Synthesis of?a?new series of?heterocyclic scaffolds for?medicinal purposes

Article abstract of DOI:10.1016/j.ejmech.2006.03.025

A new series of substituted 8-fluro-4H-pyrimido[2,1-b] [1,3]benzothiazole-4-ones () substituted 7-methyl-4H-isoxazolo[2,3-a]pyrimidin-4-ones, and substituted 2-methyl-5,6,7,8-tetrahydro-9H-isoxazolo[2,3-a]pyridopyrimidin-9-ones, compounds I-VII, have been prepared via condensation of β-keto esters with 2-aminopyridine derivatives, in the presence of polyphosphoric acid. The same technique has also been used to prepare diazepine compounds, VIII-X, by condensation of a γ-keto ester with 2-aminopyridine derivatives. Details of synthetic procedures are shown. The new compounds have been characterized by elemental analysis, GC-MS, FT-IR and NMR spectrometry. Antibacterial, antifungal and anticancer (cytotoxic) activities, for three of these compounds, have been investigated and are presented.

Full text of DOI:10.1016/j.ejmech.2006.03.025

European Journal of Medicinal Chemistry 41 (2006) 1017–1024
http://france.elsevier.com/direct/ejmech
Preliminary communication
Synthesis of a new series of heterocyclic scaffolds for medicinal purposes
a,
a
a
a
b
a
*
H.S. Hilal , M.S. Ali-Shtayeh , R. Arafat , T. Al-Tel , W. Voelter , A. Barakat
a
College of Sciences, An-Najah N. University, PO Box 7, Nablus, Palestine
b
Abteilung für Physikalische Biochemie des Physiologisch-Chemischen Instituts der Universität Tübingen, Hoppe-Seyler Straße 4, 72076 Tübingen, Germany
Received 6 June 2005; received in revised form 11 March 2006; accepted 16 March 2006
Available online 22 June 2006
Abstract
A new series of substituted 8-fluro-4H-pyrimido[2,1-b] [1,3]benzothiazole-4-ones () substituted 7-methyl-4H-isoxazolo[2,3-a]pyrimidin-4-
ones, and substituted 2-methyl-5,6,7,8-tetrahydro-9H-isoxazolo[2,3-a]pyridopyrimidin-9-ones, compounds I–VII, have been prepared via con-
densation of β-keto esters with 2-aminopyridine derivatives, in the presence of polyphosphoric acid. The same technique has also been used to
prepare diazepine compounds, VIII–X, by condensation of a γ-keto ester with 2-aminopyridine derivatives. Details of synthetic procedures are
shown. The new compounds have been characterized by elemental analysis, GC–MS, FT-IR and NMR spectrometry. Antibacterial, antifungal
and anticancer (cytotoxic) activities, for three of these compounds, have been investigated and are presented.
©
2006 Elsevier SAS. All rights reserved.
Keywords: β-Keto esters; 2-Aminopyridine; Diazepine; Heterocyclic; Antifungal; Antibacterial; Cytotoxic
1
. Introduction
by condensation of different β-keto esters with different 2-ami-
nopyridine derivatives, in the presence of polyphosphoric acid,
as exemplified in Scheme 1.
Naturally occurring hetero-polycyclic compounds are often
medically valuable [1]. Cytotoxic, anti-allergic, and anti-malar-
ial activities are documented for hetero-polycyclic compounds
such as colchicin [2,3], chalcones [3], 2-aryl-1,8-naphthyridin-
On the other hand, compounds VIII–X have been prepared
by condensing a γ-keto ester with different 2-aminopyridine
derivatives, in polyphosphoric acid, as exemplified in Scheme
2. Elemental analysis, GC–MS, FT-IR and NMR indicated the
preparation of compounds I–X in appreciably pure forms. The
structure, expected for each compound, has been confirmed.
Details of preparation and characterizations of these com-
pounds are presented in the experimental section.
4
-one [2] and others [4–7]. Anti-inflammatory [8,9], cardio-
tonic [10], antiallergic [11], antimalarial [12] and other activ-
ities [13–16] are known for heterocyclic compounds. There-
fore, it is important to find new efficient methods to
synthesize new hetero-polycyclic frameworks. Different syn-
thetic methods are known for such purpose. Examples are: cy-
clization via reaction of amino-heterocycles with acetylenic
compounds, [17,18], base catalyzed isoxazolinyl heterocycle
rearrangement [19–21] and intramolecular nucleophilic acyl
substitution [22–30]. The main objective of this work is to
synthesize and characterize a number of fused heterocyclic
compounds that may potentially have medical value, based
on structural similarities with earlier compounds.
3. Biological activity results
Three compounds, I, IV and V, have been studied for pos-
sible future biological functions.
2
. Chemistry
Two different groups of compounds have been prepared and
characterized [27,31]. Compounds I–VII have been prepared
*
Corresponding author. Fax: +970 9 238 7982.
E-mail address: hikmathilal@yahoo.com (H.S. Hilal).
Scheme 1. Structural formulas for I–VII here.
