N. Perlman et al. / Bioorg. Med. Chem. 16 (2008) 9032–9039
9037
10.8 Hz, 1H, CHCH2Ph); 2.89 (d, J = 5.9 Hz, 2H, CH2CO); 2.97 (dd,
J = 13.6, 4.2 Hz, 1H, CHCH2Ph); 3.61 (s, 3H, OCH3); 3.64–3.70 (m,
2H, NCH2); 4.22 (td, J = 10.3, 4.3 Hz, 1H, NHCHCH2); 4.24 (quintet,
J = 7.1 Hz, 1H, CH(Ala2)); 4.31 (quintet, J = 7.3 Hz, 1H, CH(Ala1));
4.92 (d, J = 12.8 Hz, 1H, PhCH2O); 4.95 (d, J = 12.8 Hz, 1H, PhCH2O);
5.45 (dt, J = 15.4, 5.7 Hz, 1H, CH@); 5.60 (dt, J = 15.5, 7.3 Hz, 1H,
@CH); 7.18–7.34 (m, 10 H, Ph); 7.52 (d, J = 8.7 1H, NH(Phe)); 8.08
(d, J = 7.6 Hz, 1H, NH(Ala1)); 8.26 (t, J = 5.5 Hz, 1H, NHCH2); 8.40 (d,
J = 6.8 Hz, 1H, NH(Ala2)); 13C NMR: d 16.81 (CH3(Ala2)); 18.30
(CH3(Ala1)); 37.9 (CHCH2Ph); 39.5 (CH2CO); 40.4 (NCH2); 47.48
(CH(Ala2)); 47.62 (CH(Ala1)); 51.84 (OCH3); 56.27 (NCHCH2);
65.15 (PhCH2O); 125.44, 129.13 (CH@CH); 126.21, 127.43,
127.65, 128.01, 128.27, 129.22, 137.04, 138.13 (Ph); 155.77
(OCON); 169.65, 171.14, 172.32, 172.96 (CON, CO2); HRMS: m/z
553.2680 (MH+. C29H37N4O7 requires 553.2660).
(CDCl3): d 1.36 (m, 6H, CHCH3 (diasteriomers 1+2)); 2.36 (dd,
J = 15, 6 Hz, 1H, CH2CO (1)); 2.39 (dd, J = 15, 5.6 Hz, 1H, CH2CO
(2)); 2.51 (d, J = 13.6 Hz, 1H, CH2CO (1)); 2.56 (d, J = 15.0 Hz, 1H,
CH2CO (2)); 2.85 (br s, 1H, NCH2CHO (2)); 2.89 (br s, 1H, NCH2CHO
(1)); 2.97 (m, 1H, OCHCH2CO (1)); 3.00 (m, 1H, CHCH2Ph (1+2));
3.07 (m, OCHCH2CO (2), CHCH2Ph (1+2)); 3.23 (m, 1H, NCH2 (2));
3.40 (m, 2H, NCH2 (1)); 3.51 (m, 1H, NCH2 (2)); 3.70 (s, 3H, OCH3
(1+2)); 4.49–4.62 (m, 3H, 3ꢂ NHCH (1+2)); 4.98 (d, J = 12.4 Hz,
1H, PhCH2O (1+2)); 5.05 (d, J = 12.4 Hz, 1H, PhCH2O (1+2)); 6.03
(m, 1H, NH); 7.18–7.39 (m, 13H, Ph+3ꢂ NH (1+2)); 13C NMR: d
17.74, 17.79, 18.38 (CHCH3); 38.38, 38.45 (CH2CO); 38.70, 38.76
(CHCH2Ph); 40.15, 40.26 (NCH2); 48.19, 48.84, 48.90 (NHCH);
52.48 (OCH3); 53.03, 53.11 (CHOCH2CO); 56.22, 56.26 (NHCH);
56.32, 56.41 (NCH2CHO); 66.93 (PhCH2O); 126.93, 127.84,
128.19, 128.50, 128.55, 129.31, 129.73, 130.08, 136.18, 136.51
(Ph); 156.22 (OCON) 168.03, 169.53, 169.59, 172.16, 172.48,
173.17, 173.23 (CO2+CON); HRMS: m/z 569.2581 (MH+.
4.2.5. Epoxidation
‘Pentaeptide’ (0.125 mmol), m-CPBA 50% (0.218 mmol), K2HPO4
C29H37N4O8 requires 569.2611).
