Journal of Medicinal Chemistry
Article
DMSO (3 mL) and DIPEA (5.6 mg, 0.043 mmol). The mixture was
stirred at room temperature for 16 h and the product was then purified
by preparative RP-HPLC [λ = 280 nm; solvent gradient, 0% B to 80%
B in 30 min; A = aqueous NH4OAc/AcOH buffer at pH = 7; B =
MeCN] to provide the product 7 as a plum-colored solid (22.3 mg,
63%). 1H NMR (500 MHz, DMSO-d6 + 1 drop of D2O): δ 7.88 (s, 1
H), 7.48 (s, 1 H), 7.36 (s, 1 H), 7.22−7.06 (m, 11 H), 4.50−4.48 (m,
2 H), 4.40−4.35 (m, 5 H), 4.20 (m, 1 H), 4.09 (t, J = 5.3 Hz, 1 H),
3.83 (s, 3 H), 3.91−3.86 (m, 8 H), 3.83−3.81 (m, 3 H), 3.78 (s, 1 H),
3.40 (m, 7 H), 3.26 (m, 1 H), 3.13−3.07 (m, 2 H), 2.98 (m, 3 H), 2.93
(m, 2 H), 2.83 (m, 1 H), 2.64−2.46 (m, 3 H), 2.43 (t, J = 6.4 Hz, 1
H), 2.27 (m, 4 H), 2.18 (t, J = 7.3 Hz, 2 H), 2.04 (m, 2 H), 1.95−1.85
(m, 8 H), 1.73 (m, 2 H), 1.26 (m, 5 H), 1.07 (m, 5 H), 0.96 (m, 2 H);
MALDI-MS (rel intensity) m/z 1642 (MH+). HRMS (+ESI) calcd for
MH+ (C76H96N11O26S2): 1642.5969, found 1642.6043 (Δm/m = 4.5
ppm). UV/vis: λmax = 280 nm. HPLC purity: 97.2% (MeCN, 100%).
Fmoc Solid Phase Peptide Synthesis of DUPA-Aoc-Phe-Phe-
Dap-Asp-Cys Reagent (12).15 H-L-Cys(Trt)-(2-ClTrt) resin (11)
(0.7 mequiv/g, 200 mg, 0.14 mmol) was swollen in CH2Cl2 (5 mL)
for 30 min, while argon was bubbled through the mixture. CH2Cl2 was
drained, and a solution of Fmoc-L-Asp(OtBu)-OH (2.5 equiv), PyBOP
(2.5 equiv), HOBt (2.5 equiv), and DIPEA (5.0 equiv) in DMF (3
mL) was added to the resin. Argon was bubbled through the mixture
for 3 h, and the solvent was then drained. The resin was washed with
DMF (5 mL × 3, in 5 min/wash, drained after each wash) and iPrOH
(5 mL × 3, in 5 min/wash, drained after each wash). A Kaiser test was
performed to give a negative result, which indicated the coupling
reaction was successful. The resin was then washed with 20%
piperidine in DMF (5 mL × 3, in 10 min/wash, drained after each
wash), DMF (5 mL × 3, in 5 min/wash, drained after each wash), and
iPrOH (5 mL × 3, in 5 min/wash, drained after each wash). A second
30−50% gradient of EtOAc in hexane, to provide the urea 16 as a clear
colorless syrup (0.94 g, 96%). 1H NMR (300 MHz, CDCl3) δ 7.34 (s,
5 H), 5.11 (s, 2 H), 5.05−5.00 (m, 2 H), 4.40−4.31 (m, 2 H), 2.49−
2.40 (m, 2 H), 2.37−2.26 (m, 2 H), 2.22- 2.05 (m, 2 H), 1.96−1.82
(m, 2 H), 1.46−1.43 (s, 27 H).
(S)-5-(tert-Butoxy)-4-(3-((S)-1,5-di-tert-butoxy-1,5-dioxopen-
tan-2-yl)ureido)-5-oxopentanoic Acid (17).15 Compound 16
(0.96 g, 1.66 mmol) was diluted in EtOAc (15 mL) and the mixture
was degassed in 5 min with argon, followed by addition of 10% Pd on
activated charcoal (20 mg), and the mixture was degassed for another
5 min. The mixture was hydrogenated at room temperature with a
hydrogen balloon for 40 h and was then filtered and washed with
EtOAc through a Celite pad. The solution was concentrated to provide
a clear colorless syrup. The syrup was triturated with hexane and
allowed to stand overnight to yield the DUPA precursor 17 as a white
semisolid (0.82 g, 100%). 1H NMR (300 MHz, CDCl3) δ 5.84 (d, J =
8.2 Hz, 1 H), 5.42 (br s, 1 H), 4.44 (m, 1 H), 4.34 (m, 1 H), 2.43−
2.39 (m, 2 H), 2.36−2.28 (m, 2 H), 2.24−2.03 (m, 2 H), 1.91−1.79
(m, 2 H), 1.48 (s, 9 H), 1.46 (s, 9 H), 1.44 (s, 9 H).
