Discovery, Total Synthesis, and SAR of Anaenamides A and B: Anticancer Cyanobacterial Depsipeptides with a Chlorinated Pharmacophore

Article abstract of DOI:10.1021/acs.orglett.0c01281

New modified depsipeptides and geometric isomers, termed anaenamides A (1a) and B (1b), along with the presumptive biosynthetic intermediate, anaenoic acid (2), were discovered from a marine cyanobacterium from Guam. Structures were confirmed by total synthesis. The alkylsalicylic acid fragment and the C-terminal α-chlorinated α,β-unsaturated ester are novelties in cyanobacterial natural products. Cancer cell viability assays indicated that the C-terminal unit serves as the pharmacophore and that the double-bond geometry impacts the cytotoxicity.

Full text of DOI:10.1021/acs.orglett.0c01281

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Letter
Discovery, Total Synthesis, and SAR of Anaenamides A and B:
Anticancer Cyanobacterial Depsipeptides with a Chlorinated
Pharmacophore
David A. Brumley, Sarath P. Gunasekera, Qi-Yin Chen, Valerie J. Paul, and Hendrik Luesch*
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ABSTRACT: New modified depsipeptides and geometric isomers, termed anaenamides A (1a) and B (1b), along with the
presumptive biosynthetic intermediate, anaenoic acid (2), were discovered from a marine cyanobacterium from Guam. Structures
were confirmed by total synthesis. The alkylsalicylic acid fragment and the C-terminal α-chlorinated α,β-unsaturated ester are
novelties in cyanobacterial natural products. Cancer cell viability assays indicated that the C-terminal unit serves as the
pharmacophore and that the double-bond geometry impacts the cytotoxicity.
reversed-phase chromatography yielding two isomerically pure
arine cyanobacteria continue to be a source of natural
Mproducts, producing a wealth of diverse biologically compounds, anaenamide A (1a) [colorless, solid, [α]25 −46
D
(c 0.29, CHCl3)] HRMS (ESI) m/z [M + H]+ calcd for
active compounds, in particular, unique peptides and
C27H39NO835/37Cl, 540.2364/542.2334, found 540.2349/
depsipeptides.1,2 Recently, our group described a family of
alkylphenols produced by an undescribed gray filamentous
cyanobacterium from the genus Hormoscilla.3 This discovery
highlighted the biosynthetic capacity of filamentous cyanobac-
teria, further prompting our search for novel and therapeuti-
cally relevant specialized metabolites. Here, a morphologically
related green filamentous cyanobacterium, Hormoscilla sp.
(16S rDNA; GenBank MT218338),4,5 yielded a structurally
distinct family of natural products, which we termed
anaenamides. Their general scaffold is distinguished by two
α-hydroxy acid residues, flanked by an alkylated salicylic
fragment and an unusual α-chlorinated α,β-unsaturated (E/Z)
ester. Their inherent novelty, with respect to cyanobacteria,
further emphasized the untapped chemical diversity associated
with understudied marine organisms.
542.2322) and anaenamide B (1b) [colorless, solid, [α]25
D
−38 (c 0.06, CHCl3)]; HRMS (ESI) m/z [M + H]+ calcd for
C27H39NO835/37Cl, 540.2364/542.2334, found 540.2351/
1
542.2330) (Figure 1). Reconciliation of the observed H and
13C NMR resonances for 1a and 1b with the HSQC spectra
indicated the presence of six methylenes (C-8 to C-11, C-4′
and C-4′′′), three methines (C-2′, C-3′, and C-2′′), four
methyl groups (C-12, C-5′, C-6′, and C-3′′), three contiguous
aromatic signals (C-4 to C-6), one isolated olefinic proton, and
two OMe groups (C-13 and C-1′′′). Additionally, the 13C
NMR spectrum indicated four nonprotonated sp2 carbons (C-
2, C-3, C-7, and C-2′′′), and four carbonyls (δc 169.4, C-1;
169.0 C-1′; 170.5, C-1′′; and 162.3, C-1′′′). Analysis of the
Received: April 11, 2020
The cyanobacterium was collected from the Anae Island reef
system in Guam. The freeze-dried material was exhaustively
extracted with EtOAc−MeOH (1:1) followed by H2O−
MeOH (1:9) to give nonpolar and polar extracts, respectively.
