Amitorines A and B, Nitrogenous Diterpene Metabolites of Theonella

Mar 23, 2016 - ... collected from the reef of Iriomote Island, Okinawa, Japan, in 2003, ...... Araujo , N. C.; Korshin , E. E.; Bickley , J. F.; Ward ...
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Amitorines A and B, Nitrogenous Diterpene Metabolites of Theonella swinhoei: Isolation, Structure Elucidation, and Asymmetric Synthesis Koichiro Ota,† Yukiko Hamamoto,† Wakiko Eda,† Kenta Tamura,† Akiyoshi Sawada,† Ayako Hoshino,† Hidemichi Mitome,‡ Kazuo Kamaike,† and Hiroaki Miyaoka*,† †

School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan College of Pharmaceutical Sciences, Matsuyama University, 4-2 Bunkyo-cho, Matsuyama, Ehime 790-8578, Japan

‡

S Supporting Information *

ABSTRACT: Two new nitrogenous prenylbisabolanes never before found in Lithistid sponges have been isolated from Theonella swinhoei. These new diterpenes, named amitorine A (1) and amitorine B (2), containing a prenylbisabolane skeleton have been characterized by spectroscopic analyses, and the relative and absolute configurations of 1 and 2 were determined by asymmetric synthesis of both diastereomers via the common bicyclic lactone 6 intermediate.

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L

RESULTS AND DISCUSSION A freeze-dried sample of T. swinhoei, collected from the reef of Iriomote Island, Okinawa, Japan, in 2003, was extracted with hexane and then acetone. The hexane and acetone extracts were concentrated to give a hexane residue (17.8 g) and acetone residue (9.54 g), respectively. The hexane residue and acetone residue were purified by repeated chromatographic separation to give new diterpene isocyanide 1 (690 mg) and formamide 2 (43.0 mg), together with known terpenoids 10-epi-tormesol,15,16 13-epi-neoverrucosan-5β-ol,17 and 10-isothiocyanato-4amorphene.18 The known terpenoids were identified by comparison of their spectroscopic data with the literature. Compound 1, isolated as an optically active, colorless oil, has the molecular formula C21H33N based on the [M + H]+ ion peak at m/z 300.2693 along with a diagnostic peak at m/z 273 due to the loss of HCN from the protonated molecule in the high-resolution ESIMS spectrum. The IR spectrum of 1 indicated the presence of an isocyano group (2127 cm−1). The 13C NMR (Table 1), 1H NMR (Table 2), and DEPT spectra showed signals indicating the presence of five methyls, seven sp3 methylenes, one sp3 methine, one sp3 nonprotonated carbon, three sp2 methines, three olefinic quaternary carbons, and one isocyano carbon. These spectroscopic data, coupled with the six degrees of unsaturation, suggested that compound 1 is a monocyclic diterpenoid possessing one isocyano group and three trisubstituted olefin [δH 5.36 (1H, m), δH 5.10 (1H, m), δH 5.09 (1H, m), δC 136.3 (C), δC 134.3 (C), δC 131.4 (C), δC 124.2 (CH), δC 122.7 (CH), δC 119.5 (CH)] moieties. COSY cross-peaks indicated sequences of C-2 to C-4, C-8 to C-10, and C-12 to C-14. The planar structure of 1 was determined on the basis of the following correlations (Figure 1) in the HMBC spectrum: H-2/C-3, C-17; H-4/C-3, C-17; H-6/

ithistid demosponges now include 13 families and 38 genera.1 These sponges possess a particular category of spicules, referred to as “desmas”, from which was derived the former name of this order, Desmophorida.2 The desmas spicules cannot be classified as either monoaxonic or tetraxonic megascleres. As a lithistid group, theonellids are characterized largely by their megascleres; the spicule component is dominated by a solid, rigid, heavily silicified skeleton, formed by an interlocking network of ornate tetraclone desmas spicules.3 Species of theonellids are also known to contain other tetractinal spicules, dichotrianes, discotriaenes, and phyllotriaenes at the surface, in addition to some monactinal spicules.3 The sponge genus Theonella (order Lithistida, family Theonellidae), which is commonly encountered on tropical reefs in the Indo-Pacific region, shows remarkable chemical diversity. Species of Theonella have since been referred to as noteworthy sources of a diverse array of peptide and polyketide secondary metabolites.4−11 Chemical studies on sponges of this genus have revealed numerous metabolites characterized by the presence of nitrogen-containing functional groups, typically in the form of amine,12,13 isocyanate,13 isothiocyanate,12 formamide,12 and isocyanide13,14 substituents attached to a sesquiterpene skeleton. In this paper, we report on the isolation, structure elucidation, and asymmetric syntheses of two new prenylbisabolane diterpene metabolites, amitorines A (1) and B (2), from Theonella swinhoei.

Received: December 3, 2015 © XXXX American Chemical Society and American Society of Pharmacognosy

A

DOI: 10.1021/acs.jnatprod.5b01069 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Table 1. 13C NMR Data for Compounds 1 and 2 (J in Hz) 2a 1

a

a

s-trans

position

δC, type

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 NC NHCHO

26.1, CH2 119.5, CH 134.3, C 30.7, CH2 23.8, CH2 41.7, CH 63.6, C (t, J = 4.5) 38.8, CH2 22.3, CH2 122.7, CH 136.3, C 39.6, CH2 26.6, CH2 124.2, CH 131.4, C 25.7, CH3 23.2, CH3 23.0, CH3 16.0, CH3 17.7, CH3 153.7, C (t, J = 4.1)

s-cis

δC, type 26.7, 120.3, 134.6, 31.3, 23.7, 43.8, 58.0, 40.3, 22.1, 123.6, 136.4, 40.0, 27.0, 124.6, 131.9, 26.1, 23.6, 21.4, 16.5, 18.1,

δC, type

CH2 CH C CH2 CH2 CH C CH2 CH2 CH C CH2 CH2 CH C CH3 CH3 CH3 CH3 CH3

163.6, CH

24.2, 120.7, 134.5, 31.5, 26.7, 41.0, 59.7, 36.5, 22.6, 124.3, 131.8, 40.1, 27.1, 124.7, 135.8, 26.1, 23.6, 21.0, 16.4, 18.1,

CH2 CH C CH2 CH2 CH C CH2 CH2 CH C CH2 CH2 CH C CH3 CH3 CH3 CH3 CH3

Figure 1. Selective COSY and HMBC correlations of 1 and 2 (s-trans rotamer).