1H-Benzo[d][1,2,3]triazol-1-yl[2-(pyridin-2-yldisulfanyl)-
ethyl] Carbonate (18).13 Compound 23 (1.00 g, 4.47 mmol) was
dissolved in CH2Cl2 (5 mL) and Et3N (0.45 g, 4.47 mmol) and added
dropwise to a solution of triphosgene (24) (0.44 g, 1.49 mmol) at 0
°C. The solution was stirred at room temperature for 1.5 h, followed
by dropwise addition of a solution of hydroxybenzotriazole (25) (0.60
g, 4.47 mmol) in CH2Cl2 (10 mL) and Et3N (0.45 g, 4.47 mmol). The
mixture was then stirred at room temperature for 16 h and then
diluted with CHCl3 to 50 mL and washed with H2O (100 mL × 3)
and brine (100 mL). The organic layer was dried over anhydrous
Na2SO4, filtered, and concentrated. The resulting yellow oil was
triturated with hexane and filtered to provide the product 18 as a white
solid (1.36 g, 87%): mp 116−118 °C. 1H NMR (300 MHz, CDCl3) δ
8.40 (d, J = 4.8 Hz, 1 H), 8.18 (d, J = 8.4 Hz, 1 H), 8.04 (d, J = 8.4 Hz,
1 H), 7.92 (t, J = 8.0 Hz, 1 H), 7.77−7.74 (m, 2 H), 7.66 (t, J = 7.8 Hz,
1 H), 7.19 (m, 1 H), 4.74 (t, J = 6.0 Hz, 2 H), 3.38 (t, J = 6.0 Hz, 2 H).
2-(Pyridin-2-yldisulfanyl)ethyl (2,3,8-Trimethoxy-6-(3-mor-
pholinopropyl)-5,11-dioxo-6,11-dihydro-5H-indeno[1,2-c]-
isoquinolin-9-yl) Carbonate (19). Compound 4 (50 mg, 0.10
mmol) was dissolved in CH2Cl2 (5 mL), followed by addition of
carbonate reagent 18 (48 mg, 0.12 mmol), DMAP (13 mg, 0.10
mmol), and Et3N (21 mg, 0.21 mmol). The mixture was stirred at
room temperature for 16 h and was then loaded directly onto a silica
gel column and purified by flash column chromatography, eluting with
3% MeOH in CHCl3, to provide the product 19 as a red solid (64.8
mg, 90%): mp 130−132 °C. 1H NMR (300 MHz, CDCl3) δ 8.51 (d, J
= 4.5 Hz, 1 H), 8.09 (s, 1 H), 7.69−7.66 (m, 3 H), 7.37 (s, 1 H),
7.16−7.12 (m, 2 H), 4.59 (q, J = 6.6 Hz, 4 H), 4.06 (s, 3 H), 4.00 (s, 3
H), 3.98 (s, 3 H), 3.68 (t, J = 4.3 Hz, 4 H), 3.18 (t, J = 6.5 Hz, 2 H),
2.59 (t, J = 6.9 Hz, 2 H), 2.47 (br s, 4 H), 2.14 (m, 2 H).
