Isolation and structure elucidation of cyclopeptide alkaloids from the leaves of Heisteria parvifolia

Article abstract of DOI:10.1016/j.phytochem.2019.112081

Heisteria parvifolia Sm. is prescribed in traditional medecine against numerous diseases in C?te d'Ivoire. Due to the shortcoming in scientifical knowledge of use of this species, our investigations revealed five undescribed cyclopeptide alkaloids added to one known derivative namely anorldianine. These compounds were elucidated by 1D and 2D-NMR experiments and comparison with literature data, and confirmed by HR-ESI-MS. Cytotoxic activity evaluation of these compounds against the chronic myeloid leukemia (K565) cell line exhibited an antiproliferative activity with cell growth inhibition from 13% to 46%.

Full text of DOI:10.1016/j.phytochem.2019.112081

Phytochemistry167(2019)112081
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Isolation and structure elucidation of cyclopeptide alkaloids from the leaves
of Heisteria parvifolia
Michel Boni Bitchia,b, Abdulmagid Alabdul Magidb, Faustin Aka Kabrana,
Philomène Akoua Yao-Kouassia, Dominique Harakatc, Hamid Morjanid, Félix Zanahi Tonziboa,*
,
Laurence Voutquenne-Nazabadiokob
a Laboratoire de Chimie Organique Biologique, UFR Sciences des Structures de La Matière et Technologie, Université Félix Houphouët-Boigny, 22 BP 582 Abidjan 22, Cote
d’Ivoire
b ICMR-UMR CNRS 7312, Equipe Chimie des Substances Naturelles, Campus Sciences, Bât. 18, BP 1039, 51687, Reims, France
c Service Commun D’Analyses, Institut de Chimie Moléculaire de Reims (ICMR), CNRS UMR 7312, Bat. 18 B.P. 1039, 51687, Reims Cedex 2, France
d BioSpecT EA7506, URCA, Faculté de Pharmacie, SFR CAP Santé, 1, Rue Du Maréchal-Juin, 51096, Reims, France
A R T I C L E I N F O
A B S T R A C T
Keywords:
Heisteria parvifolia
Olacaceae
Cyclopeptide alkaloids
Cytotoxic activity
Heisteria parvifolia Sm. is prescribed in traditional medecine against numerous diseases in Côte d'Ivoire. Due to
the shortcoming in scientifical knowledge of use of this species, our investigations revealed five undescribed
cyclopeptide alkaloids added to one known derivative namely anorldianine. These compounds were elucidated
by 1D and 2D-NMR experiments and comparison with literature data, and confirmed by HR-ESI-MS. Cytotoxic
activity evaluation of these compounds against the chronic myeloid leukemia (K565) cell line exhibited an
antiproliferative activity with cell growth inhibition from 13% to 46%.
Chronic myeloid leukemia (K565) cell line
1. Introduction
the Central African Republic and southward DR Congo and northern
Angola; possibly also in Uganda and southern Soudan (Louppe et al.,
Cyclopeptide alkaloids are widespread and occur in several families:
Asteraceae, Celastraceae, Euphorbiaceae, Fabaceae, Menispermaceae,
Olacaceae, Pandaceae, Rhamnaceae, Rubiaceae, Sterculiaceae, and
Urticaceae (El-Seedi et al., 2007; Gournelis et al., 1997; Morel et al.,
2009; Tan and Zhou, 2006). Previous studies have reported cyclopep-
tides alkaloids from Heisteria nitida (El-Seedi et al. 1999, 2005). The
cyclopeptide alkaloids sensu stricto were classified according to the
number of amino acid constituents outside and the size of the macro-
cycle (inside) as 4(13); 5(13); 4(14) and 4(15) type of alkaloids (Joullie
and Richard, 2004; Tan and Zhou, 2006). Several activities of cyclo-
peptides alkaloids have been reported, such as antimicrobial (Gournelis
et al., 1997; Morel et al., 2005), insecticidal (Sugawara et al., 1996),
sedative (Suh et al., 1997), and antiplasmodiale activity (Suksamrarn
et al., 2005).
The genus Heisteria belonging to the Olacaceae family comprises
about 65 species in tropical America and 3 in Africa; namely Heisteria
parvifolia Sm., Heisteria trillesiana Pierre ex Heckel, and Heisteria zim-
mereri Engl. Heisteria parvifolia Sm. is an evergreen shrub or small tree
up to 15 (−20) m tall; 40 (−60) cm in diameter (Malaisse et al., 2004).
