Highly Convergent Total Synthesis and Assignment of Absolute Configuration of Majusculamide D, a Potent and Selective Cytotoxic Metabolite from Moorea sp.

Article abstract of DOI:10.1021/acs.orglett.8b04050

The total synthesis of majusculamide D (MJS-D) is described, a lipopentapeptide originally isolated from Lyngbya majuscula and reisolated from a Moorea sp. MJS-D possesses selective and potent cancer cell toxicity. A scalable and convergent strategy with a minimal number of purifications produced significant quantities of MJM-D for in vivo evaluations. The absolute configuration of the 1,3-dimethyl-octanamide motif was determined by synthesis of this fragment via ZACA chemistry.

Full text of DOI:10.1021/acs.orglett.8b04050

Letter
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Cite This: Org. Lett. XXXX, XXX, XXX−XXX
Highly Convergent Total Synthesis and Assignment of Absolute
Configuration of Majusculamide D, a Potent and Selective Cytotoxic
Metabolite from Moorea sp.
Eduardo J. E. Caro-Diaz,† Frederick A. Valeriote,‡ and William H. Gerwick*,†,§
†Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography, University of California at San Diego, La
Jolla, California 92093, United States
‡Department of Internal Medicine, Henry Ford Cancer Institute, Henry Ford Health System, Detroit, Michigan 48202, United
States
§Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California at San Diego, La Jolla, California 92093, United
States
S
* Supporting Information
ABSTRACT: The total synthesis of majusculamide D (MJS-D) is
described, a lipopentapeptide originally isolated from Lyngbya
majuscula and reisolated from a Moorea sp. MJS-D possesses
selective and potent cancer cell toxicity. A scalable and convergent
strategy with a minimal number of purifications produced
significant quantities of MJM-D for in vivo evaluations. The
absolute configuration of the 1,3-dimethyl-octanamide motif was
determined by synthesis of this fragment via ZACA chemistry.
atural products (NPs) continue to be an important
Nsource of novel chemical structures that have directly
yielded or inspired a majority of our currently approved drugs.1
Marine cyanobacteria, especially Moorea sp.,2 are prolific
producers of bioactive secondary metabolites.3 Many of these
cyanobacterial NPs have promising antitumor properties in
vitro.4 Our continued exploration of these life forms for their
bioactive components has been productive in both finding new
molecules as well as new biological activities for known ones.5
In the current case, evaluation of the extract of a Moorea sp.
sample collected from Papua New Guinea in a solid-tumor cell
assay revealed promising selectivity and exquisite potency
against pancreatic and glioblastoma cell lines. This activity is
notable, given the acute increase in the mortality rate of
pancreatic cancer that is projected to surpass breast, colon, and
prostate cases in the near future.6 Investigation of the active
Figure 1. Structure of majusculamide D (MJS-D) and related
analogues.
fraction revealed majusculamide D (MJS-D, 1, Figure 1), a
lipopentapeptide isolated by Moore and Entzerof in 1988,7 to
be responsible for the in vitro bioactivity. MJS-D is structurally
related to the microcolins, a class of metabolites also isolated
from cyanobacteria (Figure 1)8 which show interesting
biological properties. This has prompted total syntheses9,10
as well as SAR studies of the microcolins.11 Interestingly, the
absolute configuration of both stereocenters within the 2,4-
dimethyloctanoate side chain of 1 were never unequivocally
determined in the original isolation work.7 Given the
promising in vitro biological profile and uncertainty about
the two stereocenters in 1, we designed a total synthesis that
would allow access to all possible stereoisomers of 1 and
provide more compounds for further anticancer investigations.
