Synthesis and antibacterial evaluation of 6-azapyrimidines with α-methylene-γ-(4-substituted phenyl)-γ-butyrolactone pharmacophores

Article abstract of DOI:10.1007/s00044-010-9438-7

A series of 6-substituted 2-[(4-methylene-5- oxo-2-(4-substituted phenyl) tetrahydrofuran-2-yl)methyl]-1, 2,4-triazine-3,5(2H,4H)-diones (5, 6) and 2,4-bis[(4-methylene- 5-oxo-2-(4-substituted phenyl) tetrahydrofuran-2-yl) methyl]-1,2,4-triazine-3,5(2H,4H)-diones (9, 10) were designed, synthesized, and evaluated for antibacterial activity against Gram-positive and Gram-negative microorganisms. The synthesis of these compounds involved the Reformatsky-type reaction between ethyl α-(bromomethyl) acrylate and the proper ketones. Among these compounds, 2-[(4-methylene-5-oxo-2-phenyl tetrahydrofuran-2-yl) methyl]- 1,2,4-triazine-3,5(2H,4H)-dione (5a) is the most active compound and shown the selective inhibition activity against Proteus vulgaris. Springer Science+Business Media, LLC 2010.

Full text of DOI:10.1007/s00044-010-9438-7

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2011) 20:1081–1090
DOI 10.1007/s00044-010-9438-7
ORIGINAL RESEARCH
Synthesis and antibacterial evaluation of 6-azapyrimidines
with a-methylene-c-(4-substituted phenyl)-c-butyrolactone
pharmacophores
•
Po-Jui Huang Kuan-Han Lee
Received: 28 June 2010 / Accepted: 15 September 2010 / Published online: 6 October 2010
Ó Springer Science+Business Media, LLC 2010
Abstract A series of 6-substituted 2-[(4-methylene-5-
oxo-2-(4-substituted phenyl) tetrahydrofuran-2-yl)methyl]-1,
2,4-triazine-3,5(2H,4H)-diones (5, 6) and 2,4-bis[(4-meth-
ylene-5-oxo-2-(4-substituted phenyl) tetrahydrofuran-2-yl)
methyl]-1,2,4-triazine-3,5(2H,4H)-diones (9, 10) were
designed, synthesized, and evaluated for antibacterial
activity against Gram-positive and Gram-negative micro-
organisms. The synthesis of these compounds involved the
Reformatsky-type reaction between ethyl a-(bromomethyl)
acrylate and the proper ketones. Among these compounds,
2-[(4-methylene-5-oxo-2-phenyl tetrahydrofuran-2-yl)me-
thyl]-1,2,4-triazine-3,5(2H,4H)-dione (5a) is the most active
compound and shown the selective inhibition activity
against Proteus vulgaris.
especially the sesquiterpene lactones, which possess wide-
ranging biological activities, including antitumor, bacteri-
cidal, fungicidal, antibiotic, and anthelmintic properties
(Lee et al., 1977; Spring et al., 1981; Hoffmann and Rabe,
1985). However, the biological activity of a-methylene-c-
butyrolactones is not only confined to the complex poly-
functional sesquiterpene lactones. For example, the parent
a-methylene-c-butyrolactone (tulipaline A), first isolated
from Erythronium americanum in 1946, was identified as a
substance with allergenic, antibiotic, and fungitoxic activ-
ities (Cavallito and Haskell, 1946; Slob, 1973; van Rossum
et al., 1998). Recently, it has also been reported that some
natural a-methylene-c-butyrolactone bearing butanolides,
which was isolated from Litsea akoensis, also have signif-
icant cytotoxicity (Chen et al., 1998). Due to the unique
structural feature as well as interesting biological activi-
ties of a-methylene-c-butyrolactones, their synthesis has
attracted renewed attentions (Maiti and Roy, 1996; Mandal
et al., 1998). A number of possible drug candidates bearing
this versatile functionality have also been synthesized with
the aim of finding effective clinical drugs (Sanyal et al.,
1986; Lee et al., 1987, 1999; Kim et al., 1997). Although
the enone (O=C–C=CH2) component in this type of lactone
is essential for their biological activities, by acting as an
alkylating agent through a Michael-type reaction with bio-
nucleophiles or sulfhydryl containing enzymes (Kupchan
et al., 1970), the substituent at c-position of the lactone also
played an important role for their pharmacological prop-
erties. We were particularly interested in synthesizing
a-methylene-c-butyrolactone bearing heterocycles and
explored their functions as potential biologically active
agents (Lee et al., 1999; Tzeng et al., 2000). The first 3D
pharmacophore for certain a-methylene-c-butyrolactones
using the Catalyst/HypoGen module was published
to explain how structurally different a-methylene-c-
Keywords a-Methylene-c-butyrolactone ꢀ
Antibacterial activity ꢀ Pharmacophore ꢀ
Molecular property
Introduction
The a-methylene-c-butyrolactone moiety is a characteris-
tic component of a large number of natural products,
P.-J. Huang
Faculty of Medicinal and Applied Chemistry,
Kaohsiung Medical University, Kaohsiung 80708,
Taiwan, ROC
e-mail: brhuang@kmu.edu.tw
K.-H. Lee (&)
Graduate Institute of Pharmaceutical Science,
Chia Nan University of Pharmacy and Science,
Tainan 71710, Taiwan, ROC
e-mail: kuanhanlee@mail.chna.edu.tw
123

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Med Chem Res (2011) 20:1081–1090
butyrolactones are able to induce the same biological
activity and to what extent will they contribute to the bio-
logical activity (Lee and Huang, 2003) (Chart 1).
