Journal of Medicinal Chemistry
ARTICLE
(S)-2-((S)-2-(((9H-Fluoren-9-yl)methoxy)carbonylamino)propanethio-
amido)-3-methylbutanoic Acid (34). Compound 33 (0.19 g, 0.4 mmol)
was added in one portion to an ice-cooled mixture of TFA (10 mL) and
DCM (4 mL), and stirring was maintained under nitrogen for 1 h. At this
point, the reaction was complete according to TLC. Evaporation and drying
in a vacuum oven (10 Pa, 35 °C, 12 h) quantitatively gave 34 as a thick
colorless oil, which appeared pure on TLC and was used directly in the next
step. 1HNMR(CDCl3): δ0.94 (d, J = 6.7 Hz, 3H), 1.00 (d, J= 6.7 Hz, 3H),
1.44 (d, J = 6.1 Hz, 3H), 2.29-2.40 (m, 1H), 4.18 (t, J = 6.9 Hz, 1H), 4.28-
4.39 (m, 2H), 4.72 (br-s, 1H), 5.08-5.16 (m, 1H), 5.88-5.91 (m, 1H),
7.23-7.29 (m, 2H), 7.34-7.39 (m, 2H), 7.54 (d, J = 6.9 Hz, 2H), 7.72 (d, J
= 7.4 Hz, 2H), 8.63 (br-s, 1H). 13C NMR (CDCl3): δ 18.65, 18.70, 22.16,
30.97, 47.07, 56.19, 62.70, 67.65, 120.07, 125.12, 127.15, 127.83, 141.23,
143.56, 156.10, 174.83, 205.94. Anal. calcd for C23H26N2O4S: C, 64.77; H,
6.14; N, 6.57. Found: C, 64.89; H, 6.26; N, 6.43.
N-Me-ETASV (35). 2-Chlorotrityl chloride resin (0.16 g, loading 1.57
mmol/g) was shaken in dry DCM for 15 min and drained. Compound
34 (0.17 g, 0.4 mmol) dissolved in DCM (2 mL) containing DIPEA
(0.35 mL) was added, and shaking was maintained for 2 h. After
draining, the resin was washed three times with DCM/MeOH/DIPEA
(17:2:1, 3 mL) and then flow washed with DCM and DMF. The resin
was treated for 2 ꢀ 5 min with piperidine/DMF (3:7, 3 mL), drained,
and washed twice with DMF. A solution of HBTU (0.38 g, 1 mmol),
Fmoc-Thr(O-tert-Bu)-OH (0.40 g, 1 mmol), and DIPEA (0.17 mL) in
DMF (1 mL) was added, and shaking was maintained for 30 min. After
draining, the resin was washed twice with DMF and treated for 2 ꢀ 5 min
with piperidine/DMF (3:7, 3 mL), drained, and washed twice with
DMF. A solution of HBTU (0.38 g, 1 mmol), Fmoc-N-Me-Glu(O-tert-
Bu)-OH (0.43 g, 1 mmol), and DIPEA (0.17 mL) in DMF (1 mL) was
added, and shaking was maintained for 30 min. After draining, the resin
was washed twice with DMF and then treated 2 ꢀ 5 min with
piperidine/DMF (3:7, 3 mL), drained, and washed twice with DMF.
An inhomogenous mixture of TFA/triisopropylsilane(TIPS)/H2O
(18:1:1, 5 mL) was added, and shaking was maintained for 2 h. The
peptide containing solution was filtered into a flask and combined with
two successive washes with DCM. After gentle evaporation in a stream of
nitrogen, the crude peptide was washed with cold ether (2 ꢀ 10 mL) to
give the crude product (0.16 g). Separation of 0.020 g by HPLC,
evaporation, and lyophilization gave 11 mg (79%) of 35 as a white solid.
1H NMR (CD3OD): δ 1.01 (d, J = 6.6 Hz, 3H), 1.04 (d, J = 6.9 Hz, 3H),
1.22 (d, J = 6.3 Hz, 3H), 1.43 (d, J = 6.6 Hz, 3H), 2.18-2.31 (m, 3H),
2.53 (t, J = 7.4 Hz, 2H), 2.67 (s, 3H), 3.98 (t, J = 6.1 Hz, 1H), 4.21 (p, J =
4.4 Hz, 1H), 4.42 (d, J = 4.4 Hz, 1H), 4.84 (q, J = 6.9 Hz, 1H), 4.95 (d, J =
6.1 Hz, 1H). HRMS (ESþ) calcd for C18H33N4O7S [M þ H]þ,
449.2070; found, m/z 449.2064.
