Design, synthesis, and biological evaluation of novel deguelin-based heat shock protein 90 (HSP90) inhibitors targeting proliferation and angiogenesis

Article abstract of DOI:10.1021/jm301488q

Deguelin exhibits potent apoptotic and antiangiogenic activities in a variety of transformed cells and cancer cells. Deguelin also exhibits potent tumor suppressive effects in xenograft tumor models for many human cancers. Our initial studies confirmed that deguelin disrupts ATP binding to HSP90 and consequently induces destabilization of its client proteins such as HIF-1α. Interestingly, a fluorescence probe assay revealed that deguelin and its analogues do not compete with ATP binding to the N-terminus of HSP90, unlike most HSP90 inhibitors. To determine the key parts of deguelin that contribute to its potent HSP90 inhibition, as well as its antiproliferative and antiangiogenic activities, we have established a structure-activity relationship (SAR) of deguelin. In the course of these studies, we identified a series of novel and potent HSP90 inhibitors. In particular, analogues 54 and 69, the B- and C-ring-truncated compounds, exhibited excellent antiproliferative activities with IC₅₀ of 140 and 490 nM in the H1299 cell line, respectively, and antiangiogenic activities in zebrafish embryos in a dose dependent manner (0.25-1.25 μM).

Full text of DOI:10.1021/jm301488q

Article
pubs.acs.org/jmc
Design, Synthesis, and Biological Evaluation of Novel Deguelin-
Based Heat Shock Protein 90 (HSP90) Inhibitors Targeting
Proliferation and Angiogenesis
Dong-Jo Chang,† Hongchan An,† Kyoung-suk Kim,† Hyun Ho Kim,† Jinkyung Jung,† Jung Min Lee,†
Nam-Jung Kim,† Young Taek Han,† Hwayoung Yun,† Sujin Lee,† Geumwoo Lee,† Seungbeom Lee,†
Ju Sung Lee,† Jong-Ho Cha,† Ji-Hyeon Park,† Ji Won Park,† Su-Chan Lee,† Sang Geon Kim,†
Jeong Hun Kim,‡,§ Ho-Young Lee,† Kyu-Won Kim,† and Young-Ger Suh*,†
†College of Pharmacy, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 151-742, Korea
‡Fight against Angiogenesis-Related Blindness (FARB) Laboratory, Clinical Research Institute, Seoul National University Hospital,
Seoul 110-744, Korea
§Department of Ophthalmology, College of Medicine, Seoul National University, Seoul 110-799, Korea
S
* Supporting Information
ABSTRACT: Deguelin exhibits potent apoptotic and antiangiogenic activities in a variety of
transformed cells and cancer cells. Deguelin also exhibits potent tumor suppressive effects in xenograft
tumor models for many human cancers. Our initial studies confirmed that deguelin disrupts ATP
binding to HSP90 and consequently induces destabilization of its client proteins such as HIF-1α.
Interestingly, a fluorescence probe assay revealed that deguelin and its analogues do not compete with
ATP binding to the N-terminus of HSP90, unlike most HSP90 inhibitors. To determine the key parts of deguelin that contribute
to its potent HSP90 inhibition, as well as its antiproliferative and antiangiogenic activities, we have established a structure−
activity relationship (SAR) of deguelin. In the course of these studies, we identified a series of novel and potent HSP90 inhibitors.
In particular, analogues 54 and 69, the B- and C-ring-truncated compounds, exhibited excellent antiproliferative activities with
IC50 of 140 and 490 nM in the H1299 cell line, respectively, and antiangiogenic activities in zebrafish embryos in a dose
dependent manner (0.25−1.25 μM).
1α, a subunit of HIF-1 with constitutively expressed HIF-1β, is
an oxygen-labile transcriptional factor that promotes angio-
genesis at positions of vascular disruption and dysfunction by
inducing the expression of vascular endothelial growth factor
(VEGF), an angiogenesis-regulatory protein. Angiogenesis is
the process of the formation of new blood vessels and is
necessary for the repair, reproduction, and development of
damaged blood vessels or metabolically active new tissues.
However, pathological angiogenesis not only plays an
important role in the growth and spread of tumors by their
metastases but also induces bleeding, vascular leakage, and
tissue destruction by abnormally rapid neovascularization.5 As a
result, pathological angiogenesis can lead to diverse angio-
genesis-dependent diseases, including diabetic retinopathy
(DR), age-related macular degeneration (AMD), and auto-
immune diseases, as well as cancer.6 Thus, HSP90 inhibition
can be considered as an effective and novel therapy against
angiogenesis-associated diseases, including cancer, with HSP90
inhibitors representing potentially promising chemotherapeutic
agents for angiogenesis-dependent diseases.
INTRODUCTION
■
Molecular chaperones, such as heat-shock proteins (HSPs), are
ubiquitous proteins that make use of ATP-dependent
conformational changes to regulate the folding of client
proteins for the purpose of activating nascent proteins,
refolding of damaged proteins, or degrading severely damaged
proteins.1 In addition, chaperone proteins can prevent client
protein aggregation and help membrane translocation of the
client protein for intracellular deposition. HSP90 is a heat
shock protein, and its molecular chaperone function is required
for the stability and activation of numerous client proteins
related to signal transduction in the cell.2 Under standard,
nonstress conditions, the amount of HSP90 comprises 1−2% of
total cellular proteins, but under stress, levels of HSP90
increase about 2-fold. Oncogenic mutations of client proteins
require increased HSP90 activity and consequently lead to the
overexpression of HSP90, a phenotype that is common in
human cancer.3 HSP90 client proteins include ErbB2, Src, Met
tyrosine kinases, mitogen-activated protein kinase kinase (MEK
1/2), Akt, Raf-1, cyclin-dependent serine kinases, steroid
hormone receptors, telomerase, metalloprotein-2 (MMP-2),
and hypoxia-inducible factor-1α (HIF-1α), all of which exist in
various signaling pathways for the survival, proliferation,
invasion, metastasis, and angiogenesis of cells and are known
to contribute to the malignant phenotype.4 Specifically, HIF-
HSP90 predominantly exists as a homodimer and is
composed of three principal domains; N-terminal, middle,
Received: June 19, 2012
© XXXX American Chemical Society
A
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Figure 1. Chemical structures of deguelin (1) and other reported HSP90 inhibitors.
a
Scheme 1. Synthesis of Intermediate 8 and Deguelin
a
Reagents and conditions: (a) BBr3, CH2Cl2, −10 °C; (b) NaBH3CN, HMPA, 70 °C, 50% for two steps; (c) (i) PhSeCl, CH2Cl2, −30 °C, (ii)
H2O2, THF, 0 °C, 61%.
and C-terminal domains.7 The N-terminal domain has an
adenine nucleotide-binding pocket that contains an unusual
structural motif known as the Bergerat fold, making this ATP-
binding domain different from the ATP-binding pockets of
chaperone HSP70 or other kinases.8 This structural singularity
suggests the possibility of discovering highly selective HSP90
inhibitors. Most HSP90 inhibitors, including geldanamycin
(2),9 the less toxic analogue 17-allylamino-17-demethoxygelda-
namycin (17-AAG),10 radicicol (3),11 the more stable oxime
analogues,12 and the synthetic compound PU3 (4),13 (Figure
1) are known to interact with the ATP-binding pocket in the
with consequent tumor growth reduction in xenograft models
of various human cancers, such as lung, prostate, head and
neck, and stomach cancers.19 In addition, we proposed that
deguelin binds to the N-terminal ATP-binding pocket of
HSP90 using a structure−function analysis with a docking
study of deguelin and HSP90.19,20 However, our recent work
has revealed no activity of deguelin and its analogues in a
fluorescence polarization assay (FP assay)21 using VER-
00045864,22 which is a fluorescence probe that binds to the
ATP-binding pocket in the N-terminal domain of HSP90. This
result suggested that deguelin does not bind to the ATP-
binding pocket of the N-terminal domain of HSP90 but might
bind to the other binding site in HSP90, distinguishing it from
other HSP90 inhibitors. Recently, Hieronymus and Brandt
reported that the HSP90 inhibitor gedunin showed unique
HSP90 modulatory attributes.23 Although the effective bio-
logical activities of deguelin as an HSP90 inhibitor have been
evaluated by several biological methods, details of the deguelin-
binding site in HSP90 remain undetermined.
In this article, we describe the design, synthesis, and
biological evaluation of a novel series of synthetic HSP90
inhibitors based on the structure of deguelin to establish the
structure−activity relationships between deguelin and HSP90
and to identify the congener responsible for the excellent
biological activities of deguelin. Our effort also involves the
identification of HSP90 inhibitors consisting of novel and
simplified scaffolds based on the elucidated congener. Initially,
24 analogues were prepared from deguelin by semisynthetic
procedures to elucidate the essential skeleton and function-
alities for potent HSP90 inhibition. Additionally, nine HSP90
N-terminal domain of HSP90, whereas novobiocin (5),14
a
natural antibiotic known as a DNA gyrase B inhibitor, has
shown effective inhibitory activity by interacting with the ATP-
binding pocket in the C-terminal domain of HSP90. These
HSP90 inhibitors showed prominent chemopreventive effects
against a diverse array of cancer cells in preclinical models by
accelerating the degradation of numerous oncogenic HSP90
client proteins; a few HSP90 inhibitors, including 17-AAG,
have entered phase II clinical trials. However, several clinical
trials of HSP90 inhibitors have revealed various negative side
effects with constitutional clinical symptoms.15
Deguelin (1), a rotenoid isolated from the African plant
Mundulea sericea (Leguminosae),16 has been reported to
prevent tobacco carcinogen-induced lung carcinogenesis by
blocking Akt activation17 and has shown potent apoptotic and
antiangiogenic activities against diverse transformed cells and
cancer cells in vitro.18 We recently reported that deguelin
interferes with the chaperone function of HSP90 by inhibiting
ATP binding, thereby inducing the destabilization of HIF-1α
B
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a
Scheme 2. Synthesis of the 9,10-Demethylated Analogues and α,β-Unsaturated Deguelin
a
Reagents and conditions: (a) BBr3, CH2Cl2, −78 to 0 °C, 16% for 9, 14% for 10, 33% for 11; (b) I2, NaOAc, EtOH, reflux, 35%.
a
Scheme 3. Synthesis of the C7-Modified Deguelin Analogues
a
Reagents and conditions: (a) NaBH4, MeOH, 0 °C, 100%. (b) R1 = Me: CH3I, t-BuOK, THF, 0 °C, 100%. R1 = Et: EtI, t-BuOK, THF, 0 °C, 71%.
R1 = n-Pr: 1-PrI, t-BuOK, THF, 0 °C, 68%. R1 = Bn: BnBr, t-BuOK, THF, 0 °C, 88%. R1 = THP: PPTS, DHP, CH2Cl2, rt, 50%. R1 = Ac: Ac2O,
DMAP, Et3N, CH2Cl2, rt, 82%. R1 = CONH2: CCl3CONCO, CH2Cl2, 0 °C, then MeOH/water, 67%. (c) (i) CH3I, CS2, NaH, THF, 0 °C; (ii)
Bu3SnH, AIBN, toluene, reflux, 49%; (d) AcOH, 100 °C, 70%; (e) NH2OH·HCl, pyridine, 70 °C, 100%; (f) R2 = Me: CH3I, t-BuOK, THF, 0 °C,
57%. R2 = Bn: BnBr, t-BuOK, THF, 0 °C, 33%.
inhibitors with a novel scaffold, designed on the basis of the
ring-truncation strategy, were synthesized via concise and
versatile synthetic procedures developed to produce a variety of
ring-truncated analogues. The synthesized compounds were
evaluated by the cell growth inhibition assay (MTS assay)
against H1299 non-small-cell lung cancer (NSCLC) and by
Western blot analysis of the HIF-1α protein, which is not only
an important client protein of HSP90 but also a VEGF-
regulatory transcriptional factor that induces angiogenesis.
Antiangiogenic activities of the analogues possessing a potent
HSP90 inhibitory activity in vitro were further evaluated using a
zebrafish angiogenesis model.
inhibition. The synthetic procedures are shown in Schemes
1−4.
Syntheses of the C9 and/or C10-demethylated analogues 9,
10, and 11 are described in Scheme 2. Interestingly, treatment
of deguelin with BBr3 at −78 °C followed by warming to 0 °C
afforded the desired analogues 9, 10, and 11 in a ratio of 1:1:2,
whereas warming the reaction mixture to ambient temperature
resulted in the production of catechol 11 as the major product.
Iodination of deguelin and in situ treatment with NaOAc
produced the α,β-unsaturated ketone 12.
The C7-modified analogues were synthesized as outlined in
Scheme 3. Reduction of deguelin with NaBH4 produced the
secondary alcohol 14 with a quantitative yield. Subsequent
alkylation with various alkyl halides or acid-catalyzed THP
protection of alcohol 14 afforded the corresponding alkyl ethers
(15−19). Acetylation of 14 with Ac2O in the presence of
DMAP and Et3N produced acetate 20. Treatment of 14 with
trichloroacetyl isocyanate followed by hydrolysis using a
mixture of methanol and water afforded the desired carbamate
21.
The Barton-McCombie radical reaction of 14 generated the
deoxygenated product 13.27 Dehydration of 14 in boiling acetic
acid produced styrene 22. Treatment of deguelin with
hydroxylamine in pyridine yielded oxime 23, which was
subsequently reacted with the appropriate alkyl halides to
produce the oxime ethers 24 and 25.
CHEMISTRY
■
Deguelin was prepared by a known procedure24 from a
commercially available natural product, rotenone (6), and the
structure of the synthesized deguelin was confirmed by
comparison of the spectral data to those of natural deguelin.25
Enantiomeric purity of the synthetic deguelin was confirmed by
HPLC and optical rotation. Most of the deguelin analogues
were synthesized from deguelin or intermediate 8, shown in
Scheme 1, while the ring-truncated analogues were prepared by
the procedures developed by us.26 We first synthesized the
deguelin analogues to determine the role that each fragment
and the overall conformation of deguelin play in HSP90
C
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a
Scheme 4. Synthesis of the Terminal Pyran Ring-Modified Analogues
a
Reagents and conditions. (a) R1 = H: PTSA, benzene, reflux, 99%; (b) (i) PhSeCl, CH2Cl2, −30 °C, (ii) H2O2, THF, 0 °C, 61%. (c) R1 = OH:
OsO4, NMO, acetone/water (4:1), 0 °C to rt, 47%. (d) R2 = Me: CH3I, Cs2CO3, CH3CN, 0 °C, 41%. R2 = allyl: allyl iodide, Cs2CO3, CH3CN, 0 °C,
39%. R2 = Bn: BnBr, Cs2CO3, CH3CN, 0 °C, 25%. R2 = COCH3: Ac2O, DMAP, CH2Cl2, rt, 44%.
Figure 2. Ring-truncation strategy for the new HSP90 inhibitors.
a
Scheme 5. Synthesis of 6-Bromo-5-methoxy-2,2-dimethyl-2H-chromene (38)
a
Reagents and conditions: (a) HNO3, AcOH, CHCl3, rt, 55%; (b) 2-methyl-3-butyn-2-ol, DBU, TFAA, CuCl2, CH3CN, 0 °C, 45%; (c) N,N-
diethylaniline, 130 °C, 91%; (d) CH3I, K2CO3, acetone, 60 °C; (e) SnCl2·2H2O, EtOH, reflux, 95% for two steps; (f) NaNO2, HBr, CuBr, water, 0
to 65 °C, 88%.
Syntheses of the terminal benzopyran-modified analogues
26−31 are outlined in Scheme 4. Acid-catalyzed cyclization of
intermediate 8 produced dihydrobenzopyran 26. Dihydrox-
ylation of deguelin with OsO4 in the presence of N-
methylmorpholine N-oxide (NMO) produced the desired
diol 27. Treatment of intermediate 8 with Cs2CO3 followed
by addition of the requisite alkyl halides at 0 °C gave the
corresponding aryl ethers (28−30). Synthesis of acetate 31
from phenol 8 was accomplished by acetylation with Ac2O in
the presence of DMAP and Et3N.
After successful completion of the syntheses of the deguelin
analogues, we turned our attention to the discovery of new
HSP90 inhibitors with a novel scaffold, avoiding the structural
complexity of deguelin. Thus, we designed new HSP90
inhibitors, which are devoid of the middle dihydrobenzopyran
units, by applying a ring-truncation strategy based on the initial
SAR study of deguelin (Figure 2). Our unified synthetic
procedure included addition of an aryl anion, which was
prepared from 38 by halogen−metal exchange reaction, to the
diverse aldehydes and seemed quite straightforward for the
syntheses of the ring-truncated compounds.
First, our effort focused on the synthesis of aryl bromide 38
as a key precursor for the aryl anion (Scheme 5). Generally, 5-
hydroxy-6-formyl-2,2-dimethyl-2H-chromenes are prepared by
a propargylation of resorcinol and subsequent regioselective
Claisen rearrangement.28 However, 5-hydroxy-6-bromo-2,2-
dimethyl-2H-chromene could not be prepared by the same
reaction sequence largely because of the poor regioselectivity of
the Claisen rearrangement. Thus, we prepared 5-hydroxy-6-
nitro-2,2-dimethyl-2H-chromene 35 via propargylation of 33
D
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a
Scheme 6. Synthesis of the B-Ring-Truncated Analogue (44)
a
Reagents and conditions: (a) (COCl)2, CH2Cl2, 0 °C to rt, then MeOH, 89%; (b) NaOMe, (CH2O)n, DMSO, rt, 58%; (c) Bundle’s reagent, CSA,
CH2Cl2, rt, 100%; (d) (i) DIBAL-H, THF, −78 °C, 88%, (ii) DMP, CH2Cl2, rt, 71%; (e) 38, n-BuLi, THF, −78 °C to rt, 72%; (f) DMP, CH2Cl2,
84%; (g) BCl3, CH2Cl2, −78 °C, 75%; (h) DIAD, PPh3, CH2Cl2, rt, 57%.
a
Scheme 7. Synthesis of the C-Ring-Truncated Analogue (51)
a
Reagents and conditions: (a) NBS, THF, −78 °C to rt, 96%; (b) allyl iodide, LHMDS, THF, −78 °C, 53%; (c) LiAlH4, THF, 0 °C to rt, 88%; (d)
NaH, BnBr, n-Bu4NBr, THF, rt, 86%; (e) OsO4, NMO, NaIO4, acetone/water = 4:1, rt; (f) NaBH4, MeOH, 0 °C, 73% for two steps; (g) Pd2(dba)3,
2-(di-tert-butylphosphino)biphenyl, t-BuONa, toluene, 50 °C, 75%; (h) (i) Pd(OH)2/C, H2, MeOH, rt; (ii) DMP, CH2Cl2, rt, 60% for two steps; (j)
6-bromo-2,2-dimethyl-2H-chromene, n-BuLi, THF, −78 °C to rt; (k) DMP, CH2Cl2, rt, 41% for two steps.
with 2-methyl-3-butyn-2-ol and a subsequent regioselective
Claisen rearrangement. Methylation of 35 followed by
reduction of the nitro group afforded the corresponding aniline
37. Finally, aniline 37 was successfully converted to the aryl
bromide 38 with a 88% yield under Sandmeyer condition using
NaNO2, CuBr, and HBr.29
The designed ring-truncated analogues containing a stereo-
genic center were synthesized as racemic mixtures; asymmetric
synthesis of those analogues showing potent inhibitory activity
was conducted later. Synthesis of the B-ring-truncated analogue
is described in Scheme 6. Treatment of 3′,4′-dimethoxyphenyl-
acetic acid with oxalyl chloride followed by an addition of
excess methanol gave methyl ester 39. An aldol reaction of 39
with paraformaldehyde followed by PMB protection of the
resulting alcohol afforded ether 40, which was converted into
aldehyde 41 upon DIBAL-H reduction and subsequent Dess−
Martin oxidation. Addition of the aryl anion of 38 to aldehyde
41 and subsequent Dess−Martin oxidation of the resulting
alcohol produced the corresponding ketone 42. Simultaneous
removal of the PMB and methyl protecting groups of 42 with
BCl3 and a subsequent intramolecular Mitsunobu etherification
using diisopropyl azodicarboxylate (DIAD) and triphenylphos-
phine afforded the desired B-ring-truncated analogue 44.
