Diterpenes from the Aerial Parts of Plectranthus ornatus Codd

Five new diterpenes derivatives named as ornatin A, B, C, D and E, in addition to six known related diterpenes were isolated from the aerial parts of cultivated specimens of Plectranthus ornatus. The structures were elucidated using a combination of 1D/2D nuclear magnetic resonance (NMR) spectroscopy, high-resolution electrospray ionization mass spectrometry (HRESIMS) and comparison with published NMR data of analogous compounds. All isolated compounds were assayed against four human cancer cell lines, and Gram-positive and Gram-negative bacteria strains. None of them showed any cytotoxic activity, but ornatin C, D, E and three related diterpenes displayed marginal bactericidal or bacteriostatic effects against the Gram-positive strains.


Introduction
The genus Plectranthus L'Her (Lamiaceae, subfamily Nepetoideae, tribe Ocimeae, subtribe Plectranthinae) comprises approximately 300 species largely distributed over the African, Asian and Australian continents. 1 Plectranthus is a large genus well known by its diversity of ethnobotanical uses, being especially indicated to treat digestive disorders, skin diseases, infections and respiratory problems. 2In general, are rich source of essential oils, terpenoids and phenol compounds. 3harmacological properties such as anti-inflammatory, 4 antioxidant, 5 antimicrobial, 6 anti-tumoral 7 and diuretic 8 have been demonstrated for several isolated compounds from Plectranthus, corroborating with its larger medicinal uses.
Plectranthus ornatus Codd (syn.Coleus comosus Hochst.ex Gurke) is a perennial and aromatic herb, widespread around the new world. 9In the northeast of Brazil, P. ornatus, popularly known as "malva santa" or "boldo miúdo", is cultivated as a very important medicinal plant, indicated as analgesic and particularly to treat gastric disorders. 10Several antimicrobial labdanes, ent-clerodanes, and halimane-type diterpenoids have been previously isolated from P. ornatus. 11s part of a multidisciplinary program, where the efforts are devoted to study medicinal plants or congeners, in order to unveil their pharmacological or biological properties, herein we describe the isolation and characterization of five new diterpenes derivatives together with six known compounds from the aerial parts of cultivated specimens of P. ornatus, as well as the results of pharmacological and biological assays with the isolated secondary metabolites (Figure 1).

General experimental procedures
Optical rotations were measured on a PerkinElmer 341 digital polarimeter.Infrared (IR) spectra were obtained on a PerkinElmer FT-IR spectrum 1000 spectrometer.Accurate mass spectra were acquired on a liquid chromatography-mass spectrometry ion-trap and time-of-flight (LCMS-IT-TOF, Shimadzu) spectrometer.Nuclear magnetic resonance (NMR) spectra were performed either on Bruker DPX-300 or DRX-500 spectrometers.Open column chromatography (CC) were carried out with silica gel (60 or 230 mesh, Merck) and Sephadex LH-20 (Phenomenex), while thin layer chromatography (TLC) were conducted on precoated silica gel aluminum sheets (60 F 254 , 0.20 mm, Merck).Semi preparative Gemini-Phenomenex C-18 column (150 × 10 mm) was used on an ultra-fast liquid chromatography (UFLC, Shimadzu) system equipped with a SPD-M20A diode array UV-Vis detector.High performance liquid chromatography (HPLC) procedures were carried out using UV PDA detection 210-400 nm, injection volume 200 µL, and flow rate of 4.72 mL min -1 .

Plant material
Plectranthus ornatus was harvested in May 2012 at the medicinal plant garden Francisco José de Abreu Matos, Universidade Federal do Ceará (UFC).The plant authentication was performed by Dr Maria Iracema B. Loiola from the Herbario Prisco Bezerra (EAC), UFC, where a voucher (No. 56806) was deposited.

