6-Imino-2-thioxo-pyrimidinones as a new class of dipeptidyl peptidase IV inhibitors

Article abstract of DOI:10.1007/s00044-010-9314-5

Dipeptidyl peptidase IV is a glycoprotein which removes N-terminal dipeptides from physiologically relevant polypeptides. An homologous series of 6-imino-2-thioxo-5-{[3,4,5-tris(methyloxy)phenyl]methyl}-2,5-dihydro-4(3H) -pyrimidinones has been tested for inhibition of DPP IV activity. The inhibitory effects at 0.1 mM were observed. Enzyme kinetic studies revealed that compounds inhibit DPP IV activity competitively. According to the molecular docking analysis, the inhibitors are anchored into the DPP IV hydrolytic site by interactions of the pyrimidinone core with Glu206, Tyr662, and Tyr547, with the alkyl chain entering the S1 pocket. We conclude that pyrimidinone-like compounds are a promising new scaffold for reversible inhibition of DPP IV. Springer Science+Business Media, LLC 2011.

Full text of DOI:10.1007/s00044-010-9314-5

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2011) 20:339–345
DOI 10.1007/s00044-010-9314-5
ORIGINAL RESEARCH
6-Imino-2-thioxo-pyrimidinones as a new class of dipeptidyl
peptidase IV inhibitors
•
Dubravko Jelic Krunoslav Nujic Visnja Stepanic
•
•
´
Krunoslav Kovacevic Donatella Verbanac
´
ˇ
´
•
ˇ
´
Received: 13 January 2009 / Accepted: 11 February 2010 / Published online: 7 March 2010
Ó Springer Science+Business Media, LLC 2011
Abstract Dipeptidyl peptidase IV is a glycoprotein
which removes N-terminal dipeptides from physiologically
relevant polypeptides. An homologous series of 6-imino-2-
thioxo-5-{[3,4,5-tris(methyloxy)phenyl]methyl}-2,5-dihydro-
4(3H)-pyrimidinones has been tested for inhibition of DPP
IV activity. The inhibitory effects at 0.1 mM were
observed. Enzyme kinetic studies revealed that compounds
inhibit DPP IV activity competitively. According to the
molecular docking analysis, the inhibitors are anchored
into the DPP IV hydrolytic site by interactions of the
pyrimidinone core with Glu206, Tyr662, and Tyr547, with
the alkyl chain entering the S1 pocket. We conclude that
pyrimidinone-like compounds are a promising new scaf-
fold for reversible inhibition of DPP IV.
Abbreviations
DPP IV
GLP-1
GIP
Dipeptidyl peptidase IV
Glucagon-like peptide-1
Glucose-dependent insulinotropic polypeptide
Structure–activity relationship
Root mean square deviation
SAR
RMSD
PDB
Protein Data Bank
GOLD
MTS
Genetic optimisation for ligand docking
3-(4,5-Dimethylthiazole-2-yl)-5-
(3-carboximethoxyphenyl)-2-(4-sulfophenyl)-
2H-tetrazole
ECACC European Collection of Cell Cultures
THP-1
HepG2
Human monocytic leukemia
Human Caucasian hepatocyte carcinoma
Keywords DPP IV inhibitor ꢀ Pyrimidinone ꢀ
Enzyme kinetics ꢀ Molecular docking ꢀ Cytotoxicity
Introduction
´
Dubravko Jelic and Donatella Verbanac equally contributed to this
work.
