Synthesis and structure-activity relationships of EGCG analogues, a recently identified Hsp90 inhibitor

Article abstract of DOI:10.1021/jo401027r

Epigallocatechin-3-gallate (EGCG), the principal polyphenol isolated from green tea, was recently shown to inhibit Hsp90; however, structure-activity relationships for this natural product have not yet been produced. Herein, we report the synthesis and biological evaluation of EGCG analogues to establish structure-activity relationships between EGCG and Hsp90. All four rings as well as the linker connecting the C- and the D-rings were systematically investigated, which led to the discovery of compounds that inhibit Hs90 and display improvement in efficacy over EGCG. Antiproliferative activity of all the analogues was determined against MCF-7 and SKBr3 cell lines and Hsp90 inhibitory activity of the four most potent analogues was further evaluated by Western blot analyses and degradation of Hsp90-dependent client proteins. The prenyl-substituted aryl ester of 3,5-dihydroxychroman-3-ol ring system was identified as a novel scaffold that exhibits Hsp90 inhibitory activity.

Full text of DOI:10.1021/jo401027r

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Article
Synthesis and Structure–Activity Relationships of
EGCG Analogues, A Recently Identified Hsp90 Inhibitor
Anuj Khandelwal, Jessica Hall, and Brian S.J. Blagg
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/jo401027r • Publication Date (Web): 08 Jul 2013
Downloaded from http://pubs.acs.org on July 9, 2013
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Synthesis and Structure activity relationships of EGCG Analogues, A Recently
Identified Hsp90 Inhibitor
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Anuj Khandelwal, Jessica Hall and Brian S. J. Blagg*
Department of Medicinal Chemistry, 1251 Wescoe Hall Drive, Malott 4070, The University of
Kansas, Lawrence, Kansas 66045ꢀ7563, United States
Email: bblagg@ku.edu
Abstract: Epigallocatechinꢀ3ꢀgallate (EGCG), the principal polyphenol isolated from green tea,
was recently shown to inhibit Hsp90, however structureꢀactivity relationships for this natural
product have not yet been produced. Herein, we report the synthesis and biological evaluation of
EGCG analogues to establish structureꢀactivity relationships between EGCG and Hsp90. All four
rings as well as the linker connecting the Cꢀ and the Dꢀrings were systematically investigated,
which led to the discovery of compounds that inhibit Hs90 and display improvement in efficacy
over EGCG. Antiꢀproliferative activity of all the analogues was determined against MCFꢀ7 and
SKBr3 cell lines and Hsp90 inhibitory activity of four most potent analogues was further
evaluated by western blot analyses and degradation of Hsp90ꢀdependent client proteins. Prenyl
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substituted aryl ester of 3,5ꢀdihydroxychromanꢀ3ꢀol ring system was identified as novel scaffold
that exhibit Hsp90 inhibitory activity.
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INTRODUCTION
Heat shock protein 90 (Hsp90) is ubiquitously expressed and essential for the folding of many
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ꢀ4
nascent polypeptides. As a molecular chaperone, Hsp90 regulates the conformational
maturation of more than 200 client proteins, including steroid hormone receptors, Akt, Rafꢀ1 and
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the Srcꢀfamily kinases. Many of these Hsp90ꢀdependent client proteins regulate signaling
pathways associated with cell survival, cell proliferation, as well as cellular transformation and
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, 7
oncogenesis. Prior studies have shown that Hsp90 is upregulated in malignant cells and that
Hsp90 inhibitors accumulate more efficiently in tumor cells than in the surrounding normal
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tissue. Consequently, Hsp90 inhibition represents a multiꢀfaceted approach toward the treatment
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, 10
of cancer.
Natural products represent a class of diverse structures that contribute to clinically
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relevant therapeutics.
They serve as lead compounds and/or scaffolds upon which molecules
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with improved efficacy and drugability can be pursued. Structureꢀactivity relationships studies
on natural products have led to the identification of structurally less complex molecules that are
clinically used today. (ꢀ)ꢀEpigallocatechinꢀ3ꢀgallate (EGCG (1)) is a polyphenolic natural
product that can be isolated from green tea leaves and has been shown to inhibit Hsp90’s
function and induce the degradation of client proteins; including telomerase, multiple kinases
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4ꢀ16
and the aryl hydrocarbon receptor (AhR).
Palermo and coworkers demonstrated through
affinity chromatography that (ꢀ)ꢀEGCG binds to amino acids 538ꢀ728 within the Hsp90 Cꢀ
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terminus and inhibits AhRꢀmediated transcription through interactions with Hsp90.
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Unfortunately, the exact mechanism by which EGCG inhibits the Hsp90 protein folding
machinery remains undetermined. Similar to EGCG, novobiocin (2) also binds Hsp90 within
amino acids 538ꢀ728 and represents another naturally occurring Cꢀterminal inhibitor (Figure 1).
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The bioavailability and lipophilicity exhibited by EGCG along with its metabolically
susceptible functionalities and modest efficacy against various cancer cell lines make EGCG a
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poor lead compound for development. However, only two natural products are known to inhibit
the Hsp90 Cꢀterminus, and therefore EGCG was pursued as a probe to further investigate the
mechanism by which Cꢀterminal inhibitors modulate the Hsp90 protein folding machinery.
Figure 1. Hsp 90 Cꢀterminal inhibitors
EGCG is well known for its antioxidant activity both in vitro and in vivo, which also
leads to epimerization and/or dimerization (Scheme 1) and contributes to its low efficacy and
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metabolic instability.
Epimerization of the methine hydrogen leads to formation of the
thermodynamically more stable anti product, GCG (Figure 2), whose activity against Hsp90 has
not been investigated. Studies by Suzuki and coꢀworkers have shown that incorporation of
hydroxyl groups onto the Bꢀring can lead to epimerization at Cꢀ2, whereas Oꢀmethylated
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derivatives at the 4ꢀposition prevent epimerization. Therefore, the design of new EGCG
analogues must take into account these prior studies in an effort to produce stable derivatives that
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are not prone to oxidation/epimerization.
To probe EGCG’s structureꢀactivity relationships
with Hsp90, three series of analogues (Scheme 2) were pursued; (I) 3’,4’,5’ꢀtrimethoxy groups
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were incorporated into the Bꢀring, (II) compounds omitting substituents on the Bꢀring were
prepared, and (III) compounds lacking the Bꢀring were also constructed. Furthermore, the
phenols on the Aꢀring were converted to methyl ethers for biological evaluation and finally, the
gallic acid moiety (Dꢀring) of EGCG was replaced with various aryl acids for elucidation of
additional SAR trends. These aryl acids were chosen to probe the effect of substitution at the 3ꢀ
and 4ꢀposition of the Dꢀring and to incorporate optimized novobiocin appendages to evaluate
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their potential for overlapping binding modes.
Scheme 1. (a) Epimerization of EGCG to GCC, (b) Autoꢀoxidation products of EGCG.
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Scheme 2. (a) Scaffolds derived from EGCG for Hsp90 inhibition, (b) Aryl acids used to replace
the gallic acid moiety.
