Isolation, synthesis and bioactivity studies of phomactin terpenoids

Article abstract of DOI:10.1038/s41557-018-0084-x

Studies of secondary metabolites (natural products) that cover their isolation, chemical synthesis and bioactivity investigation present myriad opportunities for discovery. For example, the isolation of novel secondary metabolites can inspire advances in chemical synthesis strategies to achieve their practical preparation for biological evaluation. In the process, chemical synthesis can also provide unambiguous structural characterization of the natural products. Although the isolation, chemical synthesis and bioactivity studies of natural products are mutually beneficial, they are often conducted independently. Here, we demonstrate the benefits of a collaborative study of the phomactins, diterpenoid fungal metabolites that serve as antagonists of the platelet activating factor receptor. Our isolation of novel phomactins has spurred the development of a bioinspired, unified approach that achieves the total syntheses of six congeners. We also demonstrate in vitro the beneficial effects of several phomactins in suppressing the rate of repopulation of tumour cells following gamma radiation therapy.

Full text of DOI:10.1038/s41557-018-0084-x

Articles
https://doi.org/10.1038/s41557-018-0084-x
Isolation, synthesis and bioactivity studies of
phomactin terpenoids
Yusuke Kurodaꢀ ꢀ1, Karen J. Nicacioꢀ ꢀ2, Ildefonso Alves da Silva-Jrꢀ ꢀ3, Paul R. Leger1, Stanley Chang1,
Juliana R. Gubiani2, Victor M. Deflon2, Nozomu Nagashima1, Alexander Rode1, Katherine Blackford1,
Antonio G. Ferreira4, Lara D. Setteꢀ ꢀ5, David E. Williams6, Raymond J. Andersen6, Sonia Jancarꢀ ꢀ3*,
Roberto G. S. Berlinckꢀ ꢀ2* and Richmond Sarpongꢀ ꢀ1*
Studies of secondary metabolites (natural products) that cover their isolation, chemical synthesis and bioactivity investigation
present myriad opportunities for discovery. For example, the isolation of novel secondary metabolites can inspire advances in
chemical synthesis strategies to achieve their practical preparation for biological evaluation. In the process, chemical synthesis
can also provide unambiguous structural characterization of the natural products. Although the isolation, chemical synthesis
and bioactivity studies of natural products are mutually beneficial, they are often conducted independently. Here, we demon-
strate the benefits of a collaborative study of the phomactins, diterpenoid fungal metabolites that serve as antagonists of the
platelet activating factor receptor. Our isolation of novel phomactins has spurred the development of a bioinspired, unified
approach that achieves the total syntheses of six congeners. We also demonstrate in vitro the beneficial effects of several
phomactins in suppressing the rate of repopulation of tumour cells following gamma radiation therapy.
he study of secondary metabolites (natural products) and Phoma sp. Additional phomactins, including phomactins B–G6,7,
their biological function often begins with their isolation phomacta-1(14),3,7-triene and Sch 490278, are produced by the
T
and characterization from a producing organism, followed same species and were reported between 1993 and 1995. Subsequent
by screening of the isolates for bioactivity. Secondary metabolites to these reports, Koyama’s analysis of a culture from an unidentified
that reveal interesting properties may then be chemically prepared fungus (MPUC 046) yielded the structurally related compounds that
to obtain larger quantities of materials to facilitate additional bio- were named phomactins H9, I–J10, K–M11 and N–P12. Our collec-
activity studies. Syntheses of these often complex molecules may tion and analysis of culture medium of the marine-derived fungus
also be used to support or revise their structural characterization Biatriospora sp. (CBMAI 1333)13 has now yielded phomactins Q–V
in cases where the structure cannot be unambiguously ascertained1. (1–6, Fig. 1a), resulting in the isolation, so far, of 27 phomactins.
Finally, chemical synthesis may also provide derivatives of the natu- The structures of the newly isolated phomactins 1–6 were eluci-
ral isolate not accessible through biosynthesis or late-stage modi- dated by spectroscopic analysis using one- (1D) and two-dimen-
fication that might possess unmatched bioactivity2,3. A medicinal sional (2D) NMR techniques and mass spectrometry. The structure
chemistry campaign may then be undertaken to obtain a promising of phomactin S (3) was unambiguously determined by X-ray dif-
drug candidate4. Given this holistic approach to identifying novel fraction analysis (Supplementary Fig. 39), which also corroborated
bioactive entities and pharmaceuticals based on natural products, the absolute configuration we have assigned to this compound.
it is surprising that the isolation, synthesis and functional assays Given its unusual framework and that the small amount of isolated
of secondary metabolites are seldom undertaken contemporane- material decomposed over time, the structure of phomactin T (4)
ously. Such an approach will undoubtedly create a ‘feedback loop’ was more challenging to establish. It was ultimately secured using
that would accelerate the discovery process and broaden its scope 2D NMR techniques (Supplementary Table 17) as well as by a com-
at each stage. Herein, we report a collaborative approach to study- parison of its spectral data with that of material prepared through
ing natural products in the phomactin family that spans isolation, chemical synthesis (see the ‘Chemical synthesis’ section for details).
