Uracil-Directed Ligand Tethering
A R T I C L E S
DMSO-d
6
) δ 163.95, 151.15, 148.96, 148.04, 145.89, 144.73, 142.23,
H), 4.16 (t, J ) 6.4 Hz, 2 H), 2.08 (m, 2 H); 13C NMR (125 MHz,
1
23.12, 119.83, 115.81, 113.15, 101.60, 71.94, 69.76, 28.46; UV/vis
6
DMSO-d ) δ 163.91, 151.10, 150.07, 147.41, 146.02, 144.66, 142.22,
+
λmax 273 nm; HRMS (m/z) [M + H] calcd for C15
H N O
17 4 6
349.11,
120.58, 119.15, 112.89, 112.29, 101.69, 71.86, 70.24, 28.38; UV/vis
+
found 349.11.
λ
max 278 nm; HRMS (m/z) [M + Na] calcd for C15
4 6
H15BrN O Na
The second set of oxime dimers based on the 3-(3)-13 hit discovered
in the first screening round were synthesized in an identical fashion as
described above using uracil aldehyde 3 and hydroxybenzaldehydes
449.01, found 449.01.
3-(3)-46: 1H NMR (400 MHz, DMSO-d ) δ 8.56 (d, J ) 1.2 Hz, 1
H), 7.95 (s, 1 H), 7.37 (s, 1 H), 6.74 (s, 1 H), 6.26 (bs, H), 5.78 (d, J
6
1
8-42 and the O,O′-diaminopropanediol linker (cf. Supporting Infor-
) 1.2 Hz, 1 H), 4.28 (t, J ) 6.0 Hz, 2 H), 4.16 (t, J ) 6.0 Hz, 2 H),
2.09 (m, 2 H); 13C NMR (125 MHz, DMSO-d ) δ 163.88, 160.27,
mation Table S2). The most potent inhibitor identified from this second
6
round of screening (3-(3)-27) was synthesized in larger scale and
151.05, 148.62, 148.00, 144.62, 142.22, 133.36, 122.38, 113.80, 109.23,
1
thoroughly characterized. 3-(3)-27: H NMR (400 MHz, DMSO-d
6
) δ
101.73, 71.85, 70.07, 28.39; UV/vis λ 269 nm; HRMS (m/z) [M +
max
+
9
5
.10 (bs, H), 8.21 (s, 1 H), 7.94 (s, 1 H), 6.88 (s, 1 H), 6.31 (s, 1 H),
H] calcd for C H N O 394.10, found 394.10.
1
5
16
5
8
.78 (s, 1 H), 4.28 (t, J ) 6.0 Hz, 2 H), 4.10 (t, J ) 6.0 Hz, 2 H), 2.06
Synthesis of Methyl Oxime Derivatives of 1-3, 13, and 27. The
O-methyl oxime of 3,4-dihydroxybenzaldehyde (47) is known and was
1
3
(m, 2 H); C NMR (125 MHz, DMSO-d
6
) δ 163.91, 151.04, 150.25,
1
7
49.17, 146.90, 144.63, 142.20, 138.73, 112.76, 107.78, 103.56, 101.69,
55
synthesized using 13 and O-methylhydroxylamine hydrochloride.
+
1.90, 69.77, 28.38; UV/vis λmax 286 nm; ESI (m/z) for [M + H]
O-Methyloximes 48-51 were made using a similar method.
+
calcd for C15
for C15
C H N O
15 16 4 7
H
18
N O
4 7
366, found 366; ESI (m/z) for [M + Na] calcd
4
8: To a solution of 27 (308 mg, 2.0 mmol) in 4.0 mL of EtOH-
-
17
H N
4
O
7
Na 388, found 388; ESI (m/z) for [M - H] calcd for
364, found 364.
Isolation and Purification of Oxime Dimers using HPLC. All of
H
2
O-THF (0.45/0.3/0.25) were added sodium acetate (264 mg) and
O-methylhydroxylamine hydrochloride (183 mg), and the solution was
stirred at room temperature for overnight. The solvents were removed
in vacuo, and the residue was extracted with chloroform three times.
