Synthesis of methyl 4-dihydrotrisporate B and methyl ...

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Jul 13, 2018 - previously described iodoalkene 9 [42] under Baldwin-. Lee conditions [43] without protection groups resulted in formation of ( ±)-(9Z)-methyl ...
Z. Naturforsch. 2018; 73(1–2)c: 59–66

Yoko Nakamura, Christian Paetz and Wilhelm Boland*

Synthesis of methyl 4-dihydrotrisporate B and methyl trisporate B, morphogenetic factors of Zygomycetes fungi https://doi.org/10.1515/znc-2017-0148 Received August 20, 2017; revised October 6, 2017; accepted October 12, 2017

Abstract: (9Z)-Methyl 4-dihydrotrisporate B and (9Z)methyl trisporate B, pheromones of Zygomycetes fungi, have been synthesized using Stille cross-coupling from previously described cyclohexenone precursors. Conducting the coupling without protection groups allowed for a short and stereospecific synthesis route of the late trisporoids. Stability studies for both the compounds revealed (9Z)-methyl trisporate B to be very unstable against UV irradiation. Keywords: natural products; Stille cross-coupling; total synthesis; trisporoids; Zygomycetes.

1 Introduction Trisporoids, C-18 apocarotenoids, are fungal morphogenetic factors isolated earlier from cultures of Mucor mucedo, Blakeslea trispora, and Phycomyces blakesleeanus [1–7]. The Zygomycetes’ sexual reproduction is initiated by two mating types ( +) and ( −), which are undistinguishable regarding their morphology. However, when both mating types are spatially close to each other, the development of special organs called zygophores can be observed. Eventually, zygophores transform into zygospores as the final structures of sexual reproduction [8]. Trisporoids have been identified to be involved in the sexual development processes of Zygomycetes. For example, trisporoids act during recognition of compatible mating types, regulate the early sexual morphogenesis (zygophore induction), and also influence the fungus’ metabolism, especially through carotene biosynthesis [8–12]. These intriguing *Corresponding author: Wilhelm Boland, Max Planck Institute for Chemical Ecology, Department of Bioorganic Chemistry, Hans-KnöllStraße 8, 07745 Jena, Germany, E-mail: [email protected] Yoko Nakamura: Max Planck Institute for Chemical Ecology, Department of Bioorganic Chemistry, Jena, Germany Christian Paetz: Max Planck Institute for Chemical Ecology, Biosynthesis/NMR, Jena, Germany

biological activities are controlled by structurally and functionally variable trisporoids. They were grouped into series A–E according to their substitutions at R4, R5, R6, R7 (here only series B, C and E were shown for clarity). Furthermore, they were classified into early and late trisporoids depending on the oxidation state of the ring system (R1, R2, R3) (Figure 1). Several total syntheses of trisporoids have been achieved by different strategies. Recently, concise syntheses of early trisporoids have been developed. Starting from ß- or α-ionone, Pd(0)-catalyzed cross-coupling with organozincates [13] or cross-metathesis [14] gave the target molecules as E/Z mixtures in good total yield [15, 16]. For the synthesis of late trisporoids, multiple transformation steps were needed in order to construct the oxidized cyclohexenyl ring, followed by chain elongation by means of Wittig olefination [17–26]. Generally, all previously described syntheses gave E/Z isomers in low yields even when stereochemically pure side chains served as starting materials [23]. Consequently, only limited amounts of late trisporoids were obtained by isolation from cross-cultures of B. trispora, followed by chromatographic purification procedures [27, 28]. Methyl 4-dihydrotrisporate (Figure 1) is one of the late trisporoids isolated only from the ( +)-mating type of Zygomycetes [9, 29–31]. It is believed to be a substrate of methyl 4-dihydromethyltrisporate dehydrogenase, which is one of the few known enzymes in trisporoid biosynthesis [32– 34]. Because of the instability and the low in vivo concentration of methyl 4-dihydrotrisporate, the molecule was synthesized from other trisporic acids obtained by isolation [30, 32]. However, this semi-synthetic approach gave generally diastereomeric mixtures of methyl 4-dihydrotrisporate C, and although this compound was reported as a minor component in fungus cultures [9], no spectroscopic data proving its identity were published [32–34]. Thus, a stereospecific minimum-step synthesis of late trisporoids, especially for (9Z)-methyl 4-dihydrotrisporate B (1), should be developed for future biological studies. So far, only one strategy for the stereospecific synthesis of trisporoids has been published using the Suzuki-Miyaura coupling, which resulted in the synthesis of the benzyl ether of ( ±)-trisporol B [35]. Another synthetic concept based on Suzuki’s strategy proposed an enynone 3 [36, 37] Bereitgestellt von | Max Planck eBooks Angemeldet Heruntergeladen am | 13.07.18 11:48

