Density functional theory of a new derivative of allantoin with antiplasmodial properties, antimycobacterial activities of essential oil from Cordia (Boraginaceae)

Background: some chemical and pharmacological investigations were performed on the EtOAc and/or CH 2 Cl 2 extracts of the stems of Cordia batesii (Boraginaeae); one of its components was subjected to some quantum calculations to get improvement in the understanding of its 13 C–NMR and UV properties along with geometric, electronic, thermodynamic data and reactivity descriptors. Results: a new allantoin ( 1 ) derivative named batesiin ( 2 ) was characterized from the EtOAc extract; thirteen known compounds including allantoin ( 1 ) were either isolated or identified by means of MS, NMR, LC–MS and GC–MS. GC–MS was applied on a fraction of essential oil which was composed of a mixture of fatty acids ( 9 – 14 ). Density functional theory (DFT) calculations were applied on batesiin ( 2 ). Data were simulated using B3LYP and MPW1PW91 functionals; calculated chemical shifts at B3LYP/6–31G(d,p) and MPW1PW91/6–31G+(d,p) showed much better correlations with the experimental data. Time dependent DFT applied on 2 at B3LYP/6–31G+(d,p) displayed a major absorption band at λ max = 299.01 nm using chloroform as solvent, 3.01 nm higher than the experimental value. In addition, The MeOH extract of the stems and some isolated compounds were tested in vitro against Pf 7G8 CQS and Pf Dd2 CQR strains of Plasmodium falciparum ; meanwhile, the CH 2 Cl 2 extract and the mixture of fatty acids ( 9 – 14) were tested on a resistant mycobacterial strain of Mycobacterium tuberculosis codified AC45. Stems disclosed a moderate antiplasmodial activity (IC 50 = 50 μg/mL) and the mixture 9 – 14 a potent antimycobacterial activity with a MIC = 9.52 μg/mL. Conclusion: these results learn ( 1 ); essential oil from Cordia batesii presents interesting antimycobacterial activities which need to gain more visibility; further antiplasmodial tests should rely on batesiin ( 2 ) and other components of the species. activities against M . tuberculosis arise from the mixture of FA ( 9 – 14 ) with a MIC = 9.52 μg/mL. These results can emphasize the use of C . batesii in the treatment of fever or bronchitis by riparians (unpublished data); they suggest that the bioactive principles occurring in the extract, batesiin ( 2 ) or mixture of FA ( 9 – 14 ) can be considered as useful templates for further development of new anti–malarial and anti–tubercular drugs respectively. Additional survey should however be performed on 3 to check whether it exhibits other anti-plasmodial properties. Thorough analyses on essential oils from the genus Cordia should be conducted to check whether they can be considered as candidates in the fight against tuberculosis.


