Euodenine A: A small-molecule agonist of human TLR4

Article abstract of DOI:10.1021/jm401321v

A small-molecule natural product, euodenine A (1), was identified as an agonist of the human TLR4 receptor. Euodenine A was isolated from the leaves of Euodia asteridula (Rutaceae) found in Papua New Guinea and has an unusual U-shaped structure. It was synthesized along with a series of analogues that exhibit potent and selective agonism of the TLR4 receptor. SAR development around the cyclobutane ring resulted in a 10-fold increase in potency. The natural product demonstrated an extracellular site of action, which requires the extracellular domain of TLR4 to stimulate a NF-κB reporter response. 1 is a human-selective agonist that is CD14-independent, and it requires both TLR4 and MD-2 for full efficacy. Testing for immunomodulation in PBMC cells shows the induction of the cytokines IL-8, IL-10, TNF-α, and IL-12p40 as well as suppression of IL-5 from activated PBMCs, indicating that compounds like 1 could modulate the Th2 immune response without causing lung damage.

Full text of DOI:10.1021/jm401321v

Article
pubs.acs.org/jmc
Euodenine A: A Small-Molecule Agonist of Human TLR4
Juliette E. Neve,† Hasanthi P. Wijesekera,† Sandra Duffy,† Ian D. Jenkins,† Justin A. Ripper,†
Simon J. Teague,‡ Marc Campitelli,† Agatha Garavelas,† George Nikolapoulos,† Phuc V. Le,†
Priscila de A. Leone,† Ngoc B. Pham,† Philip Shelton,‡ Neil Fraser,‡ Anthony R. Carroll,† Vicky M. Avery,†
Christopher McCrae,§ Nicola Williams,‡ and Ronald J. Quinn*,†
†Eskitis Institute for Drug Discovery, Griffith University, Nathan, 4111 Queensland, Australia
‡AstraZeneca R&D Charnwood, Bakewell Road, Loughborough, LE11 5RH Leicestershire, United Kingdom
§AstraZeneca R&D Molndal, Translational Science, Respiratory & Inflammation iMed, Pepperedsleden 1, SE-431 83 Molndal, Sweden
̈
̈
S
* Supporting Information
ABSTRACT: A small-molecule natural product, euodenine A (1), was identified as an agonist of the human TLR4 receptor.
Euodenine A was isolated from the leaves of Euodia asteridula (Rutaceae) found in Papua New Guinea and has an unusual
U-shaped structure. It was synthesized along with a series of analogues that exhibit potent and selective agonism of the TLR4
receptor. SAR development around the cyclobutane ring resulted in a 10-fold increase in potency. The natural product
demonstrated an extracellular site of action, which requires the extracellular domain of TLR4 to stimulate a NF-κB reporter
response. 1 is a human-selective agonist that is CD14-independent, and it requires both TLR4 and MD-2 for full efficacy. Testing
for immunomodulation in PBMC cells shows the induction of the cytokines IL-8, IL-10, TNF-α, and IL-12p40 as well as
suppression of IL-5 from activated PBMCs, indicating that compounds like 1 could modulate the Th2 immune response without
causing lung damage.
These cytokines enhance the migration and survival of
eosinophils and B cells and contribute to mucous cell
hyperplasia and mast cell activation. Modulation of the type-2
acquired immune response is an attractive approach in
controlling allergic disease because it offers the potential for
disease normalization and contrasts with existing steroidal and
antibody treatments on the market and in late-stage develop-
ment, which offer only symptomatic relief. There are very few
small-molecule inhibitors of the TLR4 receptor known,
examples include TAK-242 and (+)-naloxone (Figure 1). TAK-242
is a small-molecule inhibitor of protein−protein interactions of the
Toll/interleukin-1 receptor (TIR) domain of TLR4;12 however,
this compound acts as a Michael acceptor, binding covalently to
Cys747 in the intracellular domain of TLR4.13 (+)-Naloxone is an
opioid-inactive TLR4 signaling inhibitor that can reverse neuro-
pathic pain in rats.7 Very recently, dimethyl 2-(2-nitrobenzylidene)-
malonate has been found to inhibit LPS-mediated nuclear factor-
kappaB (NF-κB) activation, the cytokine production of IL-1β,
tumor necrosis factor (TNF-α), and nitric oxide (NO) in the
INTRODUCTION
■
Steroids and antibodies dominate the current anti-inflammatory
treatment for allergy and asthma. Since the discovery of the
Toll-like receptors (TLRs) in 19881 and the identification of
their role in adaptive immunity,2 the targeting of TLRs has
opened up new therapeutic possibilities for the prevention and
treatment of inflammatory diseases. TLRs are attractive targets
for the prevention and treatment of cancer, infection, allergy,
asthma, autoimmune disease, and chronic neuropathic pain.3−10
Activation of the innate immune system through mammalian
TLRs has an instructive role for the responses of the acquired
immune response and thus may influence allergic diseases, such
as asthma. The allergic response usually features a strong Th2
response, which can be counterbalanced by TLR signaling
induction; this would induce a cytokine response, activating
antigen-presenting cells and a Th1 response.11 Agonists of Toll-
like receptor 4 (TLR4) could be therapeutically useful for
treating the acute phase of asthma.4 There is evidence that the
acquired immune response in asthma is polarized to a response
dominated by Th2 lymphocytes, which are involved in the
orchestration of the allergic inflammatory response by secreting
a variety of type-2 cytokines (interleukin (IL)-4, IL-5, and IL-13).
Received: August 27, 2013
© XXXX American Chemical Society
A
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observed for trimethoxyaryl protons H-2′ and H-6′ because of
shielding by the adjacent pyranoquinolinone.
Compound 1 is closely related to melicodenines C−F
(Figure 3) that have recently been isolated from the leaves of
Melicope denhamii, a rutaceous shrub indigenous to regions
from Borneo to the Solomon Islands.15
Compound 1 appears to have full activity on the desired
readout in peripheral blood mononuclear cells (PBMCs) (IL-5
suppression) while limiting the undesired proinflammatory
effects of full/LPS-driven TLR4 agonism. Compound 1 was
also selective for TLR4, exhibiting no activity against TLR2/6
and TLRs 3, 5, 7, 8, and 9 at 10 μM (higher concentrations
would be required to determine if the level of selectivity is
significant, see the Supporting Information for details of TLR4
selectivity). In addition, 1 showed minimal cytotoxicity against
PBMCs at concentrations greater than 30 μM (Table 1). It is
Figure 1. Small-molecule inhibitors of TLR4: Tak-242 (a),
(+)-naloxone (b), and dimethyl 2-(2-nitrobenzylidene)malonate (c).
nanomolar to low micromolar range.14 However, as with
TAK-242, dimethyl 2-(2-nitrobenzylidene)malonate is a
Michael acceptor and almost certainly binds covalently to
Cys747 (the corresponding compound without the α,β-double
bond is inactive with an IC50 > 100 μM).14 TAK-242,
(+)-naloxone, and dimethyl 2-(2-nitrobenzylidene)malonate
are all TLR4 antagonists. There is clearly an urgent need for
small-molecule, druglike agonists of the TLR4 receptor.
Table 1. Activity Profiles and Toxicity of 1 and LPS
TLR receptor activity (EC50)
cell line
1
LPS
3 ng/mL
IDENTIFICATION OF A NOVEL CLASS OF TLR-4
AGONISTS
TLR4
TLR2/6
TLR3
4 μM
■
NA at 10 μM
NA at 10 μM
NA at 10 μM
NA at 10 μM
NA at 10 μM
NA at 1.5 μg/mL
NA at 1.5 μg/mL
NA at 1.5 μg/mL
NA at 1.5 μg/mL
NA at 1.5 μg/mL
Screening a small library of 750 pure natural products against a
panel of TLRs led to the identification of a potent and selective
small-molecule agonist of the TLR4 receptor. Euodenine A (1)
was originally isolated from the air-dried leaves of the tree
Euodia asteridula (Rutaceae) collected in Papua New Guinea.
The EL-Nino fires of 1997/8 destroyed the original tree from
which 1 was isolated, and subsequent recollection of the plant
from the same and different areas failed to provide further
quantities of 1, resulting in the need for total synthesis. The
molecule (C26H29NO5, relative molecular mass 435.51) consists
of two aromatic regions held together in a U-shape, as dictated
by the relative stereochemistry about the central cyclobutane
ring (Figure 2). The relative stereochemistry was determined
through ROESY correlations observed between the methyl at
8′ and H-7′ and among H-7′, H-4, and H-3, placing the methyl
and trimethoxyphenyl groups trans and the pyranoquinoline
and trimethoxyphenyl groups cis on the cyclobutane ring. The
cis relationship of the two aromatic groups was further
supported by the significant upfield shift (by ∼0.5 ppm)
TLR5
TLR7
TLR8
a
Toxicity (CC50)
PBMC
>33 μM
>1000 ng/mL
a
Toxicity data from IL-5-suppression experiments using PHA-
stimulated PBMCs at a concentration of 20 μM showed no toxic
effect for 1. In PBMCs from 4 donors, 1 was tested up to 100 μM with
a small effect at 33 μM and clear evidence of toxicity at 100 μM.
worth noting that closely related melicodenines C−F showed
limited cytotoxicity against DLD-1 human colon cancer cells.
For example, melicodenine D, the compound that most closely
resembles 1, resulted in a 30% reduction in cell viability at
50 μM but no reduction at 20 μM.15
In HEK cell line-secreted alkaline phosphatase (SEAP)
reporter assays for human, mouse, and rat TLR4, 1 was found
to be human-selective in comparison to LPS, which shows
Figure 2. Structure and 3D shape of euodenine A (1) (relative stereochemistry and MacroModel/MMFF force field with H2O solvation of the
lowest-energy conformer are shown).
Figure 3. Structures related to euodenine A, melicodenines C−F (relative stereochemistry shown).
B
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Figure 4. Extracellular site of action of euodenine A (1). A HEK293/NFκB reporter cell line stably transfected with human MD-2 and CD14 was
transiently transfected with a chimeric construct consisting of either the human or mouse TLR4 extracellular domain fused to either the human or
mouse transmembrane and intracellular domains. Cells were subsequently treated with LPS or 1, and reporter gene activity was measured 24 h later.
Data are representative of at least two independent experiments and are expressed as the mean of triplicate determinations. (A) LPS is active in all
human/mouse chimeric assays. (B) 1 is a human-selective agonist and only shows a response when the extracellular domain of human TLR4 is
present.
Figure 5. LPS activation of TLR4 via the CD14, TLR4, and MD-2 complex proceeds via MyD88 and the IRAK/TRAF6 pathway, leading to
activation of proinflammatory cytokines.
species crossover to both rat and mouse. Subsequent reporter
assays using chimeric human/mouse TLR4 receptors were used
to determine the receptor domains required for agonist
function, with LPS used as a control. In the four possible
combinations of chimeric human/mouse, intra/extracellular
TLR reporter assays, 1 only stimulated the NF-κB reporter
when the extracellular domain of human TLR4 was expressed
as a fusion to either the mouse or human intracellular domain
(Figure 4). When the murine extracellular domain was fused to
either the mouse or human intracellular domain, 1 was inactive.
From these studies, and the lack of activity of 1 in the SEAP
mouse assays, it can be concluded that 1 has an extracellular site
of action, as there is a requirement for the extracellular domain
of human TLR4 to stimulate a NF-κB reporter response.
TLR4 is a multicomponent receptor composed of TLR4,
myeloid differentiation factor 2 (MD-2), and cluster of
differentiation 14 (CD14), each of which has their own
costimulatory function (Figure 5).
Initial studies with 1 and LPS revealed that these agonists
require both MD-2 and TLR4 to function and do not work
with TLR4 alone, confirming the essential role of MD-2 in
TLR4 signaling (Figure 6).
Various rat, mouse, and human MD-2 and TLR4 combination
reporter assays were used to determine the dependency of 1 on
MD-2. LPS was active at both human and mouse TLR4
irrespective of the species of MD-2; however, reduced potency
was observed when mouse MD-2 was combined with human
TLR4 (Figure 7a). Compound 1 showed activity only when
human TLR4 was expressed with human MD-2 (Figure 7b).
Mouse MD-2 could not substitute. Therefore, 1 is human-selective
for both human proteins.
Because 1 and LPS stimulate TLR4 by different mechanisms,
cytokine profiling was undertaken. Compound 1 was compared
to LPS, and as expected, LPS showed significant induction of
IL-8, IL-12p40, IL-10, and TNF-α, with little effect on IL-2,
IL-4, and IL-5. In comparison to LPS, 1 showed reduced
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Figure 6. LPS and 1 require MD-2 and TLR4 for agonism. A HEK293/NFκB reporter cell line stably transfected with human TLR4 was transiently
transfected with constructs containing either human MD-2, human CD14, a combination of human MD-2 and CD14, or empty vector. Cells were
subsequently treated with (A) LPS or 1, and reporter gene activity was measured 24 h later. TNF-α was used as a control to stimulate maximal
reporter gene activity, and results were calculated as the percent of the maximal TNF-α response. Data are representative of at least two independent
experiments and are expressed as the mean of triplicate determinations. Signaling only occurs when both TLR4 and MD-2 are present.
Figure 7. 1 is human-selective. A HEK293 NFκB reporter cell line was transiently transfected with a construct containing either human or mouse
TLR4 together with a construct containing human or mouse MD-2 and CD14. Cells were subsequently treated with LPS or 1, and reporter gene
activity was measured 24 h later. Data are representative of at least two independent experiments and are expressed as the mean of triplicate
determinations. (A) LPS is nonselective against human and mouse TLR4. (B) 1 is human selective for both TLR4 and MD-2, and mouse MD-2
cannot substitute.
induction of IL-12p40, IL-10, and TNF-α as well as induction
of IL-8 from PBMCs and the suppression of IL-5 from
activated PBMCs. In 8/8 donors tested, IL-8 induction was
equal to that observed for LPS, whereas IL-10, TNF-α, and
IL-12p40 induction were less than that observed for LPS. In
9/9 activated PBMCs tested, IL-5 was suppressed (Figure 8).
These results suggest that 1 would have the potential to prevent
IL-5-mediated allergic inflammation while having a lower risk of
inducing IL-12- and TNF-α-mediated proinflammatory re-
sponses compared to LPS.
desired intermolecular cycloaddition product in modest yield
(36%) with excellent chemo- and regioselectivity, possibly as a
result of a π-stacking interaction between the aryl group of the
styrene and the quinolinone ring. Demethylation with
potassium iodide in acetic acid afforded the desired natural
product in 85% yield. Subsequent photoaddition of flindersine
(2a) and (E)-1,2,3-trimethoxy-5-(prop-1-enyl)benzene af-
forded the natural product in one step. This method was
used for the generation of 53 cyclobutane-based analogues of 1.
Closely related melicodenines C, D, and E (Figure 3) have also
been synthesized in our laboratory by this route.19
Most of the analogues were synthesized directly from the
corresponding flindersine analogue and functionalized styrene
(Scheme 2). This general synthetic approach enabled variation
of the pyranoquinoline, the alkyl carbon group R at C8′, and
the pendant aromatic attached at C7′. Because only a few of the
required styrenes were commercially available, most were
synthesized using one of four methods (Scheme 3) depending
on the availability of starting materials. The preferred method
of synthesis of the styrenes was from the aldehyde by reaction
with a Grignard reagent to afford the corresponding carbinol
(method A). Subsequent dehydration using excess self-
indicating silica gel in dioxane under microwave conditions20
predominately gave the trans styrene, which was purified by
flash chromatography prior to the photochemical cycloaddition.
Cyclopentyl and cyclohexyl styrenes were accessed via the
required benzylic alcohol, which had been treated with
SYNTHESIS OF 1 AND ANALOGUES
■
Compared to other known TLR4 agonists such as LPS, 1 is much
simpler in structure. Compound 1 was at first synthesized via a
photochemically induced [2 + 2] cycloaddition reaction from O-
methyl flindersine (3) and (E)-1,2,3-trimethoxy-5-(prop-1-enyl)-
benzene followed by cleavage of the methyl ether with potassium
iodide in acetic acid to afford the natural product (Scheme 1).
Initially, the photochemistry was carried out in dichloro-
methane, and the product was isolated by laborious preparative
thin-layer chromatography.16 Improved yields resulted when
the photoaddition was performed without solvent. Evaporation
of a chloroform solution of O-methyl flindersine17,18 and
(E)-1,2,3-trimethoxy-5-(prop-1-enyl)benzene in a jacketed round-
bottomed flask afforded a thin film of reagents on the walls of
the flask. Irradiation of this thin film using a medium-pressure
UV lamp (125 or 400 W) with water cooling for 16 h led to the
D
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Figure 8. Cytokine induction time-course data: IL-8 (A), IL-12p40 (B), IL-10 (C), and TNF-α (D). (E) IL-5 suppression from activated PBMCs by
1 and (F) IL-5 suppression from activated PBMCs by LPS. In PBMC, 1 suppresses PHA-induced IL-5 release and, compared to LPS, stimulates
lower levels of three out of four cytokines tested. (A−F) Human PBMCs were treated with LPS or 1 for 20 h. Levels of IL-8 (A), IL-12p40 (B),
IL-10 (C), and TNF-α (D) were measured in the cell supernatants by ELISA. (E, F) Human PBMC were stimulated for 44 h with 1 μg/mL of
phytohemeagglutinin (PHA) in the presence or absence of LPS or 1. Levels of IL-5 were measured in the cell supernatants by ELISA. Data are from
a representative experiment in PBMC from 1 of 8 donors (cytokine stimulation) or 9 donors (IL-5 suppression) and are expressed as the mean
standard deviation of triplicate determinations.
phosphorus tribromide followed by triethyl phosphite to afford
the phosphonate ester (Michaelis−Arbuzov reaction). Reaction
with cyclopentyl or cyclohexylcarbaldehyde gave the corre-
sponding styrene (Horner−Wadsworth−Emmons procedure,
method B). Nitrostyrenes produced via the Henry reaction
provided substituted styrenes upon treatment with the
corresponding alkyl iodide in the presence of triethylboron
and oxygen (method C),21 and the Suzuki coupling of
cyclopropylvinylboronic acid pinacol ester with an aryl bromide
afforded the cyclopropyl substituted styrenes (method D).
E
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a
Scheme 1. Synthesis of 1
a
Relative stereochemistry shown. Reagents and conditions: (i) UV light (hv), 125 W, neat 16 h; (ii) KI, HOAc, 50 °C, 4 h.
a
Scheme 2. General Synthetic Approach
a
Relative stereochemistry shown. Reagents and conditions: (i) UV light (hv), 125 or 400 W, neat 16 h. Yields: 2−36% (generally, 5−10%).
a
Scheme 3. Methods for Synthesis of Styrenes
a
Reagents and conditions: (i) RCH2MgX, MePh; (ii) 1,4-dioxan, silica, microwave, 160 °C, 30 min; (iii) PBr3, DCM; (iv) P(OEt)3, (Bu)4NI, 110 °C
16 h; (v) NaH, RCHO, THF, 0 °C to rt, O/N; (vi) CH3NO2, NH4OAc, 48 h, reflux; (vii) RI, B(Et)3, rt, 24 h; (viii) Pd(OAc)2, 2 M K2CO3, DME,
80 °C, 16 h. Overall yields for methods A−C were 40−60% and for D (Suzuki coupling), ∼80%.
Further functionalized styrenes were prepared to incorporate
a solubilizing group onto the pendant aryl ring by the following
methods (Scheme 4).
Modifications to the pyranoquinoline were investigated by
utilizing flindersine and further functionalized analogues. Flindersine
(2a), N-methylfindersine (2b), and 9-methylflindersine (2c) were
F
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a
Scheme 4. Synthesis of Functionalized Styrenes
a
Reagents and conditions: (i) DMAP, pyridine; (ii) K2CO3, acetone. Yields: 5a (78%), 5b (37%), 5c (20%), and 5d (96%).
a
Scheme 5. Synthesis of Flindersine Analogues
For example, using a combination of HSQC and ROESY,
correlations were observed between the methyl group on the
cyclobutane ring of 59 and H-3 and H-7′ and between H-7′
and H-3 and H-4.
In an attempt to avoid the low-yielding photochemical
addition step, the cyclobutane portion of 1 was replaced by an
acetal. The acetals (62−64) were produced in good yield from
the diol of flindersine (7) and the corresponding dimethyl
acetal (8a−c) of a functionalized benzaldehyde (Scheme 7).
The relative cis stereochemistry of the two aromatic rings was
established by NMR (ROESY correlations were observed
between the methine H at 11a and the methine H at 3a, and
between H-11a and the H-7′ and H-8′ methylene hydrogens of
the butyl group). Interestingly, the significant upfield shift (by
∼0.5 ppm) observed for the ortho aryl protons in all of the
cyclobutane analogues was not seen with the acetal analogues.
For example, with compound 62, the upfield shift was very
small (0.03 ppm), but there was a significant upfield shift
(0.4 ppm) for the para hydrogen. It was originally thought that
these acetals could mimic the shape of the cyclobutane portion
of 1, thus providing a more synthetically tractable target and
avoiding the photocycloaddition. However, the upfield shift for
the para hydrogen rather than the ortho hydrogen probably
reflects a slightly different shape or conformation for these
acetal analogues.
a
Reagents and conditions: (i) (a) Yb(OTf)3, MeCN, 80 °C, 12 h or
(b) MgSO4, pyridine, reflux 16 h. Yields: 2a (42%), 2b (98%), 2c
(95%), and 2e,f (18%).
prepared by the method of Lee et al.22 utilizing a tandem
Knoevenagel-electrocyclic reaction (Scheme 5).
9-Methoxyflindersine (2d) was purchased from Chembridge
and afforded 57−58. 9-Methylflindersine (2c) afforded 47−49.
Pruning of the pyranoquinoline core was also investigated.
Analogues 55 and 56, possessing a single methyl group at C-10,
were prepared as a mixture from the corresponding flindersine
analogues (2e,f). These were obtained using the same
procedure as that employed for flindersine but by replacing
3-methyl-2-butenal with crotonaldeyde. Further modifications
were made to the pyranoquinolinone by pruning the phenyl
ring to afford a pyranopyridinone (6) scaffold, which was used
to synthesize a few selected pyridinone analogues (59−61,
Scheme 6). The pyranopyridinone scaffold (6) was synthesized
from 2,4-dihydroxypyridine. Unfortunately, these compounds
(59−61) had no activity when tested in the TLR4 assay.
Relative to the styrene starting material, all of the analogues
(9−61) of euodenine A exhibited a characteristic upfield shift
of ortho aryl protons H-2′ and/or H-6′ by ∼0.5 ppm (range
SCREENING RESULTS AND DISCUSSION
■
The cyclobutane series showed good SAR at the TLR4 receptor
(Table 2). When the methyl group on the cyclobutane is
removed (9),16 activity is lost, whereas replacing the methyl
group by larger groups results in an increase in activity up to
R = cyclopentyl. For example, in the dimethoxyphenylcyclo-
butane series (1 and 9−43, R1 and R4 = H, R2 and R3 = OMe),
there is a regular increase from R = Me (10, EC50 3.1 μM) to Et
(19, EC50 1.2 μM) to Bu (37, EC50 1.2 μM) to i-Pr (26, EC50
1.0 μM) to cyclopropyl (31, EC50 0.63 μM) to cyclopentyl
(34, EC50 0.39 μM). Cyclopentyl compound 34 was the most
active analogue prepared. In the trimethoxphenylcyclobutane
series (1 and 9−43, R1 = H, R2−R4 = OMe), there was a similar
increase in activity with increasing size of R, but it was not as
1
0.2−0.75 ppm) in the H NMR spectrum. Meta aryl protons
H-3′ and/or H-5′ exhibited a smaller upfield shift of ∼0.3 ppm
(range 0.1 − 0.6 ppm), but para aryl protons H-4′ displayed a
negligible or very small (<0.2 ppm) upfield shift. This upfield
shift of the ortho and meta protons is due to shielding by the
adjacent pyranoquinolinone (or pyranopyridinone) and served
as a diagnostic to confirm the cis relationship of the two
aromatic groups. The cis relationship between the two aromatic
rings was confirmed by ROESY for the majority of analogues.
G
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a
Scheme 6. Synthesis of Pyridinone Analogues
a
Reagents and conditions: (i) (a) Yb(OTf)3, MeCN, 80 °C, 12 h or (b) MgSO4, pyridine, reflux 16 h; (ii) UV light (hv), 125 or 400 W, neat 16 h.
Yields: 6 (80%), 59 (6.6%), 60 (11%), and 61 (8.5%).
a
Scheme 7. Synthesis of Acetal Analogues
a
Reagents and conditions: (i) PropylMgBr, MePh, rt, 26 h; (ii) MnO2, DCM, rt, 24 h; (iii) H2SO4, MeOH, (MeO)3CH; (iv) DCM, PPTS, 18 h.
Overall yields: 62 (50%), 63 (78%), and 64 (30%).
pronounced or as regular (1, Me, EC50 3.9 μM; 20, Et, EC50
0.79 μM; 36, Bu, EC50 2.0 μM; 22, i-Pr, EC50 0.79 μM; 32,
cyclopropyl, EC50 0.63 μM; and 35, cyclopentyl, EC50 0.63 μM).
When R is larger than cyclopentyl or with any group other than
alkyl, activity at TLR4 is reduced or lost altogether (e.g., 38, COMe,
EC50 10 μM; 42, CH2OiPr, not active at 300 μM). This indicates
that the methyl group occupies a hydrophobic pocket of limited size.
Comparing the activities of the trimethoxyphenylcyclobutane
with the dimethoxyphenylcyclobutane series, the removal of
one of the meta methoxy groups had little effect on TLR4
activity (e.g., 1, EC50 3.9 μM and 10, EC50 3.1 μM; 31, EC50
0.63 μM and 32, EC50 0.63 μM), whereas introduction of
groups larger than OMe at the para position (R3) proved
detrimental to TLR4 activity (26, OMe, EC50 1.0 μM and 28,
OEt, EC50 3.9 μM; 10, OMe, EC50 3.1 μM and 17, OBn, not
active at 300 μM). Replacing the para OMe group of 1 by the
smaller H did not affect the activity (11, EC50 3.1 μM), but
replacing it by the polar OH resulted in a significant loss of
activity (18, EC50 >10 μM). Groups larger than OMe could,
however, be introduced at the meta position (R2), allowing the
introduction of solubilizing groups and resulting in analogues
with improved solubility properties and sometimes improved
activities (26, OMe, EC50 1.0 μM and 29, O(CH2)2OH, EC50
0.5 μM; 31, OMe, EC50 0.79 μM and 30, O(CH2)3OH, EC50
0.79 μM), but the larger solubilizing group O(CH2)3morpholine,
resulted in reduced activity (31, OMe, EC50 0.63 μM; 33,
O(CH2)3morpholine, EC50 3.9 μM). Replacing a meta methoxy
group by an acyl group resulted in a significant reduction in
activity (26, OMe, EC50 1.0 μM and 25, OAc, EC50 >10 μM; 27,
OCO(CH2)2CO2H, EC50 6.3 μM). Removal of both meta
methoxy groups was detrimental to activity (10, R2 and R3 =
OMe, EC50 3.1 μM and 15, R2 = H, R3 = OMe, EC50 >10 μM).
