Synthesis and SAR of lehualide B: A marine-derived natural product with potent anti-multiple myeloma activity

Article abstract of DOI:10.1021/cb300582s

We report a concise and convergent laboratory synthesis of the rare marine natural product lehualide B that has led to the discovery that (1) this compound has low nanomolar activity against human multiple myeloma cells and (2) the anticancer effects of lehualide B and its analogues are selective (i.e., they are approximately 2-3 orders of magnitude less toxic to human breast cancer cells). Synthetic lehualide B is shown to be an effective inhibitor of complex I of the mitochondrial electron transport chain, with potency similar to that observed for the terrestrial natural products piericidin A1 and rotenone, an observation that led to the discovery that piericidin A1 is also selectively cytotoxic toward human multiple myeloma cells. Interestingly, synthetic derivatives of lehualide B that resemble verticipyrone (an established complex I inhibitor composed of a γ-pyrone and a simple monounsaturated hydrophobic chain) lack the potent antimyeloma activity of the natural product. Finally, the synthesis and evaluation of a collection of lehualide-inspired analogues led to the elucidation of structure-activity relationships for this rare natural product that established important roles for the substituted γ-pyrone headgroup and the skipped polyene side chain.

Full text of DOI:10.1021/cb300582s

Articles
pubs.acs.org/acschemicalbiology
Synthesis and SAR of Lehualide B: A Marine-Derived Natural Product
with Potent Anti-Multiple Myeloma Activity
Valer Jeso,† Chunying Yang,‡ Michael D. Cameron,# John L. Cleveland,*,‡ and Glenn C. Micalizio*,†
†Departments of Chemistry, ‡Cancer Biology, and #Molecular Therapeutics, The Scripps Research Institute, Scripps Florida, Jupiter,
Florida 33458, United States
S
* Supporting Information
ABSTRACT: We report a concise and convergent laboratory synthesis of the rare marine natural product lehualide B that has
led to the discovery that (1) this compound has low nanomolar activity against human multiple myeloma cells and (2) the
anticancer effects of lehualide B and its analogues are selective (i.e., they are approximately 2−3 orders of magnitude less toxic to
human breast cancer cells). Synthetic lehualide B is shown to be an effective inhibitor of complex I of the mitochondrial electron
transport chain, with potency similar to that observed for the terrestrial natural products piericidin A1 and rotenone, an
observation that led to the discovery that piericidin A1 is also selectively cytotoxic toward human multiple myeloma cells.
Interestingly, synthetic derivatives of lehualide B that resemble verticipyrone (an established complex I inhibitor composed of a γ-
pyrone and a simple monounsaturated hydrophobic chain) lack the potent antimyeloma activity of the natural product. Finally,
the synthesis and evaluation of a collection of lehualide-inspired analogues led to the elucidation of structure−activity
relationships for this rare natural product that established important roles for the substituted γ-pyrone headgroup and the skipped
polyene side chain.
arine-derived natural products continue to provide an
exciting collection of therapeutically valuable leads that,
lehualide A (1) and monounsaturated or saturated hydrophobic
side chains are much less toxic (1−2 orders of magnitude) than
lehualides B and D.5
M
because of their usually low availability from natural sources,
require chemical synthesis to drive efforts focused on evaluating
their potential value as medicines.1−3 The lehualides (Figure
1A) are a family of pyrone-containing natural products recently
isolated from Hawaiian and Tongan marine sponges (Plakortis
sp.) that were originally shown to possess modest anticancer
properties against ovarian (IGROV-ET) and leukemia (K562)
cell lines.4,5 Notably, members of this class have disparate
toxicity profiles that stem from subtle variations in pyrone
substitution and the composition of their aliphatic tail. For
example, lehualides A−D (1−4) show varying brine shrimp
toxicity that suggests that the γ-pyrone motif imparts maximal
toxicity.4 Further, lehualides A (1) and C (3) do not display
toxicity in ovarian or leukemia cell lines that are sensitive to
lehualides B (2) and D (4) [2, GI50 = 830 nM (IGROV-ET); 4,
GI50 = 730 nM (IGROV-ET) or 230 nM (K562)]. In line with
these observations that indicate a role for the γ-pyrone in
imparting maximal toxicity, the most recently isolated
lehualides (F−I; 6−9) that contain α-pyrones similar to
While the mechanism of action for the most toxic lehualides
has heretofore not been elucidated, their common pyrone
structure is reminiscent of the pyridine core of piericidin A1
and the quinone of ubiquinone (Figure 1B). Related γ-pyrone
moieties are found in several other cytotoxic natural products
(Figure 1C)6−25 that have a wide variety of activities as
insecticidal, anticancer, antifungal, antibacterial and immuno-
suppressive agents.6−25 Some of these compounds have been
shown to block the voltage-gated potassium channel Kv1.3 and
to potently inhibit the mitochondrial electron transport chain
protein NADH-ubiquinone reductase (complex I), with IC50’s
as low as 0.3 nM (verticipyrone analogues).26 Finally, germane
to our studies, a subset of potent complex I inhibitors have
Received: October 26, 2012
Accepted: March 18, 2013
Published: April 2, 2013
© 2013 American Chemical Society
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Figure 1. Introduction to the lehualides and related natural products.