60      Nakamura et al.: Synthesis of methyl 4-dihydrotrisporate B and methyl trisporate B

2 Results and discussion 2.1 Efficient syntheses of late trisporoids

Figure 1: Early and late trisporoids (upper section), (9Z)-methyl 4-dihydrotrisporate B (1) and (9Z)-methyl trisporate B (2) (lower section).

as a starting compound, but no total synthesis of the trisporoid was reported. Here we report on the total synthesis of the late trisporoids ( ±)-(9Z)-methyl 4-dihydrotrisporate B (1) and ( ±)-(9Z)-methyl trisporate B (2) in four steps from enynone 3 using the Stille cross-coupling. In addition, we systematically examined the chemical properties and stability of both target compounds.

As shown in Scheme 1, we started from the known enynone 3 synthesized by a reported method [36, 37]. Using the Luche reduction, the inseparable alcohols 4a/4b (1.56:1) were obtained in quantitative yield. For the formation of the C8–C9 bond, we chose the Stille cross-coupling which proceeded under very mild conditions avoiding isomerization of the double bonds [38, 39]. Interestingly, hydrostannylation of 4a/4b by Bu3SnH/azobisisobutyronitrile (AIBN) in toluene at 130 °C [40] directly gave lactone 5 in 59% yield (from 4a) and hydroxystannane 6 in 98% yield (from 4b), which were easily separated by column chromatography in a mixture of silica gel and K2CO3 (9:1) [41]. We observed only E-addition of tributyltin in both cases. The reaction at 80 °C afforded only the hydroxystannane 6 in low yield without lactone 5 and hydroxystannane 7. A ring-opening reaction of lactone 5 by treatment with K2CO3 in methanol yielded hydroxystannane 7 with the desired stereochemistry for 1 in 91% based on recovered starting material (42% yield of 7 with 54% recovery of 5). No improvements were achieved by variation of the reaction conditions. For example, using NaOMe resulted

Scheme 1: Total synthesis of (9Z)-methyl 4-dihydrotrisporate B (1) and (9Z)-methyl trisporate B (2).

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Nakamura et al.: Synthesis of methyl 4-dihydrotrisporate B and methyl trisporate B      61

in only 20% yield and excess K2CO3 (10 eq.), room temperature to 80 °C, gave only 17% yield with 50% recovery of 5. Stille cross-coupling of hydroxystannane 7 with the previously described iodoalkene 9 [42] under BaldwinLee conditions [43] without protection groups resulted in formation of ( ±)-(9Z)-methyl 4-dihydrotrisporate B (1) with the desired stereochemistry in 76% yield. Without copper catalyst, the Stille cross-coupling gave no reaction. Hydroxystannane 6 was oxidized with MnO2 to stannyl enynone 8. Stille cross-coupling of 8 and iodoalkene 9 under the same conditions as above afforded ( ±)-(9Z)methyl trisporate B (2) with (7E, 9Z)-configuration in excellent yield. The syntheses were accomplished in four steps resulting in 19% overall yield for 1 (without considering the recovery of lactone 5) and in 80% for 2 from enynone 3. Since direct stannylation (Bu3SnH/AIBN in toluene) of enynone 3 failed, this alternative route for ( ±)-(9Z)-methyl trisporate B (2) proved to be very useful.

