SCHEME 2. Synthesis of the Phosphinic TRH Analogue
1.75-2.17 (m, 2H), 2.99-3.38 (m, 3H), 3.93 (t, J ) 7.5 Hz, 1H),
6.70 (s, 1H), 7.13 (d, J ) 538.3 Hz, 1H), 6.98-7.35 (m, 15H),
8.23 (s, 1H); 13C NMR (50 MHz, CDCl3) δ 14.0, 23.8, 31.1, 34.0,
34.8, 40.8, 60.1, 118.8, 127.8, 129.6, 138.3, 138.7, 142.3, 175.7;
31P NMR (81 MHz, CDCl3) δ 23.8; ESMS m/z calcd for
C28H30N2O4P (M + 1)+ 489.2, found 489.4.
4-{[2-Carboxy-3-(1-trityl-1H-imidazol-4-yl-propyl]-hydroxy-
phosphinoyl-4-(9H-fluoren-9-ylmethoxycarbonylamino)-butyr-
ic Acid Methyl Ester (7). In a solution of 6 (200 mg, 0.434 mmol)
in AcCl (7 mL) and AcOH (0.7 mL) were added FmocNH2 (104
mg, 0.434 mmol) and 4-oxo-butyric acid methyl ester (55 mg, 0.477
mmol) at 0 °C. The reaction mixture was stirred at room temperature
for 16 h. Evaporation of solvents and column purification with
CHCl3/MeOH/AcOH ) 90:3:3 afforded 7 (166 mg, 48%) as a white
solid. TLC Rf 0.52 (CHCl3/MeOH/AcOH ) 70:5:5); HPLC tR 41.9/
42.4; 1H NMR (600 MHz, CDCl3) δ 1.82-2.13 (m, 4H), 2.28-2.71
(m, 4H), 3.21-3.37 (m, 1H), 3.48-3.75 (m, 4H), 4.15-4.36 (m,
4H) 5.67-5.82 (m, 1H), 6.71-6.82 (m, 1H), 7.12-7.18 (m, 6H),
7.32-7.45 (m, 14H), 7.55-7.65 (m, 2H), 7.74-7.82 (m, 2H),
8.02-8.19 (m, 1H); 13C NMR (50 MHz, CDCl3) δ 24.0, 27.1, 28.9,
30.7, 30.9, 38.8, 39.3, 47.0, 49.5, 49.0, 51.2, 51.4, 66.9, 78.0, 119.8,
121.2, 127.0, 127.5, 127.5, 128.6, 128.9, 129.5, 132.6, 133.2, 135.7,
136.1, 140.0, 141.1, 143.8, 156.8, 173.7, 175.8; 31P NMR (81 MHz,
CDCl3) δ 37.9; ESMS m/z calcd for C46H45N3O8P (M + 1)+ 798.3,
found 798.3. HRMS (ES) m/z calcd for C46H45N3O8P (M + 1)+
798.2866, found 798.2662.
[3-(2-Carbamoyl-pyrrolidin-1-yl)-2-(1H-imidazol-4-ylmethyl)-
3-oxo-propyl]-(5-oxo-pyrrolidin-2-yl)-phosphinic Acid (8). Syn-
thesis was carried out on pins with a Rink-type linker (SynPhase
Lanterns, L-series pins, loading 15 µmol per pin, Mimotopes,
France). Proline coupling was performed as previously described
in detail.17 For phosphinic block coupling, a solution of 7 (1.1
equiv) is added to pins in dry CH2Cl2 (0.3 mL/pin) containing
DIPEA (2.1 equiv), HOBt (1.1 equiv), and EDC ·HCl17 (5 equiv).
After 16 h at room temperature, the reaction mixture was decanted,
and the pins were washed with CH2Cl2 (2 × 5 min, 1 mL/pin),
DMF (2 × 5 min, 1 mL/pin), and CH2Cl2 (2 × 5 min, 1 mL/pin).
