Thiolactomycin-Based Inhibitors of Bacterial β-Ketoacyl-ACP Synthases with in Vivo Activity

Article abstract of DOI:10.1021/acs.jmedchem.6b00236

β-Ketoacyl-ACP synthases (KAS) are key enzymes involved in the type II bacterial fatty acid biosynthesis (FASII) pathway and are putative targets for antibacterial discovery. Several natural product KAS inhibitors have previously been reported, including thiolactomycin (TLM), which is produced by Nocardia spp. Here we describe the synthesis and characterization of optically pure 5R-thiolactomycin (TLM) analogues that show improved whole cell activity against bacterial strains including methicillin-resistant Staphylococcus aureus (MRSA) and priority pathogens such as Francisella tularensis and Burkholderia pseudomallei. In addition, we identify TLM analogues with in vivo efficacy against MRSA and Klebsiella pneumoniae in animal models of infection.

Full text of DOI:10.1021/acs.jmedchem.6b00236

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Article
Thiolactomycin-based inhibitors of bacterial #-
ketoacyl-ACP synthases with in vivo activity
Gopal R Bommineni, Kanishk Kapilashrami, Jason Ellsworth Cummings, Yang Lu, Susan
E Knudson, Chendi Gu, Stephen G Walker, Richard A. Slayden, and Peter J Tonge
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00236 • Publication Date (Web): 17 May 2016
Downloaded from http://pubs.acs.org on May 19, 2016
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Thiolactomycinꢀbased inhibitors of bacterial βꢀ
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ketoacylꢀACP synthases with in vivo activity
†
‡
†‡¶
§
†
Gopal R. Bommineni , Kanishk Kapilashrami , Jason E. Cummings , Yang Lu , Susan E.
§
†
¥
§
†
Knudson , Chendi Gu , Stephen G. Walker , Richard A. Slayden *, Peter J. Tonge *
†
Institute of Chemical Biology & Drug Discovery, Department of Chemistry, ¥School of Dental
Medicine, Department of Oral Biology and Pathology, Stony Brook University, Stony Brook,
NY 11794ꢀ3400
§
Department of Microbiology, Immunology and Pathology, Colorado State University, Fort
Collins, CO 80523ꢀ2025
¶
Current Address: Molecular Pharmacology and Chemistry Program, Zuckerman Research
Center, Memorial SloanꢀKettering Cancer Center, 417 East 68th Street, New York, NY 10065
KEYWORDS: Thiolactomycin, Fatty Acid Biosynthesis, βꢀKetoacyl ACP synthase, Structure
Activity Relationships, in vitro activity, in vivo efficacy
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ABSTRACT
βꢀKetoacylꢀACP synthases (KAS) are key enzymes involved in the type II bacterial fatty acid
biosynthesis (FASII) pathway and are putative targets for antibacterial discovery. Several natural
product KAS inhibitors have previously been reported, including thiolactomycin (TLM), which
is produced by Nocardia spp. Here we describe the synthesis and characterization of optically
pure 5Rꢀthiolactomycin (TLM) analogs that show improved whole cell activity against bacterial
strains including methicillin resistant Staphylococcus aureus (MRSA) and priority pathogens
such as Francisella tularensis and Burkholderia pseudomallei. In addition, we identify TLM
analogs with in vivo efficacy against MRSA and Klebsiella pneumoniae in animal models of
infection.
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INTRODUCTION
While antibiotic resistance has always been a driving force for drug discovery, the increase in
the number and the diversity of resistant pathogens continues to pose a severe threat to public
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health. Multidrug resistant strains of organisms such as Klebsiella pneumoniae and
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ꢀ3
Acinetobacter baumannii are virtually untreatable by currently available antibiotics. Moreover,
resistance has been reported towards drugs such as vancomycin, that is considered the ‘drug of
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last resort’ for a variety of bacterial infections including Staphylococcus aureus. Thus there is a
critical need for antibacterial agents with novel modes of action that circumvent existing
resistance mechanisms.
The bacterial type II fatty acid biosynthesis pathway (FASII, Figure 1) is highly conserved,
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essential for survival and is distinct from the mammalian pathway. Thus, enzymes in this
pathway are thought to be profitable targets for the development of novel therapeutics. In support
of this proposal, the frontꢀline tuberculosis drug isoniazid is an inhibitor of the enoylꢀACP
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reductase (FabI) in the Mycobacterium tuberculosis FASII pathway,
while several FabI
inhibitors are currently in clinical or preclinical development to treat infections caused by
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methicillinꢀresistant S. aureus (MRSA).
In addition to FabI, the FASII βꢀketoacylꢀACP synthase (KAS) enzymes are also thought to be
appropriate targets for intervention. These enzymes catalyze a decarboxylative Claisen
condensation in the initiation and elongation phases of the FASII pathway, and typically bacteria
have three KAS homologs: FabB, FabF and FabH (Table 1). FabB and FabF (KASI/II) contain a
CysꢀHisꢀHis catalytic triad and catalyze the condensation of malonylꢀACP and acylꢀACP in the
elongation cycle, while FabH (KASIII) contains a CysꢀHisꢀAsn triad, and is responsible for
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initiation of the FASII cycle through the condensation of malonylꢀACP with acetylꢀCoA.
More recently, Yuan et al. identified a new class of KASI/II enzymes (FabY) which replaces
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Although target redundancy adds an additional level of complexity for drug development, the
identification of natural product KAS inhibitors lends support to the notion that this enzyme class
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is a valid target for drug discovery.
Cerulenin, from the fungus Caephalosporium
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caerulens, is a suicide inhibitor that targets FabB and FabF in E. coli, whilst platensimycin,
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from Streptomyces platensis, is effective against Gramꢀpositive bacteria such as S. aureus,
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Enterococcus faecalis and Streptococcus pneumonia. In addition, the platensimycin analog
platencin, inhibits both FabF and FabH comparably, and also is effective against various
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antibiotic resistant bacteria including vancomycinꢀresistant Enterococcus faecium. Finally
phomallenic acids, isolated from a plant fungus, are KAS inhibitors and have potent antibacterial
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activity against MRSA, Bacillus subtilis and Haemophilus influenza.
Thiolactomycin (TLM), a natural product thiolactone, is a competitive reversible inhibitor that
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binds to the malonylꢀACP binding site of the KAS enzymes. First isolated from Nocardia
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sp.,
TLM shows broad spectrum antibacterial activity against Gramꢀpositive and Gramꢀ
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negative bacteria, as well as mycobacteria.
Despite moderate MIC values, the favorable
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physicochemical properties, low cytotoxicity, high bioavailability and in vivo activity of TLM,
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have stimulated a number of inhibitor design initiatives aimed at improving the
antibacterial activity of this natural product. These synthetic efforts have mainly explored
substitutions at the thiolactone C5, O4 and C3 positions, but in many cases have failed to
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improve the activity of TLM.
In addition, there are few reports of enantiomerically pure
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TLM analogs,
and most studies have been performed with racemic mixtures of the respective
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TLM analogs despite the knowledge that (5R)ꢀTLM is the biologically active enantiomer.
Previously, we demonstrated that TLM is a timeꢀdependent inhibitor of the FabB and FabF
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acylꢀenzymes from M. tuberculosis and E. coli. Subsequently, interligand NOE NMR studies
suggested that substituents at the C4 and the C3 positions of TLM (Figure 1) would strengthen
interactions with the target enzyme, and subsequently it was demonstrated that modifications to
these positions on the thiolactone core resulted in improved binding to the M. tuberculosis FabB
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(mtFabB; KasA) in vitro.
Here, we describe the synthesis, in vitro inhibition and in vivo
efficacy of enantiopure (5R)ꢀTLM analogs. These compounds were evaluated for activity
against mtFabB, the S. aureus FabF enzyme (saFabF) and clinically relevant bacteria such as
Mycobacterium tuberculosis, methicillin sensitive S. aureus (MSSA), MRSA, Francisella
tularensis, Klebsiella pneumoniae, Burkholderia pseudomallei and Yersinia pestis. Collectively
the studies provide additional data to direct the optimization of TLM.
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RESULTS AND DISCUSSION
Chemistry:
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With the synthesis of the C4 analogs as a starting point, we followed literature precedent for
the asymmetric synthesis of (5R)ꢀTLM where the key dienyl oxathiolanone building block (32)
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was synthesized from Dꢀalanine (Scheme 1). Preparation of 32 proceeded via the formation of
a diastereomeric mixture of an oxathiolanone intermediate from which the required 2R,4S
diastereomer was isolated by crystallization from nꢀpentane at ꢀ78 °C and verified by 2D NMR.
The oxathiolanone ring of 32 was subsequently opened using treatment with cesium carbonate in
ethanol at 10 °C to release the unstable thiol (1). This was immediately acylated with selected
acid chlorides to give the respective acylated derivatives 1aꢀ4a, 12aꢀ16a and 19a (80ꢀ90% twoꢀ
step yield). Subsequent enolate formation of 1aꢀ4a, 12aꢀ16a and 19a with LiHMDS at ꢀ78 °C
initiated the thioꢀDieckmann condensation providing (5R)ꢀTLMꢀTLM 4, (5R)ꢀTLM 12ꢀ16 and
(5R)ꢀTLM 19 in 58ꢀ92% yields, respectively (Scheme 2). Subsequent elaboration at the C4
position was performed using the TLM 2 scaffold, since alkylation of TLM was unsuccessful
due to the formation of a mixture of C4, C2 Oꢀalkylation, and C3 alkylation products.
Preferential Oꢀalkylation of TLM 2 was achieved using NaH in DMF with tertꢀbutyl bromoester
and followed by hydrolysis of the ester with 25% TFA in CH Cl to obtain compound 22a.
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Compound 22a was coupled with 2ꢀazidoethanamine and 2ꢀazidoethylcarbamate under coupling
conditions to generate TLM 22 and TLM 23 in 70% and 66% yields, respectively. Finally,
TLM 24 and TLM 25 were obtained by treatment of TLM 22 with 27 in the presence of
CuSO .5H O and sodium ascorbate to form the corresponding ‘click’ adducts in 80% and 88%
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yields, respectively (Scheme 3).
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Similarly, 1 was acylated with 30 to give compounds 18a and 20a followed by Dieckmann
cyclization to provide TLM 18 and TLM 20 which were further treated with 27 under ‘click’
conditions to provide TLM 21 and TLM 17, respectively (Scheme 4).
