Phytochemistry 96 (2013) 397–403
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Cassane diterpenes from Caesalpinia platyloba Mario A. Gómez-Hurtado a, Fany E. Álvarez-Esquivel a, Gabriela Rodríguez-García a, Mauro M. Martínez-Pacheco a, Rosa M. Espinoza-Madrigal a, Teresa Pamatz-Bolaños a, José L. Salvador-Hernández a, Hugo A. García-Gutiérrez a, Carlos M. Cerda-García-Rojas b,⇑, Pedro Joseph-Nathan b, Rosa E. del Río a,⇑ a b
Instituto de Investigaciones Químico-Biológicas, Universidad Michoacana de San Nicolás de Hidalgo, Edificio B-1, Ciudad Universitaria, Morelia, Michoacán 58030, Mexico Departamento de Química, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, Apartado 14-740, México D.F. 07000, Mexico
a r t i c l e
i n f o
Article history: Received 30 March 2013 Received in revised form 19 September 2013 Available online 28 October 2013 Keywords: Caesalpinia platyloba Fabaceae Vouacapane derivatives Cassane diterpenes Absolute configuration Vibrational circular dichroism Density functional theory calculations
a b s t r a c t The dichloromethane extract from the leaves of Caesalpinia platyloba provided cassane diterpenes whose structures were determined as ( )-(5S,6R,8S,9S,10R,14R)-6-acetoxyvouacapane (1), ( )-(5S,6R,8S,9S, 10R,12Z,14R)-6-acetoxycassa-12,15-diene (3), and ( )-(5S,6R,8S,9S,10R,13E)-6-acetoxycassa-13,15-diene (4). Compound 1 was chemically correlated with ( )-(5S,6R,8S,9S,10R,14R)-6-hydroxyvouacapane (2), (+)-(5S,8S,9S,10R,14R)-6-oxovouacapane (5), and (+)-(5S,6S,8S,9S,10R,14R)-6-acetoxyvouacapane (6), the last one previously isolated from Dipteryx lacunifera. The absolute configurations of all six diterpenes 1–6 were established by comparison of DFT calculated vibrational circular dicroism spectra of 1, 2 and 5 with those obtained experimentally. In addition, several reported chemical shifts for 2 and 5 were reassigned based on two-dimensional NMR measurements. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction The genus Caesalpinia comprises more than 500 species which are mainly distributed in tropical or subtropical zones (Zanin et al., 2012). Chemical studies of the genus have shown the presence of phenolic derivatives, steroids, triterpenoids, and cassane diterpenes (Wu et al., 2011), and several species are pharmacologically relevant, showing antiulcer, anticancer, antidiabetic, anti-inflammatory, antirheumatic, antimicrobial, and cytotoxic activities (Wu et al., 2011; Zanin et al., 2012). The genus Caesalpinia is phytochemically characterized by the presence of cassane diterpenes, including vouacapane and cassadiene derivatives, which have also been described in other genera of the Fabaceae family (Mauria et al., 2012). It is well known that protonation of geranylgeranyl diphosphate (GGPP) can lead to both enantiomeric series of diterpenes, through either copalyl diphosphate or ent-copalyl diphosphate, depending on the existing biosynthetic pathway (Hong and Tantillo, 2010).
⇑ Corresponding authors. Tel.: +52 443 3265788/55 57474035; fax: +52 443 3265790/55 57477137. E-mail addresses: [email protected] (C.M. Cerda-García-Rojas), ndelrio@ umich.mx (R.E. del Río). 0031-9422/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.phytochem.2013.09.028
Therefore, it is relevant to define clearly the absolute configuration of new diterpene compounds, even more if they show possible pharmacological applications. Concerning cassane derivatives, there are reports on the isolation of members of both enantiomeric series, normal cassane diterpenes isolated from Acacia (ManríquezTorres et al., 2011), Caesalpinia (Yodsaoue et al., 2011), Dipteryx (Mendes and Silveira, 1994), and Pterodon (Arriaga et al., 2000) genera, among others, and ent-cassanes generally found as phytoalexins produced by rice plants (Yajima et al., 2004; Yajima and Mori, 2000a,b). Vibrational circular dicroism (VCD) has been used in recent times as an effective technique to determine the absolute configuration of natural compounds (Nafie, 2008). In the present work, we describe the structure elucidation of three new cassane diterpenes (1, 3 and 4, Fig. 1) isolated from the CH2Cl2 extract of Caesalpinia platyloba S. Watson (Fabaceae). Their structures and relative configurations were established by 1D and 2D NMR experiments and by the X-ray diffraction analysis of 1, while their absolute configuration was determined by VCD spectroscopy of 1 and derivatives 2 and 5, using density functional theory calculations at the B3LYP/DGDZVP level of theory. Cassane 1 was chemically correlated, through derivatives 2 and 5, with natural occurring 6 isolated from Dipteryx lacunifera (Mendes and Silveira, 1994).
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O
1
15
11 20 1 2
5
4
13 14
H 17
H
6
OR
18
H
1
2
3
4
5
1a 1b
1.02 (m) 1.67 (br d, 12.3) 1.60 (dt, 13.6, 3.2) 1.45 (m)
0.97 (m) 1.63 (m)
0.94 (m) 1.79 (m)
1.60 (m)
3a
1.19 (m)
1.18 (m)
3b
1.41 (br d, 13.1) 1.09 (s) 5.51 (td, 3.1, 1.9) 1.53 (m) 1.85 (dt, 14.3, 3.5) 2.03 (m)
1.39 (br d, 13.0) 0.98 (s) 4.48 (br s)
1.44 (dquin 13.6, 3.2) 1.18 (td, 13.0, 3.2) 1.39 (dt, 13.0, 3.2) 1.06 (s) 5.51 (q, 2.9) 1.50 (m) 1.80 (dt, 14.4, 3.2) 1.83 (m)
1.67 (dt, 14.0, 3.6) 1.49 (dquin 14.0, 3.6) 1.19 (td, 13.2, 4.1) 1.38 (br d, 13.2) 1.02 (s) 5.52 (q, 2.9) 1.13 (m) 2.24 (dt, 14.5, 3.3) 2.33 (m)
1.18 (m) 1.76 (br d, 12.9) 1.51 (m)
2b
1.05 (m) 1.73 (br d, 12.7) 1.63 (dt, 13.6, 3.2) 1.48 (m)
1.37 (m) 2.09 (m)
0.93 (m) 1.84 (m)
2.09 (m)
1.16 (m)
– 5.64 (t, 4.1)
2.01 (m) 2.36 (br d, 14.4) – 6.80 (dd, 17.0, 11.0) 5.13 (d, 17.0) 4.95 (d, 11.0) 1.70 (br s)
2a
OAc
1 : R = Ac 2:R=H
3
O H
5 6
H
H H
Position
7
H 19
8
H
10
3
H
9
Table 1 H NMR spectroscopic data of compounds 1–5 (400 MHz, CDCl3, d in ppm, J in Hz).
16
12
H
OAc
H
7a 7b
O
8
2.59 (m) 6.19 (d, 1.9) 7.23 (dt, 1.9, 0.9) –
2.61 (m) 6.19 (d, 2.0) 7.23 (br d, 1.9) –
0.96 7.0) 0.99 1.03 1.21 2.03
0.96 7.0) 1.01 1.26 1.23 –
5
4 O
HO
H
H
9 11a 11b
H H
1.54 (m) 2.60 (dd, 16.9, 7.1) 2.50 (dd, 16.9, 10.0) – –
1.57 (m) 1.72 (dt, 13.9, 3.7) 2.14 (dt, 12.5, 4.7) 1.51 (m) 2.57 (dd, 16.9, 7.1) 2.45 (dd, 16.9, 10.0) – –
OAc 6
12a 12b
H
14 15
H 7
16a
Fig. 1. Structure of diterpenes 1–7.
16b 17 18 19 20 AcO
2. Results and discussion 2.1. Isolation, identification, and structure elucidation Column chromatography separation of the CH2Cl2 extract of the leaves of C. platyloba allowed the isolation of ( )-(5S,6R, 8S,9S,10R,14R)-6-acetoxyvouacapane (1) in very good yields, and of ( )-(5S,6R,8S,9S,10R,12Z,14R)-6-acetoxycassa-12,15-diene (3) and ( )-(5S,6R,8S,9S,10R,13E)-6-acetoxycassa-13,15-diene (4) (Fig. 1) as minor products, which were separated by TLC impregnated with 20% AgNO3. Both dienes (3 and 4) appeared to be unstable, however, their chemical stability was substantially improved when they were dissolved in EtOAc and washed with Na2SO3 aq. satd. soln. to remove any traces of AgNO3 or its transformation products. The molecular formula of the new cassane 1, which belongs to the vouacapane series, was established as C22H32O3 by HRESI/APCIMS, showing [M+H]+ at m/z 345.2421 (calculated 345.2425). The optical activity displayed a levorotatory value of [a]D = 18.3 in CHCl3. Its 1H NMR spectrum (Table 1) showed two aromatic signals assigned to the furan hydrogen atoms at d 7.23 (1H, dt, J15,16 = 1.9 Hz, J11a,16 = J11b,16 = 0.9 Hz, H-16) and d 6.19 (1H, d, J15,16 = 1.9 Hz, H-15). The signal for the hydrogen atom geminal to oxygen at C-6 was observed at d 5.51 (1H, td, J6,7a = J6,7b = 3.1, J5,6 = 1.9 Hz, H-6). The acetyl methyl group appeared at d 2.03 (3H, s, OAc), while the methyl groups were observed at d 1.21 (3H, s, CH3-20), 1.03 (3H, s, CH3-19), 0.99 (3H, s, CH3-18), and 0.96 (3H, d, J14,17 = 7.0 Hz, CH3-17). Its 13C NMR spectrum (Table 2) showed: a carbonyl acetyl group at d 170.6 (C, OAc); furan ring signals, characteristic of vouacapane, at d 149.5 (C, C-12), 140.3 (CH, C-16), 122.0 (C, C-13), and 109.4 (CH, C-15), an oxymethine carbon C-6 appeared at d 69.6, and a typical resonance for C-5 at d 55.3. A NOE difference experiment showed, upon irradiation of H-6a at d
(d, (s) (s) (s) (s)
(d, (s) (s) (s)
2.38 (m) 6.23 (dd, 17.6, 10.6) 5.08 (d, 17.6) 4.91 (d, 10.6) 0.89 (d, 7.2) 0.96 (s) 1.00 (s) 1.13 (s) 2.03 (s)
0.94 1.00 1.14 2.06
(s) (s) (m) (s)
1.51 (m) 1.08 (m) 1.33 (br d, 13.1) 2.10 (s) – 2.24 (m) 2.35 (m) 2.22 (m) 1.95 (m) 2.67 (dd, 16.9, 7.0) 2.38 (dd, 16.9, 10.1) – – 2.62 (m) 6.20 (d, 1.9) 7.24 (d, 1.9) – 1.05 7.0) 0.96 1.25 0.87 –
(d, (s) (s) (s)
Table 2 C NMR spectroscopic data of compounds 1–5 (100 MHz, CDCl3, d in ppm).
