Ahp-Cyclodepsipeptide Inhibitors of Elastase: Lyngbyastatin 7 Stability, Scalable Synthesis, and Focused Library Analysis

Article abstract of DOI:10.1021/acsmedchemlett.9b00473

Due to the potency and selectivity of lyngbyastatin 7 in inhibiting neutrophil elastase, a serine protease involved in numerous diseases, this cyclodepsipeptide was considered as a promising lead and subjected to further developmental studies. Lyngbyastat

Full text of DOI:10.1021/acsmedchemlett.9b00473

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Letter
Ahp-Cyclodepsipeptide Inhibitors of Elastase: Lyngbyastatin 7
Stability, Scalable Synthesis, and Focused Library Analysis
Danmeng Luo and Hendrik Luesch*
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ABSTRACT: Due to the potency and selectivity of lyngbyastatin
7 in inhibiting neutrophil elastase, a serine protease involved in
numerous diseases, this cyclodepsipeptide was considered as a
promising lead and subjected to further developmental studies.
Lyngbyastatin 7 displayed a favorable serum and microsomal
stability profile. The large-scale synthesis of key building blocks
was performed on gram scale with improved yields and simplified
purification procedures. To tailor the complex structure, define the
minimal pharmacophore, and modulate the physicochemical
properties of the lead scaffold, the first pilot library of analogues
was designed and synthesized for structure−activity relationship
studies. We uncovered the essential role of the side chain,
indicating that the minimal structural requirements for elastase
inhibition extended beyond the 3-amino-6-hydroxy-2-piperidone (Ahp) and 2-aminobutenoic acid (Abu) moieties conventionally
known to convey antiprotease activity and elastase selectivity, respectively. Our studies will facilitate the design and development of
this class of elastase inhibitors.
KEYWORDS: Total synthesis, elastase inhibitor, human neutrophil elastase, macrocycle, cyclodepsipeptide
uman neutrophil elastase (HNE, EC 3.4.21.37), a serine
protease stored at high concentration in neutrophils, is
indicated that elastase may also play a critical role in cancer
metastasis, where it could manipulate the tumor microenviron-
ment and facilitate cancer cell migration.6 In addition, elastase
has also been linked to other inflammatory conditions, such as
rheumatoid arthritis,7,8 bowel intestinal inflammation,9 and
psoriasis.10 Therefore, developing a novel elastase inhibitor
would benefit multiple fields and has wide therapeutic
applications.
H
known to degrade a broad spectrum of extracellular matrix
(ECM) proteins, such as elastin, collagen, and fibronectin,
which provide tissue physical support and stability. Under
normal physiologic conditions, HNE is compartmentalized in
neutrophil azurophilic granules and tightly controlled by its
endogenous inhibitors, such as α1-proteinase inhibitor (α1-
PI), secretory leukocyte proteinase inhibitor (SLPI), and elafin.
However, at inflammation sites, the enormous release of
elastase and the inactivation of endogenous inhibitors may lead
to an imbalance between protease and antiprotease, which is
involved in the pathogenesis of numerous diseases, such as
chronic obstructive pulmonary disease (COPD), cystic fibrosis,
acute lung injury (ALI), acute respiratory distress syndrome
(ARDS), and pulmonary arterial hypertension (PAH).1
Blocking the activity of elastase has been considered for a
long time as a promising therapeutic strategy, and
pharmaceutical companies and academic laboratories have
been working for years in seeking for active small molecule
elastase inhibitors to treat these life-threatening diseases.2
However, so far only one small molecule elastase inhibitor,
sivelestat sodium hydrate (ONO-5046), reached the market in
Japan and South Korea, while its development in the United
States and Europe was discontinued due to the fact that its
clinical efficacy and safety could not be convincingly
demonstrated.3−5 In recent years, increasing evidence
As lyngbyastatin 7 (1),11,12 originally isolated from a marine
cyanobacterium, is one of the most potent and selective
elastase inhibitors from nature, the macrocyclic depsipeptide
scaffold was considered a promising starting point for
developing elastase inhibitor to address the unmet medical
need, complementing and contrasting other campaigns based
on topologically different (flat) small molecules. The first total
synthesis of lyngbyastatin 7 was recently achieved by our group
utilizing a novel designed synthetic strategy (shown in Figure
1).13 Compared with other methods to create similar
molecules,14,15 our strategy enabled easy access to lyngbyas-
Received: October 14, 2019
Accepted: February 18, 2020
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© XXXX American Chemical Society
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A

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Figure 1. Synthetic strategy and key components for constructing lyngbyastatin 7 (1).
