Structure–Activity Relationship of Biakamide, Selective Growth Inhibitors under Nutrient-Starved Condition from Marine Sponge

Abstract

The tumor microenvironment is considered as one of the important targets for anticancer drug discovery. In particular, nutrient deficiency may be observed in tumor microenvironment; biakamides A–D (1–4) isolated from marine sponge Petrosaspongia sp. as growth inhibitors against cancer cells adapted to glucose-deprived conditions have potential as new drugs and tools for elucidating adaptation mechanisms to these conditions. In this paper, we investigated structure–activity relationship (SAR) of biakamide to create easily accessible analog and gain insights about participation of the substructures to growth–inhibitory activity toward development of anticancer drug. This work revealed that 14,15-dinor-biakamide C (5), which is easily accessible, has similar activity to natural biakamide C (3). In addition, detailed SAR study showed the terminal acyl chain is important for interacting with target molecule and amide part including thiazole ring has acceptability to convert structures without losing activity.

Introduction

Solid tumors are physically heterogeneous tissues, and include a tumor microenvironment. Because of insufficient neovascularization, the tumor microenvironment has limited supply of oxygen and nutrients. Some cancer cells such as pancreatic cancer are known to adapt to these severe conditions and grow, together with acquiring resistance to cancer chemotherapies and radiotherapy.^1,2) Therefore selective growth inhibitors against cancer cells under nutrient-starved conditions have potential as anticancer drugs with novel mode of action, and as chemical tools for elucidating adaptation mechanisms to the conditions. Indeed, kigamycin D and arctigenin were discovered as selective growth inhibitors against cancer cells under nutrient-starved conditions,^3,4) and GBS-01, which is an arctigenin-rich extract from the fruit of Arctium lappa L., is undergoing phase II clinical trial for advanced pancreatic cancer refractory to gemcitabine.⁵⁾

We constructed a new bioassay system to search for selective growth inhibitors under nutrient-starved conditions.^6,7) We considered glucose-deprived conditions as nutrient-starved conditions in tumor microenvironment. On the guidance of this bioassay system, we have recently isolated novel polyketides named biakamides A–D (1–4, Fig. 1). Biakamides exhibit potent growth–inhibitory activity against human pancreatic cancer PANC-1 cells under glucose-deprived conditions with IC₅₀ values 0.5–4.0 µM, whereas they show weak activity under conditions containing glucose with IC₅₀ values of more than 30 µM.⁸⁾ Unfortunately, it is difficult to gain adequate amounts of these compounds from natural resources because biakamides were isolated from marine sponge which cannot be stably collected. Hence synthetic supply of biakamides is needed to investigate the potencies of the compounds and to develop them as anti-cancer drug candidates. In most cases to solve these problems, structural simplification of natural products with retained activity is of strong means. In addition, investigations on participation of the substructures of biakamide in their growth–inhibitory activity are important for developing potent drug candidates. The present study also led us to prepare a useful affinity probe to garner information about binding mode between the compound and its target molecule(s). Therefore we engaged in structure–activity relationship (SAR) study of biakamides to create easily accessible analogs and investigate the importance of the substructures to their growth–inhibitory activity. Herein, we present details of our study on syntheses and evaluations of various analogs of biakamides.

Fig. 1. Chemical Structures and Bioactivities of Biakamides

Results and Discussion

Participation of Stereochemistries of Biakamide at C-4 and C-6 Positions in Growth–Inhibitory Activity

In our previous work, all the stereoisomers at C-4 and C-6 positions of biakamide A–D (1–4) were synthesized to determine their absolute structures. As the first step of SAR study, we evaluated these stereoisomers of biakamides to investigate the effects of stereochemistries at C-4 and C-6 to the selective growth–inhibitory activity under glucose-deprived conditions. All isomers showed similar activities to natural products against PANC-1 cells under glucose-deprived condition (Fig. 2), implying that stereochemistries of biakamides at C-4 and C-6 are not important for their activity.

Fig. 2. IC₅₀ Values of Natural Products and Stereoisomers of Biakamides

We next decided to prepare 14,15-dinor-analogs such as 5 and 6, which are easily accessible, in hope that they possess similar activity. The synthetic route of 5 and 6 was based on the total synthesis by using 1,7-heptanediol (7) as starting material (Chart 1). Monosilylation of two hydroxyl groups of the diol 7 and subsequent oxidation using 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) gave an aldehyde 9.⁹⁾ Treatment of 9 with 1,3-propanedithiol and catalytic amount of iodine provided a 1,3-dithiane 10. The Corey–Seebach coupling reaction between a carbanion generated by the n-BuLi treatment of 10 and alkyl iodide 18, followed by treatment with tetra-n-butylammonium fluoride (TBAF), afforded the desired alcohol 11. Oxidation of primary hydroxyl group of 11 using tetrapropylammonium perruthenate (TPAP) gave an aldehyde 12 and subsequent Wittig homologation with a stabilized ylide 19 yielded an α,β-unsaturated ester 13. After hydrolysis of 13 with lithium hydroxide, condensation with a secondary amine 20 yielded an amide 15. Then, iodine treatment of 15 under basic conditions resulted in clean removal of 1,3-dithiane to give a ketone 16, and subsequent Wittig reaction proceeded smoothly to yield vinyl chloride 17. Finally, removal of tert-butoxycarbonyl (Boc) group and condensation with known carboxylic acid 21 afforded the desired analogs 5 and 6. These geometrical isomers could be separated by SiO₂ column chromatography. After complete purification by reversed-phase HPLC, we evaluated the activities of these analogs. As expected, compound 5 exhibited almost the same activity against PANC-1 cells under glucose-deprived conditions as that of natural biakamide C. (Growth–inhibitory activities of all synthesized analogs are summarized in Table 1.)

Chart 1. Synthesis of Analogs 5 and 6

Reagents and conditions: (a) TBDPSCl, Et₃N, CH₃CN/hexane, 60%; (b) TEMPO, PhI(OAc)₂, CH₂Cl₂, 91%; (c) 1,3-Propanedithiol, I₂, CHCl₃, quant.; (d) 18, n-BuLi, THF; (e) TBAF, THF, 70% (2 steps); (f) TPAP, NMO, MS4A, CH₂Cl₂, 70%; (g) 19, toluene, 100°C, 99%; (h) LiOH, THF/MeOH/H₂O, 99%; (i) 20, EDCI·HCl, HOBt, Et₃N, CH₂Cl₂, 78%; (j) I₂, NaHCO₃ aq., CH₃CN, 0°C, 89%; (k) (Chloromethyl)triphenylphosphonium chloride, LHMDS, THF, quant.; (l) TFA, CH₂Cl₂; (m) 21, EDCI·HCl, HOBt, Et₃N, CH₂Cl₂, 88% (2 steps).

Table 1. IC₅₀ Values of Synthetic Analogs and Natural Products*

Compound	IC50 (Glc 0 mM)
3	0.5 µM
4	0.5 µM
5	0.6 µM
6	1.5 µM
29	1.5 µM
31	3.0 µM
35	1.0 µM
40	2.5 µM
45	1.0 µM
48	10 µM
55	>100 µM
59	>100 µM

*In the medium containing 25 mM Glc (glucose), all analogs showed weak growth–inhibitory activity (IC₅₀: >30 µM).

Participation of Vinyl Chloride Moiety in Growth–inhibitory Activity

Since compound 5, 14,15-dinor-analog of biakamide C, was found to possess potent growth–inhibitory activity, next we conducted detailed SAR study through syntheses and evaluations of various analogs based on compound 5 as a scaffold.

At first, we examined the participation of vinyl chloride moiety. The primary hydroxyl group of N-Boc-10-amino-1-decanol (22)¹⁰⁾ was protected by tert-butyldimethylsilyl (TBS) ether, and subsequent methylation proceeded smoothly to give compound 24 having N-methyl group (Chart 2). TBAF treatment of 24 and one-pot TEMPO oxidation/Wittig reaction afforded α,β-unsaturated ester 26. In the same way as described above in the synthesis of 5, ternary amide and terminal acyl chain were successively introduced to give the desired analog 29, which does not have a vinyl chloride moiety. The bioactivity of analog 29, with IC₅₀ value of 1.5 µM, showed that the vinyl chloride moiety is not necessary for its activity.

Chart 2. Synthesis of Analog 29

Reagents and conditions: (a) TBSCl, Et₃N, CH₂Cl₂, 70%; (b) CH₃I, NaH, DMF, 92%; (c) TBAF, THF, 95%; (d) TPAP, NMO, MS4A, CH₂Cl₂; (e) 19, toluene, 100°C, 90% (2 steps); (f) LiOH, THF/MeOH/H₂O, 97%; (g) 20, EDCI·HCl, HOBt, Et₃N, CH₂Cl₂, 88%; (h) TFA, CH₂Cl₂; (i) 21, EDCI·HCl, HOBt, Et₃N, CH₂Cl₂, 68% (2 steps).

We next tried introduction of polar group to expect further conversion at the vinyl chloride moiety for target identification study. Condensation between ketone 16 and hydroxylamine hydrochloride, and subsequent introduction of a terminal acyl chain gave an oxime analog 31, albeit in low yield (Chart 3). Unfortunately, the analog 31 showed weak activity with five-fold IC₅₀ value over analog 5. Considering the result, we found that it might be difficult to synthesize useful analogs through conversion of the vinyl chloride moiety.

Chart 3. Synthesis of Analog 31 with Oxime Group

Reagents and conditions: (a) Hydroxylamine hydrochloride, AcONa, MeOH, 83%; (b) TFA, CH₂Cl₂; (c) 21, EDCI·HCl, HOBt, Et₃N, CH₂Cl₂, 34%.

Participation of Amide Including Thiazole Ring in Growth–Inhibitory Activity

We next examined the participation of an amide including thiazole ring. Because biakamides give characteristic broadened signals in their ¹H-NMR spectra, originating from the restricted bond rotation according to ternary amide, we decided to synthesize secondary amide analog so as to investigate the effect of the bond rotation threshold around the ternary amide in its activity. Condensation between unsaturated carboxylic acid 14 and primary amine 37 yielded secondary amide 32 (Chart 4). By analogy with the synthesis of 5, the desired secondary amide analog 35 was prepared. Furthermore, ethyl ester analog 40 as a simpler analog was also synthesized (Chart 5). Contrary to our expectation, both 35 and 40 showed similar activity with that of 5. These results indicate this amide part including thiazole ring can convert to various functional groups with retained activity. Finally, we introduced naphthalene to the position of thiazole ring as a sterically hindered group (Chart 6). The naphthalene analog 45 also showed similar activity with analog 5. Therefore we considered that this part has acceptability to convert the substructures without losing bioactivity.

Chart 4. Synthesis of 35

Reagents and conditions: (a) 37, EDCI·HCl, HOBt, Et₃N, CH₂Cl₂, 80%; (b) I₂, NaHCO₃ aq., CH₃CN, 0°C, 40%; (c) (Chloromethyl)triphenylphosphonium chloride, LHMDS, THF, 79%; (d) TFA, CH₂Cl₂; (e) 21, EDCI·HCl, HOBt, Et₃N, CH₂Cl₂, 97% (2 steps).

Chart 5. Synthesis of Analog 40

Reagents and conditions: (a) I₂, NaHCO₃ aq., CH₃CN, 0°C, 62%; (b) (Chloromethyl)triphenylphosphonium chloride, LHMDS, THF, 75%; (c) TFA, CH₂Cl₂; (d) 21, EDCI·HCl, HOBt, Et₃N, CH₂Cl₂, 72% (2 steps).

Chart 6. Synthesis of Analog 45

Reagents and conditions: (a) 47, EDCI·HCl, HOBt, Et₃N, CH₂Cl₂, 91%; (b) I₂, NaHCO₃ aq., CH₃CN, 0°C, 57%; (c) (Chloromethyl)triphenylphosphonium chloride, LHMDS, THF, 70%; (d) TFA, CH₂Cl₂; (e) 21, EDCI·HCl, HOBt, Et₃N, CH₂Cl₂, 26% (2 steps).

Participation of Terminal Acyl Chain and Whole Structure in Growth–Inhibitory Activity

Finally, we examined the effects of terminal acyl chain. After removal of Boc moiety of 17, acetylation afforded 48 as a simple acetamide analog (Chart 7). Analog 48 showed obviously decreased growth–inhibitory activity compared with that of 5. This implies that the terminal acyl chain might be the pharmacophore. Therefore we divided analog 5 into two fragments, 54 and 58, to confirm only the upper half of the structure is needed to exhibit activity (Chart 8). However, both 54 and 58 showed no activity. Thus the whole structure of biakamide is necessary for its activity, and terminal acyl chain is important for interacting with target molecule.