0223-5234/$ - see front matter © 2006 Elsevier SAS. All rights reserved.
doi:10.1016/j.ejmech.2006.03.025

1018
H.S. Hilal et al. / European Journal of Medicinal Chemistry 41 (2006) 1017–1024
Scheme 2. Structural formulas for compounds VIII–X here.
3
.1. Antibacterial and antifungal activities
Contrary to statistical results obtained from Table 1, the data
in Table 2 show statistically significant differences among the
group of compounds, including reference, with a P-va-
lue = 0.001.
Three compounds I, IV and V were all investigated for anti-
bacterial activity against Staphylococcus aureus, Proteus vul-
garis and Candida albicans. None of these compounds showed
any significant antibacterial activity. The same compounds
showed significant antifungal activity against Microsporum ca-
nis, Fusarium tricincutum, Pythium ultimum, P. aphaniderma-
tum and P. middletonii. Results are shown in Tables 1 and 2.
The inhibition effects of compounds I, IV and V, together
with the reference values, on M. canis and F. tricincutumare
are shown in Table 1. The data showed significant differences
between inhibition effects on M. canis and F. tricincutum, with
a P-value < 0.001. On the other hand, all compounds, includ-
ing the reference, showed same inhibition effects, with no sig-
nificant differences. Thus, all compounds used including the
references, had similar activity, while M. canis is less sensitive
than the other fungus counterpart.
Each compound activity was compared to the reference,
using the Dunnett’s intervals. The activity of I showed same
activity as reference activity, whereas compound IV and V
showed different activities from reference (with P-va-
lues < 0.01). Thus, compounds IV and V are statistically more
active than compound I and the reference. Furthermore, P. mid-
dletonii is more resistant than other fungus counterparts.
This shows the future potential of using the prepared com-
pounds in antifungal medical formulations. The hymexazole
[32] (3-hydroxy-5-methylisoxazole) reference, resembles the
structure of the starting material for compounds IV and V.
No correlation between structures, for I, II and V, and their
activities were constructed.
Inhibition effects of compounds I, IV and V, and refer-
ences, on P. ultimum, P. aphanider-matum and P. middletonii,
are shown in Table 2. The three fungi did not have the same
sensitivity to the group of compounds, with a P-value < 0.001.
Therefore, multiple comparisons, using Tukey’s test, had been
conducted and indicated that P. ultimum and P. aphaniderma-
tum had same sensitivity, which is different from that of P. mid-
dletonii (P-value < 0.001).
3.2. Cytotoxic activity
Three compounds IV, IX and X have been evaluated for
cytotoxic activity. The results are summarized in Table 3. Va-
lues of percent mortality of cells against compound concentra-
tion for each compound are shown in Fig. 1. Among the three
compounds used, IV and X showed significant cytotoxic activ-
ities, whereas IX failed to do so. In IV and X, percent cell
mortality increased with compound concentration. The data in-
dicate that X is potentially more favorable than IV. At 1.5 ×
Table 1
–
1
Antifungal activity results (mm day ) for compounds I, IV and V against
M. canis and F. tricincutum, compared to reference values
–
3
10 M concentration, X showed about 95% mortality, before
changing into a plateau. Activity of IV was lower than that of
X, within the concentration range used. To achieve mortality
Compound
% Inhibition (mean of three replicate plates ± S.E.)*
a
b
M. canis
F. tricincutum
68.5 ± 1.7
42.6 ± 0.6
48.9 ± 5.8
67.8 ± 1.9
–
3
I
IV
V
100.00 ± 0.0
84.5 ± 0.0
percent of 41%, high concentrations (~3.0 × 10 M) were
needed. This limits the potential value of IV in medical appli-
cations. Contrary to IV and X, compound IX showed incon-
sistent correlation between percent mortality and concentration,
100.00 ± 0.0
75.5 ± 3.9
Ref.
Compound concentrations used are shown in section 5.4.
–
4
a
with only a small peak (40%) for 6.25 × 10 M concentration.
Ref. griseofulvin;
Ref. nystatin.
b
4. Conclusions
Table 2
–
1
Antifungal activity results (mm day ) for compounds I, IV and V against
P. ultimum, P. aphanidermatum and P. middletonii compared to reference
values
Compounds I–X have been prepared. Two compounds, (III
and VIII), occurred in relatively low yields (36% and 35%,
respectively), whereas the remaining compounds occurred in
high yields. Spectral analyses confirmed the proposed struc-
tures for the synthesized compounds. Compounds I, IV and
X showed neither antibacterial activity against Staphylococcus
aureus, Proteus vulgaris nor anticandidal activity against Can-
dida albicans. When tested against Microsporum canis, Fusar-
ium tricincutum, Pythium ultimum, Pythium aphanidermatum
and Pythium middletonii, compounds I, IV and X showed sig-
Compound
% Inhibition
P. aphanidermatum
95.9 ± 0.6
100.00 ± 0.0
100.00 ± 0.0
70.0 ± 3.3
P. ultimum
80.4 ± 2.3
100.00 ± 0.0
91.9 ± 0.6
81.1 ± 5.1
P. middletonii
44.4 ± 3.5
80.0 ± 1.7
78.9 ± 1.0
24.3 ± 10.1
I
IV
V
Ref. (hymexazol)
*
Mean of three replicate plates ± S.E. Compound concentrations used are
shown in Section 5.4.