66.52, 68.37, 74.36, 81.22, 120.73, 122.00, 125.95, 127.77, 128.25, 129.52,
141.35, 144.41, 156.45, 170.04, 172.03. Anal. calcd for C30H40N2O6: C,
68.68; H, 7.68; N, 5.34. Found: C, 68.35; H, 7.70; N, 5.18.
(S)-tert-Butyl 2-((2S,3R)-2-(((9H-Fluoren-9-yl)methoxy)carbonyl-
amino)-3-tert-butoxybutanethioamido)propanoate (37). Compound
36 (2.65 g, 5 mmol) was dissolved in dry THF (100 mL), and Lawesson's
reagent (2.43 g, 6 mmol) was added in one portion. Stirring was maintained
at room temperature for 48 h under nitrogen. The resulting white slurry was
poured into ice (400 mL) and extracted with ethyl acetate (3 ꢀ 100 mL).
The pooled extracts were washed with water (25 mL), dried over magnesium
sulfate, and evaporated on Celite. The product was separated by automated
flash chromatography on a stationary silica gel column with ethyl acetate:
heptane (1:4). Evaporation of the solvent and drying in a vacuum oven (10
Pa, 30 °C, 24 h) gave the thioamide 37 (1.75 g, 65%) as white crystals; mp,
132-133 °C. 1H NMR (CDCl3): δ 1.18 (d, J = 6.1 Hz, 3H), 1.33 (s, 9H),
1.51 (s, 9H), 1.53 (d, J = 11.8 Hz, 3H), 4.25-4.30 (m, 2H), 4.37-4.46 (m,
3H), 4.86 (t, J = 6.9 Hz, 1H), 6.63 (d, J = 5.2 Hz, 1H), 7.25-7.34 (m, 2H),
7.37-7.42 (m, 2H), 7.64 (t, J= 7.0 Hz, 2H), 7.75 (d, J=7.4Hz, 2H), 9.50(d,
J = 5.5 Hz, 1H). 13C NMR (CDCl3): δ 17.05, 17.18, 28.08, 28.37, 47.20,
54.74, 62.79, 67.15, 69.06, 75.63, 82.26, 119.88, 125.13, 125.21, 126.99,
127.61, 141.16, 143.56, 155.60, 170.54, 199.66. Anal. calcd for C30H40N2O5S:
C, 66.64; H, 7.46; N, 5.18. Found: C, 66.62; H, 7.53; N, 5.07.
Fmoc-TASV-O-tert-Bu (39). Compound 37 (0.54 g, 1 mmol) was
added in one portion to an ice-cold mixture of TFA (15 mL) and DCM
(6 mL), and stirring was maintained under nitrogen for 1 h. After
evaporation and drying in a vacuum oven (10 Pa, 35 °C, 12 h), the free
acid 38 remained as a thick colorless oil. Val-O-tert-Bu HCl (0.23 g, 1.1
3
mmol) and DMF (6 mL) were added, and the resulting slurry was
stirred, while DIPEA (0.4 mL) and HATU (0.42 g, 1.1 mmol) were
successively added during cooling in an ice bath, and stirring was
maintained for 2 h at room temperature. The reaction mixture was
poured into water and extracted with ethyl acetate (3 ꢀ 30 mL). The
pooled organic phases were dried over sodium sulfate, filtered, and
evaporated on Celite. The product was separated by automated flash
chromatography on a stationary silica gel using ethyl acetate-heptane
(1:1). Evaporation of the solvent and drying in a vacuum oven (10 Pa,
1
30 °C, 24 h) gave 39 (0.45 g, 77%) as a dark-yellow oil. H NMR
(CDCl3): δ 0.82-0.93 (m, 6H), 1.01-1.27 (m, 6H), 1.41 (s, 9H), 1.99
(br-s, 1H), 2.04-2.18 (m, 1H), 3.98-4.17 (m, 3H), 4.19-4.58 (s, 3H),
4.98-5.07 (m, 1H), 6.26 (br-s, 1H), 6.84 (br-s, 1H), 7.17-7.37 (m,
4H), 7.50-7.72 (m, 4H), 9.11 (s, 1H). 13C NMR (CDCl3): δ 14.25,
14.31, 17.24, 17.72, 18.58, 28.08, 31.30, 47.01, 54.62, 57.85, 60.45, 63.80,
67.40, 68.89, 82.22, 119.84, 125.00, 126.94, 127.64, 141.13, 143.51,
156.39, 170.60, 171.14, 200.90. Anal. calcd for C31H41N3O6S: C, 63.78;
H, 7.08; N, 7.20. Found: C, 64.12; H, 7.43; N, 6.90.