3,5-(2H,4H)-diones were chosen as the carrier for
c-substituted
tones. We demonstrated the practical methods for the
synthesis of title compounds and the theoretical calculation
to show the correlation between molecular properties (MP)
and antibacterial activity.
c-aryloxymethyl-a-methylene-c-butyrolac-
Although the alkylating agents such as cisplatin, chlor-
ambucil, melphalan, and cyclophosphamide are important
and have been clinically used, they have serious drawbacks
which are common to all alkylating agents. These drugs act
by alkylating DNA but have no particular affinity for it.
This drawback could in principle be ameliorated by the
incorporation of the alkylating pharmacophore onto a DNA-
directing carrier, which would result in specially targeting
the pharmacophore to the DNA. A recent review from the
group at GlaxoSmithKline added valuable information on
multiple issues related to the physicochemical properties of
antibacterial compounds and provided insight into the
success and failure of a target-based, genomic approach
(Payne et al., 2007). The analysis of Payne and co-workers
illustrates the challenge of antibacterial drug discovery and
suggests that multiple parameters contribute to the high
attrition rate. One important parameter, in particular, has
been mentioned but not sufficiently analyzed is the physi-
cochemical property space of antibacterial agents.
Results and discussion
Preparation of monosubstituted target compounds 5 and 6
is illustrated in Scheme 1. Silylation of 6-substituted 1,2,4-
triazine-3,5(2H,4H)-diones (1) and (2) according to Langer
et al. (1958) with hexamethyldisilazane (HMDS) and a
catalytic amount of chlorotrimethylsilane furnished sily-
lated intermediates, which were used for the condensation
with 4-substituted-2-bromoacetophenones in dry acetoni-
trile. Compound 3 and 4 were then reacted with ethyl
a-(bromomethyl)acrylate and zinc powder in dry tetrahy-
drofuran (Reformatsky-type reaction) to afford 5 and 6 in
fairly good yields.
1,2,4-Triazines and their condensed derivatives occupy
a pivotal position in modern medicinal chemistry, because
of their high potential for biological activity. Thus, the
1,2,4-triazine ring is a prominent structural motif found in
numerous natural and synthetic biologically active com-
pounds (Lee et al., 1993; Wei et al., 1993; Hwang et al.,
1994). In addition, certain azanucleoside, for example, 6-
azauracil (Miller et al., 1981), bearing the 1,2,4-triazine
heterocycle, has displayed an impressive array of biologi-
cal activity, among which antiviral (Sidwell et al., 1968;
Falke and Rada, 1970) and antitumor (Creasey et al., 1963;
Walters et al., 1972). 6-Azathymine (Gaito and Prusoff,
1962) also demonstrated the potential as antimicrobial
(Prusoff and Welch, 1956; Gunther and Prusoff, 1967),
anticancer (Ellison et al., 1960), and anti-HIV (Luo et al.,
2000) agents. These activities have been cited in the sci-
entific literature. In this study, 6-substituted 1,2,4-triazine-
An alternated strategy to synthesize disubstituted target
compounds 9 and 10 was shown in Scheme 2. N-alkylation
of 1 and 2 under basic condition by the two ionizable
protons afforded dialkylated products 7 and 8. According
to Jane et al. (1997), it should be possible to obtain mon-
osubstituted (N1-substituted) products selectively by care-
fully controlling the pH value. In this article, we use excess
amount of anhydrous K2CO3 and add the 4-substituted-
2-bromoacetophenones in one portion to force the reaction
toward dialkylated products. Once the N1-monosubstituted
products formed, the acidity of the N3 proton is increased
sufficiently to undergo further alkylation (Nollet and Pan-
dit, 1969). The same Reformatsky-type reaction was
applied for the synthesis of 9 and 10 from their respective
ketone precursors 7 and 8.
In the present study, 12 compounds were synthesized
and calculated the MP as well as the compound scores
(Table 1). The parameters included in the MP were chosen
after balancing their caveats and advantages. The ROF
results showed all compounds passed the test. The data
show that the increase in molecular size and structural
complexity in more potent antibacterial activity are clearly
acceptable (O’Shea and Moser, 2008).
1,2,4-triazine
1
O
O
2
Determination of the in vitro antibacterial activities of
the target compounds was performed using the agar dif-
fusion method as depicted in Table 2. These compounds
demonstrated the selective inhibitory activity against
Bacillus cereus with MIC values from 10 to 50 lg/ml.
Overall antibacterial activity of synthesized compounds is
moderate as compared to the 6-azauracil as a reference
drug.
γ
5
β
α
4
3
R
n
n=1 or 2
Chart 1
123

Med Chem Res (2011) 20:1081–1090
1083
Scheme 1 Synthetic pathway
for the preparation of title
compounds (5, 6)
O
N
O
O
X
OSi(CH3)3
X
X
HN
Br
R
HN
HMDS
TMSCl
N
O
N
N
O
O
N
H
N
N
R
(H3C)3SiO
(1) X=H
(2) X=CH3
(3) X=H
(4) X=CH3
O
5
2
4
X
CO2Et
HN
6
1
1
N
1.
2. H+
, Zn
O
3
N
CH2Br
O
5
O
2
R
4
3
a R=H
(6) X=CH3 b R=F
c R=C6H5
(5) X=H
Scheme 2 Synthetic pathway
for the preparation of title
compounds (9, 10)
O
N
O
O
R
X
N
Br
X
N
R
N
K2CO3
DMF
HN
O
O
O
N
H
R
(1) X=H
(7) X=H
O
(2) X=CH3
(8) X=CH3
R
O
CO2Et
1
5
2
4
O
X
O
N
2
6
1.