Regioselective bromination of ester 39 with NBS at −78
°C30 followed by allylation of the resulting bromobenzene 45
with LHMDS and allyl iodide at −78 °C produced the allylated
ester 46. LAH reduction of 46 followed by protection of the
resulting primary alcohol with benzyl bromide yielded benzyl
ether 47. Oxidative allyl cleavage of 47 with OsO4 and NaIO4
and subsequent NaBH4 reduction of the resulting aldehyde
E
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a
Scheme 8. Synthesis of the B- and C-Ring-Truncated Analogues
a
Reagents and conditions: (a) DIBAL-H, THF, −78 °C. 52: 68%. 55: 61%. (b) 53: n-BuLi, 6-bromo-2,2-dimethyl-2H-chromene, THF, −78 °C to
rt. (c) 54: n-BuLi, 38, THF, −78 °C to rt; (d) DMP, CH2Cl2, rt. 53: 46% for two steps. 54: 48% for two steps. 56: 64% for two steps. (e) LDA,
CH3I, THF, rt, −78 °C 79%; (f) NaH, CH3I, THF, rt, 88%; (g) K2CO3, (CH2O)n, DMF, rt, 87%; (h) NaH, Me3SOI, DMSO, rt, 91%.
a
Scheme 9. Synthesis of (S)-2-(3,4-Dimethoxyphenyl)propanoic Acid (62)
a
Reagents and conditions: (a) (i) SOCl2, CH2Cl2, reflux, (ii) Et3N, (+)-pseudoephedrine, CH2Cl2, rt, 73%; (b) CH3I, LiCl, LDA, THF, −78 °C,
81%; (c) H2SO4, dioxane, 100 °C, 96%.
a
Scheme 10. Asymmetric Synthesis of the Optically Active Analogues 69 and 72
a
Reagents and conditions: (a) LiOH·H2O, THF/water (2:1), rt, 100%; (b) (−)-phenylglycinol, EDC, HOBt, i-Pr2NEt, CH2Cl2, rt. 65: 42%. 66:
42%. (c) H2SO4, dioxane, 100 °C. 67: 100%. 70: 100%. (d) BH3·SMe2, ether, 0 °C to rt. 68: 85%. 71: 81%. (e) DMP, CH2Cl2, rt. 68: 81%. 71: 79%.
(f) n-BuLi, CeCl3, 38, THF, −78 °C to rt. 69: 80%. 72: 48%; (g) TPAP, NMO, 4 Å molecular sieves, CH2Cl2, rt. 69: 84%. 72: 71%.
produced alcohol 48. Cyclization of 48 with t-BuONa in the
presence of Pd2(dba)3 and 2-(di-tert-butylphosphino)biphenyl
provided dihydrobenzopyrane 49,31 which was subjected to
benzyl deprotection and Dess−Martin oxidation of the
F
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Table 1. Cell Growth Inhibitions by Deguelin Analogues in the MTS Assay
a
b
All compounds were purified by column chromatography and recrystallization (>95%). IC50 values were determined as the mean of triplicate
c
experiments, and standard deviations are within 15%. No activity (NA) indicates that the analogue exhibits activity with an IC50 higher than 500
μM.
G
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resulting alcohol to yield aldehyde 50. The addition of the aryl
anion, prepared from 6-bromo-2,2-dimethyl-2H-chromene,31 to
aldehyde 50 followed by oxidation of the resultant alcohol
afforded the C-ring-truncated compound 51 (Scheme 7).
The B- and C-ring-truncated analogues were prepared as
described in Scheme 8. Aldehyde 52, which was prepared by
the DIBAL-H reduction of methyl 3′,4′-dimethoxyphenyl
acetate, was readily converted to 53 and 54 by the addition
of the aryl anion, which was prepared from 6-bromo-2,2-
dimethyl-2H-chromene and 38, followed by Dess−Martin
oxidation. The α-methylated analogue 56 was prepared by
addition of the aryl anion prepared from 38 to aldehyde 55,
which was conveniently synthesized by α-alkylation of methyl
3′,4′-dimethoxyphenyl acetate using LDA and CH3I, followed
by ester reduction. Dimethylation of 54 with excess CH3I and
NaH afforded analogue 57. Standard aldol condensation of 54
with paraformaldehyde gave enone 58, which was subjected to
Corey−Chaykovsky cyclopropanation32 to yield analogue 59.
We prepared the (S)- and (R)-enantiomers of racemate 56 to
evaluate the HSP90 inhibitory activity of each enantiomer
(Scheme 9). We initially synthesized the known acid 62 with an
(S)-configuration to confirm the stereochemistries of both
enantiomers of 56, which were prepared by our own procedure
shown in Scheme 10. Optically active amide 61 was synthesized
by the amidation of 3′,4′-dimethoxyphenylacetic acid and the
asymmetric methylation of (S,S)-pseudoephedrine amide 60.
Finally, optically active 62 was obtained in high yield by acid-
catalyzed hydrolysis.33
The synthesis of each enantiomer of 56 is outlined in
Scheme 10. The diastereomeric mixture of (−)-phenyl-
glycinolamides 65 and 66 were prepared by the hydrolysis of
racemic carboxylic ester 63, followed by an EDC-assisted amide
coupling of the resulting acid 64 with (−)-phenylglycinol.34
The diastereomeric mixture of amides 65 and 66 was readily
separated by flash chromatography, and each diastereomer was
converted to the corresponding acid, 67 or 70, by acid-
catalyzed hydrolysis. The absolute configurations of 67 and 70
were confirmed by comparison of their spectral data, including
optical rotation, with those of 62. The reduction of acids 67
and 70 with the BH3·SMe2 complex and subsequent Dess−
Martin oxidation of the resulting alcohols produced aldehydes
68 and 71. Aldehydes 68 and 71 were transformed into the
corresponding ketones 69 and 72 via sequential condensation
with an aryl anion, prepared from 38, in the presence of CeCl3
and Ley oxidation of the resulting alcohols (97 and 95% ee by
chiral HPLC, respectively).
study,19 did not show an inhibitory activity in the FP assay even
at 20 μM (data not shown). These data suggested that deguelin
binds to the other binding site of HSP90 and not to the ATP-
binding pocket in the N-terminal domain of HSP90.23 Thus, we
attempted to establish the SAR between deguelin and HSP90
to obtain more detailed information about the deguelin binding
mode. Ultimately, we desired to develop novel and effective
HSP90 inhibitors based on the SAR study. Deguelin consists of
five continuous rings including two aromatic rings, two cis-
fused dihydropyrans (at C7a and C13a), and a hydrophobic
2,2-dimethyl-2H-chromene moiety. In addition, deguelin
possesses two methoxy groups at C9 and C10 and a carbonyl
group at C7, which is considered to be important for binding to
HSP90.20
For the elucidation of the essential functions of each
fragment and the proper conformation of the ring-fused
systems required for potent HSP90 inhibition, we prepared
24 deguelin analogues. The synthesized analogues are classified
as (i) the C9 and/or C10-methoxy-modified analogues 9−11,
(ii) the conformation-changed analogues 12, 22, (iii) the C7-
carbonyl-modified analogues 13−21 and 23−25, and (iv) the
chromene-modified analogues 8 and 26−31. The biological
activities of the 24 analogues were evaluated by a cell growth
inhibition assay (MTS assay) against H1299 non-small-cell lung
cancer (NSCLC) cells. The results of the MTS assay are
presented in Table 1. Initially, the effects of two methoxy
groups at the C9 and C10-positions were analyzed. Analogues 9
and 10, possessing monohydroxy groups on the A-ring,
exhibited approximately 10-fold lower activity compared to
deguelin, and analogue 11 with dihydroxyl groups showed 100-
fold lower activity compared to deguelin. These results seem to
imply that both of the methoxy groups are essential as H-
bonding acceptors for the inhibitory activity of deguelin. The
importance of maintaining a cis-conformation of deguelin is
addressed by analogues 12 and 22, as both analogues result in a
complete loss of activity. Variation of the C7-carbonyl led to
significant changes in the inhibitory activities. Removal of the
C7-carbonyl group (13) caused significantly lower activity
(IC50 of 4.2 μM); however, interestingly, reduction of the
carbonyl to an alcohol (14) resulted in increased potency (IC50
of 10 nM) compared to that of deguelin, also revealing that the
C7-oxygen atom is crucial for cell growth inhibition. However,
the C7-alkoxy analogues 15−18 exhibited significantly
decreased activity compared to deguelin and 14, suggesting
that the bulky substituents at the C7-position prevent the
analogues from binding to HSP90. In fact, as the size of the
alkyl groups increase (15−19), inhibitory activity decreases. H-
Bonding of the C7-substituents also seems important for
inhibitory activity. Analogue 14, possessing a C7-hydroxyl
group, exhibited the most potent activity, which was 10-fold
more active than deguelin and 400-fold more active than the
corresponding methyl ether 15. Interestingly, the carbamate
analogue 21 and the oxime analogue 23, which possess H-
bonding donors, also exhibited excellent cell growth inhibitory
activities (IC50 of 110 and 300 nM, respectively). However, the
corresponding oxime ethers 24 and 25 showed low or no
activity. Overall, the presence of a bulky substituent at the C7-
position seems to decrease or eliminate inhibitory activity;
however, having H-bond donor groups at the C7-position can
compensate or overcome the decrease of activity induced by
the steric effect. We also examined the cell growth inhibitory
activities of analogues 8 and 26−31 to learn the function of the
terminal 2,2-dimethyl-2H-chromene moiety. Analogue 26,
STRUCTURE−ACTIVITY RELATIONSHIP (SAR)
BASED ON ANTIPROLIFERATIVE ACTIVITY
■
We previously reported that deguelin competes with ATP in
binding to HSP90 and sensitively suppresses the interaction
between HSP90 and its client proteins including HIF-1α.19 For
further confirmation of the HSP90 inhibitory activity of
deguelin, we carried out an FP assay on deguelin and its
analogues using VER-00045864 as a fluorescence probe for the
N-terminal ATP-binding pocket of HSP90.22 The HSP90
inhibitors in the FP assay are known to compete with the probe
in binding to the N-terminal ATP-binding pocket of HSP90,
resulting in a decrease of fluorescence polarization compared to
the probe alone. Geldanamycin has been reported to have an
IC50 of 348 nM in the FP assay using the VER-00045864
probe.22 However, deguelin, which exhibited a significant
HSP90 inhibitory activity at 100 nM level in our previous
H
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Table 2. Cell Growth Inhibition by the B- or/and C-Ring-Truncated Compounds in the MTS Assay
a
b
All compounds were purified by column chromatography and recrystallization (>95%). IC50 values were determined as the mean of triplicate
experiments, and standard deviations are within 15%.
Figure 3. Western blot analysis of HIF-1α pretreated with deguelin and the potent deguelin analogues using H1299 cell lines.
possessing the olefin-reduced pyran system, exhibited similar
inhibitory activity as deguelin. Diol 27 exhibited a complete loss
of activity. Generally, olefin modification or pyran-ring opening
in the terminal chromene moiety significantly reduced (28) or
eliminated (29−31) inhibitory activity. These results imply that
the HSP90 binding site interacting with the terminal 2,2-
dimethyl-2H-chromene moiety consists of hydrophobic resi-
dues rather than hydrophilic residues and is sensitive to the size
of the interacting counterpart.
Next, we focused on the B- and/or C-ring-truncated
compounds. On the basis of the aforementioned SAR, we
designed and prepared nine ring-truncated compounds for
which activities were also evaluated by the MTS assay. The
results are presented in Table 2.
The B- or C-ring-truncated compounds (44, 51) exhibited
significantly lower activity than deguelin, whereas compounds
54 and 56, devoid of both the B- and C-rings, showed excellent
inhibitory activities. Compound 54 exhibited the most potent
activity, with an IC50 of 140 nM, which is almost equipotent to
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Figure 4. Inhibition of HSP90 and HIF-1α binding in vitro by analogues 54 and 69.
Figure 5. Effects of analogues 54, 69, and 72 on developmental defects in zebrafish embryos. Zebrafish embryos were incubated with the tested
compounds at 50 nM, 250 nM, and 1.25 μM for 72 h.
deguelin. Noticeably, the deletion of the methoxy substituent in
the terminal chromene moiety of 54 resulted in a significant
decrease in antiproliferative activity (53), suggesting an
important role for the methoxy group in inhibitory activity.
The B- and C-ring-truncated compound 56, possessing the o-
methoxy substituent in the terminal chromene moiety and the
α-methyl substituent, exhibited moderate activity with an IC50
of 730 nM. Incorporation of the dimethyl substituents at the
benzylic position (57) resulted in a significantly decreased
activity compared to those of the monomethyl substituted
compound 56 or the nonsubstituted compound 54. The
decreased activities shown for 57 and 59 seem to imply that an
appropriate constraint induced by the substituents is necessary
in the ring-truncated compounds. The racemate 56 exhibited
lower inhibitory activity than deguelin and 54. However, we
anticipated that the enantiomer of 56 would have different
activities, as is the case for many chiral drugs.35 Thus, we
examined the cell growth inhibitory activities of each
enantiomer. As presented in Table 2, the (S)-enantiomer 69,
with an IC50 of 490 nM, was approximately twice as potent as
racemate 56, whereas the (R)-enantiomer 72 was approx-
imately 2-fold less active. Although compound 69 was less
active in vitro than 54 without a methyl substituent, it seems
that the stereochemistry is of much importance in determining
the effectiveness of the HSP90 inhibitor.
HIF-1α INHIBITORY ACTIVITIES
■
To confirm the correlation between antiproliferative activities
and HSP90 inhibitory activities of the synthesized compounds
and to validate their antiangiogenic activities, we selected
deguelin analogues and the ring-truncated compounds
exhibiting excellent cell growth inhibition for further study.
Because HIF-1α is one of the most well-known HSP90 client
proteins and its transcriptional activity of vascular endothelial
growth factor (VEGF) is important in terms of angiogenesis
regulation,19 the HIF-1α inhibitory activities of 14, 21, 54, 69,
and 72 were analyzed by Western blot analysis using the H1299
cell line. As presented in Figure 3, HIF-1α was suppressed in a
dose-dependent manner by all five test compounds. In addition,
the tested compounds showed parallel correlations between the
cell growth inhibitory activity and HIF-1α suppression.
Analogue 14, which exhibited the most potent HIF-1α
inhibition, showed higher HIF-1α suppression than the parent
deguelin. The ring-truncated compound 54 was also quite
potent in HIF-1α suppression. The B- and C-ring-truncated
compound 69 in an optically active form exhibited higher HIF-
1α inhibitory activity than its antipode 72 as observed in the
MTS assay (Table 2). Involvement of HSP90 in the HIF-1α
inhibition by analogues 54 and 69 was confirmed in vitro by
immunoprecipitation and immunoblotting assays using H1299
cell lysates. The experiments were done at 1.25 μM 54 and 69
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Figure 6. Antiangiogenic effects of 54, 69, and 72 in a zebrafish model. Zebrafish embryos were incubated with the tested compounds at 50, 250, and
1250 nM for 24 h. (a) A 72 hpf Tg(fli:EGFP)y1 larva treated with DMSO. Red box indicates SIV and negative control (DMSO). (b) Antiangiogenic
effect of 54. (c) Antiangiogenic effect of (S)-69. (d) Antiangiogenic effect of (R)-72. Scale bar, 100 μm.
because they exhibited evident HIF-1α inhibitions at the
concentration. As shown in Figure 4, analogues 54 and 69
interrupted the interaction between HIF-1α and HSP90 in
vitro, which was reversed by addition of excess ATP. These
results strongly support that analogues 54 and 69 may inhibit
the activity of HSP90 by competing with ATP at the ATP-
binding site.
interaction between HSP90 and HIF-1α using our potent
analogues suppresses angiogenesis in zebrafish. Thus, we
initially investigated conditions for the treatment stage and
concentration, which does not induce a developmental defect in
zebrafish embryos. We examined deguelin, 54, 69, and 72 in a
dose dependent manner at 50% epiboly (5.3 hpf) using
zebrafish embryos. We observed a developmental defect by
deguelin at 50 nM to 10 μM. Interestingly, analogues 54, 69,
and 72 exhibited the normal growth without affecting viability
and showed no developmental defects at 50 nM to 1.25 μM
(Supporting Information, Figure 1). However, developmental
defects in zebrafish embryos were observed upon treatment
with analogues 54, 69, and 72 at 10 μM (Supporting
Information, Figure 2). Unfortunately, the initial incubation
of zebrafish embryos in the presence of the deguelin analogues
ANTIANGIOGENIC EFFECTS IN ZEBRAFISH
EMBRYOS
■
Antiangiogenic effects of the ring-truncated compounds, which
exhibited potent in vitro activities, were further confirmed using
a transgenic zebrafish line.36 HIF-1α proteins are known to
contribute to the survival of tumor cells, inducing angiogenesis
via VEGF transcription. We envisioned that inhibition of an
K
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silica gel 60 (230−400 mesh, Merck), and preparative thin layer
chromatography was used with glass-backed silica gel plates (1 mm,
Merck). Thin layer chromatography was performed to monitor
reactions. All reactions were performed under dry argon atmosphere in
flame-dried glassware. Rotenone as starting material of deguelin was
14 and 21 at 0.05−1.25 μM did not affect angiogenesis in
zebrafish. We assumed that the chemical or metabolic instability
of analogues 14 and 21 induced the loss of activity in vivo
model, which is partly supported by the mass spectral data of
analogue 21. We tested deguelin for antiangiogenic effect using
zebrafish model. However, we could not observe any mature
zebrafish showing the normal growth even at 50 nM. Deguelin
also affected the zebrafish viability in a dose dependent manner
(Supporting Information, Figure 3). On the basis of these
results, the developmental defect by analogues 54, 69, and 72
were examined at 50 nM to 1.25 μM (24 hpf). As shown in
Figure 5, analogues 54, 69, and 72 exhibited the normal growth
of zebrafish embryos without developmental defect or
malformation at the selected concentrations.
As shown in Figure 6, the B- and C-ring-truncated
compound 54 exhibited a potent antiangiogenic effect,
indicated by the depletion of subintestinal veins (SIV), in a
dose-dependent manner at 72 hpf. Interestingly, optically active
69 showed the most potent antiangiogenic activity among the
tested compounds. However, its antipode 72, which was less
active in the MTS assay and Western blot analysis, did not
show significant antiangiogenic activity even at a high
concentration of 1.25 μM. These results supported that the
antiangiogenic effects of the deguelin analogues and the ring-
truncated compounds were induced by blocking the action of
HIF-1α via HSP90 inhibition. The excellent antiangiogenic
activity of 69, which appears more potent than 54, is not clearly
explained at present. Other mechanism of actions may partly
explain the antiangiogenic activity of 69 in zebrafish model in
addition to inhibition of an interaction between HIF-1α and
HSP90.
