(S)-Di-tert.-butyl-(((2,5-dioxopyrrolidin-1-yl)oxy)carbonyl)-l-glutamate 2
One hundred fifty milligrams (0.507 μmol) of Glu(O
t
Bu)-O
t
Bu·HCl 1 was dissolved in 2 mL of freshly distilled CH3CN. Seventy microliters of Et3N (0.507 μmol) and 142 mg (0.554 μmol) of N,N′-disuccinimidyl carbonate was added to the solution. The reaction was stirred at 25 °C for 12 h and concentrated under reduced pressure. After re-solubilization in 5 mL of EtOAc and successive washing with 10 mL of 10 % citric acid and 10 mL of brine, the organic layer was dried over Na2SO4 and concentrated under reduced pressure to afford 197 mg of a pale yellow powder. This unpurified powder contained 90 % of desired compound 2 (yield 78 %). TLC: (EtOAc/hexane, 3/1): R
f
= 0.7. 1H-NMR (400 MHz, CDCl3) δ: 1.46 (s, 9H), 1.50 (s, 9H), 1.94–2.14 (m, 1H), 2.10–2.21 (m, 1H), 2.24–2.44 (m, 2H), 2.83 (s, 4H), 4.23 (dt, 1H, J = 5.1 Hz, J = 7.7 Hz), 6.19 (d, 1H, J = 7.7 Hz). 13C-NMR (100.5 MHz, CDCl3) δ: 25.47, 27.59, 27.97, 28.00, 31.15, 54.78, 80.95, 83.09, 151.06, 169.60, 169.76, 171.95.
(S)-2-[3-((S)-1-tert.-Butylcarboxylate-(5-benzyloxycarbonylpentyl))ureido]-di-tert.-butyl pentanedioate 4
Compounds
4 and
5 and DCFPyL were previously synthesized [
15,
16]. Herein, we describe an improved methodology. The yellow powder containing 90 % of compound
2 (197 mg, 90 % purity) was dissolved without further purification in 3 mL of CH
2Cl
2 containing 100 μL of Et
3N and 187 mg (501 μmol) of μs-benzyloxycarbonyl-
l-lysine
tert.-butyl ester hydrochloride (H-Lys(Z)-O
t
Bu.HCl)
3. The reaction mixture was stirred for 12 h at 25 °C, and progress of the reaction was monitored by TLC (CH
2Cl
2/MeOH 95/5). Upon completion, the reaction mixture was concentrated under reduced pressure and the residue was purified by column chromatography (CH
2Cl
2/MeOH 95/5) to afford 252 mg (92 %) of desired compound
4 as a clear gel. TLC: (CH
2Cl
2/MeOH 95/5):
R
f
= 0.2.
1H-NMR (600 MHz, CDCl
3) δ: 1.16–1.24 (m, 1H), 1.24–1.30 (m, 1H), 1.35 (s, 9H), 1.37 (s, 9H), 1.38 (s, 9H), 1.39–1.47 (m, 2H), 1.48–1.56 (m, 1H), 1.61–1.69 (m, 1H), 1.69–1.77 (m, 1H), 1.93–2.01 (m, 1H), 2.13–2.25 (m, 2H), 3.02–3.15 (m, 2H), 4.22–4.28 (m, 1H), 4.28–4.33 (m, 1H), 4.99 (d, 1H, J = 12.5 Hz), 5.05 (d, 1H, J = 12.5 Hz), 5.37 (d, 1H, J = 8 Hz), 5.44 (s, 1H), 5.45 (d, 1H, J = 8.1 Hz), 7.20–7.24 (m, 1H), 7.24–7.30 (m, 4H).
13C-NMR (150.9 MHz, CDCl
3) δ: 21.36, 26.98, 27.00, 27.05, 27.32, 28.33, 30.56, 31.61, 39.69, 51.84, 52.25, 65.46, 79.44, 80.58, 81.14, 126.93, 127.03, 127.43, 135.76, 155.66, 156.10, 171.33, 171.57, 171.89.
m/z (ESI) C
32H
51N
3O
9 ([M + H
+]) calcd. 622.4, found 622.4.
(S)-2-[3-((S)-5-Amino-1-tert.-butoxycarbonylpentyl)ureido]pentanedioic acid di-tert.-butyl ester 5 [15, 16]
A solution of compound 4 (220 mg, 0.354 μmol) in 3 mL of MeOH was stirred with 10 mg of Pd/C under H2 atmosphere for 12 h. The reaction was filtered through celite and concentration under reduced pressure afforded 164 mg (95 %) of compound 5 as a clear gel. 1H-NMR (600 MHz, CD3OD) δ: 1.45–1.47 (m,1H), 1.47 (s, 9H), 1.47–1.49 (m, 1H) 1.49 (s, 9H) 1.50 (s, 9H), 1.57–1.70 (m, 3H), 1.78–1.86 (m, 2H), 2.04–2.10 (m, 1H), 2.28–2.40 (m, 2H), 2.79–2.88 (m, 2H), 4.18 (dd, 1H, J = 5.1 Hz, J = 9.0. Hz), 4.22 (dd, 1H, J = 5.0 Hz, J = 8.9 Hz). m/z (ESI) C24H45N7O7 ([M + H+]) calcd. 488.3, found 488.3.
