Synthesis and pharmacological screening of derivatives of isoxazolo[4,5-d]pyrimidine

Article abstract of DOI:10.1016/j.ejmech.2008.01.035

A number of derivatives of isoxazolo[4,5-d]pyrimidine were prepared with structures similar to that of purine. Condensation of the hydrazide of 4-amino-5-benzoylisoxazolo-3-carboxylic acid 2 with ethyloxalyl chloride followed by cyclization gave 3-oxdiazolo-[1,3,4]-4-amino-5-benzoylisoxazole 7 which, upon cyclization with acetonitrile followed by reactions with different amines, gave derivatives of isoxazolo[4,5-d]pyrimidine 9 and 10d-g. Compounds 8g and 10f were tested for their effects on the immune response in the mouse model. Both compounds significantly inhibited the humoral immune response in vivo to sheep erythrocytes at a dose of 100 μg, whereas in the delayed type hypersensitivity assay a suppressive activity was shown only by compound 10f. In addition, compound 8g inhibited and compound 10f stimulated the proliferative response of mouse splenocytes to concanavalin A. The results indicated that compound 10f was a universal inhibitor of the immune response, while compound 8g selectively suppressed the humoral immune response.

Full text of DOI:10.1016/j.ejmech.2008.01.035

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European Journal of Medicinal Chemistry 43 (2008) 2498e2504
http://www.elsevier.com/locate/ejmech
Original article
Synthesis and pharmacological screening of derivatives
of isoxazolo[4,5-d]pyrimidine
a
b
c
Edwin Wagner a, , Kamal Al-Kadasi , Micha1 Zimecki , Wanda Sawka-Dobrowolska
*
a Department of Drugs Technology, Wroclaw Medical University, Pl. Nankiera 1, 50-140 Wroclaw, Poland
b Institute of Immunology and Experimental Therapy, Polish Academy Sciences, Department of Experimental Therapy, Weigla 12, 53-114 Wroclaw, Poland
c University of Wroclaw, Faculty of Chemistry, F.J. Curie 14, 650-383 Wroclaw, Poland
Received 18 June 2007; received in revised form 19 December 2007; accepted 24 January 2008
Available online 6 February 2008
Abstract
A number of derivatives of isoxazolo[4,5-d]pyrimidine were prepared with structures similar to that of purine. Condensation of the hydrazide
of 4-amino-5-benzoylisoxazolo-3-carboxylic acid 2 with ethyloxalyl chloride followed by cyclization gave 3-oxdiazolo-[1,3,4]-4-amino-5-ben-
zoylisoxazole 7 which, upon cyclization with acetonitrile followed by reactions with different amines, gave derivatives of isoxazolo[4,5-d]py-
rimidine 9 and 10deg. Compounds 8g and 10f were tested for their effects on the immune response in the mouse model. Both compounds
significantly inhibited the humoral immune response in vivo to sheep erythrocytes at a dose of 100 mg, whereas in the delayed type hypersen-
sitivity assay a suppressive activity was shown only by compound 10f. In addition, compound 8g inhibited and compound 10f stimulated the
proliferative response of mouse splenocytes to concanavalin A. The results indicated that compound 10f was a universal inhibitor of the immune
response, while compound 8g selectively suppressed the humoral immune response.
Ó 2008 Published by Elsevier Masson SAS.
Keywords: Isoxazoles; Isoxazole[4,5-d]pyrimidines; Immunosuppressant; Antitumor agents
1. Introduction
only the isomer 4,5-d has not been thoroughly investigated.
To date there have been only three [14e16] reports concerning
the synthesis of derivatives of this isomer.
In this article we describe the synthesis of new isoxazoles 4,
5, 7, and 8aei and new derivatives of isoxazolo[4,5-d]pyrim-
idine 9 and 10deg. Three of the compounds showed interest-
ing biological activities.
Adenosine and inosine are found naturally in DNA and
RNA and play different roles in physiology. Adenosine, for
example, is an endogenous nucleoside which mediates a vari-
ety of important physiological processes by interaction with
specific adenosine receptors [1e3]. Inosine is an endogenous
agonist of the adenosine A3 receptor and is also an endogenous
ligand of the benzodiazepine receptors [4,5]. The purine base
has been utilized to obtain biologically active compounds. The
idea of modifying purine bases by replacing the imidazole ring
with isoxazole to obtain the corresponding structure of isoxa-
zolopyrimidine has been the subject of numerous studies.
