First total synthesis of fuzanins C, D and their analogues as anticancer agents

Abstract

The first total synthesis of fuzanins C and D, isolated from the culture supernatant of Kitasatospora sp. IFM10917, is described. Key features of this synthetic strategy involve the use of Sharpless asymmetric epoxidation, dihydroxylation, Mitsunobu reaction and Julia-Kocienski olefination. The total synthesis reported herein also confirmed the absolute configuration of fuzanin D. The optical rotations of synthesized fuzanin D and natural product were opposite in sign, leading to a revision of the reported structure as its enantiomer which is further confirmed by molecular modeling studies. In addition, we also synthesized the analogues of fuzanins C, D containing the quinoline nucleus. All of the synthesized compounds were screened for anticancer activity on four cell lines and found to be potent against HT29 colon cancer cell lines, whereas they were found to be less potent against cervical and breast cancer cell lines.

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Introduction

Organic synthesis is the art of building organic molecules through chemical reactions. The synthesis of natural products is one of the most fascinating and challenging areas of research in chemistry. Further, most of the natural products have been at the spearhead of merging the fields of organic chemistry and biology, and the fields will only continue to come together in the future. Nitrogen containing heterocycles such as pyridine, quinoline derivatives are among the most omnipresent azaheterocycles found in many natural products and have been claimed to be the most prevalent heterocycles in pharmaceutically active compounds.^1,2 Micrococcin P1, streptonigrin and nemerelline, etoricoxib, rosuvastatin and imatinib are a few examples of commercialized drugs bearing the pyridine motif. The pyridine ring is also ubiquitous in agrochemicals.³ Quinine, chloroquine, pamaquine, tafenoquine, bulaquine are some of the active pharmaceuticals containing quinoline nucleus. Quinoline derivatives exhibit broad range of biological activities such as antimalarial,⁴ antimicrobial,⁵ anticancer,⁶ antifungal,⁷ antileishmanial,⁸ anti-inflammatory,^9,10 and analgesic activity.¹⁰

Recently, new carbamate or pyridine containing natural products, fuzanins A (1), B (2), C (3a), and D (4a) were isolated by Ishibashi et al.¹¹ from the culture supernatant of Kitasatospora sp. IFM10917 (active strain from 323 actinomycete strains) obtained from a soil sample collected at Toyama city, Japan (Fig. 1). In addition, they have also evaluated cytotoxicity against human colon carcinoma DLD-1 cells, and inhibition of Wnt signal transcription for the four compounds.

Fig. 1 Fuzanins A, B, C and D.

As part of our continuing synthetic efforts towards synthesis of biologically and pharmaceutically favored heterocycles and natural products,¹² now we became interested in synthesis of biologically active fuzanin D and its stereo isomer fuzanin C. In this regard, we report the first total synthesis of fuzanin C (3a), D (4a) along with their analogues and also confirmed the absolute configuration of fuzanin D. Some compounds are selectively potent against HT29 colon cancer cell line.

Results and discussion

Fuzanins C (3a) and D (4a) are stereoisomers and differ in stereochemistry at 11^th position. The compounds are similar with scaffolds, hence similar synthetic strategies were applied to synthesize them. Retrosynthesis for fuzanins C (3a), D (4a) is depicted in Scheme 1. The analysis revealed that 3a and 4a could be prepared efficiently by a Julia-Kocienski olefination protocol using aldehyde 5a with sulfone 6 and 7 respectively, The intermediate 5a could be prepared from 2,3-dimethyl pyridine and compounds 6, 7 from ethyl sorbate (8).

Scheme 1 Retrosynthetic analysis of 3a, 4a.

The synthetic strategy for fuzanin C (3a) is described in Scheme 2. The commercially available starting material ethyl sorbate 8, was subjected to Sharpless dihydroxylation conditions using osmium tetraoxide as oxidant and (DHQD)₂PHAL as chiral ligand, giving the diol 9.^12a,13 Diol 9 was protected as acetonide and the ester functionality was reduced using DIBAL-H to obtain alcohol 10. Mitsunobu reaction of 10 with 1-phenyl-1H-tetrazole-5-thiol to get sulfide, and subsequent oxidation with H₂O₂ in presence of a molybdenum(VI) catalyst furnished the desired sulfone 6 in 82% yield. The vital coupling reaction of 6 and 5a (5a prepared from SeO₂ oxidation of 2,3-dimethyl pyridine¹⁴) was performed by the Julia-Kocienski olefination protocol¹⁵ using KHMDS as base to afford 11 as mixture of E and Z isomers.

Scheme 2 Synthesis of fuzanin C (3a). Reagents and conditions: (a) (DHQD)₂PHAL, OsO₄, CH₃SO₂NH₂, K₃Fe(CN)₆, K₂CO₃, t-BuOH/H₂O (1 : 1), 0 °C, 12 h, 84%; (b) (i) 2,2-Dimethoxypropane, p-TSA (cat), CH₂Cl₂, rt, 15 min; (ii) DIBAL-H, CH₂Cl₂, 0 °C, 1 h, 81% (for two steps); (c) (i) 1-phenyl-1H-tetrazole-5-thiol, PPh₃, DIAD, THF, 0 °C, 1 h, 90%; (ii) (NH₄)₆Mo₇O₂₄·4H₂O, EtOH, 30% H₂O₂, rt, 8h, 92%; (d) 5a, KHMDS, THF, ¬78 °C, 2 h, 81%; (e) p-TSA, MeOH, rt, 12 h, 82% (total yield).

The geometrical isomers i.e., E and Z isomers (7 : 3 ratio by ¹H NMR) were inseparable at this stage. However, final acetonide deprotection of 11 with p-TSA afforded fuzanin C (3a) and its Z-isomer 3b respectively (Scheme 2). All characterization data for synthesized fuzanin C (3a) (Fig. S2, ESI†) were in good agreement with those reported by Ishibashi et al.¹¹

Synthetic strategy for fuzanin D (4a) is described in Scheme 3. This synthesis begins with selective protection of C(5)–OH group of 9 with TBS-Cl in presence of triethylamine and DMAP, provided the desired ether 12.¹⁶ Mitsunobu inversion of hydroxyl group in 12 with formic acid in the presence of PPh₃, DEAD and subsequent hydrolysis of formyl ester using diluted NH₄OH–MeOH afforded compound 13 in 55% overall yield.¹⁷ TBS deprotection of 13 in presence of tetrabutylammoniumfluoride (TBAF) results trans-1,2-diol 14. trans-1,2-Diol 14 is protected as acetonide, DIBAL-H reduction of ester functionality, Julia-Kocienski olefination protocol, and hydrolysis or deprotection (the same synthetic sequence was followed as in Scheme 2) provided fuzanin D (4a) and its Z-isomer 4b in 6 : 4 ratio (Scheme 3).

Scheme 3 Synthesis of fuzanin D (4a). Reagents and conditions: (a) TBS-Cl, Et₃N, DMAP, CH₂Cl₂, rt, 24 h, 65%; (b) (i) PPh₃, DEAD, HCOOH, rt, 2 h; (ii) dil. NH₄OH/MeOH, 3 h, 55% (for two steps); (c) TBAF, CH₂Cl₂, 0 °C, 1 h, 85%; (d) (i) 2,2-Dimethoxypropane, p-TSA (cat), CH₂Cl₂, rt, 15 min; (ii) DIBAL-H, CH₂Cl₂, 0 °C, 1 h, 80% (for two steps); (e) (i) 1-phenyl-1H-tetrazole-5-thiol, PPh₃, DIAD, THF, 0 °C, 1 h; (ii) (NH₄)₆Mo₇O₂₄·4H₂O, EtOH, 30% H₂O₂, rt, 8 h, 82% (for two steps); (f) 5a, KHMDS, THF, ¬78 °C, 2 h, 81%; (g) p-TSA, MeOH, rt, 12 h, 82% (total yield).

