Pyrimidin-2(1H)-ones based inhibitors of Mycobacterium tuberculosis orotate phosphoribosyltransferase

Article abstract of DOI:10.1016/j.ejmech.2012.04.031

Tuberculosis (TB) is an ancient human chronic infectious disease caused mainly by Mycobacterium tuberculosis. The emergence of strains resistant to first and second line anti-TB drugs, associated with the increasing number of TB cases among HIV positive subjects, and the large number of individuals infected with latent bacilli have urged the development of new strategies to treat TB. Enzymes of nucleotide metabolism pathways provide promising molecular targets for the development of drugs, aiming at both active and latent TB. The orotate phosphoribosyltransferase (OPRT) enzyme catalyzes the synthesis of orotidine 5′-monophosphate from 5′-phospho-α-d-ribose 1′-diphosphate and orotic acid, in the de novo pyrimidine synthesis pathway. Based on the kinetic mechanism and molecular properties, here we describe the design, selection and synthesis of substrate analogs with inhibitory activity of M. tuberculosis OPRT (MtOPRT) enzyme. Steady-state kinetic measurements were employed to determine the mode of inhibition of commercially available and chemically derived compounds. The 6-Hydroxy-2-oxo-1,2-dihydropyridine-4-carboxylic acid (6) chemical compound and its derivative, 3-Benzylidene-2,6-dioxo-1,2,3,6-tetrahydropyridine-4-carboxylic acid (13), showed enzyme inhibition constants in the submicromolar range. Isothermal titration calorimetry data indicated that binding of both compounds to MtOPRT have negative enthalpy and favorable Gibbs free energy probably due to their high complementarity to the enzyme's binding pocket. Improvement of compound 13 hydrophobic character by addition of an aromatic ring substituent resulted in entropic optimization, reflected on a thermodynamic discrimination profile characteristic of high affinity ligands. These inhibitors represent lead compounds for further development of MtOPRT inhibitors with increased potency, which may be tested as anti-TB agents.

Full text of DOI:10.1016/j.ejmech.2012.04.031

European Journal of Medicinal Chemistry 54 (2012) 113e122
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Pyrimidin-2(1H)-ones based inhibitors of Mycobacterium tuberculosis orotate
phosphoribosyltransferase
,
b
,
,
b
,
,
b
,
,e
Ardala Breda a c, Pablo Machado a c, Leonardo Astolfi Rosado a d, André Arigony Souto c
,
Diógenes Santiago Santos a c, Luiz Augusto Basso a
,
b,
,b,c,
*
a Instituto Nacional de Ciência e Tecnologia em Tuberculose, Pontifícia Universidade Católica do Rio Grande do Sul, Avenida Ipiranga 6900, Prédio 92A e TECNOPUC,
90619-900 Porto Alegre, Rio Grande do Sul, Brazil
b Centro de Pesquisas em Biologia Molecular e Funcional, Pontifícia Universidade Católica do Rio Grande do Sul, Avenida Ipiranga 6900, Prédio 92A e TECNOPUC,
90619-900 Porto Alegre, Rio Grande do Sul, Brazil
c Programa de Pós-Graduação em Biologia Celular e Molecular, Pontifícia Universidade Católica do Rio Grande do Sul, Avenida Ipiranga 6900, Prédio 92A e TECNOPUC,
90619-900 Porto Alegre, Rio Grande do Sul, Brazil
d Programa de Pós-Graduação em Medicina e Ciências da Saúde, Pontifícia Universidade Católica do Rio Grande do Sul, Avenida Ipiranga 6900, Prédio 92A e TECNOPUC,
90619-900 Porto Alegre, Rio Grande do Sul, Brazil
e Faculdade de Química, Pontifícia Universidade Católica do Rio Grande do Sul, Avenida Ipiranga 6900, Prédio 92A e TECNOPUC, 90619-900 Porto Alegre, Rio Grande do Sul, Brazil
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
< Selection and optimization of
inhibitors based on MtOPRT kinetic
mechanism.
< Identification of MtOPRT inhibitors
with nanomolar inhibition constant
values.
< In vitro kinetic assays to characterize
binding mode of inhibitors.
< Thermodynamic
discrimination
profiles of inhibitors assessed by
ITC.
< Inhibitory activity of 13 is likely due
to entropy optimization.
a r t i c l e i n f o
a b s t r a c t
Article history:
Tuberculosis (TB) is an ancient human chronic infectious disease caused mainly by Mycobacterium
tuberculosis. The emergence of strains resistant to first and second line anti-TB drugs, associated with
the increasing number of TB cases among HIV positive subjects, and the large number of individuals
infected with latent bacilli have urged the development of new strategies to treat TB. Enzymes of
nucleotide metabolism pathways provide promising molecular targets for the development of drugs,
aiming at both active and latent TB. The orotate phosphoribosyltransferase (OPRT) enzyme catalyzes
Received 27 October 2011
Received in revised form
20 April 2012
Accepted 24 April 2012
Available online 30 April 2012
the synthesis of orotidine 50-monophosphate from 50-phospho- -ribose 10-diphosphate and orotic
a-D
Keywords:
Mycobacterium tuberculosis
Tuberculosis
Pyrimidine
Orotate phosphoribosyltransferase
acid, in the de novo pyrimidine synthesis pathway. Based on the kinetic mechanism and molecular
properties, here we describe the design, selection and synthesis of substrate analogs with inhibitory
activity of M. tuberculosis OPRT (MtOPRT) enzyme. Steady-state kinetic measurements were employed
to determine the mode of inhibition of commercially available and chemically derived compounds.