13
Position
1
2
3
4
5
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 AcO
42.2 18.7 43.6 33.8 55.3 69.6 36.3 31.0 45.6 37.9 21.7 149.5 122.0 31.0 109.4 140.3 17.5 33.6 23.4 17.1 170.6 21.7
42.5 18.8 43.7 34.0 56.3 67.6 40.3 30.4 45.9 37.6 21.8 149.7 122.1 31.2 109.5 140.3 17.7 33.8 24.3 17.7
41.8 18.7 43.8 33.8 55.8 70.0 36.2 30.5 44.3 37.6 24.6 128.5 141.4 31.3 138.4 109.5 14.4 33.6 23.4 16.4 170.6 21.8
40.8 18.9 44.2 33.8 56.1 70.2 36.8 36.7 54.1 37.4 21.0 26.3 129.3 135.7 135.3 110.9 15.9 33.2 23.2 16.3 170.5 21.8
38.8 18.3 42.5 32.0 65.1 211.9 46.8 38.4 45.4 41.8 22.6 148.8 121.9 30.7 109.4 140.8 17.3 32.8 21.9 14.9
5.51, enhancements for signals of H-5a (distance = 2.43 Å), H-7a (distance = 2.36 Å), equatorial H-7b (distance = 2.46 Å), and CH3-18a (closest distance = 2.14 Å) evidencing the axial orientation
M.A. Gómez-Hurtado et al. / Phytochemistry 96 (2013) 397–403
399
1 (GDFT = –1082.949402 hartrees)
Fig. 3. X-ray structure of ( )-(5S,6R,8S,9S,10R,14R)-6-acetoxyvouacapane (1).
2 (GDFT = –930.292806 hartrees)
5 (GDFT = –929.122047 hartrees) Fig. 2. DFT B3LYP/DGDZVP global minimum structures of (–)(5S,6R,8S,9S,10R,14R)-6-acetoxyvouacapane (1), (–)-(5S,6R,8S,9S,10R,14R)-6hydroxyvouacapane (2) and (+)-(5S,8S,9S,10R,14R)-6-oxovouacapane (5).
of the acetyl group at C-6 (Fig. 2). Two-dimensional NMR spectroscopy including COSY, NOESY, HETCOR, and HMBC experiments were useful for assignment of all 1H and 13C signals. In particular, the resonances for C-8 and C-14, which appear overlapped, were easily located from their one-bond HETCOR correlations with H-8 and H-14, respectively. The relative configuration of 1 was unambiguously established by X-ray diffraction analysis (Fig. 3) confirming the axial orientation of the acetyl group at C-6. Cassadiene 3 had a quasi-molecular ion [M+Na]+ at m/z 353.2451 by positive mode HRESIMS in agreement with a molecular formula C22H34O2. The optical rotation was [a]D = 5 in CHCl3. The 1H and 13C NMR spectra of 3 resembled those of 1, but showed characteristic signals for the CH(12)@C(13)–CH(15)@CH2(16) conjugated dienyl moiety instead of those for the furan ring. The exocyclic vinylic hydrogen resonances appeared at d 6.23 (1H, dd, J15,16a = 17.6, J15,16b = 10.6 Hz, H-15), 5.08 (1H, br d, J15,16a = 17.6 Hz, H-16a) and 4.91 (1H, d, J15,16b = 10.6 Hz, H-16b), while the endocyclic vinylic hydrogen appeared at d 5.64 ppm
(1H, t, J11a,12 = J11b,12 = 4.1 Hz, H-12). The signal for H-6 was observed at d 5.51 (1H, q, J5,6 = J6,7a = J6,7b = 2.9 Hz, H-6), which together with a singlet at d 2.03 evidenced the presence of an acetyl group at C-6. Also, the signal for the C-17 secondary methyl group appeared at d 0.89 (3H, d, J14,17 = 7.2 Hz, CH3-17) establishing the close structural relationship with compound 1. Its 13C NMR showed four carbons at d 141.4 (C-13), 138.4 (C-15), 128.5 (C-12) and 109.5 ppm (C-16) attributed to the dienyl system. Its HMBC spectrum showed a correlation between the methyl group CH3-17 and C-13, which confirmed the D12 unsaturation. Complete 1 H and 13C NMR spectroscopic assignments for 3, based on HSQC and HMBC correlations, are listed in Tables 1 and 2, respectively. Cassadiene 4 displayed a quasi-molecular ion [M+Na]+ at m/z 353.2451 by positive mode HRESIMS, which was consistent with the molecular formula C22H34O2 and its optical rotation was [a]D = 108 in CHCl3. The 1H and 13C NMR spectra of 4 also indicated the presence of a diterpene having a cassadiene skeleton. It showed a characteristic NMR signal pattern for an ABX system belonging to the C(14)@C(13)–CH(15)@CH2(16) conjugated dienyl moiety, where the vinylic hydrogen signals appeared at d 6.80 (1H, dd, J15,16a = 17.0, J15,16b = 11.0 Hz, H-15), 5.13 (1H, d, J15,16a = 17.0 Hz, H-16a), and 4.95 (1H, d, J15,16b = 11.0 Hz, H-16b). The hydrogen atom attached to the ester-bearing carbon atom C-6 was observed at d 5.52 (1H, q, J5,6 = J6,7a = J6,7b = 2.9 Hz, H-6). Its COSY spectrum confirmed the position of an acetyl group at C-6 that showed cross-peaks of H-6 with H-5, H-7a and H-7b in a similar way than in compound 1. The acetyl group appeared at d 2.06 (3H, s), while the vinyl methyl group C-17 was observed at d 1.70 (3H, br s). Its 13C NMR spectrum (Table 2) showed twenty two signals including a carbon typical for carbonyl ester at d 170.5 (C, OAc), and four resonances attributed to the conjugated dienyl moiety at d 135.7 (C, C-14), 135.3 (CH, C-15), 129.3 (C, C-13) and 110.9 (CH2, C-16). Several HMBC correlations were useful to confirm localization of the conjugated diene moiety, since cross-peaks between the vinyl methyl group CH3-17 and C-8, C-13, and C-14 were observed, as well as correlations of H-15 with C-12, C-13, and C-14.
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The isolation of the C6-epimer of 1, 6a-acetoxyvouacapane (6), from D. lacunifera (Mendes and Silveira, 1994), which showed H-6 shifted upfield (d 5.25) with respect to H-6 (d 5.51) in compound 1 was described. This variation is in agreement with the axial vs. equatorial orientation for H-6 on going from 6 to 1. Interestingly, a drastic change in the optical rotation sign was observed for both epimers, showing [a]D = 18.3 for 1 and [a]D = +44.5 for 6. In order to establish a chemical correlation of ( )-6b-acetoxyvouacapane (1) with the diterpenes from D. lacunifera, compound 1 was converted to ( )-6b-hydroxyvouacapane (2), [a]D = 8.4, with LiAlH4 in anhydrous THF. Oxidation of ( )-2 using pyridinium chlorochromate in CH2Cl2 afforded (+)-6-oxovouacapane (5), [a]D = +81, whose spectroscopic data were identical to those of a ketone prepared from (+)-6a-acetoxyvouacapane (6) (Mendes and Silveira, 1994). Reduction of (+)-ketone 5 with NaBH4 in EtOH proceeded with high diastereoselectivity since only the b-hydroxy isomer 2
was detected. Finally, compound ( )-2 was acetylated with pyridine and an excess of Ac2O under microwave irradiation conditions to regenerate ( )-1. The 1H and 13C NMR spectra of compounds 2 and 5 were fully assigned (see Tables 1 and 2) using COSY, NOESY, HETCOR and HMBC correlations. Several 13C NMR assignments of both compounds were revised with respect to those reported (Mendes and Silveira, 1994). In the spectrum of 2, the signals for C-1, C-7, C-8, C-10, C-18, C-19 and C-20 were reassigned as listed in Table 2. The COSY spectrum of 2 showed clear correlations for the H-5–H-6–H-7a/b–H-8–H-14–CH3-17 fragment which allows the precise assignment for C-7 and C-8 according to the C-7/H27a,7b and C-8/H-8 HETCOR cross-peaks, respectively. In the same plot, the gem-dimethyl group resonances, with C-18 at d 33.8 and C-19 at d 24.3, were assigned according to the C-18/H3-18 and C-19/H3-19 HETCOR cross-peaks, and the intense cross-correlation of CH3-20 at d 1.23 with H-8 at d 2.14 and H-11b at d 2.45 observed
(a)
Δε x 103
Δε x 103
(a)
(b) Δε x 103 Molar absorptivity, ε
(c)
(c)
(d) Molar absorptivity, ε
Molar absorptivity, ε
Molar absorptivity, ε
Δε x 103
(b)
ν− (cm-1) Fig. 4. Vibrational spectra of ( )-(5S,6R,8S,9S,10R,14R)-6-acetoxyvouacapane (1). (a) Experimental VCD in CDCl3, (b) calculated VCD at the B3LYP/DGDZVP level, (c) experimental IR in CDCl3, and (d) calculated IR at the B3LYP/DGDZVP level.
