The phytochemical investigation of five African Croton species: Croton oligandrus, Croton megalocarpus, Croton menyharthii, Croton rivularis and Croton megalobotrys

Article abstract of DOI:10.1016/j.phytol.2020.09.020

The chemistry of five African Croton taxa, Croton oligandrus Pierre ex Hutch., Croton megalocarpus Hutch., Croton menyharthii Pax, Croton rivularis Mull.Arg. and Croton megalobotrys Mull.Arg. is described. The undescribed ent-19-hydroxyisopimara-8(9),15-dien-7-one and ent-isopimara-7(8),15-dien-16,19-diol were isolated from the fruits of C. oligandrus, ent-isopimara-7(8),15-dien-19-yl octadecanoate was obtained from both the fruits and leaves, and ent-19-hydroxyisopimara-8(9),15-dien-7-one was isolated from the leaves of this species. The undescribed 3,4,15,16-diepoxy-8α-hydroxycleroda-13(16),14-dien-12S,17-olide and (5S,9R,10S)-7,13-ent-abietadien-2-one were isolated from the leaves and roots of C. megalocarpus respectively. Compounds isolated from C. menyharthii, C. rivularis and C. megalobotrys have been reported from other sources. The structures of the compounds were determined using NMR, IR and MS experiments. The absolute configurations of the ent-isopimarane, ent-abietane and ent-clerodane diterpenoids isolated were confirmed by comparing calculated and experimental electronic circular dichroism (ECD) spectra. DP4+ probability calculations were used to assign the configuration at C-8 for 3,4,15,16-diepoxy-8α-hydroxycleroda-13(16),14-dien-12S,17-olide. Epoxy-ent-clerodadiene, 3β,4β:15,16-diepoxy-13(16),14-ent-clerodadien-17,12S-olide, 3β,4β:15,16-diepoxy-8α-hydroxy-ent-cleroda-13(16),14-dien-12,17-olide, 7,13-abietadien-2-ol, (5S,9R,10S)-7,13-ent-abietadien-2-one, crotonolide E, furocrotinsulolide A, epoxychiromodine, 3β,4β:15,16-diepoxy-13(16),14-ent-clerodadiene and crotohalimaneic acid were selected for screening based on their ability to add diversity to the NCI small molecule compound collection, and were evaluated against the NCI60 panel of human tumour cell lines at 10μM level, but found inactive.

Full text of DOI:10.1016/j.phytol.2020.09.020

Phytochemistry Letters 40 (2020) 148–155
Contents lists available at ScienceDirect
Phytochemistry Letters
journal homepage: www.elsevier.com/locate/phytol
The phytochemical investigation of five African Croton species: Croton
oligandrus, Croton megalocarpus, Croton menyharthii, Croton rivularis and
Croton megalobotrys
,
b
,
,f
Moses K. Langata c,*, Elvire F.K. Djuidjed, Beth M. Ndundae, Sani M. Isyakab
,
Nathalie S. Dolanb, Gareth D. Ettridgeb, Hannah Whitmoreb, Isabel Lopeza
,
,
b
Alaa M. Alqahtanib g, Ibrahim Atikub h, Jules S. Lobed, Eduard Mas-Claretb, Neil R. Crouchc
,
,
,
,i
Jacob O. Midiwoe, Dulcie A. Mulhollandb c, Alain F.W. Kamdemb
,
,c,d
a Jodrell Laboratory, Natural Capital and Plant Health Department, Royal Botanic Gardens, Kew, Richmond, Surrey, TW9 3DS, United Kingdom
b Natural Products Research Group, Department of Chemistry, Faculty of Engineering and Physical Sciences, University of Surrey, Guildford, GU2 7XH, United Kingdom
c School of Chemistry and Physics, University of KwaZulu-Natal, Durban, 4041, South Africa
d Chemistry Laboratory, University of Douala, Faculty of Sciences, 24157, Douala, Cameroon
e Department of Chemistry, University of Nairobi, P Bag 30197-00100, Nairobi, Kenya
f Department of Chemical Sciences, Faculty of Science, Federal University of Kashere, Gombe, Nigeria
g Pharmaceutical Chemistry Department, College of Pharmacy, Umm Al-Qura University, Makkah, 21955, Saudi Arabia
h Department of Pharmaceutical and Medicinal Chemistry, Ahmadu Bello University Zaria, Nigeria
i Biodiversity Research, Monitoring and Assessment, South African National Biodiversity Institute, PO Box 52099, Berea Road, 4007, Durban, South Africa
A R T I C L E I N F O
A B S T R A C T
Keywords:
The chemistry of five African Croton taxa, Croton oligandrus Pierre ex Hutch., Croton megalocarpus Hutch., Croton
menyharthii Pax, Croton rivularis Mull.Arg. and Croton megalobotrys Mull.Arg. is described. The undescribed ent-
19-hydroxyisopimara-8(9),15-dien-7-one and ent-isopimara-7(8),15-dien-16,19-diol were isolated from the fruits
of C. oligandrus, ent-isopimara-7(8),15-dien-19-yl octadecanoate was obtained from both the fruits and leaves,
and ent-19-hydroxyisopimara-8(9),15-dien-7-one was isolated from the leaves of this species. The undescribed
Croton oligandrus
Croton megalocarpus
Croton menyharthii
Croton rivularis
Croton megalobotrys
ent-Diterpenoids
3,4,15,16-diepoxy-8α-hydroxycleroda-13(16),14-dien-12S,17-olide and (5S,9R,10S)-7,13-ent-abietadien-2-one
were isolated from the leaves and roots of C. megalocarpus respectively. Compounds isolated from C. menyharthii,
C. rivularis and C. megalobotrys have been reported from other sources. The structures of the compounds were
determined using NMR, IR and MS experiments. The absolute configurations of the ent-isopimarane, ent-abietane
and ent-clerodane diterpenoids isolated were confirmed by comparing calculated and experimental electronic
circular dichroism (ECD) spectra. DP4+ probability calculations were used to assign the configuration at C-8 for
3,4,15,16-diepoxy-8
α-hydroxycleroda-13(16),14-dien-12S,17-olide. Epoxy-ent-clerodadiene, 3β,4β:15,16-die-
poxy-13(16),14-ent-clerodadien-17,12S-olide,
3β,4β:15,16-diepoxy-8α-hydroxy-ent-cleroda-13(16),14-dien-
12,17-olide, 7,13-abietadien-2-ol, (5S,9R,10S)-7,13-ent-abietadien-2-one, crotonolide E, furocrotinsulolide A,
epoxychiromodine, 3β,4β:15,16-diepoxy-13(16),14-ent-clerodadiene and crotohalimaneic acid were selected for
screening based on their ability to add diversity to the NCI small molecule compound collection, and were
evaluated against the NCI60 panel of human tumour cell lines at 10μM level, but found inactive.