Abbreviations: TB, tuberculosis; MTB, Mycobacterium tuberculosis; OPRT, orotate phosphoribosyltransferase; OA, orotate; PRPP, a-D-5-phosphoribosyl-1-pyrophosphate;
Km, MichaeliseMenten constant; OMP, orotidine 50-monophosphate; PPi, pyrophosphate; UMP, uridine 50-monophosphate; MtOPRT, M. tuberculosis’ OPRT; FTIR, Fourier
transform infrared; ITC, isothermal titration calorimetry; UATR, universal attenuated total reflectance.
* Corresponding author. Instituto Nacional de Ciência e Tecnologia em Tuberculose, Pontifícia Universidade Católica do Rio Grande do Sul, Avenida Ipiranga 6900, Prédio
92A e TECNOPUC, 90619-900 Porto Alegre, RS, Brazil. Tel./fax: þ55 51 3320 3629.
E-mail addresses: ardalabreda@gmail.com (A. Breda), pablomachado.mail@gmail.com (P. Machado), leo.rosado@gmail.com (L.A. Rosado), arigony@pucrs.br (A.A. Souto),
diogenes@pucrs.br (D.S. Santos), luiz.basso@pucrs.br (L.A. Basso).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2012.04.031

114
A. Breda et al. / European Journal of Medicinal Chemistry 54 (2012) 113e122
Inhibitors
Pyrimidin-2(1H)-ones compounds
The 6-Hydroxy-2-oxo-1,2-dihydropyridine-4-carboxylic acid (6) chemical compound and its deriva-
tive, 3-Benzylidene-2,6-dioxo-1,2,3,6-tetrahydropyridine-4-carboxylic acid (13), showed enzyme
inhibition constants in the submicromolar range. Isothermal titration calorimetry data indicated that
binding of both compounds to MtOPRT have negative enthalpy and favorable Gibbs free energy
probably due to their high complementarity to the enzyme’s binding pocket. Improvement of
compound 13 hydrophobic character by addition of an aromatic ring substituent resulted in entropic
optimization, reflected on a thermodynamic discrimination profile characteristic of high affinity
ligands. These inhibitors represent lead compounds for further development of MtOPRT inhibitors
with increased potency, which may be tested as anti-TB agents.
Ó 2012 Elsevier Masson SAS. All rights reserved.
1. Introduction
(PRPP) to orotidine 50-monophosphate (OMP), and pyrophosphate
(PPi), in the presence of Mg2þ (Fig.1A). OMP is further decarboxylated
Tuberculosis (TB) is an ancient human chronic infectious disease
[1], caused mainly by Mycobacterium tuberculosis (MTB) [2]. TB has
been responsible for the death of over 2 million people annually,
and in 2009 there were an estimated 26 deaths per 100 000 pop-
ulation [3].
into UMP, ultimately leading to DNA and RNA synthesis. OPRT
homologs have been explored as targets for the development of
inhibitors to treat malaria [13,15e17] and toxoplasmosis [18], as
well as to cancer treatment for its role in 5-fluoruracil metabolism
[19,20]. Salmonella typhimurium OPRT inhibitors have also been
described [21].
TB treatment regimen includes six month therapy with rifam-
picin and isoniazid, supplemented with pyrazinamide and etham-
butol in the first two months [3,4]. The poor compliance to the
current treatment, owing to its length and side-effects [5], along
with HIV infection emergence, leads to the occurrence of multi
drug-resistant and extensively drug-resistant TB strains [3], for
which the standard treatment shows no bactericidal and sterilizing
activity [4,6]. Recently, a totally resistant strain, denoted as TDR-TB,
has been described as resistant to all first and second line classes of
anti-TB drugs tested [7,8]. Currently anti-TB available drugs are
considered not completely efficacious [5] due to natural disease
characteristics and emerging resistant strains that might impose
some obstacles to its treatment [4]. Thus, novel TB treatments
should be active against drug-resistant strains, and, ideally, be able
to eliminate latent forms of TB [9]. Latent TB represents a significant
infection reservoir, since almost 32% of world population is positive
on tuberculin purified protein derivative skin test [3].
The nucleotide metabolism is an essential pathway for micro-
organism viability [10], including MTB and the vaccinal strain
Mycobacterium bovis BCG [11]. In addition, de novo pyrimidine
biosynthesis has already been shown to be required for cell inva-
sion and virulence of obligatory parasites [12,13], in which
disruption of this pathway leads to avirulent pyrimidine auxo-
trophs [12]. Although the metabolism required for sustaining
mycobacterial survival during TB latency is still poorly understood,
experimental evidence points to the maintenance of nucleotide
synthesis in latent bacillus [14]. A specific inhibitor that targets
a nucleotide synthesis enzyme has thus the potential of being
effective against both active and latent TB.
Molecular, kinetic and thermodynamic characterization of
MtOPRT have recently been described [22]. MtOPRT follows
a MonoeIso Ordered Bi Bi kinetic mechanism (Fig. 1B). Determi-
nation of MtOPRT kinetic parameters and enzyme mechanism
allowed the selection and testing of several commercially available
compounds that might act as specific inhibitors of this mycobac-
terial enzyme (Fig. 2 and Table 1 e Compounds 1e8). Starting
compound selection was based on molecular similarity to
MtOPRT substrate OA. The rationale for choosing OA analogs is as
follows: PRPP MtOPRT substrate is also substrate for several other
metabolic reactions [23] resulting in non-specific enzyme inhibi-
tors; and, hopefully, an OA analog would preferentially inhibit
MtOPRT enzyme activity thereby minimizing undesirable side
effects. In addition, as MtOPRT follows an ordered kinetic mecha-
nism (Fig. 1B) with PRPP binding first, an OA analog should also
bind to MtOPRT:PRPP binary complex thereby overcoming any
rescue of enzyme activity due to PRPP substrate accumulation and
sequestering of free PRPP from the cellular pool in the form of
MtOPRT:PRPP:OA analog ternary complex, which could affect other
essential metabolic pathways, as purine synthesis and mycobac-
terial cell wall synthesis [23]. The most promising compounds were
then subjected to chemical modifications (Schemes 1 and 2), and
measurements of in vitro inhibitory activity of these derivatives
showed improved inhibitory effects.