RESULTS AND DISCUSSION
Synthesis of the Aꢀ, Bꢀ and Dꢀring modified compounds (10a–j & 11a–j) are described in
Scheme 3. Prior work by Li and coworkers provided rapid access towards preparation of the
flavonꢀ3ꢀol core, enlisting the use of a silica/sulfuric acid catalyst to couple electron rich phenols
(4a–b) with substituted cinnamyl alcohols (5a–b), which worked surprisingly well and led to
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various substituted Aꢀ and Bꢀring analogues (6a–d). Dihydroxylation of the resulting alkenes
(6a–d) with catalytic osmium tetroxide and excess NꢀmethylmorpholineꢀNꢀoxide gave the
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corresponding diol’s, 7a–d. Various methods have been reported for cyclization and
construction of the benzopyran core, however, stereochemical control at the 2,3ꢀring junction is
dependent upon substituents on the Bꢀring. Therefore, cylization of diol’s 7a–d to furnish the
2,3ꢀdihydrobenzopyran core in a stereoselective manner was pursued via two steps. Treatment of
7a–d with trimethylorthoacetate in the presence of catalytic pyridinium pꢀtoluenesulfonate, led to
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formation of the corresponding orthoesters, which upon the addition of 10% boron trifluoride
diethyl etherate produced the desired cyclic products. Without purification, the cyclized products
were subjected to solvolysis conditions to furnish alcohols 8a–d in high yields and with the anti
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configuration. The 2,3ꢀsyn products, 9a–d, were established by DessꢀMartin oxidation of the
secondary alcohols (8a–d) to the corresponding ketones, which underwent subsequent reduction
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with L–selectride to give syn products, 9a–d, respectively. These flavonꢀ3ꢀol moieties (9a–d)
served as late stage intermediates to incorporate additional substitutions onto the Dꢀring. Aryl
acids 12–16 (Scheme 2) were chosen as replacements for the metabolically susceptible gallic
ester moiety of EGCG and also represent optimized side chains identified from prior studies with
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the other Hsp90 Cꢀterminal inhibitor, novobiocin.
Coupling of the alcohols (9a–d) with
aromatic acids 12–16 enlisting 1ꢀethylꢀ3ꢀ(3ꢀdimethylaminopropyl) carbodiimide (EDCI) and 4ꢀ
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dimethylaminopyrine (DMAP) gave the corresponding esters, 10a–t. Hydrogenolysis of 10k–t
with palladium/carbon and hydrogen gas gave 11a–j in high yield.
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Scheme 3. Synthesis of EGCG analogues containing modifications to the Aꢀ,Bꢀ and Dꢀrings
Upon preparation of the Aꢀ, Bꢀ and the Dꢀring modified EGCG analogues (10a–j and
11a–j), these compounds were evaluated against MCFꢀ7 and SKBr3 breast cancer cell lines for
determination of their antiꢀproliferative activities (Table 1). The SKBr3 (estrogen receptor
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negative, Her2 overexpressing) and the MCFꢀ7 (estrogen receptor positive) cell lines were
chosen due to the fact that both Her2 and the estrogen receptor are Hsp90ꢀdependent client
proteins. Four of the Dꢀring analogues that contain two methoxy groups on the Aꢀring and no
substituents on the Bꢀring (10a–d) were inactive against both MCFꢀ7 and SKBr3 cell lines and
only compound 10e manifested significant antiꢀproliferative activity with an IC value of 25.35
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5.25 ꢁM against MCFꢀ7 and 36.1 ± 2.51 ꢁM against SKBr3 cell lines. Similar trends were
observed for compounds (10f–j) containing the 3,4,5ꢀtrimethoxy substituents on the Bꢀring, as
only 10j was found to be potent and exhibits an IC value of 19.48 ± 2.5 ꢁM and 24.87 ± 3.29
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M against MCFꢀ7 and SKBrꢀ3 cell lines, respectively.
Analogues 11a–e that contain phenols on the Aꢀring were also evaluated and found to be
more potent when compared to EGCG and analogues 10a–j. Incorporation of a methoxy group at
the meta- and the para- positions of the Dꢀring (11b and 11c) did not alter activity as compared
to unsubstituted analogue, 11a. Compound 11e was found to be the most potent of this series and
displayed an IC value of 3.99 ± 1.4 ꢁM against the MCFꢀ7 cell line. In contrast, compounds
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with 3ꢀ,4ꢀ,5ꢀtrimethoxy groups on the Bꢀring (11f–j) were less active when compared to
analogues without substitution on the Bꢀring (11a–e). This data suggests that substitutions on
the Bꢀring are detrimental to activity, whereas replacement of the gallate ester moiety with
prenyl benzoate enhances potency. In addition, the MCFꢀ7 cell line was found to be more
sensitive than the SKBr3 cell line upon administration of these analogues. Furthermore, the anti
isomer of 11e was synthesized and evaluated and found to be less active (IC = 33.7 ± 1.8
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against MCFꢀ7 cell line), confirming that synꢀstereochemistry is important for inhibitory activity.
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Table 1. Antiꢀproliferative activities produced by Aꢀ, Bꢀ and the Dꢀring modified EGCG
analogues
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Entry
(-)-EGCG
Geldanamycin
0a
0b
R , R
R R , R
R
ꢀ
ꢀ
MCFꢀ7 (IC , ꢁM) SKBr3 (IC , ꢁM)
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3,
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5
50
74.4 ± 2.19
0.05 ± 0.03
>100
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100.16 ± 0.03
0.008 ± 0.02
>100
ꢀ
ꢀ
ꢀ
ꢀ
1
1
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
H
H
H
H
H
OMe
OMe
OMe
OMe
OMe
H
H
H
H
H
OMe
OMe
OMe
OMe
OMe
a
b
c
d
e
a
b
c
d
e
a
b
c
d
e
a
b
c
d
e
>100
>100
10c
10d
>100
>100
>100
>100
1
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0f
0g
10h
0i
0j
1a
11b
1c
1d
1e
1f
11g
25.35 ± 5.25
>100
36.1 ± 2.51
>100
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1
91.18 ± 0.76
>100
>100
>100
1
88.7 ± 11.3
19.48 ± 2.5
15.26 ± 0.57
13.10 ± 0.86
13.12 ± 0.54
14.14 ± 0.7
3.99 ± 1.4
65.88 ± 2.1
45.72 ± 0.4
42.80 ± 7.30
47.31 ± 3.39
42.08 ± 1.85
>100
1
24.87 ± 3.29
18.67 ± 0.82
15.42 ± 1.04
17.26 ± 2.27
19.88 ± 3.22
21.45 ± 4.75
>100
37.92 ± 4.08
62.90 ± 0.70
71.9 ± 2.76
50.4 ± 1.39
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1
1
1
1
1h
11i
1
1j
Simultaneous with the above studies, synthesis of analogues that lack the Bꢀring were
commenced by the treatment of 3,5ꢀdibenzyloxyphenol (Scheme 4) with allyl bromide in the
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presence of potassium carbonate to give allyl ether 18a. 3,3ꢀRearrangement of the Oꢀallylated
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product (18a) gave 19a in high yield. Dihydroxylation of the resulting olefin afforded diol 20a.
Unfortunately, attempts to cyclize via the orthoester were unsuccessful as only the 5ꢀmembered
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product was formed. Therefore, an alternative strategy for the cyclization of 20a was pursued.
Treatment of the primary alcohol present in 20a with pꢀtoluenesulfonyl chloride resulted in
formation of the corresponding pꢀtoluenesulfonic ester, which underwent intramolecular
cyclization upon exposure to potassium carbonate to give a 1:1 mixure of 5ꢀ and 6ꢀmembered
rings that were separated by silica gel chromatography. Subsequent coupling of 22a with various
substituted benzoic acids produced the requisite esters, which underwent hydrogenolysis to
afford 23a–i, respectively.
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Scheme 4. Synthesis of esters of 3,5ꢀdihydroxychromanꢀ3ꢀol.
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Upon construction of analogues that lack the Bꢀring, each phenol on the Aꢀring was
systematically investigated. Therefore, derivatives 23i–s that contain only one hydroxyl at either
the 5ꢀ or the 7ꢀposition were pursued similar to that described above. Allylation of the phenol
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(
17) gave ally ether, 18b. 3,3ꢀRearrangement of the allyl ether (18b) gave a mixture of two
regioisomers, 19b and 19c, which upon dihydroxylation and subsequent ring closure gave 22b
and 22c, respectively.
Results from the antiꢀproliferative studies with compounds 23a–s are summarized in
Table 2. In addition to previously investigated substituents, the effect of hydroxyl substitution on
the Dꢀring was also explored. Many of the compounds were found to be more efficacious than
EGCG itself. This data suggests that methoxy substitution on the Dꢀring is more beneficial than
the naturally occurring phenols, which corresponds to an overall pattern represented by Oꢀalkyl
substitutions at the 3’ꢀposition are more active than those at the 4’ꢀposition. Data also suggests
that aryl and prenyl substitution on the Dꢀring produce enhanced efficacy, as 23i manifested an
IC value of 10.66 ± 1.09 ꢂM against MCFꢀ7 cells and 23.15 ± 0.25ꢂM against SKBr3 cells.
