Ent-abietane and ent-pimarane diterpenoids from Croton mubango (Euphorbiaceae)

Phytochemistry 170 (2020) 112217

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Ent-abietane and ent-pimarane diterpenoids from Croton mubango (Euphorbiaceae)

T

Sani M. Isyakaa, Moses K. Langata,b,c, Eduard Mas-Clareta, Blaise M. Mbalad, Bienvenu K. Mvingud, Dulcie A. Mulhollanda,c,∗ a

Natural Products Research Group, Department of Chemistry, Faculty of Engineering and Physical Sciences, University of Surrey, Guildford, GU2 7XH, UK Jodrell Laboratory, Natural Capital and Plant Health Department, Royal Botanic Gardens, Kew, Richmond, Surrey, TW9 3DS, United Kingdom c School of Chemistry and Physics, University of KwaZulu-Natal, Durban, 4041, South Africa d Departement de Chimie et Industry, Faculte des Sciences, Universite de Kinshasa, B.P.190 Kin XI, Congo b

ARTICLE INFO

ABSTRACT

Keywords: Croton mubango Euphorbiaceae Ent-pimarane Ent-abietane Cytotoxicity screening DP4+ calculations

Twelve ent-abietane and two ent-pimarane diterpenoids were isolated from the leaves of Croton mubango Müll. Arg. (Euphorbiaceae) collected in the Democratic Republic of the Congo. 2β-Hydroxy-ent-abieta-7,13-dien-3one, 15-hydroxy-ent-abieta-7,13-dien-3-one, 13α,15-dihydroxy-ent-abieta-8(14)-en-3-one, 2β,9,13-trihydroxyent-abieta-7-en-3-one, 2β,7β-dihydroxy-ent-abieta-8,11,13-trien-3-one, 15-hydroxy-ent-abieta-8,11,13-trien-3one and ent-pimara-8(14),15-dien-3-one and the ent-forms of the previously reported normal series diterpenoids, ent-abieta-8,11,13-trien-3-one, 7β-hydroxy-ent-abieta-8,11,13-trien-3-one, 3α-hydroxy-ent-abieta-8,11,13triene, 15-hydroxy-ent-abieta-8,11,13-triene and 6β-hydroxy-ent-abieta-8,11,13-triene are reported here for the first time. Structures were established using HRESIMS, FTIR, NMR, DP4+ probability calculations and by comparison of the experimental and calculated electronic circular dichroism (ECD) spectra. Ent-pimara-8(14), 15-dien-3-one, showed antiproliferative activity against melanoma (MALME-3M), renal (UO-31) and ovarian cancer cell lines (IGROV1) at a concentration of 10−5 M in the NCI 60 screen.

1. Introduction The genus Croton (Euphorbiaceae), comprises about 1300 species distributed in tropical and subtropical regions of the world (Salatino et al., 2007). A number of species are used as traditional medicines for various conditions, including gastric diseases (Craveiro et al., 1980), snake bites (Lima et al., 2010), wound healing (Pieters et al., 1993; Rao et al., 2007), rheumatism and ulcers (Nardi et al., 2003; Rao et al., 2007), diarrhoea and cancer (Rao et al., 2007), malaria (Thuong et al., 2012), chest complaints (Aldhaher et al., 2017), diabetes, gastrointestinal disturbances and high cholesterol (Campos et al., 2002). The genus Croton has been shown to produce a range of compounds including terpenoids, alkaloids and flavonoids, some of which have been shown to possess anti-cancer, anti-inflammatory, anti-ulcer, anti-malarial and anti-oxidant activities (Salatino et al., 2007). In our studies of African Croton species, we have isolated a wide range of diterpenoid classes including cembranoids (Langat et al., 2011; Mulholland et al., 2010), crotofolanes, halimanes, ent-clerodanes (Aldhaher et al., 2017), abietanes (Ndunda et al., 2016) and ent-kaurenoic acids (Langat et al.,

2012). We report on the isolation of twelve ent-abietanes (1–12) and two ent-pimaranes (13,14), of which compounds 2–5, 8, 9 and 14 and the ent-form of compounds 6, 7, 10, 11 and 12 have not been reported previously, from the leaf extract of Croton mubango Müll. Arg. (Euphorbiaceae), collected in the Democratic Republic of the Congo. Absolute configurations of isolated compounds were confirmed using ECD spectroscopy and DP4+ calculations. 2. Results and discussion The leaves of Croton mubango Müll. Arg., yielded twelve previously unreported diterpenoids, eleven belonging to the ent-abietane class: 2βhydroxy-ent-abieta-7,13-dien-3-one 2, 15-hydroxy-ent-abieta-7,13-dien3-one 3, 13α,15-dihydroxy-ent-abieta-8(14)-en-3-one 4, 2β,9,13-trihydroxy-ent-abieta-7-en-3-one 5, ent-abieta-8,11,13-trien-3-one 6, 7β-hydroxy-ent-abieta-8,11,13-trien-3-one 7, 2β,7β-dihydroxy-ent-abieta8,11,13-trien-3-one 8, 15-hydroxy-ent-abieta-8,11,13-trien-3-one 9, 3αhydroxy-ent-abieta-8,11,13-triene 10, 15-hydroxy-ent-abieta-8,11,13triene 11, 6β-hydroxy-ent-abieta-8,11,13-triene 12, and an ent-

∗ Corresponding author. Natural Products Research Group, Department of Chemistry, Faculty of Engineering and Physical Sciences, University of Surrey, Guildford, GU2 7XH, UK E-mail address: [email protected] (D.A. Mulholland).

https://doi.org/10.1016/j.phytochem.2019.112217 Received 25 June 2019; Received in revised form 21 November 2019; Accepted 22 November 2019 0031-9422/ © 2019 Elsevier Ltd. All rights reserved.

Phytochemistry 170 (2020) 112217

S.M. Isyaka, et al.

Fig. 1. Structures of ent-diterpenoids 1–14 isolated from Croton mubango.

