Evaluation of Jatropha isabelli natural products and their synthetic analogs as potential antimalarial therapeutic agents

Article abstract of DOI:10.1016/j.ejmech.2013.04.030

Protozoal diseases such as malaria are a leading world health concern. We screened a library of fractionated natural products to identify new potential therapeutic leads and discovered that jatrophone (a product of Jatropha isabelli) exerts significant activity against Plasmodium falciparum strains 3D7 and K1. A focused jatrophone-scaffold library was synthesized to evaluate jatrophone's mode of action and identify more selective analogs. Compounds 25 and 32 of this natural product-inspired compound library exhibited micromolar EC50 values against strains 3D7 and K1, thus providing a new antimalarial molecular scaffold. Our report describes an efficient derivatization approach used to evaluate the structure-activity relationship of jatrophone analogs in search of potential new antimalarial agents.

Full text of DOI:10.1016/j.ejmech.2013.04.030

European Journal of Medicinal Chemistry 65 (2013) 376e380
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Evaluation of Jatropha isabelli natural products and their synthetic
analogs as potential antimalarial therapeutic agents
Victor Hadi, Megan Hotard, Taotao Ling, Yandira G. Salinas, Gustavo Palacios,
*
Michele Connelly, Fatima Rivas
Department of Chemical Biology and Therapeutics, St. Jude Children’s Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105-3678, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Protozoal diseases such as malaria are a leading world health concern. We screened a library of frac-
tionated natural products to identify new potential therapeutic leads and discovered that jatrophone
(a product of Jatropha isabelli) exerts significant activity against Plasmodium falciparum strains 3D7 and
K1. A focused jatrophone-scaffold library was synthesized to evaluate jatrophone’s mode of action and
identify more selective analogs. Compounds 25 and 32 of this natural producteinspired compound
library exhibited micromolar EC50 values against strains 3D7 and K1, thus providing a new antimalarial
molecular scaffold. Our report describes an efficient derivatization approach used to evaluate the
structureeactivity relationship of jatrophone analogs in search of potential new antimalarial agents.
Published by Elsevier Masson SAS.
Received 15 February 2013
Received in revised form
9 April 2013
Accepted 11 April 2013
Available online 23 April 2013
Keywords:
Jatropha isabelli
Jatrophone
Conjugated additions
Antimalarial agents
Plasmodium falciparum
Introduction
while proguanil, atovaquone, and artemisinin derivatives are
chemically distinct [2,3]. The World Health Organization has rec-
Malaria is among the most deadly protozoal diseases, yet only a
limited number of therapeutic agents are available. Malaria is
endemic to more than 100 nations and remains one of the main
leading causes of death in children less than five years of age
worldwide [1]. Malaria is prevalent across the globe but is localized
in Africa, where its annual mortality rate is high. Although several
factors are responsible for the high malaria mortality rate, the
major contributors are poor vector control and the rise of drug
resistance [1]. In humans, malaria is caused by four plasmodium
species (Plasmodium falciparum, Plasmodium vivax, Plasmodium
ovale, and Plasmodium malariae), the most virulent of which is
P. falciparum [2].
The rise of multidrug-resistant strains poses a serious threat, as
the scaffold diversity of the available antimalarials is limited. Five
classes of antimalarial compounds are available for therapeutic
use. Two of these are chemically related d the 4-aminoquinolines
(chloroquine, quinine) and 8-minoquinolines (primaquine) d
ommended a combination regimen based on artemisinin [1], which
is the only agent P. falciparum is susceptible to. Resistance
is expected to emerge with greater use of artemisinin derivatives
[3e6], and the need for new antimalarial scaffolds is therefore more
urgent. Here we report our screening of a terrestrial natural product
to identify potential antimalarial lead compounds and our evalua-
tion of jatrophone analogs as potential therapeutic agents.
Natural products have provided a direct source of therapeutic
agents and a basis for drug development for the past 60 years [7].