2. Results
2.1. Synthesis
Orotate phosphoribosyltransferase (OPRT, EC 2.4.2.10) catalyzes
the fifth reaction in the de novo synthesis of pyrimidine nucleotides,
Compounds 5 and 6 (Table 1) were selected for further
converting orotate (OA) and
a-D-5-phosphoribosyl-1-pyrophosphate
optimization. Although compound 2 displa
yed a better inhibitory
Fig. 1. (A) OPRT catalyzed conversion of OA and PRPP into OMP and PPi by the formation of an N-glycosidic bond in the presence of Mg2þ. (B) Mono-Iso Ordered Bi Bi kinetic
mechanism of MtOPRT. E and E0 are different enzyme’s isoforms. Adapted from Ref. [22].

A. Breda et al. / European Journal of Medicinal Chemistry 54 (2012) 113e122
115
Fig. 2. Commercially available chemical compounds tested in vitro as possible MtOPRT inhibitors.
profile (18%) as compared to 5 (15%), we favored 5 over 2 since the
former presents the nucleophilic 5-position unoccupied, an
important feature for our initial strategy of introducing amino
alcohol derivatives as side chains in pyridine-2(1H)-one rings 5 and
6. Acyclic mimics of the ribooxacarbenium ion of transition state
analogues of purine nucleoside phosphorylases led to synthesis of
inhibitory compounds with nanomolar dissociation constants [24].
We surmised that these amino alcohol derivatives would mimic the
furnishing the compounds 13e14 with 15e22% yields. These low
yields can be attributed to formation of lactones derivatives in
these reactions [26]. The Mannich reaction was also employed to
obtain the amino and amino alcohol derivatives of 6 (Scheme 2).
Likewise the reactions for synthesis of 9e12, the compounds 15e16
were obtained by reaction of 6 and amino derivatives compounds
in the presence of paraformaldehyde and methanol as solvent. The
reaction mixtures were heated at 100 ꢀC for 2e3 h leading to
synthesis of the respective compounds with 43e48% yields
(Scheme 2). In all performed attempts the dialkylated compound 16
was obtained as major product although the molar ratio was 1:1
a-D-ribose leading to compounds more structurally similar to OMP
product of MtOPRT enzyme.
Compounds 9e12 were synthesized by classical Mannich reac-
tion [25] with 63e95% yields (Scheme 1). The reactions were per-
formed starting from 5 in the presence of amine or amino alcohol
and paraformaldehyde using methanol as solvent. The reaction
mixture was heated in sealed tubes at 100 ꢀC for 2e16 h. All such
compounds showed spectrometric properties corresponding to the
proposed structures; and were then tested as potential inhibitors of
MtOPRT enzyme activity. None of these compounds showed better
inhibitory activity as compared to the parent compound (Table 1).
Our efforts were then directed at studying compound 6 and their
derivatives.
pyridine-2(1H)-one
6
and dimethylamine. All synthesized
compounds 13e16 showed spectrometric properties correspond-
ing to the proposed structures.
2.2. Enzymatic activity assay and time-dependent inhibition
The reaction catalyzed by MtOPRT 26 nM in the presence of
saturated concentrations of both forward substrates was consid-
ered 100% under assay conditions. Initial inhibitory trials of
compounds 1e8 indicated that compounds 1, 3 and 4 did not show
any effect on the reaction catalyzed by MtOPRT at final
The hydrophobic character of 6 was improved by condensation
reaction of benzaldehydes in the presence of ethanol as solvent
(Scheme 2). The reactions were conducted in reflux for 16e24 h
concentrations of 100
mM (Fig. 2 and Table 1). The effect of addi-
tion of derivatives 9e16 at final concentrations of 100
mM in the
Table 1
Observed inhibitory activity, and inhibitory constants, of pyridinone-based compounds over MtOPRT catalyzed reaction.
Observed inhibitory activitya
Kis OA (
m
M)
Inhibition mode
Kii PRPP (
m
M)
Inhibition mode
Commercially available compounds e initial screening
1
2
3
4
5
6
7
8
None
18%
None
None
15%
90%
15%
10%
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
0.25 ꢁ 0.03
Competitive
e
8.2 ꢁ 0.4
Uncompetitive
e
e
e
e
e
e
e
Chemical derivatives
9
2%
e
e
e
e
e
e
e
10
11
12
13
14
15
16
2.2%
7.5%
4%
95%
60%
71%
40%
e
e
e
e
e
e
e
e
e
0.19 ꢁ 0.01
7.4 ꢁ 0.6
6.2 ꢁ 0.4
e
Competitive
Competitive
Competitive
e
6.5 ꢁ 0.2
Uncompetitive
Uncompetitive
Uncompetitive
e
140 ꢁ 9
135 ꢁ 7
e
a
Initial screening at 100 mM final concentration in the assay mixture.

116
A. Breda et al. / European Journal of Medicinal Chemistry 54 (2012) 113e122
were fitted to one set of sites binding model, yielding the ther-
modynamic constants H and Ka. S and G values calculated from
D
D
D
Eq. (3) and Kd as 1/Ka. The number of ligand molecules bound per
enzyme subunit (N) was directly derived from the one set of
sites binding model, indicating that two molecules of inhibitor
bind to biologically active homodimeric MtOPRT (Fig. 5 and
Table 2). Thermodynamic discrimination profiles indicate favorable
enthalpic contribution (ꢂ
MtOPRT:PRPP, and unfavorable entropy (positive T
favorable entropy (negative T S) for 13 (Table 2 and Fig. 6). In any
case, the predominant contribution of H resulted in favorable
G) for binding of both compounds (Table 2 and
DH) for binding of compounds 6 and 13 to
DS) for 6 and
D
D
Gibbs energy (ꢂ
D
Fig. 6).