H.S. Hilal et al. / European Journal of Medicinal Chemistry 41 (2006) 1017–1024
1019
Table 3
Cytotoxic activity (MTT assay results) of compounds IV, IX and X
5.2. Equipment
Compound Stock solution Final
concentration concentration
Absorbance
at λmax
% Mortality
of cells
Elemental analysis data were measured on a PE 2400 Perkin
Elmer series II CHNS/O analyzer. FT-IR spectra were mea-
sured on a Shimadzu 820 PC FT-IR Spectrometer. GC-MS re-
sults were recorded on a Shimadzu GC-MS-QP5000, using
chloroform and methanol as solvents. The GC runs were con-
ducted using a J&W Scientific DB-5MS 30 m long 0.25 mm in
diameter capillary column, equipped with 5% phenyl methyl
polysilane as stationery. Helium carrier gas was used at a flow
(
M)
(M)
2.90 × 10
–
2
–3
IV
6.10 × 10
0.030
0.039
0.043
0.050
0.040
0.031
0.031
0.002
0.003
0.009
0.028
0.039
41.20
23.50
15.70
1.96
12.60
39.20
39.20
96.00
94.10
82.40
45.10
22.90
–
–
–
3
4
4
1
7
3
.50 × 10
.30 × 10
.60 × 10
–
2
2
–3
IX
X
5.25 × 10
6.30 × 10
1.25 × 10
–
–
4
4
6
3
.25 × 10
.13 × 10
–
1
–
–3
rate of 28.2 ml min . The injection unit was kept at 250 °C,
and the interface temperature was 230 °C. The oven tempera-
3.00 × 10
–
–
–
–
3
4
4
4
1
7
3
1
.50 × 10
.50 × 10
.70 × 10
.90 × 10
–1
ture was initially set at 120 °C, with a ram rate 5 °C min
reaching a final temperature of 300 °C. The waiting time at
each stage was 0.5 min.
Absorbance for the reference was 0.051 at 550 nm.
13
C NMR spectra were recorded on a Bruker 75.48 MHz,
using CDCl as a solvent, courtesy of the laboratories of Jor-
3
dan University. The solvent characteristic signal was observed
1
as a triplet at 77.1 ppm. H NMR spectra were recorded on a
Bruker 300 MHz, using CDCl as a solvent.
3
5.3. Compound preparations and analyses
Compounds I–X were prepared by the reaction of the ami-
no-heterocycle with β- or γ-keto esters in the presence of PPA,
while continuously stirring for 2 hours at 120 °C [27,31]. Un-
less otherwise stated the general preparation procedure was as
follows: PPA, 3–6 g, was weighed in a 50-ml conical flask.
The amino heterocycle (0.5 g) was added. The β-, or γ-keto
ester was then added as excess (the molar ratio of the keto ester
to the amino heterocycle was 1.1/1, respectively). The mixture
was heated, while manually stirring, in an oil bath for 20 min
until a 120 °C final temperature was reached. The mixture was
kept at this temperature for 2 more hours with continuous stir-
ring [31]. The reaction was monitored periodically by thin
layer chromatography (TLC) using 5% ethylacetate/chloroform
developing solvent. After completion, the reaction was cooled
in an ice bath. Ice water was added to the mixture. The solution
was then neutralized by NaOH (4 N) solution. The precipitate
product was filtered on a sintered funnel and washed with dis-
tilled water. Non-precipitate products were alternatively ex-
tracted using the proper solvent. Details of product preparation,
description and analysis are shown below.
Fig. 1. Values of percent mortality of cells for different compound
concentrations.
nificant antifungal activities, as compared to reference com-
pounds. Compounds IV, IX and X were investigated for cyto-
toxic activity. Compounds IV and X showed significant cyto-
toxic activity, whereas IX showed low activity. Among the
tested compounds, X is the most promising candidate showing
high cytotoxic activity at relatively low concentrations.
5
. Experimental
5
.1. Chemicals
I: Prepared from 2-amino-6-fluorobenzothiazole (0.5 g,
2
.97 mmol) and ethylacetoacetate (0.4 g, 3.08 mmol) in PPA
Starting materials, 2-amino-6-fluorobenzothiazole, 1-ami-
(3.0 g). Product, recrystallized from EtOH, 0.6 g, 86% yield,
yellowish ppt., m.p. range 193–195 °C - H NMR (CDCl ,
300 MHz), H : 3H at 2.38 ppm (s); H : 1H at 6.26 ppm (s);
H : 1H at 7.22 ppm (dd, J
1
noisoquinoline, 3-amino-5-methylisoxazole, ethylacetoacetate,
ethyl-2-oxocyclopentane carboxylate, ethyl-2-oxocyclohexane-
carboxylate, ethyl-2-cyclohexanone acetate, methyl-1-benzyl-
3
a
3
= 8.3 Hz, JH6-F = 7.9 Hz)); H7:
H6-H7
6
4
3
-oxo-3-piperidinecarboxylate hydrochloride, ethyl-1-benzyl-
-oxo-4-piperidine carboxylate hydrochloride, polyphosphoric
1H at 9.05 ppm (dd, JH7-H6 = 8.8 Hz, J
= 4.7 Hz); H : 1H
H7-F 9
1
3
at 7.39 ppm (d, JH9-F = 5.8 Hz), [33] – C NMR (GASPE,
CDCl , 100 MHz, C–C decoupling), C : 23.7 ppm; C :
acid (PPA) and other solvents (chloroform, petroleum ether,
diethylether, ethylacetate and ethanol) were all purchased from
Aldrich Ltd., and were used as received. Reference com-
pounds, griseofulvin and nystatin, used in biological activity
experiments, were purchased from Aldrich Ltd, with catalogue
numbers G4753 and N3503, respectively.