chemical synthesis and investigation of bioactivity. Our studies
The phomactins, particularly phomactin A (7, Fig. 1b), emerged
have (1) yielded novel members of these marine diterpenoids that from an assay-guided fractionation of fungal metabolites to identify
are produced by a unique fungal strain, (2) established a chemi- platelet activating factor receptor (PAFR)14 antagonists. Notably, 7
cal synthesis strategy that achieves the shortest total syntheses of had no effect on collagen-induced platelet aggregation. Blocking
several congeners from carvone, and (3) led to the discovery of the PAFR has proven effective in experimental models of asthma,
beneficial function of several phomactins in suppressing the rate of renal disease, sepsis and pancreatitis, among other diseases15. It has
repopulation of tumour cells after gamma radiation therapy.
been shown previously that combination therapy employing PAFR
The phomactins (see Fig. 1a for representative members) are antagonists and radiation or chemotherapy is particularly effec-
diterpenoids first isolated in 19915 from cultures of the fungus tive in inhibiting human melanoma (B16F10 and SKmel137 cells,
1Department of Chemistry, University of California, Berkeley, CA, USA. 2Instituto de Química de São Carlos, Universidade de São Paulo, São Carlos, SP,
Brazil. 3Departamento de Imunologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, SP, Brazil. 4Departamento de Química,
Universidade Federal de São Carlos, São Carlos, SP, Brazil. 5Departamento de Bioquímica e Microbiologia, Instituto de Biociências, Universidade Estadual
Paulista Júlio de Mesquita Filho, Campus Rio Claro, Rio Claro, SP, Brazil. 6Departments of Chemistry and Earth, Ocean & Atmospheric Sciences, University
of British Columbia, Vancouver, British Columbia, Canada. *e-mail: sojancar@icb.usp.br; rgsberlinck@iqsc.usp.br; rsarpong@berkeley.edu
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a
O
O
H
Me
O
O
Me
O
Me
OH
O
Me
O
O
H
H
Me
Me
Me
Me
Me
Me
Me
Me
Me
O
O
O
O
O
O
Me
Me
OH
Me
HO
Me
Me
Me
Me
Me
Me
O
O
O
Me
Me
Me
Phomactin R (2)
Me
Me
Phomactin U (5)
Me
OH
Phomactin Q (1)
Phomactin S (3)
Phomactin T (4)
Phomactin V (6)
b
O
O
O
O
HO
Me
Me
Me
Me
Me
Me
Me
O
O
O
O
–H2O
[O]
[O]
Me
Me
Me
Me
Me
Me
O
O
O
Me
Phomactin K (13)
Me
Me
Me
Phomactin P (12)
Phomactin R (2)
Phomactin E (11)
20
Me
Me
OPP
Me
O
Type A
[O]
?
18
Me 11
Me
Me
Me
1
15
15
13
O
19
Me
[O]
16
Me
Me
Common synthetic
intermediate
Me
Me
9
6
Me
Me
Proposed common
biosynthetic intermediate
17
Me
?
Type B
[O]
Geranylgeranyl pyrophosphate
Phomacta-1(14),3,7-triene
(8)
(9)
(10)
O
O
O
O
O
H
H
OH
OH
OH
O
Me
Me
H
O
Me
OH
Me
Me
O
O
[O]
[O]
+
Me
Me
OH
Me
O
Me
Me
Me
Me
H
O
Me
Me
Me
Me
Sch 49027 (14)
OH
Phomactin T (4)
Phomactin A (7)
Phomactin G (15)
[proposed structure]
c
OH
O
Me
Me
Me
Me
Me
Me
CHO
PhO2S
+
TBSO
I
Me
Me
Me
Rearrangement of
carvone core
Me
(S )-(+)-carvone
(19)
17
18
Common synthetic
intermediate (16)
Fig. 1 | Members of the phomactin family and retrosynthetic analysis based on the plausible biosynthetic pathway. a, Newly isolated phomactin
congeners. b, Plausible biosynthetic pathways for the phomactins. c, Retrosynthesis of common synthetic intermediate 16.
respectively) and TC-1 (tissue culture number 1) murine carcinoma were achieved in racemic form, ranging in step count from 18 to
regrowth16. PAFR antagonists could therefore serve as adjuvants 37 (longest linear sequence, LLS) starting from commercially avail-
in cancer therapy by inhibiting the interaction between PAFR and able materials19–22. Only two reports of total syntheses culminating
PAF-like molecules generated during chemotherapy or irradiation17. in enantioenriched phomactin natural products have appeared: in
In addition to their function as PAFR antagonists, the intrigu- 1996, Yamada and co-workers reported an enantioselective synthe-
ing structures of the phomactins, which feature a bicyclo[9.3.1]pen- sis of phomactin D in 36 steps (LLS) and, in 2003, Halcomb and
tadecane core comprised of a cyclohexenyl ring and macrocyclic colleagues disclosed an enantiospecific synthesis of phomactin A in
‘strap’ that bridges the ring, have inspired interest in these mole- 27 steps (LLS) from (R)-(+)-pulegone23,24
.
cules from synthetic chemists for over 20 years. The many inventive
An unmet challenge in the chemical synthesis of the phomactins
approaches to the synthesis of the phomactins have been reviewed is to devise a strategy that leads to all the structural types in the
recently18. So far, six total syntheses of phomactin congeners have family—ideally from a common late-stage intermediate. To address
been reported. Four of these syntheses (of phomactins A, G and B2) this challenge, our retrosynthetic analysis, informed by the existing
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O
OH
O
1) m-CPBA
(98% yield, 1:1 d.r.)