5
0
The IC values of compounds containing only one phenolic group at the 7ꢀposition on the Aꢀ
5
0
ring resulted in decreased activity, except for 23n. Similarly, compounds with 5ꢀhydroxyl
substitution on the Aꢀring also resulted in decreased activity with the exception of 23r, which
manifested enhanced activity and an IC value of 21.6 ± 2.55 against the MCFꢀ7 cell line.
5
0
Similar to the most active analogue produced from the Bꢀring series, 11e, the most active
analogue identified in this series was 23i (IC = 10 ꢂM in MCFꢀ7 cell line), which also
5
0
incorporates the prenylated benzoate side chain.
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1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 2: Antiꢀproliferative activities produced by 3,5ꢀdihydroxychromanꢀ3ꢀol esters.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
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1
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9
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3
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8
9
0
Entry R1
3a OH OH
23b OH OH
3c
OH OH
3d OH OH
R2
R3
H
OMe
H
OMe
OMe
OH
H
R4
H
H
OMe
OMe
H
R5
H
H
H
H
OMe
H
H
MCFꢀ7 (IC , ꢁM) SKBr3 (IC , ꢁM)
50
50
98.24 ± 1.76
57.75 ± 3.12
>100
2
>100
50
>100
2
2
>100
>100
2
2
3e
3f
OH OH
OH OH
96.50 ± 3.51
61.94 ± 6.85
>100
>50
85.30 ± 5.36
>100
H
OH
23g OH OH
2
3h OH OH m OMePh OMe
H
H
H
H
H
H
H
H
H
H
H
H
21.93 ± 2.27
10.66 ± 1.09
>100
>100
>100
55.09 ± 5.53
15.94 ± 1.86
>100
>100
>100
21.6 ± 2.55
60 ± 7.38
34.84 ± 16.29
23.15 ± 0.25
>100
>100
>100
57.73 ± 4.28
25.25 ± 4.05
>100
>100
>100
41.72 ± 0.34
>100
2
3i
OH OH
Prenyl
H
OMe
H
mꢀOMePh OMe
Prenyl
H
OAc
H
H
2
3j
OH
H
H
H
H
H
23k OH
2l
OH
3m OH
3n OH
2
OMe
2
2
OAc
H
2
3o
23p
2q
H
H
H
H
H
OH
OH
OH
OMe
H
H
OMe
2
2
2
3r
3s
OH mꢀOMePh OMe
OH Prenyl OAc
In an effort to further investigate the Aꢀring, the free phenols were replaced with methyl
ethers. 5,7ꢀDimethoxychromanꢀ3ꢀol (26) was synthesized in two steps using a gold(III)ꢀ
4
1
mediated procedure as described by Zhangjie and coworkers (Scheme 5). Commencing with
commercially available 3,5ꢀdimethoxyphenol and enlistment of epichlorohydrin and sodium
hydride, produced oxirane 25, which underwent 6ꢀendo cyclization to yield 26 upon treatment
with a gold(III) chloride/silver trifluormethanesulfonate catalyst. Upon construction of the
chromanꢀ3ꢀol core (26), subsequent coupling with various substituted aryl acids to furnish the
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corresponding esters, 27a–m. The final products 28a–e were prepared via hydrogenolysis of 27i–
m.
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14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Scheme 5. Synthesis of 3,5ꢀdimethoxychromanꢀ3ꢀol esters.
In addition, investigation of the linker connecting the Bꢀ and Dꢀrings was pursued. The
ester linker was replaced with an amide functionality. These amideꢀbased analogues were
prepared from previously synthesized alcohol 26, which was transformed into azide 29 via
Mitsunobu conditions with diisopropyl azodicarboxylate, triphenylphosphine and
diphenylphosphoryl azide, followed by Staudinger reduction with triphenylphosphine to afford
42
amine 30 (Schemeꢀ6). Subsequent coupling of amine 30 with the optimal aryl acids gave the
37
corresponding amides, 31a–d.
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2
2
2
2
2
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3
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3
3
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3
3
3
3
4
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4
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4
4
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5
5
5
5
5
5
5
6
0
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9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Scheme 6. Synthesis of 3,5ꢀdimethoxychromanꢀ3ꢀol amides.
Results from antiꢀproliferative studies for compounds lacking the Bꢀring are summarized
in Table 3. The 3ꢀmethoxy substituted compound 28b was found to be the most active compound
against the MCFꢀ7 and the SKBr3 cell lines, and manifested IC values 0.775± .02 ꢁM and 0.88
50
±
0.06 ꢁM, respectively. Increasing the length of side chain resulted in decreased activity for
compound 27h. The hydroxyl group was found to be more beneficial at the 4’ꢀposition in lieu of
the 3’ꢀposition. Unfortunately, the combination of 3ꢀmethoxy and 4ꢀhydroxyl substitutions on
the Dꢀring (28e) did not improve antiꢀproliferative activity. Once again, MCFꢀ7 cells exhibited
greater sensitivity to these compounds. The IC values for 27d and 27e (Table 4) correlate
5
0
directly with prior studies using novobiocin, suggesting a beneficial effect for inclusion of aryl or
prenyl group on the Dꢀring. The linker between the Bꢀ and Dꢀring was also evaluated and
replacement of the ester with an amide (31a–d) was found detrimental.
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Table 3. Antiꢀproliferative activity produced by 3,5ꢀdimethoxychromanꢀ3ꢀol esters.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
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56
57
58
59
60
Entry
27a
R1
R2
R3
MCFꢀ7 (IC , ꢁM) SKBr3 (IC , ꢁM)
50
50
H
OMe
H
H
H
OMe
H
H
H
H
H
H
OMe
H
H
H
14.02 ± 0.91
44.605 ± 5.40
27b
0.77 ± .02
32.89 ± 2.05
31.20 ± 18.17
38.66 ± 7.71
10.89 ± 0.27
21.8 ± 3.08
8.19 ± 0.16
37.72 ± 6.75
17.51 ± 0.86
>100
0.88 ± 0.06
50.40 ± 1.39
80.13 ± 9.67
47.90 ± 0.71
36.98 ± 5.24
29.5 ± 1.5
33.35 ± 4.81
64.11 ± 13.95
17.73 ± 5.97
>100
27c
2
7d mꢀOMePh OMe
27e
7f
7g
7h
8a
28b
8c
8d
8e
Prenyl
OMe
OMe
OEt
OH
H
OH
OH
OMe
OAc
OMe
H
H
H
OH
H
OH
OH
2
2
2
2
2
OH
H
H
2
22.12 ± 1.01
51.29 ± 1.13
30.63 ± 11.89
76.50 ± 1.10
2
Table 4. Antiꢀproliferative activity produced by analogues containing amide linkers.
Entry
31a
R1
R2
MCFꢀ7 (IC , ꢁM) SKBr3 (IC , ꢁM)
50 50
H
H
H
OMe
OAc
>100
>100
3
3
3
1b
1c
1d
OMe
mꢀOMePh
Prenyl
>100
>100
>100
>100
54.5 ± 0.6
55.2 ± 1.2
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6
(A)
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(B)
Figure 2. Western blot analyses of MCFꢀ7 cell lysates for Hsp90 client protein degradation after
24h of incubation. (a) Compounds 27b, 27e, 10e and 11e at two different concentrations. “H”
high) represents a concentration 5 × IC value, whereas and “L” (low) represents a
concentration at one half the IC value as determined by antiꢀproliferative studies; (b)
Compound 11e at increasing concentrations.
