Structural Revision of Baulamycin A and Structure-Activity Relationships of Baulamycin A Derivatives

Article abstract of DOI:10.1021/acs.joc.7b01719

Total synthesis of the proposed structure of baulamycin A was performed. The spectral properties of the synthetic compound differ from those reported for the natural product. On the basis of comprehensive NMR study, we proposed two other possible structur

Full text of DOI:10.1021/acs.joc.7b01719

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Featured Article
Structural Revision of Baulamycin A and Structure-
Activity Relationships of Baulamycin A Derivatives
Sandip Sengupta, Munhyung Bae, Dong-Chan Oh, Uttam Dash,
Hak Joong Kim, Woon Young Song, Injae Shin, and Taebo Sim
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.7b01719 • Publication Date (Web): 13 Sep 2017
Downloaded from http://pubs.acs.org on September 14, 2017
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Structural Revision of Baulamycin A and Structure-Activity Relationships of
Baulamycin A Derivatives
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†
,1
‡,1
‡
†
§
Sandip Sengupta, Munhyung Bae, DongꢀChan Oh, Uttam Dash, Hak Joong Kim, Woon
§
†,
⊥
⊥,
Young Song, Injae Shin, Taebo Sim
*
†
[
] Dr. Sandip Sengupta, Dr. Uttam Dash, Dr. Taebo Sim, Chemical Kinomics Research Center, Korea Institute of Science and
Technology (KIST), 5 Hwarangro 14ꢀgil, Seongbukꢀgu, Seoul 02792, Republic of Korea
Eꢀmail: tbsim@kist.re.kr
‡
[
] Mr. Munhyung Bae, Dr. DongꢀChan Oh, Natural Products Research Institute, College of Pharmacy, Seoul National
University, 1 Gwanakꢀro, Gwanakꢀgu, Seoul 08826, Republic of Korea
§
[
] Dr. Hak Joong Kim, Mr. Woon Young Song, Department of Chemistry, Korea University, 145 Anamꢀro, Seongbukꢀgu, Seoul
2841, Republic of Korea
] Mr. Injae Shin, Dr. Taebo Sim, KUꢀKIST Graduate School of Converging Science and Technology, Korea University, 145
0
[⊥
Anamꢀro, Seongbukꢀgu, Seoul 02841, Republic of Korea
1
[
] These authors contributed equally to this work.
_
__________________________________________________________________________
ABSTRACT: Total synthesis of the proposed structure of baulamycin A was performed. The
spectral properties of the synthetic compound differ from those reported for the natural product.
On the basis of comprehensive NMR study, we proposed two other possible structures for
natural baulamycin A. Total syntheses of these two substances were performed, which enabled
assignment of the correct structure of baulamycin A. Key features of the convergent and fully
stereoꢀcontrolled route include Evans Aldol and Brown allylation reactions to construct the left
fragment, a prolinol amideꢀderived alkylation/desymmetrization to install the methyl substituted
centers in the right fragment, and finally a Carreira alkynylation to join both fragments. In
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addition, we have determined the inhibitory activities of novel baulamycin A derivatives against
the enzyme SbnE. This SAR study provides useful insight into the design of novel SbnE
inhibitors that overcome the drug resistance of pathogens, which cause lifeꢀthreatening
infections.
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___________________________________________________________________________
Introduction
Bacteria secrete low molecular weight and highꢀaffinity iron chelators called siderophores to
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acquire iron needed for their virulence. The biosynthetic pathways employed to generate these
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siderophores, such as staphyloferrin B of Staphylococcus aureus and petrobactin of Bacillus
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anthracis, involve NRPSꢀindependent siderophore (NIS) synthetases, which includes SbnE in
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staphyloferrin B and AsbA in petrobactin. Sherman et al recently isolated baulamycin A
(BmcA) and baulamycin B (BmcB) from marine microbialꢀderived extracts (NPEs) from
Streptomyces tempisquensis and showed that these substances serve as novel inhibitors of SbnE
and AsbA. BmcA is an invaluable substance as it provides a promising startingꢀpoint for
designing substances that are effective against drugꢀresistant pathogens such as MRSA and
Bacillus anthracis.
As a part of an ongoing program devoted to total synthesis of biologically active natural
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products, we performed a total synthesis of Sherman’s proposed stereostructure of BmcA (1).
A comparison of the spectroscopic properties of the synthetic and natural materials demonstrated
that the earlier proposed stereostructure of the natural product is incorrect. In the effort described
below, we established the correct stereostructure of this natural product by utilizing
comprehensive NMR studies and three total syntheses. It should be noted that, during the
6a
6b
preparation of this manuscript, Goswami et.al and Chandrasekhar et.al reported a synthesis of
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Sherman’s originally proposed stereostructure of BmcA by employing a strategy that differs
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c
from ours. Aggarwal et.al reported the correct structure of BmcA while our manuscript was under
review. The reported stereostructure of BmcA has the following relative configurations at its seven
stereogenic centers: 4R*, 6S*, 8R*, 11R*, 13R*, 14S*, and 1'R* while the absolute
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configurations of these centers were arbitrarily represented by stereostructure 1 (Figure 1).
Figure 1. Structures of BmcA and BmcB.
Results and discussion
As depicted retrosynthetically in Scheme 1, our strategy for the synthesis of 1 involved joining
fragments 1 and 2 by using the Carreira alkynylation process. In the plan, fragment 1 (12 or 12a)
would be derived from a protected form (4 or 4a) of 3,5ꢀdihydroxybenzaldehyde and fragment 2
(20) would be generated from (S)ꢀRoche ester.
Scheme 1. Retrosynthetic analysis for the synthesis of 1
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The synthetic pathway began with an Evans syn aldol reaction between 3,5ꢀdimethoxy
benzaldehyde 4 and auxiliary 5, which produced hydroxyamide 6 (86%, dr = 96:4 by NMR) and
protection as its TBS ether 7 (88%) followed by reductive cleavage promoted by treatment with
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DIBALꢀH generate aldehyde 8 (60%, 2 steps). Brown allylation of aldehyde 8 produced the
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homoallylic alcohol 9 (74%, dr = 92:8 by NMR). Removal of the TBS group in 9 formed 1,3ꢀ
diol 10 which upon reaction with 2,2ꢀDMP, PTSA formed acetonide 11 (89%, 2 steps). The
olefin group in 11 was cleaved by dihydroxylation followed by oxidative cleavage to afford
aldehyde 12 (fragment 1) (68%, 2 steps) (Scheme 2).
Scheme 2. Synthesis of fragment 1 (syn configuration)
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The synthesis of fragment 2 was depicted in Scheme 3. Primary alcohol group in 13
was converted to the corresponding iodide 14 by using TPP/Iodine. The lithium enolate,
produced from ꢀprolinol Nꢀpropionamide by using nꢀBuLi, was coupled with iodide 14 to form
,3ꢀanti dimethyl substituted amide 15 (85%, dr = 98:2 by NMR). Acid hydrolysis of 15,
followed by reduction formed primary alcohol 16 (69%, 2 steps), which was transformed to the
iodide 17. The lithium enolate derived from ꢀprolinol Nꢀpropionamide was coupled with iodide
7 to form 1,3,5ꢀantiꢀtrimethyl substituted amide 18 (86%, dr = 97:3 by NMR). Acid hydrolysis
L
1
2
1
D
1
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of 18, followed by reduction generated primary alcohol 19 (65%, 2 steps). Swern oxidation of
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9 followed by BestmannꢀOhira reaction then produced the required alkyne 20 (68%, 2 steps).