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Utilizing a previously published protocol for C3 acylation of tetronic acids, TLM 2 was
treated with a variety of anhydrides or acid chlorides (5aꢀ11a and 26a) in the presence of Et N
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and catalytic amount of DMAP in CH Cl to form kinetically controlled Oꢀacylated products.
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Subsequently, DMAP mediated transfer of the Oꢀacyl group to C3 of TLM over time led to the
thermodynamically stable C3ꢀacylated products, providing TLM 5ꢀ11 and 26 in 70%ꢀ85% yields
(
Scheme 5).
In vitro activity:
Previously we quantified the inhibition of mtFabB and C171Q mtFabB by TLM analogs 1ꢀ
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Here we expand these studies by analyzing the inhibition of mtFabB as well as FabF and
C164Q FabF from S. aureus, by additional C3 and C4 analogs (TLM 19ꢀ25). We also
determined the whole cell antibacterial activity of TLM (1) and all TLM analogs (2ꢀ25) against
M. tuberculosis, Y. pestis, F. tularensis, B. pseudomallei, MSSA and MRSA.
C4 substituents of demethyl TLM: Compounds TLM 22ꢀ25 were synthesized by attaching
substituents to the C4 position of demethyl TLM (TLM 2), rather than TLM, as TLM 2 showed
similar affinity for mtFabB compared to TLM. However, none of the analogs were found to bind
to mtFabB (Table 2), which was confirmed using WaterLOGSY based competition data (data
not shown). These data were mirrored in the poor MIC values observed for the C4 analogs
against M. tuberculosis. Moreover, the C4 analogs showed poor whole cell activity for all
bacterial strains tested (Table 2 and Table S2). Careful analysis of the crystallographic data of
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TLM and derivatives bound to mtFabB and mtC171QFabB revealed that the C4 hydroxyl is
involved in an extended waterꢀmediated hydrogen bonding network that includes Thr315, which
is conserved in FabB and FabF from different bacterial species (Figure S1). These data support
the need for a hydrogen bonding group at C4 and suggest that the hydrogen bonding network is
essential for the binding of TLM analogs to FabB.
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C3 substituents with bulky side chains: Based on the structural and interligand NOE NMR
data, analogs TLM 12ꢀ17, 19 and 21 were synthesized. Despite improvements in binding
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affinity, particularly for TLM 12 and 17, no improvements were observed in the whole cell
activity of these analogs against M. tuberculosis (Table 3). TLM 16 showed poor binding to
saFabF, the only βꢀketoacylꢀACP synthase in the S. aureus FASII elongation cycle (Table 4).
However, this analog showed a 5ꢀfold improvement in MIC against MSSA (16 µg/mL) and a 3ꢀ
fold improvement against MRSA (25 µg/mL) compared to TLM (75 µg/mL). Bulky aryl
substituents at the C3 position of the thiolactone ring were tolerated by F. tularensis as the whole
cell activity of the lead molecule was retained for the analogs TLM 12ꢀ16 with MIC values in
the range of 2ꢀ3 µg/mL. None of the analogs showed improvements in MIC values against Y.
pestis, M. tuberculosis and B. pseudomallei compared to TLM (Tables 3, 4 and S2).
C3 alkyl substituents: The loss of timeꢀdependent inhibition of acyl mtFabB upon removal of
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the methyl group at the C3 position (TLM 2), observed by Kapilashrami et al., triggered our
interest in developing SAR for C3ꢀsubstituted TLMs. Therefore, we studied analogs with alkyl
substituents of variable chain lengths at the C3 position (TLM 3, 4, 18, 20). While these analogs
showed subtle changes in binding affinity to mtFabB, the length of the alkyl substituent
modulated the drug:target residence time (t = 1/k ) with optimal t observed for TLM 3
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binding to the C171Q mtFabB acylꢀenzyme mimic.
While the MIC values of these
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compounds against M. tuberculosis were either similar to the MIC for TLM (3 µg/mL) or larger,
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the ethyl analog TLM 3 showed activity against a TLM resistant strain of H37Rv, with an MIC
value of 2.5 µg/mL. In addition, these analogs did not show improvement in whole cell activity
against Y. pestis, B. pseudomallei and S. aureus (Table 3 and Table S2).
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C3 acyl substituents: Analogs of TLM were also designed in which the substituent was linked
to the C3 position of the ring via a ketone (TLM 5ꢀ11, 26) in an attempt to more closely mimic
the diketo motif of malonylꢀACP. While all the TLM analogs with acyl substituents at C3
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showed tighter binding in vitro to mtFabB and the acylenzyme mtFabB mimic, no
improvement was observed in the whole cell activity against M. tuberculosis (Table 3). The lack
of correlation between K and MIC values could reflect issues either with drug uptake or
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efflux.
TLM 5, 6 and 11 showed tighter binding to saFabF compared to the lead molecule
(Table 4): TLM 5 had a ~ 8ꢀfold improvement in binding to both apo saFabF and C164Q
saFabF, compared to TLM (90ꢀ100 µM), with a binding affinity of 14.2 and 11.9 µM,
respectively (Table 4). However, these changes in affinity did not translate into similar
improvements in MIC with TLM 5 only showing a 2ꢀfold improvement in MIC (32 µg/mL)
compared to TLM (75 µg/mL) for MRSA. In contrast, TLM 9 and TLM 11, despite poor
binding affinities, had a 150ꢀfold improvement in whole cell activity against MSSA (MIC = 0.5
µg/mL) and a 75ꢀ and 37ꢀfold improvement against MRSA (1 µg/mL and 2 µg/mL, respectively)
compared to TLM (75 µg/mL). TLM 26, with an azido hexanoyl substituent at C3, showed a 5ꢀ
fold improvement in whole cell activity, with an MIC value of 16 µg/mL against MSSA and
MRSA (Table 3). Finally, TLM 3 showed similar whole cell activity against B. pseudomallei
(32 µg/mL) to the lead compound (8 µg/mL) (Table S2). No improvements in whole cell activity
were observed against Y. pestis.
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In vivo activity in MRSA and K. pneumoniae infection models
In their seminal work, Miyakawa et al. studied the pharmacokinetics and in vivo activity of
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TLM in animal models of K. pneumoniae and S. marcescens infection. These investigators
observed that TLM was rapidly absorbed and gave protection against systemic infection caused
by both pathogens despite poor whole cell activity (100ꢀ200 µg/mL). We consequently decided
to test the in vivo efficacy of selected analogs against MRSA, one of the pathogens studied in this
work, as well as K. pneumoniae. In agreement with previous reports, TLM together with most
analogs tested showed no toxicity below 100 µg/mL against THP1 monocytic leukemia cells
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(Table S3). For systemic infection with MRSA we chose analogs TLM 5, TLM 6 and TLM 11
which have improved K values for saFabF compared to TLM, and MIC values of 32, 62 and 2
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µg/mL against MRSA compared to an MIC value of 75 µg/mL for TLM. Although animals
treated with TLM, TLM 5 and TLM 11 showed similar survival times compared to the vehicle
control, TLM 6 gave an average survival time of 3 days compared to a survival time of 5 days
for treatment with vancomycin (Figure 2A). Subsequently, the ED of TLM 6 was determined
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systemic K. pneumoniae (KPC) infection (Figure 2B). While gentamicin, a clinically used
aminoglycoside antibiotic (MIC < 1 µg/mL), showed maximum efficacy with a mean survival
time of 6.2 days, TLM 5 and 6 showed improved survival times of 4.6 and 5.1 days respectively,
compared to 3.25 days for TLM. We tested the in vitro antibacterial activity of these analogs and
all had MIC values of >250 µg/mL in the cell based assay. Thus, similar to the studies by
Miyakawa et al., there was a disconnect between the in vitro and in vivo efficacy of the TLM
analogs.
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SUMMARY
The natural product TLM is an inhibitor of the βꢀketoacylꢀACP synthases in the bacterial FAS
II pathway. To provide a platform for the optimization of TLM, we have synthesized two series
of enantiomerically pure (5R)ꢀTLM analogs, and have evaluated their in vitro and in vivo
antibacterial activity. These efforts have focused on the relatively underexploited C3 and C4
positions of the thiolactone core. In general, the C4 analogs of TLM, showed poor biochemical
and microbiological activity which we believe can be attributed to disruption of a hydrogen
10
11
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5
2
bonding network that involves the TLM C4 hydroxyl group. Thus, elaboration of the
thiolactone core will likely require the introduction of substituents that maintain interactions with
this Hꢀbonding network. In contrast, several of the C3 analogs had improved activity compared
to the parent compound. TLM 11, with a bulky biphenylꢀhexanoyl substituent at C3, had a 13ꢀ
fold improvement in MIC (0.21 µg/mL) against F. tularensis compared to TLM (2.8 µg/mL).
Moreover, TLM 11 demonstrated 150ꢀ and 37ꢀfold improvement in MIC values against MSSA
(0.5 µg/mL) and MRSA (2 µg/mL) compared to TLM (75 µg/mL), respectively, while TLM 26,
with a hexanoyl azide substituent, showed a 5ꢀfold improvement in whole cell activity with an
MIC value of 16 µg/mL against both strains of S. aureus. The trifluoroacetyl analog TLM 6 had
improved in vitro activity towards saFabF and MRSA compared to TLM, and showed improved
survival in a systemic MRSA infection model. Finally, despite poor whole cell activity TLM 3, 5
and 6 showed improved mean survival times in a systemic K. pneumoniae infection model.
While a more in depth analysis is required to elucidate the disparity between the in vitro and in
vivo activities of our analogs against K. pneumoniae, one possibility is that the residence time of
the inhibitors on the K. pneumoniae KAS enzyme(s) may play a role in the observed in vivo
efficacy. Alternatively, the target(s) for TLM and the TLM analogs may be more vulnerable in
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vivo and/or one or more metabolites might be responsible for the observed activity. Experiments
are underway to investigate the thermodynamics and kinetics of binding of these analogs to the
βꢀketoacylꢀACP synthases from this pathogen. Our data suggest that further modifications at the
thiolactone C3 position may be a profitable source of additional TLMꢀbased inhibitors.
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EXPERIMENTAL SECTION
General Procedure I. Cesium carbonate (1.6 mmol) was added to a solution of 32 (1.6 mmol)
in EtOH (6.0 mL) at 10 °C. After all the starting material had been consumed (~ 20 min by
TLC), the mixture was poured into NH Cl (sat.)/1 N HCl (15 mL, 3:1) and extracted with Et O
4
2
(
3 x 20 mL) and then water (3 x 20 mL). The combined organics were dried (MgSO ), filtered
4
and evaporated to provide 1. Subsequently, the residue was redissolved in CH Cl (15 mL) and
2
2
cooled to 0 °C followed by addition of Et N (2.0 mmol) and the respective acid chloride (1.6
3
mmol). After 1 h, NH Cl (sat. 20 mL) was added and the mixture was extracted with CH Cl (3 x
4
2
2
15 mL). The combined organics were dried (MgSO ), filtered and evaporated. Flash
4
chromatography (5% EtOAc/hex) gave the pure compounds with yields of 80ꢀ90%.