The combined organic layers were dried over anhydrous MgSO and
4
concentrated in vacuo. The residue was purified by silica gel column
chromatography (EtOAc/hexanes) to give a product amount of 347 mg
the most active oxime heterodimers were purified by HPLC using a
Phenomenex Aqua reversed phase C-18 HPLC column (250 mm, 10
mm, 5 µm). Most of the oximes were purified using gradient elution
3
from 0 to 30% CH CN in 0.1 M aqueous TEAA over the course of 2
h using UV detection at 254 nm. An exception was oxime 3-(3)-27,
which is prone to air oxidation. In this case, 25 mM 2-mercaptoethanol
was added to both of the running buffers. The oximes all eluted with
baseline resolution in the order U-U homodimer, U-R heterodimer,
followed by the R-R homodimer. This HPLC method was also used
to confirm the expected 1:2:1 stoichiometries of homodimer and
heterodimer oxime formation, using 10 representative uracil and aryl
aldehydes from the library (see Supporting Information Figure S1).
Additional NMR evidence supporting the expected stoichiometries is
detailed in the Supporting Information Figures S2 and S3.
in 95% yield: 1H NMR (400 MHz, DMSO-d
6
) δ 9.38 (s, 1 H), 9.21
s, 1 H), 8.52 (s, 1 H), 8.18 (s, 1 H), 6.88 (s, 1 H), 6.30 (s, 1 H), 3.80
(
(
13
s, 3 H); C NMR (125 MHz, DMSO-d
6
) δ 150.17, 148.88, 146.52,
1
38.61, 112.51, 107.77, 103.46, 61.28; UV/vis λmax 239, 274 nm; HRMS
+
(
m/z) [M + H] calcd for C
9: To a solution of 1 (10.8 mg, 0.063 mmol) in hot DMF (0.5
mL) were added sodium acetate (5.2 mg, 0.063 mmol) solution in water
0.1 mL) and O-methylhydroxylamine hydrochloride (5.3 mg, 0.063
8 4
H10NO 184.06, found 184.06.
4
(
mmol), and the solution was stirred at room temperature for overnight.
The solvents were removed in vacuo, and the residue was purified by
column chromatography using 10-15% (v/v) methanol in CH Cl ,
2 2
resulting in 90% yield (10.3 mg 50/50 mixture of trans and cis geometric
isomers): 1H NMR (400 MHz, chloroform-d) δ 9.56 (s, 1 H), 7.43 (t,
Synthesis of 2-R-Substituted 3,4-Dihydroxybenzaldehydes and
the Corresponding Mixed Oximes with 3. Aldehyde 43 was
synthesized by removing the methyl groups of the commercially
53
available 3,4-dimethoxy-6-fluorobenzaldehyde using BBr
The aldehydes 44 and 45 were synthesized by removing the methylene
group of the corresponding 2-halogenated piperonal using AlCl and
N HCl. Aldehyde 46 was commercially available. These four
3 2 2
in CH Cl .
J ) 5.2 Hz,0.5 H), 7.20 (m, 1 H), 6.80 (t, J ) 4.4 Hz,0.5 H), 5.78 (m,
1
H), 4.55 (d, J ) 4.4 Hz,1 H), 4.48 (d, J ) 5.6 Hz,1 H), 3.94 (s, 1.5
3
13
H), 3.87 (s, 1.5 H); C NMR (100 MHz, DMSO-d
6
) δ 163.92, 163.87,
5
4
6
1
6
51.06, 150.99, 144.72, 144.54, 143.93, 143.53, 103.14, 102.97, 62.68,
aldehydes (43-46) were reacted with 6-formyluracil 3 and the O,O′-
diaminopropanediol linker using the procedure described above, and
+
2.37, 46.52, 43.76; UV/vis λmax 263 nm; HRMS (m/z) [M + H] calcd
184.07, found 184.07.
0: To a solution of 2 (70 mg, 0.5 mmol) in hot DMF (1 mL) were
7 10 3 3
for C H N O
the mixed oxime dimer was obtained after HPLC purification.
