A total of eight novel steroidal glycosides based on spirostan, furostan, pseudofurostan, and cholestane from the leaves of cestrum newellii

Article abstract of DOI:10.3390/molecules25194462

Previously, various steroidal glycosides were reported from plants of Cestrum species. However, phytochemical investigation has not been conducted on Cestrum newellii. A systematic phytochemical investigation of the leaves of C. newellii resulted in the isolation of eight novel steroidal glycosides (1-8), which were classified into three spirostanol glycosides (1-3), two furostanol glycosides (4 and 5), two pseudofurostanol glycosides (6 and 7), and one cholestane glycoside (8). In addition, three known cholestane glycosides (9-11) were isolated and identified. The structures of the new compounds were determined based on spectroscopic data and chemical transformations. Compounds 1 and 2 are spirostanol glycosides having hydroxy groups at C-2, C-3, C-12, and C-24 of the aglycone moiety. Although C. newellii is known to be a poisonous plant, the 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyl-2H-tetrazoliumbromide assay exhibited that none of the isolated compoundswere cytotoxic to HL-60 human promyelocytic leukemia cells.

Full text of DOI:10.3390/molecules25194462

molecules
Article
A Total of Eight Novel Steroidal Glycosides Based on
Spirostan, Furostan, Pseudofurostan, and Cholestane
from the Leaves of Cestrum newellii
Tomoki Iguchi *, Naoki Takahashi and Yoshihiro Mimaki
School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, 1432-1, Horinouchi, Hachioji,
Tokyo 192-0392, Japan; y20612@toyaku.ac.jp (N.T.); mimakiy@toyaku.ac.jp (Y.M.)
* Correspondence: iguchit@toyaku.ac.jp; Tel.: +81-42-676-4575
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 28 August 2020; Accepted: 26 September 2020; Published: 28 September 2020
Abstract: Previously, various steroidal glycosides were reported from plants of Cestrum species.
However, phytochemical investigation has not been conducted on Cestrum newellii. A systematic
phytochemical investigation of the leaves of C. newellii resulted in the isolation of eight novel
steroidal glycosides (
glycosides ( and ), two pseudofurostanol glycosides (
In addition, three known cholestane glycosides ( 11) were isolated and identified. The structures
of the new compounds were determined based on spectroscopic data and chemical transformations.
Compounds and are spirostanol glycosides having hydroxy groups at C-2, C-3, C-12, and C-24 of the
1
–8
), which were classified into three spirostanol glycosides (
1
–3
), two furostanol
4
5
6
and ), and one cholestane glycoside (
7
8).
9–
1
2
aglycone moiety. Although C. newellii is known to be a poisonous plant, the 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyl-2H-tetrazolium bromide assay exhibited that none of the isolated compounds were cytotoxic
to HL-60 human promyelocytic leukemia cells.
Keywords: Cestrum newellii; spirostanol glycoside; furostanol glycoside; pseudofurostanol glycoside;
cholestane glycoside; cytotoxic activity; HL-60 cell
1. Introduction
Plants in the genus Cestrum (Solanaceae) are native to warm subtropical and tropical areas of
America, and are now cultivated all over the world for ornamental purposes [
rich sources of steroidal glycosides, and structurally diverse steroidal glycosides have been isolated
from C. laevigatum [ ], C. schlechtendahlii [ ], C. ruizteranianum [ ], C. parqui [ ], C. diurnum [ 10],
C. sendtenerianum [11 12], and C. nocturnum [13 15]. C. newellii is reputed to be a poisonous plant.
1]. Cestrum species are
2,
3
4
5
6–8
9,
,
–
However, a literature survey suggests that no phytochemical investigation has been done on C. newellii.
Therefore, a systematic phytochemical analysis of the leaves of C. newellii was conducted with a focus
on steroidal glycosides. This paper deals with the structural determination of new compounds (1–8)
on the basis of spectroscopic data and chemical transformations. Furthermore, the cytotoxic activities
of the isolated compounds (1–11) were evaluated.
2. Results and Discussion
The MeOH extract of the leaves of C. newellii was fractionated by column chromatography (CC)
and preparative HPLC to obtain 11 compounds (
(22S,25R)-26-[( -d-glucopyranosyl)oxy]-22-hydroxycholest-5-en-3
-l-rhamnopyranosyl-(1 4)]- -d-glucopyranoside ( ) [16], (22S,25R)-26-[(
22-dihydroxycholest-5-en-3 -yl O- -l-rhamnopyranosyl- (1 2)-O-[ -l-rhamnopyranosyl- (1
-d-glucopyranoside (10) [17], and (25R)-26-[( -d-glucopyranosyl)oxy]-3 -[(O- -l-rhamnopyranosyl-
1-
11) (Figure 1). Compounds
-yl O- -l-rhamnopyranosyl-(1
-d-glucopyranosyl)oxy]-16
4)]-
9
–
11 were identified as
β
β
α
→
2)-O-
[α
→
β
9
β
β,
β
α
→
α
→
β
β
β
α
Molecules 2020, 25, 4462; doi:10.3390/molecules25194462
www.mdpi.com/journal/molecules

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(1
→
2)-O-[
α
-l-rhamnopyranosyl-(1
→
4)]-β-d-glucopyranosyl)oxy]-cholest-5-ene-16,22-dione (11) [18],
respectively.
Figure 1. Structures of 1, 1a, 2, 3, 3a, 4–11.
Compound
1
was obtained as an amorphous powder. Its molecular formula was determined
to be C39H62O16 based on high-resolution electrospray ionization-time of flight-mass spectroscopy
(HRESI-TOF-MS) and 13C-NMR data. The infrared (IR) spectrum of
exhibited absorption bands for
hydroxy groups at 3381 cm−1. The 1H- and 13C-NMR spectra of
displayed signals for two tertiary
1
1
methyl groups at δH 1.04 (s, Me-18) and 0.94 (s, Me-19); δC 20.3 (C-19) and 10.9 (C-18), two secondary
methyl groups at δH 1.42 (d, J = 6.9 Hz, Me-21) and 1.07 (d, J = 6.5 Hz, Me-27); δC 14.2 (C-21) and 13.6
(C-27), an olefinic group at δH 5.30 (br d, J = 3.6 Hz, H-6); δC 140.0 (C-5) and 121.9 (C-6), two quaternary
carbons at δC 46.1 (C-13) and 37.9 (C-10), an acetal carbon at δC 112.0 (C-22), and two anomeric
protons and carbons at δH 5.22 (d, J = 7.9 Hz) and 4.91 (d, J = 7.8 Hz); δC 106.9 and 103.2. The above
spectroscopic data imply that
1 had a spirost-5-ene diglycoside framework. Enzymatic hydrolysis
of with naringinase yielded 1a (C27H42O6) as the aglycone, and d-glucose and d-galactose as the
1
carbohydrate moieties. Treatment of 1a with Ac2O in pyridine gave tetraacetate (1b) of 1a, indicating
that 1a had four hydroxy groups. In the heteronuclear multiple bond correlation (HMBC) spectrum of