H. parvifolia occurs from Senegal and south-western Mali eastward to
2008). In Côte d'Ivoire, is locally abundant on sandy soils. Its wood is
used for construction and tool handles. In several areas, the fruits are
eaten fresh; the small oil-rich seeds are eaten fresh, roasted or cooked.
The twigs are used as chew-sticks. Various Heisteria species are used by
South-American Indians or in Africa in the treatment of rheumatism,
abscesses, headache, throat infections, swellings, nose bleedings, pain
in joints and muscles, diarrhea, hepatic infection (Kvist and Holm-
Nielsen, 1987; Russo, 1992; Tan and Zhou, 2006). In traditional med-
icine in Ghana, ground roots of H. parvifolia are applied as enema
against stomach-ache. In Congo, sap from the root bark is used as
dropps into the nose against migraine and into the eye to treat painful,
infected eyes. Stem bark is taken in Ghana, in Côte d'Ivoire and DR
Congo as cough medicine. In Gabon, bark is applied to circumcision
wounds. In Ghana and Côte d'Ivoire, leaf decoctions are taken or ap-
plied as a bath to invigorate rachitic children and to treat convulsions.
They are also used as analgesic and rubbed onto painful breasts of
young mothers, and in Sierra Leone to treat tooth-ache. In Congo, leaf
decoctions are administrated against asthma, costal pain, stomach pain,
and menstrual problems. Ground seeds are used to stupefy fish. In DR
Congo, powdered bark is an ingredient in the preparation of arrow
* Corresponding author. Tel.: +225 05 07 19 67.
E-mail addresses: abdulmagid.alabdul-magid@univ-reims.fr (A.A. Magid), tonzibz@yahoo.fr (F.Z. Tonzibo).
https://doi.org/10.1016/j.phytochem.2019.112081
Received 18 March 2019; Received in revised form 12 July 2019; Accepted 2 August 2019
0031-9422/©2019ElsevierLtd.Allrightsreserved.

M.B. Bitchi, et al.
Phytochemistry167(2019)112081
poison (Abbiw, 1990; Burkill and families, 1997; Malaisse et al., 2004).
Chemical investigations of Heisteria species have mainly revealed the
presence of triterpenes and proanthocyanidines in H. pallida (Dirsch
et al. 1992, 1993), cyclopeptide alkaloid in H. nitida (El-Seedi et al.
1999, 2005), scopolamine in H. olivae (Cairo-Valera et al., 1977), and
acetylenic fatty acids in H. accuminata (Kraus et al., 1998). Up to date,
only the composition of the seeds oil of H. parvifolia has been reported
as mainly long-chain saturated fatty acids, oleic acid and other mono
and di enoic fatty acids (Malaisse et al., 2004).
As a part of a continuing study for the discovery of medicinal Côte
d'Ivoire species, five undescribed cyclopeptide alkaloids (1–5), together
with one known compound (6), have been isolated and characterized
from the leaves of H. parvifolia. Their cytotoxicity against the chronic
myeloid leukemia K562 cells was evaluated.
Compound 1 was obtained as a white powder. Analysis of 1 by high-
resolution electrospray ionization mass spectrometry (HR-ESI-MS)
identified a pseudo-molecular ion [M + H]+ at m/z 457.2807, corre-
sponding to the molecular formula C25H36N4O4 (calcd for C25H37N4O4,
457.2815), in combination with analysis of NMR data. The 13C NMR
(Table 1) and HSQC spectra of 1 showed 25 carbon resonances for four
methyls (δC 13.9, 16.7, 17.5, and 19.5), one N-methyl (δC 31.8), three
methylenes (δC 23.3, 28.2, and 46.7), twelve methines (two of which
were olefinic carbons at δC 116.7 and 124.9 and four were aromatic
carbons sp2 at δC 120.9, 121.9, 131.0, and 131.4), two quaternary
aromatic carbons (δC 157.2 and 131.6), and three carbonyl carbons (δC
171.1, 167.5, and 165.2). The 1NMR spectrum (Table 1) displayed
signals for two olefinic protons at δH 6.53 and 6.65, a singlet N-methyl
(δH 2.68), four methyl doublets, four aromatic protons, and several
methine and methylene protons. The NMR data of compound 1
(Table 1) showed great similarity with the NMR data previously re-
ported for anorldianine (compound 6) possessing a 14-membered ring
type comprising a p-oxigenated Z-styrylamine group (Dongo et al.,