3
a
2
159.2 ppm; C : 107.3 ppm; C : 162.9 ppm; C : 125.7 ppm;
3
4
b
C : 121.4 ppm (d, J
6
= 8.6 Hz) ; C : 108.9 ppm (d, J
7 C7-F
C6-F
= 27.3 Hz); C : 161.7 ppm (d, J
= 106.2 Hz); C9:
8
C8-F
114.5 ppm (d, J
160.8 ppm. Further C NMR analysis [34] was done using
= 23.3 Hz) ; C : 132.4 ppm; C :
c d
C9-F
1
3

1020
H.S. Hilal et al. / European Journal of Medicinal Chemistry 41 (2006) 1017–1024
DEPT 135 technique, and the results were consistent with
those listed. – GC-MS (FD), parent ion m/z = 234, base peak
V: Prepared from 3-amino-5-methylisoxazole (0.5 g,
5.10 mmol) and ethyl-2-oxocyclohexane carboxylate (0.95 g,
5.6 mmol) in PPA (3.0 g). Product, recrystallized from EtOH,
2
8
8 (CO), fragment at 206 due to CO elimination. – IR, 1681.
–
1
–1
1
cm for C=O str., 1365.5 cm for CH , and 1600.8 , 1577.7
0.86 g, yield 83%, pale yellow ppt., m.p. range 98–100 °C.- H
3
−
1
–1
and 1506.3 cm for C=C str. Bands 1725 and 1800 cm ,
characteristic for reactant keto group [35], disappeared. – Anal.
C H ON SF (MW 234}, Calcd. C 56.40%, H 3.02%, N
NMR (CDCl , 300 MHz), H : 2H at 2.71 ppm (distorted t, J
a-b
3
a
= 5.9 Hz); H and H : 4H at 1.81 ppm (mult.); H : 2H at 2.65
b
c
d
ppm (distorted t, J = 5.8 Hz); H : 3H at 2.54 ppm (s); H :
1
1
7
2
d-c
e
8
1
3
1
1.96%. Found C 56.56%, H 3.54%, N 11.66%.
6.23 ppm (s). - C NMR, (GASPE, CDCl , 100 MHz, C–C
3
II: Prepared from 2-amino-6-fluorobenzothiazole (0.5 g,
.97 mmol) and ethyl-2-oxocyclopentanecarboxylate (0.51 g,
.27 mmol) in PPA (3.0 g). Product, recrystallized from EtOH,
decoupling), C2: 150.8 ppm; C : 23.2 ppm; C : 22.5 ppm; C :
21.8 ppm; C : 22.3 ppm; C : 115.1 ppm; C : 161.3; C :
d 3 4 7
a
b
c
2
3
0
153.8 ppm; C : 99.2 ppm; C : 165.3 ppm; C : 12.7 ppm. -
8
9
e
1
.4 g, 52% yield, brown ppt., m.p. range 150–152 °C - H
GC-MS (FD), parent ion m/z = 204, base peak 28 (CO), frag-
NMR (CDCl , 300 MHz), H : 2H at 2.96 ppm (t, J = 7 Hz);
ment at 189 due to CH elimination, 176 due to CO elimina-
3
a
a-b
3
H : 2H at 2.17 ppm (tt, J = Jb-c = 7.4 Hz); H : 2H at 2.93
tion and at 161 due to CH and CO elimination. – IR, strong
b
a-b
c
3
–
1
–1
ppm (t, Jb-c = 7.2 Hz); H : 1H at 7.00 (dd, J = 8.5 Hz, J6-F
band 1670.2 cm for CO Str., 1384.8 cm for CH , and at
3
6
6-7
–
1
=
7.8); H : 1H at 9.12 ppm (dd, J = 9 Hz, J7-F = 4.7); H : 1H
1637.5 cm for C=C. – Anal C H O N (MW 204) Calcd.
11 12 2 2
7
7-6
1
9
3
at 7.36 ppm (d, J9-F = 5.6 Hz). – C NMR, (GASPE, CDCl3,
C 64.68%, H 5.93%, N 13.72%. Found C 61.71%, H 6.39%, N
15.17%.