Replacing a meta methoxy group in the dimethoxyphenyl-
cyclobutane series by a halogen resulted in a modest decrease in
activity, with the effect becoming more pronounced with
increasing electronegativity (10, OMe, EC50 3.1 μM and 12, Br,
EC50 3.9 μM; 26, OMe, EC50 1.0 μM and 23, Cl, EC50 2.0 μM;
19, OMe, EC50 1.2 μM and 21, F, EC50 3.1 μM).
Alkylation of the pyridine nitrogen (R5, 44−54, Table 3) had
little effect on the intrinsic activity in most cases (10, R5 = H,
EC50 3.1 μM and 44, R5 = Me, EC50 2.5 μM; 19, R5 = H, EC50
1.2 μM and 45, R5 = Me, EC50 2.0 μM; 21, R5 = H, EC50
3.1 μM and 46, R5 = Me, EC50 3.1 μM). Other modifications to
the pyranoquinolone portion of the molecule generally had a
detrimental effect on the activity at the TLR4 receptor. For
example, removal of either one of the gem-dimethyl groups
from parent compound 1 resulted in a significant loss in activity
(1, EC50 3.9 μM and 55, 56, EC50 >10 μM). Assuming that the
NH and NMe analogues (R5 = H or Me) have comparable
activity as noted above, introduction of a methyl group at C9
(R6) results in a decrease in activity (20, R = Et, R6 = H, EC50
0.79 μM and 47, R = Et, R6 = Me, EC50 3.1 μM; 19, R = Et,
H
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Table 2. Aryl and Alkyl Modifications
a
b
EC50 TLR4 (μM)
IA
R
R1
R2
R3
R4
1
9
3.9
0.95
Me
H
H
OMe
OMe
OMe
OMe
Br
OMe
OMe
OMe
H
OMe
OMe
H
c
NA at 30 μM
H
H
H
H
Cl
H
H
OMe
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
3.1
0.69
0.46
0.28
0.44
Me
Me
Me
Me
Me
Me
Me
Me
Me
Et
3.1
OMe
H
3.9
OMe
OMe
H
3.1
OMe
H
H
NA at 71.5 μM
H
>10
H
OMe
OMe
OBn
OH
H
>10
OMe
OMe
OMe
OMe
OMe
F
H
NA at 300 μM
H
>10
H
1.2
1.01
1.05
0.49
1.18
0.47
0.2
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OEt
H
0.79
Et
OMe
H
3.1
Et
0.79
i-Pr
i-Pr
i-Pr
i-Pr
i-Pr
i-Pr
i-Pr
i-Pr
OMe
Cl
OMe
H
2.0
2.0
CH2Cl
OAc
OMe
H
>10
H
1.0
0.9
H
6.3
0.29
0.19
0.46
0.88
0.75
0.82
0.19
0.9
OCO(CH2)2CO2H
H
3.9
OMe
H
0.5
O(CH2)2OH
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
Br
H
0.79
Cypr
O(CH2)3OH
H
0.63
Cypr
OMe
H
0.63
Cypr
OMe
OMe
H
3.9
Cypr
O(CH2)3morpholine
0.39
Cyp
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
H
H
0.63
0.63
0.69
1.01
0.30
0.62
Cyp
OMe
OMe
H
2.0
Bu
1.2
Bu
10
COMe
COCypr
CO2Me
CH2OAc
CH2OiPr
CO2Et
H
7.9
H
NA at 25 μM
5.0
OMe
OMe
OMe
H
0.1
NA at 300 μM
NA at 333 μM
a
EC50 values are mean values based on between two and eight determinations. The standard deviations for −log EC50 values were <0.1 in nearly all
b
c
cases (range 0.00 to 0.23). Intrinsic activity relative to LPS. Ref 16.
R6 = H, EC50 1.2 μM and 48, R = Et, R6 = Me, EC50 3.1 μM;
22, R = i-Pr, R6 = H, EC50 0.79 μM and 49, R = i-Pr, R6 = Me,
EC50 7.9 μM). Introduction of a methoxy group at C9 (R6)
resulted in a large decrease in activity (1, R = Me, R6 = H, EC50
3.9 μM and 58, R = Me, R6 = Me, EC50 >10 μM; 26, R = i-Pr,
R6 = H, EC50 1.0 μM and 57, R = i-Pr, R6 = Me, EC50 > 10 μM).
Pyridinone analogues (benzene ring removed from the quinoli-
none) did not show any activity when tested in the TLR4 assay.
In summary, modifications to the trimethoxyaryl ring, the
cyclobutane substituent R, the quinolinone, and pyran rings
demonstrated the tight SAR requirements of the TLR4 receptor.
A small number of analogues with the cyclobutane ring
replaced by an acetal were also examined. On testing, these
analogues (62−64) did show some promising activity in the
human TLR4 SEAP assay (Table 4); however, they were less
active than most of the cyclobutane compounds and were not
further investigated.
CONCLUSIONS
■
Euodenine A, a natural product of unusual architecture, is a
human TLR4-selective agonist that is CD14-independent, and
it requires both TLR4 and MD-2 for full efficacy. Euodenine A
and a series of analogues were prepared in two to four steps
from readily available starting materials. The dimethoxyphe-
nylcyclobutane series showed good SAR at the TLR4 receptor.
Thus, when the methyl group on the cyclobutane is removed,
activity is lost, whereas replacing the methyl group by larger
groups results in an increase in activity up to R = cyclopentyl,
I
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Table 3. Pyranoquinolinone Modifications
a
b
EC50 TLR4 (μM)
IA
R
R1
R2
R3
R4
R5
R6
R7
R8
44
45
46
47
48
49
50
51
52
53
54
55
56
57/
58
2.5
0.64
0.61
0.36
0.3
Me
Et
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OMe
OMe
F
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
H
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
H
H
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
H
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
H
2.0
H
H
3.1
Et
H
H
3.1
Et
OMe
OMe
OMe
Cl
OMe
H
Me
Me
Me
H
3.1
0.29
0.41
0.53
0.31
0.81
0.67
0.69
Et
7.9
i-Pr
i-Pr
i-Pr
Cypr
Cypr
Cyp
Me
Me
i-Pr
Me
OMe
H
3.1
3.1
OAc
OMe
OMe
OMe
OMe
OMe
OMe
OMe
H
H
0.79
1.0
OMe
H
H
H
2.5
OMe
OMe
OMe
H
H
>10
>10
>10
>10
H
H
H
Me
Me
Me
H
OMe
OMe
Me
Me
OMe
H
a
EC50 values are mean values based on between two and eight determinations. The standard deviations for −log EC50 values were <0.1 in nearly all
b
cases (range 0.00 to 0.23). Intrinsic activity relative to LPS.
at 125 MHz. Chemical shifts are reported in parts per million (ppm)
relative to the solvent (DMSO-d6: 1H, 2.49; 13C, 39.51 ppm or CDCl3:
1H, 7.26; 13C, 77.0 ppm), and coupling constants (J) are reported in
Table 4. Acetal Analogues
hertz. HPLC−MS was run in ESI mode using a C18 50 mm × 4.6 mm,
5 μm analytical column using a diode array detector over multiple
wavelengths. Both HPLC−MS and HPLC were reverse-phase with a
MeCN/H2O (0.01% TFA) gradient and a flow rate of 1.5 mL/min.
The purity of all tested compounds was established as ≥95% by HPLC
analysis using two different gradients with UV detection on a
photodiode array detector (210, 254, and 280 nm). Flash
chromatography was performed using silica gel (230−400 mesh) or
neutral alumina.
Molecular Modeling. The structure of 1 was subjected to
geometry optimization in MacroModel using the MMFF force field
with H2O solvation. Default parameters and convergence criteria were
used elsewhere. The reported structure is the lowest-energy conformer
resulting from a mixed MCMM/Low-Mode search with 1000 steps
per rotatable bond (including tetrahydropyan ring bonds).
a
EC50 TLR4 (μM)
IA
R
R1
62
63
64
3.1
0.7
0.2
H
OMe
H
15.8
OMe
OMe
NA at 60 μM
OMe
a
EC50 values are mean values based on between two and eight
determinations. The standard deviations for −log EC50 values
were <0.1 in nearly all cases (range 0.00 to 0.23).
Isolation and Structure Determination of Euodenine A (1)
from Euodia Asteridula. The leaves of Euodia asteridula (Rutaceae)
were collected by Biodiversity Research Ltd. in PNG, and a voucher
sample is lodged at the Lae Herbarium, Lae, PNG.
which was the most active compound and 10-fold more active
than euodenine A. Replacing the methyl by groups larger than
cyclopentyl resulted in reduced activity. Groups larger than
OMe could be introduced into the meta position, allowing the
introduction of solubilizing groups and resulting in analogues with
improved solubility properties and sometimes improved activities.
Testing for immunomodulation in PBMC cells shows the
induction of the cytokines IL-8, IL-10, TNF-α, and IL-12p40
and suppression of IL-5 from activated PBMCs, indicating that
compounds like 1 could modulate the Th2 immune response
without causing lung damage. The results suggest that 1 would
have the potential to prevent IL-5-mediated allergic inflammation
while having a lower risk of inducing IL-12- and TNF-α-mediated
proinflammatory responses compared to LPS.
Extraction and Isolation. The air-dried material (100 g) was
ground and extracted exhaustively with methanol (1 L) and
dichloromethane saturated with ammonia (400 mL). The dichloro-
methane extract was evaporated; the dry extract was then dissolved in
the methanol extract. Mass spectrometry was performed on this
combined extract to confirm the target alkaloid compounds chosen by
the small-scale MS survey. Strong cation-exchange resin (SCX, 30 g)
was added to the extract. The mixture was shaken by hand and left for
15 min. Mass spectrometry was performed to confirm the target
alkaloids were completely bound to the SCX. The mixture was then
vacuum filtered with a sintered glass funnel. The load extract without
alkaloid was thus separated from the alkaloid-bound SCX resin. The
bound alkaloids were eluted off the SCX resin by methanol-ammonia
(10%) (300 mL) and dichloromethane saturated with ammonia
(100 mL). This alkaloid-enriched fraction was then evaporated (6 g)
and preabsorbed onto C18 (2 g) and packed dry into a small cartridge,
EXPERIMENTAL SECTION
■
Chemistry, General Methods, and Materials. 1H NMR spectra
were recorded at either 500 or 600 MHz, and 13C NMR were recorded
J
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1
Table 5. H (500 MHz) and 13C (125 MHz) NMR Data for Euodenine A in DMSO-d6
position
13C NMR
1H NMR
g-COSY
g-HMBC
ROESY
2
3
4
76.5
45.7
2.33 (m)
4, 8′
3, 7′
4a, 4, 2, 8′CH3, 8′, 7′
5, 4a, 10b, 3, 8′, 7′
7′, 4, 2′CH3, 2′CH3
7′, 3, 2′CH3
31.7
3.69 (dd, 8.4, 8.0)
11.06 (s)
4a
107.7
162.2
5
6 (NH)
4a, 6a, 10a
9
6a
137.5
114.6
129.8
120.9
121.7
114.8
155.5
135.9
105.4
151.7
135.8
151.7
105.4
49.4
7
7.15 (d, 8.5)
8
10b, 9, 8, 10a
9, 6a
9, NH
8
7.42 (dd, 8.5, 7.5)
7.13 (dd, 8.0, 7.5)
7.82 (d, 8.0)
9, 7
10, 8
9
9
9
8, 10a
10, 8
9
10
10b, 8, 7
10a
10b
1′
2′
3′
4′
5′
6′
7′
6.12 (s)
7′, 1′, 3′, 4′, 6′
8′,7′, OMe (C3′)
6.12 (s)
7′, 1′, 2′, 4′, 5′
4a, 3, 8′CH3, 8′, 1′, 2′, 6′
4, 2, 8′CH3, 7′, 1′
3, 2, 2′CH3
8′, 7′, OMe (C5′)
4,3, 8′CH3, 2′, 6′
8′CH3, 2′, 6′
13, 2′CH3
3.30 (dd, 8.4, 9.0)
2.33 (m)
1.14 (s)
4, 8′
8′CH3, 7′, 3
8′
35.2
2′CH3
2′CH3
8′CH3
OMe (C3′)
OMe (C4′)
OMe (C5′)
23.2
24.4
1.48 (s)
10b, 3, 2, 2′CH3
3, 8′, 7′
3′
4′
5′
8′, 2′CH3
8′, 7′, 2′, 6′
2′
20.2
1.13 (d, 7.0)
3.34 (s)
8′
55.0
59.8
3.53 (s)
55.0
3.34 (s)
6′
which was connected to a preparative C18 column (Betasil C18,
150 × 21.2 mm, 5 μm, Thermo Electro Corporation). This fraction
was chromatographed using mass-directed purification on a HP1100
LC−MS and eluted at 10 mL/min with a gradient solvent system
starting with 100% water (1% TFA) gradient to 60% acetonitrile (1%
TFA)/40% water (1% TFA) in 50 min, then gradient to 100%
acetonitrile (1% TFA) in the next 10 min. Pure 1 (20 mg) was eluted
at 53−55 min. Yellow oil, optically inactive, UV (MeOH) λmax (ε) 212
(28 602), 217 (27 502), 286 (4602), 317 (4711), 329 nm (3971). IR
(film) νmax 3424, 2949, 1647, 1605, 1507, 1457, 1427, 1396, 1238,
1H NMR spectrum. COSY correlation from the methyl protons on
C-8′ to δ 2.33 indicated that the carbon at 35.2 ppm must be vicinal to
the methyl group and therefore the carbons at δ 45.7 and 49.4 ppm
were each vicinal to C-8′. If the secondary methyl group was vicinal to
C-3, then it should have had a HMBC correlation to C-2. COSY
correlations were also observed from the signal at δ 2.33 to methine
signals at δ 3.30 (H-7′) and 3.69 (H-4). This could only be possible if
the proton attached to C-3 (δ 2.33) was vicinal to H-4 (δ 3.69). A
COSY correlation from H-4 to H-7′ indicated that they too were
vicinal to each other and that AR-Q500206 contained a tetrasub-
stituted cyclobutyl group. The molecule contained a symmetrical
tetrasubsituted phenyl group because a two-proton aromatic singlet at
δ 6.12 (H-2′ and H-6′) displayed both HMBC and HSQC correlations
to a carbon signal at 105.4 ppm (C-2′ and C-6′), indicating that the
two protons were meta to each other. The chemical shift of the
carbons C-2′ and C-6′ suggested that they were both ortho to oxygen
substituents, and this was confirmed by the observation of HMBC
correlations from these protons to oxygenated aromatic quaternary
carbons at δ 151.7 and 135.8 ppm. HMBC correlations were also
observed from the three methoxyl protons at δ 3.34 (2 methoxyls) and
3.53 to the oxygenated aromatic quaternary carbons at δ 135.8 and
151.7 ppm, respectively. The upfield chemical shift of one of these
oxygenated quaternary carbons C-4′ suggested that it was flanked on
either side by the other two methoxyl groups. It could therefore be
concluded that the molecule contained a 1,2,3-trimethoxylphenyl
group. This phenyl group was attached to C-7′ because HMBC
correlations were observed from H-2′ and H-6′ to C-7′. HMBC
correlations from δ 2.33 (H-3), 3.30 (H-7′), and 3.69 (H-4) to a
quaternary carbon (C-4a) at 107.7 ppm indicated that this carbon was
vicinal to C-4. The proton H-4 also correlated to an additional
downfield quaternary carbon at δ 155.5 (C-10b) and the amide
carbonyl carbon C-5 at 162.2 ppm. Both of these carbons could
logically be attached only to the carbon (C-4a) at δ 107.7 ppm, and
the chemical shift of C-10b suggested that it was oxygenated. A
downfield exchangeable proton H-6 (δ 11.06) assigned to an amide
NH also correlated to C-4a. The four remaining protons in the
1
1128, 756, 505 cm−1. For 13C and H NMR data, see Tables 5 and 6
(numbering as in 1, Scheme 2). (+)-HRESIMS m/z 436.2126 (calcd
for C26H30NO5, 436.2119).
Structure Determination. The 13C NMR spectrum (Table 5) of 1
displayed 23 carbon signals. Three of the carbons were double the
intensity of the others, suggesting that there was some symmetry in
the molecule. The carbons could be assigned to six methyls
(two coincident), four aliphatic methines, six aromatic methines
(two coincident), nine downfield quaternary carbons (two coincident),
and one oxygenated quaternary carbon from interpretation of g-HSQC
1
and H NMR data. The most downfield carbon signal at δ 162.2 was
assigned to a conjugated amide because an IR absorption band was
observed at 1647 cm−1. Out of the six methyls, three were aromatic
1
methoxy groups, two of which were coincident. Analysis of the H
NMR spectrum of euodenine A indicated that the remaining methyl
groups were two quaternary methyls at δ 1.14 and 1.48 and one was a
secondary methyl signal at δ 1.13 (d, 7 Hz). Both quaternary methyl
protons showed a HMBC correlation to the oxygenated quaternary
carbon C-2 (δ 76.5) and to an aliphatic methine C-3 at δ 45.7 ppm.
This demonstrated that the molecule contained an isopropyloxy group
vicinal to an aliphatic methine, C-3. The secondary methyl proton on
C-8′ also correlated to C-3 as well as to another two methine carbons,
C-8′ (35.2 ppm) and C-7′ (49.4 ppm), in the HMBC spectrum.
Interpretation of the COSY spectrum was complicated by the
observation that the signals for the protons attached to C-3
(45.7 ppm) and C-8′ (35.2 ppm) were coincident (δ 2.33) in the
K
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1
Table 6. H (600 MHz) and 13C (125 MHz) NMR Data for Euodenine A in Methanol-d4
position
13C NMR
1H NMR
g-COSY
g-HMBC
ROESY
2
3
4
78.7
47.7
2.39 (dd, 8.4, 9.0)
3.81 (dd, 8.4, 9.0)
4, 8′
3, 7′
4a, 4, 2, 2′CH3, 8′ CH3, 8′
5, 4a, 10b, 3, 8′, 7′
4, 2′CH3, 8′CH3, 7′
7′, 3, 2′CH3
33.5
4a
108.9
165.4
5
6 (NH)
6a
138.6
116.4
131.7
123.3
123.6
117.2
159.0
137.6
107.0
153.6
137.6
153.6
107.0
51.8
7
7.22 (d, 9.0)
8
10b, 9,10a
9, 7
8
8
7.49 (ddd, 8.4, 9.0, 1.2)
7.24 (dd, 8.4, 8.4)
7.98 (dd, 1.2, 8.4)
9, 7
10, 8
9
9, 7
10, 8
9
9
8, 7, 6a, 10a
10b, 8, 6a
10
10a
10b
1′
2′
3′
4′
5′
6′
7′
6.14 (s)
4, 7′, 1′, 3′, 4′, 6′
8′,7′, OMe (C3′)
6.14 (s)
4, 7′, 1′, 2′, 4′, 5′
4a, 4, 8′CH3, 8′, 1′, 2′, 6′
3, 7′, 2′
3, 2, 2′CH3
8′, 7′, OMe (C5′)
4,3, 8′CH3, 2′, 6′
8′CH3, 2′, 6′, 2′CH3
4, 3, 2′CH3
8′, 2′CH3
3.36 (dd, 10.2, 9.0)
2.45 (ddq, 10.2, 9.0, 6.0)
1.23 (s)
4, 8′
8′CH3, 7′, 3
8′
36.9
2′CH3
2′CH3
8′CH3
OMe (C3′)
OMe (C4′)
OMe (C5′)
24.1
25.2
1.56 (s)
10b, 3, 2, 2′CH3
20.8
1.19, (d, 6.0)
3.40 (s)
8′
3, 8′, 7′
3, 8′, 7′
2′
56.1
3′
4′
5′
61.1
3.64 (s)
56.1
3.40 (s)
6′
1H NMR spectrum were part of a 1,2-disubstituted aromatic group
because correlations were observed in the COSY spectrum from H-10
and H-8 to H-6 and H-7. One of the substituents of the disubstituted
aromatic group was the carbon at δ 155.5 ppm (C-10b) because a HMBC
correlation was observed to this carbon from H-10. The other substituent
was deduced to be a nitrogen because H-10, H-8, and the amide proton,
H-6, showed HMBC correlations to a heteroatom-substituted aromatic
quaternary carbon at δ 137.5 ppm. The remaining quaternary aromatic
carbon C-10a correlated to H-9, H-6a, and the amide proton, H-6, and
from this data, it was concluded that AR-Q500206 contained a
3-cyclobutyl-substituted quinolinone. This data indicated that 12 of the
13 degrees of unsaturation were accounted for, which dictated that the
molecule must contain an extra ring. A weak HMBC correlation from the
quaternary methyl proton on C-2 to the oxygenated quaternary carbon
C-10b suggested that a carbon oxygen bond linked the isopropyloxy
group to the oxygenated quaternary carbon C-10b, thus forming a
dihydropyran ring. The gross structure of euodenine A was therefore
assigned.
A change in deuterated solvents for NMR experiments from
DMSO-d6 to methanol-d4 helped to resolve the overlapping signals of
the quaternary methyl (δ 1.14), the secondary methyl (δ 1.13), H-3 (δ
2.33), and H8′ (δ 2.33) (Table 6). Correlations observed in a ROESY
spectrum performed in methanol-d4 were used to deduce the relative
stereochemistry of the substituents about the cyclobutyl group. The
secondary methyl and the trimethoxyphenyl group were trans to each
other because ROESY correlations were observed between the methyl
at 8′ and H-7′. H-7′, H-4, and H-3 were all on the same face of the
cyclobutyl group because ROESY correlations were observed among
these three protons. The cis relationship of the quinolinone and
trimethoxy phenyl ring helps to explain the significant upfield shift of
the aromatic protons H-2′ and H-6′ of the trimethoxyphenyl group. In
a cis configuration, the phenyl group lies in the shielding zone of the
quinolinone. Thus, the structure of euodenine A was deduced as 1.
Synthesis of 1 and Analogues. General Procedure for Synthesis
of Styrenes. Method A: (E)-1,2,3-Trimethoxy-5-(prop-1-enyl)-
benzene [5273-85-8] (4a). 3,4,5-Trimethoxybenzaldehyde
(51 mmol) was dissolved in toluene (40 mL) and cooled to 0 °C.
A solution ethylmagnesium chloride (3.0 M in Et2O, 91.8 mmol) was
added dropwise, and the reaction mixture was stirred for a further
16 h. To this was added 5% AcOH (200 mL), and the resulting
mixture extracted with EtOAc (100 mL). The organic phase was then
extracted with water (100 mL), saturated NaHCO3 (100 mL), and
brine (100 mL). The organic phase was dried (MgSO4), filtered, and
concentrated to afford 1-(3,4,5-trimethoxyphenyl)propan-1-ol (10.86
1
g, 94%). H NMR (CDCl3, 500 MHz) δ 0.97 (t, J = 7 Hz, 3H), 1.79
(m, 2H), 3.87 (s, 3H), 3.90 (s, 6H), 4.56 (t, J = 6.5 Hz, 1H), 6.61
(s, 2H). 13C NMR (CDCl3, 125 MHz) δ 10.44, 21.64, 32.16, 56.34,
61.03, 76.44, 103.12, 137.50, 138.07, 140.67, 153.47. (+) LRMS (ESI)
m/z 227 [M + H]+. (E)-(3,4,5-Trimethoxyphenyl)propan-1-ol (8 g)
was dissolved in dioxan (40 mL), and orange silica gel (Riedel-de-
Haen 13767, 40 g) was added. The resulting solution was heated
̈
under microwave conditions at 180 °C for 10 min. The reaction
mixture was filtered, concentrated to dryness, and then purified by
flash chromatography on silica gel using a gradient from 100% hexane
to 20% EtOAc over 20 min to afford (E)-1,2,3-trimethoxy-5-(prop-1-
enyl)benzene (3.86 g, 53%) as a colorless oil. 1H NMR (CDCl3,
500 MHz) δ 1.84 (d, J = 6.5 Hz, 3H), 3.82 (s, 3H), 3.90 (s, 6H), 6.19
(dt, J = 6.5, 13.5 Hz, 1H), 6.37 (d, J = 16 Hz, 1H), 6.60 (s, 2H). (+)
LRMS (ESI) m/z 209 [M + H]+.
(E)-1,3-Dimethoxy-5-(prop-1-enyl)benzene [146205-98-3] (4b).
1
From 3,5-dimethoxybenzaldehyde and ethylmagnesium chloride. H
NMR (CDCl3, 500 MHz) δ 1.74 (d, J = 6.5 Hz, 3H), 3.66 (s, 3H),
3.68 (s, 3H), 6.08 (dt, J = 6.5, 13.5 Hz, 1H), 6.20 (d, J = 16 Hz, 1H),
6.20 (s, 1H), 6.36 (s, 2H). (+) LRMS (ESI) m/z 179 [M + H]+.
(E)-2-Bromo-1-methoxy-4-(prop-1-enyl)benzene (4c). From 3-
bromo-4-methoxybenzaldehyde and ethylmagnesium bromide. 1H
NMR (DMSO-d6, 500 MHz) δ 1.83 (d, J = 7 Hz, 6H), 3.84 (s,
3H), 6.21 (m, 1H), 6.34 (d, J = 15 Hz, 1H), 7.05 (d, J = 8 Hz, 1H),
7.36 (dd, J = 1.5, 8 Hz, 1H), 7.60 (d, J = 1.5 Hz, 1H). LRMS (ESI)
m/z 228 [M79Br + H]+, 230 [M81Br + H]+.
(E)-2-Chloro-3,4-dimethoxy-1-(prop-1-enyl)benzene (4d). From
2-chloro-3,4-dimethoxy benzaldehyde and ethylmagnesium bromide.
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1H NMR (DMSO-d6, 500 MHz) δ 1.83 (d, J = 7 Hz, 6H), 3.74
(s, 3H), 3.85 (s, 3H), 6.34 (m, 2H), 7.05 (m, 2H). LRMS (ESI) m/z
213 [M35Cl + H]+, 215 [M37Cl + H]+.
(E)-4-(But-1-enyl)-1,2-dimethoxybenzene [76252-28-3] (4e).
From 3,4-dimethoxybenzaldehyde and propylmagnesium chloride.
1H NMR (d6-DMSO, 500 MHz) δ 1.05 (t, J = 6.5 Hz, 3H), 2.15
(q, J = 6.5 Hz, 2H), 3.74 (s, 3H), 3.77 (s, 3H), 6.20 (dt, J = 6.5, 13.5
Hz, 1H), 6.31 (d, J = 16 Hz, 1H), 6.87 (br s, 2H), 7.00 (br s, 1H). 13C
NMR (d6-DMSO, 125 MHz) δ 13.6, 25.3, 55.4, 55.4, 108.9, 111.8,
118.6, 128.4, 130.0, 130.4, 148.0, 148.8. (+) LRMS (ESI) m/z 193
[M + H]+.
(E)-5-(But-1-enyl)-1,2,3-trimethoxybenzene [357386-06-2] (4f).
From 3,4,5-trimethoxybenzaldehyde and propylmagnesium chloride.
