Synthesis, X-ray crystal structure study and antitumoral evaluations of 5,6-disubstituted pyrimidine derivatives

Article abstract of DOI:10.1016/j.bmc.2010.02.023

5,6-Disubstituted pyrimidine derivatives (3-20) were prepared by intramolecular cyclization reaction of α-(1-carbamyliminomethylene)-γ-butyrolactone (2) with sodium ethoxide and subsequent chemical transformation of 2-hydroxy group in C-5 side chain as we

Full text of DOI:10.1016/j.bmc.2010.02.023

Bioorganic & Medicinal Chemistry 18 (2010) 2704–2712
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, X-ray crystal structure study and antitumoral evaluations
of 5,6-disubstituted pyrimidine derivatives
a
a
b
b
c
´
Tatjana Gazivoda Kraljevic , Svjetlana Krištafor , Lidija Šuman , Marijeta Kralj , Simon M. Ametamey ,
Mario Cetina d, , Silvana Raic-Malic
a Department of Organic Chemistry, Faculty of Chemical Engineering and Technology, University of Zagreb, Maruli´cev trg 20, PO Box 177, HR-10000 Zagreb, Croatia
a,
*
*
´
´
b
-
ˇ
Division of Molecular Medicine, Ruder Boškovi´c Institute, Bijenicka 54, HR-10001 Zagreb, Croatia
c Institute for Pharmaceutical Sciences, HCI H427, Wolfgang-Pauli Strasse 10, 8093 Zürich, Switzerland
d
´
Department of Applied Chemistry, Faculty of Textile Technology, University of Zagreb, Prilaz baruna Filipovica 28a, HR-10000 Zagreb, Croatia
a r t i c l e i n f o
a b s t r a c t
Article history:
5,6-Disubstituted pyrimidine derivatives (3–20) were prepared by intramolecular cyclization reaction of
Received 5 October 2009
Revised 5 February 2010
Accepted 12 February 2010
Available online 18 February 2010
a
-(1-carbamyliminomethylene)-c-butyrolactone (2) with sodium ethoxide and subsequent chemical
transformation of 2-hydroxy group in C-5 side chain as well as lithiation reaction for introduction of acy-
clic side chain at C-6. All compounds were characterized by 1H NMR, 13C NMR and mass spectra. Struc-
tures of compounds 4, 7 and 14 were unambiguously confirmed by X-ray crystal structural analysis.
Supramolecular structures of these three compounds differ significantly. Two N–Hꢀ ꢀ ꢀO and one C–
Hꢀ ꢀ ꢀO hydrogen bonds in 4 form three-dimensional network. One O–Hꢀ ꢀ ꢀN hydrogen bond and one
Keywords:
5,6-Disubstituted pyrimidines
Lithiation
Positron-emission tomography (PET)
Cytostatic evaluations
X-ray diffraction
p
p
ꢀ ꢀ ꢀp
p
interaction self-assemble the molecules of 7 into sheets. In supramolecular aggregation of 14, only
stacking interactions participate, so forming chains. The compounds were evaluated for their cyto-
ꢀ ꢀ ꢀ
static activities against human malignant cell lines. Of all tested compounds, 2,4-dimethoxy-5-methoxy-
tritylethylpyrimidine (9) and 2,4-dichloro-5-chloroethylpyrimidine (14) exhibited the most prominent
inhibitory effects. Furthermore, compound 14 showed marked activity against human colon carcinoma
Hydrogen bonds
pꢀ ꢀ ꢀp interactions
(IC50 = 0.4 lM).
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
which can activate prodrugs, such as nucleoside analogs, to form
toxic drugs that may kill the cell.19 For example, 5-(2-fluoroeth-
yl)deoxyuridine, labeled with radioisotopes has an application in
positron emission tomography (PET) to monitor the successful
transfection of gene product in vivo during cancer-prodrug
therapy.20
Recently, we have reported that some C-5 and/or C-6 substi-
tuted pyrimidine derivatives exhibited antiviral and cytostatic
activities.21–24 Syntheses and biological evaluations of C-6 alkyl-
ated pyrimidines as model compounds for development of tra-
cer molecules in PET have been also reported.25,26 Thus, we
found that pyrimidine derivatives containing 2,3-dihydroxypro-
pyl and 1,3-dihydroxyisobutyl side chain can be efficiently
phosphorylated by HSV-1 TK and thus fulfill the necessary
requirements for in vivo imaging of the HSV-1 TK gene
expression.25
Therefore, biological activities by both 5-substituted and 6-
substituted uracil derivatives encouraged us to explore chemistry
and biological activities of novel 5,6-disubstituted uracil deriva-
tives. In this paper we present syntheses, X-ray crystal structural
study and cytostatic evaluations of pyrimidine derivatives (3–20)
bearing substituents at positions C-5 and C-6 of pyrimidine ring
(Figure 1).
Uracil derivatives substituted either at C-5 or C-6 positions, as
well as their nucleosides, have significant status in the field of
chemotherapy. 5-Substituted uracil analogs have been investi-
gated extensively for use in cancer1,2 and viral chemotherapy,3,4
as enzyme inhibitors5–8 and in the synthesis of modified nucleo-
tides.9,10 In addition, pyrimidines substituted at position C-6, as
for example, 1-[(2-hydroxyethyl)-methyl]-6-(phenylthio)thymine
(HEPT)11,12 or 3,4-dihydro-2-alkoxy-6-benzyl-4-oxopyrimidines
(DABOs)13 and their derivatives14–16 exhibited a potent and selec-
tive activity against human immunodeficiency virus type-1 (HIV-
1). Although these compounds are relatively simple from a struc-
tural point of view, they have been the subject of great deal of
chemical work.
‘Suicide’ gene therapy is one of the most promising approaches
in cancer therapy.17 The most intensively studied ‘suicide’ gene is
herpes simplex virus type 1 thymidine kinase (HSV-1 TK),18
* Corresponding authors. Tel.: +385 1 4597 213; fax: +385 1 4597 224 (S.R.-M.);
tel.: +385 1 3712 590; fax: +385 1 3712 599 (M.C.).
