Generation of anti-trypanosomal agents through concise synthesis and structural diversification of sesquiterpene analogues

Article abstract of DOI:10.1021/ja200374q

To access high-quality small-molecule libraries to screen lead candidates for neglected diseases exemplified by human African trypanosomiasis, we sought to develop a synthetic process that would produce collections of cyclic scaffolds relevant to an assortment of natural products exhibiting desirable biological activities. By extracting the common structural features among several sesquiterpenes, including artemisinin, anthecularin, and transtaganolides, we designed six types of scaffolds with systematic structural variations consisting of three types of stereochemical relationships on the sp³ ring-junctions and two distinct arrays of tricyclic frameworks. A modular and stereodivergent assembly of dienynes exploiting a versatile manifold produced a series of cyclization precursors. Divergent cyclizations of the dienynes employing tandem ring-closing metathesis reactions overrode variant reactivities of the cyclization precursors, leading to the six canonical sets of the tricyclic scaffolds incorporating a diene group. Screenings of trypanosomal activities of the canonical sets, as well as regio- and stereoisomers of the tricyclic dienes, allowed generation of several antitrypanosomal agents defining the three-dimensional shape of the pharmacophore. The candidate tricyclic dienes were selected by primary screenings and further subjected to installation of a peroxide bridge, which generated artemisinin analogues that exhibited potent in vitro anti-trypanosomal activities comparable or even superior to those of artemisinin and the approved drugs, suramin and eflornithine.

Full text of DOI:10.1021/ja200374q

ARTICLE
pubs.acs.org/JACS
Generation of Anti-trypanosomal Agents through Concise Synthesis
and Structural Diversification of Sesquiterpene Analogues
Hiroki Oguri,*,†,§ Takahisa Hiruma,† Yutaka Yamagishi,† Hideaki Oikawa,†
Aki Ishiyama,‡ Kazuhiko Otoguro,‡ Haruki Yamada,z and Satoshi Omuraz
†Division of Chemistry, Graduate School of Science and §Division of Innovative Research, Creative Research Institution,
Hokkaido University, North 21, West 10, Kita-ku, Sapporo 001-0021, Japan
‡Research Center for Tropical Diseases, Kitasato Institute for Life Sciences and zGraduate School of Infection Control Sciences,
Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan
S Supporting Information
b
ABSTRACT: To access high-quality small-molecule libraries to
screen lead candidates for neglected diseases exemplified by
human African trypanosomiasis, we sought to develop a syn-
thetic process that would produce collections of cyclic scaffolds
relevant to an assortment of natural products exhibiting desir-
able biological activities. By extracting the common structural
features among several sesquiterpenes, including artemisinin,
anthecularin, and transtaganolides, we designed six types of
scaffolds with systematic structural variations consisting of three
types of stereochemical relationships on the sp3 ring-junctions and two distinct arrays of tricyclic frameworks. A modular and
stereodivergent assembly of dienynes exploiting a versatile manifold produced a series of cyclization precursors. Divergent
cyclizations of the dienynes employing tandem ring-closing metathesis reactions overrode variant reactivities of the cyclization
precursors, leading to the six canonical sets of the tricyclic scaffolds incorporating a diene group. Screenings of trypanosomal
activities of the canonical sets, as well as regio- and stereoisomers of the tricyclic dienes, allowed generation of several anti-
trypanosomal agents defining the three-dimensional shape of the pharmacophore. The candidate tricyclic dienes were selected by
primary screenings and further subjected to installation of a peroxide bridge, which generated artemisinin analogues that exhibited
potent in vitro anti-trypanosomal activities comparable or even superior to those of artemisinin and the approved drugs, suramin and
eflornithine.
’ INTRODUCTION
with novel structures and unique modes of action. With the
intention of developing a new generation of lead candidates for
HAT, we herein report development of a concise synthetic
process leading to a collection of natural product-like scaffolds
with skeletal and stereochemical variations. This collection has
enabled structureꢀactivity relationship (SAR) studies to eluci-
date a three-dimensional (3-D) image of the pharmacophore
relevant to a series of biologically intriguing sesquiterpenes.
