New anti-malarial phenylpropanoid conjugated iridoids from Morinda morindoides

Article abstract of DOI:10.1016/j.bmcl.2010.01.095

A new phenylpropanoid conjugated iridoid together with four known congeners was isolated from Morinda morindoides, used for the therapy of malaria traditionally in some African countries, as anti-malarial principles through bioassay-guided separation. Furthermore, their absolute stereostructures were unambiguously established by a combination of modified Mosher's method and chemical correlation.

Full text of DOI:10.1016/j.bmcl.2010.01.095

Bioorganic & Medicinal Chemistry Letters 20 (2010) 1520–1523
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
New anti-malarial phenylpropanoid conjugated iridoids from Morinda
morindoides
Satoru Tamura a,d, Bruno Kilunga Kubata b,d, Syamsurizal a,d, Sawako Itagaki c,d, Toshihiro Horii c,d
,
Muzele Kalulu Taba c,d, Nobutoshi Murakami a,d,
*
a Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamada-oka, Suita, Osaka 565-0871, Japan
b Department of Biology and Environmental Management, University of Kinshasa, Kinshasa XI, Democratic Republic of the Congo
c Research Institute for Microbial Diseases, Osaka University, 3-1 Yamada-oka, Suita, Osaka 565-0871, Japan
d Faculty of Sciences, University of Kinshasa, Kinshasa XI, Democratic Republic of the Congo
a r t i c l e i n f o
a b s t r a c t
Article history:
A new phenylpropanoid conjugated iridoid together with four known congeners was isolated from Mor-
inda morindoides, used for the therapy of malaria traditionally in some African countries, as anti-malarial
principles through bioassay-guided separation. Furthermore, their absolute stereostructures were unam-
biguously established by a combination of modified Mosher’s method and chemical correlation.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 16 December 2009
Revised 13 January 2010
Accepted 16 January 2010
Available online 25 January 2010
Keywords:
Anti-malarial
Iridoid
Morinda morindoides
African medicinal plant
Malaria remains endemic in more than 90 countries, principally
in the tropical world. There are between 300 and 500 million in-
fected persons and more than 1 million deaths, mostly of children,
are attributable to the disease. For instance, in every 40 second a
child dies of malaria, resulting in a daily loss of more than 2000
young lives worldwide. Not only lack of vaccine but also emerging
resistance of a deadly form of malaria parasite, Plasmodium falcipa-
rum, to the commercially available anti-malarial drugs, has urged
search for new anti-malarials as a global demand.1,2 This circum-
stance prompted us to be devoted to exploring some new anti-
malarial candidates with high selectivities against the mammalian
host cells from natural medicinal plant resources.3–6
In some African countries, the leaves of Morinda morindoides
have been utilized for treatment of malaria.7 Recently, the extract
of the plant was found to show not only inhibition of proliferation
of P. falciparum in vitro but also in vivo anti-malarial potency
against mice infected by P. berghei.8,9 On the basis of this finding
as well as the traditional reputation, we undertook to reveal the
principles responsible for this bioactivity of the medicinal plant.
Herein, we describe a new anti-malarial phenylpropanoid conju-
gated iridoid together with the four congeners and elucidation of
their absolute stereostructures.
The MeOH extract of M. morindoides, which was collected at
Democratic Republic of the Congo at 2006, was suspended with
water, then the suspension was partitioned between n-hexane,
EtOAc, n-BuOH successively. The EtOAc layer with the most potent
activity was further separated by a combination of SiO2 column,
normal, and reversed phase-HPLC with monitoring anti-malarial
potency to furnish a new phenylpropanoid conjugated iridoid gly-
coside 110 along with the four congeners (2–5) as active
constituents.
The molecular formula of 1 was established as C29H32O15 by HR
FAB-MS indicative of a quasimolecular ion peak at m/z 619.1669
[MꢀH]ꢀ (calcd: 619.1663). The IR spectrum of 1 showed the
absorption bands due to a hydroxyl (3391 cmꢀ1), an
a,b-unsatu-
rated c a,
-lactone (1756 cmꢀ1), an ester carbonyl (1744 cmꢀ1), an
b-unsaturated ester (1707 cmꢀ1), and an ester conjugated olefin
(1642 cmꢀ1) functions, and an aromatic ring (1605 cmꢀ1). The 1H
NMR spectrum of 1 showed the signals ascribable to an anomeric
proton [d 4.54 (1H, d, J = 7.9 Hz, H-100)], an acetal proton [d 4.97
(1H, d, J = 3.1 Hz, H-1)], an oxymethine [d 5.35 (1H, br s, H-70)],
two methoxyl [d 3.89 (3H, s, 30-OCH3), 3.74 (3H, s, 4-COOCH3)],
and an acetyl [d 1.83 (3H, s, 600-OAc)] groups. Additionally, the pres-
* Corresponding author at present address: Kyoto Pharmaceutical University,
Japan. Tel.: +81 75 595 4634; fax: +81 75 595 4768.
