Synthesis and pharmacological screening of derivatives of isoxazolo[4,3-d]pyrimidine and isoxazolo[4,5-d]pyrimidine

Article abstract of DOI:10.1002/jhet.373

(Chemical Equation Presented) A number of derivatives of isoxazolo[4,3-d]pyrimidine and isoxazolo[4,5-d]pyrimidine were prepared with potential anticancer activity. Condensation of 4-amino-5-benzoyl-isoxazolo-3- carboxylic acid hydrazide with ethyl orthof

Full text of DOI:10.1002/jhet.373

May 2010
Synthesis and Pharmacological Screening of Derivatives of
Isoxazolo[4,3-d]pyrimidine and Isoxazolo[4,5-d]pyrimidine
677
Edwin Wagner,a Kamal Al-Kadasi,a Lilianna Becan,a* and Wanda Sawka-
Dobrowolskab
aDepartment of Drugs Technology, Wroclaw Medical University, Pl. Nankiera 1, 50-140 Wroclaw,
Poland
bFaculty of Chemistry, University of Wroclaw, ul. F.J. Curie 14, 50-383 Wroclaw, Poland
*E-mail: lilianna@ktl.am.wroc.pl
Received September 10, 2009
DOI 10.1002/jhet.373
Published online 11 May 2010 in Wiley InterScience (www.interscience.wiley.com).
A number of derivatives of isoxazolo[4,3-d]pyrimidine and isoxazolo[4,5-d]pyrimidine were prepared
with potential anticancer activity. Condensation of 4-amino-5-benzoyl-isoxazolo-3-carboxylic acid hy-
drazide with ethyl orthoformate and then with different amines gave a series of 3-benzoyl-7-oxo-7H-iso-
xazolo[4,3-d]pyrimidin-6-yl-aryliden-formamidine. A series of 5-aminomethyl-7-phenyl-isoxazolo[4,5-
d]pyrimidine-3-carboxamide was obtained from 4-amino-5-benzoylisoxazole-3-carboxamide with aceto-
nitrile, and chloroacetonitrile with gaseous hydrogen chloride. Some of these compounds were tested
for their cytotoxic activity.
J. Heterocyclic Chem., 47, 677 (2010).
INTRODUCTION
derivatives of both isomers contains a few same papers
in which the authors obtained isoxazolo[4,5-d]pyrimi-
dine but did not assay their biological activity [7–9].
Some derivatives of isoxazolo[4,5-d]pyrimidine were
shown to be CRF (corticotrophin releasing factor) antag-
onist and may be useful as cCMP phosphodiesterase
inhibitors [10,11]. Another derivatives of isoxazolo[4,5-
d]pyrimidine that are structurally similar to hypoxantine
can have anxiolytic activity comparable to that of diaz-
epam [12]. In contrast, compared with diazepam the
derivatives of isoxazolo[4,3-d]pyrimidine are devoid of
anticonvulsant and anxiolytic properties [13].
The bases of the 9H-purines adenosine (adenine) and
the adenosine metabolite inosine (hypoxantine) are en-
dogenous ligands of different receptors. Adenosine is an
endogenous ligand of adenosine receptors (AR). They
are subdivided into four subtypes (A1, A2A, A2B, and
A3) [1]. Adenosine receptors have been actively studied
as potential therapeutic targets in several disorders, such
as Parkinson’s disease, schizophrenia, analgesia, and is-
chemia, and as cytostatics. Inosine is a highly selective
ligand for the A3 receptor subtype [2,3] and so is a
ligand of the benzodiazepine receptor [4,5]. The nitro-
gen atoms at positions 3 and 7 of both purines and their
derivatives take part in the formation of a hydrogen
bond with these receptors [5,6]. For example, substitu-
tion of the nitrogen atom at position 7 in inosine gave
7-methylinosine, which was ineffective in binding to the
benzodiazepine receptor [5]. Therefore, we have been
working on synthetic derivatives of two isomers of iso-
xazolopyrimidine, 4,3-d and 4,5-d, which may be con-
sidered as 7,9-dideaza-7-oxa-8-aza purines, and which
may have high biological and similar receptor activity.
Only the isomers from four isomers of isoxazolopyrimi-
dine have nitrogen and oxygen heteroatoms at positions
analogous to the 9H-purines. The literature concerning
RESULTS AND DISCUSSION
Synthesis. In continuation of our program we report
the synthesis of new derivatives of the title compounds.