1H NMR (CDCl3, 500 MHz) δ 1.12 (t, J = 7.5 Hz, 3H), 2.25 (m, 2H),
3.86 (s, 3H), 3.90 (s, 6H), 6.21 (dt, J = 6.5, 13.5 Hz, 1H), 6.33 (d, J =
16 Hz, 1H), 6.60 (s, 2H). 13C NMR (CDCl3, 125 MHz) δ 14.2, 19.3,
41.5, 56.3, 61.1, 74.8, 103.0,137.4, 141.0, 153.4. (+) LRMS (ESI) m/z
223 [M + H]+.
(E)-4-(But-1-enyl)-2-fluoro-1-methoxybenzene (4g). From 3-fluo-
ro-4-methoxybenzaldehyde and isobutylmagnesium chloride. 1H NMR
(DMSO-d6, 500 MHz) δ 1.03 (t, J = 7.5 Hz, 3H), 2.16 (q, J = 7.5 Hz,
2H), 3.82 (s, 3H), 6.18 (dt, J = 6, 14 Hz, 1H), 6.30 (d, J = 14 Hz, 1H),
7.07 (dd, J = 8, 9 Hz, 1H), 7.11 (br d J 8.5 Hz, 1H), 7.25 (dd, J = 1.5,
11 Hz, 1H). 13C NMR (DMSO-d6, 500 MHz) δ 14.2, 26.0, 56.6, 113.3
(d, J = 18 Hz), 114.4 (d, J = 17 Hz), 123.0 (d, J = 12 Hz), 137.9, 131.6
(d, J = 6 Hz), 132.2, 146.7 (d, J = 11 Hz), 152.4 (d, J = 242 Hz). (+)
LRMS (ESI) m/z 181 [M + H]+.
6.71 (dd, J = 1.8, 8.4 Hz, 1H), 6.80 (s, 1H), 6.81 (d, J = 8 Hz, 1H),
8.83(s, 1H). LRMS (ESI) m/z 193 [M + H]+.
(E)-5-(Hex-1-enyl)-1,2,3-trimethoxybenzene [208192-84-1] (4n).
From 3,4,5-trimethoxybenzaldehyde and pentylmagnesium bromide.
1H NMR (DMSO-d6, 500 MHz) δ 0.91 (t, J = 6.5 Hz, 3H), 1.34 (m,
2H), 1.43 (m, 2H), 2.17 (q, J = 6.5 Hz, 2H), 3.64 (s, 3H), 3.78 (s,
6H), 6.25 (dt, J = 6.5, 13.5 Hz, 1H), 6.32 (d, J = 16 Hz, 1H), 6.68 (br
s, 2H). 13C NMR (DMSO-d6, 125 MHz) δ 13.7, 21.6, 30.9, 31.9, 55.8,
60.0, 103.2, 133.1, 136.7, 152.9. (+) LRMS (ESI) m/z 250 [M + H]+.
(E)-4-(Hex-1-enyl)-1,2-dimethoxybenzene [195192-75-7] (4o).
From 3,4-dimethoxybenzaldehyde and pentylmagnesium bromide.
1H NMR (DMSO-d6, 500 MHz) δ 0.91 (t, J = 6.5 Hz, 3H), 1.34 (m,
2H), 1.41 (m, 2H), 2.17 (q, J = 6.5 Hz, 2H), 3.74 (s, 3H), 3.78 (s,
3H), 6.15 (dt, J = 6.5, 13.5 Hz, 1H), 6.31 (d, J = 16 Hz, 1H), 6.86 (br
s, 2H), 7.00 (s, 1H). 13C NMR (DMSO-d6, 125 MHz) δ 13.7, 21.7,
31.1, 32.0, 55.3, 55.4, 109.0, 111.4, 111.7, 118.6, 128.3, 129.3, 130.4,
148.0, 148.8. (+) LRMS (ESI) m/z 221 [M + H]+.
Method B: 4-[(E)-2-Cyclopentylvinyl]-1,2-dimethoxybenzene (4p).
A solution of 3,4-dimethoxybenzyl alcohol (5.0 g, 4.26 mL, 29.7 mmol)
in DCM (136 mL) was cooled to 0 °C under an atmosphere of N2,
phosphorus tribromide (2.89 mL, 30.6 mmol) was added dropwise, and
the reaction mixture maintained at 0 °C for 90 min. The reaction
mixture was warmed to rt, stirred for a further 30 min, and then poured
onto ice and extracted with DCM (2 × 100 mL). The organic phase was
dried (MgSO4), filtered, and concentrated to dryness to afford 3,4-
dimethoxybenzyl bromide as a pale yellow solid (7 g), which was used in
the next step without further purification. 1H NMR (CD2Cl2, 500 MHz)
δ 3.83 (s, 3H), 3.84 (s, 3H), 4.52 (s, 2H), 6.81 (d, J = 1.5 Hz, 1H), 6.92
(d, J = 8 Hz, 1H), 6.94 (dd, J = 1.5, 8 Hz, 1H). A solution of 3,4-
dimethoxybenzyl bromide (7.0 g, 29.7 mmol) and tetrabutyl ammonium
iodide in triethyl phosphite (7.75 mL, 45.6 mmol) was heated at 110 °C
overnight. Ethyl bromide and excess triethyl phosphite was removed by
distillation at reduced pressure to afford an oil that was purified by flash
chromatography to afford diethyl 3,4-dimethoxybenzylphosphonate
(E)-1,2,3-Trimethoxy-5-(3-methylbut-1-enyl)benzene (4h). From
3,4,5-trimethoxybenzaldehyde and isobutylmagnesium chloride. 1H
NMR (DMSO-d6, 500 MHz) δ 1.06 (d, J = 6.5 Hz, 6H), 2.42 (m, 1H),
3.63 (s, 3H), 3.77 (s, 6H), 6.24 (m, 2H), 6.67 (s, 2H). 13C NMR
(DMSO-d6, 125 MHz) δ 13.7, 21.6, 30.9, 31.9, 55.8, 60.0, 103.2, 133.1,
136.66, 152.89. (+) LRMS (ESI) m/z 236 [M + H]+.
(E)-2-Chloro-1-methoxy-4-(3-methylbut-1-enyl)benzene (4i).
1
(6.55 g). H NMR (DMSO-d6, 500 MHz) δ 1.18 (t, J = 7 Hz, 6 H),
From 3-chloro-4-methoxybenzaldehyde and isobutylmagnesium chlo-
1
3.10 (s, 1 H), 3.15 (s, 1H), 3.72 (s, 6H), 3.92 (m, 4H), 6.79 (d, J = 1.5
Hz, 1H), 6.88 (m, 2H). (+) LRMS (ESI) m/z 289 [M + H]+.
A suspension of sodium hydride (150 mg, 3.64 mmol) in THF
(5 mL) was cooled to 0 °C. Cyclopentane carboxaldehyde (0.36 g,
3.64 mmol) and 3,4-dimethoxybenzylphosphonate (1 g, 3.47 mmol)
were added, and the reaction mixture was allowed to warm to rt and
stirred O/N. Water (10 mL) was added, and the mixture was extracted
with EtOAc (2 × 50 mL). The organic phase was washed with brine,
dried (MgSO4), filtered, and concentrated to dryness. The resulting oil
was purified by flash chromatography on silica gel (5% EtOAc in hexane)
to afford 4-[(E)-2-cyclopentylvinyl]-1,2-dimethoxybenzene as a colorless
oil (0.39 g, 48%). 1H NMR (DMSO-d6, 500 MHz) δ 1.38 (m, 2H), 1.56
(m, 2H), 1.67 (m, 2H), 1.81 (m, 2H), 2.58 (m, 1H), 3.73 (s, 3H), 3.77 (s,
3H), 6.23 (dd, J = 6.5, 15 Hz, 1H), 6.32 (d, J = 15 Hz, 1H), 6.86 (s, 2H),
7.00 (s, 1H). (+) LRMS (ESI) m/z 233 [M + H]+.
ride. H NMR (DMSO-d6, 500 MHz) δ 1.03 (d, J = 7 Hz, 6H), 2.42
(m, 1H), 3.83 (s, 3H), 6.18 (dd, J = 6.5, 13.5 Hz, 1H), 6.33 (d, J =
13.5 Hz, 1H), 7.06 (d, J = 8 Hz, 1H), 7.30 (d J 8 Hz, 1H), 7.46 (s,
1H). (+) LRMS (ESI) m/z 211 [M35Cl + H]+, 213 [M37Cl + H]+.
(E)-2-(Chloromethyl)-1-methoxy-4-(3-methylbut-1-enyl)benzene
(4j). From 3-(chloromethyl)-4-methoxybenzaldehyde and isobutyl-
magnesium chloride. 1H NMR (DMSO-d6, 600 MHz) δ 1.02 (d, J = 7
Hz, 6H), 2.42 (m, 1H), 3.83 (s, 3H), 4.68 (s, 2H), 6.13 (dd, J = 6, 13
Hz, 1H), 6.28 (d, J = 13 Hz, 1H), 6.97 (d, J = 8 Hz, 1H), 7.33 (d, J = 8
Hz, 1H), 7.45 (s, 1H). LRMS (ESI) m/z 243 [M35Cl + H]+, 245
[M37Cl + H]+
(E)-2-Methoxy-5-(3-methylbut-1-enyl)phenyl Acetate (4k). (E)-2-
methoxy-5-(3-methylbut-1-enyl)phenol (1.21 g, 6.3 mmol) was
dissolved in pyridine (2 mL). Acetic anhydride (0.893 mL, 9.5 mmol)
and a catalytic amount of DMAP were added, and the reaction mixture
stirred at rt for 16 h. The reaction mixture was partitioned between 1
N HCl (20 mL) and EtOAc (30 mL). The organic phase was washed
with brine (20 mL), dried (MgSO4), filtered, and concentrated to
afford (E)-2-methoxy-5-(3-methylbut-1-enyl)phenyl acetate as a white
4-[(E)-2-Cyclopentylvinyl]-1,2,3-trimethoxybenzene (4q). From
1
3,4,5-trimethoxy benzyl alcohol. H NMR (DMSO-d6, 600 MHz) δ
1.38 (m, 2H), 1.56 (m, 2H), 1.67 (m, 2H), 1.81 (m, 2H), 2.58
(m, 1H), 3.66 (s, 3H), 3.78 (s, 6H), 6.23 (dd, J = 6.5, 15 Hz, 1H), 6.32
(d, J = 15 Hz, 1H), 6.67 (s, 2H). (+) LRMS (ESI) m/z 263 [M + H]+.
Method C: (E)-1-Ethoxy-2-methoxy-4-(3-methylbut-1-enyl)-
benzene [195192-86-6] (4r). A suspension of 3-ethoxy-4-methox-
ybenzaldehyde (1.50 g, 8.32 mmol) and NH4OAc (0.205g, 2.66
mmol) in nitromethane (15 mL) was heated at reflux for 16 h. The
reaction mixture was cooled and filtered through a plug of SiO2 eluting
with hexane to afford (E)-1-ethoxy-2-methoxy-4- (2-nitrovinyl)
1
solid (1.14 g, 77%). H NMR (CDCl3, 600 MHz) δ 1.08 (d, J = 6.6
Hz, 6H), 2.31 (s, 3H), 2.43 (m, 1H), 3.83 (s, 3H), 6.04 (dd, J = 6, 13
Hz, 1H), 6.24 (d, J = 13 Hz, 1H), 6.89 (d, J = 8 Hz, 1H), 7.07 (d, J =
1.8 Hz, 1H), 7.15 (dd, J = 1.8, 8 Hz, 1H). LRMS (ESI) m/z 235
[M + H]+.
(E)-1,2-Dimethoxy-4-(3-methylbut-1-enyl)benzene [195192-74-6]
(4l). From 3,4,-dimethoxybenzaldehyde (30.6 mmol) and isobutyl-
1
1
magnesium chloride. H NMR (DMSO-d6, 500 MHz) δ 1.06 (d, J =
benzene as a yellow solid (1.58 g, 85%). H NMR (CDCl3, 500 MHz)
6.5 Hz, 6H), 2.42 (m, 1H), 3.63 (s, 3H), 3.78 (s, 3H), 6.15 (dd, J =
6.5, 13.5 Hz, 1H), 6.28 (d, J = 16 Hz, 1H), 6.87 (s, 2H), 7.01 (s, 1H). 13C
NMR (DMSO-d6, 125 MHz) δ 22.4, 30.8, 55.3, 55.4, 109.0, 111.7, 118.7,
126.4, 130.4, 135.2, 148.0, 148.8. (+) LRMS (ESI) m/z 206 [M + H]+.
(E)-2-Methoxy-5-(3-methylbut-1-enyl)phenol (4m). From 3-hydroxy-
4-methoxybenzaldehyde and isobutylmagnesium chloride. 1H NMR
(DMSO-d6, 600 MHz) δ 1.01 (d, J = 6.6 Hz, 6H), 2.38 (m, 1H),
3.73 (s, 3H), 5.98 (dd, J = 5.5, 13 Hz, 1H), 6.17 (d, J = 13 Hz, 1H),
δ 1.51 (m, 3H), 3.92 (s, 3H), 4.17 (s, 2H), 6.90 (d, J = 7.0 Hz, 1H),
7.00 (s, 1H), 7.17 (d, J = 7.0 Hz, 1H), 7.52 (d, J = 11 Hz, 1H), 7.96
(d, J = 11 Hz, 1H). (+) LRMS (ESI) m/z 239 [M + H]+.
2-Iodopropane (4.32 mL, 43 mmol) was added to a solution of
(E)-1-ethoxy-2-methoxy-4- (2-nitrovinyl) benzene (0.50 g, 2.24 mmol)
in THF (20 mL). Air was bubbled through the solution for 5 min,
triethylborane (6.48 mL, 1.0 M in hexanes, 6.48 mmol) was added, and
the resulting mixture stirred for 1 h, concentrated to dryness, and
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purified by flash chromatography on silica gel (hexane) to afford (E)-1-
and bromoethanol (0.54 mL, 7.6 mmol) were added, and the reaction
mixture was heated at reflux for 16 h. The reaction mixture was cooled,
concentrated to dryness and partitioned between EtOAc (30 mL) and
water (20 mL). The organic phase was dried (MgSO4), filtered,
concentrated and purified by flash chromatography on silica gel
ethoxy-2-methoxy-4- (3-methylbut-1-enyl) benzene as a colorless oil
1
(0.30 g, 61%). H NMR (CDCl3, 500 MHz) δ 1.08 (m, 3H), 1.09 (m,
3H), 1.45 (t, J = 6.0 Hz, 3H), 2.44 (m, 1H), 3.89 (s, 3H), 4.08 (q, J =
6.0 Hz, 2H), 6.90 (m, 1H), 6.27 (m, 1H), 6.80 (d, J = 7.0 Hz, 1H), 6.85
(d, J = 7.0 Hz, 1H), 6.91 (s, 1H). (+) LRMS (ESI) m/z 221 [M + H]+.
Method D: 5-[(E)-2-Cyclopropylvinyl]-1,2,3-trimethoxybenzene
(4s). Palladium acetate (0.023g, 0.1 mmol) was added to a solution
of 5-bromo-1,2,3-trimethoxybenzene (1.0 g, 4.05 mmol), cylopropyl-
vinyl boronic acid pinacol ester (1 mL, 4.86 mmol), triphenylphos-
phine (0.11 g, 4.05 mmol), and 2 M K2CO3 (5.6 mL) in 1,2-
dimethoxyethane (4.6 mL). The reaction mixture was heated at reflux
for 18 h, cooled, poured into water (10 mL), and extracted with
EtOAc (2 × 30 mL). The organic phases were combined, washed with
brine (30 mL), dried (MgSO4), filtered, and concentrated to dryness.
The resulting oil was purified by flash chromatography on silica gel
using gradient elution (0−20% EtOAc in hexane over 20 min) to
afford 5-[(E)-2-cyclopropylvinyl]-1,2,3-trimethoxybenzene (776 mg,
81%) as a light yellow oil that solidified on standing. 1H NMR
(DMSO-d6, 500 MHz) δ 0.51 (m, 2H), 0.81 (m, 2H), 1.55 (m, 1H),
3.64 (s, 3H), 3.78 (s, 6H), 5.82 (dd, J = 9.5, 16 Hz, 1H), 6.38 (d, J =
16 Hz, 1H), 6.65 (s, 2H). (+) LRMS (ESI) m/z 235 [M + H]+.
(E)-4-(2-Cyclopropylvinyl)-1,2-dimethoxybenzene (4t). From 5-
bromoveratrole and cylopropylvinyl boronic acid pinacol ester. 1H
NMR (DMSO-d6, 500 MHz) δ 0.47 (m, 2H), 0.77 (m, 2H), 1.53 (m,
1H), 3.72 (s, 3H), 3.74 (s, 3H), 5.72 (dd, J = 9, 16 Hz, 1H), 6.37
(d, J = 16 Hz, 1H), 6.80 (d, J = 8 Hz, 1H), 6.85 (d, J = 8 Hz, 1H), 6.96
(s, 1H). (+) LRMS (ESI) m/z 205 [M + H]+.
1
(EtOAc) to afford (5c) as a colorless oil (190 mg, 20%). H NMR
(DMSO-d6, 500 MHz) δ 1.06 (d, J = 7 Hz, 6 H), 2.42 (m, 1H), 3.72
(br s, 2H), 3.75 (s, 3H), 3.99 (t, J = 5 Hz, 1H), 4.81 (m, 1H), 6.14
(dd, J = 7, 16 Hz, 1H), 6.27 (d, J = 16 Hz, 1H), 6.88 (m, 2H), 7.02
(s, 1H). (+) LRMS (ESI) m/z 235 [M + H]+.
(E)-3-(5-(2-Cyclopropylvinyl)-2-methoxyphenoxy)propan-1-ol
(5d). (E)-5-(2-Cyclopropylopropylvinyl)-2-methoxyphenol (1.0 g,
5.26 mmol) was dissolved in acetone (15 mL). Potassium carbonate
(3.6 g, 2.63 mmol) and 3-bromo-1-propanol (0.92 mL, 10.5 mmol)
were added, and the reaction mixture was heated at reflux for 16 h.
The reaction mixture was cooled, concentrated to dryness, and
partitioned between EtOAc (30 mL) and water (20 mL). The organic
phase was dried (MgSO4), filtered, concentrated, and purified by flash
chromatography on silica gel (EtOAc:Hexane 7:3) to afford (5d) as a
white solid (1.25 g, 96%). 1H NMR (DMSO-d6, 500 MHz) δ 0.48 (m,
2 H), 0.76 (m, 1H), 1.52 (m, 1H), 1.85 (m, 2H), 3.56 (m, 2H), 3.72
(s, 3H), 4.01 (t, J = 5 Hz, 2H), 4.50 (t, J = 5 Hz, 1H), 5.70 (dd, J = 7,
16 Hz, 1H), 6.36 (d, J = 16 Hz, 1H), 6.80 (d, J = 8 Hz, 1H), 6.85
(d, J = 8 Hz, 1H), 6.95 (s, 1H). (+) LRMS (ESI) m/z 249 [M + H]+.
Synthesis of Flindersine and Analogues. 2,4-Dichloroquino-
line. Aniline (6.7 g, 72 mmol) and malonic acid (11.7 g, 112 mmol)
were dissolved in POCl3 (60 mL), heated at reflux for 5 h, cooled, and
poured into ice (an exothermic reaction occurred, and some product
was lost). The crude product was filtered off and purified on a short
column of silica gel using hexane (500 mL) and 90% hexane/EtOAc
(500 mL) to afford 2,4-dichloroquinoline (5.56 g, 39%) as a white
(E)-5-(2-Cyclopropylvinyl)-2-methoxyphenol (4u). From 5-bromo-
2-methoxyphenyl acetate and cylopropylvinylboronic acid pinacol
1
ester. H NMR (CDCl3, 500 MHz) δ 0.50 (m, 2H), 0.81 (m, 2H),
1
solid. H NMR (DMSO-d6, 500 MHz) δ 7.48 (s, 2H), 7.64 (t, J = 8
1.55 (m, 1H), 3.83 (s, 3H), 5.61 (dd, J = 9, 16 Hz, 1H), 6.37 (d, J = 16
Hz, 1H), 6.78 (s, 2H), 6.94 (s, 1H), 6.96 (s, 1H). 13C NMR (CDCl3,
125 MHz) δ −7.3, 14.5, 56.2, 110.9, 111.5, 118.1, 127.1, 132.0, 133.4,
145.8, 145.9. (+) LRMS (ESI) m/z 191 [M + H]+.
Hz, 1H), 7.75 (t, J = 8 Hz, 1H), 8.03 (d, J = 8 Hz, 1H), 8.17 (d, J = 8
Hz, 1H). (+) LRMS (ESI) m/z 198:200:202 (100:60:10) [M + H]+.
2,4-Dimethoxyquinoline. 2,4-Dichloroquinoline (5.56 g, 28 mmol)
was heated under reflux in a methanolic sodium methoxide solution
(6 g sodium in 150 mL of MeOH) for 20 h. The reaction mixture was
cooled and poured into ice water, and the residue was filtered off and
purified on a short column of silica gel using 95% hexane/EtOAc to
afford 2,4-dimethoxyquinoline (3.88 g, 73%) as a white solid and 4-
(E)-4-(2-Methoxy-5-(3-methylbut-1-enyl)phenoxy)-4-oxobuta-
noic acid (5a). (E)-2-Methoxy-5-(3-methylbut-1-enyl)phenol (0.54 g,
2.8 mmol) was dissolved in pyridine (3 mL). Succinic anhydride (0.56 g,
5.6 mmol) and a catalytic amount of DMAP were added, and the
reaction mixture was allowed to stir at rtRT for 16 h. A further 2 equiv
of succinic anhydride was added, and the reaction mixture was stirred for
a further 24h. The reaction mixture was partitioned between EtOAc
(30 mL) and water (10 mL), and the organic phase washed with 1 N
HCl (20 mL) and brine (20 mL). The organic phase was dried
(MgSO4), filtered, and concentrated to dryness to afford (E)-3-(2-
methoxy-5-(3-methylbut-1-enyl)phenoxy)propanoic acid as a white solid
1
chloro-2-methoxyquinoline as a white solid (1.06 g, 20%). H NMR
(DMSO-d6, 500 MHz) δ 4.01 (s, 3H), 4.10 (s, 3H), 6.25 (s, 1H), 7.36
(t, J = 8 Hz, 1H), 7.63 (t, J = 8 Hz, 1H), 7.82 (d, J = 8 Hz, 1H), 8.08
(d, J = 8 Hz, 1H). (+) LRMS (ESI) m/z 190 [M + H]+. 4-Chloro-2-
1
methoxyquinoline: H NMR (DMSO-d6, 500 MHz) δ 4.10 (s, 3H),
7.06 (s, 1H), 7.49 (t, J = 8 Hz, 1H), 7.70 (t, J = 8 Hz, 1H), 7.89 (d, J =
8 Hz, 1H), 8.13 (d, J = 8 Hz, 1H). (+) LRMS (ESI) m/z 194:196
(3:1) [M + H]+.
1
(640 mg, 78%). H NMR (DMSO-d6, 500 MHz) δ 1.02 (d, J = 7 Hz,
6 H), 2.39 (m, 3H), 2.57 (dd, J = 5.5, 6.5 Hz, 2H), 2.77 (dd, J = 5.5,
7 Hz, 2H), 3.73 (s, 3H), 5.98 dd (J 6.5, 16 Hz, 1H), 6.18 (d, J = 16 Hz,
1H), 6.73 (dd, J = 1.5, 8 Hz, 1H), 6.80 (d, J = 1.5 HZ, 1H), 6.82 (d, J =
8 Hz, 1H), 8.83 (s, 1H).
2,4-Dimethoxy-3-(3-methylbut-2-enyl)quinoline. 2,4-Dimethoxy-
quinoline (0.95 g, 5 mmol) was dissolved in dry THF (20 mL) and
cooled to 0 °C under an atmosphere of argon. n-BuLi (2.5M, 2.8 mL,
7 mmol) was added dropwise with stirring, and the reaction mixture
was stirred for a further 30 min at 0 °C. 1-Bromo-3-methylbut-2-ene
(1.27 g, 1 mL, 8.55 mmol) was added dropwise, and the reaction was
stirred for a further 30 min at 0 °C. The reaction mixture was then
warmed to rt and stirred for a further 1 h. The reaction mixture was
concentrated to dryness and purified by flash chromatography on silica
gel (9:1 hexane/EtOAc) to afford 2,4-dimethoxy-3-(3-methylbut-2-
(E)-4-(3-(5-(2-Cyclopropylvinyl)-2-methoxyphenoxy)propyl)-
morpholine (5b). (E)-5-(2-Cyclopropylvinyl)-2-methoxyphenol (1.0g,
5.3 mmol) was dissolved in acetone (5 mL). Potassium carbonate
(3.66 g, 26.5 mmol) and 4-(3-chloropropyl)morpholine (2.6 g, 15.9 mmol)
were added, and the reaction mixture was heated at reflux for 16 h. The
reaction mixture was cooled, concentrated to dryness, and partitioned
between EtOAc (30 mL) and water (20 mL). The organic phase was dried
(MgSO4), filtered, concentrated, and purified by flash chromatography on
silica gel (EtOAc) to afford (E)-4-(3-(5-(2-cyclopropylvinyl)-2-
methoxyphenoxy)propyl)morpholine as an orange oil (629 mg, 37%).
1H NMR (DMSO-d6, 500 MHz) δ 1.03 (d, J = 7 Hz, 6 H), 2.37 (m, 5H),
2.69 (m, 2H), 3.54 (m, 4H), 3.73 (s, 3H), 4.09 (t, J = 6 Hz, 2H), 6.12 dd
(J 7.2, 16.2 Hz, 1H), 6.23 (d, J = 16.2 Hz, 1H), 6.85 (s, 2H), 7.03 (s, 1H).
13C NMR (DMSO-d6, 125 MHz) δ 7.35, 14.6, 29.8, 53.9, 55.8, 56.3, 67.2,
67.5, 110.9, 112.2, 119.0, 127.3, 131.4, 133.1, 148.7, 148.9. (+) LRMS
(ESI) m/z 306 [M + H]+.
1
enyl)quinoline as a clear oil (0.870 g, 67%). H NMR (DMSO-d6,
500 MHz) δ 1.72 (s, 3H), 1.84 (s, 3H), 3.49 (d, J = 7 Hz, 2H),
3.99 (s, 3H), 4.12 (s, 3H), 5.25 (br t, J = 7 Hz, 1H), 7.39 (t, J = 7.5 Hz,
1H), 7.59 (t, J = 7.5 Hz, 1H), 7.87 (t, J = 7.5 Hz, 1H), 7.94 (d, J = 7.5 Hz,
1H). (+) LRMS (ESI) m/z 258 [M + H]+.
3-((3,3-Dimethyloxiran-2-yl)methyl)-2,4-dimethoxyquinoline.
2,4-Dimethoxy-3-(3-methylbut-2-enyl)quinoline (0.870 g, 3.4 mmol)
was dissolved in CHCl3 (10 mL), mCPBA (2.91 g, 16.9 mmol) was
added, and the reaction mixture was stirred for 4 h. The reaction
mixture was extracted with 2 N NaCO3 (4 × 50 mL), dried with MgSO4,
filtered, and concentrated to afford the crude epoxide (0.956 g, 100%),
which was used in the next reaction without further purification.
