Semisynthesis of (+)- and (−)-goniomitine from (−)- and (+)-vincadifformine

Tetrahedron 69 (2013) 1622e1627

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Semisynthesis of (þ)- and ()-goniomitine from ()- and (þ)-vincadifformine ve Aubert c, Thierry Cresteil c Guy Lewin a, *, Guillaume Bernadat b, Genevie ˇ

Laboratoire de Pharmacognosie (Univ. Paris-Sud BIOCIS UMR-8076 CNRS LabEx LERMIT), Facult e de Pharmacie, av. J.B. Cl ement, 92296 Chatenay-Malabry Cedex, France Mol ecules, Fluor ees et Chimie M edicinale (Univ. Paris-Sud BIOCIS UMR-8076 CNRS LabEx LERMIT), Facult e de Pharmacie, av. J.B. Cl ement, 92296 Chatenay-Malabry Cedex, France c ICSN, CNRS-UPR 2301, LabEx LERMIT 91190 Gif-sur-Yvette, France a

ˇ

b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 5 October 2012 Received in revised form 16 November 2012 Accepted 26 November 2012 Available online 1 December 2012

The first biomimetic semisynthesis of goniomitine has been accomplished in nine steps with 11% overall yield starting from vincadifformine. Natural ()- and unnatural (þ)-goniomitine were prepared from (þ)- and ()-vincadifformine, respectively. The evaluation of the antiproliferative effect of both enantiomers proved unnatural (þ)-goniomitine to be more potent, but this enantiomer as well as some close derivatives displayed a moderate activity only. Ó 2012 Published by Elsevier Ltd.

Keywords: Goniomitine Indole alkaloids Vincadifformine Aspidospermane Semisynthesis

1. Introduction (20R,21S)-()-Goniomitine 1 is an indole alkaloid of an unusual structural type, isolated from the root bark of Gonioma malagasy Markgr & Boiteau (Apocynaceae) in 1987 by Husson and coworkers who proposed a biogenetic filiation between 1 and Aspidosperma alkaloids, regarded as its precursors.1,2 According to this hypothesis depicted in Fig. 1, an Aspidosperma alkaloid such as vincadifformine 2 would lead to goniomitine by the following successive following steps: (a) oxidative fission of the C-5, N-4 bond; (b) decarboxylation; (c) retro-Mannich reaction providing the key intermediate C-21eN-4 iminium ion through the cleavage of C-7, C-21 bond; (d) final nucleophilic attack of the resulting indole nitrogen on this iminium. Starting from the absolute configuration of the Aspidosperma alkaloids found in the same plant, Husson et al. assigned to ()-goniomitine the enantiomeric structure having the 20S,21R configuration. However, the first enantiocontrolled total synthesis of ()-goniomitine was accomplished by Takano et al. in 1991,3 and proved unambiguously the absolute stereochemistry to be 20R,21S. This result was confirmed in 2011 by Mukai et al. who reported for the first time the total synthesis of both enantiomers, natural (20R,21S)-()-goniomitine and unnatural (20S,21R)-(þ)-goniomitine.4

* Corresponding author. Tel.: þ33 146835593; fax: þ33 146835399; e-mail addresses: [email protected], [email protected] (G. Lewin). 0040-4020/$ e see front matter Ó 2012 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.tet.2012.11.084

Fig. 1. Natural ()- and unnatural (þ)-goniomitine and Husson’s hypothesis for biogenesis of 1.2

G. Lewin et al. / Tetrahedron 69 (2013) 1622e1627

In 1995 the first biomimetic entry in the goniomitine skeleton was achieved in our laboratory. This consisted of the five-step synthesis of compound 3 from ()-vincadifformine 2.5 Our method largely followed the Husson biogenetic hypothesis to account for the formation of 1 from aspidospermane skeleton. Unfortunately, attempts to prepare unnatural (þ)-goniomitine itself from 3 were unsuccessful, as this conversion came up against the loss of the extra carbon unit of the methoxycarbonyl group. However, a close derivative, 16-hydroxymethyl-goniomitine 4, could be described (Scheme 1).6 Recent reports in the literature of three total syntheses of gonomitine,4,7e9 and mention of its interesting antiproliferative activity prompted us to reinvestigate synthesis of (þ)-goniomitine from ()-vincadifformine.

