Design of Novel Inhibitors of Human Thymidine Phosphorylase: Synthesis, Enzyme Inhibition, in Vitro Toxicity, and Impact on Human Glioblastoma Cancer

Article abstract of DOI:10.1021/acs.jmedchem.8b01305

Overexpressed human thymidine phosphorylase (hTP) has been associated with cancer aggressiveness and poor prognosis by triggering proangiogenic and antiapoptotic signaling. Designed as transition-state analogues by mimicking the oxacarbenium ion, novel pyrimidine-2,4-diones were synthesized and evaluated as inhibitors of hTP activity. The most potent compound (8g) inhibited hTP in the submicromolar range with a noncompetitive inhibition mode with both thymidine and inorganic phosphate substrates. Furthermore, compound 8g was devoid of apparent toxicity to a panel of mammalian cells, showed no genotoxicity signals, and had low probability of drug-drug interactions and moderate in vitro metabolic rates. Finally, treatment with 8g (50 mg/(kg day)) for 2 weeks (5 days/week) significantly reduced tumor growth using an in vivo glioblastoma model. To the best of our knowledge, this active compound is the most potent in vitro hTP inhibitor with a kinetic profile that cannot be reversed by the accumulation of any enzyme substrates.

Full text of DOI:10.1021/acs.jmedchem.8b01305

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Article
Design of Novel Inhibitors of Human Thymidine
Phosphorylase: Synthesis, Enzyme Inhibition, in vitro
Toxicity, and Impact on Human Glioblastoma Cancer
Nathalia Sperotto, Candida Deves Roth, Valnês S. Rodrigues-Junior, Christiano Ev Neves,
Fávero Reisdorfer Paula, Adilio da Silva Dadda, Pedro Bergo, Talita Freitas de Freitas,
Fernanda Souza Macchi, Sidnei Moura, Ana Paula Duarte de Souza, Maria Martha Campos,
Cristiano Valim Bizarro, Diogenes Santiago Santos, Luiz Augusto Basso, and Pablo Machado
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01305 • Publication Date (Web): 07 Jan 2019
Downloaded from http://pubs.acs.org on January 9, 2019
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Design of Novel Inhibitors of Human Thymidine
Phosphorylase: Synthesis, Enzyme Inhibition, in
vitro Toxicity, and Impact on Human Glioblastoma
Cancer
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Nathalia D. de Moura Sperottoa,b,†, Candida Deves Roth , Valnês S. Rodrigues-Junior ,
a,†
a
a
c
a
a
Christiano Ev Neves , Fávero Reisdorfer Paula , Adilio da Silva Dadda , Pedro Bergo , Talita
a
a,d
e
f
Freitas de Freitas , Fernanda Souza Macchi , Sidnei Moura , Ana Paula Duarte de Souza ,
Maria Martha Camposb,d,g, Cristiano Valim Bizarro , Diógenes Santiago Santos , Luiz
a,d
a,d
Augusto Bassoa,b,d,*, Pablo Machadoa,d,*
aCentro de Pesquisas em Biologia Molecular e Funcional, Instituto Nacional de Ciência e
Tecnologia em Tuberculose, Pontifícia Universidade Católica do Rio Grande do Sul, 90616-
900, Porto Alegre, RS, Brazil
bPrograma de Pós-Graduação em Medicina e Ciências da Saúde, Pontifícia Universidade
Católica do Rio Grande do Sul, 90616-900, Porto Alegre, RS, Brazil
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cLaboratório de Desenvolvimento e Controle de Qualidade em Medicamentos, Universidade
Federal do Pampa, 97508-000, Uruguaiana, RS, Brazil
dPrograma de Pós-Graduação em Biologia Celular e Molecular, Pontifícia Universidade
Católica do Rio Grande do Sul, 90616-900, Porto Alegre, RS, Brazil
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eLaboratório de Produtos Naturais e Sintéticos, Instituto de Biotecnologia, Universidade de
Caxias do Sul, 95070-560, Caxias do Sul, RS, Brazil
fLaboratório de Imunologia Clínica e Experimental, Centro Infant, Pontifícia Universidade
Católica do Rio Grande do Sul, 90616-900, Porto Alegre, RS, Brazil
gCentro de Pesquisa de Toxicologia e Farmacologia, Pontifícia Universidade Católica do Rio
Grande do Sul, 90616-900, Porto Alegre, RS, Brazil
†These authors contributed equally to this work
KEYWORDS
Human thymidine phosphorylase, angiogenesis, transition state analog, non-competitive
inhibitors, anticancer candidate
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ABSTRACT
Overexpressed human thymidine phosphorylase (hTP) has been associated with cancer
aggressiveness and poor prognosis by triggering pro-angiogenic and anti-apoptotic signaling.
Designed as transition state analogs by mimicking the oxacarbenium ion, novel pyrimidine-
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,4-diones were synthesized and evaluated as inhibitors of hTP activity. The most potent
compound (8g) inhibited hTP in the submicromolar range with a non-competitive inhibition
mode regarding both thymidine and inorganic phosphate substrates. Furthermore, compound
8
g was devoid of apparent toxicity to a panel of mammalian cells, showed no genotoxicity
signals, low probability of drug-drug interactions, and moderate in vitro metabolic rates.
