Novel pyrimidine-2,4-dione–1,2,3-triazole and furo[2,3-d]pyrimidine-2-one–1,2,3-triazole hybrids as potential anti-cancer agents: Synthesis, computational and X-ray analysis and biological evaluation

Article abstract of DOI:10.1016/j.ejmech.2016.11.028

Regioselective 1,4-disubstituted 1,2,3-triazole tethered pyrimidine-2,4-dione derivatives (5–23) were successfully prepared by the copper(I)-catalyzed click chemistry. While known palladium/copper-cocatalyzed method based on Sonogashira cross-coupling followed by the intramolecular 5-endo-dig ring closure generated novel 6-alkylfuro[2,3-d]pyrimidine-2-one–1,2,3-triazole hybrids (24b–37b), a small library of their 5-alkylethynyl analogs (24a–37a) was synthesized and described for the first time by tandem terminal alkyne dimerization and subsequent 5-endo-trig cyclization, which was additionally corroborated with computational and X-ray crystal structure analyses. The nature of substituents on alkynes and thereof homocoupled 1,3-diynes predominantly influenced the ratio of the formed products in both pathways. In vitro antiproliferative activity of prepared compounds evaluated on five human cancer cell lines revealed that N,N-1,3-bis-(1,2,3-triazole)-5-bromouracil (5–7) and 5,6-disubstituted furo[2,3-d]pyrimidine-2-one-1,2,3-triazole 34a hybrids exhibited the most pronounced cytostatic acitivities against hepatocellular carcinoma (HepG2) and cervical carcinoma (HeLa) cells with higher potencies than the reference drug 5-fluorouracil. Cytostatic effect of pyrimidine-2,4-dione-1,2,3-triazole hybrid 7 in HepG2 cells could be attributed to the Wee-1 kinase inhibition and abolishment of sphingolipid signaling mediated by acid ceramidase and sphingosine kinase 1. Importantly, this compound proved to be a non-mitochondrial toxicant, which makes it a promising candidate for further lead optimization and development of a new and more efficient agent for the treatment of hepatocellular carcinoma.

Full text of DOI:10.1016/j.ejmech.2016.11.028

Accepted Manuscript
Novel pyrimidine-2,4-dione–1,2,3-triazole and furo[2,3-d]pyrimidine-2-one–1,2,3-
triazole hybrids as potential anti-cancer agents: Synthesis, computational and X-ray
analysis and biological evaluation
Tomislav Gregorić, Mirela Sedić, Petra Grbčić, Andrea Tomljenović Paravić, Sandra
Kraljević Pavelić, Mario Cetina, Robert Vianello, Silvana Raić-Malić
PII:
S0223-5234(16)30966-7
10.1016/j.ejmech.2016.11.028
EJMECH 9061
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 26 July 2016
Revised Date: 10 November 2016
Accepted Date: 12 November 2016
Please cite this article as: T. Gregorić, M. Sedić, P. Grbčić, A.T. Paravić, S.K. Pavelić, M. Cetina,
R. Vianello, S. Raić-Malić, Novel pyrimidine-2,4-dione–1,2,3-triazole and furo[2,3-d]pyrimidine-2-
one–1,2,3-triazole hybrids as potential anti-cancer agents: Synthesis, computational and X-ray
analysis and biological evaluation, European Journal of Medicinal Chemistry (2016), doi: 10.1016/
j.ejmech.2016.11.028.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Novel pyrimidine-2,4-dione–1,2,3-triazole and furo[2,3-d]pyrimidine-
-one–1,2,3-triazole hybrids as potential anti-cancer agents: synthesis,
computational and X-ray analysis and biological evaluation
2
a
*bc
b
b
Tomislav Gregorić, Mirela Sedić, Petra Grbčić, Andrea Tomljenović Paravić,
bc
d
*e
*a
Sandra Kraljević Pavelić, Mario Cetina, Robert Vianello and Silvana Raić-Malić
a
University of Zagreb, Faculty of Chemical Engineering and Technology, Department of
Organic Chemistry, Marulićev trg 20, HR–10000 Zagreb, Croatia. Email: sraic@fkit.hr
b
University of Rijeka, Department of Biotechnology, Radmile Matejčić 2, HR–51000
Rijeka, Croatia. Email: msedic@biotech.uniri.hr
c
University of Rijeka, Centre for high-throughput technologies, Radmile Matejčić 2,
HR–51000 Rijeka, Croatia
d
University of Zagreb, Faculty of Textile Technology, Department of Applied Chemistry,
Prilaz baruna Filipovića 28a, HR–10000 Zagreb, Croatia
e
Computational Organic Chemistry and Biochemistry Group, Ruđer Bošković Institute,
Bijenička 54, HR–10000 Zagreb, Croatia. Email: robert.vianello@irb.hr
1

ACCEPTED MANUSCRIPT
Abstract. Regioselective 1,4-disubstituted 1,2,3-triazole tethered pyrimidine-2,4-dione
derivatives (5–23) were successfully prepared by the copper(I)-catalyzed click chemistry.
While known palladium/copper-cocatalyzed method based on Sonogashira cross-coupling
followed by the intramolecular 5-endo-dig ring closure generated novel 6-alkylfuro[2,3-
d]pyrimidine-2-one–1,2,3-triazole hybrids (24b–37b), a small library of their 5-alkylethynyl
analogs (24a–37a) was synthesized and described for the first time by tandem terminal alkyne
dimerization and subsequent 5-endo-trig cyclization, which was additionally corroborated
with computational and X-ray crystal structure analysis. The nature of substituents on alkynes
and thereof homocoupled 1,3-diynes predominantly influenced the ratio of the formed
products in both pathways. In vitro antiproliferative activity of prepared compounds evaluated
on five human cancer cell lines revealed that N,N-1,3-bis-(1,2,3-triazole)-5-bromouracil (5–7)
and 5,6-disubstituted furo[2,3-d]pyrimidine-2-one-1,2,3-triazole 34a hybrids exhibited the
most pronounced cytostatic acitivities against hepatocellular carcinoma (HepG2) and cervical
carcinoma (HeLa) cells with higher potencies than the reference drug 5-fluorouracil.
Cytostatic effect of pyrimidine-2,4-dione-1,2,3-triazole hybrid 7 in HepG2 cells could be
attributed to the Wee-1 kinase inhibition and abolishment of sphingolipid signaling mediated
by acid ceramidase and sphingosine kinase 1. Importantly, this compound proved to be a non-
mitochondrial toxicant, which makes it a promising candidate for further lead optimization
and development of a new and more efficient agent for the treatment of hepatocellular
carcinoma.
Keywords: furo[2,3-d]pyrimidine-2-one; 5-endo-trig cyclization; computational chemistry;
X-ray analysis; sphingolipid signaling; Wee-1 kinase
2

ACCEPTED MANUSCRIPT
1
. Introduction
Cancer is a multifaceted disease that represents one of the leading causes of
mortality in developed countries. One in eight deaths worldwide is due to cancer, and it
is the second most common cause of death exceeded only by heart diseases . In spite
[1]
of continuous advancements in treatment and prevention strategies, the successful
treatment of cancer, in particular metastases, still remains a challenge. Discovery of
new and safer anticancer agents with improved cytotoxicity in cancer cells will
improve the management of cancer [2].
Molecular hybridization, which covalently combines two or more drug
pharmacophores into a single molecule, proved to be an effective tool for designing
novel entities as potent antitumor agents [3]. Thus, the pyrimidine moiety, as one of the
most prominent structures found in nucleic acid chemistry, is a component of a number
of useful drugs and is associated with many biological, pharmaceutical and therapeutic
activities
agents introduced into the medical treatment of cancer
pyrimidine moiety with different heterocycle scaffolds gives rise to a new class of
hybrid heterocycles exerting improved biological activity . Various five-membered
heteroaromatic ring-fused pyrimidines, furopyrimidines in particular, were studied and
found to possess remarkable pharmacological properties . Moreover, some 4,5,6-
trisubstituted furo[2,3- ]pyrimidin-4-amines were discovered as non-receptor tyrosine
kinase ACK1 (activated Cdc42-associated tyrosine kinase 1) inhibitors , while 4,5-
disubstituted furo[2,3- ]pyrimidine was potent receptor tyrosine kinase c-Met inhibitor
. We found that among bicyclic furo[2,3- ]pyrimidine series, 6-butyl- and 6-
bromophenylfuro[2,3- ]pyrimidines showed the highest cytostatic activity, particularly
in malignant leukemia (L1210) and T-lymphocyte (Molt4/C8 and CEM) cells
Previous studies also demonstrated that some pyrimidine 10 and heteroaromatic ring
fused-pyrimidine 11 derivatives have the ability to inhibit key metabolic enzymes
[
4
]. Modified pyrimidine nucleosides were among the first chemotherapeutic
[5]. Furthermore, the fusion of
[3]
[6]
d
[7]
d
[8
]
d
p-
d
[9].
[
]
[
]
that regulate intracellular levels of tumor-suppressor lipids ceramide and sphingosine,
which elicit antiproliferative and apoptotic responses in various cancer cells. The same
compounds were also shown to interfere with the metabolism of pro-survival lipid
sphingosine-1-phosphate (S1P), whose major role is to regulate proliferation,
inflammation, angiogenesis and resistance to apoptotic cell death. In addition,
clinically approved drug imatinib,
N-(5-amino-2-methylphenyl)-4-(3-pyridyl)-2-
3