1989; El-Seedi et al. 1999, 2005). The presence of the p-oxigenated Z-
styrylamine group was indicated by two doublets at δH 6.53 and 6.65
(each 1H, J = 7.7 Hz; H-1 and H-2, respectively) corresponding to the
Z-double bond and four aromatic protons appearing as doublets of
doublets with J-values typical for an o,m-coupling pattern (H-12, 13,
15, and 16). The protons H-13 and H-15 (δH 7.11, dd, J = 8.7, 2.4 Hz)
showed correlation with C-1 (δC 116.7) and H-12 and H-16 (δH 7.27,
dd, J = 8.7, 2.4 Hz) with C-14 (δC 131.6) in HMBC spectrum. In cy-
clopeptide alkaloids, the H-9 (β-H of the β-hydroxy-amino acid moiety)
chemical shift value (between 5.00 and 5.50 ppm) is characteristic. In
this case, a doublets of doublets was present at δH 4.92 (J = 8.3,
1.5 Hz). In the COSY spectrum, two cross peaks were observed for H-9
(δH 4.92), more specifically with H-8 (δH 5.01, d, J = 8.3 Hz) and H-20
(δH 2.13, sept, J = 6.9 Hz). The proton signal of the CH-group in po-
sition 20 also showed cross peaks to H3-21 (δH 1.32, d, J = 6.7 Hz) and
H3-22 (δH 1.05, d, J = 6.7 Hz). In the HMBC spectrum, correlations
between C-9 (δC 83.4) and H-21 and H-22 were observed (Fig. 2). These
data agreed with previously reported data for β-hydroxyleucine (an-
orldianine). The methine and the methylene protons of proline were
observed in the 1H NMR spectrum: H-5 (δH 4.16, dd, J = 7.5, 1.9 Hz),
H2-17 (δH 1.65, m; 2.21, dd, J = 11.5–4.5 Hz), H2-18 (δH 1.75, m; 1.95,
m), and H2-19 (δH 3.55, brt, J = 9.8 Hz; 3.85, m). In the COSY spec-
trum, cross peaks were observed between H-5/H-17, H-17/H-18, and H-
18/H-19. In the HMBC spectrum, the H-5 exhibited correlations with C-
19 (δC 46.7) and with the carbonyl C-4 (δC 167.5) (Fig. 2). The methyl
doublets of N-methyl-valine (H-2′ and H-3′) appeared at δH 0.95
(J = 6.7 Hz) and 0.96 (J = 6.7 Hz) and the methine proton (H-1′) of
this moiety appeared as septuplet at δH 2.13 (J = 6.9 Hz). The HMBC
experiment showed correlations between C-24 (δ 165.2) and H-25 (δH
3.57, d, J = 5.4 Hz) and H-1′, and between the N-methyl carbon (δC
2. Results and discussion
The crude alkaloid extract prepared with an acid-base method of
air-dried and pulverized leaves of H. parvifolia was subjected to silica
gel flash chromatography, eluted with increasingly polar mixtures of
CHCl3/MeOH. Further purification was performed using semi-pre-
parative HPLC. As a result, five undescribed cyclopeptide alkaloids
(1–5) were isolated and chemically characterized, together with one
known cyclopeptide alkaloids, anorldianine (6) (El-Seedi et al., 1999).
Their structures (Fig. 1) were elucidated by 1D and 2D-NMR experi-
ments and comparison with literature data, and confirmed by HR-ESI-
MS.
The UV spectra of compounds 1–5 showed absorptions at 222–224
and 274–282 nm, wavelengths commonly assigned to peptide bonds
and aromatic residues (Dongo et al., 1989; Kang et al., 2015; Schwing
et al., 2011), while their IR spectra displayed bands at 3395 and
1682 cm−1, which are typical of amide groups (Dongo et al., 1989;
Schwing et al., 2011).
31.8) and the H-25 for this moiety. The combined use of 1D (1H and 13
C
NMR) and 2D (COSY, HSQC, and HMBC) spectra allowed an un-
ambiguous assignment of all protons and carbons of the amino acids
units (β-hydroxyleucine, proline, N-methyl-valine residues) and the p-
oxygenated Z-styrylamine group (Table 1). Moreover, the connectivity
between the constitutive parts of the molecule were ascertained by the
HMBC correlations between the carbonyl C-4/H-2, the carbonyl C-7/H-
5, the carbonyl C-24/H-8, and C-11/H-9 (Fig. 2). Moreover, the NOE
relationships H-8/H-25, H-8/H-12 and H-9/H-16 agreed with the β-
hydroxyleucine connection with the N-methyl-valine and the p-oxyge-
nated Z-styrylamine moieties whereas the NOE effects between H-8/H-
19 agreed with the connection of β-hydroxyleucine with proline
(Fig. 2). Compound 1 was named cycloheisterin A after its plant origin
(Fig. 1).