received attention as selective antitumor agents, yet the
mechanism responsible for this activity has not been
resolved.27,28
dimethoxy-substituted γ-pyrone and a hydrophobic tail that
contains three trisubstituted alkenes that are uniformly
“skipped” (nonconjugated) with respect to neighboring π-
systems (i.e., 2). While not possessing a single chiral center, the
stereochemistry and substitution of this skipped-polyunsatu-
rated tail renders lehualide B a significant challenge to modern
synthetic organic chemistry.29
The structural similarity of lehualide B to natural products
known to possess an array of medicinally relevant biological
activities, and the lack of a robust supply from natural sources
prompted us to define a concise synthetic entry to this natural
product that would allow assessment of the medicinal value of
this target. In pursuit of these goals, our efforts have led to (1)
the total synthesis of lehualide B and 25 synthetic analogues;
Recent studies by Boger and colleagues focused on
understanding the activity of piericidin A1 revealed that
complex I inhibition is necessary but not sufficient to confer
potent cytotoxicity to synthetic analogues. Hypotheses stated to
explain this included a potential differential ability to access the
cellular target, subtle distinctions between multiple binding
sites on complex I, and/or a role for additional (undefined)
biological targets.28
The most potent members of the lehualide family are
structurally related to the natural products depicted in Figure
1B and C, yet they have unique features that include a 2,3-
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(2) the discovery that lehualide B is selectively cytotoxic to
multiple myeloma cells at low nanomolar concentrations; (3)
the observation that lehualide B, and closely related synthetic
analogues, totally abolish long-term growth of chemoresistant
multiple myeloma cells; and (4) the discovery that lehualide B
is a potent inhibitor of mitochondrial complex I.
RESULTS AND DISCUSSION
■
Total Synthesis of Lehualide B. Given anticipated
difficulties associated with synthesizing the skipped polyene
tail of 2, our retrosynthetic strategy was primarily influenced by
the most challenging structural motif in this region: the C12−
C16 1,4-diene that houses both an (E)- and (Z)-trisubstituted
alkene (Figure 2A). While modern carbonyl olefination or
metal-catalyzed cross-coupling are commonly used for the
synthesis of trisubstituted alkenes, neither of these is optimal
for the challenge associated with this section of lehualide B.
Specifically, methods based on carbonyl olefination are plagued
by challenges associated with the control of stereochemistry in
establishing the trisubstituted alkenes and with difficulties
associated with advancing β,γ-unsaturated systems.30 Metal-
catalyzed cross-coupling is similarly complex because of the
multistep nature of synthetic pathways to generate the
stereodefined coupling partners (Figure 2B) and associated
problems with regio- and stereocontrol in the reaction of
intermediate π-allyl complexes.31 Given these hurdles we
sought an alternative method for preparing this stereodefined
subunit.
We recently established a reductive cross-coupling process
that proceeds by stereoselective union of allylic alcohols with
alkynes and that generates two stereodefined trisubstituted
alkenes along with a central C−C bond.32 In analyzing the
lehualide B structure this reaction seemed ideal for establishing
the C14−C15 bond with concomitant generation of each
stereodefined trisubstituted alkene of the 1,4-diene: a bond
construction that would ensue with stereoundefined and
“unactivated” π-systems (Figure 2C).