Nature continues to provide useful structural architectures that
feature selective chemical space that can potentially access new
biological targets. We initially screened 150 continental American
terrestrial plants with recorded ethnopharmacological properties
by utilizing automated high-throughput fractionation methods [8].
This screen identified several validated hits. The natural product
selected for follow-up studies was jatrophone, a compound isolated
from the Paraguayan plant “yaguaroba” or Jatropha isabelli Muell.
Arg., belonging to the Euphorbiaceae family and popularly used
medicinally [9]. When jatrophone was first structurally elucidated
[10e13], it showed promising anticancer and anti-gastrointestinal
properties, but its narrow therapeutic window has impeded its
advancement to the clinic [12e14]. Other terpene compounds have
been isolated from the rhizomes of Jatropha isabelli, primarily those
Abbreviations: PAMPA, parallel artificial membrane permeability assay; mCPBA,
meta-chloroperoxybenzoic acid; NBS, N-bromosuccinimide; [bmim][BF4], 1-butyl-
3-methylimidazolium tetrafluoroborate.
* Corresponding author. Tel.: þ1 901 595 6504; fax: þ1 901 595 5715.
E-mail address: Fatima.rivas@stjude.org (F. Rivas).
0223-5234/$ e see front matter Published by Elsevier Masson SAS.
http://dx.doi.org/10.1016/j.ejmech.2013.04.030

V. Hadi et al. / European Journal of Medicinal Chemistry 65 (2013) 376e380
377
Fig. 1. Chemical constituents of Jatropha isabelli.
shown in Fig. 1 [14]. We isolated compounds 1, 2, 3, and 5 from
Jatropha isabelli by using a modified extraction method, but the
reported terpenes (1a and 3a) were not detected; jatrophone (1)
was the main component isolated.
To investigate jatrophone’s antimalarial structureeactivity
relationship (SAR), we synthesized a focused compound library and
evaluated the compounds’ activity against both chloroquine-
sensitive (3D7) and chloroquine-resistant (K1) parasites by
following an established protocol [17]. Jatrophone’s highly conju-
gated system allows it to readily undergo 1, 2-nucleophilic addi-
tions as well as 1,4-Michael additions induced by either Grignard
reagents or activated heteroatoms (S, N, and O). Previous studies of
jatrophone with propanethiol suggested that its mechanism of
action involves a retrograde Michael reaction that unmasks a highly
electrophilic C8eC9 enone double-bond set to covalently capture
the sulfhydryl group [10].
It is plausible that available protein-bound thiol groups present
in the Malaria parasite react similarly with jatrophone. In cancer
biology studies, selective glutathione depletion by Michael accep-
tors sensitized hepatocytes to hypoxia, making glutathione deple-
tion a promising potential antitumor strategy [18]. To examine
whether jatrophone was reacting or depleting glutathione, we
studied the reaction of jatrophone with reduced glutathione and
with thiophenol. The Michael adduct 7 was obtained from the re-
action with glutathione, which was the limiting reagent. When
more than half an equivalent of glutathione was added, complex
compound mixtures were observed. In contrast, reaction with
Jatrophone belongs to a large family of terpenes that were iso-
lated from Jatropha diterpenes and that have a broad range of
biological activities, including antitumor, cytotoxic, anti-
inflammatory, molluscidal, and fungicidal properties [9]. Jatro-
phone is a macrocyclic diterpene featuring a unique oxaspiro center
plus several electrophilic centers; its chemical features place it in a
novel class of antimalarial scaffolds. Although jatrophone’s specific
biological target(s) remains elusive, its potent biological activity
warrants further study. Before further derivatization, ADME (ab-
sorption, distribution, metabolism and excretion) studies were
performed to evaluate jatrophone’s physicochemical properties.
The compound displayed good solubility (20 mg/mL at pH 3 or pH 7)
and membrane permeability (5.25 ꢀ 10ꢁ4 cm/s in PAMPA assay)
and a reasonable efflux ratio of 1.5 in CACO cells. Its clearance was
rapid as indicated by its short half-life in human and mouse hepatic
microsomes e t1/2 ¼ 0.10 h and t1/2 ¼ 0.09 h, respectively e and in
plasma: t1/2 ¼ 0.13 h and t1/2 ¼ 0.21 h, respectively. Jatrophone
showed the same (74%) binding affinity to human and mouse
plasma proteins [15,16].