1. Introduction
et al., 2020). In our continued research on the chemistry and bioactivity
of African Croton species, we investigated the phytochemistry of five
such species sourced from West, East and South Africa: Croton oligandrus
Pierre ex Hutch., Croton megalocarpus Hutch., Croton menyharthii Pax,
Of the approximately 1300 species of Croton L. (Euphorbiaceae) that
occur pantropically, some 55 occur in continental Africa (Ngumbau
* Corresponding author at: Jodrell Laboratory, Natural Capital and Plant Health Department, Royal Botanic Gardens, Kew, Richmond, Surrey, TW9 3DS, United
Kingdom.
E-mail address: m.langat@kew.org (M.K. Langat).
https://doi.org/10.1016/j.phytol.2020.09.020
Received 20 July 2020; Received in revised form 20 September 2020; Accepted 24 September 2020
Available online 26 October 2020
1874-3900/© 2020 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.

M.K. Langat et al.
Phytochemistry Letters 40 (2020) 148–155
Croton rivularis Mull.Arg. and Croton megalobotrys Mull.Arg. These five
species have been placed in at least three of the 40 sections delineated by
Webster (1993), in a genus that is chemically diverse and phylogeneti-
cally only partially resolved (Berry et al., 2005).
in valley bushveld along hot river valleys, growing to 1.5 m tall (Boon,
2010). There are no reports on the chemistry or of ethnomedicinal ap-
plications for C. rivularis. In this study we report the investigation of the
leaves of C. rivularis collected from southern KwaZulu Natal, South
Africa.
C. oligandrus is a tropical tree that grows to 15 m tall, and is restricted
to West and Central African forests (Aubreville, 1983). The stem bark of
this plant is used ethnomedicinally by Pygmies in Gabon to treat
anaemia and for colic, stomach disorders, pneumonia and splenomegaly
in Cameroon (Aubreville, 1983; Jiofack et al., 2009; Lagarde Betti et al.,
2013). There are four prior reports on the chemistry of C. oligandrus.
Agnaniet et al. (2005) described the chemical composition of an
essential oil obtained by hydrodistillation of stem bark from Gabon.
Abega et al. (2014) reported that the stem bark and leaves collected in
the Nkol-nkoumou, Central region of Cameroon possess crotonoligake-
tone, crotonadiol, imbricatadiol, crotonzambefuran B, 7-acetoxytrachi-
loban-18-oic acid and 3-O-acetylaleuritolic acid. Yannick et al. (2019)
reported the clerodane diterpenoids crotoliganfuran and 12-epi--
crotocorylifuran alongside lupeol, syringic acid, aleuritolic acid acetate,
scopoletin, geddic acid, sitosterol, vanillic acid and stigmastane-3,
6-dione from the stem bark collected in Bafut, North West region of
Cameroon. Further, stem bark of this species, collected from Mount
Eloundem in the Central Region of Cameroon, has yielded 12-epi--
megalocarpodolide D, an epimeric mixture of crotonolins A and B,
crotolins C–F, crotonzambefuran A, megalocarpodolide D, 12-epi--
crotocorylifuran, 7β-hydroxydehydroabietic acid, 7-oxodehydroabietic
acid, ferulic acid, cluytyl ferulate, hexacosanoyl ferulate, vanillin,
acetyl aleuritolic acid and lupeol (Guetchueng et al., 2018, 2019).
C. megalocarpus is a fast-growing tree that can exceed 30 m in height,
though often shorter in semi-arid areas (Kokwaro, 1976). It is found in
Kenya eastwards to the Democratic Republic of Congo and southwards
to Zimbabwe (World Checklist of Selected Plant Families, 2019). The sap
produced by the leaves of C. megalocarpus is applied topically in
southern Ethiopia to treat bleeding wounds (Sori et al., 2004). The sap is
also widely used in East Africa as an anthelmintic agent and for the
treatment of whooping cough and tonsillitis (Kokwaro, 1976). Two
clerodane diterpenoids, chiromodine, and epoxychiromodine, four tri-
terpenoids, 3β-acetyl aleuritolic acid, lupeol, 3β-acetoacetyl lupeol,
betulin and ferulic acid ester derivatives were isolated previously from
the stem bark of C. megalocarpus (Addae-Mensah et al., 1992). The seeds
of C. megalocarpus contain 30–32 % fixed oils, indicating their potential
as a source of biodiesel, especially due to their high methyl ester content
and a kinematic viscosity comparable to that of diesel. Linoleic acid
(18:2) was found to be the major fatty acid, constituting 74 % of all fatty
acids present in the oil (Wu et al., 2012). The essential oils have also
been shown to possess an Epstein-Barr virus-activating potency (Yanase
and Ito, 1984). Croton megalocarpus has been shown to have
anti-inflammatory, antifungal, antibacterial, antinociceptive, wound
healing molluscidal and toxic properties (Maroyi, 2017a).
C. megalobotrys (syn. C. gubouga S.Moore) is a medium sized tree or
shrub growing up to 15 m high, and is known as the Feverberry (Pal-
grave, 2002). It occurs from Tanzania in the north, through Central
Africa to Swaziland in the south (Festus, 2019). Throughout its distri-
bution range infusions, decoctions or macerations of its root, leaves,
fruits and seeds are used medicinally as a purgative, a febrifuge, or to
treat body pain. Preparations of the seeds of C. megalobotrys are used in
the treatment of malaria for which purpose it attracted considerable
attention from the South African medical fraternity in the late 19th
century (Maberly, 1899; Palgrave, 2002). Plant extracts have been re-
ported to possess antiplasmodial, antibacterial, anti-HIV, and free
radical scavenging activities (Maroyi, 2017b). Tietjen et al. (2018) re-
ported that the crude extracts of C. megalobotrys are used as one part of a
regimen possessing HIV latency-reversal properties. These authors iso-
lated two phorbol esters, numushen 1 and numushen 2 which conferred
HIV latency-reversal in vitro.