(E)-2-(2-Methoxy-5-(3-methylbut-1-enyl)phenoxy)ethanol (5c).
(E)-5-(2-Isopropylopropylvinyl)-2-methoxyphenol (0.73g, 3.8 mmol)
was dissolved in acetone (16 mL). Potassium carbonate (2.63 g, 19 mmol)
N
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1H NMR (CDCl3, 500 MHz) δ 1.33 (s, 3H), 1.48 (s, 3H), 2.94
(dd, J = 13, 5 Hz, 1H), 3.08 (dd, J = 11.5, 6.5 Hz, 1H), 3.13 (t, J = 7.5
Hz, 1H), 4.03 (s, 3H), 4.13 (s, 3H), 7.40 (t, J = 7.5 Hz, 1H), 7.62
(t, J = 7.5 Hz, 1H), 7.86 (t, J = 7.5 Hz, 1H), 7.95 (d, J = 7.5 Hz, 1H).
13C NMR (CDCl3, 125 MHz) δ 19.23, 24.33, 25.03, 53.95, 59.40,
62.97, 63.37, 113.73, 121.24, 122.29, 124.02, 127.59, 129.49, 146.72,
162.55, 163.03. (+) LRMS (ESI) m/z 274 [M + H]+.
7.17 (d, 1H, J = 8.5 Hz), 7.32 (d, 1H, J = 7.5 Hz), 7.54 (s, 1H), 11.37
(s, 1H).
2-Methyl-2H-pyrano[3,2-c]quinolin-5(6H)-one (2e,f). 2,4-Dihy-
droxyquinolinone (1.27 g, 7.8 mmol) and crotonaldehyde (1.1 g, 1.4
mL, 15.6 mmol) were suspended in acetonitrile (40 mL). Ytterbium
trifluoromethanesulfonate (230 mg, 0.37 mmol) was added, and the
reaction mixture was refluxed for 16 h. The reaction mixture was
cooled, filtered, and concentrated to dryness. Purification on silica
using flash chromatography afforded 2e,f (341 mg, 18%) as a yellow
solid. 1H NMR (CDCl3, 500 MHz) δ 1.58 (s, 3H), 5.29 (m, 1H), 5.64
(dd, J = 2, 9.5 Hz, 1H), 6.83 (d, J = 10 Hz, 1H), 7.23 (t, J = 8 Hz, 1H),
7.41 (d, J = 8 Hz, 1H), 7.52 (t, J = 8 Hz, 1H), 7.89 (d, J = 8 Hz, 1H),
11.82 (br s, 1H). 13C NMR (CDCl3, 125 MHz) δ 21.81, 73.83, 106.25,
115.32 116.48, 118.53, 122.56, 122.77 122.78, 131.35, 137.98, 158.44,
162.58. (+) LRMS (ESI) m/z 214 [M + H]+.
2,2-Dimethyl-2H-pyrano[3,2-c]pyridin-5(6H)-one (6). A mixture
of 3-methyl-2-butenal (2.59 mL, 27 mmol) and magnesium sulfate
(8.55 g) in pyridine (23 mL) was refluxed for a few minutes. A
solution of 2,4-dihydroxypyridine (2.5 g, 22.5 mmol) in pyridine
(14 mL) was then added slowly (over 30 min) to the above mixture
and refluxed overnight. The reaction mixture was cooled to room
temperature, and the pyridine was removed. The resulting residue was
diluted with water and extracted with DCM. The organic phase was
washed with 1 N HCl, aqueous sodium bicarbonate, and brine and
then dried over anhydrous MgSO4. The organic phase was then
concentrated to dryness to afford 2,2-dimethyl-2H-pyrano[3,2-c]-
pyridin-5(6H)-one (6) as a light yellow solid (3.18 g, 80%). 1H NMR
(500 MHz, DMSO-d6) δ 1.37 (s, 6H), 5.46 (d, J = 10 Hz, 1H), 5.81
(d, J = 7 Hz, 1H), 6.38 (d, 1H, J = 10 Hz, 1H), 7.18 (d, J = 7 Hz, 1H),
11.18 (br s, 1H). (+) LRMS (ESI) m/z 178.1 [M + H]+.
Photochemical 2 + 2 Cycloaddition. General Procedure. The
corresponding flindersine analogue (1 equiv) and styrene (2 equiv)
were dissolved in CHCl3 (2 mL) and added to either a 250 mL
jacketed round-bottomed flask or a pyrex test tube. The solvent was
evaporated to form a thin film, and the flask was then cooled and
irradiated with UV light (125 or 400 W, 16 h). The resulting brown
gum was purified by reverse-phase HPLC to afford the title compound.
(1R,2R,2aR,10aS)-3-Methoxy-1,10,10-trimethyl-2-(3,4,5-trimethoxy-
phenyl)-2,2a,10,10a-tetrahydro-1H-cyclobuta[4,5]pyrano[3,2-c]-
quinoline and (1S,2S,2aS,10aR)-3-Methoxy-1,10,10-trimethyl-2-(3,4,5-
trimethoxyphenyl)-2,2a,10,10a-tetrahydro-1H-cyclobuta[4,5]pyrano-
[3,2-c]quinoline (Racemic Precursor of 1, Scheme 1). From 5-methoxy-
2,2-dimethyl-4a,10b-dihydro-2H-pyrano[3,2-c]quinoline (3) (0.5 mmol)
and 4a (1.0 mmol). Purified by isocratic elution MeOH/H2O (1% TFA,
3:2) (102.3 mg, 36%). 1H NMR (DMSO-d6, 600 MHz) δ 1.12 (d, J = 6
Hz, 3H), 1.16 (s, 3H), 1.50 (s, 3H), 2.36 (m, 2H), 3.23
(s, 3H), 3.24 (s, 6H), 3.36 (t, J = 8 Hz, 1H), 3.51 (s, 3H), 3.71 (t, J =
6.5 Hz, 1H), 7.35 (t, J = 7.5 Hz, 1H), 7.56 (t, J = 7.5 Hz, 1H), 7.57 (d, J =
7.5 Hz, 1H), 8.03 (d, J = 8 Hz, 1H). 13C NMR (DMSO-d6, 150 MHz) δ
20.97, 24.33, 25.64, 35.24, 46.00, 50.46, 53.08, 55.72, 60.47, 76.08, 102.64,
105.88, 118.57, 122.90, 123.02, 126.18, 129.22, 135.18, 135.84, 145.38,
156.97, 151.51, 161.79. (+) LRMS (ESI) m/z 450 [M + H]+. HRMS
(ESI): calcd for C27H32NO5 [M + H]+, 450.2275; found, 450.2256.
(1R,2R,2aR,10aS)-1,10,10-Trimethyl-2-(3,4,5-trimethoxyphenyl)-
1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]pyrano[3,2-c]-
quinolin-3-one and (1S,2S,2aS,10aR)-1,10,10-Trimethyl-2-(3,4,5-
trimethoxyphenyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]-
pyrano[3,2-c]quinolin-3-one (1). The previous compound (39 mg,
0.07 mmol) was dissolved in acetic acid (2 mL). Potassium iodide
(12 mg, 0.14 mmol) was added, and the reaction mixture was heated at
50 °C for 4 h. The reaction mixture was extracted with a 10% NaHSO4
solution and CHCl3 (3 × 30 mL), dried with MgSO4, filtered, and
concentrated to dryness to afford 1 (35.4 mg, 83%) as a white solid.
1H NMR (DMSO-d6, 500 MHz) δ 1.12 (d, J = 6 Hz, 3H), 1.13 (s,
5-Methoxy-2,2-dimethyl-2H-pyrano[3,2-c]quinoline (3). A solu-
tion of 3-((3,3-dimethyloxiran-2-yl)methyl)-2,4-dimethoxyquinoline
(5.8 g, 21.2 mmol) in DMSO (150 mL) and 2 N KOH (30 mL)
was heated at 90 °C for 20 h. The reaction mixture was poured into
water and extracted with CHCl3 (2 × 100 mL). The aqueous phase
was neutralized with HCl to pH 7. Extraction with CHCl3 (3 × 100 mL)
gave a brown oil that was dried (MgSO4), filtered, and concentrated.
Purification on silica gel using flash chromatography (15% EtOAc in
hexane) gave 5-methoxy-2,2-dimethyl-2H-pyrano[3,2-c]quinoline (3)
(2.3g, 45%) and the diol, 1-(2,4-dimethoxyquinolin-3-yl)-3-methyl-
1
butane-2,3-diol (0.326 g). 3: H NMR (CDCl3, 500 MHz) δ 1.56 (s,
6H), 4.10 (s, 3H), 5.60 (d, J = 8 Hz, 1H), 6.67 (d, J = 8 Hz, 1H), 7.34
(t, J = 7.5 Hz, 1H), 7.58 (t, J = 7.5 Hz, 1H), 7.76 (t, J = 7.5 Hz, 1H),
8.03 (d, J = 7.5 Hz, 1H). (+) LRMS (ESI) m/z 242 [M + H]+. 1-(2,4-
1
Dimethoxyquinolin-3-yl)-3-methylbutane-2,3-diol: H NMR (CDCl3,
500 MHz) δ 1.50 (s, 6H), 3.94 (s, 3H), 4.14 (s, 3H), 6.86 (d, J = 15
Hz, 1H), 6.93 (d, J = 15 Hz, 1H), 7.39 (t, J = 7.5 Hz, 1H), 7.60 (t, J =
7.5 Hz, 1H), 7.83 (t, J = 7.5 Hz, 1H), 7.98 (d, J = 7.5 Hz, 1H). (+)
LRMS (ESI) m/z 274 [M + H]+.
2,2-Dimethyl-2H-pyrano[3,2-c]quinolin-5(6H)-one Flindersine
(2a). 2,4-Dihidroxyquinolinone (6.5 g, 40.3 mmol) and methylcroton-
aldehyde (6.79 g, 7.7 mL, 80.6 mmol) were suspended in acetonitrile
(400 mL). Ytterbium trifluoromethanesulfonate (2.5 g, 4.03 mmol)
was added, and the reaction mixture was refluxed for 16 h. The
reaction mixture was cooled, filtered, and concentrated to dryness.
Purification on silica using flash chromatography afforded 2a (3.8 g,
42%) as a yellow solid. 1H NMR (DMSO-d6, 500 MHz) δ 1.50
(s, 6H), 5.67 (d, J = 10 Hz, 1H), 6.53 (d, J = 10 Hz, 1H), 7.19 (t, J =
8 Hz, 1H), 7.28 (d, J = 8 Hz, 1H), 7.51 (t, J = 8 Hz, 1H), 7.77 (d, J =
8 Hz, 1H). 13C NMR (DMSO-d6, 125 MHz) δ 27.67, 78.57, 105.45,
114.05, 115.23, 116.58, 121.53, 121.87, 126.66, 130.78, 138.15, 155.36,
160.18. (+) LRMS (ESI) m/z 228 [M + H]+.
2,2,6-Trimethyl-2H-pyrano[3,2-c]quinolin-5(6H)-one (2b). A mix-
ture of 3-methyl-2-butenal (1.64 mL, 17.1 mmol) and magnesium
sulfate (5.43 g) in pyridine (15 mL) was refluxed for a few minutes. A
solution of 4-hydroxy-1-methyl-2(1H)-quinolone (2.5 g, 14.3 mmol)
in pyridine (9 mL) was then added slowly (over 20 min) to the above
mixture, and the mixture was refluxed overnight. The reaction mixture
was cooled to room temperature, and the pyridine was removed by
evaporation. The resulting residue was diluted with water and
extracted with DCM. The organic phase was washed with 1 N HCl,
aqueous sodium bicarbonate, and brine solution and dried over
anhydrous MgSO4. The organic phase was then concentrated to
dryness to afford 2,2,6-trimethyl-2H-pyrano[3,2-c]quinolin-5(6H)-one
(2b) as a brown oil (3.38 g, 98%). 1H NMR (500 MHz, DMSO-d6) δ
ppm 1.48 (s, 6H), 3.59 (s, 3H), 5.68 (d, 1H, J = 10 Hz), 6.57 (d, 1H,
J = 10 Hz), 7.27 (t, 1H, J = 7.5 Hz), 7.49 (d, 1H, J = 11 Hz), 7.62
(t, 1H, J = 16 Hz), 7.86 (d, 1H, J = 8 Hz).
2,2,9-Trimethyl-2H-pyrano[3,2-c]quinolin-5(6H)-one (2c). A mix-
ture of 3-methyl-2-butenal (1.92 mL, 20 mmol) and magnesium
sulfate (6.34 g) in pyridine (17.5 mL) was refluxed for a few minutes.
A solution of 4-hydroxy-6-methylquinolin-2(1H)-one (2.92 g, 16.7
mmol) in pyridine (10.5 mL) was then added slowly (over 20 min) to
the above mixture, and the mixture was refluxed overnight. The
reaction mixture was cooled to room temperature, and the pyridine
was removed by evaporation. The resulting residue was diluted with
water and extracted with DCM. The organic phase was washed with
1 N HCl, aqueous sodium bicarbonate, and brine solution and dried
over anhydrous MgSO4. The organic phase was then concentrated to
dryness to afford 2,2,9-trimethyl-2H-pyrano[3,2-c]quinolin-5(6H)-one
(2c) as a brown oil (3.81 g, 95%). 1H NMR (500 MHz, DMSO-d) δ 1.48
(s, 6H), 2.35 (s, 3H), 5.64 (d, 1H, J = 10 Hz), 6.51 (d, 1H, J = 9.5 Hz),
3H), 1.47 (s, 3H), 2.33 (m, 2H), 3.16 (m, 1H), 3.29 (s, 3H), 3.33 (s,
3H), 3.68 (t, J = 7.5 Hz, 1H), 4.06 (m, 1H), 6.11 (s, 2H), 7.13 (d, J =
7.5 Hz, 1H), 7.13 (t, J = 7.5 Hz, 1H), 7.42 (t, J = 7.5 Hz, 1H), 7.81 (d,
J = 8 Hz, 1H), 11.05 (s, 1H). 13C NMR (DMSO-d6, 150 MHz) δ 20.3,
23.3, 24.4, 31.7, 35.2, 45.7 49.4, 55.0, 59.8, 76.5, 105.4, 107.8, 114.6,
114.8, 120.9, 121.7, 129.8, 135.8, 135.9, 137.5, 151.7, 155.5, 162.2. (+)
LRMS (ESI) m/z 436 [M + H]+. HRMS (ESI): calcd for C26H29NO5Na
O
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[M + Na]+, 458.1938; found, 458.1928. (for other data, see the
Supporting Information).
acetonitrile/water (70:30) at 1.5 mL/min) tR = 3.459 min. HRMS
(ESI): calcd for C25H27NO4Na [M + Na]+, 428.1832; found, 428.1837.
(1R,2R,2aR,10aS)-2-(3-Bromo-4-methoxyphenyl)-10,10,dimethyl-
1-(1-methyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]pyrano-
[3,2-c]quinolin-3-one and (1S,2S,2aS,10aR)-2-(3-Bromo-4-methox-
yphenyl)-10,10,dimethyl-1-(1-methyl)-1,2,2a,4,10,10a-hexahydro-
3H-cyclobuta[4,5]pyrano[3,2-c]quinolin-3-one (12). From 2a (0.5 mmol)
and 4c (1.0 mmol). Purified by flash chromatography on neutral alumina
(DCM, then 1:1 DCM/CHCl3 then CHCl3). The fractions containing
MH+ 454/456 were combined and purified by reverse-phase HPLC with
isocratic elution MeCN/H2O (2:3) over 60 min to afford 30.1 mg (13%).
1H NMR (DMSO-d6, 500 MHz) δ 1.10 (d, J = 6.5 Hz, 3H), 1.11 (s, 3H),
1.47 (s, 3H), 2.32 (m, 2H), 3.32 (dd, J = 9, 10 Hz, 1H), 3.69 (s, 3H), 3.63
(dd, J = 7.5, 8.5 Hz, 1H), 6.74 (d, J = 8 Hz, 1H), 6.80 (dd, J = 1.8, 8 Hz,
1H), 7.05 (d, J = 1.8 Hz, 1H), 7.12 (dd, J = 7.5 Hz, 1H), 7.13 (d, J = 7.5
Hz, 1H), 7.41 (dd, J = 7.5 Hz, 1H), 7.80 (d, J = 7.5 Hz, 1H), 11.04
(s, 1H). (+) LRMS (ESI) m/z 454 [M + H79Br]+, 456 [M + H81Br]+.
HRMS (ESI): calcd for C24H25BrNO3Na [M + Na]+, 476.0832; found,
476.0831.
Synthesis of 1 from flindersine (2a, 0.6 mmol) and 4a (1.2 mmol).
Purified by flash chromatography on neutral alumina (DCM, then 1:1
DCM/CHCl3 then CHCl3). The fractions containing MH+ 436 were
combined and purified by isocratic elution with MeCN/H2O (55:45)
(41 mg, 16%), which was identical to the sample above by 1H and 13
C
NMR and MS. There were small differences in the 1H NMR between
the synthetic 1 and the natural product (euodenine A), attributable to
the fact that the natural product contained some TFA (present in the
solvent used for HPLC purification). They were shown to be identical
by mixing the two NMR samples together and rerunning the 1H NMR
spectrum. A single spectrum was obtained, identical to that of
euodenine A.
(1R,2R,2aR,10aS)-3-Methoxy-1,10,10-trimethyl-2-(3,4,-dimethoxy-
phenyl)-2,2a,10,10a-tetrahydro-1H-cyclobuta[4,5]pyrano[3,2-c]-
quinoline and (1S,2S,2aS,10aR)-3-Methoxy-1,10,10-trimethyl-2-
(3,4,-dimethoxyphenyl)-2,2a,10,10a-tetrahydro-1H-cyclobuta[4,5]-
pyrano[3,2-c]quinoline (Racemic Precursor of 10). From 5-methoxy-
2,2-dimethyl-4a,10b-dihydro-2H-pyrano[3,2-c]quinoline (3, 0.5
mmol) and 1,2-dimethoxy-4-propenylbenzene (1.0 mmol). Purified
by isocratic elution MeCN/H2O (1% TFA, 55:45) (36.4 mg, 17%). 1H
NMR (DMSO-d6, 600 MHz) δ 1.12 (d, J = 5.5 Hz, 3H), 1.17 (s, 3H),
1.52 (s, 3H), 2.38 (m, 2H), 3.06 (s, 3H), 3.26 (s, 3H), 3.23 (t, J = 9
Hz, 1H), 3.63 (s, 3H), 3.73 (t, J = 9 Hz, 1H), 5.97 (d, J = 1.5 Hz, 1H),
6.40 (dd, J = 2, 8 Hz, 1H), 6.65 (d, J = 8 Hz, 1H), 7.37 (t, J = 8 Hz,
1H), 7.59 (m, 2H), 8.04 (d, J = 8 Hz, 1H). 13C NMR (DMSO-d6, 150
MHz) δ 20.88, 24.52, 25.83, 33.12, 35.45, 46.53, 50.17, 53.38, 55.42,
56.59, 77.07, 103.68, 111.82, 111.83, 119.56, 121.37, 122.05, 123.72,
127.57, 129.83, 132.91, 145.69, 147.73, 148.26, 156.63, 162.28. (+)
LRMS (ESI) m/z 420 [M + H]+. HRMS (ESI): calcd for C26H31NO4
[M + H]+, 420.2169; found, 420.2159.
(1R,2R,2aR,10aS)-2-(2-Chloro-3,4-dimethoxyphenyl)-10,10,di-
methyl-1-(1-methyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]-
pyrano[3,2-c]quinolin-3-one and (1S,2S,2aS,10aR)-2-(2-Chloro-3,4-
dimethoxyphenyl)-10,10,dimethyl-1-(1-methyl)-1,2,2a,4,10,10a-
hexahydro-3H-cyclobuta[4,5]pyrano[3,2-c]quinolin-3-one (13).
From flindersine (2a, 0.5 mmol) and 4d (1.0 mmol). Purified by flash
chromatography on neutral palumina (DCM, then 1:1 DCM/CHCl3
then CHCl3). The fractions containing MH+ 440 were combined and
purified by reverse-phase HPLC with isocratic elution MeCN/H2O (2:3)
1
over 60 min to give 20.4 mg (9%). H NMR (DMSO-d6, 500 MHz) δ
1.14 (d, J = 6.5 Hz, 3H), 1.15 (s, 3H), 1.49 (s, 3H), 2.36 (m, 2H), 3.36
(dd, J = 9, 10 Hz, 1H), 3.63 (s, 3H), 3.69 (dd, J = 7.5, 8.5 Hz, 1H), 3.73
(s, 3H), 6.34 (m, 1H), 7.16 (dd, J = 7.5 Hz, 1H), 7.16 (d, J = 7.5 Hz,
1H), 7.45 (dd, J = 7.5 Hz, 1H), 7.82 (d, J = 7.5 Hz, 1H), 11.04 (s, 1H).
(+) LRMS (ESI) m/z 440 [M + H35Cl]+, 442 [M + H37Cl]+ HRMS
(ESI): calcd for C25H27ClNO4Na [M + Na]+, 462.1443; found, 462.1452.
(1R,2R,2aR,10aS)-1,10,10-Trimethyl-2-(phenyl)-1,2,2a,4,10,10a-
hexahydro-3H-cyclobuta[4,5]pyrano[3,2-c]quinolin-3-one and
(1S,2S,2aS,10aR)-1,10,10-Trimethyl-2-(phenyl)-1,2,2a,4,10,10a-hex-
ahydro-3H-cyclobuta[4,5]pyrano[3,2-c]quinolin-3-one: (14). From
3 and (E)-prop-1-enylbenzene. Purified by isocratic elution MeOH/
(1R,2R,2aR,10aS)-1,10,10-Trimethyl-2-(3,4-dimethoxyphenyl)-
1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]pyrano[3,2-c]-
quinolin-3-one and (1S,2S,2aS,10aR)-1,10,10-Trimethyl-2-(3,4-di-
methoxyphenyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]-
pyrano[3,2-c]quinolin-3-one (10). The previous compound (16.6 mg,
0.04 mmol) was dissolved in acetic acid (2 mL). Potassium iodide (9.4
mg, 0.056 mmol) was added, and the reaction mixture was heated at
50 °C for 20 h. The reaction mixture was partitioned between with a
10% NaHSO4 solution and CHCl3 (3 × 30 mL), and the organic layer
was then extracted with aqueous ammonia solution (10%, 20 mL),
dried with MgSO4, filtered, and concentrated to dryness to afford 10
1
H2O (1% TFA, 7:3) to afford 20 mg (5%). H NMR (CDCl3, 500
MHz) δ 1.24 (d, J = 6.5 Hz, 3H), 1.31 (s, 3H), 1.67 (s, 3H), 2.43
(t, J = 9 Hz, 1H), 2.59 (m, 1H), 3.50 (t, J = 11.5 Hz, 1H), 3.63 (s,
3H), 3.83 (t, J = 8.5 Hz, 1H), 6.77 (d, J = 7 Hz, 2H), 7.13 (t, J = 7 Hz,
2H), 7.18 (t, J = 7 Hz, 1H), 7.62 (t, J = 7.5 Hz, 1H), 7.82 (t, J = 7.5
Hz, 1H), 8.16 (d, J = 7.5 Hz, 1H), 8.25 (d, J = 7.5 Hz, 1H). 13C NMR
(CDCl3, 125 MHz) δ 19.8, 24.2 (2×), 24.2, 32.2, 34.0, 45.5, 58.8, 80.1,
103.2, 118.0, 120.6, 122.4, 126.5, 127.3, 127.4 (2×), 128.3 (2×), 133.2,
134.7, 137.9, 163.4. (+) LRMS (ESI) m/z 360 [M + H]+. HRMS
(ESI): calcd for C24H26NO2 [M + H]+, 360.1958; found, 360.1969.
This compound (10 mg, 0.028 mmol) was dissolved in acetic acid
(2 mL). Potassium iodide (9.4 mg, 0.056 mmol) was added, and the
reaction mixture was heated at 50 °C for 20 h. The reaction mixture
was extracted with a 10% NaHSO4 solution and CHCl3 (3 × 30 mL),
dried with MgSO4, filtered, and concentrated to dryness to afford 14
1
(9.3 mg, 57%) as a white solid. H NMR (DMSO-d6, 500 MHz) δ
1.16 (d, J = 6 Hz, 3H), 1.17 (s, 3H), 1.53 (s, 3H), 2.39 (m, 2H), 3.28
(s, 3H), 3.38 (m, 1H), 3.67 (s, 3H), 3.74 (t, J = 7.5 Hz, 1H), 6.39 (br
s, 1H), 6.52 (d, J = 8 Hz, 1H), 6.66 (d, J = 8 Hz, 1H), 7.19 (t, J = 8 Hz,
1H), 7.19 (d, J = 7.5 Hz, 1H), 7.78 (t, J = 7.5 Hz, 1H), 7.87 (d, J = 8
Hz, 1H), 11.07 (s, 1H). 13C NMR (DMSO-d6, 125 MHz) δ 21.66,
22.95, 25.64, 32.97, 35.96, 46.72, 49.86, 55.54, 56.14, 77.19, 108.54,
111.59, 112.59, 115.38, 115.59, 121.40, 121.74, 122.52, 130.77, 133.24,
138.18, 147.87, 148.07, 156.17, 162.66. (+) LRMS (ESI) m/z 406
[M + H]+. HRMS (ESI): calcd for C25H27NO4Na [M + Na]+, 428.1832;
found, 428.1843.
(1R,2R,2aR,10aS)-1,10,10-Trimethyl-2-(3,5-dimethoxyphenyl)-
1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]pyrano[3,2-c]-
quinolin-3-one and (1S,2S,2aS,10aR)-1,10,10-Trimethyl-2-(3,5-di-
methoxyphenyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]-
pyrano[3,2-c]quinolin-3-one (11). From flindersine (2a, 0.39 mmol)
and (E)-1,3-dimethoxy-5-(prop-1-enyl)benzene (4b, 0.79 mmol). The
residue was then partially purified by flash chromatography on neutral
alumina using dichloromethane/chloroform (1:1) and then further
1
(2.6 mg, 27%) as a white solid. H NMR (DMSO-d6, 500 MHz) δ
1.13 (d, J = 6 Hz, 3H), 1.14 (s, 3H), 1.50 (s, 3H), 2.36 (m, 2H), 2.46
(m, 1H), 3.39 (t, J = 8 Hz, 1H), 3.71 (t, J = 6.5 Hz, 1H), 6.89 (m, 2H),
7.02 (m, 3H), 7.12 (t, J = 7.5 Hz, 1H), 7.15 (t, J = 7.5 Hz, 1H), 7.42 (t,
J = 7.5 Hz, 1H), 7.82 (d, J = 8 Hz, 1H). (+) LRMS (ESI) m/z 346
[M + H]+. HRMS (ESI): calcd for C23H24NO2 [M + H]+, 346.1801;
found, 346.1791.