(d)
ν− (cm-1) Fig. 5. Vibrational spectra of ( )-(5S,6R,8S,9S,10R,14R)-6-hydroxyvouacapane (2). (a) Experimental VCD in CDCl3, (b) calculated VCD at the B3LYP/DGDZVP level, (c) experimental IR in CDCl3, and (d) calculated IR at the B3LYP/DGDZVP level.
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Table 3 Quantitative comparison data of the experimental and calculated IR and VCD spectra of compounds 1, 2 and 5.
Δε x 10 3
(a)
a b c d e
(b)
Compound
anHa
SIRb
SEc
S
1 2 5
0.981 0.977 0.979
91.2 85.8 86.2
90.4 84.9 85.7
31.8 18.3 12.7
E
d
Ce 100 100 100
Anharmonicity factor. IR spectral similarity. VCD spectral similarity for the correct enantiomer. VCD spectral similarity for the incorrect enantiomer. Confidence level for the absolute configuration determination.
Δε x 103
2.2. Absolute configuration determination
Molar absorptivity, ε
(c)
Molar absorptivity, ε
(d)
ν− (cm-1) Fig. 6. Vibrational spectra of (+)-(5S,8S,9S,10R,14R)-6-oxovouacapane (5). (a) Experimental VCD in CDCl3, (b) calculated VCD at the B3LYP/DGDZVP level, (c) experimental IR in CDCl3, and (d) calculated IR at the B3LYP/DGDZVP level.
in the NOESY spectrum. The HMBC spectrum was useful to assign C-8 at d 30.4, which displayed a cross-correlation with CH3-17 at d 0.96; the C-10 signal at d 37.6 showed cross-correlation with H-5 at d 0.98, the C-1 methylene carbon displayed a cross-correlation with the C-20 methyl protons at d 1.23, which in turn showed correlations with C-10 at d 37.6, and C-9 at d 45.9. From the literature 13C NMR spectrum of 5 (Mendes and Silveira, 1994), the C-14 signal at d 30.7 and that of C-18 at d 32.8 are now reassigned according to the C-14/H-14 and C-18/H3-18 HETCOR cross-peaks, respectively. Furthermore, in the HMBC plot, the C-14 methine carbon displayed a cross-correlation with the C-17 methyl protons and with the C-7 methylene protons at d 2.35 and d 2.24, respectively. The C-18 methyl group displayed cross-correlations with the C-19 methyl group at d 1.25 and with the H-5 methine at d 2.10.
Vibrational circular dichroism spectroscopy has been successfully used to determine the absolute configuration of natural products, including seco-oxacassane derivatives (Manríquez-Torres et al., 2011). This technique is based on comparison between the experimental VCD spectrum and the corresponding calculated curve for the proper enantiomer, obtained by density functional theory (DFT) (Polavarapu, 2012). The calculations involve the generation of weight-averaged vibrational plots including all significantly populated conformations of the analyzed molecule. From this group of compounds, furane cassanes or vouacapanes 1, 2 and 5 were selected for the VCD analysis because they appear as conformationally restricted molecules and showed adequate chemical stability. According to the calculations, each of these three molecules provided only one conformer, facilitating the computational efforts and improving the correspondence between experimental and calculated spectra. The molecular models of 1, 2 and 5 were subjected to a procedure based on conformational searching, using the Monte Carlo method, followed by single-point energy calculation using DFT at the B3LYP/6-31G(d) level of theory. The conformers were geometry optimized at the B3LYP/DGDZVP level of theory, and their thermochemical parameters, IR, and VCD frequencies were calculated. The procedure yielded conformers 1, 2 and 5 (Fig. 2) that accounted for 100% of the conformational population in all cases. Figs. 4–6 provide a comparison between the calculated and experimental IR and VCD spectra of ( )-(5S,6R,8S,9S,10R,14R)-6-acetoxyvouacapane (1), ( )-(5S,6R,8S,9S,10R,14R)-6-hydroxyvouacapane (2) and (+)-(5S,8S,9S,10R,14R)-6-oxovouacapane (5), respectively, showing a remarkable agreement. Quantitative evaluation of this concordance was achieved by applying the CompareVOA algorithm (Debie et al., 2011), which estimates the integrated overlap of the experimental and calculated data as a function of a relative shift. Application of this procedure (Table 3) gave the optimal anharmonicity factor and the VCD spectral similarity for the correct and the incorrect enantiomer (Table 3). This software gave a 100% confidence for the absolute configuration determination in the three cases. Cassadienes have been biogenetically related to vouacapanes (Yodsaoue et al., 2010), most likely through taepeenin L (7) (Cheenpracha et al., 2006) as intermediate, which upon oxidative cyclization could yield the corresponding vouacapane. Therefore, based on biogenetic reasons, dienes 3 and 4 should have the same absolute configuration than vouacapane 1. 3. Conclusions Chemical study of leaves of C. platyloba gave three new diterpenes 1, 3 and 4 whose stereostructures were determined by 1D and 2D NMR spectroscopy, X-ray diffraction analysis, and vibrational circular dichroism spectroscopy. The results provided conclusive evidence for the absolute configuration determination
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of the three compounds as ( )-(5S,6R,8S,9S,10R,14R)-6-acetoxyvouacapane (1), ( )-(5S,6R,8S,9S,10R,12Z,14R)-6-acetoxycassa-12, 15-diene (3), and ( )-(5S,6R,8S,9S,10R,13E)-6-acetoxycassa-13, 15-diene (4). Chemical correlations of 1 with diterpenes from D. lacunifera, extended the absolute configuration assignments to ( )-(5S,6R,8S,9S,10R,14R)-6-hydroxyvouacapane (2), (+)-(5S,8S,9S, 10R,14R)-6-oxovouacapane (5), and (+)-(5S,6S,8S,9S,10R,14R)-6acetoxyvouacapane (6). 4. Experimental 4.1. General experimental procedures Melting points were determined on a Fisher–Johns apparatus and are uncorrected. Optical rotations were determined in CHCl3 on a Perkin-Elmer 341 polarimeter. UV spectra were determined on a Perkin-Elmer Lambda 12 spectrophotometer. VCD and IR measurements were performed on a BioTools dualPEM ChiralIR FT spectrophotometer. NMR measurements were performed at 400 MHz for 1H and 100 MHz for 13C on a Varian Mercury-Plus 400 spectrometer from CDCl3 solutions using TMS as internal standard. LRMS were recorded on a Varian Saturn 2000 spectrometer, while HRMS were measured on an Agilent LCTOF instrument at the UCR Mass Spectrometry Facility, University of California, Riverside or on a Bruker micrOTOF-Q II Electrospray Ionization Mass Spectrometer at Centro de Nanociencias y Micro y Nanotecnologías del Instituto Politécnico Nacional, Mexico City. TLC was performed on silica gel 60 precoated glass plates (Analtech, layer thickness 0.5 mm, 10 20 cm with fluorescent indicator F254). Column chromatography was carried out on Merck silica gel 60 (230–400 mesh). 4.2. Plant material Specimens of C. platyloba S. Watson were collected from Los Charcos in the municipality of Buenavista, Michoacán state, Mexico, during September 2009, and identified by Prof. Xavier Madrigal at Facultad de Biología, Universidad Michoacana de San Nicolás de Hidalgo where a voucher specimen (No. 2401) is preserved. 4.3. Extraction and isolation
(C–O), 861 (furan); 1H NMR (CDCl3, 400 MHz) and 13C NMR (CDCl3, 100 MHz) see Tables 1 and 2; EIMS m/z (rel. int.) 345 [M+1]+ (21), 344 [M]+ (100), 284 (20), 269 (30), 147 (32), 131 (32), 119 (9), 109 (28), 108 (84); HRESI/APCIMS m/z 345.2421 [M+H]+ (calculated for C22H32O3 + H+, 345.2425). 4.3.2. ( )-(5S,6R,8S,9S,10R,14R)-6-Hydroxyvouacapane (2) 4.3.2.1. Method A. A solution of 1 (500 mg) in dry THF (10 mL) was treated with a suspension of LiAlH4 (550 mg) in dry THF (10 mL) at 25 °C under stirring for 2 h. The reaction mixture was treated with EtOAc (25 ml), acidified to pH 6 with 10% HCl, and extracted with EtOAc (100 ml). The organic layer was washed with water, dried over Na2SO4, filtered and evaporated to afford 2 (410 mg, 94%). 4.3.2.2. Method B. A solution of ketone 5 (45 mg) in EtOH (10 mL) was treated with NaBH4 (150 mg) at 25 °C for 24 h and extracted with AcOEt. The organic layer was washed with H2O and dried (Na2SO4) filtered and evaporated in vacuo to give 2 (13.1 mg, 29%): Colorless oil, [a]D = 8.4 (c 0.75, CHCl3); for 1H NMR (CDCl3, 400 MHz) and 13C NMR (CDCl3, 100 MHz) spectroscopic data, see Tables 1 and 2; EIMS m/z (rel. int.) 302 [M]+ (100), 285 (10), 270 (38), 145 (26), 133 (32), 132 (14), 131 (34), 119 (9), 109 (34), 108 (75), 105 (18), 95 (5), 91 (20), 79 (26), 55 (14). 4.3.3. ( )-(5S,6R,8S,9S,10R,12Z,14R)-6-Acetoxycassa-12,15-diene (3) Colorless oil; [a]D = 5 (c 0.23, CHCl3); UV (EtOH) kmax (log e) nm: 232 (3.85); IR (CHCl3) mmax cm 1: 1724 (C@O), 1653 (C@C), 1260 (C–O); for 1H NMR (CDCl3, 400 MHz) and 13C NMR (CDCl3, 100 MHz) spectroscopic data, see Tables 1 and 2; EIMS m/z (rel. int.) 330 [M]+ (1), 270 (100), 256 (91), 241 (7), 228 (14), 213 (12), 201 (55), 187 (44), 185 (64), 176 (42), 159 (41), 145 (42), 131 (55), 105 (78), 91 (72). HRESIMS m/z 353.2451 [M+Na]+ (calcd for C22H34O2 + Na+ 353.2451). 4.3.4. ( )-(5S,6R,8S,9S,10R,13E)-6-Acetoxycassa-13,15-diene (4) Colorless oil; [a]D = 108 (c 7.8, CHCl3); UV (EtOH) kmax (log e) nm: 242 (3.77); IR (CHCl3) mmax cm 1: 1724 (C@O), 1631 (C@C), 1027 (C–O); for 1H NMR (CDCl3, 400 MHz) and 13C NMR (CDCl3, 100 MHz) spectroscopic data, see Tables 1 and 2; EIMS m/z (rel. int.) 270 [M AcOH]+ (100), 256 (68), 201 (20), 186 (37), 173 (18), 160 (28), 146 (15), 134 (17), 119 (16), 91 (22). HRESIMS m/ z 353.2451 [M+Na]+ (calcd for C22H34O2 + Na+ 353.2451).
