Uracil-Containing Histone Deacetylase Inhibitors
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 20 6053
monium fluoride trihydrate (2.75 mmol, 0.87 g) was added, and
the resulting mixture was stirred at 70 °C for 8 h. After treatment
with cold water (100 mL), the resulting precipitate was filtered and
washed to furnish 3k (0.34 g), which was purified by crystallization.
1H NMR (CDCl3) δ 1.32 (t, 3H, CH2CH3), 3.87 (s, 2H, PhCH2),
4.24 (q, 2H, CH2CH3), 6.02 (s, 1H, C5-H), 6.14 (d, 1 H, CHd
CHCO), 7.29 (m, 5H, benzene ring), 8.29 (d, 1H, CH)CHCO),
12.56 (s, 1H, NH). Anal. C, H, N, S.
behavior for this aspect. Selected UBHAs were tested on human
myeloid leukemic U937 cells to determine the effect on cell
cycle, apoptosis, proliferation, and granulocyte differentiation.
The majority of tested compounds after 48 h at 1 µM showed
an arrest of the cell cycle in G1 phase (data not shown), whereas
only compounds 1j and 1i were able to induce G1 arrest at 24
h. The two cinnamyl-N-hydroxyamides 1j and 1x induced
apoptosis (8.8% and 8.0%, respectively) slightly lower than that
of SAHA (10%) when used at the concentration of 1 µM. At 5
µM, SAHA-treated cells underwent massive apoptosis (98%),
whereas with UBHAs, no increase of apoptosis induction was
recorded. In antiproliferative assays, at 5 µM, 1j showed the
strongest effect among the UBHAs tested on U937 cells, and it
was, however, less potent than SAHA and MS-275.
Tested as cytodifferentiating agents, at 1 µM, only 1j showed
high CD11c expression level (42.5%), again being less efficient
than SAHA. By increasing the dose (5 µM), 1i, 1o, and 1y were
able to induce granulocytic differentiation with the same potency
as that of SAHA, whereas 1j was more effective.
In U937 cells, the capability of selected UBHAs to induce
p21 expression as well as acetylate histone H3 and R-tubulin
was also evaluated. The distinct capabilities of UBHA com-
pounds to influence the cell cycle, differentiation, proliferation,
and apoptosis as well as the capacity of p21 induction and
histone H3 and R-tubulin acetylation might be interpreted as
an advancement in the correlation between chemical modeling
and biological functions.
General Procedure for the Synthesis of Ethyl Esters of 2-,
3-, and 4-(3,4-Dihydro-4-oxo-6-substituted-2-pyrimidinylthio)-
methylcinnamic Acids (3h-j,w-y). Example: Ethyl Ester of
3-(3,4-Dihydro-4-oxo-6-benzyl-2-pyrimidinylthio)methylcinnam-
ic Acid (3x). A mixture of 6-benzyl-4-hydroxy-2-mercaptopyrimi-
dine (2e) (6.87 mmol, 1.5 g), crude ethyl 3-bromomethylcinnamate
(6b)59 (7.56 mmol, 2.2 g), and anhydrous potassium carbonate (7.56
mmol, 1.0 g) in 3 mL of anhydrous DMF was stirred at room
temperature for 1 h. After treatment with cold water (100 mL), the
aqueous phase was extracted with ethyl acetate (3 × 40 mL). The
organic phase was washed with brine (3 × 40 mL), dried, and
evaporated to dryness to furnish crude 3x, which was purified by
chromatography on a silica gel column, eluting with a mixture ethyl
acetate/hexane (1:1) to give the desired product as a white solid
1
(1.2 g). H NMR (CDCl3) δ 1.33 (t, 3H, CH2CH3), 3.83 (s, 2H,
PhCH2), 4.26 (q, 2H, CH2CH3), 4.38 (s, 2H, CH2S), 5.98 (s, 1H,
C5-H), 6.40 (d, 1H, CHdCHCO), 7.33 (m, 9H, two benzene rings),
7.61 (d, 1H, CH)CHCO), 13.20 (s, 1H, NH). Anal. C, H, N, S.
General Procedure for the Synthesis of N-Hydroxy-ω-(3,4-
dihydro-4-oxo-6-substituted-2-pyrimidinylthio)alkanamides (1b-
g,l-o,p,q,a′-f ′). Example: N-Hydroxy-6-(6-benzyl-3,4-dihydro-
4-oxopyrimidin-2-ylthio)hexanamide (1o). To hydroxylamine
hydrochloride (39 mmol, 2.7 g) solution in dry ethanol (5 mL),
potassium hydroxide (39 mmol, 2.2 g) solution in dry ethanol (5
mL) was added at 40 °C. The mixture was cooled at 0 °C and then
filtered, and to the clear solution, 3o (4.2 mmol, 1.5 g) and well-
crushed potassium hydroxide (7.3 mmol, 0.4 g) were added. After
1 h, the mixture was diluted with water (50 mL), made neutral
with 2 N HCl, and filtered under vacuum. The solid 1o was
collected, dried, and recrystallized by acetonitrile/methanol. 1H
NMR (DMSO-d6) δ 1.27 (m, 2H, CH2CH2CH2S), 1.44 (m, 2H,
CH2CH2CO), 1.50 (m, 2H, CH2CH2S), 1.92 (t, 2H, CH2CO), 3.01
(t, 2H, CH2S), 3.72 (s, 2H, PhCH2), 5.92 (s, 1H, C5-H), 7.24 (m,
5H, benzene ring), 8.66 (s, 1H, NHOH), 10.33 (s, 1H, NHOH),
12.45 (s, 1H, NH uracil ring). Anal. C, H, N, S.
