Synthesis and biological evaluation of (±)-hippolachnin and analogs

Article abstract of DOI:10.1038/s41429-019-0176-x

Due its unique structure and its reported potent antifungal activity, the marine polyketide hippolachnin A (1) has attracted much attention in the synthetic community. Herein, we describe the development of a concise, diversifiable and scalable synthesis of the racemic natural product, which serves as a platform for the generation of bioactive analogues. Biological testing of our synthetic material did not confirm the reported antifungal activity of hippolachnin A but unraveled moderate activity against nematodes and microbes.

Full text of DOI:10.1038/s41429-019-0176-x

The Journal of Antibiotics
https://doi.org/10.1038/s41429-019-0176-x
SPECIAL FEATURE: ARTICLE
Synthesis and biological evaluation of ( )-hippolachnin and analogs
2
1,3
Nils Winter1 Zeljka Rupcic2 Marc Stadler
Dirk Trauner
●
●
●
Received: 3 December 2018 / Accepted: 12 January 2019
© The Author(s), under exclusive licence to the Japan Antibiotics Research Association 2019
Abstract
Due its unique structure and its reported potent antifungal activity, the marine polyketide hippolachnin A (1) has attracted
much attention in the synthetic community. Herein, we describe the development of a concise, diversifiable and scalable
synthesis of the racemic natural product, which serves as a platform for the generation of bioactive analogues. Biological
testing of our synthetic material did not confirm the reported antifungal activity of hippolachnin A but unraveled moderate
activity against nematodes and microbes.
Introduction
compound 2, the presumptive biosynthetic precursor of 1,
as well as the graciloethers (3, 5, 6), and plaktorin (4).
Attracted by its unusual structure combined with the
potent bioactivity, numerous research groups, including
ours, have developed unique synthetic solutions for this
molecule. In the last three years, four total syntheses have
been reported [9–13], including a recent biomimetic
synthesis by Tang and Enders [14]. In 2014, we launched a
program to develop a scalable and diversifiable approach to
hippolachnin A (1). Herein, we show how our strategy
evolved, discuss our studies on synthetic derivatives, and
the results of our evaluation of their biological properties.
Opportunistic infections by ubiquitous fungi represent a
major challenge to immunocompromised patients. The
basidiomycete yeast Cryptococcus neoformans, for
instance, can cause life-threatening meningitis and affect the
lungs and skin of patients with advanced acquired immu-
nodeficiency syndrome (AIDS) [1–6]. Hippolachnin A (1)
was recently isolated from the South China Sea sponge
Hippospongia lachne. The molecule features six contiguous
stereocenters embedded in a bicyclo[3.2.0]heptane frame-
work, four of which bear ethyl groups. Biochemical assays,
reported in the initial study, revealed high potency against
several pathogenic fungi, including C. neoformans (MIC =
0.41 µM) [7]. Biosynthetically, 1 is part of the plakortin
family [8] and, more precisely, part of the gracilioether
family (Fig. 1). This family also includes the unnamed
Results and discussion
As outlined in Scheme 1, we initially focused on a strategy
based non-biomimetic [2 + 2]-cycloaddition of allylic ester
8, to simultaneously form the cyclobutane and the tetra-
hydrofuran of the natural product. The vinylogous carbo-
nate should then be installed by Peterson olefination. Even
though the desired [2 + 2]-cycloaddition of an acyclic α,β-
unsaturated ester was anticipated to be difficult, we envi-
sioned that, conducting the reaction in an intramolecular
fashion, might allow for efficient trapping of the photo-
chemically formed triplet diradical.
Dedicated to Prof. Samuel J. Danishefsky with admiration and
gratitude.
Supplementary information The online version of this article (https://
doi.org/10.1038/s41429-019-0176-x) contains supplementary
material, which is available to authorized users.
* Dirk Trauner
dirktrauner@nyu.edu
The synthesis commenced with the preparation of ter-
tiary alcohol 13 from dicyclopentadiene (9) (Scheme 2).
Conjugate addition from the convex side of enone 10,
obtained from Alder-ene reaction of 9 with singlet oxygen,
afforded ketone 11 [15]. Subsequent 1,2-addition of
EtMgBr mediated by LaCl3.2LiCl yielded tertiary alcohol
12 as a single diastereoisomer [16]. While initial attempts to
1
Department of Chemistry, Ludwig-Maximilians-Universität
München, Butenandtstr. 5-13, 81377 Munich, Germany
2
Helmholtz Centre for InfectionResearch, Inhoffenstr. 7, 38124
Braunschweig, Germany
3
Department of Chemistry, New York University, Silver Center,
100 Washington Square East, Room 712, New York, NY, USA

