Cordidepsine is A Potential New Anti-HIV Depsidone from Cordia millenii, Baker

Chemical investigation of Cordia millenii, Baker resulted in the isolation of a new depsidone, cordidepsine (1), along with twelve known compounds including cyclooctasulfur (2), lup-20(29)-en-3-triacontanoate (3), 1-(26-hydroxyhexacosanoyl)glycerol (4), glyceryl-1-hexacosanoate (5) betulinic acid (6), lupenone (7), β-amyrone (8), lupeol (9), β-amyrin (10), allantoin (11), 2′-(4-hydroxyphenyl)ethylpropanoate (12) and stigmasterol glycoside (13). Hemi-synthetic reactions were carried out on two isolated compounds (5 and 6) to afford two new derivatives, that is, cordicerol A (14) and cordicerol B (15), respectively. The chemical structures of all the compounds were established based on analysis and interpretation of spectroscopic data such as electron ionization mass spectrometry (EI–MS), high resolution electrospray ionization mass spectrometry (HR-ESI–MS), fast atom bombardment mass spectrometry (FAB–MS), one dimension and two dimension nuclear magnetic resonance (1D and 2D-NMR) spectral data as well as X-ray crystallography (XRC). Lupeol ester derivatives [Lup-20(29)-en-3-triacontanoate (3)], monoglycerol derivatives [1-(26-hydroxyhexacosanoyl)glycerol (4) and glyceryl-1 hexacosanoate (5)] were isolated for the first time from Cordia genus while sulfur allotrope [cyclooctasulfur (2)] was isolated for the first time from plant origin. Biological assays cordidepsine (1) exhibited significant anti-HIV integrase activity with IC50 = 4.65 μM; EtOAc extract of stem barks, EtOAc fraction of roots and leaves were not toxic against 3T3 cells.


