Hemi-Synthesis of Chiral Imine, Benzimidazole and Benzodiazepines from Essential Oil of Ammodaucus leucotrichus subsp. leucotrichus

The hemi-synthesis of chiral imine, benzimidazole and benzodiazepine structures is reported by the condensation of (S)-(−)-perillaldehyde, the major phytochemical of Ammodaucus leucotrichus subsp. leucotrichus essential oil, with different amine derivatives of 2,3-diaminomaleonitrile, o-phenylenediamine and 3-[(2-aminoaryl)amino]dimedone. The reaction proceeds in situ at ambient temperature without prior isolation of the natural (S)-(−)-perillaldehyde. Final products precipitate in the ethanolic reaction medium. 2D NMR and single-crystal X-ray diffraction studies were used to unequivocally characterize the structures in solution and in the solid state, respectively. Chiral HPLC analysis confirms the formation of unique enantiomers and diastereomeric mixtures.


Introduction
Over the past decades, there has been a growing transition in drugs from natural materials, natural products, or their simple derivatives to more potent natural-mimicking synthetic prototypes [1]. Essential oils' components still play a major role in this combinatorial chemistry and are considered promising sources of stereospecific structures. They can indeed serve as substrates in hemi-synthesis, leading to new active molecules. For instance, (+)-carotol, the major constituent of carrot seed essential oil, was used as a starting material in the hemi-synthesis of ten cytotoxic hydroindene-derived chiral synthons [2]. A novel series of essential oils oriented chiral esters with high insecticidal activity have been synthesized based on the scaffold of the natural pyrethrin [3]. Cymbopogon schoenanthus essential oil is mainly composed of piperitone (68.2%), which was used for in situ preparation of three anti-parasitic carbasones [4].
Perillaldehyde, the major constituent of the A. leucotrichus essential oil, reacts in situ with the aforementioned amines without the need for prior purification or isolation from the oil matrix. The condensation reaction proceeds under similar operating conditions as previously published [22], by employing ethanol as a solvent at ambient temperature and under catalyst-free conditions over a period of 12 h. The reaction is easily worked up by filtration of the formed solid showing a high purity of products 1-4, isolated in moderate-to-good yields (47%-82%) after washing with ethanol, ultimately avoiding any further chromatographic purifications (Scheme 1).

Chiral HPLC and Stereochemistry
(S)-(−)-Perillaldehyde is found in essential oils in an optically active laevorotatory form due to the presence of an asymmetric carbon [13]. This carbon was chemically resistant to our amine nucleophilic attacks on perillaldehyde where chemical alterations were mainly brought to the aldehyde function to produce compounds 1-4 (including imine, imidazole and 1,4-diazepines) and maintaining the asymmetric carbon unchanged. This fact was further studied, however, by chiral-Scheme 2. Mechanistic pathway for the hemi-synthesis of imine 1, benzimidazole 2 and benzodiazepine 3-4 structures form essential oil of A. leucotrichus in situ.

Chiral HPLC and Stereochemistry
(S)-(−)-Perillaldehyde is found in essential oils in an optically active laevorotatory form due to the presence of an asymmetric carbon [13]. This carbon was chemically resistant to our amine nucleophilic attacks on perillaldehyde where chemical alterations were mainly brought to the aldehyde function to produce compounds 1-4 (including imine, imidazole and 1,4-diazepines) and maintaining the asymmetric carbon unchanged. This fact was further studied, however, by chiral-HPLC analysis performed on imine 1, imidazole 2 and 1,4-benzodiazepine 3, where the presence of a sole chromatographic peak indicated the existence of one enantiomer as noted in the case of compounds 1 and 2 ( Figure 1, see Supplementary Materials). Structurally, 1,4-benzodiazepines 3 and 4 are composed of the asymmetric perillaldehyde moiety attached to the new connecting asymmetric carbon which belongs to the diazepine ring. Stereochemical and mechanistic considerations suggest the presence of a 1:1 ratio of two diastereomers (11-R, 4 -S-and 11-S, 4 -S-isomers) (Scheme 2). This was proved with the help of chiral-HPLC, showing the presence of two chromatographic peaks with 1:1 integrating areas, ultimately supporting the formation of a diastereomeric mixture of 1,4-benzodiazepine 3 (see Supplementary Materials). According to chiral chromatographic profiles of 1-3, our hemi-synthetic process was efficient without any side products being formed, showing high purity of the unique enantiomeric forms of compounds 1 and 2, along with diastereomeric mixtures of 1,4-benzodiazepines 3 and 4.

Scheme 2.
Mechanistic pathway for the hemi-synthesis of imine 1, benzimidazole 2 and benzodiazepine 3-4 structures form essential oil of A. leucotrichus in situ.

