Pinus banksiana Lamb (jack pine) is widely distributed in North American forests and particularly in Canada, where its presence extends from Cape Breton Island, Nova Scotia, up to the Mackenzie river in the Northwest Territories [1,2,3]. Although jack pine was essentially used in the wood industry as a source of pulpwood, lumber, and round timber [3], it was also used in traditional medicines. Gum, when chewed, can fight colds [4], inner bark, soaked and softened, has been used as a poultice to heal wounds [5] and leaves were used as a fuminant to revive comatose patient and to clear congested lungs [6]. The pine oil and pine tar have been also used to make disinfectants, antiseptics and insecticides [7]. Numerous studies realized on bark, wood, needles, resin and essential oils of Pinus banksiana have reported the presence of monoterpenoids [8,9], diterpenoids [8,9,10,11], sesquiterpenoids [8,12], triterpenoids [11], phenylpropanes [13], flavonoids, lignans and stilbenes [10,14]. However, to this day, no study of the cytotoxic and antibacterial activities of the buds of jack pine was made.

Phytochemical investigation of the buds of jack pine resulted in the isolation of 15 compounds (see Figure 1), including three new diterpenoid derivatives: 7α-hydroxyabieta-8,11,13,15-tetraen-18-oic acid (1), 7β,15,18-trihydroxyabieta-8,11,13-triene (2) and 13,15-dihydroxypodocarpa-8,11,13-triene (3). The known compounds were identified as 7α,15-dihydroxyabieta-8,11,13-trien-18-al (4) [15], 7α,15-dihydroxydehydroabietic acid (5) [16], 7β,15-dihydroxydehydroabietic acid (6) [17,18], 15-hydroxydehydroabietic acid (7) [19], 18-nor-abieta-8,11,13-triene-4,7α-diol (8) [20], 18-nor-abieta-8,11,13-triene-4,7β-diol (9) [21], 7α-hydroxydehydroabietic acid (10) [17], 7β-hydroxydehydroabietic acid (11) [17,22], 7-oxodehydroabietinol (12) [23], 18-nor-4,15-dihydroxyabieta-8,11,13-trien-7-one (13) [15,23], 15,18-dihydroxyabieta-8,11,13-trien-7-one (14) [24] and 15-hydroxy-7-oxo-abieta-8,11,13-trien-18-oic acid (15) [25]. Compound 9 is a known synthetic dehydroabietane derivative that is now identified for the first time as a natural product. The structure elucidations of 1–3 were based on spectroscopic analyses, including 1D and 2D NMR spectroscopic techniques.

P. banksiana buds were extracted successively with hexanes, CH₂Cl₂ and MeOH. After solvent evaporation, each extract was investigated for in vitro cytotoxic and antibacterial activities. Cytotoxic activity evaluations were carried out on lung cancer (A549), colorectal cancer (DLD-1) and normal skin fibroblasts (WS1) human cell lines using the Hoechst assay [26]. Antibacterial activity was evaluated against Escherichia coli and Staphylococcus aureus. Results displayed in Table 1 show that the hexanes extract exerted a moderate activity against A549 (IC₅₀, 45 ± 4 µg/mL), DLD-1 (IC₅₀, 44 ± 3 µg/mL) cell lines and its antibacterial activity against S. aureus was interesting (IC₅₀, 29 ± 3 μg/mL). The CH₂Cl₂ extract showed a significant cytotoxic activity against A549 (IC₅₀, 26 ± 3 µg/mL), DLD-1 (IC₅₀, 32 ± 3 µg/mL) cell lines and a weak activity on S. aureus (64 ± 5 µg/mL). Finally, the MeOH extract exhibited no activity. Therefore, following studies were focused on the CH₂Cl₂ extract that was further separated over column chromatography on silica gel to afford eleven fractions (A–K). All fractions activities were tested against both cancer cell lines and bacterial strains. Only fraction F, G and H were found strongly cytotoxic activity against A549 with IC₅₀ values ranging from 5 to 7 µg/mL and against DLD-1 with IC₅₀ values ranging from 12 to 14 µg/mL. Interestingly, these fractions were found selective against cancer cell lines in comparison with normal cells (IC₅₀, 39 to 79 µg/mL). Remaining fractions were found inactive with IC₅₀ values greater than 100 µM. Fraction F was active against S. aureus (IC₅₀, 51 ± 2 µg/mL) while fractions G, H and K were found inactive (IC₅₀ > 100 µM). In a next step, fractions F, G, H and K were separated by a combination of chromatographic procedures to afford the three new compounds 1–3 together with 12 known compounds. The structures of the new compounds were determined as follows and the known products were identified by comparison of their spectroscopic data with values found in the literature.

