Synthesis of New Cyclomarin Derivatives and Their Biological Evaluation towards Mycobacterium Tuberculosis and Plasmodium Falciparum

Article abstract of DOI:10.1002/chem.201901640

Cyclomarins are highly potent antimycobacterial and antiplasmodial cyclopeptides isolated from a marine bacterium (Streptomyces sp.). Previous studies have identified the target proteins and elucidated a novel mode of action, however there are currently only a few studies examining the structure–activity relationship (SAR) for both pathogens. Herein, we report the synthesis and biological evaluation of 17 novel desoxycyclomarin-inspired analogues. Optimization via side chain modifications of the non-canonical amino acids led to potent lead structures for each pathogen.

Full text of DOI:10.1002/chem.201901640

A Journal of
Accepted Article
Title: Synthesis of New Cyclomarin Derivatives and Their Biological
Evaluation towards Mycobacterium Tuberculosis and
Plasmodium Falciparum
Authors: Alexander Kiefer, Chantal D. Bader, Jana Held, Anna Esser,
Jan Rybniker, Martin Empting, Rolf Müller, and Uli Kazmaier
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of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
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To be cited as: Chem. Eur. J. 10.1002/chem.201901640
Link to VoR: http://dx.doi.org/10.1002/chem.201901640
Supported by

10.1002/chem.201901640
Chemistry - A European Journal
DOI: 10.1002/chem.201xxxxxx
█ Natural Products
Synthesis of New Cyclomarin Derivatives and Their Biological
Evaluation towards Mycobacterium Tuberculosis and Plasmodium
Falciparum
Alexander Kiefer,[a] Chantal D. Bader,[b] Jana Held,[c] Anna Esser,[d] Jan Rybniker,[e] Martin
Empting,[f] Rolf Müller[b,g] and Uli Kazmaier* [a]
Abstract: Cyclomarins are highly potent antimycobacterial ning the structure-activity relationship (SAR) for both
and antiplasmodial cyclopeptides isolated from a marine pathogens. Herein, we report the synthesis and biological
bacterium (Streptomyces sp.). Previous studies have evaluation of 17 novel desoxycyclomarin-inspired analogues.
identified the target proteins and elucidated a novel mode of Optimization via side chain modifications of the non-canonical
action, however there are currently only a few studies exami- amino acids led to potent lead structures for each pathogen.
Introduction
Due to the increasing occurrence of antimicrobial resistance (AMR)
in the past 60 years, society is coming under immense pressure to
refocus on anti-infective research.[1] Unfortunately, continuous
improvement of established antibiotic classes is not enough to
overcome the prevailing resistance problem. Especially pathogens
known to develop mutations causing multiple-drug-resistance
(MDR) like Mycobacterium tuberculosis (Mtb) are only controllable
by innovative drugs with new modes of action, preferably
addressing new biological targets.[2-5] Microorganisms like bacteria
or fungi have proven a prolific source for the identification of novel
pharmacologically active and chemically diverse substances.[6-9]
Thus, natural products take a firm place in drug discovery, either
as direct drug candidates or as lead structures.[10-15]
A small family of cyclopeptides, isolated from the marine
streptomycete strain CNB-982, the cyclomarins A-C,[16] represent
a class of natural products with a remarkable antibacterial activity
against Mtb (Figure1) .[17] The mayor metabolite cyclomarin A
exhibits antitubercular activity against both growing and hypoxic
non-replicating intracellular mycobacteria in human-derived
macrophages in the low micromolar range. Sensitivity retains for
MDR strains, which implies a novel molecular target of the
compound family.[18] Target profiling studies based on this
hypothesis led to the identification of the caseinolytic protease C1
(ClpC1) as the point of intervention, whereby Shanda et al. recently
were able to demonstrate a blockade of the arginine-phosphate-
induced dynamics of the N-terminal domain (NTD) as the mode of
action for this antitubercular activity.[19] Additionally, cyclomarin A