Derivatives of isoxazolopyrimidine have significant biological
activity and display analgesic [6,7], anti-inflammatory [8],
bactericidal [9e11], circulatory [12], and anxiolytic activities
[13]. Of the four structural isomers of isoxazolopyrimidines,
2. Chemistry
The synthesis was carried out according to Scheme 1. The
starting compound 1 was obtained by Thorpe reaction accord-
ing to Gewald et al. [17]. The resulting 4-amino-5-benzoylizox-
azole-3-carboxamide 1 reacted with hydrazine hydrate to
produce hydrazide 2 with a good yield (81%) and hydrazone
1 of only 13%. Reaction of 2 with triethyl orthoformate in
the presence of acetic anhydride underwent smooth cyclization,
but did not yield derivatives of isoxazolo[4,3-d]pyrimidine 3,
but did yield 4-amino-5-benzoylo-3-oxdiazolo[1,3,4]isoxazole
* Corresponding author. Tel.: þ48 71 7840236; fax: þ48 71 7840238.
E-mail address: wagner@ktl.am.wroc.pl (E. Wagner).
0223-5234/$ - see front matter Ó 2008 Published by Elsevier Masson SAS.
doi:10.1016/j.ejmech.2008.01.035

E. Wagner et al. / European Journal of Medicinal Chemistry 43 (2008) 2498e2504
2499
O
N
N
O
H2N
H2N
NH2
NH
O
NH2
O
N
N
N
3
O
O
N
O
O
NH2
O
X
NH2
N
N
O
O
2
O
1
4
OC2H5
O
O
O
O
N
O
NH
N
O
N
O
X
HN
HN
N
O
N
H
NH
NH
O
O
O
H5C2O
NH2
NH2
6
O
N
O
O
5
5 b
O
N
NH
O
OC2H5
O
O
N
NH2
N
N
8 e
O
N
O
N
O
7
H2N
O
O
O
N
O
N
O
N
N
N
N
N
N
H3C
N
H3C
H2N
O
O
O
9
8 a-i
N
N
10 d-g
R
OC2H5
R
N
N
O
O
O
R = a-NH -(CH2)2-CH3 ; b- piperidine ; c-NH-cyclohexyl ; d-NH-CH2-CH2-morpholine ; e-NH-benzyl ;
f-3-NH-CH2-pyridine ; g-4-NH-CH2-pyridine ; h-4-methyl-piperazine ; i-4morpholine
Scheme 1.
4. Treatment of the hydrazide 2 with ethyloxalyl chloride in the
presence of anhydrous pyridine produced the corresponding
compounds 5, which reacted with piperidine but did not cyclize
derivatives of isoxazolo[4,3-e]triazepine[1,2,4] 6 and only
yielded the piperidine amide 5b. Compound 5 was readily
cyclized under acidic conditions with thionyl chloride to give
compound 7. The amine group at position 4 of compounds 2
and 5 was not reactive in these two cyclizations. X-ray crystal-
lography of compound 7 confirmed that the resulting structure
was indeed compound 7 (Fig. 1). Ester 7 reacts with different
amines to form amides 8aei, whereas ester 7 and amide 8e
were heated in acetonitrile with gaseous hydrogen chloride to
give the new derivatives isoxazolo[4,5-d]pyrimidine 9 and
10e. Ester 9 reacts with amines to form the amides isoxa-
zolo[4,5-d]pyrimidine 10deg. Compound 10e was prepared
by two ways from compounds 8e and 9 and its structure also
confirmed the new compounds.
3. Biological activity
Evaluation of the anticancer activity was performed for
compounds 4, 7, 8aei, 9, and 10deg at the National Cancer

2500
E. Wagner et al. / European Journal of Medicinal Chemistry 43 (2008) 2498e2504
the Institute of Immunology and Experimental Therapy, Wro-
claw, fed commercial granulated food, and had water ad libitum.
4.2. Reagents
Sheep red blood cells (SRBCs) were provided by the Wro-
claw Agriculture Academy. SRBCs were kept in Alsever’s
solution until use. Cyclosporine A (CsA) was from Sandoz
(Switzerland), DMSO and concanavalin A (Con A) from Sigma
RPMI 1640 medium, and fetal calf serum (FCS) was from
Gibco.
Fig. 1. A view of molecule A with the atomic numbering scheme. Ellipsoids
are at the 40% probability level.
4.3. Treatment of mice with the compounds
Institute (NCI) in Bethesda, MD, USA. Only compound 4
showed interesting activity on a panel of 62 tumor cell lines.
Data of the active compound 4 are presented in Table 1.