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1a, the angular methyl singlet at δH 1.09 showed long-range correlations with C-1 at δC 46.5, C-5 at δC
141.2, C-9 at δC 50.1, and C-10 at δC 38.6, and was assigned to Me-19. The olefinic proton at δH 5.41
attributed to H-6 exhibited HMBC correlations with C-4 at δC 40.7, C-5, and C-10. In the heteronuclear
multiple quantum coherence (HMQC) spectrum of 1a, the C-1 and C-4 carbons were correlated to the
one-bond coupled protons at δH 2.39 (dd, J = 12.6 and 4.4 Hz, H-1eq) and 1.42 (dd, J = 12.6, 12.3 Hz,
H-1ax), and 2.69 (H2-4), respectively. The H-1eq and H-1ax protons showed spin-coupling correlations
with the hydroxymethine proton at δH 4.12 (ddd, J = 12.3, 11.2, 4.4 Hz), whereas the H2-4 protons
exhibited spin-couplings with the hydroxymethine proton centered at δH 3.82 (m, W1/2 = 18.9 Hz) in
the 1H-1H correlation spectroscopy (COSY) spectrum of 1a. A spin-coupling correlation was observed
between the two hydroxymethine protons (H-2 and H-3) with a J value of 11.2 Hz. These data are
consistent with the presence of a hydroxy group at C-2 and C-3. Another angular methyl singlet at δH
1.08 assigned to Me-18 showed HMBC correlations with C-12 at δC 78.9, C-13 at δC 46.2, C-14 at δC
55.4, and C-17 at δC 62.3. The C-12 carbon was associated with the one-bond coupled proton at δH
3.58 (dd, J = 11.2 and 4.4 Hz) in the HMQC spectrum, from which spin-coupling correlations were
observed for the H2-11 methylene protons at δH 2.03 (m, H-11eq) and 1.75 (q-like, J = 11.2 Hz, H-11ax).
Thus, a hydroxy group was shown to be present at C-12. The methine proton at δH 1.85 (m) assignable
to H-25 displayed spin-coupling correlations with the Me-27 protons at δH 1.10 (d, J = 6.7 Hz), H2-26
methylene protons at δH 3.72 (dd, J = 11.2, 4.9 Hz, H-26eq) and 3.63 (dd, J = 12.3, 11.2 Hz, H-26ax),
and H-24 methine proton at δH 4.05 (ddd, J = 10.5, 10.5, 4.8 Hz). These correlations are indicative of the
presence of a hydroxy group at C-24 (Figure 2).
1
1
Figure 2. HMBC and H-1H spin-coupling correlations of 1a. Bold lines indicate the H-1H spin
couplings traced by 1H-1H COSY spectrum and arrows indicate 1H/13C long-range correlations
observed in the HMBC spectrum.
Accordingly, the planar structure of 1a was identified as spirost-5-ene-2,3,12,24-tetrol.
NOE correlations in the nuclear Overhauser enhancement spectroscopy (NOESY) and proton
spin-coupling constants allowed the stereochemistry of 1a to be determined. The B/C-trans, C/D-trans,
and D/E-cis ring junctions, and the configurations of 20
NOE correlations: between H-8 and H-11ax/H-15 /Me-18/Me-19, H-9 and H-11eq/H-12/H-14, H-14
and H-12/H-15 /H-17, H-17 and H-16/Me-21, Me-18 and H-20, and between H-20 and H-23ax
(Figure 3). The configurations of the C-2, C-3, and C-12 hydroxy groups were assigned as 2
α and 22α were confirmed by the following
α
α
α
,

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3
3β, and 12β, respectively, based on the proton spin-coupling constants, JH-1ax,H-2 = 12.3 Hz,
3
3
3JH-1eq,H-2 = 4.4 Hz, JH-11ax,H-12 = 11.2 Hz, and JH-11eq,H-12 = 4.4 Hz, and NOE correlations
were observed between H-1eq and H-2/Me-19, H-1ax and H-3/H-9, and between H-12 and
3
H-9/H-11eq/H-14/H-17 (Figure 3). The proton spin-coupling constants, JH-23ax,H-24 = 10.5 Hz,
3
3
3
3JH-23eq,H-24 = 4.8 Hz, JH-24,H-25 = 10.5 Hz, JH-25,H-26ax = 12.3 Hz, and JH-25,H-26eq = 4.9 Hz,
and NOE correlations between H-25 and H-23ax/H-26eq, and between H-24 and H-23eq/H-26ax/Me-27
were consistent with the 24S and 25S configurations (Figure 3). Thus, 1a was identified as
(24S,25S)-spirost-5-ene-2
the sugar moiety of comprised a 4-substituted
J = 7.8 Hz); δC 103.2, 73.0, 75.0, 79.9, 75.8, and 60.9 (C-10–60)] and a terminal
unit [Glc: δH 5.22 (1H, d, J = 7.9 Hz); δC 106.9, 75.7, 78.5, 72.0, 78.3, and 62.8 (C-100–600)]. In the
HMBC spectrum of
, long-range correlations were observed between H-100 of Glc (δH 5.22)
and C-40 of Gal (δC 79.9), and between H-10 of Gal (δH 4.91) and C-3 of the aglycone (δC 84.5).
Based on the above data, was identified as (24S,25S)-2 ,12 ,24-trihydroxyspirost-5-en-3 -yl
O-β-d-glucopyranosyl-(1→4)-β-d-galactopyranoside.
α
,3β,12β
,24-tetrol. The 1H-1H COSY and HMQC spectra of
-d-galactopyranosyl unit [Gal: δH 4.91 (1H, d,
-d-glucopyranosyl
1 suggest that
1
β
β
1
1
α
β
β
Figure 3. NOE correlations of 1a.
The 1H- and 13C-NMR spectral data of
2
(C39H60O16) suggest that
2
is analogous to
1
, including
was
were
the diglycoside moiety attached to C-3 of the aglycone. However, the molecular formula of
2
1
smaller than that of
compared with those of
group [δH 5.66 and 5.10 (each br s, H2-27); δC 106.4 (C-27) and 149.3 (C-25)] in
speculated that corresponded to the C-25/27 dehydroxy derivative of . This was supported
by HMBC correlations from H2-27 (δH 5.66 and 5.10) to C-24 (δC 67.0)/C-25 (δC 149.3)/C-26
δC 64.6). Accordingly, was identified as (24S)-2 ,12 ,24-trihydroxyspirosta-5,25(27)-dien-3 -yl
O-β-d-glucopyranosyl-(1→4)-β-d-galactopyranoside.
Compound
(C39H60O14) was obtained as an amorphous solid. The 1H- and 13C-NMR spectral
data of were similar to those of , including the signals of the diglycoside unit bound to C-3 of
the aglycone of . However, the molecular formula of was found to be smaller than that of by
two oxygen atoms, suggesting that the aglycone of had two less hydroxy groups than . Acid
with 1 M HCl (dioxane/H2O, 1:1) gave aglycone (3a), d-galactose, and d-glucose.
1
by two hydrogen atoms. When the H- and 13C-NMR spectra of
2
1, the Me-27 group was revealed to be displaced by an exomethylene
2
. Thus, it was
2
1
(
2
α
β
β
3
3
2
3
3
2
3
2
hydrolysis of
3
1
The H- and 13C-NMR spectra of 3a showed signals for two angular methyl groups at δH 1.07
(s, Me-19) and 0.82 (s, Me-18); δC 20.6 (C-19) and 16.3 (C-18), a secondary methyl group at δH 1.08
(d, J = 6.4 Hz, Me-21); δC 14.9 (C-21), an exomethylene group at δH 4.81 and 4.77 (each br s, H2-27);