shows an impressive activity against Plasmodium falciparum
(Pfalcp) with an inhibitory concentration in the low nanomolar
range.[20]
[a]
Dr. Alexander Kiefer, Prof. Dr. Uli Kazmaier
Organic Chemistry
Saarland University
Campus C4.2, D-66123 Saarbrücken, Germany
Fax: (+49)681-3022409
E-mail: u.kazmaier@mx.uni-saarland.de
[b]
Chantal D. Bader, Prof. Dr. Rolf Müller
Department Microbial Natural Products (MINS)
Helmholtz-Institute for Pharmaceutical Research Saarland (HIPS) -
Helmholtz Centre for Infection Research (HZI)
Campus E8.1, 66123 Saarbrücken, Germany
[c]
[d]
[e]
Dr. Jana Held
Department of Tropical Medicine
University of Tübingen
Wilhelmstraße 27, D-72074 Tübingen, Germany
Anna Esser, Priv.-Doz. Dr. Dr. Jan Rybniker
Center for Molecular Medicine Cologne
University of Cologne
Robert Koch Str. 21, D-50931 Cologne, Germany
Priv.-Doz. Dr. Dr. Jan Rybniker
Department I of Internal Medicine
University of Cologne
D-50937 Cologne, Germany and
German Center for Infection Research (DZIF)
Partner Site Bonn-Cologne, Germany
[f]
Dr. Martin Empting
Department of Drug Design and Optimization (DDOP)
Helmholtz-Institute for Pharmaceutical Research Saarland (HIPS) -
Helmholtz Centre for Infection Research (HZI)
Campus E8.1, 66123 Saarbrücken, Germany
[g]
Prof. Dr. Rolf Müller
Department of Pharmacy
Saarland University
Campus E8.1, 66123 Saarbrücken, Germany
Supporting information for this article is available on the WWW
under http://www.chemeurj.org/ or from the author.
Figure 1. Structure diversity of cyclomarins.
1
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10.1002/chem.201901640
Chemistry - A European Journal
DOI: 10.1002/chem.201xxxxxx
In contrast to the binding to the NTD of ClpC1, in this case,
cyclomarin A binds to a dimer of diadenosine triphosphate
hydrolases (PfAp3Aase) and, thereby, most likely prevents the
formation of the enzyme-substrate complex. The distinctive
bioactivities of the cyclomarins have turned great interest into the
synthesis of these natural products.[21] In 2004, the first total
synthesis of cyclomarin C was published by Yao et al.,[22,23] paving
the path for Barbie and Kazmaier for the first total synthesis of
cyclomarin A,[24] C and D[25] as well as the slightly simplified
derivative desoxycyclomarin C (Figure 1).[26]
Biological characterization of the natural products and some
semi-synthetic analogues at Novartis provided useful information
regarding the structure-activity relationship (SAR) of the cyclo-
peptides (Figure 2).[17,20] These studies reveal that removal of the
epoxide functionality on the tryptophan unit does not result in a loss
of activity, whereas substitution of the hydroxyl function of the
-hydroxyleucine moiety towards substituted amines abolishes
activity. We recently observed that desoxycyclomarin C, which is
missing the -hydroxy group on the tryptophan, is only slightly less
active compared to the hydroxylated natural products, and
modifications at the -methoxyphenylalanine unit are also well
tolerated.[27] Those rather limited SAR studies nevertheless did not
provide any information about the impact of side chain
modifications at the other non-canonical amino acids.
NTD (Figure 3a and 3b). Exploiting reported X-ray structures 5CS2
(PfAp3Aase)[20] and 3WDC (ClpC1 NTD)[18] we generated
respective models for desoxycyclomarin C (Figure 3), giving us a
rational for further chemical modifications. The residue on the
tryptophan, referred to as R’, is not in pronounced contact with
either of the targets. For PfAp3Ase the β-methyl group of the
threonine 110 side chain might form minor hydrophobic inter-
actions with the N’-inverse prenyl motif, while for ClpC1 NTD the
side chain of methionine 1 could act as a partner. Therefore, R’
might be subject to simplification attempts as it is rather solvent-
exposed in both complexes. In case of R’’, the unsaturated side
chain, pronounced contact is present when bound to PfAp3Ase
and broad changes can be expected to have an impact on activity.
For ClpC1 NTD we expected a different outcome, as in this case
the residue does not form any contacts with the protein at all.