Chart 7. Synthesis of 48

Reagents and conditions: (a) TFA, CH₂Cl₂; (b) Acetyl chloride, DMAP, Et₃N, CH₂Cl₂, quant. (2 steps).

Chart 8. Synthesis of Fragments of 55 and 59

Reagents and conditions: (a) MeONHMe·HCl, EDCI·HCl, HOBt, Et₃N, CH₂Cl₂, 89%; (b) CH₃I, NaH, DMF, 91%; (c) CH₃Li, Et₂O, 75%; (d) (Chloromethyl)triphenylphosphonium chloride, LHMDS, THF, quant.; (e) TFA, CH₂Cl₂; (f) 21, EDCI·HCl, HOBt, Et₃N, CH₂Cl₂, 25% (2 steps); (g) LiOH, THF/MeOH/H₂O, 84%; (h) 20, EDCI·HCl, HOBt, Et₃N, CH₂Cl₂, 91%.

Conclusion

We synthesized analogs of biakamide, which have selective growth–inhibitory activities, and evaluated their activities. We created an easily accessible analog 5, which showed almost the same activity with natural biakamide C (Fig. 3). In addition, SAR study based on analog 5 as scaffold revealed that the whole structure of biakamides is necessary, and terminal acyl chain is important for interacting with target molecule. On the other hand, the amide part including thiazole ring has broad acceptability, suggesting creation of an affinity probe for identification of target molecule of biakamides through conversion of the substructure around the part. Synthesis of suitable analogs and probes based on this SAR study and mechanistic analysis using them are now ongoing.

Fig. 3. Structure–Activity Relationship of Biakamide

Experimental

General

The following instruments were used to obtain physical data: Agilent NMR system (¹H-NMR: 600 MHz, ¹³C-NMR: 150 MHz) spectrometer for ¹H- and ¹³C-NMR data using tetramethylsilane or solvent residual peak (CHCl₃: δ 7.26) as internal standards; a JASCO FT/IR-5300 IR spectrometer for IR spectra; a JEOL JMS-S3000 mass spectrometer for matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) MS; and a Waters Q-Tof Ultima API mass spectrometer for electrospray ionization (ESI)-TOF MS. Silica gel (Kanto, 40–100 µm) and precoated TLC plates (Merck, 60F₂₅₄) were used for column chromatography and TLC. Spots on TLC plates were detected by spraying acidic p-anisaldehyde solution (p-anisaldehyde: 25 mL, c-H₂SO₄: 25 mL, AcOH: 5 mL, EtOH: 425 mL) or phosphomolybdic acid solution (phosphomolybdic acid: 25 g, EtOH: 500 mL) with subsequent heating. Cell culture and assay for growth–inhibitory activity were performed with a previously reported method.^6–8)

Unless otherwise noted, the entire reaction was performed under a N₂ atmosphere. After workup, the organic layer was dried over Na₂SO₄. Perfect separation of geometrical isomers of biakamide analog at vinyl chloride moiety proved difficult. Therefore the yields of the respective reactions are shown as a mixture of geometrical isomers. The parts of purified compound were used in the assay for growth–inhibitory activity and measurement of physical data.

All compounds used in the assay for growth–inhibitory activity of cancer cells were finally purified by HPLC (Cosmosil 5C₁₈-MS-II, 10 mm i.d. × 250 mm).

Chemistry

7-((tert-Butyldiphenylsilyl)oxy)heptan-1-ol (8)

Et₃N (1.6 mL, 11 mmol, 1.2 equiv.) and tert-butylchlorodiphenylsilane (TBDPSCl, 2.5 mL, 11 mmol, 1.2 equiv.) were added to a solution of 7 (1.24 g, 9.4 mmol) in CH₃CN : hexane (1 : 3, 28 mL), and the mixture was stirred at room temperature (r.t.) for 8 h. The reaction was quenched by addition of sat. NaHCO₃ aq., and the whole mixture was extracted with AcOEt. Concentration of the AcOEt extract in vacuo gave a crude product which was purified by SiO₂ column chromatography (hexane : AcOEt = 5 : 1 to 1 : 1) to give 8 (1.92 g, 60%) as a colorless oil. The spectroscopic and physical data were identical to those reported.⁹⁾

7-((tert-Butyldiphenylsilyl)oxy)heptanal (9)

Iodobenzene diacetate (1.75 g, 5.4 mmol, 1.2 equiv.) and TEMPO (73.8 mg, 0.47 mmol, 0.1 equiv.) were added to a solution of 8 (1.75 g, 4.7 mmol) in CH₂Cl₂ (30 mL), and the mixture was stirred for 3.5 h at r.t. The reaction was quenched by the addition of Na₂S₂O₃/NaHCO₃ aq., and the whole mixture was extracted with AcOEt. The AcOEt extract was successively washed with sat. NH₄Cl aq., NaHCO₃ aq., and brine. Concentration of the AcOEt extract in vacuo gave a crude product which was purified by SiO₂ column chromatography (hexane : AcOEt = 6 : 1) to give 9 (1.59 g, 91%) as a colorless oil. The spectroscopic and physical data were identical to those reported.⁹⁾

((6-(1,3-Dithian-2-yl)hexyl)oxy)(tert-butyl)diphenylsilane (10)

1,3-Propanedithiol (89 µL, 0.87 mmol, 1.3 equiv.) and iodine (17 mg, 0.067 mmol, 0.10 equiv.) were added to a solution of 9 (226 mg, 0.57 mmol) in CHCl₃ (10 mL), and the mixture was stirred at r.t. for 0.5 h. The reaction was quenched by the addition of Na₂S₂O₃/NaHCO₃ aq., and the whole mixture was extracted with AcOEt. The AcOEt extract was washed with Na₂S₂O₃/NaHCO₃ aq. and brine. Concentration of the AcOEt extract in vacuo gave a crude produt, which was purified by SiO₂ column chromatography (Hexane : AcOEt = 50 : 1) to give 10 (311 mg, quant.) as a colorless oil.

¹H-NMR (600 MHz, CDCl₃) δ: 1.05 (9H, s), 1.13–1.43 (4H, m), 1.43–1.66 (4H, m), 1.73 (2H, dd, J = 15.5, 7.1 Hz), 1.86 (1H, ddd, J = 14.3, 11.6, 8.3 Hz), 2.05–2.20 (1H, m), 2.77–2.93 (4H, m), 3.65 (2H, t, J = 6.5 Hz), 4.04 (1H, t, J = 6.9 Hz), 7.40 (6H, m), 7.67 (4H, d, J = 6.7 Hz). ¹³C-NMR (150 MHz, CDCl₃) δ: 19.4, 25.7, 26.2, 26.8, 27.0, 29.1, 30.6, 30.7, 32.6, 35.6, 47.8, 64.0, 127.7 (4C), 129.6 (2C), 134.2 (2C), 135.7 (4C). IR (KBr) cm⁻¹: 3070, 2931, 1427, 1109. MS (ESI-TOF) m/z: 481 [M + Na]⁺. High resolution (HR)-MS (ESI-TOF) m/z: 481.2031 calcd for C₂₆H₃₈OSiS₂Na; Found: 481.2020.

tert-Butyl (3-(2-(6-Hydroxyhexyl)-1,3-dithian-2-yl)propyl)(methyl)carbamate (11)

n-BuLi (1.59 M in n-hexane, 1.4 mL, 2.3 mmol, 1.5 equiv.) was added to a solution of 10 (745 mg, 1.5 mmol) in anhydrous tetrahydrofuran (THF, 20 mL), and the mixture was stirred for 20 min. A solution of 18 (710 mg, 2.4 mmol, 1.6 equiv.) in anhydrous THF (5 mL) was added dropwise to the organolithium solution via cannula at r.t. over 10 min, and the resulting mixture was stirred at r.t. for 0.5 h. Then, the reaction was quenched by the addition of H₂O, and the whole mixture was extracted with AcOEt. The AcOEt extract was washed with brine. Concentration of the AcOEt extract in vacuo, the residue was dissolved in THF (10 mL). TBAF solution (1 M in THF, 2.6 mL) was added to the solution at r.t., and the mixture was stirred at r.t. for 8 h. The reaction was quenched by the addition of sat. NH₄Cl aq., and the whole mixture was extracted with AcOEt, and the AcOEt extract was washed with sat. NH₄Cl aq. and brine. Concentration of the AcOEt extract in vacuo gave a crude product, which was purified by SiO₂ column chromatography (Hexane : AcOEt = 3 : 1) to give 11 (533 mg, 69%, 2 steps) as a colorless oil.

¹H-NMR (600 MHz, CDCl₃) δ: 1.02–1.36 (15H, m), 1.40 (2H, br s), 1.48 (2H, br s), 1.68 (4H, br s), 1.78 (2H, br s), 2.63 (4H, br s), 2.69 (3H, br s), 2.89 (1H, s), 3.07 (2H, br s), 3.44 (2H, dd, J = 11.5, 4.9 Hz). ¹³C-NMR (150 MHz, CDCl₃) δ: 22.2, 22.6, 23.6, 23.7, 25.2, 25.4, 25.7, 28.2, 29.3, 32.4, 33.9, 34.5, 34.7, 38.0, 47.8, 48.6, 52.7, 62.1, 79.1, 155.6. IR (KBr) cm⁻¹: 3442 (br), 2935, 1679, 1397, 1153, 756. MS (ESI-TOF) m/z: 414 [M + Na]⁺. HR-MS (ESI-TOF) m/z: 414.2113 calcd for C₁₉H₃₇NO₃S₂Na; Found: 414.2113.

tert-Butyl Methyl(3-(2-(6-oxohexyl)-1,3-dithian-2-yl)propyl)carbamate (12)

Molecular sieves 4A (100 mg) and N-methylmorpholine N-oxide (NMO, 47 mg, 0.40 mmol, 1.5 equiv.) were added to a solution of 11 (113 mg, 0.27 mmol) in CH₂Cl₂ (4 mL), and the mixture was stirred at r.t. for 20 min. Then, TPAP (4.7 mg, 0.013 mmol, 0.050 equiv.) was added to the solution, and the resulting mixture was stirred at r.t. for 40 min. The reaction was quenched by the addition of Na₂S₂O₃/NaHCO₃ aq., and the whole mixture was extracted with AcOEt. The AcOEt extract was washed with sat. NH₄Cl aq. and brine. Concentration of the AcOEt extract in vacuo gave a crude product which was purified by SiO₂ column chromatography (hexane : AcOEt = 5 : 1) to give 12 (78 mg, 70%) as a colorless oil.

¹H-NMR (600 MHz, CDCl₃) δ: 1.28 (2H, m), 1.38 (11H, m), 1.57 (4H, m), 1.76 (4H, br s), 1.86 (2H, br), 2.37 (2H, t, J = 7.2 Hz), 2.74 (7H, m), 3.15 (2H, br s), 9.69 (1H, s). ¹³C-NMR (150 MHz, CDCl₃) δ: 11.9, 13.5, 13.8, 15.1, 20.1, 22.3, 23.2, 24.9, 25.4, 27.9, 28.0, 28.9, 33.5, 34.5, 37.8, 47.4, 48.2, 52.3, 59.7, 60.3, 78.4, 127.2, 141.3, 155.0, 167.2. IR (KBr) cm⁻¹: 2934, 1692, 1393, 1148. MS (ESI-TOF) m/z: 412 [M + Na]⁺. HR-MS (ESI-TOF) m/z: 412.1956 calcd for C₁₉H₃₅NO₃S₂Na; Found: 412.1938.

Ethyl (E)-8-(2-(3-((tert-Butoxycarbonyl)(methyl)amino)propyl)-1,3-dithian-2-yl)-2-methyloct-2-enoate (13)

Methyl 2-(triphenyl-λ⁵-phosphanylidene)acetate (19, 298 mg, 0.82 mmol, 3.0 equiv.) was added to a solution of 12 (107 mg, 0.27 mmol) in toluene (8 mL), and the mixture stirred at 100°C for 10 h. Then, the reaction was quenched by the addition of sat.NH₄Cl aq. The whole mixture was extracted with AcOEt, and the AcOEt extract was washed with NH₄Cl aq. and brine. Concentration of the AcOEt extract in vacuo gave a crude product which was purified by SiO₂ column chromatography (hexane : AcOEt = 4 : 1) to give 13 (128 mg, 99%) as a colorless oil.

