Chemistry

Thin layer chromatography experiments (TLC) were carried out on silica gel (Merck; 60 Å F254). Spots were located with UV light (254 and 366 nm) and 1% ethanolic 4-(4-nitrobenzyl) pyridine (NBP). Analytical RP-HPLC was performed on a Waters 600S HPLC system (Waters 2489 UV/VIS detector, Waters 616 pump, Waters 717 plus autosampler, HPLC manager software from Chromax) using a Vydac C18 column (25 cm × 4.6 mm, 5 mm; Sigma). HPLC was performed with a gradient of 0.1% TFA in H₂O (A) and 0.08% TFA in MeCN (B), at a flow rate of 1 mL/min with UV detection at 220 nm, t_R in min.

MS analysis was performed on an MS Bruker micrOTOF-QIII.

Infrared spectra were recorded as KBr pellets or on film using a Bruker ALPHA spectrometer or a PerkinElmer Spectrum 100.

¹H-NMR and ¹³C-NMR, spectra were recorded on a Bruker Avance DPX 250 (250 MHz) spectrometer and Varian (300 MHz). Chemical shifts (ppm) were relative to TMS, used as an internal standard. Multiplicities are marked as s = singlet, d = doublet, t = triplet, q = quartet, qu = quintet, m = multiplet.

Boc-Aib-OH (0.406 g, 2 mmol) and NMM (0.110 mL, 1 mmol) were added to a vigorously stirred solution of DMT/NMM/TosO⁻ (0.826 g, 2 mmol) in CH₂Cl₂ (10 mL) cooled to 0 °C. Stirring was continued until the condensing reagent disappeared (TLC analysis, staining with 0.5% solution of NBP) (1 h), after which HCl*Ala-OMe (0.278 g, 2 mmol) and NMM (0.220 mL, 2 mmol) were added. The mixture was stirred for an additional 2 h at 0 °C and left overnight at room temperature. The mixture was diluted with CH₂Cl₂ (10 mL), then the solution was washed successively with water, 0.5 M aqueous NaHSO₄, water, 0.5 M aqueous NaHCO₃ and water again. The organic layer was dried with Na₂SO₄, filtered and concentrated to dryness. The residue was dried under a vacuum with P₂O₅ and KOH to constant weight, to obtain the neutral peptide. Finally, 0.559 g was obtained, corresponding to a 97% yield.

IR (film): ν = 3678, 3608, 3430, 3017, 2965, 2398, 1734, 1672, 1512, 1451, 1420 [cm⁻¹].

¹H-NMR (250 MHz; CDCl₃) δ = 1.39 (s,9H, (CH₃)₃-C-); 1.41 (d, 3H, J = 7.2 Hz, CH₃ CH-); 1.57 (s, 6H, (CH₃)₂-C-); 3.72 (s, 3H, CH₃O-); 4.36 (q, 1H, J = 7.2 Hz, CH₃-CH-) [ppm].

Anal. RP-HPLC (10–97%B in 30 min): t_R = 9.55 min (purity 98.8%).

Starting materials: Boc-Ala-OH (0.378 g, 2 mmol), HCl*Ala-OMe (0.278 g, 2 mmol), DMT/NMM/TosO⁻ (0.826 g, 2 mmol), NMM (0.330 mL, 3 mmol). Product: Boc-Ala-Ala-OMe. Activation time: 1 h, yield 0.527 g (96%).

IR (film): ν = 3119, 3008, 1740, 1718, 1675, 1504, 1450, 1376, 1314 [cm⁻¹].

¹H-NMR (250 MHz; CDCl₃) δ = 1.36 (d, 3H, J = 6.8 Hz, CH₃-CH-); 1.41 (d, 3H, J = 6.8 Hz, CH₃ CH-); 1.45 (s, 9H, (CH₃)₃-C-); 3.75 (s, 3H, CH₃O-); 4.57 (q, 1H, J = 6.8 Hz, CH₃ CH-); 4.61 (q, 1H, J = 6.8 Hz, CH₃ CH-); 4.97 (1H, bs, -NH-), 6.49 (1H, bs, -NH-) [ppm].

Anal. RP-HPLC (10–97%B in 30 min): t_R = 9.03 min (purity 98.1%).

