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We report the synthesis of novel
C5-arylalkynyl acyclic pyrimidine analogues with α,
β-unsaturated carbonyl structures, at the acyclic portion, using a
double Wittig reaction. The newly synthesized compounds were tested for their
cytostatic activity against a broad panel of cancer cells. The antiviral assays
showed that (E)-ethyl 4-(((E)-4-ethoxy-1-((2-fluorophenyl)ethynyl-uracil)-4-oxobout-2-en-1-yl)oxy)-5-hydropent-2-enoate (13a) had a noticeable activity against TK- VZV strain (EC50 20 μΜ, MCC 100 μΜ).
Keywords: C5-pyrimidine acyclic nucleosides, Cytotoxic antiviral activity
Abbreviations:
13C NMR: Carbon-13 Nuclear
Magnetic Resonance Spectroscopy; 1H NMR: Proton Magnetic Resonance
Spectroscopy; AcOH: Acetic Acid; CD3OD: Deuterated
Methanol; CDCl3:
Deuterated Chloroform; CH2Cl2: Dichloromethane; CH3CN: Acetonitrile; CuI:
Copper(I) Iodide; DMF: Dimethyl Formamide; DMSO-d6: Deuterated Dimethyl Sulfoxide; Et3N:
Triethylamine; EtOAc:
Ethyl Acetate; HMDS: Hexamethyldisilazane;
MeOH: Methanol; MW: Microwave; Na2SO4: Sodium Sulfate; NaHCO3:
Sodium Bicarbonate; Pd(PPh3)4:
Tetrakis(triphenylphosphine)palladium(0);
TLC: Thin Layer Chromatography; TMS: Tetramethylsilane;
TMSOTf: Trimethylsilyl Trifluoromethanesulfonate;
UV-Vis: Ultraviolet-Visible
Spectroscopy
INTRODUCTION
The synthesis of acyclic nucleosides such as
Acyclovir (ACV) [1] and the discovery of its important antiviral properties
have opened a new era in antiviral therapy, the study of acyclic nucleosides
with excellent biological properties against a broad band of RNA viruses.
Acyclovir is highly active against Herpes Simplex Virus (HSV), where it
prevents virus replication by interacting with the DNA polymerase after
metabolizing to its active triphosphate structure by disrupting DNA synthesis
of the virus.
Among
the numerous modified acyclic nucleosides with interesting biological
properties [2-4], C5 modified acyclic nucleosides of uracil have been investigated
as anticancer and antiviral agents [5-8]. Specifically Carmofur is used to treat colorectal cancer
[9] and inhibits acidic ceramidase (ASAH1) [10] which plays an important role
in the occurrence of metastatic breast cancer [11], while 1-[4-hydroxy-3-(hydroxymethyl)-1-butyl]-5-(1-azido-2-chloroethyl)uracil
proved to be effective in vitro
against Duck hepatitis B virus (EC50 0.31-1.55 µM) and
Cytomegalovirus (EC50 3.1 µM) [12]. Furthermore, C5-arylalkynyl nucleosides of
uracil showed antiviral activity, against Coxsackie virus B4, respiratory
syncytial virus and yellow fever virus [13-16] and cytotoxic activity against
murine leukemia, human T-lymphocyte, cervix carcinoma and hepatocellular
carcinoma cells [17].
Considering
the biological importance of acyclic nucleosides
EXPERIMENTAL SECTION
General methods
Melting points
were recorded in a Mel-Temp apparatus and are uncorrected. Thin Layer
Chromatography (TLC) was performed on Merck pre-coated 60F254 plates. Reactions
were monitored by TLC on silica gel, with detection by UV light (254 nm) or by
charring with sulfuric acid. Flash column chromatography was performed using
silica gel (240-400 mesh, Merck). 1H and 13C NMR spectra
were obtained at ambient temperature using a Bruker 300 spectrometer at 300 and
75.5 MHz, respectively using chloroform-d (CDCl3),
dimethylsulfoxide-d6 (DMSO-d6) or methanol-d4 (CD3OD)
with internal tetramethylsilane (TMS). Chemical shifts (δ) are given in ppm
measured downfield from TMS and spin-spin coupling constants in Hz. Mass
spectra were obtained on a ThermoQuestFinnigan AQA Mass Spectrometer
(electrospray ionization). All microwave irradiation experiments were carried
out in a dedicated CEM-Explorer and CEM Discover monomode microwave apparatus,
operating at a frequency of 2.45 GHz, with continuous irradiation power, from 0
to 300 W with utilization of the standard absorbance level of 300 W maximum
powers. The reactions were carried out in 10 mL glass tubes, sealed with a
Teflon septum and placed in the microwave cavity. Initially, microwave irradiation
of requisite Watts was used, and the temperature was ramped from room
temperature to the desired temperature. Once this was reached the reaction
mixture was held at this temperature for the required time. The reaction
mixture was continuously stirred during the reaction. The temperature was
measured with an IR sensor on the outer surface of the process vial. After the
irradiation period, gas jet cooling rapidly cooled the reaction vessel to
ambient temperature. Dichloromethane
was distilled from phosphorous pentoxide and stored over 4 Å molecular sieves. Acetonitrile and toluene
were distilled from calcium hydride and stored over 3 Å molecular sieves. Diethylether (Et2O) was freshly distilled
under nitrogen from sodium/benzophenone before use. Pyridine was stored over
potassium hydroxide pellets, N,N-Dimethylformamide (DMF) was stored over 3 Å molecular sieves. All reactions sensitive to oxygen or
moisture were carried out under an Argon atmosphere.
