SAR Studies of the Leupyrrins: Design and Total Synthesis of Highly Potent Simplified Leupylogs

Article abstract of DOI:10.1002/chem.202002622

Leupyrrins are highly potent antifungal agents. A structure–activity-relationship study of natural and synthetic derivatives is reported which reveals important insights into the biological relevance of several structural subunits leading to the discovery

Full text of DOI:10.1002/chem.202002622

Accepted Article
Accepted Article
Title: SAR Studies of the Leupyrrins: Design and Total Synthesis of
Highly Potent Simplified Leupylogs
Authors: Paul R. Wosniok, Christopher Knopf, Sandra Dreisigacker, J.
Manuel Orozco-Rodriguez, Bettina Hinkelmann, Peter Paul
Müller, Mark Brönstrup, and Dirk Menche
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To be cited as: Chem. Eur. J. 10.1002/chem.202002622
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01/2020

10.1002/chem.202002622
Chemistry - A European Journal
COMMUNICATION
SAR Studies of the Leupyrrins: Design and Total Synthesis of
Highly Potent Simplified Leupylogs
Paul R. Wosniok,[a] Christopher Knopf,[a] Sandra Dreisigacker,[a] J. Manuel Orozco-Rodriguez,[b] Bettina
Hinkelmann,[b] Peter P. Mueller,[b] Mark Brönstrup,[b] and Dirk Menche*[a]
Abstract: Leupyrrins are highly potent antifungal agents.
A
structure-activity-relationship study of natural and synthetic
derivatives is reported which reveals important insights into the
biological relevance of several structural subunits leading to the
discovery of highly potent but drastically simplified leupylogs that
incorporate a stable and readily available aromatic side chain. For
their synthesis a concise strategy is described that enables a short
and versatile access.
The leupyrrins present structurally complex macrodiolides from
Sorangium cellulosum.[1] As shown for leupyrrins A1 (1) and B1 (2,
Figure 1) they are characterized by an 18 membered
nonsymmetric cyclic core with an unusually substituted
ꢀ-butyrolactone ring together with a pyrrole and an oxazoline in
combination with a unique dihydrofuran side chain containing
two exocyclic alkylidenes. They exhibit not only very potent
antifungal activities but also antiproliferative and anti-HIV-
properties.[1, 2] So far the drug target has not been identified but
conventional targets are not addressed, which may suggest that
an unusual biochemical interaction site(s) may be involved.[1]
However, the further exploration has been severely hampered
by the low availability, in combination with their pronounced
structural complexity and a lability of the diene residue in the
side chain that is prone to double bond shifts. We have recently
isolated natural leupyrrins, determined their full stereochemistry
and developed a modular synthetic strategy which culminated in
the total synthesis of leupyrrins A1 and B1 (1 and 2, see Figure
1).[3–5] Herein, we report first detailed SAR studies with five
natural and six synthetic analogues that vary in the side chain,
the furan, the oxazoline, the pyrrole moieties and the diacid
fragment. Dramatically simplified leupylogs of type 3 and 4 were
developed that are characterized by a simplified aromatic side
chain and that retain potent biological activities (3).
Figure 1. Leupyrrin A1 (1) and structure related leupylogs 3 and 4. The
structural modifications are highlighted in red.
Despite considerable progress in leupyrrin total synthesis,[3-5]
preparation of the bis-alkylidene substituted furane moiety has
remained a challenge, due to the lability of this subunit and the
high number of synthetic steps required, despite the
advancement of a concise a one-pot process. Therefore, a
prime focus of this study was placed on simplification and
stabilization of this segment. As shown in Figure 1, it was
envisioned that the alkyliden units may be stabilized by
incorporation into an aromatic moiety. Furthermore, biological
activity tolerance for side chain variants of natural leupyrrins
(see below) also suggested that the aliphatic residue may also
be simplified. In combination, this led to design of analogues 3
and 23 (Scheme 1)
.
For their synthesis, the basic strategy developed during
leupyrrin total syntheses was applied, involving an sp2-sp3 cross
coupling to forge the C6-C7 bond, a cyclodehydration for
oxazolin condensation (C13-C14) in combination with an
esterification (C4’-C19) and a final macrolactonisation (C1’-C3).