Compound 2 displayed an [M + H]+ ion peak at m/z 471.2979 in
the positive HR-ESI-MS, corresponding to the molecular formula
C
26H38N4O4, suggesting an additional methyl group compared to 1. The
Fig. 1. Chemical structures of compounds 1–6.
NMR spectroscopic data of 2 were almost identical with those of 1
2

M.B. Bitchi, et al.
Phytochemistry167(2019)112081
Table 1
13C NMR spectroscopic data for compounds 1–5 (500 MHz, CD3OD).
1
2
3
4
5
δH m (J in Hz)
δC
δH m (J in Hz)
δC
δH m (J in Hz)
δC
δH m (J in Hz)
δC
δH m (J in Hz)
δC
1
2
4
5
7
8
9
11
12
13
14
15
16
17
6.53, d (7.7)
6.65, d (7.7)
–
4.16, dd (7.5,1.9)
–
5.01, d (8.3)
4.92, dd (8.3, 1.5)
–
7.27, dd (8.7, 2.4)
7.11, dd (8.7, 2.4)
–
116.7
124.9
167.5
62.5
171.1
53.1
6.53, d (7.7)
6.65, d (7.7)
–
4.14, d (7.5)
–
5.01, d (8.5)
4.93, dd (8.5, 1.9)
–
7.27, d (8.5)
7.11, m
116.8
125.0
167.5
62.6
171.0
52.0
6.53, d (7.7)
6.66, d (7.7)
–
4.15, d (7.8)
–
5.01, d (8.3)
4.91, dd (8.3, 1.2)
–
7.27, d (8.1)
7.11, m
116.3
124.9
167.5
62.5
171.1
53.1
6.53, d (7.7)
6.65, d (7.7)
–
4.12, d (8.1)
–
5.01, d (8.5)
4.92, dd (8.3, 1.3)
–
7.27, d (8.9)
7.11, m
116.8
124.9
167.4
62.6
171.0
53.0
6.50, d (7.7)
6.60, d (7.7)
–
3.72, d (8.0)
–
4.82, (overlapped)
4.82, (overlapped)
–
7.20, m
7.11, dd (8.5, 1.5)
–
7.12, dd (8.5, 1.5)
7.20, m
1.54, m
116.8
125.0
167.9
62.4
170.7
53.0
83.4
83.2
83.4
83.2
82.7
157.2
120.9
131.4
131.6
131.0
120.9
28.2
157.2
120.8
131.4
131.8
130.3
120.8
28.2
157.2
120.9
131.4
131.6
130.5
120.9
28.1
157.2
120.8
131.4
131.6
130.3
120.8
28.2
157.1
119.6
131.3
131.3
130.2
120.3
28.6
–
–
–
7.11, dd (8.7, 2.4)
7.27, dd (8.7, 2.4)
1.65, m
7.11, m
7.27, d (8.5)
1.67, m
7.11, m
7.27, d (8.1)
1.65, m
7.11, m
7.27, d (8.9)
1.64, m
2.21, dd (11.5, 4.5)
1.75, m
1.95, m
3.55, brt (9.8)
3.85, m
2.13, m
1.32, d (6.7)
1.05, d (6.7)
–
3.57, d (5.4)
N–CH3
2.21, dd (12.3, 4.9)
1.75, m
1.97, m
3.55, brt (10.8)
3.90, ddd (10.8, 7.1, 3.2)
2.12, dq (6.8, 1.7)
1.32, d (6.8)
1.07, d (6.8)
–
2.21, dd (12.1, 5.8)
1.75, m
1.95, m
3.55, brt (9.8)
3.85, m
2.13, m
1.32, d (6.8)
1.06, d (6.8)
–
3.62, d (5.0)
N–CH3
2.22, dd (12.5, 6.1)
1.75, m
1.97, m
3.58, brt (9.3)
3.85, ddd (10.1, 9.3, 7.4)
2.11, m
1.32, d (6.8)
1.07, d (6.8)
–
2.01, dd (11.3, 4.8)
1.67, m
1.82, m
3.42, brt (9.2)
3.71, m
2.10, m
1.27, d (6.8)
1.02, d (6.8)
–
4.10, dd (10.4, 4.5)
N–CH3
18
19
23.3
46.7
23.3
46.7
23.3
46.7
23.3
46.8
23.5
47.0
20
21
22
24
25
R1
28.7
19.5
13.9
165.2
66.7
28.7
19.4
14.0
164.3
72.7
28.7
19.5
13.9
165.3
66.2
28.7
19.4
14.0
164.4
72.2
28.6
19.3
14.0
165.0
68.1
3.59, d (5.3)
N–CH3
3.62, d (4.8)
N–CH3
2.68, s
31.8
2.92, s
N–CH3
2.92, s
Val
40.3
41.9
27.2
15.1
18.7
2.66, s
31.8
2.91, s
N–CH3
2.91, s
iLeu
39.7
42.2
33.9
26.3
2.97, s
N–CH3
2.97, s
Phe
3.12, dd (13.8, 10.6)
3.40, m
41.1
41.1
34.3
134.1
R2
R3
1′
Val
2.13, m
iLeu
1.88, m
30.2
16.7
17.5
2.45, m
36.8
25.1
2.16, m
2′
0.95, d (6.7)
0.96, d (6.7)
0.93, d (6.7)
0.97, d (6.7)
1.02, m
1.47, m
0.92, t (6.8)
0.96, d (6.8)
0.77, m
1.42, m
0.93, t (6.9)
0.98, d (6.9)
3′
4′
5′
6′
7′
10.3
13.1
10.4
11.5
7.20, m
7.30, m
7.21, m
7.30, m
7.20, m
129.0
128.5
127.5
