Journal of Medicinal Chemistry
Article
The resulting mixture was stirred overnight at rt after which the
solvents were evaporated, and the residue was taken up in EtOAc (100
mL). The organic layer was washed with KHSO4 (1 M, 100 mL, 3×)
and brine (100 mL). The organic layer was dried over Na2SO4, and the
solvents were evaporated. The product was purified using solid-phase
extraction, using a C8 reversed phase column [eluting first with 1
column volume buffer A (0.1% TFA in CH3CN/H2O, 5/95, v/v),
followed by elution using buffer B (0.1% TFA in CH3CN/H2O, 95/5,
488.24 [M − Boc + H]+, 975.10 [2M − Boc + H]+. HRMS: calcd for
C28H47FN3O7S [M + H] 588.3119, found 588.3130.
Boc-Ile1-Ile2-Thr(Bn)-Leu-ψ[CH2SO2]-F (17). The coupling reaction
was carried out starting from Boc-Ile-OH·1/2H2O (130 mg, 0.54
mmol) and sulfonyl fluoride 16 (319 mg, 0.54 mmol), which was first
Boc-deprotected. The resulting product was purified by column
chromatography (acetone:CH2Cl2, 6:94). Due to the low yield
obtained, column chromatography was performed again on the
impure fractions. Now an eluent gradient was used, acetone:CH2Cl2
(4:96) followed by acetone:CH2Cl2 (6:94). The combined pure
fractions were concentrated in vacuo to obtain 17 as a white solid (279
mg, 74%). HPLC retention time: 27.1 min. Rf = 0.56 (aceto-
1
v/v)] (14.3 mg, 25 μmol, 72%). H NMR (300 MHz, DMSO): δ =
0.86 [m, 24H, 8 × CH3 (4 × Leu)], 1.25−1.62 [m, 12H, 4 ×
CH2CH(CH3)2, 4 × CH2CH(CH3)2], 1.85 [s, 3H, CH3C(O)], 3.88
(m, 1H, CHaSO2F), 4.11 (m, 1H, CHbSO2F), 4.30 (m, 4H, 4 ×
NHCH), 7.94 (m, 4H, 4 × NHCH). 19F NMR (282 MHz, DMSO): δ
= −120.1. ESI MS: m/z = 565.55 [M + H]+; 587.40 [M+Na]+; 603.15
[M+K]+. HRMS: calcd for C26H50FN4O6S [M + H] 565.3435, found
565.3440.
N3Phe-Leu-Leu-Leu-ψ-[CH2SO2]-F (14). The deprotection and
coupling reactions were performed starting from peptido sulfonyl
fluoride 10 (48.1 mg, 82 μmol) and azido phenylalanine (13)14 (15.6
mg, 82 μmol). The purification by silica gel column chromatography
was done with EtOAc:hexanes (1:1) as eluent. The product was
obtained as a white solid (28.3 mg, 48 μmol, 59%). HPLC retention
time: 25.7 min. 1H NMR (500 MHz, CDCl3): δ = 0.86 [m, 18H, 6 ×
CH3 (3 × Leu)], 1.26−1.80 [m, 9H, 3 × CH2CH(CH3)2, 3 ×
CH2CH(CH3)2], 3.06 (dd, Jgem = 14.2 Hz, Jvic = 8.0 Hz, 1H, CHa-
C6H5), 3.26 (dd, Jgem = 14.0 Hz, Jvic = 3.9 Hz, 1H, CHb−C6H5), 3.58
(m, 1 H CHaSO2F), 3.77 (m, 1 H CHbSO2F), 4.26 [m, 1H, NHCH
(Leu1)], 4.34 (br s, CHN3), 4.46 [m, 2H, 2 × NHCH (Leu1,2)], 6.95
(m, 2H, 2 × NH (Leu2,3), 7.11 [d, J = 8.0 Hz, 1H, NH (Leu1)], 7.28
(m, 5H, ArCH). 19F NMR (282 MHz, DMSO): δ = −115.5. IR
(KBr): 2108 cm−1 (N3). HRMS: calcd for C27H44FN6O5S [M + H]
583.3078, found 583.3079.
Boc-Thr(Bn)-Leu-ψ[CH2SO2]-F (15). The coupling reaction was
performed using HCl salt 33 (330 mg, 1.5 mmol) and Boc-Thr(Bn)−
OH (464 mg, 1.5 mmol). The product was purified by precipitation
and crystallization instead of column chromatography. After extraction
and concentration in vacuo, the crude product was dissolved in
CH2Cl2, which was followed by addition of hexanes. After filtration,
the residue was crystallized from CH2Cl2 (50 mL) and hexanes (50
mL), which afforded 15 as a white solid (573 mg, 81%, two steps).
HPLC retention time: 25.7 min. Rf = 0.52 (acetone:CH2Cl2, 4:96).
