Diterpenes Isolated from Three Different Plectranthus Sensu Lato Species and Their Antiproliferative Activities against Gynecological and Glioblastoma Cancer Cells

Fourteen diterpenes were isolated from methanol extracts of the aerial parts ofColeus comosus,Coleus forsteri “Marginatus”, and Plectranthus ciliatus. The compounds belong to the abietane (1–4, 9–11, and 13), ent-clerodane (5–8), and ent-kaurane (14, 15) classes. Three new compounds were isolated from C. comosus, including 3-O-acetylornatin G (2), 3,12-di-O-acetylornatin G (3), ornatin B methyl ester (5), and ornatin F (4), for which we proposed a revised structure. The structures of the compounds were determined by comprehensive spectroscopic data analysis. The isolated diterpenes were examined in silico for their physicochemical and early ADME properties. Their antiproliferative effects were determined in vitro using human breast (MDA-MB-231 and MCF-7), cervical (HeLa), and glioblastoma (U-87 MG) cancer cell lines. The royleanone- and hydroquinone-type abietane diterpenes (9–13)exhibited the most potent antiproliferative activity against all cancer cell lines tested, particularly against glioblastoma cells, with IC50 values ranging from 1.1 to 15.6 μM.

Cancer is a leading cause of death worldwide, with almost 10 million deaths in 2020. 1 Although many drugs were developed for their treatment, toxic side effects and acquired therapeutic resistance pose major limitations to their clinical use.Therefore, there is an urgent need for new treatments. 2Over 60% of the current antineoplastic drugs are derived from natural sources. 3Vinblastine, vincristine, paclitaxel, and the semisynthetic derivatives, etoposide and teniposide, are some outstanding examples of how plants represent an invaluable reservoir of active anticancer compounds. 2,4lectranthus sensu lato (Lamiaceae), a large and widespread genus comprising three distinct genera [i.e., Coleus (294 species), Plectranthus sensu stricto (72 species), and Equilabium (42 species), 5 has attracted interest because of its important role in traditional medicine.Many species have been used for centuries to treat various respiratory, digestive, genitourinary, and dermatological disorders.6 The anticancer potential of this genus has been extensively investigated.Plectranthus s.l.extracts and their diterpene constituents exert remarkable antiproliferative and cytotoxic activities on various cancer cell lines.Among the diterpenes, abietanes from the subclasses of royleanones (6,7-dehydroroyleanone and 7α-acetoxy-6β-hydroxyroyleanone), hydroquinones (coleon U), and quinone methides (parviflorone D) are the most promising.7−9 However, a recent study conducted by Ito and co-workers indicated that spirocoleon-type abietane diterpenes exhibit cytotoxicity against human breast (MCF-7), pancreatic (PSN-1), and cervical (HeLa) cancer cell lines with low toxicity against a normal lung fibroblast cell line (WI-38).10 Coleus comosus Hochst. e Gurke (syn. Pectranthus comosus, Plectranthus ornatus) 5 was reported to produce spirocoleonand hydroquinone-type abietanes, 11,12 ent-clerodanes, 11−14 labdanes, 12−16 and halimanes.16 Coleus forsteri"Marginatus" Benth.(syn.Plectranthus forsteri "Marginatus") 5 is a source of royleanone-and hydroquinone-type abietane diterpenes.17−19 To our knowledge, no diterpenes have been isolated from Plectranthus ciliatus E.Mey.(syn.Plectranthus natalensis).5 In a preliminary screen, however, the methanol extract of fresh leaves exhibited cytotoxic activity toward human drug-sensitive CCRF-CEM and multidrug-resistant CEM/ADR5000 leukemia cell lines.20 As a continuation of our previous study using this genus, 17,21−23 the three above-mentioned Plectranthus s.l.species were selected to expand the available phytochemical data and to discover novel anticancer natural compounds.