2.2 C  hemical properties of late trisporoids 2.2.1 Structure elucidation The complete NMR spectroscopic assignment of ( ±)-(9Z)methyl 4-dihydrotrisporate B (1) and ( ±)-(9Z)-methyl trisporate B (2) was accomplished using 1H, 13C, 1H-1H correlation spectroscopy, 1H-13C heteronuclear singlequantum coherence and 1H-13C heteronuclear multiplebond correlation experiments. In addition, the 1H-1H rotating frame Overhauser effect spectroscopy (ROESY) spectrum (Figure 2) showed strong correlations between H-18 (–CH3), H-10 (–CH–), H-8 (–CH–) and H-11 (–CH2–), proving the 9Z-configuration in both cases. Furthermore, we observed the correlation of H-7 (–CH–) with H-17 (–CH3) and H-8 (–CH–) with H-15 (–CH3), which suggested that both molecules have the configuration shown in Figure 2. A different configuration of the t-butyl-dimethylsilyl-protected 4-dihydrotrisporin B has been reported earlier [16] . 2.2.2 S  tability against UV irradiation, ambient daylight and varying pH conditions It was recommended to store trisporoids only in the dark and under inert gas [9, 15, 23–25]. However, the stability of (1) and (2) under light and varying pH conditions has never been systematically tested. We exposed 500 μM ( ±)-(9Z)-methyl 4-dihydrotrisporate B (1) and ( ±)-(9Z)methyl trisporate B (2) solutions in phosphate buffer (pH 7.2) to ambient daylight (450 ± 50 lx) or UV light (combined

H-8 H-7

H-10 ppm

CH3-15

1.4 1.6

CH3-18 CH3-17

1.8 17

HO

CH3-14 H2O

18

7

15 8 11 O

CH2-11

10

O

2.0 14

2.2 1

O 6.6

6.4

6.2

6.0

5.8

5.6

H-7

H-8

ppm

5.4 H-10

ppm CH3-5

1.6 1.8

CH3-18 CH3-17 O

CH3-14

17

7

15 8 O 11

CH2-11

2.0

18 10 O

14 2

O 6.8

6.6

6.4

6.2

2.2

6.0

5.8

5.6

2.4 ppm

Figure 2: Details of the 1H-1H ROESY spectra of (9Z)-methyl 4-dihydrotrisporate B (1) and (9Z)-methyl trisporate B (2).

254 nm and 366 nm, 2.8 and 6.8 W m−2 nm−1, respectively) during a time course of 20 h. (9Z)-Methyl 4-dihydrotrisporate B (1) was stable against ambient light after 20 h. After UV irradiation we observed a decrease in concentration of 1 (Figure S1a). (9Z)-Methyl trisporate B (2) was unstable against ambient daylight and very unstable under UV light irradiation (Figure S1b). For both cases, we assume degradation by photo-oxidation, since only a small part of isomeric compounds was detectable while the major part of degradation products was completely lacking UV absorptions. Under variable pH conditions, (9Z)-methyl 4-dihydrotrisporate B (1) was stable between pH 5.6 and pH 9, but unstable under more acidic or stronger basic conditions (Figure S2a). The analysis of the crude mixture revealed 4,6,8-trien-10-ol (10) as the major conversion product (Figure S3). A similar structural change caused by elimination of the C-4 hydroxy group was reported previously [9, 30, 44]. (9Z)-Methyl trisporate B (2) was stable over the entire pH range (Figure S2b).

3 Conclusions We developed a short and stereospecific total synthesis of the late trisporoids ( ±)-(9Z)-methyl 4-dihydrotrisporate B Bereitgestellt von | Max Planck eBooks Angemeldet Heruntergeladen am | 13.07.18 11:48

62      Nakamura et al.: Synthesis of methyl 4-dihydrotrisporate B and methyl trisporate B (1) and ( ±)-(9Z)-methyl trisporate B (2) in four steps from the known enynone 3 using the Stille cross-coupling approach. All compounds were structurally characterized, and their stability against UV and ambient daylight, as well as under variable pH conditions, was examined. We hope this work will make late trisporoids more accessible for biological research.