General Procedure II. LiHMDS was added to a stirred solution of compounds (1aꢀ4a and
12aꢀ19a) (1.09 mmol) in THF (16.4 mL) at ꢀ78 °C (1.8 mmol, 1.0 M in THF) and the solution
was allowed to slowly warm to ꢀ5 °C. The solution was then poured into 1 N HCl (25 mL) and
extracted with EtOAc (3 x 15 mL). The combined organics were dried (MgSO ), filtered and
4
evaporated. This crude mixture was dissolved in NaHCO (sat, 15 mL) and stirred for 15 min
3
and extracted with Et O (3 x 10 mL). The aqueous layer was then acidified to pH 3 (pH paper)
2
with 1 N HCl and extracted with Et O (3 x 10 mL) and EtOAc (2 x 10 mL). The combined
2
organics were dried (MgSO ), filtered and evaporated to provide TLM ꢀTLM 4 and TLM 12 ꢀ
4
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TLM 19, which were further purified by flash chromatography to get obtain the pure compounds
in 58ꢀ92% yield.
General Procedure III. CuSO .5H O (1.0 mmol) and sodium ascorbate (1.0 mmol) were
4
2
10
11
12
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60
added to a solution of TLM 18, TLM 20, or TLM 22 (1.0 mmol) and alkyne (27) (1.0 mmol) in
nꢀbutanol/water (1:1, 3 mL) at room temperature. The reaction mixture was stirred at room
temperature for 1ꢀ2 h then filtered and concentrated, and the crude compound purified with an
ISCO combiflash to afford the corresponding click product.
General Procedure IV. Respective anhydride or acid chloride (1.5 mmol), Et N (2 mmol) and
3
DMAP (0.4 mmol) were added to a stirred solution of TLM 2 (1.0 mmol) in CH Cl (3 mL) at 0
2
2
°
C. The reaction mixture was stirred at room temperature for 2ꢀ8 h then extracted with CH Cl (3
2 2
x 10 mL). The organic phase was then washed with 1 N HCl (20 mL) and dried (MgSO4),
filtered and evaporated to provide residue, which was purified by flash chromatography or using
an ISCO combiflash.
Analytical Data:
(R)ꢀ4ꢀhydroxyꢀ3,5ꢀdimethylꢀ5ꢀ((E)ꢀ2ꢀmethylbutaꢀ1,3ꢀdienyl)thiophenꢀ2(5H)ꢀone
(TLM):
23
44
24
General Procedure II. TLM 80%. [α] D = + 173.0 (c 1.2, MeOH). Mp 119ꢀ121 ºC. [(lit. [α] D
1
=
+ 174 (c 0.6, MeOH), Mp 119.5ꢀ121 ºC)]; H NMR (500 MHz, CDCl and 5% CD OD): δ
3
3
1.64, 1.65 (2s, 6 H), 1.73 (s, 3 H), 1.91 (s, 3 H), 4.96. (d, J = 11.0 Hz, 1 H), 5.15 (d, J = 17.0 Hz,
13
1
H), 5.53 (s, 1 H), 6.26 (dd, J = 17.0, 11.0 Hz, 1 H). C NMR (125 MHz, CDCl and 5%
3
ꢀ
CD OD) δ 7.2, 11.6, 29.3, 55.1, 109.3, 113.2, 130.0, 139.1, 140.7, 180.6, 196.5. MS (EI ): m/z
3
calculated for C H O S 210.1; found 209.1 (Mꢀ1).
1
1
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6
(
R,E)ꢀEthyl 2,4ꢀdimethylꢀ2ꢀ(Acetylthio)hexaꢀ3,5ꢀdienoate (2a): General Procedure I. Flash
23
1
chromatography (5% EtOAc/hexanes) afforded 2a 84%. [α] D = + 17 (c 1.0, CH Cl ). H NMR
2
2
(600 MHz, CDCl ): δ 1.25, (t, J = 7.2 Hz, 3H), 1.84, 1.87 (2s, 6H), 1.21 (s, 3H), 4.20. (q, J = 7.2
3
0
1
2
3
4
5
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8
9
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9
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3
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7
8
9
0
Hz, 2H), 5.03 (d, J = 9.0 Hz, 1H), 5.20 (d, J = 14.5 Hz, 1H), 5.73 (s, 1H), 6.30 (dd, J = 14.5, 9.0
13
Hz, 1 H). C NMR (125 MHz, CDCl ) δ 12.7, 13.8, 25.8, 29.9, 55.2, 61.8, 113.1, 131.1, 138.1,
3
ꢀ
141.0, 171.8, 194.3. MS (EI ): m/z calculated for C H O S 242.1; found 243.1 (M + 1), 198.1
1
2
18
3
(M ꢀ 44).
(R)ꢀ4ꢀhydroxyꢀ5ꢀmethylꢀ5ꢀ((E)ꢀ2ꢀmethylbutaꢀ1,3ꢀdienyl)thiophenꢀ2(5H)ꢀone (TLM 2):
23
47
29
D
General Procedure II. TLM 2 58%. [α] D = + 44 (c 1.0 MeOH). [(lit. [α] = + 186 (c 1.01,
MeOH), 1H NMR (500 MHz, CD OD): δ 1.79 (s, 3H), 1.83 (s, 3H), 5.06 (d, J =10.5 Hz, 1H),
3
1
3
5
.27 (d, J = 17.7 Hz, 1H), 5.66 (s, 1H), 6.36 (dd, J = 17.5, 10.5 Hz). C NMR (100 MHz,
+
CD OD): δ 12.4, 30.3, 58.8, 113.7, 131.4, 140.5, 142.1, 190.1, 197.2. MS (EI ): m/z calculated
3
for C H O S 196.1; found 197.1 (M + 1).
1
0
12
2
(R,E)ꢀEthyl 2,4ꢀdimethylꢀ2ꢀ(butanoylthio)hexaꢀ3,5ꢀdienoate (3a): General Procedure I.
2
3
Flash chromatography (5% EtOAc/hexanes) afforded the product 3a 82%. [α] D = + 32 (c 1,
1
CH Cl ). H NMR (400 MHz, CDCl ): δ 0.88, (t, J = 7.2 Hz, 3 H), 1.21 (t, J = 7.2 Hz, 3 H),
2
2
3
1
.58ꢀ1.64 (m, 2 H), 1.82, 1.84 (2s, 6 H), 2.40 (t, J = 7.2 Hz, 3 H), 4.13ꢀ4.18 (m, 2 H), 5.00 (d, J =
13
1
0.8 Hz, 1 H), 5.20 (d, J = 17.2 Hz, 1 H), 5.72 (s, 1 H), 6.30 (dd, J = 17.2, 10.8 Hz, 1 H).
C
NMR (100 MHz, CDCl ) δ 12.7, 13.1, 13.8, 18.9, 25.9, 45.1, 55.0, 61.8, 113.0, 131.2, 138.1,
3
+
1
41.0, 172.0, 198.0. MS (EI ) m/z calculated for C H O S 270.1; found 271.1 (M + H).
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(
R)ꢀ3ꢀEthyl
4ꢀhydroxyꢀ5ꢀmethylꢀ5ꢀ((E)ꢀ2ꢀmethylbutaꢀ1,3ꢀdienyl)thiophenꢀ2(5H)ꢀone
(
TLM 3): General Procedure II. Flash chromatography (1:1, EtOAc/hexanes) afforded TLM
2
3
1
3
75%. [α] = + 98 (c 1, MeOH). H NMR (500 MHz, CDCl and 5% CD OD): δ 1.97 (t, J =
3
3
D
10
11
12
13
14
15
16
17
18
19
20
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22
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7
.2 Hz, 3 H), 1.68, 1.77 (2s, 6 H), 2.24 (q, J = 7.2 Hz, 2 H), 4.98 (d, J = 10.5 Hz, 1 H), 5.18 (d, J
1
3
=
17.0 Hz, 1 H), 5.53 (s, 1 H), 6.26 (dd, J = 17.0, 10.5 Hz, 1 H).
C NMR (125 MHz, CDCl3
and 5% CD OD) δ 11.7, 12.2, 16.0, 29.6, 55.0, 113.5, 115.3, 130.0, 139.4, 140.7, 180.6, 196.3.
3
ꢀ
+
MS (EI ) m/z: 223.1 (M ꢀ 1). HRMS (M + H) m/z calculated for C H O SH 225.0949; found
1
2
16
2
225.0949.
(R,E)ꢀEthyl 2,4ꢀdimethylꢀ2ꢀ(pentanoylthio)hexaꢀ3,5ꢀdienoate (4a): General Procedure I.
1
Flash chromatography (5% EtOAc/hexanes) afforded 4a 84%. H NMR (400 MHz, CDCl ): δ
3
0
1
=
.85, (t, J = 7.2 Hz, 3 H), 1.22 (t, J = 7.2 Hz, 3 H), 1.26ꢀ1.30 (m, 2 H) 1.55ꢀ1.59 (m, 2 H), 1.82,
.84 (2s, 6 H), 2.43 (t, J = 7.2 Hz, 2 H), 4.14ꢀ4.19 (m, 2 H), 5.00 (d, J = 10.8 Hz, 1 H), 5.20 (d, J
1
3
17.2 Hz, 1 H), 5.72 (s, 1 H), 6.30 (dd, J = 17.2, 10.8 Hz, 1 H). C NMR (100 MHz, CDCl ) δ
3
12.8, 13.5, 13.8, 21.8, 25.9, 43.0, 55.0, 61.8, 113.0, 131.3, 131.5, 138.1, 141.2, 172.0, 198.2. MS
+
(EI ) m/z calculated for C H O S 284.1; found 285.1 (M + 1).