Aside from eflornithine, an amino acid derivative, three of the
four prescription drugs for HAT disease are comprised of
heteroatom-incorporated/appended aromatic rings and have
sp2-rich structural features with a substantial lack of sp3 stereo-
centers. Recent studies reflect growing concern with larger
numbers of aromatic rings that can negatively affect several
drug-like properties, including lipophilicity and aqueous solubi-
lity, and thereby increase risks of failure in clinical trials.3
Synthetic drugs and pharmaceutical collections of chemical
libraries are typically prepared in cost- and labor-effective ways
African sleeping sickness, also known as human African
trypanosomiasis (HAT), is a vector-borne parasitic disease that
is recognized as one of the world’s most neglected diseases and
causes serious medical and agro-economic problems in >30 sub-
Saharan African countries.1 The protozoan parasite Trypanosoma
brucei is transmitted by blood-sucking tsetse flies, multiplies
within the bloodstream of the mammalian host, and eventually
invades the central nervous system. The disease is invariably fatal
if not treated properly. In 2006, the World Health Organization
estimated that HAT currently affects approximately 300 000
people annually and is responsible for 25 000 deaths each year.2
A vaccine-based approach, however, is unlikely due to the
antigenic variation of the parasite. Chemotherapeutic options
are also very limited, and only four drugs are approved for HAT
treatments (Figure 1): suramin (since 1916), pentamidine
(1941), melarsoprol (1949), and eflornithine (1990). Che-
motherapy with these outdated drugs is frequently limited in
efficacy, plagued by severe side-effects, and hampered by increas-
ing resistance of the parasites. Consequently, there is an urgent
need for discovering safe and effective anti-trypanosomal drugs
Received: January 14, 2011
Published: March 17, 2011
r
2011 American Chemical Society
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Figure 1. Four drugs approved for human African trypanosomiasis.
that exploit flat aromatic heterocyclic chemistry and consequently have
disk- or rod-like shapes.4 In contrast, natural products often possess
complex and globular shapes composed of fused skeletons with fewer
aromatic rings and increased numbers of stereogenic centers.5 Accord-
ing to a recent analysis by Shoichet, 83% of the core scaffolds present
among natural products are simply absent among commercially
available molecules, demonstrating that vast areas of the biologically
relevant chemical space charted by natural products remain
unexploited.6 It has been also recognized that an increased proportion
of sp3 stereocenters induces more 3-D shapes with diverse undulations
on the surfaces. This improves interaction with the biological target,
enhancing the potency and/or specificity of the drug candidate and
increasing the probability of providing new pharmacophores and
unique binding geometries.7 Furthermore, incorporation of sp3
centers multiplies the number of potential stereoisomers and thus
generates structural variations in the compound collections. Despite
this increased attention to synthetic approaches to access natural
product-inspired libraries,8ꢀ12 the de novo design and synthesis of
optimal screening collections of small molecules bearing biolo-
gical relevance and structural diversity has proved to be a
formidable challenge in modern organic synthesis.
The adaptation and survival of the parasite T. brucei in its host
involve integrated regulation of Ca2þ-ATPases, which are essen-
tial in calcium ion homeostasis. While several target proteins
were explored for the chemotherapy of trypanosomiasis,13 Ca2þ
-
Figure 2. (a) Naturally occurring sesquiterpenes: anti-malarial drug
artemisinin (1), anti-trypanosomal agent anthecularin (2), and Ca2þ
-
ATPases were shown to be essential for parasite viability and
regarded as targets for anti-trypanosomal drug development.14 In
fact, an anti-malarial drug, artemisinin 1 (Figure 2a),15,16 targets
PfATP6, the orthologue of the sarco-/endoplasmic reticulum
Ca2þ-ATPase,17 and exhibits anti-trypanosomal activity with an
effective 50% inhibitory concentration (IC50) of 5.8 μg/mL for
T. brucei rhodesiense.18 In 2007, Karioti and Tasdemir reported
isolation of a novel sesquiterpene lactone, anthecularin 2, which
exhibits anti-trypanosomal activity (IC50 = 10.1 μg/mL for T.