Finally, treatment with 8g (50 mg/kg/day) for two weeks (5 days/week) significantly reduced
tumor growth using an in vivo glioblastoma model. To the best of our knowledge, this active
compound is the most potent in vitro hTP inhibitor with a kinetic profile that cannot be
reversed by the accumulation of any enzyme substrates.
INTRODUCTION
Cancer is one of the leading causes of death worldwide, being responsible for approximately
one in six deaths. According to the World Health Organization (WHO), the disease will claim
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.6 million lives in 2018, being the second leading cause of death globally.¹ Although cancer
diagnosis and treatment have improved in the last few decades, providing a significant increase
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in patient survival rates, the clinical outcome of the disease remains clearly suboptimal with
high rates of recurrence, invasiveness, and metastases. Ideally, new therapeutic strategies
should act at different and critical stages of tumor progression to improve efficacy. Within this
context, angiogenesis has emerged as a hallmark process in cancer growth and metastasis.²
Since the early 1970s, it has been hypothesized that blocking angiogenesis could be a strategy
to contain the disease's progress.³ Since then, a large number of compounds targeting
angiogenesis pathways have been under pre-clinical development, with many molecules
currently in clinical trials or already approved for clinical use by the U.S. Food and Drug
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Administration (FDA). Among the several factors that trigger vascular growth, human
thymidine phosphorylase (hTP) stands out, as it has been described as a validated target for
the development of anti-angiogenic compounds.5,6
HTP is an enzyme involved in the pyrimidine salvage pathway, ensuring the reestablishment
7
of pyrimidine nucleotides required for DNA repair and replication. This enzyme catalyzes the
reversible phosphorolysis of thymidine (dThd) in the presence of inorganic phosphates
yielding thymine and 2-deoxy-α-D-ribose-1-phosphate (2DDR-1P). Moreover, hTP shows
deoxyribosyl transferase activity, by which the deoxyribosyl moiety is transferred from a
pyrimidine nucleoside to a different pyrimidine base, generating the formation of new
pyrimidine nucleosides.8,9 Particularly, hTP has been described to possess an identical amino
acid sequence to platelet-derived endothelial-cell growth factor (PD-ECGF) and gliostatin,
indicating that they are the same proteins. PD-ECGF has been described to stimulate
angiogenesis, while gliostatin is involved in the control of glial cell proliferation. 10-12
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hTP seems to play a pivotal role in the pathogenesis of cancer; unlike in healthy human
tissues, its expression is up-regulated in different types of tumors. In general,
microenvironments of stressing conditions, such as hypoxia and low pH, tissues submitted to
radio- and chemotherapy conditions, and the presence of different inflammatory cytokines
have resulted in increased levels of the enzyme.6,13 Elevated levels of hTP are associated with
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cancer aggressiveness and poor prognosis, mainly due to the pro-angiogenic and anti-apoptotic
functions.14-16
The pro-tumor signaling mechanism has been related to one of the products of the enzymatic
reaction catalyzed by hTP. The cleavage of N-glycosidic bond leads to 2DDR-1P, which is
converted to 2-deoxy-D-ribose (2DDR) by non-enzymatic dephosphorylation. Once outside
the cell, 2DDR triggers angiogenic and anti-apoptotic activities.17-19 This molecule affects
endothelial cell migration by activation of the integrin downstream signaling pathway.
Moreover, the augmented expression and/or secretion of other different angiogenic factors,
such as interleukins (ILs), vascular endothelial growth factor (VEGF), and matrix
metalloproteases (MMPs) in the tumor microenvironment improve angiogenesis and cancer
metastasis.20-22 Furthermore, hTP and 2DDR have been correlated with protection against
apoptosis in different types of tumor cells by hypoxia induction. Either hTP or 2DDR inhibited
hypoxia-induced pro-apoptotic signals via several mechanisms, such as the activation of
caspase-3 and caspase-9, mitochondrial transmembrane potential reduction, Bcl2 and Bcl-xL
down-regulation, mitochondrial cytochrome-c release, and HIF-1α up-regulation. 16, 23-25
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Aiming to suppress the angiogenic activity and apoptosis protection in tumor cells,
different inhibitors of hTP have been designed as potential anticancer agents. Pyrimidine- and
purine-based compounds such as 6-amino-5-bromouracil (1) and 7-deazaxanthine (2) have
been reported as inhibitors of TP activity (Figure 1).26-28 It is important to mention that several
inhibitors have been developed using Escherichia coli TP as a study model, but their inhibitory
profile on the human orthologous enzyme still needs to be analyzed. In 2015, the FDA
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approved the clinical use of Lonsurf®,
a
combination of 5-chloro-6-[1-(2-
iminopyrrolidinyl)methyl] (TPI; 3), a hTP inhibitor, and trifluorothymidine (TFT), a
chemotherapeutic agent, for the treatment of metastatic colorectal cancer (Figure 1).29,30 TPI
has been described as a competitive inhibitor of hTP-catalyzed reactions with an IC of 0.035
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µM and a K of 0.020 µM.
i
In an attempt to develop new hTP inhibitors that could hopefully be effective in the
pathogenesis of cancer, the present study reports the design and synthesis of novel pyrimidine-
based compounds. The molecules were designed to be analogous to the hTP transition state.
