Synthesis and antibacterial activity of unsaturated pyrimidine carboacyclonucleoside analogues

Article abstract of DOI:10.1135/cccc2011083

The novel pyrimidine carboacyclonucleoside analogues containing propargylated aryl side chains were synthesized via palladium-catalyzed cross-coupling reactions as a key step. The synthesized compounds were screened for their antibacterial activity agains

Full text of DOI:10.1135/cccc2011083

Unsaturated Pyrimidine Carboacyclonucleoside
1299
SYNTHESIS AND ANTIBACTERIAL ACTIVITY OF UNSATURATED
PYRIMIDINE CARBOACYCLONUCLEOSIDE ANALOGUES
a4
Jamal KRIMa1,b, Bouchra SILLAHIa2, Moha TAOURIRTEa3, Said EDDARIR and
b,
Joachim W. ENGELS *
aLaboratoire de Chimie Bioorganique et Macromoléculaire, Faculté des Sciences et Techniques,
1
2
Guéliz 40 000, Marrakech, Maroc; e-mail: krimjamal@gmail.com, bouchra.sillahi@gmail.com,
3
4
taourirte@fstgmarrakech.ac.ma, eddarir@yahoo.fr
b
Institut für Organische Chemie und Chemische Biologie, J. W. Goethe Universität,
Max-von-Laue Str. 7, 60438 Frankfurt am Main, Germany;
e-mail: joachim.engels@chemie.uni-frankfurt.de
Received April 20, 2011
Accepted June 17, 2011
Published online October 30, 2011
Dedicated to Professor Antonín Holý on the occasion of his 75th birthday.
The novel pyrimidine carboacyclonucleoside analogues containing propargylated aryl side
chains were synthesized via palladium-catalyzed cross-coupling reactions as a key step. The
synthesized compounds were screened for their antibacterial activity against four microor-
ganisms: Staphylococcus aureus (CIP 53.154; Gram positive), Enterococcus hirae (CIP 58.55;
Gram positive), Pseudomonas aeruginosa (CIP A22; Gram negative), Escherichia coli (CIP 54.8;
Gram negative). Some of the prepared products showed promising antibacterial activity
against the nosocomial E. hirae.
Keywords: N-Alkylation; Sonogashira cross-coupling; Thymine; Antibacterial activity;
Alkynes; Alkylation; Antibiotics; Nucleobases; Palladium.
The emergence of resistant strains of bacteria to major classes of anti-
microbial agents is recognized as a serious health concern1. Even though
pharmaceutical industries have produced a number of novel antibiotics in
the last three decades, resistance to these drugs by microorganisms has in-
creased dramatically. In general, bacteria have the genetic ability to trans-
mit and acquire resistance to drugs, which are utilized as therapeutic
agents2. The problem of microbial resistance is growing and the outlook for
the use of antimicrobial drugs in the future is still uncertain. Therefore, ac-
tions must be taken to reduce this problem, for example, to control the use
of antibiotics, develop research to better understand the genetic mecha-
Collect. Czech. Chem. Commun. 2011, Vol. 76, No. 11, pp. 1299–1305
© 2011 Institute of Organic Chemistry and Biochemistry
doi:10.1135/cccc2011083

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Krim, Sillahi, Taourirte, Eddarir, Engels:
nisms of resistance, and to continue studies to develop new drugs, either
synthetic or natural. The ultimate goal is to offer appropriate and efficient
antimicrobial drugs to the patient. Consequently, the search for new
chemotherapeutic agents constitutes a real challenge for microbiologists,
pharmacologists as well as medicinal chemists.
Nucleoside analogues display a wide range of biological activities, such as
antitumor, antiviral and chemotherapeutic3–5. Various acyclic and cyclic
nucleoside analogues were synthesized and evaluated for antibacterial and
antifungal activity6–11. Some prepared products showed promising anti-
microbial activity. In addition, it has been shown that the presence of con-
jugated system plays a fundamental role in determining bioactivity, due to
its ability to act as a Michael acceptor for the addition of protein functional
groups12,13
.
Efforts aimed at synthesizing and isolating new active carboacyclonucleo-
sides now require the development and elaboration of new strategies yield-
ing facile and rapid access to a large variety of compounds. In view of the
stimulating results reported for unsaturated acyclonucleosides14,15 and as
a part of our ongoing drug discovery efforts, this study aims to synthesize
novel unsaturated carboacyclonucleosides. This paper reports their syn-
thetic routes from the simple propargylated nucleobases by Sonogashira
cross-coupling. The cross-coupling reaction proceeds in the presence of
catalytic amounts of palladium complexes like Pd(PPh3)2Cl2, a catalytic
amount of copper(I) iodide, an organic amine base and DMF as solvent at
room temperature.
