European Journal of Medicinal Chemistry 97 (2015) 582e611

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European Journal of Medicinal Chemistry

journal homepage: http://www.elsevier.com/locate/ejmech

Review article

In search of uracil derivatives as bioactive agents. Uracils and fused

uracils: Synthesis, biological activity and applications
Aleksandra Pałasz*, Dariusz Ciez_
w, Poland
Department of Organic Chemistry, Faculty of Chemistry, Jagiellonian University, Ingardena 3 St, 30-060 Krako

a r t i c l e i n f o a b s t r a c t

Article history: This review article is an effort to summarize recent developments in researches providing uracil de-
Received 4 July 2014 rivatives with promising biological potential. This article also aims to discuss potential future directions
Received in revised form on the development of more potent and specific uracil analogues for various biological targets. Uracils are
19 September 2014
considered as privileged structures in drug discovery with a wide array of biological activities and
Accepted 3 October 2014
Available online 5 October 2014
synthetic accessibility. Antiviral and anti-tumour are the two most widely reported activities of uracil
analogues however they also possess herbicidal, insecticidal and bactericidal activities. Their antiviral
potential is based on the inhibition of key step in viral replication pathway resulting in potent activities
Keywords:
Uracils
against HIV, hepatitis B and C, the herpes viruses etc. Uracil derivatives such as 5-fluorouracil or 5-
Fused uracils chlorouracil were the first pharmacological active derivatives to be generated. Poor selectivity limits
5-Fluorouracil its therapeutic application, resulting in high incidences of gastrointestinal tract or central nervous
Anticancer toxicity. Numerous modifications of uracil structure have been performed to tackle these problems
Antiviral resulting in the development of derivatives exhibiting better pharmacological and pharmacokinetic
Antibacterial properties including increased bioactivity, selectivity, metabolic stability, absorption and lower toxicity.
Researches of new uracils and fused uracil derivatives as bioactive agents are related with modifications
of substituents at N1, N3, C5 and C6 positions of pyrimidine ring. This review is an endeavour to highlight
the progress in the chemistry and biological activity of the uracils, predominately after the year 2000. In
particular are presented synthetic methods and biological study for such analogues as: 5-fluorouracil or
5-chlorouracil derivatives, tegafur analogues, arabinopyranonucleosides of uracil, glucopyranonucleo-
sides of uracil, liposidomycins, caprazamycins or tunicamycins, tritylated uridine analogues, nitro or
cyano derivatives of uracil, uracil-quinazolinone, uracil-indole or uracil-isatin-conjugates, pyr-
imidinophanes containing one or two uracil units and nitrogen atoms in bridging polymethylene chains
etc. In this review is also discussed synthesis and biological activity of fused uracils having uracil ring
annulated with other heterocyclic ring.
© 2014 Elsevier Masson SAS. All rights reserved.

1. Introduction 2008, based on 12C/13C isotopic ratios of organic compounds found

The nitrogen heterocycles in general and pyrimidines in
particular are found in several biologically active natural products
and depict considerable therapeutic potential. The pyrimidine is
one of the most prominent structures found in nucleic acid
chemistry. Uracil is a common and naturally occurring pyrimidine
derivative and one of the four nucleobases in the nucleic acid of
RNA. In RNA, uracil binds to adenine via two hydrogen bonds. In
DNA, the uracil nucleobase is replaced by thymine. Uracil can be
considered as a demethylated form of thymine. Studies reported in
* Corresponding author.
E-mail address: palasz@chemia.uj.edu.pl (A. Pałasz).
in the Murchison meteorite, suggest that uracil was formed extra
terrestrially [1]. In 2009, NASA scientists reported having repro-
duced uracil from pyrimidine by exposing it to UV under space-like
conditions. This suggests that one possible natural original source
for uracil in the RNA world could have been panspermia [2]. In
2012, an analysis of data from the Cassini mission orbiting in the
Saturn system showed that Titan's surface composition may
include uracil [3].
Uracil undergoes amide-imidic acid tautomeric shifts because
any nuclear instability the molecule may have from the lack of
formal aromaticity is compensated by the cyclic-amidic stability
(Fig. 1). The amide tautomer is referred to as the lactama structure,
while the imidic acid tautomer is referred to as the lactim structure.

http://dx.doi.org/10.1016/j.ejmech.2014.10.008
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.
 A. Pałasz, D. Ciez_ / European Journal of Medicinal Chemistry 97 (2015) 582e611 583

O However, we had a problem to classify specified uracil derivative

OH
to a given chapter because in most cases described compounds
4
H
5 3N possessed few substituents in uracil ring.
N
6 2
1
N O
N OH
2. Design strategies
H
amide - lactam imide - lactim 2.1. 5-Halo-uracils and their nucleosides
Fig. 1. Structure of uracil with numbering system. Uracil tautomers.
5-Fluorouracil (5-FU) is antimetabolite of the pyrimidine
analogue type and a well-known anti-tumour agent which has
been widely used in the treatment of solid tumours such as colon or
These tautomeric forms are predominant at pH 7. The lactam breast cancers [9]. Because 5-fluorouracil is similar in shape to
structure is the most common form of uracil. Uracil is a weak acid. uracil, but does not perform the same chemistry as uracil, the drug
In RNA, uracil binds with a ribose sugar to form the ribonucle- inhibits RNA replication enzymes, thereby eliminating RNA syn-
oside uridine. When a phosphate attaches to uridine, uridine 50 - thesis and stopping the growth of cancerous cells. Although 5-FU
monophosphate is produced. There are many laboratory syntheses has had clinical success as a single agent, it has been modified by
of uracil available. The first reaction is the simplest of the syntheses, different ways to synthesize its derivatives which may improve its
by adding water to cytosine to produce uracil and ammonia therapeutic index because of its well-known side effects such as
(Scheme 1) [4]. The most common way to synthesize uracil is by the short half-life, wide distribution, low selectivity, and various toxic
condensation of maleic acid with urea in fuming sulfuric acid side effects. Recently chemists have paid more attention to the
(Scheme 1) [5]. conjugates of 5-FU with a wide spectrum of low or high-molecular-
Uracils are considered as privileged structures in drug discovery weight carriers (Fig. 2).
with a wide array of biological activities, synthetic accessibility and A series of conjugates of 5-fluorouracils (5-FU) and emodin were
ability to confer drug like properties to the compound libraries synthesized by coupling trimethyl emodin with N1, N3 dialkylated
appended on them at N1, N3, C5 and C6 positions [6]. Antiviral and 5-FU [10]. 1-Hydroxy-2-(hydroxymethyl)-6,8-dimethoxy-3-
anti-tumour are the two most widely reported activities of uracil methylanthracene-9,10-dione 3 was prepared using emodin 1 as
analogues [7]; however they also possess herbicidal, insecticidal starting material via methylation and modified Marschalk reaction
and bactericidal activities [8]. In the past two decades, a variety of (Scheme 2). The synthesis of intermediate 4 involved the methyl-
synthetic methods have been employed for the preparation of ation of 3 with dimethyl sulfate in the presence of anhydrous
functionalized uracils. K2CO3. Chlorination of 4 with thionyl chloride led to the compound
The quantity of papers describing the synthesis and biological 5. Subsequently, alkylation of 5-FU with intermediate 5 in the
activity of uracil derivatives is tremendous. We chose these publi- presence of K2CO3 and KI gave N1-substituted 5-FU 6. A final
cations which in our opinion show the main directions of research alkylation of intermediate 6 and the appropriately alkyl halides or
of uracil derivatives nowadays. We divided the next section, benzyl halides yielded the target conjugates 7 (Scheme 2).
describing different derivatives of uracil, into eight chapters: The 5-FU moiety contained various substituents at N3 e position
were linked to the 2-position of trimethyl emodin via a methylene
2.1. 5-Halo-uracils and their nucleosides linkage. Their cytotoxity against three cancer cell lines and one
2.2. Nucleosides of uracil analogues noncancerous cell were studied [10]. The in vitro cytotoxicity of the
2.3. Uracil-heterocycle hybrids conjugates 7 were evaluated over tumour cell lines HO-8910 (hu-
2.4. Tritylated uracil analogues man ovarian cancer cells), SGC-7901 (human gastric cancer cells),
2.5. Benzodioxepinyl-uracils, benzoxathiepinyl-uracils and dia- and HepG2 (human liver caner cells), in comparison with emodin
zepinyl-uracils and 5-FU. Cytotoxicity of some conjugates was also evaluated
2.6. Pyrimidinophanes containing one or two uracil units and against one normal cell line: 293 (human embryonic kidney cells).
bridging polymethylene chains The results revealed that some of conjugates exhibited better or
2.7. Other substituted uracils comparable in vitro anti-tumour activity to 5-FU and emodin and
2.8. Fused uracils low toxicity in normal cell. Compound 7 (R ¼ CH2(2-CNeC6H4)) was
shown to have a broad spectrum of anti-tumour activity against the
tested tumour cell lines and much lower toxic activity toward
normal cell compared to emodin. The structure-activity relation-
ship study showed N3-aromatic substituent was important for their
NH2 O
cytotoxic activity [10].
H
N N Arutyunyan and co-workers [11] prepared N1-mono and N1, N3-
H2O NH3
bis-substituted 5-fluoro, 5-bromo, 5-iodouracils. Heating uracils 8
N O N O
with the corresponding benzyl halides in the presence of K2CO3
H H
produced the target pyrimidines 9 as mixtures of mono- and bis-
cytosine uracil
derivatives that were separated by treatment with KOH solution
O
(Scheme 3).
H
The toxicity, anti-tumour, and antibacterial properties of the
COOH
H2N NH2 H2SO4 N synthesized compounds were investigated. Chemotherapy tests
C 2H2O CO
found that the majority of the studied compounds exhibited a
COOH O N O
statistically significant antitumour activity for sarcomas 45 and 37.
H
It was established that 5-fluoro- and 5-iodouracils exhibited more
Scheme 1. Synthesis of uracil from cytosine and by the condensation of maleic acid pronounced anti-tumour properties than 5-bromouracil de-
with urea. rivatives [11].
584 A. Pałasz, D. Ciez_ / European Journal of Medicinal Chemistry 97 (2015) 582e611

F
O
R
O O N N
HN ( )n H
H F O
N O O
O
O
O N H F
O N
H
O
5-FU O N H
N COOR2
O O
OH O R1
5-FU-podophyllotoxin 5-FU-amino acid ester
conjugates conjugates
O OH
H COOH
H F
N
H H
O N O
H H
( )n O
O O OH
H
O
5-FU cholic acid conjugates

Fig. 2. Structures of 5-FU and its conjugates.

6-Amino-5-chlorouracil and 6-amino-5-bromouracil were the
first thymidine phosphorylase inhibitors to be generated. However,
their relatively less favourable IC50 values did not allow them to be
developed into drug candidates. In the year 2000, a very potent
inhibitor namely 5-chloro-6-[1-(2-iminopyrrolidinyl)methyl]uracil
hydrochloride (TPI) (Fig. 3) was discovered by Fukushima et al. [12].
Thymidine phosphorylase (TP) is an enzyme that catalyses the
reversible phosphorolysis of pyrimidine nucleosides. Besides the
regulation of pyrimidine nucleosides, TP can also induce the
phosphorolysis of several pyrimidine nucleoside derivatives. Some
nucleoside derivatives such as capecitabine and 50 -deoxy-5-
fluorouridine were designed as prodrugs and they are being

OH O OH OCH3 O OH 1. Na2S2O4, CH2O, OCH3 O OH

Me2SO4, K2CO3
MeOH, NaOH, 0oC, 30 min
acetone, reflux, 5h 2. H2O2 OH

HO CH3 H3CO CH3 H3CO CH3

O O O
1 2 3
Me2SO4, K2CO3
acetone, reflux, 16h

OCH3 O OCH3 O OCH3 O OCH3 OCH3 O OCH3

5-FU, K2CO3, KI SOCl2, CH2Cl2
N NH DMSO, rt, 5h rt, 30 min OH
Cl

H3CO CH3 O H3CO CH3 H3CO CH3

O F O O
6 5 4
alkyl halide or benzyl halide
K2CO3, KI, DMSO, rt, overnight

OCH3 O OCH3 O
R = CH3, CH2CH3, CH2CH2CH2CH3,
R
N N CH2CH=C(CH3)2, CH2CH=CHCH3,
CH2Ph, CH2CH2Ph,
H3CO CH3 O CH2(2-F-Ph), CH2(2-CN-Ph)
O F
7

Scheme 2. Synthesis of 5-fluorouracil and emodin conjugates 7.

