Modular Solid-Phase Synthesis of Antiprotozoal Barnesin Derivatives

Article abstract of DOI:10.1021/acs.orglett.0c00723

Here, we applied and optimized a solid support (SP)-based Horner-Wadsworth-Emmons reagent to prepare SP-bound vinylogous amino acids. Subsequent SP-based peptide synthesis, global deprotection, and chemical modifications yielded 14 lipodipeptides carrying

Full text of DOI:10.1021/acs.orglett.0c00723

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Letter
Modular Solid-Phase Synthesis of Antiprotozoal Barnesin
Derivatives
́
̌
David Roman, Luka Raguz, Franc o̧ is Keiff, Florian Meyer, Fabian Barthels, Tanja Schirmeister,
Florian Kloss, and Christine Beemelmanns*
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sı Supporting Information
ABSTRACT: Here, we applied and optimized a solid support (SP)-based Horner−Wadsworth−Emmons reagent to prepare SP-
bound vinylogous amino acids. Subsequent SP-based peptide synthesis, global deprotection, and chemical modifications yielded 14
lipodipeptides carrying vinylogous amino acids, including the natural product barnesin A (1). Biological evaluation revealed that
several synthesized derivatives show micromolar to nanomolar inhibitory activity against papain-like cysteine proteases, human
cathepsin L, and rhodesain.
arnesin A (1) is the first metabolite reported from the
B
bacterial class Epsilonproteobacterium (Sulfurospirrilum
1,2
barnesii), and also the first metabolite known from anaerobic
bacteria biosynthesized by a nonribosomal peptide synthe-
3
tase−polyketide synthase (NRPS−PKS) hybrid. Barnesin A is
a lipodipeptide containing a rare unnatural vinylogous arginine
moiety, which is biosynthesized by condensation of an acetate
unit and an activated lipopeptide chain via the NRPS−PKS
4
assembly line. Such highly reactive vinylogous amino acids are
rarely found in natural products. To date, only one other
natural product containing a vinylogous arginine, miraziridine
5
−7
A (2), has been identified.
Most prominent and almost
exclusive examples of structurally related natural products
Figure 1. Structures of barnesin A (1), miraziridine A (2), and
syringolin A (3).
8
containing other vinylogous amino acids are syringolines and
9
glidobactins, which contain vinyl-Val and vinyl-Tyr units,
1
0
respectively. The electrophilic nature of the α,β-unsaturated
amide moiety (warhead) activates these natural products for
conjugate addition reactions, a key reactivity of protease
approach commencing with readily available Fmoc-Arg(Boc) -
2
1
OH.
Although the first-generation synthesis of barnesin allowed
for the absolute structure confirmation and first bioactivity
tests, a more efficient and modular synthetic approach was
1
1
inhibitors. As an example, pharmacological studies of
syringolin A (3) disclosed that the molecule covalently targets
the active site of proteasomes via a Michael addition
1
2
reaction. Similarly, first bioactivity studies of barnesin A
1) revealed selective nanomolar inhibitory activity (91.7 nM)
Received: February 25, 2020
(
13
against the cysteine protease cathepsin B, and indicated
inhibition mechanisms via a Michael-type addition reaction to
the enzymatic target (Figure 1).
1
The first total synthesis of barnesin A (1) was accomplished
in nine steps (0.8% overall yield) using a solution-based
©
XXXX American Chemical Society
https://dx.doi.org/10.1021/acs.orglett.0c00723
A
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Scheme 1. General Procedure for HWE-Based Solid-Phase Synthesis of Barnesin Derivatives
highly desirable to pursue more detailed structure−activity
studies.
Thus, we decided to combine the advantageous features of
resin-bound alkene B was obtained with excellent E-selectivity
20
(vide infra).
The general strategy for subsequent peptide coupling
solid-phase peptide (SPP) synthesis (e.g., minimal amount of
followed the principles of Fmoc/tBu solid-phase peptide
1
4,15
purification steps)
and Horner−Wadsworth−Emmons
synthetic strategy, which implied the use of N -Fmoc protected
α
(
HWE) reaction by coupling the carboxy terminus of HWE
amino acids and tBu-protected side chains. Thus, every
protective group orthogonal to Fmoc appeared suitable for
the protection of reactive moieties of the side chains and
16−18
reagent to a resin.
