Pharmacophore-Directed Retrosynthesis Applied to Ophiobolin A: Simplified Bicyclic Derivatives Displaying Anticancer Activity

Article abstract of DOI:10.1021/acs.orglett.0c02938

Pharmacophore-directed retrosynthesis applied to ophiobolin A led to bicyclic derivatives that were synthesized and display anticancer activity. Key features of the ultimate defensive synthetic strategy include a Michael addition/facially selective protonation sequence to set the critical C6 stereocenter and a ring-closing metathesis to form the cyclooctene. Cytotoxicity assays toward a breast cancer cell line (MDA-MB-231) confirm the anticipated importance of structural complexity for selectivity (vs MCF10A cells) while C3 variations modulate stability.

Full text of DOI:10.1021/acs.orglett.0c02938

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Letter
Pharmacophore-Directed Retrosynthesis Applied to Ophiobolin A:
Simplified Bicyclic Derivatives Displaying Anticancer Activity
Yongfeng Tao, Keighley N. Reisenauer, Marco Masi, Antonio Evidente, Joseph H. Taube,*
and Daniel Romo*
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ABSTRACT: Pharmacophore-directed retrosynthesis applied to
ophiobolin A led to bicyclic derivatives that were synthesized and
display anticancer activity. Key features of the ultimate defensive
synthetic strategy include a Michael addition/facially selective
protonation sequence to set the critical C6 stereocenter and a ring-
closing metathesis to form the cyclooctene. Cytotoxicity assays
toward a breast cancer cell line (MDA-MB-231) confirm the
anticipated importance of structural complexity for selectivity (vs
MCF10A cells) while C3 variations modulate stability.
he ophiobolins are fungal-derived sesterterpenoids
structures, unique bioactivities and unknown cellular targets,
and was further enhanced by our finding that OpA, but not 3-
deoxy OpA, specifically induces cell death in stem-like cells
with epithelial-mesenchymal transition (EMT) properties.15
Several structure−activity relationship (SAR) studies of the
anticancer effects of ophiobolins, derived mainly from
derivatization of OpA or congeners, led us to apply our
recently described pharmacophore-directed retrosynthesis
(PDR) strategy16,17 to elaborate on SAR information with
respect to effects on stem-like cancer cells. In particular, the
anticancer effects of various ophiobolins1,2,6 and synthetic
derivatives,10 in conjunction with our own work,15 point to the
importance of the A/B-rings as an initial minimal pharmaco-
phore consisting of a 1,4-keto unsaturated aldehyde moiety,
the C5/C6-relative stereochemistry, the C3-hydroxyl, and syn-
fused cyclooctene (Figure 1, highlighted in yellow). The
potential reactivity of the 1,4-ketoaldehyde to primary amines
including cellular protein lysines through a potential Paal−
Knorr pyrrole reaction has been reported.10,18 Isolation of a
phosphatidylethanolamine adduct from HEK293T cells treated
with OpA supports this notion.19 The natural C6-stereo-
chemistry appears vital for the observed bioactivity across
multiple cell lines with significant loss of activity observed for
T
consisting of over 50 members,1,2 with ophiobolin A
(1, OpA, Figure 1) being the first isolated and also one of the
Figure 1. Structure of selected ophiobolins (A−C) and proposed
minimal pharmacophore (yellow).
most bioactive congeners (Figure 1).3−5 This family of natural
products is drawing increasing interest2,6,7 not only because of
their high potency against various cancer cell lines1,8 (e.g.,
chronic lymphocytic leukemia (CLL), IC50 = 1 nM;8 P335,
IC50 = 63 nM;6 glioblastoma Hs683, IC50 = 620 nM9,10) for
OpA but also for their unique cellular effects and selectivity
observed. For example, both OpA (1) and B11 (2) show
selectivity toward cancer stem cells (CSCs) by significantly
reducing the CD44/CD24-positive population in an MDA-
MB-231 cell line.12 Moreover, the ability of certain ophiobolins
to induce paraptosis7,9,13 or autophagy14 makes these natural
products valuable leads toward apoptosis-resistant cancer cells.
