Efficient synthesis of (R)-harmonine – the toxic principle of the multicolored Asian lady beetle (Harmonia axyridis)†

A flexible synthetic route to (R)-harmonine ((R)-1), the toxic principle of the Asian lady beetle Harmonia axyridis (H. axyridis), via reductive olefination of the macrocyclic lactone (S)-5, is reported. High enantiomeric purity is achieved by enantioselective saponification of the lactone rac-5 with horse liver esterase. Minor modifications in the synthetic route give access to racemic and chiral harmonine (1), analogs and putative biosynthetic precursors. In addition, the antimicrobial activity of harmonine against Leishmania major (L. major) is demonstrated and provides the rationale for harmonine-based drug development against parasitic diseases.


Introduction
The multicolored Asian lady beetle H. axyridis, also known as the harlequin ladybird, is natively distributed from southern Siberia to southern China and from the Altai mountains to the Pacific coast. 1 This tree dwelling beetle, of the family Coccinellidae, is an important predator of aphids and scale insects and has been introduced for biological control in many countries. Over time populations began to establish and two decades ago H. axyridis became an invasive species in North America, Europe and South America threatening the native lady beetles. [1][2][3] The invasive success arises from intra-guild predation 2 and from H. axyridis' resistance to pathogens. 3 When a lady beetle is disturbed or attacked it releases droplets of hemolymph from the tibio-femoral joints of its legs. This behavior is referred to as reflex-bleeding. 4,5 The repellent and sometimes toxic properties of the hemolymph are due to some alkaloids, which are considered to be synthesized de novo by the beetles. 6 H. axyridis produces (R)-harmonine ((R)-1) ((17R,9Z)-octadec-9-ene-1,17-diamine) as the major defense compound (Fig. 1). 7 (R)-Harmonine ((R)-1) displays antibacterial activity against fast-growing mycobacteria, Mycobacterium tuberculosis, and Plasmodium falciparum (P. falciparum), demonstrates multistage antimalarial activity, 7 and exhibits cytotoxicity against human tumor cell lines. 8 Recently microsporidia from the hemolymph of H. axyridis were shown to infect intra-guild predators. 9 In this context, (R)-harmonine ((R)-1) was postulated to protect the harlequin beetle against self-infection. 10 Currently, harmonine ((R)-1) is considered a promising lead for clinical and agricultural use (yellow biotechnology). 11 Although harmonine ((R)-1) has been isolated 12 and synthesized previously, [12][13][14] there is a need for rapid and efficient syntheses of harmonine ((R)-1) and related molecules, since the mode of action is still unknown. In particular, for bioassays larger quantities are needed.
L. major is the causative agent of cutaneous leishmaniasis with an estimated annual incidence of 800 000-1.3 million new infections worldwide. 15,16 Current chemotherapy against leishmaniasis is limited due to the continuous development of drug resistance accompanied by severe side effects. 17 Therefore, naturally derived compounds like harmonine are investigated to identify and develop new therapies against leishmaniasis. The antiparasitic activity of harmonine against P. falciparum has already been reported and prompted the assessment of its activity against L. major. 7 Here, we report a short and flexible synthetic route to harmonine ((R)-1) in only a few steps starting from the readily available macrocyclic lactone (S)-5. One-pot olefination of (S)-5 directly provides the basic skeleton of harmonine ((R)-1) and allows the synthesis of biosynthetic precursors and structural analogs via functional group modifications. Furthermore, we report the antiparasitic activity of harmonine against the causative agent of cutaneous leishmaniasis, L. major. 18 Cultivation in the presence of (R)-1 leads to the inhibition of parasitic proliferation with a consequent early necrotic cell death phenotype.