pimarane, ent-pimara-8(14),15-dien-3-one 14 (Fig. 1), along with known compounds, ent-abieta-7,13-dien-3-one 1 (Anthonse and Bergland, 1973; Feliciano et al., 1993b), 3α-hydroxy-ent-pimara8(14),15-diene 13 (Ansell et al., 1993), 2-phyten-1-ol (Brown, 1994), caryophyllene oxide (Orihara et al., 1994), 132-hydroxy-(132-S)phaeophytin-a (Matsuo et al., 1996), sitosterol and sitosterone. The normal series analogues of the newly isolated ent-diterpenoids 6 (Topcu and Ulubelen, 1990), 7 (Delcorral et al., 1994), 10 (Urones et al., 1988),11 (Conner et al., 1980) and 12 (Chawla et al., 1991; Matsumoto et al., 1983), have been reported previously. Structures of the known compounds were confirmed by comparison with data from literature as referenced above and NMR literature data for 1 was corrected (Tables 1 and 2). The absolute configurations of 1, 2, 4 and 14 were determined through the comparison of their experimental and theoretically calculated ECD curves. This was achieved using previously described protocols (Tabekoueng et al., 2019; Tembu et al., 2014), where conformational searches of 1, 2, 4 and 14 and their enantiomers were initially undertaken using Spartan software at ground state with molecular mechanics force fields (MMFF). Conformers that were under 3 kcal/mol were subjected to TDDFT calculations using a B3LYP method at 6-31G** level built into Gaussian software (Frisch et al., 2016). This work represents only the second report of ent-abietane diterpenoids from an African Croton species: two ent-abietane diterpenoids, ent-abieta-7,13-dien-2-one and 2β-hydroxy-ent-abieta-7,13diene, were isolated previously from the Kenyan Croton megalocarpus (Alqahtani, 2015). Ent-pimarane diterpenoids are reported here for the first time from an African Croton species. Previously abietane diterpenoids have demonstrated antiulcer, antimicrobial and cardiovascular activities (Feliciano et al., 1993b). Leaves of the Brazilian Croton sphaerogynus have shown antiproliferative activity, believed to be caused by the presence of abietanes (Motta et al., 2013). Tricyclic diterpenes have also shown promising anti-proliferative activities (Motta et al., 2013; Righi et al., 2013), therefore compounds 2, 2Ac, 3, 4, 7Ac, 8, 9, 10Ac, 12 and 14 were submitted to the NCI Drug Therapeutics Programme for evaluation in the NCI 60 cancer cell line screen at one dose of 1 × 10−5 M (National Cancer Institute, 2019) (see Table 3).

Compound 1 was found to be the known ent-abieta-7,13-dien-3-one, isolated previously from Juniperus phoenicea (Feliciano et al., 1993a) and Solidago missouriensis (Anthonse and Bergland, 1973). Using 2D NMR techniques, we were able to correct literature NMR assignments (Tables 1 and 2). The NOESY spectrum confirmed the relative stereochemistry with correlations seen between the H-9/H-5 and H-5/H3-18 resonances, and H3-19 and H3-20 resonances. In order to determine the absolute configuration of 1, its theoretically calculated and experimental ECD curves were compared. ECD spectroscopy showed the n-π* transition at 290 nm (ΔE = +6) and negative Cotton effects at 205 nm (ΔE = −16) and at 197 nm (ΔE = −18) indicating 1 was an entabietane (Zhou, 1991). To confirm this, the theoretical excitation circular dichroism (ECD) spectrum of 1 was compared to the experimental ECD spectrum. A conformational search on the (5S,9S,10S)-isomer of 1 using Spartan software to evaluate the conformer distribution at ground state with molecular mechanics force fields (MMFF) was undertaken. MMFF analysis of 1 (of the ent diterpenoid series) gave six conformers, three of which were under 3 kcal/mol. The three conformers, consistent with NOESY NMR experimental data, M0001 (0.00 kcal/mol), M0002 (0.29 kcal/mol) and M0003 (0.41 kcal/mol), had Boltzmann distributions of 0.473, 0.288 and 0.238 respectively. Each of these three conformers was subjected to TDDFT calculations using a B3LYP method at 6-31G** level built into Gaussian 09 software (Frisch et al., 2016). The ECD curves for the three conformers were Boltzmann weighted and compared to the experimental ECD curve of 1 (Fig. 2) which enabled the unambiguous assignment of the chiral centres of 1 as 5S, 9S and 10S. To confirm this assignment, a theoretical ECD spectrum for the normal-enantiomer (1a) of 1, also consistent with the NOESY NMR experimental data (5R, 9R and 10R) was obtained. The theoretical ECD curve of 1a was opposite and equal in intensity to that of 1, confirming 1 was ent-abieta-7,13-dien-3-one. As a plant normally produces compounds of either the normal or ent-series, it was expected that all the abietanes isolated in this work belong to the ent-series and this was confirmed by ECD analysis of compounds 1, 2, 4 and 14 (Fig. 2). The HRESIMS for compound 2 showed an [M-H]- ion at m/z 301.2171, indicating a molecular formula of C20H30O2 for the 2

1.51 dt (5.5 14.0)

1α

3

– – – 1.67 dd (5.0, 12.0)

2.14 m 2.19am 5.46 bs (W1/2 = 11.0)

– 1.87 1.31 1.86 2.13

2.13 m – 5.81 bs (W1/2 = 5.0) – 2.25am 1.00ad (7.0) 1.02ad (7.0) 1.06 s 1.13 s 1.02 s – – – – – –

3α 3β 4 5

6α 6β 7α

7β 8 9 10 11α 11β 12α

12β 13 14α 14β 15 16 17 18 19 20 2-OH 1′ 2′ 7-OH 13-OHc 15-OHc

c

b

a

2.75 dt (5.5, 14.0)

2β

Overlapped proton resonances. 500 MHz. In DMSO.

m m m

m

4.61 dd (6.0, 13.0)

2.26am

2α

m m m

m

2.12 m – 5.80 bs (W1/2 = 5.0) – 2.24 m 1.00 d (7.0) 1.01 d (7.0) 1.14 s 1.19 s 1.11 s 3.65 bs (W1/2 = 14.6) – – – – –

– 1.89 1.28 1.86 2.12

2.12 m 2.21 m 5.45 bs (W1/2 = 11.0)

1.67 dd (5.0, 12.0)

–

2.55 dd (6.0, 13.0)

2.17 m

1.30 m

2

1β

a

1

Proton position

2.10 5.80 – 2.22 1.01 1.02 1.10 1.24 1.15 – – 2.17 – – –

– 1.92 1.30 1.83 2.10

s

m d (7.0) d (7.0) s s s

bs (W1/2 = 5.0)

m

m m m

m

2.16 m 2.16 m 5.46 bs (W1/2 = 11.0

– 1.66dd (5.0, 12.0)

–

5.62 dd (6.0, 13.0)

2.39 dd (6.0, 13.0)

1.60 m

2-Ac

Table 1 1 H NMR data (400 MHz, CDCl3) for compounds 1–8 (J in Hz).