Dipeptidyl peptidase IV (CD26/DPP IV) is a 110-kDa
glycoprotein which plays an important role in T cell acti-
vation, immune regulation, signal transduction, and apop-
tosis (Boonacker and Van Noorden Cornelis, 2003; Sato
et al., 2003). It interacts with antigen-presenting cell, and
regulates cytokine and chemokine functions (Ohnuma
et al., 2008). DPP IV appears to play an important role in
diabetes, obesity, anxiety, rheumatoid arthritis, multiple
sclerosis, cancer, autoimmune diseases, and AIDS
(Thompson et al., 2007; Pro and Dang, 2004; Pratley and
Salsali, 2007; Havre et al., 2008). DPP IV acts to
sequentially remove N-terminal dipeptides from proline-
containing peptides such as incretins [glucagon-like pep-
tide-1 (GLP-1) and glucose-dependent insulinotropic
polypeptide (GIP)] (Wiedeman, 2007; Deacon, 2007), from
´
´
´
ˇ
´
D. Jelic (&) ꢀ K. Nujic ꢀ V. Stepanic ꢀ K. Kovacevic ꢀ
D. Verbanac
GlaxoSmithKline Research Center Zagreb Ltd., Prilaz baruna
´
Filipovica 29, 10000 Zagreb, Croatia
e-mail: dubravko.x.jelic@gsk.com
Present Address:
´
V. Stepanic
Department for Molecular Medicine, Ruder Boskovic Institute,
ˇ
´
¯
P.O.B. 180, 10002 Zagreb, Croatia
Present Address:
D. Verbanac
University of Zagreb, School of Medicine, Center for
ˇ
Translational and Clinical Research, Salata 2, 10000 Zagreb,
Croatia
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Med Chem Res (2011) 20:339–345
some neuropeptides (Mentlein et al., 1993; Mentlein,
1999), and from other growth factors, and vasoactive
peptides. Incretin hormones are secreted in response to
meal ingestion, thereby enhancing postprandial insulin and
glucagons secretion, which modulate plasma glucose levels
(Thongtang and Sriwijitkamol, 2008). In type 2 diabetic
patients; the continuous infusion of GLP-1 decreases
plasma glucose and haemoglobin A1c concentrations and
improves beta-cell function (Madsbad et al., 2008). In
order to overcome its rapid inactivation, effective therapy
with exogenously derived GLP-1 would require either
continuous infusion or multiple daily injections. In order to
conquer this barrier, two approaches have been pursued:
(i) the creation of GLP-1 analogues (receptor agonists or
incretin mimetics) with a low affinity for DPP IV and,
resistance to enzymatic inactivation; and (ii) the develop-
ment of compounds that inhibit the activity of DPP IV and,
prevent inactivation of endogenous GLP-1 and GIP (Prat-
ley and Salsali, 2007). The first two of such drugs, the
incretin mimetic ExenatideÒ (Byetta-Amylin Pharmaceu-
ticals) and the DPP IV inhibitor SitagliptinÒ (Januvia-
Merck & Co.) (Kesty et al., 2008), were approved by the
FDA in 2005 and 2006, respectively. Reported inhibitors of
DPP IV have various heterocyclic structures and include
pyrrolidines (Hughes et al., 1999), piperazines (Brockunier
et al., 2004), amino-substituted pyrimidines (Peters et al.,
2004), etc. We propose 6-imino-2-thioxo-2,5-dihydro-
4(3H)-pyrimidinones as a promising scaffold for design of
new DPP IV competitive inhibitors, which differs from the
other published ones. The homologous series of eight
6-imino-2-thioxo-pyrimidinones is defined by H, alkyl, or
alkenyl chain substituents at the C5 atom of the pyrimidi-
none ring (Fig. 1). This group of compounds has been
extensively investigated by (i) DPP IV inhibition and
enzyme kinetics, (ii) molecular docking and scoring
approaches, and (iii) study of cytotoxicity in vitro.
Compounds were synthesized originally in the early
1980s to be screened for anti-infective activity. Since sig-
nificant anti-bacterial activity was not observed, com-
pounds were stored in a corporative Compound Bank.
After more than 25 years, the compounds were checked for
their identity and purity and tested in a standard screening
cascade. Remarkable stability and purity was observed
after more than 25 years of the room temperature storage
(all compounds were[95% pure, as determined by HPLC/
MS/DAD analysis, data not shown, but available on
request).
In-vitro inhibition of DPP IV activity and enzyme
kinetic experiments
Inhibition of DPP IV activity by pyrimidinone-like com-
pounds was tested using H-Gly-Pro-pNA tosylate as a
substrate. Results are shown in Table 1 as percent inhibi-
tion at 0.1 mM compound concentration. Unsubstituted
6-imino-2-thioxo-5-(3,4,5-trimethoxy-benzyl)-2,5-dihydro-
4(3H)-pyrimidinone (core scaffold) at position 5 (Com-
pound 1) did not inhibit DPP IV activity when tested at
0.1 mM. On the other hand, when methyl, ethyl, propyl,
isopropyl, allyl, and butyl radicals were inserted at position
5 of the core scaffold, significant inhibition was obtained.
