Linear TMC-95-Based Proteasome Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 12 2847
substituent at R1 position (NHBn, NHC6H4OH) or its total
absence with a free carboxylic C-end, (b) an orientation toward
T-L activity with lysine and arginine lateral chains at position
R2 or even at position R1, (c) a possible orientation toward
specific inhibition of PA activity. They are significant inhibitors
in vitro and in cell tumor lines and are metabolically stable.
These versatile compounds open a new avenue for the develop-
ment of new low molecular weight and noncovalently binding
proteasome inhibitors that act selectively on one or more of
the three proteasome activities.
pressure over 10% Pd on charcoal (9.14 mg) for 18 h. The mixture
was filtered over a pad of Celite and concentrated. After the mixture
was washed with ether, the product was afforded as a white solid
1
(46.55 mg, 81%). H NMR (300 MHz, DMSO-d6) δ 1.09-1.63
(m, 4H, (CH2)2 Arg), 1.30 (s, 9 H, (CH3)3), 2.62-3.14 (m, 6H),
4.05 (m, 1H, CHR), 4.23 (m, 3H, CH2 Ph and CHR), 4.56 (m, 1H,
CHR), 6.60 (m, 2H, aromatic CH), 6.96-7.58 (m, 11H, aromatic
H), 7.96 (m, 1H, aromatic H). 13C NMR (75 MHz, CD3OD) δ 25.9
(CH2 γ Lys), 28.7 (CH3 Boc), 28.9, 29.9(CH2 Trp or CH2 â Lys),
38.1 (CH2 Tyr), 41.9 (CH2N Lys), 44.2 (CH2NPh), 54.3, 55.9, 57.9
(CH R Lys or Trp or Tyr), 80.9 (C Boc), 110.7 (Car), 112.4, 118.0,
119.4, 119.9, 122.5, 124.6 (CH ar), 126.0 (Car), 128.1, 128.4 (CH
ar), 128.8 (Car), 129.4, 131.3 (CH ar), 138.0, 139.4 (Car), 157.9,
158.6, 161.5 (CdN or CarO or NCOO), 173.3, 173.8, 175.0
(CONH). HRMS (ESI) calcd for C38H49N8O6 [(M + H)+] 713.3775,
found 713.3778. HPLC homogeneity: >95% (system A, tR ) 10.1);
>95% (system B, tR ) 13.8).
Experimental Section
Chemistry. The syntheses of the more active compounds are
indicated below. For description of general methods and preparation
of the other compounds, see Supporting Information.
N-Boc-Tyr(Bn,3-iodo)-Ala-Trp(7-bromo)-OH (3h). A solution
of N-Boc-Tyr(Bn,3-iodo)-Ala-Trp(7-bromo)-OMe 3c (85.5 mg,
0.100 mmol) in THF (0.4 mL) was treated at 0 °C by 1 M aqueous
NaOH (0.12 mL, 0.12 mmol). After 5 h at room temperature, 1 M
aqueous HCl was added (0.36 mL, 0.36 mmol). The resulting
mixture was diluted by water and extracted by CH2Cl2 (3 × 10
mL). After drying of the organic phase over Na2SO4 and evaporation
of the solvent, the residue was subjected to flash chromatography
on silica gel (2% MeOH/CH2Cl2) to afford remaining N-Boc-Tyr-
(Bn, 3-iodo)-Ala-Trp(7-bromo)-OMe 3c (10.13 mg, 12%) and
N-Boc-Tyr(Bn, 3-iodo)-Ala-Trp(7-bromo)-OH 3h (58.3 mg, 70%,
N-Boc-Tyr(Bn)-Ala-Trp-NH(4-OH)Ph (4q). To a solution of
N-Boc-Tyr(Bn)-Ala-OH (106.4 mg, 0.24 mmol) in CH2Cl2/DMF
(1.5 mL, 1/1) at 0 °C were successively added EDC (53.8 mg, 0.28
mmol), HOBt (37.9 mg, 0.28 mmol), and Trp-NHPhOH 6g (75
mg, 0.254 mmol). The resulting mixture was allowed to warm to
room temperature overnight. The solvent was evaporated, and the
crude was triturated with water. After filtration, the solid was
collected and washed with Et2O to afford a pale-brown solid (72.8
mg, 42%). 1H NMR (300 MHz, DMSO-d6) δ 1.21 (d, J ) 6.7 Hz,
3H, CH3), 1.28 (s, 9H, (CH3)3), 2.62-3.21 (m, 4H, CH2 Tyr, CH2
Trp), 4.1 (m, 1H, CHR), 4.32 (m, 1H, CHR), 4.64 (m, 1H, CHR),
5.02 (s, 2H, CH2 (Bn)), 6.66 (d, J ) 8.4 Hz, 1H), 6.87-7.39 (m,
17H), 7.6 (d, J ) 7 Hz, 1H), 8 (d, J ) 6.9 Hz, 1H), 8.1 (d, J ) 7.1
Hz, 1H), 9.18 (s, 1 NH), 9.74 (s, 1 NH), 10.8 (s, 1H, NH). 13C
NMR (75 MHz, DMSO-d6) δ 18.2 (CH3), 27.8 (CH2 Trp), 28
((CH3)3), 36.2 (CH2 Tyr), 48.1, 53.9, 55.7 (CHR Ala, Tyr, Trp),
69 (CH2 (Bn)), 78 (C(CH3)3), 109.6, 111.1, 114.2, 114.9, 118.1,
118.4, 120.7, 121, 123.4, 127.2, 127.5, 127.6, 128.3, 130.1, 130.2,
130.3, 135.9, 137.1 (24 aromatic C), 153.3, 155.2, 156.7 (2 Car-O,
CO carbamate), 169.2, 171.4, 171.9 (3 CO amide). HRMS (ESI)
calcd for C41H45N5O7Na [(M + Na)+] 742.3217, found 742.3223.