(1R,2R,2aR,10aS)-1,10,10-Trimethyl-2-(4-methoxyphenyl)-
1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]pyrano[3,2-c]-
quinolin-3-one and (1S,2S,2aS,10aR)-1,10,10-Trimethyl-2-(4-me-
thoxyphenyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]pyrano-
[3,2-c]quinolin-3-one (15). From 3 (0.5 mmol) and trans anethole
(1.0 mmol). Purified by isocratic elution MeCN/H2O (1% TFA,
55:45) (12.6 mg, 6%). 1H NMR (DMSO-d6, 500 MHz) δ 1.12 (d, J =
5.5 Hz, 3H), 1.15 (s, 3H), 1.52 (s, 3H), 2.37 (m, 1H), 2.43 (m, 1H),
3.28 (s, 3H), 3.40 (t, J = 9 Hz, 1H), 3.62 (s, 3H), 3.73 (t, J = 9 Hz,
1
purified by isocratic elution MeCN/H2O (60:40) (10 mg, 6%). H
NMR (CDCl3, 500 MHz) δ 1.23 (s, 6H), 1.57 (s, 3H), 2.32 (app t, J =
8.5 Hz, 1H), 2.55 (m, 1H), 3.41 (app t, J = 9.5 Hz, 1H), 3.45 (s, 3H),
3.91 (m, 1H), 6.14 (s, 2H), 6.17 (s, 1H), 7.14 (d, J = 8 Hz, 1H), 7.26
(app t, J = 7.5 Hz, 1H), 7.49 (app t, J = 8 Hz, 1H), 8.00 (d, J = 8 Hz, 1H),
10.25 (br s, 1H). 13C NMR (CDCl3, 125 MHz) δ 20.64, 24.24, 25.09,
32.41, 35.12, 46.86, 50.63, 55.15, 77.95, 99.43, 106.55, 107.23, 115.95,
116.40, 122.77, 122.84, 130.79, 136.62, 142.17, 158.60, 160.22, 163.77. (+)
LRMS (ESI) m/z 406 [M + H]+. HPLC (Betasil C18 150 × 4.6 mm,
P
dx.doi.org/10.1021/jm401321v | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
1H), 6.58 (d, J = 8 Hz, 2H), 6.63 (d, J = 8 Hz, 2H), 7.36 (br t, J = 8
Hz, 1H), 7.57 (m, 2H), 8.02 (d, J = 8 Hz, 1H). (+) LRMS (ESI) m/z
390 [M + H]+. HRMS (ESI): calcd for C25H28NO3 [M + H]+,
390.2064; found, 390.2080. This compound (6.4 mg, 0.016 mmol)
was dissolved in acetic acid (2 mL). Potassium iodide (9.4 mg, 0.056
mmol) was added, and the reaction mixture was heated at 50 °C for 20
h. The reaction mixture was partitioned between a 10% NaHSO4
solution and CHCl3 (3 × 30 mL), and the organic layer was then
extracted with aqueous ammonia solution (10%, 20 mL), dried with
MgSO4, filtered, and concentrated to dryness to afford 15 (4.6 mg,
100%) as a white solid. 1H NMR (DMSO-d6, 500 MHz) δ 1.16 (d, J =
5.5 Hz, 3H), 1.17 (s, 3H), 1.53 (s, 3H), 2.37 (t, J = 9 Hz, 1H), 2.44
(m, 1H), 3.37(t, J = 9 Hz, 1H), 3.67 (s, 3H), 3.72 (t, J = 9 Hz, 1H),
6.63 (d, J = 8 Hz, 2H), 6.85 (d, J = 8 Hz, 2H), 7.18 (d, J = 8 Hz, 1H),
7.20 (t, J = 8 Hz, 1H), 7.47 (t, J = 6.5 Hz, 1H), 7.86 (d, J = 8 Hz, 1H),
11.02 (s, 1H). (+) LRMS (ESI) m/z 376 [M + H]+. HRMS (ESI):
calcd for C24H25NO3Na [M + Na]+, 398.1727; found, 398.1727.
(1R,2R,2aR,10aS)-1,10,10-Trimethyl-2-(2,4,5-trimethoxyphenyl)-
1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]pyrano[3,2-c]-
quinolin-3-one and (1S,2S,2aS,10aR)-1,10,10-Trimethyl-2-(2,4,5-
trimethoxyphenyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]-
pyrano[3,2-c]quinolin-3-one (16). Two-hundred milligrams of
flindersine (2a, 0.88 mmol) and α-asarone (1.66 mmol). Purified by
isocratic elution MeCN/H2O (1% TFA, 55:45) (43.9 mg, 0.1 mmol,
alumina (DCM, then 1:1 DCM/CHCl3, then CHCl3). The fractions
containing MH+ 420 were combined and purified by reverse-phase
HPLC with isocratic elution MeCN/H2O (3:2) to afford 12.2 mg
(6%). 1H NMR (DMSO-d6, 500 MHz) δ 0.65 (t, J = 5.5 Hz, 3H), 1.12
(s, 3H), 1.46 (s, 3H), 1.49 (m, 3H), 1.68 (m, 1H), 2.28 (m, 1H), 2.36
(t, J = 9 Hz, 1H), 3.16 (s, 3H), 3.37 (t, J = 9 Hz, 1H), 3.60 (s, 3H),
3.65 (t, J = 9 Hz, 1H), 6.28 (br s, 1H), 6.48 (d, J = 8.5 Hz, 1H), 6.58
(d, J = 8.5 Hz, 1H), 7.12(m, 2H), 7.41 (t, J = 8 Hz, 1H), 7.80 (d, J = 8
Hz, 1H), 10.97 (s, 1H). 13C NMR (DMSO-d6, 150 MHz) δ 12.59,
24.55, 26.21, 29.41, 32.33, 41.79, 44.71, 48.05, 55.42, 56.81, 77.04,
109.01, 111.33, 112.31, 115.48, 115.65, 121.49, 121.90, 122.60, 130.8,
134.39, 137.97, 147.50, 148.59, 156.02, 162.57. (+) LRMS (ESI) m/z
420 [M + H]+. HRMS (ESI): calcd for C26H29NO4Na [M + Na]+,
442.1989; found, 442.1971.
(1R,2R,2aR,10aS)-1-Ethyl-10,10-dimethyl-2-(2,3,4-trimethoxy-
phenyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]pyrano[3,2-
c]quinolin-3-one and (1S,2S,2aS,10aR)-1-Ethyl-10,10-dimethyl-2-
(2,3,4-trimethoxyphenyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta-
[4,5]pyrano[3,2-c]quinolin-3-one (20). From flindersine (2a, 0.5
mmol) and 4f (1.0 mmol). Purified by isocratic elution MeCN/H2O
1
(60:40) (29.3 mg, 13%). H NMR (DMSO-d6, 600 MHz) δ 0.65 (t,
J = 7.2 Hz, 3H), 1.12 (s, 3H), 1.45 (s, 3H), 1.49 (m, 1H), 1.65 (m,
1H), 2.27 (m, 1H), 2.36 (t, J = 9 Hz, 1H), 3.30 (s, 6H), 3.37 (t, J = 9
Hz, 1H), 3.51 (s, 3H), 3.66 (dd, J = 8, 9 Hz, 1H), 6.09 (s, 2H),
7.12(m, 2H), 7.41 (ddd, J = 1.2, 7.5, 8.4 Hz, 1H), 7.80 (dd, J = 1.2, 8.4
Hz, 1H), 11.02 (s, 1H). 13C NMR (DMSO-d6, 150 MHz) δ 12.4, 24.3,
25.8, 29.6, 32.4, 42.5, 44.5, 48.7, 56.1, 60.5, 77.6, 106.7, 107.5, 115.5,
115.6, 121.8, 122.7, 130.7, 136.8, 138.6, 139.1, 153.3, 156.0, 163.5. (+)
LRMS (ESI) m/z 450 [M + H]+. HRMS (ESI): calcd for C27H32NO5
[M + H]+, 450.2275; found, 450.2257.
1
12%). H NMR (CDCl3, 500 MHz) δ 1.19 (d, J = 6.5 Hz, 3H), 1.25
(s, 3H), 1.58 (s, 3H), 2.33 (t, J = 8.5 Hz, 1H), 2.50 (m, 1H), 3.04 (s,
3H), 3.71 (s, 3H), 3.89 (s, 3H), 3.89 (m, 1H), 3.96 (dd, J = 8, Hz,
1H), 6.02 (s, 1H), 6.40 (s, 1H), 7.19 (d, J = 8 Hz, 1H), 7.28 (t, J = 8
Hz, 1H), 7.49 (t, J = 8 Hz, 1H), 8.03 (d, J = 8 Hz, 1H), 10.51 (br s,
1H). 13C NMR (CDCl3, 125 MHz) δ 20.64, 24.12, 25.12, 32.10, 34.13,
41.64, 46.84, 56.03, 56.42, 57.73, 78.17, 78.17, 98.59, 107.19, 112.09,
116.27, 120.18, 122.70, 122.93, 130.77, 136.31, 142.53, 148.34, 153.09,
158.70, 163.58. (+) LRMS (ESI) m/z 436 [M + H]+. HRMS (ESI):
calcd for C26H29NO5 [M + H]+, 458.1938; found, 458.1928.
(1R,2R,2aR,10aS)-1-Ethyl-2-(3-fluoro-4-methoxyphenyl)-10,10-di-
methyl-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]pyrano[3,2-c]-
quinolin-3-one and (1R,2R,2aR,10aS)-1-Ethyl-2-(3-fluoro-4-methox-
yphenyl)-10,10-dimethyl-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta-
[4,5]pyrano[3,2-c]quinolin-3-one (21). From flindersine (2a, 0.5
mmol) and 4g (1.0 mmol). Purified by flash chromatography on
neutral alumina (DCM, then 1:1 DCM/CHCl3, then CHCl3). The
fractions containing MH+ 408 were combined and purified by reverse-
phase HPLC with isocratic elution MeCN/H2O (3:2) to MeCN over
(1R,2R,2aR,10aS)-1,10,10-Trimethyl-2-(3-methoxy-4-benzyloxy-
phenyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]pyrano[3,2-
c]quinolin-3-one and (1S,2S,2aS,10aR)-1,10,10-Trimethyl-2-(3-me-
thoxy-4-benzyloxyphenyl)-1,2,2a,4,10,10a-hexahydro-3H-
cyclobuta[4,5]pyrano[3,2-c]quinolin-3-one (17). From flindersine
(2a, 0.5 mmol) and isoeugenol benzylether (1.0 mmol). Purified by
flash chromatography on neutral alumina (DCM, then 1:1 DCM/
CHCl3, then CHCl3) and then reverse-phase HPLC with isocratic elution
MeCN/H2O (1% TFA, 7:3) to afford 8.3 mg (3%). 1H NMR (DMSO-
d6, 500 MHz) δ 1.10 (d, J = 6.5 Hz, 3H), 1.12 (s, 3H), 1.47 (s, 3H), 2.33
(m, 2H), 3.09 (m, 1H), 3.23 (s, 3H), 3.68 (t, J = 9 Hz, 1H), 4.90 s, 2H),
3.96 (dd, J = 8, Hz, 1H), 6.36 (s, 1H), 6.46 (d, J = 7.5 Hz, 1H), 6.70
(d, J = 7.5 Hz, 1H), 7.13 (m, 2H), 7.27−7.43 (m, 6H), 7.80 (d, J = 7.5
Hz, 1H), 11.02 (s, 1H). (+) LRMS (ESI) m/z 482 [M + H]+. HRMS
(ESI): calcd for C31H31NO4Na [M + Na]+, 504.2145; found, 504.2129.
(1R,2R,2aR,10aS)-1,10,10-Trimethyl-2-(3-methoxy-4-hydroxy-
phenyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]pyrano[3,2-
c]quinolin-3-one and (1S,2S,2aS,10aR)-1,10,10-Trimethyl-2-(3-me-
thoxy-4-hydroxyphenyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta-
[4,5]pyrano[3,2-c]quinolin-3-one (18). 17 (7 mg, 0.014 mmol) was
dissolved in MeOH, and Pd/C (10%, 2 mg) was added. Hydrogen gas
was bubbled through the reaction mixture for 20 min, and the resulting
solution was stirred under an atmosphere of hydrogen for 20 h.
Filtration through Celite and concentration afforded 18 as a white
1
60 min to afford 24 mg (12%). H NMR (DMSO-d6, 600 MHz) δ
0.65 (t, J = 8 Hz, 3H), 1.19 (s, 3H), 1.50 (s, 3H), 1.52 (m, 1H), 1.67
(m, 1H), 2.32 (m, 1H), 2.35 (m, 1H), 3.42 (t, J = 9 Hz, 1H), 3.64
(t, J = 9 Hz, 1H), 3.83 (s, 3H), 6.63 (d, J = 9.6 Hz, 1H), 6.67 (br d, J =
8 Hz, 1H), 6.78 (dd, J = 6, 8 Hz, 1H), 7.14 (d, J = 7.5 Hz, 1H), 7.15 (t,
J = 7.5 Hz, 1H), 7.43 (t, J = 8 Hz, 1H), 11.01 (s, 1H). (+) LRMS
(ESI) m/z 408 [M + H]+. HRMS (ESI): calcd for C25H26FNO3Na
[M + Na]+, 430.1789; found, 430.1776.
(1R,2R,2aR,10aS)-1,10,10-Trimethyl-2-(3,4,5-trimethoxyphenyl)-
1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]pyrano[3,2-c]-
quinolin-3-one and (1S,2S,2aS,10aR)-1,10,10-Trimethyl-2-(3,4,5-
trimethoxyphenyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]-
pyrano[3,2-c]quinolin-3-one (22). From flindersine (2a, 0.88 mmol)
and 4h (1.76 mmol). Purified by flash chromatography on neutral
alumina (DCM, then 1:1 DCM/CHCl3, then CHCl3). The fractions
containing MH+ 464 were combined and purified by reverse-phase
HPLC with isocratic elution MeCN/H2O (7:3) to afford 27.6 mg
1
(12%). H NMR (DMSO-d6, 500 MHz) δ 0.73 (d, J = 6.5 Hz, 3H),
0.88 (d, J = 6.5 Hz, 3H), 1.15 (s, 3H), 1.49 (s, 3H), 1.80 (m, 1H), 2.33
(m, 2H), 3.31 (s, 6H), 3.52 (s, 3H), 3.52 (t, J = 9 Hz, 1H), 3.67 (t, J =
9 Hz, 1H), 6.12 (s, 2H), 7.13 (d, J = 7.5 Hz, 1H), 7.14 (d, J = 7.5 Hz,
1H), 7.43 (t, J = 7.5 Hz, 1H), 7.82 (d, J = 7.5 Hz, 1H), 11.00 (s, 1H).
(+) LRMS (ESI) m/z 464 [M + H]+. HRMS (ESI): calcd for
C28H33NO5Na [M + Na]+, 486.2251; found, 486.2236.
(1R,2R,2aR,10aS)-2-(3-Chloro-4-methoxyphenyl)-10,10-dimethyl-
1-(1-methylethyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]-
pyrano[3,2-c]quinolin-3-one and (1S,2S,2aS,10aR)-2-(3-Chloro-4-
methoxyphenyl)-10,10-dimethyl-1-(1-methylethyl)-1,2,2a,4,10,10a-
hexahydro-3H-cyclobuta[4,5]pyrano[3,2-c]quinolin-3-one (23).
From flindersine (2a, 0.88 mmol) and 4i (1.76 mmol). Purified by
reverse-phase HPLC with gradient elution MeCN/H2O (3:7) to
MeCN over 60 min. Then repurified by flash chromatography
1
solid (5.4 mg, 100%). H NMR (DMSO-d6, 500 MHz) δ 1.10 (d, J =
6.5 Hz, 3H), 1.11 (s, 3H), 1.47 (s, 3H), 2.33 (m, 2H), 3.23 (m, 4H),
3.68 (t, J = 9 Hz, 1H), 6.26 (s, 1H), 6.32 (d, J = 7.5 Hz, 1H), 6.41
(d, J = 7.5 Hz, 1H), 7.13 (m, 2H), 7.41 (t, J = 7.5 Hz, 1H), 7.80 (d, J =
7.5 Hz, 1H), 10.99 (s, 1H). (+) LRMS (ESI) m/z 392 [M + H]+.
HRMS (ESI): calcd for C24H25NO4Na [M + Na]+, 414.1676; found,
414.1665.
(1R,2R,2aR,10aS)-1-Ethyl-10,10-trimethyl-2-(3,4,-dimethoxy-
phenyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]pyrano[3,2-
c]quinolin-3-one and 1S,2S,2aS,10aR)-1-Ethyl-10,10-trimethyl-2-
(3,4,-dimethoxyphenyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta-
[4,5]pyrano[3,2-c]quinolin-3-one (19). From flindersine (2a, 0.5 mmol)
and 4e (1.0 mmol). Purified by flash chromatography on neutral
Q
dx.doi.org/10.1021/jm401321v | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(2% MeOH/DCM) to give 45 mg (11.7%) as a white solid. 1H NMR
(DMSO-d6, 600 MHz) δ 0.68 (d, J = 6.8 Hz, 3H), 0.85 (d, J = 6.88 Hz,
3H), 1.12 (s, 3H), 1.48 (s, 3H), 1.80 (dd, J = 12.8, 6.5 Hz, 1H), 2.30−
2.36 (m, 1H), 2.50 (m, 1H), 3.53 (t, J = 9.4 Hz, 1H), 3.61 (t, J = 8.8
Hz, 1H), 3.69 (s, 3H), 6.70−6.78 (m, 2H), 6.88 (d, J = 1.56 Hz, 1H),
7.10−7.16 (m, 2H), 7.42 (t, J = 7.50 Hz, 1H), 7.81 (d, J = 7.82 Hz,
1H), 11.00 (s, 1H). 13C NMR (DMSO-d6, 150 MHz) δ 17.16, 20.29,
22.42, 24.42, 29.73, 30.16, 39.31, 41.19, 44.20, 54.56, 75.43, 106.79,
110.07, 113.54, 119.78, 120.68, 126.23, 128.48, 128.65, 133.65, 136.22,
151.25, 154.70, 160.80. (+) LRMS (ESI) m/z 437.8 [M + H]+. HRMS
(ESI): calcd for C26H28ClNO3Na [M + Na]+, 460.1650; found,
460.1657.
(1R,2R,2aR,10aS)-2-(3-Chloromethyl-4-methoxyphenyl)-10,10-di-
methyl-1-(1-methylethyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta-
[4,5]pyrano[3,2-c]quinolin-3-one and (1S,2S,2aS,10aR)-2-(3-Chlor-
omethyl-4-methoxyphenyl)-10,10-dimethyl-1-(1-methylethyl)-
1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]pyrano[3,2-c]-
quinolin-3-one (24). From flindersine (2a, 0.44 mmol) and 4j (0.88
mmol). Purified by reverse-phase HPLC with gradient elution MeCN/
H2O + 1% TFA (3:7) to MeCN over 60 min. Then flash chromatography
(DCM to 2% MeOH/DCM) to give 6.7 mg (3.4%) as a white solid. 1H
NMR (600 MHz, DMSO-d6) δ 0.68 (d, J = 6.9 Hz, 3H), 0.85 (d, J = 6.9
Hz, 3H), 1.13 (s, 3H), 1.49 (s, 3H), 1.81 (td, J = 13, 6.3 Hz, 1H), 2.36
(dd, J = 15.6, 9.4 Hz, 1H), 2.5 (m, 1H), 3.52 (t, J = 9.4 Hz, 1H), 3.58−
3.64 (m, 1H), 3.69 (s, 3H), 4.30−4.38 (m, 2H), 6.6 (d, J = 0.5 Hz 1H),
6.80−6.86 (m, 2H), 7.08−7.16 (m, 2H), 7.40 (t, J = 7.7 Hz, 1H), 7.81 (d,
J = 7.8 Hz, 1H), 10.94 (s, 1H). (+) LRMS (ESI) m/z 452.3 [M + H]+.
HRMS (ESI): calcd for C27H30ClNO3 [M + H]+, 452.1987; found,
452.2002.
(d, J = 6.9 Hz, 3H), 0.84 (d, J = 6.6 Hz, 3H), 1.10−1.12 (s, 3H), 1.47
(s, 3H), 1.77−1.82 (m, 1H), 2.30−2.36 (m, 1H), 2.41−2.46 (m, 2H),
2.49−2.51 (m, 1H), 2.58 (t, J = 6.9 Hz, 2H), 3.51 (t, J = 9.5 Hz, 1H),
3.58−3.64 (m, 4H), 6.57 (s, 1H), 6.64−6.70 (m, 2H), 7.09−7.14 (m,
2H), 7.40 (t, J = 7.6 Hz, 1H), 7.79 (d, J = 8.2 Hz, 1H), 10.96 (s, 1H).
(+) LRMS (ESI) m/z 520.3 [M + H]+.
(1R,2R,2aR,10aS)-1-Isopropyl-2-(4-ethoxy-3-methoxyphenyl)-
10,10-dimethyl-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]-
pyrano[3,2-c]quinolin-3-one and (1S,2S,2aS,10aR)-1-Isopropyl-2-
(4-ethoxy-3-methoxyphenyl)-10,10-dimethyl-1,2,2a,4,10,10a-hexa-
hydro-3H-cyclobuta[4,5]pyrano[3,2-c]quinolin-3-one (28). From
flindersine (2a, 0.5 mmol) and 4r (1.0 mmol). Purified by flash
chromatography on neutral alumina (DCM, then 1:1 DCM/CHCl3,
then CHCl3). The fractions containing MH+ 448 were combined and
purified by reverse-phase HPLC with isocratic elution MeCN/H2O
(2:3) over 60 min to afford 11 mg (5%). 1H NMR (DMSO-d6,
500 MHz) δ 0.71 (d, J = 6.5 Hz, 3H), 0.87 (d, J = 6.5 Hz, 1H), 1.15 (s,
3H), 1.25 (t, J = 6.5 Hz, 3H), 1.50 (s, 3H), 1.81 (m, 1H), 2.34 (m,
1H), 2.45 (m, 1H), 3.45 (m, 1H), 3.52 (t, J = 9 Hz, 1H), 3.63 (m,
1H), 3.84 (q, J = 6.5 Hz, 2H), 6.23 (d, J = 1.5 Hz, 1H), 6.43 (dd, J =
1.5, 7.5 Hz, 1H), 6.78 (d, J = 7.5 Hz, 1H), 7.16 (d, J = 7.5 Hz, 1H),
7.16 (d, J = 7.5 Hz, 1H), 7.43 (dd, J = 7.5 Hz, 1H), 7.83 (d, J = 7.5 Hz,
1H), 10.96 (s, 1H). (+) LRMS (ESI) m/z 448 [M + H]+.
(1S,2S,2aS,10aR)-2-[4-Methoxy-3-(2-hydroxyethyl)phenyl]-10,10-
dimethyl-1-(1-methylethyl)-1,2,2a,4,10,10a-hexahydro-3H-
cyclobuta[4,5]pyrano[3,2-c]quinolin-3-one and (1R,2R,2aR,10aS)-2-
[4-Methoxy-3-(2-hydroxyethyl)phenyl]-10,10-dimethyl-1-(1-methyl-
ethyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]pyrano[3,2-c]-
quinolin-3-one (29). From flindersine (2a, 0.4 mmol) and 5d (0.8
mmol). Purified by reverse-phase HPLC with gradient elution (3:7)
MeCN/H2O + 1% TFA to (3:2) MeCN/H2O + 1%TFA over 60 min
to give 32.4 mg (17%) of white solid. 1H NMR (500 MHz, DMSO-d6)
δ 0.70 (d, J = 7 Hz, 3H), 0.85 (d, J = 7 Hz, 3H), 1.13 (s, 3H), 1.48 (s,
3H), 1.77−1.81 (m, 1H), 2.29−2.36 (m, 1H), 2.49−2.54 (m, 1H),
3.1−3.16 (m, 2H), 3.41−3.50 (m, 3H), 3.61− 3.64 (m, 5H), 6.31 (s,
1H), 6.47 (d, J = 8.5 Hz, 1H), 6.57 (d, J = 8.2 Hz, 1H), 7.10−7.15 (m,
2H), 7.41 (t, J = 7.4 Hz, 1H), 7.81 (d, J = 8.5 Hz, 1H), 10.94 (s, 1H).
(+) LRMS (ESI) m/z 464.0 [M + H]+
5-[(1R,2R,2aR,10aS)-1-Isopropyl-10,10-dimethyl-3-oxo-
2,2a,3,4,10,10a-hexahydro-1H-cyclobuta[4,5]pyrano[3,2,-c]-
quinolinyl]-2-methoxyphenylacetate and 5-[(1S,2S,2aS,10aR)-1-Iso-
propyl-10,10-dimethyl-3-oxo-2,2a,3,4,10,10a-hexahydro-1H-
cyclobuta[4,5]pyrano[3,2,-c]quinolinyl]-2-methoxyphenylacetate
(25). From flindersine (2a, 0.88 mmol) and 4k (1.76 mmol). Purified
by flash chromatography on neutral alumina (DCM, then 1:1 DCM/
CHCl3, then CHCl3). The fractions containing MH+ 462 were
combined and purified by reverse-phase HPLC with gradient elution
MeCN/H2O (5:95) to MeCN/H2O (2:3) over 60 min (18.6 mg,
(1R,2R,2aR,10aS)-2-[4-Methoxy-3-(3-hydroxypropyl)phenyl]-
10,10-dimethyl-1-(1-cyclopropyl)-1,2,2a,4,10,10a-hexahydro-3H-
cyclobuta[4,5]pyrano[3,2-c]quinolin-3-one and (1S,2S,2aS,10aR)-2-
[4-Methoxy-3-(3-hydroxypropyl)phenyl]-10,10-dimethyl-1-(1-cyclo-
propyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]pyrano[3,2-c]-
quinolin-3-one (30). From flindersine (2a, 0.88 mmol) and 5c (1.76
mmol). Purified by reverse-phase HPLC with gradient elution (7:13)
MeCN/H2O + 1% TFA to (13:7) MeCN/H2O + 1%TFA over 60
min. Resulting residue was repurified by flash chromatography (1:99
MeOH/DCM to 4:96 MeOH/DCM) to give 10.4 mg (2.5%) of a
1
4.6%). H NMR (DMSO-d6, 600 MHz) δ 0.68 (d, J = 6.5 Hz, 3H),
0.86 (d, J = 6.5 Hz, 3H), 1.11 (s, 3H), 1.46 (s, 3H), 1.79 (m, 1H), 2.01
(s, 3H), 2.22 (m, 1H), 2.31 (m, 1H), 3.50 (dd, J = 9.5, 10 Hz, 1H),
3.59 (s, 3H), 3.61 (dd, J = 9, 10 Hz, 1H), 6.53 (br s, 1H), 6.68 (m,
2H), 7.11 (m, 2H), 7.39 (d, J = 7.5 Hz, 1H), 7.78 (d, J = 8 Hz). (+)
LRMS (ESI) m/z 462 [M + H]+. HRMS (ESI): calcd for C28H31NO5
Na [M + Na]+, 484.2094; found, 484.2113.