Finally, the methyl group referenced by R’’’ does not have
obvious contacts with PfAp3Ase and might therefore be a good
candidate for removal. This is different when considering the
complex with ClpC1 NTD, as in this case R’’’ is literally buried by
the rest of the ligand and firmly packs with the indole moiety of the
tryptophan derivative. At this point, it should be mentioned that
above considerations are only valid in terms of target interactions
and do not take into account the rest of the pathogens complexity.
When measuring actual biological activities of each derivative,
further effects like reduced or increased uptake, efflux, and/or
biochemical degradation mediated by our envisioned modification
might also play a significant role.[32–34] Nevertheless, the portrayed
rational provided a target-driven approach for focused modifica-
tions in more peripheral portions of the desoxycyclomarin scaffold.
a) Interaction with PfAp3Aase
b) Interaction with ClpC1 NTD
Figure 2. Overview of available SAR conclusion.[17,20,27]
Since a couple of years, our group is involved in the synthesis
of peptidic natural products and derivatives thereof for SAR studies.
[28–31]
Herein, we report the design and synthesis of 17 desoxy-
cyclomarin-inspired derivatives and their biological evaluation
towards Mtb and Pfalcp.
Results and Discussion
Figure 3. (Top) Structure-guided rational for novel desoxycyclomarin
C
derivatives addressing Pfalcp target PfAp3Aase (left) and Mtb target ClpC1 NTD
(right). Both complex structures of desoxycyclomarin C were modelled based on
published cyclomarin A complexes 5CS2 and 3WDC, respectively. Residues R’,
R’’, and R’’’ are shown in yellow, other carbon atoms in white, nitrogen in blue,
and oxygen in red. Hydrogens and solvent molecules are left out for clarity.
(Bottom) Planned structure modifications based on desoxycyclomarin C.
For the generation of the compound library we focused on signify-
cantly simplifying three of the four non-canonical amino acids.
Before starting the synthesis, plausibility of the aspired modify-
cation was checked for both target proteins PfAp3Aase and ClpC1
2
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DOI: 10.1002/chem.201xxxxxx
Based on our previously published synthesis of the cyclo-
marins, macrolactamization was planned between the N-terminal
aminohexenoic acid (or L-valine) and the C-terminal N-methyl-L-
leucine, affording a linear heptapeptide precursor. The three
variable amino acids will be coupled stepwise to a linear
tetrapeptide,24 which can be synthesized in large scale (Figure 4).
group and saponification afforded N’-tert-pentyl tryptophan 10 in
88% yield over all steps (Scheme 1).
Figure 4. Retrosynthetic disconnection of the desoxycyclomarin-derived
analogues.
Thus, our investigations commenced with the preparation of a
first analogue library stepwise varying the N’-side chain of the
tryptophan moiety followed by simplifying the aminohexenoic acid
unit. While N’-methyl tryptophan is commercial available, a Negishi
reaction served as key step for the generation of N’-isopropyltrypto-
phan 6 (Scheme 1).[35,36] The required iodoindole 2 was prepared
via selective iodination in 3-position and subsequent N-alkylation
of indole 1. For the cross coupling, β-iodo-L-alanine 3[37,38] was
transferred into the organozinc species I, which was reacted with a
slight excess of iodoindole 2. Catalytic amounts of Pd2dba3 and
QPhos provided the best result for the formation of Boc-protected
tryptophan 4. At this point, only protecting group manipulations
remained to achieve modified building block 6. After acidic removal
of the Boc group, the corresponding hydrochloride was protected
with AllocCl in the presence of pyridine. Final saponification
resulted in N’-isopropyltryptophan derivative 6. It should be noted,
that the protecting groups were carefully chosen to prevent the
decomposition later on of the hexapeptide under acidic conditions,
whereas the Alloc protecting group allows removal under mild
palladium-catalyzed conditions.[39]
Scheme 1. Synthesis of N’-isopropyltryptophan 6 and N’-tert-pentyl tryptophan
10.