The two extracts were combined, partitioned against H2O and
EtOAc, and subjected to repeated rounds of normal and
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Figure 2. (A) Elucidation of the α-chlorinated α,β-unsaturated ester
double bond configuration in natural 1a and 1b based on critical
COSY (bold lines), HMBC (green arrows), and NOESY (red double
arrow). (B) Spectral comparison of select α-chlorinated α,β-
unsaturated ester proton resonances for synthetic 1a and 1b. Note
the clear shift differentials due to shielding effects.
Figure 1. Structures of anaenamides A (1a), B (1b), and anaenoic
acid (2).
COSY and HMBC spectra revealed the presence of the alkyl
salicylic acid, 2-hydroxy-3-methylpentanoic acid (Hmpa), and
lactic acid residues (Tables S1 and S2). Additionally, strong
NOESY correlations (Table S2) between the 13-OMe (δH
3.79) to H-4 (δH 6.76) and 8-methylene (δH 2.57) to H-6 (δH
6.84) further confirmed the substitution pattern of the
aromatic ring. The partial structure of the terminal amino
ester of 1a and 1b was determined based on similar 2D NMR
correlation arguments (Tables S1 and S2) and connected to
the core scaffold based on a strong HMBC from NH (δH 7.09
in 1a and 1b) to the lactic acid carbonyl (δC 170.5 170.4
respectively, C-1′′). The distinct methoxy resonances observed
in the 1H NMR spectra for 1a (δH 3.71) and 1b (δH 3.76) were
assigned as conjugated methyl esters based on: (i) the
relatively low-field carbonyl chemical shifts (i.e., 1a δC 162.3,
and 1b δC 162.7) and (ii) observed HMBC correlations from
the methoxy protons to their respective carbonyls. Assignment
of the chlorine atom to the quaternary C-2′′′ carbon satisfied
the molecular formulas for 1a and 1b and was consistent with
the observed shifts and 2D correlations (Figure 2A). The
configuration of the olefin in 1a was first determined based on
a weak NOESY correlation between the methyl ester (δH 3.71)
and the H-3′′′ proton (δH 7.01). Additionally, the H-3′′′
proton in 1b (δH 6.43) was found to resonate upfield relative
to 1a due to shielding by the vinyl chloride. In combination,
these data support the Z and E configurations for 1a and 1b,
respectively (Figure 2B).
to confirm the presence of a free carboxylic acid, 2 was reacted
with trimethylsilyldiazomethane yielding anaenoic acid methyl
1
ester. New signals in the H and 13C NMR spectra of the
product were consistent with an additional methoxy group
(i.e., δH 3.75 and δC 52.4).
The absolute configurations of the amino acid residues of 1a,
1b, and 2 were determined via three independent acid
hydrolyses (6 M HCl, 110 °C, 12 h) in combination with
chiral HPLC. The retention times of the acid hydrolyzate
components were compared to authentic standards and
indicated that 1a, 1b, and 2 contained L-lactic acid and
(2R,3S)-Hmpa. In order to further confirm these assignments
and access sufficient quantities of material for biological
testing, synthetic routes to all three natural products were
devised and executed.
As 1a and 1b are geometric isomers, their presumptive
biosynthetic intermediate 2 served as a synthetic handle for
chemical divergence, promoting a linear synthesis (Scheme 1).
Boc-protected intermediates of 3a and 3b could be obtained in
one pot via HWE olefination starting from 4 and 5.6,7 Further
dissection of 2 to amino acid derivatives 6 and 7 accounted for
the three chiral centers.8 The alkylated aromatic fragment 8
was found to be identical to an intermediate described in the
total synthesis of the micacocidin family of natural
products.9−11
Further chemical investigation of another column fraction of
the crude extract indicated the presence of an additional
natural product, and possible biosynthetic intermediate to 1a
and 1b, anaenoic acid (2) [colorless, solid, [α]25D −35 (c 0.15,
CHCl3); HRMS (ESI) m/z [M + Na]+ calcd for C22H32O7Na,
431.2045, found 431.2041. Based on the molecular formula
and 2D NMR data, 2 was found to only differ from 1a and 1b
by the lack of the amide-linked α,β-unsaturated ester. In order
Synthesis of fragment 8, as previously described,7
commenced with the lithium-directed acetylation of 9 with
ethyl chloroformate and subsequent substitution of the tertiary
amine to yield benzyl chloride 10 (Scheme 2). In order to
install the remaining four carbons (i.e., C-9−C-12) of the alkyl
chain, 10 was converted to the phosphorus ylide and reacted
with crotonaldehyde under standard Wittig conditions yielding
11 as a mixture of dienes. Further reduction and hydrolysis of
B
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bond to (1) unfavorable ring electronics due to the ortho
alkyl/OMe substituents and (2) steric clash between the bulky
R groups on the branched amino acid and alkyl headgroup.14
Utilizing trifluoroacetic anhydride (TFAA), we were able to
access 12 on a half-gram scale with 66% yield (two steps).