1
purchased from Sigma-Aldrich. H NMR and 13C NMR spectra were
recorded on a JEOL JNM-LA 300 (300 MHz), JEOL JNM-GCX (400
MHz), or Bruker AMX-500 (500 MHz) spectrometer. Chemical shifts
are provided in parts per million (ppm, δ) downfield from
tetramethylsilane (internal standard) with coupling constant in hertz
(Hz). Multiplicity is indicated by the following abbreviations: singlet
(s), doublet (d), doublet of doublet (dd), triplet (t), quartet (q),
quintet (quin), multiplet (m), and broad (br). Optical rotations were
measured using JASCO DIP-1000 digital polarimeter at ambient
temperature using a 100 mm cell of 2 mL capacity. Mass spectra and
HRMS results were recorded on VG Trio-2 GC−MS instrument and
JEOL JMS-AX instrument, respectively. All the final compounds were
purified to more than 95% purity. The purity of the compounds was
determined by normal phase high performance liquid chromatography
(HPLC) (Gilson or Waters, Chiralpak AD-H (4.6 mm × 250 mm) or
Chiralpak OD-H (4.6 mm × 250 mm)). The detailed analytical
conditions are available in Supporting Information.
General Procedure A for Preparation of 15−18, 24, and 25.
To a solution of 14 or 23 in THF were added the alkyl halides (1.5
equiv) and t-BuOK (1.0 M in THF, 1 equiv) at 0 °C. After completion
of the reaction, which was monitored by TLC, the reaction mixture
was quenched with saturated NH4Cl solution and extracted with
EtOAc. The organic layer was dried over MgSO4, filtered, and
concentrated under reduced pressure. The crude residue was purified
by flash column chromatography on silica gel (EtOAc/n-hexane = 1:3
to 1:6) to afford the desired product.
General Procedure B for Preparation of 28−30. To a solution
of phenol 8 and the corresponding alkyl halides (2 equiv) in
acetonitrile was added Cs2CO3 (1.5 equiv) at 0 °C. After completion
of the reaction, which was monitored by TLC, the resulting mixture
was quenched with water and extracted with EtOAc. The organic layer
was washed with brine, dried over MgSO4, and concentrated under
reduced pressure. The residue was purified by flash column
chromatography on silica gel (EtOAc/n-hexane = 1:4) to afford the
desired product.
General Procedure C for an Addition of Aryl Anion to
Aldehyde. To a solution of aryl bromide (1.5 equiv) in THF was
added dropwise n-BuLi solution in n-hexane (1.4 equiv) at −78 °C.
The reaction mixture was stirred for 20 min at −78 °C, and aldehyde
(1.0 equiv) was added. The reaction mixture was stirred for an
additional 30 min and was warmed to ambient temperature. The
reaction mixture was washed with saturated NH4Cl solution and
extracted with EtOAc. The organic layer was washed with brine, dried
over MgSO4, and concentrated under reduced pressure. The residue
was purified by flash column chromatography on silica gel (EtOAc/n-
hexane = 1:4 to 1:2) to afford the corresponding alcohol.
General Procedure D for Dess−Martin Oxidation. To a
solution of alcohol in CH2Cl2 was added Dess−Martin periodinane
(3.0 equiv). After being stirred for 1 h, the reaction mixture was
diluted with CH2Cl2 and sodium thiosulfate solution (10%) was
added. The mixture was stirred for 10 min and extracted with CH2Cl2.
The organic layer was washed with saturated NaHCO3 solution, dried
over MgSO4, and concentrated under reduced pressure. The residue
was purified by flash column chromatography on silica gel (EtOAc/n-
hexane = 1:6 to 1:2) to afford the corresponding oxidized product.
(6aS,12aS)-9-Hydroxy-2,3-dimethoxy-8-(3-methyl-2-buten-
yl)-6a,12a-dihydrochromeno[3,4-b]chromen-12(6H)-one (8).
To a solution of rotenone 6 (200 mg, 0.50 mmol) in CH2Cl2 (5.0
mL) was added BBr3 solution (0.53 mL of 1.0 M solution in CH2Cl2,
0.53 mmol) at −10 °C, and the reaction mixture was stirred for 5 min.
The solvent was removed under reduced pressure, and methanol (1.0
mL) was added. The resulting white solid was filtered, and the residue
of intermediate 7 was used for the next step without further
purification. To a solution of 7 (130 mg, 0.27 mmol) in
hexamethylphosphoramide (HMPA) was added NaBH3CN (69 mg,
CONCLUSION
■
In this work, we have elucidated the structural features of
deguelin required for HSP90 inhibition using the MTS assay
and Western blot analysis of the HSP90 client protein, HIF-1α.
The established SAR revealed the importance of each fragment
and the conformation of deguelin and provided crucial
information for the development of novel and potent HSP90
inhibitors. We have identified new potent deguelin analogues
(14 and 21) through the SAR studies. Ring-truncated
compounds (54 and 69) designed based on the SAR studies
exhibited excellent HIF-1α suppression and potent cell growth
inhibition. In particular, we confirmed the excellent antiangio-
genic activities of the ring-truncated compounds 54 and 69
using the in vivo zebrafish model. Our efforts in the design,
synthesis, and biological evaluations of these novel HSP90
inhibitors have led to the development of promising
antiangiogenic agents 54 and 69, which consist of a novel
scaffold. As we mentioned with respect to the assay for
antiangiogenesis using zebrafish model, the higher antiangio-
genic activity of 69 than that of 54 at a concentration of 1.25
μM is not clearly explained at present. Further intensive studies
on the mechanism of antiangiogenesis and therapeutic
applications of 54 and 69 are in progress.
EXPERIMENTAL SECTION
■
Chemistry. General Methods. Unless otherwise described, all
commercial reagents and solvents were purchased from commercial
suppliers and used without further purification. Tetrahydrofuran was
distilled from sodium benzophenone ketyl. Dichloromethane,
acetonitrile, triethylamine, and pyridine were freshly distilled with
calcium hydride. Flash column chromatography was carried out using
L
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1.1 mmol). The reaction mixture was heated to 70 °C, stirred for 3 h,
and poured into water. The mixture was extracted with a mixture of
diethyl ether and n-hexane (3:1). The organic layer was washed with
brine, dried over MgSO4, and concentrated under reduced pressure.
The residue was purified by flash column chromatography on a silica
gel (EtOAc/n-hexane = 1:2) to give 55 mg (50%) of 8 as pale yellow
29.0, 28.6. HRMS (FAB) calcd for C22H21O6 (M + H+): 381.1338.
Found: 381.1335.
(7aS,13aS)-13,13a-Dihydro-9,10-dihydroxy-3,3-dimethyl-
3H-chromeno[3,4-b]pyrano[2,3-h]chromen-7(7aH)-one (11).
Analogue 11 was obtained by the procedure for analogue 9 and
purified by flash column chromatography on silica gel (EtOAc/n-
hexane/CH2Cl2 = 1:3:1 to 1:2:1) to afford 32 mg (33%) of pale yellow
1
solid with a melting point of 193−195 °C: H NMR (CDCl3, 500
1
solid with a melting point of 203−205 °C (decomposed): H NMR
MHz) δ 7.74 (d, 1H, J = 8.7 Hz), 6.76 (s, 1H), 6.50 (d, 1H, J = 8.7
Hz), 6.41 (s, 1H), 6.00 (s, 1H), 5.19 (m, 1H), 4.87 (t, 1H, J = 3.0 Hz),
4.60 (dd, 1H, J = 11.9, 3.1 Hz), 4.14 (d, 1H, J = 11.9 Hz), 3.80 (m,
1H), 3.75 (s, 3H), 3.70 (s, 3H), 3.34 (m 2H), 1.74 (s, 3H), 1.65 (s,
3H); 13C NMR (CDCl3, 125 MHz) δ 190.4, 162.1, 160.1, 149.3,
147.5, 143.6, 134.0, 126.9, 121.1, 114.8, 112.5, 110.7, 110.5, 108.5,
104.7, 100.8, 72.0, 66.2, 56.2, 55.7, 44.1, 25.7, 22.0, 17.7. HRMS
(FAB) calcd for C23H24O6 (M+): 396.1573. Found: 396.1575.
(acetone-d6, 400 MHz) δ 7.75 (s, 1H), 7.63 (d, 1H, J = 8.7 Hz), 7.38
(s, 1H), 6.59 (s, 1H), 6.58 (d, 1H, J = 10.1 Hz), 6.39 (d, 1H, J = 8.7
Hz), 6.29 (s, 1H), 5.65 (d, 1H, J = 10.1 Hz), 5.03 (m, 1H), 4.55 (dd,
1H, J = 12.2, 2.9 Hz), 4.20 (d, 1H, J = 12.2 Hz), 3.79 (d, 1H, J = 4.0
Hz), 1.38 (s, 3H), 1.30 (s, 3H); 13C NMR (acetone-d6, 100 MHz) δ
190.1, 160.8, 158.2, 148.4, 146.9, 140.7, 130.4, 129.3, 116.6, 114.9,
114.2, 112.1, 110.3, 106.2, 105.1, 78.8, 74.0, 67.2, 45.4, 29.0, 28.6.
HRMS (FAB) calcd for C21H19O6 (M + H+): 367.1182. Found:
367.1179.
(7aS,13aS)-9,10-Dimethoxy-3,3-dimethyl-13,13a-dihydro-
3H-chromeno[3,4-b]pyrano[2,3-h]chromen-7(7aH)-one (1, De-
guelin). To a solution of 8 (128 mg, 0.32 mmol) in CH2Cl2 (4.0 mL)
was added PhSeCl (68 mg, 0.35 mmol) at −30 °C, and the mixture
was stirred for 10 min at the same temperature. The reaction mixture
was warmed to ambient temperature and stirred for an additional 1 h.
The organic solvent was removed under reduced pressure, and the
resulting residue was dissolved in THF (4.0 mL), followed by addition
of H2O2 (30% in water, 0.06 mL) at 0 °C. After completion of the
reaction, which was monitored by TLC, EtOAc (8.0 mL) and water
(4.0 mL) were added. The organic layer was washed with 5%
NaHCO3 solution and brine. The organic layer was dried over MgSO4
and concentrated under reduced pressure. The residue was purified by
flash column chromatography on silica gel (EtOAc/n-hexane = 1:2) to
afford 78 mg (61%) of 1 as pale yellow solid with a melting point of
9,10-Dimethoxy-3,3-dimethyl-3H-chromeno[3,4-b]pyrano-
[2,3-h]chromen-7-(13H)-one (12). To a solution of deguelin 1 (50
mg, 0.13 mmol) in ethanol (2.0 mL) were added NaOAc (21 mg, 0.25
mmol) and I2 (290 mg, 1.14 mmol). The reaction mixture was refluxed
for 12 h and cooled to ambient temperature. The resulting reaction
mixture was extracted with EtOAc. The organic layer was washed with
brine, dried over MgSO4, and concentrated under reduced pressure.
The residue was crystallized from EtOAc and n-hexane (1:1) to afford
17 mg (35%) of α,β-unsaturated ketone 12 as pale yellow solid with a
1
melting point of 233−235 °C (decomposed): H NMR (CDCl3, 500
MHz) δ 8.42 (s, 1H), 8.01 (d, 1H, J = 8.7 Hz), 6.84 (d, 1H, J = 8.7
Hz), 6.73 (d, 1H, J = 10.0 Hz), 6.52 (s, 1H), 5.70 (d, 1H, J = 8.7 Hz),
4.99 (s, 2H), 3.93 (s, 3H), 3.84 (s, 3H), 1.47 (s, 6H); 13C NMR
(CDCl3, 125 MHz) δ 174.2, 157.1, 156.1, 151.0, 148.9, 146.2, 144.0,
130.5, 126.4, 118.4, 115.3, 114.6, 111.7, 110.5, 109.9, 109.0, 100.3,
77.7, 64.8, 56.2, 55.8, 28.1, 28.1. HRMS (FAB) calcd for C23H20O6
(M+): 392.1260. Found: 392.1263.
1
85−87 °C: H NMR (CDCl3, 400 MHz) δ 7.72 (d, 1H, J = 8.7 Hz),
6.77 (s, 1H), 6.62 (d, 1H, J = 10.0 Hz), 6.43 (s, 1H), 6.43 (d, 1H, J =
8.7 Hz), 5.53 (d, 1H, J = 10.0 Hz), 4.89 (m, 1H), 4.61 (dd, 1H, J =
12.0, 3.1 Hz), 4.17 (d, 1H, J = 12.0 Hz), 3.82 (d, 1H, J = 4.1 Hz), 3.78
(s, 3H), 3.75 (s, 3H), 1.43 (s, 3H), 1.36 (s, 3H); 13C NMR (CDCl3,
100 MHz) δ 189.2, 160.0, 156.9, 149.4, 147.4, 143.8, 128.6, 128.5,
115.7, 112.7, 111.4, 110.4, 109.1, 104.7, 100.9, 77.6, 72.4, 66.2, 56.3,
55.8, 44.3, 28.4, 28.1. HRMS (FAB) calcd for C23H23O6 (M + H+):
395.1495. Found: 395.1495.
(7S,7aR,13aS)-9,10-Dimethoxy-3,3-dimethyl-7,7a,13,13a-tet-
rahydro-3H-chromeno[3,4-b]pyrano[2,3-h]chromen-7-ol (14).
To a solution of deguelin 1 (300 mg, 0.76 mmol) in methanol
(10.0 mL) was added NaBH4 (43 mg, 1.13 mmol) at 0 °C. The
reaction mixture was stirred for 30 min and quenched with H2O. The
mixture was extracted with diethyl ether, and the organic layer was
washed with brine, dried over MgSO4, and concentrated under
reduced pressure. The residue was purified by flash column
chromatography on silica gel (EtOAc/n-hexane = 1:1) to afford 300
mg (100%) of alcohol 14 as white solid with a melting point of 142−
144 °C: 1H NMR (CDCl3, 400 MHz) δ 6.99 (d, 1H, J = 8.2 Hz), 6.68
(s, 1H), 6.64 (d, 1H, J = 10.0 Hz), 6.47 (s, 1H), 6.41 (d, 1H, J = 8.2
Hz), 5.56 (d, 1H, J = 10.0 Hz), 4.80 (m, 2H), 4.57 (d, 1H, J = 10.0
Hz), 4.21 (m, 1H), 3.84 (s, 3H), 3.82 (s, 3H), 3.36 (t, 1H, J = 4.9 Hz),
1.41 (s, 3H), 1.39 (s, 3H); 13C NMR (CDCl3, 100 MHz) δ 154.3,
149.6, 149.4, 147.9, 143.8, 130.0, 129.1, 116.4, 113.6, 111.3, 109.8,
109.5, 108.7, 100.7, 76.0, 69.1, 66.3, 65.0, 56.5, 55.8, 37.9, 27.8, 27.7.
HRMS (FAB) calcd for C23H24O6 (M+): 396.1560. Found: 396.1562.
(7aS,13aS)-9,10-Dimethoxy-3,3-dimethyl-7,7a,13,13a-tetra-
hydro-3H-chromeno[3,4-b]pyrano[2,3-h]chromene (13). To a
solution of 14 (40 mg, 0.10 mmol) in THF (2.0 mL) was added NaH
(16 mg of 60% dispersion in mineral oil, 0.40 mmol) at 0 °C. The
reaction mixture was stirred for 30 min at the same temperature, and
carbon disulfide (0.06 mL, 1.00 mmol) was added. After addition of
CH3I (0.06 mL, 1.00 mmol) at 0 °C, the reaction mixture was warmed
to ambient temperature and quenched with methanol (1.0 mL). The
reaction mixture was concentrated under reduced pressure. The crude
residue was extracted with EtOAc and water. The organic layer was
washed with brine, dried over MgSO4, and concentrated under
reduced pressure. The residue was purified by flash column
chromatography on silica gel (EtOAc/n-hexane = 1:6) to afford 35
mg (71%) of the xanthate intermediate. To the xanthate intermediate
(21 mg, 0.04 mmol) dissolved in dry toluene (1.0 mL) were added tri-
n-butyltin hydride (0.02 mL, 0.09 mmol) and AIBN (catalytic
amount). The reaction mixture was refluxed until completion of the
reaction (monitored by TLC) and concentrated under reduced
(7aS,13aS)-9-Hydroxy-13,13a-dihydro-10-methoxy-3,3-di-
methyl-3H-chromeno[3,4-b]pyrano[2,3-h]chromen-7(7aH)-one
(9). To a solution of deguelin 1 (100 mg, 0.25 mmol) in CH2Cl2 (7.0
mL) was added BBr3 (0.25 mL, 0.25 mmol, 1.0 M solution in CH2Cl2)
at −78 °C. The reaction mixture was stirred for 1 h and warmed to 0
°C. After being stirred for an additional 30 min, the resulting mixture
was quenched with water (5.0 mL) and extracted with CH2Cl2. The
organic layer was washed with brine, dried over MgSO4, and
concentrated under reduced pressure. The residue was purified by
flash column chromatography on silica gel (EtOAc/n-hexane/CH2Cl2
= 1:3:1 to 1:2:1) to afford 16 mg (16%) of 9 as pale yellow solid with a
1
melting point of 94−96 °C: H NMR (acetone-d6, 400 MHz) δ 7.64
(d, 1H, J = 8.7 Hz), 7.60 (s, 1H), 6.67 (s, 1H), 6.59 (d, 1H, J = 10.1
Hz), 6.40 (d, 1H, J = 8.7 Hz), 6.30 (s, 1H), 5.66 (d, 1H, J = 10.1 Hz),
5.06 (m, 1H), 4.57 (dd, 1H, J = 12.2, 2.9 Hz), 4.22 (d, 1H, J = 12.2
Hz), 3.85 (d, 1H, J = 4.0 Hz), 3.62 (s, 3H), 1.39 (s, 3H), 1.30 (s, 3H);
13C NMR (acetone-d6, 100 MHz) δ 190.2, 160.8, 158.2, 149.6, 148.6,
143.7, 130.4, 129.4, 116.6, 114.3, 112.2, 112.1, 110.3, 105.8, 105.2,
78.8, 74.0, 67.3, 57.3, 45.4, 29.0, 28.6. HRMS (FAB) calcd for
C22H21O6 (M + H+): 381.1338. Found: 381.1327.
(7aS,13aS)-10-Hydroxy-13,13a-dihydro-9-methoxy-3,3-di-
methyl-3H-chromeno[3,4-b]pyrano[2,3-h]chromen-7(7aH)-one
(10). Analogue 10 was obtained (14 mg, 14%) as pale yellow solid
with a melting point of 116−118 °C by the procedure for analogue 9:
1H NMR (acetone-d6, 300 MHz) δ 7.64 (d, 1H, J = 8.6 Hz), 7.07 (s,
1H), 6.60 (s, 1H), 6.59 (d, 1H, J = 10.1 Hz), 6.39 (d, 1H, J = 8.6 Hz),
6.38 (s, 1H), 5.67 (d, 1H, J = 10.1 Hz), 5.06 (m, 1H), 4.59 (dd, 1H, J
= 12.2, 2.9 Hz), 4.23 (d, 1H, J = 12.2 Hz), 3.82 (d, 1H, J = 4.0 Hz),
3.71 (s, 3H), 1.39 (s, 3H), 1.30 (s, 3H); 13C NMR (acetone-d6, 75
MHz) δ 190.0, 160.8, 158.2, 149.1, 148.2, 142.1, 130.4, 129.4, 116.6,
114.5, 114.3, 112.1, 110.3, 107.2, 102.1, 78.8, 73.9, 67.4, 56.6, 45.4,
M
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pressure. The residue was purified by flash column chromatography on
silica gel (EtOAc/n-hexane = 1:6) to afford 11 mg (69%) of 13 as pale
yellow oil: H NMR (CDCl3, 300 MHz) δ 6.75 (d, 1H, J = 8.2 Hz),
65.8, 64.9, 56.2, 55.8, 37.3, 27.9, 27.8. HRMS (FAB) calcd for
C30H30O6 (M+): 486.2042. Found: 486.2050.