Key step of the syntheses of the 2-amino-3,5-dimethylhex-4-
enoic acid is the introduction of the dimethylated double bond via
olefination of the corresponding -branched aldehyde. This
aldehyde is rather sensitive toward epimerization, especially under
the basic reaction conditions of the olefination. Therefore, we
decided to prevent this critical step by removal of the (Z)-methyl
group and by introduction of the (E)-double bond via ester enolate
Claisen rearrangement (Scheme 2).[42] This protocol is especially
suited for the stereoselective formation of syn -unsaturated
amino acids either from simple chiral allylic alcohols,[43,44] or in the
presence of chiral ligands.[45,46] Here, an enzymatic kinetic
resolution of 3-penten-2-ol 11 using Novozym® 435 gave rise to the
required chiral allylic alcohol.[47,48] While separation by distillation
due to the similar volatility of the undesired (R)-acetate and
enantiomerically enriched (S)-alcohol failed, the mixture was
Synthesis of N’-tert-pentyl tryptophan 10 was achieved by Pd-
catalyzed reduction of the N’-tert-prenyl building block 7[40] under
hydrogen atmosphere (1 atm). Subsequent Fmoc cleavage using
tris(2-aminoethyl)amine,[41] introduction of the N-Alloc protecting
3
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10.1002/chem.201901640
Chemistry - A European Journal
DOI: 10.1002/chem.201xxxxxx
directly subjected to Steglich esterification[49] with Alloc-Gly-OH
(12). Flash chromatography provided the preferred (S,E)-N-Alloc
glycine ester 13 with an enantiomeric excess of > 99%. Final ester
enolate Claisen rearrangement of 13 proceeded with perfect
chirality transfer, providing the ,-unsaturated amino acid 14 with
excellent enantio- and diastereoselectivity.[42]
In the next series the iteration should take place at the fifth
amino acid, the hydroxylated leucine. Here, tetrapeptide 29[24] was
used as a key intermediate (Scheme 5). Acidic removal of the N-
terminal Boc-protecting group and coupling with building block 18
resulted in pentapeptide 30 in 88% yield. Subsequent cleavage of
the Cbz protecting group and BEP mediated coupling generated
the linear hexapeptides 31a-e. Palladium-catalyzed Alloc depro-
tection and coupling using EDC/HOBt provided the heptapeptides
32a-d and 33a,b,e. To finalize the cyclopeptide synthesis, the
linear precursors were converted by macrolactamization into the
desired derivatives 34a-d and 35a,b,e. Over four steps the final
compounds were obtained in good to excellent yields after
purification by preparative HPLC.
Scheme 2. Synthesis of simplified amino hexenoic acid building block 14.
Biological evaluation
To introduce an additional structural alteration in the peptide
backbone of our analogues, the -hydroxy leucine unit was
replaced by -hydroxy norvaline. Inspired by Kaiser et al. we
started the fragment synthesis with protected glutamic acid 15,
which was first activated as mixed anhydride and then reduced by
NaBH4 (Scheme 3).[50] Masking the resulting alcohol as TBS ether
yielded 16,[51] subsequent saponification and N-methylation
generated N-Methyl--hydroxy norvaline 18 almost quantitatively
in gram scale.
All 17 generated analogues were tested for their biological
activities against chloroquine sensitive Pfalcp strain 3D7 and multi
resistant strain Dd2 as well as against Mtb wild-type strain Erdman.
The antiplasmodial activity was determined via an in-vitro drug
sensitivity assay using an enzyme-linked immunosorbent assay
(ELISA) to quantify histidine-rich protein 2 (HRP2), which is
produced exclusively by Pfalcp.[54-56] Table 1 summarizes the
generated half maximal inhibitory concentrations (IC50) of the
synthesized analogues in comparison to desoxycyclomarin C and
chloroquine (CQ), representing
a therapeutic standard as
reference. Additionally, the analogues were also tested for their
antitubercular activity. The growth inhibition of Mtb was determined
by a resazurin reduction microtiter assay (REMA).[57] In this case,
isoniazid was used as reference. Minimal inhibitory concentrations
(MIC) of the compound library are summarized in table 1 as well.
In general, the studied desoxycyclomarin C scaffold delivered
activities in the low micro- or submicromolar range against Mtb and
Pfalcp, respectively.
As expected, certain modifications at R’ were nicely tolerated.