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(-)-vincadifformine 2

5 steps

3

22% NaBH3CN in AcOH, rt OH

H HO N

N

CO2CH3

8 catalytic or transfer hydrogenation

TiCl3 in MeOH, rt 79% from 3

OH

OH

H HN

N

CO2CH3

N

H HO N

9

10 71%

H HN

N

CO2CH3

Pd-C, HCO2NH4 in MeOH, reflux, 45 min OH 4N HCl, 100°C CO2CH3

11 ( CO2CH3)-12 ( CO2CH3) in a 4/1 ratio

90%

OH

R

(R = H)

N

H N

(+)-1 (90%)

N-methylation 43% (R = CH3) 19

Scheme 2. Synthesis of (þ)-goniomitine from ()-vincadifformine.

Scheme 1. Previous first semisynthesis of a goniomitine derivative.5

2. Results and discussion Focus was made on the conditions of decarbomethoxylation, an essential step to achieve access to goniomitine. Decarbomethoxylation has been successively envisaged before and after the rearrangement key step of 5 into 3. Our previous study had shown that the decarbomethoxylation could be carried from 5 by a two-step process: (a) treatment of 5 by sodium iodide in acetic acid giving 6 through recovery of the anilinoacrylate ester chromophore; (b) acid hydrolysis of 6 to indolenine 7 by heating in strong acid medium (11 N HCl for 10 min at 105  C) via simultaneous m-chlorobenzoate ester hydrolysis and decarbomethoxylation.5 The low yield of this last step led us to test on compound 6 all the decarbomethoxylation conditions reported up to date with Aspidosperma alkaloids having an anilinoacrylate ester chromophore.10 Unfortunately, these methodsdacid hydrolysis with aqueous HCl (1 Ne6 N) at 100  C, such as saponification (reflux with 0.5 N KOH in EtOH)dnever provided clean reactions. Then we turned to the second approach and examined conditions of decarbomethoxylation after the rearrangement step into the goniomitine skeleton (Scheme 2).

Conversion of 3 into goniomitine requires reduction of aldehyde and hydroxyamine groups into primary alcohol and aminal functions, respectively, hydrogenation of C-16, C-17 bond and decarbomethoxylation. Our previous attempt to provide goniomitine had begun by reduction of 3 to the alcohol 8 (NaBH3CN in AcOH for 3 h at rt), which was then submitted to hydrogenation of the olefin before reduction of the hydroxyamine group. In our opinion, the remaining hydroxyamine group would prevent a concomitant reduction of the aminal function with cleavage of N-1, C-21 bond during the C-16, C-17 hydrogenation step. Unfortunately, 8 was proved to be almost unreactive under catalytic (H2, PdeC 10% in MeOH, rt, 1 atm, 15 h) or transfer (PdeC 10% in MeOH, reflux, 5 h) hydrogenation and did not provide the saturated esters 9. Moreover, decarbomethoxylation of 8 was also unsuccessful: saponification of 8 was easily achieved, but decarboxylation of the a,b-unsaturated carboxylic acid was ineffective (pyrolysis or copperequinoline procedure led only to a complex mixture). Reinvestigation of the synthesis of goniomitine from 3 led us to modify the order of the reactions. Since the removal of aldehyde seems more logical at the first step of the sequence (to prevent the formation of an intermediate having both aldehyde and secondary amine groups), the only possible change consisted in carrying out hydroxyamine reduction before C-16, C-17 hydrogenation. Treatment of 8 with TiCl3 in 2 N aqueous HCl (3 h at rt in MeOH) gave in 79% yield the aminal 10, which provided, by transfer hydrogenation (PdeC, ammonium formate in MeOH for 45 min at 100  C), a mixture 4:1 of 11 and 12 (71% yield). After separation of these compounds from an aliquot of the mixture by TLC (silica gel, CH2Cl2/ MeOH, 97:3), their spectral analysis proved 11 (major) and 12 (minor) to be epimers at C-16 resulting from 10 by hydrogenation of the olefin, without concomitant reductive cleavage of the aminal