2.13 – 6.10 – – 1.35 1.33 1.07 1.14 1.02 – – – – – –

– 1.89 1.31 1.89 2.09

s s s s s

d (2.4)

m

m m m

m

2.08 m 2.17 m 5.57 bs (W1/2 = 11.0)

– – 1.67 dd (5.0, 12.0)

2.75 dt (5.5, 14.0)

2.26 m

2.16 m

1.52 dt (5.5, 14.0)

3

1.40 – 5.82 – – 1.14 1.22 1.10 1.08 1.01 – – – – 3.87 3.91

2.38 1.78 1.68 1.62 1.25

s s

s s s s s

bs

m

m m m

m

m

2.70 dt (J = 5.5, 14.0) – – 1.48 dd (7.3, 11.0) 1.51 m 1.58 m 2.11 m

2.30 m

1.52 dt (5.5, 14.0) 2.16 m

4

1.54 m – 1.80a 1.80a) 1.91 sept 0.98 d (7.0) 0.96 d (7.0) 1.13 s 1.19 s 1.37 s 3.53 d (4.8) – – – – –

– 6.92 bs (W1/2 = 4.2) – 2.88 sep (7.0) 1.22ad (7.0) 1.22ad (7.0) 1.14 s 1.16 s 1.29 s – – – – – –

2.93 m – 7.17 d (8.0) – 7.01dd (2.0, 8.0)

1.81 m 1.82 m 2.87 m

2.62am 2.62am 6.16 bs (W1/2 = 5.0) – – 1.52 m 2.12 m 1.97 dd (9.5, 13.0)

– – 1.93a dd (3.0, 11.5)

2.66 dt (3.0, 11.5)

2.56 m

2.45 td (7.5, 11.5)

– – 2.13a dd (6.0, 13.0)

–

4.60 dd (6.0, 13.0)

2.13 dd (6.0, 9.5)

1.92a dd (3.0, 11.5)

1.97a dd (9.5, 13.0) a

6

5

– 7.16d (2.0) – 2.89 sep (7.0) 1.24 d (7.0) 1.24 d (7.0) 1.20 s 1.14 s 1.23 s – – – 3.10 bs (W1/2 = 2.0) – –

– – 7.22 d (8.0) – 7.18 dd (2.0, 8.0)

1.25 m 1.97 m 4.88 dd (1.4, 3.5)

– – 2.35 dd (3.0, 12.0)

2.50 ddd (4.0, 7.5, 11.5) 2.64 ddd (3.0, 7.5, 11.5) 2.70 dt (3.0, 11.5)

1.97 m

7b

7.21 dd (2.0, 8.0) – 7.10 d (2.0) – 2.87 sep (7.0) 1.24 d (7.0) 1.23d (7.0) 1.13 s 1.12 s 1.25 s – – 2.07 s – – –

2.50 ddd (4.0, 7.5, 11.5) 2.63 ddd (3.0, 7.5, 11.5) 2.72 dt (3.0, 11.5) – – 2.32 dd (3.0, 12.0) 1.99 m 2.03 m 6.04 dd (1.4, 3.5) – – 7.24 d (8.0)

2.06 m

7-Acb

– – –

2.08 2.50 5.10 3.5) – – – – 7.26 – 7.20 8.0) – – 7.24 – 2.88 1.25 1.24 1.29 1.23 1.45 – –

sep (7.0) d (7.0) d (7.0) s s s

d (2.0)

dd (2.0,

d (8.0)

m m dd (1.4,

– – – 2.15 m

1.69 dd (6.0, 13.0) 2.99 dd (6.0, 13.0) 4.74 dd (6.0, 13.0) –

8x

S.M. Isyaka, et al.

Phytochemistry 170 (2020) 112217

Phytochemistry 170 (2020) 112217

S.M. Isyaka, et al.

Table 2 1 H NMR s data (400 MHz, CDCl3) for compounds 9–14 (J in Hz). Proton

9

10a

10-Aca

11

12

1α 1β 2α 2β 3α 3β 4 5 6α 6β 7α 7β 8 9 10 11α 11β 12α 12β 13 14α 14β 15 16α 16β 17 18 19 20 1′ 2′

1.93b dd (3.0, 11.5) 2.48 m 2.59 m 2.69 dt (5.5, 14.0) – – 1.93b dd (3.0, 11.5) 1.80 m 1.80 m 2.81 m 2.81 m – 7.23 d (8.0) – 7.26 dd (2.0, 8.0) – – 7.19 d (2.0) – – 1.57bs – 1.57bs 1.17 s 1.14 s 1.29 s – –

1.54 m 2.29 dt (3.5, 10.0) 1.78 m 1.78 m – 3.30 dd (6.3, 10.0) 1.34 dd (2.3, 12.0) 1.72 m 1.87 m 2.84 m 2.91 m – 7.14 d (8.0) – 6.99 dd (2.0, 8.0) – 6.89 bs (W1/2 = 4.2) – 2.82 sept (7.0) 1.23 d (7.0) – 1.20d (7.0) 1.07 s 0.89 s 1.19 s – –

1.59 2.29 1.25 1.78 – 4.54 1.42 1.75 1.86 2.82 2.91 – 7.13 – 6.99 – 6.89 – 2.81 1.22 – 1.21 0.97 0.95 1.20 – 2.07

1.37 m 2.28 dt (3.5, 10.0) 1.61 m 1.71bm 1.21 m 1.47 m 1.35 dd (2.3, 12.0) 1.71bm 1.86 m 2.86 m 2.91 m – 7.22d (8.0) – 7.24dd (2.0, 8.0) – – 7.16 d (2.0) – – 1.56bs – 1.56bs 0.96 s 0.93 s 1.19 s – –

1.39 2.29 1.62 1.74 1.24 1.50 1.64 4.82 – 1.64 1.99 – 7.22 – 7.13 – – 7.20 – 2.87 1.25 – 1.23 0.98 0.94 1.13 – –

a b

m dt (3.5, 10.0) m m dd (6.3, 11.0) dd (2.3, 12.0) m m m m

d (8.0) dd (2.0, 8.0) bs (W1/2 = 4.2) sept (7.0) d (7.0) d (7.0) s s s s

14a

13 m m m m m m m bs (W1/2 = 11.4) m m

d (8.0) dd (2.0, 8.0) bs (W1/2 = 4.2) sept (7.0) d (7.0) d (7.0) s s s

1.18 1.67 1.58 1.63 – 3.26 1.03 1.39 1.62 2.06 2.34 1.64 1.32 1.48 1.23 1.53 5.14 – 5.72 4.89 4.96 0.99 1.01 0.82 0.74 – –

m m m m d (10.3) dd (2.7, 7.4) m m m m m m m m m bs (W1/2 = 4.0) dd (10.4, 17.3) dd (2.0, 17.3) dd (2.0, 10.4) s s s s

1.56 1.96 2.28 2.66 – – – 1.46 1.53 1.53 2.09 2.38 1.71 1.38 1.52 1.23 1.53 – 5.21 – 5.74 4.93 4.98 1.01 1.10 1.07 0.95 – –

m ddd (3.3, 5.8, 13.3) m m

m m m m m m m m m m bs (W1/2 = 4.0) dd (10.4, 17.3) dd (2.0, 17.3) dd (2.0, 10.4 s s s s

Overlapped proton resonances. 500 MHz.

compound and six degrees of unsaturation. The FTIR spectrum showed peaks at 3429 cm−1 (O–H stretch) and 1714 cm−1 (ketone C]O stretch). As with compound 1, a keto group carbon resonance (δC

216.5) was assigned as C-3 due to correlations seen in the HMBC spectrum with the H-5 (δH 1.67, dd, J = 5.0 Hz, 12.0 Hz), H3-18 (δH 1.14), and H3-19 (δH 1.19) resonances. The 13C NMR spectrum showed

Fig. 2. Calculated (ent- and normal series) and experimental ECD spectra for compounds 1, 2, 4 and 14. 4

Phytochemistry 170 (2020) 112217

S.M. Isyaka, et al.