The compounds were initially dissolved in DMSO and then
in 0.9% NaCl. The compounds were given to mice intraperito-
neally (i.p.) at doses of 10 mg or 100 mg per mouse 2 h after
immunization with SRBCs. For control, 0.2 mL of 0.9%
NaCl or appropriately diluted DMSO were used.
4. Materials and methods
4.1. Mice
4.4. Determination of the humoral immune
response to SRBCs
Twelve-week old male and female CBA mice were used for
the experiments. The mice were kept in the Animal Facility of
Mice were immunized with 0.2 mL of a 5% SRBC suspen-
sion in 0.9% NaCl i.p. Four days later the mice were sacrificed,
spleens were isolated, and the number of antibody-forming
cells (AFCs) in the spleens was determined using the local
hemolysis assay [18]. The results are shown as mean AFC
values from 5 mice per group calculated per 106 viable spleno-
cytes ꢀ standard error (SE).
Table 1
Inhibition of in vitro cell lines by compound 4a
Panel cell line
c
e
MG-MIDb
log GI50
[M]
log TGId
log LC50
[M]
Leukemia
CCRF-CEM
K-562
ꢁ4.74
ꢁ4.24
ꢁ4.25
>ꢁ4.00
>ꢁ4.00
>ꢁ4.00
>ꢁ4.00
>ꢁ4.00
>ꢁ4.00
4.5. Determination of delayed type hypersensitivity
(DTH) to SRBCs
MOLT-4
Non-small cell
lung cancer
HOP-92
ꢁ4.51
ꢁ4.47
ꢁ4.01
>ꢁ4.00
>ꢁ4.00
The test was performed according to Lagrange et al. [19].
Mice were immunized with 0.2 mL of an SRBC suspension
containing 105 erythrocytes intravenously. Four days later
the mice were given an eliciting dose of antigen, i.e. 108 eryth-
rocytes, in 0.05 mL, into the hind footpads. Twenty-four hours
later the delayed type hypersensitivity reaction was measured
as the footpad edema, using callipers. The background, non-
specific response was elicited by administration of the eliciting
dose to naive mice, which was subtracted from the response of
the sensitized mice. The results are shown as mean values of
DTH units from 5 mice ꢀSE.
NCI-H522
>ꢁ4.00
Colon cancer
HCC-2998
HCT-15
ꢁ4.57
ꢁ4.53
ꢁ4.02
>ꢁ4.00
>ꢁ4.00
>ꢁ4.00
CNS cancer
SF-539
U-251
ꢁ5.56
ꢁ4.79
ꢁ4.65
ꢁ4.16
ꢁ4.19
>ꢁ4.00
Melanoma
LOX-IMVI
SK-MEL-2
ꢁ4.54
ꢁ4.48
>ꢁ4.00
>ꢁ4.00
>ꢁ4.00
>ꢁ4.00
Renal cancer
CAKI-1
MG-MID
Delta
ꢁ4.66
ꢁ4.25
1.31
>ꢁ4.00
ꢁ4.01
0.64
>ꢁ4.00
ꢁ4.00
0.19
4.6. The proliferative response of splenocytes to
concanavalin A
Range
1.56
0.65
0.19
A single splenocyte suspension was prepared by pressing
the organs through a plastic screen into Hank’s medium. The
cells were centrifuged and treated with 0.83% ammonium
chloride to lyse erythrocytes. Then the cells were washed
twice in Hank’s medium, passed through cotton wool columns
to remove dead cells and debris, and finally resuspended in
a culture medium consisting of RMPI 1640 supplemented
with 10% FCS, L-glutamine, sodium pyruvate, and antibiotics.
Compound 8deg and 10deg were also assayed for immunologic activity in
selected tests.
a
Data obtained from the NCI’s in vitro disease-oriented human cells screen
[21,22].
b
MG-MID ¼ mean graph midpoint ¼ arithmetical mean value for all tested
cell lines.
c
The log of the molar concentration that inhibits 50% net cell growth.
The log of the molar concentration leading to total growth inhibition.
The log of the molar concentration leading to 50% net cell death.
d
e

E. Wagner et al. / European Journal of Medicinal Chemistry 43 (2008) 2498e2504
2501
The cells, 2 ꢂ 105 in 100 mL, were distributed in flat-bottom
Table 3
Effects of the compounds on the delayed type hypersensitivity to SRBCs
96-well culture plates. Con A was added in a final concentra-
tion of 2.5 mg/mL and the studied compounds at the concentra-
tion range of 0.1e10 mg/mL. After three days of culture in
a cell culture incubator, the proliferation rate was determined
by the MTT colorimetric method [20]. The results were pre-
sented as the mean optical density (OD) at 550/630 nm from
quadruplicate wells ꢀSE.