Our synthetic strategy involved two key disconnections
between positions C13−N12 and C39−N29, resulting in three
fragments of similar molecular weight that converge at a late
stage via standard peptide coupling reactions. We also sought
to manage our reaction sequences by carefully removing
unnecessary purifications and using simple aqueous workups
and filtration over silica plugs so that solvent and labor were
minimized. Furthermore, in our synthetic design of the 2,4-
dimethyloctanoic acid fragment, we required a concise set of
Received: December 19, 2018
© XXXX American Chemical Society
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DOI: 10.1021/acs.orglett.8b04050
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Letter
reactions that could access all possible stereoisomers, to
establish the absolute configuration of C40 and C42.
Scheme 2. Synthesis of Tripeptide Backbone of MJS-D
Based on the absolute configuration established for micro-
colin A and B,9,10b we inferred that the correct configuration of
1 at the unassigned carbon centers C40 and C42 should be
R,R. To accomplish the synthesis of this octanoic acid
fragment, we took advantage of the zirconium-catalyzed
asymmetric carboalumination (ZACA) chemistry described
by Negishi and co-workers.12 Beginning with commercially
available 1-hexene, a catalyst/reagent controlled carbo-
alumination reaction was performed with this alkene using
13
Me3Al and (+)-NMI2ZrCl2 (Scheme 1). The resulting
Scheme 1. Synthesis of (2R, 4R)-2,4-Dimethyl Octanoic
Acid via ZACA Vinylation/Oxidation Sequence
column purifications and provided gram-scale quantities of
intermediate 10.
For the synthesis of the pyrrolylproline fragment of 1, we
sought to improve upon the established chemistry10,11 to
access these types of motifs. Beginning with commercially
available N-Boc-cis-4-hydroxy-L-proline, we distinguished the
two functional groups via TBS protection of the alcohol and
pentafluorophenol DCC coupling to the acid (Scheme 3). A
Scheme 3. Synthesis of Pyrrolylproline Fragment via Imide
Coupling
organoaluminum intermediate was trans-metalated in situ
with Zn(OTf)2 and coupled with a freshly prepared vinyl-Pd
species. The crude (R)-4-methyl-1-octene was moved forward
with minimal purification and subjected to a second
carboalumination reaction; upon exposure to a stream of O2
and workup, crude 3 was obtained in a modest diasteromeric
ratio (dr) ratio (6:1, 13C NMR).12b Fortunately, the undesired
diastereomer was largely removed via silica gel chromatog-
raphy providing enantiomerically enriched 3 in 32% yield.
Octanol 3 was then mildly oxidized14 to the corresponding
acid in good yield.
To efficiently build the tripeptide backbone of 1, we sought
to use a peptide coupling reagent that could be removed via
aqueous workup, would provide acceptable yields, and, most
importantly, avoid epimerization of the α-carbons during
reaction. Thus, we protected commercially available Boc-N-
Me-L-valine with Dudley’s reagent,15 which after filtration
through a silica plug, was directly coupled with N-Boc-OAc-L-
threonine 6 (Scheme 2) using hexafluorophosphate azabenzo-
triazole tetramethyl uronium (HATU) as the coupling reagent.
A single-column purification was used in this three-reaction
sequence. This was followed by quantitative acidic conditions
removal of the N-Boc protecting group from dipeptide 7 and
subsequent coupling to tyrosine derivative 8, this time with
Goodman’s16 reagent. Debenzylation by standard hydro-
genation conditions quantitatively provided tripeptide acid
10. This six-step reaction sequence required only two silica
careful aqueous workup of both reactions gave high-purity
TBS-protected perfluorester 12 without the need for
purification. Imide coupling with chiral pyrrolidone 13 and
n-BuLi at low temperature smoothly provided imide 14, which
could be oxidized to enone 15 under well-established
selenoxide elimination conditions.14 Simultaneous removal of
the N-Boc and O-TBS protecting groups under strongly acidic
conditions yielded the appropriate pyrrolylproline HCl salt 16
in quantitative fashion. Overall, synthesis of this fragment was
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achieved in five synthetic steps, with only two silica column
purifications and an overall yield of 64%.