Scheme 1.
3. Discussion
cuvette was evaluated, showing that compounds 13, 14 and 15
present inhibitory activities above 60% (Table 1), which was the
cutoff here set to select inhibitors. However, only compound 13
showed better inhibitory activity than its parental molecule
(Table 1). None of the selected compounds presented time-
dependent inhibitory activity up to 30 min of pre-incubation with
MtOPRT (data not shown).
Characterization of MtOPRT [22] prompted us to propose,
synthesize and test a few potential enzyme inhibitors. Accordingly,
a series of derivatives (Schemes 1 and 2) of pyridine-2(1H)-one
(Fig. 2) were synthesized and screened for in vitro inhibitory
activity of MtOPRT enzyme (Table 1). The mode of inhibition for the
compounds that showed promising activity was evaluated
(compounds
6 and 13e15). In addition, the thermodynamic
signatures of non-covalent interactions upon complex formation
were evaluated for the two best inhibitors (6 and 13). Compounds
2, 5, 7 and 8 showed a slight inhibitory effect on MtOPRT enzyme
activity (Table 1).
It is noteworthy that inhibitory activity was increased by
the presence of electron withdrawing groups attached at 3- and
5-position on the pyridine-2(1H)-one rings of compounds that
were tested; where the compound 5-chloro-substituted 2 showed
18% of inhibitory activity whereas 1 showed no inhibitory activity
(Table 1). The same pattern could be observed for compounds 4 and
2.3. Inhibition assays
The mode of inhibition and inhibitor dissociation constants for
compounds 6 and 13e15 are given in Table 1. These compounds
showed competitive inhibition toward substrate OA ( Figs. 3 and 4,
A and C) as indicated by the pattern of intersecting lines that cross
at the same value on the y-axis of the double reciprocal plots,
suggesting that the binding of these inhibitors and OA are mutually
exclusive. When substrate OA was fixed at saturating concentration
and PRPP substrate concentration was varied in the presence of
fixed-varying concentrations of 6 and 13e15, these compounds
showed a pattern of parallel lines in double reciprocal plots (Figs. 3
and 4 B and D). Accordingly, compounds 6 and 13e15 are uncom-
petitive inhibitors towards PRPP, suggesting both that these
compounds do not bind to free enzyme form but bind to
MtOPRT:PRPP binary complex or subsequent species [27]. Fitting
the competitive inhibition data to Eq. (1) and the uncompetitive
inhibition data to Eq. (2) yielded the inhibitor dissociation constant
values for compounds 6 and 13e15 presented in Table 1.
5, where 3-nitro substituted compound 5 at 100 mM showed 15% of
inhibitory activity of MtOPRT whereas compound 4 was ineffective
in the same conditions (Table 1). The presence of these groups can
increase the acidity of the hydrogen bound to the nitrogen of
heterocyclic pyridine-2(1H)-one rings improving the putative
interactions via hydrogen bonds with MtOPRT active site. There also
seems to be a need for the presence of the nitrogen (NH) in the
pyridinone system for inhibitory activity, as compound 6 inhibitory
activity (90%) is superior as compared to compound 7 (15%) both at
100
mM (Table 1). This result is in accordance with data from
literature which showed that N1 of OA is involved in interactions
with Toxoplasma gondii OPRTactive site [18]. Interestingly, isoniazid
(8), the most prescribed anti-tuberculosis drug [3,4], showed
2.4. Isothermal titration calorimetry (ITC) binding assays
enzyme inhibitory activity of 10% at 100 mM final concentration
The best MtOPRT inhibitors (6 and its derivative 13) were
selected for further characterization of their binding mode by ITC.
Binding assays conducted at 25 ꢀC indicated that both molecules
are able to bind to MtOPRT:PRPP binary complex (Fig. 5). ITC data
(Table 1). This result is in agreement with the apparently promis-
cuous effect of isoniazid adducts on pyridine nucleotide-dependent
dehydrogenases/reductases [28].
Compounds 13e16 are chemical derivatives of 6 (Scheme 2).
Compound 13 showed better inhibitory activity (95%) than 14
(60%), suggesting that the presence of a hydroxyl group attached at
2-position of the phenyl substituent reduce the inhibitory activity
(Table 1). Therefore, modest changes in analogue structure seem to
cause relatively large changes in the ability to inhibit MtOPRT
activity. Compounds 15 and 16 showed inhibitory activity of,
respectively, 71% and 40% (Table 1). Based on these data, the dia-
lkylated compound 16 seems to display steric hindrance that could
reduce its interaction with MtOPRT. A threshold of 60% inhibitory
activity of a specific compound was set as a starting point to carry
out detailed determination of the mode of inhibition. Accordingly,
compounds 6, 13e15 (Table 1) were selected for further studies.
None of these compounds presented time-dependent inhibition,
which was assessed by measuring MtOPRT enzyme activity as
Scheme 2.