(0.181 mmol) were stirred in CH2Cl2 (10 ml) and H2O (10 ll) over
night. EtOAc was added (60 ml) and the solution was washed suc-
cessively with saturated NaHCO3, 5% Na2SO3, saturated NaHCO3,
water and brine (30 ml each), and dried over MgSO4. Evaporation
to dryness afforded the clean product almost quantitatively. The
trans isomer was purified by chromatography (47:1:2 ethyl ace-
tate/CHCl3/MeOH). A low 27% yield is due to decomposition on
the column. Thus, the cis product was not purified by chromatog-
raphy (93% yield).
4.2.6. Cbz-Phe-Gly-epoxide
The exo dipeptidyl epoxide was synthesized from the protected
dipeptide Cbz-Phe-GlyOH via the corresponding bromoketone as
previously described.14
4.2.6.1. PhCH2OCONHCH(CH2Ph)CONHCH2COCH2Br (Cbz-Phe-
GlyCH2Br) (5).
1H NMR (CDCl3): d 2.98 (dd, J = 13.5, 7.9 Hz,
1H, CHCH2Ph); 3.10 (dd, J = 13.8, 5.5 Hz, 1H, CHCH2Ph); 3.81 (s,
2H, CH2Br); 4.13 (dd, J = 19.0, 5.0 Hz, 1H, NHCH2); 4.21 (dd,
J = 19.0, 5.0 Hz, 1H, NHCH2); 4.59 (q, J = 7.3 Hz,1H,CH); 4.97 (d,
J = 12.3 Hz, 1H, CH2O); 5.03 (d, J = 12.3 Hz, 1H, CH2O); 5.87 (d,
J = 8.3 Hz, 1H, NH(phe)); 7.13–7.3 (m, 11H, NH(Gly), Ph); 13C
NMR: d 31.57 (CH2Br); 38.34 (CHCH2Ph); 46.81 (NHCH2); 55.94
(CH); 66.91 (CH2O); 126.90, 127.76, 128.05, 128.41, 128.51,
129.15, 136.00, 136.25 (Ph); 156.07 (OCON); 171.78 (CON);
197.47 (CO); HRMS: m/z 433.0770 (MH+. C20H22N2O4Br requires
433.0763).
4.2.5.1. cis PhCH2OCONHCH(CH2Ph)CONHCH2CHOCHCH2CON-
HCH(CH3)CONHCH(CH3)CO2CH3 (cis Cbz-Phe-Gly-epoxy-Gly-Ala-
Ala-OCH3) (4).
Diasteriomeric ratio (1:0.6). 1H NMR (CD3CN): d
1.29 (d, J = 7.2 Hz, 3 H, CHCH3 (major)); 1.30 (d, J = 7.2 Hz, 3H, CHCH3
(minor)); 1.31 (d, J = 7.4 Hz, 3H, CHCH3 (minor)); 1.32 (d, J = 7.2 Hz,
3H, CHCH3 (major)); 2.37 (dd, J = 15.3, 7.5 Hz, 1H, CH2CO (minor));
2.43 (dd, J = 15.3, 6.4 Hz, 1H, CH2CO (major)); 2.54 (dd, J = 15.3,
6.4 Hz, 1H, CH2CO (major)); 2.59 (dd, J = 15.1, 5.9 Hz, 1H, CH2CO
(minor)); 2.86 (dd, J = 13.7, 9.2 Hz, 1H, CHCH2Ph (major+minor));
2.90 (m, 1H, NCH2CHO (minor)); 2.95 (q, J = 4.3 Hz, 1H, NCH2CHO
(major)); 3.12 (m, CHCH2Ph (major+minor)+NCH2 (minor)); 3.20
(m, NCH2 (major)+OCHCH2CO(major+minor)); 3.48 (ddd, J = 14.2,
6.5, 5.5 Hz, 1H, NCH2 (major)); 3.58 (ddd, J = 13.8, 6.8, 5 Hz, 1H,
NCH2 (minor)); 3.65 (s, 3H, OCH3 (minor)); 3.66 (s, 3H, OCH3 (ma-
jor)); 4.33 (m, 1H, NHCHCH2); 4.37 (quintet, J = 7.3 Hz, 1H, CHCH3);
4.38 (quintet, J = 7.3 Hz, 1H, CHCH3); 4.97 (d, J = 13 Hz, 1H, PhCH2O
(minor)); 4.97 (d, J = 12.1 Hz, 1H, PhCH2O (major)); 5.04 (d,
J = 12.6 Hz, 1H, PhCH2O (minor)); 5.04 (d, J = 12.7 Hz, 1H, PhCH2O