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δC 108.7 (C-27) and 144.4 (C-25), an acetal carbon at δC 109.4 (C-22), an olefinic group at δH 5.40
(br d, J = 4.4 Hz, H-6); δC 141.2 (C-5) and 121.2 (C-6), and two vicinal hydroxy groups at δH 4.16
(ddd, J = 12.0, 11.2, 4.2 Hz, H-2) and 3.84 (m, W1/2 = 20.1 Hz, H-3); δC 72.6 (C-2) and 76.7 (C-3).
These spectroscopic data imply that 3a was spirosta-5,25(27)-diene-2
provided evidence that the O- -d-glucopyranosyl-(1 4)- -d-galactopyranosyl group was present
at C-3 of the aglycone in . Therefore, was deduced to be 2 -hydroxyspirosta-5,25(27)-dien-3 -yl
O-β-d-glucopyranosyl-(1→4)-β-d-galactopyranoside.
Compound was obtained as an amorphous solid, and its molecular formula was determined to
be C46H74O20 based on HRESI-TOF-MS and 13C-NMR data. In the 1H- and 13C-NMR spectra of
α,3β-diol. The HMBC spectrum
β
→
β
3
3
α
β
4
4,
the following signals were observed: three steroidal methyl groups at δH 1.14 (d, J = 6.9 Hz, Me-21), 0.93
(s, Me-19), and 0.76 (s, Me-18); δC 20.3 (C-19), 16.1 (C-21), and 16.0 (C-18), an exomethylene group at δH
5.34 and 5.04 (each br s, H2-27); δC 146.7 (C-25) and 111.0 (C-27), an olefinic group at δH 5.30 (br d, J = 4.6
Hz, H-6); δC 140.0 (C-5) and 121.8 (C-6), and three anomeric protons and carbons at δH 5.23 (d, J = 7.9 Hz,
H-100), 4.93 (d, J = 7.8 Hz, H-10), and 4.90 (d, J = 7.8 Hz, H-1000); δC 106.9 (C-100), 103.7 (C-1000), and 103.3
(C-10). In addition, an acetal carbon signal at δC 112.3, a methoxy proton and carbon signals at δH 3.23
(s); δC 47.3, and a positive color reaction in Ehrlich’s test suggested that
glycoside. Compound was treated with -d-glucosidase to obtain the corresponding spirostanol
glycoside ( -d-glucopyranosyl (δH 4.90) to C-26 of
) and d-glucose. A 3JC,H correlation from H-1000 of
the aglycone (δC 71.9) was observed in the HMBC spectrum of . The C-22 configuration was confirmed
by the NOE correlation observed between -OMe (δH 3.23) and H-16 (δH 4.41) of the aglycone. Thus, was
-yl
4 was a 22-methoxyfurostanol
4
β
3
β
4
α
4
determined to be 26-[(
β
-d-glucopyranosyl)oxy]-2
α
-hydroxy-22
α
-methoxyfurosta-5,25(27)-dien-3
β
O-β-d-glucopyranosyl-(1→4)-β-d-galactopyranoside.
The 1H- and 13C-NMR spectroscopic features of
5
(C52H84O23) were similar to those of 4, except
for the signals assignable to the sugar moiety attached to C-3 of the aglycone. The molecular formula
of was larger than that of by C6H10O3, corresponding to a hexosyl unit. Acid hydrolytic cleavage of
with 1 M HCl (dioxane/H2O, 1:1) afforded 3a, d-glucose, and l-rhamnose. Analysis of the 1H-1H
COSY and HMQC spectra for the sugar moieties of indicated the presence of a 2,4-disubstituted
d-glucopyranosyl unit [Glc (I): δH 4.96 (1H, d, J = 7.2 Hz, H-10); δC 100.9, 77.6, 77.6, 78.5, 76.9, and 61.0
(C-10–60)], a terminal -d-glucopyranosyl unit [Glc (II): δH 4.90 (1H, d, J = 7.8 Hz, H-10”’); δC 103.7,
75.0, 78.5, 71.6, 78.4, and 62.7 (C-10000–60000)], and two terminal
-l-rhamnopyranosyl units [Rha (I):
δH 6.33 (1H, br s, H-10’); δC 101.9, 72.2, 72.7, 73.9, 69.4, and 18.5 (C-100–600); Rha (II): δH 5.79 (1H, br s,
5
4
5
5
β-
β
α
H-1000); δC 102.7, 72.3, 72.6, 73.8, 70.3, and 18.4 (C-1000–6000)]. In the HMBC spectrum of
5, long-range
correlations were observed between H-100 of Rha (I) (δH 6.33) and C-20 of Glc (I) (δC 77.6), H-1000 of
Rha (II) (δH 5.79) and C-40 of Glc (I) (δC 78.5), H-10 of Glc (I) (δH 4.96) and C-3 of the aglycone (δC
84.9), and between H-10000 of Glc (II) (δH 4.90) and C-26 of the aglycone (δC 71.9). Therefore,
5
was
-yl
characterized as 26-[(
β
-d-glucopyranosyl)oxy]-2
α
-hydroxy-22
α
-methoxyfurosta-5,25(27)-dien-3
β
O-α-l-rhamnopyranosyl-(1→2)-O-[α-l-rhamnopyranosyl-(1→4)]-β-d-glucopyranoside.
1
The H- and 13C-NMR spectra of
6 (C45H70O19) and 7 (C51H80O22) were closely related to
those of
Instead of the secondary methyl signal for Me-21 [
(d, J = 6.9 Hz); δC 16.1] and a methoxy signal [
the signals arising from the tertiary methyl groups [
4
and
5
, respectively, except for the signals attributable to the E-ring part of the aglycone.
δH 1.14 (d, J = 6.9 Hz); δC 16.1; δH 1.14
4:
5:
4
:
δH 3.23 (s); δC 47.3;
δH 1.60 (s); δC 11.7;
δC 151.6 and 103.9;
and . Thus, and were thought to be the corresponding
, respectively. The structures of and were confirmed by
the following chemical transformations (Figure 4). Enzymatic hydrolysis of with -d-glucosidase
gave and d-glucose. Furthermore, complete acetylation of with Ac2O in pyridine afforded
5:
δH 3.24 (s); δC 47.3],
6
:
7: δH 1.59 (s); δC 11.7]
and tetrasubstituted olefinic carbons [
6:
7: δC 151.6 and 103.9] were observed
1
in the H- and 13C-NMR spectra of
6
7
6
7
pseudofurostanol glycosides of
4
and
5
6
7
6
β
3
6
dodecaacetate (6a), which agreed with t◦he peracetate of the dehydro derivative of
4 prepared by
treating
with
4 with Ac2O in pyridine at 130 C for 3 h. On the other hand, enzymatic hydrolysis of 7
β-d-glucosidase gave the spirostanol glycoside (7a), which is in agreement with the glycoside