Starting materials: Boc-Ser-OH (0.410 g, 2 mmol), HCl*Ala-OMe (0.278 g, 2 mmol), DMT/NMM/TosO⁻ (0.826 g, 2 mmol), NMM (0.330 mL, 3 mmol). Product: Boc-Ser-Ala-OMe. Activation time: 1.5 h, yield 0.540 g (93%).

IR (film): ν = 3110, 3002, 1748, 1722, 1648, 1501, 1444, 1372, 1314 [cm⁻¹].

¹H-NMR (250 MHz; CDCl₃) δ =1.38 (d, 3H, J = 7.12 Hz, CH₃-CH-); 1.43 (s, 9H, (CH₃)₃-C-); 3.65 (dd, 2H, J₁ = 6.88 Hz, J₂ = 3.22 Hz, HO-CH₂-); 3.72 (s, 3H, CH₃O-); 4.35 (q, 1H, J = 7.1 Hz, CH₃-CH-); 4.37 (t, 1H, J = 7.2 Hz, -CH-CH2-); 4.55 (t, 1H, J = 6.88 Hz, -CH₂-CH-) [ppm].

Anal. RP-HPLC (10–97%B in 30 min): t_R = 7.25 min (purity 98.7%).

Starting materials: Fmoc-Lys(Boc)-OH (0.937 g, 2 mmol), HCl*Ala-OMe (0.278 g, 2 mmol), DMT/NMM/TosO⁻ (0.826 g, 2 mmol), NMM (0.330 mL, 3 mmol). Product: Fmoc-Lys(Boc)-Ala-OMe. Activation time: 1 h, yield 1.041 g (94%).

IR (film): ν = 3401, 3087, 2994, 1753, 1748, 1710, 1674, 1546, 1452, 1373 [cm⁻¹].

¹H-NMR (250 MHz; CDCl₃) δ = 1.41 (d, 3H, J = 7.2 Hz, CH₃-CH-); 1.43 (s, 9H, (CH₃)₃C-); 1.48–1.55 (m, 4H, -CH₂-CH₂-); 1.88 (qu, 2H, J = 7.4 Hz, -CH-CH₂-); 3.21 (t, 2H, J = 7.0 Hz, NH-CH₂-); 3.71 (s, 3H, CH₃O-); 4.36 (q, 1H, J = 7.2 Hz, CH₃-CH-); 4.46 (t, 1H, J = 3.1 Hz, -CH-CH₂-); 4.62 (d, 2H, J = 3.1 Hz, -CH₂-O-); 7.36–8.11 (m, 8H, arom.) [ppm].

Anal. RP-HPLC (10–97%B in 30 min): t_R = 10.25 min (purity 98.1%).

Starting materials: Fmoc-Asp(OtBu)-OH (0.823 g, 2 mmol), HCl*Ala-OMe (0.278 g, 2 mmol), DMT/NMM/TosO⁻ (0.826 g, 2 mmol), NMM (0.330 mL, 3 mmol). Product: Fmoc-Asp(OtBu)-Ala-OMe. Activation time: 1 h, yield 0.934 g (94%).

IR (film): ν = 3410, 3090, 2990, 1750, 1740, 1710, 1675, 1540, 1450, 1375 [cm⁻¹].

¹H-NMR (250 MHz; CDCl₃) δ = 1.40 (d, 3H, J = 7.1 Hz, CH₃- CH-); 1.46 (s, 9H, -OC(CH₃)₃); 2.57 (dd, 1H, J₁ = 17.2 Hz, J₂ = 7.3 Hz, -OCO-CH₂-CH-); 2.92 (dd, 1H, J₁ = 17.2 Hz, J₂ = 4.1 Hz, -OCO-CH₂-CH-); 3.73 (s, 3H, CH₃O-); 4.26 (t, 1H, J = 7.1 Hz, -CH-CH₂O-); 4.42 (d, 2H, J = 7.1 Hz, -CH-CH₂O-); 4.48–4.60 (m, 2H, CH₃CH- + OCO-CH₂-CH-); 5.97 (d, 1H, J = 7 Hz, NH); 7.07 (d, 1H, J = 5 Hz, NH); 7.31 (t, 2H, J = 7 Hz, arom.); 7.41 (t, 2H, J = 7 Hz, arom.); 7.58 (d, 2H, J = 7 Hz, arom.); 7.77 (d, 2H, J = 7 Hz, arom.) [ppm].