VI: Prepared from3-amino-5-methylisoxazole (0.5 g,
5.10 mmol) and ethyl-1-benzyl-3-oxo-4-piperidine carboxylate
hydrochloride (1.67 g, 5.6 mmol) in PPA (6.0 g). Product, ex-
tracted with ethylacetate, recrystallized from EtOH and washed
1
2
1
1
00 MHz, C–C decoupling) C : 159 ppm; C : 34.8 ppm; C :
2 a b
1.7 ppm; C : 27.3 ppm; C : 132.2 ppm; C : 168.1 ppm; C :
c
3
4
d
32.6 ppm; C : 121.4 ppm; C : 108.9 ppm; C : 161.7 ppm; C :
6
7
8
9
14.4 ppm; C : 126.1 ppm; C : 159.3 ppm.- GC-MS (FD), par-
e
f
ent ion m/z = 260, base peak 28 (CO), fragment at 232 due to
–
1
–1
CO elimination.- IR, 1678 cm for CO str., 1463.4 cm for
with diethylether and petroleum ether, 0.8 g, yield 53%, brown
–
1
1
CH (C-H) bending and 1579.1 and 1501.5 cm for aromatic
ppt., m.p. range 142–144 °C. - H NMR (CDCl
3
, 300 MHz),
and
: 2H at
2
C=C str.- Anal. C H ON SF (MW 260), Calcd. C 59.98%, H
H
H
a
: 3H at 2.5 ppm (s); H
3
: 1H at 6.19 ppm (broad s); H
: 4H at 2.75 ppm (broad s); H : 2H at 3.5 (s); H
3.7 ppm (s); H : 5H at 7.23 ppm (mult.). C NMR, (GASPE,
7
1
3
9
2
3
.49%, N 10.77%. Found C 60.42%, H 4.63%, N 11.22%.
III: Prepared from 3-amino-5-methylisoxazole (0.5 g,
.10 mmol) and ethylacetoacetate (0.73 g, 5.6 mmol) in PPA
8
5
e
1
3
f
5
CDCl , 100 MHz, C–C decoupling), C : 12.7 ppm; C :
3
a
2
(
3.0 g). Product extracted with CHCl , and recrystallized from
153.4 ppm; C : 99.2 ppm; C : 165.6 ppm; C : 151.5 ppm; C
3 b c 5
3
EtOH, 0.3 g, 36% yield, brown ppt., m.p. range 176–178 °C -
: 57.2 ppm; C : 49.3 ppm; C : 22.7 ppm; C : 112.9 ppm; C :
7 8 d 9
159 ppm; C : 62.2 ppm; C : 137.5 ppm; C : 129.1 ppm; C :
e f1 f2 f3
1
H NMR (CDCl , 300 MHz), H : 3H at 2.38 ppm (s); H : 3H
3
a
b
at 2.57 ppm (s); H : 1H at 6.29 ppm (s); H : 1H at 6.15 ppm
128.3 ppm; C : 127.3 ppm. - GC-MS (FD), parent ion m/
3
8
f4
1
3
(
s). - C NMR, (GASPE, CDCl , 100 MHz, C–C decoupling),
z = 295, base peak 28 (CO), fragment 176 due to benzyl and
CO eliminations, 91 due to benzyl ion. – IR, strong 1679.
3
C : 24.2 ppm; C : 153.4 ppm; C : 105.3 ppm; C : 165.5 ppm;
a
2
3
4
–
1
–1
–1
C : 153.6 ppm; C : 99.5 ppm; C : 166.5 ppm; C : 12.8 ppm. -
9 cm for CO str., 1382.9 cm for CH , 1642 cm for
3
7
8
9
b
–
1
GC-MS (FD), parent ion m/z × 164, base peak 28 (CO), frag-
C=C, 1587.3 and 1541 cm for C=C (phenyl). – Anal.
ment at 121 due to elimination of CO and CH groups. – IR,
C H O N (MW 295) Calcd. C 69.12%, H 5.81%, N
14.23%. Found C 68.01%, H 6.44%, N 16.62%.
3
17 17 2 3
–
1
–1
strong band 1689.5 cm for CO str., 1371.3 cm for methyl
–
1
group, 1625.9 cm for C=C str.- Anal. C H O N (MW 164),
VII: Prepared from 3-amino-5-methylisoxazole (0.5 g,
5.10 mmol) and methyl-1-benzyl-4-oxo-3-piperidine carboxy-
late hydrochloride 1.67 g, 5.6 mmol) in PPA (6.0 g). Product,
extracted with ethylacetate, recrystallized from EtOH and
washed with dietheylether and petroleum ether, 0.53 g, yield
8
8 2 2
Calcd. C 58.52%, H 4.88%, N 17.07%. Found C 57.54%, H
5
.12%, N 17.35%.