Chiral HPLC and Stereochemistry
(S)-(−)-Perillaldehyde is found in essential oils in an optically active laevorotatory form due to the presence of an asymmetric carbon [13]. This carbon was chemically resistant to our amine nucleophilic attacks on perillaldehyde where chemical alterations were mainly brought to the aldehyde function to produce compounds 1-4 (including imine, imidazole and 1,4-diazepines) and maintaining the asymmetric carbon unchanged. This fact was further studied, however, by chiral-HPLC analysis performed on imine 1, imidazole 2 and 1,4-benzodiazepine 3, where the presence of a sole chromatographic peak indicated the existence of one enantiomer as noted in the case of compounds 1 and 2 (

Nuclear Magnetic Resonance Spectroscopy
The structures of 1-4 were characterized by 1D and 2D NMR spectroscopy (see Supplementary Materials). The 1 H-NMR spectrum of compounds 1-4 suggested the presence of the asymmetric perillalkyl unit, which is mainly characterized by aliphatic protons of methylene groups appearing as multiplets between δ H 1.0 and 3.0 ppm with their positions confirmed based on DEPT-135, HSQC and HMBC experiments ( Figure 2). Vinylic protons of the perillalkyl moiety were attributed around 4.40-5.00 ppm (for the extra-cyclic protons) and 5.00-6.70 ppm (for the intra-cyclic ones). Using DEPT-135 and HSQC experiment, all the protonated carbons were assigned, especially those from δ C 20 to 40 ppm due to aliphatic methyl and methylene groups of perillalkyl radical (see Supplementary Materials).
The The structures of 1-4 were characterized by 1D and 2D NMR spectroscopy (see Supplementary  Materials). The 1 H-NMR spectrum of compounds 1-4 suggested the presence of the asymmetric perillalkyl unit, which is mainly characterized by aliphatic protons of methylene groups appearing as multiplets between δH 1.0 and 3.0 ppm with their positions confirmed based on DEPT-135, HSQC and HMBC experiments (Figure 2). Vinylic protons of the perillalkyl moiety were attributed around 4.40-5.00 ppm (for the extra-cyclic protons) and 5.00-6.70 ppm (for the intra-cyclic ones). Using DEPT-135 and HSQC experiment, all the protonated carbons were assigned, especially those from δC 20 to 40 ppm due to aliphatic methyl and methylene groups of perillalkyl radical (see Supplementary Materials).

Single-Crystal X-ray Diffraction
Single-crystal X-ray diffraction studies were further used to determine the spatial arrangement of the new compounds 1-4 (Figures 3 and 4). Crystals of compounds 1-4 were directly obtained from the reaction batches after filtration and washing with ethanol. This is noteworthy because use of the natural (S)-(−)-perillaldehyde as a starting chiral reagent means that all the resulting structures crystallized in chiral and non-centrosymmetric space groups. The absence of strongly diffracting elements in all structures means that the absolute configurations of asymmetric carbons could not be determined from the X-ray studies, but they were nevertheless unveiled from the chiral HPLC studies performed.
Compounds 1 and 2 only have a single asymmetric carbon centre belonging to the parent perillalkyl unit, exhibiting the (S)-configuration ( Figure 3). While 1 crystallized in a noncentrosymmetric monoclinic space group P21 with the asymmetric unit having only one molecule, 2 crystallized in the non-centrosymmetric orthorhombic space group P212121, with two molecules composing the asymmetric unit. These two molecules had the same configuration for the asymmetric carbon centre with the inequivalence arising from simple rotations of the various molecular units composing the molecule. The presence of donor and acceptor units capable of engaging in strong hydrogen bonds was well evidenced in the crystal packing of both 1 and 2. In 1, the two nitrile groups and the amine moiety were engaged in strong intermolecular N-H···N hydrogen bonds, forming a R 3 3(14) graph set (dN···N = 3.012(3)-3.028(3) Å and <(NHN) = 147-168°), ultimately dictating the way the molecules closely pack in the solid state. In 2, there was a single N-H···N interaction connecting

