeric compound 10c
A) 9c (0.5 g, 0.375 mmol) was dissolved in CH2Cl2 (5 mL) and TFA (5 mL) was added. The reaction mixture was stirred at room temperature for 3 h. Then the reaction mixture was evaporated in vacuo, the residue was dissolved in ethyl acetate (5 mL) and evaporated (three times) and then in diethyl ether (5 mL) and evaporated (twice) – to remove all remaining trifluoroacetic acid. Dark-orange oil (0,4g) was used for the next step without purification.
B) (2-Cl-Z)L-Lys(Boc) (0.68 g, 1.65 mmol) and N-hydroxysuccinimide (HOSu, 0.19 g 1.66 mmol) was dissolved in THF (10 mL) followed by addition of DCC (0.34 g, 1.66 mmol). The solution was stirred for 0.5 h and added to the solution of Boc-deprotected 9c and N(Et)3 (1 mL, 7.13 mmol) in DMF (5 mL). The final solution was stirred for 48 h at room temperature, then DCU was filtered off and the solvents was evaporated in vacuo. The residue was dissolved in CHCl3 (50 mL) and washed with 10% Na2CO3 and saturated NaCl solution (twice), dried over Na2SO4 overnight, filtered and evaporated to dryness. The residue was purified by flash chromatography (CHCl3:MeOH, 15:1) to give 0.729 g of Boc protected 10c (77.1%) as a white foam.
C) Boc-10c (360 mg, 0.14 mmol) was deprotected according to procedure A). Trifluoroacetate ions were replaced by chlorides by dissolving the oily residue in HCl-saturated ethyl acetate and evaporation in vacuo (four times) to give 330 mg of 10c hexahydrochloride (99%) as a white, hygroscopic foam.
N,N',N,N’-tetrakis[(Nα-2-chlorobenzyloxycarbonyl)-L-lysil-L-phenylalanyl-3-aminopropyl]-L-α,δ-diaminopropanol hexahydrochloride (10a): Yield 1.0 g (98%); C107H142O17N18Cl4·6HCl; pale yellow foam; ESI MS 1,046 (M+2H+)2+, 1,057 (M+H++Na+)2+, 2,105 (M+H+); 1H-NMR (DMSO, 298K) δ 1–2 (bm, 32H, γ,β,δCH2 G-1 Lys, C-CH2-C core arms), 2.6-3.7 (4 bm, 37H, CH2-NH core, εCH2 G-1 Lys i βCH2 core, α, βN-(CH2)2 core, Ar-CH2-CH Phe, CH2OH & αCH core), 4.1, 4.6 (2 bm, 8H, αCH Lys & Phe, respectively), 5.16 (4s, 8H, Ar-CH2 from 2-Cl-Z), 7.2 – 7.45 (m; 36H, Ar-H); 13C-NMR δ 22.1, 22.3 (γC G-1 Lys), 23.4 (α, γC-CH2-C core), 26.6 (δC G-1 Lys), 28.7 (βC core), 30.3, 31.1 (βC G-1 Lys), 36.3, 36.5 (α, βCH2NH core), 37.7 (Ar-CH2-CH), 38.3, 38.6 (εCH2NH2 G-1 Lys), 48.8, 49.7 [α, βN-(CH2)2 core], 52.3 (βC core), 54.2, 55.3 (αC Phe and Lys, respectively), 58.8 (αC core), 60.3 (CH2OH core) 62.7, 62.8 (Ar-CH2-O), 126.3, 127.2, 128.2, 129.1, 129.3, 129.6, 129.7, 129.8 (CHAr), 132.1, 133.2 (Car-Cl), 134.3, 134.4 (Car-CH2Phe), 136.9 (Car-CH2O), 155.4, 155.5 (O-CO-NH), 171.3, 172.4 (CONH Phe and L-Lys, respectively); Anal. Calcd for C107H142O17N18Cl4·6HCl: C, 55.6; H, 6.45; N, 10.9; Cl, 15.33. Found: C, 55.38; H, 6.63; N, 10.58; Cl, 15.02.
N,N',N,N’-tetrakis[(Nα-2-chlorobenzyloxycarbonyl)-L-lysil-L-phenylalanyl-3-aminopropyl]-L-α,δ-diaminobutanol hexahydrochloride (10b): Yield 1.01 g (99%); C108H144O17N18Cl4·6HCl, white hygroscopic foam; ESI MS 1,053 (M+2H+)2+, 1,064 (M+H++Na+)2+, 2,105 (M+H+); 1H-NMR (DMSO, 298K) δ 1 – 2 (bm, 34H, βCH2 core i γ,β,δCH2 G-1 Lys, C-CH2-C core arms), 2.65-3.75 (3 bm, 37H, CH2-NH core, εCH2 G-1 Lys & γCH2 core, α, γN-(CH2)2 core, Ar-CH2-CH Phe, CH2OH and αCH core), 4.15, 4.57 (2 bm, 8H, αCH Lys and Phe, respectively), 5.11 (4s, 8H, Ar-CH2 from 2-Cl-Z), 7.2–7.45 (m; 36H, Ar-H). 13C-NMR δ 22.1, 22.2 (γC G-L-Lys), 23.5 (α, δC-CH2-C core), 26.5 (δC G-1 Lys), 28.8 (βC core), 30.2, 31.1 (βC G-1 Lys), 36.2, 36.3 (α, γCH2NH core), 37.6 (Ar-CH2-CH), 38.4, 38.5 (εCH2NH2 G-1 Lys), 48.9, 49.7 [α, γN-(CH2)2 core], 52.0 (γC core), 54.1, 55.1 (αC Phe & Lys, respectively), 58.4 (αC core), 60.1 (CH2OH core) 62.6, 62.8 (Ar-CH2-O), 126.2, 127.3, 128.1, 129.1, 129.3, 129.5, 129.7, 129.8 (CHAr), 132.2, 133.3 (Car-Cl), 134.2, 134.3 (Car-CH2Phe), 137.0 (Car-CH2O), 155.3, 155.6 (O-CO-NH), 171.2, 172.6 (CONH Phe and Lys, respectively); Anal. Calcd for C108H144O17N18Cl4*6HCl: C, 55.74; H, 6.5; N, 10.83; Cl, 15.23. Found: C, 55.5; H, 6.7; N, 10.51; Cl, 14.94.