Introduction
One of the main goals of World Health Organization (WHO) is to end the epidemics of neglected tropical diseases, tuberculosis (TB) and malaria (which remains the major public health and mortality problem in the tropics) by 2030 [1,2]. In 2018, TB infected about 10.0 million people, mainly in WHO regions of South-East Asia (44%), Africa (24%) and the Western Pacific (18%); meanwhile, about 213 million cases of malaria were found in the WHO African region. In the same year, half a million newly rifampicin-resistant TB cases were estimated. In general, 3.4% of new TB cases and 18% of formerly cured patients displayed either multidrugresistant TB or rifampicin-resistant TB (MDR/RR-TB) [3][4][5]. Chemotherapy has become less effective due to widespread drug-resistant TB, high cost, shortage of drugs for treatment of malaria. Plants are considered to be an important source of major compounds in drug development because of their successful use in treating various human ailments since millenniums. In this context, the search for new natural products from medicinal plants could provide new ways for antimalarial and antitubercular drugs. Among these plants, the root decoction of some species of the genus Cordia (Boraginaceae) is reported to be useful in the treatment of tuberculosis, bronchitis and malaria [6].
The genus Cordia (Boraginaceae) is composed of trees or shrubs and is widespread in Central and South America, India, Asia and Africa [7]. About 350 species of that genus have been identified worldwide, mainly in warmer regions [8]. Previous phytochemical investigations of plants from this genus reported the isolation and characterization of different classes of secondary metabolites including naphthoquinones, hydroquinones [8], cromenes [9], terpenenoids [10] or polyphenols [11]. Meanwhile and based on some pharmacological surveys, essential oils from C. curassivica and C. gilletii appeared as active against some microbial strains [12,13] Some biological activities and in silico investigations of C. dichotoma were recently reported [14,15]; the plant is also known to contain, apart from allantoin (1) [16,17] which has been the subject of many quantum calculations [18,19], fatty acids (FA) [20]. FA appear as energy sources for M. tuberculosis inside host tissues and are supposed to induce dormancy in Mycobacterium bacilli [21,22].
Despite the intensive work performed on some Cordia species, no or less investigation has been done on Cordia batesii species. In our continuing search for secondary metabolites with potent antiplasmodial and anti-tubercular activities, chemical investigations were carried on the stems of Cordia batesii, a forest shrub growing in the central and western regions of Cameroon. This paper describes the isolation of a new derivative of 1 named batesiin (2) along with other compounds. A detailed characterization of 2 was investigated based on NMR and UV-visible spectroscopic analyses and density functional theory (DFT) at B3LYP /6-31G(d,p) [23,24]; 6-31G+(d,p) and MPW1PW91 /6-31G+(d,p) [25,26]. DFT calculations at B3LYP/6-311G++(d,p) were also performed to check some electronic and thermodynamic properties of 2. Meanwhile, in vitro activities regarding extracts of stems and some isolated compounds against two CQR strains of Plasmodium falciparum and a resistant mycobacterial strain of Mycobacterium tuberculosis were also examined.
The HMBC spectrum exhibited noticeable correlations between protons at δH 5. Lakshmanan et al [39] confirmed through an X-ray analysis that the occurring enantiomer of 1 is its (S) one. A thorough analysis of all the spectra and comparison with data from the literature revealed that compound 2 is described for the first time as a new derivative of allantoin (1); it was identified as (S,S)-1,3-bis(2,5-dioxoimidazolidin-4-yl)urea, trivially named batesiin (2). Table 1 shows some NMR data of allantoin (1) and batesiin (2); it strengthens the agreement of a close relationship between those two compounds in terms of NMR spectroscopic data.

DFT calculations of compound 2
The structure of compound 2 was assigned based on spectroscopic analyses including, UV, 1 Hand 13 C-NMR, 1D and 2D techniques. To get supplementary detailed awareness into the structure, DFT calculations were completed. The structure of the compound with the right stereochemistry was at first optimized at B3LYP method of DFT using 6-31G(d) basis set. Secondly, conformational analysis [still at B3LYP/6-31G(d)] of the previous optimized structure was applied around the H-C(5)-N(6)-H dihedral angle; this scan led to one optimized geometry for 2, based on the cis-relationship between H-C(5)-N(6)-H ( 3 J(H,H) = 2.0 Hz). It is shown in Fig. 3 and takes in account previous reports of 1 [39,41]. The five membered ring is almost planar as observed in the case of allantoin (1) [19]. and compared with those of allantoin (1). The calculated HOMO-LUMO gap was 6.532 eV; results are summarized in Table 2.
The 1 H-and 13 C-NMR spectra of compound 2 were experimentally measured in DMSO-d6 on 500 and 125 MHz spectrometers respectively. The theoretical NMR were calculated at B3LYP/6-31G(d,p) and MPW1PW91/6-31+G(d,p) in DMSO. The chemical shifts were also simulated at B3LYP/6-31G(d), B3LYP/6-31+G(d,p), MPW1PW91/6-31G(d) and MPW1PW91/6-31G(d,p); however, the correlation with the experiment was relatively weak. GIAO (Gauge Invariant Atomic Orbital) formalism was used during these calculations, and the solvent effect was introduced through polarizable continuum model (PCM) by applying integral equation formalism (IEF). A comparison of the theoretical 13 C-NMR values at B3LYP/6-31G(d,p) and MPW1PW91/6-31+G(d,p) with the experimental ones is given in Table 3. A better correlation with the experiment can be achieved if a scaling factor is applied to the 13 C-NMR theoretical values.
UV-visible spectrum of 1 displayed two maxima at λmax 183 and 195 nm (representing the absorption bands of amide and imide functions) [19] in one case or a maximum at 265 nm in another case [39]. Despite on the fact that batesiin (2) Table 4. Excitation energy (in nm) determined in CHCl3 at 283.89 is closed to experimental value at 287 nm while simulation in MeOH exhibits an energy (in nm) at 315.59 which is comparable to the experimental λmax at 304 nm.