Anti-proliferative assays
Compounds 4-9, 12-14, were evaluated for their cytostatic activity against the human cells:
pancreatic adenocarcinoma (Capan-1), chronic myeloid leukemia (Hap-1), colorectal carcinoma (HCT-116), lung carcinoma (NCI-H460), acute
lymphoblastic leukemia (DND-41), acute myeloid leukemia (HL-60), chronic
myeloid leukemia (K-562) and non-Hodgkin lymphoma
(Z-138). All assays were performed in 96-well
microtiter plates. To each well (5-7.5) × 104 tumor cells were
added,along withand varying concentrations of the test compounds ranging from
250, 50, 10, 2, 0.4 to 0.08 µM. The tumor cells were then allowed to
proliferate at 37°C in a humidified CO2-controlled atmosphere. To
obtain optimal growth curves, 2 days of the: pancreatic adenocarcinoma (Capan-1), chronic myeloid leukemia (Hap-1), colorectal carcinoma (HCT-116), lung
carcinoma (NCI-H460), acute lymphoblastic leukemia (DND-41), acute myeloid
leukemia (HL-60) and 3 days for the chronic
myeloid leukemia (K-562) and non-Hodgkin lymphoma
(Z-138), were required. At the end of the incubation
period, the cells were counted in a Coulter counter. The IC50 (50%
inhibitory concentration) was defined as the concentration of the compound that
inhibited cell proliferation by 50%.
Antiviral activity assays
The antiviral tests were based on inhibition of the virus-induced
cytopathicity in Human Embryonic Lung (HEL) (Varicella-Zoster Virus (VZV)) cell cultures. Confluent cell cultures in microtiter 96-well plates
were inoculated with 100 cell culture inhibitory dose-50 (CCID50) of
virus (1 CCID50 being the virus dose to infect 50% of the cell
cultures). After a 1 h virus adsorption period, the residual virus was removed,
and the cell cultures were incubated in the presence of varying concentrations
(200, 40, 8, … µM) of the test compounds. Viral cytopathicity was recorded as
soon as it reached completion in the control virus-infected cell cultures,
which were not treated with the test compounds.
Synthesis of C5 alkynyl
1-(1-((1,3-dihydropropan-2-yl)oxy)-2-hydroxyethyl)-5-iodouracil (7, 9)1-(5΄-O-Trityl-ribofuranozyl)-5-iodouracil
(2)
A solution of trityl chloride (2.8 g, 9.90 mmol) in anhydrous
dichloromethane (19 mL) was added drop wise to a solution of 1 (3.0 g, 8.26
mmol) in anhydrous pyridine (35 mL) at 0°C. Following the addition, the mixture
was allowed to warm slowly to room temperature and set aside for 12 h. Methanol
(7 mL) and ethyl acetate (50 mL) were added and the mixture was washed
successively with saturated aqueous NaHCO3 (200 mL) and H2O
(100 mL). The organic phase was dried over MgSO4 and evaporated
under reduced pressure. The resulting residue was crystallized from chloroform
to give 6 (2.3 g; 82%). m.p.
162-163°C;
[α]D22=-8 (c 0.10, CHCl3); Rf=0.27 (EtOAc/Hexane, 7:3); λmax
383 nm (ε 21460); 1H-NMR (300 MHz, CD3OD) δ 8.14 (s, 1H,
H-6), 7.49-7.24 (m, 15H, Tr), 5.90 (d, 1H, J1΄,2΄=4.8 Hz, H-1΄),
5.35-4.31 (m, 2H, H-2΄, H-4΄), 4.10 (t, 1H, J=2.7, J=1.2 Hz, H-3΄), 3.41 (dd,
1H, J4΄-5a΄=3.2 Hz, J5a΄-5b΄=10.8 Hz, H-5b΄), 4.55 (dd,
1H, J4΄-5b΄=2.1 Hz, H-5a΄); 13C NMR (75.5 MHz, CDCl3)
δ 161.80, 150.83, 144.19, 143.65, 129.71, 128.34, 126.28, 97.67, 94.83, 86.93,
73.22, 70.11, 68.42, 63.92; Anal. Calcd. for C9H11IN2O6: C, 59.41; H, 4.11; N, 4.57% found: C,
59.61; H, 4.41; N, 4.97%; Mass (M+H)+: 612.08.
1-(2-Hydroxy-1-(1-hydroxy-3-(trityloxy)propan-2-yl)oxy)ethyl)-5-iodouracil (3)
Compound 2 (1.1 g, 1.8 mmol) was added to a stirred solution of NaIO4
(425 mg, 1.98 mmol) in H2O (19 mL) and MeOH (19 mL) leading to
immediate precipitation of NaIO3. After 1 h at room temperature, any
residual periodate was destroyed with a drop of ethylene glycol. The reaction
mixture was stirred for 1 h at room temperature with NaBH4 (500 mg,
18.5 mmol), neutralized with aqueous NaHCO3 and then extracted with
ethyl acetate (EtOAc) (4 × 300 mL). The organic layer was washed with NaHSO4,
dried over anhydrous Na2SO4, evaporated to dryness and
purified by column chromatography with EtOAc/Hexane (7:3) to give compound 3
(650 mg, 86%) as a syrup. [α]D22+13 (c 0.10, MeOH); Rf=0.35 (EtOAc/Hexane,
7:3); λmax 305 nm (ε21400); 1HNMR
(300 MHz, CDCl3) δ 9.97 (brs, 1H, NH), 7.84 (s, 1H, H-6), 7.39-7.25
(m, 15H,Tr), 5.99 (t, 1H, J1΄,2a΄=5.2 Hz, J1΄,2b΄=5.0
Hz, H-1΄), 3.87-3.72 (m, 7H, H-2΄, H-3΄, H-4΄, OH, OH), 3.26 (dd, 1H, J4΄-5a΄=3.7
Hz, J5a΄-5b΄=10.8
Hz, H-5a΄), 3.16 (dd, 1H, J4΄-5b΄=6.0 Hz, H-5b΄); 13C NMR (75.5 MHz, CDCl3)
δ 161.80, 150.83, 144.19, 143.65, 129.71, 128.34, 126.28, 96.25, 94.83,
83.63, 68.42, 63.92, 61.81, 61.13; Anal. Calcd for C28H27IN2O6:
C, 54.73; H, 4.43; N, 4.56. Found: C, 54.33; H, 4.83; N, 4.96; ESI-MS (m/z):
614.09 (M+H+).