Introduction
Acquired Immune Deficiency Syndrome (AIDS) is one of the major viral infections ravaging the world; around 76.1 million people have been infected since the first report in 1981 [1]. According to the report of the Cameroon Population Based HIV Impact Assessment (CAMPHIA) in 2018, viral prevalence in Cameroon is 3.4% [2] and about 37 million people around the world are currently living with the infection [1]. The absence of vaccines and associated resistance of virus to antiretroviral drugs [1] have contributed to the impairment of most patient's defense system by opportunistic bacterial infections such as tuberculosis and other bacterial pneumonia infections [3].
Bacterial infections are one of the major causes of morbidity and mortality in developing countries [4]. Though antimicrobial drugs have improved the healing of some patients with bacterial
Cordidepsine (1) Cyclooctasulfur (2) Lup-20(29)-en-3-triacontanoate (3) R: CH2OH: 1-(26-hydroxyhexacosanoyl)glycerol (4) Betulinic acid (6) R: CH3: Glyceryl-1-hexacosanoate (5) O Lupenone (7) β-amyrone (8) Lupeol (9) HO β-amyrin (10) Allantoin (11) 2′-(4hydroxyphenyl)ethylpropanoate (12) Stigmasterol glycoside ( Compound 1 was isolated as a white powder from n-hexane/EtOAc (6:4, v/v). Its molecular formula was established as C 17 3 ), two aromatic methyl signals at δ C 21.7 (C-12) and 9.8 (C-13). In addition, we observed in the downfield shield, two aromatic methine signals at δ C 117.4 (C-2) and 107.7 (C-9). This spectrum also revealed the presence of ten aromatic quaternary carbons, of which six oxygenated appeared at δ C 164.0 (C-3), 164.8 (C-4a), 142.5 (C-5a), 161.0 (C-9a), 143.4 (C-6), 154.7 (C-8) and the rest at δ C 152.7 (C-1), 110.9 (C-4), 122.8 (C-7), 111.9 (C-11a). The other signals were attributed to the lactone carbonyl at δ C 166.1 (C-11) and aldehyde function at δ C 193.9 (C-14). These data were similar to those of related depsidones previously isolated from several microorganisms [21][22][23][24] and some species of Garcinia genus [25]. The 1 H NMR spectrum (Table 1) confirmed the presence of two aromatic singlets at δ H 6.85 (1H, s, H-2) and δ H 7.08 (1H, s, H-9), suggesting two penta-substituted aromatic rings. The 1 H NMR also displayed two aromatic methyl signals at δ H 2.45 (3H, s, H-12), 2.19 (3H, s, H-13), one methoxy group signal at δ H 3.85 (3H, s, OCH 3 ) and the signal of aldehyde proton at δ H 10.43 (1H, s, H-14). The HMBC correlations ( Figure 2) of H-2 (δ H 6.85) to C-3 (δ C 164.0), C-14 (δ C 193.5), C-11a (δ C 111.9), C-12 (δ C 21.7) suggested that the aldehyde function and the aromatic methyl at δ C 21.7 are located on the same aromatic ring (I). The position of the aldehyde function was also supported by the correlation of H-14 (δ H 10.4) with C-4 (δ C 110.9) and C-4a (δ C 164.8), indicating that the aldehyde function is located at C-4 (δ C 110.9) as evidenced by the above HMBC correlations. In addition, the substituents of the second aromatic ring (II) were identified through HMBC correlations of H-9 (δ H 7.08) to C-5a (δ C 142.5), C-7 (δ C 122.8), C-8 (δ C 154.7), C-11 (δ C 166.1) and C-13 (δ C 9.8), as well as the HMBC correlations of H-13 to C-6; C-7; and C-8. The assignments of protons to carbon were further confirmed on the HSQC spectrum. However, the aldehyde correlations were shifted to around 30 ppm and the proton at δ H 10.43 ( Figure S5, Supplementary materials) due to spectral "folding" of aldehyde signal resulting from a poorly selected 13 C spectral width applied to reduce the recording time [26]. The required degrees of unsaturation suggested that the aromatic rings (I) and (II) should be linked by an ether and an ester bridge confirming a depsidone skeleton with a seven-membered ring (III) [23]. The proposed structure was reinforced by a NOESY experiment which revealed the correlations (Figure 2) between the methoxy group (δ H 3.85) and H-9 (δ H 7.08), between both methyl groups (δ H 2.19 and δ H 2.45), between aromatic proton at δ H 6.85 (H-2) and methyl protons at δ H 2.45 (H-12); and between both aromatic protons (δ H 6.85 and δ H 7.08). From an analysis of all the above data, the structure of compound 1 was established as 3,6-dihydroxy-8-methoxy-1,7-dimethyl-11-oxo-11H-dibenzo[b,e] [1,4]dioxepine-4 carbaldehyde which is a new depsidone to which the trivial name Cordidepsine was assigned. To the best of our knowledge, depsidone metabolite is reported here for the first time in the Boraginaceae family and Cordia genus.  (Table 1) of 1 exhibited the presence of 17 carbon signals, including one methoxy carbon at δC 56.3 (OCH3), two aromatic methyl signals at δC 21.7 (C-12) and 9.8 (C-13). In addition, we observed in the downfield shield, two aromatic methine signals at δC 117.4 (C-2) and 107.7 (C-9). This spectrum also revealed the presence of ten aromatic quaternary carbons, of which six oxygenated appeared at δC 164.0 (C-3), 164.8 (C-4a), 142.5 (C-5a), 161.0 (C-9a), 143.4 (C-6), 154.7 (C-8) and the rest at δC 152.7 (C-1), 110.9 (C-4), 122.8 (C-7), 111.9 (C-11a). The other signals were attributed to the lactone carbonyl at δC 166.1 (C-11) and aldehyde function at δC 193.9 (C-14). These data were similar to those of related depsidones previously isolated from several microorganisms [21][22][23][24] and some species of Garcinia genus [25]. The 1 H NMR spectrum (Table 1)  indicating that the aldehyde function is located at C-4 (δC 110.9) as evidenced by the above HMBC correlations. In addition, the substituents of the second aromatic ring (II) were identified through HMBC correlations of H-9 (δH 7.08) to C-5a (δC142.5), C-7 (δC 122.8), C-8 (δC 154.7), C-11 (δC 166.1) and C-13 (δC 9.8), as well as the HMBC correlations of H-13 to C-6; C-7; and C-8. The assignments of protons to carbon were further confirmed on the HSQC spectrum. However, the aldehyde correlations were shifted to around 30 ppm and the proton at δH 10.43 ( Figure S5, Supplementary materials) due to spectral "folding" of aldehyde signal resulting from a poorly selected 13 C spectral width applied to reduce the recording time [26]. The required degrees of unsaturation suggested that the aromatic rings (I) and (II) should be linked by an ether and an ester bridge confirming a depsidone skeleton with a seven-membered ring (III) [23]. The proposed structure was reinforced by a NOESY experiment which revealed the correlations ( Figure 2) between the methoxy group (δH 3.85) and H-9 (δH 7.08), between both methyl groups (δH 2.19 and δH 2.45), between aromatic proton at δH 6.85 (H-2) and methyl protons at δH 2.45 (H-12); and between both aromatic protons (δH 6.85 and δH 7.08). From an analysis of all the above data, the structure of compound 1 was established as 3,6-dihydroxy-8methoxy-1,7-dimethyl-11-oxo-11H-dibenzo[b,e] [1,4]dioxepine-4 carbaldehyde which is a new depsidone to which the trivial name Cordidepsine was assigned. To the best of our knowledge, depsidone metabolite is reported here for the first time in the Boraginaceae family and Cordia genus. Compound 2 was isolated as a yellow powder from ethyl acetate fraction of roots. Its HR-EI-MS spectrum exhibited a molecular ion peak at m/z 255.7766, suggesting the molecular formula of S8 (calcd 255.7766), which is an allotrope of sulfur. It also presented a difference of m/z 32 between two Compound 2 was isolated as a yellow powder from ethyl acetate fraction of roots. Its HR-EI-MS spectrum exhibited a molecular ion peak at m/z 255.7766, suggesting the molecular formula of S 8 (calcd 255.7766), which is an allotrope of sulfur. It also presented a difference of m/z 32 between two consecutive peaks on mass spectral data, suggesting a 32 S nature of the sulfur. The 32 S-NMR spectrum was not performed due to the insufficient quantity of isolated sample but its X-ray ( Figure 3) was performed and compound 2 was identified as cyclooctasulfur [27]. Cyclooctasulfur was previously reported from a fungal source [28]. To the best of our knowledge, this is the first report of cyclooctasulfur isolated from a plant source. consecutive peaks on mass spectral data, suggesting a 32 S nature of the sulfur. The 32 S-NMR spectrum was not performed due to the insufficient quantity of isolated sample but its X-ray ( Figure 3) was performed and compound 2 was identified as cyclooctasulfur [27]. Cyclooctasulfur was previously reported from a fungal source [28]. To the best of our knowledge, this is the first report of cyclooctasulfur isolated from a plant source.