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obtained by enzymatic hydrolysis
5
.
5
Tridecaacetate (7b) of
with Ac2O in pyridine at 130 C for 3 h. Accordingly,
7
was c◦onsistent with the product
prepared from
and were identified as 26-[(
O- -d-glucopyranosyl-(1 4)-
rosta-5,20(22),25(27)-trien-3 -yl O-
glucopyranoside, respectively.
5
upon treatment of
6
7
β-d-glucopyranosyl)oxy]-2
α
-hydroxyfurosta-5,20(22),25(27)-trien-3
β-yl
β
→
β-d-galactopyranoside and 26-[(
β-d-glucopyranosyl)oxy]-2
α
-hydroxyfu-
4)]- -d-
β
α
-l-rhamnopyranosyl-(1 2)-O-[
→
α
-l-rhamnopyranosyl-(1
→
β
Figure 4. Chemical transformations of 4–7.
Compound
was the same as that of
8
(C51H86O21) was obtained as an amorphous solid. The molecular formula of
1
8
9
, and the H- and 13C-NMR spectra of
8
were very similar to those
of
9
, except for the signals attributable to the side chain of the aglycone units. Compound
8
was enzymatically hydrolyzed with naringinase, yielding (22S)-cholest-5-ene-3
d-glucose, and l -rhamnose. The 1H- and 13C-NMR spectra of
-d-glucopyranosyl unit [Glc (I)], two terminal
and a terminal
-d-glucopyranosyl unit [Glc (II): δH 5.08 (1H, d, J = 7.8 Hz, H-10000); δC 98.6, 75.4,
β,22,25-triol (8a) [19],
8
imply the presence of a 2,4-disubstituted
β
α-l-rhamnopyranosyl units [Rha (I) and Rha (II)],
β

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3
78.8, 71.8, 78.0, and 62.9 (C-10000–60000)] in this molecule. In the HMBC spectrum of
8, a JC,H
correlation was observed between H-10000 of Glc (II) (δH 5.08) and C-25 of the aglycone moiety (δC 77.4).
Therefore,
O-α-l-rhamnopyranosyl-(1→2)-O-[α-l-rhamnopyranosyl-(1→4)]-β-d-glucopyranoside.
The isolated compounds ( 11) were evaluated for their cytotoxic activity toward HL-60
human promyelocytic leukemia cells using a modified 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-
2H-tetrazolium bromide (MTT) assay method. None of 11 exhibited cytotoxicity at a sample
concentration of up to 10 M. Although C. newellii is regarded to be a poisonous plant, the MTT assay
8 was determined to be (22S)-25-[(β-d-glucopyranosyl)oxy]-22-hydroxycholest-5-en-3β-yl
1–
1
–
µ
exerted that none of the isolated compounds were cytotoxic against HL-60 cells.
3. Materials and Methods
3.1. General
Optical rotations were measured on a JASCO P-1030 and a JASCO DIP-360 (JASCO, Tokyo, Japan)
automatic digital polarimeter. IR spectra were obtained using a FT/IR-620 (JASCO) spectrophotometer.
1
NMR spectral data were recorded on a DRX-500 (500 MHz for H-NMR, 125 MHz for 13C-NMR)
spectrometer using standard Bruker pulse programs at 300 K (Bruker, Karlsruhe, Germany). Chemical
shifts are given as
δ values with reference to tetramethylsilane (TMS) as an internal standard.
HRESI-TOF-MS data were obtained using a Waters Micromass LCT mass spectrometer (Waters, MA,
USA). Diaion HP-20 porous polymer polystyrene resin (Mitsubishi-Chemical, Tokyo, Japan), silica gel
Chromatrex BW-300 (Fuji-Silysia Chemical, Aichi, Japan), and ODS silica gel COSMOSIL 75C18-OPN
(Nacalai Tesque, Kyoto, Japan) were used for CC. Thin-layer chromatography (TLC) analysis was
conducted using precoated silica gel 60F254 or RP18 F254S plates (0.25 mm thick; Merck, Darmstadt,
Germany), and the spots were made visible by spraying the plates with H2SO4/H2O (1:9), followed by
heating. TLC was used to check the progress of the separation of fractions and to confirm the purity
of the isolated compounds. A Tosoh CCPM (Tosoh, Tokyo, Japan) or a Tosoh-8020 (Tosoh), Tosoh
RI-8020 (Tosoh), or Shodex OR-2 (Showa-Denko, Tokyo, Japan) detector, and a Rheodyne injection port
(Rohnert Park, CA, USA) constituted the HPLC system. A Capcell Pak C18 UG120 column (10 mm
i.d.
×
250 mm, 5
µ
m, Shiseido, Tokyo, Japan) was used for preparative HPLC. Enzymatic hydrolysis
was carried out using
β-d-glucosidase (EC 232-589-7; Sigma, St. Louis, MO, USA) or naringinase
(EC 232-962-4; Sigma, St. Louis, MO, USA).
3.2. Plant Material
The leaves of C. newellii were purchased from Sakata Seed Corporation (Kanagawa, Japan) and
grown in the medicinal botanical garden of Tokyo University of Pharmacy and Life Sciences (TUPLS).
A voucher specimen was kept at the herbarium of the TUPLS.
3.3. Extraction and Isolation
C. newellii leaves (dry weight, 3.6 kg) were extracted with MeOH at 60 ◦C for 2 h, and concentrated
under reduced pressure to obtain the MeOH extract (180 g). Then, all MeOH extract was loaded onto a
Diaion HP-20 column, and successively eluted with MeOH/H2O (3:7), EtOH, and EtOAc (each 12 L).
The EtOH eluted fraction was separated by silica gel CC and eluted with a stepwise gradient mixture of
CHCl3/MeOH (9:1, 6:1, 3:1, 1:1) to obtain three fractions (Frs. I–III). Fraction II was further divided by
ODS silica gel CC eluted with MeCN/H2O (1:3, 1:1, 3:1, 5:1) to yield seven subfractions (Frs. II-1–II-7).
Fraction II-1 was purified by silica gel CC eluted with CHCl3/MeOH/H2O (20:10:1), ODS silica gel
CC eluted with MeCN/H2O (1:3), and preparative HPLC using MeCN/H2O (5:11) to obtain
and (12 mg). Fraction II-2 was subjected to silica gel CC eluted with CHCl3/MeOH/H2O (20:10:1;
7:4:1), ODS silica gel CC eluted with MeCN/H2O (2:7), and preparative HPLC using MeCN/H2O
(1:3) to obtain (1.8 g). Fraction II-3 was separated by silica gel CC eluted with CHCl3/MeOH/H2O
(25:10:1; 20:10:1), ODS silica gel CC eluted with MeCN/H2O (5:13; 5:14), and preparative HPLC using
6 (21 mg)
7
4