Fig. 3. (a) 1H–1H COSY and key HMBC (H→C) correlations of 2; (b) Key NOESY correlations of 2.

the presence of a conjugated 7,13-diene system with resonances at δC 120.3 (C-7), δC 135.3 (C-8), δC 146.3 (C-13) and δC 122.1 (C-14), confirmed by correlations seen in the HMBC spectrum between the C-5 (δC 52.5) and H-7 (δH 5.45 bs, W1/2 = 11.0 Hz), and C-9 (δC 50.4) and both H-7 and H-14 (δH 5.80, bs, W1/2 = 5.0 Hz) resonances and correlations seen in the COSY spectrum between H-5/H2-6 (δH 2.12 m, δH 2.21 m), and the H-7 alkene resonance. The C-13 resonance showed correlations with the H3-16 and H3-17 resonances (δH 1.00, δH 1.01, both d, J = 7.0 Hz) confirming the isopropyl group at C-13 and hence the abietane structure. The 1H NMR chemical shifts differed from those of compound 1 with oxymethine resonances at δC 69.2 and δH 4.61 (dd, J = 6.0, 13.0 Hz) and a hydroxy group was placed at C-2 due to correlations seen between the oxymethine proton resonance and the C-1 (δC 48.2), C-3 (δC 216.5), C-4 (δC 47.4) and C-10 (δC 35.6) resonances in the HMBC spectrum. The configuration at C-2 was assigned using the NOESY spectrum, which showed correlations between the H3-20, H3-19 and H-2 resonances (all α) and between the H3-18 and H-5, H-5 and 2OH proton resonances (all β) (Fig. 3). Compound 2 was acetylated to produce the 2β-acetyl derivative, 2Ac, which showed the expected downfield shift of the H-2α resonance to δH 5.62. The absolute configuration of 2 was confirmed by comparing the theoretically calculated ECD and the experimental ECD data, as was done for 1 (Fig. 2). Information on the calculation of the ECD curves of 1, 2, 4 and 14 is provided in the Supplementary Data. HRESIMS of compound 3 gave the same molecular formula as for compound 2. However, the C-2β hydroxy group was not present and the doublets seen for the H3-16 and H3-17 in the 1H NMR spectra of 1 and 2 were replaced by singlets at δH 1.35 and δH 1.33, and a fully substituted oxygenated carbon resonance which could be assigned to C-15 was present at δC 72.7. Thus the structure of 3 was assigned as 15-hydroxyent-abieta-7,13-dien-3-one. HRESIMS analysis of compound 4 showed [M-H]- at m/z 319.2282 giving a molecular formula of C20H32O3 for the compound. As with compound 3, compound 4 had a keto group at C-3 (δC 216.8) and a tertiary hydroxy group at C-15 (δC 74.9). The 13C NMR spectrum showed the presence of one tri-substituted 8(14)-double bond (δC 143.1, C-8; δC 124.3, C-14), confirmed by a correlation seen between

the C-9 (δC 50.4) and C-15 (δC 74.9) resonances and the alkene H-14 broad resonance (δH 5.82). A second tertiary hydroxy group was placed at C-13 (δC 74.3) due to correlations seen in the HMBC spectrum between this resonance and the H3-16 and H3-17 singlet resonances (δH 1.14, δH 1.22). The configuration at C-13 was determined as S by rerunning NMR spectra in DMSO which enabled a correlation to be seen between the 13-OH proton resonance (δH 3.87) and the H3-20 resonance (δH 0.93) in the NOESY spectrum, allowing assignment of the hydroxyl group as α (Fig. 4). The absolute configuration of 4 was also assigned as for 1 and 2 using ECD spectroscopy (Fig. 2). Compound 5, C20H32O4, was similar to compound 2 with a keto group at C-3, hydroxy group at C-2β, a C-7 double bond and an isopropyl group at C-13 with H3-16 and H3-17 doublets (δH 0.98, 0.96, ea d, J = 7.0 Hz) seen to be coupled to a H-15 septet (δH 1.91). The two H14 resonances (δH 1.80, superimposed) showed correlations in the HMBC spectrum with the C-7 (δC 127.9), C-8 (δC 143.4), C-13 oxygenated carbon (δC 79.7), C-15 (δC 32.3) and a further oxygenated fully substituted carbon resonance (δC 80.7) which was assigned as C-9. For 5, correlations were observed in the NOESY spectrum between the H-5/ H3-18 and H3-19/H3-20 resonances, but did not allow us to establish the configuration at C-9 and C-13 (Fig. 5). As a result, four isomers were possible (Table 5). The configuration at C-9 and C-13 for compound 5 were determined by calculating the DP4+ probability of the possible isomers following the protocol previously described (Grimblat et al., 2015; Langat et al., 2018). This method computes NMR shifts of isomeric compounds using quantum chemical calculations, which are then compared to the experimental NMR data in order to establish the stereochemistry of isomeric compounds (Grimblat et al., 2015). The overall results indicated that the (9R,13R)-isomer of 5 was the most likely isomer (Table 5). Despite the 1H-DP4+ probability calculation initially suggesting the (9R,13S)-isomer as the correct isomer, this was outweighed by the contribution made by 13C-DP4+ data, which led to the final assignment of (9R, 13R) for 5. Such discrepancies between 1Hand 13C-DP4+ data were analysed by Grimblat et al. and were rationalized that a combined analysis corrects the anomaly. In the different compounds of known stereochemistry examined in their case study, the misassignments were corrected when including both sets (1H and 13C)

Fig. 4. (a) 1H–1H COSY and key HMBC (H→C) correlations of 4; (b) Key NOESY correlations of 4. 5

Phytochemistry 170 (2020) 112217

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Fig. 5. (a) 1H–1H COSY and key HMBC (H→C) correlations of 5; (b) Key NOESY correlations of 5.

of data in the probability calculations (see Table 4). NMR spectra of 6–9 indicated ent-abieta-8,11,13-trien-3-one derivatives. For compound 6, the HMBC spectrum showed correlations between the H-15 (δH 2.88, sept, J = 7.0 Hz) and C-12 (δC 124.5) and C-14 (δC 127.0) resonances, the H3-16 and H3-17 (6H, δH 1.22, d, J = 7.0 Hz) and the C-13 (δC 146.4) resonances and the H-5 (δH 1.93, dd, J = 3.0, 11.5 Hz), H-12 (δH 7.01, dd, J = 2.0, 8.0 Hz) and H-14 (δH 6.92, bs, W1/2 = 4.2 Hz) with the C-9 (δC 145.1) resonances. The H-12 resonance showed coupling in the COSY spectrum with the H-11 (δH 7.17, d, J = 8.0 Hz) and H-14 resonances. The NOESY spectrum showed correlations between the H-5/H3-18 and H3-19/H3-18 resonances as expected. The specific rotation was found to be −40. The normal series analogue has been reported from Salvia wiedemannii (Topcu and Ulubelen, 1990) but no specific rotation was reported. Compound 7 was found to be the 7β-hydroxy derivative of compound 6. The COSY spectrum showed correlations between the H-5 (δH 2.35, dd, J = 3.0, 12.0 Hz)/two H-6 (δH 1.25 m, 1.97 m) and H-7 (δH 4.88, dd, J = 1.4, 3.5 Hz) resonances. The significantly upfield-shifted chemical shift of C-5 (δC 44.1 compared to δC 50.9 in compound 6) due to the γ-gauche effect indicated that the hydroxy group at C-7 was βaxial (Clemans and Alemayehu, 1993) and this was confirmed by the correlation seen in the NOESY spectrum between the H-7 and H3-20 (δH 1.23) resonances. Acetylation of 7 gave the 7β-acetyl derivative, 7Ac. The normal series analogue of compound 7 has been reported from the leaves of Juniperus phoenicea subsp. turbinate (Delcorral et al., 1994).