Characterization data for the synthesized fuzanin D (4a) (Fig. S6, ESI†) were in good agreement with those reported by Ishibashi et al. except optical rotation. The [α]_D of the natural product is −32.9 (CHCl₃), and synthesized fuzanin D (4a) has +29.47. The original work by Ishibashi et al.¹¹ mentioned that fuzanin D as a stereoisomer to fuzanin C and further there was no attempt made to substantiate the stereochemistry of fuzanin D. The fuzanin D synthesized in this work exhibited optical rotation +29.47, whereas the sign of optical rotation is found to be opposite to the natural product reported by Ishibashi et al. In order to investigate the absolute configuration of fuzanin D, we further synthesized the other enantiomer of fuzanin D as given in Scheme 5.

The retrosynthesis of other enantiomer fuzanin D i.e., (11R,12S)-fuzanin D [(11R,12S)-4a] is depicted in Scheme 4. We envisioned the two stereocenters in (11R,12S)-4a and this could be constructed by the Sharpless asymmetric epoxidation. In this regard, the synthesis was started from 2-buten-1-ol, which was subjected to Sharpless epoxidation to afford the epoxy alcohol 18 (Scheme 5).¹⁸

Scheme 4 Retrosynthetic analysis of (11R,12S)-4a.

Scheme 5 Synthesis of (11R,12S)-4a. Reagents and conditions: (a) Ti(OⁱPr)₄, (+)-DIPT, anhydrous TBHP, CH₂Cl₂, ¬20 °C, 2 h; (b) (i) DMSO, (COCl)₂, Et₃N, CH₂Cl₂, ¬78 °C, 30 min. (ii) Ph₃P=CHCOOEt, CH₂Cl₂, rt, 24 h, 56% (for two steps); (c) BnOH, BF₃·OEt₂, DCM, ¬20 °C, 1 h, 60%; (d) AlCl₃, m-xylene, 0 °C, 15 min, 76%; (e) (i) 2,2-Dimethoxypropane, p-TSA, CH₂Cl₂, rt, 15 min. (ii) DIBAL-H, CH₂Cl₂, 0 °C, 1 h, 86% (for two steps); (f) (i) 1-phenyl-1H-tetrazole-5-thiol, PPh₃, DIAD, THF, 0 °C, 1 h. (ii) (NH₄)₆Mo₇O₂₄·4H₂O, EtOH, 30% H₂O₂, rt, 81% (for two steps); (g) 5a, KHMDS, THF, ¬78 °C, 2 h, 80%; (h) p-TSA, MeOH, rt, 12 h, 82% (total yield).

Swern oxidation of epoxy alcohol 18 and C2-Wittig homologation with [(ethoxycarbonyl)methylene]triphenyl-phosphorane, afforded requisite epoxy ester 19.¹⁹ The trans regioselective opening of epoxide of 19 was accomplished with benzyl alcohol in presence of BF₃·OEt₂ as lewis acid catalyst to get benzyl ether 20,²⁰ which was deprotected using AlCl₃ to get diol 21.^20,21 Acetonide protection of 21, DIBAL-H reduction, Julia-Kocienski olefination protocol, and hydrolysis or deprotection (the same synthetic sequence was followed as in Scheme 2) provided (11R,12S)-4a and its Z-isomer (11R,12S)-4b in 6 : 4 ratio (Scheme 5) (Fig. S11, ESI†).

The optical rotation of (11R,12S)-4a −26.9, which is in good accordance with isolated fuzanin D. This lead unambiguously to the conclusion that the natural product isolated has opposite configuration to that of reported fuzanin D (Fig. 2).

Fig. 2 Revised structure of fuzanin D.

This observation was further corroborated by computational study. Optical rotations (at 589.3 nm) of fuzanin C (3a), fuzanin D (4a) and (11R,12S)-fuzanin D [(11R,12S)-4a] were computed using B3LYP/Aug-CC-pVDZ method for geometries obtained at B3LYP/6-31G(d) basis set (Fig. 3).²² Calculated [α]_D for (3a), (4a) and (11R,12S)-4a are +66.5, +22.4 and −8.5 degrees[dm(g cm⁻³)]⁻¹ respectively (Table 1). Optical rotations of computationally calculated fuzanins and synthesized fuzanins were having same sign (Table 1). The comparison of experimental and calculated optical rotations allows us to define the stereochemistry fuzanin D reported by Ishibashi et al. as (11R,12S)-fuzanin D (Fig. 2).

Fig. 3 Energy minimized geometries of fuzanin D (4a) & (11R,12S)-4a.

Table 1 Experimental and calculated specific rotations of selected compounds

Compounds	[α]D (in ° dm^−1 g^−1 cm^3)
	Isolation paper^a	Synthesized compounds	Calculated^b
a Ref. 11.b Optical rotations were computed using B3LYP/aug-cc-pVDZ//B3LYP/6-31G(d) basis set in gas phase.
Fuzanin C (3a)	[α]^15D = +34.5	[α]^24D = +39.89	+66.5
Fuzanin D (4a)	[α]^15D = −32.9	[α]^24D = +29.47	+22.4
(11R,12S)-4a	—	[α]^24D = −26.9	−8.5

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Intrigued by this, we also synthesized different analogues (3c, 3d, 4c and 4d) using substituted 2-chloroquinoline-3-carboxaldehydes (5b and 5c)²³ by same synthetic strategy of Julia-Kocienski olefination protocol, and hydrolysis (Scheme 6) (Fig. S4, S5, S8 and S9, ESI†).

Scheme 6 Analogues of fuzanins C, D.

Furthermore, all the synthesized compounds were subjected to anticancer activity on various cell lines such as HT29 (Colon cancer), ME-180 (Cervical cancer), MCF-7 and MDA-MB-453 (Breast cancer) by employing MTT assay (details of bio assay are presented in Experimental section). For comparison purpose, the cytotoxicity of salinomycin was evaluated under the same experimental conditions. The compounds that exhibiting ≥50% cell inhibition at 100 μM were considered for determination of IC₅₀ value, which was calculated from the % cell viability (from control) versus concentration curves obtained after 24 h drug treatment from MTT assay, are shown in Table 2.

Table 2 IC₅₀ values (μM) of fuzanin compounds and salinomycin against HT29 human colon cancer cell lines

Compound	IC50/μg mL^−1
	HT-29 (colon cancer)
3a	96.2 ± 2.65
3b	85.3 ± 4.72
3c	48.5 ± 2.56
3d	76.2 ± 2.45
4a	77.9 ± 2.95
4b	98.5 ± 1.88
4c	35.3 ± 0.83
4d	27.4 ± 0.12
(11R,12S)-4a	>100
(11R,12S)-4b	>100
Salinomycin	20.5 ± 1.26

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All compounds were found to be relatively potent against colon cancer cell line and less potent against cervical and breast cancer cell lines. Among all the compounds, quinoline derivatives of fuzanin D (4c, 4d) were found to be potent with an IC₅₀ value of 35.3 ± 0.83, 27.4 ± 0.12 μM respectively. Compound 3c has exhibited moderate activity. Remaining compounds were found to be less potent against HT-29 cell lines. The reference compound salinomycin has IC₅₀ value of 20.5 ± 1.26 μM (Fig. 4).

Fig. 4 Dose response of fuzanin compounds against HT-29 cancer cell line.

Conclusion

In conclusion, we have unveiled the first stereoselective total synthesis of fuzanins C, D and their analogues. Synthesis of fuzanin D described here also serves to establish its absolute configuration. Specific optical rotations of synthesized compound and reported fuzanin D indicated opposite signs. This was confirmed by total synthesis of its enantiomer (11R,12S)-4a. Stereochemistry of reported fuzanin D (4a) should be 11R,12S instead of 11S,12R configuration. Molecular modeling studies also supported this observation. Further, fuzanins C, D and their analogues were screened for anticancer activity in four cancer cell lines. The compounds were found to exert cytotoxicity selectively on HT29 cancer cell lines. Quinoline nucleus containing analogues 3c, 4c and 4d are relatively more potent.