15
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(R)ꢀ4ꢀHydroxyꢀ5ꢀmethylꢀ5ꢀ((E)ꢀ2ꢀmethylbutaꢀ1,3ꢀdienyl)ꢀ3ꢀpropylthiophenꢀ2(5H)ꢀone
(TLM 4): General Procedure II. Flash chromatography (1:1, EtOAc/hexanes) afforded TLM 4
23
1
78%. [α] D = + 61 (c 1, MeOH). H NMR (500 MHz, CDCl and 5% CD OD): δ 1.89 (t, J = 7.2
3
3
Hz, 3 H), 2.26ꢀ2.20 (m, 2 H), 1.73, 1.85 (2s, 6 H), 2.23 (q, J = 7.2 Hz, 2 H), 5.02. (d, J = 10.5
13
Hz, 1 H), 5.22 (d, J = 17.0 Hz, 1 H), 5.58 (s, 1 H), 6.27 (dd, J = 17.0, 10.5 Hz, 1 H). C NMR
(125 MHz, CDCl and 5% CD OD) δ 12.0, 13.8, 21.1, 24.6, 29.8, 55.4, 113.6, 114.5, 129.3,
3
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5
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5
5
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ꢀ
+
1
39.9, 140.7, 181.2, 197.5. MS (EI ) m/z: 237.1 (M ꢀ 1). HRMS (M + H) m/z calculated for
C H O SH 239.1106; found 239.1102.
13 18
2
(
R)ꢀ3ꢀAcetylꢀ4ꢀhydroxyꢀ5ꢀmethylꢀ5ꢀ((E)ꢀ2ꢀmethylbutaꢀ1,3ꢀdienyl)thiophenꢀ2(5H)ꢀone
0
1
2
3
4
5
6
7
8
9
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2
3
4
5
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7
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9
0
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0
(
TLM 5): General Procedure IV. Flash chromatography (30% EtOAc/hexanes/1% AcOH)
23
1
afforded TLM 5 75%. [α] D = + 404 (c 1, MeOH). H NMR (500 MHz, CDCl and 5%
3
CD OD): δ 1.68, 1.79 (2 s, 6 H), 2.54 (s, 3 H), 5.04 (d, J = 10.0 Hz, 1 H), 5.20 (d, J = 17.0 Hz, 1
3
1
3
H), 5.64 (s, 1 H), 6.27 (dd, J = 17.0, 10.0 Hz, 1 H).
C NMR (100 MHz, CDCl and 5%
3
CD OD, peak broadening was observed due to ketoꢀenol tautomerism) δ 18.8, 19.8, 29.3, 57.7,
3
ꢀ
1
07.9, 113.9, 117.3, 130.0, 132.5, 139.5, 139.9, 193.9, 204.4. MS (EI ) m/z: 237.2 (M ꢀ 1).
+
HRMS (M + H) m/z calculated for C H O SH 239.0742; found 239.0748.
1
2
14
3
(
R)ꢀ3ꢀ(2,2,2ꢀTrifluoroacetyl)ꢀ4ꢀhydroxyꢀ5ꢀmethylꢀ5ꢀ((E)ꢀ2ꢀmethylbutaꢀ1,3ꢀ
dienyl)thiophenꢀ2(5H)ꢀone (TLM 6): General Procedure IV. Flash chromatography (40%
23
1
hexanes/10% THF/2% AcOH/EtOAc) afforded TLM 6 82%. [α] D = + 310 (c 1, MeOH). H
NMR (500 MHz, CD OD): δ 1.72, 1.75 (2 s, 6 H), 5.00 (d, J = 10.4 Hz, 1 H), 5.20 (d, J = 17.6
3
1
3
Hz, 1 H), 5.72 (s, 1 H), 6.37 (dd, J = 17.6, 10.4 Hz, 1 H). C NMR (100 MHz, CD OD, peak
3
broadening was observed due to ketoꢀenol tautomerism) δ 16.9, 18.8, 51.3, 104.0, 114.0, 118.1ꢀ
ꢀ
1
26.7 (q), 138.5, 140.1, 140.54, 193.3, 195.8, 201.7. MS (EI ) m/z: 291.1 (M ꢀ 1). HRMS (M +
+
Na) m/z calculated for C H F O SNa 315.0279; found 315.0229.
1
2
11
3
3
(R)ꢀ3ꢀButyryl
4ꢀhydroxyꢀ5ꢀmethylꢀ5ꢀ((E)ꢀ2ꢀmethylbutaꢀ1,3ꢀdienyl)thiophenꢀ2(5H)ꢀone
(
TLM 7): General Procedure IV. Flash chromatography (20% EtOAc/hexanes/1% AcOH)
23
1
afforded TLM 7 80%. [α] D = + 396 (c 1, MeOH). H NMR (400 MHz, CDCl ): δ 1.00 (t, J =
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.60 Hz, 3 H), 1.69ꢀ1.79 (m, 5 H), 1.86 (s, 3 H), 2.92ꢀ3.01 (m, 2 H), 5.08 (d, J = 10.8 Hz, 1 H),
1
3
5
.25 (d, J = 17.6 Hz, 1 H), 5.69 (s, 1 H), 6.31 (dd, J = 17.6, 10.8 Hz, 1 H). C NMR (100 MHz,
CDCl ) δ 13.1, 13.6, 18.4, 30.0, 37.6, 57.5, 108.0, 114.1, 130.0, 139.8, 140.2, 190.3, 198.3,
3
10
11
12
13
14
15
16
17
18
19
20
21
22
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25
26
27
28
29
30
31
32
33
34
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48
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60
ꢀ
+
2
05.2. MS (EI ) m/z: 265.1 (M ꢀ 1). HRMS (M + H) m/z calculated for C H O SH 267.1055;
14 18 3
found 267.1050.
(R)ꢀMethyl
2,5ꢀdihydroꢀ4ꢀhydroxyꢀ5ꢀmethylꢀ5ꢀ((E)ꢀ2ꢀmethylbutaꢀ1,3ꢀdienyl)ꢀ2ꢀ
oxothiopheneꢀ3ꢀcarboxylate (TLM 8): General Procedure IV. Flash chromatography (3:1,
2
3
1
Acetone/Toluene) afforded TLM 8 71%. [α] D = + 313 (c 1, MeOH). H NMR (400 MHz,
CD OD): δ 1.48, 1.51 (2 s, 6 H), 3.54 (s, 3 H), 4.76 (d, J = 10.8 Hz, 1 H), 4.94 (d, J = 17.2 Hz, 1
3
1
3
H), 5.45 (s, 1 H), 6.06 (dd, J = 17.2, 10.8 Hz, 1 H).
C NMR (100 MHz, CD OD, peak
3
broadening was observed due to ketoꢀenol tautomerism) δ 20.4, 23.8, 52.3, 56.3, 98.5, 113.3,
ꢀ
1
34.2, 139.7, 142.4, 170.7, 182.3, 206.2. MS (EI ) m/z: 253.1 (M ꢀ 1), 195.1 (M ꢀ 59). HRMS (M
+
+
Na) m/z calculated for C H O SNa 277.0510; found 277.0513.
12
14
4
(
R)ꢀ4ꢀHydroxyꢀ5ꢀmethylꢀ5ꢀ((E)ꢀ2ꢀmethylbutaꢀ1,3ꢀdienyl)ꢀ3ꢀpalmitoylthiophenꢀ2(5H)ꢀone
(TLM 9): General Procedure IV. Flash chromatography (1:1, EtOAc/hexanes) to afford TLM
2
D
3
1
9
75%. [α] = + 130 (c 1, CH Cl ). H NMR (400 MHz, CDCl ): δ 0.87 (t, J = 6.80 Hz, 3 H),
2
2
3
1
.80ꢀ1.31 (m, 20 H), 1.40ꢀ1.59 (m, 4 H), 1.69, 1.71 (2 s, 6 H), 2.31 (t, J = 6.80 Hz, 2 H), 2.89 (q,
J = 7.20 Hz, 2 H), 5.03 (d, J = 10.8 Hz, 1 H), 5.18 (d, J = 17.6 Hz, 1 H), 5.55 (s, 1 H), 6.27 (dd, J
13
=
17.6, 10.8 Hz, 1 H). C NMR (100 MHz, CDCl ) δ 12.8, 14.0, 22.6, 246, 25.0, 28.9, 29.2,
3
29.4, 29.6, 29.70, 29.73, 31.0, 31.9, 34.0, 40.0, 56.6, 108.7, 131.6, 139.5, 140.5, 190.3, 201.0,
+
+
2
04.6. MS (EI ) m/z: 435.2 (M + 1). HRMS (M + H) m/z calculated for C H O SH 435.2933;
26 42 3
found 435.2929.
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(
R)ꢀ3ꢀ(2ꢀCarboxybenzoyl)ꢀ4ꢀhydroxyꢀ5ꢀmethylꢀ5ꢀ((E)ꢀ2ꢀmethylbutaꢀ1,3ꢀdienyl)thiophenꢀ
2
(5H)ꢀone (TLM 10): General Procedure IV. Flash chromatography (2:1, Acetone/Toluene) to
2
D
3
1
afford TLM 10 70%. [α] = + 243 (c 1, MeOH). H NMR (500 MHz, CD OD): δ 1.77, 1.78 (2
3
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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8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
s, 6 H), 4.96 (d, J = 10.8 Hz, 1 H), 5.17 (d, J = 17.5 Hz, 1 H), 5.78 (s, 1 H), 6.32 (dd, J = 17.5,
1
3
1
0.8 Hz, 1 H), 7.10 (d, J = 6.0 Hz, 1 H), 7.35ꢀ7.04 (m, 2 H), 7.94 (br s, 1 H). C NMR (125
MHz, CD OD, peak broadening was observed due to ketoꢀenol tautomerism) δ 20.4, 49.0, 58.8,
3
ꢀ
1
10.3, 113.1, 127.4, 129.1, 130.7, 131.3, 134.8, 139.6, 173.8, 195.7, 196.8, 206.0. MS (EI ) m/z:
+
343.0 (M ꢀ 1). HRMS (M + Na) m/z calculated for C H O SNa 367.0616; found 367.0612.
1
8
16
5
(R)ꢀ3ꢀ(4ꢀPhenylꢀphenylhexanoyl)ꢀ4ꢀhydroxyꢀ5ꢀmethylꢀ5ꢀ((E)ꢀ2ꢀmethylbutaꢀ1,3ꢀ
dienyl)thiophenꢀ2(5H)ꢀone (TLM 11): General Procedure IV. Flash chromatography (20%
2
3
1
EtOAc/hexanes/1% AcOH) afforded TLM 11 72%. [α] D = + 211 (c 1, MeOH). H NMR (400
MHz, CDCl ): δ 1.29ꢀ1.39 (m, 3 H), 1.56ꢀ1.62 (m, 3 H), 1.68, 1.72 (2 s, 6 H), 2.56ꢀ2.61 (m, 2
3
H), 2.79ꢀ2.85 (m, 2 H), 4.94 (d, J = 10.0 Hz, 1 H), 5.14 (d, J = 17.6 Hz, 1 H), 5.07 (s, 1 H), 6.28
1
3
(
dd, J = 17.6, 10 Hz, 1 H), 7.15ꢀ7.53 (m, 9 H). C NMR (100 MHz, CDCl , peak broadening
3
was observed due to ketoꢀenol tautomerism) δ 13.2, 14.0, 21.0, 29.2, 29.6, 31.4, 35.4, 60.9,
+
1
26.8, 128.6, 138.4, 140.8, 141.5, 190.33, 198.37, 205.0. MS (EI ) m/z: 447.2 (M + 1), 469.2 (M
+
+
Na). HRMS (M + H) m/z calculated for C H O SH 447.1994; found 447.1989.