4 Experimental section 4.1 General methods All solvents and reagents were used as supplied from commercial sources. NMR measurements were carried out on a Bruker Avance AV-500HD spectrometer, equipped with a TCI cryoprobe using standard pulse sequences as implemented in Bruker Topspin ver. 3.5 (Bruker Biospin GmbH, Rheinstetten, Germany). Chemical shifts were referenced to the residual solvent signals for measurements in CD3CN (δH 1.94/δC 1.39) and in CDCl3 (δH 7.27/δC 77.23). HPLC-HRESIMS measurements were conducted on an Agilent Infinity 1260  system (quaternary pump G1311B, autosampler G1367E, column oven G1316A and diode array detector G1315D, Agilent Technologies GmbH, Waldbronn, Germany) coupled to a Bruker Compact ESI-OTOF spectrometer (Bruker Daltonics GmbH, Bremen, Germany). An Agilent Zorbax SB-C18 column, 150 × 4.6 mm and 3.5 μm particle size, was used. The following conditions were used for chromatographic separations: mobile phase A – H2O (0.1% v/v formic acid); mobile phase B – MeCN (0.1% v/v formic acid). Initial composition was 95% A, kept for 2 min after injection, and then 5% A after 20  min, which was kept isocratic until 30 min. Then the composition was changed to 95% A after 33  min, which was kept until 35  min. For GC-MS measurements an ISQ LT and a Trace 1310 (Thermo Fisher Scientific, Waltham, MA, USA), equipped with a ZB5 column (15 m × 0.25 mm × 0.25 μm), were used. Helium gas (flow rate 1.5 mL min−1) served as carrier gas. The GC injector (split ratio 1:10), transfer line, and ion source were set to 250 °C, 280 °C and 250 °C, respectively. Oven program conditions: from 50 °C (held for 1 min) to 300 °C (held for 1 min) with 15 °C min−1. GC mass spectra were measured in electron impact (EI) mode at 70 eV, 50–450 m/z. For open column chromatography, Merck silica gel Si60, 0.040–0.063  mm (230–400  mesh), and Florisil (60–100 mesh) were used.

4.2 Synthetic procedures 4.2.1 Methyl (1S*,4R*)-2-ethynyl-4-hydroxy-1,3dimethylcyclohex-2-ene-1-carboxylate (4a) and methyl(1S*,4S*)-2-ethynyl-4-hydroxy-1,3dimethylcyclohex-2-ene-1-carboxylate (4b) To enynone 3 (50  mg, 0.242  mmol) and CeCl3 · 7H2O (112 mg, 0.300 mmol) in abs. EtOH (2 mL), NaBH4 (9.2 mg, 0.242  mol) was added at 0 °C. The reaction mixture was stirred for 30  min, and then hydrolyzed with saturated aq. NH4Cl and extracted by methyl-tert-butyl ether (MTBE). The organic layer was washed with brine, dried over Na2SO4, and filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (n-hexane:EtOAc = 13:1–3:2) to give 4a and 4b in quantitative yield (50.4  mg, 0.242  mmol, diastereomeric mixture 4a:4b = 1.56:1 from NMR) as a colorless oil. 4a: 1H-NMR (500 MHz, CDCl3) δ ppm: 4.03 (t, J = 5.5 Hz, 1H, H-4), 3.71 (s, 3H, –OMe), 3.13 (s, 1H, H-2′), 2.17 (m, 1H, H-6a), 2.07 (s, 3H, CH3-3), 1.72 (m, 2H, H-5a/b), 1.54 (m, 1H, H-6b), 1.39 (s, 3H, CH3-1). 13C-NMR (125 MHz, CDCl3) δ ppm: 176.6 (–C=O), 145.4 (C-3), 120.8 (C-2), 82.4 (C-2′), 81.3 (C-1′), 68.6 (C-4), 52.6 (–OMe), 47.0 (C-1), 30.4 (C-6), 28.4 (C-5), 23.6 (CH3-1), 19.4 (CH3-3), 4b: 1H-NMR (500 MHz, CDCl3) δ ppm: 4.11 (t, J = 4.8 Hz, 1H, H-4), 3.69 (s, 3H, –OMe), 3.12 (s, 1H, H-2′), 2.03 (m, 1H, H-6a), 2.07 (s, 3H, CH3-3), 1.90 (m, 2H, H-5a/b), 1.70 (m, 1H, H-6b), 1.46 (s, 3H, CH3-1). 13C-NMR (125 MHz, CDCl3) δ ppm: 176.2 (–C=O), 145.2 (C-3), 121.1 (C-2), 82.6 (C-2′), 81.4 (C-1′), 68.3 (C-4), 52.4 (–OMe), 46.7 (C-1), 30.2 (C-6), 28.5 (C-5), 24.7 (CH3-1), 19.6 (CH3-3). EI-MS (70 eV): m/z (%) 208 (9), 193 (35), 152 (46), 107 (65), 91 (100); HRMS (ESI, positive mode) m/z [M + Na]+: calcd. for C12H16NaO3, 231.0992; found, 231.0990.