The synthetic pathways used to obtain the target com-
pounds are depicted in Scheme 1. The starting material
1 was obtained by the Thorpe reaction according to
Gewald et al. [14], by which it could be converted into
hydrazide 2 [15]. Hydrazide 2 with aldehydes gave the
new hydrazido-hydrazones 3-5, which were condensed
with ethyl orthoformate in the presence of the acetic an-
hydride, yielding the new derivatives of isoxazolo[4,3-
C
V
2010 HeteroCorporation

678
E. Wagner, K. Al-Kadasi, L. Becan, and W. Sawka-Dobrowolska
Vol 47
Scheme 1
d]pyrimidines 6-8. The acid hydrazide 2 was refluxed in
a mixture with only equimolar amounts of triethyl
ortho-formate, giving [4-amino-3-(1,3,4-oxadiazol-2-yl)i-
soxazol-5-yl]-(phenyl)methanone [15]. If hydrazide 2
was refluxed with an excess of triethyl orthoformate and
acetic anhydride, then the derivatives of isoxazolo[4,3-
d]pyrimidine 6-8 and 9,10 were formed, which with dif-
fer-rent amines formed new derivatives of 3-benzoyl-7-
oxo-7H-isoxazole[4,3-d]pyrimidin-6-yl-aryliden-formam-
idine 11–14. Compound 1 was heated in acetonitrile or
chloroacetonitrile with gaseous hydrogen chloride to
form the new derivatives of isoxazolo[4,5-d]pyrimidine
15–16. Mechanism of the reaction was described earlier
by Shishoo et al [16,17]. Compound 16 reacts with
amines to form the new 5-(aminemethyl)-7-phenyl-iso-
xazolo[4,5-d]pyrimidine-3-carboxamide 17–20. It is well
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May 2010 Synthesis and Pharmacological Screening of Derivatives of Isoxazolo[4,3-d]pyrimidine and
Isoxazolo[4,5-d]pyrimidine
679
The molecular structure and atom numbering scheme
are shown in Figure 1. Compound 21 crystallizes in a
noncentrosymmetric Pna21 space group with one mole-
cule per asymmetric unit. The determination of the
structure of compound 21 confirms the S configuration
of the chiral atom C7. The interatomic distances for C5-
˚
˚
N4 [1.310(3) A] and C3-N2 [1.320(3) A] are typical of
˚
carbon-nitrogen double bonds [24] (1.279–1.329 A) and
˚
˚
the C9-N4 [1.401(3) A] and C7-N6 [1.476(3) A], bond
lengths correspond to typical carbon-nitrogen single
˚
bonds (1.366–1.454 A).
The isoxazole and pyrimidine rings are essentially
planar, with maximum deviations from the calculated
˚
˚
mean plane of 0.006(2) A (C9) and 0.036(2) A (C8),
respectively. The dihedral angle between the best planes
through the isoxazole ring and the pyrimidine ring is
3.0(2)ꢂ, indicating that the whole molecule is almost
planar. The carboamide moiety deviates slightly from
the isoxazole ring plane, the N3-C1-C3-N2 and O2-C1-
C3-C9 torsion angles being ꢁ172.4(2)ꢂ, and –172.5(3)ꢂ
respectively. The chloromethyl and phenyl moieties are
nearly perpendicular, to the pyrimidine ring.
Figure 1. A view of the molecular structure of compound 21 with the
atomic numbering scheme. Ellipsoids are at the 35% probability level.
known that isoxazole rings are readily cleaved by hydro-
genation [18–21]. Thus the reduction of compound 16
followed by treatment with sodium borohydride in meth-
anol at room temperature gave compound 21 with only
one reduced double bond at position 6-7 from four. X-
ray crystallography of compound 9 (Fig. 1) confirmed
that the resulting structure was indeed compound 21 and
15,16, and 17-20.
However, amid 1 readily reacts with nitriles and
forms compounds 15,16. Of the compounds selected by
the staff members of the NCI the derivatives isoxazole
3c, isoxazole[4,3-d]pyrimidine 9, and isoxazole[4,5-
d]pyrimidine 19 were interesting.
The dihedral angles between the pyrimidine ring
plane and the Cl1-C2-C5 and C11-C16 planes are
66.6(1)ꢂ and 77.8(1)ꢂ, respectively.
In the present structure the nearly planar conformation
of the title compound is stabilized by a network of inter-
molecular and intramolecular hydrogen bonds.