Me
Me
4) [Rh(cod)OH]2, MeOH
Me
Me
Me
PhS
PhS
(75% yield)
2) Cp2TiCl2, Zn
(66% yield, 2:3 d.r.)
Me
Variations from
the above condition
Product
Me
3) PhSSPh, Bu3P
(91% yield)
Cs2CO3 instead of [Rh]
Added phosphine ligands
Benzene instead of MeOH
Carvone
NR
Decomp.
20
21
(S)-(+)-carvone
(19)
5) (NH4)6Mo7O24
H2O2
(95% yield)
OSEM
O
O
6) MeLi
7) Burgess reagent
(86% yield, 2 steps)
Me
Me
Me
9) 18, t-BuLi
(89% yield, 1:1 d.r.)
Me
PhO2S
PhO2S
PhO2S
8) SeO2 (56% yield)
10) SEMCl (88% yield)
Me
TBSO
23
Me
Me
Me
17
22
Me
11) TBAF
12) MsCl, LiBr
OSEM
Me
OH
OSEM
Me
Me
14) Na•Hg
Me
PhO2S
(75% yield, 86% brsm)
13) NaHMDS
PhO2S
Me
Me
Br
Me
Me
(53% yield, 3 steps)
Me
15) TBAF, DMPU
16) MnO2; NaBH4, CeCl3
(56% yield, 2 steps, 13:1 d.r.)
Me
24
Me
Me
25
16
Fig. 2 | Synthesis of common intermediate 16. m-CPBA, meta-chloroperoxybenzoic acid; Cp2TiCl2, bis(cyclopentadienyl)titanium dichloride; cod,
1,5-cyclooctadiene; SEM, 2-(trimethylsilyl)ethoxymethyl; TBAF, tetra-n-butylammonium fluoride; MsCl, methanesulfonyl chloride; NaHMDS, sodium
bis(trimethylsilyl)amide; DMPU, 1,3-dimethyltetrahydropyrimidin-2(1H)-one; BINAP, 2,2'-bis(diphenylphosphino)-1,1'-binaphthyl; NR, no reaction; brsm,
based on recovered starting material.
syntheses, took inspiration from the proposed biosynthesis of these oxygenation events that would have otherwise been difficult. In our
intriguing fungal secondary metabolites (Fig. 1b). Like other diter- envisioned bioinspired sequence, a series of C2 hydroxy-directed
penoids, the phomactin natural products are believed to arise from epoxidations and subsequent epoxide openings from 16 could yield
geranylgeranyl pyrophosphate (8), which is advanced to phomacta- divergent syntheses of many of the phomactin natural products.
1(14),3,7-triene (9) through a series of carbocation-initiated cycliza- Oxidized phomactatriene derivative 16 would arise from cyclohex-
tion events and attendant methyl and hydride shifts. The feasibility enyl fragment 17 and vinyl iodide 18, building on the pioneering
13
of these biosynthetic transformations is supported by C labelling studies of Yamada to construct the macrocyclic strap23.
and density functional theory calculations25,26. Although the bio-
Our analysis of the previous syntheses of the phomactins indi-
synthesis of the metabolites further down the biosynthetic pathway cates to us that while inventive and efficient syntheses of the macro-
from 9 (Fig. 1b) remains largely speculative, the oxygenation pat- cyclic strap have been achieved, a significant investment of chemical
tern of these downstream congeners suggests that 10 may serve as a steps has gone into preparing the densely functionalized cyclohex-
branching point in their biosynthesis. For example, a stereoselective enyl-derived portion of the phomactins (especially for syntheses in
hydroxylation (type A oxidation, Fig. 1b) at C15 in 10 would lead to enantioenriched form). Therefore, gains in efficiency for any new
phomactin E (11) and subsequently phomactin R (2), from which synthesis of the phomactins will primarily arise by identifying a
phomactin P (12) and phomactin K (13) may arise through a series shorter and more efficient synthetic sequence to the cyclohexenyl-
of additional enzymatic oxidations. On the other hand, phomac- derived fragment adorned with the strategically disposed functional
tin A (7), Sch 49027 (14) and phomactin T (4) probably arise from groups necessary for the preparation of congeners. In a key recogni-
phomactin G (15) via an alternative pathway starting with the selec- tion for our synthesis, we saw methylidene cyclohexene 17 (bearing
tive oxidation of 10 (type B oxidation). To emulate P450-mediated challenging vicinal tertiary and quaternary carbon centres) arising
chemoselective oxygenations in the biosynthesis of phomactins, we from (S)-(+)-carvone (19), an abundant terpene feedstock available
designed a suitably oxygenated phomactatriene derivative (16) as a as either antipode (Fig. 1c). Although carvone has been extensively
common chemical synthesis intermediate (Fig. 1c). The choice of 16 employed as a starting material for synthesis, strategies that exploit
hinged on maintaining the same overall oxidation level as 10 so as the rearrangement of its core framework to access other cyclohexe-
to facilitate access to the majority of the phomactin congeners. By nyl derivatives, of which our recent work is representative27, are rare.