Especially reduction to an isopropyl group provides a promising
simplification retaining antimycobacterial activity. In general,
manipulations at this position do not result in dramatic effects on
potency measured against Mtb and Pfalcp as well. Interestingly,
the methyl group in 25d is an appropriate balance between the
reduction of synthetic complexity and loss of activity.
Results obtained for the modifications at R’’ are in good
agreement with the initial rational. In the case of Pfalcp, where this
residue is completely buried between the target and the ligand
scaffold, large changes are not accepted. However, it is important
that the removal of the terminal methyl group in cis position of the
 -unsaturated side chain led to an equivalent or even slightly
improved activity. This fits very well to the binding pose observed
in the reported X-ray structure. Further simplifications, however,
are not advisable, as they lead to dramatic losses in activity, which
is seen in the comparison of 25a and 28a.
Removal of methyl group at R’’’ led to decrease in potency
against both pathogens. This was indeed expected in case of the
Mtb target ClpC1 NTD. This group interacts well with the target,
which is also closely packed with the indole motif of the tryptophan
core. However, it is not clear yet, why deletion of this methyl
residue impairs activity towards Pfalcp. Based on the X-ray
structure (5CS2) this group is not in direct contact with the target
protein. But it is noteworthy that it is also packaged against the
indole moiety and might, hence, be important for conformational
constraints at this position.
Scheme 3. Synthesis of N-methylated δ-hydroxy norvaline 18.
With all required non-canonical amino acids in hand, we first
focused on assembling linear precursors which vary in their
tryptophan and aminohexenoic acid motif (Scheme 4). Thus, the
N-terminal Cbz group of 19[24] was cleaved via hydrogenation,
allowing the free amine to be coupled with the N-Alloc-tryptophan
derivatives to the corresponding hexapeptides 23a-e utilizing 2-
bromo-1-ethyl-pyridinium tetrafluoroborate (BEP).[52,53] Additional
structural diversity was introduced by elongation to the heptapep-
tide series 24a-e and 27a-e using either -unsaturated amino acid
derivative 14 or N-Alloc-L-Val-OH (26). To complete the synthesis,
the methyl ester at the C-terminus was saponified with lithium
hydroxide. Subsequently, the N-termini of the heptapeptides were
deprotected under mild Pd-catalysis. Macrolactamization of both
heptapeptide series was carried out as described by Yao et al.
using two equivalents of benzotriazol-1-yl-oxytripyrrolidinophos-
phonium hexafluorophosphate (PyBOP) and DIPEA under high
dilution conditions (1 mM).[22,23] For this purpose, the C- and N-
terminally deprotected peptides were slowly added to the coupling
reagent via transfer cannula. The final step, cleavage of the TBS
ether using TBAF, led to our first small library of 10 analogues.
4
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10.1002/chem.201901640
Chemistry - A European Journal
DOI: 10.1002/chem.201xxxxxx
Scheme 4. Peptide coupling and macrolactamization of the first cyclomarin analogues.
In the case of an anti-mycobacterial activity, a higher degree of
simplification is well tolerated when combined with the removal of
the substituent at R'. In particular, compound 28e retains high
activity against Mtb but is simplified in two non-canonical amino
acids. This improves synthetic accessibility and avoids cumber-
some synthetic steps of the otherwise required building blocks. In
addition, by varying and combining the different residues, we can
achieve a clear selectivity towards Mtb or Pfalcp. In light of these
excellent basal potencies minor activity tradeoffs are acceptable in
order to improve synthetic accessibility.
5
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Chemistry - A European Journal
DOI: 10.1002/chem.201xxxxxx
Scheme 5. Synthesis of the second set of cyclomarin analogues based on key intermediate 29.
Conclusion
Experimental Section
General Procedure: Synthesis of hexapeptides using BEP coupling reagent.