Compound
Dose
(mg/mouse)
DTH units
(mean)
ꢀSE
P
Control
DMSO control
13.0
10.9
6.4
0.76
0.18
0.37
10
100
8g
10
100
7.9
4.9
0.85
0.60
NS
NS
10f
CsA
10
100
5.9
3.5
0.79
0.42
<0.05
<0.02
4.7. Statistics
10
100
6.1
5.0
0.33
0.51
<0.01
<0.01
The Student’s t-test was applied for evaluation of the data.
The results are presented as mean values ꢀ SE. The results
were regarded to be significant when p < 0.05. NS denotes
not significant.
response of mouse splenocytes to concanavalin A, a T-cell mi-
togen. The results, shown in Table 4, indicate that compound
8g inhibited, whereas compound 10f stimulated, the prolifera-
tive response of cells to the mitogen. These effects, although
small, were statistically significant.
5. Results and discussion
The hydrazide 2 and compound 5 do not yield derivatives 3
and 6 because the amino groups at position 4 have decreased
activity due to the electronegative group at position 5. How-
ever, if the oxdiazole ring was formed, the amine group at
position 4 with acetonitrile gave compound 7.
To evaluate the effects of the compounds on the humoral
immune response in vivo, mice were immunized with SRBCs
and given the compounds i.p. 2 h after immunization. Control
mice were given the solvent (DMSO) or the reference suppres-
sive drug cyclosporine A. The number of antibody-forming
cells was determined 4 days later in the spleens. The results
(Table 2) showed that both compounds (8g and 10f) signifi-
cantly suppressed the humoral immune response at the higher
dose (100 mg per mouse). This inhibition was even deeper than
that of CsA at the same dose.
To determine the effects of the compounds on the cellular
immune response in vivo, mice were sensitized with SRBCs
and given the compounds i.p. 2 h later. After 4 days the
mice were challenged with the eliciting dose of antigen
(SRBCs) and the delayed type hypersensitivity was measured
24 h later as footpad swelling. It appeared (Table 3) that only
compound 10f exhibited significant inhibition of DTH at both
doses. This effect was even stronger than that of CsA.
The compounds were also tested (concentration range:
0.1e10 mg/mL) for their ability to alter the proliferative
The differential actions of the studied compounds in the
humoral and cellular (delayed type hypersensitivity) responses
probably resulted from the differences in their structures. The
common component in the structures of both compounds, 8g
and 10f, is the oxadiazole ring. That part of the molecule
could, presumably, be responsible for the observed suppres-
sion of the humoral immune response. On the other hand,
compound 10f, which strongly suppressed the DTH response,
contains isoxazolo[4,5-d]pyrimidine, substituted at position 7
with the phenyl radical, which may be relevant in the inhibi-
tion of the signaling pathways leading to the generation of
the cellular immune response. Obviously, the isoxazole ring,
substituted at position 4 with the NH2 group and at position
5 with the benzoyl group, being the second part of the 8g
molecule, cannot affect this type of immune response. The dif-
ferences in the structures of both compounds could also be the
reason for the opposite effects of these compounds on the pro-
liferative response of splenocytes to concanavalin A. However,
Table 4
Effects of the compounds on the proliferative response of splenocytes to con-
canavalin A
Compound
Concentration
(mg/mL)
OD
(550/630 nm)
ꢀSE
P
Control
0.089
0.004
(no mitogen)
Con A only
DMSO control
Table 2
Effects of the compounds on the humoral immune response in vivo to SRBCs
0.447
0.438
0.451
0.429
0.008
0.007
0.003
0.003
0.1
1
Compound
Dose
(mg/mouse)
AFC ꢂ 106
ꢀSE
P
10
Control
DMSO control
2153
1553
1698
13.58
8g
0.1
1
0.442
0.401
0.371
0.018
0.011
0.010
NS
<0.01
<0.01
10
100
70.5
33.2
10
8g
10
100
1361
574
105.1
57.5
NS
<0.001
10f
CsA
0.1
1
0.483
0.479
0.482
0.010
0.008
0.013
<0.02
<0.05
<0.01
10f
CsA
10
100
1660
545
49.8
88.5
NS
<0.001
10
0.1
1
0.070
0.046
0.001
0.002
0.003
0.000
<0.001
<0.001
<0.001
10
100
1617
953
102.6
54.8
<0.02
<0.001
10

2502
E. Wagner et al. / European Journal of Medicinal Chemistry 43 (2008) 2498e2504
these immunomodulatory effects in vitro were modest. In sum-
mary, both compounds exhibited distinct immunosuppressive
activities; therefore further investigations applying other im-
munological tests are justified to reveal their potential thera-
peutic utility.