inverting the oxidation state at C2 (see 9, Fig. 1b, for numbering) We sought to employ such a rearrangement of the core framework
and C15 of 16 relative to 10 with the installation of a hydroxyl group of (S)-(+)-carvone that subsumes the isopropenyl group to provide
at C2, we were poised to site-selectively direct the downstream rapid access to 17.
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OH
O
O
O
H
H
H
Me
Me
Me
Me
Me
Me
Rh2(cap)4
TBHP
1) VO(OEt)3, TBHP
–78 °C (72%)
m-CPBA
O
O
O
Me
Me
Me
Me
Me
Me
13
2) DMP (90%)
then DBU
(52%)
(66%)
O
O
O
Me
16
Me
Phomactin R (2)
Me
Phomactin P (12)
Me
Phomactin K (13)
VO(OEt)3, TBHP
0 °C (65%)
RO
AcO
20
OH
H
OH
OH
O
O
OH
Me
Me
Me
H
O
Me
Me
H
Me
H
Me4NBH(OAc)3
CsOAc, 18-c-6
1) DMP, 0 °C
Warm to r.t.
O
O
O
O
Then
NaOMe
Me
OH
Me
O
14
Me
Me
Me
Me
Me
Me
27; R = Ac (66%)
Me
Me
26
29
30
28; R = H (63% after NaOMe quench)
2) Red-Al
Sch 49027 (32): 23%
Phomactin A (7): 51%
Then NaOMe
(62% from 27)
1) DMP
(2 steps from 27)
Me
O
H
O
O
O
O
OH
OH
Me
H2O
Me
Me
H
O
O
O
Me
Me
H
O
Me
Me
H
O
2) m-CPBA
Me
H
O
CH2Cl2, H2O
OH
OH
Me
Me
Me
H
O
H
O
Me
Me
Me
Me
34
31
33
Sch 49027 (32)
[revised structure]
O
Me
O
O
O
H
OH
O
H
OH
Me
Me
H
O
O
Me
OH
Me
Me
OH
Me
O
O
O
Me
MnO2
OH
Me
Me
Me
O
Me
H
O
31%, 2 steps
O
from 28
OH
Me
Sch 49027 (14)
[proposed structure]
Me
Me
Me
OH
Phomactin T (4)
35
Phomactin A (7)
Fig. 3 | Completion of the total syntheses of phomactins A, K, P, R and t and Sch 49027. TBHP, tert-butyl hydroperoxide; DMP, Dess–Martin periodinane;
cap, caprolactamate; DBU, 1,8-diazabicyclo(5.4.0)undec-7-ene; Red-Al, sodium bis(2-methoxyethoxy)aluminum hydride; 18-c-6, 18-crown-6; r.t., room
temperature.
Results and discussion
fragmentation to give carvone, (2) addition of phosphine ligands
Chemical synthesis. Our synthetic studies commenced with the including Ph3P, n-Bu3P and BINAP completely inhibited reactivity,
preparation of methylidene cyclohexene 17 (Fig. 2). Following the and (3) the use of MeOH as solvent was singularly effective. In this
precedent of ref. 28, the cyclobutanol moiety was installed through way, an overall rearrangement of the carvone core framework to
epoxidation of (S)-(+)-carvone, followed by Ti(iii)-mediated integrate the isopropenyl group was achieved via 20 to furnish 21
reductive coupling of the epoxide and carbonyl functional groups . Oxidation of the phenyl sulfide moiety of 21 gave phenyl sulfone
to provide a 2:3 ratio of cyclobutanol diastereomers, of which the 22, which was treated with MeLi followed by the Burgess dehydrat-
minor was carried forward. Mitsunobu-type displacement of the ing reagent to append C20 of the phomactins. Allylic oxidation of
resulting primary hydroxyl group gave phenyl sulfide 20. With the vinylic methyl group was accomplished using SeO2 to afford
cyclobutanol20 inhand, weinvestigatedtheopeningofthecyclobu- aldehyde 17. 1,2-Addition of a vinyl lithium nucleophile derived
tanol to install the angular methyl group (C19) of the phomactins. from known vinyl iodide 1823 gave a bis-allylic alcohol (1:1 mixture
Using an adaptation of our recently reported protocol27 that builds of diastereomers) that was protected with a 2-(trimethylsilyl)eth-
on precedent from refs 29,30, exposure of 20 to catalytic [Rh(cod) oxymethyl (SEM) group to afford 23. Treatment of 23 with TBAF
OH]2 in MeOH effects the desired C–C bond cleavage to afford unveiled the primary hydroxyl, which was converted to allylic
cyclohexene 21 in 75% yield. It is worth noting that (1) exposure bromide 24 via the mesylate. Macrocyclization of sulfone/allylic
of 20 to inorganic bases such as Cs2CO3 only promoted Grob bromide 24 was effected by treatment with NaHMDS (in line with
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give diol 27 or triol 28 (when the acetyl group was cleaved using a
NaOMe quench). With the oxygen installed at C20, we proceeded
to synthesize phomactin A. Oxidation of diol 27 using the Dess−
Martin periodinane reagent at room temperature, followed by
cleavage of the acetyl group using NaOMe, leads to ketone 30 where
hemi-ketal formation occurs spontaneously. It is of note that exclu-
sive formation of keto-alcohol 29 was observed when the reaction
temperature was kept at 0°C. The final step required stereoselective
reduction of the C14 ketone in the presence of the labile hemi-ketal.
