Thioxo-dihydroquinazolin-one Compounds as Novel Inhibitors of Myeloperoxidase

Article abstract of DOI:10.1021/acsmedchemlett.5b00287

Myeloperoxidase (MPO) is a key antimicrobial enzyme, playing a normal role in host defense, but also contributing to inflammatory conditions including neuroinflammatory diseases such as Parkinsons and Alzheimers. We synthesized and characterized more than 50 quinazolin-4(1H)-one derivatives and showed that this class of compounds inhibits MPO with IC₅₀ values as low as 100 nM. Representative compounds showed partially reversible inhibition that was competitive with respect to Amplex Red substrate and did not result in the accumulation of MPO Compound II. Members of this group show promise for therapeutic development for the treatment of diseases in which inflammation plays a pathogenic role.

Full text of DOI:10.1021/acsmedchemlett.5b00287

Letter
pubs.acs.org/acsmedchemlett
Thioxo-dihydroquinazolin-one Compounds as Novel Inhibitors of
Myeloperoxidase
Yang Li,† Thota Ganesh,‡ Becky A. Diebold,† Yerun Zhu,† James W. McCoy,† Susan M. E. Smith,§
Aiming Sun,*,† and J. David Lambeth*,†
†Department of Pathology and Laboratory Medicine, Emory University, Atlanta, Georgia 30322, United States
‡Department of Pharmacology, Emory University, Atlanta, Georgia 30322, United States
§Department of Molecular and Cellular Biology, Kennesaw State University, Kennesaw, Georgia 30144, United States
S
* Supporting Information
ABSTRACT: Myeloperoxidase (MPO) is a key antimicrobial
enzyme, playing a normal role in host defense, but also
contributing to inflammatory conditions including neuroinflam-
matory diseases such as Parkinson’s and Alzheimer’s. We
synthesized and characterized more than 50 quinazolin-4(1H)-
one derivatives and showed that this class of compounds inhibits
MPO with IC50 values as low as 100 nM. Representative
compounds showed partially reversible inhibition that was competitive with respect to Amplex Red substrate and did not
result in the accumulation of MPO Compound II. Members of this group show promise for therapeutic development for the
treatment of diseases in which inflammation plays a pathogenic role.
KEYWORDS: Myeloperoxidase, thioxo-dihydroquinazolin-one, inflammation, NADPH-oxidase, therapeutic
As part of a screen to identify inhibitors of human neutrophil
yeloperoxidase (MPO) is localized in the azurophilic
Mgranules of granulocytes. During phagocytosis of a NADPH oxidase 2 (NOX2; see PubChem AID 1274 and AID
1275), we discovered a compound that inhibited NADPH-
dependent L-012 chemiluminescence in neutrophil mem-
branes. The L-012 assay was previously reported as a sensitive
method to measure superoxide (O2−•).7 Later, we reported that
the L-012 signal requires not only a source of reactive oxygen
(e.g., a NOX) but also a peroxidase.8 We found that the
microorganism, MPO is secreted into the phagolysosome,
where it catalyzes the reaction between hydrogen peroxide and
halides to form hypohalite ions, especially hypochlorite, a
powerful microbicidal agent. Hypochlorite, a highly potent
oxidant, reacts with biomolecules, causing oxidation and/or
chlorination, for example, to form 3-chloro-tyrosine in proteins.
In addition, MPO directly catalyzes the H2O2-dependent
oxidation of biomolecules including proteins and DNA.
These reactions play a key role in host defense by neutrophils
and macrophages, resulting (along with other reactions) in the
killing of invading microbes.
