6050 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16
Robins et al.
the residue was dissolved in EtOAc. The solution was washed
(H2O), and volatiles were removed under vacuum. Recrystalli-
zation of the residue (EtOAc/Et2O) gave 22 (4.16 g, 72%). H
NMR (DMSO-d6, 500 MHz) δ 11.98 (s, 1H), 9.38 (s, 1H), 3.16
(“q”, J = 6.7 Hz, 2H), 2.19 (t, J = 7.3 Hz, 2H), 1.44-1.53 (m,
4H), 1.23-1.29 (m, 2H). HRMS (EI) m/z 228.0836 (MHþ
[C8H13F3NO3] = 228.0848).
4-Nitrophenyl 6-(Trifluoroacetamido)hexanoate (23). A mix-
ture of 22 (1.26 g, 5.55 mmol), 4-nitrophenol (930 mg, 6.69
mmol), and DCC (1.39 g, 6.73 mmol) in CH2Cl2 (10 mL) was
stirred overnight at ambient temperature. DCU was filtered and
volatiles were removed under vacuum. Flash chromatography
of the residue (20 f 30% EtOAc/hexanes) gave 23 (1.73 g,
90%). 1H NMR (CDCl3, 500 MHz) δ 8.27 (d, J = 9.5 Hz, 2H),
7.28 (d, J = 9.0 Hz, 2H), 6.47 (br s, 1H), 3.41 (“q”, J = 7.0 Hz,
2H), 2.63 (t, J = 7.3 Hz, 2H), 1.77-1.83 (m, 2H), 1.64-1.70 (m,
2H), 1.45-1.52 (m, 2H). HRMS (FAB) m/z 349.1021 (MHþ
[C14H16F3N2O5] = 349.1011).
Procedure H: General Method for Conjugation of FITC with
the Pendant Amino Group at C50. Example: 5a. A mixture of
FITC (58 mg, 90% isomer I, 0.13 mmol), crude 3 (∼0.13 mmol
calculated from its precursor 25a), and Et3N (56 μL, 41 mg, 0.41
mmol) in DMF (2 mL) was stirred with protection from light for
3 h at ambient temperature and volatiles were removed under
vacuum. Flash chromatography of the residue (10 f 12 f 15%
MeOH/CH2Cl2) gave 5a (64 mg, 56%): UV-vis (50 mM pH 7.0
phosphate buffer) max 241, 265, 367, 496 nm (ε 47 100, 39 100,
5200, 42 100), min 227, 255, 362, 409 nm (ε 38 400, 37 600, 4300,
1
1
2900). H NMR (DMSO-d6, 500 MHz) δ 10.15-10.21 (br m,
3H), 8.59 (br s, 1H), 8.44 (s, 1H), 8.26 (br s, 1H), 8.24 (s, 1H),
8.22 (s, 1H), 8.17 (d, J = 9.0 Hz, 2H), 7.74 (d, J = 8.0 Hz, 1H),
7.57 (d, J = 8.5 Hz, 2H), 7.17 (d, J = 8.0 Hz, 1H), 6.67 (d, J =
2.0 Hz, 2H), 6.55-6.61 (m, 4H), 5.92 (d, J = 5.5 Hz, 1H), 5.53
(d, J = 6.0 Hz, 1H), 5.38 (br s, 1H), 4.80 (br s, 3H), 4.18 (br s,
1H), 4.05-4.09 (m, 1H), 3.68-3.69 (br m, 2H), 3.01 (dd, J = 6.0,
14.0 Hz, 1H), 2.93 (dd, J = 6.8, 13.8 Hz, 1H), 2.74-2.80 (m,
2H). 13C NMR (DMSO-d6, 125 MHz) 180.4, 168.5, 159.8, 154.3,
152.6, 152.0, 149.0, 148.3, 146.8, 146.3, 141.2, 140.2, 129.4,
129.1, 128.0, 126.8, 124.2, 123.5, 119.7, 116.7, 112.7, 109.8,
102.2, 87.5, 84.0, 72.6, 43.5, 42.6, 33.8, 30.5. HRMS (FAB)
m/z 851.1921 (MHþ [C40H35N8O10S2] = 851.1918).