An attempted hydroxyl-directed reduction using Me4NBH(OAc)3
(ref. 35) gave complex mixtures, whereas, surprisingly, NaBH4 or
LiAlH4 brought about the exclusive formation of a compound with
analytical data fully consistent with that reported for Sch 49027.
However, our analysis of the spectral data reported for Sch 49027
indicated that it was more consistent with the allylic alcohol struc-
ture obtained from the reduction step (that is, 32) and not the
unusual enol 14. Because a pathway to convert 32 to 14 is not obvi-
ous, we propose that the structure of Sch 49027 should be revised
to 32. Allylic alcohol 32 was also obtained in 62% yield when keto-
alcohol 29 was treated with NaOMe, further corroborating its struc-
ture. Notably, reduction of ketone 30 with Red-Al affords a 2.2:1
mixture of phomactin A (7) and 32. Phomactin A probably arose
from diastereoselective carbonyl reduction of 30 to give 31 followed
by cyclization of the resultant hydroxyl onto the epoxide. To access
phomactin T, triol 28 was oxidized to keto-aldehyde 33 using the
Dess−Martin periodinane reagent. On exposure of 33 to m-CPBA,
lactol 35 formed directly through a cascade presumably initiated by
stereoselective epoxidation of the double bond followed by incorpo-
ration of the hemi-acetal in 34. Although this transformation effec-
tively constructs the phomactin T framework, the resulting lactol
proved unstable and slowly decomposed. Therefore, a superior pro-
cess was developed wherein the epoxidation step was telescoped with
a MnO2 oxidation to afford phomactin T (4). In summary, total syn-
theses of phomactin A (20 steps), phomactin K (20 steps), phomac-
tin P (19 steps), phomactin R (18 steps), phomactin T (20 steps)
and Sch 49027 (19 steps) were achieved from (S)-(+)-carvone. In
the process, the structure of phomactin T was confirmed and the
structure of Sch 49027 was revised to 32. This synthetic work set the
stage for our bioactivity studies.
Table 1 | PAFR-inhibitory concentration (IC50) of phomactins
(μM)
Phomactins
IC50
Phomactin A
Phomactin F
Phomactin I
Phomactin P
Phomactin R
Phomactin S
Phomactin U
Phomactin V
WEB 2170
3.8
2.7
3.2
3.0
2.5
2.8
10.0
3.1
3.2
Concentration of phomactins or WEB 2170 (in µM) that reduced 50% of the response to 10ꢀnM
of cPAF (IC50). Inhibitory doses were generated using three-parameter non-linear regression
analysis from nꢀ=ꢀ3 independent experiments (see the ‘Biological assays’ and ‘Statistical
analysis’ sections for more details). cPAF, carbamoyl-PAF (1-hexadecyl-2-N-methylcarbamoyl
glycerophosphocholine).
Table 2 | Inhibition of tumour cell repopulation by phomactin
congeners
Phomactins
% inhibition of RLu*
Phomactin A
Phomactin R
Phomactin S
Phomactin P
Phomactin U
Phomactin F
Phomactin V
Phomactin I
WEB 2170
55ꢀ ꢀ11
83ꢀ ꢀ7
68ꢀ ꢀ2
29ꢀ ꢀ5
34ꢀ ꢀ5
77ꢀ ꢀ3
47ꢀ ꢀ2
49ꢀ ꢀ6
71ꢀ ꢀ2
*% inhibition of RLU by 10ꢀμM of phomactin compounds or WEB 2170 relative to untreated
control irradiated at 8ꢀGy. Data are from nꢀ≥ꢀ3 independent experiments and are presented as
meanꢀ ꢀstandard error of the mean (s.e.m.).
Biological assays. Activation of PAFR in tumour cells through
chemo- or radiotherapy activates intracellular programs that pro-
mote their survival and proliferation36. It is therefore expected that
the precedent of ref. 23) to afford 25. Reductive removal of the sul- combining radiotherapy with PAFR antagonists could provide an
fone group, cleavage of the SEM group with TBAF in DMPU31, and effective combination therapy for reducing tumour cell prolifera-
oxidation of the resulting secondary hydroxyl followed by diaste- tion. En route to this eventual goal, we evaluated the PAFR antago-
reoselective NaBH4 reduction gave 16, which serves as a common nist activities of several of the phomactins obtained from cultures
synthetic intermediate to phomactin natural products in each of of Biatriospora sp. (CBMAI 1333) or through chemical synthesis.
the known structural classes.