The hydrogen bond involving the amide H31 atom is
bifurcated, H31 forming one intramolecular hydrogen
bond with the N4 atom of the pyrimidine ring and the
Cl1 atom of the chloromethyl moiety. The distances
between the N(2)-H31. . .N(4) and N2-H31. . .Cl1(ꢁx þ
Crystallographic part. X-ray diffraction studies: The
X-ray diffraction data were collected at 100 K for a
crystal of size 0.2 ꢀ 0.25 ꢀ 0.25 mm. All measure-
ments were made on a KM4 CCD computer-controlled
j-axis diffractometer with graphite-monochromated
˚
1, ꢁy þ 1, z ꢁ 1/2) atoms are 2.946(3) and 3.552(2) A,
respectively. Furthermore, the amide H6 forms an N6-
H6. . .O2(x ꢁ 1/2, ꢁy þ 1/2, z þ 1) hydrogen bond
with the O2 atom of the carboamide group. The distan-
˚
˚
MoKa(0.71073 A) radiation. The intensities were cor-
ces N6. . .O2 and H6. . .O2 are 2.908(3) and 2.22(3) A,
rected for Lorentz and polarization effects, but no correc-
tions were made for absorption. The structure was solved
by direct methods with SHELXS-97 and refined by full-
matrix least-squares methods on F2 using the SHELXL-
97 [22] program. Nonhydrogen atoms were refined with
anisotropic thermal parameters. All the H atoms were
located using a differrence Fourier map and refined.
Figure 1 was drawn using the XP program [23]. Crys-
tal data for 5-chloromethyl-7-phenyl-6,7-dihydro-isoxa-
zlo[4,5-d]pyrimidine-3-carboxamide 21: C13H11N4O2Cl,
T ¼ 100(2) K, M ¼ 290.71, orthorhombic, space group
and the angle is 139(2)ꢂ. Additionally, the amide H32
atom on N2 interacts with the ring centroid Cg of the
phenyl ring, forming a nonconventional hydrogen bond
˚
of the N2-H32. . .p (Cg) [H32. . . p, 2.71 A N2. . . p
ꢂ
˚
3.443(3) A, N2-H32. . . p 149 ; symmetry code: x þ 1/
2, ꢁy þ 1/2, zꢁ1]. The molecules are also linked by
the C7-H1. . .N4, C2-H21. . .O2, C12-H12. . .O2, and
C15-H15. . .O1[3.523(3),
˚
3.149(3),
3.437(3) A] weak hydrogen bonds to form a chain run-
3.550(3),
and
ning parallel to the axis. The distances H. . .O(N)
ꢂ
˚
[2.51(3)–2.85(3) A] and angles [126(2)–163(2) ] suggest
˚
˚
Pna21, with a ¼ 14.713(2) A, b ¼ 16.745(2) A, c ¼
some kind of weak CAH. . .X hydrogen bond.
3
˚
˚
5.236(1) A, V ¼ 1290.0(3) A , Z ¼ 4, Dc ¼ 1.4969 g
cmꢁ3, l ¼ 0.303 mmꢁ1. There were 2930 unique reflec-
tions, of which 2553 were considered as observed, with
final R ¼ 0.0439 and wR ¼ 0.079.
CCDC726160 contains the supplementary crystallo-
graphic data for this article. These data can be obtained
free of charge at www.ccdc.cam.ac.uk/const/retrie-
ving.html (or from the Cambridge Crystallographic Data
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

680
E. Wagner, K. Al-Kadasi, L. Becan, and W. Sawka-Dobrowolska
Vol 47
Table 1
Characterization data of compounds 3–20 and growth percent of some selected in vitro tumor cell lines (10 lmol).a.
Ovarian Cancer
OVCAR-8
Leukemia
RPMI-8226
Compound
R
Renal Cancer UO-31
Melanoma SK-MEL-2
3
4
CHCH3
CHC6H5
5
6
7
CHC6H4-Cl-4
CHCH3
CHC6H5
45.80
41.53
ꢁ10.37
57.66
42.59
62.54
8
9
C6H4-Cl-4
OCH3
OC2H5
83.79
45.76
25.74
ꢁ12.84
60.83
39.94
10
11
12
13
14
15
16
17
18
19
20
4-Pyridine
C6H5
63.61
C6H4Cl-4
C6H4-F-4
CH3
CH2Cl
4-Morpholine
4-Methylpiperazine
NH-4-morpholine
N(C4H9)2
42.20
55.00
ꢁ17.94
a Data obtained from the NCI’s in vitro human tumor cell screen.
obtained from Alfa Aesar (Karlsruhe, Germany), Chempur
(Piekary Sl., Poland), and Lancaster (Frankfurt am Main).
General procedure for the synthesis of compounds 3-
5. To a stirred solution of acid hydrazide 2 (2.46 g, 0.01 mol)
in 20 mL of anhydrous 1,4-dioxane heated at 80ꢂC, the appro-
priate aldehyde (0.012 mol) was added. The reaction mixture
was heated under reflux for 5 h and the solvent was rotoevapo-
rated. The resulting solid was recrystallized from the appropri-
ate solvent.
Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax:
(þ44) 1223 336033; e-mail: deposit@ccdc.cam.uk).
Cytotoxic screening. The cytotoxic studies were per-
formed at the National Cancer Institute, (NCI, Bethesda,
MD). The screening is a two-stage process beginning
with the evaluation of the compound against 60 different
human tumor cell lines representing leukemia, mela-
noma, and cancers of the lung, colon, brain, breast,
ovary, prostate, and kidney at a single dose of 10 lmol.