¹H-NMR (600 MHz, CDCl₃) δ: 0.85–1.09 (5H, m), 1.17 (11H, m), 1.36 (2H, br s), 1.45–1.72 (9H, m), 1.90 (2H, br s), 2.42–2.60 (7H, m), 2.95 (2H, br s), 3.77–3.96 (2H, dd, J = 15.5, 6.5 Hz), 6.45 (1H, br s). ¹³C-NMR (150 MHz, CDCl₃) δ: 11.8, 13.5, 13.8, 15.1, 20.1, 22.3, 23.3, 24.9, 25.4, 27.1, 27.9, 28.0, 28.7, 28.8, 28.9, 33.5, 34.5, 37.8, 47.5, 48.2, 52.3, 59.3, 59.7, 60.3, 78.4, 127.2, 132.6, 141.0, 141.3, 142.2, 155.0, 167.2. IR (KBr) cm⁻¹: 2935, 1698, 1393, 1274, 1150. MS (ESI-TOF) m/z: 496 [M + Na]⁺. HR-MS (ESI-TOF) m/z: 496.2531 calcd for C₂₄H₄₃NO₄S₂Na; Found: 496.2554.

(E)-8-(2-(3-((tert-Butoxycarbonyl)(methyl)amino)propyl)-1,3-dithian-2-yl)-2-methyloct-2-enoic Acid (14)

LiOH (23 mg, 0.95 mmol, 5.0 equiv.) was added to a solution of 13 (90 mg, 0.19 mmol) in THF : MeOH : H₂O (1 : 1 : 1, 6 mL), and the mixture was stirred at r.t. for 13 h. The reaction was quenched by the addition of 5% HCl, and the whole mixture was extracted with AcOEt. Concentration of the AcOEt extract in vacuo gave a crude product which was purified by SiO₂ column chromatography (hexane : AcOEt = 1 : 1) to give 14 (84 mg, 99%) as a colorless oil.

¹H-NMR (600 MHz, CDCl₃) δ: 1.24–1.33 (2H, m), 1.33–1.48 (13H, m), 1.60 (2H, dt, J = 14.9, 7.1 Hz), 1.76–1.83 (7H, m), 1.89 (2H, br s), 2.16 (2H, dd, J = 14.7, 7.3 Hz), 2.64–2.87 (7H, m), 3.19 (2H, br s), 6.84 (1H, t, J = 7.3 Hz), 10.22 (1H, br). ¹³C-NMR (150 MHz, CDCl₃) δ: 12.1, 22.7, 23.9, 25.4, 26.0, 28.3, 28.5, 28.8, 29.5, 34.2, 35.0, 38.4, 48.6, 53.0, 79.4, 127.2, 144.8, 155.9, 173.4. IR (KBr) cm⁻¹: 2933, 1691, 1394, 1152. MS (ESI-TOF) m/z: 468 [M + Na]⁺. HR-MS (ESI-TOF) m/z: 468.2218 calcd for C₂₂H₃₉NO₄S₂Na; Found: 468.2197.

tert-Butyl (E)-Methyl(3-(2-(7-methyl-8-(methyl(thiazol-2-ylmethyl)amino)-8-oxooct-6-en-1-yl)-1,3-dithian-2-yl)propyl)carbamate (15)

1-Hydroxybenzotriazole (HOBt, 36 mg, 0.26 mmol, 1.4 equiv.), N-methyl-1-(thiazol-2-yl)methanamine (20, 36 mg, 0.28 mmol, 1.5 equiv.), Et₃N (150 µL, 1.1 mmol, 4.0 equiv.), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI·HCl, 51 mg, 0.26 mmol, 1.4 equiv.) were added to a solution of 14 (84 mg, 0.19 mmol) in CH₂Cl₂ (5 mL), and the mixture was stirred at r.t. for 10 h. Then, the reaction was quenched by the addition of sat. NH₄Cl aq. The whole mixture was extracted with AcOEt, and the AcOEt extract was washed with sat. NH₄Cl aq., NaHCO₃ aq., and brine. Concentration of the AcOEt extract in vacuo gave a crude product which was purified by SiO₂ column chromatography (hexane : AcOEt = 1 : 2) to give 15 (82 mg, 78%) as a colorless oil.

¹H-NMR (600 MHz, CDCl₃) δ: 1.06–1.41 (15H, m), 1.46–1.58 (2H, m), 1.59–1.87 (9H, m), 1.94–2.05 (2H, m), 2.56–2.79 (7H, m), 2.91 (3H, s), 3.09 (2H, br s), 4.75 (2H, s), 5.51 (1H, br s), 7.22 (1H, br s), 7.59 (1H, br s). ¹³C-NMR (150 MHz, CDCl₃) δ: 14.0, 19.9, 22.3, 22.6, 23.6, 25.2, 25.8, 27.4, 28.3, 28.5, 28.8, 29.3, 31.9, 33.1, 33.9, 34.6, 34.8, 35.4, 36.8, 38.1, 47.4, 47.8, 48.2, 48.6, 52.7, 52.8, 79.0, 119.9, 120.1, 129.5, 130.6, 131.9, 141.9, 142.0, 142.7, 155.5, 166.1, 166.6, 172.0, 173.5. IR (KBr) cm⁻¹: 3019, 1682, 1216, 909, 756. MS (ESI-TOF) m/z: 578 [M + Na]⁺. HR-MS (ESI-TOF) m/z: 578.2521 calcd for C₂₇H₄₅N₃O₃S₃Na; Found: 578.2543.

tert-Butyl (E)-Methyl(11-methyl-12-(methyl(thiazol-2-ylmethyl)amino)-4,12-dioxododec-10-en-1-yl)carbamate (16)

Iodine (149 mg, 0.59 mmol, 4.0 equiv.) was added to a solution of 15 (82 mg, 0.15 mmol) in a mixture of CH₃CN (4 mL) and sat. NaHCO₃ aq. (2 mL) at 0°C, and the mixture was stirred at 0°C for 0.5 h. The reaction was quenched by the addition of Na₂S₂O₃/NaHCO₃ aq., and the whole mixture was extracted with AcOEt. Then, the AcOEt extract was washed with Na₂S₂O₃/NaHCO₃ aq. and brine. Concentration of the the AcOEt extract in vacuo gave a crude product which was purified by SiO₂ column chromatography (hexane : AcOEt = 1 : 3) to give 16 (61 mg, 89%) as a colorless oil.

¹H-NMR (600 MHz, CDCl₃) δ: 1.20 (2H, br s), 1.29–1.42 (11H, m), 1.49 (2H, br s), 1.66–1.76 (2H, m), 1.81 (3H, s), 2.04 (2H, br d, J = 7.1 Hz), 2.32 (4H, t, J = 7.0 Hz), 2.76 (3H, s), 2.97 (3H, s), 3.14 (2H, br s), 4.81 (2H, s), 5.57 (1H, br s), 7.28 (1H, d, J = 2.8 Hz), 7.66 (1H, br s).¹³C-NMR (150 MHz, CDCl₃) δ: 14.2, 20.1, 21.5, 21.9, 23.5, 27.4, 28.5, 28.7, 28.9, 29.2, 29.7, 34.0, 35.6, 37.0, 39.4, 39.5, 42. 7, 47.6, 48.1, 48.4, 51.8, 79.2, 79.3, 120.1, 120.3, 129.6, 130.8, 132.1, 142.1, 142.9, 155.8, 166.3, 166.9, 172.2, 173.0, 210.1, 210.4. IR (KBr) cm⁻¹: 2931, 1692, 1394, 1170. MS (ESI-TOF) m/z: 488 [M + Na]⁺. HR-MS (ESI-TOF) m/z: 488.2559 calcd for C₂₄H₃₉N₃O₄SNa; Found: 488.2559.

tert-Butyl ((4EZ,10E)-4-(Chloromethylene)-11-methyl-12-(methyl(thiazol-2-ylmethyl)amino)-12-oxododec-10-en-1-yl)(methyl)carbamate (17)

Lithium bis(trimethylsilyl)amide (LHMDS) solution (1.0 M in THF, 515 µL, 0.53 mmol, 5.0 equiv.) was added to a solution of (chloromethyl)triphenylphosphonium chloride (185 mg, 0.51 mmol, 5.0 equiv.) in anhydrous THF (3 mL), and the mixture was stirred at r.t. for 10 min. A solution of 16 (50 mg, 0.11 mmol) in anhydrous THF (1 mL) was added dropwise to the ylide solution via cannula at r.t. over 5 min, and the resulting mixture was stirred at r.t. for 1 h. Then, the reaction was quenched by the addition of sat. NH₄Cl aq. The whole mixture was extracted with AcOEt, and the AcOEt extract was washed with NH₄Cl aq. and brine. Concentration of the AcOEt extract in vacuo gave a crude product which was purified by SiO₂ column chromatography (hexane : AcOEt = 1 : 2) to give 17 (57 mg, quant.) as a colorless oil.

¹H-NMR (600 MHz, CDCl₃) δ: 1.21–1.48 (15H, m), 1.56–1.66 (2H, m), 1.86 (3H, s), 1.97–2.03 (2H, t, J = 8.0 Hz), 2.06–2.23 (4H, m), 2.77–2.88 (3H, m), 3.04 (3H, s), 3.19 (2H, br s), 4.89 (2H, s), 5.65 (1H, br s), 5.72–5.81 (1H, m), 7.34 (1H, d, J = 3.3 Hz), 7.73 (1H, d, J = 3.2 Hz). IR (KBr) cm⁻¹: 3020, 1216, 771. MS (ESI-TOF) m/z: 520 [M + Na]⁺. HR-MS (ESI-TOF) m/z: 520.2377 calcd for C₂₅H₄₀N₃O₃S³⁵ClNa; Found: 520.2368.

14,15-Dinor-biakamide C, D (5, 6)

Trifluoroacetic acid (TFA, 255 µL, 3.4 mmol, 30 equiv.) was added to a solution of 17 (57 mg, 0.11 mmol) in CH₂Cl₂ (2 mL), and the mixture was stirred for 1 h. Then, the reaction was quenched by the addition of sat. NaHCO₃ aq. The whole mixture was extracted with AcOEt, and the AcOEt extract was washed with sat. NaHCO₃ aq. and brine. Concentration of the AcOEt extract in vacuo gave an amine, which was used in the next reaction without further purification. The above crude product was dissolved to CH₂Cl₂ (2 mL). HOBt (14 mg, 0.11 mmol, 1 equiv.), 21 (12 mg, 0.11 mmol, 1 equiv.), Et₃N (90 µL, 0.65 mmol, 6.0 equiv.), and EDCI·HCl (20 mg, 0.11 mmol, 1.0 equiv.) were added to the solution, and the resulting mixture was stirred at r.t. for 15 h. Then, the reaction was quenched by the addition of sat. NH₄Cl aq. The whole mixture was extracted with AcOEt, and the AcOEt extract was successively washed with sat. NH₄Cl aq., NaHCO₃ aq., and brine. Concentration of the AcOEt extract in vacuo gave a crude product which was purified by SiO₂ column chromatography (AcOEt : MeOH = 50 : 1) to give a mixture of 5 and 6 (50 mg, 88%, two steps) as a colorless oil.

5: ¹H-NMR (600 MHz, CDCl₃) δ: 1.16–1.47 (4H, m), 1.58–1.77 (4H, m), 1.86 (3H, s), 1.99–2.14 (4H, m), 2.14–2.23 (5H, m), 2.87–3.10 (6H, m), 3.24–3.40 (2H, m), 3.60 (3H, br s), 4.88 (2H, s), 5.11–5.17 (1H, br s), 5.64 (1H, br s), 5.80 (1H, s), 7.33 (1H, d, J = 2.8 Hz), 7.72 (1H, br s). ¹³C-NMR (150 MHz, CDCl₃) δ: 14.2, 18.8, 20.2, 20.3, 22.8, 24.7, 25.3, 25.6, 25.9, 26.2, 26.3, 26.4, 26. 9, 27.3, 27.5, 27.6, 28.7, 29.1, 29.4, 29.6, 29.8, 29.9, 30.0, 31.6, 32.0, 32.1, 32.7, 32.8, 34.8, 35.8, 35.9, 36.1, 37.0, 46.8, 46.9, 47.4, 48.5, 49.7, 50.1, 54.9, 91.3, 112.4, 112.5, 112.9, 118.0, 118.1, 118.3, 118.4, 120.1, 130.8, 130.9, 132.3, 141.4, 142.0, 142.1, 142.2, 146.3, 146.4, 166.9, 168.3, 168.5, 173.9. IR (KBr) cm⁻¹: 2927, 1644, 1381, 1239, 1072. MS (ESI-TOF) m/z: 518 [M + Na]⁺. HR-MS (ESI-TOF) m/z: 518.2220 calcd for C₂₅H₃₈N₃O₃S³⁵ClNa; Found: 518.2212.