Fig. 2. Conjugated cascade cyclization reactions of jatrophone with sulfhydryl groups.

378
V. Hadi et al. / European Journal of Medicinal Chemistry 65 (2013) 376e380
With this preliminary information, we proceeded to investigate
jatrophone’s potential as an antimalarial agent and seek to broaden
its therapeutic window. Taking the systematic approach illustrated
in Fig. 3, we used conjugated additions, including 1,2 and 1,4-
Michael additions at C7, C9, and C12, C14, to impede the binding
of endogenous thiol groups and probe if they kept their activity.
Overall reduction reactions of the jatrophone olefin systems was
expected to limit its reactivity, and electrophilic additions were
made to help probe jatrophone’s chemical space and to investigate
if they retain biological activity. Each individual constituent of
Jatropha isabelli was also evaluated for antimalarial properties to
anticipate any synergism attributable to traces of other compounds
during the fractionation process.
As shown in Fig. 4, the exploration commenced with the hy-
Fig. 3. General synthetic approaches to jatrophone analogs.
drogenation of jatrophone, affording compounds
9 and 10.
Several attempts to selectively conduct epoxidation [19] across
C8eC9 were unsuccessful due to competing side reactions.
However, epoxidation of jatrophone at C3eC4 by using mCPBA
resulted in the thermally stable compound 11, presumably
through oxidative rearrangement at C12eC13. A Wittig reaction of
the enone at C-7 afforded the expected exocyclic olefin 12, and
deoxygenation of the allylic hydroxyl at C-7 with a hydride source
(Et3SiH) in the presence of a Lewis acid (BF3.Et2O) afforded
product 12a [20].
thiophenol yielded only the stable trans-annular conjugated
Michael product 8 (Fig. 2), illustrating the ease with which the
small, electron-rich thiol group was added at multiple sites. These
findings suggest that both size and electronic properties influence
the outcome of addition of thiol-bound reagent, but additional
biological evidence is needed to support whether jatrophone is
indeed reacting with a thiol-group as its mechanism of action.
Fig. 4. Focused jatrophone compound library 1e37.

V. Hadi et al. / European Journal of Medicinal Chemistry 65 (2013) 376e380
379
3D7
K1
Raji
Hep G2
Hek 293
BJ
1
2
3
4a 4b 4c 4d
5
6
7
8
9
10 11 12 12a 13 14 15 16 17 18 19 20
3D7
K1
EC50<1.92
EC50<4.05
EC50<8.09
EC50<12.5
EC50<18.7
EC50<26.0
Raji
Hep G2
Hek 293
BJ
21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37
Fig. 5. Heat map of antimalarial activity and cytotoxicity of compounds 1e37.
The reaction of jatrophone with CF3SiMe3 in the presence of a
catalytic quantity of CsF proceeded predominantly via 1,2-addition,
yielding a 2:3 mixture of compounds 13 and 14, respectively, after
hydrolysis [21]. A methyl Grignard reaction yielded the 1,2-addition
product 15 and the 1,4-conjugated addition compound 16 as
byproducts. In view of the high affinity of silicon for fluoride anion,
jatrophone was treated with trimethylsilyl cyanide, and CsF,
providing compounds 17 and 18 [22].
Metal-free cyclopropanation reactions of jatrophone with
diazomethane afforded only dihydrofuran product 19, via carbene
addition at C9 with simultaneous enolate treatment to release ni-
trogen gas [23]. Selective 1,2-reduction of enone C7 with sodium
borohydride in combination with CeCl3 resulted in the expected
diastereoisomers 20 and 22 (3:1 ratio in favor of equatorial hydride
attack) and trace amounts of jatrophone reduced at both C7 and
C14. Acetylation of the resulting hydroxyl groups yielded com-
pounds 21, 23, and 24.