Anal. RP-HPLC (10–97%B in 30 min): t_R = 10.05 min (purity 97.9%).

Starting materials: Fmoc-Trp(Boc)-OH (1.053 g, 2 mmol), HCl*Ala-OMe (0.278 g, 2 mmol), DMT/NMM/TosO⁻ (0.826 g, 2 mmol), NMM (0.330 mL, 3 mmol). Product: Fmoc-Trp(Boc)-Ala-OMe. Activation time: 1 h, yield 1.187 g (97%).

IR (film): ν = 3412, 3088, 2987, 1754, 1743, 1711, 1679, 1538, 1452, 1375 [cm⁻¹].

¹HNMR (250 MHz; CDCl₃) δ = 1.41 (d, 3H, J = 7.2 Hz, CH₃-CH-); 1.43 (s, 9H, (CH₃)₃C-); 3.15 (d, 2H, J = 6.7 Hz, -CH-CH₂-); 3.71 (s, 3H, CH₃O-); 4.91 (t, 1H, J = 6.7 Hz, CH-CH₂-); 4.36 (q, 1H, J = 7.2 Hz, CH₃-CH-); 4.46 (t, 1H, J = 3.1 Hz, -CH-CH₂-); 4.62 (d, 2H, J = 3.1 Hz, -CH₂-O-); 7.03–8.11 (m, 13H, arom.) [ppm].

Anal. RP-HPLC (10–97%B in 30 min): t_R = 11.14 min (purity 98.1%).

A Boc-protected dipeptide solution (1 mmol) dissolved in 1,4-dioxane (5 mL) was cooled in a water-ice bath with vigorously stirring, and a 4 M solution of HCl in 1,4-dioxane (5 mL) was added. Stirring was continued for 4 h. The solution was then removed in a vacuum evaporator and 1,4-dioxane (10 mL) was added to the residue. The solvent was removed again using a vacuum evaporator. This operation was repeated four times. The residue was dried in a vacuum desiccator to constant weight. The procedure gave HCl*Aib-Ala-OMe, HCl*Ala-Ala-OMe and HCl*Ser-Ala-OMe in quantitative yield. The derivatives obtained were used in the subsequent reaction steps without additional purification procedures.

A solution of Fmoc-protected dipeptide derivatives (1 mmol) dissolved in DCM (5 mL) was cooled in a water-ice bath with intensive stirring and a 25% piperidine solution in DCM (10 mL) was added. Stirring was continued for 15 min, then MeOH (20 ml) was added to the solution. Stirring was continued for 5 min. The solvent was removed using a vacuum evaporator. To the residue was added 10 mL of DCM and the solvent was removed again in a vacuum evaporator. This operation was repeated four times. The solid residue was dried under a vacuum to constant weight. The procedure gave H-Lys(Boc)-Ala-OMe, H-Asp(OtBu)-Ala-OMe and H-Trp(Boc)-Ala-OMe in quantitative yield. The derivatives obtained were used immediately in subsequent reaction steps, without additional purification procedures.

To a vigorously stirred slurry of solid NaHCO₃ (0.42 g, 5 mmol) in dichloromethane (DCM) (3 mL) cooled to 0 °C was added 2,4-dichloro-6-methoxy-1,3,5-triazine (DCMT) (0.270 g, 1.5 mmol) in DCM (3 mL), followed by the slow addition of a solution of HCl*NH-Aib-Ala-OMe (0.337 g, 1.5 mmol). Stirring was continued until complete consumption of the DCMT, as shown by the disappearance of a DCMT spot stained with 0.5% solution of NBP in ethanol at room temperature, monitored by TLC analysis. After filtration of the solid deposit, the solvent was removed using vacuum evaporation and the residue dried under a vacuum with P₂O₅ and KOH to a constant weight, giving 0.491 g of 2-chloro-4-methoxy-6-(NH-Aib-Ala-OMe)-1,3,5-triazine (4a), yield = 98.7% as colorless oil.