6: ¹H-NMR (600 MHz, CDCl₃) δ: 1.16–1.47 (4H, m), 1.58–1.77 (4H, m), 1.86 (3H, s), 2.00–2.13 (4H, m), 2.13–2.24 (5H, m), 2.89–3.08 (6H, m), 3.24–3.46 (2H, m), 3.61 (3H, s), 4.87 (2H, s), 5.17 (2H, s), 5.63 (1H, s), 5.73–5.85 (1H, m), 7.33 (1H, s), 7.72 (1H, br s). ¹³C-NMR (150 MHz, CDCl₃) δ: 14.4, 18.9, 25.1, 26.1, 27.0, 27.6, 27.7, 28.8, 29.0, 30.1, 33.7, 34.8, 36.1, 37.1, 47.5, 47.7, 48.8, 50.4, 55.0, 91.1, 91.5, 112.4, 113.1, 120.2, 130.9, 132.2, 141.6, 142.3, 166.9, 168.2, 168.5, 174.4. IR (KBr) cm⁻¹: 3019, 1643, 1216, 756. MS (ESI-TOF) m/z: 518 [M + Na]⁺. HR-MS (ESI-TOF) m/z: 518.2220 calcd for C₂₅H₃₈N₃O₃S³⁵ClNa; Found: 518.2205.

tert-Butyl (10-((tert-Butyldimethylsilyl)oxy)decyl)carbamate (23)

Et₃N (1.8 mL, 13 mmol, 4.4 equiv.), and TBSCl (759 mg, 5.0 mmol, 1.8 equiv.) were added to a solution of 22 (789 mg, 2.9 mmol) in CH₂Cl₂ (15 mL), and the mixture was stirred at r.t. for 40 h. Then, the reaction was quenched by the addition of sat. NH₄Cl aq. The whole mixture was extracted with AcOEt, and the AcOEt extract was washed with sat. NH₄Cl aq. and brine. Concentration of the AcOEt extract in vacuo gave a crude product which was purified by SiO₂ column chromatography (hexane : AcOEt = 10 : 1) to give 23 (787 mg, 70%) as a colorless oil.

¹H-NMR (600 MHz, CDCl₃) δ: –0.02 (3H, s), –0.03 (3H, s), 0.82 (9H, s), 1.20 (12H, br s), 1.29–1.57 (13H, m), 3.02 (2H, br s), 3.52 (2H, td, J = 6.4, 3.4 Hz), 4.70 (1H, br). ¹³C-NMR (150 MHz, CDCl₃) δ: –5.4, –5.2, 18.4, 25.3, 25.8, 25.9, 26.0, 26.2, 26.6, 26.8, 27.4, 27.8, 28.4, 29.1, 29.3, 29.4, 29.5, 29.6, 30.1, 32.9, 40.6, 63.3, 78.8, 156.0. IR (KBr) cm⁻¹: 3356 (br), 2929, 1702, 1514, 1253, 1100, 837. MS (ESI-TOF) m/z: 410 [M + Na]⁺. HR-MS (ESI-TOF) m/z: 410.3066 calcd for C₂₁H₄₅NO₃SiNa; Found: 410.3082.

tert-Butyl (10-((tert-Butyldimethylsilyl)oxy)decyl)(methyl)carbamate (24)

NaH (about 60% oil suspension, 80 mg, 2.0 mmol, 1.1 equiv.) was added to a solution of 23 (707 mg, 1.8 mmol) in N,N-dimethylformamide (DMF, 15 mL) at 0°C, and the mixture was stirred at 0°C for 1 h. Iodomethane (250 µL, 4.0 mmol, 2.2 equiv.) was added to the amide solution, and the resulting mixture was stirred at r.t. for 12 h. Then, the reaction was quenched by the addition of sat. NH₄Cl aq. The whole mixture was extracted with AcOEt, and the AcOEt extract was washed with NH₄Cl aq. and brine. Concentration of the AcOEt extract in vacuo gave a crude product which was purified by SiO₂ column chromatography (hexane : AcOEt = 10 : 1) to give 24 (676 mg, 92%) as a colorless oil.

¹H-NMR (600 MHz, CDCl₃) δ: 0.00 (6H, s), 0.84 (9H, s), 1.23 (12H, br s), 1.38–1.61 (13H, m), 2.78 (3H, s), 3.13 (2H, br s), 3.54 (2H, t, J = 6.6 Hz). ¹³C-NMR (150 MHz, CDCl₃) δ: –5.4, 18.3, 25.7, 25.9, 26.6, 27.5, 27.8, 28.4, 29.3, 29.5, 32.8, 33.9, 48.4, 48.7, 63.2, 78.85, 155.7. IR (KBr) cm⁻¹: 2929, 1686, 1394, 1168, 758. MS (ESI-TOF) m/z: 424 [M + Na]⁺. HR-MS (ESI-TOF) m/z: 424.3223 calcd for C₂₂H₄₇NO₃SiNa; Found: 424.3225.

tert-Butyl (10-Hydroxydecyl)(methyl)carbamate (25)

TBAF solution (1 M in THF, 4.4 mL) was added to a solution of 24 (676 mg, 1.7 mmol) in THF (15 mL) at r.t., and the mixture was stirred at r.t. for 8 h. The reaction was quenched by the addition of sat. NH₄Cl aq., and the whole mixture was extracted with AcOEt. The AcOEt extract was washed with sat. NH₄Cl aq. and brine. Concentration of the AcOEt extract in vacuo gave a crude product, which was purified by SiO₂ column chromatography (Hexane : AcOEt = 3 : 1) to give 25 (461 mg, 95%) as a colorless oil.

¹H-NMR (600 MHz, CDCl₃) δ: 1.02–1.26 (12H, m), 1.29–1.41 (11H, m), 1.44 (2H, dd, J = 13.7, 7.5 Hz), 2.71 (3H, s), 2.84 (1H, br), 3.07 (2H, br s), 3.49 (2H, td, J = 6.7, 3.1 Hz). ¹³C-NMR (150 MHz, CDCl₃) δ: 25.5, 25.7, 26.6, 27.5, 27.8, 28.0, 28.4, 29.3, 29.4, 29.5, 32.7, 33.9, 48.4, 48.7, 62.5, 67.8, 79.0, 155.8. IR (KBr) cm⁻¹: 3448 (br), 2927, 1697, 1397, 1165. MS (ESI-TOF) m/z: 310 [M + Na]⁺. HR-MS (ESI-TOF) m/z: 310.2358 calcd for C₁₆H₃₃NO₃Na; Found: 310.2371.

Ethyl (E)-12-((tert-Butoxycarbonyl)(methyl)amino)-2-methyldodec-2-enoate (26)

Iodobenzene diacetate (562 mg, 1.7 mmol, 2.0 equiv.) and TEMPO (14 mg, 0.087 mmol, 0.1 equiv.) were added to a solution of 25 (251 mg, 0.87 mmol) in CH₂Cl₂ (10 mL), and the mixture was stirred at r.t. for 13 h. Then, toluene (10 mL) and 19 (1.26 g, 3.5 mmol, 4.0 equiv.) were added to the aldehyde solution, and the resulting mixture was stirred at 100°C for 12 h. The reaction was quenched by the addition of Na₂S₂O₃/NaHCO₃ aq., and the whole mixture was extracted with AcOEt. The AcOEt extract was successively washed with Na₂S₂O₃/NaHCO₃ aq. and brine. Concentration of the AcOEt extract in vacuo gave a crude product which was purified by SiO₂ column chromatography (hexane : AcOEt = 10 : 1) to give 26 (291 mg, 90%) as a colorless oil.

¹H-NMR (600 MHz, CDCl₃) δ: 0.58–1.38 (26H, m), 1.52–2.15 (5H, m), 2.52–2.73 (3H, br s), 2.99 (2H, br s), 3.82–4.20 (2H, m), 6.46–6.64 (1H, br s). ¹³C-NMR (150 MHz, CDCl₃) δ: 12.1, 13.9, 14.0, 14.1, 17.0, 20.4, 26.4, 27.3, 27.6, 28.2, 28.4, 29.1, 29.2, 29.3, 33.7, 48.1, 48.5, 59.9, 60.6, 76.9, 77.2, 77.4, 78.6, 127.4, 133.1, 141.9, 142.8, 155.4, 167.6. IR (KBr) cm⁻¹: 2928, 1699, 1365, 1249, 1166. MS (ESI-TOF) m/z: 392 [M + Na]⁺. HR-MS (ESI-TOF) m/z: 392.2777 calcd for C₂₁H₃₉NO₄Na; Found: 392.2787.

(E)-12-((tert-Butoxycarbonyl)(methyl)amino)-2-methyldodec-2-enoic Acid (27)

LiOH (94 mg, 3.9 mmol, 5.0 equiv.) was added to a solution of 26 (291 mg, 0.79 mmol) in THF : MeOH : H₂O (1 : 1 : 1, 10 mL), and the mixture was stirred at r.t. for 24 h. The reaction was quenched by the addition of 5% HCl, and the whole mixture was extracted with AcOEt. Concentration of the AcOEt extract in vacuo gave a crude product which was purified by SiO₂ column chromatography (hexane : AcOEt = 1 : 1) to give 27 (260 mg, 97%) as a colorless oil.

¹H-NMR (600 MHz, CDCl₃) δ: 1.22 (10H, br), 1.27–1.82 (13H, m), 2.09 (2H, dd, J = 14.6, 7.3 Hz), 2.74 (2H, s), 3.09 (2H, s), 6.80 (1H, t, J = 7.3 Hz), 11.62 (1H, br). ¹³C-NMR (150 MHz, CDCl₃) δ: 11.7, 20.3, 26.4, 27.3, 27.6, 28.2, 28.3, 28.6, 29.06, 29.11, 29.2, 29.5, 33.8, 48.2, 48.6, 78.9, 126.1, 126.9, 144.6, 145.8, 155.7, 172.9. IR (KBr) cm⁻¹: 2927, 1693, 1394, 1166. MS (ESI-TOF) m/z: 364 [M + Na]⁺. HR-MS (ESI-TOF) m/z: 364.2464 calcd for C₁₉H₃₅NO₄Na; Found: 364.2452.

tert-Butyl (E)-Methyl(11-methyl-12-(methyl(thiazol-2-ylmethyl)amino)-12-oxododec-10-en-1-yl)carbamate (28)

HOBt (29 mg, 0.21 mmol, 1.5 equiv.), amine 20 (27 mg, 0.21 mmol, 1.5 equiv.), Et₃N (180 µL, 1.3 mmol, 6.0 equiv.), and EDCI·HCl (40 mg, 0.21 mmol, 1.5 equiv.) were added to a solution of 27 (48 mg, 0.14 mmol) in CH₂Cl₂ (5 mL), and the mixture was stirred at r.t. for 12 h. Then, the reaction was quenched by the addition of sat. NH₄Cl aq. The whole mixture was extracted with AcOEt, and the AcOEt extract was successively washed with sat. NH₄Cl aq., NaHCO₃ aq., and brine. Concentration of the AcOEt extract in vacuo gave a crude product which was purified by SiO₂ column chromatography (hexane : AcOEt = 1 : 1) to give 28 (56 mg, 88%) as a colorless oil.

¹H-NMR (600 MHz, CDCl₃) δ: 1.16 (10H, m), 1.28 (2H, s), 1.36 (11H, m), 1.76 (3H, s), 2.00 (2H, br s), 2.72 (3H, s), 2.94 (3H, s), 3.08 (2H, s), 4.78 (2H, s), 5.55 (1H, br s), 7.24 (1H, br s), 7.62 (1h, br s). ¹³C-NMR (150 MHz, CDCl₃) δ: 14.1, 20.0, 20.8, 26.6, 27.5, 27.8, 28.2, 28.4, 28.5, 28.7, 29.0, 29.1, 29.2, 29.3, 29.4, 29.6, 29.7, 32.3, 33.9, 35.5, 36.9, 47.5, 48.3, 48.7, 51.8, 52.8, 78.9, 119.7, 119.9, 120.1, 129.9, 130.5, 130.8, 132.4, 142.0, 142.8, 155.7, 166.2, 166.8, 172.1, 173.7. IR (KBr) cm⁻¹: 2929, 1683, 1398, 1216, 755. MS (ESI-TOF) m/z: 474 [M + Na]⁺. HR-MS (ESI-TOF) m/z: 474.2766 calcd for C₂₄H₄₁N₃O₃SNa; Found: 474.2763.