Further derivatization of compound 20 to the corresponding
imidazole-1-carbothioate by treatment with 1,10-thiocarbonyldiim-
idazole provided compound 25, and treatment with phenyl chol-
orodithioformate in the presence of 4-dimethyaminopyridine
afforded compounds 26 and 27.
Electrophilic reactions, particularly bromination, were of high
interest given the fact that bromination often enhances the bioac-
tive properties of organic compounds [24]. Because the use of
molecular bromine had been reported to afford mixtures [11], we
used other bromine-containing reagents, such as N-bromosucci-
nimide (NBS). As treatment of jatrophone with NBS in carbon tet-
rachloride produced small quantities of compound 28, we sought
an alternative reaction condition. Jatrophone was then treated with
NBS in ionic liquid medium [bmim][BF4] (known to increase po-
larization of the NeBr bond, enhancing the reagent’s reactivity and
shortening the reaction time), yielding compound 29 and the
starting material [24].
Heck cross-coupling reactions of jatrophone with p-iodoanisole,
iodobenzene, and iodo-benzotrifluoride readily afforded com-
pounds 30, 31, and 32, respectively. Finally, addition of non-carbon
soft nucleophiles (H2O, MeOH, iPrOH) to the C9 of jatrophone
proceeded efficiently to afford compounds 33, 34, and 35. Selective
hydroxylamine or aniline addition at C9 produced compounds 36
and 37 respectively, albeit in poor yields.
Some of the presented reactions did not proceed in good yields
due to the high complexity of the starting material, and its insta-
bility under basic and high temperature conditions.
The heat map corresponding to our antimalarial strains and
cytotoxicity mammalian cell lines (Raji, HepG2, HEK293, and BJ)
results for compounds 1e37 are illustrated in Fig. 5. These results
indicated that the olefin C8eC9 of jatrophone played an impor-
tant role in its activity, and subtle changes at this bond allowed
the development of selective compounds. Although most jatro-
phone analogs exerted antimalarial effects against both the 3D7
and K1 strains, few compounds (compounds 8, 15e20, 22e23, 29
and 37) were more potent than the parent compound. Although
more potent compounds were needed, we were interested pri-
marily in compounds more effective against the K1 (chloroquine-
resistant) malaria strain. Compounds 8, 25, 27, 32, and 37 met this
criterion although they had lost their extended conjugation sys-
tem, indicating that the overall scaffold contributes to compound
activity.
Surprisingly, addition of electron-rich aromatic groups at C-9
(compounds 30e31) had only a moderate effect on antimalarial
activity and showed no cytotoxicity to the tested mammalian cell
lines. However, compound 32 was a substantial lead, with potency
similar to that of jatrophone against both malaria strains (EC50: 6.07
and 5.8
Further, its physicochemical properties did not differ substantially
from those of the parent compound (solubility, 17 g/mL; perme-
mM for 3D7 and K1, respectively) and low cytotoxicity.
m
ability, 1.06 ꢀ 10ꢁ4 cm/s in PAMPA assay). Together, these jatro-
phone analogs provided an activity profile of the overall chemical
space. While the substitution pattern was important, the olefin
across C8eC9 proved to be instrumental to overall reactivity.
In conclusion, our systematic synthetic derivatization of the
natural product jatrophone yielded the promising compound leads
25 and 32, which displayed good antimalarial activity and lower
cytotoxicity than that of jatrophone in mammalian cells. Our findings
warrant further studies to identify the biological target of this highly
functionalized molecular scaffold, which possesses a chemical space
different from those of known antimalarial therapeutic agents.
Having evaluated several functionalities of jatrophone, we
turned our attention to electrophilic reactions that induce subtle
changes in the overall electronic properties of the parent com-
pound, but changes in the overall chemical space. Stereoselective
synthesis of cyclic, substituted olefins by Heck reaction of organic
halides with an
a,b-unsaturated system are fairly uncommon.