Evaluation of the cytotoxic activity was performed for
compounds 5, 6, 9, 11, and 19. Only compounds 5, 9, and
19 showed interesting activity in the panel 62 tumor cell
lines, but chiefly on the renal cancer line UO-31. Data on
the active compounds are presented in Table 1.
4-Amino-5-benzoyl-N-(ethylideneamino)isoxazole-3-carbox-
amide (3). Yield: 1.78 g (65.5%), mp 191ꢂC (1,4-dioxane); IR
(potassium bromide): 3500, 3395 (NH2), 3260 (NH), 1685, 1660
1
(CO), 1640 (C¼¼N) cmꢁ1; H NMR (DMSO-d6): d 1.94–2.00 (d,
3H, CH3, J ¼ 6.0 Hz ), 6.41 (s, 2H, NH2), 7.68 (m, 3H, arom.),
7.80–7.90 (q, 1H, CH, J ¼ 6.0 Hz), 8.00–8.10 (d, 2H, arom.),
12.06 (s, 1H, HN-CO). Anal. Calcd. for C13H12N4O3: C, 57.35;
H, 4.44; N, 20.58. Found: C, 57.21; H, 4.38; N, 20.37.
4-Amino-5-benzoyl-N-(phenylmethylenebenzylideneamino)i-
soxazole-3-carboxamide (4). Yield: 2.52 g (75.5%), mp 215C
(ethanol); IR (potassium bromide): 3420, 3305 (NH2), 3260
EXPERIMENTAL
Melting points were determined with a Boethius apparatus
and are uncorrected. The progress of the reaction and the pu-
rity of the compounds were monitored by TLC on analytical
silica gel plates (Merck F254, Darmstadt, Germany). IR spectra
were recorded on a Specord M80 spectrometer (Zeiss/Analytic
(NH), 1685, 1660 (CO), 1640 (C¼¼N) cmꢁ1
;
1H NMR
(DMSO-d6): d 6.44 (s, 2H, NH2), 7.50–8.66 (m, 10H, arom.),
8.57 (s, 1H, CH), 12.42 (s, 1H, HN-CO). Anal. Calcd. for
C18H14N4O3: C, 64.67; H, 4.22; N, 16.76. Found: C, 64.80; H,
4.23; N, 16.47.
4-Amino-5-benzoyl-isoxazole-3-carboxylic acid (4-chloro-
benzylidene)-hydrazide (5). Yield: 2.54 g (69.3%), mp 249ꢂC
(THF-ethanol 1:2); IR (potassium bromide): 3480, 3385
1
Jena, Germany) for KBr dises. H NMR spectra were recorded
with a Bruker Avance ARX-300 instrument (Bruker Analytic,
Karlsruhe, Germany; Bruker AG, Fallanden, Switzerland).
Chemical shifts are reported in ppm downfield from the inter-
nal tetramethylsilane reference and the coupling constants are
in Hz. Mass spectra were recorded on a Finningan Mat 95
GC-MS (Finningan, Bremen, Germany) with an ionization
energy of 70 eV. Elemental analyses were performed on a Per-
kin Elmer 2400 analyzer (Waltham, MA) and the results are
within 6 0.4% of the theoretical values obtained for the new
compounds. The chemicals and reagents for synthesis were
(NH2), 3210 (NH), 1705, 1660 (CO), 1650 (C¼¼N) cmꢁ1
;
1H
NMR (DMSO-d6): d 6.49 (s, 2H, NH2), 7.58–8.05 (m, 9H,
arom.), 8.55 (s, 1H, CH), 12.48 (s, 1H, HN-CO). Anal. Calcd.
for C18H13ClN4O3: C, 58.63; H, 3.55; N, 15.19. Found: C,
58.81; H, 3.48; N, 14.98.
General procedure for the synthesis of compounds 6-
8. To an equimolar mixture of 12.5 mL of acetic anhydride
and 22 mL of triethyl orthoformate was added 0.008 mol of
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May 2010 Synthesis and Pharmacological Screening of Derivatives of Isoxazolo[4,3-d]pyrimidine and
Isoxazolo[4,5-d]pyrimidine
681
N-(3-Benzoyl-7-oxo-7H-isoxazolo[4,3-d]pyrimidin-6-yl)-N0-
pyridin-4-ylmethyl-formamidine (11). Yield: 2.08 g (55.6%),
mp 195ꢂC (ethanol); IR (potassium bromide): 3300 (NH),
the compounds 3–5, or 3c and refluxed for 5 h. After concen-
tration in vacuo the residual oil was poured into 50 mL of
10% ethanolic ammonia. After 1 h of stirring at room tempera-
ture the mixture was refrigerated. The resulting solid was fil-
trated, washed with water, vacuum dried, and recrystallized
from the appropriate solvent.