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(gain of 2 um for molecular peak and persistence of the H-21 singlet in the 1H NMR spectrum). Configuration at C-16 of each epimer was inferred from NOE experiments and was fully consistent with a major addition of the hydrogens from the less hindered side of the molecule. Lastly, heating the mixture 11,12 in aqueous acid medium (4 N HCl for 1 h at 100  C) provided in 90% yield expected (þ)-goniomitine through hydrolysis of the ester function then an easy decarboxylation step assisted by the neighboring N-1, C-2 iminium.11 A transient cleavage of the N-1, C-21 aminal bond is probable, but without consequence on the final stereochemistry at C-21, since the cis ring fusion is known to fit with the thermodynamically more stable isomer (1H and 13C NMR spectra of our final compound exactly matched data of the literature for goniomitine).3 So, this reinvestigation displayed that the simple reverse of two steps has dramatically modified the course of reactions, and allowed for an easy access to (þ)-goniomitine. Connected to our previously described five-step conversion of ()-vincadifformine 2 into compound 3 (22%), these results represent the first biomimetic access to (þ)-goniomitine from an Aspidosperma precursor (11% total yield). This same nine-step sequence was then applied to (þ)-vincadifformine,12 which allowed the first semisynthesis of natural ()-goniomitine. Two questions arise from this study: the first about the highly different behaviors of hydroxyamine 8 and aminal 10 toward hydrogenation; the second related to the absolute configuration of natural ()-goniomitine. Regarding both compounds 8 and 10, a large conjugated p system might involve the indolic core, the double bond, and the carbonyl group of the ester. However, the fact that the frequency for the IR carbonyl stretch band remains almost unchanged after hydrogenation (a shift by a mere 2e3 cm1 is observed) suggests that the ester moiety is actually not conjugated with the rest of this p system. Intrigued by this finding, we decided to carry out theoretical investigations on compounds 8 and 10 at the B3LYP/6-31G* level (Fig. 2 and Supplementary data). Geometries found for these molecules are very similar. In particular, both structures display a same OeCeC-16eC-17 dihedral angle (approximately 40 ) between respective planes of the vinylindolic system and the methoxycarbonyl ester group, counteracting possibilities of conjugation, which is consistent with unchanged IR carbonyl band frequencies before and after hydrogenation. This lack of planarity seems due to steric factors, independently of a possible hydrogen bond with the close primary alcohol. Equatorial form of the hydroxyamine group in compound 8 seems favored, but exhibits no steric hindrance of the double bond (our first hypothesis). Even in the less stable axial form, the hydroxyamine group remains far from the double bond (O and C-16 or C-17 atoms are separated by 3.11 and 3.06  A respectively) and is unlikely to impact hydrogenation. This lack of interaction is supported by comparison of the chemical shifts of H-17 and C-17 signals in 8

(6.63 and 143.1 ppm) and 10 (6.65 and 142.7 ppm). HOMO and LUMO isodensity surface plots clearly show the conjugated p system (see Supplementary data) but these frontier molecular orbitals and their respective energy levels are found to be highly similar in both compounds. Estimated heats of hydrogenation do not significantly differ either. On the basis of these results, it was difficult to hypothesize a reasonable substrate-dependent explanation for this astonishing difference of reactivity between compounds 8 and 10. About the second point, natural goniomitine 1 was discovered from G. malagasy along with, particularly, five alkaloids, which are all issued from a same biogenetic secodine 13 type intermediate (Fig. 3): quebrachamine 14; aspidofractinine 15 and kopsinine 16, with an Aspidosperma skeleton; eburnamine 17 and eburnamenine 18, with an eburnane skeleton, well known to be derived from the aspidospermane skeleton.13 For quebrachamine, considered by Hesse as a probable biogenetic precursor of vincadifformine according to a biogenetic-chemotaxonomic interpretation,14 the reported [a]D 61 (c 0.8, CHCl3),13 far from literature data,15 could suggest a partial racemate with one enantiomer in excess. Comparison of absolute configuration for 15e18 indicates that aspidospermane alkaloids on the one hand, and eburnane alkaloids on the other hand, belong to the two different optical series. So, the apparently surprising occurrence in G. malagasy of both opposite optical series for alkaloids resulting from a same biogenetic scheme illustrates very well the theory that Hesse proposed for vincadifformine and closely related alkaloid biogenesis.14 According to Hesse’s conclusion, it may be conceivable that G. malagasy contains both active enzyme systems, which lead to both vincadifformine enantiomers. However, subsequent transformations would operate only in a stereospecific manner, giving aspidospermane alkaloids, 15,16 from ()-vincadifformine, and ()-goniomitine 1 and eburnane alkaloids 17 and 18 from the other enantiomer. From a biological point of view, evaluation of the antiproliferative effect of (þ)- and ()-goniomitine was undertaken on five human cancer cell lines (Table 1). A weak antiproliferative activity was observed in all cell lines at 105 M, in agreement with the updated IC50 values of ()-goniomitine reported by Waser et al. with the same cell lines in a corrigendum of their initial