2. H+
, Zn
5
CH2Br
N
3
1
4
O
3 N
1
O
O
2
R
5
3
4
a R=H
b R=F
(9) X=H
(10) X=CH3
c R=C6H5
Experimental
shifts were expressed in parts per million (d) with tetra-
methylsilane (TMS) as an internal standard. Thin-layer
chromatography (TLC) was run on pre-coated (0.2 mm)
silica gel 60 F-254 plates manufactured by EM Laborato-
ries, Inc., and short-wave UV light (254 nm) was used to
detect the UV-absorbing spots. Elemental analyses were
carried out for carbon, hydrogen, nitrogen, and oxygen on
a Heraeus CHN-O-Rapid elemental analyzer. Calculated
Chemistry
Melting points were determined on an Yanaco micro
melting-point apparatus and are uncorrected. Nuclear
magnetic resonance (NMR) (1H and 13C) spectra were
recorded on a Varian Gemini-200 spectrometer. Chemical
123

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Med Chem Res (2011) 20:1081–1090
amount of chlorotrimethylsilane (ca. 2 ml) and heated at
reflux with exclusion of moisture. Excess silylating reagent
was removed in vacuo with protection against moisture. The
residual clear oil was dissolved in dry acetonitrile (25 ml)
and cooled to 0°C. A solution of 4-substituted 2-bromo-
acetophenones (11 mmol) in dry acetonitrile (10 ml) was
added. The solution was allowed to stir for 2 h while
warming to room temperature at which TLC indicated
complete reaction. The organic phase was evaporated and
the crude oil submitted to flash column chromatography
(silica gel 230–400 mesh, CH3OH/CH2Cl2) to give 2-(2-
oxo-2-(4-substituted phenyl)ethyl)-1,2,4-triazine-3,5(2H,
4H)-diones (3) and (4). Reformatsky-type condensation of 3
and 4 react with ethyl a-(bromomethyl)acrylate and zinc
powder in dry tetrahydrofuran to give compounds 5 and 6.
To a solution of 2-(2-oxo-2-(4-substituted phenyl)ethyl)
-1,2,4-triazine-3,5(2H,4H)-dione (2 mmol) in dry THF
(60 ml) were added activated zinc power (0.17 g, 2.6 mmol),
hydroquinone (4 mg), and ethyl a-(bromomethyl)acrylate
(0.52 g, 2.6 mmol). The mixture was refluxed under nitro-
gen atmosphere for 6 h (TLC monitoring). After cooling, it
was poured into an ice-cold 5% HCl solution (200 ml) and
extracted with CH2Cl2 (3 9 75 ml). The extracts were
combined, washed with H2O, dried (Na2SO4), and evapo-
rated to give a residual solid which was crystallized from
CH2Cl2 and Et2O (1:5) to afford 6-substituted 2-[(4-meth-
ylene-5-oxo-2-(4-substituted phenyl) tetrahydrofuran-2-
yl)methyl]-1,2,4-triazine-3,5(2H,4H)-diones (5) and (6) in a
fairly good yield.
Table 1 Molecular properties and compound scores of compounds
(5, 6) and (9, 10)
Compound MW
Hacc Hdon Rotable AlogP98 No of Score
bond
N&O
5a
299.29
317.28
5
6
1
1
1
1
1
1
0
0
0
0
0
0
3
3
4
3
3
4
6
6
8
6
6
8
1.4484
1.6539
2.9668
1.5269
1.7324
3.0453
4.0249
4.4359
7.0617
4.1034
4.5144
7.14021
7
7
7
7
7
7
9
9
9
9
9
9
1
5b
5c
1
375.388 5
313.317 5
331.307 6
389.415 5
485.502 7
521.482 9
637.698 7
499.529 7
535.509 9
651.725 7
1
6a
1
6b
6c
1
1
9a
1
9b
9c
0.9
0.8
1
10a
10b
10c
0.9
0.8
and found values are within 0.4% of the theoretical
compositions.
Procedure for the synthesis of 6-substituted
2-[(4-methylene-5-oxo-2-(4-substituted phenyl)
tetrahydrofuran-2-yl)methyl]-1,2,4-triazine-3,
5(2H,4H)-dione (5, 6)
To a suspension of 6-substituted 1,2,4-triazine-3,5(2H,4H)-
diones (10 mmol) in HMDS (25 ml) was added a catalytic
Table 2 Antibacterial activities of compounds (5, 6) and (9, 10) against selected bacterial strains (MICs in lg/ml)
Compound
Microorganisms
Bordetella
bronchiseptica
Proteus
vulgaris
Bacillus
subtilis
Micrococcus
lutca
Bacillus
cereus
Staphylococcus
epidermidis
Staphylococcus
aureus
5a
NA
NA
NA
NA
NA
50
5
NA
NA
NA
NA
NA
25
NA
NA
NA
NA
NA
NA
NA
NA
50
NA
50
NA
NA
NA
NA
NA
25
NA
NA
NA
NA
NA
NA
NA
NA
50
5b
50
5c
25
50
6a
NA
NA
NA
NA
NA
25
NA
50
6b
6c
NA
NA
10
9a
NA
NA
25
NA
NA
10
NA
NA
10
9b
9c
25
10a
10b
10c
6-AU (1)
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
25
NA
25
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
25
NA
MIC minimal inhibitory concentration, determined as micrograms per milliliter. The MIC [100 lg/ml listed as NA (not detected). Bordetella
bronchiseptica ATCC 4617, Proteus vulgaris ATCC 13315, Bacillus subtilis ATCC 6633, Micrococcus lutca ATCC 9341, Bacillus cereus
ATCC 11778, Staphylococcus epidermidis ATCC 12228, Staphylococcus aureus ATCC 25923
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Med Chem Res (2011) 20:1081–1090
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2-(2-Oxo-2-phenylethyl)-1,2,4-triazine-3,5(2H,4H)-dione
(3a)
6-Methyl-2-(2-Oxo-2-(4-phenylphenyl)ethyl)-1,2,4-
triazine-3,5(2H,4H)-dione (4c)
Yield 68.2%, m.p. 138–139°C. 1H NMR (DMSO-d6,
200 MHz) d 5.332 (s, 2H, NCH2), 7.594 (d, 2H, ph),
7.651(s, 1H, C6-H), 7.733(m, 1H, ph), 8.805(d, 2H, ph),
12.823 (br, 1H, NH); 13C NMR (DMSO-d6, 50 MHz) d
45.667, 128.167, 129.046, 134.083, 134.332, 134.604,
148.833, 155.838, 191.581. HRMS (ESI, m/z) C11H9N3O3
calcd 231.0644, found 231.0641.