However, previous studies had shown that through a complex
mechanism, 1,2-disubstituted, electron-poor olefins can undergo a
Heck-type reaction in the presence of palladium (II), Ag2CO3 as the
base, and a large excess of the iodo-aromatic donor [25]. In fact,

380
V. Hadi et al. / European Journal of Medicinal Chemistry 65 (2013) 376e380
[9] R.K. Devappa, H.P.S. Makkar, K. Becker, Jatropha diterpenes: a review, J. Am.
Competing interests
Oil Chem. Soc. 88 (2011) 301e322.
[10] J.P. Mallari, W.A. Guiguemde, R.K. Guy, Antimalarial activity of thio-
semicarbazones and purine derived nitriles, Bioorg. Med. Chem. Lett. 19
(2009) 3546e3549.
The authors declare that they have no competing interests.
[11] S.M. Kupchan, C.W. Sigel, J.M. Matz, C.J. Gilmore, R.F. Bryan, Structure and
stereochemistry of jatrophone, a novel macrocyclic diterpenoid tumor in-
hibitor, J. Am. Chem. Soc. 98 (1975) 2295e2300.
Acknowledgments
[12] M.D. Taylor, A.B. Smith, G.T. Furst, S.P. Gunasekara, C.A. Bevelle, G.A. Cordell,
N.R. Farnsworth, S.M. Kupchan, H. Uchida, A.R. Branfman, R.G. Dailey,
A.T. Sneden, New antileukemic jatrophone derivatives from Jatropha gossy-
pifolia: structural and stereochemical assignment through nuclear magnetic
resonance spectroscopy, J. Am. Chem. Soc. 105 (1983) 3177e3183.
[13] G. Schmeda-Hirschmann, I. Razmilic, M. Sauvain, C. Moretti, V. Munoz, E. Ruiz,
E. Balanza, A. Fournet, Antiprotozoal activity of Jatrogrossidione from Jatropha
grossidentata and Jatrophone from Jatropha isabellii, Phytother. Res. 10 (1996)
375e378.
This study was supported by the American Lebanese Syrian
Associated Charities (ALSAC). The authors thank Ing. Mariza C.
Quintana Centurion of the Museo Nacional de Historia Natural del
Paraguay for assistance with the ethnobotanical survey and
collection and identification of Jatropha isabelli. We also thank the
High-Throughput Analytical Chemistry Core Facility at St. Jude
Children’s Research Hospital for analytical support.
[14] M. Pertino, G. Schmeda-Hirschmann, J.A. Rodriguez, C. Theoduloz, Gastro-
protective effect and cytotoxicity of terpenes from the Paraguayan crude drug
“yagua rova” (Jatropha isabelli), J. Ethnopharmacol. 111 (2007) 553e559.
[15] L. Di, E.H. Kerns, X.J. Ma, Y. Huang, G.T. Carter, Applications of high throughput
microsomal stability assay in drug discovery, Comb. Chem. High Throughput
Screen. 11 (2008) 469e476.
[16] A.M. Marino, M. Yarde, H. Patel, S. Chong, P.V. Balimane, Validation of the 96
well Caco-2 cell culture model for high throughput permeability assessment
of discovery compounds, Int. J. Pharm. 297 (2005) 235e241.
Appendix A. Supplementary data
Supplementary data related to this article can be found at
http://dx.doi.org/10.1016/j.ejmech.2013.04.030.
References
[17] K. Mitachi, Y.G. Salinas, M. Connelly, N. Jensen, T. Ling, F. Rivas, Synthesis and
structureeactivity relationship of disubstituted benzamides as a novel class of
antimalarial agents, Bioorg. Med. Chem. Lett. 22 (2012) 4536e4539.
[18] J.M. Lluis, A. Morales, C. Blasco, A. Colell, M. Mari, C. Garcia-Ruiz,
J.C. Fernandez-Checa, Critical role of mitochondrial glutathione in the survival
of hepatocytes during hypoxia, J. Biol. Chem. 280 (2005) 3224e3232.