Anal. (C41H45N5O7‚2H2O) C, H, N.
N-Boc-Tyr-Arg-Trp-NH(4-OH)Ph (4v). The same procedure
as for 4l was used, using N-Boc-Tyr(Bn)-Arg(Z2)-Trp-NH(4-OH)-
Ph 4u (60.81 mg, 0.0567 mmol) and 10% Pd on charcoal (6.43
mg) in MeOH/DMF (0.8 mL, 7/1) and affording after washing with
ether N-Boc-Tyr-Arg-Trp-NH(4-OH)Ph as a white solid (34.89 mg,
86%). 1H NMR (300 MHz, DMSO-d6) δ 1.23-1.65 (m, 4H, (CH2)2
Arg), 1.31 (s, 9 H, (CH3)3), 2.73-3.17 (m, 6H), 4.09 (m, 1H, CHR),
4.28 (m, 1H, CHR), 4.63 (m, 1H, CHR), 6.59 (m, 3H, aromatic
CH), 6.92-7.31 (m, 7H, aromatic H), 7.57 (d, J ) 7.6 Hz, 1H,
aromatic H). 13C NMR (75 MHz, CD3OD) δ 26.1 (CH2 γ Lys),
28.7 (CH3 Boc), 26.7, 28.9 (CH2 Trp or CH2 â Lys), 34.8 (CH2
Tyr), 41.9 (CH2N Lys), 53.7, 56.4, 57.6 (CH R Lys or Trp or Tyr),
80.8 (C Boc), 110.6 (Car), 112.3, 116.9 (CH ar), 117.2 (C ar), 119.4,
119.9, 122.4, 124.1, 124.6 (CH ar), 128.7 (Car), 131.3 (CH ar),
138.0 (Car), 157.8, 158.4 (CdN or CarO or NCOO), 172.2, 173.3,
174.7 (CONH). HRMS (ESI) calcd for C37H47N8O7 [(M + H)+]
715.3568, found 715.3572. HPLC homogeneity: >95% (system
A, tR ) 9.8); >95% (system B, tR ) 13.2).
1
corrected yield 82%) as a white amorphous solid. H NMR (200
MHz, CDCl3) δ 0.9 (d, J ) 6.4 Hz, 3H, CH3), 1.42 (s, 9H, (CH3)3),
2.87 (m, CH2 Tyr), 3.32 (m, 2H, CH2 Trp), 4.40 (m, 1H, CH Tyr),
4.52 (m, 1H CH Ala), 4.85 (m, 1H, CH Trp), 5.06 (s, 2H, CH2O),
5.16 (broad d, J ) 7.6 Hz, 1H, NHBoc), 6.71 (d, J ) 8 Hz, 1H,
H5 Tyr), 6.91 - 7.58 (m, 14 H, aromatic H and NH), 8.68 (s, 1H,
NHind). 13C NMR (75 MHz, acetone-d6) δ 19.4 (CH3 Ala), 29.1
(CH2 Trp), 29.5 ((CH3)3), 38.3 (CH2 Tyr), 50.4 (CH Ala), 54.7
(CH Trp), 57.4 (CH Tyr), 72.3 (CH2O), 80.5 (C(CH3)3), 87.6 (C3
Tyr), 106.1 (C7 Trp), 114.3 (CH ar), 120.0 (CH ar), 120.7 (Car),
122.0 (CH ar), 125.6 (CH ar), 126.7 (Car), 129.0 (CH ar), 129.5
(CH ar), 130.2 (CH ar), 131.3 (Car), 132.7 (CH ar), 134.2 (Car),
136.5 (Car), 138.9 (Car), 142.0 (CH ar), 157.3, 157.8 (C4 Tyr or
CO Boc), 172.9, 173.7, 174.1 (2 CO amide, CO acid). HRMS (ESI)
calcd for C35H3879BrIN4O7Na [(M + Na)+] 855.0866, found
855.0896. Anal. (C35H38BrIN4O7‚2H2O) C, H. N: calcd, 6.44;
found, 5.73. HPLC homogeneity: >97% (system A, tR )13);
>99% (system B, tR ) 17.8).