N

N 21 20

N H

CO2CH3

N H

19 18

quebrachamine 14

secodine 13

20R -(-); 20S -(+) N

N H

N

H

1819

H

N H CO2CH3

aspidofractinine 15

H N H HO

21

N

kopsinine 16

H N

N

20

19 18

eburnamine 17 Fig. 2. Calculated geometry (a) and HOMO isodensity surface (b) at 50% level for compound 8.

eburnamenine 18

Fig. 3. Aspidospermane-eburnane alkaloids isolated from G. malagasy.13

G. Lewin et al. / Tetrahedron 69 (2013) 1622e1627 Table 1 Antiproliferative activity of ()-, (þ)-goniomitine 1 and analogs Substrate

()-1 (þ)-1 3 4 8 10 11 12 19

% Inhibition growth at 105 M on several cell lines KB

A549

PC3

MCF7

HCT116

04 692 03 825 305 231 04 06 25

314 595

162 431

1510 625

313 633

communication.16 It is noteworthy that antiproliferative activity was definitively lost at 106 M. Unnatural (þ)-goniomitine displayed in the five cell lines a better activity than natural ()-goniomitine in opposition to Mukai et al.’s results on a canine kidney cell line (MDCK II), reporting a very low antiproliferative activity with IC50 ranging 67e161 mM for individual enantiomers and surprisingly 37 mM for the racemic mixture.4 To date we have no explanation for that discrepancy, except differences in cell lines used for this biological exploration.4 Then the evaluation was extended to derivatives of the most active enantiomer (þ)-1, intermediate compounds 3, 8, 10e12, and analogs 4 (previously synthesized) and 19 resulting from methylation (CH2O, NaBH3CN in AcOH, rt) of (þ)-1. As depicted in Table 1, structureeactivity relationships within goniomitine series are difficult to establish: except methylation at N-4, which clearly appears detrimental to the activity (19 vs (þ)-1), the influence of other groups cannot be easily pointed out: so, a C16 substitution can increase (4) or decrease (11 and 12) the antiproliferative effect. Lastly, in spite of 16-hydroxymethyl derivative (4) being slightly more potent than (þ)-1, our study shows that the goniomitine series displays a weak activity in the antiproliferative agent field. 3. Conclusion This study describes the first semisynthesis of both enantiomers of goniomitine. Unnatural (þ)-goniomitine, which showed the best (though moderate) antiproliferative activity, was prepared from ()-vincadifformine 2 by a nine-step synthesis in 11% yield. Compound 2 is directly available from its 14,15-dehydro analog, ()-tabersonine, which is the major alkaloid from seeds of Voacanga africana Stapf. Our process thus constitutes an original access to (þ)-goniomitine, from a readily available precursor, while it could only be obtained by total synthesis so far. This biomimetic approach exemplifies the interest of biogenesis knowledge for the choice of a starting material for organic synthesis. 4. Experimental 4.1. General experimental procedures Melting points were determined with a micro-Koffler apparatus and are uncorrected. Optical rotation measurements were conducted using an Optical Activity PolAAr 32 polarimeter. 1H and 13C NMR spectra were recorded in deuterated chloroform on a Bruker AC-300 (300 MHz) or a Bruker AM-400 (400 MHz) instrument; NOESY and 1He13C (HMQC and HMBC) experiments were performed with a Bruker AM-400. IR spectra were recorded on Vector 22 Bruker spectrometer and values are reported in cm1 units. Mass spectra (ESIMS) were recorded on Esquire-LC Bruker 00040 spectrometer (ESI source) and high resolution mass spectra (HRMS)