Our interest in the ophiobolins stems from their complex
Received: September 4, 2020
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© XXXX American Chemical Society
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the C6-epimer of OpA.1,2 Molecular modeling studies8 suggest
one rationale for significant loss of bioactivity of 6-epi-
ophiobolin congeners, namely significant alteration of the
eight-membered B-ring conformation. This altered conforma-
tion changes the orientation of the C21 aldehyde, thus
impacting any potential Paal−Knorr condensation with the
ketoaldehyde and also possibly impacting potential Michael
additions to the unsaturated aldehyde.8 Therefore, we
incorporate the cyclooctene into our current proposed
pharmacophore (Figure 1). Efforts toward a broader, deep-
seated SAR profile of the ophiobolins is precluded by structural
convergence of the natural ophiobolins. Thus, we envisioned
that application of PDR would enable a greater dissection of
minimal structural requirements for anticancer activity through
synthesis of simplified derivatives of OpA that retain structural
features known to be critical for activity. Our initial PDR-
guided studies toward this goal are described herein.
To date, three completed total syntheses of various
ophiobolin and derivatives have been reported including the
synthesis of OpA by the Nakada group who employed a late-
stage ring closing metathesis (RCM) to form the 8-membered
B-ring20,21 and ophiobolin C by the Kishi group who employed
a Nozaki−Hiyama−Kishi coupling to generate the C-ring.22
Recently, Maimone reported an elegant 9-step radical cascade
process to fashion the BC-ring system of (−)-6-epi-OpN,23 and
more recently a 15-step synthesis of (+)-6-epi-OpA employing
a similar synthetic strategy.24
Nakada employing an RCM reaction.20,21 Michael addition of
a cuprate derived from iodide 6 to cyclopentenone 5 to form
the C1−C4 bond followed by facially selective protonation
would enable control of the key C6-stereocenter. A protected
secondary alcohol at C3 would enable variations in
substitution and also stereochemistry at this center. The
described synthetic strategy sets the stage for more elaborate
tricyclic congeners or the natural product itself. Importantly,
application of PDR enables the exploration of key strategic
bond disconnections that typically would only be considered
model studies in a total synthesis effort; however, these studies
can be utilized to access simplified derivatives and provide
important SAR information as described herein.
We initiated our synthetic efforts by studying the conjugate
addition of cuprate reagents derived from known neopentyl
iodide 12a26 to enone ( )-5, to mimic the eventual
cyclopentyl cuprate that would be required for tricyclic analogs
(Scheme 1). However, several problems quickly surfaced
Scheme 1. Development of the Conjugate Addition
Sequence Leading to Enol Acetate 16
Herein, we report Stage I and an initial Stage II study of our
PDR approach toward OpA with the goal of gaining further
SAR information for this complex sesterterpene. To address
questions for the C6-stereochemistry, the C3-hydroxy group,
and required ring systems for bioactivity, we applied PDR to
arrive at simplified monocyclic (e.g., known ketoaldehyde
( )-1125) and bicyclic (e.g., ( )-10) derivatives (Figure 2), all
bearing our current hypothesized minimal pharmacophore
(Figure 2, yellow highlight).
Our synthetic strategy placed an emphasis on installing the
critical C6-stereocenter while retaining flexibility at certain
positions such as the C3-hydroxyl bearing center to invert
stereochemistry or alter substitution. For synthesis of the
challenging 8-membered B-ring, we relied on the strategy of
including the anticipated low reactivity of the neopentyl
cuprate reagent which could be overcome by forming the more
reactive higher order organocuprate using the method of
Lipshutz.27 Unfortunately, the resulting cuprate was prone to
5-exo-trig cyclization (see inset, Scheme 1),26,28 likely
accelerated by the Thorpe−Ingold (gem-dimethyl) effect.