Kaiser test was performed to give a positive result, which indicated the
cleavage of the Fmoc group was successful. The above sequence was
repeated for the coupling of Boc-L-Dap(Fmoc)-OH, Fmoc-L-Phe-OH,
Fmoc-L-Phe-OH, Fmoc-8-Aoc-OH, and the protected DUPA
precursor 17. The final product was cleaved from the resin by
washing with a TFA/H2O/TIPS/1,2-ethanedithiol cocktail
(92.5:2.5:2.5:2.5) (7.5 mL, 30 min), during which argon was bubbled
through the mixture. Another 7.5 mL portion of the cocktail was
diluted with TFA (7.5 mL) to make a 15 mL solution. This solution
was used to wash the resin twice (7.5 mL/wash, in 15 min/wash). The
filtrate was collected and concentrated. Addition of Et2O caused
precipitation of a solid. The mixture was centrifuged, and the
precipitate was collected. The crude product was purified by
preparative RP-HPLC [λ = 254 nm; solvent gradient, 0% B to 80%
B in 30 min; A = aqueous NH4OAc/AcOH buffer at pH = 5; B =
MeCN]. Pure fractions were combined, concentrated under vacuum,
and lyophilized for 48 h to yield the pure DUPA−peptide product 12
as a white solid (172 mg, 58% overall yield, or 91.3% average yield per
2-(Pyridin-2-yldisulfanyl)ethanol Hydrochloride (23).13 2-
Mercaptoethanol (20) (0.77 g, 9.9 mmol) was dissolved in CH3CN
(5 mL), and the solution was added dropwise to a solution of
methoxycarbonylsulfenyl chloride (21) (1.25 g, 9.9 mmol) in CH3CN
(8 mL) precooled at 0 °C. The pale yellow solution was stirred at 0 °C
for 30 min until it turned colorless. A solution of 2-mercaptopyridine
(22) (1.0 g, 9.0 mmol) in CH3CN (20 mL) was added dropwise to the
clear solution, and the yellow mixture was stirred at reflux for 2 h,
during which a white precipitate formed. The colorless mixture with
white precipitate was then stirred at 0 °C for 1 h and filtered. The filter
cake was washed with CH3CN to provide the product 23 as a white
1
coupling step): mp 175−178 °C. H NMR (300 MHz, DMSO-d6) δ
9.39 (d, J = 9.10 Hz, 1 H), 8.92 (d, J = 8.2 Hz, 1 H), 8.68 (m, 1 H),
8.16 (m, 1 H), 7.81 (m, 1 H), 7.71 (d, J = 5.6 Hz, 1 H), 7.29−7.10,
(m, 10 H), 6.45 (m, 1 H), 6.36 (m, 1 H), 4.43 (m, 4 H), 4.22 (q, J =
6.6 Hz, 3 H), 4.03−3.96 (m, 6 H), 3.43−3.36 (m, 2 H), 3.14−2.84 (m,
7 H), 2.63 (d, J = 6.6 Hz, 3 H), 2.20−2.18 (m, 2 H), 2.07 (m, 2 H),
2.02−1.94 (m, 1 H), 1.91−1.80 (m, 3 H), 1.74−1.68 (m, 3 H), 1.31−
1.26 (m, 4 H), 1.17−1.03 (m, 8 H); LC/MS (ES-API) m/z 1060.2
(M+) and 530.7 (M2+). HRMS (+ESI) calcd for MH+: 1060.4297;
found, 1060.4291 (Δm/m = 0.5 ppm). UV/vis: λmax = 254 nm.
5-Benzyl 1-(tert-Butyl)-(((S)-1,5-di-tert-butoxy-1,5-dioxopen-
tan-2-yl)carbamoyl)-L-glutamate (16).15 L-Glu(OtBu)-OtBu (13)
(500 mg, 1.69 mmol) and triphosgene (168 mg, 0.565 mmol) were
diluted in CH2Cl2 (25 mL) at 0 °C in argon for 5 min, and then Et3N
(376 mg, 3.72 mmol) was added. The mixture was stirred at 0 °C for 2
h to allow isocyanate 14 formation, followed by addition of L-
Glu(OBn)-OtBu (15) (613 mg, 1.86 mmol) in Et3N (244 mg, 2.42
mmol) and CH2Cl2 (5 mL). Stirring was continued at room
temperature for 16 h, and then the reaction was quenched with 1 M
HCl (50 mL). The organic layer was concentrated to a yellow syrup,
which was purified by flash column chromatography, eluting with a
1
amorphous solid (1.84 g, 92%): mp 128−130 °C. H NMR (300
MHz, CDCl3) δ 9.13 (d, J = 5.5 Hz, 1 H), 8.10 (t, J = 7.4 Hz, 1 H),
7.82 (d, J = 8.3 Hz, 1 H), 7.61 (t, J = 6.6 Hz, 1 H), 4.01 (t, J = 5.2 Hz,
2 H), 3.27 (t, J = 5.6 Hz, 2 H); ESI-MS (positive mode) m/z (rel
intensity) 188 [(MH − H2O)+, 100].
General Procedure for IC50 (Dose Dependence) Studies.
22RV1 cells were seeded in 24-well (50 000 cells/well) Falcon plates
and allowed to form monolayers over a period of 24−48 h. The old
medium was replaced with fresh medium (0.5 mL) containing
increasing concentrations of drug (either targeted or nontargeted), and
H
J. Med. Chem. XXXX, XXX, XXX−XXX