Scheme 3. Synthesis of fragment 2 (anti configuration)
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The crucial coupling reaction joining 12 and 20 was conducted by using Carreira
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alkynylation conditions (Scheme 4). This process formed propargylic alcohol 21 (72%, dr =
95:5 by NMR), whose absolute configuration at the newly formed hydroxyl substituted center
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was confirmed as being ‘S’ by utilizing the modified Mosher’s ester method (Figure 2). The
hydroxyl group in 21 was protected to form TBS ether 22 (87%), which was subjected to
complete reduction by using Pd/C promoted hydrogenation to produce saturated alcohol 23
(
75%). Primary alcohol 23 was then oxidized to form a crude aldehyde which was subsequently
reacted with EtMgBr to generate 24 (87%, 2 steps) which was a diastereomeric mixture (1:1 by
NMR). Oxidation of the secondary alcohol group in 24 afforded ketone 25 (92%), which upon
removal of both the acetonide and TBS groups using PTSA in methanol generated the BmcA
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precursor 26 (90%). Removal of methoxy groups in 26 under several conditions proved to be
fruitless except when aluminium iodide was employed, in which case BmcA (22%) was obtained
1
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but only after difficult purification.
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Figure 2. Modified Mosher’s ester analysis.
Owing to the difficulty encountered with removal of the methoxy groups, the protection
strategy was altered. Accordingly, the synthetic route was repeated using MOM protection of the
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phenolic hydroxy groups to produce aldehyde 12a. Carreira alkynylation between 12a and 20
was conducted to deliver propargylic alcohol 21a (73%, dr = 96:4 by NMR) which was protected
as its TBSꢀether 22a (89%). The MOM protected intermediate 22a was then subjected to
hydrogenation by using Pd/C to produce an inseparable mixture of the saturated alcohol 23a and
1
TBSꢀether elimination product 23' in a 8:2 ratio (confirmed by H NMR analysis). After several
unsuccessful attempts to separate 23a, the mixture was used in the next step. Contrary to
expectations, purification was not successful until the final step in the sequence involving global
removal of the MOM, acetonide and TBS groups in 25a by using PTSA in methanol to form 1
(90%).
Scheme 4. Synthesis of the proposed stereostructure of BmcA
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The absolute configurations at the stereogenic centers in 1, prepared by using the route
described above, were firmly established by using the modified Mosher’s method and HSQC
NMR analysis of the corresponding acetonide derivative (acetonide A and B, Figure 3). The
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NMR data for synthetic 1 clearly differed from those reported previously. For instance, the H
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and C NMR chemical shifts for C/Hꢀ1' (δ 76.4; δ 4.84), C/Hꢀ14 (δ 49.1; δ 1.70) and C/Hꢀ6
C
H
C
H
(
δ 28.8; δ 1.52) were observed for the synthetic material whereas C/Hꢀ1' (δ 76.5; δ 4.47),
C
H
C
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C/Hꢀ14 (δ 48.5; δ 1.88), and C/Hꢀ6 (δ 29.1; δ 1.42) were reported for natural BmcA. These
C
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C
H
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differences demonstrate that the proposed stereostructure of the natural product is incorrect. The
Hꢀ14 and Cꢀ14 chemical shift discrepancies and the results of a ROESY NMR study (Figure
S137) with the acetonide derivative of 1 (Figure 3ꢀa) suggest that the configurations at the three
consecutive 1,3ꢀhydroxy substituted centers and isoꢀbutyl substituted carbon are 11R*, 13R*,
14R* and 1'R* rather than the originally proposed 11R*, 13R*, 14S* and 1'R*.
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Figure 3. (a) Acetonide derivatization of 1. H and C NMR chemical shifts indicating a syn
relationship of the both acetonides. (b) The key ROESY correlations of the acetonide A of 1
showing the relative configuration of the isoꢀbutyl group.
In order to address the Cꢀ6 and Hꢀ6 chemical shift discrepancies, NMR spectroscopic
analysis was carried out on the trimethylheptanol derivatives 19, 38 and 42 (Figure 4) to gain
insight into the correct configurations of Cꢀ4, Cꢀ6 and Cꢀ8 in the natural product. The results
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show that the Hꢀ5 and Hꢀ7 H chemical shifts of the protons in the two enantiomeric synꢀsynꢀ
trimethylhepatanol fragments 42 and 38, and the antiꢀantiꢀtrimethylheptanol fragment 19 are
remarkably different (Figure 4).
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Figure 4. H and C chemical shifts of trimethylheptanol fragments.
The Hꢀ5 and Hꢀ7 methylene protons in the two enantiomeric synꢀsynꢀtrimethylhepatanol
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fragments, 38 and 42, display H NMR patterns that are consistent with those of the
corresponding methylene protons in natural BmcA (eg., Hꢀ5a (δ 1.39)/Hꢀ5b (δ 0.89) and Hꢀ7a
H
H
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(
δ 1.31)/H ꢀ7b (δ 0.88)). It was reported that Hꢀ5a (δ 1.73) and Hꢀ7a (δ 1.22) in the
H
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H
H
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spectrum of natural BmcA are remarkably more deshielded than are Hꢀ5b (δ 0.98) and Hꢀ7b (δH
H
0.95), respectively. In contrast, in the spectrum of the antiꢀantiꢀtrimethylheptanol fragment 19,
the chemical shifts of Hꢀ5a (δ 1.24)/Hꢀ5b (δ 1.07) and Hꢀ7a (δ 1.18)/Hꢀ7b (δ 1.01) are not
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that much different. The detailed H NMR analysis of fragments 19, 38 and 42, led us to the
conclusion that synꢀsyn relative configuration of the three methyl groups is more plausible for
natural BmcA than the proposed antiꢀanti relative stereochemistry. A combination of the
findings outlined above led us to propose the two plausible alternative stereostructures, 1a and
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b, (Figure 5) for natural BmcA.
Figure 5. Proposed and revised structure of BmcA.
To resolve this issue and, consequently, assign the correct stereostructure to natural
BmcA, we conducted total syntheses of both 1a and 1b. As depicted retrosynthetically in
Scheme 5, the plan for these syntheses involved joining 39 and 43 with aldehyde 34 using the
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Carreira
alkynylation
protocol.
Fragment
34
was
prepared
from
3,5ꢀ
bis(methoxymethoxy)benzaldehyde (4a) by employing a route similar to the one used to generate
2. The redesigned alkyne fragments, 39 and 43 were synthesized starting with the common
1
chiral precursor 36 which in turn was stereoselectively prepared from commercially available
cisꢀ4,6ꢀdimethylcyclohexanꢀ1,3ꢀdione by employing an approach that uses enzymatic
desymmetrization, Wittig olefination and Evans asymmetric alkylation.