2
8
30
3
(R,E)ꢀEthyl
2ꢀ(4ꢀchloroꢀphenylbutanoylthio)ꢀ2,4ꢀdimethylhexaꢀ3,5ꢀdienoate
(12a):
2
D
3
General Procedure I. Flash chromatography (8% EtOAc/hexanes) afforded 12a 85%. [α]
=
1
ꢀ
12 (c 1, CH Cl ). H NMR (400 MHz, CDCl ): δ 1.42, (t, J = 7.2 Hz, 3 H), 1.88, 1.90 (2s, 6 H),
2
2
3
2
.29 (m, 2 H), 2.75 (t, J = 7.2 Hz, 2 H), 2.83ꢀ2.90 (m, 2 H), 4.38. (q, J = 7.2 Hz, 2 H), 5.22 (d, J
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=
10.8 Hz, 1 H), 5.38 (d, J = 17.2 Hz, 1 H), 5.94 (s, 1 H), 6.50 (dd, J = 17.2, 10.8 Hz, 1 H), 7.23ꢀ
1
3
7.28 (m, 4 H). C NMR (125 MHz, CDCl ) δ 13.8, 26.7, 28.8, 34.5, 42.2, 52.8, 55.1, 61.9,
3
+
113.2, 128.3, 128.9, 129.8, 131.7, 139.7, 140.2, 140.5, 172.8, 197.6. MS (EI ) m/z calculated for
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
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46
47
48
49
50
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58
59
60
C H ClO S 380.1; found 381.0 (M + 1).
20
25
3
(R)ꢀ3ꢀ(4ꢀChlorophenethyl)ꢀ4ꢀhydroxyꢀ5ꢀmethylꢀ5ꢀ((E)ꢀ2ꢀmethylbutaꢀ1,3ꢀ
dienyl)thiophenꢀ2(5H)ꢀone (TLM 12). General Procedure II. Purified by flash
2
3
1
chromatography (30% EtOAc/hexanes) afforded TLM 12 86%. [α] D = + 104 (c 1, CH Cl ). H
2
2
NMR (400 MHz, CDCl ): δ 1.69. 1.81 (2 s, 6 H), 2.54ꢀ2.59 (m, 2 H), 2.74ꢀ2.80 (m, 2 H) 5.07 (d,
3
J = 11.0 Hz, 1 H), 5.24 (d, J = 17.0 Hz, 1 H), 5.54 (s 1 H), 6.28 (dd, J = 17.0, 11.0 Hz, 1 H), 7.14
1
3
(d, J = 8.0 Hz, 1 H), 7.21 (d, J = 8.0 Hz, 1 H) 7.23 (s, 1 H). C NMR (125 MHz, CDCl ) δ 12.0,
3
2
1
3
4.4, 29.7, 32.8, 55.51, 113.4, 113.9, 128.3, 128.94, 128.97, 129.8, 131.7, 139.7, 140.2, 140.5,
ꢀ
+
81.1, 196.9. MS (EI ) m/z: 333.0 (M ꢀ1). HRMS (M + H) m/z calculated for C H ClO SH
18 19
2
35.0873; found 335.0867.
(R,E)ꢀEthyl
2ꢀ(4ꢀmethylꢀphenylbutanoylthio)ꢀ2,4ꢀdimethylhexaꢀ3,5ꢀdienoate
(13a):
23
General Procedure I. Flash chromatography (7% EtOAc/hexanes) afforded 13a 80%. [α] D
=
1
ꢀ
212 (c 1, CH Cl ). H NMR (500 MHz, CDCl ): δ 1.28, (t, J = 7.5 Hz, 3 H), 1.88, 1.90 (2s, 6
2
2
3
H), 1.20ꢀ1.93 (m, 2H), 2.33 (s, 3 H), 2.25 (t, J = 7.5 Hz, 2 H), 2.61 (t, J = 7.5 Hz, 2 H), 4.23. (q,
J = 7.2 Hz, 2 H), 5.07 (d, J = 10.5 Hz, 1 H), 5.24 (d, J = 17.5 Hz, 1 H), 5.79 (s, 1 H), 6.35 (dd, J
13
=
17.5, 10.5 Hz, 1 H), 7.06ꢀ7.19 (m, 4 H). C NMR (125 MHz, CDCl ) δ 13.0, 14.0, 20.1, 26.0,
3
27.1, 34.3, 42.6, 55.2, 113.2, 128.3, 129.0, 131.4, 135.4, 138.0, 138.2, 141.0, 172.1, 198.1. MS
+
(
EI ) m/z calculated for C H O S 360.2; found 361.1 (M + 1).
21
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(
R)ꢀ3ꢀ(4ꢀMethylphenethyl)ꢀ4ꢀhydroxyꢀ5ꢀmethylꢀ5ꢀ((E)ꢀ2ꢀmethylbutaꢀ1,3ꢀdienyl)thiophenꢀ
2
(5H)ꢀone (TLM 13): Procedure II. Flash chromatography (30% EtOAc/hexanes) afforded
2
D
3
1
TLM 13 87%. [α] = + 161 (c 1, CH Cl ). H NMR (500 MHz, CDCl ): δ 1.70, 1.74 (2s, 6 H),
2
2
3
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
2
.31 (s, 3 H), 2.47ꢀ2.55 (m, 1 H), 2.60ꢀ2.54 (m, 1 H), 2.68ꢀ2.78 (m, 2 H), 5.05 (d, J = 10.5 Hz, 1
H), 5.24 (d, J = 17.5 Hz, 1 H), 5.50 (s, 1 H), 6.30 (dd, J = 17.5, 10.5 Hz, 1 H), 7.03ꢀ7.12 (m, 4
1
3
H). C NMR (125 MHz, CDCl ) δ 12.1, 21.0, 25.2, 29.7, 33.3, 55.0, 113.8, 113.9, 128.3,
3
+
1
29.14, 129.16, 129.3, 135.8, 138.4, 140.1, 140.6, 179.7, 195.4. MS (EI ) m/z: 315.1 (M + 1).
+
HRMS (M + H) m/z calculated for C H O SH 315.1419; found 315.1411.
1
9
22
2
(R,E)ꢀEthyl
2ꢀ(4ꢀfluorophenylꢀhexanoylthio)ꢀ2,4ꢀdimethylhexaꢀ3,5ꢀdienoate
(14a):
2
D
3
General Procedure I. Flash chromatography (8% EtOAc/hexanes) afforded 14a 89%. [α]
=
1
+
965 (c 1, CH Cl ). H NMR (500 MHz, CDCl ): δ 1.24, (t, J = 7.5 Hz, 3 H), 1.26ꢀ1.34 (m, 2
2
2
3
H), 1.35ꢀ1.67 (2m, 4 H), 1.85, 1.87 (2s, 6 H), 2.42ꢀ2.55 (m, 2 H), 2.56ꢀ2.59 (m, 2 H), 4.20. (q, J
= 7.2 Hz, 2 H), 5.03 (d, J = 9.0 Hz, 1 H), 5.20 (d, J = 14.5 Hz, 1 H), 5.76 (s, 1 H), 6.32 (dd, J =
13
1
1
1
4.5, 9.0 Hz, 1 H), 6.93ꢀ6.95 (m, 2 H), 7.08ꢀ7.10 (m, 2 H). C NMR (125 MHz, CDCl ) δ 12.9,
3
3.9, 25.7, 25.9, 28.6, 30.9, 34.7, 43.1, 55.1, 113.1, 114.8, 114.9, 129.5, 131.3, 137.8, 138.1,
+
41.2, 160.1, 162.0, 172.0, 198.1. MS (EI ) m/z calculated for C H FO S 392.2; found 393.1
2
2
29
3
(
M + 1), 410.2 (M + NH4).
(R)ꢀ3ꢀ(4ꢀ(4ꢀFluorophenyl)butyl)ꢀ4ꢀhydroxyꢀ5ꢀmethylꢀ5ꢀ((E)ꢀ2ꢀmethylbutaꢀ1,3ꢀ
dienyl)thiophenꢀ2(5H)ꢀone (TLM 14): General Procedure II. Flash chromatography (25%
2
3
1
EtOAc/hexanes) afforded TLM 14 82%. [α] D = + 63 (c 1, CH Cl ). H NMR (300 MHz,
2
2
CDCl ) δ 1.50ꢀ1.61 (2m, 4 H), 1.70 (s, 3 H), 1.83 (s, 3 H), 2.26 (t, J = 7.2 Hz, 2 H), 2.56 (t, J =
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.2 Hz, 2 H), 5.06 (d, J = 10.5 Hz, 1 H), 5.24 (d, J = 17.5 Hz, 1 H), 5.54 (s, 1 H) 6.27 (dd, J =
13
17.5, 10.5 Hz, 1 H), 6.89ꢀ6.95 (m, 2 H), 7.05ꢀ7.10 (m, 2 H). C NMR (125 MHz, CDCl ) δ
3
12.0, 22.3, 27.3, 29.7, 31.2, 34.7, 55.6, 114.7, 114.9, 129.5, 129.6, 138.0, 140.0, 140.6, 159.9,
10
11
12
13
14
15
16
17
18
19
20
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48
49
50
51
52
53
54
55
56
57
58
59
60
1
9
+
1
62.3, 181.3, 197.7. F NMR (400 MHz, CDCl ) δ ꢀ122.32 (m). MS (EI ) m/z: 347.1 (M + 1).
3
+
HRMS (M + H) m/z calculated for C H FO SH 347.1481; found 347.1478.