Fourteen diterpenes (1−11, 13−15) were isolated from C. comosus, C. forsteri "Marginatus", and P. ciliatus.These compounds, along with 6,7-dehydroroyleanone (12) previously isolated by our group, 17 were assessed for their druglikeness based on the predicted physicochemical and early ADME parameters.Their antiproliferative activities were evaluated using MDA-MB-231 and MCF-7 human breast cancer cell lines, the HeLa human cervical cancer cell line, and the U-87 MG human glioblastoma cell line.

■ RESULTS AND DISCUSSION
Secondary metabolites isolated from Plectranthus s.l.species, particularly diterpenes and phenolic compounds, exhibited characteristic absorption patterns in the UV spectral region. 8herefore, the chloroform-soluble phases prepared from the methanol extracts of C. comosus, C. forsteri "Marginatus", and P. ciliatus were analyzed by high-performance liquid chromatography (HPLC)-DAD for fast screening of diterpene content.Preliminary HPLC-DAD analysis of C. comosus and P. ciliatus revealed the presence of constituents, whose UV spectra showed one absorption band with a maximum in the region 215−270 nm that could be attributed to different classes of diterpenes 13,24−26 (Figures S1 and S3, Supporting Information).The chloroform-soluble phase of C. comosus was found to be a complex mixture with nine peaks between 11 and 31 min that were supposed to belong to diterpenes.On the other hand, the chloroform-soluble phases of P. ciliatus appeared as a simpler mixture, showing only two peaks (t R = 19.007and 21.538 min) attributable to diterpenes.The HPLC chromatogram of the chloroform-soluble phase of C. forsteri "Margin- atus" (Figure S2, Supporting Information) contained two peaks (t R = 20.206 and 24.081 min) with typical "royleanonetype" UV spectra, 27 having an absorption band with a maximum at ∼270 nm and another broad band with a maximum at ∼400 nm.One peak (t R = 29.832min) possessed a "diosphenol-type" UV spectrum 28 with four absorption bands characterized by maxima at ∼262, ∼ 286, ∼ 333, and ∼382 nm.
Compound 5 was present as a white, amorphous powder.The peak of the protonated molecule at m/z 425.2561 [M + H] + (calcd for C 23 H 37 O 7 , 425.25393) in the HRESIMS spectrum predicted the molecular formula C 23 H 36 O 7 with six indices of hydrogen deficiency.Tandem analysis of the 1 H NMR and HSQC spectra revealed the signals for 34 protons classified as seven methyl, four methylene, and five methine protons including one olefinic and one oxygenated methine proton (Table 1).The two remaining protons were likely part of the hydroxyl and carboxyl group.The APT spectrum exhibited 23 carbon resonances (Table 2).Three carbonyl carbons (δ C 178.7, 170.6, 166.8) and a nonprotonated sp 2 carbon (δ C 157.1) along with an olefinic carbon (δ C 118.2) indicated the presence of three carbonyl groups and one trisubstituted olefinic bond, respectively.To satisfy the six indices of hydrogen deficiency, a bicyclic ring system was proposed.In addition, the resonances assigned to an acetoxy group (δ C 170.6, δ C−H 21.1/2.01)and a methoxy group (δ C−H 51.0/3.67)indicated that 20 carbon atoms were left for the skeleton suggesting a diterpene core for compound 5.
Compared with the published NMR data for the known compound ornatin B, 12 only minor differences were observed.The presence of an additional methoxy group (δ C−H 51.0/ 3.67) and its HMBC correlation with the carbonyl carbon C-15 (δ C 166.8) indicated that compound 5 was a methyl ester of ornatin B (Figures S7, Supporting Information).The NOE cross-peaks revealed that this compound has the same relative configuration as ornatin B. 12  H 3 -19, suggested that these protons are on the same side of the bicyclic ring core with α-orientation.Similarly, the correlation of H-10/H-6a indicated that these protons are located on the opposite side of the bicyclic diterpene skeleton; thus, they are β-oriented.The NOE correlation H-10/H-8 was decisive for determining Me-17 as α-oriented (Figure S8, Supporting Information).Only a configuration for the C-11 stereogenic center could be assigned with the NOESY because of the free rotation ability of the side chain.However, based on previous reports 12−14 on ent-clerodanes isolated from C. comosus substituted with the same C-9 side chain, the configuration of C-11 was proposed as R*.The Econfiguration of the trisubstituted olefinic double bond Δ 13 (14) was evident from the characteristic resonances of Me-16 (δ H 2.18, δ C 18.8), 13,14,36 and the strong supportive NOE interaction H-12b/H-14. 16Compound 5 was identified as depicted (Figure 1) and designated as the ornatin B methyl ester.
