Synthesis and SAR of the antistaphylococcal natural product nematophin from Xenorhabdus nematophila

Article abstract of DOI:10.3762/bjoc.15.47

The repeated and improper use of antibiotics had led to an increased number of multiresistant bacteria. Therefore, new lead structures are needed. Here, the synthesis and an expanded structure–activity relationship of the simple and antistaphylococcal amide nematophin from Xenorhabdus nematophila and synthetic derivatives are described. Moreover, the synthesis of intrinsic fluorescent derivatives, incorporating azaindole moieties was achieved for the first time.

Full text of DOI:10.3762/bjoc.15.47

Synthesis and SAR of the antistaphylococcal natural product
nematophin from Xenorhabdus nematophila
Frank Wesche1,2, Hélène Adihou1,3,4, Thomas A. Wichelhaus5 and Helge B. Bode*1,6,§
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2019, 15, 535–541.
1Molekulare Biotechnologie, Goethe University Frankfurt,
Max-von-Laue-Str. 9, D-60438 Frankfurt am Main, Germany, 2present
address: Respiratory, Inflammation and Autoimmunity, Innovative
Medicines and Early Development, Biotech Unit, AstraZeneca,
Pepparedsleden 1, Mölndal 43183, Sweden, 3present address:
Cardiovascular and Metabolic Diseases, Innovative Medicines and
Early Development, Biotech Unit, AstraZeneca, Pepparedsleden 1,
Mölndal 43183, Sweden, 4present address: AstraZeneca MPI Satellite
Unit, Abteilung Chemische Biologie, Max Planck Institut für
Molekulare Physiologie, Otto-Hahn-Str. 11, 44227 Dortmund,
Germany, 5Institut für Medizinische Mikrobiologie und
doi:10.3762/bjoc.15.47
Received: 05 November 2018
Accepted: 13 February 2019
Published: 25 February 2019
Associate Editor: J. S. Dickschat
© 2019 Wesche et al.; licensee Beilstein-Institut.
License and terms: see end of document.
Krankenhaushygiene, Universitätsklinikum Frankfurt, Paul-Ehrlich-Str.
40, D-60596 Frankfurt am Main, Germany and 6Buchmann Institute
for Molecular Life Sciences (BMLS), Goethe University Frankfurt,
Max-von-Laue-Str. 15, D-60438 Frankfurt am Main, Germany
Email:
Helge B. Bode* - h.bode@bio.uni-frankfurt.de
* Corresponding author
§ Phone: 0049-69-798-29557
Keywords:
azaindole; fluorescent dye; MRSA; nematophin; Staphylococcus
aureus
Abstract
The repeated and improper use of antibiotics had led to an increased number of multiresistant bacteria. Therefore, new lead struc-
tures are needed. Here, the synthesis and an expanded structure–activity relationship of the simple and antistaphylococcal amide
nematophin from Xenorhabdus nematophila and synthetic derivatives are described. Moreover, the synthesis of intrinsic fluores-
cent derivatives, incorporating azaindole moieties was achieved for the first time.
Introduction
Microorganisms present a rich source of bioactive natural prod- which live in symbiosis with soil-living nematodes of the
ucts of pharmacological importance against an emerging genera Steinernema [2,3]. During a complex life cycle the
number of multiresistant bacteria [1]. Such examples are nematode–bacteria pair infects and kills insect larvae, whereby
Xenorhabdus sp., Gram-negative entomopathogenic bacteria Xenorhabdus produce a broad range of natural products with
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Beilstein J. Org. Chem. 2019, 15, 535–541.
antimicrobial properties [4-8]. As the Steinernema–Xeno- substitution of side chains in the α-keto carboxylic acids to
rhabdus complex is not pathogenic against humans, they are generate more derivatives of this fascinating small and bioac-
widely used as biocontrol agents in agriculture [9].
tive amide.