 A. Pałasz, D. Ciez_ / European Journal of Medicinal Chemistry 97 (2015) 582e611
O O
ArCH2Cl, K2CO3
H R o R2 R phosphoroditriazolide 23 was prepared by reaction of 4-
N DMF, 90-100 C, 4-5h N chlorophenyl phosphorodichlorate 21 with 1,2,4-triazole 22 in the
O N O N
H R1
8 9
R = H, F, Br, I
R1 = CH2(2',5'-Me2)Ph, CH2(2',4'-Me2)Ph, CH2(2'-Cl)Ph, CH2(4'-Cl)Ph
R2 = H, CH2(2',5'-Me2)Ph, CH2(2',4'-Me2)Ph, CH2(2'-Cl)Ph, CH2(4'-Cl)Ph
Scheme 3. Synthesis of N1-mono and N1, N3-bis-substituted 5-fluoro-, 5-bromo, 5-
iodouracils 9.
converted to the corresponding parent drugs by TP. It has been
reported in literature that TP also possesses other functions related
to cancer biology: it can stimulate tumor angiogenesis, induce tu-
mor metastasis and promote tumor growth by preventing
apoptosis. Therefore, TP is a target for developing inhibitors that
may be used as therapeutic agents for chemotherapy.
Sun and co-workers [13] designed 5-chlorouracil-linked-pyr-
azolo[1,5-a][1,3,5] triazines as new thymidine phosphorylase in-
hibitors. The intermediate 5-chloro-6-chloromethyluracil 14 was
synthesized by 4-step reaction (Scheme 4).
A series of second bicyclic intermediates, namely pyrazolo[1,5-
a][1,3,5]triazin-2-thioxo-4-one 15, was obtained from various
substituted 3-aminopyrazoles. These two intermediates 14 and 15
were coupled finally in the presence of sodium ethoxide and
methanol to yield the desirable target compounds 16 (Scheme 4).
The methylthio coupling spacer was found to be suitable in
enabling the interaction of the two fragments at the active site and
allosteric site of the enzyme. The best coupled compound 16 (R ¼ 4-
SF5-phenyl) inhibited the thymidine phosphorylase with an IC50
value as low as 0.36±0.1 mM [13]. In addition, this compound
demonstrated a mixed-type of enzyme inhibition kinetics, thus
suggesting that it might indeed potentially bind at two different
sites on the enzyme.
The introduction of fluorine atoms into organic molecules usu-
ally promotes dramatic changes in their biological properties. This
strategy has been used to synthesize biologically active fluorinated
nucleotides and nucleosides. Lewandowska and co-workers [14]
synthesized a series of 4-chlorophenyl N-alkyl phosphoramidates
of 30 -azido-20 ,30 -dodeoxy-5-fluorouridine 25 by phosphorylation of
30 -azido-20 ,30 -dideoxy-5-fluorouridine 20 with 4-chlorophenyl
phosphoroditriazolide 23 followed by a reaction with appropriate
amine (Scheme 5). 5-Fluoro-20 -deoxyuridine 17 was converted into
2,30 -anhydro-50 -O-benzoyl-5-fluoro-20 -deoxyuridine 18 by a one-
pot transformation involving Mitsunobu reaction. Ring opening of
derivative 18 with lithium azide in hexamethylphosphoramide
(HMPA) in the presence of p-toluenesulfonic acid (PTSA) afforded
uridine 19. 50 -O-benzoyl group was removed from compound 19 by
O O
H Cl H Cl
N N
O N O N
H N NH HCl H S
NH HCl
TPI S-(5-chlorouracil-6-ylmethyl)
thiourea hydrochloride
Fig. 3. Examples of 5-chlorouracil analogues e thymidine phosphorylase inhibitors.
585
treatment with methanolic ammonia to give 20. 4-Chlorophenyl
presence of triethylamine. Reaction of compound 23 with 20 in the
presence of pyridine afforded reactive intermediate 24 which was
treated in situ with appropriate amine to give the desired products
25 in 67e86% yield (Scheme 5).
4-Chlorophenyl phosphoroditriazolide 23 as a phosphorylating
agent was more selective than its dichloro counterpart and its use
did not result in the formation of symmetrical (5-50 )dinucleoside
phosphates. Prepared compounds 25 were evaluated for their
cytotoxic activity in three human cancer cell lines: cervical (HeLa),
oral (KB) and breast (MCF-7) using the sulforhodamine B(SRB)
assay. The highest activity in all the investigated cancer cells was
displayed by phosphoramidate 25 (R ¼ CH2CH3) with the N-ethyl
substituent and its activity was much higher than of the parent
nucleoside. Also compound 25 (R ¼ CH2CCH) with the N-propargyl
substituent exhibited good activity in all the used cell lines [14].
Kiritsis et al. [15] described the total and facile synthesis of 30 -C-
cyano and 30 -C-cyano-30 -deoxy pyrimidine pyrano nucleosides.
Reaction of 3-keto glucoside 26 with sodium cyanide gave the
desired precursor 3-C-cyano-1,2:5,6-di-O-isopropylidene-a-D-glu-
cofuranose 27 (Scheme 6). Hydrolysis followed by acetylation led to
the 1,2,3,4,6-penta-O-acetyl-3-C-cyano-D-glucopyranose 29. Com-
pound 29 was condensed with silylated 5-fluorouracil or uracil and
deacylated to afford the 1-(30 -C-cyano-b-D-glucopyranosyl)nucle-
osides 31.
Routine deoxygenation at position 30 of cyanohydrin 27, fol-
lowed by hydrolysis and acetylation led to the 3-C-cyano-3-deoxy-
1,2,4,6-tetra-O-acetyl-D-allopyranose 35. Coupling of sugar 35 with
silylated pyrimidines and subsequent deacetylation yielded the 1-
(30 -C-cyano-30 -deoxy-b-D-allopyranosyl)nucleosides 37. It was
found that 31 (X ¼ F) was endowed with a pronounced anti-
proliferative that was only 2- to 8-fold less potent than that
shown for the 5-fluorouracil. None of the compounds showed ac-
tivity against a broad panel of DNA and RNA viruses [15].
2.2. Nucleosides of uracil analogues
Nucleosides and nucleotides analogues play a pivotal role in
antiviral and anticancer therapy. They are structurally related to the
natural nucleosides bearing modifications at the base and/or at the
sugar moieties. Numerous nucleosides and nucleotides analogues
have been developed for medicinal purposes. Many of which have
been used in the clinic for the treatment of human immunodefi-
ciency virus (HIV), hepatitis B virus (HBV), herpes simplex virus
(HSV), hepatitis C virus (HCV) and various cancers as anticancer and
antiviral drugs. The preparation of molecules that mimic the
structures of nucleic acids or their building blocks has provided
many therapeutically useful compounds.
Wigerinck et al. [16] reported that uracil nucleosides bearing
thienyl or furanyl substituents exhibit interesting biological
O
H N
Cl N N
O R
S N
NH2 N N
H H
O
3H-2-(5-chlorouracil-6-methylthio)-
pyrazolo[1,5-a][1,3,5]triazin-4-ones
586 A. Pałasz, D. Ciez_ / European Journal of Medicinal Chemistry 97 (2015) 582e611

O O NaBH4 O O
H SeO2, AcOH H MeOH/H2O H SOCl2
N H
reflux N 0oC N reflux N

O N O N CHO O N O N
H H H OH H Cl
10 11 12 13

SOCl2, AcOH

O O
O
H EtONa, MeOH H Cl
N H N N
Cl N N reflux N N +
O R
S N R O N
S N
N N H Cl
H H 16
15 14
O
R = H, Me, C(Me)3, Cl, Br, I, CN, COOEt, CF3, 4-FPh, 4-ClPh,
4-CF3Ph, 4-MePh, 4-tert-BuPh, 4-MeOPh, 4-CH3CH2OPh, Ph, 4-SF5Ph

Scheme 4. Synthesis of 5-chloro-6-chloromethyluracil 14 and 3H-2-(5-chlorouracil-6-methylthio) pyrazolo[1,5-a]-1,3,5-triazin-4-ones 16.

properties. The coupling reactions between 5-iodo-20 -deoxyur-
idine 38 and the trialkylstannyl derivatives of the heterocycles 39
were carried out in a coordinating solvent (THF, dioxane) and a
Pd(II) catalyst was needed (Scheme 7). When a non-coordinating
solvent (toluene) was used, Pd (0) catalyst was used. 5-(Thien-2-
yl)-1-(b-D-arabinofuranosyl)uracil 44 was obtained in the same
way. The protective groups were removed with ammonia in
methanol. Halogenation of the furan-2-yl or thien-2-yl substituents
with Br2 in CCl4 and N-chlorosuccinimide in pyridine gave 5-
halogenated derivatives 41 (Scheme 7).
5-(Thien-2-yl)- and 5-(furan-2-yl)-20 -deoxyuridines 40 and 5-
(thien-2yl)-1-(b-D-arabinofuranosyl)uracil 44 have been found to
exhibit marked activity against herpes simplex virus type 1 (HSV-
1). Substitution of the heterocyclic ring of 5-(thien-2-yl)-20 -deox-
yuridine has resulted in 5-halogenated analogues 41, equipotent to
(E)-5-(2-bromovinyl)-20 -deoxyuridine (BVDU, brivudin) against
HSV-1 and significantly active against varicella-zoster virus (VZV)
[16].
Rai et al. [17] prepared of 5-[1-(2-haloethyl(or nitro)ethoxy-2-
iodoethyl)]-20 -deoxyuridines 46 using 5-vinyl-20 -deoxyuridine 45
as starting compounds (Scheme 8). The region specific reaction of
45 with iodine monochloride and an alcohol provided the target
compounds 46. The products 46 were prepared as mixture of two
diastereoisomers in ratio 1:1, which differ in configuration at the 1
position of the 5-substituent.
These analogues were evaluated in vitro for inhibitory activity
against thymidine-kinase (TK) positive and negative strains of
herpes simplex virus type-1. The compounds 46 were either weak
or non-inhibitory to HSV-1 replication. All compounds 46 exhibited
low host cell cytotoxicity [17].
Kim and Hong [18] synthesized a series of fluorocyclopropyl
nucleoside 55 starting from acetol using SimmonseSmith reaction

O O O O
H F F H F H F
N N N N
DIAD, PPh3
LiN3, PTSA
PhCO2H, DMF MeOH, NH3
HO O N BzO O HMPA, 120 C BzO O N
N o HO O N
O rt, 84% O O rt, 12h, 91% O
3h, 65%

OH N3 N3
17 18 19 20
N TEA, CH3CN
O O
rt 30 min
N N
Cl O P Cl Cl O P N
N
Cl N N
H
N
21 22 23
N
O O
H F H F
pyridine N N
O O
rt 1h R-NH2, rt, 1h
20 23 Cl O P O O N Cl O P O O N
O O
N NH
N R
N N3 24 N3 25

R = Me, Et, CH2CF3, CH2CH2CH3, CH2CH=CH2,

CH2CCH, CH2CH2CH2N3, CH2CH2CH2CH3,
CH2CH2CH2CH2N3' (S)-CH(CH3)CO2CH3

Scheme 5. Synthesis of 4-chlorophenyl N-alkyl phosphoramidates of 30 -azido-20 ,30 -dodeoxy-5-fluorouridine 25.

 A. Pałasz, D. Ciez_ / European Journal of Medicinal Chemistry 97 (2015) 582e611 587

NaCN, NaHCO3 Phenyl chlorothionoformate

O O OH O OC(S)OPh
O O H2O/Et2O O O Et3N, DMAP, CH3CN, 0oC O O

O O O
O O CN 27 O CN 32 O
26
+
Amberlite IR 120 (H )
OR H2O/MeOH Bu3SnH
AIBN, toluene
O 100oC
RO
RO
OR O
OR O O
CN
28 R = H O
Ac2O
CN 33 O
pyridine
29 R = Ac
+
Silylated base Amberlite IR 120 (H )
CH3CN, Me3SiOSO2CF3 H2O/MeOH

OH OH OR
X X Silylated base
N N CH3CN, Me3SiOSO2CF3 O
OAc OAc RO
O N O O N O OR
AcO AcO CN OR
AcO
OAc OAc
CN CN Ac2O 34 R = H
30 X = H, F 36 X = H, F pyridine
35 R = Ac
N 2H 4 H 2O
NH3/MeOH
AcOH, pyridine
OH OH
X X
OH N N
OH
O N O O N O
HO HO
HO
OH OH
CN CN
31 X = H, F 37 X = H, F

Scheme 6. Synthesis of 30 -C-cyano and 30 -C-cyano-30 -deoxy pyrimidine pyrano nucleosides 31 and 37.

as a key step (Scheme 9). A number of nucleosides comprising the made with the purpose of improving or enhancing the antiviral
cyclopropyl sugar moiety have been synthesized as conforma- activity of some of them [18]. Methylene-spacered cyclopropyl
tionally constrained analogues of acyclonucleosides. Regarding nucleosides have shown potent antiviral activity, particularly
cyclopropyl derivatives, several structural modifications have been against the human cytomegalovirus (HCMV) [18]. As a part of a

O O
H I O
H
N N X H
N X Y
RO O N Pd(Ph3P)4 or
RO O N
O PdCl2(Ph3P)2 O Br2 or NCS RO O N
O
X SnMe3
OR 38 39 OR 40
OR 41
R = H, 4-MeC6H4CO, CH3CO
X = O, S Y = Br, Cl

O O
H I H
N N S

RO O N RO O N
O RO Pd(Ph3P)4
O RO
S SnBu3
OR 42 43 OR 44
R = CH3CO

Scheme 7. Synthesis of 5-(thien-2-yl)- and 5-(furan-2-yl)-20 -deoxyuridines 40 and their 5-halogenated analogues 41. Synthesis of 5-(thien-2-yl)-1-(b-D-arabinofuranosyl)uracil 44.
588 A. Pałasz, D. Ciez_ / European Journal of Medicinal Chemistry 97 (2015) 582e611

O O OR methylene-a-D-glycero-hex-2-enopyrano)uracil 73. Commercially

H CH CH2 H C CH2I
N N
H condensed with silylated uracil, deacetylated and acetylated to
HO O N 1. ICl HO O N
O 2. R-OH O (Scheme 10). Two different synthetic routes were investigated for
OH 45 OH 46
R = CH2CH2Br, CH2CH2Cl, CH2CH2NO2
CH2CBr3, CH2CCl3
Scheme 8. Synthesis of 5-[1-(2-haloethyl(or nitro)ethoxy-2-iodoethyl)]-20 -deoxyur-
idines 46.
searching for antiviral agents, novel classes of nucleosides
comprising cyclopropyl backbone and trisubstituted cyclopropyl
nucleosides with a fluorine group at 10 -position were designed and
synthesized to evaluate them against various viruses because
fluorine group might act as a hydrogen bonding acceptor at the
active site of their target enzyme [18].
Compound 49 was subjected to reduction conditions using dii-
sobutylaluminium hydride (DIBAL-H) to afford the fluoroallylic
alcohol 50, which then underwent a SimmonseSmith reaction with
Zn(Et)2/CH2I2 to give compounds 51. The sugar moiety was alky-
lated via a nucleophilic substitution reaction by converting the
allylic alcohols 51 to the allylic bromide 52 and by sequential
addition of NBS to a solution of the alcohols and triphenylphos-
phine. The condensation of the allylic bromide 52 with the uracil 53
in DMF with cesium carbonate as a basic catalyst afforded the
nucleoside derivative 54. The deprotection of the tert-butyldime-
thylsilyl grup (TBDMS) using tetrabutylammonium fluoride (TBAF)
in tetrahydrofuran (THF) gave the desired fluorocyclopropyl
nucleoside 55 (Scheme 9). Prepared compounds were tested
against several viruses such as the HIV (MT-4 cells), HSV-1 (herpes
simplex virus type 1: CCL-81), HSV-2 (herpes simplex virus type 2;
CCL-81) cells, and HCMV (human cytomegalovirus; AD-169). The
uracil analogue 55 showed moderate anti-HCMV activity (10.61 mg/
mL in AD-169) [18]. The cis-like uracil analogue 55 showed higher
anti-HCMV activity compared with the trans-like derivative, indi-
cating that this virus might allow the sugar moiety to serve as a
template for phosphorylation as well as for DNA polymerase, which
is unlike other viruses.
Agelis et al. described [19] the total and facile synthesis of the
unsaturated and exomethylene pyrano nucleoside analogues, 1-
(2,3,4-trideoxy-4-methylene-6-O-trityl-a-D-glycero-hex-2-
enopyrano)uracil 66, 1-(2,3-dideoxy-a-D-glycero-hex-2-
enopyranosyl-4-ulose)uracil 72 and 1-(2,3,4-trideoxy-4-
available 1,2,3,4,6-penta-O-acetyl-a-D-mannopyranose 56 was
afford 1-(2,3-O-isopropylidene-a-D-mannopyranosyl)uracil 59
the conversion of 59 into the olefinic derivative 65. Although the
two procedures are quite similar with respect to yields and final
products, the second also leads to the keto-20 ,30 -unsaturated
analogue 73.
All new analogues were evaluated for their anticancer and
antiviral activities using several tumor cell lines and gastrointes-
tinal rotavirus. The antiviral properties of the nucleoside analogues
were examined using a colon adenocarcinoma Caco-2 cell line
infected with gastrointestinal rotavirus as a model virus and AZT
drug as positive control. The cytotoxicity (CC50) of the new com-
pounds was measured on normal human intestinal cell line (H4)
and on a series of human tumor cell, such as human colonic
adenocarcinoma cell line (Caco-2), skin melanoma cell line, and
epithelial breast cancer cell line (MCF-7). All of the compounds
showed direct antiviral effect against rotavirus infectivity. More-
over, uracil 65 was found to be potent in MCF-7 breast carcinoma
cell line. Uracil 65 being sufficiently cytotoxic on carcinoma cells
and also highly selective was identified as a lead compound for
further studies. All new molecules inhibited the growth of Caco-2
cells and showed direct antiviral effect as they were able to
inhibit rotavirus action [19].
Brulikova and co-workers [20] produced uridine analogues
modified at the 5-position with the 5-[alkoxy-(4-nitrophenyl)-
methyl] moiety 76 (Scheme 11). Bases 76 with the highest activity
were transformed into the corresponding nucleosides 79. The
Vorbruggen method which utilizes silylated nucleobases and
strong Lewis acids was used. First the starting compounds were
silylated, next silylated uracils reacted with protected sugar 77 in
the presence of TMSOTf to afford benzoylated ribonucleosides 78
that were formed as a mixture of two diastereoisomers. The di-
astereoisomers were separated and treatment of ribonucleosides
with methanolic ammonia afforded the nucleosides 79.
Bases 76 and nucleosides 79 were tested for their cytotoxic ac-
tivity in vitro against cancer cell lines including drug sensitive (CEM
and K-562) as well as drug resistant (CEM-DNR-B and K-562 TAX)
cell lines and A549 cells as representative of solid tumors. The
cytotoxic activity was slightly increased in some cases by trans-
formation of bases to nucleosides. The activity increases with chain
length in both anticancer as well as antimicrobial activity. The
cytotoxic activity of the nucleosides was not due to cell cycle al-
terations, DNA and/or RNA synthesis [20].
Yu and co-workers [21] designed a series of 20 ,30 -diethanethio-
2 ,30 ,50 -trideoxy-50 -triazoloribonucleosides of uracil and evaluated
0

OH
(EtO)2-POCHFCO2Et 48 CO2Et
DIBAl-H
PO n-BuLi, THF CH2Cl2
O PO F F
PO
H3C
CH3 CH3
47 49 50
P = TBDMS CH2I2, Zn(Et)2
O CH2Cl2
O

N NH N NH Br OH
TBAF uracil 53 PPh3, NBS
O O
F THF, rt CsCO3, DMF, rt F CH2Cl2, rt F
HO PO F PO PO
CH3 CH3 CH3 CH3
55 54 52 51

Scheme 9. Synthesis of fluorocyclopropyl bromide 52 and next fluorocyclopropyl nucleoside 55.