Using this bond construction as a central feature of our
design, the retrosynthetic strategy to lehualide B that emerged
was comprised of a simple sequence that embraced the utility of
allylic alcohols for establishing all of the trisubstituted alkenes
in this target (Figure 2D). Well-established Claisen rearrange-
ment chemistry33,34 was envisioned as a means to convert allylic
alcohol 12 to an (E)-trisubstituted alkene (as in 11), while Ti-
mediated, alkoxide-directed reductive cross-coupling could then
be employed to convert allylic alcohol 11 to the desired (Z)-
trisubstituted C12−C13 alkene of lehualide B. In this latter
process, stereochemical control was predicted in accord with
our previous studies that put forth an empirical model based on
a boat-like transition state geometry for carbometalation that
features minimization of A1,2-strain (Figure 2C; A → B →
C).32
With this retrosynthetic strategy in mind, we began with
synthesis of the requisite 2,3-dimethoxy-5,6-dimethyl γ-pyrone
13. As shown in Scheme 1A, iodosobenzene diacetate-mediated
oxidation of β-ketoester 14 in methanol provided the α-
methoxy-β-ketoester 15 in 70% yield.35 Deprotonation with
LDA (2.2 equiv), followed by addition of Weinreb amide 16,
then delivered the tricarbonyl 17 as a mixture of isomers.
Stirring this tricarbonyl in neat H2SO4 (for 21 h) then
generated the γ-pyrone 13 in 24% yield over the two-step
sequence.36
Figure 2. Development of a concise synthesis strategy for lehualide B.
Next, attempted addition of the extended enolate of 13 to
methacrolein was problematic, likely because of competitive
1,4-addition (Scheme 1B). To avoid this issue, α-(phenyl-
seleno)-isobutyraldehyde was used as the electrophile in
reaction with the anion of 13. Subsequent oxidation to the
selenoxide was followed by syn-elimination in situ to deliver the
desired allylic alcohol 12 in 73% yield (over two steps).37 Next,
Johnson orthoester Claisen rearrangement delivered the
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Scheme 1. Synthesis of Lehualide B
desired γ,δ-unsaturated aldehyde (E:Z = 10:1),38 which was
then used to generate the functionalized allylic alcohol 11 by
the addition of 2-propenylmagnesium bromide (53% yield over
two steps).
transition states for carbometalation depicted in Scheme 1C,
it is evident that the steric impediment of the Ph group can
easily be avoided by simple bond rotation (this similar steric
profile of Me and Bn is evident in their A values: Me = 1.7 kcal/
mol; Bn = 1.8 kcal/mol40).
Having the allylic alcohol 11 in hand, alkoxide-directed Ti-
mediated reductive cross-coupling with 1-phenyl-2-butyne was
then performed using slightly modified reaction conditions than
previously reported for this stereoselective coupling process.32
Initial formation of a dianion of 11 (to mask the potential
electrophilic character of the pyrone carbonyl) was followed by
exposure to a preformed Ti−alkyne complex that was
generated by treating 1-phenyl-2-butyne with a combination
of ClTi(Oi-Pr)3 (6 equiv) and c-C5H9MgCl (12 equiv).
Warming from −78 to 0 °C, followed by an aqueous quench,
delivered the coupled products in 50% combined yield.