Table 1. In Vitro IC50 Values of Majusculamide D (MJS-D,
1) to a Panel of Cancel Cell Lines
To complete the synthesis of MJS-D, the three fragments
constructed above were coupled together in two moderate
yielding condensation reactions. Given the chemo-differ-
entiation of the protecting groups on tripeptide 9, we could
selectively reveal the appropriate functional group for coupling.
Hence, acid 10 was coupled to the pyrrolylproline fragment 16,
using Goodman’s reagent16 to provide pentapepetide 17
(Scheme 4). N-Boc removal and subsequent coupling with
cancer cell line
tissue type
IC50 (nM)
PANC-1
U251N
HepG2
H125
pancreatic
brain
liver
0.32
36.8
1,396
147
lung
(Table 1 and Figure 2). These results have stimulated
additional ongoing SAR studies in the MJS-D structure class.
Scheme 4. Late-Stage Coupling of Key Fragments and
Completion of the Synthesis of 1
Figure 2. In vitro concentration−response curves for MJS-D (1) to a
panel of cancer cell lines
In conclusion, we report here a highly convergent and
efficient approach for the synthesis of the cyanobacterial
lipopeptide majusculamide D (1). Our synthetic strategy
highlights the use of ZACA chemistry to rapidly build the
chirally methylated octanoic acid side chain. We also carefully
selected reagents and conditions that facilitated convenient
workups and bypassed purifications of the synthetic
intermediates; this resulted in a chemical synthesis with
significant scale-up potential and thereby enables future
biological investigations. Finally, we determined the absolute
configuration of the C40 and C42 methyl groups (Figure 1)
within the octanoic acid side chain of 1 to be R,R, and showed
that the synthetic material is equipotent and similarly selective
to the natural product in its cytotoxicity profiles toward
PANC-1 and U251N cell lines in vitro. We envision utilizing
this efficient and scalable chemical synthesis to further explore
Structure−Activity-Relationships (SARs) of majusculamide D
as well as creation of derivatives that will allow determination
of its biochemical target and mechanism of action.
dimethyl octanoic acid 4 provided MJS-D (1) in acceptable
yield. This synthetic material perfectly matched HPLC
retention time, MS/MS fragmentation, 1H and 13C NMR
spectra, and 2D NMR data sets (COSY, HSQC), with those of
an authentic sample of natural MJS-D. Our comparison of
1
natural and synthetic 1 usng H NMR is possible given that
MJS-D does not have concentration-dependent NMR shifts
and is observed as a single rotamer (see SI-14 in the
Supporting Information). Furthermore, close inspection of
previously published NMR data for the total synthesis of
microcolin A showed substantial differences in the shift
associated with other diastereomers when other octanoic
acid stereoisomers are installed.10 Upon completion of the
total synthesis of 1, we submitted this material, along with
natural MJS-D for cancer cell cytotoxicity evaluation. We
initially utilized a solid-tumor disk-diffusion assay (DDA),4a
where a panel of cancer cell lines are grown on agar under
conditions that simulate anchorage independency.17 The
synthetic and naturally occurring samples of MJS-D had
essentially identical biological profiles exhibiting selective
cytotoxicity toward both pancreatic (PANC-1) and glioblas-
toma (U251N) cancer cell lines. Furthermore, 1 showed a
range of potencies (IC50 values; see Table 1) demonstrating a
>4000-fold difference between PANC-1 cells and HepG2 cells
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.or-
glett.8b04050.
Synthetic procedures and spectroscopic data (PDF)
AUTHOR INFORMATION
Corresponding Author
■
*E-mail: wgerwick@ucsd.edu.
C
DOI: 10.1021/acs.orglett.8b04050
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ORCID
Letter
William H. Gerwick: 0000-0003-1403-4458
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This research was supported by NIH (No. CA100851) and a
Fellowship to EJEC-D (NIH/NCI, No. T32 CA009523). We
would like to acknowledge Y. Su for MS support, A. Mrse for
NMR support, and C. C. Hughes for technical chromato-
graphic support.
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DOI: 10.1021/acs.orglett.8b04050
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