Mp = 158 °C. 1H NMR (300 MHz, CDCl3): δ = 0.86 [2d, J = 3.03 Hz,
6H, CH(CH3)2], 1.21 [d, J = 6.05 Hz, 3H, CH3 (Thr)], 1.46 [s, 9H,
(CH3)3], 1.48−1.79 [m, 3H, CH2CH(CH3)2], 3.55−3.71 (m, 2H,
CH2SO2F), 4.21 (m, 2H, NCHCHCH3), 4.46−4.63 [m, 3H, NCH
(Leu), CH2C6H5], 5.37 (br s, 1H, NHBoc), 6.87 [br s, 1H, NH
(Leu)], 7.26−7.34 (m, 5H, Ar−CH). 19F NMR (282 MHz, CDCl3): δ
= −114.8 (s). ESI MS: m/z = 375.15 [M − Boc + H]+, 748.95 [2M −
Boc + H]+. HRMS: calcd for C22H36FN2O6S [M + H] 475.2278,
found 475.2277.
1
ne:CH2Cl2, 10:90). H NMR (300 MHz, CDCl3): δ = 0.82−1.00 [m,
20H, CH(CH3)2, 4 × CH3 (Ile1,2), 2 × CH3CHaCH (Ile1,2)], 1.14−
1.26 [m, 5H, 2 × CH3CHbCH (Ile1,2), CH3 (Thr)], 1.42 [s, 9H,
(CH3)3], 1.46−1.77 [m, 3H, CH2CH(CH3)2], 1.90 [m, 1H, NCHCH
(Ile1)], 2.05 [m, 1H, NCHCH (Ile2)], 3.46−3.55 (ddd, Jgem = 14.9 Hz,
3
a
Jvic = 6.2 Hz, JH F = 5.6 Hz, 1H, CHaSO2F), 3.71−3.79 (ddd, Jgem
=
3
b
14.9 Hz, Jvic = 5.4 Hz, JH F = 2.8 Hz, 1H, CHbSO2F), 3.78 [m, 1H,
NCH (Ile1)], 4.15−4.19 [m, 1H, NCH (Ile2)], 4.33 [m, 1H, NCHCH
(Thr)], 4.42−4.58 [m, 4H, NCH (Thr), NCH (Leu), CH2C6H5], 4.80
(d, J = 5.8 Hz, 1H, NHBoc), 6.64 [s, 1H, NH (Ile2)], 6.86 [d, J = 8.0
Hz, 1H, NH (Thr)], 7.14 [d, J = 7.4 Hz, 1H, NH (Leu)], 7.25−7.30
(m, 5H, Ar−CH). 19F NMR (282 MHz, CDCl3): δ = −117.5 (s). ESI
MS: m/z = 601.38 [M − Boc + H]+, 623.44 [M − Boc + Na]+,
1201.19 [2M − Boc + H]+, 1223.10 [2M − Boc + Na]+. HRMS: calcd
for C34H58FN4O8S [M + H] 701.3959, found 701.3969.
Ac-Ile1-Ile2-Thr(Bn)-Leu-ψ[CH2SO2]-F (18). To a stirring suspension
of the TFA salt of compound 17 (174 mg, 0.25 mmol) in CH2Cl2 (2.5
mL) was added a solution of acetic anhydride in CH2Cl2 (10% v/v,
260 μL, 0.275 mmol) under N2 atmosphere. After cooling to 0 °C,
triethylamine (104 μL, 0.75 mmol) was added dropwise. In order to
improve the solubility, DMF (60 mL) was added. Additional acetic
anhydride (52 μL, 0.55 mmol) and triethylamine (104 μL, 0.75 mmol)
were added. The solution was stirred for 18 h at rt under N2
atmosphere. The solvent was partially removed by evaporation. The
remaining solution (∼5 mL) was added to diethyl ether (∼40 mL),
which led to precipitation of the product. After centrifugation (10 min,
5000 rpm), the pellet was washed with diethyl ether (3×) and air-dried
to yield 18 as a white solid (125 mg, 78%). HPLC retention time: 24.4
min. 1H NMR (500 MHz, DMSO): δ = 0.75−0.82 (m, 18H,
CH(CH3)2, 4 × CH3 (Ile1,2), 1.02−1.10 [m, 5H, 2 × CH3CHaCH
(Ile1,2), CH3 (Thr)], 1.30−1.35 [m, 1H, NCHCHa (Leu)], 1.38−1.47
[m, 2H, 2 × CH3CHbCH (Ile1,2)], 1.48−1.54 [m, 1H, NCHCHb
(Leu)], 1.58−1.61 [m, 1H, CH2CH(CH3)2], 1.65−1.76 [m, 2H, 2 ×
NCHCH (Ile1,2)], 1.84 (s, 3H, CH3CO), 3.78−3.83 (m, 1H,
CHaSO2F), 3.93 [m, 1H, NCHCH (Thr)], 4.08−4.13 (ddd, Jgem
=
b
15.0 Hz, Jvic = 7.3 Hz, 3JH F = 4.3 Hz, 1H, CHbSO2F), 4.20−4.29 [2t, J
= 8.2 Hz, 2H, 2 × NCH (Ile1,2)], 4.37−4.52 [m, 4H, NCH (Thr),
NCH (Leu), CH2C6H5], 7.25−7.33 (m, 5H, Ar−CH), 7.79 [d, J = 9.2
Hz, 1H, NH (Thr)], 7.89−7.96 [m, 3H, NH (Leu), 2 × NH (Ile1,2)].
