Synthesis, cytostatic activity and ADME properties of C-5 substituted and N-acyclic pyrimidine derivatives

Article abstract of DOI:10.1016/j.bmcl.2011.11.009

The synthesis of the novel 5-alkyl pyrimidine derivatives, 5,6-dihydrofuro[2,3-d]pyrimidines and 5-alkyl N-methoxymethyl pyrimidine derivatives and evaluation of their cytostatic activities are described. The mechanism of antiproliferative effect of 5-(2-chloroethyl)-substituted pyrimidine 3 that exerted the pronounced cytostatic activity was studied in further details on colon carcinoma (HCT116) cells. The cell cycle perturbation analysis demonstrated severe DNA damage (G2/M arrest) pointing to a potential DNA alkylating ability of 3. Preliminary ADME data of 3 and its 6-methylated structural congener (6-Me-3) showed their high permeability and good metabolic stability.

Full text of DOI:10.1016/j.bmcl.2011.11.009

Bioorganic & Medicinal Chemistry Letters 22 (2012) 308–312
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis, cytostatic activity and ADME properties of C-5 substituted
and N-acyclic pyrimidine derivatives
a
a
b
b
c
´
´
Tatjana Gazivoda Kraljevic , Mateja Klika , Marijeta Kralj , Irena Martin-Kleiner , Stella Jurmanovic ,
c
c
a,
⇑
´
´
´
Astrid Milic , Jasna Padovan , Silvana Raic-Malic
a Department of Organic Chemistry, Faculty of Chemical Engineering and Technology, University of Zagreb, Maruli´cev trg 19, HR-10000 Zagreb, Croatia
Division of Molecular Medicine, Ruder Boškovi´c Institute, Bijenicka 54, HR-10001 Zagreb, Croatia
Galapagos Research Centre Zagreb Ltd, Prilaz baruna Filipovica 29, HR-10000 Zagreb, Croatia
b
-
ˇ
c
´
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis of the novel 5-alkyl pyrimidine derivatives, 5,6-dihydrofuro[2,3-d]pyrimidines and 5-alkyl
N-methoxymethyl pyrimidine derivatives and evaluation of their cytostatic activities are described. The
mechanism of antiproliferative effect of 5-(2-chloroethyl)-substituted pyrimidine 3 that exerted the pro-
nounced cytostatic activity was studied in further details on colon carcinoma (HCT116) cells. The cell
cycle perturbation analysis demonstrated severe DNA damage (G2/M arrest) pointing to a potential
DNA alkylating ability of 3. Preliminary ADME data of 3 and its 6-methylated structural congener
(6-Me-3) showed their high permeability and good metabolic stability.
Received 3 October 2011
Revised 31 October 2011
Accepted 2 November 2011
Available online 10 November 2011
Keywords:
5-Alkyl pyrimidine derivatives
Cytostatic evaluations
Cell cycle analysis
ADME properties
Ó 2011 Elsevier Ltd. All rights reserved.