brucei rhodesiense).19,20 In addition, structurally related transta-
ATPase inhibitors, transtaganolides C and D (3, 4). (b) Structural
motifs: tricyclic 5 bearing a diene and tetracyclic 6 bearing a peroxide
bridge. (c) Schematic illustration for structural diversification of the
motifs and a synthetic approach to define the pharmacophore.
and structural diversification of structural motifs inspired by this
assortment of sesquiterpenes with desirable biological properties
toward the lead generation for trypanosomiasis. We also explored
SARs exploiting a systematic set of small molecules with a dense
matrix of stereochemical and skeletal variations.
ganolides/basiliolides 3 and 4 are reported to inhibit Ca2þ
-
ATPase, whereas their anti-protozoal activity has not been
reported.21,22 Regardless of the presence or absence of an
endoperoxide bridge installed on the framework, we paid parti-
cular attention to the structural homology of these sesquiter-
penes as a privileged scaffold for the development of novel anti-
trypanosomal agents. We also noted the promising pharmaco-
logical properties of anti-malarial trioxane 1 bearing seven
stereogenic sp3 centers, which satisfies all the requirements for
Lipinski’s rule of five.5a,23 Herein, we report the concise synthesis
’ RESULTS AND DISCUSSION
Design of Structural Motifs. Given the structural homology of
the four sesquiterpenes 1ꢀ4 sharing closely related core skeletons,
we designed a tricyclic scaffold 5 as the structural motif for
generating anti-trypanosomal agents (Figure 2a,b). In addition,
the presence of methyl substituents on the seven-membered rings
in 1 and 2 as well as diene functionalities in 2 and 3 inspired us to
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Scheme 1. Schematic Illustration of Synthetic Strategies To
Generate Six Types of Sesquiterpene Analogues
Design of a Synthetic Process Generating Stereochemical
and Skeletal Variations. To obtain concise and stereocon-
trolled access to the systematic sets of the tricyclic motif 5,24
we soughtto develop a synthetic process, as outlined inScheme 1,
fulfilling the following strategic requirements: (1) diversification
of stereochemical relationships of the sp3 ring-junctions into
three types and the cyclic arrays into two types and (2) rapid and
efficient construction of varied molecular architectures employ-
ing robust annulations to override the variances of the substrates.
To this end, we designed oxonitrile 7 as a versatile manifold to
assemble three building blocks on the three consecutive sites
(AꢀC) of the six-membered ring in a modular fashion.25
Addition of an R1 group to the carbonyl group would produce
a hydroxylalkenenitrile that effects chelation-controlled conju-
gate additions of acetylides as the R2 group by applying Fleming’s
protocol.26 Subsequent alkylation of the resulting C-magnesiated
nitrile would proceed with retention of configuration to afford
product 8 bearing R1ꢀR3 groups with high levels of stereose-
lectivities. According to the sequential installations of R1ꢀR3
groups, dienyne 10 with an anti-syn stereochemical relationship
could be synthesized. Moreover, we conceived a stereodivergent
method to access 9-(syn-syn) and 11-(anti-anti), exploiting the
hydroxyl and nitrile groups installed on 8. Either alkylation of the
secondary alcohol or introduction of an R1 group having a
terminal olefin as a Grignard reagent at site A would establish
syn or anti stereochemical relationships with the acetylene group
at site B. Similarly, installation of an olefin group as the R3 group
or manipulation of the nitrile group would construct requisite
stereochemical relationships between sites B and C.