E-mail addresses: mario.cetina@ttf.hr (M. Cetina), silvana.raic@fkit.hr
(S. Raic´-Malic´).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.02.023

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T. G. Kraljevic et al. / Bioorg. Med. Chem. 18 (2010) 2704–2712
R4
R4
OCH3
R
Cl
N
N
N
OH
OBn
R2
N
CH3
R2
N
H3CO
N
CH3
18
R2, R4 = OH, OCH3, Cl
R2 = R4 = OH, OCH3
R = OH, OAc, OMtr, F, Cl
19, 20
3-17
Figure 1. The new 5,6-disubstituted pyrimidine derivatives (3–20).
derivative 327,28 (Scheme 1). Primary hydroxyl group in 3 was pro-
tected to give 5-acetoxy-6-methyl substituted uracil derivative (4)
which was subsequently chlorinated using phosphoryl chloride
with N,N-diethylaniline yielding corresponding 2,4-dichloropyrim-
idine derivative 5. Methoxylation of 5 gave a mixture of 2,4-
dimethoxypyrimidine (6) and 2-chloro-4-methoxypyrimidine (7)
containing 2-hydroxyethyl side chain, as well as bicyclic furo[2,3-
d]pyrimidine derivative (8). Protection of hydroxyl group in
2. Results and discussion
2.1. Chemistry
a-(1-Carbamyliminomethylene)-
c
-butyrolactone (2) was pre-
pared by condensation of -acetyl-
in ethanol. Intramolecular cyclization reaction occurred when
compound 2 was treated with sodium ethoxyde to give pyrimidine
a
c-butyrolactone (1) with urea
O
O
O
O
O
OH
H
HN
(i)
(ii)
H2N
N
O
O
N
CH3
O
CH3
H
CH3
1
2
3
(iii)
OCH3
O
Cl
OTrM
OAc
OAc
N
HN
N
(iv)
TrM =
OCH3
CH3
N
5
CH3
Cl
N
H
CH3
O
N
H3CO
9
4
(v)
(vi)
O
OCH3
OCH3
OH
OH
N
N
N
+
+
CH3
N
CH3
H3CO
N
CH3
Cl
N
H3CO
6
7
8
(viii)
(vii)
OCH3
OCH3
OCH3
OCH3
F
OAc
F
OAc
N
N
N
N
+
+
CH3
N
Cl
CH3
CH3
N
H3CO
CH3
N
H3CO
N
Cl
10
11
12
13
Scheme 1. Reagents and conditions: (i) urea, HCl, EtOH; (ii) NaOEt/EtOH; (iii) acetic anhydride, pyridine; (iv) POCl3, N,N-diethylaniline, pyridine; (v) NaOMe/MeOH; (vi) 4-
methoxytriphenylmethyl chloride, triethylamine, N,N-dimethylaminopyridine, DMF; (vii) DAST, CH2Cl2; (viii) acetic anhydride, pyridine.

´
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2706
compound 6 was performed with (p-methoxytriphenyl)methyl
chloride in DMF in the presence of triethylamine and N,N-dimeth-
ylaminopyridine to give p-methoxytritylated derivative 9. Trans-
formation of hydroxyl group into fluorine was achieved with
diethylaminosulfur trifluoride (DAST) as reagent. Thus, when a
mixture of 5-(2-hydroxyethyl) substituted pyrimidine derivatives
6 and 7 reacted with DAST corresponding 5-(2-fluoroethyl) substi-
tuted pyrimidines (10 and 11) were obtained. Reaction mixture of
6 and 7 in acetic anhydride and dry pyridine gave acetylated 12
and 13, respectively (Scheme 1).
The introduction of acyclic side chain at position C-6 in pyrim-
idine derivatives 9, 10 and 12 by lithiation using lithium diisopro-
pylamide (LDA) followed by addition of benzyloxyacetaldehyde to
the lithiated intermediate failed. When compounds 10 and 12 were
submitted to C-6 condensation defluorination and deacetylation
occurred and 2,4-dimethoxy-5-(2-hydroxyethyl)-6-methylpyrimi-
dine (6) was isolated as the product. On the other hand, in the
same reaction of p-methoxytritylated derivative (9) starting com-
pound 9 was isolated. This may be caused by spatial hindrance
due to bulky trityl moiety in 9.
magnitude and multiplicity of H–H spin–spin coupling, as well as
connectivities in two-dimensional homo and heteronuclear corre-
lation spectra.
2.2. X-ray crystal structure analysis
The molecular structures of 7 and 14 are shown in Figs. 2 and 3,
respectively. Two 2-chloro-6-methylpyrimidines differ in substitu-
ent at C-5 of pyrimidine ring, which is 2-hydroxyethyl group in 7
and 2-chloroethyl group in 14. The geometrical parameters of
pyrimidine ring in these two structures are very similar, and in ac-
cord with those in closely related 2-chloro-6-methylpyrimidines.
The exception is N3–C4 bond in benzyl-(2-chloro-6-methylpyrim-
idin-4-yl)amine hemihydrate,29 which is slightly longer (ca. 0.03 Å)
due to the influence of the benzylamine group on the pyrimidine
ring geometry.
The conformation of the alkyl chain in 7 and 14 is locked by
intramolecular hydrogen bonds, C7ꢀ ꢀ ꢀO1 in 7 and C7ꢀ ꢀ ꢀCl2 in 14
(Table 1; Figs. 2 and 3). Both intramolecular hydrogen bonds form
five-membered rings which can be described by graph-set notation
as S(5).30
Chlorination reaction of 5-(2-hydroxyethyl)-6-methylpyrimi-
dine (3) proceeded smoothly and the desired trichlorinated
derivative 14 was obtained (Scheme 2). In this reaction monochlo-
rinated (15) and dichlorinated (16) pyrimidines were also isolated.
Subsequent methoxylation of 14 using sodium methoxide yielded
2,4-dimethoxypyrimidine derivative 17 with chloroethyl substitu-
ent at C-5 of pyrimidine ring and its 5-vinyl analog 18 as a product
of dehydrohalogenation. Lithiation of compound 17 with LDA
afforded organometallic intermediate that reacted in situ with
benzyloxyacetaldehyde to give 19 bearing the C-6 3-benzyloxy-
2-hydroxypropyl side chain. Removal of 2,4-dimethoxy protecting
groups with sodium iodide and trimethylchlorosilane (TMSCl)
yielded corresponding pyrimidin-2,4-dione derivative 20.
The hydroxyl hydrogen atom of 2-hydroxyethyl group in 7 par-
ticipates in supramolecular aggregation via O2ꢀ ꢀ ꢀN1 hydrogen
bond. This intermolecular interaction forms C(7) chains30 parallel
to the c axis (Table 1; Fig. 4). Hydrogen-bonded chains are weakly
linked by one
distance between the ring centroids of the coplanar pyrimidine
rings [ = 0°] is 3.5649(7) Å, the planes are separated by ca.
pꢀ ꢀ ꢀ
p
stacking interaction into (0 1 0) sheets.31 The
a
3.52 Å, and centroids offset amounts ca. 0.57 Å.
