Scalable Syntheses of Methoxyaspartate and Preparation of the Antibiotic Cystobactamid 861-2 and Highly Potent Derivatives

Article abstract of DOI:10.1021/acs.orglett.9b03143

An improved scalable synthesis of orthogonally functionalized methoxyaspartate, the chiral hinge region element in cystobactamids, is reported. This improvement sets the stage for the total synthesis of four new cystobactamids along with cystobactamid 861

Full text of DOI:10.1021/acs.orglett.9b03143

Letter
pubs.acs.org/OrgLett
Cite This: Org. Lett. XXXX, XXX, XXX−XXX
Scalable Syntheses of Methoxyaspartate and Preparation of the
Antibiotic Cystobactamid 861‑2 and Highly Potent Derivatives
Malte Moell‡er,†,§ Matthew D. Norris,†,§,† Therese Planke,† Katarina Cirnski,‡ Jennifer Herrmann,‡
̈
Rolf Muller, and Andreas Kirschning*
†Institut fu
1B, 30167 Hannover, Germany
‡Abteilung Mikrobielle Naturstoffe, Helmholtz Institut fu
̈
̈
r Organische Chemie und Biomolekulares Wirkstoffzentrum (BMWZ) der Leibniz Universitat Hannover, Schneiderberg
̈
r Pharmazeutische Forschung Saarland, Helmholtz Zentrum fur
̈
̈
̈
Infektionsforschung und Universitat des Saarlandes, Campus E8.1, 66123 Saarbrucken, Germany
S
* Supporting Information
ABSTRACT: An improved scalable synthesis of orthogonally
functionalized methoxyaspartate, the chiral hinge region
element in cystobactamids, is reported. This improvement
sets the stage for the total synthesis of four new
cystobactamids along with cystobactamid 861-2, whose
antibacterial properties are determined and compared. The
cyano derivative of cystobactamide 861-2 shows superior
antibacterial activity against Gram-negative bacteria to any
natural cystobactamide tested so far.
Scheme 1. Recent Retrosynthetic Approaches to the
Methoxyaspartate Unit in Cystobactamids
he group of cystobactamids was reported by Muller et al. in
̈
T2014 as part of a search for new secondary metabolites in
Cystobacter sp. Cbv34. The extracts inhibited the growth of
several Gram-negative and Gram-positive bacteria, and HPLC-
assisted bioactivity-guided screenings provided cystobactamids
919-1 (1) and 919-2 (2) as active compounds (Figure 1).1
These oligoamides contain p-aminobenzoic acid building blocks
and either an iso-β-methoxyasparagine or a β-methoxyaspar-
agine unit. Later additional derivatives such as the cystobacta-
mids 920-1 (3), 920-2 (4), and 862-1 (5) were reported that
structurally differ in the D- and E-rings and the hinge region.2 To
date cystobactamid 862-1 is the most active natural member
inhibiting several clinically relevant Gram-positive and Gram-
negative strains (Acinetobacter baumannii: MIC = 0.5 μg/mL,
Citrobacter freundii: MIC = 0.06 μg/mL, carbapenem-resistant
E. coli WT-III marRΔ74bp: MIC = 0.5 μg/mL, carbapenem-
resistant P. aeruginosa CRE: MIC = 1.0 μg/mL and Proteus
vulgaris: MIC = 0.25 μg/mL)2 by inhibiting bacterial type IIa
topoisomerases. To date, three total syntheses of cystobactamids
861-2, 919-2, and 920-1 have been published by the Trauner
group and by us.2,3 These endeavors were essential to revise and
Figure 1. Structures of selected cystobactamids 919-1 (1), 919-2 (2),
920-1 (3), 920-2 (4), and 861-2 (5) and new derivatives 6 and 7
reported here (rings are labeled with letters A−E).
Received: September 4, 2019
© XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.9b03143
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
Scheme 2. Scalable and Facile Synthesis of the
Methoxyaspartate Derivative 18
Scheme 5. Finalization of the Synthesis of Cystobactamid
861-2 (5) and Derivatives 6 and 7
a
a
a
Boc = tert-butyloxycarbonyl.
a
Scheme 3. Preparation of Amides 23−25
a
EEDQ = N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline; HATU
= [O-(7-azabenzotriazole-1-yl)-N,N,N′,N′-tetramethyluronium-hexa-
fluorophosphate], DIPEA = di-isopropylethyl amine, DMF =
dimethylformamide.
a
TFA = trifluoroacetic acid.
a
Scheme 4. Synthesis of Triamide 30
Scheme 6. Synthesis of Truncated Cystobactamid Derivatives
36 and 37
The phenyl ring served as a masked carboxylic acid equivalent
which was liberated by Kuhn−Roth oxidation to finally yield
fully differentiated carboxylic acid 8.
a
DIPEA = di-isopropylethyl amine, pyr = pyridine.