Scheme 5. Retrosynthetic analysis of two possible structures (1a and 1b) of BmcA
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Synthesis of 34 commenced with preparation of MOMꢀprotected aldehyde 4a from 2
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(
78%, 3 steps). Anti aldol reaction between amide 5 and MOMꢀprotected aldehyde 4a in
presence of magnesium chloride produced hydroxyamide 27 (72%, dr = 97:3 by NMR). The anti
aldol product 27 was then used in a reaction sequence that mimics the one used to prepare 12 to
provide revised aldehyde fragment 34. Accordingly, anti aldol product 27 was protected as its
TBS ether 28 (88%). Amide group in 28 was reduced by DIBALꢀH to get alcohol 29 which was
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subsequently oxidized to aldehyde 30 using IBX (76%, 2 steps). Brown allylation of aldehyde
1
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that the diastereomeric mixture was separated in the next stage of the reaction. Deprotection of
TBS group in 31 afforded 1,3ꢀdiol 32 followed by acetonide protection to provide 33 (89%, 2
steps). Dihydroxylation followed by oxidative cleavage of olefin 33 provided revised aldehyde
fragment 34 (70%, 2 steps) (Scheme 6).
Scheme 6. Synthesis of revised aldehyde fragment 34 (anti configuration)
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Both the revised alkyne fragments (39 and 43) derived from a known precursor 36 which
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was synthesized from 35 by following a reported protocol. Fragment 35, synthesized by using a
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known four step protocol starting with cisꢀ4,6ꢀdimethylcyclohexanꢀ1,3ꢀdione, was transformed
to alcohol 36. Benzyl protection of hydroxyl group in 36 was carried out to afford 37 (82%).
TBS group of 37 was deprotected to get primary alcohol 38 (85%) which was then subsequently
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oxidized under Swern condition followed by BestmannꢀOhira reaction to generate alkyne
fragment 39 with all syn configuration (69%, 2 steps). On the other hand, acetyl protection of 36,
followed by deprotection of TBS ether delivered primary alcohol 40 (78%, 2 steps). Benzyl
protection of hydroxyl group in 40 was done to afford 41 (72%), which was hydrolyzed under
basic condition to afford primary alcohol 42 (95%). Primary alcohol 42 underwent Swern
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oxidation followed by BestmannꢀOhira reaction to generate alkyne fragment 43 with all syn
configuration (66%, 2 steps) (Scheme 7).
Scheme 7. Synthesis of revised alkyne 39 and 43
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Carreira alkynylation of 34 and 43 occurred to form propargylic alcohol 44 (76%, dr =
9
5:5 by NMR). Protection of the hydroxyl group in 44 formed TBS ether 45 (82%), which was
then subjected to hydrogenation to produce a mixture (76%) of saturated alcohol 46 and its TBS
1
ether elimination product (8:2, confirmed by H NMR analysis) which was inseparable. The
mixture of 46 and its TBS ether elimination product was oxidized to generate a crude aldehyde,
which was reacted with EtMgBr to produce a secondary alcohol which upon oxidation formed a
mixture (68%, 3 steps) of ketone 47 and its TBS ether elimination product which was inseparable.
Finally, global deprotection of the mixture was achieved by using PTSA to produce 1a and 11ꢀ
deoxy derivative 48 (91%, combined yield) which was separable. Similarly, the BmcA isomer 1b
15
was synthesized by condensing aldehyde 34 with alkyne 39. The formed propargylic alcohol 49
(
75%, dr = 96:4 by NMR) was converted to its TBS ether 50 (83%), which when subjected to a
similar reaction sequence utilized to form 1a gave BmcA isomer 1b along with 11ꢀdeoxy
derivative 51 (41%, 5 steps) (Scheme 8). Comprehensive NMR analysis data (COSY, HSQC,
1
1
HMBC, Hꢀ H decoupling, and HETLOC) for 1a are available in supporting information.
Scheme 8. Synthesis of 1a and 1b
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The absolute configurations of the chiral centers on the left part of 1a were confirmed by
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3
using NMR analysis of its acetonide derivatives (acetonide A and B, Figure 6). The C chemical
shifts of the gemꢀdimethyl groups in acetonide A are 20.1 and 30.6 ppm, indicating the existence
of a synꢀrelationship between 13ꢀOH and 1'ꢀOH groups. The gemꢀdimethyl groups in acetonide
13
B have C chemical shifts of 20.3 and 30.7, indicating that 11ꢀOH and 13ꢀOH also have a synꢀ
relationship (Figure 6).
Figure 6. Acetonide derivatization of 1a. Chemical shifts indicate a syn relationship of the both
acetonides.
1
In addition, analysis of H and 2D NMR spectra of acetonide A, which contains an
isobutyl group in the 1,3ꢀdioxane ring, shows that the Hꢀ14/Hꢀ1' coupling constant is 10.5 Hz.
This large coupling shows that an axialꢀaxial and thus antiꢀrelationship exists between Hꢀ14 and
Hꢀ1' (Figure 7). As depicted in Figure 7, Hꢀ1' (δ 4.35) and Hꢀ13 (δ 3.79) exhibit ROESY
H
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correlations commonly associated with an axially oriented acetonide methyl group (δ 1.53).
H
Moreover, Hꢀ1'/H ꢀ15 (δ 1.20 and 0.99) and Hꢀ13/H ꢀ15 ROESY correlations establish that the
2
H
2
isobutyl group exists in an equatorial position, thus showing that a synꢀrelationship exists
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between Hꢀ1' and Hꢀ13. ROESY coupling between and Hꢀ16 (δ 0.95) and Hꢀ1' also support the
H
assignment of the relative configuration in the 1,3ꢀdioxane, thus confirming the 11R*, 13R*,
14R*, and 1'R* configuration of 1a. Based on the absolute configuration of Cꢀ11 in this
acetonide, established by using the modified Mosher’s method, the absolute stereochemistry was
unequivocally confirmed to be 11R, 13R, 14R, and 1'R.
Figure 7. The key ROESY correlations of the acetonide A of 1a showing the relative
configuration of the isoꢀbutyl group.
1
13
Importantly, the H and C NMR spectra of 1a, having 4R, 6R, 8R, 11R, 13R, 14R, and
4
1'R configurations, were virtually identical to those of natural BmcA even though the reported
NMR spectra of natural BmcA include unidentified impurities (Figure S163). In contrast, the
NMR data of 1b, possessing 4S, 6S, 8S, 11R, 13R, 14R, and 1'R configurations, do not match
those for natural BmcA. These observations strongly suggest that the relative configurations of
BmcA need to be revised to be 4R*, 6R*, 8R*, 11R*, 13R*, 14R*, and 1'R*. The optical rotation
20
of synthetic 1a is +10.0 (c 0.40, MeOH) whereas that of isolated natural BmcA [α] is ˗12.0 (c
D
0.20, MeOH). The magnitude of the two optical rotation values (1a and natural BmcA) was very
similar but the sign was opposite, which suggests that synthetic 1a would be enantiomer of the
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natural product. However, because the H NMR spectrum (Figure S163) of natural BmcA
shows that it contains many unidentified impurities the reported optical rotation is questionable.
As previously mentioned, the absolute configuration of natural BmcA was arbitrarily assigned in
the original report. Owing to the impurities in natural BmcA and the uncertainty of the reported
optical rotation value, the absolute stereochemistry of BmcA was unclear to us. Aggarwal et.
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11
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c
al. reported that synthetic 1a is enantiomer of the natural product while our manuscript was
under review. In the effort described above, we achieved the first total synthesis of correct
1
1
1
enantiomer (1a) of BmcA. By using H, Hꢀ H decoupling, and HETLOC NMR experiments
performed with a 900 MHz spectrometer, we have performed Jꢀbased configuration analysis
(
Figure 8) for 1a and clarified the misinterpreted Jꢀbased configuration analysis in the previous
1
1
report, which was possibly a result of overlapped H signals in a lower field region of the H
NMR caused by using a 700 MHz instrument.
