Synthesis and AChE inhibiting activity of 2, 4 substituted 6-phenyl pyrimidines

Article abstract of DOI:10.4067/S0717-97072012000300020

Novel substituted pyrimidines were synthesized from methyl 2,4-dioxo-4-phenyl-butanoate (I-A) and urea, followed by Mitsunobu coupling of I-A with benzyl or allyl alcohol to give the corresponding 2-hydroxypyrimidine ethers in good yields. Saponification

Full text of DOI:10.4067/S0717-97072012000300020

J. Chil. Chem. Soc., 57, Nº 3 (2012)
SYNTHESIS AND AChE INHIBITING ACTIVITY OF 2, 4 SUBSTITUTED 6-PHENYL PYRIMIDINES
1CRISTIAN PAZ, 2MARTIN G. PETER, 2BERND SCHMIDT, 1JOSÉ BECERRA, 3MARGARITA GUTIÉRREZ,
3LUIS ASTUDILLO, 1MARIO SILVA*
1Laboratorio de Química de Productos Naturales, Universidad de Concepción, Chile
2Institute of Chemistry, University of Potsdam, Germany
3Instituto de Química, Universidad de Talca, Chile
(Received: March 6, 012 - Accepted: June 29, 2012)
ABSTRACT
Novel substituted pyrimidines were synthesized from methyl 2,4-dioxo-4-phenyl-butanoate (I-A) and urea, followed by Mitsunobu coupling of I-A with
benzyl or allyl alcohol to give the corresponding 2-hydroxypyrimidine ethers in good yields. Saponification of I-A, followed by reaction with benzyl or allyl
amines in the presence of TBTU yielded 2-hydroxy-6-phenyl-pyrimidine 4-carboxamides. AChE and BuChE assays revealed 2-hydroxy-6-phenyl-pyrimidine-4-
carboxyallyamide as the most active compound, IC50= 90 μM, with no inhibition of BuChE.
Keywords: Pyrimidines; inhibition AChE; mitsunobu; TBTU
(final concentration of 0.03 U/mL and 0.01 U/mL, respectively) were added to
each well, and the plates were pre-incubated for 30 min at room temperature.
After the pre-incubation period, acetylthiocholine iodide (or butyrylthiocholine
iodide) was added to a final concentration of 200 μM. 5,5`-Dithio-bis(2-
nitrobenzoic acid) (DTNB) was used for the measurement of cholinesterase
activity. The hydrolysis of ACh or BCh was monitored by following the
formation of the yellow 5-thio-2-nitrobenzoate anion. The absorbance was
read in a Thermo Multiskan Ex Instrument microplate reader at 405 nm after 3
min. The enzyme activity was calculated as a percentage compared to a control
using only the buffer and enzyme solution. The compounds were assessed
in the dilution interval of 500-15.63 μg/mL, and the alkaloid galanthamine
was used as the reference compound. The ChEs inhibitory activity of each
compounds was expressed in terms of the IC50 value (μg/mL and μM required
to inhibit the hydrolysis of the substrate by 50%), as calculated from the dose-
response curve.
INTRODUCTION
Alzheimer’s disease (AD) is
the most
common age-related
neurodegenerative disease, is progressive neurodegenerative disorder
a
that affects regions of the brain that control cognition, memory, language,
speech and awareness to one’s physical surroundings1. Those alterations are
associated with regional deficits in the cholinergic system. The development
of acetylcholinesterase (AChE) inhibitor drugs has followed the finding
that cholinergic pathways in the cerebral cortex and basal forebrain are
compromised in AD2 and the resultant cholinergic deficit contributes to the
cognitive impairment of these patients3. Cholinesterase inhibitors (ChEIs) are
considered to be valuable as a therapeutic target and they have become the main
approach to symptomatic treatment; Donepezil, galantamine and rivastigmine
are the first line pharmacotherapy for mild to moderate Alzheimer’s
disease4. The drugs have slightly different pharmacological properties, but
they all work by inhibiting the breakdown of acetylcholine increasing the
availability of acetylcholine in central synapses4. Acetylcholinesterase and
butyrylcholinesterase (BuChE), the two forms of cholinesterase, co-exist
ubiquitously throughout the body. They exhibit a high catalytic power and are
very similar in structure and catalytic function. However, BuChE has a less
defined role in biological processes, although it has been postulated that it acts
as a detoxifying enzyme5.
