Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 20 7449
and washed with CH2Cl2, DMF, and CH2Cl2 (each 3ꢀ), trans-
ferred to a round-bottom flask (100 mL), and Teoc-deprotection
was performed with Bu4NF (3 equiv) in dry DMF (50 mL) at 50 °C
for 1 h and then at room temperature for 1 h. The resin was
transferred to the syringe, drained, and washed with DMF, MeOH,
DMF, and CH2Cl2 (each 3ꢀ) and finally dried overnight to yield
resin 25 (2.93 g, ∼1.2 mmol/g). The resin was preswollen in dry
DMF (5 mL). A mixture of Fmoc-Cha-OH (3 equiv), DIC
(3 equiv), and HOBt (3 equiv) in dry DMF (30 mL) was stirred
for 20 min, filtered, and added to the resin, and the mixture was
stirred gently for 20 h. The resin was transferred to the syringe and
washed with DMF, MeOH, DMF, and CH2Cl2 (each 3ꢀ). After
drying, portions of the resin (0.175 g, 0.83 mmol/g) were placed in
five Teflon filter vessels. Fmoc deprotection with 20% piperidine-
DMF (3 mL, 2ꢀ10 min) was followed by washing with DMF (2ꢀ).
The appropriate carboxylic acid (5 equiv), HOBt (5 equiv), and
DIC (5 equiv) in dry DMF (3 mL) were mixed together and after
15 min added to the resins, which were agitated at room tempera-
ture for 16 h. The resins were then drained and washed with DMF,
MeOH, DMF, and CH2Cl2 (each 3ꢀ). Cleavage of the products
from the resin was performed with 50% TFA-CH2Cl2 (5 mL for
2 h). The resins were washed with CH2Cl2 and MeOH (each 2 ꢀ
5 mL), and the combined eluates were concentrated in vacuo,
coevaporated with toluene and MeOH (each 3ꢀ5 mL), and the
residue was dried overnight. Purification of the crude material by
preparative HPLC (using a linear gradient rising from 5% of B to
100% of B during 20 min) afforded 7-11 as the tris(TFA) salts.
Compounds 9 and 10 required rechromatography (using a linear
gradient rising from 5% B to 60% B during 20 min) to obtain
sufficient purity.
Compound 7. Yield: 59 mg (54%); tR = 15.13 min (99.8% at
220 nm). HRMS: calcd for C23H45N5O2 [M þ H]þ 424.36460,
found 424.36444. 1H NMR (300 MHz, CD3OD): δ 4.25 (1H, dd,
J = 8.8 and 6.3 Hz, H-R), 3.40-3.21 (2H, br m, H-1), 3.17-2.96
(10H, br m, H-3, H-4, H-7, H-8 and H-10), 2.09 (2H, m, H-9),
1.87 (2H, p, J = 6.6 Hz, H-2), 1.84-1.64 (10H, br m, cHex Heq,
H-5, H-6, and H-20), 1.59 (2H, m, H-β), 1.40 (1H, m, H-γ),
1.34-1.14 (3H, br m, cHex Hax), 1.05-0.88 (2H, br m, cHex
Hax), 0.86-0.76 (4H, br m, H-30). 13C NMR (75 MHz, CD3OD):
δ 176.7, 176.5, 53.3, 48.2, 48.1, 46.1, 45.8, 40.4, 37.8, 36.7, 35.4,
34.9, 33.4, 27.6 (2C), 27.4, 27.2, 25.4, 24.3 (2C), 14.6, 7.7 (2C).
Compound 8. Yield: 56 mg (48%); tR = 17.78 min (>99.9%
at 210 nm). HRMS: calcd for C26H51N5O2 [M þ H]þ 466.41165,
found 466.41141. 1H NMR (300 MHz, CD3OD): δ 4.23 (1H, dd,
J = 8.8 and 6.3 Hz, H-R), 3.39-3.23 (2H, br m, H-1), 3.16-2.96
(10H, br m, H-3, H-4, H-7, H-8, and H-10), 2.29 (1H, m, H-20),
2.08 (2H, m, H-9), 1.86 (2H, p, J = 6.8 Hz, H-2), 1.84-1.64
(14H, br m, cHex Heq, H-5, and H-6), 1.57 (2H, m, H-β),
1.50-1.13 (9H, br m, H-γ and cHex Hax), 1.02-0.82 (2H, br
m, cHex Hax). 13C NMR (75 MHz, CD3OD): δ 179.4, 176.5,
52.9, 48.2, 48.1, 46.1, 45.9 (2C), 40.2, 37.8, 36.7, 35.5, 34.9, 33.2,
31.2, 30.3, 27.6 (2C), 27.4, 27.3, 26.9 (2C), 26.6, 25.4, 24.3 (2C).