(S)-2-[3-((S)-1-Carboxy-5-[3-(6-fluoropyridine)carbonyl)amino)pentyl)ureido]-pentanedioic acid (DCFPyL) [15]
In a flame-dried flask, HBTU (156 mg, 410 μmol), DIPEA (72 μL, 410 μmol), and 6-fluoronicotinic acid 6 (58 mg, 410 μmol) were added to a solution of 100 mg (0.205 μmol) of compound 5 in 4 mL of distilled CH2Cl2. The reaction was stirred at 25 °C for 3 h under nitrogen, and progress of the reaction was monitored by TLC (hexane/ethyl acetate 1/1 v/v). Upon completion, the reaction mixture was concentrated under reduced pressure and the residue was purified by column chromatography (CH2Cl2/MeOH 97/3) to afford 97 mg (78 %) of tert.-butyl ester intermediate as a white powder. TLC: (EtOAc/hexane 1/1): R
f
= 0.45. 1H-NMR (600 MHz, CDCl3) δ: 1.39 (s, 9H), 1.41–1.46 (m, 19H), 1.51–1.63 (m, 2H), 1.64–1.71 (m, 1H), 1.73–1.80 (m, 1H), 1.80–1.86 (m, 1H), 1.98–2.08 (m, 1H), 2.25–2.39 (m, 2H), 3.33–3.42 (m, 1H), 3.50–3.59 (m, 1H), 3.69–3.77 (m, 1H), 4.45–4.24 (m, 2H), 5.59 (t, 1H, J = 9.5 Hz), 5.88 (t, 1H, J = 9.8 Hz), 7.00 (d, 1H, J = 8.1Hz), 7.81–7.90 (m, 1H), 8.41 ( t, 1H, J = 7.9 Hz), 8.81 (s, 1H). 13C-NMR (150.9 MHz, CDCl3) δ: 23.36, 27.81, 27.88, 27.95, 28.04, 28.66, 31.51, 32.43, 39.93, 53.12, 53.69, 80.72, 81.64, 82.54, 109.09 (d, J = 37.6 Hz), 128.56 (d, J = 4.3 Hz) 140.96 (d, J = 8.7 Hz), 147.95 (d, J = 13.1 Hz), 157.60, 164.86 164.87 (d, J = 244 Hz), 172.16, 172.31, 173.49. m/z (ESI) C30H47FN4O8 ([M + H+]) calcd. 611.3, found 611.3.
Tert.-butyl ester intermediate was treated with 6 mL of CH2Cl2/TFA (1/1 v/v) for 8 h at 25 °C, concentrated under reduced pressure, and purified by HPLC. HPLC purification was performed on a semi-preparative Jupiter C12 column (100 Å, 10 μm, 250 × 10 mm). The eluting solvent started with a 10/90 CH3CN/(water 0.5 % TFA) gradient for 5 min at a flow rate of 2 mL min−1 followed by a gradient from 10/90 to 70/30, v/v, for 25 min. Compound DCFPyL eluted at 14.5 min, and after solvent removal, 54 mg (67 %) of DCFPyL was isolated as a white powder. 1H-NMR (400 MHz, D2O) δ: 1.42–1.56 (m, 2H), 1.60–1.73 (m, 2H), 1.73–1.83 (m, 1H), 1.85–1.95 (m, 1H), 1.95–2.05 (m, 1H), 2.13–2.24 (dt, 1H, Jd = 20.6 Hz, Jt = 7.5 Hz), 2.51 (t, 2H, J = 7.2 Hz), 3.43 (t, 2H, 6.5 Hz), 4.25 (ddd, 2H, J = 9.4 Hz, J = 5.1 Hz, J = 9.2 Hz), 7.23 (d, 1H, J = 8.2 Hz), 8.31 (dt, 1H Jt = 8.2 Hz, Jd = 2.2 Hz), 8.57 (d, 1H, J = 2.2 Hz).13C-NMR δ (125.7 MHz, D2O): 23.26, 27.11, 28.56, 31.00, 31.55, 40.68, 53.51, 54.07, 109.05 (d, J = 37.8 Hz), 128.47 (d, J = 4.4 Hz) 140.87 (d, J = 8.1 Hz), 147.98 (d, J = 12.9 Hz), 160.26, 164.62 (d, J = 240.1 Hz), 168.16, 177.12, 178.05, 178.19. m/z (HRMS) C18H22FN4O8 ([M − H+]) calcd. 441.1427, found 441.1430.