 A. Pałasz, D. Ciez_ / European Journal of Medicinal Chemistry 97 (2015) 582e611 589

OAc H OR OH
N O Me3SiOSO2CF3, HMDS, acetone, 2,2-dimethoxypropane
O O O
+ saccharine, CH3CN, 120-80oC p-toluenesulfonic acid, water
OAc OAc OR OR O O
N
OAc OAc H OR N O OH N O
56 O NH3 57: R = Ac 59
MeOH 58: R = H NH NH
Triphenylmethyl chloride
O pyridine O
OTr
OTr
O Ph3PCH3Br, NaH, OTr
i-amyl alcohol, THF O
H2C O O O
0-25 oC O O O PDC, Ac2O, CH2Cl2
N O O O
N O
62 OH N O
NH 61 NH
60 NH
O
O
TFA, MeOH O

OR OTr OH
iodine-imidazole-Ph3P, O
O O
Tol/DMF 4:1, 80oC formic acid/diethyl ether 1:1
H2C OH OH H2C H2C

N O N O N O

Triphenylmethyl chloride 63: R = H 65 66 NH

NH NH
pyridine 64: R = Tr
Ph3PCH3Br, NaH, O O
O
i-amyl alcohol, THF
0-25 oC
OH
OTr OTr
O
O O
O PDC, Ac2O, CH2Cl2
formic acid/diethyl ether 1:1O
N O
N O OH N O
73 NH
72 NH 71 NH
O
O O
Triphenylmethyl chloride
pyridine
OAc OAc OR
O O iodoform-imidazole-Ph3P, O
pyridine,Ac2O formic acid/CH2Cl2 1:1
59 O O OH OH Tol/DMF 4:1, 120oC
OAc N O OAc N O OR N O
67 68 NH3
NH NH 69: R = Ac NH
MeOH 70: R = H
O O O

Scheme 10. Synthesis of the unsaturated exomethylene 66 and keto pyrano nucleosides 73.

their antitumour activity. Compound 80 was prepared from D-
xylose via a cascade reaction and next it was converted into tosylate
81 followed by treatment with sodium azide to give azido ribo-
furanoside derivative 82 (Scheme 12). Under Huisge-Sharpless
azide-alkyne cycloaddition reaction conditions, conversion of the
azido group in 82 into the triazole ring with phenyl acetylene was
carried out with CuCl, CuBr or CuI as the catalyst. The yield for the
products 83 was higher when CueCuSO4 was used to activate this
1,3-dipolar cycloaddition reaction at room temperature. Under the
Silyl-Hilbert-Johnson glycosylation conditions, derivatives 83 were
treated with trimethylsisilylated uracil in the presence of trime-
thylsilyl trifluoromethanesulfonate (TMSOTf) to give the nucleo-
sides 84 (Scheme 12).
The antitumour activity of these novel nucleosides was tested
in vitro towards the following human cancer cell lines: human
hepatocellular liver carcinoma cell line (HepG2), two types of non-
small cell lung cancer: lung adenocarcinoma (LAC) and squamous
subdivision of epithelial cells (A549), and human cervical carci-
noma cell line (Hela). The compounds 84 with aromatic substituted
triazole rings showed significantly improved activity towards a
broad range of tumor cell lines and those without arene substitutes
were inactive. The molecular structure analysis indicated the po-
tential coplanar relationship between the phenyl moiety and the
triazole ring which results in the conjugate effects. These results
suggested that the conjugation effects of the triazole ring with the
aromatic system are essential for bioactivity [21].
Tzioumaki et al. [22] described the synthesis of pyrimidine
unsaturated keto and exomethylene arabinopyranopyranonucleo-
side analogues as potential antitumour and antiviral agents.
Commercially available 1,2,3,4-tetra-O-acetyl-D-arabinopyranose
85 was condensed with silylated uracil (U), 5-fluorouracil (5-FU) or
5-(trifluoromethyl)uracil (5-CF3U), deacetylated and acetylated to
afford 1-(3,4-O-isopropylidene-a-D-arabinopyranosyl)pyrimidine
analogues 88 (Scheme 13).
590 A. Pałasz, D. Ciez_ / European Journal of Medicinal Chemistry 97 (2015) 582e611

NO2 NO2

O CHO O O
H
N HCl, reflux, 4h H R H
Cl N ROH, reflux, 4h O N
N O
N O N O
H NO2 H H
73 74 75 76
R = CH3, (CH2)nCH3 n = 1-11,
i-Pr, sec-Bu, t-Bu

BzO OAc
O

OBz OBz
77
(CH3)3SiNHSi(CH3)3, (NH4)2SO4,
CF3SO3Si(CH3)3, 1,2-dichloroetane
rt, 2 days
NO2 NO2

O O

R H R H
O N O N
1. separation of diastereoisomers BzO N O
HO N O 2. MeOH/NH3, rt, 6 days
O O

OH OH OBz OBz
79 78

Scheme 11. Synthesis of 5-(alkoxy-(4-nitrophenyl)methyl)uracils 76 and their nucleosides 79.

The conversion of compounds 88 into the new 1-(2,3,4-
trideoxy-2-methylene-a-pent-3-enopyranosyl)nucleoside de-
rivatives of uracils 94 was described [22]. This reactions resulted
also to the 2-keto-3,4-unsaturated analogues 93. The new ana-
logues did not show inhibition of DNA and RNA virus replication in
cell culture. With the exception of the human T-lymphocyte CEM
cells that were inhibited by the 5-fluorouracil derivatives at higher
micromolar concentrations, the murine leukemia L1210, the mu-
rine mammary carcinoma FM3A, and the human cervix carcinoma
HeLa cells were inhibited in their proliferation by some compounds
at concentrations that were in the lower micromolar range. The 20 -
ketonucleoside derivatives 93 were found to be more cytostatic
than the corresponding 20 -exomethylene nucleosides 94. The 5-
fluorouracil unsaturated ketoderivative 93 (R1 ¼ F, R2 ¼ OH) and
the exomthylene derivatives 94 (R1 ¼ F, R2 ¼ OH) and 97 (R1 ¼ F,
R2 ¼ OH) showed antiproliferative activity in the lower micromolar
range. Experimental evidence revealed that these compounds may
act as novel types of 5-fluorouracil releasing prodrugs, and points
to thymidylate synthase as target for their cytostatic action [22].
Novel 5-alkynyl and alkylfurano[2,3-d]pyrimidine glucopyr-
anonucleosides have been synthesized and studied by Kantsadi
et al. [23] as inhibitors of glycogen phosphorylase (GP). Glycogen

TsCl, Et3N NaN3, DMF N3

HO OMe TsO OMe OMe
o
O CH2Cl2, rt, 87% O 105 C, 85% O

SEt SEt SEt SEt SEt SEt

80 81 82

O Alkyne, Cu, CuSO4

BuOH/H2O 1:1
NH
N N N N
N N O N OMe
MeCN, CF3SO2OSiMe3 O
R O silylated uracil R

SEt SEt SEt SEt

84 R = Ph, 4-F-Ph, 4-MeO-Ph, 2-CF3-Ph 83
4-Me-Ph, 3-Me-Ph, COOC2H5, CH2OOCCH3
thiophene-2-yl

Scheme 12. Synthesis of 20 ,30 -diethanethio-20 ,30 ,50 -trideoxy-50 -triazoloribonucleosides of uracil 84.
 A. Pałasz, D. Ciez_ / European Journal of Medicinal Chemistry 97 (2015) 582e611 591

silylated base acetone

Me3SiOSO2CF3/SnCl4 2,2-dimethoxypropane
O O O
1,2-EtCl2/CH3CN p-toluenesulfonic acid
OAc OAc OAc OH
OAc OAc N O O N O
OAc OAc O
N N
85 R1 88 R1
R1 = H, R2 = OH (U) R2 R2
R1 = F, R2 = OH (5-FU) NH3 86 R = Ac Ac2O
R1 = CF3, R2 = OH (5-CF3U) MeOH 87 R = H pyridine PDC, Ac2O
CH2Cl2

O HCOOH/CH2Cl2 O O
(1:1)
OAc OAc
OH N O O N O O N O
OH O O O
N N N
90 R1 R1 95 R1
R2 89 R2 R2

Toluene/DMF (4:1) Ph3PCH3Br, NaH,

iodoform-imidazole-Ph3P t-amyl alcohol, THF
o o
120 C, 90 min 0-25 C

PDC, Ac2O
O O O
CH2Cl2
OR
N O N O O N O
O CH2
N O
N N
R1 93 R1 96 R1
R2 R2 R2
NH3 91 R = Ac Ph3PCH3Br, NaH, HCOOH
MeOH 92 R = H t-amyl alcohol, THF CH2Cl2
0-25oC 1:1
Toluene/DMF (4:1)
O iodoform-imidazole-Ph3P O
o
80 C, 15 min

N O OH N O
CH2 CH2
OH
N N
94 R1 97 R1
R2 R2

Scheme 13. Synthesis of pyrimidine unsaturated keto and exomethylene arabinopyranopyrano nucleoside analogues 93, 94 and 97.

phosphorylase catalyses the first step of intra-cellular degradation
of glycogen to Glc-1-P [24]. As such, GP has a pivotal role in glucose
homeostasis and over last decade it has been validated as an
important target for structure-assisted design of hypoglycaemic
agents [25e27]. The efficacy of such inhibitors on blood glucose
control and hepatic glycogen balance has been confirmed from
biological studies [28e30]. All inhibitors were designed to mimic
the contacts of glucose that localize the closed position of the 280s
loop that blocks the entrance to the active site. The most potent
inhibitors for the catalytic site have hydrophobic groups pointing
towards the direction of b-pocket [25]. The synthesis of the 5-
alkynyl pyrimidine glucopyranonucleosides is illustrated in
Scheme 14. The introduction of 5-alkynyl groups was performed by
reaction of terminal alkynes with the nucleoside 1-(b-D-glucopyr-
anosyl)-5-iodouracil 98 by a Pd(0)-mediated reaction, using the
Sonogashira conditions and yielded 5-ethynyluracil derivatives 99
[23]. Fused bicyclic pyrimidine pyranonucleosides 100 were pre-
pared by electrophilic cyclization catalysed by AgNO3.
Kinetic experiments have shown that most of these compounds
were low micromolar inhibitors of the enzyme. The best inhibitor
was 1-(b-D-glucopyranosyl)-5-ethynyluracil 99 (Ki ¼ 4.7 mM) [23].
Crystallographic analysis of compounds 99 and 100 in complex
with GP revealed that inhibitors with a long 5-alkynyl group
exploited interactions with b-pocket of the active site and induced
significant conformational changes of the 280s loop compared to
GP in complex with compounds with a short 5-alkynyl group. The
results highlight the importance in the length of the aliphatic
groups used to enhance inhibitory potency for the exploitation of
the hydrophobic b-pocket. The best of the inhibitors had also a
moderate effect on glycogeneolysis in the cellular lever with an IC50
value of 291.4 mM.
2.3. Uracil-heterocycle hybrids
The incorporation of heterocyclic compounds, for example 1,2,3-
triazoles as attractive linker units between two pharmacophores to
give bifunctional drugs, have become interestingly useful and
important in constructing bioactive molecules. Kumar et al. [31]
reported the synthesis of uracil-isatin hybrids via azide-alkyne
cycloadditions and their cytotoxic evaluation against three
592 A. Pałasz, D. Ciez_ / European Journal of Medicinal Chemistry 97 (2015) 582e611

R
O
R O O
I H C
OH N C H
OH N OH N
R C CH, CuI, Pd(PPh3)4 AgNO3, acetone
O
HO N O DMF, 55oC, 90 min O rt, 2h O
HO HO N O HO N O
OH HO HO
98 OH OH
99 100
R = C3H7, C5H11, Me3Si, H

Scheme 14. Synthesis of 5-alkynyl and alkylfurano[2,3-d]pyrimidine glucopyranonucleosides 99 and 100.