A. Breda et al. / European Journal of Medicinal Chemistry 54 (2012) 113e122
117
Fig. 3. Double-reciprocal plots of inhibition assays for compounds 6 (A, B) and 13 (C, D). Pattern of intersecting lines at y-axis are prognostic of competitive inhibition towards OA,
whereas parallel lines indicate uncompetitive inhibition towards PRPP. Substrate OA was varied from 12 to 75 M in presence of PRPP 500 M (A, C), substrate PRPP was varied from
25 to 500 M in presence of OA 50 M (B, D). Each inhibitor variation range is depicted in the plots.
m
m
m
m
a function of time (up to 30 min) for enzyme pre-incubated with
either compound 6, 13, 14 or 15 (data not show).
with respect to PRPP, all ITC binding assays were carried out by
titration of each inhibitor into a solution containing MtOPRT
Inhibition assays for 6, 13e15 showed that these compounds are
competitive inhibitors with respect to OA substrate (Figs. 3 and 4, A
and C). Accordingly, compounds 6, 13e15 compete with OA for
binding to MtOPRT enzyme, as anticipated for structural mimics of
OA substrate [27]. On the other hand, compounds 6, 13e15 dis-
played uncompetitive inhibition with respect to PRPP substrate
(Figs. 3 and 4, C and D). These data suggest that these compounds
bind to MtOPRT:PRPP, which is in agreement with MtOPRT
MonoeIso Ordered Bi Bi kinetic mechanism (Fig. 1B). Hence, as
compounds 6, 13e15 are OA analogs and binding of PRPP must
precede OA binding, compounds 6, 13e15 compete with OA for
molecular interactions with the MtOPRT:PRPP binary complex. On
the other hand, as PRPP binding is followed by OA, compounds 6,
13e15 behave as uncompetitive inhibitors with respect to PRPP,
showing increased binding to increasing concentrations of
MtOPRT:PRPP binary complex as PRPP concentration rises, and
subsequent competitive inhibition towards OA binding to the
binary complex.
enzyme incubated with PRPP 500 mM (5 times its Km) and MgCl2
20 mM. MgCl2 was included in the reaction mixture as the true
substrate for MtOPRT, as well as type I PRTases, is Mg2þePRPP
complex [29]. ITC binding assays of compounds 6 and 13 in the
absence of either PRPP or MgCl2 indicated that inhibitor concen-
trations 10 times larger (for instance, compound 6 at 4 mM
concentration) than in the presence of Mg2þePRPP complex yiel-
ded minor 1 kcal/mol variation (data not shown). ITC data obtained
under these conditions could be an indicative that under a situation
where inhibitor molecules accumulate to a significantly high
intracellular concentration, they may bind to MtOPRT free enzyme
thereby overriding the enzyme kinetic mechanism. Any relevance
of these data to intracellular physiological context remains to be
tested though.
The ITC data (Fig. 5) were fitted to one set of sites binding model,
indicating that there is no difference in binding affinity between
the two active sites of the MtOPRT homodimer. Thermodynamic
data indicate a predominantly favorable (negative) enthalpy (
DH)
As compounds 14 and 15 did not show increased inhibitory
activity when compared to their parent molecule (6), binding of
lead-like compounds 6 and 13 to MtOPRT were characterized by ITC
assays. Since compounds 6 and 13 are uncompetitive inhibitors
and favorable Gibbs free energy ( G) upon binding of both
D
compounds to MtOPRT:PRPP (Table 2 and Fig. 6). Taken together,
these data are indicative that binding of compounds 6 and 13
to MtOPRT are favorable spontaneous processes mainly ruled by

118
A. Breda et al. / European Journal of Medicinal Chemistry 54 (2012) 113e122
Fig. 4. Double-reciprocal plots of inhibition assays for compounds 14 (A, B) and 15 (C, D). Variation of substrate OA (A, C), and of substrate PRPP (B, D) were in the same range as for
compounds 6 and 13 inhibition assays. Each inhibitor variation range is depicted in the plots.
formation of favorable hydrogen bonds and/or van der Waals
interactions. The favorable entropic contribution (negative T S) of
compound 13 binding may be accounted for by release of “bound”
Interestingly, the overall dissociation constant values (Kd;
Table 2) for 6 (0.14 M) and 13 (0.09 M) are in fairly good agree-
ment with the dissociation constant values (Kis; Table 1) for
competitive inhibition with respect to OA substrate (0.25 M for 6,
0.19 M for 13). Interestingly, although compounds 6 and 13
D
m
m
water molecules to bulk solvent. On the other hand, the unfavor-
m
able entropic contribution (positive T
D
S) for compound 6 implies
m
conformational changes upon MtOPRT:PRPP:6 ternary complex
formation [30]. Although these conformational changes may be
either in the enzyme or inhibitor, it is tempting to suggest that it
corresponds to protein isomerization step upon ligand binding,
which is a hallmark of enzymes that follow Iso Mechanisms [31]. It
has been reported that movements of a catalytic flexible loop are
integral to catalysis of phosphoribosyltransferase enzymes [29],
allowing closing of active site, recruitment of catalytic essential
residues, and displacement of water molecules to bulk solvent [32].
Binding of OMP product to MtOPRT displays similar thermody-
namic discrimination profile (Fig. 6), which could be explained by
nearly complete conservation of OA moiety of OMP in compound 6,
except N1 (pyridine ring in 6, pyrimidine ring in OA and OMP). It is
thus tempting to suggest that compounds 6 and 13 can make key
enzyme-ligand contacts in MtOPRT active site involving N1, C2 and
C6 ketone groups, in agreement with the three-dimensional
structures of Saccharomyces cerevisiae OPRT in complex with
ligands [33e35] and T. gondii OPRT [18].
binding processes showed a favorable enthalpic contribution, only
compound 13 displayed favorable entropic contribution (Table 2).