an isocyano group, while a C-7 signal at δC 63.6 (t, J = 4.5 Hz, coupled to 14N) was assigned to a carbon bearing a nitrogen substituent. On the basis of the above evidence, the gross structure of 1 was determined as 7-isocyanoprenylbisabola2,10,14-triene, named amitorine A.22 Compound 2, isolated as an optically active, colorless oil, has the molecular formula C21H36NO based on the protonated molecule peak [M + H]+ at m/z 318.2796 in the highresolution ESIMS spectrum. The IR spectrum of 2 suggested the presence of a formamide group (1681 cm−1). This assignment was confirmed by the 1H NMR resonance at 8.18 (0.67H, d, s-trans −NHCHO, J = 12.3 Hz), 8.10 (0.33H, m, scis −NHCHO), 5.65 (0.67H, d, s-trans −NHCHO, J = 12.3 Hz), and 5.11 (0.33H, brs, s-cis −NHCHO) ppm. 1H NMR (Table 1) and DEPT spectra showed signals indicating the presence of five methyls, seven sp3 methylenes, one sp3 methine, one sp3 nonprotonated carbon, three sp2 methines, three olefinic quaternary carbons, and one formamide carbon. These spectroscopic data, coupled with the five degrees of unsaturation, suggested that compound 2 is a monocyclic diterpenoid possessing one formamide group [δC 163.6 (s-trans −NHCHO), δC 160.7 (s-cis −NHCHO)] and three trisubstituted olefin [s-trans: δH 5.35 (0.67H, brs), δH 5.08 (0.67H, m), 5.07 (0.67H, m), δC 136.4 (C), δC 134.6 (C), δC 131.9 (C), δC 124.6 (CH), δC 123.6 (CH), δC 120.3 (CH), s-cis: δH 5.35 (0.33H, brs), δH 5.12 (0.33H, m), 5.08 (0.33H, m), δC 135.8 (C), δC 134.5 (C), δC 131.8 (C), δC 124.7 (CH), δC 124.3 (CH), δC 120.7 (CH)] moieties. COSY cross-peaks indicated sequences of C-2 to C-4, C-8 to C-10, and C-12 to C-14. The planar structure of 2 was determined on the basis of the following correlations (Figure 1) in the HMBC spectrum of the s-trans rotamer: H-2/C-3, C-17; H-4/C-3, C-17; H-6/C-18; H8/C-6, C-7, C-18; H-9/C-7, C-11; H-10/C-19; H-12/C-10, C11, C-19; H-13/C-11, C-15; H-14/C-16, C-20; NH/C-7; CHO/C-7. On the basis of the above evidence, the gross structure of 2 was determined as 7-formamidoprenylbisabola2,10,14-triene, named amitorine B.22 Furthermore, amitorine A (1) was treated with acetic acid at room temperature (rt) for 12 h to afford amitorine B (2) in 96% yield. The 1H and 13C NMR spectra and [α]D value were identical with those of natural amitorine B (2). These results clearly indicated that amitorine B (2) possesses the same absolute configuration as amitorine A (1). The possibility that amitorine B (2) may have been derived from amitorine A (1) during the extraction or separation by silica gel column chromatography was then investigated. For demonstration purposes, a solution of amitorine A (1) was mixed with scraped support material from a TLC plate and then allowed to stand for 1 week. Extraction of the support material with Et2O afforded amitorine A (1) with trace amounts of amitorine B (2). Although certain isocyanides are known to be easily converted to formamides by treatment with an acid,23 amitorine

160.7, CH

125 MHz in CDCl3.

Table 2. 1H NMR Data for Compounds 1 and 2 (J in Hz) 2a 1a

s-trans

s-cis

position

δH (J in Hz)

δH (J in Hz)

δH (J in Hz)

1a 1b 2 4 5a 5b 6 8a 8b 9 10 12a 12b 13 14 16 17 18 19 20 CHO NH

2.01, m 1.84, m 5.36, m 2.05−2.03, m 1.97, m 1.39, m 1.72, m 1.67, m 1.52, m 2.14−2.13, m 5.10, m 2.03, m 1.99, m 2.09−2.03, m 5.09, m 1.68, d (0.9) 1.66, s 1.31, d (1.9) 1.63, s 1.60, s

2.01, m 1.80, m 5.35, brs 1.98−1.97, m 1.82, m 1.27, m 1.53, m 1.56−1.55 (2H), m 2.00−1.99, m 5.07, m 1.97−1.95 (2H), m

1.78, m 1.30, m 5.35, brs 1.99−1.98, m 2.02, m 1.80, m 2.09, m 1.88, m 1.67, m 1.96−1.95, m 5.12, m 1.56−1.54 (2H), m

2.07−2.03, m 5.08, m 1.67, s 1.64, s 1.24, s 1.58, s 1.59, s 8.18, d (12.3) 5.65, d (12.3)

2.01−2.00, m 5.08, m 1.67, s 1.64, s 1.27, s 1.58, s 1.59, s 8.10, d (1.8) 5.11, brs

a

500 MHz in CDCl3.

C-18; H-8/C-6, C-7, C-18; H-9/C-7, C-11; H-10/C-19; H-12/ C-10, C-11, C-19; H-13/C-11, C-15; H-14/C-16, C-20. Furthermore, in the 13C NMR spectrum, a signal at δC 153.7 (t, J = 4.1 Hz, coupled to 14N)19−21 confirmed the presence of B

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temperature for 12 h.29 Furthermore, the addition of TMSCl to the generated lithium acetylide followed by the usual workup gave TMS-acetylene 4 as a volatile compound. Hydroboration30 of TMS-acetylene 4 and subsequent oxidation yielded carboxylic acid 5. Iodolactonization31 of carboxylic acid 5 proceeded to give the unstable iodolactone, which was then subjected to elimination to give the corresponding bicyclic lactone 6 in 70% yield over four steps from methyl ketone 3. The optical purity of bicyclic lactone 6 was determined as 92% ee, which was estimated from the 1H NMR spectra in the presence of the chiral shift reagent “Chirabite-AR”.32 To demonstrate the practical utility of Chirabite-AR, synthetic racemic lactone rac-6 was tested. Prior to the comparison between synthetic lactone 6 and rac-6 with Chirabite-AR, we examined the effect of differing amounts of Chirabite-AR on rac-6 in an effort to determine conditions that produced sufficient signal separations between 6 and its enantiomer ent-6. Consequently, a mixture of rac-6 and 100 mol % of ChirabiteAR was measured sequentially by 400 MHz 1H NMR at 25 °C in CDCl3. Signal separations were observed at 4.81 and 4.67 ppm, respectively, for the shielded olefinic methylene proton at C-17, and good enantiomeric discrimination was achieved for 6 and ent-6. NMR analysis of bicyclic lactone 6 under the same conditions indicated that the separated signals exhibited a 1/24 integration ratio. Therefore, the optical purity of 6 was determined as 92% ee. Bicyclic lactone 6 was treated with LDA in tetrahydrofuran (THF) at −78 °C and then with homogeranyl iodide33 in hexamethylphosphoric triamide (HMPA) to give α-monosubstituted lactone 7. α-Monosubstituted lactone 7 was alkylated at C-7 under conditions similar to those used for the previous reaction to give single diastereomeric lactone 8 in 94% yield. Similarly, bicyclic lactone 6 was treated with LDA in THF at −78 °C and then with iodomethane in HMPA to give αmonosubstituted lactone 9 (Scheme 3). α-Monosubstituted

A (1) was found to be resistant to hydrolysis under weakly acidic conditions. In the present study, these samples were frozen immediately after collection and then freeze-dried prior to transport. Moreover, separation by silica gel column chromatography was quickly executed following extraction with hexane and Et2O. Taken together, we concluded that it is unlikely that the extracts underwent hydrolysis and that the presence of amitorine B (2) was presumed not to be an artifact. As mentioned above, we were able to determine the planar structure of amitorines A (1) and B (2). Previously, it was reported that two bisabolane isocyanides A and B24 and bisabolane formamide C25 were isolated from marine organisms (Figure 2). These sesquiterpenoids possess a bisabolane

Figure 2. Nitrogenous bisabolane sesquiterpenoids.

skeleton with an isocyano/formamido moiety attached to a nonprotonated carbon at C-7, and their relative and absolute configurations were elucidated by single-crystal X-ray diffraction analysis following chemical conversion.24,25 Crystallization efforts following the chemical conversion to the corresponding p-bromophenylurea13 of amitorines A (1) and B (2) were unsuccessful in generating suitable single crystals for diffraction analysis. In order to unambiguously fully characterize compounds 1 and 2, we carried out the syntheses of both epimers at C-7. Subsequent coupling of the common bicyclic lactone intermediate with methyl and homogeranyl groups should generate two diastereomers and lead to the determination of the absolute configurations. The synthesis of amitorines A (1) and B (2) commenced with construction of a rigid bicyclic lactone core for the purpose of stereoselective double alkylation at C-7. The synthetic procedure for the generation of bicyclic lactone 6 from (S)methyl ketone 3 (99% ee)26,27 is described in Scheme 1.