1-(1-((1,3-Dihydroxypropan-2-yl)oxy)-2-hydroxyethyl)-5-iodouracil
(4)
To a solution containing 3 (1 g, 1.62 mmol) is added 1: 1 mixture of
formic acid (HCOOH)/diethyl ether (Et2O) (29 ml) and reflux for 30
min. The mixture is then neutralized with solid sodium bicarbonate (NaHCO3)
and extracted sequentially with sodium chloride (NaCl) and water (H2O),
dried over magnesium sulphate (MgSO4), evaporated to dryness and
purified by column chromatography with CH2Cl2/MeOH (9:1)
to give compound 4 (554 mg, 92%) as a syrup. [α]D22+9 (c 0.14, MeOH);
Rf=0.25 (CH2Cl2/MeOH 9:1); λmax 286 nm (ε18592); 1HNMR
(300 MHz, CD3OD) δ 8.12 (s, 1H, H-6), 7.55-7.11 (m, 4H, Bz),
8.99 (t, 1H, J=4.9 Hz, J=4.9 Hz N-CH-C), 3.80-3.51 (m, 7H, 3x -CH2OH,
C-CH-C); 13C NMR (75.5 MHz, CD3OD) δ 161.30, 150.83, 144.19, 96.25, 94.83, 80.63, 68.22, 60.92, 58.32;
Anal. Calcd for C9H13IN2O6: C, 29.05; H, 3.52; N, 7.53. Found: C, 29.45; H, 3.92; N, 7.93; ESI-MS
(m/z): 372.98 (M+H+).
2-(2-Acetoxy-1-(5-iodouracil)ethoxy)propan-1,3-diyldiacetylo (5)
To a solution of 4 (84 mg, 0.23 mmol) added
dry pyridine (2 ml) and acetic anhydride (1 ml). The reaction was carried out
at room temperature for 1 h, then was quenched
with MeOH at 0°C and was concentrated in vacuum. The residue was diluted with ethyl acetate (EtOAc), washed with saturated NaHSO4,
NaHCO3 and H2O. The organic
extract was dried over anhydrous Na2SO4, filtered and
evaporated to dryness
to give compound 5 (80 mg, 95%) as a white crystal.
M.P. 215-217°C; [α]D22+12 (c 0.25, CHCl3); Rf=0.22
(EtOAc/Hexane, 2:8); λmax 286 nm (ε 16459); 1HNMR
(300 MHz, CDCl3) δ 8.47 (brs, 1H, NH), 7.85 (s, 1H, H-6), 6.13
(t, 1H, J=5.1 Hz, J=5.3 Hz N-CH-C), 4.46-3.95 (m, 7H, 3x-CH2OAc,
C-CH-C), 2.13, 2.09, 2.07 (3s, 9H, 3x-OAc);13CNMR (75.5 MHz, CDCl3)
δ 170.48, 170.44. 169.98, 159.46, 150.24, 144.14, 81.34, 75.76, 68.85,
63.36, 63.05, 62.50, 21.06, 20.67, 20.49; Anal. Calcd for C15H19IN2O9: C, 36.16; H, 3.84, N 5.62. Found: C, 36.56;
H, 4.04, N 5.92; ESI-MS (m/z): 416.99 (M+H+).
General procedure
for the preparation of the C5-arylalkynyl uracil acyclic nucleosides-6,8
Mixtures of the appropriate alkynes (0.72 mmol), Pd (PPh3)4
(28 mg, 0.02 mmol), CuI (5.3 mg, 0.02 mmol), triethylamine (116 μl, 0.34 mmol)
and 2-(2-Acetoxy-1-(5-iodouracil)ethoxy)propan-1,3-diyldiacetylo (5) (100 mg, 0.24
mmol) in 1.0 mL of anhydrous DMF, were irradiated in a microwave apparatus (200
W maximum power) for 5 min at 50°С. The reaction mixture was concentrated under
reduced pressure and the crude residue was purified by flash chromatography on silica
gel. The purified material was dried in vacuo to afford the corresponding
derivatives 6, 8 in 78-81% yields.
2-(2-Acetoxy-1-(5-((2-fluorophenyl)ethynyl)-uracil)ethoxy)propan-1,3-diyl
diacetyl (6a)
75 mg, 78% as white foam; [α]D22=+12
(c 0.15, CHCl3); Rf=0.24 (EtOAc/Hexane 2:8); λmax 286 nm
(ε17254); 1HNMR (300 MHz, CDCl3)
δ 8.70 (brs, 1H, NH), 7.80 (s, 1H, H-6), 7.52 (t, 1H, J=7.5 Hz, J=7.1
Hz, Bz), 7.35 (dd, 1H, J=7.2 Hz, J=13.8 Hz, Bz), 7.14-7.07 (m, 2H, Bz), 6.20
(t, 1H, J=5.3 Hz, J=5.6 Hz N-CH-C), 4.46-3.95 (m, 7H, 3x-CH2OAc,
C-CH-C), 2.13, 2.09, 2.08 (3s, 9H, 3xOAc); 13CNMR (75.5 MHz, CDCl3)
δ 170.60, 170.48. 170.00, 160.49, 149.61, 142.08, 133.56, 130.74,
130.62, 124.09, 124.03, 115.70, 115.37, 100.69, 81.30, 75.55, 63.39, 63.03,
62.44, 21.06, 20.67, 20.50; Anal. Calcd. for C23H23FN2O9:
C, 56.33; H, 4.73; N, 5.71%; Found: C, 56.73; H, 4.33; N, 5.91%; ESI-MS (m/z): Mass
(M+H)+: 491.14.
2-(2-Acetoxy-1-(5-((2-chlorophenyl)ethynyl)-uracil)ethoxy)propan-1,3-diyldiacetyl (6b)
80 mg, 79% as white
foam; [α]D22=+15
(c 0.19, CHCl3); Rf=0.26 (EtOAc/Hexane 2:8); λmax 286 nm (ε 17845); 1HNMR (300 MHz, CDCl3)
δ 8.56 (brs, 1H, NH), 7.80 (s, 1H, H-6), 7.55 (t, 1H, J=7.5 Hz, J=7.1
Hz, Bz), 7.41 (dd, 1H, J=7.2 Hz, J=13.8 Hz, Bz), 7.30-7.22 (m, 2H, Bz), 6.19
(t, 1H, J=5.2 Hz, J=5.5 Hz N-CH-C), 4.47-3.93 (m, 7H, 3x-CH2OAc,
C-CH-C), 2.14, 2.09 (2s, 9H, 3xOAc); 13CNMR (75.5 MHz, CDCl3)
δ 170.53, 170.48. 170.00, 160.33, 149.55, 142.09, 135.76, 133.45,
129.90, 129.33, 126.54, 122.19, 100.73, 91.03, 84.66, 81.36, 75.70, 63.38,
63.05, 62.47, 20.78, 20.67, 20.51; Anal. Calcd. for C23H23ClN2O9:
C, 54.50; H, 4.57; N, 5.53%; Found: C, 54.10; H, 4.27; N, 5.93%; ESI-MS (m/z): Mass
(M+H)+: 507.11.
