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C ys al S uc u es and Cy o oxici y o en -Kau ane-Type Di e penoids om Two Aspilia
Species
© he Au ho s, 2018.
Published e sion
Yaouba, Souaibou; Valkonen, A o; Coghi, Paolo; Gao, Jiaying; Guan ai, E ic M.;
De ese, Solomon; Wong, Vincen K. W.; E délyi, Má é; Yenesew, Abiy
Yaouba, S., Valkonen, A., Coghi, P., Gao, J., Guan ai, E. M., De ese, S., Wong, V. K. W., E délyi,
M., & Yenesew, A. (2018). C ys al S uc u es and Cy o oxici y o en -Kau ane-Type Di e penoids
om Two Aspilia Species. Molecules, 23(12), A icle 3199.
h ps://doi.o g/10.3390/molecules23123199
2018
molecules
A icle
C ys al S uc u es and Cy o oxici y o en -Kau ane-Type
Di e penoids om Two Aspilia Species
Souaibou Yaouba 1, A o Valkonen 2, Paolo Coghi 3, Jiaying Gao 3, E ic M. Guan ai 4,
Solomon De ese 1, Vincen K. W. Wong 3, Má éE délyi 5,6,7,* and Abiy Yenesew 1,*
1Depa men o Chemis y, Uni e si y o Nai obi, P. O. Box 30197, 00100 Nai obi, Kenya;
[email p o ec ed] (S.Y.); [email p o ec ed] (S.D.)
2Depa men o Chemis y, Uni e si y o Jy askyla, P.O. Box 35, 40014 Jy askyla, Finland;
[email p o ec ed]
3S a e Key Labo a o y o Quali y Resea ch in Chinese Medicine/Macau Ins i u e o Applied Resea ch in
Medicine and Heal h, Macau Uni e si y o Science and Technology, Macau 999078, China;
[email p o ec ed] (P.C.); [email p o ec ed] (J.G.); [email p o ec ed] (V.K.W.W.)
4Depa men o Pha macology and Pha macognosy, School o Pha macy, Uni e si y o Nai obi,
P. O. Box 19676, 00202 Nai obi, Kenya; [email p o ec ed]
5Depa men o Chemis y–BMC, Uppsala Uni e si y, Husa ga an 3, 75237 Uppsala, Sweden
6The Swedish NMR Cen e, Medicina ega an 5, 40530 Go henbu g, Sweden
7Depa men o Chemis y and Molecula Biology, Uni e si y o Go henbu g, 40530 Go henbu g, Sweden
*Co espondence: [email p o ec ed] (M.E.); [email p o ec ed] (A.Y.);
Tel.: +46-72-999-9166 (M.E.); +254-73-383-2576 (A.Y.); Fax: +254-20-444-6138 (A.Y.)
Academic Edi o s: Isabel C.F.R. Fe ei a and Nancy D. Tu ne
Recei ed: 11 No embe 2018; Accep ed: 30 No embe 2018; Published: 4 Decembe 2018


Abs ac :
A phy ochemical in es iga ion o he oo s o Aspilia plu ise a led o he isola ion
o en -kau ane- ype di e penoids and addi ional phy ochemicals (
1
–
23
). The s uc u es o
he isola ed compounds we e elucida ed based on Nuclea Magne ic Resonance (NMR)
spec oscopic and mass spec ome ic analyses. The absolu e con igu a ions o se en o he
en -kau ane- ype di e penoids (
3
–
6
,
6b, 7
and
8
) we e de e mined by single c ys al X- ay
di ac ion s udies. Ele en o he compounds we e also isola ed om he oo s and he
ae ial pa s o Aspilia mossambicensis. The li e a u e NMR assignmen s o compounds
1
and
5
we e e ised. In a cy o oxici y assay, 12
α
-me hoxy-en -kau -9(11),16-dien-19-oic acid (
1
)
(IC
50
= 27.3
±
1.9
µ
M) and 9
β
-hyd oxy-15
α
-angeloyloxy-en -kau -16-en-19-oic acid (
3
) (IC
50
=
24.7 ±2.8 µM
) we e he mos cy o oxic agains he hepa ocellula ca cinoma (Hep-G2) cell line,
while 15
α
-angeloyloxy-16
β
,17-epoxy-en -kau an-19-oic acid (
5
) (IC
50
= 30.7
±
1.7
µ
M) was he mos
cy o oxic agains adenoca cinomic human al eola basal epi helial (A549) cells.
Keywo ds:
As e aceae; Aspilia plu ise a;Aspilia mossambicensis;en -kau ane di e penoid; X- ay c ys al
s uc u e; cy o oxici y
1. In oduc ion
The genus Aspilia belongs o he amily As e aceae. The majo i y o plan s in his amily a e
he baceous, while ees and sh ubs a e a e [
1
]. Plan s belonging o he As e aceae amily a e ound
wo ldwide, excep An a c ica [
2
]. They a e ound in coole mon ane habi a s o empe a e a eas in
opical egions, and a e no common dwelle s o ho lowland opical ain o es s [
1
,
2
].
The amily
o As e aceae is one o he la ges plan amilies and he iches in ascula plan s in he wo ld.
The amily
has abou 1,600–1,700 gene a and 24,000–30,000 species [
1
,
3
,
4
]. Plan s om he genus
Aspilia (As e aceae) occu widely in Sou h, Sou h-Wes , and Wes Kenya, om he coas o Lake
Molecules 2018,23, 3199; doi:10.3390/molecules23123199 www.mdpi.com/jou nal/molecules
Molecules 2018,23, 3199 2 o 13
Vic o ia. The genus Aspilia exhibi s biological ac i i ies, including an ibac e ial and an i ungal e ec s,
mainly a ibu ed o he p esence o kau ane- ype di e penoids [3,5] and sesqui e pene lac ones [5,6].