Experimental section

All reactions were carried out under an inert atmosphere unless mentioned otherwise, and standard syringe-septa techniques were followed. Solvents were freshly dried and purified by conventional methods prior to use. The progress of all reactions were monitored by TLC, using TLC aluminium backed sheets precoated with silicagel 60 F₂₅₄ to a thickness of 0.25 mm (Merck). Column chromatography was performed on silica gel (60–120 mesh and 100–200 mesh), and EtoAc, hexane were used as eluents. Optical rotation values were measured with a Perkin-Elmer P241 polarimeter and a JASCO DIP-360 digital polarimeter, and IR spectra were recorded with a Perkin-Elmer FT-IR spectrophotometer. ¹H and ¹³C NMR spectra were recorded with Varian Gemini 200 MHz, Bruker Avance 300 MHz, Varian Unity 400 MHz or Varian Inova 500 MHz spectrophotometers. TMS was used as an internal standard in CDCl₃. Mass spectra were recorded with a VG micromass 7070H (EI), QSTAR XL high-resolution mass spectrophotometer, a Thermo Finnigan ESI Ion trap Mass spectrophotometer.

(4R,5R,E)-Ethyl-4,5-dihydroxyhex-2-enoate (9)

To a mixture of K₃Fe(CN)₆ (28.22 g, 85.71 mmol), K₂CO₃ (11.80 g, 85.71 mmol), and (DHQD)₂PHAL (0.41 g, 0.57 mmol) in 45 mL of t-BuOH–H₂O (1 : 1), was added OsO₄ (70.56 mg, 0.28 mmol) followed by methane sulfonamide (2.71 g, 28.57 mmol) at 0 °C. After stirring for 15 min at 0 °C, ethyl sorbate 8 (4 g, 28.57 mmol) was added and stirred vigorously at 0 °C for 12 h, and then quenched with sat. sodium sulfite (35 mL). The reaction mixture was extracted with ethyl acetate (3 × 40 mL). The organic layer washed with 2 N KOH (20 mL), brine solution and dried over Na₂SO₄. Solvent removed under vacuo. The crude residue was purified by silica gel column chromatography (EtOAc–hexane, 7 : 3) to afford diol 9 (3.97 g, 84%) as light yellow oil; [α]²⁵_D + 50.21 (c 0.1, EtOH); IR (Neat): 3396, 2924, 1704, 1371, 1281, 1036 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz): δ 6.89 (dd, 1H, J = 15.5, 5.2 Hz), 6.10 (dd, 1H, J = 15.5, 1.5 Hz), 4.17 (q, 2H, J = 7.5 Hz), 4.02 (brt, 1H, J = 5.2 Hz), 3.75–3.63 (m, 1H), 1.27 (t, 3H, J = 7.5 Hz), 1.20 (d, 3H, J = 6.0 Hz); ¹³C NMR (CDCl₃, 75 MHz): δ 166.5, 146.6, 122.4, 75.6, 70.2, 60.7, 19.0, 14.1; anal. calcd for C₈H₁₄O₄ (174.19): C, 55.16; H, 8.10. Found: C, 55.15; H, 8.17%.

(E)-3-((4R,5R)-2,2,5-Trimethyl-1,3-dioxolan-4-yl)prop-2-en-1-ol (10)

To the solution of 9 (3 g, 17.24 mmol) in 30 mL CH₂Cl₂, 2,2-dimethoxypropane (5.37 g, 51.72 mmol), catalytic amount of p-TSA were added and stirred for 15 min at rt. The reaction mixture was washed with saturated aqueous NaHCO₃ solution (10 mL), dried over Na₂SO₄, concentrated under vacuo. The crude residue was purified by silica gel column chromatography (EtOAc–hexane, 1 : 9) to afford cyclic acetonide (3.46 g, 94%) as colour less oil. To the solution cyclic acetonide (3.2 g, 14.9 mmol) in 30 mL of dry CH₂Cl₂ at 0 °C, DIBAL-H (21.01 mL, 37.25 mmol, 25% solution in THF) was added, and stirred for 1 h at rt. The reaction mixture was quenched by slow addition of aq. sodium potassium tartate and stirred for 2 h. Organic layer was separated and aqueous layer was extracted by chloroform (20 mL), combined organic layer was dried over Na₂SO₄, solvent removed under vacuo. The crude residue was purified by silica gel column chromatography (EtOAc–hexane, 6 : 4) to afford 10 (2.4 g, 81%) as light yellow, viscous oil; [α]²⁴_D + 46.01 (c 0.1, CHCl₃); IR (Neat): 3395, 2923, 2851, 1449, 1259, 998 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz): δ 5.99 (dtd, 1H, J = 15.5, 5.1, 0.7 Hz), 5.70 (tdd, 1H, J = 15.5, 7.5, 1.5 Hz), 4.19 (d, 2H, J = 5.1 Hz), 3.9 (t, 1H, J = 7.5 Hz), 3.84–3.75 (m, 1H), 1.42 (s, 3H), 1.41 (s, 3H), 1.26 (d, 3H, J = 6.0 Hz); ¹³C NMR (CDCl₃, 50 MHz): δ 134.2, 127.0, 108.2, 83.1, 76.5, 62.3, 27.2, 26.8, 16.3; anal. calcd for C₉H₁₆O₃ (172.10): C, 62.77; H, 9.36. Found: C, 62.58; H, 9.57%.

1-Phenyl-5-(((E)-3-((4R,5R)-2,2,5-trimethyl-1,3-dioxolan-4-yl)allyl)sulfonyl)-1H-tetrazole (6)

To a solution of alcohol 10 (0.6 g, 3.48 mmol) and 1-phenyl-1H-tetrazole-5-thiol (0.74 g, 4.15 mmol) in 20 mL THF at 0 °C, DIAD (0.82 mL, 4.16 mmol) and triphenyl phosphine (0.84 g, 4.15 mmol) were added, and stirred for 1 h at same temperature. The reaction was quenched by addition of saturated aqueous NH₄Cl solution, extracted with ethyl acetate (2 × 20 mL), dried over Na₂SO₄, concentrated under vacuo. The crude residue was purified by silica gel column chromatography (EtOAc–hexane, 3 : 7) to afford sulphide (1.0 g, 90%) as colour less, viscous oil; [α]²⁴_D − 24.80 (c 0.1, CHCl₃); IR (Neat): 2923, 1679, 1618, 1185, 1001, 741 cm⁻¹; ¹H NMR (CDCl₃, 500 MHz): δ 7.61–7.55 (m, 5H), 6.05–5.95 (m, 1H), 5.81 (dd, 1H, J = 15.1, 6.7 Hz), 4.13–3.97 (m, 2H), 3.90 (t, 1H, J = 8.3 Hz), 3.79–3.70 (m, 1H), 1.40 (s, 3H), 1.39 (s, 3H), 1.22 (d, 3H, J = 6.7 Hz); ¹³C NMR (CDCl₃, 50 MHz): δ 153.3, 133.4, 132.2, 130.0, 129.6, 127.5, 123.6, 108.4, 82.5, 76.4, 34.4, 27.1, 26.7, 16.2; MS (ESI): m/z 355 (M + Na)⁺; HRMS (ESI): m/z 355.1200 (M + Na)⁺ (calcd for C₁₆H₂₁N₄O₂S 355.1199).

To a solution of sulphide (0.50 g, 1.50 mmol) and ammonium heptamolybdate tetrahydrate (0.55 g, 0.45 mmol) in 12 mL of ethanol at 0 °C, H₂O₂ (30% solution in water, 2.04 mL, 18.07 mmol) was added and stirred for 8 h at rt. The reaction mixture was poured in to saturated aqueous NaHCO₃ solution (40 mL) and extracted with ethyl acetate (3 × 30 mL), dried over Na₂SO₄, concentrated under vacuo. The crude residue was purified by silica gel column chromatography (EtOAc–hexane, 3 : 7) to afford sulphone 4 (0.5 g, 92%) as colour less, viscous oil; [α]²⁴_D − 28.70 (c 0.1, CHCl₃); IR (Neat): 2922, 1679, 1618, 1222, 741 cm⁻¹; ¹H NMR (CDCl₃, 500 MHz): δ 7.68–7.59 (m, 5H), 5.98 (dd, 1H, J = 15.8, 6.0 Hz), 5.93–5.83 (m, 1H), 4.45 (m, 2H), 3.93 (dd, 1H, J = 8.3, 6.0 Hz), 3.77–3.68 (m, 1H), 1.41 (s, 3H), 1.37 (s, 3H), 1.22 (d, 3H, J = 6.0 Hz); ¹³C NMR (CDCl₃, 50 MHz): δ 152.8, 140.0, 132.8, 131.4, 129.5, 125.0, 116.6, 108.9, 82.1, 76.4, 59.0, 27.1, 26.6, 16.3; MS (ESI): m/z 365 (M + H)⁺; HRMS (ESI): m/z 387.1093 (M + Na)⁺ (calcd for C₁₆H₂₀O₄N₄NaS 387.1097).