human cancer cell lines viz. HeLa (cervix), MCF-7 (breast) and
DU145 (prostate) using MTT assay. The synthetic protocol involved
an initial di-alkylation of C-5 substituted uracil 101 using propargyl
bromide 102 to yield the corresponding dipropargylated precursor
103 as shown in Scheme 15. The synthesis of N-alkyl azido isatin
derivatives 106 was based on initial base-assisted N-alkylation of
substituted isatins with dibromoalkane and subsequent reaction
with sodium azide resulting in second precursor 106. The target
compounds 107 were synthesized by utilizing azide-alkyne cyclo-
addition reaction of precursors 103 and 106 (Scheme 15).
The evaluation studies revealed the dependence of cytotoxicity
on C-5 substituents on both uracil and isatin as well as the alkyl
chain length with marked preference of Cl substituent over H, F and
CH3 along with longer alkyl chain length (n ¼ 3). The cytotoxic
profiles revealed that the compounds 107 (X ¼ Cl, R ¼ H) and 107
(X ¼ H, R ¼ Cl) have shown lowest IC50 values, 18.21 and 13.90 mM
respectively, among the test compounds against DU 145 [31]. Most
of the synthesized conjugates exhibited considerable selectivity
against MCF-7 and DU 145 cell lines.
Głowacka and co-workers [32] produced nucleoside analogues
having uracils connected to the 1,2,3-triazole ring by a methylene
linker. The 1,2,3-triazoloacyclonucleotides 113 has been obtained
from diethyl azidomethyl-, 2-azidoethyl-, 3-azidopropyl-, 4-
azidobutyl-, 2-azido-1-hydroxyethyl-, 3-azido-2-hydroxypropyl-
and 3-azido-1-hydroxypropylphosphonates 112 and N1-propargyl
uracils 111 via 1,3-dipolar cycloadditions carried out under micro-
wave irradiation (Scheme 16).
All compounds were evaluated in vitro for activity against a
broad variety of DNA and RNA viruses and cytostatic activity against
murine leukemia L1210, human T-lymphocyte CEM and human
cervix carcinoma HeLa cells. Several compounds of 113 were found
to be the most active towards T-lymphocyte CEM cell proliferation
[32].
Gawad et al. [33] described the synthesis of 3-substituted qui-
nazolin-4(3H)-ones and 3,4-dhydro-quinazolin-2(1H)-one de-
rivatives and their biological evaluation as antitumor agents. 3-
Substituted-2-thioxo-2,3-dihydro-quinazolin-4(1H)-ones 114
reacted with the appropriate phenacyl bromides 115 to yield 3-
substituted-quinazolin-4(3H)-ones 116 (Scheme 17). On the other
hand, the reaction of 114 with dimethyl sulphate in ethanolic so-
dium hydroxide afforded the 2-methylthio derivatives 117. Nucle-
ophilic displacement of the eSCH3 function of 117 by 2-amino-5-
aryl-10,30 ,40 -thiadiazoles 118, produced 3-substituted-quinazolin-
4(3H)-ones 119 (Scheme 17).
The synthesized compounds were subjected to the NCI's
disease-oriented human cell lines screening assay to be evaluated
for their in-vitro antitumor activity. They were used in the full NCI
60 cell lines panel assay which includes nine tumor subpanels
namely; leukemia, non small cell lung cancer, colon, CNS, mela-
noma, ovarian, renal, prostate, and breast cancer cells. Quinazolin-
4(3H)-ones 116 (R1 ¼ 4-CH3O-Ph, R2 ¼ Cl) and 116 (R1 ¼ 4-Cl-Ph,
R2 ¼ CH3O) which represent fused thiouracil system are broad-
spectrum antitumours showing effectiveness toward numerous
cell lines that belong to different tumor subpanels. Those two

O
O
H X K2CO3, DMF
N X
rt, 6h N
Br
O N
O N
102
H X = H, Cl, F
101 103

O dibromoalkanes O O
NaH, DMF NaN3, DMF
R R o R
60oC, 2h 60 C, 3h
O O O
N N N
H ( )n N
104 105 ( )n Br 106 3

O O X O
CuSO4 5H2O O O
N
sodium ascorbate
EtOH/H2O, rt, 8h N N N
103 106 ( )n N N
R O N ( )n
N R
107 N N
n = 2, 3 X = H, Cl, F R = H, F, Cl, Me

Scheme 15. Synthesis of 1,3-dipropargylated uracils 103, N-alkyl azido isatins 106 and uracil-isatin-conjugates 107 using click chemistry.
 A. Pałasz, D. Ciez_ / European Journal of Medicinal Chemistry 97 (2015) 582e611 593

O benzoyl chloride O O
O propargyl bromide
pyridine, CH3CN
H Bz K2CO3 aq
N rt, 12h Bz K2CO3, DMF Bz
N dioxane, rt, 24h N rt, 24h N

N O N O N O N O
H Bz H
108 109 110 111a
CuSO4 5H2O
sodium ascorbate
H2O/EtOH (1:1)
B N
MW, 40-45oC, 10 min N
(EtO)2(O)P X N3 (EtO)2(O)P X N

112 113 B
111
O O
Bz
NH N X = CH2, (CH2)2, (CH2)3, (CH2)4, CH(OH)CH2,
B=
CH2CH(OH)CH2,CH(OH)CH2CH2
N O N O

Scheme 16. Synthesis of 1,2,3-triazoloacyclonucleotides of uracil 113.

quinazoline analogues could be considered as useful templates for
future development to obtain more potent antitumor agents [33].
2.4. Tritylated uracil analogues
Tritylated deoxyuridine analogues and tritylated acyclic uridine
analogues (Fig. 4) are able to inhibit the Plasmodium falciparum
dUTPase (PfdUTPase) enzyme selectively in low micromolar range
[34]. The parasite P. falciparum, transmitted to the human host
through the female mosquito, is the main cause of severe clinical
malaria. The ubiquitous enzyme dUPT nucleotidohydrolase (dUT-
Pase) catalyses the hydrolysis of dUTP to dUMP and can be
considered as the first line of defence against incorporation of uracil
into DNA. Inhibition of this enzyme results in over-incorporation of
uracil into DNA, leading to DNA fragmentation and cell death and is
therefore lethal. By taking advantage of structural differences be-
tween the human and Plasmodium dUTPase, selective inhibitors of
the enzyme can be designed and synthesized with the aim of being
developed into novel anti-parasitic drugs.
Nguyen and co-workers [35] described the successful synthesis
of a variety of analogues of dUMP in which the substituents are
introduced at 30 - and 50 -positions, together with variation in the
heteroatom at the 50 -position. Preparation of 50 -amino-
deoxyuridines 124 was achieved by selective tosylation of the 50 -
position of deoxyuridine 120, displacement with azide, and hy-
drogenation, following a reaction with Ph3CCl (Scheme 18).
The compounds were assayed against recombinant P. falciparum
and Leishmania major enzymes and the human enzyme to give a
measure of selectivity [35]. The compounds were also tested in vitro
against the intact parasites P. falciparum and L. donovani. A number
of potent and selective inhibitors of the P. falciparum dUTPase that
show drug-like properties and represent good leads for future
development were identified. The best inhibitors was the com-
pound 50 -tritylamino-20 ,50 -dideoxyuridine 124 (X ¼ OH)
(Ki ¼ 0.2 mM) with selectivity greater than 200-fold compared to the
human enzyme. The correlation observed between the inhibition of
the enzyme and the inhibition of the parasite growth in vitro
demonstrates that the P. falciparum dUTPase constitutes a valid and
attractive novel target for the development of much-needed new
antimalarial drugs [35].
Gilbert at al [36]. reported in 2006 the discovery of novel uracil-
based acyclic compounds as inhibitors of dUTPase. Compounds
were assayed against both P. falciparum dUTPase and intact para-
sites. A good correlation was observed between enzyme inhibition

O
Br O
R1
N K2CO3 R1
+ N
R2
N S O N S
H R2
115 R1 = Et, Ph, 4-Cl-Ph, 4-CH3O-Ph116
114
R2 = Cl, CH3O
O
(Me)2SO4 H2N
N R2

S N
O O
R1 R1
N K2CO3 N S
+
N
N SCH3 N N N
117 R2 H 119
118 R1 = Et, Ph, 4-Cl-Ph, 4-CH3O-Ph
R2 = Cl, CH3O, N(Me)2

Scheme 17. Synthesis of quinazolinones derivatives 116 and 119.

594 A. Pałasz, D. Ciez_ / European Journal of Medicinal Chemistry 97 (2015) 582e611

O O O

NH NH NH

N O N O N O
O O O
N O Si O
H
HO HO HO
O
O O
NH
NH NH
N O
H N O N O
N N Si O
H

Fig. 4. Tritylated deoxyuridine analogues and tritylated acyclic uridine analogues e PfdUTPase inhibitors.

O O O

NH NH NH

HO N O TsCl, pyridine TsO N O

NaN3, DMF N3 N O
O 0-4 oC O 90oC O

X 120 X 121 X 122

X = OH, F
O O
H2, 5%Pd/C
EtOH/H2O
NH NH
H
R N N O H2N N O
O RCl, pyridine O

X 124 X 123

R = Ph3C, H, (PhO)2PO, (EtO)2P(O)CH2CO, AdamantylCO, CH3CO,

CH3(CH2)3CO, PhCO, Ts, CH3SO2, PhCH2

Scheme 18. Synthesis of tritylated deoxyuridine analogues 124.

and cellular assays. Acyclic uracil derivatives were identified that (Scheme 20). The tritylamino analogues were synthesized by
showed greater or similar potency and in general increased selec- mono-tritylation of the relevant diamines 132 followed by coupling
tivity compared to previously reported inhibitors [35]. The most to 1-methylcarboxyuracil 129 again using EDC as a coupling
active compound reported against the P. falciparum enzyme had a Ki reagent.
of 0.2 mM. Preliminary ADME studies indicated that some of the Chatelain et al. [37] synthesized triphenylmethyl alkylated
lead compounds are drug-like molecules [36]. nucleoside of uracil and 5-chlorouracil. Trityl moieties were
Tritylaminopropyl and tritylaminohexyl analogues 128 (Scheme
19) could be obtained by direct coupling of uracil with suitably Boc2O, Et3N, DCM, rt or
activated aliphatic fragment. Gilbert et al. have shown that trity- PhCH2OCOCl, Na2CO3, H2O, 0oC - rt or
lated deoxyuridine analogues and then subsequently tritylated ( )n TrCl, Et3N, DCM, 0oC R ( )n
H2N OH N OH
acyclic uridine analogues were able to inhibit the P. falciparum
H 126
dUTPase enzyme selectively in the low micromolar range [35,36]. n = 3, 4, 6
125 R = Ph3C, PhCH2O(CO), t-BuO(CO)
These compounds also inhibited parasite growth in the low
micromolar range. The structures of previously discovered selective O

inhibitors of the PfdUTPase (Fig. 4) were modified in 2009 by TsCl, pyridine, 0oC or
NH o
McCarthy et al. by insertion of an amide bond [34]. A series of tri- TsCl, Et3N, DMAP, 0 C

tylated uracil acetamide derivatives were synthesized and assessed N O

R ( )n uracil, Cs2CO3
for inhibition of the enzyme and parasite growth in vitro [34]. These N DMF, 40oC
R ( )n
N LG
compounds were weak inhibitors of the PfdUTPase. The synthesis H
H
of the tert-butyl diphenyl silyl (TBDPS) uracil acetamides 131 was 128 127
carried out by coupling the TBDPS protected amino alcohols 130 to
1-carboxymethyl uracil 129 using EDC as a coupling reagent Scheme 19. Synthesis of acyclic nucleoside analogues 128 as inhibitors of Plasmodium
falciparum dUTPase.
 A. Pałasz, D. Ciez_ / European Journal of Medicinal Chemistry 97 (2015) 582e611 595

O O
O H H2O, KOH reflux, 1h H
N HCl, 4oC, 16h N O
Cl
HO H
N O N O N
H HO 129
EDC, DMF N O
18h
O Si O NH
( )n
O
131
DMF, imidazole
( )n rt, 18h ( )n
HO NH2 Si Cl Si O NH2
n = 3, 4
130

O
H
N

TrtCl, DCM EDC, DMF H N O

( )n rt, 18h ( )n rt, 18h
H2N NH2 N NH2 129 N NH
( )n
n = 2, 3, 4 H
O
132 133

Scheme 20. Synthesis of TBDPS uracil acetamide derivatives 131 and tritylamino uracil acetamide derivatives 133.

attached at various positions of the sugar ring. Synthesis of 5-
chlorouridine 137 was attempted on acetylated uridine 135 using
ceric(IV) ammonium nitrate (CAN) and LiCl (Scheme 21). Com-
pound 136 was deprotected to afford uridine 137. Tritylation of
unprotected ribonucleosides of uridine 134 and chlorouridine 137
was done using triphenylmethyl chloride, affording the 50 -mono-
tritylated, the 20 , 50 - and 30 ,50 -bistritylated analogues 138 and 139.
Prepared analogues were evaluated for their in vitro antiviral
activities against the dengue virus (DENV) and yellow fever virus
(YFV). The most selective inhibitor was 30 ,50 -bis-O-tritylated-5-
chlorouridine 139 (R1 ¼ Tr, R2 ¼ H) affording a selectivity index of
over 90. These lipophilic compounds are exerting their effect
through inhibition of the RNA polymerase of flaviviruses. The
finding of these lipophilic structures should stimulate the interest
for structure-activity research [37].
Saudi and co-workers [38] synthesized a series of nucleoside
analogues of 30 ,50 -bistritylated uridine. Mono or bis-tritylation of 5-
fluoro-20 deoxyuridine 140 and 5-fluorouridine 143 provided the
compounds 141, 142, 143, 144 and 145, respectively (Scheme 22).
Prepared compounds were evaluated for their in vitro antiviral
activities against dengue fever virus and yellow fever virus. Among
the new series of derivatives, 30 ,50 -di-O-trityl-5-fluoro-20 -deoxy-
uridine 142 (R1 ¼ R2 ¼ Tr) was the most efficient in this series and
inhibited both yellow fever virus and dengue virus replication with
a 50% effective concentration (EC50) of 1 mg/mL without consider-
able cytotoxicity. The other fluorinated derivatives proved more
toxic [38].
2.5. Benzodioxepinyl-uracils, benzoxathiepinyl-uracils and
diazepinyl-uracils
The synthesis of a wide range of uracil derivatives linked to
saturated seven-membered moieties through N-1 of uracil ring was
carried out and this methodology allowed on access to non-
classical nucleosides with a 1,4-dioxepanyl group as the “sugar”
moiety. 1-(2,3-Dihydro-5H-1,4-benzodioxepin-3-yl)-5-
fluorouracils have proved to be good antiproliferative agents
against the MCF-7 human breast cancer cell line [39]. Such com-
pounds accumulate the cancerous cells in the G0/G1 phase. There-
fore, Saniger et al. [40] completed the synthesis and biological
evaluation of cyclic 150 and acyclic 151 5-FU O,N-acetals starting
from several salicyl alcohols 147 (Scheme 23). They have investi-
gated the Lewis acid-promoted transformation of the acyclic O,N-
acetals 148 to the corresponding cyclic O,N-acetals 150.
All the compounds 150 and 151 exhibited an IC50 against the
MCF-7 human breast cancer cell line in the micromolar range,
among which the nitro derivative 151 (R1 ¼ NO2, R2 ¼ H) was found
to be the most cytotoxic. They have demonstrated that compound
151 (R1 ¼ R2 ¼ H) induced apoptosis and G0/G1 cell cycle arrest in
the MCF-7 human breast cancer cell line, whereas 151 (R1 ¼ NO2,
R2 ¼ H) seems to act as the prodrug Ftorafur on the basis that both
compounds accumulate the tumoural cells in the S phase of the cell
cycle [40].
On the basis of molecular variations on isosteric replacements
from the prototype 1-(2,3-dihydro-5H-1,4-benzodioxepin-3-yl)-5-

O O O
O
Cl
NH NH NH Cl
NH
TrO N O 2.5 eq. TrCl, pyridine RO N O CAN, LiCl, RO N O 2.5 eq. TrCl, pyridine
O O o O o TrO N O
80oC, overnight CH3CN, 80 C 80 C, overnight
O

OR OR OR OR OR OR
OR1 OR2
R = H, Tr Acetic R1 = H, Tr
139 1 NH3, MeOH
R2 = H, Tr anhydride 134 R = H 136 R = Ac 138
rt R2 = H, Tr
pyridine, rt 135 R = Ac 137 R = H

Scheme 21. Synthesis of tritylated-5-chlorouridines 138 and tritylated-uridines 139.