added. Ethyloxalyl chloride (15 g, 0.109 mol) was added
drop wise to the mixture. The mixture was stirred at room tem-
perature for 5 h, cooled, filtered (TEA$HCl), and the solvents
were removed under reduced pressure. The residue was treated
with water, and then filtered. The resulting precipitate was
washed with water and methanol, dried, and recrystallized
from ethyl acetate. The yield was 26.3 g (79.77%). The pure
product melted at 204e205 ꢃC. IR (cmꢁ1, KBr): 3475, 3365,
3250 (NH2, NH), 1735, 1685, 1650 (C]O), 1605, 1545,
1497, 1427 (C]N, C]C, amide II, aromatics), 1230 (Ce
6. Experimental
All chemicals were obtained from Aldrich. Dry pyridine
was prepared by refluxing over sodium and storage over potas-
sium hydroxide. Melting points were determined with a Boetius
apparatus and are uncorrected. The progress of the reaction and
purity of compounds were monitored by TLC analytical silica
gel plates (Merck F254). IR spectra were recorded on a Specord
M80 spectrometer for KBr discs. 1H NMR spectra were
recorded with a Bruker Avance DRX-300 instrument; chemical
shifts are reported in ppm downfield from the internal tetrame-
thylsilane and coupling constants in Hz. Mass spectra were
recorded on a Finningan Mat 95. Elemental analyses were
performed on a Carbo Erba NA 1500 analyzer.
1
OeC). H NMR (DMSO-d6) d 11.13 (s, 2H, CONHeNHe
CO), 7.65e8.15 (m, 5H, Ar), 6.38 (s, 2H, NH2), 4.15e4.41
(q, 2H, CH2), 1.22e1.41 (t, 3H, CH3). Anal. calcd. for
C15H14N4O6 (346.30): C, 52.03; H, 4.07; N, 16.18. Found:
C, 52.23; H, 4.01; N, 16.28.
6.4. Ethyl{2-[(4-amino-5-benzoylisoxazol-3-yl)
carbonyl]hydrazine}(oxo)acetate 5b
Piperidine (0.5 g, 6 mmol) was added to a solution of com-
pound 5 (1.73 g, 5 mmol) in absolute dioxane (5 mL). The
reaction mixture was refluxed for 1 h, and then allowed to
attain room temperature. The separated solid was filtered,
washed with cold methanol, and recrystallized from absolute
ethanol. The yield of compound 5b was 1.5 g (77.72%). Mp:
207e209 ꢃC. IR (cmꢁ1, KBr): 3480, 3365, 3255 (NH2, NH),
6.1. 4-Amino-5-benzoylisoxazole-3-carbohydrazide 2
To a solution of 100 mL of dioxane, 50 mL methanol, and
20 g of 80% hydrazine hydrate was added 23.1 g (0.1 mol) of
4-amino-5-benzoylisoxazolo-3-carboxamide 1. The reaction
mixture was stirred under reflux for 6 h. The mixture was
concentrated to ca. 50 mL. After cooling, the precipitate was
filtered and washed with methanol. The solids were purified
by recrystallization from dioxane, giving 20 g of pure (TLC)
1
1695, 1665, 1650, 1640 (C]O, C]N). H NMR (DMSO-
d6) 10.91e11.04 (d, 2H, CONH), 7.69e8.19 (m, 5H, Ar),
6.41 (s, 2H, NH2), 3.38e3.53 (m, 4H, N(CH2)2), 1.38e1.82
(m, 6H, (CH2)3). Anal. calcd. for C18H19N5O5 (385.38): C,
56.10; H, 4.97; N, 18.17. Found: C, 56.40; H, 4.84; N,
18.10. The filtrate from this reaction contained (TLC) only
compound 5, no compound 6.
compound 2, yield (81.3%). Mp: 220e221 ꢃC. IR (cmꢁ1
,
KBr): 3430, 3360, 3230 (NH2, NH), 1685, 1650 (C]O),
1
700 (Ar). H NMR (DMSO-d6) d 10.27 (s, 1H, NH), 7.56e
8.07 (m, 5H, Ar), 6.35 (s, 2H, NH2 poz.4), 4.69 (s, 2H, e
NH2). Anal. calcd. for C11H10N4O3 (246.22): C, 53.66; H,
4.09; N, 22.75. Found: C, 53.69; H, 4.07; N, 22.38.