(1R,2R,2aR,10aS)-1-Isopropyl,10,10-dimethyl-2-(3,4,-dimethoxy-
phenyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]pyrano[3,2-
c]quinolin-3-one and (1S,2S,2aS,10aR)-1-Isopropyl,10,10-dimethyl-
2-(3,4,-dimethoxyphenyl)-1,2,2a,4,10,10a-hexahydro-3H-
cyclobuta[4,5]pyrano[3,2-c]quinolin-3-one (26). From flindersine
(2a, 0.88 mmol) and 4l (1.76 mmol). Purified by flash chromatography
on neutral alumina (DCM, then 1:1 DCM/CHCl3, then CHCl3). The
fractions containing MH+ 434 were combined and purified by reverse-
1
light yellow solid. H NMR (500 MHz, DMSO-d6) δ 0.01−0.05 (m,
2H), 0.26−0.36 (m, 2H), 0.90−0.94 (m, 1H), 1.14 (s, 3H), 1.47−1.57
(m, 5H), 2.06 (d, J = 8.5 Hz, 1H), 2.46 (t, J = 9 Hz, 1H), 3.10−3.12
(m, 1H), 3.25−3.28 (m, 1H), 3.36−3.41 (m, 1H), 3.49−3.52 (m, 1H),
3.60−3.62 (m, 4H), 4.36 (t, J = 5 Hz, 1H), 6.23 (s, 1H), 6.46 (d, J = 8
Hz, 1H), 6.59 (d, J = 8.2 Hz, 1H), 7.12−7.16 (m, 2H), 7.42 (t, J = 7.69
Hz, 1H), 7.81 (d, J = 7.96 Hz, 1H), 10.97 (s, 1H). (+) LRMS (ESI)
m/z 476.0 [M + H]+. HRMS (ESI): calcd for C29H34NO5 [M + H]+,
476.2432; found, 476.2424.
1
phase HPLC with isocratic elution MeCN/H2O (7:3) (14 mg, 6%). H
NMR (DMSO-d6, 500 MHz) δ 0.68 (d, J = 6.5 Hz, 3H), 0.84 (d, J = 6.5
Hz, 3H), 1.12 (s, 3H), 1.47 (s, 3H), 1.79 (m, 1H), 2.32 (m, 2H), 3.12 (m,
3H), 3.49 (t, J = 9 Hz, 1H), 3.61 (s, 3H), 3.63 (t, J = 9 Hz, 1H), 6.26 (s,
1H), 6.49 (d, J = 7.5 Hz, 1H), 6.57 (d, J = 7.5 Hz, 1H), 7.13 (d, J = 7.5
Hz, 1H), 7.13 (d, J = 7.5 Hz, 1H), 7.41 (t, J = 7.5 Hz, 1H), 7.80 (d, J =
7.5 Hz, 1H), 10.95 (s, 1H). (+) LRMS (ESI) m/z 434 [M + H]+. HRMS
(ESI): calcd for C27H31NO4Na [M + Na]+, 456.2145; found, 456.2140.
(1R,2R,2aR,10aS)-2-[4-Methoxy-3-(oxobutanoicacid)phenyl]-
10,10-dimethyl-1-(1-methylethyl)-1,2,2a,4,10,10a-hexahydro-3H-
cyclobuta[4,5]pyrano[3,2-c]quinolin-3-one and (1S,2S,2aS,10aR)-2-
[4-Methoxy-3-(oxobutanoicacid)phenyl]-10,10-dimethyl-1-(1-meth-
ylethyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]pyrano[3,2-
c]quinolin-3-one (27). From flindersine (2a, 0.55 mmol) and 5a (1.1
mmol). Purified by flash chromatography (2% up to 5% MeOH/
DCM). The resulting residue was repurified by reverse-phase HPLC
with isocratic elution MeCN/H2O + 1% TFA (1:1) to give 1.6 mg
(0.56%) of a tan solid. 1H NMR (DMSO-d6, 500 MHz) δ 0.69
(1R,2R,2aR,10aS)-2-(3,4-Dimethoxyphenyl)-10,10-dimethyl-1-(1-
cyclopropyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]pyrano-
[3,2-c]quinolin-3-one and (1S,2S,2aS,10aR)-2-(3,4-Dimethoxyphen-
yl)-10,10-dimethyl-1-(1-cyclopropyl)-1,2,2a,4,10,10a-hexahydro-
3H-cyclobuta[4,5]pyrano[3,2-c]quinolin-3-one (31). From flinder-
sine (2a, 0.88 mmol) and 4t (1.76 mmol). Purified by reverse-phase
HPLC with isocratic elution MeCN/H2O + 1% TFA (53:47) and then
flash chromatography (1% up to 3% MeOH/DCM) to give 11.9 mg
(3.1%) as a white solid. 1H NMR (DMSO-d6, 500 MHz) δ 0.01−0.08
(m, 2H), 0.25−0.30 (m, 1H), 0.33−0.39 (m, 1H), 0.89−0.96 (m, 1H),
1.14 (s, 3H), 1.53 (s, 3H), 2.06−2.12 (m, 1H), 2.47 (t, J = 8.8 Hz,
1H), 3.19 (s, 3H), 3.35−3.41 (m, 1H), 3.60−3.65 (m, 4H), 6.27
(d, J = 1.7 Hz, 1H), 6.45 (dd, J = 8.2, 1.7 Hz, 1H), 6.59 (d, J = 8.2 Hz,
1H), 7.11−7.15 (m, 2H), 7.39−7.44 (m, 1H), 7.81 (d, J = 8.2 Hz,
1H), 10.99 (s, 1H). (+) LRMS (ESI) m/z 431.8 [M + H]+. HRMS
R
dx.doi.org/10.1021/jm401321v | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(ESI): calcd for C27H29NO4Na [M + Na]+, 454.1989; found,
454.1986.
161.83. (+) LRMS (ESI) m/z 490.3 [M + H]+. HRMS (ESI): calcd for
C30H35NO5Na [M + Na]+, 512.2407; found, 512.2426.
(1R,2R,2aR,10aS)-2-(3,4,5-Trimethoxyphenyl)-10,10-dimethyl-1-
(1-cyclopropyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]-
pyrano[3,2-c]quinolin-3-one and (1S,2S,2aS,10aR)-2-(3,4,5-Trime-
thoxyphenyl)-10,10-dimethyl-1-(1-cyclopropyl)-1,2,2a,4,10,10a-
hexahydro-3H-cyclobuta[4,5]pyrano[3,2-c]quinolin-3-one (32).
From flindersine (2a, 0.88 mmol) and 4s (1.76 mmol). Purified by
reverse-phase HPLC isocratic elution MeCN/ H2O + 1% TFA (1:1)
over 60 min. Then flash chromatography (0% up to 2% MeOH/
(1R,2R,2aR,10aS)-1-Butyl,10,10-trimethyl-2-(3,4,5-trimethoxy-
phenyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]pyrano[3,2-
c]quinolin-3-one and (1S,2S,2aS,10aR)-1-Butyl,10,10-trimethyl-2-
(3,4,5-trimethoxyphenyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta-
[4,5]pyrano[3,2-c]quinolin-3-one (36). From flindersine (2a, 0.5
mmol) and 4n (1.0 mmol). Purified by isocratic elution MeCN/H2O
(60:40) (34 mg, 14%). 1H NMR (DMSO-d6, 600 MHz) δ 0.72 (t, J =
7 Hz, 3H), 0.99 (m, 1H), 1.06 (m, 1H), 1.13 (s, 3H), 1.14 (m, 2H),
1.48 (s, 3H), 1.52 (m, 1H), 1.64 (m, 1H), 2.32 (m, 1H), 2.38 (t, J = 9
Hz, 1H), 3.31 (s, 6H), 3.39 (t, J = 9 Hz, 1H), 3.53 (s, 3H), 3.69 (t, J =
9 Hz, 1H), 6.11 (d, J = 8 Hz, 2H), 7.12 (d, J = 7.5 Hz, 1H), 7.13 (t, J =
7.5 Hz, 1H), 7.43 (t, J = 7.5 Hz, 1H), 7.83 (d, J = 8 Hz, 1H), 11.03 (s,
1H). 13C NMR (DMSO-d6, 150 MHz) δ 14.60, 22.69, 24.38, 25.50,
29.23, 32.20, 36.62, 40.45, 44.75, 49.08, 55.94, 60.40, 77.19, 106.32,
108.78, 115.46, 115.52, 121.79, 122.61, 130.48, 136.36, 137.60, 137.69,
152.20, 156.16, 162.76. (+) LRMS (ESI) m/z 478 [M + H]+. HRMS
(ESI): calcd for C29H36NO5 [M + H]+, 478.2588; found, 478.2596.
(1R,2R,2aR,10aS)-1-Butyl,10,10-trimethyl-2-(3,4,-dimethoxyphen-
yl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]pyrano[3,2-c]-
quinolin-3-one and (1S,2S,2aS,10aR)-1-Butyl,10,10-trimethyl-2-
(3,4,-dimethoxyphenyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta-
[4,5]pyrano[3,2-c]quinolin-3-one (37). From flindersine (2a, 0.5
mmol) and 4o (1.0 mmol). Purified by flash chromatography on
neutral alumina (DCM, then 1:1 DCM/CHCl3, then CHCl3). The
fractions containing MH+ 448 were combined and purified by reverse-
phase HPLC with isocratic elution MeCN/H2O (7:3) (13.2 mg, 7%).
1H NMR (DMSO-d6, 500 MHz) δ 0.69 (t, J = 7 Hz, 3H), 0.94 (m,
1H), 0.99 (m, 1H), 1.11 (s, 3H), 1.11 (m, 2H), 1.48 (s, 3H), 1.49 (m,
1H), 1.63 (m, 1H), 2.34 (m, 2H), 3.14 (s, 3H), 3.36 (t, J = 9 Hz, 1H),
3.60 (s, 3H), 3.65 (t, J = 9 Hz, 1H), 6.26 (s, 1H), 6.48 (d, J = 7.5 Hz,
1H), 6.58 (d, J = 7.5 Hz, 1H), 7.12(d, J = 7.5 Hz, 1H), 7.13 (t, J = 7.5
Hz, 1H), 7.41 (t, J = 7.5 Hz, 1H), 7.81 (d, J = 8 Hz, 1H), 10.97 (s,
1H). 13C NMR (DMSO-d6, 150 MHz) δ 13.54, 21.56, 23.23, 24.60,
28.31, 35.36, 31.17, 39.23, 43.81, 47.65, 53.92, 54.92, 76.36, 107.98,
110.06, 111.08, 114.47, 114.49, 120.42, 120.72, 121.54, 129.54, 133.42,
137.11, 146.63, 146.98, 155.30, 161.43. (+) LRMS (ESI) m/z 448 [M
+ H]+. HRMS (ESI): calcd for C28H33NO4Na [M + Na]+, 470.2302;
found, 458.2289.
1
DCM) to give 8.8 mg (2.2%) as a white solid. H NMR (DMSO-d6,
500 MHz) δ 0.03−0.12 (m, 2H), 0.28−0.40 (m, 2H), 0.90−0.97 (m,
1H), 1.15 (s, 3H), 1.53 (s, 3H), 2.09 (q, J = 8.9 Hz, 1 H) 2.45−2.49
(m, 1H), 3.35−3.41 (m, 1H), 3.53 (s, 3H), 3.64 (t, J = 8.5 Hz, 1 H)
6.08 (s, 2H), 7.11−7.16 (m, 2H), 7.42 (t, J = 7.6 Hz, 1H), 7.81 (d, J =
8 Hz, 1H), 11.03 (s, 1H). (+) LRMS (ESI) m/z 461.6 [M + H]+.
HRMS (ESI): calcd for C28H31NO5Na [Na]+, 484.2094; found,
484.2085.
(1R,2R,2aR,10aS)-2-(4-Methoxy-3-(2-morpholine-4-ylpropoxy)-
phenyl)-10,10,6-trimethyl-1-(1-cyclopropyl)-1,2,2a,4,10,10a-hexa-
hydro-3H-cyclobuta[4,5]pyrano[3,2-c]quinolin-3-one and
(1S,2S,2aS,10aR)-2-(4-Methoxy-3-(2-morpholine-4-ylpropoxy)-
phenyl)-10,10,6-trimethyl-1-(1-cyclopropyl)-1,2,2a,4,10,10a-hexa-
hydro-3H-cyclobuta[4,5]pyrano[3,2-c]quinolin-3-one (33). From
flindersine (2a, 0.5 mmol) and 5b (1.0 mmol). Purified by flash
chromatography on neutral alumina (DCM, then 1:1 DCM/CHCl3,
then CHCl3). The fractions containing MH+ 545 were combined and
purified by reverse-phase HPLC with XBridge gradient elution
MeCN/H2O (10 mM NH4OAc) 95:5 to MeCN over 60 min to
afford 5.8 mg (2%). 1H NMR (DMSO-d6, 500 MHz) δ 0.02 (m, 2H),
0.27 (m, 1H), 0.35 (m, 1H), 0.92 (m, 1H), 1.13 (s, 3H), 1.53 (s, 3H),
2.07 (m, 3H), 2.47 (m, 1H), 2.54 (m, 6H), 3.60 (m, 5H), 3.62 (s, 3H),
3.98 (m, 2H), 6.46 (d, J = 1.5 Hz), 6.47 (dd, J = 1.5, 8 Hz, 1H), 6.60
(d, J = 8.5 Hz, 1H), 7.12 (d, J = 8 Hz, 1H), 7.13 (dd, J = 7.5, 8 Hz,
1H), 7.42 (dd, J = 7.5, 8.5 Hz, 1H), 7.57 (dd, J = 7.5, 8.5 Hz, 1H), 7.93
(d, J = 8 Hz, 1H). (+) LRMS (ESI) m/z 545 [M + H]+. HRMS (ESI):
calcd for C33H41N2O5 [M + H]+, 545.3010; found, 545.3010.
(1R,2R,2aR,10aS)-1-Cyclopentyl-2-(3,4-dimethoxyphenyl)-10,10-
dimethyl-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]pyrano[3,2-
c]quinolin-3-one and (1S,2S,2aS,10aR)-1-Cyclopentyl-2-(3,4-dime-
thoxyphenyl)-10,10-dimethyl-1,2,2a,4,10,10a-hexahydro-3H-
cyclobuta[4,5]pyrano[3,2-c]quinolin-3-one (34). From flindersine
(2a, 0.50 mmol) and 4p (1.0 mmol). Purified by reverse-phase
(1R,2R,2aR,10aS)-1-Ethanone-2-(3,4-dimethoxyphenyl)-10,10-di-
methyl-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]pyrano[3,2-c]-
quinolin-3-one and (1S,2S,2aS,10aR)-1-Ethanone-2-(3,4-dimethox-
yphenyl)-10,10-dimethyl-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta-
[4,5]pyrano[3,2-c]quinolin-3-one (38). From flindersine (2a, 0.5 mmol)
and (E)-4-(3,4-dimethoxyphenyl)but-3-en-2-one (1.0 mmol). Purified by
flash chromatography on neutral alumina (DCM, then 1:1 DCM/CHCl3,
then CHCl3). The fractions containing MH+ 434 were combined and
purified by reverse-phase HPLC with gradient elution MeCN/H2O (3:7) to
1
HPLC with isocratic elution MeCN/H2O (3:2) (19.4 mg, 8.5%). H
NMR (DMSO-d6, 600 MHz) δ 0.89 (m, 1H), 1.11 (m, 1H), 1.55 (m,
2H), 1.44 (m, 4H), 1.12 (s, 3H), 1.49 (s, 3H), 1.97 (m, 1H), 2.41 (m,
1H), 2.42 (m, 1H), 3.12 (s, 3H), 3.48 (m, 1H), 3.60 (s, 3H), 3.64
(t, J = 9 Hz, 1H), 6.25 (br s, 1H), 6.48 (dd, J = 1.5, 8.4 Hz, 1H), 6.57
(d, J = 8.4 Hz, 1H), 7.13 (t, J = 8 Hz, 1H), 7.14 (d, J = 8 Hz, 1H), 7.42
(t, J = 8.4 Hz, 1H), 7.81 (d, J = 8.4 Hz, 1H), 10.95 (s, 1H). 13C NMR
(CDCl3, 150 MHz) δ 24.2, 25.2, 26.2, 29.1, 31.5, 32.1, 40.5, 44.0, 45.2,
45.6, 55.0, 55.8, 78.1, 109.5, 111.1, 112.0, 115.3, 115.8, 121.5, 121.7,
122.5, 130.5, 135.9, 138.4, 147.6, 148.3, 155.8, 162.7. (+) LRMS (ESI)
m/z 460 [M + H]+. HRMS (ESI): calcd for C29H33NO4Na [M + Na]+,
482.2302; found, 482.2322.
(1R,2R,2aR,10aS)-2-(3,4,5-Trimethoxyphenyl)-10,10-dimethyl-1-
(1-cyclopentyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]-
pyrano[3,2-c]quinolin-3-one and (1S,2S,2aS,10aR)-2-(3,4,5-Trime-
thoxyphenyl)-10,10-dimethyl-1-(1-cyclopentyl)-1,2,2a,4,10,10a-
hexahydro-3H-cyclobuta[4,5]pyrano[3,2-c]quinolin-3-one (35).
From flindersine (2a, 0.88 mmol) and 4q (1.76 mmol). Purified by
reverse-phase HPLC with isocratic elution MeCN/H2O (1:1) over
60 min. Then flash chromatography (DCM to 2% MeOH/DCM) to
give 1 mg (0.23%) as a white solid. 1H NMR (DMSO-d6, 500 MHz) δ
1.12−1.19 (m, 5H), 1.39−1.48 (m, 4H), 1.51 (s, 3H), 1.56−1.63 (m,
2H), 1.95−2.01 (m, 1H), 2.37−2.51 (m, 2H), 3.29−3.41 (m, 6H),
3.49−3.53 (m, 4H), 3.68 (t, J = 8.7 Hz, 1H), 6.10 (s, 2H), 7.11−7.15
(m, 2H), 7.41−7.43 (m, 1H), 7.81 (d, J = 8 Hz, 1 H) 11.00 (s, 1H).
13C NMR (DMSO-d6, 150 MHz) δ 23.18, 24.18, 24.34, 25.31, 30.91,
1
MeCN over 60 min to give 12.3 mg (6%). H NMR (DMSO-d6, 500
MHz) δ 0.73 (d, J = 6.5 Hz, 3H), 0.88 (d, J = 6.5 Hz, 3H), 1.15 (s, 3H),
1.49 (s, 3H), 1.86 (m, 3H), 2.98 (dd, J = 9 Hz, 1H), 3.23 (s, 3H), 3.31 (m,
1H), 3.41 (dd, J = 10.5 Hz, 1H), 3.66 (s, 3H), 3.74 (dd, J = 10 Hz, 1H),
6.44 (d, J = 1.5 Hz, 1H), 6.62 (dd, J = 1.5, 8.5 Hz, 1H), 6.67 (d, J = 8.5 Hz,
1H), 7.16 (d, J = 7.5 Hz, 1H), 7.17 (dd, J = 7.5 Hz, 1H), 7.46 (dd, J = 7.5
Hz, 1H), 7.84 (d, J = 7.5 Hz, 1H), 11.07 (s, 1H). (+) LRMS (ESI) m/z 434
[M + H]+. HRMS (ESI): calcd for C26H27NO5 [M + H]+, 434.1962; found,
434.1942.
(1R,2R,2aR,10aS)-1-Cyclopropylmethanone-2-(3,4-dimethoxy-
phenyl)-10,10-dimethyl-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta-
[4,5]pyrano[3,2-c]quinolin-3-one and (1S,2S,2aS,10aR)-1-Cyclopro-
pylmethanone-2-(3,4-dimethoxyphenyl)-10,10-dimethyl-
1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]pyrano[3,2-c]-
quinolin-3-one (39). From flindersine (2a, 0.5 mmol) and (E)-1-
cyclopropyl-3-(3,4-dimethoxyphenyl)prop-2-en-1-one (1.76 mmol).
Purified by flash chromatography on neutral alumina (DCM, then
1:1 DCM/CHCl3, then CHCl3). The fractions containing MH+ 460
were combined and purified by reverse-phase HPLC with gradient
elution MeCN/H2O (3:7) to MeCN over 60 min to give 24.6 mg
(11%). 1H NMR (DMSO-d6, 600 MHz) δ 0.75 (m, 4H), 1.16 (s, 3H),
1.36 (s, 3H), 1.67 (m, 1H), 2.98 (dd, J = 8.4, 9.5 Hz, 1H), 3.20
39.51, 43.64, 44.71, 44.85, 54.76, 59.56, 76.08, 105.36, 108.24, 114.26,
114.64, 120.55, 121.38, 129.52, 135.49, 137.30, 137.63, 151.30, 155.14,
S
dx.doi.org/10.1021/jm401321v | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(s, 3H), 3.52 (dd, J = 8.4, 9.6 Hz, 1H), 3.61 (s, 3H), 3.64 (dd, J = 8.4,
9.5 Hz, 1H), 3.77 (dd, J = 8.4, 9 Hz, 1H), 6.44 (br s, 1H), 6.59 (br d,
J = 8.4 Hz, 1H), 6.63 (d, J = 8.4 Hz, 1H), 7.14 (d, J = 7.8 Hz, 1H),
7.14 (dd, J = 7.8 Hz, 1H), 7.43 (dd, J = 7.5 Hz, 1H), 7.82 (d, J = 7.5
Hz, 1H), 11.06 (s, 1H). 13C NMR (CDCl3, 150 MHz) δ 11.01, 19.73,
23.54, 25.35, 31.94, 40.41, 46.01, 50.79, 55.19, 55.91, 76.74, 108.25,
111.31, 111.95, 115.42, 115.66, 121.32, 121.68, 122.72, 130.72, 132.36,
138.25, 148.08, 148.28, 156.74, 162.87, 210.44. (+) LRMS (ESI) m/z
460 [M + H]+. HRMS (ESI): calcd for C28H29NO5Na [M + Na]+,
482.1938; found, 482.1947.
(1R,2R,2aR,10aS)-1-Carbomethoxy-10,10-dimethyl-2-(3,4,5-tri-
methoxyphenyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]-
pyrano[3,2-c]quinolin-3-one and (1S,2S,2aS,10aR)-1-Carbome-
thoxy-10,10-dimethyl-2-(3,4,5-trimethoxyphenyl)-1,2,2a,4,10,10a-
hexahydro-3H-cyclobuta[4,5]pyrano[3,2-c]quinolin-3-one (40).
From flindersine (2a, 0.5 mmol) and (E)-methyl-3-(3,4,5-trimethoxyphenyl)-
acrylate (1.0 mmol). Purified by gradient elution MeCN/H2O (1%
TFA, 3:7 for 45 min then gradient elution to 75:25 over 25 min) to
afford a white solid (5.4 mg, 2.2%). 1H NMR (DMSO-d6, 500 MHz) δ
1.18 (s, 3H), 1.46 (s, 3H), 3.02 (t, J = 8.5 Hz, 1H), 3.20 (t, J = 8.5 Hz,
1H), 3.37 (s, 6H), 3.54 (s, 3H), 3.62 (s, 3H), 3.76 (t, J = 8 Hz, 1H),
3.95 (t, J = 9 Hz, 1H), 6.19 (s, 2H), 7.13 (m, 2H), 7.46 (t, J = 7.5 Hz,
1H), 7.84 (d, J = 8 Hz, 1H), 11.14 (s, 1H). (+) LRMS (ESI) m/z 480
[M + H]+. HRMS (ESI): calcd for C27H30NO7 [M + H]+, 480.2017;
found, 480.1997.
(1R,2R,2aR,10aS)-1-Acetoxymethyl-10,10-dimethyl-2-(3,4,5-tri-
methoxyphenyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]-
pyrano[3,2-c]quinolin-3-one and (1S,2S,2aS,10aR)-1-Acetoxymeth-
yl-10,10-dimethyl-2-(3,4,5-trimethoxyphenyl)-1,2,2a,4,10,10a-hexa-
hydro-3H-cyclobuta[4,5]pyrano[3,2-c]quinolin-3-one (41). From
flindersine (2a, 0.5 mmol) and (E)-3-(3,4,5-trimethoxyphenyl)-
allylacetate (1.0 mmol). Purified by gradient elution MeCN/H2O
(1% TFA, 3:7 for 45 min then gradient elution to 75:25 over 25 min)
to afford a white solid (44.3 mg, 9%). 1H NMR (DMSO-d6, 500 MHz)
δ 1.17 (s, 3H), 1.47 (s, 3H), 1.89 (s, 3H), 2.60 (t, J = 9 Hz, 1H), 2.66
(t, J = 9 Hz, 1H), 3.34 (s, 6H), 3.51 (s, 3H), 3.58 (t, J = 9 Hz, 1H),
3.74 (t, J = 8 Hz, 1H), 6.13 (s, 2H), 7.13 (m, 2H), 7.42 (t, J = 7.5 Hz,
1H), 7.81 (d, J = 8 Hz, 1H), 11.08 (s, 1H). (+) LRMS (ESI) m/z 494
[M + H]+.
(1R,2R,2aR,10aS)-1-Isopropyloxymethyl-10,10-dimethyl-2-(3,4,5-
trimethoxyphenyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]-
pyrano[3,2-c]quinolin-3-one and (1S,2S,2aS,10aR)-1-Isopropyloxy-
methyl-10,10-dimethyl-2-(3,4,5-trimethoxyphenyl)-1,2,2a,4,10,10a-
hexahydro-3H-cyclobuta[4,5]pyrano[3,2-c]quinolin-3-one (42).
From flindersine (2a, 0.5 mmol) and (E)-5-(3-isopropoxyprop-1-
enyl)-1,2,3-trimethoxybenzene (1.0 mmol). Purified by gradient
elution MeCN/H2O (1% TFA, 35:65 for 30 min then gradient
elution to 75:25 over 30 min) to afford a white solid (34.2 mg, 13%).
1H NMR (CDCl3, 500 MHz) δ 1.13−1.22 (m, 9H), 1.45 (s, 3H), 2.96
(2 mL). Potassium iodide (0.066 mmol) was added, and the reaction
mixture was heated at 50 °C for 20 h. The reaction mixture was
extracted with a 10% NaHSO4 solution and CHCl3 (3 × 30 mL), dried
with MgSO4, filtered, and concentrated to dryness to afford 43 (20 mg,
1
83%) as a white solid. H NMR (CDCl3, 500 MHz) δ 1.18 (t, J = 7
Hz, 3H), 1.14 (s, 3H), 1.46 (s, 3H), 2.99 (t, J = 9 Hz, 1H), 3.19 (t, J =
10 Hz, 1H), 3.72 (t, J = 8 Hz, 1H), 3.99 (m, 2H), 4.08 (m, 1H), 6.89
(d, J = 7 Hz, 2H), 7.14 (t, J = 7 Hz, 2H), 7.22 (d, J = 7.5 Hz, 2H), 7.43
(t, J = 7 Hz, 1H), 7.82 (d, J = 7.5 Hz, 1H), 11.10 (s, 1H). (+) LRMS
(ESI) m/z 482:484 (1:1) [M + H]+. HRMS (ESI): calcd for C25H24
BrNO4 [M + Na]+, 504.0781; found, 504.0777.