The molecules of 14, arranged in herringbone fashion (Fig. 5), are
self-assembled by two weak aromatic
p p stacking interactions
ꢀ ꢀ ꢀ
between the pyrimidine rings of the neighboring molecules. An
interplanar angle between the rings is 0.77°, interplanar spacing is
ca. 3.54 Å, a centroid separations are 3.9066(16) and 3.9067(16) Å,
Newly prepared compounds 3–20 were characterized by 1H
NMR, 13C NMR and mass spectra. The assignment of 1H and 13C
NMR spectra was performed on the basis of the chemical shift,
and corresponding centroid-centroid offset ca. 1.66 Å. These
interactions link the molecules parallel to the a axis into chains.
p p
ꢀ ꢀ ꢀ
O
O
Cl
Cl
OH
Cl
Cl
OH
(i)
HN
HN
N
N
+
+
O
N
H
CH3
O
N
H
CH3
CH3
CH3
N
N
Cl
Cl
14
(ii)
15
16
3
OCH3
OCH3
Cl
N
N
+
CH3
CH3
N
N
H3CO
H3CO
17
18
(iii)
O
OCH3
Cl
Cl
HN
(iv)
N
OH
OH
OBn
OBn
N
O
N
H3CO
H
19
20
Scheme 2. Reagents and conditions: (i) POCl3, N,N-diethylaniline, pyridine; (ii) NaOCH3/MeOH; (iii) LDA, benzyloxyacetaldehyde; (iv) trimethylchlorosilane, NaI, acetonitrile.

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Figure 2. A molecular structure of 7, with the atom-numbering scheme. Displace-
ment ellipsoids for non-hydrogen atoms are drawn at the 30% probability level.
Intramolecular hydrogen bond is shown dashed.
Figure 4. A crystal packing diagram of 7, viewed along the a axis, showing O2ꢀ ꢀ ꢀN1
hydrogen bond that link the molecules parallel to the c axis and stacking of the
pyrimidine rings. Hydrogen bonds are indicated by dashed lines.
Figure 3. A molecular structure of 14, with the atom-numbering scheme.
Displacement ellipsoids for non-hydrogen atoms are drawn at the 30% probability
level. Intramolecular hydrogen bond is shown dashed.
In compound 4 (Fig. 6), 2-acetoxyethyl chain is bonded to the
pyrimidine ring C-5 atom. The geometry of pyrimidine-2,4-dione
is in a good agreement with similar structures in which alkyl chain
is bonded to C-5 atom of the pyrimidine ring.32,33 The planes of the
C8/O3/C10/O4/C11 atoms and pyrimidine rings’ atoms are almost
coplanar to each other. Their mean planes form an angle of
5.28(11)°.
The N1ꢀ ꢀ ꢀO1 hydrogen bond in 4 generates C(4) chains (Table 1;
Fig. 7), while N3ꢀ ꢀ ꢀO3 hydrogen bond forms R22ð8Þ centrosymmetric
dimers.30 These two N–Hꢀ ꢀ ꢀO hydrogen bonds are reinforced by
Figure 5. A crystal packing diagram of 14, viewed along the c axis, showing the
molecules arranged in herringbone fashion.
Table 1
Hydrogen-bonding geometry for compounds 4, 7 and 14
Dꢀ ꢀ ꢀA
D–H (Å)
Hꢀ ꢀ ꢀA (Å)
Dꢀ ꢀ ꢀA (Å)
D–Hꢀ ꢀ ꢀA (°)
Symmetry codes
4
N1ꢀ ꢀ ꢀO1
N3ꢀ ꢀ ꢀO2
C9ꢀ ꢀ ꢀO4
C7ꢀ ꢀ ꢀO1
O2ꢀ ꢀ ꢀN1
C7ꢀ ꢀ ꢀCl2
0.87(2)
0.89(2)
0.96
0.97
0.80(2)
0.97
1.99(2)
1.97(2)
2.41
2.43
2.15(2)
2.72
2.846(2)
2.858(2)
3.252(3)
2.7832(15)
2.9314(15)
3.076(3)
170(2)
175(2)
147
101
166(2)
103
ꢁx, ꢁ1/2 + y, 1/2 ꢁ z
ꢁx, 1ꢁy, ꢁz
1ꢁx, ½ + y, 1/2 ꢁ z
7
x, y, 1 + z
14

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Table 2
Inhibitory effects of compounds 3–15, 17, 19 and 20 on the growth of human
malignant tumor cell lines
a
Compd
Molt-4
IC50 (lM)
HCT 116
SW 620
MCF-7
H 460
3
4
5
6
7
8
9
>100
>100
>100
>100
N.T.
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
50 34
>100
>100
47 15
>100
N.T.
5
4
4
0.03
20
3
6
3
10
11
12
13
14
15
17
19
20
5-FUb
Ara-Cb
N.T.
N.T.
N.T.
>100
>100
>100
40 10
>100
>100
>100
>100
>100
65 19
>100
44
2
>100
0.4 0.2
>100
>100
5
3
2
0.2
8
7
3
0.1
Figure 6. A molecular structure of 4, with the atom-numbering scheme. Displace-
ment ellipsoids for non-hydrogen atoms are drawn at the 30% probability level.
>100
>100
19 11
>100
0.03 0.01
>100
>100
>100
>100
26 12
>100
>100
>100
76 26
>100
16
1
20
4
>100
>100
4
0.9
9
2
15
2
2
0.6
one C–Hꢀ ꢀ ꢀO hydrogen bond, C9ꢀ ꢀ ꢀO4, which link the molecules
into C(9) chains.
1
0.2
0.1 0.1
30 23
P100
0.1 0.02
a
IC50—the concentration that causes 50% growth inhibition.
5-FU—5-fluorouracil, Ara-C—cytosine arabinoside.
b
2.3. Antitumoral activities
The compounds 4–15, 17, 19 and 20 were evaluated for their
cytostatic activities against human malignant tumor cell lines:
acute lymphoblastic leukemia (Molt-4), colon carcinoma (HCT
116 and SW 620), breast carcinoma (MCF-7) and lung carcinoma
(H 460). Their activities were compared with those of 5-fluoroura-
cil (5-FU) and cytosine arabinoside (Ara-C) (Table 2).