5
1
3
-(3)-43: H NMR (400 MHz, DMSO-d
6
) δ 8.11 (s, 1 H), 7.94 (s,
added sodium acetate (41 mg, 0.5 mmol) solution in water (0.5 mL)
and O-methylhydroxylamine hydrochloride (42 mg, 0.5 mmol), and
the solution was stirred at room temperature for 4 h. The solvents were
removed in vacuo, and the residue was collected by filtration and
washed with cold water 2 × 1 mL, resulting in 76% yield (70 mg
1
H), 7.05 (d, J ) 7.2 Hz, 1 H), 6.58 (d, J ) 6.8 Hz, 1 H), 5.77 (s, 1
H), 5.10 (bs, H), 4.28 (t, J ) 6.8 Hz, 2 H), 4.16 (t, J ) 6.0 Hz, 2 H),
1
3
2
1
1
6
.07 (m, 2 H); C NMR (125 MHz, DMSO-d ) δ 163.91, 155.50,
53.09, 151.08, 149.87, 149.76, 144.65, 142.72, 142.30, 142.21, 110.91,
10.87, 108.62, 108.49, 103.21, 102.96, 101.67, 71.85, 70.06, 28.38;
F NMR (DMSO-d
68 nm; HRMS (m/z) [M + Na] calcd for C15
7/13 mixture of trans and cis geometric isomers): 1H NMR (400 MHz,
8
1
9
6
) δ -54.33, -54.35, -54.36, -54.38; UV/vis λmax
DMSO-d
6
) δ 11.40 (bs, 2 H), 8.52 (s, 0.13 H), 7.87 (s, 0.87 H), 7.74
+
2
4 6
H15FN O Na 389.09,
13
(s, 0.87 H), 7.29 (s, 0.13 H), 3.89 (s, 0.39 H), 3.80 (s, 2.61 H);
C
found 389.09.
6
NMR (100 MHz, DMSO-d ) δ 162.98, 162.36, 150.80, 150.27, 146.08,
1
3
-(3)-44: H NMR (400 MHz, DMSO-d
6
) δ 8.23 (s, 1 H), 7.94 (s,
1
42.26, 140.09, 137.31, 104.41, 103.43, 62.32, 61.44; UV/vis λmax 288
1
H), 7.18 (s, 1 H), 6.77 (s, 1 H), 5.78 (s, 1 H), 4.27 (t, J ) 5.6 Hz, 2
+
nm; HRMS (m/z) [M + H] calcd for C
H N O
6 8 3 3
170.06, found 170.06.
1
3
H), 4.16 (t, J ) 6.0 Hz, 2 H), 2.08 (m, 2 H); C NMR (125 MHz,
DMSO-d ) δ 163.90, 151.08, 149.71, 145.48, 145.16, 144.64, 142.21,
23.00, 119.09, 116.08, 112.33, 101.67, 71.85, 70.22, 28.37; UV/vis
5
1: To a solution of 3 (79 mg, 0.5 mmol) in hot DMF (2.0 mL)
6
were added sodium acetate (46 mg, 0.5 mmol) solution in water (0.5
mL) and O-methylhydroxylamine hydrochloride (46 mg, 0.5 mmol),
and the solution was stirred at 50 °C for 4 h. The solvents were removed
in vacuo, and the residue was washed by cold water. After the filtration,
1
λ
4
+
4 6
max 275 nm; HRMS (m/z) [M + Na] calcd for C15H15ClN O Na
05.06, found 405.06.
1
3
-(3)-45: H NMR (400 MHz, DMSO-d
6
) δ 8.18 (s, 1 H), 7.94 (s,
1
product was obtained in 62% yield (53 mg): H NMR (400 MHz,
1
H), 7.19 (s, 1 H), 6.93 (s, 1 H), 5.78 (s, 1 H), 4.27 (t, J ) 6.4 Hz, 2
DMSO-d
6
) δ 11.18 (s, 1 H), 10.77 (s, 1 H), 7.91 (s, 1 H), 5.77 (s, 1
H), 3.96 (s, 3 H); 1 C NMR (125 MHz, DMSO-d
3
6
) δ 163.87, 151.02,
(
53) Kirk, K. L.; Cantacuzene, D.; Nimitkitpaisan, Y.; Mcculloh, D.; Padgett,
W. L.; Daly, J. W.; Creveling, C. R. J. Med. Chem. 1979, 22, 1493-1497.
54) Reitz, A.; Avery, M. A.; Verlander, M. S.; Goodman, M. J. Org. Chem.
(
(55) Watanabe, T.; Suzuki, T.; Umezawa, Y.; Takeuchi, T.; Otsuka, M.;
1
981, 46, 4859-4863.
Umezawa, K. Tetrahedron 2000, 56, 741-752.
J. AM. CHEM. SOC.
9
VOL. 127, NO. 49, 2005 17419