2
0
23
2
(R,E)ꢀEthyl
2ꢀ(4ꢀchlorophenylꢀhexanoylthio)ꢀ2,4ꢀdimethylhexaꢀ3,5ꢀdienoate
(15a):
23
General Procedure I. Flash chromatography (8% EtOAc/hexanes) afforded 15a 85%. [α] D = ꢀ
1
1
0 (c 1, MeOH). H NMR (500 MHz, CDCl ): δ 1.27, (t, J = 7.5 Hz, 3 H), 1.33ꢀ1.37 (m, 2 H),
3
1
.61ꢀ1.68 (2m, 4 H), 1.87, 1.89 (2s, 6 H), 2.49 (t, J = 7.5 Hz, 2 H), 2.56ꢀ2.60 (m, 2 H), 4.20. (q, J
=
7.5 Hz, 2 H), 5.06 (d, J = 10.5 Hz, 1 H), 5.23 (d, J = 17.5 Hz, 1 H), 5.78 (s, 1 H), 6.34 (dd, J =
13
1
7.5, 10.5 Hz, 1 H), 7.09ꢀ7.10 (m, 2 H), 7.24ꢀ7.28 (m, 2 H). C NMR (125 MHz, CDCl ) δ 12.9,
3
13.9, 25.2, 25.9, 28.1, 30.7, 34.8, 43.1, 55.1, 113.1, 128.2, 128.3, 129.5, 129.6, 131.2, 131.3,
+
1
38.1, 141.6, 141.2, 172.0, 198.1. MS (EI ) m/z calculated for C H ClO S 408.2; found 409.1
2
2
29
3
(
M+1), 426.2 (M + NH4).
(
R)ꢀ3ꢀ(4ꢀ(4ꢀChlorophenyl)butyl)ꢀ4ꢀhydroxyꢀ5ꢀmethylꢀ5ꢀ((E)ꢀ2ꢀmethylbutaꢀ1,3ꢀ
dienyl)thiophenꢀ2(5H)ꢀone (TLM 15): General Procedure II. Purified by flash
2
3
1
chromatography (20% EtOAc/hexanes) afforded TLM 15 83%. [α] D = + 48 (c 1, MeOH). H
NMR (500 MHz, CDCl ) δ 1.50ꢀ1.62 (2m, 4 H), 1.73 (s, 3 H), 1.85 (s, 3 H), 2.28 (t, J = 7.2 Hz, 2
3
H), 2.56 (t, J = 7.2 Hz, 2 H), 5.06 (d, J = 10.5 Hz, 1 H), 5.26 (d, J = 17.5 Hz, 1 H), 5.56 (s, 1 H),
13
6.28 (dd, J = 17.5, 10.5 Hz, 1 H), 7.07ꢀ7.09 (m, 2 H), 7.22ꢀ7.25 (m, 2 H). C NMR (125 MHz,
CDCl ) δ 12.0, 22.4, 27.2, 30.2, 31.0, 34.9, 55.9, 114.2, 114.8, 128.3ꢀ129.6 (m), 131.3, 140.0,
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+
+
1
40.4, 140.7, 179.8, 198.2. MS (EI ) m/z: 363.1 (M + 1). HRMS (M + H) m/z calculated for
C H ClO SH 363.1186; found 363.1180.
20 23
2
(R,E)ꢀEthyl
2ꢀ(4ꢀphenylꢀphenylꢀhexanoylthio)ꢀ2,4ꢀdimethylhexaꢀ3,5ꢀdienoate
(16a):
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
General Procedure I. Purified by flash chromatography (7% EtOAc/hexanes) to form (16a)
23
1
8
7%. [α] D = ꢀ 158 (c 1, CH Cl ). H NMR (500 MHz, CDCl ): δ 1.27, (t, J = 7.5 Hz, 3 H),
2
2
3
1
.36ꢀ1.48 (m, 2 H), 1.59ꢀ1.72 (2m, 4 H), 1.89, 1.91 (2s, 6 H), 2.52 (t, J = 7.5 Hz, 2 H), 2.65ꢀ2.69
(
m, 2 H), 4.24. (q, J = 7.5 Hz, 2 H), 5.07 (d, J = 10.5 Hz, 1 H), 5.25 (d, J = 17.5 Hz, 1 H), 5.80
13
(s, 1 H), 6.35 (dd, J = 17.5, 10.5 Hz, 1 H), 7.25ꢀ7.61 (m, 9 H). C NMR (125 MHz, CDCl ) δ
3
1
1
4
2.9, 13.9, 25.3, 25.9, 28.4, 30.9, 35.2, 43.2, 55.2, 113.2, 126.9ꢀ128.7 (m), 131.4, 138.2, 138.6,
+
41.2, 141.4, 172.1, 198.2. MS (EI ) m/z calculated for C H O S 450.2; found 451.2 (M + 1),
28 34
3
68.2 (M + NH4).
(
R)ꢀ3ꢀ(4ꢀ(4ꢀPhenylphenyl)butyl)ꢀ4ꢀhydroxyꢀ5ꢀmethylꢀ5ꢀ((E)ꢀ2ꢀmethylbutaꢀ1,3ꢀ
dienyl)thiophenꢀ2(5H)ꢀone (TLM 16): General Procedure II. Flash chromatography (20%
2
3
1
EtOAc/hexanes) afforded TLM 16 86%. [α] D = ꢀ 91 (c 1, CH Cl ). H NMR (500 MHz,
2
2
CDCl ) δ 1.50ꢀ1.69 (2m, 4 H), 1.75 (s, 3 H), 1.86 (s, 3 H), 2.32 (t, J = 7.2 Hz, 2 H), 2.67 (t, J =
3
7.2 Hz, 2 H), 5.08 (d, J = 10.5 Hz, 1 H), 5.26 (d, J = 17.5 Hz, 1 H), 5.57 (s, 1 H), 6.30 (dd, J =
13
1
7.5, 10.5 Hz, 1 H), 7.23ꢀ7.59 (m, 9 H). C NMR (125 MHz, CDCl ) δ 12.0, 22.5, 27.4, 30.2,
3
31.1, 35.2, 55.0, 114.1, 114.8, 126.9ꢀ128.8 (m), 138.6, 140.5, 140.6, 141.0, 141.6, 179.4, 195.9.
+
+
MS (EI ) m/z: 405.1 (M + 1). HRMS (M + H) m/z calculated for C H O SH 405.1888; found
2
6
28
2
405.1895.
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ꢀ((4ꢀFluorophenylsulfonyl)amino)ꢀNꢀ((1ꢀ(4ꢀ((R)ꢀ2,5ꢀdihydroꢀ4ꢀhydroxyꢀ5ꢀmethylꢀ5ꢀ((E)ꢀ
2
ꢀmethylbutaꢀ1,3ꢀdienyl)ꢀ2ꢀoxothiophenꢀ3ꢀyl)butyl)ꢀ1Hꢀ1,2,3ꢀtriazolꢀ4ꢀ
yl)methyl)benzamide (TLM 17): General Procedure III. Chromatography on silica gel (ISCO
combiflash, 4 g gold cartridge, eluting with CH Cl → 8% MeOH in CH Cl ) afforded TLM 17
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
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46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2
2
2
2
23
1
7
5%. [α] D = + 70 (c 1, MeOH). H NMR (400 MHz, CD OD): δ 1.39ꢀ1.43 (m, 2 H), 1.67, 1.76
3
(
2 s, 6 H), 1.89ꢀ1.92 (2 s, 2 H), 2.20ꢀ2.29 (m, 2 H), 4.32ꢀ4.42 (m, 2 H), 4.56ꢀ4.62 (m, 2 H), 5.03
(
d, J = 10.8, Hz, 1 H), 5.25 (d, J = 17.6 Hz, 1 H), 5.58 (s, 1 H), 6.37 (dd, J = 17.6, 10.8 Hz, 1 H),
1
3
7.18ꢀ7.24 (m, 4 H), 7.69ꢀ7.78 (m, 2 H), 7.84ꢀ7.87 (m, 2 H). C NMR (100 MHz, CDCl ) δ 20.4,
3
2
0.5, 22.7, 25.6, 36.1, 50.3, 55.6, 113.6, 117.1, 117.3, 120.3, 123.5, 129.5, 131.0, 131.1, 132.1,
+
1
31.3, 137.0, 139.7, 141.4, 142.0, 165.0, 166.6, 167.0, 193.6, MS (EI ) m/z: 626.1 (M + 1), 627.2
+
(
M + 2). HRMS (M + H) m/z calculated for C H FN O S H 626.1907; found 626.1910.
3
0
32
5
5 2
(
R,E)ꢀEthyl 2ꢀ(6ꢀazidoꢀhexanoylthio)ꢀ2,4ꢀdimethylhexaꢀ3,5ꢀdienoate (18a): General
23
Procedure I. Flash chromatography (7% EtOAc/hexanes) afforded 18a 85%. [α] D = + 6 (c 1,
1
CH Cl ). H NMR (500 MHz, CDCl ) δ 1.23 (t, J = 7.2 Hz, 3 H), 1.39ꢀ1.34 (m, 2H), 1.58ꢀ1.52
2
2
3
(
m, 2 H), 1.67ꢀ1.60 (m, 2 H) 1.85, 1.83 (2s, 6 H), 2.46 (t, J = 7.5 Hz, 2 H), 3.22 (t, J = 6.5 Hz, 2
H), 4.18ꢀ4.16 (m, 2 H), 5.00 (d, J = 11.0 Hz, 1 H), 5.20 (d, J = 17.5 Hz, 1 H), 5.71 (s, 1 H), 6.30
13
(
dd, J = 17.5, 10.8 Hz, 1 H). C NMR (125 MHz, CDCl ) δ 12.90, 13.90, 24.9, 25.8, 25.9, 28.4,
3
+
43.0, 51.0, 55.2, 61.9, 113.2, 131.2, 131.3, 138.1, 141.1, 172.0, 197.9. MS (EI ) m/z calculated
for C H N O S 339.2; found 340.1 (M + 1).
1
6
25
3
3
(
R)ꢀ3ꢀ(4ꢀAzidobutyl)ꢀ4ꢀhydroxyꢀ5ꢀmethylꢀ5ꢀ((E)ꢀ2ꢀmethylbutaꢀ1,3ꢀdienyl)thiophenꢀ
2(5H)ꢀone (TLM 18): General Procedure II. Flash chromatography (30% EtOAc/hexanes)
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2
2
2
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3
3
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5
5
5
5
5
5
6
2
D
3
1
afforded TLM 18 75%. [α] = + 106 (c 1, CH Cl ). H NMR (400 MHz, CDCl ): δ 1.21 (t, J
2
2
3
=
7.5 Hz, 3 H), 1.73, 1.85 (2s, 6 H), 2.26 (m, 2 H), 2.30ꢀ2.28 (m, 2 H), 3.33 (t, J = 6.5 Hz, 2 H),
5.03. (d, J = 10.0 Hz, 1 H), 5.22 (d, J = 17.0 Hz, 1 H), 5.54 (s, 1 H), 6.27 (dd, J = 17.0, 10.0 Hz,
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
13
1
H). C NMR (100 MHz, CDCl ) δ 12.6, 21.9, 25.2, 28.0, 29.8, 51.4, 55.4, 113.7, 113.8, 129.4,
3
+
+
140.0, 181.1, 196.9. MS (EI ) m/z: 294.1 (M + 1). HRMS (M + H) m/z calculated for
C H N O SH 294.1276; found 294.1281.