2-(2-Acetoxy-1-(5-((1,4-dimethylphenyl)ethynyl)-uracily)ethoxy)propan-1,3-diyldiacetyl (6c)
80 mg, 79% as white
solid; M.P. 251-253°C; [α]D22=+12
(c 0.22, CHCl3); Rf=0.30 (EtOAc/Hexane 2:8); λmax 286 nm (ε 14523); 1HNMR (300 MHz, CDCl3)
δ 8.28 (brs, 1H, NH), 7.71 (s, 1H, H-6), 7.22 (t, 1H, J=4.5 Hz, J=5.9
Hz, Bz), 7.11 (d, 1H, J=4.5 Hz, Bz ), 7.06 (d, 1H, J=7.2 Hz, Bz), 6.19 (t, 1H,
J=5.6 Hz, J=5.6 Hz N-CH-C), 4.47-3.96 (m, 7H, 3x-CH2OAc, C-CH-C),
2.44, 2.29 (2s, 6H, 2x CH3)2.14, 2.08, 2.07 (3s, 9H, 3xOAc); 13CNMR (75.5 MHz, CDCl3) δ 170.53, 170.48. 170.00, 160.33, 149.55, 142.09, 135.76, 133.45,
129.90, 129.33, 126.54, 122.19, 100.73, 91.03, 84.66, 81.36, 75.70, 63.38,
63.05, 62.47, 20.74, 20.70, 20.62, 20.54, 20.21; Anal. Calcd. for C25H28N2O9:
C, 59.59; H, 5.64; N, 5.50%; Found: C, 59.59; H, 5.24; N, 5.20%; ESI-MS (m/z): Mass
(M+H)+: 501.18.
2-(2-Acetoxy-1-(5-((6-methoxynapthlene)ethynyl-uracil)ethoxy)propan-1,3-diyldiacetyl (8)
83 mg, 81% as white
solid; M.P. 286-289°C; [α]D22=-2
(c 0.16, CHCl3); Rf=0.30 (EtOAc/Hexane 2:8); λmax 286 nm (ε 16895); 1HNMR (300 MHz, CDCl3) δ 8.43 (brs, 1H, NH), 7.96 (s, 1H, H-6), 7.77 (s, 1H, napthalene), 7.70
(t, 2H, J=7.7 Hz, J=8.2 Hz, napthalene), 7.49 (d, 1H, J=8.7 Hz, napthalene),
7.16 (d, 1H, J=8.7 Hz, napthalene), 7.11 (s, 1H, napthalene), 6.20 (t, 1H,
J=5.4 Hz, J=5.6 Hz N-CH-C), 4.48-3.97 (m, 7H, 3x-CH2OAc, C-CH-C),
3.93 (s, 3H, OCH3), 2.14, 2.11, 2.09 (3s, 9H, 3xOAc); 13CNMR
(75.5 MHz, CDCl3) δ 170.53, 170.48. 170.00, 160.33, 158.92,
149.55, 142.09, 135.76, 133.45, 129.90, 129.33, 126.54, 122.19, 119.89, 117.50,
105.96, 100.73, 91.03, 84.66, 81.36, 75.70, 63.38, 63.05, 62.47, 55.41, 20.74,
20.70, 20.62; Anal. Calcd. for C28H28N2O10: C,
60.87; H, 5.11; N, 5.07%; Found: C, 60.47; H, 5.31; N, 5.27%; ESI-MS (m/z): Mass
(M+H)+: 553.17.
General procedure
for the preparation of the unprotected C5-arylalkynyl uracil acyclic
nucleosides-7,9
The protected nucleosides 6, 8 (0.12 mmol), were treated with
methanolic ammonia (saturated at 0°C, 6.7 mL). The solution was stirred
overnight at room temperature and then evaporated under reduced pressure. The
residue obtained was purified by flash column chromatography to afford the
unprotected derivatives 7, 9 in 65-85% yields, as white solids.
1-(1-((1,3-Dihydroxypropan-2-yl)oxy)-2-hydroxyethyl)-5-[(2-fluorophenyl) ethynyl] uracil (7a)
80 mg, 85% as white
solid; M.P. 189-192°C; [α]D22=+8
(c 0.24, MeOH); Rf=0.19 (CH2Cl2/MeOH 9:1); λmax 286 nm (ε 13564); 1HNMR (300 MHz, CD3OD) δ 8.12 (s, 1H, H-6), 7.55-7.11 (m, 4H, Bz), 8.99 (t,
1H, J=4.9 Hz, J=4.9 Hz N-CH-C), 3.80-3.51 (m, 7H, 3x-CH2OH, C-CH-C);
13CNMR (75.5 MHz, CD3OD) δ 162.79, 150.64, 144.70, 133.25, 130.25, 130.12,
123.99, 123.93, 115.24, 114.90, 98.71, 85.62, 84.36, 81.11, 62.21, 61.13, 60.69; Anal. Calcd. for C17H17FN2O6:
C, 56.04; H, 4.70; N, 7.69%; Found: C, 56.44; H, 4.30; N, 8.09%; ESI-MS (m/z): Mass
(M+H)+: 365.11.