Isolated and chemically synthesized phomactins were screened for
With key intermediate 16 in hand, we focused our attention PAFR antagonist activity in human epidermal carcinoma (KB) cells
on the syntheses of phomactins R, P and K. Hydroxyl-directed transfected with PAFR (KBP) by measuring interleukin 8 (IL-8)
epoxidation of 16 with VO(OEt)3 (ref. 32) and TBHP at −78 °C concentrations (see Methods and Supplementary Fig. 40a,b). A half-
followed by oxidation of the hydroxyl group gave phomactin R maximum inhibitory concentration (IC50) of 3.2μM was obtained
(2, Fig. 3). The use of VO(OEt)3 was critical to achieving good for the PAFR antagonist WEB 2170 (Supplementary Fig. 40c), which
diastereoselectivity in the epoxidation. In line with the presumed served as a control. This IC50 value is in agreement with the previ-
biosynthesis of the phomactins, epoxidation of phomactin R (2) ously determined potency for this compound in a human breast
using m-CPBA yields phomactin P (12). Furthermore, oxidation cancer cell line37. Table 1 shows the IC50 values of various phomac-
of the allylic C13 position of 12 using Rh2(cap)4 (ref. 33) and TBHP tins as PAFR antagonists. Phomactins A, S, P, F, V and I were found
affords phomactin K (13).
to be as potent as WEB 2170, displaying IC50 values between 2.8 and
Alternatively, a double epoxidation of 16 occurs following its 3.2µM, within the range of previously determined values5,8. Notably,
exposure to VO(OEt)3 and excess TBHP at 0°C to give bis-epox- phomactin R emerged as the most potent of the phomactins, with an
ide 26, setting the stage for a crucial epoxide opening to install the IC50 value of 2.5µM. At the concentrations employed in this study,
requisite hydroxy group at C20 of phomactins A, T and Sch 49027. none of the phomactins was found to adversely affect cell viability
After extensive experimentation, we found that treatment of 26 with as determined using the lactate dehydrogenase (LDH) cytotoxic-
Me4NBH(OAc)3 (ref. 34) in the presence of CsOAc and 18-crown-6 ity assay38. Overall, these in vitro studies reveal bioactivities for the
initiated SN2′ epoxide opening with acetate as the nucleophile to phomactins that approach and in some cases surpass those of an
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established PAFR antagonist (WEB 2170). However, a more com- a wide range of phomactin congeners, including the novel iso-
prehensive comparison to other phomactins such as phomactin D lates reported here. In doing so, chemical synthesis facilitated
using the same assay will need to be conducted. Also, given the lim- their unambiguous structural characterization. In addition, our
ited data set, no definitive structure–activity relationships (SARs) PAFR antagonist activity evaluation of the phomactins at an
for the phomactins could be gleaned from the results that we have early stage inspired the novel tumour cell repopulation studies
so far obtained. A summary of some SAR trends that appear to be of the phomactins. Finally, unambiguous structural elucidation
emerging are summarized in the Supplementary Information. In of phomactin T was achieved in a case where the material iso-
addition to reducing the viability of tumour cells, gamma irradia- lated from the natural source had decomposed and the mate-
tion also induces the over-production of PAFR ligands, which, by rial required to complete structural analysis was only available
a direct effect or by enhancing prostaglandin E2 formation, pro- through synthesis. Many such scenarios where only a small
mote tumour repopulation39. The investigation of the phomactins amount of a secondary metabolite is isolated, or decomposes,
as inhibitors of tumour cell repopulation was planned only at the exist. In these cases, natural products that may possess useful
completion of our PAFR antagonist assays, and required additional bioactivity go unreported or may be mischaracterized. Here, our
amounts of the natural products that had to be procured by both collaboration yielded additional material through synthesis to
re-isolation from the producing fungus and chemical synthesis. We complete unambiguous structural characterization.
used an in vitro assay to investigate the ability of several phomactin
Finally, several additional studies have been inspired by our col-
natural products to inhibit the repopulation of murine lung carci- laboration. For example, it can be envisioned that some of the late-
noma TC-1 cells following radiation (see ‘Biological assay’ section stage synthetic intermediates en route to our completed bioinspired
for details). Consistent with the PAFR antagonist activity shown in chemical syntheses of the phomactins may themselves be secondary
Table 1, most compounds induced significant inhibition of TC-1 metabolites that exist in the producing organism but are yet to be
repopulation, tracking closely with their PAFR antagonist activ- isolated. We are therefore re-examining the cultures of Biatriospora
ity. Phomactin R showed the highest TC-1 repopulation inhibitory sp. (CBMAI 1333) at different time points to see if some of these
activity in comparison to the other phomactins (Table 2), consistent synthetic intermediates can be detected as secondary metabolites.