The corresponding pentapeptide was dissolved in MeOH (0.1 M) and 10% Pd-C
(10% on charcoal) was added. The mixture was stirred for three hours at room
temperature under H2 atmosphere (1 atm) until complete conversion (TLC). After
filtration through Celite® the filtrate was concentrated in vacuo. The residue was
dissolved in CH2Cl2 (0.5 M), mixed with the corresponding acid (1.05 equiv) and
cooled down to –20 °C. NMM (2.2 equiv) and BEP (1.1 equiv) were added
successively at this temperature. The reaction mixture was slowly warmed up
0 °C. After complete conversion (TLC), the solution was diluted with CH2Cl2 and
washed with water, sat. NaHCO3 solution and brine. The organic phase was dried
over Na2SO4 and the solvent was removed under reduced pressure. The crude
product was purified by column chromatography.
We have designed and synthesized a small library of 17 novel
cyclomarin analogues using a straightforward solution-phase
approach and have evaluated their antitubercular and
antiplasmodial activities to gain a deeper insight into the SAR.
Through the course of our studies, we identified three hit
compounds (25a, 25b and 25d) with excellent parasitic growth
inhibition. One of these compounds, 25d, harbors a simplified γ,δ-
unsaturated side chain and the commercially available N'-methyl
tryptophan unit. While the bioactivity is in the same range like the
natural product-inspired desoxycyclomarin C, the synthesis of this
greatly simplified analog is significantly shortened (six synthetic
steps less and provides a good option for the development of
antimalarial drugs. We also showed that five derivatives (25a, 25c,
25d, 28c and 28e) exhibit potent anti-mycobacterial activity against
Erdman wild-type strain. Due to a remarkable simplification by
replacing two of the four non-canonical amino acids by L-valine and
L-tryptophan, especially 28e represents a highly attractive natural
product-derived lead structure for combating Mtb.
General Procedure: Synthesis of heptapeptides using EDC/HOBt coupling
reagents. The corresponding hexapeptide was dissolved in MeCN/H2O (1:1, 0.1
M) and diethylamine (5.0 equiv), TPPTS (4 mol%) and Pd(OAc)2 (2 mol%) were
added at room temperature. After one hour (LC-MS control) the solvent was
removed under reduced pressure and the residue was dissolved in CH2Cl2 (0.5
M). After cooling to 0 °C, the corresponding acid (1.10 equiv), EDC·HCl (1.1
equiv), HOBt (1.0 equiv) and NMM (2.2 equiv) were added. The reaction mixture
was warmed up room temperature and after complete conversion (LC-MS)
diluted with CH2Cl2 and washed successively sat. NaHCO3 solution, 1 N KHSO4
6
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Chemistry - A European Journal
DOI: 10.1002/chem.201xxxxxx
Table 1. Biological activities of the new cyclomarin derivatives (see Figure 4) towards Pfalcp (chloroquine sensitive 3D7 and multi-resistant Dd2 strains) as
well as Mtb (Erdman wt strain) and overall steps involved in synthesis
R‘
R‘‘
R‘‘‘
overall synthetic
steps
Entry
Compound
Pfalcp
IC50[nM]
Mtb
MIC[µM]
IC50[nM]
3D7
Dd2
wt
ref Mtb
isoniazid
CQ
0.9
ref Pfalcp
3.4
233.9
NA
desoxycyclo-
marin C
1
2
Me
Me
39.8
0.9
0.5
37
34
25a
9.0
12.9
3
4
5
6
7
25b
25c
25d
25e
28a
Me
Me
Me
Me
Me
4.4
6.5
1.7
0.4
0.9
1.5
21.2
31
36
31
31
32
iPr
Me
H
47.8
13.4
28.1
34.4
76.0
17.8
27.5
71.8
8
28b
28c
28d
28e
34a
Me
Me
Me
Me
H
57.6
200.2
300.3
256.7
421.5
36.7
1.7
0.4
1.6
0.5
2.3
33
34
29
29
32
iPr
Me
H
9
355.7
230.3
314.2
47.9
10
11
12
13
14
15
16
34b
34c
34d
35a
H
H
H
H
452.0
177.5
362.4
303.5
346.5
287.8
318.4
260.8
>32
1.8
3.4
2.6
33
34
32
30
iPr
Me
17
18
35b
35e
H
H
428.5
287.4
305.1
196.7
3.2
4.3
31
26
H
solution and brine. The organic phase was dried over Na2SO4 and the solvent
was removed under reduced pressure. Flash chromatography afforded the
desired product.
