Synthesis and evaluation of macrocyclic diarylether heptanoid natural products and their analogs

Article abstract of DOI:10.1016/j.bmcl.2011.11.025

The macrocyclic diarylether heptanoid (MDEH) natural products have been used in folk medicine for centuries. MDEHs are reported to exert anti-tumor properties by inhibiting the activation of NF-κB. Here we report the synthesis of a small MDEH library (first reported synthesis of racemic platycarynol) using a Grubbs cross metathesis/Ullmann cyclization strategy. Evaluation of the library led to the identification of MDEH 9b which sensitizes pancreatic cancer cells to gemcitabine mediated growth inhibition and apoptosis.

Full text of DOI:10.1016/j.bmcl.2011.11.025

Bioorganic & Medicinal Chemistry Letters 22 (2012) 245–248
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and evaluation of macrocyclic diarylether heptanoid natural
products and their analogs
Vashti C. Bryant a,b, G.D. Kishore Kumar b, Abijah M. Nyong a, Amarnath Natarajan a,b,
⇑
a Eppley Institute for Cancer Research, University of Nebraska Medical Center, Omaha, NE 68198, USA
b Chemical Biology Program, Department of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston, TX 77555, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The macrocyclic diarylether heptanoid (MDEH) natural products have been used in folk medicine for cen-
turies. MDEHs are reported to exert anti-tumor properties by inhibiting the activation of NF- B. Here we
report the synthesis of a small MDEH library (first reported synthesis of racemic platycarynol) using a
Grubbs cross metathesis/Ullmann cyclization strategy. Evaluation of the library led to the identification
of MDEH 9b which sensitizes pancreatic cancer cells to gemcitabine mediated growth inhibition and
apoptosis.
Received 3 September 2011
Revised 3 November 2011
Accepted 7 November 2011
Available online 11 November 2011
j
Keywords:
Macrocyclic diarylether heptanoid
Pancreatic cancer
Ó 2011 Elsevier Ltd. All rights reserved.
NF-jB
Grubbs/Ullmann
Total synthesis
Natural products are highly desired as lead candidates in the
drug discovery process. This can be attributed to not only their his-
torical role in treating and preventing human disease, but also
their structural diversity.1,2 Naturally occurring macrocyclic diary-
lether heptanoids (MDEHs) are found in plant materials that have
been used in folk medicine for centuries (Chart 1). They are a sub-
set of the larger class of diarylheptanoids and are characterized by
two aromatic rings linked via a seven carbon chain and an ether
bond to form the inherent macrocycle.3 The diarylheptanoids have
been reported to exert anti-inflammatory and anti-tumor activi-
ties.4 Macrocyclic diarylheptanoids isolated from natural sources
Chart 1. MDEH natural products.
have been reported to inhibit tumor necrosis factor-
and NO production. These natural products have also been shown
a (TNF-a)
Many chemotherapeutic agents including gemcitabine have
been reported to activate the NF-
thought to provide tumor cells with a mechanism of resistance
to such agents.8 We aimed to increase the diversity of NF-
B inhib-
itors by seeking out scaffolds found in natural sources and evaluat-
ing them as NF- B inhibitors and potential anti-cancer agents.
jB pathway and this event is
to inhibit nuclear factor-
reporter assay.5
jB (NF-jB) transcription activity in a gene
j
NF-
in pancreatic tumor progression.6 A protein–protein interaction
between NF- B and I retains NF- B in the cytoplasm. Upon
activation of the pathway by pro-inflammatory cytokines such as
TNF- , I is rapidly phosphorylated and ubiquitinated. This pro-
cess leads to the degradation of I by the proteasome and sub-
sequent translocation of NF- B into the nucleus. In the nucleus,
jB is a pleiotropic family of transcription factors implicated
j
j
jBa
j
A 15-member macrocycle with a para-substituted phenyl ring is
a common structural feature found in acerogenin MDEH natural
products. These were isolated from the stem bark of Acer nikoense
Maxim (Aceraceae) used in folk medicine to treat hepatic disorder.
The first total synthesis of the acerogenin natural product used an
intramolecular SNAr ring closure to generate the macrocycle. The
macrocyclization step required the insertion of a nitro group fol-
lowed by its removal post cyclization.3 A more direct route used
an Ullmann reaction for macrocyclization to access acerogenins C
and A.9,10 We recently reported the synthesis of a related natural
product ovalifoliolatin B which has a trans double bond in the
a
jBa
jBa
j
NF-jB activates transcription of a variety of genes including those
involved in cell proliferation, apoptosis, and chemoresistance.7
⇑
Corresponding author. Tel.: +1 402 559 3793; fax: +1 402 559 8270.
E-mail address: anatarajan@unmc.edu (A. Natarajan).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.11.025