1
(7S,7aS,13aS)-9,10-Dimethoxy-3,3-dimethyl-7-(tetrahydro-
2H-pyran-2-yloxy)-7,7a,13,13a-tetrahydro-3H-chromeno[3,4-
b]pyrano[2,3-h]chromene (19). To a mixture of 14 (30 mg, 0.08
mmol) and DHP (13 mg, 0.15 mmol) in CH2Cl2 (1.0 mL) was added
pyridinium p-toluenesulfonate (5.8 mg, 0.02 mmol), and the reaction
mixture was stirred for 1 h. The reaction was quenched with water (0.5
mL), and the resulting mixture was extracted with EtOAc. The organic
layer was dried over MgSO4 and concentrated under reduced pressure.
The residue was purified by flash column chromatography on silica gel
(EtOAc/n-hexane = 1:4) to afford 18 mg (50%, diasteromeric
6.63 (d, 1H, J = 10.0 Hz), 6.62 (s, 1H), 6.38 (s, 1H), 6.31 (d, 1H, J =
8.2 Hz), 5.52 (d, 1H, J = 10.0 Hz), 5.53 (d, 1H, J = 10.0 Hz), 4.67 (q,
1H, J = 4.7 Hz), 4.24 (d, 1H, J = 5.4 Hz), 3.79 (s, 6H), 3.26 (m, 1H),
2.99 (m, 2H), 1.38 (s, 3H), 1.37 (s, 3H); 13C NMR (CDCl3, 75 MHz)
δ 152.2, 148.9, 148.7, 147.7, 143.5, 128.9, 128.5, 116.7, 113.0, 111.4,
111.2, 109.7, 108.8, 100.6, 75.6, 69.7, 65.6, 56.6, 55.8, 31.6, 29.4, 27.8,
27.6. HRMS (FAB) calcd for C23H24O5 (M+): 380.1624. Found:
380.1631.
(7S,7aR,3aS)-7,9,10-Trimethoxy-3,3-dimethyl-7,7a,13,13a-
tetrahydro-3H-chromeno[3,4-b]pyrano[2,3-h]chromene (15).
Ether 15 was prepared from 14 according to the general procedure
A and purified by flash column chromatography on silica gel (EtOAc/
n-hexane = 1:3) to afford a colorless solid with a melting point of 66−
68 °C (21 mg, 100%): 1H NMR (CDCl3, 400 MHz) δ 6.93 (d, 1H, J =
8.2 Hz), 6.77 (s,1H), 6.64 (d, 1H, J = 10.0 Hz), 6.42 (s, 1H), 6.37 (d,
1H, J = 8.2 Hz), 5.55 (d, 1H, J = 10.0 Hz), 4.78 (m, 1H), 4.51 (t, 1H, J
= 10.0 Hz), 4.39 (d, 1H, J = 3.6 Hz), 4.22 (dd, 1H, J = 10.0, 3.9 Hz),
3.82 (s, 3H), 3.81 (s, 3H), 3.38 (m, 1H), 3.15 (s, 3H), 1.41 (s, 3H),
1.39 (s, 3H); 13C NMR (CDCl3, 100 MHz) δ 154.2, 149.1, 149.0,
148.4, 143.5, 129.3, 129.0, 116.5, 113.6, 111.5, 110.3, 110.0, 108.4,
100.4, 76.7, 75.9, 70.0, 65.8, 57.0, 56.5, 55.7, 37.2, 27.9, 27.8. HRMS
(FAB) calcd for C24H26O6 (M+): 410.1729. Found: 410.1713.
(7S,7aR,3aS)-9,10-Dimethoxy-3,3-dimethyl-7-ethoxy-
7,7a,13,13a-tetrahydro-3H-chromeno[3,4-b]pyrano[2,3-h]-
chromene (16). Ether 16 was prepared from 14 according to the
general procedure A and purified by flash column chromatography on
silica gel (EtOAc/n-hexane = 1:3) to afford a colorless solid with a
1
mixture) of 19 as a colorless oil: H NMR (CDCl3, 500 MHz) δ
7.06 (d, 1H, J = 8.2 Hz), 6.91 (d, 1H, J = 8.2 Hz), 6.84 (s, 1H), 6.72
(s, 1H), 6.63 (t, 1H, J = 9.9 Hz), 6.42 (s, 1H), 6.69 (s, 1H), 6.37 (d,
1H, J = 8.4 Hz), 6.33 (d, 1H, J = 8.2 Hz), 5.54 (t, 1H, J = 9.3 Hz), 4.85
(m, 4H), 4.75 (m, 1H), 4.63 (t, 1H, J = 10.0 Hz), 4.55 (t, 1H, J = 10.0
Hz), 4.33 (m, 1H), 4.21 (m, 2H), 3.81 (s, 3H), 3.80 (s, 3H), 3.79 (s,
3H), 3.78 (s, 3H), 3.70 (m, 1H), 3.40 (m, 3H), 3.29 (m, 1H), 3.11 (m,
1H), 1.71−1.19 (m, 12H), 1.40 (s, 12H); 13C NMR (CDCl3, 125
MHz) δ 154.2, 153.7, 149.2, 149.1, 149.0, 148.9, 148.7, 148.7, 148.2,
148.2, 143.3, 143.2, 116.5, 116.4, 113.4, 113.1, 111.8, 111.7, 110.2,
110.0, 109.6, 109.6, 109.3, 109.3, 109.1, 108.2, 100.3, 100.2, 99.1, 93.2,
75.9, 75.8, 72.4, 69.8, 69.4, 69.1, 65.8, 65.2, 61.6, 60.4, 56.5, 56.5, 55.8,
55.8, 37.2, 36.8, 30.3, 30.2, 28.0, 27.9, 27.8, 27.7, 25.4, 25.3, 18.7, 18.1.
HRMS (FAB) calcd for C28H32O7 (M+): 480.2148. Found: 480.2155.
(7S,7aS,13aS)-9,10-Dimethoxy-3,3-dimethyl-7,7a,13,13a-tet-
rahydro-3H-chromeno[3,4-b]pyrano[2,3,h]chromen-7-yl Ace-
tate (20). To a solution of 14 (30 mg, 0.08 mmol) and DMAP
(catalytic amount) in CH2Cl2 (1.0 mL) were added Et3N (0.01 mL,
0.09 mmol) and acetic anhydride (0.06 mL, 0.64 mmol) at 0 °C. The
reaction mixture was stirred for 10 min at ambient temperature and
quenched with saturated NH4Cl solution. The mixture was extracted
with CH2Cl2. The organic layer was washed with brine, dried over
MgSO4, and concentrated under reduced pressure. The residue was
purified by flash column chromatography on silica gel (EtOAc/n-
hexane = 1:5) to afford 27 mg (82%) of 20 as a white solid with a
melting point of 111−113 °C: 1H NMR (CDCl3, 500 MHz) δ 7.01 (d,
1H, J = 8.3 Hz), 6.63 (s, 1H), 6.63 (d, 1H, J = 9.9 Hz), 6.38 (s, 1H),
6.38 (d, 1H, J = 7.5 Hz), 6.24 (d, 1H, J = 4.4 Hz), 5.56 (d, 1H, J = 9.9
Hz), 4.86 (m, 1H), 4.43 (t, 1H, J = 10.2 Hz), 4.24 (m, 1H), 3.81 (s,
6H), 3.49 (m, 1H), 1.71 (s, 3H), 1.40 (s, 6H); 13C NMR (CDCl3, 125
MHz) δ 170.0, 154.6, 149.3, 148.6, 148.5, 143.4, 130.5, 129.1, 116.2,
111.7, 110.9, 109.7, 109.6, 108.6, 100.1, 76.1, 69.0, 66.7, 64.5, 56.4,
55.8, 36.4, 27.9, 27.8, 20.8. HRMS (FAB) calcd for C25H26O7 (M+):
438.1679. Found: 438.1681.
1
melting point of 133−135 °C (23 mg, 71%): H NMR (CDCl3, 300
MHz) δ 6.95 (d, 1H, J = 8.0 Hz), 6.84 (s, 1H), 6.63 (d, 1H, J = 9.9
Hz), 6.39 (s, 1H), 6.35 (d, 1H, J = 8.3 Hz), 5.53 (d, 1H, J = 9.9 Hz),
4.75 (m, 1H), 4.58 (t, 1H, J = 9.7 Hz), 4.53 (d, 1H, J = 3.8 Hz), 4.20
(dd, 1H, J = 9.9, 4.2 Hz), 3.80 (s, 3H), 3.80 (s, 3H), 3.42 (m, 2H),
3.29 (m, 1H), 1.39 (s, 3H), 1.38 (s, 3H), 1.00 (t, 3H, J = 6.8 Hz); 13C
NMR (CDCl3, 75 MHz) δ 153.9, 149.1, 148.9, 148.4, 143.3, 128.9,
128.6, 116.6, 114.4, 111.7, 110.1, 109.8, 108.5, 100.3, 75.8, 75.0, 70.1,
66.0, 64.9, 56.5, 55.7, 36.8, 29.6, 27.8, 27.8. HRMS (FAB) calcd for
C25H28O6 (M+): 424.1886. Found: 424.1894.
(7S,7aR,3aS)-9,10-Dimethoxy-3,3-dimethyl-7-propoxy-
7,7a,13,13a-tetrahydro-3H-chromeno[3,4-b]pyrano[2,3-h]-
chromene (17). Ether 17 was prepared from 14 according to the
general procedure A and purified by flash column chromatography on
silica gel (EtOAc/n-hexane = 1:5) to afford a colorless solid with a
1
melting point of 54−56 °C (15 mg, 68%): H NMR (CDCl3, 500
(7S,7aS,13aS)-9,10-Dimethoxy-3,3-dimethyl-7,7a,13,13a-tet-
rahydro-3H-chromeno[3,4-b]pyrano[2,3-h]chromen-7-yl Car-
bamate (21). To a solution of 14 (30 mg, 0.08 mmol) in CH2Cl2
(0.5 mL) was added dropwise trichloroacetyl isocyanate (0.01 mL,
0.11 mmol) at 0 °C. The reaction mixture was stirred for 20 min at 0
°C, and then methanol (1.0 mL) and water (0.2 mL) were added at 0
°C. The resulting mixture was stirred for 2.5 h and extracted with
EtOAc. The organic layer was washed with brine, dried over MgSO4,
and concentrated under reduced pressure. The residue was purified by
flash column chromatography on silica gel (EtOAc/n-hexane = 1:5) to
give 21 mg (67%) of 21 as a colorless thick oil: 1H NMR (CDCl3, 500
MHz) δ 6.87 (d, 1H, J = 8.2 Hz), 6.62 (d, 1H, J = 10.0 Hz), 6.53 (s,
1H), 6.33 (s, 1H), 6.27 (d, 1H, J = 8.2 Hz), 5.46 (d, 1H, J = 10.0 Hz),
4.74 (m, 1H), 4.56 (dd, 1H, J = 11.7, 2.7 Hz), 4.49 (d, 1H, J = 2.7 Hz),
4.18 (d, 1H, J = 11.7 Hz), 3.73 (s, 3H), 3.72 (s, 3H), 3.41 (m, 1H),
1.34 (s, 6H); 13C NMR (CDCl3, 125 MHz) δ 154.2, 149.6, 148.9,
148.3, 143.3, 130.6, 128.4, 116.6, 110.2, 110.1, 109.6, 108.6, 108.2,
100.9, 75.9, 75.1, 67.5, 65.9, 56.7, 55.8, 55.6, 35.5, 28.1, 27.6. HRMS
(FAB) calcd for C23H22O5 (M+ − CO2NH3): 378.1467. Found:
378.1474.
MHz) δ 6.94 (d, 1H, J = 8.2 Hz), 6.83 (s, 1H), 6.63 (d, 1H, J = 9.9
Hz), 6.39 (s, 1H), 6.34 (d, 1H, J = 8.2 Hz), 5.53 (d, 1H, J = 9.9 Hz),
4.77 (m, 1H), 4.57 (t, 1H, J = 9.9 Hz), 4.50 (d, 1H, J = 3.7 Hz), 4.21
(dd, 1H, J = 9.9, 4.2 Hz), 3.80 (s, 6H), 3.39 (m, 2H), 3.18 (m, 1H),
1.39 (s, 8H), 0.69 (t, 3H, J = 7.3 Hz); 13C NMR (CDCl3, 125 MHz) δ
153.9, 149.1, 148.9, 148.3, 143.3, 128.9, 128.7, 116.6, 114.3, 111.7,
110.2, 109.8, 108.4, 100.3, 75.8, 75.3, 71.3, 70.1, 65.9, 56.5, 55.8, 36.8,
29.6, 27.9, 27.8, 22.9. HRMS (FAB) calcd for C26H31O6 (M + H+):
439.2121. Found: 439.2120.
(7S,7aR,3aS)-7-Benzyloxy-9,10-dimethoxy-3,3-dimethyl-
7,7a,13,13a-tetrahydro-3H-chromeno[3,4-b]pyrano[2,3-h]-
chromene (18). Ether 18 was prepared from 14 according to the
general procedure A and purified by flash column chromatography on
silica gel (EtOAc/n-hexane = 1:6) to afford a colorless solid with a
1
melting point of 142−144 °C (22 mg, 88%): H NMR (CDCl3, 500
MHz) δ 7.22 (m, 3H), 7.00 (m, 2H), 6.87 (d, 1H, J = 8.2 Hz), 6.66 (d,
1H, J = 9.9 Hz), 6.62 (s, 1H), 6.45 (s, 1H), 6.37 (d, 1H, J = 8.2 Hz),
5.56 (d, 1H, J = 9.9 Hz), 4.81 (m, 1H), 4.62 (t, 1H, J = 9.9 Hz), 4.54
(d, 1H, J = 3.2 Hz), 4.48 (AB quartet, 2H, J = 85.0, 12.5 Hz), 4.25 (dd,
1H, J = 14.2, 4.6 Hz), 3.84 (s, 3H), 3.72 (s, 3H), 3.36 (m, 1H), 1.42 (s,
3H), 1.40 (s, 3H); 13C NMR (CDCl3, 125 MHz) δ 154.2, 149.1,
148.9, 148.7, 143.4, 137.9, 129.2, 129.0, 128.1, 127.6, 127.4, 127.2,
116.5, 113.8, 111.3, 110.1, 110.0, 108.4, 100.4, 75.9, 72.8, 69.9, 69.8,
(13aS)-9,10-Dimethoxy-3,3-dimethyl-13,13a-dihydro-3H-
chromeno[3,4-b]pyrano[2,3-h]chromene (22). A solution of 14
(3 mg, 8 μmol) in acetic acid (1.0 mL) was stirred for 2 h at 100 °C
and treated with water (2.0 mL). The mixture was extracted with
N
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diethyl ether. The organic layer was washed with brine, dried over
MgSO4, and concentrated under reduced pressure. The residue was
purified by flash column chromatography on silica gel (EtOAc/n-
hexane = 1:7) to afford 2 mg (70%) of 22 as pale yellow solid with a
melting point of 152−154 °C: 1H NMR (CDCl3, 400 MHz) δ 6.98 (s,
1H), 6.81 (d, 1H, J = 8.2 Hz), 6.60 (d, 1H, J = 10.0 Hz), 6.55 (s, 1H),
6.40 (s, 1H), 6.36 (d, 1H, J = 8.2 Hz), 5.59 (d, 1H, J = 10.0 Hz), 5.27
(m, 1H), 4.57 (dd, 1H, J = 10.0, 5.4 Hz), 4.13 (t, 1H, J = 10.0 Hz),
3.88 (s, 3H), 3.83 (s, 3H), 1.42 (s, 3H), 1.38 (s, 3H); 13C NMR
(CDCl3, 100 MHz) δ 153.3, 150.3, 149.2, 148.2, 144.7, 129.7, 126.4,
123.7, 116.6, 116.2, 111.8, 110.6, 109.8, 109.6, 105.2, 100.9, 76.1, 71.1,
67.9, 56.3, 55.9, 28.0, 27.6. HRMS (FAB) calcd for C23H22O5 (M+):
378.1467. Found: 378.1474.
(7aR,13aS)-9,10-Dimethoxy-3,3-dimethyl-13,13a-dihydro-
3H-chromeno[3,4-b]pyrano[2,3-h]chromen-7(7aH)-one Oxime
(23). To a solution of deguelin 1 (25 mg, 0.06 mmol) in pyridine (1.0
mL) was added hydroxylamine hydrochloride (14 mg, 0.19 mmol).
The reaction mixture was heated to 70 °C and stirred until the
reaction was completed (monitored by TLC). The resulting mixture
was quenched with water (0.5 mL) and extracted with CH2Cl2. The
organic layer was washed with 2 N HCl solution, water, and brine. The
organic layer was dried over MgSO4 and concentrated under reduced
pressure. The residue was purified by flash column chromatography on
silica gel (EtOAc/n-hexane = 1:2) to afford 26 mg (100%) of oxime 23
as a colorless thick oil: 1H NMR (CDCl3, 500 MHz) δ 8.26 (br, 1H),
7.59 (d, 1H, J = 8.7 Hz), 6.62 (m, 2H), 6.41 (s, 1H), 6.37 (d, 1H, J =
8.7 Hz), 5.49 (d, 1H, J = 10.0 Hz), 4.84 (d, 1H, J = 3.2 Hz), 4.61 (dd,
1H, J = 12.0, 2.4 Hz), 4.48 (m, 1H), 4.24 (d, 1H, J = 12.0 Hz), 3.79 (s,
3H), 3.72 (s, 3H), 1.39 (s, 3H), 1.34 (s, 3H); 13C NMR (CDCl3, 125
MHz) δ 155.7, 151.6, 151.3, 149.3, 147.7, 143.6, 128.6, 124.5, 116.2,
112.2, 110.7, 109.8, 108.2, 106.2, 100.6, 76.5, 69.6, 66.8, 56.4, 55.8,
31.6, 28.1, 27.8. HRMS (FAB) calcd for C23H23NO6 (M+): 409.1525.
Found: 409.1513.
was purified by flash column chromatography on silica gel (EtOAc/n-
hexane/CH2Cl2 = 1:3:1 to 1:2:1) to afford 3.5 mg (99%) of 26 as
white solid with a melting point of 145−147 °C: H NMR (CDCl3,
1
300 MHz) δ 7.61 (d, 1H, J = 8.6 Hz), 6.79 (s, 1H), 6.43 (s, 1H), 6.43
(d, 1H, J = 8.7 Hz), 4.90 (m, 1H), 4.62 (dd, 1H, J = 12.0, 3.0 Hz), 4.17
(d, 1H, J = 12.0 Hz), 3.81 (m, 1H), 3.79 (s, 3H), 3.75 (s, 3H), 2.65
(m, 2H), 1.74 (t, 2H, J = 6.6 Hz), 1.33 (s, 3H), 1.27 (s, 3H); 13C
NMR (CDCl3, 125 MHz) δ 189.4, 161.0, 159.9, 149.2, 147.3, 143.7,
126.3, 112.2, 111.6, 110.2, 108.8, 104.9, 100.7, 75.5, 72.2, 66.3, 56.2,
55.7, 44.1, 31.5, 26.9, 26.3, 16.5. HRMS (FAB) calcd for C23H25O6 (M
+ H+): 397.1651. Found: 397.1642.