Identification of Hemzi-Synthetic Derivatives
The acetylation reaction (Scheme 1) of isolated compounds 4 and 5 led to the synthesis of new derivatives, that is, cordicerol A (14) and cordicerol B (15), respectively. consecutive peaks on mass spectral data, suggesting a 32 S nature of the sulfur. The 32 S-NMR spectrum was not performed due to the insufficient quantity of isolated sample but its X-ray ( Figure 3) was performed and compound 2 was identified as cyclooctasulfur [27]. Cyclooctasulfur was previously reported from a fungal source [28]. To the best of our knowledge, this is the first report of cyclooctasulfur isolated from a plant source.

Biological activities
Many species of Cordia are used in traditional medicine for the treatment of various infectious diseases such as malaria, diarrhea, dysentery, stomach pain, fever, blood disorder and syphilis [9]. Due to time constraints, small amounts of samples and availability of assays, antibacterial, cytotoxicity activities of leaves, stem barks and roots crude extracts, fractions and anti-HIV activity of only some isolated compounds were investigated in this study.

Anti-HIV Activity
Cordidepsine (1) and allantoin (11) were tested in vitro for their inhibitory effect against HIV-1 Integrase. Cordidepsine (1) exhibited promising activity with an IC 50 value of 4.65 µM. Chicoric acid was used as a reference HIV-1 Integrase drug (IC 50 = 0.33 µM). Allantoin (11) also displayed weak activity with an IC 50 value of 412.94 µM ( Table 3). The activity of compound 1 can be justified by the fact that previous biological studies revealed that depsidones are potential antiviral agents [24]. Furthermore, with reference to compound 1, previous studies showed that an aromatic moiety next to (at least) two adjacent oxygens appears to be a structural element that is essential for activity against HIV-1 integrase [29]. In addition, the activity can also be explained by the presence of aromatic hydroxyl groups which have been reported to be effective inhibitors of integrase [29,30]. Moreover, previous studies showed that many species of Cordia are used as antiviral. To this instance, the leaves water extract of Cordia spinescens was demonstrated to be a potential inhibitor on HIV reverse transcriptase enzyme with IC 50 values of 6-8 µg/mL [27].