Compound 8 was the 2β-hydroxy derivative of compound 7. The HRESIMS showed [M+H]+ at m/z 317.2116 corresponding to a molecular formula of C20H28O3. An AMX system showing coupling between the two non-equivalent H-1 (δH 1.69; δH 2.99, both dd, J = 6.0, 13.0 Hz) and H-2 (δH 4.74, dd, J = 6.0, 13.0 Hz) resonances was seen in the COSY spectrum, and the H-2 resonance showed correlations with the C-4 (δC 46.7) and C-10 (δC 38.3) resonances in the HMBC spectrum. As with compound 2, the hydroxy group was placed at C-2β due to correlations seen in the NOESY spectrum between the H-2α resonance and the H3-20 and H3-19 resonances (Fig. 6). Compound 9 was identified as the 15-hydroxy derivative of compound 6. The H3-16/H3-17 singlet resonance occurred at δH 1.57 (6H) and C-15 occurred as a fully substituted oxygenated resonance at δC 72.5. Compound 10 was identified as the 3α-hydroxy derivative of 6, 3α-hydroxy-ent-abieta-8,11,13-triene. The H-3 resonance occurred at δH 3.30 (dd, J = 6.3, 10.0 Hz), as expected for a 3α-hydroxy group of an ent-diterpenoid (Ayafor et al., 1994), and this was confirmed due to correlations seen in the NOESY spectrum between the H3-18/H-3 and H-5/H-3 resonances. Compound 10 was acetylated to produce the 3αacetyl derivative, 10Ac, 3α-acetoxy-ent-abieta-8,11,13-triene. The normal enantiomer of compound 10 has been isolated previously from Nepeta tuberosa subsp. reticulata (Urones et al., 1988). Compound 11, 15-hydroxy-ent-abieta-8,11,13-triene, was the 3deoxy analogue of 9. The C-3 keto carbon resonance seen for compound 9 was no longer present and correlations were seen in the HMBC

Table 3 13 C NMR data (100 MHz, CDCl3) for compounds 1–8. Carbon

1

2

2-Ac

3

4

5

6

7a

7-Aca

8a

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 1′ 2′

38.3 35.0 217.0 47.4 51.7 24.4 120.9 135.5 50.3 35.0 22.9 27.6 146.0 122.3 35.1 21.1 21.6 25.2 22.4 13.7 – –

48.2 69.2 216.5 47.4 52.5 24.2 120.3 135.3 50.4 35.6 22.9 27.5 146.3 122.1 35.1 21.0 21.6 24.9 22.3 14.5 – –

44.2 71.9 209.3 48.4 52.1 24.2 120.6 135.2 50.5 36.1 22.9 27.4 146.1 122.2 35.1 21.0 21.6 25.0 21.8 14.5 170.7 21.0

38.5 35.0 216.4 48.1 51.3 24.4 120.9 135.0 50.0 35.0 22.7 25.8 145.4 123.2 72.7 28.8 28.9 25.1 22.6 13.6 – –

37.8 35.0 216.8 50.4 55.4 22.9 35.3 143.1 50.4 37.9 18.2 29.9 74.3 124.3 74.9 24.7 24.5 25.8 22.6 14.7 – –

41.0 68.7 215.4 47.4 45.3 24.4 127.9 143.4 80.7 39.7 22.1 25.3 79.7 18.6 32.3 17.3 17.6 24.6 23.0 19.4 – –

37.7 34.9 217.6 47.6 50.9 20.5 31.1 134.8 145.0 37.3 125.6 124.5 146.4 127.0 33.7 24.2 24.2 27.1 21.3 24.9 – –

37.4 34.8 217.3 47.1 44.1 29.3 68.4 135.9 145.1 37.4 125.7 128.0 147.4 127.3 33.8 24.2 24.1 26.9 21.3 24.0 – –

37.4 34.9 216.7 47.0 45.0 26.9 70.8 131.9 146.0 37.4 125.7 128.6 147.3 127.8 33.7 24.2 24.0 26.5 21.2 23.8 170.8 21.7

47.2 69.8 215.7 46.7 46.8 24.0 81.5 129.7 146.9 38.3 124.5 128.1 147.3 129.5 33.7 24.2 24.1 24.8 21.9 24.7 – –

a

125 MHz. 6

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and H3-17 resonances, and also between the H3-20 and H3-19 resonances, implying that they are on the same side of the molecule (Fig. 7). The ECD spectrum showed Cotton effects at 290 nm (ΔE = +3), at 205 nm (ΔE = −49) and at 192 nm (ΔE = −45), which were the same as those reported for 17-hydroxy-ent-pimara-8(14),15dien-3-one (Tembu et al., 2014), confirming that the compound belonged to the ent-pimarane series. The absolute configuration of 14 was also assigned by comparing the calculated and the experimental ECD curves, as was done for 1, 2 and 4. The measured ECD spectrum compares well with the theoretically calculated ECD (Fig. 2). Compounds 2, 2Ac, 3, 4, 7Ac, 8, 9, 10Ac, 12 and 14 were evaluated against the NCI 60 panel of human tumour cell lines (National Cancer Institute, 2019) which is derived from nine cancer cell types including leukaemia, lung, melanoma, colon, CNS, ovary, renal, prostate and breast cancers (Shoemaker, 2006). Compounds were evaluated at a single dose of 10−5 M. Results for the single dose screen are given in the Supplementary Material (Figures S4.1 to S4.10). Only compound 14 (Fig. S4.10) showed significant activity and selectivity against three of the 60 cancer cell lines at the single dose concentration of 10−5 M, the melanoma cancer cell line (MALME-3M, 98.9% cell death), the ovarian cancer cell line (IGROV1, 88.7% cell death) and the renal cancer cell line (UO-31, 82.1% cell death).