Of all compounds evaluated, 2,4-dimethoxy-5-methoxytrityl-
ethylpyrimidine (9) and 2,4-dichloro-5-chloroethylpyrimidine
(14) derivatives exhibited the most pronounced inhibitory effect
ritylated and acetylated derivatives. Thus, 2,4-dimethoxy-5-meth-
oxytritylethylpyrimidine (9) showed the strongest inhibitory
effect against human colon carcinoma (HCT 116: IC50 = 4
lM) and
acute lymphoblastic leukemia (Molt-4: IC50 = 5 M), while 5-acet-
l
oxy-2,4-dimethoxypyrimidine (12) displayed moderate cytostatic
activity against all tested cell lines. However, pyrimidin-2,4-dione
derivatives (3, 4, 15 and 20) did not exhibit any inhibitory effects
(IC50 >100 lM). The presence and identity of halogen substituents
have significant impact on biological activity. Thus, the formation
of halogen bonds in molecules is recognized as a kind of intermolec-
ular interaction that favorably contributes to the stability of ligand-
target complexes.34 Interestingly, increase in the chlorine content
enhanced the antiproliferative effect. Therefore, compound 14 con-
taining both aliphatic and aromatic chlorine atoms possessed the
most significant inhibitory effect. This compound inhibited colon
(IC50 in the range 0.4–50 lM). 5-Acetoxy-2,4-dimethoxypyrimidine
(12) and 5-chloroethyl-2,4-dimethoxypyrimidine with 6-(3-
benzyloxy-2-hydroxypropyl) side chain (19) showed moderate
cytostatic activities (12: IC50 ꢂ49
l
M, 19: IC50 ꢂ31
lM). It is
interesting to note that methoxytritylation and acetylation of hy-
droxyl group in 6 caused a significant increase of antitumor activity
of 9 and 12. On the contrary to this, their structural congener 6 with
unprotected 5-hydroxyethyl substituent showed no inhibitory
effect. This might be due to the greater lipophilicity of the methoxyt-
carcinoma SW 620 in submicromolar range (IC50 = 0.4
lM).
Influence of bromine in 2,4-dimethoxypyrimidine derivatives on
Figure 7. Part of the crystal structure of 4, showing the N–Hꢀ ꢀ ꢀO and C–Hꢀ ꢀ ꢀO hydrogen bonds. Hydrogen bonds are indicated by dashed lines.

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T. G. Kraljevic et al. / Bioorg. Med. Chem. 18 (2010) 2704–2712
antitumoral activity was determined in our previous paper.22 Thus,
pyrimidinederivative thatbears one aromaticand two aliphaticbro-
mine atoms showed the highest inhibitory activity against all tested
malignant tumor cell lines.
Comparison of cytostatic activities of novel compounds with
those of 5-FU and Ara-C revealed that 14 inhibited more pro-
nounced the growth of HCT 116 and MCF-7 cell lines than 5-FU.
Furthermore, inhibitory effect of this compound was approxi-
mately 20-fold higher against SW 620 cell line than that of 5-FU.
Besides, compounds 9, 14 and 19 expressed better antiproliferative
effects on SW 620 and MCF-7 cells than Ara-C.
(5.1 g, 0.03 mol). The reaction mixture was refluxed for 18 h and
water (15 mL) was added to the cooled reaction mixture. pH was
adjusted to ꢂ3 by adding few drops of 98% sulfuric acid. The solu-
tion was kept in refrigerator overnight, white crystals of compound
3 were filtered off and washed with anhydrous ether (5.1 g, 100%).
Mp = 219–221 °C. 1H NMR: d 10.88 (1H, s, NH), 10.60 (1H, s, NH),
4.53 (1H, s, OH), 3.36 (2H, t, J = 6.73 Hz, H-20), 2.37 (2H, t,
J = 6.87 Hz, H-10), 2.06 (3H, s, CH3) ppm. 13C NMR: d 164.94 (C-4),
151.30 (C-2), 149.37 (C-6), 106.37 (C-5), 60.15 (C-20), 28.47 (C-
10), 16.69 (CH3) ppm.
MS m/z 171 [MH]+. Anal. Calcd for [C7H10N2O3]: C, 49.41; H,
5.92; N, 16.46. Found: C, 49.50; H, 5.90; N, 16.43.
3. Conclusions
4.1.1.3. 5-(2-Acetoxyethyl)-6-methylpyrimidin-2,4-dione
(4). Reaction mixture of compound 3 (200 mg, 1.2 mmol) and ace-
tic anhydride (0.9 mL, 9.8 mmol) in anhydrous pyridine (5.5 mL)
was stirred for 1 h under argon atmosphere. Water was then added
and mixture was evaporated to dryness. Crude product was puri-
fied by column chromatography (CH2Cl2/CH3OH = 20:1) to give
Novel 5,6-disubstituted pyrimidine derivatives (3–20) were
synthesized and characterized. The structures of compounds 4, 7
and 14 were determined by X-ray crystal structural analysis. These
three compounds built completely different supramolecular archi-
tectures.
aggregation of 14, so forming chains. One O–Hꢀ ꢀ ꢀN hydrogen bond
and one interaction self-assemble the molecules of 7 into
pꢀ ꢀ ꢀ
p
stacking interactions participate in supramolecular
colorless crystals of compound
4 (230 mg, 90%). Mp = 190–
192 °C. 1H NMR: d 10.88 (1H, s, NH), 10.61 (1H, s, NH), 4.00 (2H,
t, J = 6.83 Hz, H-20), 2.53 (2H, t, J = 6.90 Hz, H-10), 2.08 (3H, s,
COCH3), 1.98 (3H, s, CH3) ppm. 13C NMR: d 170.72 (C@O), 164.72
(C-4), 151.17 (C-2), 149.85 (C-6), 105.17 (C-5), 62.75 (C-20), 24.31
(C-10), 21.13 (COCH3), 16.45 (CH3) ppm.
pꢀ ꢀ ꢀ
p
sheets, while two N–Hꢀ ꢀ ꢀO and one C–Hꢀ ꢀ ꢀO hydrogen bonds in 4
form three-dimensional network.
Compounds were evaluated for their cytostatic activities against
human malignant cell lines. By comparison of cytostatic activities of
novel 5,6-disubstituted pyrimidine derivatives and 5-FU and Ara-C,
we can infer that compound 14 exhibited higher inhibitory effects
against SW 620, HCT 116 and MCF-7 cells than 5-FU, while com-
pounds 9, 14 and 19 revealed better antiproliferative effects against
SW 620 and MCF-7 cells than Ara-C. Finally, compounds 9 and 14
emerged as the most interesting compounds with cytostatic activi-
ties that could be used for further structural optimization.
MS m/z 213 [MH]+. Anal. Calcd for [C9H12N2O4]: C, 50.94; H,
5.70; N, 13.20. Found: C, 50.89; H, 5.71; N, 13.25.
4.1.1.4. 5-(2-Acetoxyethyl)-2,4-dichloro-6-methylpyrimidine
(5). A mixture of compound 4 (1.3 g, 6.13 mmol), POCl3 (12.1 mL),
N,N-diethylaniline (1.2 mL) and pyridine was refluxed for 1 h. The
solvent was evaporated to dryness, and the residue was diluted
with iced water, washed with dichloromethane and dried over
Na2SO4. The oily product was purified by column chromatography
(CH2Cl2/CH3OH = 50:1) to afford compound 5 as brown oil (1.16 g,
4. Experimental
1
76%). H NMR: d 4.23 (2H, t, J = 6.66 Hz, H-20), 2.53 (2H, t,
J = 6.66 Hz, H-10), 2.58 (3H, s, COCH3), 1.98 (3H, s, CH3) ppm. 13C
NMR: d 172.56 (C@O), 170.70 (C-4), 161.95 (C-2), 156.58 (C-6),
128.06 (C-5), 61.47 (C-20), 28.17 (C-10), 22. 85 (COCH3), 21.06
(CH3) ppm.