The latter may be attributed to release of solvation water molecules
of the exocyclic aromatic ring substituent of 13 not present in 6. It is
thus tempting to suggest that the lower Kis and Kii values for 13
(Table 1) are due to entropy optimization by increasing hydropho-
bicity, which results in larger log P (0.714 for 6, and 0.938 for 13) and
log D (ꢂ3.92 for 6 and -2.58 for 13) values at pH 8.0 for 13. A general
guide for optimal gastrointestinal absorption by passive diffusion
permeability after oral dosing is to have a moderate log P (range
0e3), a requirement that is fulfilled by these compounds. Enthalpy
and entropy simultaneous optimization is one of the major goals in
drug discovery, which is not trivial due to enthalpy/entropy
compensation [35]. The ITC data suggest that this goal was partially
achieved for compound 13 as it retained the favorable enthalpic
contribution of compound 6 binding to MtOPRT:PRPP, and the
unfavorable entropic contribution of 6 was converted into a favor-
able entropic contribution for 13 binding process (Table 2 and Fig. 6).

A. Breda et al. / European Journal of Medicinal Chemistry 54 (2012) 113e122
119
Fig. 5. ITC ligand binding assays. Binding isotherms of 6 (A) and 13 (B) were fitted to sequential binding sites model.
4. Conclusion
a Microquímica MQAPF-302 apparatus. 1H NMR spectra were
acquired on a Bruker DPX 400 (Federal University of Santa Maria,
UFSM/Brazil) or Anasazi EFT-60 spectrometer (1H at 400.13 MHz or
60.13 MHz, respectively) at 25ꢀC, for DPX 400, and 30ꢀC for EFT-60, in
5 mm sample tubes. DMSO-d6 was used as solvent and TMS was used
as internal standard. High resolution mass spectra were obtained for
all compounds on a LTQ Orbitrap Discovery mass spectrometer
(Thermo Fisher Scientific). Thishybrid system meets the LTQXL linear
ion trap mass spectrometer and an Orbitrap mass analyzer. The
experiments were performed via direct infusion of sample in MeOH
The favorable enthalpic and entropic contributions of molecule
13 binding to MtOPRT:PRPP correspond to favorable thermody-
namic profiles for high affinity ligands [36]. Taken together, the data
here presented suggest that more potent MtOPRT inhibitors may
rely on optimization of compounds 6 and 13, including exploration
of lipophilicity that should address its effect on properties such
as solubility, permeability and equilibrium inhibition constants,
to name a few [37]. Interestingly, 2-aminosubstituted pyrimidine
5-carboxylate derivatives have been shown to be promising anti-
tubercular lead molecules, even though their MIC values were larger
than the isoniazid MIC [38]. As the de novo pyrimidine biosynthesis
pathway has been shown to be required for cell invasion and viru-
lence of obligatory parasites as T. gondii [12] and Plasmodium vivax
[13], MtOPRT may represent a target for the development of drugs to
be used in TB treatment and prophylaxis [14]. Efforts to determine
in vivo inhibitory effects of compounds 6 and 13 are currently
underway. The results here presented may be useful to chemical
biologists interested in designing loss-of-function (inhibitors) or
gain-of-function (activators) chemical compounds to reveal the
biological role of MtOPRT in the context of whole MTB cells.
50%/FA 0.1% (flow: 5 m
L minꢂ1) in the positive-ion mode using elec-
trospray ionization. Elemental composition calculations were
executed using the specific tool included in the Qual Browser module
of Xcalibur (Thermo Fisher Scientific, release 2.0.7) software. Fourier
transform infrared (FTIR) spectra were recorded using a universal
attenuated total reflectance (UATR) attachment on a PerkinElmer
Spectrum 100 spectrometer in the wavenumber range of
650e4000 cmꢂ1 with resolution of 4 cmꢂ1. All TLC analyses were
performed on silica gel 0.2 mm (Aldrich 60 F254) and spots revealed
by UV visualization at 360 nm.
5.2. General procedure for the synthesis of pyridine-2(1H)-one
9e12
5. Experimental section
The mixture of 4-Hydroxy-6-methyl-3-nitropyridin-2(1H)-one 5
(1.0 mmol, 0.170 g), amine or amino alcohol (1.0 mmol) and para-
formaldehyde (0.030 g) in methanol (10 mL) was heated in a sealed
tube at 100 ꢀC for the appropriate time (Scheme 1). After cooling to
room temperature the solvent was evaporated under reduced
pressure. The solid obtained was washed with distilled water
5.1. Synthesis e apparatus and analysis
Unless otherwise indicated, all common reactants and solvents
were used as obtained from commercial suppliers without
further purification. All melting points were measured using
Table 2
Thermodynamic functions and number of ligands per subunit (N) for binding of compounds 6 and 13 to MtOPRT:PRPP binary complex.
Compound
D
H (cal/mol)
ꢂT
D
S (cal/mol/deg)
D
G (cal/mol)
Ka (Mꢂ1
)
Kd
(
m
M)
N
6
13
ꢂ9.8 ꢁ 0.1 ꢃ 103
390 ꢁ 6
ꢂ9.4 ꢁ 0.1 ꢃ 103
7 ꢁ 1 ꢃ 106
0.14 ꢁ 0.03
0.09 ꢁ 0.01
0.932 ꢁ 0.005
1.05 ꢁ 0.03
ꢂ6.6 ꢁ 0.1 ꢃ 103
ꢂ3.04 ꢁ 0.02 ꢃ 103
ꢂ9.63 ꢁ 0.07 ꢃ 103
12 ꢁ 2 ꢃ 106

120
A. Breda et al. / European Journal of Medicinal Chemistry 54 (2012) 113e122
reflux in dry ethanol (10 mL) for 16 h, compound 13, and 24 h for 14.
The progress of the reaction was monitored by TLC using a solution
of EtOAc and hexane (1:1, v/v) as a mobile phase. After cooling to
room temperature the solid formed was filtered and washed with
distilled water (2 ꢃ 10 mL) and Et2O (2 ꢃ 10 mL). The products were
recrystallized from hexane and dried under reduced pressure at
38 ꢀC for 24 h.