RESULTS AND DISCUSSION
Chemistry
The first step of the synthesis was the preparation of propargylated nucleo-
bases. For this, uracil and thymine were used as starting materials that were
treated with propargyl bromide in the presence of K2CO3. All reactions were
carried out in DMF, as it is an excellent solvent for dissolving both
nucleobases16,17 (Scheme 1) and inorganic salts. All the pyrimidine deriva-
tives were exclusively alkylated at N-1 position 3 and 4 as confirmed by
1
their H NMR spectra and comparison with authentic samples16.
The second step was the introduction of various aryl iodides at the termi-
nal triple bond by Sonogashira reaction. The palladium-catalyzed cross-
coupling reaction of aryl iodides with propargyl pyrimidines proceeds effi-
ciently in the presence of catalytic amounts of palladium complexes such
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Unsaturated Pyrimidine Carboacyclonucleoside
1301
as Pd(PPh3)2Cl2, a catalytic amount of copper(I) iodide, an organic amine
base and DMF as solvent.
We examined the reaction of various aryl iodides 5a–5d with the pro-
pargyl pyrimidines 3 and 4. The presence of methoxy groups in the meta-
position on the aryl iodide seems to be advantageous. Electron donors in
para-positions, however, are inferior to electron withdrawing groups like
NO2. When p-iodoanisole was used as the aryl iodide, the Sonogashira cou-
pling reaction yield decreased to 50 or 60%. The results are summarized in
Table I.
O
O
R
R
NH
NH
Br
i
+
O
N
O
N
H
3 R = H
4 R = CH3
1 R = H
2 R = CH3
R2
R1
R3
I
ii
R4
5a-5d
O
R
NH
O
N
R1
R2
R3
6a-6d
7a-7d
R4
SCHEME 1
(i) K2CO3, DMF, r.t.; (ii) Pd(PPh3)2Cl2, CuI, Et3N, DMF, r.t.
Antimicrobial Activity
Newly synthesized compounds 6a–6d and 7a–7d were tested for anti-
microbial activity against the following bacterial strains obtained from the
Pasteur Institute Collection: Gram (+): Staphylococcus aureus (CIP 53.154),
Enterococcus hirae (CIP 58.55); Gram (–): Pseudomonas aeruginosa (CIP A22),
Escherichia coli (CIP 54.8).
The compounds were dissolved in 5% DMSO/H2O and added to the
culture medium (nutrient agar for bacteria) immediately before it was emp-
tied into the Petri dishes. The concentrations tested ranging from 27 to
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Krim, Sillahi, Taourirte, Eddarir, Engels:
217 µg/ml. Inocula of the bacteria were prepared from 18 h cultures and the
suspensions of microorganisms (spot content 2 µl of 108 cells per ml) were
inoculated onto the surface of the Petri dishes immediately after their prep-
aration (three replicates). The negative control received the same quantity
of the 5% DMSO mixed with the culture medium and bacteria. Suspension
of each microorganism was prepared and applied to plates with serially di-
TABLE I
Pd-Catalyzed reaction of aryl iodides 5a–5d with propargyl pyrimidines
Compounds
R
R1
R2
R3
R4
Yield, %
6a
6b
6c
6d
7a
7b
7c
7d
H
NO2
H
H
H
H
80
90
92
50
80
92
96
60
H
H
NO2
H
H
H
H
OCH3
H
OCH3
H
H
H
OCH3
H
CH3
CH3
CH3
CH3
NO2
H
H
H
H
NO2
H
H
H
OCH3
H
OCH3
H
H
OCH3
TABLE II
Minimum inhibitory concentration (MIC in µg/ml) of medium
Staphylococcus
aureus
Enterococcus
hirae
Pseudomonas
aeruginosa
Compounds
Escherichia coli
6a
–
–
–
27
27
–
6b
–
–
6c
54
–
–
27
–
6d
–
108
108
–
–
7a
–
–
–
7b
–
–
–
7c
–
–
54
–
7d
54
3.1
–
54
–
Amoxicillin
0.2
0.5
0.2
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Unsaturated Pyrimidine Carboacyclonucleoside
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luted compounds (DMSO/H2O) to be tested and incubated (24 h) at 37 °C.
After the incubation period the growth was visually evaluated by compari-
son with those of control plates. The minimum inhibitory concentration
(MIC) was taken as the lowest concentration that completely inhibited growth
after incubation. The MICs of the standards (Amoxicillin) were also deter-
mined in parallel experiments, to control the sensitivity of the bacteria.