3-Methyl-2-((3E)-4-((4R,5R)-2,2,5-trimethyl-1,3-dioxolan-4-yl)buta-1,3-dien-1-yl)pyridine (11)

To a solution of sulphone 6 (0.40 g, 1.09 mmol) in 10 mL THF at −78 °C, potassium bis(trimethylsilyl)amide (0.5 M in THF solution, 2.85 mL, 1.42 mmol) was added. After 30 min of stirring at same temperature, the solution of aldehyde 5a (0.14 g, 1.19 mmol) in 2 mL THF was added. The reaction mixture was stirred for further 1.5 h at −78 °C, the reaction allowed to warm to rt and was stirred for additional 2 h. Brine solution was added to the reaction mixture and extracted with ethyl acetate (3 × 10 mL), dried over Na₂SO₄, concentrated under vacuo. The crude residue was purified by silica gel column chromatography (EtOAc–hexane, 1 : 4) to afford 11 (0.23 g, 81%) (inseparable cis, trans mixture, 3 : 7) as viscous, light yellow oil; IR (Neat): 2984, 2932, 1581, 1498, 1380, 1028, 858 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz): δ 8.45 (d, 0.3H, J = 4.7 Hz), 8.40 (d, 0.7H, J = 4.1 Hz), 7.45–7.35 (m, 2H), 7.07–7.01 (m, 1H), 6.79 (d, 0.7H, J = 15.1 Hz), 6.60–6.65 (m, 1.5H), 5.92–5.76 (m, 1H), 4.02 (brt, 1H, J = 7.5 Hz), 3.84–3.75 (m, 1.5H), 2.36 (s, 2.1H), 2.31 (s, 0.9H), 1.44 (s, 4.2H), 1.41 (s, 1.8H), 1.29–1.26 (m, 3H); ¹³C NMR (CDCl₃, 50 MHz): δ 155.0, 153.4, 147.3, 146.8, 138.3, 137.8, 133.7, 132.8, 132.3, 131.7, 130.9, 129.1, 127.0, 122.3, 122.0, 116.8, 108.7, 108.5, 83.7, 77.7, 77.0, 27.5, 27.1, 19.3, 18.9, 16.8, 16.7; MS (ESI): m/z 260 (M + H)⁺; HRMS (ESI): m/z 260.1641 (M + H)⁺ (calcd for C₁₆H₂₂O₂N 260.1645).

(2R,3R,4E,6E)-7-(3-Methylpyridin-2-yl)hepta-4,6-diene-2,3-diol (3a)

To a solution of 11 (0.10 g, 0.38 mmol) in 10 mL methanol, was added p-toluenesulphonic acid (1.32 g, 0.77 mmol) and the reaction mixture stirred for 12 h at rt, and solvent removed under vacuo. The crude residue was diluted with CH₂Cl₂ and washed with saturated aqueous NaHCO₃ solution and the solvent removed under vacuo. The crude compound was purified by silica gel column chromatography (EtOAc–hexane, 1 : 1) to afford fuzanin C (3a) (0.05 g) as viscous, colour less oil, and cis isomer 3b (0.02 g) as viscous, colour less oil, (total yield 82%); [α]²⁴_D + 39.89 (c 0.055, CHCl₃); IR (Neat): 3395, 2923, 1679, 1618, 772 cm⁻¹; ¹H NMR (CDCl₃, 500 MHz): δ 8.39 (d, 1H, J = 4.7 Hz), 7.42 (d, 1H, J = 7.7 Hz), 7.37 (dd, 1H, J = 15.0, 11.0 Hz), 7.04 (dd, 1H, J = 7.7, 4.7 Hz), 6.78 (d, 1H, J = 15.0 Hz), 6.54 (dd, 1H, J = 15.3, 11.0 Hz), 5.94 (dd, 1H, J = 15.3, 6.9 Hz), 3.95 (t, 1H, J = 6.9 Hz), 3.68 (q, 1H, J = 6.6 Hz), 2.34 (s, 3H), 1.18 (d, 3H, J = 6.6 Hz); ¹³C NMR (CDCl₃, 100 MHz): δ 153.2, 146.8, 138.2, 135.7, 133.3, 132.1, 130.8, 128.0, 122.0, 77.1, 70.7, 18.8, 18.6; MS (ESI): m/z 220 (M + H)⁺; HRMS (ESI): m/z 242.1148 (M + Na)⁺ (calcd for C₁₃H₁₇O₂NNa 242.1151).

(2R,3R,4E,6Z)-7-(3-Methylpyridin-2-yl)hepta-4,6-diene-2,3-diol (3b)

Yield: 0.02 g; [α]²⁴_D + 82.23 (c 0.051, CHCl₃); IR (Neat): 3421, 2924, 1619, 1449, 772 cm⁻¹; ¹H NMR (CDCl₃, 500 MHz): δ 8.42 (d, 1H, J = 4.7 Hz), 7.45 (d, 1H, J = 7.3 Hz), 7.29 (dd, 1H, J = 15.2, 11.5 Hz), 7.06 (dd, 1H, J = 7.3, 4.7 Hz), 6.46 (d, 1H, J = 11.5 Hz), 6.38 (t, 1H, J = 11.5 Hz), 5.87 (dd, 1H, J = 15.2, 6.6 Hz), 3.93 (t, 1H, J = 6.6 Hz), 3.66 (quin, 1H, J = 6.2 Hz), 2.31 (s, 3H), 1.17 (d, 3H, J = 6.2 Hz); ¹³C NMR (CDCl₃, 100 MHz): δ 154.5, 146.2, 137.8, 136.9, 132.9, 131.9, 129.2, 126.0, 121.8, 76.7, 70.4, 19.0, 18.8; MS (ESI): m/z 220 (M + H)⁺; HRMS (ESI): m/z 220.1329 (M + H)⁺ (calcd for C₁₃H₁₈O₂N 220.1332).

(2R,3R,4E,6E)-7-(2-Chloro-6-isopropylquinolin-3-yl)hepta-4,6-diene-2,3-diol (3c)

According to the procedure 3a, the sulphone 6 (0.10 g, 0.27 mmol) and aldehyde 5b (0.07 g, 0.32 mmol), gave cyclic acetonide (inseparable cis, trans mixture, 6 : 94 by NMR) as light yellow oil. Cyclic acetonide dissolved in 10 mL of methanol, catalytic amount of p-TSA was added and stirred for 30 min at rt, and solvent removed under vacuo. The crude residue was diluted with CH₂Cl₂ and washed with saturated aqueous NaHCO₃ solution and the solvent removed under vacuo. The crude compound was purified by silica gel column chromatography (EtOAc–hexane, 1 : 1) to afford compound 3c (0.06 g, 70%, for two steps) as white solid; mp 120–122 °C; [α]²⁴_D − 89.56 (c 0.022, CHCl₃); IR (KBr): 3409, 2963, 2926, 1584, 1047, 768 cm⁻¹; ¹H NMR (CDCl₃, 500 MHz): δ 8.19 (s, 1H), 7.89 (d, 1H, J = 9.0 Hz), 7.60–7.56 (m, 2H), 6.98 (d, 1H, J = 15.9 Hz), 6.83 (dd, 1H, J = 15.9, 10.9 Hz), 6.59 (dd, 1H, J = 14.9, 10.9 Hz), 5.91 (dd, 1H, J = 14.9, 5.9 Hz), 4.01 (t, 1H, J = 5.9 Hz), 3.73 (quin, 1H, J = 5.9 Hz), 3.07 (sep, 1H, J = 6.9 Hz), 1.33 (d, 6H, J = 6.9 Hz), 1.24 (d, 3H, J = 5.9 Hz); ¹³C NMR (CDCl₃, 100 MHz): δ 149.0, 148.0, 145.5, 134.7, 133.3, 132.3, 131.9, 130.3, 129.5, 127.8, 127.6, 127.3, 123.5, 77.0, 70.8, 34.0, 23.6, 19.0; MS (ESI): m/z 332 [M + H]⁺; HRMS (ESI): m/z 332.1410 (M + H)⁺ (calcd for C₁₉H₂₃O₂NCl 332.1411).

