Thiolactomycin-based inhibitors of bacterial β-ketoacyl-ACP synthases with in vivo activity

Abstract

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) 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.

Graphical Abstract

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 health.¹ Multidrug resistant strains of organisms such as Klebsiella pneumoniae and Acinetobacter baumannii are virtually untreatable by currently available antibiotics.^1–3 Moreover, resistance has been reported towards drugs such as vancomycin, that is considered the ‘drug of last resort’ for a variety of bacterial infections including Staphylococcus aureus.⁴ 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, essential for survival and is distinct from the mammalian pathway.^5–7 Thus, enzymes in this pathway are thought to be profitable targets for the development of novel therapeutics. In support of this proposal, the front-line tuberculosis drug isoniazid is an inhibitor of the enoyl-ACP reductase (FabI) in the Mycobacterium tuberculosis FASII pathway,^8–11 while several FabI inhibitors are currently in clinical or preclinical development to treat infections caused by methicillin-resistant S. aureus (MRSA).^12–14

Figure 1.

Fatty Acid Biosynthesis pathway in E. coli. β-Ketoacyl-ACP synthases (FabB, FabF and FabH) catalyze the decarboxylative Claisen condensation of malonyl-ACP with the acyl primer. While FabB and FabF are responsible for the elongation step of FASII, FabH initiates FASII by synthesizing acetoacetyl-ACP. Thiolactomycin (TLM), a natural product thiolactone, preferentially inhibits the FabB and FabF β-ketoacyl-ACP synthases¹⁵.

In addition to FabI, the FASII β-ketoacyl-ACP 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 Cys-His-His catalytic triad and catalyze the condensation of malonyl-ACP and acyl-ACP in the elongation cycle, while FabH (KASIII) contains a Cys-His-Asn triad, and is responsible for initiation of the FASII cycle through the condensation of malonyl-ACP with acetyl-CoA.^{7, 15–18} More recently, Yuan et al. identified a new class of KASI/II enzymes (FabY) which replaces FabH in Pseudomonas aeruginosa.¹⁹

Table 1.

KAS enzyme distribution

Organism	Classification	FabB	FabF	FabH
Y. pestis	Gram-negative	+	+	+
M. tuberculosis	Gram-positive	+	+	+
K. pneumoniae	Gram-negative	+	+	+
S. aureus	Gram-positive	−	+	+
B. pseudomallei	Gram-negative	−	+	+
F. tularensis	Gram-negative	−	+	+

The β-Ketoacyl-ACP synthases present in the bacteria used in the study

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 is a valid target for drug discovery.^{7, 20–23} Cerulenin, from the fungus Caephalosporium caerulens, is a suicide inhibitor that targets FabB and FabF in E. coli,⁷ whilst platensimycin, from Streptomyces platensis,²³ is effective against Gram-positive bacteria such as S. aureus, Enterococcus faecalis and Streptococcus pneumonia.²⁰ In addition, the platensimycin analog platencin, inhibits both FabF and FabH comparably, and also is effective against various antibiotic resistant bacteria including vancomycin-resistant Enterococcus faecium.²⁴ Finally phomallenic acids, isolated from a plant fungus, are KAS inhibitors and have potent antibacterial activity against MRSA, Bacillus subtilis and Haemophilus influenza.^{21, 22}

Thiolactomycin (TLM), a natural product thiolactone, is a competitive reversible inhibitor that binds to the malonyl-ACP binding site of the KAS enzymes.¹⁵ First isolated from Nocardia sp.,^25–27 TLM shows broad spectrum antibacterial activity against Gram-positive and Gram-negative bacteria, as well as mycobacteria.^{25, 27, 28} Despite moderate MIC values, the favorable physicochemical properties, low cytotoxicity, high bioavailability and in vivo activity of TLM,^{25–27, 29–33} 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 improve the activity of TLM.^{31, 34–44} In addition, there are few reports of enantiomerically pure TLM analogs,^45–51 and most studies have been performed with racemic mixtures of the respective TLM analogs despite the knowledge that (5R)-TLM is the biologically active enantiomer.⁴³

Previously, we demonstrated that TLM is a time-dependent inhibitor of the FabB and FabF acyl-enzymes from M. tuberculosis and E. coli.³² 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 (mtFabB; KasA) in vitro.^{33, 52, 53} 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.

RESULTS AND DISCUSSION

Chemistry

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) was synthesized from D-alanine (Scheme 1).⁴⁴ 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 n-pentane 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 1a–4a, 12a–16a and 19a (80–90% two-step yield). Subsequent enolate formation of 1a–4a, 12a–16a and 19a with LiHMDS at −78 °C initiated the thio-Dieckmann condensation providing (5R)-TLM-TLM 4, (5R)-TLM 12–16 and (5R)-TLM 19 in 58–92% 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 O-alkylation, and C3 alkylation products. Preferential O-alkylation of TLM 2 was achieved using NaH in DMF with tert-butyl bromoester and followed by hydrolysis of the ester with 25% TFA in CH₂Cl₂ to obtain compound 22a. Compound 22a was coupled with 2-azidoethanamine and 2-azidoethylcarbamate 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 CuSO₄.5H₂O and sodium ascorbate to form the corresponding ‘click’ adducts in 80% and 88% yields, respectively (Scheme 3).

Scheme 1.

Synthesis of building blocks 32 and 1.

Scheme 2.

Synthesis of C3 analogs of 5R-(+)-Thiolactomycin (TLM 2 – 4, 12 – 16, 19)

Scheme 3.

Synthesis of C4 analogs of 5R-(+)-Thiolactomycin (TLM 22 – 25)

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).

Scheme 4.

Synthesis of C3 analogs of 5R-(+)-Thiolactomycin (TLM 17, 18, 20, 21)

Utilizing a previously published protocol for C3 acylation of tetronic acids,⁵⁴ TLM 2 was treated with a variety of anhydrides or acid chlorides (5a–11a and 26a) in the presence of Et₃N and catalytic amount of DMAP in CH₂Cl₂ to form kinetically controlled O-acylated products. Subsequently, DMAP mediated transfer of the O-acyl group to C3 of TLM over time led to the thermodynamically stable C3-acylated products, providing TLM 5–11 and 26 in 70%–85% yields (Scheme 5).

Scheme 5.

Synthesis of C3 analogs of 5R-(+)-Thiolactomycin (TLM 5–11, 26)

In vitro activity

Previously we quantified the inhibition of mtFabB and C171Q mtFabB by TLM analogs 1–18.^{33, 52} 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 19–25). We also determined the whole cell antibacterial activity of TLM (1) and all TLM analogs (2–25) against M. tuberculosis, Y. pestis, F. tularensis, B. pseudomallei, MSSA and MRSA.

C4 substituents of demethyl TLM

Compounds TLM 22–25 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 TLM and derivatives bound to mtFabB and mtC171QFabB revealed that the C4 hydroxyl is involved in an extended water-mediated 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.

Table 2.

In vitro activity of C4 TLM analogs

Name	Structure		Ki (μM) mtFabB		MIC (μg/mL)1
	R	R′	wt	C171Q	M. tb H37Rv	F. tu LVS	S. aureus
							RN4220 MSSA	BAA1762 MRSA
TLM	-OH	-CH3	226.0 ± 9.02	175.4 ± 3.02	3.1	2.8	75	75
TLM 2	-OH	-H	147 ± 2.22	46.9 ± 12	>100	124	150	100
TLM 22		-H	>200	>200	>100	39	>64	>64
TLM 23		-H	>200	>200	>100	54	>64	>64
TLM 24		-H	>200	ND3	100	159	>64	>64
TLM 25		-H	ND3	ND3	>100	>256	>64	>64

Binding affinity and whole cell activity of C4 analogs of TLM 2.

¹Whole cell activity (MIC) was measured using the microbroth dilution method in which the reported MIC values are 90% inhibition of bacterial growth using an Alamar Blue based colorimetric assay. M. tb H37Rv: M. tuberculosis strain H37Rv; F. tu LVS: F. tularensis live vaccine strain.

²Reported by Kapilashrami et al.³³

³ND: Not determined.