N. Winter et al.
effect thermolysis resulted in decomposition, flash vacuum
pyrolysis (FVP) cleanly provided sensitive tertiary alcohol
13 in good yield [17].
synthesis is shown in Scheme 4. We envisioned formation
of 1 by ethyl Grignard addition to cyclopentenone 17, fol-
lowed by dehydration. The β-ketoester could be derived by
Claisen condensation of methyl acetate with lactone 18.
Lactone 18 would be traced back to an intramolecular [2 +
2]-cycloaddition of ester 19.
With 13 in hand, we envisioned esterification with the
known carboxylic acid 14 (Scheme 3) [18]. Standard
esterification conditions utilizing the acid chloride only
resulted in isolation of the free acid after work up. Attempts
to generate the acylium ion only led to decomposition of the
starting materials [19]. Anhydride 15 was found to be stable
to column chromatography but could not be used for ester
formation, even at elevated temperatures. Interestingly, both
the acid chloride and the mixed anhydride were able to react
with t-BuOK to form the corresponding t-butyl ester 16.
Since the sterical hindrance of the tertiary alcohol could
not be overcome, we decided to install the fourth ethyl
group at a later stage of the synthesis. The revised retro-
Epoxidation of cyclopentadiene [20] (20) followed by
SN2׳ displacement with ethylcyanocuprate [21] provided
allylic alcohol 21 in moderate yield (Scheme 5). Quantita-
tive formation of the lithium alkoxide and subsequent
quenching with acid chloride 14 gave rise to allylic ester 19.
With 19 in hand we investigated the key [2 + 2]-
cycloaddition [22]. Unfortunately, irradiation of 19 with
310 nm LEDs in the presence of either acetone or benzo-
phenone in various solvents (MeCN, CH2Cl2, benzene) did
not result in the desired cycloadditon. Instead, we isolated
starting material as a mixture with its (E)-configured dia-
stereomer 22. While excitation of the system proceeded
smoothly as proved by the formation of 22, we wondered
whether π-bond rotation was too fast to allow for capture of
the diradical by the tethered olefin.
To prevent isomerization we decided to incorporate the
enone double bond into a ring by stitching the ends of the
ethyl groups together using a sulfur bridge. Ring expansion
of tetrahydrothiopyranone 23 using ethyl diazoacetate gave
rise to thiepanone 24 which, after reduction, mesylation,
and elimination provided ethyl ester 26 [23]. Saponification
followed by activation of the carboxylic acid as the acid
chloride and subsequent esterification accessed enoate 28 in
moderate yields (Scheme 6). To investigate the cycloaddi-
tion, we submitted 28 to the same conditions used for 19.
After irradiation, the clear solution became cloudy and
NMR analysis of the mixture indicated decomposition of
the starting material.
Fig. 1 Selected members of the plakortin family
While excitation of the molecule was possible, no pro-
ductive pathways were observed, which might be attributed
to a preferred conformation of the molecule where both
olefins are pointing into opposite directions. In order to
Scheme 1 First generation retrosynthesis of hippolachnin A based on a
[2 + 2]-cycloaddition
Scheme 2 Synthesis of allylic
alcohol 13