(1R,2R,2aR,10aS)-1,4,10,10-Tetramethyl-2-(3,4-dimethoxyphen-
yl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]pyrano[3,2-c]-
quinolin-3-one and (1S,2S,2aS,10aR)-1,4,10,10-Tetramethyl-2-(3,4-
dimethoxyphenyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]-
pyrano[3,2-c]quinolin-3-one (44). From N-Me-flindersine (2b, 0.93
mmol) and 4a (1.87 mmol). Purified by reverse-phase HPLC with
isocratic elution MeCN/H2O (55:45) over 60 min to give 15.4 mg
1
(3.7%) as a light brown solid. H NMR (DMSO-d6, 600 MHz) δ
1.10−1.17 (m, 6H), 1.49 (s, 3H), 2.30−2.37 (m, 2H), 3.23 (s, 3H),
3.30−3.32 (m, 7H), 3.51 (s, 3H), 3.71 (t, J = 7.8 Hz, 1H), 6.06 (s,
2H), 7.26 (t, J = 7.5 Hz, 1H), 7.41 (d, J = 8.2 Hz, 1H), 7.59 (t, J = 7.8
Hz, 1H), 7.95 (d, J = 7.8 Hz, 1H). 13C NMR (DMSO-d6, 150 MHz) δ
20.04, 22.91, 24.09, 32.04, 34.58, 45.39, 49.17, 54.68, 59.53, 76.29,
104.91, 107.10, 114.83, 115.49, 120.98, 121.85, 130.11, 135.63, 135.79,
138.32, 151.29, 154.14, 161.15. (+) LRMS (ESI) m/z 449.8 [M + H]+.
HRMS (ESI): calcd for C26H31NO5Na [M + Na]+, 472.2094; found,
472.2104.
(1R,2R,2aR,10aS)-2-(3,4-Dimethoxyphenyl)-1-ethyl-4,10,10-tri-
methyl-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]pyrano[3,2-c]-
quinolin-3-one and (1S,2S,2aS,10aR)-2-(3,4-Dimethoxyphenyl)-1-
ethyl-4,10,10-trimethyl-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta-
[4,5]pyrano[3,2-c]quinolin-3-one (45). From N-Me-flindersine (2b,
0.50 mmol) and 4e (1.0 mmol). Purified by flash chromatography on
neutral alumina (DCM, then 1:1 DCM/CHCl3, then CHCl3). The
fractions containing MH+ 435 were combined and purified by reverse-
phase HPLC with isocratic elution MeCN/H2O (3:2) over 60 min to
1
afford 21.1 mg (10%). H NMR (DMSO-d6, 500 MHz) δ 0.64 (t, J =
6.5 Hz, 3H), 1.51 (m, 1H), 1.67 (m, 1H), 1.13 (s, 3H), 1.47 (s, 3H),
2.29 (m, 1H), 2.37 (t, J = 9 Hz, 1H), 3.13 (s, 3H), 3.20 (s, 3H), 3.60
(s, 3H), 3.69 (t, J = 9 Hz, 1H), 6.25 (br s, 1H), 6.44 (dd, J = 1.5, 7.5
Hz, 1H), 6.58 (d, J = 7.5 Hz), 7.25 (t, J = 7.5 Hz, 1H), 7.38 (d, J = 7.5
Hz, 1H), 7.57 (t, J = 7.5 Hz, 1H), 7.95 (d, J = 7.5 Hz, 1H). 13C NMR
(CDCl3, 150 MHz) δ 12.50, 23.82, 25.32, 29.32, 30.68, 33.28, 41.82,
48.51, 55.84, 56.90, 77.68, 109.51, 112.00, 113.11, 115.02, 115.97,
122.31, 123.08, 123.82, 132.39, 135.88, 140.10, 148.68, 149.26, 156.38,
163.88. (+) LRMS (ESI) m/z 435 [M + H]+. HRMS (ESI): calcd for
C27H31NO4Na [M + Na]+, 456.2145; found, 456.2138.
(1R,2R,2aR,10aS)-1-Ethyl-2-(3-fluoro-4-methoxyphenyl)-4,10,10-
trimethyl-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]pyrano[3,2-
c]quinolin-3-one and (1S,2S,2aS,10aR)-1-Ethyl-2-(3-fluoro-4-me-
thoxyphenyl)-4,10,10-trimethyl-1,2,2a,4,10,10a-hexahydro-3H-
cyclobuta[4,5]pyrano[3,2-c]quinolin-3-one (46). From N-Me-flin-
dersine (2b, 0.50 mmol) and 4g (1.0 mmol). Purified by flash
chromatography on neutral alumina (DCM, then 1:1 DCM/CHCl3,
then CHCl3). The fractions containing MH+ 422 were combined and
purified by reverse-phase HPLC with isocratic elution MeCN/H2O
(3:2) to MeCN over 60 min to afford 9.8 mg (5%). 1H NMR (DMSO-
d6, 600 MHz) δ 0.63 (t, J = 8 Hz, 3H), 1.11 (s, 3H), 1.46 (s, 3H), 1.52
(m, 1H), 1.66 (m, 1H), 2.27 (m, 1H), 2.36 (t, J = 10 Hz, 1H), 3.20 (s,
3H), 3.43 (t, J = 9 Hz, 1H), 3.64 (t, J = 9 Hz, 1H), 3.75 (s, 3H), 6.54 (br
d, J = 9.6 Hz, 1H), 6.59 (br d, J = 8 Hz, 1H), 6.73 (dd, J = 6, 8 Hz, 1H),
7.25 (t, J = 7.5 Hz, 1H), 7.38 (d, J = 7.5 Hz, 1H), 7.56 (t, J = 8 Hz, 1H),
7.93 (d, J = 8 Hz). (+) LRMS (ESI) m/z 422 [M + H]+. HRMS (ESI):
calcd for C26H28FNO3Na [M + Na]+, 444.1945; found, 444.1951.
(1R,2R,2aR,10aS)-1-Ethyl-7,10,10-trimethyl-2-(3,4,5-trimethoxy-
phenyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]pyrano[3,2-
c]quinolin-3-one and (1S,2S,2aS,10aR)-1-Ethyl-7,10,10-trimethyl-2-
(3,4,5-trimethoxyphenyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta-
[4,5]pyrano[3,2-c]quinolin-3-one (47). From 2c (0.5 mmol) and 4a
(1.0 mmol). Purified by flash chromatography on neutral alumina
(t, J = 9 Hz, 1H), 3.10 (t, J = 9 Hz, 1H), 3.37 (s, 6H), 3.52 (s, 3H),
3.61 (m, 1H), 3.75 (m, 1H), 3.95 (t, J = 9 Hz, 1H), 6.16 (s, 2H), 7.13
(m, 2H), 7.43 (t, J = 7.5 Hz, 1H), 7.81 (d, J = 8 Hz, 1H), 11.11 (s,
1H). (+) LRMS (ESI) m/z 504 [M + H]+. HRMS (ESI): calcd for
C29H33NO7 [M + Na]+, 530.2149; found, 530.2125.
(1R,2R,2aR,10aS)-1-Carboethoxy-10,10-dimethyl-2-(3,4,5-trime-
thoxyphenyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]pyrano-
[3,2-c]quinolin-3-one and (1S,2S,2aS,10aR)-1-Carboethoxy-10,10-
dimethyl-2-(3,4,5-trimethoxyphenyl)-1,2,2a,4,10,10a-hexahydro-
3H-cyclobuta[4,5]pyrano[3,2-c]quinolin-3-one (43). From 3 and
(E)-ethyl-3-(4-bromophenyl)acrylate. Purified by isocratic elution
MeOH/H2O (1% TFA, 6:4) (59.7 mg, 12%). 1H NMR (CDCl3,
500 MHz) δ 1.11 (t, J = 7 Hz, 3H), 1.16 (s, 3H), 1.49 (s, 3H), 3.04 (t,
J = 9 Hz, 1H), 3.23 (t, J = 10 Hz, 1H), 3.28 (s, 3H), 3.79 (t, J = 8 Hz,
1H), 3.99 (m, 1H), 4.08 (m, 2H), 6.72 (d, J = 7 Hz, 2H), 7.21 (d, J = 7
Hz, 2H), 7.37 (t, J = 7 Hz, 1H), 7.58 (m, Hz, 2H), 8.02 (d, J = 8 Hz,
1H). 13C NMR (CDCl3, 125 MHz) δ 13.95, 22.90, 24.46, 31.71, 41.13,
41.37, 43.78, 52.19, 60.23, 75.74, 101.53, 118.51, 119.54, 121.46,
123.43, 126.43, 129.43, 129.77, 130.1, 137.47, 145.00, 156.34, 160.99,
172.53. (+) LRMS (ESI) m/z 496:498 (1:1) [M + H]+. HRMS (ESI):
calcd for C26H27BrNO4 [M + H]+, 496.1118; found, 496.1121. This
compound was (26.3 mg, 0.050 mmol) was dissolved in acetic acid
T
dx.doi.org/10.1021/jm401321v | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(DCM, then 1:1 DCM/CHCl3, then CHCl3). The fractions
containing MH+ 464 were combined and purified by reverse-phase
HPLC with isocratic elution MeCN/H2O (55:45) to MeCN over
60 min (12.6 mg, 6%). 1H NMR (DMSO-d6, 600 MHz) δ 0.68 (d, J =
8.5 Hz, 3H), 1.14 (s, 3H), 1.49 (s, 3H), 1.52 (m, 1H), 1.67 (m, 1H),
2.28 (m, 2H), 2.36 (s, 3H), 3.31 (s, 6H), 3.38 (t, J = 9 Hz, 1H), 3.52
(s, 3H), 3.66 (t, J = 9 Hz, 1H), 6.11 (s, 2H), 7.05 (d, J = 7.5 Hz, 1H),
7.26 (d, J = 7.5 Hz, 1H), 7.63 (br s, 1H), 10.95 (s, 1H). (+) LRMS
(ESI) m/z 464 [M + H]+. HRMS (ESI): calcd for C28H33NO5Na
[M + Na]+, 486.2251; found, 486.2273.
MH+ 476 were combined and purified by reverse-phase HPLC with
gradient elution MeCN/H2O (5:95) to MeCN/H2O (2:3) over 60 min
to afford 17.6 mg (4.5%). 1H NMR (600 MHz, DMSO-d6) δ 0.69 (d, J
= 6.6 Hz, 3H), 0.85 (d, J = 6.57 Hz, 3H), 1.13 (s, 3H), 1.48 (s, 3H),
1.79 (m, Hz, 1 H), 1.95 (s, 3H), 2.29 (m, 1H), 2.26 (m, 1H), 3.21 (s,
3H), 3.52 (m, 1H), 3.61 (s, 3H), 3.67 (m, 1H), 6.35 (s, 1 H), 6.67−6.76
(m, 2 H), 7.24 (t, J = 7.5 Hz, 1H), 7.35 (d, J = 8 Hz, 1H), 7.56 (dd, J =
7.5, 8 Hz, 1H), 7.92 (d, J = 8 Hz, 1H). (+) LRMS (ESI) m/z 476 [M +
H]+. HRMS (ESI): calcd for C29H33NO5 [M + H]+, 476.2432; found,
476.2425.
(1R,2R,2aR,10aS)-2-(3,4-Dimethoxyphenyl)-1-ethyl-7,10,10-tri-
methyl-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]pyrano[3,2-c]-
quinolin-3-one and (1S,2S,2aS,10aR)-2-(3,4-Dimethoxyphenyl)-1-
ethyl-7,10,10-trimethyl-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta-
[4,5]pyrano[3,2-c]quinolin-3-one (48). From 2c (0.83 mmol) and 4e
(1.67 mmol). Purified by flash chromatography on neutral alumina
(DCM, then 1:1 DCM/CHCl3, then CHCl3). The fractions
containing MH+ 434 were combined and purified by reverse-phase
HPLC with isocratic elution MeCN/H2O (3:2) to MeCN over 60 min
(1R,2R,2aR,10aS)-1-Cyclopropyl-2-(3,4,5-trimethoxyphenyl)-
10,10,6-trimethyl-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]-
pyrano[3,2-c]quinolin-3-one and (1S,2S,2aS,10aR)-1-Cyclopropyl-2-
(3,4,5-trimethoxyphenyl)-10,10,6-trimethyl-1,2,2a,4,10,10a-hexahy-
dro-3H-cyclobuta[4,5]pyrano[3,2-c]quinolin-3-one (52). From N-
Me-flindersine (2b, 0.5 mmol) and 4s (1.0 mmol). Purified by flash
chromatography on neutral alumina (DCM, then 1:1 DCM/CHCl3,
then CHCl3). The fractions containing MH+ 476 were combined and
purified by reverse-phase HPLC with isocratic elution MeCN/H2O
1
1
(9.7 mg, 3%). H NMR (DMSO-d6, 600 MHz) δ 0.65 (t, J = 8 Hz,
(42.5:57.5) over 60 min to give 8.6 mg (4%). H NMR (DMSO-d6,
3H), 1.13 (s, 3H), 1.48 (s, 3H), 1.53 (m, 1H), 1.67 (m, 1H), 2.31 (m,
1H), 2.34 (s, 3H), 2.49 9m, 1H), 3.20 (s, 3H), 3.38 (t, J = 9 Hz, 1H),
3.62 (s, 3H), 3.64 (t, J = 9 Hz, 1H), 6.30 (s, 1H), 6.48 (d, J = 8 Hz,
1H), 6.59 (d, J = 8 Hz, 1H), 7.06 (d, J = 7.5 Hz, 1H), 7.25 (d, J =
7.5 Hz, 1H), 7.62 (br s, 1H), 10.91 (s, 1H). (+) LRMS (ESI) m/z 434
[M + H]+. HRMS (ESI): calcd for C27H31NO4 [M + H]+, 434.2326;
found, 434.2329.
500 MHz) δ 0.07 (m, 2H), 0.30 (m, 1H), 0.36 (m, 1H), 0.93 (m, 1H),
1.15 (s, 3H), 1.53 (s, 3H), 2.06 (m, 1H), 2.47 (m, 1H), 3.20 (s, 3H),
3.28 (s, 6H), 3.39 (dd, J = 10 Hz, 1H), 3.50 (s, 3H), 3.65 (dd, J = 8.5,
10 Hz, 1H), 6.01 (s, 2H), 7.24 (dd, J = 7.5, 8 Hz, 1H), 7.38 (d, J = 8.5
Hz, 1H), 7.57 (ddd, J = 1.5, 7.5, 8.5 Hz, 1H), 7.93 (d, J = 8 Hz, 1H).
(+) LRMS (ESI) m/z 476 [M + H]+. HRMS (ESI): calcd for
C29H33NO5 [M + H]+, 476.2432; found, 476.2452.
(1R,2R,2aR,10aS)-7,10,10-Trimethyl-1-(1-methylethyl)-2-(3,4,5-
trimethoxyphenyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]-
pyrano[3,2-c]quinolin-3-one and (1S,2S,2aS,10aR)-7,10,10-Trimeth-
yl-1-(1-methylethyl)-2-(3,4,5-trimethoxyphenyl)-1,2,2a,4,10,10a-
hexahydro-3H-cyclobuta[4,5]pyrano[3,2-c]quinolin-3-one (49).
From 2c (0.83 mmol) and 4h (1.67 mmol). Purified by flash
chromatography on neutral alumina (DCM, then 1:1 DCM/CHCl3,
then CHCl3). The fractions containing MH+ 480 were combined and
purified by reverse-phase HPLC with isocratic elution MeCN/H2O
(1R,2R,2aR,10aS)-1-Cyclopropyl-2-(3,4-dimethoxyphenyl)-
10,10,6-trimethyl-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]-
pyrano[3,2-c]quinolin-3-one and (1S,2S,2aS,10aR)-1-Cyclopropyl-2-
(3,4-dimethoxyphenyl)-10,10,6-trimethyl-1,2,2a,4,10,10a-hexahy-
dro-3H-cyclobuta[4,5]pyrano[3,2-c]quinolin-3-one (53). From N-
Me-flindersine (2b, 0.5 mmol) and 4t (1.0 mmol). Purified by flash
chromatography on neutral alumina (DCM, then 1:1 DCM/CHCl3,
then CHCl3). The fractions containing MH+ 446 were combined and
purified by reverse-phase HPLC with isocratic elution MeCN/H2O
(42.5:57.5) over 60 min to afford 9.4 mg (4%). 1H NMR (DMSO-d6,
500 MHz) δ 0.03 (m, 2H), 0.27 (m, 1H), 0.35 (m, 1H), 0.91 (m, 1H),
1.14 (s, 3H), 1.53 (s, 3H), 2.06 (m, 1H), 2.47 (m, 1H), 3.15 (s, 3H),
3.20 (s, 3H), 3.38 (dd, J = 9.5, 10 Hz, 1H), 3.60 (s, 3H), 3.65 (dd, J =
8.5, 10 Hz, 1H), 6.22 (br s, 1H), 6.39 (br d, J = 8.5 Hz, 1H), 6.57
(d, J = 8.5 Hz, 1H), 7.24 (dd, J = 7.5, 8 Hz, 1H), 7.37 (d, J = 8.5 Hz,
1H), 7.56 (dd, J = 7.5, 8.5 Hz, 1H), 7.92 (d, J = 8 Hz, 1H). (+) LRMS
(ESI) m/z 446 [M + H]+. HRMS (ESI): calcd for C28H31NO5 Na
[M + Na]+, 484.2094; found, 484.2113.
(1R,2R,2aR,10aS)-1-Cyclopentyl-2-(3,4,5-trimethoxyphenyl)-
4,10,10-trimethyl-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]-
pyrano[3,2-c]quinolin-3-one and (1S,2S,2aS,10aR)-1-Cyclopentyl-2-
(3,4,5-trimethoxyphenyl)-4,10,10-trimethyl-1,2,2a,4,10,10a-hexahy-
dro-3H-cyclobuta[4,5]pyrano[3,2-c]quinolin-3-one (54). From N-
Me-flindersine (2b, 0.5 mmol) and 4q (1.0 mmol). Purified by flash
chromatography on neutral alumina (DCM, then 1:1 DCM/CHCl3,
then CHCl3). The fractions containing MH+ 504 were combined and
purified by reverse-phase HPLC with gradient elution MeCN/H2O
(5:95) to MeCN/H2O (2:3) over 60 min to afford 18.6 mg (4.6%).
1H NMR (DMSO-d6, 600 MHz) δ 95 (m, 1H), 1.16 (m, 1H), 1.16 (s,
1
(58:42) to MeCN over 60 min to afford 17.3 mg (4%). H NMR
(DMSO-d6, 600 MHz) δ 0.72 (d, J = 8 Hz, 3H), 0.88 (d, J = 8 Hz, 3H),
1.15 (s, 3H), 1.49 (s, 3H), 1.80 (m, 1H), 1.67 (m, 1H), 2.29 (m, 1H), 2.36
(s, 3H), 2.49 (m, 1H), 3.32 (s, 6H), 3.38 (t, J = 9 Hz, 1H), 3.52 (s, 3H),
3.66 (t, J = 9 Hz, 1H), 6.11 (s, 2H), 7.04 (d, J = 7.5 Hz, 1H), 7.25 (d, J =
7.5 Hz, 1H), 7.62 (br s, 1H), 10.92 (s, 1H). 13C NMR (CDCl3, 150 MHz)
δ 18.26, 20.39, 21.12, 23.22, 25.17, 30.52, 31.17, 40.94, 43.66, 45.45, 77.20,
105.34, 109.12, 114.2, 115.51, 120.93, 130.52, 130.72, 136.11, 136.23,
136.40, 155.96, 162.63. (+) LRMS (ESI) m/z 480 [M + H]+. HRMS
(ESI): calcd for C29H35NO5Na [M + Na]+, 500.2407; found, 500.2421.
(1R,2R,2aR,10aS)-2-(3-Chloro-4-methoxyphenyl)-4,10,10-tri-
methyl-1-(1-methylethyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta-
[4,5]pyrano[3,2-c]quinolin-3-one and (1S,2S,2aS,10aR)-2-(3-
Chloro-4-methoxyphenyl)-4,10,10-trimethyl-1-(1-methylethyl)-
1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]pyrano[3,2-c]-
quinolin-3-one (50). From N-Me-flindersine (2b, 0.5 mmol) and 4i
(1.0 mmol). Purified by flash chromatography on neutral alumina
(DCM, then 1:1 DCM/CHCl3, then CHCl3). The fractions
containing MH+ 452 were combined and purified by reverse-phase
HPLC with isocratic elution MeCN/H2O (3:2) over 60 min to give
1
12.4 mg (5.5%). H NMR (DMSO-d6, 600 MHz) δ 0.68 (d, J = 8.4
3H), 1.45 (m, 4H), 1.52 (s, 3H), 1.62 (m, 2H), 2.01 (m, 1H), 2.41
(ddd, J = 7.5, 9.5 Hz, 1H), 2.48 (dd, J = 8.5 Hz, 1H), 3.21 (s, 3H),
3.28 (s), 3.51 (s, 6H), 3.54 (dd, J = 8.5, 9 Hz, 1H), 3.71 (t, J = 8.5, 9
Hz, 1H), 6.06 (s, 3H), 7.27 (dd, J = 7.5, 8.5 Hz, 1H), 7.40 (d, J = 8.5
Hz, 1H), 7.59 (dd, J = 7.5, 8.5 Hz, 1H), 7.96 (d, J = 8.5 Hz). (+)
LRMS (ESI) m/z 504 [M + H]+. HRMS (ESI): calcd for C31H37NO5
[M + H]+, 504.2745; found, 504.2736.
Hz, 3H), 0.86 (d, J = 8.4 Hz, 3H), 1.15 (s, 3H), 1.49 (s, 3H), 1.52 (m,
1H), 1.81 (m, 1H), 2.30 (m, 1H), 2.51 (m, 1H), 3.20 (s, 3H), 3.55 (t,
J = 9 Hz, 1H), 3.65 (t, J = 9 Hz, 1H), 3.70 (s, 3H), 6.73 (m, 3H), 7.26
(t, J = 7.5 Hz, 1H), 7.38 (d, J = 7.5 Hz, 1H), 7.58 (t, J = 8 Hz, 1H),
7.94 (d, J = 8 Hz). (+) LRMS (ESI) m/z 452 [M + H35Cl]+, 454 [M +
H37Cl]+. HRMS (ESI): calcd for C27H30ClNO3Na [M + Na]+,
474.1806; found, 458.1795.
(1R,2R,2aR,10aS)-1,10-Dimethyl-2-(3,4,5-trimethoxyphenyl)-
1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]pyrano[3,2-c]-
quinolin-3-one and (1S,2S,2aS,10aR)-1,10-Dimethyl-2-(3,4,5-trime-
thoxyphenyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]pyrano-
[3,2-c]quinolin-3-one (55 and 56). From 2e−f (0.5 mmol) and 4a
(1.0 mmol). Purified by gradient elution MeCN/H2O (55:45 for 45
min then gradient elution to 100% MeCN over 5 min) to afford a
white solid (6.3 mg, 4%). 56: 1H NMR (d6 DMSO, 500 MHz) δ 1.08
(1R,2R,2aR,10aS)-2-(3-Acetoxy-4-methoxyphenyl)-4,10,10-tri-
methyl-1-(1-methylethyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta-
[4,5]pyrano[3,2-c]quinolin-3-one and (1S,2S,2aS,10aR)-2-(3-Ace-
toxy-4-methoxyphenyl)-4,10,10-trimethyl-1-(1-methylethyl)-
1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]pyrano[3,2-c]-
quinolin-3-one (51). From N-Me-flindersine (2b, 0.83 mmol) and 4k
(1.76 mmol). Purified by flash chromatography on neutral alumina
(DCM, then 1:1 DCM/CHCl3, then CHCl3). The fractions containing
U
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1
(d, J = 6 Hz, 3H), 1.16 (d, J = 6 Hz, 3H), 2.37 (m, 2H), 3.35 (s, 6H),
3.52 (s, 3H), 3.66 (m, 1H), 3.78 (m, 1H), 4.62 (m, 1H), 6.17 (s, 2H),
7.16 (m, 2H), 7.41 (m, 1H), 7.82 (m, 1H), 11.05 (s, 1H). (+) LRMS
(ESI) m/z 422 [M + H]+. HRMS (ESI): calcd for C25H28NO5 [M +
H]+, 422.1962; found, 422.1976. The next fraction afforded the
MeCN/H2O (1:1) to give an off-white solid (15.6 mg, 8.5%). H
NMR (500 MHz, DMSO-d6) δ 0.63 (t, J = 7.5 Hz, 3H), 1.06 (s, 3H),
1.32 (s, 3H), 1.46 (m, 1H), 1.60 (m, 1H), 2.25 (m, 2H), 3.29 (t, J =
9.5 Hz, 1H), 3.51 (s, 3H), 3.65−3.62 (m, 4H), 5.83 (d, J = 7.5 Hz,
1H), 6.35 (s, 1H), 6.35 (d, J = 8.5 Hz, 1H), 6.48 (d, J = 8.5 Hz, 1H),
7.05 (d, J = 7.5 Hz, 1H), 10.75 (br s, 1H). Using a combination of
HSQC and ROESY, correlations were observed between H-7′ (δ 3.29
ppm) and the methyl of the ethyl group (δ 0.63 ppm), H-3 (δ 2.25
ppm), H-4 (δ 3.50 ppm), and one of the methylene hydrogens (δ 1.46
ppm). LRMS (ESI) m/z 370.4 [M + H]+.
General Procedure: Preparation of Acetals 62−64. To a
solution of flindersine (2a, 1.00 g, 4.4 mmol) and 4-methylmorpholine
N-oxide (1.90 g, 16 mmol) in 2-methyl-2-propanol (100 mL),
tetrahydrofuran (30 mL), and water (10 mL) was added a solution of
2.5% osmium tetroxide in 2-methyl-2propanol (2.45 mL). The
resulting solution was stirred for 2 days. Saturated sodium hydrogen
sulfite (50 mL) was added, and the mixture was extracted with
dichloromethane (3 × 200 mL). The combined organic phase was
dried (MgSO4) and filtered, and the solvent was removed in vacuo.
The residue was purified by flash chromatography using methanol/
chloroform (2:98) to give the flindersine diol 7 as a white solid (1.10
1
alternate isomer (55) (4.5 mg, 3%). 55: H NMR (d6 DMSO, 500
MHz); 1.22 (d, J = 6 Hz, 3H), 1.43 (d, J = 6 Hz, 3H), 2.42 (m, 2H),
3.33 (s, 6H), 3.58 (s, 3H), 3.64 (m, 1H), 3.75 (m, 1H), 4.03 (m, 1H),
6.16 (s, 2H), 7.18 (m, 2H), 7.43 (m, 1H), 7.83 (m, 1H), 11.03 (s,
1H). (+) LRMS (ESI) m/z 422 [M + H]+. HRMS (ESI): calcd for
C27H30NO7Na [M + Na]+, 444.1781; found, 444.1780.