Scheme 2. Synthesis of α-Disubstituted Lactone 8a

Scheme 1. Synthesis of Bicyclic Lactone 6a (a) LDA, THF, −78 °C; homogeranyl iodide, HMPA, −78 °C, 95%; (b) LDA, THF, −78 °C; CH3I, HMPA, −78 °C, 94%.

a

lactone 9 was subjected to a second alkylation with homogeranyl iodide to give α-disubstituted lactone 10 in 81%

a (a) LDA, THF, −78 to 0 °C; (EtO)2POCl, −78 °C to rt; LDA, −78 °C to rt; TMSCl, −78 °C to rt; (b) Cy2BH, THF, rt; NaOH aq, H2O2 aq, rt; (c) NIS, CH2Cl2, rt; (d) LDA, THF, −78 °C to rt, 70% in four steps.

Scheme 3. Synthesis of α-Disubstituted Lactone 10a

Methyl ketone 3 was added to a solution of lithium diisopropylamide (LDA, 1.2 equiv) at −78 °C, and the solution was stirred at this temperature for 1 h. Diethyl chlorophosphate was then added, and the solution was warmed to rt.28 The kinetic enol phosphates were not isolated but taken on directly. This reaction mixture was subjected to elimination conditions (LDA, 3.0 equiv) at −78 °C and then stirred at this

(a) LDA, THF, −78 °C; CH3I, HMPA, −78 °C, 79%; (b) LDA, THF, −78 °C; homogeranyl iodide, HMPA, −78 °C, 81%.

a

C

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yield. Lactones 9 and 10 were then generated as single diastereomers. The relative configurations of lactones 8, 9, and 10 were determined by NOESY analysis (Figure 3). The NOE

Figure 3. Selected NOE correlations of compounds 8, 9, and 10.

Figure 4. Stereoselectivity of α-alkylation and predicted conformation of lactone 9.

correlations of 8 between H-1a and H-4b, H-1b and H3-18, and H-4a and H2-8 suggested that the homogeranyl group was pseudoaxially oriented. Therefore, the configuration at C-7 in lactone 8 was found to be S. Similarly, the NOE correlations of 9 between H-1a and H-4b, H-1b and H3-18, and H-4a and H-7 suggested that the C-18 methyl group was pseudoequatorially oriented. The NOE correlations of 10 between H-1a and H-4b and between H-4a and H3-18 suggested that the C-18 methyl group was pseudoaxially oriented. Therefore, the configuration at C-7 in lactone 10 was found to be R. Further, the following additional experiments were performed to support the stereoselectivity in a stepwise double alkylation of lactone 6. α-Monosubstituted lactone 9 was treated with LDA and homoprenyl iodide34 to give αdisubstituted lactone 11 as a single diastereomer. Lactone 11 was converted to amine 12 by a Birch reduction, followed by Curtius rearrangement and acidic hydrolysis of the corresponding isocyanate. The selection of these conversions is later described in detail. The 1H and 13C NMR spectroscopic data of (6S,7R)-amine 12 matched those reported for (6R,7S)-7amino-7,8-dihydro-α-bisabolene.35 Comparison of the specific rotation data {[α]20D −56.3 (c 3.2, CHCl3); lit.35 [α]20D +59.9 (c 3.0, CHCl3)} enabled us to establish the absolute configuration of synthetic amine 12. These results clearly show that a diastereoselectivity in the second alkylation at C-7 is strongly dependent on their intermediate conformation.

aforementioned NOE correlation between H-1a and H-4b in α-monosubstituted lactone 9 suggests that the favored conformers of the corresponding lithium enolate comprise a boat/twist-boat conformation. In each conformer derived from 7 and 9, attack of the alkyl iodide from the less hindered convex side would lead to α-disubstituted lactones 8 and 10 as single isomers. For the ring opening of bicyclic lactone 8, we examined a variety of reduction conditions. Our initial attempts included cleavage via a corresponding π-allyl palladium complex such as LiBHEt3/Pd(dppe)Cl2 or Pd(dppp)Cl2. However, each of these resulted in isomerization of the double bond. Fortunately, the best result was achieved upon treatment of bicyclic lactones with Li in liquid NH3,36 which gave carboxylic acid 13 without double-bond isomerization (Scheme 5). Carboxylic acid 13 was converted to amine 15 by a Curtius rearrangement using diphenyl phosphoryl azide (DPPA), followed by hydrolysis of corresponding isocyanate 14 using aqueous HCl/AcOH.37 Generated amine 15 was then converted into formamide 16 by acetic formic anhydride.38 Finally, dehydration with pTsCl in Scheme 5. Synthesis of Isocyanide 17a

Scheme 4. Synthesis of (6S,7R)-7-Amino-7,8-dihydro-αbisabolene 12a

a (a) LDA, THF, −78 °C; homoprenyl iodide, HMPA, −78 °C, 82%; (b) Li, liq NH3, Et2O, −78 °C; (c) DPPA, Et3N, toluene, reflux; (d) 15% HCl aq, AcOH, rt; 10% NaOH aq, 34% in three steps.

The complete diastereoselectivity associated with the second alkylation could be explained by consideration of the conformation of lithium enolate (Figure 4). Therefore, the most probable conformation of the lithium enolates possessing an oxabicyclo[3.3.1]nonene framework comprises either a chair/twist-boat or a boat/twist-boat arrangement. The

a (a) Li, liq NH3, Et2O, −78 °C; (b) DPPA, Et3N, toluene, reflux; (c) 15% HCl aq, AcOH, rt; 10% NaOH aq; (d) acetic formic anhydride, CH2Cl2, −78 °C, 81% in four steps; (e) pTsCl, pyridine, CH2Cl2, rt, 97%.

D

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the presence of pyridine38 yielded isocyanide 17 in excellent yield. Moreover, under similar conditions, bicyclic lactone 10 was converted into isocyanide 18 in five steps (Scheme 6).