Biological properties
The antimalarial potencies screening against P. falciparum Dd2 and 7G8 (CQR) strains of the MeOH extract of stems of C. batesii and compounds 2, 3, 5 and 6 were performed according to the Sybr Green I fluorescence-based assays [42]. The results are presented in Fig.7; they indicate the IC50 of extract of stems of C. batesii and the percentage of growth inhibition against Dd2 and 7G8 P. falciparum strains respectively. Figure 7a showed that the MeOH extract of stems discloses an IC50 = 50 μg/mL against Dd2 P. falciparum strain which can be considered as a moderate activity. It appears from Fig. 7b that, apart from artemisinin (95.75% of inhibition) used as reference, the CH3OH extract has the highest antiplasmodial activity with 88.24% inhibition percentage followed by 2 with approximately 78% of growth inhibition against Dd2 strain. Compounds 3 and 5 showed high activity > 65% of inhibition, exhibiting a growth inhibition of Dd2 strain P. falciparum with percentage of 66.43% and 72.99% respectively.
From the anti-mycobacterial test results (Table 5), it should be noticed that the mixture of FA (A1) exhibited a good anti-tubercular activity with a Minimal Inhibitory Concentration (MIC) of 9.52 μg/mL. According to Cantrell et al. [43], isolated compounds that exhibit a MIC ≤ 64 μg/mL are considered promising. For crude extracts, the MIC should be ≤ 125 μg/mL [44]. The extract showed poor inhibitory activity against Mycobacterium tuberculosis, exhibiting a MIC and a Minimal Bactericidal Concentration (MBC) of 1250 and 2500 μg/mL respectively.

Discussion
The high value of the HOMO-LUMO band gap is indicative of a relative stability of the molecule towards oxidation reactions. Plots of frontier orbitals demonstrate that HOMOs and LUMOs are globally focused over the entire molecule; meanwhile, in the case of HOMOs, the ureidyl moiety is less concerned whereas the positive charge is detected over it in LUMOs. The theoretical 13 C-NMR values are, on the average, higher than the experimental values. Regardless of the difference in the absolute values, the theoretical values match nicely with the experimental data. Based on the simulated UV-visible spectrum, batesiin (2) should most likely appear as an intermediate between various iminols and iminolates groups maybe due to additional stability compared to 1 (Fig. 8).
Two protons seem to be located somewhere between, in each case, a nitrogen and an anionic oxygen of the same iminolate group, at a site nearer to oxygen (distance < 1.4 Ǻ) than to nitrogen (distance > 2.3 Ǻ) (Fig. 8). Hence, a virtual loss of symmetry becomes noticeable within 2, inducing a change in molecular orbitals (MOs) with an impact on electronic transitions (Fig. 9).
MOs LUMO and LUMO+1 are localized on an imidazole fragment (especially in the region covering a proton and the anionic oxygen which is near to it) when the other one seems totally unoccupied. When moving from MOs HOMO-1 to HOMO-3, a decrease in the occurrence of MOs in the two imidazole fragments is remarkable and the major electronic density can be found on the anionic oxygen. Table 4 expresses the nature of electronic transitions which are in accordance with the corresponding λmax.
Results from bioassays against 7G8 P. falciparum strain reveal that an increase in concentration (10 to 100 μg/mL) exhibits an increase in percentages of inhibition for MeOH extract of stems, 2 and 3 but a decrease (with a negative percentage) for 5, which should indicate that the latter is inactive at high concentrations. The moderate activity of the MeOH extract can suggest insufficient synergistic or additive effects of possible anti-plasmodial secondary metabolites from C. batesii.
In contrast to the mixture of FA, the weak anti-mycobacterial properties of the crude extract suggest the occurrence within C. batesii of components with very poor anti-mycobacterial effects.
Moreover, a report from literature reflects that mycobacteria have a lipid-rich hydrophobic cell wall and are often susceptible to less polar compounds [45]. According to Peterson & Shanholtzer [46], bacteriostatic activity has been defined as a ratio of MBC to MIC of > 4. Thus, essential oil exhibited bactericidal activity.

Conclusion
Batesiin (2) has been characterized for the first time and its structure was confirmed by DFT at B3LYP/6-31G(d,p) and 6-31G+(d,p) and MPW1PW91/6-31G+(d,p) from this study. Additional data corresponding to HOMO, LUMO, enthalpy, entropy or some reactivity descriptors like IP or EA were also simulated, this time at B3LYP/6-311G++(d,p); moreover, no comparison with the experiment in this case could be made. Simulated energies (in nm) match nicely with experimental data after molecular modelling. The MeOH crude extract of the stems showed a moderate activity against Dd2 P. falciparum strain with IC50 = 50 μg/mL. The anti-plasmodial properties of batesiin

General
Melting points were uncorrected and were measured on a Mettler Toledo instrument. IR spectra were recorded on an Alpha FT-IR spectrometer from Bruker, while 1D and 2D NMR spectra were obtained on a Bruker DRX 500 (500 MHz for 1 H and 125 MHz for 13 C spectra) spectrometer Silica gel 60 (230-400 mesh E. Merck, Darmstadt, Germany) and Sephadex® LH-20 were employed for CC, the solvent mixing systems for elution were mainly CH2Cl2/MeOH for the phytochemical study with increasing polarity and pure MeOH, while precoated aluminum sheets silica gel 60 F254 were used for TLC [47].