4.1. General methods
Melting points (uncorrected) were determined with Kofler mi-
cro hot-stage (Reichert, Wien). Precoated Merck Silica Gel 60F-
254 plates were used for thin layer chromatography (TLC) and
the spots were detected under UV light (254 nm). Column chroma-
tography (CLC) was performed using silica gel (0.063–0.2 mm) Flu-
ka; glass column was slurry-packed under gravity. 1H and 13C NMR
spectra were acquired on Bruker 300 MHz NMR spectrometer. All
data were recorded in DMSO-d6 at 298 K. Chemical shifts were ref-
erenced to the residual solvent signal of DMSO at d 2.50 ppm for 1H
and d 39.50 ppm for 13C. Individual resonances were assigned on
the basis of their chemical shifts, signal intensities, multiplicity
of resonances and H–H coupling constants.
MS m/z 248 250 252 [M+, M+2, M+4]+. Anal. Calcd for
[C9H10N2O2 Cl2]: C, 43.40; H, 4.05; N, 11.25. Found: C, 43.36; H,
4.06; N, 11.22.
4.1.1.5. 5-(2-Hydroxyethyl)-2,4-dimethoxy-6-methylpyrimidine
(6), 2-chloro-5-(2-hydroxyethyl)-4-methoxy-6-methylpyrimi-
dine (7) and 2-methoxy-4-methyl-5,6-dihydrofuro[2,3-d]pyrim-
idine (8). To a solution of sodium (40 mg, 1.74 mmol) in absolute
methanol (30 mL) compound 5 (200 mg, 0.8 mmol) was added and
reaction mixture was refluxed for 3 h. The solvent was removed in
vacuo and crude product was chromatographed on silica column
(CH2Cl2/CH3OH = 60:1) to afford oily compound 6 (53.8 mg, 34%),
4.1.1. Procedures for the preparation of compounds
4.1.1.1.
a
-(1-Carbamyliminomethylmethylene)-
c-butyrolac-
-acetyl- -butyrolactone (1) (60 g,
7
(42.6 mg, 26%, mp = 101–103 °C) and bicyclic derivative 8
tone (2). To a solution of
a
c
(28.3 mg, 21%, mp = 105–107 °C) as white solids.
Compound 6: 1H NMR: d 4.64 (1H, t, J = 5.58 Hz, OH), 3.87 (3H,
s, OCH3), 3.83 (3H, s, OCH3), 3.44 (2H, q, J = 6.60 Hz, H-20), 2.63 (2H,
t, J = 7.14 Hz, H-10), 2.34 (3H, s, CH3) ppm. 13C NMR: d 169.48 (C-4),
162.79 (C-6), 158.14 (C-2), 109.56 (C-5), 60.00 (C-20), 55.20 (OCH3),
55.07 (OCH3), 28.64 (C-10), 21.83 (CH3) ppm.
0.46 mol) and urea (32 g, 0.54 mol) in ethanol (10 mL) 4–5 drops of
36% hydrochloric acid were added and the reaction mixture was
heated at 70 °C for 20 h. The precipitate was collected by filtration
to yield compound 2 as the white powder (51 g, 65%). Mp = 170–
172 °C. 1H NMR: d 9.78 (1H, s, NH), 6.75 (2H, s, NH2), 4.28 (2H, t,
J = 7.72 Hz, OCH2), 2.83 (2H, t, J = 7.67, CH2), 2.27 (3H, s, CH3) ppm.
13C NMR: d 172.63 (C@O lactone), 160.26 (C@O), 150.62 (C-20),
97.09 (C-2), 65.66 (C-4), 25.72 (C-3), 19.08 (C-30) ppm.
MS m/z 199 [MH]+. Anal. Calcd for [C9H14N2O3]: C, 54.53; H,
7.12; N, 14.13. Found: C, 54.42; H, 7.12; N, 14.14.
Compound 7: 1H NMR: d 4.71 (1H, t, J = 5.61 Hz, OH), 3.92 (3H,
s, OCH3), 3.71 (2H, q, J = 6.64 Hz, H-20), 2.69 (2H, t, J = 6.77 Hz, H-
10), 2.41 (3H, s, CH3) ppm. 13C NMR: d 168.66 (C-4), 168.05 (C-6),
155.33 (C-2), 115.64 (C-5), 59.03 (C-20), 54.74 (OCH3), 28.26 (C-
10), 21.18 (CH3) ppm.
4.1.1.2. 5-(2-Hydroxyethyl)-6-methylpyrimidin-2,4-dione
(3). To absolute ethanol (30 mL) sodium (0.87 g, 0.038 mol) was
added in small portions followed by addition of compound 2

´
T. G. Kraljevic et al. / Bioorg. Med. Chem. 18 (2010) 2704–2712
2710
MS m/z 202 204 [M+, M+2]. Anal. Calcd for [C8H11N2O2Cl]: C,
temperature for 1 h. After addition of water solvents were removed
under reduced pressure and crude product was purified by column
chromatography (CH2Cl2/CH3OH = 60:1) to afford oily products 12
(268 mg, 81%) and 13 (27 mg, 93%).
47.42; H, 5.47; N, 13.82. Found: C, 47.37; H, 5.46; N, 13.83.
Compound 8: 1H NMR: d 3.81 (3H, s, OCH3), 4.65 (2H, t,
J = 8.58 Hz, H-20), 2.80 (2H, t, J = 6.87 Hz, H-10), 2.35 (3H, s, CH3)
ppm. 13C NMR: d 166.44 (C-4), 168.34 (C-6), 159.38 (C-2), 108.26
(C-5), 66.20 (C-20), 54.32 (OCH3), 44.82 (C-10), 21.97 (CH3) ppm.
MS m/z 167 [MH]+. Anal. Calcd for [C8H11N2O2Cl]: C, 57.82; H,
6.07; N, 16.86. Found: C, 57.94; H, 6.07; N, 16.88.
1
Compound 12: H NMR: d 4.09 (2H, t, J = 6.84 Hz, H-20), 3.89
(3H, s, OCH3), 3.85 (3H, s, OCH3), 2.80 (2H, t, J = 6.87 Hz, H-10),
2.35 (3H, s, CH3OAc), 1.97 (3H, s, CH3), ppm. 13C NMR: d 171.12
(C@OAc), 160.63 (C-4), 165.23 (C-6), 164.91(C-2), 110.30 (C-5),
60.84 (C-20), 56.16 (OCH3), 55.84 (OCH3), 28.22 (CH3OAc), 27.73
(C-10), 20.45 (CH3) ppm.