(7aS,13aS)-1,2-Dihydroxy-9,10-dimethoxy-3,3-dimethyl-
2,3,13,13a-tetrahydro-1H-chromeno[3,4-b]pyrano[2,3-h]-
chromen-7(7aH)-one (27). To a solution of deguelin 1 (20 mg, 0.05
mmol) in a mixture of acetone and water (4.0 mL, 4:1) were added N-
methylmorpholine N-oxide (18 mg, 0.15 mmol) and OsO4 (20 μL of
0.1 M solution in toluene, 2 μmol) at 0 °C. After being stirred for 10
min, the reaction mixture was warmed to ambient temperature and
stirred for an additional 72 h. The reaction mixture was quenched with
saturated sodium sulfite solution at 0 °C, filtered through Celite pad,
and extracted with EtOAc. The organic layer was dried over MgSO4
and concentrated under reduced pressure. The residue was purified by
flash column chromatography on silica gel (EtOAc/n-hexane = 2:1 to
EtOAc only) to afford 10 mg (47%, diastereomeric mixture) of 27 as
1
pale yellow solid with a melting point of 87−89 °C. Isomer A: H
NMR (CDCl3, 400 MHz) δ 7.76 (d, 1H, J = 8.8 Hz), 6.48 (m, 3H),
4.98 (d, 1H, J = 4.7 Hz), 4.46 (m, 3H), 4.42 (s, 1H), 3.80 (s, 3H), 3.76
(t, 1H, J = 4.7 Hz), 3.71 (s, 3H), 3.45 (br, 1H), 3.34 (d, 1H, J = 4.5
Hz), 1.40 (s, 3H), 1.31 (s, 3H); 13C NMR (CDCl3, 100 MHz) δ
190.7, 160.6, 160.4, 151.2, 148.2, 144.3, 128.7, 113.0, 110.5, 110.0,
109.1, 108.6, 101.1, 79.1, 76.3, 70.4, 67.3, 63.7, 61.9, 56.3, 55.9, 24.3,
1
22.5. Isomer B: H NMR (CDCl3, 400 MHz) δ 7.77 (d, 1H, J = 8.8
Hz), 6.47 (m, 3H), 4.97 (d, 1H, J = 4.7 Hz), 4.59 (m, 3H), 4.43 (s,
1H), 3.80 (s, 3H), 3.73 (m, 1H), 3.70 (s, 3H), 3.53 (br, 1H), 3.30 (d,
1H, J = 4.5 Hz), 1.43 (s, 3H), 1.27 (s, 3H); 13C NMR (CDCl3, 100
MHz) δ 190.8, 160.8, 160.6, 151.2, 148.2, 144.1, 128.8, 113.2, 110.8,
109.7, 109.2, 108.4, 101.0, 79.0, 76.7, 70.3, 67.2, 63.8, 62.0, 56.3, 55.8,
24.5, 22.5. HRMS (FAB) calcd for C23H24O8 (M+): 428.1471. Found:
428.1463.
(7aR,13aS)-9,10-Dimethoxy-3,3-dimethyl-13,13a-dihydro-
3H-chromeno[3,4-b]pyrano[2,3-h]chromen-7(7aH)-one O-
Methyloxime (24). The oxime ether 24 was prepared from 23
according to the general procedure A and purified by flash column
chromatography on silica gel (EtOAc/n-hexane = 1:4) to afford a
1
white solid with a melting point of 120−122 °C (12 mg, 57%): H
NMR (CDCl3, 500 MHz) δ 7.68 (d, 1H, J = 8.7 Hz), 6.61 (d, 1H, J =
10.0 Hz), 6.51 (s, 1H), 6.38 (m, 2H), 5.48 (d, 1H, J = 10.0 Hz), 4.71
(d, 1H, J = 3.3 Hz), 4.59 (dd, 1H, J = 12.0, 2.4 Hz), 4.45 (m, 1H), 4.21
(d, 1H, J = 12.0 Hz), 4.03 (s, 3H), 3.77 (s, 3H), 3.73 (s, 3H), 1.39 (s,
3H), 1.33 (s, 3H); 13C NMR (CDCl3, 125 MHz) δ 155.6, 151.2,
150.0, 149.2, 147.6, 143.5, 128.5, 124.7, 116.3, 111.9, 110.6, 109.7,
108.4, 106.3, 100.5, 76.5, 69.6, 66.9, 61.9, 56.3, 55.8, 32.3, 28.1, 27.8.
HRMS (FAB) calcd for C24H25NO6 (M+): 423.1682. Found:
423.1677.
2,3,9-Trimethoxy-8-(3-methylbut-2-enyl)-6a,12a-dihydro-
6H-chromeno[3,4-b]chromen-12-one (28). Ether 28 was prepared
from 8 according to the general procedure B and purified by flash
column chromatography on silica gel (EtOAc/n-hexane = 1:4) to
afford a white solid with a melting point of 130−132 °C (16 mg,
1
41%): H NMR (CDCl3, 500 MHz) δ 7.80 (d, 1H, J = 8.8 Hz), 6.74
(s, 1H), 6.56 (d, 1H, J = 8.9 Hz), 6.41 (s, 1H), 5.11 (m, 1H), 4.87 (m,
1H), 4.58 (dd, 1H, J = 11.9, 3.3 Hz), 4.15 (d, 1H, J = 11.9 Hz), 3.83 (s,
3H), 3.81 (d, 1H, J = 4.1 Hz), 3.77 (s, 3H), 3.74 (s, 3H), 3.28 (m,
2H), 1.74 (s, 3H), 1.61 (s, 3H); 13C NMR (CDCl3, 125 MHz) δ
190.0, 163.6, 159.2, 149.3, 147.4, 143.6, 131.8, 126.9, 121.6, 117.4,
113.1, 110.3, 105.1, 104.6, 100.7, 71.8, 66.2, 56.2, 55.8, 55.8, 44.3, 25.7,
21.9, 17.6. HRMS (FAB) calcd for C24H26O6(M+): 410.1729. Found:
410.1735.
(7aR,13aS)-9,10-Dimethoxy-3,3-dimethyl-13,13a-dihydro-
3H-chromeno[3,4-b]pyrano[2,3-h]chromen-7(7aH)-one O-Ben-
zyloxime (25). The oxime ether 25 was prepared from 23 according
to the general procedure A and purified by flash column
chromatography on silica gel (EtOAc/n-hexane = 1:5) to afford
white solid with a melting point of 80−82 °C (11 mg, 33%): 1H NMR
(CDCl3, 500 MHz) δ 7.71 (d, 1H, J = 8.7 Hz), 7.46 (d, 2H, J = 7.1
Hz), 7.31 (m, 3H), 6.62 (d, 1H, J = 10.0 Hz), 6.46 (s, 1H), 6.37 (m,
2H), 5.48 (d, 1H, J = 10.0 Hz), 5.27 (AB quartet, 2H, J = 50.7, 12.0
Hz), 4.76 (d, 1H, J = 3.0 Hz), 4.58 (dd, 1H, J = 12.0, 2.2 Hz), 4.44 (m,
1H), 4.20 (d, 1H, J = 12.0 Hz), 3.78 (s, 3H), 3.48 (s, 3H), 1.40 (s,
3H), 1.33 (s, 3H); 13C NMR (CDCl3, 125 MHz) δ 155.6, 151.2,
150.1, 149.1, 147.5, 143.5, 137.5, 128.7, 128.7, 128.5, 128.4, 128.4,
128.1, 124.8, 116.3, 111.7, 110.6, 109.7, 108.5, 106.4, 100.5, 76.6, 76.5,
69.6, 66.8, 56.0, 55.7, 32.3, 28.1, 27.8. HRMS (FAB) calcd for
C30H29NO6 (M+): 499.1995. Found: 499.1999.
(7aS,13aS)-9,10-Dimethoxy-3,3-dimethyl-2,3,13,13a-tetra-
hydro-1H-chromeno[3,4-b]pyrano[2,3-h]chromen-7(7aH)-one
(26). A mixture of 8 (4 mg, 10 μmol) and p-toluenesulfonic acid (0.5
mg, 2.5 μmol) in benzene (1.0 mL) was stirred at 80 °C for 1 h. The
reaction mixture was quenched with saturated NaHCO3 (1.0 mL)
solution and extracted with diethyl ether. The organic layer was dried
over MgSO4 and concentrated under reduced pressure. The residue
9-Allyloxy-2,3-dimethoxy-8-(3-methylbut-2-enyl)-6a,12a-di-
hydro-6H-chromeno[3,4-b]chromen-12-one (29). Ether 29 was
prepared from 8 according to the general procedure B and purified by
flash column chromatography on silica gel (EtOAc/n-hexane = 1:6) to
afford a pale yellow solid with a melting point of 151−153 °C (12 mg,
1
39%): H NMR (CDCl3, 400 MHz) δ 7.78 (d, 1H, J = 8.9 Hz), 6.75
(s, 1H), 6.53 (d, 1H, J = 8.9 Hz), 6.42 (s, 1H), 5.98 (m, 1H), 5.30 (m,
2H), 5.15 (m, 1H), 4.88 (m, 1H), 4.58 (m, 3H), 4.16 (d, 1H, J = 11.8
Hz), 3.81 (d, 1H, J = 2.0 Hz), 3.78 (s, 3H), 3.74 (s, 3H), 3.33 (m,
2H), 1.74 (s, 3H), 1.62 (s, 3H); 13C NMR (CDCl3, 100 MHz) δ
190.0, 162.6, 159.4, 149.4, 147.5, 143.7, 132.5, 131.7, 126.8, 121.6,
117.8, 117.6, 113.2, 110.5, 106.2, 104.7, 100.8, 72.0, 69.0, 66.3, 56.3,
55.8, 44.4, 25.7, 22.1, 17.8. HRMS (FAB) calcd for C26H28O6 (M+):
436.1886. Found: 436.1883.
9-Benzyloxy-2,3-dimethoxy-8-(3-methylbut-2-enyl)-6a,12a-
dihydro-6H-chromeno[3,4-b]chromen-12-one (30). Ether 30
was prepared from 8 by the general procedure B and purified by
O
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flash column chromatography on silica gel (EtOAc/n-hexane = 1:6) to
afford a white solid with a melting point of 160−162 °C (12 mg,
°C: 1H NMR (CDCl3, 300 MHz) δ 7.90 (d, 1H, J = 9.4 Hz), 6.70 (d,
1H, J = 10.0 Hz), 6.38 (d, 1H, J = 9.4 Hz), 5.63 (d, 1H, J = 10.0 Hz),
1.45 (s, 6H). HRMS (FAB) calcd for C11H12NO4 (M + H+):
222.0766. Found: 222.0775.
1
25%): H NMR (CDCl3, 400 MHz) δ 7.78 (d, 1H, J = 8.8 Hz), 7.31
(m, 5H), 6.74 (s, 1H), 6.60 (d, 1H, J = 8.8 Hz), 6.41 (s, 1H), 5.15 (m,
3H), 4.48 (m, 1H), 4.61 (dd, 1H, J = 11.9, 3.0 Hz), 4.16 (d, 1H, J =
11.9 Hz), 3.81 (d, 1H, J = 4.1 Hz), 3.78 (s, 3H), 3.73 (s, 3H), 3.35 (m,
2H), 1.66 (s, 3H), 1.61 (s, 3H); 13C NMR (CDCl3, 75 MHz) δ 190.0,
162.7, 159.3, 149.4, 147.5, 143.6, 136.3, 131.8, 129.7, 128.6, 128.0,
127.1, 126.9, 122.8, 121.7, 117.9, 113.4, 110.5, 106.3, 104.7, 100.8,
72.0, 70.3, 66.3, 56.3, 55.8, 44.4, 25.7, 22.2, 17.7. HRMS (FAB) calcd
for C30H31O6 (M + H+): 487.2121. Found: 487.2113.
Acetic Acid 2,3-Dimethoxy-8-(3-methylbut-2-enyl)-12-oxo-
6,6a,12,12a-tetrahydrochromeno[3,4-b]chromen-9-yl Ester
(31). To a solution of 8 (25 mg, 0.06 mmol) and DMAP (catalytic
amount) in CH2Cl2 (1.0 mL) were added Et3N (0.01 mL, 0.07 mmol)
and acetic anhydride (0.05 mL, 0.53 mmol) at 0 °C. The reaction
mixture was stirred for 10 min at ambient temperature and quenched
with saturated NH4Cl solution. The mixture was extracted with
CH2Cl2, washed with brine, dried over MgSO4, and concentrated
under reduced pressure. The residue was purified by flash column
chromatography on silica gel (EtOAc/n-hexane = 1:4) to afford 14 mg
(44%) of 31 as a pale yellow oil: 1H NMR (CDCl3, 400 MHz) δ 7.80
(d, 1H, J = 8.6 Hz), 6.70 (d, 1H, J = 8.6 Hz), 6.68 (s, 1H), 6.42 (s,
1H), 5.02 (m, 1H), 4.93 (m, 1H), 4.60 (dd, 1H, J = 12.0, 3.2 Hz), 4.17
(d, 1H, J = 12.0 Hz), 3.87 (d, 1H, J = 4.0 Hz), 3.78 (s, 3H), 3.74 (s,
3H), 3.24 (m, 2H), 2.27 (s, 3H), 1.72 (s, 3H), 1.62 (s, 3H); 13C NMR
(CDCl3, 75 MHz) δ 190.1, 168.6, 159.8, 154.9, 149.7, 147.6, 143.9,
132.5, 125.9, 123.0, 120.7, 116.4, 110.5, 104.0, 100.9, 72.3, 66.1, 56.3,
55.8, 44.4, 29.6, 25.6, 23.0, 20.8, 17.7. HRMS (FAB) calcd for
C25H26O7 (M+): 438.1679. Found: 438.1681.
5-Methoxy-2,2-dimethyl-6-nitro-2H-chromene (36). A mix-
ture of phenol 35 (294 mg, 1.33 mmol), K2CO3 (553 mg, 4.0 mmol),
and CH3I (0.25 mL, 3.98 mmol) in acetone (5.0 mL) was heated to 55
°C and stirred overnight. The mixture was concentrated, treated with
water, and extracted with EtOAc. The organic layer was washed with
water, dried over MgSO4, and concentrated under reduced pressure to
afford the crude 36, which was used for the next reaction without
1
further purification: H NMR (CDCl3, 300 MHz) δ 7.77 (d, 1H, J =
8.9 Hz), 6.59 (m, 2H), 5.72 (d, 1H, J = 10.0 Hz), 3.89 (s, 3H), 1.44 (s,
6H). HRMS (FAB) calcd for C12H14NO4 (M + H+): 236.0923.
Found: 236.0924.
5-Methoxy-2,2-dimethyl-2H-chromen-6-amine (37). A mix-
ture of 36 (318 mg, 1.35 mmol) and SnCl2·2H2O (1.47 g, 6.52 mmol)
in ethanol (13.0 mL) was refluxed for 1 h. The reaction mixture was
concentrated under reduced pressure, and the residue was extracted
with EtOAc. The organic layer was washed with 2 N NaOH (20.0 mL)
and water, dried over MgSO4, and concentrated under reduced
pressure. The residue was purified by flash column chromatography on
silica gel (EtOAc/n-hexane = 1:4) to afford aniline 37 (258 mg, 95%
1
for 2 steps) as a yellow oil: H NMR (CDCl3, 300 MHz) δ 6.54 (m,
2H), 6.43 (d, 1H, J = 8.4 Hz), 5.63 (d, 1H, J = 9.9 Hz), 3.78 (s, 3H),
3.46 (br, 2H), 1.37 (s, 6H). HRMS (FAB) calcd for C12H15NO2 (M+):
205.1103. Found: 205.1104.
6-Bromo-5-methoxy-2,2-dimethyl-2H-chromene (38). Hy-
drogen bromide (1.6 mL, 48% in water) was slowly added to a
solution of aniline 37 (260 mg, 1.27 mmol) in water (5.0 mL) that was
cooled in advance to 0 °C. The resulting reaction mixture was stirred
vigorously for 10 min at the same temperature. A solution of NaNO2
(92 mg, 1.30 mmol) in water (1.0 mL) was slowly added, and the
reaction mixture was kept below 5 °C. To a solution of CuBr (190 mg,
1.30 mmol) in water (5.0 mL) was added dropwise the above diazo
solution at 65 °C. The reaction mixture was stirred until the reaction
was completed. The resulting reaction mixture was cooled to ambient
temperature and extracted with EtOAc. The organic layer was washed
with brine, dried over MgSO4, and concentrated under reduced
pressure. The residue was purified by flash column chromatography on
silica gel (EtOAc/n-hexane = 1:12) to yield the aryl bromide 38 (365
mg, 88%) as a pale yellow oil: 1H NMR (CDCl3, 500 MHz) δ 7.16 (d,
1H, J = 8.6 Hz), 6.52 (d, 1H, J = 10.0 Hz), 6.42 (d, 1H, J = 8.6 Hz),
5.59 (d, 1H, J = 10.0 Hz), 3.74 (s, 3H), 1.35 (s, 6H); 13C NMR
(CDCl3, 125 MHz) δ 153.3, 152.7, 132.1, 131.2, 116.9, 116.6, 113.9,
107.3, 76.1, 61.5, 30.9, 27.7. HRMS (FAB) calcd for C12H13BrO2
(M+): 268.0099. Found: 268.0107.
Methyl 2-(3,4-Dimethoxyphenyl)acetate (39). To a solution of
3′,4′-dimethoxyphenylacetic acid (500 mg, 2.54 mmol) and dry DMF
(catalytic amount) in CH2Cl2 (12 mL) was added oxalyl chloride (0.68
mL, 7.64 mmol) dropwise at 0 °C. After the mixture was stirred for 30
min at ambient temperature, absolute methanol (10 mL) was added
and the mixture was stirred for 10 min. The reaction mixture was
quenched with water and extracted with EtOAc. The organic layer was
washed with saturated NaHCO3 solution, dried over MgSO4, and
concentrated under reduced pressure. The residue was purified by
flash column chromatography on silica gel (EtOAc/n-hexane = 1:3) to
afford the desired product 39 (476 mg, 89%) as a yellow oil: 1H NMR
(CDCl3, 300 MHz) δ 6.79 (s, 3H), 3.86 (s, 3H), 3.84 (s, 3H), 3.67 (s,
3H), 3.55 (s, 2H).
4-Nitrobenzene-1,3-diol (33). To a solution of resorcinol 32 (5.0
g, 44.96 mmol) in a mixture (2:1, 270 mL) of chloroform and acetic
acid was slowly added a solution of nitric acid (3.6 mL) in acetic acid
(70 mL). After being stirred for 1 h, the reaction mixture was
quenched with water (100 mL) and extracted with CH2Cl2. The
organic layer was dried over MgSO4 and concentrated under reduced
pressure. The residue was purified by flash column chromatography on
silica gel (EtOAc/n-hexane = 1:4 to EtOAc/n-hexane/CH2Cl2
=
1:4:2) to afford 3.8 g (55%) of nitroresorcinol 33 as yellow solid with a
1
melting point of 120−122 °C: H NMR (CD3OD, 300 MHz) δ 7.99
(d, 1H, J = 9.1 Hz), 6.43 (m, 2H).
5-(2-Methylbut-3-yn-2-yloxy)-2-nitrophenol (34). To a sol-
ution of 2-methyl-3-butyn-2-o1 (0.36 mL, 3.70 mmol) in acetonitrile
(18 mL) at 0 °C was added DBU (0.63 mL, 4.19 mmol), followed by a
dropwise addition of trifluoroacetic anhydride (0.58 mL, 4.19 mmol)
over 30 min. The resulting yellow solution was stirred at 0 °C for 40
min. In a separate flask, a solution of phenol 33 (500 mg, 3.22 mmol)
in acetonitrile (18 mL) at 0 °C was treated with DBU (0.63 mL, 4.19
mmol), followed by an addition of CuC12 (9 mg, 0.06 mmol). To this
mixture at 0 °C was added dropwise the first acetonitrile solution over
40 min. The reaction mixture was stirred overnight at 0 °C and
concentrated under reduced pressure. The residue was poured into
water, and the aqueous solution was extracted with EtOAc. The
organic layer was washed successively with 2 N HCI, 2 N KOH, and
brine. The organic layer was dried over MgSO4 and concentrated
under reduced pressure. The residue was purified by flash column
chromatography on silica gel (EtOAc/n-hexane = 1:20 to 1:2) to
afford 320 mg (45%) of 34 as yellow oil: 1H NMR (CDCl3, 300 MHz)
δ 10.92 (s, 1H), 8.00 (d, 1H, J = 9.5 Hz), 6.99 (d, 1H, J = 2.6 Hz), 6.69
(dd, 1H, J = 9.5, 2.6 Hz), 2.69 (s, 1H), 1.72 (s, 6H). HRMS (FAB)
calcd for C11H12NO4 (M + H+): 222.0766. Found: 222.0773.