Additionally, inhibition studies with the characterization of kinetic mechanisms for the most
potent molecule and evaluation of the interaction mode using molecular docking were
performed. Finally, early toxicity experiments, pharmacokinetic study, and the effect on
xenograft tumor growth in vivo for the most effective hTP inhibitor are also shown.
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IC50 = 17 M
IC50 = 40 M
IC50 = 0.035 M
Figure 1. Chemical structures and IC of some pyrimidine- and purine-based inhibitors of
50
hTP-catalyzed reactions.
RESULTS AND DISCUSSION
Design and Synthesis
The strategy for obtaining new compounds with inhibitory capacity on hTP was carried
out in three distinct phases: i) the synthesis of analogs to the transition state of the activated
complex produced in the enzymatic catalysis; ii) the choice of halogens as substituents on the
5
-position of the heterocyclic ring uracil; and iii) the use of different amino alcohols,
mimicking 2DDR in an acyclic form.
Obtaining compounds analogous to the transition states of enzymatically catalyzed
reactions has led to the development of the most potent inhibitors to date.31,32 The hTP-
catalyzed reaction is considered, from a chemical point of view, a nucleophilic substitution
reaction that can be of 1st or 2nd order (S 1 or S 2, respectively) (Figure 2). Two studies have
N
N
described that thymidine arsenolysis catalyzed by hTP presents a concerted SN2
mechanism.33,34 The first proposed mechanism was symmetrical with the nucleophile
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(
arsenate) and the leaving group (thymine) showing approximately the same binding order.33
In this study, a histidine-tagged protein was used that could modify the interpretation of the
results.34 Other work corroborated the S 2 concerted mechanism; however, it was
N
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demonstrated that the transition state presents a carbocationic character with the likely
3
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formation of the ribooxocarbenium ion. This fact was explained by the different orders of
binding between the 1-N-ribosidic bond (C-N = 2.45 Å) and the distance of the arsenate
relative to 2DDR (C-O = 2.95 Å) approaching the opposite side. On the other hand, it has been
proposed that the hydrolysis of thymidine catalyzed by hTP undergoes an S 1 transition state
N
with the formation of a ribooxocarbonium intermediate without the participation of the
nucleophile.35 In this mechanism, the determinant step of the reaction speed was the
nucleophilic attack of the water to the reaction intermediate. It is noteworthy that lack of
inhibitory activity of the compounds geometrically and electronically similar to the S 2 transition
N
3
6
state has supported a transition state with ribocationic character. Therefore, herein, the
objective was to synthesize compounds that could take advantage of the carbocation character
presented by both the S 1 and S 2-like transition states proposed for hTP (Figure 2).
N
N
The second round was the choice of halogen at the 5-position of the heterocyclic uracil ring,
substituting the methyl present at that position in the thymidine. Many compounds described
as hTP inhibitors include chlorine (Cl), bromine (Br) or fluorine (F) attached to the 5-position
of the above heterocyclic ring.37-40 It is noteworthy that the high electronegativity of the
halogenated substituents increases polarization of the heterocyclic ring, stabilizing the possible
negative charge formed on the uracil ring similar to the transition states of hTP (S 1 or S 2
N
N
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mechanism). Nevertheless, this polarization reduces the pKa of the heterocycle as it stabilizes
its conjugate base. With increasing acidity, the hydrogen-bonding donor group as 3-NH forms
more effective interactions, allowing greater stabilization of the complex through the presence
of more efficient hydrogen bonds at the active site of the protein. Furthermore, the presence
of halogens could establish halogens bonding with electron density donor groups through their
sigma-hole.41
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Finally, similar to that which has already been described,42,43 the use of acyclic amino
alcohols can mimic the oxocarbenium formed in the transition state catalyzed by hTP (Figure
2
). This strategy of molecular simplification has already been shown to be efficient for
maintaining inhibitory efficiency and facilitating chemical synthesis. The pKa of the side chain
NH) of the amino alcohols can produce a positively-charged center at physiological pH,
similar to the oxocarbenium formed in the transition state for hTP.
(
O
O
CH3
CH3
S 2-like TS
O
HN
HN


X
S 1 TS
N
N
HN
OH
OH
O
N
O
N

O
N
O
O
O

OH
HO
O P OH
O
OH
OH
X = Cl, Br, I
Figure 2. S 1- and S 2-like transition states proposed for the hTP-catalyzed reaction.
N
N
Compounds were designed using amino groups on the side chain to allow the formation of a
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positively-charged center similar to oxocarbenium formed at the transition state of the
enzymatic reaction. The amino alcohols were used to mimic the open form of 2-deoxy-D-
ribose attached to thymidine.