Compound 1, a white amorphous powder, was assigned as C₂₀H₂₆O₃ by positive HRESIMS (m/z 337.1766 calcd for C₂₀H₂₆O₃Na⁺ 337.1774). Its IR spectrum showed the presence of a hydroxyl (3391 cm⁻¹) and an olefinic bond (1639 cm⁻¹). The ¹H-NMR spectrum (Table 2) showed an AMX aromatic ring system [δ_H 7.46 (1H, d, J = 1.6 Hz, H-14), 7.39 (1H, dd, J = 8.4, 1.6 Hz, H-12) and 7.23 (1H, d, J = 8.4 Hz, H-11)], an oxymethine proton at δ_H 4.81 (1H, d, J = 3.5 Hz, H-7), an isopropenyl group [δ_H 2.14 (3H, s, Me-17), 5.06 (1H, s, H-16Z) and 5.36 (1H, s, H-16E)] and two methyl group singlets [δ_H 1.18 (3H, s, Me-20) and 1.30 (3H, s, Me-19)]. The ¹³C-NMR spectrum of 1 (Table 3) showed a signal characteristic of a carboxylic acid group (δ_C 182.3, C-18), eight unsaturated carbons (δ_C 148.3, 142.6, 139.1, 135.8, 127.5, 125.6, 124.2, 112.1) and one hydroxylated carbon (δ_C 68.2, C-7). HMBC correlations (see Figure 2) observed between H₃-19 and C-3, C-4, C-5 and C-18 (δ_C 36.3, 46.9, 39.6, 182.3, respectively), together with the presence of an aromatic ring, suggested that the molecule was a dehydroabietane [17]. The hydroxyl group was assigned at position 7 because of its ¹H-¹H COSY correlation with H-6 (δ_H 1.72 and 2.14, both overlapped) (see Figure 2).

The multiplicity of H-7 was a broad doublet (J = 3.5 Hz) suggesting that the configuration is α. The configurations of other stereocenters were assessed by comparing the ¹³C chemical shifts of 1 with those of 7α-hydroxydehydroabietic acid [17]. Hence, compound 1 was established as 7α-hydroxyabieta-8,11,13,15-tetraen-18-oic acid.

Compound 2 was assigned the molecular formula C₂₀H₃₀O₃, as established from its HRESIMS (m/z 341.2077 calcd for C₂₀H₃₀O₃Na⁺ 341.2087). The IR spectrum showed absorbances consistent with hydroxyl (3411 cm⁻¹) and olefinic (1650 cm⁻¹) groups. The ¹H-NMR spectrum of 2 (Table 2) showed the presence of three AMX aromatic protons [δ_H 7.64 (1H, d, J = 1.9 Hz, H-14), 7.31 (1H, dd, J = 8.3, 1.9 Hz, H-12), 7.21 (1H, d, J = 8.3 Hz, H-11)], one oxygenated methine at δ_H 4.86 (1H, t, J = 8.9 Hz, H-7), one oxygenated methylene [δ_H 3.50, 3.20 (each 1H, d, J = 10.9 Hz, H-18a,b)], and three singlets methyls [δ_H 1.57 (6H, s, Me-16 and 17), 1.29, 0.89 (each 3H, s, Me-20 and 18)]. ¹³C-NMR signals (Table 3) were observed at δ_C 72.5 (C-15), 70.9 (C-7) and 71.6 (C-18) for tertiary, secondary and primary alcohol groups respectively, along with six other signals assigned to an aromatic ring. The methyl groups Me-16 and Me-17 showed HMBC correlations with C-15 and C-13 (δ_C 146.5), indicative of a hydroxypropyl branched at C-13 (see Figure 2). Long-range correlations were observed between H₃-19 and H₂-18 with C-3, C-4 and C-5 (34.7, 37.5, 42.3, respectively) suggesting that the hydroxyl function of the primary alcohol was attached to C-18. The β-configuration of the hydroxyl group at C-7 was determined from the ¹H-NMR spectrum, in which a triplet at δ_H 4.86 (J = 8.9 Hz) due to an axial proton was observed [18]. Cross peaks in the NOESY spectrum (see Figure 3) were observed between H₃-19 and H₃-20 and between H₂-18 and H-5, supporting the relative stereochemistry depicted. Thus, compound 2 was identifed as 7β,15,18-trihydroxyabieta-8,11,13-triene.