Aspilia plu ise a Schwein has been used in adi ional medicine o ea lace a ions, b uises and
bu ns, and i is epu ed o aid in he healing o cu aneous lesions [
7
]. The plan is ound in Kenya
and is commonly known as ‘Dwa Aspilia’ [
7
]. The p esence o di e penoids om ae ial pa s o
A. plu ise a
has been epo ed p e iously [
8
], and ou o hese di e penoids exhibi ed mode a e ac i i y
agains chlo oquine-sensi i e (D6) and chlo oquine- esis an (W2) s ains o Plasmodium alcipa um.
The aqueous ex ac o he plan was also epo ed by he same au ho s o exhibi hypoglycemic
p ope ies in alloxanized mice. A. plu ise a is locally known in Kenya as Muu i (Kikuyu), Wu i (Kamba),
Ol-oiyabase (Maasai), and Shilambila (Luhya). Many communi ies in Kenya, as well as some in he es
o Eas e n and Sou he n A ica, use he plan e hnomedically o ea wounds [7].
Aspilia mossambicensis (Oli .) Wild is a sh ub na i e o cen al and Eas e n opical A ica.
The plan
is ound in he Democ a ic Republic o Congo, E hiopia, Kenya, Malawi, Mozambique,
Tanzania, Uganda, Zambia, and Zimbabwe [
9
]. In Eas e n A ica, he plan is well known o
he ea men o cys i is, gono hea, abdominal pain, in es inal wo ms, and skin in ec ions [
9
–
12
].
The hiophene de i a i es, hia ub ines A and B, ha e p e iously been isola ed om Aspilia
mossambicensis [
9
]. The oo s o his plan exhibi ed an ibac e ial ac i i y, which was sugges ed o
explain i s use by wild chimpanzees [
9
,
11
]. He ein, we epo he phy ochemical in es iga ion and he
cy o oxici y s udy o he cons i uen s o Aspilia plu ise a Schwein and Aspilia mossambicensis (Oli .)
Wild (As e aceae).
2. Resul s and Discussion
Compound
1
, [
α]20
D−
88
◦
, was isola ed as colo less c ys als (m.p. 184–186
◦
C) om he
CH
2
Cl
2
/MeOH (1:1) ex ac o he oo s o Aspilia plu ise a. HRMS (Figu e S7, Supplemen a y Ma e ial)
showed a [M–H]
−
ion peak a m/z = 329.2191, which is in ag eemen wi h he molecula o mula
C
21
H
30
O
3
. The NMR spec a (Table 1) indica ed ha his compound is a kau ene di e penoid (Figu e 1).
The
1
H–NMR spec um u he e ealed he p esence o h ee ole inic p o ons, namely H-11 (
δH
5.30),
H-17a (
δH
4.84) and H-17b (
δH
4.94), sugges ing wo double bonds. The
13
C–NMR chemical shi s o
C-16 (δC152.9) and C-17 (δC108.1) a e ypical o a e minal double bond in an en -kau ene skele on.
Molecules 2018, 23, x 2 o 13
The genus Aspilia exhibi s biological ac i i ies, including an ibac e ial and an i ungal e ec s, mainly
a ibu ed o he p esence o kau ane- ype di e penoids [3,5] and sesqui e pene lac ones [5,6].
Aspilia plu ise a Schwein has been used in adi ional medicine o ea lace a ions, b uises and
bu ns, and i is epu ed o aid in he healing o cu aneous lesions [7]. The plan is ound in Kenya and
is commonly known as ‘Dwa Aspilia’ [7]. The p esence o di e penoids om ae ial pa s o A.
plu ise a has been epo ed p e iously [8], and ou o hese di e penoids exhibi ed mode a e ac i i y
agains chlo oquine-sensi i e (D6) and chlo oquine- esis an (W2) s ains o Plasmodium alcipa um.
The aqueous ex ac o he plan was also epo ed by he same au ho s o exhibi hypoglycemic
p ope ies in alloxanized mice. A. plu ise a is locally known in Kenya as Muu i (Kikuyu), Wu i
(Kamba), Ol-oiyabase (Maasai), and Shilambila (Luhya). Many communi ies in Kenya, as well as some
in he es o Eas e n and Sou he n A ica, use he plan e hnomedically o ea wounds [7].
Aspilia mossambicensis (Oli .) Wild is a sh ub na i e o cen al and Eas e n opical A ica. The
plan is ound in he Democ a ic Republic o Congo, E hiopia, Kenya, Malawi, Mozambique,
Tanzania, Uganda, Zambia, and Zimbabwe [9]. In Eas e n A ica, he plan is well known o he
ea men o cys i is, gono hea, abdominal pain, in es inal wo ms, and skin in ec ions [9–12]. The
hiophene de i a i es, hia ub ines A and B, ha e p e iously been isola ed om Aspilia
mossambicensis [9]. The oo s o his plan exhibi ed an ibac e ial ac i i y, which was sugges ed o
explain i s use by wild chimpanzees [9,11]. He ein, we epo he phy ochemical in es iga ion and
he cy o oxici y s udy o he cons i uen s o Aspilia plu ise a Schwein and Aspilia mossambicensis
(Oli .) Wild (As e aceae).
2. Resul s and Discussion
Figu e 1. S uc u es o compounds 1–8.
Compound 1, [α]20
D−88°, was isola ed as colo less c ys als (m.p. 184–186 °C) om he
CH2Cl2/MeOH (1:1) ex ac o he oo s o Aspilia plu ise a. HRMS (Figu e S7, Supplemen a y
Ma e ial) showed a [M–H]− ion peak a m/z = 329.2191, which is in ag eemen wi h he molecula
o mula C21H30O3. The NMR spec a (Table 1) indica ed ha his compound is a kau ene di e penoid
(Figu e 1). The 1H–NMR spec um u he e ealed he p esence o h ee ole inic p o ons, namely H-
H
H
3
CO H
1
1
35
7
9
12 13
15
17
20
OHO
H
2
H
OO
OH
3
OOH
OH
OO
H
OCH
3
O
4
OHO
OO
H
H
O
5
H
OHO
6
6a
OHO
H
6b
OO
H
OHO
7
OHO
H
8
OH
H
H
H
OHO
H
OCH
3
OH
O
18
19
OHO
OO
H
H
O
OHO
OO
H
H
O
5a 5b 5'
4'
1'
1'
4'
5'
1'
4'
5'
Figu e 1. S uc u es o compounds 1–8.