Isolation of compounds 1−4 confirmed that spirocoleontype abietane diterpenes esterified with acetic acid at different positions are the major secondary metabolites produced by C. comosus.A comparison of their 13 C NMR spectroscopic data also suggests that chemical shift of C-16 may be considered diagnostic for determining the arrangement in ring C. While trans-spirocoleons 1−3 displayed the C-16-assigned signal at δ C 25.4, 26.7, and 26.8, respectively, the corresponding signal of cis-spirocoleon 4 was observed at δ C 13.4.This result is consistent with the data published for cis-and transspirocoleons with the absolute configuration (12R,13S,15R) and (12R,13S,15S), respectively, although their 13 C NMR spectra were recorded in (CD 3 ) 2 CO. 35 Interestingly, C. comosus is the only known Plectranthus s.l.species that biosynthesizes ent-clerodane diterpenes having either a 6/6fused ent-clerodane scaffold as present in the structures of 7 and 8 or a modified 5/6-fused (4 → 2)-abeo-ent-clerodane ring system present in 5 and 6.All the ent-clerodanes isolated from C. comosus contain a C-9 side chain with an E-configured Δ 13 (14) double bond and a 15-carboxyl group capable of forming methyl esters. 11−14 While abietanes 9−13 have already been reported to be biosynthesized by C. forsteri "Marginatus" 17−19 and are among the most widespread representatives of Plectranthus s.l.diterpenes, 9 ent-kauranes 14 and 15 were isolated only from Plectranthus strigosus Benth.ex E.Mey. 5,37hus, their isolation from P. ciliatus was the first report of diterpenes in this species.
Fifteen diterpene compounds isolated from three different plants (C.comosus, C. forsteri "Marginatus", and P. ciliatus) were subjected to in silico physicochemical and early ADME characterization (Table 3) and cell proliferation assays (Table 4).First, the compounds were evaluated based on the Lipinski rule of five (Ro5), 38,39 which is associated with classical druglikeness, and the central nervous system multiparameter optimization 40 descriptor for BBB permeability.In addition, aqueous solubility, Caco-2 penetration, and Pgp efflux risk were also predicted as screening parameters using the ACD/ Percepta software package. 41ased on the physicochemical data, compounds 5 and 12 did not conform to Ro5 because of their high lipophilicity (log P: 5.9 and 5.4, respectively) and the relatively high molecular weight of compound 4 (M w : 507).However, an increased log D pH7.4 value was also assigned to 13 because of its reduced acidic character (pK a,acid : 7.9, log D pH7.4 :4.6).For the spirocoleon-type abietanes 1−4, increased topological polar surface area (TPSA) values were identified, indicating a lower Table 4. Antiproliferative Effect of the Isolated Diterpenes on Human Gynecological (MDA-MD-231, MCF-7, and HeLa) and Glioblastoma (U-87 MG) Cell Lines by MTT Assay for 72 h a Limit for antiproliferative effect�IC 50 < 10 μM (green values).b Lipophilic ligand efficiency (LLE) parameters were calculated by the following equation: LLE = pIC 50 − log P (using Table 3. data).Classification system for LLE: green (lower promiscuity risk): IC 50 < 10 μM and LLE ≥ 1.5, yellow (moderate promiscuity risk): IC50 < 10 μM and 1.5 > LLE ≥ 1, red (higher promiscuity risk): IC 50 < 10 μM and 1.0 > LLE.Values not marked with a color do not fulfill any of the classification criteria.c Ambiguous fitting, confidence interval cannot be calculated due to the high slope of the regression curve, the provided values represent experimental dilutions below and above the IC 50 ; growth inhibitory values were −3.7 and 63.7% at 12.5 and 25 μM, respectively.Cis = Cisplatin, TMZ = Temozolomide.