No antibacterial activity of 6a–6d and 7a–7d was detected against Staphy-
lococcus aureus and Pseudomonas aeruginosa strains. All these compounds
except 7b showed antibacterial activity (MIC ≈ 27–108 µg/ml) against
Enterococcus hirae, a strain, known to be present in hospitals (Table II).
In addition compounds 6c and 7d exhibit some activity against E. coli
(MIC ≈ 54 µg/ml).
CONCLUSIONS
In summary, the palladium-catalyzed cross-coupling reaction of various
aryl iodides with propargyl pyrimidines was achieved by using Sonogashira
reactions. Newly synthesized compounds 6a–6d and 7a–7d were tested for
their antimicrobial activity. No antibacterial activity of 6a–6d and 7a–7d
was detected against Staphylococcus aureus and Pseudomonas aeruginosa
strains, some of them exhibit a good activity against Enterococcus hirae
strains (MIC ≈ 27 µg/ml) and E. coli (MIC ≈ 54 µg/ml).
EXPERIMENTAL
Melting points were determined in open capillary tubes and are uncorrected. NMR spectra
were recorded at 300 MHz (1H, 13C) Bruker (in DMSO-d6, CDCl3) using TMS as an internal
reference. All chemical shifts (δ) are expressed in ppm and coupling constant (J) are given in
Hz. Mass spectra were obtained using ESI/MS and (FAB+). DMF was distilled prior to use and
stored over 4Å molecular sieves. Precoated Merck Silica Gel 60F-254 plates were used for
thin layer chromatography (TLC) and the spots were detected under UV light (254 nm). Col-
umn chromatography (CLC) was performed using silica gel (0.063–0.2 mm) Fluka. All re-
agents used were purchased from Aldrich.
Synthesis of the Monopropargyl Pyrimidines. General Procedure
The mixture of 1 mmol of the pyrimidine base (thymine and uracil), 0.5 mmol of K2CO3
and 1 mmol of propargyl bromide in 20 ml of anhydrous DMF was stirred at room tempera-
ture for 24 h. After removal of the solvent under reduced pressure, the obtained residue was
purified on silica gel column (CH2Cl2 and MeOH 99:1).
N-1-Propargyl thymine (3): Yield 56%; solid, m.p. 154–156 °C. 1H NMR (DMSO-d6): 1.75 (s,
3 H, CH3); 3.37 (t, 1 H, CH, J = 2.2); 4.46 (d, 2 H, CH2N, J = 7.8); 7.55 (s, 1 H, H-6); 11.35 (s,
1 H, NH). 13C NMR (DMSO-d6): 11.87; 36.30; 75.58; 78.61; 109.38; 140.04; 150.33; 164.09.
FAB-MS, m/z: calculated for C8H8N2O2 [M + H]+ 165.06, found 165.
Collect. Czech. Chem. Commun. 2011, Vol. 76, No. 11, pp. 1299–1305

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Krim, Sillahi, Taourirte, Eddarir, Engels:
N-1-Propargyl uracil (4): Yield 54%; solid, m.p. 164–166°C. 1H NMR (DMSO-d6): 3.43 (t,
1 H, CH, J = 2.3); 4.57 (d, 2 H, CH2N, J = 2.3); 5.7 (d, 1 H, H-5, J = 7.8); 7.75 (d, 1 H, H-6,
J = 7.8); 11.44 (s, 1 H, NH). 13C NMR (DMSO-d6): 36.60; 75.79; 78.42; 101.67; 144.42;
150.35; 163.50. FAB-MS, m/z: calculated for C7H6N2O2 [M + H]+ 151.04, found 151.
Sonogashira Cross-Coupling. General Procedure
Aryl iodides 5a–5d (5 mmol) were dissolved in a mixture of dry DMF (15 ml), dry Et3N
(3 equiv.) and propargyl pyrimidine (2 equiv.). CuI (0.2 equiv.) and Pd (PPh3)2Cl2
(0.1 equiv.) were then added and the reaction mixture was stirred at room temperature until
complete (typically 5–20 h, checked by TLC). The solvent was removed by rotary evapora-
tion, and the residue was purified on silica gel with dichloromethane and methanol (95:5)
to give the desired compound.