(E)-12-((E)-3-Methoxy-N-methylbut-2-enamido)-N,2-dimethyl-N-(thiazol-2-ylmethyl)dodec-2-enamide (29)

TFA (165 µL, 2.2 mmol, 50 equiv.) was added to a solution of 28 (20 mg, 0.044 mmol) in CH₂Cl₂ (2 mL), and the mixture was stirred at r.t. for 1 h. Then, the reaction was quenched by the addition of sat. NaHCO₃ aq. The whole mixture was extracted with AcOEt, and the AcOEt extract was washed with sat. NaHCO₃ aq. and brine. Concentration of the AcOEt extract in vacuo gave an amine, which was used in the next reaction without further purification. The above crude product was dissolved to CH₂Cl₂ (1 mL). HOBt (10 mg, 0.062 mmol, 1.4 equiv.), 21 (23 mg, 0.18 mmol, 4.0 equiv.), Et₃N (52 µL, 0.37 mmol, 6.0 equiv.), and EDCI·HCl (12 mg, 0.062 mmol, 1.4 equiv.) were added to the solution, and the resulting mixture was stirred for 12 h at r.t. Then, the reaction was quenched by the addition of sat. NH₄Cl aq. The whole mixture was extracted with AcOEt, and the AcOEt extract was successively washed with sat. NH₄Cl aq., NaHCO₃ aq., and brine. Concentration of the AcOEt extract in vacuo gave a crude product which was purified by SiO₂ column chromatography (hexane : AcOEt = 1 : 3) to give 29 (14 mg, 69%, two steps) as a colorless oil.

¹H-NMR (600 MHz, CDCl₃) δ: 1.08–1.42 (12H, m), 1.44–1.57 (2H, m), 1.83 (3H, s), 2.01–2.10 (2H, m), 2.11–2.23 (3H, m), 2.81–3.08 (6H, m), 3.27 (1H, br s), 3.35 (1H, br s), 3.57 (3H, s), 4.85 (2H, s), 5.14 (1H, d, J = 9.9 Hz), 5.62 (1H, br s), 7.30 (1H, br s), 7.69 (1H, br s). ¹³C-NMR (150 MHz, CDCl₃) δ: 14.8, 18.8, 23.1, 23.8, 26.8, 27.0, 27.6, 27.7, 28.5, 28.9, 29.4, 29.5, 29.6, 30.4, 31.0, 33.6, 35.7, 36.0, 38.8, 47.7, 50.6, 54.85, 54.90, 68.2, 91.4, 91.5, 120.1, 128.9, 130.6, 130.7, 131.0, 132.5, 132.6, 142.2, 166.4, 166.9, 167.8, 168.0, 168.1, 168.2, 172.3, 173.8. IR (KBr) cm⁻¹: 2926, 1648, 1381, 1238, 1140, 1073. MS (ESI-TOF) m/z: 472 [M + Na]⁺. HR-MS (ESI-TOF) m/z: 472.2610 calcd for C₂₄H₃₉N₃O₃SNa; Found: 472.2600.

tert-Butyl ((10E)-4-(Hydroxyimino)-11-methyl-12-(methyl(thiazol-2-ylmethyl)amino)-12-oxododec-10-en-1-yl)(methyl)carbamate (30)

Hydroxylamine hydrochloride (2.7 mg, 0.026 mmol, 1.5 equiv.) and AcONa (3.2 mg, 0.039 mmol) were added to a solution of 16 in MeOH (0.5 mL), and the mixture was stirred at r.t. for 1 h. Concentration in vacuo and purification of the residue by SiO₂ column chromatography (hexane : AcOEt = 1 : 3) gave 30 (10 mg, 83%) as a colorless oil.

¹H-NMR (600 MHz, CDCl₃) δ: 1.18–1.51 (15H, m), 1.64–1.77 (2H, m), 1.83 (3H, s), 2.03–2.22 (4H, m), 2.22–2.41 (2H, m), 2.82 (3H, br s), 3.02 (3H, br s), 3.20 (2H, br s), 4.87 (3H, d, J = 4.8 Hz), 5.62 (1H, d, J = 6.2 Hz), 7.31 (1H, br s), 7.71 (1H, br s), 8.78 (1H, br). ¹³C-NMR (150 MHz, CDCl₃) δ: 14.2, 20.0, 20.1, 22.8, 23.7, 24.1, 24.3, 24.6, 25.0, 25.6, 26.0, 27.51, 27.54, 27.6, 28.56, 28.58, 28.6, 28.8, 29.4, 29.6, 29.8, 31.7, 34.0, 34.3, 37.1, 48.2, 48.5, 48.7, 49.1, 53.0, 79.4, 120.2, 130.9, 132.3, 142.2, 155.9, 160.7, 167.0, 174.1. IR (KBr) cm⁻¹: 3332 (br), 2929, 1692, 1396, 1169, 755. MS (ESI-TOF) m/z: 503 [M + Na]⁺. HR-MS (ESI-TOF) m/z: 503.2668 calcd for C₂₄H₄₀N₄O₄SNa; Found: 503.2669.

(2E)-9-(Hydroxyimino)-12-((E)-3-methoxy-N-methylbut-2-enamido)-N,2-dimethyl-N-(thiazol-2-ylmethyl)dodec-2-enamide (31)

TFA (49 µL, 0.43 mmol, 20 equiv.) was added to a solution of 30 (10 mg, 0.022 mmol) in CH₂Cl₂ (1 mL), and the mixture was stirred at r.t. for 3 h. Then, the reaction was quenched by the addition of sat. NaHCO₃ aq. The whole mixture was extracted with AcOEt, and the AcOEt extract was washed with sat. NaHCO₃ aq. and brine. Concentration of the AcOEt extract in vacuo gave an amine, which was used in the next reaction without further purification. The above crude product was dissolved to CH₂Cl₂ (1 mL). HOBt (4.3 mg, 0.029 mmol, 1.3 equiv.), 21 (2.2 mg, 0.019 mmol, 0.9 equiv.), Et₃N (12 µL, 0.85 mmol, 3.9 equiv.), and EDCI·HCl (6.1 mg, 0.029 mmol, 1.3 equiv.) were added to the solution, and the resulting mixture was stirred at r.t. for 9 h. Then, the reaction was quenched by the addition of sat. NH₄Cl aq. The whole mixture was extracted with AcOEt, and the AcOEt extract was successively washed with sat. NH₄Cl aq., NaHCO₃ aq., and brine. Concentration of the AcOEt extract in vacuo gave a crude product which was purified by SiO₂ column chromatography (AcOEt : MeOH = 30 : 1) to give 31 (3.5 mg, 34%, two steps) as a colorless oil.

¹H-NMR (600 MHz, CDCl₃) δ: 1.12–1.55 (6H, m), 1.69–1.93 (5H, m), 1.96–2.46 (9H, m), 2.86–3.10 (6H, m), 3.30–3.47 (2H, m), 3.53–3.64 (3H, m), 4.83–4.94 (2H, m), 5.16 (1H, br s), 5.63 (1H, br s), 7.32 (1H, br s), 7.72 (1H, br s), 8.42 (1H, br s). ¹³C-NMR (150 MHz, CDCl₃) δ: 14.3, 18.9, 23.6, 24.3, 24.6, 25.0, 25.2, 25.6, 25.9, 27.5, 27.6, 28.6, 29.2, 29.3, 29.8, 30.9, 31.6, 33.6, 34.0, 36.06, 36.14, 37.1, 47.4, 47.7, 48.5, 49.9, 50.6, 55.0, 91.2, 91.4, 120.2, 130.9, 132.4, 142.2, 142.4, 160.3, 161.1, 168.2, 168.6, 174.1. IR (KBr) cm⁻¹: 3306 (br), 2925, 1641, 1384, 1240. MS (ESI-TOF) m/z: 501 [M + Na]⁺. HR-MS (ESI-TOF) m/z: 501.2511 calcd for C₂₄H₃₈N₄O₄SNa; Found: 501.2503.

tert-Butyl (E)-Methyl(3-(2-(7-methyl-8-oxo-8-((thiazol-2-ylmethyl)amino)oct-6-en-1-yl)-1,3-dithian-2-yl)propyl)carbamate (32)

HOBt (38 mg, 0.28 mmol, 1.4 equiv.), 2-(aminomethyl)thiazole (37, 35 mg, 0.30 mmol, 1.5 equiv.), Et₃N (160 µL, 1.1 mmol, 4.0 equiv.), and EDCI·HCl (54 mg, 0.28 mmol, 1.4 equiv.) were added to a solution of 14 (90 mg, 0.20 mmol) in CH₂Cl₂ (5 mL), and the mixture was stirred at r.t. for 10 h. Then, the reaction was quenched by the addition of sat. NH₄Cl aq. The whole mixture was extracted with AcOEt, and the AcOEt extract was successively washed with sat. NH₄Cl aq., NaHCO₃ aq., and brine. Concentration of the AcOEt extract in vacuo gave a crude product which was purified by SiO₂ column chromatography (hexane : AcOEt = 1 : 3) to give 32 (87 mg, 80%) as a colorless oil.

¹H-NMR (500 MHz, CDCl₃) δ: 1.24 (2H, d, J = 7.1 Hz), 1.31–1.38 (13H, m), 1.49–1.76 (7H, m), 1.78 (3H, s), 1.84 (2H, d, J = 4.6 Hz), 2.06 (2H, dd, J = 14.5, 7.2 Hz), 2.69 (4H, d, J = 4.7 Hz), 2.75 (3H, s), 3.13 (2H, br s), 4.70 (2H, d, J = 5.7 Hz), 6.35 (1H, t, J = 7.1 Hz), 7.00 (1H, br s), 7.20 (1H, d, J = 3.2 Hz), 7.60 (1H, d, J = 3.1 Hz). ¹³C-NMR (150 MHz, CDCl₃) δ: 12.6, 22.4, 22.7, 23.8, 25.3, 25.7, 25.9, 26.0, 27.7, 28.0, 28.2, 28.4, 29.1, 29.3, 34.0, 34.1, 35.0, 38.2, 41.0, 47.9, 48.7, 52.8, 60.3, 79.1, 119.5, 130.2, 137.0, 142.0, 142.1, 155.6, 167.9, 169.2. IR (KBr) cm⁻¹: 3330 (br), 2933, 1692, 1523, 1394, 1150. MS (ESI-TOF) m/z: 564 [M + Na]⁺. HR-MS (ESI-TOF) m/z: 564.2364 calcd for C₂₆H₄₃N₃O₃S₃Na; Found: 564.2391.

tert-Butyl (E)-Methyl(11-methyl-4,12-dioxo-12-((thiazol-2-ylmethyl)amino)dodec-10-en-1-yl)carbamate (33)

Iodine (77 mg, 0.30 mmol, 2.0 equiv.) was added to a solution of 32 (82 mg, 0.15 mmol) in a mixture of CH₃CN (6 mL) and sat. NaHCO₃ aq. (3 mL) at 0°C, and the mixture was stirred at 0°C for 3 h. The reaction was quenched by the addition of Na₂S₂O₃/NaHCO₃ aq., and the whole mixture was extracted with AcOEt. Then, the AcOEt extract was washed with Na₂S₂O₃/NaHCO₃ aq. and brine. Concentration of the AcOEt extract in vacuo gave a crude product which was purified by SiO₂ column chromatography (hexane : AcOEt = 1 : 4) to give 33 (27 mg, 40%) as a colorless oil.