19F NMR (282 MHz, DMSO): δ = −119.8 (s). HRMS: calcd for
C31H52FN4O7S [M + H] 643.3541, found 643.3538.
Boc-Ile-Thr(Bn)-Leu-ψ[CH2SO2]-F (16). The coupling reaction was
carried out starting from Boc-Ile-OH·1/2H2O (233 mg, 0.97 mmol)
and sulfonyl fluoride 15 (462 mg, 0.97 mmol), which was first Boc-
deprotected. The resulting product was purified by column
chromatography using an eluent gradient in three steps, first
CH2Cl2, second acetone:CH2Cl2 (1:99), and last acetone:CH2Cl2
(50:50). Concentration in vacuo afforded 16 as a white solid (409
mg, 72%). HPLC retention time: 26.5 min. Rf = 0.61 (aceto-
Ac-Ile1-Ile2-Thr-Leu-ψ[CH2SO2]-F (19). To a solution of Ac-Ile-Ile-
Thr-Leu-ψ[CH2SO2]-F (18) (50 mg, 77.8 μmol) in CH2Cl2 (10 mL)
was added a solution of HBr in acetic acid (33%, 3.0 mL). After
stirring for 2 h at rt, the mixture was concentrated in vacuo.
Preparative HPLC was used for purification. The crude material was
dissolved in a minimal amount of buffer A and loaded onto a C8
Alltima column. The product was eluted with a flow rate of 11.5 mL/
min using a linear gradient to 100% buffer B (0.1% TFA in CH3CN/
H2O, 95/5, v/v) in 85 min from 100% buffer A (0.1% TFA in
CH3CN/H2O, 5/95, v/v). Fractions of 4.6 mL were taken.
Epoxomicin-derived sulfonyl fluoride 19 was obtained as a white
1
ne:CH2Cl2, 10:90). H NMR (300 MHz, CDCl3): δ = 0.85−1.00 [m,
13H, CH(CH3)2, 2 × CH3 (Ile), CH3CHaCH (Ile)], 1.10−1.29 [m,
4H, CH3CHbCH (Ile), CH3 (Thr)], 1.36 [s, 9H, (CH3)3], 1.43−1.74
[m, 3H, CH2CH(CH3)2], 1.92−2.17 [m, 1H, NCHCH (Ile)], 3.48−
a
3.57 (ddd, Jgem = 14.9 Hz, Jvic = 6.2 Hz, 3JH F = 5.5 Hz, 1H, CHaSO2F),
3
b
1
3.70−3.78 (ddd, Jgem = 14.9 Hz, Jvic = 5.5 Hz, JH F = 2.6 Hz, 1H,
CHbSO2F), 3.92−3.96 [t, J = 5.0 Hz, 1H, NCH (Ile)], 4.32 [m, 1H,
NCHCH (Thr)], 4.40−4.87 [m, 4H, NCH (Thr), NCH (Leu),
CH2C6H5], 4.86 (d, J = 5.0 Hz, 1H, NHBoc), 6.86, 7.20 [2d, J = 7.7
Hz, J = 7.2 Hz, 2H, NH (Leu), NH (Thr)], 7.23−7.36 (m, 5H, Ar−
CH). 19F NMR (282 MHz, CDCl3): δ = −117.5 (s). ESI MS: m/z =
solid (15 mg, 34%). HPLC retention time: 30.5 min. H NMR (500
MHz, DMSO): δ = 0.79−0.85 [m, 18H, CH(CH3)2, 4 × CH3 (Ile1,2)],
1.00−1.07 [m, 5H, 2 × CH3CHaCH (Ile1,2), CH3 (Thr)], 1.33 [m, 1H,
NCHCHa (Leu)], 1.42 [m, 2H, 2 × CH3CHbCH (Ile1,2)], 1.53 [m,
1H, NCHCHb (Leu)], 1.64−1.75 [m, 3H, CH2CH(CH3)2, 2 ×
NCHCH (Ile1,2)], 1.85 (s, 3H, CH3CO), 3.85−3.93 [m, 2H,
G
dx.doi.org/10.1021/jm301443r | J. Med. Chem. XXXX, XXX, XXX−XXX