Table 4 13 C NMR data (100 MHz, CDCl3) for compounds 9–14. Carbon position

9

10a

10-Aca

11

12

13

14a

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 1′ 2′

37.7 34.8 217.5 47.6 50.8 20.4 31.2 134.9 146.0 37.3 125.6 122.6 146.7 125.1 72.5 31.9 31.9 27.1 21.3 24.8 – –

37.5 28.3 79.0 39.2 50.1 19.1 31.0 135.0 147.0 37.2 124.6 124.2 145.9 127.0 33.7 24.1 24.2 28.4 15.6 25.1 – –

36.8 24.6 80.9 38.1 50.2 19.0 30.8 134.8 146.0 37.5 124.6 124.2 145.9 127.0 33.7 24.1 24.2 28.4 16.8 25.2 171.2 21.5

39.0 19.5 41.9 33.7 50.6 19.3 30.8 135.2 145.9 37.8 124.5 122.1 148.9 125.1 72.5 31.8 31.8 33.5 21.8 25.1 – –

38.7 19.5 41.8 33.2 44.9 68.6 28.9 136.3 147.7 38.1 124.7 126.8 146.6 128.0 33.2 24.3 24.3 33.4 21.9 24.1 – –

37.3 27.8 79.4 39.2 54.3 22.4 35.9 138.2 51.4 38.4 19.3 35.9 38.8 128.3 147.5 113.0 29.6 28.7 15.9 15.0 – –

37.9 35.1 217.1 48.1 55.7 23.3 35.6 137.4 50.7 38.3 19.5 35.8 38.9 129.2 147.4 113.2 29.6 25.9 22.6 14.7 – –

a

125 MHz.

3. Experimental section

spectrum between a methylene carbon resonance at δC 41.9 (C-3) and the H-5 (δH 1.35, dd, J = 2.3, 12.0 Hz), H3-18 (δH 0.96) and H3-19 (δH 0.93) resonances. The normal series analogue has been isolated from Pinus monticola (Conner et al., 1980). Compound 12 differed from 11 in having a hydrogen instead of a hydroxy group at C-15 shown by the coupled H3-16/H-15/H3-17 system and had a 6β-hydroxy substituent shown by correlations seen in the HMBC spectrum between H-6 (δH 4.82, bs, W1/2 = 11.4 Hz) and the C-4 (δC 33.2), C-10 (δC 38.1) and C-8 (δC 136.3) resonances. The NOESY spectrum showed correlations between the H-6 resonance and H3-20 (δH1.13), and H3-19 (δH 0.94) resonances enabling the configuration of the hydroxy group to be established as β. The normal series analogue has been reported from Vitex negundo (Chawla et al., 1991) and also synthesised (Matsumoto et al., 1983). Compound 13 was found to be the known 3α-hydroxy-ent-pimara8(14),15-diene (Ansell et al., 1993), and compound 14 was found to be its 3-keto derivative. A keto group (δC 217.1) was placed at C-3 due to correlations seen with the H-5 (δH 1.46) and H3-18 (δH 1.10) and H3-19 (δH 1.07) resonances in the HMBC spectrum. The NOESY spectrum showed correlations between the H3-18 and H-5, H-5 and H-9, and H-9

3.1. General experimental procedure NMR experiments were conducted on either a 400 MHz or 500 MHz Bruker AVANCE III NMR spectrometer. Spectra were recorded in CDCl3 and, in some cases, DMSO‑d6. CDCl3 was referenced according to the central line at δ 7.26 in the 1H NMR spectrum and at δ 77.23 in the 13C NMR spectrum. DMSO was referenced according to the central line at δ 2.50 in the 1H NMR spectrum and at δ 39.51 in the 13C NMR spectrum. The spectra were processed using Bruker NMR Academic Topspin software. HRESIMS spectra were recorded using an Agilent 1260 Infinity II coupled to an Agilent 6550 Quadrupole Time-of-Flight mass spectrometer using electrospray ionisation. LC conditions were as follows: inj vol. 1.00 μL, column Agilent Extend-C18, flow rate 1.0 mL/min, mobile phase (positive mode) Solvent A: CH3CN (0.1% formic acid), Solvent B: water (0.1% formic acid), mobile phase (negative mode) Solvent A: CH3CN, Solvent B: water; gradient: 0–3 min, 5% A; 3–3.5 min, 100% A; 3.5–4 min, 5% A. GC-MS analysis was carried out using an Agilent Technologies 6890N and GC system coupled to an Agilent Technologies HP5973 mass electron detector with samples dissolved in CH2Cl2.

Table 5 DP4+ probability analysis for the four possible isomers of 5. (sDP4+ and uDP4+ refer to DP4+ calculation using scaled and unscaled chemical shifts respectively). Probability (%)

sDP4+ (H data) sDP4+ (C data) sDP4+ (all data) uDP4+ (H data) uDP4+ (C data) uDP4+ (all data) DP4+ (H data) DP4+ (C data) DP4+ (all data)

0.01 0.88 0.00 0.02 0.00 0.00 0.00 0.00 0.00

90.76 0.18 3.13 71.63 4.67 13.88 96.60 0.02 0.54

0.33 43.58 2.71 2.76 16.03 1.84 0.01 13.73 0.06

7

8.91 55.36 94.16 25.58 79.30 84.28 3.39 86.25 99.39

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Fig. 6. (a) 1H–1H COSY and key HMBC (H→C) correlations of 8; (b) Key NOESY correlations of 8.

Fig. 7. (a) 1H–1H COSY and key HMBC (H→C) correlations of 14; (b) Key NOESY correlations of 14.

Infra-red (IR) spectra were recorded in the range 600–4000 cm−1 using a PerkinElmer Spectrum Two FT-IR Spectrometer. Optical rotations were measured using a JASCO-P-1020 polarimeter using quartz cuvettes of 1 cm path length. ECD spectra were measured on an Applied Photophysics Chirascan CD spectrometer using a 1 mm cell with CH3CN as solvent.

chromatography was carried out using different column sizes (1–3 cm diameter), which were packed with silica gel (Merck Art. 9385) in selected solvent systems or Sephadex (LH 20) in CH3OH/CH2Cl2. A detailed separation scheme is provided in the Supplementary Data. Purity of compounds was monitored via thin layer chromatography (TLC) using pre-coated aluminium-backed plates (silica gel 60 F254) and compounds were visualised by UV radiation at 254 nm and then using an anisaldehyde spray reagent (1% p-anisaldehyde: 2% H2SO4: 97% cold CH3OH) followed by heating.

3.2. Plant material Leaves of Croton mubango Müll. Arg. (Euphorbiaceae) were collected in April 2017 from the Kikwit forest, 5ο02′19″S and 18ο49′05″E, Kwilu province, Democratic Republic of the Congo by the group of Prof. Blaise M. Mbala. A voucher specimen, H. Breyne 3241, has been retained at the INRA Herbarium, University of Kinshasa.

3.4. Computational methods 3.4.1. DP4+ calculations Systematic conformational searches of the different isomers were carried out via the Spartan’16 software using molecular mechanics force field calculations (MMFF). Conformers under 2.0 kcal/mol energy cutoff were selected and optimized using the DFT method at the B3LYP/631G* level (gas phase). The magnetic shielding constants (σ) were computed using the gauge-including atomic orbital (GIAO) method at the mPW1PW91/6-31 + G** level of theory. The calculations were carried out in solution (IEFPCM, solvent: CHCl3). The computed shielding tensors were Boltzmann averaged for the selected conformations and introduced to the Excel spreadsheet provided by Grimblat et al. (2015) to facilitate DP4+ probability calculations.