Antibacterial assay
The antibacterial activity of the diterpenes was evaluated against four bacteria: Staphylococcus aureus ATCC 25923, S. epidermidis ATCC 12128, Pseudomonas aeruginosa ATCC 9027 and Escherichia coli ATCC 11303.Before experimental procedures, each bacterial species was grown in tryptic soy agar (TSA, Himedia, USA) for 24 h at 37 °C.The cells were inoculated in tryptic soy broth (TSB, Himedia, USA) and incubated for 18 h at 37 °C under constant agitation.Subsequently, cell's concentration of each bacterium was adjusted to 1 × 10 6 cell mL -1 using spectrophotometry and calibration curves previously determined.The minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) were determined by the broth microdilution method according with the guidelines from the National Committee for Clinical Laboratory Standards, M7-A6 (NCCLS, 2003), 13 with some modifications.Briefly, different concentrations of the compounds (7.8 to 500 µg mL -1 ) were prepared in TSB (with 4% of dimethyl sulfoxide) and aliquots of 100 µL of each compound were mixed with bacterial suspensions (100 µL) in the 96-well polystyrene plates.The plates were incubated at 37 °C during 24 h under constant agitation.The MIC value was established as the lowest concentration of compound able to inhibit the visible growth of microorganism.MBC value was determined by transferring 10 µL from each well without visible growth into TSA plates.MBC was considered the lowest concentration that completely inhibited microbial growth in the plates.

22
-43.96° (c 0.  1 and 2) of 1 were similar to those reported for the diterpenes 11-acetoxyneoclerodane, which were previously isolated of the P. ornatus. 19The only difference between these two compounds was the replacement of the carboxyl function at C-15 (d C 171.7) for a methyl ester (d C 166.7, 51.0 / d H 3.66).The double bond C-13/C-14 was defined as E based on chemical shifts (see Table 1) in agreement with previous diterpenes isolated of P. ornatus. 16As can be seen in the Experimental section, no MeOH has been used during any extraction or chromatographic procedures, thus, eliminating the possibility of 1 being an artifact.The final structure of 1 was confirmed by the heteronuclear multiple bond correlation (HMBC) spectrum through the selected long-range correlations depicted by arrows in Figure 2 3), also in agreement with the stereochemistry reported previously for 11R* diterpenes from P. ornatus. 16,18 1 and 2), where the difference between these compounds were the presence of a double bond at C2/C3 and an aldehyde function at C-2, instead of a carboxyl acid.This proposition was supported by the HMBC spectrum, which exhibited correlations for the aldehyde proton d H 9.93 (H-3) with the carbon signals at d C 137.9 (C-2) and d C 28.1 (C-1), as well as correlations of d H 1.99 (s, 3H, Me-18) with d C 137.9 (C-2) and d C 51.7 (C-5).It is worthy of notice that both structures of 2 and 4 were also supported by comparison of the 1 H and 13 C NMR data of these with those reported to the rearranged (4→2)-abeo-clerodane diterpenes. 22he relative stereochemistry of 4, as depicted in Figure 3, was defined by NOESY and literature data, 16,18 which was similar to compound 2. Thus, the structure of compound 4, a new (4→2)-abeo-clerodane diterpene, was established and named as ornatin D.