MPO-dependent reactions are also damaging to host tissues,
can be pro-inflammatory, and can play a role in the initiation
and progression of inflammatory diseases. For example, MPO
itself and its oxidation/chlorination products such as 3-
chlorotyrosine are detected in atherosclerotic plaques where
they may play a pathogenic role. MPO has also been implicated
in other diseases including stroke, renal injury, lung
inflammation, Alzheimer’s disease, Parkinson’s disease, and
others.1 Disease occurrence and severity may also be influenced
by MPO overexpression. For example, the MPO allele bearing a
−463G/A polymorphism results in increased transcription and
abnormally high MPO levels.2 This polymorphism is associated
with increased risk of coronary artery disease, sarcoidosis,
Alzheimer’s disease, and several cancers. Thus, there is ample
rationale to develop small molecule inhibitors of MPO that may
prove useful for drug development.3−6
−•
compound failed to inhibit NOX2-generated O2 but instead
targeted MPO (vide infra). The present study characterizes the
inhibition and explores structural features that correlate with
bioactivity among members of this group of MPO inhibitors.
Results. Medicinal Chemistry. The hit compound 1c (see
Table 1) inhibited L-012 oxidation (IC50 of 0.8 μM, Table 2,
Supporting Information (SI)). Medicinal chemistry was
undertaken to improve potency and to investigate structure−
activity relationships (SARs). Modifications were made in the
left-hand and middle rings as well as in the side chain. Based on
synthetic strategy, the thioxodihydroquinazolinone com-
pounds9−12 described here are subdivided into Series 1 and
Series 2 (further subdivided into Series 2A and 2B, Figure 1).
The modifications focused mainly on three regions (X, Y, and
Z) of molecules (Figure 1).
Series 1 compounds were synthesized as in Scheme 1 from
commercially available isatoic anhydrides (A), which were
treated with alkylamine containing varying alkyl chain lengths
Received: February 10, 2015
Accepted: August 31, 2015
© XXXX American Chemical Society
A
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a
Table 1. IC50 Values for Inhibition of MPO and Xanthine Oxidase (XO) Activity
a
MPO (Amplex Red assay) and XO activities were measured under the same pH and buffer conditions (see Methods, SI), using a concentration
range up to 100 μM for each inhibitor. IC50 values represent the average SEM of two to six determinations. N.I., no inhibition. % maximum
inhibition values (% MI) are indicated in parentheses below the IC50 values.
B
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Figure 1. General structures of Series 1 and 2 compounds and
compound 1c.
Scheme 1. General Synthesis of Series 1 Compounds
Figure 2. Concentration dependence for inhibition of human MPO
and horseradish peroxidase (HRP). Activity was measured using the
Amplex Red method at the indicated concentrations of 1c (squares)
and 2b (circles). Assays were performed with 0.17 nM MPO (filled
symbols) or 20 nM HRP (open symbols). Points show the mean
SEM of three determinations.
and varying terminal units (amines or other substitute groups)
to generate 2-aminobenzamides (B). B was then treated in situ
with NaOH and CS2 to generate the final Series 1 compounds.
Compounds were purified using silica column chromatography
(SI) and/or recrystallization.
Series 2 compounds were synthesized according to Scheme
2, beginning with a benzamine (C), which was treated with
of the compound, and by 2b with an IC50 of 0.1 μM and 90%
maximum inhibition. These were also tested for assay
interference using horseradish peroxidase in place of MPO in
the Amplex Red assay (Figure 2); no inhibition was seen even
at high concentrations of compounds. Summary results for 36
representative compounds are shown in Table 1. Table 1 also
provides data on possible assay interference, using xanthine/
xanthine oxidase as a source for oxidants; in general, the
compounds did not show assay interference in the concen-
tration range that inhibited MPO.
Scheme 2. General Synthesis of Series 2 Compounds
Structure−Activity Correlations. Inspection of Table 1
allows some conclusions with regard to structural features
that are important for optimal inhibition of MPO. First, sulfur
was essential for bioactivity since replacement of sulfur with
oxygen reduced or eliminated inhibition (compounds 1a vs
1b). Within limits, the side-chain (X in Scheme 1) or the
presence of additional carbons on the terminal nitrogen had
small effects on potency, suggesting that the binding site can
accommodate modest variations in structure. However,
increasing the aliphatic side-chain to five carbons (compound
1e-L, Table 1) completely eliminated activity, suggesting a size
limitation to the binding cavity. A side-chain containing two to
three carbons appeared to be optimal (e.g., compare 1a with
1e-L, 1i, 1j, 1k, and 1m). Analogues with a 3-carbon link and a
piperazine tail (1p) or a piperidine tail (1n) showed modestly
increased potency.