Yield 83.7%, m.p. 142–143°C. 1H NMR (DMSO-d6,
200 MHz) d 2.106 (s, 3H, CH3), 5.478 (s, 2H, NCH2),
7.482 (m, 3H, ph), 7.762 (d, 2H, ph), 7.881 (d, 2H, ph),
8.123 (d, 2H, ph), 12.305 (br, 1H, NH); 13C NMR (DMSO-
d6, 50 MHz) d 15.948, 56.514, 127.015, 128.537, 128.783,
129.083, 132.957, 138.623, 143.289, 145.342, 148.947,
157.196, 192.581. HRMS (ESI, m/z) C18H15N3O3 calcd
321.1113, found 321.1110.
2-(2-Oxo-2-(4-fluorophenyl)ethyl)-1,2,4-triazine-
3,5(2H,4H)-dione (3b)
2-[(4-Methylene-5-oxo-2-phenyl tetrahydrofuran-2-
yl)methyl]-1,2,4-triazine-3,5(2H,4H)-dione (5a)
Yield 76.5%, m.p. 101–102°C. 1H NMR (DMSO-d6,
200 MHz) d 5.499 (s, 2H, NCH2), 7.419 (d, 2H, ph), 7.574(s,
1H, C6-H), 8.136(d, 2H, ph), 12.378 (br, 1H, NH); 13C NMR
(DMSO-d6, 50 MHz) d 56.722, 115.915, 116.207, 131.182,
131.312, 135.825, 148.348, 157.065, 191.591. HRMS (ESI,
m/z) C11H8N3O3 calcd 249.0550, found 249.0546.
Yield 72.5%, m.p. 129–130°C. 1H NMR (DMSO-d6,
200 MHz) d 3.024 (d, 1H, CH2), 3.499 (d, 1H, CH2), 4.092
(d, 1H, NCH2), 4.413 (d, 1H, NCH2), 5.472 (s, 1H, CH2),
6.037 (s, 1H, CH2), 7.125 (s, 1H, CH), 7.189–7.337 (m, 5H,
ph), 12.213 (br, 1H, NH); 13C NMR (DMSO-d6, 50 MHz)
d 29.651, 58.427, 85.789, 123.154, 124.805, 128.805,
128.879, 133.124, 135.236, 139.812, 145.528, 159.341,
163.864 HRMS (ESI, m/z) C15H13N3O4 calcd 299.0906,
found 299.0903.
2-(2-Oxo-2-(4-phenylphenyl)ethyl)-1,2,4-triazine-
3,5(2H,4H)-dione (3c)
Yield 74.4%, m.p. 105–106°C. 1H NMR (DMSO-d6,
200 MHz) d 5.534 (s, 2H, NCH2), 7.497 (m, 3H, ph), 7.588
(s, 1H, C6-H), 7.774 (d, 2H, ph), 7.884 (d, 2H, ph), 8.128 (d,
2H, ph), 12.406(br, 1H, NH);13C NMR (DMSO-d6, 50 MHz)
d 56.799, 127.046, 128.576, 128.845, 129.106, 132.896,
135.802, 138.631, 145.426, 148.371, 192.450. HRMS (ESI,
m/z) C17H13N3O3 calcd 307.0957, found 307.0954.
2-[(4-Methylene-5-oxo-2-(4-fluorophenyl)
tetrahydrofuran-2-yl)methyl]-1,2,4-triazine-3,5(2H,4H)-
dione (5b)
Yield 79.3%, m.p. 102–103°C. 1H NMR (DMSO-d6,
200 MHz) d 3.196 (d, 1H, CH2), 3.593 (d, 1H, CH2), 4.253
(d, 1H, NCH2), 4.324 (d, 1H, NCH2), 5.696 (s, 1H, CH2),
5.989 (s, 1H, CH2), 7.570 (s, 1H, CH), 7.228 (dd, 2H, ph),
7.431 (dd, 2H, ph), 12.218 (br, 1H, NH) 13C NMR (DMSO-
d6, 50 MHz) d 29.575, 57.994, 85.985, 114.276, 122.817,
126.758, 126.814, 133.245, 135.996, 145.349, 154.292,
159.333, 164.468 HRMS (ESI, m/z) C15H12FN3O4 calcd
317.0812, found 317.0815.
6-Methyl-2-(2-oxo-2-phenylethyl)-1,2,4-triazine-
3,5(2H,4H)-dione (4a)
Yield 81.5%, m.p. 118–119°C. 1H NMR (DMSO-d6,
200 MHz) d 2.097(s, 3H, CH3), 5.447 (s, 2H, NCH2), 7.567
(d, 2H, ph), 7.692 (m, 1H, ph), 8.026 (d, 2H, ph), 12.726
(br, 1H, NH); 13C NMR (DMSO-d6, 50 MHz) d 15.964,
56.522, 128.045, 128.929, 134.134, 143.312, 148.955,
157.203, 193.050. HRMS (ESI, m/z) C12H11N3O3 calcd
245.0800, found 245.0803.
