4664 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 12
Souto et al.
0.005 mmol) in 1,2-dichloroethane (0.1 mL) was added. The
mixture was briefly degassed and then stirred at 90 °C overnight.
The solvent was removed, and the residue was purified by column
chromatography (silica gel, 10:10:1 hexane/EtOAc/MeOH) to
afford 6 mg (27%) of a dark-yellow oil identified as largazole (4a).
1H NMR (400.16 MHz, CDCl3): δ 7.85 (s, 1H), 7.19 (d, J =
8.8 Hz, 1H), 6.64 (br, 1H), 5.86 (td, J = 15.0, 6.8 Hz, 1H), 5.68 (t,
J = 8.8 Hz, 1H), 5.56 (dd, J = 15.8, 6.8 Hz, 1H), 5.34 (dd, J =
17.7, 9.1 Hz, 1H), 4.63 (dd, J = 9.0, 3.1 Hz, 1H), 4.30 (d, J =
17.1 Hz, 1H), 4.12 (d, J = 10.9 Hz, 1H), 3.34 (d, J = 11.4 Hz, 1H),
3.1-3.0 (m, 1H), 2.93 (t, J = 7.3 Hz, 2H), 2.69 (dd, J = 16.3,
1.5 Hz, 1H), 2.55 (t, J = 7.5 Hz, 2H), 2.34 (q, J = 7.2 Hz, 2H),
2.2-2.1 (m, 1H), 1.78 (s, 3H), 1.7-1.6 (m, 2H), 1.4-1.2 (m, 8H),
0.90 (t, J = 6.5 Hz, 3H), 0.70 (d, J = 6.9 Hz, 3H), 0.54 (d, J =
6.8 Hz, 3H) ppm. 13C NMR (100.62 MHz, CDCl3): δ 199.3 (s),
173.0 (s), 169.6 (s), 168.7 (s), 168.2 (s), 165.5 (s), 147.1 (s), 132.8
(d), 128.4 (d), 124.7 (d), 83.6 (s), 71.9 (d), 57.9 (d), 44.2 (t), 43.3 (t),
41.1 (t), 40.5(t), 34.1(d), 32.3 (t), 31.6(t), 28.9(t, 2ꢀ), 27.9 (t), 25.7
(t), 23.8 (q), 22.6 (t), 18.8 (q), 16.7 (q), 14.1 (q) ppm. MS (ESIþ):
645 ([M þ Na]þ, 57), 623 ([M þ H]þ, 100), 413 (15), 321 (51).
HRMS (ESIþ): calcd for C29H43N4O5S3, 623.2390; found,
623.2379.
(3S,20S)-tert-Butyl 3-(2-Amino-3-methylbutanoyloxy)pent-4-
enoate (32).37 Following the general procedure described above
for the cleavage of Fmoc-carbamates, the reaction of (3S,20S)-
tert-butyl-3-(-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-
methylbutanoyloxy)-pent-4-enoate (26) (0.6 g, 1.22 mmol) and
piperidine (0.48 mL, 4.86 mmol) in CH3CN (23 mL) afforded,
after purification by column chromatography (silica gel, 50:50
hexane/EtOAc) 320 mg (97%) of a colorless oil identified
as (3S,20S)-tert-butyl 3-(2-amino-3-methylbutanoyloxy)pent-4-
enoate (32). 1H NMR (400.16 MHz, CDCl3): δ 5.85 (ddd, J =
17.1, 10.5, 6.6 Hz, 1H), 5.68 (dd, J = 13.6, 6.7 Hz, 1H), 5.36 (td,
J = 17.2, 1.1 Hz, 1H), 5.24 (td, J = 10.5, 1.0 Hz, 1H), 3.30 (d, J =
4.8 Hz, 1H), 2.65 (dd, J = 15.4, 8.0 Hz, 1H), 2.55 (dd, J = 15.4,
5.7 Hz, 1H), 2.2-2.0 (m, 1H), 1.65 (br, 2H), 1.46 (s, 9H), 1.00 (d,
J = 6.9 Hz, 3H), 0.91 (d, J = 6.9 Hz, 3H) ppm. 13C NMR (100.62
MHz, CDCl3): δ 174.2 (s), 168.5 (s), 134.7 (d), 117.5 (t), 80.8 (s),
71.1 (d), 59.5 (d), 40.2 (t), 31.6 (d), 27.7 (q, 3ꢀ), 18.4 (q), 16.6 (q)
ppm. IR (NaCl): ν 3500-3300 (br, N-H), 2963 (s, C-H), 2923
(s, C-H), 2850 (s, C-H), 1735 (s, CdO), 1458 (m, CdC), 1368
(m), 1157 (s) cm-1. MS (ESIþ): 294 ([M þ Na]þ, 5), 272 ([M þ
H]þ, 100). HRMS (ESIþ): calcd for C14H26NO4, 272.1856;
found, 272.1869.