[19] M.A. Gonzalez, S. Ghosh, F. Rivas, D. Fischer, E.A. Theodorakis, Synthesis of (þ)
- and (-)-isocarvone, Tetrahedron 45 (2004) 5039e5041.
[20] D.G. Batt, G.D. Maynard, J.J. Petraitis, J.E. Shaw, W. Galbraith, R.R. Harris,
2-Substituted-1-naphthols as potent 5-lipoxygenase inhibitors with topical
anti-inflammatory activity, J. Med. Chem. 33 (1990) 360e370.
[21] R.P. Singh, J.M. Shreeve, Nucleophilic trifluoromethylation reactions of organic
compounds with (trifluoromethyl)trimethylsilane, Tetrahedron 56 (2000)
7613e7632.
[22] J. Yang, Y. Wang, S. Wu, F.X. Chen, The highly efficient 1,4-addition of TMSCN
to aromatic enones catalyzed by CsF with water as additive, Synlett (2009)
3365e3367.
[23] H. Lebel, J.F. Marcoux, C. Molinaro, A.B. Charette, Stereoselective cyclo-
propanation reactions, Chem. Rev. 103 (2003) 977e1050.
[24] J.S. Yadav, B.V.S. Reddy, G. Baishya, S.J. Harshavardhan, C.J. Chary,
M.K. Gupta, Green approach for the conversion of olefins into vic-halohy-
drins using N-halosuccinimides in ionic liquids, Tetrahedron 46 (2005)
3569e3572.
[1] World Health Organization, World Malaria Report (2011). Geneva, http://
www.who.int/malaria/world_malaria_report_2011/en/ (accessed 13.01.2013).
[2] E.I. Lueze, S.L. Croft, D.C. Warhurst, Activity of pyronaridine and mepacrine
against twelve strains of Plasmodium falciparum in vitro, J. Antimicrob. Che-
mother. 37 (1996) 511e518.
[3] C.J. Sutherland, S.D. Polley (Eds.), Genetics and Evolution of Infectious Dis-
eases, Elsevier, 2011, pp. 607e627.
[4] M.E. Sinka, J.M. Bangs, S. Manguin, M. Coetzee, C.M. Mbogo, J. Hemingway,
A.P. Patil, W.H. Temperley, P.W. Gething, C.W. Kabaria, R.M. Okara,
T.V. Boeckel, H.C.J. Godfray, R.E. Harbach, S.I. Hay, The dominant vectors of
human malaria in Africa, Europe and the Middle East: occurrence data, dis-
tribution maps and bionomic précis, Parasit. Vectors 3 (2010) 117.
[5] S. Bröer, K. Kirk, Chloroquine transport via the malaria parasite’s chloroquine
resistance transporter, Science 325 (2009) 1680e1682.
[6] V. Lakshmanan, P.G. Bray, D. Verdier-Pinard, D.J. Johnson, P. Horrocks,
R.A. Muhle, G.E. Alakpa, R.H. Hughes, S.A. Ward, D.J. Krogstad, A.B. Sidhu,
D.A. Fidock, A critical role for PfCRT K76T in Plasmodium falciparum verapamil-
reversible chloroquine resistance, EMBO J. 24 (2005) 2294e2305.
[7] D.J. Newman, G.M. Cragg, Natural products as sources of new drugs over the
30 years from 1981 to 2010, J. Nat. Prod. 75 (2012) 311e335.
[8] Y. Tu, C. Jeffries, H. Ruan, C. Nelson, D. Smithson, A.A. Shelat, K.M. Brown,
X.C. Li, J.P. Hester, T. Smillie, I.A. Khan, L. Walker, L.,K. Guy, B. Yan, An auto-
mated high-throughput system to fractionate plant natural products for drug
discovery, J. Nat. Prod. 73 (4) (2010) 751e754.
[25] P. Mauleon, Palladium-catalyzed cascade reaction of
a,b-unsaturated sulfones
with aryl iodides, Eur. J. Chem. 9 (2003) 1511e1520.