Analysis: for C₁₂H₁₈ClN₅O₄ (331.76): calculated: C 43.4%, H 5.5%, Cl 10.7%, N 21.1%, found: C 43.2%, H 5.6%, Cl 10.7%, N 21.2%.

¹H-NMR (250 MHz; CDCl₃) δ = 1.36 (s, 6H, ((CH₃)₂-C-); 1.41 (s, 3H, d, J = 7.2 Hz, CH₃-CH-); 3.72 (s, 3H, CH₃O-); 4.02 (s, 3H, CH₃O-); 4.36 (q, 1H, J = 7.2 Hz, CH₃-CH-) [ppm].

¹³C-NMR (75 MHz, CDCl₃): δ = 17.7, 27.1, 48.1, 52.3, 54.6, 58.1, 159.7, 166.0, 172.9, 175.6 [ppm].

Starting material: solid NaHCO₃ (0.42 g, 5 mmol), DCMT (0.270 g, 1.5 mmol), HCl*H-Ala-Ala-OMe (0.316 g, 1.5 mmol). Product: 0.465 g 2-chloro-4-methoxy-6-(NH-Ala-Ala-OMe)-1,3,5-triazine (4b), yield = 97.6%, colorless oil.

Analysis: for C₁₁H₁₆ClN₅O₄ (317.73): calculated: C 41.6%, H 5.1%, Cl 11.2%, N 22%, found: C 41.5%, H 5.2%, Cl 11.2%, N 22.1%.

¹H-NMR (250 MHz; CDCl₃) δ = 1.39 (d, 3H, J = 7.0 Hz, CH₃-CH-); 1.41 (d, 3H, J = 7.2 Hz, CH₃-CH-); 3.71 (s, 3H, CH₃O-); 4.02 (s, 3H, CH₃O-); 4.35 (q, 1H, J = 7.2 Hz, CH₃-CH-); 4.38 (q, 1H, J = 7.0 Hz, CH₃-CH-) [ppm].

¹³C-NMR (75 MHz, CDCl₃): δ = 17.4, 17.6, 47.8, 49.2, 52.3, 54.6, 159.8, 166.0, 172.2, 172.8 [ppm].

Starting material: solid NaHCO₃ (0.42 g, 5 mmol), DCMT (0.270 g, 1.5 mmol), HCl*H-Ser-Ala-OMe (0.340 g, 1.5 mmol). Product: 0.480 g 2-chloro-4-methoxy-6-(NH-Ser-Ala-OMe)-1,3,5-triazine (4c), yield = 95.8%, colorless oil.

Analysis: for C₁₁H₁₆ClN₅O₅ (333.73): calculated: C 39.6%, H 4.8%, Cl 10.6%, N 21%, found: C 39.7%, H 4.7%, Cl 10.6%, N 21%.

¹H-NMR (250 MHz; CDCl₃) δ = 1.41 (d, 3H, J = 7.2 Hz, CH₃-CH-); 3.67 (dd, 2H, J₁ = 6.8 Hz, J₂ = 3.2 Hz, -CH-CH₂-OH); 3.78 (s, 3H, CH₃O-); 4.05 (s, 3H, CH₃O-); 4.35 (1, 1H, J = 7.2 Hz, CH₃-CH-); 4.48 (t, 1H, J = 6.8 Hz, -CH-CH₂-) [ppm].

¹³C-NMR (75 MHz, CDCl₃): δ = 17.6, 38.3, 47.8, 52.3, 54.6, 61.4, 159.5, 166.0171.3, 172.9 [ppm].

Starting material: solid NaHCO₃ (0.42 g, 5 mmol), DCMT (0.270 g, 1.5 mmol), H-Lys(Boc)-Ala-OMe (0.497 g, 1.5 mmol). Product: 0.685 g 2-chloro-4-methoxy-6-(NH-Lys(Boc)-Ala-OMe)-1,3,5-triazine (4d), yield = 96.2%, colorless oil.

Analysis: for C₁₉H₃₁ClN₆O₆ (474.94): calculated: C 48%, H 6.6%, Cl 7.5%, N 17.7%, found: C 48.1%, H 6.7%, Cl 7.4%, N 17.6%.