Despite the success of this coupling process and the concise
synthetic sequence (six steps from the simple γ-pyrone), the
natural product was formed as an inseparable 1.3:1 mixture of
regioisomers (2 and 19), where the minor product was derived
from C−C bond formation occurring α- to the Bn substituent
of the alkyne to deliver isolehualide B. This low selectivity was
expected, as regiochemical control in functionalizing Ti−alkyne
complexes typically requires substantial steric or electronic
differentiation of the alkyne termini.39 In the competing
To circumvent this problem, a straightforward solution was
adopted that exploited the well-appreciated regioselectivity
associated with Ti-mediated reductive cross-coupling reactions
of TMS-substituted alkynes.39 Specifically, metallacycle-medi-
ated reductive cross-coupling of allylic alcohol 11 with TMS-
propyne (20, Scheme 1D) delivered the TMS-alkene-
containing 1,4-diene product 21 in 61% yield (Z:E = 7:1; rs
≥ 20:1). Subsequent conversion to the vinyliodide [NIS,
ClCH2CN:EtOAc (2:1)],41 followed by Pd-catalyzed cross-
coupling with benzylzinc bromide generated lehualide B in 64%
yield over the final two-step sequence.42
Anticancer Properties of Lehualide B. The concise
synthesis of lehualide B fueled efforts to prepare sufficient
quantities of the natural product to evaluate its anticancer
properties. Our initial assessment of the effects of 0.1−10 μM
doses of lehualide B on a cast of tumor cell lines was performed
with MTT assays using estrogen receptor-positive (ER+)
(MCF7, T47D) and triple negative (MDA-MB-231, HS578)
breast cancer cells, and three multiple myeloma (MM) cell lines
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Figure 3. Lehualide B has potent and selective activity against multiple myeloma (MM) cells, impairs MM cell growth and survival, and is a potent
inhibitor of complex I of the mitochondrial electron transport chain. (A) MTT assays were performed on MDA-MB-231 triple-negative breast cancer
cells and on NCI H929 and U266 MM cells, which were treated with the indicated doses of lehualide B (3 days of culture). (B) The EC50 values of
lehualide B for the indicated MM cells (left) and bone marrow-derived primary mouse B cells were determined using 11-point dose−response MTT
assays. For the ER+ breast cancer cells MCF7 and T47 D (right) the EC50 was >1 μM (five-point dose−response MTT assays). Error bars are the
SD of the mean (n = 3). (C) The indicated tumor cell lines were incubated with 1 μM lehualide B or vehicle. Cell numbers (top) and viability
(bottom) were determined after 4 days lehualide B (30 nM). Error bars are the SD of the mean (n = 2). (D) Lehualide B blocks complex I.
Mitochondrial complex I activity was measured using the MitoTox Complex I OXPHOS Activity assay kit. Error bars are the SD of the mean (n = 4)
(E) Mitochondrial complex I inhibitors have potent anti-MM activity. U266 MM cells were treated with the indicated doses of lehualide B, piericidin
A1, or rotenone for 3 days, and MTT assays were performed. Error bars are the SD of the mean (n = 3).
(NCI H929, U266, and RPMI-8266).43 While there were
essentially no effects of lehualide B on the growth of triple-
negative breast cancer, and only modest effects on ER+ breast
cancer cells, there were profound effects on multiple myeloma
cells (Figure 3A). Indeed, MTT assays established very potent
activity for lehualide B in all MM tumor cell lines investigated,
with EC50 values of 2.1, 2.9, and 13 nM for RPMI-8226, U266
and NCI H929 MM cells, respectively. Interestingly, lehualide
B was 3 orders of magnitude less effective vs MCF7 and T47D
ER+ breast cancer cells (>1 μM, Figure 3) and >1 order of
magnitude less toxic to mouse bone marrow-derived primary B
cells cultured on stroma and in IL7 medium (EC50 of 92 nM,
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Figure 4. Lehualide B abolishes OXPHOS in multiple myeloma cells. (A,B) U266 (left panels) and MDA-MB-231 cells (right panels) (in 96 wells)
were treated with vehicle (orange line) or with lehualide B (30 nM, teal line) for 1 h and then assessed for OCR (A) or ECAR (B) using the
Seahorse Bioscience XF96 analyzer.45 Measurements were determined every 7 min for six replicates, and error bars are standard error of the mean.
After basal measurements cells were treated with the ATP synthase inhibitor oligomycin (green arrow), which induces increases in glycolysis (i.e.,
ECAR), followed by treatment with the mitochondrial uncoupling agent FCCP (white arrow), which measures mitochondrial respiratory reserves.
Results shown are representative of four independent experiments. Note that lehualide B abolishes OCR and mitochondrial respiratory capacity. (C)
Summary of OCR (left) and ECAR (right) analyses of U266 MM and MDA-MB-231 breast cancer cells. Note that the basal rates of OCR are ∼3-
fold higher in U266 MM cells than in MDA-MB-231 cells. Rotenone treatment allows measurement of mitochondrial-independent oxygen
consumption, which was minimal in both tumor cell lines. Mean values were calculated before (basal) or after oligomycin, FCCP or rotenone
treatment.