Synthetic nucleoside mimics with modified nucleobase and/or
sugar moieties are of considerable importance in the search for
new structural leads exhibiting biologically interesting activity.1
Some nucleoside analogues substituted at various positions on
the heterocycle, are known to have potent biological properties
and have been investigated, for instance, as antiviral agents
(against HSV, VZV, CMV, HIV, HBV and HCV), non-radioactive fluo-
rescent labels for DNA and as anticancer drugs.2 Extensive studies
have been carried out on 5-substituted uracil analogues for use in
cancer and viral chemotherapy, as enzyme inhibitors.3 Notable
among them are 5-fluorouracil (5-FU) and the corresponding
20-deoxyribonucleoside (FdU) which have been used in cancer
chemotherapy for decades.4 Furthermore, bicyclic furo[2,3-
d]pyrimidine nucleosides have demonstrated antiviral and antileu-
kemic activity which has led to increased interest in preparation of
corresponding nucleoside analogues.5 Among fluorinated
pyrimidine nucleoside analogues, 20-deoxy-20-fluoro-5-methyl-1-
as leads for gene expression imaging by positron emission tomog-
raphy (PET).7 In view of the importance of 5-substituted uracil
derivatives in cancer chemotherapy8 and as antiviral agents9 we
became interested in the synthesis of the novel C-5 and/or C-6
substituted pyrimidine derivatives.10
As part of our ongoing research in drug discovery we initiated
the preparation of 5-alkyl pyrimidine derivatives (3–6, 8, 9, 11
and 17), 5,6-dihydrofuro[2,3-d]pyrimidines (7 and 10) and 5-alkyl
N-methoxymethyl pyrimidine derivatives (12–16). Construction of
the pyrimidine ring was performed using the classical intramolec-
ular cyclization of
tone (1) with sodium ethoxide to afford 5-(2-hydroxyethyl)uracil
(2) following a literature method.11 Initially, the sodium
hydroxymethylene- -butyrolactone was prepared by reaction of
-butyrolactone and methylformate in dry ether with the presence
a-(1-carbamyliminomethylene)-c-butyrolac-
a-
c
c
of sodium methoxide, which was subsequently reacted with urea
to give 2. Transformation of hydroxyl and carbonyl functionalities
to chlorine was performed using phosphoryl chloride affording
chlorinated pyrimidine derivative 3 (Scheme 1).11 The primary
hydroxyl group of compound 2 was protected to give 5-(2-acetoxy-
ethyl) substituted uracil 4. Chlorination of 4 with phosphoryl
chloride and N,N-diethylaniline yielded 2,4-dichloropyrimidines
with 2-acetoxyethyl (5) and 2-hydroxyethyl (6) side-chain as well
as bicyclic 5,6-dihydrofuro[2,3-d]pyrimidine derivative (7).
Methoxylation of 5 gave desired 2,4-dimethoxypyrimidine (8) with
2-hydroxyethyl side-chain in moderate yield. 2-Chloro-4-meth-
oxypyrimidine (9) and 2-methoxy-5,6-dihydrofuro[2,3-d]pyrimi-
dine (10) were also isolated as minor products. Fluorination of 9
b-D-arabinofuranosyluracil (FMAU) and other small side-chain
5-substituted derivatives are phosphorylated with different effi-
cacy by human and other mammalian nucleoside kinases including
thymidine kinases TK1 and/or TK2; viral kinases such as herpes
simplex virus type 1 and 2 (HSV1-TK and HSV2-TK).6 Thymine
derivatives with 6-(1,3-dihydroxyisobutenyl) and 6-(1,3-dihydr-
oxyisobutyl) side-chain, as ligands for HSV1-TK, were developed
⇑
Corresponding author.
E-mail address: sraic@fkit.hr (S. Raic´-Malic´).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.11.009

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T. G. Kraljevic et al. / Bioorg. Med. Chem. Lett. 22 (2012) 308–312
O
O
Cl
O
OH
Cl
O
NH2
HN
O
i
N
ii
iii
O
O
N
H
N
Cl
N
H
2
3
1
iv
O
Cl
O
Cl
OAc
OAc
OH
HN
v
N
N
N
+
+
O
N
H
Cl
Cl
Cl
N
N
N
7
4
6
5
vi
OCH3
O
OCH3
OH
OH
N
N
N
+
+
H3CO
N
Cl
N
N
H3CO
8
9
10
vii
OCH3
F
N
Cl
N
11
Scheme 1. Reagents and conditions: (i) NaOMe, ether, HCOOCH3, rt, 24 h; urea, 3 M HCl, 4 °C, 24 h; (ii) NaOEt, EtOH, reflux, 6 h; (iii) POCl3, N,N-diethylaniline, reflux, 1 h; (iv)
Ac2O, pyridine, reflux, 1 h; (v) POCl3, reflux, 1 h; (vi) NaOMe, reflux, 1 h; (vii) DAST, CH2Cl2, ꢁ75 °C, 0.5 h.