(1R,2R,2aR,10aS)-7-Methoxy-10,10-dimethyl-1-(1-methylethyl)-
2-(3,4-dimethoxyphenyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta-
[4,5]pyrano[3,2-c]quinolin-3-one and (1S,2S,2aS,10aR)-7-Methoxy-
10,10-dimethyl-1-(1-methylethyl)-2-(3,4-dimethoxyphenyl)-
1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]pyrano[3,2-c]-
quinolin-3-one (57). 2d (0.15 mmol) and 4l (0.30 mmol). Purified by
isocratic elution MeCN/H2O (1% TFA, 55:45 for 60 min) to afford a
1
white solid (10.9 mg, 16%). H NMR (CDCl3, 500 MHz) δ 1.12 (s,
H), 1.12 (d, J = 7 Hz, 3H), 1.47 (s, 3H), 2.32 (m, 2H), 3.28 (m, 1H),
3.35 (s, 6H), 3.52 (s, 3H), 3.76 (m, 1H), 3.78 (s, 3H), 6.11 (s, 2H),
7.09 (m, 2H), 7.25 (m, 1H), 10.95 (s, 1H). (+) LRMS (ESI) m/z 466
[M + H]+. HRMS (ESI): calcd for C28H34NO5 [M + H]+, 466.2224;
found, 466.2224.
(1R,2R,2aR,10aS)-7-Methoxy-1,10,10-trimethyl-2-(3,4,5-trime-
thoxyphenyl)-1,2,2a,4,10,10a-hexahydro-3H-cyclobuta[4,5]pyrano-
[3,2-c]quinolin-3-one and (1S,2S,2aS,10aR)-7-Methoxy-1,10,10-tri-
methyl-2-(3,4,5-trimethoxyphenyl)-1,2,2a,4,10,10a-hexahydro-3H-
cyclobuta[4,5]pyrano[3,2-c]quinolin-3-one (58). From 2d (0.19
mmol) and 4a (0.38 mmol). Purified by isocratic elution MeCN/
H2O (1% TFA, 2:3 for 60 min) to afford a white solid (12.7 mg, 14%).
1H NMR (CDCl3, 500 MHz) δ 0.69 (d, J = 6.5 Hz, 3H), 0.85 (d, J =
1
g, 96%). H NMR (CDCl3, 500 MHz) δ 1.37 (s, 3H), 1.46 (s, 3H),
3.67 (dd, J = 4.5, 4.5 Hz, 1H), 4.78 (d, J = 4.5 Hz, 1H), 4.78 (dd J =
1.5, 4.5 Hz, 1H), 5.63 (d, J = 1.5 Hz, 1H), 7.18 (app t, J = 8 Hz, 1H),
7.31 (d, J = 8.5 Hz, 1H), 7.52 (app t, J = 8 Hz, 1H), 7.76 (d, J = 7.5
Hz, 1H), 11.56 (s, 1H). 13C NMR (CDCl3, 125 MHz) δ 23.25, 25.96,
62.71, 70.67, 80.97, 106.78, 115.05, 115.93, 122.29, 123.28, 131.48,
138.54, 156.14, 164.36. (+) LRMS (ESI) m/z 262 [M + H]+. HPLC
(Betasil C18 150 × 4.6 mm, 0−95% acetonitrile (0.1% TFA) in water
(0.1% TFA)/9 min at 1.5 mL/min). tR = 7.040 min.
1-(3,5-Dimethoxyphenyl)butan-1-ol. 3,5-Dimethoxybenzalde-
hyde (2 g, 12 mmol) was dissolved in toluene (30 mL) and cooled
to 0 °C. Propylmagnesium chloride (2.0 M in Et2O, 30 mL, 60 mmol)
was added dropwise, and the reaction mixture was allowed to warm to
rt and stirred for a further 16 h. To this was added 5% AcOH (30 mL),
and the resulting mixture was extracted with EtOAc (80 mL). The
organic phase was then extracted with water (50 mL), saturated
NaHCO3 (50 mL), and brine (50 mL). The organic phase was dried
(MgSO4), filtered, and concentrated to afford 1-(3,5-dimethoxyphenyl)-
butan-1-ol (2.22 g, 88%) as a colorless oil. 1H NMR (DMSO-d6,
500 MHz) δ 0.85 (t, J = 7 Hz, 3H), 1.23 (m, 1H), 1.34 (m, 1H), 1.57
(m, 1H), 1.68 (m, 1H), 3.70 (s, 6H), 4.50 (m, 1H), 6.28 (dd, J = 1.5, 2
Hz, 1H), 6.41 (d, J = 2.5 Hz, 2H). 13C NMR (DMSO-d6, 125 MHz) δ
14.19, 19.27, 41.37, 74.68, 99.58, 104.08, 147.90, 161.10. (+) LRMS
(ESI) m/z 211 [M + H]+.
6.5 Hz, 3H), 1.13 (s, 3H), 1.48 (s, 3H), 1.80 (m, 1H), 2.30 (m, 2H), 3.15
(s, 3H), 3.49 (m, 1H), 3.59 (s, 3H), 3.63 (m, 1H), 3.79 (s, 3H), 6.27 (s,
1H), 6.48 (d, J 8 Hz, 1H), 6.57 (d, J = 8 Hz, 1H), 7.07 (m, 2H), 7.24 (m,
1H), 10.84 (s, 1H). (+) LRMS (ESI) m/z 464 [M + H]+. HRMS (ESI):
calcd for C27H32NO6 [M + H]+, 464.2432; found, 464.2432.
(1R,2R,2aR,8aS)-2-(3,4,5-Trimethoxyphenyl)-1,8,8-trimethyl-
1,2,2a,4,8,8a-hexahydro-3H-cyclobuta[4,5]pyrano[3,2-c]pyridine-3-
one and (1S,2S,2aS,8aR)-2-(3,4,5-Trimethoxyphenyl)-1,8,8-trimeth-
yl-1,2,2a,4,8,8a-hexahydro-3H-cyclobuta[4,5]pyrano[3,2-c]-
pyridine-3-one (59). From 6 (0.088 g, 0.5 mmol) and 4a (0.21 g, 1
mmol). Purified by reverse-phase HPLC with isocratic elution MeCN/
H2O (1:1) to give 12.8 mg (6.6%) of an off-white solid. 1H NMR (500
MHz, DMSO-d6) δ 1.06 (s, 3H), 1.09 (s, 3H), 1.33 (s, 3H), 2.18 (m,
1H), 2.21 (m, 1H), 3.17 (t, J = 8.5 Hz, 1H), 3.75−3.50 (m, 10H), 5.83
(d, J = 7 Hz, 1H), 6.16 (apparent s, 2H), 7.07 (d, 1H, J = 7 Hz), 10.75
(bs, 1H). Using a combination of HSQC and ROESY, correlations
were observed between the methyl group on the cyclobutane ring (δ
1.07 ppm) and H-3 (δ 2.18 ppm) and H-7′ (δ 3.17 ppm) and between
H-7′ and H-3 and H-4 (δ 3.50 ppm). LRMS (ESI) m/z 386.4 [M + H]+.
(1R,2R,2aR,8aS)-1-Ethyl-2-(3,4,5-trimethoxyphenyl)-8,8-dimeth-
yl-1,2,2a,4,8,8a-hexahydro-3H-cyclobuta[4,5]pyrano[3,2-c]-
pyridine-3-one and (1S,2S,2aS,8aR)-1-Ethyl-2-(3,4,5-trimethoxy-
phenyl)-8,8-dimethyl-1,2,2a,4,8,8a-hexahydro-3H-cyclobuta[4,5]-
pyrano[3,2-c]pyridine-3-one (60). From 6 (0.088 g, 0.5 mmol) and 4f
(0.21 g, 1 mmol). Purified by reverse-phase HPLC with isocratic
elution MeCN/H2O (1:1) to give (22 mg, 11%). 1H NMR (500 MHz,
DMSO-d6) δ 0.66 (t, J = 7.5 Hz, 3H), 1.07 (s, 3H), 1.32 (s, 3H), 1.45
(m, 1H), 1.60 (m, 1H), 2.20 (m, 1H), 2.26 (m, 1H), 2.19 (m, 2H),
3.30 (t, J = 9 Hz, 1H), 3.77−3.59 (m, 10H), 5.83 (t, J = 5.5 Hz, 1H),
6.16 (apparent s, 2H), 7.06 (d, J = 6.5 Hz, 1H), 10.75 (bs, 1H). Using
a combination of HSQC and ROESY, correlations were observed
between one of the methylene hydrogens (δ 1.45 ppm) and H-3 (δ
2.26 ppm) and between H-3 and H-4 (δ 3.50 ppm). LRMS (ESI) m/z
400.4 [M + H]+.
1-(3,5-Dimethoxyphenyl)butan-1-one. A solution of 1-(3,5-
dimethoxyphenyl)butan-1-ol (2.22 g, 10.6 mmol) and manganese(IV)
oxide (14.59 g, 176 mmol) in dichloromethane (20 mL) was stirred
for 18 h. The reaction mixture was then filtered through a plug of
silica, the residue was washed with chloroform, and the solvent was
removed from the combined filtrate in vacuo. The residue was purified
by flash chromatography using ethyl acetate/hexane (1:9) to give 1-
1
(3,5-dimethoxyphenyl)butan-1-one (1.74 g, 79%). H NMR (DMSO-
d6, 500 MHz) δ 0.94 (t, J = 7 Hz, 3H), 1.63 (app hx, J = 7 Hz, 2H),
2.98 (t, J = 7 Hz, 2H), 3.82 (s, 6H), 6.76 (app t, J = 2 Hz, 1H), 7.08
(d, J = 2 Hz, 2H). 13C NMR (DMSO-d6, 125 MHz) δ 14.29, 17.98,
40.56, 56.20, 105.55, 106.35, 139.63, 161.32, 200.38. (+) LRMS (ESI)
m/z 209 [M + H]+.
2-Butyl-2-(3,5-dimethoxyphenyl)-11,11-dimethyl-3a,5,11,11a-tet-
rahydro-1,3-dioxolo[4,5]pyrano[3,2-c]quinolin-4-one (62). To a
solution of 1-(3,5-dimethoxyphenyl)butan-1-one (790 mg, 3.79
mmol) in methanol (4 mL) were added trimethyl orthoformate
(2.00 mL, 18.28 mmol) and concentrated sulfuric acid (1 drop), and
the resulting mixture was stirred under nitrogen for 18 h. Saturated
sodium hydrogen carbonate (20 mL) and dichloromethane (50 mL)
were then added, and the resulting mixture was filtered through a 1PS
filter paper. The solvent was then removed from the organic phase in
vacuo to give acetal 8a. Without purification, 8a was dissolved in
dichloromethane (2 mL), and 7 (100 mg, 0.38 mmol) and pyridinium
p-toulenesulfonate (220 mg) were added. The resulting solution was
(1R,2R,2aR,8aS)-1-Ethyl-2-(3,4-dimethoxyphenyl)-8,8-dimethyl-
1,2,2a,4,8,8a-hexahydro-3H-cyclobuta[4,5]pyrano[3,2-c]pyridine-3-
one and (1S,2S,2aS,8aR)-1-Ethyl-2-(3,4-dimethoxyphenyl)-8,8-di-
methyl-1,2,2a,4,8,8a-hexahydro-3H-cyclobuta[4,5]pyrano[3,2-c]-
pyridine-3-one (61). From 6 (0.088 g, 0.5 mmol) and 4e (0.21 g,
1 mmol). Purified by reverse-phase HPLC with isocratic elution
V
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allowed to stir for 18 h, after which saturated sodium hydrogen
carbonate (20 mL) and dichloromethane (50 mL) were added. The
mixture was filtered through a 1PS filter paper, and the solvent was
removed from the organic phase in vacuo. The residue was purified by
5% CO2. LPS (Escherichia coli 0111:B4 (Ultrapure, Invivogen) was
used as a positive control. The SEAP released was quantified using p-
nitrophenyl phosphate as a substrate, and the absorbance at 405 nm
was measured at 30 s intervals over several minutes using a microplate
reader. Data is expressed as Vmax calculated over the linear portion of
the kinetic absorbance measurements.
Transient Transfections with Human CD14 and MD-2. TLR4-
expressing HEK293 cells (stably transfected with human TLR4
(pUNO expression plasmid) plus pNiFty2-SEAP reporter plasmid
were transiently transfected using either pUNO-human MD-2, pUNO-
human CD14, pDUO-CD14/MD-2, or pUNO-(empty) and then
seeded in tissue culture-treated clear flat-bottom polystyrene 96-well
plates (Costar, 3598) at 10 000 cells per well in 100 μL of media
(DMEM, 10% FCS, and 2 mM L-glutamine). Cells were incubated
overnight at 37 °C/5% CO2 in a humidified incubator. Compound
treatments and SEAP assay were performed as above.
1
flash chromatography using chloroform to give 62 (86 mg, 50%). H
NMR (DMSO-d6, 500 MHz) δ 0.86 (t, J = 7 Hz, 3H), 1.21 (s, 3H),
1.32 (m, 2H), 1.66 (s, 3H), 1.75 (m, 2H), 3.51 (s, 6H), 4.40 (d, J = 5.5
Hz, 1H), 5.36 (d, J = 5.5 Hz, 1H), 5.82 (s, 1H), 6.38 (s, 2H), 7.01
(app t, J = 7.5 Hz, 1H), 7.09 (d, J = 8.5 Hz, 1H), 7.39 (app t, J = 7.5
Hz, 1H), 7.49 (d, J = 7.5 Hz, 1H), 11.18 (s, 1H). 13C NMR (DMSO-
d6, 125 MHz) δ 14.64, 17.19, 21.94, 25.85, 44.05, 55.43, 68.57, 78.47,
79.33, 99.16, 103.29, 105.55, 110.92, 114.37, 115.26, 121.40, 122.91,
131.21, 138.95, 147.15, 156.49, 159.88, 162.48. ROESY correlations
were observed between the 11a methine H (δ 4.40 ppm) and the 3a
methine H (δ 5.36 ppm) and between the 11a methine H (δ 4.40
ppm) and the methylene hydrogens of the butyl group (δ 1.75 and
1.32 ppm), confirming the cis relationship of the dimethoxyphenyl and
the pyranoquinolinone ring systems. (+) LRMS (ESI) m/z 474 [M +
Na]+. HRMS (ESI): calcd for C26H30NO6 [M + H]+, 452.2068; found,
452.2069.
Transient Transfections with Different Combinations of Human
and Mouse TLR4, CD14, and MD-2. HEK293 cells stably transfected
with pNiFty2-SEAP reporter plasmid were transiently transfected with
either human or mouse TLR4 (pUNO expression vector) in
combination with either human or mouse CD14/MD-2 (pDUO
expression vector) and then seeded in tissue culture-treated clear flat-
bottom polystyrene 96-well plates (Costar, 3598) at 30 000 cells per
well in 100 μL of media (DMEM, 10% FCS, and 2 mM L-glutamine).
Cells were incubated overnight at 37 °C/5% CO2 in a humidified
incubator. Compound treatments and SEAP assay were performed as
above. Results are expressed as the percent of the maximal response to
TNF-α in the same experiment.
Testing Human/Mouse Chimeric TLR4 Receptors. Chimeric TLR4
constructs were made that expressed the human TLR4 extracellular
domain fused to the mouse transmembrane and cytoplasmic domains
or the mouse TLR4 extracellular domain fused to the human
transmembrane and cytoplasmic domains. Site-directed mutagenesis of
human and mouse TLR4 was performed to introduce a PciI restriction
site at a conserved position in the coding region for the extracellular
domain, close to the predicted transmembrane domain, without affecting
the amino acid sequence. Constructs encoding the chimeric receptors
were generated in the pUNO expression vector using this restriction site
to join the coding sequences for the human and mouse domains.
HEK293 cells stably transfected with human CD14/MD-2 (pDUO
expression vector) and the pNiFty2-SEAP reporter plasmid were
transiently transfected with either human, mouse, human/mouse, or
mouse/human TLR4 construct and then seeded in tissue culture-
treated clear flat-bottom polystyrene 96-well plates (Costar, 3598) at
30 000 cells per well in 100 μL of media (as described above). Cells
were incubated overnight at 37 °C/5% CO2 in a humidified incubator.
Compound treatments and SEAP assay were performed as above.
TLR Selectivity Testing. HEK293 cells stably expressing pNiFty2-
SEAP reporter plasmid and a pUNO or pDUO expression plasmid
containing either human TLR2, 2/6, 3, 5, 7 or 8 were maintained as
above for the human TLR4/CD14/MD-2 cells. Cells were seeded in
tissue culture-treated clear polystyrene 384-well plates at 7500 cells per
well. Compound treatment was performed as above. The SEAP
released was quantified using DDAO phosphate as a substrate, and
fluorescence was measured at an excitation of 620 nm and emission of
665 nm in a PerkinElmer Envision spectrophotometer. Positive control
compounds for each cell line are given in Table 7.
2-Butyl-2-(3,4-dimethoxyphenyl)-11,11-dimethyl-3a,5,11,11a-tet-
rahydro-1,3-dioxolo[4,5]pyrano[3,2-c]quinolin-4-one (63). From 1-
(3,4-dimethoxyphenyl)butan-1-one (790 mg, 3.79 mmol) and 7 (100
1
mg, 0.38 mmol) was obtained 63 (171 mg, 99%). H NMR (DMSO-
d6, 500 MHz) δ 0.85 (t, J = 7 Hz, 3H), 1.20 (s, 3H), 1.32 (m, 2H),
1.65 (s, 3H), 1.78 (m, 2H), 3.37 (s, 3H), 3.57 (s, 3H), 4.40 (d, J = 6.5
Hz, 1H), 5.35 (d, J = 6.5 Hz, 1H), 6.46 (d, J = 8.5 Hz, 1H), 6.75
(dd, J = 8.5, 1.5 Hz, 1H), 6.80 (d, J = 1.5 Hz, 1H), 7.01 (app t, J = 7.5
Hz, 1H), 7.09 (d, J = 8.5 Hz, 1H), 7.37 (app t, J = 8 Hz, 1H), 7.50 (d, J
= 8 Hz, 1H), 11.18 (s, 1H). 13C NMR (DMSO-d6, 125 MHz) δ 14.66,
17.35, 22.04, 25.78, 44.17, 55.71, 56.18, 68.50, 78.61, 79.25, 105.82,
109.40, 111.04, 111.82, 114.47, 115.32, 117.46, 121.51, 122.90, 131.18,
137.50, 138.86, 148.12, 148.19, 156.34, 162.47. ROESY correlations
were observed between the 11a methine H (δ 4.40 ppm) and the 3a
methine H (δ 5.35 ppm) and between the 11a methine H (δ 4.40
ppm) and the methylene hydrogens of the butyl group (δ 1.78 and
1.32 ppm), confirming the cis relationship of the dimethoxyaryl and
the pyranoquinolinone ring systems. (+) LRMS (ESI) m/z 474
[M + Na]+. HRMS (ESI): calcd for C26H30NO6 [M + H]+, 452.2068;
found, 452.2072.
2-Butyl-2-(3,4,5-trimethoxyphenyl)-11,11-dimethyl-3a,5,11,11a-
tetrahydro-1,3-dioxolo[4,5]pyrano[3,2-c]quinolin-4-one (64). From
1-(3,4,5-trimethoxyphenyl)butan-1-one (238 mg, 3.29 mmol) and 7
(100 mg, 0.38 mmol) was obtained 64 (56 mg, 30%). 1H NMR
(DMSO-d6, 500 MHz) δ 0.87 (t, J = 7.5 Hz, 3H), 1.19 (s, 3H), 1.37
(m, 2H), 1.70 (s, 3H), 1.74 (m, 2H), 2.96 (s, 3H), 3.63 (s, 6H), 4.39
(d, J = 6.5 Hz, 1H), 5.36 (d, J = 6.5 Hz, 1H), 6.51 (s, 2H), 6.99 (app t,
J = 7.5 Hz, 1H), 7.05 (d, J = 8 Hz, 1H), 7.33 (app t, J = 7.5 Hz, 1H),
7.47 (d, J = 8 Hz, 1H), 11.16 (s, 1H). 13C NMR (DMSO-d6, 125
MHz) δ 19.39, 21.83, 26.67, 30.68, 48.99, 60.86, 64.71, 73.29, 83.36,
84.34, 107.40, 110.49, 115.62, 119.12, 120.04, 126.25, 127.44, 135.84,
141.39, 143.64, 145.63, 156.75, 161.22, 167.13. ROESY correlations
were observed between the 11a methine H (δ 4.39 ppm) and the 3a
methine H (δ 5.36 ppm) and between the 11a methine H (δ 4.39 ppm)
and H-8′ (methylene hydrogens of the butyl group, δ 1.37 ppm),
confirming the cis relationship of the trimethoxyaryl and the
pyranoquinolinone ring systems. (+) LRMS (ESI) m/z 482 [M + H]+,
504 [M + Na]+. HRMS (ESI): calcd for C27H32NO7 [M + H]+,
482.2173; found, 482.2168.
Table 7
Biological Assays. Human TLR4 Reporter Assays. Stable TLR4/
CD14/MD-2 Transfectants. HEK293-human TLR4/CD14/MD-2
cells, stably transfected with human TLR4 (pUNO expression vector),
CD14, MD-2 (pDUO expression vector), and pNiFty2-SEAP reporter
plasmid were maintained in Dulbecco’s modified Eagle’s medium
supplemented with 10% FCS (v/v), 2 mM L-glutamine, nonessential
amino acids, 10 μg/mL blasticidin S, 50 μg/mL hygromycin, and
100 μg/mL zeocin. Cells were seeded in tissue culture-treated clear
flat-bottom polystyrene 96- or 384-well plates (Costar, 3598) at 1 ×
104 cells/well. Dose−response curves were generated by addition of
test compounds and incubation for 20 h at 37 °C in an atmosphere of
TLR2, 2/6
TLR3
Pam3cysk4 (Invivogen, France)
Poly(I:C) (Invivogen, France)
Flagellin (Invivogen, France)
Ultrapure LPS (Invivogen, France)
R848 (AstraZeneca, UK)
TLR5
TLR4
TLR7 and 8
Human PBMC Preparation. Blood from healthy, consenting
volunteers was collected into heparin, layered onto Lymphoprep
(Axis-Shield Diagnostics), and centrifuged at 700g for 25 min. The
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dx.doi.org/10.1021/jm401321v | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
PBMC layer was removed, diluted to 50 mL with PBS, and centrifuged
at 400g for 10 min. The supernatant was removed, and the pellet was
resuspended in 50 mL of PBS and centrifuged at 300g for 5 min. The
cells were then washed in 50 mL of PBS and recovered by centrifuging
at 200g for 5 min. PBMCs were resuspended in assay medium (RPMI
1640 with 25 mM HEPES, 10% FCS (v/v), 2 mM L-glutamine,
10 U/mL penicillin, and 10 μg/mL streptomycin).
PBMC Incubations. Twenty microliters of test compound or assay
medium with 1% DMSO (v/v), vehicle control, were added to each
well of a 96-well polystyrene tissue culture-treated clear flat-bottom
plate (Costar 3598) followed by 180 μL of PBMC cell suspension
(prepared as above) in assay medium (200 000 cells). PBMCs and
compounds were incubated at 37 °C in an atmosphere of air/CO2
(95:5 v/v) for 20 h for cytokine stimulation and 44 h for IL-5
suppression studies (see below).
Cytokine Determinations. Cytokines were determined from PBMC
supernatants using the following commercial kits (OptEIA kits, BD,
Oxford, UK) following the manufacturer’s instructions: human IL-5
(555202), human TNF-α (555212), human IL-12p40 (555171),
human IL-8 (555244), and human IL-10 (555157).
Human PBMC IL-5 Suppression Assay. Human PBMCs were
prepared and plated out with compounds as described above.
Phytohemeagglutinin (PHA) (Sigma, Dorset, UK) was added at a
final concentration of 1 μg/mL, and the PBMCs were then incubated
for 44 h before the supernatant was removed for determination of the
amount of IL-5 produced.
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ASSOCIATED CONTENT
* Supporting Information
■
S
Representative NMR data for the 10 most active compounds from
the cyclobutane series (compounds 1, 20, 22, 26, 31, 32, 34, 35,
52, and 53) and two of the acetal analogues (compounds 63 and
64). Selectivity data for euodenine A, LPS, and the positive controls
across the TLR lines. This material is available free of charge via the
Internet at http://pubs.acs.org.
(13) Peri, F.; Piazza, M.; Calabrese, V.; Damore, G.; Cighetti, R.
Exploring the LPS/TLR4 signal pathway with small molecules.
Biochem. Soc. Trans. 2010, 38, 1390−1395.
(14) Zhang, S.; Cheng, K.; Wang, X.; Yin, H. Selection, synthesis, and
anti-inflammatory evaluation of the arylidene malonate derivatives as
TLR4 signaling inhibitors. Bioorg. Med. Chem. 2012, 20, 6073−6079.
(15) Nakashima, K.; Oyama, M.; Ito, T.; Akao, Y.; Witono, J. R.;
Darnaedi, D.; Tanaka, T.; Murata, J.; Iinuma, M. Novel quinolinone
alkaloids bearing a lignoid moiety and related constituents in the leaves
of Melicope denhamii. Tetrahedron 2012, 68, 2421−2428.
(16) Teague, S. J. Unpublished results. 2006.
(17) Bowman, R. M.; Grundon, M. F.; James, K. J. Quinoline
alkaloids. Part XVI. 2,2-Dimethylpyranoquinolines from base-catalysed
rearrangement of isoprenyl epoxides. Synthesis and biogenesis of
flindersine. J. Chem. Soc., Perkin Trans. 1 1973, 1055−1059.
(18) Jones, K.; Roset, X.; Rossiter, S.; Whitfield, P. Demethylation of
2,4-dimethoxyquinolines: the synthesis of atanine. Org. Biomol. Chem.
2003, 1, 4380−4383.
AUTHOR INFORMATION
Corresponding Author
*Phone: +61 7 37356009. Fax: +61 7 37356001. E-mail:
R.Quinn@griffith.edu.au.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We thank Hoan T. Vu for HRMS measurements and Mark
Furber for assistance in the preparation of this manuscript.
■
(19) Holla, H.; Jenkins, I. D.; Neve, J. E.; Pouwer, R. H.; Pham, N.;
Teague, S. J.; Quinn, R. J. Synthesis of melicodenines C, D and E.
Tetrahedron Lett. 2012, 53, 7101−7103.
(20) Sharma, A.; Joshi, B. P.; Sinha, A. K. An effective system to
synthesize hypolipidemic active alpha-asarone and related methoxy-
lated (E)-arylalkenes. Bull. Chem. Soc. Jpn. 2004, 77, 2231−2235.
(21) Liu, J. T.; Jang, Y. J.; Shih, Y. K.; Hu, S. R.; Chu, C. M.; Yao, C.
F. Novel synthesis of alkenes via triethylborane-induced free-radical
reactions of alkyl iodides and beta-nitrostyrenes. J. Org. Chem. 2001,
66, 6021−6028.
(22) Wang, X.; Lee, Y. R. Efficient synthesis of substituted
pyranoquinolinones from 2,4-dihydroxyquinoline: Total synthesis of
zanthosimuline, cis-3′,4′-dihydroxy-3′,4′-dihydroflindersine, and orix-
alone D. Synthesis 2007, 3044−3050.
ABBREVIATIONS USED
■
CD14, cluster of differentiation 14; MD-2, MD-2 is a 160-
amino acid protein that is associated with TLR4 on the cell
surface and enables TLR4 to respond to LPS; PBMC,
peripheral blood mononuclear cells; SEAP, secreted embryonic
alkaline phosphatase
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