246
V. C. Bryant et al. / Bioorg. Med. Chem. Lett. 22 (2012) 245–248
contact of the alkenes templated on the diarylether backbone.
Therefore, we reversed the sequence with olefin cross-metathesis
between the alkene analogs (5) and styrene analogs (6) before
the Ullmann reaction mediated macrocyclization. This approach
yielded the desired MDEH core that was elaborated to the desired
natural products and their analogs.
Briefly, methyl-3-(4-methoxyphenyl)propanoate 1a and met
hyl-3-(4-hydroxyphenyl)propanoate 1b were brominated to gener-
ate 2a and 2b, respectively, (Scheme 1). The ester groups on the bro-
minated compounds were hydrolyzed to corresponding acids (3)
under basic conditions. In a two-step process, the acids (3) were con-
verted to the Weinreb amides (4) and subsequently reacted with the
Grignard reagent 3-butenylmagnesium bromide to generate the de-
sired alkene fragments (5).
The linear diarylheptanoid fragment (8) required for Ullmann
cyclization was generated in moderate yields in a two-step se-
quence, that is, Grubbs II catalyzed cross metathesis of 5 with 6
to form 7, followed by reduction of the resulting double bond via
catalytic hydrogenation. As expected, in addition to the desired
cross-coupled products, we isolated homodimers of both the al-
kene fragments and the styrene fragments.
Macrocyclization of fragments 8a and 8c using copper oxide in a
sealed tube resulted in meta–para linked MDEH analog 9a and the
natural product galeon 9c (Scheme 1). Reduction of the ketone in
9a and 9c resulted in racemic platycarynol 10a and MDEH analog
10c. On the other hand Ullmann cyclization of fragments 8b and
8d resulted in meta–meta linked diarylether MDEH analogs 9b
and 9d. Reduction of the ketone in 9b yielded analog 10b. Removal
of the MOM protecting group in 9d yielded a monomethylated
analog 10d while treating 9b with aluminium chloride resulted
in another analog 10e. This structure was originally reported as
Chart 2. Retrosynthetic analysis of MDEH natural products and their analogs.
macrocycle using a Grubbs cross metathesis/Ullmann cyclization
strategy.11 Here we extend this strategy to generate an MDEH
library.
In this report we describe the first total synthesis of the MDEH
natural product platycarynol. We used a Grubbs cross metathesis/
Ullmann cyclization strategy to generate the natural product and a
small library of MDEH analogs. The library was evaluated in NF-jB
assays and the best MDEH analog 9b was found to sensitize pan-
creatic cancer cells to gemcitabine mediated growth inhibition
and apoptosis.
Synthesis of MDEH analogs with variations in the substitution
patterns on the aryl rings, the heptanoid chain, and the diarylether
linkage were envisioned as summarized in the retrosynthetic anal-
ysis described in Chart 2. Ullmann cross coupling followed by ring
closing metathesis would result in the structural framework that
can be manipulated to generate the desired library.
We were able to successfully generate the linear compounds
using Ullmann cross coupling. However, despite our best efforts
with varying solvents, dilution, temperature and catalysts, we
failed to generate the ring closed products.11 A reason for this fail-
ure could be attributed to the reduced probability of end-to-end
Scheme 1. Synthesis of MDEHs. Reagents and conditions: (a) 2a: Br2, AlCl3, CH2Cl2, 0 °C, 97%; 2b: Br2, CH2Cl2, 0 °C, 98%; (b) MOMCl, K2CO3, acetone, 0 °C, 98%; (c) 3a: 2 N
NaOH, MeOH, room temp, 98%; 3c: LiOH, THF/H2O, 95%; (d) (i) CDMT, NMM, THF; (ii) MeNH–OMeÁHCl, room temp.; (e) 3-butenylmagnesium bromide, THF, 0 °C, 65–70% over
two steps; (f) 7a, 7c: Grubbs II, CH2Cl2, reflux, 4 h, 26–30%; 7b, 7d: Grubbs II, toluene, reflux, 2 h, 30–53%; (g) 8a, 8c: Pd/C, CH2Cl2/MeOH, 175 psi; 8b, 8d: Pd/C, CH2Cl2, room
temp, 55–82%; (h) CuO, K2CO3, pyridine, sealed tube,
D, 12–52%; (i) NaBH4, MeOH, 84–94%; (j) AlCl3, CH2Cl2, reflux, 41%; (k) 6 N HCl, MeOH, 80%.