2. Results and discussion
In this study, we report the phytochemical investigation of five Af-
rican Croton taxa, C. oligandrus, C. megalocarpus, C. menyharthii, Croton
rivularis. and C. megalobotrys.
Eight compounds, including ent-isopimara-8,15-dien-7-one (1), the
undescribed ent-19-hydroxyisopimara-8(9),15-dien-7-one (2), ent-iso-
pimara-7,15-dien-19-ol (3), the undescribed ent-isopimara-7(8),15-
dien-16,19-diol (4) and ent-isopimara-7(8),15-dien-19-yl octadecanoate
(5) (Fig. 1), methyl palmitate, octyl ferulate and lupenone were isolated
from the fruits of C. oligandrus from Cameroon. Five compounds,
including the undescribed ent-19-hydroxyisopimara-8(9),15-dien-7-one
(2), ent-isopimara-7(8),15-dien-19-yl octadecanoate (5), the known ent-
isopimara-7,15-dien-19-ol (3), lupeol and β-caryophyllene oxide, were
isolated from the leaves and nine known compounds, ent-isopimara-
7,15-dien-19-oic acid (Atta-Ur-Rahman et al., 2005), ent-isopimara-7,
15-diene (Radulgin et al., 1977), ent-isopimara-8(14),15-diene (Mafu
et al., 2016), ent-isopimara-8(14),15-dien-7β-ol (Ravu et al., 2015),
crotocorylifuran, megalocarpodolide D (Guetchueng et al., 2018, 2019;
Ndunda et al., 2016), acetyl aleurotolic acid, lupeol, lupenone and octyl
ferulate, were isolated from the stem bark.
This is the second report of ent-isopimarane diterpenoids from a
member of the Croton genus but the first report from this plant. Previ-
ously, isopimara-7,15-dien-3β-ol was isolated from the African
C. zambesicus (Block et al., 2005) and ent-pimarane was reported from the
African C. mubango (Isyaka et al., 2020). From the leaves and the roots of
C. megalocarpus collected from Kenya we isolated ent-clerodane and
ent-abietane diterpenoids. From the leaves, we report three ent-clerodane
C. menyharthii is a large shrub/small tree of dry forests and semi-arid
habitats, growing to 4 m tall. It occurs from southern Ethiopia south-
wards through East and southern Africa to South Africa (Schmelzer,
2007). A decoction of the dried or fresh roots is taken to treat malaria,
influenza and dysmenorrhoea. The leaves soaked in water are drunk to
treat tapeworm and hepatitis, and the smoke from the burnt leaves is
inhaled to ease menstruation pains and labour pains (Schmelzer, 2007).
In KwaZulu-Natal in South Africa, smoke from the dried plant material
of C. menyharthii is used traditionally to repel mosquitoes (Mavundza
et al., 2011). Most parts of the plants are used medicinally in Somalia in
the management of intestinal obstruction, dysmenorrhoea, hepatitis and
ascites (Samuelsson et al., 1992). The leaves of C. menyharthii growing in
the Umzinyathi District, KwaZulu-Natal, South Africa have earlier
yielded the three flavonols myricetrin 3-O-rhamnoside (myricetrin),
quercetin 3-O-rhamnoside and quercetin, along with an indole alkaloid,
(E)-N-(4-hydroxycinnamoyl)-5-hydroxytryptamine (Aderogba et al.,
2013).
diterpenoids,
epoxy-ent-clerodadiene,
3β,4β:15,16-diepoxy-13(16),
C. rivularis is a shrub endemic to eastern South Africa, where it occurs
Fig. 1. Ent-isopimarane diterpenoids isolated from C. oligandrus.
149

M.K. Langat et al.
Phytochemistry Letters 40 (2020) 148–155
14-ent-clerodadien-17,12S-olide (6) and the undescribed 3β,4β:15,
δH 1.26, m) resonances, and the C-9 carbon resonance showed correla-
tions with the 3H-20, two H-12 (δH 2.33, m and δH 2.00, m) and two H-
14 resonances. Proton resonances ascribed to the Δ15 AMX system were
seen at δ 5.66 (H-15, J = 10.8, 17.5 Hz), δ 4.92 (H-16cis, J = 10.8,
16-diepoxy-8α-hydroxy-ent-cleroda-13(16),14-dien-12,17-olide
(7)
(Fig. 2), a labdane, kolavenol, lupeol and sitosterol. From the roots we
isolated 3β,4β:15,16-diepoxy-13(16),14-ent-clerodadien-17,12S-olide
H
H
(6), crotonolide E, furocrotinsulolide A, epoxychiromodine, 3β,4β:15,
16-diepoxy-13(16),14-ent-clerodadiene and trans-annonene, crotohali-
maneic acid, ent-7,13-abietadien-2-ol (8) the undescribed (5S,9R,10S)-7,
13-abietadien-2-one (9) (Fig. 2) and lupeol. From the leaves of
C. menyharthii we report the isolation of 5-hydroxy-3,7,4’-trimethoxy-
flavone, 5,3’-dihydroxy-3,7,4’-trimethoxyflavone, 4-hydroxy-1-me-
thyl-2-pyrrolidinone, caryophyllene oxide and sitosterol. From the
leaves of C. rivularis we report the isolation of squalene and the acyclic
diterpenoid, phytol, and from the stem bark of C. megalobotrys we report
cycloartenone, lupeol acetate and geranyl geraniol.
1.2 Hz), and δH 4.84 (H-16trans, J = 1.2, 17.5 Hz). The NOESY spectrum
was used to assign the configuration at C-13. As was observed for
compound 1, correlations were observed between 3H-18 and H-5, 3H-18
and H-6α, H-6α and H-15 resonances on one face of the molecule and
between the 2H-19 and 3H-20, 2H-19 and H-6β, 3H-20 and 3H-17, and
between H-6β and 3H-17 resonances on the other face of the molecule.
This data indicated that the configuration at C-13 was S. Thus compound
2 was the 19-hydroxy derivative of 1, ent-19-hydroxyisopimara-8(9),15-
dien-7-one, and is previously undescribed.