The level of inhibition lay between 40 and 47%. Com-
pound 5 was chosen for the determination of the type of
DPP IV inhibition because of its good solubility in buffer.
Time course experiments showed a linear increase in DPP
IV activity during the first few minutes. Consequently,
initial velocities were measured for 4 min and expressed as
a change in absorbance at 405 nm with time (Fig. 2).
The calculated enzyme kinetic parameters in the
absence of compound were Km = 0.7 mM and
Vmax = 5.6 9 10-4. The Km value is slightly higher than,
but close to the previously published Km values for this
substrate (Abbott et al., 2000). Values in the presence of
0.3 mM of Compound
5 were Km = 2.2 mM and
Results and discussion
Vmax = 6.9 9 10-4. A difference in Km values, with and
without compound, and almost no difference in Vmax val-
ues are characteristics of competitive inhibition, for which
Kmꢁ ¼ aKm; where a = 1 ? [I]/Ki (Marangoni, 2003).
These results suggest that Compound 5 is a weak
(Ki = 160 lM) competitive inhibitor of DPP IV. In order
to confirm this and to gain a more detailed understanding of
Synthesis, stability, and purity of the compounds
Compounds were synthesized in two steps (Fig. 1), in
accordance with the procedure described previously
ˇ ´
(Gluncic et al., 1986).
R
Fig. 1 Structures and synthesis
of a series of 6-imino-2-thioxo-
5-{[3,4,5-tris(methyloxy)
phenyl]methyl}-2,5-dihydro-
4(3H)-pyrimidinone compounds
1
2
H
O
O
O
CH3
C2H5
C3H7
iso-C3H7
CH2-CH=CH2
C4H9
3,4,5-trimethoxybenzyl
N
HN
Thiourea
H
N
3
4
5
6
7
8
O
O
S
C2H5ONa, C2H5OH
reflux 6 hr
O
R
R
N
H
O
O
O
1 - 8
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Med Chem Res (2011) 20:339–345
341
We reproduced successfully the experimental poses of
˚
Table 1 A series of pyrimidinone-like compounds with different
side-chain-modifications
known inhibitors (RMSD \1.5 A) with DPP IV crystal
structures available from the PDB (Bernstein et al., 1977;
Berman et al., 2003) using GOLD (Jones et al., 1997;
Hartshorn et al., 2007, Thoma et al., 2003; Peters et al.,
2004). Binding modes of pyrrolidine nitriles NVP-DPP728
(Hughes et al., 1999), and FE999011 (Sudre et al., 2002),
reversible covalent DPP IV inhibitors, were satisfactorily
predicted without applying covalent bond constraint.
GOLD also successfully differentiated active from inac-
tive compounds in reproducing the results of wet screen-
ing of an in-house compound library in the DPP IV
enzymatic assay. The calculations were performed using
the two different binding site conformations of the crystal
structures with PDB code 1NU8 (Thoma et al., 2003), and
1RWQ (Peters et al., 2004). In these structures, DPP IV is
complexed with competitive reversible non-covalent
inhibitors of different chemical classes. In 1NU8, DPP IV
is complexed with Ile-Pro-Ile (Diprotin), the pyrrolidine
ring lying within the S1 pocket. In 1RWQ, it binds a
4-aminomethylpyrimidine inhibitor, and the dichloro-
phenyl ring enters the S1 pocket. In solution, the tested
pyrimidinones may exist in an equilibrium complex of
various ionic and tautomeric forms. Here, we considered
only the most prevalent basic tautomer form [as attested
by the software MN.TAUTOMER (Molecular Networks
GmbH, 2004)] in its neutral and mono-protonated ioni-
zation states. According to ACD/pKa (ACD/Labs, 2006),
these molecules should be predominantly in the neutral
ionization form ([80%) in neutral solutions. Since ste-
reochemistry of the ring C5 atom was not known, two
possible enantiomers per molecular species were formed.