Trauner’s approach commenced with (2R,3R)-tartrate 14
which was transformed into azide 13. Next, the diester was
transformed into the reactive anhydride 12, which served as
acylating agent for p-aminobenzoate bearing nucleophiles. This
step turned out to be unselective, as two electrophilic sites are
present in anhydride 12. The lack of regioselectivity was
overcome, as the resulting regioisomeric products converge to
the same regioisomer in the subsequent reaction.
As part of a medicinal chemistry program on cystobactamids,
which includes the preparation of larger amounts of a possible
lead structure, we now report on an improved synthesis of the
methoxyaspartate unit and include this synthesis in the total
synthesis of new cystobactamids 6 and 7.
prove the constitution and stereochemical configuration of the
group of cystobactamids.4
A structural key element is the (2S,3R)-methoxyaspartate
hinge element, which is embedded inside a larger oligoamide
chain that is composed of p-aminobenzoic acid building blocks.
To date two synthetic approaches toward the methoxyasparate
unit have been reported. One of the key synthetic issues is the
topic of orthogonality of functionalization with respect to the
two carboxylic acids and the nitrogen bearing functionality.
Our approach relied on methyl cinnamate 11 as starting
material, which was transformed into diol 10 utilizing the
Sharpless asymmetric dihydroxylation followed by standard
steps to introduce the nitrogen functionality as azide 9 (Scheme
1).
We had to focus our attention again onto the methoxy-
aspartate unit, as both published syntheses are flawed. Although
we achieved full differentiation of both carboxylic termini, the
Kuhn−Roth oxidation could only be conducted on scales up to
B
DOI: 10.1021/acs.orglett.9b03143
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Organic Letters
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Table 1. Antibacterial Activity of 6, 7, 36, and 37 Compared to Cystobactamid 861-2 (5) and Ciprofloxacin (CP) against a Panel
a
of Gram-Negative Bacteria and S. aureus
Strain
5
6
7
36
37
64
8
4
CP
S. aureus Newmann
E. coli BW25113
E. coli ΔacrB
1
0.125
≤0.03
≤0.03
≤0.03
≤0.03
1
0.125
0.125
≤0.03
0.125
0.25
8
>64
>64
>64
>64
>64
>64
>64
0.2
0.005
≤0.003
0.003
0.005
0.2
0.25
≤0.03
≤0.03
0.25
1
E. coli ΔtolC
2
E. coli DSM-1116
P. aeruginosa PA14
P. aeruginosa PA14 ΔmexAB
>64
>64
>64
0.25
0.125
2
0.01
a
Given are MIC values in μg/mL.
0.5 g for achieving best yields. Furthermore, our first generation
synthesis requires several chromatographic purification steps.
The Trauner approach is high yielding but reveals selectivity
issues due too improper differentiation of the two carbonyl
groups in anhydride 12. The modified approach that we present
here follows the first steps of the tartrate route in which azide 13
is a key intermediate (Scheme 2).
We optimized the first steps toward large scale application and
without the necessity of chromatographic purification (see
Supporting Information).5 After azide reduction, acidic
conditions led to hydrolysis of the two ethyl esters and the
reaction mixture was neutralized by addition of propylene oxide.
Then, position selective methylation of the carboxyl group that
is not adjacent to the protonated amino group yielded
monoester 17. Finally, the amino group was protected and the
methyl ester was directly transformed into the amide. It is worth
noting that this sequence can be performed on a multigram scale
starting from 30 g of tartrate 14, finally yielding 10 g of the chiral
building block 18. Furthermore, only one purification
(recrystallization) is necessary after the acidic hydrolysis of the
esters to separate the desired product from the side products
that are reagent-derived. We wish to stress that it is principally
possible to carry out the synthesis to methoxyaspartate
derivative 18 on a kilogram scale.
Next, we used building block 18 for the synthesis of
cystobactamid 861-2 (5) and two new derivatives 6 and 7 that
are modified in the A ring. We altered the mode of assembly in
that the western (rings A and B) and eastern arene units (rings C
to E) are added as fully assembled building blocks to the central
methoxyasparate unit 18. Our previously reported strategy was
rather lengthy in that ring C was attached first followed by ring B.
Then rings D and E were introduced in one step and finally ring
A.2
To achieve this, the corresponding amides 23−25 were
prepared first. These are available by coupling tert-butyl 4-
aminobenzoate 22 with benzoyl chlorides 19−21 under mild
conditions followed by tert-butyl ester hydrolysis (Scheme 3).