Figure 2. In vitro stability of lyngbyastatin 7 (1) under various conditions. Lyngbyastatin 7 (1) was incubated as indicated and extracted with
EtOAc, subjected to LC-MS, and monitored by using compound-specific MRM with harmine as internal standard. (A) Stability in human serum.
(B) Stability in human microsomes in the absence of NADPH. (C) Stability in human microsomes in the presence of NADPH. Data are presented
as mean SD (n = 3).
a
Scheme 1. Synthesis of Building Block II (4)
a
Note: key optimization labeled in red.
tatin 7-based analogues with diverse side chains, as the
essential macrocyclic core (2) was constructed first and the
pendant side chain (6) was attached at late stage. It also
facilitated the exploration of drug physicochemical property via
diversifying the pendant side chain, which also paved the way
for solubility optimization and formulation development.
Poor stability under physiological conditions is among the
most serious but frequently occurring issues affecting
therapeutic outcome. The resulting inadequate drug exposure
and poor pharmacological response would not only impose the
requirement of more frequent/higher daily doses (lower
patient compliance and higher safety risk) but also, oftentimes,
lead to the discontinuation of drug development.16−20
Therefore, early data on the stability profile would provide
in-time liability warnings and allow the removal of susceptible
moieties/the blockage of metabolic sites during structural
optimization.20−24 Here, the serum and microsomal stability of
lyngbyastatin 7 (1) was first investigated in vitro to provide
insights into the potential stability issues inherent in (depsi)-
peptide based molecules and assess the lead status of this
intriguing scaffold. According to the results shown in Figure 2,
lyngbyastatin 7 (1) was remarkably stable in human serum and
remained intact over 24 h at 37 °C. Notably, the embedded
amide and ester bonds proved to be resistant to hydrolysis
catalyzed by serum enzymes. To evaluate the drug stability in
liver, the principal site of drug metabolism, liver microsomes
were incorporated here as an enzyme source, which contain
the major drug-metabolizing enzymes cytochrome P450 and
B
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a
Scheme 2. Synthesis of Building Block III (5)
a
Note: key optimization labeled in red.
a
Scheme 3. Synthesis of Macrocyclic Core (2)
a
Note: key optimization labeled in red.
UDP-glucuronosyltransferase. No NADPH-independent me-
tabolism was observed when incubating lyngbyastatin 7 (1)
with pooled human microsomes for up to 24 h. Furthermore,
this compound was demonstrated to possesses a moderate
microsomal metabolism rate (in the presence of NADPH
cofactor) with t1/2 ∼ 2 h. It is likely that the modified amino
acids and the cyclized scaffold conferred good stability and
resistance toward proteolytic degradation, and those features
are also considered as the hallmarks of cyanobacterial
metabolites, including those in preclinical development.25−28
The excellent stability profile, especially for a depsipeptide-type
molecule with such large size (molecular weight over 900 Da),
strengthened the lead status for lyngbyastatin 7 (1) and
justified further efforts.
scalable to generate grams of materials. The four building
blocks were successfully prepared in large amounts (3 (4.9 g),
4 (51.5 g), 5 (10.2 g), and 6 (side chain, 1.6 g)), and the
macrocyclic core (2 (1.0 g)) was obtained on gram-scale. An
optimized synthetic methodology for constructing the macro-
cycle (2) was developed with the aim to further improve the
reaction yields and simplify the purification procedures. Here,
only the steps with significant improvement are discussed.
First of all, the reaction conditions for several yield-limiting
steps were optimized. For example, the step of deprotecting
the benzyl group from 7 (Scheme 1) previously had a fairly low
yield (24%).13 Changing the type of catalyst or altering the
dose of catalyst was ineffective in increasing the reaction yield.