Compound 3 has the molecular formula, C₁₇H₂₄O₂, as shown from its positive HRESIMS at m/z 283.1670 (calcd for C₁₇H₂₄O₂Na⁺ 283.1668). Analysis of the ¹H-NMR spectrum (Table 2) indicated the presence of three protons in a trisubstituted aromatic ring [δ_H 7.04 (1H, d, J = 8.6 Hz), 6.51 (1H, dd, J = 8.6, 2.7 Hz) and 6.42 (1H, d, J = 2.7 Hz)], one oxygenated methylene [δ_H 3.43 and 3.09 (2H, d, J = 7.5 Hz)], and two singlets methyls at δ_H 1.17 and 0.84 (both 3H, s, Me-17 and Me-16, respectively). The ¹³C-NMR spectrum (Table 3) showed 17 carbon signals, including three olefinic carbons and two oxygenated carbons. The resonances at δ_H 3.43, 3.09 and δ_C 72.0 support the presence of a secondary alcohol group. Long-range correlations from H₂-15 with C-3, C-4, C-5 and C-16 where observed in the HMBC spectrum (see Figure 2), indicating the position of the alcohol group. The ¹³C signal at δ_C 155.5 suggested the presence of a phenolic group. The location of this hydroxyl group at C-13 was confirmed by observation of C–H long range correlations from H-11 to C-8, C-10 and C-13 in the HMBC spectrum. The presence of the two alcohols was confirmed by the absorption bands at 3448 cm⁻¹ in the FTIR spectrum of 3. These data, together with other results of COSY and HMBC analysis (see Figure 2), confirmed that compound 3 is 13,15-dihydroxypodocarpa-8,11,13-triene.

Cytotoxic and antibacterial activities of all compounds were evaluated against two human cancer cell lines, lung carcinoma (A549) and colorectal adenocarcinoma (DLD-1) and two bacterial strains, S. aureus and E. coli (Table 4). With the exception of compounds 12 and 13, all compounds were found inactive against both cancer cell lines tested, IC₅₀ > 100 µM. Compound 12 exhibited a moderate cytotoxicity against A549 and DLD-1 with IC₅₀ of 34 and 47 µM, respectively. Compound 13 was also found active against A549 (IC₅₀, 46 µM) but not to DLD-1. These results are in good agreement with those reported by Barrero [27] for compound 12. As far as antibacterial activity was concerned, all compounds were inactive which is also in agreement with previously published results for compounds 7, 11, 12 and 15 [28,29,30]. Compounds 12 and 13 were in part responsible of the cytotoxic activity of the CH₂Cl₂ extract of bud from P. banksiana. However, additional studies will be conducted to identify other active compounds.

Three new diterpenoids, namely 7α-hydroxyabieta-8,11,13,15-tetraen-18-oic acid (1), 7β,15,18-trihydroxyabieta-8,11,13-triene (2), 13,15-dihydroxypodocarpa-8,11,13-triene (3), and twelve known compounds, were isolated from the buds of Pinus banksiana Lamb. All isolated compounds were tested for their antibacterial and cytotoxic activities, and among all the isolated compounds, only compounds, 7-oxo dehydroabietinol (12) and 18-nor-4,15-dihydroxyabieta-8,11,13-trien-7-one (13), showed moderate cytotoxicity against lung carcinoma A549. From the above results, the cytotoxic activities of the isolated compounds do not explain the strong activities of the CH₂Cl₂ extract and further studies are needed to identify the active cytotoxic compounds from CH2Cl2 extract or other extracts.