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were obtained using a Finnigan MAT 95Q instrument. Flash chromatographies were performed with silica gel 60 (9385 Merck) or aluminum oxide 90 (1097 Merck). Preparative TLC was performed with 60F254 silica gel (5715 Merck). 4.1.1. (20S,21R)-16,17-Dehydro-16-methoxycarbonyl-goniomitine (10). To a solution of 3 (368 mg, 1 mmol) in AcOH (50 mL), NaBH3CN (360 mg) was added and the mixture was kept at room temperature for 2.5 h. The solution was diluted with iced water, treated with 2 N aqueous NaOH until pH 6, and extracted three times with CH2Cl2. The organic layer was washed with water, dried over Na2SO4, filtered, and evaporated. The quantitative dried residue [homogeneous in TLC (silica gel CH2Cl2/MeOH 98:2)] was dissolved in MeOH (50 mL), added with TiCl3 (1.55 M solution in 2 M hydrochloric acid, 2 mL, 3.1 mmol), then the mixture was kept under nitrogen for 20 h at room temperature. The solution was diluted with iced water, treated with NaHCO3 until pH 6, and extracted three times with CH2Cl2. The organic layer was washed with water, dried over Na2SO4, filtered, and evaporated. The dried residue was purified by flash chromatography on alumina (CH2Cl2/ MeOH 99.5:0.5) and yielded 10 as a pure amorphous compound (278 mg, 79%). Amorphous; 1H NMR (400 MHz, CDCl3) d 7.63 (d, J¼8.0 Hz, 1H, H-9), 7.34 (d, J¼8.2 Hz, 1H, H-12), 7.25 (m, 1H, H-11), 7.10 (m 1H, H-10), 6.65 (s, 1H, H-17), 4.83 (s, 1H, H-21), 3.93e3.89 (m, 2H, 2H-5), 3.90 (s, 3H, OCH3), 3.15e3.02 (m, 3H, 2H-6 and 1H3), 2.79e2.72 (m, 1H, 1H-3), 2.18e2.13 (m, 1H, 1H-15), 1.70e1.63 (m, 1H, 1H-14), 1.61e1.48 (m, 2H, 1H-14 and 1H-15), 1.37e1.28 (m, 1H, 1H-19), 1.23e1.16 (m, 1H, 1H-19), 0.77 (t, J¼7.6 Hz, 3H, 3H-18). 13C NMR (100 MHz, CDCl3) 166.8 (CO2CH3), 142.7 (C-17), 135.8 (C-13), 129.0 (C-8), 126.2 (C-2), 123.4 (C-11), 119.8 (C-9 and C-10), 111.3 (C7), 108.2 (C-12), 69.3 (C-21), 63.0 (C-5), 52.3 (CO2CH3), 44.8 (C-3), 41.2 (C-20), 33.7 (C-15), 31.5 (C-19), 28.4 (C-6), 23.9 (C-14), 7.7 (C18); C-16 not detected. IR 3480e3260, 1721, 1599 cm1. ESIMS(þ) m/z 355 [MþH]þ; HRMS calcd for C21H26N2O3 354.1937, found 354.1942. 