Crystal structure of akuammicine, an indole alkaloid from Catharanthus roseus

The structure of akuammicine, an alkaloid isolated from the Madagascar periwinkle, was confirmed and its absolute configuration determined.


Chemical context
The Madagascar periwinkle or rosy periwinkle (Catharanthus roseus L. G. Don), a member of the family Apocynaceae, is one of the most intensively studied medicinal plants (Sottomayor et al., 1998;Sreevalli et al., 2004). Aerial parts of the plant contain between 0.2 and 1% of a mixture of more than 120 alkaloids (van Der Heijden et al., 2004). The most abundant are the monomers such as catharanthine and vindoline (Renault et al., 1999). The dimeric alkaloids that result from the joining of two compounds can display interesting pharmaceutical activities. Thus vinblastine and vincristine are used in the chemotherapy of leukemia and in the treatment of Hodgkin's disease (Verma et al., 2007). Additionally, ajmalicine, a monomeric indole alkaloid present in the root of C. roseus, is an antihypertensive alkaloid (Noble, 1990). In view of their medical and commercial value, the appropriate methods of extraction and purification have been well studied.
We have undertaken the X-ray crystal structure determination of the title compound in order to establish its absolute stereochemistry. One-dimensional ( 1 H, 13 C, DEPT135) and two-dimensional NMR (HSQC, HMBC, 1 H/ 1 H-COSY, 1 H/ 1 H-NOESY) experiments clearly assigned the proton and carbon resonances and are consistent with the constitution of ISSN 2056-9890 akuammicine (Buckingham et al., 2010). As the final purification step was performed with an alkaline solvent mixture, the NMR data of akuammicine correspond to the free base, and can be linked to the determined stereochemistry.

Structural commentary
The title compound crystallizes in space group P1 with two independent molecules (Fig. 1). The two molecules are closely similar; a least-squares fit of all non-H atoms gives an r.m.s. deviation of 0.065 Å , whereby the largest deviation is 0.29 Å for the methyl carbon C18. The absolute configuration is established as S at C3 and C15 and R at C7. Intramolecular classical N1-H01Á Á ÁO1 hydrogen bonds are observed ( Table 1). The C18-H18AÁ Á ÁO2 contacts in both molecules may represent a significant intramolecular interaction.
The five-membered ring involving N4 displays an envelope conformation, with C5 lying outside the plane of the other four atoms. The cyclohexene ring is a 'skew-boat' or 1,3diplanar form, with torsion angles of approximately zero about C3-C7 and C2 C16. Finally, the six-membered ring involving N4 shows a form intermediate between boat and skew-boat; the torsion angle about C15-C20 is approximately zero, but that about C3-N4 (which would also be zero for an ideal boat) is about 24 . See Table 2 for details.

Database survey
The most similar natural product to have been investigated by X-ray structure analysis is probably isovoacangine (Soriano-García et al., 1991), refcode KORZOG.

Isolation and crystallization
The title compound was isolated using a combination of highperformance countercurrent chromatography (HPCCC) (Ito, 2005), preparative C18 high-performance liquid chromatography (HPLC) and silica-gel column chromatography (Ito, 2005). Seedlings of F1 Titan Rose Catharanthus roseus, purchased from a commercial provider of pharmaceutical plants (Gä rtnerei Volk GmbH Pflanzenhandel, Braunschweig, Germany), were planted and grown outside from June to July 2015 on a mixture of standard garden soil and sand (2:1). Aerial plant parts were harvested and lyophilized, and dried tissue material was then milled by a bead mill [Mixer Mill MM 200 (RETSCH,Haan,Germany) at a vibrational frequency of 25 Hz for 1 min]. The dried powder was immersed in water adjusted to pH 2 by trifluoroacetic acid (TFA), homogenized by a T-25 digital ULTRA-TURRAX (IKA, Staufen, Germany) at maximum speed (25 000 rpm for 10 min) and shaken overnight for extraction. Plant particles were centrifuged off (30 min, 8000 rpm). The acidic extract was lyophilized and redissolved in 1 l chloroform. A solution of NaOH was added (1 l, 200 mM) and the solutions were vigorously mixed for alkaloid extraction. The phases were centrifuged (8000 rpm, 15 min) and the chloroform phase was dried for indole-alkaloid recovery.
À3.6 (3) C14 0 -C15 0 -C20 0 -C21 0 À1.6 (4) Figure 1 The structure of the title compound in the crystal. Ellipsoids represent 30% probability levels. Both independent molecules are shown, but for clarity only one is labelled. The second molecule has the same numbering but with primes. Dashed lines represent hydrogen bonds.
tion velocity was set to 1600 rpm (240 g field), and the flow rate of the aqueous mobile phase (5.0 ml min À1 ) (head-to-tail mode) resulted in a stationary phase retention of 60% after system equilibration. For metabolite profiling, aliquots of the recovered HPCCC fractions were injected in sequence into an ESI-ion trap MS/ MS (HCT Ultra ETD II, Bruker Daltonics, Bremen, Germany) in a standard protocol described by Jerz et al. (2014) and the target alkaloid akuammicine was detected with [M+H] + at m/z 323 in fractions 61 to 69 (elution volume 304-345 ml). The combined fractions were re-chromatographed by preparative HPLC (Wellchrom K-1001, Knauer Gerä tebau Berlin, Germany) using a C18 column (Prontosil C18Aq, 25 Â 250 mm) and an isocratic flow rate of 4.5 ml min À1 (acetonitrile:water, 60:40 with 1% TFA). Alkaloids were monitored using a UV detector (Wellchrom K-2600, Knauer Gerä tebau, Berlin, Germany) at 254, 280 and 300 nm.
Akuammicine crystals grew in tube fractions during slow evaporation of the solvents, and an appropriate colourless crystal was chosen for X-ray analysis.

Refinement
Crystal data, data collection and structure refinement details are summarized in Table 3. N-bound H atoms were refined freely. Methyls were refined as idealized rigid groups, with C-H = 0.98 Å and H-C-H = 109.5 . Other H atoms were included using a riding model starting from calculated positions, with aromatic C-H = 0.95 Å , methylene C-H = 0.99 Å and methine C-H = 1.00 Å , with U iso (H) = 1.5U eq (C) for methyl H atoms and 1.2U eq (C) for other H atoms. The Flack parameter of 0.10 (13)    SHELXS97 (Sheldrick, 2015); program(s) used to refine structure: SHELXL2017 (Sheldrick, 2015); molecular graphics: XP (Siemens, 1994); software used to prepare material for publication: SHELXL2017 (Sheldrick, 2015).

Special details
Geometry. All esds (except the esd in the dihedral angle between two l.s. planes) are estimated using the full covariance matrix. The cell esds are taken into account individually in the estimation of esds in distances, angles and torsion angles; correlations between esds in cell parameters are only used when they are defined by crystal symmetry. An approximate (isotropic) treatment of cell esds is used for estimating esds involving l.s. planes.