1-(1-((1,3-Dihydroxypropan-2-yl)oxy)-2-hydroxyethyl)-5-[(2-chlorophenyl)
ethynyl] uracil (7b)
84 mg, 85% as white
solid; M.P. 195-197°C; [α]D22=+9
(c 0.23, MeOH); Rf=0.22 (CH2Cl2/MeOH 9:1); λmax 286 nm (ε 15732); 1HNMR (300 MHz, CD3OD) δ 8.12 (s, 1H, H-6), 7.59-7.27 (m, 4H, Bz), 5.99 (t,
1H, J1΄-2a΄=4.9 Hz, J1΄-2b΄=4.9 Hz N-CH-C), 3.79-3.53
(m, 7H, 3x-CH2OH, C-CH-C); 13CNMR (75.5 MHz, CD3OD) δ 164.16, 152.08, 146.15, 136.60, 134.53, 130.85,
127.90, 124.21, 100.19, 90.49, 87.23, 85.88, 82.58, 63.70, 62.81, 62.20; Anal. Calcd. for C17H17ClN2O6:
C, 53.62; H, 4.50; N, 7.36%; Found: C, 54.02; H, 4.30; N, 7.76%; ESI-MS (m/z): Mass
(M+H)+: 381.08.
1-(1-((1,3-Dihydroxypropan-2-yl)oxy)-2-hydroxyethyl)-5-[(1,4-dimethylphenyl)
ethynyl] uracil (7c)
88 mg, 85% as white
solid; M.P. 220-221°C; [α]D22=+12
(c 0.18, MeOH); Rf=0.28 (CH2Cl2/MeOH 9:1); λmax 282 nm (ε 18563); 1HNMR (300 MHz, CD3OD) δ 8.07 (s, 1H, H-6), 7.25 (s, 1H, ArH), 7.10 (d, 1H,
J=7.6, Bz), 7.04 (d, 1H, Bz), 5.99 (t, 1H, J=4.9 Hz, J=4.9 Hz N-CH-C), 3.80-3.52
(m, 7H, 3x-CH2OH, C-CH-C), 2.40, 2.26 (2s, 6H, 2xCH3); 13CNMR
(75.5 MHz, CD3OD) δ 164.16, 152.08, 146.15,
136.60, 134.53, 130.85, 127.90, 124.21, 100.19, 90.49, 87.23, 85.88, 82.58,
63.70, 62.81, 62.20, 19.37, 18.94.; Anal. Calcd. for C19H22N2O6:
C, 60.95; H, 5.92; N, 7.48%; Found: C, 60.55; H, 5.62; N, 7.08%; ESI-MS (m/z): Mass
(M+H)+: 375.15.
1-(1-((1,3-Dihydroxypropan-2-yl)oxy)-2-hydroxyethyl)-5-[(6-methoxynapthalene)
ethynyl] uracil (9)
88 mg, 65% as white
solid; M.P. 162-163°C; [α]D22=+15
(c 0.17, MeOH); Rf=0.15 (CH2Cl2/MeOH 9:1); λmax 286 nm (ε 19654); 1HNMR (300 MHz, CD3OD) δ 8.12 (s, 1H, H-6), 7.94-7.12 (m, 6H, naphthalene),
5.99 (t, 1H, J=5.15 Hz, J=5.13 Hz N-CH-C), 3.90 (s, 3H, OCH3), 3.82-3.53
(m, 7H, 3x-CH2OH, C-CH-C); 13CNMR (75.5 MHz, CD3OD)
δ 164.57, 160.07, 152.13,
145.48, 135.85, 132.14, 130. 38, 129.95, 129.69, 128.09, 120.60, 119.17,
106.90, 100.77, 94.59, 85.79, 82.55, 81.41, 63.69, 62.82, 62.16, 55.89; Anal. Calcd. for C22H22N2O7:
C, 61.97; H, 5.20; N, 6.57%; Found: C, 62.27; H, 5.60; N, 6.97%; ESI-MS (m/z): Mass
(M+H)+: 427.14.
Synthesis of (E)-ethyl 4-(((E)-4-ethoxy-1-(5-iodo-uracil)-4-oxobout-2-en-1-yl)oxy)-5-(trityloxy)pent-2-enoate (11)
A solution of sodium periodate (0.23 g, 1.09
mmol) in water (5 mL) was slowly added to a cooled stirred solution of the
protected nucleoside 2 (0.612 g, 1 mmol) in methanol (10 mL). The reaction mixture was
stirred at room temperature for 1 h and then filtered to remove the salts. The
solution was diluted with ethyl acetate (15 mL), washed with a saturated
solution of NaCl (2 × 15 mL) and dried over MgSO4. The solvent was
removed by evaporation under reduced pressure to yield a white powder of the
corresponding dialdehyde derivative. The crude dialdehyde was dissolved in
freshly distilled tetrahydrofuran (5 mL) and (ethoxycarbonylmethylene) triphenylphosphorane
(0.871 g, 2.5 mmol) added and the mixture
was heated at 40°C for 1 h under nitrogen. The reaction mixture washed twice
with a saturated solution of ammonium chloride (2 × 30 mL), dried over MgSO4,
evaporated to a yellow syrup and purified by column chromatography (ethyl
acetate/hexane, 2:8) to afford the protected nucleoside 11 (0.435 g, 53%) as a foam. [α]D22 +22 (c 0.25, CHCl3);
Rf=0.52 (EtOAc/Hexane, 2:8); λmax 286 nm (ε 24538); 1HNMR
(300 MHz, CDCl3) δ 8.62 (s, 1H, ΝΗ), 7.61 (s, 1H, H-6), 7.41-7.25 (m, 15H,
trityl), 6.72 (dd, 1H, J=3.5 Hz, J=15.6 Hz, Η-3), 6.69 (dd, 1H,
J=6.7 Hz, J=15.8 Hz, Η-2), 6.35 (dd, 1H, J=6.7 Hz, J=15.8 Hz, Η-2΄), 6.29 (t, 1H, J=3.4 Hz, Η-1), 6.11 (dd, 1H, J=1.2 Hz, J=15.8 Hz, Η-3΄), 4.28 (dd, 2H, J=6.1 Hz, J=13.3 Hz, CH2), 4.21 (dd, 2H,
J=7.1 Hz, J=14.2 Hz, CH2), 4.03-3.99 (m, 1H, H-4), 3.38 (dd, 1H,
J=7.4 Hz, J=11.0 Hz, -Η-5), 3.23 (dd, 1H, J=3.4 Hz, Η-5΄), 1.35 (t, 3H, J=7.2 Hz, CH3), 1.29 (t, 3H, J=7.2 Hz, CH3);
13CNMR (75.5 MHz, CDCl3) δ 165.32, 164.96,
159.38, 150.07, 144.07, 143.36, 141.21, 139.76, 128.64, 128.47, 128.05, 127.96,
127.90, 127.30, 127.23, 127.14, 126.10, 125.27, 87.38, 79.79, 77.24, 70.25,
65.56, 61.26, 60.90, 14.15; Anal. Calcd for C36H35ΙN2O8: C, 57.61; H, 4.70, N 3.73. Found: C, 58.01;
H, 5.10, N 4.13; ESI-MS (m/z): 751.14 (M+H+).