1-(3-(2-Nitrophenyl)prop-2-ynyl)uracil (6a): Yield 80%; m.p. 199 °C. 1H NMR (DMSO-d6):
4.38 (d, 2 H, CH2-N, J = 2.3); 6.13 (d, 1 H, H-5, J = 7.8); 7.65–7.9 (m, 5 H, 4CH and H-6);
11.4 (s, 1 H, NH). FAB-MS, m/z: calculated for C13H9N3O4 [M + H]+ 272.06, found 272.
1-(3-(4-Nitrophenyl)prop-2-ynyl)uracil (6b): Yield 90%; m.p. > 250 °C. 1H NMR (DMSO-d6):
4.83 (s, 2 H, CH2-N); 5.65 (d, 1 H, H-5, J = 7.8); 7.75 (d, 2 H, 2CH, J = 8.7); 7.84 (d, 1 H,
H-6, J = 7.8); 8.22 (d, 2 H, 2CH, J = 8.7); 11.4 (s, 1 H, NH). FAB-MS, m/z: calculated for
C
13H9N3O4 [M + H]+ 272.06, found 272.
1-(3-(3,5-Dimethoxyphenyl)prop-2-ynyl)uracil (6c): Yield 92%; m.p. 141 °C. 1H NMR
(DMSO-d6): 4.75 (s, 2 H, CH2-N); 3.77 (s, 6 H, 2 × OCH3); 5.65 (d, 1 H, H-5, J = 7.8); 6.53 (s,
1 H, CH); 6.59 (s, 2 H, 2CH); 7.78 (d, 1 H, H-6, J = 7.8); 11.45 (s, 1 H, NH). FAB-MS, m/z:
calculated for C15H14N2O4 [M + H]+ 287.10, found 287.
1-(3-(4-Methoxyphenyl)prop-2-ynyl)uracil (6d): Yield 50%; m.p.
>
250 °C. 1H NMR
(DMSO-d6): 3.76 (s, 3 H, OCH3); 4.72 (s, 2 H, CH2-N); 5.65 (d, 1 H, H-5, J = 7.8); 6.9 (d, 2 H,
2CH, J = 9); 7.39 (d, 2 H, 2CH, J = 9); 7.8 (d, 1 H, H-6, J = 7.8); 11.4 (s, 1 H, NH). FAB-MS,
m/z: calculated for C14H12N2O3 [M + H]+ 257.08, found 257.
1-(3-(2-Nitrophenyl)prop-2-ynyl)thymine (7a): Yield 80%; m.p. 208 °C. 1H NMR (DMSO-d6):
1.9 (s, 3 H, CH3); 4.38 (s, 2 H, CH2-N); 7.58–7.81 (m, 5 H, 4CH and H-6); 11.4 (s, 1 H, NH).
FAB-MS, m/z: calculated for C14H11N3O4 [M + H]+ 286.07, found 286.
1-(3-(4-Nitrophenyl)prop-2-ynyl)thymine (7b): Yield 92%; m.p. 242 °C. 1H NMR (DMSO-d6):
1.87 (s, 3 H, CH3); 4.39 (s, 2 H, CH2-N); 7.73 (d, 2 H, 2CH, J = 9); 8.02 (s, 1 H, H-6); 8.22 (d,
2 H, 2CH, J = 9); 11.42 (s, 1 H, NH). FAB-MS, m/z: calculated for C14H11N3O4 [M + H]+
286.07, found 286.
1-(3-(3,5-Dimethoxyphenyl)prop-2-ynyl)thymine (7c): Yield 96%; m.p. 128 °C. 1H NMR
(DMSO-d6): 1.78 (s, 3 H, CH3); 3.74 (s, 6 H, 2 × OCH3); 4.75 (s, 2 H, CH2-N); 6.53 (s, 1 H,
CH); 6.65 (s, 2 H, 2CH); 7.64 (s, 1 H, H-6); 11.4 (s, 1 H, NH). FAB-MS, m/z: calculated for
C
16H16N2O4 [M + H]+ 301.11, found 301.
1-(3-(4-Methoxyphenyl)prop-2-ynyl)thymine (7d): Yield 60%; m.p. 207 °C. 1H NMR
(DMSO-d6): 1.79 (s, 3 H, CH3); 3.76 (s, 3 H, OCH3); 4.70 (s, 2 H, CH2-N); 6.78 (d, 2 H, 2CH,
J = 8.7); 7.21 (d, 2 H, 2CH, J = 8.7); 7.59 (m, 1 H, H-6); 11.4 (s, 1 H, NH). FAB-MS, m/z:
calculated for C15H14N2O3 [M + H]+ 271.10, found 271.
We thank Prof. F. Benkhalti and Dr. H. Bouaamama for supporting the biochemical and
pharmacological data (experiments).
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