Antimicrobial Activity
The different extracts and fractions were tested for their antimicrobial activities (Table 4). EtOAc extracts of roots and stem barks showed good and significant activity against Gram positive bacteria (Bacillus subtilis and Staphylococcus aureus). These samples were inactive on Gram negative (Escherichia coli, Pseudomonas aeruginosa and Salmonella typhi) whereas EtOAc extract of leaves was not active against all these tested bacteria. This result is in concordance with the previous studies which revealed that species of Cordia genus are potential antibacterial agents [9]. The stem bark ethanolic extract of C. alliodora, neutral fraction of leaves of C. cylindrostachya, leaves and flowers methanolic extracts of C. boissieri exhibited antimicrobial activity against Gram positive and negative bacteria [9]. Furthermore, previous studies reported that betulinic acid (6), lupenone (7), lupeol (9) and β-amyrin (10) exhibit antibacterial activities [31][32][33] and these reported findings might also explain the observed activities of the extracts.

Cytotoxicity Activity
The percentage cell viability of ethyl acetate extract of stem barks, ethyl acetate fraction of roots and leaves were 99%, 96% and 95% respectively, compared to reference (Cyclohexamide 93%). Based on these findings, all these extracts did not exhibit obvious cytotoxicity activity against 3T3 cell (Human cells) and therefore, were not submitted to further IC 50 studies. To the best of our knowledge, this is the first report on the toxicity profile of Cordia millenii, Baker in the literature.

General Experimental Procedures
The NMR spectra ( 1 H and 13 C) were recorded on four different Bruker instruments including 400 MHz, 500 MHz, 600 MHz, 800 MHz. Chemical shifts are given in δ (ppm) value relative to TMS as internal standard. Deuterated solvents were used to dissolve the samples for NMR experiments. The HR-ESI-MS spectra were obtained from Bruker Compact QToF and MAXIS II mass spectrometers. EI-MS and FAB-MS data were recorded on a Jeol JMS HX 110 mass spectrometer. Silica gel (230-400 meshes) was used for column chromatography. Thin Layer Chromatography (TLC) and preparative TLC were performed on precoated silica gel plates (60 F 254 , Macherey-Nagel) using various solvent systems as eluent. Spots were visualized using UV light (λ max 254 and 366 nm) and diluted sulphuric acid (10%).

Plant Materials
The plant materials (leaves, stem bark and root) of Cordia millenii, Baker were collected from Batoufam, in the West region of Cameroon in May 2015. A voucher specimen (N • 35142/HNC) was deposited in the National Herbarium of Cameroon in Yaoundé.

Acetylation Reaction of Compounds 4 and 5
A mixture of 10 mg of compound 4, 1 ml of acetic anhydride and 1 ml of pyridine was stirred at room temperature for 12 h. After the end of the reaction, a normal workup procedure was followed: 10 mL chloroform and 10 mL distilled water were added to the reaction mixture, the organic layer was separated and dried under reduce pressure. The resulted product was purified by CC with n-hexane/EtOAc (9:1, v/v) to afford compound 14 (6 mg). The same reaction conditions were applied to compound 5 to afford compound 15 (4.8 mg).

