Optimization of O3-acyl kojic acid derivatives as potent and selective human neutrophil elastase inhibitors

Article abstract of DOI:10.1021/jm4011725

Human neutrophil elastase (HNE) is an attractive target for treating chronic and acute inflammatory lung diseases. An optimization campaign of the kojic acid scaffold to develop new potent HNE inhibitors is reported. O ₃-Pivaloyl derivatives we

Full text of DOI:10.1021/jm4011725

Brief Article
pubs.acs.org/jmc
Optimization of O3‑Acyl Kojic Acid Derivatives as Potent and
Selective Human Neutrophil Elastase Inhibitors
Susana D. Lucas,* Lídia M. Gonca
̧ lves, Luís A. R. Carvalho, Henrique F. Correia,
Eduardo M. R. Da Costa, Romina A. Guedes, Rui Moreira, and Rita C. Guedes
́
Instituto de Investigaca̧ o do Medicamento (iMed.ULisboa), Faculdade de Farmacia, Universidade de Lisboa, Av. Prof. Gama Pinto,
̃
1649-003 Lisboa, Portugal
S
* Supporting Information
ABSTRACT: Human neutrophil elastase (HNE) is an attractive target for treating chronic and acute inflammatory lung
diseases. An optimization campaign of the kojic acid scaffold to develop new potent HNE inhibitors is reported. O3-Pivaloyl
derivatives were shown to be the most potent inhibitors with IC5o values down to 80 nM. These compounds presented excellent
selectivity and cytotoxicity profiles with suitable ligand efficiency.
Japan and Korea, and its administration is limited to infusion
because of its poor pharmacokinetics. Hence, there is an urgent
need of an effective HNE inhibitor with appropriate
pharmacokinetic profile.1
INTRODUCTION
■
Human neutrophil elastase (HNE) is a serine protease that
plays an important role in lung disease initiation and
progression wherein an excess of extracellular HNE is
produced. The imbalance between HNE and its endogenous
inhibitors leads to unregulated elastolytic activity observed in
life-threatening diseases as chronic obstructive lung disease,
cystic fibrosis, acute respiratory distress syndrome, and acute
lung injury.1 Recent studies indicate that HNE-related
processes are involved in non-small-cell lung cancer pro-
gression.2 Targeting HNE to reestablish the protease/
antiprotease balance is an attractive approach for treating
chronic and acute inflammatory lung diseases. A large number
of low molecular weight HNE inhibitors, covering a wide range
of compound families, have been reported.1,3 Many of them
entered clinical trials, but only one HNE inhibitor, sivelestat (1,
ONO-5046, Figure 1), was launched onto the market. Sivelestat
is currently used for treatment of ALI/ARDS exclusively in
Our group has been engaged in the discovery and
development of HNE inhibitors based on new scaffolds.4
Recently we developed a virtual screening protocol that led to
the discovery of the kojic acid hit 2 (Figure 1), which is a 18
μM acyl enzyme inhibitor that was shown to be selective for
HNE when compared with parent proteases. However, 2
presented poor stability toward hydrolytic plasma and micro-
somal enzymes,5 a result consistent with that reported in the
late 80s for closely related long chain bis-ester HNE inhibitors.6
We thus envisaged a hit-to-lead optimization campaign, where
the kojic acid scaffold was modified to improve activity and
druggability. Herein we report the structure−activity profiling
of kojic acid derivatives and their pyridone bioisosteres (Figure
1), together with evaluation of their selectivity and metabolic
stability.
RESULTS AND DISCUSSION
■
Kojic acid derivatives 8a−i were designed to incorporate
different small and hydrophobic ester moieties to promote S1
recognition, and thioether-linked building blocks were intro-
duced as recognition pattern on the opposite counterpart of the
acylating function toward the S1′ site. Additionally, we also
studied the effect of the isosteric N-aryl-γ-pyridone scaffold, i.e.,
8j−q, on the inhibitory potency against HNE. The synthetic
route to afford the desired 8j−q was accomplished starting
from commercially available kojic acid that was transformed
into the corresponding γ-pyrimidones by reaction with aniline
in aqueous acidic conditions (Scheme 1).7 Scaffolds 3 and 4
were transformed to the corresponding chlorides, 5a,b,8 that
were further reacted with thiols 6a−e to afford intermediates
7a−h in good yields. The target molecules 8a−q were obtained
by reaction of 7a−h with the appropriate acyl chloride required
Figure 1. Launched HNE inhibitor from ONO-Pharmaceutical, 1, and
hit 2, generated by a virtual screening protocol; kojic acid derived lead
optimization scheme.