N-Boc-Tyr-Ala-Trp-OMe (4b). N-Boc-Tyr(Bn)-Ala-Trp-OMe
4a (119 mg, 0.185 mmol) in solution in MeOH (4 mL) was
hydrogenated overnight at atmospheric pressure in the presence of
10% Pd on charcoal (30 mg). After filtration, evaporation of the
solvent, and trituration with ether, N-Boc-Tyr-Ala-Trp-OMe (77
1
mg, 76%) was obtained as a solid. H NMR (300 MHz, DMSO-
d6) δ 1.19 (d, J ) 7 Hz, 3H, CH3), 1.29 (s, 9H, (CH3)3), 2.55 and
2.80 (ABX system, 2H, CH2â), 3.13 and 3.16 (ABX system, 2H,
CH2 â), 3.55 (s, 3H, OCH3), 4.05 (m, 1H, CHR), 4.35 (m, 1H,
CHR), 4.50 (m, 1H, CHR), 6.63 (d, J ) 8.3 Hz, 2H), 6.83 (d, J )
8.6 Hz, 1H), 7.03 (m, 4H), 7.16 (d, J ) 2.1 Hz, 1H), 7.34 (d, J )
7.9 Hz, 1H), 7.47 (d, J ) 7.8 Hz, 1H), 7.92 (d, J ) 7.4 Hz, 1H,
NH), 8.32 (d, J ) 7.3 Hz, 1H, NH), 9.14 (s, 1H, OH), 10.86
(s, 1NH). 13C NMR (75 MHz, DMSO-d6) δ 18.4 (CH3), 26.9
(CH2 Trp), 28.1 ((CH3)3), 36.4 (CH2 Tyr), 47.8, 51.7, 53.0, 55.9
(CHR Ala, Tyr, Trp or OCH3), 77.9 (C(CH3)3), 109.1 (aromatic
C), 111.4, 114.8, 117.9, 118.4, 120.9, 123.7 (aromatic CH), 127.0,
128.2 (aromatic C), 130.0 (aromatic CH), 136.0 (aromatic C), 155.2,
156.6 (Car-O or CO carbamate), 171.4, 172.0, 172.2 (2 CO
amide or CO ester). HRMS (ESI) calcd for C29H36N4O7Na [(M +
Na)+] 575.2482, found 575.2480. Anal. (C29H36N4O7‚H2O) C,
H, N.
Tyr(Bn)-Lys-Trp-NHCH2Ph (4aa). N-Boc-Tyr(Bn)-Lys(Boc)-
Trp-NHCH2Ph‚2H2O (80.23 mg, 0.0881 mmol) was reacted with
3 M anhydrous HCl in MeOH (1.5 m L) overnight. After solvent
evaporation and washing of the residue with ether, a solid was
obtained (33.3 mg). Purification of this crude product by MPLC
over RP silica (C18, 3 g) afforded after lyophilization Tyr(Bn)-
Lys-Trp-NHCH2Ph‚ 3HCl‚2H2O as a white solid (17 mg, 24%).
1H NMR (300 MHz, CD3OD) δ 1.30 (m, 2H, CH2 Lys), 1.49-
1.72 (m, 4H, CH2 Lys), 2.73-2.83 (m, 3H, CH of ABX system
and CH2 Lys), 2.95-3.21 (m, 3H, CH of ABX system and CH2),
4.00 (dd, J ) 7.5 Hz, J ) 6.0 Hz, 1H, CHR), 4.11 and 4.20 (AB
system, J ) 15 Hz, 2H, NCH2Ph), 4.29 (t, J ) 7 Hz, 1H, CHR
Lys), 4.58 (t, J ) 7.2 Hz, 1H, CHR), 4.89 (m, 2H, CH2 (Bn)), 6.80
(d, J ) 8.6 Hz, 2H), 6.89-7.30 (m, 15H), 7.54 (d, J ) 7.6 Hz,
N-Boc-Tyr-Arg-Trp-NHCH2Ph (4l). A solution of N-Boc-Tyr-
(Bn)-Arg(Z2)-Trp-NHCH2Ph 4k (86.15 mg, 0.0804 mmol) in
MeOH/DMF (1.4 mL, 1/0.4) was hydrogenated at atmospheric