This side reaction is unavoidable even at −78 °C unless the
olefin is trisubstituted, directing us toward a defensive
strategy29 and the use of iodide 1330 bearing a pendant
trisubstituted alkene known to lower the rate of intramolecular
cyclization.31 Lithium−halogen exchange32 followed by
addition of (2-thienyl)Cu(CN)Li27 converted iodide 13 into
a reactive organocuprate species leading to conjugate addition
without detectable 5-exo-trig cyclization at −78 °C (Scheme
1). Use of TMSCl33,34 was also beneficial for this conjugate
addition but led to a TMS enol ether that was too labile for
isolation. This problem was circumvented by treating the
crude, intermediate silyl enol ether ( )-15 directly with MeLi
followed by addition of Ac2O. This led to in situ conversion to
the more stable and readily purified enol acetate ( )-16.35
Overall, this process entailed a one-pot conjugate addition/
double-enolate trapping for the key C1−C5 bond construc-
tion. However, enol acetate ( )-16 failed to undergo the
subsequent desired RCM reaction36−39 (not shown) to deliver
the targeted cyclooctene despite trying a variety of conditions.
We anticipated that the syn-substituted dienyl cyclopentane
( )-18 would undergo the RCM reaction more readily.
Toward this end, we next attempted to introduce the C6
stereocenter prior to the RCM through a facially selective
Figure 2. (a) Pharmacophore-directed retrosynthesis applied to OpA.
(b) Staged syntheses of OpA derivatives including monocyclic ( )-11
and bicyclic ( )-10 derivatives bearing minimal pharmacophore
described herein.
B
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protonation of a derived enolate following treatment of the
enol acetate with MeLi. However, enolate generation and
protonation led to complete isomerization of the alkene to the
more conjugated position leading to tetrasubstituted alkene
( )-17 rather than the desired syn-substituted cyclopentane
( )-18. To preclude this olefin isomerization, we decided to
again employ a defensive strategy29 by refining the structure of
the substrates without altering the overall synthetic plan. After
extensive exploration, we settled on introduction of a phenyl
ring at C8 (OpA numbering) to preclude C7−C8 olefin
isomerization (cf. enone ( )-26, Scheme 2) during installation
Enone ( )-26 was then subjected to the one-pot cuprate
addition silylation sequence developed previously; however, a
direct protonation of the intermediate enolate was also
investigated to eliminate a step (Scheme 3). Following the
same procedure developed previously for the conjugate
addition−silylation/desilylation sequence beginning with
iodide 27, a kinetic protonation delivered a single diastereomer
of the trisubstituted cyclopentanone ( )-29 upon quenching
with methyl acetoacetate (28) as the proton source.47 This
sequence forged the required C1−C5 bond and also sets the
C6 stereocenter with high diastereoselectivity. As expected,
minimal olefin isomerization was observed due to the presence
of the conjugated phenyl ring. This complex Michael addition/
kinetic protonation sequence affording cyclopentanone ( )-29
was scalable (up to 8 g scale) and proceeded with high
diastereoselectivity (>19:1) in 77% yield. Diastereoselective
reduction of the ketone with L-selectride gave alcohol ( )-30
(>19:1, dr) and protection as the benzyl ether gave
cyclopentane ( )-31. At this stage, the refined substrate
served us well to prevent problematic issues encountered
previously. However, in order to enable the proposed RCM
reaction, terminal monosubstituted alkenes are required for
optimal reactivity and efficiency which we verified through
extensive experimentation.48 Thus, benzyl deprotection
followed by ozonolysis revealed 1,8-ketoaldehyde ( )-32.
While direct cyclization of ketoaldehydes by a McMurry
reaction49,50 to form 8-membered rings have been reported,51
several attempts were unsuccessful. Thus, toward applying
RCM for cyclooctene formation with a substrate related to
Nakada’s, we converted the ketoaldehyde ( )-32 to the bis-
terminal alkene ( )-33. However, this bis-olefination required
extensive optimization, as the ketone was found to be inert to
many common methylenation conditions.52,53 After extensive
studies, we were delighted to find that conditions developed by
Takai54−57 delivered the desired bis-alkene ( )-33 in 52%
yield. Following PMB ether deprotection to further reduce
steric bulk around one alkene following the precedent of
Nakada,20,21 RCM of bis-olefin ( )-34 successfully established
the 5/8 bicyclic core.58−60 Benzyl ether deprotection with
Freeman’s reagent,61,62 Swern oxidation,63,64 and TBS-ether
deprotection provided the first bicyclic OpA derivative ( )-38
which was best purified on Fluorosil. This bicyclic OpA
derivative shares the entire A/B-ring system with the natural
product OpA, differing only by having an epimeric, secondary
alcohol at C3.