(major)); 5.95 (d, J = 7.9 Hz, 1H, NHCHCH2 (major)); 6.03 (d,
J = 8.4 Hz, 1H, NHCHCH2 (minor)); 6.92 (d, J = 5.9 Hz, 1H, NHCHCH3
(major+minor)); 6.96 (bd, J = 5 Hz, 1H, NHCHCH3 (major)); 7.02
(bd, J = 6 Hz, 1H, NHCHCH3 (minor)); 7.22–7.35 (m, 11H, Ph+NHCH2
(major+minor)); 13C NMR: d 17.69, 17.74, 18.27, 18.29 (CHCH3);
35.56 (CH2CO (minor)); 35.70 (CH2CO (major)); 38.25 (NCH2 (min-
or)); 38.48 (NCH2 (major)); 38.90 (CHCH2Ph); 49.04 (CHCH3 (ma-
jor)); 49.08, 49.67 (CHCH3 (minor)); 49.74 (CHCH3 (major)); 52.79
(OCH3 (major)); 52.82 (OCH3 (minor)); 54.00 (CHOCH2CO (major));
54.06 (CHOCH2CO (minor)); 54.91 (NCH2CHO (minor)); 55.13
(NCH2CHO (major)); 57.45 (NCHCH2 (minor)); 57.53 (NCHCH2 (ma-
jor)); 67.53 (PhCH2O); 127.64, 128.55, 128.86, 129.35, 129.37,
129.45, 130.31, 130.33, 138.53 (Ph); 156.95 (OCON) 170.35 (CO2
(minor)); 170.45 (CO2 (major)); 172.47 (CON (minor)); 172.54,
173.09 (CON (major)); 174.93 (CON (minor)); HRMS: m/z
569.2637 (MH+. C29H37N4O8 requires 569.2611).
4.2.6.2. PhCH2OCONHCH(CH2Ph)CONHCH2CHOCH2.
(Cbz-Phe-
Gly-epoxide) (6).
1H NMR (CDCl3): d 2.28 (dd, J = 4.6, 2.7 Hz,
1H, CH2O (minor)); 2.46 (dd, J = 4.1, 2.6 Hz, 1H, CH2O (major));
2.62 (t, J = 4.1 Hz, 1H, CH2O (minor)); 2.67 (t, J = 4.0 Hz, 1H, CH2O
(major)); 2.90 (m, 1 H, CHO (major)); 2.96 (m, 1H, CHO (minor));
3.05 (d, J = 6.5 Hz, 2H, CHCH2Ph); 3.20 (dt, J = 14.4, 5.6 Hz, 1H,
NHCH2 (major)); 3.24 (m, 1H, NHCH2 (minor)); 3.56 (ddd,
J = 14.6, 6.0, 3.0 Hz, 1H, NHCH2); 4.44 (dt, J = 11.7, 7.0 Hz, 1H,
CHCH2Ph); 5.03 (d, J = 12.4 Hz, 1H, PhCH2); 5.06 (d, J = 12.4 Hz,
1H, PhCH2); 5.60 (d, J = 8.7 Hz, 1H, NH(Phe) (minor)); 5.62 (d,
J = 8.0 Hz, 1H, NH(Phe) (major)); 6.38 (m, 1H, NH(Gly)); 7.16–
7.33 (m, 10H, Ph); 13C NMR: d 38.66 (CHCH2Ph); 40.19 (NHCH2
(minor)); 40.32 (NHCH2 (major)); 44.77 (CH2O (minor)); 44.90
(CH2O (major)); 50.17 (CHO (minor)); 50.22 (CHO (major)); 56.30
(CHCH2Ph); 67.01 (PhCH2); 127.00, 127.95, 128.14, 128.47,
128.61, 129.21, 136.03, 136.30 (Ph); 155.90 (OCON); 171.32
(CON); HRMS: m/z 355.1620 (MH+. C20H23N2O4 requires 355.1648).
4.3. Inhibition activity
Enzyme kinetics was measured as previously described.8a
4.4. Computational modeling
4.4.1. The model construction
Since the epoxy inhibitors occupy both S and S0 subsites in the
papain active site, we used the 2cio.pdb file with 1.5 Å resolution23
as the initial template for the construction of the target non-cova-
lent complexes of our inhibitors with papain. Crystal water and
4.2.5.2. Trans
CONHCH(CH3)CONH CH(CH3)CO2CH3 (trans Cbz-Phe-Gly-epoxy-
Gly-Ala-Ala-OCH3) (4).
Diasteriomeric ratio (1:1). 1H NMR
PhCH2OCONHCH(CH2Ph)CONHCH2CHOCHCH2-