D. Trafalis et al. / Steroids 115 (2016) 1–8
5
44.5, 42.7, 42.1, 36.9, 36.7, 36.2, 35.5, 33.2, 27.6, 23.5, 21.4, 21.3,
21.0, 12.1. FT-IR (KBr): 3453, 2930, 2848, 1732, 1653, 1606,
1480, 1446, 1374, 1249, 1130, 1041, 875; HRMS m/z for
13C NMR (126 MHz, DMSO-d6) d 167.9, 154.9, 137.0, 130.1, 125.9,
114.9, 112.7, 52.5, 43.6, 43.3, 37.9, 34.2, 29.0, 26.8, 25.9, 24.8,
22.5, 17.3; FT-IR (KBr): 3413, 3261, 2959, 2925, 2851, 1655,
1560, 1455, 1238, 1117, 1107; HRMS m/z for C18H24NO2 [M+H]+
calcd 286.1807, found 286.1810.
C
21H32NO3 [M+H]+ calcd 346.2382, found 346.2384.
4.4. 3-Aza-17b-hydroxy-A-homo-4a-androsten-4-one, 6
4.7. Lactam 17a-aza- -homoestrone, 10
D
3-Aza-17b-acetoxy-A-homo-androsten-4-one,
S2
117 mg
(0.341 mmol) was dissolved in 4.9 mL MeOH and LiOH (1 N,
2 mL) were added dropwise. The mixture was stirred at room tem-
perature for 1 h. The reaction was quenched with NH4Cl and the
mixture was extracted with dichloromethane (3 Â 10 mL). The
organic layers were dried (Na2SO4) and concentrated under
reduced pressure to afford 87 mg of 3-aza-17b-hydroxy-A-homo-
Estrone oxime S3 (108 mg, 0.378 mmol) were dissolved in
6.4 mL of dry dioxane. The mixture was cooled to 0 °C and thionyl
chloride (0.7 mL) was added dropwise. The mixture was allowed to
reach room temperature and stirred for 24 h. The reaction was
quenched with NaHCO3 and the mixture was extracted with
dichloromethane (3 Â 20 mL). The organic layers were dried (Na2-
SO4) and concentrated under reduced pressure to afford the crude
product that was further purified by chromatography on SiO2 (elu-
ent; hexane: ethyl acetate = 2:1) to afford 42 mg of lactam 10 (56%
based on recovered starting material) accompanied by recovered
starting material [32 mg of starting material (0.112 mmol)]. The
spectral data were in accordance with those reported in the litera-
ture [29,35]. 10: mp > 300 °C; 1H NMR (500 MHz, DMSO-d6) d 9.01
(s, 1H), 7.53 (s, 1H), 7.05 (d, J = 8.0 Hz, 1H), 6.51 (d, J = 8.4 Hz, 1H),
6.44 (s, 1H), 2.72 (m, 2H), 2.38–2.11 (m, 4H), 2.06–1.75 (m, 4H),
1.60–1.32 (m, 2H), 1.21 (m, 3H), 1.07 (s, 3H); 13C NMR (126 MHz,
DMSO-d6) d 169.9, 154.9, 136.9, 130.0, 125.9, 114.6, 112.8, 53.6,
45.9, 42.8, 39.1, 39.06, 30.6, 29.3, 26.4, 25.7, 21.8, 19.5; FT-IR
(KBr): 3386, 2933, 2865, 1631, 1584, 1500, 1455, 1383, 1293,
1255, 1176, 1159, 1135, 972; HRMS m/z for C18H24NO2 [M+H]+
calcd 286.1807, found 286.1810.
4a-androsten-4-one 6 in 92% yield [27]. 6: mp = 283–286 °C;
23
[a]
À16.4 (c 0.58, CHCl3); 1H NMR (300 MHz, CDCl3) d 6.20 (s,
D
1H), 5.73 (s, 1H), 3.64 (t, J = 8.5 Hz, 1H), 3.23 (m, 1H), 3.14 (m,
1H), 2.48 (td, J = 13.2, 4.0 Hz, 1H), 2.14 (m, 1H), 2.07 (m, 1H),
1.98 (dd, J = 14.6, 8.0 Hz, 1H), 1.90–1.77 (m, 2H), 1.67–1.53 (m,
4H), 1.50–1.40 (m, 1H), 1.37 (dd, J = 13.0, 4.0 Hz, 1H), 1.29 (ddd,
J = 17.5, 11.7, 5.2 Hz, 2H), 1.15 (s, 3H), 1.10–0.82 (m, 3H), 0.77
(m, 3H); 13C NMR (75 MHz, CDCl3) d 169.9, 160.9, 119.0, 81.6,
53.4, 50.5, 44.5, 42.1, 42.1, 36.7, 36.5, 36.3, 35.6, 33.2, 30.5, 23.3,
21.4, 21.4, 11.1; FT-IR (KBr): 3313, 2960, 2928, 2846, 1654, 1648,
1611, 1467, 1258, 1125, 1055; HRMS m/z for C19H30NO2 [M+H]+
calcd 304.2277, found 304.2279.
4.5. Conjugate ENGA-L06E, 8
3-Aza-17b-hydroxy-A-homo-4a-androsten-4-one,
6
87 mg
(0.287 mmol) was dissolved in 28 mL of dry dichloromethane.
Then, 3-(4-(bis(2-chloroethyl)amino)phenoxy)propanoic acid
(106 mg, 0.573 mmol), DCC (119 mg, 0.574 mmol) and a catalytic
amount of DMAP were added. After the resulting solution was stir-
red at room temperature for 24 h the solvent was evaporated and
the residue was purified by flash column chromatography on SiO2
4.8. ENGA-L08E (11)
Lactam 10 (42 mg, 0.147 mmol) was dissolved in 14 mL of dry
DMF. Then, 3-(4-(bis(2-chloroethyl)amino)phenoxy)propanoic
acid (90 mg, 0.293 mmol), DCC (61 mg, 0.293 mmol) and a cat-
alytic amount of DMAP were added. After the resulting solution
was stirred at room temperature for 24 h the solvent was evapo-
rated and the residue was purified by flash column chromatogra-
(eluent; hexane:ethyl acetate = 1:2) to give conjugate 8 (191 mg,
23
99%). 8: mp = 53–56 °C; [
a
]
+10.5 (c = 0.91 CHCl3); 1H NMR
D
(500 MHz, CDCl3) d 6.92 (s, 1H), 6.83 (d, J = 8.8 Hz, 2H), 6.66 (d,
J = 8.6 Hz, 2H), 5.72 (s, 1H), 4.66 (t, J = 8.4 Hz, 1H), 4.17 (t,
J = 6.0 Hz, 2H), 3.63 (m, 4H), 3.59 (m, 4H), 3.32–3.04 (m, 2H),
2.75 (t, J = 6.1 Hz, 2H), 2.48 (m, 1H), 2.27 (m, 1H), 2.15 (m, 2H),
1.50–1.98 (m, 10H), 1.33 (m, 2H), 1.14 (s, 3H), 1.05 (m, 1H), 0.80
(s, 3H); 13C NMR (126 MHz, CDCl3) d 171.0, 170.4, 161.3, 151.3,
140.8, 118.8, 116.3, 114.4, 82.7, 64.4, 54.2, 53.2, 50.2, 44.5, 42.7,
41.9, 40.7, 36.7, 36.2, 35.3, 33.8, 33.1, 27.5, 25.6, 24.9, 23.4, 21.3,
12.1; FT-IR (KBr): 3450, 2925, 1731, 1651, 1607, 1512, 1469,
1353, 1238, 1181, 1041, 869, 813; HRMS m/z for C32H45Cl2N2O4
[M+H]+ calcd 591.2756, found 591.2760.
phy on SiO2 (eluent; dichloromethane:acetone = 2:1) to give
23
conjugate 11 (56 mg, 68%). 11: glass, [
a]
+73.5 (c 0.90, CHCl3);
D
1H NMR (500 MHz, CDCl3) d 7.25 (d, J = 6.0 Hz, 1H), 6.89 (d,
J = 8.8 Hz, 2H), 6.85 (s, 1H), 6.82 (s, 1H), 6.68 (d, J = 8.8 Hz, 2H),
6.31 (s, 1H), 4.30 (t, J = 6.1 Hz, 2H), 3.62 (dt, J = 29.2, 6.6 Hz, 8H),
2.97 (dd, J = 15.1, 9.0 Hz, 2H), 2.88 (m, 2H), 2.58–2.36 (m, 4H),
2.23–2.00 (m, 2H), 1.92–1.66 (m, 3H), 1.60–1.29 (m, 4H), 1.19 (s,
3H); 13C NMR (126 MHz, CDCl3) d 171.7, 169.8, 151.3, 148.5,
141.0, 137.8, 137.2, 126.1, 121.3, 118.7, 116.5, 114.5, 64.4, 54.4,
54.2, 46.6, 43.4, 40.7, 39.9, 38.9, 34.9, 30.5, 29.5, 26.5, 25.9, 22.1,
19.8; FT-IR (KBr): 3329, 2927, 2850, 1757, 1626, 1577, 1512,
1437, 1311, 1244, 1157, 1088, 1045, 892; HRMS m/z for C31H39Cl2-
N2O4 [M+H]+ calcd 574.2287, found 574.2289.
4.6. Estrone oxime, S3
To a solution of estrone 9 (100 mg, 0.37 mmol) in 2.2 mL abso-
lute ethanol was added hydroxylamine hydrochloride (62 mg,
0.88 mmol) and pyridine (1.2 mL). The mixture was refluxed for
6 h. Then, water was added and the mixture was extracted with
ethyl acetate (3 Â 10 mL). The organic layers were dried (Na2SO4)
and concentrated under reduced pressure to afford the crude pro-
duct that was further purified by chromatography on SiO2 (eluent;
hexane:ethyl acetate = 3:1) to afford 105 mg of estrone oxime S3
(100%) as a white solid. The spectral data were accordance with
those reported in the literature [34]. S3: mp = 250–253 °C; 1H
NMR (500 MHz, DMSO-d6) d 10.08 (s, 1H), 9.00 (s, 1H), 7.05 (d,
J = 8.2 Hz, 1H), 6.51 (d, J = 8.2 Hz, 1H), 6.44 (s, 1H), 4.04 (s, 2H),
3.57 (s, 1H), 2.73 (m, 2H), 2.44–2.21 (m, 2H), 2.16 (m, 1H), 1.96–
1.73 (m, 2H), 1.58–1.43 (m, 1H), 1.45–1.17 (m, 4H), 0.85 (s, 3H).
4.9. 17a-Hydroxyandrost-4-ene-3,11-dione, S4
A solution of adrenosterone (500 mg, 1.65 mmol) in 100 mL in
MeOH was cooled down to 0 °C. To this solution, 50 mg
(1.32 mmol) NaBH4 were slowly added. Then, the reaction mixture
was allowed to reach room temperature. After 1 h, the solvent was
removed and the mixture was extracted with ethyl acetate
(3 Â 50 mL). The organic layers were dried (Na2SO4) and concen-
trated under reduced pressure. Ethyl acetate was added and the
precipitate was collected by filtration to give 497 mg of S4 as a
23
white solid (99% yield) [30]. S4: mp = 146–149 °C; [
a
]
+184.1
D
(c = 1.67 CHCl3); 1H NMR (500 MHz, CDCl3) d 5.57 (s, 1H), 3.97
(d, J = 7.0 Hz, 1H), 3.72 (t, J = 8.3 Hz, 2H), 3.37 (s, 1H), 2.60 (d,
J = 13.1 Hz, 1H), 2.45–1.96 (m, 5H), 1.67 (ddd, J = 77.4, 27.8,