E-mail addresses: murakami@phs.osaka-u.ac.jp, murakami@poppy.kyoto-phu.
ac.jp (N. Murakami).
ence of a 1,3,4-trisubstituted aromatic ring, two a-substituted a,b-
unsaturated enones, and a nonconjugated olefin was suggested by
1
the following H NMR data; d 7.01 (1H, br s, H-20), 6.79 (1H, d,
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.01.095

S. Tamura et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1520–1523
1521
J = 7.9 Hz, H-50), 6.89 (1H, br d, J = 7.9 Hz, H-60), 7.51 (1H, br s,
H-3), 7.28 (1H, br s, H-10), 6.48 (1H, dd, J = 5.5, 2.4 Hz, H-6), 5.59
(1H, dd, J = 5.5, 1.8 Hz, H-7).
On the other hand, two acetals [dc 94.0 (C-1), 100.4 (C-100)] and
three carbonyl [dc 172.3 (C-90), 168.3 (C-11), 172.8 (600-OAc)] car-
bon signals appeared in the 13C NMR spectrum of 1. Furthermore,
the 13C NMR spectrum showed the presence of one oxymethine
carbon [dc 70.0 (C-70)] in the aglycone moiety. Intense analysis of
the NMR data between 1 and 3 revealed that both data resemble
each other except for those around C-600 (Table 1). Namely, the def-
inite acylation shift11 around C-600 was observed with respect to 1.
In addition, the HMBC correlation appeared from H-600 to the car-
bonyl carbon in the acetyl moiety. Consequently, the anti-malarial
principle 1 was elucidated to be the 600-O-acetyl congener of 3. The
known compounds 2–5 were identified by direct comparison of
their spectral data with those reported in the literature.12 Although
the relative configuration of 2–5 except for C-8 and C-70 were
determined by the coupling constants of protons signals in the
1H NMR spectra, no obvious establishment for absolute configura-
tions of 2–5 were conducted.
Determination of the absolute configurations at position C-1 and
C-70 of 1–5 was achieved in the following manner. In the NOESY
spectra of 3 and 4, definite correlations between H-1 and H-10 ap-
peared. Thus, therelative configurations at C-8 of 3 and 4 were deter-
mined as shown in Figure 1. Treatment of 1 and 2 with NaOMe in
MeOH, respectively, afforded 3 and 4, this indicating that the above
two pairs of congeners possessed the same absolute configuration.
Accordingly, the absolute configurations of aglycone moieties of 3
and 4 were at first elucidated by modified Mosher’s method.13 Meth-
ylation of the aromatic hydroxyl groups in 3 and 4 with trimethylsi-
lyldiazomethane followed by enzymatic hydrolysis with
naringinase provided the two corresponding hemiacetals 8 and 9.
Figure 1. Anti-malarial phenylpropanoid conjugated iridoids from M. morindoides.
bodiimide hydrochloride (EDCIꢁHCl) and 4-dimethylaminopyridine
(DMAP) furnished di-(S)-MTPA ester 10a in 88% yield. In the same
manner, di-(R)-MTPA ester 10b was also provided in 98% yield. Dis-
tribution of difference in the chemical shifts of 10a and 10b estab-
lished 1R,70S configurations of 8 as depicted in Figure 2. According
to the same protocol, the aglycone 9 was found to possess the same
absolute configurations as 8. By a combination of the above de-
scribed findings, the absolute stereostructures of the aglycone moi-
eties in 3 and 4 were unambiguously established.
Next, the stereochemistry of sugar portion was determined by
13
application of the C NMR glycosylation shift rule of 1,100-disac-
charides. In this rule, RR and SS disaccharides showed smaller shifts
on two hemiacetal carbon signals by less than 3.5 ppm, while SR
disaccharide induced larger
Ddc values by more than 4 ppm in
pyridine-d5.14 With regard to the acetal glucoside 6 (dC-1:94.0,
00
dC-1 :101.1) analogous to the disaccharide, both glycosylation shifts
00
for 8 (dC-1:92.5) and glucose (dC-1 :98.9) were less than 3.5 ppm. In
00
the case of 7 (dC-1:94.4, dC-1 :101.3), nearly similar behavior was
observed for 9 (dC-1:92.5) and glucose. These outcomes are charac-
teristic of the RR- or SS-dihemiacetal combinations. Taking 1R con-
figurations of the aglycone moieties of 6 and 7 into account, each
Condensation of (S)-a-methoxy-a-(trifluoromethyl)phenylacetic
acid (MTPA) with 8 using 1-ethyl-3-(3-dimethylaminopropyl)car-
sugar residue was determined to be D-glucose with 1R configura-
tion. Consequently, the absolute stereostructures of 1–4 were
definitely elucidated as depicted in Figure 1. On the other hand,
2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) oxidation of 4
furnished 5 in 98% yield. On the basis of this chemical correlation,
the absolute stereostructure of 5 was unambiguously constructed
(Scheme 1).