Through investigating potential side reactions, a transester-
ification product was identified, where the C-terminus Tce-
ester was transformed into a methyl ester, presumably
triggered by the use of methanol (MeOH) as a solvent in
the reaction and workup process. By switching the solvent to
tetrahydrofuran (THF) and avoiding MeOH, the yield was
In order to address the supply issue of lyngbyastatin 7 (1)
and facilitate the generation of lyngbyastatin 7-based
analogues, a large-scale synthesis of the key components of
lyngbyastatin 7 (1) was performed. All the synthetic conditions
in building the key components have been proven to be
C
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Figure 3. Structure of the essential macrocyclic core (2), the natural product lyngbyastatin 7 (1), and the synthetic analogues 15, 16, 17, and 18, as
well as their corresponding cLogD values at pH 7.4 (calculated in Instant JChem) and their IC50 values in blocking elastase activity.
dramatically increased to approximately 100%. As for other
major improvements, upon screening multiple coupling
reagents, the reaction yield of synthesizing 4 (Scheme 1) was
improved from 42% to 91% by using PyAOP instead of BOP-
Cl. For the generation of 5 (Scheme 2), using properly dried
dimethylformamide (DMF) and avoiding moisture from the
environment were identified as the essential factors, which
enabled the reaction yield to increase from 61% to 96%.
Second, unnecessary column chromatography steps were
eliminated to simplify the purification procedures. For
example, the crude product of 8 (Scheme 1), 5 (Scheme 2)
and the deprotected 4 (Scheme 3) could be used directly in
the next step without purification while not affecting reaction
yields. In some other cases, column chromatography
purification was critical to achieve a high yield for the
following reaction. For instance, after removing the protecting
groups from 14 (Scheme 3), a purification step was required to
get rid of the remaining allyl group scavenger Me2NH·BH3, as
this reagent could react with the exposed C-terminus of this
molecule and therefore interfere with the subsequent macro-
cyclization.
Third, a simplified purification method was developed for
the step to obtain 11 (Scheme 2). Previously, column
chromatography was required for this step, which was quite
time-consuming as the desired product was prone to be
retained on a silica gel column.13 Taking advantage of the
acidic property of this compound, a purification method using
solvent-partitioning was developed. The crude reaction
mixture was first basified using 10% Na2CO3 1,4-dioxane:H2O
1:1 solution, followed by washing with EtOAc to remove
impurities. Then the aqueous phase was acidified with 1 M
HCl to pH 2−3 and extracted with EtOAc to enrich the
desired product back into the organic phase. After evaporating
in vacuo, the product was pure enough for use in the next
reaction. Furthermore, for the purification of 13, 14, and 2
(Scheme 3), the hexane−acetone solvent system more
efficiently separated the desired product from other potential
impurities than the previously reported hexane−EtOAc
system.13
With large amounts of the macrocyclic core (2) in hand, the
construction of a focused library of lyngbyastatin 7-based
analogues could be initiated. With the aim to trim off the
complex structure, minimize the molecular weight, and
investigate the role of the macrocyclic core as the sole factor
in affording elastase inhibitory activity, two simplified versions
of lyngbyastatin 7 were synthesized. Taking advantage of the
attached tert-butoxycarbonyl (Boc) group, which originally
served as the protecting group of the Thr amine, analogue 15
(Figure 3) was first designed and the synthesis could be easily
performed within just 3 steps. In order to mimic an amide-
liked short side chain, an acetyl group replaced the Boc group
to afford analogue 16 (Figure 3) smoothly via 5 steps.
Solubility issues (involving approximately 75% of drug
candidates currently under development) adversely affect
preclinical/clinical studies.29,30 Poor aqueous solubility would
not only lead to erratic assay results (artificially weak potency,
flawed good selectivity, faulty clean off-target activities, etc.)
