N. Kaur et al. / Bioorg. Med. Chem. 14 (2006) 5981–5988
5987
2-Ar-H), 7.34 (s, 1H, 5-Ar-H), 4.84 (m, 1H, a-CH), 4.52
(m, 1H, a-CH), 4.43 (m, 1H, a-CH), 4.22 (q, 2H, N–
CH2, J = 7.1 Hz), 3.64 (m, 2H, CH2), 3.08 (m, 2H, Im-
CH2), 2.40–1.28 (m, 8H, 4· CH2), 0.89 (t, 3H, CH3,
J = 7.2 Hz); MS (ESI): m/z 391 (M+1); Anal. Calcd
for C18H26N6O4 (390.2): C, 55.37; H, 6.71; N, 21.52.
Found: C, 55.45; H, 6.92; N, 21.73; Rf = 0.57
[CH3OH/25% NH4OH/CHCl3 (20:2:78)]; HPLC:
tR = 5.74 min, purity: 98.2%.
Yield: 62%; mp 95–96 ꢁC; 1H NMR (CD3OD): d 7.86 (s,
1H, 2-Ar-H), 7.14 (s, 1H, 5-Ar-H), 6.17 (m, 1H, CH),
5.44 (m, 2H, CH2), 4.81 (m, 2H, N–CH2), 4.41 (m,
1H, CH), 4.29 (m, 1H, CH), 3.91 (s, 3H, OCH3), 3.24
(m, 2H, Im-CH2), 2.64 (m, 2H, CH2), 2.48 (m, 2H,
CH2); MS (ESI): m/z 321 (M+1); Anal. Calcd for
C15H20N4O4 (320.3): C, 56.24; H, 6.29; N, 17.49. Found:
C, 56.55; H, 6.09; N, 17.33.
4.6. Synthesis of (2S)-3-(1-allyl-1H-4-imidazolyl)-2-[(2S)-
5-oxoazolan-2-ylcarboxamido]propanoic acid [L-pGlu-(1-
allyl)-L-His-OH] (30)
4.4.2. (2S)-1-{(2S)-3-(1-Propyl-1H-4-imidazolyl)-2-[(2S)-
5-oxoazolan-2-ylcarboxamido]propanoyl}azolane-2-car-
boxamide [L-pGlu-(1-propyl)-L-His-L-Pro-NH2] (4). Mp
97–98 ꢁC (dec); H NMR (CD3OD): d 8.74 (s, 1H, 2-
Ar-H), 7.46 (s, 1H, 5-Ar-H), 4.94 (m, 1H, a-CH), 4.46
(m, 1H, a-CH), 4.13 (m, 2H, N–CH2,), 3.75 (m, 1H,
a-CH), 3.29 (m, 2H, Im-CH2), 2.30 (m, 2H, CH2),
2.04–1.86 (m, 6H, 3· CH2), 1.17 (t, 3H, CH3,
J = 7.1 Hz), 0.97 (m, 4H, 2· CH2); MS (ESI): m/z 405
(M+1); Anal. Calcd for C19H28N6O4 (404.2): C, 56.42;
H, 6.98; N, 20.78. Found: C, 56.09; H, 6.78; N, 21.11;
Rf = 0.60 [CH3OH/25% NH4OH/CHCl3 (20:2:78)];
HPLC: tR = 5.97 min, purity: 97.6%.
1
L-pGlu-(1-allyl)-L-His-OMe (29, 1 mmol) was dissolved
in a mixture of CH3OH (50 mL) and 0.5 N NaOH
(40 mL). The solution was stirred at 0 ꢁC for 30 min,
and after that water (35 mL) was added to the solution.
The pH of the solution was then adjusted to 6.0 with
Dowex (50 · 2–200, H+ form) ion-exchange resin. The
resin was removed by filtration, and the filtrate was
evaporated under reduced pressure to afford L-pGlu-
(1-allyl)-L-His-OH 30.
Yield: 63%; mp 132–134 ꢁC (dec); 1H NMR (CD3OD): d
7.35 (s, 1H, 2-Ar-H), 6.88 (s, 1H, 5-Ar-H), 6.00 (m, 1H,
CH2), 5.38 (m, 2H, CH2), 4.71 (m, 2H, N–CH2), 4.58
(m, 1H, a-CH), 4.15 (m, 1H, a-CH), 2.98 (m, 2H, Im-
CH2), 2.64 (m, 2H, CH2), 2.48 (m, 2H, CH2); MS
(ESI): m/z 307 (M+1); Anal. Calcd for C14H18N4O4
(306.3): C, 54.89; H, 5.92; N, 18.29. Found: C, 55.15;
H, 5.77; N, 17.95.
4.4.3. (2S)-1-{(2S)-3-(1-Isopropyl-1H-4-imidazolyl)-2-[(2S)-
5-oxoazolan-2-ylcarboxamido]propanoyl}azolane-2-car-
boxamide [L-pGlu-(1-isopropyl)-L-His-L-Pro-NH2] (5).