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The compounds were synthesized in two chemically simple steps from 6-(chloromethyl)-
pyrimidine-2,4-(1H,3H)-dione (4). The first step was carried out from the reaction of
compound 4 with the corresponding N-halosuccinimide 5 in the presence of
dimethylformamide (DMF) as a solvent (Scheme 1). The halogenated products 6a-c were
obtained in a yield of 68–87%. The second step was performed from the S 2 nucleophilic
N
substitution reaction of compounds 6a-c using amino alcohols or secondary amines (7). Two
synthetic procedures were used to obtain the desired products (Scheme 1). The first method
used water as solvent according to techniques already reported.37-38 This environmental benign
protocol furnished 8a-n after reaction times of 6–20 h with yields ranging from 13–77%. The
low yields obtained for some molecules was consistent with similar procedures previously
described.37-38 The second method employed DMF as solvent in the presence of potassium
carbonate (K CO ) as base. The reactants were stirred for 16–18 h, furnishing products with
2
3
28-85% yields. All compounds showed spectroscopy and spectrometric data consistent with
the proposed structures (Main text and supporting information).
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O
O
O
R1
X
X
i
ii or iii
R1
N
HN
HN
HN
R2
HN
O
O
N
X
6
8-87%
13-85%
Cl
Cl
O
N
H
O
N
H
O
N
H
R2
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5
6a-c
7
8a-n
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Yield (%)a Yield (%)b IC (µM)
c
Entry
X
Cl
Br
I
Yield (%)
Entry
8a
X
R1 R2
5
0
6
a
71
68
87
Cl
Cl
H
H
CH CH OH
13
33
28
45
39 ± 5
2
2
6
b
8b
1.0 ± 0.2
OH
6
c
OH
8
8
c
Br H
Br H
CH CH OH
32
23
49
52
24 ± 2
2
2
d
5.6 ± 0.9
OH
OH
OH
OH
8
e
Br H
20
47
>100
8
8
f
I
I
H
H
CH CH OH
21
75
45
77
1.2 ± 0.1
2
2
g
0.12 ± 0.01
OH
OH
OH
8
8
h
i
I
I
H
26
42
57
58
40 ± 7
36 ± 7
OH
OH
Me
OH
8
8
8
8
j
I
I
I
I
Me Me
Pr Pr
75
27
21
71
79
55
46
76
8.4 ± 0.5
>100
k
l
CH (CH ) CH
28 ± 5
87 ± 12
2
2 3
2
m
CH CH OCH CH
2
2
2
2
8
n
I
H
77
85
34 ± 4
N
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Scheme 1. Reactants and conditions: i = DMF, 25-100 °C, 1 h; ii = H O, 25-35 °C, 6-20 h; iii =
2
a
b
c
K CO , DMF, 25 °C, 16-18 h. Procedure ii; Procedure iii; Inhibitory activity of pyrimidine-
2
3
2
,4-diones 8a-n over hTP-catalyzed reactions.
1
1
1
1
1
1
1
1
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1
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8
9
0
In vitro inhibition studies of hTP
The IC screening was employed to assess the relative potency of compounds against hTP
5
0
activity and select the best compound for further studies.
The synthesized compounds 8a-n inhibited the hTP activity with IC values ranging from
5
0
0
.12 μM to 87 μM (Scheme 1). The inhibitory activity of compounds varied widely according
to the different halogens at the 5-position of the heterocyclic ring and according to the
1
2
substituents at R and R . Of the two compounds having the Cl as halogen substituent at the
5
-position of the uracil ring, compound 8b was 39-fold more effective than compound 8a. The
same trend was observed for bromo-containing compounds. Indeed, molecule 8d inhibited the
enzymatic activity of hTP with a potency that was nearly 4-fold greater than that of 8c. This
denotes the importance of the insertion of a second hydroxyl in the side chain of compounds
8
as the ethanolamine derivatives (8a and 8c) were less effective as inhibitors of hTP than
compounds derived from 2-aminopropane-1,3-diol (8b and 8d). This second hydroxyl formed,
according to docking simulations (see next section), a donor-acceptor pair of hydrogen bonds
with hTP, which should be related to the better activity of these compounds.
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6
The greatest inhibitory effect was observed for iodine-containing compounds at the 5-
position of the uracil ring. It is interesting to note that iodine better positioned the structure
8
g at the active site of hTP than chlorine and bromine derivatives 8b and 8d according to
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docking findings. In addition, iodine was the halogen with the higher molecular volume and
polarizability used in the series of synthesized compounds. These properties may be related to
the better effectiveness of the inhibitory action providing a better fit with the enzyme catalytic
site and better stabilization of negative charge on the heterocyclic ring. Furthermore, iodine
4
1
presents the largest sigma-hole among the halogens evaluated. Thus, compounds containing
this atom could perform halogen bond interactions more effectively. Once more, substitution
using a 2-aminopropane-1,3-diol derivative (8g) led to the compound having the greatest
inhibitory effect over hTP-catalyzed reactions. Structure 8g showed an inhibitory effect that
was 10-fold higher than 8f (derived from ethanolamine). This second hydroxyl formed,
according to docking simulations (see next section), a donor-acceptor pair of hydrogen bonds
with hTP which should be related to the better activity elicited by the 2-aminopropane-1,3-
diol derivatives 8b, 8d, and 8g. By contrast, the presence of the 3-aminopropane-1,2-diol
2
substituent in R considerably decreased the inhibitory activity, leading to pyrimidine-2,4-
dione 8h which exhibited an IC of 40 ± 7 μM (Scheme 1). An unexpected finding was
5
0
presented by compound 8e, which was inactive at a concentration of 100 μM. Compared to
compounds 8d and 8g, this structural modification, by repositioning the hydroxyl from
position β to position γ, considering the nitrogen, reduced the inhibition capacity of the
enzyme by more than 300-fold. It was apparent from experiments that the position of a second
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hydroxyl exerts a significant effect on stabilizing the interaction of the inhibitor with the
protein.