Compound 3 was identified as ent-isopimara-715-dien-19-ol, previ-
ously isolated from Sagittaria latifolia Willd. (Alismataceae) (Ravu et al.,
2015) and compound 4 was its undescribed 6β-hydroxy derivative. The
protonated HR-ESIMS spectrum for compound 4 gave a molecular ion
peak at m/z 303.2325 [M+H]+ (calculated m/z 303.2324), corre-
sponding to a molecular formula of C20H30O2 for the compound. The IR
–
The structure of compound 1 was determined to be the known ent-
isopimara-8,15-dien-7-one (1) previously reported from Salvia parryi A.
Gray (Lamiaceae) (Touche et al., 1997) and Vellozia nanuzae L.B.Sm. &
Ayensu (Velloziaceae) (Pinto et al., 1988). Electronic circular dichroism
studies were used to determine the absolute configurations at the chiral
centres of 1 and to confirm that ent-isopimaranes were isolated in this
study. A conformational analysis and TDDFT calculations were under-
taken for ent-isopimara-8,15-dien-7-one (1) and its normal enantiomer,
isopimara-8,15-dien-7-one (1a) following the protocol reported previ-
ously (Isyaka et al., 2020; Langat et al., 2014, 2018; Tembu et al., 2014)
and their simulated ECD curve were compared to the experimental data
for 1 (Fig. 3). Compound 1 was confirmed to belong to the ent series of
isopimarane diterpenoids (Fig. 1). The specific rotation value for com-
spectrum for compound 4 gave absorption bands at 3434 cmꢀ 1 (O
H
stretch), 3023 cmꢀ 1 (=C–H stretch) and 1639 cmꢀ 1 (C C stretch). As
–
–
observed with compounds 2 and 3, the methyl group at C-19 had been
oxidised to a primary alcohol, shown by the pair of H-19 doublets at δH
3.81, d, J=8.2 Hz and δ 3.65, (J = 8.2 Hz). The C-19 (δ 81.4) reso-
H
C
nance showed a correlation in the HMBC spectrum with the H-5 reso-
nance (δ 1.29, d, J = 4.8 Hz), which showed coupling in the COSY
H
spectrum with the oxymethine H-6 resonance (δ 4.30, brd, W1/2
=
pound 1 was [
α
] -8◦, which was comparable to those reported by Touche
10.6 Hz), which showed further coupling with thHe H-7 alkene proton
et al. (1997) and Ponnapalli et al. (2013).
resonance (δ 5.67, brs, J = 4.8 Hz), showing the presence of a Δ7
H
The HR-ESIMS spectrum of compound 2 gave a protonated molec-
ular ion peak at m/z 303.2321 [M+H]+ (calculated m/z 303.2324),
corresponding to a molecular formula of C20H30O2 for the compound.
double bond. The NOESY spectrum showed correlations between the
H-6 resonance and the H-5, H-9 (δ 1.67 m) and 3H-18 (δ 1.15, s)
H
proton resonances, indicating that the hydroxy group at CH-6 was β.
Compound 4 was identified as ent-isopimara-7(8),15-dien-6β,19-diol.
Compound 5 was found to be a C-19 octadecanoyl ester derivative of
compound 3. Compound 5 was isolated as a yellow oil and its HR-ESIMS
gave a sodiated molecular ion peak at m/z 577.5195 [M + Na]+
(calculated 577.49605), corresponding to C38H66NaO2. The IR spectrum
gave absorption bands 3023 cmꢀ 1 (=C–H stretch), 2924 cm-1 and
2853 cm-1 (C–H stretch) and 1734 (carbonyl stretch). The HMBC spec-
trum for 5 showed that the oxymethylene proton resonances at δH 4.37
(d J = 10.9 Hz) and δH 3.96 (d J = 10.9 Hz) of 2H-19 were correlated to a
carbonyl carbon resonance at δ 174.3 of an ester for the octadecanoic
acid. Alkaline hydrolysis of compound 5 using 2 N methanolic solution
of NaOH followed by column chromatography yielded 3 and octadeca-
noic acid. Compound 5 was the undescribed ent-isopimara-7(8),15-dien-
19-yl octadecanoate.
The IR spectrum gave absorption bands at 3430 cmꢀ 1 (O H stretch),
–
3023 cm-1
(
C
H stretch), broad peak at 1645 cm (for the keto
ꢀ 1
–
–
–
group and the double bond of the α-β unsaturated ketone stretch). The
1H NMR spectrum (Table 1) showed that compound 2 had three methyl
group proton singlets, compared to four in compound 1 and the C-19
methyl group had been oxidised to a primary alcohol with the two H-19
protons occurring as a pair of doublets at δ 3.56 and δ 3.80
(J = 10.9 Hz). The two H-19 proton resonances sHhowed correlations in
the HMBC spectrum with the C-4 (δC 39.9), C-5 (δC 50.6) and C-18 (δ
26.4) resonances. The two H-19 proton resonances showed correlationCs
with the 3H-20 (δ 1.09) resonance in the NOESY spectrum. As with
H
compound 1, thereHwas an
α,β-unsaturated carbonyl system with carbon
resonances for C-7, C-8 and C-9 occurring at δC 199.9, δC 128.9, and δ
C
166.0 respectively. The C-7 carbon resonance showed correlations with
the H-5 (δH 1.79, dd J = 14.5, 3.6 Hz) and two H-14 (δH 1.32, m and δ
Compound 6 was determined to be the known 3β,4β:15,16-diepoxy-
13(16),14-ent-clerodadien-17,12S-olide previously reported from
Croton persimilis Müll.Arg. (as syn. C. oblongifolius Roxb.) (Pudhom and
Sommit, 2011) and compound 7 was its undescribed 8-hydroxy deriv-
H
2.33, m) resonances, the C-8 carbon resonance showed correlations with
the two H-6 (δH 2.45, dd, J = 14.5, 17.6) and two H-11 (δH 2.20, m and
ative, 3β,4β:15,16-diepoxy-8α-hydroxy-13(16),14-ent-clerodadien-17,
12S-olide. The HR-ESIMS spectrum for compound 7 gave a sodiated
molecular ion peak at m/z 369.0945 [M + Na]+ (calculated m/z
369.1678), corresponding to a molecular formula of C20H26O5 for 7. The
13C NMR spectrum for 7 showed a fully substituted oxygenated carbon
resonance at δ 75.7 in CDCl3 and δ 74.2 in DMSO which was missing in 6.