(S)-tert-Butyl 2-((2S,3R)-2-(((9H-Fluoren-9-yl)methoxy)carbonyl-
amino)-3-tert-butoxybutanamido)propanoate (36). Triethylamine
(3 mL) and HBTU (3.95 g, 10.4 mmol) were added to an ice-cooled
slurry of Fmoc-Thr(O-tert-Bu)-OH (3.97 g, 10 mmol) and Ala-O-tert-
Fmoc-E(O-tert-Bu)TSAV-O-tert-Bu (40). Compound 39 (0.29 g,
0.5 mmol) was dissolved in a dimethylamine solution (2.0 M in MeOH,
4 mL, 8 mmol), and stirring was maintained for 2 h. After evaporation in
a stream of air, THF (4 mL) was added and successively evaporated in a
stream of air in order to remove traces of dimethylamine. The residue
was placed in a vacuum oven (10 Pa, 30 °C, 24 h), dissolved in DMF
(3 mL), and cooled in an ice bath, while Fmoc-N-Me-Glu(O-tert-Bu)-
OH (0.21 g, 0.55 mmol), DIPEA (0.2 mL), and HATU (0.21 g, 0.55
mmol) were successively added. After it was stirred at room temperature
for 2 h, the reaction mixture was poured into water (40 mL) and
extracted with ethyl acetate (3 ꢀ 30 mL). The pooled organic phases
were dried over sodium sulfate, filtered, and evaporated on Celite. The
product was separated by automated flash chromatography on a
stationary silica gel using ethyl acetate-heptane (1:1). Evaporation of
the solvent and drying in a vacuum oven (10 Pa, 30 °C, 24 h) gave 40
Bu HCl (1.89 g, 10 mmol) in acetonitrile (40 mL). After the solution
3
was stirred at room temperature for 2 h, the resulting white slurry was
poured into brine (250 mL) and extracted with ethyl acetate (3 ꢀ 100
mL). The pooled extracts were successively washed with hydrochloric
acid (2 M, 10 mL) and saturated sodium hydrogen carbonate (10 mL).
After evaporation on Celite, the product was separated by automated
flash chromatography on a stationary silica gel using ethyl acetate/
heptane (1:4). Evaporation of the solvent and drying in a vacuum oven
(10 Pa, 30 °C, 24 h) gave the protected dipeptide 36 (4.56 g, 98%) as a
white crystalline mass; mp, 70-72 °C. 1H NMR (CDCl3): δ 1.07 (d, J
= 7.6 Hz, 3H), 1.10 (s, 9H), 1.26 (d, J = 7.2 Hz, 3H), 1.37 (s, 9H),
3.82-3.86 (m, 1H), 4.01-4.07 (m, 1H), 4.14-4.17 (m, 3H), 4.23-
4.34 (m, 1H), 7.00 (d, J = 9.4 Hz, 1H), 7.29 (t, J = 7.3 Hz, 2H), 7.38 (t,
J = 7.3 Hz, 2H), 7.70-7.74 (m, 2H), 7.85 (d, J = 7.4 Hz, 2H), 8.04 (d,
J = 6.9 Hz, 1H). 13C NMR (CDCl3): δ 18.18, 20.32, 47.47, 49.03, 60.34,
1
(0.13 g, 33%) as a thick colorless oil. H NMR (DMSO-d6): δ 0.85
(d, J = 3.3 Hz, 3H), 0.87 (d, J = 3.0 Hz, 3H), 1.03 (d, J = 5.8 Hz, 3H), 1.36
(s, 18H), 1.79 (br-s, 1H), 1.96-2.07 (m, 3H), 2.66 (s, 3H), 2.68-2.76
(m, 3H), 4.02 (t, J = 6.6 Hz, 2H), 4.27-4.40 (m, 4H), 4.50-4.65
1342
dx.doi.org/10.1021/jm1013924 |J. Med. Chem. 2011, 54, 1333–1346