Molecules 2018,23, 3199 3 o 13
Table 1. The 1H (800 MHz) and 13C-NMR (200 MHz) da a o compound 1acqui ed in CDCl3.
Posi ion δCLi . [13] * δCδH, mul . (Jin Hz) HMBC (2J,3J)
138.17 40.6 1.14 ddd (13.5, 9.5, 4.2) C-2, C-3, C-10, C-20
1.90 ddd (13.5, 3.5, 1.4) C-2, C-3, C-10, C-20
218.35 20.0 1.43 dddd (14.2, 9.5, 3.9, 3.5) C-1, C-3, C-4, C-5, C-10
1.79 ddddd (14.2, 11.1, 4.2, 3.5, 1.4) C-1, C-4, C-5
329.03 38.1 0.93 ddd (13.4, 11.1, 3.9) C-1, C-2, C-4, C-18, C-19
2.08 ddd (13.4, 3.5, 3.5) C-1, C-4, C-5, C-7
4 43.43 44.6
5 43.81 46.1 1.56 dd (11.1, 8.5) C-4, C-7, C-9, C-10, C-18, C-19, C-20
6 20.07 18.3 1.82 dddd (14.2, 10.0, 8.5, 2.5) C-3, C-4, C-5, C-7, C-10
2.43 dddd (14.2, 11.1, 9.5, 3.5) C-4, C-5, C-8
7 40.60 28.9 1.42 ddd (13.8, 3.5, 2.5) C-5, C-6, C-8, C-9, C-15
1.95 ddd (13.8, 10.0, 9.5) C-6, C-8, C-9, C-14, C-15
8 44.66 43.4
9 160.28 160.2
10 38.94 38.9
11 115.42 115.3 5.30 dd (4.3, 1.4) C-8, C-9, C-10, C-12, C-13, C-15, C-20
12 81.79 81.7 3.38 dd (4.3, 2.9) C-9, C-11, C-13, C-16, C-20, OCH3-12
13 46.17 43.7 2.89 dd (2.9, 1.4) C-10, C-11, C-12, C-15, C-16
14 40.60 40.5 1.31 dd (10.8, 4.3) C-7, C-8, C-9, C-12, C-13, C-15
1.58 dd (10.8, 2.5) C-9, C-12, C-13, C-15, C-16
15 47.17 47.1 2.08 dd (15.4, 4.3) C-7, C-8, C-9, C-16, C-17
2.35 dd (15.4, 2.5) C-7, C-9, C-13, C-14, C-16, C-17
16 153.00 152.9
17 108.12 108.1 4.84 dd (3.0, 1.6) C-12, C-13, C-15, C-16
4.94 dd (3.0, 1.6) C-12, C-13, C-15, C-16
18 28.22 28.2 1.17 sC-3, C-4, C-5, C-8, C-19
19 182.98 183.2
20 23.41 23.4 1.01 sC-1, C-5, C-9, C-10
OCH3-12 56.53 56.5 3.34 sC-12
* CDCl3a 100 MHz [13].
The second double bond was placed be ween C-9 (
δC
160.2) and C-11 (
δH
5.30;
δC
115.3)
by compa ison o he NMR da a wi h ha ound in he li e a u e [
13
–
15
]. Signals indica ing
he p esence o a me hoxy (
δH
3.34,
δC
56.5) and a ca boxylic acid (
δC
183.2) subs i uen we e
obse ed.
The He e onuclea
Mul iple Bond Co ela ions (HMBCs) o CH
3
-18 (
δH
1.17), H-3 (
δH0.93
),
and H-5 (
δH
1.56) wi h he ca boxy esonance C-19 (
δC
183.2) sugges ed he loca ion o he ca boxy
g oup (C-19) a C-4. Ou o he h ee me hyl g oups expec ed in kau ene di e penoid, only wo,
i.e., CH
3
-18 (
δH
1.17,
δC
28.2) and CH
3
-20 (
δH
1.01,
δC
23.4), we e obse ed. This co obo a ed
he sugges ion o he hi d me hyl g oup being oxidized o a ca boxylic acid (C-19,
δC
183.2).