Synthesis and biological evaluation of ( )-hippolachnin and analogs
provide the molecule more flexibility, β-ketoester 32 was
prepared (Scheme 7). Activation of acid 30 with CDI and
subsequent Claisen condensation with t-BuOAc, followed
by ketalization, gave dioxanone 31 [24]. Retro [4 + 2]-
cycloaddition and trapping of the resulting ketene with
alcohol 19 then afforded β-ketoester 32 [25]. Irradiation of
32 under the previously established conditions resulted in a
complex mixture from which we were unable to isolate the
desired bicyclo[3.2.0]heptane 33.
Frustrated by our inability to effect (2 + 2) cycloadditons
we decided to turn to a different type of photochemistry
(Scheme 8). In our new retrosynthetic analysis, we planned
to close the tetrahydofuran ring in 1 by O-alkylation of an
enolized β-ketoester 34 [26]. The vicinal ethyl groups
should be introduced by addition of an ethyl nucleophile
and an ethyl electrophile to the Michael acceptor 35.
Compound 35 was derived from the known bicyclo[3.2.0]
heptadiene 36, which can be obtained from tropolone ether
37 (Scheme 8) [27].
Scheme 3 Attempted esterification of 8
Our synthesis commences with the photochemical con-
version of 37 into methoxy bicyclo[3.2.0]heptadienone 36,
which was originally reported by Dauben et. al [27]. Irra-
diation of 37 induced a disrotatory 4π-electrocyclization to
Claisen
condensation
dehydration
OMe
OMe
H
H
O
O
O
O
O
H
H
Grignard addition
oxidation
1
17
[2+2]-cyclo-
addition
O
O
H
O
H
O
H
esterification
18
19
Scheme 4 Revised retrosynthesis of 1
Scheme 5 Synthesis of allylic ester 19 and formation of ester 22
Scheme 6 Synthesis of thiepan 28 and attempted [2 + 2]-cycloaddition

N. Winter et al.
give 38, which then undergoes an excited state rearrange-
ment to yield 36. Notably, even though 38 is an isolatable
intermediate, 36 was the only product obtained after full
conversion of 37. Ethyl cuprate addition to 36 occurred
exclusively from the convex side [15] of the molecule and
gave, after Wittig olefination [28] and acid mediated clea-
vage of the enol ether [29] ketone 40 as a 10:1 mixture of E-
and Z- isomers (major isomer shown). Formation of the
vinyl triflate [30, 31] and subsequent carbomethoxylation
[32] then gave methyl ester 41 (Scheme 9).
With 41 in hand, the stage was set for the installation of
the vicinal ethyl groups (Scheme 10). We first envisioned
the conjugate addition of an ethyl cuprate and subsequent
alkylation at the α-position of the ester. While the conjugate
addition occurred solely from the convex side of the
molecule, acidic quenching of the resulting ester enolate
gave an inconsequential mixture of diastereomers with
respect to the ester group. Reformation of the enolate using
LDA and subsequent trapping with ethyl iodide resulted in
2:1 mixture of diastereomers [33], favoring the desired one.
We attributed this result to the opposing stereochemical bias
provided by the bicyclic core and the newly introduced
ethyl group. Although, the major isomer could be converted
into an advanced intermediate 44 of the Wood-Brown
synthesis [10], we thought that this low level of selectivity
was unacceptable for an efficient synthesis.
To overcome this problem, we turned toward the 1,3-
dipolar cycloaddition of a thiocarbonyl ylide followed by
reductive desulfurization to add, in effect, two ethyl radicals
across the strained and electron poor double bond of 41
[34–38]. The requisite thiocarbonyl ylide 48 could be
Scheme 7 Synthesis of β-ketoester 32 and attempted [2 + 2]-
cycloaddition
three component
coupling
Scheme 8 Retrosynthetic
analysis of 1 based on an excited
state rearrangement
COOMe
OMe
OMe
H
O
H
H
H
H
O
O
O
H
1
34
35
chelation-controlled
enolate trapping
excited state
rearangement
OMe
O
OMe
H
H
O
36
37
Scheme 9 Synthesis of methyl
ester 41