Air dried leaves (90 g) of C. platyloba were extracted with n-hexane (1 L 3) at room temperature for 3 days and then the process was performed with CH2Cl2 (1 L 3). Filtration and evaporation of the CH2Cl2 extract afforded a dark green viscous oil (8.9 g, 9.9%), from which a portion (4.2 g) was subjected to silica gel column chromatography (cc) with hexane–CH2Cl2 mixtures as eluent. Fractions 35–39 (hexane–CH2Cl2 99:1) afforded mixtures of cassadienes 3 and 4, while fractions 40–50 (hexane–CH2Cl2 99:1 to 19:1) gave pure vouacapane 1 (190 mg). The mixture of cassadienes 3 and 4 (36 mg) was applied on TLC plates impregnated with 20% AgNO3, developing with hexane–EtOAc 9:1, followed by extraction with EtOAc and filtration. The organic layers were washed with Na2SO3 aq. satd. soln. (3) and H2O (3), dried over anh. Na2SO4, filtered and evaporated to afford 3 (5 mg, Rf = 0.43) and 4 (12 mg, Rf = 0.31). Compound 3 was reapplied to a silica gel microcolumn (0.5 10 cm) eluting with pentane, hexane (10 ml), and hexane–EtOAc 99:1 (20 ml) to yield pure 3 (3 mg) from the last fractions.
4.3.5. (+)-(5S,8S,9S,10R,14R)-6-Oxovouacapane (5) A solution of 2 (410 mg) in CH2Cl2 (10 mL) was treated with freshly prepared PCC (615 mg) in CH2Cl2 (45 mL) at 25 °C for 1 h. The reaction mixture was evaporated and the crude product was subjected to silica gel CC, eluting with hexane to give 5 (155 mg, 38%): Colorless oil, [a]D = +81 (c 0.75, CHCl3); IR (CHCl3) mmax cm 1: 2929, 2871, 2846, 1709, 1508, 1463, 1392, 900; for 1 H NMR (CDCl3, 400 MHz) and 13C NMR (CDCl3, 100 MHz) spectroscopic data, see Tables 1 and 2; EIMS m/z (rel. int.) 300 [M]+ (76), 282 (1), 267 (4), 229 (5), 217 (5), 159 (9), 147 (12), 133 (67), 108 (100), 91 (11), 81 (12).
4.3.1. ( )-(5S,6R,8S,9S,10R,14R)-6-Acetoxyvouacapane (1) Colorless crystals (CH2Cl2–hexane); m.p. 114–116 °C; [a]D = 18.3 (c 3.8, CHCl3); UV (EtOH) kmax (log e) nm: 211 (3.86), 252 (3.29); IR (CHCl3) mmax cm 1: 1724 (C@O), 1631 (C@C), 1250
4.4. Single crystal X-ray diffraction analysis of 1
4.3.6. Acetylation of ( )-6-hydroxyvouacapane (2) A solution of 2 (50 mg) was treated with pyridine (1.5 mL) and Ac2O (3 mL) for 15 min in a microwave oven (100 W, 80 °C), and extracted with AcOEt. The organic layer was successively washed with 10% HCl, H2O, NaHCO3 saturated solution, and H2O, dried (Na2SO4) filtered and evaporated to give 1 (51 mg, 90%).
X-ray data for 1 were collected on a Bruker-Nonius CAD4 diffractometer using Cu Ka radiation (k = 1.54184 Å) at 293(2) K in
M.A. Gómez-Hurtado et al. / Phytochemistry 96 (2013) 397–403
the x-2h scan mode. Unit cell refinements using 25 machine centered reflections were done using the CAD4 Express v2.0 software. Crystal data were C22H32O3, M = 344.48, orthorhombic, space group P212121, a = 8.443(1) Å, b = 10.453(3) Å, c = 22.386(2) Å, V = 1975.8(7) Å3, Z = 4, q = 1.158 mg/mm3, l(Cu Ka) = 0.589 mm 1, total reflections = 1589, unique reflections 1511 (Rint 0.0001%), observed reflections 1450. The structure was solved by direct methods using the SHELXS-97 program included in the WinGX v1.70.01 crystallographic software package. For the structural refinement, the non-hydrogen atoms were treated anisotropically, and the hydrogen atoms, included in the structure factor calculation, were refined isotropically. The final R indices were [I > 2r(I)] R1 = 3.8% and wR2 = 10.5%. The largest difference peak and hole were 0.105 and 0.102 e Å3. Crystallographic data (excluding structure factors) have been deposited under CCDC deposition number 951443 at the Cambridge Crystallographic Data Centre. Copies of the data can be obtained free of charge on application to the CCDC, 12 Union Road, Cambridge CB2 IEZ, UK. Fax: +44 (0)1223 336033 or e-mail: [email protected].
4.5. VCD measurements Samples of 1, 2 and 5 (12.4, 13.1, and 15.2 mg) were dissolved in CDCl3 (150 lL) and placed in a BaF2 cell with a pathlength of 100 lm. Data were acquired at a resolution of 4 cm 1 for 20 h each. The stability of the molecules was monitored by 1H NMR spectroscopy immediately prior and after VCD measurements.
4.6. Molecular modeling and VCD calculations The conformational search for 1, 2 and 5 was carried out in the Spartan’04 program (Wavefunction, Inc., Irvine, CA, USA) using the Monte Carlo protocol with the MMFF94 force-field to give only one main conformer, in all cases, which were subjected to single point energy DFT calculations using the B3LYP/ 6-31G(d) level of theory. Geometry optimization of these three structures, using the Gaussian 03W program (Gaussian, Inc., Wallingford, CT, USA) at the DFT B3LYP/DGDZVP level of theory, and calculation of the vibrational frequencies yielded the minimum energy structures shown in Fig. 2. The optimized structures were used to calculate the thermochemical parameters estimated at 298 K and 1 atm. The VCD and IR frequencies were plotted using Lorentzian bandshapes and bandwidths of 6 cm 1. The level of agreement between experimental and calculated VCD spectra was estimated with the CompareVOA algorithm (BioTools Co., Jupiter, FL, USA; Debie et al., 2011). Molecular visualization was done with the GaussianView 3.0 program.
403
Acknowledgements Financial support from Universidad Michoacana de San Nicolás de Hidalgo (PROMEP UMSNH-PTC-332) and from CONACYT, Mexico (Grant Nos. 152994 and CB-167952) is acknowledged. FEAE, RMEM, TPB and JLSH are grateful to CONACYT for scholarships. The authors thank Prof. Xavier Madrigal, Facultad de Biología, Universidad Michoacana de San Nicolás de Hidalgo for identifying the plant material. References Arriaga, A.M.C., Castro, M.A.B., Silveira, E.R., Braz-Filho, R., 2000. Further diterpenoids isolated from Pterodon polygaleflorus. J. Braz. Chem. Soc. 11, 187– 190. Cheenpracha, S., Karalai, C., Ponglimanont, C., Chantrapromma, K., Laphookhieo, S., 2006. Cassane-type diterpenes from the seeds of Caesalpinia crista. Helv. Chim. Acta 89, 1062–1066. Debie, E., De Gussem, E., Dukor, R.K., Herrebout, W., Nafie, L.A., Bultinck, P., 2011. A confidence level algorithm for the determination of absolute configuration using vibrational circular dichroism or Raman optical activity. ChemPhysChem 12, 1542–1549. Hong, Y.J., Tantillo, D.J., 2010. Formation of beyerene, kaurene, trachylobane, and atiserene diterpenes by rearrangements that avoid secondary carbocations. J. Am. Chem. Soc. 132, 5375–5386. Manríquez-Torres, J.J., Torres-Valencia, J.M., Gómez-Hurtado, M.A., Motilva, V., García-Mauriño, S., Ávila, J., Talero, E., Cerda-García-Rojas, C.M., Joseph-Nathan, P., 2011. Absolute configuration of 7,8-seco-7,8-oxacassane diterpenoids from Acacia schaffneri. J. Nat. Prod. 74, 1946–1951. Mauria, R., Ravi, M., Singh, S., Yadav, P.P., 2012. A review on cassane and norcassane diterpenes and their pharmacological studies. Fitoterapia 83, 272–280. Mendes, F.N.P., Silveira, E.R., 1994. Fatty acids, sesqui- and diterpenoids from seeds of Dipteryx lacunifera. Phytochemistry 35, 1499–1503. Nafie, L.A., 2008. Vibrational circular dichroism: a new tool for the solution-state determination of the structure and absolute configuration of chiral natural product molecules. Nat. Prod. Commun. 3, 451–466. Polavarapu, P.L., 2012. Determination of the structures of chiral natural products using vibrational circular dichroism. In: Berova, N., Polavarapu, P.L., Nakanishi, K., Woody, R.W. (Eds.), Comprehensive Chiroptical Spectroscopy, vol. 2: Stereochemical Analysis of Synthetic Compounds, Natural Products, and Biomolecules. Wiley, New York, p. 387 (Chapter 11). Wu, M., Wang, Y.F., Zhang, M.L., Huo, C.H., Dong, M., Shi, Q.W., Kiyota, H., 2011. Chemical constituents of plants from the genus Caesalpinia. Chem. Biodivers. 8, 1370–1399. Yajima, A., Mori, K., 2000a. Absolute configuration of phytocassanes as proposed on the basis of the CD spectrum of synthetic (+)-2-deoxyphytocassane A. Tetrahedron Lett. 41, 351–354. Yajima, A., Mori, K., 2000b. Synthesis and absolute configuration of ( )phytocassane D, a diterpene phytoalexin isolated from the rice plant, Oryza sativa. Eur. J. Org. Chem., 4079–4091. Yajima, A., Mori, K., Yabuta, G., 2004. Total synthesis of ent-cassa-12,15-diene, a putative precursor of rice phytoalexins, phytocassanes A–E. Tetrahedron Lett. 45, 167–169. Yodsaoue, O., Karalai, C., Ponglimanont, C., Tewtrakul, S., Chantrapromma, S., 2010. Potential anti-inflammatory diterpenoids from the roots of Caesalpinia mimosoides Lamk. Phytochemistry 71, 1756–1764. Yodsaoue, O., Karalai, C., Ponglimanont, C., Tewtrakul, S., Chantrapromma, S., 2011. Pulcherrins D-R, potential anti-inflammatory diterpenoids from the roots of Caesalpinia pulcherrima. Tetrahedron 67, 6838–6846. Zanin, J.L.B., de Carvalho, B.A., Martineli, P.S., dos Santos, M.H., Lago, J.H.G., Sartorelli, P., Viegas Jr., C., Soares, M.G., 2012. The genus Caesalpinia L. (Caesalpiniaceae): phytochemical and pharmacological characteristics. Molecules 17, 7887–7902.
Contents lists available at ScienceDirect
Phytochemistry journal homepage: www.elsevier.com/locate/phytochem
Cassane diterpenes from Caesalpinia platyloba Mario A. Gómez-Hurtado a, Fany E. Álvarez-Esquivel a, Gabriela Rodríguez-García a, Mauro M. Martínez-Pacheco a, Rosa M. Espinoza-Madrigal a, Teresa Pamatz-Bolaños a, José L. Salvador-Hernández a, Hugo A. García-Gutiérrez a, Carlos M. Cerda-García-Rojas b,⇑, Pedro Joseph-Nathan b, Rosa E. del Río a,⇑ a b
Instituto de Investigaciones Químico-Biológicas, Universidad Michoacana de San Nicolás de Hidalgo, Edificio B-1, Ciudad Universitaria, Morelia, Michoacán 58030, Mexico Departamento de Química, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, Apartado 14-740, México D.F. 07000, Mexico
a r t i c l e
i n f o
Article history: Received 30 March 2013 Received in revised form 19 September 2013 Available online 28 October 2013 Keywords: Caesalpinia platyloba Fabaceae Vouacapane derivatives Cassane diterpenes Absolute configuration Vibrational circular dichroism Density functional theory calculations
a b s t r a c t The dichloromethane extract from the leaves of Caesalpinia platyloba provided cassane diterpenes whose structures were determined as ( )-(5S,6R,8S,9S,10R,14R)-6-acetoxyvouacapane (1), ( )-(5S,6R,8S,9S, 10R,12Z,14R)-6-acetoxycassa-12,15-diene (3), and ( )-(5S,6R,8S,9S,10R,13E)-6-acetoxycassa-13,15-diene (4). Compound 1 was chemically correlated with ( )-(5S,6R,8S,9S,10R,14R)-6-hydroxyvouacapane (2), (+)-(5S,8S,9S,10R,14R)-6-oxovouacapane (5), and (+)-(5S,6S,8S,9S,10R,14R)-6-acetoxyvouacapane (6), the last one previously isolated from Dipteryx lacunifera. The absolute configurations of all six diterpenes 1–6 were established by comparison of DFT calculated vibrational circular dicroism spectra of 1, 2 and 5 with those obtained experimentally. In addition, several reported chemical shifts for 2 and 5 were reassigned based on two-dimensional NMR measurements. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction The genus Caesalpinia comprises more than 500 species which are mainly distributed in tropical or subtropical zones (Zanin et al., 2012). Chemical studies of the genus have shown the presence of phenolic derivatives, steroids, triterpenoids, and cassane diterpenes (Wu et al., 2011), and several species are pharmacologically relevant, showing antiulcer, anticancer, antidiabetic, anti-inflammatory, antirheumatic, antimicrobial, and cytotoxic activities (Wu et al., 2011; Zanin et al., 2012). The genus Caesalpinia is phytochemically characterized by the presence of cassane diterpenes, including vouacapane and cassadiene derivatives, which have also been described in other genera of the Fabaceae family (Mauria et al., 2012). It is well known that protonation of geranylgeranyl diphosphate (GGPP) can lead to both enantiomeric series of diterpenes, through either copalyl diphosphate or ent-copalyl diphosphate, depending on the existing biosynthetic pathway (Hong and Tantillo, 2010).
⇑ Corresponding authors. Tel.: +52 443 3265788/55 57474035; fax: +52 443 3265790/55 57477137. E-mail addresses: [email protected] (C.M. Cerda-García-Rojas), ndelrio@ umich.mx (R.E. del Río). 0031-9422/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.phytochem.2013.09.028
Therefore, it is relevant to define clearly the absolute configuration of new diterpene compounds, even more if they show possible pharmacological applications. Concerning cassane derivatives, there are reports on the isolation of members of both enantiomeric series, normal cassane diterpenes isolated from Acacia (ManríquezTorres et al., 2011), Caesalpinia (Yodsaoue et al., 2011), Dipteryx (Mendes and Silveira, 1994), and Pterodon (Arriaga et al., 2000) genera, among others, and ent-cassanes generally found as phytoalexins produced by rice plants (Yajima et al., 2004; Yajima and Mori, 2000a,b). Vibrational circular dicroism (VCD) has been used in recent times as an effective technique to determine the absolute configuration of natural compounds (Nafie, 2008). In the present work, we describe the structure elucidation of three new cassane diterpenes (1, 3 and 4, Fig. 1) isolated from the CH2Cl2 extract of Caesalpinia platyloba S. Watson (Fabaceae). Their structures and relative configurations were established by 1D and 2D NMR experiments and by the X-ray diffraction analysis of 1, while their absolute configuration was determined by VCD spectroscopy of 1 and derivatives 2 and 5, using density functional theory calculations at the B3LYP/DGDZVP level of theory. Cassane 1 was chemically correlated, through derivatives 2 and 5, with natural occurring 6 isolated from Dipteryx lacunifera (Mendes and Silveira, 1994).
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O
1
15
11 20 1 2
5
4
13 14
H 17
H
6
OR
18
H
1
2
3
4
5
1a 1b
1.02 (m) 1.67 (br d, 12.3) 1.60 (dt, 13.6, 3.2) 1.45 (m)
0.97 (m) 1.63 (m)
0.94 (m) 1.79 (m)
1.60 (m)
3a
1.19 (m)
1.18 (m)
3b
1.41 (br d, 13.1) 1.09 (s) 5.51 (td, 3.1, 1.9) 1.53 (m) 1.85 (dt, 14.3, 3.5) 2.03 (m)
1.39 (br d, 13.0) 0.98 (s) 4.48 (br s)
1.44 (dquin 13.6, 3.2) 1.18 (td, 13.0, 3.2) 1.39 (dt, 13.0, 3.2) 1.06 (s) 5.51 (q, 2.9) 1.50 (m) 1.80 (dt, 14.4, 3.2) 1.83 (m)
1.67 (dt, 14.0, 3.6) 1.49 (dquin 14.0, 3.6) 1.19 (td, 13.2, 4.1) 1.38 (br d, 13.2) 1.02 (s) 5.52 (q, 2.9) 1.13 (m) 2.24 (dt, 14.5, 3.3) 2.33 (m)
1.18 (m) 1.76 (br d, 12.9) 1.51 (m)
2b
1.05 (m) 1.73 (br d, 12.7) 1.63 (dt, 13.6, 3.2) 1.48 (m)
1.37 (m) 2.09 (m)
0.93 (m) 1.84 (m)
2.09 (m)
1.16 (m)
– 5.64 (t, 4.1)
2.01 (m) 2.36 (br d, 14.4) – 6.80 (dd, 17.0, 11.0) 5.13 (d, 17.0) 4.95 (d, 11.0) 1.70 (br s)
2a
OAc
1 : R = Ac 2:R=H
3
O H
5 6
H
H H
Position
7
H 19
8
H
10
3
H
9
Table 1 H NMR spectroscopic data of compounds 1–5 (400 MHz, CDCl3, d in ppm, J in Hz).
16
12
H
OAc
H
7a 7b
O
8
2.59 (m) 6.19 (d, 1.9) 7.23 (dt, 1.9, 0.9) –
2.61 (m) 6.19 (d, 2.0) 7.23 (br d, 1.9) –
0.96 7.0) 0.99 1.03 1.21 2.03
0.96 7.0) 1.01 1.26 1.23 –
5
4 O
HO
H
H
9 11a 11b
H H
1.54 (m) 2.60 (dd, 16.9, 7.1) 2.50 (dd, 16.9, 10.0) – –
1.57 (m) 1.72 (dt, 13.9, 3.7) 2.14 (dt, 12.5, 4.7) 1.51 (m) 2.57 (dd, 16.9, 7.1) 2.45 (dd, 16.9, 10.0) – –
OAc 6
12a 12b
H
14 15
H 7
16a
Fig. 1. Structure of diterpenes 1–7.
16b 17 18 19 20 AcO
2. Results and discussion 2.1. Isolation, identification, and structure elucidation Column chromatography separation of the CH2Cl2 extract of the leaves of C. platyloba allowed the isolation of ( )-(5S,6R, 8S,9S,10R,14R)-6-acetoxyvouacapane (1) in very good yields, and of ( )-(5S,6R,8S,9S,10R,12Z,14R)-6-acetoxycassa-12,15-diene (3) and ( )-(5S,6R,8S,9S,10R,13E)-6-acetoxycassa-13,15-diene (4) (Fig. 1) as minor products, which were separated by TLC impregnated with 20% AgNO3. Both dienes (3 and 4) appeared to be unstable, however, their chemical stability was substantially improved when they were dissolved in EtOAc and washed with Na2SO3 aq. satd. soln. to remove any traces of AgNO3 or its transformation products. The molecular formula of the new cassane 1, which belongs to the vouacapane series, was established as C22H32O3 by HRESI/APCIMS, showing [M+H]+ at m/z 345.2421 (calculated 345.2425). The optical activity displayed a levorotatory value of [a]D = 18.3 in CHCl3. Its 1H NMR spectrum (Table 1) showed two aromatic signals assigned to the furan hydrogen atoms at d 7.23 (1H, dt, J15,16 = 1.9 Hz, J11a,16 = J11b,16 = 0.9 Hz, H-16) and d 6.19 (1H, d, J15,16 = 1.9 Hz, H-15). The signal for the hydrogen atom geminal to oxygen at C-6 was observed at d 5.51 (1H, td, J6,7a = J6,7b = 3.1, J5,6 = 1.9 Hz, H-6). The acetyl methyl group appeared at d 2.03 (3H, s, OAc), while the methyl groups were observed at d 1.21 (3H, s, CH3-20), 1.03 (3H, s, CH3-19), 0.99 (3H, s, CH3-18), and 0.96 (3H, d, J14,17 = 7.0 Hz, CH3-17). Its 13C NMR spectrum (Table 2) showed: a carbonyl acetyl group at d 170.6 (C, OAc); furan ring signals, characteristic of vouacapane, at d 149.5 (C, C-12), 140.3 (CH, C-16), 122.0 (C, C-13), and 109.4 (CH, C-15), an oxymethine carbon C-6 appeared at d 69.6, and a typical resonance for C-5 at d 55.3. A NOE difference experiment showed, upon irradiation of H-6a at d
(d, (s) (s) (s) (s)
(d, (s) (s) (s)
2.38 (m) 6.23 (dd, 17.6, 10.6) 5.08 (d, 17.6) 4.91 (d, 10.6) 0.89 (d, 7.2) 0.96 (s) 1.00 (s) 1.13 (s) 2.03 (s)
0.94 1.00 1.14 2.06
(s) (s) (m) (s)
1.51 (m) 1.08 (m) 1.33 (br d, 13.1) 2.10 (s) – 2.24 (m) 2.35 (m) 2.22 (m) 1.95 (m) 2.67 (dd, 16.9, 7.0) 2.38 (dd, 16.9, 10.1) – – 2.62 (m) 6.20 (d, 1.9) 7.24 (d, 1.9) – 1.05 7.0) 0.96 1.25 0.87 –
(d, (s) (s) (s)
Table 2 C NMR spectroscopic data of compounds 1–5 (100 MHz, CDCl3, d in ppm).
13
Position
1
2
3
4
5
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 AcO
42.2 18.7 43.6 33.8 55.3 69.6 36.3 31.0 45.6 37.9 21.7 149.5 122.0 31.0 109.4 140.3 17.5 33.6 23.4 17.1 170.6 21.7
42.5 18.8 43.7 34.0 56.3 67.6 40.3 30.4 45.9 37.6 21.8 149.7 122.1 31.2 109.5 140.3 17.7 33.8 24.3 17.7
41.8 18.7 43.8 33.8 55.8 70.0 36.2 30.5 44.3 37.6 24.6 128.5 141.4 31.3 138.4 109.5 14.4 33.6 23.4 16.4 170.6 21.8
40.8 18.9 44.2 33.8 56.1 70.2 36.8 36.7 54.1 37.4 21.0 26.3 129.3 135.7 135.3 110.9 15.9 33.2 23.2 16.3 170.5 21.8
38.8 18.3 42.5 32.0 65.1 211.9 46.8 38.4 45.4 41.8 22.6 148.8 121.9 30.7 109.4 140.8 17.3 32.8 21.9 14.9
5.51, enhancements for signals of H-5a (distance = 2.43 Å), H-7a (distance = 2.36 Å), equatorial H-7b (distance = 2.46 Å), and CH3-18a (closest distance = 2.14 Å) evidencing the axial orientation
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399
1 (GDFT = –1082.949402 hartrees)
Fig. 3. X-ray structure of ( )-(5S,6R,8S,9S,10R,14R)-6-acetoxyvouacapane (1).
2 (GDFT = –930.292806 hartrees)
5 (GDFT = –929.122047 hartrees) Fig. 2. DFT B3LYP/DGDZVP global minimum structures of (–)(5S,6R,8S,9S,10R,14R)-6-acetoxyvouacapane (1), (–)-(5S,6R,8S,9S,10R,14R)-6hydroxyvouacapane (2) and (+)-(5S,8S,9S,10R,14R)-6-oxovouacapane (5).
of the acetyl group at C-6 (Fig. 2). Two-dimensional NMR spectroscopy including COSY, NOESY, HETCOR, and HMBC experiments were useful for assignment of all 1H and 13C signals. In particular, the resonances for C-8 and C-14, which appear overlapped, were easily located from their one-bond HETCOR correlations with H-8 and H-14, respectively. The relative configuration of 1 was unambiguously established by X-ray diffraction analysis (Fig. 3) confirming the axial orientation of the acetyl group at C-6. Cassadiene 3 had a quasi-molecular ion [M+Na]+ at m/z 353.2451 by positive mode HRESIMS in agreement with a molecular formula C22H34O2. The optical rotation was [a]D = 5 in CHCl3. The 1H and 13C NMR spectra of 3 resembled those of 1, but showed characteristic signals for the CH(12)@C(13)–CH(15)@CH2(16) conjugated dienyl moiety instead of those for the furan ring. The exocyclic vinylic hydrogen resonances appeared at d 6.23 (1H, dd, J15,16a = 17.6, J15,16b = 10.6 Hz, H-15), 5.08 (1H, br d, J15,16a = 17.6 Hz, H-16a) and 4.91 (1H, d, J15,16b = 10.6 Hz, H-16b), while the endocyclic vinylic hydrogen appeared at d 5.64 ppm
(1H, t, J11a,12 = J11b,12 = 4.1 Hz, H-12). The signal for H-6 was observed at d 5.51 (1H, q, J5,6 = J6,7a = J6,7b = 2.9 Hz, H-6), which together with a singlet at d 2.03 evidenced the presence of an acetyl group at C-6. Also, the signal for the C-17 secondary methyl group appeared at d 0.89 (3H, d, J14,17 = 7.2 Hz, CH3-17) establishing the close structural relationship with compound 1. Its 13C NMR showed four carbons at d 141.4 (C-13), 138.4 (C-15), 128.5 (C-12) and 109.5 ppm (C-16) attributed to the dienyl system. Its HMBC spectrum showed a correlation between the methyl group CH3-17 and C-13, which confirmed the D12 unsaturation. Complete 1 H and 13C NMR spectroscopic assignments for 3, based on HSQC and HMBC correlations, are listed in Tables 1 and 2, respectively. Cassadiene 4 displayed a quasi-molecular ion [M+Na]+ at m/z 353.2451 by positive mode HRESIMS, which was consistent with the molecular formula C22H34O2 and its optical rotation was [a]D = 108 in CHCl3. The 1H and 13C NMR spectra of 4 also indicated the presence of a diterpene having a cassadiene skeleton. It showed a characteristic NMR signal pattern for an ABX system belonging to the C(14)@C(13)–CH(15)@CH2(16) conjugated dienyl moiety, where the vinylic hydrogen signals appeared at d 6.80 (1H, dd, J15,16a = 17.0, J15,16b = 11.0 Hz, H-15), 5.13 (1H, d, J15,16a = 17.0 Hz, H-16a), and 4.95 (1H, d, J15,16b = 11.0 Hz, H-16b). The hydrogen atom attached to the ester-bearing carbon atom C-6 was observed at d 5.52 (1H, q, J5,6 = J6,7a = J6,7b = 2.9 Hz, H-6). Its COSY spectrum confirmed the position of an acetyl group at C-6 that showed cross-peaks of H-6 with H-5, H-7a and H-7b in a similar way than in compound 1. The acetyl group appeared at d 2.06 (3H, s), while the vinyl methyl group C-17 was observed at d 1.70 (3H, br s). Its 13C NMR spectrum (Table 2) showed twenty two signals including a carbon typical for carbonyl ester at d 170.5 (C, OAc), and four resonances attributed to the conjugated dienyl moiety at d 135.7 (C, C-14), 135.3 (CH, C-15), 129.3 (C, C-13) and 110.9 (CH2, C-16). Several HMBC correlations were useful to confirm localization of the conjugated diene moiety, since cross-peaks between the vinyl methyl group CH3-17 and C-8, C-13, and C-14 were observed, as well as correlations of H-15 with C-12, C-13, and C-14.
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The isolation of the C6-epimer of 1, 6a-acetoxyvouacapane (6), from D. lacunifera (Mendes and Silveira, 1994), which showed H-6 shifted upfield (d 5.25) with respect to H-6 (d 5.51) in compound 1 was described. This variation is in agreement with the axial vs. equatorial orientation for H-6 on going from 6 to 1. Interestingly, a drastic change in the optical rotation sign was observed for both epimers, showing [a]D = 18.3 for 1 and [a]D = +44.5 for 6. In order to establish a chemical correlation of ( )-6b-acetoxyvouacapane (1) with the diterpenes from D. lacunifera, compound 1 was converted to ( )-6b-hydroxyvouacapane (2), [a]D = 8.4, with LiAlH4 in anhydrous THF. Oxidation of ( )-2 using pyridinium chlorochromate in CH2Cl2 afforded (+)-6-oxovouacapane (5), [a]D = +81, whose spectroscopic data were identical to those of a ketone prepared from (+)-6a-acetoxyvouacapane (6) (Mendes and Silveira, 1994). Reduction of (+)-ketone 5 with NaBH4 in EtOH proceeded with high diastereoselectivity since only the b-hydroxy isomer 2
was detected. Finally, compound ( )-2 was acetylated with pyridine and an excess of Ac2O under microwave irradiation conditions to regenerate ( )-1. The 1H and 13C NMR spectra of compounds 2 and 5 were fully assigned (see Tables 1 and 2) using COSY, NOESY, HETCOR and HMBC correlations. Several 13C NMR assignments of both compounds were revised with respect to those reported (Mendes and Silveira, 1994). In the spectrum of 2, the signals for C-1, C-7, C-8, C-10, C-18, C-19 and C-20 were reassigned as listed in Table 2. The COSY spectrum of 2 showed clear correlations for the H-5–H-6–H-7a/b–H-8–H-14–CH3-17 fragment which allows the precise assignment for C-7 and C-8 according to the C-7/H27a,7b and C-8/H-8 HETCOR cross-peaks, respectively. In the same plot, the gem-dimethyl group resonances, with C-18 at d 33.8 and C-19 at d 24.3, were assigned according to the C-18/H3-18 and C-19/H3-19 HETCOR cross-peaks, and the intense cross-correlation of CH3-20 at d 1.23 with H-8 at d 2.14 and H-11b at d 2.45 observed
(a)
Δε x 103
Δε x 103
(a)
(b) Δε x 103 Molar absorptivity, ε
(c)
(c)
(d) Molar absorptivity, ε
Molar absorptivity, ε
Molar absorptivity, ε
Δε x 103
(b)
ν− (cm-1) Fig. 4. Vibrational spectra of ( )-(5S,6R,8S,9S,10R,14R)-6-acetoxyvouacapane (1). (a) Experimental VCD in CDCl3, (b) calculated VCD at the B3LYP/DGDZVP level, (c) experimental IR in CDCl3, and (d) calculated IR at the B3LYP/DGDZVP level.
(d)
ν− (cm-1) Fig. 5. Vibrational spectra of ( )-(5S,6R,8S,9S,10R,14R)-6-hydroxyvouacapane (2). (a) Experimental VCD in CDCl3, (b) calculated VCD at the B3LYP/DGDZVP level, (c) experimental IR in CDCl3, and (d) calculated IR at the B3LYP/DGDZVP level.
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Table 3 Quantitative comparison data of the experimental and calculated IR and VCD spectra of compounds 1, 2 and 5.
Δε x 10 3
(a)
a b c d e
(b)
Compound
anHa
SIRb
SEc
S
1 2 5
0.981 0.977 0.979
91.2 85.8 86.2
90.4 84.9 85.7
31.8 18.3 12.7
E
d
Ce 100 100 100
Anharmonicity factor. IR spectral similarity. VCD spectral similarity for the correct enantiomer. VCD spectral similarity for the incorrect enantiomer. Confidence level for the absolute configuration determination.
Δε x 103
2.2. Absolute configuration determination
Molar absorptivity, ε
(c)
Molar absorptivity, ε
(d)
ν− (cm-1) Fig. 6. Vibrational spectra of (+)-(5S,8S,9S,10R,14R)-6-oxovouacapane (5). (a) Experimental VCD in CDCl3, (b) calculated VCD at the B3LYP/DGDZVP level, (c) experimental IR in CDCl3, and (d) calculated IR at the B3LYP/DGDZVP level.
in the NOESY spectrum. The HMBC spectrum was useful to assign C-8 at d 30.4, which displayed a cross-correlation with CH3-17 at d 0.96; the C-10 signal at d 37.6 showed cross-correlation with H-5 at d 0.98, the C-1 methylene carbon displayed a cross-correlation with the C-20 methyl protons at d 1.23, which in turn showed correlations with C-10 at d 37.6, and C-9 at d 45.9. From the literature 13C NMR spectrum of 5 (Mendes and Silveira, 1994), the C-14 signal at d 30.7 and that of C-18 at d 32.8 are now reassigned according to the C-14/H-14 and C-18/H3-18 HETCOR cross-peaks, respectively. Furthermore, in the HMBC plot, the C-14 methine carbon displayed a cross-correlation with the C-17 methyl protons and with the C-7 methylene protons at d 2.35 and d 2.24, respectively. The C-18 methyl group displayed cross-correlations with the C-19 methyl group at d 1.25 and with the H-5 methine at d 2.10.
Vibrational circular dichroism spectroscopy has been successfully used to determine the absolute configuration of natural products, including seco-oxacassane derivatives (Manríquez-Torres et al., 2011). This technique is based on comparison between the experimental VCD spectrum and the corresponding calculated curve for the proper enantiomer, obtained by density functional theory (DFT) (Polavarapu, 2012). The calculations involve the generation of weight-averaged vibrational plots including all significantly populated conformations of the analyzed molecule. From this group of compounds, furane cassanes or vouacapanes 1, 2 and 5 were selected for the VCD analysis because they appear as conformationally restricted molecules and showed adequate chemical stability. According to the calculations, each of these three molecules provided only one conformer, facilitating the computational efforts and improving the correspondence between experimental and calculated spectra. The molecular models of 1, 2 and 5 were subjected to a procedure based on conformational searching, using the Monte Carlo method, followed by single-point energy calculation using DFT at the B3LYP/6-31G(d) level of theory. The conformers were geometry optimized at the B3LYP/DGDZVP level of theory, and their thermochemical parameters, IR, and VCD frequencies were calculated. The procedure yielded conformers 1, 2 and 5 (Fig. 2) that accounted for 100% of the conformational population in all cases. Figs. 4–6 provide a comparison between the calculated and experimental IR and VCD spectra of ( )-(5S,6R,8S,9S,10R,14R)-6-acetoxyvouacapane (1), ( )-(5S,6R,8S,9S,10R,14R)-6-hydroxyvouacapane (2) and (+)-(5S,8S,9S,10R,14R)-6-oxovouacapane (5), respectively, showing a remarkable agreement. Quantitative evaluation of this concordance was achieved by applying the CompareVOA algorithm (Debie et al., 2011), which estimates the integrated overlap of the experimental and calculated data as a function of a relative shift. Application of this procedure (Table 3) gave the optimal anharmonicity factor and the VCD spectral similarity for the correct and the incorrect enantiomer (Table 3). This software gave a 100% confidence for the absolute configuration determination in the three cases. Cassadienes have been biogenetically related to vouacapanes (Yodsaoue et al., 2010), most likely through taepeenin L (7) (Cheenpracha et al., 2006) as intermediate, which upon oxidative cyclization could yield the corresponding vouacapane. Therefore, based on biogenetic reasons, dienes 3 and 4 should have the same absolute configuration than vouacapane 1. 3. Conclusions Chemical study of leaves of C. platyloba gave three new diterpenes 1, 3 and 4 whose stereostructures were determined by 1D and 2D NMR spectroscopy, X-ray diffraction analysis, and vibrational circular dichroism spectroscopy. The results provided conclusive evidence for the absolute configuration determination
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of the three compounds as ( )-(5S,6R,8S,9S,10R,14R)-6-acetoxyvouacapane (1), ( )-(5S,6R,8S,9S,10R,12Z,14R)-6-acetoxycassa-12, 15-diene (3), and ( )-(5S,6R,8S,9S,10R,13E)-6-acetoxycassa-13, 15-diene (4). Chemical correlations of 1 with diterpenes from D. lacunifera, extended the absolute configuration assignments to ( )-(5S,6R,8S,9S,10R,14R)-6-hydroxyvouacapane (2), (+)-(5S,8S,9S, 10R,14R)-6-oxovouacapane (5), and (+)-(5S,6S,8S,9S,10R,14R)-6acetoxyvouacapane (6). 4. Experimental 4.1. General experimental procedures Melting points were determined on a Fisher–Johns apparatus and are uncorrected. Optical rotations were determined in CHCl3 on a Perkin-Elmer 341 polarimeter. UV spectra were determined on a Perkin-Elmer Lambda 12 spectrophotometer. VCD and IR measurements were performed on a BioTools dualPEM ChiralIR FT spectrophotometer. NMR measurements were performed at 400 MHz for 1H and 100 MHz for 13C on a Varian Mercury-Plus 400 spectrometer from CDCl3 solutions using TMS as internal standard. LRMS were recorded on a Varian Saturn 2000 spectrometer, while HRMS were measured on an Agilent LCTOF instrument at the UCR Mass Spectrometry Facility, University of California, Riverside or on a Bruker micrOTOF-Q II Electrospray Ionization Mass Spectrometer at Centro de Nanociencias y Micro y Nanotecnologías del Instituto Politécnico Nacional, Mexico City. TLC was performed on silica gel 60 precoated glass plates (Analtech, layer thickness 0.5 mm, 10 20 cm with fluorescent indicator F254). Column chromatography was carried out on Merck silica gel 60 (230–400 mesh). 4.2. Plant material Specimens of C. platyloba S. Watson were collected from Los Charcos in the municipality of Buenavista, Michoacán state, Mexico, during September 2009, and identified by Prof. Xavier Madrigal at Facultad de Biología, Universidad Michoacana de San Nicolás de Hidalgo where a voucher specimen (No. 2401) is preserved. 4.3. Extraction and isolation