with its higher PAFR antagonist activity. Similarly, phomactin U With authentic samples now available from chemical synthesis,
showed the lowest repopulation inhibition activity, which correlates detection of the natural isolates may be accelerated, thus provid-
with its poor PAFR antagonist activity. Overall, our results suggest ing insight into the range of naturally occurring phomactin natural
that irradiated TC-1 cells do indeed produce factor(s) that promote products. Finally, the preliminary biological results described here
repopulation. In addition, we have demonstrated that tumour cell portend excellent bioactivity for derivatives of the phomactins that
repopulation is inhibited by the PAFR antagonist WEB 2170 as well may be accessed through chemical synthesis. These studies (both in
as several phomactin natural products (albeit at modest potencies), vitro and in vivo) constitute a major portion of our future studies of
with phomactin R showing superior results. Given the emerging the phomactins.
significance of PAFR antagonists in inhibiting irradiated tumour
cell repopulation in in vivo systems39, our observations here warrant Methods
Assay of PAFR antagonistic activity. Immortalized human epithelial cells (KBMs)
additional study. Our future work will seek to make more significant
that do not express PAFR were transfected with the PAF-receptor (KBP). Ten,
disruptive changes to the structures of the phomactins to gain more
2×105 KBP cells were plated in 24-well plates in Dulbecco’s modifed Eagle’s
insight into their SARs.
medium (DMEM, GIBCO) supplemented with 10% FCS, penicillin (100Uml−1)
and streptomycin (100µgml−1). Afer 24h, these cells were stimulated with 10nM
methylcarbamyl PAF C16 (cPAF). Afer 6h, supernatants were collected for IL-8
measurement (readout of PAFR activation40) using BD OptEIA ELISA sets (BD
Biosciences). Control PAFR antagonist WEB 2170 and phomactin samples were
added to the KBP cells, in doses ranging from 1 to 100μM (in DMSO), 30min
before cPAF addition. We established 10nM as the concentration of cPAF that
induces 50% of the maximal response. Tis concentration of cPAF was then used to
determine the concentration of WEB 2170 required to cause 50% inhibition (IC50)
(S40). Te same concentration of cPAF was then used to calculate the IC50 of the
phomactins (Table 1). Of note, cPAF was used in this study because it is resistant to
PAF acetyl hydrolase, a cellular enzyme that degrades PAF.
Conclusions
Natural product chemistry remains critical to the discovery of
small molecules that possess unique bioactivities. By pursuing the
isolation, chemical synthesis and biological evaluation of natural
products and their derivatives in tandem, a more effective and com-
prehensive approach to identifying small molecules that possess
significant bioactivity can be established. We have demonstrated
the success of this approach with the phomactin diterpenoids.
Novel congeners of the phomactins (Q–V, 1–6) were obtained from
media extracts of Biatriospora sp. (CBMAI 1333), a fungus isolated
from a marine sponge collected off the Brazilian coastline. A bioin-
spired chemical synthesis approach enabled the efficient prepara-
tion of some of these newly isolated phomactin natural products
as well as previously isolated congeners that are representative of
each phomactin subclass. Our chemical syntheses feature the use of
(S)-(+)-carvone as a starting material, a unique Rh-catalysed
cyclobutanol opening/protonation that formally achieves a rear-
rangement of the carvone framework to incorporate the isoprope-
nyl substituent, and late-stage diversification to access a range of
phomactin congeners. Biological activity investigations revealed
notable PAFR antagonist activity for several of the phomactin natu-
ral products. Although no clear SAR correlations emerged, in the
majority of cases, the PAFR antagonist activity correlates with the
ability of these molecules to inhibit the repopulation of tumour cells
that have undergone gamma irradiation.
Tumour cell repopulation assay. The method used has been described
previously39. The TC-1 cell line (lung epithelium of murine carcinoma) was
used. For the assay, a layer of irradiated (8Gy) or non-irradiated TC-1 cells that
express PAFR (TC-1 cells, 2×105) were co-cultured with 103 luciferase-labelled
TC-1 cells (TC-1 Fluc+). Cell irradiation was conducted using an IBL 136 cell
and animal gamma radiator at a dose rate of 251.7cGymin−1. The 8Gy dose was
selected on the basis of previous studies and represents the best dose for induction
of repopulation phenomena in vitro39,41. After nine days, the proliferation of the
TC-1 Fluc+ cells was evaluated as relative luminescence units (RLU) (S41 A). The
TC-1 Fluc+ cells grew significantly faster when seeded onto irradiated TC-1 than
when seeded onto non-irradiated cells (S41 B). Notably, when seeded onto non-
irradiated cells, the growth of the TC-1 Fluc+ cells was very slow (fluorescence
levels were below detection). Fluc+ TC-1 cells were treated with PAFR antagonists
or phomactins, 30min before irradiation and each 2 days until 9 days of culture.
Results are presented as RLU or % inhibition of RLU relative to the non-treated
group. Of note, all cell lines used in this work were regularly tested for Mycoplasma
and were free of this contaminant.
Cell viability assay. The concentrations of phomactins used in this study were
all assayed for viability to avoid using concentrations that are toxic to the cells.