(2R,3R,4E,6E)-7-(2-Chloro-7-methylquinolin-3-yl)hepta-4,6-diene-2,3-diol (3d)

According to the procedure 3c, the sulphone 6 (0.10 g, 0.27 mmol) and requisite aldehyde 5c (0.07 g, 0.35 mmol) gave the compound 3d (0.06 g, 71%, for two steps) as white solid; mp 130–131 °C; [α]²⁴_D − 91.18 (c 0.024, CHCl₃); IR (KBr): 3397, 2925, 1623, 1049, 756 cm⁻¹; ¹H NMR (CDCl₃, 500 MHz): δ 8.19 (s, 1H), 7.73 (br s, 1H), 7.68 (d, 1H, J = 8.3, Hz), 7.37 (dd, 1H, J = 8.3, 1.13 Hz), 6.99 (d, 1H, J = 15.5 Hz), 6.83 (dd, 1H, J = 15.5, 10.3 Hz), 6.60 (dd, 1H, J = 15.3, 10.3 Hz), 5.91 (dd, 1H, J = 15.3, 6.8 Hz), 4.01 (t, 1H, J = 6.8 Hz), 3.73 (quin, 1H, J = 6.4 Hz), 2.54 (s, 3H), 1.24 (d, 3H, J = 6.4 Hz); ¹³C NMR (CDCl₃, 100 MHz): δ 149.8, 146.9, 141.0, 134.5, 133.3, 132.4, 131.7, 129.5, 128.8, 127.8, 127.2, 127.1, 125.4, 77.1, 70.8, 21.9, 19.0; MS (ESI): m/z 304 (M + H)⁺; HRMS (ESI): m/z 304.1096 [M + H]⁺ (calcd for C₁₇H₁₉O₂NCl 304.1098).

(4R,5R,E)-Ethyl 5-((tert-butyldimethylsilyl)oxy)-4-hydroxyhex-2-enoate (12)

To a solution of the diol 9 (3.0 g, 17.5 mmol) in CH₂Cl₂ (30 mL) at 0 °C, Et₃N (4.1 mL, 29.8 mmol), DMAP (1.06 g, 0.88 mmol) and TBSCl (3.96 g, 26.3 mmol) were added simultaneously, and the mixture was stirred for 24 h at rt. The reaction mixture was diluted with CH₂Cl₂, successively washed with 10% aq. HCl and sat. NaHCO₃, dried over Na₂SO₄, filtered, and concentrated in vacuo. The resulting oil was purified by silicagel column chromatography (EtOAc–hexane, 1 : 4) to afford the TBS ether (12) (3.29 g, 65%) as a colorless oil; [α]²⁵_D − 0.71 (c 0.051, EtOH); IR (Neat): 3475, 2932, 2858, 1721, 1257, 838 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz): δ 6.88 (dd, 1H, J = 15.8, 4.5 Hz), 6.10 (dd, 1H, J = 15.8, 1.8 Hz), 4.19 (q, 2H, J = 7.1 Hz), 4.02–3.98 (m, 1H), 3.79–3.71 (m, 1H), 2.57 (brs, 1H), 1.29 (t, 3H, J = 7.1 Hz), 1.20 (d, 3H, J = 6.0 Hz), 0.88 (s, 9H), 0.07 (s, 3H), 0.05 (s, 3H); ¹³C NMR (CDCl₃, 75 MHz): δ 166.2, 147.2, 121.9, 75.2, 71.0, 60.3, 25.7, 20.1, 17.9, 14.2, −4.3, −4.9; MS (ESI): m/z 289 (M + H)⁺.

(4S,5R,E)-Ethyl-5-((tert-butyldimethylsilyl)oxy)-4-hydroxyhex-2-enoate (13)

Under inert atmosphere, to a solution of PPh₃ (0.62 g, 2.38 mmol) in 5 mL benzene at 0 °C, DEAD (0.16 mL, 2.38 mmol), TBS ether (12) (0.274 g, 0.95 mmol) and formic acid (0.11 mL, 2.85 mmol) were added simultaneously and the mixture was stirred for 2 h at rt. The solvent was evaporated in vacuo, and the crude residue was dissolved in NH₄OH–MeOH (1 : 1, 3.0 mL) at 0 °C and stirred for 3 h at rt. This reaction mixture was diluted with Et₂O, successively washed with 10% HCl solution and sat. NaHCO₃, dried over Na₂SO₄, and concentrated in vacuo. The resulting oil was purified by silica gel column chromatography (EtOAc–hexane, 1 : 4) to afford alcohol 13 (0.15 g, 55%) as a colorless oil; [α]²⁵_D − 27.1 (c 0.051, EtOH); IR (Neat): 3466, 2930, 2857, 1720, 1259, 836 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz): δ 6.87 (dd, 1H, J = 15.6, 4.6 Hz), 6.08 (dd, 1H, J = 15.6, 2.3 Hz), 4.24–4.21 (m, 1H), 4.19 (q, 2H, J = 7.0 Hz), 3.93–3.88 (m, 1H), 2.43 (brs, 1H), 1.28 (t, 3H, J = 7.0 Hz), 1.08 (d, 3H, J = 6.2 Hz), 0.89 (s, 9H), 0.07 (s, 6H); ¹³C NMR (CDCl₃, 70 MHz): δ 166.3, 145.5, 121.7, 74.8, 70.7, 60.3, 25.7, 17.9, 17.7, 14.2, −4.4, −4.9; MS (ESI): m/z 289 (M + H)⁺.

(4S,5R,E)-Ethyl-4,5-dihydroxyhex-2-enoate (14)

To a solution of 13 (0.5 g, 1.73 mmol) in 10 mL THF at 0 °C, was added TBAF (1.0 M, 5.2 mL, 5.19 mmol) and the resulting mixture was stirred for 1 h. The reaction was quenched with water (25 mL) and extracted with CH₂Cl₂ (2 × 25 mL). The combined organic extracts were dried over Na₂SO₄, concentrated in vacuo. The crude reside was purified by silica gel column chromatography to yield diol 14 (0.25 g, 85%); [α]²⁴_D − 9.15 (c 0.047, CHCl₃); IR (Neat): 3429, 2981, 2931, 1714, 1659, 1278, 984 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz): δ 6.94 (dd, 1H, J = 15.1, 4.5 Hz), 6.11 (d, 1H, J = 15.1 Hz), 4.30 (brs, 1H), 4.20 (q, 2H, J = 6.8 Hz), 3.99–3.91 (m, 1H), 1.28 (t, 3H, J = 6.8 Hz), 1.16 (d, 3H, J = 6.0 Hz); ¹³C NMR (CDCl₃, 75 MHz): δ 166.6, 145.8, 122.37, 74.5, 69.9, 60.6, 17.4, 14.1; anal. calcd for C₈H₁₄O₄ (174.19): C, 55.16; H, 8.10. Found: C, 55.19; H, 8.14%.

(E)-3-((4S,5R)-2,2,5-Trimethyl-1,3-dioxolan-4-yl)prop-2-en-1-ol (15)

According to the procedure 10, compound 14 (2.4 g, 13.79 mmol) gave alcohol 15 (1.90 g, 80%); [α]²⁴_D − 15.1 (c 0.1, CHCl₃); IR (Neat): 3431, 2984, 1380, 1219, 772 cm⁻¹; ¹H NMR (CDCl₃, 500 MHz): δ 5.90 (td, 1H, J = 15.4, 5.1 Hz), 5.68 (tdd, 1H, J = 15.4, 7.7, 1.5 Hz), 4.52 (brt, 1H, J = 7.7 Hz), 4.32 (quin, 1H, J = 6.4 Hz), 4.17 (dd, 2H, J = 5.1, 1.5 Hz), 1.47 (s, 3H), 1.35 (s, 3H), 1.13 (d, 3H, J = 6.4 Hz); ¹³C NMR (CDCl₃, 100 MHz): δ 133.5, 127.1, 108.0, 79.0, 74.0, 62.8, 28.2, 25.5, 16.1; anal. calcd for C₉H₁₆O₃ (172.10): C, 62.77; H, 9.36. Found: C, 62.61; H, 9.59%.