The me hoxy
g oup OCH
3
-12 (
δH3.34
) showed HMBC co ela ion wi h C-12 (
δC
81.7), whe eas
H-12 (
δH
3.38) showed HMBC co ela ion wi h C-9 (
δC
160.2), C-11 (
δC
115.3), C-13 (
δC
43.7), C-16
(
δC
152.9), and OCH
3
-12 (
δC
56.5). Fu he mo e, CH
3
-20 (
δH
1.01) showed HMBC co ela ion wi h
C-1 (
δC40.6
), C-5 (
δC
46.1), he ole inic ca bon C-9 (
δC
160.2), and C-10 (
δC
38.9). This con i med
ha he second double bond in he molecule is loca ed a C-9. Mo eo e , he HMBC co ela ion o
CH
2
-14 (
δH
1.31, 1.58) wi h a deshielded ca bon C-12 (
δC
81.7) is in ag eemen wi h OCH
3
being
connec ed o C-12. The abo e indings con i med he iden i y o compound
1
as a C-12-me hoxy
subs i u ed en -kau -9(11),16-dienoic acid de i a i e. The ela i e con igu a ion a C-12 was deduced
om he Nuclea O e hause E ec (NOE) o OCH
3
-12 (
δH
3.34) o H-13 (
δH
2.89) (Figu e 2),
indica ing hem o be syn-o ien ed, and hence OCH
3
-12 o be
α
-o ien ed. I should be no ed ha
H-12
(δH3.38
) also showed a weak NOE o H-13 (
δH
2.89), which is expec ed in a s ained ing
sys em. The p oposed con igu a ion a C-12 is u he co obo a ed by he NOE o H-12 (
δH3.38
)
wi h H-14b (
δH
1.58). The NOE o H-12 (
δH
3.38 ppm) wi h H-17b (
δH
4.94 ppm) suppo ed
H-12 o be
β
-o ien ed, and hence OCH
3
-12 o be
α
-o ien ed. Based on he abo e spec oscopic
e idence, compound
1
, 12
α
-me hoxy-en -kau -9(11),16-dien-19-oic acid, (Figu e 1) was iden i ied
as (4R,4aS,6aS,9R,10S,11bR)-10-me hoxy-4,11b-dime hyl-8-me hylene-1,2,3,4,4a,5,6,7,8,9,10,11b-
dodecahyd o-6a,9-me hanocyclohep a[a]naph halene-4-ca boxylic acid. This compound has
p e iously been epo ed bo h as syn he ic de i a i e [
13
] and as a na u al p oduc [
14
,
15
]. Howe e ,
Molecules 2018,23, 3199 4 o 13
ou NMR da a assignmen di e s om ha epo ed in he li e a u e [
13
] o i s C-3 and C-7.
The accu acy
o he co ec ed assignmen , gi en in Table 1, is co obo a ed by he HMBC co ela ions
o H-3 and H-7 (Table 1), by he HSQC c osspeaks o CH
2
-3 (
δH
0.93/2.08) o C-3 (
δC
38.1) along wi h
he TOCSY(To al Co ela ion Spec oscopy) co ela ions o CH
2
-3 o CH
2
-1 (
δH
1.14/1.90) and CH
2
-2
(
δH
1.43/1.79), and by he HSQC (He e onuclea Single Quan um Co ela ion) c osspeaks o CH
2
-7
(
δH
1.42/1.95) o C-7 (
δC
28.9) along wi h he TOCSY co ela ions o CH
2
-7 o CH
2
-6 (
δH1.82/2.43
)
and H-5 (
δH
1.56) (Figu es S4–S6, Supplemen a y Ma e ial). Besides he compound ha ing been
epo ed ea lie , i is unlikely o be an ex ac ion a i ac as he ex ac ion (wi h CH
2
Cl
2
/MeOH, 1:1)
has been pe o med a low empe a u e a neu al pH ha does no p omo e o ma ion o me hyl
e he s. The compound has been de ec ed in he c ude ex ac indica ing ha he compound is a na u al
p oduc and has no o med du ing he ch oma og aphic isola ion.
Molecules 2018, 23, x 4 o 13
expec ed in a s ained ing sys em. The p oposed con igu a ion a C-12 is u he co obo a ed by he
NOE o H-12 (δH 3.38) wi h H-14b (δH 1.58). The NOE o H-12 (δH 3.38 ppm) wi h H-17b (δH 4.94 ppm)
suppo ed H-12 o be β-o ien ed, and hence OCH3-12 o be α-o ien ed. Based on he abo e
spec oscopic e idence, compound 1, 12α-me hoxy-en -kau -9(11),16-dien-19-oic acid, (Figu e 1) was
iden i ied as (4R,4aS,6aS,9R,10S,11bR)-10-me hoxy-4,11b-dime hyl-8-me hylene-
1,2,3,4,4a,5,6,7,8,9,10,11b-dodecahyd o-6a,9-me hanocyclohep a[a]naph halene-4-ca boxylic acid.
This compound has p e iously been epo ed bo h as syn he ic de i a i e [13] and as a na u al
p oduc [14,15]. Howe e , ou NMR da a assignmen di e s om ha epo ed in he li e a u e [13]
o i s C-3 and C-7. The accu acy o he co ec ed assignmen , gi en in Table 1, is co obo a ed by he
HMBC co ela ions o H-3 and H-7 (Table 1), by he HSQC c osspeaks o CH2-3 (δH 0.93/2.08) o C-3
(δC 38.1) along wi h he TOCSY(To al Co ela ion Spec oscopy) co ela ions o CH2-3 o CH2-1 (δH
1.14/1.90) and CH2-2 (δH 1.43/1.79), and by he HSQC (He e onuclea Single Quan um Co ela ion)
c osspeaks o CH2-7 (δH 1.42/1.95) o C-7 (δC 28.9) along wi h he TOCSY co ela ions o CH2-7 o CH2-
6 (δH 1.82/2.43) and H-5 (δH 1.56) (Figu es S4–6, Supplemen a y Ma e ial). Besides he compound
ha ing been epo ed ea lie , i is unlikely o be an ex ac ion a i ac as he ex ac ion (wi h
CH2Cl2/MeOH, 1:1) has been pe o med a low empe a u e a neu al pH ha does no p omo e
o ma ion o me hyl e he s. The compound has been de ec ed in he c ude ex ac indica ing ha he
compound is a na u al p oduc and has no o med du ing he ch oma og aphic isola ion.
Figu e 2. Some o he key NOE co ela ions obse ed o compound 1. The NOESY (Nuclea
O e hause E ec Spec oscopy) spec um (800 MHz, CDCl3, 25 °C, 700 ms mixing ime) is shown in
he Supplemen a y Ma e ials.