For implementation of this approach, Eastern fragments 10 and
11 were required. They were obtained in a short approach from
known epoxyalcohols 5[6] and 6,[7] by coupling with diacid 7[5]
using the Shiina protocol[8] and regio- and stereoselective
epoxide opening of derived esters 8 and 9 under Brønsted
acidic conditions with NH4Cl and NaN3 as a nitrogen source
(Scheme 1).[9] Small amounts of likewise obtained C2-esters
could be removed by chromatography. Subsequent azide
reduction by hydrogenation in presence of Pd/C gave amino
alcohols 12 and 13 which may be used without further
purification. Alternatively, also a Staudinger reduction with
polymer-bound triphenylphosphine may be applied, leading to
quantitative yields for 12 (see SI section). For further elaboration,
azabenzotriazole 16 was prepared from pyrrole 14 and -lactone
15 following our previously reported procedure,[3-5] involving a
[a]
Dr. P. R. Wosniok,[+] C. Knopf,[+] Dr. S. Dreisigacker,[+] Prof. Dr. D.
Menche,
Kekulé-Institut für Organische Chemie und Biochemie
Universität Bonn
Gerhard-Domagk-Str. 1
D-53121 Bonn, Germany
E-mail: dirk.menche@uni-bonn.de
Respective contributions: PW: synthesis of leupylogs 3, 23 and 50;
CK: synthesis of 4 and 36; SD: isolation of natural leupyrrins and
synthesis of 56.
J. M. Orozco-Rodriguez, B. Hinkelmann, Prof. Dr. P. P. Müller, Prof.
Dr. M. Brönstrup,
[+]
[b]
Helmholtz Centre for Infection Research
Inhoffenstraße 7
D-38124 Braunschweig, Germany
Current address:
[c]
Dr. P. R. Wosniok: Eurofins Umwelt West GmbH Wesseling,
Germany
Dr. S. Dreisigacker: Pharmaceutical Industry, Germany.
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10.1002/chem.202002622
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well-scalable tandem sequence for 15. Subsequent amide
formation of 16 with either 12 and 13 under basic conditions
followed by cyclodehydration with DAST[10] gave oxazolines 17
and 18 in high yield (90 and 87%). For conversion to seco-acids
19 and 20, cleavage of the tert-butyl esters was realized with
TMSOTf/lutidine[11] followed by selective acetate saponification
using K2CO3 in a THF/MeOH/H2O mixture. Macrolactonization
was then realized by applying the Shiina protocol[8] under high
dilution conditions to give macrolactons 21 and 22, and finally
TASF mediated deprotection yielded the desired leupylogs 3
and 23.
for leupyrrin synthesis[5] involving Pd(PtBu3)2, Cs2CO3 in
THF/H2O were directly applicable to access 26 in 99% yield,
coupling with thiophene 25 required adjustment of parameters.
Finally, optimal results to obtain 27 in quantitative yield were
realized with Pd(PPh3)4 in presence of K3PO4 in THF/water at
60 °C.[13] Subsequently, removal of the tert-butyl ester with
TMSOTf/lutidine,[11] conversion of derived crude acids to active
esters 28 and 29 and cyclodehydration[3] with amine 12 gave
oxazolines 30 and 31 in high yield. In contrast to previously
reported route (Scheme 1), application of conditions for
cleavage of tert-butyl ester (TMSOTf) and acetate esters
(K2CO3) were unreliable or led to decomposition of 30 and 31.
After considerable attempts,
a
deprotection sequence
incorporating magnesium methoxide[14] for acetate cleavage at
0°C and zinc bromide[15] in dichloromethane at room
temperature for tert-butyl removal enabled a reliable access to
seco-acids 32 and 33. Subsequent Shiina macrolactonization
gave macrocycles 34 (73%) and 35 (94%) in a good and
excellent yield. Finally, removal of the remaining primary TBS-
group proceeded smoothly by utilizing TBAF in THF at 0 °C to
reveal leupylogs 4 in 96% and 36 in 61% yield, which proved
more reliable as compared to TASF.
Scheme 1. Concise three-step synthesis of simplified Eastern building
blocks 12 and 13 and completion of the synthesis of leupylogs 3 and 23.
Encouraged by the potent biological activities of these two
analogues (see below), we opted for further modifications and
studied the biological role of the pyrrole by replacing it with a
furan and a thiophen, giving analogues 4 and 36. As shown in
Scheme 2, their synthesis was based on initial cross coupling of
bromides 24 and 25[12] with 15. While the conditions developed
Scheme 2. Synthesis of Western fragments 26 and 27 and completion
of the synthesis of leupylogs 4 and 34.
Inspired by a certain remaining activity of 4 in combination with
a high biological tolerance of the diester fragment observed for
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natural leuyprrins, more truncated leupylog 51 (Scheme 3) was
targeted, which contains an oxazol instead of the oxazoline and
a simplified diacid building block. For its synthesis, a modified
Northern building block (43) was required. Its synthesis was
initiated by treatment of α-chloro-β-keto-ester 38[16] with a
mixture of commercially available acid (37) and Hünig’s base in
acetonitrile[17] to give β-keto-ester 39. Subsequent microwave
assisted heating in acetic acid in presence of ammonium
trifluoroacetate[18] gave desired oxazole 40 together with
imidazole 41, which could be removed by chromatography.