Compound 9. Yield: 60 mg (48%); tR = 19.75 min (>99.9%
at 220 nm). HRMS: calcd for C32H49N5O2 [M þ H]þ 536.39590,
found 536.39593. 1H NMR (300 MHz, CD3OD): δ 7.98 (2H, d,
J = 8.8 Hz, BiPh ArH), 7.75 (2H, d, J = 8.8 Hz, BiPh ArH), 7.68
(2H, br d, J = 7.2 Hz, BiPh Ph), 7.47 (2H, br t, J = 7.2 Hz, BiPh
Ph), 7.38 (1H, br t, J = 7.2 Hz, BiPh Ph), 4.52 (1H, dd, J = 10.1
and 5.4 Hz, H-R), 3.47-3.25 (2H, br m, H-1), 3.10-2.96 (10H,
br m, H-3, H-4, H-7, H-8, and H-10), 2.05 (2H, m, H-9), 1.91
(2H, p, J = 6.7 Hz, H-2), 1.87-1.64 (11H, br m, H-β, cHex Heq,
H-5 and H-6), 1.47 (1H, m, H-γ), 1.38-1.16 (3H, br m, cHex
Hax), 1.14-0.90 (2H, br m, cHex Hax). 13C NMR (75 MHz,
CD3OD): δ 176.5, 170.0, 145.7, 140.9, 133.6, 129.5 (2C), 129.1
(3C), 128.0 (2C), 127.9 (2C), 53.9, 48.2, 48.1, 46.1, 45.8, 40.1,
37.7, 36.8, 35.7, 34.8, 33.4, 27.6 (2C), 27.4, 27.3, 25.4, 24.3 (2C).
Compound 10. Yield: 54 mg (44%); tR = 18.53 min (>99.9%
at 220 nm). HRMS: calcd for C31H49N5O2 [M þ H]þ 524.39590,
found 524.39593. 1H NMR (300 MHz, CD3OD): δ 8.07 (1H, m,
ArH), 7.91-7.79 (2H, br m, ArH), 7.57-7.43 (4H, br m, ArH),
4.24(1H, dd, J = 9.6 and 5.5 Hz, H-R), 4.07(2H, m, -CH2=Ar),
3.39-3.18 (2H, br m, H-1), 3.09-2.70 (10H, br m, H-3, H-4, H-7,
H-8, and H-10), 2.04 (2H, m, H-9), 1.79 (2H, p, J = 6.7 Hz, H-2),
1.72-1.50 (11H, br m, H-β, cHex Heq, H-5 and H-6), 1.34-0.76
(6H, br m, H-γ and cHex Hax). 13C NMR (75 MHz, CD3OD): δ
176.1, 174.1, 135.2, 133.4, 133.0, 129.7, 129.1, 128.8, 127.3, 126.8,
126.5, 125.0, 53.4, 48.2, 47.9, 46.0, 45.8, 41.0, 40.0, 37.8, 36.7, 35.3,
34.9, 33.1, 27.5 (2C), 27.4, 27.1, 25.4, 24.2, 24.1.
Compound 11. Yield: 54 mg (42%); tR = 19.59 min (99.5% at
220 nm). HRMS: calcd for C31H57N5O2 [M þ H]þ 532.45850,
found 532.45845. 1H NMR (300 MHz, CD3OD): δ 4.26 (1H, dd,
J=10.3 and 5.1 Hz, H-R), 3.38-3.24 (2H, br m, H-1), 3.16-2.97
(10H, br m, H-3, H-4, H-7, H-8, and H-10), 2.10 (2H, m, H-9),
2.08-1.91 (5H, br m, CH2-Adm and Adm-H), 1.87 (2H, p, J =
6.9 Hz, H-2), 1.84-1.48 (23H, br m, H-β, cHex Heq, H-5, H-6,
and Adm-H), 1.43 (1H, m, H-γ), 1.34-1.10 (3H, br m, cHex
Hax), 1.06-0.83 (2H, br m, cHex Hax). 13C NMR (75 MHz,
CD3OD): δ 176.4, 174.0, 52.8, 51.6, 48.2, 48.1, 46.2, 45.8, 43.8
(3C), 39.9, 37.9 (3C), 37.8, 36.8, 35.3, 35.0, 34.0, 32.8, 30.2 (3C),
27.6 (3C), 27.3, 25.4, 24.4, 24.3.