Table 1
1H and 13C NMR data for 1a
Position
1H (600 MHz)
13C (150 MHz)
1
3
4
5
6
7
8
9
10
11
10
20
30
40
50
60
70
80
90
100
200
300
400
500
600
4.97 (d, 3.1 Hz)
7.51 (br s)
94.0
152.1
111.5
39.4
140.7
130.3
97.8
Table 2 summarizes anti-malarial potency against P. falciparum15
together with cytotoxicity against the mammalian host KB 3-1
cells16 with regard to 1–5. All theisolates exhibitedlittle cytotoxicity
3.86 (br d, 7.9 Hz)
6.48 (dd, 5.5, 2.4 Hz)
5.59 (dd, 5.5, 1.8 Hz)
against KB 3-1 cells even at the concentration of 150
lar, the 70-ketocongener (5) displayed the most potent inhibitory
effect for proliferation of the malaria parasite (IC50 = 0.04 M) with-
lM. In particu-
2.98 (dd, 7.9, 3.1 Hz)
7.28 (br s)
51.0
l
149.8
168.3
134.0
111.6
149.1
147.7
116.2
121.3
70.0
138.3
172.3
100.4
74.2
out showing any cytotoxicity. From the structure activity relation-
ship analysis of the five congeners, the 600-acetyl, 30-methoxyl, and
7.01 (br s)
6.79 (d, 7.9 Hz)
6.89 (br d, 7.9 Hz)
5.35 (br s)
4.54 (d, 7.9 Hz)
3.13 (dd, 7.9, 8.6 Hz)
3.42 (m)
77.7
71.4
75.6
64.6
3.33 (m)
3.24 (dd, 9.8, 6.1 Hz)
4.20 (dd, 12.2, 6.1 Hz)
4.15 (br d, 12.2 Hz)
3.74 (s)
3.89 (s)
1.83 (s)
4-COOCH3
30-OCH3
600-OAc
52.0
56.6
172.8
20.6
a
The spectra were taken in CD3OD.
Figure 2. Distribution of
Dd in the MTPA esters of 8 and 9.

1522
S. Tamura et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1520–1523
Scheme 1. Reagents and conditions: (a) NaOMe, MeOH, 89% for 3, 91% for 4; (b) TMSCHN2, MeOH–Et2O, quant. for 6, quant. for 7; (c) naringinase, acetate buffer (pH 5), 79%
for 8, 77% for 9; (d) (S)- or (R)-MTPA, EDCIꢁHCl, DMAP, CH2Cl2, 79% for 10a, 91% for 10b, 88% for 11a, 98% for 11b; (e) DDQ, 1,4-dioxane, 98%.
Table 2
3. Murakami, N.; Umezome, T.; Mahmud, T.; Sugimoto, M.; Kobayashi, M.;
Antimalarial and cytotoxic activity of 1–5
Wataya, Y.; Kim, H.-S. Bioorg. Med. Chem. Lett. 1998, 8, 459.
4. Kim, H.-S.; Shibata, Y.; Ko, N.; Ikemoto, N.; Ishizuka, Y.; Murakami, N.;
Compound
Antimalarial activity (IC50
,
l
M)
Cytotoxicity (%, 150 lM)
Sugimoto, M.; Kobayashi, M.; Wataya, Y. Parasitol. Int. 2000, 48, 271.
5. Murakami, N.; Mostaqul, H. M.; Tamura, S.; Itagaki, S.; Horii, T.; Kobayashi, M.
Bioorg. Med. Chem. Lett. 2001, 11, 2445.
6. Murakami, N.; Sugimoto, M.; Kawanishi, M.; Tamura, S.; Kim, H.-S.; Begum, K.;
Wataya, Y.; Kobayashi, M. J. Med. Chem. 2003, 46, 638.
1
2
3
4
5
0.1
4.1
21.9
0.8
9.3
4.3
13.1
13.4
6.1
7. Kambu, K. Plantes Medicinales Africaines; CRP: Kinshasa, 1990. pp 20–22.
8. Tona, L.; Mesia, K.; Ngimbi, N. P.; Cimangga, K.; Bruyne, T.; Apers, S.; Hermans,
N.; Totte, J.; Pieters, L.; Vlietinck, A. J. Ann. Trop. Med. Parasitol. 2001, 95, 47.