1-Phenyl-5-(((E)-3-((4S,5R)-2,2,5-trimethyl-1,3-dioxolan-4-yl)allyl)sulfonyl)-1H-tetrazole (7)

According to the procedure 6, the alcohol 15 (0.5 g, 2.9 mmol) gave sulphone 7 (0.86 g, 82%); [α]²⁴_D − 3.14 (c 0.05, CHCl₃); IR (Neat): 2922, 1679, 1618, 1524, 1347, 741 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz): δ 7.68–7.56 (m, 5H), 5.96 (dd, 1H, J = 15.8, 6.8 Hz), 5.86–5.76 (m, 1H), 4.54–4.38 (m, 3H), 4.32 (dd, 1H, J = 12.8, 6.7 Hz), 1.45 (s, 3H), 1.34 (s, 3H), 1.03 (d, 3H, J = 6.7 Hz); ¹³C NMR (CDCl₃, 75 MHz): δ 152.9, 140.3, 132.9, 131.4, 129.6, 125.1, 116.1, 108.5, 78.1, 73.8, 59.0, 28.0, 25.3, 15.8; MS (ESI): m/z 365 (M + H)⁺; HRMS (ESI): m/z [M + H]⁺ 387.1094 (M + Na)⁺ (calculated for C₁₆H₂₀N₄O₄NaS 387.1097).

3-Methyl-2-((3E)-4-((4S,5R)-2,2,5-trimethyl-1,3-dioxolan-4-yl)buta-1,3-dien-1-yl)pyridine (16)

According to the procedure 11, the sulphone 6 (0.38 g, 1.04 mmol) and 5a (0.13 g, 1.14 mmol), gave 16 (0.22 g, 81%) (inseparable cis, trans mixture, 4 : 6) as colourless, viscous oil; IR (Neat): 2923, 2851, 1667, 1382, 1048, 771 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz): δ 8.45 (d, 0.4H, J = 4.5 Hz), 8.42 (d, 0.6H, J = 4.5 Hz), 7.48–7.36 (m, 2H), 7.08–7.02 (m, 1H), 6.81 (d, 0.6H, J = 15.1 Hz), 6.56–6.39 (m, 1.6H), 5.96–5.81 (m, 1H), 4.65–4.59 (m, 1H), 4.41–4.30 (m, 1H), 2.36 (s, 1.8H), 2.30 (s, 1.2H), 1.53 (s, 1.8H), 1.50 (s, 1.2H), 1.39 (s, 1.8H), 1.36 (s, 1.2H), 1.16 (t, 3H, J = 6.0 Hz); ¹³C NMR (CDCl₃, 75 MHz): δ 154.7, 153.2, 146.9, 146.4, 138.1, 137.6, 133.3, 132.8, 132.6, 132.3, 130.9, 128.3, 126.3, 122.0, 121.7, 108.0, 107.9, 79.4, 79.1, 74.37, 74.32, 28.2, 25.4, 19.0, 18.7, 16.17; MS (ESI): m/z 260 (M + H)⁺; HRMS (ESI): m/z 260.1640 (M + H)⁺ (calcd for C₁₆H₂₂O₂N 260.1645).

(2R,3S,4E,6E)-7-(3-Methylpyridin-2-yl)hepta-4,6-diene-2,3-diol (4a)

According to the procedure 3a, compound 16 (0.1 g, 0.38 mmol) gave fuzanin D (4a) (0.05 g) as viscous, colour less oil, and cis isomer 4b (0.02 g) as viscous, colour less oil, (total yield 82%); [α]²⁴_D + 29.47 (c 0.045, CHCl₃); IR (Neat): 3424, 2924, 2854, 1621, 1458, 1078, 771 cm⁻¹; ¹H NMR (CDCl₃, 500 MHz): δ 8.40 (d, 1H, J = 4.1 Hz), 7.45–7.36 (m, 2H), 7.04 (dd, 1H, J = 7.2, 4.1 Hz), 6.79 (d, 1H, J = 14.5 Hz), 6.54 (dd, 1H, J = 14.5, 10.4 Hz), 6.01 (dd, 1H, J = 14.5, 6.2 Hz), 4.20 (dd, 1H, J = 6.2, 4.1 Hz), 3.90 (dd, 1H, J = 6.2, 4.1 Hz), 2.34 (s, 3H), 1.16 (d, 1H, J = 6.2 Hz); ¹³C NMR (CDCl₃, 100 MHz): δ 153.3, 146.9, 138.2, 134.6, 133.2, 132.3, 130.7, 128.1, 122.0, 75.9, 70.3, 18.7, 17.6; MS (ESI): m/z 242 (M + Na)⁺; HRMS (ESI): m/z 242.1148 (M + Na)⁺ (calcd for C₁₃H₁₇O₂NNa 242.1151).

(2R,3S,4E,6Z)-7-(3-Methylpyridin-2-yl)hepta-4,6-diene-2,3-diol (4b)

Yield: (0.02 g, 90% overall yield); [α]²⁴_D + 59.15 (c 0.04, CHCl₃); IR (Neat): 3405, 2924, 1449, 995 cm⁻¹; ¹H NMR (CDCl₃, 500 MHz): δ 8.45 (d, 1H, J = 5.2 Hz), 7.46 (d, 1H, J = 7.2 Hz), 7.39 (dd, 1H, J = 15.6, 11.4 Hz), 7.07 (dd, 1H, J = 7.2, 5.2 Hz), 6.52 (d, 1H, J = 11.4 Hz), 6.44 (t, 1H, J = 11.4 Hz), 5.97 (dd, 1H, J = 15.6, 7.2 Hz), 4.18 (dd, 1H, J = 7.27, 4.1 Hz), 3.9 (dd, 1H, J = 6.2, 4.1 Hz), 2.31 (s, 3H), 1.16 (d, 3H, J = 6.2 Hz); ¹³C NMR (CDCl₃, 100 MHz): δ 154.5, 146.2, 138.0, 135.7, 133.1, 132.0, 129.9, 125.9, 121.9, 75.9, 70.1, 19.0, 17.7; MS (ESI): m/z 220 (M + H)⁺; HRMS (ESI): m/z 220.1329 (M + H)⁺ (calcd for C₁₃H₁₈O₂N 220.1332).

(2R,3S,4E,6E)-7-(2-Chloro-6-isopropylquinolin-3-yl)hepta-4,6-diene-2,3-diol (4c)

According to the procedure 3c, the sulphone 7 (0.10 g, 0.27 mmol) and aldehyde 5b (0.07 g, 0.32 mmol), gave compound 4c (0.07 g, 70% overall yield); mp 121–122 °C; [α]²⁴_D − 24.0 (c 0.03, CHCl₃); IR (KBr): 3423, 2962, 2924, 1585, 1046, 757 cm⁻¹; ¹H NMR (CDCl₃, 500 MHz): δ 8.22 (S, 1H), 7.90 (d, 1H, J = 8.9 Hz), 7.60–7.58 (m, 2H), 7.01 (d, 1H, J = 15.9 Hz), 6.87 (dd, 1H, J = 15.9, 6.9 Hz), 6.59 (dd, 1H, J = 15.9, 9.8 Hz), 5.99 (dd, 1H, J = 15.9, 6.7 Hz), 4.24 (dd, 1H, J = 6.7, 4.5 Hz), 3.98–3.94 (m, 1H), 3.08 (sep, 1H, J = 6.9 Hz), 1.34 (d, 6H, J = 6.9 Hz), 1.20 (d, 3H, J = 6.9 Hz); ¹³C NMR (CDCl₃, 100 MHz): δ 149.1, 148.0, 145.6, 133.4, 133.3, 132.6, 132.0, 130.3, 129.6, 128.0, 127.8, 127.4, 123.5, 75.9, 70.3, 34.0, 23.7, 17.7; MS (ESI): m/z 332 (M + H)⁺; HRMS (ESI): m/z 332.1409 (M + H)⁺ (calcd for C₁₉H₂₃O₂NCl 332.1411).