Results and discussion
According to Scheme 1 the harmonine backbone can be obtained from the lactone (S)-5 and the phosphonium salt 4 by a Wittig-type reaction. The chiral lactone (S)-5 is available with high ee and excellent yield by enantioselective hydrolysis with horse liver esterase. Furthermore, synthetic intermediates like ((S)-3) are promising candidates for biosynthetic and pharmaceutical studies. 19 By modifying the chain length of the phosphonium salt or the ring size of the lactone, analogs with different positions of the double bond and molecular size become available, thus opening a new synthetic route to harmonine-like compounds using a unified procedure.
The progress of the reaction was monitored using a GC-MS equipped with a chiral column (β-6TBDM) for resolution of the enantiomers (Fig. 2).
For the evaluation of the antileishmanial activity of harmonine, the AlamarBlue® assay with L. major promastigotes and the Britelite™ assay with Luciferase-transgenic L. major amastigotes were employed ( Table 1). The cytotoxic effect of harmonine against host cells was investigated in bone marrowderived macrophages (BMDM). The half maximal inhibitory concentration (IC 50 ) of harmonine was detected at 14.2 µM against L. major promastigotes and at 2.4 µM against the intra-cellular and clinically relevant L. major amastigote form. When compared to Miltefosine (1-hexadecylphosphocholine, positive control) with an IC 50 value of 36.2 µM against L. major promastigotes and 33.0 µM against L. major amastigotes, 29 harmonine showed leishmanicidal activities at significantly lower concentrations. Miltefosine showed an IC 50 value of 65.5 µM and harmonine showed a value of 36.5 µM against BMDM. The antileishmanial efficacy of a tested drug compared to its cytotoxicity against host cells is defined as the selectivity index (SI). SIs > 20 are considered excellent, as high antileishmanial activity and low cytotoxicity are desirable prerequisites for drug development. 30 Here, harmonine shows a very good SI of 15.2 towards L. major which is considerably higher than the SI of 2.0 of Miltefosine.
The antileishmanial effect of harmonine as shown in Table 1 was further investigated. The cell morphology of L. major promastigotes was studied by means of transmitted light microscopy upon treatment with harmonine for 6 h, 10 h, and 24 h (Fig. 3). The visual examination of promastigotes incubated with harmonine showed rapid changes of the cell morphology. After 24 h only dead cells were observed upon harmonine-treatment whereas in the presence of dimethyl sulfoxide (DMSO) no significant changes in the cell morphology of the parasites were observed. Rounding of cells upon harmonine treatment was induced after 6 h of culture and dead cells were visible after 10 h of cultivation. DMSOtreated control L. major promastigotes show upon 6 h and 10 h of culture the characteristic flagellated and slender shape of the viable and unaffected parasite (Fig. 3).
The type of cell death induced by harmonine was investigated using flow cytometric approaches. The loss of membrane integrity in necrotic cells and the translocation of phosphatidylserine (PS) to the outside of the cellular membrane of apoptotic cells can be determined by Annexin V (AV, binds PS) and PI (DNA-binding) staining. 31 Double staining with AV-fluorescein isothiocyanate (FITC) and PI allows the discrimination between four Leishmania cell death phenotypes as described elsewhere: live (AV − /PI − ), late necrotic/late apoptotic (AV + /PI + ), early necrotic (AV − /PI + ) and early apoptotic cells (AV + /PI − ). 31,32 Harmonine-treatment for 24 h induced early necrotic cell death in 31.6% and late necrotic/late apoptotic cell death in 51.2% of all the treated cells (Fig. 3). This means that after 24 h of harmonine-treatment a total of 82.8% of cells were   (Fig. 3). Cell death during harmonine-treatment was observed to increase over time, as after 6 h a total of 26.2% and after 10 h a total of 40.3% of cells were dead, as indicated by PI staining. Early necrosis is characterized by PI-binding to the DNA of cells which have lost their cell membrane integrity. Harmonine was found to induce early necrotic cell death in L. major, as a clear population of 24.5% early necrotic cells was detected after 10 h of cultivation (Fig. 3). The increase of late necrotic/late apoptotic cells to 51.15% after 24 h is a consequence of an early necrotic cell death phenotype, as the rupture of the cell membrane allows AV to bind to PS in the dead parasite.