(
50
5
0
After determination of antiꢀproliferative activity for EGCG analogues, four representative
examples were chosen for subsequent western blot analyses to confirm Hsp90 inhibition, based
on each class of scaffold investigated. Since Hsp90 inhibition results in the induction of client
protein degradation via the ubiquitinꢀproteasome pathway, immunoblots are used to confirm
Hsp90 inhibitory activity. As shown in Figure 2, 11e, 27e and 10e induced the degradation of
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Hsp90 client proteins Her2, Raf and pAkt at concentrations that mirror the concentration needed
to exhibit antiꢀproliferative activity, thereby linking Hsp90 inhibition to cell viability. Analog
27b failed to induce client protein degradation, demonstrating that this compound manifests antiꢀ
10
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21
22
23
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26
27
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proliferative activity through a mechanism independent of Hsp90 inhibition. However a related
compound containing the prenylated benzoate side chain, 27e, was shown to exhibit Hsp90
inhibitory activity. Further investigation of 11e at increasing concentrations demonstrated client
protein degradation in a doseꢀdependent manner, while actin levels remained the same. Actin is
not an Hsp90ꢀdependent protein and is therefore unaffected by Hsp90 inhibition. Similar to other
Hsp90 Cꢀterminal inhibitors, the level of Hsp90 was unaffected.
CONCLUSIONS
In summary, we have synthesized and evaluated the first structureꢀactivity relationships between
EGCG and Hsp90 (Figure 3). The results obtained suggest that phenolic groups on the Aꢀring are
beneficial for Hsp90 inhibition, while phenolic substituents on the Dꢀring are detrimental. The
inclusion of a novobiocinꢀderived prenyl benzoate was found to be a suitable replacement for the
gallic acid moiety present on EGCG, and suggests that both novobiocin and the EGCG may bind
similarly to the Hsp90 Cꢀterminus. Results from these studies have led to the development of
analogue 11e, which exhibits a 18ꢀfold improvement over EGCG and can serve as a probe for
further biological investigations.
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9
0
1
2
3
4
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6
7
8
9
0
Figure 3: Summary of EGCG structureꢀactivity relationships.
EXPERIMENTAL SECTION
All reactions were performed in ovenꢀdried glassware under argon atmosphere unless otherwise
stated. Dichloromethane (DCM), tetrahydrofuran (THF), and toluene were passed through a
column of activated alumina prior to use. Anhydrous methanol, acetonitrile, dimethylformamide
(DMF), and dimethoxyethane (DME) were purchased and used without further purification. (ꢀ)ꢀ
EGCG (≥95%) was purchased from SigmaꢀAldrich and used as obtained. Flash column
1
chromatography was performed using silica gel (40 – 63 ꢂm particle size). The H (500 MHz
13
and 400 MHz) and CꢀNMR (proton 125 MHz and 100 MHz) spectra were recorded on 500
MHz and 400 MHz spectrometer. Data are reported as p = pentet, q = quartet, t = triplet, d =
doublet, s = singlet, bs = broad singlet, m = multiplet; coupling constant (s) in Hz. Infrared
spectra were obtained using FT/IR spectrometer. High resolution mass spectral data were
obtained on a time of flight mass spectrometer and analysis was performed using electrospray
1
13
ionization. The purity of all compounds was determined to be >95% by H and C NMR
spectra, unless otherwise noted.
3,5ꢀBis(benzyloxy)phenol (4b) and (E)ꢀ3ꢀ(3,4,5ꢀtrimethoxyphenyl)propꢀ2ꢀenꢀ1ꢀol (5b) and 3ꢀ
32, 43–44
(benzyloxy)phenol (17) were prepared following literature procedures.
Reactions of
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phenols (4a–b) with cinnamyl alcohols (5a–b) to yield compounds 6a–d were accomplished via
3
2
the protocol described by Li et al.
-Cinnamyl-3,5-dimethoxyphenol (6a). A solution of 3,5ꢀdimethoxy phenol (2.3 g, 14.91
2
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
mmol) and cinnamyl alcohol (2.0 g, 14.91 mmol) in a solvent mixture of dichloromethane (30
mL) and carbon disulfide (30 mL) was treated with 25% H SO /SiO catalyst (2.4 g, 5.96 mmol)
2
4
2
at rt. The resulting mixture was stirred for 4 h and then filtered through a plug of SiO (40 – 63
2
ꢂm particle size). Solvent was removed and the residue purified by flash chromatography (SiO2,
1
1:9 EtOAc/Hexanes) to give 6a (1.735 g, 43.15 %) as an amorphous light yellow solid: H NMR
(
500 MHz, CDCl ) δ 7.37 – 7.26 (m, 2H), 7.30 (dd, J = 7.2, 1.7 Hz, 2H), 7.23 – 7.17 (m, 1H),
3
6.48 (dt, J = 16.0, 1.7 Hz, 1H), 6.34 (dt, J = 15.9, 6.3 Hz, 1H), 6.15 (d, J = 2.3 Hz, 1H), 6.11 (d,
J = 2.3 Hz, 1H), 5.06 (s, 1H), 3.82 (s, 3H), 3.80 (s, 3H), 3.56 (d, J = 1.6 Hz, 1H), 3.55 (d, J =
13
1
1
1
.6 Hz, 1H); C NMR (125 MHz, CDCl ) δ 159.9, 159.0, 155.9, 137.4, 130.6, 128.6 (2), 128.6,
3
28.5, 127.3, 126.3, 106.1, 93.9, 91.8, 56.0, 55.5, 26.4; IR (KBr)νmax 3367, 1614, 1596, 1454,
ꢀ1
+
423, 1201, 1147, 1097, 1053, 811, 736, 692 cm ; HRMS (ESI+) m/z [M+H ] calcd for
C H O , 271.1334, found 271.1336.
17
19
3
3,5-Bis(benzyloxy)-2-cinnamylphenol (6b). A solution of 3,5ꢀbis(benzyloxy)phenol (3.3 g, 9.98
mmol) and cinnamyl alcohol (1.34 g, 9.98 mmol) in a solvent mixture of dichloromethane (20
mL) and carbon disulfide (20 mL) was treated with 25% H SO /SiO catalyst (1.59g, 3.99 mmol)
2
4
2
at rt. The resulting mixture was stirred for 4 h and then filtered through a plug of SiO (40 – 63
2
ꢂm particle size). Solvent was removed and the residue purified by flash chromatography (SiO2,
1
1
:8 EtOAc/Hexanes) to give 6b (1.425 g, 33.7 %) as amorphous light yellow solid: H NMR
(
400 MHz, CDCl ) δ 7.48 – 7.29 (m, 15H), 6.53 – 6.44 (m, 1H), 6.39 – 6.30 (m, 1H), 6.29 (d, J
3
13
=
2.2 Hz, 1H), 6.18 (d, J = 2.3 Hz, 1H), 5.03 (m, 5H), 3.60 (dd, J = 6.5, 1.6 Hz, 2H); C NMR
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4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(
125 MHz, CDCl ) δ 159.0, 158.1, 155.9, 137.5, 137.2, 137.0 (2), 128.8 (2), 128.7 (2), 128.6 (3),
3
1
28.5, 128.2, 128.0, 127.8, 127.5 (2), 127.3, 126.3 (2), 107.0, 95.3, 93.9, 70.5, 70.3, 26.7; IR
ꢀ1
(KBr)νmax 3419, 3028, 2925, 1618, 1596, 1452, 1436,1375, 1147, 1091, 734, 696 cm ; HRMS
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
(ESI+) m/z [M+H ] calcd for C H O , 423.1960, found 423.1966.