SPS of Philanthotoxin Analogues containing Diamino Acid
Residues. General Procedure E. In a syringe (20 mL, fitted with
a filter and valve), 2-chlorotrityl chloride resin (2.38 g, 1.55
mmol/g) was swelled in dry CH2Cl2. Upon draining, the resin
was treated with 10% iPr2EtN in CH2Cl2 for 5 min and was
then washed with CH2Cl2 (2 ꢀ 5 min). A solution of H2N-
(CH2)3NBoc(CH2)4NBoc(CH2)3NHTeoc24 (2.22 g, 1.1 ꢀ 3.69
mmol) and iPr2EtN (3.2 mL, 5 ꢀ 3.69 mmol) in CH2Cl2 (25 mL)
was added to the resin in a round-bottom flask (50 mL). The
suspension was stirred gently for 2 days. The resin was trans-
ferred to the syringe and washed with CH2Cl2 (2 ꢀ 15 mL, each
5 min). Capping of unreacted linker groups was performed with
iPr2EtN-MeOH-CH2Cl2 (5:15:80, 2 ꢀ 15 mL, each 3 min). The
product wasthen dried overnight to yield the loadedresin (3.78g,
74%, 0.73 mmol/g). In a round-bottomed flask (100 mL), Teoc-
deprotection was performed with Bu4NF (4.66 g, 5.3 equiv) in
dry DMF (50 mL) at 50 °C for 1 h and then at room temperature
for 4 h. The resin was transferred to the syringe, drained. and
washed with DMF, MeOH, DMF, and CH2Cl2 (each 3ꢀ), and
then dried overnight to yield resin 27 (3.53 g, 0.78 mmol/g).
Batches of the resin (200 mg, 0.156 mmol) in Teflon filter
vessels were preswollen in DMF (5 mL) for 15 min and then
drained. Protected diamino acid building block (18, 19, 20,
or 21, 3 equiv) in DMF (3 mL) was mixed with DIC (3 equiv)
and HOBt (3 equiv) in dry DMF (3 mL), and after 15 min the
solution was added to the resin and the suspension was agitated
at room temperature for 16 h. The resin was drained and washed
with MeOH and DMF (each 3 ꢀ 5 mL for 5 min), and then
Fmoc deprotection was performed with 20% piperidine-DMF
(3 mL, 2 ꢀ 10 min), followed by washing with DMF (5ꢀ). The
resin was drained and dried (oil pump) for 16 h. Cyclohexane-
carboxylic acid (3 equiv), HOBt (3 equiv), and DIC (3 equiv) in
dry DMF (3 mL) were added to the resin, and the suspension
was agitated at room temperature for 16 h. Upon draining and
washing with DMF (5ꢀ), the Teoc group was removed by
treatment with Bu4NF (3 equiv) in dry DMF (6 mL) at 50 °C
for 1 h followed by 2 h at room temperature. The resin was
drained and washed with DMF, MeOH, DMF, and CH2Cl2
(each 3ꢀ). PhCH2COOH (3 equiv), HOBt (3 equiv), and DIC
(3 equiv) in dry DMF (6 mL) were added, and then the mixture
was agitated at room temperature for 16 h. The resin was washed
with DMF, MeOH, DMF, and CH2Cl2 (3ꢀ), and cleavage was
performed with 20% TFA in CH2Cl2 (3 mL for 30 min followed
by 2 ꢀ 3 mL for 15 min). The resin was washed with CH2Cl2 and
MeOH (each 2.5 mL) and the combined eluates were concen-
trated in vacuo, and the residue was coevaporated successively
with MeOH and toluene (each 3 ꢀ 2.5 mL) and freeze-dried for
16 h. Purification of the crude by preparative HPLC (using a
linear gradient rising from 5% of B to 100% of B during 20 min)
afforded 12-15 as the tris(TFA) salts.