1
1725, 1680 (C¼¼O), 1645 (C¼¼N) cmꢁ1; H NMR (DMSO-d6):
d 4.38 (s, 1H, NH), 4.61 (s, 2H, CH2), 6.56 (s, 1H, CH), 7.27–
7.42 (m, 3H, arom.), 7.56–7.68 (m, 3H, arom. þ CH at pos.
5), 8.05–8.12 (m, 2H, arom.), 8.50–8.57 (dd, 2H, arom.). Anal.
Calcd. for C19H14N6O3: C, 60.96; H, 3.77; N, 22.45. Found:
C, 60.74; H, 3.65; N, 22.18.
N-(3-Benzoyl-7-oxo-7H-isoxazolo[4,3-d]pyrimidin-6-yl)-N0-
benzyl-formamidine (12). Yield: 2.20 g (59%), mp 164ꢂC
(ethanol); IR (potassium bromide): 3300 (NH), 1715, 1680
3-Benzoyl-6-ethylideneamino-6H-isoxazolo[4,3-d]pyrimidin-
7-one (6). Yield: 2.23 g (79.1%), mp 180ꢂC (1,4-dioxane); IR
(potassium bromide): 1740, 1660 (CO), 1650 (C¼¼N) cmꢁ1
;
1H NMR (DMSO-d6): d 2.18–2.20 (d, 3H, CH3, J ¼ 6.0 Hz),
7.59–7.64 (m, 3H, arom.), 7.74–7.80 (q, 1H, CH J ¼ 6.0 Hz),
8.02–8.05 (m, 2H, arom.), 8.57 (s, 1H, pos. 5) Anal. Calcd. for
C14H10N4O3: C, 59.57; H, 3.57; N, 19.85. Found: C, 59.38; H,
3.64; N, 20.04.
(C¼¼O), 1645 (C¼¼N) cmꢁ1
;
1H NMR (DMSO-d6): d 4.68 (s,
1H, NH), 5.19 (s, 2H, CH2), 6.72 (s, 1H, CH), 7.23–7.40 (m,
6H, arom.), 7.59–7.78 (m, 3H, arom. and H at pos. 5), 8.03–
8.10 (m, 2H, arom.). Anal. Calcd. for C20H15N5O3: C, 64.34;
H, 4.05; N, 18.76. Found: C, 64.56; H, 4.11; N, 18.89.
3-Benzoyl-6-(benzylidene-amino)-6H-isoxazolo[4,3-d]pyri-
midin-7-one (7). Yield: 2.49 g (72.5%), mp 205ꢂC (ethanol);
ir (potassium bromide): 1720, 1700 (CO), 1650 (C¼¼N) cmꢁ1
;
1H NMR (DMSO-d6): d 7.55–8.07 (m, 10H, arom.), 8.48 (s,
1H, CH), 9.15 (s, 1H, CH, pos. 5). Anal. Calcd. for
C19H12N4O3: C, 66.28; H, 3.51; N, 16.27. Found: C, 66.29; H,
3.58; N, 16.11.
N-(3-Benzoyl-7-oxo-7H-isoxazolo[4,3-d]pyrimidin-6-yl)-N0-
[(4-chloro-benzyl)-formami-dine (13). Yield: 2.03 g (49.8%),
mp 169ꢂC (ethanol); IR (potassium bromide): 3310 (NH),
1
1710, 1690 (C¼¼O), 1640 (C¼¼N) cmꢁ1; H NMR (DMSO-d6):
3-Benzoyl-6-[(4-chloro-benzylidene)-amino]-6H-isoxazolo[4,3-
g
(59.4%), mp 235ꢂC
d 4.33 (s, 1H, NH), 4.56 (s, 2H, CH2), 6.55 (s, 1H, CH), 7.18–
7.46 (m, 5H, arom.), 7.56–7.68 (m, 3H, arom. and CH at pos.
5), 8.03–8.19 (m, 2H, arom.). Anal. Calcd. for C20H14ClN5O3:
C, 58.90; H, 3.46; N, 17.17. Found: C, 59.08; H, 3.51; N,
17.30.
d]pyrimidin-7-one (8). Yield: 2.24
(THF); IR (potassium bromide): 1715, 1695 (C¼¼O), 1650
(C¼¼N) cmꢁ1
;
1H NMR (DMSO-d6): d 7.61–8.11 (m, 9H,
arom.), 8.47 (s, 1H, CH), 9.20 (s, 1H, CH pos. 5). Anal. Calcd.
for C19H11 Cl N4O3: C, 60.25; H, 2.93; N, 14.79. Found: C,
60.46; H, 3.06; N, 15.03.