The use of HMBC and COSY spectra confirmed the presence of a hydroxy
group at C-8. A proton resonance at δH 5.91 (s) ascribed to the hydroxy
group proton showed correlation in the HMBC spectrum with C-17
(δC = 172.8), C-8 (δC = 74.2), C-9 (δC = 40.4) and C-7 (δC = 28.9) res-
onances. The NOESY spectrum showed a correlation between the 8ꢀ OH
proton resonance and the 3H-20 (δ 1.00, s) and 3H-19 (δ 0.98, s)
H
resonances, and thus was on the
α
-fHace of the molecule. The absolute
configuration at C-8 for compound 7 was determined by DP4+ proba-
bility calculations following the protocol previously described (Grimblat
et al., 2015; Langat et al., 2018) (Table 2). This method computes NMR
Fig. 2. Ent-clerodane (6,7) and ent-abietane (8,9) diterpenoids isolated from C.
megalocarpus.
150

M.K. Langat et al.
Phytochemistry Letters 40 (2020) 148–155
Fig. 3. Comparison of experimental and TDDFT-calculated ECD spectra for compound 1 (ent-enantiomer) with different functionals.
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. The results indi-
cated that the (8R)-isomer of 7 was the most likely isomer (Table 2).
(15)-dien-3-one (Sadgrove et al., 2019).
Compounds 6-9, crotonolide E, furocrotinsulolide A, epoxychir-
omodine, 3β,4β:15,16-diepoxy-13(16),14-ent-clerodadiene and croto-
halimaneic acid were evaluated against the NCI 60 panel of human
tumour cell lines (National Cancer Institute, 2019). The tested com-
pounds were found to be inactive at this concentration.
Compound 7 was the undescribed 3β,4β:15,16-diepoxy-8
α
-hydroxy-13
(16),14-ent-clerodadien-17,12S-olide.
Compound 8 was identified as the known 7,13-ent-abietadien-2
α
-ol
previously reported from Solidago missouriensis Nutt. (Asteraceae)
(Anthonsen and Bergland, 1970), and compound 9 was the 2-keto de-
rivative of compound 8. The HR-ESIMS spectrum for 9 gave a sodiated
[M + Na]+ ion at m/z 309.2188 (calculated m/z 309.2194) which
indicated a molecular formula of C20H30O for the compound and a
double bond equivalence of six. The FTIR spectrum showed absorption
bands at 2959, 2923 and 2871 cmꢀ 1 for C–H aliphatic stretches and
3. Experimental section
3.1. General experimental procedures
Optical rotations were measured on a JASCO P-1020 polarimeter.
ECD spectra were obtained on an Applied Photophysics Chirascan CD
spectrometer using a 1 mm cell and acetonitrile as the solvent. FTIR
spectra were recorded using a Perkin-Elmer (2000) spectrometer. 1D
and 2D NMR spectra were recorded in CDCl3 on a 500 MHz Bruker
AVANCE NMR instrument at room temperature. Chemical shifts (δ) are
expressed in ppm and were referenced against the solvent resonances at
7.26 and 77.23 ppm for 1H and 13C NMR respectively. HR-ESI mass
spectra were recorded a Micromass Quattro Ultima mass spectrometer
using a Waters Alliance HPLC to introduce samples (University of Sur-
rey) or a Waters Xevo G2-S (National Mass Spectrometry Facility,
Swansea). GC–MS analysis was carried out using an Agilent Technolo-
gies 6890 N and GC system coupled to an Agilent Technologies HP5973
mass electron detector with samples dissolved in CH2Cl2 (University of
Surrey). Flash chromatography were undertaken using Grace Reveleris
X2 Flash Column Chromatography instrument. Grace Reveleris © silica
120 g cartridges were used during fractionation. 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 MeOH) fol-
lowed by heating. Final purifications used preparative thin layer chro-
matography (Merck 818133) and gravity column chromatography that
was carried out using different column sizes (1ꢀ 2 cm diameter), which
were packed with silica gel (Merck Art. 9385) in selected solvent systems
or Sephadex (LH 20) in CH3OH/CH2Cl2.
1711 cm-1 for C O stretch of a keto group (Silverstein and Webster,
–
–
1997). The 13C NMR spectrum showed the presence of twenty carbon
resonances for 9, including a keto group at δC 211.9 that were consistent
with a tricylic abietanoid structure related to compound 8 but with the
hydroxy group at C-2 replaced by a keto group. The 1H NMR spectrum
(Table 1) showed three methyl group proton resonances at δ 1.05
H
(3H-18), at δH 0.95 (3H-19) and at δH 0.82 (3H-20), along with reso-
nances assigned to two non-equivalent isopropyl methyl groups at δ
1.02 (d, J = 6.5 Hz) and at δH 1.01 (d, J = 6.5 Hz) (3H-16/3H-17). ThHe
two 3H-18 and 3H-19 proton resonances showed correlations in the
HMBC spectrum with C-3, C-4 and C-5 (δC 50.2) resonances, whereas the
3H-20 resonance showed correlations in the HMBC spectrum with C-1
(δ 54.1), C-10, C-5 and C-9 resonances. The corresponding proton
reCsonances for the two H-1 and two H-3 resonances occurred at δH 2.44
(d, J = 11.9 Hz) and δ 2.22 (d, J = 11.9 Hz) (2H-1) and δ 2.10 (d,
H
H
J = 12.5 Hz) and δ 2.37 (d, J = 12.5 Hz) (2H-3), respectively and
showed correlationHwith a C-2 (δ 211.9) keto group in the HMBC
C
spectrum, The NOESY spectrum displayed correlations between the
3H-18 (δH 1.05, s) and the H-5 resonances (δH 1.84, dd, J = 4.5, 11.9 Hz)
and between the H-5 and the H-9 resonances (δH 2.13, dd J = 2.4, 11.9),
confirming the β-configuration of H-5, H-9 and 3H-18. Furthermore, the
3H-19 (δH 0.95, s) resonance correlated with the 3H-20 (δH 0.82, s)
resonance in the NOESY spectrum, confirming α-configuration of 3H-19
and 3H-20. Compound 9 was determined to be 7,13-abietadien-2-one.