Compound 5, a colorless oil, had its molecular formula C 22 H 32 O 4 determined from the ion peak [M + K] + at m/z 399.2495 in the HRESIMS.Its IR spectrum exhibited UV absorption bands at 3393 and 1691 cm -1 for conjugated carboxylic acid, and 1734 cm -1 for an ester function.The Vol. 28, No. 6, 2017 1 H NMR spectrum showed signals for olefinic protons at d H 6.05 (d, 1H, J 9.5 Hz, H-3), 5.80 (m, 1H, H-2), 5.66 (s, 1H, H-14), 4.80 (s, 1H, H-18a) and 4.66 (s, 1H, H-18b), the latter two corresponding to vinyl protons of an exocyclic double bond.In addition, it displayed signals for an oxymethine proton at d H 5.33 (d, 1H, J 10.3 Hz, H-11) and five methyl groups, including a methyl attached to an olefinic carbon at d H 2.11 (s, 3H, Me-16) and a methyl of an acetoxyl group at d H 2.01 (s, 3H, C-11').The 13 C NMR spectrum exhibited signals to 22 carbons atoms assigned as five methyls, five methylenes, six methines and six non-hydrogenated carbons, including four carbon-carbon double bonds and two carboxyl carbons at d C 171.0 (C-15) and d C 170.8 (C-11').The 1 H and 13 C NMR data of 5 were similar to those reported initially to 1-4, showing differences only for the A ring.A system comprising an exocyclic double bond of conjugated diene located at A ring was confirmed through the long range correlations of the allylic protons d H 2.40 (m, 2H, H-1) with d C 128.1 (C-2) and d C 129.1 (C-3) and correlations of the exomethylene protons d H 4.80; 4.66 (s, 1H-18ab) with C-3 and C-5 (Figure 2).Finally, the relative stereochemistry of 5 was confirmed by NOESY spectrum, similar to those of compounds 1-4.Based on the aforementioned data, the complete structure of 5 was established as a new ent-clerodane diterpene which was named ornatin E.
Regarding the antibacterial activity, compounds 1 through 11 were evaluated by MIC and MBC assay against clinically relevant bacteria.The results showed that some diterpenes were bacteriostatic or bactericidal against Gram-positive bacteria.Compounds 3, 4 and 6 showed MIC for S. aureus at concentrations of 250 µg mL -1 and 7 and 8 at 500 µg mL -1 .Furthermore, S. epidermidis was susceptible only to compound 5 at 500 µg mL -1 .Interestingly, only compound 3 showed bactericidal effects on S. aureus at 500 µg mL -1 .On the other hand, no compounds were effective against Gram-negative bacteria.The antibacterial results found here corroborates with other studies reporting on the antimicrobial activity of diterpenes. 23Moreover, the effective concentration of the diterpenes used in this study were similar to those found for other works. 24According Urzúa et al. 25 the antimicrobial activity of diterpenes occurs by destabilization of the plasma membrane caused by the interaction with the lipid bilayer fatty acids present in its structure.Moreover, some studies showed that diterpenes exhibit antibacterial activity only against Gram-positive bacteria, but not against Gram-negative bacteria.Probably, the outer membrane of the Gram-negative bacteria decreases the permeability of the diterpenes and increases resistance to the action of these compounds.