2-[(4-Methylene-5-oxo-2-(4-phenylphenyl)
tetrahydrofuran-2-yl)methyl]-1,2,4-triazine-3,5(2H,4H)-
dione (5c)
6-Methyl-2-(2-Oxo-2-(4-fluorophenyl)ethyl)-1,2,4-triazine-
3,5(2H,4H)-dione (4b)
Yield 76.1%, m.p. 133–134°C. 1H NMR (CDCl3-d,
200 MHz) d 3.176 (d, 1H, CH2), 3.636 (d, 1H, CH2), 4.231
(d, 1H, NCH2), 4.557 (d, 1H, NCH2), 5.606 (s, 1H, CH2),
6.170 (s, 1H, CH2), 7.570(s, 1H, CH), 7.344–7.665 (m, 9H,
ph-ph), 12.203 (br, 1H, NH) 13C NMR (CDCl3-d, 50 MHz)
d 29.652, 58.398, 85.065, 122.026, 125.304, 127.041,
127.511, 127.739, 128.869, 133.945, 135.606, 138.952,
140.006, 141.028, 149.823, 160.253, 164.362. HRMS (ESI,
m/z) C21H17N3O4 calcd 375.1219, found 375.1215.
Yield 79.5%, m.p. 109–110°C. 1H NMR (DMSO-d6,
200 MHz) d 2.094(s, 3H, CH3), 5.444 (s, 2H, NCH2), 7.401
(d, 2H, ph), 8.129 (d, 2H, ph), 12.288 (br, 1H, NH); 13C
NMR (DMSO-d6, 50 MHz) d 15.941, 56.438, 116.176,
130.936, 143.328, 148.939, 157.188, 163.776, 167.128,
191.720. HRMS (ESI, m/z) C12H10FN3O3 calcd 263.0706,
found 263.0703.
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6-Methyl-2-[(4-methylene-5-oxo-2-phenyl tetrahydrofuran-
2-yl)methyl]-1,2,4-triazine-3,5(2H,4H)-dione (6a)
water (100 ml) and extracted with CH2Cl2 (3 9 20 ml). The
organic phase was washed with H2O, dried (Na2SO4), and
evaporated to give 2,4,-bis(2-oxo-2-(4-substituted pheny-
l)ethyl)-1,2,4-triazine-3,5(2H,4H)-diones (7) and (8). The
same Reformatsky-type condensation procedure was used to
convert 7a–c to 2,4-bis[2-(4-substituted phenyl)-2-oxoeth-
yl]-1,2,4-triazine-3,5(2H,4H)-dione (9a–c) and 8a–c to 2,
4-bis[2-(4-substituted phenyl)-2-oxoethyl]-6-methyl-1,2,4-
triazine-3,5(2H,4H)-dione (10a–c), respectively.
Yield 81.7%, m.p. 127–128°C. 1H NMR (DMSO-d6,
200 MHz) d 1.993 (s, 3H, CH3), 3.110–3.168 (d, 1H, CH2),
3.571–3.627 (d, 1H, CH2), 4.130–4.179 (d, 1H, NCH2),
4.316–4.363 (d, 1H, NCH2), 5.635 (s, 1H, CH2), 6.936 (s,
1H, CH2), 7.356–7.434 (m, 5H, ph), 12.864 (br, 1H, NH).
13C NMR (DMSO-d6, 50 MHz) d 16.539, 35.556, 56.999,
85.469, 121.213, 125.696, 127.230, 128.161, 135.625,
142.369, 151.082, 155.212, 163.238, 165.833 HRMS (ESI,
m/z) C16H15N3O4 calcd 313.1063, found 313.1059.
2,4-Bis(2-oxo-2-phenyl ethyl)-1,2,4-triazine-3,5(2H,4H)-
dione (7a)
Yield 82.9%, m.p. 125–126°C. 1H NMR (DMSO-d6,
200 MHz) d 5.417 (s, 2H, NCH2), 5.627 (s, 2H, NCH2),
7.789 (d, 4H, ph), 7.752 (m, 2H, ph), 7.848 (s, 1H, C6-H),
8.061 (d, 4H, ph); 13C NMR (DMSO-d6, 50 MHz) d
46.552, 57.752, 128.114, 128.937, 128.975, 133.957,
134.011, 134.218, 134.280, 134.667, 148.271, 155.320,
191.112, 192.504. HRMS (ESI, m/z) C19H15N3O4 calcd
349.1063, found 349.1060.
6-Methyl-2-[(4-methylene-5-oxo-2-(4-fluorophenyl)
tetrahydrofuran-2-yl)methyl]-1,2,4-triazine-3,5(2H,4H)-
dione (6b)
Yield 86.9%, m.p. 108–109°C. 1H NMR (DMSO-d6,
200 MHz) d 1.989 (s, 3H, CH3), 3.136–3.192 (d, 1H, CH2),
3.589–3.647 (d, 1H, CH2), 4.138–4.186 (d, 1H, NCH2),
4.290–4.338 (d, 1H, NCH2), 5.668 (s, 1H, CH2), 5.957 (s,
1H, CH2), 7.216–7.276 (dd, 2H, ph), 7.441–7.489 (dd, 2H,
ph), 12.126 (br, 1H, NH). 13C NMR (DMSO-d6, 50 MHz)
d 16.649, 35.6276, 56.236, 85.132, 116.242, 123.596,
126.124, 127.290, 133.621, 141.999, 152.093, 156.221,
164.269, 168.844 HRMS (ESI, m/z) C16H14N3O4F calcd
331.0968, found 331.0965.
2,4-Bis(2-oxo-2-(4-fluorophenyl)ethyl)-1,2,4-triazine-
3,5(2H,4H)-dione (7b)
Yield 88.7%, m.p. 112–113°C. 1H NMR (DMSO-d6,
200 MHz) d 5.405 (s, 2H, NCH2), 5.618 (s, 2H, NCH2),
7.426 (d, 4H, ph), 7.843 (s, 1H, C6-H), 8.174 (d, 4H, ph);
13C NMR (DMSO-d6, 50 MHz) d 46.413, 57.622, 115.863,
116.155, 130.746, 131.146, 131.284, 134.636, 148.197,
155.239, 163.803, 167.155, 189.772, 191.140. HRMS (ESI,
m/z) C19H1F23N3O4 calcd 385.0874, found 385.0870.