The coupling step was repeated if necessary, until negative chloranil
test. Removal of the Fmoc group was accomplished by 20%
piperidine/DMF (1 mL/pin, 2 × 30 min, rt) and washings. Lactam
formation was by addition of pip/DMF 2:1 (1 mL/pin), until
negative Kaiser test (ca. 48 h) at room temperature. Washings were
as generally. Cleavage from pins was by TFA/TIS/H2O 95:2.5:2.5
(1 mL/pin). Concentration in vacuo, precipitation with dry Et2O,
and lyophilization afforded 8 (50 mg for 10 pins, 84%) as a white
solid. TLC Rf 0.21 (CH3CN/H2O ) 1:1); HPLC tR 4.2/5.0/6.4; 1H
NMR (200 MHz, CD3OD) δ 1.61-2.45 (m, 8H), 2.82-4.05 (m,
8H), 4.22-4.49 (m, 1H) 7.22-7.41 (m, 1H), 8.72-8.81 (m, 1H);
13C NMR (50 MHz, CD3OD) δ 21.9, 22.1, 22.3, 23.1, 23.7, 23.9,
25.3, 25.6, 26.0, 29.3, 31.0, 31.3, 33.3, 38.7, 45.7, 61.2, 61.4, 62.5,
118.3, 118.9, 127.3, 129.3, 130.5, 135.0, 172.5, 177.1, 177.6, 177.8,
181.4; 31P NMR (81 MHz, CD3OD) δ 38.4/40.0/40.5; ESMS m/z
calcd for C16H25N5O5P (M + 1)+ 398.2, found 398.1. HRMS
(ES) m/z calcd for C16H25N5O5P (M + 1)+ 398.1515, found
398.1261.
added HCHO (2.145 g, 71.5 mmol) and piperidine (9.37 g,
110mmol), and the reaction mixture was refluxed until evolution
of CO2 stops (∼2 h). Removal of solvent, dissolution in AcOEt,
washing with 0.5 M HCl to pH 1 and brine, drying over Na2SO4,
solvent removal, and column purification with CH2Cl2/MeOH )
97:3 afforded 5 (18.4 g, 79%) as a white solid. TLC Rf 0.61 (CHCl3/
1
MeOH ) 95:5); H NMR (200 MHz, CDCl3) δ 1.14 (t, J ) 7.6
Hz, 3H), 2.75 (t, J ) 7.4 Hz, 1H), 3.00 (t, J ) 7.4 Hz, 1H), 4.04
(q, J ) 7.6 Hz, 2H), 6.03 (s, 1H), 6.27 (s, 1H), 6.71 (s, 1H),
6.98-7.35 (m, 15H), 7.97 (d, J ) 5.8 Hz, 1H); 13C NMR (50 MHz,
CDCl3) δ 14.0, 28.6, 60.7, 77.4, 120.4, 128.4, 128.7, 129.4, 134.1,
135.4, 135.8, 140.2, 165.9; ESMS m/z calcd for C28H27N2O2 (M +
1)+ 423.2, found 423.3. HRMS (ES) m/z calcd for C28H27N2O2 (M
+ 1)+ 423.1994, found 423.1831.
Acknowledgment. This work was supported by funds from
the Max-Planck-Institut fu¨r Experimentelle Endokrinologie, Han-
nover, Germany and the Special Account for Research Grants of
National and Kapodistrian University of Athens, Greece.
3-Hydroxyphosphinoyl-2-(1-trityl-1H-imidazol-4-ylmethyl)-
+
propionic Acid Ethyl Ester (6). A suspension of H2P(O)(O-NH4
)
(1.18 g, 14.2 mmol) and HMDS (2.29 g, 14.2 mmol) was warmed
to 110 °C under Ar for 3 h. After cooling to room temperature,
anhydrous CH2Cl2 (20 mL) was added, and a solution of 5 (1.2 gr,
2.84 mmol) in CH2Cl2 (7 mL) was added dropwise at 0 °C. Stirring
was continued at room temperature for 22 h, and then the reaction
was quenched with EtOH (5 mL). Evaporation of solvent, dissolu-
tion in AcOEt (100 mL), washing with H2O (1 × 10 mL), drying
over Na2SO4, and solvent removal afforded 6 (1.22 g, 88%) as a
white solid. TLC Rf 0.58 (CHCl3/MeOH/AcOH ) 70:5:5); HPLC
Supporting Information Available: General considerations;
full experimental procedures for compounds 1-4; 1H, 13C, and
31P NMR and HPLC chromatograms for compounds 6-8; and
HRMS copies for compounds 5, 7, and 8. This material is
1
tR 29.2; H NMR (200 MHz, CDCl3) δ 1.07 (t, J ) 7.5 Hz, 3H),
JO8014215
J. Org. Chem. Vol. 73, No. 21, 2008 8593