Received: August 1, 2013
Published: November 13, 2013
© 2013 American Chemical Society
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dx.doi.org/10.1021/jm4011725 | J. Med. Chem. 2013, 56, 9802−9806

Journal of Medicinal Chemistry
Brief Article
a
Scheme 1. Synthesis of Kojic Acid Derivatives 8a−q
Table 1. Structures of Kojic Acid Derivatives and HNE
Activity
a
Reagents: (a) NH2Ph, HCl, H2O, reflux; (b) SOCl2; (c) 6, TEA,
DMF, rt; (d) R2COCl, TEA, DMAP, DCM.
t
to incorporate Bu, sec-pentyl, and phenyl esters suitable for
hydrophobic interaction with the S1 pocket (Table 1).
To optimize the starting hit 2, we started by synthesizing 8a
t
(Table 1), with a Bu counterpart in the acylating moiety
leading to a 40-fold improvement in activity. This is in line with
the well established preference for small hydrophobic residues
at the S1 HNE pocket. Potent acyl enzyme inhibitors frequently
have tBu, as 1, or diethyl, as the oxo-β-lactams developed in our
group,4b in analogy with natural substrates where P1-Val is
preferred over an Ala or Phe.9 We also decided to incorporate
building blocks 6b−e (Scheme 1), as they proved to be good
recognition elements for potent HNE inhibitors with oxo-β-
lactam scaffolds.4b Compounds with building block 6b, lacking
the amide linkage between the thiodiazole and phenyl rings
(8b−e), led to poor activities when compared with 8a. In
contrast, the tBu esters with a benzoxazole, 8f, or benzothiazole,
8g, counterparts led to very promising HNE inhibitors, with
IC50 values of 0.67 and 0.11 μM, respectively (Table 1).
Considering the effect of the acylating moiety on the
t
inhibitory potency, inspection of Table 1 reveals that the Bu
moiety leads to potent pyrone-based inhibitors when compared
with the corresponding diethyl and aryl counterparts (e.g., 8b
vs 8c−e and 8g vs 8h), respectively, which is again in
agreement with the preference for small hydrophobic residues
at the S1 HNE pocket. On the other hand a more lipophilic
benzothiazole counterpart is preferable over the corresponding
benzoxazole where the activity gain may be explained by the
hydrophobic character of possible interaction with Phe192 at
the S1 HNE site.
We envisaged that arylpyridones could improve HNE
inhibition by adding an extra recognition moiety, and thus, a
small library of related arylpyridones was synthesized. Never-
theless, only a few compounds showed slightly improved
activity when compared with the corresponding pyrones. Again,
better IC50 values were obtained with benzothiazole over a
benzoxazole moiety. For compounds bearing the benzoxazole
unit, 8j−l, the preference for the tBu moiety is observed as it is
for the pyrone series. When we have the benzothiazole
counterpart, the pyrimidones that were shown to be more
active present diethyl and phenyl acylating units, 8n,o.
Compounds 8i and 8q, containing a carboxylic acid at the
benzoxazole recognition moiety, were synthesized to improve
aqueous solubility. Fortunately, this structural modification
improved potency up to 15 times (8q vs 8j), suggesting that a
carboxylate group may lead to additional interactions with the
enzyme surface. A better understanding of the molecular
interaction with the HNE active site was achieved by molecular
docking studies into HNE active site using GOLD 5.1
software.10 The 3D coordinates of the enzyme structure were
obtained from Protein Data Bank, selecting the structure with
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Journal of Medicinal Chemistry
Brief Article
accession code 1HNE (X-ray coordinates at 1.84 Å resolution,
previously validated for docking procedures5). Comparison of
the docking poses for the pyrone derivative 8i and its pyridone
counterpart 8q (Figure 2) revealed that the N-aryl moiety of 8q
is directed toward the S2 pocket.