N-(3-Benzoyl-7-oxo-7H-isoxazolo[4,3-d]pyrimidin-6-yl)-N0-
(4-fluoro-benzyl)-formami-dine (14). Yield: 1.76 g (45.0%),
mp 2202ꢂC (ethanol); IR (potassium bromide): 3280 (NH),
Methyl N-(3-Benzoyl-7-oxo-7H-isoxazolo[4,3-d]pyrimidin-
6-yl)-formimidate (9). A solution of 24.6 g (0.1 mol) of the
acid hydrazide 2 in an equimolar mixture of 75 mL acetic an-
hydride and 87 mL of trimethyl orthoformate was refluxed for
4 hours. Then the reaction mixture was concentrated in vacuo,
filtered, and recrystallized to give compound 9. Yield: 2.26 g
(76.0%), mp 226ꢂC (methanol); IR (potassium bromide):1760,
1
1720, 1690 (C¼¼O), 1645 (C¼¼N) cmꢁ1; H NMR (DMSO-d6):
d 4.32 (s, 1H, NH), 4.55 (s, 2H, CH2), 6.54 (s, 1H, CH), 7.15–
7.41 (m, 5H, arom.), 7.56–7.71 (m, 3H, arom. and CH at pos.
5), 8.06–8.08 (m, 2H, arom.). Anal. Calcd. for C20H14FN5O3:
C, 61.38; H, 3.61; N, 17.89. Found: C, 61.49; H, 3.70; N,
18.08.
1680 (C¼¼O), 1660,1650 (C¼¼N), 1160 (CAOAC) cmꢁ1
;
1H
5-Methyl-7-phenyl-isoxazolo[4,5-d]pyrimidine-3-carboxamide
(15). To a solution of amide 1 (2.31 g, 0.01 mol) in anhydrous
1,4-dioxane (40 mL) was added anhydrous acetonitrile (7 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. Then the reaction mix-
ture was concentrated, cooled, and the resulting solid was fil-
tered, washed with water, dried, and recrystallized from etha-
nol. Yield: 2.17 g (85.3%), mp 227ꢂC (ethanol); IR (potassium
bromide): 3250 (CONH2), 1690 (C¼¼O), 1600 (C¼¼N), 760
NMR (DMSO-d6): d 2.09 (s, 3H, CH3), 7.59–8.06 (m, 5H,
arom.), 8.19 (s, 1H, pos. 5), 11.41 (s, 1H, pos. 6). Anal. Calcd.
for C14H10N4O4: C, 56.38; H, 3.38; N, 18.78. Found: C,
56.12; H, 3.22; N, 18.69.
Ethyl N-(3-Benzoyl-7-oxo-7H-isoxazolo[4,3-d]pyrimidin-6-
yl)formimidate (10). A solution of 2.46 g (0.01 mol) of the
acid hydrazide 2 in a mixture of 12.5 mL acetic anhydride and
22 mL of trimethyl orthoformate was refluxed for 4 h. Then
the reaction mixture was concentrated in vacuo, filtered, and
recrystallized to give compound 10. Yield: 2.12 g (68.0%), mp
222ꢂC (1,4-dioxane); IR (potassium bromide): 1710, 1690
(phenyl) cmꢁ1 1H NMR (DMSO-d6): d 3.00 (s, 3H, CH3),
;
7.70–7.73 (m, 3H, arom.), 8.48–8.49 (m, 2H, arom.), 8.50–
8.52 (ss, 2H, NH2). Anal. Calcd. for C13H10N4O2: C, 61.41; H,
3.96; N, 22.04. Found: C, 61.62; H, 4.01; N, 21.84.
(C¼¼O), 1650 (C¼¼N), 1155 (CAOAC) cmꢁ1
;
1H NMR
(DMSO-d6): d 1.34–1.39 (t, 3H, CH3), 4.33–4.40 (q, 2H,
CH2), 7.59–8.05 (m, 5H, arom.), 8.23 (s, 1H, pos. 5), 8.56 (s,
1H, pos. 6). Anal. Calcd. for C15H12N4O4: C, 57.69; H, 3.87;
N, 17.94. Found: C, 57.86; H, 3.83; N, 17.87.
5-Chloromethyl-7-phenyl-isoxazolo[4,5-d]pyrimidine-3-car-
boxamide (16). Compound 16 was prepared in the same man-
ner as described above for compound 15 except that 23.1 g
(0.1 mol) of amide 1 was added to a mixture of 330 mL anhy-
drous 1,4-dioxane and 90 mL anhydrous chloroacetonitrile.
Yield: 2.33 g (81.0%), mp 226ꢂC (benzene); Compound 16
was obtained by another method described in our previous pa-
per [25].