4.1.2. (16S,20S,21R)-16-Methoxycarbonyl-goniomitine (11) and (16R,20S,21R)-16-methoxycarbonyl-goniomitine (12). A solution of 10 (250 mg, 0.7 mmol) in MeOH (50 mL) was added with ammonium formate (252 mg, 4 mmol) and 10% PdeC (250 mg) then stirred at reflux under nitrogen for 45 min. The reaction mixture was filtered then diluted with water. Extraction of the aqueous phase with CH2Cl2 (three times), then a standard work-up of the organic phase led to a dried residue (216 mg). Purification by flash chromatography on alumina (CH2Cl2/MeOH, 99.75:0.25) provided a mixture of 11 and 12 (177 mg, 71%). TLC on silica gel (CH2Cl2/ MeOH 97:3) of an aliquot (20 mg) of this mixture led to isolation of 11 (12 mg) and 12 (3 mg) as pure amorphous compounds. Compound 11. Amorphous; 1H NMR (400 MHz, CDCl3) d 7.58 (d, J¼7.9 Hz, 1H, H-9), 7.32 (d, J¼8.1 Hz, 1H, H-12), 7.19 (m, 1H, H-11), 7.11 (m 1H, H-10), 4.77 (s, 1H, H-21), 4.18 (dd, J¼8.2 and 1.3 Hz, H-16), 3.94e3.82 (m, 2H, 2H-5), 3.72 (s, 3H, OCH3), 2.96e2.81 (m, 4H, 1H3, 2H-6 and 1H-17), 2.74 (m, J¼11.8 and 2.8 Hz, 1H, 1H-3), 2.06 (d, J¼14.5, 1H, H-17), 1.87e1.80 (m, 1H, 1H-15), 1.73e1.62 (m, 2H, 1H-14 and 1H-19), 1.55e1.40 (m, 2H, 1H-14 and 1H-15), 1.32e1.23 (m, 1H, 1H-19), 0.82 (t, J¼7.5 Hz, 3H, 3H-18). 13C NMR (100 MHz, CDCl3) 174.2 (CO2CH3), 135.9 (C-13), 129.3 (C-8), 128.8 (C-2), 121.6 (C-11), 119.9 (C-10), 119.1 C-9), 110.7 (C-7), 108.7 (C-12), 71.7 (C-21), 62.1 (C-5), 52.4 (CO2CH3), 45.3 (C-3), 36.7 (C-16), 35.4 (C-20), 33.5 (C15), 28.9 (C-19), 28.3 (C-6), 26.0 (C-17), 21.3 (C-14), 6.8 (C-18). IR 3480e3380, 1723 cm1. ESIMS(þ) m/z 357 [MþH]þ; HRMS calcd for C21H28N2O3 356.2093, found 356.2097. Compound 12. Amorphous; 1 H NMR (400 MHz, CDCl3) d 7.56 (d, J¼7.8 Hz, 1H, H-9), 7.34 (d, J¼8.1 Hz, 1H, H-12), 7.19 (m, 1H, H-11), 7.10 (m 1H, H-10), 4.77 (s, 1H, H-21), 4.08 (dd, J¼10.0 and 8.0 Hz, H-16), 3.90e3.78 (m, 2H, 2H-5), 3.77 (s, 3H, OCH3), 3.18e3.11 (m, 1H, H-3), 2.94e2.89 (m, 2H, 2H-6),

