E. Sadikoglou et al. / European Journal of Medicinal Chemistry 44 (2009) 3175–3187
3185
4.5.5. Dimethyl N-[(2E,4E,6E)-5-methyl-7-(2,6,6-
(1H, d, J 16 Hz, H-8), 5.90 (1H, s, H-2), 4.69 (1H, t, J 5.8 Hz, H-30), 2.84
(2H, dABq, J 5.8 and 16.4 Hz, H-20), 2.30 (3H, s, H-16), 2.02 (2H, t, J
5.2 Hz, H-12), 1.99 (3H, s, H-17), 1.70 (3H, s, H-18), 1.69–1.60 (2H, m,
H-13), 1.50–1.47 (2H, m, H-14), 1.03 (6H, s, H-19 and H-20) ppm. 13C
trimethylcyclohex-1-enyl)hepta-2,4,6-trienoyl]aspartate (32)
Yield: 82% (0.33 g), yellow oil, Rf (I) 0.34, MS (ESIþ): m/z 829.38
(2M þ Na), 442.20 (M þ K), 426.19 (M þ Na), 404.24 (M þ H). IR
(KBr): cmꢁ1 3288, 1740, 1658. 1H NMR (CDCl3):
d
7.70 (1H, dd, J 12
NMR: d 178.5, 177.9, 171.6, 152.3, 142.0, 141.6, 139.6, 134.3, 133.6,
and 16 Hz, H-3), 6.51 (1H, d, J 8.2 Hz, NHCO), 6.36 (1H, d, J 16 Hz, H-
7), 6.14 (1H, d, J 16 Hz, H-6), 6.13 (2H, d, J 12 Hz, H-4), 5.89 (1H, d, J
16 Hz, H-2), 4.97 (1H, dt, J 4.4 and 8.2 Hz, H-30), 3.78 (3H, s, OCH3),
3.70 (3H, s, OCH3), 3.09 (1H, dd, J 4.4 and 17.2 Hz, H-20a), 2.93 (1H,
dd, J 4.4 and 17.2 Hz, H-20b), 2.04 (3H, s, H-14), 2.05-1.95 (2H,
unresolved t, H-10),1.71 (3H, s, H-15),1.64–1.60 (2H, m, H-11),1.48–
1.45 (2H, m, H-12), 1.03 (6H, s, H-16 and H-17) ppm, 13C NMR:
133.4, 133.0, 131.5, 125.2, 53.5, 43.3, 41.0, 37.8, 36.5, 31.9 (two C),
24.5, 22.8, 16.4, 15.4 ppm. Anal. Calcd. for C24H33NO5: C, 69.37; H,
8.00; N, 3.37. Found: C, 69.21; H, 7.88; N, 3.57.
4.6.2. N-[(2E,4E,6E)-5-Methyl-7-(2,6,6-trimethylcyclohex-1-
enyl)hepta-2,4,6-trienoyl]aspartic acid (4)
Yield: 64% (0.26 g), yellow solid, m.p. 134–136 ꢀC, Rf (A) 0.32, MS
(ESIþ): m/z 773.65 (2M þ Na), 414.62 (M þ K), 398.64 (M þ Na),
376.63 (M þ H). IR (KBr): cmꢁ1 3400–2870, 1730, 1664. 1H NMR
d
171.7, 171.3, 166.0, 143.8, 138.2, 137.6, 136.8 (two C), 130.4, 127.1,
121.8, 52.8, 52.0, 48.6, 39.6, 36.2, 34.3, 33.1, 28.9 (two C), 21.7, 19.2,
13.0 ppm.
(CDCl3): d 7.61 (1H, unresolved dd, H-3), 7.47 (1H, br s, NHCO), 6.31
(1H, d, J 16 Hz, H-7), 6.09 (1H, d, J 16 Hz, H-6), 6.07 (1H, d, J 12 Hz, H-
4), 5.97 (1H, d, J 16 Hz, H-2), 4.91 (1H, br s, H-30), 2.93 (2H, ABq, J
15.8 Hz, H-20), 1.99 (2H, unresolved t, H-10), 1.94 (3H, s, H-14), 1.67
(3H, s, H-15), 1.62–1.53 (2H, m, H-11), 1.48–1.39 (2H, m, H-12), 0.99
4.5.6. Trimethyl N-[(2E,4E,6E)-5-methyl-7-(2,6,6-
trimethylcyclohex-1-enyl)hepta-2,4,6-trienoyl]aspartyl-aspartate
(33)
Yield: 81% (0.43 g), yellow oil, Rf (H) 0.29, MS (ESIþ): m/z 1104.17
(2M þ K), 1087.20 (2M þ Na), 571.10 (M þ K), 555.16 (M þ Na),
533.20 (M þ H). IR (KBr): cmꢁ1 3276, 1738, 1644. 1H NMR (CDCl3):
(6H, s, H-16 and H-17) ppm, 13C NMR:
d 175.0 (two C), 167.7, 144.2,
138.7, 137.4, 136.8, 130.5 (two C), 127.3, 121.6, 49.3, 39.6, 36.6, 34.2,
33.1, 28.9 (two C), 21.8,19.2,12.9 ppm. Anal. Calcd. for C21H29NO5: C,
67.18; H, 7.79; N, 3.73. Found: C, 67.88; H, 7.50; N, 3.98.