with diethylaminosulfur trifluoride (DAST) as the fluorinating
reagent afforded 2-chloro-4-methoxypyrimidine 11 with 2-fluoro-
ethyl substituent at C-5.
compared to the reference compound 5-fluorouracil. In general,
compounds 4, 6 and 8–16 showed no inhibitory activity on tu-
mor-cell growth up to 100 lM (data not shown). However, moder-
N-methoxymethylation of 2 was accomplished with methoxy-
methyl chloride (MOMCl) as alkylating reagent and potassium
carbonate to yield N-1-MOM (12a), N-3-MOM (12b) and N,N-1,3-
diMOM (12c) 5-(2-hydroxyethyl)pyrimidine derivatives in 24%,
15% and 3% yields, respectively (Scheme 2). To the best of our
knowledge there are only a few reports describing the N-meth-
oxymethylated pyrimidine derivatives.12
ate inhibition of cell growth was obtained with compounds 5, 7
and to a certain extent 17 (Table 1).
While 2,4-dichloropyrimidine (6) with 5-(2-hydroxyethyl) side
chain did not show any inhibitory effect, its acetylated structural
analog 5 exhibited cytostatic activity (IC50 ꢀ30
lM). Similar activ-
ity (IC50 ꢀ27 M) was found for bicyclic 2-chloro-5,6-dihydro
l
furo[2,3-d]pyrimidine (7). The 2,4-dimethoxypyrimidine (17) with
5-(2-(methoxymethoxy)ethyl) side chain showed only slight inhib-
itory effect against colon carcinoma (HCT116). Of all compounds
tested, 5-(2-chloroethyl)-2,4-dichloropyrimidine (3) exerted the
most potent antiproliferative activity (in the low micromolar
range), especially and rather selectively towards HCT116 colon
The fluorine in side-chain of 12a was introduced using DAST
affording N-MOM pyrimidine derivative 13 containing 2-fluoroeth-
yl at C-5 position. N-methoxymethylation of acetylated analog 4
also gave N-1-MOM (14a) and N-3-MOM (14b) regioisomers as
well as N,N-1,3-diMOM (14c) in 22%, 2% and 17% yields, respec-
tively. We can deduce that N-alkylation of uracil with both
hydroxyethyl and acetoxyethyl substituents gave target N-1-
MOM uracil derivatives in rather low yield (ꢀ20%). Thus, we
cancer cell line (IC50 = 0.8 lM) (Table 1 and Fig. 1). Similar results
showing significant impact of chlorine atoms on biological activity
were described previously.13 Interestingly, the increase in the chlo-
rine content also enhanced the antiproliferative effects of 2,4-di-
chloro-6-methylpyrimidine structural congener bearing the same
5-(2-chloroethyl)-substituent.13
In order to shed more light on the mechanism of action of the
most active compound 3, we attempted the flow cytometry analy-
sis of potential cell cycle perturbations. The compound was tested
at two concentrations being close or slightly higher than its IC50
applied
a more efficient N-methoxymethylation using same
reaction conditions but starting from 2,4-dimethoxy-5-(2-
hydroxyethyl)uracil (8) to give N-1-MOM (15) in improved yield
of 46%. Compounds 16 and 17 with 2-(methoxymethoxy)ethyl
substituent at C-5 were also isolated as byproducts.