6-Trimethylammonium-nicotinic acid 2,3,5,6-tetrafluorophenyl ester triflate salt 8 (adapted from reference [21])
One gram (6.34 mmol) of 6-chloronicotinic acid 7; 1.1 g (6.5 mmol) of 2,3,5,6 tetrafluorophenol; and 1.31 g (6.34 mmol) of
N,
N′-dicyclohexylcarbodiimide (DCC) were stirred in dioxane (40 mL) for 5 h at 25 °C. Progress of the reaction was monitored by TLC (EtOAc/hexane 4/1). Upon completion, the reaction mixture was filtered and concentrated under reduced pressure and the residue was purified by recrystallization in hot hexane to afford 1.35 g (70 %) of the 6-chloronicotinic acid active ester intermediate as a white powder [
18]. TLC: (EtOAc/hexane 4/1):
R
f
=
0.28.
1H-NMR (600 MHz, CDCl
3) δ 7.11 (tt, 1H, J = 7.1 Hz, J = 9.9 Hz), 7.57 (d, 1H, J = 8.3 Hz), 8.43 (dd, 1H, J = 8.3 Hz, J = 2.7 Hz), 9.21 (d, 1H, J = 2.7Hz).
m/z (ESI) C
12H
4ClF
4NO
2 ([M + H
+]) calcd. 305.0, found 304.9.
6-Chloronicotinic acid active ester intermediate (130 mg) [
18] was dissolved in 3 mL of a 1 M Me
3N solution in THF and stirred 2 h at 25 °C. After 5 min, a white precipitate was formed. After completion of the reaction, the precipitate was collected by filtration and washed with diethyl ether and cold CH
2Cl
2. The obtained white powder was suspended in 5 mL of CH
2Cl
2 containing 2 % TMSOTf and sonicated for 10 min. The reaction mixture was concentrated under reduced pressure and washed with diethyl ether to afford 140 mg (68 % over two steps) of a gray powder after drying.
1H-NMR (600 MHz, CD
3CN) δ 7.43 (tt, 1H, J = 7.4 Hz, J = 10.5 Hz), 8.07 (dd, 1H, J = 8.6 Hz, J = 0.8 Hz), 8.85 (dd, 1H, J = 8.6 Hz, J = 2.3 Hz), 9.34 (dd, 1H, J = 2.3 Hz, J = 0.8 Hz).
m/z (ESI) C
15H
13F
4N
2O
2 ([M
+]) calcd. 330.1, found 330.0.
(S)-2-[3-((S)1-Carboxy-5-((6-trimethylammonium-pyridine-3-carbonyl)-amino)-pentyl)-ureido]-pentanedioic acid trifluoroacetate salt 9
To a solution of 80 mg (164 μmol) of compound 5 in CH2Cl2 (4 mL) was added compound 8 (100 mg, 209 μmol) and 100 μL of DIPEA (572 μmol). The reaction was stirred for 2 h at 25 °C and then concentrated under reduced pressure.
Progress of the reaction was monitored by TLC: (CH2Cl2/MeOH 4/1): R
f
= 0.26. HPLC purification was performed on a semi-preparative Jupiter C12 column (100 Å, 10 μm, 250 × 10 mm). The eluting solvent started with a gradient from 5/95 to 70/30 acetonitrile/(water 0.5 % TFA) for 20 min at a flow rate of 2 mL min−1. Then the eluent was kept at 70/30 acetonitrile/(water 0.5 % TFA) for 10 min to elute the desired compound at 25.8 min. After removal of the solvent under reduced pressure gave 96 mg (77 %) of desired compound 9 as a white powder. 1H-NMR (600 MHz, D2O) δ: 1.32 (s, 9H), 1.34 (s, 9H), 1.35(s, 9H), 1.35–1.39 (m, 2H) 1.55–1.66 (m, 3H), 1.70–1.82 (m, 2H), 1.95–2.03 (m, 1H), 2.30 (M, 2H), 3.36 (t, 2H, J = 6.8 Hz), 3.57 (s, 9H), 4.02 (ddd, 2H, J = 9.5 Hz, J = 8.7 Hz, J = 5.1 Hz), 7.94 (d, 1H, J = 8.8 Hz), 8.35 (dd, 1H Jt = 8.8 Hz, Jd = 2.3 Hz), 8.57 (d, 1H, J = 2.3 Hz). 13C-NMR (125.7 MHz, D2O) δ: 23.35, 27.63, 28.19, 28.20, 28.31, 28.78, 31.92, 32.58, 40.80, 54.34, 54.99, 56.06, 83.47, 84.18, 84.36, 115.48, 118.47, 133.37, 141.14, 148.90, 160.16, 167.46, 174.55, 175.14, 175.33. m/z (HRMS) C33H56NO8 ([M+]) calcd. 650.4123, found 650.4116. Mp = 56 °C.