6-Methyl-2-[(4-methylene-5-oxo-2-(4-phenylphenyl)
tetrahydrofuran-2-yl)methyl]-1,2,4-triazine-3,5(2H,4H)-
dione (6c)
Yield 83.6%, m.p. 151–152°C. 1H NMR (CDCl3-d,
200 MHz) d 1.996 (s, 3H, CH3), 3.158–3.215 (d, 1H, CH2),
3.591–3.648 (d, 1H, CH2), 4.172–4.221 (d, 1H, NCH2),
4.357–4.406 (d, 1H, NCH2), 5.661 (s, 1H, CH2), 5.951 (s, 1H,
CH2), 7.378–7.523 (m, 5H, Ph-Ph), 7.662–7.738 (m, 4H, ph-
ph), 12.149 (br, 1H, NH). 13C NMR (CDCl3-d, 50 MHz) d
16.507, 30.341, 57.342, 86.450, 120.782, 125.950, 126.786,
126.805, 127.773, 129.076, 135.055, 136.325, 136.692,
137.342, 139.994, 149.861, 153.374, 155.463 HRMS (ESI,
m/z) C22H19N3O4 calcd 389.1376, found 389.1373.
2,4-Bis(2-oxo-2-(4-phenylphenyl)ethyl)-1,2,4-triazine-
3,5(2H,4H)-dione (7c)
Yield 79.5%, m.p. 106–107°C. 1H NMR (DMSO-d6,
200 MHz) d 5.452 (s, 2H, NCH2), 5.661 (s, 2H, NCH2),
7.498 (m, 6H, ph), 7.784 (d, 4H, ph), 7.865 (s, 1H, C6-H),
7.899 (d, 4H, ph), 8.162 (m, 4H, ph); 13C NMR (DMSO-d6,
50 MHz) d 46.640, 57.841, 127.142, 128.696, 128.995,
129.222, 132.888, 134.790, 138.707, 145.643, 148.419,
155.476, 190.842, 192.226. HRMS (ESI, m/z) C31H23N3O4
calcd 501.1689, found 501.1686.
Procedure for the synthesis of 6-substituted 2,4-bis[(4-
methylene-5-oxo-2-(4-substituted phenyl)
tetrahydrofuran-2-yl)methyl]-1,2,4-triazine-
3,5(2H,4H)-dione (9, 10)
6-Methyl-2,4-bis(2-oxo-2-phenylethyl)-1,2,4-triazine-
3,5(2H,4H)-dione (8a)
Yield 81.0%, m.p. 133–134°C. 1H NMR (DMSO-d6,
200 MHz) d 2.204 (s, 3H, CH3), 5.418 (s, 2H, NCH2),
5.574 (s, 2H, NCH2), 7.590 (m, 4H, ph), 7.708 (m, 2H, ph),
8.073 (d, 4H, ph); 13C NMR (DMSO-d6, 50 MHz) d
16.524, 46.782, 57.453, 128.040, 127.071, 128.901,
1,2,4-Triazine-3,5(2H,4H)-dione (6-azauracil) (1, 10 mmol)
was treated with excess amount of K2CO3 (22 mmol) and
4-substituted 2-bromoacetophenones (22 mmol) in dry
DMF (20 ml). The resulting mixture was stirred at room
temperature for 48 h (TLC monitoring) then poured into ice-
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Med Chem Res (2011) 20:1081–1090
1087
128.947, 134.006, 134.083, 134.129, 134.206, 142.201,
148.581, 155.546, 191.201, 192.616. HRMS (ESI, m/z)
C20H17N3O4 calcd 361.1219, found 361.1215.
159.567, 166.146. HRMS (ESI, m/z) C27H21N3O6F2 calcd
521.1398, found 521.1395.
2,4-Bis[(4-methylene-5-oxo-2-(4-phenylphenyl)
tetrahydrofuran-2-yl)methyl]-1,2,4-triazine-3,5(2H,4H)-
dione (9c)
6-Methyl-2,4-bis(2-oxo-2-(4-fluorophenyl)ethyl)-1,2,4-
triazine-3,5(2H,4H)-dione (8b)
Yield 72.9%, m.p. 149–150°C. 1H NMR (DMSO-d6,
200 MHz) d 2.196 (s, 3H, CH3), 5.414 (s, 2H, NCH2),
5.569 (s, 2H, NCH2), 7.412 (m, 4H, ph), 8.162 (m, 4H, ph);
13C NMR (DMSO-d6, 50 MHz) d 16.539, 46.721, 57.422,
115.849, 116.816, 116.632, 130.823, 131.161, 131.284,
142.293, 148.597, 155.562, 163.818, 167.185, 167.216,
189.941, 191.324. HRMS (ESI, m/z) C20H15F2N3O4 calcd
399.1031, found 399.1027.
Yield 72.7%, m.p. 102–103°C. 1H NMR (CDCl3-d,
200 MHz) d 3.173 (d, 2H, CH2), 3.643 (d, 2H, CH2), 4.235
(d, 2H, NCH), 4.545 (d, 2H, NCH), 7.230 (s, 1H, CH),
7.340–7.640 (m, 18H, CH, ph-ph). 13C NMR (CDCl3-d,
50 MHz) d 36.962, 58.972, 85.979, 124.154, 125.816,
126.783, 126.864, 127.129, 128.805, 128.879, 133.124,
135.236, 135.893, 136.216, 139.327, 140.228, 145.528,
147.336, 157.242, 167.468. HRMS (ESI, m/z) C39H31N3O6
calcd 637.2213, found 637.2209.