RESULTS AND DISCUSSION
2, 4-Dioxobutanoates are interesting starting materials for the synthesis
of ligands and organometallic complexes of anti-HIV-1 integrase10. Amides
and hydrazides of 2, 4-dioxobutanoic acid possess antimicrobial and analgesic
activity11. The β diketone I-A has been synthesized in 84% yield from the
acetophenone and dimethyloxalate in dimethoxyethane, using NaH as base,
scheme 1. Compound I-A shows an interchangeable proton at 15.83 ppm and
just one ketone carbon at 190.81 ppm suggesting that I-A occurs only as the
β-hydroxyketone.
Hydrolysis of I-A in aqueous NaOH for 10-15 min gave the corresponding
acid II-B in 80% yield. II-B is known to possess promising activity against
HIV-1 integrase7 and slow-binding inhibition of KDPG aldolase12 which is a
target for new bacteriostatic or bactericidal drugs. Other reaction conditions,
such as NaOH in MeOH, afforded II-B in a yield of ca. 50%, together with
unreacted starting material.
Looking for new active compounds we have isolated several natural
alkaloids from Aristotelia chilensis6, which have displayed broad range of
pharmacology activities. Recently investigations have shown that substituted
pyrimidines are potent ChE inhibitors7 of Acetyl and Butylcholine. Therefore
it seems interesting to synthesize 6-phenylpyrimidines as specific ChE,
substituted at position 2 and 4 and evaluated their enzymatic activity.
EXPERIMENTAL
Melting points were determined on a Melting Point SMP10 (Stuart) and are
uncorrected. The 1H NMR spectra were determined using a Bruker ARX 300
instrument, operating at 300.1 MHz (1H) and 75.5 MHz (13C) or Bruker ARX
500 operating at 500 MHz (1H) and 125 MHz (13C). High-Resolution ESI mass
spectra (HR-MS) were measured on a Q-TOF mass spectrometer Micromass
(Manchester). EI low-resolution MS spectra were measured on Trace DSQII
GC/MS-system Axel Semrau GmbH & Co). Column chromatography was
performed using Merck silica gel 60 (0.063–0.200 mm). TLC was carried out
on a Merck silica gel 60 PF254. Solvents used in this study were distilled prior
to use and dried over appropriate drying agents.
Scheme 1
Pyrimidine I-A1 was synthesized from β-hydroxy ketone I-A in 78%
yield, using an excess of urea in MeOH under reflux in the presence of
catalytic amounts of H2SO4. Ethers I-A2 and I-A3 were obtained from I-A1
under Mitsunobu conditions, using diisopropylazodicarboxylate (DIAD) and
Triphenylphosphine in dimethylformamide. Column chromatography afforded
the products in ca. 87% yield, scheme 2.
Enzyme assays
The in vitro measurement of AChE and BuChE inhibition were carried out
using a colorimetric method8 adapted to 96-well microtiter plates9. The AChE
was obtained from Electrophorus electricus (C3389-500UN), type VI-S,
Sigma Chemical Co., St. Louis, MO), and BuChE from equine serum (C1057-
1KU, Sigma Chemical Co., St. Louis, MO). Fifty μL of test sample dissolved
in 100 mM phosphate buffer pH 7.6, and 50 μL, of AChE or BuChE solution
e-mail: njsilva@udec.cl
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J. Chil. Chem. Soc., 57, Nº 3 (2012)
ppm: 55.41; 99.45; 127.55; 129.16; 130.19; 135.16; 162.61; 169.09; 190.81.