This strategy would allow us to
synthesize any required vinylogous amino acid on solid
support and enable subsequent SPP-based peptide elongation
steps.
allowed the selective deprotection of the N -amine and
α
regioselective coupling. In general, a qualitative ninhydrin
test was used to monitor the reaction progress.
As a first proof of principle, we aimed for the synthesis of
barnesin A (1), followed by the synthesis of C4, C10-
stereoisomers (5, 6), acyl chain length derivatives (7−10), and
derivatives containing different amino acids (12−17). To
maintain the essential structural protease recognition features,
we focused on the exchange of vinyl arginine against lysine,
and phenylalanine against tyrosine. Fully reduced barnesin
derivative 11 should be obtained by hydrogenationfrom 1 or 8,
respectively.
Coupling with the Fmoc-protected amino acid was
performed using the highly effective coupling reagent
21
COMU and DIPEA in DMF (product C). Eventually, the
resin-bound peptide was coupled with the respective fatty acid
(
product D) and the product was cleaved off as fully protected
modified lipo-dipeptide E using HFIP/DCM (1:4) within 15
min. Notably, longer exposure of D to more acidic conditions,
e.g., HFIP/DCM (1:1 to 1:3), led to the partial cleavage of the
Boc-protective groups and isolation of a mixture of mono- and
diprotected derivatives. The obtained lipodipeptides were
globally deprotected using 95% TFA in DCM and purified
using reverse-phase preparative high pressure liquid chroma-
tography (HPLC) to yield multimilligram amounts of
analytical pure (E)-barnesin derivatives (Figure 2). Here, it
needs to be noted that reaction sequences containing arginine
derivatives were prone to side reactions (e.g., partial Boc
deprotection) and epimerization if conditions were not
accurately followed. To separate the resulting diastereomers
and monoprotected species required occasionally an additional
HPLC purification step resulting in overall lower yields of the
desired end product (see the Supporting Information). Methyl
esters (13, 14) were obtained after treatment of cleaved and
fully protected peptides (15 and 16) with TMS-diazomethane,
followed by global deprotection.
Among several possible resin candidates, we selected 2-
chlorotrityl chloride resin (CTC resin) for subsequent studies
as mild cleavage conditions are required to release the peptide
scaffold carrying a vinylogous amino acid from the resin. The
envisaged SPP−based Horner−Wadsworth−Emmons (HWE)
reaction required the preparation of phosphonate A loaded on
1
6−18
to the solid-phase (CTC) (Scheme 1),
which was
achieved using standard coupling conditions (theoretical
loading of 0.8 mmol phosphonate per gram resin, Supporting
Information). Here, it is worth noting that the newly formed
resin could be stored for several months at 4 °C until further
use. The synthesis of the resin-bound vinylogous arginine
required the preparation of triple protected D- or L-Fmoc-
Arg(Boc) -OMe, which was achieved according to a previously
2
described four step synthesis starting from commercially
1
,19
available L- or D-ornithine hydrochloride.
The methyl
In summary, the synthesis of 14 barnesin derivatives was
achieved in only seven or eight synthetic steps (instead of
ester was then reduced using a moderate excess of DIBAL(H)
at −78 °C to ensure high conversion and minimal over
reduction (Scheme 1, Supporting Information). For the
preparation of vinylogous lysine derivatives commercially
available N-Boc-Lys-Fmoc was transformed in an analogous
fashion.
1
previous nine steps), and only one final purification protocol
was generally required to yield analytical pure samples.
We then tested the inhibitory activity of all barnesin
derivatives against two pharmacologically important papain-
like cysteine proteases, human cathepsin L (hCatL) and
22
Due to their inherent instability amino acid aldehydes 4
were immediately subjected to modified HWE reaction
conditions using resin-bound phosphonate A and reaction
rhodesain (Rd) (Table 1, reference compounds: Table S2).
Cathepsin L is a lysosomal endopeptidase, which is involved in
the initiation of physiologically important protein degradation
16−18
23
scales varied between 0.5 and 3.0 g of resin.
The resulting
reactions. An increased expression of hCatL and other
B
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selective inhibition of cysteine proteases in Trypanosoma
represents a promising strategy for the development of new
drugs with antiparasitic activity.