predicted bioavailability using the Egan filter. 42The increased polarity associated with TPSA for these compounds and their low predicted aqueous solubility (≤10 μg/mL) may be contradictory, but the predicted poor solubility may be explained by a rigid close-to-planar structure of spirocoleontype abietanes.Combining log BB (≥−0.2) as a pharmacokinetic parameter and Wager's optimization (CNS MPO ≥ 4.5) for distribution in the CNS, ent-clerodanes 6, 8, abietanes 9−10, and ent-kauranes 14−15 are suitable candidates.An increased risk related to intestinal absorption was identified for compound 13 (Caco-2 − P e × 10 −6 cm/s < 10).For compounds 1−4, the Pgp substrate positivity indicated by the ADME predictor should also be emphasized, which suggests an elevated risk for efflux transport and related pharmacokinetic consequences.This is consistent with previous studies reporting the alteration of Pgp function by abietane diterpenes. 23,43,44he antiproliferative effect of the isolated diterpenes was evaluated against selected human gynecological (MDA-MB-231, MCF-7, and HeLa) and glioblastoma (U-87 MG) cancer cell lines (Table 4).Spirocoleon-type abietanes (1−4) were weak or inactive as antiproliferative agents against these cells.Ornatin G and F (1 and 4) are already known diterpenes, but to our knowledge, their effects have not been determined.Compounds 5−8 with an ent-clerodane skeleton did not show any effect on any of the cell lines at the tested concentrations.This is consistent with previous reports for compounds 6 and 8, which exerted cytotoxicity neither on soft tissues nor on leukemia cancer cells. 12The isopropylsubstituted royleanone-and hydroquinone-type abietanes 9− 13 exhibited the highest antiproliferative activity.These compounds (9−13) were previously isolated from various Plectranthus species, 8,45,46 and their antiproliferative effects were observed to be lower than the screening criterion of IC 50 ≤ 10 μM in these cell lines.Finally, ent-kaurane diterpenes (14−15) exhibited moderate antiproliferative effects on both gynecological and glioblastoma cells.
In general, isopropyl-substituted abietanes from the royleanone and hydroquinone subclasses exhibited selective and significantly higher antiproliferative effects against glioblastoma compared with those of the gynecological cancer cell lines.Thus, while compounds 9−10 were not effective against gynecological cancer cells and IC 50 values of compounds 11−12 ranged from 13.5 to 30.1 μM, all four abietanes (9−12) were effective against glioblastoma cell lines with IC 50 values below 10 μM.The IC 50 values of compound 13 were also below 10 μM for HeLa and MCF-7 cancer cell lines.The results of compound 13 against MCF-7 were consistent with those of previous reports. 8,45Royleanone diterpenes (9−12) containing a p-benzoquinoid C-ring exhibited stronger antiproliferative effects on glioblastoma compared with hydroquinone coleon U (13) containing an aromatic C-ring.Therefore, the p-benzoquinone moiety of the isopropyl-substituted abietanes is important for their antiproliferative activity.Previously, the antiproliferative effect of compound 11 was determined using another CNS tumor cell line, SF-268. 45It showed a potent activity with IC 50 values of 8.6 μM.Another abietane diterpene, compound 9, exhibited a 1 order of magnitude weaker effect on cell proliferation.This suggests that the higher lipophilicity of 11 may facilitate cell membrane penetration.Based on our results, we made a similar observation given that compound 11 demonstrated markedly higher antiproliferative activity against all of the cell lines compared with compound 9.