The effect of substituents on the left side phenyl ring was
evaluated. A halide at position 6 of the left-hand phenyl ring
frequently improved bioactivity (e.g., compare 1a with 1c and
1e in Table 1). Halides in the 7- or 8-positions on the same ring
or the presence of two halides failed to improve activity
compared with a halide at position 6 (e.g., see 1d, 1g, 1h). For
example, a fluoro group at position 6 (1e, Table 1) improved
activity, compared with 1a, whereas compound 1g with fluoro
group at both 6- and 7-positions was less potent than the 6-
monofluoro derivative. A chloro group at the 8-position (1h)
eliminated inhibitory activity. Replacement of 6-fluoro
substituent with a 6,7-dimethoxyl substitutes (2g) or dioxane
group (2h) decreased activity compared with 2b suggesting
somewhat stringent structural requirements at the binding site
for the left side of the molecule.
thiophosgene in the presence of triethylamine to provide
isothiocyanate (D). D was refluxed with an alkyl hydrazine and
triethylamine in THF to generate Series 2A compounds.
Treatment of D with hydrazine and triethylamine followed by a
substituted chlorocarbonyl reagent generated series 2B
compounds (Scheme 2).
Approximately 38 Series 1 thioxodihydroquinazolinone
analogues were first synthesized (some of them shown in
Table 2, SI). Some compounds containing nitrogen at position
X (Table 2, SI) showed good activity in the screening assay and
formed the basis for designing Series 2A and 2B compounds
(Figure 1).
In preliminary studies (Table 2, SI), compounds were tested
by measuring L-012 oxidation, in a cell-free system from human
neutrophils (METHODS).13 The xanthine/xanthine oxidase
assay system14 was used to eliminate false positives from assay
interference. Representative compounds in both series were
−•
then tested for inhibition of O2 generation by NOX2 using
NADPH-dependent, SOD-inhibitable cytochrome c reduction,
15
−•
an assay that is specific for O2
.
Because no inhibition was
seen (not shown), some of the more promising compounds
were then tested for inhibition of purified human MPO. All
compounds tested showed inhibition of MPO, indicating that
for these compounds, the initial assay using neutrophil
membranes and L-012 was detecting inhibition of MPO and
not NOX2. Examples of MPO inhibition curves for two
compounds from series 1 (1c) and series 2 (2b) are shown in
Figure 2. Recombinant MPO was inhibited by 1c with an IC50
of 0.8 μM and 60% maximum inhibition at high concentrations
A free amino group directly linked to one of the ring
nitrogens in place of the N,N-dimethylaminopropyl side chain
sometimes improved potency (e.g., compare 3f vs 1e), while a
dimethylamino (3a) or a hydroxyl (3g) group weakened the
C
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potency compared with the free amino group. Ethyl (3b) and
isopropyl (3c) groups linked to the ring nitrogen decreased
potency (Table 1). Based on the improved activity of
compounds with nitrogen directly linked to the ring nitrogen,
Series 2A and 2B compounds were synthesized to maintain this
structural feature while varying the side chain and left-side
substituents (2a−2m, Table 1). Structural features of Series 2
inhibitors include an N−N linkage (Series 2A) or N−N−CO
(Series 2B). An attached aromatic ring distal to the N−N
linkage improved potency compared with an aliphatic side-
chain (e.g., compare 2a and 2a-E with others in Series 2). As a
group, compounds with this feature showed IC50 values for
MPO inhibition that were consistently less than 400 nM. Some
(2b, 2c, 2e, 2j, and 2k) showed IC50 values of 100−200 nM
with 90% maximum inhibition. With regard to the importance
of the benzamide linker, a sulfonamide linkage (2j) did not
have a significant effect on potency. Analogues with a phenyl
amine link (2k and 2l) instead of an amide showed similar
potency compared with compounds containing an amide
linkage. Inhibitor 2k with o-chloro and p-trifluoromethyl
substituent groups, and 2l with a p-fluoro group showed
excellent potency against purified MPO (Table 1). Compounds
1c, 2b, 2k, and 2i were also evaluated using the taurine
chlorination assay (Methods, SI), and all were found to be
active, with IC50 values about 2−3-fold higher than with the
Amplex Red assay.