128.5
129.0
except for one additional methyl group (Table 1). The detailed analysis
of the 2D-NMR spectra led to the identification, as in 1, of the amino
acids units (β-hydroxyleucine, proline, and valine residues) and the p-
oxygenated Z-styrylamine group (Table 1). The HMBC cross-peaks of
the methyl signals at δH 2.92 (6H, s) to C-25 (δC 72.7) of the valine
residue indicated that the terminal amino acid in 2 is N,N-dimethyl-
valine. Compound 2 was named cycloheisterin B.
isoleucine residue suggested that the terminal amino acid in 4 is N,N-
dimethyl-isoleucine. Compound 4 was named cycloheisterin D (Fig. 1).
Cycloheisterin E (5) exhibited an [M + Na]+ ion peak at m/z
541.2799 in the HR-ESI-MS spectrum, consistent with the molecular
formula of C30H38N4O4. Comparing the NMR data of 5 with those of
1–4 (Table 1) and detailed analysis of the 2D-NMR spectra showed that
it had the same macrocycle (inside) composed of the amino acids units
(β-hydroxyleucine and proline) and the p-oxygenated Z-styrylamine
group (Table 1). The 1H and 13C-NMR spectra of 5 exhibited signals
corresponding to an aromatic amino acid [ δH 7.20–7.30, 5H]. Ex-
tensive 2D-NMR analysis enabled the full assignments of the N,N-di-
methyl phenylalanine. (Tuenter et al., 2017). The presence of the N,N-
dimethyl groups was confirmed by the HMBC correlation between the
methyl signals at δH 2.97 (6H, s) and C-25 (δC 68.1) of the phenylala-
nine residue. The HMBC correlation between H-8 (δH 4.82) of the β-
hydroxyleucine and the C-24 (δC 165.0) of the N,N-dimethyl phenyla-
lanine confirmed it to be the terminal amino acid moiety. Compound 5
was named cycloheisterin E (Fig. 1).
The stereochemistry of the cyclopeptide alkaloids 1–6, was pro-
posed from the 1H NMR coupling constants, 13C NMR data, and NOESY
analysis and by determining the absolute configuration of the amino
acids by chiral HPLC after acid hydrolysis. With this purpose, com-
pounds 1–6 were hydrolyzed and their amino acids analyzed through
the chiral HPLC. In cycloheisterin A-E and 6, proline has the L config-
uration and N-methyl-valine, N,N-dimethyl-valine, N-methyl-iso-
leucine, N,N-dimethyl-isoleucine, and N,N-dimethyl phenylalanine in
cycloheisterin A-E, respectively and N,N-dimethyl-leucine in 6 were in
Compound 3 displayed an [M + Na]+ ion peak at m/z 493.2785 in
the positive HR-ESI-MS, corresponding to the molecular formula
C
26H38N4O4, suggesting an additional methylene group compared to 1.
The 1H and 13C NMR values of 3 were almost superimposable on those
of 1 (Table 1) excepting those of the N-methyl-valine residue in 1. In-
stead, an N-methyl-isoleucine residue was identified as summarized in
Table 1 (Tuenter et al., 2017). 1H–1H COSY analysis confirmed the
presence of an isoleucine residue. The HMBC cross-peak of H-8 of hy-
droxyleucine (δH-8 5.01) to C-24 of isoleucine residue (δC-24 165.3) and
H-25 (δH-25 3.62) to C-24 and the N-methyl carbon (δC 31.8) to H-25 for
this moiety confirmed that the terminal amino acid is N-methyl-iso-
leucine. Compound 3 was named cycloheisterin C (Fig. 1).