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2.86e2.75 (m, 2H, 1H-3 and 1H-17), 1.87e1.70 (m, 3H, 1H-14, 1H-15, 1H-17), 1.58e1.43 (m, 2H, 1H-14 and 1H-15), 1.28e1.13 (m, 1H, 1H19), 1.10e1.00 (m, 1H, 1H-19), 0.82 (t, J¼7.6 Hz, 3H, 3H-18). 13C NMR (100 MHz, CDCl3) 174.9 (CO2CH3), 135.4 (C-13), 128.5 (C-8)*, 128.3 (C-2)*, 121.6 (C-11), 119.7 (C-10), 118.7 C-9), 108.5 (C-12), 108.3 (C-7), 71.2 (C-21), 62.4 (C-5), 52.6 (CO2CH3), 45.5 (C-3), 37.9 (C-16), 35.9 (C-20), 33.6 (C-15), 29.3 (C-19), 28.0 (C-6), 26.9 (C-17), 21.5 (C-14), 7.1 (C-18); *interchangeable assignments. IR 3480e3380, 1724 cm1. ESIMS(þ) m/z 357 [MþH]þ; HRMS calcd for C21H28N2O3 356.2093, found 356.2098. 4.1.3. (þ)-(20S,21R)-Goniomitine (1). A solution of the mixture 11,12 (153 mg, 0.43 mmol) in 4 N aqueous hydrochloric acid (7 mL) was stirred at 100  C under nitrogen for 1 h. The reaction mixture was diluted with iced water, carefully brought to pH 7 with 2 N aqueous NaOH, then extracted three times with CH2Cl2. Standard work-up of the organic phase then filtration on silica gel (CH2Cl2/ MeOH, 97:3) led to a dried crystallized residue of pure (þ)-goniomitine 1 (115 mg, 90%). Colorless crystals: mp 141e143  C (lit.4 mp 140e143  C); [a]D þ75.8 (c 0.22, CHCl3) [lit.4 [a]D þ72.9 (c 0.05, CHCl3)]. 1H NMR (400 MHz, CDCl3) d 7.52 (d, J¼7.6 Hz, 1H, H-9), 7.29 (d, J¼7.9 Hz, 1H, H-12), 7.14 (m, 1H, H-11), 7.08 (m 1H, H-10), 4.79 (s, 1H, H-21), 3.83 (t, J¼6.5 Hz, 2H, 2H-5), 3.08e2.98 (m, 2H, 1H-3 and 1H-16), 2.94 (td, J¼6.4 and 2.2 Hz, 2H, 2H-6), 2.90e2.75 (m, 2H, 1H3 and 1H-16), 2.57e2.48 (m, 1H, 1H-17), 1.93e1.84 (m, 1H, 1H-15), 1.75e1.65 (m, 1H, 1H-14), 1.64e1.55 (m, 1H, 1H-19), 1.55e1.43 (m, 3H, H-14, H-15 and H-17), 1.26e1.16 (m, 1H, H-19), 0.88 (t, J¼7.5 Hz, 3H, 3H-18). 13C NMR (100 MHz, CDCl3) 135.4 (C-13), 132.8 (C-2), 129.1 (C-8), 120.6 (C-11), 119.6 (C-10), 118.1 C-9), 108.3 (C-12), 106.0 (C-7), 71.6 (C-21), 62.6 (C-5), 45.6 (C-3), 35.1 (C-20), 34.0 (C-15), 28.6 (C-19), 27.7 (C-6), 21.7 (C-14)*, 21.6 (C-17)*, 18.6 (C-16), 7.1 (C18); interchangeable assignments. ESIMS(þ) m/z 299 [MþH]þ; HRMS calcd for C19H26N2O 298.2039, found 298.2043. 4.1.4. ()-(20R,21S)-Goniomitine (1). Colorless crystals (from ether): mp 145e147  C (lit.1 mp 150  C; lit.3 mp 149e150  C; lit.4 mp 144e145  C); [a]D 78.9 (c 0.22, CHCl3) [lit.1 [a]D 80 (c 0.9, CHCl3); lit.3 [a]D 87.1 (c 0.42, CHCl3); lit.4 [a]D 78.1 (c 0.14, CHCl3)]. 4.1.5. (20S,21R)-4-Methyl-goniomitine (19). To a solution of (þ)-1 (20 mg, 0.067 mmol) in AcOH (1.5 mL), aqueous 35% formaldehyde (0.25 mL) and NaBH3CN (20 mg) were successively added, then the mixture was kept at room temperature for 3 h. The solution was diluted with iced water, treated with 2 N aqueous NaOH until pH 6, and extracted three times with CH2Cl2. The organic layer was washed with water, dried over Na2SO4, filtered, and evaporated. The dried residue was purified by TLC on silica gel (CH2Cl2/MeOH 98:2) and yielded 19 as a pure amorphous compound (9 mg, 43%). Amorphous; 1H NMR (400 MHz, CDCl3) characteristic peaks at d 7.52 (d, J¼7.6 Hz, 1H, H-9), 7.37 (d, J¼7.9 Hz, 1H, H-12), 7.17e7.07 (m, 2H, H-10 and H-11), 4.06 (s, 1H, H-21), 3.85 (t, J¼6.5 Hz, 2H, 2H5), 0.72 (t, J¼7.5 Hz, 3H, 3H-18). 13C NMR (100 MHz, CDCl3) 137.4, 133.8, 128.3, 120.2, 119.2, 117.8, 109.1, 77.7, 62.7, 56.1, 43.4, 37.7, 33.5, 30.1, 27.7, 23.4, 21.1, 18.0, 7.5; one carbon (C-7) not detected. ESIMS(þ) m/z 313 [MþH]þ; HRMS calcd for C20H28N2O 312.2195, found 312.2189. 4.2. Computational methods Conformations of the reactants and products were optimized without constraint using DFT17 method with the hybrid Becke3LYP functional18 and the 6-31G* base19 as implemented in the Gaussian 09 software package.20 Vibrational analysis was performed at the same level of theory upon geometrical optimization convergence and local minima were characterized by the absence of imaginary