IV: Prepared from 3-amino-5-methylisoxazole (0.5 g,
.10 mmol) and ethyl-2-oxocyclopentanecarboxylate (0.87 g,
.6 mmol) in PPA (3.0 g). Product, recrystallized from EtOH,
5
5
0
1
35%, yellow ppt., m.p. range 176–177 °C. - H NMR
1
.84 g, 87% yield, brown ppt., m.p.. range 147–150 °C - H
(CDCl , 300 MHz), H : 3H at 2.52 ppm (s); H : 1H at 6.22
3
a
3
NMR (CDCl , 300 MHz), H and H : 4H at 2.92 ppm (t, J
ppm (s); H : 2H at 2.75 ppm (t, J
= 5.2 Hz); H : 2H at
3
a
c
a,c-b
5
H5-H6
6
=
7.5 Hz); H : 2H at 2.16 ppm (q, (J
= 7.5 Hz); H : 3H at
2.82 ppm (t, JH6-H5 = 5.0 Hz); H : 2H at 3.61 ppm (s); H : 2H
8 e
b
b-a,c
d
1
3
13
2
1
2
1
.57 ppm (s); H : 1H at 6.31 (s).- C NMR, (GASPE, CDCl3,
at 3.74 ppm (s); H : 5H at 7.35 ppm (mult.). - C NMR,
8
f
00 MHz, C-C decoupling), C2: 152.1 ppm; Ca: 35 ppm; Cb:
2.4 ppm; Cc: 27.4 ppm; C3: 117.6; C4: 154 ppm; C7:
66.1 ppm; C8: 99.4 ppm; C9: 170.4 ppm; Cd: 12.9 ppm. -
(GASPE, CDCl , 100 MHz, C-C decoupling), C : 12.7 ppm,
3
a
C : 152.3 ppm; C : 99.2 ppm; C : 165.7 ppm, C : 151.5 ppm;
2
3
b
c
C : 32.3 ppm; C : 49.2 ppm; C : 49.9 ppm; C : 113.2 ppm; C
9
5
6
8
d
GC-MS (FD), parent ion m/z = 190, base peak 28 (CO), frag-
: 159.7 ppm; C : 62.3 ppm; C : 137.9 ppm; C : 129.1 ppm;
e f1 f2
ment at 147 due to elimination of CO and CH groups.- IR,
C : 128.4 ppm; C : 127.2 ppm. - GC-MS (FD), parent ion m/
f3 f4
z = 295, base peak 28 (CO), fragment 176 due to benzyl and
3
–1
–
1
strong band 1674.1 cm for CO str., 1384.8 cm for methyl
–1
–1
(
bend.), 1421.4 and 1438.8 cm for CH (bend.), 1622 cm
benzyl and CO eliminations, fragment 91 benzyl, fragment 204
2
–
1
for C=C str. – Anal. C H O N (MW 190) Calcd. C 63.14%,
due to benzyl elimination. – IR, strong 1666.4 cm for CO
10 10 2 2
–
1
–1
H 5.31%, N 14.73%. Found 51.01%, H 7.08%, N 12.51%.
str., 1359.7 cm for CH3, 1581.5 and 1529.4 cm for phenyl.

H.S. Hilal et al. / European Journal of Medicinal Chemistry 41 (2006) 1017–1024
1021
–
Anal. C H O N (MW 295). Calcd. C 69.12%, H 5.81%,
C decoupling), C : 62.2 ppm; C : 33.8 ppm; C : 28 ppm; C :
2 a b c
1
7
17
2
3
N 14.23%. Found C 68.55%, H 6.46%, N 15.87%.
23.1 ppm; C : 28.8 ppm; C : 157.4 ppm; C : 117.6 ppm; C :
d 3 4 5
VIII: Prepared from 2-amino-6-fluorobenzothiazole (0.5 g,
165.5 ppm; C : 169.5 ppm; C : 95.2 ppm; C : 169.8 ppm; C :
8 9 10 e
2
3
0
.97 mmol) and ethyl-2-cyclohexanone acetate (0.6 g,
.27 mmol) in PPA (3.0 g). Product, recrystallized from EtOH,
.68 g, yield 79%, pale yellow ppt., m.p. range 158–160 °C. -
12.6 ppm. - GC-MS (FD), parent ion m/z = 218, base peak 28
(CO), fragment 190 due to CO elimination, fragment m/z 175
–1
due to CO and CH elimination. – IR, strong 1697.2 cm for
3
1
–1
–1
H NMR (CDCl , 300 MHz), H : 1H at 4.7 ppm (dd, J
CO str., 1456.2 cm for CH bend., 1654.8 and 1635.5 cm for
3
2
H2-Ha
2
=
5.7 Hz, JH2-Ha’ = 11.0 Hz); H –H : 8H in the range 1.15–
C=C str. – Anal. C H O N (MW 218). Calcd. C 66.03%, H
a
d
12 14 2 2
3
7
7
7
.35 ppm (mult.); H : 1H at 5.92 ppm (broad s); H : 1H at
6.48%, N 12.84%. Found C 65.42%, H 6.83%, N 14.54%.