¹H-NMR (600 MHz, CDCl₃) δ: 1.23 (2H, br), 1.37 (11H, m), 1.49 (2H, br s), 1.71 (2H, br), 1.80 (3H, s), 2.08 (2H, br s), 2.20–2.45 (4H, br), 2.74 (3H, s), 3.13 (2H, br s), 4.74 (2H, br), 6.36 (1H, br s), 6.97–7.02 (1H, br), 7.24 (1H, br s), 7.63 (1H, br s). ¹³C-NMR (150 MHz, CDCl₃) δ: 12.7, 21.5, 21.8, 23.5, 28.1, 28.4, 28.5, 28.8, 29.9, 34.0, 38.3, 39.5, 41.0, 42.6, 47.5, 48.1, 63.4, 79.2, 119.7, 130.3, 137.1, 141.9, 155.9, 168.1, 169.3, 210.1, 210.4. IR (KBr) cm⁻¹: 3341 (br), 2931, 1692, 1523, 1395, 1168. MS (ESI-TOF) m/z: 474 [M + Na]⁺. HR-MS (ESI-TOF) m/z: 474.2402 calcd for C₂₃H₃₇N₃O₄SNa; Found: 474.2403.

tert-Butyl ((10E)-4-(Chloromethylene)-11-methyl-12-oxo-12-((thiazol-2-ylmethyl)amino)dodec-10-en-1-yl)(methyl)carbamate (34)

LHMDS solution (1.0 M in THF, 170 µL, 0.17 mmol, 2.8 equiv.) was added to a solution of (chloromethyl)triphenylphosphonium chloride (63 mg, 0.18 mmol, 3.0 equiv.) in anhydrous THF (3 mL), and the mixture was stirred at r.t. for 1 h. A solution of 33 (27 mg, 0.060 mmol) in anhydrous THF (0.5 mL) was added dropwise to the ylide solution via cannula at r.t., and the resulting mixture was stirred at r.t. for 4 h. Then, the reaction was quenched by the addition of sat. NH₄Cl aq. The whole mixture was extracted with AcOEt, and the AcOEt extract was washed with NH₄Cl aq. and brine. Concentration of the AcOEt extract in vacuo gave a crude product which was purified by SiO₂ column chromatography (hexane : AcOEt = 1 : 3) to give 34 (23 mg, 79%) as a colorless oil.

¹H-NMR (600 MHz, CDCl₃) δ: 1.20–1.44 (15H, m), 1.58 (2H, br), 1.84 (3H, s), 1.96–2.04 (2H, m), 2.08–2.19 (4H, m), 2.80 (3H, m), 3.15 (2H, br s), 4.77 (2H, d, J = 5.5 Hz), 5.76 (1H, br s), 6.37–6.44 (1H, br), 6.81 (1H, br s), 7.26 (1H, d, J = 2.4 Hz), 7.67 (1H, br s). ¹³C-NMR (150 MHz, CDCl₃) δ: 12.7, 25.1, 25.5, 25.9, 26.9, 27.4, 27.5, 28.3, 28.4, 28.52, 28.54, 29.0, 29.2, 30.0, 31.9, 34.3, 34.8, 41.2, 48.2, 48.5, 48.9, 79.3, 79.4, 112.4, 119.7, 128.5, 128.6, 130.2, 130.3, 132.0, 132.07, 132.14, 137.3, 137.4, 141.9, 142.2, 155.8, 155.8, 167.8, 169.2, 169.3. IR (KBr) cm⁻¹: 3331 (br), 2930, 1693, 1523, 1394, 1166. MS (ESI-TOF) m/z: 506 [M + Na]⁺. HR-MS (ESI-TOF) m/z: 506.2220 calcd for C₂₄H₃₈N₃O₃S³⁵ClNa; Found: 506.2195.

tert-Butyl ((10E)-4-(Chloromethylene)-11-methyl-12-oxo-12-((thiazol-2-ylmethyl)amino)dodec-10-en-1-yl)(methyl)carbamate (35)

TFA (65 µL, 0.87 mmol, 20 equiv.) was added to a solution of 34 (21 mg, 0.044 mmol) in CH₂Cl₂ (1 mL), and the mixture was stirred at r.t. for 1 h. Then, the reaction was quenched by the addition of sat. NaHCO₃ aq. The whole mixture was extracted with AcOEt, and the AcOEt extract was washed with sat. NaHCO₃ aq. and brine. Concentration of the AcOEt extract in vacuo gave an amine, which was used in the next reaction without further purification. The above crude product was dissolved to CH₂Cl₂ (1 mL). HOBt (11 mg, 0.078 mmol, 1.8 equiv.), 21 (9.0 mg, 0.078 mmol, 1.8 equiv.), Et₃N (29 µL, 0.21 mmol, 4.8 equiv.), and EDCI·HCl (15 mg, 0.078 mmol, 1.8 equiv.) were added to the solution, and the resulting mixture was stirred at r.t. for 24 h. Then, the reaction was quenched by the addition of sat. NH₄Cl aq. The whole mixture was extracted with AcOEt, and the AcOEt extract was washed with sat. NH₄Cl aq., NaHCO₃ aq., and brine. Concentration of the AcOEt extract in vacuo gave a crude product which was purified by SiO₂ column chromatography (hexane : AcOEt = 1 : 5) to give 35 and 36 (23 mg, 97%, two steps) as a colorless oil.

35: ¹H-NMR (500 MHz, CDCl₃) δ: 1.02–1.70 (8H, br), 1.87 (3H, s), 2.06 (3H, dd, J = 10.0, 5.1 Hz), 2.10–2.27 (6H, br), 2.97 (3H, br), 3.20–3.44 (2H, br), 3.60 (3H, s), 4.82 (2H, d, J = 5.7 Hz), 5.14 (1H, s), 5.81 (1H, s), 6.44 (1H, t, J = 7.4 Hz), 6.69 (1H, br s), 7.29 (1H, d, J = 3.3 Hz), 7.71 (1H, d, J = 3.3 Hz). ¹³C-NMR (150 MHz, CDCl₃) δ: 12.8, 18.9, 27.0, 28.4, 28.6, 29.2, 30.0, 32.3, 36.2, 41.3, 47.5, 49.9, 55.0, 77.4, 91.3, 112.5, 119.6, 130.3, 137.5, 142.4, 165.6, 168.7, 169.4. IR (KBr) cm⁻¹: 3326 (br), 2931, 1635, 1522, 1360, 1139. In Mass mesurement, the spectrum of 3-oxobutanoic amide derivative which decomposed from compound 35 was obtained. MS (ESI-TOF) m/z: 490 [M + Na]⁺. HR-MS (ESI-TOF) m/z: 490.1910 calcd for C₂₃H₃₄N₃O₃S³⁵ClNa; Found: 490.1910.

Ethyl (E)-12-((tert-Butoxycarbonyl)(methyl)amino)-2-methyl-9-oxododec-2-enoate (38)

Iodine (49 mg, 0.19 mmol, 3.4 equiv.) was added to a solution of 15 (27 mg, 0.057 mmol) in a mixture of CH₃CN (2 mL) and sat. NaHCO₃ aq. (1 mL) at 0°C, and the mixture was stirred at 0°C for 1 h. The reaction was quenched by the addition of Na₂S₂O₃/NaHCO₃ aq., and the whole mixture was extracted with AcOEt. Then, the AcOEt extract was washed with Na₂S₂O₃/NaHCO₃ aq. and brine. Concentration of the AcOEt extract in vacuo gave a crude product which was purified by SiO₂ column chromatography (hexane : AcOEt = 3 : 1) to give 38 (14 mg, 62%) as a colorless oil.

¹H-NMR (600 MHz, CDCl₃) δ: 1.21–1.32 (5H, m), 1.43 (11H, s), 1.52–1.60 (2H, m), 1.67–1.82 (5H, m), 2.10–2.17 (2H, m), 2.34–2.42 (4H, m), 2.80 (3H, s), 3.19 (2H, s), 4.16 (2H, q, J = 7.1 Hz), 6.71 (1H, t, J = 7.3 Hz). ¹³C-NMR (150 MHz, CDCl₃) δ: 12.5, 14.4, 14.5, 21.6, 21.9, 23.6, 26.1, 28.5, 28.6, 28.7, 29.0, 29.5, 34.1, 39.6, 42.8, 47.7, 48.2, 60.1, 60.5, 79.4, 128.0, 142.1, 142.8, 155.9, 168.4, 210.2, 210.6. IR (KBr) cm⁻¹: 2932, 1697, 1365, 1171. MS (ESI-TOF) m/z: 406 [M + Na]⁺. HR-MS (ESI-TOF) m/z: 406.2569 calcd for C₂₁H₃₇NO₅Na; Found: 406.2566.

Ethyl (2E)-12-((tert-Butoxycarbonyl)(methyl)amino)-9-(chloromethylene)-2-methyldodec-2-enoate (39)

LHMDS solution (1.0 M in THF, 170 µL, 0.17 mmol, 4.8 equiv.) was added to a solution of (chloromethyl)triphenylphosphonium chloride (61 mg, 0.18 mmol, 5.0 equiv.) in anhydrous THF (2 mL), and the mixture was stirred for 1 h at r.t. A solution of 38 (14 mg, 0.035 mmol) in anhydrous THF (0.5 mL) was added dropwise to the ylide solution via cannula at r.t., and the resulting mixture was stirred at r.t. for 1 h. Then, the reaction was quenched by the addition of sat. NH₄Cl aq., and the whole mixture was extracted with AcOEt, and the AcOEt extract was washed with NH₄Cl aq. and brine. Concentration of the AcOEt extract in vacuo gave a crude product which was purified by SiO₂ column chromatography (hexane : AcOEt = 5 : 1) to give 39 (11 mg, 75%) as a colorless oil.

¹H-NMR (600 MHz, CDCl₃) δ: 1.29 (3H, t, J = 7.1 Hz), 1.39–1.51 (11H, m), 1.55–1.69 (6H, m), 1.82 (3H, s), 2.03 (2H, t, J = 7.0 Hz), 2.12–2.25 (4H, m), 2.83 (3H, s), 3.18 (2H, br s), 4.19 (2H, q, J = 7.1 Hz), 5.81 (s, 1H), 6.72–6.78 (1H, m). ¹³C-NMR (150 MHz, CDCl₃) δ: 12.5, 14.5, 19.2, 20.8, 26.7, 27.0, 27.6, 28.5, 28.6, 28.7, 29.1, 29.3, 29.9, 30.1, 32.0, 34.3, 34.6, 48.4, 48.6, 49.0, 60.6, 79.4, 112.5, 127.9, 128.0, 129.8, 134.9, 135.3, 142.2, 155.9, 168.4. IR (KBr) cm⁻¹: 2930, 1672, 1428, 1113, 704. MS (ESI-TOF) m/z: 438 [M + Na]⁺. HR-MS (ESI-TOF) m/z: 438.2387 calcd for C₂₂H₃₈NO₄³⁵ClNa; Found: 438.2389.

Ethyl (2E,9E)-9-(Chloromethylene)-12-((E)-3-methoxy-N-methylbut-2-enamido)-2-methyldodec-2-enoate (40)

TFA (80 µL, 1.1 mmol, 47 equiv.) was added to a solution of 39 (11 mg, 0.026 mmol) in CH₂Cl₂ (1 mL), and the mixture was stirred at r.t. for 1.5 h. Then, the reaction was quenched by the addition of sat. NaHCO₃ aq. The whole mixture was extracted with AcOEt, and the AcOEt extract was washed with sat. NaHCO₃ aq. and brine. Concentration of the AcOEt extract in vacuo gave an amine, which was used in the next reaction without further purification. The above crude product was dissolved to CH₂Cl₂ (1 mL). HOBt (5.0 mg, 0.037 mmol, 1.6 equiv.), 21 (4.1 mg, 0.035 mmol, 1.6 equiv.), Et₃N (31 µL, 0.22 mmol, 9.8 equiv.), and EDCI·HCl (7.1 mg, 0.037 mmol, 1.6 equiv.) were added to the solution, and the resulting mixture was stirred at r.t. for 7 h. Then, the reaction was quenched by the addition of sat. NH₄Cl aq. The whole mixture was extracted with AcOEt, and the AcOEt extract was washed with sat. NH₄Cl aq., NaHCO₃ aq., and brine. Concentration of the AcOEt extract in vacuo gave a crude product which was purified by SiO₂ column chromatography (hexane : AcOEt = 3 : 1) to give 40 and 41 (6.8 mg, 72%, two steps) as a colorless oil.