Plant material
The plant material was collected on March 2014 at Koumoul in center region of Cameroon. The identity of plant material was confirmed by the taxonomist Victor Nana. A voucher sample (14106 SRF) is deposited at the National Herbarium of Cameroon, Yaounde.

Extraction and isolation
The stems were dried in shade and cut into small pieces and then submitted for further studies.
80% of air dried pieces of stems of C. batesii (500 g) were extracted with MeOH (5 x 500 mL, 30 min each) using an Elma® sonic S 100 at r. t. The extract was filtered; the filtrate was evaporated to dryness in a Rotavapor. The residue obtained from the MeOH extract (about 53 g) was dissolved into hexane-water 80:20 (100 mL) during one day; the resulting hexane-water gum (42 g) was dissolved in a mixture of CHCl3-H2O 80:20 (100 mL) once again in a period of 24 hours; the resulting CHCl3-H2O extract (36 g) was dissolved in CH3COOH-H2O 70:30 (100 mL) during 24 hours. The final extract (26g) was submitted to further CC analyses. After extraction, the crude MeOH extract (26 g) was subjected to column chromatography of LH-20 (2.5 cm, 50 cm, eluent MeOH). Four main fractions were obtained: A (10.36 g), B (4.6 g), C (3.0 g) and D (7.0 g).

Computational Details
All calculations were performed with Gaussian 09 suite of programs [48] and UV-visible curves were generated by GaussSum [49]. Geometries were optimized at hybrid B3LYP method using 6- axe has been scanned at 15 degrees step, to find the lowest energy conformers. A minimum at the dihedral angle of 22.9° was retained because of its conformation closed to the structure of allantoin (1). Afterwards, it was submitted to geometry optimization at B3LYP/6-31G(d) level of theory to provide the optimized geometry of 2 (Fig. 3). The optimized structure was confirmed by frequency analysis at the same level (B3LYP/6-31G(d)) as a true minimum (no imaginary frequency). Six methods were evaluated for the simulation of 1 H-and 13

In vitro cultivation of P. falciparum strains
PfDd2 and Pf7G8 strains of P. falciparum were used in vitro in blood stage culture to test the antimalarial efficacy of methanol extract of stems of C. batesii and isolated compounds 2, 3 and 5.
The culture was maintained at the Laboratory of parasitology, Centre Pasteur du Cameroon. P.
falciparum culture was maintained according to the method described by Trager and Jensen [50] with light modifications. P. falciparum Dd2 and 7G8 cultures were maintained in fresh O +ve human erythrocytes suspended at 4% haematocrit in RPMI 1640 (Sigma Aldrich -France) containing 0.2% sodium bicarbonate, 0.5% Albumax, 45 μg/L hypoxanthine and 50 μg/L gentamicin, and incubated at 37°C under a gas mixture 5% O2, 5% CO2, and 90% N2. Every day, infected erythrocytes were transferred into fresh complete medium to propagate the culture.

Drug dilutions
Artemisinin (Sigma Aldrich -France) and isolated compounds were prepared in DMSO. All stocks were then diluted with culture medium to achieve the required concentrations. The final solution of all plant extracts, isolated compounds and artemisinin contained 0.4% DMSO, which was found to be non-toxic to the parasites. Drugs and test compounds were then placed in 96-well flat bottom tissue culture grade plates.

Assay for anti-plasmodial activity
The stems of C. batesii were evaluated for their antimalarial activity against P. falciparum strains Dd2 and 7G8. For drug screening, SYBR green I-based fluorescence assay was setup as described by Smilkstein et al [42]. Where Abscontrol is the absorbance of untreated well and Absextracts/isolated compounds is the absorbance of extracts or compounds well.

Ethics approval
Not applicable.

Consent for publication
Not applicable.

Availability of data and materials
All data generated or analyzed during this study are included in this published article (and its supplementary information files).

Competing interests
The authors declare that they have no competing interests.

Funding
The authors acknowledge financial support from the Ministry of Higher Education, Cameroon and from the University of Yaounde I grant committee.