Molecules 2020, 25, 4462
8 of 14
MeCN/H2O (10:27) to yield
1
(49 mg) and
2
(8.3 mg). Fraction II-5 was applied to silica gel CC eluted
with CHCl3/MeOH/H2O (30:10:1; 25:10:1; 20:10:1; 7:4:1) and ODS silica gel CC eluted with MeCN/H2O
(1:1; 1:2; 5:12; 2:5; 10:27; 1:3), and preparative HPLC using MeCN/H2O (5:12) to afford (22 mg),
(29 mg), (20 mg), 10 (65 mg), and 11 (71 mg). Fraction II-7 was chromatographed on silica gel and
5
8
9
ODS silica gel eluted with CHCl3/MeOH/H2O (30:10:1) and MeCN/H2O (1:1), respectively, to furnish
3 (25 mg).
3.4. Structural Characterization
25
Compound
1
: Amorphous solid; [
α
]
−
62.5 (c = 0.10, MeOH); IR (film)
ν
max: 3381 (OH), 2907
D
(CH) cm−1; HRESI-TOF-MS m/z: 787.4146 [M + H]+ (calcd. for C39H63O16: 787.4116). H-NMR spectral
data (500 MHz, C5D5N): δH 5.30 (1H, br d, J = 3.6 Hz, H-6), 5.22 (1H, d, J = 7.9 Hz, H-100), 4.91 (1H,
d, J = 7.8 Hz, H-10), 4.63 (1H, br d, J = 3.5 Hz, H-40), 4.61 (1H, q-like, J = 8.4 Hz, H-16), 4.56 (1H, dd,
J = 10.9, 8.0 Hz, H-60a), 4.53 (1H, dd, J = 11.2, 2.4 Hz, H-600a), 4.41 (1H, dd, J = 9.3, 7.8 Hz, H-20), 4.25
(1H, dd, J = 9.3, 3.5 Hz, H-30), 4.23 (1H, dd, J = 9.5, 8.6 Hz, H-30’), 4.22 (1H, dd, J = 10.9, 4.2 Hz, H-60b),
4.19 (1H, dd, J = 11.2, 4.7 Hz, H-600b), 4.09 (1H, m, H-50), 4.08 (1H, dd, J = 8.6, 7.9 Hz, H-200), 4.07 (1H,
dd, J = 9.5, 8.8 Hz, H-400), 4.03 (1H, m, H-24), 4.01 (1H, m, H-2), 3.96 (1H, ddd, J = 8.8, 4.7, 2.4 Hz,
H-500), 3.79 (1H, m, H-3), 3.70 (1H, dd, J = 11.2, 4.8 Hz, H-26eq), 3.60 (1H, dd, J = 12.2, 11.2 Hz, H-26ax),
3.55 (1H, dd, J = 11.0, 4.2 Hz, H-12), 1.42 (3H, d, J = 6.9 Hz, Me-21), 1.07 (3H, d, J = 6.5 Hz, Me-27), 1.04
(3H, s, Me-18), 0.94 (3H, s, Me-19). For 13C-NMR spectral data, see Table 1. For NMR spectral data,
see Supplementary Materials.
1
Table 1. 13C-NMR (125MHz, C5D5N) spectral assignments for 1, 1a, 2, 3, 3a, 4–8.
Positions
1
1a
2
3
3a
4
5
6
7
8
1
2
3
4
5
6
7
8
9
45.7
69.9
84.5
37.4
140.0
121.9
31.9
30.2
49.8
37.9
31.4
78.7
46.1
55.2
31.7
81.4
62.2
10.9
20.3
43.2
14.2
112.0
41.8
70.6
39.8
65.2
13.6
46.5
72.5
76.7
40.7
141.2
121.3
32.0
30.3
50.1
38.6
31.5
78.9
46.2
55.4
31.8
81.5
62.3
11.0
20.6
43.2
14.2
112.1
41.9
70.6
39.9
65.3
13.6
45.7
69.9
84.5
37.5
140.0
121.9
31.9
30.2
49.8
38.0
31.4
78.7
46.2
55.2
31.7
81.8
62.2
11.0
20.3
43.0
14.1
112.0
43.5
67.0
149.3
64.6
106.4
45.8
70.0
84.5
37.5
140.0
121.8
32.1
31.0
50.1
37.8
21.1
39.6
40.4
56.4
32.0
81.3
62.7
16.2
20.3
41.7
14.9
109.4
33.1
28.8
144.3
64.9
108.7
46.5
72.6
76.7
40.8
141.2
121.2
32.2
31.1
50.3
38.4
21.2
39.7
40.4
56.5
32.1
81.4
62.8
16.3
20.6
41.8
14.9
109.4
33.1
28.9
144.4
64.9
108.7
45.8
70.0
84.5
37.5
140.0
121.8
32.0
30.9
50.1
37.8
21.0
39.5
40.3
56.3
32.1
81.3
63.9
16.0
20.3
40.7
16.1
112.3
31.5
28.0
146.7
71.9
111.0
47.3
Gal
45.9
70.1
84.9
37.1
139.8
121.9
32.0
31.0
50.1
37.8
21.0
39.5
40.3
56.3
32.1
81.3
63.9
16.1
20.3
40.7
16.1
112.3
31.5
28.0
146.7
71.9
111.0
47.3
Glc (I)
100.9
45.8
70.0
84.6
37.6
140.0
121.8
32.2
30.8
50.1
37.8
21.3
39.5
43.2
54.7
34.4
84.4
64.4
14.1
20.4
103.9
11.7
151.6
24.6
31.0
146.1
71.6
111.6
46.0
70.1
84.9
37.1
139.8
121.9
32.3
30.8
50.1
37.8
21.2
39.5
43.2
54.7
34.4
84.4
64.4
14.0
20.3
103.9
11.7
151.6
24.6
31.0
146.1
71.6
111.6
37.5
30.2
78.1
39.0
140.8
122.0
32.2
32.1
50.4
36.9
21.3
40.1
42.3
57.0
24.5
28.2
53.1
12.0
19.4
41.9
12.5
73.2
30.5
39.3
77.4
27.1
27.1
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
OMe
Gal
103.2
Gal
103.3
Gal
103.3
Gal
103.4
Glc (I) Glc (I)
100.9 100.2
10
103.3

Molecules 2020, 25, 4462
9 of 14
Table 1. Cont.
3a
Positions
1
1a
2
3
4
5
6
7
8
20
30
40
50
60
73.0
75.0
79.9
75.8
60.9
73.0
75.1
80.0
75.9
60.9
73.0
75.1
80.0
75.9
60.9
73.0
75.0
80.0
75.8
60.9
77.6
77.6
78.5
76.9
61.0
Rha
(I)
73.0
75.1
80.0
75.9
60.9
77.7
77.7
78.5
77.0
61.0
Rha
(I)
77.8
77.9
78.5
76.9
61.2
Rha
(I)
Glc
Glc
Glc
Glc (I)
Glc (I)
100
200
300
400
500
600
106.9
75.7
78.5
72.0
78.3
62.8
106.9
75.7
78.6
72.1
78.4
62.9
106.9
75.7
78.6
72.0
78.4
62.9
106.9
75.7
78.6
72.0
78.3
62.9
Glc
101.9
72.2
72.7
73.9
69.4
18.5
Rha
(II)
107.0
75.7
78.6
72.1
78.4
63.0
Glc
101.9
72.3
72.7
74.0
69.5
18.5
Rha
(II)
102.0
72.5
72.8
74.1
69.5
18.6
Rha
(II)
(II)
(II)
1000
2000
3000
4000
5000
6000
103.7
75.0
78.5
71.6
78.4
62.7
102.7
72.3
72.6
73.8
70.3
18.4
Glc
103.7
75.1
78.5
71.6
78.4
62.7
102.7
72.4
72.6
73.8
70.3
18.4
Glc
102.8
72.4
72.7
73.9
70.4
18.5
Glc
(II)
(II)
(II)
10000
20000
30000
40000
50000
60000
103.7
75.0
78.5
71.6
78.4
62.7
103.7
75.1
78.5
71.6
78.4
62.6
98.6
75.4
78.8
71.8
78.0
62.9
25
Compound 1a: Amorphous solid; [
α
]
−
44.2 (c = 0.10, MeOH); IR (film) νmax: 3364 (OH),
D
2924 and 2872 (CH) cm−1; HRESI-TOF-MS m/z: 463.3084 [M + H]+ (calcd. for C27H43O6: 463.3060).
1H-NMR spectral data (500 MHz, C5D5N): δH 5.41 (1H, br d, J = 4.8 Hz, H-6), 4.63 (1H, q-like, J = 7.5
Hz, H-16), 4.12 (1H, ddd, J = 12.3, 11.2, 4.4 Hz, H-2), 4.05 (1H, ddd, J = 10.5, 10.5, 4.8 Hz, H-24), 3.82
(1H, m, W1/2 = 18.9 Hz, H-3), 3.72 (1H, dd, J = 11.2, 4.9 Hz, H-26eq), 3.63 (1H, dd, J = 12.3, 11.2 Hz,
H-26ax), 3.58 (1H, dd, J = 11.2, 4.4 Hz, H-12), 1.45 (3H, d, J = 6.9 Hz, Me-21), 1.10 (3H, d, J = 6.7 Hz,
Me-27), 1.09 (3H, s, Me-19), 1.08 (3H, s, Me-18). For 13C-NMR spectral data, see Table 1. For NMR
spectral data, see Supplementary Materials.
1
Tetraacetate of compound 1a
(
1b): H-NMR spectral data (500 MHz, C5D5N): δH 5.34 (1H, br d,
J = 4.8 Hz, H-6), 5.04 (1H, m, H-3), 4.84 (1H, dd, J = 11.2, 4.5 Hz, H-12), 1.16 (3H, d, J = 6.8 Hz, Me-21), 1.06
(3H, s, Me-18), 0.92 (3H, s, Me-19), 0.80 (3H, d, J = 6.5 Hz, Me-27), 2.20, 2.10, 2.09 × 2 (each 3H, Ac × 4).
25
Compound
2
: Amorphous solid; [
α
]
−
78.0 (c = 0.10, MeOH); IR (film)
ν
max: 3381 (OH), 2921
D
(CH) cm−1; HRESI-TOF-MS m/z: 785.3943 [M + H]+ (calcd. for C39H61O16: 785.3960). H-NMR spectral
data (500 MHz, C5D5N): δH 5.66 (1H, br s, H-27a), 5.31 (1H, br d, J = 4.8 Hz, H-6), 5.24 (1H, d, J = 7.9
Hz, H-100), 5.10 (1H, br s, H-27b), 5.06 (1H, dd, J = 11.3, 6.2 Hz, H-24), 4.92 (1H, d, J = 7.7 Hz, H-10), 4.60
(1H, q-like, J = 6.9 Hz, H-16), 4.53 (1H, d, J = 12.8 Hz, H-26eq), 4.23 (1H, d, J = 12.8 Hz, H-26ax), 3.98
(1H, m, H-2), 3.80 (1H, m, H-3), 3.55 (1H, dd, J = 11.0, 4.4 Hz, H-12), 1.37 (3H, d, J = 6.9 Hz, Me-21), 1.05
(3H, s, Me-18), 0.95 (3H, s, Me-19). For 13C-NMR spectral data, see Table 1. For NMR spectral data,
see Supplementary Materials.
1