Cycloheisterin D (4) displayed an [M + H]+ ion peak at m/z
485.3138 in the positive HR-ESI-MS, corresponding to the molecular
formula C27H40N4O4, suggesting an additional methyl group compared
to 3. Comparing the NMR data of 4 with those of 3 (Table 1) and the
analysis of the 2D-NMR spectra led to the identification, as in 3, of the
amino acids units (β-hydroxyleucine, proline and isoleucine residues)
and the p-oxygenated Z-styrylamine group (Table 1). The HMBC cross-
peaks of the methyl signals at δH 2.91 (6H, s) to C-25 (δC 72.2) of the
3

M.B. Bitchi, et al.
Phytochemistry167(2019)112081
Fig. 3. Representatives and approximates NMR data for threo and erythro β-
hydroxyleucine in cyclopeptide alkaloids.
Furthermore, the J value of the 1H NMR signal attributed to the methyl
group at position C-22 was 6.7 Hz, indicative for a pseudoaxial/equa-
torial coupling, typical for L-erythro-β-hydroxyleucine (Abu-Zarga et al.,
1995; Gournelis et al., 1997; Tuenter et al., 2016). In addition, the
cross-peak observed in the NOESY spectra of 1–5 between H-9 and H-
20, H-9/H-21 and H-8/H-22 and the lack of the NOESY interaction
between H-9 and H-8, suggests the L-erythro configuration for the β-
hydroxyleucine moiety (Fig. 2). Furthermore, the NOESY effect ob-
served between H-25 and H-1′ indicated that these protons are co-fa-
cially oriented.
Fig. 2. Selected key HMBC and NOESY interactions for compound 1.
the L form. The 13C-NMR chemical shift values of the α-amino acid of
the macrocycle (proline in all five alkaloids) and the terminal units (N-
methyl-valine in 1, N,N-dimethyl-valine in 2, N-methyl-isoleucine in 3,
N,N-dimethyl-isoleucine in 4, N,N-dimethyl-phenylalanine in 5, and
N,N-dimethyl-leucine in 6) match well with those previously reported
for similar compounds and was in agreement with the fact that the
majority of plant cyclopeptides are composed of L-amino acids(El-Seedi
et al., 2005; Kang et al., 2015; Maldaner et al., 2011; Medina et al.,
2016; Suksamrarn et al., 2005; Tuenter et al., 2017).
The configuration of the β-hydroxyleucine was established based on
the available NMR data. In the case of the erythro form, Jα,β ca. 8.0 Hz,
whereas for threo compounds Jα,β ca. 2.0 Hz (Fig. 3) (Dias et al., 2007;
Gournelis et al., 1997; Mostardeiro et al., 2013; Tuenter et al., 2016).
The coupling constant of the doublet corresponding to H-9 (Jα,β) of
compounds 1–5 ca. 8.3 Hz, clearly indicative of an erythro configura-
tion. 13C NMR spectroscopy is used for the elucidation of the absolute
configuration of the β-hydroxy amino acids. For both L-threo and D-threo
series, the signal of the α carbon appears at ca. δC 55.0, wheras for the β
carbon, its signal appears at ca. δC 82.0 for the D-threo and ca. δC 86.0
for the L-threo (Fig. 3) (Mostardeiro et al., 2013). For the L-erythro series,
the signal of the α carbon (C-8) appears at ca. δC 55.0, wheras for the D-
erythro it appears at ca. δC 53.0. Important information is also observed
for the β carbon (C-9): in the L-erythro series, the signal appears at ca. δC
81.5, whereas for the D-erythro configuration it appears at ca. δC 87.0
(Abu-Zarga et al., 1995; Caro et al., 2012; Dongo et al., 1989; Gournelis
et al., 1997; Medina et al., 2016; Mostardeiro et al., 2013; Tuenter
et al., 2016). These data show that the chemical shift of the β carbon is
most indicative for the L and D forms of a β-hydroxy amino acids (Δδ
4–5 ppm) than α carbon (Δδ 0–3 ppm). The chemical shift of C-9 in
compounds 1–5 was around δC-9 83.3, clearly suggestive for the L-ery-
thro form, whereas the chemical shift of C-8 was around δC 53.0.