Mp 93–94 ꢁC (dec); H NMR (CD3OD): d 8.66 (s, 1H,
2-Ar-H), 7.51 (s, 1H, 5-Ar-H), 4.57 (m, 1H, a-CH),
4.44 (m, 1H, a-CH), 4.21 (m, 1H, a-CH), 3.76 (m, 2H,
Im-CH2), 3.11 (m, 1H, N–CH), 2.43 (m, 2H, CH2),
1.99 (m, 2H, CH2), 1.52 (d, 6H, 2· CH3, J = 5.3 Hz),
1.28–0.85 (m, 6H, 3· CH2); MS (ESI): m/z 405 (M+1);
Anal. Calcd for C19H28N6O4 (404.2): C, 56.42; H,
6.98; N, 20.78. Found: C, 56.84; H, 6.75; N, 20.99;
Rf = 0.60 [CH3OH/25% NH4OH/CHCl3 (20:2:78)];
HPLC: tR = 5.88 min, purity: 98.0%.
1
4.7. Synthesis of (2S)-1-{(2S)-3-(1-allyl-1H-4-imidazolyl)-
2-[(2S)-5-oxoazolan-2-ylcarboxamido]propanoyl}azolane-
2-carboxamide [L-pGlu-(1-allyl)-L-His-L-Pro-NH2] (7)
Dipeptide acid (30, 1 mmol) was dissolved in DMF
(25 mL), and HONB (1 mmol) was added to the solution
followed by addition of DIC (1 mmol) under cooling at
4 ꢁC. Stirring of the reaction mixture continued for anoth-
er 5 min at 4 ꢁC. L-ProNH2 was thenaddedin one portion.
The reaction mixture was stirred for additional 36 h at
4 ꢁC. Solvent evaporated under reduced pressure to afford
crude product. Flash column chromatography using
CHCl3/CH3OH(4:1) asthesolventsystem gaveTRH ana-
logue 7.
4.4.4. (2S)-1-{(2S)-3-(1-Benzyl-1H-4-imidazolyl)-2-[(2S)-
5-oxoazolan-2-ylcarboxamido]propanoyl}azolane-2-car-
boxamide [L-pGlu-(1-benzyl)-L-His-L-Pro-NH2] (6). Mp
1
121–122 ꢁC (dec); H NMR (CD3OD): d 7.76 (s, 1H,
2-Ar-H), 7.40 (s, 5H, C6H5), 7.12 (s, 1H, 5-Ar-H), 5.35
(s, 2H, CH2), 4.82 (m, 1H, a-CH), 4.24 (m, 1H, a-
CH), 3.97 (m, 1H, a-CH), 2.97 (m, 2H, Im-CH2),
2.13–1.66 (m, 10H, 5· CH2); MS (ESI): m/z 453
(M+1); Anal. Calcd for C23H28N6O4 (452.2): C, 61.05;
H, 6.24; N, 18.57. Found: C, 61.41; H, 6.55; N, 18.23;
Rf = 0.62 [CH3OH/25% NH4OH/CHCl3 (20:2:78)];
HPLC: tR = 5.27 min, purity: 97.0%.
Yield: 30%; mp 112-114 ꢁC (dec); 1H NMR (CD3OD): d
7.58 (s, 1H, 2-Ar-H), 6.99 (s, 1H, 5-Ar-H), 6.91 (br s,
1H, NH), 5.98 (m, 1H, CH), 5.21 (m, 2H, CH2), 4.61
(m, 2H, N–CH2), 4.55 (m, 1H, a-CH), 4.39 (m, 1H, a-
CH), 4.18 (m, 1H, a-CH), 3.74 (m, 2H, Im-CH2), 2.95
(m, 2H, CH2), 2.29 (m, 4H, 2· CH2), 1.96 (m, 4H, 2·
CH2); MS (ESI): m/z 403 (M+1); Anal. Calcd for
C19H26N6O4 (402.2): C, 57.68; H, 6.78; N, 20.18. Found:
C, 57.61; H, 6.59; N, 20.31; Rf = 0.55 [CH3OH/25%
NH4OH/CHCl3 (20:2:78)]; HPLC: tR = 6.15 min, purity:
98.0%.
4.5. Synthesis of methyl (2S)-3-(1-allyl-1H-4-imidazolyl)-
2-[(2S)-5-oxoazolan-2-ylcarbox-amido]propanoate [L-pGlu-
(1-allyl)-L-His-OMe] (29)
L-pGlu-OTcp (28, 1 mmol) was added dropwise to 1-al-
lyl-L-histidine methyl ester (17, 1 mmol) in EtOAc
(50 mL) and reaction mixture was stirred at 4 ꢁC for
36 h. Complete removal of the solvent under reduced
pressure gave crude product. Column chromatographic
purification on silica gel (230–400 mesh) using EtOAc–
CH3OH (94:6) as eluant provided dipeptide methyl ester
29.
4.8. Receptor binding assay
HEK293EM cells stably expressing either TRH-R1 or
TRH-R2 were grown in Dulbecco’s modified Eagle’s