4.2.2 (1R*,4S*)-4,6-dimethyl-5-((E)-2-(tributylstannyl) vinyl)-2-oxabicyclo[2.2.2]oct-5-en-3-one (5), methyl (1S*,4S*)-4-hydroxy-1,3-dimethyl-2((E)-2-(tributylstannyl)vinyl)-cyclohex-2-ene-1carboxylate (6) To a mixture of 4a and 4b (32 mg, 0.154 mmol, diastereomeric mixture 4a:4b = 1.56:1) and 0.2  M AIBN (77 μL, 0.015 mmol in toluene) in anhydrous toluene (3.7 mL) was added n-Bu3SnH (166 μL, 0.616  mmol). The mixture was heated to 130 °C and stirred at 130 °C for 1 h. Subsequently, the solvent was evaporated and the residue was purified by column chromatography (silica gel:K2CO3 = 9:1, n-hexane/

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Nakamura et al.: Synthesis of methyl 4-dihydrotrisporate B and methyl trisporate B      63

EtOAc = 20:1 to 5:1) to give 5 [25.7  mg, 0.055  mmol, 59% from 4a (0.094 mmol)] as a colorless oil and 6 [29.9 mg, 0.059  mmol, 98% from 4b (0.06  mmol)] as a slightly yellow oil. (5): 1H-NMR (500  MHz, MeCN-d3) δ ppm: 6.32 (d, J = 19.6 Hz, 3JSnH = 31.9 Hz, 1H, H-1′), 6.19 (d, J = 19.6 Hz, 2 J117SnH = 36.9  Hz, 2J119SnH = 35.3  Hz, 1H, H-2′), 4.96 (m, 1H, H-4), 2.08 (m, 1H, H-2a), 1.91 (bs, 3H, CH3-6), 1.68 (m, 1H, H-2b), 1.61 (m, 1H, H-3a), 1.54 (m, 6H, H-2″), 1.48 (m, 1H, H-3b), 1.33 (s, 3H, CH3-4), 1.31 (m, 6H, H-3″), 0.95 (m, 6H, H-1″), 0.89 (m, 9H, H-4″). 13C-NMR (125  MHz, MeCN-d3) δ ppm: 177.5 (–C=O), 140.5 (2JSnC = 4.4Hz, C-1′), 140.3 (C-5), 138.0 (1J117SnC = 179.8  Hz, 1J119SnC = 171.7  Hz, C-2′), 135.5 (C-6), 79.9 (C-1), 46.1 (C-4), 30.0 (2JSnC = 10.7  Hz, C-2″), 29.5 (C-3), 28.0 (3JSnC = 25.8 Hz, C-3″), 26.4 (C-2), 17.9 (CH3-4), 16.5 (CH36), 14.0 (C-4″), 10.3 (1J117SnC = 173.2  Hz, 1J119SnC = 165.6  Hz, C-1″). HRMS (ESI, positive mode) m/z [M + Na]+: calcd. for C23H40NaO2Sn, 491.1947; found, 491.1946 (see Supplementary Information). (6): 1H-NMR (500 MHz, MeCN-d3) δ ppm: 6.47 (d, J = 19.9 Hz, 3JSnH = 34.2 Hz, 1H, H-1′), 5.95 (d, J = 19.9 Hz, 2J117SnH = 37.3 Hz, 2J119SnH = 35.6 Hz, 1H, H-2′), 3.99 (m, 1H, H-4), 3.56 (s, 3H, –OMe), 2.87 (d, J = 6.7  Hz, 1H, –OH), 1.88 (m, 1H, H-5a), 1.86 (m, 1H, H-6a), 1.83 (s, 3H, CH33), 1.69 (m, 1H, H-6b), 1.58 (m, 1H, H-5b), 1.51 (m, 6H, H-2″), 1.31 (s, 3H, CH3-1), 1.31 (m, 6H, H-3″), 0.91 (m, 6H, H-1″), 0.88 (m, 9H, H-4″). 13C-NMR (125  MHz, MeCN-d3) δ ppm: 179.0 (–C=O), 144.9 (2JSnC = 3.9  Hz, C-1′), 137.3 (C-2), 136.1 (C-3), 133.0 (1J117SnC = 189.3  Hz, 1J119SnC = 180.9  Hz, C-2′), 70.4 (C-4), 52.4 (–OMe), 46.7 (C-1), 33.5 (C-6), 30.1 (2JSnC = 10.5 Hz, C-2″), 29.2 (C-5), 28.0 (3JSnC = 25.5 Hz, C-3″), 24.5 (CH3-1), 17.7 (CH3-3), 14.2 (C-4″), 10.4 (1J117SnC = 171.1 Hz, 1J119SnC = 164.1 Hz, C-1″). HRMS (ESI, positive mode) m/z [M + Na]+: calcd. for C24H44NaO3Sn, 523.2209; found, 523.2204 (see Supplementary Information).