In order to increase the structural space of the compound series studied, the 8i-n derivatives
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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0
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8
9
0
2
containing cyclic and acyclic alkyl groups in R were synthesized. In these compounds, the
iodine atom was maintained at the 5-position of the heterocyclic ring as it performed the best
activity when compared to chlorine and bromine. None of these tested compounds exhibited
a better inhibitory effectiveness on reactions catalyzed by the hTP enzyme than compound 8g
(Scheme 1). The apparent steric effect exerted by compound 8k compared to its counterpart
8
g should be noted. Changing the dimethyl to dipropyl substituent abolished the inhibitory
effectiveness since the 8k compound did not exert any action when tested at a concentration
of 100 μM, whereas 8j inhibited the enzymatic activity of hTP with an IC of 8.4 ± 0.5 μM.
50
Based on the inhibition data, compound 8g was selected as the lead of this series of molecules
and used in subsequent assays.
Prior to assessing the mode of inhibition of the most active compound, it was confirmed that
its inhibitory activity is not time-dependent up to 40 min of pre-incubation with hTP (data
not shown). The mode of inhibition of pyrimidine-2,4-dione 8g on the phosphorolysis activity,
during the conversion of thymidine (dThd) nucleoside into thymine, was deduced by
Lineweaver-Burk plots. The reciprocal of the rate of reaction was plotted against the reciprocal
of substrate concentration, and the effect of inhibitor on both K and V was monitored. The
m
max
in vitro inhibition constant values (K and K ) were determined by plotting the slope of each
ii
is
line in the Lineweaver-Burk plots against the different concentrations of 8g and data fitting to
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an appropriate equation (Eq.(2)). The K of 8g was determined for both substrates. When the
i
inorganic phosphate substrate (P ) was fixed at a non-saturating concentration and dThd was
i
varied in the presence of fixed-varying concentrations of compound 8g, the double reciprocal
plots gave a family of lines that intercept on the left of the y-axis (Figure 3; A). These results
indicated that compound 8g inhibited hTP in a non-competitive manner with respect to dThd.
Moreover, the analysis of the kinetic constants showed that the Vmax of the enzyme decreased,
0
1
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0
while the K increased in the presence of increasing concentrations of compound 8g, which is
m
4
4
a typical effect of a non-competitive inhibitor with K < K . This analysis was consistent with
is
ii
K and K values obtained when fitting the data to Eq. (2), yielding K of 0.091 ± 0.034 µM and
is
ii
is
K = 0.291 ± 0.88 µM. The inhibitory profile suggests that compound 8g displays binding
ii
affinity for both the free enzyme (or enzyme-Pi binary complex) and the enzyme-Pi-dThd
ternary complex; however, the inhibitor preferentially binds to the free enzyme. It is
important to mention that the non-competitive inhibition elicited for 8g does not invalidate
the design of the compound as a possible transition state analog inhibitor, since the observed
inhibition mode does not exclude that this molecule can interact at the dThd binding site.44
Likewise, when P was the varied substrate in the presence of non-saturating concentrations
i
of dThd, compound 8g also displayed a non-competitive mode of inhibition, as indicated on
the double-reciprocal plots (Figure 3; B). Fitting data to Eq.(2) yielded a K value of 0.200 ±
is
0
.114 µM and a K value of 0.090 ± 0.009 µM. Compound 8g exhibited K > K , indicating a
ii
is
ii
binding preference for the enzyme-dThd-Pi ternary complex in comparison to binding to
either free enzyme or enzyme-dThd binary complex. In addition, the presence of increasing
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concentrations of inhibitor decreased both Vmax and K , which reflected, once more, a non-
m
competitive signature with K > K . Interestingly, a rapid-equilibrium random bi-bi kinetic
is
ii
mechanism has been proposed for hTP, in which both substrates can bind to free enzyme to
form the catalytically competent ternary complex; also, both products can randomly dissociate
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to yield free enzyme. Compound 8g inhibited the enzyme reaction in a non-competitive
fashion with respect to both dThd and P , in which the inhibitor can bind, not only to the
i
enzyme-substrate complex, but also to the free enzyme without obeying a binding order,
which is in agreement with the random bi-bi kinetic mechanism proposed for hTP.
Figure 3. Determination of HTP-inhibition mechanism for compound 8g. The Lineweaver-
Burk plot displays a pattern of intersection to the left of the y-axis towards dThd (A) and Pi
(B), which are diagnostic of non-competitive inhibition.
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Molecular docking studies
Docking studies were carried out to try to determine the key intermolecular interactions
that occur upon enzyme−inhibitor complex formation. In addition, these assessments sought
further evidence of the interaction site of synthesized pyrimidine-2,4-diones into hTP as non-
competitive inhibition mode presented by lead compound 8g may generate some doubt.