CH2Cl2 (2mM). PyBOP (2.0 equiv) and DIPEA (2.2 equiv) were also dissolved in
CH2Cl2 (4 mM). The peptide solution was transferred to the coupling reagent
solution via transfer cannula within 6ours. After 24 h stirring at room temperature,
the reaction solution was washed with 1 N KHSO4 solution, sat. NaHCO3 solution
and brine. After drying over Na2SO4 and evaporation of the solvent, the residue
was dissolved in THF (0.5 M) and mixed with 2.2 equiv TBAF (1 M in THF) at
room temperature. After complete conversion (LC-MS), the solvent was removed
under reduced pressure and the residue was filtered via a short silica gel column
with EtOAc and then purified via preparative HPLC.
General Procedure: Cyclization and TBS deprotection. LiOH (1.5 equiv, 1.0
M in H2O) was added to a solution of a heptapeptide in THF (0.1 M) at 0 °C and
was stirred overnight at room temperature. After complete conversion (LC-MS),
the solvent was removed under reduced pressure the residue dissolved in
MeCN/H2O (1:1, 0.1 M) and treated at room temperature with diethylamine (5.0
equiv), TPPTS (4 mol%) and Pd(OAc)2 (2 mol%, 0.02 M in MeCN). After complete
conversion (LC-MS) the solvent was removed and the residue was diluted in
7
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10.1002/chem.201901640
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DOI: 10.1002/chem.201xxxxxx
Modeling
[7]
D. Reker, A. M. Perna, T. Rodrigues, P. Schneider, M.Reutlinger, B.
Mönch, A. Koeberle, C. Lamers, M. Gabler, H. Steinmetz, et al. Nat.
Chem. 2014, 6, 1072.
Molecular Operating System (MOE, version 2015.10, Chemical Computing
Group)[54] was used for all in silico operations. Models of protein-ligand
complexes between desoxycyclomarin C and both targets PfAp3Aase and ClpC1
NTD were generated based on the published crystal structures 5CS2 and 3WDC,
respectively, with closely related cyclomarin A as ligand. The unit cell of 5CS2
does not directly provide the shown binding mode of the cyclomarin scaffold to
the target and the crystal lattice had to be extended beyond the asymmetric unit
using the crystal builder function of MOE and unnecessary cyclomarin as well as
protein duplicates arising from this operation had to be removed. Then, the β-
hydroxy function on the tryptophan unit was removed and the epoxy motif was
replaced by a double bond. Subsequently, the built-in QuickPrep function with
standard parameters and AMBER10:EHT forcefield was applied to yield the
complex structure of desoxycyclomarin C bound to PfAp3Aase. In case of 3WDC,
ligand modifications were applied directly after loading the coordinates of 3WDC
into the software. These operations comprised removal of the β-hydroxy function
on the tryptophan core as well as removal of the linkage between Met1 and the
epoxide-bearing moiety. Then, the double bond feature was installed and the
superfluous oxygen was deleted without changing positions of the carbon atoms.
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Keywords: Cyclomarins • malaria • multiple-drug-resistance •
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9
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10.1002/chem.201901640
Chemistry - A European Journal
DOI: 10.1002/chem.201xxxxxx
Entry for the Table of Contents [Please choose one layout only]
FULL PAPER
Cyclomarins are highly potent
█ Natural Products
antimycobacterial and antiplasmodial
cyclopeptides isolated from a marine
bacterium (Streptomyces sp.). The
synthesis and biological evaluation of
17 novel desoxycyclomarin-inspired
analogues is reported. Side chain
modifications of the non-canonical
amino acids led to potent lead
Dr. Alexander Kiefer, Chantal D. Bader,
Dr. Jana Held, Anna Esser, Priv.-Doz.
Dr. Dr. Jan Rybniker, Dr. Martin
Empting, Prof. Dr. Rolf Müller, Prof. Dr.
Uli Kazmaier*
■■ – ■■
structures for each pathogen.
Synthesis of New Cyclomarin Deriva-
tives and Their Biological Evaluation
towards Mycobacterium Tuberculosis
and Plasmodium Falciparum
10
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