Synthesis and biological evaluation of ( )-hippolachnin and analogs
Table 1 Attempted Claisen condensation of 49.
#
1
2
3
4
5
substrate
MeOAc
MeOAc
MeOAc
base
LDA
temp
–78 °C to RT
RT
result
no reaction
no reaction
no reaction
no reaction
Scheme 10 Synthesis of 44 via three component coupling
MgDA
NaH
60 °C
MeOAc KOt-Bu
50 °C
t-BuOAc t-BuLi –78 °C to RT decomposition
n-BuLi 0 °C decomposition
6
dipolar cycloaddition gave tricycle 53 as a single diaster-
eomer, albeit in slightly lower yield. Blaise reaction of 53
with zinc organyl 54 then yielded the desired en-amine 55
[50]. Unfortunately, the vinylogous carbamate 55 proved to
be completely resistant to hydrolysis with starting material
recovered under standard conditions [51, 52]. Harsher
conditions led to decomposition of 55.
Scheme 11 Synthesis of thiocarbonylylide 48 and 1,3-cycloaddition to
form 49
Although methyl ester 41 was resistant to a crossed-
Claisen condensation, it could be reduced to the primary
alcohol and mildly reoxidized. The resulting aldehyde 56
was able to undergo an aldol addition [53] with the enolate
generated from methyl acetate and gave, after Dess Martin
oxidaition, the desired β-ketoester 56. Formation of the tin
enolate followed by trapping of the simultaneously gener-
ated tertiary carbocation [26] gave rise to (Z)-configured
vinylogous carbonate 57 [54]. Its structure could be con-
firmed by X-ray structure analysis. Reductive desulfuriza-
tion with Raney nickel in THF [55–57] gave access to the
generated in situ from sulfoxide 47 following Achiwa’s
method [39–44]. Compound 47, in turn, was derived from
(chloromethyl)trimethylsilane 45 by alkylation with methyl
iodide followed by nucleophilic displacement with sodium
sulfide and subsequent oxidation (Scheme 11).
Dropwise addition of 48 to a hot solution of 41 indeed
resulted in the clean formation of tetrahydrothiophen 49,
which was obtained as a single diastereosiomer. Single
crystal X-ray structure analysis showed that the methyl
groups adopt a trans-configuration with respect to the het-
erocyclic ring. Notably, the reaction also proceeds under
high pressure conditions (12 kbar) at room temperature,
albeit with slightly lower yield.
With the key intermediate in hand we turned our focus to
the homologation of the ester group to the corresponding β-
ketoester. Unfortunately, all attempts [45–48] to effect a
Claisen condensation failed, presumably due to steric hin-
derance (Table 1).
We sought to overcome this problem by using a less
hindered electrophile settling on the cyano group with its
sp-hybridized carbon. To this end we prepared nitrile 52
from vinyl triflate 51 by Pd-catalyzed cross coupling with
sodium cyanide [49] (Scheme 12). To our delight, the 1,3-
natural product, hippolachnin A, as
(Scheme 13).
a
racemate
Piao et. al. reported strong antifungal activity of hippo-
lachnin A [7]. With a good synthetic entry at hand, we
decided to develop analogues that would allow us to map
structure-activity relationships and would show improved
physicochemical properties. Since 1 is poorly soluble in
aqueous solutions, we chose to synthesize more polar
derivatives by oxidizing the sulfur of the advanced inter-
mediate 57 to either the sulfoxide or the sulfone
(Scheme 14). Notably, treatment of 57 with excess m-
CPBA at room temperature led to partial isomerization of
the double bond to afford a mixture of 58 and 59. Partial
oxidation of 57 gave sulfoxide 60 as a 5:1 mixture of