Figure 3. Determination of reversibility of MPO inhibition; ABAH (6
μM), SHA (100 μM), 1c (50 μM), and 2k (50 μM), all in DMSO (1%
final) or DMSO alone (Control), were incubated with MPO bound to
wells of an ELISA plate for 1 h in the presence of 50 μM H2O2. Wells
were either washed to remove inhibitor (gray bars) or remained
unwashed (black bars) prior to measuring MPO activity as in
Methods, SI. Results show mean MPO activity (ΔA652 nm/min values
SEM, n = 3).
and compound 2k also recovered significant activity upon
washing. This indicates that in contrast to several other MPO
inhibitors,16 representative members of this chemical series do
not irreversibly inactivate MPO.
Effects of Representative Inhibitors on Absorption Spectra
of Myeloperoxidase During Turnover. MPO (Fe3+) reacts
with H2O2 to generate Compound I, which can react with
chloride to generate HOCl, regenerating MPO (Fe3+), or it can
react with a 1-electron-donor to produce Compound II. A
second electron returns the enzyme to MPO (Fe3+). Some
compounds inhibit MPO-dependent chlorination by acting as
an efficient substrate for Compound I but not Compound II,
trapping the enzyme as Compound II (removing it from the
chlorination cycle).19 Under our conditions, H2O2 generated a
small quantity of Compound II (solid line, Figure 4), which was
Inhibitor 1c was soluble in water to 90 μg/mL, but the
solubility of 2b, 2c, and 2e were fairly modest, around 30 μg/
mL (data not shown), which might prove problematic for
future drug development. To increase solubility, a derivative
with a methylene piperidinyl group (2i) was synthesized. This
derivative maintained good potency (0.4 μM IC50), and its
solubility was improved 50-fold to 1500 μg/mL, suggesting that
this strategy may be useful for future development of these
compounds.
Kinetic Mechanism of Inhibition. The kinetic mechanism of
inhibition of MPO-dependent oxidation of Amplex Red was
investigated for the inhibitors 1c and 2b. When H2O2
concentration was varied at several fixed inhibitor concen-
trations, the apparent Vmax changed but the Km for H2O2 did
not, indicating noncompetitive inhibition. Figure 1, SI, shows a
representative experiment using inhibitor 1c, and the same
result was obtained for 2b. This shows that the inhibitors do
not block the access of H2O2 to heme. In contrast, when
Amplex Red concentration was varied at various inhibitor
concentrations, the Vmax remained the same, but the Km for
Amplex Red shifted to higher concentrations. Figure 2, SI,
shows a representative kinetic analysis for 2b, and the same
result was seen for 1c. These results support a model in which
the inhibitor binds at the substrate-binding site, preventing
access of Amplex Red.
Reversibility of Inhibition. Several known inhibitors of MPO
(including the control inhibitor 4-aminobenzoic acid hydrazide
ABAH) covalently modify MPO and therefore act as
irreversible inhibitors.16,17 Reversibility was investigated using
an ELISA assay in which plate-bound MPO is incubated with
the inhibitor, and the ELISA plate is then washed to remove
any inhibitor that is noncovalently bound, followed by assay of
activity. As shown in Figure 3, ABAH nearly fully inhibits
activity, and only a small amount of activity is regained upon
washing the plate. In contrast, a reversible inhibitor of MPO,18
salicylhydroxamic acid (SHA), partially inhibited the enzyme,
which then regained full activity after washing. Compound 1c
Figure 4. Effect of compounds 1c, 2b, 3f, and 5-fluorotryptamine on
MPO intermediates. Difference spectra resulting from the addition of
250 μM H2O2 to purified MPO (1.2 μM) in the absence or presence
of 10 μM inhibitor, 1% DMSO, and 150 mM NaCl in 50 mM
potassium phosphate buffer, pH 7.4.