Conclusion
Five new diterpenes, along with six known ones, were isolated from the aerial parts of Plecthranthus ornatus.Diterpenes are the most characteristic compounds of the genus Plecthranthus, especially those having the abietane and labdane skeletons.However, previous work on P. ornatus have reported it, instead, as a prolific source of clerodane   and halimane diterpenes which are not well common to the genus.The results reported in the present work indeed corroborate the tendency of P. ornatus to behave differently of the other species belonging to Plecthranthus.Nine (1-5, 7, 8, 10, 11), out of eleven diterpenes reported here belong to the clerodane class, while the two others belong to the labdane ( 9) and abietane (6) classes, respectively.Two of the isolated ent-clerodanes (2, 4) present an unusual (4→2)-abeo-clerodane rearranged, while no halimane has been isolated in the present work.Unfortunately, none of the eleven isolated compounds showed any cytotoxic action, and the antimicrobial activity was too weak in order to be considered effective.
The 1 H NMR spectrum showed signals to olefinic protons at d H 5.64 (s, 1H, H-14) and 5.19 (s, 1H, H-3), to an oxymethine proton at d H 5.44 (d, 1H, J 10.9 Hz, H-11) and a methoxyl at d H 3.66.In addition, were observed six methyl signals, two of which attached to sp 2 carbons at 1, CH 2 Cl 2 ), had its molecular formula assigned as C 23 H 36 O 4 based on the [M + Na] + ion peak at m/z 399.2508 (calcd.399.2506,D +0.50 ppm) in the HRESI mass spectrum.
, while its relative stereochemistry was established by the nuclear Overhauser spectrum (NOESY) (see Supplementary Information).The trans-configuration for the A and B rings of the decaline moiety was confirmed by the dipolar coupling effect (NOE) between H-8/H-10 and Me-19/Me-20 both set in a 1,3-diaxial relationship.The observed NOE's for Me-20 with Me-17 and Me-11' (methyl of the acetoxyl group) confirmed the a-position inferred for both AcO-11' and Me-17 (Figure Its 1 H NMR spectrum displayed signals for an olefinic proton at d H 5.66 (s, 1H, H-14), an oxymethine proton at d H 5.10 (d, 1H, J 10.3 Hz, H-11), and six methyl groups, including one attached to a sp 2 carbon at d H 2.20 (s, 3H, H-16) and another one of an acetoxyl group at d The 13 C NMR spectrum showed signals for 22 carbon atoms, accounting for six methyls, five methylenes, five methines including one olefinic d C 119.6 (C-14) and one oxymethine carbons d C 77.6 (C-3).The 13 C NMR and DEPT spectra displayed six non-hydrogenated carbons, including one olefinic carbon d C 158.6 (C-13), an acetoxyl d C 172.8 (C-11') and a conjugated acid carboxyl d C 169.7 (C-15).The 13 C NMR data of 3 were similar to those of 1, except by the appearance of two carbon signals at d C 77.6 and d C 76.9 corresponding to the hydroxylation of C-3 and C-4, instead the carbon-carbon double bond.Based on the HSQC spectrum the carbon signal at d C 77.6 exhibited correlation with the proton signal at d H 3.50 (br t, J 2.3 Hz, H-3).Comparing these data with those of 3β,4β and 3a,4βdihydroxylated ent-clerodane diterpenes from literature, 20,21 the structure of compound 3 was established as the 3a,4β derivative which was named as ornatin C. Cl 2 ), had its molecular formula C 23 H 38 O 6 determined by HRESIMS through the ion peak [M + Na] + at m/z 399.2114 (calcd.for C 22 H 32 O 5 Na, 399.2142,D +1.75 ppm).The 1 H NMR spectrum showed signals for an aldehyde proton at d H 9.93 (s, 1H, H-3), an olefinic proton at d H 5.64 (s, 1H, H-14), an oxymethine proton at d H 5.12 (dd, 1H, J 9.6, 2.6 Hz, H-11), and six methyl groups, two of which attached to sp 2 carbons at d H 2.12 (s, 3H, H-16) and 1.99 (s, 3H, H-18), and another one of an acetyl group at d H 1.96 (s, 3H, Me-11').The 13 C NMR spectrum exhibited 22 carbon signals, classified into six methyls, four methylenes, four methines and seven non-hydrogenated carbons, including two acetoxy carbonyl d C 170.
16,18 compound 1 was assigned as the ent-clerodane diterpene designated as ornatin A, in allusion to the species.Compound 2, obtained as a colorless resin, [a] D 22 −34.44°(c0.06,CH 2 Cl 2 ), had its molecular formula determined as C 22 H 34 O 7 based on the [M + Na] + ion peak found at m/z 433.2197 (calcd.433.2202,D+0.46ppm) in the HRESIMS.H 2.15 (m, 1H, H-1a) with the methine carbons at d C 51.4 (C-2) and 45.7 (C-10), and with the carboxyl at d C 180.3 (C-3), as well as correlation of the d H 1.35 (s, 3H, Me-18) with the quaternary carbon at d C 49.5 (C-5) and with an oxygenated non-hydrogenated carbon at 82.7 (C-4) allowed to suggest a rearranged five membered ring bearing a carboxyl group at C-2 and a hydroxyl at C-4.Finally, the relative stereochemistry of 2 was established by the NOESY spectrum which showed dipolar couplings for H-2/Me-18 and Me-18/Me-19 indicating a β-position to the carboxyl acid at C-2, while the NOE's of H-8/H-10 and Me-19/ Me-20 supported a trans fusion of the A/B rings, as depicted in Figure3.The stereochemistry of C-11 was suggested as R* in accordance with previously diterpenes isolated from P. ornatus.16,18Thus, the structure of compound 2