4.2.3 M  ethyl (1S*,4R*)-4-hydroxy-1,3-dimethyl-2((E)-2-(tributylstannyl)vinyl)-cyclohex-2-ene-1carboxylate (7) To a solution of 5 (21.5  mg, 0.046  mmol) in anhydrous MeOH (0.4 mL), K2CO3 (15.9 mg, 0.115 mmol) was added at room temperature under Ar. The mixture was stirred for 2  h at room temperature. The mixture was directly subjected to column chromatography (silica gel:K2CO3 = 9:1, n-hexane:EtOAc = 10:1–4:1) to give recovered 5 (11.6  mg, 0.025  mmol, 54%) and 7 (9.7  mg, 0.0194  mmol, 42%) as a slightly yellow oil. 7: 1H-NMR (500  MHz, MeCN-d3) δ  ppm: 6.51 (d, J = 19.9  Hz, 3JSnH = 33.9  Hz, 1H, H-1′), 5.98 (d, J = 19.9  Hz, 2J117SnH = 36.4  Hz, 2J119SnH = 35.1  Hz, 1H, H-2′),

3.91 (m, 1H, H-4), 3.57 (s, 3H, –OMe), 2.90 (d, J = 6.1  Hz, 1H, –OH), 2.09 (m, 1H, H-6a), 1.85 (s, 3H, CH3-3), 1.80 (m, 1H, H-5a), 1.67 (m, 1H, H-5b), 1.50 (m, 6H, H-2″), 1.44 (m, 1H, H-6b), 1.30 (m, 6H, H-3″), 1.28 (s, 3H, CH3-1), 0.90 (m, 6H, H-1″), 0.88 (m, 9H, H-4″). 13C-NMR (125  MHz, MeCNd3) δ ppm: 179.1 (–C=O), 144.8 (2JSnC = 4.1  Hz, C-1′), 137.3 (C-2), 134.9 (C-3), 133.0 (1J117SnC = 189.7 Hz, 1J119SnC = 180.6 Hz, C-2′), 69.3 (C-4), 52.9 (–OMe), 46.7 (C-1), 32.0 (C-6), 30.0 (2JSnC = 10.6 Hz, C-2″), 28.5 (C-5), 28.0 (3JSnC = 25.8 Hz, C-3″), 23.1 (CH3-1), 18.8 (CH3-3), 14.1 (C-4″), 10.3 (1J117SnC = 171.7 Hz, 1 J119SnC = 164.3  Hz, C-1″). HRMS (ESI, positive mode) m/z [M + Na]+: calcd. for C24H44NaO3Sn, 523.2209; found, 523.2225 (see Supplementary Information).

4.2.4 Methyl (1S*,4R*)-4-hydroxy-1,3-dimethyl2-((1E,3Z)-3-methyl-7-oxo-1,3-octadienyl)cyclohex-2-ene-1-carboxylate ((9Z)-methyl 4-dihydrotrisporate B) (1) Reaction and workup were carried out under dim light. 7 (13.6  mg, 0.027  mmol), iodoalkene 9 [42] (7  mg, 0.030 mmol), Pd(PPh3)4 (1.6 mg, 0.0014 mmol), CuI (1.9 mg, 0.010 mmol) and CsF (18.5 mg, 0.12 mmol) were added to a flask. The flask was repeatedly (five times) flushed with Ar gas and subsequently evacuated to remove traces of water and oxygen. Then, anhydrous DMF (1  mL) was added. The mixture was stirred for 30  min at 35 °C, and then diluted with MTBE and filtered through filter paper. Water was added to the filtrate, and the aqueous layer was extracted twice with MTBE. The combined organic phases were washed with brine, dried over Na2SO4 and concentrated in vacuo. The residue was purified by column chromatography (Florisil, n-hexane:EtOAc = 10:1 to 3:2) to give 1 (6.6  mg, 0.0206  mmol, 76%) as a colorless oil. 1 H-NMR (500  MHz, MeCN-d3) δ ppm: 6.44 (d, J = 16.4  Hz, 1H, H-8), 6.29 (d, J = 16.4 Hz, 1H, H-7), 5.33 (dd, J = 7.5/7.5 Hz, 1H, H-10), 3.93 (m, 1H, H-4), 3.59 (s, 3H, –OMe), 2.95 (d, J = 5.9 Hz, 1H, –OH), 2.48 (dd, J = 7.2/7.2 Hz, 2H, H-12), 2.30 (m, 2H, H-11), 2.14 (m, 1H, H-6a), 2.07 (s, 3H, H-14), 1.89 (s, 3H, H-17), 1.84 (m, 1H, H-5a), 1.80 (bs, 3H, H-18), 1.70 (m, 1H, H-5b), 1.49 (m, 1H, H-6b), 1.33 (s, 3H, H-15). 13C-NMR (125  MHz, MeCN-d3) δ ppm: 208.9 (C-13), 179.2 (–C=O), 136.4 (C-3), 135.0 (C-2), 134.1 (C-9), 130.6 (C-10), 128.2 (C-8), 126.4 (C-7), 69.0 (C-4), 52.6 (–OMe), 47.0 (C-1), 44.0 (C-12), 32.3 (C-6), 30.0 (C-14), 28.5 (C-5), 23.1 (C-15), 22.6 (C-11), 20.4 (C-18), 18.7 (C-17). EI-MS (70 eV): m/z (%) 320 (10), 171 (76), 119 (98), 105 (78), 91 (100); HRMS (ESI, positive mode) m/z [M + Na]+: calcd. for C19H28NaO4, 343.1880; found, 343.1887.