596 A. Pałasz, D. Ciez_ / European Journal of Medicinal Chemistry 97 (2015) 582e611

O O
F F
NH NH
either 1.2 or 2.5 eq of
HO N O triphenylmethyl chloride R1O N O
O O
pyridine, 85oC, 16h

OH OR2
140
141 R1 = Tr, R2 = H
142 R1 = R2 = Tr
O
O
F
NH F
either 1.2 or 2.5 eq of NH
triphenylmethyl chloride
HO N O
O pyridine, 85oC, 16h N O
R1O
O

OH OH
143 OR2 OR3

144 R1 = Tr, R2 = H, R3 = H
145 R1 = R2 = Tr, R3 = H
146 R1 = R3 = Tr, R2 = H

Scheme 22. Synthesis of tritylated derivatives of 5-fluoro-20 -deoxyuridine 141, 142 and 5-fluorouridine 144, 145 and 146.

fluorouracil, Nunez and co-workers [41] prepared a series of 3-(2,3-
dihydro-5H-1,4-benzoxathiepin-3-yl)-uracil or thymine O,N-ace-
tals. The commercially available o-(hydroxymethyl)thiophenol 152
was selective alkylated using bromoacetaldehyde dimethyl acetal
in the presence of sodium hydride (Scheme 24). Subsequent
cyclization of the resulting hydroxyacetal 153 using p-toluene-
sulfonic acid produced the sulfur-containing acetal 154. The last
step was the condensation reaction between the seven-membered
acetal 154 and the natural pyrimidine bases uracil and thymine 155.
For this procedure Nunez et al. used tin(IV) chloride in dichloro-
methane, trimethylchlorosilane (TCS) and 1,1,1,3,3,3-
hexamethyldisilazane (HMDS) in acetonitrile. The oxidation of
sulfides to sulfoxides or sulfones was made using hydrogen
peroxide in the presence of scandium trifluoromethansulfonate or
potassium peroxymonosulfate (OXONE) (Scheme 24).
O,N-Acetals 156 have been screened for their anticancer acivity.
They were assayed for their in vitro antiproliferative activity against
the MCF-7 cell line. Some of them were found to be inhibitors of the
MCF-7 cell growth [41].
Mravljak and co-workers [42] described new inhibitors of the
bacterial transferase MraY. A scaffold strategy based on the dia-
zepanone central core of liposidomycins, natural inhibitors of MraY
has been developed. Several families of natural antibiotics
including liposidomycins, caprazamycins or tunicamycins (Fig. 5)
have been identified which display high in vitro inhibition of the
MraY enzymatic activity, but modest antibacterial activity probably
due to their high hydrophilicity limiting their passive diffusion
through membranes [43].
A scaffold strategy involved the introduction of key structural
fragments required for biological activity on enantiopure dia-
zepanones 157 by reductive amination, esterification and glyco-
sylation (Scheme 25). Biological evaluation of these compounds on
MraY enzyme revealed interesting inhibitory activity for com-
pounds 165 or 169 displaying three fragments on the scaffold: a
palmitoyl chain, an aminoribose part and an alkyluracil moiety. The
inhibitors were also evaluated on MurG [42].
Biological evaluation of the resulting inhibitors on both MraY
and MurG activities resulted in IC50 values in the 100 mM range for
the best compounds and showed that among the introduced frag-
ments, three of them are important for activity since their absence
led to lower activities, while the presence of a second aminoribose
leads to weaker inhibitors. Interestingly the biological results sug-
gested that the presence of a hydrophobic residue on the primary
alcohol function of the scaffold is preferable to that of a free alcohol

R2 R2 OMe
OH BrCH2CH(OMe)2
NaH, anhydrous DMF O
OMe
OH OH
R1 R1
147 148
R1 = H, OMe, Cl, Br, NO2 5-FU, HMDS, TCS
R2 = H, OMe SnCl4/CH2Cl2, MeCN BF3 OEt2, anhydrous Et2O

O
F
HN
R2 O R2
R2 O N 5-FU, HMDS, TCS
O NH O
O SnCl4/CH2Cl2, MeCN
N O OMe OMe
R1 O OH R1 O
F R1 149
150 151

Scheme 23. Synthesis of cyclic 150 and acyclic 151 5-FU O,N-acetals.
 A. Pałasz, D. Ciez_ / European Journal of Medicinal Chemistry 97 (2015) 582e611 597

OMe
SH BrCH2CH(OMe)2 p-TsOH S
S
NaH, DMF OMe toluene
OH OMe
OH
O
152 153 154

O
O H o
X 1. pyrimidine, HMDS, TCS, SnCl4, MeOH, 45 C R H
N N
2. H2O2, Sc(OTf)3, CH2Cl2/EtOH or OXONE, MeOH/H2O
N O
O N O
X = S, SO, SO2
156 R H
R = H, Me
155

Scheme 24. Synthesis of 3-(2,3-dihydro-5H-1,4-benzoxathiepin-3-yl)-uracil or thymine O, N-acetals 156.

for MraY inhibition. Furthermore, a free primary amine on the of 1-mono- or 1,3-bis(bromoalkyl)uracils with a considerable
ribose seemed to be crucial to inhibit the MurG activity. Therefore, excess of appropriate amine.
this study led to reaching some insight into the requirements for Quaternization of the bridging nitrogen atom with o-nitro-
inhibition of the trans-membrane protein MraY [42]. benzyl bromide, benzyl bromide, n-decyl bromide gave rise to
water-soluble pyrimidinophanes. The in vitro antibacterial and
antifungal activity of the synthesized pyrimidinophanes was
2.6. Pyrimidinophanes containing one or two uracil units and investigated against several pathogenic representative Gram-
bridging polymethylene chains negative bacteria (Pseudomonas aeruginosa 9027 and Escherichia
coli F-50), Gram-positive bacteria (Staphylococcus aureus 209p,
The synthesis of macrocyclic compounds containing pyrimidine Bacillus subtilis 6633 and Enterococcus faecalis ATCC 8043), patho-
rings, pyrimidinophanes, is rapidly developing field of pyrimidines genic fungi (Aspergillus niger BKMF-1119, Trichophyton menta-
chemistry. These macrocycles can contain different number of py- grophytes var. gypseum 1773 and Aspergillus fumigatus AF-27) and
rimidine units linked to each other by hydrocarbon or polyether yeast (Candida Albicans 885-653). Antibacterial and antifungal ac-
spacers through either the N-1 and N-3 atoms or carbon atoms of tivity of pyrimidinophanes increases with the increase of poly-
pyrimidine rings or substituents at pyrimidine rings. In most of methylene N(pyr)-N-chain length and dramatically increases upon
known pyrimidinophanes uracil derivatives fragments were the introduction of n-decyl substituent at nitrogen atoms in
introduced as pyrimidine units because the chemical features of spacers. Pyrimidinophanes with 5 and 6 methylene groups in
uracils allow to vary the structure of the macrocyclic compounds N(pyr)-N-chain and n-decyl substituent showed significant bacte-
synthesized. riostatic, fungistatic, bactericidal, fungicidal activity which com-
Semenov and co-workers [44] described the reactions of 1,3- parable with standard antibacterial and antifungal drugs [44].
bis(a,ubromoalkyl)-6-methyluracils 171 with 1,3- Nikolaev et al. [45] synthesized pyrimidinophanes containing
bis(a,uethylaminoalkyl)-6-methyluracils 172 (Scheme 26). These one 5(6)-alkyl substituted uracil moiety and a 10- or 12-methylene
reactions afforded pyrimidinophanes containing one or two uracil bridge including a sulfur atom. Pyrimidinophanes 176 with
units and nitrogen atoms in bridging polymethylene chains. 1- bridging S atom were synthesized using 1,3-bis(u-bromoalkyl)
Mono- or 1,3-bis(aminoalkyl)uracils were prepared by amination uracils 175 with sodium sulfide as the cyclizing agent (Scheme 27).
The bridging S atom of macrocycles was converted into a sulfonium
ion by reaction of pyrimidinophanes 176 with the methyl or nonyl
HO3SO OH
esters of p-toluenesulfonic acid. Amphiphilic pyrimidinophanes
NH2 177 with SþCH3 and SþC9H19 groupings in macrocycles with 10 and
R O Me 12 methylene groups were prepared (Scheme 27).
O O O
N Antimicrobal activity against P. aeruginosa 9027, E. coli F-50, S.
1 O O
R O2C O aureus 209p, Bacillus cereus 8035, and E. faecalis ATCC 8043 was
N NH
O measured. Amphiphilic pyrimidinophanes 177 with 5-decyl-6-
HO2C N
O O methyluracil moieties had high levels of bacteriostatic activity
Me HO OH against Gram-positive bacteria. Introduction of a lipophilic sub-
Liposidomycins: R = H
1 stituent into the uracil moiety increases the bacteriostatic activity
Caprazamycins: R1 = methylglucoside of this type of pyrimidinophane. The minimum inhibitory con-
centration of the macrocycle containing a methyl group in the
O OH sulfonium group against S. aureus was 0.75 mg/ml [45].
OH O
HO
R N O 2.7. Other substituted uracils
O N NH
H
O
Numerous modifications of the uracil structure have been per-
HO O
AcHN OH formed which resulted in the development of derivatives exhibiting
HO better pharmacological and pharmacokinetic properties including
HO O increased bioactivity, selectivity, metabolic stability, absorption and
Tunicamycins
HO lower toxicity. For example, 6-amino-5-chlorouracil is known as a
potent inhibitor of thymidine phosphorylases (TP). The efforts to
Fig. 5. Uracil derivatives e natural inhibitors of MraY. enhance the inhibitory activity of this uracil derivative by
598 A. Pałasz, D. Ciez_ / European Journal of Medicinal Chemistry 97 (2015) 582e611

O
H
N
O
H H N O
HO N N
NaBH(OAc)3 ( )5
HO N
OBn + N O 87%
TBDPSO N OBn
O (CH2)4-CHO TBDPSO
H N
O
157 158 H
159
C15H31COOH
DCC, DMAP
O O
H H
N N
N O N O
( )5 ( )5
C15H31CO2 N nBu4NF
C15H31CO2 N
THF
OH 96% OP
HO N TBDPSO
O N
O
H H
166
BCl3 160: P = Bn (87%)
O F 161: P = H (92%)
N3 CH2Cl2

BF3 OEt2 O F
N3
O O
O BF3 OEt2
H O O
N O

N O H
N
( )5
C15H31CO2 N N O
O O ( )5
A C15H31CO2 N
N
O
O O H O O
A O O N A
O
TBDPSO H
O O O O

(Ph2P-CH2)2 167: A = N3 (62%) (Ph2P-CH2)2 162: A = N3 (97%)

THF, H2O 168: A = NH2 (96%) THF, H2O 163: A = NH2 (92%)

TFA H2O
TFA H2O (57%)
O
O
H
H N
N
N O
N O
( )5
( )5 C15H31CO2 N
C15H31CO2 N
O O
O O NH2
NH2 N
N O
O PO H
O O H
OH OH
H2N OH OH
169 NH4F 164: P = TBDPS (57%)
DMF 165: P = H (87%)
HO OH

Scheme 25. Synthesis of inhibitors with three 165 or four 169 key fragments from diazepanone 157.

substitution of the amino group with different amines were made.
5,6-Disubstituted uracils with significant inhibitory activity against
human and E. coli TP were reported by Nenecka et al. [46]. They
employed a simple method for the synthesis of target uracils 179
and 180 based on a selective nucleophilic substitution of the
chlorine atom at the position 6 of 5,6-dichlorouracil or 5-bromo-6-
chlorouracil by various amines and diamines (Scheme 28).
5-Bromo-bis-uracil derivatives with piperazine-1,4-diyl and
ethylenediamino linkers were identified as the most potent
inhibitors of human TPs and E.coli enzyme in this study. Authors of
this study supposed that one of the nitrogen atoms in the linker
interacts with the serine oxygen of the enzyme (S117), and
enhancing the inhibitory activity of bis-uracil derivatives 180. The
second uracil moiety could enhance the probability of interaction
with the enzyme [46].
Noll and co-workers [47] examined the antiproliferative activity
on human tumor cell lines of a series of modified uracil derivatives
and their corresponding acyclonucleosides. They synthesized of a
 A. Pałasz, D. Ciez_ / European Journal of Medicinal Chemistry 97 (2015) 582e611 599

H3C H H3C (CH2)6Br CH3

1. Na, n-BuOH, reflux, 15h N 40-fold excess of NH2Et
N
2. Br(CH2)6Br, DMF, 50-60oC O
i-PrOH, rt, 7 days H3C (CH2)6NH
O
N
N N
O
O 170 H O 171 (CH2)6Br
N
H3C O O CH3 O 172 (CH2)6NH

N N N N CH3
(CH2)6 (CH2)6 (CH2)6 (CH2)6
O K2CO3, MeCN
N O N N N o
O O 60-70 C, 12h
H3C CH3 H3C CH3
(CH2)6 (CH2)6 (CH2)6 (CH2)6
N N N N

H3C O H3C O
cis-173 trans-174

Scheme 26. The example of synthesis of pyrimidinophanes with six methylene groups with cis-173 and trans-174 arrangement of the carbonyl groups.

O (CH2)nBr O O
(CH2)n (CH2)n
N N R2OTs N
Na2S
R1 O R1 O S R1 O S R2
N N N OTs
(CH2)n (CH2)n
H3C (CH2)nBr H3C H3C
175 176 177

n = 5, 6
R1 = H, n-C10H21
R2 = CH3, C9H19

Scheme 27. Synthesis of pyrimidinophanes 177 containing one 5(6)-alkylsubstituted uracil moiety and a 10- or 12-methylene bridge including a sulfur atom.

series of uracil derivatives, differing in properties of C5-positioned
side chains, like chemical reactivity and bulkiness. Most of these
compounds were also prepared in a form of N1 e substituted acyclic
uridine analogues, to explore the impact of acyclic sugar analogues
on biological activity. 5-(Chloroacetyl)aminouracil 183 was syn-
thesized by reaction of 5-aminouracil 181 and chloroacetyl chloride
182 (Scheme 29). The acetylaminouracil 185 was prepared by direct
condensation of the persilylated uracil 183 with amine 184.
Synthesized compounds have been screened for their anticancer
activity. They were assayed for their in vitro antiproliferative
activity against HeLa, MiaPaCa-2, SW620, MCF-7 and H460 cells.
Modification of the N1-position of the uracil molecule was accom-
plished by direct condensation of the appropriate uracils 186 with
1,3-dibenzyloxy-2-chloro-methoxypropane 187 (Scheme 29).
Comparison of structure-activity relationship revealed the impor-
tance of chemical reactivity of the substituent attached to the C5-
position of uracil for the biological activity of studied compounds.
The results obtained for the most active compounds, 5-(chlor-
oacetylamino)uracil 183 and its acyclic sugar analogue 190, sug-
gested that formation of a covalent bond between reactive

O O
X H X H
N Amine, EtOH N

Cl N O R N O
HCl
H H
178 179
X = Cl, Br
R = NH2, piperidin-1-yl, pyrrolidin-1-yl, azetidin-1-yl,hydrazino,
Diamine morpholin-4-yl, cyclopropylamino, 2-(hydroxymethyl)piperidin-1-yl,
EtOH 3-(hydroxymethyl)piperidin-1-yl, 4-(hydroxymethyl)piperidin-1-yl