2,2-Dimethyl-6-nitro-2H-chromen-5-ol (35). A solution of 34
(310 mg, 1.40 mmol) in N,N-diethylaniline (28 mL) was heated to
130 °C and stirred until the reaction was completed (monitored by
TLC). The reaction mixture was poured into ice−water and extracted
with EtOAc. The organic layer was washed with 2 N HCl and water.
The organic layer was dried over MgSO4 and concentrated under
reduced pressure. The residue was purified by flash column
chromatography on silica gel (EtOAc/n-hexane = 1:15) to afford 35
(281 mg, 91%) as a pale yellow solid with a melting point of 135−137
Methyl 2-(3,4-Dimethoxyphenyl)-3-(4-methoxybenzyloxy)-
propanoate (40). Ester 39 (500 mg, 2.38 mmol) and paraformalde-
hyde (76 mg, 2.50 mmol) were suspended in DMSO (5 mL), and
NaOMe (6.8 mg, 0.12 mmol) was added. The reaction mixture was
stirred overnight and poured into ice-cold water (10 mL). The
resulting mixture was neutralized with 2 N HCl and extracted with
EtOAc. The organic layer was washed with water and brine, dried over
MgSO4, and concentrated under reduced pressure. The residue was
purified by flash column chromatography on silica gel (EtOAc/n-
hexane = 1:1) to afford 331 mg (58%) of the aldol product as a pale
P
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1
yellow thick oil: H NMR (CDCl3, 300 MHz) δ 6.74 (m, 3H), 4.05
(3S)-3-(3,4-Dimethoxyphenyl)-8,8-dimethyl-2,3-dihydro-
4H,8H-pyrano[2,3-f ]chromen-4-one (44). To a solution of 43 (10
mg, 0.03 mmol) in THF (1.0 mL) was added in one portion a solution
of PPh3 (65 mg, 0.25 mmol) and diisopropyl azodicarboxylate (0.02
mL, 0.12 mmol) in THF (0.5 mL). The reaction mixture was stirred
until completion (monitored by TLC) and concentrated under
reduced pressure. The residue was purified by flash column
chromatography on silica gel (EtOAc/n-hexane = 1:4) to afford 5.5
mg (57%) of 44 as white solid with a melting point of 109−111 °C:
1H NMR (CDCl3, 500 MHz) δ 7.74 (d, 1H, J = 8.7 Hz), 6.82 (s, 2H),
6.77 (s, 1H), 6.61 (d, 1H, J = 10.0 Hz), 6.47 (d, 1H, J = 8.7 Hz), 5.58
(d, 1H, J = 10.0 Hz), 4.94 (m, 1H), 4.63 (m, 2H), 3.83 (s, 3H), 3.83
(s, 3H), 1.44 (s, 6H); 13C NMR (CDCl3, 125 MHz) δ 190.9, 159.5,
157.6, 149.1, 148.5, 128.9, 128.6, 127.8, 120.6, 115.6, 114.7, 111.8,
111.4, 111.2, 109.1, 77.5, 55.8, 51.3, 29.6, 28.2, 28.2, 21.7. HRMS
(FAB) calcd for C22H23O5 (M + H+): 367.1545. Found: 367.1552.
Methyl 2-(2-Bromo-4,5-dimethoxyphenyl)acetate (45). To a
solution of methyl ester 39 (500 mg, 2.38 mmol) in THF (12 mL) was
added NBS (449 mg, 2.50 mmol) at −78 °C. The reaction mixture
was stirred for 30 min and warmed to ambient temperature. The
reaction mixture was filtered, washed with EtOAc, and concentrated
under reduced pressure. The residue was purified by flash column
chromatography on silica gel (EtOAc/n-hexane = 1:3) to afford 45
(660 mg, 96%) as a pale yellow solid with a melting point of 63−65
°C: 1H NMR (CDCl3, 300 MHz) δ 6.96 (s, 1H), 6.72 (s, 1H), 3.79 (s,
6H), 3.66 (s, 2H), 3.65 (s, 3H). HRMS (FAB) calcd for C11H13BrO4
(M+): 287.9997. Found: 287.9995.
(m, 1H), 3.81 (s, 3H), 3.79 (s, 3H), 3.73 (m, 2H), 3.65 (s, 3H).
HRMS (FAB) calcd for C12H16O5 (M+): 240.0998. Found: 240.1013.
To a solution of the above aldol product (36 mg, 0.15 mmol) in
CH2Cl2 (1.0 mL) were added p-methoxybenzyl 2,2,2-trichloroaceta-
midate (84 mg, 0.30 mmol) and a catalytic amount of CSA. The
reaction mixture was stirred overnight and quenched with saturated
NaHCO3 solution. The resulting mixture was extracted with CH2Cl2.
The organic layer was washed with brine, dried over MgSO4, and
evaporated under reduced pressure. The residue was purified by flash
column chromatography on silica gel (EtOAc/n-hexane = 1:3) to
1
afford 54 mg (100%) of 40 as a colorless oil: H NMR (CDCl3, 500
MHz) δ 7.19 (d, 2H, J = 8.5 Hz), 6.84 (d, 2H, J = 8.5 Hz), 6.78 (m,
3H), 4.46 (AB quartet, 2H, J = 33.4, 11.7 Hz), 3.98 (m, 1H), 3.83 (s,
6H), 3.80 (m, 2H), 3.77 (s, 3H), 3.67 (s, 3H). HRMS (FAB) calcd for
C20H24O6 (M+): 360.1573. Found: 360.1569.
2-(3,4-Dimethoxyphenyl)-3-(4-methoxybenzyloxy)propanal
(41). To a solution of methyl ester intermediate 40 (20 mg, 0.06
mmol) in THF (1.0 mL) was added methanol (0.003 mL, 0.07 mmol).
The reaction mixture was cooled to −78 °C, and DIBAL-H (0.16 mL
of 1.0 M solution in toluene, 0.16 mmol) was slowly added. The
reaction mixture was stirred at −78 °C for 30 min and warmed to
ambient temperature. Rochelle’s solution was carefully added to the
reaction mixture over 15 min, and the mixture was stirred vigorously
for 1 h at 0 °C. The reaction mixture was poured into water and
extracted with EtOAc. The organic layer was dried over MgSO4 and
concentrated under reduced pressure. The crude residue was purified
by flash column chromatography on silica gel (EtOAc/n-hexane = 1:1)
to afford the corresponding alcohol (16 mg, 88%): 1H NMR
(CDCl3,500 MHz) δ 7.21 (d, 2H, J = 8.5 Hz), 6.85 (d, 2H, J = 8.5
Hz), 6.77 (d, 1H, J = 8.6 Hz), 6.73 (m, 2H), 4.46 (s, 2H), 3.93 (m,
1H), 3.83 (s. 6H), 3.81 (m, 1H), 3.78 (s, 3H), 3.72 (m, 2H), 3.10
(quin, 1H, J = 6.5 Hz). HRMS (FAB) calcd for C19H24O5 (M+):
332.1624. Found: 332.1628.
The aldehyde 41 (11 mg, 71%) was prepared from the above
alcohol according to the general procedure D and was purified by flash
column chromatography on silica gel (EtOAc/n-hexane = 1:2) to
afford 11 mg (71%) of pure 41 as a colorless oil: 1H NMR (CDCl3,300
MHz) δ 9.66 (d, 1H, J = 1.7 Hz), 7.14 (d, 1H, J = 8.4 Hz), 6.78 (m,
3H), 6.66 (m. 2H), 4.40 (d, 2H, J = 3.5 Hz), 3.99 (dd, 1H, J = 8.6, 6.9
Hz), 3.80 (s, 3H), 3.77 (s, 3H), 3.73 (s, 3H), 3.70 (m, 2H).
(S)-2-(3,4-Dimethoxyphenyl)-1-(5-methoxy-2,2-dimethyl-
2H-chromen-6-yl)-3-(4-methoxybenzyloxy)propan-1-one (42).
Coupling of aldehyde 41 and aryl bromide 38 by the general
procedure C afforded the alcohol intermediate (34 mg, 72%,
diastereomeric mixture), which was purified by flash column
chromatography on silica gel (EtOAc/n-hexane = 1:2).
Ketone 42 was prepared from the above alcohol by the general
procedure D and purified by flash column chromatography on silica
gel (EtOAc/n-hexane = 1:3) to give pure 42 (26 mg, 84%): 1H NMR
(CDCl3, 300 MHz) δ 7.34 (d, 1H, J = 8.6 Hz), 7.11 (d, 2H, J = 8.6
Hz), 6.73 (m, 5H), 6.50 (d, 1H, J = 10.0 Hz), 6.45 (d, 1H, J = 8.2 Hz),
5.57 (d, 1H, J = 9.9 Hz), 4.83 (dd, 1H, J = 8.9, 5.1 Hz), 4.39 (q, 2H, J
= 11.5 Hz), 4.12 (t, 1H, J = 9.1 Hz), 3.75 (s, 6H), 3.71 (s, 3H), 3.57
(dd, 1H, J = 9.1, 5.1 Hz), 3.52 (s, 3H), 1.34 (s, 6H). HRMS (FAB)
calcd for C31H35O7 (M + H+): 519.2383. Found: 519.2373.
(S)-2-(3,4-Dimethoxyphenyl)-3-hydroxy-1-(5-hydroxy-2,2-
dimethyl-2H-chromen-6-yl)propan-1-one (43). To a solution of
42 (23 mg, 0.04 mmol) in CH2Cl2 (1.0 mL) was added BCl3 (0.16 mL
of 1.0 M solution in CH2Cl2, 0.16 mmol) at −78 °C. After being
stirred for 1 h, the reaction mixture was quenched with saturated
NH4Cl solution and extracted with CH2Cl2. The organic layer was
dried over MgSO4 and concentrated under reduced pressure. The
residue was purified by flash column chromatography on silica gel
(EtOAc/n-hexane = 1:2) to afford 12 mg (75%) of 43 as pale yellow
thick oil: 1H NMR (CDCl3, 300 MHz) δ 7.40 (d, 1H, J = 8.7 Hz), 6.76
(m, 3H), 6.53 (d, 1H, J = 11.1 Hz), 6.49 (d, 1H, J = 10.5 Hz), 5.63 (d,
1H, J = 9.9 Hz), 4.75 (dd, 1H, J = 8.7, 4.8 Hz), 3.81 (s, 3H), 3.80 (s,
3H), 3.77 (m, 2H), 1.39 (s, 3H), 1.38 (s, 3H). HRMS (FAB) calcd for
C22H24O6 (M+): 384.1573. Found: 384.1570.
Methyl 2-(2-Bromo-4,5-dimethoxyphenyl)pent-4-enoate
(46). To a solution of ester 45 (150 mg, 0.52 mmol) in THF (2.0
mL) was added LHMDS (0.63 mL of 1.0 M solution in THF, 0.63
mmol) at −78 °C. The reaction mixture was stirred for 20 min, and
allyl iodide (0.049 mL, 0.52 mmol) was added dropwise. The mixture
was stirred for 1 h, and saturated NH4Cl solution was added. The
mixture was poured into water and extracted with EtOAc. The organic
layer was dried over MgSO4 and concentrated under reduced pressure.
The residue was purified by flash column chromatography on silica gel
1
(EtOAc/n-hexane = 1:8) to afford 90 mg (53%) of 46: H NMR
(CDCl3, 300 MHz) δ 6.94 (s, 1H), 6.81 (s, 1H), 5.68 (m, 1H), 4.98
(m, 2H), 4.11 (dd, 1H, J = 8.2, 7.2 Hz), 3.78 (s, 6H), 3.61 (s, 3H),
2.69 (m, 1H), 2.42 (m, 1H). HRMS (FAB) calcd for C14H17BrO4
(M+): 328.0310. Found: 328.0316.
1-(1-(Benzyloxy)pent-4-en-2-yl)-2-bromo-4,5-dimethoxy-
benzene (47). To a solution of methyl ester 46 (84 mg, 0.25 mmol)
in THF (2.0 mL) was slowly added LiAlH4 (10 mg, 0.25 mmol) at 0
°C. The mixture was warmed to ambient temperature and stirred for 1
h. The mixture was then cooled to 0 °C, and saturated NaHCO3
solution was carefully added over 30 min. The mixture was stirred
vigorously for 1 h and poured into water. The mixture was extracted
with EtOAc. The organic layer was dried over MgSO4 and
concentrated under reduced pressure. The residue was purified by
flash column chromatography on silica gel (EtOAc/n-hexane = 1:6) to
1
afford the alcohol intermediate (68 mg, 88%) as a colorless oil: H
NMR (CDCl3, 500 MHz) δ 7.01 (s, 1H), 6.73 (s, 1H), 5.73 (m, 1H),
5.03 (d, 1H, J = 17.1 Hz), 4.97 (d, 1H, J = 10.1 Hz), 3.84 (s, 3H), 3.83
(s, 3H), 3.77 (d, 2H, J = 5.7 Hz), 3.41 (quin, 1H, J = 7.1 Hz), 2.50
(quin, 1H, J = 7.1 Hz), 2.37 (quin, 1H, J = 7.1 Hz). HRMS (FAB)
calcd for C13H17BrO3 (M+): 300.0361. Found: 300.0356.
To a solution of the above alcohol (400 mg, 1.33 mmol) in THF
(7.0 mL) was added NaH (64 mg, 1.59 mmol, 60% in mineral oil) at 0
°C. The reaction mixture was stirred for 30 min at ambient
temperature, and n-Bu4NBr (23 mg, 0.07 mmol) and benzyl bromide
(0.19 mL, 0.59 mmol) were added. After being stirred overnight, the
resulting mixture was treated with saturated NH4Cl (5.0 mL) solution
and extracted with EtOAc. The organic layer was washed with brine,
dried over MgSO4, and concentrated under reduced pressure. The
residue was purified by flash column chromatography on silica gel
1
(EtOAc/n-hexane = 1:8) to afford 448 mg (86%) of 47: H NMR
(CDCl3, 300 MHz) δ 7.23 (m, 5H), 6.94 (s, 1H), 6.71 (s, 1H), 5.65
(m, 1H), 4.93 (m, 2H), 4.43 (s, 2H), 3.78 (s, 3H), 3.72 (s, 3H), 3.53
Q
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(m, 3H), 2.53 (m, 1H), 2.30 (m, 1H). HRMS (FAB) calcd for
C20H23BrO3 (M+): 390.0831. Found: 390.0839.
NMR (CDCl3, 125 MHz) δ 199.6, 157.7, 149.4, 149.2, 143.3, 131.3,
130.5, 129.0, 127.4, 121.5, 120.9, 116.3, 112.4, 110.6, 101.1, 77.7, 63.4,
56.4, 55.8, 41.6, 28.4, 28.4, 26.5. HRMS (FAB) calcd for C23H24O5
(M+): 380.1624. Found: 380.1621.
4-(Benzyloxy)-3-(2-bromo-4,5-dimethoxyphenyl)butan-1-ol
(48). To a solution of olefin 47 (252 mg, 0.64 mmol) in a mixture of
acetone and water (4:1, 25 mL) were added NMO (233 mg, 1.93
mmol) and OsO4 (0.32 mL of 0.1 M solution in toluene, 0.03 mmol)
at 0 °C. The reaction mixture was stirred at ambient temperature for 6
h and extracted with EtOAc. The organic layer was washed with
saturated sodium sulfite solution, dried over MgSO4, and concentrated
under reduced pressure. The residue was dissolved in a mixture of
acetone and water (4:1, 25 mL), and NaIO4 (413 mg, 1.93 mmol) was
added. The reaction mixture was stirred for 0.5 h and extracted with
EtOAc. The organic layer was washed with saturated sodium
thiosulfate solution and concentrated under reduced pressure to give
the crude aldehyde, which was used for the next step without further
purification.
2-(3,4-Dimethoxyphenyl)acetaldehyde (52). To a solution of
methyl 3′,4′-dimethoxyphenylacetate (1.94 g, 9.24 mmol) in dry
diethyl ether was added dropwise DIBAL-H (11.1 mL of 1.0 M
solution in THF, 11.1 mmol) at −78 °C. The reaction mixture was
stirred for 1 h at the same temperature, and Rochelle’s solution was
carefully added over 15 min. The mixture was stirred vigorously for 1 h
at 0 °C and then poured into water. The mixture was extracted with
EtOAc. The organic layer was dried over MgSO4 and concentrated
under reduced pressure. The residue was purified by flash column
chromatography on silica gel (EtOAc/n-hexane = 1:2) to afford
aldehyde 52 as a yellow oil (1.13 g, 68%): 1H NMR (CDCl3,300
MHz) δ 9.66 (t, 1H, J = 2.5 Hz), 6.80 (d, 1H, J = 8.0 Hz), 6.68 (m,
1H), 6.64 (d, 1H, J = 1.8 Hz), 3.81 (s, 6H), 3.56 (d, 2H, J = 2.5 Hz).
2-(3,4-Dimethoxyphenyl)-1-(2,2-dimethyl-2H-chromen-6-
yl)ethanone (53). Ketone 53 was prepared from 6-bromo-2,2-
dimethyl-2H-chromene and aldehyde 52 according to the general
procedures C and D and purified by flash column chromatography on
silica gel (EtOAc/n-hexane = 1:3) to afford 37 mg (46% for 2 steps) of
To a solution of the above aldehyde (0.64 mmol) in methanol (6.0
mL) was added NaBH4 (49 mg, 1.29 mmol) at 0 °C. The reaction
mixture was stirred at the same temperature for 1 h, quenched with
saturated NH4Cl solution, and extracted with EtOAc. The organic
layer was dried over MgSO4 and concentrated under reduced pressure.
The residue was purified by flash column chromatography on silica gel
(EtOAc/n-hexane = 1:1) to give 184 mg (73% for two steps) of 48 as
1
pure 53 as pale yellow solid with a melting point of 111−113 °C: H
1
a yellow oil: H NMR (CDCl3, 300 MHz) δ 7.23 (m, 5H), 6.94 (s,
NMR (CDCl3,400 MHz) δ 7.76 (dd, 1H, J = 8.4, 2.2 Hz), 7.62 (d, 1H,
J = 2.2 Hz), 6.76 (m, 4H), 6.29 (d, 1H, J = 9.8 Hz), 5.61 (d, 1H, J =
9.8 Hz), 4.10 (s, 2H), 3.80 (s, 3H), 3.79 (s, 3H), 1.40 (s, 6H); 13C
NMR (CDCl3, 100 MHz) δ 196.3, 157.3, 148.8, 147.7, 131.0, 130.4,
129.4, 127.3, 127.1, 121.5, 121.4, 120.6, 116.0, 112.3, 111.2, 77.4, 55.7,
55.7, 44.5, 28.2, 28.2. HRMS (FAB) calcd for C21H23O4 (M + H+):
339.1596. Found: 339.1595.
1H), 6.69 (s, 1H), 4.47 (s, 2H), 3.78 (s, 3H), 3.73 (s, 3H), 3.54 (m,
4H), 2.01 (m, 2H), 1.81 (m, 1H). HRMS (FAB) calcd for C19H23BrO4
(M+): 394.0780. Found: 394.0774.
4-(Benzyloxymethyl)-6,7-dimethoxychroman (49). To a sol-
ution of Pd2(dba)3 (13 mg, 0.01 mmol) and 2-(di-tert-
butylphosphino)biphenyl (7 mg, 0.02 mmol) in dry toluene (10
mL) were added alcohol 48 (379 mg, 0.96 mmol) and t-BuONa (124
mg, 1.25 mmol). The reaction mixture was heated to 50−55 °C and
stirred overnight. The reaction mixture was cooled to ambient
temperature, diluted with EtOAc (10 mL), and filtered through Celite.