40: ¹H-NMR (600 MHz, CDCl₃) δ: 1.09–1.78 (12H, m), 1.82 (3H, s), 2.06 (2H, m), 2.11–2.28 (6H, m), 2.94–3.00 (3H, m), 3.29–3.38 (2H, br), 3.60 (3H, s), 4.18 (2H, q, J = 7.1 Hz), 5.15 (1H, s), 5.81 (1H, s), 6.74 (1H, td, J = 7.5, 1.3 Hz). ¹³C-NMR (150 MHz, CDCl₃) δ: 12.5, 14.4, 18.9, 25.7, 26.2, 27.0, 28.5, 28.7, 29.3, 30.1, 47.5, 55.0, 60.6, 76.8, 77.4, 77.5, 91.3, 112.5, 142.2, 142.4, 168.5, 168.6. IR (KBr) cm⁻¹: 2929, 1651, 1442, 1241, 1119. MS (ESI-TOF) m/z: 436 [M + Na]⁺. HR-MS (ESI-TOF) m/z: 436.2231 calcd for C₂₂H₃₆NO₄³⁵ClNa; Found: 436.2214.

tert-Butyl (E)-Methyl(3-(2-(7-methyl-8-(methyl(naphthalen-1-ylmethyl)amino)-8-oxooct-6-en-1-yl)-1,3-dithian-2-yl)propyl)carbamate (42)

HOBt (43 mg, 0.32 mmol, 1.4 equiv.), N-methyl-1-naphthylmethylamine (47, 78 mg, 0.45 mmol, 2.0 equiv.), Et₃N (265 µL, 1.9 mmol, 8.4 equiv.), and EDCI·HCl (61 mg, 0.32 mmol, 1.4 equiv.) were added to a solution of 14 (101 mg, 0.23 mmol) in CH₂Cl₂ (5 mL), and the mixture was stirred at r.t. for 8 h. Then, the reaction was quenched by the addition of sat. NH₄Cl aq. The whole mixture was extracted with AcOEt, and the AcOEt extract was successively washed with sat. NH₄Cl aq., NaHCO₃ aq., and brine. Concentration of the AcOEt extract in vacuo gave a crude product which was purified by SiO₂ column chromatography (hexane : AcOEt = 3 : 1) to give 42 (124 mg, 91%) as a colorless oil.

¹H-NMR (600 MHz, CDCl₃) δ: 0.73–1.48 (15H, m), 1.51–1.62 (2H, br s), 1.65–1.92 (9H, m), 1.93–2.08 (2H, br s), 2.60–2.74 (10H, br), 3.16 (2H, br s), 5.02 (2H, s), 5.51 (1H, br s), 7.24–7.35 (1H, br s), 7.35–7.55 (3H, m), 7.72–8.01 (3H, m). ¹³C-NMR (150 MHz, CDCl₃) δ: 14.2, 22.7, 23.7, 25.2, 25.3, 25.4, 25.8, 27.3, 28.4, 28.5, 29.3, 34.0, 34.8, 35.8, 38.2, 48.7, 52.8, 79.1, 104.9, 123.8, 125.1, 125.8, 125.9, 126.3, 126.4, 128.5, 131.1, 132.4, 133.7, 155.6, 173.3. IR (KBr) cm⁻¹: 2936, 1685, 1616, 1398, 1216, 1153, 755. MS (ESI-TOF) m/z: 621 [M + Na]⁺. HR-MS (ESI-TOF) m/z: 621.3161 calcd for C₃₄H₅₀N₂O₃S₂Na; Found: 621.3172.

tert-Butyl (E)-Methyl(11-methyl-12-(methyl(naphthalen-1-ylmethyl)amino)-4,12-dioxododec-10-en-1-yl)carbamate (43)

Iodine (105 mg, 0.41 mmol, 2.0 equiv.) was added to a solution of 42 (124 mg, 0.21 mmol) in a mixture of CH₃CN (6 mL) and sat. NaHCO₃ aq. (3 mL) at 0°C, and the mixture was stirred at 0°C for 1 h. The reaction was quenched by the addition of Na₂S₂O₃/NaHCO₃ aq., and the whole mixture was extracted with AcOEt. Then, the AcOEt extract was washed with Na₂S₂O₃/NaHCO₃ aq. and brine. Concentration of the AcOEt extract in vacuo gave a crude product which was purified by SiO₂ column chromatography (hexane : AcOEt = 1 : 1) to give 43 (60 mg, 57%) as a colorless oil.

¹H-NMR (600 MHz, CDCl₃) δ: 0.61–1.63 (15H, m), 1.63–1.78 (2H, m), 1.83 (3H, s), 1.97 (2H, m), 2.31 (4H, br s), 2.56–3.09 (6H, m), 3.16 (2H, br s), 5.03 (2H, s), 5.51 (1H, br s), 7.32 (1H, br s), 7.41 (1H, br s), 7.44–7.56 (2H, m), 7.77 (1H, d, J = 8.2 Hz), 7.84 (1H, d, J = 7.2 Hz), 8.03 (1H, br s). ¹³C-NMR (150 MHz, CDCl₃) δ: 14.3, 21.7, 23.4, 27.3, 28.4, 28.6, 28.8, 29.7, 34.0, 35.9, 39.4, 42.6, 47.76, 48.00, 52.6, 79.3, 122.3, 122.8, 123.9, 125.3, 126.0, 126.4, 127.1, 128.3, 128.7, 131.1, 131.5, 132.5, 133.8, 155.8, 173.4, 210.1. IR (KBr) cm⁻¹: 2930, 1692, 1622, 1396, 1168. MS (ESI-TOF) m/z: 531 [M + Na]⁺. HR-MS (ESI-TOF) m/z: 531.3199 calcd for C₃₁H₄₄N₂O₄Na; Found: 531.3174.

tert-Butyl ((10E)-4-(Chloromethylene)-11-methyl-12-(methyl(naphthalen-1-ylmethyl)amino)-12-oxododec-10-en-1-yl)(methyl)carbamate (44)

LHMDS solution (1.0 M in THF, 230 µL, 0.23 mmol, 3.0 equiv.) was added to a solution of (chloromethyl)triphenylphosphonium chloride (80 mg, 0.23 mmol, 3.0 equiv.) in anhydrous THF (2 mL), and the mixture was stirred at r.t. for 0.5 h. A solution of 43 (39 mg, 0.077 mmol) in anhydrous THF (1 mL) was added dropwise to the ylide solution via cannula at r.t. over 5 min, and the resulting mixture was stirred at r.t. for 2 h. Then, the reaction was quenched by the addition of sat. NH₄Cl aq. The whole mixture was extracted with AcOEt, and the AcOEt extract was washed with NH₄Cl aq. and brine. Concentration of the AcOEt extract in vacuo gave a crude product which was purified by SiO₂ column chromatography (hexane : AcOEt = 1 : 1) to give 44 (29 mg, 70%) as a colorless oil.

¹H-NMR (600 MHz, CDCl₃) δ: 0.74–1.50 (15H, m), 1.56 (2H, br s), 1.71–2.24 (9H, m), 2.60–3.26 (8H, m), 5.07 (2H, s), 5.42–5.83 (2H, m), 7.29–7.54 (4H, m), 7.75–7.90 (2H, m), 8.06 (1H, br s). ¹³C-NMR (150 MHz, CDCl₃) δ: 14.4, 25.3, 25.8, 26.8, 27.4, 27.5, 28.4, 28.5, 28.6, 28.7, 29.1, 30.0, 31.9, 34.3, 34.7, 36.0, 48.4, 52.7, 79.3, 79.4, 112.4, 124.0, 125.3, 126.1, 126.5, 127.3, 128.4, 128.8, 131.3, 132.6, 133.9, 142.0, 155.80, 155.82, 173.5. IR (KBr) cm⁻¹: 2931, 1686, 1616, 1397, 1216, 754. MS (ESI-TOF) m/z: 563 [M + Na]⁺. HR-MS (ESI-TOF) m/z: 563.3016 calcd for C₃₂H₄₅N₂O₃³⁵ClNa; Found: 563.3020.

(2E,9E)-9-(Chloromethylene)-12-((E)-3-methoxy-N-methylbut-2-enamido)-N,2-dimethyl-N-(naphthalen-1-ylmethyl)dodec-2-enamide (45)

TFA (83 µL, 1.1 mmol, 20 equiv.) was added to a solution of 44 (29 mg, 0.057 mmol) in CH₂Cl₂ (1 mL), and the mixture was stirred at r.t. for 1 h. Then, the reaction was quenched by the addition of sat. NaHCO₃ aq. The whole mixture was extracted with AcOEt, and the AcOEt extract was washed with sat. NaHCO₃ aq. and brine. Concentration of the AcOEt extract in vacuo gave an amine, which was used in the next reaction without further purification. The above crude product was dissolved to CH₂Cl₂ (1 mL). HOBt (11 mg, 0.080 mmol, 1.4 equiv.), 21 (9.3 mg, 0.080 mmol, 1.4 equiv.), Et₃N (32 µL, 0.23 mmol, 4.0 equiv.), and EDCI·HCl (15 mg, 0.080 mmol, 1.4 equiv.) were added to the solution, and the resulting mixture was stirred at r.t. for 2.5 h. Then, the reaction was quenched by the addition of sat. NH₄Cl aq. The whole mixture was extracted with AcOEt, and the AcOEt extract was successively washed with sat. NH₄Cl aq., NaHCO₃ aq., and brine. Concentration of the AcOEt extract in vacuo gave a crude product which was purified by SiO₂ column chromatography (hexane : AcOEt = 1 : 2) to give 45 and 46 (8.0 mg, 26%, two steps) as a colorless oil.

45: ¹H-NMR (500 MHz, CDCl₃) δ: 1.25–1.66 (8H, br), 1.86 (3H, s), 2.03 (6H, br), 2.18 (3H, s), 2.58–3.12 (6H, br), 3.32 (2H, br), 3.61 (3H, s), 5.07 (2H, s), 5.15 (1H, d, J = 5.9 Hz), 5.57 (1H, s), 5.77 (1H, s), 7.32–7.96 (9H, m). ¹³C-NMR (150 MHz, CDCl₃) δ: 11.1, 14.2, 14.4, 18.9, 23.1, 23.7, 23.8, 25.7, 26.2, 26.9, 27.6, 28.7, 29.0, 29.8, 30.0, 30.5, 31.6, 32.2, 33.5, 36.1, 38.8, 47.4, 49.8, 55.0, 68.3, 91.0, 91.3, 112.4, 113.0, 124.0, 125.4, 126.1, 126.6, 127.2, 128.5, 128.9, 131.0, 131.4, 131.9, 132.5, 133.9, 164.0, 167.9, 174.1. IR (KBr) cm⁻¹: 2930, 1728, 1275, 1123. MS (ESI-TOF) m/z: 561 [M + Na]⁺. HR-MS (ESI-TOF) m/z: 561.2860 calcd for C₃₂H₄₃N₂O₃³⁵ClNa; Found: 561.2850.

(2E,9E)-9-(Chloromethylene)-N,2-dimethyl-12-(N-methylacetamido)-N-(thiazol-2-ylmethyl)dodec-2-enamide (48)

TFA (46 µL, 0.62 mmol, 30 equiv.) was added to a solution of 17 (10 mg, 0.020 mmol) in CH₂Cl₂ (1 mL), and the mixture was stirred at r.t. for 1 h. Then, the reaction was quenched by the addition of sat. NaHCO₃ aq. The whole mixture was extracted with AcOEt, and the AcOEt extract was washed with sat. NaHCO₃ aq. and brine. Concentration of the AcOEt extract in vacuo gave an amine, which was used in the next reaction without further purification. The above crude product was dissolved to CH₂Cl₂ (1 mL). 4-Dimethylaminopyridine (DMAP, 3.8 mg, 0.031 mmol, 1.5 equiv.), Et₃N (60 µL, 0.43 mmol, 21 equiv.), and acetyl chloride (10 µL, 0.14 mmol, 6.9 equiv.) were added to the solution, and the resulting mixture was stirred for 1 h at r.t. Then, the reaction was quenched by the addition of sat. NaHCO₃ aq. The whole mixture was extracted with AcOEt, and the AcOEt extract was washed with sat. NaHCO₃ aq., and brine. Concentration of the AcOEt extract in vacuo gave a crude product which was purified by SiO₂ column chromatography (hexane : AcOEt = 1 : 5) to give 48 and 49 (9.1 mg, quant., two steps) as a colorless oil.