14
19
3
2
(R,E)ꢀEthyl
2ꢀ(4ꢀbromophenylꢀhexanoylthio)ꢀ2,4ꢀdimethylhexaꢀ3,5ꢀdienoate
(19a):
23
General Procedure I. Flash chromatography (8% EtOAc/hexanes) afforded 19a 80%. [α] D
=
1
ꢀ
70 (c 1, MeOH). H NMR (500 MHz, CDCl ): δ 1.27, (t, J = 7.5 Hz, 3 H), 1.30ꢀ1.36 (m, 2 H),
3
1.59ꢀ1.68 (2m, 4 H), 1.87, 1.89 (2s, 6 H), 2.49 (t, J = 7.5 Hz, 2 H), 2.52ꢀ2.60 (m, 2 H), 4.20. (q, J
7.5 Hz, 2 H), 5.06 (d, J = 10.5 Hz, 1H), 5.24 (d, J = 17.5 Hz, 1 H), 5.78 (s, 1 H), 6.34 (dd, J =
=
1
3
1
7.5, 10.5 Hz, 1 H), 7.04ꢀ7.06 (m, 2 H), 7.39ꢀ7.41 (m, 2 H). C NMR (125 MHz, CDCl ) δ 12.9,
3
1
3.9, 25.2, 25.9, 28.2, 30.7, 35.0, 43.2, 55.2, 113.2, 119.3, 130.0ꢀ131.4 (m), 138.2, 141.6, 172.0,
+
1
98.2. MS (EI ) m/z calculated for C H BrO S 452.1; found 453.1 and 455.1 (M + 1), 470.1
2
2
29
3
and 472.1 (M + NH4).
(
R)ꢀ3ꢀ(4ꢀ(4ꢀBromophenyl)butyl)ꢀ4ꢀhydroxyꢀ5ꢀmethylꢀ5ꢀ((E)ꢀ2ꢀmethylbutaꢀ1,3ꢀ
dienyl)thiophenꢀ2(5H)ꢀone (TLM 19): General Procedure II. Flash chromatography (20%
23
1
EtOAc/hexanes) afforded TLM 19 78%. [α] D = + 57 (c 1, CH Cl ). H NMR (500 MHz,
2
2
CDCl ) δ 1.50ꢀ1.64 (2m, 4 H), 1.73 (s, 3 H), 1.85 (s, 3 H), 2.28 (t, J = 7.2 Hz, 2 H), 2.56 (t, J =
3
7
.2 Hz, 2 H), 5.09 (d, J = 10.5 Hz, 1 H), 5.26 (d, J = 17.5 Hz, 1 H), 5.56 (s, 1 H), 6.29 (dd, J =
1
3
1
7.5, 10.5 Hz, 1 H), 7.02ꢀ7.04 (m, 2 H), 7.37ꢀ7.39 (m, 2 H). C NMR (125 MHz, CDCl ) δ 12.0,
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1.0, 27.2, 30.2, 31.0, 34.9, 55.0, 114.1, 114.7, 119.3, 128.8ꢀ130.1 (m), 131.3, 140.4, 140.6,
+
+
141.4, 179.5, 195.8. MS (EI ) m/z: 407.0 (M + 1). HRMS (M + H) m/z calculated for
C H BrO SH 407.0680; found 407.0670.
20
23
2
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
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31
32
33
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59
60
(
R,E)ꢀEthyl 2ꢀ(5ꢀazidoꢀpentanoylthio)ꢀ2,4ꢀdimethylhexaꢀ3,5ꢀdienoate (20a): General
23
Procedure I. Flash chromatography (7% EtOAc/hexanes) afforded 20a 90%. [α] D = ꢀ 3.6 (c 1,
1
CH Cl ). H NMR (500 MHz, CDCl ) δ 1.22 (t, J = 7.2 Hz, 3 H), 1.55ꢀ1.58 (m, 2 H), 1.67ꢀ1.70
2
2
3
(
m, 2 H) 1.82, 1.84 (2s, 6 H), 2.46ꢀ2.49 (m, 2 H), 3.23 (t, J = 6.5 Hz, 2 H), 4.14ꢀ4.19 (m, 2 H),
5.00 (d, J = 11.0 Hz, 1 H), 5.20 (d, J = 17.5 Hz, 1 H), 5.71 (s, 1 H), 6.30 (dd, J = 17.5, 10.8 Hz, 1
13
H). C NMR (125 MHz, CDCl ) δ 12.85, 13.8, 25.9 27.8, 42.4, 50.8, 55.2, 61.9, 113.2, 131.13,
3
+
131.16, 138.2, 141.0, 172.0, 197.5. MS (EI ) m/z calculated C H N O S 325.2; found 326.0
1
5
23
3
3
(
M + 1).
(R)ꢀ3ꢀ(3ꢀAzidopropyl)ꢀ4ꢀhydroxyꢀ5ꢀmethylꢀ5ꢀ((E)ꢀ2ꢀmethylbutaꢀ1,3ꢀdienyl)thiophenꢀ
2(5H)ꢀone (TLM 20): General Procedure II. Flash chromatography (30% EtOAc/hexanes)
2
D
3
1
afforded TLM 20 75%. [α] = + 13.6 (c 1, MeOH). H NMR (500 MHz, CD OD): δ 1.21 (t, J
3
=
7.5 Hz, 2 H), 1.71, 1.78 (2s, 6 H), 2.26 (m, 2 H), 3.26 (m, 2 H), 5.02. (d, J = 10.0 Hz, 1 H),
1
3
5.20 (d, J = 17.0 Hz, 1 H), 5.54 (s, 1 H), 6.27 (dd, J = 17.0, 10.0 Hz, 1 H). C NMR (125 MHz,
MeOHꢀd4) δ 19.4, 26.6, 29.254.7, 112.1, 113.0, 114.5, 129.3, 139.2, 140.3, 181.6, 195.9. MS
ꢀ
+
(EI ) m/z: 278.0 (M ꢀ 1). HRMS (M + H) m/z calculated for C H N O SH 280.1120; found
1
3
17
3
2
2
80.1121.
(2R)ꢀNꢀ((1ꢀ(4ꢀ((R)ꢀ2,5ꢀDihydroꢀ4ꢀhydroxyꢀ5ꢀmethylꢀ5ꢀ((E)ꢀ2ꢀmethylbutaꢀ1,3ꢀdienyl)ꢀ2ꢀ
oxothiophenꢀ3ꢀyl)butyl)ꢀ1Hꢀ1,2,3ꢀtriazolꢀ4ꢀyl)methyl)ꢀ2,4ꢀdihydroxyꢀ3,3ꢀ
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5
5
5
6
dimethylbutanamide (TLM 21): General Procedure III. Chromatography on silica gel (ISCO
combiflash, 4 g gold cartridge, eluting with CH Cl → 8% MeOH in CH Cl ) afforded TLM 21
2
2
2
2
2
D
3
1
8
5%. [α] = + 82 (c 1, MeOH). H NMR (400 MHz, CD OD): δ 0.92 (br s, 6 H), 1.43 (br s, 2
3
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
H), 1.70, 1.78 (2 s, 6 H), 1.86ꢀ1.91 (m, 2H), 2.24 (br s, 2 H), 3.38ꢀ3.52 (m, 2 H), 3.95 (br s, 1 H),
.34ꢀ4.56 (m, 4 H), 5.03 (d, J = 10.8, Hz, 1 H), 5.25 (d, J = 17.6 Hz, 1 H), 5.60 (s, 1 H), 6.37 (dd,
4
1
3
J = 17.6, 10.8 Hz, 1 H), 7.89 (s, 1H). C NMR (100 MHz, CDCl ) δ 14.5, 19.8, 21.3, 22.8,
3
25.8, 30.6, 35.6 40.3, 51.1, 71.9, 74.3, 106.7, 113.4, 123.2, 132.3, 139.9, 140.6, 142.1, 171.3,
+
+
188.7, 197.2. MS (EI ) m/z: 461.2 (M ꢀ 17), 479.3 (M + NH ). HRMS (M + H) m/z calculated
4
for C H N O SH 479.2328; found 479.2325.
2
3
34
4
5
2ꢀ((R)ꢀ2,5ꢀDihydroꢀ2ꢀmethylꢀ2ꢀ((E)ꢀ2ꢀmethylbutaꢀ1,3ꢀdienyl)ꢀ5ꢀoxothiophenꢀ3ꢀyloxy)ꢀ Nꢀ
(2ꢀazidoethyl)acetamide (TLM 22): NaH (60% dispersed in oil) (148 mg, 6.16 mmol) was
added to a stirred solution of TLM 2 (0.5 g, (2.55 mmol) in DMF (10 mL) at room temperature
and the mixture was stirred for a further 30 min. tertꢀbutyl bromoacetate (0.75 mL, 5.10 mmol)
was then added and the mixture stirred overnight. After completion, the reaction mixture was
extracted with EtOAc (3 x 10 mL) and the combined organics were dried (MgSO ), filtered and
4
evaporated. The crude material was purified by flash chromatography (5% MeOH/CH Cl ) to
2
2
obtain a Oꢀalkylated TLM 2 derivative, which was redissolved in CH Cl (10 mL). TFA (2.5
2
2
mL) was added and the solvent was removed by evaporation after stirring for 24 h at room
temperature. Purification by flash chromatography (1:1 EtOAc/hexanes/2% AcOH) to afford
ꢀ
2
2a in 90% yield. MS (EI ): 253.1 (M ꢀ 1). Et N (287 µL, 2 mmol) and EDC (230 mg, 1.2 mmol)
3
were added to a solution of 22a (254 mg, 1.0 mmol) and 2ꢀazidoethanamine (103 mg, 1.2 mmol)
in DMF (2 mL) at room temperature and stirred overnight. After completion, the reaction
mixture was extracted with CH Cl (2 x 10 mL) and the combined organic phase was washed
2
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with 1 N HCl (10 mL), dried (MgSO ), filtered and evaporated to obtain the crude material,
4
which was subsequently purified by flash chromatography (1:1, EtOAc/hexanes) to yield TLM
2
D
3
1
2
2 70%. [α] = + 131 (c 1, CH Cl ). H NMR (400 MHz, CDCl ): δ 1.65, 1.79 (2 s, 6 H), 3.34
2
2
3
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
(
br s, 4 H), 4.41 (s, 1 H), 4.98 (d, J = 10.8 Hz, 1 H), 5.15 (d, J = 17.2 Hz, 1 H), 5.55 (s, 1 H),
1
3
6.21 (dd, J = 17.2, 10.8 Hz, 1 H), 7.14 (br s, 1 H), C NMR (100 MHz, CDCl ) δ 11.9, 29.4,
3
+
3
8.4, 50.0, 57.0, 69.9, 102.1, 113.7, 128.82, 128.87, 139.5, 166.0, 184.7, 193.8. MS (EI ) m/z:
+
3
23.1 (M + 1), 345.1 (M + Na). HRMS (M + H) m/z calculated for C H N O SH 323.1178;
1
4
18
4
3
found 323.1179.