ACCEPTED MANUSCRIPT
pyrimidineamine, down-regulates the activity of sphingosine kinase 1 (SK1), an
enzyme that catalyzes the phosphorylation of sphingosine to S1P in imatinib-sensitive
chronic myeloid leukemia cells, which precedes the onset of apoptosis
[12].
Furthermore, the antineoplastic drug carmofur, as the representative of the -acyclic 5-
N
fluorouracil class, was shown to be a potent inhibitor of acid ceramidase (ASAH), an
enzyme that catalyzes the lysosomal degradation of ceramide, which contributes to the
antiproliferative effects of carmofur
Despite the biological importance of furopyrimidine derivatives, the synthetic
methodology used for the -heteroannulation process is limited. Therefore, the need
[13].
O
persists for more versatile and efficient procedure. Palladium-catalyzed annulation of
functionalized aryl halides and alkynes is the most common method to access a wide
variety of carbo- and hetero-cycles
catalysts, such as AgNO , Zn(Cl)
cyclization of alkynylated nucleosides to furopyrimidines
[
14
]
. Instead of using copper, application of other
(OCOCF O cluster, also enabled the
15 . Moreover, having in
3
2
or Zn
4
3 6
)
[
]
mind the development of environmentally friendly synthetic tools, one-pot protocol to
directly access various furopyrimidines under the aqueous conditions with low catalyst
loading was applied [16]. In the last decade, 1,2,3-triazole and its derivatives have
attracted a great deal of interest due to their synthetic accessibility by click chemistry
and wide application in drug discovery, particularly in the early discovery phase and
the lead optimization processes
as moderate dipole character, hydrogen bonding capability, rigidity and stability under
in vivo conditions, are clearly responsible for their enhanced biological activities 18].
[17]. The favorable features of 1,2,3-triazole ring, such
[
Moreover, the incorporation of 1,2,3-triazoles between two pharmacophores to give
bifunctional drugs is increasingly becoming useful and important in constructing
bioactive molecules
pyrimidine nucleosides
,2,3-triazole as a substituent at either the pyrimidine ring
sugar mimic 23 were endowed with pronounced cytostatic activity. Taking into
consideration the aforementioned data and in continuation of our recent efforts towards
the synthesis of 1,2,3-triazole-appended -heterocycle pharmacophores 24 , we
envisaged that pyrimidine, furo[2,3- ]pyrimidine and 1,2,3-triazole pharmacophores, if
[
19
]
. Thus, it was found that some 1,2,3-triazole tethered
, 1,2,3-triazole pyrimidine nucleoside conjugates with the
21 , sugar moiety 22 or
[20
]
1
[
]
[
]
[
]
N
[
]
d
linked together, would generate novel molecular entities which are likely to exhibit
promising biological properties. To the best of our knowledge, this is the first report on
4

ACCEPTED MANUSCRIPT
1,2,3-triazole-appended furo[2,3-
d]pyrimidines. Although the synthetic method to
provide 6-substituted furo[2,3-
d
]pyrimidines is well-documented 9,14a,b , the
[
]
synthesis of their 5-alkylethynyl analogs is still elusive. Herein, we describe for the
first time synthetic methodology that enables an access to the construction of furo[2,3-
d]pyrimidine-2-one (24a–37a) with 5-alkylethynyl-6-alkyl substituents through
palladium/copper-cocatalyzed symmetrical diyne formation and 5-endo-trig cyclization
sequence. Precise reaction mechanisms leading to both sets of compounds were
rationalized with the aid of computational chemistry methods at the DFT level with
implicit solvation. Moreover, the apoptosis induction and mitochondrial toxicity were
analyzed for the selected 1,2,3-triazole–pyrimidine hybrids. Additional biological
studies were carried out to investigate their inhibitory effects on cell proliferation,
growth and survival mechanisms governed by sphingolipid metabolic enzymes acid
ceramidase (ASAH) and sphingosine kinase 1 (SK1), as well as those mediated by the
cell cycle regulatory enzyme Wee-1 tyrosine kinase which controls the G2 checkpoint
in response to DNA damage.
2. Results and discussion
2.1. Chemistry
N
-Propargylation of 5-bromo- and 5-iodouracil with propargyl bromide and
sodium hydride gave a mixture of -1,3-disubstituted ( and ) and -1-substituted
and ) prop-2-ynyl uracil derivatives (Scheme 1). -1,3-Disubstituted ( ) and
-1-monosubstituted ( 12) 1,2,3-triazole derivatives of 5-bromouracil were
N,
N
1
3
N
(2
4
N,
N
5–8
N
9–
subsequently prepared by copper(I)-catalyzed Huisgen 1,3-dipolar cycloaddition
reaction of terminal alkyne with corresponding azide (Scheme 1). The synthesis of
N
,
N
-1,3-disubstituted 1,2,3-triazole–5-bromouracil hybrids (
copper(II) sulfate, sodium ascorbate as a reducing agent, and various azides, including
-flourophenyl, -flourophenyl, -chlorophenyl and 3,5-dichlorophenyl azide, in the
yield ranging from 30–58%. The 1,3-dipolar cycloaddition of -1-propargyl 5-
bromouracil derivative ( ) and aromatic azides was carried out using copper(II) sulfate
and metallic copper as a reducing agent under the microwave irradiation to give -1-
monosubstituted 1,2,3-triazole–5-bromouracil hybrids ( 12) in higher yield (75–84%)
relative to their -1,3-disubstituted analogs. Click reaction of 5-iodo-
di(prop-2-ynyl)pyrimidine-2,4-dione ( ) and aromatic azides afforded
5–8) was performed using
p
o
p
N
2
N
9
–
N,
N
N
,
,
N
-1,3-
-1,3-
3
N
N
5

ACCEPTED MANUSCRIPT
disubstituted 1,2,3-triazole–5-iodouracil hybrids (13
triazole)-substituted uracils (14 16 and 18) bearing prop-2-ynyl side chain at
the uracil ring, as minor products. Reaction of -1-propargyl 5-iodouracil (
,
15
,
17 and 19) and
N
-1-(1,2,3-
-3 of
) with
,
N
N
4
aromatic azides gave 1,2,3-triazole–5-iodouracil hybrids (20
–23) (Scheme 1).
O
O
3
"
1"
R1
2"
Br
Br
N
4
N
2
5
6
HN
N
N
O
N
O
N
1'
2'
3'
N
R1
N R1
N
N
N
N
5
−8
8' 9'
8' 9'
9−12
4'
7'
4'
7
'
5
, 9: R = F
1
6, 10:
R =
1
6
'
5'
6'
5'
Cl
F
7
, 11: R
1
= Cl
8, 12: R =
1
Cl
ii)
iii)
O
O
O
3"
1"
2
"
R5
R5
4
R5
N
2
HN
5
6
i)
HN
+
O
N
O
N
O
N
H
2'
1'
3'
1, 3
1, 2: R
, 4: R
5
= Br
= I
2, 4
3
5
iii)
iii)
O
N
O
N
O
R1
I
I
I
N
N
N
N
HN
N
+
O
O
O
N
N
R1
N R
1
N R1
N
N
N
N
N
N
1
3, 15, 17, 19
14, 16, 18
20−23
1
3, 14, 20: R = F
15, 16, 21: R =
1
1
9' 10'
F
8
'
1
7, 18, 22: R = Cl
19, 23: R =
4'
1
1
5'
7
'
6'
Scheme 1. Reagents and conditions: (i) propargyl bromide, NaH, DMF, Ar atmosphere, r.t.,
2
4 h; (ii) azide, sodium ascorbate, CuSO x 5 H O, DMF, r.t. , 24 h. (iii) azide, Cu, 1M CuSO
4 2
4
2
solution, tert-butanol : H O = 1 : 1, MW 300 W, 80 °C, 45 min.
6

ACCEPTED MANUSCRIPT
In connection with our previous findings on bioactive bicyclic pyrimidine
nucleosides
dimerization and subsequent transition copper-mediated intramolecular cyclization for
the synthesis of the 5-alkylethynyl-6-alkylfuro[2,3- ]pyrimidine-2-one derivatives
Scheme 2). Therefore, the coupling of 1,2,3-triazole–5-iodouracil hybrids (20–23)
[9], we further investigated the feasibility of the terminal alkyne
d
(
with terminal alkynes including cyclopropylethyne, 5-chloropent-1-yne, 1-ethynyl-4-
pentylbenzene and oct-1-yne and the presence of palladium catalyst
tetrakis(triphenylphosphine)palladium(0) [Pd(PPh
N,N-DIPEA) and Cu(I) iodide as cocatalyst afforded target 5-alkylethynyl-6-
alkylfuro[2,3- ]pyrimidine-2-one derivatives (24a 37a) and their 6-substituted analogs
24b 37b) (Scheme 2).
3 4
) ], N,N-diisopropylethylamine
(
d
–
(
–
Scheme 2. Reagents and conditions: (i) corresponding terminal alkyne, CuI, (PPh
3
)
4
Pd, N,N-
o
DIPEA, toluene, 80 C, 24 h.
7