To construct the six structural motifs 12ꢀ17, we planned tandem
ring-closing olefin metathesis reactions of dienynes,27,28 leading to
tricyclic systems with concomitant incorporation of diene function-
alities into the skeletons (Scheme 1). We performed divergent
cyclizations by controlling the modes of tandem cyclizations of
dienynes, leading to products with distinct cyclic arrays. A less-
substituted olefin facilitates initial ruthenium carbene complex
formation. Therefore, the tandem ring-closure of dienynes with
sterically differentiated olefins would proceed in a regio-controlled
manner via cyclization from sites A to B followed by B to C leading
to a tricyclic diene, whereas a reversed substitution pattern of olefins
at sites A and C could invert the cyclization mode to produce a
distinct tricycle. The overall synthetic process consisted of (1)
preparation of manifold and building blocks (build), (2) modular
and stereodivergent assembly of the three components on the
manifold (couple), and (3) divergent cyclizations of dienynes
(pair). This strategy demonstrates the applicability of the
buildꢀcoupleꢀpair (BCP) strategy9d,25,29 in library developments
to generate scaffold diversity in a programmable fashion.
Synthesis of Tricyclic Skeletons. On the basis of our strategic
plan, we carried out the synthesis of the six distinct tricycles
incorporating internal dienes starting from manifold 7
(Scheme 2). First, we developed synthetic pathways 1 and 2,
leading to type I and II dienes, 12 and 13, with cis-cis-fused
tricyclic skeletons. The C1 carbonyl group of 7 was reduced with
NaBH4ꢀCeCl3 to yield racemic hydroxylalkenenitrile 18. The
chelation-controlled conjugate addition of acetylide 19 and
subsequent stereocontrolled alkylation of the resulting anion
with 20 produced 22 in 61% yield. Because allylations of the
alcohol 22 under basic conditions caused side reactions such as
isomerization of the acetylene into an allene, we employed acid-
catalyzed conditions exploiting trichloroacetimidate 24. Subsequent
removal of the trimethylsilyl group gave the dienyne 26 in 75%
install methyl and diene groups on the scaffold. More impor-
tantly, in order to systematically generate 3-D structural diversity,
we designed six types of tricyclic frameworks as canonical sets of
structural motifs as illustrated in Figure 2c. To this end, we
planned to diversify two factors that regulate molecular archi-
tecture: (1) three variations of stereochemical relationships (cis-
cis, trans-cis, and trans-trans) for the three consecutive sp3 ring-
junctions highlighted in red, blue, and green and (2) two distinct
arrays of the seven-membered ring against the ring-junctions. By
gaining access to a series of tricyclic sesquiterpene analogues
bearing rigid skeletons, we planned to perform SAR studies
estimating the optimum stereochemical properties and cyclic
arrays of the pharmacophore concurrent with screening for anti-
trypanosomal activities. Moreover, we envisaged further manip-
ulations of the optimal structural motifs selected in the primary
screenings. As illustrated in Figure 2b,c, installation of a peroxide
bridge exploiting the diene groups embedded in the tricyclic
cores would produce the artemisinin-like tetracyclic motif 6 by
incorporating two additional sp3 ring-junctions. This would lead
to the development of a flexibly expandable synthetic process to
afford multiple types of targeted motifs with elevated oxidation
levels and structural diversities and complexities.
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Scheme 2. Systematic Synthetic Process Leading to Six Types of Tricyclic Dienes (Type IꢀVI)
yield (two steps). The dienyne 27, with a 2-methylallyl ether at
site A and a 3-butenyl group at site C, was synthesized in a similar
fashion. Upon treatment of 26 with 10 mol % Grubbs second-
generation catalyst 28 in benzene under reflux for 3.5 h, tandem
ring-closing metathesis (RCM) of the dienyne system proceeded
smoothly to afford the desired tricyclic diene 12 in 90% yield.