4
In order to revise the reported configurations at stereocenters of natural baulamycin A,
we focused to clarify what caused misinterpretation in Jꢀbased configuration analysis in the
4
1
1
1
previous report. The H, Hꢀ H decoupling, and HETLOC NMR experiments of 1a performed in
3
3
2
900 MHz NMR spectrometer enabled us to obtain accurate J , J , and J values and
HH
CH
CH
4
3
compare them with those in literature (Figure 8). Based on our careful measurement of J ,
HH
3
2
J , and J values and Jꢀbased configuration analysis, we identified critical errors in the
CH
CH
4
previous report. Besides minor errors like alleged typos, wrong interpretation of J based
configuration analysis of the stereogenic centers around Cꢀ6 and Cꢀ14 resulted in wrong
3
assignments for the configurations of Cꢀ6 and Cꢀ14. First, we corrected JH14,H13 value from 7.7
1
1
Hz to 4.0 Hz based on the Hꢀ H decoupling experiment at Hꢀ15, which was not performed in the
4
2
previous report. Furthermore, J
value was revised from 7.0 Hz to 2.0 Hz, establishing the
C13H14
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rotamer in Figure 8ꢀb. Additional decoupling experiments were performed at Hꢀ10, Hꢀ11, Hꢀ12,
3
3
3
and Hꢀ13 to correct the J
(7.2 Hz to 3.5 Hz), J
(3.1 Hz to 9.5 Hz), J
(8.0
H13,H12a
H13,H12b
H11,H12a
3
Hz to 4.5 Hz), and J
(3.2 Hz to 8.5 Hz) values. Careful analysis of HETLOC data
H11,H12b
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enabled to identify J
, J
, and J as 1.0, 4.5, and 1.5 Hz, which were
C11,H12a
C13,H12a
C13,H12b
4
misassigned as 6.2, 1.8, and 6.8 Hz respectively in the previous report. The antiꢀrelationship
2
between Hꢀ13 and Hꢀ12b and large value of JC13,H12b constructed the revised rotamer depicted in
Figure 8ꢀc. The Hꢀ11/Hꢀ13, Hꢀ12a/Hꢀ13, Hꢀ12a/Hꢀ14, and Hꢀ12b/Hꢀ14 ROESY correlations
supported this revised rotamer. The small coupling constant (4.5 Hz) of Hꢀ11/Hꢀ12a and the
large coupling constant (8.5 Hz) of Hꢀ11/Hꢀ12b, which were previously reported in the opposite
3
3
way [large (8.0 Hz) for J
and small (3.2 Hz) for JH11,H12b] were key evidence for the
H11,H12a
construction of the revised rotamer in Figure 8ꢀd. Furthermore, several critical errors of coupling
4
constants in the previous report were found in Cꢀ7ꢀCꢀ6 (Figure 8ꢀi) and Cꢀ5ꢀCꢀ4 (Figure 8ꢀk)
1
1
rotamers. For the extraction of accurate coupling constants, a suite of Hꢀ H decoupling NMR
3
experiments were performed for H ꢀ7, Hꢀ6, H ꢀ5, and Hꢀ4. Based on the newly measured J
2
2
HH
3
3
3
values, J
(3.2 Hz) and J
(3.0 Hz) were revised to 8.5 and 6.6 Hz, respectively. J
H7a,H6
H7b,H6
C5H7b
3
4
and J
values (6.0 Hz and 3.5 Hz), which were not reported in the previous report, were
C20H7b
also clearly measured in our HETLOC NMR data. Thus, antiꢀrelationships between Hꢀ7a/Hꢀ6
and Cꢀ5/Hꢀ7b were established in Figure 8ꢀi. The gauche relationships of Hꢀ7b/Hꢀ6, Cꢀ8/Hꢀ6, Cꢀ
5/Hꢀ7a, Cꢀ20/Hꢀ7a, and Cꢀ20/Hꢀ7b were also identified by the coupling constants, completing
the rotamer depicted in Figure 8ꢀi. Additional proton decoupling experiments for Hꢀ4, Hꢀ5a, Hꢀ
4
3
3
5
b, H ꢀ19 revealed that reported J
(3.2 Hz) and J
(8.0 Hz) values had to be revised
3
H5a,H4
H5b,H4
to 8.5 and 5.0 Hz, respectively. Careful analysis of HETLOC data resulted in the correction of
3
JC21,H5a (11.0 Hz) to 4.0 Hz. Thus, the antiꢀrelationship between Hꢀ5a and Hꢀ4 was established
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in Figure 8ꢀk. The relationships between Hꢀ5b/Hꢀ4, Cꢀ6/Hꢀ4, C21/Hꢀ5a, and Cꢀ21/Hꢀ5b were
determined as gauche, completing the rotamer depicted in Figure 8ꢀk. Consequently, our
1
1
1
comprehensive Jꢀbased configuration analysis utilizing H, Hꢀ H decoupling and HETLOC
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NMR experiments at high fields (800/900 MHz) allowed for the revision of JHH and J , thus
CH
4
confirming the configurations of synthetic baulamycin A (1a). In the previous report of natural
baulamycin A, the coupling constants were measured at lower magnetic field without performing
1
1
Hꢀ H decoupling experiments. This may have caused the mismeasurement of the coupling
constants and thus resulted in the confusion for establishing the relative configurations because
1
of highly overlapped H signals in baulamycin A. Errors in coupling constant measurement in
4
the previous report are comprehensively noted in Figure S164.
Figure 8. Jꢀbased configuration analysis of 1a. Arrows represents ROESY correlations.
As part of these studies, twelve BmcA derivatives, including enantiomer of BmcA (1a),
22
were subjected to SAR analysis to assess inhibitory activities against SbnE. Synthesis of
1
5
baulamycin A derivatives 55 and 57 is depicted in Scheme 9. Carreira alkynylation between
aldehyde fragment 12a and benzylꢀprotected alkyne fragment 52 derived from 7ꢀoctyneꢀ1ꢀol was
achieved to deliver propargylic alcohol 53 (74%, dr = 96:4 by NMR). MOM and acetonide
group in 53 was deprotected by using PTSA to afford polyhydroxy compound 55 (84%).
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Propargylic hydroxyl group in 53 was protected as its TBS ether 54 (81%). Hydrogenation of 54
2
3
using Pd/C was carried out to provide saturated primary alcohol 56 (81%). A similar sequence
of reactions like baulamycin A preparation was adopted to deliver 57, a derivative of baulamycin
A (61%, 4 steps). Baulamycin A derivatives 55 and 57 were submitted for SAR study (Table 1).
Scheme 9. Synthesis of baulamycin A derivatives 55 and 57
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Synthesis of other baulamycin A derivatives 58, 59, 60 and 61 are outlined in Scheme 10.
All of protecting groups (MOM, acetonide, and TBS) in 21a, 23a and 23' were deprotected to
afford compound 58, 59 and 60 respectively and similar sequence of reactions (oxidation,
Grignard reaction, oxidation, deprotection) like baulamycin A synthesis were applied on 23' to
provide 61 as a baulamycin A derivative (60%, 4 steps) (Scheme 10).