Compounds 3–17 were evaluated for their cytostatic activities
against three human malignant tumor cell lines: breast carcinoma
(MCF-7), colon carcinoma (HCT116), lung carcinoma (H 460) and
(1 and 5 lM) on HCT116 cells (Table 2). Interestingly, the results

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T. G. Kraljevic et al. / Bioorg. Med. Chem. Lett. 22 (2012) 308–312
O
F
HN
O
N
O
13
ii
O
N
O
O
O
OH
OH
OH
OH
O
N
HN
i
HN
O
N
+
+
O
O
N
O
N
H
O
N
H
O
O
2
12a
12c
12b
O
O
O
O
OAc
OAc
OAc
OAc
HN
O
N
HN
O
N
i
+
+
O
N
O
N
O
N
H
O
N
H
O
O
4
14b
14c
14a
OCH3
OCH3
OCH3
OCH3
OH
O
O
O
O
OH
N
N
N
N
i
+
+
O
N
O
N
H3CO
N
H3CO
N
O
O
8
17
15
16
Scheme 2. Reagents and conditions: (i) K2CO3, DMF, MOMCl, ꢁ15 °C, 0.5 h, rt, overnight; (ii) DAST, CH2Cl2, ꢁ75 °C, 0.5 h.
Table 1
Table 2
Inhibitory effects of 6-Me-3, 3, 5, 7 and 17 on the growth of malignant tumor cell lines
The effects of compound 3 at 1 and 5
after the 24- and 48-hour treatments
l
M on the cell cycle distribution of HCT116 cells
Percentage of cellsa (%)
a
Compd
IC50
(l
M)
Treatment
HCT116
0.2
0.8 0.2
29 13
13 10
75 17
MCF-7
H 460
SubG1
G1
S
G2/M
6-Me-313
3
5
7
2
8
10
30
45
7
1
5
6
3
3
0.01
0.001
24 h
48 h
Control
1.8
2.5
8.1
32.5
33.5
7.8
50.0
48.8
14.6
17.4
17.2
77.6
31 0.0
22 0.05
>100
1
5
l
l
M
M
17
>100
14 0.3
Control
1.5
1.7
8.5
42.3
41.7
24.6
31.0
31.5
14.8
26.6
26.7
60.7
5-FUb
4
0.8
3
0.3
1
5
l
l
M
M
a
IC50: 50% inhibitory concentration, or compound concentration required to
inhibit tumor cell proliferation by 50%.
a
The numbers represent the percentages of cells in respective cell cycle phase
b
5-FU: 5-fluorouracil.
(G1, S and G2/M), along with the percentage of cells in the subG1 (dead cells)
obtained by flow cytometry.
demonstrated that 3 at the higher concentration (5 lM) induced
strong G2/M phase arrest at both time points, that is the majority
of cells could not progress through mitosis and eventually died by
apoptosis, as evidenced by accumulation of subG1 cells, represent-
ing apoptotic cells. Accumulation of cells in G2/M phases (a G2/M
phase arrest) points to a damage in the cell, which occurs during
the S phase (DNA replication), or before mitosis (aberrant mitotic
spindle formation). The G2 checkpoint senses unreplicated DNA,
generates a signal leading to cell cycle arrest and therefore pre-
venting the initiation of M phase before completion of S phase.
Thus, it is usually triggered by agents that induce DNA- or mitotic
spindle-damage. Since compound 3 is structurally similar to a
Figure 1. Concentration–response profiles for compound 3 tested on tumor cell
lines in vitro. PG = percentage of growth.