¹H-NMR (250 MHz; CDCl₃) δ = 1.26–1.57 (m, 4H, -CH₂-CH₂-); 1.41 (d, 3H, d, J = 7.2 Hz, CH₃-CH-); 1.43 (s, 9H, (CH₃)₃C-); 1.93 (dt, 2H, J₁ = 7.5 Hz, J₂ = 5.1 Hz, -CH-CH₂-); 3.22 (t, 2H, J = 7.1 Hz, -CH₂-NH-); 3.71 (s, 3H, CH₃O-); 4.05 (s, 3H, CH₃O-); 4.28 (t, 1H, J = 7.5 Hz, -CH-CH₂-); 4.36 (q, 1H, J = 7.2 Hz, CH₃-CH-) [ppm].

¹³C-NMR (75 MHz, CDCl₃): δ = 17.7, 21.4, 28.2, 29.3, 29.6, 40.5, 47.8, 52.3, 53.6, 54.6, 80.5, 156.3, 159.5, 166.0, 172.5, 172.9 [ppm].

Starting material: solid NaHCO₃ (0.42 g, 5 mmol), DCMT (0.270 g, 1.5 mmol), H-Asp(OtBu-Ala-OMe (0.411 g, 1.5 mmol). Product: 0.607 g 2-chloro-4-methoxy-6-(NH-Asp(OtBu)-Ala-OMe)-1,3,5-triazine (4e), yield = 96.9%, colorless oil.

Analysis: for C₁₆H₂₄ClN₅O₆ (417.84): calculated: C 46%, H 5.8%, Cl 8.5%, N 16.8%, found: C 46%, H 5.9%, Cl 8.5%, N 16.8%.

¹H-NMR (250 MHz; CDCl₃) δ = 1.38 (s, 9H, (CH₃)₃C-); 1.41 (d, 3H, J = 7.2 Hz, CH₃-CH-); 3.01 (d, 2H, J = 6.3 Hz, -CH-CH₂-); 3.72 (s, 3H, CH₃O-); 4.02 (s, 3H, CH₃O-); 4.36 (1, 1H, J = 7.2 Hz, CH₃-CH-); 5.02 (t, 1H, J = 6.3 Hz, -CH-CH₂-) [ppm].

¹³C-NMR (75 MHz, CDCl₃): δ = 17.7, 28.1, 37.9, 47.8, 52.3, 53.6, 54.6, 81.4, 159.6, 166.0, 172.3, 172.5, 172.9 [ppm].

Starting material: solid NaHCO₃ (0.42 g, 5 mmol), DCMT (0.270 g, 1.5 mmol), H-Trp(Boc)-Ala-OMe (0.584 g, 1.5 mmol). Product: 0.776 g 2-chloro-4-methoxy-6-(NH-Trp(Boc)-Ala-OMe)-1,3,5-triazine (4f), yield = 97.1%, colorless oil.

Analysis: for C₂₄H₂₉ClN₆O₆ (532.98): calculated: C 54.1%, H 5.5%, Cl 6.7%, N 15.8%, found: C 54%, H 5.4%, Cl 6.7%, N 15.7%.

¹H-NMR (250 MHz; CDCl₃) δ = 1.41 (d, 3H, J = 7.2 Hz, CH₃-CH); 1.44 (s, 9H, (CH₃)₃C-); 3.15 (d, 2H, J = 6.7 Hz, -CH-CH₂-); 3.71 (s, 3H, CH₃O-); 4.02 (s, 3H, CH₃O-); 4.37 (q, 1H, J = 7.2 Hz, CH₃-CH-); 4.91 (t, 1H, J = 6.7 Hz, CH-CH₂-); 7.03–7.98 (m, 5H, arom) [ppm].

¹³C-NMR (75 MHz, CDCl₃): δ = 17.7, 27.7, 28.2, 47.9, 52.3, 53.6, 54.6, 80.6, 115.4, 117.1, 118.8, 120.1, 122.8, 123.7, 129.6, 135.4, 149.2, 166.0, 162.1, 172.3, 172.9 [ppm].