To select the most promising lead compound(s) and reduce the inherent risk of promiscuity of anticancer agents, we calculated the respective lipophilic ligand efficiency (LLE = pIC 50 − log P) values. 47,48Adhering to the double criteria, IC 50 ≤ 10 μM and LLE ≥ 1.5, compounds 9 and 11 were identified as primary candidates against the U-87 MG human glioblastoma cell line.These compounds were predicted to have the lowest off-target or toxicological risk.With a slightly higher risk of promiscuity, compound 10 may be considered to be a secondary hit.Although hydroquinone coleon U (13) did not meet our LLE criterion because of its marginally increased lipophilicity, it may be considered the most effective member of the isopropyl-substituted abietanes against gynecological cancer cells, as it exhibited efficacy greater than or equal to that of the positive control cisplatin.
In conclusion, the isolation of diterpenes 1−15 provides further insight into the phytochemistry of the genus Plectranthus s.l. and confirms that it is a rich source of structurally diverse diterpenes.The promising antiproliferative activity, particularly against glioblastoma cells, suggests that these compounds are potential anticancer leads worthy of further investigation.
■ EXPERIMENTAL SECTION General Experimental Procedures.Optical rotations were measured in CHCl 3 using an AUTOPOL IV polarimeter (Rudolph Research Analytical, Hackettstown, NJ, USA) with a 0.8 mL polarimetric cell at 23 °C (instrument room temperature).1D and 2D NMR data were acquired with a JEOL ECZR 400 MHz NMR spectrometer (JEOL, Tokyo, Japan) operating at 400 MHz for 1 H and 100 MHz for 13 C, a Bruker AVANCE DRX 500 MHz spectrometer (Bruker, Billerica, MA, USA) at 500 MHz for 1 H and 125 MHz for 13 C, and a Bruker AVANCE III HD 700 MHz spectrometer (Bruker, Billerica, MA, USA) at 700 MHz for 1 H and 175 MHz for 13 C.The spectra were recorded in CDCl 3 and signals for the residual solvent (δ H 7.26 ppm; δ C 77.16 ppm) were used as reference points.HRESIMS analyses were carried out using an LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) by direct sample injection and on an Agilent 1100 LC−MS instrument (Agilent Technologies, Santa Clara, CA, USA) coupled with a Thermo Q-Exactive Plus Orbitrap analyzer (Thermo Fisher Scientific, Waltham, MA, USA) operating in positive and negative ionization modes.Column chromatography was performed using Silica gel 60, particle size 40−63 μm, and a 230−400 mesh particle size (Sigma-Aldrich, St. Louis, MO, USA).The fractions obtained by column chromatography were monitored by thin layer chromatography with precoated aluminum TLC plates Silica gel 60 F 254 , 20 × 20 cm, 200 μm (Merck KGaA, Darmstadt, Germany) at different ratios of CHCl 3 −EtOAc (15:1−1:1, v/v) as the mobile phase.The compounds were visualized by irradiation with UV light at 254 and 366 nm.Flash chromatography was performed using a Combiflash Rf+ instrument (Teledyne ISCO, Lincoln, NE, USA) equipped with a diode array and evaporative light scattering detectors.The apparatus was used with columns manually filled with MP EcoChromTM Polyamide, particle size 50−160 μm (MP Biomedicals Germany, GmbH, Eschwege, Germany).Analytical-scale, reversed-phase HPLC measurements were performed using two HPLC instruments.An Agilent 1100 HPLC instrument equipped with an Agilent 1100 Series diode array detector (Agilent Technologies, Santa Clara, CA, USA) was used with an analytical HPLC column Ascentis Express RP-Amide, 100 mm × 2.1 mm, particle size 2. Cell Culture.Human gynecological cancer cell lines (MDA-MB-231, MCF-7, and HeLa) and the human glioblastoma U-87 MG cell line were purchased from the ECACC (European Collection of Cell Cultures, Salisbury, UK).Cells were grown in Eagle's Modified Essential Medium supplemented with 10% heat-inactivated fetal bovine serum, 1% nonessential amino acids, and 1% penicillin−streptomycin−amphotericin B mixture in a humidified atmosphere containing 5% CO 2 at 37 °C.To maintain the U-87 MG cell line, the basic medium was supplemented with 1% L-glutamine and 1% sodium pyruvate.All media and supplements were obtained from Capricorn Scientific Ltd. (Ebsdorfergrund, Germany).Cells in the near-confluent phase of growth were used for the assays described below.