Molecules 2020, 25, 4462
10 of 14
25
Compound
3: Amorphous solid; [
α
]
−
54.6 (c = 0.10, MeOH); IR (film)
ν
max: 3369 (OH),
D
2927 and 2852 (CH) cm−1; HRESI-TOF-MS m/z: 753.4058 [M + H]+ (calcd. for C39H61O14: 753.4061).
1H-NMR spectral data (500 MHz, C5D5N): δH 5.30 (1H, br d, J = 4.7 Hz, H-6), 5.23 (1H, d, J = 7.9 Hz,
H-100), 4.93 (1H, d, J = 7.8 Hz, H-10), 4.80 (1H, br s, H-27a), 4.77 (1H, br s, H-27b), 4.51 (1H, q-like,
J = 7.9 Hz, H-16), 4.43 (1H, d, J = 12.4 Hz, H-26ax), 4.05 (1H, m, H-2), 4.02 (1H, d, J = 12.4 Hz, H-26eq),
3.82 (1H, m, H-3), 1.06 (3H, d, J = 7.0 Hz, Me-21), 0.94 (3H, s, Me-19), 0.79 (3H, s, Me-18). For 13C-NMR
spectral data, see Table 1. For NMR spectral data, see Supplementary Materials.
25
Compound 3a: Amorphous solid; [
α
]
−
78.6 (c = 0.10, MeOH); IR (film) νmax: 3351 (OH),
D
2926 and 2852 (CH) cm−1; HRESI-TOF-MS m/z: 429.3010 [M + H]+ (calcd. for C27H41O4: 429.3005).
1H-NMR spectral data (500 MHz, C5D5N): δH 5.40 (1H, br d, J = 4.4 Hz, H-6), 4.81 (1H, br s, H-27a),
4.77 (1H, br s, H-27b), 4.52 (1H, q-like, J = 7.1 Hz, H-16), 4.44 (1H, d, J = 12.1 Hz, H-26ax), 4.16 (1H,
ddd, J = 12.0, 11.2, 4.2 Hz, H-2), 4.02 (1H, d, J = 12.1 Hz, H-26eq), 3.84 (1H, m, W1/2 = 20.1 Hz, H-3),
1.08 (3H, d, J = 6.4 Hz, Me-21), 1.07 (3H, s, Me-19), 0.82 (3H, s, Me-18). For 13C-NMR spectral data,
see Table 1. For NMR spectral data, see Supplementary Materials.
25
Compound
4: Amorphous solid; [
α
]
−
53.4 (c = 0.10, MeOH); IR (film)
ν
max: 3369 (OH),
D
2936 and 2899 (CH) cm−1; HRESI-TOF-MS m/z: 915.4611 [M + H
−
MeOH]+ (calcd. for C45H71O19:
1
915.4590). H-NMR spectral data (500 MHz, C5D5N): δH 5.34 (1H, br s, H-27a), 5.30 (1H, br d, J = 4.6
Hz, H-6), 5.23 (1H, d, J = 7.9 Hz, H-100), 5.04 (1H, br s, H-27b), 4.93 (1H, d, J = 7.8 Hz, H-10), 4.90 (1H, d,
J = 7.8 Hz, H-1000), 4.61 (1H, d, J = 12.8 Hz, H-26a), 4.53 (1H, dd, J = 11.9, 2.4 Hz, H-6000a), 4.41 (1H, m,
H-16), 4.36 (1H, dd, J = 11.9, 5.6 Hz, H-6000b), 4.35 (1H, d, J = 12.8 Hz, H-26b), 4.26 (1H, dd, J = 8.7,
8.7 Hz, H-3000), 4.21 (1H, dd, J = 8.7, 8.7 Hz, H-4000), 4.06 (1H, dd, J = 8.7, 7.8 Hz, H-2000), 4.04 (1H, m,
H-2), 3.94 (1H, m, H-5000), 3.81 (1H, m, H-3), 3.23 (3H, s, OMe), 1.14 (3H, d, J = 6.9 Hz, Me-21), 0.93
(3H, s, Me-19), 0.76 (3H, s, Me-18). For 13C-NMR spectral data, see Table 1. For NMR spectral data,
see Supplementary Materials.
25
Compound
5
: Amorphous solid; [
α
]
−
52.0 (c = 0.10, MeOH); IR (film)
νmax: 3382 (OH), 2924
D
(CH) cm−1; HRESI-TOF-MS m/z: 1045.5264 [M + H
−
MeOH]+ (calcd. for C51H81O22: 1045.5220).
1H-NMR spectral data (500 MHz, C5D5N): δH 6.33 (1H, br s, H-100), 5.79 (1H, br s, H-1000), 5.34 (1H, br
s, H-27a), 5.32 (1H, br d, J = 4.4 Hz, H-6), 5.05 (1H, br s, H-27b), 4.96 (1H, d, J = 7.2 Hz, H-10), 4.90
(1H, d, J = 7.8 Hz, H-10000), 4.86 (1H, m, H-500), 4.85 (1H, m, H-5000), 4.81 (1H, br s, H-200), 4.65 (1H, br s,
H-2000), 4.62 (1H, d, J = 12.7 Hz, H-26a), 4.61 (1H, dd, J = 9.0, 3.7 Hz, H-300), 4.53 (1H, dd, J = 12.5, 3.4
Hz, H-60000a), 4.52 (1H, dd, J = 9.3, 3.4 Hz, H-3000), 4.42 (1H, q-like, J = 7.3 Hz, H-16), 4.37 (1H, br d,
J = 12.5 Hz, H-60000b), 4.36 (1H, d, J = 12.7 Hz, H-26b), 4.35 (1H, dd, J = 9.0, 9.0 Hz, H-400), 4.33 (1H, dd,
J = 8.9, 8.9 Hz, H-40), 4.32 (1H, dd, J = 9.3, 9.3 Hz, H-4000), 4.25 (1H, dd, J = 8.9, 8.9 Hz, H-40000), 4.24 (1H,
dd, J = 8.9, 8.9 Hz, H-30000), 4.21 (1H, br d, J = 12.9 Hz, H-60a), 4.20 (1H, dd, J = 8.9, 8.4 Hz, H-30), 4.17
(1H, dd, J = 8.4, 7.2 Hz, H-20), 4.16 (1H, m, H-2), 4.07 (1H, dd, J = 12.9, 4.6 Hz, H-60b), 4.06 (1H, dd, J
= 8.9, 7.8 Hz, H-20000), 3.93 (1H, m, H-50000), 3.82 (1H, m, H-3), 3.70 (1H, m, H-50), 3.24 (3H, s, OMe),
1.67 (3H, d, J = 6.2 Hz, Me-600), 1.60 (3H, d, J = 6.2 Hz, Me-6000), 1.14 (3H, d, J = 6.9 Hz, Me-21), 1.04
(3H, s, Me-19), 0.77 (3H, s, Me-18). For 13C-NMR spectral data, see Table 1. For NMR spectral data,
see Supplementary Materials.
25
Compound
6
: Amorphous solid; [
α
]
−
44.7 (c = 0.10, MeOH); IR (film)
ν
max: 3360 (OH), 2900
D
(CH) cm−1; HRESI-TOF-MS m/z: 915.4541 [M + H]+ (calcd. for C45H71O19: 915.4590). H-NMR spectral
data (500 MHz, C5D5N): δH 5.36 (1H, br s, H-27a), 5.31 (1H, br d, J = 4.6 Hz, H-6), 5.25 (1H, d, J = 7.9
Hz, H-100), 5.05 (1H, br s, H-27b), 4.93 (1H, d, J = 7.7 Hz, H-10), 4.90 (1H, d, J = 7.8 Hz, H-1000), 4.78 (1H,
q-like, J = 7.7 Hz, H-16), 4.59 (1H, d, J = 12.5 Hz, H-26a), 4.35 (1H, d, J = 12.5 Hz, H-26b), 4.11 (1H,
m, H-2), 3.82 (1H, m, H-3), 1.60 (3H, s, Me-21), 0.95 (3H, s, Me-19), 0.67 (3H, s, Me-18). For 13C-NMR
spectral data, see Table 1. For NMR spectral data, see Supplementary Materials.
1