Scheme 10. Synthesis of baulamycin A derivatives 58, 59, 60 and 61
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24
SbnE inhibition assay results, obtained by employing a malachite greenꢀbased method
are outlined in Table 1. In contrast to Sherman and coꢀworkers report that natural BmcA has an
4
IC value of 4.8 µM, 1 possessing the originally proposed stereostructure of this substance has
5
0
virtually no activity (IC >50 µM). In contrast, 1a, whose stereostructure is now known to be
5
0
that of enantiomer of natural BmcA, has an IC value of 14.4 µM.
5
0
Table 1: IC values of BmcA derivatives against SbnE.
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0
Compound
IC (uM)
Compound
IC (uM)
50
50
>
50
20.98
25.73
>50
>50
1
4.40
7.27
4
27.77
7.16
>50
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The results of the SAR study show that the existence of branched methyl groups on the
hydrocarbon tail (Cꢀ1 through Cꢀ10) has little to no effect on inhibitory activity. Based on a
comparison of the IC values of 1, 26, 57 and 59 with those of 1a and 1b, the (R)ꢀconfiguration
5
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at Cꢀ14 seems to be required for inhibitory activity for BmcA derivatives having three hydroxyl
groups at Cꢀ11, Cꢀ13 and Cꢀ1' positions and saturated hydrocarbon tails (Cꢀ1 through Cꢀ10).
However, BmcA derivatives 48, 51, 60 and 61 that do not contain the Cꢀ11 hydroxyl group
possess inhibitory activities regardless of the stereochemistry at Cꢀ14. Interestingly, the
acetylene derivatives 55 and 58 exhibit appreciable potencies despite having the (S)ꢀ
configuration at Cꢀ14 and three hydroxyl groups at Cꢀ11, Cꢀ13 and Cꢀ1'. These compounds
contain a chainꢀrigidifying modification caused by incorporation of a triple bond between Cꢀ9
and Cꢀ10, which may contribute to control of the hydrocarbon tail orientation for optimal
interaction in the active site of SbnE. It should be noted that 55 has the highest potency among
all of the tested derivatives. A more comprehensive SAR study probing the consequences of
specific stereochemical and structural changes is underway and will be discussed in due course.
Conclusions
In summary, we have accomplished a synthesis of the earlier reported stereostructure of BmcA
and found that discrepancies exist in the spectroscopic properties between the synthetic material
and the natural BmcA. Two possibly correct stereostructures for BmcA, identified on the basis of
comprehensive NMR analysis, were prepared by total synthesis. The synthetic pathways employ
stereoselective Evans Aldol, Brown allylation and Carreira alkynylation reactions, and a prolinol
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amide derived alkylation/desymmetrization protocol for installing three chiral centers at Cꢀ4, Cꢀ6
and Cꢀ8. Comprehensive NMR studies with these two synthetic materials (1 and 1a) enabled the
assignment of the correct enantiomer (1a) of BmcA. Moreover, inhibitory activities of novel
BmcA derivatives against SbnE were determined. The SAR data provide insight about how the
configuration of Cꢀ14, the existence of OHꢀ11 and chainꢀrigidifying group affect inhibitory
activities against SbnE. It is worth noting that the propargylic alcohol 55, having 14S
configuration, exhibits the highest SbnE inhibitory activity even though it lacks three methyl
groups at Cꢀ4, Cꢀ6 and Cꢀ8 and a ketone group, suggesting that the presence of a chainꢀ
rigidifying triple bond contributes to the inhibitory activity of members of this class of
substances. This structural revision with efficient synthetic strategy and SAR study lay the
groundwork for discovering new BmcA derivatives that overcome drug resistant pathogens that
cause lifeꢀthreatening infections.
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Experimental Section
General Experimental Methods: All reactions were carried out under an inert atmosphere of
argon or nitrogen using standard syringe, septa, and cannula techniques unless otherwise
mentioned. Reactions were monitored by using TLC with 0.25 mm E. Merck precoated silica gel
plates (60 F ). Reaction progress was monitored by using TLC with a UV lamp, ninhydrin, or
2
54
pꢀanisaldehyde stain for detection purposes. Commercially available reagents were used without
further purification. All solvents were purified by using standard techniques. Purification of
products was carried out by using silica gel column chromatography using Kieselgel 60 Art.
9385 (230ꢀ400 mesh). The purity of all compounds was determined to be over 95% by using a
Waters LCMS system (Waters 2998 Photodiode Array Detector, Waters 3100 Mass Detector,
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Waters SFO System Fluidics Organizer, Water 2545 Binary Gradient Module, Waters Reagent
Manager, Waters 2767 Sample Manager) using SunFireTM C18 column (4.6 x 50 mm, 5 µm
particle size): solvent gradient = 60% (or 95%) A at 0 min, 1% A at 5 min. Solvent A = 0.035%
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TFA in H O; Solvent B = 0.035% TFA in MeOH; flow rate: 3.0 (or 2.5) mL/min. H and C
2
1
NMR spectra were obtained using a Bruker 400 MHz FTꢀNMR (400 MHz for H and 100 MHz
1
3
1
13
for C) spectrometer. H, C, and 2D nuclear magnetic resonance (NMR) spectra were also
recorded on Bruker Avance 600ꢀMHz spectrometers at the National Center for Interꢀuniversity
Research Facilities at Seoul National University (NCIRF) and on a Bruker Avance II 900ꢀMHz
NMR spectrometer at the Korea Basic Science Institute (KBSI) at Ochang. Standard
abbreviations are used for denoting the signal multiplicities. Infrared spectra were measured on
FTꢀIR Nicolet iS10 spectrometer. Samples were recorded under neat or as KBr optics. Highꢀ
resolution mass spectra (HRMS) were recorded on a QTOF mass spectrometer. Optical rotations
were measured by a JASCO Pꢀ200 polarimeter with a 1 cm cell. UV spectra were acquired with
a Perkin Elmer Lambda 35 UV/VIS spectrometer. Electrospray ionization (ESI) low–resolution
LC/MS data were acquired on an Agilent Technologies 6130 quadrupole mass spectrometer
coupled with an Agilent Technologies 1200–series HPLC. Semiꢀpreparative HPLC separations
were performed with an HPLC system composed of a Gilson 305 pump and a Gilson UV/VISꢀ
1
55 detector.
Methyl 3,5-Bis(methoxymethoxy)benzoate (3a). To
dihydroxybenzoate 2 (10 g, 59.4 mmol) in anhydrous CH Cl (200 mL) was added
a
solution of methyl 3,5ꢀ
2
2
diisopropylethylamine (41.8 mL, 240 mmol), followed by MOMCl (10.6 mL, 120 mmol)
o
dropwise at 0 C. The mixture was warmed to room temperature and stirred for 12 h. The
mixture was then quenched with water (30 mL) and diluted with CH Cl (50 mL). The organic
2
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layer was separated and washed with saturated NaHCO solution (30 mL) and extracted with
3
CH Cl (3 x 100 mL). The combined organic layer was washed with brine, dried over MgSO
4
2
2
and concentrated under reduced pressure. The resulting residue was subjected to silica gel
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
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43
44
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46
47
48
49
50
51
52
53
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57
58
59
60
column chromatography to afford compound 3a (13.9 g, 92%) as colorless oil. [Known literature
25
25 1
method] ; R = 0.8 (30% EtOAc/hexane). H NMR (400 MHz, CDCl3):
δ
7.35 (d, J = 2.3 Hz,
f
2H), 6.90 (t, J = 2.3 Hz, 1H), 5.18 (s, 4H), 3.88 (s, 3H), 3.47 (s, 6H); LCMS (ESI): 257 (M +
+
H) .