6-N-(4-Nitrobenzyl)-50-S-{2-[6-(trifluoroacetamido)hexanamido]-
ethyl}-50-thioadenosine (27a). A mixture of crude 3 (∼0.36 mmol
calculated from its precursor 25a), 23 (125 mg, 0.36 mmol), and
Et3N (61 μL, 44 mg, 0.44 mmol) in DMF (3 mL) was stirred
overnight at ambient temperature and volatiles were evaporated
under vacuum. Flash chromatography of the residue (5 f 10%
MeOH/CH2Cl2) gave 27a (110 mg, 47%): UV (MeOH) max
5b: Treatment of a mixture of FITC (50 mg, 90% isomer I,
0.12 mmol), crude 26b (∼ 0.12 mmol calculated from its
precursor 25b), and Et3N (48 μL, 35 mg, 0.35 mmol) in DMF
by procedure H gave 5b (96 mg, 95%; flash chromatography
with 10 f 15 f 20% MeOH/CH2Cl2): UV-vis (50 mM pH 7.0
phosphate buffer) max 241, 264 (sh), 320 (sh), 367 (sh), 496 nm
(ε 51 700, 40 100, 6500, 4200, 39 800), min 226, 406 nm (ε 39 800,
2900). 1H NMR (DMSO-d6, 500 MHz) δ 10.11 (br s, 2H), 10.05
(br s, 1H), 9.78 (s, 1H), 8.38 (s, 1H), 8.34 (br s, 1H), 8.22 (s, 2H),
8.15 (br s, 1H), 7.73 (d, J = 7.5 Hz, 1H), 7.49 (d, J = 9.0 Hz, 2H),
7.24 (d, J = 8.5 Hz, 2H), 7.17 (d, J = 8.0 Hz, 1H), 6.67 (d, J =
2.5 Hz, 2H), 6.55-6.61 (m, 4H), 5.91 (d, J = 6.0 Hz, 1H), 5.51
(d, J = 5.5 Hz, 1H), 5.33 (d, J = 5.0 Hz, 1H), 4.76-4.78 (m, 1H),
4.63 (br s, 2H), 4.16-4.18 (m, 1H), 4.04-4.08 (m, 1H),
3.69-3.70 (br m, 2H), 3.01 (dd, J = 6.0, 14.0 Hz, 1H), 2.92
(dd, J = 7.3, 13.8 Hz, 1H), 2.75-2.81 (m, 2H), 2.27 (q, J = 7.5
Hz, 2H), 1.05 (t, J = 7.5 Hz, 3H). 13C NMR (DMSO-d6, 125
MHz) δ 180.4, 171.8, 168.5, 159.6, 154.4, 152.6, 151.9, 148.8,
147.2, 141.2, 139.9, 138.0, 134.5, 129.6, 129.1, 127.5, 126.6,
124.1, 119.6, 118.9, 116.6, 112.6, 109.7, 102.2, 87.4, 83.9,
72.64, 72.62, 43.5, 42.5, 33.8, 30.5, 29.4, 9.7. HRMS (FAB)
m/z 877.2430 (MHþ [C43H41N8O9S2] = 877.2438).
7a: Treatment of a mixture of FITC (33 mg, 90% isomer I,
0.077 mmol), crude 28a (∼ 0.078 mmol, calculated from its
precursor 27a), and Et3N (33 μL, 24 mg, 0.24 mmol) in DMF
by procedure H gave 7a (64 mg, 86%; flash chromatography
with 10 f 15 f 20% MeOH/CH2Cl2): UV-vis (50 mM pH 7.0
phosphate buffer) max 240, 265, 367, 496 nm (ε 53 700, 43 100,
7200, 47 500), min 227, 256, 362, 409 nm (ε 45 600, 42 000, 5800,
4000). 1H NMR (DMSO-d6, 500 MHz) δ 10.13 (br s, 2H), 10.04
(br s, 1H), 8.59 (br s, 1H), 8.43 (s, 1H), 8.27 (s, 1H), 8.22 (s, 1H),
8.16-8.18 (m, 3H), 7.91 (t, J = 5.8 Hz, 1H), 7.74 (d, J = 7.0 Hz,
1H), 7.58 (d, J = 8.5 Hz, 2H), 7.17 (d, J = 8.5 Hz, 1H), 6.67 (d,
J = 2.5 Hz, 2H), 6.55-6.61 (m, 4H), 5.91 (d, J = 6.0 Hz, 1H),
5.52 (d, J = 6.5 Hz, 1H), 5.35 (d, J = 4.5 Hz, 1H), 4.81 (br s, 2H),
4.76-4.77 (br m, 1H), 4.14-4.15 (br m, 1H), 4.00-4.03 (m, 1H),
3.47-3.48 (br m, 2H), 3.17-3.21 (m, 2H), 2.93 (dd, J = 5.8, 13.8
Hz, 1H), 2.84 (dd, J = 6.8, 13.8 Hz, 1H), 2.56 (dt, J = 2.5, 7.0
Hz, 2H), 2.07 (t, J = 7.5 Hz, 2H), 1.49-1.58 (m, 4H), 1.26-1.32
(m, 2H). 13C NMR (DMSO-d6, 125 MHz) δ 180.3, 172.1, 168.6,
159.6, 154.3, 152.6, 151.9, 149.0, 148.3, 146.8, 146.3, 141.5,
140.2, 129.3, 129.1, 128.0, 126.6, 124.1, 123.5, 119.7, 116.3,
112.6, 109.8, 102.2, 87.4, 83.9, 72.64, 72.57, 43.7, 42.6, 38.5,
35.3, 33.9, 31.4, 28.2, 26.2, 25.0. HRMS (FAB) m/z 964.2756
(MHþ [C46H46N9O11S2] = 964.2758).