General procedure for the synthesis of compounds 17-
20. A stirred mixture of compound 16 (0.576 g, 0.002 mol),
0.05 g KI, and the corresponding amine in 30 mL of anhy-
drous toluene was heated to ca. 80ꢂC for 6 h. After cooling,
General procedure for the synthesis of compounds 11-
14. To a stirred solution of compound 9 (0.596g, 0.002 mol)
in 15 mL of anhydrous 1,4-dioxane was added the appropriate
amine (0.004 mol) and heated at 80ꢂC for 4 h. The hot reac-
tion mixture was treated with charcoal and filtered. Concentra-
tion of the filtrate to a small volume and addition of 5 mL
ethanol resulted in the separation of a colorless solid which
was collected by filtration, dried, washed with methanol, and
recrystallized from ethanol.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

682
E. Wagner, K. Al-Kadasi, L. Becan, and W. Sawka-Dobrowolska
Vol 47
REFERENCES AND NOTES
water (30 mL) was added and the reaction mixture was
extracted with dichloromethane. The combined organic phases
were washed with water, dried (Na2SO4), and concentrated to
give a residue, which was recrystallized.
5-Morpholin-4-ylmethyl-7-phenyl-isoxazolo[4,5-d]pyrimi-
dine-3-carboxamide (17). Yield: 2.71 g (80.1%), mp 208ꢂC
(methanol); IR (potassium bromide): 3280 (NH2), 1715
[1] Fredholm, B. B.; Abbacchio, M. P.; Burnstock, G.; Daly, J.
W.; Harden, T. K.; Jacobson, K. A.; Leff, P.; Williams, M. Pharmacol
Rev 1994, 46, 143.
[2] Merighi, S.; Mirandola, P.; Varani, K.; Gessi, S.; Leung, F.;
Baraldi, P. G.; Tabrizi, M. A.; Borea, P. A. Pharmacol Ther 2003,
100, 31.
(C¼¼O), 1620 (C¼¼N), 1120 (CAOAC) cmꢁ1
;
1H NMR
[3] Miller, Ch.E. Curr Topies Med Chem 2003, 3, 445.
[4] Asano, T.; Spector, S. Proc Natl Acad Sci USA 1979, 76,
977.
(DMSO-d6): d 2.57–2.60 (m, 4H, CH2ACH2), 3.94 (s, 2H,
CH2), 7.58–7.68 (m, 3H, arom.), 8.41–8.45 (m, 4H, 2H, arom.
and 2H, NH2). Anal. Calcd. for C17H17N5O3: C, 60.17; H,
5.05; N, 20.64. Found: C, 59.79; H, 5.15; N, 20.88.
[5] Skolnick, P.; Syapin, P. J.; Paugh, B. A.; Moncada, Y.;
Marangos, P. J.; Paul, S.M. Proc Natl Acad Sci USA 1979, 76, 1515.
[6] Cosyn, L.; Gao, Z. G.; Van Rompaey, P.; Lu, Ch.; Jacob-
son, K. A.; Van Calenbergh, S. Bioorg Med Chem 2006, 14, 1403.
[7] Desimoni, G.; Gru¨nanger, P.; Finzi, P.V. Tetrahedron 1967,
23, 675.
5-(4-Methyl-piperazin-1-ylmethyl)-7-phenyl-isoxazolo[4,5-
d]pyrimidine-3-carboxamide (18). Yield: 2.16 g (61.5%), mp
188ꢂC (methanol); Compound 18 was obtained by another
method described in our previously paper [13], the physical
and analytical data are identical.
[8] Laurent, S.; Barbieux-Flammang, M.; Van Haverbeke, Y.;
Flammang, R.; Wentrup, C. Bull Soc Chim Belg 1994, 103, 181.
[9] Aliabiev, S. B.; Ivanov, J. S.; Ilyin, A. P.; Kravchenko, D.
V.; Ivachtchenko, A. V. Synth Lett Org Chem 2007, 203.
[10] Niewochner, U.; Haning, H.; Lampe, T.; Es-Sayed, M.;
Schmidt, G.; Bischoff, E.; Dembowsky, K.; Perzborn, E.; Schlem-
meret, K. H. Ger Offen DE 19,962,925, 24 (2001).
[11] Frietze, W. E. US 1998–97,386 (1998).