Natural products produced by bacteria play an important role in Results and Discussion
the bacteria/nematode/insect life cycle and most natural prod- We first synthesized 1 and 1-methylnematophin (3) as stan-
ucts are non-ribosomal peptides (NRP), e.g., rhabdopeptides dards to confirm preliminary results. We then initiated the syn-
[10,11] and polyketide–NRP hybrids, like xenocoumacins thesis of derivatives 2, and 4–12. Briefly, the appropriate α-keto
[12,13]. The quite simple amide nematophin (1) is another well- carboxylic acid was coupled to the respective amine. Amide
known member of natural products common in all Xenorhabdus bond formation was achieved using 1-ethyl-3-(3-dimethyl-
nematophila strains and was first described by Li et al. in 1997 aminopropyl)carbodiimide hydrochloride (EDC·HCl),
for their antimicrobial properties [14]. Simply, 1 is the conden- 1-hydroxybenzotriazole (HOBt) and N,N-diisopropylethyl-
sation product of 3-methyl-2-oxopentanoic acid and tryptamine amine (DIPEA) in DMF under microwave irradiation
and showed good activities against different Gram-positive (Scheme 1). Racemic 1 was synthesized using tryptamine and
pathogens like Staphylococcus aureus, including methicillin- (rac)-3-methyl-2-oxopentanoic acid. Similarly, compounds 2–7
resistant S. aureus (MRSA) comparable with activities of were synthesized using different amines including 2-phenyl-
vancomycin. Recently, the biosynthesis of 1 was elucidated by ethylamine (for 2), 1-methyltryptamine (for 3), 2-(2-
our group as well as the 2-phenylethylamine derivative 2 with naphthyl)ethylamine hydrochloride (for 4), 2-(1-naphthyl)ethyl-
an α-keto amide moiety, which could be identified upon hetero- amine hydrochloride (for 5), 2-(1H-inden-3-yl)ethylamine
logous expression of the appropriate gene cluster in E. coli [15]. hydrobromide (for 6) [21], and 2-(1-benzofuran-3-yl)ethyl-
Moreover, elongated nematophin derivatives, namely neval- amine (for 7), respectively. Compounds 8–12 were synthesized
tophins from Xenorhabdus PB62.4, were described incorporat- by coupling tryptamine with different α-keto carboxylic acids,
ing an additional valine. As 1 and nevaltophines act as pro- including 3-furylglyoxylic acid (for 8), 3-indoleglyoxylic acid
phenoloxidase activators, it is suggested that they have a specif- (for 9), phenylglyoxylic acid (for 10), and isomeric
ic role in the bacteria/nematode/insect symbiosis.
3-methylpent-2-enoic acid (for 11 and 12) [22], respectively.
Compounds 11 and 12 were separated during the isolation
Little or nothing is known about the mode of action of this process and initially constructed to enable target identification.
simple amide against S. aureus. Structure–bioactivity studies Compounds 11 and 12 might act as Michael acceptor (α,β-
revealed that the α-keto moiety and the amide moiety itself are unsaturated carbonyl) and attach irreversibly and covalently to a
required for bioactivity [16]. These findings suggest a specific potential target [23]. After purification and characterization, the
interaction of this electrophilic moiety with a nucleophile. Such above mentioned compounds were tested against different path-
an interaction was previously reported for the macrocyclic ogenic strains, i.e., methicillin-susceptible S. aureus (MSSA),
peptide cyclotheonamide A, isolated from marine sponge methicillin-resistant S. aureus (MRSA), Enterococcus faecalis,
Theonella sp. Cyclotheonamide A is described as a potent in- and Micrococcus luteus. The yields and bioactivities are sum-
hibitor of various proteases, in particular trypsin and thrombin marized in Table 1 (and Table S1, Supporting Information
[17-19]. Hereby, the α-keto amide covalently binds to the serine File 1).
oxygen in the active site under formation of a stable tetrahedral
hemiketal. Furthermore, substitution of the indole hydrogen by
alkyl, aryl or benzyl improves the in vitro antistaphylococcal
activity. In contrast, the incorporation of smaller heterocycles
like pyridine and imidazole as well as isosteric benzimidazole
instead of the indole moieties lead to a loss of antibacterial ac-
Scheme 1: General procedure for the synthesis of nematophin and
tivity. Kennedy et al. could synthesize a 2-phenyl derivative
that showed nanomolar activity against S. aureus [20]. To the
detriment of this compound class, all derivatives lose their anti-
bacterial activity in the presence of serum in vitro in serial broth
related derivatives. i) 1.5 equiv α-keto carboxylic acid, 1.0 equiv amine,
1.5 equiv EDC·HCl, 1.5 equiv HOBt, 2.0 equiv DIPEA in DMF
(c 0.1 M), 25 W, 75 °C, 20 min.