O X X O

H N linker N H linker = piperazine-1,4-diyl

N N etylenodiamino
2HCl
O H H O
180

Scheme 28. Synthesis of 5,6-disubstituted uracils 179 and bis-uracil derivatives 180.
600 A. Pałasz, D. Ciez_ / European Journal of Medicinal Chemistry 97 (2015) 582e611

substituent and several possible targets within the thymidylate
synthase mechanism (sulphur of the cysteine residue, basic part of
the enzyme, N,N-methylene tetrahydrofolate or its reactive imi-
nium forms) is the most probable mode of action. C5-substituted
uracil derivative 185 (5-[bis-(2-p-methoxybenzylthioethyl)amine]
acetylaminouracil) exhibited high antiproliferative activity against
HeLa and MiaPaCa-2 cell lines [47].
The ANN (artificial neutral networks) e QSAR (quantitative
structure-activity relationship) model developed was used to pre-
dict the biological activity of some cyclopentyl pyrimidine deriva-
tive analogues of tegafur (Scheme 30), a largely known anticancer
drug [48]. Gonzalez-Diaz and co-workers predicted the biological
activity only for simple analogues from which CH3, F, Cl, Br, and I
substituted at 5-carbon of the uracil skeleton was selected. This
selection was based on the simplicity and synthetic accessibility.
Compounds 193 were prepared by condensation of the trime-
thylsilylated base 191 (uracil, thymine, and fluorouracil) with bro-
mocyclopentane 192. Compounds 194 and 195 were prepared from
193a using N-chlorosuccinimide or N-bromosuccinimide in acetic
acid, or I2 in nitric acid and dioxane (Scheme 30).
The predictions coincided with the biological test result where
all the compounds showed detectable biological activity in the
three studied cellular lines namely L 1210/0. Molt4/C8, and CEM/0.
The most interesting activity on the leukemia line L1210/0 was
presented by the F-tegafur analogue 193c. However, results in the
human lymphocyte lines were more discrete. Interestingly, the Br-
tegafur analogue 194b resulted more selective for the human
lymphocyte lines than the leukemia line. This result confirmed the
potentialities of the MARCH-INSIDE approach to model biological
data and guide drug discovery in bioorganic medicinal chemistry
[48].
Cui and co-workers [49] produced uracil derivatives as potential
PfPNP inhibitors. Plasmodium falciparum purine nucleoside phos-
phorylase PfPNP has a central role in purine salvage and inhibitors
of the enzyme have been shown to have antiplasmodial activity.
The first series of compounds was based on known human uridine
phosphorylase inhibitors whilst the second series of compounds
were uracil equivalents of purine-based PNP transition state in-
hibitors. The first series of compounds were prepared as shown in
Scheme 31. Barbituric acid 196 was condensed with aromatic
aldehyde to give the derivatives 197. The compounds 197 were
reduced with sodium borohydride. (2-Acetoxyethoxy)methyl bro-
mide 201 was prepared by reaction of acetyl bromide 199 and 1,3-
dioxolane 200. Compound 198 (R ¼ OBn) was first silylated and
then stirred with the bromide 201 to give the required ester 202.
The ester 202 was deprotected to give the final product 203.
The second series of inhibitors 206 and 208 was prepared as
shown in Scheme 32 [49]. Uracil 204 reacted with 1,2-

O O
S CH2 OMe
H NH2 NaOH/H2O H NH C CH2Cl
N O o N
+ Cl CH2 C 0-22 C O
Cl + N
O N O N
H H
181 182 183 S CH2 OMe
184
S CH2 OMe 1. hexamethyldisilazane
NH4(SO4)2, reflux, 3h
O 2.bis(2-p-methoxybenzylthioethyl)-amine 4
o
H NH C CH2N Et3N, MeCN, 90 C, 4h
N
O
O N S CH2 OMe
H
185

O O O
H R1 H R1 PdO H R1
N BzO N N
cyclohexene
+ O
O N R2 BzO Cl O N R2 O N R2
H
O 189 O
186 187 188
R1 = H, OH, NH2, I
R2 = H, CH3
OBz OBz OH OH
O O
NaOH/H2O
H NH2 O o
H NH C CH2Cl
N 0-22 C N
+ Cl CH2 C O
182 Cl
O N O N

O O

OH OH OH OH
189 R1 = NH2, R2 = H 190

Scheme 29. Synthesis of C5-substituted uracil derivatives 183 and 185 and uridine derivatives 189 and 190.
 A. Pałasz, D. Ciez_ / European Journal of Medicinal Chemistry 97 (2015) 582e611 601

O
F
NH

N O tegafur

O
R
OTMS NH
Br 1.CH2Cl2, reflux
R 2. MeOH/ H2O 6:1
N N O
+
N OTMS 193a, R = H
193b, R = Me
191 192 193c, R = F
193
O O O
R I
NH NH I2, HNO3 NH
NXS (X=Cl, Br)
AcOH, reflux o
dioxane, 100 C
N O N O N O

194a, R = Cl
194 194b, R = Br 193a 195

Scheme 30. Synthesis of cyclopentylpyrimidine derivative analogues of tegafur.

dibromoethane, 1,3-dibromopropane or 1,4-dibromobutane in Immuno Deficiency Syndrome (AIDS). 6-(3,5-Dimethylbenzyl)-1-

presence of CsCO2. The dibromoalkanes could alkylate either N1 or
N3 of the uracil 204. Compounds 205 or 207 were then reacted with
pyrrolidine to give the final compounds 206 or 208.
These two series of compounds were assayed for inhibition of
both PfPNP activity and growth of P. falciparum. The transition state
analogues were found to be moderate inhibitors of PfPNP (most
potent compound, Ki ¼ 6 mM) [49].
In the recent years, the HIV-1 non-nucleoside reverse tran-
scriptase inhibitors have been of great interest for the design of
drugs against HIV-1 reverse transcriptase, the enzyme being an
important factor in the development of the disease Acquired
(ethoxy-methyl)-5-isopropyluracil(GCA-186) has been investigated
as a drug candidate (Scheme 33) [50]. The two methyl groups on
the 6-benzyl moiety have been shown to improve the binding
stability of the drug to the NNRT1-binding site in reverse tran-
scriptase of drug mediated mutant HIV-1 viruses. The two methyl
substituents on GCA-186 were though to undergo metabolism by
the cytochrom P450 system in the liver. Pedersen and co-workers
[50] described the HIV-1 inhibitors closely related to GCA-186.
The methyl groups were replaced with isosteric chloro-atoms to
avoid metabolism due to the two methyl groups. Reaction of 3,5-
dichlorophenylacetonitrile 209 with the zinc organometallic

O O O
H aromatic aldehyde H R NaBH4, EtOH H R
N CH3COOH, reflux N 2h, rt N

O N O O N O O N O
H 196 H 197 H 198 R = H, OBn
R = H, OBn

O
O O reflux, 70oC 1. chloromethylsilane, hexamethyldisilazane,
O Br
O reflux, 80oC, overnight, Ar
Br 2. (2-acetoxyethoxy)methyl bromide,AlCl3,
199 200 201
1,2-dichloroethane, 48h, rt, Ar
O O
H R H R
N N
O N O O N O
NaOMe, MeOH, rt
O O
203
202

OH O O

Scheme 31. Synthesis of the first series of inhibitors of human uracil phosphorylase 198 and 203.
602 A. Pałasz, D. Ciez_ / European Journal of Medicinal Chemistry 97 (2015) 582e611

R1 N
O O O
Br(CH2)nBr n = 2-4 EtOH H
H o
H
N CsCO3, DMF, 40 C, 48h N o
150 C, 1 min, microwave N

O N O N O N
H 204 (CH2)n
(CH2)n 205
Br R1 N
Br(CH2)2Br
n = 2-4 206
CsCO3, DMF, 40oC, 48h
R1 = H, CH2OH
H
N R1
O R1 O
Br EtOH N
N o N
150 C, 1 min, microwave
O N O N
H H
207 208 R1 = H, CH2OH

Scheme 32. Synthesis of the second series of inhibitors of human uracil phosphorylase 206 and 208.

reagent from ethyl 2-bromobutyrate gave ethyl 4-(3,5-
dichlorophenyl)-2-ethyl-3-oxobutyrate 210 which was reacted
with thiourea in the presence of sodium ethoxide to give the 2-
thiouracil 211 (Scheme 33). Desulfuration of 211 with aqueous
chloroacetic acid afforded 6-(3,5-dichlorobenzyl)-5-ethyluracil
212. The uracil 212 was silylated with N,O-bis-(trimethylsilyl)
acetamide (BSA) and then treated with bis oxysubstituted meth-
anes 213 under the Vorbruggen conditions using trimethylsilyl
trifluoromethanesulfonate (TMS-triflate) as a catalyst to give the
corresponding compounds 214 (Scheme 33).
The isosteric chloro derivatives show tenfold less activity
against HIV-1 than their corresponding methyl derivatives. This
showed that chlorine is not the substituents of choice for replacing
the methyl groups in GCA-186 in order to reduce its biodegradable
properties [50].
Mager and co-workers [51a] investigated 5,6-substituted 1-[(2-
hydroxyethoxy)methyl]uracils (Fig. 6). The dioxypyrimidine ring of
these uracils is an extended “partial p system” with ring distortion
mainly due to the N1 atom. On basis of PM3-MM þ geometry
optimization authors suggested a lipophilic “butterfly-like”
orientation which was also found in other non-nucleoside in-
hibitors that interact with the HIV-1 reverse transcriptase. The
synthesis of 5,6-substituted 1-[(2-hydroxyethoxy)methyl]uracils
was described in 1992 [51b].
Cluster analysis of the biological activity profile and multivariate
QSAR has shown that maximization of the antiviral response and
minimization of the undesired cytotoxicity is possible. Related to
the C-6 thiophenyl ring substituents and to modifications at the C-5
position, the antiviral response depends on hydrogen-bonding
forces, steric parameters, and electronic properties. The cytotox-
icity depends on the lipophilicity and steric parameters [51a].
Illan-Cabeza et al. [52a] reported the synthesis and molecular
structures of (6-amino-1-methyl-5-nitrosouracilato-N3)-triphe-
nylphosphine-gold(I) with interesting abilities to inhibit tumor
growth in an animal model of experimental glioma. The synthesis
of the precursor organic ligand 6-amino-1-methyl-5-nitrosouracil
(MANU) (Fig. 7) was described earlier [52b]. The complex
[Au(MANUH-1)PPh3] was obtained by mixing [AuCl(PPh3) and
MANU (1:4) in a methanolic alkaline medium.

Cl Cl O Cl
EtCH(Br)CO2Et H2NCSNH2
CN O H Et
Zn, THF EtONa/EtOH N
CH2 C CH COOEt
Et S N Cl
Cl 209 Cl 210 211
H

ClCH2COOH

O Cl O Cl
H Et 1. BSA, MeOH H Et
N o N
2. TMS triflate, -40 C
ROCH2OR
O N Cl O N Cl
214 213 H 212
RO O
H
N R = CH2CH3, CH2CH CH2, CH2CH C(CH3)2,
CH2(CH3)C CH2, CH2CH CHPh,
O N CH2(CH3)C CHPh, CH2C CH, indanyl

O GCA - 186

Scheme 33. Synthesis of 6-(3,5-dichlorolbenzyl)- analogues of uracil 214.

 A. Pałasz, D. Ciez_ / European Journal of Medicinal Chemistry 97 (2015) 582e611 603

O
X = O, S 5' R1 down-regulation of anti-apoptotic genes such as Bd-2. In contrast,
R2 H R= S several proapoptotic genes related with the endoplasmic reticulum
N (ER)-stress-induced apoptosis, such as BBC3 and Noxa, appeared
3'
R1 = H, 3-Me, 3-Et, 3-tert-Bu, 3-CF3, 3-F, 3-Cl, 3-Br, 3-I, 3-NO2, up-regulated. These results seemed to show that the mechanism of
R N X
3-OH, 3,5-diMe, 3,5-diCl, 3,5-diOMe, 3-COOMe, 3-Ac, 3-CN
CH2OCH2CH2OH action and selectivity of uracils presented in Fig. 8 was via the
R2 = Me, Et, Pr, iso-Pr,CH2 CH CH2 activation of ER stress-induced apoptosis. The selective activity of
this compound against tumour cells via the ER stress-induced
Fig. 6. Basic structure of 5,6-substituted 1-[(2-hydroxyethoxy)methyl]uracils.
apoptosis supposes a great advantage for future therapeutic use
against breast cancer [53].
Morgan et al. [55] discussed the potential role played by human
O thymine DNA glycosylase (TDG) in the cytotxic effects of ClU and
BrU incorporation into DNA, which can occur under inflammatory
H N
N O conditions and in the cytotoxicity of FU, a widely used anticancer
agent (Fig. 9). They reported that TDG rapidly excises 5-
halogenated uracils, exhibiting much greater activity for CpG.FU,
O N NH2 CpG.ClU, and CpG.BrU than for CpG.T.
They examined the effects of altering the CpG context on the
CH3 excision activity for U, T, FU, ClU, and BrU. TDG excises thymine
from G.T mispairs and removes a variety of damaged bases (X) with
Fig. 7. Structure of the 6-amino-1-methyl-5-nitrosouracil (MANU).
a preference for lesions in a CpG.X context. They showed that the
maximal activity (kmax) for G.X substrates depends significantly on
R1 OMe the 50 base pair. For example, kmax decreases by 6-, 11-, and 82-fold
for TpG.ClU, GpG.ClU, and ApG.ClU, respectively, as compared with
N R2
N CpG.ClU. For the other G.X substrates, the 50 -neighbour effects have
a similar trend but vary in magnitude. The activity for G.FU, G.ClU,
O N O and G.BrU, with any 50 -flanking pair, meets and in most cases
OH H significantly exceeds the CpG.T activity [55].

R1 = H, PhCO, 4-NO2-PhSO2
2-NO2-PhSO2, 2-NH2-PhSO2
R2 = H, F
Fig. 8. Acyclic O,N-acetals of uracil and 5-fluorouracil.
Anti-tumor properties of this compound, effects on both
enzyme and non-enzyme antioxidant defense systems and the
response of several biochemical biomarkers have been analysed.
After seven days of treatment, the gold compound decreased the
tumor growth to ca. one-tenth and reduced oxidative stress bio-
markers compared to animals treated with the vehicle. Also, gold
compound maintained non-enzyme antioxidant defense systems
as in non-tumor animals and increased enzyme antioxidant de-
fenses, such as superoxide dismutase and glutathione peroxidase
activities, and decreased catalase activity. The analysis of blood
serum parameters indicated few adverse effects [52a].
Caba and co workers [53] have selected an anthranilic alcohol-
derived acyclic 5-fluorouracil O,N-acetal (Fig. 8) to carry out the
anti-cancer studies. Previous they reported the cytotoxic activities
against the MCF-7 human breast cancer line of acyclic O,N-acetals
[54]. Selected uracils showed activity as a potent growth inhibitor
of the tumour cell line MCF-7 at a very low concentration.
Uracil (R1 ¼ SO2eC6H4-oNH2, R2 ¼ F) had no activity against
classic pro-apoptotic genes such as p53, and even induced the
2.8. Fused uracils
Fused uracils as fused heterobicyclic system, constitutes an
important contribution in medicinal chemistry and a wide variety
of attractive pharmacological effects was attributed to them. Fused
uracils, such as pyrano[2,3-d]pyrimidines, pyrido[2,3-d]pyrimi-
dines, thieno[2,3-d]pyrimidines, pyrazo[3,4-d]pyrimidines or pyr-
imido[4,5-d]pyrimidines are reported to have a wide range of
biological activities such as antiallergic, antihypertensive, cardio-
tonic, bronchodilator, antibronchitic or anti-tumor activity [56].
Moreover, thieno[2,3-d]pyrimidines or pyrazo[3,4-d]pyrimidines
are potential bioactive molecules as they bear structural analogues
and isoelectronic relations to purines. In view of the above, the
synthesis and biological activity of novel analogues of fused uracils
is presented below.
Fustero et al. [57] described synthesis and biological evaluation
of bicyclic fluorinated uracils possessing ring, which is fused at the
C-5 and C-6 or N-1 and C-6 positions of the uracil moiety. Nitrile
216 was obtained from 2,2-difluoropent-4-enoic acid 215 by ethyl
ester formation, transformation to the corresponding primary
amide, and dehydratation (Scheme 34).
Next, compound 216 was treated with the lithium enolates, to
afford b-enamino esters 217. Compounds 217 were deprotonated
with NaH, and further addition of isocyanate gave uracils 218.
Uracils 218 were precursors of bicyclic uracils 219 and 220 (Scheme

O O O O O
H3C X C
NH NH H NH HO NH

N O N O N O N O

thymine 5-halo-U 5-formyl-U 5-hydroxymethyl-U

(5-methyl-U) X = F, Cl, Br, I

Fig. 9. Uracils removed by human thymine DNA glycosylase (hTDG).