C3 substituents with bulky side chains

Based on the structural and interligand NOE NMR data, analogs TLM 12–17, 19 and 21 were synthesized. Despite improvements in binding affinity, particularly for TLM 12 and 17,³³ 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 β-ketoacyl-ACP synthase in the S. aureus FASII elongation cycle (Table 4). However, this analog showed a 5-fold improvement in MIC against MSSA (16 μg/mL) and a 3-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 12–16 with MIC values in the range of 2–3 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).

Table 3.

Whole Cell Activity of C3 TLM analogs

Name	Structure R=	MIC (μg/mL)1
		M. tb H37Rv	F. tu LVS	Y. ps A1122	S. aureus
					RN4220 MSSA	BAA1762 MRSA
TLM	CH3	3.1	2.8	8.0	75	75
TLM 2	H	>100	124	250	150	100
TLM 3	CH3CH2	2.5	6.9	145	150	125
TLM 4	CH3(CH2)2	25	25.8	>250	>200	>200
TLM 5		12.5	8.8	>250	64	32
TLM 6		100	98.1	>250	>64	64
TLM 7		100	1.8	>250	16	32
TLM 8		100	58.2	>250	>64	>64
TLM 9		100	3.9	>250	0.5	1
TLM 10		50	15.9	>250	32	32
TLM 11		50	0.21	>250	0.5	2
TLM 12		100	3.1	>250	64	100
TLM 13		ND	3.4	>250	>64	>100
TLM 14		>100	3.1	>250	>64	75
TLM 15		100	1.7	>250	64	50
TLM 16		12.5	1.9	>250	16	25
TLM 17		100	250	>250	>64	>100
TLM 18		100	15.3	>250	>64	>100
TLM 19		12.5	2.2	>250	32	50
TLM 20		100	23.6	>250	>64	>100
TLM 21		>100	250	>250	>64	>100
TLM 26		ND	ND	ND	16	16

¹Whole cell activity (MIC) was measured using the microbroth dilution method in which the reported MIC values are 90% inhibition of bacterial growth using an Alamar Blue based colorimetric assay. M. tb H37Rv: M. tuberculosis strain H37Rv; F. tu LVS: F. tularensis live vaccine strain; Y. ps A1122: Y. pestis strain A1122.

Table 4.

Inhibition of saFabF by TLM analogs

Inhibitors	wt saFabF Kd (μM)	C165Q saFabF Kd (μM)
TLM	107.0 ± 3.0	88.0 ± 2.0
TLM 5	14.2 ± 0.8	11.9 ± 0.6
TLM 6	58.4 ± 0.7	56.2 ± 0.7
TLM 9	>200	ND
TLM 11	35.2 ± 1.0	33.0 ± 0.7
TLM 16	131.0 ± 1.0	ND
TLM 18	107.2 ± 1.4	ND
TLM 19	92 ± 1.0	ND

Dissociation constants were determined using the fluorescence based assay described previously³². The inhibitor stock solutions, dissolved in DMSO or 50mM Tris-HCl, 150mM NaCl pH 8.5, were titrated into the enzyme (1 μM) in 50 mM Tris-HCl, 150mM NaCl, 1mM DTT, pH 8.5. Changes in fluorescence were monitored using excitation and emission wavelengths of 280 nm and 337 nm, respectively. By convention, K_d is used to identify the dissociation constant for wild-type KAS enzymes, while K_i and K_i* are used for the initial and final equilibrium constants for inhibitors binding to acyl KAS enzyme mimic. ND: Not determined.

C3 alkyl substituents

The loss of time-dependent inhibition of acyl mtFabB upon removal of the methyl group at the C3 position (TLM 2), observed by Kapilashrami et al.,³³ triggered our interest in developing SAR for C3-substituted 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_R = 1/k_off) with optimal t_R observed for TLM 3 binding to the C171Q mtFabB acyl-enzyme mimic.^{33, 52} While the MIC values of these compounds against M. tuberculosis were either similar to the MIC for TLM (3 μg/mL) or larger, the ethyl analog TLM 3 showed activity against a TLM resistant strain of H37Rv,²⁹ 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).

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 5–11, 26) in an attempt to more closely mimic the diketo motif of malonyl-ACP. While all the TLM analogs with acyl substituents at C3 showed tighter binding in vitro to mtFabB and the acylenzyme mtFabB mimic,³³ no improvement was observed in the whole cell activity against M. tuberculosis (Table 3). The lack of correlation between K_i and MIC values could reflect issues either with drug uptake or efflux.^{40, 43} TLM 5, 6 and 11 showed tighter binding to saFabF compared to the lead molecule (Table 4): TLM 5 had a ~ 8-fold improvement in binding to both apo saFabF and C164Q saFabF, compared to TLM (90–100 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 2-fold 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 150-fold improvement in whole cell activity against MSSA (MIC = 0.5 μg/mL) and a 75- and 37-fold 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 5-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.

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 TLM in animal models of K. pneumoniae and S. marcescens infection.²⁷ These investigators observed that TLM was rapidly absorbed and gave protection against systemic infection caused by both pathogens despite poor whole cell activity (100–200 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 (Table S3). For systemic infection with MRSA we chose analogs TLM 5, TLM 6 and TLM 11 which have improved K_d values for saFabF compared to TLM, and MIC values of 32, 62 and 2 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 ED₅₀ of TLM 6 was determined to be ~125 mg/kg, compared to an ED₅₀ of <30 mg/kg for vancomycin (Figure S2).

Figure 2.

Average survival time of mice infected with, A) Methicillin resistant S. aureus BAA1762 and treated with vehicle (●, n=7), TLM (■, n=7), TLM 5 (▲, n=6), TLM 6 (▼, n=6), TLM 11 (◆, n=6) and vancomycin (○, n=6); B) K. pneumoniae KPC2ST258 and treated with vehicle (●, n=10), TLM (■, n=8), TLM 3 (▲, n=8), TLM 5 (▼, n=8), TLM 6 (◆, n=8) and Gentamicin (▽, n=10). The infected animals were monitored twice a day for 5 days post infection for the MRSA infection model and 7 days for the K. pneumoniae infection model. The average survival time was calculated at the end of each study. The one-tailed t test was used to calculate the p value of each data set with respect to the vehicle control.

We then evaluated the activity of TLM 3, TLM 5 and TLM 6 in a mouse model of 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.

SUMMARY

The natural product TLM is an inhibitor of the β-ketoacyl-ACP 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 bonding network that involves the TLM C4 hydroxyl group.⁵² Thus, elaboration of the thiolactone core will likely require the introduction of substituents that maintain interactions with this H-bonding network. In contrast, several of the C3 analogs had improved activity compared to the parent compound. TLM 11, with a bulky biphenyl-hexanoyl substituent at C3, had a 13-fold improvement in MIC (0.21 μg/mL) against F. tularensis compared to TLM (2.8 μg/mL). Moreover, TLM 11 demonstrated 150- and 37-fold 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 5-fold 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 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 β-ketoacyl-ACP synthases from this pathogen. Our data suggest that further modifications at the thiolactone C3 position may be a profitable source of additional TLM-based inhibitors.

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 NH₄Cl (sat.)/1 N HCl (15 mL, 3:1) and extracted with Et₂O (3 × 20 mL) and then water (3 × 20 mL). The combined organics were dried (MgSO₄), filtered and evaporated to provide 1. Subsequently, the residue was redissolved in CH₂Cl₂ (15 mL) and cooled to 0 °C followed by addition of Et₃N (2.0 mmol) and the respective acid chloride (1.6 mmol). After 1 h, NH₄Cl (sat. 20 mL) was added and the mixture was extracted with CH₂Cl₂ (3 × 15 mL). The combined organics were dried (MgSO₄), filtered and evaporated. Flash chromatography (5% EtOAc/hex) gave the pure compounds with yields of 80–90%.