N,N',N,N’-tetrakis[(Nα-2-chlorobenzyloxycarbonyl)-L-lysil-L-phenylalanyl-3-aminopropyl]-L-α,δ-diaminopentanol hexahydrochloride (10c): Yield: 99%; C109H146O17N18Cl4*6HCl, white hygroscopic foam; ESI MS 1,051 (M-18+2)2+, 1,060 (M+2)2+, 1,071 (M+1+23)2+, 2,119 (M+1)+; 1H-NMR (298K, DMSO); NMR: δ 1 – 2 (bm, 36H, core γ, βCH2, G2 γ,β,δCH2, core C-CH2-C), 2.7-3.8 (3 bm, 37H, core CH2-NH, G2 εCH2, core δCH2, core α, δN-(CH2)2, G1 Ar-CH2-CH, core CH2OH and αCH), 4.1, 4.55 (2 bm, 8H, G1 and G2 αCH), 5.15 (m, 8H, G2 Ar-CH2), 7.25 (m; 36H, Ar-H), 8.15 (bm, 21H, N-H). 13C=NMR δ 22.2, 22.3 (G2 γC), 23.2 (core α, δC-CH2-C), 23.9 (core γC), 26.4 (G2 δC), 28.4 (core βC), 30.3, 31.0 (G2 βC), 35.9, 36.2 (core α, δ CH2NH), 37.7 (G1 Ar-CH2-CH), 38.3, 38.6 (G2 εCH2NH2), 48.9, 49.6 [core α, δN-(CH2)2], 52.1 (core δC), 54.1, 55.0 (G1 and G2 αC), 57.9 (core αC), 60.3 (core CH2OH) 62.7, 62.9 (G2 CH2Ar), 126.2, 127.3, 128.0, 129.1, 129.4, 129.6, 129.7, 129.7 (CHAr), 132.1, 133.3 (G2 Car-Cl), 134.3, 134.4 (G1 Car-CH2), 136.9 (G2 Car-CH2), 155.2, 155.7 (G2 O-CO-NH), 171.3, 172.8 (G1 and G2 CO-NH); Anal. Calcd for C109H146O17N18Cl4·6HCl: C, 55.92; H, 6.54; N, 10.77; Cl, 15.14. Found: C, 55.68; H, 6.65; N, 10.43; Cl, 14.96.
N,N',N,N’-tetrakis[(Nα-2-chlorobenzyloxycarbonyl)-L-lysil-L-phenylalanyl-3-aminopropyl]-L-α,ε-diaminohexanol hexahydrochloride (10d): Yield: 99%; C110H148O17N18Cl4*6HCl, white hygroscopic foam; ESI MS 1,058 (M-18+2)2+, 1,067 (M+2)2+, 1,078 (M+1+23)2+, 2,133 (M+1)+; 1H-NMR (298K, DMSO) δ 1–2 (bm, 38H, core and G2 γ,β,δCH2, core C-CH2-C), 2.6-3.8 (3 bm, 37H, core CH2-NH, core and G2 εCH2, core α, εN-(CH2)2, G1 Ar-CH2-CH, core CH2OH, core αCH), 4.0, 4.49 (2 bm, 8H, G1 and G2 αCH), 5.10 (m, 8H, G2 Ar-CH2), 7.20 (m; 36H, G1 and G2 Ar-H), 8.11–8.7 (3 bm, 21H, N-H); 13C-NMR δ 22.2, 22.3 (G2 γC), 23.0 (core α, εC-CH2-C), 24.4 (core γC), 25.2 (core δC), 26.3, 26.4 (G2 δC), 28.6 (core βC), 30.0, 31.2 (G2 βC), 36.0, 36.2 (core α, ε CH2NH), 37.7 (G1 Ar-CH2-CH), 38.3, 38.4 (G2 εCH2NH2), 48.5, 49.8 [core α, εN-(CH2)2], 51.3 (core εC), 53.7, 54.0, 54.7 (G1 and G2 αC), 57.4 (core αC), 60.4 (core CH2OH) 62.8, 62.9 (G2 CH2Ar), 126.2, 127.2, 127.9, 129.2, 129.5, 129.6, 129.7 (G1 and G2 CHAr), 132.1, 133.2 (G2 Car-Cl), 134.2, 134.3 (G1 Car-CH2), 137.6 (G2 Car-CH2), 155.6, 155.8 (G2 O-CO-NH), 171.0, 171.4, 172.7 (G1 and G2 CO-NH); Anal. Calcd for C110H148O17N18Cl4*6HCl: C, 56.1; H, 6.6; N, 10.7; Cl, 15.05. Found: C, 55.65; H, 6.76; N, 10.35 Cl, 14.91.