frequency. Thermodynamic quantities at 298.15 K were calculated using the zero-point and thermal energy corrections derived from unscaled frequencies. Figures are rendered with UCSF Chimera.21 4.3. Biological evaluation Cell culture. Human cell lines were purchased from ATCC or ECACC or obtained from the NCI. The human cell line KB was cultured in D-MEM medium supplemented with 10% fetal calf serum, in the presence of penicillin, streptomycin, and fungizone in 75 cm2 flasks under 5% CO2, whereas all other cell lines were cultured in complete RPMI medium. Cell proliferation assay. Cells (600 cells/ well) were plated in 96-well tissue culture microplates in 200 mL of medium and treated 24 h later with compounds dissolved in DMSO at concentrations that ranged 0.5 nM to 10 mM with a Biomek 3000 automation workstation (Beckman-Coulter). Control cells received the same volume of DMSO (1% final volume). After 72 h exposure to the drug, MTS reagent (Celltiter 96AQeous One, Promega) was added and incubated for 3 h at 37  C. Experiments were performed in triplicate: the absorbance was monitored at 490 nm and results were expressed as the inhibition of cell proliferation calculated as the ratio [(1(OD490 treated/OD490 control))100]. Acknowledgements We thank J.-C. Jullian and E. Morvan for NMR measurements, the  Paris-Sud for providing computing IT department from Universite resources and A. Pearson for English corrections of the manuscript. Supplementary data Copies of 1H and 13C NMR spectra (with HSQC, HMBC, NOESY) allowing assignments of all signals and computational results. Supplementary data associated with this article can be found in the online version, at http://dx.doi.org/10.1016/j.tet.2012.11.084. References and notes 1. Randriambola, L.; Quirion, J. C.; Kan-Fan, C.; Husson, H. P. Tetrahedron Lett. 1987, 28, 2123e2126. 2. Biogenetic numbering system proposed by: Le Men, J.; Taylor, W. I. Experientia 1965, 21, 508e510. 3. Takano, S.; Sato, T.; Inomata, K.; Ogasawara, K. J. Chem. Soc., Chem. Commun. 1991, 462e464. 4. Mizutani, M.; Inagaki, F.; Nakanishi, T.; Yanagihara, C.; Tamai, I.; Mukai, C. Org. Lett. 2011, 13, 1796e1799. 5. Lewin, G.; Schaeffer, C.; Lambert, P. H. J. Org. Chem. 1995, 60, 3282e3287. 6. Lewin, G.; Schaeffer, C. Nat. Prod. Lett. 1995, 7, 227e234. 7. Morales, C. L.; Pagenkopf, B. L. Org. Lett. 2008, 10, 157e159. 8. De Simone, F.; Gertsch, J.; Waser, J. Angew. Chem., Int. Ed. 2010, 49, 5767e5770. jicek, J. Collect. 9. (a) For a review on syntheses of goniomitine till 2011, see: Ha Czech. Chem. Commun. 2011, 76, 2023e2083; (b) For a very recent synthesis of goniomitine, see: Jiao, L.; Herdtweck, E.; Bach, T. J. Am. Chem. Soc. 2012, 134, 14563e14572. 10. (a) Zsadon, B.; Otta, K. Acta Chim. Hung. 1971, 69, 87e95; (b) Hajicek, J.; Trojanek, J. Tetrahedron Lett. 1981, 22, 1823e1826; (c) Blowers, J. W.; Saxton, J. E.; Swanson, A. G. Tetrahedron 1986, 42, 6071e6095; (d) Brennan, J. P.; Saxton, J. E. Tetrahedron 1986, 42, 6719e6734; (e) Pilarcik, T.; Havlicek, J.; Hajicek, J. Tetrahedron Lett. 2005, 46, 7909e7911. 11. (a) Bennasar, M.-L.; Alvarez, M.; Lavilla, R.; Zulaica, E.; Bosch, J. J. Org. Chem. 1990, 55, 1156e1168; (b) Noland, W. E.; Xia, G.-M.; Gee, K. R.; Konkel, M. J.; Wahlstrom, M. J.; Condoluci, J. J.; Rieger, D. L. Tetrahedron 1996, 52, 4555e4572. 12. Thanks to Dr. J. Hannart (Omnichem Belgium) for the precious gift of (þ)-vincadifformine.  Paris-Sud: Orsay, France, 1987. 13. Randriambola, L. Ph.D. dissertation. Universite 14. Hesse, M. Alkaloids Nature’s Curse or Blessing?; Wiley-VCH: Weinheim, 2002, pp 189e194; It is noteworthy that previous studies ( Scott, A. I. Acc. Chem. Res. 1970, 3, 151e157 ) rather suggest for vincadifformine and quebrachamine skeletons distinct biogenetic pathways from a same secodine type intermediate; For a recent review on indole alkaloid biosynthesis, see O’Connor, S.; Maresh, J. J. Nat. Prod. Rep. 2006, 23, 532e547. 15. Amat, M.; Lozano, O.; Escolano, C.; Molins, E.; Bosch, J. J. Org. Chem. 2007, 72, 4431e4439. 16. De Simone, F.; Gertsch, J.; Waser, J. Angew. Chem., Int. Ed. 2011, 50, 4038.

G. Lewin et al. / Tetrahedron 69 (2013) 1622e1627 17. (a) Kohn, W.; Sham, L. J. Phys. Rev. 1965, 140, A1133eA1138; (b) Hohenberg, P.; Khon, W. Phys. Rev. 1964, 136, B864eB871. 18. (a) Becke, A. D. J. Chem. Phys. 1993, 98, 5648e5652; (b) Lee, C.; Yang, W.; Parr, R. G. Phys. Rev. B 1988, 37, 785e789. 19. Hehre, W. J.; Radom, L.; Schleyer, P. v. R.; Pople, J. A. Ab Initio Molecular Orbital Theory; Wiley: New York, NY, 1986. 20. Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci, B.; Petersson, G. A.; Nakatsuji, H.; Caricato, M.; Li, X.; Hratchian, H. P.; Izmaylov, A. F.; Bloino, J.; Zheng, G.; Sonnenberg, J. L.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.; Montgomery, J. A., Jr.; Peralta, J. E.; Ogliaro, F.; Bearpark, M.; Heyd, J. J.; Brothers, E.; Kudin, K. N.; Staroverov, V. N.; Kobayashi, R.; Normand, J.; Raghavachari, K.; Rendell, A.;

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