10.1002/cbic.201800588
ChemBioChem
COMMUNICATION
Cladosporin Derivatives Obtained by Biotransformation Provide
Guidance for the Focused Derivatization of this Antimalarial Lead
Compound
Andreas Fredenhagen*[a], Kirsten Schroer[a], Harald Schröder[a], Dominic Hoepfner[b], Mathieu Ligibel[a],
Liliane Porchet Zemp[a], Caroline Radoch[a], Ernst Freund[a], Aldo Meishammer[a]
Abstract: Cladosporin, a natural product known for decades, has
recently been discovered to display potent and selective
antiplasmodial activity by inhibition of lysyl-tRNA synthetase. It was
subjected to a panel of oxidative biotransformations with one fungal
drawbacks of cladosporin as a drug candidate. For this purpose
an extensive biotransformation program was undertaken.
Cladosporin was incubated with 14 recombinant (rec.)
human (h) CYPs co-expressed in E. coli with rec. h P450-
reductase as described earlier, as well as with two bacterial
CYPs.[9,10] Only the bacterial CYP 102A1 produced larger
amounts of oxidation products (Table 1; other data are not
shown). In addition, it was incubated with 96 microbial strains,
approximately half of them fungi and half actinomycetes, that are
known for their ability to oxidize drug-like compounds. The
samples were analyzed with LC-MS/MS under conditions that
allowed the separation of all formed oxidation products with the
exception of compounds 8 and 9 that were differentiated by MS.
Strains with the highest conversion towards oxidized products
are compiled in Table 1.
and two actinomycetes strains and
a triple mutant bacterial
CYP102A1 yielding eight, mostly hydroxylated, derivatives. These
new compounds covered a wide chemical space and contained two
pairs of epimers in the tetrahydropyran ring. Although less potent
than the parental compound, all analogs showed activity in a cell-
based synthetase assay demonstrating uptake and on-target activity
in living cells with varying degrees of selectivity for the enzyme from
Plasmodium falciparum highlighting sites suitable for synthesis of
future cladosporin analogs. Compounds with adjacent hydroxyl
functions showed different MS/MS fragmentation that can be
explained by a, in some cases, regioselective loss of water followed
by a retro-Diels-Alder reaction.
From the screening experiments the Streptomyces aureofaciens
strain (ATCC 13189) was selected for a preparative production of
compound 2, A. niger (ATCC 11394) for compound 3 and Amycolata
autotrophica (ATCC 55293) for compounds 4, 5, 6 and 7. The
bacterial CYP 102A1 (also called BM3) is widely evaluated for the
production of human metabolites due to its high catalytic activity.[11] It
originates from Bacillus megaterium. The triple mutant
R47L/F87V/L188Q has three mutations close to the active site and
was designed by the group of Vermeulen for increased activity and
less substrate selectivity.[12] Moreover it is expressed in E.coli that
offers a higher cell wall permeability than B. megaterium. From
cladosporin wild type B. megaterium formed only compound 4 in
relatively low yield and CYP 102A1 formed 4 as well and moreover
compounds 8 and 9 (Table 1). The time course of production and the
structures suggested that 4 could be unstable upon exposure to
CYP102A1 and react further to 8 and 9. Therefore 4 and 8 were
incubated with CYP102A1 on analytical scale. Compound 4 reacted
within 3 hours to 48 % 8 and 17 % 9. Within the same time frame
ketone 8 reacted to 44 % 9. Thus a reaction sequence was
established whereby 9 was formed via 8 and both compounds from
compound 4.
Cladosporin, also called asperentin, was isolated from
Aspergillus
flavus
and
Cladosporium
cladosporioides
independently by Canadian and British researchers around
1970.[1-2] The relatively weak antibiotic activity was attributed to
the inhibition of uptake processes, notably the uptake of uracil
and leucine.[3] The absolute configuration of the center 3 was
assigned by comparison of the CD spectrum to the one of
mellein B and the absolute structure of the tetrahydropyran
moiety was determined by X-ray crystallography.[2,4] At that time
4’-hydroxyasperentin and 5’-hydroxyasperentin were isolated as
minor fermentation products, but the stereochemistry of the
hydroxyl function were not assigned.[2,5-6] Very recently the
structure of 5’-hydroxyasperentin was shown to be 5’(S) with X-
ray diffraction analysis.[7] Only recently cladosporin gained new
interest, as it was found to be
a potent and selective
antiplasmodial compound with a novel mode of action.[8] Its
potential as novel antimalarial lead compound raised the need to
have closely related compounds in hand to investigate the
structure-activity relationship and - hopefully - to overcome the
The biotransformation reactions were performed on a 0.6 liter to 2
liters scale and purification was achieved with reversed-phase
chromatography. From these four preparative biotransformation
reactions the following amounts were obtained: A total of 38.1 mg of
2 (from S. aureofaciens), 8.7 mg of 3 (from Asp. niger), 24.5 mg of 4
(from A. autotrophica and CYP102A1), 9.5 mg of 5 (from A.
autotrophica and CYP102A1), 15.8 mg of 6 (from A. autotrophica),
3.5 mg of 7 (from A. autotrophica), 11.2 mg of 8 (from CYP102A1)
and 1.6 mg of 9 (from CYP102A1) were obtained. NMR experiments
such as 1H, COSY, ROESY, DEPT-HSQC and HMBC allowed
elucidating the structure and relative configuration as discussed in
the supporting information (Scheme 1). Specifically 3 was identical
[a]
Dr. Andreas Fredenhagen, Dr. Kirsten Schroer, Dr. Harald
Schröder, Mathieu Ligibel, Liliane Porchet Zemp, Caroline Radoch,
Dr. Ernst Freund, Aldo Meishammer
Novartis Institutes for BioMedical Research, Global Discovery
Chemistry WKL-122.P.37, 4002 Basel, Switzerland.
E-mail: andreas.fredenhagen@novartis.com
PD Dr. Dominic Hoepfner, Novartis Institutes for BioMedical
Research, Chemical Biology & Therapeutics, 4002 Basel,
Switzerland
[b]
Supporting information for this article is given via a link at the end of
the document.
This article is protected by copyright. All rights reserved.