3.3. Extraction and isolation Plant material was cut into small pieces, air dried, then ground using a laboratory hammer mill with a sieve diameter of 1 mm, and stored in a well ventilated environment. Ground leaves (900 g) were extracted on a shaker at room temperature, with CH2Cl2 (2.5 L) for 48 h and then filtered. This process was repeated with fresh CH2Cl2 (2.5 L) for a further 48 h and then filtered. The extracts were combined and evaporated to dryness using a rotary evaporator at 40 °C to yield a dark brown gum (45 g). The plant extract (30 g) was dissolved in CH2Cl2 and mixed with silica gel, dried, crushed to a powder and packed into a Biotage SNAP 100 g flash purification cartridge. Flash chromatography (Biotage SP1 system) was carried out firstly using a hexane/CH2Cl2 step gradient starting with 100% hexane and gradually increasing the concentration of CH2Cl2 to 100% and secondly, starting from 100% CH2Cl2 and increasing the polarity to 50:50 CH2Cl2:EtOAc. Compounds were detected using a wavelength of 254 nm. Final purifications used gravity column and preparative thin layer chromatography (Merck 818133). Column

3.4.2. ECD analysis Systematic conformational searches of ent- and normal-isomers of the diterpenoids were carried out via the Spartan’16 program using molecular mechanics force field calculations (MMFF). Conformers under 3.0 kcal/mol were optimized using the DFT method at the B3LYP/6-31 + G** level. ECD spectra were simulated using Time Dependant Density Functional Theory (TDDFT) at the B3LYP/631 + G** level (Gaussian 09). A polarizable continuum model (IEFPCM, solvent: acetonitrile) was applied in the calculations to mimic 8

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+H]+ (calcd. for C20H30O + H, m/z 287.2369). Ent-pimara-8(14),15-dien-3-one (14): white solid; [ ]22 D -40 (c 0.10 CH2Cl2); IR (NaCl) νmax (cm−1): 3082, 2955, 2871, 1706; ECD (CH3CN) λ (Δε) 290 nm (+3), 205 nm (−49), 191 nm (−45); 1H and 13C NMR are given in Tables 1 and 2; HRESIMS m/z 309.2197 [M+Na]+ (calcd. for C20H30O + Na, m/z 309.2189).

the effects of the solvent used in the experimental ECD spectra. The theoretical ECD spectra of the conformers were Boltzmann weighted and compared to the respective experimental ECD spectra (Nugroho and Morita, 2014). 3.5. Compound characterisation

Declaration of competing interest

Ent-abieta-7,13-dien-3-one (1): yellow solid; [ ]22 D -20 (c 0.20, CH2Cl2); IR (NaCl) νmax (cm-1): 3055, 2965, 2930, 2869, 1705; ECD (CH3CN) λ (Δε) 290 nm (+7), 205 nm (−16), 197 nm (−18); 1H, 13C NMR and 2D NMR data are given in Tables 1 and 2; HRESIMS m/z 287.2373 [M+H]+ (calcd. C20H30O + H, m/z 287.2375). 2β-Hydroxy-ent-abieta-7,13-dien-3-one (2): yellow solid; [ ]22 D -25 (c 0.20 CH2Cl2); IR (NaCl) νmax (cm-1): 3429, 3057, 2964, 2874, 1714, 1463; ECD (CH3CN) λ (Δε) 290 nm (+8), 215 nm (−14), 197 nm (−18); 1H and 13C NMR are given in Tables 1 and 2; HRESIMS m/z 301.2171 [M-H]- (calcd. C20H30O2– H, m/z 301.2173). 15-Hydroxy-ent-abieta-7,13-dien-3-one (3): yellow solid; [ ]22 D -45 (c 0.10 CH2Cl2); IR (NaCl) νmax (cm−1): 3428, 3057, 2970, 2934, 2876, 1712, 1263; 1H and 13C NMR are given in Tables 1 and 2; HRESIMS m/z 320.2590 [M + NH4]+ (calcd. for C20H30O2 + NH4, m/z 320.2584). 13α,15-Dihydroxy-ent-abieta-8(14)-en-3- (4): yellow solid; [ ]22 D -55 (c 0.20 CH2Cl2); IR (NaCl) νmax (cm-1): 3428, 2970, 2936, 2869, 1705; ECD (CH3CN) λ (Δε) 290 nm (+0.6), 202 nm (−13); 1H and 13C NMR are given in Tables 1 and 2; HRESIMS m/z 319.2282 [M-H]- (calcd. for C20H32O3 – H, m/z 319.2279). 2β,9,13-Trihydroxy-ent-abieta-7-en-3-one (5): yellow solid; [ ]22 D -40 (c 0.40 CH2Cl2); IR (NaCl) νmax (cm-1): 3428, 3057, 2966, 2935, 2874, 1713, 1463; ECD (CH3CN) λ (Δε) 290 nm (+1), 215 nm (−3); 1H and 13 C NMR are given in Tables 1 and 2; HRESIMS m/z 359.2178 [M +Na]+ (calcd. for C20H32O4 + Na, m/z 359.2193). Ent-abieta-8,11,13-trien-3-one (6): yellow solid; [ ]22 D -40 (c 0.10 CH2Cl2); IR (NaCl) νmax (cm−1): 2961, 2919, 2850, 1706, 1462, 1262; 1 H and 13C NMR are given in Tables 1 and 2; GCMS tR = 23.9 min m/z (EI) 284.1 (M+, 44%), 269 ([M-CH3]+, 81%), 227 (100%), 185 (31%), 143 (36%), 91 (17%); HRESIMS m/z 285.2219 [M+H]+ (calcd. C20H28O + H, m/z 285.2218). 7β-Hydroxy-ent-abieta-8,11,13-trien-3-one (7): yellow solid; [ ]22 D -5 (c 0.40 CH2Cl2); IR (NaCl) νmax (cm−1): 3431, 3055, 2964, 2930, 2874, 1706, 1462, 1386, 1265, 736; 1H and 13C NMR are given in Tables 1 and 2; GCMS tR = 26.0 min m/z (EI) 300 (M+, 1%), 282 (12%), 225 (9%), 197 (100%), 183 (16%), 141 (15%); HRESIMS m/z 301.2169 [MH]- (calcd. C20H28O2+H, m/z 301.2168). 2β,7β-Dihydroxy-ent-abieta-8,11,13-trien-3-one (8): yellow solid; -1 [ ]22 D -40 (c 0.10 CH2Cl2); IR (NaCl) νmax (cm ): 3436, 2963, 2931, 2874, 1715, 1679, 1261; ECD (CH3CN) λ (Δε) 290 nm (+3), 215 nm (−4); 1H and 13C NMR are given in Tables 1 and 2; HRESIMS m/z 317.2116 [M +H]+ (calcd. for C20H28O3 + H, m/z 317.2111). 15-Hydroxy-ent-abieta-8,11,13-trien-3-one (9): yellow solid; [ ]22 D -10 (c 0.30 CH2Cl2); IR (NaCl) νmax (cm−1): 3436, 2969, 2933, 2870, 1705; 1 H and 13C NMR are given in Tables 1 and 2; HRESIMS m/z301.2172 [M+H]+ (calcd. for C20H28O2 + H, m/z 301.2162). 3α-Hydroxy-ent-abieta-8,11,13-triene (10): yellow solid; [ ]22 D -5 (c 0.20 CH2Cl2); IR (NaCl) νmax (cm−1): 3419, 3053, 2962, 2870, 1710, 1678, 1265, 1091, 1033, 738; 1H and 13C NMR are given in Tables 1 and 2; GCMS tR = 24.4 min m/z (EI) 286 (M+, 29%), 271 ([M-CH3]+, 35%), 253 (100%), 185 (28%), 159 (26%); HRESIMS m/z 285.2214 [MH]- (calcd. C20H30O – H, m/z 285.2218). 15-Hydroxy-ent-abieta-8,11,13-triene (11): yellow solid; [ ]22 D -20 (c 0.10 CH2Cl2); IR (NaCl) νmax (cm−1): 3392, 2927, 2867, 1264, 738; GCMS tR = 23.8 min m/z (EI) 286 (M+, 1%), 268 (68%), 253 (100%), 197 (21%), 183 (53%), 171 (86%), 167 (80%); HRESIMS m/z 269.2267 [M-H2O + H]- (calcd. C20H30O – H2O + H, m/z 269.2269). 6β-Hydroxy-ent-abieta-8,11,13-triene (12): yellow solid; [ ]22 D -5 (c 0.20 CH2Cl2); IR (NaCl) νmax (cm−1): 3416, 2959, 2927, 2868, 1461; 1H and 13C NMR are given in Tables 1 and 2; HRESIMS m/z 287.2361 [M