The filtrate was concentrated under reduced pressure and the residue
was purified by flash column chromatography on silica gel (EtOAc/n-
2-(3,4-Dimethoxyphenyl)-1-(5-methoxy-2,2-dimethyl-2H-
chromen-6-yl)ethanone (54). Ketone 54 was prepared from aryl
bromide 38 and aldehyde 52 according to the general procedures C
and D and purified by flash column chromatography on silica gel
(EtOAc/n-hexane = 1:4) to afford 80 mg (48% for 2 steps) of pure 54
1
as a pale yellow solid with a melting point of 90−92 °C: H NMR
1
hexane = 1:6) to afford 226 mg (75%) of 49: H NMR (CDCl3, 300
(CDCl3, 300 MHz) δ 7.43 (d, 1H, J = 8.4 Hz), 6.72 (s, 3H), 6.54 (d,
1H, J = 9.9 Hz), 6.52 (d, 1H, J = 8.4 Hz), 5.62 (d, 1H, J = 9.9 Hz),
4.12 (s, 2H), 3.77 (s, 6H), 3.69 (s, 3H), 1.37 (s, 6H); 13C NMR
(CDCl3, 75 MHz) δ 198.7, 157.7, 156.3, 148.8, 147.8, 131.1, 130.5,
127.6, 124.8, 121.7, 116.5, 114.8, 112.8, 112.6, 111.2, 63.2, 55.8, 55.8,
50.3, 50.2, 28.1, 28.0. HRMS (FAB) calcd for C22H25O5 (M + H+):
369.1702. Found: 369.1705.
MHz) δ 7.24 (m, 5H), 6.61 (s, 1H), 6.30 (s, 1H), 4.48 (q, 2H, J = 11.9
Hz), 4.03 (m, 2H), 3.73 (s, 3H), 3.70 (s, 3H), 3.61 (m, 1H), 3.49 (m,
1H), 2.98 (m, 1H), 1.95 (m, 2H). HRMS (FAB) calcd for C19H22O4
(M+): 314.1518. Found: 314.1518.
6,7-Dimethoxychroman-4-carbaldehyde (50). The mixture of
49 (191 mg, 0.61 mmol) and 20% Pd(OH)2/C (38 mg) in methanol
(5.0 mL) was stirred under hydrogen gas for 5 h at ambient
temperature. The reaction mixture was filtered through a short pad of
Celite, and the pad was washed with methanol (10 mL). The filtrate
was concentrated under reduced pressure. The residue was purified by
flash column chromatography on silica gel (EtOAc/n-hexane = 1:1) to
afford 158 mg (100%) of alcohol: 1H NMR (CDCl3, 300 MHz) δ 6.61
(s, 1H), 6.33 (s, 1H), 4.08 (m, 2H), 3.78 (m, 2H), 3.74 (s, 6H), 2.85
(m, 1H), 1.98 (m, 2H); LRMS (FAB) m/z 225 (M + H+).
2-(3,4-Dimethoxyphenyl)propanal (55). To a solution of
methyl 3′,4′-dimethoxyphenylacetate (4.76 g, 22.6 mmol) in THF
(5 mL) was added dropwise LDA (18.1 mL of 2.0 M solution in THF,
36.2 mmol) at −78 °C. The reaction mixture was stirred for 30 min at
the same temperature, and methyl iodide (2.82 mL, 45.3 mmol) was
added. The mixture was stirred for 1 h, acidified with 2 N HCl and
extracted with EtOAc. The organic layer was washed with brine, dried
over MgSO4, and concentrated under reduced pressure. The residue
was purified by silica gel column chromatography (EtOAc/n-hexane =
1:4) to give methyl 2-(3,4-dimethoxyphenyl)propanoate (4.01 g, 79%)
The aldehyde 50 was prepared from the above alcohol according to
the general procedure D and purified by flash column chromatography
on silica gel (EtOAc/n-hexane = 1:2) to afford 15 mg (60%) of pure
1
1
as a colorless oil: H NMR (CDCl3, 500 MHz) δ 6.80 (m, 3H), 3.85
50: H NMR (CDCl3, 300 MHz) δ 9.67 (d, 1H, J = 1.8 Hz), 6.58 (s,
(s, 3H), 3.83 (s, 3H), 3.64 (q, 1H, J = 7.1 Hz), 3.63 (s, 3H), 1.46 (d,
3H, J = 7.1 Hz); 13C NMR (CDCl3, 125 MHz) δ 175.7, 148.9, 148.1,
133.0, 119.5, 111.2, 110.6, 55.8, 55.8, 51.9, 44.9, 18.6. HRMS (FAB)
calcd for C12H16O4 (M+): 224.1049. Found: 224.1052.
1H), 6.42 (s, 1H), 4.13 (m, 3H), 3.81 (s, 6H), 2.32 (m, 1H), 2.06 (m,
1H).
(6,7-Dimethoxychroman-4-yl)(2,2-dimethyl-2H-chromen-6-
yl)methanone (51). Coupling of aldehyde 50 with 6-bromo-2,2-
dimethyl-2H-chromene according to the general procedure C afforded
the alcohol intermediate (18 mg, 75%), which was purified by flash
column chromatography on silica gel (EtOAc/n-hexane = 1:4). HRMS
(FAB) calcd for C23H26O5(M+): 382.1780. Found: 382.1793.
Ketone 51 was prepared from the above alcohol according to the
general procedure D and purified by flash column chromatography on
silica gel (EtOAc/n-hexane = 1:6) to give 9.9 mg (55%) of pure 51 as
colorless oil: 1H NMR (CDCl3, 500 MHz) δ 7.79 (dd, 1H, J = 8.5, 2.1
Hz), 7.66 (d, 1H, J = 1.9 Hz), 6.81 (d, 1H, J = 8.4 Hz), 6.42 (s, 1H),
6.34 (m, 2H), 5.66 (d, 1H, J = 9.9 Hz), 4.61 (t, 1H, J = 5.8 Hz), 4.17
(m, 2H), 3.81 (s, 3H), 3.65 (s, 3H), 2.21 (m, 2H), 1.45 (s, 6H); 13C
To a solution of methyl 2-(3,4-dimethoxyphenyl)propanoate (3.0 g,
13.4 mmol) in THF (35 mL) was added dropwise DIBAL-H (16.0 mL
of 1.0 M solution in THF, 16.0 mmol) at −78 °C. The reaction
mixture was stirred for 1 h at the same temperature, and Rochelle’s
solution was carefully added over 15 min. The mixture was stirred
vigorously for 1 h at 0 °C and poured into water. The mixture was
extracted with EtOAc, and the organic layer was dried over MgSO4
and concentrated under reduced pressure. The residue was purified by
flash column chromatography on silica gel (EtOAc/n-hexane = 1:2) to
afford 55 as a yellow oil (1.58 g, 61%): 1H NMR (CDCl3, 300 MHz) δ
9.63 (d, 1H, J = 1.5 Hz), 6.86 (d, 1H, J = 8.2 Hz), 6.74 (m, 1H), 6.66
R
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(d, 1H, J = 2.0 Hz), 3.85 (s. 6H), 3.56 (q, 1H, J = 6.9 Hz), 1.40 (d, 3H,
J = 6.9 Hz).
mL) was added LiOH·H2O (230 mg, 5.35 mmol), and the mixture was
stirred for 5 h. The reaction mixture was extracted with EtOAc, and 2
N NaOH (20 mL) was added to the organic layer. The aqueous layer
was acidified with aqueous 2 N HCl and extracted with EtOAc. The
organic layer was washed with brine, dried over MgSO4, and
concentrated under reduced pressure. The residue was used directly
2-(3,4-Dimethoxyphenyl)-1-(5-methoxy-2,2-dimethyl-2H-
chromen-6-yl)propan-1-one (56). Ketone 56 was prepared from
aryl bromide 38 and aldehyde 55 according to the general procedures
C and D and purified by flash column chromatography on silica gel
(EtOAc/n-hexane = 1:4) to afford 11 mg (64% for 2 steps) of pure 56
as a colorless thick oil: 1H NMR (CDCl3, 500 MHz) δ 7.27 (d, 1H, J =
8.5 Hz), 6.76 (m, 3H), 6.55 (d, 1H, J = 10.0 Hz), 6.48 (d, 1H, J = 8.5
Hz), 5.62 (d, 1H, J = 10.0 Hz), 4.60 (q, 1H, J = 6.9 Hz), 3.81 (s, 3H),
3.80 (s, 3H), 3.65 (s, 3H), 1.48 (d, 3H, J = 6.9 Hz), 1.39 (s, 6H); 13C
NMR (CDCl3, 75 MHz) δ 202.5, 157.1, 155.7, 149.0, 147.9, 133.8,
130.7, 130.5, 125.5, 120.2, 116.5, 112.3, 111.2, 77.2, 76.7, 63.3, 55.8,
55.7, 50.0, 43.1, 28.0, 28.0, 19.0. HRMS (FAB) calcd for C23H27O5 (M
+ H+): 383.1858. Found: 383.1853.
2-(3,4-Dimethoxyphenyl)-1-(5-methoxy-2,2-dimethyl-2H-
chromen-6-yl)-2-methylpropan-1-one (57). To a solution of 54
(15 mg, 0.04 mmol) in THF (1.0 mL) were added NaH (5 mg, 0.12
mmol, 60% in mineral oil) and CH3I (0.02 mL, 0.16 mmol). The
reaction mixture was stirred until the reaction was completed
(monitored by TLC), and extraction was with EtOAc. The organic
layer was washed with brine, dried over MgSO4, and concentrated
under reduced pressure. The residue was purified by flash column
chromatography on silica gel (EtOAc/n-hexane = 1:4) to afford 14 mg
(88%) of 57 as pale yellow solid with a melting point of 111−113 °C:
1H NMR (CDCl3, 400 MHz) δ 6.87 (m, 3H), 6.52 (d, 1H, J = 9.9
1
for the next reaction: H NMR (CDCl3, 300 MHz) δ 6.83 (m, 3H),
3.85 (s, 3H), 3.84 (s, 3H), 3.66 (q, 1H, J = 7.3 Hz), 1.48 (d, 3H, J =
7.3 Hz). HRMS (FAB) calcd for C11H14O4 (M+): 210.0892. Found:
210.0907.
(S)-2-(3,4-Dimethoxyphenyl)-N-((R)-2-hydroxy-1-
phenylethyl)propanamide (65) and (R)-2-(3,4-Dimethoxy-
phenyl)-N-((R)-2-hydroxy-1-phenylethyl)propanamide (66).
To a solution of 64 (282 mg, 1.34 mmol) in CH2Cl2 (10 mL) were
added N-(3-dimethylaminoproryl)-N′-ethylcarbodiimide hydrochlor-
ide (287 mg, 1.47 mmol), (R)-(−)-2-phenylglycinol (207 mg, 1.47
mmol), HOBt (230 mg, 1.47 mmol), and diisopropylethylamine (0.26
mL, 1.47 mmol) at 0 °C. The reaction mixture was stirred overnight at
ambient temperature, quenched with saturated NH4Cl solution, and
diluted with CH2Cl2. The organic layer was washed with water, dried
over MgSO4, and concentrated under reduced pressure. The residue
was purified by flash column chromatography on silica gel (EtOAc/
CH2Cl2/n-hexane = 3:2:1) to afford 187 mg (42%) of 65 and 186 mg
(42%) of 66 as a pale yellow solid with a melting point of 115−117 °C
(65) and 133−135 °C (66).
1
(S)-Diastereomer (65): H NMR (CDCl3, 300 MHz) δ 7.26 (m,
Hz), 6.21 (q, 2H, J = 5.4 Hz), 5.61 (d, 1H, J = 9.9 Hz), 3.88 (s, 3H),
3.83 (s, 3H), 3.70 (s, 3H), 1.50 (s, 6H), 1.37 (s, 6H); 13C NMR
(CDCl3, 100 MHz) δ 206.6, 155.1, 154.1, 148.9, 147.9, 136.2, 130.5,
127.8, 126.7, 118.2, 116.6, 115.0, 111.1, 111.0, 109.8, 77.2, 76.2, 63.6,
55.8, 55.8, 51.6, 27.9, 27.8, 26.3. HRMS (FAB) calcd for C24H29O5 (M
+ H+): 397.2015. Found: 397.2015.
3H), 7.03 (m, 2H), 6.81 (m, 2H), 6.73 (s, 1H), 6.05 (m, 1H), 5.02 (m,
1H), 3.86 (s, 3H), 3.80 (m, 2H), 3.78 (s, 3H), 3.58 (q, 1H, J = 7.1 Hz)
1.48 (d, 3H, J = 7.1 Hz). HRMS (FAB) calcd for C19H24NO4 (M +
H+): 330.1705. Found: 330.1696.
1
(R)-Diastereomer (66): H NMR (CDCl3, 300 MHz) δ 7.23 (m,
3H), 7.10 (m, 2H), 6.78 (m, 3H), 5.97 (m, 1H), 4.96 (m, 1H), 3.82 (s,
3H), 3.80 (s, 3H), 3.72 (d, 2H, J = 5.0 Hz), 3.50 (q, 1H, J = 7.1 Hz)
1.46 (d, 3H, J = 7.1 Hz). HRMS (FAB) calcd for C19H24NO4 (M +
H+): 330.1705. Found: 330.1712.
2-(3,4-Dimethoxyphenyl)-1-(5-methoxy-2,2-dimethyl-2H-
chromen-6-yl)prop-2-en-1-one (58). To a solution of ketone 54
(20 mg, 0.05 mmol) in anhydrous DMF (1 mL) were added K2CO3
(15 mg, 0.11 mmol) and paraformaldehyde (3 mg, 0.08 mmol). The
reaction mixture was stirred for 4 h and then extracted with EtOAc.
The organic layer was washed with saturated NH4Cl solution, brine,
dried over MgSO4, and concentrated under reduced pressure. The
residue was purified by flash column chromatography on silica gel
(EtOAc/n-hexane = 1:4) to afford 18 mg (87%) of 58 as a colorless
oil: 1H NMR (CDCl3, 300 MHz) δ 7.30 (d, 1H, J = 8.6 Hz), 6.95 (m,
2H), 6.78 (d, 1H, J = 8.0 Hz), 6.51 (m, 2H), 5.90 (s, 1H), 5.60 (d, 1H,
J = 10.0 Hz), 5.55 (s, 1H), 3.81 (s, 3H), 3.80 (s, 3H), 3.70 (s, 3H),
1.38 (s, 6H); 13C NMR (CDCl3, 100 MHz) δ 196.5, 157.3, 156.2,
149.4, 149.1, 148.6, 131.7, 130.4, 129.7, 125.0, 121.7, 120.2, 116.5,
114.8, 111.8, 110.9, 110.7, 77.2, 76.8, 63.1, 55.8, 28.0, 27.9. HRMS
(FAB) calcd for C23H25O5 (M + H+): 381.1702. Found: 381.1709.
1-(3,4-Dimethoxyphenyl)cyclopropyl)(5-methoxy-2,2-di-
methyl-2H-chromen-6-yl)methanone (59). To anhydrous DMSO
(0.5 mL) were added NaH (1.2 mg, 0.03 mmol, 60% in mineral oil)
and trimethylsulfoxonium iodide (6.5 mg, 0.03 mmol). The resulting
mixture was stirred for 40 min, followed by an addition of a solution of
α,β-unsaturated ketone 58 (10 mg, 0.03 mmol) in anhydrous DMSO
(0.5 mL). After being stirred for 1 h, the reaction mixture was
quenched with a saturated NH4Cl solution and extracted with EtOAc.
The organic layer was washed with saturated NH4Cl solution, brine,
dried over MgSO4, and concentrated under reduced pressure. The
residue was purified by flash column chromatography on silica gel
(S)-2-(3,4-Dimethoxyphenyl)propanoic Acid (67). To a
solution of 65 (186 mg, 0.56 mmol) in 1,4-dioxane (5.0 mL) was
slowly added H2SO4 (5.0 mL, 4.0 M in water) at 0 °C. The reaction
mixture was refluxed for 2 h, cooled to ambient temperature, and
diluted with EtOAc. The organic layer was washed with water, dried
over MgSO4, and concentrated under reduced pressure to afford
carboxylic acid 67 (117 mg, 100%) as a brown oil: 1H NMR (CDCl3,
300 MHz) δ 6.83 (m, 3H), 3.86 (s, 3H), 3.84 (s, 3H), 3.67 (q, 1H, J =
7.1 Hz), 1.47 (d, 3H, J = 7.1 Hz). HRMS (FAB) calcd for C11H14O4
(M+): 210.0892. Found: 210.0903; [α]D25 +70° (c 0.2, CH2Cl2).
(S)-2-(3,4-Dimethoxyphenyl)propanal (68). A solution of (S)-
67 (115 mg, 0.55 mmol) in dry diethyl ether (8.0 mL) was cooled to 0
°C, and BH3·SMe2 complex (1.5 mL, 3.0 mmol) was added dropwise.
The reaction mixture was stirred for 1 h at the same temperature and
then stirred 3 h at ambient temperature. The mixture was quenched
with water at 0 °C, and 2 N NaOH was added dropwise until the
evolution of H2 gas was not observed. The aqueous layer was extracted
with ether, and the organic layer was washed with brine, dried over
MgSO4, and concentrated under reduced pressure. The residue was
purified by flash column chromatography on silica gel (EtOAc/n-
hexane = 1:2) to afford 104 mg (85%) of the alcohol intermediate as a
colorless oil. The enantiomeric excess (95% ee) was determined by
chiral HPLC (Chiralcel OD-H, isopropanol/n-hexane = 1:3, 1.0 mL/
min): 1H NMR (CDCl3, 500 MHz) δ 6.78 (m, 3H), 3.87 (s, 3H), 3.85
(s, 3H), 3.66 (m, 2H), 2.88 (sex, 1H, J = 6.9 Hz), 1.24 (d, 3H, J = 6.9
Hz). HRMS (FAB) calcd for C11H16O3 (M+): 196.1099. Found:
196.1106.
1
(EtOAc/n-hexane = 1:4) to afford 9 mg (91%) of 59 as white oil: H
NMR (CDCl3, 500 MHz) δ 6.98 (d, 1H, J = 8.4 Hz), 6.78 (m, 2H),
6.67 (d, 1H, J = 8.4 Hz), 6.48 (d, 1H, J = 10.0 Hz), 6.35 (d, 1H, J = 8.4
Hz), 5.58 (d, 1H, J = 10.0 Hz), 3.78 (s, 3H), 3.75 (s, 3H), 3.73 (s,
3H), 1.67 (q, 2H, J = 4.0 Hz), 1.35 (s, 6H), 1.28 (q, 2H, J = 4.0 Hz);
13C NMR (CDCl3, 125 MHz) δ 203.6, 155.4, 153.9, 148.4, 147.8,
133.0, 130.4, 128.7, 126.3, 121.4, 116.5, 114.7, 113.6, 111.4, 110.6,
76.2, 63.1, 55.7, 55.7, 37.1, 27.8, 27.8, 17.7, 17.7. HRMS (FAB) calcd
for C24H26O5 (M+): 394.1780. Found: 394.1774.
(S)-Aldehyde 68 was prepared from the above alcohol according to
the general procedure D and purified by flash column chromatography
on silica gel (EtOAc/n-hexane = 1:3) to afford 21 mg (85%) of pure
1
(S)-68 as a pale yellow oil: H NMR (CDCl3, 300 MHz) δ 9.63 (d,
1H, J = 1.5 Hz), 6.86 (d, 1H, J = 8.2 Hz), 6.74 (dd, 1H, J = 8.2, 2.0
Hz), 6.66 (d, 1H, J = 2.0 Hz), 3.86 (s, 6H), 3.55 (q, 1H, J = 7.1 Hz),
1.40 (d, 3H, J = 7.1 Hz).