We used an established fluorescence-based assay to
determine the activity of all synthesized barnesin derivatives
27
against hCatL and Rd, and an initial screening at inhibitor
concentrations of 20 μM was performed to identify ligands
with an inhibition of hCatL and Rd above 60%. For active
compounds, continuous assays at different inhibitor concen-
trations were carried out. All inhibitors with more than 60%
inhibition in the initial screening were found to act in a time-
dependent manner and compared to reference standards
(
Supporting Information, Table S2). During kinetic analyses of
the substrate conversion, the maximum inactivation rates kinact,
the apparent dissociation constants Ki , and the second-order
app
rates of inhibition ksecond were determined. The reversible
enzyme−inhibitor complex constants K were calculated
i
2
8
according to the Cheng-Prusoff equation. To evaluate the
potential of all compounds regarding their systemic application
in mammalians, we determined in vitro pharmacokinetic
properties in metabolic assays using human liver microsomes
(
HLM) and glutathione (GSH).
As depicted in Table 1, barnesin A (1) revealed K values of
i
1
.5 (±0.39) and 0.081 (±0.013) μM against hCatL and Rd,
respectively. The stereochemistry at position C-4 and C-10 was
found to be of crucial importance as diastereoisomer 5
(
6
4R,10S) showed only very low activity against rhodesain, and
(4S,10R) was found to be inactive (nd) against both
Figure 2. Synthesized barnesin derivatives (1, 5−17) using an HWE-
based solid-phase synthesis approach (overall yields after seven or
eight steps, respectively).
enzymes. In accordance with previous findings, the fully
reduced barnesin A derivative 11 showed no inhibitory activity
against both proteases. In a next step, different acyl derivatives
of barnesin A (7−10) were tested. The results illustrated that
the chain length, but not saturation degree, is of significant
importance for activity. While long-chain derivative 7, propenyl
derivatives 9 and acyl derivative 10 showed significant
reduction in activity, octanyl derivative 8 revealed properties
similar to barnesin A. Furthermore, the exchange of tyrosine
(1) against phenylalanine (12) did not significantly alter the
activity. As shown in Table 1, all tested vinyl-Arg derivatives
(1−12) were found to be stable in our microsomal and
proteases, however, has been reported in many cancer types
and tumor-associated fibroblasts and macrophages, thus
rendering them promising drug targets. Rhodesain (Rd), also
a clan CA papain-family cysteine protease plays a central role
in the life cycle of Trypanosoma brucei and its related
2
4
subspecies, which are parasitic hemoflagellate protists and
2
5
the major cause of Human African Trypanosomiasis (HAT),
26
more commonly known as “African sleeping sickness”. The
Table 1. Inhibitory Activity (K Values) of Synthesized Compounds against Cathepsin L and Rhodesain and Their Metabolic
i
Stability
cat L
Rd
ksecond (M− s−1
1
)
Ki (μM)
ksecond (M−1 s−1
)
HLM (%)
a
GSH (%)
b
compd
Ki (μM)
1
5
6
7
8
9
0
1
2
3
4
5
6
7
1.5 ± 0.39
nd
nd
nd
7.0 ± 2.1
nd
2216 ± 347
nd
nd
nd
749 ± 85
nd
105 ± 10
nd
1,110 ± 601
3004 ± 292
103802 ± 1573
1704 ± 167
22436 ± 897
904 ± 43
0.081 ± 0.013
3.6 ± 1.0
nd
144427 ± 23974
2130 ± 144
nd
83
68
87
90
92
≥99
≥99
≥98
79
25
<5
94
≥99
99
93
≥99
88
≥99
≥99
≥99
53
76
≥99
97
nd
nd
0.080 ± 0.012
2.8 ± 1.1
0.91 ± 0.34
nd
0.075 ± 0.017
0.21 ± 0.049
0.024 ± 0.005
0.048 ± 0.01
0.012 ± 0.005
0.045 ± 0.02
84579 ± 7358
6847 ± 1627
4862 ± 736
nd
104631 ± 14066
54716 ± 5982
797250 ± 151069
157543 ± 25248
673809 ± 32619
239099 ± 85338
1
1
1
1
1
1
1
1
35 ± 7.6
nd
6.4 ± 2.2
1.9 ± 0.4
0.047 ± 0.005
4.3 ± 1.0
0.11 ± 0.02
4.2 ± 0.39
≥99
≥99
80
98
a
b
Microsomal stability assays using human liver microsomes (HLM). Glutathione (GSH) stability assays. Values are given in % based on relative
peak areas of the extracted ion chromatograms after 90 min (assay) versus time of initiation (100%).