X: Prepared from 1-aminoisoquinoline (0.5 g, 3.47 mmol)
and ethyl-2-cyclohexanone acetate (0.7 g, 5.6 mmol) in PPA
(3.0 g). Product, recrystallized from EtOH, 0.9 g, yield
4
7
.13 ppm (dd, JH7-H8 = 8.2 Hz, JH7-F = 8.9 Hz); H : 1H at
8
.72 ppm (dd, JH8-H7 = 8.6 Hz, JH8-F = 4.6 Hz); H : 1H at
10
1
3
.49 ppm (d, JH10
= 5.8 Hz). - C NMR, (GASPE,
–F
1
CDCl , 100 MHz, C–C decoupling), C : 63.9 ppm; C :
97.8%, brown ppt., m.p. range 190–192 °C. H NMR
3
2
a
3
1
4.1 ppm; C : 23.1 ppm; C : 28 ppm; C : 29.1 ppm, C :
(CDCl , 300 MHz) H –H : 8H in the range 0.78–2.31 ppm
b
c
d
3
3
a
d
45.6 ppm; C : 116.9 ppm; C : 169.7 ppm; C : 133 ppm (
(multi.); H : 4.98 ppm (dd, J
= 5 Hz, JH2-Ha’ = 10 Hz));
H2-Ha
4
5
e
2
JCe-F = 10.9 Hz); C : 121.7 ppm (JC7-F = 8.3 Hz); C8:
H : 1H at 5.82 ppm (s); H –H : 6H in the range 7.19–
4 7 12
7
1
3
1
07.5 ppm (JC8-F = 26.6 Hz); C : 156.9 ppm (JC9-F = 135.7
8.38 ppm (multi.). – C NMR (GASPE, CDCl , 100 MHz)
9
3
Hz); C : 114.1 ppm (J
= 24.5 Hz); C : 119.7 ppm (J
C : 63.5 ppm; C : 33.4 ppm; C : 23 ppm; C : 27.2 ppm; C :
1
0
C10-F
f
Cf-F
2
a
b
c
d
=
33.2 Hz); C : 166.9 ppm. - GC-MS (FD), parent ion m/
g
28.5 ppm; C : 120 ppm; C : 126.5 ppm; C : 117.5 ppm; C :
4 7 8 9
z = 288, base peak 28 (CO), fragment 260 due to CO elimina-
1
26.8 ppm; C : 141 ppm; C : 127.1 ppm; C : 130.5 ppm.
10 11 12
–
1
–1
tion. - IR, strong 1687 cm for CO str., 1458.1 cm for CH2
(MW 266).
–
1
bend., 1635.5 cm for C=C (7-membered ring), 1606.6 and
It should be noted that the structures for compounds XIII,
IX and X are not unequivocally elucidated, and further analy-
tical study is underway to elucidate structures for these and
other compounds of similar nature.
–1
1
541 cm for C=C str. (benzene). – Anal. C H ON SF
15 13 2
(
MW 288). Calcd. C 62.47%, H 4.55%, N 9.72%. Found C
5
3.89%, 3.81%, N 14.52%.
IX: Prepared from 3-amino-5-methylisoxazole (0.5 g,
.10 mmol) and ethyl-2-cyclohexanone acetate (1.03 g,
.6 mmol) in PPA (3.0 g). Product, recrystallized from EtOH,
5
5
0
5
.4. Biological activity experiments
1
.6 g, yield 54%, brown ppt., m.p. range 87–90 °C. - H NMR
5.4.1. Antibacterial and antifungal activity
(CDCl , 300 MHz), H -H : 8H in the range 1.12–3.25 ppm
3 a d
(
=
3
mult.); H : 1H at 4.39 ppm (dd, J
= 3.4 Hz, JH2-Ha’
5.4.1.1. Test microorganisms. Two bacteria (S. aureus and
P. vulgaris), one isolate of yeast (Candida albicans), two iso-
lates of human pathogenic dermatophytes (Microsporum canis
2
H2-Ha
10.7 Hz); H : 1H at 6.84 ppm (s); H : 1H at 5.83 (s); H :
4 9 e
13
H at 2.4 ppm (s). C NMR, (GASPE, CDCl , 100 MHz, C-
3
Table 4
Test microorganisms and source of collection
Microorganism
Number
Source/researcher
Reference antibiotic (concentration)
Bacteria
Staphylococcus aureus
Proteus vulgaris
a
ATCC 25923
ATCC 13315
ATCC
ATCC
Ampicillin (10 mg per disc)
Gentamicin (10 mg per disc)
Yeast
b
Candida albicans (Robin) Berkhout
Dermatophytes
FCCAU R10
Foot swab, patient/Mr. S. Abu-Ghdieb
Nystatin (10 mg per disc)
c
–1
Microsporum canis Bodin
FCCAU S14
FCCAU S15
Tinea capitis clinical specimens in Nablus
area, patient/Mr. Abu-Ghdieb
Tinea capitis clinical specimens in Nablus
area, patient/Mr. Abu-Ghdieb
Griseofluvin (0.6 μg ml )
–
1
Microsporum gypseum (Bodin) Guiart
and Grigorakis
Nystatin (0.6 μg ml )
Phytopathogenic fungi
Fusarium tricinctum (Corda) Sacc.
Pythium ultimum var ultimum Trow
–
1
FCCAU M10
FCCAU H5R3
Water pool in Nablus/Mr. T. Khalid
Water and soil samples in Nablus area/
Professor Ali-Shtayeh
Water and soil samples in Nablus area/
Professor Ali-Shtayeh
Water and soil samples in Nablus area/
Professor Ali-Shtayeh
Water and soil samples in Nablus area/
Dr. A-H Hamdan
Nystatin (5 mg ml )
–
–
1
1
Hymexazol (25 μg ml
Hymexazol (25 μg ml
)
)
P. aphanidermatum (Edson) Fitzp.
P. middletonii Sparrow
FCCAU H739
–
1
FCCAU PH 122
FCCAU AIE 2005
Hymexazol (25 g ml )
–
1
Phytophthora citrophthora
Metalaxyl (10 μg ml )
(
R. E. Smith and E. H. Smith)
a
American Type Culture Collection.