d
7.72 (1H, dd, J 12 and 16 Hz, H-3), 7.51 (1H, d, J 8.6 Hz, NHCO), 6.76
(1H, d, J 8 Hz, NHCO), 6.38 (1H, d, J 16 Hz, H-7), 6.15 (1H, d, J 16 Hz,
H-6), 6.14 (1H, d, J 12 Hz, H-4), 5.90 (1H, d, J 16 Hz, H-2), 4.94 (1H,
ddd, J 4.4, 6.8 and 8 Hz, H-60), 4.85 (1H, dt, J 4.6 and 8.6 Hz, H-30),
3.76 (3H, s, OCH3), 3.75 (3H, s, OCH3), 3.71 (3H, s, OCH3), 3.07 (1H,
dd, J 4.4 and 17.2 Hz, H-70a), 3.01 (1H, dd, J 4.6 and 16.8 Hz, H-20a),
2.84 (1H, dd, J 4.6 and 16.8 Hz, H-20b), 2.71 (1H, dd, J 6.8 and 17.2 Hz,
H-70b), 2.05 (3H, s, H-14), 2.04 (2H, t, J 6.4 Hz, H-10), 1.73 (3H, s, H-
15), 1.66-1.59 (2H, m, H-11), 1.51-1.46 (2H, m, H-12), 1.04 (6H, s, H-
4.6.3. N-(all-trans-Retinoyl)aspartyl-aspartic acid (5)
Yield: 57% (0.33 g), yellow solid, m.p. 118–120 ꢀC, Rf (A) 0.29, MS
(ESIþ): m/z 1083.61 (2M þ Na), 569.41 (M þ K), 553.42 (M þ Na),
531.46 (M þ H). IR (KBr): cmꢁ1 3400–2856, 1730, 1654. 1H NMR
(CDCl3):
d 7.00 (1H, dd, J 12 and 16 Hz, H-5), 6.74 (1H, d, J 8.4 Hz,
NHCO), 6.33 (1H, d, J 16 Hz, H-4), 6.27 (1H, d, J 16 Hz, H-9), 6.16 (1H,
d, J 12 Hz, H-6), 6.13 (1H, d, J 16 Hz, H-8), 5.90 (1H, s, H-2), 4.80–4.73
(2H, m, H-30 and H-60), 2.85–2.79 (4H, m, H-20 and H-70), 2.29 (3H, s,
H-16), 2.04 (2H, t, J 6 Hz, H-12), 2.01 (3H, s, H-17), 1.71 (3H, s, H-18),
1.68–1.61 (2H, m, H-13), 1.51–1.46 (2H, m, H-14), 1.03 (6H, s, H-19
16 and H-17) ppm. 13C NMR:
d 172.5, 171.0, 170.7, 170.4, 166.3, 144.1,
138.5, 137.5, 136.7, 130.6 (two C), 127.0, 121.5, 52.9, 52.2, 52.1, 49.1,
48.7, 39.6, 35.9, 35.7, 34.2, 33.1, 28.9 (two C), 21.8, 19.2, 13.0 ppm.
and H-20) ppm. 13C NMR:
d 176.5, 176.0, 174.61, 174.5, 171.9, 152.7,
4.6. General method for the saponification of fully
protected conjugates
142.1, 141.6, 141.5, 139.5, 133.6, 133.1, 131.6, 124.9, 124.5, 52.9, 52.7,
46.4, 43.3, 40.8, 37.8, 36.5, 31.9 (two C), 24.4, 22.8, 16.5, 15.3 ppm.
Anal. Calcd. for C28H38N2O8: C, 63.38; H, 7.22; N, 5.28. Found: C,
63.58; H, 7.35; N, 5.01.