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64      Nakamura et al.: Synthesis of methyl 4-dihydrotrisporate B and methyl trisporate B 4.2.5 Methyl 4-oxo-1,3-dimethyl-2-((E)-2(tributylstannyl)vinyl)-cyclohex-2-ene-1carboxylate (8) To a mixture of 6 (18.7  mg, 0.037  mmol) in anhydrous CH2Cl2 (1  mL), MnO2 (65  mg, 0.748  mmol) was added at room temperature under Ar gas. The mixture was stirred for 2 h, and then filtered through Celite and concentrated in vacuo. The residue was purified by column chromatography (silica gel:K2CO3 = 9:1, n-hexane:EtOAc = 10:1) to give 8 (16.6  mg, 0.033  mmol, 89%) as a colorless oil. 1 H-NMR (500 MHz, MeCN-d3) δ ppm: 6.67 (d, J = 20.1 Hz, 3 JSnH = 32.1 Hz, 1H, H-1′), 6.42 (d, J = 20.1 Hz, 2J117SnH = 33.4 Hz, 2 J119SnH = 32.1 Hz, 1H, H-2′), 3.62 (s, 3H, –OMe), 2.45 (m, 2H, H-5a/b), 2.32 (m, 1H, H-6a), 1.92 (m, 1H, H-6b), 1.83 (s, 3H, CH3-3), 1.52 (m, 6H, H-2″), 1.45 (s, 3H, CH3-1), 1.31 (m, 6H, H-3″), 0.96 (m, 6H, H-1″), 0.89 (m, 9H, H-4″). 13C-NMR (125 MHz, MeCN-d3) δ ppm: 199.0 (C-4), 177.1 (–C=O), 155.2 (C-2), 143.9 (2JSnC = 4.5  Hz, C-1′), 140.6 (1J117SnC = 172.0  Hz, 1 J119SnC = 164.0  Hz, C-2′), 132.0 (C-3), 52.9 (–OMe), 47.6 (C-1), 35.4 (C-6), 34.4 (C-5), 30.0 (2JSnC = 10.5 Hz, C-2″), 28.1 (3JSnC = 25.6  Hz, C-3″), 23.4 (CH3-1), 14.0 (C-4″), 12.3 (CH33), 10.4 (1J117SnC = 174.1  Hz, 1J119SnC = 165.3  Hz, C-1″); HRMS (ESI, positive mode) m/z [M + Na]+: calcd. for C24H42NaO3Sn, 521.2053; found, 521.2067 (see Supplementary Information).