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T. G. Kraljevic et al. / Bioorg. Med. Chem. Lett. 22 (2012) 308–312
Table 3
The apparent permeability (Papp) values for 3 and 6-Me-3 (with and without P-gp inhibitor)
Compd
Papp (AB) (cm/s)
Papp (BA) (cm/s)
Efflux ratio
Papp (AB) (cm/s)
Papp (BA) (cm/s)
Efflux ratio
ꢁElacridara
+Elacridar
3
20.8 ꢂ 10ꢁ6
35.8 ꢂ 10ꢁ6
25.8 ꢂ 10ꢁ6
20.9 ꢂ 10ꢁ6
1.2
0.6
25.6 ꢂ 10ꢁ6
21.6 ꢂ 10ꢁ6
29.6 ꢂ 10ꢁ6
31.1 ꢂ 10ꢁ6
1.2
1.4
6-Me-3
a
Elacridar (GF120918) as P-gp inhibitor.16
Table 4
Stability of 3 and 6-Me-3 in plasma and liver microsomes of various species
Compd
Plasma stability % remaining (24 h) (t1/2 (min))
Microsomal stability % remaining (60 min) (t1/2 (min))
Human
Rat
Mouse
Human
Rat
Mouse
3
0
0
0
65
73
33
(153)
20
(24)
3
(91)
22
(104)
76
(134)
74
(34)
41
6-Me-3
(600)
(184)
(478)
(145)
(134)
(46)
pyrimidine antagonist 5-FU, this mechanism of cell cycle perturba-
tion is quite peculiar. Namely, antimetabolite drugs, such as pyri
midine antagonists arrest/delay the cell cycle in G1, or early S
phase.14 Nevertheless, the chloroethyl-substituent in 3, which is
present in other known alkylating agents (such as: nitrogen mus-
tards, or uracil mustard—uramustine) might be responsible for
alkylation of DNA, thus causing a more severe DNA damage, which
induces G2/M arrest and apoptosis. More detailed analysis of DNA
alkylation/cross linking/damage ability of 3 is underway.
all evaluated compounds, pyrimidine derivative 3 that bears two
aromatic and one aliphatic chlorine atoms showed the most potent
inhibitory activity against malignant tumor cell lines tested, partic-
ularly against the colon cancer (HCT116) cell line. Interestingly, the
cell cycle perturbation analysis demonstrated severe DNA damage
(G2/M arrest) pointing to a potential DNA alkylating ability of chlo-
roethyl-substituted pyrimidine 3. Furthermore, this compound
exhibited favourable in vitro ADME properties, that is high perme-
ability and good metabolic stability in liver microsomes of human
and rat.
Compound 3 and its 6-methylated structural analog (6-Me-3)
with pronounced cytostatic activities were selected for preliminary
ADME assays including permeability and P-glycoprotein (P-gp)
substrate assessment, stability in plasma (rat, human and mouse),
as well as metabolic stability in liver microsomes (rat, human and
mouse). These properties are important for new molecules design
and affect their bioavailability in a great manner. Permeability as-
say was designed to determine permeability of selected com-
pounds through MDCKII-MDR1 cell monolayers, as well as for
their identification as potential P-gp supstrate.15 MDCKII-MDR1
cells are Madin–Darby canine kidney cells with overexpressed
MDR1 (human multidrug resistance 1) gene. This gene encodes
an integral membrane protein P-gp that functions as ATP-depen-
dant efflux pump, which could have a significant influence on
compounds permeability. Both compounds 3 and 6-Me-3 showed
to be highly permeable compounds with Papp values above
20.0 ꢂ 10ꢁ6 cm/s (Table 3). Besides, efflux ratios did not exceed
2, indicating that both compounds are not P-gp substrates. Results
of plasma stability showed that 6-Me-3 was more stable in all
tested species comparing to 3 (Table 4). Moreover, this compound
exhibited significantly higher (four to eightfold) half-life values
than those of 3, suggesting that methyl substituent at C-6 of pyri
midine scaffold in 6-Me-3 has impact on plasma stability. On the
contrary to this, determination of metabolic stability in liver
microsomes, monitoring exclusively phase I metabolism, showed
similar metabolic stability of tested compounds. In addition, both
compounds exhibited higher stability in human and rat micro-
somes than in mouse microsomes.
Acknowledgments
Support of this study by the Ministry of Science, Education and
Sports of the Republic of Croatia (projects #125-0982464-2925
and 098-0982464-2514) is gratefully acknowledged.
Supplementary data
Supplementary data (experimental procedures, compound
characterization data, methods for antitumor activity assay, cell
cycle analysis and ADME assay) associated with this article can
be found, in the online version, at doi:10.1016/j.bmcl.2011.11.009.
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