3. Conclusion
In summary, six compounds were isolated from the crude alkaloid
extract of H. parvifolia leaves, among them five previously undescribed
cyclopeptide alkaloids from the 4(14) type, 4 amino acid constituents
outside and the 14-atoms of the macrocycle (inside). Their structures
were established by different spectroscopic methods including 1D and
2D-NMR experiments as well as HR-ESI-MS analysis. Compound 6
(anorldianine) that has a unique substructure containing proline, was
previously isolated from Heisteria nitida (El-Seedi et al., 1999). Com-
pounds 1–5 were derivatives of anorldianine and differed in only the
terminal amino acid which was N-methyl-valine in 1, N,N-dimethyl-
valine in 2, N-methyl-isoleucine in 3, N,N-dimethyl-isoleucine in 4, and
N,N-dimethyl-phenylalanine in 5. Cyclopeptide alkaloids have only
been reported from a few families of the plant kingdom, in fact, they
seem to be quite rare and present in small quantities. This kind of cy-
clopeptide alkaloids was isolated only in Canthium anorldianum (Ru-
biaceae) and Heisteria nitida (Olacaceae). Further phytochemical in-
vestigation on Heisteria species are needed to verify wether
anorldianine derivative cyclopeptide alkaloids could be considered as a
taxonomic markers for the genus Heisteria. The cytotoxic activity of
compounds 1–6 against the chronic myeloid leukemia (K562) cell line
was evaluated. Only compounds 2,
4 and 6 exhibited an anti-
proliferative activity at the concentration 100 μM with cell growth in-
hibition of 46%, 44%, and 43%, respectively, whereas compounds 1, 3,
and 5 showed cell growth inhibition of 13%, 19%, and 36%, respec-
tively at the same concentration.
4

M.B. Bitchi, et al.
Phytochemistry167(2019)112081
4. Experimental
4.3.3. Cycloheisterin C (3)
White amorphous powder; [α]2D0 = −135 (c 0.31; MeOH); UV
(MeOH) λmax (abs.) 224 (1.38), 276 (0.37); IR vmax 3388, 2965, 1686,
1506, 1206, 1133, 985, 719; 1H and 13C NMR, see Table 1; HR-ESI-MS
4.1. General experimental procedures
Optical rotations were measured on a PerkinElmer model 341 po-
larimeter (589 nm, 20 °C). IR spectra were obtained on a Nicolet Avatar
320 FT-IR spectrometer with KBr disks. NMR spectra were acquired in
CD3OD on Bruker Avance DRX III 500 instruments (1H at 500 MHz and
13C at 125 MHz). Standard pulse sequences and parameters were used
to obtain 1D- (1H and 13C) and 2D- (COSY, ROESY, HSQC and HMBC)
(positive ion mode) m/z 493.2785 [M
26H38N4O4Na, 493.2791).
+
Na]+ (calcd for
C
4.3.4. Cycloheisterin D (4)
White amorphous powder; [α]2D0 = −179 (c 0.23; MeOH); UV
(MeOH) λmax (abs.) 222 (3.21), 280 (0.3); IR vmax 3395, 2972, 1682,
1508, 1205, 1133, 720; 1H and 13C NMR, see Table 1; HR-ESI-MS
(positive ion mode) m/z 485.3138 [M + H]+ (calcd for C27H41N4O4,
485.3128).
NMR spectra. HR-ESI-MS experiments were performed using
a
Micromass Q-TOF high-resolution mass spectrometer (Manchester, UK).
Mass spectra were recorded in the positive-ion mode in the range m/z
100–2000, with a mass resolution of 20000 and an acceleration voltage
of 0.7 kV. Flash chromatography was conducted on a Grace Reveleris
system equipped with dual UV and ELSD detection using Grace® car-
tridges (Silica gel or RP-18). A prepacked RP-C18 column (Phenomenex
250 × 15 mm, Luna 5 μ) was used for semi-preparative HPLC. The
eluting mobile phase consisted of H2O with TFA (0.0025%) and CH3CN
with a flow rate of 5 mL/min and the chromatogram was monitored at
210, 250, 270, and 300 nm. TLC was performed on precoated silica gel
60 F254 Merck and compounds were visualized by spraying the dried
plates with Dragendorff's reagent.
4.3.5. Cycloheisterin E (5)
White amorphous powder; [α]2D0 = −91 (c 0.41; MeOH); UV
(MeOH) λmax (abs.) 222 (0.91), 274 (0.5); IR vmax 3439, 2969, 1681,
1508, 1204, 1136, 700; 1H and 13C NMR, see Table 1; HR-ESI-MS
(positive ion mode) m/z 541.2799 [M
30H38N4O4Na, 541.2791).