The experimental ECD curve of 9 showed positive Cotton effects at
238 (+ 26) and 199 (+ 36), and a negative Cotton effect at 293 (- 1.7). A
conformational analysis and TDDFT calculations were undertaken for 7,
13-ent-abietadien-2-one (9) and 7,13-abietadien-2-one (9a) following
the protocol described previously (Langat et al., 2018, 2014; Tembu
et al., 2014) and their simulated ECD curves were compared to the
experimental data for 9, confirming it was an ent abietane (Fig. 2). A
search in the literature showed that a normal series abietane, 7,13-abie-
tadien-3-one has been reported previously from the gymnosperm Juni-
perus phoenicea L. (Cupressaceae) (Feliciano et al., 1993) and
S. missouriensis (Anthonsen et al., 1973) with a positive optical rotation
of + 111. 7,13-Abietadiene has also been reported from S. missouriensis
(Anthonsen et al., 1973). Often confused for C. megalocarpusi, the South
African C. gratissimus has yielded an abietanoid ent-abiet-8(14), 13
3.2. Plant material
Fruits, leaves and stem bark of C. oligandrus Pierre ex Hutch. were
collected from the central part of Yaounde city, Cameroon in 2012, and
identified by Mr Nana Victor, a botanist at the National Herbarium of
´
Yaounde, Cameroon where voucher specimen No. 65776/HNC has been
deposited. The leaves and roots of C. megalocarpus Hutch. were collected
from a plant at the University of Nairobi, Kenya, and identified by Mr
Patrick Chalo Mutiso, a botanist at the University of Nairobi Herbarium,
where voucher specimen No. BN/2012/02 was deposited. The stem bark
of C. megalobotrys Mull.Arg. was collected from Nelspruit, Mpumalanga
Province, South Africa in 2006, the leaves of C. menyharthii Pax collected
from Mabobhane District on the Tugela River, KwaZulu Natal Province,
South Africa in 2009, and the leaves of C. rivularis Mull.Arg. collected
151

Table 1
1H (500 MHz) and 13C (125 MHz) NMR chemical shift s for compounds 2, 4, 5, 7 and 9.
No
2 in CDCl3
4 in CDCl3
5 in CDCl3
7 in CDCl3
δC
7 in DMSO
9 in CDCl3
δC
δC
δH (J in Hz)
δC
δH (J in Hz)
δC
δH (J in Hz)
δH (J in Hz)
δC
δH (J in Hz)
δH (J in Hz)
1
β
2
β
3
β
4
5
6
β
7
β
8
9
α
α
α
35.7
1.85, m
34.6
1.54, m
39.8
1.83, m
16.8
1.18, m
1.34, m
1.62, m
2.11, m
2.89, s
16.3
1.13, m
54.1
2.44 d, J = 11.9
2.22 d, J = 11.9
–
1.22, m
1.25, m
1.03, m
1.15, m
18.4
35.2
1.54, m
18.8
31.7
1.53, m
18.6
36.4
1.45, m
28.4
62.1
27.6
60.8
1.73, m
211.9
56.7
1.56, m
1.67, m
1.40, m
1.94, br s (W1/2 = 22.5)
1.84, m
1.82, m
1.76, m
2.88, br s (W1/2 = 4.1)
2.10 d, J = 12.5
2.37 d, J = 12.5
–
1.02, m
1.40, m
1.47, m
–
38.4
50.6
35.6
–
41.4
54.9
75.1
–
36.3
51.7
27.4
–
65.8
36.8
34.0
–
–
64.8
36.1
33.4
–
39.6 C
50.2
1.79, dd, (14.2, 3.5)
2.58, dd, (17.6, 3.5)
2.45, d, (14.5, 17.6)
–
1.29, d, (4.7)
4.31, br s, (W1/2 = 13.0)
1.31, m
–
1.84 d, J = 4.5, 11.9
2.24 d, J = 6.2
2.02 m
α
α
2.03, m
1.62, m
1.51
24.4
2.02, m
1.73, m
1.54
199.9
120.9
5.67, br s, (W1/2 = 12.6)
121.5
5.35, br s (W1/2 = 13.9)
29.8
1.81, m
28.9
2.17 dt J = 3.3, 13.8
120.9
5.45 br s W1/2 = 10.7
2.28, m
1.23
128.9
166.0
39.9
–
142.2
49.8
32.8
20.0
–
135.9
52.4
35.6
20.6
–
75.7
41.0
53.2
48.3
–
–
74.2
40.4
52.6
47.3
135.3
51.2
41.6
22.9
–
–
1.67, m
1.66, m
–
2.13 d, J = 2.4, 11.9
10
11
β
–
–
–
0.84, m
0.76, dd, J = 2.2, 11.5
–
α
α
23.2
2.19, m
1.61, m
1.54, m
1.63, m
1.63
1.68 d, J = 11.9
1.22 d, J = 11.9
1.40 m
1.25, m
1.50, m
1.34, m
1.83, s
1.77
12
β
33.5
2.33, m
35.9
1.52, m
36.7
1.35, m
70.7
6.10, dd, J = 11.7, 1.8
69.4
6.06 J = 2.0, 11.6
27.4
2.01, m
1.37, m
1.04, m
2.06 m
13
14
β
34.6
33.9
–
37.0
45.8
–
36.7
46.3
–
125.1
108.5
–
125.1
109.0
–
145.7
122.2
–
α
1.32, m
2.07, m
1.89, m
6.37, s
6.61 J = 1.6, 0.8
5.80 s
2.33, m
2.03, m
1.93, m
15
145.3
111.8
5.66, dd, (10.8, 17.5)
4.92, d, (10.8, 1.2)
4.84, d, (17.5, 1.2)
1.01, s
150.2
109.7
5.81, dd, (10.8, 17.5)
4.94, d, (17.5, 0.5)
4.84, d, (10.8, 0.5)
0.90, s
150.6
109.5
5.80, dd, (10.8, 17.5)
4.93, dd, (17.5, 1.3)
4.88, dd, (10.8, 1.3)
0.86, s
139.7
143.6
7.43, s
140.2
143.7
7.76 br s (W1/2 = 3.4)
7.65 dd J = 1.6, 1.6
35.0
21.0
2.19 m
16a
b
7.38, d J=1.4
1.02 d, J = 6.5
17
28.3
26.4
65.4
22.1
29.9
81.4
21.7
27.7
66.9
173.5
19.7
16.2
–
172.8
19.3
15.9
–
21.5
32.9
22.9
1.01 d, J = 6.5
1.05 s
18
0.98, s
1.15, s
0.96, s
1.14, s
1.08, s
1.08, s
1.00, s
19a
b
3.56, d, (10.7)
3.80, d, (10.7)
1.09, s
3.65, d, (8.2)
3.81, d, (8.2)
1.08, s
4.37, d, (10.9)
3.96, d, (10.9)
0.86, s
0.95 s
20
18.3
19.8
16.3
18.8
1.10, s
18.6
0.99, s
14.6
0.82 s
1′
174.4
25.2
–
2′
1.57, m
3′-16′
18′
8-OH
22.9ꢀ 29.4
14.3
1.29 m
0.87, t, (6.8)
5.91, s

M.K. Langat et al.