For all the six C5-substituted pyrimidinone derivatives, the
predicted first GOLD/GoldScore ranked poses of both
neutral and mono-protonated pyrimidinones in both 1NU8
and 1RWQ binding sites satisfy three recognition inter-
actions: alkyl/alkenyl chain enters the S1 pocket, HN=C–
NH fragment exhibits anchoring H-bond interactions with
Glu206 and Tyr662, and the bulky trimethoxybenzyl
group occupies the S2 pocket, providing favorable inter-
actions with Arg125. The O=C–NH fragment of the
pyrimidinone ring can interact with the Tyr547 amino acid
residue, while the sulphur atom is orientated toward free
space (Fig. 3).
Compound
R
% of DPP IV inhibition
1
2
3
4
5
6
7
8
Hydrogen (H)
0
40
40
47
45
47
44
51
Methyl (CH3)
Ethyl (C2H5)
Propyl (C3H7)
Isopropyl (iso-C3H7)
Allyl (CH2–CH=CH2)
Butyl (C4H9)
3,4,5-Trimethoxybenzyl
Percent inhibition of DPP IV is shown at 0.1 mM compound
concentration
7.0×10-4
6.0×10-4
5.0×10-4
4.0×10-4
3.0×10-4
2.0×10-4
1.0×10-4
0
0.0
2.5
5.0
7.5 10.0 12.5 15.0 17.5
H-Gly-Pro-pNA (mM)
Fig. 2 Determination of the type of inhibition of DPP IV by
Compound 5. Filled square, without compound; filled triangle,
0.3 mM Compound 5. Non-linear regression curve fitting was used
for determination of kinetic parameters
compound-enzyme interactions, in silico studies were
performed.
In silico studies
The catalytic site of DPP IV is buried in a small pocket
within a large cavity placed at the interface of its two
domains, the b-propeller and a/b-hydrolase domains. It
has been found that residues surrounding the scissile bond
P1–P10 of a dipeptidyl-polypeptide mainly determine the
substrate selectivity of DPP IV (Aertgeerts et al., 2004).
For substrate recognition, the substrate must enter the
hydrophobic pocket S1, close to Ser630, and interact with
glutamates Glu205 and/or Glu206 (Glu-Glu motif), that
was shown to be essential for enzymatic activity (Abbott
et al., 1999). Interactions of ligands with side chains
constituting the spacious S2 pocket, especially ones with
hydrophobic, long or bulky side chains, generally showed
an enhanced binding (Brandt, 2000; Peters et al., 2004).
In order for such a binding position to be achieved, the C5
atom of the pyrimidinone ring must have the S configuration
(i.e., R in the case of Compound 5). The docking calculations
were performed under the assumption of reversible non-
covalent binding. The possibility of reversible covalent
binding through interaction of Ser630 and 6-imino group of
the compounds considered cannot be neglected (Kim et al.,
2006). However, the polar 6-imino 2-thioxo pyrimidinone
heterocycle is quite unlikely to enter the S1 pocket, and,
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Med Chem Res (2011) 20:339–345
Fig. 3 Binding conformation of the pyrimidinones within the
hydrolytic site of DPP IV. Interacting amino acid residues Glu206,
Tyr662, Tyr547, and Arg125 are shown together with Compound 7
Fig. 4 Comparison of binding modes of the pyrimidinones, repre-
sented by Compound 5 and the 1RWQ ligand aminomethylpyrimi-
dine, within the peptide-binding site of DPP IV
hence, such a binding mechanism is expected to be less
probable. With regard to the energetics of the ligand–DPP IV
interaction, the first-ranked poses at the GoldScore fitness
scale also scored highly for the other scoring function
considered, i.e., consensus score. In addition to the pub-
lished DPP IV inhibitors, the best score (consensus score of
3) was obtained for pyrimidinones with the longest chains
(Compounds 4 and 7). In the compound set considered, the
poorest score was obtained with the non-substituted Com-
pound 1. Its binding mode is analogous to the binding mode
of its C5-substituted derivatives (Compounds 2–7).
Although Compound 8 showed similar percentage of inhi-
bition of DPP IV, its binding mode could not be determined
unambiguously from docking analysis. Based on SAR for
published DPP IV competitive inhibitors (Aertgeerts et al.,
2004; Abbott et al., 1999; Brandt, 2000; Peters et al., 2004),
and molecular modeling results for the tested pyrimidinones
(Fig. 4), it is expected that substitution of pyrimidinones at
the C5 ring position with pyrrolidine or phenyl rings should
significantly increase their potency.