Synthesis of (E)-ethyl
4-(((E)-4-ethoxy-1-(5-iodo-uracil)-4-oxobout-2-en-1-yl)oxy)-5-hydroxypent-2-enoate (12)
Compound 12 was synthesized from 11 by a
similar procedure to that described for the preparation of 4. The crude product
was purified by flash column chromatography (EtOAc/Hexane, 3:7) to give
analogue 12 (88 mg, 95%) as a white solid. M.P. 182-184°C; [a]D22=23 (c
0.31, CHCl3); Rf=0.35 (EtOAc/ Hexane, 3:7); λmax 286 nm
(ε 24167)); 1HNMR (300
MHz, CDCl3) δ 8.80 (s, 1H, ΝΗ), 7.71 (s, 1H, H-6), 6.79 (dd, 1H, J=6.7 Hz,
J=15.3 Hz, Η-2 ), 6.75 (dd, 1H, J=3.9 Hz, J=15.8 Hz, Η-3), 6.38 (t, 1H,
J=3.3 Hz,Η-1), 6.35 (dd, 1H, J=1.2 Hz, J=15.8 Hz, Η-3΄), 6.11 (dd, 1H, J=1.2 Hz, J=15.8 Hz, Η-2΄), 4.32 (s, 1H, OH), 4.28-4.18 (m, 4H, 2xCH2, H-4), 3.74
(dd, 1H, J=2.9 Hz, J=11.9 Hz, -Η-5), 3.67 (dd, 1H, J=7.5 Hz, Η-5΄), 1.34 (t, 3H, J=7.2 Hz, CH3), 1.31 (t, 3H, J=7.2 Hz, CH3);
13CNMR (75.5 MHz, CDCl3) δ 165.48, 165.01,
159.96, 150.61, 144.68, 144.04, 139.63, 126.23, 125.15, 80.59, 79.07, 69.76,
64.25, 61.29, 60.98, 14.14; Anal. Calcd.for C17H21ΙN2O8: C, 40.17; H, 4.16, N 5.51. Found: C, 40.57;
H, 4.36, N 5.81; ESI-MS (m/z): 509.03 (M+H+).
General procedure
for the preparation of the C5-arylalkynyl (E)-ethyl 4-(((E)-4-ethoxy-1-(5-iodo-uracil)-4-oxobout-2-en-1-yl)oxy)-5-hydroxypent-2-enoate (13, 14)
Mixtures of the appropriate alkynes (0.72 mmol), Pd (PPh3)4
(28 mg, 0.02 mmol), CuI (5.3 mg, 0.02 mmol), triethylamine (116 μl, 0.34 mmol)
and (E)-ethyl
4-(((E)-4-ethoxy-1-(5-iodo-uracil)-4-oxobout-2-en-1-yl)oxy)-5-hydroxypent-2-enoate (12) (100 mg, 0.20
mmol) in 1.0 mL of anhydrous DMF, were irradiated in a microwave apparatus (200
W maximum power) for 5 min at 50°С. The reaction mixture was concentrated under
reduced pressure and the crude residue was purified by flash chromatography on
silica gel. The purified material was dried in vacuo to afford the
corresponding derivatives 13, 14 in 69-82% yields.
(E)-ethyl 4-(((E)-4-ethoxy-1-((2-fluorophenyl)ethynyl-uracil)-4-oxobout-2-en-1-yl)oxy)-5-hydropent-2-enoate (13a)
75 mg, 74% as white
foam; [α]D22=+21
(c 0.14, CHCl3); Rf=0.36 (EtOAc/Hexane3:7); λmax 286 nm (ε 19547); 1HNMR (300 MHz, CDCl3) δ 8.83 (s, 1H, ΝΗ), 7.66 (s, 1H, H-6), 7.51 (dd, 1H, J=8.2 Hz,
J=7.4 Hz, Bz), 7.32 (dd, 1H, J=8.2 Hz, J=7.4 Hz, Bz), 7.01 (dd, 2H, J=6.7 Hz,
J=15.3 Hz, Bz), 6.81 (dd, 1H, J=3.9 Hz, J=15.8 Hz, Η-3), 6.77(dd, 1H, J=3.9 Hz, J=15.8 Hz, Η-2), 6.46 (t, 1H, J=3.3 Hz, Η-1), 6.39 (dd, 1H, J=1.2 Hz, J=15.8 Hz, Η-3΄), 6.13 (dd, 1H, J=1.2 Hz, J=15.8 Hz, Η-2΄), 4.36 (s, 1H, OH), 4.28-4.20 (m, 5H, 2xCH2,
H-4), 3.74 (dd, 1H, J=2.9 Hz, J=11.9 Hz , Η-5), 3.67 (dd, 1H, J=7.5 Hz, Η-5΄), 1.33 (t, 3H, J=7.2 Hz, CH3),
1.30 (t, 3H, J=7.2 Hz, CH3); 13CNMR (75.5 MHz, CDCl3)
δ 165.48, 165.01, 160.07, 159.96, 150.61,
144.68, 144.04, 139.63, 133.11, 126.23, 126.08, 125.15, 123.09, 115.01, 109.32,
93.25, 86.43, 80.59, 79.07, 69.76, 64.25, 61.29, 60.98, 14.14; Anal. Calcd. for C25H25FN2O8:
C, 60.00; H, 5.03; N, 5.60%; Found: C, 60.20; H, 5.13; N, 5.90%; ESI-MS (m/z): Mass
(M+H)+: 501.16.