2
9
27
3
(E)-3,5-Dimethoxy-2-(3-(3,4,5-trimethoxyphenyl)allyl)phenol (6c). A solution of 3,5ꢀ
dimethoxy phenol (2.06 g, 13.4 mmol) and (E)ꢀ3,4,5ꢀtrimethoxycinnamyl (3.0 g, 13.4 mmol) in
a solvent mixture of dichloromethane (26 mL) and carbon disulfide (26 mL) was treated with
25% H SO /SiO catalyst (2.2g, 5.36 mmol) at rt. The resulting mixture was stirred for 4 h and
2
4
2
then filtered through a plug of SiO (40 – 63 ꢂm particle size). Solvent was removed and the
2
residue purified by flash chromatography (SiO , 1:2 EtOAc/Hexanes) to give 6c as an
2
1
amorphous light yellow solid: (1.660 g, 39.4 %): H NMR (500 MHz, CDCl ) δ 6.56 (s, 2H),
3
6
.38 (dt, J = 15.8, 1.7 Hz, 1H), 6.23 (dt, J = 15.8, 6.2 Hz, 1H), 6.14 (d, J = 2.4 Hz, 1H), 6.10 (d,
J = 2.3 Hz, 1H), 5.09 (s, 1H), 3.85 (s, 6H), 3.83 (s, 3H), 3.81 (s, 3H), 3.79 (s, 3H), 3.54 (dd, J =
1
3
6
.2, 1.7 Hz, 2H); C NMR (125 MHz, CDCl ) δ 159.9, 159.0, 155.9, 153.4 (2), 137.6, 133.2,
3
1
30.4, 128.1, 106.1, 103.3 (2), 93.9, 91.7, 61.1, 56.2 (2), 56.0, 55.5, 26.2; IR (KBr)νmax 3379,
ꢀ1
3379, 2937, 1620, 1593, 1506, 1421, 1361, 1330, 1201, 1147, 1053, 817, 707 cm ; HRMS
+
(ESI+) m/z [M+H ] calcd for C H O , 361.1651, found 361.1657.
2
0
25
6
(E)-3,5-Bis(benzyloxy)-2-(3-(3,4,5-trimethoxyphenyl)allyl)phenol (6d). A solution 3,5ꢀ
bis(benzyloxy)phenol (5.2 g, 6.97 mmol) and (E)ꢀ3,4,5 trimethoxycinnamyl alcohol (3.81 g,
1
6.97 mmol) in a solvent mixture of dichloromethane (33 mL) and carbon disulfide (33 mL) was
treated with 25% H SO /SiO catalyst (1.11 g, 2.8 mmol) at rt. The resulting mixture was stirred
2
4
2
for 4 h and then filtered through a plug of SiO (40 – 63 ꢂm particle size). Solvent was removed
2
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and the residue purified by flash chromatography (SiO , 1:3 EtOAc/Hexanes) to give 6d (1.970
2
1
g, 22.6 %) as an amorphous light yellow solid: H NMR (500 MHz, CDCl ) δ 7.47 – 7.28 (m,
3
1
0H), 6.54 (s, 2H), 6.39 (dt, J = 15.8, 1.7 Hz, 1H), 6.30 (d, J = 2.3 Hz, 1H), 6.24 (dt, J = 15.8,
.3 Hz, 1H), 6.19 (d, J = 2.3 Hz, 1H), 5.05 (s, 2H), 5.03 (s, 1H), 5.02 (s, 2H), 3.90 – 3.80 (m,
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56
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6
1
3
9H), 3.65 – 3.56 (m, 2H). C NMR (125 MHz, CDCl ) δ 159.1, 158.2, 155.9, 153.5 (2), 137.6,
3
1
37.3, 137.1, 133.3, 130.7, 128.9 (2), 128.8 (2), 128.7, 128.3 (2), 128.1 (2), 127.8 (2), 127.5,
07.0, 103.4, 95.3, 93.9, 70.6, 70.4, 61.2, 56.3 (2), 26.6. IR (KBr)νmax 3400, 2937, 1614, 1585,
1
ꢀ1
+
1454, 1328, 1238, 1126, 1001, 736, 696 cm . HRMS (ESI+) m/z [M+Na ] calcd for
C H NaO , 535.2097, found 535.2100.
32
32
6
3
-(2-Hydroxy-4,6-dimethoxyphenyl)-1-phenylpropane-1,2-diol (7a). NꢀMethylmorpholineꢀNꢀ
oxide (1.26g, 10.76 mmol) was added to a solution of 6a (1.7g, 6.33 mmol) in a solvent mixture
of tetrahydrofuran (18 mL) and H O (12 mL). The resulting solution was stirred for 15 min at rt
2
before the addition osmium tetraoxide (0.1 mmol, 4% solution in water). The mixture was stirred
for 14 h at rt before quenching with 20% of sodium metabisulphite (15 mL). The aqueous layer
was extracted with ethyl acetate (3 × 25 mL) and the combined organic layers were washed with
saturated sodium chloride solution (50 mL), dried over anhydrous Na SO , filtered and
2
4
concentrated. The residue was purified by flash chromatography (SiO , 1:2 EtOAc/Hexanes) to
2
1
afford 7a (1.55 g, 81 %) as a colorless oil: H NMR: (500 MHz, CDCl ) δ 7.98 (brs, 1H), 7.43 –
3
7.37 (m, 2H), 7.37 – 7.32 (m, 3H), 6.17 (d, J = 2.4 Hz, 1H), 6.03 (d, J = 2.4 Hz, 1H), 4.55 (d, J
=
6.6 Hz, 1H), 4.04 (ddd, J = 7.4, 6.5, 3.8 Hz, 1H), 3.77 (s, 3H), 3.59 (s, 3H), 3.23 (brs, 1H),
1
3
2
.84 (dd, J = 14.8, 3.8 Hz, 1H), 2.74 (dd, J = 14.8, 7.4 Hz, 1H), 2.46 (brs, 1H); C NMR (125
MHz, CDCl ) δ 160.2, 158.9, 157.5, 140.6, 128.7 (2), 128.5 (2), 127.2, 105.5, 76.9, 76.5, 94.6,
3
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1.5, 55.5 (2), 26.2; IR (KBr)νmax 3348, 2837, 1622, 1593, 1496, 1456, 1338, 1199, 1147, 1105,
ꢀ1
ꢀ
cm ; HRMS (ESIꢀ) m/z [MꢀH ] calcd for C H O , 303.1233, found 303.1227.
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3-(2,4-Bis(benzyloxy)-6-hydroxyphenyl)-1-phenylpropane-1,2-diol (7b). Nꢀ
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MethylmorpholineꢀNꢀoxide (393 mg, 3.36 mmol) was added to a solution of 6a (0.9g, 2.1 mmol)
in a solvent mixture of tetrahydrofuran (9 mL) and H O (6 mL). The resulting solution was
2
stirred for 15 min at rt before the addition osmium tetraoxide (0.02 mmol, 4% solution in water).
The mixture was stirred for 14 h at rt before quenching with 20% of sodium metabisulphite (10
mL). The aqueous layer was extracted with ethyl acetate (3 × 20 mL) and the combined organic
layers were washed with saturated sodium chloride solution (40 mL), dried over anhydrous
Na SO , filtered and concentrated. The residue was purified by flash chromatography (SiO , 1:3
2
4
2
1
EtOAc/Hexanes) to afford 7b (0.78g, 80.1 %) as a colorless oil: H NMR (500 MHz, CDCl ) δ
3
8
6
6
2
.03 (s, 1H), 7.46 – 7.36 (m, 5H), 7.36 – 7.29 (m, 6H), 7.27 – 7.25 (m, 2H), 7.19 – 7.06 (m, 2H),
.29 (d, J = 2.3 Hz, 1H), 6.21 (d, J = 2.4 Hz, 1H), 5.01 (s, 2H), 4.90 – 4.82 (m, 2H), 4.56 (d, J =
.8 Hz, 1H), 4.04 (ddd, J = 8.5, 6.7, 3.5 Hz, 1H), 3.32 (s, 1H), 2.93 (dd, J = 14.7, 3.5 Hz, 1H),
13
.75 (dd, J = 14.6, 8.4 Hz, 1H), 2.46 (s, 1H); C NMR (125 MHz, CDCl ) δ 159.3, 158.0, 157.7,
3
140.4, 137.1, 137.0, 128.8 (5), 128.7 (2), 128.6, 128.2, 127.8 (4), 127.2 (2), 127.0 (2), 106.3,
96.1, 93.6, 70.3 (2), 26.6; IR (KBr)νmax 3363, 3330 3087, 3031, 1701, 1620, 1598, 1452, 1375,
1
+
1147, 1099, 815, 698 cmꢀ ; HRMS (ESI+) m/z [M+H ] calcd for C H O , 457.2015, found
29 29 5
457.2028.