Accordingly, ring-closure of 27 produced tricyclic diene 13 in
excellent yield (90%). The modes of ring-closure were success-
fully controlled by exploiting the group-selective ruthenium
carbene complex formation of monosubstituted olefins in the
presence of disubstituted olefins. Thus, we achieved divergent
and stereocontrolled access to tricyclic dienes 12 and 13 having
cis-cis-fused ring-junctions in five steps from 7.
Chelation-controlled conjugate additionꢀalkylation and subse-
quent removal of the silyl group produced 33 in 71% yield (two
steps) as the major product.31 Similar stepwise transformations
gave 34, in which the substitution pattern of terminal olefins at
sites A and C was exchanged compared to that of 33 employing
Grignard reagent 30 and alkyl iodide 21 in place of 29 and 20,
respectively. While ring-closure of 33 required a high catalyst
loading (66 mol %) and resulted in a modest yield (52%)
of the desired 14 with trans-cis ring-junctions, cyclization of
34 with 20 mol % catalyst produced 15 in satisfactory yield
(75%). The structure of 15 was confirmed on the basis of X-ray
analysis.32
Next, tricyclic dienes, 16 and 17, with trans-trans ring-junc-
tions were synthesized via pathways 5 and 6. Three-component
assembly of 7, 29, and 19, followed by protonation of the
resultant carbanion in one pot, stereoselectively produced 35
Next, pathways 3 and 4 were explored to access trans-cis-fused
tricyclic type III and IV dienes, 14 and 15, respectively. Addition
of Grignard reagent 29 to 7 afforded alcohol 31 in 55% yield.30
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Table 1. In Vitro Anti-trypanosomal Activities of Synthetic Sesquiterpene Analogues and Approved Drugs against T. brucei brucei
GUTat 3.1a
a Culture of trypanosome (2.0ꢀ2.5 ꢁ 104 trypanosomes/mL for GUTat 3.1 strain) was used. The cytotoxicities were evaluated with MRC-5 cells, and
the selectivity index (SI) for trypanosomiasis was calculated as (IC50 for MRC-5)/(IC50 for T. brucei brucei). ND means “not determined”. b Existing anti-
trypanosomal drugs. c Reported in ref 19 against T. brucei rhodesiense.
in 46% yield. Reduction of the nitrile 35 gave an aldehyde in 88%
yield, which was treated with 2-methylallyl Grignard reagent to
yield a diastereomeric mixture of diols in an approximately 1:1
ratio. Separation of the diastereomers and removal of the silyl
group produced the dienynes 37a and 37b. The dienynes 38a
and 38b with the substitution patterns of terminal olefins at sites
A and C exchanged were synthesized in a similar fashion except
for stepwise additions of the Grignard reagents (7 f 32 f 36).
Whereas tandem ring-closures of 37a and 37b to afford the
tricyclic dienes 16a and 16b resulted in moderate yields
(39ꢀ49%), cyclizations of 38a and 38b proceeded smoothly
to produce 17a and 17b in good yields (80ꢀ86%). Structures of
37a and 17b were unambiguously determined on the basis of
X-ray analysis of their derivatives.24 Thus, the six types of tricycles
bearing internal dienes were successfully constructed in a systematic
fashion. We developed this synthetic process to demonstrate the
synergistic effects of stereoisomerism of sp3 ring-junctions and
the divergent cyclizations of dienynes to generate 3-D structural
diversity in conformationally rigid tricycles. This synthetic strategy
created structural diversity without a substantial increase in the
molecular weights of the six canonical tricycles (average MW ≈
235), consistent with a key aspect of Lipinski’s rule: restriction of
molecular weight range (MW < 500).23 This is in contrast to the
typical strategy in medicinal and combinatorial chemistry of
appending building blocks to a common skeleton to efficiently
generate analogues of a target structure.
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Figure 3. SAR study of synthetic sesquiterpene analogues for in vitro anti-trypanosomal activities. The dashed line divides types of the canonical
scaffolds except for the lane of peroxides: left of the line are the potencies for type I, III, and V dienes; right of the line are those for type II, IV, and VI
dienes.