3,5-Bis(methoxymethoxy)benzaldehyde (4a). To a stirred solution of methyl 3,5ꢀ
bis(methoxymethoxy)benzoate 3a (12 g, 46.4 mmol) in anhydrous THF (120 mL) was added
dropwise a solution of lithium aluminium hydride (56 mL of a 1.0 M solution in THF, 55.7
mmol). The mixture was stirred at ambient temperature for 4 h and treated dropwise sequentially
with water (1 mL), 15% aqueous NaOH (1 mL) and water (3 mL). The mixture was stirred for
additional 1 h and filtered and the resulting solid was washed with THF. The filtrate was
concentrated to provide 3,5ꢀbis(methoxymethoxy)benzyl alcohol as a colorless liquid which was
directly used in the next step without further purification.
To a solution of PCC (13.1 g, 61.2 mmol) and sodium acetate (17.3 g, 21.4 mmol) in CH Cl
2
2
(100 mL) was added a solution of 3,5ꢀbis(methoxymethoxy)benzyl alcohol (7 g, 30.6 mmol) in
CH Cl (8 mL). The mixture was stirred under nitrogen for 2 h and treated with ether (300 mL).
2
2
The brown mixture was filtered through filter paper over Celite. The filtrate was concentrated to
provide brown oil which was subjected to silica gel column chromatography to afford compound
25
4a (8.9 g, 85% over two steps) as a colorless oil. [known literature method] ; R = 0.4 (20%
f
1
EtOAc/hexane); H NMR (400 MHz, CDCl ):
δ 9.89 (s, 1H), 7.19 (d, J = 2.3 Hz, 2H), 6.96 (t, J
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5
5
5
5
5
5
5
5
5
6
13
=
2.3 Hz, 1H), 5.19 (s, 4H), 3.47 (s, 6H); C NMR (100 MHz, CDCl ):
δ
191.5, 158.7, 138.4,
3
+
111.1, 110.3, 94.4, 77.3, 77.0, 76.6, 56.1; LCMS (ESI): 227 (M + H) .
(
R)-4-Benzyl-3-(4-methylpentanoyl)oxazolidin-2-one (5). To a stirred solution of 4ꢀ
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
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
o
methylvaleric acid (4 g, 34.4 mmol) in anhydrous THF (100 mL) at ˗20 C was added Et N (12
3
mL, 86.1 mmol) followed by pivaloyl chloride (4.2 mL, 34.4 mmol). The mixture was stirred for
o
1
2
h at ˗20 C and treated sequentially with LiCl (2.2 g, 51.6 mmol) and (R)ꢀ4ꢀbenzyloxazolidinꢀ
o
ꢀone (6.7 g, 37.8 mmol) at same temperature. The mixture was stirred for 1 h at ˗20 C and for 2
o
h at 0 C and quenched with saturated NH Cl solution (30 mL). The mixture was diluted with
4
EtOAc (50 mL) and extracted with EtOAc (3 x 50 mL). The combined organic layer was washed
with brine, dried over MgSO , filtered and concentrated under reduced pressure. The residue was
4
subjected to silica gel column chromatography to afford 5 (8.5 g, 90%) as a yellow liquid. R =
f
2
4
1
0
.3 (30% EtOAc/hexane); [α]D = +45.4 (c 0.55, CHCl ); H NMR (400 MHz, CDCl ):
δ
7.35ꢀ
3
3
7
.31 (m, 2H), 7.29ꢀ7.25 (m, 1H), 7.22ꢀ7.19 (m, 2H), 4.69ꢀ4.64 (m, 1H), 4.21ꢀ4.09 (m, 2H), 3.28
(
dd, J = 13.4, 3.3 Hz, 1H), 3.02ꢀ2.86 (m, 2H), 2.76 (dd, J = 13.4, 9.6 Hz, 1H), 1.70ꢀ1.52 (m, 3H),
1
3
0.93 (d, J = 6.4 Hz, 6H); C NMR (100 MHz, CDCl ):
δ
173.6, 153.4, 135.3, 129.4, 128.9,
3
1
27.3, 66.1, 55.1, 37.9, 33.6, 33.1, 27.6, 22.3(2); HRMS (ESI): calcd. for C H O NNa [M +
1
6
21
3
+
Na] 298.1419; found 298.1415.
R)-4-Benzyl-3-((R)-2-((R)-(3,5-dimethoxyphenyl)(hydroxy)methyl)-4-
(
methylpentanoyl)oxazolidin-2-one (6). To a 0.2ꢀ0.5 M solution of 5 (9.0 g, 32.7 mmol) in
o
anhydrous CH Cl (100 mL) under argon at 0 C was added dibutylboron triflate (39.2 mL of a
2
2
1
.0 M solution in CH Cl , 39.2 mmol), followed by diisopropylethylamine (7.4 mL, 42.5 mmol)
2 2
o
o
dropwise. After stirred for 30 min at 0 C, the mixture was cooled to ˗78 C and treated with
o
benzaldehyde 4 (6 g, 36 mmol). The mixture was then stirred for 30 min at ˗78 C and for 1.5 h
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at room temperature. The boron complex was quenched with phosphate buffer (pH~7, 50 mL)
and oxidized with a mixture of 30% H O and methanol (100 mL of a 2:1 solution) for 1 h at 0
2
2
o
C. The mixture was stirred for 1 h at room temperature and the resulting mixture was
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48
49
50
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concentrated to slurry. The residue was extracted with ether (3 x 100 mL) and the combined
organic extracts were washed with 5% aqueous NaHCO solution (30 mL), brine (30 mL), and
3
then dried over MgSO , filtered and concentrated under reduced pressure. The residue was
4
subjected to silica gel column chromatography to afford 6 (13.7 g, 86%) as a colorless liquid. Rf
2
4
1
= 0.2 (20% EtOAc/hexane); [α]D = −206.6 (c 1.08, CHCl ); H NMR (400 MHz, CDCl ):
δ
3
3
7.34ꢀ7.27 (m, 3H), 7.19ꢀ7.17 (m, 2H), 6.56 (d, J = 2.1 Hz, 2H), 6.35 (t, J = 2.3 Hz, 1H), 4.76 (q,
J = 3.1 Hz, 1H), 4.49ꢀ4.45 (m, 1H), 4.43ꢀ4.36 (m, 1H), 4.01 (dd, J = 9.0, 2.2 Hz, 1H), 3.77 (s,
6H), 3.22 (dd, J = 13.4, 3.4 Hz, 1H), 2.68 (dd, J = 13.4, 9.8 Hz, 1H), 2.35 (d, J = 3.3 Hz, 1H),
1
3
2
.04ꢀ1.94 (m, 1H), 1.58ꢀ1.49 (m, 3H), 0.91 (d, J = 2.6 Hz, 3H), 0.89 (d, J = 2.4 Hz, 3H); C
174.6, 160.4, 152.9, 144.1, 135.0, 129.2, 128.7, 127.1, 103.7, 100.1,
5.7, 65.7, 55.5, 55.1, 48.4, 37.7, 36.7, 26.5, 23.5, 21.7; HRMS (ESI): calcd. for C H O NNa
NMR (100 MHz, CDCl3):
δ
7
2
5
31
6
+
[M + Na] 464.2049; found 464.2044.