604 A. Pałasz, D. Ciez_ / European Journal of Medicinal Chemistry 97 (2015) 582e611

OLi
1. EtOH, Dowex
2. NH4OH OEt NH2 O
COOH 3. P2O5 CN R1
OEt
F F o
F F THF, -78 C F F R1
215 216 217
R2NCO, NaH
DMF
O O O
H R2 R2
N N H R2
F N N N N
F
O and ( )n
O O
( )n
F F R1 F F R1
220 219 218
R1 = H, CH=CH2, R2 = 4-MeO-Ph, 4-F-Ph, CH(CH3)Ph
CH2-CH=CH2 CH2CH3, CH2COOEt

Scheme 34. Synthesis of bicyclic fluorinated uracils 219 and 220 by ring-closing metathesis (RCM) reaction.

34). The ring-closing metathesis (RCM) reactions of compound 218
were carried out in the presence of Grubbs' second generation
catalyst and bicyclic fluorinated uracils 219 and 220 were obtained.
The selective formation of olefin regioisomers in the metathesis
process can be controlled according to the reaction conditions.
These bicyclic uracils were tested as acaricides against Tetranychus
urticae, a parasite of crops and common houseplants, using the
commercially available miticide tebafenpyrad as reference stan-
dard. Only few compounds were active at a concentration of
4.0e5.0 mg/mL, in terms of both mortality and fecundity inhibition
[57].
Herold et al. [58] synthesized derivatives of 4-aryl-5,6,7,8-
tetrahydro-pyrido[1,2-c]pyrimidine possessing a 3-(4-piperidyl)-
1H-indole residue or its 5-methoxy or 2-methyl derivative. The
nitriles 222 were prepared by C-arylation of the arylacetonitriles
221 with 2-bromopyridine (Scheme 35). The nitriles 222 were
hydrolysed using a mixture of sulfuric acid and acetic acid to pro-
duce amides 223. Compounds 224 were formed by the intermo-
lecular cyclization of 223 with diethyl carbonate. The nitrogen in
the imide group of compounds 224 was alkylated with 1,4-
dibromobutane to form derivatives 225, which were hydrogenat-
ed to form compounds 226. The target compounds 228 were

CH2CN R1 R1
R1 2-bromopyridine CH3COOH,
KOH, DMSO, heat H2SO4, heat
R2 CHCN R2 CHCONH2

N N
R2 221
222 223

diethyl carbonate
EtONa, EtOH, heat

R2 R2 R2

R1 R1 R1
O H2, 10% Pt/C O 1,4-dibromobutane O
Br 60 atm, 50oC K2CO3, acetone, heat
N N Br
N N N NH
O 226 O 225 O 224

R2 R3

H
N
R3 R1
MeCN, K2CO3 NH
KI, heat O
N R4
N N
R4 N
H 227 O 228

Scheme 35. Synthesis of 4-aryl-5,6,7,8-tetrahydro-pyrido[1,2-c]pyrimidines 228.

 A. Pałasz, D. Ciez_ / European Journal of Medicinal Chemistry 97 (2015) 582e611 605

obtained by the condensation of bromobutyl derivatives 226 with
the piperidyl-indole 227 (Scheme 35).
These compounds were considered to have a double mechanism
of action. In vitro receptor binding studies of synthesized ligands
showed a very high to minimal binding affinity for 5-HT1A re-
ceptors, as well as a moderate to weak binding affinity for SERT
[58]. The Ki values for both 5-HT1A receptor and SERT showed that
compounds 228 (R1 ¼ R3 ¼ R4 ¼ H, R2 ¼ OCH3), 228 (R1 ¼ R4 ¼ H,
R2 ¼ R3 ¼ OCH3), 228 (R1 ¼ F, R2 ¼ R3 ¼ R4 ¼ H), and 228 (R1 ¼ H,
R2 ¼ F, R3 ¼ OCH3, R4 ¼ H) had the highest affinity among new
derivatives. The authors established that the presence of the 3-(4-
piperidyl)-1H-indole residue or its 5-methoxy derivative, as well
as the fluorine atom at the ortho/para position or eOCH3/eCH3
group at the para position of the phenyl group in the aryl ring of the
pyrido[1,2-c]pyrimidine system, are at an advantage with regard to
binding affinity. The presence of the 2-methyl-3-(4-piperidyl)-1H-
indole group results in a drastic affinity decrease with regard to 5-
HT1A receptors and a significant reduction in the SERT-inhibiting
action of relevant compounds [58].
Nieto et al. [59] produced 9-deazaxanthine (9-dAX) derivatives
having uracil ring fused with pyrrole ring. Along with purine nu-
cleosides analogues, xanthines derivatives constitute one of the
most widely exploited classes of adenosine receptor ligands. Se-
lective A2B receptor antagonists can play a role in important pa-
thologies such as neurological and hypersensitive disorders, and
diabetes [59]. The target was to develop of selective A2B antagonists
as potential anti-asthmatic agents. Some 9-deazaxanthine deriva-
tive represent a novel class of adenosine receptor ligands endowed
with nanomolar affinities at hA2B adenosine receptor. The key in-
termediates for the synthesis of compounds 233 were 9-dAX car-
boxylic acid derivatives 232, and these were prepared by the
condensation of appropriate uracil derivatives 229 with the cor-
responding para-substituted benzaldehydes 230 to afford the 6-
styryluracils 231, followed by cyclization to the 9-dAXs 232 and
saponification to the free carboxylic acid (Scheme 36). Condensa-
tion of 9-dAX carboxylic acids 232 and the appropriate amines was
conducted in DMF in the presence of base TEA, 1-[3-
dimethylaminopropyl]-3-ethylcarbodiimide hydrochloride (EDC)
and 1-hydroxybenzotriazole (HOBt).
These compounds were evaluated for their binding affinities for
the four human recombinant adenosine receptors, A1-A3 subtypes.
A number of the 9-deazaxanthines 233 showed moderate to high
affinity for hA2B receptors, with compound 233 (R1 ¼ Pr, R2 ¼ H,
R ¼ 4-fluorophenylaminocarbonyl, R4 ¼ H) showing a 32-fold
selectivity for A2B over A1 and a 2750-fold selectivity for A2B over
A2A [59].
Rashad et al. prepared uracil derivatives containing thieno[2,3-
d]pyrimidine ring systems [60]. A large number of fused thieno[2,3-
d]pyrimidine derivatives exhibited biological activities as potential
antimicrobial, antiviral, analgesic, anti-inflammatory and anti-
canceractivities [60]. The synthesis of novel analogues of thieno
[2,3-d]pyrimidine derivatives was described. Hydrolysis of the
cyano group of compounds 234 was done with using of a mixture of
orthophosphoric acid/polyphosphoric acid (1:1) at temperature
90  C (Scheme 37). Amide 235 underwent cyclization with carbon
disulfide to give the corresponding 9-thioxo derivative 236. Alka-
line treatment of compound 236 with some chloroalkyl reagents
like 2-chloroethyl methyl ether, chloroacetaldehyde dimethyl
acetal or 2-chloroethanol afforded product assigned to the struc-
ture of the unexpected thieno[2,3-d]pyrimidinedione derivative
237. When the potassium salt of compound 236 was coupled with
some bromoalkyl reagents like bromoethane, 2-bromoethyl methyl
ether or 2-bromoethyl ethyl ether, the corresponding S-alkyl de-
rivatives 238, were formed (Scheme 37).
The prepared products were tested for antiviral activity against
H5N1 influenza virus by determination of both EC50 and LD50 and
confirmed by plaque reduction assay on MDCK cells. Compounds
235, 236 and 237 showed the highest effect compared with the
other tested compounds. High activity of thienopyrimidine de-
rivatives 236 and 237 may be due to the structure resemblance of
these compounds to thiouracil and uracil, respectively. Compound
237 has the highest anti-H5N1 activity compared with the other
tested compounds with percentage of virus reduction varying from
92 at concentration of 5 mge98 at concentration of 40 mg [60].
Solano and co-workers [61] reported the synthesis of the fused
uracils - pyrimidine monoadducts of bis-chalcones prepared the
reaction between 6-amine-1,3-dimethyl uracil and bis-chalcones.
Over the last years, the interest for chalcones-like compounds has
been continually renewed given the diversity of their effects [62].
Their antineoplastic activity is mediated in part by induction of a
programmed cell death (PCD) type 1 or apoptosis. Chalcones and
some of their biosynthetic derivatives such as flavopiridol, baicelein
and quercetin, induce apoptosis in vitro [62a,63]. Even when cur-
rent anticancer drugs are used to induce apoptosis, tumor cells may
evade this PCD, so it is important to develop new strategies to
induce different PCDs. The first step of the synthesis consisted in
the generation of a,b-unsaturated compounds 241, these bis chal-
cones were obtained in moderate yields (40e50%) by aldolic
condensation between ketones 239 and aldehydes 240 or 243.
Subsequently, the synthesis of pyrimidine mono-adducts of bis-
chalcones 242 or 245 was performed through the Michael addition

CHO
O O
R4
R1 NO2 dioxane, piperidine R1 NO2 R4
N DMAP, reflux, 5-96h N
O
O N O N
CO2R3
R2 O CO2R3 231
R2
229 230 1. HCOOH, Na2S2O4
R1 = H, Me, Pr R3 = H, Et reflux, 18h
R2 = H, Me, Pr R4 = H, OMe 2. NaOH, EtOH
reflux, 1h
O H R4
Appropriate amine O H R4
R1 N
N EDC, HOBt, TEA R1 N
O DMF, rt, 5-8 days N
R O
O N
O N CO2R3
R2 233 R = 4-halophenylaminocarbonyl 232
R2

Scheme 36. Synthesis of 9-deazaxanthine derivatives (9-dAX) 233.

606 A. Pałasz, D. Ciez_ / European Journal of Medicinal Chemistry 97 (2015) 582e611

O
CN CONH2 NH
PPA/OPA KOH/EtOH S
o
90 C CS2, heat
NH2 NH2 NH
S S S
234 235 236

O KOH/EtOH KOH/EtOH
NH bromo alkyl reagent chloro alkyl reagent
heat heat
SR
N
O
S
238 NH
R = CH3, CH2CH2OCH3 O
CH2CH2OCH2CH3 NH
S
237

Scheme 37. Synthesis of thieno[2,3-d]pyrimidines 237 and 238.

of 6-amino-1,3-dimethyl-2,4-uracil to bis chalcones 241 or 244
(Scheme 38).
The cytotoxic activity of pyrimidine monoadducts of bis-chal-
cone was assessed using the MTT (3-(4,5-dimethylthiazol-3-yl)-
2,5-diphenyltetrazolium bromide) assay on; cervical cell lines HeLa,
C-33 and CaLo, breast cancer MCF-7, myelogenous leukemia K-562,
colorectal cancer SW480 and SW620, and on normal epithelial cell
of embryonic kidney 293Q; all cell lines used had a human origin.
Examined compounds displayed cytotoxicity with a massive
vacuolation in different human cell lines in vitro. The cytotoxicity is
favoured by having a 1-methylpyrrol-2-yl group in the hetero-
arylidene moiety. The compounds induced G1 phase cell cycle ar-
rest, and their cytotoxicity went without morphological and
biochemical evidence of apoptotic cell death in HeLa cells. In
addition, the results showed that this vacuole formation does not
require de novo protein synthesis and the content vacuolar is acidic.
Prepared compounds could be of pivotal importance in the study of
non-apoptotic cell death with vacuole formation and could be
useful in research into new autophagy inhibitors agents [61].
Sun et al. [64] constructed the fused uracils - pyrazolo[1,5-a]
[1,3,5]triazine nucleus using a reaction that annulated the 1,3,5-
triazine ring onto a pyrazole scaffold. Prepared 1,3-dihydro-pyr-
azolo[1,5-a][1,3,5]triazin-2-thioxo-4-ones exhibited various extent
of inhibitory activity against thymidine phosphorylase (TP). TP is an
enzyme that promotes tumor growth and metastasis and therefore
is an attractive drugable target. 7-Deazaxanthine (7DX) has been
found to demonstrate inhibitory activity against TP, however its
structural modifications may lead to more potent purine like TP
inhibitors. The authors use 7DX as the lead compound and checked
if 1,3-dihydro-pyrazolo[1,5-a][1,3,5]triazin-2,4-diones and 1,3-
dihydro-pyrazolo[1,5-a][1,3,5]triazin-2-thioxo-4-ones may possess
TP inhibitory activity comparable to 7DX. The pyrazolo[1,5-a][1,3,5]
triazine scaffold has served as a good template for developing
enzyme inhibitors as therapeutic agents. With appropriate struc-
tural modifications, this scaffold can be readily used to fulfill
pharmacophoric requirements in the design stage of drug discov-
ery. By reacting respectively the substituted 3-amino pyrazoles
with either ethoxycarbonyl isocyanate or ethoxycarbonyl isothio-
cyanate: both the 2,4-dione series and the 2-thioxo-4-one series
can be readily synthesized in two steps (Scheme 39). Target com-
pounds 1,3-dihydro-pyrazolo[1,5-a][1,3,5]triazin-2,4-diones 255
and 258 were synthesized via a two-step synthetic scheme. The
corresponding amines 252, 253 and 256 were reacted separately
with ethoxycarbonyl isocyanate at room temperature in DMF to
give N-ethoxycarbonyl-N’-(pyrazol-3-yl) ureas 254 and 257. This
was followed subsequently by an intramolecular ring annulation
reaction under the catalysis of sodium ethoxide to generate the
target compounds 255 and 258. 1,3-Dihydropyrazolo[1,5-a][1,3,5]
triazin-2-thioxo-4-ones 260 and 262 were synthesized similarly by
a two-step synthesis (Scheme 39). The corresponding amines 249,

N N

O O 6-amino-1,3-dimethyl N O
NaOH, H2O -2,4-pyrimidinedione
EtOH, rt Triton B, THF, rt
+
N N N N
Y N CHO Y
Y
Y = CH2, O 241 242 O
239 240
N N