Animal Material. The sponge specimens of Theonella swinhoei were obtained from the coral reef of Iriomote Island, Okinawa, Japan, in July 2003. A voucher specimen has been deposited at Tokyo University of Pharmacy and Life Sciences (S-03-08). Extraction and Isolation. The freeze-dried samples (400 g), derived from the wet specimens (3.0 kg), were cut into small pieces and extracted with hexane (5.0 L × 3) and then acetone (5.0 L × 3). The hexane extracts and acetone extracts were concentrated to give a hexane residue (17.8 g) and an acetone residue (9.54 g), respectively. The hexane residue was chromatographed on silica gel using a hexane/ EtOAc (1:1, 1:3, to 0:1) gradient and MeOH as eluent to produce fractions 1 (6.60 g), 2 (10-epi-tormesol, 5.52 g), and 3 (5.60 g). Fraction 1 was subjected to flash silica gel column chromatography (elution with hexane/Et2O, 15:1) to give fractions 1-1 (2.54 g), 1-2 (1.92 g), 1-3 (0.90 g), 1-4 (102 mg), and 1-5 (1.11 g). Fraction 1-2 was subjected to flash silica gel column chromatography (elution with hexane/Et2O, 30:1) to give 10-isothiocyanato-4-amorphene (121 mg). Fraction 1-3 was subjected to flash silica gel column chromatography (elution with hexane/Et2O, 15:1) to give amitorine A (1) (690 mg). The acetone residue was chromatographed on silica gel using a hexane/EtOAc (3:1 to 0:1) gradient and MeOH as eluent to produce fractions 4 (758 mg), 5 (1.36 g) 6 (1.34 g), 7 (1.36 g), 8 (2.25 g), and 9 (1.70 g). Fraction 7 was subjected to flash silica gel column chromatography (elution with CHCl3/acetone, 99:1) to give fractions 7-1 (34.9 mg), 7-2 (1.00 g), and 7-3 (276 mg). Fraction 7-2 was subjected to flash ODS column chromatography (elution with CH3CN/acetone, 19:1) to give 13-epi-neoverrucosan 5β-ol (205 mg). Fraction 8 was subjected to flash silica gel column chromatography (elution with CHCl3/EtOAc, 9:1) to give fractions 8-1 (105 mg), 8-2 (115 mg), 8-3 (197 mg), 8-4 (1.05 g), and 8-5 (736 mg). Fraction 8-2 was subjected to flash silica gel column chromatography (elution with hexane/acetone, 4:1) to give fractions 8-2-1 (21.8 mg), 8-2-2 (80.2 mg), and 8-2-3 (10.2 mg). Fraction 8-2-2 was subjected to Sephadex LH-20 column chromatography (elution with CHCl3/MeOH, 1:1) to give fractions 8-2-2-1 (1.1 mg), 8-2-2-2 (59.8 mg), and 8-2-2-3 (15.9 mg). Fraction 8-2-2-2 was subjected to flash silica gel column chromatography (elution with hexane/EtOAc, 2:1) to give fractions 8-2-2-2-1 (3.0 mg), 8-2-2-2-2 (53.3 mg), and 8-22-2-3 (3.1 mg). Fraction 8-2-2-2-2 was subjected to Sephadex LH-20 column chromatography (elution with CHCl3/MeOH, 1:1) to give amitorine B (2) (43.0 mg). Amitorine A (1): colorless oil; [α]20D −36.6 (c 1.42, CHCl3); IR (neat) νmax 2925, 2127, 1448, 1381 cm−1; 1H NMR (CDCl3, 500 MHz), Table 2; 13C NMR (CDCl3, 125 MHz), Table 1; ESIMS m/z 300 [M + H]+ (100), 273 [M − NC]+ (40); HRESIMS m/z 300.2693 [M + H]+ (calcd for C21H34N, 300.2691). Amitorine B (2): colorless oil; [α]26D −35.0 (c 0.39, CHCl3); IR (neat) νmax 3290, 2964, 2924, 1681, 1540, 1454 cm−1; 1H NMR (CDCl3, 500 MHz), Table 2; 13C NMR (CDCl3, 125 MHz), Table 1; ESIMS m/z 318 [M + H]+ (100), 273 [M − NHCHO]+ (40); HRESIMS m/z 318.2796 [M + H]+ (calcd for C21H36NO, 318.2797). Acid-Catalyzed Hydrolysis of Amitorine A (1) to Amitorine B (2). To amitorine A (1) (90.1 mg, 0.301 mmol) was added glacial AcOH (3.00 mL). The mixture was stirred at rt for 12 h. The reaction mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (elution with hexane/EtOAc, 4:1) to give amitorine B (2) (91.4 mg, 96% yield): [α]26D −33.8 (c 0.50, CHCl3). TLC-Support-Mediated Hydrolysis of Amitorine A (1) to Amitorine B (2). To a solution of amitorine A (1) (2.0 mg) in Et2O (0.500 mL) was added the support material stripped off from a TLC plate (Merck). After evaporation of the solvent in vacuo, the silica gel support was left for 1 week. Extraction with Et2O gave a colorless oil, which proved to be identical with amitorine A (1) with trace amounts of amitorine B (2) (1H NMR). (1S,5S)-8-Methylene-2-oxabicyclo[3.3.1]nonan-3-one (6). nBuLi (2.60 M solution in hexane, 1.00 mL, 2.60 mmol) was added to a solution of diisopropylamine (0.400 mL, 2.85 mmol) in THF (3.90 mL) at −78 °C, and the resulting mixture was stirred at 0 °C for 30 min. After cooling to −78 °C, a solution of methyl ketone 3 (300 mg,

Scheme 6. Synthesis of Isocyanide 18a

a (a) Li, liq NH3, Et2O, −78 °C; (b) DPPA, Et3N, toluene, reflux; (c) 15% HCl aq, AcOH, rt; 10% NaOH aq; (d) acetic formic anhydride, CH2Cl2, −78 °C, 68% in four steps; (e) pTsCl, pyridine, CH2Cl2, rt, 95%.

The 1H and 13C NMR spectroscopic data of isocyanides 17 and 18 were compared with those of natural amitorine A (1). The 1H NMR data of natural amitorine A (1) and isocyanides 17 and 18 were very similar. However, the data for isocyanide 17 were identical with those of natural amitorine A (1), while the data for isocyanide 18 clearly differed at C-8 and C-17 in the 13C NMR spectrum. These results indicated that the structure of (6S,7S)-isocyanide 17 represented the actual structure of natural amitorine A (1). Comparison of the specific rotation data {natural amitorine A (1): [α]20D −36.6 (c 1.42, CHCl3); isocyanide 17: [α]20D −33.8 (c 0.27, CHCl3)} enabled us to establish the absolute configuration of 1 as being identical to that of synthetic isocyanide 17. Furthermore, amitorine B (2) possesses the same absolute configuration of amitorine A (1) on the basis of the aforementioned hydrolysis experiments. Consequently, the absolute configuration of the two stereogenic centers in amitorines A (1) and B (2) was determined to be 6S and 7S. In conclusion, two nitrogenous diterpenoids, amitorine A (1) and amitorine B (2), were isolated from T. swinhoei. The total syntheses of amitorines A (1) and B (2) were performed by constructing the prenylbisabolane skeleton through a stepwise double alkylation via the common bicyclic lactone 6 intermediate. This stereoselective process allowed for an unambiguous determination of the absolute configurations of amitorines A (1) and B (2). The biological activity of the two new diterpenoids is currently under investigation.

■

EXPERIMENTAL SECTION

General Experimental Procedures. Melting points (mp) were measured using a Yanaco MP-S3 melting point apparatus and are uncorrected. Optical rotations were measured with a JASCO P-1030 polarimeter. IR spectra were recorded with a JASCO FT-IR/620 spectrometer. 1H and 13C NMR spectra were recorded on Bruker Biospin AVANCE III HD 400 Nanobay, DRX-500, and AV-600 spectrometers. Chemical shifts are given on the δ (ppm) scale using tetramethylsilane (TMS) as the internal standard. HRESIMS spectra were obtained using a Micromass LCT spectrometer. Elemental analysis data were obtained using an Elementar Vario EL. Reactions were monitored by thin layer chromatography on glass plates coated with a fluorescent indicator with a 254 nm excitation wavelength (Merck Merck-5554-7). Flash column chromatography was performed using Kanto Chemical silica gel 60N (spherical, natural) 40−50 μm, Wakosil 25C18, and Sephadex LH-20. All reagents (Aldrich, Kanto, TCI, and Wako) and solvents were of commercial quality and were used as received. E