4.2.6 Methyl 1,3-dimethyl-2-((1E,3Z)-3-methyl-7oxo-1,3-octadienyl)-4-oxo-cyclohex-2-ene-1carboxylate ((9Z)-methyl trisporate B) (2) Reaction and workup were carried out under dim light. 8 (9.1  mg, 0.018  mmol), iodoalkene 9 [42] (4.8  mg, 0.020  mmol), Pd(PPh3)4 (1.6  mg, 0.0009  mmol), CuI (1.25  mg, 0.0066  mmol) and CsF (12.5  mg, 0.082  mmol) were added to a flask. The flask was repeatedly (five times) flushed with Ar gas and subsequently evacuated to remove traces of water and oxygen. Then, anhydrous DMF (1 mL) was added. The mixture was stirred for 30 min at 35 °C, and another portion of 9 [6.3  mg, 0.026  mmol, in degassed, anhydrous DMF (0.2 mL)] was added to the mixture. After 20 min of stirring, the mixture was filtered through filter paper with MTBE. To the organic layer, water was added and the resulting aqueous layer was extracted with MTBE. The combined organic phases were washed with brine, dried over Na2SO4 and concentrated in vacuo. The residue was purified by column chromatography (Florisil, n-hexane:EtOAc = 10:1 to 5:1) to give 2 (5.4  mg, 0.0169 mmol, 93%) as a colorless oil. 1H-NMR (500 MHz,

MeCN-d3) δ ppm: 6.84 (d, J = 16.4  Hz, 1H, H-8), 6.45 (d, J = 16.4 Hz, 1H, H-7), 5.54 (dd, J = 7.6/7.6 Hz, 1H, H-10), 3.64 (s, 3H, –OMe), 2.52 (m, 2H, H-12), 2.47 (m, 2H, H-5a/b), 2.34 (m, 1H, H-6a), 2.34 (m, 2H, H-11), 2.08 (s, 3H, H-14), 1.95 (m, 1H, H-6b), 1.89 (s, 3H, H-17), 1.85 (bs, 3H, H-18), 1.53 (s, 3H, H-15). 13C-NMR (125  MHz, MeCN-d3) δ ppm: 208.7 (C-13), 198.0 (C-4), 177.6 (–C=O), 153.1 (C-2), 134.7 (C-10), 133.5 (C-9), 133.2 (C-3), 132.8 (C-8), 126.0 (C-7), 53.0 (–OMe), 47.5 (C-1), 43.5 (C-12), 35.3 (C-6), 33.8 (C-5), 30.2 (C-14), 22.8 (C-15), 22.6 (C-11), 20.1 (C-18), 12.3 (C-17). EI-MS (70 eV): m/z (%) 318 (26), 173 (54), 159 (64), 145 (52), 91 (100); HRMS (ESI, positive mode) m/z [M + H]+: calcd. for C19H27O4, 319.1904; found, 319.1911.

4.3 Chemical stability test Stability tests were performed using stereochemically pure ( ±)-(9Z)-methyl 4-dihydrotrisporate B (1) and (±)-(9Z)-methyl trisporate B (2), containing about 5% of the E-isomer. For the photostability experiments, 500 μM solutions of each compound in phosphate buffer (pH 7.2) containing 1% MeCN were prepared. One milliliter of each solution was transferred into quartz cells and exposed to UV light (distance 7 cm, combined 254 nm and 366 nm, 2.8 and 6.8 W m−2 nm−1, respectively), to ambient daylight (450 ± 50 lx) or kept in the dark until the experimental time was finished. Temperature was kept at 24 °C. Samples were taken during a course of 20  h, and directly analyzed by LC-HRMS. Individual compounds (peak areas) were measured based on UV (DAD 279-281 nm for 1 and 329–331 nm for 2, respectively). For variable pH conditions, a 500 μM solution of each compound was prepared as described for the photostability experiment. Six different buffers were used (pH 2, 4, 5.6, 7.2, 9 and 12). To prevent additional degrading effects from light each solution was kept in the dark at 24 °C. After 1 h the solution was extracted using 500 μL of MTBE, and the organic phase was separated from the buffer by means of a phase separation cartridge (Macherey-Nagel PTL, 1 mL volume). Subsequently, the separated MTBE extract was evaporated using argon gas and the residue was reconstituted with 100 μL MeCN and subjected to HPLC-HRESIMS analysis. The relative concentrations of 1 and 2 during the course of the experiments were determined based on the peak area at wavelengths specified above. Acknowledgments: We thank Daniel Veit (MPI-CE) for light intensity measurements, and the Max Planck Society for supporting this research.

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Nakamura et al.: Synthesis of methyl 4-dihydrotrisporate B and methyl trisporate B      65

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Supplemental Material: The online version of this article offers supplementary material (https://doi.org/10.1515/znc-2017-0148).

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