General Procedure II

LiHMDS was added to a stirred solution of compounds (1a–4a and 12a–19a) (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 × 15 mL). The combined organics were dried (MgSO₄), filtered and evaporated. This crude mixture was dissolved in NaHCO₃ (sat, 15 mL) and stirred for 15 min and extracted with Et₂O (3 × 10 mL). The aqueous layer was then acidified to pH 3 (pH paper) with 1 N HCl and extracted with Et₂O (3 × 10 mL) and EtOAc (2 × 10 mL). The combined organics were dried (MgSO₄), filtered and evaporated to provide TLM -TLM 4 and TLM 12 – TLM 19, which were further purified by flash chromatography to get obtain the pure compounds in 58–92% yield.

General Procedure III

CuSO₄.5H₂O (1.0 mmol) and sodium ascorbate (1.0 mmol) were added to a solution of TLM 18, TLM 20, or TLM 22 (1.0 mmol) and alkyne (27) (1.0 mmol) in n-butanol/water (1:1, 3 mL) at room temperature. The reaction mixture was stirred at room temperature for 1–2 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), Et₃N (2 mmol) and DMAP (0.4 mmol) were added to a stirred solution of TLM 2 (1.0 mmol) in CH₂Cl₂ (3 mL) at 0 °C. The reaction mixture was stirred at room temperature for 2–8 h then extracted with CH₂Cl₂ (3 × 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)-4-hydroxy-3,5-dimethyl-5-((E)-2-methylbuta-1,3-dienyl)thiophen-2(5H)-one (TLM): General Procedure II

TLM 80%. ${[\mathrm{\alpha }]}_D^{23}$ = + 173.0 (c 1.2, MeOH). Mp 119–121 °C. [(lit.⁴⁴ ${[\mathrm{\alpha }]}_D^{24}$ = + 174 (c 0.6, MeOH), Mp 119.5–121 °C)]; ¹H NMR (500 MHz, CDCl₃ and 5% CD₃OD): δ 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, 1 H), 5.53 (s, 1 H), 6.26 (dd, J = 17.0, 11.0 Hz, 1 H). ¹³C NMR (125 MHz, CDCl₃ and 5% CD₃OD) 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 calculated for C₁₁H₁₄O₂S 210.1; found 209.1 (M−1).

(R,E)-Ethyl 2,4-dimethyl-2-(Acetylthio)hexa-3,5-dienoate (2a): General Procedure I

Flash chromatography (5% EtOAc/hexanes) afforded 2a 84%. ${[\mathrm{\alpha }]}_D^{23}$ = + 17 (c 1.0, CH₂Cl₂). ¹H NMR (600 MHz, CDCl₃): 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 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 Hz, 1 H). ¹³C NMR (125 MHz, CDCl₃) δ 12.7, 13.8, 25.8, 29.9, 55.2, 61.8, 113.1, 131.1, 138.1, 141.0, 171.8, 194.3. MS (EI⁻): m/z calculated for C₁₂H₁₈O₃S 242.1; found 243.1 (M + 1), 198.1 (M − 44).

(R)-4-hydroxy-5-methyl-5-((E)-2-methylbuta-1,3-dienyl)thiophen-2(5H)-one (TLM 2): General Procedure II

TLM 2 58%. ${[\mathrm{\alpha }]}_D^{23}$ = + 44 (c 1.0 MeOH). [(lit.⁴⁷ ${[\mathrm{\alpha }]}_D^{29}$ = + 186 (c 1.01, MeOH), 1H NMR (500 MHz, CD₃OD): δ 1.79 (s, 3H), 1.83 (s, 3H), 5.06 (d, J =10.5 Hz, 1H), 5.27 (d, J = 17.7 Hz, 1H), 5.66 (s, 1H), 6.36 (dd, J = 17.5, 10.5 Hz). ¹³C NMR (100 MHz, CD₃OD): δ 12.4, 30.3, 58.8, 113.7, 131.4, 140.5, 142.1, 190.1, 197.2. MS (EI⁺): m/z calculated for C₁₀H₁₂O₂S 196.1; found 197.1 (M + 1).

(R,E)-Ethyl 2,4-dimethyl-2-(butanoylthio)hexa-3,5-dienoate (3a): General Procedure I

Flash chromatography (5% EtOAc/hexanes) afforded the product 3a 82%. ${[\mathrm{\alpha }]}_D^{23}$ = + 32 (c 1, CH₂Cl₂). ¹H NMR (400 MHz, CDCl₃): δ 0.88, (t, J = 7.2 Hz, 3 H), 1.21 (t, J = 7.2 Hz, 3 H), 1.58–1.64 (m, 2 H), 1.82, 1.84 (2s, 6 H), 2.40 (t, J = 7.2 Hz, 3 H), 4.13–4.18 (m, 2 H), 5.00 (d, J = 10.8 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). ¹³C NMR (100 MHz, CDCl₃) δ 12.7, 13.1, 13.8, 18.9, 25.9, 45.1, 55.0, 61.8, 113.0, 131.2, 138.1, 141.0, 172.0, 198.0. MS (EI⁺) m/z calculated for C₁₄H₂₂O₃S 270.1; found 271.1 (M + H).

(R)-3-Ethyl 4-hydroxy-5-methyl-5-((E)-2-methylbuta-1,3-dienyl)thiophen-2(5H)-one (TLM 3): General Procedure II

Flash chromatography (1:1, EtOAc/hexanes) afforded TLM 3 75%. ${[\mathrm{\alpha }]}_D^{23}$ = + 98 (c 1, MeOH). ¹H NMR (500 MHz, CDCl₃ and 5% CD₃OD): δ 1.97 (t, J = 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 = 17.0 Hz, 1 H), 5.53 (s, 1 H), 6.26 (dd, J = 17.0, 10.5 Hz, 1 H). ¹³C NMR (125 MHz, CDCl₃ and 5% CD₃OD) 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. MS (EI⁻) m/z: 223.1 (M − 1). HRMS (M + H)⁺ m/z calculated for C₁₂H₁₆O₂SH 225.0949; found 225.0949.

(R,E)-Ethyl 2,4-dimethyl-2-(pentanoylthio)hexa-3,5-dienoate (4a): General Procedure I

Flash chromatography (5% EtOAc/hexanes) afforded 4a 84%. ¹H NMR (400 MHz, CDCl₃): δ 0.85, (t, J = 7.2 Hz, 3 H), 1.22 (t, J = 7.2 Hz, 3 H), 1.26–1.30 (m, 2 H) 1.55–1.59 (m, 2 H), 1.82, 1.84 (2s, 6 H), 2.43 (t, J = 7.2 Hz, 2 H), 4.14–4.19 (m, 2 H), 5.00 (d, J = 10.8 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). ¹³C NMR (100 MHz, CDCl₃) δ 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 C₁₅H₂₄O₃S 284.1; found 285.1 (M + 1).

(R)-4-Hydroxy-5-methyl-5-((E)-2-methylbuta-1,3-dienyl)-3-propylthiophen-2(5H)-one (TLM 4): General Procedure II

Flash chromatography (1:1, EtOAc/hexanes) afforded TLM 4 78%. ${[\mathrm{\alpha }]}_D^{23}$ = + 61 (c 1, MeOH). ¹H NMR (500 MHz, CDCl₃ and 5% CD₃OD): δ 1.89 (t, J = 7.2 Hz, 3 H), 2.26–2.20 (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 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). ¹³C NMR (125 MHz, CDCl₃ and 5% CD₃OD) δ 12.0, 13.8, 21.1, 24.6, 29.8, 55.4, 113.6, 114.5, 129.3, 139.9, 140.7, 181.2, 197.5. MS (EI⁻) m/z: 237.1 (M − 1). HRMS (M + H)⁺ m/z calculated for C₁₃H₁₈O₂SH 239.1106; found 239.1102.

(R)-3-Acetyl-4-hydroxy-5-methyl-5-((E)-2-methylbuta-1,3-dienyl)thiophen-2(5H)-one (TLM 5): General Procedure IV

Flash chromatography (30% EtOAc/hexanes/1% AcOH) afforded TLM 5 75%. ${[\mathrm{\alpha }]}_D^{23}$ = + 404 (c 1, MeOH). ¹H NMR (500 MHz, CDCl₃ and 5% CD₃OD): 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 H), 5.64 (s, 1 H), 6.27 (dd, J = 17.0, 10.0 Hz, 1 H). ¹³C NMR (100 MHz, CDCl₃ and 5% CD₃OD, peak broadening was observed due to keto-enol tautomerism) δ 18.8, 19.8, 29.3, 57.7, 107.9, 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 C₁₂H₁₄O₃SH 239.0742; found 239.0748.