(2R,3S,4E,6E)-7-(2-Chloro-7-methylquinolin-3-yl)hepta-4,6-diene-2,3-diol (4d)

According to the procedure 3c, the sulphone 7 (0.10 g, 0.27 mmol) and aldehyde 5c (0.06 g, 0.32 mmol) gave compound 4d (0.07 g, 70% overall yield); mp 134–136 °C; [α]²⁴_D − 75.14 (c 0.03, CHCl₃); IR (Neat): 3418, 2922, 1622, 1337, 986 cm⁻¹; ¹H NMR (CDCl₃, 500 MHz): δ 8.20 (s, 1H), 7.73 (brs, 1H), 7.68 (d, 1H, J = 8.3 Hz), 7.36 (d, 1H, J = 8.3 Hz), 6.99 (d, 1H, J = 15.1 Hz), 6.84 (dd, 1H, J = 15.1, 10.5 Hz), 6.57 (dd, 1H, J = 15.1, 10.5 Hz), 5.98 (dd, 1H, J = 15.1, 6.7 Hz), 4.24 (dd, 1H, J = 6.7, 3.0 Hz), 4.01–3.92 (m, 1H), 2.54 (s, 3H), 1.20 (d, 3H, J = 6.7 Hz); ¹³C NMR (CDCl₃, 100 MHz): δ 149.9, 147.0, 141.0, 133.3, 132.6, 131.8, 129.5, 128.8, 127.8, 127.7, 127.2, 127.1, 126.0, 75.9, 70.3, 21.9, 17.7; MS (ESI): m/z 304 (M + H)⁺; HRMS (ESI): m/z 304.1097 (M + H)⁺ (calculated for C₁₇H₁₈N₃O₆ 304.1098).

(E)-Ethyl-3-((2S,3S)-3-methyloxiran-2-yl)acrylate (19)

To a solution of oxalyl chloride (0.87 mL, 9.96 mmol) in 30 mL of CH₂Cl₂ at −78 °C, DMSO (1.41 mL, 19.92 mmol) was added dropwise. The solution was stirred for 10 min and a solution of epoxy alcohol 18 (0.73 mg, 8.3 mmol) in 20 mL of CH₂Cl₂ was added. After 20 min at same temperature, triethylamine (3.47 mL, 24.7 mmol) was added. After 5 min, the reaction mixture of was allowed to warm to rt during 30 min. A solution of (carbethoxymethylene)triphenylphosphorane (7.63 g, 20.8 mmol) in 10 mL of CH₂Cl₂ was added and the reaction mixture was stirred for 24 h. Reaction mixture was quenched with aq. NH₄Cl, and extracted with CH₂Cl₂ (2 × 10 mL). The combined organic layer was dried over Na₂SO₄, crude residue was purified by silica gel column chromatography (EtOAc–hexane, 1 : 4) to afford epoxide 19 (0.55 g, 56%); [α]²⁴_D − 15.54 (c 0.04, CHCl₃); IR (Neat): 2927, 1721, 1455, 1272, 1178, 772 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz): δ 6.67 (dd, 1H, J = 15.6, 7.0 Hz), 6.12 (d, 1H, J = 15.6 Hz), 4.20 (q, 2H, J = 7.17 Hz), 3.18 (dd, 1H, J = 8.7, 7.0 Hz), 2.97 (qd, 1H, J = 5.1, 2.07 Hz), 1.39 (d, 3H, J = 5.1 Hz), 1.28 (t, 3H, J = 7.17 Hz); ¹³C NMR (CDCl₃, 75 MHz): δ 165.5, 144.5, 123.5, 60.4, 57.2, 57.1, 17.3, 14.0; anal. calcd for C₈H₁₂O₃ (156.07): C, 61.52; H, 7.74. Found: C, 61.62; H, 7.82%.

(4R,5S,E)-Ethyl-4-(benzyloxy)-5-hydroxyhex-2-enoate (20)

To a solution of epoxide ester 19 (3.14 g, 20.15 mmol), benzyl alcohol (4.19 mL, 40.3 mmol) in CH₂Cl₂ (40 mL) at −20 °C, BF₃·OEt₂ (2.5 mL, 40.3 mmol) was added and whole mixture was stirred at rt for 1 h, then diluted with H₂O and extracted with ether. The ether layer was washed with saturated brine and dried over Na₂SO₄. The organic layer was concentrated under vacuo. Crude residue was purified by silica gel column chromatography (EtOAc–hexane, 3 : 2) to afford 20 (3.03 g, 60%) as colorless oil; [α]²⁵_D − 51.51 (c 1.0, CHCl₃); IR (Neat): 3508, 2983, 1721, 1264, 836 cm⁻¹; ¹H NMR (CDCl₃, 500 MHz): δ 7.40–7.30 (m, 5H), 6.91 (dd, 1H, J = 15.8, 6.8 Hz), 6.08 (d, 1H, J = 15.8 Hz), 4.66 (d, 1H, J = 11.3 Hz), 4.42 (d, 1H, J = 11.3), 4.23 (q, 2H, J = 7.5 Hz), 4.01–3.90 (m, 2H), 1.31 (t, 3H, J = 7.5 Hz), 1.16 (d, 3H, J = 6.0 Hz); ¹³C NMR (CDCl₃, 75 MHz): δ 165.7, 143.8, 137.6, 128.4, 127.8, 127.7, 124.7, 81.9, 71.3, 69.2, 60.6, 17.9, 14.2; anal. calcd for C₁₅H₂₀O₄ (264.13): C, 68.16; H, 7.63. Found: C, 68.22; H, 7.69%.

(4R,5S,E)-Ethyl 4,5-dihydroxyhex-2-enoate (21)

To a well-stirred solution of AlCl₃ (0.99 g, 7.5 mmol) in 10 mL CH₂Cl₂ at 0 °C was added a solution of 20 (0.40 g, 1.5 mmol) in 5 mL m-xylene and the reaction mixture was stirred for 15 min at the same temperature. The reaction mixture was poured in ice cold water and extracted with ether. The ether layer was washed with saturated NaCl solution and dried over Na₂SO₄. The ether layer was concentrated under vacuo. Crude residue was purified by silica gel column chromatography (EtOAc–hexane, 2 : 1) to give 21 (0.29 g, 76%) as a colorless viscous oil; [α]²⁵_D + 9.01 (c 0.09, CHCl₃); IR (Neat): 3417, 2981, 1706, 1276 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz): δ 6.94 (dd, 1H, J = 15.6, 5.0 Hz), 6.11 (dd, 1H, J = 15.6, 1.5 Hz), 4.31–4.27 (m, 1H), 4.20 (q, 2H, J = 7.1 Hz), 3.98–3.90 (m, 1H), 1.29 (t, 3H, J = 7.1 Hz), 1.14 (d, 3H, J = 6.6 Hz); ¹³C NMR (CDCl₃, 50 MHz): δ 166.5, 145.8, 122.2, 74.5, 69.9, 60.5, 17.2, 14.0; anal. calcd for C₈H₁₄O₄ (174.19): C, 55.16; H, 8.10. Found: C, 55.12; H, 8.19%.