EI-MS: 206
Methyl 2-hydroxy-6-phenylpyrimidine-4-carboxylate (I-A1):
A
mixture of compound I-A (3.0g, 14.6 mmol) and urea (2.8g, 47 mmol) were
dissolved in 100mL of methanol and ca. 100 μL of conc. sulfuric acid was
added. The solution was stirred overnight. The product precipitated as a white
solid (2.8g, 78%) mp = 195-196ºC. 1H-NMR (DMSO-d6, 500 MHz) ppm: 3.91
(3H, s); 7.59 (3H, m); 7.72 (1H, s); 8.13 (2H, d, J= 7.2 Hz); 12.47 (1H, s, br).
13C-NMR (DMSO-d6, 125 MHz) ppm: 53.50; 105.98; 127.92; 129.51; 132.40;
135.23; 155.43; 162.54; 163.78; 168.22.
Methyl 2-(benzyloxy)-6-phenylpyrimidine-4-carboxylate (I-A2):
DIAD (1.49g, 7.4mmol) in 10mL of dry THF was cooled in an ice bath, then
triphenylphospine (1.93g, 7.4mmol) dissolved in 10 mL of THF was added,
the mixture was stirred for 15 min, during which a white solid was formed.
A mixture of I-A1 (1.13g, 4.9mmol) and benzyl alcohol (0.78g, 7.2mmol)
dissolved in 15mL of dry dimethylformamide was added to the suspension.
The reaction was stirred for 5 h from 0 ºC to room temperature. The solvents
were removed in vacuo and the crude product was purified by silica gel column
with hexane/EtOAc 30%. White solid (1.35g, 86%) mp = 98°C. 1H-NMR
(CDCl3, 300 MHz) ppm: 4.03 (3H, s); 5.60 (2H, s); 7.27-7.42 (3H, m); 7.46-
7.62 (5H, m); 8.07 (1H, s); 8.16 (2H, dd, J= 2.1, 7.6 Hz). 13C-NMR (CDCl3,
75 MHz) ppm: 53.30; 69.80; 110.59; 127.63; 128.22; 129.12; 131.87; 135.98;
136.45; 158.01; 164.96; 165.87; 168.78. ESI-MS: M+1: 321.1137
Scheme 2
Reaction of 4-phenyl-2,4-dioxobutanoic acid (II-B) with an excess of
urea and catalytic amounts of H2SO4 in refluxing toluene gave 2-hydroxy-
6-phenylpyrimidine-4-carboxylic acid (II-BI) in yields of ca. 55%. II-BI
precipitated after the reaction as a white solid, soluble in dimethylsulfoxide
or dimethylformamide. Amide coupling of II-B1 with benzyl or allyl amines
with O-(Benzotriazol-1-yl)-N,N,N’,N’-tetramethyluroniumtetrafluoroborate
(TBTU) in DMF gave compounds II-B2, II-B3, and II-B4, respectively, in ca.
40% yield, scheme 3. Attempts to prepare secondary amides with diethyl- or
dibenzylamine were unsuccessful, as only traces of product were detected
Methyl 2-(allyloxy)-6-phenylpyrimidine-4-carboxylate (I-A3): The
compound was prepared following the same procedure as described for I-A2,
using allyl alcohol (0.427g, 7.3mmol). The crude product was purified by CC
with hexane-ethyl acetate 20%, giving a white solid (1.15 g, 87%) mp = 102 -
103°C. 1H-NMR (MeOD, 300 MHz) ppm: 4.00 (3H, s); 4.91 (2H, s); 5.25 (2H,
m); 6.00 (1H, m); 7.44 (1H, s); 7.57 (3H, m); 8.15 (2H, m). 13C-NMR (MeOD,
75 MHz) ppm: 43.14; 52.92; 103.29; 117.69; 127.74; 128.69; 131.95; 132.44;
135.13; 148.32; 157.10; 161.52; 171.68. ESI-MS: M+1: 271.1389.