Simulations were performed with free enzyme using the coordinate obtained from the crystal
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46
structure of the hTP-5-iodouracil complex (PDB:2WK6). Docking using free enzyme allowed
direct comparison with the interaction mode performed by 5-iodouracil, a simplified analogue
of compound 8g, at the active site of hTP.
Experiments showed that compound 8g is stabilized at the hTP binding site through
interactions with residues His116, Ser117, Thr118, Leu148, Tyr199, Arg202, Ser217, Ile218,
and Lys221 (Figure 4). Incidentally, the residues His116, Arg202, Ser217, and Lys221, which
stabilized the heterocyclic 5-iodouracil at the 8g binary complex, are the same as those which
4
6
make strong contacts at hTP-5-iodouracil co-crystal structure. Carbonyl groups at the 2- and
-position of 8g act as hydrogen bonds acceptors from Lys221, Arg202, and His116. The
4
distance for acceptor-donor pairs were obtained with 3.02 and 2.65 Å for Arg202, 2.66 Å for
Lys221, and 2.75 Å for His116 from carbonyl groups. Another hydrogen bond of the
heterocyclic ring at the active site of hTP was observed between 3-NH and Ser217 with a
distance of 2.68 Å. The 1-NH of uracil ring also established hydrogen bond contact with
imidazole from His116 at distance of 3.05 Å. Considering the side chain of compound 8g,
hydrogen bonds and van der Waals interactions were obtained between the 1,3-
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dihydroxypropanylamino group and amino acid residues Ser117, Thr118, and Tyr199. Amino
group formed hydrogen bonds between the amino alcohol side chain and the active site of hTP
using the backbone of Ser117 and Thr118 residues. Amino donor group was positioned 2.70
and 3.10 Å from Ser117 and Thr118, respectively. In addition, hydrogen bonds between the
4-hydroxy group of Tyr199 and the hydroxyl group of 8g was observed with distance of 2.75
and 3.10 Å. Importantly, Tyr199 does not appear in the crystallographic studies among the
amino acids that stabilize the 5-iodouracil or TPI at the catalytic site of hTP.46,47 This difference
may be related to the mode of enzymatic inhibition presented by structure 8g when compared
to TPI. One of these hydroxyl groups of the side chain of 8g that act as a hydrogen acceptor
upon binding to Tyr199 participated as a hydrogen bond donor at distance of 3.11 Å from the
donor group (τ-NH) present at His116. The intermolecular interactions of the two hydroxyl
groups of the 1,3-dihydroxypropanylamino system depicted by docking experiments explain,
in theory, the better hTP inhibition profile of pyrimidine-2,4-diones 8b, 8d, and 8g in
comparison with molecules 8a, 8c, and 8f, which contain ethanolamine as a side chain.
Interestingly, the molecular docking was able to predict the best potency presented by
compound 8g. The best pose of molecules 8b, 8d, and 8g showed an affinity energy of -80.2, -
1
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-
1
8
3.3, and -99.8 Kcal.mol , respectively. Unlike 8b and 8d, iodine-containing compound 8g
positioned the amino alcohol side chain in a position that allowed strong interactions with
Tyr199 and Thr118 (Supporting information).
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Figure 4. Binding mode of compound 8g at active site of hTP (PDB: 2WK6) using Gold 5.5
molecular software. Graphic visualization obtained using PyMOL (v.0.99).
Early toxicity and in vitro metabolic stability investigations
The cytotoxicity of hTP inhibitor 8g on normal and tumor cells was evaluated using
both MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) and neutral
red uptake (NRU) methods to assess cell viability. The MTT assay is able to evaluate the
functional integrity of mitochondria based on the ability for the enzymatic reduction of
tetrazolium salt MTT by mitochondrial dehydrogenases to formazan crystal by living cells.48
49
The NRU assay is based on the accumulation of the dye in the lysosome of viable cells. The
in vitro incubation with various concentrations of pyrimidine-2,4-dione 8g (25, 50, 100, 250,
and 500 μM) did not significantly affect the viability of HepG2, Vero, HaCat, RAW 264.7, and
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U-87 MG cells (Table 1). Only at the highest concentration evaluated (1,000 μM) 8g caused a
statistically significant decrease in cell viabilities for all lineages evaluated using both MTT and
NRU methodologies (Table 1).
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In order to estimate genotoxicity, the induction of DNA single- and double-strand
breaks and alkali-labile sites in HepG2 cells was evaluated using the alkaline comet assay.
Results showed that the lead compound 8g did not cause DNA-strand breaks, even at the
highest concentration tested of 1,000 μM (Figure 5). These results indicate a likely safety
profile for 8g based on cytotoxic and genotoxic evaluations.
It is noteworthy that the viability of the glioblastoma cell line U-87 MG was evaluated in
the presence of 25, 50, 100, 250, 500, and 1,000 μM of pyrimidine-2,4-dione 8g. In the
experimental conditions tested, 8g did not affect the in vitro cellular viability of U-87 MG.