9. Tona, L.; Cimangga, R. K.; Mesia, K.; Musuamba, C. T.; Bruyne, T. D.; Apers, S.;
Hernan, N.; Miert, S. V.; Pieters, L.; Totte, J.; Vlietinck, A. J. J. Ethnopharmacol.
2004, 93, 27.
0.04
70-ketonic carbonyl functionalities enhanced anti-malarial potency
independent of cytotoxicity against the host cells.
In conclusion, bioassay-guided separation of the MeOH extract
of M. morindoides disclosed the new phenylpropanoid conjugated
iridoid 1 together with the known four congeners 2–5 as the
anti-malarial candidates. To date, the two anti-malarial iridoids,
showing IC50 of about 40
sources.17,18 In comparison with the two predecessors, it is worth-
while that all the isolates except for potently inhibited
proliferation of the parasites with little cytotoxicity against the
host mammalian cells. Furthermore, it should be noted that the
most potent congener 5 is directly prepared in high yield by DDQ
oxidation from the most abundant constituent 2 without prior pro-
tection of the hydroxyl groups. Exploration for more potent deriv-
atives accompanied by efficacy in mouse model is currently under
investigation by use of the five principles as the scaffolds.
10. A white powder, ½a D27 +28.0 (c 0.9, MeOH), IR mmax (KBr) cmꢀ1: 3391, 1756,
ꢂ
1744, 1707, 1642, 1605, FAB-MS (m/z): 619 [MꢀH]ꢀ, HR FAB-MS (m/z): calcd
for C29H31O15; 619.1663, found;619.1669, 1H and 13C NMR: see Table 1.
11. Yamasaki, K.; Kasai, R.; Masaki, Y.; Okihara, M.; Tanaka, O.; Oshio, H.; Takagi, S.;
Yamaki, M.; Masuda, K.; Nonaka, G.; Tsuboi, M.; Nishioka, I. Tetrahedron Lett.
1977, 1231.
lg/mL, have been found from natural re-
12. Cimanga, K.; Hermans, N.; Apers, S.; Miert, S. V.; Heuvel, H.; Claeys, M.; Pieters,
L.; Arnold, V. J. Nat. Prod. 2003, 66, 97.
13. Ohtani, I.; Kusumi, T.; Kashman, Y.; Kakisawa, H. J. Am. Chem. Soc. 1991, 113,
4092.
14. Nishizawa, M.; Kodama, S.; Yamane, Y.; Kayano, K.; Hatakeyama, S.; Yamada,
H. Chem. Pharm. Bull. 1994, 42, 982.
15. A strain of P. falciparum (CDC1, cycloguanil-resistant from Gambia) was used in
sensitivitytest. P. falciparumwas cultivatedusinga4%hematocritoftypeOhuman
red blood cells suspended in RPMI 1640 medium (Gibco, NY), supplemented with
3
heat-inactivated 10% type O human serum, 5.95 mg/mL of HEPES, 0.5 mg/mL of
glutamine, 2.0 mg/mL of NaHCO3, 50 g/mL of hypoxanthine, and 10 g/mL of
gentamycin sulfate, under a 5% CO2, 5% O2, and 90% N2 atmosphere at 37 °C. After
synchronization by sorbitol treatment, 50 L of parasite culture at the ring stage
L-
l
l
l
(0.5% parasitemia and 4% hematocrit) was added to each well in 96-well
microculture plates. The test samples were dissolved in DMSO and diluted to
Acknowledgments
the appropriate concentration using the complete medium, then 50 lL of each
This study was financially supported by Grants-in-Aid for Scien-
tific Research from the Ministry of Education, Science, Sports, and
Culture of Japan. The authors are also grateful to the Shorai Foun-
dation for Science and Technology and the San-Ei Gen Foundation
for Food Chemical Research for financial support.
sample solution was inoculated. The final concentration of DMSO in the culture
was 1%. After incubation at 37 °C for 48 h, the proliferation of P. falciparum was
assessed by Giemsa-stained smear by observing 10,000 erythrocytes per one thin
bloodfilmintriplicate.Inthisanti-malarialassay, quinine,usedasananti-malarial
control, inhibited the proliferation of P. falciparum in a concentration-dependent
manner with IC50 of 40 ng/mL and IC90 of 90 ng/mL.
16. Cytotoxicity against human epidermoid carcinoma KB cells (KB-3-1) was
evaluated by means of MTT colorimetric assay performed in 96-well plates.
KB-3-1 cells were cultured in RPMI 1640 medium supplemented with 10%
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S. Tamura et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1520–1523
1523
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