(E)-3-(4R,5R)-2,2,5-(E)-3-((4R,5S)-2,2,5-Trimethyl-1,3-dioxolan-4-yl)prop-2-en-1-ol (22)

According to the procedure 10, diol 21 (0.26 g, 1.49 mmol) gave alcohol 22 (0.21 g, 86%) as colourless, viscous liquid; [α]²⁴_D + 21.20 (c 0.1, CHCl₃); IR (Neat): 3422, 2985, 1376, 1217, 772 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz): δ 5.92 (td, 1H, J = 15.4, 5.1 Hz), 5.70 (tdd, 1H, J = 15.4, 7,7, 1.3 Hz), 4.53 (t, 1H, J = 7.7 Hz), 4.34 (quin, 1H, J = 6.4 Hz), 4.19 (m, 2H), 1.49 (s, 3H), 1.36 (s, 3H), 1.15 (d, 3H, J = 6.4 Hz); ¹³C NMR (CDCl₃, 100 MHz): δ 133.5, 127.1, 108.0, 79.0, 74.0, 62.8, 28.2, 25.5, 16.1.

1-Phenyl-5-(((E)-3-((4R,5S)-2,2,5-trimethyl-1,3-dioxolan-4-yl)allyl)sulfonyl)-1H-tetrazole (17)

According to the procedure 6, compound 22 (0.1 g, 0.58 mmol) gave sulphone 17 (0.17 g, 81%); [α]²⁴_D + 1.12 (c 0.1, CHCl₃); IR (Neat): 2984, 1497, 1347, 1153, 763 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz): δ 7.69–7.56 (m, 5H), 5.95 (dd, 1H, J = 15.1, 6.0 Hz), 5.85–5.75 (m, 1H), 4.52–4.42 (m, 3H), 4.32 (t, 1H, J = 6.7 Hz), 1.44 (s, 3H), 1.33 (s, 3H), 1.02 (d, 3H, 6.7 Hz); ¹³C NMR (CDCl₃, 100 MHz): δ 152.9, 140.3, 131.4, 129.6, 125.1, 116.1, 108.5, 78.0, 73.8, 59.0, 28.0, 25.3, 15.8; MS (ESI): m/z 364 (M + H)⁺; HRMS (ESI): m/z 387.1093 [M + Na]⁺ (calculated for C₁₆H₂₀N₄O₄NaS 387.1097).

(2S,3R,4E,6E)-7-(3-Methylpyridin-2-yl)hepta-4,6-diene-2,3-diol (11R,12S)-4a

According to the procedure 3a, compound 23 (0.05 g, 0.19 mmol) gave (11R,12S)-4a (0.02 g), and cis isomer (11R,12S)-4b (0.01 g) as viscous, color less oils, (total yield 82%); [α]²⁴_D − 26.9 (c 0.04, CHCl₃); IR (Neat): 3414, 2924, 2853, 1731, 1617, 995 cm⁻¹; ¹H NMR (CDCl₃, 500 MHz): δ 8.42 (d, 1H, J = 4.3 Hz), 7.49–7.40 (m, 2H), 7.06 (dd, 1H, J = 7.5, 4.3 Hz), 6.80 (d, 1H, J = 15.1 Hz), 6.56 (dd, 1H, J = 15.1, 11.1 Hz), 6.02 (dd, 1H, J = 15.4, 6.2 Hz), 4.23 (dd, 1H, J = 6.2, 3.3 Hz), 3.93 (dd, 1H, J = 6.2, 3.3 Hz), 2.36 (s, 3H), 1.16 (d, 3H, J = 6.2 Hz); ¹³C NMR (CDCl₃, 100 MHz): δ 153.2, 146.9, 138.2, 134.3, 133.0, 132.4, 130.8, 128.2, 122.1, 75.9, 70.2, 18.7, 17.6; MS (ESI): m/z 220 (M + H)⁺; HRMS (ESI): m/z 220.1327 [M + H]⁺ (calculated for C₁₃H₁₈O₂N 220.1332).

(2S,3R,4E,6Z)-7-(3-Methylpyridin-2-yl)hepta-4,6-diene-2,3-diol (11R,12S)-4b

Yield: 0.01 g, as viscous, colour less oil; [α]²⁴_D − 59.6 (c 0.03, CHCl₃); IR (Neat): 3419, 2924, 1628, 1457, 762 cm⁻¹; ¹H NMR (CDCl₃, 500 MHz): δ 8.43 (d, 1H, J = 4.0 Hz), 7.46 (d, 1H, J = 7.1 Hz), 7.30 (dd, 1H, J = 15.3, 11.2 Hz), 7.07 (dd, 1H, J = 7.1, 4.0 Hz), 6.50 (d, 1H, J = 11.2 Hz), 6.43 (t, 1H, J = 11.2 Hz), 5.97 (dd, 1H, J = 7.1, 15.3 Hz), 4.16 (dd, 1H, J = 6.1, 4.0 Hz), 3.89 (dd, 1H, J = 4.0, 6.1 Hz), 2.30 (s, 3H), 1.15 (d, 3H, J = 6.1 Hz); ¹³C NMR (CDCl₃, 100 MHz): δ 154.6, 146.4, 137.8, 135.3, 132.8, 130.2, 128.5, 126.3, 121.9, 76.0, 70.1, 19.0, 17.8; Mass (ESI-MS): m/z 220 (M + H)⁺; HRMS (ESI): m/z 220.1327 (M + H)⁺ (calculated for C₁₃H₁₈O₂N 220.1332).

Molecular modeling

The calculations of this investigation have been carried out by following the procedure given below. Fuzanin C (3a), fuzanin D (4a) and (11R,12S)-4a were considered for computational prediction of optical rotations. Initially, geometry optimization was performed at B3LYP/6-31G (d) basis set and frequency calculations were done for lowest energy conformer in order to ascertain the minima. Optical rotation was calculated at 589.3 nm using B3LYP/Aug-CC-pVDZ method in gas phase for lowest energy conformer of respective molecules.²⁴ B3LYP/Aug-CC-pVDZ method was opted based on the literature.²² The solvent effect has been omitted for geometry optimizations and optical rotation calculations, since a methodology does not yet exist which predicts the solvent effects with uniform reliability.²⁵ All these calculations were performed using Gaussian 09 software.²⁶

Biological activity

Cell culture. HT-29 (Colon cancer) cell line was grown as adherent in RPMI medium, ME-180 (Cervical cancer), MCF-7 (Breast cancer) and MDA-MB-453 (Breast cancer) cell line were grown as adherent in DMEM medium supplemented with 10% fetal bovine serum, 100 μg mL⁻¹ penicillin, 200 μg mL⁻¹ streptomycin, 2 mM L-glutamine, and culture was maintained in a humidified atmosphere with 5% CO₂. All in vitro experiments were performed during the exponential phase of cell growth.

Preparation of samples for cytotoxicity

20 mM stock solution for compounds was prepared in DMSO, from the above stock various dilutions were made with sterile PBS to get required concentration.

Cytotoxicity screening using MTT assay

MTT assay was performed according to the method of Naidu et al.²⁷ MTT assay is a standard colorimetric assay for measuring cellular proliferation. MTT is a tetrazolium salt, which is yellow in color and is photosensitive. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] is taken by the living cells and reduced by a mitochondrial dehydrogenase enzyme to a purple formazan product that is impermeable to the cell membrane. Solubilisation with solvents like DMSO leads to liberation of product and amount of purple formazan product is directly related to the cell viability. 1 × 10⁴ cells (counted by trypan blue exclusion dye method) in 96-well plates were incubated with series of concentrations of compounds for 48 h at 37 °C in DMEM with 10% FBS medium. Then the above media was replaced with 90 μL of fresh serum free media and 10 μL of MTT reagent (5 mg mL⁻¹) and plates were incubated at 37 °C for 4 h, there after the above media was replaced with 200 μL of DMSO and incubated at 37 °C for 10 min. The absorbance at 570 nm was measured on a spectrophotometer (spectra max, Molecular devices). IC₅₀ values were determined from plot: % cell viability (from control) versus concentration.

Acknowledgements

The authors thank the Director CSIR-IICT and Project Director NIPER-Hyderabad for support. VJR thank ORIGIN-0108 for funding. SNK, CH.N.S.S.P.K, E.S thank CSIR-New Delhi, for the fellowships. This is main lab project (MLP) of IICT.

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Footnote

† Electronic supplementary information (ESI) available: ¹H NMR spectra and ¹³C NMR spectra for all final compounds, molecular modeling studies, Cartesian coordinates of fuzanins C, D, (11R,12S)-fuzanin D. See DOI: 10.1039/c3ra47263a

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