5-(Morpholin-4-ylaminomethyl)-7-phenyl-isoxazolo[4,5-d]py-
rimidine-3-carboxamide (19). Yield: 2.48
g (70.4%), mp
228ꢂC (methanol); Yield: 2.48 g (70.4%), mp 228ꢂC (metha-
nol); IR (potassium bromide): 3300, 3280 (NH, NH2), 1705
(C¼¼O), 1630 (C¼¼N), 1115 (CAOAC) cmꢁ1
;
1H NMR
(DMSO-d6): d 2.21–2.23 (m, 4H, CH2ACH2), 3.57–3.61 (m,
4H, CH2ACH2), 3.94 (s, 2H, CH2), 4.31 (s, 1H, NH), 7.65–
7.67 (m, 3H, arom.), 8.36 (s, 1H, NH), 8.42–8.44 (m, 2H,
arom.), 8.48 (s, 1H, NH). Anal. Calcd. for C17H18N6O3: C,
57.62; H, 5.12; N, 23.72. Found: C, 57.38; H, 5.18; N, 24.01.
5-Dibutylaminomethyl-7-phenyl-isoxazolo[4,5-d]pyrimidine-
3-carboxamide (20). Yield: 2.84g (74.6%), mp 130ꢂC (1,4-
dioxane); IR (potassium bromide): 3300, 3200 (NH2), 1680
[12] Wagner, E.; Becan, L.; Nowakowska, E. Bioorg Med Chem
2004, 12, 265.
[13] Pore˛ba, K.; Wagner, E.; Jakowicz, I.; Balicka, D. II. Acta
Pol Pharm Drug Res 1994, 51, 355–358.
¨
[14] Gewald, K.; Bellmann, P.; Jansh, H. J. Liebigs Ann Chem
1980, 10, 1623.
[15] Wagner, E.; Al-Kadasi, K.; Zimecki, M.; Sawka-Dobrowol-
ska, W. Eur J Med Chem 2008, 43, 2498.
;
(C¼¼O), 1630 (C¼¼N) cmꢁ1 1H NMR (DMSO-d6): d 0.82 (t,
6H, 2xCH3), 1.20–1.32 (m, 4H, CH2CH2), 1.41–1.50 (m, 4H,
CH2CH2), 2.49–2.57 (m, 4H, N(CH2)2), 4.03 (s, 2H, CH2),
7.66–7.68 (m, 3H, arom.), 8.36 (s, 1H, NH), 8.44–8.47 (m, 2H,
arom.), 8.49 (s, 1H, NH). Anal. Calcd. for C21H27N5O2: C,
66.12; H, 7.13; N, 18.39. Found: C, 66.36; H, 7.06; N, 18.64.
5-Chloromethyl-7-phenyl-6,7-dihydro-isoxazolo[4,5-d]pyrimi-
dine-3-carboxamide (21). Sodium borohydride (2 g) was added
portionwise to a stirred solution of compound 16 (1.44 g,
0.005 mol) in 90 mL of methanol at room temperature under
nitrogen. The reaction mixture was stirred for 12 h, concen-
trated to ca. 30 mL in vacuo, and refrigerated. The solid was
filtered, washed with water, vacuum dried, and recrystallized.
Yield: 1.48 g (51.0%), mp 177ꢂC (methanol); Anal. Calcd. for
C13H11ClN4O2: C, 53.71; H, 3.81; N, 19.27. Found: C, 53.43;
H, 3.82; N, 19.43.
[16] Dave, K. G.; Shishoo, C. J.; Devani, M. B.; Kalyanaraman,
R.; Ananthan, S.; Ulas, G. V.; Bhadti, V. S. J Heterocyclic Chem
1980, 17, 1497.
[17] Shishoo, C. J.; Devani, M. B.; Ananthan, S.; Ulas, G. V.;
Bhadti, V.S. Tetrahedron Lett 1984, 25, 1291.
[18] Perold, G. W.; von Reiche, F. V. K. J Am Chem Soc 1957,
79, 465.
[19] LeBel, N. A.; Whand, J. J. J Am Chem Soc 1959, 81,
6334.
[20] Saxema, R.; Singh, V.; Batra, S. Tetrahedron 2004, 60,
10311.
[21] Taylor, E. C.; Garcia, E. E. J Org Chem 1964, 29, 2116.
[22] Sheldrick, G. M. SHELXS-97 and SHELXL-97: Programs
for the Solution and Refinement of Crystal Structures, University of
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Gottingen, Germany, 1997.
[23] Sheldrick, G. M. SHELXTL-NTV5.1, Brucker-AXS, 1999.
[24] Allen, F. H.; Kennard, O.; Watson, D. G.; Brammer, L.;
Orpen, A. G. J Chem Soc Perkin Trans 2 1987, 1.
Acknowledgment. The authors are grateful to the staff members
of the National Cancer Institute (NCI, Bethesda, MD), for carry-
ing out the anticancer screening of the newly synthesized
compounds.
[25] Wagner, E.; Pore˛ba, K.; Jakowicz, I.; Balicka, D.; Rutkow-
´
ska, M.; Ke˛dzierska-Gozdzik, L.; Szela˛g, A. Il Farmaco 1995, 50, 183.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet