Cleidbrevoids A–C, new clerodane diterpenoids from Cleidion brevipetiolatum

Fitoterapia 83 (2012) 1100–1104

Contents lists available at SciVerse ScienceDirect

Fitoterapia journal homepage: www.elsevier.com/locate/fitote

Cleidbrevoids A–C, new clerodane diterpenoids from Cleidion brevipetiolatum Yuan-Yuan Cheng a, b, Shi-Fei Li a, b, Yu Zhang a, Gui-Hua Tang a, b, Ying-Tong Di a, Xiao-Jiang Hao a, Shun-Lin Li a,⁎, Hong-Ping He a,⁎ a State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, People's Republic of China b Graduate University of Chinese Academy of Sciences, Beijing 100039, People's Republic of China

a r t i c l e

i n f o

Article history: Received 11 March 2012 Accepted in revised form 25 April 2012 Available online 3 May 2012

a b s t r a c t Three new clerodane-type diterpenoids with a rare sulfoxide group, cleidbrevoids A–C (1–3), were isolated from the twigs and leaves of Cleidion brevipetiolatum. Their structures were elucidated on the basis of spectroscopic methods and the absolute configuration of compounds 1 and 3 was determined by X-ray analysis. Compound 3 showed weak inhibitory against HIV-1. © 2012 Elsevier B.V. All rights reserved.

Keywords: Cleidion brevipetiolatum Clerodane-type diterpenoids Sulfoxide Cleidbrevoids A–C

1. Introduction The genus Cleidion (Euphorbiaceae) comprising 25 species distributes mainly in the tropical southeast regions of Asia. In the southwest of China, three species, Cleidion brevipetiolatum, C. javanicum and C. bracteosum growing at an altitude of 200–1500 m, are used as folk medicine to treat inflammation, diuresis, and acute and chronic hepatitis [1,2]. Up to date, a series of chemical constituents, such as ellagitannins, phenylpropanoids, triterpenoids and diterpenoids have been reported from the genus Cleidion [3]. As a part of our continuous search for novel and potentially bioactive secondary metabolites from Euphorbiaceae family [4], three new clerodane-type diterpenoids, cleidbrevoids A–C (1–3), with a rare sulfoxide moiety, were isolated from C. brevipetiolatum. The chiral centers of sulfoxide in cleidbrevoids A (1) and C (3) were determined by X-ray analysis. Sulfur-containing diterpenoids were reported previously, for example, clerodane diterpenoid cordifolide A

⁎ Corresponding authors. Tel.: + 86 871 5223263; fax: + 86 871 5223070. E-mail addresses: [email protected] (S-L. Li), [email protected] (H-P. He). 0367-326X/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2012.04.028

with immunomodulatory activity [5], and atisine diterpenoid serofendic acid A with potent neuroprotective activity [6]. 2. Experimental 2.1. General experimental procedure X-ray data were collected using a Bruker APEX DUO instrument. Optical rotations were determined on a Jasco P-1020 polarimeter. UV data were measured using a Shimadzu UV2401PC spectrometer, and IR spectra were recorded on a BRUKER TENSOR27 spectrometer with KBr pellets. ESIMS was carried out on an API QSTAR Pulsar mass spectrometer. 1D and 2D NMR spectra were measured on a Bruker AM-400, DRX500 spectrometer. Column chromatography was performed on Silica gel H (10–40 m; Qingdao Marine Chemical Factory), MCI gel (75–150 m, Mitsubishi Chemical Corporation, Tokyo, Japan) and Sephadex LH-20 (40–70 m, Amersham Pharmacia Biotech AB, Uppsala, Sweden). MPLC was performed on Büchi Sepacore System (Büchi Labortechnik AG, Switzerland), and columns were packed with Chromatorex C-18 (40–75 m, Fuji Silysia Chemical Ltd., Japan). Preparative HPLC was performed by using an Agilent 1200 series system equipped with a Zorbax XDB-C18, 9.4 mm × 150 mm column. Precoated silica gel GF254

Y-Y. Cheng et al. / Fitoterapia 83 (2012) 1100–1104

and HF254 plates (Qingdao Haiyang Chemical Plant, Qingdao, People's Republic of China) were used for TLC.

2.2. Plant material The twigs and leaves of C. brevipetiolatum were collected in Xishuangbanna of Yunnan Province, People's Republic of China, in October 2010, and identified by Mr. Chen Yu, Kunming Institute of Botany, Chinese Academy of Sciences. A voucher specimen (H20101008) was deposited in the Herbarium of Kunming Institute of Botany.

2.3. Extraction and isolation The air-dried, powdered twigs and leaves (14.0 kg) of C. brevipetiolatum were extracted three times with MeOH under reflux. After removal of the solvent by evaporation in vacuum, the residue was suspended in water and then partitioned with EtOAc. One twentieth (9.0 g) of the EtOAc portion (180.0 g) was fractionated with petroleum ether/acetone in a silica gel column to give seven fractions (A–G). Fraction D (1.5 g) was subjected to MCI gel, Sephadex LH-20 and repeatedly silica gel CC to yield compound 1 (6.0 mg). Fraction E (2.5 g) was repeatedly chromatographed on silica gel, MPLC, Sephadex LH-20 and finally purified by semi-preparative HPLC to afford compounds 2 (3.5 mg) and 3 (5.0 mg) (Fig. 1).

2.4. Cleidbrevoid A (1) Colorless prism crystals, mp 190.0–191.0 °C; [a]D26 +23.5 (c 0.20, CH3COCH3); UV (MeOH) λmax (log ε) 204 (4.03) nm; IR (KBr) νmax 3543, 3423, 2944, 1720, 1705, 1631, 1436, 1227, 1211, 1107, 1032, 989, 875, 780, 601 cm − 1; 1H and 13C NMR data see Table 1; ESIMS m/z 437 [M + H] +; HRESIMS m/z 459.1450 [M + Na] + (calcd for C22H28O7SNa, 459.1453).

2.5. Cleidbrevoid B (2) White amorphous power, [a]D26 −32.1. (c 0.20, CH3COCH3); UV (MeOH) λmax (log ε) 204 (4.00) nm; IR (KBr) νmax 3509, 3433, 2920, 1795, 1708, 1639, 1459, 1227, 1269, 1209, 1107, 1032, 989, 875, 780, 601 cm− 1; 1H and 13C NMR data see Table 1; ESIMS m/z 437 [M +H]+, 459 [M+ Na]+; HRESIMS m/z 459.1460 [M+ Na]+ (calcd for C22H28O7SNa, 459.1453).

1101

2.6. Cleidbrevoid C (3) Colorless block crystals, mp 191.0–192.0 °C; [a]D26 +12.8 (c 0.20, CH3COCH3); UV (MeOH) λmax (log ε) 275 (2.27), 204 (3.83) nm; IR (KBr) νmax 3543, 3423, 2944, 1720, 1705, 1631, 1436, 1227, 1211, 1129, 1094, 1021, 987, 874, 783, 686, 601 cm − 1; 1H and 13C NMR data see Table 1; ESIMS m/z 439 [M + H] +; HRESIMS m/z 461.1602 [M + Na] + (calcd for C22H30O7SNa,461.1609). 2.7. X-ray structure determination of compound 1 The compound 1 was recrystallized from methanol. C22H28O7S·CH3OH, Mr 468.55, colorless prism crystals, orthorhombic system, space group P212121, with a = 7.5599 (4) Å, b = 14.0258 (7) Å, c = 22.3956 (11) Å, α = β = γ = 90°, V = 2374.7 (2) Å 3, Dcald = 1.311 g/cm 3 for Z = 4, F (000) = 1000, μ = 1.599 mm− 1. Crystal dimensions: 0.64 × 0.23 × 0.23 mm. 18162 reflections measured (θ max = 68.12°), 4018 were unique (Rint = 0.0388) and of these 3994 had |F| 2 ≥ 2σ|F| 2 for which final R1, wR2 (w=1/σ|F|2), and S (goodness-of-fit) values were 0.0353, 0.0957 and 1.065, respectively, for 297 parameters, remaining difference electron density 0.374 and −0.247 e-Å− 3. 2.8. X-ray structure determination of compound 3 The compound 3 was recrystallized from methanol. C22H30O7S, Mr 438.52, colorless block crystals orthorhombic system, space group P212121, with a = 11.3573 (4) Å, b = 13.2349 (4) Å, c = 13.7885 (5) Å, α = β = γ = 90°, V = 2072.59 (12) Å3, Dcald = 1.405 g/cm3 for Z = 4, F (000) = 936, μ = 1.755 mm − 1. Crystal dimensions: 0.60×0.48×0.36 mm. 15273 reflections measured (θ max=68.18°), 3628 were unique (Rint =0.0386) and of these 3614 had |F|2 ≥2σ|F|2 for which final R1, wR2 (w=1/σ|F|2), and S (goodness-of-fit) values were 0.0302, 0.0783 and 1.067, respectively, for 277 parameters, remaining difference electron density 0.240 and −0.225 e-Å− 3. Data were collected using a Bruker APEX DUO diffractometer using Cu-Kα radiation. Crystal structure solution and refinement were achieved using direct methods as implemented in SHELXL-97 (Sheldrick, University of Gottingen (Germany), 2000) and visualized using the XP program. Crystallographic data (excluding structure factor tables) for 1 and 3 have been deposited with the Cambridge Crystallographic Data Center as supplementary publication NO.CCDC 845970 and NO.CCDC 845971 respectively. Copies of the data

Fig. 1. Molecular structures of 1–3 (1 and 1a stand for a same compound).

1102

Y-Y. Cheng et al. / Fitoterapia 83 (2012) 1100–1104

Table 1 1 H and 13C NMR data for cleidbrevoids A (1), B (2) and C (3).a Position

1 δH, mult, (J in Hz)

δC, type

δH, mult, (J in Hz)

δC, type

δH, mult, (J in Hz)

δC, type

1a 1b 2β 2α 3 4 5 6β 6α 7β 7α 8 9 10 11β 11α 12 13 14 15 16 17 18 19 20 SOMe OMe OH-4

5.92, t (3.7)

120.7, CH

5.73, t (3.8)

119.7, CH

21.9, CH2

2.85, ddd (18.4, 10.0, 3.7) 2.50, m 3.28, dd (10.0, 8.4)

19.6, CH2

2.54, ddd (13.8, 9.0, 3.8) 2.13, m 3.55, t (9.1)

25.2, CH2

2.11, m 1.68, m 1.58, dt (9.6, 3.6) 2.17, m 3.04, t (6.4)

a

1.71, 1.36, 2.47, 2.08, 2.54,

2

ddd (12.8, 7.6, 0.8) dd (12.8, 8.4) m dt (4.0, 2.0) dd (7.6, 4.0)

2.03, dd (14.6, 11.8) 2.60, dd (14.6, 2.8) 5.53, dd (11.8, 2.8)

CH C C CH2

19.3, CH2 45.5, CH 37.9, C 142.6, C 42.9, CH2 71.6, CH 126.7, C 109.6, CH 144.5, CH 140.7, CH 174.6, C 174.7, C 23.2, CH3 34.3, CH3 36.8, CH3 52.7, CH3

6.60, d (1.6) 7.59, t (1.6) 7.65, s

1.17, 1.31, 2.52, 3.68, 4.67,

60.5, 78.9, 43.7, 26.3,

s s s s s

1.74, 1.52, 2.42, 2.04, 2.50,

3

ddd (13.4, 7.6, 2.2) m m m m

2.00, dd (14.6, 11.9) 2.52, m 5.46, dd (11.9, 2.9) 6.56, d (1.6) 7.56, t (1.6) 7.61, s

1.20, 1.26, 2.73, 3.71, 5.43,

NMR data were recorded in CD3COCD3, δ in ppm, 1H NMR at 400 MHz,

s s s s s 13

can be obtained free of charge on application to CCDC, 12 Union Road, Cambridge CB 1EZ, UK [fax: Int. + 44 (0) (1223) 336 033; e-mail: [email protected]]. 2.9. Anti-HIV-1 assay Compounds 1–3 were tested for inhibitory activity against HIV-1 by MTT method [7] and the results were summarized in Table 2. Compound 3 showed weak activity to prevent the cytopathic effects of HIV-1 in C8166 cells with an EC50 of 16.27 μg/mL, and the TI value of more than 11.39. 3. Results and discussion Cleidbrevoid A (1), was obtained as colorless prism crystals (methanol). The molecular formula C22H28O7S, was deduced from its positive HRESIMS at m/z 459.1450 [M+ Na]+ (calcd

Table 2 Anti-HIV-1 activities of compounds 1–3. Compounds

Anti-HIV-1 activity

Cytotoxicity

Therapy index (TI)

EC50 (μg/mL)

CC50 (μg/mL)

CC50/EC50

1 2 3 AZT

84.88 39.28 16.27 0.0032

>200 >200 >200 1139.47

>2.10 >3.62 >11.39 351 688.27

61.1, 82.0, 43.4, 26.3,

CH2 C C CH2

19.6, CH2 45.7, CH 37.8, C 143.4, C 42.8, CH2 71.6, CH 126.7, C 109.6, CH 144.5, CH 140.7, CH 173.4, C 174.5, C 23.9, CH3 34.1, CH3 38.3, CH3 51.9, CH3

1.85, m 1.76, m 2.33, m 2.03, m 2.42, dd (8.8, 5.6) 2.04, m 1.81, dd (15.2, 12.4) 2.35, dd (15.2, 4.4) 5.73, dd (12.4, 4.4) 6.56, dd (2.0, 0.8) 7.55, t (2.0) 7.63, m

1.29, s 1.11, s 2.50, s 3.78, s 4.79, s

28.0, CH2 65.9, 81.4, 44.0, 21.2,

CH C C CH2

19.4, CH2 47.5, 36.3, 44.5, 43.0,

CH C CH CH2

70.8, CH 126.8, C 109.6, CH 144.5, CH 140.9, CH 173.8, C 174.5, C 26.5, CH3 31.2, CH3 41.3, CH3 52.6, CH3

C NMR at 100 MHz.

for C22H28O7SNa, 459.1453) with 9 degrees of unsaturation. Its IR spectrum displayed the absorption bands of hydroxyl (3543 cm− 1), ester carbonyl (1720 cm− 1), and δ-lactone (1705 cm− 1) functionalities. The 1H NMR spectrum (Table 1) showed two tertiary methyl signals (δH 1.17 and δH 1.31), one carboxymethyl signal (δH 3.68) and one methyl signal (δH 2.52). In addition, the signals at δH 7.65 (1H, br. s), 7.59 (1H, t, 1.6), and 6.60 (1H, d, 1.6) were assigned to two α- and one β-position protons of a β-substituted furan moiety. The 13C NMR (Table 1) showed 22 carbon resonances comprising four methyls (including one methoxy), four methylenes, seven methines (one oxygenated and four olefinic ones), and seven quaternary carbons (two ester, two olefinic, and one oxygenated ones), as classified by DEPT and HSQC spectra. The aforementioned evidence indicated that it was a clerodane-type diterpenoid [8]. The 1H– 1H COSY spectrum revealed that 1 possessed four fragments, a (C-1 to C-3), b (C-6 to C-8), c (C-11 to C-12), and d (C-14 to C-15) as shown in Fig. 2. The HMBC correlations of H3-19 to C-4, C-5, C-6, and C-10, H3-20 to C-8, C-9, C-10, and C-11, and H-1 to C-5 established the connectivity of rings A and B. The three rings, two ester groups, and three olefinic bonds accounted for the eight degrees of unsaturation, indicative of the formation of one extra ring (ring C). The furan ring (ring D) was located at C-12 by the HMBC correlations of H-12/C-14 and C-16. The OH proton signal resonance at δH 4.67 (s, 1H) was assigned at C-4 by the HMBC correlation of OH-4 to C-4. In addition, a methoxy group attached to C-18 by HMBC correlations of the methoxyl proton to C-18. The remaining atoms were linked into a methylsulfoxyl group that was supported by IR absorption at 1032 cm − 1 [9]

Y-Y. Cheng et al. / Fitoterapia 83 (2012) 1100–1104

1103

Fig. 2. Selected COSY (bold) and HMBC (arrow) correlations for 1 and 3.

and was located at C-3 from the HMBC correlation of the methylsulfoxyl protons (δH 2.52) to C-3 (δC 60.5) [10]. Therefore, the planar structure of 1 was established. The relative configuration of 1 was subsequently defined by analysis of its ROESY correlations. In ROESY spectrum (Fig. 3), the correlations of H-12/H3-19, H3-19/4-OH, and H3-19/H-3 indicated that H-12, H-3, 4-OH, and H3-19 were co-facial and were assigned as α-oriented, while H-8 and H3-20 were β-oriented based on the ROESY cross-peaks of H-8/H3-20. Finally, the single-crystal X-ray structure of 1

(Fig. 4) by using Flack parameter [0.064(19)] [11] confirmed the supposed structure and determined unambiguously the absolute configuration of 1 as (3S, 4R, 5R, 8R, 9S, 12S), with the (S)-sulfoxide. Cleidbrevoid B (2), had the same molecular formula, C22H28O7S, as 1, based on the HRESIMS spectrum at m/z 459.1460 [M + Na] + (calcd for C22H28O7SNa, 459.1453). The 1 H-NMR and 13C-NMR data (Table 1) of 2 was closely similar to those of 1 except that C-2 and C-4 were downfield shifted (ΔδC +5.6, and +3.1) and C-3 was upfield shifted (ΔδC −0.6),

Fig. 3. Key ROESY (arrow) correlations for 1 and 2.

Fig. 4. X-ray crystal structures of compounds 1 and 3.

1104

Y-Y. Cheng et al. / Fitoterapia 83 (2012) 1100–1104

respectively. The H-3 proton signal of 2 (δH 3.55, t, J = 9.1 Hz) was different from that of 1 (δH 3.28, dd, J = 10.0, 8.4 Hz). The above data indicated 2 and 3 could be a pair of epimers differing at C-3. The ROESY correlation between H-3 and H-2β (δH 2.54, ddd) and the absence of the correlation of H3-19 and H-3 implied that H-3 in 2 took a β-orientation (Fig. 3). Therefore, the structure of compound 2 was determined as shown, and the stereochemistry elucidated as 3R, 4R, 5R, 8R, 9S, 10S, and 12S, with the (S)-sulfoxide. Cleidbrevoid C (3), colorless block crystals, had a molecular formula of C22H30O7S, as determined by the HRESIMS at m/z 461.1602 [M + Na] + (calcd for C22H30O7SNa, 461.1609) with one unsaturation degree less and 2 mass units higher than those of 1 suggested that the Δ 1(10) double bond was hydrogenated. Comparison of the NMR data of 3 with those of 1 (Table 1) indicated that both shared the same basic skeleton. This assignment was confirmed by the presence of a methylene and a methine signal (δC 21.9 and 44.5) in 3, which was further confirmed by the 2D NMR (HSQC, 1H– 1H COSY, and HMBC) (Fig. 2). In the ROESY spectrum, the correlation of H-10/H3-19 showed that they were α-orientation, and the others were identical with those of 1. In order to confirm the relative and absolute structure of compound 3, the single-crystal X-ray structure of 3 by using Flack parameter [0.089(14)] [9] (Fig. 4) revealed its stereochemistry to be 3S, 4R, 5R, 8R, 9S, 10S, and 12S, as well as the configuration of sulfoxide to be S. Acknowledgments This work was financially supported by the National Natural Science Foundation of China (21072199), the Ministry of Science and Technology of China (2009CB522300 and 2009CB940900), the Natural Science Foundation of Yunnan Province (2009CD112), the Foundation of Chinese Academy of Sciences to H. P. He, and the Young Academic and Technical Leader Raising Foundation of Yunnan Province (2010CI047). The authors thank Yu Chen, for the collection and identification of the plant material and Prof. Zheng Yongtang of Kunming Institute of Zoology for his pioneering work on bioactive natural products. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.fitote.2012.04.028. References [1] Institute of Botany. The Chinese Academy of Sciences, Flora Reipublicae Popular is Sinicae, Vol. 44. Beijing: Science Press; 1996. p. 74–5. part 2.