Addi ional compounds (Figu es 1 and 3) isola ed om he oo s o Aspilia plu ise a include (16R)-
hyd oxy-en -kau an-19-oic acid (2) [16,17], 9β-hyd oxy-15α-angeloyloxy-en -kau -16-en-19-oic acid
(3) [18], me hyl-9β-hyd oxy-15α-angeloyloxy-en -kau -16-en-19-oa e (4) [19], 15α-angeloyloxy-
16β,17-epoxy-en -kau an-19-oic acid (5) [20], en -kau -9(11),16-dien-19-oic acid (6) [21], 15α-
angeloyloxy-en -kau -16-en-19-oic acid (7) [22], en -kau -9(11),16-dien-12-one (9) [23] and me hyl-en -
kau -16-en-19-oa e (10) [24]. The ae ial pa o Aspilia plu ise a con ained en -kau -16-en-19-oic acid
(11) [21,25], en -kau -16-en-19-ol (12) [26], lanos e ol (13) [27], s igmas a-5,22(E)-dien-3β-ol (14) [28],
3β-hyd oxy-olean-12-en-29-oic acid (15) [29], and ca issone (16) [30].
Simila phy ochemical in es iga ion o he oo s o Aspilia mossambicensis esul ed in he isola ion
o me hyl-15α-angeloyloxy-en -kau -16-en-19-oa e (17) [24,31], 12-oxo-en -kau -9(11),16-dien-19-oic
acid (18) [32], (16S)-en -kau an-19-oic acid (8) [33], oleanolic acid (19) [34] (Figu e 3), and compounds
3–5. The ae ial pa o A. mossambicensis a o ded compound 6 [21,26], 3β-ace yloxy-olean-12-ene (20)
[35,36] en -kau -9(11),16-diene (21) [37], 15a-hyd oxy-kau -9(11),16-diene (22) [38], and me hyl
cinnama e (23) [39].
The c ys al s uc u es o compounds 3–8 (Figu e 4) a e also epo ed he e, whe e he iden i ies
o he compounds we e con i med, and he absolu e con igu a ions es ablished. In he c ys al s a e,
compound 3 exhibi ed a con inuous ne wo k, in ol ing in e molecula O(9)-H···O(19a) and O(19b)-
H···O(21) hyd ogen bonds. Simila O(9)-H···O(19a) hyd ogen bonding mo i was ound in compound
4, which also showed s a ic diso de exhibi ing wo di e en spa ial o ien a ions o (Z)-2-me hylbu -
Figu e 2.
Some o he key NOE co ela ions obse ed o compound
1
. The NOESY (Nuclea
O e hause E ec Spec oscopy) spec um (800 MHz, CDCl3, 25 ◦C, 700 ms mixing ime) is shown in
he Supplemen a y Ma e ials.
Addi ional compounds (Figu es 1and 3) isola ed om he oo s o Aspilia plu ise a include
(16R)-hyd oxy-en -kau an-19-oic acid (
2
) [
16
,
17
], 9
β
-hyd oxy-15
α
-angeloyloxy-en -kau -16-en-19-oic
acid (
3
) [
18
], me hyl-9
β
-hyd oxy-15
α
-angeloyloxy-en -kau -16-en-19-oa e (
4
) [
19
],
15
α
-angeloyloxy-16
β
,17-epoxy-en -kau an-19-oic acid (
5
) [
20
], en -kau -9(11),16-dien-19-oic
acid (
6
) [
21
], 15
α
-angeloyloxy-en -kau -16-en-19-oic acid (
7
) [
22
], en -kau -9(11),16-dien-12-one
(
9
) [
23
] and me hyl-en -kau -16-en-19-oa e (
10
) [
24
]. The ae ial pa o Aspilia plu ise a con ained
en -kau -16-en-19-oic acid (
11
) [
21
,
25
], en -kau -16-en-19-ol (
12
) [
26
], lanos e ol (
13
) [
27
],
s igmas a-5,22(E)-dien-3
β
-ol (
14
) [
28
], 3
β
-hyd oxy-olean-12-en-29-oic acid (
15
) [
29
], and ca issone
(16) [30].
Simila phy ochemical in es iga ion o he oo s o Aspilia mossambicensis esul ed in he isola ion
o me hyl-15
α
-angeloyloxy-en -kau -16-en-19-oa e (
17
) [
24
,
31
], 12-oxo-en -kau -9(11),16-dien-19-oic
acid (
18
) [
32
], (16S)-en -kau an-19-oic acid (
8
) [
33
], oleanolic acid (
19
) [
34
] (Figu e 3), and compounds
3
–
5
. The ae ial pa o A. mossambicensis a o ded compound
6
[
21
,
26
], 3
β
-ace yloxy-olean-12-ene
(
20
) [
35
,
36
]en -kau -9(11),16-diene (21) [
37
], 15a-hyd oxy-kau -9(11),16-diene (
22
) [
38
], and me hyl
cinnama e (23) [39].
The c ys al s uc u es o compounds
3
–
8
(Figu e 4) a e also epo ed he e, whe e he iden i ies
o he compounds we e con i med, and he absolu e con igu a ions es ablished. In he c ys al
s a e, compound
3
exhibi ed a con inuous ne wo k, in ol ing in e molecula O(9)-H
···
O(19a) and
O(19b)-H
···
O(21) hyd ogen bonds. Simila O(9)-H
···
O(19a) hyd ogen bonding mo i was ound
in compound
4
, which also showed s a ic diso de exhibi ing wo di e en spa ial o ien a ions o
(Z)-2-me hylbu -2-enoyl g oup in an app oxima ely 1:1 a io. Compounds
5
–
8
showed common double
hyd ogen bonding mo i s o ca boxylic acids leading o he o ma ion o hyd ogen-bonded pai s.

Molecules 2018,23, 3199 5 o 13
Molecules 2018, 23, x 5 o 13
2-enoyl g oup in an app oxima ely 1:1 a io. Compounds 5–8 showed common double hyd ogen
bonding mo i s o ca boxylic acids leading o he o ma ion o hyd ogen-bonded pai s.
Figu e 3. S uc u es o compounds 9–23.