(E)-ethyl 4-(((E)-4-ethoxy-1-((2-chlorophenyl)ethynyl-uracil)-4-oxobout-2-en-1-yl)oxy)-5-hydropent-2-enoate (13b)
75 mg, 78% as white
foam; [α]D22=+19
(c 0.15, CHCl3); Rf=0.42 (EtOAc/ Hexane 3:7); λmax 286 nm (ε 21547); 1HNMR (300 MHz, CDCl3) δ8.70 (s, 1H, ΝΗ), 7.66 (s, 1H, H-6), 7.56 (dd, 1H, J=8.2 Hz,
J=7.4 Hz, Bz), 7.40 (dd, 1H, J=8.2 Hz, J=7.4 Hz, Bz), 7.21 (dd, 2H, J=6.7 Hz,
J=15.3 Hz, Bz), 6.80 (dd, 1H, J=3.9 Hz, J=15.8 Hz, Η-3), 6.78 (dd, 1H, J=3.9 Hz, J=15.8 Hz, Η-2), 6.45 (t, 1H, J=3.3 Hz, Η-1), 6.39 (dd, 1H, J=1.2 Hz, J=15.8 Hz, Η-3΄), 6.14 (dd, 1H, J=1.2 Hz, J=15.8 Hz, Η-2΄), 4.34 (s, 1H, OH), 4.28-4.21 (m, 5H, 2xCH2,
H-4), 3.74 (dd, 1H, J=2.9 Hz, J=11.9 Hz, H-5), 3.67 (dd, 1H, J=7.5 Hz,H-5΄), 1.34 (t, 3H, J=7.2 Hz, CH3), 1.31 (t, 3H, J=7.2 Hz, CH3);
13CNMR (75.5 MHz, CDCl3) δ 165.48, 165.01, 160.07, 159.96, 150.61, 144.68, 144.04, 139.63,
133.11, 126.23, 126.08, 125.15, 123.09, 115.01, 109.32, 93.25, 86.43, 80.59,
79.07, 69.76, 64.25, 61.29, 60.98, 14.14; Anal. Calcd. for C25H25ClN2O8:
C, 58.09; H, 4.87; N, 5.42%; Found: C, 58.49; H, 4.47; N, 5.82%; ESI-MS (m/z): Mass
(M+H)+: 517.13.
(E)-ethyl4-(((E)-4-ethoxy-1-((1,4-dimethylphenyl)ethynyl-uracil)-4-oxobout-2-en-1-yl)oxy)-5-hydropent-2-enoate (13c)
79 mg, 82% as white
foam; [α]D22=+24
(c 0.18, CHCl3); Rf=0.42 (EtOAc/Hexane 3:7); λmax 286 nm (ε 21574); 1HNMR (300 MHz, CDCl3) δ 8.83 (s, 1H, ΝΗ), 7.57 (s, 1H, H-6), 7.27 (s, 1H, Bz), 7.07
(d, 1H, J=7.7 Hz, Bz), 7.02 (d, 1H, J=7.7 Hz, Bz), 6.80 (dd, 1H, J=3.9 Hz,
J=15.8 Hz, Η-3), 6.77 (dd, 1H, J=3.9 Hz, J=15.8 Hz, Η-2), 6.45 (t, 1H, J=3.3 Hz, Η-1), 6.39 (dd, 1H, J=1.2 Hz, J=15.8 Hz, Η-3΄), 6.14 (dd, 1H, J=1.2 Hz, J=15.8 Hz, Η-2΄), 4.35 (s, 1H, OH), 4.26-4.20 (m, 5H, 2xCH2,
H-4), 3.73 (dd, 1H, J=2.9 Hz, J=11.9 Hz Η-5), 3.67 (dd, 1H, J=7.5 Hz,Η-5΄), 2.42, 2.27 (2s, 6H, 2xCH3), 1.32
(t, 3H, J=7.2 Hz, CH3), 1.29 (t, 3H, J=7.2 Hz, CH3); 13CNMR
(75.5 MHz, CDCl3) δ 165.48, 165.01, 160.07, 159.96, 150.61,
144.68, 144.04, 139.63, 133.11, 126.23, 126.08, 125.15, 123.09, 115.01, 109.32,
93.25, 86.43, 80.59, 79.07, 69.76, 64.25, 61.29, 60.98, 20.75, 20.22, 14.34,
14.14; Anal. Calcd.
for C27H30N2O8: C, 63.52; H, 5.92;
N, 5.49%; Found: C, 63.92; H, 5.62; N, 5.89%; ESI-MS (m/z): Mass (M+H)+: 511.20.
(E)-ethyl4-(((E)-4-ethoxy-1-((6-methoxynaphtalene)ethynyl-uracil)-4-oxobout-2-en-1-yl)oxy)-5-hydropent-2-enoate (14)
68 mg, 69% as white
solid; M.P. 192-193°C; [α]D22=+24
(c 0.10, CHCl3); Rf=0.28 (EtOAc/Hexane 3:7); λmax 286 nm (ε 26413); 1HNMR (300 MHz, CDCl3) δ 8.83 (s, 1H, ΝΗ), 7.57 (s, 1H, H-6), 7.27 (s, 1H, Bz), 7.07
(d, 1H, J=7.7 Hz, Bz), 7.02 (d, 1H, J=7.7 Hz, Bz), 6.80 (dd, 1H, J=3.9 Hz,
J=15.8 Hz, Η-3), 6.77 (dd, 1H, J=3.9 Hz, J=15.8 Hz, Η-2), 6.45 (t, 1H, J=3.3 Hz, Η-1), 6.39 (dd, 1H, J=1.2 Hz, J=15.8 Hz, Η-3΄), 6.14 (dd, 1H, J=1.2 Hz, J=15.8 Hz, Η-2΄), 4.00 (s, 3H, OCH3), 4.35 (s, 1H,
OH), 4.26-4.20 (m, 5H, 2xCH2, H-4), 3.73 (dd, 1H, J=2.9 Hz, J=11.9
Hz , Η-5), 3.67 (dd, 1H, J=7.5 Hz, Η-5΄), 1.32 (t, 3H, J=7.2 Hz, CH3),
1.29 (t, 3H, J=7.2 Hz, CH3); 13CNMR (75.5 MHz, CDCl3)
δ 165.48, 165.01, 160.07, 159.96, 150.61,
144.68, 144.04, 139.63, 133.11, 129.64, 129.16, 126.23, 126.08, 125.15, 123.09,
115.01, 109.32, 105. 93, 93.25, 86.43, 80.59, 79.07, 69.76, 64.25, 61.29,
60.98, 55.82, 14.28; Anal. Calcd. for C30H30N2O9: C, 64.05;
H, 5.38; N, 4.98%; Found: C, 64.25; H, 5.58; N, 4.78%; ESI-MS (m/z): Mass (M+H)+: 563.20.