[2] Xie ZW, Yu YC. Index Chinese traditional and herbal drugs. Beijing: People's Medical Publishing House; 1989. p. 371. [3] (a) Lu W, Zou J, Chen J, Zhu Y, Ya Q, Zhao W. Two new compounds from Cleidion brevipetiolatum. Fitoterapia 2007;78:614–6. (b) Ju P, Wang YF, Wang Q, Luo SD. A new ellagitannin from Cleidion bracteosum. Chin Chem Lett 2007;18:291–2. (c) Li C, Zhou H, Yang X, Zhao J, Li L. The chemical study on Cleidion brevipetiolatum. Nat Prod Res Dev 2004;16:514–5. (d) Naengchomnong W, Pinho PM, Kijjoa A, Sawangwong P, Gonzalez MJ, Silva AMS, et al. Clerodanes and other constituents of Cleidion spiciflorum. Phytochemistry 2006;67:1029–33. (e) Li CH, Yang XD, Zhao JF, Chen W, Yang J, Li L. Studies on chemical constituents of Cleidion brevipetiolatum. Yunnan Daxue Xuebao (Ziran Kexueban) 2005;27:249–51. (f) Liu B, Lu W, Ya Q, Chen J. Study on the chemical constituent from Cleidion brevipetiolatum in Guangxi. Guangxi Kexue 2005;12:214–5. [4] (a) Gao S, Liu HY, Wang YH, He HP, Wang JS, Di YT, et al. Lathyranone A: a diterpenoid possessing an unprecedented skeleton from Euphorbia lathyris. Org Lett 2007;9:3453–5. (b) Xiao HT, He HP, Wang YH, Yang XW, Hu XJ, Hao XY, et al. Two new norbisabolane sesquiterpenoid glycosides from Glochidion coccineum. Asian Nat Prod Res 2008;10:1–5. (c) Xiao HT, Hao XY, Yang XW, Wang YH, Lu Y, Zhang Y, et al. Bisabolane-type sesquiterpenoids from the rhizomes of Glochidion coccineum. Helv Chim Acta 2007;90:164–70. (d) Tan CJ, Di YT, Wang YH, Zhang Y, Si YK, Zhang Q, et al. Three new indole alkaloids from Trigonostemon lii. Org Lett 2010;12:2370–3. (e) Hu XJ, Wang YH, Kong LY, He HP, Gao S, Liu HY, et al. New phenanthrenes from Trigonostemon lii Y.T. Chang. Tetrahedron Lett 2009;50:2917–9. (f) Li SF, Di YT, Li SL, Zhang Y, Yang FM, Sun QY, et al. Trigonosins A–F, daphnane diterpenoids from Trigonostemon thyrsoideum. J Nat Prod 2011;74:464–9. (g) S.F.Li SF, Y.T.Di YT, H.P.He HP, Y.Zhang Y, Y.H.Wang YH, J.L.Yin JL et al. Trigonoines A and B, two novel alkaloids from the leaves of Trigonostemon lii. Tetrahedron Lett 2011;52:3186–8. [5] P.Li P, T.Cesca T, M.L.D.Claudio MLD, R.Nirmala R, C.G.Judith CG, R.S.Abhay RS. Cordifolide A, a sulfur-containing clerodane diterpene glycoside from Tinospora cordifolia. Org Lett 2012;14:2118–21. [6] Toshiaki K, Naoki A, Hiroyuki N, Nariyasu M, Taro T, Ryota T, et al. Isolation of a diterpenoid substance with potent neuroprotective activity from fetal calf serum. Proc Natl Acad Sci U S A 2002;99:3288–93. [7] (a) Zheng YT, Zhang F, Ben KL, Wang JH. In vitro immunotoxicity and cytotoxicity of trichosanthin against uuman normal immunocytes and leukemia-lymphoma cells. Immunopharmacol Immunotoxicol 1995;17:69. (b) Zheng YT, Ben KL, Jin SW. Alpha-momorcharin inhibits HIV-1 replication in acutely but not chronically infected T-lymphocytes. Acta Pharmacol Sin 1999;20:239. [8] L.Mambu L, V.Ramanandraibe V, M.T.Martin MT, A.Blond A, P.Grellier P, F.Frappier F. Constituents of Burasaïa madagascarensis: a new clerodane-type diterpene. Planta Med 2002;68:377–9. [9] (a) Kubec R, Musah RA. Cysteine sulfoxide derivatives in Petiveria alliacea. Phytochemistry 2001;58:981–5. (b) Tang GH, Chen DM, Qiu BY, Sheng L, Wang YH, Hu GW, et al. Cytotoxic amide alkaloids from Piper boehmeriaefolium. J Nat Prod 2011;74:45–9. [10] R.Kawecki R. New, recoverable and highly effective sulfinyl chiral auxiliary. Tetrahedron: Asymmetry 2003;14:2827–32. [11] Flack HD. On enantiomorph-polarity estimation. Acta Crystallogr Sect A 1983;39:876–81.