but also negatively affect the pharmacokinetic profile of the
molecules of interest. In addition, drug physicochemical
property is also known to be a key to develop formulations
for pulmonary targeted delivery. Therefore, the key consid-
eration in designing analogues is to differentiate their
D
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physicochemical property. Several lyngbyastatin 7-based
analogues were designed by changing the length of the
terminal lipid chain, replacing the side chain amide of Gln with
other polar functional groups, and attaching hydrophilicity/
lipophilicity-modulating groups at the terminus of the pendant
side chain. Molecular docking using Autodock Vina31 was
applied to virtually evaluate the interaction between the
designed analogues and the HNE binding site (Figure S1),
using the cocrystal structure of lyngbyastatin 7 and porcine
pancreatic elastase (PPE)12 as a starting point and to validate
the docking approach. Several hits with improved affinity were
obtained, and we first selected two analogues to corroborate
our strategy to attach various side chain at late stage and to
generate diverse analogues in a few steps. In addition to
binding affinity of the designed molecules, we also took their
cLogD into account to probe the nature of the HNE binding
pocket. Moreover, if this molecule were to be developed for
inhalation, hydrophilicity/lipophilicity would also affect the
absorption rate and bioavailability.32 Therefore, analogues 17
and 18 characterized by a cyclohexyl and morpholine group,
respectively (Figures 3), were prioritized for synthesis due to
their differential molecular properties. The synthetic strategy to
generate these two molecules was similar to the previously
reported one for obtaining lyngbyastatin 7 (1) (see Supporting
Information for details).13
The HNE inhibitory activities of 15, 16, 17, and 18 were
evaluated in parallel with lyngbyastatin 7 (1) at the enzyme
level (see Figure 3 for IC50 values and dose−response curves in
Figure 4A for the most active compounds), and some critical
information could be obtained from the structure−activity
relationship (SAR) analysis. The first important message is that
the side chain is actually essential to afford the elastase
inhibitory activity. As the side chain was shortened to an acetyl
group (compound 16), the activity decreased over 300-fold. It
is the first time that the minimal cyclodepsipeptide structure of
effective elastase inhibition was defined. Although 3-amino-6-
hydroxy-2-piperidone (Ahp) was the known pharmacophore
and the macrocyclic conformation was known to be essential,
the macrocycle alone (in acetylated form) was insufficient to
substantially inhibit the enzyme. At least a minimal side chain
was required to achieve recognition and binding toward HNE.
Second, the elastase binding pocket could not tolerate a bulky
functional group, especially at the conjunction site of the side
chain and the macrocycle. As the side chain was switched to a
Boc group (compound 15), the activity was completely lost;
according to the result derived from molecular docking, this
structure also displayed a decreased binding affinity to HNE
(−6.7 kcal/mol) compared with lyngbyastatin 7 (−7.2 kcal/
mol). When bulky groups were attached at the terminal site
(compounds 17 and 18), the potency only decreased slightly.
The next message is, the HNE binding pocket prefers a more
lipophilic terminal chain, as the analogue with higher
lipophilicity (compound 17) retained more activity than the
one with lower lipophilicity (compound 18). It was previously
known that the Abu unit adjacent to Ahp contributes to the
selectivity for elastase.6,11,12,33,34
Figure 4. In vitro biological evaluation of lyngbyastatin 7 (1),
analogue 17, and analogue 18. (A) In the HNE enzyme assay, HNE
was first incubated with lyngbyastatin 7 (1), analogue 17, or analogue
18, respectively, for 15 min in 0.1 M Tris−0.5 M NaCl (pH 7.5), and
then N-(OMe-succinyl)-Ala-Ala-Pro-Val-p-nitroanilide was used as
substrate to monitor the enzyme activity. The IC50 value for each
compound is 29 2, 103 6, and 197 15 nM, respectively. Data
are presented as mean
SD (n = 3), relative to 0.5% DMSO
treatment + vehicle. (B) In the cell viability assay, BEAS-2B cells were
cotreated with the tested compound and 100 nM HNE for 24 h. Cell
viability was monitored using MTT reagent. The EC50 values for
lyngbyastatin 7 (1), analogue 17, and analogue 18 were determined as
20 4, 91 17, and 185 33 nM, respectively. Data are presented
as mean SD (n = 3), relative to 0.5% DMSO treatment + vehicle.
(C) In order to monitor the changes in transcript level of IL1B in the
presence of different compounds at various concentrations, total RNA
was extracted after BEAS-2B cells were cotreated with lyngbyastatin 7
(1), analogue 17 and analogue 18 or solvent control and 100 nM
HNE or vehicle for 3 h. After cDNA synthesis, qPCR was carried out
while using GAPDH as endogenous control. Data are presented as
mean SD, *P < 0.05, ****P < 0.0001 compared to HNE-treated
cells using ANOVA, Dunnett’s t test (n = 3).