RESULTS AND DISCUSSION
Chemistry
Our first synthetic efforts focused on the
preparation of the C5-substituted uracil 1-(1-((1,3-dihydroxyprapan-2-yl)oxy)-2-hydroxyethyl)
(Figure 1). Uridine 1 was treated in
pyridine with an excess of trityl chloride (TrCl), in the presence of a
catalytic amount of 4,4-dimethylaminopyridine (DMAP) to give the corresponding
trityl derivative 2 in 82% yield [26]. The oxidative cleavage of the cis-diol
in the 2΄,3΄-position of compound 2 followed by borohydride reduction of the
resulting aldehyde (one pot), furnished the corresponding acyclic nucleoside 3.
Deprotection of the 5΄-O-trityl 3 by treatment with formic acid (HCOOH) in
diethylether (Et2O) gave the acyclic nucleoside of iodouracil 4
[27]. The next step of the synthesis involves the acetylation of the hydroxyl
groups of the nucleoside 4 using acetic anhydride in the presence of pyridine
led to acetylated derivative 5. In order to extract more detailed
structure-activity relationships, diverse alkyne substituents R were selected,
which included a phenyl ring substituted with halogens (6a, R=2-fluoro, 6b,
R=2-chloro) or methyl groups (6c, R=2,5-dimethyl) and a polycyclic aromatic
hydrocarbon substituted with a methoxyl group (8, R=6-methoxynaphthalene). In a
typical experiment, the acetylated acyclic nucleoside of 5-iodouracil (5) was
mixed with N,N-dimethylformamide (DMF), the appropriate alkyne, triethylamine
(base), copper(I) iodide (CuI) (co-catalyst) and tetrakis(triphenylphosphine)
palladium (0) (Pd(PPh3)4) (catalyst) and were irradiated
at 50°C for 5 min. After removing all the volatile materials in vacuo, the
solid obtained was purified by flash chromatography to provide the C5-alkynyl
acyclic nucleosides 6, 8, which upon treatment with saturated methanolic
ammonia afforded the unprotected derivatives 7 and 9, in good yields (65-85%).
The next objective was to
synthesize C5 modified acyclic nucleosides of uracil, wherein the acyclic
moiety would have been introduced into α, β-unsaturated carbonyl structures
that have been shown to confer particular biological activity on the molecules
carrying them. The
synthesis of the C5-substituted uracil α,
β-unsaturated carbonyl acyclic nucleosides is outlined in Figure 2. The oxidative cleavage of the
cis-diol in the 2΄,3΄-position of compound 2 was achieved with sodium periodate
in a mixture of methanol/water. The dialdehyde were unstable, hence after
isolation they were directly subjected to a double Wittig olefination, using (ethoxycarbonylmethylene) triphenylphosphorane in
tetrahydrofurane (THF) at 40°C for 1 h afforded compound 11in 58% yield. It was
of particular note, that under such reaction conditions no diastereoisomeric by
products observed, by 1ΗNMR and COSY spectroscopy, indicating that
complete chiral integrity was retained at carbon atoms C1 and C4 of 11, the
iodouracil base retains the β configuration and finally the protons of the two
double bonds are in trans position as we observe large coupling constants (15.8
Hz) [28-30]. Deprotection of the 5΄-O-trityl 11 by treatment with formic
acid (HCOOH) in diethylether (Et2O) gave the acyclic nucleoside of
iodouracil 12. Using the same one-pot Sonogashira protocol as previously
discussed, the novel aryl alkynyl acyclic nucleosides 13 and 14 were obtained,
in satisfying yields (69-82%).
Biological evaluation
Compounds 4-9, 12-14,
were evaluated for their cytostatic activity against the human cells:
pancreatic adenocarcinoma (Capan-1), chronic myeloid leukemia (Hap-1), colorectal carcinoma (HCT-116), lung
carcinoma (NCI-H460), acute lymphoblastic leukemia (DND-41), acute myeloid leukemia
(HL-60), chronic myeloid leukemia (K-562) and non-Hodgkin lymphoma (Z-138) and their
antiviral activity varicella-zoster
virus (VZV) in human embryonic lung (HEL) cell cultures.The results of cytotoxic and antiviral
activity are shown in Tables 1 and 2,
respectively. Unfortunately none of the tested compound showed any
significant cytostatic activity at a broad panel of cancer cell lines. The antiviral assays showed that (E)-ethyl
4-(((E)-4-ethoxy-1-((2-fluorophenyl)ethynyl-uracil)-4-oxobout-2-en-1-yl)oxy)-5-hydropent-2-enoate
(13a) had a
noticeable activity against TK- VZV strain (EC50 20 μΜ, MCC 100 μΜ).
CONCLUSION
In the present
study, we report the synthesis of novel C5-arylalkynyl pyrimidine acyclic
nucleosides, by developing highly efficient synthetic routes. Nucleoside (E)-ethyl
4-(((E)-4-ethoxy-1-((2-fluorophenyl)ethynyl-uracil)-4-oxobout-2-en-1-yl)oxy)-5-hydropent-2-enoate
(13a) had a noticeable activity against TK- VZV strain (EC50 20 μΜ, MCC 100 μΜ) (Table 2).
CONFLICT OF INTEREST
The authors confirm that this article content has no conflict of
interest.
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Table 1 -
Table 2