R)-4-Benzyl-3-((R)-2-((R)-((tert-butyldimethylsilyl)oxy)(3,5-dimethoxyphenyl)methyl)-4-
(
methylpentanoyl)oxazolidin-2-one (7). To a solution of 6 (8 g, 18.1 mmol) in anhydrous
CH Cl (50 mL) was added 2,6ꢀlutidine (8.4 mL, 72.4 mmol), followed by TBSOTf (8.3 mL,
2
2
o
o
3
6.2 mmol) slowly at 0 C. The mixture was stirred for 1 h at 0 C and quenched with water (15
mL). The layers were separated and the aqueous layer was extracted with CH Cl (3 x 50 mL).
2
2
The combined organic layer was washed with saturated NaHCO solution (30 mL) and dried
3
over MgSO , concentrated under reduced pressure to give crude residue which was subjected to
4
silica gel column chromatography to afford 7 (8.8 g, 88%) as a yellow liquid. R = 0.3 (30%
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5
5
5
5
5
5
5
5
5
6
24
1
EtOAc/hexane); [α]D = +218.5 (c 1.09, CHCl ); H NMR (400 MHz, CDCl ):
δ 7.32ꢀ7.23 (m,
3
3
3H), 7.19ꢀ7.14 (m, 2H), 6.49 (d, J = 2.6 Hz, 2H), 6.30 (t, J = 2.2 Hz, 1H), 4.54 (d, J = 7.8 Hz,
H), 4.36ꢀ4.31 (m, 1H), 4.17ꢀ4.12 (m, 1H), 3.88 (dd, J = 8.9, 1.6 Hz, 1H), 3.74 (s, 6H), 3.48 (t, J
8.1 Hz, 1H), 3.11 (dd, J = 13.3, 3.2 Hz, 1H), 2.67 (dd, J = 13.3, 9.5 Hz, 1H), 1.99ꢀ1.91 (m, 1H),
1
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
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
=
1.68ꢀ1.59 (m, 1H), 0.93 (d, J = 6.4 Hz, 6H), 0.91ꢀ0.89 (m, 1H), 0.88 (s, 9H), 0.03 (s, 3H), ˗0.18
13
(
s, 3H); C NMR (100 MHz, CDCl ):
δ
174.3, 160.3, 152.8, 145.1, 135.4, 129.4, 128.8, 127.2,
3
104.2, 100.2, 77.8, 65.7, 55.9, 55.4, 50.2, 38.1, 37.9, 26.8, 25.7, 23.7, 21.9, 18.1, ˗4.6, ˗5.2;
+
HRMS (ESI): calcd. for C H O NSiNa [M + Na] 578.2914; found 578.2918.
31
45
6
(
R)-2-((R)-((tert-Butyldimethylsilyl)oxy)(3,5-dimethoxyphenyl)methyl)-4-methylpentanal
(8). To a solution of 7 (8 g, 14.4 mmol) in anhydrous CH Cl (100 mL) was added DIBALꢀH
2
2
o
(
25.6 mL, 36 mmol, 20% solution in toluene) slowly for 15 min at ˗78 C. The mixture was
o
stirred for 30 min at ˗78 C before being quenched with methanol (10 mL) and aqueous saturated
sodium potassium tartrate solution (50 mL). The mixture was passed through a small pad of
Celite and then extracted with CH Cl (3 x 50 mL). The combined organic layer was dried over
2
2
MgSO , concentrated under reduced pressure to get crude residue which was used for next step
4
without further purification.
In the next step, to a solution of crude residue in MeOH: H O (4:1, 40 mL) was added solid
2
o
K CO (8 g, 58 mmol) at 0 C and the mixture was stirred for 1 h at the same temperature. The
2
3
mixture was filtered through a pad of Celite and the filter cake was washed with CH Cl (20 mL).
2
2
The filtrate was washed with water (10 mL) and brine (10 mL), dried over MgSO and
4
concentrated under reduced pressure. The residue was subjected to silica gel column
chromatography to give compound 8 (3.3 g, 60% over two steps) as a colorless liquid. R = 0.8
f
24
1
(20% EtOAc/hexane); [α]D = +51.8 (c 0.89, CHCl ); H NMR (400 MHz, CDCl ):
δ 9.67 (d, J
3
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=
2.8 Hz, 1H), 6.43 (d, J = 2.1 Hz, 2H), 6.34 (t, J = 2.2 Hz, 1H), 4.93 (d, J = 4.7 Hz, 1H), 3.77 (s,
6H), 2.59ꢀ2.52 (m, 1H), 1.74ꢀ1.66 (m, 1H), 1.49ꢀ1.38 (m, 1H), 1.33ꢀ1.25 (m, 1H), 0.90 (s, 9H),
1
3
0.83 (d, J = 6.5 Hz, 3H), 0.74 (d, J = 6.5 Hz, 3H), 0.03 (s, 3H), ˗0.13 (s, 3H); C NMR (100
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
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37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
MHz, CDCl3):
δ
205.0, 160.6, 144.7, 104.3, 99.3, 74.8, 58.2, 55.3, 33.0, 25.7, 23.3, 21.7, 18.1,
+
˗4.6, ˗5.3; HRMS (ESI): calcd. for C H O SiNa [M + Na] 403.2281; found 403.2272.
2
1
36
4
(4R,5S)-5-((R)-((tert-Butyldimethylsilyl)oxy)(3,5-dimethoxyphenyl)methyl)-7-methyloct-1-
en-4-ol (9). To a stirred solution of ( )ꢀipc BOMe (3.3 g, 10.4 mmol) in anhydrous Et O (20
+
2
2
o
mL) was added allylmagnesium bromide (7.9 mL of 1.0 M solution in ether, 7.89 mmol,) at 0 C.
o
The mixture was stirred at room temperature for 1 h before being cooled to ˗78 C. The mixture
o
was treated dropwise with aldehyde 8 (2.0 g, 5.26 mmol) at ˗78 C and stirred for 1 h at same
temperature and allowed to warm to room temperature. The mixture was treated with an aqueous
o
solution of NaOH (2 M in H O, 10 mL) and 30% H O solution (5 mL) at 0 C. The biphasic
2
2
2
solution was separated and the aqueous layer was extracted with Et O (3 x 30 mL). The
2
combined organic layer was dried over MgSO and concentrated under reduced pressure. The
4
residue was subjected to silica gel column chromatography to give compound 9 (3.2 g, 74%) as a
2
4
1
colorless liquid. R = 0.2 (10% EtOAc/hexane); [α]D = + 29.1 (c 0.5, CHCl ); H NMR (400
f
3
MHz, CDCl3):
δ 6.48 (d, J = 2.1 Hz, 2H), 6.33 (t, J = 2.2 Hz,1H), 5.84ꢀ5.73 (m, 1H), 5.12ꢀ5.06
(m, 2H), 4.78 (d, J = 4.4 Hz, 1H), 3.88 (t, J = 7.5 Hz, 1H), 3.78 (s, 6H), 2.31ꢀ2.22 (m,1H), 2.18ꢀ
2.11 (m, 1H), 1.67ꢀ1.63 (m, 1H), 1.56 (brs, 1H), 1.46ꢀ1.38 (m, 1H), 1.29ꢀ1.19 (m, 2H), 0.92 (s,
1
3
9H), 0.78 (d, J = 6.2 Hz, 3H), 0.72 (d, J = 6.2 Hz, 3H), 0.07 (s, 3H), ˗0.17 (s, 3H); C NMR
(100 MHz, CDCl3):
δ
160.5, 146.2, 135.6, 117.2, 104.3, 99.1, 77.8, 72.4, 55.2, 48.0, 39.6, 32.3,
+
27.0, 25.8, 23.2, 22.4, 18.0, ˗4.5, ˗5.1; HRMS (ESI): calcd. for C H O SiNa [M + Na]
24 42 4
445.2750; found 445.2745.