Figure 3B). Thus, there is significant selectivity associated with
the cytotoxic profile of lehualide B.
of piericidin A1, we tested if lehualide B was an inhibitor of
complex I of the mitochondrial electron transport chain.
Indeed, in vitro assays established that, like piericidin A1,
lehualide B is a potent complex I inhibitor (IC50 = 30 nM;
Figure 3D).
These findings suggested that other complex I inhibitors may
display similar antimyeloma activity. Indeed, piericidin A1 and
rotenone also block the proliferation of U266 and RPMI-8226
MM cells (Figure 3E and data not shown). Related to the
profile of lehualide B, these agents were similarly ineffective vs
breast cancer cells (data not shown). Thus, mitochondrial
complex I inhibitors such as lehualide B and piericidin A1 are
selective anticancer agents and have high potency against
multiple myeloma. In addition to discovering this interesting
To address the mechanism by which lehualide B
compromised MM cells, its effects were assessed on cell
growth using standard growth assays and upon cell survival by
trypan blue dye exclusion.44 Human MM cells and Raji B
lymphoma were plated in culture and then treated with
lehualide B for two days. Notably, treatment with the natural
product led to rapid and marked reductions in cell number that
were accompanied by a loss in cell viability (Figure 3C) and cell
shrinkage (especially in NCI H929 MM) typical of apoptotic
cell death.
Given the structural similarity of the γ-pyrone group of
lehualide B with the quinone of ubiquinone and the heterocycle
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Figure 5. Synthesis and evaluation of Series I analogues of lehualide B: Significance of the hydrophobic region.
anticancer activity of lehualide B, to our knowledge this is the
first report that describes the potent and selective anti-MM
properties associated with piericidin A1.
To assess why MM cells are so sensitive to complex I
inhibitors such as lehualide B while others such MDA-MB-231
breast cancer cells are not, we evaluated the metabolic status of
these tumor cells and how this is affected by treatment with
lehualide B using the Seahorse Bioscience XF96 Analyzer.45
Specifically, we assessed the effects of lehualide B on the oxygen
consumption rate (OCR, a measure of oxidative phosphor-
ylation [OXPHOS]) and extracellular acidification rate (ECAR,
a measure of aerobic glycolysis). Notably, these analyses
demonstrated that U266 MM cells have much higher rates of
OCR than MDA-MB-231 breast cancer cells and that lehualide
B treatment abolishes OCR and depletes mitochondrial energy
reserves, as demonstrated by treatment of cells with the
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Figure 6. (A) Synthesis and evaluation of Series III analogues of lehualide B: Significance of substitution about the γ-pyrone hydrophilic head.
Lehualide B and key analogues compromise growth and survival of multiple myeloma. (B,C) NCI H929 and U266 cells were cultured for 3 days
with vehicle or the indicated doses of lehualide B or key analogues and cell number (B) and percent viable cells (i.e., those excluding trypan blue)
(C) were determined by counting with a hemocytometer. (D) The indicated MM cells were plated in methylcellulose containing the indicated
agents (500 nM). Colony numbers were determined on day 10. Error bars are the SD.
mitochondrial uncoupling agent FCCP (Figure 4). Further-
more, lehualide B causes a marked shift in metabolism to ECAR
(Figure 4B,C). We conclude that U266 MM cells rely on
OXPHOS (OCR) to support their metabolism, which is
disabled by lehualide B, whereas the metabolism of MDA-MB-
231 breast cancer cells rather relies on aerobic glycolysis.
Synthesis and Activity of Lehualide B Analogues. The
potent and selective anti-MM profile of lehualide B, and the
lack of substantive structure−activity relationships for this rare
marine-derived natural product, prompted analyses of the
molecular features essential for its antimyeloma activity. We
investigated the role that three key regions of the natural
product play in imparting anti-MM properties: (1) the aromatic
tail, (2) the hydrophobic linker, and (3) the γ-pyrone
hydrophilic headgroup.