Anti-trypanosomal Activities of the Six Canonical Sets of
Tricyclic Dienes. With the six kinds of tricyclic dienes 12ꢀ17 in
hand, in vitro anti-trypanosomal activities were evaluated em-
ploying a GUTat 3.1 strain of T. brucei brucei (Table 1, entries
1ꢀ8).33 The cis-cis-fused 12 (type I) possessing a cyclic ether and
a nitrile exhibited the most potent activity (IC50 = 0.55 μg/mL),
and 13 (type II) with its distinct cyclic array also showed
convincing activity (IC50 = 1.1 μg/mL). The trans-cis-fused
dienes 14 (type III) and 15 (type IV) having a tertiary alcohol
and a nitrile showed moderate activities. Regarding the type V
and VI dienes, 16 and 17, bearing trans-trans-fused skeleton with
two hydroxyl groups, only 16a having a 1,4-anti-diol exhibited
substantial activity (IC50 = 1.92 μg/mL), whereas the other
three dienes 16b, 17a, and 17b showed negligible activities.
Comparisons of the anti-trypanosomal activities between the six
types of canonical scaffolds are graphically illustrated in Figure 3,
clearly showing that type I and II dienes, 12 and 13, exhibited
dominant potencies over the others. On the basis of the evalua-
tion of cytotoxities of the synthetic molecules against a mamma-
lian cell line (MRC-5 cells), we calculated a selectivity index [SI =
(IC50 for MRC-5 cells)/(IC50 for T. brucei strain)] as a means to
assess combined potencies of both anti-trypanosomal and cyto-
toxic activities, as shown in Table 1. It is worth mentioning that
the dienes 12 (type I) and 13 (type II) possessed SIs comparable
to those of two approved drugs for HAT, suramin and eflor-
nithine (Table 1, entries 16 and 17).
Diversification of Dienes 12 and 13 and Further SAR
Studies. Taking the preliminary SAR study into account, we
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Scheme 3. Synthesis of Endoperoxides and Their Equivalents Starting from Type I and II Dienes
turned our attention to generating regioisomers of 12 and 13 by
transpositions of the diene groups incorporated on the cis-
cis-fused skeletons (Scheme 3). Isomerization of s-trans diene 12
upon treatment with p-toluenesulfonic acid in toluene at 80 °C
produced the s-cis dienes 39 (13%) and 40 (11%) with recovery
of the starting material 12 (16%). In addition, epimerization of
the central sp3 ring-junction competed to furnish 41 (22%)
having the trans-trans-fused skeleton, and the regioisomeric s-cis
diene 42 (26%) was also obtained. Whereas isomerization of 12
provided variations of conjugated dienes under thermodynami-
cally controlled conditions, the conversion of 13 with distinct
cyclic arrays proceeded in a regio-controlled manner to form s-cis
diene 48 in 45% yield as the major product employing the
identical Brønsted acid mediator. After screenings of various
conditions for the diene transposition, we found that SbCl5 was
the best mediator, affording 48 in 66% yield below 0 °C with no
more than 0.4 equiv of SbCl5. In comparison, the next best
mediators involving TiCl4, AlCl3, and GaCl3 required at least 1.5
equiv to form 48 in moderate yields (14ꢀ49%).
and 48) were decreased compared to those of the corresponding
s-trans dienes (12, 13, and 41). In addition, lactone derivative 47
synthesized by oxidation of 12 with PCC34 also showed dimin-
ished activity. Although it is difficult to directly compare the
trypanosomal activities of anthecularin (2: IC50 = 10.1 μg/mL
against T. brucei rhodesiense; Table 1, entry 18) reported by
Karioti and Tasdemir because of inconsistencies of the T. brucei
strains used, the racemic sesquiterpene analogues bearing an
s-trans diene moiety (12, 13, and 41) exhibited either equal or
increased anti-trypanosomal activities. Consequently, the SAR
study employing tricyclic dienes with systematic variations of
cyclic arrays and stereo- and regioisomerism allowed us to reveal
novel pharmacophores with 3-D images relevant to the chemical
space defined by 12, 13, and 41.