tertꢀButylꢀ2ꢀ(2ꢀ((R)ꢀ2,5ꢀdihydroꢀ2ꢀmethylꢀ2ꢀ((E)ꢀ2ꢀmethylbutaꢀ1,3ꢀdienyl)ꢀ5ꢀoxothiophenꢀ
ꢀyloxy)acetamido)ethylcarbamate (TLM 23): Et N (287 µL, 2 mmol) and EDC (230 mg, 1.2
3
3
mmol) were added to a solution of 22a (254 mg, 1.0 mmol) and tertꢀbutyl 2ꢀazidoethylcarbamate
192 mg, 1.2 mmol) in DMF (4 mL) at room temperature after which the solution was stirred
overnight. After completion, the reaction mixture was extracted with CH Cl (2 x 10 mL) and the
(
2
2
combined organic phase was washed with 1 N HCl (10 mL), dried (MgSO ), filtered and
4
evaporated to obtain the crude product, which was then purified by flash chromatography (1:1,
23
1
EtOAc/hexanes) to yield TLM 23 in 66% yield. [α] D = ꢀ 116 (c 1, CH Cl ). H NMR (500
2
2
MHz, CDCl ): δ 1.37 (s, 9 H), 1.73, 1.89 (2 s, 6 H), 3.19ꢀ3.23 (m, 2 H), 3.31ꢀ3.42 (m, 2 H), 4.43
3
(s, 2 H), 5.04 (d, J = 11.0 Hz, 1 H), 5.08 (br s, 1 H), 5.24 (d, J = 17.5 Hz, 1 H), 5.55 (s, 1 H),
1
3
6
.30 (dd, J = 17.5, 11.0 Hz, 1 H), 7.18 (br s, 1 H), C NMR (125 MHz, CDCl ) δ 28.1, 29.5,
3
3
9.9, 40.7, 57.0, 70.3, 79.6, 102.4, 113.8, 129.3, 139.4, 140.5, 156.8, 165.8, 184.4, 193.0. MS
+
+
(EI ) m/z: 397.1 (M + 1). HRMS (M + H) m/z calculated for C H N O SH 397.1797; found
1
9
28
2
5
3
97.1804.
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2
2
2
2
2
2
3
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(
2R)ꢀNꢀ((1ꢀ(2ꢀ(2ꢀ((R)ꢀ2,5ꢀDihydroꢀ2ꢀmethylꢀ2ꢀ((E)ꢀ2ꢀmethylbutaꢀ1,3ꢀdienyl)ꢀ5ꢀ
oxothiophenꢀ3ꢀyloxy)acetamido)ethyl)ꢀ1Hꢀ1,2,3ꢀtriazolꢀ4ꢀyl)methyl)ꢀ2,4ꢀdihydroxyꢀ3,3ꢀ
dimethylbutanamide (TLM 24): General Procedure III. Chromatography on silica gel (ISCO
combiflash, 4 g gold cartridge, eluting with CH Cl → 7% MeOH inCH Cl ) afforded TLM 24 in
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
2
2
2
2
23
1
8
0% yield. [α] D = + 94 (c 1, MeOH). H NMR (500 MHz, CD OD, 10% CDCl ): δ 1.05 (s, 6
3
3
H), 1.89, 2.01 (2 s, 6 H), 3.40ꢀ3.49 (m, 2 H), 3.73ꢀ3.77 (m, 1 H), 3.95 (s, 1 H), 4.45ꢀ4.59 (m, 8
H), 5.08 (d, J = 10.75 Hz, 1 H), 5.28 (d, J = 17.5 Hz, 1 H), 5.47 (s, 1 H), 5.77 (s, 1 H), 6.38 (dd,
1
3
J = 17.5, 10.75 Hz, 1 H), 7.78 (br s, 1 H), 8.30 (s, 1 H). C NMR (125 MHz, CD OD, 10%
3
CDCl ) δ 11.5, 14.3, 14.5, 19.8, 20.2, 28.8, 33.8, 39.1, 48.3, 57.3, 69.1, 70.0, 76.2, 101.9, 112.8,
3
ꢀ
1
23.2, 129.5, 139.2, 140.6, 167.2, 174.7, 186.0, 194.5. MS (EI ) m/z: 490.2 (M ꢀ 17), 508.2 (M +
+
1
). HRMS (M + H) m/z calculated for C H N O SH 508.2230; found 508.2230.
2
3
33
5
6
Nꢀ((1ꢀ(2ꢀ(2ꢀ((R)ꢀ2,5ꢀDihydroꢀ2ꢀmethylꢀ2ꢀ((E)ꢀ2ꢀmethylbutaꢀ1,3ꢀdienyl)ꢀ5ꢀoxothiophenꢀ3ꢀ
yloxy)acetamido)ethyl)ꢀ1Hꢀ1,2,3ꢀtriazolꢀ4ꢀyl)methyl)ꢀ4ꢀ((4ꢀfluorophenylsulfonyl)ꢀ
amino)benzamide (TLM 25): General Procedure III. Chromatography on silica gel (ISCO
combiflash, 4 g gold cartridge, eluting with CH Cl → 7% MeOH in CH Cl ) afforded TLM 25
2
2
2
2
23
1
in 88% yield. [α] D = + 71 (c 1, MeOH). H NMR (500 MHz, CD OD, 10% CDCl ): δ 1.75,
3
3
1
.85 (2 s, 6 H), 3.72ꢀ3.76 (m, 2 H), 4.50ꢀ4.58 (m, 8 H), 5.07 (d, J = 10.5 Hz, 1 H), 5.24 (d, J =
7.5 Hz, 1 H), 5.41 (s, 1 H), 5.71 (s, 1 H), 6.30 (d, J = 17.5, 10.5 Hz, 1 H), 7.13ꢀ7.19 (m, 4 H),
1
1
3
7.73 (m, 1 H), 7.83ꢀ7.85 (m, 3 H), 8.05 (s, 1 H). C NMR (125 MHz, CD OD, 10% CDCl ) δ
3
3
1
2.8, 20.7, 29.9, 35.9, 39.9, 40.0, 50.0, 58.3, 70.9, 102.9, 114.2, 120.1, 129.4, 130.2, 130.6,
+
130.7, 136.4, 140.2, 141.4, 141.7, 165.0, 167.1, 167.9, 168.0, 168.7, 186.8, 195.7. MS (EI ) m/z:
+
6
55.2 (M + 1). HRMS (M + H) m/z calculated for C H FN O S H 655.1809; found 655.1812.
3
0
31
6
6 2
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(
R)ꢀ3ꢀ(6ꢀAzidohexanoyl)ꢀ4ꢀhydroxyꢀ5ꢀmethylꢀ5ꢀ((E)ꢀ2ꢀmethylbutaꢀ1,3ꢀdienyl)thiophenꢀ
2
(5H)ꢀone (TLM 26): General Procedure IV. Flash chromatography (30% EtOAc/hexanes/1%
2
3
1
AcOH) afforded TLM 26 85%. [α] D = + 183 (c 1, MeOH). H NMR (500 MHz, CDCl ) δ
3
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
1
.37ꢀ1.41 (m, 2 H), 1.56ꢀ1.65 (m, 4 H), 1.68, 1.72 (2 s, 6 H), 2.72ꢀ2.90 (m, 2 H), 3.23ꢀ2.28 (m, 2
H), 5.03 (d, J = 10.5 Hz, 1 H), 5.14 (d, J = 17.5 Hz, 1 H), 5.63 (s, 1 H), 6.28 (dd, J = 17.6, 10 Hz,
1
3
1
H). C NMR (100 MHz, CDCl , peaks broadening was observed due to ketoꢀenol
3
tautomerism) δ 13.2, 14.0, 24.1, 26.2, 28.4, 29.6, 33.8, 39.6, 57.2, 113.3, 138.4, 140.8, 141.5,
+
+
179.8, 194.8, 204.7. MS (EI ) m/z: 336.1 (M + 1), 353.1 (M + NH ). HRMS (M + H) m/z
4
calculated for C H N O SH 336.1382; found 336.1375.
1
6
21
3
3
Materials and Methods: All commercially available chemicals and dry solvents (except THF
which was distilled from sodium benzophenone ketyl) were purchased and used without further
purification unless noted otherwise. Airꢀ and moistureꢀsensitive reactions were run under inert
1
13
atmosphere (N or Ar) using flameꢀdried glassware. H and C NMR spectra were acquired on
2
a Varian and/or Bruker 400, 500 and 600 MHz NMR spectrometer. Chemical shifts are
expressed in parts per million (δ value) downfield from tetramethylsilane using tetramethylsilane
1
13
(
δ = 0) and/or relative to residual solvents such as CHCl (7.26 ppm, H; 77.0 ppm, C), MeOHꢀ
3
1
13
1
13
d (3.31 ppm, H; 49.1 ppm, C), or acetoneꢀd (2.04 ppm, H; 29.9 ppm, C). Splitting patterns
4
6
are indicated as s, singlet; brs broad singlet; d, doublet; t, triplet; q, quartet; m, multiplet; dt,
doublet of triplet. TLC was performed on Merck preꢀcoated silica gel 60 F254 plates and all the
spots were visualized using UV light followed by staining with phosphomolybdic acid. Column
chromatography was performed using silica gel 60 (Merck, 230ꢀ400 mesh) and/or an ISCO
Combiflash instrument using ISCO redisep flash cartridges (particle size: 35−70 µm).
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