Scheme 2. Refined Scalable Synthesis of the Refined Enone
Substrate 26
of the critical C6 stereocenter (for further description of this
defensive strategy see Scheme S1 in the Supporting
Information (SI)). Our revised synthesis began by developing
an efficient route to cyclopentenone ( )-26 (Scheme 3).
Scheme 3. Synthesis of Bicyclic OpA Derivatives ( )-38
and ( )-10
Toward gaining more information regarding the importance
of the C3-teriary alcohol, we also targeted derivative ( )-10
bearing a tertiary alcohol with natural configuration at C3
(Scheme 3b). While use of a later stage, common intermediate
such as benzyl ether ( )-35 would appear prudent, attempts to
selectively manipulate the C3 alcohol required extensive
protecting group manipulations or led to extensive β-
elimination. We therefore opted to begin with alcohol
( )-33 toward C3-derivatives, and an abbreviated synthetic
scheme is shown (Scheme 3b; for the detailed synthetic
sequence, see Scheme S2 in SI). Methyl Grignard addition to
the derived ketone ( )-39 proceeded diastereoselectively (dr
>20:1) to deliver tertiary alcohol ( )-40. Subsequent synthetic
steps toward this bicyclic derivative followed a similar strategy
to that described above for OpA derivative ( )-38; however,
the final step, a bis-oxidation step to afford the ketoaldehyde,
was found to be quite challenging, so it is described briefly
here. This bis-oxidation was plagued by C6-epimerization or
Sonogashira coupling40 of PMB-protected propargyl alcohol
19 with iodobenzene followed by regioselective hydroboration
with pinacol diborane,41 both on multidecagram scale,
delivered vinyl borane 23. Hydrolysis to the more reactive
boronic acid 24,42 followed by Suzuki−Miyaura coupling43−45
with racemic, known cyclopentenyl bromide ( )-25,46 gave
enone ( )-26 on a multigram scale in 83% yield.
C
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a
Table 1. Cell Viability Data of Synthesized, Simplified Analogs of Ophiobolin A and Natural Congeners
a
b
IC50 values are denoted in mean standard deviation of at least 3 independent replicates. Ketoaldehyde ( )-11 was unstable in DMSO, and
thus a freshly prepared sample was required to preclude inconsistencies observed when using stored DMSO stock solutions.
C3-dehydration through β-elimination under a variety of
oxidative conditions screened. Ultimately, we found that
selectivity between the cancer vs noncancer cell line, ∼4.9 vs
4.2 μM, respectively.
Hu
̈
nig’s base in lieu of the more commonly employed Et3N
In summary, we completed the first stage and initiated
studies of the second stage of a PDR approach toward OpA to
access bicyclic analogs bearing the AB-ring system. To
circumvent synthetic challenges including an inherent 5-exo-
trig cyclization of a neopentyl alkyllithium and an undesired
olefin isomerization, we adopted a defensive strategy by careful
refinement of intermediates that ultimately enabled an RCM to
form the required cyclooctene ring and maintain the critical
C6-stereochemistry. Cell viability assays toward MDA-MB-231
breast cancer cells confirm our hypothesis that significant
cytotoxicity observed for the ophiobolins originates from
structural features of the A/B-ring system. In addition to the
SAR studies of the ophiobolins, the work described represents
model studies of key reactions envisioned toward more
elaborate OpA derivatives, a useful byproduct of the PDR
approach. This study further highlights the advantage of PDR
to access SAR information at the early stages of a total
synthesis effort and inform the design of more elaborate
derivatives in the later stages.
was key to a clean Swern bis-oxidation but still afforded a
moderate yield (33%). Furthermore, it should be noted that
the resulting ketoaldehyde ( )-10, while stable once purified,
was quite sensitive to many stationary phases for purification.