We then studied the stability of 8g, 8i, and 8q in aqueous
buffers, human plasma, and rat liver microsomes. The starting
hit compound, 2, presented poor stability toward hydrolytic
enzymes, and the use of Bu esters was envisaged to improve
activity and to afford a protective effect against hydrolysis.
However, HNE inhibitor 8g did not show significant
improvement, being even less stable toward rat microsomes,
when compared with the hit compound 2 (Table 3). Hence,
5
t
Table 3. Stability Assays and Corresponding Half-Lives for
Decomposition in pH 7.4 Phosphate Buffer, 70% (v/v)
Human Plasma, and Pooled Rat Liver Microsomes at 37 °C
t1/2 (min)
pH 7.4 buffer
human plasma
rat microsomes
25
8g
8i
8q
1
stable
6.7 0.7
10.8 0.4
8.9 0.9
32.9 0.5
stable
1.4 0.1
<1
stable
stable
20.7 0.3
82.5 0.0
19.9 0.7
t1/2 = 4 h
stable
Figure 2. GOLD docking poses for 8i (violet) and 8q (cyano) in HNE
active site. The figure was made using MOE 2012.10 software.11
the next step was the introduction of a carboxylic moiety that,
as mentioned, improved the activity toward HNE and also
allowed us to improve stability parameters. The pyrone 8i
proved to be stable in pH 7.4 aqueous buffer while being
hydrolyzed in human plasma with a half-life of 9 min.
Surprisingly, an almost 20-fold increase in microsomal stability
was observed, although with a discrete half-life of 20.7 min.
When we compare with a pyridone containing a carboxylic
moiety, 8q, we observed a significant improvement for plasma
and microsomal stability, with a remarkable 4 times higher
stability toward rat microsomes than the commercial inhibitor
1. This result likely reflects the increase in pKa of the enol-like
3-OH group from the pyrone (pKa = 7.9)12 toward pyridone
(8.6)12 leaving group, and also the addition of the N−Ph
moiety may block the hydrolytic activity. Nevertheless for 8q
we observed chemical susceptibility, and the half-time of this
compound in phosphate buffer solution (pH 7.4) was only 4 h.
Overall these results indicate that inhibitors 8g, 8i, and 8q may
be adequate for inhalatory route of administration, while further
development is necessary to pursue oral bioavailability.
A perfect lock pose favored by π−π interaction of the
benzoxazole with Phe192 is observed (Figure 2). Phe192
usually displays some flexibility and acts as a S1 pocket lid;
hence, the opening of the lid will be blocked in the presence of
these derivatives. Moreover, an extra interaction with a core
residue, Arg147, by hydrogen-bonding with the carboxylate is
observed (Figure 2) and may explain the increase of activity of
the synthesized compounds to lower nanomolar values.
We further studied the selectivity of the potent inhibitors 8g,
8i, and 8q against the human neutrophil serine proteases,
proteinase 3, cathepsin G, urokinase, kallikrein, thrombin,
trypsin, and chymotrypsin, and against porcine pancreatic
elastase, PPE (Table 2). Compounds 8g, 8i, and 8q presented
an excellent selectivity profile, being selective over most of the
serine proteases tested. Compound 8g showed some activity
toward PR3 (selectivity index SI = 54). Compound 8i was also
active to a lower extent against PR3 (SI = 264). On the other
hand, phenylpyridone 8q was inactive against PR3 and only
showed residual activity toward CatG (SI = 237) and thrombin
(SI = 105). These are important results in the field, as protease
covalent inhibitors are often declined for development because
of its promiscuity to parent proteases. Our data indicate that
kojic acid derivatives present desirable selectivity profile, and
further development is on the way to improve recognition
toward S2 and S1′ binding sites, as it seems that different
building blocks on the S2 and S1′ recognition counterpart of
this molecules modulate selectivity over the reactive acylating
moiety. Cytotoxicity assays were also performed, and an
absence of toxicity was observed after exposure of mouse cell
lines NIH3T3 and human cell lines HEK293T to 8g, 8i, 8q,
and 1 for a 24 h period (Table 2).
CONCLUSIONS
■
We have developed new potent HNE inhibitors after a hit-to-
lead optimization campaign of a kojic acid derivative discovered
by a virtual screening protocol. The first insight on the SAR of
kojic acid derived acylating HNE inhibitors was obtained.