DOI: 10.1021/acs.jnatprod.5b01069 J. Nat. Prod. XXXX, XXX, XXX−XXX

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5.05 (2H, m), 4.91 (1H, s), 4.87 (1H, m), 4.85 (1H, m), 2.54 (1H, m), 2.30−1.90 (11H, m), 1.70−1.67 (3H, m), 1.63−1.44 (4H, m), 1.61 (3H, s), 1.60 (3H, s); 13C NMR (CDCl3, 100 MHz) δ 174.4 (C), 174.3 (C), 145.1 (C) × 2, 136.5 (C), 136.4 (C), 131.6 (C), 131.4 (C), 124.13 (CH), 124.08 (CH), 124.0 (CH), 123.1 (CH), 111.3 (CH2) × 2, 80.3 (CH) × 2, 43.1 (CH), 43.0 (CH), 39.7 (CH2) × 2, 34.13 (CH2), 34.12 (CH2) × 2, 32.0 (CH2), 28.5 (CH) × 2, 28.2 (CH2), 27.9 (CH2), 26.7 (CH2), 26.6 (CH2), 25.8 (CH2) × 2, 25.7 (CH3) × 2, 25.1 (CH2), 25.0 (CH2), 23.3 (CH3), 17.7 (CH3), 17.6 (CH3), 16.1 (CH3); ESIMS m/z 325 [M + Na]+ (100); HRESIMS m/z 325.2144 [M + Na]+ (calcd for C20H30O2Na, 325.2144); anal. C 79.37, H 9.84%, calcd for C20H30O2, C 79.42, H 10.00%. (1S,4S,5S)-4-((E)-4,8-Dimethylnona-3,7-dien-1-yl)-4-methyl-8methylene-2-oxabicyclo[3.3.1]nonan-3-one (8). nBuLi (2.65 M solution in hexane, 1.65 mL, 4.37 mmol) was added to a solution of diisopropylamine (0.653 mL, 4.66 mmol) in THF (10.0 mL) at −78 °C, and the resulting mixture was stirred at 0 °C for 30 min. After cooling to −78 °C, a solution of lactone 7 (434 mg, 1.37 mmol) in THF (3.70 mL) was introduced, stirring continued at this temperature for 1 h, and then premixed MeI (0.852 mL, 13.7 mmol) and HMPA (3.58 mL, 20.6 mmol) was introduced to the mixture. After stirring for 2 h at the same temperature, the reaction mixture was diluted with Et2O, washed with a saturated aqueous NH4Cl solution, H2O, and brine, dried over anhydrous MgSO4 and Na2SO4, and then concentrated in vacuo. The residue was purified with flash column chromatography on silica gel (hexane/EtOAc, 12:1) to give lactone 8 (408 mg, 94% yield) as a colorless oil: Rf 0.25 (hexane/EtOAc, 12:1); [α]25D +108 (c 0.37, CHCl3); IR (neat) νmax = 2931, 1727, 1449 cm−1; 1 H NMR (CDCl3, 600 MHz) δ 5.14 (1H, t, J = 6.0 Hz), 5.09 (1H, m), 4.90 (1H, s, H-17a), 4.87 (1H, d, J = 4.1 Hz, H-2), 4.84 (1H, s, H17b), 2.50 (1H, m, H-1a), 2.28 (1H, m, H-4a), 2.23 (1H, m, H-4b), 2.10−2.03 (4H, m), 2.02−2.00 (2H, m, H-8), 1.98−1.92 (2H, m), 1.86 (1H, m), 1.74 (1H, m), 1.68 (3H, s), 1.64−1.54 (2H, m), 1.61 (3H, s), 1.60 (3H, s), 1.39 (3H, s, H-18); 13C NMR (CDCl3, 150 MHz) δ 177.8 (C), 145.2 (C), 135.6 (C), 131.5 (C), 124.2 (CH), 123.6 (CH), 111.1 (CH2), 80.7 (CH), 43.1 (C), 39.7 (CH2), 34.8 (CH2), 34.4 (CH), 30.2 (CH2), 28.3 (CH2), 26.7 (CH2), 26.2 (CH2), 26.0 (CH3), 25.7 (CH3), 21.0 (CH2), 17.7 (CH3), 16.1 (CH3); ESIMS m/z 339 [M + Na]+ (100); HRESIMS m/z 339.2301 [M + Na]+ (calcd for C21H32O2Na, 339.2300); anal. C 79.73, H 10.39%, calcd for C21H32O2, C 79.70, H 10.19%. (1S,4R,5S)-4-Methyl-8-methylene-2-oxabicyclo[3.3.1]nonan-3one (9). nBuLi (2.65 M solution in hexane, 1.91 mL, 5.06 mmol) was added to a solution of diisopropylamine (0.729 mL, 5.20 mmol) in THF (14.0 mL) at −78 °C, and the resulting mixture was stirred at 0 °C for 30 min. After cooling to −78 °C, a solution of bicyclic lactone 6 (233 mg, 1.53 mmol) in THF (1.30 mL) was introduced, stirring continued at this temperature for 1 h, and then premixed MeI (0.952 mL, 15.3 mmol) and HMPA (4.00 mL, 23.0 mmol) was introduced to the mixture. After stirring for 1 h at the same temperature, the reaction mixture was diluted with Et2O, washed with saturated aqueous NH4Cl solution, H2O, and brine, dried over anhydrous MgSO4 and Na2SO4, and then concentrated in vacuo. The residue was purified with flash column chromatography on silica gel (hexane/EtOAc, 4:1) to give lactone 9 (201 mg, 79% yield) as a white, needle-like, crystalline solid: Rf 0.40 (hexane/EtOAc, 2:1); mp 88−89 °C; [α]25D +174 (c 0.77, CHCl3); IR (KBr) νmax 2951, 2916, 1710, 1450 cm−1; 1H NMR (CDCl3, 400 MHz) δ 4.90 (1H, d, J = 1.0 Hz, H-9a), 4.88 (1H, m, H2), 4.85 (1H, d, J = 1.4 Hz, H-9b), 2.72 (1H, quint, J = 7.3 Hz, H-7), 2.27 (1H, m, H-4a), 2.22 (1H, m, H-1a), 2.18 (1H, m, H-6), 2.15 (1H, m, H-1b), 2.01 (1H, m, H-5a), 1.91 (1H, m, H-4b), 1.58 (1H, m, H5b), 1.32 (3H, d, J = 7.3 Hz, H-18); 13C NMR (CDCl3, 100 MHz) δ 174.7 (C), 144.9 (C), 111.3 (CH2), 80.8 (CH), 38.8 (CH), 34.1 (CH2), 31.4 (CH), 26.6 (CH2), 25.3 (CH2), 14.0 (CH3); ESIMS m/z 189 [M + Na]+ (100); HRESIMS m/z 189.0892 [M + Na]+ (calcd for C10H14O2Na, 189.0891); anal. C 71.99, H 8.54%, calcd for C10H14O2, C 72.26, H 8.49%. (1S,5S)-4-((E)-4,8-Dimethylnona-3,7-dien-1-yl)-8-methylene-2oxabicyclo[3.3.1]nonan-3-one (10). nBuLi (1.65 M solution in hexane, 3.70 mL, 6.11 mmol) was added to a solution of