ACCEPTED MANUSCRIPT
Generally, an excess amount of alkyne (3 equiv) and N,N-DIPEA base (6 equiv)
in copper-cocatalyzed conditions under heating provided 5,6-disubstituted furo[2,3-
d
]pyrimidine-2-ones (24a–37a), as the major or sole product. The only exceptions
were reactions of 4-(2-fluorophenyl)-1,2,3-triazole appended 5-iodouracil (21) with 5-
chloropen-1-yne and oct-1-yne, which gave only 6-substituted furo[2,3- ]pyrimidine-
-ones 27b and 29b. Possible synthetic pathways A and B for the formation of both
furopyrimidine rings in 24a 37a and 24b 37b series from pyrimidine-2,4-dione–1,2,3-
triazole hybrides 20 23 presumably started via oxidative addition of 5-iodouracil
derivatives 20 23 to Pd(0) (Int ), as depicted in Scheme 3.
d
2
–
–
–
–
–1
Scheme 3. Proposed mechanism for the formation of 5,6-disubstituted 24a–37a and 6-
substituted 24b–37b furopyrimidine–1,2,3-triazole hybrids.
The presence of Cu(I) salt is generally believed to facilitate the transfer of the
alkynyl group to the Pd(II) catalyst in Int
acetylide species and the subsequent trans-metalation of this group to Pd(II) in Int
Finally, reductive elimination of Int 3B gave 5-alkynyluracil derivatives, which
–
2B by the in situ generation of copper
–3B
.
–
subsequently underwent 5-endo-dig cyclization to afford 6-substituted furopyrimidine
8

ACCEPTED MANUSCRIPT
series 24b
–37b by copper-promoted reaction. Several studies have already shown that
in the typical Sonogashira protocol employing cocatalytic Cu(I) salts, copper increased
the reaction rate, but also in situ generation of copper acetylides induced Glaser-type
oxidative homocoupling of the terminal acetylene to yield symmetrical 1,3-diynes [25].
Therefore, instead of coordination of copper acetylide that occurred in pathway B,
symmetrical head-to-head diynes can be anticipated to be involved, as an intermediate,
in an alternative pathway A. Thus, coordination of the internal diyne to the metal
center of the uracil palladium intermediate Int
uracil-Pd bond to give enyne palladium complex Int
Next step was reductive elimination and intramolecular 5-endo-trig cyclization to form
,6-disubstituted furopyrimidine series 24a 37a and regenerate Pd(0) (Scheme 3). So
far, 5-endo-trig cyclization has been rarely explored, due to its disfavored cyclization
pattern according to Baldwin’s rules 26 . Therefore, our interest was further focused
–2A and subsequent insertion into the
–3A might occur in pathway A.
5
–
[
]
on the elucidation of the proposed mechanism (Scheme 3) with the aid of computations
at the DFT level.
2
.2. Computational analysis
To examine the reaction pathways leading to the formation of target 6-
substituted furo[2,3-d]pyrimidine-2-ones and their unexpected 5-alkylethynyl
derivatives, we performed a computational analysis using the B97D DFT functional in
conjuction with the 6–31+G(d) basis set for carbon, nitrogen, oxygen and hydrogen
atoms and the SDD pseudopotentials for iodine and copper atoms, all immersed in the
implicit SMD solvation corresponding to pure toluene. The molecules studied
computationally are presented in Fig. 1.
Fig. 1. Structure and atom numbering of relevant molecules studied computationally.
9

ACCEPTED MANUSCRIPT
We initiated our analysis with the system M1, since the mechanism of its
formation from the corresponding 5-iodide M5 is known and well described in the
literature [14]. M1 is a model system for a class of compounds Int–4B (Scheme 3),
where the substituent on the N1 atom has been truncated to the methyl group, since the
length of the alkyl chain at this position does not significantly influence the kind of
reactivity investigated in this work. N3–H tautomer is the most stable form of M1
(M1–t1, Fig. S1, Supplementary information). Both O–H tautomers are 13.9 (M1–t2)
–
1
and 20.8 kcal mol (M1-t3) less stable than M1-t1. Interestingly, a system in which
N–H group tautomerizes to the C7 atom of the acetylene group, M1-t4, spontaneously
rearranges to the target product M2. However, our calculations reveal the barrier to
–1
transfer a proton from M1–t1 to M1–t4 exceeds 160 kcal mol making this process
highly unfavorable. Therefore, a simple tautomerization of M1 could not rationalize
the M2 formation.
The cyclization of neutral M1 is not feasible as during the approach of the O4
atom towards C8, the energy of the system only increases without any indication for
–
transition states or stable intermediates. N3–H deprotonation gives M1 , which could
–
undergo the mentioned cyclization to give the anionic M2 ; however the calculated
‡
–1
barrier for this process appears too high (ꢀ
G
= 23.8 kcal mol ), while the reaction
= 23.0 kcal mol is too endergonic for this reaction to be feasible.
This goes along with the fact that the calculated p value for the necessary N3–H
deprotonation in toluene assumes p M1 N3 = 59.7, being too high to occur under
–1
free energy of ꢀG
R
K
a
a
K (
)
normal conditions. Therefore, we can conclude that the formation of the cyclic product
–
M2 from either M1 or deprotonated M1 is very unlikely.
The presence of Cu(I) ions changes the reactivity of M1. Cu(I) ion most
preferentially binds between the carbonyl oxygen O4 and the C7–C8 triple bond (M1
–
+
Cu ) with the corresponding Cu–O4, Cu–C7 and Cu–C8 distances of 2.114, 2.037 and
–1
2
.172 Å, respectively, and the binding free energy of ꢀGBIND = –29.9 kcal mol . The
+
cyclization of M1
–
Cu follows the mechanism depicted in Fig. 2. It requires 13.8 kcal
–1
mol to arrive at transition state structure characterized with one imaginary frequency
–
1
of νIMAG = 196i cm . During that process, Cu(I) ion moves to the other side of the
acetylene bond and is predominantly bonded to C7 with the distance of 1.895 Å. This
twists the angle C8–C7–C5 to 116.0º bringing the C8 atom close to O4 at 2.057 Å.
+
Following the O4–C8 bond formation, the system relaxes to monocationic M2–Cu
with the O4–C8 and Cu–C7 bonds assuming 1.490 and 1.885 Å, respectively. The
10

ACCEPTED MANUSCRIPT
+
latter bond is crucial for the stability of M2–Cu , since the removal of the Cu(I) ion or
even placing it to alternative binding positions, including N1, N3, O2, and O4 atoms,
restores the system back to the acyclic form M1. Interestingly, the rearrangement of
+
+
M2–Cu involving the N3 proton and the Cu(I) ions gives M3–Cu that is by 18.3
–
1
+
kcal mol more stable than M2–Cu (Fig. 2).
Fig. 2. Free energy profile for the Cu(I)-catalyzed cyclization of 5-alkylethynyl uracils
towards 6-substituted furo[2,3-d]pyrimidine-2-ones.
a
This rearrangement is facilitated by the fact that the calculated pK value for
+
M2–Cu is 19.2. To put this number in the proper context, we estimated the influence
of Cu(I) on A1 acetylene deprotonation (Fig. 1), which occurs in toluene during the
a
Sonogashira coupling reaction. pK for isolated A1 is 77.4 clearly indicating the
predominance of the unionized form. Nevertheless, following Cu(I) binding, the pKa
value drops to 19.1, which could be taken as the acidity constant corresponding to a
feasible process in toluene under our reaction conditions. Therefore, the calculated
+
–
+
pKa
(
M2–Cu )N3 = 19.2 indicates a large amount of deprotonated M2 –Cu , which
+
facilitates the rearrangement to M3–Cu , where the calculated binding free energy of
–1
–1
Cu(I) ions is ꢀ
G
BIND = –32.4 kcal mol . Therefore, it requires 32.4 kcal mol to arrive
at the first type of products M2, being the target 6-substituted furo[2,3-
d]pyrimidine-2-
–1
ones. Still, the overall change in the reaction free energy is ꢀ
G
R
= –6.8 kcal mol ,
‡
which, together with the rate limiting cyclization step associated with ꢀ
G
= 13.8 kcal
–1
mol makes this the most likely pathway.
11