C ys al s uc u e o compound 6 was de e mined a 120 K, which co esponds o he p e iously
epo ed s uc u e a oom empe a u e [40]. C ys al s uc u e analysis o a syn he ic de i a i e o
compound 6 led o he iden i ica ion o en -kau -9(11)-en-19-oic acid (6b). In he s uc u e 6b, he e is
a sligh (~10%) diso de in he main ing sys em, whe e ca bons C7–C17 ha e di e en posi ions
gi ing a shade o di e en con o ma ions o he co esponding alipha ic ings. The da a quali y o
compound 7 is sligh ly de icien and he absolu e s uc u e o i could no be jus i ied on he basis o
da a. The X- ay di ac ion da a pa ame e s, he mal ellipsoid diag ams, and hyd ogen bonding
geome ies a e p esen ed in he Supplemen a y In o ma ion sec ion o his a icle.
In mos o he en -kau ane- ype di e penoids (excep o compounds 9, 21, and 22) isola ed om
he wo Aspilia species, A. plu ise a and A. mossambicensis, he α-me hyl g oup a C-4 (C-19) is oxidized
in o ca boxylic acid o me hyl es e g oups, which could be a cha ac e is ic ea u e o he genus Aspilia
[3,8]. The e a e also examples whe e oxida ion has occu ed a C-12 (compounds 1, 9 and 18), C-16
(compound 2), C-9 (compounds 3 and 4), and C-15 (compounds 4, 5, 6, and 18). In ag eemen wi h
he li e a u e [36], oxida ion has no been obse ed a o he ca bon a oms in en -kau ane- ype
di e penoids o Aspilia species.
Some en -kau ane- ype di e penoids, including 16,17-epoxy-15β- igloyloxy-en -kau an-18-oic
acid (5a) and 16,17-epoxy-15β-senecioyloxy-en -kau an-18-oic acid (5b), we e epo ed ea lie om
Aspilia plu ise a [8]. These compounds we e epo ed o ha e he 15β- igloyloxy and 15β-senecioyloxy
g oups, espec i ely, occupying he less a o able o ien a ion [8]; howe e , he au ho s ha e no
p o ided e idence o hese p oposals. Ou single c ys al X- ay analyses ha e shown ha he C-15
subs i uen o compounds 3, 4, 5, and 7 is an angeloyloxy g oup occupying he mo e a ou able-15α-
posi ion (Figu e 4). In ac , he p oposed s e eochemical assignmen o en -kau ane- ype di e penoids
epo ed om his genus, pa icula ly in highly unc ionalized compounds, lacks e idence. We ha e
Figu e 3. S uc u es o compounds 9–23.
C ys al s uc u e o compound
6
was de e mined a 120 K, which co esponds o he p e iously
epo ed s uc u e a oom empe a u e [
40
]. C ys al s uc u e analysis o a syn he ic de i a i e o
compound
6
led o he iden i ica ion o en -kau -9(11)-en-19-oic acid (
6b
). In he s uc u e
6b
, he e
is a sligh (~10%) diso de in he main ing sys em, whe e ca bons C7–C17 ha e di e en posi ions
gi ing a shade o di e en con o ma ions o he co esponding alipha ic ings. The da a quali y o
compound
7
is sligh ly de icien and he absolu e s uc u e o i could no be jus i ied on he basis
o da a. The X- ay di ac ion da a pa ame e s, he mal ellipsoid diag ams, and hyd ogen bonding
geome ies a e p esen ed in he Supplemen a y In o ma ion sec ion o his a icle.
In mos o he en -kau ane- ype di e penoids (excep o compounds
9
,
21,
and
22
) isola ed om
he wo Aspilia species, A. plu ise a and A.mossambicensis, he
α
-me hyl g oup a C-4 (C-19) is oxidized
in o ca boxylic acid o me hyl es e g oups, which could be a cha ac e is ic ea u e o he genus
Aspilia [
3
,
8
]. The e a e also examples whe e oxida ion has occu ed a C-12 (compounds
1, 9
and
18
),
C-16 (compound
2
), C-9 (compounds
3
and
4
), and C-15 (compounds
4
,
5
,
6,
and
18
). In ag eemen
wi h he li e a u e [
36
], oxida ion has no been obse ed a o he ca bon a oms in en -kau ane- ype
di e penoids o Aspilia species.
Some en -kau ane- ype di e penoids, including 16,17-epoxy-15
β
- igloyloxy-en -kau an-18-oic acid
(
5a
) and 16,17-epoxy-15
β
-senecioyloxy-en -kau an-18-oic acid (
5b
), we e epo ed ea lie om Aspilia
plu ise a [
8
]. These compounds we e epo ed o ha e he 15
β
- igloyloxy and 15
β
-senecioyloxy g oups,
espec i ely, occupying he less a o able o ien a ion [
8
]; howe e , he au ho s ha e no p o ided
e idence o hese p oposals. Ou single c ys al X- ay analyses ha e shown ha he C-15 subs i uen
o compounds
3
,
4
,
5,
and
7
is an angeloyloxy g oup occupying he mo e a ou able-15
α
-posi ion
(Figu e 4). In ac , he p oposed s e eochemical assignmen o en -kau ane- ype di e penoids epo ed
om his genus, pa icula ly in highly unc ionalized compounds, lacks e idence. We ha e illed his
knowledge gap by de e mining he absolu e con igu a ion o se en en -kau ane- ype di e penoids,
as shown in Figu e 4, using single c ys al X- ay analyses.
Molecules 2018,23, 3199 6 o 13
Molecules 2018, 23, x 6 o 13
illed his knowledge gap by de e mining he absolu e con igu a ion o se en en -kau ane- ype
di e penoids, as shown in Figu e 4, using single c ys al X- ay analyses.
Figu e 4. C ys al s uc u e ep esen a ions o compounds 3–8. The s uc u es we e deposi ed wi h he
ollowing CCDC (Camb idge C ys allog aphic Da a Cen e) codes: 3 (1868318), 4 (1868319), 5
(1868321), 6 (1868320), 6b (1868324), 7 (1868323), and 8 (1868322).