Lastly, in vitro drug-drug interactions were determined for compound 8g. As angiogenesis
inhibitors have been used as adjuvants in cancer chemotherapy, knowing the potential drug-
drug interactions is crucial in early drug discovery. The drug-drug interactions were evaluated
by assaying compound 8g against a panel of human CYP450 isoforms (3A4, 1A2, 2C9, 2C19,
2D6, and 2E1). The results denoted a reduced risk of drug-drug interactions as the IC of the
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compound was >100 µM for all of the above isoforms (Supporting information). Finally, in
vitro intrinsic clearance analysis using human S9 fraction revealed that pyrimidine-2,4-dione
8
g exhibit moderate rates of metabolism with Cl of 13.0 mL/min/kg and half-life of 28.1 min
int
(Supporting information).
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Figure 5. Induction of DNA damage measured with the alkaline comet assay in HepG2 cells
incubated with pyrimidine-2,4-dione 8g. NC = control group; Methyl methanesulfonate
(MMS) = positive control group. Results are expressed as damage index and represent the mean
±
standard error of the mean for three independent experiments. Statistical analysis was
performed by one-way analysis of variance followed by Dunnett’s Multiple Comparison Test.
Data significant in relation to control group at ***p<0.001.
Pharmacokinetics and in vivo anti-tumor activity
Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor
among adults, with an average survival of approximately 15 months.50,51 The standard protocol
available for the treatment of GBM consists of maximal safe surgical resection, followed by
radiotherapy, plus concomitant and adjuvant chemotherapy with the alkylating agent
temozolamide (TMZ).52,53 However, at least 50 % of TMZ-treated patients do not respond to
this agent. Moreover, hematological malignancies such as acute lymphoblastic leukemia, acute
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myeloid leukemia and myelodysplastic syndrome have been reported to occur after treatment
with TMZ. 53-55
Importantly, hTP is overexpressed in GBM, while undetectable or trace levels are found in
the normal human brain.56-58 The occurrence of microvascular proliferation, invasion and
resistance to apoptosis are hallmarks of glioblastoma.58,59 Anti-angiogenic therapy is
considered an important target for GBM treatment, and several clinical trials enrolling anti-
angiogenic agents are in progress.52,60-62 The novel strategies for GBM treatment are focused
on the modulation of vessel permeability, hypoxia, and tumor-induced edema.54,58,60
Pharmacokinetics studies were performed in mice after intraperitoneal (IP) administration
of 8g (50 mg/kg) (Table 2). Maximum plasma concentration (Cmax) of 34.8 µg/mL was achieved
after 0.08 h (Tmax). The data corroborated the in vitro metabolic stability indicating a
rapid/moderate elimination rate with elimination rate constant (Ke) and elimination half-life
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(t ) values of 1.38 h and 0.5 h, respectively. In addition, area under the concentration-time
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(AUC0→t and AUC0→∞) revealed a promising profile with values of 16.9 and 17.4 µg h/L,
respectively. The obtained pharmacokinetic data supported subsequent in vivo effectiveness
assays.
Table 2. Pharmacokinetic parameters determined after intraperitoneal (IP) administration of
8
g (50 mg/kg) to mice (non-compartmental analysis).
Parameters
50 mg/kg IP (n = 21)
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Cmax (µg/mL)
Tmax (h)
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AUC0→∞ (µg h/L)
AUMC0→t (µg h /L)
AUMC0→∞ (µg h /L)
MRT (h)
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Cmax: peak concentration; Tmax: time to peak plasma concentration; Ke: elimination rate
constant; t : elimination half-life; AUC: area under the concentration-time curve from 0 to
1/2
time t; AUC0→∞: area under the plasma concentration time curve from time zero to infinity;
AUMC0→t: area under the first moment of the plasma concentration time-curve; AUMC0→∞
:
area under the first moment of the plasma concentration time-curve extrapolated to infinity;
MRT: mean residence time; (n = 3 animals/ sampling time point).
To evaluate the in vivo anti-tumoral activity of the lead compound 8g, human GBM tumor
cells (U-87 MG) were subcutaneously injected into the flank of nude mice. After tumors
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reached a volume of approximately 50 mm , the animals were randomly divided and received
intraperitoneal administrations of NaCl 0.9% (control group), the compound 8g, TMZ or a
combination of TMZ plus 8g, for 14 days. The results revealed a significant decrease of tumor
growth in mice treated with pyrimidine-2,4-dione 8g, TMZ, or with the combination of both,
when compared to the control group (Figure 6). The reduction of tumor growth rate started
after 11 days of treatment, and it was maintained until euthanasia (14 days treatment).
Importantly, tumor-bearing nude mice treated with 8g did not show significant body weight
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alterations, while animals receiving TMZ showed a slight decrease (less than 20% BWC) in
body weight. These observations indicate that both 8g alone or in combination may not cause
important toxic effects (Supporting information).
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Figure 6. Effects of lead compound 8g on the relative tumor volume (RTV) of U 87-MG human
glioblastoma tumor implanted into the right flank of nude mice. NC = vehicle-treated group;
TMZ = temozolamide. Statistical analysis was performed by two-way analysis of variance
followed by Bonferroni post-tests. Data significant in relation to control group at *p<0.05,
**p<0.01 and *** p<0.001.
Immunohistochemistry (IHC) and histological analysis supported the capacity of the
compound 8g to act on tumor angiogenesis, by reducing the immunolabeling for VEGF, the
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major angiogenic factor present in GBM, and for CD31, a marker of vascular endothelium.