(Z)-2-Hydroxy-4-oxo-4-phenylbut-2-enoic acid (II-B): Hydrolysis of
I-A (5.5g, 26.7mmol) was carried out with NaOH (1.1g, 26.7mmol) in 20mL of
water and stirring at 50ºC for 15 min. The reaction was filtered and the rection
mixture was extracted three times with 10 mL of ethyl acetate. The water layer
was acidified with HCl until pH 2. The product precipitated as a white solid,
it was filtered off and dried (4.1g, 80%) , mp = 160ºC. 1H-NMR (CDCl3, 300
MHz) ppm: 6.75 (1H, br); 7.52 (2H, m); 7.53 (1H, d, J= 4.2 Hz); 7.87 (2H, d,
J= 4.5 Hz). 13C-NMR (CDCl3, 75 MHz) ppm: 98.04; 127.48; 128.00; 128.42;
128.90; 133.05; 135.44; 163.81. EI-MS: 192
2-Hydroxy-6-phenylpyrimidine-4-carboxylic acid (II-B1): A mixture
of II-B (1.0g, 5.2mmol), urea (0.98g, 16.3mmol) and ca. 100 μL of conc.
sulfuric acid were dissolved in 100 mL of toluene and stirred overnight, during
which a brown solid precipitated, which was filtered off and washed with cold
methanol. White solid (0.7g, 62.2%), decomposes at ca. 215°C, 1H-NMR
(DMSO d6, 300 MHz) ppm: 7.25-7.60 (4H, m, overlap); 8.13 (2H, dd, J= 2;
6 Mz). 13C-NMR (DMSO d6, 75 MHz) ppm: 103.8; 128.40; 129.83; 132.75;
136.14; 154.67; 160.61; 164.00; 169.86. ESI-MS: M+1: 217.0711
Scheme 3
Compounds I-A2; I-A3; II-B2; II-B3; II-B4, as well as pyrimidines
I-A1 and II-B1 were evaluated as inhibitors of AChE (Table 1). All amides
showed higher activity than pyrimidine carboxylic acid, II-B1. The most active
compound was the allyl amide II-B3 with an IC50 of 90.1 μM. All compounds
were also tested with BuChE, but did not show significant inhibition (IC50
1000 μM).
>
Table 1. Activity of 6-phenyl pyrimidine 2,4 substituted toward the
enzyme AChE.
2-Hydroxy-N-(3-metoxyphenyl)-6-phenylpyrimidine-4-carboxamide
(II-B2): A mixture of 2-hydroxy-6-phenylpyrimidine-4-carcarboxylic acid
(150mg, 0.69mmol) and N,N-Diisopropylethylamine (180mg, 1.4mmol) were
dissolved in 10mL of dry DMF, then O-(Benzotriazol-1-yl)-N,N,N’,N’-tetram
ethyluroniumtetrafluoroborate (TBTU) (268 mg, 0.83mmol) was added. The
mixture was stirred for 30 min at 0ºC, and 3-methoxyaniline (94mg, 0.76mmol)
was added. The reaction was stirred for 3 h, then the reaction was quenched
with H2O, and the DMF was removed in vacuo (5 mmbar and 60ºC), the crude
was purified by CC with hexane-ethyl acetate 50%. Yellow solid (100mg,
AChE IC
mg/mL
5μ0 M
Compound
Entry
1
2
3
4
5
6
7
I-A1
I-A2
I-A3
II-B1
II-B2
II-B3
II-B4
0.024
104.3
0.089
0.032
0.081
0.059
0.023
0.063
1.1x10-3
278.0
118.0
375.0
184.0
90.1
1
45%), decomposes at ca. 205ºC. H-NMR (DMSO d6, 300 MHz) ppm: 3.77
185.8
3.0
(3H, s); 6.76 (1H, dd, J= 3.0; 9.0 Hz); 7.31 (1H, dd, J= 9; 15 Hz); 7.4 – 7.7
(6H, m, overlap); 7.80 (2H, d, J= 9 Hz); 10.51 (1H, s). 13C-NMR (DMSO d6,
75 MHz) ppm: 55.95; 107.03; 110.45; 110.95; 113.48; 120.86; 126.23; 128.19;
128.36; 129.93; 130.42; 132.75; 134.87139.87; 143.75; 160.30. ESI-MS, M+1:
322.1339
*Galantamine
* Internal control, n=3.