FCCAU: Fungal Culture Collection of An-Najah University.
Nablus is largest city in the West Bank, 60 km north of Jerusalem.
b
c

1022
H.S. Hilal et al. / European Journal of Medicinal Chemistry 41 (2006) 1017–1024
and Microsporum gypseum), and five isolates of plant patho-
genic fungi (Fusarium tricinctum , Pythium ultimum var ulti-
mum, P. aphanidermatum, P. middletonii and Phytophthora
citrophthora), were employed in this study (Table 4).
(I)
8-Fluoro-2-methyl-4H-pyrimido [2,1b][1,3]benzothiazole-4-
one.
5
.4.2. Screening for antimicrobial activities
The test compounds were dissolved in 10% aqueous
dimethyl sulfoxide (DMSO) to a final concentration of
–
1
2
00 mg ml and sterilized by filtration through a 0.45 um
membrane filter. Antimicrobial tests were then carried out
by the disc diffusion method [36] using an inoculum contain-
6
8
–1
ing 10 bacterial cells or 10 yeast cells ml to spread on
Muller-Hinton agar plates (1 ml inoculum per plate). Six
mm diameter discs were each impregnated with 50 µl of ex-
(II)
–
1
8-Fluoro-2,3-trimethylene-4H-pyrimido
zothiazole-4-one.
[2,1b][1,3]ben-
tract (10 mg per disc) at a concentration of 200 mg ml and
placed on the inoculated agar and incubated at 37 °C for 24 h
for bacteria and for 48 h for Candida albicans. On each plate,
an appropriate reference antibiotic disc was applied depend-
ing on the test microorganisms. Each test was carried out in
triplicates.
5
.4.3. Screening for antifungal activity
Antifungal activity tests were carried out by the poisoned-
food technique method [37]. Test isolates were inoculated onto
SDA or CMA plates and incubated at 25 °C for 1–4 days for
Pythium and Phytophthora, and for 7–14 days for Microspor-
um and Fusarium, to obtain young, actively growing cultures
consisting of mycelia and conidia. The required amount of the
test compound or reference antimycotic drug was dissolved in
(III)
2,7-Dimethyl-4H-isoxazolo[2,3-a]pyrimidine-4-one.
2
ml sterile distilled water or 10% aqueous DMSO, sterilized
by filtration through a 0.45 μm membrane filter, and then
mixed in requisite amount of pre-sterilized SDA or CMA med-
–
1
ium to give a final concentration of 15 μg ml . A mycelial
disc of 6-mm diameter, cut out from the periphery of 1–4 day
old cultures, was aseptically inoculated onto the medium. In
controls, sterile DMSO or distilled water was used in place of
the compound as negative control and reference antibiotics as a
positive control. The inoculated plates were then incubated at
2
5 °C and colony diameter measured and recorded after 7 days
for dermatophytes and keratinophilic fungi, and one day for
Pythium and Phytophthora species. Percentage of mycelial in-
hibition was calculated as follows: % mycelial inhibition =
(IV)
7-Methyl-2,3-trimethylene -4H-isoxazolo[2, 3-a]pyrimidine-
(
(dc–dt)/dc) × 100; where: dc, colony diameter in control (–
4-one.
ve); dt, colony diameter in treatment. Three replicate plates
were used for each treatment [37–39].
5
.4.4. Statistical analysis
Data were analyzed and treatments compared using analysis
of variance with Duncan multiple range test (P < 0.05).
5
.4.5. Cytotoxic activity
The MTT assay method [40,41] has been used to study the
cytotoxic activity of compounds IV, IX and X, against human
Caucasian bone marrow neuroblastoma (SK-N-SH) cells (Ta-
ble 3). Experiments were conducted in the Hospital of the Uni-
versity of Tuebingen. Compound concentrations used are
shown in Table 3.

H.S. Hilal et al. / European Journal of Medicinal Chemistry 41 (2006) 1017–1024
1023
(
V)
(IX)
7
-Methyl-2,3-tetramethylene-4H-isoxazolo[2, 3-a]pyrimi-
5H-8-methyl-2,3-tetramethyleneisoxazolo[2,3-a][1,3]diaze-
pin-5-one.
dine-4-one.
(
5
X)
(
6
VI)
-Benzyl-2-methyl-5,6,7,8-tetrahydro-9H-isoxazolo[2,3-a]
pyrido[3,4d]pyrimidine-9-one.
H-2,3-tetramethyleneisoquinolo[2,1-a][1,3]diazepin-5-one.
Acknowledgements
(
7
VII)
-Benzyl-2-methyl-5,6,7,8-tetrahydro-9H-isoxazolo[2,3-a]
pyrido[4,3d]pyrimidine-9-one.
Technical help from N. Zatar and R. Zaid (ANU), Dr. M.
Abu Zarga and K. Bargathi (Jordan University) and A.R. Ma-
tinez (University of Tuebingen) is acknowledged.
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