This is to confirm that there are no conflict of interests here and all funding sources have been acknowledged. Acknowledgements SMI gratefully acknowledges PhD studentship funding from the Tertiary Education Trust Fund and the Federal University of Kashere, Nigeria, as well as the University of Surrey. We thank the Developmental Therapeutics Programme (DTP) of the National Cancer Institute, U.S.A. for compound screening. This research was funded by a Royal Society-Royal Society of Chemistry International Exchanges award IE170047. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.phytochem.2019.112217. References Aldhaher, A., Langat, M., Ndunda, B., Chirchir, D., Midiwo, J.O., Njue, A., Schwikkard, S., Carew, M., Mulholland, D., 2017. Diterpenoids from the roots of Croton dichogamus pax. Phytochemistry 144, 1–8. Alqahtani, A.M., 2015. Phytochemical Investigation of Members of the Asparagaceae and Euphorbiaceae Families. PhD Thesis. University of Surrey. Ansell, S.M., Pegel, K.H., Taylor, D.A.H., 1993. Diterpenes from the timber of 20 Erythroxylum species. Phytochemistry 32, 953–959. Anthonse, T., Bergland, G., 1973. Constituents of Solidago species 3. The constitution and sterochemistry of diterpenoids from Solidago missouriensis nutt. Acta Chem. Scand. 27, 1073–1082. Ayafor, J.F., Tchuendem, M.H.K., Nyasse, B., Tillequin, F., Anke, H., 1994. Aframodial and other bioactive diterpenoids from Aframomum species. Pure Appl. Chem. 66, 2327–2330. Brown, G.D., 1994. Phytene-1,2-diol from Artemisia annua. Phytochemistry 36, 1553–1554. Campos, A.R., Albuquerque, F.A.A., Rao, V.S.N., Maciel, M.A.M., Pinto, A.C., 2002. Investigations on the antinociceptive activity of crude extracts from Croton cajucara leaves in mice. Fitoterapia 73, 116–120. Chawla, A.S., Sharma, A.K., Handa, S.S., Dhar, K.L., 1991. Chemical investigation and antiinflammatory activity of Vitex negundo seeds: Part I. Indian J. Chem., Sect. B 30, 773–776. Clemans, G.B., Alemayehu, M., 1993. The gamma gauche substituent effect in 13C NMR. Tetrahedron Lett. 34, 1563–1566. Conner, A.H., Nagasampagi, B.A., Rowe, J.W., 1980. Terpenoid and other extractives of western white-pine bark. Phytochemistry 19, 1121–1131. Craveiro, A.A., Andrade, C.H.S., Matos, F.J.A., Alencar, J.W., Dantas, T.N.C., 1980. Fixed and volatile constituents of Croton aff. nepetifolius. J. Nat. Prod. 43, 756–757. Delcorral, J.M.M., Gordaliza, M., Salinero, M.A., Sanfeliciano, A., 1994. 13C NMR data for abieta-8,11,13-triene diterpenoids. Magn. Reson. Chem. 32, 774–781. Feliciano, A.S., Delcorral, J.M.M., Gordaliza, M., Salinero, M.A., 1993a. 13C NMR data for abieta-7,13-diene diterpenoids. Magn. Reson. Chem. 31, 841–844. Feliciano, A.S., Gordaliza, M., Delcorral, J.M.M., Castro, M.A., Garciagravalos, M.D., Ruizlazaro, P., 1993b. Antineoplastic and antiviral activities of some cyclolignans. Planta Med. 59, 246–249. Frisch, M.J., Trucks, G.W., Schlegel, H.B., Scuseria, G.E., Robb, M.A., Cheeseman, J.R., Scalmani, G., Barone, V., Petersson, G.A., Nakatsuji, H., Li, X., Caricato, M., Marenich, A.V., Bloino, J., Janesko, B.G., Gomperts, R., Mennucci, B., Hratchian, H.P., Ortiz, J.V., Izmaylov, A.F., Sonnenberg, J.L., Williams, Ding, F., Lipparini, F., Egidi, F., Goings, J., Peng, B., Petrone, A., Henderson, T., Ranasinghe, D., Zakrzewski, V.G., Gao, J., Rega, N., Zheng, G., Liang, W., Hada, M., Ehara, M., Toyota, K., Fukuda, R., Hasegawa, J., Ishida, M., Nakajima, T., Honda, Y., Kitao, O., Nakai, H., Vreven, T., Throssell, K., Montgomery Jr., J.A., Peralta, J.E., Ogliaro, F., Bearpark, M.J., Heyd, J.J., Brothers, E.N., Kudin, K.N., Staroverov, V.N., Keith, T.A., Kobayashi, R., Normand, J., Raghavachari, K., Rendell, A.P., Burant, J.C., Iyengar, S.S., Tomasi, J., Cossi, M., Millam, J.M., Klene, M., Adamo, C., Cammi, R., Ochterski, J.W., Martin, R.L., Morokuma, K., Farkas, O., Foresman, J.B., Fox, D.J., 2016. Gaussian 09. Rev. A.02, Wallingford, CT. Grimblat, N., Zanardi, M.M., Sarotti, A.M., 2015. Beyond DP4: an improved probability

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