Experimental Section
Chemistry. Melting points were determined on a Buchi 530
melting point apparatus and are uncorrected. Infrared (IR) spectra
(KBr) were recorded on a Perkin-Elmer Spectrum One instrument.
1H NMR spectra were recorded at 400 MHz on a Bruker AC 400
spectrometer; chemical shifts are reported in δ (ppm) units relative
to the internal reference, tetramethylsilane (Me4Si). All compounds
were routinely checked by TLC and 1H NMR. TLC was performed
on aluminum-backed silica gel plates (Merck DC, Alufolien
Kieselgel 60 F254) with spots visualized by UV light. All solvents
were reagent grade and, when necessary, were purified and dried
by standard methods. The concentration of solutions after reactions
and extractions involved the use of a rotary evaporator operating
at reduced pressure of ca. 20 Torr. Organic solutions were dried
over anhydrous sodium sulfate. Analytical results are within
(0.40% of the theoretical values. A SAHA sample for biological
assays was prepared as previously reported by us.61 All chemicals
were purchased from Aldrich Chimica, Milan (Italy) or from
Lancaster Synthesis GmbH, Milan (Italy) and were of the highest
purity.
General Procedure for the Synthesis of 3-(3,4-Dihydro-4-oxo-
6-substituted-2-pyrimidinylthio)-2-propenoic Acids (4a,b,h),
6-(3,4-Dihydro-4-oxo-6-(un)substituted-2-pyrimidinylthio)-
hexanoic Acids (4c-g,i-k), and 2-, 3-, and 4-(3,4-Dihydro-6-
substituted-4-oxopyrimidin-2-ylthio)methylcinnamic Acids (4l-
q). Example: 6-(3,4-Dihydro-4-oxopyrimidin-2-ylthio)hexanoic
Acid (4i). A mixture of 3g′ (1.1 mmol, 0.3 g), 2 N KOH (8.8 mmol,
0.49 g), and EtOH (5 mL) was stirred at room temperature for 18
h. The solution was poured into water (50 mL) and extracted with
ethyl acetate (2 × 20 mL). HCl (2 N) was added to the aqueous
layer until the pH 5, and the precipitate was filtered and recrystal-
General Procedure for the Synthesis of Ethyl ω-(3,4-Dihydro-
4-oxo-6-(un)substituted-2-pyrimidinylthio)alkanoates (3b-g,l-
q,v-d′). Example: Ethyl 6-(6-Benzyl-3,4-dihydro-4-oxopyrimidin-
2-ylthio)hexanoate (3o). A mixture of 6-benzyl-4-hydroxy-2-
mercaptopyrimidine (2e) (9.16 mmol, 2.0 g), ethyl 6-bromohexanoate
(10 mmol, 1.8 mL), and anhydrous potassium carbonate (10 mmol,
1.4 g) in 3 mL of anhydrous DMF was stirred at room temperature
for 1 h. After treatment with cold water (100 mL), the obtained
precipitate was filtered and washed to furnish 3o (1.6 g), which
1
lized to yield the title compound 4i (0.23 g) as a pure solid. H
NMR (DMSO-d6) δ 1.32 (m, 2H, CH2CH2CH2S), 1.49 (m, 2H,
CH2CH2CO), 1.61 (m, 2H, CH2CH2S), 1.93 (t, 2H, CH2CO), 3.06
(t, 2H, CH2S), 6.07 (s, 1H, C5-H), 7.83 (s, 1H, C6-H), 12.2 (s,
1H, COOH). Anal. C, H, N, S.
General Procedure for the Synthesis of N-Hydroxy-3-(3,4-
dihydro-4-oxo-6-substituted-2-pyrimidinylthio)-2-propen-
amides (1a,k,z), N-Hydroxy-6-(3,4-dihydro-4-oxo-6-(un)sub-
stituted-2-pyrimidinylthio)hexanamides (1p-t,g′-i′), and N-
Hydroxy-2-, -3-, and -4-(3,4-dihydro-6-substituted-4-oxopyri-
midin-2-ylthio)-methylcinnamylamides (1h-j,w-y). Example:
N-Hydroxy-6-(3,4-dihydro-4-oxopyrimidin-2-ylthio)hexana-
mide (1g′). To a 0 °C cooled solution of 4i (0.9 mmol, 0.22 g) in
dry tetrahydrofuran (5 mL), ethyl chloroformate (2.2 mmol, 0.21
mL) and triethylamine (2.3 mmol, 0.33 mL) were added, and the
mixture was stirred for 10 min. The solid was filtered off, and to
the filtrate was added O-(2-methoxy-2-propyl)hydroxylamine56 (5.4
1
was purified by crystallization. H NMR (CDCl3) δ 1.25 (t, 3H,
CH2CH3), 1.40 (m, 2H, CH2CH2CH2CO), 1.63 (m, 4H, CH2CH2-
CO and CH2CH2S), 2.28 (t, 2H, CH2CO), 3.13 (t, 2H, CH2S), 3.80
(s, 2H, PhCH2), 4.13 (q, 2H, CH2CH3), 5.96 (s, 1H, C5-H), 7.28
(m, 5H, benzene ring), 12.87 (s, 1H, NH). Anal. C, H, N, S.
General Procedure for the Synthesis of Ethyl 3-(3,4-Dihydro-
4-oxo-6-substituted-2-pyrimidinylthio)-2-propenoates (3a,k,z).
Example: Ethyl 3-(6-Benzyl-3,4-dihydro-4-oxopyrimidin-2-
ylthio)-2-propenoate (3k). To a solution of 6-benzyl-4-hydroxy-
2-mercaptopyrimidine (2e) (2.29 mmol, 0.50 g) and ethyl propiolate
(2.75 mmol, 0.28 mL) in anhydrous THF (10 mL), tetrabutylam-