ACCEPTED MANUSCRIPT
If we consider the formation of the unexpected 5-alkylethynyl derivatives of M2
from any stationary point on the pathway depicted in Fig. 2, the results would reveal
unfeasible processes. Essentially, we would need to substitute the hydrogen atom on
C7 in M2 with another acetylide anion, which would introduce two negative charges
into M2 to give di-anionic molecule, unless the mentioned C7 hydrogen atom is
abstracted as the hydride anion, which is something we did not observe. This limitation
is what renders most of the investigated mechanisms highly improbable as being
associated with either high barriers or unfavorable endergonicities of the overall
–
reaction. For example, the approach of deprotonated A1 to the C7 atom in M2 does
+
not produce stable intermediates. Even positively charged M3–Cu , with Cu(I) bound
–
to the N3 site, is not electrophilic enough to bind A1 to the stable C7 intermediate. If
+
–
+
–
we consider M2–Cu , it can efficiently bind A1 to M2–Cu –A1 with the binding
–1
‡
free energy as high as ꢀ
G
BIND = –77.7 kcal mol . From there, it takes ꢀ
G
= 28.2 kcal
–1
–1
mol to arrive at the transition state for the C–C bond formation (νIMAG = 343i cm ),
which appears too high for the feasible process, on top of the fact that the reaction is
–1
both highly endergonic, ꢀ
R
G = 10.6 kcal mol and reverts the product back to the
acyclic M4. The only potentially chemically feasible pathway is when one considers
–
+
the acyclic M1 –Cu (Fig. S2, Supplementary information), where Cu(I) is bound with
–1
–
ꢀ
GBIND = –78.3 kcal mol . The approach of the acetylide anion A1 is endergonic (ꢀG
–
1
=
5.8 kcal mol ) from which the nuclephilic attack to C7 is associated with the barrier
‡
–1
–1
–1
of ꢀ
G
= 20.2 kcal mol (νIMAG = 267
i
cm ) and ꢀ
G
R
= –4.3 kcal mol . This gives
+
–1
anionic intermediate, which increases the Cu binding (ꢀ
G
BIND = –103.5 kcal mol ).
‡
–1
–1
Subsequent cyclization requires ꢀ
G = 21.8 kcal mol (νIMAG = 325i cm ) producing
the unexpected 5-alkylethynyl derivative with copper ion bound to N3 with ꢀGBIND = –
–
1
1
28.2 kcal mol . However, if one removes the Cu(I) ion from the product, it leaves a
dianion (Fig. S2, Supplementary information), which is not what we observed in our
experiment or spectroscopic measurements including the crystal structure analysis. In
addition, the overall pathway in Fig. S2 is highly unfavorable, as the rate-limiting first
‡
–1
step has a large total barrier (ꢀ
G
= 26.0 kcal mol ) and very unfavorable
–1
endergonicity (ꢀ = 51.3 kcal mol ) rendering this mechanism very unlikely.
G
R
Therefore, we can conclude that the unexpected 5-alkylethynyl derivatives M9 cannot
be obtained within the same pathway as the expected M2 shown in Fig. 2.
12

ACCEPTED MANUSCRIPT
In order to elucidate the precise mechanistic scenario that would give bicyclic 5-
alkylethynyl derivatives, we must recall that Pd(0), instead of catalyzing hetero-
coupling among alkyne A1 and 5-iodoouracil M5 to give M1, could facilitate the
homo-coupling of two A1 molecules to yield symmetrical 1,3-diyne A2 (Fig. 1) [27],
which can, as our results will show, enter the reaction with M5 to give target products
M9. Still, the reaction of neutral 5-iodouracil M5 with A2 is feasible, but unlikely. The
–1
transition state for the C2(A2)–C5(M5) bond formation is 42.6 kcal mol above the
–
1
reactants (νIMAG = 455
i cm ) with C2(A2)–C5(M5) and C5(M5)–I bonds of 1.928 and
2.527 Å, respectively. This very high barrier is slightly reduced in N3-deprotonated
–
‡
–1
–1
M5 to ꢀ
G
= 38.7 kcal mol (ν
= 334i cm ) and the transition state becomes
IMAG
more compact with C2(A2)–C5(M5) and C5(M5)–I bonds of 1.742 and 2.344 Å,
respectively. However, the calculated barrier is still too high for the efficient process.
This goes along with the calculated high pKa(M5)N3 = 56.8. We note in passing that
any attempts to model the reaction starting with the C1(A2)–O4 bond formation in
–
either M5 or M5 did not give any stable intermediates or the corresponding transition
state structures. The presence of Cu(I) ions reduces the pKa value of M5 to 16.4 with
–1
Cu(I) bound to the deprotonated N3 atom at 1.907 Å and ꢀGBIND = –80.9 kcal mol .
This would suggest that, under reaction conditions, M5 deprotonation is very feasible
process when Cu(I) ions are present. However, when Cu(I) is bound to the
–
deprotonated M5 , it deprives the electron density from 5-iodouracil making the whole
system less reactive towards A2, in addition to the fact that the most stable reactant
–
complex involves Cu(I) binding the 1,3-diyne A2 to the opposite site of M5 (as in
M6, Fig. 1). This makes the subsequent reaction between the two systems highly
‡
–1
–1
unlikely. For example, from M6 it requires ꢀ
G
= 61.5 kcal mol (ν
= 455i cm )
IMAG
–
to reach the transition state for the C2(A2)–C5(M5 ) bond formation, which is way too
high. Therefore, one must look into the reactivity of neutral M5, which allows M7 as
the most stable reactant complex, where Cu(I) is bonded to C1 and C2 atoms of A2 at
1.945 and 2.317 Å, respectively, with Cu(I)–C1–C2 plane being practically parallel to
the plane of the six-membered ring of M5. With this, the Cu(I) ion activates C1–C2
triple bond for the nucleophilic attack and two scenarios are possible: the approach of
the carbonyl O4(M5) atom towards the C1(A2) atom (Fig. S3, Supplementary
information), and the C5(M5) atom towards the C2(A2) atom (Fig. 3). As our results
demonstrate, the first option is less feasible because of the following. The O4(M5)–
‡
–1
C1(A2) bond formation is associated with a barrier of ꢀ
G = 15.3 kcal mol (νIMAG =
13

ACCEPTED MANUSCRIPT
–
1
268i cm ) to give the adduct with Cu(I) bound to the C2(A2) atom at 1.902 Å, from
which all attempts to model the subsequent ring closure failed and resulted in the
formation of CuI and separated M5 and A2. For the latter reaction to be possible, an
+
intramolecular rearrangement must occur, involving a shift of Cu from C2 to C4 in
–
1
A2, but this gives 4.6 kcal mol less stable isomer. The subsequent barrier is high,
‡
–1
–1
ꢀG
= 24.8 kcal mol (νIMAG = 313
exothermic, the overall barrier for this process assumes ꢀ
rendering this process unlikely. After the formation of M8, the system needs to lose the
CuI molecule and be deprotonated at the N3–H position to give the final M9
i cm ), and although the reaction is highly
‡
–1
G
= 42.3 kcal mol (Fig. 3),
.
–1
Collectively, this requires 26.1 kcal mol , and the details of that will be presented in
the next section describing feasible mechanism, where the same requirements exist.
Fig. 3. Free energy profile for the formation of the 5-alkylethynyl-6-alkyl-furo[2,3-
d]pyrimidine-2-ones initiated by the carbon–carbon bond formation.
The most feasible mechanism producing M9 from M5 and A2 is Cu(I)-
catalyzed process depicted in Fig. 3, which is initiated with the C5(M5)–C2(A2) bond
–1
formation. Bringing all three components to M7 in favorable (18.5 kcal mol ), with
the C5(M5)–C2(A2) distance of 3.602 Å. In the transition state, the latter is reduced to
1.999 Å, while the C5(M5)–I bond elongates from 2.134 to 2.191 Å, and the Cu(I) ion
–1
is bound to C1(A2) at 1.925 Å. The barrier for this is 21.9 kcal mol (νIMAG = 405
i
–
1
cm ). Interestingly, after the transition state, the system relaxes through a series of
events occurring in a concerted manner, involving the C5(M5)–C2(A2) bond
14

ACCEPTED MANUSCRIPT
–
formation, abstraction of the I leaving group by the Cu(I) ion to give CuI, and the
five-membered ring closure through the O4(M5)–C1(A2) bond formation. This highly
–1
exorgenic sequence (ꢀ
G = –68.5 kcal mol ) produces bicyclic compound with CuI
bound to the C7–C8 double bond, which is further stabilized to M8 by the
intramolecular rearrangement of the CuI molecule. In order to evaluate whether M8 is
first deprotonated or loses CuI to afford the final M9, one needs to consider the
corresponding p
M8) = 11.0 and ꢀ
M8, the binding of CuI changes to ꢀGBIND = –11.1 kcal mol , while if one removes
CuI from M8, the p is increased to p M8) = 14.1. This analysis indicates the pK
a
a
K values and CuI binding free energies. For M8, these assume
–
1
a
pK (
GBIND = –8.3 kcal mol . On the other hand, if one deprotonates
–1
K
a
a
K (
values undergo larger change in a direction of increasing the energy requirements for
these processes to occur, thus suggesting M8 is first deprotonated and then loses CuI.
Since no measured autoprotolysis constant of toluene exists in the literature, we
followed observations by Kocaoba and co-worker
zero. Therefore, from the value of p M8) = 11.0 we estimated that it requires 15.0
kcal mol for deprotonating M8 in toluene leading to a system requiring further 11.1
[28] who suggested it is close to
a
K (
–
1
–
1
kcal mol to remove CuI (Fig. 3) to give the unexpected 5-alkylethynyl-6-substituted
furo[2,3- ]pyrimidine-2-ones M9. On the overall, this mechanism is associated with a
R
barrier of ꢀ = 21.9 kcal mol and very favorable exergonicity of ꢀG = –49.5 kcal
d
‡
–1
G
–1
mol , both making it highly probable. Comparing it to the process leading to the
expected M2 derivatives (Fig. 2), we conclude that the formation of unexpected M9 is
–
1
related with 8.1 kcal mol higher activation free energy, meaning that it is around six
orders of magnitude slower process, being a predominant factor affecting the
distribution of M2 and M9 products. Still, with a particular choice of substituents on
the reacting alkynes A1 and thereof homocoupled 1,3-diynes A2, more favored
exergonicity of the pathway producing systems of the M9-type could compensate or
even overcome differences in the kinetic parameters of both processes and lead to a
different ratio of final products.
2
.3. X-ray crystal structure analysis
The formation of the 5,6-disubstituted furo[2,3-d]pyrimidine-2-one was
unambiguously confirmed by the X-ray structure of 34a (Fig. 4a), where two
cyclopropane rings are bonded to bicyclic furo[2,3- ]pyrimidine-2-one scaffold. One
ring is directly bonded to the C6 atom of the bicyclic ring, while the second one is
d
15