To a vigorously stirred solution of 2-chloro-4-methoxy-6-(NH-Aib-Ala-OMe)-1,3,5-triazine (4a) (0.332 g, 1 mmol) in DCM (10 mL) cooled to 0 °C in an ice-water bath was added 1,4-diazabicyclo[2.2.2]octane (DABCO, 5) (0.112 g, 1 mmol). Stirring at 0 °C was continued until complete consumption of 4a, as shown by TLC analysis (Rf = 0.45, DCM; staining with 0.5% solution of NBP in ethanol). The cooling bath was removed and stirring was continued at room temperature until all 6a salt was consumed, as shown by TLC analysis (Rf = 0.00, pure DCM, colored with 0.5% solution of NBP in ethanol). The solvent was removed by evaporation and the residue was dried under a vacuum with P₂O₅ and KOH to constant weight, giving 0.442 g 2-[4-(2-chloroethyl) piperazin-1-yl]-4-methoxy-6-(NH-Aib-Ala-OMe)-1,3,5-triazine (7a), yield = 99.1%, colorless oil.

Analysis: for C₁₈H₃₀ClN₇O₄ (443.93): calculated: C 48.7%, H 6.8%, Cl 8%, N 22.1%, found: C 48.6%, H 6.7%, Cl 8%, N 22%.

¹H-NMR (250 MHz; CDCl₃) δ = 1.36 (s, 6H, (CH₃)₂-C-); 1.41 (d, 3H, J = 7.2 Hz, CH₃-CH-); 2.66 (t, 2x2H, J = 7.8 Hz, -N-CH₂-); 2.85 (t, 2H, J = 6.2 Hz, -N-CH₂-); 3.60 (t, 2x2H, J = 7.8 Hz, -N-CH₂-); 3.86 (2, 2H, J = 6.2 Hz, Cl-CH₂-); 3.71 (s, 3H, CH₃O-); 4.02 (s, 3H, CH₃O-); 4.36 (q, 1H, J = 7.2 Hz, CH₃-CH-) [ppm].

¹³C-NMR (75 MHz, CDCl₃): δ = 17.7, 27.1, 41.2, 44.1, 48.1, 51.8, 52.3, 53.1, 54.6, 58.1, 167.8, 171.7, 172.9, 175.6 [ppm].

LC/MS: 444.9373 ([M + H]⁺, C₁₈H₃₁ClN₇O₄⁺; calc. 443.93).

Starting material: 2-chloro-4-methoxy-6-(NH-Ala-Ala-OMe)-1,3,5-triazine (4b) (0.318 g, 1 mmol), DABCO (5) (0.112 g, 1 mmol). Product: 0.419 g 2-[4-(2-chloroethyl)piperazin-1-yl]-4-methoxy-6-(NH-Ala-Ala-OMe)-1,3,5-triazine (7b), yield = 97.4%, colorless oil.

Analysis: for C₁₇H₂₈ClN₇O₄ (429.91): calculated: C 47.5%, H 6.6%,Cl 8.2%, N 22.8%, found: C 47.6%, H 6.7%,Cl 8.2%, N 22.7%.

¹H-NMR (250 MHz; CDCl₃) δ = 1.37 (d, 3H, J = 7.0 Hz, CH₃-CH-); 1.41 (d, 3H, J = 7.2 Hz, CH₃-CH-); 2.66 (t, 2x2H, J = 7.8 Hz, -N-CH₂-); 2.85 (t, 2H, J = 6.2 Hz, -N-CH₂-); 3.60 (t, 2x2H, J = 7.8 Hz, -N-CH₂-); 3.86 (2, 2H, J = 6.2 Hz, Cl-CH₂-); 3.71 (s, 3H, CH₃O-); 4.02 (s, 3H, CH₃O-); 4.35 (q, 1H, J = 7.2 Hz, CH₃-CH-); 4.38 (q, 1H, J = 7.0 Hz, CH₃-CH-) [ppm].

¹³C-NMR (75 MHz, CDCl₃): δ = 17.4, 17.7, 41.2, 44.0, 47.8, 49.2, 51.8, 52.3, 53.0, 163.4, 171.1, 172.1, 172.9 [ppm].

MS: 430.9118 ([M + H]⁺, C₁₇H₂₉ClN₇O₄+; calc. 429.91).