(R)-3-(2,2,2-Trifluoroacetyl)-4-hydroxy-5-methyl-5-((E)-2-methylbuta-1,3-dienyl)thiophen-2(5H)-one (TLM 6): General Procedure IV

Flash chromatography (40% hexanes/10% THF/2% AcOH/EtOAc) afforded TLM 6 82%. ${[\mathrm{\alpha }]}_D^{23}$ = + 310 (c 1, MeOH). ¹H NMR (500 MHz, CD₃OD): δ 1.72, 1.75 (2 s, 6 H), 5.00 (d, J = 10.4 Hz, 1 H), 5.20 (d, J = 17.6 Hz, 1 H), 5.72 (s, 1 H), 6.37 (dd, J = 17.6, 10.4 Hz, 1 H). ¹³C NMR (100 MHz, CD₃OD, peak broadening was observed due to keto-enol tautomerism) δ 16.9, 18.8, 51.3, 104.0, 114.0, 118.1–126.7 (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 C₁₂H₁₁F₃O₃SNa 315.0279; found 315.0229.

(R)-3-Butyryl 4-hydroxy-5-methyl-5-((E)-2-methylbuta-1,3-dienyl)thiophen-2(5H)-one (TLM 7): General Procedure IV

Flash chromatography (20% EtOAc/hexanes/1% AcOH) afforded TLM 7 80%. ${[\mathrm{\alpha }]}_D^{23}$ = + 396 (c 1, MeOH). ¹H NMR (400 MHz, CDCl₃): δ 1.00 (t, J = 7.60 Hz, 3 H), 1.69–1.79 (m, 5 H), 1.86 (s, 3 H), 2.92–3.01 (m, 2 H), 5.08 (d, J = 10.8 Hz, 1 H), 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). ¹³C NMR (100 MHz, CDCl₃) 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, 205.2. MS (EI⁻) m/z: 265.1 (M − 1). HRMS (M + H)⁺ m/z calculated for C₁₄H₁₈O₃SH 267.1055; found 267.1050.

(R)-Methyl 2,5-dihydro-4-hydroxy-5-methyl-5-((E)-2-methylbuta-1,3-dienyl)-2-oxothiophene-3-carboxylate (TLM 8): General Procedure IV

Flash chromatography (3:1, Acetone/Toluene) afforded TLM 8 71%. ${[\mathrm{\alpha }]}_D^{23}$ = + 313 (c 1, MeOH). ¹H NMR (400 MHz, CD₃OD): 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 H), 5.45 (s, 1 H), 6.06 (dd, J = 17.2, 10.8 Hz, 1 H). ¹³C NMR (100 MHz, CD₃OD, peak broadening was observed due to keto-enol tautomerism) δ 20.4, 23.8, 52.3, 56.3, 98.5, 113.3, 134.2, 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 C₁₂H₁₄O₄SNa 277.0510; found 277.0513.

(R)-4-Hydroxy-5-methyl-5-((E)-2-methylbuta-1,3-dienyl)-3-palmitoylthiophen-2(5H)-one (TLM 9): General Procedure IV

Flash chromatography (1:1, EtOAc/hexanes) to afford TLM 9 75%. ${[\mathrm{\alpha }]}_D^{23}$ = + 130 (c 1, CH₂Cl₂). ¹H NMR (400 MHz, CDCl₃): δ 0.87 (t, J = 6.80 Hz, 3 H), 1.80-1.31 (m, 20 H), 1.40–1.59 (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 = 17.6, 10.8 Hz, 1 H). ¹³C NMR (100 MHz, CDCl₃) δ 12.8, 14.0, 22.6, 246, 25.0, 28.9, 29.2, 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, 204.6. MS (EI⁺) m/z: 435.2 (M + 1). HRMS (M + H)⁺ m/z calculated for C₂₆H₄₂O₃SH 435.2933; found 435.2929.

(R)-3-(2-Carboxybenzoyl)-4-hydroxy-5-methyl-5-((E)-2-methylbuta-1,3-dienyl)thiophen-2(5H)-one (TLM 10): General Procedure IV

Flash chromatography (2:1, Acetone/Toluene) to afford TLM 10 70%. ${[\mathrm{\alpha }]}_D^{23}$ = + 243 (c 1, MeOH). ¹H NMR (500 MHz, CD₃OD): δ 1.77, 1.78 (2 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, 10.8 Hz, 1 H), 7.10 (d, J = 6.0 Hz, 1 H), 7.35-7.04 (m, 2 H), 7.94 (br s, 1 H). ¹³C NMR (125 MHz, CD₃OD, peak broadening was observed due to keto-enol tautomerism) δ 20.4, 49.0, 58.8, 110.3, 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 C₁₈H₁₆O₅SNa 367.0616; found 367.0612.

(R)-3-(4-Phenyl-phenylhexanoyl)-4-hydroxy-5-methyl-5-((E)-2-methylbuta-1,3- dienyl)thiophen-2(5H)-one (TLM 11): General Procedure IV

Flash chromatography (20% EtOAc/hexanes/1% AcOH) afforded TLM 11 72%. ${[\mathrm{\alpha }]}_D^{23}$ = + 211 (c 1, MeOH). ¹H NMR (400 MHz, CDCl₃): δ 1.29–1.39 (m, 3 H), 1.56–1.62 (m, 3 H), 1.68, 1.72 (2 s, 6 H), 2.56–2.61 (m, 2 H), 2.79–2.85 (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 (dd, J = 17.6, 10 Hz, 1 H), 7.15–7.53 (m, 9 H). ¹³C NMR (100 MHz, CDCl₃, peak broadening was observed due to keto-enol tautomerism) δ 13.2, 14.0, 21.0, 29.2, 29.6, 31.4, 35.4, 60.9, 126.8, 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 C₂₈H₃₀O₃SH 447.1994; found 447.1989.

(R,E)-Ethyl 2-(4-chloro-phenylbutanoylthio)-2,4-dimethylhexa-3,5-dienoate (12a): General Procedure I

Flash chromatography (8% EtOAc/hexanes) afforded 12a 85%. ${[\mathrm{\alpha }]}_D^{23}$ = − 12 (c 1, CH₂Cl₂). ¹H NMR (400 MHz, CDCl₃): δ 1.42, (t, J = 7.2 Hz, 3 H), 1.88, 1.90 (2s, 6 H), 2.29 (m, 2 H), 2.75 (t, J = 7.2 Hz, 2 H), 2.83–2.90 (m, 2 H), 4.38. (q, J = 7.2 Hz, 2 H), 5.22 (d, J = 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), 7.23–7.28 (m, 4 H). ¹³C NMR (125 MHz, CDCl₃) δ 13.8, 26.7, 28.8, 34.5, 42.2, 52.8, 55.1, 61.9, 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 C₂₀H₂₅ClO₃S 380.1; found 381.0 (M + 1).

(R)-3-(4-Chlorophenethyl)-4-hydroxy-5-methyl-5-((E)-2-methylbuta-1,3- dienyl)thiophen-2(5H)-one (TLM 12). General Procedure II

Purified by flash chromatography (30% EtOAc/hexanes) afforded TLM 12 86%. ${[\mathrm{\alpha }]}_D^{23}$ = + 104 (c 1, CH₂Cl₂). ¹H NMR (400 MHz, CDCl₃): δ 1.69. 1.81 (2 s, 6 H), 2.54–2.59 (m, 2 H), 2.74–2.80 (m, 2 H) 5.07 (d, 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 (d, J = 8.0 Hz, 1 H), 7.21 (d, J = 8.0 Hz, 1 H) 7.23 (s, 1 H). ¹³C NMR (125 MHz, CDCl₃) δ 12.0, 24.4, 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, 181.1, 196.9. MS (EI⁻) m/z: 333.0 (M − 1). HRMS (M + H)⁺ m/z calculated for C₁₈H₁₉ClO₂SH 335.0873; found 335.0867.