The 13C-NMR da a o compound 5 (Table 2) is in close ag eemen o ha p e iously epo ed in
he li e a u e [20], excep o he chemical shi assignmen o C-2 and C-12 (Table 2). Rela ed
s uc u es, 5a and 5b, ha e been p oposed o wo compounds ea lie epo ed [8] om Aspilia
plu ise a. The 13C-NMR assignmen (Table 2) o hese compounds di e s om ou assignmen , which
is based on 2D NMR co ela ions, despi e he common 16,17-epoxy-15-oxy-en -kau an-18-oic acid
skele on (Figu e 1). The 13C-NMR chemical shi s o C-4’ (δC 27.4) and C-5’ (δC 20.8) in compound 5a
do no suppo a igloyloxy g oup a C-15, as p oposed in Re e ence [8]; me hyl ca bon a oms in such
g oup a e expec ed o esona e a ~14 ppm ( o C-4’) and a ~11 ppm ( o C-5’), based on chemical
shi p edic ion [41] and p e ious li e a u e [14]. The NMR spec a o compound 5b ha a e gi en in
Figu e 4.
C ys al s uc u e ep esen a ions o compounds
3
–
8
. The s uc u es we e deposi ed wi h he
ollowing CCDC (Camb idge C ys allog aphic Da a Cen e) codes:
3
(1868318),
4
(1868319),
5
(1868321),
6(1868320), 6b (1868324), 7(1868323), and 8(1868322).
The
13
C-NMR da a o compound
5
(Table 2) is in close ag eemen o ha p e iously epo ed
in he li e a u e [
20
], excep o he chemical shi assignmen o C-2 and C-12 (Table 2). Rela ed
s uc u es,
5a
and
5b
, ha e been p oposed o wo compounds ea lie epo ed [
8
] om Aspilia plu ise a.
The
13
C-NMR assignmen (Table 2) o hese compounds di e s om ou assignmen , which is based
on 2D NMR co ela ions, despi e he common 16,17-epoxy-15-oxy-en -kau an-18-oic acid skele on
(Figu e 1). The
13
C-NMR chemical shi s o C-4’ (
δC
27.4) and C-5’ (
δC
20.8) in compound
5a
do
no suppo a igloyloxy g oup a C-15, as p oposed in Re e ence [
8
]; me hyl ca bon a oms in such
g oup a e expec ed o esona e a ~14 ppm ( o C-4’) and a ~11 ppm ( o C-5’), based on chemical
shi p edic ion [
41
] and p e ious li e a u e [
14
]. The NMR spec a o compound
5b
ha a e gi en in
he suppo ing in o ma ion o [
8
] a e o low quali y and do no allow con i ma ion o he p oposed
Molecules 2018,23, 3199 7 o 13
assignmen . I should be no ed ha he numbe ing used in his pape does no ollow he li e a u e
con en ion [
3
]. Hence, he ca boxylic g oup o
5b
and o i s s uc u al analogues should no be
assigned as C-18, bu a he as C-19, ollowing e e ence [
3
]. O e all, se e al de ails epo ed [
8
]
o hese compounds appea deba able, and consequen ly so a e he p oposed s uc u es. To a oid
such unce ain ies, he NMR assignmen s o all compounds discussed in his pape a e p esen ed in
Tables S1 and S2 in he Supplemen a y Ma e ial.
Table 2.
The li e a u e epo ed NMR da a o
5
,
5a,
and
5b
and he
1
H (800 MHz) and
13
C-NMR
(200 MHz) da a o compound 5acqui ed in CDCl3.
Posi ion 5 [20] 5a [8] 5b [8] 5 5
δCδCδCδCδH, mul . (Jin Hz)
141.2 40.6 40.6 40.6 0.80 ddd (7.2, 7.1, 1.3)
1.86* dd (2.9, 1.4)
228.9 19.8 19.0 19.7 1.55 ddd (7.3, 3.6, 2.4)
1.75 dd (3.7, 3.6)
337.7 36.7 36.4 37.6 0.96 ddd (13.7, 13.6, 4.3)
2.11 dd (13.7, 3.1)
4 43.6 46.9 47.8 43.5
5 56.7 20.3 56.6 56.5 1.16 dd (9.1, 7.1)
6 19.0 41.2 20.3 20.8 1.76 ddd (5.7, 3.4, 2.1)
1.86* ddd (3.4, 3.4, 2.7)
7 35.4 47.8 41.2 35.3 1.25 ddd (14.4, 13.9, 4.4)
1.79 ddd (13.8, 13.2, 4.3)
8 47.9 52.9 43.6 47.8
9 52.9 43.6 53.0 52.8 1.28 dd (13.8, 3.8)
10 39.8 56.6 39.8 39.7
11 19.8 20.8 19.8 18.9 1.40 ddd (13.8, 3.4, 3.4, 3.1)
1.81 dd (13.8, 4.3)
12 20.8 28.9 28.9 28.8 1.50 ddd (13.5, 7.8, 7.2)
13 41.2 36.4 35.1 41.1 1.82 dd (13.8, 4.4)
14 36.5 37.7 37.7 36.4 1.68 dd (14.5, 3.3)
1.97 dd (13.1, 3.4)
15 81.9 81.2 81.2 81.9 4.73 b s
16 66.3 66.4 66.4 66.3
17 49.6 49.6 49.6 49.6 2.78 dd (5.6, 1.3)
3.09 dd (5.8, 1.3)
18 28.8 28.9 28.9 28.7 1.28 s
19 182.3 182.6 182.6 182.7
20 15.7 15.8 16.0 15.9 1.03 s
1’ 167.9 166.5 166.5 167.8
2’ 128.1 129.0 115.9 128.0
3’ 137.3 137.1 156.8 137.3 5.96 q(7.1)
4’ 15.9 27.4 20.8 15.7 1.96 d(1.9)
5’ 20.6 20.8 27.4 20.6 s
Mos o he compounds isola ed in his s udy we e assayed agains wo no mal (BEAS-2B and
LO
2
) and wo cance (A549 and Hep-G2, Table 3) cell lines. O he i een compounds es ed,
1
,
3
,
5
,
9,
and
18
showed cy o oxici y owa ds some o he cell lines. Compound
1
showed selec i e
ac i i y agains he human hepa oma (HepG2) cance cell line wi hou signi ican oxici y o o he
cell lines (IC
50
abo e
100 µM
). Compound
3
was mode a ely cy o oxic agains he Hep-G2 cance
cell line (
IC50 = 24.7 ±2.8
), bu also showed weak cy o oxici y owa ds he no mal cell lines LO
2
(IC
50
= 57.2
±
1.2) and BEAS-2B (IC
50
= 89.9
±
2.0
µ
M), indica ing low selec i i y. Compound
5
(
IC50 = 30.7 ±1.7 µM
) was he mos ac i e agains he A549 cell line, ollowed by compound
18
(
IC50 = 80.5 ±1.8 µM
).