Installation of the Endoperoxide Bridge and Its Equiva-
lents Leading to Artemisinin Analogues. Encouraged by the
efficacious activities of s-trans dienes (12, 13, and 41), we next
examined the installation of endoperoxides on the corresponding
s-cis dienes (39, 40, 42, and 48) to furnish analogues of
artemisinin 1 (43ꢀ46 and 49, Scheme 3). Photo-oxidation of
39 with an sp2 ring-junction produced two separable diastereo-
mers, 43 and 44, in 43% and 26% yields, respectively. Diaster-
eocontrolled addition of singlet oxygen to cis-cis-fused 40 and 48
occurred at the sterically less-hindered face to afford endoper-
oxide 45 and trioxane 49 in good yields, whereas photo-oxidation
of trans-trans-fused 42 proceeded with the opposite face selec-
tivity, leading to 46 in 66% yield. X-ray analyses of the crystalline
43 and 45 as well as NMR analyses including NOE experiments
confirmed the structure of the endoperoxides.35 Among the four
s-cis dienes, 48 bearing a seven-membered cyclic enol ether
Further SAR studies were conducted with these stereo- and regio-
isomeric dienes (Table 1, entries 9ꢀ14). The transꢀtrans-fused
41 having a cyclic ether and a nitrile exhibited appreciable activity
(IC50 = 1.02 μg/mL), comparable to those of 12 and 13 (entries 1
and 2). Interestingly, the racemic s-trans dienes 41 and 16a (entry 5)
had equivalent activities and shared similar skeletal and stereo-
chemical properties, possessing oxygen and nitrile functional
groups in either 1,3-anti- or 1,4-anti relationships in the vicinity
of the transꢀtrans-fused ring-junctions, whereas the activity was
abolished with diastereomer 16b having a 1,4-syn diol (entry 6).
Anti-trypanosomal activities of the four s-cis dienes (39, 40, 42,
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Table 2. In Vitro Anti-trypanosomal Activities of Artemisinin 1 and Its Synthetic Analogues against T. brucei brucei GUTat 3.1a
a Culture of trypanosome (2.0ꢀ2.5 ꢁ 104 trypanosomes/mL for GUTat 3.1 strain) was used. The cytotoxicities were evaluated with MRC-5 cells, and
the selectivity index (SI) for trypanosomiasis was calculated as (IC50 for MRC-5)/(IC50 for T. brucei brucei). ND means “not determined”.
exhibited the most proficient reactivity toward the cycloaddition.
This reactivity prompted us to perform hetero-DielsꢀAlder
reactions with azo and nitroso compounds to install “ꢀNꢀNꢀ”
and “ꢀNꢀOꢀ” bridges in place of peroxides.36 Diastereoselec-
tive cycloaddition of 48 with diallyl azodicarboxylate proceeded
below room temperature to produce 50 in 74% yield, although
removal of the carbamate ester group to produce the diaza-
analogue corresponding to 49 proved to be difficult. Similarly,
upon treatment of 48 with an acyl nitroso dienophile generated
in situ via oxidation of the corresponding hydroxycarbamate, the
hetero-DielsꢀAlder reaction occurred in a regio- and stereocon-
trolled manner to afford 51 in 78% yield. Treatment with
piperidine to remove the Fmoc group, however, caused cleavage
of the N,O-acetal, giving rise to crystalline 52 in 62% yield. X-ray
analysis of 52 revealed a unique structure composed of a spiro-
fused oxazine ring.35
dienes 13 and 41. Compounds 50 and 51 bearing ꢀNꢀNꢀ and
ꢀNꢀOꢀ bonds share an identical framework with peroxide 49
and retained moderate activities, whereas spirocyclic 52 exhib-
ited a loss of activity. Thus, all of the artemisinin analogues
bearing peroxide bridges showed appreciable anti-trypanosomal
activities (IC50 < 1.4 μg/mL; Figure 3).37 Notably, the peroxides
43 and 44 showed higher SIs compared to those for suramin and
eflornithine, and 44 satisfies the hit criteria for trypanosomiasis
(IC50 < 0.2 μg/mL, SI >100).38 Thus, we demonstrated that SAR
studies of tetracyclic sesquiterpene analogues with increased sp3
character and 3-D structural diversity allowed us to define novel
pharmacophores for the development of anti-trypanosomal lead
candidates.