(R,E)-Ethyl 2-(4-methyl-phenylbutanoylthio)-2,4-dimethylhexa-3,5-dienoate (13a): General Procedure I

Flash chromatography (7% EtOAc/hexanes) afforded 13a 80%. ${[\mathrm{\alpha }]}_D^{23}$ = − 212 (c 1, CH₂Cl₂). ¹H NMR (500 MHz, CDCl₃): δ 1.28, (t, J = 7.5 Hz, 3 H), 1.88, 1.90 (2s, 6 H), 1.20–1.93 (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 = 17.5, 10.5 Hz, 1 H), 7.06–7.19 (m, 4 H). ¹³C NMR (125 MHz, CDCl₃) δ 13.0, 14.0, 20.1, 26.0, 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 C₂₁H₂₈O₃S 360.2; found 361.1 (M + 1).

(R)-3-(4-Methylphenethyl)-4-hydroxy-5-methyl-5-((E)-2-methylbuta-1,3-dienyl)thiophen-2(5H)-one (TLM 13): Procedure II

Flash chromatography (30% EtOAc/hexanes) afforded TLM 13 87%. ${[\mathrm{\alpha }]}_D^{23}$ = + 161 (c 1, CH₂Cl₂). ¹H NMR (500 MHz, CDCl₃): δ 1.70, 1.74 (2s, 6 H), 2.31 (s, 3 H), 2.47–2.55 (m, 1 H), 2.60-2.54 (m, 1 H), 2.68–2.78 (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), 7.03–7.12 (m, 4 H). ¹³C NMR (125 MHz, CDCl₃) δ 12.1, 21.0, 25.2, 29.7, 33.3, 55.0, 113.8, 113.9, 128.3, 129.14, 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 C₁₉H₂₂O₂SH 315.1419; found 315.1411.

(R,E)-Ethyl 2-(4-fluorophenyl-hexanoylthio)-2,4-dimethylhexa-3,5-dienoate (14a): General Procedure I

Flash chromatography (8% EtOAc/hexanes) afforded 14a 89%. ${[\mathrm{\alpha }]}_D^{23}$ = + 965 (c 1, CH₂Cl₂). ¹H NMR (500 MHz, CDCl₃): δ 1.24, (t, J = 7.5 Hz, 3 H), 1.26–1.34 (m, 2 H), 1.35–1.67 (2m, 4 H), 1.85, 1.87 (2s, 6 H), 2.42–2.55 (m, 2 H), 2.56–2.59 (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 = 14.5, 9.0 Hz, 1 H), 6.93–6.95 (m, 2 H), 7.08–7.10 (m, 2 H). ¹³C NMR (125 MHz, CDCl₃) δ 12.9, 13.9, 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, 141.2, 160.1, 162.0, 172.0, 198.1. MS (EI⁺) m/z calculated for C₂₂H₂₉FO₃S 392.2; found 393.1 (M + 1), 410.2 (M + NH₄).

(R)-3-(4-(4-Fluorophenyl)butyl)-4-hydroxy-5-methyl-5-((E)-2-methylbuta-1,3-dienyl)thiophen-2(5H)-one (TLM 14): General Procedure II

Flash chromatography (25% EtOAc/hexanes) afforded TLM 14 82%. ${[\mathrm{\alpha }]}_D^{23}$ = + 63 (c 1, CH₂Cl₂). ¹H NMR (300 MHz, CDCl₃) δ 1.50–1.61 (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 = 7.2 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 = 17.5, 10.5 Hz, 1 H), 6.89–6.95 (m, 2 H), 7.05–7.10 (m, 2 H). ¹³C NMR (125 MHz, CDCl₃) δ 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, 162.3, 181.3, 197.7. ¹⁹F NMR (400 MHz, CDCl₃) δ -122.32 (m). MS (EI⁺) m/z: 347.1 (M + 1). HRMS (M + H)⁺ m/z calculated for C₂₀H₂₃FO₂SH 347.1481; found 347.1478.

(R,E)-Ethyl 2-(4-chlorophenyl-hexanoylthio)-2,4-dimethylhexa-3,5-dienoate (15a): General Procedure I

Flash chromatography (8% EtOAc/hexanes) afforded 15a 85%. ${[\mathrm{\alpha }]}_D^{23}$ = − 10 (c 1, MeOH). ¹H NMR (500 MHz, CDCl₃): δ 1.27, (t, J = 7.5 Hz, 3 H), 1.33–1.37 (m, 2 H), 1.61–1.68 (2m, 4 H), 1.87, 1.89 (2s, 6 H), 2.49 (t, J = 7.5 Hz, 2 H), 2.56–2.60 (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 = 17.5, 10.5 Hz, 1 H), 7.09–7.10 (m, 2 H), 7.24–7.28 (m, 2 H). ¹³C NMR (125 MHz, CDCl₃) δ 12.9, 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, 138.1, 141.6, 141.2, 172.0, 198.1. MS (EI⁺) m/z calculated for C₂₂H₂₉ClO₃S 408.2; found 409.1 (M+1), 426.2 (M + NH₄).

(R)-3-(4-(4-Chlorophenyl)butyl)-4-hydroxy-5-methyl-5-((E)-2-methylbuta-1,3-dienyl)thiophen-2(5H)-one (TLM 15): General Procedure II

Purified by flash chromatography (20% EtOAc/hexanes) afforded TLM 15 83%. ${[\mathrm{\alpha }]}_D^{23}$ = + 48 (c 1, MeOH). ¹H NMR (500 MHz, CDCl₃) δ 1.50–1.62 (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 = 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), 6.28 (dd, J = 17.5, 10.5 Hz, 1 H), 7.07–7.09 (m, 2 H), 7.22–7.25 (m, 2 H). ¹³C NMR (125 MHz, CDCl₃) δ 12.0, 22.4, 27.2, 30.2, 31.0, 34.9, 55.9, 114.2, 114.8, 128.3–129.6 (m), 131.3, 140.0, 140.4, 140.7, 179.8, 198.2. MS (EI⁺) m/z: 363.1 (M + 1). HRMS (M + H)⁺ m/z calculated for C₂₀H₂₃ClO₂SH 363.1186; found 363.1180.

(R,E)-Ethyl 2-(4-phenyl-phenyl-hexanoylthio)-2,4-dimethylhexa-3,5-dienoate (16a): General Procedure I

Purified by flash chromatography (7% EtOAc/hexanes) to form (16a) 87%. ${[\mathrm{\alpha }]}_D^{23}$ = − 158 (c 1, CH₂Cl₂). ¹H NMR (500 MHz, CDCl₃): δ 1.27, (t, J = 7.5 Hz, 3 H), 1.36–1.48 (m, 2 H), 1.59–1.72 (2m, 4 H), 1.89, 1.91 (2s, 6 H), 2.52 (t, J = 7.5 Hz, 2 H), 2.65–2.69 (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 (s, 1 H), 6.35 (dd, J = 17.5, 10.5 Hz, 1 H), 7.25–7.61 (m, 9 H). ¹³C NMR (125 MHz, CDCl₃) δ 12.9, 13.9, 25.3, 25.9, 28.4, 30.9, 35.2, 43.2, 55.2, 113.2, 126.9–128.7 (m), 131.4, 138.2, 138.6, 141.2, 141.4, 172.1, 198.2. MS (EI⁺) m/z calculated for C₂₈H₃₄O₃S 450.2; found 451.2 (M + 1), 468.2 (M + NH₄).