V. C. Bryant et al. / Bioorg. Med. Chem. Lett. 22 (2012) 245–248
247
Figure 1. Engelhardione analog 9b inhibits NF-jB transcription activity and sensitizes pancreatic tumor cells to gemcitabine mediated growth inhibition and apoptosis. (A)
9b Inhibits NF- B transcription activity in stable
j
j
B-luciferase expressing A549 cells at 20 M. The table summarizes the results from dose response experiments. (B) and (C)
l
9b Sensitizes Hs 766T and PANC-1 cells to gemcitabine mediated growth inhibition (data representative of three independent experiments). (D) 9b Sensitizes PANC-1 cells to
gemcitabine mediated caspase activation (data representative of two independent experiments). All data are expressed as the mean SD and ⁄P < 0.5, ⁄⁄P < 0.1, ⁄⁄⁄P < 0.01.
the natural product engelhardione and was recently revised based
on elegant work by the Sun group.13 This is the first report describ-
ing the synthesis of racemic platycarynol.
A cell line specifically designed to monitor the transcription
activity of NF- B in response to TNF- was used to screen the
MDEH library. Parthenolide, a known NF- B inhibitor and a re-
cently reported NF- B inhibitor noscapine, an isoquinoline alkaloid
j
a
j
Analysis of the 1H NMR spectra of the alcohols 10a and 10c
showed doubling of the methoxy peaks on the para-disubstituted
phenyl ring in the macrocycle. This suggests the generation of
atropisomeric mixtures of platycarynol (10a) and MDEH analog
10c. Careful chromatography allowed us to separate a small
amount of one of the two atropisomers in platycarynol. The spec-
tral data of the separated material matched reported natural prod-
uct R(À)-platycarynol.12
j
natural product with anti-tussive activity were used as positive
controls.14–16
Results from the
jB-luciferase reporter assay screen are
summarized in Figure 1A (and Fig. S2). We selected 9b and platy-
carynol, the most active meta–meta and meta–para diarylether
compounds, respectively, for dose response studies (Figs. 1A and
S3). The IC50 data show that analog 9b was (i) more active than
platycarynol, (ii) ꢀfour fold more active than noscapine, (iii)
comparable or marginally better than the linear diarylheptanoid
To add to the diversity we generated analogs of the intermedi-
ates from the recently reported ovalifoliolatin B synthesis (Fig. S1).
These include the reduction of the keto group on the linker in
methyl acerogenin C (12) to generate analog 13. We also elabo-
rated acerogenin C to the known compound aceroside following
literature procedure.3 To explore the size effects on the heptanoid
linker, we functionalized the double bond in ovalifoliolatin B to
generate epoxide and aziridine containing compounds 14 and 15
as racemic mixtures. In summary, we assembled a small library
of 18 MDEH compounds (see Fig. S1 in Supplementary data)
including seven natural products for evaluation as inhibitors of
curcumin, another well characterized NF-
ꢀthree fold less active than parthenolide. To further demonstrate
the ability of 9b to inhibit NF- B activation, we evaluated the
expression levels of I after TNF- stimulation of HEK 293 cells
by Western blotting (Fig. S4). Cells treated with 9b show a decrease
in I protein expression at 60 and 120 min, suggesting that 9b
inhibits NF- B mediated transcription of the I gene.
jB inhibitor, and (iv)
j
jB
a
a
jBa
j
jBa
Pancreatic cancer (PC) is one of the most lethal human cancers,
with a 5-year survival rate of <4% and a median survival of less
than six months. The clinically silent onset of PC has been attrib-
NF-jB activity.

248
V. C. Bryant et al. / Bioorg. Med. Chem. Lett. 22 (2012) 245–248
uted to the up-regulation of inflammatory signals and subsequent
activation of NF- B activity has been impli-
B.17 Constitutive NF-
cated in PC progression.18,19 It is also well known that many che-
motherapeutic agents induce the NF- pathway and this
Acknowledgment
j
j
This work was supported in part by NIH (T90DK070109 training
fellowship for V.C.B. and R01CA127239).
jB
activation is implicated as a possible mechanism of chemoresis-
tance by tumor cells. Therefore we decided to examine whether
inhibition of NF-jB by 9b could lead to PC cell growth inhibition
Supplementary data
and/or sensitize cells to chemotherapeutics. To test this, we se-
lected commonly used PC cell lines, Hs766T and PANC-1, and
examined the ability of 9b to inhibit cell growth over a five-day
period, alone and in combination with the chemotherapeutic agent
gemcitabine (Fig. 1B and C).
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.11.025.
References and notes
Our results show that 9b alone did not exhibit growth inhibitory
effects (dark triangles vs dark circles). However, 9b did sensitize
Hs766T and PANC-1 tumor cells to gemcitabine mediated growth
inhibition (Fig. 1B and C). Increased caspase activity in cells is indic-
ative of apoptosis. To determine whether the growth inhibitory ef-
fect seen with the combination of 9b and gemcitabine involved
apoptosis, PANC-1 cells were treated with 9b alone or in combina-
tion with gemcitabine and caspase 3/7 activity was measured after
48 h ( Fig. 1D). Consistent with the growth inhibitory effects, we
found increased levels of caspase activation with the combination
of 9b and gemcitabine, when compared to gemcitabine or 9b alone.
Together these data suggest that 9b sensitizes PC cells to gemcita-
bine mediated growth inhibition and apoptosis.
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In conclusion, we report the first total synthesis of the natural
product platycarynol. We evaluated a small library of MDEH analogs
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as NF-jB inhibitors. This led to the identification of a meta–meta
linked analog 9b as a low micromolar inhibitor. Growth inhibition
and apoptosis studies showed that 9b sensitizes pancreatic cancer
cells to gemcitabine, a commonly used chemotherapeutic agent
for pancreatic cancer. We are currently exploring 9b as an adjuvant
in combination therapy with other chemotherapeutic agents in a
panel of cancer cell lines.