Triamide 30 was prepared by a newly developed modified
route compared to existing ones, with the goal of minimizing
purification steps and enabling large scale protocols (Scheme 4).
It commenced from the known tetrasusbtituted building block
26, which is avalable in six steps (17% overall yield) from 2,3-
dihydroxybenzaldehyde.2 The C- and E-ring (building blocks 22
and 19) were introduced in a repetitive manner activated as acyl
chlorides followed by reduction of the nitro groups in arenes 27
and 29, respectively, to yield the corresponding anilines 28 and
30. Indeed, this sequence can be performed on a multigram scale
with only one chromatographic purification step for 30 at the
very end of the sequence.
As was reported by Sussmuth for the structurally closely
̈
related albicidins,6 HATU and EEDQ are well suited for amide
formation of aromatic amides with α-amino acids. We utilized
both coupling agents too, EEDQ for linking triamide 30 to the
methoxyaspartate building block 18 and HATU for introducing
the diamide units 23−25 after removal of the Boc-group
(Scheme 5).
With these advanced synthetic products in hand, the
syntheses were finalized by Pd(0)-catalyzed cleavage of the
allyl ether group and acid-mediated hydrolysis of the tert-butyl
ester7 to afford cystobactamid 861-2 (5) and derivatives 6 and 7,
respectively. In our previous synthesis to cystobactamid 861-2
(5) we used allyl protection of the carboxylic acid in ring E. In
the present work, the tert-butyl ester was chosen as the
hydrolysis proceeds smoothly and purification of the final
cystobactamids is dramatically simplified. In fact, no chromato-
graphic purification is required as the final product is only
washed with diethyl ether. Finally, it should be noted that the
first amide coupling of the diamides 23−25 with the
methoxyasparate unit 18 followed by Boc-deprotection could
also be performed on a gram scale.
Finally, tetraamides 31 and 32, respectively, served to prepare
truncated cystobactamid 861-2 analogues 36 and 37 (Scheme
6). Conditions were applied that served to prepare the
cystobactamids 5−7 as desribed in Scheme 5, in order to test
the biological properties of structurally simpler cystobactamids.
The new cystobactamids 6 and 7 as well as the truncated
analogues 36 and 37 were tested against a panel of bacteria,
including intrinsically multiresistant Pseudomonas aeruginosa
(Table 1), and directly compared with cystobactamid 861-2 (5)
and ciprofloxacin (CP).
Both full-length cystobactamids show similar antibacterial
activity to the natural product 5. Remarkably, in the case of the
cyano derivative 6 a derivative is at hand, which is slightly more
active than the most active cystobactamid 5 isolated from
Cystobacter sp. Cbv34 to date and it shows comparable potency
to ciprofloxacin, the gold standard for antibiotics that inhibit the
bacterial gyrase.
In this panel of tested compounds, the trifluoro derivative 7 is
a less potent antibiotic, while the truncated cystobactamid
derivatives that lack the A and B rings (as in 36) or only the B
ring (as in 37) do not exert antibacterial properties anymore.
These results clearly show that the presence of the A and B rings
is essential and that substitution of the nitro group in
cystobactamid 861-2 (5) by cyanide leads to improved
antibacterial activity.8
In summary, we report an improved synthetic approach to
methoxy aspartate, the central chiral element present in the
cystobactamids, and provide a short and high yielding synthesis
of cystobactamid 861-2 (5). This synthetic endeavor paved the
C
DOI: 10.1021/acs.orglett.9b03143
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Organic Letters
Letter
̈
(8) The group of M. Bronstrup independently prepared cyanide 6 and
also found strong antibacterial activities: Testolin, G., Ph.D. thesis,
way to prepare several new cystobactamids with different
substitutions in the 4-position of ring A. Remarkably, the cyano
derivative 6 is the most potent cystobactamid derivative against
a panel of different Gram-negative pathogens, including
multiresistant mutants of bacterial pathogens reported to date.
This work opens the door for a broad medicinal chemistry
program on this class of natural antibiotics.
̈
Leibniz Universitat Hannover, 2018.
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.orglett.9b03143.
Detailed experimental procedures and spectral data
(PDF)
AUTHOR INFORMATION
Corresponding Author
■
*E-mail: andreas.kirschning@oci.uni-hannover.de.
ORCID
Andreas Kirschning: 0000-0001-5431-6930
Author Contributions
§M.M. and M.D.N. contributed equally to the work presented.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
This work was supported by the German Center for Infection
Research (DZIF) and the Bundesministerium fu
Forschung (BMBF; project OpCyBac). M.D.N. thanks the
Alexander-von Humboldt Foundation for a scholarship.
■
̈
r Bildung und
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DOI: 10.1021/acs.orglett.9b03143
Org. Lett. XXXX, XXX, XXX−XXX