(R)-3-(4-(4-Phenylphenyl)butyl)-4-hydroxy-5-methyl-5-((E)-2-methylbuta-1,3-dienyl)thiophen-2(5H)-one (TLM 16): General Procedure II

Flash chromatography (20% EtOAc/hexanes) afforded TLM 16 86%. ${[\mathrm{\alpha }]}_D^{23}$ = − 91 (c 1, CH₂Cl₂). ¹H NMR (500 MHz, CDCl₃) δ 1.50–1.69 (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 = 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 = 17.5, 10.5 Hz, 1 H), 7.23–7.59 (m, 9 H). ¹³C NMR (125 MHz, CDCl₃) δ 12.0, 22.5, 27.4, 30.2, 31.1, 35.2, 55.0, 114.1, 114.8, 126.9–128.8 (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 C₂₆H₂₈O₂SH 405.1888; found 405.1895.

4-((4-Fluorophenylsulfonyl)amino)-N-((1-(4-((R)-2,5-dihydro-4-hydroxy-5-methyl-5-((E)-2-methylbuta-1,3-dienyl)-2-oxothiophen-3-yl)butyl)-1H-1,2,3-triazol-4-yl)methyl)benzamide (TLM 17): General Procedure III

Chromatography on silica gel (ISCO combiflash, 4 g gold cartridge, eluting with CH₂Cl₂→ 8% MeOH in CH₂Cl₂) afforded TLM 17 75%. ${[\mathrm{\alpha }]}_D^{23}$ = + 70 (c 1, MeOH). ¹H NMR (400 MHz, CD₃OD): δ 1.39–1.43 (m, 2 H), 1.67, 1.76 (2 s, 6 H), 1.89–1.92 (2 s, 2 H), 2.20–2.29 (m, 2 H), 4.32–4.42 (m, 2 H), 4.56–4.62 (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), 7.18–7.24 (m, 4 H), 7.69–7.78 (m, 2 H), 7.84–7.87 (m, 2 H). ¹³C NMR (100 MHz, CDCl₃) δ 20.4, 20.5, 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, 131.3, 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 C₃₀H₃₂FN₅O₅S₂H 626.1907; found 626.1910.

(R,E)-Ethyl 2-(6-azido-hexanoylthio)-2,4-dimethylhexa-3,5-dienoate (18a): General Procedure I

Flash chromatography (7% EtOAc/hexanes) afforded 18a 85%. ${[\mathrm{\alpha }]}_D^{23}$ = + 6 (c 1, CH₂Cl₂). ¹H NMR (500 MHz, CDCl₃) δ 1.23 (t, J = 7.2 Hz, 3 H), 1.39-1.34 (m, 2H), 1.58-1.52 (m, 2 H), 1.67-1.60 (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), 4.18-4.16 (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 (dd, J = 17.5, 10.8 Hz, 1 H). ¹³C NMR (125 MHz, CDCl₃) δ 12.90, 13.90, 24.9, 25.8, 25.9, 28.4, 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 C₁₆H₂₅N₃O₃S 339.2; found 340.1 (M + 1).

(R)-3-(4-Azidobutyl)-4-hydroxy-5-methyl-5-((E)-2-methylbuta-1,3-dienyl)thiophen-2(5H)-one (TLM 18): General Procedure II

Flash chromatography (30% EtOAc/hexanes) afforded TLM 18 75%. ${[\mathrm{\alpha }]}_D^{23}$ = + 106 (c 1, CH₂Cl₂). ¹H NMR (400 MHz, CDCl₃): δ 1.21 (t, J = 7.5 Hz, 3 H), 1.73, 1.85 (2s, 6 H), 2.26 (m, 2 H), 2.30-2.28 (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, 1 H). ¹³C NMR (100 MHz, CDCl₃) δ 12.6, 21.9, 25.2, 28.0, 29.8, 51.4, 55.4, 113.7, 113.8, 129.4, 140.0, 181.1, 196.9. MS (EI⁺) m/z: 294.1 (M + 1). HRMS (M + H)⁺ m/z calculated for C₁₄H₁₉N₃O₂SH 294.1276; found 294.1281.

(R,E)-Ethyl 2-(4-bromophenyl-hexanoylthio)-2,4-dimethylhexa-3,5-dienoate (19a): General Procedure I

Flash chromatography (8% EtOAc/hexanes) afforded 19a 80%. ${[\mathrm{\alpha }]}_D^{23}$ = − 70 (c 1, MeOH). ¹H NMR (500 MHz, CDCl₃): δ 1.27, (t, J = 7.5 Hz, 3 H), 1.30–1.36 (m, 2 H), 1.59–1.68 (2m, 4 H), 1.87, 1.89 (2s, 6 H), 2.49 (t, J = 7.5 Hz, 2 H), 2.52–2.60 (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 = 17.5, 10.5 Hz, 1 H), 7.04–7.06 (m, 2 H), 7.39–7.41 (m, 2 H). ¹³C NMR (125 MHz, CDCl₃) δ 12.9, 13.9, 25.2, 25.9, 28.2, 30.7, 35.0, 43.2, 55.2, 113.2, 119.3, 130.0–131.4 (m), 138.2, 141.6, 172.0, 198.2. MS (EI⁺) m/z calculated for C₂₂H₂₉BrO₃S 452.1; found 453.1 and 455.1 (M + 1), 470.1 and 472.1 (M + NH₄).

(R)-3-(4-(4-Bromophenyl)butyl)-4-hydroxy-5-methyl-5-((E)-2-methylbuta-1,3-dienyl)thiophen-2(5H)-one (TLM 19): General Procedure II

Flash chromatography (20% EtOAc/hexanes) afforded TLM 19 78%. ${[\mathrm{\alpha }]}_D^{23}$ = + 57 (c 1, CH₂Cl₂). ¹H NMR (500 MHz, CDCl₃) δ 1.50–1.64 (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 = 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 = 17.5, 10.5 Hz, 1 H), 7.02–7.04 (m, 2 H), 7.37–7.39 (m, 2 H). ¹³C NMR (125 MHz, CDCl₃) δ 12.0, 21.0, 27.2, 30.2, 31.0, 34.9, 55.0, 114.1, 114.7, 119.3, 128.8–130.1 (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 C₂₀H₂₃BrO₂SH 407.0680; found 407.0670.

(R,E)-Ethyl 2-(5-azido-pentanoylthio)-2,4-dimethylhexa-3,5-dienoate (20a): General Procedure I

Flash chromatography (7% EtOAc/hexanes) afforded 20a 90%. ${[\mathrm{\alpha }]}_D^{23}$ = − 3.6 (c 1, CH₂Cl₂). ¹H NMR (500 MHz, CDCl₃) δ 1.22 (t, J = 7.2 Hz, 3 H), 1.55–1.58 (m, 2 H), 1.67–1.70 (m, 2 H) 1.82, 1.84 (2s, 6 H), 2.46–2.49 (m, 2 H), 3.23 (t, J = 6.5 Hz, 2 H), 4.14–4.19 (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 H). ¹³C NMR (125 MHz, CDCl₃) δ 12.85, 13.8, 25.9 27.8, 42.4, 50.8, 55.2, 61.9, 113.2, 131.13, 131.16, 138.2, 141.0, 172.0, 197.5. MS (EI⁺) m/z calculated C₁₅H₂₃N₃O₃S 325.2; found 326.0 (M + 1).

(R)-3-(3-Azidopropyl)-4-hydroxy-5-methyl-5-((E)-2-methylbuta-1,3-dienyl)thiophen-2(5H)-one (TLM 20): General Procedure II

Flash chromatography (30% EtOAc/hexanes) afforded TLM 20 75%. ${[\mathrm{\alpha }]}_D^{23}$ = + 13.6 (c 1, MeOH). ¹H NMR (500 MHz, CD₃OD): δ 1.21 (t, J = 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), 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). ¹³C NMR (125 MHz, MeOH-d4) 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 C₁₃H₁₇N₃O₂SH 280.1120; found 280.1121.