Molecules 2020, 25, 4462
11 of 14
1
Dodecaacetate of Compound
6 (6a): H-NMR spectral data (500 MHz, C5D5N): δH 5.41 (1H, br s,
H-27a), 5.40 (1H, br s, H-6), 5.06 (1H, br s, H-27b), 4.49 (1H, d, J = 12.5 Hz, H-26a), 4.30 (1H, d, J = 12.5
Hz, H-26b), 1.60 (3H, s, Me-21), 0.94 (3H, s, Me-19), 0.81 (3H, s, Me-18), 2.34, 2.17, 2.14, 2.12
2.05, 2.03, 2.00 × 3, 1.98 (each 3H, Ac × 12).
×
2, 2.09,
25
Compound
7
: Amorphous solid; [
α
]
−
51.0 (c = 0.10, MeOH); IR (film) νmax: 3380 (OH), 2919
D
(CH) cm−1; HRESI-TOF-MS m/z: 1045.5173 [M + H]+ (calcd. for C51H81O22: 1045.5219). 1H-NMR
spectral data (500 MHz, C5D5N): δH 6.34 (1H, br s, H-100), 5.80 (1H, br s, H-1000), 5.36 (1H, br s, H-27a),
5.34 (1H, br d, J = 5.1 Hz, H-6), 5.04 (1H, br s, H-27b), 4.97 (1H, d, J = 6.6 Hz, H-10), 4.90 (1H, d,
J = 7.8 Hz, H-10000), 4.78 (1H, q-like, J = 7.9 Hz, H-16), 4.59 (1H, d, J = 12.0 Hz, H-26a), 4.35 (1H, d,
J = 12.0 Hz, H-26b), 4.16 (1H, m, H-2), 3.82 (1H, m, H-3), 1.67 (3H, d, J = 6.2 Hz, Me-600), 1.60 (3H, d,
J = 6.1 Hz, Me-6000), 1.59 (3H, s, Me-21), 1.06 (3H, s, Me-19), 0.67 (3H, s, Me-18). For 13C-NMR spectral
data, see Table 1. For NMR spectral data, see Supplementary Materials.
25
Compound 7a: Amorphous solid; [
α
]
−
3.9 (c = 0.10, MeOH); IR (film) νmax: 3376 (OH),
D
2924 and 2852 (CH) cm−1; HRESI-TOF-MS m/z: 883.4738 [M + H]+ (calcd. for C45H71O17: 883.4691).
1H-NMR spectral data (500 MHz, C5D5N): δH 6.36 (1H, br s, H-100), 5.81 (1H, br s, H-1000), 5.35 (1H, br
d, J = 4.6 Hz, H-6), 4.98 (1H, d, J = 7.2 Hz, H-10), 4.81 (1H, br s, H-27a), 4.78 (1H, br s, H-27b), 4.53 (1H,
m, H-16), 4.45 (1H, d, J = 12.1 Hz, H-26a), 4.16 (1H, m, H-2), 4.02 (1H, d, J = 12.1 Hz, H-26b), 3.83 (1H,
m, H-3), 1.68 (3H, d, J = 6.2 Hz, Me-600), 1.61 (3H, d, J = 6.2 Hz, Me-6000), 1.07 (3H, d, J = 6.9 Hz, Me-21),
1.06 (3H, s, Me-19), 0.80 (3H, s, Me-18). 13C-NMR spectral data (125 MHz, C5D5N): δC 45.9, 70.2, 85.0,
37.1, 139.9, 121.9, 32.2, 31.1, 50.1, 37.9, 21.1, 39.6, 40.4, 56.4, 32.1, 81.4, 62.8, 16.2, 20.3, 41.8, 14.9, 109.4,
33.1, 28.9, 144.3, 64.9, 108.7 (C-1-27), 100.9, 77.7, 77.6, 78.6, 77.0, 61.0 (C-10-60), 101.9, 72.3, 72.7, 74.0, 69.5,
18.5 (C-100-600), 102.8, 72.4, 72.6, 73.8, 70.4, 18.4 (C-1000-6000).
1
Tridecaacetate of compound 7 (7b): H-NMR spectral data (500 MHz, C5D5N): δH 5.49 (1H, br s,
H-6), 5.43 (1H, br s, H-27a), 5.34 (1H, br s, H-27b), 4.52 (1H, d, J = 13.1 Hz, H-26a), 4.32 (1H, d, J = 13.1
Hz, H-26b), 1.62 (3H, s, Me-21), 0.94 (3H, s, Me-19), 0.86 (3H, s, Me-18), 2.35, 2.19, 2.18, 2.16, 2.14, 2.09,
2.06, 2.04, 2.03 (×2), 2.01 (×2), 1.96 (each 3H, Ac × 13).
25
Compound
8
: Amorphous solid; [
α
]
−
48.9 (c = 0.10, MeOH); IR (film) νmax: 3381 (OH), 2932
D
(CH) cm−1; HRESI-TOF-MS m/z: 1035.5762 [M + H]+ (calcd. for C51H87O21: 1035.5740). 1H-NMR
spectral data (500 MHz, C5D5N): δH 6.38 (1H, br s, H-100), 5.86 (1H, br s, H-1000), 5.34 (1H, br d,
J = 4.9 Hz, H-6), 5.08 (1H, d, J = 7.8 Hz, H-10000), 4.95 (1H, d, J = 7.7 Hz, H-10). 3.91 (1H, m, H-22), 3.87
(1H, m, H-3), 1.77 (3H, d, J = 6.2 Hz, H-600), 1.62 (3H, d, J = 6.2 Hz, H-6000), 1.47 (3H
×
2, each s, Me-26
and Me-27), 1.15 (3H, d, J = 6.7 Hz, Me-21), 1.07 (3H, s, Me-18), 0.70 (3H, s, Me-19). For 13C-NMR
spectral data, see Table 1. For NMR spectral data, see Supplementary Materials.
Enzymatic hydrolysis of
treated with naringinase ( : 168 mg,
h. Each reaction mixture was purified by silica gel CC eluted with CHCl3/MeOH/H2O (
9:1:0) to obtain 1a (10.1 mg) from 8a (3.0 mg) from , and sugar fractions (6.1 mg from
), respectively. The sugar fraction was analyzed using HPLC under the following conditions: detection,
refractive index, and optical rotation; column, Capcell Pak NH2 UG80 (4.6 mm i.d. 250 mm, 5 m,
1
and
4
8
–
8
: Compounds
: 102 mg) in AcOH/AcOK buffer (pH 4.3, 3.0 mL) at 28 ◦C for 132
; 9:1:0, 7:4:1,
, 2.4 mg from
1 (20 mg) and 8 (9.8 mg) were independently
1
1
8;
1,
8
1
8
×
µ
Shiseido); solvent, MeCN/H2O (17:3); flow rate, 1.0 mL/min. d-Galactose, d-glucose, and l-rhamnose
were identified by comparing their retention times and optical rotations with those of authentic samples:
d-galactose (12.13, positive optical rotation), d-glucose (13.62, positive optical rotation), and l-rhamnose
(7.48, negative optical rotation). Compounds
independently treated with -d-glucosidase (
(pH 5.0, 3.0 mL) at 28 ◦C for 20 h. Each reaction mixture was chromatographed on silica gel eluted
with CHCl3/MeOH/H2O ( , and ; 20:10:1, ; 9:1:0) to collect (17.4 mg) from 7a (3.4 mg) from 5, 3
4
(29.7 mg),
5
(10.1 mg),
6
(5.6 mg), and
7 (3.1 mg) were
β
4
: 25 mg, : 15 mg,
5
6
: 8.8 mg,
7
: 10 mg) in AcOH/AcONa
4,
6
7
5
3
4,