Conclusion
The 6-imino-2-thioxo-pyrimidinone series of compounds
showed inhibition of dipeptidyl peptidase IV activity at a
concentration of 0.1 mM. Enzyme kinetic experiments
revealed that the enzyme inhibition was competitive,
indicating that the compounds bind to the peptide-binding
site. The possible binding mode of the pyrimidinones to the
hydrolytic site of DPP IV was proposed by combining
knowledge of binding modes of known DPP IV ligands and
molecular docking and scoring results for the homologous
chemical series. The most likely binding mode for this
group of compounds involves insertion of the alkyl/alkenyl
chain into the S1 pocket, the HN=C–NH fragment making
an H-bond network with Glu206 and Tyr662, the 6-car-
bonyl oxygen interacting with Tyr547, and the trimeth-
oxybenzyl-group interacting with Arg125 in the S2 pocket.
According to the proposed binding mode, S-enantiomeric
forms (R in the case of Compound 5) of the pyrimidinones
are preferable, and it is expected that replacement of alkyl/
alkenyl chains with a pyrrolidine or phenyl ring at the C5
atom will significantly increase their potency for inhibition
of DPP IV activity. Neither compound showed any cyto-
toxic effect or inhibition of cell growth of THP-1 and
HepG2 cell lines at concentrations up to 100 lM. In con-
clusion, we propose 6-imino-2-thioxo-5-{[3,4,5-tris(meth-
yloxy)phenyl]methyl}-2,5-dihydro-4(3H)-pyrimidinone as
a new anchoring fragment in which substitution at C5
position is likely to optimize inhibition of DPP IV.
Cytotoxicity testing of the tested compounds
Dose–response curves for evaluation of cytotoxicity have
been made for two human immortal cell lines—THP-1 and
HepG2 cells, measured in terms of the mitochondrial
succinate dehydrogenase activity of living cells, expressed
as the percentage of the optical density (OD) values of non-
treated cells. None of the compounds showed any cytotoxic
effect or inhibition of cell growth of THP-1 or HepG2 cells
at concentrations up to 0.1 mM.
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Med Chem Res (2011) 20:339–345
343
˚
Experimental protocols
Ser630 and a radius of 10 A. Planar N-atom, amide bonds,
and free ring corners of compounds were allowed to flip.
Ten conformations were generated per compound and
ranked according to GoldScore fitness function. Input
conformations were pre-minimized by Tripos force field
with Gasteiger–Hu¨ckel charges and distance-dependent
dielectric constant e=e(4r) (Tripos Inc., 2003). Geometrical
optimizations were performed with at most 500 iterations,
Chemistry of pyrimidinone-like group of compounds
Compounds were synthesized in two steps, as described
ˇ ´
previously (Gluncic et al., 1986). In brief, the ethyl ester of
(3,4,5-trimethoxybenzyl)-cyanoacetic acid (Fig. 1, R=H)
was used as the starting material, being first alkylated with
corresponding halides in the presence of sodium alcohol-
ate, to give ethyl esters of 2,2-disubstituted cyanoacetic
acid. The reaction was performed in ethanol in the presence
of sodium ethanolate, for 1 h at boiling temperature. In the
second step, prepared esters were condensed with thiourea
in ethanol with sodium ethanolate, for 6 h, at boiling
temperature. Ethanol was then distilled off, the residue
dissolved in water and acidified to pH 6 at 0°C with diluted
hydrochloric acid to precipitate the compounds.
˚
using a gradient of 0.05 kcal/molA as a terminating crite-
rion. The three best-ranked positions were re-scored using
ChemScore (Baxter et al., 1998) and ASP (Astex Statistical
Potential) (Mooij and Verdonk, 2005) fitness functions
available in GOLD (Jones et al., 1995). GoldScore,
ChemScore, and ASP score were each converted to Z-score
and binned by taking 0 as the cut-off value within SpotFire
DecisionSite software, v. 8.2.1 (Spotfire 2005). The con-
sensus score was calculated by summing the three binned
scores and the best-ranked positions had the consensus
score 3. Consensus scoring was done with seven known
inhibitors within the scored compound set: Ile-Pro-Ile
(Thoma et al., 2003), NVP-DPP728 (Hughes et al., 1999),
FE999011 (Sudre et al., 2002), (S)-isoleucine thiazolidide
P32/98 (Sorbera et al., 2001), cyclohexylglycine pyrroli-
dide, aminomethylpyrimidine representative (Peters et al.,
2004), and substituted piperazine representative (Brocku-
nier et al., 2004).