(2R)-N-((1-(4-((R)-2,5-Dihydro-4-hydroxy-5-methyl-5-((E)-2-methylbuta-1,3-dienyl)-2-oxothiophen-3-yl)butyl)-1H-1,2,3-triazol-4-yl)methyl)-2,4-dihydroxy-3,3-dimethylbutanamide (TLM 21): General Procedure III

Chromatography on silica gel (ISCO combiflash, 4 g gold cartridge, eluting with CH₂Cl₂→ 8% MeOH in CH₂Cl₂) afforded TLM 21 85%. ${[\mathrm{\alpha }]}_D^{23}$ = + 82 (c 1, MeOH). ¹H NMR (400 MHz, CD₃OD): δ 0.92 (br s, 6 H), 1.43 (br s, 2 H), 1.70, 1.78 (2 s, 6 H), 1.86–1.91 (m, 2H), 2.24 (br s, 2 H), 3.38–3.52 (m, 2 H), 3.95 (br s, 1 H), 4.34–4.56 (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, J = 17.6, 10.8 Hz, 1 H), 7.89 (s, 1H). ¹³C NMR (100 MHz, CDCl₃) δ 14.5, 19.8, 21.3, 22.8, 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 + NH₄). HRMS (M + H)⁺ m/z calculated for C₂₃H₃₄N₄O₅SH 479.2328; found 479.2325.

2-((R)-2,5-Dihydro-2-methyl-2-((E)-2-methylbuta-1,3-dienyl)-5-oxothiophen-3-yloxy)- N-(2-azidoethyl)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. tert-butyl 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 × 10 mL) and the combined organics were dried (MgSO₄), filtered and evaporated. The crude material was purified by flash chromatography (5% MeOH/CH₂Cl₂) to obtain a O-alkylated TLM 2 derivative, which was redissolved in CH₂Cl₂ (10 mL). TFA (2.5 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 22a in 90% yield. MS (EI⁻): 253.1 (M − 1). Et₃N (287 μL, 2 mmol) and EDC (230 mg, 1.2 mmol) were added to a solution of 22a (254 mg, 1.0 mmol) and 2-azidoethanamine (103 mg, 1.2 mmol) in DMF (2 mL) at room temperature and stirred overnight. After completion, the reaction mixture was extracted with CH₂Cl₂ (2 × 10 mL) and the combined organic phase was washed with 1 N HCl (10 mL), dried (MgSO₄), filtered and evaporated to obtain the crude material, which was subsequently purified by flash chromatography (1:1, EtOAc/hexanes) to yield TLM 22 70%. ${[\mathrm{\alpha }]}_D^{23}$ = + 131 (c 1, CH₂Cl₂). ¹H NMR (400 MHz, CDCl₃): δ 1.65, 1.79 (2 s, 6 H), 3.34 (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), 6.21 (dd, J = 17.2, 10.8 Hz, 1 H), 7.14 (br s, 1 H), ¹³C NMR (100 MHz, CDCl₃) δ 11.9, 29.4, 38.4, 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: 323.1 (M + 1), 345.1 (M + Na). HRMS (M + H)⁺ m/z calculated for C₁₄H₁₈N₄O₃SH 323.1178; found 323.1179.

tert-Butyl-2-(2-((R)-2,5-dihydro-2-methyl-2-((E)-2-methylbuta-1,3-dienyl)-5-oxothiophen-3-yloxy)acetamido)ethylcarbamate (TLM 23)

Et₃N (287 μL, 2 mmol) and EDC (230 mg, 1.2 mmol) were added to a solution of 22a (254 mg, 1.0 mmol) and tert-butyl 2-azidoethylcarbamate (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 CH₂Cl₂ (2 × 10 mL) and the combined organic phase was washed with 1 N HCl (10 mL), dried (MgSO₄), filtered and evaporated to obtain the crude product, which was then purified by flash chromatography (1:1, EtOAc/hexanes) to yield TLM 23 in 66% yield. ${[\mathrm{\alpha }]}_D^{23}$ = − 116 (c 1, CH₂Cl₂). ¹H NMR (500 MHz, CDCl₃): δ 1.37 (s, 9 H), 1.73, 1.89 (2 s, 6 H), 3.19–3.23 (m, 2 H), 3.31–3.42 (m, 2 H), 4.43 (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), 6.30 (dd, J = 17.5, 11.0 Hz, 1 H), 7.18 (br s, 1 H), ¹³C NMR (125 MHz, CDCl₃) δ 28.1, 29.5, 39.9, 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 C₁₉H₂₈N₂O₅SH 397.1797; found 397.1804.

(2R)-N-((1-(2-(2-((R)-2,5-Dihydro-2-methyl-2-((E)-2-methylbuta-1,3-dienyl)-5-oxothiophen-3-yloxy)acetamido)ethyl)-1H-1,2,3-triazol-4-yl)methyl)-2,4-dihydroxy-3,3-dimethylbutanamide (TLM 24): General Procedure III

Chromatography on silica gel (ISCO combiflash, 4 g gold cartridge, eluting with CH₂Cl₂→ 7% MeOH inCH₂Cl₂) afforded TLM 24 in 80% yield. ${[\mathrm{\alpha }]}_D^{23}$ = + 94 (c 1, MeOH). ¹H NMR (500 MHz, CD₃OD, 10% CDCl₃): δ 1.05 (s, 6 H), 1.89, 2.01 (2 s, 6 H), 3.40–3.49 (m, 2 H), 3.73–3.77 (m, 1 H), 3.95 (s, 1 H), 4.45–4.59 (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, J = 17.5, 10.75 Hz, 1 H), 7.78 (br s, 1 H), 8.30 (s, 1 H). ¹³C NMR (125 MHz, CD₃OD, 10% CDCl₃) 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, 123.2, 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 C₂₃H₃₃N₅O₆SH 508.2230; found 508.2230.

N-((1-(2-(2-((R)-2,5-Dihydro-2-methyl-2-((E)-2-methylbuta-1,3-dienyl)-5-oxothiophen-3-yloxy)acetamido)ethyl)-1H-1,2,3-triazol-4-yl)methyl)-4-((4-fluorophenylsulfonyl)-amino)benzamide (TLM 25): General Procedure III

Chromatography on silica gel (ISCO combiflash, 4 g gold cartridge, eluting with CH₂Cl₂→ 7% MeOH in CH₂Cl₂) afforded TLM 25 in 88% yield. ${[\mathrm{\alpha }]}_D^{23}$ = + 71 (c 1, MeOH). ¹H NMR (500 MHz, CD₃OD, 10% CDCl₃): δ 1.75, 1.85 (2 s, 6 H), 3.72–3.76 (m, 2 H), 4.50–4.58 (m, 8 H), 5.07 (d, J = 10.5 Hz, 1 H), 5.24 (d, J = 17.5 Hz, 1 H), 5.41 (s, 1 H), 5.71 (s, 1 H), 6.30 (d, J = 17.5, 10.5 Hz, 1 H), 7.13–7.19 (m, 4 H), 7.73 (m, 1 H), 7.83–7.85 (m, 3 H), 8.05 (s, 1 H). ¹³C NMR (125 MHz, CD₃OD, 10% CDCl₃) δ 12.8, 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: 655.2 (M + 1). HRMS (M + H)⁺ m/z calculated for C₃₀H₃₁FN₆O₆S₂H 655.1809; found 655.1812.

(R)-3-(6-Azidohexanoyl)-4-hydroxy-5-methyl-5-((E)-2-methylbuta-1,3-dienyl)thiophen-2(5H)-one (TLM 26): General Procedure IV

Flash chromatography (30% EtOAc/hexanes/1% AcOH) afforded TLM 26 85%. ${[\mathrm{\alpha }]}_D^{23}$ = + 183 (c 1, MeOH). ¹H NMR (500 MHz, CDCl₃) δ 1.37–1.41 (m, 2 H), 1.56–1.65 (m, 4 H), 1.68, 1.72 (2 s, 6 H), 2.72–2.90 (m, 2 H), 3.23–2.28 (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 H). ¹³C NMR (100 MHz, CDCl₃, peaks broadening was observed due to keto-enol 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 + NH₄). HRMS (M + H)⁺ m/z calculated for C₁₆H₂₁N₃O₃SH 336.1382; found 336.1375.