Anaenoic acid (2) was synthesized from 12 and 6 under
standard DCC/DMAP coupling conditions and subsequent
deprotection to the free acid. Boc Intermediates of 3a and 3b
were first synthesized via HWE olefination from commercially
purchased 4 and freshly prepared 5, and the products were
subsequently deprotected under acidic conditions.7 Utilizing 2
as a point of chemical divergence, intermediates 3a and 3b
were found to react in the presence of EDC/HOBt to yield
Scheme 1. Retrosynthetic Analysis of Anaenamide Scaffold
1
anaenamide A (1a) and anaenamide B (1b). The H and 13C
NMR data for the three isolated natural products and the
synthetic compounds perfectly matched (Figures S16−S21).
Additionally, optical rotations for the synthetic compounds
were consistent with the isolated natural products, confirming
the correct absolute configuration.
Since marine cyanobacteria are prolific producers of
cytotoxic anticancer agents15−19 with different mechanisms of
action, including templates for payloads of FDA-approved
antibody−drug conjugates,19,20 we subjected the three
compounds initially to cancer cell viability assays. Compounds
1a and 1b were first tested alongside 2 for antiproliferative
activity to HCT116 colorectal cancer cells (Figure 3). While 2
only displayed slight activity at the highest concentration
tested (∼20% inhibition at 80 μM), 1a and 1b showed low
micromolar activity, implicating the C-terminal residue as the
pharmacophore. Additionally, we observed a SAR due to
double-bond geometry as 1a elicited 2-fold increased potency
relative to 1b (IC50 2.8 vs 4.8 μM). We sought to further
explore this SAR via compound 1c, which notably lacked the
11 furnished the first fragment of the scaffold on gram scale.
To our surprise, the esterification of 8 with 7 was notably
challenging and did not occur under standard conditions (e.g.,
Yamaguchi esterification, Steglich coupling, and acid chloride
activation).12,13 We attributed the difficulty of forging this
Scheme 2. Total Synthetic Route to 1a−c and 2
C
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pharmacophore for the cytotoxic activity. We hypothesize
that this highly unusual modification could function as a
Michael acceptor, driving the observed SAR. In light of our
identification of the halogenated α,β-unsaturated ester as a
synthetically “tunable” pharmacophore, our ongoing work will
explore the biological impacts of C-terminal residue function-
alization.
ASSOCIATED CONTENT
* Supporting Information
■
sı
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.orglett.0c01281.
Figure 3. Dose−response curves for synthetic 1a, 1b, 1c, and 2 in
HCT116 cell viability assays using MTT. Error bars represent
standard deviations of mean IC50 values based on three technical
replicates.
1
Experimental procedures, NMR Tables S1 and S2, H,
13C, COSY, HMBC, 2D NOESY, and HSQC NMR
spectra of natural 1a, 1b, and 2 in addition to their
synthetic equivalents and intermediates in CDCl3, and
Scheme S1 (PDF)
halogenation and α,β-unsaturation (Scheme 2). Ablation of
these structural features resulted in a ∼10-fold decrease in
cytotoxicity. While this compound was still marginally active
(IC50 37.5 μM), we hypothesize that at higher concentrations
either alternative cytotoxic mechanisms are observed in this
cell line or that the presence of the conjugated π system
reinforces target engagement. The unsaturated C-terminal unit
could impact inherent binding affinity or selectivity via
covalent or noncovalent interactions. Compound 1c may
later function as a control in our mechanistic studies.