ACCEPTED MANUSCRIPT
bonded to the C5 atom via the ethynyl linker. The third substituent of the
furopyrimidine, (1-benzyl-1,2,3-triazol-4-yl)methyl moiety, is connected to the N3
atom. Search of Cambridge Structural Database
[
29
]
revealed that this is the first
]pyrimidine-2-one,
structure comprising three main structural elements, furo[2,3-
d
cyclopropane and triazole rings. The cyclopropane C9−C11 ring and five-membered
O2/C5/C6/C7/C8 ring are almost perpendicular to each other. The dihedral angle
o
between the mean planes of the rings is 84.6(2) . Furo[2,3-
d]pyrimidine-2-one ring is
planar, with the largest ring atom deviation from the mean plane of 0.019(2) Å.
Because of bicyclic ring formation, the N1
−
C7−C8 bond angle in the six-membered
o
N1/C2/N3/C4/C7/C8 ring is significantly widened and amounts 128.92(18) .
a)
b)
Fig. 4. (a) Molecular structure of 34a, with the atom-numbering scheme. Displacement
ellipsoids for non-hydrogen atoms are drawn at the 30% probability level. (b) A part of the
crystal structure of 34a, showing C
−H···O and C−H···N intermolecular hydrogen bonds.
Only major component of disordered atoms is presented in both figures.
o
However, the sum of all endocyclic bond angles in the ring is ca. 720 , as expected for
the six-membered aromatic ring. As strong hydrogen-bonding donors are absent in this
compound, the molecules of 34a are linked only by weak interactions, two C
−H···O hydrogen
bonds, one C H···N hydrogen bond, one C H··· and one ··· interaction (Table S1,
−
−
π
π
π
Supplementary information). Two of the abovementioned interactions, the C20···N1 and
C20···O1 hydrogen bonds form dimers (Fig. 4b), which are further linked by the C9···O1
16

ACCEPTED MANUSCRIPT
hydrogen bond and one C
−
H···
π
interaction into two-dimensional sheets (Fig. S4 and S5,
··· interaction extends two-dimensional sheets into three-
Supplementary information). One
π
π
dimensional network (Fig. S6; for more detailed description of intermolecular interactions see
Supplementary information).
2
.4. In vitro antiproliferative activity screening
The results of antiproliferative evaluations of 5–37a,b carried out in human tumor cell
lines including lung adenocarcinoma (A549), hepatocellular carcinoma (HepG2), ductal
pancreatic adenocarcinoma (CFPAC-1), cervical carcinoma (HeLa) and metastatic colorectal
adenocarcinoma (SW620), as well as in normal human lung fibroblasts (WI38) and mouse
embryonic fibroblasts (3T3), are presented in Table 1.
Table 1
The growth-inhibition effects in vitro of
5
–
37a,b on selected tumor cell lines and normal
fibroblasts.
a
IC50 (µM)
Compound
c
A549 Hep-G2 CFPAC-1 HeLa SW620 WI38/NIH 3T3 logP
b
5
6
7
8
9
25.45
5.33
7.41
4.47
7.11
3.62
6.87
4.53
12.58
1.64
9.82
7.86
5.63
>100
0.07
0.59
0.37
4.30
3.67
b
b
b
3.67
4.48
5.59
1.52
1.52
1.92
2.48
4.20
2.50
4.20
2.50
5.00
2.90
4.03
2.05
5.92
6.13
95.79 42.73
64.00 68.93
>100
>100
>100
39.54
5.51
>100
b
b
72.90 >100
93.86 100
33.28 >100
8.42 9.87
41.89 94.82
4.50 6.63
49.75 >100
>100 >100
96.49 >100
4.74 18.29
15.07 >100
7.20 >100
50.79
59.34
1
0
1
2
3
4
5
6
7
8
9
0
>100
40.93 19.14
80.68 7.66
78.65 53.49
3.06 5.11
53.35 36.10
>100
≥
b
1
1
1
1
1
1
1
1
1
2
6.70
1.16
b
51.19
4.21
<0.01
<0.01
2.22
55.94
>100
>100
5.53
56.16
71.07
4.69
>100
>100
13.12
5.77
0.16
0.03
0.04
41.31
78.82
>100
83.74
<0.01
3.78
>100
17

ACCEPTED MANUSCRIPT
2
2
2
1
2
3
95.12
50.60
>100
>100
>100
>100
>100
77.15
>100
95.79
6.49
>100
6.79
>100
34.56
>100
73.12
<0.01
55.25
0.07
2.05
2.45
1.96
4.17
2.15
6.62
3.02
2.87
9.96
4.09
3.42
2.73
4.57
3.27
10.36
7.02
2.93
2.24
4.09
2.78
9.87
6.54
4.01
-0.9
>100
24a
39.43 36.94
68.39 14.58
41.83 69.59
24b
6.99
>100
>100
<0.01
>100
45.92
76.46
7.34
2
2
5a
6a
>100
>100
>100
54.47
81.75
>100
>100
35.44
>100
>100
>100
>100
>100
4.59
>100
38.28 39.06
69.79 79.78
38.65 72.48
51.20 49.14
66.45 >100
2
7b
8a
9b
0a
0b
1a
1b
2
9.00
57.09
>100
2
>100
>100
>100
69.20
3.15
3
39.01 39.17
16.07 36.93
3
60.95
>100
>100
72.53
>100
>100
>100
>100
>100
>100
72.54
>100
>100
0.29
3
3
51.98 89.83
60.22 >100
3
3
3
2a
3a
4a
>100
>100
4.81
>100
>100
2.67
>100
>100
6.51
>100
>100
9.72
3
4b
5a
5b
45.00 49.06
26.85 25.54
27.94 27.62
48.45
32.83
23.47
>100
27.30
67.03
0.14
49.98 54.73
25.35 33.86
29.66
17.60
5.20
3
3
9.58
75.52
3
3
6a
7a
>100
>100
73.03 >100
23.39 32.60
59.36 93.47
2.65
21.94 25.84
51.04 68.70
23.39
62.78
0.94
37b
5
-FU
2.80
8.90
8.81
0.08
a
5
0% inhibitory concentration or compound concentration required to inhibit tumor cell proliferation
b
c
by 50%. 5–12 were tested on 3T3 cell line. Values of n-octanol/water partition coefficients logP for
synthesized compounds were calculated by two algorithms with their default settings: ChemAxon
algorithm available within MarvinView Ver. 5.2.6.
5
-Fluorouracil was used as the reference drug. From tested N,N-1,3-bis(1-aryl-
substituted-1,2,3-triazole)–5-bromouracil hybrids, compounds containing p-fluoro- ( ), o-
fluoro- ( ), p-chlorophenyl-substituted 1,2,3-triazole ( ) exhibited marked cytostatic activities
against all evaluated tumor cell lines. Among these, the most potent growth inhibitory activity
was observed with in HeLa cells (IC50 = 1.64 µM). Compounds and proved to be
selective towards HepG2 cells with IC50 values of 4.47 and 3.62 µM, respectively. Notably,
5
6
7
6
5
7
18