HRMS (ESIþ): calcd for C15H22N3O4S2 372.1046; found,
372.1039.
(3S,20S,400R)-tert-Butyl 3-(2-(2-(2-(tert-Butoxycarbonylamino-
methyl-thiazol-4-yl)-4-ethyl-4,5-dihydrothiazole-4-carboxamido)-
3-methylbutanoyloxy)pent-4-enoate (34c). General procedure for
the formation of peptide bonds. To a solution of (3S,20S)-tert-
butyl 3-(2-amino-3-methylbutanoyloxy)pent-4-enoate (32) (0.02 g,
0.075 mmol) and (R)-2-(2-tert-butoxycarbonylaminomethyl-
thiazol-4-yl)-4-ethyl-4,5-dihydrothiazole-4-carboxylic acid (33)
(0.028 g, 0.075 mmol) in DMF (0.5 mL) were added HATU
(0.034 g, 0.09 mmol) and DIPEA (0.039 mL, 0.225 mmol) and the
reaction was stirred at room temperature for 2 h. A phosphate
buffer (pH = 7, 0.5 mL) was added, and the mixture was
extracted with EtOAc (3ꢀ). The combined organic layers were
washed with H2O (2ꢀ), brine (2ꢀ), and dried (Na2SO4) and the
solvent was removed under vacuum. The residue was purified by
column chromatography (silica gel, 80:20 hexane/EtOAc) to
afford 14 mg (89%) of a colorless oil identified as a mixture of
conformers of (3S,20S,400R)-tert-butyl 3-(2-(2-(2-(tert-butoxycar-
bonylaminomethyl-thiazol-4-yl)-4-ethyl-4,5-dihydrothiazole-4-
carboxamido)-3-methylbutanoyloxy)pent-4-enoate (34c). [R]25
D
-29.3° (c 1.067, MeOH). 1H NMR (400.16 MHz, CDCl3) (major
conformer): δ 7.99 (s, 1H), 7.14 (d, J = 9.0 Hz, 1H), 5.81 (ddd,
J = 17.2, 10.4, 6.8 Hz, 1H), 5.66 (dd, J = 13.7, 6.8 Hz, 1H), 5.35
(d, J = 17.2 Hz, 1H), 5.23 (d, J = 10.4 Hz, 1H), 4.63 (d, J =
6.1 Hz, 2H), 4.55 (dd, J = 9.2, 4.6 Hz, 1H), 3.81 (d, J = 11.7 Hz,
1H), 3.34 (d, J = 11.7 Hz, 1H), 2.69 (dd, J = 15.5, 7.9 Hz, 1H),
2.57 (dd, J = 15.5, 9.5 Hz, 1H), 2.19 (dq, J = 11.4, 6.8 Hz, 1H),
2.04 (dq, J = 15.0, 7.5 Hz, 1H), 1.93 (qd, J = 14.6, 7.4 Hz, 1H),
1.47 (s, 9H), 1.43 (s, 9H), 0.99 (t, J = 5.9 Hz, 3H), 0.86 (d, J =
6.8 Hz, 3H), 0.79 (d, J = 6.9 Hz, 3H) ppm. 13C NMR (100.62
MHz, CDCl3) (major conformer): δ 174.0 (s), 170.5 (s), 168.7 (s),
160.8 (s), 155.7 (s), 134.6 (d), 123.9 (d), 121.2 (s), 118.5 (t), 89.2 (s),
81.3 (s), 80.5 (s), 77.3 (d), 72.3 (d), 56.8 (d), 42.3 (t), 40.6 (t), 39.7
(t), 32.4 (t), 31.1 (d), 28.3 (q, 3ꢀ), 28.0 (q, 3ꢀ), 19.1 (q), 17.4 (q),
8.8 (q) ppm. IR (NaCl): ν 3500-3200 (br, N-H), 2920 (s, C-H),
2850 (s, C-H), 1733 (m, CdO), 1716 (m, CdO), 1683 (m, CdO)
cm-1. MS (ESIþ): 647 ([M þ Na]þ, 37), 625 ([M þ H]þ, 100), 534
(22), 512 (18). HRMS (ESIþ): calcd for C29H45N4O7S2, 625.2724;
found, 625.2728.