6.5. Ethyl-5-(4-amino-5-benzoylisoxazol-3-yl)
[1,3,4]oxadiazole-2-carboxylate 7
6.2. [4-Amino-3-(1,3,4-oxadiazol-2-yl)isoxazol-5-yl]
(phenyl)methanone 4
To a solution of the ester 5 (18 g, 0.052 mol) in absolute
dioxane (150 mL) was added thionyl chloride (100 mL). The
reaction mixture was refluxed for 15 h, and then allowed to
attain room temperature. The solvents were removed under
reduced pressure. The residue was dissolved in ethyl acetate
(120 mL) and treated with 4 g of Norit, boiled for a few
minutes, and filtered hot. The filtrate was kept in a refrigerator
for 12e16 h. The precipitated compound 7 was filtered and
washed with ethanol. The product formed colorless prisms
melting at 199e200 ꢃC. The yield was 11.6 g (67.9%). IR
(cmꢁ1, KBr): 3465, 3360 (NH2), 1745, 1650, 1640 (C]O,
The acid hydrazide 2 (2.46 g, 0.01 mol) was dissolved in
30 ml of absolute dioxane. Triethyl orthoformate (3 mL) was
then added while stirring and the reaction mixture was heated
under reflux for 5 h. The mixture was cooled and the separated
yellow product was filtered, washed with methanol, dried, and
recrystallized from dioxane, giving 2.1 g (yield: 82.03%) of
pure (TLC) compound 4. Mp: 222e223 ꢃC. IR (cmꢁ1, KBr):
3435, 3335 (NH2), 1660, 1620, 1605 (C]O, C]N), 710
1
(Ar). H NMR (DMSO-d6) d 7.68e8.40 (m, 6H, aromatics),
6.43 (s, 2H, NH2). Anal. calcd. for C12H8N4O3 (256.22): C,
56.25; H, 3.15; N, 21.87. Found: C, 56.38; H, 3.41; N, 22.08.
1
C]N). H NMR (DMSO-d6) d 7.60e8.40 (m, 5H, Ar), 6.43
(s, 2H, NH2), 4.48e4.77 (q, 2H, CH2), 1.43e1.61 (t, 3H,
CH3). Anal. calcd. for C15H2N4O5 (328.28): C, 54.88; H,
3.68; N, 17.07. Found: C, 54.93; H, 3.66; N, 16.89. MS, m/z
(%): 328 (Mþ, 82.0), 300.0 (18.0), 283.0 (3.5), 181.0 (5.0),
268.0 (44.0), 162.0 (95.0), 140.0 (27.0), 134.0 (78.0), 107.0
(100), 106 (82.0), 77.0 (75.0).
6.3. Ethyl{2-[(4-amino-5-benzoylisoxazol-3-yl)
carbonyl]hydrazine}(oxo)acetate 5
The acid hydrazide 2 (24.6 g, 0.1 mol) was dissolved in
200 mL of absolute dioxane and 10.1 g of triethylamine was
Atom labeling is shown in Fig. 1.

E. Wagner et al. / European Journal of Medicinal Chemistry 43 (2008) 2498e2504
2503
6.6. 5-(4-Amino-5-benzoylisoxazol-3-yl)
[1,3,4]oxadiazole-2-carboxamide 8aei
3H, CH3). Anal. calcd. for C17H13N5O4 (351.32): C, 58.12;
H, 3.73; N, 19.94. Found: C, 57.95; H, 3.57; N, 20.01.
To a solution of the ester 7 (1 g, 3 mmol) in absolute etha-
nol (5 mL) was added the appropriate amines (4 mmol). The
reaction mixture was refluxed for 2e3 h, and then allowed
to attain room temperature. The separated solid was filtered,
washed with cold ethanol, and recrystallized. Physical and
analytical data are listed in Table 1. IR (cmꢁ1): 3475, 3365,
3240 (NH2, NHCO), 1680, 1650, 1640 (C]O, C]N), 710
(Ar).
6.8. Amidoderivatives of 3-(1,3,4-oxadiazol-2-yl)-5-
methyl-7-phenylisoxazolo[4,5-d]-pyrimidine 10deg
6.8.1. Method A
The amide 8e (0.79 g, 2 mmol) was dissolved in (10 mL) of
anhydrous dioxane and (3 mL) anhydrous acetonitrile. Dry
gaseous hydrogen chloride was bubbled into the solution for
6 h at room temperature. Stirring was maintained at room
temperature for 6 days. The solvents were removed under
reduced pressure, and the residue was treated with water,
and then filtered. The resulting precipitate was washed with
water, dried, and recrystallized.