and agar dilution method [16,20]. Even with the use of charged As previously described, the minimum inhibitory concentration
groups as modifiers, serum-protein binding could not be (MIC) of 1 improves by alkylation of the indole moiety as seen
avoided. However, we were interested in expanding the afore- for 3 [16] even 1 and 3 show a good activity against MRSA. Al-
mentioned structure–activity studies regarding the substitution though for all other tested compounds no better activity than
of the indole moiety by different aromatic systems as well as that of the original natural product 1 was observed, 6 showed an
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Beilstein J. Org. Chem. 2019, 15, 535–541.
Table 1: Summary of synthesized nematophin derivatives (1–12) and their bioactivity (MIC in µg/mL) against S. aureus (MSSA and MRSA).
MIC (µg/mL)b
S. aureus (MSSA)
ATCC 29213
MRSA (MRSA)
ATCC 43300
structure
yielda
46%
0.5
0.5
32
1
2
56%
74%
16
0.125
0.031
3
4
48%
70%
2
1
4
4
5
6
26%
54%
59%
84%
1
1
>128
>128
>64
>64
7
8
9
64
>64
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Beilstein J. Org. Chem. 2019, 15, 535–541.
Table 1: Summary of synthesized nematophin derivatives (1–12) and their bioactivity (MIC in µg/mL) against S. aureus (MSSA and MRSA).
(continued)
47%
59%c
59%c
8
8
10
11
12
>128
>128
>128
>128
aPurification using column chromatography on silica gel. bMedian of three experiments. cDerivatives were synthesized using an isomeric mixture of
3-methylpent-2-enoic acid.
activity comparable to 1 against MRSA. Since only a slight ac- converted in a Friedel–Crafts acylation with chloroacetyl chlo-
tivity could be observed for some compounds against addition- ride (ClCH2COCl) and aluminium chloride (AlCl3) in DCM to
ally tested bacteria, i.e., M. luteus ATCC 9431 and E. faecalis give compounds 14 and 21. Subsequent reduction was achieved
ATCC 29212, a staphylococcal-specific target is suggested. All with triethylsilane (Et3SiH) in TFA to give 15 and 22. For the
derivatives with α-keto-β-methylvaleric moiety (1–7) were synthesis of the primary amine, halides 15 and 22 were con-
active against S. aureus. Substitution of the side chain in the verted in a Gabriel synthesis with potassium phthalimide in
α-keto amide significantly affects the bioactivity, as seen for DMF to the appropriate phthalimides 16 and 23 [29]. These
compounds 8, 9 and 10. Furthermore, attempts to identify a intermediary compounds 16 and 23 also allowed an N-methyla-
possible target by the incorporation of an α,β-unsaturated car- tion of the azaindole moiety with sodium hydride (NaH) and
bonyl moiety, as it should bind irreversible to a potential target, methyl iodide (MeI) to yield 17 and 24. By ethanolic hydrazi-
instead of the α-keto amide in 11 and 12 are stalled as it led to a nolysis and microwave irradiation the phthalimides (16, 17, 23,
complete loss of bioactivity.
and 24) were deprotected yielding the desired primary amines
in good yields (18, 19, 25, and 26) (Scheme 2 and Scheme 3).