4.1.1.6. 2,4-Dimethoxy-5-[2-(4-methoxytriphenylmeth-
oxy)ethyl]-6-methylpyrimidine (9). The reaction mixture of
MS m/z 241 [MH]+. Anal. Calcd for [C11H16N2O4]: C, 54.99; H,
6.71; N, 11.66. Found: C, 55.15; H, 6.70; N, 11.68.
compound
6 (565 mg, 2.85 mmol), 4-methoxytriphenylmethyl
chloride (2.19 g, 7.2 mmol), triethylamine (2.36 mL) and N,N-
dimethylaminopyridine (40 mg, 0.33 mmol) in anhydrous DMF
(5 mL) was stirred under argon atmosphere at 50 °C for 2 h. Sol-
vents were evaporated and residual oil was purified by column
chromatography (CH2Cl2/CH3OH = 50:1) yielding oily compound
9 (515.4 mg, 39%). 1H NMR: d 7.26–7.27 (10H, m, Ph), 7.12 (2H,
d, J = 8.82 Hz, Ph), 6.84 (2H, d, J = 8.85 Hz, Ph), 3.84 (3H, s, OCH3),
3.80 (3H, s, OCH3), 3.73 (3H, s, OCH3), 3.12 (2H, t, J = 6.36 Hz, H-
20), 2.76 (2H, t, J = 6.24 Hz, H-10), 2.30 (3H, s, CH3) ppm. 13C NMR:
d 169.50 (C-4), 166.82 (C-6), 162.93 (C-2), 158.56 (Cquat-OCH3),
144.85 (Ph-quat), 135.60 (Cquat-Tr), 130.28 (CH-Ph), 128.22–
128.26 (CH-Ph), 127. 25 (CH-Ph), 113.51 (CH-Ph-OCH3 Tr),
109.44 (C-5), 86.30 (C-quat 300), 61.99 (C-20), 55.48 (OCH3), 54.52
(OCH3), 54.18 (OCH3), 25.47 (C-10), 21.80 (CH3) ppm.
1H NMR: d 4.14 (2H, t, J = 6.63 Hz, H-20), 3.94 (3H, s, OCH3), 2.86
(2H, t, J = 6.66 Hz, H-10), 2.42 (3H, s, CH3), 1.97 (3H, s, CH3OAc)
ppm. 13C NMR: d 170.34 (C@OAc), 162.55 (C-4), 163.12 (C-6),
158.36 (C-2), 112.17 (C-5), 59.78 (C-20), 58.33 (OCH3), 30.49
(CH3OAc), 26.44 (C-10), 20.41 (CH3) ppm
MS m/z 204 206 [M+, M+2]. Anal. Calcd for [C10H13N2O3Cl]: C,
49.09; H, 5.36; N, 11.45. Found: C, 48.89; H, 5.34; N, 11.47.
4.1.1.9. 2,4-Dichloro-5-(2-chloroethyl)-6-methylpyrimidine
(14), 5-(2-chloroethyl)-6-methylpyrimidin-2,4-dione (15) and
2,4-dichloro-5-(2-hydroxyethyl)-6-methylpyrimidine (16). The
reaction mixture of compound
3 (1.155 g, 6.8 mmol), POCl3
(13.45 mL) and N,N-diethylaniline (1.35 mL) was refluxed for 4 h.
An excess of POCl3 was removed under reduced pressure. The res-
idue was poured onto ice, washed with dichloromethane and dried
over Na2SO4. The crude product was purified by column chroma-
tography to afford compound 14 (0.893 g, 59%, mp = 207–210 °C)
as yellow solid, and oily compounds 15 (6 mg, 0.5%) and 16
(48 mg, 4%).
MS m/z 471 [MH]+. Anal. Calcd for [C29H30N2O4]: C, 74.02; H,
6.43; N, 5.95. Found: C, 73.87; H, 6.45; N, 5.94.
4.1.1.7. 5-(2-Fluoroethyl)-2,4-dimethoxy-6-methylpyrimidine
(10) and 2-chloro-5-(2-fluoroethyl)-4-methoxy-6-methylpyrim-
1
idine (11). The solution of compounds
6
and
7
(200 mg,
Compound 14: H NMR: d 3.85 (2H, t, J = 7.21 Hz, H-20), 3.23
1.03 mmol) in dry CH2Cl2 (6 mL) was cooled to -78 °C and stirred
for 15 min under argon atmosphere. Then, DAST (0.2 mL,
1.45 mmol) was added dropwise and reaction mixture was stirred
at ꢁ78 °C for additional 15 min after which cooling bath was re-
moved. After 3 h of stirring at room temperature, saturated aque-
ous solution of NaHCO3 (5 mL) was added and reaction was
partitioned. Organic layer was separated, dried over MgSO4 and
evaporated to dryness. Raw product was purified by column chro-
matography (CH2Cl2/CH3OH = 30:1) to afford compounds 10
(108.3 mg, 59%) and 11 (9.3 mg, 41%) as pale yellow oils.
Compound 10: 1H NMR: d 4.57 (1H, t, JH–F = 6.44 Hz, H-20), 4.41
(1H, t, JH–F = 6.41 Hz, H-20), 3.89 (3H, s, OCH3), 3.84 (3H, s, OCH3),
2.92 (1H, t, JH–F = 6.45 Hz, H-10), 2.85 (1H, t, JH–F = 6.42 Hz, H-10),
2.34 (3H, s, CH3) ppm. 13C NMR: d 169.55 (C-4), 167.47 (C-6),
163.19 (C-2), 107.53 (d, JC–F = 6.92 Hz, C-5), 82.46 (d, JC–
F = 165.86 Hz, C-20), 54.55 (OCH3), 54.37 (OCH3), 26.08 (d, JC–
F = 21.16 Hz, C-10), 21.75 (d, JC–F = 1.32 Hz, CH3) ppm.
(2H, t, J = 7.23 Hz, H-10), 2.60 (3H, s, CH3) ppm. 13C NMR: d
172.64 (C-4), 162.03 (C-2), 156.79 (C-6), 128.09 (C-5), 42.17 (C-
20), 31.46 (C-10), 23.02 (CH3) ppm.
MS m/z 224 226 228 230 [M+, M+2, M+4, M+6]. Anal. Calcd for
[C7H7N2Cl3]: C, 37.28; H, 3.13; N, 12.42. Found: C, 37.39; H, 3.13; N,
12.37.
Compound 15: 1H NMR: d 11.03 (1H, s, NH), 10.75 (1H, s, NH),
3.65 (2H, m, H-20), 2.67 (2H, t, J = 4.11 Hz, H-10), 2.10 (3H, s, CH3)
ppm. 13C NMR: d 164.65 (C-4), 151.17 (C-2), 150.49 (C-6), 105.55
(C-5), 43.76 (C-20), 28.28 (C-10), 16.67 (CH3) ppm.