We measured LDH released into the media from damaged cells as a biomarker
for cellular cytotoxicity and cytolysis. A colorimetric viability assay using
commercially available kits (BioClin) was used according to the manufacturer’s
instructions.
Several aspects of this work would not have been possible had
the isolation, synthesis and bioactivity assays not been conducted
in tandem. For example, we subjectively chose to pursue a bio-
inspired synthetic strategy with an eye towards gaining access to
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NATure CHemISTry
Articles
Statistical analysis. Data were analysed using GraphPad Prism 4.0 software. For each
individual cell-based assay, compounds were run in duplicate (binding) or triplicate
and normalized to the untreated cells. Dose–response curves were generated using
three-parameter nonlinear regression analysis. Data are from n≥3 independent
experiments and are presented as mean s.e.m. or as indicated in the legend.
25. Tokiwano, T., Fukushi, E., Endo, T. & Oikawa, H. Biosynthesis of phomactins:
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2713–2722 (2005).
Materials. Methylcarbamyl PAF (cPAF) was obtained from Cayman Chemical. The
reference compound WEB 2170 was obtained from Boehringer Ingelheim, Pharma
KG and was prepared in DMSO as a 5mM stock solution.
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Reporting Summary. Further information on experimental design is available in
the Nature Research Reporting Summary linked to this article.
Data availability. The characterization data for all new chemical compounds are
provided in the Supplementary Information. The.cif file for phomactin S (3) has
been deposited at the Cambridge Crystallographic Data Centre (CCDC 1830519).
Received: 25 October 2017; Accepted: 15 May 2018;
Published: xx xx xxxx
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Acknowledgements
The authors thank K. Koyama (Meiji Pharmaceutical University) for providing spectral
data for phomactin P and S. Dreher and A. Buevich (Merck Pharmaceuticals) for the
1H NMR spectrum of Sch 49027. R.S. thanks the National Science Foundation (CHE-
1566430) for financial support. Y.K. thanks the Japan Society for the Promotion of
Science (JSPS) for an Overseas Research Fellowship. P.R.L. thanks the National Science
Foundation for a graduate research fellowship. S.C. acknowledges a National Science
and Engineering Council–Canada (NSERC) Postdoctoral Fellowship. R.G.S.B and J.R.G.
thank FAPESP (Fundaçao de Amparo a Pesquisa de São Paulo) for financial support
(BIOTA-BIOprospecTA 2013/50228-8, 2015/01017-0 and 2017/06014-4) and K.J.N.
thanks CNPq for a PhD scholarship. S.J. and I.A.S.J. are grateful to FAPESP for financial
support and a graduate research fellowship. N.N. thanks JSPS for a travel fellowship. K.B.
is grateful to the Amgen Scholar Program (UC Berkeley) for support. R.J.A. thanks the
NSERC for funding. The KBP cells were supplied by J.B. Travers (Indiana University,
Indianapolis, IN) and the TC-1 cell line was donated to us by T.-C. Wu (Johns Hopkins,
Baltimore). The authors thank K. Owens for insightful discussions regarding the
structure of Sch 49027.
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Author contributions
R.G.S.B. (isolation and identification), Y.K. (chemical synthesis), R.S. (chemical
synthesis) and S.J. (biological assays) wrote each of the corresponding sections and
R.S. composed the manuscript. P.R.L., S.C. and R.S. conceived the general plan for the
chemical synthesis of phomactin R. Y.K. and R.S. designed the plan for the chemical
syntheses of phomactins A, K, P, T and Sch 49027. P.R.L. and S.C. carried out the
initial studies on the cyclobutanol opening, cyclohexenone functionalization, and
macrocyclization that provided a part of the basis of the reported syntheses. Y.K.
conducted the chemical reactions reported herein and compiled the Supplementary
Information. N.N. conducted the large-scale preparation of 18 and 20 to support front-
line synthetic studies. A.R. and K.B. carried out exploratory studies on the cyclobutanol
21. Huang, J., Wu, C. & Wulf, W. D. Total synthesis of ( )-phomactin B2 via an
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B-alkyl Suzuki macrocyclization. J. Am. Chem. Soc. 125, 1712–1713 (2003).
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intermediates and subsequent functionalizations. K.J.N. isolated the new phomactins
and elucidated their structures jointly with J.R.G. V.M.D. performed X-ray diffraction
analysis. A.G.F., D.E.W. and R.J.A. provided support and performed NMR analyses of
the new phomactins. L.D.S. provided and identified the fungal strain. I.A.S.J. and S.J.
performed the assays for PAFR antagonistic activity and the repopulation assays with
cancer cell lineages.
Additional information
Supplementary information is available for this paper at https://doi.org/10.1038/
s41557-018-0084-x.
Reprints and permissions information is available at www.nature.com/reprints.
Correspondence and requests for materials should be addressed to S.J. or R.G.S.B.
or R.S.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
Competing interests
The authors declare no competing interests.
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Sonia Jancar, Roberto G. S. Berlinck, Richmond
Sarpong
Corresponding author(s):
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