3-(2-Hydroxy-4,6-dimethoxyphenyl)-1-(3,4,5-trimethoxyphenyl)propane-1,2-diol (7c). Nꢀ
methylmorpholineꢀNꢀoxide (702 mg, 6 mmol) was added to a solution of 6c (1.350 g, 3.75
mmol) in a solvent mixture of tetrahydrofuran (12 mL) and H O (8 mL). The resulting solution
2
was stirred for 15 min at rt before the addition osmium tetraoxide (0.04 mmol, 4% solution in
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water). The mixture was stirred for 14 h at rt before quenching with 20% of sodium
metabisulphite (12 mL). The aqueous layer was extracted with EtOAc (3 × 25 mL) and the
combined organic layers were washed with saturated sodium chloride solution (50 mL), dried
over anhydrous Na SO , filtered and concentrated. The residue was purified by flash
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59
60
2
4
chromatography (SiO , 1:10 Acetone/Dichloromethane) to afford 7c (1.33 g, 90.4 %) as a
2
1
colorless oil: H NMR (500 MHz, CDCl ) δ 8.08 (s, 1H), 6.54 (s, 2H), 6.14 (d, J = 2.4 Hz, 1H),
3
6.03 (d, J = 2.4 Hz, 1H), 4.47 (d, J = 6.0 Hz, 1H), 3.98 (ddd, J = 8.0, 6.1, 3.8 Hz, 1H), 3.84 (s,
6H), 3.82 (s, 3H), 3.75 (s, 3H), 3.62 (s, 3H), 3.44 (brs, 1H), 3.10 – 2.92 (m, 1H), 2.85 (dd, J =
13
1
4.7, 3.8 Hz, 1H), 2.73 (dd, J = 14.7, 7.8 Hz, 1H); C NMR (125 MHz, CDCl ) δ 160.1, 159.0,
3
157.3, 153.4 (2), 137.6, 136.5, 105.6, 103.9 (2), 94.6, 91.4, 76.9, 76.7, 61.0, 56.3 (2), 55.7, 55.5,
ꢀ
1
26.5; IR (KBr)νmax 3405, 2932, 1620, 1591, 1498, 1439, 1379, 1218, 1146, 1029, 817 cm ;
+
HRMS (ESI+) m/z [M+H ] calcd for C H O , 395.1706, found 395.1719.
2
0
27
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3-(2,4-Bis(benzyloxy)-6-hydroxyphenyl)-1-(3,4,5-trimethoxyphenyl)propane-1,2-diol (7d).
NꢀmethylmorpholineꢀNꢀoxide (444 mg, 3.79 mmol) was added to a solution of 6c (1.0 g, 2.36
mmol) in a solvent mixture of tetrahydrofuran (7.5 mL) and H O (5 mL). The resulting solution
2
was stirred for 15 min at rt before the addition osmium tetraoxide (0.02 mmol, 4% solution in
water). The mixture was stirred for 14 h at rt before quenching with 20% of sodium
metabisulphite (10 mL). The aqueous layer was extracted with ethyl acetate (3 × 20 mL) and the
combined organic layers were washed with saturated sodium chloride solution (40 mL), dried
over anhydrous Na SO , filtered and concentrated. The residue was purified by flash
2
4
chromatography (SiO , 1:10 Acetone/Dichloromethane) to afford 7d (595 g, 56.7 %) as an
2
1
amorphous light yellow solid: H NMR (500 MHz, CDCl ) δ 8.00 (brs, 1H), 7.47 – 7.28 (m,
3
10H), 6.54 (s, 2H), 6.28 (d, J = 2.3 Hz, 1H), 6.22 (d, J = 2.3 Hz, 1H), 5.06 – 4.95 (m, 4H), 4.91
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(
d, J = 3.0 Hz, 1H), 4.52 (d, J = 6.0 Hz, 1H), 3.77 (d, J = 10.2 Hz, 9H), 3.25 (brs, 1H), 3.01 –
1
3
2
1
1
.95 (m, 1H), 2.83 (dd, J = 14.6, 8.3 Hz, 1H), 2.74 (brs, 1H); C NMR (125 MHz, CDCl ) δ
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59.4, 158.1, 157.6, 153.5 (2), 137.1, 137.0, 136.4, 129.0, 128.8, 128.7, 128.5, 128.4, 128.3 (2),
28.1, 127.8, 127.5, 127.4, 127.3, 126.9, 106.3, 103.8 (2), 96.2, 93.7, 70.3 (2), 61.0, 56.3, 56.3,
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0
ꢀ1
27.0; IR (KBr)νmax 3446, 2935, 2837, 1591, 1498, 1456, 1328, 1232, 1126, 1004, 736cm ;
ꢀ
HRMS (ESIꢀ) m/z [MꢀH ] calcd for C H O , 547.2332, found 547.2347.
3
2
33
8
5
,7-Dimethoxy-2-phenylchroman-3-ol (8a). Trimethyl orthoacetate (2.50 mmol, 300 ꢂl) and
pyridinium pꢀtoluenesulfonate (9 mg, 0.036 mmol) were added to a solution of 7a (600 mg, 1.92
mmol) in dichloromethane (36 mL) at rt. The resulting mixture was stirred for 30 min at rt and
then cooled to 0 °C before the dropwise addition of borontrifluoride diethyletherate (25 ꢂl, 0.192
mmol). The reaction mixture was warmed to rt and stirred for another 15 min before quenching
with aqueous acetone (4 mL). Solvent was removed and the residue was dissolved methanol (32
mL). Potassium carbonate (225 mg, 1.84 mmol) was added and mixture stirred for 6 h at rt.
Methanol was removed, water (25 mL) was added and the products extracted with ethyl acetate
(2 × 30 mL). Organic layers were combined and washed with saturated sodium chloride solution
(60 mL). The organic phase was dried over anhydrous Na SO and filtered. Solvent was removed
2
4
and residue was purified via flash chromatography (SiO , 1:4 EtOAc/Hexanes) to yield
2
1
compound 8a (422 mg, 77.7 %) as a colorless oil: H NMR (500 MHz, CDCl ) δ 7.47 – 7.34 (m,
3
5H), 6.16 (d, J = 2.3 Hz, 1H), 6.12 (d, J = 2.3 Hz, 1H), 4.79 (d, J = 7.8 Hz, 1H), 4.11 (td, J =
8
8
1
.1, 5.5 Hz, 1H), 3.81 (s, 3H), 3.77 (s, 3H), 3.00 (dd, J = 16.4, 5.5 Hz, 1H), 2.63 (dd, J = 16.4,
13
.4 Hz, 1H), 1.71 (s, 1H); C NMR (125 MHz, CDCl ) δ 159.7, 158.8, 155.1, 138.1, 128.8,
3
28.6, 127.1, 101.4, 93.0, 91.9, 81.7, 68.2, 55.5, 55.4, 27.2; IR (KBr)νmax 3446, 2937, 2839,
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618, 1593, 1496, 1213, 1143, 1120, 1051, 1022, 813, 761, 689 cm ; HRMS (ESI+) m/z [M+H ]
calcd for C H O , 287.1283, found 287.1270.