markedly increased by 5-fluorotryptamine (5-FTA), known to
inhibit MPO by trapping the enzyme as Compound II.19
Inhibitors 1c, 2b, or 3f caused a slight decrease rather than an
increase in Compound II (Figure 4), indicating that their
mechanism of inhibition does not involve trapping of MPO as
Compound II.
Summary. Thioxo-dihydroquinazolin-one derivatives were
identified as novel inhibitors of MPO. These compounds are
D
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similar in some ways to the thioxanthine class of MPO
inhibitors17,20−24 in that both have a thiourea moiety as part of
a ring system, but the thioxanthines act as covalent inhibitors of
MPO.17 Members of this class of compounds may prove useful
for the future development of drugs targeting diseases in which
inflammation plays a role, including neurodegenerative diseases,
atherosclerosis, and others. These compounds, like the
aromatic hydroxamates,18 differ from many other MPO
inhibitors,16,17,19,24,25 in that they act reversibly rather than
irreversibly and do so without the generation of MPO
Compound II. Because inhibition by compounds that act by
trapping the enzyme as Compound II might be reversed by
endogenous reductants, e.g., ascorbate,26 they may lose
effectiveness in vivo. The inhibitors described here do not
trap the enzyme as Compound II and may provide advantages
in some clinical settings.
SHA, salicylhydroxamic acid; SOD, superoxide dismutase;
TLC, thin layer chromatography; TMB, tetramethylbenzidine;
XO, xanthine oxidase
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ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsmedchem-
lett.5b00287.
Experimental details along with the chemical and
physical characterization of the compounds (PDF)
AUTHOR INFORMATION
Corresponding Authors
*Tel: +1-404-727-5875. E-mail: noxdoc@mac.com.
*Tel: +1-404-931-0703. E-mail: aimingsun@yahoo.com.
■
Author Contributions
All authors contributed to the writing of the manuscript. Y.L.,
T.G., and A.S. synthesized, purified, and characterized the
compounds. Y.L., T.G., A.S., and J.D.L. designed compounds.
B.D., Y.Z., J.M., S.S., and J.D.L. carried out and/or designed
enzymatic assays. J.D.L. was responsible for the oversight of the
project. All authors have given approval to the final version of
the manuscript.
Funding
Supported by NIH Grant R33 AI102197.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Dr. Minoru Tamura for the p67Np47N chimera
plasmid. We thank Ben Lambeth for writing the computer
program for storage and retrieval of compounds and associated
data.
ABBREVIATIONS
■
ABAH, aminobenzoic acid hydrazide; DMSO, dimethyl
sulfoxide; DTPA, diethylenetriamine pentaacetic acid; FAD,
flavin adenine dinucleotide; 5-FTA, 5-fluorotryptamine; HRP,
horseradish peroxidase; MPO, myeloperoxidase; NADPH,
nicotinamide adenine dinucleotide phosphate; NOX2,
NADPH oxidase 2; p47phox, cytosolic 47 000 Da protein,
subunit of the NOX2 complex; p67Np47N, a chimeric protein
of the N-terminal segment of the p67phox and p47phox
subunits; p67phox, cytosolic 67 000 Da protein, subunit of the
NOX2 complex; phox, phagocyte NOX; PMA, phorbal 12-
myristate 13-acetate; PPB, potassium phosphate buffer, pH 7.4;
ROS, reactive oxygen species; SDS, sodium dodecyl sulfate;
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J.; Paton, L. N.; Jameson, G. N.; Eriksson, H.; Kettle, A. J. 2-
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