Conclusion
In conclusion a highly efficient and flexible synthetic route to chiral (R)-harmonine ((R)-1) is reported. A highly enantioselective hydrolysis of rac-5 affords both the remaining lactone (S)-5 and the hydroxy acid (R)-9 in high yield and excellent enantiomeric purities. Reductive olefination of the lactone (S)-5 with readily available phosphonium ylides gives direct access to the backbone of harmonine ((R)-1). Subsequent functional group modification provides derivatives and analogs of the natural product for structure-activity studies and mechanistic analyses to unravel the mode of action of the ladybeetle alkaloid harmonine ((R)-1). The pronounced activity of harmonine ((R)-1) against mycobacteria or the malaria parasite P. falciparum as is reported elsewhere, 7   Germany) or Lichroprep RP-18 silica gel (40-63 µm, Merck, Darmstadt, Germany) was used. Thin layer chromatography was conducted on silica gel 60 F 254 aluminum sheets (Merck, Darmstadt, Germany). The compounds were detected using Hanessian's stain, vanillin or potassium permanganate stain. Except for diethyl ether all solvents purchased were of HPLC grade and used without further purification. Diethyl ether was distilled prior to use. Anhydrous solvents were purchased as such. Horse liver esterase was obtained as lyophilized powder (0.5-1.0 U mg −1 , Sigma Aldrich, Saint Louis, MO, USA).
HRMS m/z calcd for C 18  (9Z)-17-Hydroxyoctadec-9-enamide (rac-8). Isopropyl (9Z)-17hydroxyoctadec-9-enoate (rac-6) (237 mg, 0.70 mmol) was dissolved at 0°C in MeOH (2.25 ml). Mg 3 N 2 (353 mg, 3.48 mmol) was quickly added in one portion, the reaction vessel was thoroughly closed and the suspension was allowed to warm to room temperature. Stirring was continued for 1 hour. The reaction was heated to 80°C and stirring was continued for 24.5 hours. After cooling to room temperature, CHCl 3 (25 ml) and water (25 ml) were added. The aqueous phase was neutralized with 3 M HCl, the layers were separated and the aqueous phase was extracted with CHCl 3 (2 × 25 ml). The organic layers were combined and the solvent was removed under reduced pressure. The residue was purified by column chromatography on RP-18 silica gel using H 2 O-MeOH (1 : 9) for elution. CHCl 3 was added and the solvents were removed under reduced pressure. This procedure was repeated three times with CHCl 3 and three times with benzene to remove the last traces of water. The residue was concentrated under high vacuum to yield rac-8 as a colorless, sticky oil. (182 mg, 0.61 13  (9Z)-18-Aminooctadec-9-en-2-ol (rac-2). LiAlH 4 (106 mg, 3.00 mmol) was suspended with stirring in dry THF (3 ml) and cooled to 0°C. (9Z)-17-Hydroxyoctadec-9-enamide (rac-8) (168 mg, 0.56 mmol), dissolved in dry THF (2 ml), was added dropwise. The reaction was allowed to warm to room temperature, stirred for 4 hours at room temperature and for 19.5 hours at 45°C. After cooling to room temperature, water (10 ml) and CHCl 3 (10 ml) were added. The layers were separated and the aqueous phase was extracted with CHCl 3 (5 × 10 ml). The organic extracts were combined and the solvents were removed under reduced pressure. The product was partitioned between MTBE (80 ml) and 2% HCl (80 ml). The aqueous phase was extracted with MTBE (3 × 20 ml) and the combined organic extracts were washed with 2% HCl (35 ml). Both aqueous solutions were combined and MTBE (50 ml) and NH 3 (25% aqueous solution, 80 ml) were added. The layers were separated and the aqueous phase was extracted with MTBE (3 × 80 ml). The combined organic extracts were dried over Na 2 SO 4 and the solvent was evaporated under reduced pressure yielding rac-2 as oily, colorless wax (151 mg, 0.53 mmol, 95%).
HRMS m/z calcd for C 18

Enzymatic hydrolysis of lactone (rac-5)
Racemic 9-methyloxonan-2-one (rac-5) (3.50 g, 22.4 mmol) was suspended in NaH 2 PO 4 buffer (0.1 M, pH = 7.2, 100 ml) and stirred for 15 minutes. Then horse liver esterase (350 mg, lyophilized powder) was added and the pH was kept constant by dropwise addition of 0.5 M NaOH during the whole reaction time. After 12 hours, another portion of horse liver esterase (70 mg) was added and stirring was continued for 4 hours. Then Celite (3.50 g) and ice (7.00 g) were added, the suspension was stirred for 5 minutes and the solids were filtered off. observation of phenotypic changes within the parasite. The stained cells were analyzed by transmitted light microscopy under a 50× objective on a Nikon ECLIPSE 50i microscope equipped with a digital camera (Nikon, Tokyo, Japan). The images were processed using NIS Elements D software (Nikon).
Determination of the cell death phenotype by flow cytometric analysis L. major promastigotes were either treated for 6 h, 10 h, and 24 h with 30 µM harmonine or 1% DMSO as the solvent control. Cell staining was performed using an Annexin V-FITC Apoptosis detection kit (Sigma-Aldrich, Saint Louis, MO, USA) according to the manufacturer's protocol. The stained samples were immediately analyzed by flow cytometry using a MACS Quant Analyzer (Miltenyi Biotech, Bergisch Gladbach, Germany).