To the best of our knowledge, no one has ever tried to incorpo- With all building blocks available, the synthesis of the appro-
rate azaindole moieties in small natural products and tried to use priate fluorescent derivatives was performed as before, using
them for fluorescence imaging. This approach may circumvent (rac)-3-methyl-2-oxopentanoic acid. Yields after coupling and
the above-mentioned impasse and should allow at least localiza- bioactivities are summarized in Table 2.
tion studies in S. aureus cells. Azaindoles are isosteric to indole,
whereas one of the endocyclic methines is substituted with Whereas the biological data, for example against S. aureus or
nitrogen, and thus leads to an increased and red-shifted fluores- MRSA shows a decreased activity for all azaindole derivatives
cence [24-26]. Budisa and co-workers have already used (compare 1 with 27 or 28) as observed for isosteric benzimida-
azatryptophans to study proteins with intrinsic green and blue zoles [20], an increased activity for all methylated derivatives
fluorescence [27,28]. The azaindole moiety allows a linker-less (compare pairs of 1/3, 27/28, and 29/30) could be observed for
incorporation of a fluorescent label with minimal disturbance.
at least MSSA. This observation may correlate to the molecular
polarity, and thus hydrophilicity of the cyclic moiety, whereas
Therefore, four fluorescent derivatives of nematophin were de- almost all hydrophobic moieties showed good antistaphylo-
signed and their synthesis initiated. The syntheses of the appro- coccal activity as seen for naphthyl (4 and 5) and indene (6).
priate azatryptamine derivatives (17, 18, 25, and 26) were However, the increase of hydrophobicity influences the passive
achieved from the non-expensive and commercially available 4- membrane diffusion and therefore might influence how the
and 7-azaindole (13 and 20), respectively. First, 13 and 20 were compounds get to their actual target [30]. Moreover, a specific
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Beilstein J. Org. Chem. 2019, 15, 535–541.
Scheme 2: Synthesis of azatryptamines (4-azatryptamine (4ATRA) and 1-methyl-4-azatryptamine (1M4ATRA)). i) 5.0 equiv AlCl3, 5.0 equiv chloro-
acetyl chloride in DCM, overnight, rt 67%; ii) 7.0 equiv Et3SiH in TFA, overnight, rt, 93%; iii) 1.1 equiv potassium phthalimide, in DMF, 100 °C, 21%;
iv) 1.2 equiv NaH, 1.0 equiv MeI in DMF, overnight, rt, 62%; v) 5.0 equiv N2H4·H2O in EtOH, 90 °C, 2 h, 25 W, 99% to quant.
Scheme 3: Synthesis of azatryptamines (7-azatryptamine (7ATRA) and 1-methyl-7-azatryptamine (1M7ATRA)). i) 5.0 equiv AlCl3, 5.0 equiv
ClCH2COCl in DCM, overnight, rt 32%; ii) 7.0 equiv Et3SiH in TFA, overnight, rt, 93%; iii) 1.1 equiv potassium phthalimide, in DMF, 100 °C, 56%;
iv) 1.2 equiv NaH, 1.0 equiv MeI in DMF, overnight, rt, 57%; v) 5.0 equiv N2H4·H2O in EtOH, 2 h, 90 °C, 25 W, 92% to quant.
minimal size of the cyclic moiety must be fulfilled as, e.g., 2 imaging. Furthermore, derivatives 3 and 6 will be further
with a phenyl moiety is less active in vitro.
studied for identification of their actual target in S. aureus. This
could be a serine protease that can also be addressed in the
future by more stable compounds. Moreover, it might be
Conclusion
Despite their lower bioactivities, the azaindole derivatives are possible to use nematophin and its derivatives for topical treat-
currently under investigation upon their use for fluorescence ment of S. aureus infections.
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Beilstein J. Org. Chem. 2019, 15, 535–541.
Table 2: Summary of synthesized nematophin derivatives (27–30) and their bioactivity (MIC in µg/mL) against S. aureus (MSSA and MRSA).
MIC (µg/mL)b
S. aureus (MSSA)
ATCC 29213
MRSA (MRSA)
ATCC 43300
structure
yielda
32%
32
16
16
4
64
>128
64
27
16%
72%
28%
28
29
32
30
aPurification using column chromatography on silica gel. bMedian of three experiments.
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[https://www.beilstein-journals.org/bjoc/content/
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ORCID® iDs
Frank Wesche - https://orcid.org/0000-0001-6340-3155
Helge B. Bode - https://orcid.org/0000-0001-6048-5909
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