MS m/z 188 190 [M+, M+2]. Anal. Calcd for [C7H9N2O2Cl]: C,
44.58; H, 4.81; N, 14.85. Found: C, 44.71; H, 4.82; N, 14.84.
Compound 16: 1H NMR: d 4.74 (1H, t, J = 8.61 Hz, OH), 3.61 (2H,
t, J = 6.90 Hz, H-20), 2.76 (2H, t, J = 7.29 Hz, H-10), 2.34 (3H, s, CH3)
ppm. 13C NMR: d 170.58 (C-4), 161.84 (C-2), 155.47 (C-6), 116.23
(C-5), 54.20 (C-20), 27.51 (C-10), 22.99 (CH3) ppm.
MS m/z 206 208 210 [M+, M+2, M+4]. Anal. Calcd for
[C7H8N2OCl2]: C, 40.60; H, 3.89; N, 13.53. Found: C, 40.48; H,
3.89; N, 13.56.
MS m/z 201 [MH]+. Anal. Calcd for [C9H13N2O2F]: C, 53.99; H,
6.54; N, 13.99. Found: C, 54.04; H, 6.55; N, 14.02.
Compound 11: 1H NMR: d 4.64 (1H, t, JH–F = 6.32 Hz, H-20), 4.48
(1H, t, JH–F = 6.33 Hz, H-20), 3.94 (3H, s, OCH3), 3.00 (1H, t, JH–
F = 6.32 Hz, H-10), 2.92 (1H, t, JH–F = 6.32 Hz, H-10), 2.41 (3H, s,
CH3) ppm. 13C NMR: d 169.23 (C-4), 168.83 (C-6), 156.50 (C-2),
114.17 (d, JC–F = 5.98 Hz, C-5), 82.08 (d, JC–F = 165.75, C-20), 55.43
(OCH3), 26.26 (d, JC–F = 21.22 Hz, C-10), 21.56 (d, JC–F = 0.91 Hz,
CH3) ppm.
4.1.1.10. 5-(2-Chloroethyl)-2,4-dimethoxy-6-methylpyrimidine
(17) and 2,4-dimethoxy-6-methyl-5-vinyl-pyrimidine (18). To a
solution of sodium (45 mg, 1.95 mmol) in anhydrous MeOH (3 mL)
compound 14 (190 mg, 0.84 mmol) was added and the mixture
was heated at reflux for 3 h. After evaporation of solvents, water
was added and the oily layer was extracted with CH2Cl2 and dried
over Na2SO4. Filtered solution was concentrated, the residue was
kept in refrigerator overnight and crystals of compound 17 were
filtered off (81 mg, 45%, mp = 40–42 °C). Mother liquor was evapo-
rated to dryness and the residue was purified on silica gel column
(CH2Cl2/CH3OH = 60:1) to afford compound 18 as colorless oil
(56 mg, 37%).
MS m/z 204 206 [M+, M+2]+. Anal. Calcd for [C8H10N2OFCl]: C,
46.96; H, 4.93; N, 13.69. Found: C, 46.91; H, 4.93; N, 13.72.
4.1.1.8. 5-(2-Acetoxyethyl)-2,4-dimethoxy-6-methylpyrimidine
(12) and 5-(2-acetoxyethyl)-2-chloro-4-methoxy-6-methylpyr-
imidine (13). The compounds 6 and 7 (300 mg, 1.52 mmol) were
dissolved in anhydrous pyridine (7 mL) and acetic anhydride
(1.18 mL) was added. The reaction mixture was stirred at room
Compound 17: 1H NMR: d 3.93 (3H, s, OCH3), 3.88 (3H, s, OCH3),
3.70 (2H, t, J = 7.27 Hz, H-20), 2.95 (2H, t, J = 7.27 Hz, H-10), 2.41 (3H,

´
2711
T. G. Kraljevic et al. / Bioorg. Med. Chem. 18 (2010) 2704–2712
s, CH3) ppm. 13C NMR: d 169.53 (C-4), 167.39 (C-6), 163.21 (C-2),
110.26 (C-5), 54.73 (OCH3), 54.63 (OCH3), 43.46 (C-20), 28.26 (C-
10), 22.91 (CH3) ppm.
66.16 (CH2O), 54.63 (OCH3), 54.44 (OCH3), 43.47 (C-20), 33.61
(Pyr-CH2), 23.75 (C-10) ppm.
MS m/z 366 368 [M+, M+2]. Anal. Calcd for [C18H23N2O4Cl]: C,
58.93; H, 6.32; N, 7.64. Found: C, 59.05; H, 6.32; N, 7.65.
MS m/z 216 218 [M+, M+2]. Anal. Calcd for [C9H13N2O2Cl]: C,
49.89; H, 6.05; N, 12.93. Found: C, 50.04; H, 6.06; N, 12.92.
Compound 18: 1H NMR: d 6.64 (1H, dd, J = 17.76, 11.85 Hz, CH-
10), 5.74 (1H, dd, J = 2.07, 17.76 Hz, CH-20), 5.45 (1H, dd, J = 2.06,
11.84 Hz, CH-20), 3.90 (3H, s, OCH3), 3.85 (3H, s, OCH3), 2.37 (3H,
s, CH3) ppm. 13C NMR: d 168.83 (C4), 166.53 (C-6), 162.61 (C-2),
127.95 (C10), 119.71 (C20), 108.74 (C-5), 54.69 (OCH3), 54.44
(OCH3), 21.88 (CH3) ppm.
4.1.1.12. 6-(3-Benzyloxy-2-hydroxypropyl)-5-(2-chloro-
ethyl)pyrimidin-2,4-dione (20). A mixture of compound 19
(120 mg, 0.33 mmol), chlorotrimethylsilane (0.16 mL, 1.2 mmol)
and NaI (180 mg, 1.2 mmol) in dry acetonitrile (6 mL) was stirred
at room temperature under argon atmosphere for 18 h and then
at 60 °C for 3 h. The solvent was evaporated under reduced pres-
sure, and the residue was purified by column chromatography
(CH2Cl2/CH3OH = 15:1) to give compound 20 (40.5 mg, 36%) as
white solid. Mp = 101–103 °C. 1H NMR: d 11.03 (1H, s, NH), 10.76
(1H, s, NH), 7.28–7.37 (5H, m, Ph), 5.76 (1H, s, OH), 4.48 (2H, m,
CH2Ph), 3.61 (2H, t, J = 7.25 Hz, CH2Cl), 3.42–3.47 (2H, m, CH2),
2.64 (2H, t, J = 7.15 Hz, CH2), 2.10 (2H, s, pyr CH2), 3.80 (1H, m,
CH) ppm. 13C NMR: d 164.57 (C-4), 156.22 (C-6), 151.20 (C-2),
135.31 (Ph-quat), 127.81–128.56 (Ph), 109.12 (C-5), 98.44 (CH),
70.60 (CH2Ph), 64.37 (CH2O), 44.28 (C-20), 32.89 (Pyr-CH2), 22.80
(C-10) ppm.