Compounds 8g, 8i, and 8q were shown to be the most potent
with activities of 0.11, 0.18, and 0.08 μM, respectively, and
presented good selectivity and cytotoxicity profiles with suitable
ligand efficiency13 (LE of 0.39, 0.34, and 0.29, respectively).
The stability over hydrolytic enzymes from plasma and
microsomes was improved when compared with the starting
Table 2. Selectivity and Cytotoxicity assays
IC50 (μM)
HNE
PR3
Cat G
>100
urokinase
thrombin
kallikrein
trypsin
chymotrypsin
PPE
NIH 3T3
HEK 293T
8g
8i
8q
1
0.11
0.18
0.08
0.01
5.9 1.0
47.5 1.0
>100
>50
>50
>50
>2
>50
>50
>50
>50
>2
>50
>50
>50
>2
>50
>50
>50
>2
>100
>100
>50
>100
>100
>100
>100
>100
>100
>100
>100
>100
>50
19 1.2
>100
8.4 1.2
>2
>100
>50
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Journal of Medicinal Chemistry
Brief Article
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hit, and we could obtain higher stability toward microsomes
than the commercial sivelestat 1. Nevertheless, this is a problem
that still needs to be addressed for further development of
HNE inhibitors in order to overcome metabolic liability.
EXPERIMENTAL SECTION
■
6-((Benzo[d]thiazol-2-ylthio)methyl)-4-oxo-4H-pyran-3-yl
Pivalate, 8g. To a solution of intermediate 7d in DCM (0.05 M)
were added TEA (1.2 equiv) and DMAP (cat.). The corresponding
acyl chloride was added (1.2 equiv), and the mixture was stirred at
room temperature for 3 h, with completion observed by TLC. The
mixture was poured into 10% HCl and the product extracted with
DCM. Desired product was precipitated off with DCM/n-hexane to
afford a white solid: 78% yield; mp 150−151 °C; 1H NMR (400 MHz,
DMSO) δ 8.52 (1H, s, CH), 8.05 (1H, d, J = 7.9 Hz, CHarom), 7.90
(1H, d, J = 8.1 Hz, CHarom), 7.50 (2H, t, J = 7.7 Hz, 2CHarom), 7.41
(2H, t, J = 7.6 Hz, 2CHarom), 6.63 (1H, s, CH), 4.67 (2H, s, CH2),
1.25 (9H, s, CH3-Piv); 13C-APT NMR (101 MHz, CDCl3) δ 181.8
(Cq), 175.3 (Cq), 172.0 (Cq), 164.65 (2Cq), 152.8 (Cq), 149.8
(CH), 141.0 (Cq), 135.4 (Cq), 127.0 (CH), 125.4 (CH), 122.5 (CH),
121.9 (CH), 115.8 (CH), 33.9 (CH2), 27.24 (CH3). Anal. Calcd for
C18H17NO4S2: C, 57.58; H, 4.56; N, 3.73%. Found: C, 57.99; H, 4.82;
N, 3.67%.
ASSOCIATED CONTENT
* Supporting Information
■
S
Synthesis of kojic acid derivatives and compound character-
ization; detailed protocols of activity, cytotoxicity, and stability
assays. This material is available free of charge via the Internet
at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Phone: +351217946477. E-mail: sdlucas@ff.ul.pt.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
The authors thank the Fundaca
■
̧
o para a Cien
̂
cia e Tecnologia
̃
for financial support, Grants Pest-OE/SAL/UI4013/2011,
REDE/1518/REM/2005, SFRH/BPD/64265/2009 (postdoc-
toral grant, S.D.L.).
ABBREVIATIONS USED
■
HNE, human neutrophil elastase; DMF, dimethylformamide;
TEA, triethylamine; DCM, dichloromethane; DMAP, dime-
thylaminopyridine; PPE, porcine pancreatic elastase; PR3,
proteinase 3; CatG, cathepsin G; NIH3T3, mouse embryonic
fibroblast cell line; HEK293T, human embryonic kidney
epithelial cell line
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for N-aryl-gamma-pyridones from gamma-pyrones. J. Heterocycl. Chem.
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Journal of Medicinal Chemistry
Brief Article
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