ACCEPTED MANUSCRIPT
introduction of 3,5-dichlorophenyl-substituted 1,2,3-triazole in
8 led to a decrease in potency.
On the contrary, within the N-1-(1-aryl-substituted-1,2,3-triazole)–5-bromouracil hybrids,
compound bearing 3,5-dichlorophenyl-substituted 1,2,3-triazole (12) displayed strong
cytostatic effects, particularly in CFPAC-1 cells with IC50 = 5.51 µM. Furthermore, 1,2,3-
triazole–5-iodouracil hybrids 14
, 18, 20 and 22 containing p-fluoro- and p-chlorophenyl-
substituted 1,2,3-triazole exhibited strong inhibitory activities (IC = 4.50–7.20 µM) against
5
0
HeLa cell line. Additionally, 3-propargylpyrimidine–1,2,3-triazole hybrids 14 and 18 showed
marked cytostatic effects (14: IC50 = 3.06 µM; 18: IC50 = 4.69 µM) in A549 cells. Amongst
the N,N-1,3-bis(1,2,3-triazole)-substituted 5-iodouracils, only 1-benzyl-substituted 1,2,3-
triazole–pyrimidine-2,4-dione hybrid (19) inhibited the growth of Hep-G2 cells (IC50 = 5.77
µM). Among the furopyrimidine–1,2,3-triazole series, 34a with 5-cyclopropylethynyl and 6-
cyclopropyl substituents at bicyclic moiety showed to be the most potent, displaying the
highest cytostatic effect (IC50 = 2.67 µM) against HepG2 cells. Both bicyclic furo[2,3-
d]pyrimidine congeners 34a and 34b had better antiproliferative effects than their 5-
iodouracil–1,2,3-triazole analog 23 that showed poor antitumor activity in all evaluated cancer
cell lines. Importantly, our data revealed that the formation of bicyclic furopyrimidine
scaffold in 24
,
26
–
28
,
30
,
34
,
35
,
37 led to the improvement in activities relative to
23. Besides, comparison of cytostatic effects for 5,6-
corresponding 5-iodouracil analogs 20
–
disubstituted and 6-substituted furopyrimidines revealed that symmetrical 5-alkylethynyl-6-
alkyl-substituted compounds (30a, 34a, 37a) had better antiproliferative activities than those
with 6-alkyl substituent (30b, 34b, 37b). Thus, the activity of 5,6-disubstituted
furopyrimidine 34a was ~ 10-fold higher than that of 6-substituted analog 34b. On the other
hand, 6-(3-chloropropyl)-substituted furo[2,3-d]pyrimidines (24b and 35b) were more potent
against CFPAC-1 and HeLa cell lines than corresponding 5,6-disubstituted 24a and 35a
.
However, compounds showing cytostatic activity against cancer cells were also cytotoxic to
normal human lung (WI38) and mouse embryonic (3T3) fibroblasts. Calculated octanol/water
partition coefficients, logPs take values between 2.5 and 4.7 for the most active compounds. It
can be observed that 5,6-disubstitued furopyrimidines had higher lipophilic properties than
corresponding 6-substituted analogs that may contribute to their enhanced activity.
2
.5. The prediction of activity spectra for substances (PASS) analysis
We applied the Prediction of Activity Spectra for Substances (PASS) to explore the
biological potential of selected compounds and to prioritize them for further in vitro studies.
The PASS is an in silico tool used for predicting biological activity spectra for natural and
19

ACCEPTED MANUSCRIPT
synthetic substances, which is based on the structure-activity relationships knowledgebase for
more than 260 000 compounds with known biological activities including drugs, drug
candidates, pharmaceutical leads and toxic systems [30]. The PASS predicts the probable
biological potential of the compound based on its structure and reveals the predicted activities
as the probability of activity (Pa) and inactivity (Pi). The higher Pa value, the lower is the
predicted probability of obtaining false positives in biological testing. The PASS analysis of
compounds with the most potent antiproliferative activities across the panel of tested tumor
cell lines showed high activity score for Wee-1 tyrosine kinase inhibitory effect of 5–7 with
Pa values of 0.56, 0.53 and 0.52, respectively (Table S2, Supplementary information). The
obtained in silico results were further confirmed in vitro by Western blot method to validate
the probable protein target of selected compounds, as described in the following section.
2
.6. Western blot validation of potential protein targets
Results of the PASS prediction prompted us to examine in vitro inhibitory effect of 5–
7
on the activity of Wee-1 tyrosine kinase, an enzyme belonging to the Ser/Thr family of
protein kinases and a negative regulator of the G2/M checkpoint that averts mitotic
progression by inhibiting the activity of cycle-dependent kinase 1 (CDK1) 31 . It was found
[
]
that small molecule compounds with anticancer activities based on pyrimidine and
heteroaromatic ring fused-pyrimidine moiety inhibited Wee-1 kinase at nanomolar
concentrations, e.g. pyrido[2,3-d]pyrimidine (PD0166285)
derivative (MK-1775) 33 , the latter being investigated in clinical studies either as
monotherapy or in combination with standard chemotherapeutics. Since the most potent
cytostatic effects of were detected in HepG2 and HeLa cells, these cell lines were used
for Western blot analyses of Wee-1 kinase activity. Obtained results clearly showed that
[32] and pyrazolo-pyrimidine
[
]
5–7
7
significantly (p < 0.05) reduced the expression level of phospho-Wee-1 kinase indicating
strong inhibition of its activity (Fig. 5).
The same trend, although to a lesser extent, was also evident with 5. Surprisingly, 6
elicited quite the opposite effect on Wee-1 kinase activity as deduced from marked elevation
of its phospho-form. These results could be ascribed to the type of halogen substituent and its
position at phenyl-substituted 1,2,3-triazole subunit in 5–7. Thus, p-chlorophenyl substituent
at the N-1 position of 1,2,3-triazole ring is critical for the inhibitory activity of N,N-1,3-
disubstituted 1,2,3-triazole–5-bromouracil hybrids towards Wee-1 kinase. Interestingly, o-
fluoro-substitution in
6 was detrimental for inhibitory activity of Wee-1 tyrosine kinase. A
20

ACCEPTED MANUSCRIPT
study carried out by Palmer et al.
[34] describing Wee-1 inhibitors based on 4-
phenylpyrrolo[3,4-c]carbazole-1,3-dione template demonstrated that Wee-1 potency and
selectivity were improved by the incorporation of lipophilic functionality at the 2'-position of
the 4-phenyl ring. Similarly, correlation between lipophilicity and inhibitory activity towards
Wee-1 kinase could be observed for tested
triazole scaffold in , which led to the improvement of its inhibitory activity, also increased
the lipophilicity (logP = 4.48) in comparison to p-fluoro- and o-fluoro-substituted (logP =
.67) 1,2,3-triazole counterparts and , respectively.
5–7. Thus, p-chlorophenyl-substituted 1,2,3-
7
3
5
6
a)
b)
Fig. 5. Western blot analysis of predicted protein targets of 5–7. a) Representative Western
blots are shown detecting the cellular levels of phospho-Wee1 kinase, phospho-Sphingosine
kinase 1 (SK1) and acid ceramidase (ASAH) before and after treatment of HepG2 and HeLa
cells with indicated compounds at their 2 x IC50 values for 48 h. Approximate molecular
weights (kDa) are indicated. b) Relative protein expressions, as determined by densitometric
analysis of protein bands and normalized to the alpha-tubulin loading control. Two
independent experiments were performed with similar results. Data are presented as mean
values ± SD.
Based on these observations, it is plausible that mechanism other than Wee-1 kinase
inhibition accounts for cytostatic effect of
6 in HeLa cells. Previous studies have
21

ACCEPTED MANUSCRIPT
demonstrated that enzymes regulating sphingolipid synthesis and turnover are important
mediators of G2/M transition and thereby mitotic entry and progression. For example, down-
regulation of sphingosine kinase 1 in MDA-MB-231 breast carcinoma cells is associated with
mitotic defects characterized by compromised function of spindle checkpoint and cytokinesis
failure
breast and lung cancer cells could be attributed to induction of M phase arrest, which was
associated with down-regulation of Wee-1 kinase 36 . In order to ascertain whether
[35]. Similarly, antiproliferative effects of ceramide kinase inhibitor NVP-231 in
[
]
alterations in the Wee-1 kinase activity induced by tested compounds are linked with
perturbations in sphingolipid metabolism, we examined the activity of sphingosine kinase 1
(SK1) and acid ceramidase (ASAH) in Hela and HepG2 cells. Interestingly, 7, which proved
to be potent Wee-1 kinase inhibitor, interferes with sphingolipid metabolism in HepG2 cells,
as this compound was able to induce a significant decline in the expression levels of both
phospho-SK1 and ASAH pointing to abrogation of their activities (Fig. 5). On the contrary,
and did not exert notable effects on the expression levels of phospho-SK1 and ASAH in
HepG2 and HeLa cells, respectively, which suggests that these two enzymes are not the key
mediators of cellular response to and . Altogether, obtained results suggest that Wee-1
kinase inhibition by could be correlated with the impairment in sphingolipid metabolism
mirrored by negative regulation of SK1 and ASAH activity, whereas and lacking strong
5
6
5
6
7
5
6
inhibitory activity towards Wee-1 did not have impact on critical regulatory points in
sphingolipid pathway. Consistent with the results obtained for inhibitory activity of Wee-1
tyrosine kinase of
5
–
7, replacement of p-fluoro and o-fluoro substituent in
5
and
6 with p-
chloro substituent at the 1-phenyl-1,2,3-triazole subunit in
of SK1 and ASAH activities.
7 was crucial to achieve abrogation
2
.7. Apoptosis detection
Recent study demonstrated that Wee-1 kinase inhibitor pyrido[2,3-d]pyrimidine
PD166285) induced apoptosis in human hepatocellular carcinoma cells in a concentration-
dependent manner 37 . In order to investigate whether antiproliferative effects of
identified as the Wee-1 kinase inhibitor, as well as the effects of and could be associated
with induction of apoptosis, Annexin V assay was performed as previously described 10
(
[
]
7,
5
6
[
]. 7
exerted a profound effect in the induction of apoptosis in HepG2 cells in comparison with
untreated cells as evidenced from marked reduction in the viable cell population by 44.2%
with concomitant increase in early apoptotic and secondary necrotic cells by 34.2% and 10%,
22