N-Allyl-2-hydroxy-6-phenylpyrimidine-4-carboxamide (II-B3): The
compound was prepared following the same procedure as described for II-B2,
using allylamine (87mg, 2.0mmol). White solid (140 mg, 40%), decomposes
at ca. 150°C. 1H-NMR (MeOD, 300 MHz) ppm: 4.04 (2H, m); 5.20 (2H, m);
5.94 (1H, m); 7.48 – 7.65 (4H, m); 7.96 (2H, d, J= 3, 6 Hz). 13C-NMR (MeOD,
75 MHz) ppm: 43.14; 111.57; 116.90; 118.82; 127.28; 128.42; 128.80; 130.54;
133.55; 134.97; 164.39; 173.15. ESI-MS: M+1: 256.1131
N-(2-Chlorobenzyl)-2-hydroxy-6-phenylpyrimidine-4-carboxamide
(II-B4): The compound was prepared following the same procedure as
described for IIB2, using 2-chlorobenzyl amine (216 mg, 1.53mmol). White
(Z)-Methyl 2-hydroxy-4-oxo-4-phenylbut-2-enoate (I-A): A mixture
of acetophenone (5g, 41.7 mmol) and dimethyloxalate (5.9g, 50 mmol) were
stirred in 150mL of anhydrous dimethoxyethane, then sodium hydride (1.2g,
50 mmol) was added. The mixture was stirred under reflux for 4 h. The reaction
was cooled and quenched with water 100mL and HCl until pH 2. The mixture
was extracted 3 times with EtOAc, and the organic layer was evaporated in
vacuo. The compound precipitated as a white solid (7.2g, 84%). 1H-NMR
(CDCl3, 300 MHz) ppm: 3.94 (3H, s); 7.10 (1H, s); 7.50 (2H, m); 7.61 (1H,
m); 8.00 (2H, m), 15.83 (1H, s, interchangeable). 13C-NMR (CDCl3, 75 MHz)
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J. Chil. Chem. Soc., 57, Nº 3 (2012)
solid (200mg, 43%), decomposes at ca. 205 ºC. 1H-NMR (DMSO d6, 300
MHz) ppm: 4.58 (2H, d, J= 3 Hz); 7.29-7.35 (2H, m); 7.42 (1H, t, J= 3Hz);
7.53 – 7.60 (4H, m); 7.73 (1H, d, J= 6 Hz); 8.00 (1H, d, J= 3 Hz); 8.07 (1H, d,
J= 3 Hz); 9.38 (1H, s). 13C-NMR (DMSO d6, 75 MHz) ppm: 41.01; 110.08;
119.60; 125. 00; 127.67; 127.79; 127.95; 128.29; 129.08; 129.18; 129.55;
129.61; 133.00; 136.19; 143.30; 163.37. ESI-MS: M+1: 340.1096
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CONCLUSIONS
The investigation described in this communication reports the synthesis
of substituted pyrimidines in few steps with goods yields and simplicity of
operations. Separately we modified the pyrimidine substituent at position
2 through Mitsunobu reaction and the carboxy group at position 4 by
amidation. All compounds were evaluated as AChE inhibitors showing that
position 4 is the most important for enzyme-inhibition. N-Allyl-2-hydroxy-6-
phenylpyrimidine-4-carboxamide (II-B3) is the most active compound against
AChE with IC50 90.1μM. It is highly selective for AChE, not showing activity
against BuChE.
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ACKNOWLEDGEMENTS
Authors like to thank the financial support from Universidad de Concepción
and CONICYT for its Doctoral Scholarship Program.
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