N. Winter et al.
Scheme 12 Synthesis of
enamine 55 and attempted
hydrolysis
Scheme 13 Total synthesis of 1
diastereomers. We also synthesized the butyrolactone 61 by
acid mediated cyclization of 41.
inhibited the growth of Caenorhabditis elegans, but again
with weak potency (Table 2).
With the racemic natural product and several synthetic
analogous in hand, we evaluated the antifungal, anti-
microbial and the nematicidal activity of 1, 57, 44, 61, 59,
58, and 60. We were surprised to find that ( )− 1 showed
no antifungal activity, in particular against C. neoformans
(Table 2). This observation was recently independently
reported by Wood [58]. It seems unlikely that the unnatural
enantiomer somehow neutralizes the biological effect of
(+)-hippolachnin A. Our synthetic analogs of 1 were also
inactive against the fungi listed in Table 2, except for
butyrolactone 61, which showed modest activity against C.
neoformans. Compounds 1, 57, 44, and 61 exhibited weak
antimicrobial activity (Table 2 and supporting information)
and no or very weak cytotoxicity (supporting information).
Interestingly, all analogs, including the natural product,
Conclusion
Herein, we have presented the evolution of our campaign
for the total synthesis of hippolachnin A. Starting from
tropolone methyl ether 37, we developed a robust and
scalable synthetic route which enabled us to synthesize not
only more than 100 mg of the natural product but also a
variety of synthetic derivatives. The bicyclic carbon core
was constructed by photoisomerization of 37. The four ethyl
groups were introduced by diastereoselective cuprate addi-
tion, Wittig olefination, and the 1,3-dipolar cycloaddition of
a thiocarbonyl ylide. The latter represents a rarely used
method to overcome steric hindrance and, in effect, link a

Synthesis and biological evaluation of ( )-hippolachnin and analogs
Scheme 14 Synthesis of
synthetic analogs of 1
Table 2 Antifungal,
antimicrobial, and nematicidal
Test organism
DSM
1
57
44
61
59
58
60
Reference (µg/ml)
activity activity of 1 and its
analogs
Fungi
Alternaria solani
2947
819
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
16.6c
67.0-33.3c
67.0c
4.2c
8.3c
Aspergillus fumigatus
Botrytis cinerea
/
877
/
Candida albicans
Cryptococcus neoformans
Fusarium oxysporum
Phytophthora drechslerif
Sclerotinia sclerotiorum
Bacteria
1665
15466
62297
62679
1946
/
67.0
/
67.0c
c
c
Staphylococcus aureus
Staphylococcus aureus MRSA
Bacillus subtilis
346
8.3
33.3
8.3
/
33.3
/
33.3
/
/
/
/
/
/
/
/
/
/
/
/
0.21-0.1a
0.83b
4.2a
11822
10
/
/
/
/
/
33.3
/
8.3
/
Escherichia coli
1116
3.3-0.83a
Nematode
Caenorhabditis elegans
–
12.5
10
10
50
10
10
25
1.0e
In vitro antibacterial, anti-oomycete nematicidal activity of substances 1, 57, 44, 61, 59, 58, 60 and our
control drugs. For determination of antibacterial and antifungal activity, substances were dissolved in MeOH
(10 mg/ml) and then further diluted to a final concentration of 1 mg/ml. 2 and 20 µl of this solution was tested
against different test organisms (67.0-0.052 µg/ml final concentration). Alternatively, for determination of
nematicidal activity, substances were dissolved in MeOH (1.5 mg/mL) and an aliquot thereof transferred to
24 well plate, where each well contained 1 mL of M9 buffer, to reach final concentrations in a range from
100–1 µg/mL [61]. For all assays MeOH was used as negative control and showed no activity against the
selected test strains. Results of antibacterial and antifungal assays were expressed as MIC: Minimum
inhibitory concentration µg/ml, while nematicidal activity was assessed as LD50: lethal dose (concentration)
causing over 50 % immobility of nematodes. The cell density was adjusted to 8 × 106 cells/ml
N.I. no inhibition, DSMZ German collection of microorganisms and cell cultures, Braunschweig
aOxytetracyclin hydrochloride
bVancomycin
cNystatin
dKanamycin
eIvermecitin
fspores from agar plates were applied without justification

N. Winter et al.
nucleophile to an electrophile. The heterocyclic ring was
closed via trapping of carbocation generated in situ by a tin
enolate. Reports about the antifungal activity of hippo-
lachnin A could not be confirmed. This is in keeping with
several other recent cases where the purported bioactivity of
natural products did not match the activity of their synthetic
versions [59, 60]. Of course, this serves as yet another
justification for total synthesis, which often provides
material in purer form and on a larger scale than isolation
from natural sources.
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