O 6-amino-1,3-dimethyl N O
CHO NaOH, H O O
2 -2,4-pyrimidinedione
EtOH, rt Triton B, THF, rt
+
Y S S S S S
Y Y
Y = CH2, O 243 244 245
239

Scheme 38. Synthesis of the pyrimidine monoadducts of chalcones 242 and 245.
 A. Pałasz, D. Ciez_ / European Journal of Medicinal Chemistry 97 (2015) 582e611 607

252, 253, and 256 were reacted separately with ethoxycarbonyl
isothiocyanate at room temperature in DMF to give N-ethox-
ycarbonyl-N0 -(pyrazol-3-yl)thioureas 259 and 261. An intra-
molecular ring annulation reaction of compounds 259 and 261 was
carried out under the catalysis of sodium ethoxide to produce the
target compounds 260 and 262.
Prepared compounds were evaluated for inhibitory activity us-
ing a modified TP bioassay. The bioassay used recombinant E. coli
TP, expressed in E. coli as the enzyme and thymidine as the sub-
strate. The bioisosteric replacement of oxygen with sulfur at posi-
tion 2 of the pyrazolo[1,5-a][1,3,5]triazine scaffold was found to be
essential for the TP inhibitory activity of this type of compounds.
The insertion of a phenyl ring at either position 7 or 8 was enough
to enhance the TP inhibition, especially when the aryl group was
located at position 8. It was also found that when substituents
introduced at position 4 of the attached phenyl ring, were more
electron withdrawing, the IC50 values would decrease further. The
best compound 262 (R ¼ 4-F5S -phenyl), which bears a para-
substituted pentafluorosulfur group, showed an IC50 value of
0.04 mM, which was around 800 times more potent than the 7DX
(IC50 ¼ 32 mM) under the same bioassay conditions. In addition, 262
(R ¼ 4-F5S-phenyl) was found to be a non-competitive inhibitor
thus suggested that it might interact with TP at a position different
from the substrate binding site [64].
Bera and co-workers [65] designed and synthesized fused uracil
derivatives - 1,2,4-triazolo[1,5-a][1,3,5]triazin-5,7-diones 269, 270

CH3 N
CH3O CH3 HN
N NH2
CH N CH3
KCN, EtOH CH3O CH3
Br NH2-NH2, EtOH
reflux CN DMF, reflux CN reflux
R R
246 R
247 248 249
R = I, SF5 R

O O N
KCN, EtOH HN
CN NH2-NH2, EtOH NH2
Br
50oC reflux

250 251 252

ethoxycarbonyl O
NH isocyanate EtOOC EtONa, EtOH
N DMF, rt NH N
NH HN N N
R R reflux R
H2N O N O N
252, 253 H 254 H 255
R = H, Me, C(Me)3
CF3, Ph, substituted phenyl
ethoxycarbonyl O
NH isocyanate EtOOC EtONa, EtOH
N DMF, rt NH N
NH HN N N
reflux

H2N O N O N
R H R H R
257 258 O
256
R = H, Me, C(Me)3 H
N
CF3, Ph, substituted phenyl
O N N
ethoxycarbonyl O
isothiocyanate H H
NH EtOOC EtONa, EtOH
N DMF, rt NH N
NH HN N N 7-DX
R R reflux R
H2N S N S N
252, 253 H 259 H 260
R = H, Me, C(Me)3
CF3, Ph, substituted phenyl
ethoxycarbonyl O
NH isothiocyanate EtOOC EtONa, EtOH
N DMF, rt NH N
NH HN N N
reflux

H2N S N S N
R H R H R
261 262
249, 256
R = H, Me, C(Me)3
CF3, Ph, substituted phenyl

Scheme 39. Synthesis of 1,3-dihydro-pyrazolo[1,5-a][1,3,5]triazin-2,4-diones 255, 258 and 1,3-dihydropyrazolo[1,5-a][1,3,5]triazin-2-thioxo-4-ones 260 and 262.
608 A. Pałasz, D. Ciez_ / European Journal of Medicinal Chemistry 97 (2015) 582e611

and its 5-thioxo analogues 271, 272 which contained different
substituents at meta- and/or para-positions of 2-phenyl or 2-benzyl
ring attached to the fused ring structure (Scheme 40).
Synthetic approach was utilized to produce 5-amino-1,2,4-
triazoles 266. The two-step reaction involved the synthesis of
amidoguanidines 265 from readily available substituted acid chlo-
rides 264 or hydrazides 263, followed by microwave-assisted
cyclocondensation in water (Scheme 40). The reaction of 5-
amino-1,2,4-triazoles 266 with ethyl iso-cyanoformate or ethox-
ycarbonyl isothiocyanate in DMF afforded the urea 267 and thio-
urea 268 derivatives which underwent intramolecular
heterocyclization in the presence of base, resulting in the formation
of target compounds 269, 270, 271 and 272. The pharmacological
evaluation demonstrated that 5-thioxo analogues exhibited a
varying degree of inhibitory activity towards thymidine phos-
phorylase, comparable or better than reference compound, 7-
deazaxanthine (7-DX) (IC50 value ¼ 42.63 mM). Compounds 271
(R ¼ 3,4-Cl2C6H3) and 272 (R ¼ 3,4-Cl2C6H3), those that bear di-
chloro group on the phenyl ring, displayed a mixed-type of inhib-
itory mechanism in the presence of variable concentrations of
thymidine (dThd). Several compounds were found to have a
noticeable inhibitory effect on the expression of angiogenesis
markers, including VEGF and MMP-9 in MDA-MB-231 breast cancer
cells. These compounds constitute a new direction for design and
synthesis of novel TP inhibitors with promising anti-angiogenic
property [65].
Evdokimov et al. [66] prepared the camptothecin-inspired fused
uracils - 1H-indeno[20 ,10 :5,6]dihydropyrido[2,3-d]pyrimidine ana-
logues 276 as well as their oxidized planar counterparts 277 uti-
lizing a practical multicomponent synthetic protocol (Scheme 41).
Natural-product-mimetic scaffolds were synthesized by one-step
multicomponent synthesis. 1H-Indeno[20 ,10 :5,6]dihydropyrido
[2,3-d]pyrimidines 276 were accessible by refluxing 6-aminouracil
273, indane-1,3-dione 274 and selected aldehydes 275 in a mixture
of acetic acid and ethylene glycol. Dihydropyridines 276 were
further chemically oxidized. Chloroanil was found to be excellent
oxidizing agent, and indeneopyridines 277 precipitated from
refluxing DMF solutions within a few minutes.
Camptothecin is natural product but due to limited water sol-
ubility and high levels of toxicity, the clinical trials aimed at eval-
uation of camptothecin as an anticancer drug were suspended.
The synthesized compounds exhibited submicromolar anti-
proliferative potencies toward a panel of human cancer cell lines:
HeLa human cervical cancer, Jurkat human T-cell leukemia, MCF-7
human mammary carcinoma, A-549 human NSCLC, Lovo, human
colon cancer, U373 human glioblastoma, SKMEL-28 melanoma, PC-
3 human prostate cancer. Biochemical experiments were consistent
with the dihydropyridine library members undergoing intracellular
oxidation to the corresponding planar pyridines, which then inhibit
topoisomerase II activity, leading to inhibition of proliferation and
cell death. The compounds 276 and 277 represent a convenient
starting point for anticancer drug discovery [66].
According to our interest of uracils we also occupied in the
synthesis of fused uracils of potential pharmacological activity.
Pałasz et al. described convenient and efficient procedures for the
preparation of fused uracils - pyrano[2,3-d]pyrimidine derivatives
[67e71]. Pyran derivatives are common structural subunits in a
variety of important natural products, including carbohydrates,
alkaloids, polyether antibiotics, pheromones, and iridoids. The
preparation of the compounds containing a pyran ring fused to an
uracil ring poses a significant synthetic challenge. Fused uracils
such as pyrano[2,3-d]pyrimidines can be efficiently synthesized by
hetero-Diels-Alder reactions of 5-arylidene derivatives of barbituric
acids with the vinyl ethers. We demonstrated that 5-arylidene-N,N-
dimethylbarbituric acids 278 underwent smooth hetero-Diels-
Alder reactions with enol ethers 279 to afford cis and trans di-
astereoisomers of 7-alkoxy-5-aryl-2H-pyrano[2,3-d]pyrimidine-
2,4-diones 280 in excellent yields (84e95%) (Scheme 42) [67].

O
H N
N N ( )n
R
O N N
O
H 269, 270
R
( )n NHNH2
NaOH, 80% ethanol (aq)
n = 0, 1 263 o
100 C, 20 min
N NH O
S-methylisothiourea sulphate
NaOH, rt, 72h R COOEt
ethyl isocyano formate ( )n N N
N
DMF, rt, 5h
H H
H2N NH2 H2O, MW
O o
N NH n = 0, 1 267
180 C, 5-7 min
R N R
( )n ( )n N NH2
N N NH S
H 266
R COOEt
ethoxycarbonyl isothiocyanate ( )n N N N
n = 0, 1 265
DMF, rt, 5h H H
n = 0, 1 268
aminoguanidine hydrochloride
o NaOH, 80% ethanol (aq)
180 C, NaOH, 20 min o
R = Ph, substituted Ph 100 C, 20 min
n = 0, 1
O O
269, 271: n = 0
R 270, 272: n = 1 H
( )n Cl N
N N ( )n
n = 0, 1 264 R
S N N
H 271, 272

Scheme 40. Synthesis of 1,2,4-triazolo[1,3,5]triazin-5,7-diones 269, 270 and 5-thioxo analogues 271 and 272.
 A. Pałasz, D. Ciez_ / European Journal of Medicinal Chemistry 97 (2015) 582e611 609

O
A B C N
N D
E O
Camptothecin
O OH O

O O R O O R O
AcOH/glycol (2:1) chloranil, DMF
H H H
N O 274 o N reflux, 3-5 min, 95-98% N
120 C, 60-90%
O
O N NH2 O N N O N N
H R H H H H H
273 275 276 277

R = 4-MeO-Ph; 4-Quinolinyl; 3,4-Me2-Ph; 3,4-(MeO)2-Ph; 2,4-Cl2Ph; 4-isopropyl-Ph;

4-EtO-Ph; 4-CF3Ph; 2,3,4-Cl3-Ph; 4-MeS-Ph; 4-Me-Ph; 4-F-Ph; 3-Br,4-F-Ph;
3-Br,4-MeO,5-MeO-Ph; 3-Br,4-F-Ph, 4-CF3O-Ph

Scheme 41. Preparation of fused uracils e dihydro- 276 and indenoyrido[2,3-d]pyrimidines 277 by MCR protocol.

Scheme 42. Different fused uracils - pyrano[2,3-d]pyrimidines of potential pharmacological activity, prepared by hetero-Diels-Alder cycloadditions.
610 A. Pałasz, D. Ciez_ / European Journal of Medicinal Chemistry 97 (2015) 582e611
Cycloadducts with cis-configuration were the major products. It
was also examined that, three-component one-pot reactions of
N,N-dimethylbarbituric acid, aromatic and heteroaromatic alde-
hydes, and enol ethers in the presence of piperidine gave uracils in
very good yields (87e95%) [67]. Moreover, solvent-free hetero-Di-
els-Alder reactions of 5-arylidene derivatives of barbituric acids
278 with ethyl vinyl ether were investigated at room temperature
and pyrano[2,3-d]pyrimidines 280 were obtained in excellent
yields (Scheme 42) [68]. Three-component one-pot syntheses of
fused uracils 280 were also performed in aqueous suspensions [68].
“On-water” cycloadditions were characterized by high diaster-
eoselectivity in contrast to reactions carried out in homogeneous
organic media.
Next, we introduced oxazolidin-2-one ring as substituent at 7
position of pyrano[2,3-d]pyrimidines by hetero-Diels-Alder cyclo-
additions [69]. We demonstrated that reactions of 5-arylidene-1,3-
dimethylbarbituric acids 278 with N-vinyl-oxazolidin-2-one affor-
ded mixtures of 2H-pyrano[2,3-d]pyrimidine-2,4-diones trans 281
and uracils 282 resulted from an elimination of oxazolidin-2-one
(Scheme 42). Designing of structural modifications of pyrano[2,3-
d]pyrimidine scaffold we synthesized spirouracils and dis-
pirouracils [70]. Inverse-electron demand hetero-Diels-Alder cy-
cloadditions of sterically hindered cycloalkylidene derivatives of
N,N-dimethylbarbituric acid 285 with enol ethers or cyclic enol
ether 287 were investigated (Scheme 42). Cycloalkylidene de-
rivatives of N,N-dimethylbarbituric acid were synthesized by
Knoevenagel condensation of N,N-dimethylbarbituric acid 283
with appropriate cycloalkanone 284 by refluxing in toluene or
xylene in the presence of b-alanine and acetic acid as catalyst.
Cycloaddition reactions were performed in toluene solution at
110  C for 24 h. Spirouracils 286 and dispirouracils 288 were ob-
tained in good 78e93% yields. In our recent work we developed a
convenient and efficient procedure for the preparation of fused
uracils containing a sugar moiety [71]. The reaction sequence was:
Knoevenagel condensation of unprotected sugars and N,N-dime-
thylbarbituric acid 283, acetylation of C-glycosides and hetero-
Diels-Alder reaction. O-Acetylated 1,3-dimethyl-2,4,6-trioxo-pyr-
imidin-5-ylidene derivatives 290 were used as new heterodienes in
the synthesis of fused uracils - pyrano[2,3-d]pyrimidines 291 and
292 with a sugar moiety (Scheme 42). Solvent-free hetero-Diels-
Alder cycloadditions of O-acetylated pyrimidin-5-ylidene alditols
290 with enol ethers and cyclic enol ether were investigated at
room temperature. New, enantiomerically pure cis and trans di-
astereoisomers of pyrano[2,3-d]pyrimidines 291 and 292 with
alditol moiety were obtained. The same pyrimidin-5-ylidene aldi-
tols 290 underwent conjugate Michael addition-cyclizations with
malononitrile at room temperature to afford optically active uracils
293 - diastereoisomers of pyrano[2,3-d]pyrimidine-6-carbonitriles
with a sugar moiety (Scheme 42) [71]. All fused uracils presented
on Scheme 42 have not been evaluated for their pharmacological
activity yet.
3. Conclusion
Drug development has been a principal driving force in the field
of medicinal chemistry. A significant attention of scientists has
been paid to the development of synthesis of heterocyclic com-
pounds containing nitrogen atoms. The uracil derivatives are one of
the most researched areas in medicinal chemistry. The new uracil
conjugates exhibit promising activities like anticancer, antiviral,
antibacterial, antiparasitic or can act as hypoglycaemic agents. In
this review, we have introduced readers to a broad range of highly
functionalized uracil derivatives. This article is focused on the
showing of reaction conditions, substrate scope and development
of new reaction types. The structure-activity relationship of the
reported uracil derivatives revealed that the choice of a suitable
substitution pattern including the presence of fluorine atom or
chlorine atom or some heterocyclic moieties, on the basic skeleton
plays a key role in regulating the biological potential of the syn-
thesized uracils. We sincerely hope that this article will stimulate
further research in uracil derivatives synthesis and will encourage
scientists to design novel approaches. The authors apologize to the
researches which, for one reason or another, have not been
mentioned in this review.
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