1
Compound 8a: H NMR (DMSO-d6) d 7.55e8.29 (m, 5H,
Ar), 7.12e7.20 (t, 1H, CONH), 6.48 (s, 2H, NH2), 3.44e3.54
(m, 2H, NeCH2e), 1.66e1.78 (m, 2H, CH2), 1.01e1.06 (t,
3H, CH3). MS, m/z (%): 341 (Mþ, 34.0), 313 (11.0), 255
(7.0), 228 (9.0), 181 (35.0), 161 (17.0), 134 (20.0), 106
(44.0), 105 (100), 96.0 (43), 86.0 (13.0), 77.0 (6.0).
6.8.2. Method B
1
Compound 8b: H NMR (DMSO-d6) d 7.50e8.28 (m, 5H,
Ar), 6.39 (s, 2H, NH2) 3.73e4.16 (m, 4H, N(CH2)2), 1.70e
To a solution of ester 9 (0.70 g, 2 mmol) in anhydrous tet-
rahydrofurane, appropriate amines (3 mmol) were added. The
resulting suspension was heated under reflux for 2 h, and then
allowed to attain room temperature. The precipitate formed
was filtered off, washed with ethanol and water, dried, and re-
crystallized. Physical and analytical data are listed in Table 5.
Compound 10d: IR (cmꢁ1, KBr) 3330 (NH), 1690 (C]O),
1635, 1605, 1552 (C]N, C]C, aromatics), 1215 (CeOeC).
1H NMR (DMSO-d6) d 9.75 (s, 1H, CONH), 7.75e8.21 (m,
5H, Ar), 3.40e3.64 (m, 6H, CH2eOeCH2 þ COeNeCH2),
2.98 (s, 3H, CH3), 2.47e2.64 (m, 6H, N](CH2)3).
Compound 10e: IR (cmꢁ1 KBr) 3335 (NH), 1690, 1640
(C]O, C]N), 710 (Ar). 1H NMR (DMSO-d6) d 10.13e
10.28 (t, 1H, CONH), 7.43e8.63 (m, 10H, Ar2), 4.53e4.63
(d, 2H, CH2), 3.00 (s, 3H, CH3).
1.76 (m, 6H, (CH2)3).
1
Compound 8i: H NMR (DMSO-d6) d 7.49e8.13 (m, 5H,
Ar), 6.54 (s, 2H, NH2), 3.96e4.00 (m, 4H, CH2eOeCH2),
3.61e3.71 (m, 4H, N(CH2)2).
6.7. Ethyl-5-(5-methyl-7-phenylisoxazolo[4,5-
d]pyrimidin-3-yl)[1,3,4]oxadiazole-2-carboxy-late 9
To a solution of the ester 7 (9.84 g, 0.03 mol) in anhydrous
dioxane (100 mL) was added anhydrous acetonitrile (25 mL)
and dry gaseous hydrogen chloride was bubbled into the solu-
tion for 6 h at room temperature. The reaction mixture was
stirred at room temperature for 6 days. After the reaction the
mixture was concentrated, cooled, and the separated solid
was filtered, washed with water, dried, and recrystallized
from ethyl acetate. The yield was 8.86 g (84.11%). The pure
product melted at 191e192 ꢃC. IR (cmꢁ1, KBr): 1745, 1640
(C]O, C]N), 1230 (CeOeC). 1H NMR (DMSO-d6)
d 8.16e8.24 (m, 2H, o,o0Ar), 7.65e7.73 (m, 3H, m,p,m0 Ar),
4.51e4.79 (q, 2H, CH2), 3.05 (s, 3H, CH3) <1.45e1.63 (t,
Compound 10f: IR (cmꢁ1, KBr) 3300 (NH), 1687, 1640,
1605 (C]O, C]N), 705 (Ar). 1H NMR (DMSO-d6)
d
10.18e10.22 (t, 1H, CONH), 7.40e8.69 (m, 9H,
C6H5 þ C5H4N), 4.57e4.63 (d, 2H, NeCH2), 3.02 (s, 3H,
CH3).
Compound 10g: IR (cmꢁ1, KBr) 3305 (NH), 1680, 1640,
1
1603 (C]O, C]N). H NMR (DMSO-d6) d 10.20e10.25
Table 5
Physical and analytical data of compounds 8aei, 10deg
Cpd. No.