17 19
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,7-Bis(benzyloxy)-2-phenylchroman-3-ol (8b): Trimethyl orthoacetate (1.48 mmol, 188 ꢂl)
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and pyridinium pꢀtoluenesulfonate (6 mg, .012 mmol) were added to a solution of 7b (560 mg,
1.22 mmol) in dichloromethane (24 mL) at rt. The resulting mixture was stirred for 30 min and
cooled to 0 °C before the addition of borontrifluoride diethyletherate (18 ꢂl, 0.24 mmol)
dropwise. The reaction mixture was warmed to rt and stirred for another 15 min before
quenching with aqueous acetone (4 mL). Solvent was removed and the residue was dissolved
methanol (18 mL). Potassium carbonate (185 mg, 1.34 mmol) was added and mixture stirred for
6
h at rt. Methanol was removed, water (20 mL) was added and the products extracted with ethyl
acetate (2 × 25 mL). The combined organic layers and washed with saturated sodium chloride
solution (60 mL). The organic phase was dried over anhydrous Na SO and filtered. Solvent was
2
4
removed and the residue was purified via flash chromatography (SiO , 1:4 EtOAc/Hexanes) to
2
1
yield compound 8b (420 mg, 78.2 %) as a pale yellow oil: H NMR (400 MHz, CDCl ) δ 7.61 –
3
7.23 (m, 15H), 6.28 – 6.09 (m, 2H), 5.09 – 4.76 (m, 4H), 4.73 (d, J = 7.9 Hz, 1H), 4.07 (td, J =
8.4, 5.6 Hz, 1H), 3.05 (dd, J = 16.5, 5.5 Hz, 1H), 2.65 (dd, J = 16.4, 8.6 Hz, 1H); IR (KBr)νmax
ꢀ1
+
3
460, 2912, 1617, 1592, 1375, 1145, 1126, 1076, 973, 813, 696 cm ; HRMS (ESI+) m/z [M+H ]
calcd for C H O , 439.1909, found 439.1897.
2
9
27
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5,7-Dimethoxy-2-(3,4,5-trimethoxyphenyl)chroman-3-ol (8c). Trimethyl orthoacetate (1.92
mmol, 250 ꢂl) and pyridinium pꢀtoluenesulfonate (10 mg, 0.032 mmol) were added to a solution
of 7c (620 mg, 1.6 mmol) in dichloromethane (32 mL) at rt. The resulting mixture was stirred for
30 min at rt and then cooled to 0 °C before the dropwise addition of borontrifluoride
diethyletherate (20 ꢂl, 0.16 mmol). The reaction mixture was warmed to rt and stirred for
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another 15 min before quenching with aqueous acetone (4 mL). Solvent was removed and the
residue dissolved in methanol (32 mL). Potassium carbonate (240 mg, 1.76 mmol) was added
and mixture stirred for 6 h at rt. Methanol was removed, water (25 mL) was added and the
products extracted with ethyl acetate (2 × 30 mL). The combined organic layers were washed
with saturated sodium chloride solution (50 mL). The organic phase was dried over anhydrous
Na SO and filtered. Solvent was removed and the residue was purified via flash
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chromatography (SiO , 1:4 EtOAc/Hexanes) to yield compound 8c (460 mg, 77.8) as a colorless
2
1
oil: H NMR (500 MHz, CDCl ) δ 6.68 (s, 2H), 6.15 (d, J = 2.3 Hz, 1H), 6.12 (d, J = 2.3 Hz,
3
1H), 4.63 (d, J = 8.5 Hz, 1H), 4.07 (ddd, J = 9.3, 8.5, 5.8 Hz, 1H), 3.87 (s, 6H), 3.85 (s, 3H),
1
3
3
.82 (s, 3H), 3.76 (s, 3H), 3.11 (dd, J = 16.3, 5.8 Hz, 1H), 2.60 (dd, J = 16.3, 9.3 Hz, 1H); C
NMR (125 MHz, CDCl ) δ 159.9, 158.9, 155.4, 153.7 (2), 138.2, 133.6, 104.3 (2), 101.9, 93.2,
3
92.2, 82.4, 68.5, 61.0, 56.3 (2), 55.7, 55.6, 28.0; IR (KBr)νmax 3438, 3001, 2916, 2848, 1622,
ꢀ1
1
593, 1496, 1622, 2593, 1456, 1361, 1215, 1145, 1120, 810, 667 cm ; HRMS (ESI+) m/z
+
[
M+Na ] calcd for C H NaO , 399.1420, found 399.1414.
2
0
24
7
5,7-Bis(benzyloxy)-2-(3,4,5-trimethoxyphenyl)chroman-3-ol (8d). Trimethyl orthoacetate
(0.94 mmol, 120 ꢂl) and pyridinium pꢀtoluene sulfonate (4 mg, 0.016 mmol) were added to a
solution of 7d (425 mg, 0.78 mmol) in dichloromethane (16 mL) at rt. The resulting mixture was
stirred for 30 min at rt and then cooled to 0 °C before the dropwise addition of borontrifluoride
diethyletherate (11 ꢂl, 0.08 mmol) dropwise. The reaction mixture was warmed to rt and stirred
for another 15 min before quenching with aqueous acetone (3 mL). Solvent was removed and the
residue dissolved in methanol (16 mL). Potassium carbonate (118 mg, 0.85 mmol) was added
and mixture stirred for 6 h at rt. Methanol was removed, water (20 mL) was added and the
products extracted with ethyl acetate (2 × 25 mL). The combined organic layers were washed
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with saturated sodium chloride solution (30 mL). The organic phase was dried over anhydrous
Na SO and filtered. Solvent was removed and the residue was purified via flash
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chromatography (SiO , 1:4 EtOAc/Hexanes) to afford 8d (265 mg, 63.3 %) as a pale yellow oil:
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H NMR (500 MHz, CDCl ) δ 7.49 – 7.37 (m, 8H), 7.37 – 7.30 (m, 2H), 6.69 (s, 2H), 6.30 (d, J
3
= 2.3 Hz, 1H), 6.26 (d, J = 2.3 Hz, 1H), 5.11 – 4.96 (m, 4H), 4.65 (d, J = 8.5 Hz, 1H), 4.10 (td, J
=
8.9, 5.8 Hz, 1H), 3.88 (s, 6H), 3.86 (s, 3H), 3.22 (dd, J = 16.3, 5.8 Hz, 1H), 2.69 (dd, J = 16.4,
1
3
9
.3 Hz, 1H), 1.82 (s, 1H); C NMR (125 MHz, CDCl ) δ 159.0, 157.9, 155.4, 153.7 (2), 137.1,
3
137.0 (2), 133.5, 128.8 (2), 128.7 (2), 128.2, 128.1, 127.7, 127.3 (3), 104.3 (2), 102.6, 94.5, 94.1,
8
2.4, 70.3, 70.1, 68.5, 61.0, 56.3 (2), 28.2; IR (KBr)νmax 3481, 2935, 1618, 1593, 1498, 1460,
ꢀ1
+
1
421, 1346, 1145, 1128, 1022, 829, 752, 734 cm ; HRMS (ESI+) m/z [M+H ] calcd for
C H O , 529.2226, found 529.2234.
32
33
7
Transformations of antiꢀalcohols to synꢀalcohols was accomplished via following the procedure
2
6
described by Tuckmantel et. al.
,7-Dimethoxy-2-phenylchroman-3-ol (9a). Obtained as a colorless oil (232 mg, 55%): H
1
5
NMR (400 MHz, CDCl ) δ 7.58 – 7.52 (m, 2H), 7.45 (dd, J = 8.4, 6.7 Hz, 2H), 7.43 – 7.33 (m,
3
1H), 6.23 (d, J = 2.3 Hz, 1H), 6.15 (d, J = 2.3 Hz, 1H), 5.05 (s, 1H), 4.34 (s, 1H), 3.82 (s, 3H),
1
3
3
.80 (d, J = 0.7 Hz, 3H), 3.04 – 2.82 (m, 2H), 1.73 (brs, 1H); C NMR (125 MHz, CDCl ) δ
3
1
59.9, 159.5, 155.4, 138.4, 129.0, 128.8, 128.3, 126.5, 126.4, 100.4, 93.5, 92.4, 78.8, 66.6, 55.7,
55.6, 28.3; IR (KBr)νmax 3451, 1952, 2923, 2854, 1618, 1593, 1203, 1145, 1118, 1058, 968, 811,
ꢀ1
+
7
46, 700 cm ; HRMS (ESI+) m/z [M+H ] C H O , 287.1283, found 287.1277.