C
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glutathione-based stability assays (HLM and GSH) over a
period of more than 90 min (Supporting Information). This is
quite surprising as the intrinsic mechanism-based reactivity of
unsaturated barnesins would imply interference with nucleo-
philes or oxidative metabolic degradation pathways. In case of
lysine derivatives 13−17, both methyl esters (13, 14) showed
higher inhibitory activity against both cysteine proteases
compared to the free acids (15−17); presumably due to an
increased electrophilicity of the vinylogous warhead. At the
same time, the ester moiety reduced the metabolic stability,
presumably due to spontaneous trans-acylation reactions.
Thus, overall, derivative 16 was determined as the most
active and stable derivative. We then analyzed literature reports
of other natural products with similar structural features with
respect to protease inhibitory activities against Cat L and Rd.
Although inhibitory activities values across different assays and
literature reports needs to be handled with extreme care, our
survey (Table S2) showed that barnesin derivative 14 had
similar inhibitory activity against CatL as the fungal dipeptide
Authors
David Roman − Leibniz Institute for Natural-Product Research
and Infection Biology - Hans Knoll Institute (HKI), 07745
Jena, Germany
Luka Raguz − Leibniz Institute for Natural-Product Research
and Infection Biology - Hans Knoll Institute (HKI), 07745
Jena, Germany; orcid.org/0000-0001-5383-3012
Francois Keiff − Leibniz Institute for Natural-Product Research
and Infection Biology - Hans Knoll Institute (HKI), 07745
Jena, Germany
Florian Meyer − Leibniz Institute for Natural-Product Research
and Infection Biology - Hans Knoll Institute (HKI), 07745
Jena, Germany
́
̈
̌
̈
̧
̈
̈
Fabian Barthels − Institute of Pharmaceutical and Biomedical
Sciences, Johannes Gutenberg University Mainz, 55128 Mainz,
Germany
Tanja Schirmeister − Institute of Pharmaceutical and
Biomedical Sciences, Johannes Gutenberg University Mainz,
5
5128 Mainz, Germany
Florian Kloss − Leibniz Institute for Natural-Product Research
and Infection Biology - Hans Knoll Institute (HKI), 07745
Jena, Germany
2
9
E-64 (E-64: IC 0.05 μM versus 14: IC 0.093 μM), while a
5
0
50
structural analogue of E-64, named kojistatin A, was found to
̈
30
be 2 orders of magnitude more potent (0.0048 μM).
Interestingly, both fungal protease inhibitors carry a epox-
ysuccinate moiety as warhead. However, to the best of our
knowledge, none of the related reports included inhibitory
activity studies against rhodesain (Rd) or stability assays to
evaluate their systemic application.
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.orglett.0c00723
Notes
In conclusion, we have developed a new and efficient
synthesis of barnesin and derivatives using a largely underex-
plored HWE reaction on solid support. Only seven synthetic
steps and one purification procedure were needed to yield
derivatives on a multimilligram scale. Subsequent biological
studies of all synthesized compounds showed an intriguing
metabolic stability and clear structure−activity relationships
against human cathepsin L (hCatL) and rhodesain (Rd). The
replacement of tyrosine to phenylalanine and vinyl arginine to
vinyl lysine generally increased inhibitory activities of barnesin
derivatives, without significant loss of metabolic stability.
Overall, these encouraging bioassay results will guide future
studies on this intriguing natural product scaffold. The
modularity of the synthetic approach presented further sets
the stage for extensive combinatorial studies necessary for the
targeted generation of leads in the field of cysteine protease
inhibitors.
The authors declare the following competing financial
interest(s): The authors declare a financial conflict of interest.
A patent application has been filed (registration number: EP
18 160 274.9: Barnesin A, derivatives and uses thereof) at the
European patent office.
ACKNOWLEDGMENTS
■
We are grateful for financial support from the German
Research Foundation (DFG, BE 4799/2-1). C.B. greatly
acknowledges funding by the ERC (ERC Starting Grant
project 802736 MORPHEUS). F.M. and F.K. have been
supported by InfectControl 2020 (FKZ 03ZZ0803A, FKZ
0
3ZZ0826A, and FKZ 03ZZ0835A). We wish to thank Ms.
Heike Heinecke (Leibniz Institute for Natural Product
Research and Infection Biology - Hans Knoll Institute, Jena)
̈
for measurement of NMR spectra.
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sı Supporting Information
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