In addition, the potency of 17 and 18 was further assessed at
the cellular and transcriptional level side by side with
lyngbyastatin 7 (1) (Figures 4B and 4C). Compared with 1,
17 displayed similar potency in protecting bronchial epithelial
cells against elastase-induced antiproliferation, as measured by
MTT assay, and in upregulating transcript levels of pro-
inflammatory cytokine IL1B, as measured by reverse tran-
E
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Funding
scription and quantitative polymerase chain reaction (RT-
qPCR), while 18 was less effective. Their performance is in
agreement with the trend observed at the enzyme level (Figure
4A).
This research was supported by the UF College of Pharmacy
PROSPER grant and the National Institutes of Health grant
R01CA172310.
In conclusion, an optimized and scalable synthetic method-
ology was developed for large-scale synthesis of all the key
components of lyngbyastatin 7 (1) with improved reaction
yields and simplified purification procedures. All four building
blocks were obtained in large amounts and the critical
macrocyclic core was smoothly obtained on gram-scale. To
translate natural products into pharmacotherapeutics, it is also
critical to take the stability issue into concern at early stage to
avoid costly and risky development. Lyngbyastatin 7 was
shown to be stable in serum and possess a reasonable
microsomal metabolic rate, which further highlighted its drug-
likeness and reaffirmed its lead status. A pilot library of
lyngbyastatin 7 (1) analogues was constructed to tailor the
molecules and modulate the lipophilicity/hydrophilicity
through pendant side chain manipulation. For the first time
synthetic analogues were easily accessed within 3−5 steps from
a common key intermediate, validating the design of our
synthetic strategy, which enabled the attachment of the side
chain at late stage and provides opportunities for straightfor-
ward diversification. Moreover, equipped with the improved
understanding of the HNE binding pocket deduced via SAR
study, the molecular design of lyngbyastatin 7-based analogues
could be navigated in a more precise manner in the future. In
addition, the entry to three active molecules with different
lipophilicity/hydrophilicity also provides the opportunity to
develop formulations for different routes of administration for
pulmonary delivered therapeutics or for other disease
indications with overactive HNE.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We thank J. Rocca (Advanced Magnetic Resonance Imaging
and Spectroscopy) for assistance with NMR data acquisition.
■
ABBREVIATIONS
BOP-Cl bis(2-oxo-3-oxazolidinyl)phosphinic chloride
DEPC diethyl phosphorocyanidate
■
DIEA N,N-diisopropylethylamine
DMAP 4-(dimethylamino)pyridine
EDCI·HCl N-ethyl-N’-(3-(dimethylamino)propyl) carbodii-
mide hydrochloride
HATU 1-[bis(dimethylamino)methylene]-1H-1,2,3-
triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate
MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide
NADPH nicotinamide adenine dinucleotide phosphate
P y A O P ( 7 - a z a b e n z o t r i a z o l - 1 - y l o x y ) -
trispyrrolidinophosphonium hexafluorophosphate
TBDPS tert-butyldiphenylsilyl
TBS tert-butyldimethylsilyl
Tce trichloroethyl
Trt triphenylmethyl
UDP uridine diphosphate
REFERENCES
■
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acsmedchemlett.9b00473.
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AUTHOR INFORMATION
Corresponding Author
■
Hendrik Luesch − Department of Medicinal Chemistry and
Center for Natural Products, Drug Discovery and Development
(CNPD3), University of Florida, Gainesville, Florida 32610,
United States; orcid.org/0000-0002-4091-7492;
Phone: +1-352-273-7738; Email: luesch@cop.ufl.edu;
Fax: +1-352-273-7741
Author
Danmeng Luo − Department of Medicinal Chemistry and
Center for Natural Products, Drug Discovery and Development
(CNPD3), University of Florida, Gainesville, Florida 32610,
United States
Complete contact information is available at:
https://pubs.acs.org/10.1021/acsmedchemlett.9b00473
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
F
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