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,7-Bis(benzyloxy)-2-phenylchroman-3-ol (9b) . Obtained as a pale yellow oil (198 mg, 47%).
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H NMR (400 MHz, CDCl ) δ 7.91 (s, 1H), 7.65 – 7.31 (m, 15H), 6.23 (d, J = 2.3 Hz, 1H), 6.16
3
(d, J = 2.3 Hz, 1H), 5.62 (dt, J = 7.6, 4.9 Hz, 1H), 5.13 (d, J = 5.3 Hz, 1H), 4.99 (d, J = 1.9 Hz,
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H), 3.25 (dd, J = 14.6, 4.9 Hz, 1H), 2.89 (dd, J = 14.6, 8.0 Hz, 1H); IR (KBr)νmax 3449, 2954,
ꢀ1
+
2842, 1618, 1593, 1498, 1458, 1198, 1145, 1120, 1080, 729 cm ; HRMS (ESI+) m/z [M+Na ]
calcd for C H NaO , 461.1729, found 461.1724.
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,7-Dimethoxy-2-(3,4,5-trimethoxyphenyl)chroman-3-ol (9c): Obtained as a colorless oil (175
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mg, 43%). H NMR (500 MHz, CDCl ) δ 6.75 (s, 2H), 6.21 (d, J = 2.3 Hz, 1H), 6.12 (d, J = 2.4
3
Hz, 1H), 4.93 (s, 1H), 4.44 – 4.23 (m, 1H), 3.89 (s, 6H), 3.85 (s, 3H), 3.80 (s, 3H), 3.78 (s, 3H),
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3.00 – 2.93 (m, 1H), 2.89 (dd, J = 17.3, 4.4 Hz, 1H), 1.88 (brs, 1H); C NMR (125 MHz,
CDCl ) δ 159.8, 159.4, 155.2, 153.6 (2), 137.5, 134.2, 103.4 (2), 100.4, 93.5, 92.4, 78.8, 66.6,
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1.0, 56.3 (2), 55.6, 55.5, 28.2; IR (KBr)νmax 3460, 2997, 2939, 2839, 1620, 1593, 1498, 1456,
ꢀ1
1419, 1357, 1330, 1317, 1236, 1197, 1145, 1120, 1081, 939, 815, 729 cm ; HRMS (ESI+) m/z
+
[M+H ] calcd for C H O , 377.1600, found 377.1593.
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,7-Bis(benzyloxy)-2-(3,4,5-trimethoxyphenyl)chroman-3-ol (9d): Obtained as an amorphous
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pale yellow solid (72 mg, 68 %): H NMR (500 MHz, CDCl ) δ 7.44 – 7.34 (m, 10H), 6.75 (s,
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2H), 6.32 (d, J = 2.3 Hz, 1H), 6.30 (d, J = 2.3 Hz, 1H), 5.06 – 5.00 (m, 4H), 4.97 (s, 1H), 4.30
(d, J = 4.3 Hz, 1H), 3.91 (s, 6H), 3.87 (s, 3H), 3.07 (dd, J = 17.4, 2.5 Hz, 1H), 2.98 (dd, J =
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17.3, 4.5 Hz, 1H), 1.78 (brs, 1H); C NMR (125 MHz, CDCl ) δ 159.0, 158.5, 155.3, 153.7 (2),
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37.2 (2), 137.1, 134.1, 128.8 (2), 128.7 (2), 128.2, 128.1, 127.8 (2), 127.4 (2), 103.4 (2), 101.1,
4.9, 94.4, 78.9, 70.4, 70.2, 66.8, 61.1, 56.4 (2), 28.5; IR (KBr)νmax 3461, 2925, 2834, 1593,
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1458, 1375, 1236, 1145, 1126, 1078, 1010, 813, 738, 696 cm ; HRMS (ESI+) m/z [M+H ] calcd
for C H O , 529.2226, found 529.2234.
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5,7-Dimethoxy-2-phenylchroman-3-yl benzoate (10a). Benzoyl chloride (8 ꢂl, 0.07 mmol) in
dichloromethane (0.5 mL) was added to a solution of 9a (10 mg, 0.035 mmol) and 4ꢀ
dimethylaminopyridine (11 mg, 0.08 mmol) in dichloromethane (1mL) at 0 °C. The resulting
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mixture was stirred for 6 h at rt. Solvent was removed and the residue purified via flash
chromatography (SiO , 1:8 EtOAc/Hexanes) to give the desired ester 10a as an amorphous white
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solid: (11 mg, 88.8%): H NMR (500 MHz, CDCl ) δ 7.95 – 7.87 (m, 2H), 7.56 – 7.47 (m, 3H),
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.41 – 7.28 (m, 5H), 6.27 (d, J = 2.3 Hz, 1H), 6.12 (d, J = 2.3 Hz, 1H), 5.69 (ddd, J = 4.1, 3.2,
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1.4 Hz, 1H), 5.21 (m, 1H), 3.82 (s, 3H), 3.78 (s, 3H), 3.15 – 3.02 (m, 2H); C NMR (125 MHz,
CDCl ) δ 165.9, 159.9, 159.1, 155.7, 138.0, 133.1, 130.2, 129.9 (2), 128.5 (3), 128.3 (2), 126.7,
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00.4, 93.5, 92.1, 78.0, 68.8, 55.6 (2), 26.1; IR (KBr)νmax 2956, 1935, 2839, 1714, 1593, 1458,
ꢀ1
+
1419, 1361, 1257, 1147, 1124, 1101, 1029, 1006, 846, 813, 769 cm ; HRMS (ESI+) m/z [M+H ]
calcd for C H O , 391.1545, found 391.1538.
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5,7-Dimethoxy-2-phenylchroman-3-yl 3-methoxybenzoate (10b). A solution of 9a (8 mg,
0.027 mmol) in dichloromethane (0.5 mL) was added to a solution of 3ꢀmethoxybenzoic acid (8
mg, 0.05 mmol), Nꢀ(3ꢀdimethylaminoꢀpropyl)ꢀN’ꢀethylcarbodiimide hydrochloride (9.5 mg, 0.05
mmol) and 4ꢀdimethylaminopyridine ( 6 mg, 0.05 mmol) in dichloromethane (1 mL) at 0 °C.
The resulting mixture was stirred for 6 h at rt and then diluted with dichloromethane (5 mL). The
organic phase was washed with saturated sodium bicarbonate solution (2 × 4 mL). The organic
layer was dried over anhydrous sodium sulfate, filtered and solvent removed. The residue was
purified via flash chromatography (SiO , 1:8 EtOAc/Hexanes) to give the desired ester 10b (9
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1
mg, 76.9%), as a colorless oil: H NMR (500 MHz, CDCl ) δ 7.56 – 7.46 (m, 3H), 7.42 (dd, J =
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2.7, 1.5 Hz, 1H), 7.38 – 7.30 (m, 2H), 7.29 (t, J = 2.6 Hz, 1H), 7.27ꢀ7.21 (m, 1H), 7.04 (ddd, J =
8.3, 2.7, 1.0 Hz, 1H), 6.26 (d, J = 2.3 Hz, 1H), 6.12 (d, J = 2.3 Hz, 1H), 5.67 (ddd, J = 4.1, 3.2,
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1.4 Hz, 1H), 5.21 (s, 1H), 3.81 (s, 3H), 3.80 (s, 3H), 3.78 (s, 3H), 3.10 – 3.04 (m, 2H); C NMR
(125 MHz, CDCl ) δ 165.8, 159.9, 159.6, 159.1, 155.7, 138.0, 131.5, 129.5 (2), 128.5 (2), 128.3,
3
1
26.7, 122.3, 119.6, 114.4, 100.3, 93.5, 92.1, 77.9, 69.0, 55.6 (3), 26.0; IR (KBr)νmax 2925, 2837,
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