MS m/z 181 [MH]+. Anal. Calcd for [C9H12N2O2]: C, 59.99; H,
6.71; N, 15.55. Found: C, 60.17; H, 6.70; N, 15.53.
4.1.1.11. 6-(3-Benzyloxy-2-hydroxypropyl)-5-(2-chloroethyl)-
2,4-dimethoxypyrimidine (19). The solution of compound 17
(100 mg, 0.46 mmol) in anhydrous THF (10 mL) was cooled at
ꢁ70 °C and lithium diisopropylamide (LDA, 0.1 mL, 2 M in THF/
heptane/ethylbenzene) was added dropwise to the reaction mix-
ture. The temperature was then raised to -55 °C and the reaction
mixture was stirred for 30 min. Benzyloxyacetaldehyde (0.1 g,
0.55 mmol) in THF (1 mL) was added and the mixture was addi-
tionally stirred for 3 h and then at room temperature for 2 h. The
solution was neutralized with glacial acetic acid and stirred further
for 15 min. The solvent was evaporated and the residual yellow
oily product was extracted with CH2Cl2 and water. Organic layer
was dried over MgSO4 and purified by column chromatography
(petroleum ether/ethyl acetate = 7:1). After column chromatogra-
phy, compound 19 was isolated (132.3 mg, 78%) as pale yellow
oil. 1H NMR: d 7.27–7.32 (5H, m, Ph), 5.58 (1H, dd, J = 2.28,
6.18 Hz, OH), 4.48 (4H, m, OCH2), 3.90 (3H, s, OCH3), 3.85 (3H, s,
OCH3), 3.82 (1H, m, CH), 3.77 (2H, m, CH2-Pyr), 3.70 (2H, t,
J = 7.29 Hz, H-20), 2.95 (2H, t, J = 7.29 Hz, H-10) ppm. 13C NMR: d
169.54 (C-4), 167.39 (C-2), 163.21 (C-6), 138.85 (Ph-quat),
127.78–128.88 (Ph), 107.42 (C-5), 97.63 (CH), 72.62 (OCH2Ph),
MS m/z 338 340 [M+, M+2]. Anal. Calcd for [C16H19N2O4Cl]: C,
56.72; H, 5.65; N, 8.27. Found: C, 56.68; H, 5.65; N, 8.29.
4.2. X-Ray Determination of compounds 4, 7 and 14
Single crystals of 4 and 7 suitable for X-ray single crystal anal-
ysis were obtained at room temperature by partial evaporation
from methanol solution. Single crystals of 14 were obtained from
dichloromethane solution applying the same recrystallization
method. The intensities were collected at 295 K on a Oxford
Diffraction Xcalibur2 diffractometer using graphite-monochromat-
35
ed MoK
a radiation (k = 0.71073 Å). CRYSALIS programs were used
for data collection and reduction. The intensities for 4 and 14 were
corrected for absorption using the multi-scan absorption
Table 3
X-ray crystallographic data for compounds 4, 7 and 14
Compound
4
7
14
Formula
C9H12N2O4
212.21
0.13 ꢃ 0.16 ꢃ 0.68
Colorless, prism
Monoclinic
P 21/c
C8H11ClN2O2
202.64
0.25 ꢃ 0.29 ꢃ 0.54
C7H7Cl3N2
225.50
0.10 ꢃ 0.31 ꢃ 0.53
Formula weight
Crystal size/mm
Crystal color, shape
Crystal system
Space group
Triclinic
Orthorhombic
P b c a
ꢀ
P1
Unit cell dimensions
a/
b/
c/
11.7336(6)
5.0612(3)
18.2071(10)
90
114.113(4)
90
986.90(9)
4
1.428
7.5752(2)
7.9618(2)
7.9984(2)
82.878(2)
79.812(2)
81.739(2)
467.46(2)
2
1.440
0.377
x
3.86–28.00
ꢁ9 6 h 6 9
ꢁ10 6 k 6 10
ꢁ10 6 l 6 10
12,894
7.8128(4)
13.9748(5)
17.1690(6)
90
90
90
1874.55(13)
8
1.598
0.921
x
3.82–28.00
ꢁ10 6 h 6 9
ꢁ18 6 k 6 18
ꢁ22 6 l 6 22
12,818
a
/°
b/°
c
/°
V/3
Z
Dcalcd/g cmꢁ3
Absorption coefficient
Scan mode
h range/°
l
/mmꢁ1
0.114
x
and /
4.21–27.99
ꢁ15 6 h 6 15
ꢁ6 6 k 6 6
ꢁ24 6 l 6 22
13,349
Index ranges
Collected reflections No.
Independent reflections No./Rint.
Reflections No. I P 2 (I)
2379/0.0471
1462
2248/0.0198
1785
2238/0.0384
1506
r
Data/restraints/parameters
2379/0/146
0.962
2248/0/124
1.020
2238/0/110
1.026
Goodness-of-fit on F2, S
R [I P 2
wR [I P 2
Max. and min. el. density/e ꢁ3
r
(I)]/R [all data]
0.0535/0.0899
0.1408/0.1677
0.344/ꢁ0.236
0.0334/0.0425
0.1130/0.1165
0.273/ꢁ0.155
0.0572/0.0793
0.1764/0.1945
0.794/ꢁ0.372
r
(I)]/wR [all data]

´
T. G. Kraljevic et al. / Bioorg. Med. Chem. 18 (2010) 2704–2712
2712
correction method (CrysAlis Red35). The crystal structures were
solved by direct methods.36 All non-hydrogen atoms were refined
anisotropically by full-matrix least-squares calculations based on
F2.36 The N1 and N3 hydrogen atoms in 4 and hydroxyl hydrogen
atom in 7 were found in a difference Fourier map and their coordi-
nates and isotropic thermal parameters have been refined freely.
All other hydrogen atoms in 4 and 7, as well as all hydrogen atoms
in 14 were included in calculated positions as riding atoms with
References and notes
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37
SHELXL9736 defaults. PLATON program was used for structure analy-
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749761 (4), 749762 (7) and 749763 (14) contain the supplemen-
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4.3. Antitumor activity assays
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Acknowledgment
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Support for this study was provided by the Ministry of Science
of the Republic of Croatia (Projects #125-0982464-2925, #119-
1193079-3069 and #098-0982464-2514).