1
271 nm, min 232 nm. H NMR (MeOH-d4, 500 MHz) δ 8.25
(s, 1H), 8.21 (s, 1H), 8.11 (d, J = 9.0 Hz, 2H), 7.54 (d, J = 8.5
Hz, 2H), 5.98 (d, J = 4.5 Hz, 1H), 4.75 (t, J = 5.0 Hz, 1H), 4.30
(t, J = 4.8 Hz, 1H), 4.18 (dd, J = 5.5, 10.5 Hz, 1H), 3.28-3.31
(m, 2H), 3.22 (t, J = 7.0 Hz, 2H), 2.97 (dd, J = 5.3, 13.8 Hz, 1H),
2.91 (dd, J = 6.5, 14.0 Hz, 1H), 2.64 (dt, J = 3.0, 7.0 Hz, 2H),
2.13 (t, J = 7.5 Hz, 2H), 1.49-1.60 (m, 4H), 1.25-1.32 (m, 2H).
13C NMR (MeOH-d4, 125 MHz) δ 176.2, 159.6 (q, J = 36.7 Hz),
156.2, 154.1, 150.3, 148.7, 148.6, 141.3, 129.4, 124.7, 121.2, 117.7
(q, J = 286.1 Hz), 90.2, 85.9, 75.0, 74.2, 44.5, 40.7, 40.2, 37.0,
35.2, 33.2, 29.7, 27.5, 26.6. HRMS (FAB) m/z 693.2037 (MNaþ
[C27H33F3N8O7SNa] = 693.2043).
6-N-[4-(Propanamido)benzyl]-50-S-{2-[6-(trifluoroacetamido)-
hexanamido]ethyl}-50-thioadenosine (27b). A mixture of crude
26b (∼0.20 mmol calculated from its precursor 25b), 23 (82 mg,
0.24 mmol), and Et3N (33 μL, 24 mg, 0.24 mmol) in DMF
(3 mL) was stirred overnight at ambient temperature and
volatiles were evaporated under vacuum. Flash chromato-
graphy of the residue (5 f 10% MeOH/CH2Cl2) gave 27b
(130 mg, 96%): UV (MeOH) max 266 nm, min 255 nm. 1H
NMR (DMSO-d6, 500 MHz) δ 9.78 (s, 1H), 9.38 (br s, 1H), 8.37
(s, 1H), 8.33 (br s, 1H), 8.22 (s, 1H), 7.87 (t, J = 5.8 Hz, 1H), 7.49
(d, J = 8.5 Hz, 2H), 7.25 (d, J = 9.0 Hz, 2H), 5.90 (d, J = 6.0 Hz,
1H), 5.49 (d, J = 6.5 Hz, 1H), 5.31 (d, J = 5.0 Hz, 1H),
4.73-4.75 (m, 1H), 4.64 (br s, 2H), 4.14 (dd, J = 4.5, 9.0 Hz,
1H), 4.00 (dt, J = 4.0, 6.5 Hz, 1H), 3.13-3.20 (m, 4H), 2.92 (dd,
J = 5.8, 13.8 Hz, 1H), 2.83 (dd, J = 6.8, 13.8 Hz, 1H), 2.56 (dt,
J = 2.0, 7.0 Hz, 2H), 2.28 (q, J = 7.5 Hz, 2H), 2.03 (t, J = 7.3
Hz, 2H), 1.43-1.50 (m, 4H), 1.18-1.23 (m, 2H), 1.05 (t, J = 7.5
Hz, 3H). 13C NMR (DMSO-d6, 125 MHz) δ 172.0, 171.8, 156.1
(q, J = 35.8 Hz), 154.4, 152.6, 148.8, 139.8, 137.9, 134.4, 127.5,
119.6, 118.9, 116.0 (q, J = 288.5 Hz), 87.4, 83.8, 72.63, 72.55,
45.7, 42.5, 38.5, 35.2, 33.9, 31.4, 29.4, 28.0, 25.8, 24.8, 9.6.
HRMS (FAB) m/z 719.2557 (MNaþ [C30H39F3N8O6SNa] =
719.2563).
50-S-{2-[(6-Amino)hexanamido]ethyl}-6-N-(4-nitrobenzyl)-50-
thioadenosine (28a). Treatment of 27a (55 mg, 0.082 mmol) by
procedure G gave a residue containing 28a. HRMS (FAB) m/z
575.2394 (MHþ [C25H35N8O6S]=575.2400) that was used direc-
tly for conjugation with FITC.
50-S-{2-[(6-Amino)hexanamido]ethyl}-6-N-[(4-propanamido)-
benzyl]-50-thioadenosine (28b). Treatment of 27b (60 mg, 0.086
mmol) by procedure G gave a residue containing 28b. HRMS
(FAB) m/z 623.2753 (MNaþ [C28H40N8O5SNa] = 623.2740)
that was used directly for conjugation with FITC.
7b: Treatment of a mixture of FITC (36 mg, 90% isomer I,
0.082 mmol), crude 28b (∼0.081 mmol calculated from its