2.17 mmol) in THF (1.00 mL) was added, stirring continued at this temperature for 1 h prior to addition of diethyl chlorophosphate (0.380 mL, 2.63 mmol), and the solution was warmed to rt over 1 h. After cooling to −78 °C, the preprepared LDA (0.65 M solution in THF/hexane, 10.0 mL, 6.50 mmol) was introduced and then allowed to warm to rt for 12 h. After cooling to −78 °C, TMSCl (0.410 mL, 3.25 mmol) was added. After stirring for 2 h at the same temperature, the reaction mixture was diluted with Et2O, washed with saturated aqueous NH4Cl solution, H2O, and brine, dried over anhydrous MgSO4 and Na2SO4, and then concentrated in vacuo. The residue was passed through a pad of silica gel (petroleum ether) and then concentrated in vacuo to give crude TMS-acetylene 4. To a solution of cyclohexene (1.41 mL, 13.9 mmol) in THF (9.00 mL) at 0 °C was added BH3·THF (0.92 M solution in THF, 7.08 mL, 6.51 mmol). The reaction mixture was warmed to rt and stirred for 1 h. To this solution the above crude TMS-acetylene 4 in THF (1.90 mL) was added at 0 °C, and then the mixture was stirred for 2 h. The reaction mixture was oxidized by the addition of MeOH (2.41 mL), NaOH (3.0 M solution in H2O, 2.41 mL), and 30% aqueous H2O2 solution (2.41 mL) at 0 °C and then warmed to rt. After stirring for 12 h at rt, the reaction mixture was neutralized with HCl (1.0 M solution in H2O, 10.0 mL). The acidified mixture was extracted with Et2O, washed with H2O and brine, dried over anhydrous MgSO4 and Na2SO4, and then concentrated in vacuo. The residue was passed through a pad of silica gel (hexane/ EtOAc, 4:1) and then concentrated in vacuo to give crude carboxylic acid 5. To a stirring solution of the above crude carboxylic acid 5 in CH2Cl2 (21.7 mL) was added N-iodosuccinimide (NIS) (1.95 g, 8.68 mmol) at 0 °C, and then the solution was allowed to warm to rt. After stirring for 3 h, the mixture was diluted with Et2O, washed with saturated aqueous Na2S2O3 solution, H2O, and brine, dried over anhydrous MgSO4 and Na2SO4, and then concentrated in vacuo to give a crude iodolactone. nBuLi (1.60 M solution in hexane, 2.58 mL, 4.13 mmol) was added to a solution of diisopropylamine (0.608 mL, 4.34 mmol) in THF (9.00 mL) at −78 °C, and the resulting mixture was stirred at 0 °C for 30 min. After cooling to −78 °C, a solution of the above crude iodolactone in THF (1.90 mL) was introduced, stirring continued at this temperature for 5 h, and then the solution was allowed to warm to rt over 1 h. After stirring for 30 min, the reaction mixture was diluted with Et2O, washed with a saturated aqueous NH4Cl solution, H2O, and brine, dried over anhydrous MgSO4 and Na2SO4, and then concentrated in vacuo. The residue was purified with flash column chromatography on silica gel (hexane/EtOAc, 2:1) to give bicyclic lactone 6 (230 mg, 70% yield for four steps) as a white, needle-like, crystalline solid: Rf 0.30 (hexane/EtOAc, 2:1); mp 85−86 °C; [α]25D +31.5 (c 0.21, CHCl3); IR (KBr) νmax 2935, 1722, 1656, 1448, 1373 cm−1; 1H NMR (CDCl3, 400 MHz) δ 4.94 (1H, m), 4.92 (1H, m), 4.88 (1H, m), 2.81 (1H, ddd, J = 0.92, 7.1, 18.8 Hz), 2.56 (1H, ddd, J = 0.96, 2.2, 18.8 Hz), 2.40−2.28 (3H, m), 2.16 (1H, ddt, J = 6.7, 13.6, 2.7 Hz), 1.86−1.79 (2H, m), 1.72 (1H, m); 13C NMR (CDCl3, 100 MHz) δ 171.1 (C), 144.6 (C), 111.9 (CH2), 80.6 (CH), 35.8 (CH2), 32.25 (CH2), 32.17 (CH2), 25.9 (CH), 25.6 (CH2); ESIMS m/z 175 [M + Na]+ (100); HRESIMS m/z 175.0734 [M + Na]+ (calcd for C9H12O2Na, 175.0735); anal. C 70.88, H 8.15%, calcd for C9H12O2, C 71.03, H 7.95%. (1S,5S)-4-((E)-4,8-Dimethylnona-3,7-dien-1-yl)-8-methylene-2oxabicyclo[3.3.1]nonan-3-one (7). nBuLi (2.65 M solution in hexane, 1.31 mL, 3.47 mmol) was added to a solution of diisopropylamine (0.508 mL, 3.62 mmol) in THF (13.0 mL) at −78 °C, and the resulting mixture was stirred at 0 °C for 30 min. After cooling to −78 °C, a solution of bicyclic lactone 6 (230 mg, 1.51 mmol) in THF (2.10 mL) was introduced, stirring continued at this temperature for 1 h, and then premixed homogeranyl iodide (1.48 g, 5.32 mmol) and HMPA (3.05 mL, 17.5 mmol) was introduced to the mixture. After stirring for 1 h at the same temperature, the reaction mixture was diluted with Et2O, washed with saturated aqueous NH4Cl solution, H2O, and brine, dried over anhydrous MgSO4 and Na2SO4, and then concentrated in vacuo. The residue was purified with flash column chromatography on silica gel (hexane/EtOAc, 12:1) to give lactone 7 (434 mg, 95% yield) as a colorless oil: Rf 0.30 (hexane/EtOAc, 10:1); IR (neat) νmax = 2929, 2859, 1730, 1446 cm−1; 1H NMR (CDCl3, 400 MHz) δ 5.15− F

DOI: 10.1021/acs.jnatprod.5b01069 J. Nat. Prod. XXXX, XXX, XXX−XXX

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diisopropylamine (0.900 mL, 6.42 mmol) in THF (10.0 mL) at −78 °C, and the resulting mixture was stirred at 0 °C for 30 min. After cooling to −78 °C, a solution of lactone 9 (315 mg, 1.90 mmol) in THF (2.00 mL) was introduced and stirred at rt for 30 min, and then premixed homogeranyl iodide (1.50 g, 5.39 mmol) and HMPA (3.10 mL, 17.8 mmol) was introduced to the mixture. After stirring for 2 h at the same temperature, the reaction mixture was diluted with Et2O, washed with saturated aqueous NH4Cl solution, H2O, and brine, dried over anhydrous MgSO4 and Na2SO4, and then concentrated in vacuo. The residue was purified with flash column chromatography on silica gel (hexane/EtOAc, 12:1) to give lactone 10 (487 mg, 81% yield) as a colorless oil: Rf 0.35 (hexane/EtOAc, 12:1); [α]25D +112 (c 0.28, CHCl3); IR (neat) νmax 2930, 1726, 1451, 1380 cm−1; 1H NMR (CDCl3, 600 MHz) δ 5.07−5.05 (2H, m), 4.90 (1H, s, H-17a), 4.85 (1H, d, J = 4.4 Hz, H-2), 4.84 (1H, s, H-17b), 2.53 (1H, m, H-1a), 2.28 (1H, m, H-4a), 2.21 (1H, m, H-4b), 2.06−1.95 (8H, m), 1.75 (1H, m), 1.72−1.65 (2H, m, H-8), 1.67 (3H, s), 1.59 (1H, m, H-1b), 1.59 (3H, s), 1.59 (3H, s), 1.31 (3H, s); 13C NMR (CDCl3, 150 MHz) δ 177.6 (C), 145.3 (C), 136.0 (C), 131.4 (C), 124.2 (CH), 123.1 (CH), 111.2 (CH2), 81.2 (CH), 43.8 (C), 40.4 (CH2), 39.6 (CH2), 34.3 (CH), 30.1 (CH2), 28.6 (CH2), 26.6 (CH2), 26.0 (CH2), 25.7 (CH3), 22.4 (CH2), 20.6 (CH3), 17.7 (CH3), 16.0 (CH3); ESIMS m/z 339 [M + Na]+ (100); HRESIMS m/z 339.2301 [M + Na]+ (calcd for C21H32O2Na, 339.2300); anal. C 79.44, H 10.04%, calcd for C21H32O2, C 79.70, H 10.19%. (1S,4R,5S)-4-Methyl-8-methylene-4-(4-methylpent-3-en-1-yl)-2oxabicyclo[3.3.1]nonan-3-one (11). nBuLi (2.66 M solution in hexane, 2.60 mL, 6.92 mmol) was added to a solution of diisopropylamine (1.00 mL, 7.14 mmol) in THF (15.0 mL) at −78 °C, and the resulting mixture was stirred at 0 °C for 30 min. After cooling to −78 °C, a solution of lactone 9 (244 mg, 1.46 mmol) in THF (2.00 mL) was introduced and stirred at rt for 30 min, and then premixed homoprenyl iodide (0.920 g, 4.38 mmol) and HMPA (2.60 mL, 14.9 mmol) was introduced to the mixture. After stirring for 12 h at the same temperature, the reaction mixture was diluted with Et2O, washed with a saturated aqueous NH4Cl solution, H2O, and brine, dried over anhydrous MgSO4 and Na2SO4, and then concentrated in vacuo. The residue was purified with flash column chromatography on silica gel (hexane/EtOAc, 7:1) to give lactone 11 (297 mg, 82% yield) as a white, needle-like, crystalline solid: Rf 0.50 (hexane/EtOAc, 4:1); mp 46−47 °C; [α]22D +134 (c 2.22, CHCl3); IR (KBr) νmax 2936, 2866, 1724, 1452, 1378 cm−1; 1H NMR (CDCl3, 400 MHz) δ 5.05 (1H, ddt, J = 1.2, 1.3, 7.0 Hz), 4.90 (1H, s), 4.86 (1H, d, J = 4.5 Hz), 4.83 (1H, d, J = 0.9 Hz), 2.53 (1H, ddt, J = 4.4, 14.0, 2.9 Hz), 2.30− 2.18 (2H, m), 2.08−1.92 (4H, m), 1.79−1.54 (4H, m), 1.67 (3H, s), 1.60 (3H, s), 1.31 (3H, s); 13C NMR (CDCl3, 100 MHz) δ 177.5 (C), 145.2 (C), 132.3 (C), 123.2 (CH), 111.2 (CH2), 81.1 (CH), 43.7 (C), 40.3 (CH2), 34.3 (CH), 30.0 (CH2), 28.5 (CH2), 26.0 (CH2), 25.6 (CH3), 22.4 (CH2), 20.5 (CH3), 17.6 (CH3); ESIMS m/z 271 [M + Na]+ (100); HRESIMS m/z 271.1666 [M + Na]+ (calcd for C16H24O2Na, 271.1674). (R)-6-Methyl-2-((S)-4-methylcyclohex-3-en-1-yl)hept-5-en-2amine (12). A solution of lactone 11 (251 mg, 1.01 mmol) in Et2O (2.00 mL) was added to a preprepared Li (70.1 mg, 10.1 mmol)/liquid ammonia (10.1 mL) solution at −78 °C. After stirring for 20 min, NH4Cl (1.28 g, 23.9 mmol) was added to the mixture, and excess NH3 was removed by warming. The reaction mixture was diluted with Et2O, washed with H2O and brine, dried over anhydrous MgSO4 and Na2SO4, and then concentrated in vacuo. The residue was passed through a pad of silica gel (hexane/EtOAc, 4:1) and then concentrated in vacuo to give crude carboxylic acid. To a stirring solution of the carboxylic acid in toluene (10.1 mL) were added Et3N (0.253 mL, 1.82 mmol) and DPPA (0.392 mL, 1.81 mmol) at 0 °C, and then the solution was allowed to warm to rt. After stirring for 2 h, the mixture was refluxed for an additional 2 h. The reaction mixture was cooled to rt and then diluted with Et2O, washed with H2O and brine, dried over anhydrous MgSO4 and Na2SO4, and then concentrated in vacuo to give a crude isocyanate. To a stirring solution of the isocyanate in glacial acetic acid (0.337 mL, 5.89 mmol) was added HCl (3.0 M solution in H2O, 0.337 mL, 1.01 mmol) at rt for 18 h. The reaction mixture was