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(
1R,2S,3R)-1-(3,5-Dimethoxyphenyl)-2-isobutylhex-5-ene-1,3-diol (10). To a stirred solution
of silyl compound 9 (3 g, 7.10 mmol) in anhydrous THF (20 mL) was added TBAF (12.8 mL of
M solution in THF, 12.79 mmol) at 0 °C. The mixture was stirred for 4 h at room temperature,
1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
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0
1
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4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
diluted with saturated NaHCO solution (5 mL). Organic layer was separated and washed with
3
brine (5 mL), dried over MgSO and concentrated under reduced pressure. The residue was
4
subjected to silica gel column chromatography to give compound 10 (2.0 g, 93%) as a colorless
2
4
1
liquid. R = 0.15 (20% EtOAc/hexane); [α]D = +1.9 (c 0.69, CHCl ); H NMR (400 MHz,
f
3
CDCl3):
δ
6.51 (d, J = 2.2 Hz, 2H), 6.33 (t, J = 2.2 Hz, 1H), 5.87ꢀ5.77 (m, 1H), 5.18ꢀ5.14 (m,
2
H), 4.97 (brs, 1H), 4.04ꢀ4.01 (m, 1H), 3.78 (s, 6H), 3.25 (d, J = 1.7 Hz, 1H), 2.56 (d, J = 2.2 Hz,
1H), 2.32ꢀ2.21 (m, 2H), 1.74ꢀ1.70 (m, 1H), 1.44ꢀ1.35 (m, 1H), 1.28ꢀ1.21 (m, 1H), 1.01ꢀ0.93 (m,
13
1
H), 0.72 (d, J = 6.6 Hz, 3H), 0.52 (d, J = 6.6 Hz, 3H); C NMR (100 MHz, CDCl ):
δ
160.6,
3
1
46.0, 135.0, 118.2, 103.7, 99.1, 78.1, 75.5, 55.3, 47.0, 39.9, 30.2, 27.3, 22.6, 22.5; HRMS
+
(ESI): calcd. for C H O Na [M + Na] 331.1885; found 331.1885.
1
8
28
4
(4R,5S,6R)-4-Allyl-6-(3,5-dimethoxyphenyl)-5-isobutyl-2,2-dimethyl-1,3-dioxane (11). To a
stirred solution of 10 (2.0 g, 6.49 mmol) in CH Cl (20 mL) was added 2,2ꢀdimethoxy propane
2
2
o
(4 mL, 30 mmol) followed by PTSA (cat.) at 0 C. The mixture was allowed to warm to room
temperature and stirred for 30 min before being quenched with water (10 mL). The organic layer
was extracted with CH Cl (3 x 10 mL) and the combined organic layer was washed with brine
2
2
(5 mL), dried over MgSO and concentrated under reduced pressure. The residue was subjected
4
to silica gel column chromatography to afford 11 (2.2 g, 96%) as a colorless liquid. R = 0.8
f
24
1
(20% EtOAc/hexane); [α]D = +49.4 (c 0.75, CHCl ); H NMR (400 MHz, CDCl ):
δ
6.50 (dd, J
3
3
=
2.4, 0.6 Hz, 2H), 6.33 (t, J = 2.2 Hz, 1H), 5.91ꢀ5.80 (m, 1H), 5.15ꢀ5.05 (m, 2H), 4.99 (d, J =
1.8 Hz, 1H), 4.10 (dt, J = 7.8, 2.1 Hz, 1H), 3.79 (s, 6H), 2.40ꢀ2.29 (m, 1H), 2.22ꢀ2.14 (m, 1H),
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.50ꢀ1.49 (m, 1H), 1.50 (s, 3H), 1.49 (s, 3H), 1.25ꢀ1.14 (m, 2H), 0.88ꢀ0.78 (m, 1H), 0.69 (d, J =
13
6.5 Hz, 3H), 0.45 (d, J = 6.5 Hz, 3H); C NMR (100 MHz, CDCl ):
δ
160.6, 143.7, 135.1, 116.7,
3
103.9, 99.3, 98.8, 75.2, 74.2, 55.3, 40.9, 37.5, 30.2, 29.9, 27.2, 22.8, 22.5, 19.5; HRMS (ESI):
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
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45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
+
calcd. for C H O Na [M + Na] 371.2198; found 371.2181.
2
1
32
4
2-((4R,5S,6R)-6-(3,5-Dimethoxyphenyl)-5-isobutyl-2,2-dimethyl-1,3-dioxan-4-
yl)acetaldehyde (12). To a stirred solution of compound 11 (2 g, 5.75 mmol) in THF:H O (3:1,
2
2
0 mL) was added NMO (1.2 g, 10.35 mmol) followed by osmium tetroxide (600 µL, 0.06
o
mmol) at 25 C. The mixture was stirred at the same temperature for 2 h and quenched with
aqueous Na S O solution (5 mL). The mixture was extracted with EtOAc (3 x 10 mL) and the
2
2
4
combined organic layer was washed with brine (5 mL), dried over MgSO and concentrated
4
under reduced pressure to get crude diol which was directly used for next step without further
purification.
To a stirred solution of crude diol in THF:H O (2:1, 20 mL) was added NaIO (1.9 g, 9.2 mmol)
2
4
o
portion wise at 0 C. The mixture was stirred for 2 h at room temperature and then diluted with
saturated NaHCO solution (5 mL). The layers were separated and the organic layer was washed
3
with brine (5 mL), dried over MgSO and concentrated under reduced pressure. The residue was
4
subjected to silica gel column chromatography to give compound 12 (1.37 g, 68% over two
24
1
steps) as a colorless liquid. R = 0.4 (20% EtOAc/hexane); [α]D = +172.1 (c 3.47, CHCl ); H
f
3
NMR (400 MHz, CDCl3):
δ 9.79 (t, J = 1.7 Hz, 1H), 6.49 (d, J = 1.7 Hz, 2H), 6.34 (t, J = 2.2 Hz,
1
H), 5.07 (d, J = 2.0 Hz, 1H), 4.71ꢀ4.67 (m, 1H), 3.78 (s, 6H), 2.71 (ddd, J = 16.8, 8.8, 1.8 Hz,
1H), 2.45 (ddd, J = 16.9, 4.5, 1.5 Hz, 1H), 1.59ꢀ1.55 (m, 1H), 1.52 (s, 3H), 1.47 (s, 3H), 1.22ꢀ
13
1
.17 (m, 2H), 0.83ꢀ0.71 (m, 1H), 0.66 (d, J = 6.4 Hz, 3H), 0.45 (d, J = 6.4 Hz, 3H); C NMR
(100 MHz, CDCl3):
δ
200.9, 160.6, 143.1, 103.8, 99.6, 98.9, 74.8, 69.0, 55.3, 47.1, 40.8, 30.4,
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