6-Methyl-2,4-bis(2-oxo-2-(4-phenylphenyl)ethyl)-1,2,4-
triazine-3,5(2H,4H)-dione (8c)
6-Methyl-2,4-bis[(4-methylene-5-oxo-2-phenyl
tetrahydrofuran-2-yl)methyl]-1,2,4-triazine-3,5(2H,4H)-
dione (10a)
Yield 87.2%, m.p. 126–127°C. 1H NMR (DMSO-d6,
200 MHz) d 2.222 (s, 3H, CH3), 5.460 (s, 2H, NCH2), 5.612
(s, 2H, NCH2), 7.498 (m, 6H, ph), 7.781 (d, 4H, ph), 7.894 (d,
4H, ph), 8.165 (m, 4H, ph); 13C NMR (DMSO-d6, 50 MHz) d
16.555, 46.813, 57.484, 127.010, 127.087, 128.548, 128.840,
129.070, 132.853, 132.899, 138.603, 142.247, 145.383,
145.476, 148.628, 155.577, 190.786, 192.185. HRMS (ESI,
m/z) C32H25N3O4 calcd 515.1845, found 515.1842.
Yield 80.5%, m.p. 156–157°C. 1H NMR (DMSO-d6,
200 MHz) d 2.108 (s, 3H, CH3), 3.168 (dt, 2H, CH2), 3.560
(dt, 2H, CH2), 4.228 (d, 2H, NCH), 4.488 (d, 2H, NCH),
5.720 (s, 2H, CH2), 5.995 (s, 2H, CH2), 7.371 (m, 2H, ph),
7.600 (m, 4H, ph), 8.084 (d, 4H, ph). 13C NMR (DMSO-d6,
50 MHz) d 16.693, 36.617, 57.182, 85.684, 121.213,
122.315, 125.696, 126.081, 127.051, 128.263, 135.526,
135.625, 137.742, 138.294, 140.099, 142.369, 151.682,
156.324, 163.183, 165.965. HRMS (ESI, m/z) C28H25N3O6
calcd 499.1743, found 499.1740.
2,4-Bis[(4-methylene-5-oxo-2-phenyl tetrahydrofuran-2-
yl)methyl]-1,2,4-triazine-3,5(2H,4H)-dione (9a)
Yield 78.5%, m.p. 136–137°C. 1H NMR (DMSO-d6,
200 MHz) d 3.268 (dt, 2H, CH2), 3.660 (dt, 2H, CH2), 4.468
(d, 2H, NCH), 4.738 (d, 2H, NCH), 5.490 (s, 2H, CH2), 5.845
(s, 2H, CH2), 7.24 (s, 1H, CH), 7.323 (m, 2H, ph), 7.521 (m,
4H, ph), 7.832 (d, 4H, ph). 13C NMR (DMSO-d6, 50 MHz) d
36.327, 58.286, 85.885, 123.244, 125.816, 126.864,
127.129, 128.805, 133.124, 135.236, 135.893, 136.216,
139.327, 140.228, 145.439, 157.316, 168.358. HRMS (ESI,
m/z) C27H23N3O6 calcd 485.1587, found 485.1584.
6-Methyl-2,4-bis[(4-methylene-5-oxo-2-(4-fluorophenyl)
tetrahydrofuran-2-yl)methyl]-1,2,4-triazine-3,5(2H,4H)-
dione (10b)
Yield 84.9%, m.p. 132–133°C. 1H NMR (DMSO-d6,
200 MHz) d 2.078 (s, 3H, CH3), 3.260 (dt, 2H, CH2), 3.504
(dt, 2H, CH2), 4.270 (d, 2H, NCH), 4.582 (d, 2H, NCH),
5.681 (s, 2H, CH2), 5.999 (s, 2H, CH2), 7.590 (d, 4H, ph),
7.947 (d, 4H, ph). 13C NMR (DMSO-d6, 50 MHz) d 16.927,
37.627, 57.362, 85.425, 116.624, 116.885, 123.669,
123.993, 126.124, 127.290, 133.621, 140.688, 141.999,
143.294, 152.093, 156.221, 164.396, 168.643. HRMS (ESI,
m/z) C28H23N3O6F2 calcd 535.1555, found 535.1552.
2,4-Bis[(4-methylene-5-oxo-2-(4-fluorophenyl)
tetrahydrofuran-2-yl)methyl]-1,2,4-triazine-3,5(2H,4H)-
dione (9b)
Yield 76.3%, m.p. 109–110°C. 1H NMR (DMSO-d6,
200 MHz) d 3.550 (dt, 2H, CH2), 3.794 (dt, 2H, CH2), 4.461
(d, 2H, NCH), 4.682 (d, 2H, NCH), 5.681 (s, 2H, CH2),
5.999 (s, 2H, CH2), 7.240 (s, 1H, CH), 7.590 (d, 4H, ph),
7.951 (d, 4H, ph). 13C NMR (DMSO-d6, 50 MHz) d 36.257,
58.346, 85.857, 114.386, 114.513, 123.817, 123.982,
126.368, 126.789, 132.541, 135.986, 145.568, 155.383,
6-Methyl-2,4-bis[(4-methylene-5-oxo-2-(4-phenylphenyl)
tetrahydrofuran-2-yl)methyl]-1,2,4-triazine-3,5(2H,4H)-
dione (10c)
Yield 85.2%, m.p. 169–170°C. 1H NMR (CDCl3-d,
200 MHz) d 2.125 (s, 3H, CH3), 3.146 (d, 2H, CH2), 3.213
(d, 2H, CH2), 4.197 (d, 2H, NCH), 4.589 (d, 2H, NCH),
123

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Med Chem Res (2011) 20:1081–1090
5.618 (s, 2H, CH2), 6.163 (s, 2H, CH2), 7.455–7.643 (m,
18H, 29 ph-ph). 13C NMR (CDCl3-d, 50 MHz) d 16.818,
37.527, 57.945, 85.970, 125.626, 126.672, 126.972,
127.041, 127.087, 127.641, 128.578, 128.932, 132.837,
134.421, 138.683, 139.483, 139.649, 140.002, 142.047,
148.874, 155.248, 168.339. HRMS (ESI, m/z) C40H33N3O6
calcd 651.2369, found 651.2366.