(C–O), 861 (furan); 1H NMR (CDCl3, 400 MHz) and 13C NMR (CDCl3, 100 MHz) see Tables 1 and 2; EIMS m/z (rel. int.) 345 [M+1]+ (21), 344 [M]+ (100), 284 (20), 269 (30), 147 (32), 131 (32), 119 (9), 109 (28), 108 (84); HRESI/APCIMS m/z 345.2421 [M+H]+ (calculated for C22H32O3 + H+, 345.2425). 4.3.2. ( )-(5S,6R,8S,9S,10R,14R)-6-Hydroxyvouacapane (2) 4.3.2.1. Method A. A solution of 1 (500 mg) in dry THF (10 mL) was treated with a suspension of LiAlH4 (550 mg) in dry THF (10 mL) at 25 °C under stirring for 2 h. The reaction mixture was treated with EtOAc (25 ml), acidified to pH 6 with 10% HCl, and extracted with EtOAc (100 ml). The organic layer was washed with water, dried over Na2SO4, filtered and evaporated to afford 2 (410 mg, 94%). 4.3.2.2. Method B. A solution of ketone 5 (45 mg) in EtOH (10 mL) was treated with NaBH4 (150 mg) at 25 °C for 24 h and extracted with AcOEt. The organic layer was washed with H2O and dried (Na2SO4) filtered and evaporated in vacuo to give 2 (13.1 mg, 29%): Colorless oil, [a]D = 8.4 (c 0.75, CHCl3); for 1H NMR (CDCl3, 400 MHz) and 13C NMR (CDCl3, 100 MHz) spectroscopic data, see Tables 1 and 2; EIMS m/z (rel. int.) 302 [M]+ (100), 285 (10), 270 (38), 145 (26), 133 (32), 132 (14), 131 (34), 119 (9), 109 (34), 108 (75), 105 (18), 95 (5), 91 (20), 79 (26), 55 (14). 4.3.3. ( )-(5S,6R,8S,9S,10R,12Z,14R)-6-Acetoxycassa-12,15-diene (3) Colorless oil; [a]D = 5 (c 0.23, CHCl3); UV (EtOH) kmax (log e) nm: 232 (3.85); IR (CHCl3) mmax cm 1: 1724 (C@O), 1653 (C@C), 1260 (C–O); for 1H NMR (CDCl3, 400 MHz) and 13C NMR (CDCl3, 100 MHz) spectroscopic data, see Tables 1 and 2; EIMS m/z (rel. int.) 330 [M]+ (1), 270 (100), 256 (91), 241 (7), 228 (14), 213 (12), 201 (55), 187 (44), 185 (64), 176 (42), 159 (41), 145 (42), 131 (55), 105 (78), 91 (72). HRESIMS m/z 353.2451 [M+Na]+ (calcd for C22H34O2 + Na+ 353.2451). 4.3.4. ( )-(5S,6R,8S,9S,10R,13E)-6-Acetoxycassa-13,15-diene (4) Colorless oil; [a]D = 108 (c 7.8, CHCl3); UV (EtOH) kmax (log e) nm: 242 (3.77); IR (CHCl3) mmax cm 1: 1724 (C@O), 1631 (C@C), 1027 (C–O); for 1H NMR (CDCl3, 400 MHz) and 13C NMR (CDCl3, 100 MHz) spectroscopic data, see Tables 1 and 2; EIMS m/z (rel. int.) 270 [M AcOH]+ (100), 256 (68), 201 (20), 186 (37), 173 (18), 160 (28), 146 (15), 134 (17), 119 (16), 91 (22). HRESIMS m/ z 353.2451 [M+Na]+ (calcd for C22H34O2 + Na+ 353.2451).
Air dried leaves (90 g) of C. platyloba were extracted with n-hexane (1 L 3) at room temperature for 3 days and then the process was performed with CH2Cl2 (1 L 3). Filtration and evaporation of the CH2Cl2 extract afforded a dark green viscous oil (8.9 g, 9.9%), from which a portion (4.2 g) was subjected to silica gel column chromatography (cc) with hexane–CH2Cl2 mixtures as eluent. Fractions 35–39 (hexane–CH2Cl2 99:1) afforded mixtures of cassadienes 3 and 4, while fractions 40–50 (hexane–CH2Cl2 99:1 to 19:1) gave pure vouacapane 1 (190 mg). The mixture of cassadienes 3 and 4 (36 mg) was applied on TLC plates impregnated with 20% AgNO3, developing with hexane–EtOAc 9:1, followed by extraction with EtOAc and filtration. The organic layers were washed with Na2SO3 aq. satd. soln. (3) and H2O (3), dried over anh. Na2SO4, filtered and evaporated to afford 3 (5 mg, Rf = 0.43) and 4 (12 mg, Rf = 0.31). Compound 3 was reapplied to a silica gel microcolumn (0.5 10 cm) eluting with pentane, hexane (10 ml), and hexane–EtOAc 99:1 (20 ml) to yield pure 3 (3 mg) from the last fractions.
4.3.5. (+)-(5S,8S,9S,10R,14R)-6-Oxovouacapane (5) A solution of 2 (410 mg) in CH2Cl2 (10 mL) was treated with freshly prepared PCC (615 mg) in CH2Cl2 (45 mL) at 25 °C for 1 h. The reaction mixture was evaporated and the crude product was subjected to silica gel CC, eluting with hexane to give 5 (155 mg, 38%): Colorless oil, [a]D = +81 (c 0.75, CHCl3); IR (CHCl3) mmax cm 1: 2929, 2871, 2846, 1709, 1508, 1463, 1392, 900; for 1 H NMR (CDCl3, 400 MHz) and 13C NMR (CDCl3, 100 MHz) spectroscopic data, see Tables 1 and 2; EIMS m/z (rel. int.) 300 [M]+ (76), 282 (1), 267 (4), 229 (5), 217 (5), 159 (9), 147 (12), 133 (67), 108 (100), 91 (11), 81 (12).
4.3.1. ( )-(5S,6R,8S,9S,10R,14R)-6-Acetoxyvouacapane (1) Colorless crystals (CH2Cl2–hexane); m.p. 114–116 °C; [a]D = 18.3 (c 3.8, CHCl3); UV (EtOH) kmax (log e) nm: 211 (3.86), 252 (3.29); IR (CHCl3) mmax cm 1: 1724 (C@O), 1631 (C@C), 1250
4.4. Single crystal X-ray diffraction analysis of 1
4.3.6. Acetylation of ( )-6-hydroxyvouacapane (2) A solution of 2 (50 mg) was treated with pyridine (1.5 mL) and Ac2O (3 mL) for 15 min in a microwave oven (100 W, 80 °C), and extracted with AcOEt. The organic layer was successively washed with 10% HCl, H2O, NaHCO3 saturated solution, and H2O, dried (Na2SO4) filtered and evaporated to give 1 (51 mg, 90%).
X-ray data for 1 were collected on a Bruker-Nonius CAD4 diffractometer using Cu Ka radiation (k = 1.54184 Å) at 293(2) K in
M.A. Gómez-Hurtado et al. / Phytochemistry 96 (2013) 397–403
the x-2h scan mode. Unit cell refinements using 25 machine centered reflections were done using the CAD4 Express v2.0 software. Crystal data were C22H32O3, M = 344.48, orthorhombic, space group P212121, a = 8.443(1) Å, b = 10.453(3) Å, c = 22.386(2) Å, V = 1975.8(7) Å3, Z = 4, q = 1.158 mg/mm3, l(Cu Ka) = 0.589 mm 1, total reflections = 1589, unique reflections 1511 (Rint 0.0001%), observed reflections 1450. The structure was solved by direct methods using the SHELXS-97 program included in the WinGX v1.70.01 crystallographic software package. For the structural refinement, the non-hydrogen atoms were treated anisotropically, and the hydrogen atoms, included in the structure factor calculation, were refined isotropically. The final R indices were [I > 2r(I)] R1 = 3.8% and wR2 = 10.5%. The largest difference peak and hole were 0.105 and 0.102 e Å3. Crystallographic data (excluding structure factors) have been deposited under CCDC deposition number 951443 at the Cambridge Crystallographic Data Centre. Copies of the data can be obtained free of charge on application to the CCDC, 12 Union Road, Cambridge CB2 IEZ, UK. Fax: +44 (0)1223 336033 or e-mail: [email protected].
4.5. VCD measurements Samples of 1, 2 and 5 (12.4, 13.1, and 15.2 mg) were dissolved in CDCl3 (150 lL) and placed in a BaF2 cell with a pathlength of 100 lm. Data were acquired at a resolution of 4 cm 1 for 20 h each. The stability of the molecules was monitored by 1H NMR spectroscopy immediately prior and after VCD measurements.
4.6. Molecular modeling and VCD calculations The conformational search for 1, 2 and 5 was carried out in the Spartan’04 program (Wavefunction, Inc., Irvine, CA, USA) using the Monte Carlo protocol with the MMFF94 force-field to give only one main conformer, in all cases, which were subjected to single point energy DFT calculations using the B3LYP/ 6-31G(d) level of theory. Geometry optimization of these three structures, using the Gaussian 03W program (Gaussian, Inc., Wallingford, CT, USA) at the DFT B3LYP/DGDZVP level of theory, and calculation of the vibrational frequencies yielded the minimum energy structures shown in Fig. 2. The optimized structures were used to calculate the thermochemical parameters estimated at 298 K and 1 atm. The VCD and IR frequencies were plotted using Lorentzian bandshapes and bandwidths of 6 cm 1. The level of agreement between experimental and calculated VCD spectra was estimated with the CompareVOA algorithm (BioTools Co., Jupiter, FL, USA; Debie et al., 2011). Molecular visualization was done with the GaussianView 3.0 program.
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