Phytochemistry Letters 40 (2020) 148–155
evaporator at 50 oC for n-hexane and 40 oC for CH2Cl2 to give 21.3 g of
the n-hexane extract and 27.2 g of CH2Cl2 extract. The roots were
extracted using 1:1, CH2Cl2:CH3OH (2.0 L) on a shaker for 24 h, filtered
and evaporated to dryness using a rotary evaporator at 50 oC to give
37.6 g of the CH2Cl2:CH3OH extract. Epoxy-ent-clerodadiene,
Table 2
DP4+ probability analysis for the two possible isomers of 7. (sDP4+ and uDP4+
refer to DP4+ calculation using scaled and unscaled chemical shifts
respectively).
Probability (%)
3β,4β:15,16-diepoxy-13(16),14-ent-clerodadien-17,12S-olide
3β,4β:15,16-diepoxy-8 -hydroxy-ent-cleroda-13(16),14-dien-12,17-
(6),
α
olide, kolavenol, lupeol and sitosterol were isolated from the leaves of
this plant. From the roots we isolated 3β,4β:15,16-diepoxy-13(16),14-
ent-clerodadien-17,12S-olide (6), crotonolide E, furocrotinsulolide A,
epoxychiromodine, 3β,4β:15,16-diepoxy-13(16),14-ent-clerodadiene,
trans-annonene, crotohalimaneic acid, 7,13-abietadien-2-ol (8),
(5S,9R,10S)-ent-7,13-abietadien-2-one (9) and lupeol. A detailed pro-
tocol of isolation and purifications of compounds is given as supple-
mentary material.
sDP4+ (H data)
99.95
92.22
100.00
0.15
0.05
sDP4+ (C data)
sDP4+ (all data)
uDP4+ (H data)
uDP4+ (C data)
uDP4+ (all data)
DP4+ (H data)
DP4+ (C data)
DP4þ (all data)
7.78
0.00
99.85
28.83
99.64
25.84
3.30
71.17
0.36
3.3.3. Extraction and isolation of compounds from C. megalobotrys
Stem bark (900.0 g) from C. megalobotrys was extracted on a shaker
at room temperature with methanol (1.5 L) for 24 h and then filtered.
The extract was evaporated to dryness using a rotary evaporator at 50 oC
to give 42.3 g. Cycloartenone, lupeol acetate and geranyl geraniol were
isolated from this plant. A detailed protocol of isolation and purifica-
tions of compounds is given as supplementary material.
74.16
96.70
98.82
1.18
from the Mzimkulu River valley, KwaZulu Natal Province, South Africa
in 2009. Croton megalobotrys, C. menyharthii and C. rivularis were iden-
tified by Prof. Neil Crouch at the KwaZulu-Natal Herbarium (NH) in
Durban, and voucher specimens Crouch 1109 NH, Crouch 1282 BNRH
and Crouch 1252 BNRH respectively retained for verification purposes.
3.3.4. Extraction and isolation of compounds from C. menyharthii
Leaves (340.0 g) from C. menyharthii were extracted on a shaker at
room temperature separately with CH2Cl2 (1.0 L) and EtOAc (1.0 L) for
24 h and then filtered. The extracts were evaporated to dryness using a
rotary evaporator at 50 oC to give 3.6 g of CH2Cl2 extract and 8.9 g of
ethyl acetate extract. 5-Hydroxy-3,7,4′-trimethoxyflavone, 5,3′-dihy-
droxy-3,7,4′-trimethoxyflavone, 4-hydroxy-1-methyl-2-pyrrolidinone,
caryophyllene oxide and sitosterol were isolated from this plant. A
detailed protocol of isolation and purifications of compounds is given as
supplementary material.
3.3. Extraction and isolation
Plant materials were 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.
3.3.1. Extraction and isolation of compounds from C. oligandrus
3.3.5. Extraction and isolation of compounds from C. rivularis
Leaves (300.0 g) of C. rivularis were extracted on a shaker at room
temperature with with CH2Cl2 (1.5 L) methanol (1.0 L) for 24 h and
then filtered. The extract was evaporated to dryness using a rotary
evaporator at 50 oC to give 6.5 g. Squalene and phytol were isolated
from this plant. A detailed protocol of isolation and purifications of
compounds is given as supplementary material.
Ground fruits (676.1 g), leaves (378.2 g) and stem bark (900.0 g) of
C. oligandrus were extracted separately on a shaker at room temperature,
with CH2Cl2 (1.5 L) for 24 h and then filtered. This process was suc-
cessively repeated with CH3OH (1.5 L) for a further 24 h and then
filtered. The extracts were evaporated to dryness using a rotary evapo-
rator at 40 oC for CH2Cl2 and 50 oC for CH3OH. The fruits gave 12.6 g of
CH2Cl2 extract and 28.7 g of CH3OH extract, the leaves gave 7.8 g of
CH2Cl2 extract and 14.6 g of CH3OH extract and the stem bark gave
22.0 g of CH2Cl2 extract and 37.6 g of CH3OH extract. Ent-isopimara-
8,15-dien-7-one (1), ent-19-hydroxyisopimara-8(9),15-dien-7-one (2),
ent-isopimara-7,15-dien-19-ol (3), ent-isopimara-7(8),15-dien-16,19-
diol (4), ent-isopimara-7(8),15-dien-19-yl octadecanoate (5), methyl
palmitate, octyl ferulate and lupenone were isolated from the fruits of
this plant. Ent-19-hydroxyisopimara-8(9),15-dien-7-one (2), ent-iso-
pimara-7(8),15-dien-19-yl octadecanoate (5), ent-isopimara-7,15-dien-
19-ol (3), lupeol and β-caryophyllene oxide, were isolated from the
leaves of this plant and ent-isopimara-7,15-dien-19-oic acid (Atta-Ur--
Rahman et al., 2005), ent-isopimara-7,15-diene (Radulgin et al., 1977),
ent-isopimara-8(14),15-diene (Mafu et al., 2016), ent-isopimara-8(14),
15-dien-7β-ol (Ravu et al., 2015), crotocorylifuran, megalocarpodolide
D (Guetchueng et al., 2018, 2019; Ndunda et al., 2016), acetyl aleur-
otolic acid, lupeol, lupenone and octyl ferulate, were isolated from the
stem bark of this plant. A detailed protocol of isolation and purifications
of compounds is given as supplementary material.