AUTHOR INFORMATION
Corresponding Author
■
Hendrik Luesch − Department of Medicinal Chemistry and
Center for Natural Products, Drug Discovery and Development
(CNPD3), University of Florida, Gainesville, Florida 32610,
United States; orcid.org/0000-0002-4091-7492;
Phone: (352) 273-7738; Email: luesch@cop.ufl.edu;
Fax: (352) 273-7741
On the basis of their structural features, the anaenamides are
likely biosynthesized from a PKS/NRPS hybrid pathway
containing undescribed cyanobacterial enzymology (Scheme
S1). Putatively, we propose that the initiation module in the
biosynthesis of 1a-2 includes a fatty acid AMP ligase domain
and type 1 PKS, yielding the alkyl salicylic acid residue.21,22
Interestingly, the same 5-carbon alkyl salicylic acid fragment
observed in the anaenamides also serves as an intermediate in
the biosynthesis of the micacocidins, and originates from an
iterative type 1 PKS.10,11 Studies of cyanobacterial lipopeptides
(e.g., hassallidins and puwainaphycins) and their gene clusters
have uncovered similar FAAL/PKS initiation domain mo-
tifs.23−25 As (2R,3S)-HMPA is a derivative of D-allo-isoleucine,
the first NRPS module would include the corresponding
adenylation, and α-ketoreductase domains.26 The L-lactic acid
residue could be directly incorporated from a single NRPS
module or derived from L-alanine via a 2-oxopropanoic acid
intermediate. Given the high abundance of anaenoic acid (2)
(0.13% dry weight), it is our opinion that 2 is a biosynthetic
intermediate for 1a and 1b. The four-carbon backbone of the
chlorinated α,β-unsaturated ester could originate from glycine
and acetate. This chemistry is exemplified by the BaeJ gene,
coding for the PKS/NRPS bacillaene, where glycine resides are
modified and extended by downstream PKS modules.27,28 As
this functional group has never been reported in cyanobacteria,
our efforts to characterize the underling cellular machinery is
ongoing.
Authors
David A. Brumley − Department of Medicinal Chemistry and
Center for Natural Products, Drug Discovery and Development
(CNPD3), University of Florida, Gainesville, Florida 32610,
United States
Sarath P. Gunasekera − Smithsonian Marine Station, Ft. Pierce,
Florida 34949, United States
Qi-Yin Chen − Department of Medicinal Chemistry and Center
for Natural Products, Drug Discovery and Development
(CNPD3), University of Florida, Gainesville, Florida 32610,
United States
Valerie J. Paul − Smithsonian Marine Station, Ft. Pierce, Florida
34949, United States; orcid.org/0000-0002-4691-1569
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.orglett.0c01281
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This research was supported by the National Institutes of
Health, NCI Grant No. R01CA172310. We thank the Harbor
Branch Oceanographic Institute at Florida Atlantic University
spectroscopy facility for 600 MHz NMR spectrometer time
and optical rotation measurements for purified natural
products. We are grateful to J. M. Sneed at the Smithsonian
Marine Station and J. Biggs and the staff of the University of
Guam Marine Laboratory for assistance with collections. Our
16S rDNA sequencing was made possible by the efforts of T.
Sauvage and L. dos Santos. We additionally acknowledge V. A.
Folimonova (University of Florida) for her assistance with
HPLC purification of synthetic compounds, data analysis, and
manuscript editing. Our cell culture experiments benefited
from consultations with R. Ratnayake (University of Florida;
In summary, we have described the isolation of the
anaenamide family of natural products (1a, 1b, and 2) from
a green filamentous cyanobacterium Hormoscilla sp. These new
compounds were successfully synthesized, confirming our
structural assignments. Our strategic linear synthesis will
provide a platform for the rapid generation of chemically
diverse, biological probes, as demonstrated by the success of
1c. While 1a and 1b displayed moderate cytotoxicity against
HCT116 cancer cells, our results have demonstrated that the
halogenated α,β-unsaturated ester moiety serves as the
D
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(17) Chen, Q. Y.; Liu, Y.; Cai, W.; Luesch, H. Improved Total
Synthesis and Biological Evaluation of Potent Apratoxin S4 Based
Anticancer Agents with Differential Stability and Further Enhanced
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Our proposed biosynthetic scheme greatly benefited from the
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Department of Agriculture Division of Aquatic and Wildlife
Resources for permits. This is contribution number 1139 from
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