5.3.1. 3-Benzylidene-2,6-dioxo-1,2,3,6-tetrahydropyridine-4-
carboxylic acid (13)
Pale yellow powder; yield: 0.053 g (22%); M.p. 254e258 ꢀC
(Dec); 1H NMR (60 MHz, DMSO-d6):
d 6.46 (s, 1H, CH), 7.09e7.22
(m, 5H, Ph), 7.35 (s, 1H, CH); FTIR (UATR, cmꢂ1): 3073, 1669, 1572,
1480, 1465, 1221, 758; HRMS (ESI) calcd for C13H9NO4 þ H:
244.0604. Found 244.0603 (M þ H)þ.
Fig. 6. Enthalpy (
D
H), entropy (ꢂT
DS) and Gibbs free energy (DG) thermodynamic
5.3.2. 3-(2-Hydroxybenzylidene)-2,6-dioxo-1,2,3,6-
tetrahydropyridine-4-carboxylic acid (14)
profile for 6, 13 and substrate OMP binding to MtOPRT. ITC data for OMP binding was
previously described elsewhere (in press, Molecular BioSystems, doi:10.1039/
C1MB05402C) and is given here for comparison only.
Beige powder; yield: 0.039 g (15%); M.p. 232e234 ꢀC; 1H NMR
(60 MHz, DMSO-d6):
d 6.48 (s, 1H, CH), 6.98e7.37 (m, 4H, C6H5),
7.45 (s, 1H, CH); FTIR (UATR, cmꢂ1): 3069, 1666, 1568, 1486, 1455,
1224, 752; HRMS (ESI) calcd for C11H17N3O6 þ H: 288.1190. Found
288.1204 (M þ H)þ.
(3 ꢃ 10 mL) and cold hexane (2 ꢃ 10 mL). When necessary, the
products were recrystallized from 10% EtOAc in hexane, yielding the
analytically pure pyridine-2(1H)-ones 9e12. After, the products
were dried under reduced pressure at 38 ꢀC for at least 24 h.
5.4. General procedure for the synthesis of pyridine-2(1H)-one
15e16
5.2.1. 5-((Dimethylamino)methyl)-4-hydroxy-6-methyl-3-
nitropyridin-2(1H)-one (9)
Yellow powder; yield: 0.216 g (95%); M.p. 143e145 ꢀC; 1H NMR
The mixture of 2,6-Dihydroxypyridine-4-carboxylic acid
6
(1.0 mmol, 0.155 g), amine or amino alcohol (1.0 mmol) and para-
formaldehyde (0.030 g) in methanol (10 mL) was heated in a sealed
tube at 100 ꢀC for 3 h, compound 15, and 2 h for 16. After cooling to
room temperature the solvent was evaporated under reduced
pressure. The solid obtained was diluted in distilled water (20 mL)
(60 MHz, DMSO-d6):
d 2.03 (s, 3H, CH3), 2.65 (s, 6H, N(CH3)2), 3.81
(s, 2H, CH2), 11.01 (br, 1H, NH); FTIR (UATR, cmꢂ1): 3041, 2817, 1629,
1533, 1403, 1274, 1167, 793; HRMS (ESI) calcd for C9H13N3O4 þ H:
228.0979. Found 228.0981 (M þ H)þ.
and filtered (0.22
mm). After, the water was evaporated under
5.2.2. 4-Hydroxy-5-((2-hydroxyethylamino)methyl)-6-methyl-
3-nitropyridin-2(1H)-one (10)
reduced pressure. Finally, the product was washed with cold
hexane (2 ꢃ 10 mL) and dried under reduced pressure at 30 ꢀC for
24 h.
Yellow powder; yield: 0.182 g (75%); M.p. 139e141 ꢀC; 1H NMR
(400 MHz, DMSO-d6):
d 2.16 (s, 3H, CH3), 2.94 (t, 2H, CH2), 3.45 (t,
2H, CH2), 3.88 (s, 2H, CH2), 8.00 (br, 2H), 10.09 (br, 1H, NH); FTIR
(UATR, cmꢂ1): 3379, 3158, 2954, 2803, 1609, 1529, 1416, 1266, 1190,
1068, 803, 788; HRMS (ESI) calcd for C9H13N3O5 þ H: 244.0928.
Found 244.0929 (M þ H)þ.
5.4.1. 6-Hydroxy-5-((2-hydroxyethylamino)methyl)-2-oxo-1,2-
dihydropyridine-4-carboxylic acid (15)
Dark-red solid; yield: 0.098 g (43%); M.p. 48e50 ꢀC; 1H NMR
(400 MHz, DMSO-d6):
d 2.81 (t, 2H, CH2), 3.55 (t, 2H, CH2), 3.68 (s,
2H, CH2), 5.19 (br, 2H), 6.53 (s, 1H, CH); FTIR (UATR, cmꢂ1): 2843,
1561, 1450, 1411, 1327, 1176, 1078, 991, 763; HRMS (ESI) calcd for
C9H12N2O5 þ H: 229.0819. Found 229.0816 (M þ H)þ.
5.2.3. 5-((2,3-Dihydroxypropylamino)methyl)-4-hydroxy-6-
methyl-3-nitropyridin-2(1H)-one (11)
Light yellow powder; yield: 0.172 g (63%); M.p. 109e111 ꢀC; 1H
NMR (400 MHz, DMSO-d6):
d 2.06 (s, 3H, CH3), 2.78e2.81 (dd, 2H,
5.4.2. 3,5-Bis((dimethylamino)methyl)-6-hydroxy-2-oxo-1,2-
dihydropyridine-4-carboxylic acid (16)
CH2), 3.03e3.07 (m, 1H, CH), 3.42e3.45 (dd, 2H, CH2), 3.86 (s, 2H,
CH2), 5.33 (br, 2H), 10.02 (br, 2H); FTIR (UATR, cmꢂ1): 3235, 3004,
2806, 1638, 1518, 1435, 1252, 1187, 1035, 776; HRMS (ESI) calcd for
C10H15N3O6 þ H: 274.1034. Found 274.1039 (M þ H)þ.