Inhibition of DPP IV and enzyme kinetics
DPP IV enzyme (R&D systems, USA, MN) kinetics and
the activity of the compounds were tested using H-Gly-Pro-
pNA tosylate (Bachem, Switzerland) as a substrate. Com-
pound 5 (Table 1) was used for the determination of the
enzyme inhibition type. The enzymatic reaction was per-
formed in 100 ll of buffer containing 50 mM Tris,
150 mM NaCl at pH 7.9. Percent of inhibition and the type
of inhibition were determined after 60-min reaction with
100 lM of each compound; 200 ng enzyme, 300 lM of
Compound 5, and 0.25–8 mM substrate. Initial velocities
were determined by measuring absorbance at 405 nm
during the first 4 min of the enzyme reaction. The non-
linear regression (curve fit) curve was created using the
Michaelis–Menten equation (1). Changes in kinetic
parameters (Km and Vmax), after compound interaction with
the enzyme, were used as parameters for the determination
of the type of inhibition (Marangoni, 2003). Data analysis
was performed using GraphPad Prism software version
3.02 (GraphPad software, San Diego, USA)
Cell lines and cytotoxicity assay
Cell lines were purchased from the ECACC—THP-1,
monocyte, ECACC-88081201 (Tsuchiya et al., 1980), and
HepG2, epithelial, ECACC-85011430 (Aden et al., 1979).
Cells were maintained in complete RPMI 1640 medium
(Institute of Immunology, Zagreb, Croatia) supplemented
with 10% Fetal Bovine Serum (BioWest, S04382S1810) at
37°C in a 5% CO2 atmosphere. Cytotoxicity assay was
performed by using the MTS CellTiter 96Ò AQueous One
Solution Cell Proliferation Assay (G358B, 18824201,
Promega, USA). Each culture in the 96-well plate con-
tained 50,000 (for HepG2) or 75,000 (for THP-1) cells.
Cultures exposed to tested compounds were incubated for
24 h at 37°C in 5% CO2. Thereafter, 15 ll of MTS reagent
(Mosmann, 1983) was added directly to the cell lines. After
an additional 2 h of incubation at 37°C in 5% CO2, the
absorbance was recorded at 490 nm using a spectropho-
tometric plate reader (Ultra, TECAN, USA). The method
was programmed for the TECAN robotic system in
GEMINI pipetting software (Verbanac et al., 2005).
m ¼ Vmax ½Sꢂ=ðKm þ ½SꢂÞ;
where Km is the concentration of substrate required to
reach half-maximum velocity; Vmax is the maximum
reaction velocity; and [S] is the substrate concentration.
ð1Þ
In silico studies
The binding of the pyrimidinones to DPP IV was explored
by molecular docking and a scoring method using the
program GOLD, Version 2.1 (Jones et al., 1995). It was
assumed that the pyrimidinones bind in reversible non-
covalent way. The binding site was defined as a sphere with
an origin at the hydroxyl O-atom of the amino-acid residue
Acknowledgments Special thanks to Professor Michael J. Parnham
for his help in editing and critical reviewing of this manuscript, as
well as to Dr. Andrew Leach for useful remarks. The authors are
ˇ
´
grateful to Snjezana Dragojevic for analytical characterization of the
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Med Chem Res (2011) 20:339–345
tested compounds. We also appreciate the excellent technical assis-
ˇ
tance provided by Ana Cvetkovic, Klara Markusic and Zeljka Tolic.
Kesty NC, Roth JD, Maggs D (2008) Hormone-based therapies in the
regulation of fuel metabolism and body weight. Expert Opin Biol
Ther 8:1733–1747
´
ˇ ´
´
Kim YB, Kopcho LM, Kirby MS, Hamann LG, Weigelt CA, Metzler
WJ, Marcinkeviciene (2006) Mechanism of Gly-Pro-pNA
cleavage catalyzed by dipeptidyl peptidase-IV and its inhibition
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