ACCEPTED MANUSCRIPT
respectively (Table 2, Fig. 6).
5 had weaker pro-apoptotic effect in the same cells as
evidenced by a moderate decline in viable cells by 21.3% accompanied by the increase of
early apoptotic cells by 26% as compared to control cells (Table 2).
Table 2
Results of Annexin V assays for apoptosis detection of
5–7.
a
a
HepG2 cells (%)
HeLa cells (%)
Control
0
5
7
Control
2.3
6
Late apoptotic/primary necrotic cells
Viable cells
0
0
1.8
94.2
0.6
72.9 50
26.6 34.8
0.5 15.2
75
71.3
23.9
3
Early apoptotic cells
20.4
2.3
Secondary necrotic cells
5.2
a
The percentages of viable cells (PI-/Ann V-), early apoptotic cells (PI-/Ann V+), late apoptotic/primary necrotic
cells (PI+/Ann V+) and secondary necrotic cells (PI+) after 48 h treatment with 5–7 at their 2 x IC50 values.
a) Untreated HepG2 cells
b) HepG2 cells treated with 7
Fig. 6. Detection of apoptosis induced by
7 in HepG2 cells using Annexin V-FITC assay.
Cells were visualized by fluorescence microscope at 40x magnification before and after
treatment with 2 x IC value of for 48 h. PI staining was used as a nuclear marker. Shown
7
5
0
here are bright-field images (upper left micrographs), early apoptotic cells (PI-/Ann V+, upper
right micrographs), secondary necrotic cells (PI+, lower left micrographs) and late
apoptotic/primary necrotic cells (PI+/Ann V+, lower right micrographs).
In HeLa cells,
6 did not induce dramatic changes in cell death response. Thus, we
observed slight decline in viable cell population by 3.7% related with an increase in early
23

ACCEPTED MANUSCRIPT
apoptotic and secondary necrotic cells by 3.5% and 0.7%, respectively. This weak anti-
apoptotic effect of
inhibitory activity towards enzymes involved in the regulation of cell growth and survival.
Obtained results suggest that apoptosis is not a major cell death mechanism with , and that
6 is in agreement with Western blot results showing an absence of its
6
probably some other modes of cell death mediate cytostatic effects of this system in HeLa
cells.
2
.8. Mitochondrial toxicity
Drug-induced mitochondrial impairment is associated with severe toxicities in
mammals, especially those associated with the liver, skeletal and cardiac muscle and the
central nervous system 38 . Several drugs withdrawn from the market such as cerivastatin
Baycol), troglitazone (Rezulin) and nefazodone (Serzone) have been shown to severely
[
]
(
interfere with mitochondrial function. Therefore, identification of mitochondrial toxicants
early in the drug discovery process is highly desirable as to avoid potential adverse effects
that may not be obvious in the preclinical and clinical studies due to absence of specific
histopathologic changes, and to prevent financial loss that may arise from post-market
withdrawal. In the present study, we investigated potential mitochondrial toxicity of
5–7 using
previously established protocol 39 . This method relies on the inherent feature of highly
[
]
proliferative cells such as HepG2 cells to use glycolysis for ATP production when grown in
the presence of glucose rather than mitochondrial oxidative phosphorylation despite abundant
oxygen and functional mitochondria. When glucose in cell culture media is replaced with
galactose, cancer cells are forced to revert back to mitochondrial oxidative phosphorylation to
survive because oxidation of galactose to pyruvate via glycolysis yields no net ATP. Thus,
cells grown in galactose become more susceptible to drugs that cause mitochondrial insult.
Our results clearly showed that there were no striking differences in the response between
HepG2 cells grown in glucose versus those grown in galactose for all three tested compounds,
in particular there was not a significant reduction in IC50 values for galactose-grown cells
indicative of mitochondrial toxicity (Table 3). This implies that mitochondrial dysfunction is
not a primary mechanism leading to cell death induced by 5–7, i.e. apoptosis and necrosis
induced by these compounds could be triggered by some other cellular events that do not
involve mitochondrial dysfunction directly.
24

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Table 3
Mitochondrial toxicity testing in HepG2 cells grown in the media containing either glucose or
galactose.
a
a
Compound IC50 (µM) glucose IC50 (µM) galactose
5
6
7
4.78
7.11
3.82
4.57
4.67
7.10
a
Decreased IC50 values in galactose- versus glucose-containing media represent a compound with properties of
potential mitochondrial toxicant.
3
. Conclusions
Both N,N-1,3- (
pyrimidine-2,4-diones (
5
–
8
,
13
14
,
15
16
,
17 and 19) and
N-1-(1,2,3-triazole) tethered
9
–
12
,
,
,
18 and 20 23) containing halogen-substituted and
–
nonsubtituted aromatic subunits were synthesized by copper(I)-catalyzed Huisgen 1,3-
dipolar cycloaddition, among which microwave-assisted click reactions proved to be
more efficient than conventional ones. Furthermore, we have applied palladium-
catalyzed cross-coupling of 5-iodouracil–1,2,3-triazole hybrids (20
the presence of the CuI catalyst and heteroannulation method to generate furo[2,3-
]pyrimidine series 24a,b 37a,b. The stereostructure of 5-cyclopropylethynyl-6-
cyclopropyl-substituted furo[2,3- ]pyrimidine derivative (34a) was unambiguously
confirmed by X-ray crystal structure analysis. The results of the computational analysis
revealed that the 6-substituted bicyclic series (24b 37b) was produced through the
Cu(I)-catalyzed cyclization of the corresponding 5-alkynyluracils associated with a
–23) with alkyne in
d
–
d
–
‡
–1
–1
barrier of ꢀ
G
= 13.8 kcal mol and a reaction energy of ꢀ
G
R
= –13.8 kcal mol ,
‡
whereas the 5-alkylethynyl analogs (24a
–37a) were linked with a higher barrier of ꢀ
G
–
1
=
21.9 kcal mol , yet much favorable change in the reaction energy of ꢀ
G
R
= –49.5
kcal mol . Among the 1,2,3-triazole tethered pyrimidine-2,4-dione derivatives ( 23),
N,N-1,3-bis(1-phenyl-substituted-1,2,3-triazole)–5-bromouracil hybrids containing
fluoro- ( ), -fluoro- ( ) and -chlorophenyl ( ) moiety exhibited the most
–
1
5–
p-
5
o
6
p
7
pronounced cytostatic activities against hepatocellular and cervical carcinoma cells
with potencies better than those of the reference drug 5-fluorouracil. Strong growth
inhibitory activity of
apoptosis and secondary necrosis in these cells. Cell death induced by
by cellular events other than mitochondrial dysfunction, as this compound was not
7
against HepG2 cells could be associated with induction of early
7
was triggered
25

ACCEPTED MANUSCRIPT
shown to be mitochondrial toxicant. Major cytostatic effects of
7 could be attributed to
inhibition of Wee-1 kinase as well as sphingosine kinase 1 and acid ceramidase that
regulate the balance between pro-apoptotic and pro-survival sphingolipids. Taking into
account strong antitumor activity and no mitochondrial liabilities detected with 7, it
represents a good starting point for the development of new and more efficient drugs
for treating hepatocellular carcinoma.
4. Experimentals
4
.1. Materials and methods
All solvents were dried/purified following recommended drying agents and/or
distilled over 3 Å molecular sieves. For monitoring the progress of a reaction and for
comparison purpose, thin layer chromatography (TLC) was performed on pre-coated
Merck silica gel 60F-254 plates using an appropriate solvent system and the spots were
detected under UV light (254 nm). For column chromatography silica gel (Fluka
,
0.063-0.2 mm) was employed, glass column was slurry-packed under gravity. Melting
points (uncorrected) were determined with Kofler micro hot-stage (Reichert, Wien).
Microwave-assisted syntheses were performed in a Milestone start S microwave oven
using glass cuvettes at 80 °C and 300 W under the pressure of 1 bar. Elemental
analyses were performed in the Central Analytic Service, Ruđer Bošković Institute,
1
Zagreb. All elemental compositions were within the 0.4% of the calculated values. H
1
3
and C NMR spectra were acquired on a Bruker 300 and 600 MHz NMR
spectrometer. All data were recorded in DMSO-
d
6
at 298 K. Chemical shifts were
1
referenced to the residual solvent signal of DMSO at
δ
2.50 ppm for H and
δ
39.50
1
3
ppm for C. Individual resonances were assigned based on their chemical shifts, signal
intensities, multiplicity of resonances and H H coupling constants.
−
4
.2. Procedures for the preparation of compounds
4.2.1. 5-Bromo-N-1,N-3-di(prop-2-yn-1-yl)pyrimidine-2,4-dione (1) and 5-bromo-N-1-
(prop-2-yn-1-yl)pyrimidine-2,4-dione (2)
To the solution of 5-bromouracil (1 g, 5.2 mmol) in dimethylformamide (DMF) (20
mL), NaH (125.6 mg, 5.2 mmol) were added and reaction mixture was stirred for 2h.
Then 3-bromopropyne (0.71 mL, 6.3 mmol) was added and the mixture was stirred for
26