Ethyl-thiazoline Macrocycle 36c. General procedure for the
synthesis of macrocycles (method B). (S)-tert-butyl 3-((S)-2-
((R)-2-(2-tert-butoxycarbonylaminomethylthiazol-4-yl)-4-ethyl-
4,5-dihydrothiazole-4-carboxamido)-3-methylbutanoyloxy)pent-
4-enoate (34c) (0.02 g, 0.034 mmol) and triethylsilane (6.8 μL,
0.043 mmol) in CH2Cl2 (0.2 mL) and TFA (0.08 mL, 0.85 mmol)
was stirred at 0 °C. After 4 h stirring, the solvents were removed
and the residue was used in the next step without further purifica-
tion. To a cooled (0 °C) solution of HATU (0.045 g, 0.119 mmol)
and DIPEA (0.058 mL, 0.34 mmol) in THF (8.8 mL) was added a
solution of the previously synthesized product (0.034 mmol) in
THF (1.6 mL) and the reaction was stirred for 15 h, allowing it to
reach room temperature. H2O was added, and the layers were
separated. The aqueous layer was extracted with EtOAc (3ꢀ), the
combined organic layers were washed with brine (2ꢀ), dried
(Na2SO4), and the solvent was evaporated under vacuum. The
residue was purified by column chromatography (silica gel,
10:10:1 hexane:EtOAc/MeOH) to afford 18 mg (99%) of a color-
(R)-2-(2-tert-Butoxycarbonylaminomethyl-thiazol-4-yl)-4-ethyl-
4,5-dihydrothiazole-4-carboxylic acid (33c). General procedure
for the condensation of nitriles and alkylcysteines. To a solution
of 2-(tert-butoxycarbonylaminomethyl)-thiazole-4-carbonitrile
(14) (0.043 g, 0.18 mmol) and ethylcysteine hydrochloride (24c)
(0.053 g, 0.29 mmol) in MeOH (2.3 mL) and phosphate buffer
(pH = 7, 1.4 mL) was added NaHCO3 (0.076 g, 0.9mmol) and the
reaction was stirred for 24 h at 70 °C. H2O (1 mL) and a saturated
aqueous NaHCO3 solution (1 mL) was added and the mixture was
extracted with EtOAc (3ꢀ). The aqueous layer was acidified until
pH = 1 by addition of KHSO4 and then extracted with EtOAc
(4ꢀ). The combined organic layers were washed with brine, dried
(Na2SO4) and the solvent was evaporated. The residue was
purified by column chromatography (C18 silica gel, CH3CN) to
afford 30 mg (49%) of a colorless foam identified as (R)-2-(2-tert-
less foam identified as the ethyl-thiazoline macrocycle 36c. [R]25
D
þ39.0° (c 0.24, MeOH). 1H NMR (400.16 MHz, CD3OD): δ 8.14
(s, 1H), 7.41 (d, J = 9.2 Hz, 1H), 5.96 (ddd, J = 16.6, 10.6, 5.8 Hz,
1H), 5.8-5.6 (m, 1H), 5.30 (dt, J = 17.3, 1.1 Hz, 1H), 5.25 (dt,
J = 10.6, 1.0Hz, 1H), 5.16 (d, J = 17.6 Hz, 1H), 4.6-4.5 (m, 1H),
4.39 (d, J = 17.6 Hz, 1H), 3.87 (d, J = 11.6 Hz, 1H), 3.45 (d, J =
11.6 Hz, 1H), 2.98 (dd, J = 16.5, 11.2 Hz, 1H), 2.79 (dd, J = 16.5,
2.7 Hz, 1H), 2.24 (qd, J = 14.7, 7.4 Hz, 1H), 2.12 (dtd, J = 13.7,
6.9, 3.4 Hz, 1H), 2.1-1.9 (m, 1H), 1.13 (t, J = 7.4 Hz, 3H), 0.74
(d, J = 6.9 Hz, 3H), 0.51 (d, J = 6.9 Hz, 3H) ppm. 13C NMR
(100.62 MHz, CDCl3): δ 174.2 (s), 170.9 (s), 168.8 (s), 168.4 (s),
166.2 (s), 147.0 (s), 135.1 (d), 125.0 (d), 116.0 (t), 88.1 (s), 72.5 (d),
butoxycarbonylaminomethyl-thiazol-4-yl)-4-ethyl-4,5-dihydrothia-
1
zole-4-carboxylic acid (33c). [R]27 -2.97° (c 0.31, MeOH). H
D
NMR (400.16 MHz, CDCl3): δ 8.11 (s, 1H), 5.39 (br, 1H), 4.65 (d,
J = 5.8 Hz, 2H), 3.91 (d, J = 11.8 Hz, 1H), 3.42 (d, J = 11.8 Hz,
1H), 2.10 (q, J = 7.4 Hz, 2H), 1.49 (s, 9H), 1.07 (t, J = 7.4 Hz,
3H) ppm. 13C NMR (100.62 MHz, CDCl3): δ 174.3 (s), 170.3 (s),
166.0 (s), 155.7 (s), 147.4 (s), 123.5 (d), 88.3 (s), 80.6 (s), 42.3 (t),
38.6 (t), 31.8 (t), 28.3 (q, 3ꢀ), 8.6 (q) ppm. IR (NaCl): ν
3500-3200 (br, N-H), 2972 (m, C-H), 2927 (m, C-H), 1705
(s, CdO), 1654 (m, CdC), 1515 (s, CdC), 1249 (m), 1169 (s)
cm-1. MS (ESIþ): 391 ([M þ Na]þ, 16), 372 ([M þ H]þ, 100).