Starting material: 2-chloro-4-methoxy-6-(NH-Ser-Ala-OMe)-1,3,5-triazine (4c) (0.334 g, 1 mmol), DABCO (5) (0.112 g, 1 mmol). Product: 0.438 g 2-[4-(2-chloroethyl)piperazin-1-yl]-4-methoxy-6-(NH-Ser-Ala-OMe)-1,3,5-triazine (7c), yield = 98.2%, colorless oil.

Analysis: for C₁₇H₂₈ClN₇O₅ (445.90): calculated: C 45.8%, H 6.3%, Cl 8%, N 22%, found: C 45.7%, H 6.4%, Cl 8%, N 22%.

¹H-NMR (250 MHz; CDCl₃) δ = 1.40 (d, 3H, J = 7.2 Hz, CH₃-CH); 2.66 (t, 2x2H, J = 7.8 Hz, -N-CH₂-); 2.85 (t, 2H, J = 6.2 Hz, -N-CH₂-); 3.60 (t, 2x2H, J = 7.8 Hz, -N-CH₂-); 3.68 (d, 2H, J = 6.8 Hz, -CH₂-OH); 3.86 (2, 2H, J = 6.2 Hz, Cl-CH₂-); 3.71 (s, 3H, CH₃O-); 4.02 (s, 3H, CH₃O-); 4.35 (q, 1H, J = 7.2 Hz, CH₃-CH-); 4.47 (t, 1H, J = 6.8 Hz, -CH-CH₂-OH) [ppm].

¹³C-NMR (75 MHz, CDCl₃): δ = 17.6, 38.3, 41.2, 44.0, 47.8, 51.8, 52.3, 53.0, 54.6, 61.4, 163.5, 171.3, 171.7, 172.9 [ppm].

MS: 446.9342 ([M + H]⁺, C₁₇H₂₉ClN₇O₅⁺; calc. 445.90).

Starting material: 2-chloro-4-methoxy-6-(NH-Lys(Boc)-Ala-OMe)-1,3,5-triazine (4d) (0.475 g, 1 mmol), DABCO (5) (0.112 g, 1 mmol). Product: 0.580 g 2-[4-(2-chloroethyl)piperazin-1-yl]-4-methoxy-6-(NH-Lys(Boc)-Ala-OMe)-1,3,5-triazine (7d), yield = 98.8%, colorless oil.

Analysis: for C₂₅H₄₃ClN₈O₆ (587.11): calculated: C 51.1%, H 7.4%, Cl 6%, N 19.1%, found C 51.1%, H 7.4%, Cl 6%, N 19.1%.

¹H-NMR (250 MHz; CDCl₃) δ = 1.26–1.57 (m, 4H, -CH₂-CH₂-); 1.41 (d, 3H, d, J = 7.2 Hz, CH₃-CH-); 1.43 (s, 9H, (CH₃)₃C-); 1.93 (dt, 2H, J₁ = 7.5 Hz, J₂ = 5.1 Hz, -CH-CH₂-); 2.66 (t, 2x2H, J = 7.8 Hz, -N-CH₂-); 2.85 (t, 2H, J = 6.2 Hz, -N-CH₂-); 3.22 (t, 2H, J = 7.1 Hz, -CH₂-NH-); 3.60 (t, 2x2H, J = 7.8 Hz, -N-CH₂-); 3.86 (2, 2H, J = 6.2 Hz, Cl-CH₂-); 3.71 (s, 3H, CH₃O-); 4.02 (s, 3H, CH₃O-); 4.28 (t, 1H, J = 7.5 Hz, -CH-CH²-); 4.36 (q, 1H, J = 7.2 Hz, CH₃-CH-) [ppm].

¹³C-NMR (75 MHz, CDCl₃): δ = 17.7, 21.4, 28.2, 29.3, 29.9, 40.5, 41.2, 44.0, 47.8, 51.8, 52.3, 53.1, 53.6, 54.6, 80.1, 156.2, 162.2, 171.7, 172.5, 172.9 [ppm].

MS: 588.2016 ([M + H]⁺, C₂₅H₄₄ClN₈O₆⁺; calc. 587.11).