After reduction of ester 40 with DIBAL-H, resulting alcohol was
protected as acetate 42. Selective side chain bromination with
NBS then gave furfuryl bromide 43 (80%) together with small
amount of 44 (7%), derived from double bromination.[19] This
side product could be removed at a later stage of aldehyde
stage (46, see SI section).
before full conversion by addition of saturated NH4Cl. This
strategy gave mainly desired diol 47 together with variable
amounts of easily separable mono-deprotected 48, which may
be submitted to another round of ester cleavage. Gratifyingly,
the pivotal one-pot macrolactonization of diol 47 could then be
realized with succinic acid anhydride (49) applying the Shiina
protocol,[8] treating a highly diluted solution of anhydride 49,
MNBA and DMAP in dichloromethane very slowly over several
hours with a solution of diole 47 giving the desired macrodiolide
50 in respectable yield (44%), considering the synthetic
challenge of this tandem process. Finally, removal of the
remaining TBS-group proceeded smoothly with TBAF buffered
with AcOH to yield truncated leupylog 51.
Biological assays
Antifungal activities were first assayed for natural leupyrrins
1, 2, 52−55 and triol 56 (Figure 2), which was obtained by
saponification of leupyrrin B1 (see SI-section).
Figure 2. Overview of natural leupyrrins 52−55 and triol 56.
As outlined in Table 1, all natural leupyrrins show similarly
highly efficacious fungal cell growth inhibitory effects in the low
µg/mL range towards the yeast Rhodotorula glutinis, and the two
molds Botrytis cinerea and Mucor hiemalis (both of which are
important plant pathogens) in agreement with the data originally
published for 1.[1] In addition, none of them showed activity
against the yeast Saccharomyces cerevisiae or the Gram-
positive bacterium Staphylococcus aureus.
Scheme 3. Synthesis of Northern building block 43 and completion of
the synthesis of leupylog 51.
The next step required a challenging sp2-sp3 cross coupling with
boronate 15. Gratifyingly, by applying our initially optimized
conditions[5] (see above), the desired product 45 could be
isolated in 58% yield. For introduction of symmetric diacid a
one-pot esterification-macrolactonization was then evaluated,
requiring diol 47. Subsequent saponification studies to access
47 from 45 using K2CO3 in MeOH indicated the primary alcohol
to be cleaved first, followed by slower liberation of the
secondary hydroxyl group. However, as extended reaction
times led to unexpected decompositions, it proved beneficial to
keep them very short (6 minutes) and to stop the reaction
These data suggest that slight modifications of the diacid
fragment and aliphatic side chain have only a minor effect on
antifungal activity. On the other hand, triol 56 showed no activity
against all tested bacterial and fungal organisms, revealing the
whole macrocycle to be essential.
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glabrata that is resistant against natural leupyrrins, was also
resistant to 23 (see SI section). While the insensitive organisms
are all capable of fermenting glucose, members of the genus
Table 1. Biological activity of natural leupyrrins (1, 2, 52−55) and Triol 56.
MIC [µg/mL]
Rhodotorula
possess
a
respiratory
(non-fermentative)
[a]
A1
A2
B1
B2
C
D
Triol
metabolism. These differences in metabolism might account for
the variations in susceptibility observed. In order to assess
whether leupylogs have a specific antifungal activity, or also
affect the growth of mammalian cells, the natural leupyrrins 1
and 2 and synthetic leupylogs 3, 4, 23, 36 and 51 were tested in
an antiproliferative assay with the murine fibroblast cell line L929.
Natural leupyrrins 1 and 2 and antifungal leupylogs 3, 23 and 51
also exhibited antiproliferative potencies with similar IC50 values,
which indicates that the molecular target is likely to be
evolutionary conserved for these simplified analogs. In contrast,
leupylogs 4 and 36 were inactive, but also had poor antifungal
activity.
Compound
(1)
(53)
(2)
(54)
(52)
(55)
56
Rhodotorula
glutinis
0.25
0.47
0.47
0.97
0.78
0.47
>40
Botrytis cinerea
Mucor hiemalis
0.80
0.30
1.25
0.24
1.25
1.25
1.25
0.31
1.25
0.47
1.25
0.31
>40
>40
Saccharomyces
cerevisiae
n.d.
n.d.