Molecules 2020, 25, 4462
12 of 14
(3.8 mg) from
6,
7a (1.0 mg) from
7
, and their sugar fractions. HPLC analysis of the sugar fractions
under the same conditions as those of 1 exhibited the presence of d-galactose in 4 and 6, d-glucose in
4–7, and l-rhamnose in 5 and 7.
Acid hydrolysis of
3 and 5: Compounds 3 (18.2 mg) and 5 (10.1 mg) were independently treated
with 1 M HCl (dioxane/H2O, 1:1, 3.0 mL) at 95 ◦C for 1 h under Ar atmosphere. The reaction solution
was neutralized by passing through an Amberlite IRA-93 ZU (Organo, Tokyo, Japan) column and
separated using a Sep-Pak C18 cartridge (Waters) eluted with MeOH/H2O (1:4) to yield sugar fractions
(3.1 mg from
fractions were analyzed by HPLC under the same conditions as those of
d-galactose in , d-glucose in and , and l-rhamnose in . The aglycone fractions were independently
3
and 1.2 mg from
5) and finally MeOH alone to obtain aglycone fractions. The sugar
1
showed the presence of
3
3
5
5
subjected to silica gel CC eluted with CHCl3/MeOH (19:1) to furnish 3a (8.3 mg) from
from 5.
3, and 3a (2.8 mg)
Acetylation of
4
–
7
and 1a: Compounds
4
(100 mg) and
5
(4.◦5 mg) were independently applied
to acetylation with Ac2O (2.0 mL) in pyridine (2.0 mL) at 130 C for 3 h. The reaction solutions
were distributed using Et2O (10 mL
subjected to silica gel CC eluted with hexane hexane/Me2CO (1:1) to obtain 6a (73. 0 mg) from
×
2). After concentration of the Et2O soluble phases, those were
, and 7b
4
(3.4 mg) from 5. Compounds 6 (10.2 mg), 7 (8.7 mg), and 1a (8.8 mg) were independently acetylated
with Ac2O (1.0 mL) in pyridine (1.0 mL) at 28 ◦C for 20 h. The reaction solutions were distributed and
purified, as well as 4, to afford 6a (4.6 mg) from 6, 7b (4.1 mg) from 7, and 1b (4.0 mg) from 1a.
3.5. Evaluation of Cytotoxic Activity
Cytotoxic activity of 1–11 against HL-60 cells (JCRB 0085; Human Science Research Resources
Bank, Osaka, Japan) was examined by a modified MTT assay method as previously described [20].
In short, HL-60 cells were incubated at 37 ◦C for 24 h in RPMI-1640 medium with 10% heat-inactivated
fetal bovine serum. The cell viability was evaluated using the MTT method.
4. Conclusions
A systematic phytochemical analysis of the leaves of C. newellii was conducted with a focus on
steroidal glycosides. As a result, three new spirostanol glycosides (
and ), two new pseudofurostanol glycosides ( and ), one new cholestane glycoside (
known cholestane glycosides ( 11) were isolated. Compounds and are spirostanol glycosides
1
–3
), two new furostanol glycosides
(4
5
6
7
8), and three
9–
1
2
having hydroxy groups at C-2, C-3, C-12, and C-24 of the aglycone moiety. Although C. newellii is
known to be a poisonous plant, the MTT assay showed that none of the isolated compounds were
cytotoxic toward HL-60 cells.
Supplementary Materials: The following are available online. Figures S1–S52 showed NMR spectral data of
1a, 2, 3, 3a, 4–11.
1,
Author Contributions: T.I., N.T., and Y.M. conceived and designed the experiments; T.I., N.T., and Y.M. performed
the experiments; T.I. and Y.M. wrote the paper. All authors have read and agreed to the published version of
the manuscript.
Funding: This research received no external funding.
Conflicts of Interest: The authors declare no conflict of interest.

Molecules 2020, 25, 4462
13 of 14
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Sample Availability: Samples of the compounds are not available from the authors.
©
2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).