Starting material: 2-chloro-4-methoxy-6-(NH-Asp(OtBu)-Ala-OMe)-1,3,5-triazine (4e) (0.418 g, 1 mmol), DABCO (5) (0.112 g, 1 mmol). Product: 0.520 g 2-[4-(2-chloroethyl)piperazin-1-yl]-4-methoxy-6-(NH-Asp(OtBu)-Ala-OMe)-1,3,5-triazine (7e), yield = 98.2%, colorless oil.

Analysis: for C₂₂H₃₆ClN₇O₆ (530.02): calculated: C 49.9%, H 6.8%, Cl 6.7%, N 18.5%, found: C 49.8%, H 6.7%, Cl 6.7%, N 18.5%.

¹H-NMR (250 MHz; CDCl₃) δ = 1.38 (s, 9H, (CH₃)₃C-); 1.41 (d, 3H, J = 7.2 Hz, CH₃-CH-); 3.01 (d, 2H, J = 6.3 Hz, -CH-CH₂-); 2.66 (t, 2x2H, J = 7.8 Hz, -N-CH₂-); 2.85 (t, 2H, J = 6.2 Hz, -N-CH₂-); 3.60 (t, 2x2H, J = 7.8 Hz, -N-CH₂-); 3.86 (2, 2H, J = 6.2 Hz, Cl-CH₂-); 3.71 (s, 3H, CH₃O-); 4.02 (s, 3H, CH₃O-); 4.36 (1, 1H, J = 7.2 Hz, CH₃-CH-); 5.02 (t, 1H, J = 6.3 Hz, -CH-CH₂-) [ppm].

¹³C-NMR (75 MHz, CDCl₃): δ = 17.7, 37.9, 41.1, 44.0, 47.8, 51.8, 52.3, 53.1, 53.5, 54.6, 81.1, 162.2, 171.7, 172.1, 172.5, 172.9 [ppm].

MS: 531.1256 ([M + H]⁺, C₂₂H₃₇ClN₇O₆⁺; calc. 530.02).

Starting material: 2-chloro-4-methoxy-6-(NH-Trp(Boc)-Ala-OMe)-1,3,5-triazine (4f) (0.533 g, 1 mmol), DABCO (5) (0.112 g, 1 mmol). Product: 0.638 g 2-[4-(2-chloroethyl)piperazin-1-yl]-4-methoxy-6-(NH-Trp(Boc)-Ala-OMe)-1,3,5-triazine (7f), yield = 98.9%, colorless oil.

Analysis: for C₃₀H₄₁ClN₈O₆ (645.15): calculated: C 55.9%, H 6.4%, Cl 5.5%, N 17.4%, found: C 55.9%, H 6.4%, Cl 5.5%, N 17.4%.

¹H-NMR (250 MHz; CDCl₃) δ = 1.41 (d, 3H, J = 7.2 Hz, CH₃-CH-), 1.44 (9H, s, (CH₃)₃-C-), 2.66 (t, 2x2H, J = 7.8 Hz, -N-CH₂-); 2.85 (t, 2H, J = 6.2 Hz, -N-CH₂-); 3.15 (d, 2H, J = 6.7 Hz, -CH₂-CH-); 3.60 (t, 2x2H, J = 7.8 Hz, -N-CH₂-); 3.86 (2, 2H, J = 6.2 Hz, Cl-CH₂-); 3.72 (s, 3H, CH₃O-); 4.02 (s, 3H, CH₃O-); 4.36 (q, 1H, J = 7.2 Hz, CH₃-CH-); 4.91 (t, 1H, J = 6.7 Hz, -CH₂-CH-); 7.03–7.98 (m, 5H, arom) [ppm].

¹³C-NMR (75 MHz, CDCl₃): δ = 17.7, 27.7, 28.1, 41.2, 44.1, 47.9, 51.8, 52.3, 53.0, 53.6, 54.5, 81.1, 115,1, 117.1, 118.7, 120.6, 122.7, 123.7, 129.6, 135.4, 149.5, 163.3, 172.4, 172.9 [ppm].

MS: 646.15864 ([M + H]⁺, C₃₀H₄₂ClN₈O₆⁺; calc. 645.15).