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Series I analogues were prepared to evaluate the significance
of the entire hydrophobic tail of lehualide B (including the
aromatic terminus). These analogues were generated as
described in Figure 5A and follow either (1) nucleophilic
addition of a pyrone-derived anion to an allylic halide (eq 1);
(2) nucleophilic addition of a pyrone-derived anion to an
aldehyde (eq 2); or (3) nucleophilic addition to a pyrone-
containing unsaturated aldehyde (eq 3). Analogues were
prepared with prenyl, geranyl and farnesyl side chains (22−
24), as well as varying saturated/unsaturated motifs (25−27
and 11). Notably, while all of these analogues retained the
substituted γ-pyrone of lehualide B, none of them showed
significant antimyeloma activity (all had EC50 values >1 μM in
U266 MM cells; Figure 5B), indicating a significant role for the
stereodefined skipped polyene subunit of the natural product.
Series II analogues assessed the effect of alterations to the tail
of the hydrophobic linker (Figure 5C) while maintaining the
functionalized 2,3-dimethoxy-5,6-dimethyl-γ-pyrone core and
the hydrophobic polyunsaturated linker. Analogues were
prepared from the general sequence shown in Figure 5C,
defined by Grignard addition to an unsaturated pyrone-
containing aldehyde, followed by Ti-mediated alkoxide-directed
reductive cross-coupling. While derivatives 28−30 showed
substantially diminished anti-MM activity (EC50’s of 100−600
nM), a variety of other substitutions were tolerated in this
region. The most active analogues prepared include the C15-
and C16-biphenyls (31 and 35; EC50’s of 10−12 nM) and the
C12-Et substituted variant 36 (EC50 = 13 nM) (Figure 5D).
Other analogues possessing notable activity include the C14-Ph
(32; EC50 60 nM), the C-15 protio (33; EC50 30 nM), the C15-
TMS (21; EC50 20 nM) and the C15 chloropentyl derivative
(34; EC50 60 nM).
Finally, Series III analogues were synthesized to determine
the effects of subtle structural perturbations about the pyrone
headgroup (Figure 6A). The synthesis of each analogue within
this series was accomplished in a fashion akin to that described
for lehualide B synthesis (Scheme 1D), where the variation in
structure of the pyrone was introduced at the beginning of each
sequence.
As illustrated, deletion of the 2,3-dimethoxy substituent of
lehualide B essentially abolished antimyeloma activity, where
compounds 37 and 38 displayed markedly reduced abilities to
inhibit the growth of U266 MM cells (EC50 > 1 μM). Notably,
substitution α- to the pyrone also obliterated the cytotoxic
properties of lehualide B. For example, analogue 41 that houses
a prenyl substituent at this position did not inhibit MM or
lymphoma cell growth at concentrations greater than 1 μM.
The most potent Series III analogues were those that simply
replaced the C3 methyl ether of the pyrone with an ethyl
group. Analogues 42−44 were the most active members of this
series, with EC50 values vs U266 MM cells of 19, 3, and 20 nM,
respectively.
000 deaths per year.47 Morbidity associated with this
malignancy is very high, with patients experiencing anemia,
chronic bone pain and fractures, and an overall 5-year survival
still below 40% despite aggressive chemotherapy, up-front bone
marrow transplantation, the novel use of agents such as
thalidomide, and the development of new drugs such as
bortezomib.
The marine-derived natural product lehualide B was
originally reported to have modest activity against IGROV-
ET ovarian cancer cells.4 While no other biological properties
had been assigned to this compound, its modest activity, unique
chemical structure, and lack of availability from natural sources
stimulated our interest in exploring a means to prepare this
compound (and related derivatives) in the laboratory to fuel
efforts focused on thoroughly evaluating its efficacy as an
anticancer agent.
Notably, we achieved a concise synthesis of this target in just
eight steps from a simple γ-pyrone through a sequence that
highlights the unique and divergent stereochemical course of
allylic alcohol−alkyne reductive cross-coupling when compared
to the Claisen rearrangement. Both processes were used back-
to-back to establish the entire stereodefined hydrophobic tail of
lehualide B and generated each of the three trisubstituted
alkenes with just four carbon−carbon bond forming reactions
(Claisen rearrangement, Grignard addition, Ti-mediated
reductive cross-coupling and Negishi coupling).