2-(3,4-Dimethoxyphenyl)propanoic Acid (64). To a solution of
ester 63 (400 mg, 1.78 mmol) in a mixture of THF and water (2:1, 9.0
S
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Journal of Medicinal Chemistry
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(S)-2-(3,4-Dimethoxyphenyl)-1-(5-methoxy-2,2-dimethyl-
2H-chromen-6-yl)propan-1-one (69). CeCl3·7H2O (189 mg, 0.52
mmol) in a one-necked flask was heated gradually to 135−140 °C
under reduced pressure (0.1 Torr) and stirred for 2 h. While the flask
was still hot, argon gas was injected into the flask, which was then
cooled in an ice bath. THF (1.0 mL) was added all at once with
vigorous stirring. The ice bath was removed, and the suspension was
stirred overnight at ambient temperature. The aryl anion, which was
generated by an addition of n-BuLi (0.13 mL of 1.6 M solution in n-
hexane, 0.21 mmol) to 38 (61 mg, 0.23 mmol) in THF (1.0 mL) at
−78 °C, was added at −78 °C. After the mixture was stirred for 1.5 h
at −78 °C, aldehyde 68 (20 mg, 0.10 mmol) was added and the
reaction mixture was stirred for 30 min. The reaction mixture was
quenched with saturated NH4Cl solution and extracted with EtOAc.
The organic layer was washed with saturated NaHCO3 solution, brine,
dried over MgSO4, and concentrated under reduced pressure. The
residue was purified by flash column chromatography on silica gel
(EtOAc/n-hexane = 1:3) to afford the corresponding alcohol (32 mg,
(s, 3H), 3.66 (m, 2H), 2.88 (sex, 1H, J = 6.9 Hz), 1.24 (d, 3H, J = 6.9
Hz). HRMS (FAB) calcd for C11H16O3 (M+): 196.1099. Found:
196.1112.
(R)-Aldehyde 71 was prepared from the above alcohol according to
the general procedure D and purified by flash column chromatography
on silica gel (EtOAc/n-hexane = 1:3) to give 16 mg (79%) of pure
1
(R)-71 as a pale yellow oil: H NMR (CDCl3, 300 MHz) δ 9.63 (d,
1H, J = 1.5 Hz), 6.86 (d, 1H, J = 8.2 Hz), 6.74 (dd, 1H, J = 8.2, 2.0
Hz), 6.66 (d, 1H, J = 2.0 Hz), 3.86 (s, 6H), 3.55 (q, 1H, J = 7.1 Hz),
1.40 (d, 3H, J = 7.1 Hz).
(R)-2-(3,4-Dimethoxyphenyl)-1-(5-methoxy-2,2-dimethyl-
2H-chromen-6-yl)propan-1-one (72). The alcohol intermediate
(12 mg, 48%) was prepared as a colorless oil from 38 (36 mg, 0.14
mmol) and aldehyde 71 (13 mg, 0.07 mmol) by the same procedure
for the alcohol intermediate of ketone 69. Isomer A: 1H NMR
(acetone-d6, 300 MHz) δ 7.22 (d, 1H, J = 8.4 Hz), 6.73 (m, 3H), 6.50
(m, 2H), 5.69 (d, 1H, J = 9.9 Hz), 4.95 (m, 1H), 3.70 (s, 6H), 3.61 (s,
3H), 3.07 (quin, 1H, J = 6.8 Hz), 1,35 (s, 6H), 1.28 (d, 3H, J = 6.9
Hz). Isomer B: 1H NMR (acetone-d6, 500 MHz) δ 7.02 (d, 1H, J = 8.4
Hz), 6.75 (m, 3H), 6.55 (d, 1H, J = 10.0 Hz), 6.43 (d, 1H, J = 8.5 Hz),
5.70 (d, 1H, J = 9.9 Hz), 4.93 (dd, 1H, J = 7.5, 4.0 Hz), 3.72 (s, 3H),
3.72 (s, 3H), 3.71 (s, 3H), 2.95 (quin, 1H, J = 7.2 Hz), 1.34 (d, 6H, J =
11.8 Hz), 1.06 (d, 3H, J = 7.2 Hz).
1
80%, diastereomeric mixture) as colorless oil. Isomer A: H NMR
(acetone-d6, 300 MHz) δ 7.22 (d, 1H, J = 8.4 Hz), 6.73 (m, 3H), 6.50
(m, 2H), 5.69 (d, 1H, J = 9.9 Hz), 4.95 (m, 1H), 3.70 (s, 6H), 3.61 (s,
3H), 3.07 (quin, 1H, J = 6.8 Hz), 1,35 (s, 6H), 1.28 (d, 3H, J = 6.9
Hz). Isomer B: 1H NMR (acetone-d6, 500 MHz) δ 7.02 (d, 1H, J = 8.4
Hz), 6.75 (m, 3H), 6.55 (d, 1H, J = 10.0 Hz), 6.43 (d, 1H, J = 8.5 Hz),
5.70 (d, 1H, J = 9.9 Hz), 4.93 (dd, 1H, J = 7.5, 4.0 Hz), 3.72 (s, 3H),
3.72 (s, 3H), 3.71 (s, 3H), 2.95 (quin, 1H, J = 7.2 Hz), 1,34 (d, 6H, J =
11.8 Hz), 1.06 (d, 3H, J = 7.2 Hz).
Ketone 72 (10 mg, 71%) was prepared as a colorless oil from the
above alcohol (14 mg, 0.036 mmol) by the same procedure as for
ketone 69. The enantiomeric excess (95% ee) was determined by
chiral HPLC (Chiralpak AD-H, isopropanol/n-hexane = 1:9, 1.0 mL/
1
To a solution of the above alcohol (32 mg, 0.08 mmol) in CH2C12
(2.0 mL) were added 4 Å molecular sieves (41.0 mg) and N-
methylmorpholine N-oxide (15 mg, 0.13 mmol). The reaction mixture
was stirred for 10 min, and tetrapropylammonium perruthenate (3 mg,
0.01 mmol) was added. When the reaction was completed (monitored
by TLC), the reaction mixture was diluted with CH2Cl2. The mixture
was washed with 10% sodium sulfite solution, brine, and saturated
copper(II) sulfate solution. The organic layer was dried over MgSO4
and concentrated under reduced pressure. The residue was purified by
flash column chromatography on silica gel (EtOAc/n-hexane = 1:4) to
afford ketone 69 as a colorless oil (27 mg, 84%). The enantiomeric
excess (97% ee) was determined by chiral HPLC (Chiralpak AD-H,
min): H NMR (CDCl3, 500 MHz) δ 7.27 (d, 1H, J = 8.5 Hz), 6.76
(m, 3H), 6.55 (d, 1H, J = 10.0 Hz), 6.48 (d, 1H, J = 8.5 Hz), 5.62 (d,
1H, J = 10.0 Hz), 4.60 (q, 1H, J = 6.9 Hz), 3.81 (s, 3H), 3.80 (s, 3H),
3.65 (s, 3H), 1.48 (d, 3H, J = 6.9 Hz), 1.39 (s, 6H); 13C NMR
(CDCl3, 75 MHz) δ 202.5, 157.1, 155.7, 149.0, 147.9, 133.8, 130.7,
130.5, 125.5, 120.2, 116.5, 112.3, 111.2, 77.2, 76.7, 63.3, 55.8, 55.7,
50.0, 43.1, 28.0, 28.0, 19.0. HRMS (FAB) calcd for C23H27O5 (M +
H+): 383.1858. Found: 383.1849.
Biological Assay. MTS Assay and Western Blot Analysis of
HIF-1α Expression. To analyze the antiproliferation effect of
deguelin and its analogues against the non-small-cell lung cancer
(NSCLC), H1299 cells were plated in 96-well plates at a density of 5
× 103 cells per well. Next day, the cells were treated with 0.1% DMSO
as a dilution control or with various concentrations of deguelin and the
tested analogues. Cell proliferation was assessed by the 3-(4,5-
dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-
2H-tetrazolium (MTS) assay after 3 days. At least three replicate wells
were used for each analysis. Western blot analysis of HIF-1α was
utilized to evaluate the HSP90 inhibitory activities of the analogues
after the same process. H1299 cells were untreated or treated with
deguelin and the analogues at each concentration for 72 h, followed by
incubation under hypoxic conditions (1% O2) for 12 h. The resulting
cell lysates were used for Western blot analysis with a monoclonal
antibody against HIF-1α (BD Pharmingen).
Immunoprecipition and Immunoblotting Assay. In vitro assay
was performed using the lysates of H1299 cells that had been treated
with 100 μM CoCl2 for 5 h. The cell lysates were incubated with 1.25
μM of analogue 54 or 69 in the presence or absence of 20 mM ATP
for 30 min at 37 °C. The resulting samples were incubated with anti-
HIF-1α antibody overnight at 4 °C. The antigen−antibody complex
was then precipitated following incubation for 2 h at 4 °C with protein
G-agarose. The immune complex was solubilized in 2× Laemmli buffer
and boiled for 5 min. The samples were resolved and analyzed using
6% SDS−PAGE and then transferred to nitrocellulose membrane.
They were then immunoblotted with the antibody directed against
HSP90.
Antiangiogenic Effect on Zebrafish Embryos. Zebrafish
embryos were used to determine the antiangiogenic effects of the
tested compounds. The transgenic line Tg(fli1:EGFP)y1 zebrafish were
obtained from the Zebrafish International Resource Center, University
of Oregon, Eugene, OR (Lawson and Weinstein, 2002). Zebrafish
embryos were generated by natural pairwise mating and raised at 28.5
°C in Danieau’s solution. The Tg(fli1:EGFP)y1 embryos were
1
isopropanol/n-hexane = 1:9, 1.0 mL/min): H NMR (CDCl3, 500
MHz) δ 7.27 (d, 1H, J = 8.5 Hz), 6.76 (m, 3H), 6.55 (d, 1H, J = 10.0
Hz), 6.48 (d, 1H, J = 8.5 Hz), 5.62 (d, 1H, J = 10.0 Hz), 4.60 (q, 1H, J
= 6.9 Hz), 3.81 (s, 3H), 3.80 (s, 3H), 3.65 (s, 3H), 1.48 (d, 3H, J = 6.9
Hz), 1.39 (s, 6H); 13C NMR (CDCl3, 75 MHz) δ 202.5, 157.1, 155.7,
149.0, 147.9, 133.8, 130.7, 130.5, 125.5, 120.2, 116.5, 112.3, 111.2,
77.2, 76.7, 63.3, 55.8, 55.7, 50.0, 43.1, 28.0, 28.0, 19.0. HRMS (FAB)
calcd for C23H27O5 (M + H+): 383.1858. Found: 383.1853.
(R)-2-(3,4-Dimethoxyphenyl)propanoic Acid (70). To a
solution of 66 (217 mg, 0.66 mmol) in 1,4-dioxane (5.6 mL) was
slowly added H2SO4 (5.6 mL, 4.0 M in water) at 0 °C. The reaction
mixture was refluxed for 2 h, cooled to ambient temperature, and
diluted with EtOAc. The organic layer was washed with water, dried
over MgSO4, and concentrated under reduced pressure to afford acid
1
70 (138 mg, 100%) as a brown oil: H NMR (CDCl3, 300 MHz) δ
6.83 (m, 3H), 3.86 (s, 3H), 3.84 (s, 3H), 3.67 (q, 1H, J = 7.1 Hz), 1.47
(d, 3H, J = 7.1 Hz). HRMS (FAB) calcd for C11H14O4 (M+):
210.0892. Found: 210.0911; [α]2D5 −73° (c 0.2, CH2Cl2).
(R)-2-(3,4-Dimethoxyphenyl)propanal (71). To a stirred
solution of acid (R)-70 (138 mg, 0.66 mmol) in dry diethyl ether
(10 mL) was added dropwise BH3·SMe2 complex (1.8 mL, 3.61
mmol) at 0 °C. The reaction mixture was stirred for 1 h at the same
temperature and then for 3 h at ambient temperature. The reaction
mixture was quenched with water and then 2 N NaOH at 0 °C. The
aqueous layer was extracted with ether, and the organic layer was
washed with brine, dried over MgSO4, and concentrated in reduced
pressure. The residue was purified by flash column chromatography on
silica gel (EtOAc/n-hexane = 1:2) to afford 104 mg (81%) of alcohol
as colorless oil. The enantiomeric excess (97% ee) was determined by
chiral HPLC (Chiralcel OD-H, isopropanol/n-hexane = 1:3, 1.0 mL/
min): 1H NMR (CDCl3, 500 MHz) δ 6.78 (m, 3H), 3.87 (s, 3H), 3.85
T
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Article
maintained in Danieau’s solution at 28.5 °C, and the embryos were
sorted for viability and the developmental stage (shield stage) at 6 hpf
(hours postfertilization). The embryos were treated with 1-phenyl-2-
thiourea (Sigma) to inhibit pigment formation at 12 hpf. The embryos
were placed into each well of a 24-well plate containing 1 mL of
Danieau’s solution with or without the tested compounds at 24 hpf.
The Danieau’s solution was washed out at 48 hpf and examined for
viability and gross morphological abnormalities. At 72 hpf, the
embryos were anesthetized in tricaine (Sigma) and mounted in 6%
methylcellulose (Sigma). The embryos were then photographed under
a Leica DM5000B fluorescence microscope (Leica Microsystems,
Wetzlar GmbH, Germany).
overexpression via a HER3, phosphatidylinositol 3′-kinase-AKT-
dependent pathway. Cancer Res. 2002, 62, 3132−3137. (d) Basso, A.
D.; Solit, D. B.; Chiosis, G.; Giri, B.; Tsichlis, P.; Rosen, N. Akt forms
an intracellular complex with heat shock protein 90 (Hsp90) and
Cdc37 and is destabilized by inhibitors of Hsp90 function. J. Biol.
Chem. 2002, 277, 39858−39866. (e) Isaacs, J. S.; Jung, Y. J.;
Mimnaugh, E. G.; Martinez, A.; Cuttitta, F.; Neckers, L. M. Hsp90
regulates a von Hippel Lindau-independent hypoxia-inducible factor-
1alpha-degradative pathway. J. Biol. Chem. 2002, 277, 29936−29944.
(f) Beliakoff, J.; Bagatell, R.; Paine-Murrieta, G.; Taylor, C. W.;
Lykkesfeldt, A. E.; Whitesell, L. Hormone-refractory breast cancer
remains sensitive to the antitumor activity of heat shock protein 90
inhibitors. Clin. Cancer Res. 2003, 9, 4961−4971. (g) Eustace, B. K.;
Sakurai, T.; Stewart, J. K.; Yimlamai, D.; Unger, C.; Zehetmeier, C.;
Lain, B.; Torella, C.; Henning, S. W.; Beste, G.; Scroggins, B. T.;
Neckers, L.; Ilag, L. L.; Jay, D. G. Functional proteomic screens reveal
an essential extracellular role for hsp90 alpha in cancer cell
invasiveness. Nat. Cell Biol. 2004, 6, 507−514.
ASSOCIATED CONTENT
* Supporting Information
■
S
HPLC data of the final compounds and supplementary figures
on the zebrafish angiogenesis model. This material is available
free of charge via the Internet at http://pubs.acs.org.
(5) Kim, J. H.; Yu, Y. S.; Park, K. H.; Kang, H. J.; Lee, H. Y.; Kim, K.
W. Antiangiogenic effect of deguelin on choroidal neovascularization.
J. Pharmacol. Exp. Ther. 2008, 324, 643.
AUTHOR INFORMATION
Corresponding Author
*Phone: 82-2-880-7875. Fax: 82-2-888-0649. E-mail: ygsuh@
snu.ac.kr.
■
(6) Folkman, J. Angiogenesis: an organizing principle for drug
discovery? Nat. Rev. Drug Discovery 2007, 6, 273−286.
(7) Pearl, L. H.; Prodromou, C. Structure and in vivo function of
Hsp90. Curr. Opin. Struct. Biol. 2000, 10, 46−51.
(8) Stebbins, C. E.; Russo, A. A.; Schneider, C.; Rosen, N.; Hartl, F.
U.; Pavletich, N. P. Crystal structure of an Hsp90-geldanamycin
complex: targeting of a protein chaperone by an antitumor agent. Cell
1997, 89, 239−250.
(9) (a) Whitesell, L.; Mimnaugh, E. G.; Decosta, B.; Myers, C. E.;
Neckers, L. M. Inhibition of heat-shock protein Hsp90-PP60(v-src)
heteroprotein complex-formation by benzoquinone ansamycins:
essential role for stress proteins in oncogenic transformation. Proc.
Natl. Acad. Sci. U.S.A. 1994, 91, 8324−8328. (b) Supko, J. G.;
Hickman, R. L.; Grever, M. R.; Malspeis, L. Preclinical and
pharmacological evaluation of geldanamycin as an antitumor agent.
Cancer Chemother. Pharmacol. 1995, 36, 305−315. (c) Neckers, L.;
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by a National Research Foundation
grant funded by the Korea Government (MEST) (Grant No.
2012-0006019) and by the Global Core Research Center
(GCRC) grant (Grant No. 2012-0001187) from the National
Research Foundation (NRF), Ministry of Education, Science
and Technology (MEST), Republic of Korea.
ABBREVIATIONS USED
■
HSP, heat shock protein; HIF, hypoxia-inducible factor; VEGF,
vascular endothelial growth factor; DR, diabetic retinopathy;
AMD, age-related macular degeneration; FP, fluorescence
polarization; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy-
methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; NSCLC,
non-small-cell lung cancer; DHP, dihydropyran; PTSA, p-
toluenesulfonic acid; DMP, Dess−Martin periodinane; DIAD,
diisopropyl azodicarboxylate; EDC, 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide; HOBt, 1-hydroxybenzo-
triazole; SIV, subintestinal vein; hpf, hour postfertilization;
CSA, camphor sulfonic acid
(10) Schulte, T. W.; Neckers, L. M. The benzoquinone ansamycin
17-allylamino-17-demethoxygeldanamycin binds to HSP90 and shares
important biologic activities with geldanamycin. Cancer Chemother.
Pharmacol. 1998, 42, 273−279.
(11) (a) Roe, S. M.; Prodromou, C.; O’Brien, R.; Ladbury, J. E.;
Piper, P. W.; Pearl, L. H. Structural basis for inhibition of the Hsp90
molecular chaperone by the antitumor antibiotics radicicol and
geldanamycin. J. Med. Chem. 1999, 42, 260−266. (b) Schulte, T. W.;
Akinaga, S.; Soga, S.; Sullivan, W.; Stensgard, B.; Toft, D.; Neckers, L.
M. Antibiotic radicicol binds to the N-terminal domain of Hsp90 and
shares important biologic activities with geldanamycin. Cell Stress
Chaperones. 1998, 3, 100−108. (c) Soga, S.; Kozawa, T.; Narumi, H.;
Akinaga, S.; Irie, K.; Matsumoto, K.; Sharma, S. V.; Nakano, H.;
Mizukami, T.; Hara, M. Radicicol leads to selective depletion of Raf
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(12) Soga, S.; Shiotsu, Y.; Akinaga, S.; Sharma, S. V. Development of
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Zheng, F. F.; Sepp-Lorenzino, L.; Rosen, N. A small molecule designed
to bind to the adenine nucleotide pocket of Hsp90 causes Her2
degradation and the growth arrest and differentiation of breast cancer
cells. Chem. Biol. 2001, 8, 289−299. (b) Chiosis, G.; Lucas, B.; Shtil,
A.; Huezo, H.; Rosen, N. Development of a purine-scaffold novel class
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induce the degradation of Her2 tyrosine kinase. Bioorg. Med. Chem.
2002, 10, 3555−3564. (c) Chiosis, G.; Rosen, N.; Sepp-Lorenzino, L.
LY294002-geldanamycin heterodimers as selective inhibitors of the
PI3K and PI3K-related family. Bioorg. Med. Chem. Lett. 2001, 11, 909−
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