Black solid; yield: 0.131 g (48%); M.p. 101e103 ꢀC; 1H NMR
(60 MHz, DMSO-d6): d 2.70 (s, 9H, 2N(CH3)2), 3.98 (s, 2H, CH2); FTIR
(UATR, cmꢂ1): 3052, 2802, 1583, 1465, 1357, 1299, 1022, 759; HRMS
(ESI) calcd for C12H19N3O4 þ H: 270.1448. Found 270.1442 (M þ H)þ.
5.2.4. 5-((1,3-Dihydroxy-2-methylpropan-2-ylamino)methyl)-4-
hydroxy-6-methyl-3-nitropyridin-2(1H)-one (12)
Yellow powder; yield: 0.235 g (82%); M.p. 91e93 ꢀC; 1H NMR
5.5. Enzymatic activity assay
(400 MHz, DMSO-d6):
d 1.35 (s, 3H, CH3), 2.10 (s, 3H, CH3), 3.32 (s,
4H, 2CH2), 3.91 (s, 2H, CH2), 10.01 (br, 1H, NH); FTIR (UATR, cmꢂ1):
3167, 2941, 1610, 1515, 1399, 1268, 1178, 1055, 824, 787; HRMS (ESI)
calcd for C11H17N3O6 þ H: 288.1190. Found 288.1204 (M þ H)þ.
All chemicals were purchased from Sigma Aldrich. Homoge-
neous recombinant MtOPRT was obtained as previously
described [22]. Effect of each compound on MtOPRT activity was
measured for the forward phosphoribosyltransferase reaction
(OA þ PRPP / OMP þ PPi) by monitoring the decrease in OA
concentration. The assay was performed in quartz cuvettes with
UVevisible Shimadzu spectrophotometer UV2550 equipped with
a temperature-controlled cuvette holder. The reaction mixture
5.3. General procedure for the synthesis of pyridine-2(1H)-one
13e14
The mixture of 2,6-Dihydroxypyridine-4-carboxylic acid
6
(500
m
L) contained Tris HCl 50 mM MgCl2 20 mM pH 8.0, OA 50
mM,
(1.0 mmol, 0.155 g) and aldehyde (1.0 mmol) was stirred under
PRPP 500
m
M and MtOPRT 26 nM. Reaction was started by the

A. Breda et al. / European Journal of Medicinal Chemistry 54 (2012) 113e122
121
addition of enzyme, and a linear progress curve of absorbance
change at 295 nm by 60 s at 25 ꢀC was followed. The extinction
coefficient of this conversion was 3950 Mꢂ1 cmꢂ1 [39]. Inhibitors
were then added to reaction mixture at final concentration of
into buffer) were performed to subtract the heats of dilution and
mixing for each experiment prior to data analysis. ITC data were
fitted to Eq. (3),
D
G ¼
D
H ꢂ T
D
S ¼ RTlnKa;
(3)
100 mM and enzyme activity was measured as before. All enzymatic
activity and further assays were performed in duplicate or tripli-
cate. Selection of inhibitors for further assays was based on the
difference of enzyme activity in absence and presence of inhibitor
in which
DH represents the enthalpy change,
DG the Gibbs free
energy change,
DS the entropy change, T the temperature of the
experiment in Kelvin, R the gas constant (1.987 cal Kꢂ1 molꢂ1), and
Ka the equilibrium association constant. The equilibrium dissocia-
tion constant, Kd, was calculated by the inverse of Ka. All data were
evaluated utilizing the Origin 7 SR4 software (Microcal, Inc.,
100 mM, and a cutoff was set to a 60% inhibitory activity.
5.6. Time-dependent inhibition
Northampton, MA). Entropy values are presented as ꢂT
DS.
Evaluation of time-dependent inhibitory activity was evaluated
at the same enzymatic assay conditions as described above.
Aliquots corresponding to 26 nM of MtOPRT were taken at different
times (up to 30 min) from pre-incubation mixtures containing
Acknowledgments
This work was supported by funds of National Institute of
Science and Technology on Tuberculosis (INCT-TB), MCT-CNPq,
Ministry of Health e Department of Science and Technology
(DECIT) e Secretary of Health Policy (Brazil) to D.S.S. and L.A.B.
L.A.B. and D.S.S. also acknowledge financial support awarded by
FAPERGS-CNPq-PRONEX-2009. A.A.S (CNPq 306764/2010-5), L.A.B.
(CNPq, 520182/99-5) and D.S.S. (CNPq, 304051/1975-06) are
Research Career Awardees of the National Research Council of
Brazil (CNPq). A.B. acknowledges scholarship awarded by BNDES.
P.M. and L.A.R. acknowledge scholarships awarded by CNPq.
MtOPRT and inhibitor at final concentration of 1
were added to a reaction mixture containing OA 50
500 M, and OA / OMP conversion under steady-state conditions
mM. These aliquots
mM, PRPP
m
was monitored as a linear decrease in absorbance at 295 nm.
5.7. Inhibition assays
Inhibition assays were carried out in 500 mL of reaction mixtures
containing Tris HCl 50 mM MgCl2 20 mM, 26 nM MtOPRT, at 25 ꢀC,
monitoring disappearance of OA at 295 nm for 60 s. In the inhibi-
tion assays one substrate concentration was constant at saturating
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m
M and PRPP ¼ 500
mM)
M, OA:
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