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. Air- and moisture-sensitive reactions were run under inert atmosphere (N₂ or Ar) using flame-dried glassware. ¹H and ¹³C NMR spectra were acquired on 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 (δ = 0) and/or relative to residual solvents such as CHCl₃ (7.26 ppm, ¹H; 77.0 ppm, ¹³C), MeOH-d₄ (3.31 ppm, ¹H; 49.1 ppm, ¹³C), or acetone-d₆ (2.04 ppm, ¹H; 29.9 ppm, ¹³C). Splitting patterns 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 pre-coated 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, 230–400 mesh) and/or an ISCO Combiflash instrument using ISCO redisep flash cartridges (particle size: 35–70 μm). Measurements of optical rotations were performed using a Perkin Elmer 241 automatic digital polarimeter. Mass spectra were obtained using electrospray (ES) ionization techniques. High-resolution mass spectra were obtained from the mass spectrometry laboratory at the University of Illinois, Urbana Champaign, Urbana, IL. TLM and TLM 2 analytical data was agreement with the reported data.^{44, 47} All the C3 acylated (TLM 5–11 and 26) compounds showed extended keto-enol tautomerism,⁵⁵ and peak broadening was generally observed in ¹³C NMR Spectrum. The purity of all final compounds was determined to be ≥95% by reverse phase HPLC (Agilent HPLC, C-18 column, with MeOH/H₂O as the mobile phase).

Cloning and protein purification

The S. aureus fabF gene was cloned into a pET23a vector using KpnI and EcoRI restriction sites. The intrinsic NdeI cut site was removed using Quikchange mutagenesis and subsequently the gene was cloned into pET15b between NdeI and BamHI sites. Due to problems in obtaining soluble protein with the original construct, the I5K mutation was introduced into the NdeI cloning primer. Subsequently, the C165Q mutation was introduced using Quikchange mutagenesis. The primers used in this study are described in Table S1. The plasmids encoding mtFabB (pFPCA)³² and saFabF were transformed into electro-competent M. smegmatis MC²155 and chemically competent E. coli BL21 (DE3) PlysS cell lines, respectively. The proteins were purified using standard affinity chromatography as described previously.^{32, 33} Briefly, the cells were resuspended in 40 mL binding buffer (20 mM Tris-HCl, 800 mM NaCl, 5 mM imidazole, pH 7.9) and lysed by sonication. The cellular debris was removed by ultracentrifugation at 40,000 rpm for 1 h at 4 °C, after which the supernatant was filtered and loaded onto a His-bind column. The column was then washed with 40 mL binding buffer, 80 mL wash buffer (20 mM Tris-HCl, 150 mM NaCl, 60 mM imidazole, pH 7.9) and subsequently eluted (20 mM Tris-HCl, 800 mM NaCl, 1 M imidazole, pH 7.9). The fractions containing protein were pooled and loaded onto a Superdex 200 column equilibrated with 30 mM Tris-HCl, 150 mM NaCl, pH 8.5. The purified proteins were analyzed with SDS-PAGE.

Direct Binding experiments

The binding kinetics and thermodynamics of select TLM analogs were conducted by monitoring quenching of intrinsic tryptophan fluorescence of mtFabB, C171Q-mtFabB, saFabF and C165Q-saFabF as described previously.³³

MIC determination

Protocols followed for MIC determinations for B. pseudomallei, Y. pestis, F. tularensis, S. aureus (MSSA, MRSA), and K. pneumoniae were similar except for the growth media. Compounds were added to the 96-well plate starting at 512 μg/mL in the first column and serially diluted 1:2 to column 12 for a final concentration of 0.25 μg/mL in CAMHB (or MMH for F. tularensis). MIC plates were incubated at 37°C for 18–24 h at which time 10 μL of Alamar Blue (Invitrogen, Carlsbad, California) was added to each well and plates were incubated for an additional 4 h. Reduction of Alamar Blue was determined by absorbance readings at wavelengths of 570 nm and 600 nm using a microplate reader (Biotek, Winooski, VT). Percent growth reduction was calculated and MIC₉₀ values were determined by plotting the percentage inhibition calculated from spectrophotometric readings over the drug concentration series. Bacterial growth and MIC values were confirmed by optical density. Non-linear regression analysis was run on % growth inhibition curves to determine MIC₉₀ values.

A modified 96-well Alamar blue assay (MABA) used for the generation of MICs for M. tuberculosis H37Rv has been described previously.⁵⁶ Briefly, M. tuberculosis was grown to early-mid log phase in Middlebrook 7H9 media supplemented with 10% OADC and 0.05% Tween 80. Compounds were serially diluted 1:2 in 96 well microtiter plates. Bacteria were added to the plates and grown at 37°C for 6 days, Alamar blue was added and the plates were incubated for an additional 24 h. The MIC was the drug concentration that inhibited visible growth and the reduction of Alamar blue in all the replicates.

In vivo studies

In vivo efficacy of TLM analogues was first investigated against a systemic infection model. Six-week old, pathogen free, male Swiss Webster mice weighing 27 to 30 g were used in this study. Animals were housed in the Division of Laboratory Animal Resources (DLAR) at Stony Brook University under BSL2 conditions and were provided ad libitum access to food and water through the entire experimental course. The animal study was approved by the Institutional Animal Care and Use Committee (IACUC) at Stony Brook University.

Methicillin resistant Staphylococcus aureus systemic infection model

The mice were rendered neutropenic by injecting cyclophosphamide 4 days and 1 day prior to MRSA infection. MRSA strain BAA 1762 was cultivated in Muller Hilton Broth to late exponential phase after which the bacteria were harvested and resuspended in Brain Heart Infusion (BHI) broth to give a concentration of 5×10⁷ cells/mL. Systemic infection was initiated by injecting 0.5 mL of bacterial inoculum intraperitoneally. TLM analogues were dissolved in water/ethanol while vancomycin, as a positive control, was dissolved in water. Drug deliveries were given by subcutaneous injection 1 h and 25 h post infection with the dosage of 100 mg/kg/inj. The animals had access to food and water during the whole experiment. Survival was assessed every 12 h post infection until the end of the study.

Klebsiella pneumoniae systemic infection model

A starting culture of K. pneumoniae KPC2ST258 was incubated in MH II broth to mid log phase (~10⁸ cfu/mL). Bacterial cells were harvested by centrifugation at 11,000 rpm for 3 min. The inoculums were prepared by diluting cell pellets with freshly sterilized brain heart infusion (BHI) broth to 5×10⁷ cfu/mL. Systemic infection was initiated by administering 100 μL of inoculum (5×10⁶ cfu) via intraperitoneal (IP) injection. Infected animals received either TLM-analogues or clinical antibiotics. All drugs were dissolved in the same vehicle which consisted of saline/PEG400/ethanol (40 / 20 / 40). Drugs were administered 1 h, 12 h and 24 h post infection via subcutaneous (SC) injection of 200 μL containing 100 mg/kg/inj. Animals were monitored every 12 h following infection for 7 days. Dead animals were removed from the study immediately. Mice surviving at the end of the experiment were euthanized by CO₂ inhalation as recommended by the American Veterinary Medical Association (AVMA). Survival curves were plotted based on the day that dead animals were discovered. Average survival time of each group was calculated.

Toxicity Studies

THP1, a human monocytic cell line cell line, was cultured in RPMI 1640 media supplemented with 10% FBS, 2 mM L-Glutamine, 1 mM sodium pyruvate, penicillin (100 units/mL) and streptomycin (100 μg/mL).at 37 °C, 5% CO₂. The assay was performed in a 96 well plate format with a seeding density of 6.25 × 10⁴ cells /well. The compounds were added at a starting concentration of 100 μg/mL and diluted 1:2 until 12.5 μg/mL. serially diluted 1:2 in 96 well microtiter plates. The cells were allowed to grow for 2 days. Alamar blue was added and the plates were incubated for an additional 24 hours. The LD₅₀ was monitored based on reduction of Alamar blue in all the replicates.

ABBREVIATIONS

FASII

Fatty acid biosynthesis type II pathway

KAS

β-Ketoacyl-ACP synthase

ACP

acyl carrier protein

CoA

Coenzyme A

TLM

thiolactomycin

MIC

minimum inhibitory concentration

MSSA

methicillin sensitive S. aureus

MRSA

methicillin resistant S. aureus