Antiproliferative Assay.The inhibitory effect of the 15 isolated compounds on cell proliferation was characterized by performing colorimetric MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. 49Briefly, 5 × 10 3 cells per well were seeded in 96-well plates.After overnight incubation, the cells were treated with eight different concentrations of the compounds ranging from 50 to 0.39 μM.After 72 h of incubation, the cells were treated with MTT solution and incubated as recommended.The supernatant was removed from the wells, and the formazan crystals were dissolved in 100 μL dimethyl sulfoxide.The absorbance values at 545 nm for each sample were determined using a microplate UV−Vis reader (SPECTROstar Nano, BMG Labtech GmbH, Offenburg, Germany).At least two independent experiments were performed in triplicate.The resulting data were transformed into inhibitory percentages, which were used for the determination of IC 50 values of the tested compounds using GraphPad Prism 9.5.1.software (GraphPad Software, San Diego, CA, USA).95% confidence intervals (95% CI) for the IC 50 values were also calculated to demonstrate the reliability of the experiments.

Data Availability Statement
The raw NMR spectra for compounds 2−5 are freely available on Zenodo with DOI: 10.5281/zenodo.10531934.

Table 2 .
(calcd for C 26 H 35 O 9 , 491.22811) indicated the molecular formula C 26 H 34 O 9 , with ten indices of hydrogen deficiency.An assessment of the NMR spectra revealed that compound 3 is a derivative of 1 and contains the same skeleton (dicarbonyl 13 C NMR Data for Compounds 1−5 (δ in ppm, Measured in CDCl 3 ) Compound 3 appeared as a yellowish, amorphous powder.The HRESIMS ion at m/z 491.2277 [M + H] + a Spectra taken at 100 MHz.b Spectra taken at 175 MHz.cThe signal appeared to overlap with the solvent signal and its chemical shift was deduced from HSQC.d Carbonyl carbons indistinguishable in HMBC (the signals of 7-OAc and 12-OAc are interchangeable).moiety:δC196.3, 194.3, 155.1, and 140.7; methylcyclopropane ring: δ C 34.9, 26.8, 21.43 and 13.0), three acetoxy groups (δ C 170.9, 21.37, δ H 2.08; δ C 170.0, 21.6, δ H 2.06; δ C 169.9, 20.9, δ H 2.13), and one hydroxyl group (δ H 2.71).The main differences between compounds 1 and 3 were the additional resonances of the methyl groups (δ C−H 21.37/2.08 and 20.9/ 2.13) and the carbonyl carbons (δ C 170.9 and 169.9) as well as downfield-shifted protons H-3 (δ H 4.53, Δδ = +1.21)andH-12(δ H 4.89, Δδ = +0.90),whichwereevidence of two additional acetoxy groups at C-3 and C-12.These results were verified by the HMBC connectivities of the methyl protons at δ H 2.08 and H-3, with the carbonyl carbon at δ C 170.9 and the methyl protons at δ H 2.13 and H-12 with the carbonyl carbon at δ C 169.9 (FigureS7, Supporting Information).NOESY permitted the assignment of the same relative configuration for 3 as that of 1 and 2 (Figures S8, Supporting Information).

Table 3 .
Predicted Physicochemical and Early ADME Parameters of Diterpene Compounds for Drug-Likeness Classification a Risk for drug-and CNS-agent-likeness (moderate�yellow, high�red).b Lipinski Ro5 violation; HBD = hydrogen bond donor; HBA = hydrogen bond acceptor.