To an ice-cold solution of 1.1 mmol of esters 28–33 in MeOH
(5.4 mL) was added a solution of NaOH (0.053 g, 1.32 mmol/group
to be saponified) in H2O (0.6 mL). The resulting mixture was stirred
at RT for 1 h (3–4 h for 28–30), ice-cold H2O (6 mL) was added and
the pH of the solution was carefully adjusted to 7 by the dropwise
addition of a 1 N HCl aqueous solution. The solvents evaporated
under reduced pressure, at a bath temperature less than 40 ꢀC, the
residue was dissolved in H2O (20 mL), cooled to 0 ꢀC and the
solution was brought to pH 3.5 by the dropwise addition of a 1 N
HCl aqueous solution. The resulting yellow precipitate was filtered,
washed with cold H2O, dried under vacuo and finally recrystallized
from MeCN/H2O to give pure product as yellow solid. In the case of
Lys, after the final acidification, the solution was evaporated under
reduced pressure to dryness; a small volume of MeOH was added
and refrigerated overnight. The precipitated NaCl was filtered off
and the filtrate was re-subjected to the same procedure twice to
remove NaCl. The residue was isolated, as a yellow foam, through
preparative RP-HPLC using as eluant a gradient of MeCN (0.08%
TFA)/H2O (0.08% TFA), from 10% to 100% MeCN, followed by pooling
and lyophilization of the fractions containing the product.
4.6.4. N-[(2E,4E,6E)-5-Methyl-7-(2,6,6-trimethylcyclohex-1-
enyl)hepta-2,4,6-trienoyl]aspartyl-aspartic acid (6)
Yield: 61% (0.33 g), yellow solid, m.p. 124–126 ꢀC, Rf (A) 0.15, MS
(ESIþ): m/z 529.16 (M þ K), 513.25 (M þ Na), 491.24 (M þ H). IR
(KBr): cmꢁ1 3328, 3200–2400, 1730, 1642. 1H NMR (CD3OD):
d 7.64
(1H, ddd, J 1.6, 12 and 16 Hz, H-3), 6.39 (1H, d, J 16 Hz, H-7), 6.19
(1H, d, J 12 Hz, H-4), 6.16 (1H, d, J 16 Hz, H-6), 6.08 (1H, d, J 16 Hz, H-
2), 4.83 (1H, td, J 2 and 5.6 Hz, H-60), 4.74 (1H, dt, J 3.2 and 6 Hz, H-
30), 2.88–2.78 (4H, m, H-20 and H-70), 2.04 (3H, s, H-14), 2.04 (2H, t, J
7.6 Hz, H-10), 1.71 (3H, s, H-15), 1.68–1.61 (2H, m, H-12), 1.51–1.44
(2H, m, H-11), 1.03 (6H, s, H-16 and H-17) ppm. 13C NMR:
d 176.8,
176.6, 174.6, 174.4, 171.4, 146.9, 141.4, 141.3, 141.0, 133.7, 131.3, 126.3,
126.0, 53.2, 52.8, 43.3, 40.8, 39.5, 37.8, 36.5, 31.9 (two C), 24.5, 22.8,
15.5 ppm. Anal. Calcd. for C25H34N2O8: C, 61.21; H, 6.99; N, 5.71.
Found: C, 61.00; H, 6.75; N, 5.99.
4.6.5. Hydrochloride of Na-(all-trans-retinoyl)lysine (8)
Yield: 54% (0.28 g), yellow foam, Rf (A) 0.15, MS (ESIþ): m/z
880.84 (2M þ Na), 467.72 (M þ K), 453.66 (M þ Na), 429.10 (M þ H).
4.6.1. N-(all-trans-Retinoyl)aspartic acid (3)
IR (KBr): cmꢁ1 3400–2882, 1725, 1638. 1H NMR (CDCl3):
d 7.71 (3H,
Yield: 58% (0.27 g), yellow solid, m.p. 131–133 ꢀC, Rf (A) 0.35, MS
(ESIþ): m/z 869.39 (2M þ K), 454.38 (M þ K), 438.28 (M þ Na),
416.25 (M þ H). IR (KBr): cmꢁ1 3400–2866, 1730, 1626, 1606. 1H
br s, NHþ3 ), 6.89 (1H, dd, J 12 and 16 Hz, H-5), 6.24 (1H, d, J 16 Hz, H-
4), 6.22 (1H, d, J 16 Hz, H-9), 6.17 (1H, d, J 8.4 Hz, NHCO), 6.16 (1H, d,
J 12 Hz, H-6), 6.15 (1H, d, J 16 Hz, H-8), 5.67 (1H, s, H-2), 4.37 (1H, dt,
4.8 and 8.4 Hz, H-20), 2.92 (2H, m, H-60), 2.30 (3H, s, H-16), 1.97 (2H,
t, J 6.4 Hz, H-12), 1.94 (3H, s, H-17), 1.89–1.84 (2H, m, H-30),
NMR (CDCl3):
d 6.99 (1H, dd, J 12 and 16 Hz, H-5), 6.34 (1H, d, J
16 Hz, H-4), 6.27 (1H, d, J 16 Hz, H-9), 6.16 (1H, d, J 12 Hz, H-6), 6.13