neutralized with NaOH (3.0 M solution in H2O, 10.0 mL, 30.0 mmol), diluted with Et2O, washed with H2O and brine, dried over anhydrous MgSO4 and Na2SO4, and then concentrated in vacuo. The residue was purified with flash column chromatography on silica gel (EtOAc) to give amine 12 (76.3 mg, 34% yield for three steps) as a colorless oil: Rf 0.10 (EtOAc); [α]20D −56.3 (c 3.2, CHCl3); IR (neat) νmax 3370, 3310, 2963, 1449, 1375 cm−1; 1H NMR (CDCl3, 400 MHz) δ 5.39 (1H, brs), 5.10 (1H, t, J = 7.0 Hz), 2.04−1.92 (3H, m), 1.92−1.75 (2H, m), 1.74−1.57 (2H, m), 1.68 (3H, s), 1.64 (3H, s), 1.61 (3H, s), 1.55−1.36 (2H, m), 1.34−1.22 (2H, m), 1.05 (3H, s); 13C NMR (C6D6, 100 MHz) δ 133.4 (C), 130.7 (C), 125.8 (CH), 121.7 (CH), 52.8 (C), 43.5 (CH), 41.2 (CH2), 31.6 (CH2), 26.4 (CH2), 25.9 (CH3), 25.4 (CH3), 24.2 (CH2), 23.6 (CH3), 22.8 (CH2), 17.7 (CH3); ESIMS m/z 222 [M + H]+ (100); HRESIMS m/z 222.2219 [M + H]+ (calcd for C15H28N, 222.2222). N-((S,E)-6,10-Dimethyl-2-((S)-4-methylcyclohex-3-en-1-yl)undeca-5,9-dien-2-yl)formamide (16). A solution of lactone 8 (148 mg, 0.468 mmol) in Et2O (0.936 mL) was added to a preprepared Li (32.5 mg, 4.68 mmol)/liquid ammonia (4.68 mL) solution at −78 °C. After stirring for 20 min, NH4Cl (2.50 g, 46.8 mmol) was added to the mixture and excess NH3 was removed by warming. The reaction mixture was diluted with Et2O, washed with H2O and brine, dried over anhydrous MgSO4 and Na2SO4, and then concentrated in vacuo. The residue was passed through a pad of silica gel (hexane/EtOAc, 4:1) and then concentrated in vacuo to give crude carboxylic acid 13. To a stirring solution of the carboxylic acid 13 in toluene (4.68 mL) were added Et3N (0.117 mL, 0.839 mmol) and DPPA (0.182 mL, 0.845 mmol) at 0 °C, and then the solution was allowed to warm to rt. After stirring for 2 h, the mixture was refluxed for an additional 2 h. The reaction mixture was cooled to rt and then diluted with Et2O, washed with H2O and brine, dried over anhydrous MgSO4 and Na2SO4, and then concentrated in vacuo to give crude isocyanate 14. To a stirring solution of the isocyanate 14 in glacial acetic acid (0.333 mL, 5.82 mmol) was added HCl (3.0 M solution in H2O, 0.333 mL, 0.999 mmol) at rt for 18 h. The reaction mixture was neutralized with NaOH (3.0 M solution in H2O, 0.666 mL, 2.00 mmol), diluted with Et2O, washed with H2O and brine, dried over anhydrous MgSO4 and Na2SO4, and then concentrated in vacuo to give crude amine 15. To a stirring solution of the amine 15 in CH2Cl2 (9.36 mL) was added freshly prepared acetic formic anhydride (824 mg, 9.36 mmol) at −78 °C for 30 min. The mixture was concentrated in vacuo, and the residue was purified with flash column chromatography on silica gel (hexane/ EtOAc, 4:1) to give formamide 16 (120 mg, 81% yield for four steps) as a colorless oil; Rf 0.25 (hexane/EtOAc, 4:1); [α]26D −31.9 (c 0.54, CHCl3); ESIMS m/z 340 [M + Na]+ (100); HRESIMS m/z 340.2618 [M + Na]+ (calcd for C21H35NONa, 340.2616); anal. C 79.42, H 11.21, N 4.59%, calcd for C21H35NO, C 79.44, H 11.11, N 4.41%. (S)-4-((S,E)-2-Isocyano-6,10-dimethylundeca-5,9-dien-2-yl)-1methylcyclohex-1-ene (17). To a stirring solution of formamide 16 (120 mg, 0.378 mmol) in CH2Cl2 (2.70 mL) were added pyridine (0.0608 mL, 0.756 mmol) and pTsCl (144 mg, 0.755 mmol) at rt. After stirring for 24 h, the reaction mixture was concentrated in vacuo and the residue was purified with flash column chromatography on silica gel (hexane/EtOAc, 20:1) to give isocyanide 17 (110 mg, 97% yield) as a colorless oil: Rf 0.60 (hexane/EtOAc, 10:1); [α]20D −33.8 (c 0.27, CHCl3); ESIMS m/z 322 [M + Na]+ (100); HRESIMS m/z 322.2512 [M + Na]+ (calcd for C21H33NNa, 322.2511); anal. C 84.09, H 11.19, N 4.65%, calcd for C21H33N, C 84.22, H 11.11, N 4.68%. N-((R,E)-6,10-Dimethyl-2-((S)-4-methylcyclohex-3-en-1-yl)undeca-5,9-dien-2-yl)formamide. A solution of lactone 10 (83.8 mg, 0.265 mmol) in Et2O (0.530 mL) was added to a preprepared Li (18.4 mg, 2.65 mmol)/liquid ammonia (2.70 mL) solution at −78 °C. After stirring for 30 min, NH4Cl (1.42 g, 26.5 mmol) was added to the mixture and excess NH3 was removed by warming. The reaction mixture was diluted with Et2O, washed with H2O and brine, dried over anhydrous MgSO4 and Na2SO4, and then concentrated in vacuo. The residue was passed through a pad of silica gel (hexane/EtOAc, 4:1) and then concentrated in vacuo to give a crude carboxylic acid. To a stirring solution of the carboxylic acid in toluene (2.70 mL) were added Et3N (0.0670 mL, 0.480 mmol) and DPPA (0.103 mL, 0.478 G