Crystallized from
Mp
Yield
Formula
Molecular
weight
Calc. (%)
C
Found (%)
C
H
N
H
N
8a
8b
8c
Ethyl acetate
Ethanol
Ethanol
206e207
168e169
257e258
186e187.5
200e201
190e181
213e214
160e161.5
246e248
209e210
227e228
76.92
67.86
60.24
72.93
86.00
75.63
83.19
77.32
62.17
69.36
A. 70.47
B. 77.76
68.03
73.13
C16H15N5O4
C18H17N5O4
C19H19N5O4
C19H20N6O5
C20H15N5O4
C19H14N6O4
C19H14N6O4
C18H18N6O4
C17H15N5O5
C21H21N7O4
C22H16N6O3
341.32
367.36
381.39
412.40
389.37
390.36
390.36
382.37
369.33
435.44
412.41
56.30
58.85
59.84
55.34
61.69
58.46
58.46
56.54
55.29
57.93
64.07
4.43
4.66
5.02
4.89
3.88
3.61
3.61
4.74
4.09
4.86
3.91
20.52
19.06
18.36
20.38
17.99
21.53
21.53
21.98
18.96
22.52
20.38
56.49
58.70
60.15
55.13
62.01
58.70
58.86
56.48
54.99
58.12
64.38
4.40
4.59
4.96
4.90
3.75
3.54
3.59
4.76
4.03
4.84
4.00
20.65
19.02
18.22
20.53
18.24
22.05
21.82
22.16
18.72
22.76
20.62
8d
8e
Ethanol
Propanol
Ethanol
Ethanol
8f
8g
8h
8i
Ethanol
Acethyl acetate
Ethanol
Acethyl acetate
10d
10e
10f
10g
Ethanol
Ethanol
251e252
224e225
C21H15N7O3
C21H15N7O3
413.41
413.39
61.01
61.01
3.66
3.66
23.72
23.72
60.86
61.17
3.70
3.61
23.49
23.91

2504
E. Wagner et al. / European Journal of Medicinal Chemistry 43 (2008) 2498e2504
(t, 1H, CONH), 7.44e8.69 (m, 9H, C6H5 þ C5H4N), 4.59e
4.66 (d, 2H, NeCH2), 2.96 (s, 3H, CH3).
atoms are the acceptors for the amino groups (N(5), N(51)).
The distances between the N(5)/N(3) and N(51)/N(31)
˚
atoms are 3.116(2) and 3.100(2) A, respectively.
6.9. Crystallographic part
In the structure there are two intramolecular hydrogen
bonds between amino N(5), N51 and O(4), O(41) oxygen
atoms. The distances between the N(5)/(O4) and N(51)/
6.9.1. X-ray diffraction studies
˚
The X-ray diffraction data were collected at 100 K for
a crystal of size 0.15 ꢂ 0.25 ꢂ 0.25 mm. All measurements
were made on a KM4 CCD computer-controlled k-axis diffrac-
O(41) atoms are 2.827(2) and 2.814(2) A, respectively.
Furthermore, hydrogen bonding involving the amino H(5)
and H(51) atoms are bifurcated and forms two intramolecular
H-bonds with N(4) and N(41) atoms of the oxadiazole rings.
The distances between the N(5)/N(4) and N(51)/N(41)
˚
tometer with graphite-monochromated Mo Ka (0.71073 A)
radiation. The intensities were corrected for Lorentz and polar-
ization effects, but no corrections were made for absorption.
The structure was solved by direct methods with SHELXS97
and refined on F2 by full-matrix least-square methods using
the SHELXL97 [23] program. Non-hydrogen atoms were re-
fined with anisotropic thermal parameters. The carbon-bonded
H-atoms were included in calculating the positions and refined
using a riding model with isotropic displacement parameters
equal to 1.2 Ueq or 1.5 Ueq of the attached C atoms. The H-
atoms of the amino groups were located from a difference
Fourier map and refined.
˚
are 2.970(2) and 2.938(2) A, respectively.
Acknowledgements
The authors are very grateful to the staff members of the
National Cancer Institute (NCI), Bethesda, MD, USA, for car-
rying out the anticancer screening of the newly synthesized
compounds.
References
Fig. 1 is a view of one of the two independent molecules
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˚
ꢃ
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c ¼ 14.041(3) A, a ¼ 88.49(3), b ¼ 75.6ꢁ13(3) , g ¼ 88.89(3) ,
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There are two independent molecules A and B in the asym-
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