The o he
compounds es ed, i.e.,
2
,
4
,
6
,
7, 10
,
11
,
14,
o
17
, did no exhibi
signi ican cy o oxici y (IC
50
abo e 100
µ
M).
The lack
o cy o oxici y agains he wo no mal cell lines
o hese di e penoids is aluable in o ma ion, conside ing ha some kau ane- ype di e penoids om
Aspilia species ha e showed an ibac e ial and an i ungal e ec s [5].
Molecules 2018,23, 3199 8 o 13
Table 3.
Cy o oxici y (IC
50
,
µ
M) o compounds isola ed om Aspilia species agains a ious cell lines.
Compound No mal Cell Lines Cance Cell Lines
BEAS-2B LO2A549 Hep-G2
1>100 >100 >100 27.3 ±1.9
2>100 >100 >100 >100
389.9 ±2.0 57.2 ±1.2 >100 24.7 ±2.8
4>100 >100 >100 >100
5>100 >100 30.7 ±1.7 >100
6>100 >100 >100 >100
6a >100 >100 >100 >100
6b >100 >100 >100 >100
7>100 >100 >100 >100
9>100 75.3 ±2.8 >100 >100
10 >100 >100 >100 >100
11 >100 >100 >100 >100
14 >100 >100 >100 >100
17 >100 >100 >100 >100
18 38.6 ±2.5 30.0 ±1.7 80.5 ±1.8 81.3 ±0.3
Pacli axel <0.1 <0.1 0.0033 0.19
3. Ma e ials and Me hods
3.1. Gene al Expe imen al P ocedu es
NMR spec a we e acqui ed on a B uke A ance II 600 MHz, a B uke A ance III HD 800 MHz
(B uke BioSpin AG, Fällanden, Swi ze land) o a Va ian Uni y 500 MHz (Va ian Inc, Palo Al o, CA,
USA) NMR spec ome e , using he esidual sol en peaks as a e e ence. The spec a we e p ocessed
using he so wa e Mes ReNo a ( e sion 10.0, Mes elab Resea ch S.L., San iago de Compos ela, Spain)
Coupling cons an s (J) a e gi en in Hz. EI-MS and LC-MS we e ca ied ou using 70 eV ioniza ion
elec on ol age on a Mic omass GC-TOF spec ome e (Mic omass, Wy henshawe, Wa e s Inc., UK).
TLC (Thin Laye Ch oma og aphy) was ca ied ou on Me ck p e-coa ed silica gel 60 F
254
pla es (Me ck,
Da ms ad , Ge many). P epa a i e TLC was pe o med on 20
×
20 cm glass pla es, p e-coa ed wi h
silica gel 60F
254
wi h hicknesses o 0.25 o 1 mm. Column ch oma og aphy was un on silica gel 60 Å
(70–230 mesh). Gel il a ion was pe o med on Sephadex LH-20(Me ck, Da ms ad , Ge many).
3.2. X- ay Di ac ion Analyses
The single c ys al X- ay di ac ion da a we e collec ed using Agilen Supe -No a (Agilen
Technologies, W ocław, Poland) dual wa eleng h di ac ome e wi h a mic o- ocus X- ay sou ce and
mul ilaye op ics monoch oma ized Cu-K
α
(
λ
= 1.54184 Å) adia ion. P og am C ysAlisP o [
42
] was
used o he da a collec ion and educ ion. The in ensi ies we e co ec ed o abso p ion using analy ical
ace index abso p ion co ec ion me hod. The s uc u es we e sol ed wi h in insic phasing me hod
(SHELXT [
43
]) and e ined by ull-ma ix leas squa es on F
2
wi h SHELXL-2018/3 [
44
]. Aniso opic
displacemen pa ame e s we e assigned o non-H a oms. All C-H hyd ogen a oms we e e ined using
iding models. Hyd oxy hyd ogens we e ound om elec on densi y maps and es ained o he
p ope dis ance om oxygen a om (0.84 Å). All hyd ogen a oms we e e ined wi h U
eq
(H) o 1.5
×
U
eq
(C,O) o hyd oxy and e minal me hyl g oups o 1.2
×
U
eq
(C) o o he C-H g oups. Fu he
geome ic leas -squa es es ain s (s = 0.02) we e applied o s uc u es
4
,
6b,
and
7
o ob ain mo e
chemically easonable bond dis ances be ween diso de ed a oms. Aniso opic displacemen pa ame e s
o ew diso de ed o e minal a oms we e es ained (s = 0.01, s = 0.02) o be mo e equal in s uc u es
3
,
4
,
6b,
and
7
. CCDC 1868318-1868324 con ains he supplemen a y c ys allog aphic da a o his pape .
These da a can be ob ained ee o cha ge ia h p://www.ccdc.cam.ac.uk/con s/ e ie ing.h ml
(o om he CCDC, 12 Union Road, Camb idge CB2 1EZ, UK; Fax: +44 1223 336033; E-mail:
[email p o ec ed]).