Single-Crystal X-ray Diffraction
Single-crystal X-ray diffraction studies were further used to determine the spatial arrangement of the new compounds 1-4 (Figures 3 and 4). Crystals of compounds 1-4 were directly obtained from the reaction batches after filtration and washing with ethanol. This is noteworthy because use of the natural (S)-(−)-perillaldehyde as a starting chiral reagent means that all the resulting structures crystallized in chiral and non-centrosymmetric space groups. The absence of strongly diffracting elements in all structures means that the absolute configurations of asymmetric carbons could not be determined from the X-ray studies, but they were nevertheless unveiled from the chiral HPLC studies performed.
Compounds 1 and 2 only have a single asymmetric carbon centre belonging to the parent perillalkyl unit, exhibiting the (S)-configuration ( Figure 3). While 1 crystallized in a non-centrosymmetric monoclinic space group P2 1 with the asymmetric unit having only one molecule, 2 crystallized in the non-centrosymmetric orthorhombic space group P2 1 2 1 2 1 , with two molecules composing the asymmetric unit. These two molecules had the same configuration for the asymmetric carbon centre with the inequivalence arising from simple rotations of the various molecular units composing the molecule. The presence of donor and acceptor units capable of engaging in strong hydrogen bonds was well evidenced in the crystal packing of both 1 and 2. In 1, the two nitrile groups and the amine moiety were engaged in strong intermolecular N-H···N hydrogen bonds, forming a R 3 3 (14) graph set (d N···N = 3.012(3)-3.028(3) Å and <(NHN) = 147-168 • ), ultimately dictating the way the molecules closely pack in the solid state. In 2, there was a single N-H···N interaction connecting the two crystallographically independent molecular units, leading to the formation of a C 2 2 (8) graph set motif (d N···N = 2.853(4) -2.854(4) Å and <(NHN) = 163 • ) [25].
Compounds 3 and 4 both crystallized in the non-centrosymmetric monoclinic I2 space group, with the asymmetric units being composed of a pair of (S,S)-and (R,S)-diastereomers (in Figure 4 only the (S,S)-diastereomers are depicted for the two compounds). According to our previous mechanistic descriptions, the diazepine cyclisation leads to the formation of an extra asymmetric carbon (C-11), with the one from perillaldehyde (C-4 ) always maintaining its original (S)-configuration. We further note that chiral HPLC clearly showed the presence of a mixture of two diastereomers, which agrees with the crystallographic studies performed. The configuration of all the stereocentres was determined to be (11-R,4 -S) and (11-S,4 -S). These results are also in agreement with the aforementioned 2D-NMR investigations.
Molecules 2019, 24, x FOR PEER REVIEW 6 of 10 the two crystallographically independent molecular units, leading to the formation of a C 2 2(8) graph set motif (dN···N = 2.853(4) -2.854(4) Å and <(NHN) = 163°) [25].  Compounds 3 and 4 both crystallized in the non-centrosymmetric monoclinic I2 space group, with the asymmetric units being composed of a pair of (S,S)-and (R,S)-diastereomers (in Figure 4 only the (S,S)-diastereomers are depicted for the two compounds). According to our previous Asymmetric carbon centres are depicted by an asterisk and the bonds in light yellow. Non-hydrogen atoms are represented as thermal ellipsoids drawn at the 50% probability level and hydrogen atoms as small spheres with arbitrary radii.

General Remarks
Melting points were measured with a Büchi Melting Point B-540 apparatus (BÜCHI Labortechnik AG, Flawil, Switzerland). NMR spectra were recorded on a Bruker AVANCE 500 spectrometer (Bruker, Wissembourg, France, 400 MHz for 1 H and 100 MHz for 13 C), in DMSO-d 6 as solvent. Chemical shifts (δ) were reported in ppm and coupling constants (J) in Hz and the internal standard was TMS. Unequivocal 13 C assignments were made with the aid of 2D gHSQC and gHMBC experiments (delays for one-bond and long-range J C/H couplings were optimized for 145 and 7 Hz, respectively). High-resolution mass spectra (ESI + -HRMS) were measured with a micrOTOF-Q 98 spectrometer (Bruker Daltonics, Hamburg, Germany). GC-MS analysis of A. leucotrichus subsp. leucotrichus essential oil was performed on a Hewlett-Packard computerized system comprising a 6890 gas chromatograph coupled with a 5973A mass spectrometer (Agilent Technologies, Santa Clara, CA, United States). All chemicals and solvents were purchased from commercial sources and were used as received. Chiral-HPLC separation of compounds 1-3 was performed on chiral stationary phase at 25 • C using a CHIRALPAK ® IA (Chiral Technologies Europe, Illkirch, France, amylose-tris(3,5-dimethylphenylcarbamate) immobilized on 5 µm silica-gel, 250 mm × 4.6 mm ID). The mobile phase used was hexane/acetone (isocratic mode, 50:50 (v/v)) at a flow rate of 1.0 mL/min. The UV detector was set at 220 nm ( Figure S21 in the Supplementary Information). An injection of 20 µL of 1.0 g/L concentrated samples of dissolved compounds 1-3 was used in the mobile phase.

Plant Material and Extraction Procedure
The aerial parts of A. leucotrichus were collected from Ghardaia (Septentrional Algerian Sahara) in April 2017. They were identified by the botanists of the National Agronomic Institute in El-Harrach Algeria. Air-dried fruits of A. leucotrichus were submitted to water distillation with a Clevenger-type apparatus (Merck KGaA, Darmstadt, Germany) for 3 h. The final product yielded 2.23% of deep-blue liquid oil. The obtained essential oil was dried over anhydrous sodium sulphate and, after filtration, it was stored at +4 • C.