HIV-1 Integrase Strand Transfer Reaction Assay
The HIV-1 subtype C integrase (CIN) strand transfer inhibition assay was adapted from previously described method [34]. 20 nM double-stranded biotinylated donor DNA (5 -5Biotin TEG/ACCCTTTTAGTCAGTGTGGAAAATCTCTAGCA-3 annealed to 5 ACTGCTAGAGATTTTCC ACACTGACTAAAAG-3 ) was immobilized in wells of streptavidin coated 96-well microtiter plates (R&D Systems, USA). Following incubation at room temperature for 40 min and a stringent wash step, 5 µg/mL purified recombinant HIV-1 CIN in buffer 1 (50 mm NaCl, 25 mM Hepes, 25 mM MnCl 2 , 5 mM β-mercaptoethanol, 50 µg/mL BSA, pH 7.5) was added to individual wells. Test samples and chicoric acid were added to individual wells to a final concentration of 20 µM (pure compounds and chicoric acid) and 20 µg/mL (extracts). Recombinant HIV-1 subtype C IN was assembled onto the preprocessed donor DNA through incubation for 45 min at room temperature. Strand transfer reaction was initiated through the addition of 10 nM (final concentration) double-stranded FITC-labelled target DNA (5 -TGACCAAGGGCTAATTCACT/36-FAM/−3 annealed to 5 -AGTGAATTAGCCCTTGGTCA−/36-FAM/−3 ) in integrase buffer 2 (same as buffer 1, except 25 mm MnCl 2 replaced with 2.5 mm MgCl 2 ). After an incubation period of 60 min at 37 • C, the plates were washed using PBS containing 0.05% Tween 20 and 0.01% BSA, followed by the addition of peroxidase-conjugated sheep anti-FITC antibody (ThermoScientific, USA), diluted 1:1000 in the same PBS buffer. Finally, the plates were washed and peroxidase substrate (Sure Blue Reserve TM , KPL, USA) was added to allow for detection at 620 nm using a Synergy MX (BioTek®) plate reader. Absorbance values were converted to percentage enzyme activity relative to the readings obtained from control wells (enzyme without inhibitor).

Antibacterial Activity
This assay was used to screen the antibacterial activity of the extracts and it was evaluated using the microplate alamar blue method [35,36]. Organisms, grown in Mueller Hinton medium (Oxoid Limited, UK), were inoculated in Mueller Hinton Broth (MHB) (Oxoid Limited, UK) and were incubated overnight at 37 • C. Fully grown turbid bacterial cultures were then diluted to adjust with 0.5 McFarland Turbidity Index (equivalent to 1.5 × 10 8 CFU/mL). Stock solutions (60 mg/mL) of different extracts were prepared in DMSO and 10 µL each of these stock solutions were placed in wells of flat bottom, polystyrene, sterile 96-wells micro titer plate except the positive control wells (media + bacteria). This gave 3000 µg/mL concentrations of extracts in the final 200 µL solution. Finally, bacterial suspension (3 × 10 6 CFU/mL) was added in each well. Plates were sealed with parafilm and incubated at 37 • C for 18-24 h. Next day, 20 µL of 0.02% resazurin sodium salt dye (Chem-Impex-Int'L Inc.) was added to each well and was incubated in a shaking incubator at 80 rpm and 37 • C for 2-3 h. The color change from blue to reddish pink indicated the growth of bacteria. For quantitative analysis, plates were read at 570 nm and 600 nm in a Multiskan™ GO microplate spectrophotometer, (ThermoScientific, USA). The % inhibition of bacterial growth was calculated using the formula: % inhibition = 100 − (% difference in the reduction between treated and positive control bacteria)

Cytotoxicity Activity
It was evaluated in 96-well flat-bottomed micro plates by using the standard MTT (3-[4, 5-dimethylthiazole-2-yl]-2, 5-diphenyl-tetrazolium bromide) colorimetric assay [37]. For this purpose, 3T3 (mouse fibroblast) cells were cultured in Dulbecco's Modified Eagle Medium, supplemented with 5% of fetal bovine serum (FBS), 100 IU/mL of penicillin and 100 µg/mL of streptomycin in 75 cm 2 flasks and kept in 5% CO 2 incubator at 37 • C. Exponentially growing cells were harvested, counted with a hemocytometer and diluted with a particular medium. Cell culture with the concentration of 5 × 10 4 cells/mL was prepared and introduced (100 µL/well) into 96-well plates. After overnight incubation, medium was removed and 200 µL of fresh medium was added with different concentration of compounds (1-30µM). After 48h, 200 µL MTT (0.5 mg/mL) was added to each well and incubated further for 4h. Subsequently, 100 µL of DMSO was added to each well. The extent of MTT reduction to formazan within cells was calculated by measuring the absorbance at 540 nm, using a micro plate reader (Spectra Max plus, Molecular Devices, CA, USA). The cytotoxicity was recorded as concentration causing 50% growth inhibition (IC 50 ) for 3T3 cells. The percentage inhibition was calculated by using the following formula: %