In addition, 8g was able to reduce the density of blood vessels present in the tumor tissue,
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compared to control group (Figure 7). The anti-tumor activity of the combined therapy was
not statistically different when compared to the treatment with TMZ alone. Moreover, we
have observed reduced VEGF staining and number of vessels in the group treated with the
combination TMZ and 8g. The TMZ group, even having a reduced tumor growth, did not show
any difference in the histochemical analysis, when compared to the control group (Figure 7).
The immunostaining for Ki-67 (cell proliferation marker), or capase-3 (the apoptosis marker)
was not significantly different among the experimental groups (Supporting information). The
low total percentage of apoptotic tumor cells present in U-87 MG cells could partially explain
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our outcome for caspase-3 labeling; however, further studies are needed to clarify this point.
Moreover, the lack of efficacy of our compound in the proliferation of tumor cells
demonstrated by Ki-67 staining, is in agreement with the in vitro results (Table 1), as the
pyrimidine-2,4-dione 8g was inactive against U-87 MG cell.65
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Figure 7. Immunohistochemistry and histological analysis. Mean of positive areas of
immunolabeling for (A) VEGF (angiogenesis marker), (B) CD31 (vascular endothelium marker)
and (C) number of blood vessels present in the tumor tissue, compared to control group. (D)
Representative images of IHC labeling of VEGF, CD31 and HE staining in different treatment
groups. NC= control group, TMZ= temozolamide. Results are expressed as optical density or
number of vessels and represent the mean ± standard error of the mean for 4 images captured
per tumor (x 200). Statistical analysis was performed by one-way analysis of variance followed
by Dunnett’s Multiple Comparison Test. Data significant in relation to control group at *p<0.05
and **p<0.01.
CONCLUSIONS
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Herein, we have designed and synthesized potent inhibitors of human thymidine
phosphorylase (hTP). The design was based on the possible oxocarbenium character of the
transition state proposed for enzyme-catalyzed phosphorolysis. In addition, the synthetic
process involved simple and easily accessible reagents and reactants. The most potent
compound was able to inhibit the enzyme with IC and K in the submicromolar range with
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i
a non-competitive inhibition mode with respect to both substrates (thymidine and inorganic
phosphate). To the best of our knowledge, this is, to date, the most potent inhibitor of hTP
with an inhibition mechanism that cannot be reversed by the accumulation of both substrates
(thymidine and inorganic phosphate). Competitive inhibition results in building up of
substrate concentrations that can overcome the inhibitor effect and ensuing increase in
enzyme activity. Unlike competitive inhibitors, non-competitive inhibitors cannot be
overcome by high concentrations of substrate as the inhibitor can bind to enzyme-substrate
complexes and decrease enzyme activity. Moreover, the most effective compound exhibited
low cytotoxicity against HepG2, Vero, HaCat, RAW 264.7, and U-87 MG lineages, absence of
genotoxicity in the alkaline comet assay, low probability of drug-drug interactions accessed
through CYP450 inhibition studies, and moderate in vitro metabolic rates. Finally, in a model
of glioblastoma cancer xenografts, the lead compound 8g was able to significantly reduce
tumor growth when administered to nude mice. The reduction in tumor volume growth was
comparable to that presented by temozolomide, a first-line drug for glioblastoma multiforme
treatment. These results demonstrate that this class of compounds may provide drug candidates
for the treatment of various solid cancers, targeting angiogenesis, the anti-apoptotic activity
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stimulated by hTP, and/or its role in fluoropyrimidine-based therapy. Additionally, the
overexpression of hTP is involved in a variety of pathological complications such as psoriasis,
atherosclerosis, rheumatoid arthritis, inflammatory bowel disease and mitochondrial
neurogastrointestinal encephalomyopathy. In diseases such as these, hTP inhibitors can play
an important role and, hopefully, bring about an improvement in the patients' quality of life.
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EXPERIMENTAL SECTION
Chemistry and structure: apparatus and analysis
The commercially available reactants and solvents were obtained from commercial suppliers
and were used without any purification. The reactions were monitored by thin-layer
chromatography (TLC) with Merck TLC Silica gel 60 F . The melting points were measured
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using a Microquímica MQAPF-302 apparatus. H and C NMR spectra were acquired on a
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Anasazi EFT-60 ( H at 60.13 MHz) or Avance III HD Bruker ( C at 400.13 MHz) spectrometer.
Chemical shifts (δ) were expressed in parts per million (ppm) relative to DMSO-d , which was
6
used as the solvent, and to TMS, which was used as an internal standard. High-resolution mass
spectra (HRMS) were obtained for all of the compounds on an LTQ Orbitrap Discovery mass
spectrometer (Thermo Fisher Scientific, Bremer, Germany). This system combines an LTQ XL
linear ion-trap mass spectrometer and an Orbitrap mass analyzer. The analyses were performed
through the direct infusion of the sample in MeOH/H O (1:1) with 0.1% formic acid (flow rate
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0 µL/min) in a positive-ion mode using electrospray ionization (ESI). For elemental
composition, calculations used the specific tool included in the Qual Browser module of
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