48: ¹H-NMR (600 MHz, CDCl₃) δ: 1.11–1.46 (6H, m), 1.53–1.72 (2H, m), 1.85 (3H, s), 1.97–2.24 (9H, m), 2.86–2.92 (3H, m), 2.93–3.09 (3H, m), 3.18–3.38 (2H, m), 4.86 (2H, s), 5.63 (1H, br s), 5.74–5.83 (1H, m), 7.32 (1H, br s), 7.71 (1H, br s). ¹³C-NMR (150 MHz, CDCl₃) δ: 14.4, 21.4, 22.1, 24.8, 25.4, 25.9, 26.3, 26.9, 27.0, 27.4, 27.48, 27.53, 27.65, 27.67, 28.70, 28.72, 29.0, 29.2, 29.8, 29.97, 30.02, 31.8, 32.1, 33.3, 34.8, 36.3, 37.1, 47.3, 48.6, 50.3, 112.6, 113.1, 113.2, 120.2, 130.8, 131.0, 132.4, 141.3, 142.0, 142.4, 166.9, 170.4, 170.6, 173.9. IR (KBr) cm⁻¹: 2928, 2857, 1635, 1395. MS (ESI-TOF) m/z: 462 [M + Na]⁺. HR-MS (ESI-TOF) m/z: 462.1958 calcd for C₂₂H₃₄N₃O₂S³⁵ClNa; Found: 462.1936.

tert-Butyl (4-(Methoxy(methyl)amino)-4-oxobutyl)carbamate (51)

HOBt (186 mg, 1.4 mmol, 1.4 equiv.), MeONHMe·HCl (137 mg, 1.4 mmol, 1.4 equiv.), Et₃N (548 mL, 3.9 mmol, 4.0 equiv.), and EDCI·HCl (264 mg, 1.4 mmol, 1.4 equiv.) were added to a solution of 50 (200 mg, 0.98 mmol) in CH₂Cl₂ (5 mL), and the mixture was stirred at r.t. for 12 h. Then, the reaction was quenched by the addition of sat. NaHCO₃ aq. The whole mixture was extracted with AcOEt, and the AcOEt extract was washed with sat. NaHCO₃ aq., and brine. Concentration of the AcOEt extract in vacuo gave a crude product which was purified by SiO₂ column chromatography (hexane : AcOEt = 1 : 1) to give 51 (216 mg, 89%) as a colorless oil.

¹H-NMR (600 MHz, CDCl₃) δ: 1.40 (3H, s), 1.79 (2H, m), 2.44 (2H, s), 2.95–3.30 (5H, m), 3.65 (3H, s), 4.76 (1H, s). ¹³C-NMR (150 MHz, CDCl₃) δ: 24.9, 28.5, 29.3, 32.3, 40.4, 61.3, 79.2, 156.1, 174.2. IR (KBr) cm⁻¹: 3347 (br), 2975, 1711, 1523, 1173. MS (ESI-TOF) m/z: 269 [M + Na]⁺. HR-MS (ESI-TOF) m/z: 269.1477 calcd for C₁₁H₂₂N₂O₄Na; Found: 269.1464.

tert-Butyl (4-(Methoxy(methyl)amino)-4-oxobutyl)(methyl)carbamate (52)

NaH (about 60% oil suspension, 37 mg, 0.93 mmol, 1.5 equiv.) was added to a solution of 51 (154 mg, 0.62 mmol) in DMF (3 mL) at 0°C, and the mixture was stirred at 0°C for 15 min. Iodomethane (388 µL, 6.2 mmol, 10 equiv.) was added to the amide solution, and the resulting mixture was stirred at r.t. for 12 h. Then, the reaction was quenched by the addition of sat. NH₄Cl aq. The whole mixture was extracted with AcOEt, and the AcOEt extract was washed with NH₄Cl aq. and brine. Concentration of the AcOEt extract in vacuo gave a crude product which was purified by SiO₂ column chromatography (hexane : AcOEt = 1 : 1) to give 52 (148 mg, 91%) as a colorless oil.

¹H-NMR (600 MHz, CDCl₃) δ: 1.41 (9H, s), 1.71–1.87 (2H, m), 2.38 (2H, s), 2.81 (3H, s), 3.13 (3H, s), 3.23 (2H, t, J = 6.9 Hz), 3.63 (3H, s). ¹³C-NMR (150 MHz, CDCl₃) δ: 22.7, 28.5, 28.9, 32.2, 34.1, 48.3, 61.2, 79.3, 155.9, 174.1. IR (KBr) cm⁻¹: 2974, 1693, 1392, 1172. MS (ESI-TOF) m/z: 283 [M + Na]⁺. HR-MS (ESI-TOF) m/z: 283.1634 calcd for C₁₂H₂₄N₂O₄Na; Found: 283.1631.

tert-Butyl Methyl(4-oxopentyl)carbamate (53)

CH₃Li (3.0 M in Et₂O, 128 µL, 0.38 mmol, 1.1 equiv.) was added to a solution of 52 (91 mg, 0.35 mmol) in Et₂O (1 mL) at 0°C, and the mixture was stirred at 0°C for 0.5 h. Then, the reaction was quenched by the addition of sat. NH₄Cl aq. The whole mixture was extracted with AcOEt, and the AcOEt extract was washed with NH₄Cl aq. and brine. Concentration of the AcOEt extract in vacuo gave a crude product which was purified by SiO₂ column chromatography (hexane : AcOEt = 3 : 1) to give 53 (56 mg, 75%) as a colorless oil. The spectroscopic and physical data were identical to those reported.¹¹⁾

tert-Butyl (5-Chloro-4-methylpent-4-en-1-yl)(methyl)carbamate (54)

LHMDS solution (1.0 M in THF, 436 µL, 0.44 mmol, 2.9 equiv.) was added to a solution of (chloromethyl)triphenylphosphonium chloride (156 mg, 0.45 mmol, 3.0 equiv.) in anhydrous THF (1 mL), and the mixture was stirred at r.t. for 15 min. A solution of 53 (32 mg, 0.15 mmol) in anhydrous THF (0.5 mL) was added dropwise to the ylide solution via cannula at r.t., and the resulting mixture was stirred at r.t. for 0.5 h. Then, the reaction was quenched by the addition of sat. NH₄Cl aq. The whole mixture was extracted with AcOEt, and the AcOEt extract was washed with NH₄Cl aq. and brine. Concentration of the AcOEt extract in vacuo gave a crude product which was purified by SiO₂ column chromatography (hexane : AcOEt = 8 : 1) to give 54 (40 mg, quant.) as a colorless oil.

¹H-NMR (600 MHz, CDCl₃) δ: 1.40 (9H, s), 1.58 (2H, quint, J = 7.6 Hz), 1.67–1.77 (3H, m), 1.99 (1H, t, J = 7.6 Hz), 2.09–2.20 (1H, t, J = 7.6 Hz), 2.79 (3H, br s), 3.13 (2H, br s), 5.68–5.80 (1H, m). ¹³C-NMR (150 MHz, CDCl₃) δ: 16.4, 20.8, 24.7, 25.1, 25.6, 25.7, 28.3, 28.5, 29.1, 34.2, 48.1, 48.2, 48.4, 48.8, 79.2, 79.3, 112.0, 112.3, 137.9, 155.7. IR (KBr) cm⁻¹: 2975, 1696, 1393, 1166. MS (ESI-TOF) m/z: 270 [M + Na]⁺. HR-MS (ESI-TOF) m/z: 270.1237 calcd for C₁₂H₂₂NO₂³⁵ClNa; Found: 270.1249.

(E)-N-((E)-5-Chloro-4-methylpent-4-en-1-yl)-3-methoxy-N-methylbut-2-enamide (55)

TFA (120 µL, 1.6 mmol, 10 equiv.) was added to a solution of 54 (40 mg, 0.16 mmol) in CH₂Cl₂ (1 mL), and the mixture was stirred at r.t. for 2.5 h. Then, the reaction was quenched by the addition of sat. NaHCO₃ aq. The whole mixture was extracted with AcOEt, and the AcOEt extract was washed with sat. NaHCO₃ aq. and brine. Concentration of the AcOEt extract in vacuo gave an amine, which was used in the next reaction without further purification. The above crude product was dissolved to CH₂Cl₂ (1 mL). HOBt (32 mg, 0.24 mmol, 1.5 equiv.), 21 (28 mg, 0.24 mmol, 1.5 equiv.), Et₃N (180 mL, 1.3 mmol, 8.0 equiv.), and EDCI·HCl (46 mg, 0.24 mmol, 1.5 equiv.) were added to the solution, and the resulting mixture was stirred at r.t. for 1 h. Then, the reaction was quenched by the addition of sat. NaHCO₃ aq. The whole mixture was extracted with AcOEt, and the AcOEt extract was washed with sat. NaHCO₃ aq., and brine. Concentration of the AcOEt extract in vacuo gave a crude product which was purified by SiO₂ column chromatography (hexane : AcOEt = 2 : 1) to give 55 and 56 (9.7 mg, 25%, two steps) as a colorless oil.

55: ¹H-NMR (600 MHz, CDCl₃) δ: 1.68 (2H, m), 1.77 (3H, s), 2.06 (2H, t, J = 7.6 Hz), 2.18 (3H, s), 2.85–2.99 (3H, m), 3.32 (2H, m), 3.59 (3H, s), 5.14 (1H, s), 5.81 (1H, s). ¹³C-NMR (150 MHz, CDCl₃) δ: 16.5, 18.9, 25.5, 26.0, 33.5, 34.0, 34.6, 36.1, 47.4, 49.7, 55.0, 91.0, 91.3, 112.3, 112.9, 137.5, 138.2, 168.2, 168.3, 168.7. IR (KBr) cm⁻¹: 2932, 1650, 1380, 1240, 1158. MS (ESI-TOF) m/z: 268 [M + Na]⁺. HR-MS (ESI-TOF) m/z: 268.1080 calcd for C₁₂H₂₀NO₂³⁵ClNa; Found: 268.1088.

(E)-2-Methylhept-2-enoic Acid (58)

LiOH (83 mg, 3.5 mmol, 2.0 equiv.) was added to a solution of 57 (295 mg, 1.7 mmol) in THF : MeOH : H₂O (1 : 1 : 1, 9 mL), and the mixture was stirred at r.t. for 20 h. The reaction was quenched by the addition of 5% HCl, and the whole mixture was extracted with AcOEt. Then, the AcOEt extract was washed with brine. Concentration of the AcOEt extract in vacuo gave a crude product which was purified by SiO₂ column chromatography (hexane : AcOEt = 5 : 1) to give 58 (208 mg, 84%) as a colorless oil.

¹H-NMR (600 MHz, CDCl₃) δ: 0.87 (3H, t, J = 7.3 Hz), 1.25–1.44 (4H, m), 1.79 (3H, s), 2.11–2.20 (2H, m), 6.89 (1H, t, J = 7.6 Hz), 12.62 (1H, s). ¹³C-NMR (150 MHz, CDCl₃) δ: 11.8, 13.7, 22.5, 28.6, 30.6, 127.1, 145.3, 174.1. IR (KBr) cm⁻¹: 2959 (br), 1686, 1281. In mass mesurement, the spectra of sodium salt of compound 78 was obtained. MS (ESI-TOF) m/z: 187 [M + Na]⁺. HR-MS (ESI-TOF) m/z: 187.0711 calcd for C₈H₁₃O₂Na₂; Found: 187.0715.

(E)-N,2-Dimethyl-N-(thiazol-2-ylmethyl)hept-2-enamide (59)

HOBt (85 mg, 0.63 mmol, 1.4 equiv.), amine 20 (114 mg, 0.89 mmol, 2.0 equiv.), Et₃N (523 µL, 3.8 mmol, 6.0 equiv.), and EDCI·HCl (120 mg, 0.63 mmol, 1.4 equiv.) were added to a solution of 58 (64 mg, 0.45 mmol) in CH₂Cl₂ (5 mL), and the mixture was stirred at r.t. for 24 h. Then, the reaction was quenched by the addition of sat. NaHCO₃ aq. The whole mixture was extracted with AcOEt, and the AcOEt extract was washed with sat. NaHCO₃ aq., and brine. Concentration of the AcOEt extract in vacuo gave a crude product which was purified by SiO₂ column chromatography (hexane : AcOEt = 1 : 1) to give 59 (103 mg, 91%) as a colorless oil.

¹H-NMR (600 MHz, CDCl₃) δ: 0.81 (3H, t, J = 6.8 Hz), 1.24 (2H, br s), 1.30 (2H, br s), 1.79 (3H, s), 2.03 (2H, dd, J = 14.0, 6.9 Hz), 2.97 (3H, s), 4.81 (2H, br s), 5.58 (1H, s), 7.26 (1H, br s), 7.64 (1H, br s). ¹³C-NMR (150 MHz, CDCl₃) δ: 13.7, 14.0, 22.1, 27.1, 30.7, 36.1, 47.8, 119.9, 129.8, 130.3, 132.2, 141.9, 166.6, 173.6. IR (KBr) cm⁻¹: 2927, 1631, 1393, 1074. MS (ESI-TOF) m/z: 275 [M + Na]⁺. HR-MS (ESI-TOF) m/z: 275.1194 calcd for C₁₃H₂₀N₂OSNa; Found: 275.1201.

Acknowledgments

The authors are grateful to JSPS KAKENHI (Grant Nos. JP26242074, JP15K07996 and JP18K05363) and Hoansha Foundation for financial support. This study was supported by the Platform Project for Supporting Drug Discovery and Life Science Research (Platform for Drug Discovery, Informatics, and Structural Life Science) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) and the Japan Agency for Medical Research and Development (AMED).

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

The online version of this article contains supplementary materials.

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