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
>40
Staphylococcus
aureus
In summary, a simplified series of leupyrrin analogues has
[a] Value from ref. [1].
been developed which are characterized by
a drastically
simplified aromatic side chain. A first set of relevant SAR data
for the leupyrrins has been obtained with natural and synthetic
derivatives, focusing on variations of the macrocyclic ring, the
pyrrole and oxazoline fragment, the side chain and the diacid
We then evaluated antifungal activities of leupylogs 3, 4, 23,
36 and 51 in direct comparison to leupyrrins A1 (1) and B1 (2), as
well as with Amphotericin B (AmB) as a control using the yeasts
Candida albicans, a human pathogen, or Rhodotorula glutinis as
test organisms. As shown in Table 2, aryl-substituted leupylogs 3
and 23 displayed potent IC50 values in the same range as the
parent natural products against R. glutinis, demonstrating that
substitution of the dihydrofuran side chain by an aryl group is
well-tolerated. In contrast, furan leupylog 4 was less active and
the thiophene analogue 36 was essentially inactive,
demonstrating that the original pyrrole may not be easily
substituted. Notably, the pyrrole N-H of the original heterocycle
building block. They revealed
a high importance of the
macrocyclic core, a certain flexibility of the diacid fragment and
high tolerance of the side chain. Two of these leupylogs retain
the potent biological activity of the parent natural products. This
demonstrates that the overall structure can be simplified, which
is quite remarkable as only few such examples have been
described in the context of complex natural macrolides.[20] The
most potent leupylogs 3 and 23 are characterized by a
substitution of the synthetically challenging and chemically labile
bis-alkylidene diene-substituted dihydrofuran side chain of the
authentic leupyrrins by a mere aryl substituent, which also
replace the fragment. These leupylogs are much more readily
available as compared to the parent natural product (17 steps
for 3 vs 24 steps for 1 in the longest linear sequences from
commercial material), involving a much more readily available
Eastern fragment (6 steps for 12 from commercial material vs.
18 steps for the authentic Eastern fragment of leupyrrin A1).
Furthermore, these studies suggest that the pyrrole-oxazoline
subunit seems to be a crucial element of their pharmacophore,
which may be rationalized by solution conformation
considerations. Due to the facilitated practical access, many
more leupylogs may now be envisioned, enabling more detailed
biological studies of these potent antifungal agents. The
increased availability and the potential modifications will aid the
drug target identification and will help to evaluate practical
applications in basic research and medicine.
exerts
oxazoline, leading to an N,N-syn orientation of the two respective
nitrogen atoms, which has pronounced effect on the
a characteristic hydrogen bond to the neighboring
a
macrocyclic conformation in solution and solid state.[3] Potentially,
a hydrogen bond donor like the pyrrole is also be necessary for
the correct conformation of the molecule at the target. The entire
exchange of the pyrrole-oxazoline structure by a furan-oxazole
unit as in leupylog 51 led to complete loss of activity. These data
suggest that the pyrrole-oxazoline subunit and the macrocyclic
ring constitute important parts of the pharmacophore.
Table 2. Biological activity of natural leupyrrin A1 (1) and B1 (2) and synthetic
leupylogs 3, 4, 23, 36 and 51 with Amphotericin B (AmB) as a control.
IC50 [µg/mL]
Compound
1
2
3
23
4
36
51
AmB
2.45
Rhodotorula
glutinis
0.29
0.14
0.23
0.21
10
>16
>16
Candida
albicans
>64
>64
>64
>64
>16
>40
>16
>40
>64
0.33
n.d.
Acknowledgements
Mouse
fibroblasts
(L929)
0.95
2.09
9.82
5.08
5.69
Financial support by the DFG (ME 2756/7-1, TRR 261 (B07) and
GK 850: scholarship to: S. D.) is gratefully acknowledged. We
thank Andreas J. Schneider for excellent HPLC support and
Maximilian Guhlke for excellent synthetic support (both
University of Bonn).
Furthermore, none of the compounds tested were active
against C. albicans. As 23 was the most active and potent
leupylog, we evaluated it further and found it to be highly active
against R. minuta and R. mucilaginosa and against
Zygosaccharomyces bailii (MIC’s of 1 µg/ml and <0.125 µg/ml,
respectively). The growth of Zygosaccharomyces bailii was
impaired, but not completely inhibited at concentrations up to 16
µg/ml. As a control for unspecific toxicity, the yeast Candida
Keywords: antifungal agents • Leupylogs • natural products •
total synthesis • SAR studies
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Entry for the Table of Contents
Macrolides*
Novel simplified leupyrrins retaining
powerful antifungal potencies were
efficiently obtained by a concise
sp2-sp3 cross coupling and macro-
lactonization.
Paul R. Wosniok, Christopher Knopf,
Sandra Dreisigacker, J. Manuel
Orozco-Rodriguez, Bettina
Hinkelmann, Peter P. Mueller, Mark
Brönstrup, and Dirk Menche
SAR Studies of the Leupyrrins:
Design and Total Synthesis of
Highly Potent Simplified Leupylogs
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