DOI: 10.1021/acs.jnatprod.5b01069 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

mmol) at 0 °C and then allowed to warm to rt. After stirring for 2 h, the mixture was refluxed for an additional 2 h. The reaction mixture was cooled to rt and then diluted with Et2O, washed with H2O and brine, dried over anhydrous MgSO4 and Na2SO4, and then concentrated in vacuo to give a crude isocyanate. To a stirring solution of the isocyanate in glacial acetic acid (0.088 mL, 1.54 mmol) was added HCl (3.0 M solution in H2O, 0.088 mL, 0.264 mmol) at rt for 12 h. The reaction mixture was neutralized with NaOH (3.0 M solution in H2O, 0.132 mL, 0.396 mmol), diluted with Et2O, washed with H2O and brine, dried over anhydrous MgSO4 and Na2SO4, and then concentrated in vacuo to give a crude amine. To a stirring solution of the amine in CH2Cl2 (5.30 mL) was added freshly prepared acetic formic anhydride (467 mg, 5.30 mmol) at −78 °C for 15 min. The mixture was concentrated in vacuo, and the residue was purified with flash column chromatography on silica gel (hexane/EtOAc, 4:1) to give a formamide product (57.2 mg, 68% yield for four steps) as a colorless oil: Rf 0.25 (hexane/EtOAc, 4:1); [α]25D −58.5 (c 0.044, CHCl3); IR (neat) νmax = 3297, 2923, 1680, 1537, 1450 cm−1; 1H NMR (CDCl3, 500 MHz) δ 8.17 (0.60H, d, J = 12.4 Hz), 8.08 (0.40H, d, J = 1.9 Hz), 5.66 (0.60H, d, J = 12.4 Hz), 5.36 (1H, s), 5.12 (0.40H, t, J = 6.0 Hz), 5.10−5.06 (2H, m), 2.14 (0.4H, m), 2.08−1.90 (8.6H, m), 1.78 (1H, m), 1.70−1.60 (2H, m), 1.68 (3H, s), 1.64 (3H, s), 1.61 (3H, s), 1.60 (3H, s), 1.59−1.48 (2H, m), 1.28 (1.8H, s), 1.27 (1H, m), 1.27 (1.2H, s); 13C NMR (CDCl3, 125 MHz) δ 163.1 (CH), 160.3 (CH), 136.1 (C), 135.4 (C), 134.1 (C), 134.0 (C), 131.5 (C), 131.4 (C), 124.3 (CH), 124.1 (CH), 123.9 (CH), 123.1 (CH), 120.3 (CH), 120.0 (CH), 59.3 (C), 57.4 (C), 43.8 (CH), 40.7 (CH), 39.7 (CH2), 39.6 (CH2), 39.1 (CH2), 36.2 (CH2), 31.1 (CH2), 30.9 (CH2), 26.7 (CH2), 26.6 (CH2), 26.3 (CH2), 26.1 (CH2), 25.7 (CH3) × 2, 24.0 (CH2), 23.8 (CH2), 23.2 (CH3), 23.1 (CH3), 22.3 (CH2), 21.8 (CH3), 21.7 (CH2), 20.9 (CH3), 17.7 (CH3) × 2, 16.04 (CH3), 15.99 (CH3); ESIMS m/z 340 [M + Na]+ (100); HRESIMS m/z 340.2618 [M + Na]+ (calcd for C21H35NONa, 340.2616); anal. C 79.53, H 10.94, N 4.38%, calcd for C21H35NO, C 79.44, H 11.11, N 4.41%. (S)-4-((R,E)-2-Isocyano-6,10-dimethylundeca-5,9-dien-2-yl)-1methylcyclohex-1-ene (18). To a stirring solution of the above formamide (42.9 mg, 0.135 mmol) in CH2Cl2 (2.70 mL) were added pyridine (0.0220 mL, 0.270 mmol) and pTsCl (51.5 mg, 0.270 mmol) at rt. After stirring for 24 h, the reaction mixture was concentrated in vacuo and the residue was purified with flash column chromatography on silica gel (hexane/EtOAc, 20:1) to give isocyanide 18 (38.3 mg, 95% yield) as a colorless oil: Rf 0.60 (hexane/EtOAc 10:1); [α]20D −57.5 (c 0.38, CHCl3); IR (neat) νmax = 2965, 2925, 2126, 1450, 1379 cm−1; 1H NMR (CDCl3, 500 MHz) δ 5.38 (1H, dd, J = 1.9, 2.8 Hz), 5.13−5.06 (2H, m), 2.18−1.90 (10H, m), 1.85 (1H, m), 1.68 (3H, d, J = 1.0 Hz), 1.67 (1H, m), 1.66 (3H, s), 1.63 (3H, s), 1.60 (3H, s), 1.55 (1H, m), 1.36 (2H, m), 1.35 (3H, s); 13C NMR (CDCl3, 125 MHz) δ 153.8 (C, t, J = 4.3 Hz), 136.3 (C), 133.9 (C), 131.4 (C), 124.2 (CH), 122.7 (CH), 119.8 (CH), 63.5 (C, t, J = 4.5 Hz), 41.9 (C), 39.6 (CH2), 38.3 (CH2), 30.7 (CH2), 26.6 (CH2), 26.3 (CH2), 25.7 (CH3), 23.60 (CH2), 23.57 (CH3), 23.1 (CH3), 22.4 (CH2), 17.7 (CH3), 16.0 (CH3); ESIMS m/z 300 [M + H]+ (100); HRESIMS m/z 300.2690 [M + H]+ (calcd for C21H34N, 300.2691); anal. C 84.41, H 11.20, N 4.56%, calcd for C21H33N, C 84.22, H 11.11, N 4.68%.

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Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Dr. K. Ogawa of the Z. Nakai Laboratory for identifying the sponge.

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S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.5b01069. 1D and 2D NMR data of amitorine A (1), amitorine B (2), and compounds 6−18; a photo of Theonella swinhoei “S-03-08” (PDF)

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*Tel: +81-42-676-3080. Fax: +81-42-676-3073. E-mail: [email protected] (H. Miyaoka). H

DOI: 10.1021/acs.jnatprod.5b01069 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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DOI: 10.1021/acs.jnatprod.5b01069 J. Nat. Prod. XXXX, XXX, XXX−XXX