Silica gel deactivated with Et3N, basic alumina, and Fluorosil
all resulted in extensive decomposition. Use of reversed-phase
semiprep HPLC was uniquely successful in enabling
purification of OpA derivative ( )-10. This is in stark contrast
to the stability observed for the C3-secondary alcohol-bearing
derivative ( )-38 and points to the importance of the C3-
substitution pattern for stability of OpA derivatives. While
OpA itself is more stable to silica gel than ( )-38 and ( )-10,
it is also prone to C6 epimerization and C3 dehydration under
acidic or basic conditions leading to significant loss in
bioactivity.1,2,6
We then assayed the cytotoxicity of the racemic, simplified
OpA derivatives synthesized toward a breast cancer cell line
(MDA-MB-231) and a noncancerous cell line (MCF-10A), to
probe selectivity, if any, in comparison to OpA and a few
naturally occurring OpA congeners (Table 1). While
significantly less potent than OpA, simplified bicyclic OpA
derivatives ( )-10 and ( )-38 bearing the proposed
pharmacophore displayed cytotoxicity (∼7 and 11 μM,
respectively) against MDA-MB-231 cells. The activity of the
highly simplified and unstable ketoaldehyde ( )-11 was
unsurprisingly less active (∼44 μM). The selectivity observed
with OpA between a cancer vs noncancer cell line (∼69 vs 200
nM, ∼3-fold difference) is erased with the structurally
simplified bicyclic derivatives ( )-10 and ( )-38. The similar
potency of these simplified bicyclic derivatives is noteworthy
and suggests that the C3-stereochemistry as either a secondary
or tertiary alcohol has little effect on bioactivity. Overall, the
described SAR data with racemic bicyclic derivatives of OpA
suggest that cytotoxicity and selectivity increase in parallel with
increasing complexity as expected.
ASSOCIATED CONTENT
* Supporting Information
■
sı
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.orglett.0c02938.
Experimental procedures and compound characteriza-
tion data (1H and 13C and select 2D (NOESY, HMBC,
HSQC) NMR spectra) for all new compounds reported
(PDF)
AUTHOR INFORMATION
Corresponding Authors
■
Daniel Romo − Department of Chemistry and Biochemistry,
Baylor University, Waco, Texas 76798, United States;
orcid.org/0000-0003-3805-092X; Email: Daniel_Romo@
baylor.edu
A few closely related congeners of OpA were also assayed.
Direct comparison of OpA and (+)-6-epi-OpA suggests the
importance of the C6 relative stereochemistry for cytotoxicity
toward this breast cancer cell line leading to an ∼12-fold drop
in activity (∼69 nM vs ∼0.8 μM, Table 1). Other structural
changes also impact cytotoxicity including loss of the C3-
hydroxyl through β-elimination in conjunction with epimeriza-
tion at C6 leading to an 8-fold drop in activity (cf. (+)-3-
anhydro-6-epi-OpA). Retaining the C6-stereochemistry but
dehydration in combination with reduction of the aldehyde to
the primary alcohol as found in (+)-OpI leads to a significant
loss in cytotoxicity (∼10-fold drop vs OpA) with insignificant
Joseph H. Taube − Department of Biology, Baylor University,
Waco, Texas 76798, United States; Email: Joseph_Taube@
baylor.edu
Authors
Yongfeng Tao − Department of Chemistry and Biochemistry,
Baylor University, Waco, Texas 76798, United States
Keighley N. Reisenauer − Department of Biology, Baylor
University, Waco, Texas 76798, United States
Marco Masi − Dipartimento di Scienze Chimiche, Complesso
Universitario Monte Sant’ Angelo, Naples, Italy; orcid.org/
0000-0003-0609-8902
D
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(13) Bury, M.; Girault, A.; Megalizzi, V.; Spiegl-Kreinecker, S.;
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Antonio Evidente − Dipartimento di Scienze Chimiche,
Complesso Universitario Monte Sant’ Angelo, Naples, Italy;
orcid.org/0000-0001-9110-1656
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.orglett.0c02938
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We gratefully acknowledge support by the National Institute of
General Medical Sciences of the National Institutes of Health
(R37 GM052964 and R35 GM134910, D.R.), the Robert A.
Welch Foundation (AA-1280, DR), and the Cancer Prevention
& Research Institute of Texas (RP180771, J.H.T.). We thank
Prof. Alexander Kornienko (Texas State University) for
continued fruitful discussions and collaboration on the
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Produzioni Alimentari, CNR, Bari, Italy for a supply of culture
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