’ CONCLUSIONS
Anti-trypanosomal Activities of Artemisinin Analogues.
On the basis of the considerable anti-trypanosomal activity
of artemisinin 1 against T. brucei brucei GUTat 3.1 strain
(IC50 = 0.94 μg/mL), which we confirmed (Table 2, entry 1),
collections of artemisinin analogues possessing regio- and stereo-
isomeric variations of peroxide bridges were evaluated (entries
2ꢀ9). The peroxide 44 synthesized from the diene 12 (type I)
exhibited the most significant activity (IC50 = 0.16 μg/mL),
which was superior to that of artemisinin 1. The diastereomeric
peroxide 43 also exhibited potent activity (IC50 = 0.38 μg/mL),
and 45 bearing a regioisomeric peroxide showed diminished but
appreciable activity (IC50 = 1.39 μg/mL). The other peroxides
46 and 49 also displayed anti-trypanosomal activities with IC50
values of 1.18 and 1.15 μg/mL, respectively, which are almost
equivalent to the efficacies of 1 and the corresponding s-trans
Our approach to produce collections of fused molecules with
structural novelty and complexity features concise access and
diversification of privileged structural motifs generated from a
proper selection of natural products with structural and func-
tional homologies. The high hit rate and the discovery of new
anti-trypanosomal agents demonstrated the effectiveness of this
approach and the potential to generate novel lead compounds
composed of natural product-like elaborated cores with densely
integrated sp3-hybridized stereocenters and heteroatom-containing
substituents.39 Although SAR studies of fused molecules have often
been limited to alterations of substituents and functionalities
attached to an identical core, we performed SAR studies exploit-
ing canonical sets of tricyclic skeletons with systematic variations
of the sp3 ring-junctions and cyclic arrays, which provided insight
into the stereoscopic view of pharmacophores. Accordingly, the
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synthetic approach exploring chemical space relevant to natural
products is a complementary means for screenings of small
molecules from natural sources and will play an increasingly
important and indispensable role in the development of biolo-
gically active agents without prior structural information about
the biological targets.
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Supporting Information. Experimental procedures and
b
1H and 13C NMR spectra of compounds 12ꢀ18, 22, 23, 26, 27,
31ꢀ52; complete refs 13c and 29m; CIF files for 43 and 52. This
material is available free of charge via the Internet at http://pubs.
acs.org.
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’ AUTHOR INFORMATION
Corresponding Author
oguri@sci.hokudai.ac.jp
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’ ACKNOWLEDGMENT
We thank Prof. Takanori Suzuki (Hokkaido University), Prof.
Kiyoshi Tsuge (Osaka University), and Ms. Yuko Fujimura
(Shionogi & Co., Ltd.) for performing the X-ray analyses and
Prof. Toshiaki Sunazuka (Kitasato University) for valuable
discussions. This work was supported by a Grant-in-Aid for
Young Scientists (A) 19681022 to H. Oguri and in part by the
Takeda Science Foundation, The Naito Foundation, Northern
Advancement Center for Science and Technology, the Drugs for
Neglected Diseases initiative (DNDi), a grant from the All
Kitasato Project Study (AKPS), and the Science and Technology
Research Partnership for Sustainable Development Program
(STREP) of the Japan Science and Technology Agency (JST).
We are grateful to Ms. Miyuki Namatame (Kitasato University)
for technical assistance.
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