+
Na]+ (calcd for
C
4.4. General procedure for determination of amino acid configurations
The absolute configurations of amino acids were determined by
chiral HPLC after acid hydrolysis according to literature (Mostardeiro
et al., 2013; Silva et al., 1996; Wang et al., 2017). Briefly, each solution
of 1–5 (0.5 mg) in 6 N HCl (0.4 mL) was heated at 110 °C for 24 h and
then concentrated to dryness. The residue was dissolved in H2O
(200 μL) to obtain the test solution, 10 μL of which was injected into
chiral HPLC system with a Chiralpak IC column (250 mm × 4.6 mm
I.D., 5 μm) maintained at 35 °C and detected at 254 nm: Isopropanol/n-
hexane (90:10, v/v) containig 0.1% TFA was used as the mobile phase
at a flow rate of 0.8 mL/min.
4.2. Plant material
The leaves of Heisteria parvifolia Sm. were collected in Agboville
forest in August 2016. They are identified by Pr. Akke Assi in the na-
tional center florestic of Félix Houphouët-Boigny University of Côte
d’Ivoire (Ake assi 11049).
4.3. Extraction and isolation
The dried powdered leaves of H. parvifolia (1 kg) were wetted with
50% aq. NH4OH (500 mL), macerated overnight and then percolated
with 15 L of EtOAc. The organic solvent was concentrated under re-
duced pressure. The crude extract (26 g) was suspended in 2 L of EtOAc
and extracted with an aqueous 2% H2SO4 solution (3 × 2 L). The acid
phase was made alkaline with aqueous NH3 and extracted with 3 × 2 L
of CHCl3. The CHCl3 solution was washed with H2O (2 L), dried
(Na2SO4) and evaporated in vacuo to give 500 mg of crude alkaloid
extract (yield 0.05%). The crude alkaloid extract was subjected to silica
gel flash chromatography eluted with increasingly polar CHCl3/MeOH
(100:00–95:05) for 25 min, to yield 26 fractions (F1-26). Fractions F6,
F8, F10, F12, F14 and F17 were subjected separately to semipreparative
HPLC RP-18 chromatography, by eluting with an isocratic gradient
(28% CH3CN). Compound 4 (tR 13.2 min, 31 mg) was obtained from
fractions F6 and F8, compound 5 (tR 14.9 min, 4 mg) from fraction F10,
compound 6 (tR 10.6 min, 6 mg) from fraction F12, compounds 2 (tR
14.6 min, 6 mg) and 3 (tR 17.3 min, 4 mg) from fraction F14, and
compound 1 (tR 11.3 min, 5 mg) from fraction F17.
5. Cytotoxicity bioassay by MTS
K562 cells (chronic myeloid leukemia) were trypsinized, harvested,
and spread onto 96-well flat-bottom plates at a density of 1000 cells per
well, and then incubated for 24 h in RPMI 1640 Medium supplemented
with 10% fetal bovine serum and antibiotics. After culture, the cells
were treated with compounds 1–6 for 72 h. The cell cultures were then
analyzed
yphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt (MTS) according
to the manufacturer's instructions (Promega Corporation,
Charbonnières, France). Camptothecin was used as positive control.
MTS is bioreduced by cells into colored formazan product.
using
3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethox-
a
Absorbance was analyzed at a wavelength of 540 nm with a Multiskan
Ex microplate absorbance reader (Thermo Scientific, Paris, France).
Percentage of cell growth was calculated as 100% × (absorbance of the
treated cells)/(absorbance of the negative control cells). Control cells
were treated with complete culture medium containing 0.2% DMSO.
The values represent averages of three independent experiments.
4.3.1. Cycloheisterin A (1)
White amorphous powder; [α]2D0 = −148 (c 0.5; MeOH); UV
(MeOH) λmax (abs.) 222 (1.66), 274 (0.33); IR vmax 3395, 2972, 1682,
1508, 1205, 1133, 984, 720; 1H and 13C NMR, see Table 1; HR-ESI-MS
(positive ion mode) m/z 457.2807 [M + H]+ (calcd for C25H37N4O4,
457.2815).
Acknowledgements
The authors are grateful to Conseil Regional Champagne Ardenne,
Conseil General de la Marne, Ministry of Higher Education, Research
and Innovation (France) (MESRI) and EU-programme FEDER to the
PlAneT CPER project for financial support as well as the Ministry of
Research of Côte d’Ivoire.
4.3.2. Cycloheisterin B (2)
White amorphous powder; [α]2D0 = −187 (c 0.52; MeOH); UV
(MeOH) λmax (abs.) 222 (0.10), 282 (0.01); IR vmax 3439, 2969, 1681,
1508, 1204, 1136, 700; 1H and 13C NMR, see Table 1; HR-ESI-MS
(positive ion mode) m/z 471.2979 [M + H]+ (calcd for C26H39N4O4,
471.2971).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.phytochem.2019.112081.
5

M.B. Bitchi, et al.
Phytochemistry167(2019)112081
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