ACCEPTED MANUSCRIPT
24 h at room temperature. After 24 h solvent was evaporated and residue was purified
by column chromatography on silica gel using CH
2
Cl
2
: CH
3
OH = 150 : 1, as an
o
o
eluent.
1
(215 mg, 15%, m.p. = 129–130 C) and
2
(453 mg, 38%, m.p. = 199–200 C)
1
were isolated as white powder.
), 4.6 (4H, dd, H-1', H-1'',
1H, t, H-3'', = 2.4 Hz) ppm. C-NMR (75 MHz, DMSO) (δ/ppm): 158.4 (C-4), 149.6
C-2), 143.8 (C-6), 94.9 (C-5), 78.9 (C-2'), 78.4 (C-2''), 77.0 (C-3'), 74.0 (C-3''), 38.9
1: H-NMR (300 MHz, DMSO) (δ/ppm): 8.4 (1H, s, H-
6
J = 2.7 Hz, J = 2.4 Hz), 3.5 (1H, t, H-3', J = 2.4 Hz), 3.2
1
3
(
(
(
J
7 2 2
C-1'), 31.9 (C-1'') ppm. Anal. calcd. for C10H BrN O : C, 44.97; H, 2.64; N, 10.49%.
1
Found: C, 44.89; H, 2.65; N, 10.51%.
2
: H-NMR (300 MHz, DMSO) (δ/ppm): 11.9
(1H, s, N-H), 8.3 (1H, s, H-6), 4.5 (2H, d, H-1', J = 2.7 Hz), 3.5 (1H, t, H-3', J = 2.5
1
3
Hz) ppm. C-NMR (75 MHz, DMSO) (δ/ppm): 159.5 (C-4), 149.7 (C-2), 144.1 (C-6),
95.3 (C-5), 78.2 (C-2'), 76.1 (C-3'), 37.1 (C-1') ppm. Anal. calcd. for C H BrN O : C,
7 5 2 2
36.71; H, 2.20; N, 12.23%. Found: C, 36.77; H, 2.21; N, 12.26%.
4.2.2. 5-Iodo-N-1,N-3-di(prop-2-yn-1-yl)pyrimidine-2,4-dione (3) and 5-iodo-N-1-
(prop-2-yn-1-yl)pyrimidine-2,4-dione (4)
3
and 4 were synthesized following the procedure for the preparation of 1 and 2.
Reagents: 5-iodouracil (5 g, 21 mmol), NaH (483 mg, 21 mmol) 3-bromopropyne (2.8
mL, 25 mmol), DMF (40 mL). After column chromatography using CH
Cl
2 2
: CH
3
OH
o
=
70 : 1, as an eluent compounds
3
(3 g, 46%, m.p. = 199–200 C) and
4
(924 mg,
o
1
1
6%, m.p. = 129–130 C) were isolated as white powder.
3
: H-NMR (300 MHz,
= 1.8 Hz), 3.5 (1H, t, H-3',
= 2.4 Hz) ppm. C-NMR (150 MHz, DMSO) (δ/ppm):
59.0 (C-4), 149.5 (C-2), 147.8 (C-6), 78.6 (C-2'), 78.1 (C-2''), 76.3 (C-3'), 73.4 (C-3''),
DMSO) (δ/ppm): 8.4 (1H, s, H-6), 4.6 (4H, t, H-1', H-1'',
J
13
J
1
6
2
= 2.4 Hz), 3.2 (1H, t, H-3'', J
7 2 2
7.5 (C-5), 38.2 (C-1'), 31.4 (C-1'') ppm. Anal. calcd. for C10H IN O : C, 38.24; H,
1
.25; N, 8.92%. Found: C, 38.18; H, 2.25; N, 8.95%.
4
: H-NMR (300 MHz, DMSO)
(
δ/ppm): 11.7 (1H, s, NH), 8.2 (1H, s, H-6), 4.5 (2H, d, H-1',
J
= 2.4 Hz), 3.4 (1H, t, H-
= 2.4 Hz) ppm. C-NMR (150 MHz, DMSO) (δ/ppm): 160.9 (C-4), 150 (C-2),
48.7 (C-6), 78.3 (C-2'), 75.9 (C-3'), 68.9 (C-5), 36.9 (C-1') ppm. Anal. calcd. for
IN : C, 30.46; H, 1.83; N, 10.15%. Found: C, 30.40; H, 1.82; N, 10.20%.
1
3
3
', J
1
C
7
H
5
2 2
O
4.2.3. General procedure for the synthesis of 5-bromo-N-1,N-3-di[4-(1-aryl-1,2,3-
triazol-4-yl)methyl]pyrimidine-2,4-dione derivatives (
5–8)
27

ACCEPTED MANUSCRIPT
1
was dissolved in DMF and correspoding aromatic azide (2.1 eq), water solution (1
4 2
mL) of sodium ascorbate (0.3 eq) and water solution (1 mL) of CuSO x 5 H O (0.03
eq) were added. The reaction mixture was stirred for 24 h at room temperature. The
progress of the reaction was monitored by TLC. Upon completion, the solvent was
removed under reduced pressure and the residue was purified by column
chromatography.
4
.2.3.1. 5-Bromo-N-1,N-3-di{ [1-(4-fluorophenyl)-1,2,3-triazol-4-
4-
pyrimidine-2,4-dione (
was synthesized according to the general procedure from
-azido-4-fluorobenzene (0.62 mL, 0.31 mmol), sodium ascorbate (9.9 mg, 0.05
yl
]methyl
}
5)
5
1 (40 mg, 0.15 mmol) using
1
-3
4 2
mmol), CuSO x 5 H O (1.1 mg, 4.5 x 10 mmol) and DMF (4 mL). Purification by
column chromatography (CH
2
Cl
2
1
: CH
3
OH = 120 : 1) afforded
5
(42 mg, 52%, m.p. =
o
1
3
5
7
80–181 C) as yellow powder. H-NMR (300 MHz, DMSO) (δ/ppm): 8.8 (1H, s, H-
'), 8.5 (1H, H-3'') 8.5 (1H, H-6), 7.9 (4H, m, H-6',8', H-6'',8''), 7.5 (4H, m, H-5',9', H-
13
'',9''), 5.2 (4H, m, H-1', H-1'') ppm. C-NMR (150 MHz, DMSO) (δ/ppm): 162.5 (C-
', d, = 245.7 Hz, C-F), 162.4 (C-7'', d, 232.5 Hz, C-F), 158 (C-4), 150.1 (C-2), 144.2
= 2.7 Hz, C-F), 122.4 (C-3'),
= 9.2 Hz, C-F), 116.7 (C-6',8', C-6'',8'', d,
4.1 Hz, C-F), 94.5 (C-5), 44.0 (C-1'), 31.4 (C-1'') ppm. Anal. calcd. for
: C, 48.81; H, 2.79; N, 20.70%. Found: C, 48.87; H, 2.78; N, 20.68%.
J
(C-6), 143.5 (C-2'), 143.1 (C-2''), 133.1 (C-4', C-4'', d, J
122.3 (C-3''), 122.2 (C-5',9', C-5'',9'', d,
J
J =
2
22 2 8 2
C H15BrF N O
4
.2.3.2. 5-Bromo-N-1,N-3-di{ [1-(2-fluorophenyl)-1,2,3-triazol-4-
4-
pyrimidine-2,4-dione (
was synthesized according to the general procedure using
yl
]methyl
}
6)
6
1 (70 mg, 0.26 mmol), 1-
azido-2-fluorobenzene (1.08 mL, 0.54 mmol), sodium ascorbate (15.8 mg, 0.08 mmol)
-
3
and CuSO x 5 H O (1.9 mg, 7.8 x 10 mmol) in DMF (5 mL). Purification by column
4
2
2 2 3
chromatography (CH Cl : CH OH = 70 : 1) afforded (82 mg, 58%, m.p. = 165–166
o
1
C) as yellow powder. H-NMR (300 MHz, DMSO) (δ/ppm): 8.9 (1H, s, H-3'), 8.7
(1H, s, H-3''), 8.6 (1H, s, H-6), 7.9–7.7 (4H, m, Ph-H), 7.7 (2H, m, Ph-H), 7.4–7.2 (2H,
13
m, Ph-H), 5.2 (4H, H-1', H-1'') ppm. C-NMR (75 MHz, DMSO) (δ/ppm): 162.9 (C-
5
', C-5'', d,
J
= 243.75 Hz, C-F), 159.1 (C-4), 150.6 (C-2), 144.6 (C-6), 144.2 (C-2'),
= 21 Hz), 132.3 (C-8', C-8'', d, J = 5.7 Hz, C-F),
144.0 (C-2''), 138.2 (C-4', C-4'', d,
J
28