3.4. Physical data
3.4.1. Ent-19-hydroxyisopimara-8(9),15-dien-7-one (2)
Clear oil. [
α
] -8◦ (c 0.004 CH2Cl2). The IR spectrum gave absorption
bands at 3430 cmꢀ 1 (O H), 3023 cm-1 (=C–H stretch), 2916 cmꢀ 1 and
–
2848 cmꢀ 1 (C–H stretch), 1645 cmꢀ 1
(
α
-β unsaturated ketone stretch),
1462 and 1382 cmꢀ 1 (C–H stretch) and 918 cm-1 (=C–H stretch). 1H
NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3): Table 1. HR-
ESIMS: m/z 303.2321 ([M+H]+, C20H31O2, calc. 303.2324).
3.4.2. Ent-isopimara-7(8),15-dien-16,19-diol (4)
Yellow oil. [α
] -16◦ (c 0.005 CH2Cl2). The IR spectrum for compound
–
gave absorption bands at 3434 cm-1 (O H), 3023 cm-1 (=C–H), 2916
–
–
and 2848 cm-1 (C–H), 1639 cm-1 (C C), 1461 and 1382 cm-1 (C–H),
1215 cm 1 (C O), 758 and 669 cm-1 (=C–H). 1H NMR (500 MHz,
ꢀ
–
CDCl3) and 13C NMR (125 MHz, CDCl3): Table 1. HR-ESIMS: m/z
303.2325 ([M+H]+, C20H29O2, calc. 303.2324).
3.3.2. Extraction and isolation of compounds from C. megalocarpus
Leaves (913.0 g) and roots (1027.0 g) of C. megalocarpus were
extracted on a shaker at room temperature separately. The leaves were
extracted using n-hexane (1.5 L) for 24 h and then filtered. This process
was successively repeated with CH2Cl2 (1.5 L) for a further 24 h and
then filtered. The extracts were evaporated to dryness using a rotary
3.4.3. Ent-isopimara-7(8),15-dien-19-yl octadecanoate (5)
Yellow oil. [
α
] -13◦ (c 0.004 CH2Cl2). The IR spectrum gave ab-
sorption bands at 3435 cmꢀ 1 (O H stretch), 2924 and 2854 cmꢀ 1 (C–H
–
stretch), 1639 cmꢀ 1 (C C stretch), 1458 and 1385 cmꢀ 1 (C–H stretch).
–
–
1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3): Table 1.
153

M.K. Langat et al.
Phytochemistry Letters 40 (2020) 148–155
HR-ESI-MS: m/z 577.5195 ([M + Na]+, C38H66ONa+, calc. 577.4961).
Acknowledgements
3.4.4. 3β,4β:15,16-diepoxy-8
α
-hydroxy-ent-cleroda-13(16),14-dien-
A. F. Kamdem Waffo wishes to thank the University of KwaZulu-
Natal, South Africa and the University of Surrey, UK, the National
Research Foundation (NRF) and the Royal Society for financial aid. A. F.
Kamdem Waffo gratefully acknowledges a postdoctoral fellowship from
the Royal Society. BN wishes to thank the University of Surrey, Guild-
ford, UK, for providing laboratory space from where this work was done
and also for NMR, IR and MS analysis. BN also thanks the University of
Nairobi, International Foundation of Sciences (IFS), International Sci-
ence Program (ISP-Sweden), Organization for Prevention of Chemical
Weapons (OPCW), Natural Products Research Network for East and
Central Africa (NAPRECA) for funds to support her PhD studies and
supporting the research work that produced these results. The authors
thank Mr. Victor Nana and Mr P.C. Mutiso for collection of the Camer-
oonian and Kenyan plant material respectively.
1217-olide (7)
Yellow oil. [α] = + 9.8 (c = 0.0043, CHCl3). The IR spectrum gave
–
absorption bands at 3413 (O H stretch), 3000 (=C–H stretch), 2923
and 2857 cmꢀ 1 (C–H aliphatic stretch) and 1730 cm-1 (C O stretch). H
1
–
–
NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3): Table 1. HR-
ESI-MS: m/z 369.0945 (C20H26O5Na), calc. 369.1678.
3.4.5. Ent-7,13-abietadien-2-one (9)
Yellow gum. [α] = - 30 (c =0.06 g/mL, CHCl3). The IR spectrum
gave absorption bands at 2959, 2923 and 2871 cmꢀ 1 (C–H aliphatic
stretch) and 1711 cm-1 (C O stretch). ECD curve: 293 nm (- 1), 238 nm
–
–
(+ 26) 199 nm (+ 3). 1H NMR (500 MHz, CDCl3) and 13C NMR
(125 MHz, CDCl3): Table 1. HR-ESI-MS: m/z 309.2188 (C20H30O Na)
calculated 286.22967.
Appendix A. Supplementary data
3.5. Computational methods
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.phytol.2020.09.020.
3.5.1. ECD calculations
The conformational search was carried out for (1) and (1a) at the
molecular mechanics level of theory employing the MMFF94x force field
incorporated in the Molecular Operating Environment (MOE) v2019.01
CCG® software package. Conformers under 3 kcal/mol were further
optimized using Gaussian09 software (Frisch et al., 2016), first applying
the B3LYP functional at 6ꢀ 31G+(d,p) level of calculation and then
improving the basis set to 6ꢀ 311G+(d,p) using a polarized continuous
model (iefpcm, solvent = acetonitrile). Boltzmann distribution was
applied to the set of low-energy minima obtained after the optimization,
and the selected conformers were subjected to TDDFT calculations using
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Declaration of Competing Interest
The authors declare that they have no conflict of interest.
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