MP rules have the score of 1.0 or the summations will be
deducted from 1.0 (Eq. 2).
Antibacterial evaluation
The bacterial strains used in this investigation were cultures
obtained from the American Type Culture collection
(ATCC), Bethesda, USA. All strains were maintained in
semi-solid agar before use. Compounds (5, 6) and (9, 10)
were tested against Gram-positive (Bordetella bronchisep-
tica ATCC 4617 and Proteus vulgaris ATCC 13315) and
Gram-negative (Bacillus subtilis ATCC 6633, Micrococcus
lutca ATCC 9341, Bacillus cereus ATCC 11778, Staphy-
lococcus epidermidis ATCC 12228 and Staphylococcus
aureus ATCC 25923). Antibacterial activity was assayed by
the agar dilution method (Wiegand et al., 2008) using
Mueller-Hinton medium (Difco Laboratories). Prior to
testing, the bacteria were inoculated into 2 ml of tryptic soy
broth (Difco laboratories) and incubated for 16–18 h at
31°C. The cultures were then adjusted with sterile normal
saline to 2–5 9 107 colony forming units (CUF) per ml.
Tested compounds (1,000 lg) were first dissolved in 200-ll
DMSO, then 800 ll of sterilized distilled water was added
to end up with a final concentration of the stock solution of
1,000 lg/ml. Compound containing media was prepared by
adding 1 volume of its diluted solution (in DMSO) into 9
volumes of autoclaved Mueller-Hinton agar that had been
cooled to 50°C. Agar plates containing compound and
control plates without compound were spot inoculated with
l04 CFU with a Steers Replicator within 30 min after
preparation the inocular. The agar plates were allowed to
stand until the inoculums spots were absorbed and were
then incubated at 37°C for 20–22 h. The minimal inhibitory
concentration (MIC) of targets were determined by com-
parison to 6-Azauracil (1) as reference compound using the
serial dilution technique with the concentrations in the
range 100–5 lg/ml. The MIC is defined as the lowest
concentration of compound at which no visible growth of
organisms was observed.
Molecular properties calculation
The chemical structures shown in the article were built with
CHEMDRAW, and 2D structure was converted to 3D
structures using CHEM 3D software (Cambridge Soft Corp.,
USA, http://www.cambridgesoft.com). All the calculations
for the MP used the Discovery Studio 2.0, Accelrys Inc.,
USA (http://accelrys.com/products/discovery-studio) and
were carried out on a Windows-based workstation.
The scoring function used to assess the MP of the target
compounds (5, 6) and (9, 10) is expressed as follows:
MP ¼ MW [ 100 ꢁ 5 þ Hacc [ 2 ꢁ 5
þ Hdon [ 5 þ AlogP98 [ 5
þ Rotatable Bond [ 3 ꢁ 5
þ 5 \ N& O \ 15:
ð1Þ
The metric includes identification of reactive functional
groups defined by the Lipinski’s rule of five (ROF)
(Lipinski, 2000) and structural descriptors as defined by
Xu (Xu and Stevenson, 2000). The ‘‘rule of five’’ derived its
name from the cutoff values from the parameters that
measured. These cutoff values are all close to five or a
multiple of five. In Eq. 1, the number of hydrogen bond
acceptors (Hacc) is the number of N, O, S, F, Cl, Br, and I
atoms in the molecule. The number of hydrogen bond
donors (Hdon) is the number of hydrogen atoms attached to
N, O, or S. The AlogP98 descriptor is an implementation of
the atom-based AlogP method using the latest published set
of parameter (Ghose and Crippen, 1986) to calculate the
octanol–water partition coefficient. The number of rotatable
bonds is the sum of bonds that can be considered rotatable.
The N&O is the number of N atoms and O atoms. The ROF
states that poor absorption or permeation is more likely
when the descriptors are violated. Compound classes that
are substrates for biological transporters are exceptions to
the rule.
Conclusion
In the present study, a series of 6-substituted 2-[(4-methy-
lene-5-oxo-2-(4-substituted phenyl) tetrahydrofuran-2-yl)
methyl]-1,2,4-triazine-3,5(2H,4H)-diones (5, 6) and 2,4-bis
[(4-methylene-5-oxo-2-(4-substituted phenyl) tetrahydro-
furan-2-yl)methyl]-1,2,4-triazine-3,5(2H,4H)-diones(9, 10)
were synthesized and calculated the MP as well as the
compound scores. Among these a-methylene-c-butyrolac-
tones, 2-[(4-methylene-5-oxo-2-phenyl tetrahydrofuran-2-
yl)methyl]-1,2,4-triazine-3,5(2H,4H)-dione (5a) is the most
The summation of these violation rules leads to the
following of the compound scoring function:
Compound Score ¼ 1:0 ꢂ ð0:1 ꢁ MPÞ:
ð2Þ
For each compound, we have calculated the six metrics.
For example, is the molecular weight of the compound
greater than 500 (MW [ 5 9 100)? If yes, 1 is entered into
the column, otherwise, 0. Compounds that satisfying all the
123

Med Chem Res (2011) 20:1081–1090
1089
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candidates for potent and versatile antibacterial agents are
undergoing.
Acknowledgments We are grateful to the National Center for High-
performance Computing and the Center of Resources, Research and
Development of Kaohsiung Medical University for computer time
and facilities the technical supports.
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