The concise nature of this synthesis fueled our pursuits that
have defined the substantial anti-multiple myeloma properties
of lehualide B. In short order it was discovered that lehualide B
possesses rather profound and selective anti-multiple myeloma
activity, having low nanomolar EC50 activity in human MM and
Raji B lymphoma and low or no activity against human breast
cancer.
Our studies established that lehualide B has similar activity to
piericidin A1 as a potent inhibitor of complex I. Establishing
this relationship led to the discovery that piericidin A1 also has
selective activity against MM. These findings add to the
interesting anticancer profile of piericidin A1 that, to date,
includes toxicity against human NSCLC, leukemia and colon
carcinoma.28,48−50 We conclude that at least some mitochon-
drial complex I inhibitors, specifically lehualide B and piericidin
A1, are selective anticancer agents that have profound cytotoxic
activity against chemoresistant multiple myeloma cells. Further,
our studies suggest that tumor types that rely on OXPHOS to
support their metabolism, such as MM, will be exquisitely
sensitive to complex I inhibitors such as lehualide B. Finally, the
sensitivity of MM cells to complex I inhibitors may also reflect
the massive production of immunoglobulins and inherently
high activity of the endoplasmic reticulum (ER) in this tumor
type.51 Specifically, mitochondria function to balance Ca2+
pools in the lumen of ER, and their inhibition would lead to
Ca2+ loss and cell death.
Antimyeloma Effects of Lehualide B Analogues. The
effects of lehualide B and top analogues showing antimyeloma
and lymphoma activity in MTT assays suggested that they
would also compromise the long-term, anchorage-independent
growth of MM in methylcellulose colony assays.46 Indeed,
treatment with lehualide B, 36, 42, or 43 completely abolished
colony formation of NCI H929 and U266 MM cells (Figure
6B−D), further supporting their potential use as antimyeloma
agents.
Our in vivo evaluations of lehualide B revealed that dosing i.p.
(1 mg/kg) in sub-Q transplant models of RPMI 8226 and
U266 MM cells led to tumor drug concentrations that were an
order of magnitude higher than the EC50 value of lehualide B in
cellular assays. While promising, much higher concentrations
were, however, found in fat, kidney and heart. The relevance of
tissue distribution data are difficult to extrapolate across species,
yet these findings indicate that the DMPK profile for lehualide
B (and perhaps its analogues) needs substantial improvement
or that a targeted drug delivery strategy needs to be established
before advancing as a clinically relevant antimyeloma agent.
Over 70 000 patients in the United States currently suffer
from multiple myeloma (MM), with 20 000 new cases and 10
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The compelling anti-multiple myeloma profile of lehualide B
prompted the investigation of structure−activity relationships
associated with the natural product. Over 20 analogues were
prepared to evaluate the role of three regions of the natural
product skeleton (the hydrophobic tail terminus, the hydro-
phobic polyunsaturated linker, and the hydrophilic headgroup)
in anticancer activity. These studies led to the general summary
of SAR illustrated in Figure 7 and include the following
ACKNOWLEDGMENTS
■
We gratefully acknowledge financial support of this work by
grants from the National Institutes of Health/NIGMS
(GM80266, GM80266-04S1, to G.C.M.) and NCI
(CA076379, to J.L.C.), and by monies from the State of
Florida to Scripps Florida.
REFERENCES
■
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Figure 7. Summary of lehualide B analogues: SAR for anti-MM
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ASSOCIATED CONTENT
■
S
* Supporting Information
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N., and Tsuda, E. (2002) SNF4435C and D, novel immunosup-
pressants produced by a strain of Streptomyces spectabilis. III.
Immunosuppressive efficacy. J. Antibiot. 55, 71−77.
Experimental procedures and tabulated spectroscopic data for
new compounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
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Anzai, K., Harayama, S., Doi, T., Takahashi, T., Nagasawa, K.,
Natsume, T., Takagi, M., and Shin-ya, K. (2007) New aureothin
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AUTHOR INFORMATION
Corresponding Author
*E-mail: micalizio@scripps.edu; jcleve@scripps.edu.
Notes
■
(16) Kurosawa, K., Takahashi, K., and ans Tsuda, E. (2001)
SNF4435C and D, novel immunosuppressants produced by a strain of
The authors declare no competing financial interest.
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